Microencapsulation, Activité antioxydante.

Cette thèse s'inscrit dans la cadre de la thématique du Laboratoire de Pharmacognosie et du Laboratoire de Pharmacie Galénique de l'UFR Sciences de Santé, circonsription Pharmacie, au sein de l'Université de Bourgogne Franche-Comté, afin de trouver de nouvelles molécules naturelles bioactives à encaspuler. D'une part, nous nous sommes concentrés sur la recherche de saponines naturelles de plantes issues de la biodiversité vietnamienne et de l'horticulture française du genres Dracaena, Cordyline (Asparagaceae) et Weigela (Caprifoliaceae). Les travaux menés ont conduit à isolement de 35 saponines naturelles en utilisant différentes techniques chromatographiques. Les structures ont été déterminées par des méthodes de spectrométrie de masse en source ESI et de spectroscopie RMN. Parmi les 17 composés purs obtenus des 3 espèces appartenant au genre Weigela, 9 sont des glycosides de l'acide oléanolique et de l'hédéragénine de structure nouvelle. A partir des espèces Dracaena braunii et Cordyline fruticosa "Fairchild red", nous avons isolé et caractérisé 18 saponines stéroïdiques dont 7 nouvelles de type spirostane et 6 nouvelles de type furostane. Les activités cytotoxiques de la majorité des saponines isolées ont été évaluées sur trois lignées cellulaires : CT26 (cellules tumorales coliques murines), B16 (cellules de mélanome murin) et HepG2 (cellules d'hépatocarcinome) par le test de prolifération cellulaire MTS. Des relations structure/ activité ont ainsi été proposées. D'autre part, nous avons sélectionné une molécule naturelle bien connue afin de mettre au point les essais d'encaspulation. La curcumine possède des propriétés thérapeutiques très intéressantes mais elle présente à la fois une faible solubilité et une faible biodisponibilité, limitant l'administration par voie orale. Dans cette partie de la thèse, nous avons cherché à améliorer la stabilité et la biodisponibilité de la curcumine ansi qu'une libération contrôlée dans le tractus gastro-intestinal. Des billes de pectinate de calcium ont préparées en utilisant la gélification ionique en présence de différents tensioactifs. Elles ont été caractérisées grâce à leur propriétés physico-chimiques et leur profils de dissolution in vitro. Leur activité antioxydante a été évaluée par le test DPPH. Le Kolliphor ® HS 15 est le tensioactif le plus prometteur pour optimiser les propriétés de la curcumine.

Title : Valorization of natural drug products: from extraction to encapsulation

Keywords : Saponins, Cytotoxicity, Weigela, Cordyline, Dracaena, Curcumin, Microencapsulation, Antioxidant actitivy. This thesis was carried out at the Laboratory of Pharmacognosy and the Laboratory of Pharmaceutical technology, at the UFR Sciences de Santé, circonscription Pharmacy, in the University of Burgundy Franche-Comté, to find new natural molecules to encapsulate. First of all, we focused on the natural saponins from plants of the Vietnam biodiversity and the French horticulture, belonging to the three genera Dracaena, Cordyline (Asparagaceae) and Weigela (Caprifoliaceae). The work led to the successful isolation and elucidation of 35 natural saponins using various chromatographic techniques. The structures were determined by ESI mass spectrometry and NMR spectroscopy. Among the 17 pure compounds obtained from three species of the Weigela genus, 9 oleanolic acid and hederagenin glycosides are previously undescribed ones. From the two species Dracaena braunii and Cordyline fruticosa "Fairchild red", we isolated and characterized 18 steroidal saponins including 7 new spirostane-types and 6 new furostane-types ones. The cytotoxic activities of the majority of isolated saponins were evaluated against mouse colon cancer (CT26 cells), mouse melanoma (B16 cells) and human liver cancer (HepG2 cells) by MTS assays. The structure / activity relationships were also proposed.

On the other hand, we selected a wellknown natural molecule to develop encapsulation tests. Among the natural products, curcumin has very interesting therapeutic properties but exhibits both a poor solubility and a low bioavailability, limiting the administration by the oral route. The purpose of this study was to improve the solubility and bioavailability of curcumin as well as simultaneously achieve controlled release in gastrointestinal tract. Pectinate gel beads were prepared based on ionotropic gelation method with the presence of vaious surfactants. After drying, these beads were investigated for physicochemical characteristics (morphological aspects, encapsulation efficiency, stability, physical state), dissolution kinetics (in vitro release) and antioxidant activity was determined with DPPH assay. Kolliphor ® HS 15 seems to be the best promising surfactant to increase stability and bioavailability of curcumin.

AKNOWLEDGEMENTS

My sincere appreciation is addressed to my Ph.D supervisor Prof. Odile Chambin. I would like to express my deep and sincere thankfulness for giving me the opportunity to work on my thesis in such creative research environment. Her continuous support, guidance and encouragements help me stand on my feet and climb through the ups and downs of my PhD journey which would has never been this far without her wise advices. I am also deeply grateful to my second supervisor, Prof. Anne-Claire Mitaine-Offer, who coordinated, guided and inspired me through my research work. With her enthusiasm, cheerful character and excellent encouragement, she always showed me how to see problems during my research and taught me how to find solutions. I owe my most sincere gratitude to Prof. Marie-Aleth Lacaille-Dubois, Director of Laboratory of Pharmacognosy, Université de Bourgogne. She was always willing to help with her experience and supported me with tips and analytical tricks throughout my research.

I also express my thankfulness to Prof. Marie-Pierre Flament and Prof. Yves Wache, two members of thesis supervisory committee. They offered advices about and assessment of my research.

I would like to thank all colleagues in Université de Bourgogne for providing a stimulating and nice environment. I also warmly thank the Vietnamese Government, for providing me the scholarship, and my colleagues in Department of Biology, University of Education, Thainguyen University, Vietnam for their encouragement.

The laboratory of PAM, especially to Physico-chemistry of Food and Wine (PCAV) team, Agrosup Dijon and Dr. Gaëlle Roudaut are acknowledged for inspiring me at the beginning of PhD journey. Special thanks go to Mr. Bastien Petit, for being a great lab mate throughout my whole PhD journey in Dijon. Bastien, my friend, I will never forget the words "Paradise" and "Cafélisto" for the rest of my life. Thank you for all the crazy moments we have ever done! I also warmly thank Mr. David Pertuit, a great technician, for supporting me during the fraction and isolation process. I am sincerely grateful for his patience and long hours spent to teach me how to use all equipments. Futhermore, I am also greatful to the colleagues of laboratory of Pharmacognosy: Dr.

Tomofumi Miyamoto, Dr. Chiaki Tanaka, Dr. Thomas Paululat for the measurements of the diverse NMR and MS experiments. Thankfulness is given to Dr. Bertrand Collin and Dr. Pierre-Simon Bellaye for help in cytotoxic activity.

To my lovely wife…

My wonderful wife Huyen could not be here during my PhD journey but she has always encouraged me and offered me the support that I needed to complete this thesis. I would have dropped out this journey and would not have written these words if I had lived without her. Huyen, my darling, you showed me what it means to love and to be loved, I am a lucky person with you. In the deepness of my heart, I give you a great love and appreciation for your faithful patience and all the moments we shared together, I thank you for being a half of my life.

…my sons…

Nam, my second son, I started my PhD journey while you were still in your mother's womb. I am sorry you, my son, for one reason that I could not be there when you were born. It can be the biggest regret of my life.

To Lam, my first son, I still remember the day I had to say goodbye to you. It has been an unforgettable in my mind. I hope you will understand my situation and excuse my absence at home.

…and my family!

My gratefulness address to my parents who have been supporting me in each and every step and breath I take, to my mother Ngan who gives me encouragement through her neverended good wished, pray and emotional support, and to my father Hong whose kindness, inspiration always be in my heart. My gratitude is also forwarded to my big brother Ha and his wife Thao for their continuous tenderness and encouragement. To my mother-in-law Chien, my aunts-in-law Khay and Chin, my sister-in-law Ha and her husband Dat, receive my deep gratitude and love for your dedication and trust in me.

I thank to my aunt Hoa and two my uncles Tuan and Dung, you showed me the importance of education and helped me find the right way on every step of my life.

Last, but not least, to Madam Agnès Roy, receive my great gratitude and love for your supports during my whole schooling. Not only did you supply me with energy and motivation every time I depressed but also you taught me languages and the significance of life and love. Agnès, you have always been the one to make me better. For your love and endless support, I am happy to dedicate this thesis to you.

GENERAL INTRODUCTION

Nowadays, chemistry and biology laboratories around the world are studying on natural herbal medicines due to their safe to treat various diseases. There are several evidences to prove the use of traditional medicine in different cultures. Despite this historical importance of plants and considerable contemporary research into the identification of new naturally occurring chemical compounds, many of those are still not revealed both chemical structures and pharmacological activities. Saponin, a naturally occurring surface active glycoside, has been reported in recent years of publications about their properties. These compounds were discovered for their biological activities, from traditional uses to pharmaceutical applications. Saponins possess a various of pharmaceutical potentials, such as antitumor, anti-inflammatory, antiviral, antioxidant and antibacterial [START_REF] Moghimipour | Saponin: Properties, Methods of Evaluation and Applications[END_REF]. In this context, the Laboratory of Pharmacognosy, EA 4267, at the University of Burgundy Franche-Comté, is focusing on research of biologically active saponins from various plant families. Our research strategy consists in the selection of families and genera known for their richness in saponins, based essentially on chemotaxonomic criteria. Then, after extraction, isolation and structural characterization, the natural molecules are subjected to in vitro biological evaluation mainly in the field of cancerology. Due to these steps, three genera Dracaena, Cordyline and Weigela of two families Asparagaceae and Caprifoliaceae, were chosen for phytochemical investigation. It is interesting to note that many traditional uses of several species belonging to these genera present a potential in the pharmaceutical domain. Saponins were extracted and identified by using various chromatographic methods. These compounds were further evaluated for their cytotoxic activities and the analysis of relationships between structure and activity were carried out.

Aglycon (or sapogenin), an hydrophobic portion of saponin, has gained importance as a novel drug. But, their major limitation of its poor aqueous solubility leads to a relatively short half-life, low bioavailability and less permeability and degradation during human digestion. These problems are able to be overcome by various encapsulation method [START_REF] Anand | Bioavailability of Curcumin: Problems and Promises[END_REF]. However, extracted saponins is not enough to provide a good quantity, so encapsulation are not carried out with this compound. Fortunately, curcumin, the principal curcuminoid found in turmeric which exhibits a wide spectrum of biological and pharmacological effects, is also a hydrophobic natural compound with low water solubility quite similar to saponins. We pointed out here an idea of encapsulation of curcumin instead of sapogenin in order to improve the solubility and bioavailability of hydrophobic compounds. This study was done using ionotropic gelation method with the presence of surfactants. Physicochemical characteristics, dissolution kinetics and antioxidant activity were further evaluated for efficacy of encapsulation to enhance the solubility and protect antioxidant capacity of curcumin, in view of future food applications. This present thesis is structured by two parts:

-The first part will be carried out on saponin including three chapters. The first chapter will concern a literature review about botanical study and previous phytochemical works. The second chapter will give phytochemical investigations on selected plants. The third chapter corresponds to biological study on isolated compounds, in collaboration with Centre Georges-François Leclerc, ICMUB UMR CNRS 6302, using MTS colorimetric assay on different colon cancer cell lines.

-The second part will give a study on encapsulation of curcumin including three chapters.

The first chapter will inform about chemical profile of curcumin and general background about some encapsulation methods. The second chapter will present the methodological and material tools used to manufacture beads, together with investigations of physicochemical characteristics, dissolution kinetics and antioxidant activity of beads. The third chapter will show the results and discussion.

A general conclusion also will be carried out at the end of this thesis providing an overview on results and propose perspectives. 3. 13 C and 1 H NMR spectroscopic data of the aglycon moieties of 1-6 in pyridine-d5 ....... Table 4. 13 C and 1 H NMR spectroscopic data of the sugar moieties of 1-6 in pyridine-d5 ........... Table 5. 13 8. 13 C and 1 H NMR spectroscopic data of the aglycon moieties of 18-26 in pyridine-d5 . Table 9. 13 C and 1 H NMR spectroscopic data of the sugar moieties of 18-26 in pyridine-d5 ..... Saponins possess strong pharmaceutical properties as anti-inflammatory, antimicrobial, anticancer, anti-cardiovascular, adjuvants and absorption enhancer [START_REF] Moghimipour | Saponin: Properties, Methods of Evaluation and Applications[END_REF]. Saponins are also known to possess mineral complexes of iron, zinc, and calcium. The beneficial effect of absorption activity is a significant consideration to sort of saponins [START_REF] Milgate | The nutritional & biological significance of saponins[END_REF] Cordyline fruticosa "Fairchild red" is a cultivated dwarf evergreen shrub, belongs to a genus rich in saponins according to literature data [START_REF] Mimaki | Steroidal glucosides from leaves of Cordyline stricta[END_REF](Mimaki et al., , 1998a As a part of the Dracaena genus, this plant may contain saponins playing an interesting role in bioactivities.

In our continuing research for new biologically active saponins and completing the chemotaxonomic data about saponins from the genus Weigela (Caprifoliaceae), three species including W. x "Bristol ruby", W. florida "Pink poppet" and W. florida "Jean's gold" were phytochemically investigated, together with two species of the Asparagaceae family, Cordyline fruticosa "Fairchild red" and Dracaena braunii.

1. Botanical study 2009). They are distributed worldwide but the origin was probably in the Northern Hemisphere. Plants in Dipsacales have opposite, often gland-toothed leaves, petals fused into a corolla tube and inferior ovaries. Most members of the order are shrubby, but there are a few herbaceous members as well [START_REF] Britanica | Britanica[END_REF].

Family Caprifoliaceae

Caprifoliaceae, one of two families of order Dipsacales, contains 42 genera and over 890 species contributed largely in warm temperate areas in the Northern Hemisphere (Britanica). They are trees or herbs that can be recognized by their opposite leaves and often rather weakly monosymmetric flowers with a more or less radially symmetric calyx; the ovary is inferior and the fruits are often few or one seeded. The bark in the woody taxa often comes off in thin flakes (Missouri Botanical Garden). The species Weigela florida has ovate-oblong in leaves with 5-15 cm in length which an acuminate tip and a serrated margin are included. The flowers have a length of 2-4 cm, with a five-lobed white, pink, or red corolla which are produced in small corymbs of several together in early summer. The fruits appear like dry capsules containing small winged seeds [START_REF] Lamarck | Encyclopédie méthodique: Botanique[END_REF].

Order Asparagales

Asparagales, an order of monocot plants, has been classified by Angiosperm Phylogeny Group. It was introduced firstly by Huber in 1977 [START_REF] Huber | The Treatment of the Monocotyledons in an Evolutionary System of Classification[END_REF] and later by Krikorian

Family Asparagaceae

Placed in the order Asparagales, Asparagaceae is a family of flowering plants which includes 114 genera with 2900 known species (Christenhusz and Byng, 2016). Species in this family are herbaceous plants, with rhizomatous roots, fibrous, or thickened in tuber.

They develop branched, erect or climbing stems, thorny and sometimes high as in Asparagus umbellatus. Their leaves are small, thorny and completely devoid of stipules.

The flowers are small, occur cyclically which have 6 tepals, 2 whorls of 3 stamens and a gynoecium with 3 carpels. These plants have superior ovaries and three compartments that mature into dry capsules. This family posscess phytomelanin which create black colored seed coats. The fruits are small berries (Willamette Botany, 2001).

Genus Dracaena

The botanical name of Dracaena comes from ancient Greek "drakaina" which means dragon. It refers to a deep red resin and comprises approximately 60 species which are found nearly in tropical and subtropical of Africa [START_REF] Marrero | A new species of the wild dragon tree, Dracaena (Dracaenaceae) from Gran Canaria and its taxonomic and biogeographic implications[END_REF]

Genus Cordyline

Cordyline is derived from the ancient Greek "kordyle", meaning club which is a reference to the enlarged underground stems or rhizomes [START_REF] Ho | Cordyline obtecta[END_REF] [START_REF] Naidu | Natural Food Antimicrobial Systems[END_REF]. Most saponins have haemolytic properties and are toxic to most cold-blooded animals. [START_REF] Herlt | Two Major Saponins from Seeds of Barringtonia asiatica : Putative Antifeedants toward Epilachna sp. Larvae[END_REF] reported that saponins extracted from seeds of Barringtonia asiatica Kurz., a "fish killer tree" in tropical Asia and Pacific, exhibit piscicidal activity to fish when thrown into water.

In structure, saponin contain sugar chain(s) attached with an aglycon (or sapogenin). These sugars could be attached to aglycon one, two or three side chains and the term monodesmosidic, bi-desmosidic and tri-desmosidic are named for these saponins, respectively (in Greek, desmos means chain) [START_REF] Naidu | Natural Food Antimicrobial Systems[END_REF]. Aglycons (or sapogenins) are divided into two groups based on their skeleton: steroidal and triterpenoid. Steroidal saponins almost present in the monocotyledonous angiosperms, while triterpenoid saponins occur mainly in dicotyledonous angiosperms [START_REF] Bruneton | Pharmacognosie, phytochimie, plantes médicinales[END_REF] Due to the chemical properties and soap-like foaming, saponins are also used in food industry and cosmetics. In example, the saponin of Quillaja saponaria is accepted to be used in food as "Generally Recognized as Safe" (GRAS) in the USA [START_REF] Zhao | Efficient protocol for isolation and purification of different soyasaponins from soy hypocotyls[END_REF].

As pointed out above, saponins can be divided into two groups due to the nature of their In nature, the saponins mainly consist of (25S)-spirostane derivatives (neosaponins), (25R)spirostane derivatives (isosaponins), and (25S)-and (25R)-furostane derivatives. are also reported. The hydroxyl groups can be acylated, and this leads to the formation of ester saponins. Acidic components in such cases are formic, acetic, n-and iso-butyric, isovalerianic, α-methyl butyric, angelic, tiglic, benzoic, cinnamic, and ferulic acid, and, in some cases, sulfuric acid.

Me

Cordyline rubra [START_REF] Yang | Two furostane sapogenins from Cordyline rubra[END_REF] Cordyline rubra [START_REF] Yang | Two furostane sapogenins from Cordyline rubra[END_REF] Cordyline stricta [START_REF] Mimaki | Steroidal glucosides from leaves of Cordyline stricta[END_REF] Cordyline stricta [START_REF] Mimaki | Steroidal glucosides from leaves of Cordyline stricta[END_REF] Cordyline stricta [START_REF] Mimaki | Steroidal glucosides from leaves of Cordyline stricta[END_REF] Cordyline stricta [START_REF] Mimaki | Steroidal glucosides from leaves of Cordyline stricta[END_REF] Cordyline stricta [START_REF] Mimaki | Steroidal glucosides from leaves of Cordyline stricta[END_REF])

R H OH

Cordyline stricta [START_REF] Mimaki | Steroidal glucosides from leaves of Cordyline stricta[END_REF] Cordyline stricta (Mimaki et al., 1998a) Cordyline stricta (Mimaki et al., 1998a) Cordyline stricta (Mimaki et al., 1998a) Cordyline stricta (Mimaki et al., 1998a) Cordyline stricta (Mimaki et al., 1998a) Cordyline fruticosa (Fouedjou et

R1 R2 R3 H H α-L-Rha H H α-L-Rha α-L-Rha β-D-Fuc

Methods of isolation

Several chromatographic techniques are used to identify and separate the crude extract and obtain pure saponins including TLC (Thin Layer Chromatography), HPTLC (High Performance Thin Layer Chromatography), VLC (Vacuum liquid chromatography), MPLC (Medium Pressure Liquid Chromatography) and HPLC (High Pressure Liquid Chromatography).

Chromatographic methods of analysis a. Thin Layer Chromatography (TLC)

TLC is a simple, quick and inexpensive procedure used to identify how many components are in a mixture. A TLC plate is a sheet of glass coated with a thin layer of a solid absorbent.

A small amount of mixture which need to be analysed is spotted near the bottom of this plate. The TLC plate is placed in a rectangular TLC developing tank with a solvent. This solvent is the mobile phase and it slowly rises on the TLC plate by capillary action. The TLC plates are observed on UV light from 254 to 366 nm before revelation.

Revelation

Vanillin sulfuric reagent: Solution consisting of 1 g vanillin, 2 mL sulfuric acid and 95% ethanol up to 100 mL.

After soaking the plates in a dip tank containing the reagent, the plates are then dried at 110°C for a few minutes. Several colorations appear depending on the type of compounds.

Detection spots of saponin

The saponin spot forms mainly green on TLC plate, but in some cases, it is the spot of sugar.

b. High Performance Thin Layer Chromatography (HPTLC)

High-performance thin-layer chromatography (HPTLC) is an enhanced form of thin-layer chromatography (TLC). Various enhancements can be made to the basic method of thinlayer chromatography to automate the different steps, to increase the resolution achieved and to allow more accurate quantitative measurements. Chromatographic conditions for HPTLC are similar to TLC (Reich and Maire-Widmer, 2019).

Preparative chromatographic methods

a. Column Chromatography (CC)

Column Chromatography is a method used to purify individual chemical compounds from mixtures of compounds depending on molecular mass or special structure. A column is filled by a stationary phase and an organic solvent or a mixture of solvents flows down through the column. Sample is placed on the top of column and the eluent is then added. -Mobile phase: MeOH 100%, EtOH 100%

Components

b. Vacuum-liquid chromatography (VLC)

VLC may be considered as a preparative TLV run as a column with a vacuum applied to speed up eluent flow rates. VLC is mainly used for the fractionation of natural products prior to other separation steps such as RPC, MPLC and HPLC [START_REF] Sticher | Natural product isolation[END_REF]. VLC is carried out by using a sintered glass and a vacuum pump. Stationary phase is washed by MeOH and equilibrated by the solvent used as an eluent. Sample is dissolved in water (if stationary phase is reversed phase) or in mixture Chloroform: Methanol: Water (if stationary phase is normal phase).

Chromatographic conditions -Stationary phase:

Silica for normal phase: 60 Å, 15-40 µm, Merck.

Silica for reversed phase: RP-18 Spherical C18 (300 Å, 75-200 µm, Silicycle).

-Mobile phase:

In case of normal phase:

CHCl3/MeOH/H2O 75/25/3; 70/30/5, 60/32/7. Tannin are stayed on the silica and interesting compounds are eluted.

In case of reversed phase RP-18: MeOH/H2O 0/1, 1/1, 1/0; EtOH/H2O 0/1, 1/1, 1/0. In this case, saponins are eluted in the methanolic or ethanolic solution, while sugars are eluted in water.

c. Flash chromatography

Flash chromatography is a rapid form of preparative column chromatography based upon an air pressure driven hybrid of medium and short column chromatography optimized for rapid separation. This is a process in which a vertical column (plastic or glass) is placed and that column is packed with the solid stationary phase from which the mobile phase is passed through it by gravity or some external pressure.

e. High Pressure Liquid Chromatography (HPLC)

HPLC is the more useful technique used to analyse substances of plant under a maximum pressure of 300 bars in analytical mode and 100 bars in semi-preparative mode. This technique is applied to isolate, separate and purify compounds in a mixture, as well as to display botanical relationships between different species based on a chromatographic comparison of their chemical compositions [START_REF] Hostettmann | Natural products | Liquid Chromatography[END_REF].

-Apparatus: Agilent Technologies Seri 1260 Infinity.

-Analytical mode: Column Luna 5μ C18(2) 100A 250 x 4.6 mm, 5 μm, flow rate 1mL/min.

-Semi-preparative mode: Column Luna 5μ C18(2) 100A 250 x 10 mm, 5 μm, flow rate 3mL/min.

-Eluent: Acetonitrile-Water (TFA 0.01%).

Methods of structural determination

Mass spectrometry (MS)

Mass spectrometry (MS) is an analytical method used to identify the mass of charged substances when they move in an electric filed. This technique is useful to identify molecular and isotopic composition of elements and detemine structure of compounds. A mass spectrometer generates multiple ions from the sample under investigation, it then separates them according to their specific mass-to-charge ratio (m/z), and then records the relative abundance of each ion type. A mass spectrum of the molecule is thus produced. It provides this result as a plot of ion abundance versus mass-to-charge ratio [START_REF] Hoffmann | Mass Spectrometry: Principles and Applications[END_REF].

Electro-Spray Ionization (ESI)

Electro-Spray Ionization (ESI) is a technique used in mass spectrometry for production of ions using an electrospray in which a high voltage is applied to a liquid to create an aerosol.

It is applied to structural determination because of its rapidity, sensitivity, higher accuracy -From 0.5 to 3 ppm: the protons of aglycon, with methyl, methylene and methane groups.

-From 3 to 4.5 ppm: the osidic protons except anomers.

-From 4.5 to 6 ppm: the anomeric protons (No.1 of the sugars) in the form of enlarged doublets or singlets and the ethylenic protons of aglycon (Massiot and Lavaud, 1995). 13 C NMR

In the same way as the 1 H NMR spectrum, the 13 C spectrum is divided into the following three resonance zones:

-From 10 to 60 ppm: the carbons of aglycon.

-From 60 to 90 ppm: the osidic carbons (except anomers) and some hydroxyl carbons of aglycon.

-From 90 to 110 ppm: the carbons of anomers.

-Above 110 ppm: ethylenic carbons, carbons of carboxylic acids, aldehydes, esters and ketones [START_REF] Agrawal | NMR spectroscopy of steroidal sapogenins and steroidal saponins: An update[END_REF][START_REF] Agrawal | Carbon-13 NMR spectroscopy of steroidal sapogenins and steroidal saponins[END_REF][START_REF] Massiot | Structural elucidation of saponins[END_REF].

b. Two-dimensional nuclear magnetic resonance spectroscopy (2D NMR)

Heteronuclear Single Quantum Correlation (HSQC)

The HSQC spectrum allows to interpret precisely the direct H-C ( 1 JH-C) heteronuclear couplings. In case of comparison to 1D

13 C NMR and 1D 1 H NMR spectra, the 1 H, 13 C HSQC spectrum reduces spectral overlap which highly benefits compound identification. It allows to identify CH, CH2, CH3 groups with the chemical shift assignment [START_REF] Elyashberg | Identification and structure elucidation by NMR spectroscopy[END_REF].

Heteronuclear Multiple-Bond Correlation (HMBC)

The data provided by HMBC show the long-range H-C heteronuclear couplings with 2 JH-C or 3 JH-C coupling constants. This spectrum is very useful for determining the structure of the aglycon, but above all for elucidating the sequence of sugars in the oligosaccharide chains and their position of attachment to the aglycon [START_REF] Reynolds | Choosing the Best Pulse Sequences, Acquisition Parameters, Postacquisition Processing Strategies, and Probes for Natural Product Structure Elucidation by NMR Spectroscopy[END_REF].

Correlated Spectroscopy (COSY)

The COSY demonstrates the protons with 2 JH-H geminal couplings and 3 JH-H vicinal couplings. They are used to determine the structure of sugars and to verify the position of certain protons of the aglycon [START_REF] Elyashberg | Identification and structure elucidation by NMR spectroscopy[END_REF].

Total Correlation Spectroscopy (TOCSY)

TOCSY allows to observe the correlations of all the protons of the same spin system, as for the COSY experiment, to extended through bond H-H correlation [START_REF] Elyashberg | Identification and structure elucidation by NMR spectroscopy[END_REF]. The ROESY spectrum is used to determine the signals coming from protons that are close in space. This spectrum can verify the data analysed by HMBC [START_REF] Elyashberg | Identification and structure elucidation by NMR spectroscopy[END_REF].

Acid hydrolysis and GC analysis

Each pure compound (3 mg) was hydrolysed with 2N aq. CF3COOH (5 mL) for 3 h at 95°C.

After extraction with CH2Cl2 (3 x 5 mL), the aqueous layer was repeatedly evaporated to dryness with MeOH until neutral, and then analysed by TLC over silica gel (CHCl3/MeOH/H2O 8/5/1) by comparison with authentic samples. Furthermore, the sugars residue was dissolved in anhydrous pyridine (100 L), and L-cysteine methyl ester hydrochloride (0.06 mol/L) was added. The mixture was stirred at 60 °C for 1 h, then 150

L of HMDS-TMCS (hexamethyldisilazane/trimethylchlorosilane 3/1) was added, and the mixture was stirred at 60 °C for another 30 min. The precipitate was centrifuged off, and the supernatant was concentrated under a N2 stream. The residue was partitioned between n-hexane and H2O (0.1 mL each), and the hexane layer (1 L) was analysed by GC [START_REF] Hara | Gas-Liquid Chromatographic Separation of Aldose Enantiomers as Trimethylsilyl Ethers of Methyl 2-(Polyhydroxyalkyl) -thiazolidine-4 (R) -carboxylates[END_REF]. The absolute configurations were determined by comparing the retention times with thiazolidine derivatives prepared in a similar way from standard sugars (Sigma-Aldrich).

Phytochemical investigation 2.1. Phytochemical study of Weigela x "Bristol Ruby"

Seven oleanane-type triterpenoid saponins were obtained from an aqueous-ethanolic extract of the roots of Weigela x "Bristol Ruby" and fractionated by vacuum-liquid chromatography (VLC) and purified by several solid/liquid chromatographic methods. Six of them are previously undescribed (1-6) in addition to one known compound (7).

Isolation and purification

The dried and powdered roots of Weigela x "Bristol Ruby" (

NMR spectroscopy

Structure of aglycon

The aglycon was identified by analysing spectra NMR 1 H, 13 C, HSQC and HMBC. In spectra of NMR 1 H and 13 C, characteristic signals of oleanolic acid were showed:

-7 methyl groups with singlet signals at δH 1.28, 1.12, 0.86, 0.99, 1.32, 0.97 and 1.02 confirmed the presence of triterpene.

-1 olefinic proton at δH 5.48 (br t, J = 3.6 Hz) corresponds to proton H-12.

The 13 C NMR spectrum showed:

-5 tertiary carbons (CH), 10 secondary carbons (CH2), 7 primary carbons (CH3) and 8 quaternary carbons (C).

-2 signals at δC 122.5 and 144.8 correspond to the double bond Δ 12 .

The 1 H and 13

Structure of sugar moieties

The monosaccharides were identified by acid hydrolysis and extensive 2D NMR analysis as α-L-arabinopyranosyl (Ara), α-L-rhamnopyranosyl (Rha), β-D-xylopyranosyl (Xyl) and β-D-glucopyranosyl (Glc) in the case of 1-3, and Rha, Xyl and Glc in the case of 4-7. The absolute configurations of the sugars were determined to be D for glucose (Glc), and xylose (Xyl), and L for arabinose (Ara) and rhamnose (Rha), by GC analysis (see Section 1.3.4).

The relatively large 3 JH-1,H-2 values of the Glc, Xyl, and Ara (6.0-8.0 Hz) indicated a β anomeric orientation for Glc and Xyl, and an α anomeric orientation for Ara. For compound 3, the broad singlet observed for the anomeric proton of the terminal arabinopyranosyl moiety is consistent with the 1 C4 conformation of Ara with an α-orientation of the anomeric center [START_REF] Ishii | The configuration and conformation of the arabinose moiety in platycodins, saponins isolated from Platycodon grandiflorum, and misaponins from Madhuca longifolia based on Carbon-13 and Hydrogen-1 NMR spectroscopic evidence: the total structures of the saponins[END_REF]. The large 1 JH-1,C-1 values of the Rha (165-168 Hz), confirmed that the anomeric protons were equatorial (α-pyranoid anomeric form).

For the sugar part of the molecule, the The sequence of the oligosaccharide was established by analysing the HMBC and ROESY spectra:

α-L-arabinopyranosyl showed to be attached to the C-3 position of the aglycon by observation of an HMBC correlation between δH 4.88 (Ara I H-1) and δC 88.7 (C-3) (Fig. 19).

-The HMBC correlation between δH 4.55 (Ara I H-2) and δC 101.

NMR spectroscopy

Structure of aglycon

The analysis of the NMR spectra of compound 2 showed that the aglycon was almost identified to those of compound 1. The correlations of HSQC, HMBC and ROESY allowed to identify the genin of compound 2 as the oleanolic acid which was characterised to compound 1 (Table 3).

Structure of sugar moieties

The molecular weight of compound The sequence of the oligosaccharide was established by analysing the HMBC and ROESY spectra:

α-L-arabinopyranosyl showed to be attached to the C-3 position of the aglycon by observation of an HMBC correlation between δH 4.88 (Ara I H-1) and δC 88.7 (C-3) (Fig. 28).

-The HMBC correlation between δH 4.54 (Ara I H-2) and δC 101.

Conclusion

The structure of 2 was elucidated as

3-O-β-D-xylopyranosyl-(1→4)-[β-D-glucopyranosyl- (1→3)]-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl- (1→2)-α-L-arabinopyranosyloleanolic acid.

NMR spectroscopy

Structure of aglycon

The analysis of the NMR spectra of compound 3 showed that the aglycon was almost identified to those of compounds 1 and 2. The correlations of HSQC, HMBC and ROESY allowed to identify the genin of compound 2 as the oleanolic acid which was characterised to compounds 1 and 2 (Table 3).

Structure of sugar moieties

The molecular weight of compound 3 is lower than 2 by 30 amu, corresponding to a pentosyl group instead of an hexosyl one. The HSQC spectrum of the sugar part showed According to the coupling constant of their anomeric protons, (d, J = 6.2 Hz) for Ara I-1 and (br s) for Ara II-1, the 4 C1 conformation was proposed for Ara I, and 1 C4 for Ara II.

-1 α-L-rhamnopyranosyl Rha-1 at δH 6.16 (br s), The sequence of the oligosaccharide was established by analysing the HMBC and ROESY spectra:

α-L-arabinopyranosyl showed to be attached to the C-3 position of the aglycon by observation of an HMBC correlation between δH 4.88 (Ara I H-1) and δC 88.8 (C-3) (Fig. 34).

-The HMBC correlation between δH 4.88 (Ara I H-2) and δC 101.

Conclusion

Thus, on the basis of the above results, the structure of compound 3 was established as 3-

O-β-D-xylopyranosyl-(1→4)-[α-L-arabinopyranosyl-(1→3)]-β-D-glucopyranosyl-(1→4)-β- D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-arabinopyranosyloleanolic acid.

Mass spectrometry

For compound 4, the same molecular formula as 2 (C63H102O29) was obtained according to its HR-ESIMS (positive-ion mode) spectrum which displayed a pseudo-molecular ion peak at m/z 1345.6409 [M+Na] + , and its ESIMS (negative-ion mode) spectrum with m/z 1322.1

[M-H] -.

NMR spectroscopy

Structure of aglycon

The analysis of the NMR spectra of compound 4 showed that the aglycon was almost identified to those of compounds 1, 2 and 3. The correlations of HSQC, HMBC and ROESY allowed to identify the genin of compound 4 as the oleanolic acid which was characterised to compounds 1, 2 and 3 (Table 3).

Structure of sugar moieties

The HSQC spectrum of the sugar part showed six cross-peaks at δH/δC 4.

NMR spectroscopy

Structure of aglycon

The analysis of the NMR spectra of compound 5 showed that the aglycon was almost identified to those of compounds 1, 2, 3 and 4. The correlations of HSQC, HMBC and ROESY allowed to identify the genin of compound 5 as the oleanolic acid which was characterised to compounds 1, 2, 3 and 4 (Table 3).

Structure of sugar moieties

The HSQC spectrum of the sugar part showed six cross-peaks at δH/δC 4.

Mass spectrometry

The HR-ESIMS (positive-ion mode) spectrum of compound 6 showed a pseudo-molecular ion peak HR-ESIMS (positive-ion mode) at m/z 1346.3724 [M+Na] + , indicating a molecular weight of 1322 and a molecular formula of C63H102O29.

NMR spectroscopy

Structure of aglycon

The analysis of the NMR spectra of compound 6 showed that the aglycon was almost identified to those of compounds 1, 2, 3, 4 and 5. The correlations of HSQC, HMBC and ROESY allowed to identify the genin of compound 6 as the oleanolic acid which was characterised to compounds 1, 2, 3, 4 and 5 (Table 3).

Structure of sugar moieties

Six sugar units were detected by HSQC correlations in the sugar region between protons at Table 3. 13 C and 1 H NMR spectroscopic data of the aglycon moieties of 1-6 in pyridine-d5 (δ in ppm, J in Hz)

NMR spectroscopy

Structure of aglycon

The analysis of the NMR spectra of compound 8 showed that the aglycon was almost identified to those of compounds isolated from W. x "Bristol Ruby". The correlations of HSQC, HMBC and ROESY allowed to identify the genin of compound 8 as the oleanolic acid which was characterised to compounds 1, 2, 3, 4 and 5 (Table 3 and5). The downfield shift at δC 88.5 and upfield shift at δC 180.0, due to the glycosylation shift, suggested that compound 8 was a monodesmosidic glycoside.

Structure of sugar moieties

The monosaccharides were identified by acid hydrolysis and extensive 2D NMR analysis as α-L-arabinopyranosyl (Ara), α-L-rhamnopyranosyl (Rha), β-D-xylopyranosyl (Xyl) and β-D-glucopyranosyl (Glc) in the case of 8, 11 and Rha, Xyl and Glc in the case of 9, 10.

The absolute configurations of the sugars were determined to be D for glucose (Glc), and xylose (Xyl), and L for arabinose (Ara) and rhamnose (Rha), by GC analysis (see Section -The HMBC correlation between δH 5.85 (Ara H-1) and δC 79.8 (Glc C-3) and in ROESY at δH 5.85 (Ara H-1) and δH 4.43 (Glc H-3) proved that Ara was attached to the Glc at C-3.

81

-The HMBC correlation between δH 5.28 (Xyl III H-1) and δC 73.8 (Glc C-4) and in ROESY at δH 5.28 (Xyl III H-1) and δH 4.42 (Glc H-4) proved that Xyl III was attached to the Glc at C-4. Overlapped proton signals are reported without designated multiplicity.

Conclusion

Phytochemical study of Weigela florida "Jean's Gold"

Phytochemical investigation on aqueous-ethanolic extract of the aerial parts of Weigela florida "Jean's Gold" led to the isolation of two previously undescribed triterpenoid saponins (12, 13) along with four known compounds (14-17).

Isolation and purification

The dried and powdered of aerial parts of Weigela florida "Jean's Gold" (56.

Structural determination of isolated saponins

All compounds 12-17 were isolated as white amorphous powders and assigned as bidesmosidic structures by means of the 1D and 2D spectra ( 1 H and 13 C NMR, 1 H-1 H COSY, TOCSY, ROESY, HSQC and HMBC).

NMR spectroscopy

Structure of aglycon

The structure of aglycon was identified by analysing spectra NMR 1 H, 13 C, HSQC and HMBC. In spectra of NMR 1 H and 13 C, characteristic signals of oleanolic acid were showed:

-7 methyl groups with singlet signals at δH 1.15, 0.87, 0.85, 1.02, 1.21, 0.83 and 0.80 confirmed the presence of triterpene.

-1 olefinic proton at δH 5.40 (br t, J = 3.6 Hz) corresponds to proton H-12.

The 13 C NMR spectrum showed:

-5 tertiary carbons (CH), 10 secondary carbons (CH2), 7 primary carbons (CH3) and 8 quaternary carbons (C).

-2 signals at δC 124.1 and 145.4 correspond to the double bond Δ 12 .

The 1 H and 13

NMR spectroscopy

Structure of aglycon

The analysis of the NMR spectra of compound 13 showed that the aglycon was almost identified to those of compound 12. The correlations of HSQC, HMBC and ROESY allowed to identify the genin of compound 13 as the oleanolic acid which was characterised to compound 12 (Table 6).

Structure of sugar moieties

The molecular weight of compound The sequence of the oligosaccharide was established by analysing the HMBC and ROESY spectra:

96

-The HMBC correlation between δH 4.87 (Ara H-1) and δC 90.5 (C-3) and in ROESY at δH 4.87 (Ara H-1) and δH 3.12 (H-3) proved that Ara was attached to the C-3 position of the aglycon (Fig. 80).

-The HMBC correlation between δH 4.54 (Ara H-2) and δC 106. Table 6. 13 C and 1 H NMR spectroscopic data of the aglycon moieties of compounds 12 and 13 in pyridine-d5 (δ in ppm, J in Hz) Overlapped proton signals are reported without designated multiplicity.

Table 7. 13 C and 1 H NMR spectroscopic data of the sugar moieties of compounds 12 and 13 in pyridine-d5

Isolation and purification

The dried and powdered of roots of Cordyline fruticosa "Fairchild red" (74.

Structural determination of isolated saponins a. Compound 18 (CFR3b1)

Mass spectrometry

The HR-ESIMS (positive-ion mode) spectrum of compound 18 showed a pseudo-molecular ion peak [M+Na] + at m/z 615.3536, indicating a molecular weight of 592 and a molecular formula of C33H52O9.

NMR spectroscopy

The structure of aglycon was identified by analysing spectra NMR 1 H, 13 C, HSQC and HMBC. The 1 H and 13 C spectra of the aglycon part of 18 (Table 8) were very characteristic of spirostanol glycoside which showed:

-2 methyl groups with singlet signal and another one with doublet signal at δH 0.83, 1.07 and 1.06 (d, J = 6.4 Hz), respectively.

- Accordingly, the aglycon of 18 was elucidated as 5α-spirost-25( 27)-ene-1β,3β,4α-triol.

Structure of sugar moieties

The monosaccharides were identified by acid hydrolysis and extensive 2D NMR analysis

NMR spectroscopy

Structure of aglycon

The analysis of the NMR spectra of compound 19 showed that the aglycon was almost identified to those of compounds 18. The correlations of HSQC, HMBC and ROESY allowed to identify the genin of compound 19 as spirostanol glycoside which was characterised to compounds 18 (Table 8).

Structure of sugar moieties

The molecular weight of compound 19 is lower than 18 14 amu corresponding to a pentosyl group instead of an hexosyl one. The 2D NMR spectroscopic analyses enabled the full assignments of the resonances of one sugar as β-D-xylopyranosyl (δH 5.04, Xyl H-1). The sequence of the oligosaccharide was established by analysing the HMBC and ROESY spectra:

-The HMBC correlation between δH 5.04 (Xyl H-1) and δC 80.9 (C-1) and a ROESY correlation between δH 5.04 (Xyl H-1) and δH 4.00 (d, J = 11.7, 4.1 Hz, H-1) proved that Xyl was attached to the C-1 position of the aglycon (Fig. 98).

Conclusion

Based on the above evidences, the structure of 19 was characterized as 5α-spirost-( 25

NMR spectroscopy

Structure of aglycon

The spectral features of compound 20 in the region of aglycon were quite similar to 18, excepted the signals due to the A ring carbons. The oxymethine signal at δC 74.7 (C-4) in 18 was replaced by a methylene carbon signal at δC 39.2, confirmed by the HSQC correlation at δC 39.2/δH 1.74, 1.77, and the shielded value of C-3 at δC 67.7. Accordingly, the aglycon of 20 was characterized as 5α-spirost-25(27)-ene-1β,3β-diol.

Structure of sugar moieties

The 2D NMR spectroscopic analyses enabled the full assignments of the resonances of these sugars as:

-1 β-D-fucopyranosyl Fuc-1 at δH 4.71 (d, J = 7.6 Hz).

-1 α-L-rhamnopyranosyl Rha-1 at δH 6.35 (br s).

The sequence of the oligosaccharide was established by analysing the HMBC and ROESY spectra:

-The HMBC correlation between δH 4.71 (Fuc H-1) and δC 83.1 (C-1) and in ROESY at δH 4.71 (Fuc H-1) and δH 3.80 (dd, J = 11.7, 3.5 Hz, H-1), proved that Fuc was linked to the C-1 position of the aglycon (Fig. 104).

-The HMBC correlation between δH 6.35 (Rha H-1) and δC 74.4 (Fuc C-2) and in ROESY at δH 6.35 (Rha H-1) and δH 4.49 (Fuc H-2) proved that Rha was attached to the Fuc at C-2.

Conclusion

NMR spectroscopy

Structure of aglycon

The analysis of the NMR spectra of compound 21 showed that the aglycon was the same as compound 20. The correlations of HSQC, HMBC and ROESY allowed to identify the genin of compound 21 as a spirostanol glycoside which was characterised to compounds 20 (Table 8).

Structure of sugar moieties

The molecular weight of compound 21 differed than 20 by only 14 amu, corresponding to a pentosyl group instead of an hexosyl one. Two cross-peaks were showed in the HSQC indicating the presence of two sugar units, and their structures were established using the 2D NMR spectra:

-1 α-L-arabinopyranosyl Ara-1 at δH 4.73 (d, J = 7.0 Hz).

-1 α-L-rhamnopyranosyl Rha-1 at δH 6.32 (br s).

The sequence of the oligosaccharide was established by analysing the HMBC and ROESY spectra:

-The HMBC correlation between δH 4.73 (Ara H-1) and δC 82.2 (C-1) and in ROESY at δH 4.73 (Ara H-1) and δH 3.87 (d, J = 11.7, 3.5 Hz, H-1) proved that Ara was attached to the C-1 position of the aglycon (Fig. 111).

-The HMBC correlation between δH 6.32 (Rha H-1) and δC 75.0 (Ara C-2) and in ROESY at δH 6.32 (Rha H-1) and δH 4.55 (Ara H-2) proved that Rha was attached to the Ara at C-2.

Conclusion

Mass spectrometry

The HR-ESIMS (positive-ion mode) spectrum of compound 22 showed a pseudo-molecular ion peak [M+Na] + at m/z 761.4122, indicating a molecular weight of 738 and a molecular formula of C39H62O13.

NMR spectroscopy

Structure of aglycon

The analysis of the NMR spectra of compound 22 showed that the aglycon was quite similar to compound 18.

Structure of sugar moieties

The molecular weight of compound 22 differs from 18 by only 146 amu, corresponding to a supplementary pentosyl group. By 2D NMR analyses, two sugar units were identified as:

-1 β-D-fucopyranosyl Fuc-1 at δH 4.72 (d, J = 7.6 Hz).

-1 α-L-rhamnopyranosyl Rha-1 at δH 6.40 (br s).

The sequence of the oligosaccharide was established by analysing the HMBC and ROESY spectra:

-The HMBC correlation between δH 4.72 (Fuc H-1) and δC 82.4 (C-1) and in ROESY at δH 4.72 (Fuc H-1) and δH 3.94 (dd, J = 11.7, 3.5, H-1) revealed the glycosidic linkage of Fuc at the C-1 position of the aglycon. (Fig. 117).

-The HMBC correlation between δH 6.40 (Rha H-1) and δC 74.4 (Fuc C-2) and in ROESY at δH 6.40 (Rha H-1) and δH 4.51 (dd, J = 9.4, 7.6 Hz, Fuc H-2) proved the (1→2) linkage between Rha and Fuc.

Conclusion

The structure of 22 was characterized as 5α-spirost-( 25

NMR spectroscopy

Structure of aglycon

The analysis of HSQC, HMBC and ROESY spectra of compound 23 showed that the aglycon was the same as 22. (Table 8).

Structure of sugar moieties

The sugar part was shown to be similar to those of compound 22 using the correlations observed in the HMBC, COSY, TOCY and ROESY spectra as :

128 -1 β-D-fucopyranosyl Fuc-1 at δH 4.70 (d, J = 7.6 Hz).

-1 α-L-rhamnopyranosyl Rha-1 at δH 6.35 (br s).

The sequence of the oligosaccharide was established by analysing the HMBC and ROESY spectra:

-The HMBC correlation between δH 4.70 (Fuc H-1) and δC 82.2 (C-1) and in ROESY at δH 4.70 (Fuc H-1) and δH 3.92 (dd, J = 11.7, 4.1 H-1) proved that Fuc was attached to the C-1 position of the aglycon (Fig. 124).

-The HMBC correlation between δH 6.35 (Rha H-1) and δC 75.

NMR spectroscopy

Structure of aglycon

The analysis of the NMR spectra of compound 24 showed that the aglycon was quite similar to those of compounds 23 (Table 8).

Structure of sugar moieties

The molecular weight of compound 24 differs from 23 by only 130 amu, corresponding to a supplementary hexosyl group. The part of the HSQC spectrum corresponding to the osidic chain showed cross-peaks at δH/δC 4.77 (d, J = 7.6 Hz)/99.7, 6.33 (br s)/101.3 and 5.54 (br s)/100.2. After analysing the spectre of HSQC, HMBC, COSY, TOCSY and ROESY, the sugar moieties of compound 24 was identified as:

-1 β-D-fucopyranosyl Fuc-1 at δH 4.70 (d, J = 7.6 Hz).

-2 α-L-rhamnopyranosyl : Rha I-1 at δH 6.33 (br s) and Rha II-1 at δH 5.54 (br s)

The sequence of the oligosaccharide was established by analysing the HMBC and ROESY spectra:

-The HMBC correlation between δH 4.77 (Fuc H-1) and δC 79.8 (Agly C-1) and in ROESY at δH 4.77 (Fuc H-1) and δH 4.44 (H-1), proved that Fuc was attached to the C-1 position of the aglycon (Fig. 130).

-The HMBC correlation between δH 6.33 (Rha I H-1) and δC 75.5 (Fuc C-2) and in ROESY at δH 6.33 (Rha H-1) and δH 4.52 (Fuc H-2) proved that Rha I was attached to the Fuc at C-2.

-The HMBC correlation between δH 5.54 (Rha II H-1) and δC 74.8 (C-3) and in ROESY at (Fuc C-4) revealed that the hydroxy group at Fuc C-4 was corresponded to a SO3H substituent [START_REF] Fouedjou | Steroidal saponins from the leaves of Cordyline fruticosa (L.) A. Chev. and their cytotoxic and antimicrobial activity[END_REF].

Conclusion

The structure of 24 was determined as 5α-spirost-( 25

Mass spectrometry

The HR-ESIMS (positive-ion mode) spectrum of compound 25 showed a pseudo-molecular ion peak [M+Na] + at m/z 795.4147, indicating a molecular weight of 772 and a molecular formula of C41H68O17.

NMR spectroscopy

Structure of aglycon

The structure of aglycon was identified by analysing spectra NMR 1 H, 13 C, HSQC and HMBC. The 1 H and 13 C spectra of the aglycon part of 25 (Table 8) were very characteristic of furostanol glycoside which showed:

-2 tertiary methyl protons at δH 0.82 (s, H3-18), 1.06 (s, H3-19) and a secondary methyl -Exomethylene protons at δH 5.07 and 5.37 (H-27, each 1H, br s).

The 13 C NMR spectrum showed:

-10 tertiary carbons (CH), 10 secondary carbons (CH2), 3 primary carbons (CH3) and 4 quaternary carbons (C). The multiplicity of the H-2β at δH 2.17 as a quadruplet (J = 11.1 Hz) suggested an axial/axial coupling with H-1 and H-3 and hence an α-axial orientation of both H-

Structure of sugar moieties

The -The HMBC correlation between δH 4.91 (Glc H-1) and δC 71.9 (C-26) and in ROESY at

Isolation and purification

The dried and powdered aerial parts of Cordyline fruticosa "Fairchild Red" (103.

Structure of sugar moieties

The monosaccharides were identified by acid hydrolysis and extensive 2D NMR analysis

NMR spectroscopy

Structure of aglycon

The analysis of the NMR spectra of compound 31 showed that the aglycon was similar to compound 30 (Table 10).

Structure of sugar moieties

The -1 α-L-rhamnopyranosyl Rha-1 at δH 6.33 (br s).

-1 β-D-glucopyranosyl Glc-1 at δH 4.89 (d, J = 7.6 Hz).

The sequence of the oligosaccharide was established by analysing the HMBC and ROESY spectra:

-The HMBC correlation between δH 4.82 (Fuc H-1) and δC 80.6 (C-1) and in the ROESY spectrum at δH 4.82 (Fuc H-1) and δH 4.44 (dd, J = 11.7, 3.5 Hz, H-1) proved that Fuc was attached to the C-1 position of the aglycon.

-The HMBC correlation between δH 6.33 (Rha H-1) and δC 74.

Correlation between the structure and cytotoxicity of saponins

There are close relationships between the chemical structure of saponins and their biological activities. Observations from numerous studies confirm that the structure of the aglycon and the sugar moiety influences to biological activity of saponins (Chwalek et al.,182 2006; [START_REF] Mimaki | Cytotoxic activities and structure-cytotoxic relationships of steroidal saponins[END_REF]. Herein, those correlations were summered due to the previously reported cytotoxicity.

In case of steroidal saponins

-Although the introduction of a hydroxyl group at C-17α position of the aglycon slightly reduced their cytotoxicities, an hydroxyl group at C-27 position led to a considerable decrease in the cytotoxic activity. Furthermore, the attachment of an α-L-Rha group at C-2 position of the glycosidic moiety led to the exhibition of a potent activity [START_REF] Mimaki | Cytotoxic activities and structure-cytotoxic relationships of steroidal saponins[END_REF]).

- [START_REF] Tran | New Spirostanol Steroids and Steroidal Saponins from Roots and Rhizomes of Dracaena angustifolia and Their Antiproliferative Activity[END_REF] suggested that the presence of Fuc and Xyl linked at C-24 position of the aglycon of spirostanol saponins seemed to be important for the cytotoxic activity against HT-1080 fibrosarcoma cells. Furthermore, without the Fuc, Xyl and acetyl groups, a spirostanol saponin showed a significant activity against colon 26-L5 carcinoma and B-16

BL6 melanoma cell lines.

-Steroidal saponins with a glycosyl group linked at C-1 position of the aglycon showed no activity with the exceptions of those possessing some acyl groups at the glycosyl moiety. It could be assumed that the presence of a certain degree of lipophilicity in the sugar moiety is essential for exhibiting the cytotoxic activity [START_REF] Mimaki | Cytotoxic activities and structure-cytotoxic relationships of steroidal saponins[END_REF]. This suggestion was confirmed by Rezgui et al.,

-The presence of an acetyl group at Glc C-6 position of glycosidic moiety may considerably decreased the cytotoxicity against HT-29 cells and had no effect against HCT116 cells -A very interesting observation comes from in vivo experiments, as no direct correlation was seen with in vitro cytotoxicity: Oleanolic acid derivatives showed much stronger cytotoxicities in vitro than those of hederagenin, and had an inferior in vivo antitumor activity. This may be due to the differences in bioavailability of these structures, so the presence of a hydroxyl at C-23 seems to be more effective for in vivo antitumor activity (Sobolewska et al., 2020).

Cytotoxic mechanism of saponins

Saponins are potential cytotoxic agents, with different mechanisms of action. Their cytotoxic effects may be due to either apoptosis inducement or nonapoptotic cell death stimulation. There is a number of well-known processes which lead to the same effect with different mechanisms of action. The most important mechanisms are summarized in Table

12 [START_REF] Podolak | Saponins as cytotoxic agents: a review[END_REF].

Experiment protocol

Cells were purchased from ATCC (USA); Fetal Bovine Serum (FBS) was from Dutscher.

RPMI 1640 medium was from Corning. MTS colorimetric assay was from Abcam.

Microplate reader was from Spark ® , Tecan.

This experiment protocol was carried out according to the manufacturer's instruction (Abcam Cambridge). Cells were seeded at the density of 5x10

Results of cytotoxic study on isolated and selected saponins

The cytotoxic tests were done in Centre Georges-François Leclerc, ICMUB UMR CNRS 6302. The results were expressed as concentrations of compound producing 50% toxicity (IC50 value). Fluorouracil (5-FU) was used as positive control.

Compounds 1-3, 6 and 7, monodesmosidic and bidesmosidic saponins, were tested to evaluate their cytotoxic activity against a mouse colon cancer cell line (CT26), in concentrations ranging from 1 to 50 µM (Fig. 186 ). All the tested glycosides showed potent cytotoxicities, with IC50 from 1.09 to 1.97 µM, comparing to 5-FU with an IC50 of 11.7 µM (Table 11). First of all, to propose clear structure / activity relationships, oleanolic acid is important for the cytotoxicity. In the literature, a substitution for example by a primary The cytotoxicities of the isolated compounds 8, 10, 11, 13, 15, 17 against mouse colon cancer (CT26 cells), mouse melanoma (B16 cells) and human liver cancer (HepG2 cells)

were evaluated by MTS assay, in concentrations ranging from 1 to 50 µM (Fig. 188 ). The results of their cytotoxicities were shown in Table 12. From the family Caprifoliaceae:

-Weigela x "Bristol ruby": 7 oleanane-type triterpenoid saponins were isolated including 6

previously undescribed in addition to 1 known.

-Weigela florida "Pink poppet": 3 oleanane-type and 1 hederagenin-type triterpenoid saponins were isolated including 1 previously undescribed in addition to 3 knowns.

-Weigela florida "Jean's gold": 2 oleanane-type and 4 hederagenin-type triterpenoid saponins were isolated including 2 previously undescribed in addition to 4 knowns.

From the family Asparagaceae:

-Cordyline fruticosa "Fairchild red": 9 spirostane-type and 6 furostane-type steroidal saponins were isolated including 13 previously undescribed in addition to 2 knowns.

-Dracaena braunii: Three known compounds were purified from the roots. Moreover, this work opens new perspectives:

Diosgenin, a genin of the saponin dioscin which was firstly extracted from the rhizome of Dioscorea tokoro Makino [START_REF] Honda | Untersuchungen über die Saponinsubstanzen der Dioscorea Tokoro Makino[END_REF] and also successfully purified from the roots of Dracaena braunii, possess interesting potentials. This compound exhibits good anticancer activities including liver cancer [START_REF] Cruz | Diosgenin induces genotoxic and mutagenic effects on HepG2 cells[END_REF]

INTRODUCTION

Curcumin (CAS Registry Number 458-37-7), the principal curcuminoid found in turmeric, is generally considered as its most active constituent [START_REF] Sharma | Curcumin: The story so far[END_REF]. Other major curcuminoids present in turmeric are: demethoxycurcumin (curcumin II, n° CAS 22608-11-3), bisdemethoxycurcumin (curcumin III, n° CAS 33171-05-0), and the recently identified cyclocurcumin, n° CAS 153127-42-5 [START_REF] Kiuchi | Nematocidal activity of turmeric: synergistic action of curcuminoids[END_REF]. More and more evidences point out towards curcumin having potential health benefits including antioxidant, anti-inflammatory, antimicrobial, anti-amyloid and antitumor properties [START_REF] Aggarwal | 10 Curcumin-Biological and Medicinal Properties[END_REF][START_REF] Chattopadhyay | Turmeric and curcumin: Biological actions and medicinal applications[END_REF].

In acidic and neutral conditions (pH 3-7), the major constituents present are curcumin molecules in bis-keto form where curcumin acts as a proton donor (pKa 8.1), inducing a crucial role in the antioxidant activity [START_REF] Wang | Stability of curcumin in buffer solutions and characterization of its degradation products[END_REF]. In vitro, curcumin can significantly inhibit the generation of reactive oxygen species (ROS) like superoxide anions, H2O2 and nitrite radical generation by activated macrophages, which play an important inflammatory activity [START_REF] Joe | Role of capsaicin, curcumin and dietary n -3 fatty acids in lowering the generation of reactive oxygen species in rat peritoneal macrophages[END_REF]. Curcumin also lowers the production of ROS in vivo [START_REF] Chattopadhyay | Turmeric and curcumin: Biological actions and medicinal applications[END_REF] [START_REF] Nguyen | Physicochemical state influences in vitro release profile of curcumin from pectin beads[END_REF]. The main advantage of encapsulation is that curcumin will be protected from oxidation, and its solubility increased improving therefore its bioavailability and making controlled release in gastrointestinal tract achievable [START_REF] Anand | Bioavailability of Curcumin: Problems and Promises[END_REF].

Polymers are often used as carriers due to their non-toxicity and safe history of human consumption, thus ionotropic gelation is a common method used to entrap drug with hydrophilic polymer. This method is based on polyelectrolytes ability to crosslink in the presence of counter ions to form hydrogel beads [START_REF] Pillay | In vitro release modulation from crosslinked pellets for site-specific drug delivery to the gastrointestinal tract: II. Physicochemical characterization of calcium-alginate, calcium-pectinate and calcium-alginatepectinate pellets[END_REF]. Pectins gather a family of oligosaccharides and polysaccharides that have common features but are diverse in their fine structures [START_REF] Willats | Pectin: new insights into an old polymer are starting to gel[END_REF]. The degree of esterification (DE) and degree of amidation (DA), both expressed as a percentage of carboxyl groups (esterified or amidated), are important means to classify pectins [START_REF] Chambin | Colonspecific drug delivery: Influence of solution reticulation properties upon pectin beads performance[END_REF]. Interestingly, complexes are formed due to the combination of low methoxyl pectin (DE < 50%) and divalent cations (calcium ions) caused by reducing the solubility and inducing non-covalent associations of carbohydrate chains [START_REF] Sriamornsak | Calcium pectinate gel beads for controlled release drug delivery[END_REF]. Therefore, calcium pectinate has been investigated as an insoluble hydrophilic coating for sustainedrelease delivery by an interfacial complexation process [START_REF] Günter | Calcium pectinate gel beads obtained from callus cultures pectins as promising systems for colon-targeted drug delivery[END_REF].

A surfactant (surface active agent) is an amphiphilic molecule including a hydrophobic tail and a hydrophilic head in structure [START_REF] Santos | Amphiphilic Molecules in Drug Delivery Systems[END_REF]. The main problems of curcumin come from its poor solubility in water and lowly pharmacokinetic profile, which could be significantly improved by using surfactants. antioxidant properties releasing free α-tocopherol which then localizers in the cell membrane and protects the membrane from lipid peroxidation and damage [START_REF] Yan | Tocopheryl polyethylene glycol succinate as a safe, antioxidant surfactant for processing carbon nanotubes and fullerenes[END_REF].

Proteins and surfactants can be used individually to positively impact drug delivery. In some cases, proteins are analogous to surfactants forming self-assembled stable structures.

Due to this ability, β-Lactoglobulin (BLG), a major whey protein in bovine milk, has shown to increase solubility and especially, antioxidant activity of curcumin [START_REF] Li | Binding of curcumin to β-lactoglobulin and its effect on antioxidant characteristics of curcumin[END_REF][START_REF] Sneharani | Interaction of Curcumin with β-Lactoglobulin-Stability, Spectroscopic Analysis, and Molecular Modeling of the Complex[END_REF].

The purpose of this study was to improve the solubility and bioavailability of curcumin without affecting its antioxidant activity and simultaneously achieve controlled release in gastrointestinal tract by encapsulation with ionotropic gelation method thanks to the combination of pectin with various surfactants.

oxygens [START_REF] Sharma | Curcumin: The story so far[END_REF]. However, in situations (pH > 8) where the enolate form is dominant, curcumin acts instead as an electron donor [START_REF] Jovanovic | H-atom transfer is a preferred antioxidant mechanism of curcumin[END_REF]. Curcumin is relatively insoluble in water due to its hydrophobic nature, but soluble in acetone, ethanol and dimethylsulphoxide. The use of curcumin is thereby limited by its low water solubility, high decomposition rate in alkaline media and photodegradation in organic solvents [START_REF] Tønnesen | Studies of curcumin and curcuminoids. XXVII. Cyclodextrin complexation: solubility, chemical and photochemical stability[END_REF].

Curcumin is unstable at basic pH, and degrades within 30 min to trans-6-(40-hydroxy-30methoxyphenyl)-2,4-dioxo-5-hexanal, ferulic acid, feruloylmethane and vanillin [START_REF] Lin | Recent studies on the biofunctions and biotransformations of curcumin[END_REF]. This degradation is blocked by the presence of foetal calf serum or human blood, or addition of antioxidants such as ascorbic acid, N-acetylcysteine or glutathione in culture media or phosphate buffer above pH 7 [START_REF] Wang | Stability of curcumin in buffer solutions and characterization of its degradation products[END_REF]. activity. [START_REF] Ravindranath | Absorption and tissue distribution of curcumin in rats[END_REF] showed that after oral administration of 400 mg of curcumin to rats, only traces of unchanged drug were found in the liver and kidney. At 30 min, 90% of curcumin was found in the stomach and small intestine, but only 1% was present at 24 h [START_REF] Ravindranath | Metabolism of curcumn-studies with [3H]curcumin[END_REF].

Pharmacological activity of curcumin

Curcumin, the principal curcuminoid found in turmeric, is generally considered as its most active constituent [START_REF] Sharma | Curcumin: The story so far[END_REF]. More and more evidences point out towards curcumin having potential health benefits including antioxidant, anti-inflammatory, antimicrobial, anti-amyloid and antitumor properties [START_REF] Aggarwal | 10 Curcumin-Biological and Medicinal Properties[END_REF][START_REF] Chattopadhyay | Turmeric and curcumin: Biological actions and medicinal applications[END_REF] evidencing the beneficial properties of curcumin to the organism (Fig. 191).

Encapsulation in food industry

Encapsulation may be defined as a process to entrap one substance (active agent) within another substance (wall material). The encapsulated substance can be called the core, fill, active, internal or payload phase, while the substance encapsulating was often called the coating, membrane, shell, capsule, carrier material, external phase, or matrix [START_REF] Fang | Encapsulation of polyphenolsa review[END_REF][START_REF] Wandrey | Materials for encapsulation[END_REF].

In the food industry, encapsulation process can be applied for a variety of reasons.

Some methods of microencapsulation

Microencapsulation is widely used in food and pharmaceutical industries as an alternative technology for enhancing entrapment, protection, controlled release and functionalization of bioactive substances. In the literature, a variety of microencapsulation techniques were used to improve solubility and bioavailability of active gradients, such as spray drying, solvent evaporation, coacervation, supercritical fluid, inclusion complexation and ionotropic gelation. Although a variety of alternative microencapsulation techniques is available, no single method is suitable for encapsulating different types of core material.

Ultimately, the best method will depend upon the type of core material, the required particle size, the permeability of the shell wall, and the different properties of the microcapsule.

To this day, various curcumin encapsulation techniques are divided into 3 basic groups: chemical, physical and physicochemical methods. Each method has its own advantages as well as disadvantages, so they are listed and described basically according to their principles in Table 13:

Table 13. Some methods of curcumin encapsulation Method Characteristics Advantages Disadvantages Ref.

Physical methods

Spray drying

Coacervation

This method is based on the ability of cationic and anionic water-soluble polymers to interact in water to form a liquid, neutral, polymer-rich phase called coacervate. The complex coacervation process is the separation of an aqueous polymeric solution into two miscible liquid phases: a dense coacervate phase and a dilute equilibrium phase. The dense coacervate phase wraps as a uniform layer around dispersed core materials. The core material, which can be oily droplets, is dispersed into the aqueous solution of the two polymers. The coating in liquid form is removed from a polymer solution, coats the material to be encapsulated, solidified and collected by centrifugation or filtration (Fig. 194). Drying can be accomplished by spray or fluidized bed drying [START_REF] Gibbs Inteaz Alli | Encapsulation in the food industry: a review[END_REF]. This is an immobilization rather than an encapsulation technology and is therefore mostly proposed and applied for bioactive food molecules rather than for bioactive living cells. The technique is applied for flavours, oils but also for some water-soluble bioactive molecules

Fluid bed coating

A modified spray dry methodology that enlarges the field of application is the fluid bed coating methodology. In this technology, the bioactive food components are suspended in air and the matrix molecules are sprayed onto the bioactive components and form a capsule (Fig. 195). The choice for matrix molecules is broader than for traditional spray drying. It may be fats, proteins, carbohydrates but also emulsifiers. It may even be applied to give spray dried products or a sensitive core of for example oils as a second coating. It is also useful for applying an additional layer of molecules for targeted release in the gut. In

Chitosan

Chitosan, a partially deacetylated product of chitin, is a copolymer consisting of β-(1→4)-2-acetamido-D-glucose and β-(1→4)-2-amino-D-glucose units (Fig. 198), with the latter usually exceeding 80% [START_REF] Yoon | Thermostable chitosanase from Bacillus sp. strain CK4: cloning and expression of the gene and characterization of the enzyme[END_REF]. This biopolymer is a glucosaminoglycan and is composed of two common sugars, glucosamine and N-acetylglucosamine, the proportion of which depends on the alkaline treatment [START_REF] Bakshi | Chitosan as an environment friendly biomateriala review on recent modifications and applications[END_REF]. Chitosan, despite its crystallinity, is insoluble in water at pH > 7; however, it is soluble in dilute acids owing to the presence of a protonated amino groups in acidic conditions (pH < 6.0). Furthermore, the presence of reactive functional groups such as amino, primary, and secondary hydroxyl

Pectin

Pectin is plant derived molecules which are mostly linear polymers of mainly α-(1-4)-linked D-galacturonic acid residues and 1,2-linked L-rhamnose residues (Fig. 201A). These polysaccharides are advantageous for targeted delivery because they remain intact in the stomach and the small intestine. The application of pectin as unmodified molecule is limited due to its high solubility in water. Pectin is capable of forming gels by different mechanisms regardless of their degree of methylation:

-Highly methylated pectin (HM) is capable of forming a gel under the conditions of low water activity, typically held by high concentrations of sugar and at acidic pH (between 2.2 and 2.8). The gelling mechanism in this case corresponds to the formation of hydrophobic interactions between the methyl groups and of hydrogen bonds between the carboxylic groups of the non-esterified galaturonic acid residues and the secondary alcohols [START_REF] Bakshi | Chitosan as an environment friendly biomateriala review on recent modifications and applications[END_REF].

-Low methylated pectin (LM) is capable of forming a gel in the presence of multivalent cations and more particularly with Ca 2+ (Fig. 201B). This gelling mechanism is based on the interactions between cations and deprotonated carboxylic groups of galacturonic acids.

This association forms an "egg box" type gel due to Van der Waals bonds, hydrogen bonds and electrostatic bonds. The ability of LM pectin to associate with calcium depends on the degree of methylation but also on the distribution of non-esterified galacturonic acids. After enzymatic de-esterification of pectin, the formation of bonds with calcium is higher than Surfactants exist in many different forms in nature, classified according to the chemical nature of their polar head group. Surfactants with a negatively charged head group are referred to anionic, whereas cationic surfactants contain a positively charged head group.

Among of those, nonionic surfactants are the most common type of surface active agent used in preparing vesicles due to the superior benefits they impart with respect to stability, compatibility and toxicity compared to their anionic, amphoteric or cationic counterparts [START_REF] Santos | Amphiphilic Molecules in Drug Delivery Systems[END_REF]. Nonionic surfactants consist of both polar and non-polar segments and possess high interfacial activity. The formation of bilayer vesicles instead of micelles is dependent on the hydrophilic-lipophilic balance (HLB) of the surfactant, the chemical structure of components and the critical packing parameter. The chain length and size of the hydrophilic head group of the nonionic surfactant along with the HLB value of a surfactant plays a key role in entrapment efficiency by controlling drug entrapment of the vesicle it forms. Therefore, surfactant systems play an important role in modern drug delivery since they allow, for instance, the control of drug uptake and release rate and minimization of drug degradation and toxicity. An effective synergism between surfactant systems and drugs is nowadays recognized as a key issue to assure therapeutic efficiency [START_REF] Santos | Amphiphilic Molecules in Drug Delivery Systems[END_REF].

Materials

Curcumin (purity > 65%) was obtained from Sigma-Aldrich, Germany

where PD is percentage of degradation of curcumin, Cmax is maximum concentration of curcumin solubilised and C24 is concentration of curcumin after 24 days.

Encapsulation of curcumin by ionotropic gelation in pectin beads with different surfactants

Curcumin was encapsulated by ionotropic gelation according to a method of [START_REF] Chambin | Colonspecific drug delivery: Influence of solution reticulation properties upon pectin beads performance[END_REF]. 1.6 g of amidated low-methoxyl pectin was weighed and dissolved in 20 mL of deionized water (pH = 6.8), stirred at high speed (700 rpm) for 2 hours at room temperature.

Depending on the type of beads, surfactant was dissolved in 20 mL of deionized water (Table 14). The surfactant levels used were selected making sure the viscosity of the pectinate solution kept visually unchanged, and still permitted to prepare beads. 500 mg of 𝑃𝑅 (%) = 𝑀𝑡 𝑀𝑐 𝑥 100

PRpercentage of release of curcumin during time (%)

Mtmass of curcumin released in certain time (mg)

Mcmass of curcumin encapsulated in 400 mg of dry beads (mg)

Mathematical modeling of curcumin release from beads was applied according to Korsmeyer-Peppas model (Eq.( 3)) to determine the release behaviour:

𝑀 𝑡 𝑀 ∞ = 𝑘 × 𝑡 𝑛 (3)
where Mt/M∞ is absolute cumulative amounts of drug released at time t and infinity, k is constant incorporating structural and geometrical characteristics of the device and n is exponent indicative of the mechanism of drug release (Siepmann and Siepmann, 2008). Percentage of crystalline curcumin in beads was calculated according to Eq.( 4):

Differential Scanning Calorimetry (DSC) study

P CC = ∆H 𝐶 ∆H f * 100 (4)
where PCC is percentage of crystalline curcumin in bead (%), ∆HC is enthalpy of crystallization of curcumin in bead and ∆Hf is the enthalpy of melting for a fully crystalline curcumin powder. Antioxidant activity of curcumin was expressed as percentage of radical scavenging which represented difference between control and sample after 2 hours (Eq. ( 5)):

DPPH radical scavenging assay

𝑃𝐼 (%) = 𝐴 𝑐𝑜 -𝐴 𝐴𝑡 𝐴 𝑐𝑜 × 100 (5)
where PI was percentage of radical scavenging (inhibition), Aco was absorbance (515 nm) of control at t = 0 min and AAt was absorbance (515 nm) of curcumin at t = 2 h (Ak and Gülçin, 2008).

Physicochemical parameters 1.Solubility studies

Solubility study was performed to determine which of the surfactants is the most effective to disperse curcumin (Table 16). Kolliphor ® HS 15 and TPGS ® proved to be more effective than BiPro ® BLG to solubilize curcumin. Those surfactants are non-ionic solubilizers, possessing the favourable hydrophilic-lipophilic balance (HLB ≈ 13-16) and are currently used for manufacturing aqueous parenteral preparations with vitamins and other lipophilic compounds [START_REF] Reintjes | Solubility Enhancement with BASF Pharma Polymers: Solubilizer Compendium[END_REF]. The water solubility of curcumin was highly increased with these surfactants. Our experimental value for solubility in deionized water (1.12 mg/L) was higher than the one reported by [START_REF] Tønnesen | Solubility, chemical and photochemical stability of curcumin in surfactant solutions[END_REF] explained by the specificity of their interaction with curcumin [START_REF] Seo | Preparation and pharmacokinetic evaluation of curcumin solid dispersion using Solutol® HS15 as a carrier[END_REF]. Solubility of curcumin in EtOH 99.5 % has been seen as 6700 times higher than in water [START_REF] Carvalho | Production, solubility and antioxidant activity of curcumin nanosuspension[END_REF], however, ethanol is not an appropriate solvent for pectin-curcumin microparticles because of precipitation due to the interaction between ethanol and pectin. The 2 nd lowest solubility of curcumin was in the BiPro ® BLG solution (15.7 ± 1.4 mg/L).

However, it was 1427 times higher than in water. BLG was used as stabilizer in order to improve curcumin's bioavailability due to its stability in acidic pH and the presence of specific receptors in small intestine. Furthermore, BLG has previously been selected to

Short term stability study

To measure its stability over time in those conditions, short term stability was experimented by monitoring curcumin in deionized water (pH = 6.8) and 5 g/L Kolliphor ® HS 15 in different environmental conditions. The percentage of curcumin degradation was measured at various time points in a period of 24 days.

Encapsulation efficiency (EE) and encapsulation yield (EY)

EE and EY can be considered as two important parameters to evaluate the curcumin loading in the polymeric matrixes. The results were presented in Table 17.

Firstly, EE was estimated as a percentage of curcumin entrapped in pectin beads in comparison to the curcumin amount initially used for the bead preparation. Since curcumin is hydrophobic, the EE for all types of beads was very high (>75%) showing the interest of pectinate gel as a potential carrier in pharmaceutical and food industries [START_REF] Chambin | Colonspecific drug delivery: Influence of solution reticulation properties upon pectin beads performance[END_REF]. Curcumin beads have the highest EE due to no surfactant use. Kolliphor ® beads, on the other side, have the lowest EE because of the increased curcumin solubility which induced a higher curcumin diffusion to calcium chloride solution during production process. Results for EE were in correlation with those obtained by Nguyen et al., 2014. In case of TPGS ® bead, a high EE was achieved surprisingly, not affected by its high solubility (601.9 mg/L). This result was interesting in comparison with Kolliphor ® beads. This could be explained by the property of TPGS ® which is an amphiphilic copolymer used for preparation of micelles (CMC = 0.02% w/v) [START_REF] Keshari | Curcumin loaded TPGS micelles for nose to brain drug delivery: in vitro and in vivo studies[END_REF]. Owing this ability, the loaded curcumin TPGS micelles were formed and so dissolved curcumin could not diffuse out of the matrix into the CaCl2 solution.

Commercial BiPro ® BLG was used for production of beads due to its efficacy in increasing curcumin solubility. The percentage of encapsulation was high (90.5%) because of a lower solubility limiting the diffusion out the capsules during the microcapsules production process.

On the other hand, EY is an indicator of curcumin share in the total mass of particles. This parameter is proportional to encapsulation efficiency (Table 17). Therefore, both

Kolliphor ® and TPGS ® beads showed lower EY (6.3% and 9.0%) than other beads due to their ability to solubilize curcumin during production.

Morphologic description of beads by scanning electron microscopy

The size, morphology, surface topology and internal structure of curcumin powder and pectinate beads were carried out in the pictures obtained by SEM (Fig. 203). Pectinate beads formed instantaneously when the dispersion was dripped onto the CaCl2 solution. After drying in oven, their size ranged from 2-3mm. Both types of beads were almost regular ovoid in shape, but the Kolliphor ® and TPGS ® beads (Fig.

Dissolution study in simulated intestinal medium

Results of release in simulated intestinal medium are used to understand the behavior of nutraceuticals released in the intestinal tract. It is important in the formulation system As the aqueous solubility of curcumin is very low, 5 g/L Kolliphor ® HS 15 was added to PBS 7.4 medium for another dissolution study based on the results of solubility test. Figure 205 shows the dissolution of curcumin from a pectinate matrix of different types of beads.

The release from Kolliphor ® HS 15 beads started immediately after the start of dissolution experiment. A significant release of curcumin was observed after 30 minutes of dissolution, with a steep slope of release for 180 minutes. At that point, 93% of release was achieved.

When the beads got in contact with the dissolution medium, surface curcumin rapidly dissolved resulting in an increase of curcumin release and reduced lag time [START_REF] Nguyen | Physicochemical state influences in vitro release profile of curcumin from pectin beads[END_REF].

Curcumin beads, on the contrary, showed a profile with a lag time. During the first 30 minutes, there was no significant release of curcumin and it took 7 hours to reach a release of only 80 %. This result could be explained by the low water solubility of curcumin in The glassy pectin polymer of dried beads swelled by contact with the release medium due to water diffusion into the system. The increase of water content led to an increased mobility of curcumin and polymer chains. Kolliphor ® and TPGS ® beads showed a faster water uptake than the other ones as a result of their morphological characteristics. Indeed BiPro ® BLG and Curcumin beads displayed a slow water uptake. The swelling contributed to the diffusion of encapsulated curcumin into the release medium.

Differential scanning calorimetry (DSC) study

The effect of surfactant on the thermal characteristics and crystanillinity of curcumin was then experimented except for BiPro ® BLG beads due to its low improvement of curcumin solubility. Curcumin showed a melting peak at 173.46 °C which was in accordance with previously published results [START_REF] Nguyen | Physicochemical state influences in vitro release profile of curcumin from pectin beads[END_REF]. Pectin powder exhibited a transition at 80.56 °C which was not visible for the pectin-based beads except those with TPGS ® beads.

Kolliphor ® HS 15 showed a melting peak at 32.63 °C also visible for Kolliphor ® beads.

Similarly, TPGS ® exhibited a melting peak at 40.06 °C also detected for TPGS ® bead at lower temperature (32.62°C) (Fig. 206A). The lower melting point of curcumin was due to its interaction with the pectinate matrix [START_REF] Nguyen | Physicochemical state influences in vitro release profile of curcumin from pectin beads[END_REF]. For Curcumin beads, 60.1% of curcumin was in crystalline state. In case of TPGS beads, 17.3 % of curcumin was still crystalline, while the melting peak of curcumin disappeared in Kolliphor ® beads, confirmed a better dissolution of curcumin in the presence of surfactants. As a result, curcumin in these beads is expected to be mainly in the amorphous state following the encapsulation process. These results were in good probably occurred during manufacturing process even if protection against light was set.

After 330 minutes of dissolution experiment, the percentage of inhibition seemed unchanged in Kolliphor ® and TPGS ® beads indicating the efficacy of curcumin to donate its hydrogen atom to radicals with these surfactants (Fig. 207B). In case of TPGS ® beads, it could be explained by its antioxidant properties [START_REF] Yan | Tocopheryl polyethylene glycol succinate as a safe, antioxidant surfactant for processing carbon nanotubes and fullerenes[END_REF]. The other beads showed This barrier can be solved by encapsulation in "egg-box" matrix with surfactants and pectin. But, the most problem in spite of using surfactant in encapsulation is how to find not only the good surfactant but also the right ratio between materials used to encapsulate curcumin successfully. In this study, curcumin was successfully encapsulated by inotropic gelation with different surfactants: Kolliphor ® HS 15, TPGS ® and BiPro ® BLG. It was interesting to show that Kolliphor ® HS 15 and TPGS ® increased significantly solubility of curcumin in comparison to water or PBS medium. Since solubility of curcumin was higher with Kolliphor ® , these beads had the lowest EE and EY due to a higher amount of curcumin loss during encapsulation process. Curcumin, BiPro ® BLG and TPGS ® beads have EE and EY above 90%, with no significant curcumin loss during production. Curcumin was in the crystalline state in the powder form, but it was transformed to amorphous state during the manufacturing process of Kolliphor ® and TPGS ® beads. According to these characteristics, curcumin release from Kolliphor ® and TPGS ® beads was faster and stronger than from the other beads in intestinal model medium.

Mathematical models have shown that the type of curcumin release from the pectin matrix particles is Supercase II transport in case of BiPro ® BLG and Curcumin beads possessed a smaller and more compact structure leading to a slow water uptake. To the Kolliphor ® and TPGS ® beads, their release mechanism followed the anomalous transport which showed a faster water uptake than the other ones relating to their morphological characteristics. The antioxidant activity of curcumin was decreased in a few extend (10%) after encapsulation with surfactants except for Kolliphor ® beads which protected better curcumin. During dissolution experiment, Kolliphor ® HS 15 and TPGS ® allowed to maintain antioxidant properties of curcumin. Therefore, the use of surfactant encapsulation formulations played important roles through not only enhancing drug solubility and encapsulation efficiency, but also affecting physical state, release kinetics and protection for antioxidant capacity of L curcumin. So, the choice of surfactant is essential to obtain the properties and functionalities of pectinate curcumin beads. Kolliphor ® HS 15 seemed to be the more promising surfactant for both curcumin protection during manufacturing process and release.

which posscess significant biological and pharmacological activities, was chosen to assess the interest of encapsulation for natural products. An encapsulation process was carried out using ionotropic gelation method with the presence of different surfactants. Different beads were manufactured successfully and characterized for physicochemical characteristics.

High encapsulation efficiency was achieved in all case of beads produced. The crystallinity of curcumin was tranformed into amorphous state during the production process based on DSC profile results. The improvement of curcumin solubility was demontrated with Kolliphor ® and TPGS ® and the matrix encapsulation has a great potential to control the release properties in intestinal medium. This study exhibited that surfactants are significantly effective in order to enhance the solubility and protect antioxidant capacity of curcumin and that their choices are essential.

The same strategy could be applied for microencapsulation of other bioactive ingredients, particularly natural compounds as saponin, in the development of functional foods and pharmaceutical products.

This work gives opening perspectives:

-Study on biological properties of diosgenin as a model of aglycone and set up a suitable encapsulation method.

-Try to discover new saponins with biological activities from other plants in Asian countries.

-Investigate new encapsulation techniques to preserve the natural compounds and optimize their bioavailability.

-Evaluate the interest to use saponin as surfactant in curcumin encapsulation with pectin.

Finally, to someone

  who have not been here for any reason, I want you know that you have been always the ones to help me to reach this far in my career. Thank you for being a part of my life. Duc Hung NGUYEN Dijon, 2020 Dedication I am so proud to dedicate this thesis To my lovely wife and my children who have always encouraged me throughout my PhD journey… To my whole family... To Agnès Roy.

Figure 1 .

 1 Figure 1. Phylogenetic classification of order Dipsacales ...................................................

Figure 2 .

 2 Figure 2. Diagram of Caprifoliaceae flower part .................................................................

Figure 3 .

 3 Figure 3. Weigela x "Bristol Ruby" .....................................................................................

Figure 4 .

 4 Figure 4. Weigela florida "Jean's Gold" ..............................................................................

Figure 5 .

 5 Figure 5. Weigela florida "Pink Poppet" ..............................................................................

Figure 6 .

 6 Figure 6. Phylogenetic classification of order Asparagales .................................................

Figure 7 .

 7 Figure 7. Dracaena braunii ..................................................................................................

Figure 8 .

 8 Figure 8. Cordyline fruticosa "Fairchild red" ....................................................................

Figure 9 .

 9 Figure 9. Structure of spirostane aglycon and furostane aglycon ......................................

Figure 10 .

 10 Figure 10. Structure of oleanane-(a), dammarane-(b), ursane-(c), lupane-(d) and hopane-type aglycon (e) ..............................................................................................

Figure 11 .

 11 Figure 11. Available different sugars in saponins ..............................................................

Figure 12 .

 12 Figure 12. Some triterpenoid saponins elucidated from species of Weigela genus ...........

Figure 13 .

 13 Figure 13. Steroidal saponins elucidated from species of Cordyline genus .......................

Figure 14 .

 14 Figure 14. Steroidal saponins elucidated from species of Dracaena genus .......................

Figure 15 .

 15 Figure 15. Mechanism of electrospray ionization ..............................................................

Figure 16 .

 16 Figure 16. Correlations of HSQC and HMBC between α-L-rhamnose and α-L-arabinose ....

Figure 17 .

 17 Figure 17. Correlations of COSY, TOCSY and ROESY ...................................................

Figure 18 .

 18 Figure 18. Purification scheme of compounds in roots of Weigela x "Bristol Ruby" .......

Figure 19 .

 19 Figure 19. Structure of compound 1 ...................................................................................

Figure 20 .

 20 Figure 20. HSQC spectrum of compound 1 .......................................................................

Figure 21 .

 21 Figure 21. HSQC spectrum of sugar anomers of compound 1 ..........................................

Figure 22 .

 22 Figure 22. HMBC spectrum of sugar moieties of compound 1 .........................................

Figure 23 .

 23 Figure 23. HMBC spectrum of sugar moieties of compound 1 .........................................

Figure 24 .

 24 Figure 24. ROESY spectrum of sugar moieties of compound 1 ........................................

Figure 25 .

 25 Figure 25. ROESY spectrum of sugar moieties of compound 1 ........................................

Figure 26 .

 26 Figure 26. Mass spectrum of compound 1 .........................................................................

Figure 27 .

 27 Figure 27. Structure of compound 2 ...................................................................................

Figure 28 .

 28 Figure 28. HSQC spectrum of compound 2 .......................................................................

Figure 29 .

 29 Figure 29. HSQC spectrum of sugar anomers of compound 2 ..........................................

Figure 30 .

 30 Figure 30. HMBC spectrum of sugar moieties of compound 2 .........................................

Figure 31 .

 31 Figure 31. ROESY spectrum of sugar moieties of compound 2 ........................................

Figure 32 .

 32 Figure 32. ROESY spectrum of sugar moieties of compound 2 ........................................

Figure 33 .

 33 Figure 33. Mass spectrum of compound 2 .........................................................................

Figure 34 .

 34 Figure 34. Structure of compound 3 ...................................................................................

Figure 35 .

 35 Figure 35. HSQC spectrum of compound 3 .......................................................................

Figure 36 .

 36 Figure 36. HSQC spectrum of sugar anomers of compound 3 ..........................................

Figure 37 .

 37 Figure 37. HMBC spectrum of sugar moieties of compound 3 .........................................

Figure 38 .

 38 Figure 38. HMBC spectrum of sugar moieties of compound 3 .........................................

Figure 39 .

 39 Figure 39. HMBC spectrum of sugar moieties of compound 3 .........................................

Figure 40 .

 40 Figure 40. ROESY spectrum of sugar moieties of compound 3 ........................................

Figure 41 .

 41 Figure 41. Mass spectrum of compound 3 .........................................................................

Figure 42 .

 42 Figure 42. Structure of compound 4 ...................................................................................

Figure 43 .

 43 Figure 43. HSQC spectrum of compound 4 .......................................................................

Figure 44 .

 44 Figure 44. HSQC spectrum of sugar anomers of compound 4 ..........................................

Figure 45 .

 45 Figure 45. HMBC spectrum of sugar moieties of compound 4 .........................................

Figure 46 .

 46 Figure 46. ROESY spectrum of sugar moieties of compound 4 ........................................

Figure 47 .

 47 Figure 47. Mass spectrum of compound 4 .........................................................................

Figure 48 .

 48 Figure 48. Structure of compound 5 ...................................................................................

Figure 49 .

 49 Figure 49. HSQC spectrum of compound 5 .......................................................................

Figure 50 .

 50 Figure 50. HSQC spectrum of sugar anomers of compound 5 ..........................................

Figure 51 .

 51 Figure 51. HMBC spectrum of sugar moieties of compound 5 .........................................

Figure 52 .

 52 Figure 52. ROESY spectrum of sugar moieties of compound 5 ........................................

Figure 53 .

 53 Figure 53. ROESY spectrum of sugar moieties of compound 5 ........................................

Figure 54 .

 54 Figure 54. Mass spectrum of compound 5 .........................................................................

Figure 55 .

 55 Figure 55. Structure of compound 6 ...................................................................................

Figure 56 .

 56 Figure 56. HSQC spectrum of compound 6 .......................................................................

Figure 57 .

 57 Figure 57. HSQC spectrum of sugar anomers of compound 6 ..........................................

Figure 58 .

 58 Figure 58. HMBC spectrum of sugar moieties of compound 6 .........................................

Figure 59 .

 59 Figure 59. ROESY spectrum of sugar moieties of compound 6 ........................................

Figure 60 .

 60 Figure 60. Mass spectrum of compound 6 .........................................................................

Figure 61 .

 61 Figure 61. Structure of compound 7 ...................................................................................

Figure 62 .

 62 Figure 62. Purification of compounds in roots of Weigela florida "Pink Poppet" ............

Figure 63 .

 63 Figure 63. Purification of compounds in aerial parts of Weigela florida "Pink Poppet" ...

Figure 64 .

 64 Figure 64. Structure of compound 8 ...................................................................................

Figure 65 .

 65 Figure 65. HSQC spectrum of compound 8 .......................................................................

Figure 66 .

 66 Figure 66. HSQC spectrum of sugar anomers of compound 8 ..........................................

Figure 67 .

 67 Figure 67. HMBC spectrum of sugar moieties of compound 8 .........................................

Figure 68 .

 68 Figure 68. ROESY spectrum of sugar moieties of compound 8 ........................................

Figure 69 .

 69 Figure 69. Mass spectrum of compound 8 .........................................................................

Figure 70 .

 70 Figure 70. Structure of compound 9 ...................................................................................

Figure 71 .

 71 Figure 71. Structure of compound 10 .................................................................................

Figure 72 .

 72 Figure 72. Structure of compound 11 .................................................................................

Figure 73 .

 73 Figure 73. Purification of compounds in leaves of W. florida "Jean's Gold" ....................

Figure 74 .

 74 Figure 74. Structure of compound 12 .................................................................................

Figure 75 .

 75 Figure 75. HSQC spectrum of compound 12 .....................................................................

Figure 76 .

 76 Figure 76. HSQC spectrum of sugar anomers of compound 12 ........................................

Figure 77 .

 77 Figure 77. HMBC spectrum of sugar moieties of compound 12 .......................................

Figure 78 .

 78 Figure 78. ROESY spectrum of sugar moieties of compound 12 ......................................

Figure 79 .

 79 Figure 79. Mass spectrum of compound 12 .......................................................................

Figure 80 .

 80 Figure 80. Structure of compound 13 .................................................................................

Figure 81 .

 81 Figure 81. HSQC spectrum of compound 13 .....................................................................

Figure 82 .

 82 Figure 82. HSQC spectrum of sugar anomers of compound 13 ........................................

Figure 83 .

 83 Figure 83. HMBC spectrum of sugar moieties of compound 13 .......................................

Figure 84 .

 84 Figure 84. ROESY spectrum of sugar moieties of compound 13 ......................................

Figure 85 .

 85 Figure 85. Mass spectrum of compound 13 .......................................................................

Figure 86 .

 86 Figure 86. Structure of compound 14 .................................................................................

Figure 87 .

 87 Figure 87. Structure of compound 15 ...............................................................................

Figure 88 .

 88 Figure 88. Structure of compound 16 ...............................................................................

Figure 89 .

 89 Figure 89. Structure of compound 17 ...............................................................................

Figure 90 .

 90 Figure 90. Purification of compounds in roots of Cordyline fruticosa "Fairchild red" ...

Figure 91 .

 91 Figure 91. Structure of compound 18 ...............................................................................

Figure 92 .

 92 Figure 92. HSQC spectrum of compound 18 ...................................................................

Figure 93 .

 93 Figure 93. HSQC spectrum of compound 18 ...................................................................

Figure 94 .

 94 Figure 94. HMBC spectrum of sugar moieties of compound 18 .....................................

Figure 95 .

 95 Figure 95. ROESY spectrum of aglycon of compound 18...............................................

Figure 96 .

 96 Figure 96. ROESY spectrum of sugar moieties of compound 18 ....................................

Figure 97 .

 97 Figure 97. Mass spectrum of compound 18 .....................................................................

Figure 98 .

 98 Figure 98. Structure of compound 19 ...............................................................................

Figure 99 .

 99 Figure 99. HSQC spectrum of compound 19 ...................................................................

Figure 100 .

 100 Figure 100. HSQC spectrum of sugar moieties of compound 19 ....................................

Figure 101 .

 101 Figure 101. HMBC spectrum of sugar moieties of compound 19 ...................................

Figure 102 .

 102 Figure 102. ROESY spectrum of sugar moieties of compound 19 ..................................

Figure 103 .

 103 Figure 103. Mass spectrum of compound 19 ...................................................................

Figure 104 .

 104 Figure 104. Structure of compound 20 .............................................................................

Figure 105 .

 105 Figure 105. HSQC spectrum of compound 20 .................................................................

Figure 106 .

 106 Figure 106. HSQC spectrum of sugar moieties of compound 20 ....................................

Figure 107 .

 107 Figure 107. HMBC spectrum of sugar moieties of compound 20 ...................................

Figure 108 .

 108 Figure 108. HMBC spectrum of sugar moieties of compound 20 ...................................

Figure 109 .

 109 Figure 109. ROESY spectrum of sugar moieties of compound 20 ..................................

Figure 110 .

 110 Figure 110. Mass spectrum of compound 20 ...................................................................

Figure 111 .

 111 Figure 111. Structure of compound 21 .............................................................................

Figure 112 .

 112 Figure 112. HSQC spectrum of compound 21 .................................................................

Figure 113 .

 113 Figure 113. HSQC spectrum of sugar moieties of compound 21 ....................................

Figure 114 .

 114 Figure 114. HMBC spectrum of sugar moieties of compound 21 ................................... Figure 115. ROESY spectrum of sugar moieties of compound 22 ..................................

Figure 116 .

 116 Figure 116. Mass spectrum of compound 22 ...................................................................

Figure 117 .

 117 Figure 117. Structure of compound 22 .............................................................................

Figure 118 .

 118 Figure 118. HSQC spectrum of compound 22 .................................................................

Figure 119 .

 119 Figure 119. HSQC spectrum of sugar moieties of compound 22 ....................................

Figure 120 .

 120 Figure 120. HMBC spectrum of sugar moieties of compound 22 ...................................

Figure 121 .

 121 Figure 121. ROESY spectrum of sugar moieties of compound 22 ..................................

Figure 122 .

 122 Figure 122. ROESY spectrum of sugar moieties of compound 22 ..................................

Figure 123 .

 123 Figure 123. Mass spectrum of compound 22 ...................................................................

Figure 124 .

 124 Figure 124. Structure of compound 23 .............................................................................

Figure 125 .

 125 Figure 125. HSQC spectrum of compound 23 .................................................................

Figure 126 .

 126 Figure 126. HSQC spectrum of sugar moieties of compound 23 ....................................

Figure 127 .

 127 Figure 127. HSQC spectrum of sugar moieties of compound 23 ....................................

Figure 128 .

 128 Figure 128. ROESY spectrum of sugar moieties of compound 23 ..................................

Figure 129 .

 129 Figure 129. Mass spectrum of compound 23 ...................................................................

Figure 130 .

 130 Figure 130. Structure of compound 24 .............................................................................

Figure 131 .

 131 Figure 131. HSQC spectrum of compound 24 .................................................................

Figure 132 .

 132 Figure 132. HSQC spectrum of sugar moieties of compound 24 ....................................

Figure 133 .

 133 Figure 133. HMBC spectrum of sugar moieties of compound 24 ...................................

Figure 134 .

 134 Figure 134. ROESY spectrum of sugar moieties of compound 24 ..................................

Figure 135 .

 135 Figure 135. Mass spectrum of compound 24 ...................................................................

Figure 136 .

 136 Figure 136. Structure of compound 25 .............................................................................

Figure 137 .

 137 Figure 137. HSQC spectrum of compound 25 .................................................................

Figure 138 .

 138 Figure 138. HSQC spectrum of sugar anomers of compound 25 ....................................

Figure 139 .

 139 Figure 139. HSQC spectrum of sugar moieties of compound 25 ....................................

Figure 140 .

 140 Figure 140. HMBC spectrum of sugar moieties of compound 25 ...................................

Figure 141 .

 141 Figure 141. ROESY spectrum of sugar moieties of compound 25 ..................................

Figure 142 .

 142 Figure 142. Mass spectrum of compound 25 ...................................................................

Figure 143 .

 143 Figure 143. Structure of compound 26 .............................................................................

Figure 144 .

 144 Figure 144. HSQC spectrum of compound 26 .................................................................Figure 145. HSQC spectrum of sugar anomers of compound 26 ....................................

Figure 146 .

 146 Figure 146. HSQC spectrum of sugar moieties of compound 26 ....................................

Figure 147 .

 147 Figure 147. HMBC spectrum of sugar moieties of compound 26 ...................................

Figure 148 .

 148 Figure 148. ROESY spectrum of sugar moieties of compound 26 ..................................

Figure 149 .

 149 Figure 149. Mass spectrum of compound 26 ...................................................................

Figure 150 .

 150 Figure 150. Structure of compound 27 .............................................................................

Figure 151 .

 151 Figure 151. Structure of compound 28 .............................................................................

Figure 152 .

 152 Figure 152. Purification of compounds in aerial parts of C. fruticosa "Fairchild red" ....

Figure 153 .

 153 Figure 153. Structure of compound 29 .............................................................................

Figure 154 .

 154 Figure 154. HSQC spectrum of compound 29 .................................................................

Figure 155 .

 155 Figure 155. HSQC spectrum of sugar anomers of compound 29 ....................................

Figure 156 .

 156 Figure 156. HSQC spectrum of sugar moieties of compound 29 ....................................

Figure 157 .

 157 Figure 157. HMBC spectrum of sugar moieties of compound 29 ...................................

Figure 158 .

 158 Figure 158. ROESY spectrum of sugar moieties of compound 29 ..................................

Figure 159 .

 159 Figure 159. Mass spectrum of compound 29 ...................................................................

Figure 160 .

 160 Figure 160. Structure of compound 30 .............................................................................

Figure 161 .

 161 Figure 161. HSQC spectrum of compound 30 .................................................................

Figure 162 .

 162 Figure 162. HSQC spectrum of sugar anomers of compound 30 ....................................

Figure 163 .

 163 Figure 163. HSQC spectrum of sugar moieties of compound 30 ....................................

Figure 164 .

 164 Figure 164. HMBC spectrum of sugar moieties of compound 30 ...................................

Figure 165 .

 165 Figure 165. ROESY spectrum of sugar moieties of compound 30 ..................................

Figure 166 .

 166 Figure 166. Mass spectrum of compound 30 ...................................................................

Figure 167 .

 167 Figure 167. Structure of compound 31 .............................................................................

Figure 168 .

 168 Figure 168. HSQC spectrum of compound 31 .................................................................

Figure 169 .

 169 Figure 169. HSQC spectrum of sugar anomers of compound 31 ....................................

Figure 170 .

 170 Figure 170. HSQC spectrum of sugar moieties of compound 31 ....................................

Figure 171 .

 171 Figure 171. HMBC spectrum of compound 31 ................................................................

Figure 172 .

 172 Figure 172. ROESY spectrum of compound 31 ...............................................................

Figure 173 .

 173 Figure 173. Mass spectrum of compound 31 ...................................................................

Figure 174 .

 174 Figure 174. Structure of compound 32 .............................................................................Figure 175. HSQC spectrum of compound 32 .................................................................

Figure 176 .

 176 Figure 176. HSQC spectrum of sugar anomers of compound 32 ....................................

Figure 177 .

 177 Figure 177. HSQC spectrum of sugar moieties of compound 32 ....................................

Figure 178 .

 178 Figure 178. HMBC spectrum of sugar moieties of compound 32 ...................................

Figure 179 .

 179 Figure 179. ROESY spectrum of sugar moieties of compound 32 ..................................

Figure 180 .

 180 Figure 180. Mass spectrum of compound 32 ...................................................................

Figure 181 .

 181 Figure 181. Purification of compounds in roots of Dracaena braunii .............................

Figure 182 .

 182 Figure 182. Structure of compound 33 .............................................................................

Figure 183 .

 183 Figure 183. Structure of compound 34 .............................................................................

Figure 184 .

 184 Figure 184. Structure of compound 35 .............................................................................

Figure 185 .

 185 Figure 185. MTS assay summary .....................................................................................

Figure 187 .

 187 Figure 187. Structure compounds 1-3, 6 and 7 ................................................................

Figure 188 .

 188 Figure 188. Evaluation of the cytotoxic activity of compounds 8, 10, 11, 13, 15, 17 against CT26, B16 and HepG2, in concentrations ranging from 1 to 50 µM....................................

Figure 189 .

 189 Figure 189. Structure compounds 8, 10, 11, 13, 15 and 17 ..............................................

  C and 1 H NMR spectroscopic data of the aglycon and sugar moieties of 8 in pyridine-d5............................................................................................................................................. Table 6. 13 C and 1 H NMR spectroscopic data of the aglycon moieties of compounds 12 and 13 in pyridine-d5 .............................................................................................................................. Table 7. 13 C and 1 H NMR spectroscopic data of the sugar moieties of compounds 12 and 13 in pyridine-d5 .............................................................................................................................. Table

  (4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium) 5-FU Fluorouracil INTRODUCTION Saponins are a class of phytochemical compounds which are found mainly but not exclusively in plants and normally foam when exposed in aqueous solution. In their structure, saponins are amphiphilic compounds composed of sugar chain(s) attached with an aglycon and as the result, this characteristic is cause of the foaming ability of saponins. The sugar chain(s) contains one or more linear oligosaccharides (L-arabinose, D-galactose, D-glucose, D-galactose, L-rhamnose and D-fructose) while aglycon (or sapogenin) is either a steroid (C27) or a triterpenoid (C30). When a single sugar chain normally attached at C-3 of aglycon, it is a mono-desmosidic saponin, while the other has two sugar chains which the one linked to C-3 and another one linked to C-22 of aglycon defined as bi-desmosidic saponins (Chaieb, 2010). Tri-desmosidic saponins have three sugar chains and are usually rare among others (Majinda, 2012). In plants, saponins can be found in different parts of a number of plants, such as root, trunk, leaf, flower and seed, in both wild plants and cultivated crops. Their concentration, quantification and qualification depend on not only the cultivar, age and distribution, but also the part of plant species. Van et al. (2015) compared the content of saponin in different underground parts of Panax vietnamensis including radix, rhizome and roots.

Figure 1 .

 1 Figure 1. Phylogenetic classification of order Dipsacales (Missouri Botanical Garden)

Figure 2 .

 2 Figure 2. Diagram of Caprifoliaceae flower part (Watson, L., and Dallwitz, M.Z, 1992)1.1.2. Genus Weigela

1. 1 . 3 .

 13 Some horticultural species of genus Weigela a. Weigela x "Bristol Ruby" Weigela x "Bristol Ruby" is a hybrid deciduous shrub of upright habit with narrowly ovate, dark green leaves. Flowers are ruby-red produced and cover the foliage. The bell-shaped flowers attract hummingbirds. This plants can grow up to 120-140cm in height and 120-180 cm in width and may flower during the summer into fall(Gardenia, 2019).

Figure 3 .

 3 Figure 3. Weigela x "Bristol Ruby" b. Weigela florida "Jean's Gold"

Figure 4 .

 4 Figure 4. Weigela florida "Jean's Gold"

Figure 5 .

 5 Figure 5. Weigela florida "Pink Poppet"

( 1986 )

 1986 and then by theAPG 1998APG , 2003APG , 2009APG and 2016. . In the past, many families of the order were distributed in old order Liliales which contains almost of monocot plants. But, it has then redistributed by DNA sequence analysis to three orders including Liliales, Asparagales and Dioscoreales that Asparagales becomes the largest order of monocots with 14 families, 1122 genera and 36205 species (Missouri Botanical Garden, 2019b).

Figure 6 .

 6 Figure 6. Phylogenetic classification of order Asparagales (Missouri Botanical Garden, 2019b)

  grow as understory plants in rainforests. It is an upright shrub growing to 1.5 m in height, with leaves 15-25 cm in length and 1.5-4 cm breadth at the base. Indoor height rarely exceeds 90cm. This species can grow in water and have a bright green, cane-like stem, green leaves and roots with different colors (black, white, orange, red) (PlantsRescue, 2019).

Figure 7 .

 7 Figure 7. Dracaena braunii

Figure 9 .

 9 Figure 9. Structure of spirostane aglycon (a) and furostane aglycon (b) (P. Munafo Jr and Gianfagna, 2014)

  contain aglycons with 30 carbon atoms. The most commonly occurring core structures are pentacyclic oleananes and tetracyclic dammarans. Other aglycons of triterpenoid saponins are of the ursane-, lupane-, and hopane-type. The ring systems A/B/C/D/E for oleanane and ursane derivatives are linked together in the order trans-trans-trans-trans, for lupane derivatives in the order trans-trans-trans-cis, and for the rings A/B/C/D in dammarane derivatives in the order trans-trans-trans.

Figure 10 .

 10 Figure 10. Structure of oleanane-(a), dammarane-(b), ursane-(c), lupane-(d) and hopane-type aglycon (e) (Moses et al., 2014) Substituents on the frequently occurring aglycons derived from the oleanane structure of saponins are showed in Table 2. The hydroxyl group at position C-3 is found in all structures; very often hydroxyl groups are also reported at positions C-16, C-21, and C-22, and less often in positions C-2 and C-15. The methyl groups in positions C-23, C-24, C-28, C-29, and C-30 can be oxidized to CH2OH-or COOH-moieties, and in some cases also to a CHO-group. Epoxide groups, keto functions and double bonds between C-12 and C-13

Figure 11 .

 11 Figure 11. Available different sugars in saponins

2 SO3HFigure 13 .

 213 Figure 13. Steroidal saponins elucidated from species of Cordyline genus 2.4. Saponins isolated from genus Dracaena The botanical name of Dracaena comes from ancient Greek "drakaina" which means dragon. It refers to a deep red resin and comprises approximately 60 species which are found nearly in tropical and subtropical of Africa (Marrero, 1998). The genus Dracaena contains both spirostane-type and furostane-type steroidal saponins possessing bioactivities. Different steroidal saponins elucidated from species of Cordyline genus are showed in Fig.14.

H

  Dracaena Dracaena draco L. (Hernández et al., 2006) Dracaena surculosa (Yokosuka et al., 2000) Dracaena surculosa (Yokosuka et al., 2000) Dracaena surculosa (Yokosuka et al., 2002) R β-D-Fucp β-D-Glcp Dracaena surculosa (Yokosuka et al., 2002) R β-D-Glcp β-D-Fucp Dracaena cambodiana (Shen et al., 2014)

  outcome depends upon a balance among three polarities -that of the plate, the development solvent and the spot material. Different components in the original spot, having different polarities, will move different distances from the original spot location and show up as separate spots (Oleszek and Bialy, 2006). Chromatographic conditions -Stationary phase: Silica plate for normal phase: 60 Å F254, Silicycle. Silica plate high performance HPTLC: 60 Å F254, Merck. -Mobile phase: Chloroform (CHCl3)-Methanol (MeOH)-Water (H2O) CHCl3/MeOH/H2O 85/15/2; 75/25/3; 70/30/5; 65/31/6; 60/32/7

  of the sample are separated from each other by partitioning between the stationary packing material and the mobile eluent. Molecules with different polarity partition to different extents are moved through the column at different rates. The eluent is collected in fractions. Fractions are typically analysed by TLC to see if separation of the components was successful (Hostettmann and Wolfender, 2000). Chromatographic conditions -Stationary phase: Sephadex LH-20 is prepared by hydroxypropylation of Sephadex G-25 to give a Dextran gel with both hydrophilic and lipophilic properties. It is stable in all solvents which are not strongly acidic and do not contain strong oxidizing agents. Separation depends on molecular size (gel filtration), adsorption, partition, ion exclusion and retardation, and probably other mechanisms still unknown (Murphy and D'Aux, 1975).

  Stationary phase is washed by MeOH and equilibrated by the solvent used as an eluent. Sample is dissolved in mixture Chloroform: Methanol: Water (Hostettmann and Wolfender, 2000). Chromatographic conditions -Material: Stationary phase: RediSep Rf Normal phase Silica Flash Columns (60 Å, 35-70µm).-Mobile phase: CHCl3/MeOH/H2O 70:30:5; 60:32:7 (v/v).d. Medium Pressure Liquid Chromatography (MPLC)Medium-pressure liquid chromatography (MPLC) is one of the various preparative column chromatography techniques which separate samples under pressure to render the use of smaller particle size supports possible and increases the diversity of usable stationary phases. MPLC allows purification of large compound quantities, faster and improved separations. Packing of material with lower particle size under pressure enhances separation quality and moreover the solid phase can be re-used[START_REF] Hostettmann | Medium pressure liquid chromatography A2 -Wilson, Ian D. BT -Encyclopedia of Separation Science[END_REF]. Chromatographic conditions -Material: + Separation column using silica: 110 x 15 mm, 460 x 25 mm, 460 x 15 mm, 230 x 15 mm. + Pump for equipment MPLC normal phase: HPLC pump 426 (Alltech); Fraction collector: Biophamacia. + Pump for equipment MPLC reverse-phase RP-18: Büchi ® Pump manager C-605, Büchi ® Pump module C-601; Fraction collector: C-660, 4 rack, 240 tubes. -Stationary phase Silica for normal phase: 60 Å, 15-40 µm, Merck. Silica for reversed phase: RP-18 Spherical C18 (300 Å, 75-200 µm, Silicycle). -Mobile phase Silica for normal phase: CHCl3/MeOH/H2O 75/25/3; 70/30/5, 60/32/7 Silica for reversed phase RP-18: Several gradients of MeOH/H2O and EtOH/H2O can be utilized (20/80, 30/70, 40/60, 50/50).

  than others traditional techniques of mass determination[START_REF] Cech | Practical Implications of Some Recent Studies in Electrospray Ionization Fundamentals[END_REF][START_REF] Ho | Electrospray Ionisation Mass Spectrometry: Principles and Clinical Applications[END_REF].

Figure 15 . 1 . 3 . 3 .

 15133 Figure 15. Mechanism of electrospray ionization (Cech and Enke, 2001)1.3.3. Nuclear magnetic resonance (NMR) spectrometry

Figure 17 .Figure 16 .

 1716 Figure 17. Correlations of COSY (orange), TOCSY (pink) and ROESY (green) Rotating Frame Overhauser Effect Spectroscopy (ROESY)

  49 g) were submitted to an ultrasound assisted extraction three times with EtOH/H2O (75/35, 3 x 1L, 1 h each). The resulting extracts were combined and concentrated under vacuum to give a crude extract (3 g), which was fractionated by VLC (Silica gel 60, CHCl3/MeOH/H2O 70/30/5) yielding 12 fractions (F1-F12). The fractions F3 and F4 were combined (780 mg) and then submitted to CC (Sephadex LH-20, MeOH) to give 4 fractions. The fraction rich in saponins (103 mg) was fractionated by MPLC on silica gel 60 (CHCl3/MeOH/H2O 80/20/2, 70/30/5) yielding compounds 1 (4.4 mg), 2 (3.8 mg), 3 (3.7 mg), 4 (3.6 mg), 5 (3.6 mg), and 7 (8.6 mg). The combined fractions F5 and F6 (130.4 mg) were fractionated by MPLC on silica gel 60 (CHCl3/MeOH/H2O 80/20/2, 70/30/5) to give compound 6 (3.1 mg).

Figure 18 .

 18 Figure 18. Purification scheme of compounds in roots of Weigela x "Bristol Ruby"

  C NMR signals of the aglycon assigned from the 2D NMR spectra, were in good agreement with those of oleanolic acid commonly encountered in the Weigela genus (Andriamisaina et al., 2018; Champy-Tixier et al., 2018; Rezgui et al., 2016). The downfield shift at δC 88.7 and upfield shift at δC 180.2, due to the glycosylation shift, suggested that compound 1 was a monodesmosidic glycoside.

  1 H NMR spectrum of 1 showed five anomeric proton signals at δH 4.88 (d, J = 6.0 Hz), 4.99 (d, J = 7.6 Hz), 5.09 (d, J = 7.6 Hz), 5.26 (d, J = 7.6 Hz) and 6.18 (br s), which gave correlations in the HSQC spectrum with their corresponding anomeric carbons at δC 105.0, 103.2, 105.5, 107.0 and 101.5, respectively. All protons and carbons signals were identified by 2D NMR experiments, starting mainly by the TOCSY experiment (Table 3). Five sugar moieties are identified: -1 α-L-arabinopyranosyl Ara-1 at δH 4.88 (d, J = 6.0 Hz), -1 α-L-rhamnopyranosyl Rha-1 at δH 6.18 (br s), -1 β-D-glucopyranosyl Glc-1 at δH 4.99 (d, J = 7.6 Hz), -2 β-D-xylopyranosyl Xyl I-1 at δH 5.26 (d, J = 7.6 Hz) and Xyl II-1 at δH 5.09 (d, J = 7.6 Hz).

5 (

 5 Rha C-1) and in ROESY at δH 4.55 (Ara I H-2) and δH 6.18 (Rha H-1) proved that Rha was attached to the Ara at C-2. at δH 5.26 (Xyl I H-1) and δH 4.67 (Rha H-3) proved that Xyl I was attached to the Rha at C-3. -The HMBC correlation between δH 4.99 (Glc H-1) and δC 77.6 (Xyl I C-4) and in ROESY at δH 4.99 (Glc H-1) and δH 4.27 (Xyl I H-4) proved that Glc was attached to the Xyl I at C-4. -The HMBC correlation between δH 5.09 (Xyl II H-1) and δC 80.7 (Glc C-4) and in ROESY at δH 5.09 (Xyl II H-1) and δH 4.26 (Glc H-4) proved that Xyl II was attached to the Glc at C-4. Conclusion Based on the above results, the structure of 1 was elucidated as 3-O-β-D-xylopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)α-L-arabinopyranosyloleanolic acid.

Figure 19 . 1 Figure 20 . 1 Figure 21 . 1 RhaFigure 24 . 1 Figure 25 . 1 AraFigure 26 .Figure 27 . 2 Compound 2 ,

 191201211241251262722 Figure 19. Structure of compound 1

2

 2 differs from 1 by only 162 amu, corresponding to a supplementary hexosyl group. The part of the HSQC spectrum corresponding to the osidic chain showed cross-peaks at δH/δC 4.88/105.0, 4.94/102.8, 5.25 (d, J = 7.2 Hz)/107.0, 5.48 (d, J = 6.4 Hz)/102.9, 5.52 (d, J = 7.6 Hz)/104.7 and 6.16 (br s)/101.6, indicating the presence of six sugar units. After analysing the spectre of HSQC, HMBC, COSY, TOCSY and ROESY, the sugar moieties of compound 2 was identified as: -1 α-L-arabinopyranosyl Ara-1 at δH 4.88 (d, J = 6.0 Hz), -1 α-L-rhamnopyranosyl Rha-1 at δH 6.16 (br s), -2 β-D-glucopyranosyl Glc I-1 at δH 4.94 and Glc II-1 at δH 5.52 (d, J = 7.6 Hz) -2 β-D-xylopyranosyl Xyl I-1 at δH 5.25 (d, J = 7.2 Hz) and Xyl II-1 at δH 5.09 (d, J = 6.4 Hz).

  6 (Rha C-1) and in ROESY at δH 4.54 (Ara I H-2) and δH 6.16 (Rha H-1) proved that Rha was attached to the Ara at C-2. -The HMBC correlation between δH 5.25 (Xyl I H-1) and δC 83.1 (Rha C-3) and in ROESY at δH 5.25 (Xyl I H-1) and δH 4.67 (Rha H-3) proved that Xyl I was attached to the Rha at C-3. -The HMBC correlation between δH 4.94 (Glc I H-1) and δC 77.9 (Xyl I C-4) and in ROESY at δH 4.94 (Glc H-1) and δH 4.22 (Xyl I H-4) proved that Glc I was attached to the Xyl I at C-4. -The HMBC correlation between δH 5.52 (Glc II H-1) and δC 82.2 (Glc I C-3) and in ROESY at δH 5.52 (Glc II H-1) and δH 4.45 (Glc I H-3) proved that Glc II was attached to the Glc I at C-3. -The HMBC correlation between δH 5.48 (Xyl II H-1) and δC 74.2 (Glc I C-4) and in ROESY at δH 5.48 (Xyl II H-1) and δH 4.54 (Glc I H-4) proved that Xyl II was attached to the Glc I at C-4.

Figure 28 . 2 Figure 29 .RhaFigure 32 . 2 Figure 33 .

 2822932233 Figure 28. HSQC spectrum of compound 2

  cross-peaks at δH/δC 4.88 (d, J = 6.2 Hz)/105.0, 4.90 (d, J = 7.8 Hz)/102.9, 5.24 (d, J = 7.6 Hz)/107.0, 5.35 (d, J = 6.8 Hz)/103.7, 5.92 (br s)/103.4, and 6.16 (br s)/101.6, indicating the presence of six sugar units. Starting mainly the TOCY and NOESY spectra, the sugar moieties of compound 3 was identified as: -2 α-L-arabinopyranosyl Ara I-1 at δH 4.88 (d, J = 6.2 Hz) and Ara II-1 at δH 5.92 (br s).

- 1 β

 1 -D-glucopyranosyl Glc-1 at δH 4.90 (d, J = 7.8 Hz), -2 β-D-xylopyranosyl Xyl I-1 at δH 5.24 (d, J = 7.6 Hz) and Xyl II-1 at δH 5.35 (d, J = 6.8 Hz).

  6 (Rha C-1) and in ROESY at δH 4.88 (Ara I H-2) and δH 6.16 (Rha H-1) proved that Rha was attached to the Ara at C-2. -The HMBC correlation between δH 5.24 (Xyl I H-1) and δC 83.0 (Rha C-3) and in ROESY at δH 5.24 (Xyl I H-1) and δH 4.66 (Rha H-3) proved that Xyl I was attached to the Rha at C-3. -The HMBC correlation between δH 4.90 (Glc H-1) and δC 77.4 (Xyl I C-4) and in ROESY at δH 4.90 (Glc H-1) and δH 4.21 (Xyl I H-4) proved that Glc was attached to the Xyl I at C-4. -The HMBC correlation between δH 5.92 (Ara II H-1) and δC 79.8 (Glc C-3) and in ROESY at δH 5.92 (Ara II H-1) and δH 4.44 (Glc H-3) proved that Xyl II was attached to the Glc at C-3. -The HMBC correlation between δH 5.35 (Xyl II H-1) and δC 74.0 (Glc I C-4) and in ROESY at δH 5.35 (Xyl II H-1) and δH 4.48 (Glc H-4) proved that Xyl II was attached to the Glc at C-4.

Figure 34 .Figure 35 .

 3435 Figure 34. Structure of compound 3

57 Figure 42 .Figure 43 .

 574243 Figure 42. Structure of compound 4

  82 (d, J = 7.6 Hz)/106.1, 4.86 (d, J = 7.6 Hz)/103.8, 4.97/103.2, 5.03 (d, J = 8.0 Hz)/105.1, 5.28 (d, J = 7.2 Hz)/107.1, and 6.50 (br s)/101.6, indicating the presence of six sugar units. Starting mainly the TOCY and ROESY spectra, the sugar moieties of compound 5 was identified as: -1 α-L-rhamnopyranosyl Rha-1 at δH 6.50 (br s), -1 β-D-glucopyranosyl Glc-1 at δH 4.97, -4 β-D-xylopyranosyl Xyl I-1 at δH 4.82 (d, J = 7.6 Hz), Xyl II-1 at δH 5.28 (d, J = 7.2 Hz), Xyl III-1 at δH 5.03 (d, J = 8.0 Hz) and Xyl IV-1 at δH 4.86 (d, J = 7.6 Hz). The sequence of the oligosaccharide was established by analysing the HMBC and ROESY spectra:-The HMBC correlation between δH 4.82 (Xyl I H-1) and δC 88.5 (C-3) and in ROESY at

  δH 4.82 (Xyl I H-1) and δH 3.30 (H-3) proved that Xyl I was attached to the C-3 position of the aglycon (Fig.48).ROESY at δH 4.22 (Xyl I H-2) and δH 6.50 (Rha H-1) proved that Rha was attached to the Xyl I at C-2.-The HMBC correlation between δH 5.28 (Xyl II H-1) and δC 83.1 (Rha C-3) and in ROESY at δH 5.28 (Xyl II H-1) and δH 4.72 (Rha H-3) proved that Xyl II was attached to the Rha at C-3. -The HMBC correlation between δH 4.97 (Glc H-1) and δC 77.6 (Xyl II C-4) and in ROESY at δH 4.97 (Glc H-1) and δH 4.26 (Xyl II H-4) proved that Glc was attached to the Xyl II at C-4. -The HMBC correlation between δH 5.03 (Xyl III H-1) and δC 80.5 (Glc C-4) and in ROESY at δH 5.03 (Xyl III H-1) and δH 4.21 (Glc H-4) proved that Xyl III was attached to the Glc at C-4. -The HMBC correlation between δH 4.86 (Xyl IV H-1) and δC 76.1 (Xyl III C-4) and in ROESY at δH 4.86 (Xyl IV H-1) and δH 4.22 (Xyl III H-4) proved that Xyl IV was attached to the Xyl III at C-4. Conclusion Due to the evidences above, the structure of 5 was elucidated as 3-O-β-D-xylopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-β-D-xylopyranosyloleanolic acid.

Figure 48 .Figure 49 . 5 Figure 50 .Zone

 4849550 Figure 48. Structure of compound 5

δH 4 .

 4 69 (d, J = 5.2 Hz), 4.79 (d, J = 7.6 Hz), 5.14 (d, J = 6.9 Hz), 5.19 (d, J = 7.6 Hz), 6.00 (d, J = 7.3 Hz) and 6.33 (br s), and carbons at δC 104.8, 101.2, 102.2, 105.3, 92.1 and 100.1, respectively. The 2D NMR spectroscopic analyses enabled the full assignments of the resonances of these sugars as: -1 α-L-rhamnopyranosyl Rha-1 at δH 6.33 (br s), -2 β-D-glucopyranosyl Glc I-1 at δH 4.79 and Glc II-1 at δH 6.00, -3 β-D-xylopyranosyl Xyl I-1 at δH 4.69 (d, J = 6.0 Hz), Xyl II-1 at δH 5.19 (d, J = 7.6 Hz) and Xyl III-1 at δH 5.14 (d, J = 6.9 Hz). The sequence of the oligosaccharide was established by analysing the HMBC and NOESY spectra: -The HMBC correlation between δH 4.69 (Xyl I H-1) and δC 87.4 (C-3) and in NOESY at δH 4.69 (Xyl I H-1) and δH 3.23 (H-3) proved that Xyl I was attached to the C-3 position of the aglycon (Fig.55). NOESY at δH 4.09 (Xyl I H-2) and δH 6.33 (Rha H-1) proved that Rha was attached to the Xyl I at C-2. -The HMBC correlation between δH 5.19 (Xyl II H-1) and δC 80.9 (Rha C-3) and in NOESY at δH 5.19 (Xyl II H-1) and δH 4.63 (Rha H-3) proved that Xyl II was attached to the Rha at C-3. -The HMBC correlation between δH 4.79 (Glc I H-1) and δC 76.1 (Xyl II C-4) and in NOESY at δH 4.79 (Glc I H-1) and δH 4.14 (Xyl II H-4) proved that Glc I was attached to the Xyl II at C-4. -The HMBC correlation between δH 5.14 (Xyl III H-1) and δC 79.0 (Glc C-4) and in NOESY at δH 5.14 (Xyl III H-1) and δH 4.31 (Glc H-4) proved that Xyl III was attached to the Glc I at C-4. -The shielded chemical shift at δC 92.1 (Glc II C-1) and the shielded at δC 177.0 (C-28), suggested an ester linkage of Glc II with C-28 of the aglycon. Conclusion These evidences led to the elucidation of 6 as 3-O-β-D-xylopyranosyl-(1→4)-β-Dglucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-β-Dxylopyranosyloleanolic acid 28-O-β-D-glucopyranosyl ester.

Figure 55 .Figure 56 . 6 Figure 57 .Figure 60 .Figure 61 .

 55566576061 Figure 55. Structure of compound 6

Figure 62 .Figure 63 .

 6263 Figure 62. Purification of compounds in roots of Weigela florida "Pink Poppet"

  ESIMS (positive-ion mode) spectrum of compound 8 showed a pseudo-molecular ion peak [M+Na] + at 1315.6029 m/z, indicating a molecular weight of 1292 and a molecular formula of C62H100O28.

1. 3 . 4 )

 34 . The relatively large3 JH-1, H-2 values of the Glc, Xyl, and Ara (5.0-8.2 Hz) indicated a ß anomeric orientation for Glc and Xyl, and an α anomeric orientation for Ara. The large1 JH-1,C-1 values of the Rha (165-168 Hz), confirmed that the anomeric protons were equatorial (α-pyranoid anomeric form).The sugar parts of 8 contains six residues as evidenced by1 H spectrum which displayed six anomeric protons at δH 4.80 (d, J = 4.7 Hz), 4.91 (d, J = 7.6 Hz), 5.28 (d, J = 7.0 Hz), 5.31 (d, J = 7.6 Hz), 5.85 (d, J = 4.6 Hz) and 6.48 (br s) which gave correlation, in the HSQC spectrum, with six anomeric carbon signals at δC 105.8, 102.5, 103.4, 106.4, 103.2 and 101.2, respectively. Starting mainly the TOCY and ROESY spectra, the sugar moieties of compound 8 was identified as: -1 α-L-arabinopyranosyl Ara-1 at δH 5.85 (d, J = 4.6 Hz), -1 α-L-rhamnopyranosyl Rha-1 at δH 6.48 (br s), -1 β-D-glucopyranosyl Glc-1 at δH 4.91 (d, J = 7.6 Hz), -3 β-D-xylopyranosyl Xyl I-1 at δH 4.80 (d, J = 4.7 Hz), Xyl II-1 at δH 5.31 (d, J = 7.6 Hz) and Xyl III-1 at δH 4.91 (d, J = 7.6 Hz). The sequence of the oligosaccharide was established by analysing the HMBC and ROESY spectra: -The HMBC correlation between δH 4.80 (Xyl I H-1) and δC 88.5 (C-3) and in ROESY atδH 4.80 (Xyl I H-1) and δH 3.35 (H-3) proved that Xyl I was attached to the C-3 position of the aglycon (Fig.64). -The HMBC correlation between δH 4.21 (Xyl I H-2) and δC 101.2 (Rha C-1) and in ROESY at δH 4.21 (Xyl I H-2) and δH 6.48 (Rha H-1) proved that Rha was attached to the Xyl I at C-2. -The HMBC correlation between δH 5.31 (Xyl II H-1) and δC 82.1 (Rha C-3) and in ROESY at δH 5.31 (Xyl II H-1) and δH 4.75 (Rha H-3) proved that Xyl II was attached to the Rha at C-3. -The HMBC correlation between δH 4.91 (Glc H-1) and δC 77.2 (Xyl II C-4) and in ROESY at δH 4.91 (Glc H-1) and δH 4.26 (Xyl II H-4) proved that Glc was attached to the Xyl II at C-4.

  Based on the above results, the structure of 8 was elucidated as 3-O-β-D-xylopyranosyl-(1→4)-[α-L-arabinopyranosyl-(1→3)]-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-xylopyranosyloleanolic acid.

Figure 64 . 8 Figure 65 . 8 Figure 66 . 8 ZoneFigure 69 .Figure 70 .

 6486586686970 Figure 64. Structure of compound 8

Figure 71 .

 71 Figure 71. Structure of compound 10 d. Compound 11 (WEF200918)

  8 g) were submitted to microwaves three times with the solvent EtOH/H2O (200 W, 60°C, 45 min, 75/35 400 mL each). After filtration and evaporation, 21.5 g of crude of extract were obtained. An aliquot (17.5 g) was fractionated using successive VLC on silica gel 60, (CHCl3/MeOH/H2O 60/32/7), and on RP-18 silica gel (H2O, EtOH/H2O 50/50, EtOH), yielding 4 fractions (A1-A4). Fraction A3 (309.1 mg) was submitted to MPLC on silica gel 60 (CHCl3/MeOH/H2O 70/30/5) yielding two pure compounds, 13 (3.2 mg) and 15 (2.8 mg) among the 5 fractions obtained (A3.1 to A3.5). The remaining extract (4.0 g) was fractionated by flash chromatography (CHCl3/MeOH/H2O 60/32/7) to give 4 fractions. The fractions rich in saponins were further separated using MPLC on silica gel 60 (CHCl3/MeOH/H2O 80/20/2, 75/25/3) yielding compound 12 (3.2 mg) and compound 14 (8.0 mg) which was previously obtained from the leaves extract of W. florida "Pink Poppet". The remaining fractions were combined and chromatographed again on silica gel 89 60 by MPLC (CHCl3/MeOH/H2O 70/30/5), yielding compound 16 (4.4 mg), compound 17 (8.6 mg) and compound 14 (2.8 mg).

Figure 73 .

 73 Figure 73. Purification of compounds in leaves of W. florida "Jean's Gold"

a. Compound 12

 12 (WEFJM1) Mass spectrometry ion peak HR-ESIMS (positive-ion mode) at m/z 1051.5427 [M+Na] + , indicating a molecular weight of 1028 and a molecular formula of C52H84O20.

92 Figure 74 . 12 Figure 75 . 12 ZoneCompound 13 ,

 927412751213 Figure 74. Structure of compound 12

  13 differs from 12 by only 162 amu, corresponding to a supplementary hexosyl group. The HSQC spectrum of sugar part of 7 showed five crosspeaks at δH/δC 4.78 (d, J = 7.6 Hz)/106.4, 4.87 (d, J = 5.8 Hz)/106.0, 5.13 (d, J = 7.6 Hz)/106.5, 6.00 (d, J = 8.2 Hz)/ 96.0 and 6.35 (br s)/102.6, indicating the presence of five sugar units. After analysing the spectre of HSQC, HMBC, COSY, TOCSY and ROESY, the sugar moieties of compound 13 was characterised as: -1 α-L-arabinopyranosyl Ara-1 at δH 4.87 (d, J = 5.8 Hz), -1 α-L-rhamnopyranosyl Rha-1 at δH 6.35 (br s), -2 β-D-glucopyranosyl Glc I-1 at δH 5.13 (d, J = 7.6 Hz) and Glc II-1 at δH 6.00 (d, J = 8.2 Hz), -1 β-D-xylopyranosyl Xyl-1 at δH 4.78 (d, J = 7.6 Hz).

5 (

 5 Glc I C-1) and in ROESY at δH 4.54 (Ara H-2) and δH 5.13 (Glc I H-1) proved that Glc I was attached to the Ara at C-2. -The shielded chemical shift at δC 96.0 (Glc II C-1) and the shielded at δC 178.1 (C-28) suggested an ester linkage of Glc II with C-28 of the aglycon. -The HMBC correlation between δH 4.28 (Glc II H-2) and δC 102.6 (Rha C-1) and in ROESY at δH 4.28 (Glc II H-2) and δH 6.35 (Rha H-1) proved that Rha was attached to the Glc II at C-2. -The HMBC correlation between δH 4.78 (Xyl H-1) and δC 70.0 (Glc II C-6) and in ROESY at δH 4.78 (Xyl H-1) and δH 4.19 (Glc II H-6) proved that Xyl was attached to the Glc II at C-6. Conclusion Based on the above evidences, the structure of compound 13 was elucidated as 3-O-β-Dglucopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-β-D-xylopyranosyl-(1→6)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranosyl ester.

Figure 80 . 13 Figure 81 . 13 Figure 82 . 13 ZoneFigure 85 .

 80138113821385 Figure 80. Structure of compound 13

Figure 86 .

 86 Figure 86. Structure of compound 14

Figure 87 .

 87 Figure 87. Structure of compound 15 e. Compound 16 (WEFJR2)

Figure 88 .

 88 Figure 88. Structure of compound 16 f. Compound 17 (WEFJ0711)

Figure 89 .

 89 Figure 89. Structure of compound 17

Figure 90 .

 90 Figure 90. Purification of compounds in roots of Cordyline fruticosa "Fairchild red"

( 107 Figure 91 . 18 Figure 92 .Figure 93 . 18 Figure 94 . 18 FucFigure 95 . 18 Figure 96 .

 10791189293189418951896 Figure 91. Structure of compound 18

FucFigure 97 .

 97 Figure 97. Mass spectrum of compound 18 b. Compound 19 (CFR3b3)

  )27ene-1β,3β,4α-triol-1-O-β-D-xylopyranoside.

Figure 98 .Figure 99 . 19 Figure 100 .Figure 101 . 19 Figure 102 . 19 XylFigure 103 .

 9899191001011910219103 Figure 98. Structure of compound 19

  These evidences led to the elucidation of 20 as 5α-spirost-25(27)-ene-1β,3β-diol-1-O-α-Lrhamnopyranosyl-(1→2)-β-D-fucopyranoside. The aglycon with a 3α-hydroxyl group was previously isolated by enzymatic hydrolysis from the stems of Dracaena concinna (Mimaki et al., 1998b), but this is the first native isolation of 3β-hydroxyl group on the spirostanol glycoside.

Figure 104 . 20 Figure 105 . 20 Figure 106 . 20 ZoneFigure 107 . 20 Figure 108 . 20 Fuc H- 1 /C- 1 Rha H- 1 /Fuc C- 2 Figure 109 . 20 Figure 110 . 1 Fuc H- 1 /

 104201052010620107201082011121092011011 Figure 104. Structure of compound 20

  All the data above allowed to elucidate the structure of 21 as 5α-spirost-25(27)-ene-1β,3βdiol-1-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside. As for 20, the 3α-hydroxyl 120 derivative was previously isolated by enzymatic hydrolysis from the stems of Dracaena concinna(Mimaki et al., 1998b).

Figure 111 . 21 Figure 112 .Figure 113 . 21 Figure 114 . 2 RhaFigure 115 . 22 Figure 116 . 2 Ara

 11121112113211142115221162 Figure 111. Structure of compound 21

  )27-ene-1β,3β,4α-triol-1-O-α-Lrhamnopyranosyl-(1→2)-β-D-fucopyranoside. The 3α-hydroxyl derivative was previously 124 isolated by enzymatic hydrolysis from the stems of Dracaena concinna (Mimaki et al., 1998b).

Figure 117 . 22 Figure 118 . HSQC spectrum of compound 22 ZoneFigure 119 . 22 Figure 120 . 22 Fuc H- 1 Fuc H- 2 Fuc H- 3 Fuc H- 4 Fuc 1 Figure 123 .

 1172211822119221202212341123 Figure 117. Structure of compound 22

5 ( 3 (

 53 Fuc C-2) and in ROESY at δH 6.35 (Rha H-1) and δH 4.52 (dd, J = 9.4, 7.6 Hz, Fuc H-2) proved that Rha was attached to the Fuc at C-2.-The downfield shifts observed in the HSQC spectrum at δH/δC 5.13 (br s, Fuc H-4)/78.Fuc C-4) revealed that the hydroxy group at Fuc C-4 was substituted. The molecular weight of 23 (m/z 818) differs than 22 (m/z 738) by 80 amu corresponding to a SO3H substituent[START_REF] Fouedjou | Steroidal saponins from the leaves of Cordyline fruticosa (L.) A. Chev. and their cytotoxic and antimicrobial activity[END_REF], which reveals a fucopyranosyl-4-sulfate.ConclusionThe structure of 23 was characterized as 5α-spirost-(25)27-ene-1β,3β,4α-triol-1-O-α-Lrhamnopyranosyl-(1→2)-(4-O-sulfo)-β-D-fucopyranoside.

Figure 124 . 23 129Figure 125 . HSQC spectrum of compound 23 Figure 126 . 23 ZoneFigure 127 . 23 Figure 128 . 23 RhaFigure 129 .

 1242312523126231272312823129 Figure 124. Structure of compound 23

δH 5 . 54 (

 554 Rha II H-1) and δH 3.77 (H-3) proved that Rha I was attached to the C-3 position of the aglycon (Iwagoe et al., 1987; Tschesche et al., 1973).. -The downfield shifts observed in the HSQC spectrum at δH/δC 5.16 (br s, Fuc H-4)/78.4

Figure 130 . 24 Figure 131 . 24 Figure 133 . 24 Rha

 130241312413324 Figure 130. Structure of compound 24

group at δH 1 .

 1 05 (d, J = 7.0 Hz, H3-21), -3 oxygen bearing methine proton signals at δH 3.99 (dd, J = 11.1, 3.5 Hz, H-1), 3.93 (H-3) and 3.72 (H-4) (Mimaki et al., 1998b).

- 8

 8 characterisitic carbons of a 22-OH furostane-type genin at δC 40.5 (C-20), 15.9 (C-21), 110.2 (C-22), 37.5 (C-23), 27.8 (C-24), 146.5 (C-25), 71.9 (C-26) and 111.5 (C-27). The ROESY correlation between δH/δH 3.99 (H-1)/1.05 (H3-21) and the HMBC correlation at δH 1.06 (s, H3-19)/δC 81.3 (C-1) revealed the location of a hydroxyl group at the C-1 position in a β-orientation. The downfield shift to δC 81.3 suggested a glycosidic linkage.

1 H

 1 NMR spectrum of compound 25 in the sugar region signals of two anomeric protons at δH/δC 4.83 (d, J = 7.6 Hz)/101.2 and δH/δC 4.91 (d, J = 7.6 Hz)/103.3 indicating the presence of two sugar units which were identified as: -1 β-D-fucopyranosyl Fuc-1 at δH 4.83 (d, J = 7.6 Hz). -1 β-D-glucopyranosyl Glc-1 at δH 4.91 (d, J = 7.6 Hz). The sequence of the oligosaccharide was established by analysing the HMBC, TOCSY and ROESY spectra: -The HMBC correlation between δH 4.83 (Fuc H-1) and δC 81.3 (C-1) and in ROESY at δH 4.83 (Fuc H-1) and δH 3.99 (dd, J = 11.7, 3.5 Hz, H-1) revealed the glycosidic linkage of Fuc at the C-1 position of the aglycon (Fig.136).

δH 4 . 91 (Figure 136 . 25 Figure 137 . HSQC spectrum of compound 25 ZoneFigure 138 . 25 Figure 139 . 25 Glc H- 1 FucFuc H- 2 Figure 140 . 25 Figure 141 . 25 FucFigure 142 .

 49113625137251382513925121402514125142 Figure 136. Structure of compound 25

Figure 143 . 26 Figure 144 . HSQC spectrum of compound 26 Figure 145 . 26 ZoneFigure 146 . 26 Figure 147 . 26 Glc 22 Figure 148 . 26 Figure 149 .

 14326144261452614626147262214826149 Figure 143. Structure of compound 26

Figure 150 .

 150 Figure 150. Structure of compound 27 k. Compound 28 (CFRCR-2)

Figure 151 .

 151 Figure 151. Structure of compound 28

  9 g) were submitted to microwaves three times with the solvent EtOH/H2O (200 W, 60°C, 30 min, 75/35, 300 mL each). After filtration and evaporation, 15.3 g of the crude extract were obtained. An aliquot of the extract (7.5 g) was submitted to VLC (RP-18 silica gel, H2O, EtOH/H2O 50/50, EtOH) to give 3 fractions (A-C). Fraction B (1.9 g), rich in saponins, was fractionated by flash chromatography (CHCl3/MeOH/H2O 70/30/5) to give 2 fractions. The fraction rich in saponins was further separated using MPLC on silica gel 60 (15-40 µm, CHCl3/MeOH/H2O 80/20/2, 75/25/3) yielding compound 29 (14.0 mg) and 30 (6.3 mg). The remaining fractions were combined and chromatographed on successive MPLC on silica gel RP-18 (MeOH/H2O 40/60→49/51) yielding compounds 31 (6.1 mg) and 32 (17.6 mg).

Figure 152 . 2 . 5 . 2 .

 152252 Figure 152. Purification of compounds in aerial parts of C. fruticosa "Fairchild Red"2.5.2. Structural determination of isolated saponins

(

  COSY, TOCY, ROESY, HSQC, HMBC) as α-L-rhamnopyranosyl (Rha), β-Dfucopyranosyl (Fuc) and β-D-glucopyranosyl (Glc). The absolute configurations of the sugars were determined to be D for glucose (Glc) and fucose (Fuc), and L rhamnose (Rha), by GC analysis (see Section 1.3.4). The relatively large 3 JH-1,H-2 values of the Glc and Fuc (6.0-8.0 Hz) indicated a ß anomeric orientation for Glc and Fuc. The large 3 JH-1,C-1 values of the Rha (165-168 Hz), confirmed that the anomeric protons were equatorial (α-pyranoid anomeric form). The HSQC spectrum of compound 29 in the sugar region indicates signals of two anomeric protons of two sugar units which were identified, using COSY, TOCSY, HMBC and ROESY spectra as: -2 β-D-glucopyranosyl: Glc I-1 at δH/δC 5.03 (d, J = 8.2 Hz)/100.1 and Glc II-1 at δH/δC 4.96 (d, J = 7.6 Hz)/104.6. The sequence of the oligosaccharide was established by analysing the HMBC and ROESY spectra:-The HMBC correlation between δH 5.03 (Glc I H-1) and δC 81.1 (C-1) and in ROESY at δH 5.03 (Glc I H-1) and δH 3.99 (dd, J = 11.7, 3.5 Hz, H-1) proved that Glc I was attached to the C-1 position of the aglycon (Fig.160).-The HMBC correlation between δH 4.96 (Glc II H-1) and δC 73.9 (C-26) proved that Glc II was attached to the C-26 position of the aglycon. All the evidences above revealed the structure of compound 29 was 26-O-β-Dglucopyranosyl-5α-furost-(25)27-ene-1β,3β,22α,26-tetrol-1-O-β-D-glucopyranoside.

Figure 153 . 29 Figure 154 .Figure 155 . 29 Figure 156 . 29 GlcFigure 157 . 29 Figure 158 . 29 Glc 1 Figure 159 .

 15329154155291562915729158291159 Figure 153. Structure of compound 29

Figure 160 . 30 Figure 161 . HSQC spectrum of compound 30 Figure 162 . 30 ZoneFigure 163 . 30 Figure 164 . 30 Glc 1 Figure 165 . 30 Figure 166 .

 1603016130162301633016430116530166 Figure 160. Structure of compound 30

  1 H NMR spectrum of compound 31, in the sugar region signals, showed signals of three anomeric protons at δH/δC 4.82 (d, J = 7.6 Hz)/99.7, δH/δC 6.33 (br s)/101.1 and δH/δC 4.89 (d, J = 7.6 Hz)/103.3, indicating the presence of three sugar units: -1 β-D-fucopyranosyl Fuc-1 at δH 4.82 (d, J = 7.6 Hz).

3 (

 3 Fuc C-2) and in ROESY at δH 6.33 (Rha H-1) and δH 4.50 (Fuc H-2) proved that Rha was attached to the C-2 position of the Fuc. -The HMBC correlation between δH 4.89 (Glc H-1) and δC 72.0 (C-26) and the ROESY correlation between 4.89 (Glc H-1)/δH 4.65 (H-26) proved that Glc was attached to the C-26 position of the aglycon. All the evidences above revealed the structure of compound 31 as 26-O-β-Dglucopyranosyl-5α-furost-(25)27-ene-1β,3β,22α,26-tetrol-1-O-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranoside.

Figure 167 . 31 Figure 168 .Figure 169 . 31 Figure 170 . 31 Figure 171 . HMBC spectrum of compound 31 Figure 172 . 31 Rha 1 Figure 173 .

 16731168169311703117131172311173 Figure 167. Structure of compound 31

Figure 174 . 32 Figure 175 . HSQC spectrum of compound 32 ZoneFigure 176 . 32 Figure 177 .

 174321753217632177 Figure 174. Structure of compound 32

Figure 181 .

 181 Figure 181. Purification of compounds in roots of Dracaena braunii 2.6.2. Structural determination of isolated saponins

Figure 182 .

 182 Figure 182. Structure of compound 33 b. Compound 34 (DSU4a1)

Figure 183 .

 183 Figure 183. Structure of compound 34 c. Compound 35 (DSU6C)

Figure 184 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 2 . 1 . 2 .

 1841111111212 Figure 184. Structure of compound 35

( 1 . 3 . 2 .

 132 [START_REF] Kougan | Steroidal Saponins from Two Species of Dracaena[END_REF].Tang et al. (2014) assumed that the presence of an acetyl group at C-4 of the α-L-Rha exhibited less cytotoxicity than the one without the acetyl group. -Furostanol saponins hardly exhibited specific properties of saponins, but the furostanol glycosides bearing α-L-Rha-(1→2)-β-D-Glc or an α-L-Rha-(1→2)-[α-L-Rha-(1→4)]-β-D-Glc at C-3 position of the aglycon were found to be cytotoxic to HL-60 cells (Mimaki et al., 2001). In case of triterpenoid saponins -The presence of an α-L-Rha-(1→2)-α-L-Ara or α-L-Rha-(1→2)-α-L-Xyl sequence at the C-3 position of the aglycon is a structural requirement essential for potent cytotoxic activity of oleanane saponins (Bang et al., 2005; Gauthier et al., 2009a). Moreover, trisaccharides with a third sugar linked at C-4 position of Ara or C-3 position of Rha exhibited stronger cytotoxicities than disaccharides. But, a change in the sugar moiety influences cytotoxic activity (Rezgui et al., 2016).-The cytotoxicity of the lupane-type saponins were much weaker than those of oleananes, when an α-L-Rha-(1→2)-α-L-Ara sequence was present in a branched sugar moiety at C-3 of the aglycon(Gauthier et al., 2009b).-The existence of a free COOH at C-28 is important for antitumor activity, conformed with some reports[START_REF] Chwalek | Structure-activity relationships of some hederagenin diglycosides: Haemolysis, cytotoxicity and apoptosis induction[END_REF][START_REF] Rezgui | Oleanolic acid and hederagenin glycosides from Weigela stelzneri[END_REF], but was contradictory to Gauthier et al. (2009a) who showed that some bidesmosidic betulinic acid saponins glycosylated at C-28 were highly cytotoxic.-Comparison of activities exerted by saponins derived from oleanolic acid and hederagenin made the authors postulate that the hydroxyl group at C-23 has a negative effect on cytotoxicity[START_REF] Barthomeuf | In vitro activity of hederacolchisid A1 compared with other saponins from Hedera colchica against proliferation of human carcinoma and melanoma cells[END_REF][START_REF] Park | Kalopanaxsaponin A is a basic saponin structure for the anti-tumor activity of hederagenin monodesmosides[END_REF]. It could be explained by the attribution to the electron donating effect of two lone-paired electrons of hydroxyl group toward C-3 of the aglycon[START_REF] Bang | Antitumor activity of Pulsatilla koreana saponins and their structure-activity relationship[END_REF].

Table 10 . 1 .

 101 Mechanisms of cytotoxic activity of The principle of MTS colorimetric assayMTS tetrazolium compound is reduced by viable cells, leading to generate a soluble colored formazan in cell culture media. NAD(P)H-dependent dehydrogenase enzymes in metabolically active cells has an important role in this conversion. The absorbance can be measured at 490-500 nm. The assay can be used to measure of cell proliferation and analyse of pharmaceutical compounds. Comparing to the routine MTT assay, this colorimetric one can be performed by adding the reagent directly into the cell culture media, with out washing or solubilization step and can be carried out in a 96-well microtiter plate (Abcam Cambrige, 2019).

Figure 185 .

 185 Figure 185. MTS assay summary

3. 1 .

 1 Cytotoxic study on the saponins isolated from Weigela x "Bristol Ruby" Since saponins isolated from Weigela stelzneri exhibited significant cytotoxic activities on SW480 human cell lines and EMT-6 mouse cell lines (Rezgui et al., 2016), we have investigated the cytotoxicity of the compounds 1-3, 6, 7 by using MTS colorimetric assay on the mouse colon cancer cell line CT26 with different concentrations from 1 to 50 µM.

C r u d e e x tr a c t C o m p o u n d 6 C o m p o u n d 1 C o m p o u n d 7 C o m p o u n d 3 C o m p o u n d 2 5Figure 186 .

 61732186 Figure 186. Evaluation of the cytotoxic activity of compounds 1-3, 6 and 7 against colon cancer cells (CT-26), in concentrations ranging from 1 to 50 µM.

  alcoholic function at C-23, as hederagenin, seems to decrease the activity (Bang et al., 2005; Rezgui et al., 2016). For the osidic part, the presence of the common sequences α-Lrhamnopyranosyl-(1→2)-β-D-xylopyranosyloleanolic or α-L-rhamnopyranosyl-(1→2)arabinopyranosyloleanolic acid, have a role in the cytotoxicity (Bang et al., 2005; Rezgui et al., 2016), but in our study, we can propose more precisely the key sequence β-Dxylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-β-D-xylopyranosyloleanolic acid or β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-arabinopyranosyloleanolic acid.Moreover, a linear oligosaccharide chain linked at the C-3 position in 1 and 7, instead of a branched one in 2, 3 and 6, seems to weakly decrease the activity. Compound 6 shows the more potent activity, even if its structure contains a linear oligosaccharide chain and an esterification at the position 28, two structural.

Figure 187 . 7 3. 2 .

 18772 Figure 187. Structure compounds 1-3, 6 and 7

V

  Preliminary biological tests were based on their structural diversity including the nature of the aglycones and sugars, the type of inter-glycosidic bond and the type of saccharide sequence. Five saponins extracted from Weigela x "Bristol ruby" were using to evaluate their cytotoxic activity against CT26 cell lines. Six other isolated from two other Weigela species, Weigela florida "Pink poppet" and Weigela florida "Jean's gold", were tested for their cytotoxicity against not only CT26 cell lines, but also two other cancer cell lines B16 and HepG2 in comparison with 5-FU used as positive control. In both cases, the presence of the sequence β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-β-Dxylopyranosyloleanolic acid or β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)arabinopyranosyloleanolic acid played an important role in cytotoxic activity. In contrary, the cytotoxic activity of hederagenin-type triterpenoid saponins were inactive in vitro, but those saponins exhibited an inferior in vivo antitumor activity due to literature. Thus, it will interresting to evaluate the in vivo cytotoxic activity of this type saponins in the further study.

Figure 200 .

 200 Figure 200. Chemical structure of dextran .......................................................................

Figure 201 .

 201 Figure 201. Chemical structure of pectin (A) and eggbox model showing .....................

Figure 202 .

 202 Figure 202. Stability of curcumin in deionized water (pH = 6.8) containing ..................

Figure 203 .

 203 Figure 203. SEM micrographs of pectinate beads and curcumin powder with views of surface (1) and internal beads (2) from Curcumin beads (a), BiPro ® BLG beads (b), Kolliphor ® beads (c), TPGS ® beads (d) and Curcumin powder (e). ................................

Figure 204 .

 204 Figure 204. Curcumin release from a pectinate matrix of different types of beads .........

Figure 205 .

 205 Figure 205. Curcumin release from a pectinate matrix of different types of beads .........

Figure 206 .

 206 Figure 206. DSC thermograms of curcumin (a), pectin (b), Kolliphor ® HS 15 (c), ........

Figure 207 .

 207 Figure 207. DPPH radical scavenging activity of curcumin powder and curcumin from pectinate beads after 48 hours of extraction (A) and 330 minutes of dissolution (B)......

Figure 208 .

 208 Figure 208. Calibration curve of solubilized curcumin in deionized water .....................

Figure 209 .

 209 Figure 209. Calibration curve of solubilized curcumin in EtOH 96% .............................

Figure 210 .

 210 Figure 210. Calibration curve of solubilized curcumin in EtOH 96% .............................

Figure 190 .

 190 Figure 190. Tautomerism of curcumin under aqueous solutions (Sharma et al., 2005)

Figure 191 .

 191 Figure 191. Pharmacological activity of curcumin (Aggarwal et al., 2006)

Encapsulation is a useful

  tool to improve delivery of bioactive molecules and living cells into foods (de Vos et al., 2010;[START_REF] Wandrey | Materials for encapsulation[END_REF]. Encapsulation aims to preserve stability of the bioactive compounds during processing and storage and to prevent undesirable interactions with food matrix. Another benefit of encapsulation is less evaporation and degradation of volatile actives, such as aroma. Furthermore, encapsulation is used to mask unpleasant feelings during eating, such as bitter taste and astringency of polyphenols. This technology may provide barriers between sensitive bioactive materials and the environment, and thus, to allow taste and aroma differentiation, mask bad tasting or smelling, stabilize food ingredients or increase their bioavailability[START_REF] Nedovic | An overview of encapsulation technologies for food applications[END_REF].

Figure 192 .

 192 Figure 192. Schematic presentation of the spray drying method (Munin and Edwards-Lévy, 2011)

Figure 193 .

 193 Figure 193. Schematic presentation of the emulsification-solvent removal methods (Munin and Edwards-Lévy, 2011)

(

  de Vos et al., 2010).

Figure 194 .

 194 Figure 194. Schematic representation of the coacervation process. (a) Core material dispersion in solution of shell polymer; (b) separation of coacervation from solution; (c) coating of core material by microdroplets of coacervate; (d) coalescence of coacervate to form continuous shell around core particles (Van Parys, 2006).

  principle the core is always solid (de Vos et al., 2010).

Figure 195 .

 195 Figure 195. Schematic presentation of the fluid bed coater. (a) Top spray; (b) bottom spray; (c) tangential spray[START_REF] Van Parys | Smart Textiles Using Microencapsulation Technology[END_REF]

Figure 197 .

 197 Figure 197. Schematic presentation of the ionotropic gelation method (Suri et al., 2013)

  groups makes chitosan particularly amenable to structural modification to yield materials of different mechanical and physical properties. Owing to this simplicity of modification, negligible toxicity and biodegradability, mucoadhesive property, chitosan and its derivatives are being extensively used by pharmaceutical industries (Yadav and Karthikeyan, 2019).

Figure 198 .

 198 Figure 198. Chemical structure of chitosan (Okuyama et al., 2000)4.2.2. Alginate

Figure 199 .

 199 Figure 199. Chemical structure of alginate (Yadav and Karthikeyan, 2019)

Figure 200 .

 200 Figure 200. Chemical structure of dextran (Yadav & Karthikeyan, 2019)

  after chemical de-esterification (Daas et al., 1999; Fraeye et al., 2010; Grant et al., 1973).

Figure 201 .

 201 Figure 201. Chemical structure of pectin (A) and eggbox model showing calcium-induced junction zones (B)[START_REF] Flutto | Pectin | Properties and Determination[END_REF]

  DSC analysis was applied to study the physical forms of encapsulated curcumin within the different beads. DSC analyzer (Q20, TA Instruments, USA) was calibrated with Indium for temperature and enthalpy (Tm= 156.6 °C, ΔHf = 28.71 J/g). A quantity of each type of beads was weighed and placed in a sealed aluminum pan. Scans were recorded between 20 °C and 190 °C at heating rate of 10 °C/min under nitrogen (flow rate of 20 mL/min). All experiments were performed in triplicate.

  deliver curcumin to cancer cells and improve antioxidant activity by different authors (Aditya et al., 2015; Li et al., 2013).

Darkness 25 °C 96 Figure 202 .240

 2596202 Figure 202. Stability of curcumin in deionized water (pH = 6.8) containing 5 g/L Kolliphor ® HS 15 in duration of 24 days in different environmental conditions

  C and D) exhibited a rounder shape and a smooth surface than the other ones which displayed a rough surface. All beads were characterised by a dense inner structure. Interestingly, curcumin crystals were only found and observed in BiPro ® BLG beads (observation of 10 beads) as a result of the lowest solubility in BLG based capsules.

Figure 203 .

 203 Figure 203. SEM micrographs of pectinate beads and curcumin powder with views of surface (1) and internal beads (2) from Curcumin beads (a), BiPro ® BLG beads (b), Kolliphor ® beads (c), TPGS ® beads (d) and Curcumin powder (e).

  Fig.204 and 205.

Figure 204 .

 204 Figure 204. Curcumin release from a pectinate matrix of different types of beads during 4 hours in PBS 7.4 medium

Figure 205 .

 205 Figure 205. Curcumin release from a pectinate matrix of different types of beads during 7 hours in PBS 7.4 containing 5 g/L Kolliphor ® HS 15

Figure 206 .

 206 Figure 206. DSC thermograms of curcumin (a), pectin (b), Kolliphor ® HS 15 (c), TPGS ® (d), Blank bead (e), Curcumin bead (f), Kolliphor ® bead (g), TPGS ® bead (h).

  agreement with those of[START_REF] Seo | Preparation and pharmacokinetic evaluation of curcumin solid dispersion using Solutol® HS15 as a carrier[END_REF][START_REF] Song | Characterization, in Vivo and in Vitro Evaluation of Solid Dispersion of Curcumin Containing d-α-Tocopheryl Polyethylene Glycol 1000 Succinate and Mannitol[END_REF] which explained by the dissolution of curcumin in the melted Kolliphor ® HS 15 and TPGS ® when thermal ananlysis was carried out.6. DPPH radical scavenging assayDPPH assay has been widely used to evaluate the free radical scavenging effectiveness of various antioxidant substances[START_REF] Floegel | Comparison of ABTS/DPPH assays to measure antioxidant capacity in popular antioxidant-rich US foods[END_REF]. The method is based on the reduction of DPPH in alcoholic solution in the presence of a hydrogen-donating antioxidant inducing the formation of the yellow colored non-radical form (DPPH-H) in the reaction[START_REF] Ak | Antioxidant and radical scavenging properties of curcumin[END_REF]. A significant decrease was reported in the concentration of DPPH radical due to the scavenging ability of curcumin[START_REF] Tapal | Complexation of curcumin with soy protein isolate and its implications on solubility and stability of curcumin[END_REF].

Figure 207 .

 207 Figure 207. DPPH radical scavenging activity of curcumin powder and curcumin from pectinate beads after 48 hours of extraction (A) and 330 minutes of dissolution (B).

  by virtually insoluble in water, poor absorption with crystalline state are one of the main reasons why curcumin has been unsuccessful to use in therapeutic in spite of its pharmacological properties.

Table 1 .

 1 Structural features of spirostane derivatives for saponins ...............................................

Table 2 .

 2 Types of aglycons substituted at carbon positions ..........................................................

	Table

Table 10 .

 10 Mechanisms of cytotoxic activity of saponins .................................................

Table 11 .

 11 IC50 of compounds 1-3, 6 and 7 against colon cancer cells (CT-26) ..............

	MS	Mass spectrometry
	RP	Reversed phase
	NP	Normal phase
	ESI	Electro spray ionization
	NMR	Nuclear magnetic resonance
	IR	Infrared spectroscopy
	1 H NMR	Nuclear magnetic resonance of proton

can be predominantly red. It has parallel veins. This plant has pink flower and blooms from spring to fall. Fruit are small, round berries (Looking at plant, 2014). Figure 8. Cordyline fruticosa "Fairchild Red" 2. Previous phytochemical analysis 2.1. Saponin generalities

	. This genus is found natively in Saponins, which is derived from Latin word "sapo" which means "soap" and have a form
	Western Pacific Ocean region which consists of about 26 species of woody monocots plants soap-like foams upon shaking, are known as a non-volatile and surfactant compound. Due
	which is accepted by World Checklist of Selected Plant Families (Zonneveld, 2019). to this property, saponin dissolve easily in water and form soap-like. Some of plants which
	These are evergreen shrubs and tree-like, woody perennials. The larger perennials resemble have this ability are named with a word "sapo", such examples soapwort (Saponaria
	palm trees. Long, leathery leaves are produced in tufts or rosettes. Flowers are cup-shaped officinalis L.) (Bruneton, 2016; Sparg et al., 2004), soapberry
	and sweet-smelling, sometimes produced in large terminal panicles. Round white, red, blue,
	or purple berries follow. Cabbage palms are good as houseplants or grown in a greenhouse.
	In warmer areas, they can be used as specimen plants, in a border, or in a courtyard garden
	(Finegardening, 2019).
	Species Cordyline fruticosa "Fairchild Red"
	Cordyline fruticosa "Fairchild Red" is a dwarf evergreen shrub that averages 60-90 cm in
	height with a spread of 45-60 cm. It has an upright, fountain-shaped growth habit. Leaves
	are evergreen, linear in shape, narrow, spiral in arrangement, simple, somewhat glossy and
	medium green with pink margins. Some older foliage can become very dark green with
	pink or red margins. As plants get older and depending on the amount of light, newer foliage

  ).

	methyl ester (Mitaine-Offer et al., 2001). Another example, Momordica charantia utilized
	to reduce blood glucose and lipids, protect β cells, enhance insulin sensitivity and reduce
	oxidative stress contains saponins (Keller et al., 2011).
	Lacaille-Dubois and Wagner (1996) reported biological and pharmacological activities of
	saponins in several reviews. The biological role of saponins are not completely figured.
	Saponins are considered as a part of plants' defense systems and often have antifungal and
	antimicrobial activities in molecules of plants (Morrissey and Osbourn, 1999). Besides that,
	a wide number of plants containing saponins are used in traditional medicine, in example,
	root of Panax notoginseng (Burk.) F. H. Chen. which is utilized in treating injury (Zhang
	et al., 2013). The genus Bupleurum, officially recognized in Chinese and Japanese
	pharmacopoeia, is utilized widely to treat different diseases. The main anti-inflammatory
	compounds in dry root of Bupleurum fruticescens L. (Apiaceae) are saikosaponins (J Just
	et al., 1998). Active constituents of Achyranthes bidentata, a Chinese traditional medicine
	which used for strengthening bones and muscles and ensuring proper downward flow of
	blood, have been shown to be saponins named Bidentatoside II and chikusetsusaponin V

Table 1 .

 1 Structural features of spirostane derivatives for saponins

	There are

Type of sapogenin Substituent at different C-positions 1β 3β 25 Double bonds Configuration at Reference C-5 C-25

	Australigenin OH OH =CH2	α		(Blunden et al., 1984)
	Diosgenin	CH3	Δ 5	R	(Tsukamoto and Ueno, 1936)

Table 2 .

 2 Types of aglycons substituted at carbon positions[START_REF] Thakur | Angiosperm Phylogeny Group III (APG III). An update of The Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG III[END_REF]

			Substitution at carbon positions
	Type of sapogenin	3β	R 1	R 2	R 3	R 4	R 5	∆ 12
	Hederagenin	OH	CH2OH	H	COOH	CH3	H	+
	Oleanolic acid	OH	CH3	H	COOH	CH3	H	+
	b. Sugars (carbohydrate chains)

2.2. Saponins isolated from genus Weigela

			R
	Weigela hortensis (Murayama et al., 2003) (Champy-Tixier et al., 2018) H Ac R Weigela florida "rumba"	subsessilis Weigela	H H R1 H	β-D-Glc β-D-Xyl R2
		(Won et		H
		al., 2015)
				H
		R1	R2	R3
	Weigela x "kosteriana variegata" (Andriamisaina et al., 2018)		H	H
			H	H
			H	H
	Weigela stelzneri			H
	Plants of genus Weigela contains 10 species but over 200 cultivar names are recorded and (Rezgui et al., 2016)
	still in cultivation. Several species of Weigela plants were investigated and structures of
	compounds isolated were also elucidated which are triterpenoid saponins of oleanane-type
	and hederagenin-type. Fig.12 shows different triterpenoid saponins elucidated from species OH
	of Weigela genus.
	17

R Figure 12. Some triterpenoid saponins elucidated from species of Weigela genus 2.3. Saponins isolated from genus Cordyline Plants of genus Cordyline are found natively in Western Pacific Ocean region which consists of about 26 species of woody monocots plants which is accepted by World Checklist of Selected Plant Families. Some species of Cordyline plants were investigated

		R1 R2	R3	R4	R5
	Cordyline cannifolia (Jewers et al.,	OH OH OH H Ac Ac	OH H H	H Me Me	Me H H	Cordyline cannifolia (Jewers et al., 1974)	R1 Me H	R2 H
	1974)	Ac OH	H	Me	H
		H	OH	H	Me	H
	and structures of compounds isolated were also elucidated which are steroidal saponins of
	spirostane-type and furostane-type. Different steroidal saponins elucidated from species of
	Cordyline genus are showed in Fig.13.

Figure 14. Steroidal saponins elucidated from species of Dracaena genus CHAPTER 2 PHYTOCHEMICAL STUDY 1. Materials and methods 1.1. Materials and extraction

  60°C, 30 min, 200W, 300 mL of solvent EtOH/H2O 75/35 x 3 times). After evaporation of the solvents under vacuum, 24.88 g of roots' residue and 15.34 g of aerial parts' residue were obtained.

	Weigela x "Bristol Ruby" was provided in 2016 from Jardiland ® (Chenôve, France). A
	voucher specimen (N° 20160110) was deposited in the herbarium of the Laboratory of
	angustifolia (Huang et herbarium of the Laboratory of Pharmacognosy, Université de Bourgogne Franche-Comté, H S4 S5 Roxb. 2014) S4 H OH H CH3 H OS5 H H H CH3 "Pink Poppet" and N° 2018/05/2 for W. florida "Jean's Gold") were deposited in the H Dracaena R1 R2 H Dracaena cambodiana (Shen et al., R1 R2 R3 R4 R5 Dracaena marginata Dracaena marginata The roots of D. braunii (6.16 g) were extracted by microwaves (60°C, 30 min, 200W, 250 Pharmacognosy, Université de Bourgogne Franche-Comté, Dijon, France. R6 S4 H H OH CH2OH (Rezgui et al., 2013) (Rezgui et al., 2013) mL of solvent EtOH/H2O 75/35 x 2 times). After filtration and evaporation, 1.22 g dried H S4 H H H CH2OH H S4 H H H CH3 H Weigela florida "Pink Poppet" and Weigela florida "Jean's Gold" were provided in 2018 from Jardiland ® (Chenôve, France). The voucher specimens (N° 2018/05/1 for W. florida crude extract was collected.
	al., 2013) Dracaena thalioides Dijon, France.	R1 H	R2 H
	H Xyl H Cordyline fruticosa "Fairchild Red" was collected in a flower shop in Thainguyen city, (Tang et al., 2014) Rha Dracaena thalioides Rha (Tang et al., 2014)
	Vietnam in 2018. The voucher specimen (N° 2018/10/8) was deposited in the herbarium of
	the Laboratory of Pharmacognosy, Université de Bourgogne Franche-Comté, Dijon,
	France.
	Dracaena braunii (synonym: Dracaena sanderiana hort. ex Mast.) used in this study were
			Dracaena thalioides
	R collected in a garden-shop in Can Tho, Vietnam. A small dried specimen of Dracaena R R1 R2 (Tang et al., 2014)
	Dracaena cambodiana (Shen et al., 2014) Dracaena thalioides (Tang et al., 2014) braunii was deposited in our laboratory (N° 2019/03/20). Dracaena H H Rha H cambodiana (Shen et al., 2014) Rha Xyl The dried and powdered roots of Weigela x "Bristol Ruby" (49 g) were submitted to
	ultrasounds three times with 1 L of the solvent EtOH/H2O (75/35), 30 min/time. The solvent
	was evaporated to give 7 g of crude extract.
	The dried and powdered of aerial parts of Weigela florida "Jean's Gold" (56.78 g) were
	R1 submitted to a microwaves extraction three times with the solvent EtOH/H2O (75/35, 400 R2 R3
	Dracaena cambodiana (Shen et al., 2014) mL each one, 200 W, 60°C, 45 min). After filtration and evaporation, 21.49 g of crude R1 R2 Dracaena cambodiana (Shen et al., 2014) R1 R2 Glc Ac Ac R3 SO3H H H H H S2 H S3 Fuc Ac Ac R1 R2 R3 Dracaena thalioides (Tang et al., 2014) Fuc Ac H Fuc H H Dracaena thalioides Rha Ac H Rha extract was yielded. Ac Ac S2 SO3H H Glc Ac H (Tang et al., 2014) H H H The dried roots (26.52 g) and aerial parts (28.97 g) of Weigela florida "Pink Poppet" were S2 S2 S3 H Ac H Rha H H extracted separately by microwaves three times for each part with 300 mL of solvent

EtOH/H2O (60°C, 30 min, 200W, 75/35, 250 mL of solvent x 3 times). After removal of the solvent, 2.25 g of roots' residue and 7.7 g of aerial parts' residue were obtained.

The dried roots (74.18 g) and aerial parts (103.87 g) of Cordyline fruticosa "Fairchild red" were submitted separately to microwaves (

Table 4 .

 4 13 C and 1 H NMR spectroscopic data of the sugar moieties of 1-6 in pyridine-d5 (δ in ppm, J in Hz)

		1		2		3		4		5		6
	δC	δH	δC	δH	δC	δH	δC	δH	δC	δH	δC	δH
	38.9	0.96 m,	38.9	0.98 m,	38.9	0.98 m,	39.0	0.98 m,	38.9	0.98 m,	38.0	0.89 m,
		1.50 m		1.52 m		1.51 m		1.52 m		1.52 m		1.40 m
	26.6	1.85 m,	26.6	1.85 m,	26.6	1.85 m,	26.8	1. 89 m	26.9	1.89 m,	26.6	1.91 m,
		2.11 m		2.10 m		2.10 m		2.14 m		2.15 m		2.09 m
	88.7	3.30 dd	88.7	3.30	88.8	3.30	88.3	3.31 dd	88.5	3.30	87.4	3.23
		(12.1, 3.6)						(12.4, 4.0)
	39.6	-	39.6	-	39.6	-	39.6	-	39.6	-	39.0	-
	56.0	0.83 br d	56.0	0.83	56.0	0.82 br d	56.2	0.84	56.2	0.83	55.2	0.75
		(11.7)
						(12.0)
	18.5 1.29, 1.50 m	18.5 1.29, 1.51 m	18.6 1.30, 1.50 m	18.6 1.30, 1.50 m	18.5 1.28, 1.48	nd	nd
	33.2 1.28, 1.47 m	33.2 1.28, 1.48 m	33.2 1.28, 1.48 m	33.3 1.28, 1.48 m	33.2 1.28, 1.48	33.2 1.32, 1.50 m
	39.8	-	39.8	-	39.8	-	39.8	-	39.8	-	39.2	-
	48.1	1.68 dd	48.1	1.67	48.1	1.68 dd	48.1	1.68	48.1	1.67	47.3	1.59
		(15.2, 8.8)
						(17.2, 8.8)
	10 37.1	-	37.1	-	37.1	-	37.1	-	37.1	-	37.0	-
	11 23.8	1.89, 1.94	23.8	1.90, 1.92	23.8 1.90, 1.92	23.8 1.90, 1.92	23.8 1.90, 1.92	23.9 1.90, 1.93
	12 122.5	5.48 br t	122.5	5.48 br t	122.5 5.48 br t	122.5 5.47 br t	122.5	5.48 br t	122.5 5.40 br t
		(3.6)		(4.4)				(3.6)		(4.4)		(3.6)
						(3.6)

Overlapped proton signals are reported without designated multiplicity. 73

Table 5 .

 5 13 C and 1 H NMR spectroscopic data of the aglycon and sugar moieties of 8 in pyridine-d5 (δ in ppm, J in Hz)

			Aglycon			Sugar moieties
		δC	δH		δC	δH
	1	38.5	1.53 m, 0.98 m	Ara-1	103.2	5.85 d (4.6)
	2	26.5	1.88 m, 2.13 m	2	72.8	4.19
	3	88.5	3.35	3	74.6	4.22
	4	39.4	-	4	70.2	4.14
	5	56.0	0.86 overlapped	5	66.4	3.74, 4.48
	6	18.5	1.21, 1.55 m	Rha-1	101.2	6.48 br s
	7	33.0	1.30, 1.39	2	71.4	4.96 br s
	8	39.5	-	3	82.1	4.75 dd (9.3, 2.8)
	9	47.9	1.67 dd (15.8, 8.8)	4	72.5	4.51 dd (9.3, 9.3)
	10	37.0	-	5	69.5	4.73 dq (9.3, 5.2)
	11	23.6	1.92, 2.10	6	18.5	1.65 d (5.2)
	12 122.0	5.49 br t (3.6)	Xyl I-1 105.8	4.80 d (4.7)
	13 145.0	-	2	77.1	4.21
	14	42.0	-	3	77.1	4.18
	15	28.3	1.37, 2.21	4	70.5	4.19
	16	23.6	1.92 m, 2.20	5	64.6	3.75, 4.68
	17	47.9	-	Xyl II-1 106.4	5.31 d (7.6)
	18	42.1	3.34	2	74.6	4.09
	19	46.5	1.30, 1.82	3	75.5	4.16
	20	30.8	-	4	77.2	4.26
	21	34.2	1.20, 1.46 m	5	64.4	3.66 t (10.5), 4.41
	22	33.0	1.82 m, 2.05 m	Xyl III-1 103.4	5.28 d (7.0)
	23	27.9	1.37 s	2	74.7	4.10
	24	17.1	1.21 s	3	79.1	4.21

Phytochemical study of roots of Cordyline fruticosa "Fairchild red"

  Phytochemical investigation of an aqueous-ethanolic extract of the roots of Cordyline fruticosa "Fairchild red" led to the isolation of 11 compounds. Nine of them are previously undescribed steroidal glycosides(18)(19)(20)(21)(22)(23)(24)(25)(26) in addition to two known compounds(27, 28).

		12		13
	δC	δH	δC	δH
	Ara-1 108.4	4.73 d (6.4)	106.0	4.87 d (5.8)
	73.9	4.38	81.2	4.54 t (5.8)
	75.6	4.17	74.7	4.33
	70.5	4.33	69.5	4.34 m
	67.8	3.79 dd (12.3, 1.2), 4.27	66.2	3.76 dd (11.1, 1.2), 4.24
	Rha-1 102.6	6.39 br s	102.6	6.35 br s
	73.3	4.72 br s	73.2	4.72 br s
	73.5	4.48 dd (9.3, 2.9)	73.5	4.47 dd (9.3, 2.9)
	74.9	4.28	74.9	4.28
	71.2	4.44	71.1	4.42
	20.0	1.69 d (5.8)	19.9	1.69 d (5.8)
	Xyl-1 106.6	4.79 d (7.0)	106.4	4.78 d (7.6)
	75.8	3.89	76.0	3.88
	79.0	4.08	79.0	4.06
	72.3	4.09	72.1	4.08
	68.1	3.57, 4.22	68.1	3.55 t (11.1), 4.21
	Glc I-1 96.0	6.02 d (7.6)	106.5	5.13 d (7.6)
	76.9	4.29	77.2	4.00
	80.2	4.19	79.1	4.14
	71.9	4.27	72.5	4.15
	78.6	4.01	79.3	3.75 m
	70.1	4.19, 4.55	63.7	4.28, 4.36
	Glc II-1		96.0	6.00 d (8.2)
			77.0	4.28
			80.1	4.18
			71.8	4.24
			78.7	4.01 m
			70.0	4.19, 4.54

Overlapped proton signals are reported without designated multiplicity. dt: not determined 103 2.4.

  3 oxygen bearing methine proton signals at δH 4.00 (dd, J = 11.7, 4.1 Hz, H-1), 3.94 (td,

	J= 11.7, 4.1 Hz, H-3) and 3.72 (t, J = 11.7 Hz, H-4) (Mimaki et al., 1998b).
	-1 exo-methylene group at δH 4.81 and 4.85 (H-27, each 1H, s).
	The 13 C NMR spectrum showed:
	-10 tertiary carbons (CH), 10 secondary carbons (CH2), 3 primary carbons (CH3) and 4
	quaternary carbons (C).
	multiplicity of the H-2β at δH 2.18 as a quadruplet (J = 11.7 Hz) suggested a geminal
	coupling with H-2α and an axial/axial coupling with H-1 and H-3 and hence an α-axial
	orientation of H-3. This suggestion was confirmed by ROESY correlations at δH 4.00 (H-
	1α)/δH 2.93 (H-2α), δH 2.93 (H-2α)/δH 3.94 (H-3α) and at δH 3.94 (H-3α)/δH 1.29 (H-5α).
	Furthermore, the signals at δC 74.7 / δH 3.72 (t, J = 11.7 Hz) in the HSQC spectrum revealed
	the presence of an additional hydroxyl group at the 4 position. The H-4, coupled to H-3 (δH

-6 carbons of F ring at δC 109.7 (C-22), 32.9 (C-23), 28.7 (C-24), 144.0 (C-25), 64.8 (C-26) and 108.7 (C-27) showed a presence of one bearing exo-methylene group at C-25. In the COSY spectrum, the signal of an oxygen bearing a methine proton at δH 4.00 (dd, J = 11.7, 4.1 Hz, H-1) correlated to the δH 2.18 (q, J = 11.7 Hz, H-2β) and δH 2.93 (dt, J = 11.7, 4.1 Hz, H-2α), with the ROESY correlation between δH 4.00 (dd, J = 11.7, 4.1 Hz) and δH 1.04 (d, J = 6.4 Hz, H3-21) and the HMBC correlation between δH 1.07 (s, H3-19) and δC 81.2, revealed the location of a hydroxyl group at the C-1 position with a βorientation. The downfield shift of C-1 δC 81.2 suggested a glycosidic linkage. The 3.94, J = 11.7 Hz), was defined in a β-axial orientation through the ROESY correlation at δH 3.72/δH 1.07 (s, H3-19). The hydroxyl group was thus assigned as 4α-equatorial.

  α-equatorial through the ROESY correlation at δH 3.72 (H-4)/δH 1.06 (s, H3-19). On the other hand, the configuration of hydroxyl group at C-22 was established to be α from the ROESY correlations between β-H-20 (δH 1.96) and H-23a (δH 2.29) and H-23b (δH 2.22) (Perrone et al., 2009).

1 and H-3. This was confirmed by ROESY cross-peaks at δH 3.99 (H-1)/δH 2.92 (H-2α), δH 2.92 (H-2α)/δH 3.93 (H-3) and at δH 3.93 (H-3)/δH 1.28 (H-5). The H-4 was defined as β-axial and thus the hydroxyl group as

Table 8 .

 8 13 C and 1 H NMR spectroscopic data of the aglycon moieties of 18-26 in pyridine-d5 (δ in ppm, J in Hz)

			18 CFR3b1		19 CFR3b3		20 CFR3b4	21 CFR2801		22 CFR3R5	23 CFRA5-1_1	24 CFRA5-1_2		25 CFRC2a	26 CFRC2b
		δC	δH	δC	δH	δC	δH	δC	δH	δC	δH	δC	δH	δC	δH	δC	δH	δC	δH
	1	81.2	4.00	80.9	4.00	83.1	3.80	82.2	3.87	82.4	3.94	82.2	3.92	79.8	4.44	81.3	3.99	81.1	4.00
			dd (11.7, 4.1)		dd (11.7, 4.1)		dd (11.7, 3.5)		dd (11.7, 3.5)		dd (11.7, 3.5)		dd (11.7, 4.1)		dd (11.7, 4.1)		dd (11.1, 3.5)		dd (11.1, 3.5)
	2	34.6	2.18 q (11.7)	34.3	2.18 q (11.7)	37.4	2.24 q (11.7)	35.5	2.16 q (11.7)	34.6	2.39 q (11.7)	34.8	2.36 q (11.7)	33.1	2.25 q (11.7)	34.6	2.17 q (11.1)	34.7	2.17 q (11.1)
			2.93 dt (11.7, 4.1)		2.92 dt (11.7, 4.1)		2.72		2.79		2.75 dt (11.7, 4.1)		2.74		2.60		2.92		2.92
	3	73.3 3.94 td (11.7, 4.1) 73.2 3.94 td (11.7, 4.1) 67.7 3.94 tt (8.4, 6.4)	73.0	3.94	72.8 3.94 td (11.7, 4.1) 73.5	3.89	74.8	3.77	73.4	3.93	73.3	3.92
	4	74.7	3.72 t (11.7)	74.7	3.73 t (11.7)	39.2	1.74, 1.77	39.0	1.69, 1.75	74.7	3.79	74.7	3.76	39.0	1.69, 1.75	74.7	3.72	74.7	3.71
	5	49.3	1.29	49.5	1.30	42.9	1.05	42.5	0.99	49.4	1.28	49.3	1.26	42.5	0.98	49.3	1.28	49.4	1.28
	6	29.7	1.26, 1.33	29.7	1.26, 1.33	29.7	1.26, 1.33	29.7	1.28, 1.32	29.7	1.24, 1.34	29.7	1.28, 1.34	29.7	1.28, 1.34	28.8	1.26, 1.32	29.7	1.29, 1.33
	7	32.0	0.86, 1.68	32.0	0.86, 1.68	32.2	0.85, 1.56	32.2	0.84, 1.54	32.1	0.85, 1.68	32.1	0.84, 1.66	32.1	0.84, 1.66	32.0	0.84, 1.67	32.1	0.83, 1.67
	8	36.0	1.50 m	36.0	1.47 m	36.4	1.54 m	36.2	1.52 m	36.1	1.56 m	36.2	1.52 m	36.2	1.52 m	36.1	1.46 m	35.9	1.48 m
	9	55.2	1.04	55.0	1.02	55.0	1.02	54.9	1.01	55.3	1.07	55.3	1.16	55.3	1.16	55.2	1.02	55.2	1.02
		42.4	-	42.4	-	41.3	-	41.3	-	42.4	-	42.4	-	42.4	-	42.4	-	42.4	-
		23.2	1.36	23.3	1.40	23.4	1.41	23.6	1.44, 3.14	23.1	1.40	23.1	1.42, 3.14	23.1	1.42, 3.14	23.2	1.36, 3.10	23.1	1.36, 3.10
			3.12 dd (13.2, 3.0)		3.03 dd (13.4, 3.0)		3.19 dd (13.4, 3.0)				3.18 dd (12.9, 3.0)
		40.5	1.20, 1.65	40.8	1.16, 1.62	40.4	1.24, 1.60	40.4	1.20, 1.60	40.4	1.23, 1.60	40.2	1.18, 1.63	40.2	1.18, 1.63	40.5	1.20, 1.64	40.5	1.20, 1.66
		40.2	-	40.2	-	40.2	-	40.2	-	40.2	-	40.1	-	40.1	-	40.2	-	40.3	-
		56.6	1.10	56.5	1.07	56.8	1.14	56.7	1.12	56.4	1.10	56.8	1.06	56.8	1.06	56.5	1.05	56.5	1.04
		32.1 1.44 td (12.7, 5.8)	32.1 1.46 td (12.3, 5.8)	32.1 1.46 td (12.6, 5.8)	32.1	1.44, 2.05	32.2	1.45, 2.02	32.2	1.42, 2.00	32.2	1.42, 2.00	32.0	1.40, 1.98	32.1	1.39, 1.98
			2.03 td (11.4, 5.8)		2.05 td (12.3, 5.8)		2.06 td (11.7, 5.8)
		81.3	4.56 q (7.0)	81.4	4.57 q (7.0)	81.3	4.56 q (7.3)	81.3	4.56 q (7.0)	81.4	4.55 q (7.0)	81.4	4.54 q (7.0)	81.4	4.54 q (7.0)	81.4	4.46 q (7.0)	81.3	4.44 q (7.0)
		62.9 1.83 dd (7.0, 6.4) 62.9	1.83 overlapped	62.9 1.84 dd (7.3, 6.6) 62.9	1.83	62.8	1.83	62.8	1.82	62.8	1.82	64.0	1.78	64.1	1.76
		16.7	0.83 s	16.6	0.82 s	17.0	0.85 s	16.8	0.86 s	16.7	0.85 s	16.6	0.84 s	16.6	0.83 s	16.6	0.82 s	16.8	0.81 s
		9.3	1.07 s	9.2	1.07 s	8.5	1.25 s	8.5	1.20 s	9.7	1.30 s	9.7	1.29 s	8.7	1.30 s	9.3	1.06 s	9.3	1.06 s
		41.7	1.96 t (6.4)	41.9	1.97 t (6.4)	41.7	1.96 t (6.4)	41.8	1.96 t (6.4)	41.7	1.96 t (6.4)	41.7	1.95 t (7.0)	41.7	1.95 t (7.0)	40.5	1.96 t (7.0)	40.5	2.24 t (6.4)
		14.7	1.06 d (6.4)	14.7	1.06 d (6.4)	14.7	1.03 d (6.4)	14.7	1.05 d (6.4)	14.7	1.03 d (6.4)	14.7	1.04 d (7.0)	14.7	1.04 d (7.0)	15.9	1.05 d (7.0)	16.0	1.12 d (6.4)
		109.7	-	109.9	-	109.4	-	109.3	-	109.4	-	109.4	-	109.4	-	110.2	-	112.4	-

Table 9 .

 9 13 C and 1 H NMR spectroscopic data of the sugar moieties of 18-26 in pyridine-d5 (δ in ppm, J in Hz)

		18 CFR3b1		19 CFR3b3	20 CFR3b4	21 CFR2801	22 CFR3R5	23 CFRA5-1_1	24 CFRA5-1_2		25 CFR2a		26 CFR2b
	δC	δH	δC	δH	δC	δH	δC	δH	δC	δH	δC	δH	δC	δH	δC	δH	δC	δH
	Fuc-1 101.2 4.83 d (7.6)			99.9	4.71 d (7.6)			100.0	4.72 d (7.6)	99.7	4.70 d (7.6)	99.7	4.77 d (7.6)	101.2	4.83 d (7.6) 101.2 4.82 d (7.6)
	72.0	4.33			74.4	4.49			74.4	4.51	75.5	4.52	75.5	4.52	72.0	4.33	71.9	4.32
		dd (9.4, 7.6)				dd (9.4, 7.6)				dd (9.4, 7.6)		dd (9.4, 7.6)		dd (9.4, 7.6)		dd (9.4, 7.6)		dd (9.4, 7.6)
	74.9	4.13			76.5	4.12			76.5	4.13	76.2	4.18	76.2	4.20	74.9	4.14	74.9	4.12
		dd (9.4, 3.5)				dd (9.4, 2.9)				dd (9.4, 2.9)						dd (9.4, 2.9)		dd (9.4, 2.9)
	72.2 4.08 br d (3.5)			72.9 4.00 br d (2.9)			72.9 4.00 br d (2.9)	78.3	5.13 br s	78.4	5.16 br s	72.2 4.08 br d (2.9) 72.1 4.07 br d (2.9)
	71.0 3.76 br q (6.4)			70.9 3.72 br q (6.4)			70.9 3.73 br q (6.4)	70.4	3.75	70.3	3.85	71.1 3.76 br q (6.4) 71.0 3.75 br q (6.4)
	17.1	1.56 d (6.4)			17.0	1.54 d (6.4)			17.0	1.54 d (6.4)	17.1	1.68 d (6.4)	17.2	1.74 d (6.4)	17.1	1.56 d (6.4)	17.1	1.56 d (6.4)
	Xyl-1		100.0	5.04 d (7.0)
			75.1 4.03 dd (7.0, 8.2)
			77.9 4.29 overlapped
			72.3	4.10
			67.5 3.75 t (11.1),
				4.32
				dd (11.1, 4.6)
	Ara-1						100.1	4.73 d (7.0)
							75.0	4.55
							75.8	4.15
							69.9	4.24
							67.3	3.70
								dd (12.6, 0.5),
								4.29
	Rha I-1				101.2	6.35 br s	101.3	6.32 br s	101.2	6.40 br s	101.3	6.35 br s	101.3	6.33 br s
					72.1	4.76 br s	72.1	4.78 br s	72.1	4.78 br s	72.1	4.78 br s	72.1	4.78 br s
					71.9	4.65	71.9	4.67	71.9	4.67	72.1	4.60	72.1	4.60
						dd (9.4, 3.5)		dd (9.4, 2.9)		dd (9.4, 2.9)		dd (9.4, 3.5)		dd (9.4, 3.5)

. Phytochemical study of aerial parts of Cordyline fruticosa "Fairchild Red"

  Phytochemical investigation on aqueous-ethanolic extract of the aerial parts of Cordyline fruticosa "Fairchild Red" led to the isolation of 4 previously undescribed steroidal glycosides[START_REF] Floegel | Comparison of ABTS/DPPH assays to measure antioxidant capacity in popular antioxidant-rich US foods[END_REF](30)(31)(32).

Table 10 .

 10 13 C and 1 H NMR spectroscopic data of the aglycon moieties of 29-32 in pyridine-d5 (δ in ppm, J in Hz)

			29 CFA3		30 CFA5		31 CFA6		32 CFA4
		δC	δH	δC	δH	δC	δH	δC	δH
	1	81.1	3.99 dd (11.7, 3.5)	80.9	3.98	80.6	4.44 dd (11.7, 3.5)	80.7	4.43
	2	34.2	2.02, 2.87	37.0	1.99 q (11.7),	34.8	2.20, 2.56	35.0	2.19, 2.53
					2.86
	3	66.0	4.38	65.7	4.34	65.8	4.37	65.7	4.32
	4	36.9	1.57, 1.63	37.1	1.60, 1.63	37.0	1.64, 1.66	36.9	1.60, 1.64
	5	39.0	1.98	39.1	2.01	39.2	2.00	39.2	2.02
	6	28.5	1.26,1.30	28.5	1.24, 1.28	28.5	1.28, 1.32	28.5	1.26, 1.32
	7	32.0	0.74, 1.44	32.1	0.82, 1.53	32.2	0.85, 1.54	32.0	0.86, 1.55
	8	36.1	1.42	36.1	1.46	36.3	1.58	36.3	1.52
	9	54.4	0.90	55.1	1.18	55.0	1.19	55.0	1.20
		42.1	-	42.6	-	42.3	-	42.3	-
		23.8	1.40, 3.00	23.8	1.38, 3.00	23.9	1.47, 3.22	23.5	1.44, 3.20

Table 11 .

 11 13 C and 1 H NMR spectroscopic data of the sugar moieties of 29-32 in pyridine-d5 (δ in ppm, J in Hz)

		.96	56.5	1.05	56.7	1.08	56.7	1.08
	32.1	1.36, 1.99	32.0	1.42, 1.98	32.3	1.45, 2.02	32.1	1.41, 1.98
	81.1	4.81	81.1	4.92 q (8.2)	81.2	4.98 q (7.0)	81.3	4.93 q (7.0)
	62.3	1.88	62.4	1.84	63.5	1.99	64.1	1.76
	16.6	0.81 s	16.5	0.79 s	16.8	0.92 s	16.9	0.85 s
	8.0	1.02 s	8.0	1.24 s	7.5	1.24 s	7.52	1.24 s
	38.8	2.21 t (6.4)	41.2	1.98 t (6.4)	40.6	2.27	40.5	2.24
	14.8	1.13 d (6.4)	14.8	1.08 d (6.4)	15.9	1.28 d (6.4)	15.9	1.11 d (6.4)
	120.5	-	109.3	-	110.4	-	112.4	-
	40.4	2.42, 2.68	37.5	2.20, 2.26	37.4	2.24, 2.32	31.3	2.04, 2.14
	29.1	2.15, 2.29	28.5	2.59, 2.70	28.0	2.70, 2.76	28.1	2.68, 2.72
	148.8	-	148.9	-	146.7	-	146.4	-
	73.9	4.15, 4.49	71.9	4.36 br d (12.3),	72.0	4.40 br d (12.3),	71.8	4.36 br d (12.3),
				4.61 br d (12.3)		4.65 br d (12.3)		4.62 br d (12.3)
	112.0	5.08 br s	110.9	5.05 br s,	111.1	5.08 br s,	111.1	5.05 br s
		5.38 br s		5.33 br s		5.36 br s		5.34 br s
	OMe						47.2	3.29 s
	Overlapped proton signals are reported without designated multiplicity.

Table 11 .

 11 IC50 of compounds 1-3, 6 and 7 against colon cancer cells (CT-26)

	Compounds	IC50 (µM)
	1	1.97
	2	1.22
	3	1.48
	6	1.09
	7	1.87
	5-FU	11.7
	5-FU was used as positive control.

Table 12 .

 12 Cytotoxic activity of compounds 8

, 10, 11, 13, 15, 17

  Evaluation of the cytotoxic activity of compounds 8

		against three mouse cancer cell lines
			IC50 (µM)
	Compounds
		CT26	B16	HepG2
	8	2.45	2.35	2.77
	10	2.19	2.32	2.83
	11	̶	̶	̶
	13	̶	̶	̶
	15	̶	̶	̶
	17	̶	̶	̶
	" ̶ ": no data is available due to low toxicity.
	Figure 188.

, 10, 11, 13, 15, 17

  flora is one of the richest biodiversity on earth, with an abundant vegetation and its high rate of endemism. However, there are still very few studies that value this particularity of the country. The present thesis included a study of the chemical constituents of one species harvested in Vietnam and three others provided by a company located in France, belonging to two different plant families. These plants were Weigela x "Bristol Ruby", Weigela florida "Pink poppet" and Weigela florida "Jean's gold" (Caprifoliaceae), Cordyline fruticosa "Fairchild red" and Dracaena braunii (Asparagaceae). The choice of species was based, following a thorough literature search, on the saponin content of certain species of the same genus.

	IC50 (µM) at 2.35 (B16), 2.45 (CT26), 2.78 (HepG2) for 8, and 2.32 (B16), 2.19 (CT26),
	2.83 (HepG2) for 10 (Table 12), according to the presence of sequence β-D-xylopyranosyl-
	(1→3)-α-L-rhamnopyranosyl-(1→2)-β-D-xylopyranosyloleanolic	acid	which	was
	previously described in W. x "Bristol Ruby".
	The phytochemical study allowed the isolation of 35 natural compounds using several
	chromatographic techniques (Flash chromatography, Vacuum liquid chromatography,
	Column chromatography, Medium pressure liquid chromatography). Their characterization
	was carried out using spectroscopic techniques (Mass spectroscopy and Nuclear Magnetic
	Resonance). The isolated molecules of the Caprifoliaceae family are triterpenoid saponins
	while the others of the Asparagaceae family are steroidal saponins. These compounds are
	distributed as follows:
	against CT26 (A), B16 (B) and HepG2 (C), in concentrations ranging from 1 to 50 µM.

ietnam

  Figure 190. Tautomerism of curcumin under aqueous solutions .....................................Figure 191. Pharmacological activity of curcumin ..........................................................Figure 192. Schematic presentation of the spray drying method .....................................Figure 193. Schematic presentation of the emulsification-solvent removal methods......Figure 194. Schematic representation of the coacervation process..................................Figure 195. Schematic presentation of the fluid bed coater. ............................................Figure 196. (A) Types of cyclodextrins; (B) Schematic presentation ..............................Figure 197. Schematic presentation of the ionotropic gelation method ...........................Figure 198. Chemical structure of chitosan ......................................................................Figure 199. Chemical structure of alginate ......................................................................

	, colon carcinoma (Raju and Bird, 2007),
	hepatocellular carcinoma (Chen et al., 2018), breast cancer (He et al., 2014), prostate cancer
	(Hamid et al., 2017), gastric cancer (Dong et al., 2018). The anticancer mechanism of
	diosgenin is related to the modulation of multiple cell signaling that plays a significant role
	in proliferation, differentiation, migration, apoptosis, oncogenesis, and angiogenesis (Raju
	and Rao, 2012). But, like others natural plant compounds, diosgenin has less toxicity than
	chemical drugs, and its bioactivities can be limited because of low water solubility and poor
	absorption. In order to overcome limitations and enhance therapeutic efficacy of diosgenin,
	a new method is required to not only enhances its positive bioavailability but also decreases
	its negative characteristics. This perspective will focus on diosgenin encapsulation.

  They are employed to increase solubility, protect curcumin against gastrointestinal degradation, enhance colloidal stability and oral bioavailability from the preparation (Araiza-Calahorra et al., 2018; Cheng et al., 2018; Nguyen et al., 2014). As FDA proved surfactants, Kolliphor ® HS 15 and TPGS ® are the al., 2014; Seo et al., 2012; Zhang et al., 2012). Furthermore, TPGS ® is reported to possess

  . Amidated lowmethoxyl pectin (Unipectine ® 305 C, DA = 20-24%, DE = 20-28%, pKa = 3.5), used as a polymer carrier, was purchased from Cargill, France. Kolliphor ® HS 15 (Macrogol 15

	x -curcumin concentration in mg/L
	2.2. Short term stability study
	20 mg of curcumin powder was dissolved in 20 mL of deionized water (pH = 6.8)
	Hydroxystearate, melting point: 30 °C, hydrophilic-lipophilic balance ≈ 14-16) and TPGS ® containing 5 g/L of Kolliphor ® HS 15. Curcumin concentration in solution was measured
	(Vitamin E Polyethylene Glycol Succinate, hydrophilic-lipophilic balance ≈ 13.2, melting spectrophotometrically (426 nm) at t0 and after at different time intervals in duration of 24
	days. Stability was determined at three different environmental conditions: darkness at 25 point: 37-41 °C) were supplied by BASF, Germany. BiPro ® beta-lactoglobulin (BiPro ® o C, darkness at 37 o C and daylight at 25 o C. Blank was deionized water containing 5 g/L BLG) was obtained from Davisco, Switzerland. Ethanol (EtOH) 96% (v/v), hydrochloric acid (HCl) 1M and sodium hydroxide (NaOH) 1 M were obtained from VRW International, Kolliphor ® HS 15 concentration. All experiments were done in triplicate.
	France. Calcium chloride dihydrate (purity > 99 %) and potassium phosphate dibasic All calculations were made according to equation from calibration curve obtained using
	(KH2PO4) 0.1 M, 2,2-Diphenyl-1-picrylhydrazyl (DPPH, CAS number = 1898-66-4) were dissolved curcumin in phosphate buffer pH 7.4 containing 5 g/L of Kolliphor ® HS 15
	obtained from Sigma-Aldrich (Germany). All the reagents were analytical grade. concentration.
	2. Physicochemical parameters Calibration curve equation: y = 0.1268x -0.0255 (R 2 = 0.9998) (Fig.208)
	2.1. Solubility study y -absorbance measured at 426 nm
	The solubility of curcumin in different surfactants was measured spectrophotometrically at x -curcumin concentration in mg/L
	426 nm. The method was conducted according to Nguyen et al. (2014). 1 g of each Percentage of degradation was determined by Eq.(1):
	surfactant (5%, w/v) was weighed and dissolved in 20 mL of deionized water. Surfactants used were Kolliphor ® HS 15, TPGS ® and BiPro ® BLG. An excess amount of curcumin (20 𝑃𝐷 (%) = 𝐶 𝑚𝑎𝑥 -𝐶 24 𝐶 𝑚𝑎𝑥 × 100
	mg) was added and dispersed in these solutions. They were then stirred for 15 minutes at
	high rate (500 rpm), sonicated for 15 minutes (37 Hz) and stirred for another 15 minutes.
	After storing 12 hours in the dark, 2 mL of centrifuged (5000 rpm, 5 minutes) supernatant
	was taken and dissolved in 2 mL of EtOH 96 % (v/v). The mixture was finally diluted with
	EtOH 96% (v/v) before measuring the absorbance at 426 nm. Blank was deionized water
	in EtOH 96 % (v/v). All experiments were done in triplicate.
	To calculate concentration of solubilized curcumin, equation from calibration curve was
	used. Calibration curve was obtained by dissolving curcumin powder in EtOH 96%.
	Calibration curve equation: y = 0.1869x + 0.0077 (R 2 = 0.999) (Fig.209)
	y -absorbance measured at 426 nm
	231

DPPH method applied on extraction and dissolution medium samples

  Bulk solution was prepared by dissolving 36.8 mg of curcumin in 100 mL of EtOH 96 % (v/v). The solution was diluted to different concentrations (1.0, 1.25, 1.5, 1.75, 2.0, 2.5, 3.0, 3.5 x 10-5 M) with EtOH 96 % (v/v). 5 mL of DPPH in EtOH 96 % solution (concentration 0.0678 g/L w/v, meaning 1.7*10 -4 M) were added to 5 mL of each curcumin solution at different concentrations. DPPH radical has a deep purple color in solution, and becomes colorless or pale yellow when neutralized. This property permits visual monitoring of the reaction, and the number of initial radicals can be counted from the change in the optical absorption at 515 nm (Alger, 1997). Absorbance was measured at 515 nm for 2 hours at different time intervals. Blank was ethanol and control was DPPH solution in EtOH 96 %.During experiments with extraction of curcumin from the beads, after 48 hours of extraction, 1 mL of extraction medium was taken and diluted with 4 mL of EtOH 96 %. 5 mL of DPPH solution was then added and measured spectrophotometrically at 515 nm for 2 hours at different time intervals.

	6.2. During dissolution kinetics studies, after 330 minutes of dissolution, 1 mL of dissolution
	medium is taken and diluted with 4 mL of EtOH 96%. 5 mL of DPPH solution was then
	added and measured spectrophotometrically at 515 nm for 2 hours in different time
	intervals.
	Concentrations of extraction and dissolution medium were shown before adding of DPPH
	solution in Table 15 and measured spectrophotometrically at 426 nm. Blank in both cases
	was 1 mL of dissolution or extraction medium without curcumin in 9 mL of EtOH 96%.
	Control was 1 mL of dissolution or extraction medium without curcumin diluted with 4 mL
	of EtOH 96% and 5 mL of DPPH in EtOH 96% solution.

6.1. DPPH method applied on curcumin powder

Table 15 .

 15 Concentration of extraction and dissolution medium

		before adding DPPH solution
	Beads	Extraction medium concentration (mg/L)	Dissolution medium concentration (mg/L)
	Curcumin beads	47.3 ± 5.1	48.4 ± 3.5
	Kolliphor ® beads	27.9 ± 3.0	22.6 ± 0.5
	BiPro ® BLG beads	51.8 ± 3.1	41.0 ± 2.0
	TPGS ® beads	38.6 ± 0.3	28.7 ± 2.2

  and higher than that reported by Carvalho et al., 2015 (1.34 mg/L). The slightly different values between Kolliphor ® HS 15 (562.7 ± 1.1 mg/L) and TPGS ® (601.9 ± 3.0 mg/L) (Nguyen et al., 2014; Song et al., 2016) could be

Table 16 .

 16 Solubility of curcumin in deionized water with different mediumsOtherwise, curcumin in PBS 7.4 solution was solubilized much more than in water (82.3 ± 6.2 mg/L). The pH dependence of curcumin solubility has been described bySong et al., 2016 as constant at pH 1.2 and 4.5, higher 25.1 times at pH 6.8 and at pH 7.4.

	Surfactant and solvents	Curcumin concentration (mg/L)
	Deionized water a	0.011
	PBS 7.4	82.3 ± 6.2
	Ethanol (99.5 %) b	8895.9 ± 737.2
	BiPro ® BLG	15.7 ± 1.4
	Kolliphor ® HS 15	562.7 ± 1.1
	TPGS ®	601.9 ± 3.0
	a. Tønnesen, 2002
	b. Carvalho et al., 2015

Table 17 .

 17 Encapsulation efficiency and encapsulation yield of beads

	Beads	EE (%)	EY (%)
	Curcumin beads	91.7 ± 4.1	16.2 ± 0.7
	BiPro ® BLG beads	90.5 ± 2.1	13.5 ± 0.3
	Kolliphor ® beads	76.7 ± 5.5	6.3 ± 0.2
	TPGS ® beads	90.7 ± 1.4	9.0 ± 0.2

Dracaena marginata[START_REF] Rezgui | Oleanolic acid and hederagenin glycosides from Weigela stelzneri[END_REF]

Figure 189. Structure compounds 8, 10, 11, 13, 15 and 17 The bidesmosidic oleanolic derivative 13 along to those hederagenin 11, 15, 17 were inactive cell lines. The appearance of the ester group at C-28 and/or the primary alcohol function at C-23 led to a negative effect which was previously described by (Bang et al., 2005) and confirmed by Rezgui et al., 2016. On the other hand, two monodesmosidic

Abbreviations and Symbols

Ara

CHAPTER 1 BOTANICAL STUDIES AND PREVIOUS PHYTOCHEMICAL WORKS

Phytochemical study of roots of Dracaena braunii

Phytochemical investigation on aqueous-ethanolic extract of the roots of Dracaena braunii led to the isolation of 3 steroidal glycosides (33)(34)(35). All of them have been previously described in litterature.

Isolation and purification

The dried and powdered of roots of Dracaena braunii (6. ). This feature is desired from the standpoint of sensorial and textural characteristics of final products. Although spray-dryers are widespread in the food industry, there are several disadvantages of this technique such complexity of the equipment, nonuniform conditions in the drying chamber and it is not always easy to control particle size.

The principle of spray drying is dissolve the core in a dispersion of carrier material. The misture is then subsequently atomized in heated air leading to an evaporation of water. The powdered particles are then separated after they fall to the bottom of the drier (Fig. 192). About 80-90% of encapsulates are spray-dried ones, rest of them are mostly prepared by spray-chilling, freeze-drying, melt extrusion and melt injection [START_REF] Nedovic | An overview of encapsulation technologies for food applications[END_REF].

Inclusion complexation

Inclusion complexation consists of entrapment of active compound by a polymer through physicochemical interactions. In this technique, β-cyclodextrin is used since the centre is hydrophobic while the outer surface is hydrophilic due to its seven glucose units linked 1 to 4 (Fig. 196). In the centre of the cyclodextrin, water molecules are replaced by less polar All calculations are made according to equation from calibration curve made using dissolved curcumin in PBS 7.4 containing 5 g/L of Kolliphor ® HS 15 concentration (Fig. 208).

Encapsulation efficiency (EE) and encapsulation yield (EY) were calculated according to Eq.( 2):

where Mex is mass of curcumin encapsulated, Mo is starting mass of curcumin used for encapsulation and Mdb is mass of dried beads.

Morphologic description of beads by scanning electron microscopy

Morphology of dried pectinate beads and curcumin powder were studied using scanning electron microscopy (SEM) to determine the size, surface and internal structure. The samples were fixed on metal stub double-sided sticky tape and fractured with a scalpel (Swann Morton, England B.S.). Subsequently, the samples were coated with a thin gold layer (100-150 Å) under vacuum using an ion sputter coater (POLARON Thermo VG Scientific, East Grinstead, Sussex, UK) and examined using a SEM FEI La B6 (Eindhoven, Netherlands), at an accelerating voltage of 5 kV with an electron detector for low vacuum conditions.

Dissolution studies in simulated intestinal medium

Curcumin release from the beads was tracked during 7 hours in conditions of human intestine (pH 7.4 and 37 o C). Dissolution studies were not conducted in gastric conditions since no significant release of curcumin at pH 1. All calculations are made according to equation from calibration curve made using dissolved curcumin in PBS 7.4 containing 5 g/L of Kolliphor ® HS 15 concentration (Fig. 209).

CHAPTER 3 RESULTS AND DISCUSSION

absence of surfactant. TPGS ® beads showed an interesting release of curcumin: slower than Kolliphor ® beads after first 30 minutes but not different after 7 hours of dissolution.

BiPro ® BLG beads showed a release profile similar to curcumin beads without any surfactant. Conclusively, the presence of surfactants in formulation played a very important role in curcumin release from beads [START_REF] Cheng | Functionalization of nanodiamond with vitamin E TPGS to facilitate oral absorption of curcumin[END_REF][START_REF] Nguyen | Physicochemical state influences in vitro release profile of curcumin from pectin beads[END_REF]Sookkasem et al., 2015). This effect seems to be linked to the increase of curcumin solubility and so could drive the choice of surfactants to modulate curcumin release. To determine the mechanism of curcumin release from the particles, mathematical modeling of using the Korsmeyer-Peppas model (Eq. 3) was applied the release profile. A satisfactory linearity (R 2 = 0.86 -0.95) was obtained for all profiles. Depending on geometry of drug delivery system, the indicator of release profile, n, has different values.

For spheric polymeric delivery system, n has been defined: as 0.43 for Fickian diffusion, 0.43 < n < 0.85 for anomalous transport and n = 0.85 for polymer swelling [START_REF] Siepmann | Mathematical modeling of drug delivery[END_REF]. For the Kolliphor ® and TPGS ® beads, the calculated n values were found equal to 0.72 and 0.84, respectively, indicating a release mechanism following an anomalous transport mode. In case of BiPro ® BLG and Curcumin beads, their release exponents n were higher than 1 indicating the supercase II transport of release mechanism.

In all cases, the Fickian curcumin release rate was non-Fickian and depended on the chemical reaction with dissolution medium (i.e. pectin depolymerisation), physical mass transfer process of curcumin (i.e. diffusion) and its physical state [START_REF] Nguyen | Physicochemical state influences in vitro release profile of curcumin from pectin beads[END_REF].

The swelling of the different beads was also observed during the dissolution experiment.

This stage was firstly shown for the curcumin release from the beads by visual observation.

a decreasing percentage of inhibition around 9-17%. It could be explained by the lower curcumin solubility. Curcumin beads and BiPro ® BLG beads leading to a dispersion of crystalline curcumin which could not take part in the radical scavenging process.

APPENDIX