Merci Philippe

Bernard

Gwénola

Thong, Mohamad, Chandru, Benjamin Merci À Cuong

Alain Annic

Halymenia Agardh

Durvillei (bory

Algues marines d'importance économique aux Philippines : évaluation de six espèces

Keywords: Caulerpa, Ulva, Sargassum, Halymenia, nutrition, biological activities Algues marines d'importance économique aux Philippines : évaluation de six espèces Rexie Magdugo 2020

transition des bourses

Acknowledgements

This study was funded by CHED-PhilFrance scholarships K12 transition program and in cooperation with Campus France, Embassy of France to the Philippines, and LBCM in University of South Brittany, Vannes, France. This study would not be possible without the help of many remarkable people whom I am forever grateful.

I am grateful to my thesis committee members: to Professor Nathalie Bourgougnon, my thesis adviser, and to Dr. Gilles Bedoux, my co-supervisor who welcomed me to their laboratory, for being there to monitor the status of my research work and for taking time to correct my dissertation paper for providing all the necessary support in the conduct of this study. Although I may not show it, and I am like a cloud floating freely in the sky, having my own world and decisions, but your suggestions and criticisms surely would not gone to waste for I remembered, treasured, taking notes, and learned from every bit of it. And for that, I will always give my respect, help and support to whatever request that you need, whether academic or non-academic, in the near future. And to Prof. Nathalie, thank you for being my Saint Thomas.

I am also grateful to Dr. Anicia Q. Hurtado, my supervisor to the Philippines for providing me alga samples, and for always checking on me as like a mother figure, who always wanted her children to succeed by simply just being there to cheer me up.

Thanks to Anne-Sophie and Romain, my first ever teachers in using some apparatuses. Thanks also to Nolwenn, Marie, Chrystel, and Kevin, I thank you all for your help during my study. I want you to know that I really learned a lot from your respective expertise. Thanks to Anaïs, Alice, and Widya. Thanks also to Hugo for politics, music, history, and whatever wholesome topics that we can discuss.

Thanks also for your help for some important analyses. Thank you, Philippe, Bérnard, and Gwénola for always helping me every time I needed your help.

To Dr. Laurent Vandajon, thank you for the friendship, for the trust, for believing. Thank you for sharing your wonderful ideas, and thank you for being true.

Thanks to Cuong, Thong, Mohamad, Chandru, Benjamin, Alain, Annic, Giséle, Tini, Moana, John, Gary and to all my friends in France. Thanks also to Filipino community in Bretagne together with their families, for welcoming me as their own family. Thanks to Abel, ate Jessie, Pascal, ate Marissa, to Ronrick for welcoming me at their home when I have nowhere to go. Thanks also to my family here in Europe; to Aunt Margie, aunt Thess, to ate Bibeth, and to my little cousin Dirly, thanks to all your support. I know there are still a lot of people to be mentioned, with all my heart, I truly cherished everyone's help during my stay here in France and I am grateful for that.

Big thanks to all.

Dedicated to the one who broke my heart,

-especially to the one who healed it-"Life is short and funny" RPMagdugo "Simplicity is the ultimate sophistication" Leonardo da Vinci "It is true intelligence for a man to take a subject that is mysterious and great in itself and to unfold and simplify it so that a child can understand it" John Taylor Special Acknowledgment To: Les Philippines sont connues pour sa riche flore, et ses 1291 taxons d'algues macrobenthiques marines qui représentent une production végétale naturelle importante et diversifiée. Sur 350 espèces considérées comme d'importance économique, seulement 5% d'entre elles sont utilisées, la plupart doivent encore être développées.

Commision on Higher Education K12 transition program and to the Embassy of France to the Philippines for its Foreign Postgraduate Scholarship under PhilFrance scholarships program

Les macroalgues vertes (Caulerpa et Ulva), brunes (Sargassum spp.) et rouges (Halymenia spp.) présentent de nombreuses application dans l'alimentation humaine ou animale, source de vitamines et de minéraux. Cependant, les propriétés nutritionnelles et les activités biologiques des espèces Caulerpa, Ulva, Sargassum et Halymenia aux Philippines sont peu connues. Par conséquent, il est intéressant de valoriser davantage ces algues pour diverses applications industrielles, telles que l'alimentaire, la pharmacologie et la cosmétique. Cette étude a été menée afin de fournir des informations supplémentaires sur le potentiel des espèces sauvages de Caulerpa, Ulva, Sargassum et Halymenia des Philippines. La présente étude traite (1) de la composition biochimique, des lipides, des protéines, des acides aminés, de la composition minérale comme valeurs nutritionnelles, et pour déterminer sa composition en acides gras à partir des lipides totaux; (2) caractérisation des polysaccharides de la paroi cellulaire, nature des sucres, spectres infrarouges, compositions pigmentaires; et (3) de l'évaluation des activités antivirales et antioxydantes des polysaccharides pariétaux dans un contexte de santé.

Mots-clés : Caulerpa; Ulva; Sargassum; Halymenia; nutrition; activités biologiques vi interest for the identification and discrimination of brown algal polysaccharides from S. polycystum (adapted from [START_REF] Vandanjon | Seasonal Variation of Sargassum Muticum Biochemical Composition Determined by Fourier Transform Infra-Red Spectroscopy[END_REF][START_REF] Sun | The stability and bioaccessibility of fucoxanthin in spray-dried microcapsules based on various biopolymers[END_REF][START_REF] Palanisamy | Investigation of antioxidant and anticancer potential of fucoidan from Sargassum polycystum[END_REF][START_REF] Kok | Physicochemical properties of edible alginate film from Malaysian Sargassum polycystum C. Agardh[END_REF][START_REF] Artemisia | The Properties of Brown Marine Algae Sargassum turbinarioides and Sargassum ilicifolium Collected from Yogyakarta, Indonesia[END_REF].

XXXI

List of infrared (IR) vibrational modes characteristic to polysaccharides used in determining spectral bands of interest for the identification and discrimination of brown algal polysaccharides from S. ilicifolium (adapted from [START_REF] Artemisia | The Properties of Brown Marine Algae Sargassum turbinarioides and Sargassum ilicifolium Collected from Yogyakarta, Indonesia[END_REF][START_REF] Kok | Physicochemical properties of edible alginate film from Malaysian Sargassum polycystum C. Agardh[END_REF][START_REF] Palanisamy | Investigation of antioxidant and anticancer potential of fucoidan from Sargassum polycystum[END_REF][START_REF] Vandanjon | Seasonal Variation of Sargassum Muticum Biochemical Composition Determined by Fourier Transform Infra-Red Spectroscopy[END_REF][START_REF] Marudhupandi | In vitro anticancer activity of fucoidan from Turbinaria conoides against A549 cell lines[END_REF][START_REF] Ale | Fucose-Containing Sulfated Polysaccharides from Brown Seaweeds Inhibit Proliferation of Melanoma Cells and Induce Apoptosis by Activation of Caspase-3 in Vitro[END_REF]. Plant specimen of C. racemosa (Forsskål) displayed at the local public market in Langihan, Butuan City, Philippines (personal documentation).

LIST OF FIGURES

2 Plant specimen of Ulva lactuca (Linnaeus) collected at Lawigan, Bislig City, Philippines (personal documentation).

3 Plant specimen of S. polycystum (C. Agardh) collected at Lamanok island, Anda, Bohol, Philippines (personal documentation).

4

Plant specimen of S. ilicifolium (Turner) C. Agardh, collected at Currimao, Ilocos Norte, Philippines (personal documentation).

5

Plant specimen of H. durvillei (Bory de Saint-Vincent) collected at Currimao, Ilocos Norte, Philippines (personal documentation).

6

Plant specimen of H. dilatata (Zanardini) collected at Lawigan, Bislig City, Philippines (personal documentation).

7 Evolutionary tree of the eukaryotes shows the position of red, green, and brown algae and the divergence of red algae, dinoflagellates, and brown algae (cited by [START_REF] Chandler | Structural Color in Marine Algae[END_REF], and taken from the paper of [START_REF] Cock | The Ectocarpus genome and the independent evolution of multicellularity in brown algae[END_REF]).

8

The new tree of eukaryotes showing Archaeplastida, and Stramenopilathe lineage of marine algae 9 Schematic cell wall present in green seaweeds, with natural polysaccharides [START_REF] Lahaye | Structure and functional properties of ulvan, a polysaccharide from green seaweeds[END_REF]. 12 Cell wall model of red algae from the order Halymeniales [START_REF] Stiger-Pouvreau | Carbohydrates from Seaweeds[END_REF].

13 Utilization of algae in France (production and imports) according to CEVA 2009 [START_REF] Mesnildrey | Seaweed Industry in Europe[END_REF].

14 Perspective map of the world and sampling locations in the Philippines showing the site collection of selected species of: green seaweeds (A) Caulerpa racemosa var. 19 Principle and reaction of the protein assay by the method of [START_REF] Smith | Measurement of protein using bicinchoninic acid[END_REF] (Burlot, 2016).

20

Principle and reaction of the determination of total sugars by the method of [START_REF] Dubois | Colorimetric Method for Determination of Sugars and Related Substances[END_REF] (Burlot, 2016).

21

Principle and reaction of the dosage of uronic acids by the method of [START_REF] Blumenkrantz | New method for quantitative determination of uronic acids[END_REF] modified by Filisetti-Cozzy et al. (1991) (Burlot, 2016).

22

Colorimetric determination of total phenols by the Folin-Ciocalteu [START_REF] Hardouin | Download citation of Production of water-soluble extracts from Ulva sp. using enzymatic hydrolysis processes: characterization, upgrading and potential development[END_REF]Burlot, 2016).

23

Principle and reaction of the determination of polysaccharidelinked sulfate groups by the method of [START_REF] Jaques | A microelectrophoresis method for heparin[END_REF]) (Burlot, 2016).

24

Incineration of the organic material of the samples in a muffle furnace at 585 °C (A), and obtaining the mineral material (B).

25

The first treatment of total or partition lipid extraction common to all groups of algae (A), washing and drying (B), and evaporation (C).

26

The second treatment of total lipid extraction common to all groups of algae: (A) Saponification, and (B) Esterification.

(adapted from [START_REF] Terme | Selective extraction of lipid classes from Solieria chordalis and Sargassum muticum using supercritical carbon dioxide and conventional solid-liquid methods[END_REF].

27

Extraction and isolation of polysaccharides common to all groups of algae.

28 Schematic diagram of sequential extraction of polysaccharides of Caulerpa racemosa (Forsskål) with inorganic solvents.

29

Routes of purification of sulfated polysaccharides, by successive precipitations and by dialysis of Ulva lactuca (Linnaeus).

30 Schematic diagram of sequential extraction of sulfated polysaccharides from S. polycystum and S. ilicifolium with inorganic solvents.

x 31 Schematic diagram of extraction and purification of sulfated polysaccharides from H. durvillei and H. dilatata with inorganic solvents (adapted from AS. Burlot, 2016).

32 CarboPac PA1 column. The quaternary amine functions of latex microbeads bind to SO3 groupings -the substrate of the column [START_REF] Rohrer | Analysis of Carbohydrates by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection[END_REF].

33

Operation of the PAD detector. 34 HPAEC-PAD system. 35

Chromatogram showing the retention time of the different standards of mixture 2 (A) and its peak summary

General introduction

Marine has a lot to offer to humanity, some of which are yet to be explored.

The Philippines, made up of 7,641 groups of Islands, which are mostly made up of coastal areas, estuaries, ponds lakes, mountainous areas, are areas of growth where alga thrives [START_REF] Rao | Indian seaweed resources and sustainable utilization: Scenario at the dawn of a new century[END_REF]. Research on seaweeds opened the door to the right knowledge and use [START_REF] Trono | The seaweed resources of the Philippines[END_REF]. It was also found out that there are over 350 species of seaweeds that are valued in the market [START_REF] Trono | Diversity of the seaweed flora of the Philippines and its utilization[END_REF]. A significant business venture to individuals who reside in the Philippines is one of the significant producers, along with China and Indonesia as the leading one (FAO, 2018;[START_REF] Trono | The seaweed resources of the Philippines[END_REF]. The strategic geographical location of the Philippines in the heart of the Coral Triangle as it is considered as the global epicenter of marine biodiversity [START_REF] Hehre | Differential Response of Fish Assemblages to Coral Reef-Based Seaweed Farming[END_REF], produces a large number of seaweeds and has proven to be favorable for the commercial farming of eucheumatoids [START_REF] Trono | The seaweed resources of the Philippines[END_REF].

The Philippines is known for its rich flora, and its marine algae are significant and diversified natural vegetable production. There are 1,291 taxa of marine macrobenthic algae with 306 published names of taxa (including species, varieties, and forms) of greens, 234 names of browns, and 751 names reds currently listed respectively on the Philippines littoral zones [START_REF] Ang | A verification of reports of marine algal species from the Philippines[END_REF]. Many of these species (350 species) are of economic importance as food, sources of industrial products such as polysaccharides, bioactive and nutritional natural products, and growth-promoting substances. However, about 5% of these are economically important; most have still to be developed [START_REF] Trono | Diversity of the seaweed flora of the Philippines and its utilization[END_REF].

Out from almost 30 genera of macroalgae that are cultivated worldwide, species from 17 genera are commercially cultivated which includes; Agardhiella, Eucheuma, Gelidium, Gigartina, Gracilaria, Hydropuntia, Hypnea, Kappaphycus, Meristotheca, Porphyra/Pyropia (Rhodophyta), Saccharina, Laminaria, Undaria, Cladosiphon (Phaeophyceae), Monostroma, Ulva, and Caulerpa (Chlorophyta).

Agardhiella, Gelidium, Gigartina, Porphyra/Pyropia, Saccharina, Laminaria, Undaria, Monostroma, and Ulva are cultivated in the temperate zone, and Eucheuma, Gracilaria, Hydropuntia, Hypnea, Kappaphycus, Cladosiphon, and Caulerpa are mainly grown in the tropics and subtropics (de [START_REF] De Gaillande | Caulerpa consumption, nutritional value and farming in the Indo-Pacific region[END_REF]).

In the Philippines, most species have been harvested in the Pacific coast since the early 60's when American Seaweed Company was forced to seek an alternative source of dried seaweeds due to Indonesia's (primary source) political problems.

Since then, farming technology was developed, and it is a sustainable harvest up to today for just a few species such as Kappaphycus alvarezii, and Eucheuma denticulatum, and non-traditional seaweed resources such as Caulerpa lentillifera

and Caulerpa racemosa as an export product, and some species of Gracilaria, while all others are gathered from wild stocks [START_REF] Trono | Diversity of the seaweed flora of the Philippines and its utilization[END_REF][START_REF] Trono | The seaweed resources of the Philippines[END_REF]. The Philippines used to be a world leader for the production of eucheumatoids but, due to ease of technology as well as other factors affecting production such as the frequent occurrence of typhoons, the Philippines slid down to second place next to Indonesia with more than 80% of the world's Eucheuma and Kappaphycus [START_REF] Trono | The seaweed resources of the Philippines[END_REF]. Other seaweed resources are likewise explored for their highvalue products, such as the brown seaweed Sargassum, and red seaweed Halymenia are sources of natural extracts in the form of biostimulants, hydrocolloids (alginate, sulfated polysaccharides rich in fucose or carrageenan) and pigments (fucoxanthine or phycoerythrin). Halymenia for example have essential, value-added industrial applications that could boost the local carrageenan industry for its lambda-type colloid, but also find commercial applications for its natural pigment phycoerythrin especially when a viable and cost-effective culture technique can be developed [START_REF] Trono | The seaweed resources of the Philippines[END_REF].

Contribution to Philippine economy

Asia as a prime producer of seaweed farming, large groups of islands came to the point of doing lucrative ways to get onto the top market of producers. The Philippines' total export of seaweeds and seaweed products has grown tremendously, which amounted to US$198 million in 2016. Recently, most seaweed products are exported in processed forms representing 65% of the total seaweed exports [START_REF] Trono | The seaweed resources of the Philippines[END_REF]. This has sustained fishers as an alternative income other than fishing, which gathered up considerable evidence that the activity is a profitable business venture amongst them, thus boosted the economy of the regions [START_REF] Crawford | Seaweed Farming: An Alternative Livelihood for Small-Scale Fishers[END_REF][START_REF] Sievanen | Weeding through assumptions of livelihood approaches in ICM: Seaweed farming in the Philippines and Indonesia[END_REF]. People from the Asian regions, especially the Philippines, have depended on their needs on the sea. Due to the fact, the coastal areas have a massive seaweed supply; thus, seaweed farming is one of the livelihoods a fisher can have for sustainable economic development in the countryside [START_REF] Trono | The seaweed resources of the Philippines[END_REF]. Unless alternative methods are used, subsistence fishing will decrease. In the Indo-Pacific region, seaweed farming consists mostly of small subsistence farms (~1 ha 2 ) [START_REF] Hehre | Differential Response of Fish Assemblages to Coral Reef-Based Seaweed Farming[END_REF].

In the country, 'hard data' were difficult to obtain from official sources since seaweed shipping channels were complex. However, the Seaweed Industry Association of the Philippines (SIAP) has pooled its commercial data to support common causes [START_REF] Siap | SIAP | Seaweed Industry Association of the Philippines[END_REF]. SIAP members reported total production of all types of dried seaweeds in the Philippines, with an average of almost 88,000 MT per annum, from 2011-2015. The Philippine government reported for 2015 that live seaweed production was in the order of 1,566,161 MT; this was a year when SIAP reported 101,900 MT dw production. This indicates a wet-to-dry ratio of approximately 15.4 to 1.0, which is more than 1.5 times the level of wet: dry ratio experienced by the industry. Despite unavailable precise numbers, general trends are still evident from consensus estimates production volumes and price trends.

Industry estimates of global Kappaphycus production, over the past decade, hovered in the range of 180,000 ±20,000 MT per year. This is roughly equivalent to 1.3-1.7 million MT fresh weight if one assumes a wet-to-dry ratio of 8.5:1. These numbers are consistent with industry production estimates; however, they are not consistent with FAO (2014) estimates supplied by government sources [START_REF] Hurtado | Tropical Seaweed Farming Trends, Problems and Opportunities: Focus on Kappaphycus[END_REF]. A study about seaweed farming as an alternative occupation for fishers [START_REF] Hill | The Interaction between Seaweed Farming as an Alternative Occupation and Fisher Numbers in the Central Philippines[END_REF]) perceived a decrease of fishing due to the availability of seaweed farming in some villages of the Philippines. Access to this has dramatically boosted the Philippines' economy and fed the poverty line. Thus, Seaweed farming is beneficial in the Philippines, but due to declines, it may be hard to maintain as seaweed farming emphasizes an opportunistic nature.

Seaweed farming

In the early 1950s, fishers began farming these green algae C. racemosa and C. lentillifera in fishponds initially used for the aquaculture of milkfish (Chanos chanos) cleared mangrove areas in the island of Mactan, Cebu, Philippines. The ponds were flooded to a depth of 0.5 to 0.6 m depending on the tidal range, and sluice gates-controlled water level and renewal. Pond's dams were constructed with dried black mud and clusters of coral stone. The area of the ponds ranges from 1000 m 2 to about 1 ha. Planting was done by burying (2 cm) or by merely spreading rhizoidal thalli or young plants in the mud or on the bottom, then harvesting after around 45 days by uprooting the plants from the mud leaving several plants to propagate new growth-each harvest lasting several days. The freshly collected seaweeds could be stored in baskets for up to 48 h (de [START_REF] De Gaillande | Caulerpa consumption, nutritional value and farming in the Indo-Pacific region[END_REF]. About 30 million T (wet weight) of red, brown, and green seaweeds were harvested in 2016, mainly for human food, animal feed, and hydrocolloids production. The most collected macroalgae from the wild and sold on the markets are Caulerpa species.

In contrast, the most widely cultivated species include Japanese kelp (Laminaria japonica), Eucheuma seaweeds, Kappaphycus alvarezii, and Undaria pinnatifida (FAO, 2018). However, farms can use a range of culture techniques.

Because of the increasing demand for food, mainly aquatic food in most regions of the world, the aquaculture industry became popular since the 1970s. It continued to have a significant contribution, especially for Caulerpa with total annual crop harvested in Fiji, Samoa, and Tonga Islands combined represented 123 T of wet material with a value of US$266,492. This suggests that, for every edible and potentially cultivatable species of interest, it should be genetically characterized by rapid molecular traceability methods [START_REF] De Gaillande | Caulerpa consumption, nutritional value and farming in the Indo-Pacific region[END_REF]. However, in Ulva species, the cultivation method is similar to Monostroma, a non-clonal marine plant that does not have fragmentation ability. In the natural seeding method, rope nets are submerged in places where this alga grows naturally, and once germination starts, the nets are taken and brought to the farm. In the artificial seeding method, gamete release and collection are induced from matured thalli with a combination of brief drying and floating in seawater with intense illumination. Gametes from thalli fragmentation used for its artificial seeding are allowed to settle on culture nets in dark conditions, and these nets are taken to the farm once germinated. These germinated algae are then cultivated on poles fixed in shallow, calm areas of the sea or in estuaries, where the nets are periodically exposed to air during low-tide.

Tank cultivation or also known as the "germling cluster method," has also been developed. Information on phylogenetics and phylogeography is known, making it one of the well-studied green algal lineages [START_REF] Bast | An Illustrated Review on Cultivation and Life History of Agronomically Important Seaplants[END_REF].

Due to the high potential yields of seaweeds' biomass, its utilization as raw materials in a biorefinery is a promising alternative to fossil resources to produce energy and chemicals. Seaweeds as an alternative source of energy could help reduce or mitigate greenhouse gas (GHG) emissions (by 42-48%) compared to the use of natural gas [START_REF] Pechsiri | Energy performance and greenhouse gas emissions of kelp cultivation for biogas and fertilizer recovery in Sweden[END_REF]) and thus enhance energy security. Every year, about 1 million T of algal phycocolloids are extracted in these industries worldwide [START_REF] Bixler | A decade of change in the seaweed hydrocolloids industry[END_REF], which nearly equal the number of industrial wastes disposed of in landfills and open sea, causing fouling smell and water pollution. Thus, the waste from these industries, which contains high amounts of carbohydrates, maybe a source of raw material for bioethanol or biogas production [START_REF] Teh | Dilute sulfuric acid hydrolysis of red macroalgae Eucheuma denticulatum with microwave-assisted heating for biochar production and sugar recovery[END_REF] . So technically, in this scenario, bioethanol production from seaweeds and industrial seaweed waste will reduce the usage of freshwater, synthetic fertilizers, and that agricultural land and will produce high-value commodities in an environmentally friendly biorefinery manner [START_REF] Baweja | Biology of Seaweeds[END_REF][START_REF] Maneein | A Review of Seaweed Pre-Treatment Methods for Enhanced Biofuel Production by Anaerobic Digestion or Fermentation[END_REF].

There have been many other reports in the recent years of ethanol production from various seaweeds such as Ulva pertusa, U. lactuca, Gelidium amansii, G.

elegans, Sargassum fulvellum, S. sagamianum, Chigaso crassifolia, Saccharina japonica, S. latissimi, Laminaria japonica, L. digitata, L. hyperborean, Nizimuddinia zanardini, Euchema sp., Gracilaria verrucosa, and G. Salicornia [START_REF] Baweja | Biology of Seaweeds[END_REF]. Bioconversion of algal material into bioethanol and other valuable co-products depends on the technologies available for converting biomass to fuels and has been applied in limited ways. For example, the high-water content of seaweed (ca. 80-90%) will harm the energy balance of applications dependent on dry biomass. This makes seaweed (wet biomass) undesirable for processes like; direct combustion, pyrolysis, and gasification, but suitable for processes that depend on the use of wet biomass. These methods include hydrothermal liquefaction for bio-oil production, fermentation for ethanol production, and anaerobic digestion (AD) for biogas production. All three methods are still under development to produce biofuel from seaweed [START_REF] Maneein | A Review of Seaweed Pre-Treatment Methods for Enhanced Biofuel Production by Anaerobic Digestion or Fermentation[END_REF]).

In the past years, there have been continuing efforts to optimize pretreatments to achieve better yields and lower costs, whether using microalgae or seaweed. Pre-treatments of biomass that modify the bioavailability of polysaccharides for their hydrolysis to sugars. Different pre-treatment methods (mechanical, thermal, enzymatic, chemical, biological) to improve cellular access to polysaccharide-hydrolysis agents were tested and improved [START_REF] Barbot | Anaerobic Digestion of Laminaria japonica Waste from Industrial Production Residues in Laboratory-and Pilot-Scale[END_REF][START_REF] Maneein | A Review of Seaweed Pre-Treatment Methods for Enhanced Biofuel Production by Anaerobic Digestion or Fermentation[END_REF]. Other factors to consider during different pre-treatment methods are the season of collection and location of seaweed biomass, salt content, heavy metal content, polyphenols, structural make-up, and the relative content of the different polymeric carbohydrates. The biogas production, sugars from hydrolyzed polysaccharides were converted to acetate, CO2, and H2 by acidogenesis and acetogenesis, and proceed to methane and CO2 by methanogenesis. While for ethanol production, glucose and galactose from the hydrolysis of the major polymers in red seaweed (e.g., carrageenan and agar), in brown seaweed (e.g., laminarin and fucoidan along with glucuronic and mannitol), and in green seaweed (e.g., cellulose, starch, and ulvan) were converted by the natural microbial process to pyruvate by glycolysis in anaerobic respiration, and then proceed to ethanol and CO2 by alcoholic fermentation [START_REF] Maneein | A Review of Seaweed Pre-Treatment Methods for Enhanced Biofuel Production by Anaerobic Digestion or Fermentation[END_REF].

Currently, seaweed biomass is harvested for high-value natural products and food only. A biorefinery approach can sustainably produce and maximize biofuel and process co-products on a condition to meet the required and appropriate pretreatments for different seaweeds. The future of biofuel production from seaweeds depends on developing new technologies, with an appropriate and market-friendly regulatory environment [START_REF] Baweja | Biology of Seaweeds[END_REF]). Therefore, it is possible to design suitable seaweed pre-treatments that will maximize the sustainability and energy return of investments (EROI) for biofuel production at the lowest cost [START_REF] Maneein | A Review of Seaweed Pre-Treatment Methods for Enhanced Biofuel Production by Anaerobic Digestion or Fermentation[END_REF].

Ecological impact of seaweed farming

Problems on aquaculture worldwide have been afloat for some time as sectors failed to regulate or due to poor management of the areas. These problems are caused by the expansion of the cultured regions, spike of aquaculture installations, and the increased feed resources produced away from the immediate area [START_REF] Soto | Applying an ecosystem-based approach to aquaculture: Principles, scales and some management measures[END_REF]. Human activities such as aquaculture will lead to some loss of biodiversity; this may be a minimal change or crucial to an extent, is a concept by Odum, who pioneered the relationship between human activity and the natural process of biodiversity [START_REF] Odum | Seaweeds as a Source of Proteins for Use in Pharmaceuticals and High-Value Applications[END_REF]. Ecological issues are the focal point of interest rather than the changed ecosystem to an agro-ecosystem.

Seaweed aquaculture is a complex sector conditioned by environmental, biological, and external factors, relying on cultivation techniques and sites which may face environmental issues, and subject to legislation and market demand [START_REF] Barbier | PEGASUS-PHYCORMORPH European Guidelines for a Sustainable Aquaculture of Seaweeds[END_REF]. As seaweeds are common in Asia, factors such as nutrient levels, phytoplankton blooms, temperature, turbidity, and fluctuations are the significant ones that may affect the yield of the product [START_REF] Beveridge | Aquaculture and the environment: the supply of and demand for environmental goods and services by Asian aquaculture and the implications for sustainability[END_REF].

Continuing to the effects on the study on impacts of effluents of aquaculture, it has been sought out that it contains negative impacts on the particular area where it has been done, this is due to particulate matters which are desecrated by the types of machinery; this may be inorganic or organic components that may harm the existing living organisms [START_REF] Piedrahita | Reducing the potential environmental impact of tank aquaculture effluents through intensification and recirculation[END_REF]. Due to aquaculture installations, the place's ecosystem might be at risk due to alteration of ecosystem structure and function, as natural bioturbation may change due to ecological disturbances that may cause greater damage to the locality [START_REF] Piedrahita | Reducing the potential environmental impact of tank aquaculture effluents through intensification and recirculation[END_REF][START_REF] Barbier | PEGASUS-PHYCORMORPH European Guidelines for a Sustainable Aquaculture of Seaweeds[END_REF].

Because of increasing aquatic food demand in the early 1960s, aquaculture farms were booming in the Philippines, and many of these farms were in mangrove areas. At present, the conversion of mangroves into ponds has been banned because of its environmental impact, and therefore less Caulerpa production [START_REF] Tanduyan | Off bottom culture of Caulerpa lentillifera in three different water levels in the marine waters of San Francisco, Cebu, Philippines[END_REF]. Also, harvesting of the brown seaweed Sargassum from the wild has been banned since 2014 due to its ecological importance [START_REF] Trono | The seaweed resources of the Philippines[END_REF].

Because of this prohibition, cultivation of Caulerpa was tried in open and natural waters at different culture media, at different water levels, at different sites using:

1. plastic screen cage, 2. tubular plastic screen, 3. nylon screen cage, and 4. tubular nylon screen at the surface, middle, and bottom water at muddy, sandy, or rocky sites [START_REF] Tanduyan | Off bottom culture of Caulerpa lentillifera in three different water levels in the marine waters of San Francisco, Cebu, Philippines[END_REF]. At the same time, it hurt the livelihood opportunities of some sectors and the young Sargassum industry [START_REF] Trono | The seaweed resources of the Philippines[END_REF]. In the study of Tanduyan et al. the plastic screen cage is the best culture method, the middle layer is the best water level, and there is no significant difference in the culture sites [START_REF] Tanduyan | Off bottom culture of Caulerpa lentillifera in three different water levels in the marine waters of San Francisco, Cebu, Philippines[END_REF]. The need to protect natural habitats while providing sufficient raw materials for an industry that is looking for opportunities to utilize local resources to produce value-added products must be balanced and based on scientifically proven evidence [START_REF] Barbier | PEGASUS-PHYCORMORPH European Guidelines for a Sustainable Aquaculture of Seaweeds[END_REF][START_REF] Trono | The seaweed resources of the Philippines[END_REF]. This can only be addressed by developing appropriate sustainable culture technologies that are on-going [START_REF] Trono | The seaweed resources of the Philippines[END_REF].

Seaweeds, unlike terrestrial plants, do not compete for arable land, do not require the use of freshwater, fertilizers, or pesticides to grow much faster than land plants. The seaweeds' cell wall consists of alginates, carrageenan, agar, cellulose, hemicellulose, fucoidan, ulvan, and protein [START_REF] Trono | Diversity of the seaweed flora of the Philippines and its utilization[END_REF][START_REF] Stiger-Pouvreau | Carbohydrates from Seaweeds[END_REF]. Aquaculture is one of the main livelihoods and fastest-growing producers of the world. It is more than ever, harder to prevent environmental degradation due to aquaculture intensification. This would most likely affect a large area to the places in Asia, where it is more common [START_REF] Ahmed | Sustainable Intensification of Aquaculture[END_REF]. Sites such as the eastern countries may have a hard time doing this since they are new to the aquaculture world and barely have traditions in using the pollutants as products. A new ecological concept in agriculture addresses these problems, which are the Ecological intensification concepts. It is made to address the challenges of producing the human population's sufficient needs and the thought of conserving the environment at the same time. The goal of this concept is to protect and preserve the remaining resources and the quality of living of humans [START_REF] Aubin | Implementing ecological intensification in fish farming: Definition and principles from contrasting experiences[END_REF].

Harvesting due to intensification has provided with the right produce provided but soon may change since the population is continuously rising; thus, demands are higher. The concept was later adopted in the eastern regions of the globe, Asia.

China, the leading seafood producer, treats effluents as products rather than pollutants, commonly used as enhancements of natural stocks. The two primary forms of an Integrated multi-trophic Aquaculture or IMTA are sea-ranching and suspended aquaculture. This tradition opened a path to intensive aquaculture on the scale and intensity of Chinese waters [START_REF] Leung | More rapid and severe disease outbreaks for aquaculture at the tropics: implications for food security[END_REF]. However, this does not apply to the western world as they are recent to aquaculture and have no traditions on the usage of effluents as inputs, which it is difficult for them in following IMTA.

Nonetheless, seaweed farming may impact fishing behavior due to pieces of machinery, expansion of areas, overproduction of goods, and disturbance in the ecosystem. Seaweed farming often leads to inmigration, which attracted outsiders in Tawi-Tawi and Sibutu, Philippines [START_REF] Sievanen | Weeding through assumptions of livelihood approaches in ICM: Seaweed farming in the Philippines and Indonesia[END_REF]. To prevent overexploitation of the natural beds, most seaweed industries sourced research to improve knowledge and understanding of the seaweed diversity, sustainable harvesting techniques, and the development of efficient culture systems to cope up with increasing global demands. As this became a thing, Sulu archipelago begins advertising and attracting people to participate in a new way of earning money [START_REF] Trono | The seaweed resources of the Philippines[END_REF].

Seaweed species selected in this study

The selected species of green (C. racemosa and U. lactuca), brown (S. The genus Sargassum is the most dominant genus in the Philippines. The reason for selecting S. polycystum as specimen was because of its easier identification than other Sargassum species, while S. ilicifolium was utilized as food. Although exploitation of this genus Sargassum began in the 1970s for the production of organic liquid fertilizer, it was just recently tested for possible cultivation [START_REF] Trono | The seaweed resources of the Philippines[END_REF]. Both H. durvillei and H. dilatata were utilized as food in the Philippines [START_REF] Trono | The seaweed resources of the Philippines[END_REF]. The H. durvillei is one of the seven emerging red seaweeds from the Philippines as a potential alternative source of lambda-carrageenan, and r-phycoerythrin [START_REF] Trono | The seaweed resources of the Philippines[END_REF][START_REF] Hurtado | Chapter Two -Selected red seaweeds from the Philippines with emerging high-value applications[END_REF].

Less research on Halymenia species in the Philippines [START_REF] Trono | The seaweed resources of the Philippines[END_REF].

Green seaweeds (C. racemosa and U. lactuca)

The thallus of Caulerpa is formed by a giant multinucleated cell (coenocytic organization), which develops into a complex branching siphon. Caulerpa is characterized by this siphon (called trabeculae) in an inward projection of the wall.

The thallus is composed of upright fronts arising from creeping stolons anchored by branched rhizoids. These vertical branches bear distinctive branchlets (called metamers) and are used for morphological species identification. (de Gaillande, 2017). Specimen of C. racemosa shown in Fig. 1. The thalli of Ulva are composed of two layers of cells in which each cell contains a cup-shaped chloroplast [START_REF] Baweja | Biology of Seaweeds[END_REF]. The plant is greenish to yellowish, often twisted and crumpled in appearance, and with irregular perforations [START_REF] Hurtado-Ponce | Seaweeds of Panay[END_REF]. This macroalga of the genus Ulva is also known as Sea lettuce [START_REF] Bast | An Illustrated Review on Cultivation and Life History of Agronomically Important Seaplants[END_REF]. Macroalgae having a resemblance to lettuce are, in fact, U. fasciata, U. lactuca, or that Ulva genus that has distromatic blades. At the same time, commercially cultivated species include U. prolifera (=Enteromorpha prolifera) and U. intestinalis tubular walls with walls of the tube one cell thick. Thalli are coarsely filamentous or foliaceous at 5-12 cm diameter [START_REF] Hurtado-Ponce | Seaweeds of Panay[END_REF]) that can be branched or unbranched and have disc-like holdfasts developed from basal cells that attach firmly on suitable substrata like pebbles or rocks. Plant specimen of Ulva lactuca shown in Fig. 2.

Classification of green algae

Classification of green algae

Brown seaweeds (S. polycystum and S. ilicifolium)

In the Philippines, the genus Sargassum is commonly known as Samo (Cebuano), while S. polycystum is called Aragan (Iloku), and Kulapu (Hiligaynon) [START_REF] Hurtado-Ponce | Seaweeds of Panay[END_REF]). The S. polycystum has a cylindrical, small spiny thallus with a disc-shaped holdfast and air bladders in every corner. Typical characteristics of S. polycystum are their small, pointed and jagged blades. The lower part of the main axis giving rise to stolon-like horizontal branches, often bearing secondary haptera from which a new plantlet may arise [START_REF] Mattio | 190 Years of Sargassum Taxonomy, Facing the Advent of DNA Phylogenies[END_REF]. Consequently, they inhabit mostly along the coast with 0.5-5 m. Plant specimen of S. polycystum shown in Fig. 3.

According to Guiry and Guiry (2020)

Red seaweeds (H. durvillei and H. dilatata)

The H. durvillei is one of the new species developed for cultivation in the Philippines. It is characterized by large, bushy thalli that may grow up to 35 cm. It is soft, cartilaginous, and slimy when fresh and found in upper subtidal attached to the rocky substrate by its discoid holdfast [START_REF] Trono | The seaweed resources of the Philippines[END_REF]. It is abundant

Objectives of the study

Philippines is a source of enormous number of rich natural resource, especially the marine environment. And yet, little is known about the nutritional properties, health values and biological activities of the wild green (C. racemosa and U. fasciata), brown (S. polycystum and S. ilicifolium), and red (H. durvillei and H. dilatata) economically important seaweeds species from the Philippines.

Therefore, this study was conducted to give additional information on the potential of these wild seaweed species for various industrial applications, and it is divided into two parts.

Part I: The first part focuses on "Nutritional values" by:

1. determining the biochemical composition, especially proteins and its amino acids, ash and its mineral composition; and

Part II: The second part focuses on the "Health value and biological activities" by: 2. extracting polysaccharides fractions using appropriate methods for each seaweed;

3. determining each of its polysaccharides fraction yields;

4. characterization of cell wall polysaccharides fractions;

5. determining its polysaccharides sugar compositions, infrared spectra;

6. evaluation of the antiviral and antioxidant (DPPH radical scavenging) activities of polysaccharides; and 7. analyses of lipids and its fatty acids, and pigments composition.

Chapter 1 Review of Literature [START_REF] Graham | Deep-water kelp refugia as potential hotspots of tropical marine diversity and productivity[END_REF]) and growth is usually vertical, looking for sunlight [START_REF] Gomez-Zavaglia | The Potential of Seaweeds as a Source of Functional Ingredients of Prebiotic and Antioxidant Value[END_REF]. Some algae are microscopic such as the phytoplankton that provides the base for most marine food chains. Some are enormous like the giant kelp, and most are medium-sized that come in colors of red, green, and brown that randomly wash up on beaches and shorelines everywhere [START_REF] Guiry | HOW MANY SPECIES OF ALGAE ARE THERE?[END_REF]. These primary producers (protista microorganisms) are responsible for 30-50% of the oxygen in the atmosphere [START_REF] Gomez-Zavaglia | The Potential of Seaweeds as a Source of Functional Ingredients of Prebiotic and Antioxidant Value[END_REF]. Aside from its function as ecosystem engineers in reef-building communities, macroalgae also serve as a refuge for fish and invertebrates, serve as a food source for human consumption, and are harvested to extract useful natural products such as hydrocolloids and pigments. The multitude of seaweeds applications in various fields and industries led to a global increase in demand for the biomass of a variety of seaweed species [START_REF] Trono | The seaweed resources of the Philippines[END_REF]. All lineages of these aquatic groups have primary plastids, organelles that originated directly from cyanobacteria [START_REF] Burki | The New Tree of Eukaryotes[END_REF].

Taxonomy and classification

More than 30,000 species of algae have been described (Guiry 2020;[START_REF] Gomez-Zavaglia | The Potential of Seaweeds as a Source of Functional Ingredients of Prebiotic and Antioxidant Value[END_REF]). In the biological sense of the word, algae are used to designate a vast, varied, and heterogeneous group of organisms, at present, classification is not well defined, and taxonomic status may vary according to the authors, under constant and rapid revision at all levels [START_REF] Gomez-Zavaglia | The Potential of Seaweeds as a Source of Functional Ingredients of Prebiotic and Antioxidant Value[END_REF]. The groupings of the macroalgae: green (Chlorophyta), the red (Rhodophyta), both belonged to kingdom Plantae; and brown (Ochrophyta) of kingdom Protista, as well as microalgae kingdom, were grouped according to their pigment composition, and through their photosynthetic activity to carry out autotrophic feeding. Other authors include some green macroalgae in another differentiated group and at a taxonomic level equivalent to Chlorophyta, which are responsible for life expanding beyond the oceans, and are precursor of terrestrial plants. Table I showed the summary of the phylogeny of these four large groups of macroalga. The taxonomic classification of algae classes is very complicated due to the number of existing varieties and many applicable classification criteria [START_REF] Gomez-Zavaglia | The Potential of Seaweeds as a Source of Functional Ingredients of Prebiotic and Antioxidant Value[END_REF], such as the nature and locations of the reserve glucans, the nature of their cell walls, the number of periplastidial membranes, the shape of the mitochondrial crystal, the presence and ultra-structure of flagella, the type of mitosis, the mode of nutrition, reproduction, and habitat [START_REF] De Reviers | Morphology and systematics of the genus Halymenia C. Agardh (Halymeniales, Rhodophyta) in the Philippines[END_REF]. Seaweed is the common name for approximately 15,000 species of marine plants and algae that grow in the ocean and rivers, lakes, and other water bodies [START_REF] Chandler | Structural Color in Marine Algae[END_REF][START_REF] Gomez-Zavaglia | The Potential of Seaweeds as a Source of Functional Ingredients of Prebiotic and Antioxidant Value[END_REF]. By the early 2000s, a model of the tree emerged that divided almost all known eukaryote diversity among five to eight significant taxa, usually referred to as 'supergroups' [START_REF] Burki | The New Tree of Eukaryotes[END_REF]. The category of the supergroup was a purely informal one, denoting extensive assemblages that contain, for example, the traditional 'kingdoms' like Metazoa and Fungi as subclades. A typical list of these groups included Archaeplastida that is also known as Plantae. Archaeplastida is distinguished by the presence of primary plastids, also known as the photosynthetic organelles deriving directly from cyanobacteria by primary endosymbiosis around 1500 Mya [START_REF] Chandler | Structural Color in Marine Algae[END_REF][START_REF] Burki | The New Tree of Eukaryotes[END_REF]. In comparison, the divergence of the brown algae (Phaeophyceae), as a consequence of secondary endosymbiosis, or the occurrence of a separate distinct iridescent bodies (with structural coloration)

suggest that it may have evolved independently in brown algae occurred relatively recently, at around 200 Mya (Fig. 7) [START_REF] Chandler | Structural Color in Marine Algae[END_REF]. The three main groups with primary plastids are the green algae and land plants, the red algae, and glaucophyte algae [START_REF] Burki | The New Tree of Eukaryotes[END_REF]). Today, Archaeplastida is generally considered a supergroup, although most phylogenomic analyses do not actively support its monophyly (Fig. 8). Hence, the three taxa that comprise Archaeplastida are the Chloroplastida (green algae and land plants), Rhodophyta (red algae), and Glaucophyta. All three lineages have primary plastids, which are photosynthetic organelles that originated directly from cyanobacteria [START_REF] Burki | The New Tree of Eukaryotes[END_REF]. The clade 'SAR' (Stramenopiles, Alveolates, and Rhizaria) has been estimated to comprise up to half of all eukaryote species [START_REF] Burki | The New Tree of Eukaryotes[END_REF]. Most macroalgae come from either brown alga (class Phaeophyceae) or red algae (phylum Rhodophyta). A few marine macroalgae occur in the phylum Charophyta since about 600 large green algal taxa are in the phylum Chlorophyta (C. [START_REF] Dawes | Macroalgae systematics[END_REF]. Macroalgae in these four groups have more fundamental differences (cytological, chemical, and life histories) between them (C. J. [START_REF] Dawes | Response of the tropical red seaweed Gracilaria cornea to temperature, salinity and irradiance[END_REF].

Since then, advancements in phycology have resulted in significant changes in the classification of algae [START_REF] Wynne | Addendum to the synoptic review of red algal genera[END_REF], and the number of seaweed species has increased. There are 3330 species of green algae, 2060 brown algae, and 7102 red algae worldwide [START_REF] Stiger-Pouvreau | Carbohydrates from Seaweeds[END_REF].

Biochemical composition

Among marine macroorganisms, seaweed gained considerable significance due to its utilization as a precious raw material for the extraction of various valuable materials, such as polyphenols, organic pigments, proteins, unsaturated fatty acids, and polysaccharides, and other biochemical compounds [START_REF] Al-Nahdi | The Effect of Extraction Conditions on Chemical and Thermal Characteristics of Kappa-Carrageenan Extracted from Hypnea bryoides[END_REF].

Seaweeds are among the wealthiest sources of known and novel bioactive compounds, and the biological activity of compounds derived from this group has been extensively reviewed (Bedoux et al. 2016;[START_REF] Hurtado | Chapter Two -Selected red seaweeds from the Philippines with emerging high-value applications[END_REF][START_REF] Theophilus | Chapter Six -Dictyotaceae (Dictyotales, Phaeophyceae) species from French Polynesia: Current knowledge and future research[END_REF]. However, the biochemical composition of algae varies according to species, location and season of collection, growth conditions or sensitivity of methods used were seen in lipids and fatty acids [START_REF] Kelman | Antioxidant Activity of Hawaiian Marine Algae[END_REF][START_REF] Kok | Physicochemical properties of edible alginate film from Malaysian Sargassum polycystum C. Agardh[END_REF][START_REF] Artemisia | The Properties of Brown Marine Algae Sargassum turbinarioides and Sargassum ilicifolium Collected from Yogyakarta, Indonesia[END_REF][START_REF] Hurtado | Chapter Two -Selected red seaweeds from the Philippines with emerging high-value applications[END_REF], in polysaccharide yields [START_REF] Chattopadhyay | Polysaccharides from Caulerpa racemosa: Purification and structural features[END_REF][START_REF] Ji | Separation of the polysaccharides in Caulerpa racemosa and their chemical composition and antitumor activity[END_REF][START_REF] Kok | Physicochemical properties of edible alginate film from Malaysian Sargassum polycystum C. Agardh[END_REF][START_REF] Artemisia | The Properties of Brown Marine Algae Sargassum turbinarioides and Sargassum ilicifolium Collected from Yogyakarta, Indonesia[END_REF], in minerals and ash contents [START_REF] Kelman | Antioxidant Activity of Hawaiian Marine Algae[END_REF][START_REF] Alam Bhuiyan | Proximate Chemical Composition of Sea Grapes Caulerpa racemosa (J. Agardh, 1873) Collected from a Sub-Tropical Coast[END_REF][START_REF] Garcia | Nutritional Potential of Four Seaweed Species Collected in the Barbate Estuary (Gulf of Cadiz, Spain)[END_REF][START_REF] De Gaillande | Caulerpa consumption, nutritional value and farming in the Indo-Pacific region[END_REF][START_REF] Wanyonyi | Kappaphycus alvarezii as a Food Supplement Prevents Diet-Induced Metabolic Syndrome in Rats[END_REF]Wells et al. 2017;[START_REF] Kok | Physicochemical properties of edible alginate film from Malaysian Sargassum polycystum C. Agardh[END_REF][START_REF] Artemisia | The Properties of Brown Marine Algae Sargassum turbinarioides and Sargassum ilicifolium Collected from Yogyakarta, Indonesia[END_REF], in β-carotene and other pigments (Paul et al. 2013), and proteins and amino acids [START_REF] Kelman | Antioxidant Activity of Hawaiian Marine Algae[END_REF][START_REF] Anh | Potential uses of gut weed Enteromorpha spp. as a feed for herbivorous fish[END_REF][START_REF] Hardouin | Enzyme-assisted extraction (EAE) for the production of antiviral and antioxidant extracts from the green seaweed Ulva armoricana (Ulvales, Ulvophyceae)[END_REF][START_REF] Garcia | Nutritional Potential of Four Seaweed Species Collected in the Barbate Estuary (Gulf of Cadiz, Spain)[END_REF][START_REF] De Gaillande | Caulerpa consumption, nutritional value and farming in the Indo-Pacific region[END_REF][START_REF] Kok | Physicochemical properties of edible alginate film from Malaysian Sargassum polycystum C. Agardh[END_REF][START_REF] Artemisia | The Properties of Brown Marine Algae Sargassum turbinarioides and Sargassum ilicifolium Collected from Yogyakarta, Indonesia[END_REF]. Macroalgae have many uses; food for human consumption, ingredients in cosmetics and fertilizers, treated products for extracting gelling agents, or animal feed additives [START_REF] De Gaillande | Caulerpa consumption, nutritional value and farming in the Indo-Pacific region[END_REF]. They are mainly used as food in the form of fresh vegetables. Their popularity tends to be on the increase due to the general awareness of health-conscious consumers of the advantages of its natural products.

Minerals

Seaweeds are absorbed from the sea, an incomparable wealth of mineral elements and are known as excellent sources of vitamins and minerals, especially potassium and iodine (Leandro et al. 2020). However, nutritional generalization about algal mineral contents is difficult because of sometimes significant seasonal, geographic, and taxonomic variations in mineral contents of marine algae (Wells et al. 2017). Green algae Caulerpa and Ulva present many uses as human or animal food, source of minerals like (Ca, K, Mg, Na, Cu, Fe, Zn), or vitamins E. Brown algae, particularly from the genera Laminaria, are a great source of iodine. The highest concentration of iodine is in L. japonica, 5.6, and 3.0 mg kg -1 from all investigated algae [START_REF] Mišurcová | Chapter 7: Chemical Composition of Seaweeds[END_REF]. The high ash content can reach up to 50.2%

of dw in H. floresii and represent a high content in minerals [START_REF] Garcia | Nutritional Potential of Four Seaweed Species Collected in the Barbate Estuary (Gulf of Cadiz, Spain)[END_REF].

Other red seaweed species such as Gracilaria spp., Gelidium sp., and Porphyra sp.

reported varying ash contents from 7.72% to 29.06% (Alam Bhuiyan and Qureshi, 2016), and 58% from K. alvarezii [START_REF] Wanyonyi | Kappaphycus alvarezii as a Food Supplement Prevents Diet-Induced Metabolic Syndrome in Rats[END_REF]. Table II showed mineral composition results from different groups of seaweeds. The percentages of the recommended daily intake covered by an intake of 8g of dry seaweed are also presented as well as the daily recommended intake allowance (MacArtain et al.

2007).

Proteins

Seaweed and microalgae are considered a viable source of protein. Some seaweed and microalgae species are known to contain protein levels similar to those of traditional protein sources, such as meat, egg, soybean, and milk, as cited by [START_REF] Bleakley | Algal Proteins: Extraction, Application, and Challenges Concerning Production[END_REF]. Algae use for protein production has several benefits over traditional high-protein crop use in terms of productivity and nutritional value [START_REF] Bleakley | Algal Proteins: Extraction, Application, and Challenges Concerning Production[END_REF]. Proteins of marine algae have attracted less attention, and their structure and biological properties are not as widely documented as their polysaccharides [START_REF] Chojnacka | Biologically Active Compounds in Seaweed Extracts -the Prospects for the Application[END_REF]. However, recently, they have gained a great deal of interest due to their functional properties and bioactivity potential for further application [START_REF] Michalak | Algae as production systems of bioactive compounds[END_REF][START_REF] Xu | Recent Advances in Marine Algae Polysaccharides: Isolati,on, Structure, and Activities[END_REF].

Proteins are shown in Table III. Other nutritional values differ from phylum to phylum and from species to species in seaweeds ( 0.02-9.0 0.7-1.1 0.6-1.9 0.6-1.6 7.8≥22.0 0.02-81.3 0-2.5 0-5.8 0-0.8 C. lentillifera a 0.02-7.0 0.3-3.9 0.6-5.9 0.4-4.1 0.3≥100 0.02-21.4 0.002-3.5 0.001-7.9 0-0. (Øverland et al. 2019). However, their protein content in different species is dependent on the season. It is linked to their surrounding variables, including nutrient supply [START_REF] Gaillard | Amino acid profiles of nine seaweed species and their in-situ degradability in dairy cows[END_REF], or even the sensitivity of methods used [START_REF] Gaillard | Amino acid profiles of nine seaweed species and their in-situ degradability in dairy cows[END_REF][START_REF] Øverland | Marine macroalgae as sources of protein and bioactive compounds in feed for monogastric animals[END_REF]. [START_REF] Gaillard | Amino acid profiles of nine seaweed species and their in-situ degradability in dairy cows[END_REF] reported that Porphyra sp. had an average crude protein (CP) content (33.4% dw), which is comparable to high protein seeds such as soybeans (35.0% dw) indicates a suitable substitute for soybeans in terms of CP content. Furthermore, the brown seaweeds (Alaria sp., Laminaria sp., and Saccharina sp.) had CP content that ranges from 6.8 to 16.4% dw and further explained that the higher CP content might be influence by the amount of sunlight and nutrients during spring season compared to autumn season [START_REF] Gaillard | Amino acid profiles of nine seaweed species and their in-situ degradability in dairy cows[END_REF]. Crude protein content is measured based on the assessment of crude protein from overall nitrogen content using the traditional nitrogen-to-protein (N-Prot) conversion factor of 6.25, while protein (true protein) is quantified using the sum of amino acid residues [START_REF] Biancarosa | Amino acid composition, protein content, and nitrogen-to-protein conversion factors of 21 seaweed species from Norwegian waters[END_REF]. While histidine (0.11-4.11% dw), methionine (0.17-5.12% dw), and tyrosine (0.44-3.9% dw) were the limiting amino acids in all seaweeds except H. porphyroides.

The abundance of glutamic and aspartic acids is responsible for the characteristic flavor and taste of seaweeds [START_REF] Gaillard | Amino acid profiles of nine seaweed species and their in-situ degradability in dairy cows[END_REF]. These results suggest that the seaweed species mentioned above could be potentially used as a nutritious and functional food item for human consumption (Alam Bhuiyan and Qureshi 2016). 6.2 5.1 6.6 5.8 6.9 5.0 2.6 1.4 9.2 5.8 12.7 6.5 6.4 7.6 3.8 5.1 C. fastigiata c 4.8 6.1 7.1 4.0 8.7 6.6 2.2 1.5 10.1 6.2 10.7 7.1 5.5 6.2 3.9 7.7 Ulva fasciata c 5.1 5.7 5.1 3.9 7.6 5.1 2.4 0.9 13.0 5.8 12.6 6.5 5.6 8.5 3.3 4.6 Alaria sp. d 4. 88 5.59 5.44 3.75 6.18 4.22 1.42 1.95 11.32 5.14 31.19 5.25 4.25 17.58 -3.62 Laminaria sp. d 5.69 6.35 5.65 4.48 7.55 5.03 1.75 2.0 13.68 5.55 15.31 6.13 4.81 12.23 -4.95 Saccharina sp. d 5.92 6.54 5.49 4.4 7.56 4.94 1.53 2.11 13.23 5.51 22.33 6.17 4.75 12.18 -4 In the paper of [START_REF] Hamed | Marine Bioactive Compounds and Their Health Benefits: A Review: Importance of functional seafood…[END_REF], the green alga Ulva rigida contains Leu, Phe, and Val as primary essential amino acids. EAA/NEAA ratio for red and green seaweeds at around 0.7 and lower ratio for brown seaweeds.

Sugars

In general, total sugars were abundant components compared to proteins, phenols, and flavonoids [START_REF] Hao | Chemical composition and immunostimulatory properties of green alga Caulerpa racemosa var Peltata[END_REF]). In the last decades, sulfated polysaccharides from algae have attracted much attention as functional additives in the pharmaceutical field, food, and cosmetic industries. The major sulfated polysaccharides found in marine algae are ulvan (green algae), sulfated polysaccharide rich in fucosefucan and fucoidan-(brown algae), and carrageenan (red algae) [START_REF] Cunha | Sulfated Seaweed Polysaccharides as Multifunctional Materials in Drug Delivery Applications[END_REF].

Cell wall

Seaweed cell walls represent at least 50% of its dry weight and typically consist of a fibrillar skeleton and an amorphous embedding matrix [START_REF] Stiger-Pouvreau | Carbohydrates from Seaweeds[END_REF]. Marine algae are well-known by their chemically complex and heterogeneous structure, i.e., sulfated and branched polysaccharides associated with proteins and various bound ions, including calcium and potassium. The number and chemical structure of these polymers vary according to the specific algal species [START_REF] Cunha | Sulfated Seaweed Polysaccharides as Multifunctional Materials in Drug Delivery Applications[END_REF].

Green seaweeds

In green algae, it has polymer assemblage cell walls similar to the cellulose, pectins, hemicelluloses, and exceptionally arabinogalactan proteins (AGPs).

Ulvophyceae seaweeds have abundant fibrillar constituents' cell wall components.

Most may change from cellulose to β-mannans to β-xylans during different life cycle phases; these also produce complex sulfated polysaccharides, AGPs, and extensin. Chlorophyceae green algae had a wide array of walls ranging from cellulose-pectin complexes to ones made of hydroxyproline-rich glycoproteins [START_REF] Domozych | The Cell Walls of Green Algae: A Journey through Evolution and Diversity[END_REF]. The schematic diagram of the green seaweed [START_REF] Lahaye | Structure and functional properties of ulvan, a polysaccharide from green seaweeds[END_REF] is shown in Fig. 9. Cell wall polymers common to green seaweed, particularly for Caulerpa and Ulva species is shown in Table V (Holdt and Kraan, 2011).

Figure 9. Schematic cell wall present in green seaweeds, with natural polysaccharides [START_REF] Lahaye | Structure and functional properties of ulvan, a polysaccharide from green seaweeds[END_REF].

Polysaccharides, especially extracellular polysaccharides derived from Caulerpa, are complex in composition and structurally diverse. It has water-soluble polysaccharides (mostly glucans and sulfated polysaccharides). These sulfated polysaccharides are mainly of sulfated galactose (commonly of xylose, glucose, and mannose). The cell wall of C. racemosa is described as a branched polymer containing (3-linked galactose, terminal-and 4-linked xylose, as well as 4-and 3,4linked arabinose residues). When sulfate groups are present, are linked to (C-3 of 4-linked arabinose and C-6 of 3-linked galactose) units [START_REF] Chattopadhyay | Polysaccharides from Caulerpa racemosa: Purification and structural features[END_REF][START_REF] Arata | Chemical structure and anticoagulant activity of highly pyruvylated sulfated galactans from tropical green seaweeds of the order Bryopsidales[END_REF]. The seaweed Ulva (Ulvales) also has a complex composition and structure sulfated polysaccharide ulvan, a potential source of new functional biopolymer. The name ulvan is derived from the original terms ulvin and ulvacin introduced by Kylin, 1946 as cited [START_REF] Lahaye | Structure and functional properties of ulvan, a polysaccharide from green seaweeds[END_REF] about different fractions of U. lactuca water-soluble sulfated polysaccharides from members of the Ulvales. The pioneering work of Brading et al. 1954 andMcKinnel andPercival 1962, as cited by [START_REF] Lahaye | Structure and functional properties of ulvan, a polysaccharide from green seaweeds[END_REF], established that sulfate, rhamnose, xylose, and glucuronic acid are the main constituents of ulvan. Thus, its main composition is made of disaccharide (sulfated rhamnose and glucuronic acid, iduronic acid, or xylose) repeating sequences. The two major repeating disaccharides are aldobiuronic acids as type A: ulvanobiuronic acid 3-sulfate (A3s) and type B: ulvanobiuronic acid 3-sulfate (B3s) is shown in Fig. 10 (Lahaye and Robic, 2007;[START_REF] Cunha | Sulfated Seaweed Polysaccharides as Multifunctional Materials in Drug Delivery Applications[END_REF]. In surface view, the cells are usually hexagonal or pentagonal, which remain loosely arranged [START_REF] Baweja | Biology of Seaweeds[END_REF].

Brown seaweeds

This cell wall structure in brown algae is even more complicated as they consist of an amorphous matrix of acid polysaccharides linked to each other by proteins (Fig. 11). These acid polysaccharides are mainly composed of sulfated polysaccharides rich in fucosefucan and fucoidan-and alginic acid, which confer structural toughness and flexibility to seaweed [START_REF] Cunha | Sulfated Seaweed Polysaccharides as Multifunctional Materials in Drug Delivery Applications[END_REF]. This physical barrier has limited the efficiency of the solid-liquid extraction technique of biologically active compounds from the marine algae [START_REF] Wijesinghe | Biological activities and potential cosmeceutical applications of bioactive components from brown seaweeds: A review | Request PDF[END_REF]. Sulfated polysaccharides rich in fucose usually constitutes about 5-10% of dry algal biomass, depending on the species, the part of the thallus being used, and the harvesting period [START_REF] Cunha | Sulfated Seaweed Polysaccharides as Multifunctional Materials in Drug Delivery Applications[END_REF]. Alginate generally makes up to 40% of the algae dry weight [START_REF] Dobrinčić | Advanced Technologies for the Extraction of Marine Brown Algal Polysaccharides[END_REF]. Fucoidan can differ in structure among and within algal species, and extraction conditions may alter the natural structure, and produce distinct fucoidan forms that may affect bioactivity and physicochemical properties [START_REF] Cunha | Sulfated Seaweed Polysaccharides as Multifunctional Materials in Drug Delivery Applications[END_REF]. Table VI showed the amorphous component of the brown algal cell wall is made up of alginic acid and sulfated polysaccharides rich in fucose. In contrast, their mucilage and cuticle polysaccharides are mainly sulfated polysaccharides rich in fucose, alginate, and laminarin [START_REF] Cunha | Sulfated Seaweed Polysaccharides as Multifunctional Materials in Drug Delivery Applications[END_REF]. In general, the cell wall and storage carbohydrate in brown algae varied strongly with the season, maturity, environmental factors and are species-specific, which increases the difficulty for industrial processing. For example, the Phaeophyceae Ascophyllum nodosum and Laminaria digitata have a composition of alginic acid, sulfated polysaccharides rich in fucose, laminarin equivalent to the ratio of 6:3:1 and 6:1:3, respectively [START_REF] Ravanal | Chapter 4-Production of Bioethanol from Brown Algae[END_REF]. Besides structural polysaccharides, algae also contain storage polysaccharides, notably laminarin and mannitol, shown in Table VI [START_REF] Stiger-Pouvreau | Carbohydrates from Seaweeds[END_REF]. Sulfated polysaccharides rich in fucose primarily consists of α-L-fucose units (usually referred to as α-L-fucopyranose). The sulfation pattern of the sugar backbone is species-related, and sulfation of α-L-fucose may occur at positions C-2 and C-4 and, at the rare position, C-3 structure [START_REF] Cunha | Sulfated Seaweed Polysaccharides as Multifunctional Materials in Drug Delivery Applications[END_REF]. Besides fucose and sulfate, fucoidan may also contain additional sugar constituents (mannose, galactose, glucose, xylose, uronic acids, and acetyl groups. Alginic acid consists of (1-4) linked β-D-mannuronic acid (M) and ɑ-Lguluronic acid (G) residues. In the mixed salts form known as alginates-they contain several cations such as Na + , K + , Mg 2+ , and Ca 2+ [START_REF] Puspita | Chapter Five -Indonesian Sargassum species bioprospecting: Potential applications of bioactive compounds and challenge for sustainable development[END_REF].

Alginic acids may differ in the M/G ratio depending on the species, the part of the thallus being used, and the harvesting period [START_REF] Artemisia | The Properties of Brown Marine Algae Sargassum turbinarioides and Sargassum ilicifolium Collected from Yogyakarta, Indonesia[END_REF][START_REF] Puspita | Chapter Five -Indonesian Sargassum species bioprospecting: Potential applications of bioactive compounds and challenge for sustainable development[END_REF]. Also, the molecules may differ in block composition, since uronic acid residues are usually arranged in two homopolymeric blocks (poly-M and poly-G), interspersed with the alternating structure of MG-blocks. Alginates have been used

as thickeners, stabilizers, and gelling agents in food and pharmaceutical industries due to their hydrophilic and viscous properties [START_REF] Puspita | Chapter Five -Indonesian Sargassum species bioprospecting: Potential applications of bioactive compounds and challenge for sustainable development[END_REF].

Storage carbohydrates

Laminarin (~34%), mannitol (~30%),

Several species with their total polysaccharides content (Table VII) from order Chordariales and Laminariales contain sulfated polysaccharides rich in fucose with a linear backbone composed of (1-3)-linked α-L-fucose residues. In contrast, order Fucales mostly display a backbone composed of alternating (1-3)-and (1-4)linked α-L-fucose residues [START_REF] Cunha | Sulfated Seaweed Polysaccharides as Multifunctional Materials in Drug Delivery Applications[END_REF]. Seaweed from order Fucales have been reported to have abundant amounts of fucose and galactose, called galactofucans, and mainly composed of (1-6)-β-D-galactose and/or (1-2)-β-Dmannose units. In these cases, sulfated polysaccharides rich in fucose molecules differ in composition and terms of glycosidic bond positions [START_REF] Cunha | Sulfated Seaweed Polysaccharides as Multifunctional Materials in Drug Delivery Applications[END_REF]. In general, fucoidan polysaccharides may be branched, presenting a variety of substituting groups and side-chain compositions. The typical positions referred to link sulfate groups (C-2, C-3 or C-4) may also be occupied by neutral, and partially sulphated residues of glucuronic acid, mannose, galactose, and xylose have been further reported as side chain (e.g., Sargassum sp., Fucus serratus, Punctaria plantaginea, Ascophyllum nodosum) [START_REF] Cunha | Sulfated Seaweed Polysaccharides as Multifunctional Materials in Drug Delivery Applications[END_REF]. Polysaccharides contents (% of dry weight) of different species of Sargassum is shown in Table VII.

Red seaweeds

Carrageenans is a generic name for a family of galactans [START_REF] Cunha | Sulfated Seaweed Polysaccharides as Multifunctional Materials in Drug Delivery Applications[END_REF], a family of linear sulfated polysaccharides. They are found and extracted from the cell wall of certain species of red seaweeds [START_REF] Al-Nahdi | The Effect of Extraction Conditions on Chemical and Thermal Characteristics of Kappa-Carrageenan Extracted from Hypnea bryoides[END_REF].

Rhodophyta is characterized by their content on non-fibrillar and sulfated polysaccharides such as carrageenan which is up to 20-38% [START_REF] Lalegerie | Chapter Eight -The stressful life of red and brown seaweeds on the temperate intertidal zone: effect of abiotic and biotic parameters on the physiology of macroalgae and content variability of particular metabolites[END_REF], agars and complex sulfated galactans, which are the main components of their cell walls [START_REF] Fenoradosoa | Highly sulphated galactan from Halymenia durvillei (Halymeniales, Rhodophyta), a red seaweed of Madagascar marine coasts[END_REF], shown in Fig. 12 and Table VIII. These polysaccharides, well known for their gelling, stabilizing, and thickening properties in food, pharmaceutical, and cosmetic industry [START_REF] Cunha | Sulfated Seaweed Polysaccharides as Multifunctional Materials in Drug Delivery Applications[END_REF], are made up of linear chains of alternating 3-linked-galactopyranosyl and 4-linked galactopyranosyl residues. The 3-linked units always belong to series, whereas the 4-linked ones may have the D or L configurations, often occurring as a 3,6anhydrogalactopyranosyl moiety after enzymatic or alkaline treatment. Many rhodophycean galactans have been described depending on their structural features.

Generally, the large carrageenan family is obtained from different species of Rhodophyta, including Gigartina, Chondrus crispus, Eucheuma, and Hypnea [START_REF] Fenoradosoa | Highly sulphated galactan from Halymenia durvillei (Halymeniales, Rhodophyta), a red seaweed of Madagascar marine coasts[END_REF]. The source of carrageenan was the red seaweed

Chondrus crispus which contains a mixture of kappa and lambda carrageenan that cannot be separated during commercial extraction. Today, most of the carrageenan is extracted from K. alvarezii and E. denticulatum, a predominant source of kappaand iota-carrageenan, respectively [START_REF] Cunha | Sulfated Seaweed Polysaccharides as Multifunctional Materials in Drug Delivery Applications[END_REF].

Lipids content and fatty acids

Generally, seaweeds are characterized by a relatively low content of saturated fatty acids (SFA) and substantial amounts of polyunsaturated fatty acids (PUFAs), which are considered beneficial for human health as long as they are not oxidized or converted into saturated lipids [START_REF] Hamed | Marine Bioactive Compounds and Their Health Benefits: A Review: Importance of functional seafood…[END_REF].

Brown algae contain relatively lower contents of lipids having less than 5% of dry weight. Quantitative analysis revealed that the total lipid content of a major brown algae family, Sargassaceae, was higher in subarctic zones (approximately 5% of dry weight) than tropical zones (0.9-1.8% of dry weight Total fatty acid concentrations vary among species, accounting for 1-8 in % of dry weight, showing significant differences in the fatty acid profiles, which can also be depending on the storage conditions (time and temperature) and the solvent also influences the yields and composition of the lipid extracts [START_REF] Torres | Integral Utilization of Red Seaweed for Bioactive Production[END_REF]. Sargassum is rich in fatty acids with 20 carbon atoms, such as eicosapentaenoic acid (EPA, C20:5n-3), and arachidonic acid (AA, C20:4n-6).

Certain species show different content of these fatty acids. As an example, S.

kjellmanianum is a rich source of ω6, whereas S. thunbergii has a high content of ω3 [START_REF] Burtin | Nutritional value of seaweeds[END_REF].

PUFAs generally contain high levels of omega-3 (ω3) and omega-6 (ω6) (Holdt and Kraan 2011). Seafood is the primary sources of long-chain PUFAs [START_REF] Lordan | Marine Bioactives as Functional Food Ingredients: Potential to Reduce the Incidence of Chronic Diseases[END_REF], and humans should get them from food since humans are incapable of synthesizing PUFAs with more than 18 carbons, e.g., long-chain fatty acids such as eicosapentaenoic acid (EPA, C20:5n-3) and arachidonic acid (AA, C20:4n-6). EPA and AA are mostly found in seafood and have the following health benefits which include the regulation of blood clotting, blood pressure, and develop the function of the brain and nervous systems, decrease the risk of many chronic diseases [START_REF] Hamed | Marine Bioactive Compounds and Their Health Benefits: A Review: Importance of functional seafood…[END_REF], and regulate inflammatory responses by producing inflammation mediators called eicosanoids [START_REF] Lordan | Marine Bioactives as Functional Food Ingredients: Potential to Reduce the Incidence of Chronic Diseases[END_REF] fasciata, and Ulva rigida by which DHA levels is reported to be between 4-5% [START_REF] Kumari | Tropical marine macroalgae as potential sources of nutritionally important PUFAs[END_REF] and Ulva pertusa as anti-allergic [START_REF] Hamed | Marine Bioactive Compounds and Their Health Benefits: A Review: Importance of functional seafood…[END_REF].

However, the Halymeniales species contain significantly higher levels of polyunsaturated fatty acids than vegetables [START_REF] Torres | Integral Utilization of Red Seaweed for Bioactive Production[END_REF][START_REF] Hurtado | Chapter Two -Selected red seaweeds from the Philippines with emerging high-value applications[END_REF], considered essential nutritional components in humans and animals [START_REF] Hurtado | Chapter Two -Selected red seaweeds from the Philippines with emerging high-value applications[END_REF] and have been proposed as a chemotaxonomic tool to differentiate macroalgae [START_REF] Torres | Integral Utilization of Red Seaweed for Bioactive Production[END_REF]. Fatty acid data revealed chemotaxonomic characteristics with C18 PUFAs (LA, ALA, and stearidonic acid or STA) being higher in Chlorophyta, C20 PUFAs (AA and EPA) in Rhodophyta, while depicted even distribution of C18 and C20 PUFAs in Phaeophyta. Moreover, the green, red, and brown macroalgae acquired their ability of producing long-chain PUFAs from the coupling of the chloroplastic desaturase enzymatic systems of their respective endosymbionts and FA desaturase and elongase system of the eukaryotic host [START_REF] Kumari | Fatty acid profiling of tropical marine macroalgae: An analysis from chemotaxonomic and nutritional perspectives[END_REF]. Some macroalgae present a low ω6/ω3 ratio; the ω3 polyunsaturated fatty acids (PUFAs) cannot be synthesized by humans and are thus obtained only through dietary sources [START_REF] Torres | Integral Utilization of Red Seaweed for Bioactive Production[END_REF]. Their therapeutic role, especially EPA and other unsaturated fatty acids during pregnancy, has been crucial

for transport across the placenta for the production of prostaglandins [START_REF] Ramu Ganesan | A comparison of nutritional value of underexploited edible seaweeds with recommended dietary allowances[END_REF]. EPA is also shown to reduce blood cholesterol and protect against cardiovascular and coronary heart diseases, anti-inflammatory, antithrombotic and anti-arrhythmic properties [START_REF] Kumari | Tropical marine macroalgae as potential sources of nutritionally important PUFAs[END_REF][START_REF] Torres | Integral Utilization of Red Seaweed for Bioactive Production[END_REF].

High DHA and unsaturated fatty acids will improve the fetus's visual acuity, mental development, and psychomotor skills (Ramu Ganesan et al. 2020).

Applications

For centuries, marine macroalgae within the entire algal group have been harvested in a wide variety along with coastal communities. Macroalgae were frequently used for domestic human consumption and are harvested to extract useful natural products such as hydrocolloids and pigments [START_REF] Delaney | Society and Seaweed: Understanding the Past and Present[END_REF][START_REF] Trono | The seaweed resources of the Philippines[END_REF]. Macroalgae is a lucrative business, driven by economic rather than environmental conditions [START_REF] Delaney | Society and Seaweed: Understanding the Past and Present[END_REF]). As the human population increases, so are the need to find alternative natural resources that could replace the demand for consumptive forms of needs (Henchion et al. 2017). It has compelled humanity to search more into the depth of the ocean [START_REF] Murty | Seaweeds: The Wealth of Oceans[END_REF]. Aside from consumptive forms, macroalgae include nonconsumptive forms such as medicine, fertilizer and animal feed, inputs into the industrial process, and other purposes such as building materials [START_REF] Delaney | Society and Seaweed: Understanding the Past and Present[END_REF]. The multitude of applications of seaweeds in various fields and industries led to a global increase in demand for the biomass of a variety of seaweed species. [START_REF] Trono | The seaweed resources of the Philippines[END_REF].

Algae have been subjected to many researches these past years, and some of them have been introduced and used in biotechnological, nutraceutical, and pharmaceutical applications. Biochemical constitutions of algae, especially the valorization of bioactives, is emphasized [START_REF] Torres | Integral Utilization of Red Seaweed for Bioactive Production[END_REF]). These algae constituents, including proteins (with EAA) and minerals (iron and calcium), lipids, vitamins, and fibers, provide high nutritional values that are beneficial for humans.

Because of their high nutritional and nutraceutical value, algae are consumed traditionally as food, as herbal medicine (Okolie et al. 2018), as an alternative to agricultural products to address the nutritional deficiencies of the current food (Leandro et al. 2020). Furthermore, they are also used as fodder, fertilizer (Okolie et al. 2018), fungicides, herbicides, dietary supplements, and as a resource of phycocolloids such as agar, alginate, and carrageenan for various industrial applications [START_REF] Peng | Chapter 5-Chemical composition of seaweeds[END_REF]. It must be remembered that seaweeds and their extracts are very complex and, as such, there is no one compound which conveys the many beneficial properties, instead of that various seaweeds and their extracts contain many compounds which may work synergistically and antagonistically with biological processes [START_REF] Hurtado | Chapter Two -Selected red seaweeds from the Philippines with emerging high-value applications[END_REF].

Algae as vegetable in human consumption

Of the 221-seaweed species worldwide that have commercial value, only about ten are intensely cultivated, and use also as food such as brown seaweed (Phaeophyceae-Saccharina japonica, Undaria pinnatifida, and Sargassum fusiforme); red seaweed (Rhodophyta-Porphyra spp., Eucheuma ssp., Kappaphycus alvarezii, and Gracilaria spp.); and green seaweed (Chlorophyta-Ulva clathrata, Monostroma nitidum, and Caulerpa spp.) (Pereira 2018a).

Seaweeds in East Asia have been documented to have been harvested as consumptive and non-consumptive for thousands of years (6000 BCE-300 BCE) in Japan, (600 BC) in China, (AD 918-1392) in Korea [START_REF] Dicks | Biomass Utilization: Economic Analysis of a Systems Approach[END_REF][START_REF] Delaney | Society and Seaweed: Understanding the Past and Present[END_REF], and the rest of the Pacific islands. The introduction of seaweeds in Europe as human food began in the 15th century, especially by littoral populations suffering from famine. Now, seaweeds as human food is widely distributed throughout the world, especially as a resource for the production of food, food additives, and nutritional supplements in Asia, Europe, North and South America, Africa, and Pacific Islands nations [START_REF] Fleurence | Seaweeds as Food[END_REF].

Although nutritional values can vary and are widely affected by physical and environmental factors, edible macroalgae are mostly rich in bioactive antioxidants, soluble dietary fibers, proteins, minerals, vitamins, phytochemicals, and polyunsaturated fatty acids [START_REF] Hamed | Marine Bioactive Compounds and Their Health Benefits: A Review: Importance of functional seafood…[END_REF][START_REF] De Gaillande | Caulerpa consumption, nutritional value and farming in the Indo-Pacific region[END_REF].

Seaweeds are also very rich in essential minerals and trace elements required for the human diet compared to terrestrial foods due to their ability to maintain inorganic atoms from seawater [START_REF] Hamed | Marine Bioactive Compounds and Their Health Benefits: A Review: Importance of functional seafood…[END_REF]. Macroalgae have many uses;

food for human consumption, ingredients in the manufacture of cosmetics and fertilizers, treated products for extracting gelling agents, or animal feed additives [START_REF] De Gaillande | Caulerpa consumption, nutritional value and farming in the Indo-Pacific region[END_REF]. In brown algae, Sargassum fusiforme, also known as Hijiki is served as food, named as "vegetable for longevity" in food industries of Japan. It is a side dish prepared by soaking and boiling the dried algae with water and served with soy sauce [START_REF] Liu | Towards a better understanding of medicinal uses of the brown seaweed Sargassum in Traditional Chinese Medicine: A phytochemical and pharmacological review[END_REF]. S. polycystum has been eaten as sea vegetables in Asian countries such as Malaysia and Vietnam [START_REF] Hong | Seaweeds from Vietnam used for functional food, medicine and biofertilizer[END_REF][START_REF] Matanjun | Nutrient content of tropical edible seaweeds, Eucheuma cottonii, Caulerpa lentillifera and Sargassum polycystum[END_REF]. Macroalgae are mainly used as food in the form of fresh vegetables, and their popularity tends to be on the increase due to the general awareness of health-conscious consumers of the advantages of its natural products.

Annual production of seaweed for direct (sea vegetables) and indirect (phycocolloids) human consumption is estimated at 2 million tonnes of dry matter, and 90% of this production is from China, Korea, Japan, Vietnam, Chile, the Philippines, Norway, France, Spain, and the United Kingdom [START_REF] Fleurence | Seaweeds as Food[END_REF].

The Philippines is the third-largest producer of seaweed in the world following China and Indonesia, where Eucheuma species is the most commercially significant. In 2015, 90% of the Philippines' farmed output comprised Eucheuma species, such as E. cottonii (Kappaphycus) and E. denticulatum. Also, about 80%

of the E. cottonii (Kappaphycus) production comes from the Philippines (777 963 tons), and about 30% traded in dry matter (FAO, 2018). Several edible species of the green, brown, and red seaweeds' nutrient contents are shown in Table X. The brown algae processing industry in Europe is divided into two main categories, first is the alginic acid production, and second is the production for agriculture such as fertilizer and animal feeds. Valuable products such as agar and carrageenan extracts are not immediately recognizable as human food since it is used in particular as thickening agents in beverages (de [START_REF] De Gaillande | Caulerpa consumption, nutritional value and farming in the Indo-Pacific region[END_REF]. For more than four decades. There has been a lot of published works presented on economically important red seaweeds, including the carrageenophytesespecially the genera Kappaphycus and Eucheuma [START_REF] Hurtado | Tropical Seaweed Farming Trends, Problems and Opportunities: Focus on Kappaphycus[END_REF]. The amounts of total protein and profiles of amino acids in red seaweeds make them interesting sources for industrial food and even aquaculture feed sectors [START_REF] Hamed | Marine Bioactive Compounds and Their Health Benefits: A Review: Importance of functional seafood…[END_REF][START_REF] Baweja | Biology of Seaweeds[END_REF]. Commercial carrageenans (used at the food level) have a high molecular weight, usually ranging from 400 to 600 kDa, with a minimum of 100 kDa [START_REF] Baweja | Biology of Seaweeds[END_REF]. Molecular forms of carrageenan (kappa, lambda, and iota) can be tailored for a wide variety of uses, as additives in the manufacture of dessert gels, especially for dairy products, fish gels, pizzas, barbecues, sauces, and medical and industrial applications [START_REF] Dawes | Macroalgae systematics[END_REF][START_REF] Fleurence | Seaweeds as Food[END_REF]. Agar consists of two polymers (agarose and agaropectin) used in food processing such as baked goods, ice cream, candies, juices, wine, and vinegar [START_REF] Dawes | Macroalgae systematics[END_REF][START_REF] Fleurence | Seaweeds as Food[END_REF]. Carrageenan contributes 15-20% in the use of food hydrocolloids globally; the most important algal sources are C. crispus harvested in the North Atlantic, and species of Eucheuma and Kappaphycus, farmed in the Philippines (C. [START_REF] Dawes | Macroalgae systematics[END_REF]FAO, 2018). These species, C. crispus (in Ireland and the British Isles) and Eucheuma, and Kappaphycus (in the Philippines) are used directly as sea vegetables, in stews, or salads. The K. alvarezii (Doty) contains kappa-carrageenan, while E. isiforme (C. Agardh) contains iotacarrageenan [START_REF] Dawes | Macroalgae systematics[END_REF].

Active ingredients (cosmetics and health)

Viral infections, as this present pandemic that the world is facing for COVID-19, count as one of the most predominant cause of death in human life and the economy worldwide. So far this year, COVID-19 has killed more than 1.5 million people and forecasters predict that by the end of the year 2020, the pandemic death toll could rise to 1.9 million. If that happens, COVID-19 would rank as the 6 th -deadliest disease in the world (WHO, 2019). Seaweeds are known rich source for active antiviral metabolites. Extracted polysaccharides exhibit antiviral activity against a broad spectrum of viruses including; Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV), Vesicular stomatitis virus (VSV),

Cytomegalovirus (CMV) [START_REF] Bouhlal | Antiviral Activities of Sulfated Polysaccharides Isolated from Sphaerococcus coronopifolius (Rhodophytha, Gigartinales) and Boergeseniella thuyoides (Rhodophyta, Ceramiales)[END_REF][START_REF] Wang | Overview on Biological Activities and Molecular Characteristics of Sulfated Polysaccharides from Marine Green Algae in Recent Years[END_REF][START_REF] Mahomoodally | Marine Algae: A Potential Resource of Anti-HSV Molecules[END_REF].

Algae proteins and their derivatives have anti-inflammatory, antioxidant, antitumor, anti-aging, and protective activity. They are also applied as a moisturizer to hair and skin. Amino acids are generally used as skin moisturizers since many are constituents of the natural moisturizing factor (NMF) in human skin (Pereira, 2018b). Xanthophylls (astaxanthin, fucoxanthin, loraxanthin, lutein, violaxanthin), and carotene (α-carotene, β-carotene, lycopene) are widely used as natural dyes and vigorous antioxidants with antitumor, anti-inflammatory and radical sequestering properties [START_REF] Stengel | Algal chemodiversity and bioactivity: Sources of natural variability and implications for commercial application[END_REF]Pereira 2018a). Pro-vitamin A activity can also be found in carotenoids where it can remain active when standard vitamin C cannot, especially under low oxygen condition; β-carotene, protect the skin and eyes from photo-oxidation against UV radiation and prevent eye disease such as cataracts;

Astaxanthin performs a variety of activities, e.g., UV-mediated photo-oxidation, tumors, and inflammatory processes; and fucoxanthin, a typical xanthophyll of brown algae with extraordinary antioxidant activity. Its main bioactivities are antimalarial, antitumor, anti-obesity, anti-inflammatory, antidiabetic, and antiangiogenic activities. It also has protective effects on the brain, skin, liver, blood vessels, bones, and eyes [START_REF] Joshi | Applications of Algae in Cosmetics: An Overview[END_REF]Pereira 2018a).

The fatty acid alpha-linolenic acid (ALA), the precursor to EPA and DHA were reported to inhibit the production of eicosanoids, lowering blood pressure, and blood triglyceride levels [START_REF] Oomah | Health benefits of phytochemicals from selected Canadian crops[END_REF] while docosapentaenoic acid (DPA) was shown to inhibit platelet aggregation and has strong endothelial cell migration abilities [START_REF] Kaur | Docosapentaenoic acid (22:5n-3): a review of its biological effects[END_REF]. Commercial ALA or hybrid liposomes of ALA ethyl ester (HL-ALAE) seemed to provide benefits against several health problems, e.g., decrease in blood cholesterol levels, reduction in the risk of heart attacks, and a growth reduction in breast, colon, and prostate cancer [START_REF] Tanaka | Remarkably high inhibitory effects of docosahexaenoic acid incorporated into hybrid liposomes on the growth of tumor cells along with apoptosis[END_REF]. Researchers have suggested that elevated blood pressure in adulthood may be associated with peri-natal omega-3 fatty acid deficiency and that early exposure to dietary, long-chain omega-3 played a critical role in supporting heart health and reducing CVD risk in later life [START_REF] Papanikolaou | adults are not meeting recommended levels for fish and omega-3 fatty acid intake: results of an analysis using observational data from NHANES 2003-2008[END_REF]. Given the situation above, then it is safe to assume that consumption of recommended amounts of omega-3s as ALA in food, or in a supplement, would be generally beneficial to consumers.

Caulerpa and Ulva species

Antioxidant compounds from macroalgae (Caulerpa and Ulva spp. which have antifungal and antitumoral activities, and the β-sitosterol which is a plant stanol known to reduce blood levels of cholesterol as it inhibits cholesterol absorption in the intestine was also isolated from C. racemosa [START_REF] Ragasa | Secondary metabolites from Caulerpa racemosa[END_REF].

A cytotoxic activity is at the concentration of 60-90 µM leading to the destruction of human hematopoietic progenitors (melanocytes, keratinocytes and fibroblasts) but not toxic to normal melanocytes at <10.5 µM and normal keratinocytes at 12.6 µM [START_REF] Kumar | Toxicological effects of marine seaweeds: A cautious insight for human consumption[END_REF]. It is important to remember the importance of monitoring Caulerpa species' toxicity before consumption (de Gaillande et al.

2017

), as they exhibited significant increase in their toxic metabolites when eaten by predators, are then transferred to the marine food chain, resulting into toxicity to its predators [START_REF] Kumar | Toxicological effects of marine seaweeds: A cautious insight for human consumption[END_REF].

Sargassum species

In brown algae, recent pharmacological research discovered that alginates isolated from Sargassum demonstrated anticancer (in vivo), antiviral (in vitro), and hypolipidemic (in vivo) activity [START_REF] Mao | Preliminary studies on the chemical characterization and antihyperlipidemic activity of polysaccharide from the brown alga Sargassum fusiforme[END_REF][START_REF] De Sousa | In vivo growth-inhibition of Sarcoma 180 tumor by alginates from brown seaweed Sargassum vulgare[END_REF][START_REF] Sinha | Polysaccharides from Sargassum tenerrimum: Structural features, chemical modification and antiviral activity[END_REF]. Pharmacological properties of Sargassum crude extracts or isolated pure compounds have been widely recognized, including antibacterial, antifungal, antiviral, anticoagulant, antioxidant, antimelanogenic, anti-inflammatory, hypoglycemic, hepatoprotective and neuroprotective activities (Puspita et al. 2017;[START_REF] Dobrinčić | Advanced Technologies for the Extraction of Marine Brown Algal Polysaccharides[END_REF]. This suggests that Sargassum is a rich source of health and promoting agents. Before these exceptional findings, Sargassum has been traditionally used as medicine in ancient times in China to treat goiter. Sargassum was also used exclusively for medicinal purposes in ancient times in Monte Verde, South America [START_REF] Dillehay | Monte Verde: Seaweed, food, medicine, and the peopling of South America[END_REF]. Sargassum vulgare C. Agardh and Sargassum thunbergii Yendo from Japan have been used as anthelmintics [START_REF] Chapman | Seaweeds and their Uses[END_REF][START_REF] Kang | Antipyretic, analgesic, and anti-inflammatory activities of the seaweed Sargassum fulvellum and Sargassum thunbergii in mice[END_REF]). Records about the traditional use of Sargassum in medicine have made this genus interesting to explore. Thus, Sargassum has been the most extensively as alternative natural resources with economic value.

Identified bioactive meroterpenoids such as 2-methyl-6-(3-methyl-7oxo-2,5octadienyl)-1,4-benzoquinone and sargafuran [START_REF] Kamei | The novel anti-Propionibacterium acnes compound, Sargafuran, found in the marine brown alga Sargassum macrocarpum[END_REF]) with significant antibacterial activity (2 μg mL -1 < MIC < 15 μg mL -1 ) could provide scientific evidence for the traditional therapeutic claims of Sargassum. The pharmacological activity of Sargassum extracts and research of its bioactive constituents provide scientific evidence that underpins the traditional therapeutical claims made for Sargassum such as furuncle and abscesses [START_REF] Liu | Towards a better understanding of medicinal uses of the brown seaweed Sargassum in Traditional Chinese Medicine: A phytochemical and pharmacological review[END_REF]. The iodine in Sargassum was believed to play an important role in therapeutical function.

However, the role of iodine may have been overestimated, and other bioactive metabolites may have made more significant contributions toward Sargassum's therapeutic claims [START_REF] Liu | Towards a better understanding of medicinal uses of the brown seaweed Sargassum in Traditional Chinese Medicine: A phytochemical and pharmacological review[END_REF]. Research suggests that Sargassum may play a role as an immunomodulator [START_REF] Yende | Therapeutic potential and health benefits of Sargassum species[END_REF]). The antimalarial activity has been found in Sargassum natans (Linnaeus) Gaillon [START_REF] Orhan | Turkish freshwater and marine macrophyte extracts show in vitro antiprotozoal activity and inhibit FabI, a key enzyme of Plasmodium falciparum fatty acid biosynthesis[END_REF]. S. hemiphyllum lipophilic fraction of the dichloromethane/methanol extract exerted potent in vitro antiplasmodial activity (IC50 = 2.8 μg mL -1 ; [START_REF] Lategan | Antiplasmodial and antimicrobial activities of South African marine algal extracts[END_REF]. [START_REF] Samee | Anti-allergic effects of ethanol extracts from brown seaweeds[END_REF] showed that semipurified hydroethanolic extracts from Sargassum growing in Pakistan or China had an antihyaluronidase activity proportional to the contents of the phlorotannin contents the ethyl acetate fractions. Also, the IC50 of the extract from Sargassum tennerimum was similar to that of catechin. Sargassum in India is mostly exploited for its alginate production in hydrocolloid industries either for local or export [START_REF] Rao | Indian seaweed resources and sustainable utilization: Scenario at the dawn of a new century[END_REF].

Halymenia species

Some particular red seaweed is reported to contain antihypertensive, anticoagulant, and antihemorrhagic compounds. Several scientific studies on the collective biomasses of carrageenophytes demonstrate their potential as sources of structurally diverse, biologically active compounds with potentially multiple pharmaceutical and biomedical applications (Pereira, 2018a). They also contain specific lectins, enzymes, and derivatives, such as peptides, which could present bioactive potential. Peptides from Pyropia columbina were reported as immunosuppressive and antihypertensive [START_REF] Cian | Bioactive properties of peptides obtained by enzymatic hydrolysis from protein by-products of Porphyra columbina[END_REF]. Lectins from five species of red seaweeds of Eucheuma were reported to have strong hemagglutination activity.

Lectins, in general, have also been reported to present many advantages, such as detection of disease-related to glycan synthesis, detection of infectious agents (viruses, bacteria, fungi, parasites), an antibiotic, mitogenic, and cytotoxic activities [START_REF] Dumay | Proteins and Pigments[END_REF].

Recent studies on human gut microbes or host/microbe inter-dependence (dysbiosis) were reported to be associated with various pathologies, e.g., inflammation, obesity, hypertension, endothelial dysfunction, diabetes, cardiovascular disease (CVD), colitis, and neurological disorders [START_REF] Cornish | A mini-review on the microbial continuum: consideration of a link between judicious consumption of a varied diet of macroalgae and human health and nutrition[END_REF]). Since CVD is highly influenced by human food digestion, carbohydrate fermentation, energy harvesting and storage, gene expression, the secondary generation of beneficial compounds (cross-feeding), and the facilitation of metabolic functions, makes applications and utilization of therapeutic dietary impacts of seaweeds to be viable and essential for intervention strategies related to food manufacturing and consumption [START_REF] Cornish | A role for dietary macroalgae in the amelioration of certain risk factors associated with cardiovascular disease[END_REF][START_REF] Cornish | A mini-review on the microbial continuum: consideration of a link between judicious consumption of a varied diet of macroalgae and human health and nutrition[END_REF].

A study of the human intestine (Caco-2) and hepatic (HepG2) cell lines showed apoptotic induction using k-carrageenan [from Kappaphycus alvarezii (cottonii)]. At the same time, no toxicity was observed in corresponding normal and cancerous intestinal and liver cell-lines, using non-degraded carrageenan [START_REF] Ariffin | Cytotoxicity effect of degraded and undegraded kappa and iota carrageenan in human intestine and liver cell lines[END_REF]. According to [START_REF] Murad | Algal sulfated carrageenan inhibits proliferation of MDA-MB-231 cells via apoptosis regulatory genes[END_REF], a pro-apoptotic activity of carrageenan [from Eucheuma denticulatum (spinosum)] was shown to be mediated by caspase-3, caspase-9, p53, Bax, and Bcl-2 genes within an MDA-MB-231 metastatic, human, breast cancer cell-line. Generally, the reported antiviral compounds from seaweeds include (Hurtado et al., 2019):

(a) carrageenans (such as -carrageenan and partially cyclicized /-carrageenan)

against HSV-1 and 2 from Gigartina skottsbergii [START_REF] Carlucci | Antiherpetic and anticoagulant properties of carrageenans from the red seaweed Gigartina skottsbergii and their cyclized derivatives: Correlation between structure and biological activity[END_REF], from

Gymnogongrus griffithsiae and Cryptonemia crenulata [START_REF] Talarico | The antiviral activity of sulfated polysaccharides against dengue virus is dependent on virus serotype and host cell[END_REF], (b) DL-galactan hybrids, against HSV-1 and 2 from G. griffithsiae and C. crenulata [START_REF] Talarico | The antiviral activity of sulfated polysaccharides against dengue virus is dependent on virus serotype and host cell[END_REF], and HSV-2 and Dengue virus type 2 (DEN-2) from G.

torulosus [START_REF] Pujol | Novel DL-Galactan Hybrids from the Red Seaweed Gymnogongrus Torulosus are Potent Inhibitors of Herpes Simplex Virus and Dengue Virus[END_REF]

2009), and

(f) galactan sulfate, and sulfated glucurono-galactan (from Schizymenia binderi), against HSV-1 and HSV-2, HIV, RSV, HCMV, Dengue virus, Pseudorabies virus, and Influenza A and B virii [START_REF] Pujol | Novel DL-Galactan Hybrids from the Red Seaweed Gymnogongrus Torulosus are Potent Inhibitors of Herpes Simplex Virus and Dengue Virus[END_REF][START_REF] Talarico | The antiviral activity of sulfated polysaccharides against dengue virus is dependent on virus serotype and host cell[END_REF].

Studies revealed that the above compounds, with particular emphasis on carrageenans, inhibited the cytopathic effect of HIV. They prevented HIV-induced syncytium (giant cell) formation, as well as other sexually transmitted viruses, such as genital warts, human papillomavirus (HPV), and different strains of Herpes simplex virus Type 1 and 2 (HSV-1 and 2) [START_REF] Mendis | Present and future prospects of seaweeds in developing functional foods[END_REF]. Reportedly k, ʎ, and -carrageenan inhibited the human papillomavirus (HPV) infection process [START_REF] Buck | Carrageenan Is a Potent Inhibitor of Papillomavirus Infection[END_REF], and are potent inhibitors of the multiplication of the Dengue virii, Type-2 and Type-3 (DENV-2 and 3) [START_REF] Talarico | The antiviral activity of sulfated polysaccharides against dengue virus is dependent on virus serotype and host cell[END_REF]).

However, it must be remembered that seaweeds and their extracts are very complex. Although, even one compound conveys many beneficial properties; more so of that, various seaweeds and their extracts contain many compounds that may work synergistically or antagonistically with biological processes [START_REF] Morán-Santibañez | Synergistic Effects of Sulfated Polysaccharides from Mexican Seaweeds against Measles Virus[END_REF]. Some of the bioactivity of extracts is a direct result of the chemical interactions between raw materials and the extraction processes. Several examples of health benefits with its functional ingredients taken from different species of green, brown, and red seaweeds that could atleast represent the present study seaweed samples are shown in Table XI.

Table XI.

Health benefits summary of green, brown, and red seaweeds (Hurtado et al., 2019). Chapter 2 Materials and Methods

Species

Sampling areas and abiotic factors

In this work, sampling areas were chosen in order to determine and compare the biochemical composition of different species of seaweed and their extracts along with their biological activities. The Philippines is divided into three (3) main group of islands: Luzon islands, the largest island located on the northern part of the archipelago; Visayas islands, are the scattered islands in the central Philippines;

and the Mindanao islands, the second largest group located in the southern part of the archipelago. Illustration of the sampling location is described in Fig. 14 showing the selected seaweed species.

Overview of the studied location

Samples preparation, drying, and grinding

All seaweed samples were handpicked by cutting or pulling off the basal part of the thalli near the holdfast. Each sample of seaweed species collected from different patches or stations in each chosen site were pooled according to its kind.

Samples were immediately washed with seawater after sampling and were put in a covered styroboxes to keep away from direct sunlight. Collected algae from all selected sites were rinsed thoroughly with running freshwater from the remaining sand and epiphytes and left to dry under the shade avoiding sunlight for 1 to 2-weeks. The dried samples were then put in ziplock and was then transported to the Laboratoire de Biotechnologie et Chimie Marines (LBCM), in Université Bretagne Sud (UBS), Campus de Tohannic, Vannes, France.

Transported materials were then ground or cut into small pieces with either scissors or blender and were then freeze-dried in LBCM, and were stored in the dark for further analyses. The water quality in all areas was taken in-situ for its salinity, temperature, pH, and dissolved oxygen. The summary of sampling and collection were shown in Table XII, and summary of processed samples and its respective analyses were shown in Table XIII.

HWE ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Lawigan, Bislig, Mindanao (August 15, 2017) 1OH  ✓  ✓ ✓ ✓ ✓     4OH  ✓  ✓ ✓ ✓ ✓     INS  ✓  ✓ ✓ ✓ ✓     U. lactuca (Linn.) DS ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Lawigan, Bislig, Mindanao (August 15, 2017) DA  ✓  ✓ ✓ ✓ ✓     PP1  ✓  ✓ ✓ ✓ ✓     PP2  ✓  ✓ ✓ ✓ ✓     PP3  ✓  ✓ ✓ ✓ ✓     S. polycystum Sp CaCl2 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓  Anda, Bohol, Visayas (August 22, 2017) Sp HCl  ✓  ✓ ✓ ✓ ✓    Sp Na2CO3  ✓  ✓ ✓ ✓ ✓    Sp KOH  ✓  ✓ ✓ ✓ ✓    S. ilicifolium Si CaCl2 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓  Currimao, I.N., Luzon (July 2017 Si HCl  ✓  ✓ ✓ ✓ ✓   Si Na2CO3  ✓  ✓ ✓ ✓ ✓   Si KOH  ✓  ✓ ✓ ✓ ✓   H. durvillei Hdur ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ H. dilatata Hdil ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Anda, Bohol, Visayas (August 22, 2017)

Biochemical analyses

Acid and water extraction for the characterization of the raw material

Acid hydrolysis: to characterize the raw matter, 10 mg of freeze-dried matter were mixed with 5 mL of HCl 1 M in a sealed vial at 100 °C for 2 h, after which 5 mL NaOH 1 M were added. The final solution was to measure the protein, neutral sugar, uronic acid, and polyphenol contents. This solution was kept at 4 °C and should be used within a week for further analysis. The biochemical composition of C. racemosa (Cr), U. fasciata (Uf), S. polycystum (Sp), S. ilicifolium (Si), H. durvillei (Hdur), and H. dilatata (Hdil) was defined as percentage proportions of each chemical compound found in the total dry weight of each raw material. All analyses were done in triplicate.

Aqueous hydrolysis: Ten mg of freeze-dried, finely ground material in a mortar and pestle were introduced to 5mL of ultrapure H 2 O in different vials. All prepared samples were then put in the heating block of 100 °C for 2 h with agitation every 30 min to optimize the process. Samples were left to cool for 5 min. In order to complete the concentration of the samples into 1 mg mL -1 , 5 mL of ultrapure H 2 O was then added to each different vial. This solution was kept at 4 °C and should be used within a week for further analysis. The designated analysis for this solution is for the determination of sulfate groups (Burlot, 2016). Sulfated groups' content was determined by the Azure A method that reacts specifically with sulfates linked to the polysaccharides [START_REF] Jaques | A microelectrophoresis method for heparin[END_REF]). For this estimation, sulfated dextran (17%) (0-100 μg mL -1 ) was used as the standard.

The final solution was used to measure the protein, uronic acid, neutral sugar, and polyphenol contents (Burlot, 2016).

Proteins and amino-acids content

The principle of this assay, according to the method of [START_REF] Smith | Measurement of protein using bicinchoninic acid[END_REF], is detailed in Fig. 19 Figure 19. Principle and reaction of the protein assay by the method of Smith et al.

(1985) (Burlot, 2016).

Principle: Dosage of total protein uses the BCA (BicinChoninic Acid) method based on the reaction of Biuret reagent, which results in the reduction of Cu 2+ ion to Cu + by the protein in the alkaline medium. The cuprous ions will then be chelated by 4,4'-dicarboxy-2,2'-biquinoline (bicinchoninic acid, BCA) to form an absorbent chromophore λ = 562 nm.

Standards, reagents and samples:

-BSA solution (bovine serum albumin) (2 mg mL -1 , Pierce BCA Protein Assay Kit, Thermo Scientific, 23227) to be diluted in ultra-pure water (Purelab Ultra, ELGA, France) at 1 mg mL -1 .

-Pierce BCA Protein Assay Kit, (Thermo Scientific, 23228) -Sample to be analyzed at 1 mg mL -1 , the dilution factor to be determined so that the ODs measured are among those in the standard range.

% = [ (𝑶𝑫 -𝒃) 𝒂 ⁄ ] × 𝑫 × 𝟏 𝟏𝟎𝟎𝟎 ⁄ × 𝟏𝟎0
where:

-OD is the optical density of the designated analysis, -b is the intercept of the calibration curve, -a is the slope of the calibration curve, -D is 1/factor of dilution, -1/1000 is the conversion of mg from µg, 100 is from 1 mg samples and conversion to % [START_REF] Smith | Measurement of protein using bicinchoninic acid[END_REF][START_REF] Kessler | Interference by lipids in the determination of protein using bicinchoninic acid[END_REF][START_REF] Wiechelman | Investigation of the bicinchoninic acid protein assay: Identification of the groups responsible for color formation[END_REF][START_REF] Brown | Biochemical composition of microalgae from the green algal classes Chlorophyceae and Prasinophyceae. 1. Amino acids, sugars and pigments[END_REF].

Amino-acids content

The amino acid composition of the raw seaweed materials (5-25 nM of protein) and or its purified fractions were determined by gas chromatography (GC) The Amino acid samples were prepared according to the procedure recommended by the kit of EZ ™ faast assay (Phenomenex, Torrance, California, USA). The preparation consisted of a solid-phase extraction step followed by derivatization and, finally, a liquid/liquid extraction. Amino acid samples were analyzed on gas chromatography coupled to a flame ionization detector (GC-FID, TRACE TM 1300, Thermo-Fisher Scientific TM , Milan, Italy). The system consisted of a Zebron ZB-AAA-GC column (10 m x 0.25 mm, Agilent, CA, USA). Two μL of samples were introduced into the injector (250 °C.) with a split ration of 1:10 and separated into using the following program: 110 °C to 320°C (increase 32 °C min -1 ), held for 2 minutes. The temperature detector was set at 320°C and the gas pressure at 1.7bar.

Nitrogen gas was used as the carrier gas. The signals were recorded using Chromeleon TM 7.2 software (DIONEX TM , Thermo-Fisher Scientific TM , Sunnyvale, CA., USA). Amino acids have been identified by their time of retention and quantified by their response factor relative to Norvaline internal standard added at a concentration of 200 μmol L -1 .

Total sugars content analysis

The principle of the determination of total sugars, according to the method of [START_REF] Dubois | Colorimetric Method for Determination of Sugars and Related Substances[END_REF], is shown in Fig. 20 below. (Burlot, 2016).

Principle: The principle of this analysis is based on dehydration of hexoses and pentoses (in Fig. 10, glucose is exemplified as hexose) under the action of hot and concentrated mineral acid cyclization forming derivatives of furfural like 5hydroxymethylfurfural, which reacts with phenol. So, a yellow complex is formed if there is a presence of sugars. This complex concentration is measured by spectrophotometer (UV-1800 Spectrophotometer, Shimadzu Europe Gmbh, Germany).

Standards, reagents, and samples:

-D-Glucose monohydrate (Fisher Scientific) in ultra-pure water (Purelab Ultra, ELGA, France) at 1mg mL -1 .

-75% phenol solution (Fisher Scientific), 96% sulfuric acid (Across organics, 302070010)

-Sample to be analyzed at 1 mg mL -1 from acid extraction, the dilution factor of which is determined so that the measured ODs are among those in the standard range.

Procedure: One mL of samples or standard was put in the tubes, then added with 50 µL of phenol 75% and followed by agitation by using the vortex. 2.5 mL of sulfuric acid was added, and the mixture was then cooled (exothermic reaction) in the cold water of 20 °C for 10 min. After 10 min, all samples were agitated and placed in the water bath of 30°C for 10 min. Optical density is read against the blank at 490 nm. A standard range of glucose is performed before each series of analyzes (0, 20, 40, 60, 80, and 100 μg mL -1 . All samples were made in triplicate [START_REF] Dubois | Colorimetric Method for Determination of Sugars and Related Substances[END_REF]).

The concentration and total sugar content are calculated with the rate of dry matter of the sample, according to the following equation:

% = [ (𝑶𝑫 -𝒃) 𝒂 ⁄ ] × 𝑫 × 𝟏 𝟏𝟎𝟎𝟎 ⁄ × 𝟏𝟎0
where:

-OD is the optical density of the designated analysis, -b is the intercept of the calibration curve, -a is the slope of the calibration curve, -D is 1/factor of dilution, -1/1000 is the conversion of mg from µg, 100 is from 1 mg samples and conversion to % [START_REF] Smith | Measurement of protein using bicinchoninic acid[END_REF][START_REF] Kessler | Interference by lipids in the determination of protein using bicinchoninic acid[END_REF][START_REF] Wiechelman | Investigation of the bicinchoninic acid protein assay: Identification of the groups responsible for color formation[END_REF][START_REF] Brown | Biochemical composition of microalgae from the green algal classes Chlorophyceae and Prasinophyceae. 1. Amino acids, sugars and pigments[END_REF].

Uronic acid content analysis

The dosage of uronic acids, inspired by the method of Blumenkrantz and Asboe-Hansen (Burlot, 2016).

Principle: Under the action of a hot concentrated strong acid, the uronic acids undergo dehydration than a cyclization. 5-formylfuroic acid derivatives are formed.

These derivatives in contact with meta-hydroxydiphenyl (MHDP) form a chromophore of color pink absorbent at λ = 525 nm. The coloring is more intense in the presence of borate but remains very sensitive to interferences due to neutral sugars, which are stained in the presence of concentrated sulfuric acid. This is why potassium sulfamate is added to limit these interferences.

Uronic acid could react to the presence of meta-hydroxydiphenyl (MHDP), and this reaction creates a pink coloration. To enhance the coloration, tetraborate is added, and the addition of sulfamic acid minimizes the interferences of neutral sugars detection in the presence of sulfuric acid [START_REF] Blumenkrantz | New method for quantitative determination of uronic acids[END_REF].

Standards, reagents, and samples:

-Glucuronic acid (Sigma-Aldrich G5269) in ultrapure water (Purelab Ultra, ELGA, France) at 1 mg mL -1 .

-The solution of potassium sulfamate 98% at 4 M (Alfa Aesar, B24447), solution of tetraborate 75 M sodium (Merk, 524 A12408) diluted in 96% sulfuric acid, solution of 0.15% MHDP in 0.5% sodium hydroxide (Fisher Scientific 11983223) -Sample to be analyzed at 1 mg mL -1 from acid extraction, the dilution factor of is determined so that the measured ODs are among those in the standard range.

Procedure: Diluted samples solution or standard of 200 µL are put in vials and added with 20 µL of 98% sulfamic acid 4 M. Before introducing 1.2 mL of sodium tetraborate 75 mM, the solution was agitated and re-agitated afterward. All samples were capped and incubated for 20 min at 80 °C then put in the ice for 5 min. Then, 40 µL of MHDP 0.15% was then added. Leave the solution at ambient temperature for 10 min for the reaction to develop before reading the optical density (OD). The optical density was read at 525 nm. Glucuronic acid with concentrations ranged from 0-200 µg mL -1 served as standards for the calibration curve. A standard range of glucuronic acid is carried out before each analysis (0, 20, 40, 60, 80, and 100 μg mL -1 ) [START_REF] Guillaume | Caractérisation biochimique d'exopolymères d'origine algale du bassin de Marennes-Oléron et étude des propriétés physico-chimiques de surface de micro-organismes impliquées dans leur adhésion[END_REF].

The concentration and total sugar content are calculated with the rate of dry matter of the sample, according to the following equation:

% = [ (𝑶𝑫 -𝒃) 𝒂 ⁄ ] × 𝑫 × 𝟏 𝟏𝟎𝟎𝟎 ⁄ × 𝟏𝟎0
where:

-OD is the optical density of the designated analysis, -b is the intercept of the calibration curve, -a is the slope of the calibration curve, -D is 1/factor of dilution, -1/1000 is the conversion of mg from µg, 100 is from 1 mg samples and conversion to % [START_REF] Smith | Measurement of protein using bicinchoninic acid[END_REF][START_REF] Kessler | Interference by lipids in the determination of protein using bicinchoninic acid[END_REF][START_REF] Wiechelman | Investigation of the bicinchoninic acid protein assay: Identification of the groups responsible for color formation[END_REF][START_REF] Brown | Biochemical composition of microalgae from the green algal classes Chlorophyceae and Prasinophyceae. 1. Amino acids, sugars and pigments[END_REF].

Polyphenol content analysis

The principle of the determination of polyphenols, according to the method of Turkmen et al. 2005, is shown in Fig. 22 below.

Figure 22.

Colorimetric determination of total phenols by the Folin-Ciocalteu [START_REF] Hardouin | Download citation of Production of water-soluble extracts from Ulva sp. using enzymatic hydrolysis processes: characterization, upgrading and potential development[END_REF]Burlot, 2016).

Principle: The samples' polyphenol content was determined by a method adapted from Folin-Ciocalteu (Turkmen et al. 2005). This assay method is based on the oxidation of the phenolic compounds by the Folin-Ciocalteu reagent. The reagent is composed of a mixture of phosphotungstic acid and phosphomolybdic acid. The reduction of these acids by phenolic compounds causes the appearance of a mixture of oxides of tungsten and molybdenum, blue. Gallic acid is used as a standard for calibration, and the results are expressed in gallic acid equivalents (EAG) per gram of extract (Wang et al. 2010).

Folin-Ciocalteu is a commonly used method for measuring the polyphenol content. This assay is based on the reduction-oxidation (redox) reactions, which are usually considered to be relatively stoichiometric and on the redox potential of the polyphenol hydroxyl group [START_REF] Hagerman | Choosing appropriate methods and standards for assaying tannin[END_REF].

Standards, reagents, and samples:

-Sodium carbonate 5%: 200 g of Na 2 CO 3 dissolved in 1L ultrapure water (Purelab Ultra, ELGA, France).

-Gallic acid solution at 1mg mL -1 : 10 mg Gallic acid in 50% methanol to make 10 mL.

-50% Folin-Ciocalteu reagent solution in distilled water: 10 mL reagent with 10 mL distilled water. Note! 100% Folin-Ciocalteu reagent is used for the microplate method.

-Sample to be analyzed at 1 mg mL -1 , the dilution factor to be determined so that the ODs measured are among those in the standard range.

The procedure of 20 µL of samples (0.25 mg mL -1 ) or standard was added with 130 µL of ultrapure water. Followed by 40 µL of Folin-Ciocalteu was then introduced to the samples or standard solution. In the end, 40 µL of 200g Na 2 CO 3 solution was added. The reaction was then incubated at 70 °C for 10 min and put in the box of ice to stop the reaction for 5 min. Then the optical density was read against the blank prepared at 620 nm. A standard range of gallic acid is carried out before each series of analyses (0, 20, 40, 60, 80, and 100 μg mL -1 ). All samples were made in triplicate.

The contents of the phenolic groups are calculated according to the following equation:

% = [ (𝑶𝑫 -𝒃) 𝒂 ⁄ ] × 𝑫 × 𝟏 𝟏𝟎𝟎𝟎 ⁄ × 𝟏𝟎0
where:

-OD is the optical density of the designated analysis, -b is the intercept of the calibration curve, -a is the slope of the calibration curve, -D is 1/factor of dilution, -1/1000 is the conversion of mg from µg, 100 is from 1 mg samples and conversion to % [START_REF] Smith | Measurement of protein using bicinchoninic acid[END_REF][START_REF] Kessler | Interference by lipids in the determination of protein using bicinchoninic acid[END_REF][START_REF] Wiechelman | Investigation of the bicinchoninic acid protein assay: Identification of the groups responsible for color formation[END_REF][START_REF] Brown | Biochemical composition of microalgae from the green algal classes Chlorophyceae and Prasinophyceae. 1. Amino acids, sugars and pigments[END_REF].

Sulfates group content analysis

The amount of sulfates bound to the polysaccharides was determined by the use of the colorimetric assay method developed by [START_REF] Jaques | A microelectrophoresis method for heparin[END_REF]. The (Burlot, 2016).

Principle:

The reaction, shown in Fig. 2-10, shows that in an aqueous medium, 3amino-7-(dimethylamino) phenothiazin-5-ium chloride (Azure A) will complex the sulfate groups. The chromophore complex emits a violet color measurable at a spectrophotometer at λ = 535 nm.

Standards, reagents and samples:

-A solution of 17% dextran sulfate (Sigma-Aldrich D6049) in ultra-pure water (Purelab Ultra, ELGA, France) at 1 mg mL -1 .

-Azure A solution (Sigma-Aldrich A6270) at 10 mg L -1 is prepared in ultrapure water.

-Sample to be analyzed at 1 mg mL -1 from extraction with ultrapure water, including the dilution is to be determined so that the measured ODs are among those in the range standard.

Procedure: In a 96-well microplate, 20 μL of the sample (diluted if necessary) and the standards are mixed with 200 μL of Azure A aqueous solution at 10 mg L -1 . The concentration of sulfate groups is determined from the linear line of the range standard made with 17% sulfate dextran (0, 10, 20, 30, 40, 50 μg mL -1 ). Optical density is read against the blank at 535 nm. All samples were made in triplicate.

The contents of the sulfate groups linked to the polysaccharides are calculated according to the following equation:

% = [ (𝑶𝑫 -𝒃) (𝒂 × 𝑫 × 𝟎. 𝟏𝟕) ⁄ ] /[𝟑𝟐 × 𝟗𝟔 × 𝟎. 𝟏]
where:

-The average OD is calculated by subtracting the OD from the blank (0 μg mL -1 of sulfated dextran, -b is the intercept of the calibration curve, -a is the slope of the calibration curve, -D is 1/factor of dilution, and the dilution factor is taken into account with a sample solution at 1 g L -1 , -The multiplying factor 0.17 is used to obtain the sulfur concentration in the sample, knowing that dextran is sulfated to 17%, -32 is the molar mass in g mol -1 of sulfur, -In this reaction, it is considered that one mole of sulfur equals one mole of sulfate. Multiply by 96 to change to the mass concentration of sulfate, -The multiplicative factor 0.1 is a unit conversion factor.

Ash content analysis

Ash is the general term used to describe the inorganic matter in fuel. In biomass fuels, the ash content may originate from the biomass itself, e.g., materials that the plant absorbed from the water during its growth. In food analysis, ash content represents the total mineral content. Ashing is the first step in preparing a food sample for specific elemental analysis. Since certain foods are high in particular minerals, ash becomes important [START_REF] Marshall | Ash Analysis[END_REF].

The principle is to incinerate materials at high temperatures (585 °C or higher). A hundred mg of dry material was put in the oven of 100 °C for 30 min then burnt on the Bunsen flame for 10 min. Later, the samples were kept in the incinerator (Carbolites FurnacesCSF, UK) at 585 °C for 3 h (Fig. 24A). Before measure their final weight, all samples were put in the desiccators for 1 h (Fig. 24B).

% = 𝑴 𝒇 𝑴 𝒊 × 𝟏𝟎𝟎

where: Mf= final mass of algae (g);

Mi= initial mass of algae (g)

Figure 24. Incineration of the organic material of the samples in a muffle furnace at 585 °C (A), and obtaining the mineral material (B).

Lipid and fatty acid methyl ester (FAME) analysis

The total lipids extraction protocol was based following the method of [START_REF] Bligh | A Rapid Method of Total Lipid Extraction and Purification[END_REF] and [START_REF] Kendel | Lipid Composition, Fatty Acids and Sterols in the Seaweeds Ulva armoricana, and Solieria chordalis from Brittany (France): An Analysis from Nutritional, Chemotaxonomic, and Antiproliferative Activity Perspectives[END_REF] with modifications (Fig. 25). Lipids were extracted from 10 grams of freeze-dried algae (crushed) with a 30-mL mixture of chloroform/methanol (1:1, v/v) and were stirred for over 12 hours at room temperature under agitation. The process was repeated three times. The extract was filtered using a Büchner funnel and washed with saturated brine (100 mL), dried over magnesium sulfate, and evaporated under reduced pressure. Lipid content was determined gravimetrically as a percentage of the seaweed dry weight before it was saponified, and esterified according to the method of [START_REF] Terme | Selective extraction of lipid classes from Solieria chordalis and Sargassum muticum using supercritical carbon dioxide and conventional solid-liquid methods[END_REF].

The proportion of lipids relative to the dry mass corresponds to the weight ratio of the total lipid extract and biomass. The percentage (%) of lipid was calculated according to the following equation: Esterification: FAs were then esterified with methanolic hydrogen chloride mixture into the round flask with a magnetic stirrer (Fig. 26B). Five percent sodium chloride with water was then added for washing and was extracted by hexane. Extraction was done four times. Each volume added is thrice the amount of the initial methanolic hydrogen chloride mixture. The organic phase was added with 2% sodium bicarbonate in water, dried over magnesium sulfate, and evaporated under reduced pressure. Finally, the obtained fatty acids and its FAME were then diluted to hexane at a ratio of 50 mg mL-1 and were analyzed by gas chromatography coupled with flame ionization detection (GC-FID, TRACE™ 1300, Thermo-Fischer Scientific™, Milan, Italy), equipped with a capillary column CP-Sil 5 CB (60 m × 0.25 mm × 0.25 µm), the carrier gas was nitrogen (0.5 mL•min -1 ). The injector and detector were set at 250 °C, and a temperature gradient was used for

Nutritional quality indexes

The nutritional quality indexes were assessed according to different parameters calculated with the concentration of fatty acids.

The equations of the indexes are the following:

(1) C18/C20 PUFA ratio,

𝐶18 𝐶20 = ∑ 𝐶18𝑃𝑈𝐹𝐴 ∑ 𝐶20𝑃𝑈𝐹𝐴
(2) ω6/ω3 ratio,

𝜔6/𝜔3 = ∑ 𝑃𝑈𝐹𝐴𝜔6 ∑ 𝑃𝑈𝐹𝐴𝜔3

A. Saponification

Evaporation of organic FA fraction

B. Esterification

Evaporation

GC-FID

(3) Atherogenicity Index (AI) [START_REF] Matos | Chemical Characterization of Six Microalgae with Potential Utility for Food Application[END_REF],

𝐴𝐼 = 𝐶12: 0 + 4 × 𝐶14: 0 + 𝐶16: 0 ∑ 𝑀𝑈𝐹𝐴 + ∑ 𝑃𝑈𝐹𝐴𝜔6 + ∑ 𝑃𝑈𝐹𝐴𝜔3 (4)
Thrombogenicity Index (TI), Unsaturation Index (UI) was calculated by multiplying the number of double bonds by the percentage of each fatty acid, followed by summing up their contributions [START_REF] Kumari | Fatty acid profiling of tropical marine macroalgae: An analysis from chemotaxonomic and nutritional perspectives[END_REF].

𝑇𝐼 = 𝐶14: 0 + 𝐶16: 0 + 𝐶18: 0 0.5 × ∑ 𝑀𝑈𝐹𝐴 + 0.5 × ∑ 𝑃𝑈𝐹𝐴𝜔6 + 3 × ∑ 𝑃𝑈𝐹𝐴𝜔3 + ∑ 𝑃𝑈𝐹𝐴𝜔3 ∑ 𝑃𝑈𝐹𝐴𝜔6 (

Characterization of polysaccharides

Ground freeze-dried seaweed samples, e.g., 15 g (Fig. 27

Caulerpa racemosa and Ulva lactuca cell wall polysaccharides

Extraction was performed, following a simplified and adapted protocol for C. racemosa [START_REF] Ghosh | In vitro anti-herpetic activity of sulfated polysaccharide fractions from Caulerpa racemosa[END_REF]) and U. fasciata [START_REF] Lahaye | Structure and functional properties of ulvan, a polysaccharide from green seaweeds[END_REF]. The extraction of Caulerpa polysaccharides was done in several stages. Each stage was separated by filtration, where its residue was precipitated in ethanol and dialyzed with a 12-14 kDa Spectra/Por membrane. A first enriched extract was obtained from 0.5 kg of freeze-dried algae (at, i.e., 10 g dw), by depigmentation.

C. racemosa hot water extraction (HWE)

The depigmented algal powder (DAP) (e.g., 4 g) from C. racemosa (Fig. 28) is first mixed with 200 mL distilled water at 80 °C, pH 6.5 for 30 minutes, and separation of the insoluble particles and the aqueous fraction was obtained by filtration on Buchner cloth. This process was repeated three times (3x), giving the hot water extracted (HWE) fraction (0.52g). The hot water insoluble residue is then washed twice or successively extracted in a strong alkali (1-and 4M-KOH/NaBH4) to isolate hemicellulosic polysaccharides. This mixture is initially at a concentration 1 M or 500 mL of (1M KOH + 0.4% NaBH4 at 30-35 °C for 4h, and 4-6 °C for 16 h) giving rise to fraction 1OH, and then at 4M concentration or 500 mL of (4M KOH + 0.4% NaBH4 at 30-35 °C for 4 h, and 4-6 °C for 16 h) resulting to 4OH fraction. Alkaline extracts were acidified with HCl to pH 6, and all extracts dialyzed extensively against water using a membrane having a molecular weight cut-off of 12 kDa. Soluble materials were recovered by diluting the retentate with water and ethanol at (1:5, v/v). The final residue was suspended in water, acidified to pH 5.2, dialyzed, and lyophilized to produce the insoluble residue (INS).

Figure 28. Schematic diagram of sequential extraction of polysaccharides of

Caulerpa racemosa (Forsskål) with inorganic solvents.

U. lactuca Ulvan extraction

The depigmented algal powder (e.g., 10 g) for U. lactuca (Fig. 29) is first Route 1: Successive precipitation was done with water and 99% ethanol (1: 5. v: v) starting from 1.37 g dw of crude ulvans for 24 hours at +4 °C. The precipitates were separated by filtration to obtain Ulvan PP1 fraction, and the liquid fraction was then concentrated by rotary evaporation and was then dissolved under the same previous condition. Second, precipitation was done to recover Ulvan PP2 fraction, and its liquid fraction was then concentrated by rotary evaporation and was named Ulvan PP3 fraction. These three precipitates or isolated polysaccharides were then dried and characterized.

Route 2: Dialysis was performed on a diluted crude extract 1.0 g Ulvan on a membrane 12-14 kDa for five days' agitation in 4.5 L of distilled water. To ensure maximum efficiency of dialysis, distilled water was renewed every 6 hours during the first two days, then every 12 hours for 3-5 days. At the end of the dialysis, the purified extract was heterogeneous, revealing a soluble fraction (Ulvan DS) and aggregates fraction (Ulvan DA) inside the membrane. Both fractions were separated by decantation, then freeze-dried and analyzed separately.

Halymenia durvillei and Halymenia dilatata polysaccharides

Extraction was performed, following a simplified and adapted protocol for red algae Solieria chordalis by [START_REF] Burlot | Response Surface Methodology for Enzyme-Assisted Extraction of Water-Soluble Antiviral Compounds from the Proliferative Macroalga Solieria chordalis[END_REF]. The extraction of Halymenia polysaccharides was done in a simple linear sequence by filtration, agitation, and ethanol precipitation. A first enriched extract was obtained from 0.5 kg of freezedried algae (at, i.e., 10 g dw), by depigmentation (Fig. 31).

The depigmented algal powder (DAP) (e.g., 5 g) from H. durvillei or H.

Monosaccharides profile by HPAEC-PAD

The Figure 32. CarboPac PA1 column. The quaternary amine functions of latex microbeads bind to SO3 groupings -the substrate of the column [START_REF] Rohrer | Analysis of Carbohydrates by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection[END_REF].

Latex microbeads

The column separates the sugars according to their pKa. The latter depends on hydroxyl groups of sugars, their number, and their position. Microbeads quaternary amines, which are positively charged, will attract the -OH groups of sugars. By introducing the eluent, here NaOH, there will be an exchange of anions between the soda and the hydroxyl groups of the sugars. Thus, monosaccharides, with the lowest pKa, interacting most strongly with the stationary phase, will be eluted last.

Pulsed Amperometric Detection (PAD) allows quantifying the monosaccharides after their passage in the column. Carbohydrates are detected by measuring the electric current generated by the oxidation of their hydroxyl groups, which are 0.10 volts on the surface of a gold electrode. Then, to clean the gold electrode and to continue the analysis of other sugars, a variation cycle of voltaic potentials is applied, as shown in Fig. 33. Finally, immediately after, a potential E4 is applied for 60 ms to reduce the native gold surface of the electrode and to allow better oxidation of sugars. This wave of potential is repeated throughout the chromatography.

The HPAEC-PAD consists of several modules shown in Fig. 34. glucose (G5767, Sigma-Aldrich), xylose (X1075, Sigma-Aldrich), ribose (R7500, Sigma-Aldrich), and glucuronic acid (G5269, Sigma-Aldrich) at 1000 ppm or 50 mg 50mL -1 of pure water. Two (2) mixtures of these sugars were made to make a calibration curve (Table XIV). Calibration of standards has been performed over a concentration range of 1. 95, 3.90, 7.80, 15.6, 31.25, 62.5, and 125 ppm. It is important to note that each of these sugars will have a final concentration (Cf) of 125 ppm except for deoxyribose, which has a final concentration (Cf) of 50 ppm.

The concentration of 125 ppm was based on the maximum concentration of sugars that the HPAEC can detect, or its sensitivity limit. The two mixtures created were based on its retention time (rt), and the peak of detection to separate each peak or avoid overlapping of peaks. Mixture one (1) were; deoxyribose, glucoheptose, rhamnose, glucosamine, galactose, mannose, and fructose. Mixture two (2) were;

deoxyribose, arabinose, glucose, xylose, ribose, and glucuronic acid. The solution of deoxyribose for 50 ppm, was done by taking 1 mL of deoxyribose from 1000 ppm mother solution and was added with 19 mL pure water to make 20 mL solution.

To determine how much volume must be taken from each prepared mother solution to make the mixture, we must decide how much our final volume (Vf) is, and in this The elution of monosaccharides was mainly in the isocratic mode. Sodium acetate is added after 30 minutes to elute the compounds in the column. Indeed, the acetate ions, more electronegative, will be exchanged by binding to quaternary amine microbeads.

Fourier transform infrared (FT-IR) spectroscopy

FT-IR is a vibrational spectroscopic technique and is based on the principle that most molecules absorb light in the infrared wavelength region of the electromagnetic spectrum. This absorption corresponds precisely to the chemical bonds present in molecules. FT-IR can facilitate to confirm the identity of a pure compound by using the unique collection of absorption bands [START_REF] Fernando | FTIR characterization and antioxidant activity of water-soluble crude polysaccharides of Sri Lankan marine algae[END_REF]. Spectra of polysaccharides extracted by conventional technique from green run before each analysis. The detected peaks were then compared with algal polysaccharide standards with their respective structural characteristics.

Minerals by flame atomic absorption spectrophotometry (AAS)

Major mineral elements (Ca, Mg, Na, and K) and trace elements (Fe, Zn, Cu, and Mn) were analyzed using a Perkin Elmer AAnalyst 200 atomic absorption spectrophotometer (AAS) with a single hollow cathode lamp for each element and an air-acetylene burner. The 200-mg raw samples of dried seaweed were placed into digestion vessels with 1.3 mL of 1 M HCl was added 19.7 mL of ultrapure water (18.2MΩ) for final sample concentration at 10 mg mL -1 and incubated 48 hours at 118 °C. Samples were filtered (0.22 μm, Minisart High Flow; Sartorius Stedim Biotech, Göttingen, Germany). Filtered samples were diluted in a 100-mL flask with distilled water at 1-mL sample added 99-mL distilled water to obtain a 1 mg mL -1 concentration.

Preparation of standards was done by diluting 20 mL of each element solution into 200ml of distilled water; then, a standard range has been established

depending on the suggested wavelengths with its respective standard range (i.e., for Zn, 213.86nm at 0.15-0.75 mg L -1 ). Quantification was performed using standard ranges depending on the element.

Analysis of pigments

Pigment analysis was based on the method used by [START_REF] Silkina | Antifouling activity of macroalgal extracts on Fragilaria pinnata (Bacillariophyceae): A comparison with Diuron[END_REF] with some modifications. The analysis was performed by high-performance liquid chromatography (HPLC) on a Nucleodur C18 column (EC 250/4.6 100-5 C18ec

Macherey-Nagel). A two-solvent elution (solvent A 100 %, 0-27 min; solvent B 100 %, 27-42 min) was used at a flow rate of 1.4 mL min -1 to separate the pigments.

Pigment extraction from raw material (20 mg) was diluted with methanol (2 mL, at 4 °C) and was sonicated (pulse 2, amplitude 100) for 2 minutes in test tubes (inserted in water and ice). After sonication, samples were kept in the dark cold room (4 °C) 24 hours before the injection of the sample (100 µL). After 24 hours, the diluted methanol extracts were centrifuged (36,000 x g, 4 min.), and then filtered (0.22 µm, Minisart High Flow; Sartorius Stedim Biotech, Göttingen, Germany) before analysis. Eluent preparation of Solvent A consisted of methanol (510 mL), acetonitrile (360 mL), and ammonium acetate (23.145 g) dissolved in water (130 mL); and solvent B was acetonitrile (30%), and ethyl acetate (70%). Pigments were detected and characterized by diode array measurements of their absorption spectra recorded between 300 and 600 nm. Chromatograms were recorded at a wavelength of 440 nm, and standards of chlorophyll a, b, and βcarotene (Sigma-Aldrich) were used for pigment identification and quantification. Pigment concentrations were expressed as mg g -1 .

Screening of biological activities

Evaluation of cytotoxicity and antiviral activity by cell viability

Each sample's antiviral activity is evaluated in parallel with the cytotoxicity [START_REF] Burlot | Response Surface Methodology for Enzyme-Assisted Extraction of Water-Soluble Antiviral Compounds from the Proliferative Macroalga Solieria chordalis[END_REF][START_REF] Hardouin | Enzyme-assisted extraction (EAE) for the production of antiviral and antioxidant extracts from the green seaweed Ulva armoricana (Ulvales, Ulvophyceae)[END_REF]. These tests are performed on previously extracted and freeze-dried polysaccharides and carried out in vitro using the Vero cells, a green monkey kidney fibroblast cell line (line n° ATCC CCL81). All of the manipulations are performed in Eagle's Minimal Essential Medium (MEM) culture (Appendix 1). The medium was renewed daily; cells need to be serviced twice a week (Appendix 2) and enumerated before each test to have an equivalent number of cells for each sample (Appendix 3).

The cell viability assessment method's principle is based on the staining of cells by the neutral red dye that only enters living cells by integrating, especially lysosomes [START_REF] Langlois | A rapid and automated colorimetric assay for evaluating the sensitivity of Herpes simplex strains to antiviral drugs[END_REF]). So, cell viability is quantifiable in spectrophotometer by reading an optical density at a wavelength fixed at ƛ = 540 nm.

The freeze-dried extracts were homogenized in MEM medium enriched with 10%

of DMSO, reaching a final concentration of 1 mg mL -1 . Five concentrations of one same extract are evaluated: 200 μg mL -1 ; 100 μg mL -1 ; 50 μg mL -1 ; 10 μg mL -1 and 1 μg mL -1 . The dilutions are carried out in cascade in enriched MEM. Each sample is made in quadruplets.

One hundred μL of cell suspension at 350,000 Vero mammalian cells mL (75 cm 2 culture flasks, 350,000 cells mL -1 ) incubated at 95/5 air/CO2, 37 °C for three days. After incubation, the suspension was frozen and thawed two times before centrifugation at low velocity (10,000 x g, 10 min) to collect the supernatant.

The liquid phase containing the virus suspension was stored at -80 °C before use.

Cytotoxic and antiherpetic activity evaluation by cell viability:

The fractions were prepared in MEM supplemented medium (8% FCS), and 50 μL was distributed into 96 wells microplate (micro-test III tissue culture plate, Nuclon Intermed, France).

A dilution series included five assays (quadruplicate) ranging from 1 to 200 μg mL - 1 extract concentrations. A 100 μL of cellular suspension diluted to a concentration at 3.5 x 10 5 Vero cells mL -1 in Eagle's MEM (8% FCS) is added to the wells. The cell suspension was infected by the addition of two different viral suspensions at a multiplicity of infection (MOI) of 0.001 ID50 per cell. The microplates were incubated at 37 °C, 5% of CO2 for three days. The cells were examined daily under an inverted microscope to determine the minimum concentration of extracts that induced alterations in cell morphology, including swelling, shrinkage, granularity, and detachment. Cytotoxicity by cell viability was tested using the neutral red dye method [START_REF] Langlois | A rapid and automated colorimetric assay for evaluating the sensitivity of Herpes simplex strains to antiviral drugs[END_REF]. Optical density (OD) was measured at 540 nm using a spectrophotometer (SpectraCountTM, Packard). The 50% cytotoxic concentration (CC50) was defined as the concentration that reduced the OD of treated cells to 50% of that of untreated cells. CC50 values were expressed as the percentage of destruction (%D):

CC 50 = ( OD 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑐𝑒𝑙𝑙𝑠 -OD 𝑐𝑒𝑙𝑙𝑠+𝑠𝑎𝑚𝑝𝑙𝑒 OD 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑐𝑒𝑙𝑙𝑠 ) x 100
where:

ODcontrol cells= were the OD values of the untreated cells, and

ODcells+sample= were the OD values of the treated cells [START_REF] Langlois | A rapid and automated colorimetric assay for evaluating the sensitivity of Herpes simplex strains to antiviral drugs[END_REF]).

After incubation, antiviral activity was evaluated by the neutral red dye ODcontrol cells+drug= were the OD of the infected cells control [START_REF] Langlois | A rapid and automated colorimetric assay for evaluating the sensitivity of Herpes simplex strains to antiviral drugs[END_REF]).

Antioxidant activity

DPPH radical scavenging assay

Antioxidant 2,2-diphenyl-1-picrylhydrazyl or DPPH radical scavenging activity was determined according to [START_REF] Terme | Radical scavenging activity of lipids from seaweeds isolated by solid-liquid extraction and supercritical fluids. Oilseeds and fats Crops and Lipids[END_REF]. DPPH assay is a stable free radical and is based on reduction reaction or the theory that a hydrogen donor is an antioxidant (Škrovánková et al. 2012). It measures compounds that are radical scavengers or reacts with compounds that can donate hydrogen atoms. DPPH shows a strong absorption maximum at 517 nm. There are two general types of assays widely used for different antioxidant studies. To the first group belong assays that are associated with electron or radical scavenging, including the DPPH assay, Trolox equivalent antioxidant capacity (TEAC) assay, and FRAP assay, while the other types are assays associated with lipid peroxidations, including the thiobarbituric acid assay and β-carotene bleaching assay (Škrovánková et al. 2012).

The DPPH method is widely used due to relatively short time required for the analysis (Škrovánková et al. 2012). A series of butylated hydroxyl-anisole (BHA), butylated hydroxyl-toluene (BHT) (respectively 2(3)-t-Butyl-4-hydroxyanisole, and 2,6-Di-tert-butyl-4-methylphenol, Sigma, Saint Quentin Fallavier, France) solutionsserves as affirmative standardis prepared in different concentrations

(2-50 µg mL -1 ) in methanol. DPPH solution of 0.25 mM is introduced to 100 µL samples solution. Samples solution of hydrolysates from C. racemosa, U. lactuca, S. polycystum, S. ilicifolium, H. durvillei, and H. dilatata were made in different concentration by diluting the solution stock in water (0-5000 µg mL -1 ). Before reading the optical density at 517 nm, all samples are incubated at 40 °C for 30 min.

Then, the percentage of inhibition is calculated by using the following formula:

𝐼 (%) = [ (𝐴 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 -𝐴 𝑠𝑎𝑚𝑝𝑙𝑒 ) 𝐴 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 ⁄ ] × 100
where:

A control = absorbance of control, and A sample = absorbance of samples IC 50 of samples is determined based on the regression obtained from dose response curve. IC is defined as the concentration sufficient to obtain 50% of a maximum scavenging capacity.

Statistical Analyses

Statistical analysis used: All measurements were done in triplicate for each alga (n=3), except for GC-FID (fatty acids, amino-acids) and HPAEC (monosaccharides) analyses were done only in duplicate. All values were reported as mean±standard deviation (SD). Student t-test was used to compare the degree of effectivity of EC50 or IC50 between different doses and control for each fraction of C. racemosa, U. lactuca, S. polycystum, S. ilicifolium, H. durvillei, and H. dilatata species.

Chapter 3 Results and Discussion

Green Seaweed (Caulerpa racemosa and Ulva lactuca)

Results and Discussion

The purpose of this part is to identify and compare the composition of the polysaccharides extracted from C. racemosa and U. lactuca, and also their nutritional and biological properties.

Cell wall polysaccharides purification

First treatment

The objective of this first treatment was to remove salts, small proteins, phlorotannins, and pigments. The yields were calculated by the ratio between the initial mass and the final mass of dry algae. Extraction yield in percent after three solvents (ethanol, chloroform/methanol, and acetone) are shown in Table XV. We obtain 89.92% and 89.65 % for C. racemosa and U. lactuca, respectively. Initial and final weights of raw C. racemosa and U. lactuca used to calculate extraction yield in percent after three solvents were shown in appendix 5.

Table XV.

Percent extraction efficiency of Philippine green seaweeds Caulerpa racemosa and Ulva lactuca.

Polysaccharide extraction

The IX).

In comparison with the study of [START_REF] Ghosh | In vitro anti-herpetic activity of sulfated polysaccharide fractions from Caulerpa racemosa[END_REF] on C. racemosa polysaccharide extraction, the authors reported higher values with (HWE= 20%, 1OH=12%, 4OH=16%, and INS= 40% dw) in similar extraction conditions. In the study of [START_REF] Chattopadhyay | Polysaccharides from Caulerpa racemosa: Purification and structural features[END_REF], the HWE fraction was purified from C.

racemosa by anion exchange chromatography to obtain three sub-fractions with its corresponding values (F1=20%, F2=11%, and F3=69%) of total material recovered.

Another study reported a 7 to 8% yield of crude polysaccharide from C. racemosa after ethanol precipitation by hot water extraction [START_REF] Ji | Separation of the polysaccharides in Caulerpa racemosa and their chemical composition and antitumor activity[END_REF]).

The differences between [START_REF] Ghosh | In vitro anti-herpetic activity of sulfated polysaccharide fractions from Caulerpa racemosa[END_REF] From crude Ulvan, we had obtained five Ulvan fractions: three from successive precipitation (PP1=63.41%, PP2=28.51%, and PP3=4.55%), and two from dialysis (DS=91.22%, and DA=8.35%). In the thesis manuscript of Hardouin in 2015 (http://www.theses.fr/2015LORIS373#), the author recorded for successive precipitation (PP1=33%, PP2=57%, and PP3=6%) and dialyzed extracts (DS=76.70%, and DA=23.30%). Despite the similar method used, the present study differs in the amounts of PP1, and PP2, while PP3 is somewhat similar. DS fraction is 15% higher, and DA is 15% lower than that of [START_REF] Hardouin | Download citation of Production of water-soluble extracts from Ulva sp. using enzymatic hydrolysis processes: characterization, upgrading and potential development[END_REF] http://www.theses.fr/2015LORIS373#). These different results may be due to the amount of the crude Ulvan that is being used, beginning with the extraction and purification (from 13 g: 10, 3 g [START_REF] Hardouin | Download citation of Production of water-soluble extracts from Ulva sp. using enzymatic hydrolysis processes: characterization, upgrading and potential development[END_REF], and from 2.37 g: 1.37, 1 g (present study) used for successive precipitation and dialysis, respectively). Different factors, such as species, season, and place of the collection again influence the yields [START_REF] Ji | Separation of the polysaccharides in Caulerpa racemosa and their chemical composition and antitumor activity[END_REF][START_REF] Chattopadhyay | Polysaccharides from Caulerpa racemosa: Purification and structural features[END_REF].

Biochemical composition analysis of extracts

The The present study sugar composition is somewhat similar to the results of [START_REF] Ghosh | In vitro anti-herpetic activity of sulfated polysaccharide fractions from Caulerpa racemosa[END_REF] using the same method of extraction from C. racemosa, where it showed the presence of glucose, xylose, galactose, smaller amounts of mannose, and traces of rhamnose, arabinose, and fucose. We obtain high percentages of glucose and galactose in fractions Cr crude, and 1OH which is in line with [START_REF] Ghosh | In vitro anti-herpetic activity of sulfated polysaccharide fractions from Caulerpa racemosa[END_REF], and other dominant sugars such as mannose, and xylose are also present in all fractions. However, although dominant, these sugars are just in small proportions, especially fractions 4OH and INS compared to [START_REF] Ghosh | In vitro anti-herpetic activity of sulfated polysaccharide fractions from Caulerpa racemosa[END_REF]. In [START_REF] Ghosh | In vitro anti-herpetic activity of sulfated polysaccharide fractions from Caulerpa racemosa[END_REF], the insoluble residue's primary sugar is xylose, indicating the presence of a xylan. In the present study, we observe xylose in all fractions, and it is even the primary sugar in the raw C. racemosa sample. Another study of [START_REF] Shevchenko | Polysaccharides and sterols from green algae Caulerpa lentillifera and C. sertularioides[END_REF] Eight monosaccharides have been identified in raw U. lactuca, 11 in DS, 10 in DA, 10 in PP1, 9 in PP2, and 10 in PP3. Primary sugar units of raw U. lactuca, were smaller amounts (<10%) of glucose, rhamnose, glucuronic acid, and xylose; and a trace amount (<1.0%) of galactose, mannose, and fructose. Fraction PP1 had rhamnose (5.87%) and glucose (3.73%) as main sugars; and trace values that ranged of a different nature aside from Ulvans [START_REF] Lahaye | Structure and functional properties of ulvan, a polysaccharide from green seaweeds[END_REF]. Both [START_REF] Lahaye | Structure and functional properties of ulvan, a polysaccharide from green seaweeds[END_REF] and [START_REF] Hardouin | Enzyme-assisted extraction (EAE) for the production of antiviral and antioxidant extracts from the green seaweed Ulva armoricana (Ulvales, Ulvophyceae)[END_REF] reported the presence of β-glucans, polymers of glucose in their samples. This result could also explain the dominant levels of glucose in all fractions in the present study.

Fourier Transform Infra-Red (FT-IR) spectroscopy

Each type of polysaccharide having different spectral number characteristics, the wavenumbers of the spectral bands are therefore specific to each polymer. The values indicated by lines or arrows are the spectral bands' characteristics of Ulvans shown in Fig 37 . The polysaccharide used as the standard for the U. lactuca fractions measurements was the Ulvan DS produced and characterized (Fig. 37).

The value specified on the spectrum indicates the specific wavenumbers for the identification of Ulvans and is expressed in cm -1 .

Figure 37.

Infra-red spectrum of the Ulvan DS sample on the 2000-500 cm -1 zone.

Ulvan DS was also used as a standard for polysaccharide differentiation of U. lactuca in the present study. Infrared spectroscopy provides useful information on the position of sulfate groups of polysaccharides [START_REF] Ghosh | In vitro anti-herpetic activity of sulfated polysaccharide fractions from Caulerpa racemosa[END_REF]). The calculated FTIR spectra of green seaweeds C. racemosa (Cr crude, 1OH, 4OH, and INS) and U. lactuca (PP1, PP2, PP3, DS, and DA), spectral bands of interest for polysaccharide extracts were shown in Fig. 38 and39.

The IR spectrum of C. racemosa extracts (Fig. 38) within 2000-500 cm -1 zone or the fingerprint region for polysaccharides were used for data analysis.

Extracts showed spectral bands of high intensity between 1025 and 1000 cm -1 , while bands of medium or low intensity at [1641-1630 cm -1 ], [1561-1546 cm -1 ], and [1245-1240 cm -1 ], in all extracts. Moreover, low-intensity peak at 871 cm -1 was shown in fraction Cr crude, and at 793 cm -1 in fraction INS. Table XX summarizes some of the major IR vibrational modes of C. racemosa polysaccharides [START_REF] Ghosh | In vitro anti-herpetic activity of sulfated polysaccharide fractions from Caulerpa racemosa[END_REF]Marungrueng and Pavasant, 2007;[START_REF] Robic | Determination of the chemical composition of ulvan, a cell wall polysaccharide from Ulva spp. (Ulvales, Chlorophyta) by FT-IR and chemometrics[END_REF][START_REF] Gómez-Ordóñez | FTIR-ATR spectroscopy as a tool for polysaccharide identification in edible brown and red seaweeds[END_REF][START_REF] Sarada | Cadmium removal by macro algae Caulerpa fastigiata: Characterization, kinetic, isotherm and thermodynamic studies[END_REF][START_REF] Fernando | FTIR characterization and antioxidant activity of water-soluble crude polysaccharides of Sri Lankan marine algae[END_REF][START_REF] Barbosa | In Vitro Immunostimulating Activity of Sulfated Polysaccharides from Caulerpa cupressoides Var. Flabellata[END_REF][START_REF] Figueira | Structural Characterization of Ulvan Polysaccharide from Cultivated and Collected Ulva fasciata (Chlorophyta)[END_REF]. 2014), and associated with 3,6-anhydrogalactose and the sulfation of C-4, C-6 of galactose units and C-2 of 3,6-anhydrogalactose [START_REF] Fernando | FTIR characterization and antioxidant activity of water-soluble crude polysaccharides of Sri Lankan marine algae[END_REF]. The band at 793 cm -1 is of Si-C group, and the bands present below 800 cm -1 are fingerprint zone of phosphate and sulfur functional groups and N containing bioligands [START_REF] Sarada | Cadmium removal by macro algae Caulerpa fastigiata: Characterization, kinetic, isotherm and thermodynamic studies[END_REF]). This silica particle was observed attached to the thallus during collection, and even after cleaning, and drying of samples. A similar peak was reported in [START_REF] Sarada | Cadmium removal by macro algae Caulerpa fastigiata: Characterization, kinetic, isotherm and thermodynamic studies[END_REF] for Caulerpa fastigiata, where the presence of siliceous from diatomaceous earth in algal waste and composite material. The spectrum of U. lactuca Ulvans (Fig. 37 and 39) within 2000-500 cm -1 zone or the fingerprint region for polysaccharides were used for data analysis.

Extracts from Ulvan DS used as the standard, have specific spectral bands of high in all fractions, corresponds to glycosidic linkages, which have also been reported in previous studies [START_REF] Robic | Determination of the chemical composition of ulvan, a cell wall polysaccharide from Ulva spp. (Ulvales, Chlorophyta) by FT-IR and chemometrics[END_REF][START_REF] Hardouin | Download citation of Production of water-soluble extracts from Ulva sp. using enzymatic hydrolysis processes: characterization, upgrading and potential development[END_REF][START_REF] Figueira | Structural Characterization of Ulvan Polysaccharide from Cultivated and Collected Ulva fasciata (Chlorophyta)[END_REF]). The bands around 845 cm -1 (in all Ulvan fractions), around 785 cm -1 (fractions DS, DA, and PP2) and probably a shift of intensity bands at around 740 cm -1 (fractions PP1 and PP3) are characteristics of the presence of sulfate groups on the molecule. At According to [START_REF] Olasehinde | Chemical characterization of sulfated polysaccharides from Gracilaria gracilis and Ulva lactuca and their radical scavenging, metal chelating, and cholinesterase inhibitory activities[END_REF], the presence of sulfate groups in algal polysaccharides is the origin of their biological activities. In U. lactuca, the maximum efficacy or the cellular protection of fraction DA (75.24%) is slightly higher than DS (71.25%) and PP2 (70.34%), while much lesser found in PP1 (30.81%) and PP3 (14.17%). In terms of potency or the effectivity, fraction DA achieved 50% at 88.32 µg mL -1 sample (EC50: 88.32 µg mL -1 ) is more potent than PP2 (EC50: 122.13 µg mL -1 ) and DS (EC50: 136.82 µg mL -1 ). ) that appeared to be more potent and effective compared to [START_REF] Hardouin | Enzyme-assisted extraction (EAE) for the production of antiviral and antioxidant extracts from the green seaweed Ulva armoricana (Ulvales, Ulvophyceae)[END_REF]. Glucose (0.99-12.42%) and rhamnose (0.54-6.44%) content, although in a smaller proportion, were the major sugar constituents in the present study for U. lactuca fractions, which is also in accordance to [START_REF] Hardouin | Enzyme-assisted extraction (EAE) for the production of antiviral and antioxidant extracts from the green seaweed Ulva armoricana (Ulvales, Ulvophyceae)[END_REF]. From the thesis results of [START_REF] Hardouin | Download citation of Production of water-soluble extracts from Ulva sp. using enzymatic hydrolysis processes: characterization, upgrading and potential development[END_REF] using similar extraction methods, EC50 activity is not only dictated on Ulvans or polysaccharides alone but also of its protein contents. [START_REF] Hardouin | Download citation of Production of water-soluble extracts from Ulva sp. using enzymatic hydrolysis processes: characterization, upgrading and potential development[END_REF] reported (EC50) values in fraction PP1 (EC50 <400.0 ug mL -1 ), and DA (EC50=68.6 ug mL -1 ) with proteins content of 22.3%, and 41.7%, respectively. In the present study of U. lactuca extracts, no pattern of sulfate content found to relate it to EC50 values. However, the protein content of the fractions with antiviral activities against HSV-1 was high in the present study. Variations in the antiviral activity of ulvan from different sources indicate a significant effect of the structure.

However, only a few studies probe the antiviral activity of ulvan, and consequently, the understanding of its structure-activity relationships is restricted [START_REF] Kidgell | Ulvan: A systematic review of extraction, composition and function[END_REF].

Antioxidant activity, DPPH radical scavenging activity

The free radical scavenging of the blank and the fractions of C. racemosa et al. 2018). In the present study of C. racemosa fractions, however, no pattern of relationships found between antioxidant activities and its sulfates (2.46-10.58% dw), uronic acids (0.1-1.5% dw), or total polyphenols (2.1-6.2% dw) contents.

The DPPH scavenging activity in the present study for all U. lactuca extracts except fraction PP1 is in accordance to (<50%) in sulfated polysaccharides from Ulva lactuca against DPPH [START_REF] Olasehinde | Chemical characterization of sulfated polysaccharides from Gracilaria gracilis and Ulva lactuca and their radical scavenging, metal chelating, and cholinesterase inhibitory activities[END_REF], and ulvan extracts from U. pertusa against superoxide radical [START_REF] Qi | Antioxidant activity of different molecular weight sulfated polysaccharides from Ulva pertusa Kjellm (Chlorophyta)[END_REF]. However, the concentration used for polysaccharides from Ulva lactuca against DPPH (333.33 µg mL -1 ) was 15 times lower [START_REF] Olasehinde | Chemical characterization of sulfated polysaccharides from Gracilaria gracilis and Ulva lactuca and their radical scavenging, metal chelating, and cholinesterase inhibitory activities[END_REF], and therefore, had stronger antiradical activity compared to the present study U. lactuca extracts. [START_REF] Qi | Antioxidant activity of different molecular weight sulfated polysaccharides from Ulva pertusa Kjellm (Chlorophyta)[END_REF] also reported stronger antiradical activity of ulvan extracts from U. pertusa with different Mw (157.7-28.2 kDa); against superoxide (<50%, IC50=22.1 µg mL -1 ), against hydroxyl (5-60%, IC50=1-2.8 mg mL -1 ), and against Fe 2+ -chelating (23.9-94.3%) activities all at 0.6-3.4 mg mL -1 . [START_REF] Shao | In intro antioxidant activities of different sulfated polysaccharides from chlorophytan seaweeds Ulva fasciata[END_REF] reported ulvan extracts from U. lactuca with different Mw (264-2 kDa), showed highest scavenged against superoxide (66.17%), and hydroxyl (82.56%, IC50=6.03.8 mg mL -1 ) radicals at 2-10 mg mL -1 concentration were at lowest sulfate content (11.67% dw). Also, the strongest inhibition (IC50: 805.59 µg mL -1 ) in the present study had the lowest sulfate content (5.44% dw), which is somewhat in accordance to [START_REF] Shao | In intro antioxidant activities of different sulfated polysaccharides from chlorophytan seaweeds Ulva fasciata[END_REF] but is contrary to [START_REF] Qi | Antioxidant activity of different molecular weight sulfated polysaccharides from Ulva pertusa Kjellm (Chlorophyta)[END_REF]. These activities mentioned, were said to be influenced: by protein and sulfate contents, or conjugated proteins to polysaccharides in Ulva lactuca polysaccharides [START_REF] Olasehinde | Chemical characterization of sulfated polysaccharides from Gracilaria gracilis and Ulva lactuca and their radical scavenging, metal chelating, and cholinesterase inhibitory activities[END_REF]; by low molecular weights in U. pertusa polysaccharides [START_REF] Qi | Antioxidant activity of different molecular weight sulfated polysaccharides from Ulva pertusa Kjellm (Chlorophyta)[END_REF]; by combined factors of Mw, sulfates content, and proteins content in U. lactuca extracts with different Mw [START_REF] Shao | In intro antioxidant activities of different sulfated polysaccharides from chlorophytan seaweeds Ulva fasciata[END_REF]. No pattern of relationships found between antioxidant activities and proteins, sulfates, uronic acids, or polyphenols content of U. lactuca extracts in the present study. These activities would need to be studied.

At the opposite, Caulerpa cupressoides var. flabellata has also been reported to contain sulfated polysaccharides with total antioxidant capacity and ferrous chelating activity [START_REF] Costa | Characterization of ulvan extracts to assess the effect of different steps in the extraction procedure[END_REF]) but showed no activity in superoxide and hydroxyl radical scavenging. Possibly, the antiradical activity could be related to phenolic compounds.

The C. racemosa and U. lactuca extracts from the present study are considered significant since they are derived from natural resources; therefore, they can be utilized to develop various functional and health foods. There are seven species of Caulerpa utilized as food in the Philippines, four of which are used for medical applications [START_REF] Trono | The seaweed resources of the Philippines[END_REF], and Ulva sp. was one of the main species farmed and sold on European market [START_REF] Barbier | PEGASUS-PHYCORMORPH European Guidelines for a Sustainable Aquaculture of Seaweeds[END_REF]. However, various factors mentioned must be understood better, and therefore extracts from C. racemosa must be studied further with a priority of investigating the structure of sulfated polysaccharide and antiviral and antioxidant mechanisms of action.

Conclusion and perspectives

In Neutral sugars content for raw S. polycystum (35.4%) and raw S. ilicifolium (24.2%) are the most abundant component in the present study, as was also the case in most of the seaweeds in general [START_REF] Hao | Chemical composition and immunostimulatory properties of green alga Caulerpa racemosa var Peltata[END_REF]. A lower amount of neutral sugars is reported (23.2%) from Sargassum fluitans [START_REF] Bedoux | Antiviral and Cytotoxic Activities of Polysaccharides Extracted from Four Tropical Seaweed Species[END_REF], and (4.9-13.6%) from Sargassum muticum [START_REF] Vandanjon | Seasonal Variation of Sargassum Muticum Biochemical Composition Determined by Fourier Transform Infra-Red Spectroscopy[END_REF] than the present study.

Higher ash content (42.4%) was reported from S. polycystum [START_REF] Matanjun | Nutrient content of tropical edible seaweeds, Eucheuma cottonii, Caulerpa lentillifera and Sargassum polycystum[END_REF] of the same species, and (37%) from Sacchoriza polyschides [START_REF] Garcia | Nutritional Potential of Four Seaweed Species Collected in the Barbate Estuary (Gulf of Cadiz, Spain)[END_REF] Lipid contents for raw S. polycystum (2.86%) and raw S. ilicifolium (3.66%) in the present study were a little over the concentration range value (0.9-2.1% dw) for edible brown seaweeds L. digitata [START_REF] Fleurence | Seaweeds as Food[END_REF]. [START_REF] Matanjun | Nutrient content of tropical edible seaweeds, Eucheuma cottonii, Caulerpa lentillifera and Sargassum polycystum[END_REF] reported about 8-times lower lipid content (0.29%) from S. polycystum compared to the same species in the present study. Lipid contents from S. muticum (3.15, 3.0, and 2.84% dw) with corresponding extraction procedures (chloroform/methanol, chloroform, and chloroform/methanol with centrifugation, respectively) [START_REF] Terme | Selective extraction of lipid classes from Solieria chordalis and Sargassum muticum using supercritical carbon dioxide and conventional solid-liquid methods[END_REF], and (3.24% dw) from Colpomenia sinuosa [START_REF] Ragonese | Characterisation of lipid fraction of marine macroalgae by means of chromatography techniques coupled to mass spectrometry[END_REF] were found similar to the present study. Also, higher lipid contents found in (6.27%)

Dictyota dichotoma [START_REF] Ragonese | Characterisation of lipid fraction of marine macroalgae by means of chromatography techniques coupled to mass spectrometry[END_REF], and (8.2%) Sacchoriza polyschides [START_REF] Garcia | Nutritional Potential of Four Seaweed Species Collected in the Barbate Estuary (Gulf of Cadiz, Spain)[END_REF]) compared to the present study. These variations of results of carbohydrates, ash, proteins, uronic acids, polyphenols, and lipids from both present and previous studies in brown seaweeds, could be affected by geography, season, or growth conditions as stated in previous papers [START_REF] Kok | Physicochemical properties of edible alginate film from Malaysian Sargassum polycystum C. Agardh[END_REF][START_REF] Artemisia | The Properties of Brown Marine Algae Sargassum turbinarioides and Sargassum ilicifolium Collected from Yogyakarta, Indonesia[END_REF].

Conclusion and perspectives

In conclusion of this first part of the study for the biochemical analyses, the raw materials from S. polycystum and S. ilicifolium are mainly composed of carbohydrates (62.0%, 42.4%), and significant presence of ash (24.9%, 17.0%) and proteins (24.2%, 17.8%), respectively. Both species are rich in lipids (2.86%, 3.66%) and are higher than the concentration range value for edible brown seaweeds L. digitata. We acknowledge the intra-species variation of biochemical results. However, the biochemical composition of raw S. polycystum and raw S.

ilicifolium were within the values specified for brown seaweeds. Thus, there is a potential for these species to be used as a raw material in food industry development or human health. Although S. polycystum is not traditionally utilized as food unlike the S. ilicifolium, it is also important to evaluate the heavy metal and microbiological contents of this seaweeds in order to established reputation as food.

Cell wall polysaccharides purification

First treatment

Extraction yield in percent after three solvents (ethanol, chloroform/methanol, and acetone) are shown in Table XXV. We obtain respectively 71.57% and 79.94% for S. polycystum and S. ilicifolium. Initial and final weights of raw S. polycystum and S. ilicifolium that were used to calculate extraction yield in percent after three solvents were shown in appendix 5.

Table XXV. Percent extraction efficiency of Philippine brown seaweeds

Sargassum polycystum and Sargassum ilicifolium. [START_REF] Kok | Physicochemical properties of edible alginate film from Malaysian Sargassum polycystum C. Agardh[END_REF].

Polysaccharide extraction

Species

with range values (24.5-2.5%); and fraction Si HCl (yield, 2.55%) and composition trend: proteins>neutral sugars>uronic acids>polyphenols>sulfates with range values (25.5-6.98% dw). These variations of biochemical results of from both present and previous studies in brown seaweeds extracts, could be affected by geography, season, or growth conditions as stated in previous papers [START_REF] Artemisia | The Properties of Brown Marine Algae Sargassum turbinarioides and Sargassum ilicifolium Collected from Yogyakarta, Indonesia[END_REF][START_REF] Kok | Physicochemical properties of edible alginate film from Malaysian Sargassum polycystum C. Agardh[END_REF]. However, the biochemical composition of S. polycystum and raw S.

ilicifolium extracts in the present study were within the values specified for brown seaweeds. Thus, there is a potential for these species to be used as a raw material in food industry development or human health, especially as there is an on-going development of appropriate sustainable culture technologies for Sargassum species in the Philippines [START_REF] Trono | The seaweed resources of the Philippines[END_REF].

Monosaccharide composition analysis

The unit sugar composition of raw and purified fractions (CaCl2, HCl, [START_REF] Lim | Characterisation of fucoidan extracted from Malaysian Sargassum binderi[END_REF]).

Several Sargassum studies reported higher monosaccharide contents than the present study Sp fractions: crude fucoidan fraction from S. polycystum had 8times higher fucose content (46.8%), 3-times higher galactose (14.3%) 2-times higher glucose (11.5%) [START_REF] Palanisamy | Isolation of fucoidan from Sargassum polycystum brown algae: Structural characterization, in vitro antioxidant and anticancer activity[END_REF]. [START_REF] Sinha | Polysaccharides from Sargassum tenerrimum: Structural features, chemical modification and antiviral activity[END_REF] reported crude fucoidan, sulfated fucoidan, and impure alginate samples from S. tenerrimum had 10-times higher contents in fucose (73, 70, and 68%), 20-times higher xylose (15, 16, and 17%), and slightly higher galactose (9, 9, and 11%), respectively. Also reported from S. duplicatum 10-times higher of fucose (51.3%), and galactose (48.6%) contents [START_REF] Usoltseva | Polysaccharides from brown algae Sargassum duplicatum: the structure and anticancer activity in vitro[END_REF]; crude fucoidan from S. binderi had 6times higher fucose (34.5%), and 2-times higher galactose (12.14%) [START_REF] Lim | Characterisation of fucoidan extracted from Malaysian Sargassum binderi[END_REF]; as well as hot water extracted polysaccharides from S. fusiforme had 6-times higher fucose (34.95%), galactose (35.09%), and mannose (12.41%), while 2-times higher glucose (13.41%) [START_REF] Wu | Analysis of the monosaccharide composition of water-soluble polysaccharides from Sargassum fusiforme by high performance liquid chromatography/electrospray ionisation mass spectrometry[END_REF]. Other sugar units such as mannitol, arabinose, rhamnose, and xylose were in trace amounts in all Sp fractions in the present study as was also reported in the literatures [START_REF] Ale | Fucose-Containing Sulfated Polysaccharides from Brown Seaweeds Inhibit Proliferation of Melanoma Cells and Induce Apoptosis by Activation of Caspase-3 in Vitro[END_REF][START_REF] Li | Fractionation, preliminary structural characterization and bioactivities of polysaccharides from Sargassum pallidum[END_REF]Ye et al. 2018).

While, there are ten monosaccharides identified present in raw S. ilicifolium, XXIX).

11

Table XXIX.

Unit sugar composition of raw and extracted fractions from the Philippines brown seaweed S. ilicifolium.

-= not detected; The values are expressed in g/100g of total sugars in the fractions.

All Si fractions in the present study had fucose, galactose, glucose, and mannose as major sugar units. These major sugars in all Si fractions are similar or slightly higher than the reported mean range values for fucose (8.2-13%), galactose (5.4-11.5%), and mannose (3.7-4.7%) by [START_REF] Thadhani | Comparative analysis of sugar and mineral content of Sargassum spp. collected from different coasts of Sri Lanka[END_REF]. However, [START_REF] Thadhani | Comparative analysis of sugar and mineral content of Sargassum spp. collected from different coasts of Sri Lanka[END_REF] had 5-times higher glucose content (25.5-33.5%) than fraction Si HCl (4.7%), 2-times higher than fraction Si KOH, similar glucose content with fraction Si CaCl2 (33.69%), and more than 20-times higher than fraction Si Na2CO3 (1.18%) in the present study. [START_REF] Palanisamy | Isolation of fucoidan from Sargassum polycystum brown algae: Structural characterization, in vitro antioxidant and anticancer activity[END_REF] reported similar mannose content (5.6%) from crude fucoidan fraction of S. polycystum to all Si fractions, and 3-times higher fucose (46.8%) than FCSPs fraction (Si CaCl2

S. ilicifolium

and Si HCl), and upto 9-times higher fucose than fraction Si Na2CO3 and Si KOH, in the present study. In contrast, [START_REF] Palanisamy | Isolation of fucoidan from Sargassum polycystum brown algae: Structural characterization, in vitro antioxidant and anticancer activity[END_REF] reported 3-times lower glucose (11.5%) than fraction (Si CaCl2), 2-times higher than (Si HCl), upto 8-times higher than Si Na2CO3, and similar content to Si KOH.

Several Sargassum species reported mostly higher monosaccharide contents. Crude fucoidan and sulfated fucoidan samples: from S. tenerrimum with fucose (73, and 70%) respectively, had 4-times higher than FCSPs fraction (Si CaCl2 and Si HCl) and 10-times higher than fractions Si Na2CO3 and Si KOH.

While slightly lower galactose (9, and 9%) than FCSPs fraction (Si CaCl2 and Si HCl) and 2-times higher than fractions Si Na2CO3 and Si KOH, respectively [START_REF] Sinha | Polysaccharides from Sargassum tenerrimum: Structural features, chemical modification and antiviral activity[END_REF]. [START_REF] Usoltseva | Polysaccharides from brown algae Sargassum duplicatum: the structure and anticancer activity in vitro[END_REF] also reported 3-times higher fucose (51.3%) and galactose (48.6%) from fucoidan extract in S. duplicatum; also, crude fucoidan from S. binderi reported 2-times higher fucose (34.5%), and a similar result of galactose (12.14%) [START_REF] Lim | Characterisation of fucoidan extracted from Malaysian Sargassum binderi[END_REF]. Also, hot water extracts from S. fusiforme reported 2 to 7-times higher fucose (34.95%), galactose (35.09%), and mannose (12.41%) [START_REF] Wu | Analysis of the monosaccharide composition of water-soluble polysaccharides from Sargassum fusiforme by high performance liquid chromatography/electrospray ionisation mass spectrometry[END_REF]) than all Si fractions in the present study. Although this hot water extracted polysaccharides from S. fusiforme reported almost 3-times lower glucose (13.41%) [START_REF] Wu | Analysis of the monosaccharide composition of water-soluble polysaccharides from Sargassum fusiforme by high performance liquid chromatography/electrospray ionisation mass spectrometry[END_REF]) than fraction Si CaCl2, it is also 3-times higher than fraction Si HCl, similar with fraction Si KOH, and way higher than fraction Si Na2CO3 in the present study.

All Si fractions in the present study had very low glucuronic acid (about 2.0%), except for fraction Si HCl (14.94%) than the range value of 13.7-24.2% [START_REF] Thadhani | Comparative analysis of sugar and mineral content of Sargassum spp. collected from different coasts of Sri Lanka[END_REF]. Other sugar units such as mannitol, arabinose, rhamnose, and xylose were in trace amounts in all Sp fractions in the present study as was also reported in the literatures [START_REF] Ale | Fucose-Containing Sulfated Polysaccharides from Brown Seaweeds Inhibit Proliferation of Melanoma Cells and Induce Apoptosis by Activation of Caspase-3 in Vitro[END_REF][START_REF] Li | Fractionation, preliminary structural characterization and bioactivities of polysaccharides from Sargassum pallidum[END_REF]Ye et al. 2018).

The sugar profiles found that all of the fractions from S. polycystum and S.

ilicifolium in the present study contained mostly the same types of sugars in different proportions. Several factors affect fucoidan, and alginate contents, which include; the type of samples used, whether between or across the same species, life cycle, period, and location of the collection. These factors were also observed in the literature [START_REF] Thadhani | Comparative analysis of sugar and mineral content of Sargassum spp. collected from different coasts of Sri Lanka[END_REF].

Fourier Transform Infra-Red (FT-IR) spectroscopy

The calculated FTIR spectra of brown seaweeds S. polycystum (Sp CaCl2, Sp HCl, Sp Na2CO3, Sp KOH) and S. ilicifolium (Si CaCl2, Si HCl, Si Na2CO3, Si KOH) spectral bands of interest were shown in Fig. 40 and 41, respectively. The IR spectrum of S. polycystum extracts (Fig. 40) within 2000-500 cm -1 zone or the fingerprint region for polysaccharides were used for data analysis. Extracts showed spectral bands of high intensity at [1412-1408 cm -1 ] for Sp CaCl2, and Sp Na2CO3;

at [1026-1025 cm -1 ] for Sp CaCl2, Sp HCl, and Sp Na2CO3; at 1596 cm -1 for Sp

Na2CO3; and at 1484 cm -1 for Sp KOH. While bands of medium or low intensity between [1641-1637 cm -1 and 535-526 cm -1 ] for Sp CaCl2, and Sp HCl; at [1325-1321 cm -1 and 883-873 cm -1 ] for Sp CaCl2, Sp HCl, Sp KOH; at 1407 cm -1 for Sp HCl; at [1063, and 815 cm -1 ] for Sp Na2CO3. Table XXX summarizes some of the major IR vibrational modes of S. polycystum polysaccharides [START_REF] Marudhupandi | In vitro anticancer activity of fucoidan from Turbinaria conoides against A549 cell lines[END_REF][START_REF] Vandanjon | Seasonal Variation of Sargassum Muticum Biochemical Composition Determined by Fourier Transform Infra-Red Spectroscopy[END_REF][START_REF] Palanisamy | Investigation of antioxidant and anticancer potential of fucoidan from Sargassum polycystum[END_REF][START_REF] Kok | Physicochemical properties of edible alginate film from Malaysian Sargassum polycystum C. Agardh[END_REF][START_REF] Artemisia | The Properties of Brown Marine Algae Sargassum turbinarioides and Sargassum ilicifolium Collected from Yogyakarta, Indonesia[END_REF]. This similar peak at 1409.70 cm -1 was also recorded in the study of [START_REF] Kok | Physicochemical properties of edible alginate film from Malaysian Sargassum polycystum C. Agardh[END_REF] for the sodium alginate extract from S. polycystum. Moreover, Kok and Wong added that the absorption bands showed a decrease in intensity when the alginate was cross-linked with calcium. This decrease in intensity for this peak was The IR spectrum of S. ilicifolium extracts (Fig. 41) within 2000-500 cm -1 zone or the fingerprint region for polysaccharides were used for data analysis.

Extracts showed spectral bands of high intensity at [1032-1025 cm -1 ] in all fractions; at 1597 cm -1 for Sp HCl, Si Na2CO3, and Si KOH; and at 1408 cm -1 for Si Na2CO3, and Si KOH. While bands of medium or low intensity at [1612, and 1546 cm -1 ] for Si CaCl2; at [1419-1414 cm -1 and 1255-1249 cm -1 ] for Si CaCl2, and

Si HCl; at [831-816 cm -1 ] in all fractions; and [962-943 cm -1 ] for Si CaCl2 and Sp Na2CO3, and Si KOH. Table XXXI summarizes some of the major IR vibrational modes of S. polycystum polysaccharides [START_REF] Ale | Fucose-Containing Sulfated Polysaccharides from Brown Seaweeds Inhibit Proliferation of Melanoma Cells and Induce Apoptosis by Activation of Caspase-3 in Vitro[END_REF][START_REF] Marudhupandi | In vitro anticancer activity of fucoidan from Turbinaria conoides against A549 cell lines[END_REF][START_REF] Vandanjon | Seasonal Variation of Sargassum Muticum Biochemical Composition Determined by Fourier Transform Infra-Red Spectroscopy[END_REF][START_REF] Sun | The stability and bioaccessibility of fucoxanthin in spray-dried microcapsules based on various biopolymers[END_REF][START_REF] Palanisamy | Investigation of antioxidant and anticancer potential of fucoidan from Sargassum polycystum[END_REF][START_REF] Kok | Physicochemical properties of edible alginate film from Malaysian Sargassum polycystum C. Agardh[END_REF][START_REF] Artemisia | The Properties of Brown Marine Algae Sargassum turbinarioides and Sargassum ilicifolium Collected from Yogyakarta, Indonesia[END_REF]. ilicifolium fractions were due to the symmetrical and asymmetrical stretching of carboxylate groups, or the C-H scissoring and bending. This similar peak was also recorded in the study of [START_REF] Kok | Physicochemical properties of edible alginate film from Malaysian Sargassum polycystum C. Agardh[END_REF] for the sodium alginate extract from S. polycystum, and peaks at [1452-1339 cm -1 ] were typical for carotenoids (fucoxanthin) extracted from Undaria pinnatifida [START_REF] Sun | The stability and bioaccessibility of fucoxanthin in spray-dried microcapsules based on various biopolymers[END_REF]. Decrease of intensity for this particular peak was also observed in the present study from fraction Si Na2CO3 (an alginate rich) decreasing in fraction Si KOH (composed of cellulose, hemicellulose, and little fucose), and followed by fucose rich fractions Si extracts were due to stretching vibrations of pyranose rings, which was also present in the study of [START_REF] Kok | Physicochemical properties of edible alginate film from Malaysian Sargassum polycystum C. Agardh[END_REF] for sodium alginate fraction of S. polycystum and [START_REF] Marudhupandi | In vitro anticancer activity of fucoidan from Turbinaria conoides against A549 cell lines[END_REF] for fucoidan fraction of T. conoides. The band at 962 cm -1 of fraction Si CaCl2 is attributed to the bending vibration of =CH, which was also reported in [START_REF] Artemisia | The Properties of Brown Marine Algae Sargassum turbinarioides and Sargassum ilicifolium Collected from Yogyakarta, Indonesia[END_REF] for all fucoidan fractions of Sargassum ilicifolium. The band at [946-943cm -1 ] fractions Si Na2CO3 and Si KOH is attributed to the stretching vibration of uronic acid, which was also found in [START_REF] Kok | Physicochemical properties of edible alginate film from Malaysian Sargassum polycystum C. Agardh[END_REF]. The band at [831-815 cm -1 ] in all fractions is due to the complex pattern of substitution at the C-S-O. This similar peak has been reported in the isolated fucoidan of S. ilicifolium and Sargassum turbinarioides [START_REF] Artemisia | The Properties of Brown Marine Algae Sargassum turbinarioides and Sargassum ilicifolium Collected from Yogyakarta, Indonesia[END_REF] and Sargassum henslowianum in the study of [START_REF] Ale | Fucose-Containing Sulfated Polysaccharides from Brown Seaweeds Inhibit Proliferation of Melanoma Cells and Induce Apoptosis by Activation of Caspase-3 in Vitro[END_REF].

The purified extracts from S. polycystum (Sp) and S. ilicifolium (Si) in the present study contained ester sulfates (at 831-815 cm -1 , 535-526 cm -1 ) and demonstrated antioxidant and antiviral properties against HSV-1. C-S-O Sulfates group at the equatorial C-2 or C-3 positions; complex pattern of substitution of C-S-O

Minerals analysis

The mineral contents expressed as content of each mineral relative to the seaweed dry weight (% of dw) of S. polycystum and S. ilicifolium are given in Table XXXII. The percentages of the recommended daily intake covered by intake of 8 g of dry seaweed are also presented, as is the daily recommended intake allowance (Mac Artain, 2007). The S. polycystum had a high total macroelement content (29.42% dw), potassium as the highest content (17.13%), followed by sodium (10.0%). Its microelement total content (1.41% dw), had iron (1.17%) as the highest. While, S. ilicifolium macroelement total content (5.43% dw) are rich in potassium (2.10%), followed by calcium (1.54%). Its microelement total content

(2.06% dw), had high iron (1.63%). Eating 8 g of dry S. polycystum represents between 6.3% and 1553.33% of the recommended daily intake (RDI) for minerals.

These percentages were high, especially for the microelements. For S. ilicifolium, 8 g of dry seaweed represents 2175.56% of the RDI of iron. The Na/K ratios in S.

polycystum and S. ilicifolium were 0.58 and 0.56, respectively. a RDI = Recommended daily intake and upper limit intake values for an adult male with a bodyweight of 70 kg according to the World Health Organization (2002); b Upper limit intake according to National Health and Medical Research Council (2006); % RDI is based on a daily intake of 8 g dry weight of seaweed; -not detected; RDA: recommended daily allowance. Data are mean ± SD (n=2). SD = standard deviation.

Mineral content is one of the main nutritional characteristics of seaweeds taken up directly from their environment. Seaweeds absorb an incomparable wealth of mineral elements from the sea and are known as excellent sources of vitamins and minerals, especially potassium and iodine (Leandro et al. 2020). However, nutritional generalization about algal mineral content is difficult because of large seasonality, geographic location, taxonomic variations, and laboratory manipulations (Wells et al. 2017). The high ash content can reach up to 42% of dw in S. polycystum and represent a high content in minerals [START_REF] Matanjun | Nutrient content of tropical edible seaweeds, Eucheuma cottonii, Caulerpa lentillifera and Sargassum polycystum[END_REF].

The present study showed that S. polycystum ash content (24.9%) was in accordance with (25%) from three Sargassum spp. (S. polycystum, S. wightii, and S. crassifolium) [START_REF] Thadhani | Comparative analysis of sugar and mineral content of Sargassum spp. collected from different coasts of Sri Lanka[END_REF], and S. ilicifolium ash content (17%) was in accordance with (15.9%) from S. ilicifolium [START_REF] Pise | Biochemical Composition of Seaweeds along Central West Coast of India[END_REF]. Other

Sargassum species reported varying ash contents such as 25% from S. wightii, 14.2% from S. vulgare, 44.29% from S. filipendula (Alam Bhuiyan and Qureshi, 2016), and 30.7% from Saccorhiza polyschides [START_REF] Garcia | Nutritional Potential of Four Seaweed Species Collected in the Barbate Estuary (Gulf of Cadiz, Spain)[END_REF].

Macroelements of S. polycystum in the present study showed that Na, K, and Mg had higher values than previous results of 1.36, 8.37, and 0.48%, respectively, while Ca was 4-times lower than 3.79% from previous results of the same species [START_REF] Matanjun | Nutrient content of tropical edible seaweeds, Eucheuma cottonii, Caulerpa lentillifera and Sargassum polycystum[END_REF]. In contrast, macroelements of S. ilicifolium in the present study showed that K, Mg, and Ca had lower values than 4.64, 4.32, and 3.34% respectively, while Na was not determined in previous results of the same species [START_REF] Pise | Biochemical Composition of Seaweeds along Central West Coast of India[END_REF]. Also, the present study macroelements of S. polycystum had higher values than S. ilicifolium except for Ca. Macroelements contents from S. wightii was considered small [START_REF] Syad | Seaweeds as nutritional supplements: Analysis of nutritional profile, physicochemical properties and proximate composition of G. acerosa and S. wightii[END_REF]) compared to S. polycystum and S. ilicifolium in the present study. However, S. polyschides reported comparable contents of Na (2.65%), K (10.17%), Ca (1.12%), and Mg (0.56%) [START_REF] Garcia | Nutritional Potential of Four Seaweed Species Collected in the Barbate Estuary (Gulf of Cadiz, Spain)[END_REF] with the present study S. polycystum except for the obvious difference in Na content (2.65%); and it is also somewhat similar with the present study S. ilicifolium except for K content (10.17%). The ratio of Na/K in S. polycystum and S. ilicifolium in the present study were similar and were considered small (<1.5) based on the previous report. It is also found higher than 0.16 of the same species S. polycystum [START_REF] Matanjun | Nutrient content of tropical edible seaweeds, Eucheuma cottonii, Caulerpa lentillifera and Sargassum polycystum[END_REF], or higher than 0.34 from different Sargassum spp. [START_REF] Thadhani | Comparative analysis of sugar and mineral content of Sargassum spp. collected from different coasts of Sri Lanka[END_REF], and 0.26 from S. polyschides [START_REF] Garcia | Nutritional Potential of Four Seaweed Species Collected in the Barbate Estuary (Gulf of Cadiz, Spain)[END_REF]). However, a higher Na/K ratio (1.45) was reported from S. wightii [START_REF] Syad | Seaweeds as nutritional supplements: Analysis of nutritional profile, physicochemical properties and proximate composition of G. acerosa and S. wightii[END_REF]) than both species in the present study. Dietary salt intake was seen to be related to the increase in blood pressure with age, and Na/K ratio was a risk factor for mortality from CVDs [START_REF] Seko | Estimation of salt intake and sodium-topotassium ratios assessed by urinary excretion among Japanese elementary school children[END_REF]). The Na/K ratio in the S. polycystum and S. ilicifolium in the present study were considered normal or low (<4.0) based on the urinary Na/K ratio and different food items consumed [START_REF] Seko | Estimation of salt intake and sodium-topotassium ratios assessed by urinary excretion among Japanese elementary school children[END_REF]. Thus, consumption of S. polycystum and S. ilicifolium seaweeds or its nutritional derivatives can help balance high Na/K ratio diets common today.

The microelements (Fe, Zn, Mn, and Cu) were all in higher amounts from S. polycystum in the present study compared with previous results of the same species [START_REF] Matanjun | Nutrient content of tropical edible seaweeds, Eucheuma cottonii, Caulerpa lentillifera and Sargassum polycystum[END_REF]). While the microelements in S. ilicifolium of the present study also showed higher values except for Mn (0.002%), which was lower than (0.009%) of the same species [START_REF] Pise | Biochemical Composition of Seaweeds along Central West Coast of India[END_REF]. Microelements of both species in the present study were also found higher than the different Sargassum spp., except for Mn (0.002%), which had a similar amount [START_REF] Thadhani | Comparative analysis of sugar and mineral content of Sargassum spp. collected from different coasts of Sri Lanka[END_REF], and also higher than S. polyschides [START_REF] Garcia | Nutritional Potential of Four Seaweed Species Collected in the Barbate Estuary (Gulf of Cadiz, Spain)[END_REF]. Iron is essential for cell functions (oxygen and electron transport, and DNA synthesis), while, manganese helps in protein, lipid, and carbohydrate metabolism [START_REF] Mišurcová | Chapter 7: Chemical Composition of Seaweeds[END_REF]. According to [START_REF] Garcia | Nutritional Potential of Four Seaweed Species Collected in the Barbate Estuary (Gulf of Cadiz, Spain)[END_REF], microelement concentrations vary with species, which could be due to morphological and physiological affinities for different metals. As the average daily consumption of dry seaweeds in Asia is 8 g, and based on the results in the present study, mineral contents are within safe levels and are potential functional foods for humans. For example, an 8 g portion of S. polycystum or S.

ilicifolium represents 9% and 15%, respectively of the RDI of calcium as reported in other seaweeds, while the same quantity of cheddar cheese provides only 5% of the RDI (Mc Artain, 2007;[START_REF] Finglas | McCance and Widdowson's The Composition of Foods[END_REF]. Results of daily recommended intake allowance showed that the use of not more than 3.86 g of S. polycystum or 2.76 g of S. ilicifolium would not compromise health due to the iron content of this seaweed. The accumulation of excess iron may generate oxidative stress and can promote and increase cardiovascular risk [START_REF] Pietrangelo | Physiology of iron transport and the hemochromatosis gene[END_REF][START_REF] Martı Ńez-Navarrete | Iron deficiency and iron fortified foods-a review[END_REF][START_REF] Puntarulo | Iron, oxidative stress and human health[END_REF]. However, the biological availability of minerals is influenced by diet composition by synergistic and antagonistic interactions [START_REF] Watanabe | Trace minerals in fish nutrition[END_REF]. Moreover, only between 1 and 53% of iron was absorbed by the human body from food [START_REF] Skikne | The Effect of Food Consistency on Iron Absorption[END_REF].

Pigments

Total pigment content of S. polycystum (197.43±28.32 mg g -1 dw) is seventeen times higher than S. ilicifolium (11.49±2.28 mg g -1 dw) shown in Table XXXIII. Chlorophyll a, fucoxanthin, and -carotene were detected in both seaweeds. Chlorophyll a, and β-carotene were much higher in S. polycystum (6.25±1.31, 188.89±26.70 mg g -1 dw) than S. ilicifolium (1.04±0.18, 7.84±1.67 mg g -1 dw) respectively. Total chlorophyll in S. polycystum (8.54±1.62 mg g -1 dw) is more than twice the amount in S. ilicifolium (3.65±0.60 mg g -1 dw). Of all the pigments, β-carotene dominates in both studied species. The amount of β-carotene in S. polycystum is thirty times higher than of the chlorophyll a, and almost eighty-three times higher than of fucoxanthin. In contrast, β-carotene in S. ilicifolium is seven times higher than the amount of the chlorophyll a, and three times higher than of fucoxanthin. [START_REF] Hidayati | Antioxidant Activities, Total Phenolic Compound and Pigment Contents of Tropical Sargassum sp. Extract, Macerated in Different Solvents Polarity[END_REF] reported higher contents of chlorophyll a (18.23 mg g -1 ) and carotenoid (32.56 mg g -1 ) from ethyl acetate extract Sargassum sp. sample compared to the present study S. ilicifolium.

However, its chlorophyll a content was about 3-times higher, and carotenoid content was about 6-times lower than the present study, S. polycystum. Also, very low contents of chlorophyll a (0.0065 mg g -1 fw), and chlorophyll b (0.0037 mg g -1 fw) was reported from fresh weight sample S. wightii [START_REF] Syad | Seaweeds as nutritional supplements: Analysis of nutritional profile, physicochemical properties and proximate composition of G. acerosa and S. wightii[END_REF], and comparable content of chlorophyll a [(3.13 mg g -1 ) after a 4-weeks photon flux treatment], and [(3.86 mg g -1 ) after a 4-weeks at 8 h, 6 °C condition] compared to S. polycystum and S. ilicifolium of the present study. Several Sargassum species reported varying results of carotenoids: the fucoxanthin contents (27.4 mg g -1 ) from Sargassum polycystum was 10 times higher than fucoxanthin (2.2, 2.6 mg g -1 ) from the present study S. polycystum and S. ilicifolium, respectively [START_REF] Balasubramaniam | Carotenoid composition and antioxidant potential of Eucheuma denticulatum, Sargassum polycystum and Caulerpa lentillifera[END_REF], fucoxanthin (7.4 mg g -1 ) from Sargassum binderi [START_REF] Wu | Analysis of the monosaccharide composition of water-soluble polysaccharides from Sargassum fusiforme by high performance liquid chromatography/electrospray ionisation mass spectrometry[END_REF]) was 3 times higher than fucoxanthin (2.6 mg g -1 ) and similar with -carotene content from the present study S. ilicifolium; and the very low contents from S. siliquosum

(1.41 mg g -1 ), S. polycystum (0.31 mg g -1 ) (Nagappan et al. 2017); the transfucoxanthin (0.107 mg g -1 ) and minor pigments (Chlorophyll a and -carotene)

from Sargassum sp. [START_REF] Indrawati | Encapsulation of Brown Seaweed Pigment by Freeze Drying: Characterization and its Stability during Storage[END_REF] compared to the present study S.

polycystum and S. ilicifolium.

The -carotene contents from Sargassum obtusifolium (0.0359 mg g -1 ), and

Sargassum echinocarpum (0.0581 mg g -1 ) are similar to -carotene content found in spinach (0.040 mg g -1 ) (McDermid, 2003). Another study reported lower carotene contents from S. polycystum (0.0004 mg g -1 ) [START_REF] Balasubramaniam | Carotenoid composition and antioxidant potential of Eucheuma denticulatum, Sargassum polycystum and Caulerpa lentillifera[END_REF], from Ascophyllum nodosum (0.003 mg 8g -1 ), Laminaria digitata (0.782 mg 8g -1 ), and (0.988 mg 8g -1 ) Undaria pinnatifida [START_REF] Macartain | Nutritional Value of Edible Seaweeds[END_REF]). The carotene contents in the present study S. polycystum and S. ilicifolium were at least 100 times higher than found in spinach and to the brown seaweeds reported in the literature (McDermid, 2003 ;[START_REF] Macartain | Nutritional Value of Edible Seaweeds[END_REF]). The pigment β-carotene is said to modulate UVA-induced gene expression, protect the skin and eyes from photo-oxidation against UV light, and prevent eye disease in humans, e.g., cataracts (Pereira, 2018). β-carotene, which, besides its anticancer activity, has been reported to be absorbed 10-times more easily by the body than the synthetic one [START_REF] Christaki | Functional properties of carotenoids originating from algae: Functional properties of algal carotenoids[END_REF]. Pigments from macroalgae are also important in this industrial range (Leandro et al. 2020).

Biological activity

Cytotoxicity and antiviral activity evaluation

No cytotoxic effect of polysaccharides (CC50 greater than 200 μg mL -1 ) on the Vero cells was observed for both S. polycystum and S. ilicifolium extracts in ranged of concentrations assayed. All fractions had low EC50 values in S.

polycystum and S. ilicifolium (Table XXXIV). Efficacy or the cellular protection of S. polycystum fractions showed that fraction Sp Na2CO3 had maximum efficacy of (94.0%), is slightly higher than Sp CaCl2 (91.78%), followed by Sp HCl (90.55%), and the least was Sp KOH (73.21%), all at 200 µg mL -1 after 72 h infection. The potency or the effectivity of the Sp HCl at 50% concentration, only needs 30.08 µg mL -1 of sample (EC50: 30.08 µg mL -1 ), which is slightly higher than Sp CaCl2 (EC50:

31.63 µg mL -1 ), followed by Sp Na2CO3 (EC50: 33.25 µg mL -1 ) and almost two times potent than Sp KOH (EC50: 67.02 µg mL -1 ). The maximum efficacy of Zovirax control is 87.62% at 5 µg mL -1 and potency of 0.49 µg mL -1 . Efficacy and potency evaluation were shown in appendix 11 and 12, while its statistical analysis was shown in appendix 17.3 and 17.4 for S. polycystum and S. ilicifolium fractions, respectively.

Fucoidans or fucan sulfates, and alginates (main acidic polysaccharides) are predominantly found in the cell wall of brown algae showed antiviral activity against HSV [START_REF] Mahomoodally | Marine Algae: A Potential Resource of Anti-HSV Molecules[END_REF]. A previous study reported antiviral activities of anionic polysaccharides (NaAlg, and fucoidan) and their derivatives (sulfated NaAlg, and sulfated fucoidan) from Sargassum tenerrimum had EC50 value that ranges from 0.5 to 15 µg mL -1 against HSV-1 ( [START_REF] Sinha | Polysaccharides from Sargassum tenerrimum: Structural features, chemical modification and antiviral activity[END_REF].

Fucoidans extracted from three different brown seaweeds (Sargassum polycystum, Sargassum mcclurei, and Turbinaria ornata) reported antiviral activity against HSV with EC50 value that ranges from 0.33 to 0.7 µg mL -1 [START_REF] Thuy | Anti-HIV activity of fucoidans from three brown seaweed species[END_REF]) [START_REF] Thuy | Anti-HIV activity of fucoidans from three brown seaweed species[END_REF]. All S. polycystum extracts in the present study possess antiviral activities against HSV-1 with values range (EC50=30.08-67.02 µg mL -1 ) that are higher and are less potent compared to the results (EC50=0.5-15 µg mL -1 ) of Sinha polycystum extracts in the present study had a range value of (3.2-6.4 % dw), which is comparable to its raw material value of 5.6% dw. No pattern or relationship was found between polyphenols contents and EC50 values in S. polycystum extract and is in accordance with Puspita et al. (2017). [START_REF] Thuy | Anti-HIV activity of fucoidans from three brown seaweed species[END_REF] had sulfates contents range value of (23.4-30.5 % dw), and sugar constituents with large portions of fucose (20.3-40% dw), galactose (13.7-25.6% dw), and small portions of mannose, xylose, and glucose (trace amounts-2.6% dw). Thuy et al. reported that antiviral activity was not significantly different, and the amount of 2-and 4-sulfated fucose in fucoidans were all different, suggest no relationship in antiviral activity against HIV.

In terms of sulfates content, S. polycystum extracts in the present study had a range value of (6.36-12.68 % dw), which is comparable to its raw material value of 10% dw. Sugar constituents (fucose, galactose, glucose, mannose, and xylose) in S. polycystum extracts are in very small proportions that range from 5.62 to 6.87%.

No pattern or relationship was found between sulfates contents and EC50 values in S. polycystum extracts and is in accordance with [START_REF] Thuy | Anti-HIV activity of fucoidans from three brown seaweed species[END_REF] and Puspita et al. (2017). Sugars constituents in S. polycystum extracts in the present study had very small amounts compared to [START_REF] Thuy | Anti-HIV activity of fucoidans from three brown seaweed species[END_REF]. In the present study of S.

polycystum extracts, no pattern of sulfate content found to relate it to EC50 values.

Protein's content of the fractions with antiviral activities against HSV-1 was in a sufficient amount in the present study. In S. ilicifolium, the maximum efficacy or the cellular protection of fraction Si KOH (118.91%) is higher than Si Na2CO3 (105.59%), followed by Si HCl (97.86%), and Si CaCl2 (83.0%). In terms of potency or the effectivity, fraction Si

CaCl2 achieved 50% at 6.63 µg mL -1 sample (EC50: 6.63 µg mL -1 ) is two to three times more potent than Si HCL (17.29 µg ml -1 ), Si KOH (17.44 µg mL -1 ), and Si Na2CO3 (19.70 µg mL -1 ). The maximum efficacy of Zovirax control is 102.64% at 5 µg mL -1 and potency of 0.32 µg ml -1 . All the S. ilicifolium extracts in the present study possess antiviral activities against HSV-1 with values range of (EC50=6.63-19.7 µg mL -1 ). These values are 2 to 5-times lesser and more potent than S.

polycystum extracts in the present study, but a little higher and are less potent compared to the results of [START_REF] Sinha | Polysaccharides from Sargassum tenerrimum: Structural features, chemical modification and antiviral activity[END_REF][START_REF] Thuy | Anti-HIV activity of fucoidans from three brown seaweed species[END_REF]. The total polyphenols content of S. ilicifolium extracts in the present study had a range value of (1.8-7.2 % dw), which is comparably less to its raw material value of 11.1% dw.

No pattern or relationship was found between polyphenols contents and EC50 values in S. ilicifolium extracts and is in accordance with Puspita et al. (2017). In terms of sulfates content, S. ilicifolium extracts in the present study had a range value of (6.96-15.21 % dw), which is comparable to its raw material value of 8.8% dw. Also, no pattern or relationship was found between sulfates contents and EC50 values in S. ilicifolium extracts and is in accordance to [START_REF] Thuy | Anti-HIV activity of fucoidans from three brown seaweed species[END_REF], and Puspita et al. (2017). Sugar constituents (fucose, glucose, galactose, and mannose) in S.

ilicifolium extracts are in different proportions that range from 3.93 to 33.69% dw.

Sugars constituents in S. ilicifolium extracts of the present study had minimal amounts compared to [START_REF] Thuy | Anti-HIV activity of fucoidans from three brown seaweed species[END_REF]. In the present study of S. ilicifolium extracts, no pattern of sulfate content found to relate it to EC50 values. Protein's content of the S. ilicifolium fractions with antiviral activities against HSV-1 was in sufficient amount, as was for S. polycystum in the present study.

As shown in the previous part of this chapter (4.2.3. Biochemical composition of extracts), the extracts obtained have biochemical contents that differ with their raw material. Aside from sulfates, monosaccharides composition [START_REF] Mahomoodally | Marine Algae: A Potential Resource of Anti-HSV Molecules[END_REF], protein, and other polysaccharides [START_REF] Hardouin | Enzyme-assisted extraction (EAE) for the production of antiviral and antioxidant extracts from the green seaweed Ulva armoricana (Ulvales, Ulvophyceae)[END_REF]Puspita et al. 2017) could have an impact on the antiviral activity reported in the extracts of S. polycystum and S. ilicifolium. The variation of antiherpetic activity results of fucoidans, alginates, and their derivatives between the present and previous studies could be influenced by several factors such as monosaccharide composition, sulfate content, polysaccharides chemical structure and its molecular weight, location and period of collection, and sensitiveness of methods used [START_REF] Kidgell | Ulvan: A systematic review of extraction, composition and function[END_REF]. These activities may be due to other compounds such as proteins and polysaccharides, as stated by Puspita et al. (2017). The Sargassum polycystum and Sargassum ilicifolium extracts results showed great potential in developing as an antiviral drug. However, these various factors mentioned must be understood better and therefore extracts from Sargassum polycystum, and Sargassum ilicifolium must be studied further.

Antioxidant activity, DPPH radical scavenging activity

The free radical scavenging of the blank and the fractions of S. polycystum and S. ilicifolium polysaccharide extracts were estimated by the decrease in absorbance due to the reduction of the 2,2-diphenyl-1-picrylhydrazyl radical by the extracts. Percent maximum scavenging ability and inhibition activity of S.

polycystum and S. ilicifolium polysaccharide extracts at (5 mg mL -1 ) were shown in Antioxidant activity varied significantly among 30 species of Hawaiian algae from 27 genera, and the brown algae as a group had the highest mean antioxidant activity, followed by green algae, and red algae with FRAP mean values of 3.55±3.16, 2.29±2.34, and 1.59±1.17 µM µg -1 extract, respectively [START_REF] Kelman | Antioxidant Activity of Hawaiian Marine Algae[END_REF]. This present study antioxidant activity for brown algae S. polycystum and S. ilicifolium is somewhat in accordance to what is reported by [START_REF] Kelman | Antioxidant Activity of Hawaiian Marine Algae[END_REF] where its IC50 mean value is low (476.71 µg mL -1 at 5 mg mL -1 ). Note that the dose concentration used for red algae is twice as high as brown and green algae; that is why the IC50 mean value appears small. Thus, antioxidant activity in the present study is most effective in brown algae, followed by green algae and red algae. This result is in accordance to [START_REF] Kelman | Antioxidant Activity of Hawaiian Marine Algae[END_REF], where antioxidant activities of algae collected from shallow waters (0-3m) were more active than from those in the deep waters, where brown algae were also collected in shallow waters (0-3 m) than green algae (0-5 m), and red algae (5-10 m) in the present study.

In the present study, fraction Sp HCl (IC50=105 µg mL -1 ) had the best antiradical activity, which is comparable to ethyl acetate extract (IC50=102.4 µg mL -1 ) and is higher to Aquadest extract (at IC50=72.95 µg mL -1 ), which had the best antiradical activity, according to [START_REF] Hidayati | Antioxidant Activities, Total Phenolic Compound and Pigment Contents of Tropical Sargassum sp. Extract, Macerated in Different Solvents Polarity[END_REF]. Other fractions with its IC50 values from Sp KOH>Sp Na2CO3>Sp CaCl2 (IC50=242.35-384.27 µg mL -1 ) and fractions Si KOH (IC50=364.74 µg mL -1 ) were comparable to methanol extract (IC50=270 µg mL -1 ), and n-hexane extract (IC50=320 µg mL -1 ) that was considered weak antioxidants by [START_REF] Hidayati | Antioxidant Activities, Total Phenolic Compound and Pigment Contents of Tropical Sargassum sp. Extract, Macerated in Different Solvents Polarity[END_REF]. The remaining fractions from the present study, Si Na2CO3 (IC50=708.92 µg mL -1 ), and Si HCl (IC50=1134.83 µg mL - 1 ) were considered moderate antioxidants (IC50=700-1000 µg mL -1 ) according to Puspita et al. (2017).

In terms of percent scavenged against DPPH, fraction Sp CaCl2>Sp KOH (>50% at 5 mg mL -1 ) were in accordance with (55.94% at 1000 µg mL -1 ) of purified fucoidan extracts by DEAE-cellulose from Sargassum polycystum [START_REF] Palanisamy | Investigation of antioxidant and anticancer potential of fucoidan from Sargassum polycystum[END_REF], and all samples in the Sp and Si fractions of the present study were also dose-dependent as in the study of [START_REF] Palanisamy | Investigation of antioxidant and anticancer potential of fucoidan from Sargassum polycystum[END_REF]. All the rest of the Sp and Si fractions in the present study had a scavenging percentage of less than <50% at 5 mg mL -1 concentration, which is lower and 5-times weaker in dose concentrations used, therefore, less effective compared to [START_REF] Palanisamy | Investigation of antioxidant and anticancer potential of fucoidan from Sargassum polycystum[END_REF].

All samples from the present study Sp and Si fractions have IC50 values of at least 4-times lower than IC50=4.3-5.13 mg mL -1 from S. glaucescens [START_REF] Sanjeewa | Bioactive potentials of sulfated polysaccharides isolated from brown seaweed Sargassum spp. in related to human health applications: A review[END_REF] and are therefore more effective antioxidant. While S. horneri had better DPPH scavenging activity (42.6-86.74% at 2.5 mg mL -1 ) at lower dose concentration than those in the present study, and therefore, were better antioxidant; and the S. plagiophyllum extract DPPH scavenged (36% at 5 mg mL -1 ) [START_REF] Sanjeewa | Bioactive potentials of sulfated polysaccharides isolated from brown seaweed Sargassum spp. in related to human health applications: A review[END_REF] the present study were within the range value (13.2-56% dw) for selected red seaweeds from the Philippines [START_REF] Hurtado | Chapter Two -Selected red seaweeds from the Philippines with emerging high-value applications[END_REF]). However, it is found to be lower than reported in the literature [START_REF] Kho | for H. durvillei (1.29%), and H. dilatata (0.29%) of the same species, while H. maculata (1.09%) was reported higher than H. dilatata. Wanyonyi et al. (2017) reported lipid content (0.62% dw) from K. alvarezii, which is similar to raw H. dilatata, but 10-times lower than raw H. durvillei in the present study[END_REF] for H. durvillei (53.65%), H. dilatata (40.53%) of the same species, and H. maculata (51.18%). Slightly higher carbohydrates contents reported (38.3%) from K. alvarezii [START_REF] Wanyonyi | Kappaphycus alvarezii as a Food Supplement Prevents Diet-Induced Metabolic Syndrome in Rats[END_REF], and (34.5%) from Solieria filiformis [START_REF] Bedoux | Antiviral and Cytotoxic Activities of Polysaccharides Extracted from Four Tropical Seaweed Species[END_REF], while reported lower (15.5%) from Halymenia floresii [START_REF] Garcia | Nutritional Potential of Four Seaweed Species Collected in the Barbate Estuary (Gulf of Cadiz, Spain)[END_REF], than the raw H. durvillei and raw H. dilatata in the present study respectively.

These variations of results from carbohydrates, ash proteins, and lipids from both present and previous studies in red seaweeds could be affected by geography, season, or growth conditions [START_REF] Kelman | Antioxidant Activity of Hawaiian Marine Algae[END_REF]. However, the biochemical composition of raw H. durvillei and raw H. dilatata in the present study were within the values specified for red seaweeds. Thus, there is a potential for these species to be used as a raw material in food industry development or human health.

Amino acids composition of raw material

The protein contents in red macroalgae, in general, are much higher compared to brown and green groups, accounting to 10-50% dw, and also higher than known high-protein vegetables such as soybean (40.3% dw) [START_REF] Hurtado | Chapter Two -Selected red seaweeds from the Philippines with emerging high-value applications[END_REF]. The amino acid composition was investigated to understand the nutritional value of the raw seaweeds Halymenia durvillei and Halymenia dilatata from the Philippines. The sulfur amino acids (methionine, cysteine), when synthesized, are closely related to taurine and its derivatives, which have active biological activities against heart attack, and ameliorating diabetic complications (Okolie et al. 2018;[START_REF] Hao | Chemical composition and immunostimulatory properties of green alga Caulerpa racemosa var Peltata[END_REF]. Gas chromatography-flame ionization detection (GC-FID) was used to identify and quantify 13, and 14 amino acids (AA) for H. durvillei and H.

dilatata, respectively and is presented in Table XXXVII. The amino acid composition of ovalbumin, a reference protein from the egg white, is also shown in provided information on the protein composition of the seaweeds and were expressed as a percentage of the total AA detected. The content of AA in the samples is divided into three categories: high (>10%), medium (5-10%), and low (<5%) [START_REF] Lourenço | Amino acid composition, protein content and calculation of nitrogen-to-protein conversion factors for 19 tropical seaweeds[END_REF].

In the present study, five dominant AA that constitutes more than or around 10% of the extracted proteins were found for the raw H. durvillei and H. dilatata. to (4-6%) from ovalbumin [START_REF] Kazir | Extraction of proteins from two marine macroalgae, Ulva sp. and Gracilaria sp., for food application, and evaluating digestibility, amino acid composition and antioxidant properties of the protein concentrates[END_REF] or (25-30%) from soymeal [START_REF] Makkar | Seaweeds for livestock diets: A review[END_REF]. The EAA/NEAA ratio has been calculated for both algae. This value, which indicates the quality of proteins, must tend towards or be greater than 1 to contribute to an optimal EAA intake. For the present study, the amino acid contents for H. durvillei and H. dilatata ranges from 0.18 to 20.56% (Table XXXVII).

Observed EAA/NEAA ratios were 0.62 in H. durvillei, and 0.87 in H. dilatata. The adequate intake of the EAAs/NEAAs ratio is crucial to preserve cell integrity and whole-body metabolism [START_REF] Romano | Influence of Diets with Varying Essential/Nonessential Amino Acid Ratios on Mouse Lifespan[END_REF]. According to [START_REF] Romano | Influence of Diets with Varying Essential/Nonessential Amino Acid Ratios on Mouse Lifespan[END_REF] that the percentage survival of mice fed with different diets was negatively affected when EAA was absent (0%/NEAAs) or deficient (30%/NEAAs). The present study H. durvillei and H. dilatata had EAAs values of 38.08 and 46.51%, respectively, that are within the reported range of EAAs content among red algae (25-50% of the total AA) and are said similar to other protein sources such as casein, ovalbumin, and leguminous plants [START_REF] Torres | Integral Utilization of Red Seaweed for Bioactive Production[END_REF]. The EAAs/NEAAs ratio in the present study for H. durvillei (0.62) is within the reported range ratio (0.49-0.68) from 9 different seaweeds, while H. dilatata (0.87) is even more significant and better than the reported range ratio [START_REF] Gaillard | Amino acid profiles of nine seaweed species and their in-situ degradability in dairy cows[END_REF].

Moreover, EAAs/NEAAs ratio is in accordance with the reference value of 0.6 recommended by FAO/WHO [START_REF] Hao | Chemical composition and immunostimulatory properties of green alga Caulerpa racemosa var Peltata[END_REF]. It is also considered favorable or nutritive compared to reference data ovalbumin and in its EAAs/NEAAs values. In the present study, four dominant AA (lysine, leucine, aspartic acid, and glutamic acid) that were found for H. durvillei and five dominant AA (lysine, leucine, methionine, aspartic acid, and glutamic acid) were found for H. dilatata were also identified as dominant AA from different red seaweeds in the literature [START_REF] Hamed | Marine Bioactive Compounds and Their Health Benefits: A Review: Importance of functional seafood…[END_REF][START_REF] Garcia | Nutritional Potential of Four Seaweed Species Collected in the Barbate Estuary (Gulf of Cadiz, Spain)[END_REF]Okolie et al. 2018). Aspartic and glutamic acids (up to 15% of the total AA) from Pyropia tenera [START_REF] Hamed | Marine Bioactive Compounds and Their Health Benefits: A Review: Importance of functional seafood…[END_REF]Okolie et al. 2018) was reported 2-times lower than the present study aspartic and glutamic acids for H. durvillei (33.79%) and H. dilatata (31.97%). However, no aspartic acid in H. floresii [START_REF] Garcia | Nutritional Potential of Four Seaweed Species Collected in the Barbate Estuary (Gulf of Cadiz, Spain)[END_REF]) and its glutamic content (6.80%) is contrary to both species in the present study. Respective leucine and lysine contents (8.7, 6.0%) from Pyropia tenera [START_REF] Hamed | Marine Bioactive Compounds and Their Health Benefits: A Review: Importance of functional seafood…[END_REF], and (5.0, 8.47%) from Halymenia floresii [START_REF] Garcia | Nutritional Potential of Four Seaweed Species Collected in the Barbate Estuary (Gulf of Cadiz, Spain)[END_REF] was slightly lower than the present study leucine and lysine contents (9.81, 11.03%) from H. durvillei, and (9.08, 9.16%) H. dilatata.

Methionine content, the highest EAAs, and only found in the present study raw H. dilatata (15.55%) is in accordance reported in Palmaria palmata [START_REF] Hamed | Marine Bioactive Compounds and Their Health Benefits: A Review: Importance of functional seafood…[END_REF], but in contrast as reported (4.0%) in H. floresii [START_REF] Garcia | Nutritional Potential of Four Seaweed Species Collected in the Barbate Estuary (Gulf of Cadiz, Spain)[END_REF]. In terms of NEAAs, aspartic and glutamic acids are the major components of both H. durvillei and H. dilatata. Aspartic and glutamic acids are responsible for distinctive flavor and taste for seaweeds [START_REF] De Gaillande | Caulerpa consumption, nutritional value and farming in the Indo-Pacific region[END_REF][START_REF] Hao | Chemical composition and immunostimulatory properties of green alga Caulerpa racemosa var Peltata[END_REF], suggesting that both H durvillei and H. dilatata are potential source flavor enhancers of food products. Also, glutamic acid plays a vital role in maintaining brain function and mental activity, while aspartic acid helps to initiate the Krebs cycle and urea cycle (Okolie et al. 2018). However, recent study dares to recognize the fact that glutamate/MSG passed to fetuses and neonates causes brain damage and disrupts the endocrine system will provide the researchers with new knowledge to apply to their studies of glutamate-related abnormalitiesincluding obesity and reproductive disorders [START_REF] Samuels | Dose dependent toxicity of glutamic acid: A review[END_REF]. The present study had poor content (<5%) in the EAAs threonine (<0.5%), isoleucine (<4.0%), and phenylalanine (<4.0%) are in accordance with the range results (0.08-4.11%) from Pyropia tenera and H. floresi [START_REF] Hamed | Marine Bioactive Compounds and Their Health Benefits: A Review: Importance of functional seafood…[END_REF][START_REF] Garcia | Nutritional Potential of Four Seaweed Species Collected in the Barbate Estuary (Gulf of Cadiz, Spain)[END_REF]Okolie et al. 2018). However, methionine content was reported poor from P. tenera, which is in high amount (15.55%) from the present study H. dilatata.

The presence of methionine in H. dilatata, therefore, indicates that it could be a promising natural source of a taurine-like compound and its derivatives with human health benefits. Variation of results between past and present study on respective amino acid content could be due to the type of species used, season of collection, and seaweed life cycle, which produce different types of proteins during the year, or it could be the extraction method used [START_REF] Anh | Potential uses of gut weed Enteromorpha spp. as a feed for herbivorous fish[END_REF][START_REF] Hardouin | Enzyme-assisted extraction (EAE) for the production of antiviral and antioxidant extracts from the green seaweed Ulva armoricana (Ulvales, Ulvophyceae)[END_REF][START_REF] Garcia | Nutritional Potential of Four Seaweed Species Collected in the Barbate Estuary (Gulf of Cadiz, Spain)[END_REF][START_REF] De Gaillande | Caulerpa consumption, nutritional value and farming in the Indo-Pacific region[END_REF].

Lipid composition

The Fatty Acid (FA) composition of H. durvillei and H. dilatata in the present study are shown in Table XXXVIII. Saturated fatty acids (SFAs) in H. durvillei are the primary fatty acids with a value of 51.15% of the total fatty acids (TFAs), while its unsaturated fatty acids such as; polyunsaturated fatty acids (PUFAs), and monounsaturated fatty acids (MUFAs) have (9.81, and 15.75%), respectively. In contrast, unsaturated fatty acids in H. dilatata constitute about 35%; PUFAs (12.0%), and MUFAs (23.28%), while its SFAs is (32.19%). Unidentified FAs were (23.29%) in H. durvillei and (32.53%) in H. dilatata. In H. durvillei, palmitic acid (C16:0) was the most abundant FA with a 30.34% TFA value. There was less diversity in unsaturated FA with six different MUFAs with content values that ranged (from <0.3 to 11.68%), and four different PUFAs with content values that ranged (from <0.6 to 4.9%). Unsaturated fatty acids from H. dilatata represented a variety of structures with 14 different FA identified by GC ranging from 0.16 to 14.51% of total FA. The main FA detected was oleic acid (C18:1n-9, 14.51% TFA). H. durvillei had smaller value (0.2) compared to H. dilatata (1.43). The higher the value, the higher the protection properties [START_REF] Turan | Fatty acid profile and proximate composition of the thornback ray (Raja clavata, L. 1758) from the Sinop coast in the Black Sea[END_REF]. [START_REF] Hurtado | Chapter Two -Selected red seaweeds from the Philippines with emerging high-value applications[END_REF]. The most important of PUFA are the essential fatty acids eicosapentaenoic acid (EPA; C20:5n-3) and docosahexaenoic acid (DHA; C22:6n-3) along with their precursors α-linolenic (ALA; C18:3n-3) and linoleic acids (LA; C18:2n-6). γ and α-Linolenic (C18:3n-6 and n-3) acids are the most abundant PUFA in H. durvillei while ALA (C18:3n-3) and DHA (C22:6n-3) [START_REF] Manam | Phytochemical, amino acid, fatty acid and vitamin investigation of marine seaweeds Colpomenia sinuosa and Halymenia porphyroides collected along Southeast coast of Tamilnadu, India[END_REF]. LA (28.6%) was the major PUFA in H. floresii, which was too high compared to both species in the present study, while EPA and DHA were not detected [START_REF] Garcia | Nutritional Potential of Four Seaweed Species Collected in the Barbate Estuary (Gulf of Cadiz, Spain)[END_REF] as well as in H. durvillei in the present study. ALA (2.29%) and DHA (0.93%) PUFAs from A. taxiformis [START_REF] Ragonese | Characterisation of lipid fraction of marine macroalgae by means of chromatography techniques coupled to mass spectrometry[END_REF] were lower than H. dilatata (5.32% and 2.24%) in the present study, respectively. Polyunsaturated fatty acids with more than 18 carbons, e.g., EPA, DHA, and arachidonic acid (AA; C20:4n-6), which cannot be synthesized naturally are present in red seaweeds such as Pyropia dioica, Gracilaria gracilis, and Palmaria palmata [START_REF] Hurtado | Chapter Two -Selected red seaweeds from the Philippines with emerging high-value applications[END_REF].

Besides, PUFAs and carotenoids are most noteworthy as functional foods [START_REF] Mccauley | Effects of nutrients and processing on the nutritionally important metabolites of Ulva sp. (Chlorophyta)[END_REF]. They have been reported to cause the production of different eicosanoids, and are known to regulate cellular processes. EPA, AA, and DHA have several health benefits such as effect on immune-regulation, cancer prevention, pre-and post-natal development of the brain and retina, and regulate inflammation and immune reaction on essential functions in skin growth and protection [START_REF] Hurtado | Chapter Two -Selected red seaweeds from the Philippines with emerging high-value applications[END_REF][START_REF] Manam | Phytochemical, amino acid, fatty acid and vitamin investigation of marine seaweeds Colpomenia sinuosa and Halymenia porphyroides collected along Southeast coast of Tamilnadu, India[END_REF] reported that the highest SFA content in H. porphyroides was Stearic acid (C18:0) at 29.12% and was way too high than the present study (H. durvillei (0.73%), and H. dilatata (4.69%)), while highest MUFA, the Oleic acid (C18:1n9c) at 31.21% was way too high than H. durvillei (1.62%), but in accordance to H. dilatata (14.51%) as the main MUFA in the present study. The main SFA in H. floresii at 5.2-5.6% for heptadecanoic (C17:0), stearic, and arachidic (C20:0) acids, while palmitic acid (C16:0) at 1.2% [START_REF] Garcia | Nutritional Potential of Four Seaweed Species Collected in the Barbate Estuary (Gulf of Cadiz, Spain)[END_REF] were way too small compared to the main SFA (C16:0) for H. durvillei (30.34%) and H. dilatata (13.89%) in the present study. On the other hand, the main MUFA of H. floresii was Myristoleic (C14:1) at 32.6% [START_REF] Garcia | Nutritional Potential of Four Seaweed Species Collected in the Barbate Estuary (Gulf of Cadiz, Spain)[END_REF] which was in accordance to present study H. durvillei as main MUFA (11.68%) but only in different proportions, while H. dilatata main MUFA, the Oleic acid (C18:1n9c) was not detected in H. floresii. The main SFA from A. taxiformis at 38.05%, the palmitic acid [START_REF] Ragonese | Characterisation of lipid fraction of marine macroalgae by means of chromatography techniques coupled to mass spectrometry[END_REF]) was in accordance with the main SFA in H. durvillei and at lower proportion in H. dilatata for the present study. The highest MUFA from A. taxiformis, palmitoleic acid (C16:1) at 5.6%, was slightly higher than 1.36%, and 2.62% in H. durvillei and H. dilatata, respectively in the present study. However, the major MUFA in the present study H. durvillei (C14:1) was not detected in A. taxiformis, and the major MUFA in present study H. dilatata (C18:1n9c) was 3-times higher than in A. taxiformis.

The nutritional quality indexes (Table XXXIX) can be assessed according to parameters calculated with the concentration of fatty acids. The FAO/WHO considered that a PUFA/SFA ratio below 0.45 is undesirable, as the SFA potentially increases the blood cholesterol level (FAO/ WHO, 2008). The present study H. durvillei and H. dilatata with 0.19 and 0.37 ratios, respectively, do not potentially conform for consumption in terms of PUFA/SFA ratio. For health, high h/H ratios are considered more beneficial, as they are directly related to high PUFA content [START_REF] Matos | Chemical Characterization of Six Microalgae with Potential Utility for Food Application[END_REF]. The results (1.43) for H. dilatata agree with h/H values for marine fishes like sardine or mackerel that are recognized for beneficial health consumption, while (0.2) for H. durvillei does not conform with the report [START_REF] Fernandes | Nutritional and lipid profiles in marine fish species from Brazil[END_REF]. AI and TI are related to the protection against coronary artery diseases. Lower AI and TI values indicate more excellent protection [START_REF] Turan | Fatty acid profile and proximate composition of the thornback ray (Raja clavata, L. 1758) from the Sinop coast in the Black Sea[END_REF]. The value of AI and TI for H. dilatata (0.54, and 0.5), respectively in the present study are in accordance to palatable C. racemosa species from the Philippines (Magdugo et al. 2020), or lower than those of meat (1.08-1.58) and milk (2.1) (Kumar, 2011), while H. durvillei (1.96, and 1.52) was somewhat similar to meat and milk. Moreover, ω6/ω3 ratio (<10) with 1.81 and 0.46, respectively, confirms the potential health benefits of the consumption of H. durvillei and H. dilatata from the Philippines regarding the lipid distribution [START_REF] Chan | Chemical composition and physicochemical properties of tropical red seaweed, Gracilaria changii[END_REF]. Although PUFA/SFA ratio for both studied species was <0.45, this values was also attributed to other SFAs with health benefits (i.e., lauric, pentadecanoic, and heptadecanoic acids) which prompts apoptosis in cancer cells [START_REF] Lappano | The lauric acid-activated signaling prompts apoptosis in cancer cells[END_REF], or promotes against cardiometabolic diseases [START_REF] Venn-Watson | Efficacy of dietary odd-chain saturated fatty acid pentadecanoic acid parallels broad associated health benefits in humans: could it be essential?[END_REF]. Also, H. durvillei and H. dilatata from the Philippines should be considered a goodhealth promoter because of the presence of C22:1n-9 (erucic) and C24:1n-9 (nervonic) acids, compounds which are said to prevent demyelinating disease [START_REF] Sargent | Nervonic acid and demyelinating disease[END_REF].

Conclusion and perspectives

In conclusion of this first part of the study for the biochemical analyses, the raw materials from H. durvillei and H. dilatata are mainly composed of ash or minerals (41.0%, 46.3%), followed by carbohydrates (30.59%, 25.2%), and proteins (5.3%, 5.5%), respectively. Both species have lipids content that are within the range value from the selected red seaweeds of the Philippines. H. durvillei and H. dilatata had ω6/ω3 ratio (<10) of 1.81 and 0.46, respectively, confirms the potential health benefits of the consumption requirements of both species regarding the lipid distribution. Although PUFA/SFA ratio for both H. durvillei and H. dilatata was <0.45 and do not conform to FAO/WHO requirement, these values were also attributed to other SFAs with health benefits (i.e., lauric, pentadecanoic, and heptadecanoic acids) which prompts apoptosis in cancer cells, or promotes against cardiometabolic diseases. Also, H. durvillei and H. dilatata from the Philippines should be considered a good-health promoter because of the presence of C22:1n-9 (erucic) and C24:1n-9 (nervonic) acids, compounds which are said to prevent demyelinating disease.

We acknowledge the intra-species variation of biochemical results.

However, the biochemical composition of raw H. durvillei and raw H. dilatata in the present study were within the values specified for red seaweeds. Thus, there is a potential for these species to be used as a raw material in food industry development or human health.

Cell wall polysaccharides purification

First treatment

Extraction yield in percent after three solvents (ethanol, chloroform/methanol, and acetone) are shown in Extraction yields between the present and previous studies from seaweeds

Halymenia vary significantly for different reasons: may be due to the amount of the crude extract that is being used, beginning with the extraction and purifications, or other factors such as species used, season and place of collection, as stated in previous papers [START_REF] Kok | Physicochemical properties of edible alginate film from Malaysian Sargassum polycystum C. Agardh[END_REF][START_REF] Artemisia | The Properties of Brown Marine Algae Sargassum turbinarioides and Sargassum ilicifolium Collected from Yogyakarta, Indonesia[END_REF].

Biochemical composition analysis of extracts

The biochemical composition of H. durvillei (Hdur extract) and H. dilatata (Hdil extract) carrageenan extracts were shown in Table XLII.

The H. durvillei carrageenan fraction (Hdur extract) is rich in neutral sugars (17.6%), followed by sulfates (15.73%), and with small amounts of proteins (7%), polyphenols (4%), and uronic acids (0.7% et al. 2019). However, the biochemical composition of Hdur extract and Hdil extract in the present study were within the values specified for red seaweeds. Thus, there is a potential for these species to be used as a raw material in food industry development or human health.

Monosaccharide composition analysis

The unit sugar composition of raw and purified fractions of H. durvillei (Hdur extract) and H. dilatata (Hdil extract) was determined by High-Performance Anion Exchange Chromatography (HPAEC). The results were given in Table XLIII and expressed as a percentage of sugars relative to the total sugars detected. were also observed in the literature [START_REF] Fidelis | Proteolysis, NaOH and ultrasoundenhanced extraction of anticoagulant and antioxidant sulfated polysaccharides from the edible seaweed, Gracilaria birdiae[END_REF].

Fourier Transform Infra-Red (FT-IR) spectroscopy

The calculated FTIR spectra of red seaweeds H. durvillei fraction (Hdur) and H. dilatata fraction (Hdil) spectral bands of interest were shown in Fig. 43. The IR spectrum of Hdur, and Hdil extracts within 2000-500 cm -1 zone or the fingerprint region for polysaccharides were used for data analysis.

The IR spectrum of H. durvillei extract (Hdur) and H. dilatata extract (Hdil)

showed spectral bands of high intensity at [1224-1221 cm -1 ], while bands of medium or low intensity at [1640-1636 cm -1 ], [1007, 987 cm -1 ], [940-935 cm -1 ],

[814-812 cm -1 ], [769-766 cm -1 ], and 576 cm -1 . Gracilaria corticata var ramalinoides, Gracilaria foliifera, Ahnfeltiopsis pygmaea, Gracilaria corticata, Jania adhaerens, and Gracilaria edulis) in the study of [START_REF] Fernando | FTIR characterization and antioxidant activity of water-soluble crude polysaccharides of Sri Lankan marine algae[END_REF]. Small spectral bands at [576-575 cm -1 ] in the present study fractions were due to asymmetric and symmetric O=S=O deformation of sulfates, was also found in [START_REF] Kim | Anticancer Activity of Sulfated Polysaccharides Isolated from the Antarctic Red Seaweed Iridaea cordata[END_REF]. High, medium and small intensity bands between 1200 and 970 cm -1 are mainly caused by CC and CO stretching in the pyranose ring and COC stretching of the glycosidic bonds. Intense absorption in this region is common to all types of polysaccharides [START_REF] Kim | Anticancer Activity of Sulfated Polysaccharides Isolated from the Antarctic Red Seaweed Iridaea cordata[END_REF]. The high ash content can reach up to 50.2% of dw in H. floresii and represent a high content in minerals [START_REF] Garcia | Nutritional Potential of Four Seaweed Species Collected in the Barbate Estuary (Gulf of Cadiz, Spain)[END_REF]. The present study showed that H. durvillei ash content (41%), and H. dilatata ash content (46.3%) was a little lower than 50.2% from H. floresii [START_REF] Garcia | Nutritional Potential of Four Seaweed Species Collected in the Barbate Estuary (Gulf of Cadiz, Spain)[END_REF], but is in accordance with 39.54% from H. durvillei, and was twice as high than 21.92% from H. dilatata of the same species from Iligan bay, Philippines [START_REF] Kho | for H. durvillei (1.29%), and H. dilatata (0.29%) of the same species, while H. maculata (1.09%) was reported higher than H. dilatata. Wanyonyi et al. (2017) reported lipid content (0.62% dw) from K. alvarezii, which is similar to raw H. dilatata, but 10-times lower than raw H. durvillei in the present study[END_REF]. Other red seaweed species such as Gracilaria spp., Gelidium sp., and Pyropia sp. reported varying ash contents from 7.72% to 29.06% (Alam Bhuiyan and Qureshi, 2016), and 58% from K.

alvarezii [START_REF] Wanyonyi | Kappaphycus alvarezii as a Food Supplement Prevents Diet-Induced Metabolic Syndrome in Rats[END_REF]).

In the present study, H. dilatata had higher macroelements (Na, K, Ca, and Mg) than H. durvillei. In the study of [START_REF] Garcia | Nutritional Potential of Four Seaweed Species Collected in the Barbate Estuary (Gulf of Cadiz, Spain)[END_REF], macroelements in H.

floresii reported almost 2-times lower Na (5.54%), slightly higher K (5.96%), 2times lower Ca (0.73%), and 2-times higher Mg (0.9%) than of H. durvillei in the present study. While, macroelements in H. floresii reported 3-times lower Na, slightly lower K, about 4-times lower Ca, and about 2-times lower Mg (Garcia et al. 2016) than (19.64, 7.76, 2.77, and 1.53%) from H. dilatata in the present study, respectively. Several red seaweeds reported higher macroelements contents: of K (13.15%) from E. cottonii [START_REF] Matanjun | Nutrient content of tropical edible seaweeds, Eucheuma cottonii, Caulerpa lentillifera and Sargassum polycystum[END_REF], of Ca (4.3%), Mg (3.3%) from G. corticata [START_REF] Pise | Biochemical Composition of Seaweeds along Central West Coast of India[END_REF], and K (20%), and Ca (2.89%) from K.

alvarezii [START_REF] Wanyonyi | Kappaphycus alvarezii as a Food Supplement Prevents Diet-Induced Metabolic Syndrome in Rats[END_REF] than the respective macroelements for both H.

durvillei and H. dilatata in the present study.

The ratio of Na/K in H. durvillei (2.06) and H. dilatata (2.53) in the present study are considered big (>1.5) based on the previous report, and was 2-times higher than 0.93 from H. floresii [START_REF] Garcia | Nutritional Potential of Four Seaweed Species Collected in the Barbate Estuary (Gulf of Cadiz, Spain)[END_REF], way higher than 0.14 from E. cottonii [START_REF] Matanjun | Nutrient content of tropical edible seaweeds, Eucheuma cottonii, Caulerpa lentillifera and Sargassum polycystum[END_REF], 0.25 from G. acerosa [START_REF] Syad | Seaweeds as nutritional supplements: Analysis of nutritional profile, physicochemical properties and proximate composition of G. acerosa and S. wightii[END_REF], higher than 0.4 from P. umbilicalis, and 0.22 from P. palmata [START_REF] Macartain | Nutritional Value of Edible Seaweeds[END_REF]), and 0.19 from popular edible red seaweed K. alvarezii [START_REF] Wanyonyi | Kappaphycus alvarezii as a Food Supplement Prevents Diet-Induced Metabolic Syndrome in Rats[END_REF]. However, a similar Na/K ratio with 1.90 was reported from C. crispus [START_REF] Macartain | Nutritional Value of Edible Seaweeds[END_REF].

Dietary salt intake was seen to be related to the increase in blood pressure with age, and Na/K ratio was a risk mortality factor for CVDs or cardiovascular diseases.

However, H. durvillei and H. dilatata Na/K ratio in the present study were still considered normal or low (<4.0) based on the urinary Na/K ratio and different food items consumed [START_REF] Seko | Estimation of salt intake and sodium-topotassium ratios assessed by urinary excretion among Japanese elementary school children[END_REF]. Thus, consumption of H. durvillei and H. dilatata seaweeds or its nutritional derivatives can help balance high Na/K ratio diets common today. The microelements (Fe, Zn, Mn, and Cu) were all in higher amounts in H. durvillei and H. dilatata in the present study, than the trace amounts reported from H. floresii [START_REF] Garcia | Nutritional Potential of Four Seaweed Species Collected in the Barbate Estuary (Gulf of Cadiz, Spain)[END_REF], from E. cottonii [START_REF] Matanjun | Nutrient content of tropical edible seaweeds, Eucheuma cottonii, Caulerpa lentillifera and Sargassum polycystum[END_REF]), from G. corticata [except that Mn (0.0036%) was found similar] (Pise and Sabale, 2010), from G. acerosa [START_REF] Syad | Seaweeds as nutritional supplements: Analysis of nutritional profile, physicochemical properties and proximate composition of G. acerosa and S. wightii[END_REF], and K. alvarezii [START_REF] Wanyonyi | Kappaphycus alvarezii as a Food Supplement Prevents Diet-Induced Metabolic Syndrome in Rats[END_REF].

Iron is essential for cell functions (oxygen and electron transport, and DNA synthesis), manganese helps in protein, lipid, and carbohydrate metabolism [START_REF] Mišurcová | Chapter 7: Chemical Composition of Seaweeds[END_REF]. According to [START_REF] Garcia | Nutritional Potential of Four Seaweed Species Collected in the Barbate Estuary (Gulf of Cadiz, Spain)[END_REF], microelement concentrations vary with species, which could be due to morphological and physiological affinities for different metals. As the average daily consumption of dry seaweeds in Asia is 8 g, based on the results in the present study, mineral contents are within safe levels and are potential functional foods for humans. For example, an 8 g portion of H.

durvillei and H. dilatata represented around 9% and 26%, respectively of the RDI of calcium as in other seaweeds, while the same quantity of cheddar cheese provides only 5% of the RDI [START_REF] Finglas | McCance and Widdowson's The Composition of Foods[END_REF]Mc Artain, 2007). Results of daily recommended intake allowance showed that eating of not more than 16.67 g of H.

durvillei or 3.64 g of H. dilatata would not compromise health, because of the iron content of this seaweed. Accumulation of excess iron may generate oxidative stress, can promote and increase cardiovascular risk [START_REF] Pietrangelo | Physiology of iron transport and the hemochromatosis gene[END_REF][START_REF] Martı Ńez-Navarrete | Iron deficiency and iron fortified foods-a review[END_REF][START_REF] Puntarulo | Iron, oxidative stress and human health[END_REF]. However, the biological availability of minerals is influenced by the diet composition by synergistic and antagonistic interactions [START_REF] Watanabe | Trace minerals in fish nutrition[END_REF]. Moreover, only between 1 and 53% of iron was absorbed by the human body from food [START_REF] Skikne | The Effect of Food Consistency on Iron Absorption[END_REF].

Pigments

Total pigment contents of H. durvillei (60.41±5.0 mg g -1 dw) is eight times higher than H. dilatata (7.21±0.02 mg g -1 dw) shown in Table XLVI. Chlorophyll a, and -carotene were detected in H. durvillei, while chlorophyll a, and chlorophyll b were detected in H. dilatata. The amount of chlorophyll a in H. durvillei (5.93±5.0 mg g -1 dw), is more than eight times higher than H. dilatata (0.68±0.023 mg g -1 dw). Total chlorophyll in H. durvillei (5.94±5.0 mg g -1 dw) is slightly lower than in H. dilatata (7.21±0.02 mg g -1 dw). The amount of β-carotene (54.47±0.0 mg g -1 dw) or the primary pigment in H. durvillei is nine times higher than of the chlorophyll a (5.93±5.0 mg g -1 dw), while chlorophyll d was not detected. The amount of chlorophyll d (6.53±0.0 mg g -1 dw) or the primary pigment in H. dilatata is nine times higher than of the chlorophyll a (0.68±0.02 mg g -1 dw), while carotene in H. dilatata was not detected.

Table XLVI.

Pigment content (mg g -1 dw) of Philippine red seaweeds Halymenia durvillei and Halymenia dilatata.

Data are mean ± SD (n=2). SD = standard deviation. -= not detected.

H. durvillei chlorophyll a content was higher than H. dilatata (0.68 mg g -1 )

in the present study. Several red seaweed species reported low chlorophyll content;

(0.25-0.35 mg g -1 fw) from fresh weight sample Pyropia haitanensis after using different light qualities (H. [START_REF] Wu | Effect of Different Light Qualities on Growth, Pigment Content, Chlorophyll Fluorescence, and Antioxidant Enzyme Activity in the Red Alga Pyropia haitanensis (Bangiales, Rhodophyta[END_REF]. Also, low chlorophyll content (0.150 mg g - 1 fw) from H. floresii (Freile-Peligrin and Robledo, 2009), low (0.288 mg g -1 ) from denticulatum, and Gracilaria Salicornia) were all very low (0.05-1.31 mg g -1 )

compared to the -carotene content (54.47 mg g -1 ) from H. durvillei in the present study (McDermid, 2003;Godinez-Ortega et al. 2008;Freile-Peligrin and Robledo, 2009;[START_REF] Chan | Chemical composition and physicochemical properties of tropical red seaweed, Gracilaria changii[END_REF][START_REF] Macartain | Nutritional Value of Edible Seaweeds[END_REF]Nunes et al. 2017;Lalegarie et al. 2019;[START_REF] Manam | Phytochemical, amino acid, fatty acid and vitamin investigation of marine seaweeds Colpomenia sinuosa and Halymenia porphyroides collected along Southeast coast of Tamilnadu, India[END_REF]. Chlorophylls and carotenoids had the potential effects of reducing DPPH radicals as antioxidants or in food development. Phycoerythrobilin extracted from Pyropia sp. reported antioxidant activity by scavenging peroxyl radicals in vitro (Okolie et al. 2018). However, the IC50 value of H. durvillei and H. dilatata in the present study, although active, but are considered weak. The -carotene content from H. durvillei (54.47 mg g -1 ) was way too high than found in spinach (0.040 mg g -1 ) and other vegetables (McDermid, 2003;[START_REF] Macartain | Nutritional Value of Edible Seaweeds[END_REF], while no β-carotene detected in H. dilatata in the present study. The pigment β-carotene is said to modulate UVA-induced gene expression, protect the skin and eyes from photo-oxidation against UV light, and prevent eye disease in humans, e.g., cataracts (Pereira, 2018). β-carotene, which, besides its anticancer activity, has been reported to be absorbed 10-times more easily by the body than the synthetic one [START_REF] Christaki | Functional properties of carotenoids originating from algae: Functional properties of algal carotenoids[END_REF]. Pigments from macroalgae are also crucial in this industrial range (Leandro et al. 2020).

Biological activity

Cytotoxicity and antiviral activity evaluation

No cytotoxic effect of polysaccharides (CC50 greater than 200 μg mL -1 ) on Red algae as a potential source of antiviral agents against HSV and other viruses were already established by many authors [START_REF] Mahomoodally | Marine Algae: A Potential Resource of Anti-HSV Molecules[END_REF].

Linear chains of galactopyranosyl residues (λ-and ιcarrageenans) are potent inhibitors against dengue viruses DENV-2 and 3 [START_REF] Bedoux | Antiviral and Cytotoxic Activities of Polysaccharides Extracted from Four Tropical Seaweed Species[END_REF]. The water extract from red marine alga Polysiphonia denudata selectively inhibits HSV-1 and 2 reproduction with EC50 value range from 8.7 to 47.7 mg mL -1 [START_REF] Mahomoodally | Marine Algae: A Potential Resource of Anti-HSV Molecules[END_REF]). Another study using a similar extraction and analysis condition with the present study reported extract from Solieria filiformis had antiviral activity (EC50=136 µg mL -1 ) against HSV-1 ( [START_REF] Bedoux | Antiviral and Cytotoxic Activities of Polysaccharides Extracted from Four Tropical Seaweed Species[END_REF]). In the present study, H.

durvillei and H. dilatata extracts possess antiviral activities against HSV-1 with EC50 values (5.89, and 1.21 µg mL -1 ) that are way lesser and are more potent compared to the literature [START_REF] Bedoux | Antiviral and Cytotoxic Activities of Polysaccharides Extracted from Four Tropical Seaweed Species[END_REF][START_REF] Mahomoodally | Marine Algae: A Potential Resource of Anti-HSV Molecules[END_REF]. Also, according to Bedoux et al. that Solieria filiformis extract with 15.6% protein, 7.5% sulfates, and 13.3% polysaccharides sulfate content in dw suggest that aside from degree of sulfation, other factors such as molecular weight, protein content, and position of sulfate groups along the chain may be responsible for this antiviral activity [START_REF] Bedoux | Antiviral and Cytotoxic Activities of Polysaccharides Extracted from Four Tropical Seaweed Species[END_REF]. In terms of sulfates content, H. durvillei and H.

dilatata extract in the present study had comparable sulfate content that was also found in the literature [START_REF] Bedoux | Antiviral and Cytotoxic Activities of Polysaccharides Extracted from Four Tropical Seaweed Species[END_REF]. Galactopyranose residues as potent antiviral inhibitors from carrageenans [START_REF] Bedoux | Antiviral and Cytotoxic Activities of Polysaccharides Extracted from Four Tropical Seaweed Species[END_REF][START_REF] Mahomoodally | Marine Algae: A Potential Resource of Anti-HSV Molecules[END_REF] are also found in the present study. The variation of antiherpetic activity results of carrageenans and their derivatives between the present and previous studies could be influenced by monosaccharide composition, sulfate content, polysaccharides chemical structure, molecular weight, location and period of collection, and sensitiveness of methods used. These activities may be due to the presence of other compounds such as proteins and polysaccharides [START_REF] Bedoux | Antiviral and Cytotoxic Activities of Polysaccharides Extracted from Four Tropical Seaweed Species[END_REF]Maya Puspita et al. 2017;[START_REF] Sun | Degradation of Polysaccharides from Grateloupia filicina and Their Antiviral Activity to Avian Leucosis Virus Subgroup[END_REF][START_REF] Kidgell | Ulvan: A systematic review of extraction, composition and function[END_REF][START_REF] Mahomoodally | Marine Algae: A Potential Resource of Anti-HSV Molecules[END_REF]. The H. durvillei and H. dilatata extracts results showed great potential in developing as an anti-HSV, or anti-DENV drug. However, these various factors mentioned must be understood better, and therefore extracts from H. durvillei and H. dilatata must be studied further.

with range values of 18.8-33.9%, and H. dilatata extract (Hdil extract) with range values of 14.49-24.13% both at 500-10000 µg mL -1 are at least 10-times weaker in terms of doses used compared to [START_REF] Vinosha | Biogenic synthesis of gold nanoparticles from Halymenia dilatata for pharmaceutical applications: Antioxidant, anticancer and antibacterial activities[END_REF]. However, if we just consider the dose of up to 1000 µg mL -1 in the present study which is the maximum dose used by [START_REF] Vinosha | Biogenic synthesis of gold nanoparticles from Halymenia dilatata for pharmaceutical applications: Antioxidant, anticancer and antibacterial activities[END_REF], then our antioxidant activities for (Hdur extract) and

(Hdil extract) were just somewhat similar with Hd-extract which is also weak from [START_REF] Vinosha | Biogenic synthesis of gold nanoparticles from Halymenia dilatata for pharmaceutical applications: Antioxidant, anticancer and antibacterial activities[END_REF].

Another study of antioxidant activity of ethanol extract and ethyl acetate extract from Eucheuma cottonii and Eucheuma spinosum from Indonesia had scavenging activity against DPPH and IC50 value [START_REF] Putri | Phytochemical analysis and antioxidant activity of marine algae Eucheuma Sp[END_REF]. Where, reported for E. cottonii ethanol extract (72.41% at 50 µg mL -1 , IC50=35.5 µg mL -1 );

E. cottonii ethyl acetate extract (1.29% at 50 µg mL -1 , IC50=435.36 µg mL -1 ); while E. spinosum ethanol extract (5.3% at 500 µg mL -1 , IC50=1386.79 µg mL -1 ); and E. spinosum ethyl acetate extract (50.4% at 500 µg mL -1 , IC50=430.5 µg mL -1 ). Among different extracts from E. cottonii and E. spinosum, ethanol extract from E. cottonii had the strongest extract against DPPH [START_REF] Putri | Phytochemical analysis and antioxidant activity of marine algae Eucheuma Sp[END_REF]. Despite using 20 to 100 times higher in dose concentrations in comparison with the study of [START_REF] Putri | Phytochemical analysis and antioxidant activity of marine algae Eucheuma Sp[END_REF], the present study ethanol extracts (Hdur extract and Hdil extract) still had <50% scavenged against DPPH which is lower than E. cottonii ethanol extract (72.41% at 50 µg mL -1 ) and E. spinosum ethyl acetate extract (50.4% at 500 µg mL - 1 ). However, the present study (Hdur extract and Hdil extract) scavenged against DPPH are up to 20 times higher compared to E. cottonii ethyl acetate extract (1.29% at 50 µg mL -1 ), and E. spinosum ethanol extract (5.3% at 500 µg mL -1 ) with comparable IC50 values. [START_REF] Souza | Antioxidant Potential of Two Red Seaweeds from the Brazilian Coasts[END_REF][START_REF] Souza | Antioxidant Potential of Two Red Seaweeds from the Brazilian Coasts[END_REF] reported ethanolic extracts of gracilaria birdiae (Gb) exhibit better scavenging activity (approx. 60%, at 5mg mL -1 ). However, a weaker antioxidant activity (IC50=0.76 mg mL -1 ) compared to the ethanolic extracts of (Hdur extract), and (Hdil extract) with (IC50<0.5 mg mL -1 ) in the present study. These results suggest that antioxidant activity is affected by methods of extraction used, among other factors, even among similar test species. The weak values of antioxidant activity for (Hdur extract) and (Hdil extract) in the present study is probably due to its group, as Red algae group reported to have the smallest FRAP mean value of 1.59±1.17 µM µg -1 extract compared to brown and green algae (3.55±3.16, and 2.29±2.34) µM µg -1 extract, respectively [START_REF] Kelman | Antioxidant Activity of Hawaiian Marine Algae[END_REF]. Another reason of weak values of antioxidant activity is due to its location and depth of the collection, where [START_REF] Kelman | Antioxidant Activity of Hawaiian Marine Algae[END_REF] reported antioxidant activities of algae collected from shallow waters (0-3m) were more active than those algae collected in the deep with mean FRAP values of 2.26±2.29, and 0.21±0.13 µM µg -1 extract, respectively. The H. durvillei and H.

dilatata in the present study were collected in the subtidal zones at depths of approximately 5 to 10 meters. The variation of antioxidant activity, DPPH radical scavenging activity results of carrageenans, between the present and previous studies, could be influenced by several factors. Previous papers reported a high correlation of DPPH radical scavenging activity versus total polyphenols, low Mw polysaccharides, pigments, proteins, or peptides [START_REF] Souza | Antioxidant Potential of Two Red Seaweeds from the Brazilian Coasts[END_REF] or sensitiveness of methods used, or period, location, and depth of collection [START_REF] Kelman | Antioxidant Activity of Hawaiian Marine Algae[END_REF].

The present study of Hdil extract is better than Hdur extract. Although both extracts are weak antioxidants, they still are considered significant since these extracts are derived from natural resources.

Conclusion and perspectives

In The presence, degree, and distribution of the sulfate groups are important in determining the biological activity of polysaccharides, and therefore must be studied further for these extracts in the future.

Chapter 6 Conclusion

The Philippines is a source of an enormous number of rich natural resources, especially the marine environment. Nevertheless, little is known about the nutritional properties or biological activities of the wild green (C. racemosa and U. lactuca), brown (S. polycystum and S. ilicifolium), and red (H. durvillei and H. dilatata) seaweed species from the Philippines. Thus, we initially characterized these macroalgae to understand their biochemical composition better. Thus, it is a potential for these wild seaweeds species to be used as a raw material in the food industry, however, microbial and ecotoxicological analyses are needed to complete the study.

Major Sugar units (% dw)

Glc (6.0-54.8), Xyl (0.4-4.4), Gal (0.9-18.4) Glc (0.9-12.4), Rham (0.5-6.4), GlcA (0.08-0.4) Fuc (4.9-6.8), Glc (0.7-22.8), Gal (2.7-4.5), Man (1.8-4.6), Xyl (0.6-1.7) Fuc (5.4-15.9), Glc (1.2-33.7), Pigments analysis (mg g -1 dw) had high pigments and carotene contents that can be used in the food industry and human health development.

-carotene contents were at least 100 times higher than found in spinach and to the brown seaweeds h, i had higher Chl a than Pyropia haitanensis, Halymenia floresii, Asparagopsis taxiformis, Chondrus crispus, Grateloupia lanceola j, k, l . -carotene contents were at least 100 times higher than found in spinach h, i Values are means ± SD (n=9) except for lipids; a= [START_REF] Hamed | Marine Bioactive Compounds and Their Health Benefits: A Review: Importance of functional seafood…[END_REF]b= Lappano et al. 2017;c= Venn-Watson et al. 2020;d= Sargent et al. 1994;e= Kumar et al. 2010;f= Matos et al. 2016;g= Turan 2007;h= McDermid, 2003;[START_REF] Macartain | Nutritional Value of Edible Seaweeds[END_REF]j= Freile-Peligrin and Robledo, 2009;k= Wu, 2016;l= Nunes et al. 2017;n.d., not detected; -, not determined; RDA, recommended daily allowance; EAAs, essential amino acids; NEAAs, non-essential amino acids.

With this preliminary biochemical characterization of the raw material, we proceeded to extract these seaweed samples' polysaccharides. The extraction yields of the green, brown, and red seaweeds of this study are affected by the species used, geographic location, and seasonal growth and even by the sensitiveness of the methods used. Biochemical composition of C. racemosa and U. lactuca (successive precipitation) extracts is mainly composed of sulfates group (8.18-10.58%, 15.79-20.03%) and proteins (3.7-11.0%, 7.1-19.6%), respectively; and sulfates group (5.44-16.0%), proteins (18.3-18.9%), and uronic acids (7.0-9.5%), respectively for dialyzed fractions of U. lactuca. The biochemical composition of S. polycystum and S. ilicifolium extracts is mainly composed of neutral sugars (8.8-23.1%, 18.5-33.2%), sulfates group (6.36-12.68%, 6.98-15.21%), and proteins (8.9-11.8%, 8.7-25.5%) dw of alga, respectively. Furthermore, the red algae H. durvillei and H. dilatata extracts are mainly composed of neutral sugars (17.6, 12.2%), sulfates group (15.73, 15.22%), and proteins (7.0, 8.7%) dw of alga, respectively. FTIR spectrum of these macroalgal extracts confirmed the presence of sulfate ester, sugars, and proteins. The C. racemosa polysaccharide is mainly composed of sulfated galactan with arabinose, glucose, and xylose sugar units. The use of strong alkali (1-and 4M-KOH) to isolate hemicellulosic polysaccharides from C. racemosa HWE fraction for biochemical characterization is not necessary since all the information found in these fractions are already found in HWE fraction (see [START_REF] Hurtado | Chapter Two -Selected red seaweeds from the Philippines with emerging high-value applications[END_REF]. The FTIR spectrum of U. lactuca extracts confirmed the presence of sulfate groups and sugars, where U. fasciata polysaccharide is mainly composed of Ulvan with rhamnose and glucose as the dominant sugar units. S. polycystum polysaccharide is mainly composed of fucoidan, alginate, and cellulose/hemicellulose at a ratio of 3:2:1 with fucose, glucose, galactose, mannose, and xylose as the dominant sugar units. S. ilicifolium cell wall polysaccharides are composed of alginate, fucoidan, and cellulose/hemicellulose at a ratio of 6:2:1 with fucose, glucose, galactose, mannose, and xylose as the dominant sugar units. H. durvillei and H. dilatata polysaccharides are mainly composed of sulfated Lambdacarrageenan with galactose, arabinose, and rhamnose as the dominant sugar units.

Therefore, this implies that the red seaweeds H. durvillei and H. dilatata are natural sources of Lambda-carrageenan. The C. racemosa, U. lactuca, S. polycystum, S. ilicifolium, H. durvillei, and H. dilatata fractions showed in vitro antiherpetic activity without cytotoxicity. Polysaccharides from S. ilicifolium fractions are more potent than S. polycystum fractions. Polysaccharides from H. dilatata fraction is five times more potent (EC50: 1.21 µg mL -1 ) than H. durvillei fraction (EC50: 5.89 µg mL -1 ). The C. racemosa and U. lactuca antioxidant results (DPPH scavenging assay) suggest that fresh seaweed should be preferred for human consumption. The S. polycystum and S. ilicifolium antioxidant results had better activity than the green (C. racemosa and U. lactuca) and the red (H. durvillei and H. dilatata) seaweeds in the present study. Suggest that the seaweeds are safe for human consumption.

Although both H. durvillei and H. dilatata extracts are weak antioxidants, they still are considered significant since these extracts are derived from natural resources.

The presence, degree, and distribution of sulfate groups are important in determining the biological activity of polysaccharides, and to analyze its antiviral potency in relation to structure and position of sulfate groups, therefore, must be studied further for these extracts in the future.

The C. racemosa and U. lactuca are rich in lipids at 4.5% dw and 4.1% dw, respectively. At the same time, U. lactuca was evident by its high amount of palmitic acid, presence of PUFAs (C20:4n-6 and C20:5n-3), and a considerable amount of C22:1n-9 (erucic) and C24:1n-9 (nervonic) acids [START_REF] Hurtado | Chapter Two -Selected red seaweeds from the Philippines with emerging high-value applications[END_REF].

The red algae, H. durvillei and H. dilatata lipids are within the range value of selected red seaweeds from the Philippines. Also, both H. durvillei and H. dilatata had ω6/ω3 ratio (<10) of 1.81, 0.46), respectively confirms the potential health benefits of the consumption requirements. Although PUFA/SFA ratio for both H. durvillei and H. dilatata was <0.45, they shall be considered a good-health promoter because of the presence of C22:1n-9 (erucic) and C24:1n-9 (nervonic) acids. These compounds are said to prevent demyelinating disease. The C. racemosa and U. lactuca had high pigments and carotene contents that can be used in the food industry and human health development. Also, S. polycystum and S. ilicifolium had -carotene contents of at least 100 times higher than found in spinach. Besides its anticancer activity, -carotene has been reported to be absorbed 10-times more easily by the body than the synthetic one. Thus, S. polycystum and S. ilicifolium are excellent sources of pigments used in the food industry and human health development. Also, H. durvillei and H. dilatata are excellent sources of pigments.

  Figure No. Page 1 Plant specimen of C. racemosa (Forsskål) displayed at the local public market in Langihan, Butuan City, Philippines (personal documentation).

10

  The main repeating disaccharide units of ulvan. A.[→4)alpha-D-Glcp-(1→4)-beta-L-Rhap3S-(1→] n; B. [→4)alpha-L-Idop-(1→4)-alpha-L-Rhap3S-(1→] n.11Cell wall model of brown algae from the order Fucales[START_REF] Deniaud-Bouët | Chemical and enzymatic fractionation of cell walls from Fucales: Insights into the structure of the extracellular matrix of brown algae[END_REF]).

  polycystum and S. ilicifolium), and red (H. durvillei and H. dilatata) seaweeds collected from different parts of the country were based on their popularity or abundance, economic, and biochemical potential. The C. racemosa was one of the most popular seaweed salads in the Philippines. It was cultured in the Philippines since the 1960s together with C. lentillifera and is still sustainable upto today[START_REF] Trono | The seaweed resources of the Philippines[END_REF]. Caulerpa is the most collected seaweed species from the wild and directly sold to the market (de Gaillande et al. 2017). The genus Ulva was seasonally abundant and considered as nuisance in the beach shores of the Philippines. This genus was encouraged by the Philippine government to be developed as human and animal feed, and as potential source of biogas feedstock (SIAP 2018; http://business.inquirer.net/227401/ph-seaweed-gears-resurgence).

Figure 1 .

 1 Figure 1. Plant specimen of C. racemosa (Forsskål) displayed at the local public market in Langihan, Butuan City, Philippines (personal documentation).

Figure 2 .

 2 Figure 2. Plant specimen of Ulva lactuca (Linnaeus) collected at Lawigan, Bislig City, Philippines (personal documentation).

Figure 3 .

 3 Figure 3. Plant specimen of S. polycystum (C. Agardh) collected at Lamanok island, Anda, Bohol, Philippines (personal documentation).

Figure 4 .

 4 Figure 4. Plant specimen of S. ilicifolium (Turner) C. Agardh, collected at Currimao, Ilocos Norte, Philippines (personal documentation).

Figure 5 .

 5 Figure 5. Plant specimen of H. durvillei (Bory de Saint-Vincent) collected at Currimao, Ilocos Norte, Philippines (personal documentation).

Figure 6 .

 6 Figure 6. Plant specimen of H. dilatata (Zanardini) collected at Lawigan, Bislig City, Philippines (personal documentation).

Figure 7 .

 7 Figure 7. Evolutionary tree of the eukaryotes shows the position of red, green, and brown algae and the divergence of red algae, dinoflagellates, and brown algae (cited by Chandler et al. 2017, and taken from the paper of Cock et al. 2010).

  This summary is based on a consensus of recent phylogenomic studies. The colored groupings correspond to the current 'supergroups.' Unresolved branching orders among lineages are shown as multifurcations. Broken lines reflect lesser uncertainties about the monophyly of certain groups. Star symbols denote taxa that were considered supergroups in early versions of the supergroup model; thus, all original supergroups except Archaeplastida have either disappeared or been subsumed into new taxa. The circles showed major lineages with no molecular data when the supergroup model emerged, most often because they had not yet been discovered.

Figure 8 .

 8 Figure 8. The new tree of eukaryotes showing Archaeplastida, and Stramenopila -the lineage of marine algae[START_REF] Burki | The New Tree of Eukaryotes[END_REF]).

Figure 10 .

 10 Figure 10. The main repeating disaccharide units of ulvan. A. [→4)-alpha-D-Glcp-(1→4)-beta-L-Rhap3S-(1→] n; B. [→4)-alpha-L-Idop-(1→4)-alpha-L-Rhap3S-(1→] n.

Figure 11 .

 11 Figure 11. Cell wall model of brown algae from the order Fucales (Deniaud-Bouët et al. 2014).

Figure 12 .

 12 Figure 12. Cell wall model of red algae from the order Halymeniales (Stiger-Pouvreau et al. 2016).

(

  [START_REF] Fenoradosoa | Highly sulphated galactan from Halymenia durvillei (Halymeniales, Rhodophyta), a red seaweed of Madagascar marine coasts[END_REF]. The principal feature of the highly sulfated carrageenan is D-galactose and anhydro-D-galactose, while the less sulfated agars the Dgalactose, L-galactose, or anhydro-L-galactose[START_REF] Stiger-Pouvreau | Carbohydrates from Seaweeds[END_REF].

  Colloids have been applied to multiple industrial applications, such as food processing, cosmetics, and pharmaceuticals. Alginate, carrageenan, and agar are widely known for their gelling, stabilizing, and thickening properties. These products have been commercialized as additives, with an average of 1 million tons of wet algae that are used every year to extract these three compounds, as shown in Fig.13[START_REF] Frangoudes | Etats Des Lieux de La Filière Des Macro-Algues En Europe[END_REF].

Figure 13 .

 13 Figure 13. Utilization of algae in France (production and imports) according to CEVA 2009 (Mesnildrey et al. 2012).

  ) have earned a reputation for protecting healthy cells against various diseases, aging processes, and considerable commercial potential in medicine, food production, and cosmetics industry (de Gaillande et al. 2017; Olasehinde et al. 2019). Several studies isolating C. racemosa as cited by de Gaillande et al. (2017) in her paper that Liu et al. (2013) found caulerprenylols A and B (new prenylated para-xylenes)

  toxic constituent called caulerpicin (from C. racemosa) has anesthetizing sensation and causes numbness of the tongue, lips and cold sensation in the fingers and the feet[START_REF] Kumar | Toxicological effects of marine seaweeds: A cautious insight for human consumption[END_REF]. Another compound Caulerpin has painkilling and anti-inflammatory properties (C. racemosa, C. serrulata and C. sertularioides)[START_REF] De Gaillande | Caulerpa consumption, nutritional value and farming in the Indo-Pacific region[END_REF], however, it also produces mild anesthetic action, difficulty in breathing, sedation and loss of balance[START_REF] Kumar | Toxicological effects of marine seaweeds: A cautious insight for human consumption[END_REF]. Simultaneously, Caulerpenyne is also known as a repellant against other organisms (grazers, epiphytes, competitors) and exhibiting anti-neoplastic, antibacterial, and anti-proliferative activities (de Gaillande et al. 2017). Tropical species of Caulerpa are highly variable in their production of caulerpenyne; for example, in Guam are higher than those from the Mediterranean alga C. sertularioides. However, caulerpenyne also represents the most abundant cytotoxic sesquiterpenoid in Caulerpa taxifolia, showed ichthyotoxic activity, which blocked cleavage of developing sea urchin eggs and can induce apoptosis in mammalian (neuroblastoma) cells. Caulerpenyne also shows neurological activity in invertebrate model organisms (leeches) by modifying the electrophysiological properties of touch mechanosensory cells. These electrophysiological effects on neurons might explain human poisoning incidents after the fish Sarpa salpa, which eats C. taxifolia, was consumed as having neurological symptoms of amnesia, vertigo, and hallucinations. These findings may have implications about the neuroecological effects of caulerpenyne on other marine consumers. Caulerpenyne's

  , (c) kainic and domoic acids, as anthelmintic agents from Digenea simplex and Chondria armata (Freile-Pelegrín and Tasdemir 2019), (d) major glycolipids: MGDG (from Hydrolithon reinboldii), DGDG (from Sargassum horneri), and SQDG (from Gigartina tenella, Palmaria palmata)as antibacterial, antitumor, antiviral and anti-protozoal agents[START_REF] Plouguerné | Glycolipids from seaweeds and their potential biotechnological applications[END_REF], (e) xylomannan sulfate, against HSV-1 from Sebdenia polydactyla(Ghosh et al.

Figure 14 .

 14 Figure 14. Perspective map of the world and sampling locations in the Philippines showing the site collection of selected species of green seaweeds (A) Caulerpa racemosa var. Forsskal, and (B) Ulva lactuca Linnaeus; brown seaweeds (C) Sargassum polycystum and (D) Sargassum ilicifolium; and red seaweeds (E) Halymenia durvillei and (F) Halymenia dilatata. It was collected from June 25 to August 16, 2017.

  a tropical archipelagic country with more than 7,000 islands located in the Indo-Pacific region at 12.8797° N and 121.7740° E. It has a tropical climate is, for the most part, hot and humid all year round. There are two seasons, the wet season (from June to November) and the dry season (from December to May). Philippines marine water covers an area of 220 million hectares, a shelf area of 18.6 million hectares, with a coastline length of 36,289 kilometers (http://nast.ph/index.php/downloads/). It has an average temperature between 25.5 and 28.3 °C, with mean annual precipitation that varies (from 965 to 4,064 mm) occurred highest during the rainy season between June and November. Baguio City, eastern Samar, and eastern Surigao receive the highest amount of rainfall, while the southern part of Cotabato receives the least amount of rain. The combination of warm temperature and high relative, absolute humidity gives rise to sensible high temperatures throughout the archipelago. Maximum levels of temperature and humidity are mostly felt during March to May (https://www1.pagasa.dost.gov.ph/index.php/climate-of-thephilippines). There were different sampling sites chosen to represent the main group of islands of Luzon, the Visayas, Mindanao (Fig. 15), and primary groups of green, brown, and red seaweed. From the Luzon, in Currimao coast, Ilocos Norte (18.021 N and 120.483 E), collected Sargassum ilicifolium (brown), and Halymenia durvillei (red). From the Visayas, in Lamanok island, Anda, Bohol (9.804° N and 124.596° E) collected Sargassum polycystum (brown). From the Mindanao, in the coastal area of Lawigan, Bislig City, Surigao del Sur (8.226° N and 126.431° E) collected Caulerpa racemosa var. Forsskal (green), Ulva lactuca Linnaeus (green), and Halymenia dilatata Delile (red).

Figure 15 .

 15 Figure 15. Sampling areas in the different parts of the Philippines (Above) during low tide at; Currimao coast, Ilocos Norte in Luzon (A), Lamanok Island, Anda, Bohol in Visayas (B), and Lawigan coast, Bislig City in Mindanao (C). The collection is from June 25 to August 16, 2017.

Algae

  Sargassum ilicifolium and Halymenia durvillei were collected last July 2017 in the subtidal area of Currimao coast, Province of Ilocos Norte, from 6:00 in the morning up to 3:00 p.m. (Fig.16).

Figure 16 .

 16 Figure 16. Sampling and collection of S. ilicifolium and H. durvillei during low tide in the coastal area of Currimao, Province of Ilocos Norte in Luzon. The collection was in July 2017.

Figure 17 .

 17 Figure 17. Sampling and collection of Sargassum polycystum during low tide in the coastal area of Lamanok Island, Anda, Island Province of Bohol in the Visayas. The collection was on August 22, 2017.

Figure 18 .

 18 Figure 18. Sampling and collection of Caulerpa racemosa, Ulva lactuca, and Halymenia dilatata during low tide in the coastal area of barangay Lawigan, Bislig, Province of Surigao del Sur in Mindanao. The collection was 15-16 August 2017.

Figure 20 .

 20 Figure 20. Principle and reaction of the determination of total sugars by the method of Dubois et al. (1956) (Burlot, 2016).

  (1973) modified by Filisetti-Cozzy et al. (1991) and cited by Burlot (2016) is shown in Fig. 21 below.

Figure 21 .

 21 Figure 21. Principle and reaction of the dosage of uronic acids by the method of Blumenkrantz and Asboe-Hansen (1973) modified by Filisetti-Cozzy et al. (1991) (Burlot, 2016).

  principle and Chemical reactions are shown in Fig.23below.

Figure 23 .

 23 Figure 23. Principle and reaction of the determination of polysaccharide-linked sulfate groups by the method of Jaques et al. (1968) (Burlot, 2016).

  the mass of dry C. racemosa Forsskal and U. lactuca Linnaeus = 10.0 g each; Mass of the lipid content of C. racemosa Forsskal = 448.5 mg; Mass of the lipid content of U. lactuca Linnaeus = 408.6 mg.

Figure 25 .

 25 Figure 25. The first treatment of total or partition lipid extraction common to all groups of algae (A), washing and drying (B), and evaporation (C).

  FAME analysis: the temperature was held at 170 °C for 4 min and programmed to 300 °C at 4 °C min -1 . The FAs were then identified by comparing their equivalent chain lengths (ECLs) values and their retention time with commercial mixtures (Supelco 37 Component FAME Mix, ThermoScientific, Illkirch, France). The ECLs of FAs were determined by expressing their elution positions relative to known straight-chain saturated FAs.

Figure 26 .

 26 Figure 26. The second treatment of total lipid extraction common to all groups of algae: (A) Saponification and (B) Esterification (adapted from Terme et al. 2017).

  5)Fatty acids hypocholesterolemic/hypercholesterolemic (h/H), and ℎ/𝐻 = 𝐶18: 1𝜔9 + 𝐶18: 2𝜔6 + 𝐶20: 4𝜔6 + 𝐶18: 3𝜔3 + 𝐶20:

  ), are inserted into a Soxhlet cartridge and were extracted sequentially inside the Soxhlet extractor. An extraction for 1 h or until the cartridge turns off-white or white, performed with 96% ethanol, of which 300 mL were first introduced into the flask with pumice. Switch on the ballon heater just enough for the solvent to condense). Second extraction with a Soxhlet extractor is carried out for 1h (same above) with 300 mL chloroform/methanol mixture (1:1, v/v). The third extraction with a Soxhlet extractor was carried out for 1 h with 300 mL 100% acetone. Let the cartridge dry (air, hair drier, or oven-dry) in between each extraction. Solvents from each extraction were filtered and evaporated under reduced pressure. Record each initial and final weight before and after extraction and evaporation. Degraded residues of pigments, lipids and proteins are obtained from these three sequential extractions. These extracts are then used for polysaccharides extraction in each samples.

Figure 27 .

 27 Figure 27. Extraction and isolation of polysaccharides common to all groups of algae.

Figure 29 .

 29 Figure 29. Routes of purification of sulfated polysaccharides, by successive precipitations and by dialysis of Ulva lactuca (Linnaeus).

  polycystum and Sargassum ilicifolium polysaccharidesThe extraction of polysaccharides for brown algae was performed following and adapted the protocol from[START_REF] Deniaud-Bouët | A review about brown algal cell walls and fucose-containing sulfated polysaccharides: Cell wall context, biomedical properties and key research challenges[END_REF]. The depigmented algal powder was purified sequentially for the extraction of polysaccharides. The extraction of Sargassum polysaccharides was done in several stages, and each stage was separated by filtration where its residue was precipitated in four (4) different solutions and its liquid filtrate were dialyzed with a 12-14 kDa Spectra/Por membrane (Fig.30).

Figure 30 .

 30 Figure 30. Schematic diagram of sequential extraction of sulfated polysaccharides from S. polycystum and S. ilicifolium with inorganic solvents.

Figure 31 .

 31 Figure 31. Schematic diagram of extraction and purification of sulfated polysaccharides from H. durvillei and H. dilatata with inorganic solvents (adapted from AS. Burlot, 2016).

  simple sugar composition of the samples was determined by High-Performance Anion Exchange Chromatography (HPAEC) with Pulsed Amperometric Detection (PAD) (Thermo Dionex, Sunnyvale, CA., USA) base on Pliego-Cortés et al. (2019) with brief modifications. Monosaccharides were identified and quantified based on their standard curves at different concentrations (1.95-125 ppm). Results were expressed as micrograms of monosaccharides per milligram of dry weight (μg mg -1 dw).Principle: HPAEC is based on sugars' property, which becomes weakly acidic in a very alkaline medium (pH> 12). This type of chromatography allows a selective separation of the monosaccharides on a stationary anion exchange phase, stable at high pH. The column CarboPac PA1 (Dionex, Thermo Scientific) is suitable for this type of analysis. It constitutes the stationary phase and is composed of a 10 μm polystyrene/divinylbenzene substrate of a diameter that agglomerated 350 nm latex microbeads, having quaternary amines functions, which bind to SO3 groups -the surface of the substrate, such as shown in Fig.32.

Figure 33 .

 33 Figure 33. Operation of the PAD detector. Potential E1 is applied for 400 ms.During this time, the intensity of the current related to sugars oxidation (integration) is measured. Potential E2, at -2.00V, cleans the gold electrode by decomposing the oxidation products of sugars. Once the electrode is clean, the potential E3 goes up to 0.60 V to "reactivate" it. Finally, immediately after, a potential E4 is applied for 60 ms to reduce the native gold surface of the electrode and to allow better oxidation of sugars. This wave of potential is repeated throughout the chromatography.

Figure 34 .

 34 Figure 34. HPAEC-PAD system. The samples are in a sampler where they are injected independently of one after the other. They injected the eluent, which, beforehand, propelled the system pumps-the set between the column where the sugars are separated. At the column outlet, the sugars are quantified by the PAD. The quantifications of the sugars present in the samples are calculated by integrations and using standards (fucose, rhamnose, arabinose, glucosamine, galactose, glucose, mannose, xylose, fructose, ribose, glucoheptose, glucuronic acid, and (standard internal) deoxyribose).

  case, Vf= 1.5 mL. Using the formula (Ci)(Vi) = (Cf)(Vf) and its given values. Where; Ci= initial concentration or 1000 ppm, Vi= initial volume or unknown, Cf= final concentraion or 125 ppm for all except for deoxyribose, and Vf= final volume or 1.5 mL.

  Before analysis, each dry matter samples (4 mg dw) undergo complete acid hydrolysis for 48 hours at 100 °C using 110 μL of 1 M hydrochloric acid (Carlo Erba, 7647-01-0) and 1 mL of ultrapure water in a sealed glass ampoule. The samples were then neutralized with 110 μL of 1M sodium hydroxide to each sample, and 780 μL of ultrapure water containing deoxyribose (internal standard) to a final concentration of 50ppm. The 2 g L -1 neutralized samples were then filtered (0.22 μm) and are inserted into specific vials for HPAEC-PAD analysis.The separation of the sugars present in the samples is carried out on the Carbo column Pac PA1 at a flow rate of 1 mL min -1 and a pressure of 110 bar. The simple sugars are identified by comparing injected standards (50 μM) and eluted under the same conditions (glucoheptose, rhamnose, glucosamine, galactose, mannose, fructose, arabinose, glucose, xylose, ribose, and glucuronic acid). Twenty-five (25) μL of sample or standard are injected. The elution program (Fig. 36) requires three eluents: Solution A, 0.1M NaOH; Solution B, 1M NaOAc in 0.1M NaOH; and Solution C, ultrapure water. The program elution started with 30 min of isocratic elution (Sol A: Sol C 20:80) followed by a gradient of sodium acetate (Sol B) from 0 to 100 % in 5 min supplemented by 5 min of isocratic elution to 100% of solution B and finally a return to initial conditions for 20 min (Sol A: Sol C, 20:80). The retention times obtained for the standards and the height of the peaks obtained for known concentrations allow the identification and quantification of sugars present in the samples.

Figure 36 .

 36 Figure 36. HPAEC-PAD elution program. The elution of monosaccharides wasmainly in the isocratic mode. Sodium acetate is added after 30 minutes to elute the compounds in the column. Indeed, the acetate ions, more electronegative, will be exchanged by binding to quaternary amine microbeads.

  seaweeds C. racemosa (Cr crude, 1OH, 4OH, and INS), and U. lactuca (PP1, PP2, PP3, DS, and DA); from brown seaweeds S. polycystum (Sp CaCl2, Sp HCl, Sp Na2CO3, Sp KOH) and S. ilicifolium fractions (Si CaCl2, Si HCl, Si Na2CO3, Si KOH); and from red seaweeds, H. durvillei (Hdur) and H. dilatata (Hdil) were determined using a Nicolet™ iS™ 5 FT-IR spectrometer (Thermo Scientific™, Madison, WI, USA) with diamond crystal plate. The spectra were recorded in reflexive mode ranging from 4000-500 cm -1 wavenumber as the percentage transmittance was collected with 16 subsequent scannings of samples. About 2-mg of dry extracts were introduced onto the crystal plate and were then acquired as the percentage transmittance. A background scan with the diamond plate in place was

  -1 in enriched Eagle's MEM are inserted into all wells (Appendix 4). In all the wells of the 12th column (Cell Control), 100 μL of enriched MEM are added. In all the wells of the 11th column (Virus control), 50 μL enriched MEM are added. The evaluation of cytotoxicity is carried out in the wells in columns 1 to 10 and lines A to D, which are added 50 μL of enriched MEM. In all the wells of an entire column from A to H, 50 μL of the same sample at a given concentration, previously prepared, are added. In all the wells of column 11 and the wells of columns 1 to 10 and lines from E to H, were infected by adding 50 μL of HSV-1 viral suspension, diluted to obtain an MOI (multiplicity of infection) equal to 0.001 ID50 per cell. In this way, each sample is made in quintuplet for each concentration and vero cells were optimally protected when polysaccharides are deposited at the same with the viruses. The microplate is then placed in an oven at 37 °C under 5% CO2 atmosphere and saturated with water vapor for 72 hours.The neutral red revelation of cell viability: Fifty μL of the neutral red solution is introduced into all wells. The microplate is again placed in an oven at 37 °C for 45 minutes. A step of washing is then performed and repeated twice successively with a solution PSB (Phosphate-buffered saline) at 450 mOsm at pH 7.4 and a citrateethanol buffer. The bottom of the wells is scraped to homogenize the neutral red released in cells by the citrate-ethanol buffer. The ODs of all the wells are then read at 540 nm spectrophotometer.Cells and Virus: Vero cell lines (line n° ATCC CCL81) were cultivated in Eagle's MEM supplemented with 8% fetal calf serum (FCS, Eurobio, France), 1% Lglutamine (200 mM), and 1% PCS (penicillin (10,000 U), colimycin (25,000 U) and streptomycin (10 mg). The culture was performed at 37 °C under a 5% CO2 atmosphere, and the medium was renewed daily. Pr. Agut provided herpes simplex virus type 1 (HSV-1; family Herpesviridae) (Laboratoire de Dynamique, épidémiologie et traitement des infections virales de la Pitié Salpêtrière Paris, France). The virus collection was obtained by infection of Vero cells monolayers

  method. The antiherpetic compound acyclovir[9-(2-hydroxyethoxymethyl) guanine] was used as a reference inhibitor. The 50% effective antiviral concentration (EC50) was expressed as the concentration achieved by 50% protection of virus-infected cells from virus-induced destruction. The OD was related directly to the percentage of viable cells, which was inversely related to the cytopathic effect (CPE). The linear regression was determined for each assay based on cell controls (0% CPE) and virus controls (100% CPE). Data were expressed as a percentage of protection (%P): EC 50 = ( OD 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑐𝑒𝑙𝑙𝑠+𝑣𝑖𝑟𝑢𝑠+𝑑𝑟𝑢𝑔 -OD 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑣𝑖𝑟𝑢𝑠 OD 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑐𝑒𝑙𝑙𝑠+𝑑𝑟𝑢𝑔 -OD 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑣𝑖𝑟𝑢𝑠 ) x 100 where: ODcontrol cells+virus+drug= were the OD values of the test sample, ODcontrol virus= were the OD values of the virus control, and

  using the same extraction (except INS) from C. lentillifera and C. sertularioides showed sugar composition of glucose, galactose, mannose, and xylose. Glucose dominates among these sugars in all fractions except in fraction 4OH C. sertularioides, which is dominated by galactose and a lesser portion of glucose. These results of Shevchenko et al. (2009) supported the present study in which all of its sugar composition was also found in different proportions, and glucose was also the dominant sugar in all fractions.

Figure 38 .

 38 Figure 38. FT-IR spectra of C. racemosa extracts (Cr crude, 1OH, 4OH, and INS). The FTIR intensity bands in the ranges of [1260-930 cm -1 ] and [850-800 cm -1 ] are distinguishing characteristics between different types of main galactans as these regions are known as stretching vibrations of the sulfate esters and the 3,6anhydrogalactose ring. All spectral bands between [1260 and 1220 cm -1 ] are related to the sulfation level in general (Rochas et al. 2009; Barbosa et al. 2019). The spectral bands at [1245-1240 cm -1 ] and at [1025-1000 cm -1 ] in all C. racemosa extracts were due to stretching of -SO3 and C-O in ring groups (Gómez-Ordóñez and Rupérez 2011; Sarada et al. 2014). The medium and weak intensity bands at [1640-1630 cm -1 ] in all extracts were due to carboxylic acid vibrations. The peaks at [1561-1540 cm -1 ] bands in all extracts indicate amide N-H bending vibrations,where the previous C. racemosa extracts reported the presence of N-H involving in metal adsorption binding process[START_REF] Javaid | Removal of heavy metals by adsorption on Pleurotus ostreatus[END_REF][START_REF] Sarada | Cadmium removal by macro algae Caulerpa fastigiata: Characterization, kinetic, isotherm and thermodynamic studies[END_REF]. The bands at 871 and 793 cm -1 found in Cr crude and INS extracts are characteristics of the presence of sulfate groups on the molecule. At 871 cm -1 , this indicates the presence of S=O and C-S-O bands from ester sulfonate[START_REF] Ghosh | In vitro anti-herpetic activity of sulfated polysaccharide fractions from Caulerpa racemosa[END_REF][START_REF] Sarada | Cadmium removal by macro algae Caulerpa fastigiata: Characterization, kinetic, isotherm and thermodynamic studies[END_REF]

  [START_REF] Figueira | Structural Characterization of Ulvan Polysaccharide from Cultivated and Collected Ulva fasciata (Chlorophyta)[END_REF]).

Figure 39 .

 39 Figure 39. FT-IR spectra of U. lactuca extracts (DS, DA, PP1, PP2, and PP3).

  [START_REF] Figueira | Structural Characterization of Ulvan Polysaccharide from Cultivated and Collected Ulva fasciata (Chlorophyta)[END_REF]. The lower intensity band at about 930 cm -1 for fractions DS and DA indicates the bonds' deformation vibrations. This peak also indicates the presence of 3,6-anhydro-D-galactose from which was reported in Ulva rigida extract extracted by distillable ionic liquid (DIL)[START_REF] Pezoa-Conte | Deconstruction of the green alga Ulva rigida in ionic liquids: Closing the mass balance[END_REF].

  and U. lactuca polysaccharide extracts were estimated by the decrease in absorbance due to the reduction of the 2,2-DiPhenyl-1-PicrylHydrazyl (DPPH) radical by the extracts. DPPH is a stable synthetic free radical chemical compound used for measuring the free radical scavenging activities of compounds or extracts.DPPH is scavenged by antioxidant molecules through the donation of hydrogen, forming the reduced diphenyl picrylhydrazine (DPPH-H), thus neutralizing the reactive chain and reducing the risk of cellular oxidative damage[START_REF] Terme | Radical scavenging activity of lipids from seaweeds isolated by solid-liquid extraction and supercritical fluids. Oilseeds and fats Crops and Lipids[END_REF][START_REF] Vinosha | Biogenic synthesis of gold nanoparticles from Halymenia dilatata for pharmaceutical applications: Antioxidant, anticancer and antibacterial activities[END_REF]. The Butylated Hydroxyanisole (BHA) and Butylated Hydroxytoluene (BHT) standards presented an inhibiting concentration (IC50) of 7.05±0.59 and 5.32±0.59 μg ml -1 , respectively. Percent maximum scavenging ability and inhibition activity of C. racemosa and U. lactuca polysaccharide extracts at (5 mg mL -1 ) were shown in

Figure 40 .

 40 Figure 40. FT-IR spectra of S. polycystum extracts (Sp CaCl2, Sp HCl, Sp Na2CO3, and Sp KOH).

  observed in the present study, decreasing from fraction Sp Na2CO3 (an alginate rich) to fucose rich fractions Sp CaCl2 and Sp HCl. The bands at [1026-1025 cm -1 ] in all extracts except Sp KOH were due to stretching vibrations of pyranoid rings with C-O-C stretches of the glycosidic bonds, was also present in the study of[START_REF] Marudhupandi | In vitro anticancer activity of fucoidan from Turbinaria conoides against A549 cell lines[END_REF] for fucoidan fraction of T. conoides and[START_REF] Kok | Physicochemical properties of edible alginate film from Malaysian Sargassum polycystum C. Agardh[END_REF] for sodium alginate fraction of S. polycystum. The band at [815 cm -1 ] at fraction Sp Na2CO3 is due to the presence of sulfates group at the equatorial C-2 or C-3 positions, which has been reported in the isolated fucoidan of Sargassum henslowianum at 817 cm -1[START_REF] Ale | Fucose-Containing Sulfated Polysaccharides from Brown Seaweeds Inhibit Proliferation of Melanoma Cells and Induce Apoptosis by Activation of Caspase-3 in Vitro[END_REF]. The peaks at [535-526 cm -1 ] bands in fractions Sp CaCl2 and Sp HCl indicates the presence of the C-O-S secondary axial sulfate group at C-4 of fructopyranose residue, where the similar peak was also found in[START_REF] Palanisamy | Investigation of antioxidant and anticancer potential of fucoidan from Sargassum polycystum[END_REF].

Figure 41 .

 41 Figure 41. FT-IR spectra of S. ilicifolium extracts (Si CaCl2, Si HCl, Si Na2CO3, and Si KOH).

  HCl and Si CaCl2. As mentioned earlier in S. polycystum fractions,[START_REF] Kok | Physicochemical properties of edible alginate film from Malaysian Sargassum polycystum C. Agardh[END_REF] record the same observation decrease in intensity of absorption bands when the alginate was cross-linked with calcium. The bands at [1255-1249 cm -1 ] in fucose rich fractions Si HCl and Si CaCl2 were due to S=O stretching, which was also reported in the study of[START_REF] Artemisia | The Properties of Brown Marine Algae Sargassum turbinarioides and Sargassum ilicifolium Collected from Yogyakarta, Indonesia[END_REF] from Sargassum ilicifolium fractions extracted at different temperature (60-90 °C) conditions. The fractions Si HCl and Si CaCl2 in the present study were extracted at 70 °C. At [1032-1025 cm -1 ] in all

  et al.(2010), and (EC50=0.33-0.7 µg mL -1 )[START_REF] Thuy | Anti-HIV activity of fucoidans from three brown seaweed species[END_REF]. According toPuspita et al. (2017), the antiviral activity of brown algae is usually shown by polysaccharides, and some by phlorotannins. The total polyphenols content of S.

Chapter 5

 5 Red Seaweed (Halymenia durvillei and Halymenia dilatata)

H

  . dilatata were similar to casein, ovalbumin, and leguminous plants. The mineral contents of these seaweeds (green, brown, and red) in the present study are within safe levels and are potential functional foods for humans. The author acknowledged the intra-species variation of their biochemical composition for the green, brown, and red algae of this present work where they were within the values specified.

Table of contents

 of

	5.4 Cell wall polysaccharides purification
		5.4.1	First treatment
	Resume	5.4.2	Polysaccharide extraction	i
	Abstract	5.4.3	Biochemical composition analysis of extracts	ii
	Scientific contribution 5.4.4 Monosaccharide composition analysis	iii
	List of tables List of figures 5.4.5 List of appendices 5.5 Mineral analysis Fourier Transformed Infra-Red (FT-IR) spectroscopy List of abbreviations 5.6 Pigments 5.7 Biological activity	iv viii xi xiii
		5.7.1	Cytotoxicity and antiviral activity evaluation
		5.7.2	Antioxidant activity, DPPH radical scavenging activity
	6 Conclusion
	7 References
	8 Appendices

Algues marines d'importance économique aux Philippines : évaluation de six espèces Rexie Magdugo 2020

  Table (B).

	36 37 38 39 40 41 42 43 11 Appendix No. HPAEC-PAD elution program. Infra-red spectrum of the Ulvan DS sample on the 2000-500 cm-1 zone. Ulvan DS was also used as a standard for polysaccharide differenciation of U. lactuca in the present study. FT-IR spectra of C. racemosa extracts (Cr crude, 1OH, 4OH, and INS). FT-IR spectra of U. lactuca extracts (DS, DA, PP1, PP2, and PP3). FT-IR spectra of S. polycystum extracts (Sp CaCl2, Sp HCl, Sp Na2CO3, and Sp KOH). FT-IR spectra of S. ilicifolium extracts (Si CaCl2, Si HCl, Si Na2CO3, and Si KOH). Mean % values for C18, C20, and C22 PUFA red seaweed H. durvillei and H. dilatata in the present study; TFA= total fatty acid. Efficacy and potency evaluation of (A) S. polycystum extracts. BHT standards. FT-IR spectra of H. durvillei and H. dilatata polysaccharide Statistical Analysis LIST OF APPENDICES 17.1 CC50 and EC50 of C. racemosa polysaccharide fractions and 1 its zovirax control MEM culture medium 2 17.2 CC50 and EC50 of U. lactuca polysaccharide fractions and Cell maintenance 3 its zovirax control. Cell enumeration 4 17.3 CC50 and EC50 of S. polycystum polysaccharide fractions Organization diagram of a microplate for cytotoxicity and and its zovirax control viral tests 5 17.4 CC50 and EC50 of S. ilicifolium polysaccharide fractions The initial and final dry weights of green (C. racemosa and and its zovirax control U. lactuca), brown (S. polycystum and S. ilicifolium), and 17.5 CC50 and EC50 of H. durvillei and H. dilatata red (H. durvillei and H. dilatata) seaweeds used to calculate polysaccharide fractions and its zovirax control percent extraction efficiency yield after three solvents. 6 17.6 Antiradical scavenging activity (IC50) for C. racemosa The initial and final dry weights of green (C. racemosa and polysaccharide fractions (Cr crude, 1OH, 4OH, and INS) U. lactuca), brown (S. polycystum and S. ilicifolium), and with BHA and BHT standards red (H. durvillei and H. dilatata) seaweeds used to calculate 17.7 Antiradical scavenging activity (IC50) for U. lactuca the extract yield at each purification step. 7 polysaccharide fractions ((DS, DA, PP1, PP2, and PP3) with Efficacy and potency evaluation of (A) C. racemosa extracts its BHA and BHT standards. (Cr crude, 1OH, and INS) compared with (B) zovirax 17.8 Antiradical scavenging activity (IC50) for S. polycystum standard. 8 polycystum polysaccharide fractions (CaCl2, HCl, Na2CO3, Efficacy and potency evaluation of (A) U. lactuca extracts and KOH) with BHA and BHT standards (Cr crude, 1OH, and INS) compared with (B) zovirax 17.9 Antiradical scavenging activity (IC50) for S. ilicifolium standard. 9 polycystum polysaccharide fractions (CaCl2, HCl, Na2CO3, Free radical scavenging activity of (A) C. racemosa extracts and KOH) with BHA and BHT standards (Cr crude, 1OH, 4OH, and INS) compared with (B) BHA 17.10 Antiradical scavenging activity (IC50) for H. durvillei and BHT standards. 10 (Hdur), and H. dilatata (Hdil) polycystum polysaccharide Free radical scavenging activity of (A) U. lactuca extracts (DS, DA, PP1, PP2, and PP3) compared with (B) BHA and fractions with BHA and BHT standards	Page
		extracts (CaCl2, HCl, Na2CO3, and KOH) compared with
		(B) zovirax standard.
	12	Efficacy and potency evaluation of (A) S. ilicifolium extracts
		(CaCl2, HCl, Na2CO3, and KOH) compared with (B)
		zovirax standard.
	13	Free radical scavenging activity of (A) S. polycystum
		extracts (CaCl2, HCl, Na2CO3, and KOH) compared with
		(B) BHA and BHT standards.
	14	Free radical scavenging activity of (A) S. ilicifolium extracts
		(CaCl2, HCl, Na2CO3, and KOH) compared with (B) BHA
		and BHT standards.
	15	Efficacy and potency evaluation of (A) H. durvillei and H.
		dilatata extracts (Hdur and Hdil) compared with (B) zovirax
		standard.
	16	Free radical scavenging activity of (A) H. durvillei (Hdur),
		and H. dilatata (Hdil) extracts compared with (B) BHA and
		BHT standards.
			xi

GENERAL INTRODUCTION

Algues marines d'importance économique aux Philippines : évaluation de six espèces Rexie Magdugo 2020

Table I .

 I Phylogenetic summary of the most common macroalgae (Gomez-Zavaglia et al. 2019).

	Kingdom Phylum/Division Classes	Orders
	Chromista Ochrophyta	Phaeophyceae	Ascoseirales;
			Desmarestiales;
			Discosporangiales;
			Dictyotales
			Ectocarpales;
			Fucales;
			Laminariales;
			Nemodermatales
			Ralfsiales
	Charophyta	Charophyceae;
		Chlorokybophyceae;
		Coleochaetophyceae;
		Klebsormidiophyceae;
		Mesostigmatophyceae;	Bryopsidales;
		Zygnematophyceae.	Cladophorales;
	Plantae		Dasycladales;
	Chlorophyta	Ulvophyceae	Oltmannsiellopsidaes;
			Trentepohliales;
	Rhodophyta	Bangiophyceae;	Ulotrichales; Ulvales
		Compsopogonophyceae;
		Florideophyceae;
		Porphyridiophyceae;
		Rhodellophyceae;
		Stylonematophyceae.

Table II .

 II Composition of minerals (% dw), recommended daily intakes and recommended daily allowances from different seaweed groups

a= de

[START_REF] De Gaillande | Caulerpa consumption, nutritional value and farming in the Indo-Pacific region[END_REF] b= Arporn and Chirapart, 2014; c= Matanjun et al. 2009; d= Pise and Sabale. 2010; e= Thadlani et al. 2019; f= Garcia et al. 2016; g= Wanyonyi et al. 2017; h= MacArtain et al. 2007

; tr= trace amount. *= Recommended daily intake values for macroelements and microelements for an adult male with a body weight of 70 kg according to the World Health Organization (WHO 2002); **= Upper limit intake according to National

Health and Medical Research council (2006)

.

Table

  III showed that C. racemosa contains a higher amount of proteins (19.72±0.77%), compared to other green, brown, and red algae (Alam Bhuiyan and

	Qureshi 2016). The protein content of brown algae is generally low, usually ranging
	between 2.4-16.8% of dry weight compared to the green (3.2-35.2%) and red algae
	(6.4-37.6%)

Table III .

 III Proximate

	The total amino acid composition of several green, brown, and red seaweed
	species is shown in Table IV. Table IV showed a higher concentration of aspartic
	acid (9.2-13.68% dw), and glutamic acid (10.7-31.19% dw) was observed in C.
	racemosa, C. fastigiata, U. fasciata, Alaria sp., Laminaria sp., Saccharina sp., and
	Porphyra sp. Alanine (12.18-17.58% dw) was also dominant in Alaria sp.,
	Laminaria sp., Saccharina sp., Porphyra sp., and Arginine (35.57% dw) H. floresii.
		protein results (% dw) of different species of green, brown,
	and red seaweeds.
	Green seaweeds	Protein	Brown seaweeds	Protein	Red seaweeds	Protein Reference
	C. lentillifera 12.49	S. polycystum 5.4	H. durvillei 13	a, c, d
	C. racemosa	0.6-18.3 S. pallidum	0.1-1.2 H. dilatata 10	b, d, g
	C. racemosa	19.72	S. filipendula 8.72	H. maculata 11	a, d
	U. rigida	6.40	S. vulgare	15.76	G. cornea	5.47	a
	U. lactuca	7.06	S. polycystum 4.7	G. changii	6.9	a, e
	U. pertusa	15.4	S. fusiforme	1.01	G. changii	12.57	a, f, h
	U. intestinalis 17.9					a
	a= Alam Bhuiyan and Qureshi, 2016; b= de Gaillande et al. 2017; c= Matanjun et al. 2009; d= Kho et al. 2016;
	e= Palanisamy et al. 2018 (crude fucoidan); f= Ye et al. 2018 (polysaccharide); g= Li et al. 2018 (purified
	polysaccharides by DEAE-sepharose fast flow); h= Chan and Matanjun, 2018.

Table IV .

 IV Total amino acid composition of several species of green, brown, and red seaweeds.

		Essential amino acids (EAA)	Nonessential amino acids (NEAA)
		THR VAL LYS ILE LEU PHE HIS MET ASP SER GLU GLY ARG ALA TYR PRO
	C. racemosa a	0.51 0.57 0.33 0.43 0.69 0.64 0.11 0.17 0.94 0.45 1.13 0.61 0.49 0.49 0.44 0.57
	C. racemosa b

  Hamed et al. also recorded that His content in U. rigida, an essential amino acid for infants, is at levels close to those in legumes and eggs. Algal polysaccharides have numerous human health benefits that can be easily isolated and are ingredients in functional foods.Gaillard et al.

	(2018) reported a rich concentration of aspartic and glutamic acids, and low
	methionine and histidine in all seaweeds tested. Compared with soybean, most of
	the seaweed species were deficient with EAA except for sulfur amino acids (Met,
	and Cys). Proline contents were low (1.68-4.78 g/16gN), and is known to
	accumulate in large quantities in response to environmental stressors, as a nontoxic
	and protective osmolyte under saline conditions (Gaillard et al. 2018). Gaillard et
	al. (2018) also reported EAA in different proportions (24-42% TAA) with

Table V .

 V Major storage carbohydrate and cell wall polymers present in C. racemosa and U. fasciata[START_REF] Lahaye | Structure and functional properties of ulvan, a polysaccharide from green seaweeds[END_REF][START_REF] Stengel | Algal chemodiversity and bioactivity: Sources of natural variability and implications for commercial application[END_REF]

	Polysaccharide	C. racemosa	U. fasciata
	Fibrillar	β-(1,4)-D-mannans	Cellulose
	polysaccharides of the	β-(1,3)-D-xylans
	crystalline phase
	Hemicelluloses	Xyloglucan, Mannans, Glucuronan, β-(1,3)-glucans
	Carboxylic	Sulfated	Ulvans
	polysaccharides of the	glucuronoxylorhamnans
	matrix phase
	Sulfated	Sulfated	Ulvans, Sulfated
	polysaccharides of the	xyloarabinogalactans	xylorhamnoglucuronans
	matrix phase
	Storage carbohydrates Starch

Table VI .

 VI Primary storage carbohydrate and cell wall polymers present in Sargassum species (Deniaud-Bouet et al. 2014).

Polysaccharide S. polycystum S. ilicifolium Fibrillar polysaccharides of the crystalline phase Cellulose (mostly xylose) Presence of hlorotannins Hemicelluloses Hexose, pentose, and uronic acids polysaccharides of the matrix phase Sulfated rich fucose

	Polysaccharides of the	rich alginate (~40%)
	matrix phase	mostly associated to
		phenols

Table VII .

 VII Content of total polysaccharides in some Sargassum spp. (% dw) a=[START_REF] Li | Fractionation, preliminary structural characterization and bioactivities of polysaccharides from Sargassum pallidum[END_REF] b= Hidayati et al. 2019;[START_REF] Puspita | Total phenolic content and biological activities of enzymatic extracts from Sargassum muticum (Yendo) Fensholt[END_REF] d= Ye et al. 2018; and e= Sinha et al. 2010; f= Palanisamy et al. 2018; and g= Artemisia et al. 2019.

	Genera	(% dw) Total polysaccharides	Extraction	References
	S. pallidum	21.3, 32.4, 5.3, 8.9	DEAE-sepharose	A
	Sargassum sp. 8.5	Aquadest	B
	S. polycystum 8.3	Methanolic	C
	S. ilicifolium	3.1	Methanolic	C
	S. aquifolium 5.7	Methanolic	C
	S. fusiforme	6.59	Hot water	D
	S. tenerrimum 33.0	0.1 M HCl; K2CO3	E
	S. polycystum 22.3, 16.9, 19.4, 5.3	DEAE-cellulose	F
	S. ilicifolium	2.89-3.48	Hot water	G

Table VIII .

 VIII Primary storage carbohydrate and cell wall polymers present in H. durvillei and H. dilatata[START_REF] Morán-Santibañez | Synergistic Effects of Sulfated Polysaccharides from Mexican Seaweeds against Measles Virus[END_REF][START_REF] Fenoradosoa | Highly sulphated galactan from Halymenia durvillei (Halymeniales, Rhodophyta), a red seaweed of Madagascar marine coasts[END_REF]

	2009).
	Polysaccharide	H. durvillei	H. dilatata
	Fibrillar polysaccharides Cellulose, β-(1,4)-D-mannans, β-(1,3)-D-xylans
	Hemicelluloses	Glucomannan, Mixed Linkage Glucan sulfates; β-
		(1,3), (1,4)-D-xylans
	polysaccharides of the	Kappa-carrageenan,
	matrix phase	Lambda-
		carrageenan
	Storage carbohydrates	Floridean starch
	The repeating units may be modified in several ways; for example, the
	repeating carrabiose structure of carrageenans may be altered by replacing the 3,6-
	anhydro-D-galactose with D-galactopyranose residues. The glycosyl units may be
	substituted by sulfate hemiesters in various patterns, or more rarely by methyl
	ethers, or by the 4,6-O-carboxyethylidene group of pyruvic acid. Similarly, the
	agars' agarobiose repeating structure may be masked by replacing 3,6-anhydro-L-
	galactose with L-galactose or adding methyl ethers, sulfate hemiesters, and,
	exceptionally, pyruvic acid ketal at specific sites on either glycosyl unit (Stiger-
	Pouvreau et al. 2016).

Table IX .

 IX Total lipid (TL) content, polyunsaturated and saturated fatty acids (PUFA/SFA) ratio, and distribution in some brown and red seaweed extracts, including Sargassum sp. and Halymenia sp.

	Algae	TL (% of dw)	PUFA/SFA	ω6/ω3	References
	U. pinnatifida	5	-	-	a
	S. horneri	0.82-0.96	-	-	b
	Alaria esculenta	1.3-1.5	-	-	c
	L. digitata	0.8-1.1	-	-	c
	L. hyperbora	1.1-1.4	-	-	c
	F. vesiculosus	2.6-3.5	-	-	c
	Pelvetia canaliculata	3.7-5.8	-	-	c
	A. nodosum	4.5	-	-	c
	F. serratus	3.7	-	-	d
	S. natans Acanthophora total lipid content, brown algae are a rich source of polyunsaturated fatty acids 1.0 --d ). Despite their low 6.8-10.4 mg g -1 fw 0.79-0.94 0.9-1.8 e Bortryocladia 2.3-5.2 mg g -1 fw 0.49-0.54 1.7-3.6 e
	(PUFAs) (van Ginneken et al. 2011). In red seaweed, lipids and fatty acids are Gracilaria 2.9-9.7 mg g -1 fw 0.15-2.13 0.6-1.9 e
	Grateloupia present in low amounts, generally 1-5% of the dry weight (Torres et al. 2019). The 5.0-6.4, 13.6 dw 0.74-1.4 0.5-1.9 e; f Halymenia 10-18.8 dw 1.37-1.8 1.7-5 e; f
	mean total lipid content of Halymeniales species can vary (0.1-3.9% dw). Kumari Porphyra 11.2-12.4 dw 2.4-2.5 1.2-9.1 f; g
	Palmaria et al. 2013 compiled the total lipid content and fatty acid distribution of different 14-46 dw 0.49-1.1 0.21-0.41 h; i a= Billakanti et al. 2013; b= Hossain et al. 2003; c= Maehre et al. 2014; d= van Ginneken et al.
	2011; e= Kumari et al. 2013; f= Chen and Chou, 2002; g= Patra et al. 2017; h= Kumari et al. 2010; seaweeds and suggested that the variations observed between different species of i= Maehre et al. 2014.
	the same genus were more likely to be due to the inter-specific/intra-generic
	variations rather than to geographical and environmental conditions as apparent
	from the minor variations found with the environmental parameters for the studied
	collection sites.
	Table X shows the total lipid content in several species of brown and red
	seaweeds, including Sargassum and Halymenia species. Macroalgae also contain
	various other lipids and lipids like compounds such as sterols, phospholipids, and
	glycolipids, but red seaweeds have a high ω-3 fatty acids content, being a rich
	source of α-linolenic acid (ALA) [18:3(n-3)], AA, eicosapentaenoic acid (EPA)
	[20:5(n-3)], and docosahexaenoic acid (DHA) [22:6(n-3)]), and most species
	showed a nutritionally beneficial ω6/ω3 ratio (Torres et al. 2019) (Table IX).

Table X .

 X Nutrient composition of different edible green, brown, and red seaweed species (% dw).

	Species	Protein	Lipid	Carbohy drate	Ash	Moisture	Reference
	Caulerpa racemosa 19.72	7.65	48.97	12.15	15.37	a
	C. lentillifera	12.49	0.86	59.27	24.21	25.31	a
	C. cupressoides	20.8	3.8	47.4	11.3	nd	b
	C. racemosa	0.6-18.3 0.1-3.8 3.6-83.2 14.5-55.1 88.8-91.5	b
	C. sertularioides	3.1; 11.96 2.3; 0.92 nd	2.9; 35.54 82.4, 88.2 ww b, d
	Codium fragili	8-11	0.5-1.5	39-67	21-39	91.4	c
	Ulva rigida	6.40	0.30	18.10	52.00	-	a
	U. reticulata	21.06	0.75	55.77	17.58	22.51	a
	U. lactuca	7.06	1.64	14.60	55.40	10.60	a
	U. pertusa	15.4	4.8	-	27.2	6.0	a
	U. intestinalis	17.9	8.0	-	27.6	6.3	a
	U. expansa	4.12	0.65	-	35.66	84.59 ww	d
	Fucus vesiculosus	3-14	1.9	46.8	14-30	-	c
	L. digitata	5-9	0.9-2.1	42-62	19-36	-	e
	Saccharina japonica	7.5	1.0	51.9	26.63	-	c
	Sargassum fusiforme	11.6	1.4	30.6	19.77	-	c
	Pyropia tenera	33-47	0.7	44.3	20.5	-	c
	P. umbilicalis	29-39	0.3	43	12	-	c
	P. yezoensis	31-44	2.1	44.4	7.8	-	c
	H. durvillei	13.02	1.29	53.65	39.54	12.4	f
	H. dilatata	9.99	0.29	40.53	21.92	8.85	f
	K. alvarezii	1.34	0.62	38.3	58	-	g

dw= dry weight; ww= wet weight of seaweeds; -= no data; nd= not determined. a= Alam Bhuiyan and Qureshi 2016; b= de

[START_REF] De Gaillande | Caulerpa consumption, nutritional value and farming in the Indo-Pacific region[END_REF] c= Pereira et al. 2016; d= Osuna-Ruiz et al. 2019; e= Fleurence, 2016; f= Kho et al. 2016; g= Wanyonyi et al. 2017

.

5.2 Algae as a source of phycocolloid

Table XII .

 XII List of seaweed samples, location, date of collection, and mean water quality of the sampling area based on in-situ measurements.

		S. ilicifolium S. polycystum	C. recemosa
		H. durvillei		U. fasciata
				H. dilatata
			Place Collected
		Currimao	Anda, Bohol	Lawigan, Bislig
	Water Quality	(Luzon)	(Visayas)	(Mindanao)
	Salinity (‰)	34	36	34
	Temperature (°C)	29	28	27
	pH	7.0	8.0	8.16
	Dissolved oxygen (DO/ mg L -1 )	8.5	8.0	6.5
			Date and Species Collected
		July 2017	22 August 2017 15-16 August 2017

Table XIII .

 XIII Summary table of samples processed for each analysis in the present study.

			Biochemical	Sugar units		Anti-	Anti-	Fatty		Amino	Location and
	Species	Extract	composition	(HPAEC)	FTIR Pigments	viral	oxidant Lipid	acids	Minerals	Acid	Date collected
			raw extract raw extract	HPLC	HSV-1 DPPH	GC-FID	AAS	GC-FID
	C. recemosa

  after acid hydrolysis of the samples with 6 M HCl 110 °C for 24 h, for microcharacterization of proteins. While this method gives good results for a majority of amino acids, there are several amino acids that are either partially or completely destroyed by such method. While 6 M HCl acid hydrolysis is the most

common precedure, there are several limitations to this method. ASN and GLN are deamidated to ASP and GLU, and thus are quantitated as a mixture. Peptide bonds of of hydrophobic amino acids (VAL, ILE, LEU) may be difficult to break and require additional hydrolysis time (upto 72 hours). Residual oxygen in the hydrolysis vessel can increase the thermal breakdown of hydrogen and sulfur containing amino acids (typical recoveries for SER, THR, HYP, and TYR range between 50-90%, MET ranges from 25-75%). Reducing hydrolysis time improves recoveries but reduces other amino acid yields (see above). GLY yields tend to exceed 100% (especially for low level samples) due to background protein contamination. Finally, both TRP and CYS are completely destroyed by acid hydrolysis and must be analyzed by alternate methods. The above listed limitations are based on hydrolysis chemistry and are not related at all to the EZ: faast process.

Table XIV .

 XIV Standard sugar mixtures used for calibration curves. Chromatogram showing the retention time of the different standards of mixture 2 (A) and its peak summary Table (B).

	Serial dilution was then made according to the determined concentration
	ranged. Monosaccharides have been identified and quantified using calibrations of
	standards. The standard solutions were prepared in ultrapure water and filtered at
	0.22 μm. Fig. 35 shows an example of a chromatogram of Mixture 2 standards, used
	at a given concentration (125 ppm), and its peak summary for each standard
	monosaccharide.
	No. Ret.Time Peak Name		Height	Area	Rel.Area Amount	Type
	ED_1 ED_1	ED_1			ED_1	ED_1	ED_1	ED_1	ED_1
	min					nC	nC*min	%
	1 1.76	Mixture 1 n.a.	(μL)		7.48	Mixture 2 2.26 0.26	(μL) n.a.	BMB
	1 2 6.02 3 10.80 Deoxyribose Desoxyribose Arabinose	75		178.15 369.82	Deoxyribose 38.35 4.38 171.28 19.56 124.822 50.00 75	BMB BMB
	2 4 15.02 Glucoheptose Glucose	187.5	335.03	Arabinose 195.12 22.29 124.784 187.5	BM
	3 5 16.65 Rhamnose Xylose	187.5	332.37	Glucose 227.04	187.5 25.93 124.762	Mb*
	4 6 21.58 Glucosamine Ribose 7 24.64 n.a.	187.5	178.31 591.00	Xylose 163.67 24.13	18.69 124.801 187.5 2.76 n.a.	bMb* bM *
	5 8 25.93 Galactose Glucuronic acid 187.5	573.24	Ribose 48.66	187.5 5.56 125.177	M *
	6 9 26.28 Mannose n.a.	187.5	29.28	Glucuronic acid 2.15 0.25	187.5 n.a.	MB*
	7 10 33.67 Fructose n.a.	187.5	6.62	Pure water 2.86 0.33	487.5 n.a.	BMB
	+ Total: Total Figure 35. Pure water	300 1500	2601.35 875.576	100.00 624.346 1500

Table XVI .

 XVI Extraction yield and purification of polysaccharides from Philippine green seaweeds Caulerpa racemosa and Ulva lactuca.

	Species EXTRACTION	Solvents	Yields (%) Yields (% dw)
	C. racemosa C. racemosa	Ethanol	89.92
	Depigmented	Chloroform/Methanol	89.92
	HWE	Acetone	13.0
	U. lactuca 1OH 4OH	Ethanol	89.65	6.5 8.0
	INS	Chloroform/Methanol	28.0
	U. lactuca	Acetone
	Depigmented		89.65
	Successive Precipitation
	Ulvan PP1		63.41
	Ulvan PP2		28.51
	Ulvan PP3		4.55
	Dialysis
	Ulvan DS		91.22
	Ulvan DA		8.35

cell wall polysaccharide was extracted from Depigmented Algal Powder (DAP). In C. racemosa, the soluble Hot Water Extracted fraction (designated HWE) and then successively fractions (1-and 4M-KOH) extracted with strong alkali to isolate hemicellulosic polysaccharides, and finally, the insoluble fraction (INS) were prepared. In U. lactuca, crude ulvan was extracted separately in two groups: successive precipitation fractions (PP1, PP2, and PP3), and by dialysis fractions (DS and DA). The yields of the different extracted fractions are given in Table XVI. Initial and final weights of raw C. racemosa and U. lactuca that were used to calculate extraction yield in percent at each purification step were shown in appendix 6. Overall yield= expressed as a percentage of total dry algae at the start. Purification yield= expressed as a percentage of the dry mass used at each stage. From HWE (see Magdugo et al. 2020), we had obtained three more sequential fractions; (1OH=6.5%, 4OH=8.0%, and INS=28% dw) (Table

Table XVII .

 XVII Biochemical composition of polysaccharide extracts from Philippine green seaweeds Caulerpa racemosa and Ulva lactuca.

	The purification of U. lactuca raw Ulvan, had five Ulvan fractions mainly for its raw materials and each fraction were; raw=87.55±7.47, Cr
	composed of sulfates and proteins in successive precipitation fractions (PP1, PP2, crude=11.68±0.06, 1OH=201.50±0.0, 4OH=33.93±0.0, and INS=67.49±0.0 (Table
	and PP3), while proteins, sulfates, and uronic acids in dialyzed fractions (aggregates XVIII).
	biochemical composition of C. racemosa (HWE, 1OH, 4OH, and INS) DA, and soluble DS). Fraction PP1 (yield, 63.41%) had sulfates (15.79%), proteins
	and U. lactuca (PP1, PP2, PP3, DS, and DA) extracts are shown in Table XVII. (13.5%) contents, followed by small amounts of uronic acids, neutral sugars, and
	The composition of isolated fractions (1OH, 4OH, and INS) revealed that polyphenols. Fraction PP2 (yield, 28.51%) had proteins (19.6%), followed by
	1OH fraction is rich in neutral sugars (16.2%) followed by sulfates (10.58%), and sulfates (16.45%), neutral sugars (12.8%), small amounts of uronic acids, and
	proteins (7.8%); with small amounts of polyphenols and uronic acids; 4OH fraction polyphenols. Fraction PP3 (yield, 4.55%) had sulfate (20.03%) followed by neutral
	is composed of proteins (11.0%) and sulfates (8.19%), with small amounts of sugars (7.2%), proteins (7.1%), and small amounts of polyphenols and uronic
	polyphenols, neutral sugars, and uronic acids. Insoluble polysaccharide (INS) is acids-Dialyzed fractions with yield values of 91.22 and 8.35% for Ulvan DS and
	abundant in sulfates (10.21%), followed by small amounts of neutral sugars, Ulvan DA, respectively. Fraction DS is rich in proteins (18.3%) and sulfates
	proteins, polyphenols, and uronic acids. (16.0%) followed by uronic acids (9.5%), neutral sugars, and a small amount of
	polyphenols. Fraction DA is rich in proteins (18.9%) followed by uronic acids,
	neutral sugars, sulfates, and a small amount of polyphenols. The amount of proteins
	(7.11-19.57%), neutral sugars (5.0-12.8%), sulfates (5.44-20.03%), and uronic
	acids (2.2-9.5%) from the present study are quite small or different from the thesis
	results of Hardouin, (2015, http://www.theses.fr/2015LORIS373#) using similar
	methods of extraction especially fractions PP1 (22.3%), and DA (41.7%) in
	proteins; all fractions in neutral sugars; fraction DS (31.8%) in sulfates; and
	fractions PP2 (18.1%), PP3 (13.9%), and DS (25.3%) in uronic acids. He stated in
	ALGAE his results that EC50 activity is not only dictated on Ulvans or polysaccharides alone Content (% dry algal material)
	Yield but also of its protein contents, which he also observed at fractions PP1 (EC50<400.0 Neutral Sulfates Uronic
	(%) Proteins ug mL -1 ), and DA (EC50=68.6 ug mL -1 ). Sulfates content in the present study is sugars group acids Polyphenol
	C. racemosa HWE within the values published in the literature (Costa et al. 2012; Hernández-Garibay 13.0 19.9±0.5 31.2±4.9 13.0±1.5 3.6±0.4 4.9±1.6
	1OH et al. 2011) and somewhat similar to the results of Hardouin et al. (2016) except for 6.5 7.8±1.7 16.2±1.4 10.58±0.7 0.5±0.2 3.0±0.7
	4OH INS fraction DA. Proteins content in the present study are way higher (up to 20 times) 8.0 11.0±0.4 1.2±0.1 8.19±0.1 0.1±0.0 3.8±0.0 28.0 3.7±0.5 4.0±0.1 10.21±0.1 0.9±0.4 2.1±0.9
	U. lactuca than Costa et al. (2012), up to 2 times than Hernández-Garibay et al. (2011), and
	Ulvan PP1 up to 2 to 3 times higher than Hardouin et al. (2016) except for fraction PP3 which 63.41 13.5±2.4 5.0±0.2 15.79±0.3 6.2±0.3 2.3±0.8
	Ulvan PP2 Ulvan PP3 is almost similar to his results. The uronic acid content in the present study except 28.5 19.6±1.5 12.8±0.8 16.45±0.4 7.0±1.4 2.5±0.9 4.5 7.1±1.0 7.2±0.9 20.03±0.4 2.2±0.1 2.8±0.5
	Ulvan DS	91.2 18.3±2.6 6.7±0.7	16.0±0.2	9.5±0.2 3.2±1.4
	Ulvan DA	8.4	18.9±0.8 6.1±0.2	5.44±0.7	7.0±0.3 1.7±0.9

Another study from C. racemosa var peltata fraction WEC (Water Extracted Caulerpa) extracted by hot water at 85 °C for 3 h reported 0.67% protein, 0.19% polyphenols, and 3.4% total sugar

[START_REF] Hao | Chemical composition and immunostimulatory properties of green alga Caulerpa racemosa var Peltata[END_REF]

, which are quite small compared to the present study. In general, total sugars were abundant components compared to proteins, phenols, and flavonoids

[START_REF] Hao | Chemical composition and immunostimulatory properties of green alga Caulerpa racemosa var Peltata[END_REF]

.

for fraction PP3 are slightly lower or just comparable to

[START_REF] Hernández-Garibay | Isolation and chemical characterization of algal polysaccharides from the green seaweed Ulva clathrata (Roth) C. Agardh[END_REF]

, and up to 2 to 3 times lower compared to

[START_REF] Hardouin | Enzyme-assisted extraction (EAE) for the production of antiviral and antioxidant extracts from the green seaweed Ulva armoricana (Ulvales, Ulvophyceae)[END_REF]

. Neutral

Table XVIII .

 XVIII Unit sugar composition of raw and extracted fractions from the Philippines green seaweed C. racemosa.

				C. racemosa
	Monosaccharide	Raw	Cr crude 1OH	4OH	INS
				(% of total sugars)
	Mannitol	-	-	0.12±0.00	0.24±0.00 0.50±0.00
	Fucose	1.49±0.06 2.12±0.00 1.09±0.00	0.17±0.01 0.12±0.08
	Rhamnose	0.37±0.01 1.07±0.00 0.43±0.00	0.09±0.00 2.19±0.01
	Arabinose	0.22±0.03 0.68±0.00 0.62±0.00	0.09±0.00 0.09±0.02
	Glucosamine	0.46±0.04 2.08±0.00 0.36±0.00	0.09±0.00 0.09±0.03
	Galactose	4.98±0.34 18.40±0.11 8.16±0.21	1.39±0.00 0.99±0.00
	Glucose	7.88±0.52 54.80±0.15 50.41±0.81 6.89±0.21 6.01±0.14
	Mannose	2.41±0.32 1.88±0.01 7.73±0.21	1.15±0.04 0.68±0.08
	Xylose	10.03±0.94 3.38±0.01 4.47±0.16	0.56±0.02 0.44±0.00
	Fructose	0.83±0.20	-	2.66±0.00	0.90±0.34 0.56±0.09
	Ribose	1.41±0.02	-	-	-	-
	Glucuronic acid	0.23±0.00 3.90±0.00 0.87±0.05	-	0.65±0.04
	Others	4.81±0.40	-	3.66±0.12	1.99±0.08 14.65±0.10
	Total (

µg mg -1 ) dw 87.55±7.47 11.68±0.06 201.50±0.12 33.93±0.05 67.49±0.05

  -= not detected; The values are expressed in g 100g -1 of total sugars in the fractions.

Table XIX .

 XIX Unit sugar composition of raw and extracted fractions from the Philippines green seaweed U. lactuca.

				U. lactuca
	Monosaccharide Raw	DS	DA	PP1	PP2	PP3
				(% of total sugars)
	Mannitol	-	-	-	-	-	0.63±0.05
	Fucose	-	0.23±0.09	0.29±0.13	0.20±0.09 0.20±0.00	-
	Rhamnose	5.02±0.20 5.13±3.11	3.56±2.03	5.87±1.28 6.44±0.75 0.54±0.08
	Arabinose	-	0.01±0.00	0.01±0.00	0.01±0.00 0.03±0.00 0.02±0.00
	Glucosamine	-	0.01±0.01	0.07±0.04	0.02±0.01 0.02±0.00 0.04±0.01
	Galactose	0.29±0.01 0.43±0.16	0.49±0.21	0.18±0.00 0.29±0.02 0.64±0.08
	Glucose	6.52±0.16 0.99±0.27	6.40±2.21	3.73±0.96 12.42±1.02 2.55±0.32
	Mannose	0.13±0.01 0.15±0.07	0.40±0.19	0.15±0.06 0.19±0.00 0.19±0.02
	Xylose	1.06±0.02 0.15±0.13	0.18±0.16	0.20±0.16 0.34±0.00 0.06±0.02
	Fructose	0.03±0.00 0.27±0.00	0.51±0.01	0.15±0.00	-	0.29±0.19
	Ribose	0.04±0.00 0.16±0.00	-	-	-	-
	Glucuronic acid	1.26±0.05 0.17±0.08	0.32±0.12	0.43±0.00 0.43±0.06 0.08±0.03
	Others	2.42±0.10 3.27±0.44	2.94±0.66	3.16±0.63 4.10±0.92 13.02±0.62
	Total (

µg mg -1 ) dw 41.83±1.45 26.91±11.43 37.94±14.40 35.03±8.21 60.62±7.46 45.19±3.59 -

  = not detected; The values are expressed in g 100g -1 of total sugars in the fractions.

Table XX .

 XX List

	Region/Band (cm -1 )	Type of bond	Identification
	Common to Caulerpa species
	1780-1670	C=O	Carboxyl group
	1650, 1400	C=O-OH	Carbonyl of a carboxylic acid group
	1549	-NH	Amide N-H bending vibrations
	1420	C-N	C-N stretching
	1248	-SO3	-SO3 stretching
	1200-1040	S=O	Sulfonyl
	1033	-C-O	-C-O benzene ring stretching
	854	S=O; C-O-S S=O; C-O-S bands from ester sulfonate, 3,6-
			anhydrogalactose and the sulfation of C-4, C-6 of
			galactose units and C-2 of 3,6-anhydrogalactose
	791.72	Si-C	Presence of siliceous group

of InfraRed (IR) vibrational modes characteristic to polysaccharides used in determining spectral bands of interest for the identification and discrimination of green algal polysaccharides from C. racemosa (adapted from

[START_REF] Ghosh | In vitro anti-herpetic activity of sulfated polysaccharide fractions from Caulerpa racemosa[END_REF] Marungrueng and Pavasant, 2007;[START_REF] Robic | Determination of the chemical composition of ulvan, a cell wall polysaccharide from Ulva spp. (Ulvales, Chlorophyta) by FT-IR and chemometrics[END_REF][START_REF] Gómez-Ordóñez | FTIR-ATR spectroscopy as a tool for polysaccharide identification in edible brown and red seaweeds[END_REF][START_REF] Sarada | Cadmium removal by macro algae Caulerpa fastigiata: Characterization, kinetic, isotherm and thermodynamic studies[END_REF][START_REF] Fernando | FTIR characterization and antioxidant activity of water-soluble crude polysaccharides of Sri Lankan marine algae[END_REF][START_REF] Barbosa | In Vitro Immunostimulating Activity of Sulfated Polysaccharides from Caulerpa cupressoides Var. Flabellata[END_REF][START_REF] Figueira | Structural Characterization of Ulvan Polysaccharide from Cultivated and Collected Ulva fasciata (Chlorophyta)[END_REF]

Table XXI .

 XXI List of InfraReds (IR) vibrational modes characteristic to polysaccharides used in determining spectral bands of interest for the identification and discrimination of green algal polysaccharides

	As shown in the previous part of this chapter (3.1.3. Biochemical anhydrogalactose. Moreover, no direct correlation found in the small amounts of
	composition of extracts), the extracts obtained have biochemical contents that differ rhamnose and xylose with antiviral activities in each C. racemosa fraction. Several
	with its raw material (see Magdugo et al. 2020). Aside from sulfates, factors could influence the variation of antiherpetic activity results between the
	monosaccharides composition (Wang et al. 2014), the amount of proteins present and previous studies such as; monosaccharide composition, sulfate content,
	(Hardouin et al. 2016), and its AA could impact the antiviral activity reported in polysaccharides chemical structure and its molecular weight, location, and period
	the extracts of C. racemosa and U. lactuca. of collection, and the sensitiveness of methods used. The Caulerpa racemosa
	After three days of treatment, microscopically visible alteration of normal extracts results showed great potential in developing as an antiviral drug. However,
	cell morphology was observed, and viability assay showed destruction of the cell these various factors mentioned must be understood better, and therefore extracts
	layer. No cytotoxic effect of polysaccharides (CC50 greater than 200 μg mL -1 ) on from Caulerpa racemosa must be studied further.
	Region/Band (cm -1 )	Type of bond	Identification
	Common to Ulva species
	1650	COOH The asymmetrical stretch of a carboxyl group
	1618, 1608	O-H	Presence of OH groups within uronic acid bands
	1400	COOH The symmetrical stretch of a carboxyl group
	1235, 1225, 1260	S-O	Elongation of S=O (sulfate esters)
	1200-1000		Sugar ring vibrations overlap with stretch vibrations of
			(C-OH) side groups and (C-O-C) glycosidic bonds
	1055	C-O	Likely due to C-O stretching from two main sugars
			(rhamnose and glucuronic acid)
	975 (980)		Glycosidic linkages
	933-928		3,6-anhydro-D-galactose
	847-853 (845)	C-O-S	C-O-S elongation in axial position (sugar cycles)
	(789-770), 785, 748 C-O-S	C-O-S deformation in equatorial position (sugar cycles)
	1 strains (EC50=2.2-4.2 µg mL -1 ). The HWE fraction from Ghosh et al. (2004) was
	considered a selective inhibitor of thymidine kinase (TK) acyclovir-resistant HSV-
	1 strains in Vero cells. Wang et al. (2014) reported antiviral activities against HSV-
	3.2 Biological activity 1 of sulfated polysaccharides from Caulerpa brachypus (EC50=9.6 µg mL -1 ) having
	a direct correlation with the degree of sulfation (0.5), and sugar units found
	3.2.1 (rhamnose, xylose, and glucose). In the present study, major sugar units (glucose, Cytotoxicity and antiviral activity evaluation

from U. lactuca (adapted from

[START_REF] Robic | Determination of the chemical composition of ulvan, a cell wall polysaccharide from Ulva spp. (Ulvales, Chlorophyta) by FT-IR and chemometrics[END_REF] Suresh et al. 2012;[START_REF] Pezoa-Conte | Deconstruction of the green alga Ulva rigida in ionic liquids: Closing the mass balance[END_REF][START_REF] Fernando | FTIR characterization and antioxidant activity of water-soluble crude polysaccharides of Sri Lankan marine algae[END_REF][START_REF] Vinosha | Biogenic synthesis of gold nanoparticles from Halymenia dilatata for pharmaceutical applications: Antioxidant, anticancer and antibacterial activities[END_REF][START_REF] Olasehinde | Chemical characterization of sulfated polysaccharides from Gracilaria gracilis and Ulva lactuca and their radical scavenging, metal chelating, and cholinesterase inhibitory activities[END_REF][START_REF] Figueira | Structural Characterization of Ulvan Polysaccharide from Cultivated and Collected Ulva fasciata (Chlorophyta)[END_REF]

the Vero cells was observed for both C. racemosa, and U. lactuca extracts in ranged of concentrations assayed. All fractions had low EC50 values except fractions PP1 and PP3 in U. lactuca. Fraction 4OH from C. racemosa was insoluble and thus, cannot be tested (Table

XXII

). Efficacy or the cellular protection of C. racemosa fractions showed that fraction Cr crude had maximum efficacy of 88.20%, is higher than 1OH (82.36%), and is higher than INS (63.85%) all at 200 µg mL -1 after 72 h infection. The potency or the effectivity of the Cr crude at 50% concentration, only needs 25.33 µg mL -1 of sample (EC50: 25.33 µg mL -1 ), which is slightly higher than 1OH (EC50: 32.15 µg mL -1 ), and four times potent than INS (EC50: 112.26 µg mL - 1 ). Efficacy and potency evaluation are shown in appendix 7 and 8, while its statistical analysis is shown in appendix 17.1 and 17.2 for C. racemosa and U. lactuca fractions, respectively. These antiviral activities mentioned here in the present study for C. racemosa extracts, although active, yet appear to be less potent compared to

[START_REF] Ghosh | In vitro anti-herpetic activity of sulfated polysaccharide fractions from Caulerpa racemosa[END_REF]

on HWE extract from C. racemosa against HSVgalactose, and mannose) were found highest to lowest in this trend: fraction Cr crude> 1OH>INS. Also, FTIR confirms the presence of sulfate groups and the 3,6-

Table XXII .

 XXII Evaluation of cytotoxicity and antiviral activity by cellular viability of Philippine green seaweeds Caulerpa racemosa and Ulva lactuca.

			CC50 (µg mL -1 )	EC50 (µg mL -1 )
	SPECIES	FRACTION	Destruction	Protection
	C. racemosa	Cr crude	>200	25.33±5.13
		1OH	>200	32.15±20.26
		INS	>200	112.26±43.45
	Zovirax		>200	0.47±0.07
	U. lactuca	DS	>200	136.82±15.54
		DA	>200	88.32±12.36
		PP1	>200	>200
		PP2	>200	122.13±14.9
		PP3	>200	>200
	Zovirax		>200	0.37±0.01
	CC50: The 50% Cytotoxic Concentration reduced the absorbance of mock-infected cells to 50% of
	that of controls. EC50: The 50% antiviral Effective Concentration: concentration that achieved 50%
	protection of virus-infected cells from the HSV-induced destruction. Fraction Cr crude, 1OH, and
	INS are polysaccharides that were successively extracted from HWE insoluble residue. Fraction DS
	and DA are dialyzed polysaccharides, while fraction PP1, PP2, and PP3 are polysaccharides that
	were successively extracted from crude Ulvan of U. lactuca.

  [START_REF] Kidgell | Ulvan: A systematic review of extraction, composition and function[END_REF] reported that the antiviral activity of Ulvan extends to different enveloped viruses of HPV, Newcastle disease, Japanese encephalitis,

	West Nile, dengue, yellow fever, influenza, and Measles Virus. In the present study,
	the antiviral activities for U. lactuca extracts had range values of EC50 from 88.32
	to 136.82 µg mL -1 ) According to the methodology used to evaluate the antiviral
	effects of ulvans against viral targets, the concentration of ulvan required to inhibit
	viral yield by 50% are mixed ranging from weak (EC50 >150 µg mL -1 ) (Jiao et al.

2012;

[START_REF] Lopes | Green seaweed Enteromorpha compressa (Chlorophyta, Ulvaceae) derived sulphated polysaccharides inhibit Herpes simplex virus[END_REF]

to significant (EC50 =0.1-30 µg mL -1 )

[START_REF] Chiu | Inhibition of Japanese Encephalitis Virus Infection by the Sulfated Polysaccharide Extracts from Ulva lactuca[END_REF][START_REF] Aguilar-Briseño | Sulphated Polysaccharides from Ulva clathrata and Cladosiphon okamuranus Seaweeds both Inhibit Viral Attachment/Entry and Cell-Cell Fusion, in NDV Infection[END_REF][START_REF] Morán-Santibañez | Synergistic Effects of Sulfated Polysaccharides from Mexican Seaweeds against Measles Virus[END_REF]

. The present study of U. lactuca fractions had EC50 values range (88.32-136.82 µg mL -1

Table XXIII .

 XXIII

	Antioxidant activity at 50% (IC50) for C. racemosa polysaccharides
	fractions was found highest to lowest in this trend: fraction 4OH>1OH>INS>Cr
	crude with (203.07, 268, 548.7, and 5253.47 µg mL -1 ), respectively. While
	antioxidant activity at 50% (IC50) for U. lactuca fractions were found highest to
	lowest in this trend: fraction DA>PP3>DS>PP1>PP2 with (805.6, 970.9, 2072.2,
	2883.2, and 2918.2 µg mL -1 ), respectively. All polysaccharides extract from C.
	racemosa, and U. lactuca had <50% scavenging ability except fractions 4OH and
	PP1, which are slightly over 50%. All C. racemosa fractions except fraction Cr
	crude had lower (IC50) values compared to U. lactuca fractions. The low IC50 value
	indicates that the sample has a strong antiradical activity. Free radical scavenging
	activity evaluation was shown in appendix 9 and 10, while its statistical analysis

was shown in appendix 17.6 and 17.7 for C. racemosa and U. lactuca polysaccharide fractions with its BHA and BHT standards.

Table XXIII .

 XXIII Summary of antioxidant activity, scavenging assay (DPPH) of Philippine green seaweeds Caulerpa racemosa and Ulva lactuca.

	synthetic antioxidants have been reported to be toxic and carcinogenic in animal
	models (Sanjeewa et al. 2018). Fernando et al. (2017) reported DPPH activity with
	IC50: >2 mg mL -1 from crude polysaccharide of C. racemosa, and that sulfates
	(10.51% dw), or total polyphenols (4.38% dw) content, influenced scavenging
	IC50 (µg mL -1 ) 7.05±0.27 activity. This polysaccharide from C. racemosa had galactans and mannans from BHA (0-50 conc)
	BHT (0-50 conc) its major sugars (15.36-32.71% dw), glucose, galactose, mannose, and xylose. 5.32±0.18
	C. racemosa Cr crude However, the dose concentration in the present study for C. racemosa fractions was 5253.47±0.55 1OH 267.99±0.24 all twice as low (0.25-5 mg mL -1 ) except for fraction crude. Thus, fraction 4OH,
	4OH	203.07±0.25
	INS	548.69±0.15
	U. lactuca
	Uf DS	2072.16±0.57
	Uf DA	805.59±0.28
	Uf PP1	2883.26±10.19
	Uf PP2	2918.20±10.52
	Uf PP3	970.90±0.31
	of Reactive Oxygen Species ROS-induced membrane phospholipids peroxidation
	caused dysfunction of vital cellular processes such as membrane transport and
	mitochondrial respiration. Seaweeds and their compounds are known to possess
	high antioxidant properties and are effective in protecting living organisms against
	oxidative damages caused by ROS (Vinosha et al. 2019). Discovery and screening
	of antioxidant compounds with antiviral properties are promising since the

IC50: corresponding to the concentration sufficient to obtain 50% of a maximum scavenging capacity. Cr crude: crude polysaccharide extract; fraction 1OH, 4OH, and INS: are polysaccharides extracted from HWE insoluble residue; fraction DS, and DA: are dialyzed polysaccharides; fraction PP1, PP2, and PP3: are successively extracted polysaccharides from crude Ulvan of U. lactuca. BHA: Butylated HydroxyAnisole; BHT: Butylated Hydroxytoluene. Viral infections are often promoted by oxidative processes, favoring replication in infected cells, induction, and inhibition of cell proliferation (Sansone et al. 2020). In patients affected by herpes simplex, it was observed that an increase treatment of viral diseases requires the suppression of viral replication and cell survival promotion. Therefore, commercial antioxidants have been in high demand, and most of them are synthesized, including BHA and BHT. However, these 1OH, and INS have better antioxidant activity than C. lentillifera polysaccharides of Tian et al. (2019) and C. racemosa crude polysaccharides of Fernando et al. (2017). Galactans and mannans were also present in C. racemosa fractions in the present study, which is in agreement with Fernando et al. (2017). Another study of crude polysaccharide (methanol extraction) from C. racemosa reported a 2-times higher DPPH scavenging activity (10.40-88.87%) at a lesser dose concentration of 31.25-2000 µg mL -1 (Lavanya et al. 2015) than C. racemosa fractions in the present study. Another study correlated an increase in antioxidant activity (DPPH scavenge:

84.18, 84.75, 87.79, and 88.60%)

with the increase in total polyphenols content (0, 1, 5, and 10% levels) in biscuits supplemented with C. racemosa powder

(Kumar

Brown Seaweed (Sargassum polycystum and Sargassum ilicifolium) 4.1 Biochemical composition analysis of raw material

  conclusion, the extraction yield results of C. racemosa are affected by geographic location, and even by the sensitiveness of the methods used. The biochemical composition of raw material from brown seaweed Sargassum polycystum and Sargassum ilicifolium for the present study were given in Table XXIV. The raw S. polycystum biochemical composition showed highest to lowest contents in this trend: neutral sugars>ash>proteins>uronic acids> sulfates>polyphenols>lipids. While the biochemical composition of raw S.

	fractions; and sulfates group (range 5.44-16.0%), proteins (range 18.3-18.9%), and
	uronic acids (range 7.0-9.5%) for dialyzed fractions. The FTIR spectrum of U.
	lactuca extracts confirmed the presence of sulfate groups and sugars, where U.
	lactuca polysaccharides is mainly composed of Ulvan with rhamnose and glucose
	as the dominant sugar units. All U. lactuca fractions showed in vitro antiherpetic
	activity without cytotoxicity.
	The C. racemosa and U. lactuca antioxidant results suggest that fresh
	Biochemical composition of C. racemosa extracts is mainly composed of sulfates seaweed should be preferred for human consumption. It is also important to
	group (range 8.18-10.58%) and proteins (range 3.7-11.0%). Previous data of this
	species reported rich in lipids at 4.5% dw and showed real nutritional value for
	While, the extraction yield results of U. lactuca in the present study
	confirmed the effectiveness of the extraction method used based on the previous
	papers. Despite similar extraction methods used, different yield was observed and
	is due to the amount of crude ulvan used at the beginning of extraction and
	purification (successive precipitation and dialysis). Other contributing factors are
	geographic location, and even the sensitiveness of the methods used. Biochemical
	composition of U. lactuca extracts is mainly composed of sulfates group (range
	15.79-20.03%) and proteins (range 7.1-19.6%) for successive precipitation

consumption requirements (see Magdugo et al. 2020). FTIR spectrum of C. racemosa extracts confirmed the presence of sulfate ester, sugars, and proteins, where, C. racemosa polysaccharide is mainly composed of sulfated heteropolysaccharides with arabinose, glucose, and xylose sugar units. The use of strong alkali (1-and 4M-KOH) to isolate hemicellulosic polysaccharides from C. racemosa HWE fraction, for the purpose of biochemical characterization is not necessary since all the information found in these fractions are already found in HWE fraction (see Magdugo et al. 2020). The mineral contents in the previous study for C. racemosa and U. lactuca are within safe levels and are potential functional foods for humans. Also, C. racemosa and U. lactuca had high pigments and carotene contents that can be used in the food industry and human health development. The C. racemosa fractions showed in vitro antiherpetic activity without cytotoxicity.

evaluate the heavy metal and microbiological contents of this seaweeds in order to established reputation as food. The extracts from both species are considered significant since they are derived from natural resources; therefore, they can be utilized to develop various functional and health foods. The presence, degree, and distribution of the sulfate groups are important in determining the biological activity and to analyze its anti-viral potency in relation to structure and position of sulfate groups of polysaccharides

[START_REF] Figueira | Structural Characterization of Ulvan Polysaccharide from Cultivated and Collected Ulva fasciata (Chlorophyta)[END_REF]

, and therefore must be studied further for these extracts in the future.

Chapter 4

ilicifolium showed highest to lowest contents in this trend: neutral sugars>proteins>ash>polyphenols>uronic acids>sulfates>lipids. Carbohydrate contents, including sulfated polysaccharides (corresponding to uronic acids, sulfate groups, and neutral sugars) in raw S. polycystum and raw S. ilicifolium, were about 62.0% and 42.4%, respectively.

Table XXIV .

 XXIV Biochemical composition of Philippine brown seaweeds Sargassum polycystum and Sargassum ilicifolium.

	ALGAE		Content (% dry algal material)
		Neutral	Sulfate	Uronic
	Proteins	sugars	groups	acids	Polyphenols Ash	Lipids
	S. polycystum 24.2±7.4 35.4±2.5 10.0±0.3 16.6±5.1 5.6±1.5	24.9±8.41 2.86
	S. ilicifolium 17.8±2.9 24.2±1.0 8.88±0.2 9.4±1.4 11.1±1.1	17.0±2.96 3.66

Values are means ± SD (n=9) except for lipids.

Table XXVI .

 XXVI The cell wall polysaccharides for S. polycystum and S. ilicifolium were extracted from Depigmented Algal Powder (DAP) with an initial weight of 10 g.From that 10 g, we extracted sequentially using four different solvents and obtained four different fractions(CaCl2, HCl, Na2CO3, and KOH). The yields of the different extracted fractions for S. polycystum and S. ilicifolium are given inTable XXVI. Initial and final weights of raw S. polycystum and S. ilicifolium that were used to calculate extraction yield in percent at each purification step were shown in appendix 6. The extraction yield of polysaccharides from S. polycystum fractions was found highest to lowest in this trend: Sp CaCl2>Sp Na2CO3>Sp KOH>Sp HCl. While the extraction yield of polysaccharides from S. ilicifolium fractions was found highest to lowest in this trend: Si Na2CO3>Si CaCl2>Si KOH>Sp HCl, respectively (Table XXVII). The fractions CaCl2 and HCL are Fucoses Containing Sulfated Polysaccharides (FCSPs), while Na2CO3 is composed of alginates extract, and KOH is of cellulose and hemicellulose extracts (Deniaud-Bouët et al. 2017). Extraction yield and purification of polysaccharides from Philippine brown seaweeds Sargassum polycystum and Sargassum ilicifolium.

	polycystum (15.44%) is almost twice as high than in S. ilicifolium (8.55%).
	However, the amount of fractions Na2CO3 (alginates) is in contrast and is about 3-
	times lower in Sp Na2CO3 than in Si Na2CO3. Fraction KOH (cellulose and
	hemicellulose) with remaining FCSPs were approximately the same (6.23, and
	5.50%) in both species, respectively (Table XXVI).
	Fucoidan extracts by DEAE-cellulose from Sargassum polycystum
	(Palanisamy et al. 2018) reported higher yields (22.3, 16.9, and 19.4%) and also
	lower yield (5.3%) compared to (15.4%) FCSPs from Sp CaCl2 + Sp HCl of the
	same species, and (8.6%) FCSPs from Si CaCl2 + Si HCl of the present study.
	Another study of crude fucoidan extracts subjected to different temperatures (60-
	90 °C), reported lower yields (6.0-7.36%) from S. turbinarioides, and (2.89-3.49%)
	EXTRACTION	Solvents	Yields (%) Yield (% dw)
	S. polycystum S. polycystum (Sp)	Ethanol	71.57
	Depigmented	Chloroform/Methanol	71.57
	Sp CaCl2	Acetone	13.83
	S. ilicifolium Sp HCl Sp Na2CO3 The alginate yield (15.85%) from S. polycystum (Kok and Wong, 2018) was Ethanol 79.94 1.61 10.77 Chloroform/Methanol Sp KOH 6.23 about 50% higher than alginate rich fraction Sp Na2CO3 (10.08%) of the same Acetone S. ilicifolium (Si) Depigmented 79.94 species and was 2-times lower than fraction Si Na2CO3 (29.6%) in the present study.
	Si CaCl2 Sinha et al. (2010) reported impure alginate yield (33%) from Sargassum 6.00
	Si HCl Si Na2CO3 tenerrimum, which is similar to fraction Si Na2CO3 but is 3-times higher than Sp 2.55 29.62
	Si KOH Na2CO3 in the present study. Another study of crude alginates yield (23%) from 5.50
	Overall yield= expressed as a percentage of total dry algae at the start. Sargassum natans, and (17%) from Sargassum vulgare (Rhein-Knudsen et al.
	Purification yield= expressed as a percentage of the dry mass used at each stage. The highest yields obtained (>10% dw) from the present study were: Si 2017) were reported lower than fraction Si Na2CO3, and higher than fraction Sp

Na2CO3 (29.6%), Sp CaCl2 (13.8%), and Sp Na2CO3 (10.08%), and the rest are small (1.6-6.23%). The amount of FCSPs (CaCl2 and HCl fractions) in S. from S. ilicifolium

[START_REF] Artemisia | The Properties of Brown Marine Algae Sargassum turbinarioides and Sargassum ilicifolium Collected from Yogyakarta, Indonesia[END_REF]

compared to (15.4%) FCSPs from Sp CaCl2 + Sp HCl, and (8.6%) FCSPs from Si CaCl2 + Si HCl of the same S. ilicifolium species in the present study. Fucoidan extracts usually consisted of about 5-10% from dry brown seaweed (Artemisia et al. 2019). However, Usoltseva et al. (2017) reported a meager yield of 0.28% laminarin, and 0.15% fucoidan from Sargassum duplicatum. In contrast, Puspita et al. (2017) produced better yield (22.8%) from the water extract from S. polycystum than the present study Sp CaCl2 (13.8%) and Sp Na2CO3 (10.08%) of the same species. However, lower yields of water extracts from S. ilicifolium (14.7%), and from S. aquifolium (17.3 %) (Puspita et al. 2017) than (29.6%) from Si Na2CO3 in the present study. Na2CO3 in the present study. However, alginate yield varies with species, seasonal growth, pretreatment, and extraction methods

Table XXVII .

 XXVII Biochemical composition of polysaccharide extracts from Philippine brown seaweeds Sargassum polycystum and Sargassum ilicifolium. (2017) reported similar polyphenols contents of hot water extract (2.6%) from S. muticum compared to all fractions of Sp and Si in the present study.

	et al. 2019). The present study neutral sugar contents FCSPs fraction Sp HCl
	(17.0%), and Sp CaCl2 (8.8%) was somewhat in accordance to F3=19.71%, and
	F4=11.53% from purified crude fucoidan fractions of S. polycystum (Palanisamy et
	al. 2018). However, F1=23.51% and F2=51.12% of Palanisamy et al. (2018) were
	almost 3-times higher than FCSPs fraction Sp CaCl2, and fraction Sp HCl in the
	present study of the same species. Also, from Palanisamy et al. (2018), reported
	similar sugar contents from two fractions (F1=23.51% and F3=19.71%), but
	ALGAE reported 2-times higher in F2=51.12%, and 2-times lower in F4=11.53% compared Content (% dry algal material)
	Yield with the present study FCSPs fractions [Si CaCl2 (22.2%) and Si HCl (18.5%)], Neutral Sulfates Uronic
	(%) Proteins cellulose/hemicellulose rich fractions [Sp KOH (23.1%), and Si KOH (24.5%)]. sugars group acids Polyphenol S. polycystum
	Sp CaCl2	13.83 11.8±0.7 8.8±0.8 6.36±0.3	7.1±0.7 3.2±1.6
	Sp HCL	1.61 10.0±0.2 17.0±0.7 11.62± 0.6 7.3±0.6 6.4±1.9
	Sp Na2CO3	10.77 9.6±0.3	22.1±0.2 12.0±0.2	8.5±0.4 5.4±1.2
	Sp KOH	6.23 8.9±0.4	23.1±1.1 12.68±0.5 9.4±0.2 3.2±0.0
	S. ilicifolium
	Si CaCl2	6.0	9.9±0.4	22.2±1.9 15.21±0.2 6.7±0.4 2.0±1.2
	Si HCL	2.55 25.5±2.5 18.5±0.5 6.98±0.4	8.4±0.3 7.2±0.4
	Si Na2CO3 Si KOH than alginate rich fractions [Sp Na2CO3 (22.1%), and Si Na2CO3 (33.2%)] in the 29.62 8.7±0.6 33.2±0.8 10.37±0.4 8.9±0.6 1.8±1.5 5.5 9.6±0.5 24.5±0.7 12.58±0.9 13.0±0.7 2.5±2.4
	present study.
	The highest sulfates contents in the present study were fractions Si CaCl2
	(15.21%), Sp KOH (12.68%), Si KOH (12.58%), and Sp Na2CO3 (12.0%), the rest
	are <12% or (6.36-11.62%). Polysaccharides extracts reported comparable sulfates
	contents (17.5%) from S. fluitans (Bedoux et al. 2017), and (13.18%) from S.
	The highest protein contents were fractions Si HCl (25.5%), Sp CaCl2 (11.8%), and fusiforme (Ye et al. 2018) compared to all fractions in the present study except
	Sp HCl (10.0%), the rest are <10% or (8.7-9.9%). The protein content in crude FCSPs fraction Sp CaCl2 (6.36) and Si HCl (6.98%) which was about 2-times lower.
	fucoidan from S. polycystum reported 2-times lower (4.7%) (Palanisamy et al. Similar sulfates result in purified polysaccharides (F1=7.2% and F3=6.5%) by
	2018), than all S. polycystum and S. ilicifolium fractions in the present study except DEAE-sepharose fast flow from Sargassum pallidum (Li et al. 2017) was reported
	fucoidan fraction Si HCl (25.5%), which is 5-times higher. with Sp CaCl2 and Si HCl in the present study. While fraction F4=16.2% was
	The highest neutral sugars contents were fractions Si Na2CO3 (33.2%), Si similar to the rest of the fractions, and fraction F2=24.6% was up to 4-times higher
	KOH (24.5%), Sp KOH (23.1%), Si CaCl2 (22.2%), and Sp Na2CO3 (22.1%), the than all fractions in the present study (Li et al. 2017). Palanisamy et al. (2018)

All fractions in the present study have high protein contents than 1.01% from S. fusiforme polysaccharides (Ye et al. 2018), and 0.1-1.2% from S. pallidum purified polysaccharide fractions by DEAE-sepharose fast flow

[START_REF] Li | Fractionation, preliminary structural characterization and bioactivities of polysaccharides from Sargassum pallidum[END_REF]

. rest are <20% or (8.8-18.5%). In the present study, neutral sugars were also found abundant components than proteins, and phenols, as was stated in the literature

(Hao

Another study reported up to 3-times higher total sugar contents from (fucoidan=59%, and sulfated fucoidan=38%) S. tenerrimum

[START_REF] Sinha | Polysaccharides from Sargassum tenerrimum: Structural features, chemical modification and antiviral activity[END_REF]

compared to all FCSPs fractions from Sp and Si, and to cellulose/hemicellulose rich fraction Sp KOH (23.1%) and Si KOH (24.5%) in the present study.

While Sinha et al. (2010)

reported about 2-times higher total sugar contents (impure alg=51%, sodium alginate or NaAlg=64%, and sulfated NaAlg=42%) from S. tenerrimum reported similar sulfates contents of purified crude fucoidan (F1=14.21, F3=11.36, and F4=6.19%) from S. polycystum with the present study FCSPs fractions

[Sp HCl et al.

Table XXX .

 XXX List of InfraRed (IR) vibrational modes characteristic to polysaccharides used in determining spectral bands of interest for the identification and discrimination of brown algal polysaccharides from Sargassum polycystum (adapted from[START_REF] Vandanjon | Seasonal Variation of Sargassum Muticum Biochemical Composition Determined by Fourier Transform Infra-Red Spectroscopy[END_REF][START_REF] Sun | The stability and bioaccessibility of fucoxanthin in spray-dried microcapsules based on various biopolymers[END_REF][START_REF] Palanisamy | Investigation of antioxidant and anticancer potential of fucoidan from Sargassum polycystum[END_REF][START_REF] Kok | Physicochemical properties of edible alginate film from Malaysian Sargassum polycystum C. Agardh[END_REF][START_REF] Artemisia | The Properties of Brown Marine Algae Sargassum turbinarioides and Sargassum ilicifolium Collected from Yogyakarta, Indonesia[END_REF].

	Region/Band (cm -1 )	Type of bond	Identification
	Common to Sargassum polycystum
	3425 (3451-3374.12) O-H	OH stretching vibrations
	2960	C-H	aliphatic C-H stretching
	2944.05, 2923-2839 C-H	Stretching vibrations of C-H
	2927	C=O	C=O stretching vibration of O-acetyl group
	1705-1595	C-C	C-C stretching vibrations in the aromatic ring
	1654.2, 1605.3		The symmetric and asymmetric stretch of
			carboxylate
	1641	CH	CH vibration of the polysaccharides (fucose, D-
			glucose, D-mannose, D -xylose and galacturonic
			acid)
	1409.70 (1452-1339) C-H	The symmetric and asymmetric stretch of
			carboxylate; C-H scissoring and bending,
			typical for carotenoids
	1261	CH3	Fucose, O-acetyl
	1230	S=O	Stretching vibration of esterified sulfate
	1090.1, 1030.72,		Stretching vibrations of pyranose rings
	1091.8, 1025.5
	1056	S=O	Stretching of alkyl sulfoxide
	945.5	C-O	Stretching vibrations of uronic acid
	840.36, 810.67		Characteristic peak of mannuronic acid
	802		Anomeric region of carbohydrates.
	543	C-O-S	Secondary axial sulfate group at C-4 of fructopyranose residue

Table XXXI .

 XXXI List of InfraRed (IR) vibrational modes characteristic to polysaccharides used in determining spectral bands of interest for the identification and discrimination of brown algal polysaccharides from Sargassum ilicifolium (adapted from[START_REF] Ale | Fucose-Containing Sulfated Polysaccharides from Brown Seaweeds Inhibit Proliferation of Melanoma Cells and Induce Apoptosis by Activation of Caspase-3 in Vitro[END_REF][START_REF] Marudhupandi | In vitro anticancer activity of fucoidan from Turbinaria conoides against A549 cell lines[END_REF][START_REF] Vandanjon | Seasonal Variation of Sargassum Muticum Biochemical Composition Determined by Fourier Transform Infra-Red Spectroscopy[END_REF][START_REF] Sun | The stability and bioaccessibility of fucoxanthin in spray-dried microcapsules based on various biopolymers[END_REF][START_REF] Palanisamy | Investigation of antioxidant and anticancer potential of fucoidan from Sargassum polycystum[END_REF][START_REF] Kok | Physicochemical properties of edible alginate film from Malaysian Sargassum polycystum C. Agardh[END_REF][START_REF] Artemisia | The Properties of Brown Marine Algae Sargassum turbinarioides and Sargassum ilicifolium Collected from Yogyakarta, Indonesia[END_REF].

	Region/Band (cm -1 )	Type of bond Identification
	Common to Sargassum ilicifolium
	1705-1595	C-C	C-C stretching vibrations in the aromatic ring
	1635	C=O	Stretching vibration
	1409.7 (1452-	C-H	Symmetric and asymmetric stretch of carboxylate;
	1339)		C-H scissoring and bending, typical for carotenoids
	1323	C-H	Bending
	1250 (1255-1240)	S=O, C-O	Asymmetric S=O stretching vibration and symmetric C-O vibration associated with the CO-SO3 group
	1230	S=O	Stretching vibration
	1200-900		Carbohydrates
	1137	C-O	Stretching
	1027		Carbohydrate
	963, 833	=CH, =CH2	Bending vibration of =CH and =CH2
	Other Sargassum species
	1615.6	O-C-O	Asymmetric stretching of carboxylate O-C-O vibration
	1593	C-H	CH bending
	1250	O=S=O; S=O Asymmetric O=S=O stretching vibration of sulfate
			group; Stretching vibration
	1028 (1030-	C-C; C-O	C-C and C-O stretching vibrations of pyranose ring
	1025)		with C-O-C stretches of the glycosidic bonds
	961		Carbohydrate
	817 (840-815)

Table XXXII .

 XXXII Composition of macroelements and microelements (% dw), recommended daily intakes, and recommended daily allowances of Philippine brown seaweeds Sargassum polycystum and Sargassum ilicifolium.

	Species			Macroelements		Microelements
		Na	K	Ca	Mg	Na/K Fe	Zn	Mn	Cu
	RDI a (g day -1 )	2.0	3.8	0.84	0.350	0.006	0.012	0.0055	0.0017
	Upper limit intake b (g day -1 )	2.3	-	2.5	0.35	0.045	0.040	-	0.01
	S. polycystum	10.0±0 17.13±0 0.94±0	1.36±0 0.58 1.17±0.02 0.13±0	0.004±0 0.11±0
	% RDI	40.0	36.06	8.93	31.01	1553.33	83.33	6.30	533.33
	RDA (g day -1 )	23.0	-	266.52	25.80	3.86	32.00	-	8.82
	S. ilicifolium	1.17±0 2.10±0 1.54±0 0.62±0	0.56 1.63±0.03 0.37±0	0.002±0 0.06±0
	% RDI	4.68	4.42	14.64	14.23	2175.56	246.67	2.91	266.67
	RDA (g day -1 )	196.58 -	162.65	56.22	2.76	10.81	-	17.65

Table XXXIII .

 XXXIII Pigment content (mg g -1 dw) of Philippine brown seaweeds Sargassum polycystum and Sargassum ilicifolium.

					Total	Total
	Seaweeds	Chlorophyll a Fucoxanthin -Carotene	chlorophyll	pigments
	S. polycystum 6.25±1.31	2.28±0.30	188.89±26.7 6.25±1.31 197.43±28.3
	S. ilicifolium 1.04±0.18	2.60±0.41	7.84±1.67	1.04±0.18 11.49±2.28
	Data are mean ± SD (n=2). SD = standard deviation

Table XXXIV .

 XXXIV Evaluation of cytotoxicity and antiviral activity by cellular viability of Philippine brown seaweeds Sargassum polycystum and : The 50% Cytotoxic Concentration. The concentration that reduced the absorbance of mockinfected cells to 50% of that of controls. EC50: The 50% antiviral Effective Concentration: concentration that achieved 50% protection of virus-infected cells from the HSV-induced destruction. CaCl2 and HCl= are sequential sulfated polysaccharide extracts rich in fucose; Na2CO3= extracts containing alginates with remaining fucoidan; KOH= are cellulose, hemicellulosic residues with remaining fucoidan.

		Sargassum ilicifolium.
			CC50 (µg mL -1 )	EC50 (µg mL -1 )
	SPECIES	FRACTION	Destruction	Protection
	S. polycystum	Sp CaCl2	>200	31.63±0.61
		Sp HCl	>200	30.08±0.83
		Sp Na2CO3	>200	33.25±2.3
		Sp KOH	>200	67.02±0.36
	Zovirax		>200	0.49±1.2
	S. ilicifolium	Si CaCl2	>200	6.63±1.64
		Si HCl	>200	17.29±0.64
		Si Na2CO3	>200	19.707±0.49
		Si KOH	>200	17.44±0.58
	Zovirax		>200	0.32±0.25

CC50

Table XXXV .

 XXXV

	BHA (0-50 conc)	7.05±0.27
	BHT (0-50 conc)	5.32±0.18
	S. polycystum
	Sp CaCl2	384.27±0.18
	Sp HCl	105.49±0.17
	Sp Na2CO3	277.74±0.21
	Sp KOH	242.35±0.63
	S. ilicifolium
	Si CaCl2	595.27±0.27
	Si HCl Its antioxidant activity at 50% (IC50) for S. polycystum polysaccharides 1134.83±0.48 Si Na2CO3 708.92±0.29 fractions was found highest to lowest in this trend: fraction Sp HCl>Sp KOH>Sp Si KOH 364.74±0.28
	Na2CO3>Sp CaCl2 with (105.49, 242.35, 277.74, and 384.27 µg mL -1 ), IC50: corresponding to the concentration sufficient to obtain 50% of a maximum scavenging
	capacity. CaCl2 and HCl= are sequential sulfated polysaccharide extracts rich in fucose; Na2CO3= respectively. While S. ilicifolium antioxidant activity at 50% (IC50) for extracts containing alginates with remaining fucoidan; KOH= are cellulose, hemicellulosic residues
	polysaccharides fractions were found highest to lowest in this trend: fraction Si with remaining fucoidan; BHA = Butylated HydroxyAnisole; BHT = Butylated Hydroxytoluene
	KOH>Si CaCl2>Si Na2CO3>Si HCl with (364.74, 595.27, 708.92, and 1134.83 µg
	mL -1 ), respectively. Polysaccharides extracts from S. polycystum had higher
	scavenging ability at fractions CaCl2 and KOH, and lower (IC50) values in all
	extracts compared to S. ilicifolium fractions. The low IC50 value indicates that the
	sample has a strong antiradical activity. Free radical scavenging activity evaluation
	was shown in appendix 13 and 14, while its statistical analysis was shown in
	appendix 17.8 and 17.9 for S. polycystum and S. ilicifolium fractions, respectively,
	with its BHA and BHT standards.
	Table XXXV. Summary of Antioxidant activity, scavenging assay (DPPH) of
	Philippine brown seaweeds Sargassum polycystum, and Sargassum
	ilicifolium.
		IC50 (µg mL -1 )

  is in accordance to all Sp and Si fractions except Sp CaCl2 and Sp KOH in the present study.All the Sp and Si fractions in the present study are also in accordance with[START_REF] Pádua | Bioactive compounds from brown seaweeds: Phloroglucinol, fucoxanthin and fucoidan as promising therapeutic agents against breast cancer[END_REF] that seaweeds containing fucoidan are dominant in L-fucose. , and xylose as the dominant sugar units. The S. polycystum and S. ilicifolium mineral contents are within safe levels and are potential functional foods for humans. Also, S. polycystum and S. ilicifolium had -carotene contents of at least 100 times higher than found in spinach. Besides its anticancer activity, carotene has been reported to be absorbed 10-times more easily by the body than the synthetic one[START_REF] Christaki | Functional properties of carotenoids originating from algae: Functional properties of algal carotenoids[END_REF]. Thus, S. polycystum and S. ilicifolium are excellent sources of pigments that can be used in the food industry and human health development.The S. polycystum and S. ilicifolium fractions showed in vitro antiherpetic activity without cytotoxicity. Polysaccharides from S. ilicifolium fractions are more potent than S. polycystum fractions. The S. polycystum and S. ilicifolium antioxidant results (DPPH scavenging assay) had better activity than the green (C. racemosa and U. lactuca), and the red (H. durvillei and H. dilatata) seaweeds in the present study, suggest that the seaweeds are safe for human consumption.The presence, degree, and distribution of the sulfate groups are important in determining the biological activity and to analyze its anti-viral potency in relation to structure and position of sulfate groups of polysaccharides, and therefore must be studied further for these extracts in the future. The extracts from both species are considered significant since they are derived from natural resources; therefore, they can be utilized to develop various functional and health foods.

	fucoidan, in which according to Palanisamy et al. (2018) that fucoidan fraction of
	S. polycystum contains fucose.
	The variation of antioxidant activity results between the present study (Sp
	and Si extracts) and previous studies from Sargassum species could be influenced
	by several factors such as season and location of collection, total polyphenols, low
	Mw polysaccharides, proteins or peptides, sulfate esters linked with sugar
	constituents, or sensitiveness of methods used. The DPPH scavenging activity of
	Sp and Si extracts is considered significant since these extracts are derived from
	natural resources; therefore, they can be utilized to develop various functional and
	healthy foods.
	Conclusion and perspectives
	In conclusion, the extraction yield results of S. polycystum and S. ilicifolium
	are affected by species used, geographic location, seasonal growth, and even by the
	sensitiveness of the methods used. Biochemical composition for both S. polycystum
	and S. ilicifolium extracts is mainly composed of neutral sugars, sulfates group, and
	proteins, where S. polycystum had range values of (8.8-23.1%), (6.36-12.68%), and
	(8.9-11.8%) dw of alga, respectively. While, S. ilicifolium extracts had range values
	of (18.5-33.2%), (6.98-15.21%), and (8.7-25.5%) dw of alga, respectively.
	FTIR spectrum of S. polycystum and S. ilicifolium extracts confirmed the
	presence of ester sulfates, and sugars, where, S. polycystum polysaccharides is
	mainly composed of fucoidan, alginate, and cellulose/hemicellulose at a ratio of
	Pádua et al. (2015) added that fucoxanthin, an abundant carotenoid in brown 3:2:1 with fucose, glucose, galactose, mannose, and xylose as the dominant sugar
	seaweeds have much higher antioxidant activities than any other antioxidant, units. While, S. ilicifolium cell wall polysaccharides are composed of alginate,
	including tocopherols, and content varies during season and life cycle of seaweed. fucoidan, and cellulose/hemicellulose at a ratio of 6:2:1 with fucose, glucose,
	FTIR analyses at [1641-1637 cm -1 ] for Sp CaCl2, and Sp HCl fractions and at [1612-galactose, mannose
	1596 cm -1 ] for all Si fractions in the present study confirms the presence of

Table

  XXXVII for comparison. Amino acids have been classified according to their quality as essential (EAAs) or non-essential (NEAAs) amino acids. Results

Table XXXVII .

 XXXVII Amino acid composition of Halymenia durvillei and Halymenia Values for the ovalbumin composition in amino acids were adapted from[START_REF] Kazir | Extraction of proteins from two marine macroalgae, Ulva sp. and Gracilaria sp., for food application, and evaluating digestibility, amino acid composition and antioxidant properties of the protein concentrates[END_REF].

		Leucine	9.81	9.08	8.16
		Phenylalanine	4.13	3.03	5.39
		Histidine	-	-	5.39
		Methionine	-	15.55	4.08
		Total EAA	38.08	46.51	50.52
		Aspartic acid	13.23	11.84	8.16
		Serine	3.51	2.32	8.95
	Non-Essential Amino Acids (NEAA)	Glutamic acid Glycine Alanine Tyrosine Proline	20.56 7.30 6.25 6.72 4.34	20.11 4.98 5.44 4.90 3.89	13.03 4.47 8.82 2.37 3.69
		Hydroxyproline			-
		Total NEAA	61.92	53.49	49.48
		EAA/NEAA ratio	0.62	0.87	1.02
	-= not detected; a
		dilatata expressed in percentage (%) of the total amino acids
		detected by gas chromatography.
			H. durvillei	H. dilatata	Ovalbumin a
			(% total AA)	(% total AA)	(% total AA)
	Essential Amino Acids (EAA)	Threonine Valine Lysine Isoleucine	0.46 7.96 11.03 4.69	0.18 6.40 9.16 3.13	3.95 7.10 10.14 6.32

Table XXXVIII .

 XXXVIII Fatty acid composition (% of total fatty acids) of Philippine red seaweed Halymenia durvillei and Halymenia dilatata.

	C12:0 (Lauric) (TI) are related to the protection against coronary artery diseases were found to have 12.00 9.99±0.62 2.57±0.01
	C13:0 (Tridecanoic) C14:0 (Myristic) higher values in H. durvillei than H. dilatata. While its high fatty acid 13.00 0.32±0.00 0.40±0.00 14.00 2.45±0.16 0.55±0.00
	C15:0 (Pentadecanoic) hypocholesterolemic/Hypercholesterolemic ratio (h/H) are considered more 15.00 3.48±0.26 0.40±0.00
	C16:0 (Palmitic) C17:0 (Heptadecanoic) beneficial as these are directly related to high PUFA content (Matos et al. 2016), 16.00 30.34±0.96 13.89±0.47 17.00 0.37±0.01 0.47±0.00
	C18:0 (Stearic)	18.00	0.73±0.00	4.69±0.03
	C20:0 (arachidic)	20.00	2.43±0.02	nd
	C22:0 (Behenic)	22.00	0.23±0.00	1.12±0.00
	C23:0 (Tricosanoic)	23.00	0.23±0.00	-
	C24:0 (Lignoceric)	24.00	-	2.82±0.02
	Total SAFs		51.15±1.23	32.19±0.55
		Monounsaturated fatty acids (MUFAs)
	C14:1 (Myristoleic)	14.11	11.68±0.60	3.01±0.02
	C15:1 (Cis-10-pentadecanoic)	15.00	-	0.38±0.00
	C16:1 (Palmitoleic)	16.00	1.36±0.01	2.62±0.00
	C17:1 (Cis-10-Heptadecenoic)	17.29	0.16±0.00	1.68±0.00
	C18:1n-9c (Oleic)	18.00	1.62±0.06	14.51±0.39
	C20:1n-9 (cis-11-Eicosenoic)	20.00	-	0.54±0.00
	C22:1n-9 (Erucic)	21.72	0.65±0.01	0.16±0.00
	C24:1n-9 (Nervonic)	23.52	0.28±0.00	0.38±0.00
	Total MUFAs		15.75±0.64	23.28±0.47
		Polyunsaturated fatty acids (PUFAs)
	C18:2n-6c (Linoleic)	18.00	0.59±0.09	0.85±0.00
	C18:2n-6t (Linolelaidic)	18.00	0.83±0.03	nd
	C18:3n-6 (γ-Linolenic)	18.03	4.90±0.46	0.78±0.00
	C18:3n-3 (α-Linolenic)	18.05	3.50±0.12	5.32±0.03
	C20:2 (cis-11. 14-Eicosadienoic) 20.00	-	1.00±0.00
	C20:3n-6 (Eicosatrienoic)	20.00	-	1.81±0.00
	C22:6n-3 (Docosahexaenoic)	22.00	-	2.24±0.01
	Total PUFAs		9.81±0.51	12.00±0.22
	Σ PUFAs ω6		6.32±0.49	3.44±0.05
	Σ PUFAs ω3		3.50±0.25	7.56±0.14
	Σ Ratio ω6/ω3		1.81±0.08	0.46±0.00
	Σ ω3/Σ ω6		0.55±0.00	2.20±0.00
	Unidentified fatty acids		23.29±0.75	32.53±0.39
	FA = Total Fatty Acid; -= not detected; SFA = Saturated Fatty Acid; MUFA = Monounsaturated
	Fatty Acid; PUFA = Polyunsaturated Fatty Acid.
	The nutritional quality indexes calculated with the fatty acid profiles of H.
	Fatty Acid (FAs) durvillei and H. dilatata are presented in Table XXXIX. The ratio of C18/C20 ECL H. durvillei (%) H. dilatata (%)
	Saturated fatty acids (SFAs) PUFA was only found in H. dilatata species with a value 2.47, which indicates a
	C6:0 (Caproic) C8:0 (Caprylic) lesser amount of C20 PUFAs (AA and EPA). The PUFA/SFA ratio of H. durvillei 6.00 0.41±0.02 0.62±0.00 8.00 0.16±0.00 -C10:0 (Capric) 10.00 -1.97±0.00 was 0.19, whereas it was 0.37 for H. dilatata. Ratios based on the functional effects
	C11:0 (Undecanoic) of FA had also been calculated. Atherogenic index (AI), and thrombogenic index 11.00 -2.68±0.01

Table XXXIX .

 XXXIX Nutritional quality indexes of fatty acids from Halymenia durvillei and Halymenia dilatata. Mean % values for C18, C20, and C22 PUFA red seaweed H. durvillei and H. dilatata in the present study; TFA= total fatty acid.

	Seaweed	C18/C20 ω6/ω3 AI	TI	h/H PUFA/SFA UI
	H. durvillei -H. dilatata 2.47 AI: Atherogenic hypocholesterolemic/Hypercholesterolemic ratio; PUFA: Polyunsaturated Fatty Acid; SFA: 1.81 1.96 1.52 0.2 0.19 44.60 0.46 0.54 0.5 1.43 0.37 64.14 Index; TI: Thrombogenic Index; h/H: fatty acid Figure 42. The mean total lipid content of Halymeniales species can vary (0.1-3.9%
	Saturated Fatty Acid; UI: Unsaturation Index; -= not detected dw) with a high content of PUFA considered essential nutritional components in
	humans and animals. Several red seaweed species are reported to have mean total
	A comparison of total average fatty acids (%) in C18, C20, and C22 PUFAs between H. durvillei and H. dilatata is shown in Fig. 42. Total average fatty acids (%) in C18 PUFAs were found higher in H. durvillei (9.82%) than in H. dilatata (6.95%). There was no C20, and C22 PUFAs detected in H. durvillei while in H. dilatata was found to be about 2-3%. lipid for A. al. 2014) of total fatty acids. We observed lower PUFA contents (9.81 and 12.0%)
	in the present study for H. durvillei and H. dilatata, respectively, compared to the
	previous results. The total lipid and fatty acids composition may vary during the
	life cycles of various seaweeds, which may be seasonal, geographical, or growth
	conditions

taxiformis (4-12%), G. filicina (22%), H. durvillei (1.3%), H. dilatata (0.3%), and H. maculata (1.1%)

[START_REF] Hurtado | Chapter Two -Selected red seaweeds from the Philippines with emerging high-value applications[END_REF]

). In the present study, we observed higher lipid contents (7.1 and 0.6% dw) for H. durvillei and H. dilatata of the same species. The highest PUFA content was measured in H. porphyroides at 51.47%

[START_REF] Manam | Phytochemical, amino acid, fatty acid and vitamin investigation of marine seaweeds Colpomenia sinuosa and Halymenia porphyroides collected along Southeast coast of Tamilnadu, India[END_REF]

, H. sinensis at 42.2%

[START_REF] Hurtado | Chapter Two -Selected red seaweeds from the Philippines with emerging high-value applications[END_REF]

, H. floresii at 32.6%

[START_REF] Garcia | Nutritional Potential of Four Seaweed Species Collected in the Barbate Estuary (Gulf of Cadiz, Spain)[END_REF]

, and A. taxiformis at 33.47%

(Ragonese et

  are the most abundant PUFA in H. dilatata of the present study. LA (C18:2n-6) was detected in H. durvillei and H. dilatata for the present study but only in trace amounts. EPA was not detected in both H. durvillei and H. dilatata, and DHA was not detected in H. durvillei in the present study. Although in different proportions, the present study H. durvillei is in accordance with H. porphyroides, where ALA (31.21%) and LA (16.14%) was its main PUFAs

		12
	% FA (% TOTAL FA)	3 6 9	H. durvillei H. dilatata
		0
		C18 PUFA	C20 PUFA	C22 PUFA

Table XL .

 XL Table XL. In H. durvillei, we obtain 95.95%, and in H. dilatata, we obtain 97.42%. Initial and final weights of raw H. durvillei and H. dilatata that were used to calculate extraction yield in percent after three solvents were shown in appendix 5. Percent extraction efficiency of Philippine red seaweeds Halymenia durvillei and Halymenia dilatata. Hdil extract). The yields of the extracted fraction for H. durvillei and H. dilatata are given in Table XLI. The initial and final weights of raw H. durvillei and H. dilatata that were used to calculate the extraction yield in percent at each purification step were shown in appendix 6.

	Species H. durvillei H. dilatata 5.4.2 Polysaccharide extraction Solvents Ethanol Chloroform/Methanol Acetone Ethanol Chloroform/Methanol Acetone The cell wall polysaccharide for H. durvillei and H. dilatata were extracted Yields (%) 95.95 97.42 from Depigmented algal powder with the initial weights of 24.02 g and 30 g, respectively. From that 24.02 g and 30 g, after a series of hot water extraction and ethanol precipitation, we obtained the final fraction yield of polysaccharides (Hdur extract, and The extraction yield of polysaccharides from H. durvillei and H. dilatata fractions was 5.41% and 26.33% dw, respectively (Table XLI).

Table XLI .

 XLI Extraction yield and purification of polysaccharides from Philippine red seaweeds Halymenia durvillei and Halymenia dilatata.Halymenia as grown in culture. This study suggests that H. durvillei and H. dilatata could potentially be used as raw material for carrageenan production in the country.

	EXTRACTION	Yield (% dw)
	H. durvillei
	Hdur extract	5.41
	H. dilatata
	Hdil extract	26.33
	durvillei, which is 5-times higher than Hdur extract (5.41%) of the same species in
	the present study. While H. dilatata yield (9.74%) is about 3-times lower compared
	to Hdil extract (26.33%) of the same species in the present study, and H. maculata
	reported (11.50%) which is 2-times higher and 2-times lower than Hdur extract, and
	Hdil extract, respectively. Moreover, the Halymenia species from Kho et al. (2016)
	were collected from Iligan Bay, Philippines, and extracted by hydration, followed
	by hot water extraction and precipitation, and oven-dried at 60 °C, 24 h and 105 °C,
	2 h. Another study by Fenoradosoa et al. (2009) reported that alkali-treated
	carrageenan from H. durvillei had a 15% yield or 2-times higher than Hdur extract
	of the same species in the present study. While Hdil extract in the present study
	was within the range yield reported by Trono and Largo, (2019) that a total of 21-
	47% g -1 of the air-dry weight of crude λ-like carrageenan can be extracted from

In the present study, the amount of polysaccharides yield in H. durvillei (5.41%) was almost 5-times lower than polysaccharides yield in H. dilatata (26.33), and comprised of about 15% and 76%, respectively of the carrageenan content found in Kappaphycus alvarezii

[START_REF] Wanyonyi | Kappaphycus alvarezii as a Food Supplement Prevents Diet-Induced Metabolic Syndrome in Rats[END_REF]

. The H. durvillei, H. dilatata, and H. maculata from

[START_REF] Kho | for H. durvillei (1.29%), and H. dilatata (0.29%) of the same species, while H. maculata (1.09%) was reported higher than H. dilatata. Wanyonyi et al. (2017) reported lipid content (0.62% dw) from K. alvarezii, which is similar to raw H. dilatata, but 10-times lower than raw H. durvillei in the present study[END_REF]

reported a 28.41% yield for H.

Table XLII .

 XLII Biochemical composition of polysaccharide extracts from Philippine red seaweeds Halymenia durvillei and Halymenia dilatata.

	The protein contents from Hdur extract (7.0%) and Hdil extract (8.7%) in extracts from hot water fraction (1%). The polyphenols contents from Hdur extract
	the present study were below the range value (9.4-47% dw) for selected red (4%) and Hdil extract (3.4%) in the present study was lower than (9.02%) from
	seaweeds from the Philippines (Hurtado et al. 2020). Lower protein contents were Halymenia porphyroides (Manam and Subbiah, 2020).
	also reported (1.45%) from crude polysaccharide Grateloupia filicina, and (0.18-Carbohydrates contents for Hdur extract (34.07%) and Hdil extract
	0.96%) from purified fractions G. filicina (Sun et al. 2017) than the present study. (27.92%) in the present study were within the range value (13.2-56% dw) for
	However, Burlot et al. (2016) reported somewhat similar protein content (4.5%) selected red seaweeds from the Philippines (Hurtado et al. 2020). However, the
	from hot water fraction from Solieria chordalis compared to the present study. present study carbohydrates were found to be lower than the carbohydrates reported
	Sulfates contents for Hdur extract (15.73%) and Hdil extract (15.22%) in in crude extract Gymnogongrus griffithsiae (55.2%) and Cryptonemia crenulata
	the present study were within the range value (13.2-56% dw) for selected red (59.3%); for purified fractions G. griffithsiae (39.2-54%) and C. crenulata (61-
	seaweeds from the Philippines (Hurtado et al. 2020). Also, similar sulfates content 61.3%), and for soluble fraction G. griffithsiae (73.5%) and C. crenulata (63%)
	was reported (15.3%) from crude polysaccharide Solieria filiformis, and (16.5%)
				). While the H. dilatata carrageenan
	fraction (Hdil extract) is rich in sulfates (15.22%), followed by neutral sugars
	(12.2%), and with small amounts of proteins (8.7%), polyphenols (3.4%), and
	uronic acids (0.5%). Sulfated polysaccharides or carbohydrates (corresponding to
	uronic acids, sulfate groups, and neutral sugars) in H. durvillei (Hdur extract) and
	H. dilatata (Hdil extract) are of about 34.07% and 27.92%, respectively.
	Burlot et al. (2016) reported a similar neutral sugar content (12.8%) from
	hot-water fraction from S. chordalis compared to Hdur and Hdil extracts in the
	present study. However, total sugars reported higher contents (55.22%) from crude
	ALGAE polysaccharides, and (41-49.74%) from purified fractions from G. filicina (Sun et Content (% dry algal material)
	Proteins al. 2017), than (17.6%, and 12.2%) from Hdur extract and Hdil extract in the present Neutral sugars Sulfates group Uronic acids Polyphenol
	H. durvillei study, respectively.
	Hdur extract	7.0±1.3	17.6±0.5	15.73±0.4 0.7±0.0 4.0±0.5
	H. dilatata
	Hdil extract	8.7±0.9	12.2±1.5	15.22±0.2 0.5±0.2 3.4±0.3

from Rhodymenia pseudopalmata

[START_REF] Bedoux | Antiviral and Cytotoxic Activities of Polysaccharides Extracted from Four Tropical Seaweed Species[END_REF]

, while slightly higher sulfates (21.52%) from crude polysaccharide, and (16.86-20.99%) purified fractions from G. filicina

[START_REF] Sun | Degradation of Polysaccharides from Grateloupia filicina and Their Antiviral Activity to Avian Leucosis Virus Subgroup[END_REF]

.

[START_REF] Talarico | Anti-herpes simplex virus activity of sulfated galactans from the red seaweeds Gymnogongrus griffithsiae and Cryptonemia crenulata[END_REF]

reported 2-times higher sulfates contents of 33.1%, and 28% from crude extracts from G. griffithsiae and C. crenulata. Also, according to

[START_REF] Talarico | Anti-herpes simplex virus activity of sulfated galactans from the red seaweeds Gymnogongrus griffithsiae and Cryptonemia crenulata[END_REF]

, that purified fractions from G. griffithsiae and C. crenulata, produced sulfates contents (16.1-29.4%) and (17.5-28.3%), respectively that were similar to the present study. Conversely, soluble fractions from G. griffithsiae and C. crenulata

[START_REF] Talarico | Anti-herpes simplex virus activity of sulfated galactans from the red seaweeds Gymnogongrus griffithsiae and Cryptonemia crenulata[END_REF]

), reported lower sulfates (9.1%), and higher sulfates (28.7%) than Hdur and Hdil extracts in the present study.

[START_REF] Burlot | Response Surface Methodology for Enzyme-Assisted Extraction of Water-Soluble Antiviral Compounds from the Proliferative Macroalga Solieria chordalis[END_REF]

reported lower sulfates contents (1.6%) from hot water fraction from Solieria chordalis compared to (Hdur and Hdil extracts) in the present study. Uronic acids contents for Hdur extract (0.7%) and Hdil extract (0.5%) in the present study showed similar results to

[START_REF] Burlot | Response Surface Methodology for Enzyme-Assisted Extraction of Water-Soluble Antiviral Compounds from the Proliferative Macroalga Solieria chordalis[END_REF]

from S. chordalis

[START_REF] Talarico | Anti-herpes simplex virus activity of sulfated galactans from the red seaweeds Gymnogongrus griffithsiae and Cryptonemia crenulata[END_REF]

). In contrast,

[START_REF] Burlot | Response Surface Methodology for Enzyme-Assisted Extraction of Water-Soluble Antiviral Compounds from the Proliferative Macroalga Solieria chordalis[END_REF]

reported lower carbohydrates contents of hot water fraction (15.4%) from Solieria chordalis extracts than the present study.

These variations of results of carbohydrates, proteins, and lipids from both present and previous studies in red seaweeds could be affected by geography, season, or growth conditions

[START_REF] Kelman | Antioxidant Activity of Hawaiian Marine Algae[END_REF][START_REF] Kok | Physicochemical properties of edible alginate film from Malaysian Sargassum polycystum C. Agardh[END_REF][START_REF] Artemisia | The Properties of Brown Marine Algae Sargassum turbinarioides and Sargassum ilicifolium Collected from Yogyakarta, Indonesia[END_REF]

Table XLIII .

 XLIII There are only four monosaccharides identified present in raw H. durvillei, 6 in Hdur extract, 7 in raw H. dilatata, and 5 in Hdil extract. Main contents of Unit sugar composition of raw and extracted fractions from Philippines red seaweed Halymenia durvillei and Halymenia dilatata. The sugar profiles found that all of the samples and its extracts from H. durvillei (Hdur extract) and H. dilatata (Hdil extract) contained mainly the same types of sugars and are therefore sulfated galactans with a slight difference in proportions. Several factors affect monosaccharide contents, including the type of samples used, whether between or across the same species, life cycle, period and location of collection, and methods of extraction used. These factors

	Fenoradosoa et al. (2009) reported similar monosaccharide contents of the present study was within the range value (66.9-80.5%) reported by Amorim et
	xylose (2.8, and 1.82%), and fucose (2.1, and 1.6%), from native polysaccharide al. (2011). Wang et al. (2013) reported fucose (76.34%) as the main sugar unit from
	monosaccharides in raw H. durvillei were: small amounts (<10%) of xylose, fucose, fractions (water fraction and sodium hydroxide fraction, respectively) from H. purified polysaccharides of Pyropia haitanensis, followed by galactose (12.98%),
	trace amounts (<1.0%) of glucoheptose, glucuronic acid, and others (94.24%). The durvillei, with xylose (2.34-241%), and fucose (1.43-1.84%) contents for all xylose (7.63%), and 1.53% each for glucose and mannose. Fucose content (76.34%)
	Hdur extract monosaccharides contents were, small amounts (<10%) of xylose, fractions in the present study. Similar results of xylose (2.7, and 2.3%), and fucose reported by Wang et al. (2013) is way too big compared to fucose content (1.43-
	fucose, fructose, glucoheptose, trace amounts (<1.0%) of glucuronic acid, (2.0, and 1.7%) was also reported from desulfated polysaccharide fractions 1.84%) found in the present study. Its xylose content (7.63%) was 3-times higher
	glucosamine, and others (93.17%). While raw H. dilatata monosaccharides were, (Fenoradosoa et al. 2009) compared to the present study Hdur extract. Xylose was than the present study.
	in small amounts (<10%) of xylose, fucose, fructose, trace amounts (<1.0%) of not detected in the Hdil extract of the present study.
	glucoheptose, glucuronic acid, glucosamine, rhamnose, and others (93.57%). The Galactose was the primary sugar (57.3-65%) in all samples from H. durvillei
	Hdil extract monosaccharides were, small amounts (<10%) of fucose, fructose, as reported by Fenoradosoa et al. (2009), followed by glucose (14.75-16%), then
	glucoheptose; trace amounts (<1.0%) of glucuronic acid, glucosamine; and others arabinose (15.3-16%), and anhydrogalactose (6.2-9.8%) by which all of these
	(94.88%). The total monosaccharides contents for its raw materials, and each significant sugars were not detected in the present study. However, the unknown
	fraction were; raw H. durvillei=11.22±0.86, Hdur=13.57±0.47, raw H. dilatata major sugars in the present study for raw H. durvillei and polysaccharide fraction
	=9.79±1.85, and Hdil=15.35±0.53 (Table XLIII). Hdur extract were about 77%, 8%, and 5% at (13.79, 10.27, and 6.66 min.) retention
	times (rt), respectively. Also, the unknown major sugars in raw H. dilatata and Hdil
	extract were found to be about 76%, 8%, and 7% at (13.84, 10.27, and 6.66 min.)
	retention times, respectively. The retention times for standard sugars (galactose,
	glucose, arabinose, and rhamnose) in the present study were recorded at 14.54,
	H. durvillei 14.82, 10.6, and 8.64 min. respectively. Given this information above, it might be H. dilatata
	Raw possible that; (77%, at rt=13.79 min.), (8% at rt=10.27 min.), and (5% at rt=6.66 Hdur extract Raw Hdil extract Monosaccharide (% of total sugars) Fucose 1.84±0.07 1.45±0.18 1.43±0.88 1.66±0.11 min.) are representing galactose, arabinose, and rhamnose sugars and their retention
	Galactose times shifted for these sugar components. The same also follows with raw H. 77.0±0.2 76.0±0.2
	Arabinose Rhamnose dilatata and Hdil extract in the present study. The spectral bands at [940-935 cm -1 ] 8.0±0.2 8.0±0.0 -7.0±0.0 0.11±0.02 7.0±0.0
	Glucosamine for both Hdur and Hdil fractions confirm the presence of 3,6-anhydro-D-galactose -0.23±0.01 0.34±0.05 0.36±0.00
	Xylose Fructose residues.	2.34±0.24 2.25±0.05 -1.30±0.09	2.41±0.49 1.01±0.20	-1.26±0.15
	Glucoheptose Another study, hot water extracted fractions at different temperatures (25, 0.68±0.03 1.04±0.11 0.76±0.21 1.16±0.05
	Acide glucuronique 0.44±0.01 0.56±0.02 Total (µg mg -1 ) dw 11.22±0.86 13.57±0.47 80, and 80 °C) extracted from H. floresii reported sugar contents of xylose (2.7%), 0.38±0.05 0.64±0.02 9.79±1.85 15.35±0.53
	-= not detected; The values are expressed in g/100g of total sugars in the fractions. and glucose (5.1%) which are only detected on the first fraction (25 °C), and
	galactose content range (66.9-80.5%) in all fractions (Amorim et al. 2011). This
	reported xylose (2.7%) is also similar to the present study except for Hdil extract
	which it is not detected, and the galactose contents (about 70%) of all samples in

Table XLIV

 XLIV Hypnea bryoides[START_REF] Al-Nahdi | The Effect of Extraction Conditions on Chemical and Thermal Characteristics of Kappa-Carrageenan Extracted from Hypnea bryoides[END_REF]. Also, this peak has been detected in very low intensities, which also has been confirmed even in the alkali-modified fraction from[START_REF] Fenoradosoa | Highly sulphated galactan from Halymenia durvillei (Halymeniales, Rhodophyta), a red seaweed of Madagascar marine coasts[END_REF]. The medium intensity bands at [814-812 cm -1 ] for both Hdur and Hdil fractions in the present study indicate the presence of the C-O-SO4 group on C6 of galactose reported in the study of[START_REF] Al-Nahdi | The Effect of Extraction Conditions on Chemical and Thermal Characteristics of Kappa-Carrageenan Extracted from Hypnea bryoides[END_REF] for Hypnea bryoides extracts. Al-Nahdi adds that this peak implies the existence of μcarrageenan in the raw seaweed, which is a natural precursor of K-carrageenan that is present in the seaweed. Therefore, this implies that the red seaweeds H. durvillei and H. dilatata are natural sources of K-carrageenan. The small spectral bands at [769-766 cm -1 ] in the present study fractions, indicates the presence of skeletal bending of galactose rings. These similar peaks were also observed and reported in small intensity, for several polysaccharide extracts of red seaweeds (such as;

	(2016) reported that the intensity of these bands is proportional to the degree of
	summarizes some of sulfation. Moreover, sulfate contents account for 15% for each Hdur and Hdil
	the major IR vibrational modes of H. durvillei fraction (Hdur) and H. dilatata fractions in the present study. The spectral bands at [940-935 cm -1 ] from both Hdur
	fraction (Hdil) polysaccharides (Fenoradosoa et al. 2009; Kim et al. 2016; Fernando and Hdil fractions indicates the presence of 3,6-anhydro-D-galactose residues, has

et al. 2017; Al-Nahdi et al. 2019).

Figure 43. FT-IR spectra of H. durvillei and H. dilatata polysaccharide extracts.

The high-intensity spectral bands at [1224-1221 cm -1 ] from both Hdur and Hdil fractions were attributed to O=S=O stretching vibration of sulfate esters with some contribution from the vibration of COH, CC, and CO. This similar peaks also of high intensity were reported in the previous study for Iridaea cordata fractions (gelling, and soluble)

[START_REF] Kim | Anticancer Activity of Sulfated Polysaccharides Isolated from the Antarctic Red Seaweed Iridaea cordata[END_REF]

, also in native and alkali-extracted polysaccharides from H. durvillei

[START_REF] Fenoradosoa | Highly sulphated galactan from Halymenia durvillei (Halymeniales, Rhodophyta), a red seaweed of Madagascar marine coasts[END_REF]

, and in carrageenan extract from Hypnea bryoides

[START_REF] Al-Nahdi | The Effect of Extraction Conditions on Chemical and Thermal Characteristics of Kappa-Carrageenan Extracted from Hypnea bryoides[END_REF]

. Accordingly, Kim et al. also been found in all galactan fractions (desulfated, native, and alkali-modified) from H. durvillei

[START_REF] Fenoradosoa | Highly sulphated galactan from Halymenia durvillei (Halymeniales, Rhodophyta), a red seaweed of Madagascar marine coasts[END_REF]

, and in carrageenan extract from

Table XLIV .

 XLIV List of InfraRed (IR) vibrational modes characteristic to polysaccharides used in determining spectral bands of interest for the identification and discrimination of red algal polysaccharides from Halymenia durvillei and Halymenia dilatata (adapted from[START_REF] Fenoradosoa | Highly sulphated galactan from Halymenia durvillei (Halymeniales, Rhodophyta), a red seaweed of Madagascar marine coasts[END_REF][START_REF] Kim | Anticancer Activity of Sulfated Polysaccharides Isolated from the Antarctic Red Seaweed Iridaea cordata[END_REF][START_REF] Fernando | FTIR characterization and antioxidant activity of water-soluble crude polysaccharides of Sri Lankan marine algae[END_REF][START_REF] Al-Nahdi | The Effect of Extraction Conditions on Chemical and Thermal Characteristics of Kappa-Carrageenan Extracted from Hypnea bryoides[END_REF].

	allowance (Mac Artain, 2007). The species H. durvillei had a high total
	macroelement content (16.20% dw) with sodium (9.96%) as the highest, followed
	by potassium, calcium, and magnesium. Its microelements total content with
	(0.80% dw), had zinc, iron, copper, and manganese. While H. dilatata
	macroelements total content (31.70% dw) are rich in sodium (19.64%), followed Region/Band (cm -1 ) Type of bond Identification by potassium, calcium, and magnesium. Its microelement total content (1.64% dw),
	Common to Halymenia durvillei 1253 S=O had high iron (1.24%), followed by zinc, copper, and manganese. Eating 8 g of dry Stretching vibration of sulfate groups 1375 Sulfate ester substitution H. durvillei represents between 1.45% and 360.0% of the recommended daily
	933 intake (RDI) for minerals. These percentages were high, especially for the C-O-C 3,6-anhydro-D-galactose residues
	820 microelements. For H. dilatata, 8 g of dry seaweed represents 1646.67% of the RDI λ-carrageenans; sulfate ester substitution at O-6 at 4-and 3-linked galactose residues 835, 823 O-2 and O-6 Equatorial sulfate esters at O-2 and O-6 gal in iron. The Na/K ratios in H. durvillei and H. dilatata were 2.06 and 2.53,
	respectively.	gal residues	respectively
	Other Halymenia and related red species
	1651, 1643	O-H	Indicates the presence of water
	(1270-1210), 1219, 1211	O=S=O	Asymmetric stretching vibration of sulfate esters with some contribution from the vibration of COH, CC, and CO
	1200-970	CC and CO; COC stretch	CC and CO stretch in pyranoid ring, and COC stretching in glycosidic bonds
	1035 (1010-1090) C-O-C	Stretching vibrations of glycoside bridge
	820-810, 867		D-galactose-6-sulfate (D6S) or the presence of C-
	(shoulder)		O-SO4 group on C6 of galactose
	770		Skeletal bending of galactose rings
	586-578	O=S=O	Asymmetric and symmetric O=S=O deformation of sulfates
	5.5 Mineral analysis
	The mineral contents expressed as content of each mineral relative to the

seaweed dry weight (% of dw) of H. durvillei and H. dilatata are given in Table

XLV

. The percentages of the recommended daily intake covered by intake of 8 g of dry seaweed are also presented as well as the daily recommended intake

Table XLV .

 XLV Composition of macroelements and microelements (% dw), recommended daily intakes, and recommended daily allowances of Philippine red seaweeds Halymenia durvillei and Halymenia dilatata. Recommended daily intake and upper limit intake values for a male adult with a bodyweight of 70 kg according to the World Health Organization (2002); b Upper limit intake according to NationalHealth and Medical Research Council (2006); % RDI is based on a daily intake of 8 g dry weight of seaweed; RDA: recommended daily allowance; Data are mean ± SD (n=2). SD = standard deviation

	Species			Macroelements			Microelements
		Na	K	Ca	Mg	Na/K Fe	Zn	Mn	Cu
	RDI a (g day -1 )	2.0	3.8	0.84	0.350	0.006	0.012	0.0055 0.0017
	Upper limit intake b (g day -1 )	2.3	/	2.5	0.35	0.045	0.040	/	0.01
	H. durvillei	9.96±0 4.84±0 1.74±0.01 0.42±0 2.06 0.27±0	0.47± 0.03 0.001±0 0.06±0
	% RDI	39.84	10.19 9.34	9.67	360.00	310.00	1.45	282.35
	RDA (g day -1 )	23.09	/	254.84	82.74	16.67	8.60	/	16.67
	H. dilatata	19.64±0 7.76±0 2.77± 0.02 1.53±0 2.53 1.24±0.03 0.33±0.02 0.002±0 0.07±0
	% RDI	78.56	16.34 26.41	34.97	1646.67 218.33	2.91	337.25
	RDA (g day -1 )	11.71	/	90.16	22.88	3.64	12.21	/	13.95

a RDI =

  Grateloupia lanceola(Nunes et al. 2017). While, meager contents of chlorophyll a and b (0.00158, and 0.00189 mg g -1 fw) from Gelidiella acerosa[START_REF] Syad | Seaweeds as nutritional supplements: Analysis of nutritional profile, physicochemical properties and proximate composition of G. acerosa and S. wightii[END_REF]; and (0.5778, and 0.0011 mg g -1 fw) from Gracilaria changii, respectively (Chan and Matanjun, 2017) compared with chlorophyll a from H. durvillei (5.93, and 0 mg g -1 ), but are somewhat similar compared with chlorophyll a from H. dilatata (0.68, and 6.58 mg g -1 ) in the present study.Several varying results of carotenoids contents from different red seaweeds(e.g., H. floresii, G. changii, A. taxiformis, C. crispus, G. lanceola, Pyropia dioica, H. porphyroides, P. umbilicalis, P. palmata, Gracilaria spp., P. vietnamensis, E.

			Chlorophyll
		Chlorophyll			Total	Total
	Seaweeds	a	unidentified -Carotene	chlorophyll	pigments
	H. durvillei 5.93±4.99	-	54.47±0.00 5.94±5.00	60.41±5.00
	H. dilatata 0.68±0.02	6.53± 0.00	-	7.21±0.02	7.21±0.02

A. taxiformis, low (0.625 mg g -1 ) from C. crispus, and low (1.84 mg g -1 ) from

Table XLVII .

 XLVII the Vero cells was observed for both H. durvillei and H. dilatata extracts in the range of the concentrations tested. Both H. durvillei and H. dilatata extracts had low EC50 values or lower than those of CC50, signifies antiherpetic activity (TableXLVII). Efficacy or the cellular protection of H. durvillei and H. dilatata extracts showed that H. dilatata extract had maximum efficacy of 136.24% is higher than H. durvillei extract 114.0% at 200 µg mL -1 after 72 h infection. The potency or the effectivity of H. dilatata extract at 50% concentration, only needs 1.21 µg mL -1 of sample (EC50: 1.21 µg mL -1 ), which is almost five times more potent than H. durvillei extract (EC50: 5.89 µg mL -1 ). Efficacy and potency evaluation were shown in appendix 15, while its statistical analysis was shown in appendix 17.5 for both H. durvillei and H. dilatata extracts. Evaluation of cytotoxicity and antiviral activity by cellular viability of Philippine red seaweeds Halymenia durvillei and Halymenia dilatata.

CC50 (µg mL -1 ) EC50 (µg mL -1 ) SPECIES

  : The 50% Cytotoxic Concentration. The concentration that reduced the absorbance of mockinfected cells to 50% of that of controls. EC50: The 50% antiviral Effective Concentration: concentration that achieved 50% protection of virus-infected cells from the HSV-induced destruction.

		FRACTION	Destruction	Protection
	H. durvillei	Hdur	>200	5.89±1.13
	H. dilatata	Hdil	>200	1.21±0.97
	Zovirax		>200	0.37±0.01

CC50

  conclusion, the extraction yield results of H. durvillei and H. dilatata are affected by species used, geographic location, seasonal growth, and even by the sensitiveness of the methods used. Biochemical composition for both H. durvillei and H. dilatata extracts is mainly composed of neutral sugars (17.6, 12.2%), sulfates group (15.73, 15.22%), and proteins (7.0, 8.7%) dw of alga, respectively. FTIR spectrum of H. durvillei and H. dilatata extracts confirmed the presence of ester sulfates, and sugars, where, H. durvillei and H. dilatata polysaccharides is mainly composed of carrageenan with galactose, arabinose, and rhamnose as the dominant sugar units. The H. durvillei and H. dilatata polysaccharides implies the presence of K-carrageenan, due to the medium intensity bands at [814-812 cm -1 ] that indicates the presence of its natural precursor, μcarrageenan. Therefore, this implies that the red seaweeds H. durvillei and H. dilatata are natural sources of K-carrageenan. Based on the results, the H. durvillei and H. dilatata mineral contents are within safe levels and are potential functional foods for humans. Also, H. durvillei and H. dilatata are excellent sources of pigments that can be used in the food industry and human health development. The H. durvillei and H. dilatata fractions showed in vitro antiherpetic activity without cytotoxicity. Polysaccharides from H. dilatata fraction is five times more potent (EC50: 1.21 µg mL -1 ) than H. durvillei fraction (EC50: 5.89 µg mL -1 ). Although both H. durvillei and H. dilatata extracts are weak antioxidants (DPPH scavenging assay), they still are considered significant since these extracts are derived from natural resources and therefore can be cultivated, extracted, and can be a renewable source of bioactive metabolites.

  TableXLIXshowed the summary of results in the present study.The C. racemosa and U. lactuca species reported have high amounts of protein. The C. racemosa offers real nutritional value, while U. lactuca could also be considered good for consumption. The total EAAs of C. racemosa and U. lactuca species were comparable to FAO/WHO requirement values. The fresh C. racemosa and U. lactuca seaweed species should be preferred for human consumption. The brown algae, S. polycystum, and S. ilicifolium are mainly composed of carbohydrates (62.0%, 42.4%), significant presence of ash (24.9%, 17.0%), and proteins (24.2%, 17.8%), respectively. Moreover, the red algae, H. durvillei and H. dilatata are mainly composed of ash or minerals (41.0%, 46.3%), followed by carbohydrates (30.59%, 25.2%), respectively. The total EAAs of H. durvillei and

Table XLIX .

 XLIX Summary results of the major analysis of all seaweed samples in the present study.

		GREEN	BROWN	RED
		C. racemosa	U. lactuca	S. polycystum	S. ilicifolium	H. durvillei	H. dilatata
	Proteins (% dw)	19.9	11.1	24.2	17.8	5.3	5.5
	Total EAAs (%)	45.2	42.1	n.d.	n.d.	38.0	46.5
	Total NEAAs	54.7	57.8	n.d.	n.d.	61.9	53.4
	EAA/NEAA	0.8	0.7	n.d.	n.d.	0.6	0.8
	Ash (% dw)	29.4	23.3	24.9	17.0	41.0	46.3
	Na (%)	7.5	4.7	-	-	9.9	19.6
	K (%)	-	-	17.1	2.1	-	-
	Fe (%)	0.5 or (9.2 g/day RDA)	1.8 or (2.5 g/day RDA)	1.2 or (3.8 g/day RDA)	1.6 or (2.7 g/day RDA)	0.3 or (16.6 g/day RDA)	1.2 or (3.6 g/day RDA)
	Extraction Yields (% dw)	HWE (13.0), 1OH (6.5), 4OH (8.0), INS (28.0)	DS (63.41), DA (28.5), PP1 (4.5), PP3 (8.4) PP2 (91.2),	Sp CaCl2 (13.8), Sp KOH (6.2) Sp HCl (1.6), Sp Na2CO3 (10.7),	Si CaCl2 (6.0), Si KOH (5.5) Si HCl (2.5), Si Na2CO3 (29.6),	H. dur. Extract (5.4)	H. dil. Extract (26.3)
	Polysaccharides biochemical composition	mainly of sulfates group, and proteins	mainly of sulfates group, proteins, and uronic acids	mainly of neutral sugars, sulfates group, and proteins	mainly of neutral sugars, sulfates group, and proteins

Algues marines d'importance économique aux Philippines : évaluation de six espèces Rexie Magdugo 2020

Remerciements

Cette étude a été financée par le programme de

LIST OF TABLES

List of abbreviations

AA: Amino Acids sugars content in the present study are mostly 2 to 3 times lower than [START_REF] Hardouin | Enzyme-assisted extraction (EAE) for the production of antiviral and antioxidant extracts from the green seaweed Ulva armoricana (Ulvales, Ulvophyceae)[END_REF]. The present study's total phenolic contents are 3 to 6 times higher than [START_REF] Hardouin | Enzyme-assisted extraction (EAE) for the production of antiviral and antioxidant extracts from the green seaweed Ulva armoricana (Ulvales, Ulvophyceae)[END_REF].

Monosaccharide composition analysis

The unit sugar composition of crude and purified fractions of C. racemosa and Ulvan of U. lactuca fractions were determined by High-Performance Anion Exchange Chromatography (HPAEC). The results are given in Table XVIII and XIX and expressed as a percentage of sugars relative to the total sugars detected.

Eleven monosaccharides have been identified present in raw C. racemosa, 9 in Cr crude, 11 in 1OH, 10 in 4OH, and 11 in INS. Main contents of monosaccharides in raw C. racemosa were xylose with 10.03%, and glucose with 7,88%, small amounts (<10%) of glucose, galactose, mannose, fucose, and ribose;

and trace amount (<1.0%) of fructose, glucosamine, rhamnose, glucuronic acid, and arabinose. The Cr crude fraction had glucose (54.8%), and galactose (18.4%) as major monosaccharides, with small amounts (<10%) of glucuronic acid, xylose, fucose, glucosamine, mannose, and rhamnose; and a trace amount of arabinose.

Identified monosaccharides from isolated fractions (1OH=11, 4OH=10, and INS=11) revealed that 1OH is rich in glucose (50.41%); followed by small amounts (<10%) of galactose, mannose, xylose, fructose, and fucose; and trace amounts (<1.0%) of glucuronic acid, arabinose, and rhamnose. Fraction 4OH only had small amounts (<10%) of glucose, galactose, and mannose; and trace amounts (<1.0%) of fructose, xylose, mannitol, fucose, rhamnose, arabinose, and glucosamine, that varies (from 0.09 to 0.9%). Insoluble polysaccharide (INS) had small amounts (<10%) of glucose, and rhamnose; and trace amounts (<1.0%) of galactose, mannose, glucuronic acid, fructose, mannitol, xylose, fucose, arabinose, and glucosamine that ranged from 0.12 to 0.99%. The total monosaccharides contents Moreover, the present study fractions Si Na2CO3, Sp CaCl2, and Sp Na2CO3 produced better yields than the following: Aquadest extract from Indonesian Sargassum (8.5%) (Hidayati et al. 2019), methanolic extract (8.3, 3.1, and 5.7%) from S. polycystum, S. ilicifolium, and S. aquifolium, respectively (Puspita et al. 2017), and hot water extract (6.59%) from S. fusiforme (Ye et al. 2018). While, fucoidan fraction from S. tenerrimum produced better yield (31%) [START_REF] Sinha | Polysaccharides from Sargassum tenerrimum: Structural features, chemical modification and antiviral activity[END_REF] than FCSPs fractions Sp CaCl2 + Sp HCl, or Si CaCl2 + Si HCl in the present study.

Biochemical composition analysis of extracts

Biochemical composition of S. polycystum (Sp) and S. ilicifolium (Si) extracts (CaCl2, HCl, Na2CO3, and KOH) were shown in All Sp fractions in the present study had sugar units that are somehow similar or slightly lower than the reported mean range values for fucose (8.2-13%), galactose (5.4-11.5%), mannose (3.7-4.7%), and xylose (1.3-3.9%); while reported

Biochemical composition analysis of raw material

Determining the biochemical composition and carrageenan content from seaweed Halymenia durvillei and Halymenia dilatata for the present study may help in providing data for its use as potential natural source raw material for carrageenan industry. The biochemical composition of raw material from red seaweed

Halymenia durvillei and Halymenia dilatata for the present study were given in Table XXXVI.

Halymenia durvillei composition showed a high amount of ash (41.0%), followed by neutral sugars (21.2%), and smaller amounts (<10%; (7.2-2.08%)) of sulfate groups, lipids, proteins, polyphenols, and uronic acids content of the dry weight, respectively. While Halymenia dilatata composition was rich in ash (46.3%), followed by neutral sugars (16.2%), and smaller amounts (<10%; (6.7-2.32%)) of sulfate groups, proteins, polyphenols, and uronic acids of the dry weight of alga, respectively. Carbohydrates including sulfated polysaccharides (corresponding to uronic acids, sulfate groups, and neutral sugars) in H. durvillei and H. dilatata are about 30.59% and 25.2%, respectively. Ash content for raw H. durvillei (41%) and raw H. dilatata (46.3%) are the most abundant component in the present study. [START_REF] Kho | for H. durvillei (1.29%), and H. dilatata (0.29%) of the same species, while H. maculata (1.09%) was reported higher than H. dilatata. Wanyonyi et al. (2017) reported lipid content (0.62% dw) from K. alvarezii, which is similar to raw H. dilatata, but 10-times lower than raw H. durvillei in the present study[END_REF] reported a similar ash content (39.54%) from H. durvillei of the same species, a lower ash content (21.92%) from H. dilatata of the same species, and also a lower ash content

Antioxidant activity, DPPH radical scavenging activity

Percent maximum scavenging ability and inhibition activity of H. durvillei and H. dilatata polysaccharide extracts at (10 mg mL -1 ) were shown in Table XLVIII.

Scavenging ability for H. durvillei polysaccharide extract (Hdur extract)

showed that it is higher than H. dilatata polysaccharide extract (Hdil extract) with values of 33.9%, and 24.13% at 10 mg mL

Appendix 2. Cell maintenance

The cells are cultured in 30 mL of enriched MEM medium in cellular culture flasks (Falcon, Corming). They are incubated at 37°C in an oven containing 5% of CO2 atmosphere and saturated with water vapor. The medium was renewed daily.