Perencat Sebagai

Protease Ns2b/Ns3

Hanya

D F Sesetengah

Peria Katak

( M Charantia

) Isi

Dan Biji Momordica

( M Minyak

Sementara

M Charantia Didapati Merencat

Terbitan Pirazolil Tertukarganti Benziliden

Lc-Ms Peratusan

Sebatian 90a-T Menjalani Kajian In

ATAS BUAH MOMORDICA CHARANTIA DAN SINTESIS TERBITAN PIRAZOLIL TERTUKARGANTI BENZILIDEN INDANON

Keywords: Momordica charantia, pyrazolyl substituted indanone derivatives, DENV-

ii ACKNOWLEDGEMENT All praise, glory and thanks give to Allah s.w.t for His amazing grace and generous that helps me throughout the whole process of completing this research I would like to take this opportunity to express my gratitude to my beloved supervisor, Prof. Dato' Dr. Hasnah Osman, for her guidance, knowledge, support and advice along the way of finishing this research.

I want to thank my supervisor from the University of Lorraine, France, Prof.

Dr. Dominique Laurain-Mattar and Prof. Dr. Nicolas Brosse for their mentorship, guidance, trust, and patience during my work in their lab (CNRS and LERMAB), in administrative work in France as well as in writing my report and thesis. The experiences obtained during my stay in France will not be forgotten.

I wish to take this chance to thank Prof. Dr. Afidah and Dr. Hazwan for their guidance and help in path a way for my dual PhD program and administrative works. Thank you so much and I am so grateful for your support and help.

iii The important FTIR and 1 H, 13 C NMR data of compounds 89a-t Table 4.9

The physical properties of synthesized compounds 90a-t Table 4.10

The FTIR absorption band of compounds 90a-t Table 4.11

The chemical shifts of 1 H NMR and 13 C NMR spectra of compounds 90a-t Table 4.12 The FEB of ligand-protein docking Table 4. [START_REF])-3a,4dihydroindeno-1,2-c-2-pyrazolylmethanone, 90[END_REF] The hydrogen bonding details of compound 90p

ABSTRACT

There are neither curative nor preventive therapies of dengue fever (DF). Only supplemental medical care and fluid therapy are available to fight DF. Some patients opt for prevention and treatment measure used by the ancient people or folk remedy to treat sickness. One of folk remedy used among patients with dengue fever in the Malaysia hospital is consumption of frog meat soup with bitter melon (Momordica charantia). The fruits flesh and seeds of Momordica charantia (M. charantia) were utilized in this study. The oil of M. charantia seeds were extracted via aqueous enzymatic extraction (AEE) and the oil yield increased with optimum condition to extract the seed oil was to use HEL1:X7 (5:1.25). The seeds oil as well as the lignin, hemicelullose, soluble sugars, and uronic acids content were different accordance to the type of enzyme cocktail ratio. On the other hand, the M. charantia fruits flesh ethyl acetate extract was found to inhibit 38.11 ± 1.06 % of the DENV-2 NS2B/NS3

protease. Based on the GC-MS analysis the trace of gallic acid was found and was selected the basic structural frameworks for synthesis of the DENV-2 NS2B/NS3 protease inhibitor. The pyrazolyl substituted indanone derivatives, 90a-t were synthesized and characterized using FTIR, NMR, and LC-MS analysis. An appropriate percentage of yield of the compounds were obtained. The compounds 90a-t underwent in silico study using AutoDock4.2 to identify the binding mode and conformation with xxi the Wichapong homology protein crystal structure. Five compounds were found to obtained free energy of binding (FEB) less than -7.55 kcal/mol. Compound 90p

showed eight hydrogen bond interactions, one π-π stacking interaction, and one π-alkyl interaction with the amino acid residue in the DENV-2 NS2B/NS3 protease. Lastly, the in vitro studies of top five docked synthesized compound towards DENV-2 NS2B/NS3 protease by using fluorogenic peptide substrate Boc-Gly-Arg-7-amino-4methylcoumarin were conducted. The panduratin A was used as the positive control.

Compound 90p showed a high DENV-2 NS2B/NS3 protease inhibitor activity (65.61 ± 0.98 %), while panduratin A (47.59 ± 1.03 %), and gallic acid (25.11 ± 1.11 %). The IC50 value for compound 90p was observed to be 78.87 µM which is lower compared to the panduratin A, 290.27 µM. The compound 90p is concluded to be a potent hitto-led compound in the development of DENV-2 NS2B/NS3 protease inhibitor.

xxii & Kouri, 2003;[START_REF] Lindenbach | Flaviviridae: The Viruses and Their Replication[END_REF][START_REF] Lanciotti | Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction[END_REF]. There are no exact symptoms of DF have been identified. However, the high fever ranging from 39.5 to 41 ºC than usually continues for 2-7 days accompanied by muscle and joint pain could be a sign. The DF may occur within 3 to 14 days after a bite from an infected mosquito. The infected human felt a loss of appetite and

LES

Aedes aegypti Aedes albopictus

vomiting. Some infected human experienced swollen glands or rashes (WHO, 2009& MoHM, 2015).

Approximately, 72,356 dengue cases were reported in Malaysia from January to July 2019, showing 89.5 % increment from the same period on 2018, including 138 death cases (Bernama, 2019[START_REF] Bahagian | iDengue. Kementerian Kesihatan Malaysia[END_REF]. In Malaysia, all four dengue serotypes can be isolated at a time. However, the main dengue serotypes found in Malaysia were dengue virus type 1 (DENV-1) and dengue virus type 2 (DENV-2) (Suppiah et al., 2018[START_REF] Rahman | All Serotypes of Dengue Viruses Circulating in Kuala Lumpur, Malaysia[END_REF]. The treatment of DF available nowadays is limited to supplemental medical care and fluid replacement, as there is no specific treatment.

Problem statement

As there is neither curative nor preventive therapies of DF and only one licensed dengue vaccine, Dengvaxia by Sanofi Pasteur was approved by regulatory authorities for trial use in a highly endemic area (WHO, 2017). Apart from that, only supportive measure and judicious fluid therapy are available for DF (Rajapakse et al., 2012).

Some patients opt for a traditional folks' remedy to cure DF by improving their platelet count. In Pakistan, a dengue patient was treated with 25 mL aqueous extract of Carica papaya (C. papaya) leave, twice per day and found that in short time, it of increased the platelet count [START_REF] Ahmad | Dengue fever treatment with Carica papaya leaves extracts[END_REF]. The studies showed that C. papaya leaves contained some active compounds that can enhance hemopoiesis and thrombopoiesis [START_REF] Dharmarathna | Does Carica papaya leaf-extract increase the platelet count? An experimental study in a murine model[END_REF].

People in Philippines used Euphorbia hirta (E. hirta) to treat dengue as it is believed to have anti-viral properties which will speed up the recovery process and prevent the DF to develop into critical phase (Perera et al., 2018;[START_REF] De Guzman | Ethnopharmacological studies on the uses of Euphorbia hirta in the treatment of dengue in selected indigenous communities in pangasinan (Philippines)[END_REF]. The E. hirta contained phenolics and flavonoids that are well-known compounds able to increase the platelet.

The traditional Chinese practician, Dr. Yong Kian Fui said that there is no cure for dengue fever in Traditional Chinese Medicine (TCM). However, a herbal concoction called "qin wen bai tu san" a soup made by slow cooking of Momordica charantia L. (M. charantia) and frog meat or any type of meat for two hours can be consumed as one dose a day for three consecutive days. The M. charantia is used to detox the body by removing the heat, while the meat will neutralises the M. charantia, so that it does not become too cooling (Tan, 2014).

With the absence of promising anti-dengue drugs or vaccine options, there are ongoing needs to seek other prevention and treatment measures. As lots of researcher focus on the use of C. papaya leaves and E. hirta, this study focused on the anti-dengue activity of M. charantia fruits or also known as bitter melon, balsam pear, bitter squash or bitter gourd. The local Malay name for this plant is "peria katak" (Ong & Azliza, 2015). M. charantia plant from the family of Cucurbitaceae, possess a thin climbing vine with a long-stalked leaf and oblong fusiform-cylindrical, irregularly warty, shaped fruit (Figure 1 M. charantia is well-known for its anti-diabetic activities [START_REF] Joseph | Antidiabetic effects of Momordica charantia (bitter melon) and its medicinal potency[END_REF], anti-tumor activities [START_REF] Yun | Mono-PEGylation of Alpha-MMC and MAP30 from Momordica charantia L.: Production, Identification and Anti-Tumor Activity[END_REF], anti-inflammatory activities [START_REF] Shivanagoudra | In vitro and in silico elucidation of antidiabetic and anti-inflammatory activities of bioactive compounds from Momordica charantia L. Bioorganic &amp[END_REF], and wound healing promoter [START_REF] Alippilakkotte | Fabrication of PLA/Ag nanofibers by green synthesis method using Momordica charantia fruit extract for wound dressing applications[END_REF]. M. charantia contains biologically active compounds that include glycosides, saponins, alkaloids, fixed oils, triterpenes, proteins, phenolic acid, and steroids [START_REF] Sharma | A chemical and medicinal potency of Momordica charantia[END_REF][START_REF] Dandawate | Bitter melon: A panacea for inflammation and cancer[END_REF]. However, there are lack of studies on the lignocellulosic biomass of M. charantia seeds.

M. charantia is worldwide known for its effectiveness in treating diabetes. As its chemically contains a compound that is very much similar to insulin (Omokhua- & Van Staden, 2020). Researchers have shown that when it is taken continuously for some period has the ability to substitute the insulin in the body. It also contains steroidal saponins called charantin, peptides similar to that of peptides and certain alkaloids that effectively control sugar level in blood (Kumar & Bhowmik, 2010).

Uyi

In addition, the M. charantia is rich in supportive bioactive molecules in the treatment of cardiovascular diseases through inhibition of proliferation of vascular smooth muscle cells. M. charantia has high level of fatty acids. It contains fatty acids, that reduce the risk of developing cardiovascular disease such as omega-3. Besides,

(a) (b)

the MAP30, a 30 kDa protein isolated from seeds, has been proposed as the agent responsible for antiviral and antineoplastic properties [START_REF] Aydin | A Review : Momordica charantia L .' s Biological Active Components and Its Potential Use in Traditional Therapies[END_REF].

As plants provide a wealth of small molecules with drug-like properties and can be used a proven template for the development of new scaffolds of drugs that might be helpful in the optimization of the drugs discovery process [START_REF] Verpoorte | Exploration of nature's chemodiversity: the role of secondary metabolites as leads in drug development[END_REF]Atanasov, 2015).

Rationale of the study

The only available licensed dengue vaccine, Dengvaxia efficacy varied by serotype. The vaccine efficacy was higher against serotype 3 and 4 (71.6 % and 76.9

% respectively) than for serotype 1 and 2 (54.7 % and 43.0%) (WHO, 2018). Besides, a warning was released by Sanofi on Nov 29, 2017, that Dengvaxia could increase the risk of severe dengue in particular circumstances, following a review of the data from the clinical trials (The, 2018). This led to a suspension of the dengue vaccination programme in the Philippines.

As DENV-1 and DENV-2 are the most domain dengue virus serotypes in Malaysia, it is crucial to find an anti-dengue drug or vaccine which is suitable for both DENV-1 and DENV-2. The knowledge of the virus infection mechanisms is crucial to facilitate the search and development of the most effective drugs from potential antidengue plants or compounds. The use of M. charantia and other anti-dengue compound based on literature may speed up the quest in searching for a hit-to-led compound.

The use of fruits in the extraction process will lead to a production of waste.

Approximately 122.80 hectares of farmland is planted with M. charantia that can produce a total of 1372.46 metric tonnes of M. charantia fruits every year. In accordance, about 548.984 metric tonnes of M. charantia seed that can be used to produce seed oil are being discarded every year in Malaysia (DoA, 2014). These are a waste of natural resources as the extraction of oil from the seed can easily be carried.

Hence, the study on seed oil as well as the lignocellulosic biomass of M. charantia seeds will be an added value in picking a right material for the development of a sustainable product using natural resources.

Hypothesis of the study

The hypothesis of the study was listed below:

The seed part:

1. The AEE will increase the M. charantia seed oil percentage of yield.

2. The AEE will extract oil with a slightly modification in the biochemical properties.

3. The AEE will change the biochemical properties of the seed lignocellulosic biomass.

The fruit flesh part:

1. M. charantia extract have an inhibitory activity towards DENV-2 NS2B/NS3 protease.

2. The pyrazolyl substituted benzylidene indanone derivatives will have a higher inhibitory activity towards DENV-2 NS2B/NS3 protease than the M. charantia extract.

Objectives of the study

The objectives of the study were consists of the seed part and the fruit flesh part. The objective were as follow:

The seed part:

1. To extract the seeds oil of M. charantia and study the composition of the seed lignocellulosic biomass.

The fruit flesh part:

1. To test the activity of M. charantia fruits extract (methanol, ethyl acetate and n-hexane) towards DENV-2 NS2B/NS3 protease.

2. To identify the chemical composition of active extract 3. To synthesize and characterize the pyrazolyl substituted indanone derivatives derivatize from small molecule present in the selected M. charantia fruit extract.

4. To conduct in silico molecular docking screening of the synthesized compounds.

5. To perform in vitro studies towards DENV-2 NS2B/NS3 protease of synthesized compounds.

Scope of the study

The research was conducted at Universiti Sains Malaysia, Penang, Malaysia and Université de Lorraine, Nancy, France. Therefore, only available instruments within the research facilities in both universities were used in this research.

The specific scopes of studies were:

The seed part (at Université de Lorraine, Nancy, France) 1. To extract the seeds oil of M. charantia, using aqueous enzymatic extraction method, in order to study effect of aqueous enzymatic extraction using a different ratio of enzyme cocktails HEL1 and X7 towards the production of M. charantia seed oil using gas chromatography (GC) and gas chromatographymass spectroscopy (GC-MS). qualities, and the number is expected to increase [START_REF] Foster | Balancing Nature and Wellness -Malaysian Traditions of "Ramuan" The History, Culture, Biodiversity and Scientific Assimilation of Medicinal Plants in Malaysia[END_REF][START_REF] Maarten | The number of known plants species in the world and its annual increase[END_REF][START_REF] Ali | Review on some Malaysian traditional medicinal plants with therapeutic properties[END_REF].

One of the frequently use tropical plant is "peria katak" or M. charantia . This tropical plant is growing wildly in tropical and subtropical Africa, Asia, America and the Caribbean and cultivated for edible fruits (Purnima & Harshani, 1999). The traditional uses of M. charantia vary widely by region throughout the world, but typically included the use as a digestive aid, anti-viral, antibacterial, parasiticide, blood sugar regulator, blood pressure regulator and hormonal balancer (Kumar & Bhowmik, 2010;[START_REF] Ahmad | Momordica charantia: for Traditional Uses and Pharmacological Actions[END_REF][START_REF] Grover | Pharmacological actions and potential uses of Momordica charantia: A review[END_REF][START_REF] Beloin | Ethnomedicinal uses of Momordica charantia (Cucurbitaceae) in Togo and relation to its phytochemistry and biological activity[END_REF].

A study conducted by Ali and co-worker (2012) at Kangkar Pulai Region, Johor Bahru, Malaysia showed that the fruit of M. charantia are consumed as a juice to cure diabetes as well as to treat high blood pressure. In addition, a limited study among patients with diabetes mellitus found that 7.9 % patient consumed M. charantia as a complementary alternative medicine [START_REF] Ching | Complementary alternative medicine use among patients with type 2 diabetes mellitus in the primary care setting: a cross-sectional study in Malaysia[END_REF].

The estimations of nutritional composition of M. charantia are 90. 00 % water, 0.200 % fat, 4.20 % carbohydrates, 1.60 % protein, 0.03 % calcium, 2.57 % vitamins, and 1.4% fibres (Saeed et al., 2018). Scientific research on M. charantia has been continuously performed since 1962 and from there onwards various chemical constituents as well as new health benefits are being revealed.

The phytochemical studies on of M. charantia fruit

M. charantia fruits have been studied chemically and were found to contain biologically active compounds that include glycosides, saponins, alkaloids, fixed oils, triterpenes, proteins, phenolic acid, and steroids [START_REF] Sharma | A chemical and medicinal potency of Momordica charantia[END_REF][START_REF] Dandawate | Bitter melon: A panacea for inflammation and cancer[END_REF].

Cucurbitane type triterpene was identified as the major chemical constituent of M. charantia (Figure 2.1). The reported structures of cucurbitane type triterpenoids isolated from M. charantia are found to have a basic tetracyclic nucleus with substitutions of methyl, hydroxy, or sugar moieties [START_REF] Perez | Chapter 3: Separation and Identification of Cucurbitane-Type Triterpenoids from Bitter Melon[END_REF]. The first compound isolated from M. charantia was cucurbitane type triterpenoids namely momordicoside A [1] and momordicoside B [2] [START_REF] Okabe | Studies on the constituents of Momordica charantia L. I. Isolation and characterization of momordicosides A and B, glycosides of a pentahydroxy-cucurbitane triterpene[END_REF]. [START_REF] Fatope | New cucurbitane triterpenoids from Momordica charantia[END_REF].

Other cucurbitane type triterpene isolated and identified from M. charantia were 5β, 19-epoxycucurbita-6,23-diene-3β,19,25-triol [9], momordicoside P [10] (Yan et al., 2007), (19R,23E)-5β,19-epoxy-19-methoxycucurbita-6,23,25-trien-3β-ol [11], hydroxy-7β,25-dimethoxycucurbita-5,23-dien-19-al [12b] [START_REF] Kimura | Cucurbitane-type triterpenoids from the fruit of Momordica charantia[END_REF].

(23E)-3β-hydroxy-7β-methoxycucurbita-5,23,25-trien-19-al [12a], (23E)-3β-
In (3β,7β,9β, 10α,23E)-25-methoxy-9-methyl-19-norlanosta-5,23-diene-3,7-diol [8e] were isolated and elucidated by Chang and co-worker [START_REF] Chang | Cucurbitane-Type Triterpenoids from Momordica charantia[END_REF] believed to be the antidiabetic components of Taiwanese M. charantia fruits. from the acid hydrolysed ethanol extract of M. charantia seeds [START_REF] Cao | A New Cucurbitane Triterpene in Acid-treated Ethanol Extract from Momordica charantia[END_REF].

O 11 H R 1 R 3 H O O HO R 2 12a-b 12a) R 1 = =CH 2 ; R 2 = H; R 3 = CH 3 12b) R 1 = =CH 2 ; R 2 = CH 3 ; R 3 = -OCH 3 H R 4 H O HO R 3 13a-c R 1
13c) R 1 = -OCH 3 ; R 2 = H; R 3 = -OH; R 4 = H 13b) R 1 = =O; R 2 = H; R 3 = H; R 4 = H 13a) R 1 =H; R 2 = H; R 3 = H; R 4 = - O HO OH HO H H 5 O OH HO O HO HO OH O OH H H 6 O OH O O O O H H 7 R 3 R 5 R 1 R 4 H H R 2 8a) R 1 = OH; R 2 = OH; R 3 = H; R 4 = -OH; R 5 = H 8a-e 8b) R 1 = =O; R 2 = =O; R 3 = =CH 2 ; R 4 = -; R 5 = CH 3 8c) R 1 = =O; R 2 = =O; R 3 = CH 3 ; R 4 = OH; R 5 = CH 3 8d) R 1 = OH; R 2 = OH; R 3 = =CH 2 ; R 4 = -; R 5 = CH 8e) R 1 = OH; R 2 = OH; R 3 = CH 3 ; R 4 = CH 3 ; R 5 = -OCH 3 15
Total phenol contents of 42.36 ± 0.75 mg per gram from M. charantia fruits extract was determined using the Folin-Ciocalteu method was reported by by Ibrahim and co-worker (2010). The phenolic acid fraction analysis of 100 g M. charantia dry sample using high performance liquid chromatography (HPLC) showed that gallic acid [15] (26.7 ± 0.73 mg) was the major phenolic acid followed by caffeic acid [16]

(5. ( [START_REF] Harinantenaina | Momordica charantia constituents and antidiabetic screening of the isolated major compounds[END_REF][START_REF] Horax | Extraction, quantification, and antioxidant activities of phenolics from pericarp and seeds of bitter melons (Momordica charantia) harvested at three maturity stages (Immature, Mature, and Ripe)[END_REF][START_REF] Kenny | Antioxidant properties and quantitative UPLC-MS analysis of phenolic compounds from extracts of fenugreek (Trigonella foenum-graecum) seeds and bitter melon (Momordica charantia) fruit[END_REF]. Figure 2.2 showed the structures of phenolic acids found in M. charantia. Gallic acid could be used as the structure along of this synthesis it is the major phenolic compound of M. charantia fruit extract. Apart from that, some researchers reported on the anti-viral activity of gallic acid and its derivatives.

The phytochemical studies of M. charantia seeds

The seeds of M. charantia are usually discarded without any further uses. Few studies have shown that the seeds of M. charantia contained high content of omega-3 polyunsaturated fatty acids such as α-linolenic acid [28], eicosapentaenoic acid [29],

and stearidonic acid [30] [START_REF] Liu | In vivo studies of the biosynthesis of vernolic acid in the seed of Vernonia galamensis[END_REF][START_REF] Yasui | Bitter gourd seed fatty acid rich in 9c,11t,13t-conjugated linolenic acid induces apoptosis and up-regulates the GADD45, p53 and PPARgamma in human colon cancer Caco-2 cells[END_REF] and apiol [57] (Figure 2.4) from M. charantia seed has been isolated and tested for its anti-bacterial and antifungal activities [START_REF] Braca | Chemical composition and antimicrobial activity of Momordica charantia seed essential oil[END_REF].

2.2

The extraction method of M. charantia

The fruits flesh extraction via maceration method

Maceration is an extractive technique that is conducted at room temperature. It consists of immersing a plant in a liquid (water, oil, alcohol, or solvent) inside an airtight container, for a variable time based on the plant material and liquid used. The solvent must be chosen based upon the chemical nature of the compounds contained within the plant.

The study on antioxidation activity of M. charantia fruit extracts under different maceration periods of 6, 12, 24 and 48 h in different organic solvent was conducted. The result indicated that nitric oxide scavenging activity of antioxidation test was higher using ethanolic extract followed by aqueous, acetone, ethyl acetate, petroleum ether and hexane extracts at 12 h [START_REF] Malik | Effect of extraction time and solvent power on phytochemical screening and antioxidant activity of Momordica charantia L. fruit extracts[END_REF]. Kaneria and coworker worked on identifying the metabolome pattern and study the biological efficacy of almond (Terminalia catappa L.) peels found that the methanolic maceration extraction method proved to be the best extraction method for the extraction of antioxidant [START_REF] Kaneria | Nontargeted metabolomics approach to determine metabolites profile and antioxidant study of Tropical Almond (Terminalia catappa L.) fruit peels using GC-QTOF-MS and LC-QTOF-MS[END_REF].

In addition, Yeo and co-worker (2014) revealed the importance of appropriate maceration periods in combination with different extraction solvents on the anti-microbial activity of M. charantia fruit extract. Thus, the results revealed that the active anti-microbial compounds from fruit of M. charantia are mostly extracted in the non-polar to intermediate polarity solvent. The Tukey's multiple comparison tests proven that findings as n-hexane, petroleum ether and ethyl acetate demonstrated better anti-microbial activity as compared to other solvents used. Hence, the semi to non-polar solvent showed a better potency in extracting the active compounds from M. charantia.

The M. charantia seed oil extraction method

Various methods have been used to extract oil from M. charantia seeds. The traditional method of oil extraction from the seeds was the maceration of the seed with a non-polar solvent. [START_REF] Verma | The component fatty acids of the oil from the seed of Momordica charantia[END_REF], obtained a clear reddish-brown oil yielding of 26.5 % by extracting the fresh seeds with petroleum ether. On the other hand, very little quantity of essential oil was obtained (yield: 0.012%) by hydro distillation of dried seeds using the Clevenger-type apparatus [START_REF] Braca | Chemical composition and antimicrobial activity of Momordica charantia seed essential oil[END_REF].

Two methods were used by Yoshime and co-workers (2016) to extract the M. charantia seed oil. The research concluded that the Soxhlet extraction method (40 % w/w) resulted in higher yield than the Folch extraction method (16 % w/w). An adequate amount of seed oil yielding of 36.89 % was obtained by using the supercritical CO2 extraction method (Liu et al., 2010b).

With the increasing demand for oilseed from agricultural plants, oil extraction technology has become more advance such as the alternative solvent extraction methods including supercritical fluids [START_REF] Sodeifian | Properties of Portulaca oleracea seed oil via supercritical fluid extraction: Experimental and optimization[END_REF], enzyme-assisted aqueous extraction (AEE) [START_REF] Yusoff | Aqueous enzymatic extraction of Moringa oleifera oil[END_REF], and microwave-assisted extraction [START_REF] Kumar | Microwave Assisted Extraction of Oil from Pongamia pinnata Seeds[END_REF].

2.2.2(a) The enzyme-assisted aqueous extraction (AEE) oil extraction method

The AEE is getting some attention nowadays due to its efficient, sustainable, and eco-friendly characteristics. This extraction process depends on the specific properties of the enzyme itself. The enzyme, acting as a catalyst under optimum experimental conditions, disrupts the plant cell wall by hydrolysation to release the intracellular components [START_REF] Sheldon | Enzyme immobilisation in biocatalysis: Why, what and how[END_REF]. As the plant cell walls are a complex structure which mainly made of cellulose, hemicellulose, lignin and protein in addition to pectin [START_REF] Waldron | Plant cell walls and food quality[END_REF][START_REF] Carpita | Structural models of primary cell walls in flowering plants: Consistency of molecular structure with the physical properties of the walls during growth[END_REF].

Rosenthal reported that the cell wall degradation enzyme for rapeseed consists of a combination of pectinase, cellulase, and hemicellulase (Rosenthal et al., 1196). In a previous study, Latif and Anwar reported that the active mechanism of enzyme cocktail (Alcalase 2.4L, Viscozyme L, Protex 7L, Natuzyme, and Kemzyme.) during AEE was the degradation of cellulose, pectin, and protein. Thus, the destructing of structural cell wall integrity led to higher sesame oil recovery; 16.5-24.8% for the AEE was observed compared to only 12.3% for the control (without enzyme treatment) (Latif & Anwar, 2011). The deployment of AEE in this study could resulting in a higher quality M. charantia seeds oil.

2.3

The indanone with gallic acid moieties derivatives

Gallic acid

Gallic acid is a phenolic compound that is easily found in various plants and is a primary phenolic compound present in M. charantia [START_REF] Ibrahim | Antioxidant potential and phenolic acid content of certain cucurbitaceous plants cultivated in Egypt[END_REF]. Gallic acid is extensively used in medicine and chemical research as well as pharmaceutical, cosmetic, and food industries [START_REF] Brewer | Natural Antioxidants: Sources, Compounds, Mechanisms of Action, and Potential Applications[END_REF]. The three hydroxyl groups and a carboxylic acid group attached to a benzene ring are the two functional groups that make up a gallic acid compound as presented in Figure 2.5. The ortho position of the hydroxyl groups resulted in a coplanar bent configuration make it prone to antioxidative activities [START_REF] Badhani | Gallic acid: A versatile antioxidant with promising therapeutic and industrial applications[END_REF]. The coplanar backbones enable the formation of π-π stacks that facilitate the intermolecular bonding [START_REF] Zhan | More than conformational "twisting" or "coplanarity": molecular strategies for designing high efficiency nonfullerene organic solar cells[END_REF]. The presence of polyphenolic functionality in gallic acid is induced by the high oxygen derived free radical scavenging moieties present in the compound and make it capable of acting as a Bronsted acid with carboxylic group shows the highest acidity value compared to the three hydroxyl groups [START_REF] Singh | Anticancer activity of gallic acid template-based benzylidene indanone derivative as microtubule destabilizer[END_REF][START_REF] Škorňa | Gallic acid: thermodynamics of the homolytic and heterolytic phenolic O-H bonds splitting-off[END_REF].

The indanone

1-indanone [58] is a polynuclear hydrocarbon with an unstable five-membered ring cyclopentanone fused to benzene ring (Figure 2.6). The two-active sites in 1-indanone reacted according to a specific reaction condition. The ionic reaction condition resulted in the substituent at the α carbon, while the reaction in a radical condition happen at the ß carbon [START_REF] Fatma Yelda | The Syntheses of Indanones and Indenones Via Rhodium Catalyzed[END_REF]. The indanone possess structural motifs that are frequently found in various types of natural compounds and act as one of the essential classes in organic chemistry as it can be an intermediate in the synthesis of a variety of molecules.

The synthesis of indanone-based derivatives and its pharmacological studies

Several synthetic strategies have been developed inspired by the useful moiety of gallic acid and indanone. These compounds have been studied mainly for their anti-Alzheimer' activity [START_REF] Cho | Inhibition of acetylcholinesterase by gallic acid-grafted-chitosans[END_REF][START_REF] Patil | Recent developments in biological activities of indanones[END_REF][START_REF] Sugimoto | Donepezil Hydrochloride (E2020) and Other Acetylcholinesterase Inhibitors[END_REF][START_REF] Huang | Multitarget-directed benzylideneindanone derivatives: Anti-β-amyloid (Aβ) aggregation, antioxidant, metal chelation, and monoamine oxidase B (MAO-B) inhibition properties against Alzheimer's disease[END_REF] and anti-cancer activity [START_REF] Saxena | Gallic acid-based indanone derivatives as anticancer agents[END_REF][START_REF] Chanda | Anticancer activity, toxicity and pharmacokinetic profile of an indanone derivative[END_REF], monoamine oxidase inhibitor [START_REF] Nel | 2-Heteroarylidene-1indanone derivatives as inhibitors of monoamine oxidase[END_REF], efflux pump inhibition [START_REF] Dwivedi | Gallic acid-based indanone derivative interacts synergistically with tetracycline by inhibiting efflux pump in multidrug resistant E. coli[END_REF]anti-tumor (Garcia-Rivera et al., 2011).

The anti-cancer compound synthesized by Saxena and co-worker (2008) Scheme 2.1: Synthesis of 4,5,6-trimethoxy-3-(3,4,5-trimethoxyphenyl)-2,3-dihydro-1H-inden-1-one. *Reagents and conditions: (i) 20% aq alkali, dimethyl sulphate, refluxed for 3 h, 60%; (ii) CH3I, Mg turnings, THF, 20 min at RT then reflux for 1 h, 42%; (iii) 3% aq methanolic NaOH, RT, overnight 16-18 h, 62-84% respective acetophenones and aldehydes used; (iv) TFA, refluxed in a sealed tube, 3-4 h, 28-52% [START_REF] Saxena | Gallic acid-based indanone derivatives as anticancer agents[END_REF].

The synthesized gallic acid-based indanone derivative, 4,5,6-trimethoxy-3-(3,4,5-trimethoxyphenyl)-2,3-dihydro-1H-inden-1-one [64] showed potent anticancer activity against hormone-dependent breast cancer, oral and liver cancer cell lines. The in vitro study of compound 64 towards MCF-7 hormone-dependent breast cancer cell lines showed a promising activity with IC50 = 2.2. The trimethoxyphenyl units identical to gallic acid in both the rings in the compound 64 suggested to have a potent anticancer activity which increased the activity towards MCF-7, HEPG2 and WRL68 human cancer cell line [START_REF] Saxena | Gallic acid-based indanone derivatives as anticancer agents[END_REF].

The widely used Claisen-Schmidt condensation method has been utilized by Xiao and co-worker (2018) to synthesize an anti-inflammatory agent for the treatment of acute lung injury. As showed in Scheme 2.2, the commercially available 6-hydroxy- (b) various benzaldehydes, EtOH, 20% (w/v) NaOH, room temperature, overnight; (c) 1.0 mol/L HCl, EtOH, room temperature, 5 h; (d) HCl (gas), EtOH, room temperature, overnight [START_REF] Xiao | Design, synthesis, and structureactivity relationships of 2-benzylidene-1-indanone derivatives as antiinflammatory agents for treatment of acute lung injury[END_REF].

O HO O O THP O O THP R 1 O HO R 1 a b c d 65 66 67a-b, 67e-t 68a-t
The anti-inflammatory activity was measured as inhibition of lipopolysaccharide, LPS induced interleukin-6, IL-6 and tumour necrosis factor-α, TNF-α. The cytokine-inhibitory effects of the 6-hydroxyl-2-benzylidene-1-indanone derivatives 68a-t was determined and found that compound 68d had the highest percentage inhibition of 69.28 ± 1.58 and 83.73 ± 4.05 towards lipopolysaccharide respectively. The structure activities relationship of synthesized compounds was determined and illustrated as presented in Figure 2.7.

Figure 2.7:

The structure activity relationship of 6-hydroxyl-2-benzylidene-1indanone derivatives 68a-t [START_REF] Xiao | Design, synthesis, and structureactivity relationships of 2-benzylidene-1-indanone derivatives as antiinflammatory agents for treatment of acute lung injury[END_REF].

A most famous indanone compound called donepezil [69] is the most effective AChE inhibitor in Alzheimer's disease that is currently available on the market. Figure 2.8 showed the structure of donepezil. The attempt to synthesize compounds with dual activity on AChE and BACE-1 of Alzheimer's disease was reported by modifying the donepezil analogue by introducing a double bond on the indanone moiety [START_REF] Costanzo | Design, Synthesis, and Evaluation of Donepezil-Like Compounds as AChE and BACE-1 Inhibitors[END_REF] ii) dry DMF, iodocyclohexane, reflux [START_REF] Costanzo | Design, Synthesis, and Evaluation of Donepezil-Like Compounds as AChE and BACE-1 Inhibitors[END_REF].

In method development phase, Costanzo and co-worker (2016) found about 20 % of the hydroxyl impurities in the synthesis of compound 72f when using homogeneous catalyst, alkali metal hydroxides or alkoxides in methanol which required additional purification steps. Hence, the homogeneous catalyst was changed to Amberlyst A-26, a commercially available heterogenous catalyst. The synthesized compounds were tested against both AChE and BACE-1 enzymes. Compounds 72f

R 1 R 2 R 3 O + N O R 5 R 4 R 1 R 2 R 3 O N R 4 R 5 70 71 72a-o i ii
and 72i displayed promising dual activity and lower IC50 value compared to the significantly low activities of compounds 72n and 72o, which suggested due to an unfavourable hindrance from the N-benzyl piperidine moiety for the compound to reach the active site of the enzymes. with 0.01 mol 4-ethoxybenzaldehyde in 10 mL methanol. Piperidine was added as a catalyst and the reaction was refluxed at 75°C-85 °C for 12 h. The reaction mixture was then cooled to a room temperature and the precipitated formed was filtered and washed with a cold methanol, before being recrystallized using hot ethanol. The reaction was depicted as in Scheme 2.4.

Scheme 2.4:

The synthesis of indanone-based chalcone compound. *Reagents and conditions: i) Piperidine, 75°C-85°C, reflux, 12 h [START_REF] Costanzo | Design, Synthesis, and Evaluation of Donepezil-Like Compounds as AChE and BACE-1 Inhibitors[END_REF].

Hence, merging both important compounds, the gallic acid and indanone could led into a formation of new derivatives which possess a high anti-dengue activity.

O + O O O O 58 73 i 2.

Dengue virus

The increasing risk of being infected with dengue virus are proportional to the increasing of the virus sources for the uninfected mosquitoes as human acted as the main carrier and multipliers of the virus. Environmental management and the pesticide use have been introduced for vector control and dengue prevention to reduce humanvector contact. Some of the methods are eliminating vector larval habitats, installing mosquito screen on windows, doors, and using mosquito nets while sleeping (WHO, 2009;Scritable, 2011;[START_REF] Mahmud | Environmental management for dengue control: A systematic review protocol[END_REF].

A minimal change of the total global area at the risk of dengue between 2015 and 2080 was predicted by Messina and co-worker (2019). The climate change resulted in a rising in temperature that intensify the dengue concern in the endemic areas, speed up the viral amplification and increased the vector survival. Prolonged the transmission seasons and increased the number of infected humans that increased the public health burden of the dengue endemic countries need to face. An additional of 2.25 billion people is estimated to be at risk of dengue in 2080 compared to 2015, bringing the total population at risk to over 6.1 billion or 60 % of the world population.

Dengue virus infection in Malaysia

The Ministry of Health (MoHM) surveillance monitoring showed that a particular serotype can be dominated for at least two years before being replaced by other serotypes (Figure 2.9). The serotype shifts led to a reoccurring of dengue outbreak and deaths due to reduction in the herd immunity to the new virus serotypes (Mudin, 2015). In a descriptive time series study by Bujang and co-worker (2017) using the dengue surveillance system data developed by Vector Borne Disease Section, MoH, it is forecasted that in 20 years the dengue cases will be increase up to 1561.3 % compared with the dengue cases in 2014 which was 108,698 cases. The increased number of dengue cases in Malaysia were due to the continuous urbanisation process and development, migration or movement from one place to another, and poor environmental management. As most of the factors are hard to be controlled, only manipulation and intervention with the environment cleanliness, vector control and changing in the human behaviour are the key to reduce the dengue cases [START_REF] Zaki | Public perception and attitude towards dengue prevention activity and response to dengue early warning in Malaysia[END_REF][START_REF] Ong | Dengue vector control in Malaysia: A review for current and alternative strategies[END_REF]. The integrated management strategy for dengue prevention and control program has been implemented by the Ministry of Health since 2011 to control the factors contributing to the disease transmission [START_REF] Singh | The many ways to control Aedes and reduce dengue. The Star[END_REF].

Dengue virus treatment

The critical period of DF is when patients undergo sudden deterioration which is between the first 48-72 hours of infection. The chance of fatal outcome is minimal if the patient managed to go through this phase. Hospitalisation is required when there is a need for fluid replacement under close supervision or if the platelets level is low which need further monitoring for bleeding tendencies. The recovered patients will be temporary immune against the same serotype but not against other dengue virus serotypes (WHO, 2012; [START_REF] Uno | Dengue virus and the host innate immune response[END_REF].

The first licensed dengue vaccine, Dengvaxia by Sanofi Pasteur the first dengue vaccine to market, which is now already licensed in several Asian and Latin American countries [START_REF] Guy | From research to phase III : Preclinical , industrial and clinical development of the Sanofi Pasteur tetravalent dengue vaccine[END_REF][START_REF] Guy | Preclinical and clinical development of YFV 17D-based chimeric vaccines against dengue, West Nile and Japanese encephalitis viruses[END_REF]. Overall, Dengvaxia had satisfactory safety and immunogenicity in both in vitro and in vivo preclinical models (including nonhuman primates), and potential environmental risks as defined above were also demonstrated to be absent or negligible [START_REF] Guy | Development of the Sanofi Pasteur tetravalent dengue vaccine: one more step forward[END_REF]. The trials demonstrated vaccine efficacy against all four serotypes, with higher efficacy rates against serotypes 3 and 4 than serotypes 1 and 2, consistent with the earlier phase II trial in Thailand [START_REF] Sabchareon | Protective efficacy of the recombinant, live-attenuated, CYD tetravalent dengue vaccine in Thai schoolchildren: a randomised, controlled phase 2b trial[END_REF][START_REF] Guy | A recombinant live attenuated tetravalent vaccine for the prevention of dengue[END_REF]. Therefore, the quest in searching for most effective anti-dengue drugs or dengue vaccine is still ongoing.

The anti-dengue screening using DENV-2 NS2B/NS3 protease enzyme

The anti-dengue screening using DENV-2 NS2B/NS3 protease enzyme was carried out by observing the cleavage of the 6-amino-4-methyl-coumarin part

[58] of the peptide substrate by hydrolysis process with the donor/acceptor pair liberates fluorescence which allowed a qualitative measurement of the enzyme activity using a fluorometer [START_REF] Schulz | A sensitive fluorescent assay for measuring the cysteine protease activity of Der p 1, a major allergen from the dust mite Dermatophagoides pteronyssinus[END_REF]& Carmona et al., 2009).

Such activity resulted in the formation of bonded interaction between enzyme and peptide part of the substrate. However no fluorescence been exhibited with the presence of the inhibitor as the inhibitor formed a bonded interaction with the enzyme and ceased the peptide cleavage. The protease assay mechanism without and with the presence of inhibitor were depicted in Scheme 2.5 and 2.6 respectively.

Scheme 2.5: The protease assay mechanism without inhibitor.

Scheme 2.6: The protease assay mechanism with inhibitor. In the study of structure-guided discovery of a novel non peptide inhibitor of dengue virus NS2B-NS3 Protease, Li and co-worker reported on

compound 1,2,3,4-tetrahydro-N-[2-methyl-4-(4-methyl-1-piperazinyl)phenyl]-2- (phenylmethyl)-benzo[b][1,6]naphthyridine-10-carboxamide [83] (Figure 2.14) with
a good DENV protease inhibitor and fulfilled the pockets of the binding site. The lone pair of compound 83 was able to accept hydrogen in the hydroxyl group of Tyr161 and form a tolerable interaction. Additionally, the benzene ring generated potent π-π stacking with amino acid residue. Besides, the pyrazol-3-one moiety also enhance the binding mode through a hydrogen bond between the carbonyl group amino acid residue [START_REF] Li | Structure-guided discovery of a novel non-peptide inhibitor of dengue virus NS2B-NS3 protease[END_REF].

36

In conclusion, the presence of lone pair molecule such as, nitrogen will increase the possibility of hydrogen bond formation. Besides, the benzene ring will generated potent π-π stacking with amino acid residue. Hence, the synthesized compounds could be fit into the binding pocket of the NS2B/NS3 protease and lower the free energy of binding. Therefore, the inhibitory activity of the synthesized compounds will be increased.

CHAPTER 3 RESEARCH METHODOLOGY

Chemical and materials

The solvent, chemical and materials used were of analytical grades and used without undergoing further purification or distillation. All the apparatus used were washed with distilled water and rinsed with acetone to ensure the apparatus is clean and to avoid any impurities.

Characterization methods

Melting point

The melting point was determined by using Stuart Scientific SMP1 melting point apparatus with the temperature range of 50-300 °C at School of Chemical Sciences, Universiti Sains Malaysia.

Fourier-transform infrared (FTIR) spectroscopy

The IR spectra of the compounds were obtained using Frontier ® FTIR-ATR spectrometer (PerkinElmer, England). The compounds were placed on the crystal top plate and analysed in the wavelength of 4000-650 cm -1 .

Nuclear magnetic resonance (NMR) spectroscopy

The 1D ( 1 H, 13 C, and DEPT-135) and 2D (COSY, and HMBC) NMR spectra were recorded using Bruker Avance 500 MHz spectrometers at School of Chemical Sciences, Universiti Sains Malaysia. The compounds (± 15 mg) were dissolved in deuterated solvent; deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3) or deuterated methanol (CD3OH) into NMR glass tube. The calculation of chemical shifts (d, ppm) was refer to the TMS signal at 0.00 ppm, and the coupling constants were written in hertz (Hz).

Gas chromatography (GC) analysis

The M. charantia extract screening was carried out by Agilent 7890A system (Agilent, U.S.A) in School of Chemical Sciences, USM. The samples diluted with 1 mL of n-pentane and 1 μL of sample was injected on DB-5 column (30 × 0.25 mm, 0.25 μm film thickness). The injector and detector temperature were fixed at 270 °C and 280 °C, respectively. Pure helium gas was used as carrier gas at the flow rate of 1 mL/min. The oven temperature was programmed at 60 °C, increased to 280 °C with the heating rate of 5 °C/min and held for 10 minutes.

The oil analysis was carried out by using Shimadzu GC-2010 plus instrument (Shimadzu, Japan) equipped with Zebron capillary GC column ZB-Wax, 60 m × 0.25 mm × 0.25 mm at University of Lorraine, France. Pure helium gas was used as carrier gas, pressurized and flowed at 141.2 kPA and 11.7 mL/min respectively. Then, 1 μL of sample was injected into GC and the oil was separated using temperature programming, starting with 120 °C, held for 2 minutes, increased to 180 °C with heating rate of 40 °C/ minutes, held for another 2 minutes before been heated to 220 °C with heating rate of 3 °C/ minutes, and held for 25 minutes.

Gas chromatography-mass spectrometry (GC-MS)

The GC-MS analysis for M. charantia extracts were performed using Perkin Elmer Clarus 600T system (PerkinElmer, England) which operated at 70eV with a mass range of m/z 50-500 at School of Chemical Sciences, USM. The injection was done on HP-5 semi-polar capillary column (30 m × 320 μm) with the same temperature programmed used in GC analysis.

Liquid chromatography -mass spectrometry (LC-MS)

The molecular mass of selected synthesized compounds was determined by Agilent Technologies 6224 TOF LC/MS spectrometer (Agilent, U.S.A) at School of Pharmaceutical Sciences, USM. Samples were prepared as solutions of 0.1 to 1.0 mL in HPLC grade acetonitrile. The measurements were carried out in the positive mode.

The studies of M. charantia fruits flesh and seed

The studies of M. charantia fruits flesh and seed are divided into two parts.

First part involved the extraction of M. charantia seed oil using hydro distillation method and Aqueous Enzyme Extraction (AEE) method as well as the studies of its lignocellulosic biomass. The second part involved the preparation of M. charantia fruits pulp extract for anti-dengue screening. Figure 3.1 summarized the study framework.

M. charantia fruit

Seed Oil extraction via AEE and study of biomass

Fruit flesh Extract and preliminary anti-dengue screening

Active extract analysis via GC-MS Synthesized of anti-dengue compounds based on small compound presence in the active extract

The in silico and in vitro study of the synthesized compounds towards DENV-2 NS2B/NS3 protease

Sample preparation

The unripe M. charantia fruits were purchased from a vegetable farm located at Ara Kuda, Seberang Perai Utara, Penang, Malaysia between June to August 2015 (USM Herbarium: 11727, deposited by Syafinaz Bt Zainol on 14/09/2016). Only unripe and fresh fruits were selected for extraction. The fruits were washed thoroughly with tap water to remove any unwanted contamination and cut to separate the flesh with the seed. After that, the fruits (flesh) and seed were sun-dried at room temperature. The fruits and seed were turned over every 24 hours to ensure completely dried. The dried fruits and seed were grounded into small pieces before being kept in a dried polyethylene bags until further used.

3.3.1(a) PART 1: Aqueous enzyme extraction (AEE) of M. charantia seed oil

The AEE was performed to extract oil from the M. charantia seed according to the method as described by Muniglia et al. (2011). The enzyme cocktails, HEL1

and X7 were blended in the Laboratoire D'ingénierie des Biomolécules (Nancy, France), which contained cellulase, betaglucosidase, endocellulase, exocellulase, arabinase, xylanase, galactanase, endopolygalacturonase, hemicellulase, proteinase, pectinase, and pectine methylesterase at different concentrations.

Initially, the freshly ground dried M. charantia seed were mixed with distilled water in a ratio of 1:4 into a pre-programmed bioreactor (Celligen Plus, New Brunswick Scientific, U.S.A) with continuous stirring. Then, the enzyme cocktails were added consecutively into the bioreactor once the temperature of the bioreactor reached 50 °C starting with HEL1 followed by X7 at their different ratios as tabulated in Table 3.1.

Table 3.1: Seed samples and their enzyme cocktail ratios.

Seed samples Enzyme (%)/Seed weight (g)

HEL1 X7 M0 - - M1 5 - M2 5 1.25 M3 5 2.5 M4 5 5
The reaction was left to carry on for four hours in the bioreactor followed by centrifugation (0.158 kilohertz, 20 minutes, 20 °C) to separate the products. The final products of this extraction process were: 1) oil, 2) seed residue, and 3) aqueous part.

The oil and seed residue obtained was proceed for analysis via GC and identification of the seed lignocellulosic biomass respectively. The aqueous part was discarded.

3.3.1(a)(i) The analysis of M. charantia seed oil

The seed oil samples were initially derivatized to fatty acid methyl esters (FAMEs) using esterification process as described by Metcalfe and Schmitz (1961) before further analyses to identify their biochemical compositions using gas chromatography (GC). The polar properties of underivatized fatty acid normally lead to the formation of hydrogen bonding, which results in adsorption issue. Hence, reducing their polarity by derivatisation make them more amenable for analysis. Each derivatized oil (FAME) was analysed to identify its biochemical composition using flame ionisation detector gas chromatography.

3.3.1(a)(ii) The Soxhlet extraction of the seed residue

The dried seed residue was further extracted using Soxhlet extraction method to ensure all extractive components were removed prior the hydrolysis to determine their lignin and acid-soluble sugar contents. The presence of extractive components will hinder the hydrolysis process by cross-reacting with acid and condense to acidinsoluble components resulting in inaccurate analysis.

An amount of 50 g of each dried seed residue sample was loaded into an extraction thimble, which was placed into a Soxhlet extractor (Pyrex, U.S.A) fitted with a condenser (Pyrex, U.S.A) on an isomantle heater (Fisherbrand, U.S.A) to execute the Soxhlet extraction. Then, 250 mL of n-hexane was added into a 500 mL round bottom flask fixed to the Soxhlet extractor. The solvent was heated at 150 °C and began to evaporate. The condensate dripped into the reservoir containing the thimble. The solvent from the reservoir was poured into a round bottom flask once the solvent level reached the siphon. The process was continued for five hours. Then, n-hexane was evaporated using a rotary evaporator (Buchi, Switzerland). The final products of Soxhlet extraction process were: 1) extracted-free materials (EFM) and 2) extractive materials. The extractive materials were discarded and the EFM was weighed and kept for further analysis.

3.3.1(a)(iii) Determinations of lignin, soluble sugars, and uronic acid of EFM

An amount of 175 mg of each dried EFM sample was placed in a conical bottom centrifuge tube, and 1.5 mL of 72 % sulfuric acid was added dropwise. The mixture was placed into a water bath and heated at 30 ºC for one hour with periodical stirring using a glass rod. A volume of 42 mL of distilled water was added into the mixture to achieve a concentration of 4 % sulfuric acid by mass. The mixture was then autoclaved at 121 ºC for one hour. The mixture was allowed to cool down to room temperature before being filtered. The filtrate was then diluted with 56.5 mL of distilled water in a 100 mL volumetric flask. The resulting solution was further analysed by high performance anion exchange chromatography with pulsed amperometry detection (HPAEC-PAD) (PerkinElmer, U.S.A) to determine its soluble sugar and uronic acid contents. The solid residue (lignin) was dried and weighed. The seed sample codes for soluble sugar and uronic acid analysis is tabulated in Table 3.2. Note: M refers to the EFM, which had undergone AEE. While, L refers to the soluble sugar and uronic acid sample of the EFM.

3.3.1(a)(iv) Determination of soluble sugar and uronic acid contents of EFM

The separation and quantification of soluble sugars and uronic acids of the EFM were carried out using HPAEC-PAD operating on Dionex ICS-3000 system, which consisted of a gradient pump SP, a passer AS, an electrochemical detector ED The eluents were degassed with helium gas for 20 minutes before being injected. After each analysis, the column was flushed for 10 minutes with 250 mM NaOH solution and restored for 10 minutes to the initial conditions of the analysis.

The samples were injected with an entire loop of 25 μm and the separations were carried out at 35 °C at a flow rate of 0.4 mL/min. The pulse sequence for pulsed amperometry detection consisted of potentials of +100 mV (0-200 ms), +100 mV integration (200-400 ms), +100 mV (400-410 ms), -2000 mV (410-420 ms), +600

mV (430 ms), and -100 mV (440-500 ms).

3.3.1(a)(v) Hemicellulose extraction from EFM

Initially, the EFM was subjected to bleaching process to produce holocellulose.

Sodium chlorite was used as the bleaching agent due to its moderately strong oxidation properties. The bleaching process was conducted by milling 1.5 g of each dried treated EFM sample into 0.05 mm size before being dispersed slowly into 125 mL deionized water in a 250 mL round bottom flask. Acetic acid glacial, 1 mL was added into the mixture with an additional 1 g of sodium chlorite. The mixture was refluxed at 80 °C for two hours. Then, another 1 mL of acetic acid glacial and 1 g of sodium chlorite

were added, consecutively into the reaction mixture with continuous stirring in the same conditions for two hours. The process was repeated until the colour of the residue turned to white to ensure that the bleaching process was completed. The reaction mixture was filtered and washed thoroughly with deionized water. The holocellulose obtained was collected and dried overnight in a fume hood, which was then further reprecipitated into hemicellulose.

An amount 200 mL of 2 % w/v sodium hydroxide aqueous solution was added into 10 g of each holocellulose sample in a round bottom flask and stirred for two hours at 80 °C. Then, an additional of 200 mL of 2 % w/v sodium hydroxide aqueous solution was added into the reaction mixture and stirred for another two hours. The mixture pH was adjusted to 5-6 by adding 20 % w/v acetic acid solution. The reprecipitation process of the mixture was repeated using 800 mL ethanol. The precipitate formed was recovered by centrifugation. The percentage yield of hemicellulose was calculated and analysed using Fourier-transform infrared (FTIR) spectroscopy as well as size exclusion chromatography (SEC).

3.3.1(a)(vi) The SEC analysis of hemicellulose

The molecular weight distribution of hemicellulose was determined by SEC.

The analysis was performed using a Hewlett-Packard 1090 series HPLC system consisting of an autosampler, a UV detector, and three columns of Styragel HR1, HR3, and HR4 (Waters Inc.) linked in series using tetrahydrofuran (THF) as the eluent. The hemicellulose was dissolved in 1 mg/mL of THF, and the solution was filtered through a 0.45 mm filter. Then, 20 mL of the filtered solution was injected into the system and was detected using a UV detector at 280 nm. The data were collected using Agilent ChemStation rev. A.10.01 and analysed using Agilent GPC Addon rev. A.02.02 software (Agilent, U.S.A).

3.3.1(b) PART 2: Preparation of M. charantia fruit flesh extract

The crushed dried fruits flesh (0.5 kg) were then exhaustively extracted by maceration sequentially in n-hexane, ethyl acetate and methanol twice in triplicate for 7-14 days at room temperature (25-30 °C) in fume hood. The mixtures were shaken and stirred on a daily basis. The extracts were then filtered using Whatman filter paper

No. 1 (Whatman, England) and the solvent was removed by rotary evaporator (Büchi, Germany) under reduced pressure. The extract was then kept refrigerated in a sealed sample bottle before proceed with anti-dengue screening and the most active extract will underwent GC-MS analysis.

Anti-dengue testing of M. charantia fruits flesh extract

The in vitro assays testing for M. charantia fruits flesh extracts were performed following the method by Yusof et al. (2000). An enzymatic reaction mixture with a total volume of 100 µL with the concentration of 200 ppm was prepared in black 96 well plates as presented in Figure 3.2. Each well in the black 96 well plates consist 94.9 µL of Tris-HCl buffer solution at pH 8.5, 3.1 µL of the enzyme, and 1 µL of extract (200 mg/L in DMSO). The mixtures were incubated and shook at 37 °C for 10 minutes. Then, 1 µL substrate 10 mM in DMSO (Peptide Institute, Japan) was added into the mixture to initiate the reaction. The mixture was then incubated and shook for another 60 minutes at 37 °C using Ambient shaker incubator SI-50 (Protech, Malaysia). The assay was triplicated. The extract with the most anti-dengue activity will be further analysed using GC-MS to identify it compositions.

3.4

The synthesis of pyrazolyl substituted benzylidene indanone derivatives

The reaction summary was depicted as Scheme 3.1 below.

Scheme 3.1: The synthesis of pyrazolyl substituted benzylidene indanone derivatives The commercially available 3,4,5-trimethoxy benzoic acid [84], undergo esterification using sulfuric acid and ethanol to produce ethyl 3,4,5trimethoxybenzoate [85] (Li et al., 2017 and[START_REF] Schejbal | Acylfunctionalized molybdenum compounds [(η3-C3H5)(η5-Cp')Mo(CO)2]: An experimental study including the X-ray structure of a rare endo conformer[END_REF]. The reaction scheme was illustrated in Scheme 3.2 below.

O O O O OH O H H 2 SO 4 O O O O O 85 84 NH 2 NH 2. H 2 O CH 3 CH 2 OH O O O O H N NH 2 86 R R O R 1 R 2 R 3 O 88a-b 87a-j + CH 3 OH 10 % w/v KOH + R R O R 1 R 2 R 3 89a-t N N O O CH 3 O CH 3 O CH 3 R R R 1 R 2 R 3 90a-t
Benzoic acid (0.1 mmol) was added in ethanol (1.0 mmol) with presence of sulfuric acid (0.01 mmol). The solution was heated under reflux. After 3 h, the reaction was monitored by TLC using ethyl acetate and hexane (3:7) system. Stirring was continued for about 12 hours or until the TLC indicated the completion of reaction.

The excess ethanol was removed by using rotary evaporator. The resulting solution was then mixed with 10 mL ethyl acetate and sodium chloride solution (10 % w/v.).

After that, the organic phase was separated by using separating funnel. The excess solvent was removed from the organic phase using rotary evaporator. The precipitated was dried under vacuum to obtained compound 85. Chemical structures of the compound were confirmed by 1 H NMR, 13 C NMR, and FTIR. monohydrate (5.00 mmol) was added into the solution and stirred overnight until a white precipitate was formed (Scheme 3.3). The precipitate [86] was filtered and washed using cold ethanol [START_REF] Kamal | Synthesis, biological evaluation, and molecular modeling of (E)-2-aryl-5-styryl-1,3,4-oxadiazole derivatives as acetylcholine esterase inhibitors[END_REF]. Chemical structures of the compound were confirmed by 1 H NMR, and 13 (C=O ester), 1181 (C-O, ester); 1 H NMR (500 MHz, DMSO-d6): dH, ppm, 1.3 (3H, t, J = 7.09, CH3), 3.7 (3H, s, OCH3), 3.8 (6H, s, OCH3), 4.3 (2H, q, J = 7.09, CH2), 7.2 (2H, s, CH). 13 C NMR (125 MHz, DMSO-d6): dC, ppm, 14.6 (C12), 56.5 (C7, C9), 60.6 (C11), 61.3 (C8), 106.9 (C2, C6), 125.5 (C1), 142.2 (C4), 153.2 (C3, C5), 165.9

(C10). O O O O O NH 2 NH 2. H 2 O CH 3 CH 2 OH O O O O H N NH 2 86

The synthesis of benzylidene indanone derivatives, 89a-t

An indanone (2.0 mmol) [87a-b] was dissolved and stirred in 10 % w/v of potassium hydroxide (KOH) in methanol for about 15 minutes. After that, a different type of aromatic aldehyde (2.0 mmol) [88a-b] was added into the solution and the reaction mixture was stirred for an hour or until the reaction was completed under reflux (Korotchenko et al., 2014[START_REF] Ishimaru | Cinchona alkaloid catalyzed enantioselective fluorination of allyl silanes, silyl enol ethers, and oxindoles[END_REF]. Scheme 3.4 depicted the reaction scheme to synthesis the benzylidene indanone derivatives [89a-t]. The reaction was monitored using TLC until it completed. The reaction mixture pH was then adjusted to 5-6 by adding 10 % w/v hydrochloric acid solution. The precipitated was filtered using vacuum filtration, washed using cold methanol and dried under vacuum. The synthesized compounds were further recrystallized using single-solvent

3.4.2(a) (E)-2-benzylidene-1-indanone, 89a

C-6, C-4', R R O R 1 R 2 R 3 R R O R 1 R 2 R 3 O 88a-j 89a-t + CH 3 OH 10 % w/v KOH 87a-b Where 87a = R = H 89a: R 1 = H; R 2 = H; R 3 = H 89b: R 1 = H; R 2 = H; R 3 = OCH 3 89c: R 1 = H; R 2 = H; R 3 = CH 3 89d: R 1 = H; R 2 = H; R 3 = Cl 89e: R 1 = H; R 2 = H; R 3 = CH(CH 3 ) 2 89f: R 1 = H; R 2 = H; R 3 = N(CH 3 ) 2 89g: R 1 = H; R 2 = H; R 3 = NC 4 H 8 O 89h: R 1 = H; R 2 = H; R 3 = NC 4 H 10 89i: R 1 = OCH 3 ; R 2 = H; R 3 = OCH 3 89j: R 1 = H; R 2 = NO 2 ; R 3 = OCH 3 Where 87b = R = OCH 3 89k: R 1 = H; R 2 = H; R 3 = H 89l: R 1 = H; R 2 = H; R 3 = OCH 3 89m: R 1 = H; R 2 = H; R 3 = CH 3 89n: R 1 = H; R 2 = H; R 3 = Cl 89o: R 1 = H; R 2 = H; R 3 = CH(CH 3 ) 2 89p: R 1 = H; R 2 = H; R 3 = N(CH 3 ) 2 89q: R 1 = H; R 2 = H; R 3 = NC 4 H 8 O 89r: R 1 = H; R 2 = H; R 3 = NC 4 H 10 89s: R 1 = OCH 3 ; R 2 = H; R 3 = OCH 3 89t: R 1 = H; R 2 = NO 2 ; R 3 =

3.4.2(d) (E)-5'-chlorolbenzylidine-1-indanone, 89d

Colour: White solid; Weight: 481.30 mg, 94.48 % (yield); mp. 137-139 °C;

3.4.2(e) (E)-2-(5'-isopropylbenzylidene)-1-indanone, 89e

Colour

3.4.2(f) (E)-2-(5'-dimethylaminobenzylidene)-1-indanone, 89f

Colour (C-1).

3.4.2(g) (E)-2-[5'-(4"-morpholinobenzylidene)]-1-indanone, 89g

Colour: Yellow solid; Weight: 571.

3.4.2(h) (E)-2-(5'-piperidinylbenzylidene)-1-indanone, 89h

Colour: Cream solid; Weight: 550.33 mg, 90.69 % (yield); mp. 178-181 °C; N 2" 6" 1" 3" 4" 5"

3.4.2(i) (E)-2-(3',5'-dimethoxybenzylidene)-1-indanone, 89i

Colour: Yellow solid; Weight: 471.64 mg, 84.12 % (yield); mp. 181-189 °C;

3.4.2(j) (E)-2-(4'-nitro-5'methoxybenzylidene)-1-indanone, 89j

3.4.2(k) (E)-2-benzylidine-5,6-dimethoxy-1-indanone, 89k

Colour

3.4.2(l) (E)-(5'-methoxybenzylidene)-5,6-dimethoxy-1-indanone, 89l

Colour: Light yellow solid; Weight: 292.60 mg, 94. [START_REF] References Abdulkarim | Some physico-chemical properties of Moringa oleifera seed oil extracted using solvent and aqueous enzymatic methods[END_REF]

3.4.2(m) (E)-(5'-methylbenzylidine)-5,6-dimethoxy-1-indanone, 89m

Colour:

3.4.2(n) (E)-(5'-chlorobenzylidine)-5,6-dimethoxy-1-indanone, 89n

Colour

3.4.2(o) (E)-2-(5'-isopropylbenzylidene)-5,6-dimethoxy-1-indanone, 89o

Colour

3.4.2(p) (E)-2-(5'-dimethylaminobenzylidene)-5,6-dimethoxy-1-indanone, 89p

Colour 149.03 (C-5'), 151.17 (C-6), 155.17 (C-5), 192.14 (C-1).

3.4.2(q) (E)-2-[5'-(4"-morpholinobenzylidene)]-5,6-dimethoxy-1-indanone, 89q

Colour

3.4.2(s) (E)-2-(3',5'-dimethoxybenzylidene)-5,6-dimethoxy-1-indanone, 89s

3.4.2(t) (E)-2-(4'-nitro-5'methoxybenzylidene)-5,6-dimethoxy-1-indanone, 89t

Colour

The synthesis of pyrazolyl substituted benzylidene indanone derivatives

The previously synthesized benzylidene indanone derivatives, 89a-t

(1.0 mmol) were further reacted with compound 86 (1.0 mmol) in the presence of 10 mL acetic acid under reflux for 12 h as depicted in Scheme 3.5.

Scheme 3.5:

The synthesis of pyrazolyl substituted benzylidene indanone derivatives.

The excess solvent was removed under reduced pressure. The reaction mixture was then poured into the crushed ice. The precipitated was filtered using gravitational filtration, washed using cold methanol and dried in the fume hood. The synthesized compounds were further recrystallized by single-solvent recrystallization technique using ethanol. The compounds were then undergone characterization analysis to determine their physicochemical properties. 31.85, 39.32, 56.25, 60.80, 68.05, 77.45, 105.87, 122.41, 123.01, 124.09, 125.32, 126.83, 127.34, 128.40, 134.55, 139.10, 140.50, 149.60, 149.60, 153.20, 173.10, 192.35. 3.93 (s, 6H, -OCH3), 4.98 (s, 1H, ArH), 7.17-7.26 (m, 6H, ArH), 7.33-7.41 (m, 5H, ArH); 13 C NMR (125 MHz, DMSO-d6): dC, ppm, 23.61, 30.11, 33.81, 41.89, 58.25, 60.13, 77.91, 107.81, 120.40, 124.31, 125.56, 126.49, 126.87, 127.91, 128.41, 136.73, 138.11, 140.23, 148.98, 149.17, 153.28, 158.11, 172.10, 193.14. (s, 3H, -OCH3), 5.01 (s, 1H, ArH), 7. 6H,ArH), 7.69-7.73 (m, 5H, ArH); 13 C NMR (125 MHz, DMSO-d6): dC, ppm, 28.37, 35.11, 40.18, 58.73, 60.11, 77.24, 105.81, 119.37, 120.89, 123.47, 125.25, 136.37, 137.18, 139.85, 141.11, 142.71, 143. 89, 146.01, 148.01, 153.28, 155.87, 169.73, 192.04. ppm, 20.89, 29.69, 56.28, 60.90, 65.80, 77.55, 104.66, 123.37, 126.13, 126.25, 127.68, 132.41, 133.24, 134.46, 138.20, 141.72, 149.42, 153.24, 164.27, 167.74, 193.92.

R R O R 1 R 2 R 3 89a-t N N O O CH 3 O CH 3 O CH 3 R R R 1 R 2 R 3 90a

3.4.3(j) 3-(3'-nitro-4'methoxyphenyl)-(3,4,5-trimethoxyphenyl)-3a,4-dihydroind eno-1,2-c-2-pyrazolylmethanone, 90j

Colour 32.10, 56.78, 60.17, 63.71, 77.81, 105.71, 114.01, 124.52, 126.19, 126.59, 127.85, 128.27, 130.71, 134.80, 135.30, 136.41, 137.84, 140.18, 149.81, 153.47, 160.73, 163.59, 193.56. 28.11, 32.18, 55.38, 56.17, 56.26, 77.33, 105.17, 107.23, 113.70, 114.43, 128.39, 130.33, 131.31, 132.29, 133.18, 144.66, 149.65, 155.27, 160.65, 193.25. (m, 7H, ArH); 13 C NMR (125 MHz, DMSO-d6): dC, ppm, 21. 70, 30.14, 56.31, 56.99, 64.05, 77.24, 105.15, 108.11, 109.73, 124.84, 126.68, 127.55, 129.16, 131.69, 136.89, 137.10, 140.51, 149.17, 153.25, 155.33, 170.44, 193.81.

3.4.3(q)

3-( 4 ppm, 20.79, 29.78, 32.49, 56.73, 58.93, 60.31, 61.07, 66.81, 77.59, 105.73, 125.43, 127.68, 132.41, 133.74, 135.16, 138.97, 141.72, 153.24, 155.67, 163.01, 168.97, 192.89. 28.85, 37.87, 56.08, 56.79, 57.02, 60.71, 60.96, 77.55, 105.15, 108.11, 109.73, 114.93, 122.37, 123.68, 124.71, 127.51, 133.23, 136.27, 138.51, 140.55, 148.46, 154.17, 155.98, 156.30, 160.81, 164.29, 193.01.

3.4.3(t) 3-(3'-nitro

3.5

The in silico studies towards DENV-2 NS2B/NS3 protease

The synthesized compound structure was sketched using Marvin Sketch 19.4.0 (ChemAxon, 2019) and converted into a 3D structure using HyperChem pro 8.0 software (www.hyper.com). The molecular docking was performed using AutoDock4.2 to identify the binding mode and conformation of the synthesized compound with the protease [START_REF] Morris | AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility[END_REF]. The homology protein crystal structure generated by Wichapong and co-worker was used (2010). AutoDock Tools (ADT) version 1.5.6 was used in the preparation of docking stimulation. The dimension of 74 × 43 × 64 in x,y,z coordination were employed as the grid box size to cover the protease active site.

3.6

The in vitro assays studies towards DENV-2 NS2B/NS3 protease

The in vitro assay preliminary screening studies for five top docked synthesized compounds were performed following method by Yusof et al. (2000). An enzymatic reaction mixture with a total volume of 100 µL was prepared in black 96 well plates. Each well in the black 96 well plates were consisting of 94.9 µL Tris-HCl buffer solution at pH 8.5, 3.1 µL of the enzyme, and 1 µL of synthesized compound (200 mg/L in DMSO). The mixtures were incubated and shook at 37 °C for 10 minutes.

After that, 1 µL substrate 10 mM in DMSO (Peptide Institute, Japan) was added into the mixture to initiate the reaction. The mixture was then incubated and shook for another 60 minutes at 37 °C using Ambient shaker incubatorSI-50 (Protech, Malaysia).

The assay was triplicated or more.

A selected compound was chosen for IC50 studies as presented in Figure 3.3.

The concentration series of a synthesized compound in DMSO (6.25 ppm, 12.5 ppm, 25 ppm, 50 ppm, 100 ppm, 200 ppm) were prepared and the in vitro assay was performed using the procedure as mention above. In the control experiment, without the addition of enzyme cocktail (M0), only 3.32 ± 0.38 % of seed oil was extracted. The highest percentage seed oil (6.26 ± 0.53 %) was extracted from M2 using a combination of 5 % HEL1 to 1.25 % X7 enzyme cocktails, followed by M4 (5.03 ± 0.28) using a combination of an equal ratio of HEL1 and X7 enzyme cocktails. Based on the results obtained, the amount of the extracted oil showed that the increment of enzyme cocktail X7 to HEL1 ratio used in the extraction was not proportional to the percentage of oil being extracted. This can be seen from the percentage oil being extracted do not show any significant increase by the increment of enzyme ratio after M2.

The results indicated that cellulase, proteinase, and pectinase present in the enzyme cocktails HEL1 and X7 could facilitate the breakdown of cellular and subcellular structures of the seed to promote oil release. The enzymes could have weakened the vegetal cells in the seed to help released the oil (Baby & Ranganathan, 2016). In a previous study, Latif and Anwar (2011) reported that the active mechanism of a selected enzyme cocktail during enzymatic aqueous extraction was the degradation of cellulose, pectin, and protein, thus, destructing the structural cell wall integrity of the seed cells [START_REF] Jiao | Microwave-assisted aqueous enzymatic extraction of oil from pumpkin seeds and evaluation of its physicochemical properties, fatty acid compositions and antioxidant activities[END_REF]. The cross-sections of immature seeds of M. charantia and the shape of cotyledon was presented in Figure 4.1.

As depicted in Figure 4.1(a) above, the M. charantia seed were built up off one outer layer called aril and five distinct layers made up a cotyledon. The first layer was epidermis which consists of a single layer of prismatic palisade cells. The second layer was hypodermis, a tissue made up of four layers of small, isodiametric, tightly closed sclerenchymatized cells, without intercellular spaces. The third layer was sclerenchyma that consists of exceedingly thick-walled cells. The fourth layer was made up of layers of spongy parenchyma called aerenchyma. The last layer was a parenchyma cells, containing chlorophyll, forms the inner tissue, called chlorenchyma ( [START_REF] Giuliani | Micromorphology and anatomy of fruits and seeds of bitter melon (Momordica charantia L., Cucurbitaceae)[END_REF].

All the layer made up a leaf-like shaped cotyledon cell, which contained abundant lipophilic cellular droplets, and oil and protein granules as depicted in Figure 4.1(b). The plant cell walls are a complex structure which mainly made of cellulose, hemicellulose, lignin and protein in addition to pectin [START_REF] Carpita | Structural models of primary cell walls in flowering plants: Consistency of molecular structure with the physical properties of the walls during growth[END_REF][START_REF] Buckeridge | Mobilisation of storage cell wall polysaccharides in seeds[END_REF][START_REF] Waldron | Plant cell walls and food quality[END_REF][START_REF] Park | A revised architecture of primary cell walls based on biomechanical changes induced by substrate-specific endoglucanases[END_REF][START_REF] Soto | Architecture, structure and chemistry of plant cell walls and their constituents[END_REF]. Hy-hypodermis; Sc-sclerenchyma [START_REF] Giuliani | Micromorphology and anatomy of fruits and seeds of bitter melon (Momordica charantia L., Cucurbitaceae)[END_REF].

The hydrolytic enzyme such as cellulases, hemicellulases, and pectinases in the AEE is to break the structure of cotyledon cell walls [START_REF] Adler-Nissen | Enzymatic Hydrolysis of Proteins for Increased Solubility[END_REF][START_REF] Giovane | Pectin methylesterase inhibitor[END_REF][START_REF] Ghosh | Enzymatic hydrolysis of whey and its analysis[END_REF]. The enzyme made the cell walls structure more permeable [START_REF] Rosenthal | Aqueous and enzymatic processes for edible oil extraction[END_REF]. In addition, the incorporation of enzymes may cleave the protein networks of the cotyledon cells and oleosin-based membranes that surround the lipid bodies of the seed cell wall resulting in oil released (Kamatou & Viljoen, 2017;[START_REF] Baharum | Analysis of the chemical composition of the essential oil of Polygonum minus Huds. Using two-dimensional gas chromatography-time-of-flight mass spectrometry (GC-TOF MS)[END_REF][START_REF] Kalia | Using Enzymes for Oil Recovery from Edible Seeds[END_REF].

The analysis of M. Charantia seed oil

The extracted seed oil samples (M0, M1, M2, M3, and M4) were subjected to derivatisation into FAMEs before identification of their biochemical compositions by GC-FID since the FAMEs are more volatile and more easily analysed. GC-FID has been widely used in the separation and detection of FAMEs. The GC chromatogram revealed the presence of some common and non-essential FAMEs, as shown in Table 4.2. Seven known fatty acids were identified. Three unknown fatty acids were also detected. However, three unknown peaks of free fatty acid were observed from the chromatogram that is co-eluting almost at the same time. It is suggested that some compounds may co-elute more significant in size and elute together at the end during separation (Kamatou & Viljeon, 2017;[START_REF] Baharum | Analysis of the chemical composition of the essential oil of Polygonum minus Huds. Using two-dimensional gas chromatography-time-of-flight mass spectrometry (GC-TOF MS)[END_REF]. Based on the composition of M. charantia seed oil (Table 4.2), the percentage of elaidic and palmitic acids increased with the introduction of the enzyme cocktails.

In the control experiment (M0), only 0.47 % of elaidic acid was present in the extracted oil. With the introduction of the enzyme cocktails, the percentage of elaidic acid increased to 8.06%, 20.42%, 28.06%, and 29.63% for M1, M2, M3, and M4, respectively. The presence of the enzyme cocktails showed a similar trend for palmitic acid, albeit lesser increment.

The oil release could be due to the presence of cellulase in the enzyme cocktail, which disrupted other polysaccharide constituents in the seed cell wall. The cellulase presence in HEL1 and X7 promoted an efficient cellulose degradation. The cellulase mechanism, described as the cleavage of internal glucosidic bonds within an unbroken glucan chain by endoglucanase enzyme, could result in the creation of a non-reducing chain of cellobiohydrolase substrate, cleaving the cellobiose dimers from the glucan chain that was released in the solution [START_REF] White | Chapter 23: Visualisation of Cellulase and Cellulose Dedegradation[END_REF]. Furthermore, β-glucosidase split the cellobiose dimer into glucose monomer, concluding the cellulose hydrolysis process [START_REF] White | Enzymatic hydrolysis of cellulose: Visual characterization of the process[END_REF]. The combination of enzymes played a dominant effect in disrupting the seed cell walls, which was also reported in other findings (Wu et al., 2017).

These results also showed that the oil composition differed with different X7 enzyme ratios. In M4, the percentage distribution of elaidic acid, vaccenic acid, palmitic acid, stearidonic acid, and paullinic acid increased with increasing ratio of X7. The results suggested that the enzyme cocktail X7 supported the reaction of enzyme cocktail HEL1 to hydrolyse the seed cellular structures as each enzyme cocktail consisted of different types of an enzyme mixture.

Besides, the biochemical composition of extracted oil also differ from the previous studies. Ali and co-worker extracted the seed oil with the compositions of palmatic acid (4.84 ± 0.13 %), stearic acid (20.21 ± 0.40 %), linoleic acid (6.37 ± 0.46 %) ,and eleostearic acid (53.22 ± 0.36 %) [START_REF] Ali | Characteristics of seed oils and nutritional compositions of seeds from different varieties of Momordica charantia Linn. cultivated in Bangladesh[END_REF]. Whilst, the seed oil being extracted using AEE have a higher amount of palmitic acid. In addition, the saturated fatty acid, stearic acid was only detected in M2. In addition, the omega-3 fatty acid detected by Ali and co-worker was eleostearic acid whilst the oil extracted using AEE was the highly unsaturated stearidonic acid.

This finding was in agreement with the findings of earlier researchers who showed that enzyme assisted extraction can lead to an increase in oil yield as well as slightly modification in the biochemical properties of the extracted oil (Zuorro et al., 2018). It can be concluded that the M. charantia seed oil obtained from the AEE exhibited slight changes in the integrity and percentage distribution of the active constituents.

Determinations of lignin, soluble sugars, and uronic acid of EFM

The lignin, soluble sugar, and uronic acid contents of the EFM were determined. Lignin consists of a very complex structure which is difficult to cleave (Liu et al., 2016). The lignin content of the EFM; M0, M1 (15.91 %) > M2 (15.06 %)

> M4 (14.71 %) > M3 (14.54 %) did not show any significant changes with the presence of enzymes in the AEE. Different types of soluble sugars and uronic acid were released and further analysed with HPAEC-PAD.

The main soluble sugar composition of EFM was glucose and rhamnose, respectively. No definite trend was observed between the percentage of enzyme ratios used with the percentage distribution of soluble sugars and uronic acid obtained. The results are presented in arabinose, rhamnose, mannose, and fucose) with the help of the enzyme cocktails [START_REF] Pedrolli | Pectin and Pectinases: Production, Characterization and Industrial Application of Microbial Pectinolytic Enzymes[END_REF]Segato et al., 2014;[START_REF] Zhong | Interactions of the complete cellobiohydrolase I from Trichodera reesei with microcrystalline cellulose Iβ[END_REF]. Despite significant knowledge in the synergism among cellulase and enzyme cocktail components, a full mechanistic understanding of lignocellulose hydrolysis by cellulolytic enzymes is still limited and needs further refinement (Nill et al., 2018).

The variation in the hemicellulose composition showed that as polysaccharides of simpler structure than cellulose, hemicellulose was also prone to breakdown with the presence of hemicellulase (Zhao et al., 2018).

The hemicellulase obtained were further analysed using FTIR spectroscopy as shown in Figure 4.2. The FTIR spectrum showed that there were differences in the peak intensity and between the absorption bands obtained. The absorption of 3272, 2927, 1628, 1416, 1239, and 1038 cm -1 peaks represented the characteristic absorption of hemicellulose [START_REF] Popescu | Characterization of fungal degraded lime wood by FT-IR and 2D IR correlation spectroscopy[END_REF]. the presence of Csp 3 -H bond stretching vibration. The band ~1737 cm -1 referred to the conjugated C=O stretching band of a carbonyl group, be attributed to the presence of the acetyl moiety in the hemicellulose. The absorption that appeared in ~1628 cm -1 is usually associated with the absorbed water [START_REF] Peng | Graded Ethanol Fractionation and Structural Characterization of Alkali-Extractable Hemicelluloses from Olea europaea L[END_REF]. A strong band that appeared at ~1038 cm -1 was assigned to C-O bending in sugar (Naidu et al., 2018).

The extracted hemicelluloses were then subjected to size exclusion chromatography analysis to evaluate the effects of the enzymes on their molecular mass distribution. Each extracted sample was observed to have slightly different molecular mass distribution trend. The difference in the molecular mass distribution trend could be due to the effects of different enzymes towards the enzymatic cleavage of cellulose chain that transformed the hemicelullose into a hemicellulose chains with different unit of sugar depends on the type of enzyme presence in the enzyme cocktails used.

The M0 had the highest weight average (Mw) of 447148-457148 followed by M4 (Mw: 36186-378116), M3 (Mw: 120339), M1 (Mw: 87865), and lastly M2 (Mw: 55915). These results indicated that the treatment degraded the macromolecular structure of hemicellulose to a noticeable extent with the incorporation of enzyme [START_REF] Izydorczyk | Cereal arabinoxylans: advances in structure and physicochemical properties[END_REF].

In addition, the high hemicellulose polydispersity (Mw/Mn) from M0 Mw/Mn of 1.15-7.17, to M2 Mw/Mn of 6.06, to M4 Mw/Mn of 1.9-21.72, to M1 Mw/Mn of 107.71, and lastly to M3 Mw/Mn of 284.30 indicated a wide molecular weight distribution which showed that the hemicellulose obtained was in a big polymer form that might be caused by hemicellulose chain aggregation [START_REF] Eremeeva | Size-exclusion chromatography of enzymatically treated cellulose and related polysaccharides: A review[END_REF]. 101

Based on the FTIR spectrum the introduction of enzyme does not affect the hemicellulose backbone structure despite difference molecular mass distribution was observed in SEC analysis. In addition, it can be concluded that the optimum seed samples and their enzyme cocktail ratios for the HEL1: X7 were found to be 5:1.25 as in M2.

The studies of M. charantia fruits flesh

In this study, the n-hexane, ethyl acetate, and methanol extract of M. charantia fruit underwent preliminary in vitro screening of the anti-dengue activity. The composition of the active extract was screen by using GC-MS.

Preparation of M. charantia fruit flesh extract

The M. charantia fruits were sun-dried prior to extraction as the drying process removed the water and halt any enzymatic activity present in the plant cell [START_REF] Mozhaev | Dengue incidence and the prevention and control program in Malaysia[END_REF]. This step was to minimize the hydrogen bonding interaction of water in the fruits with its metabolites that disrupted the solvent extraction process and affected the amount of solute been extracted. [START_REF] Brini | How Water's Properties Are Encoded in Its Molecular Structure and Energies[END_REF].

The drying temperature also played an essential role as Anwar and co-workers concluded that the drying temperature of below 60 °C did not adversely affect the total phenolic content of cauliflower extract [START_REF] Anwar | Effect of drying method and extraction solvent on the total phenolics and antioxidant activity of cauliflower (Brassica oleracea L.) Extracts[END_REF]. However, the total phenolic content of cauliflower extract decreased significantly when the drying temperature was set above 70 °C. Hence, the sun-dried temperature (31-39 °C) of M. charantia fruits preserved its chemicals composition.

The dried fruits were grounded into a course size before the extraction process to create more space for the solvent to penetrate the cells and extract the secondary metabolites. The grounded condition would increase the particle surface area and increased the solvent and fruit contact point [START_REF] Al-Sumri | Extraction of Date Palm Seed Oil by Soxhlet Apparatus[END_REF]. However, if the dried fruits being grounded too fine, it will cause the excessive absorption of solute in solid and difficulty in subsequent filtration [START_REF] Zhang | Techniques for extraction and isolation of natural products: A comprehensive review[END_REF].

The solvent-to-solid ratio of 10:1 was used. The solvent-to-solid ratio also played an essential role in the extraction process. The higher solvent-to-solid ratio will increase the concentration gradient and diffusion rate. Hence, the equilibrium constant will be affected with the increments of the extractive yield. However, a high solventto-solid ratio will cause excessive extraction solvent, and a longer time is required for concentration using a rotary evaporator [START_REF] Predescu | The influence of solid-to-solvent ratio and extraction method on total phenolic content, flavonoid content and antioxidant properties of some ethanolic plant extracts[END_REF].

The mixtures were then shaken and stirred to facilitate the extraction process and enhanced its diffusivity [START_REF] Mohdaly | Methods for the extraction of metabolites from plant tissues[END_REF]. The extraction process was carried out at room temperature. Even though high temperature increased the solubility and diffusivity, too high temperature may cause the loss of solvents, the extraction of the undesirable impurities, and the decomposition of thermolabile compounds, such as anthocyanins [START_REF] Seidel | Initial and Bulk Extraction[END_REF][START_REF] Selvamuthukumaran | Recent advances in extraction of antioxidants from plant by-products processing industries[END_REF].

The quantity of the extract is corresponding to the type of solvents being used base on the law of similarity and intermiscibility. Solvents with a polarity value near to the polarity of the solute are likely to perform better and vice versa [START_REF] Burke | Solubility Parameters : Theory and Application[END_REF].

The quantity of extractive material extracted from the M. charantia fruits with the use of non-polar to polar solvent implies the variation of phytochemical compounds [START_REF] Muhamad | Influence of solvent polarity and conditions on extraction of antioxidant, flavonoids and phenolic content from Averrhoa bilimbi[END_REF].

As plants contained a variety of bioactive compounds with difference solubility properties in a different solvent, the optimal solvent used for extraction depends on the particular plant materials, and the compounds that are to be isolated (Truong et al., 2019;[START_REF] Mahdi-Pour | Antioxidant activity of methanol extracts of different parts of Lantana camara[END_REF][START_REF] Masoko | Chapter 2: Extraction of plant material[END_REF]. The use of a non-polar solvent showed the lowest amount of extractive being extract with the percentage of yield of 0.49 ± 0.03 % followed by extractive extract by ethyl acetate, 1.48 ± 0.11 %, and methanol, 3.4 7±0 .04 %, as presented in Table 4.4. !"#$%& () 0+4 )+5#&6 )7"6% 56"0 (2$) × 100%

4.2.1(a) Anti-dengue screening of M. charantia fruits flesh extract

In the M. charantia fruit flesh extracts anti-dengue screening study, the extracts were incorporated into the protease reaction to a final concentration of 200 ppm. Each extract was tested in triplicate and each set was repeated twice.

In this study, the ethyl acetate extract showed an highest inhibition activity.

The methanol extract showed the least inhibitory activity towards DENV-2 NS2B/NS3

protease with only 10.37 ± 1.13 %. The semi-polar M. charantia extract from ethyl acetate showed the highest DENV-2 NS2B/NS3 protease inhibitory activity of 38.11 ± 1.06 %. The difference in the inhibitory activity obtained was suggested due to the type of bioactive compounds being extracted in a different type of solvent [START_REF] Altemimi | Phytochemicals: Extraction, Isolation, and Identification of Bioactive Compounds from Plant Extracts[END_REF]Farahmandfar et al., 2019). The result of this preliminary anti-dengue screening is shown in Table 4.5. The ethyl acetate extract with the most DENV-2 NS2B/NS3 protease inhibitory activity underwent screening using GC-MS to identify its bioactive components.

4.2.1(b) The GC-MS screening of M. charantia fruit flesh extract

In choosing ethyl acetate as one of the solvents utilized during the extraction process, the factor such as its polarity index of 4.4 P´ was taken into consideration. As a semi-polar solvent was used, it was anticipated that ethyl acetate extract might contain many bioactive compounds such as alkaloids, phenols, steroids, flavonoids, saponins, and terpenoids [START_REF] Pham | Investigation of Biological Activities of Wild Bitter (Momordica charantia Linn. Var. Abbreviata Ser.)[END_REF][START_REF] Harlita | The Antibacterial Activity of Dayak Onion (Eleutherine palmifolia (L.) Merr) toward Pathogenic Bacteria[END_REF][START_REF] Arega | Phytochemical Studies of the Ethyl Acetate Extract of the Fruit of Piper capense[END_REF]Jia et al., 2017).

The ethyl acetate extract could be analysed using either LC-MS or GC-MS. In this study, the most widely used techniques GC-MS was used to identify the bioactive compounds presence in the ethyl acetate extract of M. charantia fruits flesh. GC-MS is exclusively used for the analysis of esters, fatty acids, alcohols, aldehydes, terpenes.

Intrinsically, with the use of the flame ionization detector and the electron capture detector (which have very high sensitivities) gas chromatography can quantitatively determine materials present at very low concentrations [START_REF] Al-Rubaye | A review: Uses of gas chromatography-mass spectrometry (GC-MS) technique for analysis of bioactive natural compounds of some plants[END_REF]. ) . Whereas, the remaining compounds were present in a trace amount with the peak area less than 1 % including gallic acid (rt. 5.017 min, 0.54 %).

As been observed from the chromatogram (Figure 4.4), the baseline are not straight and there is high bleed, suggested due to leak in the system or oxidation of the stationary phase of the column despite the sample being injected for more than three times. However, this bleed does not visible as peaks and not affecting the qualitative analysis [START_REF] Rood | Gas chromatography problem solving and troubleshooting[END_REF][START_REF] Stein | An integrated method for spectrum extraction and compound identification from gas chromatography/mass spectrometry data[END_REF]. Some peaks seem to be broad suggested due to the incomplete vaporisation of the analyte despite using 5 °C per minutes temperature gradient as the extract usually containing a complex mixtures of volatile compounds [START_REF] Vial | Combination of dynamic time warping and multivariate analysis for the comparison of comprehensive two-dimensional gas chromatograms: application to plant extracts[END_REF][START_REF] Hites | Gas Chromatography Mass Spectroscopy[END_REF].

It shall be noted that the compounds present, their identities, and the number of identified compounds were not absolute and limited as per material only utilized in this study only. Although the peak of gallic acid was observed in less 1 %. Jia and coworker (2017), reported that gallic acid to be the main phenolic acids presence in M. charantia fruit flesh.

In addition, the dengue fever treatment with a pentaherbal medicine;

Denguenil Vati, significantly recovered the virus by increasing the total platelet numbers and total red blood cell count. The high performance liquid chromatography (HPLC) analysis detected gallic acid as one of the Denguenil formulation [START_REF] Balkrishna | Validation of a novel zebrafish model of dengue virus (DENV-3) pathology using the Penta herbal medicine Denguenil Vati[END_REF].

Besides, the gallic acid and its derivatives appeared to be the most readily available chemical that is easily found in the laboratory [START_REF] Ow | Gallic Acid and Gallic Acid Derivatives: Effects on Drug Metabolizing Enzymes[END_REF][START_REF] Choubey | Medicinal importance of gallic acid and its ester derivatives: a patent review[END_REF][START_REF] Belin | Synthetic gallic acid derivatives as models for a comprehensive study of antioxidant activity[END_REF]. However, among all the compounds, gallic acid is the ideal compound be a structure analogue for hemisynthesis. Hence, this study focused on the synthesis of therapeutically promising gallic acid derivatives as the scaffolder to produce a hit-to-led compounds with anti-dengue properties. Sulfuric acid is chosen as the catalyst to facilitate the nucleophilic attack of the alcohol at the carbonyl carbon of the carboxylic acid [START_REF] Ahmad | Name Reactions of Functional Group Transformations[END_REF]. Besides, the reaction was carried out in the excess solvent to drive the equilibrium shifted to the product to obtained more ester by the Le Chatelier's Principle [START_REF] Sandler | 10 -Esters[END_REF].

The synthesized compound 85 afforded in a good yield of 85.99 % as a colourless crystal with a melting point of 50-53 °C. Schejbal and co-worker (2014) reported a colourless crystal been synthesized with the melting point of 54-55 °C. The compound was characterized using, FTIR, 1 H-NMR, and 13 C-NMR.

4.3.1(a) The characterization of ethyl 3,4,5-trimethoxybenzoate, 85

The FTIR was used to characterize the structure of chemical compound as it has the ability to identify the functional group of the chemical compound. The comparison between the FTIR spectrum of starting material, compound 84 and synthesized compound 85 were depicted in In the preparation of compound 85, the starting material 84 underwent esterification process, which led to the removal of hydrogen and the formation of ester group resulting in the appearance of a sharp α,β-unsaturated ester C=O band at 111 1715 cm -1 , which replace the C=O acid band at 1698 cm -1 of compound 84. Besides, the broad -OH absorption at ~3020 cm -1 disappeared indicating that the esterification step is completed. In addition, the O-C band can be observed at the 1181 cm -1 and 1227 cm -1 [START_REF] Pavia | Introduction to spectroscopy[END_REF].

In compound 84, the resonance between the carbonyl group with it neighbouring oxygen atom of -OH acid group resulted in weakening of the C=O vibration. Hence the wavenumber/frequency of C=O acid decreased compared with the wavenumber/frequency of C=O ester. 113 obtained were in agreement with the previous studies [START_REF] Schejbal | Acylfunctionalized molybdenum compounds [(η3-C3H5)(η5-Cp')Mo(CO)2]: An experimental study including the X-ray structure of a rare endo conformer[END_REF][START_REF] Verma | Flavone-based novel antidiabetic and antidyslipidemic agents[END_REF]Li et al., 2017). The compound 85 underwent further nucleophilic acyl substitution reaction with hydrazine monohydrate to obtain the desired compound 86. In the first step, the prior synthesized compound 85 carbonyl carbon is attacked by the lone pairs from the nitrogen atom of the hydrazine molecule to form a tetrahedral intermediate. The ethoxy group attached to the new tetrahedral carbon atom leaved with the negative charge as it is a weaker base than the amino group. The extra hydrogen atom at the hydrazine leaved with a positive charge [START_REF] Smith | March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure[END_REF][START_REF] Bruice | Part 6: Carbonyl Compounds and Amines[END_REF], led to the The synthesized compound 86, yielding a good result of 85.31 % as a light brown crystalline solid with a melting point of 157-160 °C. Kamal and co-worker (2014) reported that compound 86 was obtained as a crystalline solid with a melting point of 158-160 °C. The compound was characterized using, FTIR, 1 H-NMR, and 13 C-NMR.

4.3.2(a) The characterization 3,4,5-trimethoxybenzohydrazide, 86

The important absorption bands in IR spectrum of compound 86 were observed at 3364 (N-H), 3319 (N-H), 1659 (amide C=O), 1385(C-N). The major difference between the two FTIR spectrum are the appearance of primary and secondary amine peak in compound 86 at 3364 cm -1 and 3319 cm -1 . Besides, the disappearance of a In addition, only two signals are observed at the range δc 56.00-62.00, which represent the carbon attached to a methoxy group signal in the 13 C-NMR spectrum of compound 86. Besides, the methyl group signal at δc 14.64 disappeared. This indicate the success of acylation of amine process. The characterization results of compound 86 were in agreement with the previous studies [START_REF] Kamal | Synthesis, biological evaluation, and molecular modeling of (E)-2-aryl-5-styryl-1,3,4-oxadiazole derivatives as acetylcholine esterase inhibitors[END_REF][START_REF] Ahmed | New 1,2,4-triazole-Chalcone hybrids induce Caspase-3 dependent apoptosis in A549 human lung adenocarcinoma cells[END_REF][START_REF] Murty | Synthesis and biological evaluation of novel resver atroloxadiazole hybrid heterocycles as potential antiproliferative agent[END_REF].

The synthesis of benzylidene indanone derivatives, 89a-t

In step 3, the benzylidene indanone derivatives [89a-t] were synthesized using aldol condensation method. The aromatic aldehyde were reacted with aromatic ketone in the presence of potassium hydroxide as a basic catalyst. Its required an aldehyde or ketone that contains at least one α-hydrogen. The α-carbon became nucleophilic when it is deprotonated by a base. The carbonyl carbon is electrophilic. The two oppositely charged species were bring together by Coulomb Law to form a C-C bond [START_REF] Bruice | Part 6: Carbonyl Compounds and Amines[END_REF].

The reaction start with the deprotonation of aromatic ketone [88a-b] by the hydroxide ion to generate an enolate ion. The enolate was formed at the indanone as the α-hydrogen exhibited acidity properties in the presence of a carbonyl group. Hence, strong base was chosen as the catalyst in the reaction to produce the hydroxide ion that attacked the acidic α-hydrogen in the ketone, which led to the formation of reactive enolate ion [START_REF] Clayden | Organic chemistry[END_REF]. The enolate ion was able to undergo an electron rearrangement to obtain a more stable enolate ion. After that, the enolate ion underwent a nucleophilic addition of the enolate ion to the carbonyl group of the aromatic aldehyde [87a-j]. The electron rich carbanion acted as a nucleophile and attacked the electron-deficient carbon located at the carbonyl group of the aldehyde by nucleophilic addition reaction. The alkoxide ion was formed. The alkoxide ion underwent protonation by extracting the proton from the methanol, resulting in the aldol product formation. Since, this reaction was carried out under reflux, the heated reaction mixture of ß-hydroxyl carbonyl underwent an E2 reaction to form a double bond between the α and ß position [START_REF]Organic Chemistry Practice Problems and Problem Sets: Aldehyde and ketone[END_REF][START_REF] Vollhardt | Organic chemistry structure and function[END_REF]. The reactions mechanism as depicted in Figure 4.13. Table 4.7 summarized the percentage of yield and the physical properties of compounds 89a-t. This reaction was a water sensitive reaction, where water was eliminated as one of the end product. The used of ethanolic potassium hydroxide avoid the presence of water that facilitate the reaction to shift towards the left (the starting material) due to the decrease in dehydration reaction rate with the presence of water [START_REF] Sandler | 10 -Esters[END_REF].

O R R OH H H O R R H O R R H enolate ion O R R H O H R 1 R 2 R 3 87a-j 88a-b alkoxide ion O R R O H H R 1 R 2 R 3 OCH 3 H ß-hydroxyl carbonyl O R R OH H R 1 R 2 R 3 OH E 2 O R R R 1 R 2 R 3 89a-t OH + + H 2 O heat
The reactions took up to 24 hours to be completed. It is suggested that aldehyde is used as the electron-donating substituent, which has a less electrophilic character.

Hence, it slowed down the rate of reaction [START_REF] Ali | Antimycobacterial Activity: Synthesis and Biological Evaluation of Novel Substituted (3E,5E)-3,5-diarylidene-1phenethylpiperidine-4-one Derivatives[END_REF]. Despite the prolonged reaction time, the synthesized benzylidene indanone derivatives, 89a-t afforded in a good yield in the range of 60-95 %.

The compounds were characterized using, FTIR, 1 H-NMR, and 13 C-NMR. The compounds with a suitable crystal size and form were submitted for X-ray crystallography analysis.

4.3.3(a) The characterization of benzylidene indanone derivatives, 89a-t

There are four key FTIR absorption bands in compounds 89a-t; the Csp 3 -H bonds vibration at the range of 2910-2920 cm -1 , the strong C=O stretching at 1690-1695 cm -1 , the -CH aromatic stretching at 1580-1620 cm -1 and the C-H out-ofplane bending vibration of aromatic ring substituents at 600-900 cm -1 (Table 4.8).

Compound 89b is the representative compound from the benzylidene indanone derivatives. The FTIR spectrum of compound 89b (Figure 4.15) displayed an absorption band at 2914 cm -1 , which assigned to the Csp 3 -H bonds vibration. The strong peak of conjugated C=O stretching indicated the carbonyl moiety of ketone was observed at 1694 cm -1 . In addition, the absorption of -CH aromatic stretching can be seen at 1575-1629 cm -1 . Apart from that, the absorption at 692-836 cm -1 located at the fingerprint region can be attributed to the C-H out-of-plane bending vibration of aromatic ring [START_REF] Wang | An infrared study of the out-of-plane C-H bending vibrations of monosubstituted naphthalenes[END_REF]. 4.8.

In compound 89b, a singlet peak appear at the downfield region, δH 7.51 attributed to the -C=CH proton of H-1' that formed during the condensation of indanone with anisaldehyde. A broad methylene peak of H-3 appeared at δH 4.05 and the sharp singlet peak of H-8' was observed at δH 3.82 attributed to -OCH3 in the 1 H NMR spectrum of compound 89b as depicted in Figure 4.15 [START_REF] Sabri | Synthesis, characterisation and vasolidation properties of indanone-based chalcones[END_REF]. In 122 addition, the doublet peaks at δH 7.055-7.073 with J = 9.0 attributed to the protonproton coupling in aromatic ring located at the benzene moiety of H-3' and H-7'. The triplet peaks were observed at δH 7.455-7.484 with J = 9.0 attributed to H-4. While multiplet peaks at δH 7. 657-7.709 and 7.736-7.780 that attributed H-4', H-6', H-5, H-6, and H-7. The triplet and multiplet peaks formed were suggested due to the proton of indanone group of is influenced by the spinning arrangement of the protons in the adjacent aromatic aldehyde group (Lampman et al., 2010). The spectroscopy data showed that the compounds 89a-t have been formed.

The compounds were recrystallized to obtain crystal. Compounds 89k, and 89p with a sturdy and big crystal size were submitted for x-ray crystallography study. The x-ray crystallography study showed that the crystal structures obtained proven the formation of compounds 89k, and 89p with the atom were located in accordance with the proposed structure. Besides, the crystal structures showed that the compound obtained were in the trans configuration. The formation of pyrazolyl substituted indanone derivatives, 90a-t involved a formation of N heterocycles of compound 86 and benzylidene indanone derivatives, 89a-t. The reaction associated with the formation of N heterocycles using acetic acid as a catalyst [START_REF] Ali | Discovery of novel methanone derivatives acting as antimycobacterial agents[END_REF].

The reaction mechanism started with the protonation of benzylidene indanone derivatives, 89a-t with the acetic acid. The hydrogen ion attached to the oxygen with the lone pairs located at the carbonyl group to form a carbocation. The carbocation was than underwent rearrangement to form a more stable state through structural reorganisation shifts within the molecule to form its structural isomer [START_REF] Vogel | Carbocation Chemistry[END_REF].

The carbocation was then attacked by the electronegative nucleophile located [START_REF] Dinnocenzo | Deprotonation of Tertiary Amine Cation Radicals. A Direct Experimental Approach[END_REF]. Consequently, the double bond formed between C-10 and N-1. Thus, the compound was rearranged to a more stable form by delocalization of π-bond. Next, acidic hydrogen from the amine moiety was eliminated as the nitrogen need to form three covalent bonds to achieve its stable configuration. Hence, the acetic acid catalyst was regenerated (Clayden et al., 2001).

The mechanism was presented in Figure 4.19.

Figure 4.19:

The formation of compounds 90a-t

R R O H O O 89a-t R R O H O O + R 1 R 2 R 2 R 1 R 2 R 3 R R O H R 1 R 2 R 3 R R O H R 1 R 2 R 3 carbocation isomer R R O H H 2 N NH O O O O R 1 R 2 R 3 86 R R O H H 2 N H N O O O O R 1 R 2 R 3 R R O H N H N O O O O R 1 R 2 R 3 H intermediate cation H R R O H N H N O O O O R 1 R 2 R 3 H H R R N H N O O O O R 1 R 2 R 3 H R R NH HN R 1 R 2 R 3 O O O O resonance structure -H 2 O R R NH N R 1 R 2 R 3 O O O O H 7 6 5 12 9 8 4 11 10 3 N 2 N 1 1' R R 3' 4' 5' 6' R 3 1''' O 2' 1'' 6'' 5'' 4'' 3'' 2'' O 7'' O O 9'' 90a-t R 1 R 2 HO O +
The compounds 90a-t afforded an appropriated amount of product being synthesized with the percentage of yield in the range of 40-85 % and the melting point in the range of 175-215 ºC. Table 4.9 summarised the percentage of yield and the physical properties of compounds 90a-t.

The percentage yields of the obtained products were in the range of 40-85 %.

It is suggested that some of the product might be loss during the sample recovery, such as during pouring, filtering or column chromatography, resulted in the low yield. In addition, the low products percentage of yield could be due to an incomplete reactions, in which some of the reactants do not react to form the product.

It were observed that the percentage yield of compounds 90h, 90j, 90n, 90s, and 90t were bellow 50 %. Based on observation, the compounds with lower yield tend to have a substituents other than hydrogen at R1, R2, and R3 in the benzylidene moiety, making it a bulk compounds. Bulkier substituents significantly slows the reaction because larger groups effectively increased the steric repulsion with the incoming nucleophile. Thus, the reaction took longer time (Liu et al., 2010a). Figure 4.20 depicted the structure of compounds with the percentage yield lower than 50 %. and 89a-t. This reaction involved a ring formation which transformed the primary and secondary amine in compound 86 to tertiary amine in compound 90a-t. Hence, two -NH and -NH2 absorption band at 3364 cm -1 and 3319 cm -1 will be absence in compound 90a-t FTIR spectra.

The FTIR spectrum of compound 90b manifest an absorption band at 2938 cm -1 attributed to the Csp 3 -H bonds vibration. In compound 90b, the C=O absorption strength at 1697 cm -1 decreased compared to the C=O absorption strength of compound 78b. It is suggested due to the loss of conjugation from one side at the

N N O O O N O N N O O O O NO 2 O 79h 79j N N O O O Cl O O O 79n N N O O O O O N N O O O O NO 2 O O O O O O 79s 79t 90j 90h 90n 90s 90t
48.55 % 49.62 % 44. 28 % 47.43 % 41.45 % 133 C=O moiety in compound 90b is located next to amine moiety [START_REF] Hassaneen | Facial regioselective synthesis of novel bioactive spiropyrrolidine/pyrrolizineoxindole derivatives via a three components reaction as potential antimicrobial agents[END_REF]& Sheat et al., 2018). While, the strong out of plane C-H bending appeared at 820 cm - 1 indicated that the present of para disubstituted ring in compound 90b (Lampman et al., 2010).

The comparison between the FTIR spectrum of compound 86, 89b and synthesized compound 90b were presented in Figure 4.21 and the other absorption band for compounds 90a-t were tabulated in Table 4.10. The LC-MS spectrum ( The structural elucidation of compounds 90a-t were accomplished by using one and two dimensional NMR spectroscopic study. In the main difference between the 1 H NMR spectrum of compounds 90a-t and the reactant compound 86 was the disappearance of N-H and N-H2 shift. There is no chemical shifts observed at δH 9.744 and δH 4.470 indicating the success in the cyclization of amines from primary and secondary amine into tertiary amine. The 1 H and 13 C chemical shift of the compounds were reported in Table 4.11. 4.11: The chemical shifts of 1 H NMR and 13 C NMR spectrum of compounds 90a-t.

Compound

Chemical shift NMR (δ, ppm)

H-3 C-3 C-11 C-4 C-1' C-1'' C-1''' 90a
9 8 7 3 2 1 N 1' N 7" 6' 5' 4' 3' 2' O O O O 12 10 8" 1"' 6" 1" 2" 3" 4" 5" 9" 11 R R R 1 9' R 2 R 3 137
Table 4.11: The chemical shifts of 1 H NMR and 13 C NMR spectra of compounds 90a-t (continue).

Compound

Chemical shift NMR (δ, ppm)

H-3 C-3 C-11 C-4 C-1' C-1'' C-1''' 90f
9 8 7 3 2 1 N 1' N 7" 6' 5' 4' 3' 2' O O O O 12 10 8" 1"' 6" 1" 2" 3" 4" 5" 9" 11 R R R 1 9' R 2 R 3
Table 4.11: The chemical shifts of 1 H NMR and 13 C NMR spectra of compounds 90a-t (continue).

Compound

Chemical shift NMR (δ, ppm)

H-3 C-3 C-11 C-4 C-1' C-1'' C-1''' 90k
N 1' N 7" 6' 5' 4' 3' 2' O O O O 12 10 8" 1"' 6" 1" 2" 3" 4" 5" 9" 11 R R R 1 9' R 2 R 3 139
Table 4.11: The chemical shifts of 1 H NMR and 13 C NMR spectra of compounds 90a-t(continue).

Compound

Chemical shift NMR (δ, ppm) C=N was assigned to δC 168.20 [START_REF] Pavia | Introduction to spectroscopy[END_REF].

H-3 C-3 C-11 C-4 C-1' C-1'' C-1''' 90p
9 8 7 3 2 1 N 1' N 7" 6' 5' 4' 3' 2' O O O O 12 10 8" 1"' 6" 1" 2" 3" 4" 5" 9" 11 R R R 1 9' R 2 R 3
However, important signal of C-3 in the range of δC 70-80 which indicated the attachment of compound 86 and compound 89b to form compound 90b was not observed in the 13 C NMR spectrum suggested that the signal was overlapped with the CDCl3 signal that appeared at δC = 77 ppm.

In 135 DEPT NMR spectrum (Figure 4.26), the C-3 signal of compound 90b appeared at δC 77.24. In addition, the spectrum showed the signal at δC 32.51 which belonged to the aromatic carbon (C-4).

All prominent signal that indicated the formation of compound 90b have been assigned and in accordance with the proposed structure. However, due to the presence of more than one aromatic ring in compound 90b, the signals of carbons and protons for aromatic region were not assigned in details. The in silico studies was conducted for all the synthesized compounds with DENV-2 NS2B/NS3 protease, and its binding mode and conformation were determined. AutoDock4.2 procedure was used extensively in predicting the interaction of ligands with the bio-macromolecular target.

The ligand preparation

In this study, a total of 20 compounds [90a-t] were prepared for docking. The 3D structure of the synthesized compounds was built initially starting with the construction of 2D structures (.mol) using MarvinSketch 19.4.0 (ChemAxon, 2019).

The 2D structures were then underwent geometry optimisation and energy minimisation using a molecular mechanic method with MM+ force field embedded in HyperChem pro 8.0 software (www.hyper.com) to obtain the 3D structure in .pdb format. Lastly, the AutoDock suited, raccoon.py was utilized to convert the .pdb file into a .pdbqt formatted ligand which required by the AutoDock4.2 [START_REF] Forli | Computational protein-ligand docking and virtual drug screening with the AutoDock suite[END_REF].

The energy minimisation steps are necessary to reduce the gradient of the ligand potential energy to zero. Besides, this refined the ligand conformations in the crystal structures to best fit the electron density map [START_REF] Wang | Preference of small molecules for local minimum conformations when binding to proteins[END_REF]. All molecules were in equilibrium bond distance of 1.242 Å after geometry optimisation resulting in an ideal structure behaviour. Besides, the equilibrium bond distance increased structure stability [START_REF] Brown | Using HyperChem[END_REF].

The DENV-2 NS2B/NS3 protease preparation

In this study, the homology protein crystal structure generated by Wichapong and co-worker were used. Some crystal structures were reported to have some missing amino acid residue or cofactor that hinder a precise interaction analysis of a complete NS2B/NS3 protein and potential inhibitors [START_REF] Erbel | Structural basis for the activation of flaviviral NS3 proteases from dengue and West Nile virus[END_REF]& Wichapong et al., 2010). In addition, the available crystal structure 2FOM does not show a direct Cterminal domain of DENV-2 NS2B interaction with the substrate-binding site of the NS3 protease, which made it a non-suitable target structure for analysing the binding interaction with a potential inhibitor [START_REF] Aleshin | Structural evidence for regulation and specificity of flaviviral proteases and evolution of the Flaviviridae fold[END_REF]. Hence, the use of homology model, a reliable 3D model generated by from its amino acid sequence is acceptable [START_REF] Vyas | Homology modeling a fast tool for drug discovery: Current perspectives[END_REF].

In the preparation for docking simulation, only protease was retained as a part of the macromolecule file, while the Bz-Nle-Lys-Arg-Arg-H was extracted and saved as a positive control ligand. The crystal water molecules were removed from the macromolecule file to ease the simulation process and to clear the binding site from possible water molecules that can distort the search [START_REF] Wong | Accounting for water molecules in drug design[END_REF]. After that, the ADT was utilized in adding the hydrogen atoms into the macromolecule system, calculated the charge and merged the non-polar hydrogens with the carbon atoms as some hydrogen atom might be missing in the crystal structure. In addition, the added hydrogen atom allowed the establishment of hydrogen bonds that may be present between the macromolecule and the potential inhibitor or ligand tested in accordance with the docking algorithm [START_REF] Ferreira | Molecular docking and structure-based drug design strategies[END_REF]. The macromolecule was saved in .pdbqt format prior to docking simulation.

The positive control docking

In positive control docking, the Bz-Nle-Lys-Arg-Arg-H was extracted and re-docked into the binding site in order to evaluate the success of the docking method in reproducing the experimentally-known complex. In this study, the centre of the grid box was determined using the centre of Bz-Nle-Lys-Arg-Arg-H ligand at 22.781, 43.373, -0.358 in x,y,z coordinates, a grid box size of 74 × 43 × 64 and spacing of 0.375 Å to cover the protease active site. The Lamarckian genetic algorithm was employed in Autodock 4.2 with a covalent map parameter was used during positive control docking simulation to constrain the molecular geometry of the peptide inhibitor [START_REF] Morris | AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility[END_REF][START_REF] Bernama ; Bianco | Covalent docking using autodock: Two-point attractor and flexible side chain methods[END_REF].

The re-docked was carried out successfully with the root mean square deviation (RMSD) value of 1.23 Å. Hevener and co-worker (2009) The scores were ranked by the most active down to the least. The activity was based on the binding interaction, in which, the more negative the score, the stronger the binding. The intermolecular interaction between the inhibitor (synthesized compound)

and the surrounding amino acid residue at the binding pocket of DENV-2 NS2B/NS3

protease played an important role in determining the NS2B/NS3pro-mediated cleavage of the capsid, intNS3, 2A/2B, 4B/5, 3/4A and 2B/3 substrates. Two criteria probed the strength of the binding interaction specifically; (i) a proton donor-acceptor distance of ≤3.5 Å, and (ii) a donor-H acceptor bond angle of ≥120 º [START_REF] Yotmanee | Binding specificity of polypeptide substrates in NS2B/NS3pro serine protease of dengue virus type 2: A molecular dynamics Study[END_REF][START_REF] Li | Strength of hydrogen bond network takes crucial roles in the dissociation process of inhibitors from the hiv-1 protease binding pocket[END_REF].

The hydrogen bonding was the most stable and strongest intermolecular forces.

Hence, the stability of the hydrogen bond network between the synthesized compounds and the amino acid residues dominates the binding strength and therefore affecting the rate of inhibitors [START_REF] Lin | Dengue Protease Substrate Recognition: Binding of the Prime Side[END_REF]. It is suggested that the shorter the hydrogen bond distance, the stronger the hydrogen bonding interaction. Whilst, the donor-H acceptor bond angle of ≥120º is required for the bond to receive favourable energy and avoids including the non-physical H-bonds with angles near 90 º [START_REF] Thorpe | Flexibility in Biomolecules[END_REF].

The synthesized compound 90p showed eight hydrogen bond interactions, one π-π stacking interaction, and one π-alkyl interaction with the amino acid residue in the DENV-2 NS2B/NS3 protease. It was noted that the interactions formed at this site were mostly contributed from the NS3 residues with different interaction type and strengths. The residue NS3 protein is essential for viral replication and maturation of virions. Hence, more interaction with the residue suggested giving more promising result in inhibitory activity (Yin et al., 2006).

The presence of the aromatic group located at the benzylidene moiety in the synthesized compound 90p enhances the possibilities of van der Waals interaction.

The π-π stacking interaction and π-alkyl interaction between the His51 and the benzylidene moiety in compound 90p suggested affecting DENV-2 NS2B/NS3 replication as His51 is one of the catalytic triads of NS3 residue, which required to cleavage the NS2B residue for virus replication activities (Noble et al., 2012[START_REF] Niyomrattanakit | Identification of Residues in the Dengue Virus Type 2 NS2B Cofactor That Are Critical for NS3 Protease Activation[END_REF]. The geometry of this stacking interaction suggests that the electrostatics cloud localization play a role in the attraction between both aromatic systems (Zhao & Zhang, 2016).

The synthesized compound 90p formed hydrogen bonds with one of the catalytic triad amino acid residues were located in NS3 serine protease, His51, as well as other amino acid residues namely, Gly153, Asn152, Phe130, Pro132, Ser131, and Phe130. Only one hydrogen bonding interaction occurred between synthesized compound 90p with the amino acid in NS2B residue, which is Met84. The details on the hydrogen bonding interaction were tabulated in Table 4.13 below. There are four hydrogen bond interactions with the distance lower than 3.5 Å between the compound 90p with the amino acid residue in the DENV-2 NS2B/NS3

protease. The shortest hydrogen bond distance of 2.56 Å with a bond angle of 139.93º

was formed between the synthesized compound 90p and amino moiety in NS3Gly153.

The oxygen atom in the carbonyl group moiety of the synthesized compound 90p

suggested acting as the H-acceptor, while the hydrogen attached to the nitrogen atom present in the simple amino acid of glycine suggested acting as the H-donor (Itoh et al., 2019).

The top five compounds with the lowest FEB namely, 90c, 90e, 90g, 90p, and 90r from the in silico studies were chosen to examine it's in vitro activity towards DENV-2 NS2B/NS3 protease. Generally, the difference between compounds 90c, 90e, 90g, 90p, and 90r are the substitute at the para position in the benzene ring. The ability of the substituent form the intermolecular bonding with the amino acid in NS2B/NS3

protease determined the binding energy. Synthesized compound 90p have a dimethylamino-group, the nitrogen could act as the hydrogen bond donor to enhance the intermolecular bonding with the amino acid in NS2B/NS3 protease. While, the bulk structure of synthesized compounds 90g and 90r with morpholine and piperidine substituents respectively, increased the steric hindrance that affected the binding energy.

Overall, the synthesized compound 90p displayed a promising in silico activity towards DENV-2 NS2B/NS3 protease with lots of intermolecular bonding formed between the synthesized compound and macromolecule.

Inhibitor

In addition, the dimethoxy group in the indanone moiety of synthesized compounds 90r and 90p played a role in increasing the electron donating potential of the compound. It increased the percentage of intermolecular bonding formation with the catalytic triad, which will halt the dengue virus replication (Yin et al., 2006). Thus, the synthesized compounds 90r and 90p obtained the top two highest inhibitory activity, surpassing panduratin A.

The presence of alkyl group with a hydrophobic property in the synthesized compounds 90c and 90e are not tolerated and lowered the inhibitory activity. This finding is in agreement with the finding of Steuer and co-worker (2011) in evaluating the inhibitory activity of α-ketoamides as DENV-2 protease inhibitors.

The active functional group present in the synthesized compound suggested playing a role in the formation of bonded interaction between enzyme and peptide part of the substrate. No fluorescence has been exhibited with the presence of the inhibitor as the inhibitor formed a bonded interaction with the enzyme and ceased the peptide cleavage (refer to the Chapter 2, page 30, Scheme 2.6).

The most potent synthesized compound, 90p, underwent further analysis to determined it IC50 value and the dose-response curved were plotted in Figure 4.31. protease using molecular docking showed that the p-methoxy group of the phenyl moiety may possess an excellent steric fit for the catalytic triad, which resulted in good inhibitory activity.

Another factor affecting the inhibitory activity was the number of electron withdrawing group possess in the compound. Compound 90p have several electron withdrawing groups in its moiety. The number of electron withdrawing group, such as sulfonyl, aldehyde, nitro, methoxy, halogen and other increased the inhibitory activity of the quinoline derivatives towards the DENV-2 NS2B/NS3 protease [START_REF] Deng | Discovery of novel small molecule inhibitors of dengue viral NS2B-NS3 protease using virtual screening and scaffold hopping[END_REF].

In summary, the finding of this study showed the results is in agreement with the previous works. The synthesized compound 90p displayed a good DENV-2 NS2B/NS3 protease inhibitory activity performed following method by Yusof et al. (2000) by using synthetic fluorogenic peptide Boc-Gly-Arg-Arg-AMC. It can be concluded that synthesized compound 90p could be a potent inhibitor of DENV-2 NS2B/NS3 protease.

CHAPTER 5

CONCLUSION AND RECOMMENDATION

Conclusion

This study highlighted the potential of M. charantia fruits. The fruits flesh and seeds part were both utilized.

The M. charantia seeds oil can be extracted. It can be concluded that the enzyme assisted extraction can lead to an increase in oil yield and hemicelullose.

Besides, slightly modification in the biochemical properties of the extracted oil were detected. Apart from that, the lignocellulosic biomass seed composition of seeds underwent aqueous enzymatic extraction seems to be have different amount of soluble sugars, and uronic acids.

The M. charantia fruits flesh ethyl acetate extract displayed a promising antidengue activity towards DENV-2 NS2B/NS3 protease. The GC-MS chromatogram profile of the ethyl acetate extract revealed the presence of gallic acid, a highly antiviral compound. The gallic acid was chosen for hemisynthesis of DENV-2 NS2B/NS3 protease inhibitor.

The pyrazolyl substituted benzylidene indanone derivatives, 90a-t were successfully synthesized by the formation of N heterocycles using acetic acid. The synthesized compounds showed a promising DENV-2 NS2B/NS3 protease inhibitory in both in silico and in vitro analysis. The compound 90p showed lower IC50 value of 78.87 µM compared with the panduratin A, 290.27 µM. It can be concluded that the compound 90p could be a potent hit-to-led compound in the search of DENV-2 NS2B/NS3 protease inhibitor.

In summary, the M. charantia showed a versatile characteristic. The seed oil that contained omega-3 fatty acid and seed's lignocellulosic biomass could be a useful in developing a sustainable product as it possess hemicellulose, soluble sugar and uronic acid. Meanwhile, the fruits flesh could be a good source of chemical constituents with the anti-dengue properties.

Recommendations for Future Studies

Broadening the scope of this study will produce multidisciplinary knowledge contributions. Several additional works are recommended, such as:

1. In objective 1: To test the activity of M. charantia fruits ethyl acetate extract towards DENV-2 NS2B/NS3 protease.

a. The M. charantia fruits could undergo isolation to identify the compound that acts as DENV-2 NS2B/NS3 protease inhibitor. b. The use of hemicellulose in producing a sustainable product such as biodegradables plastic or thin film.

3.

In objective 3: To synthesize and characterize the indanone derivatives derived from small molecule present in the M. charantia fruit extract.

a. Compound 90p having the highest DENV-2 NS2B/NS3 protease inhibitory activity. In synthesizing compound 90p, only 63.17 % yield was obtained.

The further method optimization to increase the yield of 90p can be initiated.

Introduction

La dengue (DF), une infection virale transmise par les moustiques, est passée de 2.2 millions en 2010 à plus de 3.34 millions en 2016 dans le monde entier (WHO, 2019). Les moustiques femelles infectées d'Aedes aegypti et d'Aedes albopictus (Figure 1.1) ont transmis le virus de la dengue aux humains par leurs piqûres. Les virus de la dengue peuvent être classés en quatre sérotypes en fonction des antigènes présents à la surface du virus, qui sont les virus de la dengue de type 1, 2, 3 et 4 (Guzman & Kouri, 2003). Aucun symptôme exact de FD n'a été identifié. Cependant, la forte fièvre allant de 39.5 à 41 ºC qui persiste habituellement pendant 2 à 7 jours accompagnée de douleurs musculaires et articulaires pourrait en être le signe. La FD peut survenir dans les 3 à 14 jours suivant une piqûre de moustique infecté. L'homme infecté a ressenti une perte d'appétit et des vomissements. Certains humains infectés ont eu des glandes enflées ou des éruptions cutanées (MoHM, 2015).

Environ 72,356 cas de dengue ont été signalés en Malaisie entre janvier et juillet 2019, soit une augmentation de 89.5 % par rapport à la même période en 2018, dont 138 cas mortels (Bernama, 2019). En Malaisie, les quatre sérotypes de dengue peuvent être isolés en même temps. Cependant, les principaux sérotypes de dengue trouvés en Malaisie étaient le virus de la dengue de type 1 (DENV-1) et le virus de la dengue de type 2 (DENV-2) (Suppiah et al., 2018). Le traitement de la dengue disponible aujourd'hui se limite à des soins médicaux complémentaires et au remplacement des liquides, car il n'existe pas de traitement spécifique.

Comme il n'existe ni traitement curatif ni traitement préventif de la dengue et qu'il n'existe qu'un seul vaccin contre la dengue autorisé, le vaccin Dengvaxia de Sanofi Pasteur a été approuvé par les autorités réglementaires pour une utilisation à titre d'essai dans une zone de forte endémie (WHO, 2017). En dehors de cela, seules des mesures de soutien et une thérapie fluide judicieuse sont disponibles pour le DF (Rajapakse et al., 2012).

Certains patients optent pour un remède traditionnel pour guérir la FD en améliorant leur numération plaquettaire. Au Pakistan, un patient atteint de dengue a été traité avec 25 ml d'extrait aqueux de feuilles de Carica papaya (C. papaya), deux fois par jour, et a constaté qu'en peu de temps, il avait augmenté le nombre de plaquettes [START_REF] Ahmad | Dengue fever treatment with Carica papaya leaves extracts[END_REF]. Les études ont montré que les feuilles de C. papaya contenaient certains composés actifs qui peuvent augmenter l'hémopoïèse et la thrombopoïèse [START_REF] Dharmarathna | Does Carica papaya leaf-extract increase the platelet count? An experimental study in a murine model[END_REF].

Aux Philippines, les gens utilisaient l'Euphorbia hirta (E. hirta) pour traiter la dengue car on pense qu'il possède des propriétés antivirales qui accéléreront le processus de guérison et empêcheront le DF de passer en phase critique (Perera et al., 2018). L'E. hirta contient des phénoliques et des flavonoïdes qui sont des composés bien connus capables d'augmenter la plaquette.

Le praticien traditionnel chinois, le Dr Yong Kian Fui, a déclaré qu'il n'existe pas de remède contre la dengue dans la médecine traditionnelle chinoise (MTC). Cependant, une concoction à base de plantes appelée "qin wen bai tu san", une soupe faite par la cuisson lente de Momordica charantia L. (M. charantia) et de viande de grenouille ou de tout autre type de viande pendant deux heures, peut être consommée en une seule dose par jour pendant trois jours consécutifs. Le M. charantia est utilisé pour désintoxiquer le corps en éliminant la chaleur, tandis que la viande neutralise le M. charantia, afin qu'il ne se refroidisse pas trop (Tan, 2014).

En l'absence de médicaments anti-dengue ou de vaccins prometteurs, il est nécessaire de rechercher d'autres mesures de prévention et de traitement. Comme de nombreux chercheurs se concentrent sur l'utilisation des feuilles de C. papaya et d'E. hirta, cette étude s'est intéressée à l'activité anti-dengue des fruits de M. charantia ou aussi connus sous le nom de melon amer, de poire baumier, de courge amère ou de gourde amère. Le nom malais local de cette plante est "peria katak" (Ong & Azliza, 2015). M. charantia, plante de la famille des Cucurbitacées, possède une vigne grimpante mince avec une feuille à long pétiole et un fruit oblong fusiformecylindrique, de forme irrégulière et verruqueuse (Figure 1.2). Van Staden, 2020). Les chercheurs ont montré que lorsqu'il est pris de façon continue pendant une certaine période, il a la capacité de remplacer l'insuline dans l'organisme. Il contient également des saponines stéroïdiennes appelées charantines, des peptides similaires à ceux des peptides et certains alcaloïdes qui contrôlent efficacement le niveau de sucre dans le sang (Kumar & Bhowmik, 2010). Comme les plantes fournissent une multitude de petites molécules aux propriétés semblables à celles des médicaments, elles peuvent servir de modèle éprouvé pour le développement de nouveaux échafaudages de médicaments qui pourraient être utiles à l'optimisation du processus de découverte de médicaments (Atanasov, 2015).

Matériaux et méthode 2.1 Matériaux

Le solvant, le produit chimique et les matériaux utilisés étaient de qualité analytique et ont été utilisés sans subir de purification ou de distillation supplémentaire. Tous les appareils utilisés ont été lavés à l'eau distillée et rincés à l'acétone pour garantir la propreté de l'appareil et éviter toute impureté

Méthodes

Les études de la chair et de la graine des fruits de M. charantia sont divisées en deux parties. La première partie concerne l'extraction de l'huile de graines de M. charantia par hydrodistillation et par extraction enzymatique aqueuse (AEE) ainsi que l'étude de sa biomasse lignocellulosique. La seconde partie a consisté à préparer un extrait de pulpe de fruits de M. charantia pour le criblage anti-dengue. La Figure 2.1 résume le cadre de l'étude.

Préparation de l'échantillon

Les fruits non mûrs de M. charantia ont été achetés dans une ferme maraîchère située à Ara Kuda, Seberang Perai Utara, Penang, Malaisie, entre juin et août 2015. Les fruits ont été lavés à fond à l'eau du robinet pour éliminer toute contamination indésirable et coupés pour séparer la chair avec le pépin. Ensuite, les fruits (chair) et les pépins ont été séchés au soleil à température ambiante. Les fruits et les pépins ont été retournés toutes les 24 heures afin d'assurer un séchage complet. Les fruits et les graines séchés ont été broyés en petits morceaux avant d'être conservés dans des sacs en polyéthylène séchés jusqu'à leur utilisation ultérieure.

2.2.1(a) PARTIE 1: Extraction enzymatique aqueuse (AEE) de l'huile de graines de M. charantia L'AEE a été réalisée pour extraire l'huile de la graine de M. charantia selon la méthode décrite par Muniglia et al. (2011). Les cocktails d'enzymes, HEL1 et X7, ont été mélangés au Laboratoire d'ingénierie des Biomolécules (Nancy, France), qui contenait de la cellulase, de la bétaglucosidase, de l'endocellulase, de l'exocellulase, de l'arabinase, de la xylanase, de la galactanase, de l'endopolygalacturonase, de l'hémicellulase, de la protéinase, de la pectinase et de la pectine méthylestérase à différentes concentrations.

Au départ, les graines séchées de M. charantia fraîchement moulues ont été mélangées à de l'eau distillée dans un rapport de 1:4 dans un bioréacteur préprogrammé (Celligen Plus, New Brunswick Scientific, États-Unis) avec agitation continue. Ensuite, les cocktails d'enzymes ont été ajoutés consécutivement dans le bioréacteur une fois que la température a atteint 50 °C selon différents rapports comme indiqué dans le tableau 2.1. On a laissé la réaction se poursuivre pendant quatre heures dans le bioréacteur, puis on a procédé à une centrifugation (0.158 kilohertz, 20 minutes, 20 °C) pour séparer les produits. Les produits finaux de ce processus d'extraction ont été: 1) huile, 2) résidu de graines et 3) partie aqueuse. L'huile et les résidus de graines obtenus ont été soumis à une analyse par GC et à l'identification de la biomasse lignocellulosique des graines, respectivement. La partie aqueuse a été rejetée.

2.2.1(a)(i) L'analyse de l'huile de graines de M. charantia

Les échantillons d'huile de graines ont d'abord été dérivés en esters méthyliques d'acides gras (EMAG) par le procédé d'estérification décrit par Metcalfe et Schmitz (1961), avant de faire l'objet d'analyses complémentaires pour identifier leur composition biochimique par chromatographie en phase gazeuse (CG). Chaque huile dérivée (FAME) a été analysée pour identifier sa composition biochimique par chromatographie en phase gazeuse avec détecteur à ionisation de flamme.

2.2.1(a)(ii) L'analyse de la biomasse lignocellulosique des graines de M. charantia

Les résidus de graines séchées ont ensuite été extraits selon la méthode Soxhlet pour s'assurer que tous les composants extractifs ont été éliminés avant l'hydrolyse afin de déterminer leur teneur en lignine, hémicellulose, acide uronique et sucre soluble dans l'acide. La présence de composants extractifs entravera le processus d'hydrolyse en réagissant avec l'acide et en se condensant en composants insolubles dans l'acide, ce qui entraînera une analyse inexacte.

2.2.1(b) PARTIE 2: Préparation de l'extrait de chair du fruit de M. charantia

La chair des fruits secs broyés (0.5 kg) a ensuite été extraite de manière exhaustive par macération séquentielle dans du n-hexane, de l'acétate d'éthyle et du méthanol deux fois en triple exemplaire pendant 7-14 jours à température ambiante (25-30 °C) sous hotte. Les mélanges ont été secoués et agités quotidiennement. Les extraits étaient ensuite filtrés et le solvant était éliminé par un évaporateur rotatif sous pression réduite. L'extrait a ensuite été conservé au réfrigérateur dans une bouteille d'échantillon scellée avant de procéder au criblage anti-dengue et l'extrait le plus actif sera soumis à une analyse par GC-MS.

Test anti-dengue de l'extrait de chair de fruits de M. charantia

Les essais in vitro pour les extraits de chair de fruits de M. charantia ont été réalisés selon la méthode de Yusof et al. (2000). Un mélange réactionnel enzymatique d'un volume total de 100 L à la concentration de 200 ppm a été préparé dans des plaques noires à 96 puits. Chaque puits des plaques noires à 96 puits est constitué de 94.9 L de solution tampon de Tris-HCl à pH 8.5, de 3.1 L de l'enzyme et de 1 L d'extrait (200 mg/L en DMSO). Les mélanges ont été incubés et agités à 37 C pendant 10 minutes. Ensuite, 1 L de substrat 10 mM dans du DMSO (Peptide Institute, Japon) a été ajouté dans le mélange pour amorcer la réaction. Le mélange a ensuite été incubé et secoué pendant 60 minutes supplémentaires à 37 C en utilisant l'incubateur à agitation SI-50 (Protech, Malaisie). L'essai a été triplé. L'extrait ayant la plus grande activité anti-dengue sera analysé plus avant en utilisant la GC-MS pour identifier ses compositions. (Li et al., 2017). Le schéma de réaction a été illustré dans le schéma 2.1 ci-dessous. De l'acide benzoïque (0.1 mmol) a été ajouté dans l'éthanol (1.0 mmol) en présence d'acide sulfurique (0.01 mmol). La solution a été chauffée au reflux. Après 3 h, la réaction a été contrôlée par CCM à l'aide d'un système d'acétate d'éthyle et d'hexane (3:7). L'agitation a été poursuivie pendant environ 12 heures ou jusqu'à ce que la CCM indique la fin de la réaction. L'excès d'éthanol a été éliminé à l'aide d'un évaporateur rotatif. La solution obtenue a ensuite été mélangée à 10 ml de solution d'acétate d'éthyle et de chlorure de sodium (10 % p/v.). Ensuite, la phase organique a été séparée à l'aide d'un entonnoir de séparation. L'excès de solvant a été éliminé de la phase organique à l'aide d'un évaporateur rotatif. Le précipité a été séché sous vide pour obtenir le composé 85. Les structures chimiques du composé ont été confirmées par RMN 1 H, RMN 13

85 R R O R 1 R 2 R 3 R R O R 1 R 2 R 3 O 88a-j 89a-t + CH 3 OH 10 % w/v KOH 87a-b Where 87a = R = H 89a: R 1 = H; R 2 = H; R 3 = H 89b: R 1 = H; R 2 = H; R 3 = OCH 3 89c: R 1 = H; R 2 = H; R 3 = CH 3 89d: R 1 = H; R 2 = H; R 3 = Cl 89e: R 1 = H; R 2 = H; R 3 = CH(CH 3 ) 2 89f: R 1 = H; R 2 = H; R 3 = N(CH 3 ) 2 89g: R 1 = H; R 2 = H; R 3 = NC 4 H 8 O 89h: R 1 = H; R 2 = H; R 3 = NC 4 H 10 89i: R 1 = OCH 3 ; R 2 = H; R 3 = OCH 3 89j: R 1 = H; R 2 = NO 2 ; R 3 = OCH 3 Where 87b = R = OCH 3 89k: R 1 = H; R 2 = H; R 3 = H 89l: R 1 = H; R 2 = H; R 3 = OCH 3 89m: R 1 = H; R 2 = H; R 3 = CH 3 89n: R 1 = H; R 2 = H; R 3 = Cl 89o: R 1 = H; R 2 = H; R 3 = CH(CH 3 ) 2 89p: R 1 = H; R 2 = H; R 3 = N(CH 3 ) 2 89q: R 1 = H; R 2 = H; R 3 = NC 4 H 8 O 89r: R 1 = H; R 2 = H; R 3 = NC 4 H 10 89s: R 1 = OCH 3 ; R 2 = H; R 3 = OCH 3 89t: R 1 = H; R 2 = NO 2 ; R 3 = OCH 3

2.2.3(c) La synthèse de dérivés de benzylidène indanone substitués par un groupe pyrazolyle

Les dérivés de benzylidène indanone précédemment synthétisés, 89a-t (1.0 mmol) ont ensuite été mis à réagir avec le composé 86 (1.0 mmol) en présence de 10 ml d'acide acétique sous reflux pendant 12 h, comme indiqué dans le schéma 2.4. L'excès de solvant a été éliminé sous pression réduite. Le mélange réactionnel a ensuite été versé dans la glace concassée. Le précipité a été filtré par filtration gravitationnelle, lavé au méthanol froid et séché sous la hotte. Les composés synthétisés ont ensuite été recristallisés par une technique de recristallisation à solvant unique utilisant de l'éthanol. Les composés ont ensuite été soumis à une analyse de caractérisation afin de déterminer leurs propriétés physico-chimiques. Scheme 2.4: La synthèse de dérivés de benzylidène indanone substitués par un groupe pyrazolyle.

2.2.3(d) Les études in silico vers la protéase DENV-2 NS2B/NS3

La structure composée a été esquissée à l'aide de Marvin Sketch 19.4.0 (ChemAxon, 2019) et convertie en une structure 3D à l'aide du logiciel HyperChem pro 8.0 (www.hyper.com). L'arrimage moléculaire a été réalisé à l'aide d'AutoDock4.2. La structure cristalline de la protéine d'homologie générée par Wichapong et son collègue a été utilisée (2010). La version 1.5.6 d'AutoDock Tools (ADT) a été utilisée pour la préparation de la stimulation de l'amarrage. La dimension de 74 x 43 x 64 en coordination x,y,z a été utilisée comme taille de la grille.

2.2.3(e) Les études d'essais in vitro sur la protéase DENV-2 NS2B/NS3

Un composé sélectionné a été choisi pour les études IC50. Les séries de concentrations d'un composé de DMSO (6.25 ppm, 12.5 ppm, 25 ppm, 50 ppm, 100 ppm, 200 ppm) ont été préparées et le dosage a été effectué selon la procédure mentionnée à la section 2.2.2.

3.

Résultats et discussion 3.1 Les études sur l'huile de graines de M. charantia et la biomasse lignocellulosique L'huile de graines de M. charantia a été extraite par la méthode d'extraction enzymatique aqueuse (AEE). La composition de l'huile de graines a été déterminée à l'aide de la GC-MS. La composition de la biomasse lignocellulosique des graines a été identifiée en termes de lignine, de sucres solubles, d'acide uronique et de teneur en hémicélulose.

Extraction enzymatique aqueuse (AEE) de l'huile de graines de M. charantia

L'idée d'utiliser l'enzyme dans le processus d'extraction aqueuse était de faciliter la dégradation des structures cellulaires et sous-cellulaires de la graine et d'augmenter la dissolution du composé soluble présent dans la graine dans la matrice d'eau ainsi que de favoriser la libération de l'huile (Ricochon & Muniglia, 2010). Dans l'expérience témoin, sans ajout de cocktail d'enzymes (M0), seulement 3.32 ± 0.38 % de l'huile de graines a été extraite.

R R O R 1 R 2 R 3 89a-t N N O O CH 3 O CH 3 O CH 3 R R R 1 R 2 R 3 90a-t

× 100%

Les résultats ont indiqué que la cellulase, la protéinase et la pectinase présentes dans les cocktails enzymatiques HEL1 et X7 pourraient faciliter la dégradation des structures cellulaires et sous-cellulaires de la graine pour favoriser la libération d'huile. Les enzymes pourraient avoir affaibli les cellules végétales de la graine pour favoriser la libération de l'huile (Baby & Ranganathan, 2016). Dans une étude précédente, Latif et Anwar (2011) ont rapporté que le mécanisme actif d'un cocktail d'enzymes sélectionnées pendant l'extraction aqueuse enzymatique était la dégradation de la cellulose, de la pectine et des protéines, détruisant ainsi l'intégrité de la paroi cellulaire structurelle des cellules de la graine.

L'analyse de l'huile de graines de M. charantia

Les échantillons d'huile de graines extraits (M0, M1, M2, M3, et M4) ont été soumis à une dérivatisation en FAME avant l'identification de leurs compositions biochimiques par GC-FID car les FAME sont plus volatiles et plus faciles à analyser. Le GC-FID a été largement utilisé pour la séparation et la détection des FAME. Sept acides gras connus ont été identifiés. Trois acides gras inconnus ont également été détectés. Cependant, trois pics inconnus d'acide gras libre ont été observés sur le chromatogramme qui est en coélution presque en même temps. Il est suggéré que certains composés peuvent coélutions plus importantes en taille et éluer ensemble à la fin pendant la séparation (Kamatou & Viljeon, 2017). D'après la composition de l'huile de graines de M. charantia (Tableau 3.2), le pourcentage d'acides élaïdique et palmitique a augmenté avec l'introduction des cocktails enzymatiques. Dans l'expérience témoin (M0), seulement 0,47 % d' Note: Values are % of total fatty acid expressed as mean ± SD of three separate determinations La libération d'huile pourrait être due à la présence de cellulase dans le cocktail enzymatique, qui a perturbé d'autres constituants polysaccharidiques dans la paroi cellulaire des graines. La présence de cellulase dans HEL1 et X7 a favorisé une dégradation efficace de la cellulose. Le mécanisme de la cellulase, décrit comme le clivage des liaisons glucosidiques internes au sein d'une chaîne de glucane ininterrompue par l'enzyme endoglucanase, pourrait entraîner la création d'une chaîne non réductrice de substrat de la cellobiohydrolase, clivant les dimères de cellobiose de la chaîne de glucane qui était libérée dans la solution. En outre, βglucosidase a scindé le dimère de cellobiose en monomère de glucose, concluant ainsi le processus d'hydrolyse de la cellulose. La combinaison d'enzymes a joué un rôle dominant dans la perturbation des parois cellulaires des graines, ce qui a également été signalé dans d'autres conclusions (Wu et al., 2017(Wu et al., ). et al., 2018)). On peut conclure que l'huile de graines de M. charantia obtenue à partir de l'AEE présentait de légères modifications de l'intégrité et de la répartition en pourcentage des composants actifs.

Détermination de la lignine, des sucres solubles et de l'acide uronique de l'EFM

La teneur en lignine de l'EFM; M0, M1 (15.91 %) > M2 (15.06 %) > M4 (14.71 %) > M3 (14.54 %) n'a pas montré de changements significatifs avec la présence d'enzymes dans l'AEE. Différents types de sucres solubles et d'acide uronique ont été libérés et ont fait l'objet d'une analyse plus poussée avec le HPAEC-PAD. La principale composition en sucres solubles de l'EFM était le glucose et le rhamnose, respectivement. Aucune tendance précise n'a été observée entre le pourcentage des ratios d'enzymes utilisés et le pourcentage de distribution des sucres solubles et de l'acide uronique obtenu. M0L a montré la plus faible quantité d'acide galacturonique (8.42 %). Le pourcentage de galacturonique acide augmentait avec la présence de cocktails enzymatiques dans M3L (16.12 %) < M4L (16.37 %) < M1L (22.00 %) < M2L (22.25 %). Ces résultats suggèrent que la présence de pectinase dans les cocktails enzymatiques a facilité la dégradation des polysaccharides pectiques de la paroi cellulaire en molécules plus simples comme l'acide galacturonique (Welker et al., 2015). La concentration de glucose dans le M0 n'était que de 57 %. Le pourcentage de glucose a augmenté avec l'introduction des cocktails d'enzymes; M3 (65.42 %) < M4 (66.77 %) < M1 (71.34 %) < M2 (76.20 %). Ces résultats indiquent qu'une plus grande partie de la cellulose de la paroi cellulaire a été clivée en différents types de sucres (glucose, xylose, galactose, arabinose, rhamnose, mannose et fucose) à l'aide des cocktails enzymatiques (Segato et al., 2014).

L'analyse FTIR de l'hémicellulose

La quantité d'hémicellulose extraite de la biomasse de lignocellulose des graines de M. charantia varie en fonction de la présence de l'enzyme pendant l'AEE; M3L (0.29 %) < M4L (0.38 %) < M1L (0.41 %) < M2L (0.58 %). Seulement 0.22 % de l'hémicelullose a été récupéré dans M0L. Malgré des connaissances importantes sur la synergie entre les composants de la cellulase et du cocktail enzymatique, une compréhension mécanique complète de l'hydrolyse de la lignocellulose par les enzymes cellulolytiques est encore limitée et doit être affinée (Nill et al., 2018). La variation de la composition de l'hémicellulose a montré qu'en tant que polysaccharides de structure plus simple que la cellulose, l'hémicellulose était également sujette à la dégradation en présence d'hémicellulase (Zhao et al., 2018). L'hémicellulase obtenue a été analysée plus en détail par spectroscopie FTIR, comme le montre la figure 3.1. Le spectre FTIR a montré qu'il y avait des différences dans le pic d'intensité et entre les bandes d'absorption obtenues. L'absorption des pics de 3272, 2927, 1628, 1416, 1239 et 1038 cm -1 représentait l'absorption caractéristique de l'hémicellulose [START_REF] Popescu | Characterization of fungal degraded lime wood by FT-IR and 2D IR correlation spectroscopy[END_REF]. La large bande d'absorption à ~3272 cm -1 représente généralement le O-H ou l'absorption d'un grand nombre de molécules individuelles, que l'hydrogène est lié à un composé légèrement différent. En revanche, la bande d'absorption ~2927 cm -1 indique la présence d'une vibration d'étirement de la liaison Csp3-H. La bande ~1737 cm -1 se réfère à la bande d'étirement C=O conjuguée d'un groupe carbonyle, à attribuer à la présence de la partie acétyle dans l'hémicellulose. L'absorption qui est apparue à ~1628 cm -1 est généralement associée à l'eau absorbée. Une forte bande qui est apparue à ~1038 cm -1 a été attribuée à la flexion de C-O dans le sucre (Naidu et al., 2018). D'après le spectre FTIR, l'introduction de l'enzyme n'affecte pas la structure du squelette de l'hémicellulose. En outre, on peut conclure que les échantillons de semences optimaux et leurs rapports de cocktail enzymatique pour le HEL1:X7 se sont avérés être de 5:1.25 comme dans M2.

Les études de la chair des fruits de M. charantia

Dans cette étude, l'extrait de n-hexane, d'acétate d'éthyle et de méthanol du fruit de M. charantia a fait l'objet d'un dépistage préliminaire in vitro de l'activité anti-dengue. La composition de l'extrait actif a été analysée par GC-MS.

Préparation de l'extrait de chair du fruit de M. charantia

La quantité de l'extrait correspond au type de solvants utilisés sur la base de la loi de similitude et d'intermissibilité. Les solvants dont la valeur de la polarité est proche de celle du soluté sont susceptibles d'être plus performants et vice versa. La quantité de matière extractive extraite des fruits de M. charantia avec l'utilisation d'un solvant non polaire à polaire implique la variation des composés phytochimiques. Comme les plantes contiennent une variété de composés bioactifs ayant des propriétés de solubilité différentes dans un solvant différent, le solvant optimal utilisé pour l'extraction dépend des matières végétales particulières, et des composés qui doivent être isolés (Truong et al., 2019). L'utilisation d'un solvant non polaire a montré que la plus faible quantité de produit extractif était l'extrait avec un pourcentage de rendement de 0.49 ± 0.03 %, suivi de l'extrait extractif par l'acétate d'éthyle, 1.48 ± 0.11 %, et le méthanol, 3.47 ± 0.04 %, comme présenté dans le tableau 3.2. Weight of dry fruits flesh used (mg) × 100% 3.2.1 a) Screening anti-dengue de l'extrait de chair du fruit de M. charantia Dans l'étude de screening anti-dengue des extraits de chair du fruit de M. charantia, les extraits ont été incorporés dans la réaction de protéase à une concentration finale de 200 ppm. Dans cette étude, l'extrait d'acétate d'éthyle a montré une activité d'inhibition maximale. L'extrait de méthanol a montré la moindre activité inhibitrice envers la protéase DENV-2 NS2B/NS3 avec seulement 10.37 ± 1.13 %. L'extrait semi-polaire de M. charantia provenant de l'acétate d'éthyle a montré la plus forte activité inhibitrice de la protéase DENV-2 NS2B/NS3 avec 38.11 ± 1.06 %. La différence dans l'activité inhibitrice obtenue a été suggérée en raison du type de composés bioactifs extraits dans un autre type de solvant (Farahmandfar et al., 2019). L'extrait d'acétate d'éthyle ayant la plus grande activité inhibitrice de la protéase DENV-2 NS2B/NS3 a été soumis à un criblage par GC-MS afin d'identifier ses composants bioactifs.

3.2.1(b) Le screening par GC-MS de l'extrait de chair de fruit de M. charantia

La figure 3.2 montre divers pics indiquant la présence de différents composés dans l'extrait d'acétate d'éthyle du fruit de M. charantia. Le profil du chromatogramme GC-MS a révélé la présence de 32 pics différents et a été caractérisé et identifié par comparaison de leurs modèles de fraction de masse avec des données similaires de la bibliothèque du National Institute of Standards and Technology (NIST) ou des données publiées.

Les principaux composés ont été présentés sous forme de tableau dans le tableau 3.3. Les composés ayant une surface de pic de 1 à 2 % étaient le 1,4-bis(triméthylsilyl)benzène (rt. 44.395 min, 1.953 %), l'hexaméthyl-cyclotrisiloxane (rt. 44.288 min, 1. 9 %), 25-désacétoxy cucurbitacine b (rt. 44.670 min, 1.574 %), acide butyl-ester acétique (rt. 2.485 min, 1.539 %), 3,5-bis-triméthylsilyl-2,4,6-cycloheptatriène-1-one (rt. 33.29, 1.434 %), 1,2bis(triméthylsily)benzène (rt. 44.923 min, 1.433 %), ester bis(2-éthylhexyle) de l'acide décanedioïque (rt. 40.552 min, 1.346 %), phtalate de diisooctyle (rt. 36. 806 min, 1,297 %), Ntrifluoroacétyl-1-aminononadécane (rt. 47.265 min, 1.297 %), rétine (rt. 33.660 min, 1.23 %), et 2,6-dihydroxyacétophénone (rt. 47.825, 1.035 %). Les autres composés étaient présents à l'état de traces avec une surface de pic inférieure à 1 %, y compris l'acide gallique (rt. 5.017 min, 0.54 %). Bien que le pic de l'acide gallique ait été observé en moins de 1 %. Jia et son collaborateur (2017), ont indiqué que l'acide gallique était le principal acide phénolique de la chair du fruit de M. charantia.

La synthèse de dérivés de benzylidène indanone substitués par un groupe pyrazolyle

Les composés 90a-t ont permis de synthétiser une quantité appropriée de produit avec un pourcentage de rendement compris entre 40 et 85 % et un point de fusion compris entre 175 et 215 ºC. Les pourcentages de rendement des produits obtenus étaient compris entre 40 et 85 %. Il est possible qu'une partie du produit ait été perdue lors de la récupération de l'échantillon, par exemple lors de la coulée, de la filtration ou de la chromatographie sur colonne, ce qui a entraîné un faible rendement. En outre, le faible pourcentage de rendement des produits pourrait être dû à des réactions incomplètes, dans lesquelles certains réactifs ne réagissent pas pour former le produit.

Il a été observé que le pourcentage de rendement des composés 90h, 90j, 90n, 90s et 90t était inférieur à 50 %. D'après les observations, les composés ayant un rendement plus faible ont tendance à avoir un substituant autre que l'hydrogène au niveau de R1, R2 et R3 dans la fraction benzylidène, ce qui en fait des composés en vrac. Les substituants plus volumineux ralentissent considérablement la réaction parce que les groupes plus grands ont effectivement augmenté la répulsion stérique avec le nucléophile entrant. Ainsi, la réaction a pris plus de temps (Liu et al., 2010a).

Une des méthodes de surveillance de la formation des dérivés d'indanone pyrazolyle substituée [90a-t] consiste à comparer les spectres FTIR des composés 86 et 89a-t. Cette réaction implique une étape de formation de cycle qui transforme l'amine primaire et secondaire du composé 86 en amine tertiaire du composé 90a-t. Par conséquent, deux bandes d'absorption moyennes -NH et -NH2 à 3364 cm -1 et 3319 cm -1 seront absentes du spectre FTIR du composé 90a-t.

Le spectre FTIR du composé 90b manifeste une bande d'absorption à 2938 cm -1 attribuée à la vibration des liaisons Csp2-H. Dans le composé 90b, la force d'absorption de C=O à 1697 cm -1 a diminué par rapport à la force d'absorption de C=O du composé 89b. Il est suggéré qu'en raison de la perte de conjugaison d'un côté, la fraction C=O du composé 90b est située à côté de la fraction amine (Sheat et al., 2018). Alors que la forte flexion hors du plan C-H est apparue à 820 cm -1 , elle indique la présence d'un cycle para disubstitué dans le composé 90b (Lampman et al., 2010). L'analyse LC-MS a indiqué le pic d'ion moléculaire [MH] + , ( + ESI) à m/z 459.4460 qui a confirmé la masse moléculaire de la structure.

L'élucidation de la structure des composés 90a-t a été réalisée en utilisant une étude spectroscopique RMN unidimensionnelle et bidimensionnelle. La principale différence entre le spectre RMN 1 H des composés 90a-t et le composé réactif 86 était la disparition du décalage N-H et N-H2. Aucun décalage chimique n'a été observé sur δH = 9.744 ppm et δH = 4.470, ce qui indique que le succès de la cyclisation des amines de l'amine primaire et secondaire en amine tertiaire.

Les études in silico vers la DENV-2 protéase NS2B/NS3

Les études in silico ont été menées pour tous les composés synthétisés avec la DENV-2 protéase NS2B/NS3, et son mode de liaison et sa conformation ont été déterminés. Le redécoupage a été effectué avec succès avec une valeur de déviation quadratique moyenne (RMSD) de 1.23 Å. Hevener et son collègue (2009) ont déclaré que les programmes capables de poser la valeur RMSD dans la gamme de 1.5-2 Å, selon la taille du ligand, sont considérés comme ayant réussi. Les paramètres d'amarrage obtenus ont été utilisés dans les études d'amarrage de la DENV-2 protéase NS2B/NS3 du ligand à l'aide des composés synthétisés. La Le composé 90p a montré huit interactions de liaison hydrogène, une interaction d'empilement π-π, et une interaction π-alkyle avec le résidu d'acide aminé dans la protéase DENV-2 NS2B/NS3. Il a été noté que les interactions formées sur ce site provenaient principalement des résidus NS3 avec différents types et forces d'interaction. Le résidu de la protéine NS3 est essentiel pour la réplication virale et la maturation des virions. Par conséquent, une plus grande interaction avec le résidu suggère de donner des résultats plus prometteurs en matière d'activité inhibitrice (Yin et al., 2006).

La présence du groupe aromatique situé au niveau de la fraction benzylidène dans le composé 90p augmente les possibilités d'interaction de van der Waals. L'interaction par empilement π-π et l'interaction par alkyle π entre le His51 et la fraction benzylidène dans le composé 90p suggèrent d'affecter la réplication du DENV-2 NS2B/NS3, car le His51 est l'une des triades catalytiques du résidu NS3, qui a nécessité le clivage du résidu NS2B pour les activités de réplication du virus (Noble et al., 2012). La géométrie de cette interaction d'empilement suggère que la localisation du nuage électrostatique joue un rôle dans l'attraction entre les deux systèmes aromatiques (Zhao & Zhang, 2016).

Le composé 90p a formé des liaisons hydrogène avec l'un des résidus d'acides aminés de la triade catalytique qui étaient situés dans la sérine-protéase NS3, His51, ainsi que d'autres résidus d'acides aminés, à savoir Gly153, Asn152, Phe130, Pro132, Ser131 et Phe130. Une seule interaction de liaison hydrogène s'est produite entre le composé 90p et l'acide aminé du résidu NS2B, qui est Met84. L'atome d'oxygène dans le groupe carbonyle du composé 90p a suggéré d'agir comme accepteur H, tandis que l'hydrogène attaché à l'atome d'azote présent dans l'acide aminé simple de la glycine a suggéré d'agir comme donneur H (Itoh et al., 2019).

Les cinq composés ayant le plus faible taux de FEB, à savoir 90c, 90e, 90g, 90p et 90r, ont été choisis dans les études in silico pour examiner leur activité in vitro vis-à-vis de la protéase DENV-2 NS2B/NS3. En général, la différence entre les composés 90c, 90e, 90g, 90p, et 90r est le substitut en position para dans le cycle benzénique. La capacité du substituant à former la liaison intermoléculaire avec l'acide aminé dans la protéase NS2B/NS3 détermine l'énergie de liaison. Le composé 90p a un groupe diméthyl-amino, l'azote pourrait agir comme donneur de liaison hydrogène pour améliorer la liaison intermoléculaire avec l'acide aminé dans la protéase NS2B/NS3.

Les études in vitro sur la protéase NS2B/NS3 du DENV-2

Le criblage de l'activité des cinq composés les plus actifs vis-à-vis de la protéase DENV-2 NS2B/NS3 en utilisant le substrat peptidique fluorogène Boc-Gly-Arg-7-amino-4méthylcoumarine a été effectué, et le composé le plus actif a procédé à une analyse des valeurs de la concentration inhibitrice à cinquante pour cent (IC50). La panduratine A a été utilisée comme témoin positif dans cette étude.

Le composé 90p a montré une activité inhibitrice de la protéase DENV-2 NS2B/NS3 assez élevée (65.61 ± 0.98 %), suivi du composé 90r avec 53.62 ± 1.05 %, du composé 90g avec 48.03 ± 1.01 %, de la panduratine A avec 47.59 ± 1.03 %, de l'acide gallique avec 25.11 ± 1.11 %, du composé 90e avec 20.06 ± 0.99 % et enfin du composé 90c avec seulement 16.11 ± 0.95 %.

Les composés sélectionnés ont montré une activité prometteuse d'inhibiteur de DENV-2 protéase NS2B/NS3 avec trois composés synthétisés, à savoir le composé 90p, 90r, et 90g ont obtenu un pourcentage d'inhibition supérieur à celui de la panduratine A et de l'acide gallique. Le composé le plus puissant, 90p, a fait l'objet d'une analyse plus poussée pour déterminer sa valeur IC50 et les courbes dose-réponse ont été résumées dans la figure 3.5. Sur la courbe, la valeur de la IC50 pour le composé 90p a été observée à 42 ppm, ce qui est inférieur à la valeur de 118 ppm pour la panduratine A. Les concentrations obtenues ont été converties en µM à l'aide de l'équation suivante 2 protéase NS2B/NS3, réalisée selon la méthode de Yusof et al. (2000) en utilisant le peptide fluorogène synthétique Boc-Gly-Arg-Arg-AMC. On peut conclure que le composé 90p pourrait être un puissant inhibiteur de la DENV-2 protéase NS2B/NS3.

Conclusion et recommandation 4.1 Conclusion

Cette étude a mis en évidence le potentiel des fruits de M. charantia. La partie chair et la partie pépins du fruit ont été utilisées. L'huile des graines de M. charantia peut être extraite. On peut conclure que l'extraction assistée par des enzymes peut conduire à une augmentation du rendement en huile et de l'hémicellulose. En outre, de légères modifications des propriétés biochimiques de l'huile extraite ont été détectées. En dehors de cela, la composition de la biomasse lignocellulosique des graines ayant subi une extraction enzymatique aqueuse semble avoir une quantité différente de sucres solubles et d'acides uroniques. L'extrait d'acétate d'éthyle de la chair du fruit de M. charantia a montré une activité anti-dengue prometteuse envers la DENV-2 protéase NS2B/NS3. Le profil du chromatogramme GC-MS de l'extrait d'acétate d'éthyle a révélé la présence d'acide gallique, un composé hautement antiviral. L'acide gallique a été choisi pour l'hémisynthèse de l'inhibiteur de la DENV-2 protéase NS2B/NS3. Les dérivés de benzylidène indanone pyrazolyl substitués, 90a-t, ont été synthétisés avec succès par la formation d'hétérocycles N à l'aide d'acide acétique. Les composés synthétisés ont montré un inhibiteur de la protéase DENV-2 NS2B/NS3 prometteur, tant en analyse in silico qu'in vitro. Le composé 90p a montré une valeur IC50 plus faible de 78.87 µM par rapport à la panduratine A, 290.27 µM. On peut conclure que le composé 90p pourrait être un puissant composé "hit-to-led" dans la recherche de l'inhibiteur de protéase NS2B/NS3 du DENV-2.

Recommandations pour les études futures

L'élargissement de la portée de cette étude produira des contributions de connaissances multidisciplinaires. Les fruits de M. charantia pourraient être isolés afin d'identifier le composé qui agit comme inhibiteur de la DENV-2 protéase NS2B/NS3. L'extraction enzymatique aqueuse dans cette étude a utilisé le cocktail d'enzymes HEL1 et X7. Dans les études futures, une seule enzyme telle que la cellulase, l'endocellulase, l'arabinase, la xylanase, la galactanase, l'hémicellulase, la protéinase ou la pectinase pourra être utilisée dans l'extraction enzymatique aqueuse pour obtenir une quantité appropriée d'hémicellulose. En outre, l'utilisation de l'hémicellulose dans la fabrication d'un produit durable tel qu'un plastique biodégradable ou un film mince. Alors que le composé 90p ayant l'activité inhibitrice de la DENV-2 protéase NS2B/NS3 la plus élevée n'a obtenu qu'un rendement de 63.17 %. Il est possible de lancer une nouvelle optimisation de la méthode pour augmenter le rendement du 90p. En outre, le composé synthétisé pourrait être soumis à une étude qualitative de la relation structure-activité afin d'obtenir des informations détaillées sur chaque groupe fonctionnel en fonction de sa fonction. D'autres analyses telles que l'étude de toxicité, la CMI et l'analyse in vivo peuvent être effectuées pour étudier le potentiel du composé 90p en tant qu'inhibiteur de DENV-2 protéase NS2B/NS3. [START_REF] Ahmad | Dengue fever treatment with Carica papaya leaves extracts[END_REF]

Références

  Besides, I would like to thank all the School of Chemical Sciences lecturers and staff, the Institute of Postgraduate Studies (IPS) staff, the staff of Malaysian Institute of Pharmaceuticals and Nutraceuticals (IPharm), as well as University of Lorraine, France staff for their hospitality, supports, assistances and helps. Special thanks to the France Embassy in Malaysia, Minister of Foreign Affair France, Campus France and Ministry of Education Malaysia under MyPhD program for the scholarship and financial support. Last but not the least, I would like to thank my parent (Abah & Mak) and family members (especially; Kak Diah, Ieah & Ariffudin) for their patience, encouragement, understanding and care during the hardship and the difficult times.

  anti-dengue screening using DENV-2 NS2B/NS3 protease enzyme 2.4.4 The compounds with anti-dengue properties M. charantia seed oil 3.3.1(a)(ii) The soxhlet extraction of the seed residue v 3.3.1(a)(iii) Determinations of lignin, soluble sugars, and uronic acid of EFM 3.3.1(a)(iv) Determination of soluble sugar and uronic acid contents of EFM 3.3.1(a)(v) Hemicellulose extraction of EFM 3.3.1(a)(vi) The SEC analysis of hemicellulose 3.3.1(b) PART 2: Preparation of M. charantia fruit flesh extract 3.3.2 Anti-dengue testing of M. charantia fruits flesh extract 3.4 The synthesis of pyrazolyl substituted benzylidene indanone derivatives 3.4.1 The synthesis of ethyl 3,4,5-trimethoxybenzoate, 85 and 4.2(k) (E)-2-benzylidine-5,6-dimethoxy-1-indanone, 4'-dimethylaminophenyl)-(3,4,5-trimethoxy phenyl)-3a,4-dihydroinde no-1,2-c-2-pyrazolyl methanone, pyrazolyl methanone, 90r 3.4.3(s) 3-(2',4'-dimethoxyphenyl)-(3,4,5-trimethoxy phenyl)-(6,7-dimethoxy) -3a,4-dihydroindeno-1,2c-2-pyrazolyl methanone, studies of M. charantia seeds oil and lignocellulosic biomass 89 4.1.1 Aqueous enzyme extraction (AEE) of M. charantia seed oil 89 4.1.2 The analysis of M. charantia seed oil 92 4.1.3 Determinations of lignin, soluble sugars, and uronic acid of EFM 95 4.1.4 The FTIR and SEC analysis of hemicellulose 98 4.2 The studies of M. charantia fruits flesh 4.2.1 Preparation of M. charantia fruit flesh extract 4.2.1(a) Anti-dengue screening of M. charantia fruits flesh extract 4.2.1(b) The GC-MS screening of M. charantia fruit flesh extract 4.3 The synthesis of pyrazolyl substituted benzylidene indanone derivatives 4.3.1 The synthesis of ethyl 3,4,5-trimethoxybenzoate, synthesis of pyrazolyl substituted benzylidene indanone derivatives, 90a-t 4.3.4(a) The characterization of pyrazolyl substituted benzylidene indanone derivatives, 90a-t ix 4.4 The in silico Studies towards DENV-2

Figure 1. 1 FigureFigureFigureFigureNS2B

 1 Figure 1.1 Images of the two predominant arthropod vectors of dengue virus, Aedes albopictus and Aedes aegypti 1 Figure 1.2 (a) M. charantia plant and fruit (b) Cross section of M. charantia fruit 4 Figure 1.3 The studies of M. charantia fruits and seed flowchart Figure 2.1 Some cucurbitane type triterpene isolated and identified from M. charantia Figure 2.2 Phenolic acids found in M. charantia Figure 2.3 The chemical constituents found in M. charantia seed Figure 2.4 The chemical constituents present in M. charantia seeds essential oil Figure 2.5 The gallic acid structure Figure 2.6 The 1-indanone structure Figure 2.7 The structure activity relationship of 6-hydroxyl-2benzylidene-1-indanone derivatives 68a-t Figure 2.8 The structure of called donepezil [69] Figure 2.9 The dengue virus serotypes shift in Malaysia Figure 2.10 The structure of synthetic fluorogenic peptide Boc-Gly-Arg-Arg-AMC Figure 2.11 The structure of 6-amino-4-methylcoumarin (AMC) Figure 2.12 The benzothiazole derivatives [74-80] Figure 2.13 The structure of epigallocatechin gallate [81] and kaempferol [82] Figure 2.14 The structure of compound 83 Figure 3.1 The summary of M. charantia fruits and seed study Figure 3.2 The DENV-2 NS2B/NS3 protease assay procedure using M. charantia fruit extract Figure 3.3 The IC50 DENV-2 NS2B/NS3 protease assay procedure using the synthesized compound

  existe pas de thérapies curatives ou préventives de la dengue (DF). Seuls des soins médicaux complémentaires et une thérapie par les fluides sont disponibles pour lutter contre la dengue. Certains patients optent pour des mesures de prévention et de traitement utilisées par les peuples anciens ou des remèdes populaires pour traiter la maladie. L'un des remèdes populaires utilisés par les patients atteints de dengue à l'hôpital de Malaisie est la consommation de soupe de viande de grenouille au melon amer (Momordica charantia). La chair des fruits et les graines du Momordica charantia (M. charantia) ont été utilisées dans cette étude. L'huile des graines de M. charantia a été extraite par extraction enzymatique aqueuse (AEE) et le rendement en huile a augmenté avec la condition optimale pour extraire l'huile de graines était d'utiliser HEL1:X7 (5:1.25). L'huile de graines ainsi que la teneur en lignine, hémicelullose, sucres solubles et acides uroniques étaient différentes selon le type de cocktail enzymatique. D'autre part, l'extrait d'acétate d'éthyle de la chair du fruit de M. charantia a inhibé 38.11±1.06 % de la DENV-2 protéase NS2B/NS3. Sur la base de l'analyse GC-MS, on a trouvé la trace d'acide gallique et on a sélectionné les cadres structurels de base pour la synthèse de l'inhibiteur de protéase DENV-2 NS2B/NS3. Les dérivés de l'indanone substituée par le pyrazolyle, 90a-t, ont été synthétisés et caractérisés par analyse FTIR, RMN et LC-MS. Un pourcentage approprié de xxiii rendement des composés a été obtenu. Les composés 90a-t ont été soumis à une étude in silico en utilisant AutoDock4.2 pour identifier le mode de liaison et la conformation avec la structure cristalline de la protéine d'homologie de Wichapong. Cinq composés ont obtenu une énergie libre de liaison (FEB) inférieure à -7.55 kcal/mol. Le composé 90p a montré huit interactions de liaison hydrogène, une interaction d'empilement π-π, et une interaction π-alkyle avec le résidu d'acide aminé dans la DENV-2protéase NS2B/NS3. Enfin, les études in vitro des cinq principaux composés synthétisés par ancrage vers la DENV-2 protéase NS2B/NS3 en utilisant le substrat peptidique fluorogène Boc-Gly-Arg-7-amino-4-méthylcoumarine ont été menées. La panduratine A a été utilisée comme témoin positif. Le composé 90p a montré une forte activité d'inhibiteur de la DENV-2 protéase NS2B/NS3 (65.61±0.98 %), tandis que la panduratine A (47.59±1.03 %) et l'acide gallique (25.11±1.11 %) ont été utilisés comme témoins positifs. La valeur de la CI50 pour le composé 90p a été observée à 78.87 µM, ce qui est inférieur à la valeur de 290.27 µM pour la panduratine A. Le composé 90p est considéré comme un puissant composé de type "hit-to-led" dans le développement de l'inhibiteur de DENV-2 protéase NS2B/NS3. Dengue fever (DF), a mosquito-borne viral infection increased from 2.2 million in 2010 to over 3.34 million in 2016 worldwide (WHO, 2019). The infected female mosquitoes of Aedes aegypti and Aedes albopictus (Figure 1.1) transmitted the dengue virus to humans through the bites. The dengue viruses can be categorized into four serotypes based on the antigens on the surface of the virus, which are dengue virus type 1, 2, 3 and 4 (Guzman

Figure 1 . 1 :

 11 Figure 1.1: Images of the two predominant arthropod vectors of dengue virus, Aedes albopictus and Aedes aegypti (Wikimedia commons, 2010 & 2005).

Figure 1 . 2 :

 12 Figure 1.2: (a) M. charantia plant and fruit (b) Cross section of M. charantia fruit (Komakech, 2018).

  Figure 1.3: The studies of M. charantia fruits and seed flowchart.

Fatope

  and co-worker isolated six cucurbitane type triterpene from M. charantia fruits namely, 3,7-bis(acetyloxy)-25-methoxy-(3β,7β,9β,10α,23E)-19norlanosta-5,23-diene-9-carboxaldehyde [3], 3,7-dihydroxy-25-methoxy (3β,7β,9β, 10α,23E)-19-norlanosta-5,23-diene-9-carboxaldehyde [4], (+)-Momordicin I [5], Momordicin II [6], 3,7-bis(acetyloxy)-25-hydroxy-(3β,7β,9β,10α,23E)-19-norla nosta-5,23-diene-9-carboxaldehyde [7], 3β,7β,25-trihydroxycucurbita-5,23(E)-dien-19-al [8a]

  addition, 9-methyl-(9β,10α,23E)-19-norlanosta-5,23,25-triene-3,7-dione [14b], 25hydroxy-9-methyl-(9β,10α,23E)-19-norlanosta-5,23-diene-3,7-dione [8c], (3β,7β,9β,10α,23E)-9-methyl-19-norlanosta-5,23,25-triene-3,7-diol [8d], and

Akihisa

  et al. (2007) tested the cancer chemo preventive effects of cucurbitanetype triterpenoids namely (19R,23E)-5β,19-epoxy-19-methoxycucurbita-6,23,25trien-3β-ol [13a] and (23E)-3β-Hydroxy-7β,25-dimethoxycucurbita-5,23-dien-19-al [14] from the methanolic extract of M. charantia fruits. The isolated compounds exhibited a moderate inhibitory activity in mouse skin carcinogenesis tests. Other two cucurbitane type triterpenoids were isolated and identified as 5β,19-epoxy-cucurbita-6,22E,24-trien-3β-ol [13b] and cucurbita-6,22(E),24-trien-3β-ol-19,5β-olide [13c]

Figure 2 . 1 :

 21 Figure 2.1: Some cucurbitane type triterpene isolated and identified from M. charantia.

Figure 2 . 2 :

 22 Figure 2.2: Phenolic acids found in M. charantia.

Figure 2 .

 2 Figure 2.3 showed some of the chemical constituents found in M. charantia seed.

Figure 2 . 3 :

 23 Figure 2.3: The chemical constituents found in M. charantia seed.

Figure 2 . 4 :

 24 Figure 2.4: The chemical constituents present in M. charantia seeds essential oil.

Figure 2 . 5 :

 25 Figure 2.5: The gallic acid structure.

Figure 2 . 6 :

 26 Figure 2.6: The 1-indanone structure.

  was depicted in Scheme 2.1. The synthetic strategies involved the methylation process of gallic acid [15] using dimethyl sulphate in 20% aqueous alkali to form a 3,4,5trimethyl gallic acid methyl ester [59], which further underwent Grignard reaction with methyl magnesium iodide to yield the desired substrate 3,4,5-trimethoxyacetophenone [60] and 2-(3,4,5-trimethoxyphenyl)propan-2-ol [61] as the minor compound. The acetophenone [60] and [62] were reacted and condensed in 3% aqueous methanolic sodium hydroxide to get a compound [63]. The compound [63] underwent reflux in trifluoroacetic acid to obtain desired compound 4,5,6-trimethoxy-3-(3,4,5trimethoxyphenyl)-2,3-dihydro-1H-inden-1-one [64].

  [65] was protected with tetrahydropyranyl (THP) resulted in 6-[(tetrahydro-2H-pyran-2-yl)oxy]-1-indanone [66] and further reacted with various benzaldehyde to obtain THP protected 2-benzylidene-1-indanone intermediates [67a-b, 68e-t] via Claisen-Schmidt condensation in the presence of ethanolic sodium hydroxide. The deprotection process was carried out by using hydrochloric acid in ethanol resulting in the formation of the desired 2-benzylidene-1-indanone derivatives [68a-b, 68e-t]. Whilst, the single step synthesis of the compound 65 with 3,4-dihydroxybenzaldehyde and 4-hydroxy-3-methoxybenzaldehyde with the present of HCl gas and ethanol as the solvent yielding compound 68c and 68d respectively.

Figure 2 . 8 :

 28 Figure 2.8: The structure of called donepezil [69].

Figure 2 . 9 :

 29 Figure 2.9: The dengue virus serotypes shift in Malaysia (Mudin, 2015).

  fluorophore parts of the synthetic fluorogenic peptide Boc-Gly-Arg-Arg-AMC (Figure 2.10) undergoes cleavage into 6-amino-4-methylcoumarin [58] (Figure 2.11). The AMC substance possesses the ability to exhibit fluorescence as it contained an electron donor, amino groups that can transfer charge as depicted in Figure 2.11. The subsequent appearance of fluorescence is due to the results in loss of energy transfer by the diffusion of the donor-containing substratefragment away from the acceptor-containing substrate fragment, which happened substrate is cleaved by the enzyme[START_REF] Fields | Using Fluorogenic Peptide Substrates to Assay Matrix Metalloproteinases Gregg[END_REF].

Figure 2 . 10 :

 210 Figure 2.10: The structure of synthetic fluorogenic peptide Boc-Gly-Arg-Arg-AMC.

Figure 2 . 11 :

 211 Figure 2.11: The structure of 6-amino-4-methylcoumarin (AMC).

  compounds with anti-dengue propertiesWu and co-worker (2015) synthesized seven novel compounds with a selective and non-competitive inhibition towards the serotype 2 and 3 of dengue virus in vitro and in cells. The benzothiazole derivatives [74-80] (Figure2.12) were reported exhibiting 50% inhibitory concentrations in the low-micromolar and sub-micromolar range in cells.

Figure 2 . 12 :

 212 Figure 2.12: The benzothiazole derivatives [74-80].

Figure 2 . 13 :

 213 Figure 2.13: The structure of epigallocatechin gallate [81] and kaempferol [82]

Figure 2 . 14 :

 214 Figure 2.14: The structure of compound 83

Figure 3 . 1 :

 31 Figure 3.1: The summary of M. charantia fruits and seed study

  operating with a gold electrode, a reference electrode Ag/AgCl, and Chromeleon version 6.8 (Dionex Corp, USA). A Carbopac PA20 column (3 × 150 mm, Dionex) with a guard column (3 × 30 mm, Dionex) was used in the stationary phase. The mobile phase was made of water, 250 mM NaOH in water, and 1 M NaOAc/20 mM NaOH in water. A diluted 46-48 % sodium hydroxide solution and anhydrous sodium acetate in ultrapure water were used as eluents.

Figure 3 . 2 :

 32 Figure 3.2:The DENV-2 NS2B/NS3 protease assay procedure using M. charantia fruit extract.

Scheme 3. 2 :

 2 Scheme 3.2: The synthesis of ethyl 3,4,5-trimethoxybenzoate [85] intermediate.

  Scheme 3.3: The synthesis of 3,4,5-trimethoxybenzohydrazide [86].

  brown crystalline solid; Weight: 2.41 g, 85.31 % (yield); mp. 157-160 °C; Molecular weight: 226.0953 gmol -1 ; C10H14N2O4; FTIR ATR (Vmax/cm -1 ): 3364 (N-H), 3319 (N-H), 1659 (C=O), 1385(C-N); 1 H NMR (500 MHz, DMSO-d6): dH, ppm, 3.7 (6H, s, OCH3), 3.8 (3H, s, OCH3), 4.5 (2H, s, NH2), 7.1 (2H, s, CH), 9.7 (1H, s, NH); 13 C NMR (125 MHz, DMSO-d6): dC, ppm, 56.1 (C7, C9), 60.5 (C8), 104.9 (C2, C6), 128.8 (C1), 140.2 (C4), 152.1 (C3, C5), 165.9 (C10).

  Scheme 3.4: The synthesis of benzylidene indanone derivatives.

  Colour: White solid; Weight: 230.00 mg, 72.21 % (yield); mp. 105-109 °C; Molecular weight: 220.2642 gmol -1 ; FTIR ATR (Vmax/cm -1 ): 2910 (Csp 3 -H), 1693 (C=O ketone), 1576 (C=C aromatic); 1 H NMR (500 MHz, DMSO-d6): dH, ppm,), 4.14 (s, 2H, ArH2), 7.45-7.53 (m, 5H, ArH), 7.56 (s, 1H, H-1', =CH-Ar), 7.68-7.74 (m, 3H, ArH), 7.79 (d, 1H, J = 7.5, ArH); 13 C NMR (125 MHz, DMSO-d6): dC, ppm, 32.38 (C-3), 124.10 (C-1'), 127.19 (C-7), 128.19 (C-5), 129.49 (

  ),130.31 (C-5'), 131.23 (C-4, C-3', C-7'), 133.30 (C-2'), 135.42 (C-2), 135.59 (C-8), 137.65 (C-9), 193.56 (C-1).3.4.2(b) (E)-5'-methoxybenzylidene-1-indanone, 89bColour: White solid; Weight: 434.40 mg, 86.78 % (yield); mp. 138-140 °C; Molecular weight: 250.2899 gmol -1 ; FTIR ATR (Vmax/cm -1 ): 2914 (Csp 3 -H), 1694 (C=O ketone), 1573 (C=C aromatic), 1244 (C-O); 1 H NMR (500 MHz, DMSO-d6): dH, ppm,), 3.82 (s, 3H, -OCH3), 4.05 (s, 2H, ArH2), 7.07 (d, 2H, J=9.0, ArH), 7.45 (t, 1H, ArH), 7.50 (s, 1H, =CH-Ar), 7.66-7.71 (m, 2H, ArH), 7.76 (s, 1H, ArH), 7.77 (s, 1H, ArH), 7.78 (s, 1H, ArH); 13 C NMR (125 MHz, DMSO-d6): dC, ppm, 32.37 (C-3), 55.83 (C-8'), 115.04 (C-4', C-6'), 123.93 (C-1'), 127.08 (C-7), 127.98 (C-5), 128.07 (C-6), 133.02 (C-4), 133.15 (C-3', C-7'), 133.30 (C-2'), 135.06 (C-2), 137.94 (C-8), 150.30 (C-9), 161.11 (C-5'), 193.71 (C-1). solid; Weight: 439.00 mg, 93.68 % (yield); mp. 137-139 °C; Molecular weight: 234.2905 gmol -1 ; FTIR ATR (Vmax/cm -1 ): 2916 (Csp 3 -H), 1694 (C=O ketone), 1574 (C=C aromatic); 1 H NMR (500 MHz, DMSO-d6): dH, ppm,2.42 (s, 3H, ArCH3), 3.99 (s, 2H, ArH2), 7.28(d, 2H, J=8.0, ArH), 2H, ArH), 7.43 (t, 1H, J=7.4, ArH), 2H, ArH), 7.63 (s, 1H, =CH-Ar) 7.81(d, 1H, J=7.5);13 C NMR (125 MHz, DMSO-d6): dC, ppm, 2.104 (C-8'), 35.37 (C-3), 123.60 (C-1'), 124.11 (C-7), 124.93 (C-5), 125.09 (C-6), 126.90 (C-4), 128.78 (C-4', C-6'), 131.27 (C-3', C-7'), 132.45 (C-2'), 134.89 (C-2), 139.50 (C-8), 141.85 (C-5'), 147.30 (C-9), 193.35 (C-1).

  Molecular weight: 254.7094 gmol -1 ; FTIR ATR (Vmax/cm -1 ): 2913 (Csp 3 -H), 1694 (C=O ketone), 1581 (C=C aromatic), 865 (C-Cl); 1 H NMR (500 MHz, DMSO-d6): dH, ppm, 3.85 (s, 2H, ArH2), 7.40-7.44 (m, 2H, ArH), 7.49 (d, 2H, J=7.5, ArH), 7.49 (d, 2H, J=7.5, ArH), 7.52 (t, 1H, J=7.0, ArH), 7.56 (s, 1H, =CH-Ar), 7.76 (d, 1H, ArH); 13 C NMR (125 MHz, DMSO-d6): dC, ppm, 34.44 (C-3), 123.02 (C-1'), 123.98 (C-7), 124.52 (C-5), 125.35 (C-6), 126.12 (C-4), 129.58 (C-4', C-6'), 130.64 (C-3', C-7'), 132.21 (C-2'), 134.70 (C-2), 137.45 (C-5'), 140.39 (C-8), 147.41 (C-9), 194.44 (C-1).

  ppm, 3.43 (d, 4H, J=5.0, ArH2), 3.83 (d, 4H, J=5.0, ArH2), 4.05 (s, 2H, ArH2), 6.77 (d, 2H, J=7.5, ArH), 7.20 (d, 2H, J=8.0, ArH), 7.54 (s, 1H, =CH-Ar), 7.74 (d, 1H, J=8.0, ArH), 7.81-7.83 (m, 3H, ArH); 13 C NMR (125 MHz, DMSO-d6): dC, ppm, 34.24 (C-3), 48.18 (C-2", C-6"), 66.62 (C-3", C-5"), 112.16 (C-1'), 123.86 (C-7), 125.60 (C-5), 127.08 (C-4), 128.01 (C-4', C-6'), 130.54 (C-6), 131.88 (C-3', C-7'), 132.26 (C-2'), 133.60 (C-2), 139.50 (C-8), 148.53 (C-5'), 150.80 (C-9), 194.09 (C-1).

  Molecular weight: 303.3946 gmol -1 ; FTIR ATR (Vmax/cm -1 ): 2917 (Csp 3 -H), 1692 (C=O ketone), 1574 (C=C aromatic); 1 H NMR (500 MHz, DMSO-d6): dH, ppm, 1.64-1.66 (m, 1H, ArH), 1.71 (d, 4H, J=5.0, ArH2), 3.39 (d, 4H, J=5.0, ArH2), 3.83 (s, 2H, ArH2), 7.51 (s, 1H, =CH-Ar), 7,62 (d, 2H, J=9.0, ArH), 7.67-7.69 (m, 3H, ArH); 13 C NMR (125 MHz, DMSO-d6): dC, ppm, 24.44 (C-4"), 25.38 (C-3", C-5"), 32.58 (C-3), 48.50 (C-2", C-6"), 114.85 (C-1'), 123.73 (C-7), 125.99 (C-5), 127.03 (C-4), 127.99 (C-4', C-6'), 130.76 (C-6), 133.15 (C-3', C-7'), 134.12 (C-2'), 134.69 (C-2), 138.32 (C-8), 150.06 (C-5'), 152.30 (C-9), 193.48 (C-1).

  Molecular weight: 280.3156 gmol -1 ; FTIR ATR (Vmax/cm -1 ): 2915 (Csp 3 -H), 1693 (C=O ketone), 1610 (C=C aromatic), 1238 (C-O); 1 H NMR (500 MHz, DMSO-d6): dH, ppm, 3.86 (s, 3H, -OCH3), 3.91 (s, 3H, -OCH3), 4.05 (s, 2H, ArH2), 6.68-6.69 (m, 3H, ArH), 7.53 (s, 1H, =CH-Ar), 7.75-7.77 (m, 3H, ArH), 7.88-7.90 (m, 2H, ArH); 13 C NMR (125 MHz, DMSO-d6): dC, ppm, 37.01 (C-3), 56.01 (C-9'), 56.38 (C-8'), 96.27 (C-4'), 109.15 (C-6'), 118.17 (C-1'), 123.60 (C-7), 125.90 (C-4), 128.67 (C-5), 130.71 (C-7'), 133.60 (C-6), 139.15 (C-2'), 141.33 (C-2), 145.36 (C-8), 148.33 (C-9), 156.63 (C-3'), 161.01 (C-5'), 193.87 (C-1).

  Colour: Cream solid; Weight: 494.70 mg, 83.74 % (yield); mp. 189-191 °C; Molecular weight: 295.2876 gmol -1 ; FTIR ATR (Vmax/cm -1 ): 2914 (Csp 3 -H), 1694 (C=O ketone), 1578 (C=C aromatic), 1507(N-O), 1240 (C-O); 1

  (d, 2H, J=7.1), 7.53 (s, 1H, 7.68 (d, 2H, J=8.2, ArH);13 C NMR (125 MHz, DMSO-d6): dC, ppm 32.01 (C-3), 55.80 (C-8'), 56.10 (C-10), 56.44 (C-11), 104.94 (C-7), 108.58 (C-4), 115.03 (C-4', C-6'), 128.12 (C-1'), 130.52 (C-2'), 131.66 (C-8), 132.86 (C-3', C-7'), 133.81 (C-9), 145.52 (C-2), 149.72 (C-6), 155.62 (C-5), 160.84 (C-5'), 192.64 (C-1).

  13 C NMR (125 MHz, DMSO-d6): dC, ppm, 23.58 (C-9', C-10'), 33.60 (C-8'), 35.33 (C-3), 56.15 (C-10, C-11), 105.37 (C-7), 112.05 (C-4), 126.30 (C-4', C-6'), 127.76 (C-3', C-7'), 130.27 (C-1'), 132.26 (C-2'), 134.05 (C-2), 137.43 (C-9), 138.21 (C-8), 139.03 (C-5'), 140.38 (C-6), 155.34 (C-5), 193.83 (C-1).

  : Orange Solid; Weight: 247.70 mg, 76.59 % (yield); mp. 165-180 °C; Molecular weight 323.3827 gmol -1 ; FTIR ATR (Vmax/cm -1 ): 2913 (Csp 3 -H), 1695 (C=O ketone), 1573 (C=C aromatic), 1189 (C-O); 1 H NMR (500 MHz, DMSO-d6):dH, ppm, 3.01 (s, 6H, -CH3), 3.33 (s, 6H, -OCH3), 3.83 (s, 2H, ArH2), 6.79(d, 2H, ArH), 7.20 (s, 1H, ArH), 7.21 (s, 1H, ArH), 7.36 (s, 1H, =CH-Ar), 7.58(d, 2H, J=8.8, ArH);13 C NMR (125 MHz, DMSO-d6): dC, ppm, 32.28 (C-3), 56.11 (C-8', C-9'), 56.41 (C-10, C-11), 104.95 (C-7), 108.59 (C-4), 112.49 (C-4', C-6'), 122.92 (C-1'), 130.89 (C-2'), 131.09 (C-8), 132.70 (C-2), 132.78 (C-3', C-7'), 144.80 (C-9),

  solid; Weight: 307.20 mg, 84.52 % (yield); mp. 200-210 °C; Molecular weight 323.3827 gmol -1 ; FTIR ATR (Vmax/cm -1 ): 2916 (Csp 3 -H), 1694 (C=O ketone), 1587 (C=C aromatic), 1223 (C-O); 1 H NMR (500 MHz, DMSO-d6): dH, ppm, 1.59 (s, 6H, ArH2), 2.51 (s, 4H, ArH2), 3.37 (s, 6H, -OCH3), 3.93 (s, 2H, ArH2), 6.80 (s, 1H, ArH2), 7.01 (dd, 2H, J=2.1, ArH), 7.10 (s, 1H, ArH), 7.22 (d, 2H, J=8.3, ArH), 7.35 (s, 1H, =CH-Ar); 13 C NMR (125 MHz, DMSO-d6): dC, ppm, 24.39 (C-4"), 25.53 (C-3", C-5"), 32.25 (C-3), 51.83 (C-2", C-6"), 56.73 (C-10, C-11), 104.97 (C-7), 108.60 (C-3), 113.52 (C-1'), 108.12 (C-4), 114.97 (C-4', C-6'), 131.14 (C-2'), 132.26 (C-3', C-7'), 132.73 (C-9), 133.95 (C-2), 144.92 (C-8), 148.80 (C-6), 152.09 (C-5'), 155.27 (C-5), 192.26 (C-1).

  Colour: Yellow solid; Weight: 80.58 % (yield); mp. 200-205 °C; Molecular weight 340.3670 gmol -1 ; FTIR ATR (Vmax/cm -1 ): 2917 (Csp 3 -H), 1694 (C=O ketone), 1608 (C=C aromatic), 1226 (C-O); 1 H NMR (500 MHz, DMSO-d6): , 3.86 (s, 6H, -OCH3), 3.88 (s, 3H, -OCH3), 3.95 (s, 3H, -OCH3), 3.99 (s, 2H, ArH2), 6.48 (s, 1H, ArH), 6.54 (dd, 1H, J=2.2, ArH), 6.95 (s, 1H, ArH2), 7.33 (s, 1H, =CH-Ar), 7.59(d, 1H, J=8.6, ArH), 8.01 (s, 1H, ArH);13 C NMR (125 MHz, DMSO-d6): dC, ppm, 32.25 (C-3), 55.43 (C-9'), 55.58 (C-8'), 56.13 (C-10), 56.21 (C-11), 98.32 (C-4'), 105.09 (C-6'), 105.17 (C-7), 107.20 (C-4), 117.70 (C-1'), 126.74 (C-2'), 130.62 (C-7'), 131.48 (C-8), 132.94 (C-9), 144.65 (C-2), 149.47 (C-6), 154.99 (C-5), 160.57 (C-3'), 162.24 (C-5'), 193.22 (C-1).

:

  Cream solid; Weight: 268.70 mg, 75.62 % (yield); mp. 235-240 °C; Molecular weight 355.3390 gmol -1 ; FTIR ATR (Vmax/cm -1 ): 2917 (Csp 3 -H), 1693 (C=O ketone), 1592 (C=C aromatic), 1502 (N-O), 11870 (C-O); 1 H NMR (500 MHz, DMSO-d6): dH, ppm, 3.78 (s, 3H, -OCH3), 3.78 (s, 3H, -OCH3), 3.75 (s, 2H, ArH2), 4.02 (s, 3H, -OCH3), 6.91 (s, 1H, ArH), 7.28 (s, 1H, =CH-Ar), 7.35 (d, 1H, J=8.8, ArH), 7.68 (d, 1H, J=3.0, ArH), 8.01 (s, 1H, ArH), 8.33 (s, 1H, ArH); 13 C NMR (125 MHz, DMSO-d6): dC, ppm, 34.7

  yellow solid; Weight: 164.62 mg, 76.84 % (yield); mp. 200-205 °C; Molecular weight 428.4788 gmol -1 ; EI-MS (m/z): 429.4847 (M +1 ); FTIR ATR (Vmax/cm -1 ): 1678 (C=O), 1588 (C=N), 1320 (C-N); 1 H NMR (500 MHz, DMSO-d6): dH, ppm, 2.36-2.39 (m, 2H, ArH2), 3.72-3.73 (t, 1H, J=3.8, ArH), 3.83 (s, 9H, -OCH3), 5.06 (1H, d, J=7.0, ArH), 6.99 (2H, s, ArH), 7.09-7.24 (m, 9H, ArH); 13 C NMR (125 MHz, DMSO-d6): dC, ppm,

  solid; Weight: 192.70 mg, 84.05 % (yield); mp. 179-185 °C; Molecular weight 458.5057 gmol -1 ; EI-MS (m/z): 459.4460 (M +1 ); solid; Weight: 157.70 mg, 67.03 % (yield); mp. 178-183 °C; Molecular weight 470.5595 gmol -1 ; EI-MS (m/z): 471.2600 (M +1 ); FTIR ATR (Vmax/cm -1 ): 1672 (C=O), 1592 (C=N), 1320 (C-N); 1 H NMR (500 MHz, DMSO-d6): dH, ppm, 1.26 (s, 6H, -CH3), 2.98 (m, 3H, -CH), 3.71 (s, 3H, -OCH3),

  orange solid; Weight: 171.70 mg, 72.82 % (yield); mp. 178-182 °C; Molecular weight 471.5476 gmol -1 ; EI-MS (m/z): 473.1800 (M +1 ); FTIR ATR (Vmax/cm -1 ): 1675 (C=O), 1590 (C=N), 1321 (C-N); 1 H NMR (500 MHz, DMSO-d6): dH, ppm, 2.71 (s, 2H, ArH), 3.10 (s, 6H, -CH3), 3.87 (s, 6H, -OCH3), 3.98

  brown solid; Weight: 204.10 mg, 79.48 % (yield); mp. 176-184 °C; Molecular weight 513.5842 gmol -1 ; EI-MS (m/z): 514.1100 (M +1 ); Anal. for C30H31N3O5; % calculated (% found): C: 70.16 (68.93), H: 6.08 (5.59), N: 8.18 (7.86). IR (ATR, cm -1 ) umax: 1672 (C=O), 1590 (C=N), 1320 (C-N); 1 H NMR (500 MHz, CDCl3): dH, ppm, 1.27 (s, 4H, ArH), 2.14 (s, 2H, ArH2), 3.87-3.90 (m, 9H, -OCH3), 4.04 (s, 1H, ArH), 7.09 (s, 2H, ArH), 7.43-7.46 (m, 3H, ArH), 7.58-7.70 (m, 5H, ArH), 7.92 (d, 2H, J=7.6, ArH); 13 C NMR (125 MHz, CDCl3): dH,

  yellow solid; Weight: 179.30 mg, 69.15 % (yield); mp. 198-210 °C; Molecular weight 518.5577 gmol -1 ; EI-MS (m/z): 490.1500 (M +1 ); FTIR ATR (Vmax/cm -1 ): 1672 (C=O), 1592 (C=N), 1320 (C-N); 1 H NMR (500 MHz, CDCl3): dH, ppm, 1.76 (s, 2H, ArH2), 3.88-4.01 (m, 15H, -OCH3), 4.55 (s, 1H, ArH), 5.52 (s, 1H, ArH), 6.99 (d, 2H, J=8.2, ArH), 7.28 (s, 1H, ArH), 7.58-7.64 (m, 7H, ArH); 13 C NMR (125 MHz, CDCl3): dH, ppm,

  solid; Weight: 129.70 mg, 51.62 % (yield); mp. 180-187 °C; Molecular weight 502.5583 gmol -1 ; EI-MS (m/z): 503.4131 (M +1 ); FTIR ATR (Vmax/cm -1 ): 1678 (C=O), 1588 (C=N), 1320 (C-N); 1 H NMR (500 MHz, DMSO-d6): dH, ppm, 2.35 (s, 3H, -CH3), 3.23 (s, 2H, ArH2), 3.74 (s, 1H, ArH), 3.82 (s, 6H, -OCH3), 3.98 (s, 9H, -OCH3), 5.07 (s, 1H, ArH), 6.93 (s, 1H, ArH), 7.11-7.21

  solid; Weight: 176.70 mg, 61.61 % (yield); mp. 175-185 °C; Molecular weight 572.6283 gmol -1 ; EI-MS (m/z): 571.3100 (M +1 ); Anal. for C32H35N3O7; % calculated (% found): C: 67.00 (67.28), H: 6.15 (5.29), N: 7.33 (6.40); IR (ATR, cm -1 ) umax: 1675 (C=O), 1590 (C=N), 1320 (C-N); 1 H NMR (500 MHz, DMSO-d6): dH, ppm, 1.24 (s, 1H, ArH), 2.76 (s, 2H, ArH2), 3.74 (s, 6H, -OCH3), 3.90 (s, 9H, -OCH3), 4.05 (s, 1H, ArH), 7.10 (s, 2H, ArH), 7.39-7.45 (m, 3H, ArH), 7.50-7.53 (m, 5H, ArH), 7.87-7.91 (m, 3H, ArH); 13 C NMR (125 MHz, DMSO-d6): dC,

  brown solid; Weight: 61.81 mg, 41.45 % (yield); mp. 208-213 °C; Molecular weight 563.553 gmol -1 ; EI-MS (m/z): 562.8913 (M +1 ); FTIR ATR (Vmax/cm -1 ): 1672 (C=O), 1585 (C=N), 1320 (C-N); 1 H NMR (500 MHz, CDCl3): dH, ppm, 2.89 (s, 2H, ArH2), 3.77 (s, 6H, -OCH3), 3.82 (s, 6H, -OCH3), .94 (s, 6H, -OCH3), 4.51 (s, 1H, ArH), 5.41 (s, 1H, ArH), 6.93-7.00 (m, 2H, ArH), 7.49-7.58 (m, 5H, ArH); 13 C NMR (125 MHz, CDCl3): dH, ppm,

  Figure 3.3: The IC50 DENV-2 NS2B/NS3 protease assay procedure using the synthesized compound.

Figure 4 . 1 :

 41 Figure 4.1: (a) Light micrographs of cross-sections of immature seed of M. charantia (b) cotyledon with abundant lipophilic cellular droplets. Scale bars = (a) 10 μm, (b) 25μm. Ae-aerenchyma; Ar-aril; Ch-chlorenchyma; Ep-epidermis;Hy-hypodermis; Sc-sclerenchyma[START_REF] Giuliani | Micromorphology and anatomy of fruits and seeds of bitter melon (Momordica charantia L., Cucurbitaceae)[END_REF].

Figure 4 . 2 :

 42 Figure 4.2: The FTIR spectrum of hemicellulose

  Figure 4.3 shows the size exclusion chromatogram of the extracted samples.

Figure 4 . 3 :

 43 Figure 4.3: The size exclusion chromatogram of hemicelluloses from different M. charantia seed extracted free materials.

Figure 4 .

 4 Figure 4.4 showed various peaks indicating the presents of different

Figure 4 . 4 :

 44 Figure 4.4: The GC-MS chromatogram of M. charantia fruit ethyl acetate extract.

  Figure 4.5.

Figure 4 . 5 :

 45 Figure 4.5: The formation of compound 85

  Figure 4.6. The important absorption bands in IR spectrum of compound 85 were observed at 2940 (Csp 3 -H), 1715 (C=O ester), 1181 (C-O, ester). The different in absorption band characteristic were observed at 2940-3020 cm -1 for both compounds. The FTIR spectrum of compound 84 absorption band was broader indicating at 2940-3020 cm -1 indicated the presence of O-H stretching frequencies with the overlapped signal of Csp 3 -H stretching.

Figure 4 . 6 :

 46 Figure 4.6: The comparison between FTIR spectrum of compound 84 (starting material) and synthesized compound 85

Figure 4 . 7 :

 47 Figure 4.7: The 1 H NMR spectrum of compound 85

Figure 4 . 8 :

 48 Figure 4.8: The 13 C NMR spectrum of compound 85

  compound 86 and ethanol as the side product. The reaction mechanism were illustrated in the Figure4.9.

Figure 4 . 9 :

 49 Figure 4.9: The reaction mechanism for the formation of compound 86

  band of compound 85 at 1715 cm -1 and the appearance by the sharp C=O amide band at 1659 cm -1 showed that amide was formed.The conjugation of carbonyl group in compound 86 with the neighbouring nitrogen atom of amide, -NH-NH2 weakening the C=O vibration. Hence, the wavenumber/frequency of C=O amide was lower compared with the wavenumber/frequency of C=O ester in compound 85. The comparison between the FTIR spectrum of compound 85 and synthesized compound 86 were depict in Figure4.10.

Figure 4 . 10 :

 410 Figure 4.10: The comparison of compound 85 and compound 86 FTIR spectra.

Figure

  Figure 4.11: The 1 H NMR spectrum of compound 86

Figure 4 . 13 :

 413 Figure 4.13: The formation of compounds 89a-t

Figure 4 . 14 :

 414 Figure 4.14: The FTIR spectrum of compound 89b

Figure 4 . 15 :

 415 Figure 4.15: The 1 H NMR spectrum of compound 89b

Figure 4 .

 4 Figure 4.16: The 135 DEPT NMR spectrum of compound 89b

  Figure 4.18 depicted the X-ray crystallography structure of compound 89k, and 89p.

Figure 4 . 18 :

 418 Figure 4.18: The X-ray crystallography structure of compound 89k, and 89p.

  in the compound 86 moiety to form an intermediate cation. Next, the acidic hydrogen attached to the nitrogen was removed and the intermediate underwent subsequent water elimination to form an intermediate carbocation. The intermediate carbocation underwent further rearrangement to form a more stable state called carbocation isomer. The carbocation isomer was captured by the nucleophilic electron from the amine moiety to form a pyrazolyl scaffolding consist of an aromatic five member ring called pyrazolyl cation intermediate. It is due to the subsequently molecular dipole moment can be high as C-N bond is strongly polarised towards nitrogen (McMurray, 2010). The hydride shift occurred at the pyrazolyl cation intermediate with the hydrogen ion shifted to C-11

Figure

  Figure 4.20: The compounds with lowest percentage yield

Figure 4 . 21 :

 421 Figure 4.21: The comparison between FTIR spectrum of compound 86, 89b and synthesized compound 90a-t

  Figure 4.22) indicated the molecular ion peak [MH] + , (+ESI) at m/z 459.4460 which further confirmed the molecular mass of the structure.

Figure 4 . 22 :

 422 Figure 4.22: The LC-MS spectrum of compound 90b

  Molecular formula: C27H26N2O5 Observed Molar mass [MH] +1 : 459.4460 Calculated Molar mass: 458.5057

Figure 4 . 23 :Figure 4 . 24 :

 423424 Figure 4.23: The 1 H NMR spectrum for compound 90b

Figure 4 . 25 :Figure 4

 4254 Figure 4.25: The 13 C NMR spectrum for compound 90b

  stated that the programs that able to poses the RMSD value in the range of 1.5-2 Å, depending on ligand size, are considered to have performed successfully. The overlay docked conformation between the Wichapong homology model and Bz-Nle-Lys-Arg-Arg-H referenced ligand is shown in Figure 4.27.

Figure 4 . 27 :

 427 Figure 4.27: The overlay docked conformation between the Wichapong homology model and Bz-Nle-Lys-Arg-Arg-H referenced ligand (-original ligand conformation; -docked ligand conformation)

  Figure 4.29 depicted the structure of the top five compounds with the lowest FEB.

Figure 4 . 29 :

 429 Figure 4.29: The top five compounds with the lowest FEB

Figure 4 . 30 :

 430 Figure 4.30: The anti-dengue screening of the selected synthesized compounds Note: The final concentration of synthesized compounds was 200 ppm.

Figure 4 .

 4 Figure 4.31: Dose-response curves of synthesized compound 90p and panduratin A on DENV-2 NS2B/NS3 proteases.

2.

  In objective 2: To extract the seed oil of M. charantia and study the composition of the seed lignocellulosic biomass. a. The aqueous enzymatic extraction in this study used the enzyme cocktail HEL1 and X7. In future studies, a single enzyme such as cellulase, endocellulase, arabinase, xylanase, galactanase, hemicellulase, proteinase, or pectinase can be used in the aqueous enzymatic extraction to obtain an appropriate amount of hemicellulose.

  4. In objective 4: To conduct in silico molecular docking screening of the synthesized compounds. a. The compound could undergo a qualitative structure-activity relationship study to get details insight into each functional group per its function. b. Other crystal structures or protease from dengue virus type-1 can be used to analyse it's in binding interaction. 5. In objective 5: To perform in vitro studies towards DENV-2 NS2B/NS3 protease of synthesized compounds. a. Further analysis such as toxicity study, MIC, and in vivo analysis can be done to investigate compound 90p potential as DENV-2 NS2B/NS3 protease inhibitor.

Figure 1 . 1 :

 11 Figure 1.1: Images des deux principaux arthropodes vecteurs du virus de la dengue, Aedes albopictus et Aedes aegypti (Wikimedia commons, 2010 & 2005).

Figure 1 . 2 :

 12 Figure 1.2: (a) Plante et fruit de M. charantia (b) Coupe transversale du fruit de M. charantia(Komakech, 2018).

  Le graphique de présentation des fruits et des graines de M. charantia

  triméthoxybenzoïque disponible dans le commerce [84] est estérifié à l'aide d'acide sulfurique et d'éthanol pour produire du 3,4,5-triméthoxybenzoate d'éthyle [85]

Figure 3 . 1 :

 31 Figure 3.1: Le spectre FTIR de l'hémicellulose

Tableau 3. 2 :

 2 Le pourcentage de rendement pour l'extraction des fruits de M. charantia extraction process was triplicated. The percentage of fruits flesh extracted = Weight of extract obtained (mg)

Figure 3 . 2 :

 32 Figure 3.2: Le chromatogramme GC-MS de l'extrait d'acétate d'éthyle du fruit de M. charantia.

  figure 3.3 montre la conformation amarrée par superposition entre le modèle d'homologie de Wichapong et le ligand référencé Bz-Nle-Lys-Arg-Arg-H.

Figure 3 . 3 :

 33 Figure 3.3: La superposition de la conformation ancrée entre le modèle d'homologie de Wichapong et le ligand référencé Bz-Nle-Lys-Arg-Arg-H (-conformation du ligand original; -conformation du ligand ancré)

Figure 3 . 4 :

 34 Figure 3.4: L'interaction de liaison de 90p avec la DENV-2 protéase NS2B/NS3.(--hydrogen bond; -π-π stacking; -π-alkyl)

Figure 3 . 5 :

 35 Figure 3.5: Courbes dose-réponse du composé 90p et de la panduratine A sur les DENV-2 protéases NS2B/NS3.

Table 3

 3

	.1	Seed samples and their enzyme cocktail ratios	42
	Table 3.2	Seed sample codes for acid soluble sugar analysis and their
		descriptions	44
	Table 4.1	Percentages of seed oil recovered by aqueous enzymatic
		extraction method using combination of different enzyme
		cocktail ratios	90
	Table 4.2	The FAMEs compositions (%) of M. charantia seed oils	93
	Table 4.3	Sugar analysis of EFM by HPAEC-PAD	97
	Table 4.4	The percentage of yield for M. charantia fruits extraction
	Table 4.5	The inhibition activity of M. charantia fruits extract
		towards DENV-2 NS2B/NS3 protease

Table 4

 4

.6 The major phytochemicals identified in the M. charantia fruit ethyl acetate extract by GC-MS Table

4

.7

The physical properties of synthesized compounds 89a-t Table

4

.8

31], linoleic acid [32], oleic acid [33], arachidic acid [34], and stearic acid [35] were

  found in M.

	. In the study of biodiesel
	mixture of fatty acid methyl esters (FAMEs) with methanol, Škrbić and co-worker
	(2015) stated that some of the major FAMEs such as, palmitic acid [charantia seed oil. According to Chen et al., the α-eleostearic acid [36] present in bitter
	melon seed oil could reduce hepatic triglyceride accumulation (Chen et al., 2016).

Scheme 2.3: The synthesis of donepezil analogues.

						. The
	general eco-friendly synthetic pathway for the synthesis of donepezil analogues is
	described in Scheme 2.3.
	72a : R1= R4=R5=H; R2=OH; R3=OCH3 72i : R1= R3=R4=R5=H; R2=OCH3
	72b : R1= R4=R5=H; R2= OCH3; R3=OH 72j : R1= R2=R4=R5=H; R3=OCH2O-
	72c : R1= R3= R4=R5=H; R2=OH	72k : R1= R2= R5=H; R3= R4=OCH3
	72d : R1= OH; R2=R3= R4=R5=H	72l : R1= R3= R5=H; R2= R4=OCH3
	72e : R1= R2= R4=R5=H; R3=OH	72m : R1= R4=OCH3: R2= R3=R5= H
	72f	:	R1= R4=R5=H; R2= R3=OCH3	72n	:	R1=R3=H; R2= OCH3; R4= R5=OCH2O-
	72g	:	R1= R3=R4=R5=H; R2=OCH3	72o	:	R1= OCH3; R2=R3=H; R4=
						R5=OCH2O-
	72h : R2= R3=R4=R5=H; R1=OCH3
			Reagents and conditions: i) Amberlyst A-26, Methanol, 40-50°C,))) 250W;

*

Table 3 . 2 :

 32 Seed sample codes for acid soluble sugar analysis and their descriptions.

	Sample codes	Enzyme (%)/Sample Weight
		HEL1	X7
	M0L	-	-
	M1L	5	-
	M2L	5	1.25
	M3L	5	2.5
	M4L	5	5

  : Yellow solid; Weight: 463.30 mg, 87.95 % (yield); mp. 164-168 °C;

	Molecular weight: 263.3313 gmol -1 ; FTIR ATR (Vmax/cm -1 ): 2911 (Csp 3 -H), 1696
	(C=O ketone), 1575 (C=C aromatic), 1339 (C-H); 1 H NMR (500 MHz, DMSO-d6):
	dH, ppm, 3.02 (s, 6H, N-CH3), 4.03 (s, 2H, ArH2), 6.80 (d, 2H, J=9.0, ArH), 7.51 (s,
	1H, =CH-Ar), 7.63 (d, 2H, J=8.5, ArH), 7.67-7.68 (m, 3H, ArH), 7.74 (d, 1H, J=7.6,
	ArH);

13

C NMR (125 MHz, DMSO-d6): dC, ppm, 32.61 (C-3), 112.46 (C-4', C-6'), 122.68 (C-1'), 123.67 (C-7), 126.99 (C-5), 127.95 (C-4), 129.95 (C-6), 133.19 (C-3', C-7'), 134.56 (C-2'), 138.47 (C-2), 145.53 (C-5'), 149.98 (C-8), 151.79 (C-9), 193.39

  % (yield); mp. 210-216 °C;

	Molecular weight 310.3413 gmol -1 ; FTIR ATR (Vmax/cm -1 ): 2914 (Csp 3 -H), 1694
	(C=O ketone), 1628 (C=C aromatic), 1210 (C-O); 1 H NMR (500 MHz, DMSO-d6):
	dH, ppm, 3.79 (s, 6H, -OCH3), 3.87 (s, 3H, -CH3), 3.92 (s, 2H, ArH2), 7.02 (s, 1H,
	ArH), 7.04 (s, 1H, ArH), 7.17

  White solid; Weight: 245.40 mg, 83.37 % (yield);

	-OCH3), 7.20 (s, 1H, ArH), 7.21 (s, 1H, ArH), 7.30 (d, 2H, J=6.9, ArH), 7.58 (s, 1H,
	6 5 =CH-Ar), 7.63 (d, 2H, J=8.2, ArH); 13 C NMR (125 MHz, DMSO-d6): dC, ppm, 21.49 4 9 8 O 7 3 2 1 H O 10 (C-8'), 32.09 (C-3), 56.10 (C-10), 56.44 (C-11), 104.98 (C-7), 108.50 (C-4), 130.09 1' 4' 3' 2' 7' 11 O (C-4', C-6'), 130.50 (C-2'), 131.01 (C-3', C-7'), 131.56 (C-8), 132.82 (C-9), 135.37
	6' 5' (C-2), 139.92 (C-1'), 145.49 (C-5'), 149.76 (C-6), 155.68 (C-5), 192.35 (C-1). O
								8'
	11	O O	6 5	4 7	9 8	O 3 1 2	7'	H 1' 2'	3'	4'
								6'	5'
										8'
	Molecular weight 294.3419 gmol -1 ; FTIR ATR (Vmax/cm -1 ): 29147(Csp 3 -H), 1695
	(C=O ketone), 1574 (C=C aromatic), 1223 (C-O); 1 H NMR (500 MHz, DMSO-d6):
	dH, ppm, 2.36 (s, 3H, ArCH3), 3.36 (s, 2H, ArH2), 3.90 (s, 3H, -OCH3), 3.97 (s, 3H,
					62

  : Light yellow Solid; Weight: 266.83 mg, 82.76 % (yield); mp. 168-175 °C;

	Molecular weight 322.3945 gmol -1 ; FTIR ATR (Vmax/cm -1 ): 2914 (Csp 3 -H), 1694
	(C=O ketone), 1575 (C=C aromatic), 1181 (C-O); 1 H NMR (500 MHz, DMSO-d6):
	dH, ppm, 1.27 (d, 6H, J=7.0, -CH3), 2.92-2.98 (m, 1H, -CH) 3.93 (s, 6H, -OCH3),
	3.99 (s, 2H, ArCH2), 6.97 (s, 1H, ArH), 7.27 (s, 1H, ArH), 7.30 (s, 1H, =CH-Ar),

7.33

(d, 2H, J=6

.9 ,ArH), 7.59

(d, 2H, J=8.3, ArH)

;

  : Light brown solid; Weight: 124.93 mg, 49.62 % (yield); mp. 205-208 °C;

	Molecular weight 503.5033 gmol -1 ;	EI-MS (m/z): 505.1142 (M +1 );

FTIR ATR (Vmax/cm -1 ): 1678 (C=O), 1588 (C=N), 1320 (C-N); 1 H NMR (500 MHz, CDCl3): dH, ppm, 2.31 (s, 2H, ArH2), 3.81 (s, 6H, -OCH3), 3.85-3.88 (m, 6H, -OCH3), 4.31 (s, 1H, ArH), 5.56 (s, 1H, ArH), 7.18-7.21

(d, 2H, J=8.8, ArH), 6H, ArH), 7.92 (d, 1H, J=7.6, ArH)

;

13

C NMR (125 MHz, CDCl3): dH, ppm, 29.62,

Table 4

 4

	.2: The FAMEs compositions (%) of M. charantia seed oils
			Compositions (%)
	Fatty Acid
		M0	M1	M2	M3	M4
	Palmitic Acid (C16)	8.84 ± 0.12	7.21 ± 0.09	17.97 ± 0.11	17.26 ± 0.10	11.23 ± 0.09
	Stearic Acid (C18)	-	-	3.28 ± 0.13	-	-
	Elaidic acid	0.47 ±	8.06 ±	20.42 ±	28.06 ±	29.63 ±
	(C18:1n9t)	0.11	0.08	0.09	0.11	0.08
	Oleic acid (C18:1n9c)	35.80 ± 0.08	17.31 ± 0.11	-	-	-
	Vaccenic acid	2.84	5.02 ±	2.52 ±	5.02 ±	7.58 ±
	(C18:1n7)	±0.10	0.09	0.09	0.11	0.10
	Stearidonic acid	13.55 ±	13.42 ±	19.43 ±	16.42 ±	12.83 ±
	(C18:4n3)	0.11	0.11	0.08	0.08	0.09
	Paullinic acid	6.50 ±	6.19 ±	13.51 ±	5.19 ±	2.41 ±
	(C20.1)	0.12	0.12	0.11	0.09	0.10
	Unknown 1	10.66 ± 0.09	10.14 ± 0.09	7.81 ± 0.10	15.14 ± 0.09	15.31 ± 0.10
	Unknown 2	9.55 ± 0.11	9.96 ± 0.10	6.00 ± 0.10	4.96 ± 0.10	8.69 ± 0.11
	Unknown 3	11.79 ± 0.12	8.83 ± 0.11	9.07 ± 0.09	7.98 ± 0.11	12.32 ± 0.11

Note: Values are % of total fatty acid expressed as mean ± SD of three separate determinations

Table 4

 4

	.3.
	M0L showed the lowest amount of acid galacturonic (8.42 %). The percentage
	of acid galacturonic increased with the presence of enzyme cocktails in M3L (16.12
	%) < M4L (16.37 %) < M1L (22.00 %) < M2L (22.25 %). These results suggested that
	the presence of pectinase in the enzyme cocktails facilitated the breakdown of cell wall
	pectic polysaccharides into simpler molecules like acid galacturonic (Abdulkarim et
	al., 2005; Welker et al., 2015; Biz et al., 2014).
	The concentration of glucose in M0 was only 57 %. The percentage of glucose
	increased with the introduction of enzyme cocktails; M3 (65.42 %) < M4 (66.77 %) <

M1 (71.34 %) < M2 (76.20 %). These results indicated that more cellulose from the cell wall was cleaved into different types of sugars (glucose, xylose, galactose,

Table 4

 4

	.3: Sugar analysis of EFM by HPAEC-PAD

Table 4 . 4 :

 44 The percentage of yield for M. charantia fruits extraction

		Percentage of
	Extract
		yield (%)
	n-hexane	0.49 ± 0.03
	Ethyl acetate	1.48 ± 0.11
	Methanol	3.47 ± 0.04

Note: The extraction process was triplicated.

The percentage of fruits flesh extracted =

!"#$%& () "*&+,-& (.&,#/"0 (2$)

Table 4

 4

	.5: The inhibition activity of M. charantia fruits extract towards DENV-2
	NS2B/NS3 protease.
	Extract	% Inhibition
	n-hexane	21.73 ± 1.01
	Ethyl acetate 38.11 ± 1.06
	Methanol	10.37 ± 1.13

Note: The assay was triplicated with the final concentration of 200 ppm.

Table 4

 4

	.6.

Table 4 .6: The

 4

	No.	Retention time (min)	Peak total area (%)	Compound name	CAS No.	Library match (%)
	1	48.521	5.707	10,12,14-nonacosatriynoic acid	126115-85-3	53.0
	2	43.915	5.439	(5α)-3-Ethyl-3-hydroxyandrostan-17-one	57344-99-7	49.1
	3	47.573	4.891	(3β,5S,22E)-3,5-cyclostigmasta-6,22-diene	107304-12-1	56.1
	4	34.533	2.111	bis(2-ethylhexyl) ester hexanedioic acid	103-23-1	63.7

major phytochemicals identified in the M. charantia fruit ethyl acetate extract by GC-MS.

Table 4 . 7 :

 47 The physical properties of synthesized compounds 89a-t

	Compounds	Formula	Yield (%)	Colour	Melting point (ºC)
	89a	C16H12O	72.21	White	105-109
	89b	C17H14O2	86.78	White	138-140
	89c	C17H14O	93.68	White	137-139
	89d	C16H11ClO	94.48	White	137-139
	89e	C19H18O	64.87	Cream	134-137
	89f	C18H17NO	87.95	Yellow	164-168
	89g	C20H19NO2	93.62	Yellow	177-181
	89h	C21H21NO	90.69	Cream	178-181
	89i	C18H16O3	84.12	Yellow	181-185
	89j	C17H13NO4	83.74	Cream	189-191
	89k	C18H16O3	84.90	Light yellow	178-181
	89l	C19H18O4	94.28	Light yellow	210-216
	89m	C19H18O3	83.37	White	198-210
	89n	C18H15O3Cl	78.62	Cream	178-181
	89o	C21H22O3	82.76	Light yellow	168-175
	89p	C20H21NO3	76.59	Orange	165-180
	89q	C20H23NO4	86.20	Light orange	195-199
	89r	C20H21NO3	84.52	Brown	200-203
	89s	C20H20O5	80.58	Yellow	200-205
	89t	C19H17NO6	75.62	Cream	233-237

Table 4 . 8 :

 48 The important FTIR and 1 H,13 C NMR data of compounds 89a-t

	FTIR (cm -1 )
	Compound

7.53 192.64 145.52 155.62 149.72 128.12 130.52 89m 1695 1179-1223 1574-1630 694-836 7.58 192.35 149.76 155.68 149.76 139.92 130.50 89n 1693 1180-1225 1574-1628 692-838 7.51 193.70 134.51 155.40 148.80 118.63 134.51 89o 1694 1181-1224 1575-1629 691-838 7.30 193.83 134.05 155.34 140.38 130.27 132.26 89p 1695 1180-1225 1573-1627 694-836 7.36 192.14 132.70 155.17 151.17 122.92 130.89 89q 1695 1180-1226 1575-1628 692-837 7.53 193.25 132.54 155.11 144.54 127.01 131.14

Table 4 . 9 :

 49 The physical properties of synthesized compounds 90a-t

	Compounds	Formula	Yield (%)	Colour	Melting point (ºC)	Molecular Weight (M +1 )
	90a	C26H24N2O4	76.84	Light yellow 200-205	429.4847
	90b	C27H26N2O5	84.05	Cream	179-185	460.6440
	90c	C27H26N2O4	77.78	White	189-193	443.1400
	90d	C26H23ClN2O4	83.45	Cream	179-185	463.8210
	90e	C29H30N2O4	67.03	Cream	178-183	471.2600
	90f	C28H29N3O4	72.82	Dark orange	178-182	473.1800
	90g	C30H31N3O5	79.48	Light brown	176-184	514.1100
	90h	C31H33N3O4	48.55	Light brown	183-187	512.6138
	90i	C28H28N2O6	51.03	Yellow	198-210	489.1384
	90j	C27H25N3O7	49.62	Light brown	205-208	505.1142
	90k	C28H28N2O6	70.70	Cream	195-200	489.5395
	90l	C29H30N2O7	69.15	Light yellow 201-204	490.1500
	90m	C29H30N2O6	51.62	White	180-187	503.4131
	90n	C28H27ClN2O6	44.28	Cream	175-183	523.1600
	90o	C31H34N2O4	71.65	Cream	180-183	499.0600
	90p	C30H33N3O6	63.17	Light orange 183-187	532.4800
	90q	C32H35N3O7	61.61	Cream	183-186	571.3100
	90r	C33H37N3O6	59.68	Cream	179-185	574.2900
	90s	C30H32N2O8	47.43	Yellow	179-185	549.1167
	90t	C29H29N3O9	41.45	Light brown	208-213	562.8913

Table 4

 4

	.10: The FTIR absorption band of compounds 90a-t

Table 4 .

 4 12:The FEB of ligand-protein docking.

	Ligand	FEB (kcal/mol)
	Positive Control (Peptide Inhibitor)	-6.58
	Gallic Acid	-5.73
	90p	-8.12
	90c	-7.75
	90e	-7.71
	90g	-7.63
	90r	-7.61
	90h	-7.53
	90o	-7.51
	90q	-7.54
	90d	-7.36
	90j	-7.21
	90a	-7.14
	90i	-7.13
	90t	-7.09
	90s	-7.01
	90b	-6.99
	90k	-6.95
	90n	-6.90
	90l	-6.87
	90m	-6.82
	90f	-6.71

Table 4 . 13 :

 413 The hydrogen bonding details of compound 90p.

	Name	H-bond distance (Å)	H-bond angle (º)
	NS3:Gly153:HN-90p:O	2.56	139.93
	90p:C-NS3:Asn152:OD1	3.10	149.89
	90p:C-NS3:Phe130:O	3.38	149.78
	NS3:Pro132:CA-90p:O	3.47	110.96
	90p:C-NS3:His51:O	3.51	138.78
	90p:C-NS3:Ser131:O	3.54	116.99
	90p:C-NS2B:Met84:O	3.61	170.88
	90p:C-NS3:Phe130:O	3.74	131.52

Table 2 . 1 :

 21 Échantillons de semences et leurs rapports de cocktail d'enzymes.

	Seed samples	Enzyme (%)/Seed weight (g)
		HEL1	X7
	M0	-	-
	M1	5	-
	M2	5	1.25
	M3	5	2.5
	M4	5	5

b) La synthèse des dérivés de la benzylidène indanone, 89a-t

  C et FTIR. La synthèse de l'intermédiaire 3,4,5-triméthoxybenzoate d'éthyle [85].Le compose 85 a ensuite réagi avec du monohydrate d'hydrazine pour obtenir du 3,4,5triméthoxybenzohydrazide [86]. Le composé 85 (1.00 mmol) a été dissous dans 10 ml d'éthanol et agité pendant 30 minutes. Ensuite, du monohydrate d'hydrazine (5.00 mmol) a été ajouté à la solution et agité pendant la nuit jusqu'à la formation d'un précipité blanc (Schéma 2.2). Le précipité [86] a été filtré et lavé à l'éthanol froid[START_REF] Kamal | Synthesis, biological evaluation, and molecular modeling of (E)-2-aryl-5-styryl-1,3,4-oxadiazole derivatives as acetylcholine esterase inhibitors[END_REF]. Les structures chimiques du composé ont été confirmées par RMN 1 H, et RMN13 C. Une indanone (2.0 mmol) [87a-b] a été dissoute et agitée dans 10 % p/v d'hydroxyde de potassium (KOH) dans du méthanol pendant environ 15 minutes. Ensuite, un autre type d'aldéhyde aromatique (2.0 mmol) [88a-b] a été ajouté à la solution et le mélange réactionnel a été agité pendant une heure ou jusqu'à la fin de la réaction sous reflux(Korotchenko et al., 2014). Le schéma 2.3 décrit le schéma de réaction pour la synthèse des dérivés de la benzylidène indanone [89a-t]. La réaction a été surveillée à l'aide de la CCM jusqu'à son achèvement. Le pH du mélange réactionnel a ensuite été ajusté à 5-6 en ajoutant une solution d'acide chlorhydrique à 10 % p/v. Le précipité a été filtré par filtration sous vide, lavé avec du méthanol froid et séché sous vide. Les composés synthétisés ont ensuite été soumis à une analyse de caractérisation afin de déterminer leurs propriétés physico-chimiques.

	O O			O	H N NH 2
			NH 2 NH 2. H 2 O
	O	O	CH 3 CH 2 OH	O	O
	O			O
					86
	Scheme 2.2: La synthèse du 3,4,5-triméthoxybenzohydrazide [86].
	O 84 O OH O 2.2.3(Scheme 2.3: La synthèse des dérivés de la benzylidène indanone O 85 Scheme 2.1: O O H O O H 2 SO 4 O O

  Le pourcentage le plus élevé d'huile de graines (6.26 ± 0.53 %) a été extrait de M2 en utilisant une combinaison de 5 % de HEL1 à 1.25 % de cocktails enzymatiques X7, suivi de M4 (5.03 ± 0.28) en utilisant une combinaison d'un rapport égal de HEL1 et de cocktails enzymatiques X7. Sur la base des résultats obtenus, la quantité d'huile extraite a montré que l'augmentation du rapport entre les cocktails d'enzymes X7 et HEL1 utilisés dans l'extraction n'était pas proportionnelle au pourcentage d'huile extraite. Cela peut être observé à partir du pourcentage d'huile extraite ne montrent pas d'augmentation significative par l'augmentation du rapport d'enzymes après M2. Le tableau 3.1 indique la quantité de cocktail d'enzymes introduite dans le processus de réaction et les pourcentages d'huile de graines récupérés par la méthode d'extraction enzymatique aqueuse utilisant la combinaison de différents rapports de cocktail d'enzymes.

	O NH Tableau 3.1: Pourcentages d'huile de graines récupérés par la méthode d'extraction NH 2 enzymatique aqueuse utilisant la combinaison de différents rapports de cocktail d'enzymes
	O	O Seed samples +	Acetic acid Reflux Enzyme (%)/Seed weight (g) HEL1 X7	Oil extracted (%)
	O	M0	-	-		3.32 ± 0.38
	86 M1	5	-		4.67 ± 0.11
		M2	5	1.25	6.26 ± 0.53
		M3	5	2.5	4.10 ± 0.44
		M4	5	5		5.03 ± 0.28
	Note: The extraction process was triplicated.
		The percentage of seed oil extracted =	Weight of oil obtained (mg) Weight of dry seed used (mg)

  acide élaïdique était présent dans l'huile extraite. Avec l'introduction des cocktails enzymatiques, le pourcentage d'acide élaïdique est passé à 8.06%, 20.42%, 28.06% et 29.63% pour M1, M2, M3 et M4, respectivement. La présence des cocktails d'enzymes a montré une tendance similaire pour l'acide palmitique, bien que l'augmentation soit moindre. Les compositions des FAME (%) des huiles de graines de M. charantia

	Tableau 3.2: Fatty Acid	M0	M1	Compositions (%) M2	M3	M4
	Palmitic Acid (C16)	8.84 ± 0.12 7.21 ± 0.09 17.97 ± 0.11 17.26 ± 0.10 11.23 ± 0.09
	Stearic Acid (C18)	-	-	3.28 ± 0.13	-	-
	Elaidic acid(C18:1n9t)	0.47 ± 0.11 8.06 ± 0.08 20.42 ± 0.09 28.06 ± 0.11 29.63 ± 0.08
	Oleic acid (C18:1n9c)	35.80 ± 0.08 17.31 ± 0.11	-	-	-
	Vaccenic acid (C18:1n7)	2.84 ±0.10	5.02 ± 0.09 2.52 ± 0.09 5.02 ± 0.11	7.58 ± 0.10
	Stearidonic acid (C18:4n3) 13.55 ± 0.11 13.42 ± 0.11 19.43 ± 0.08 16.42 ± 0.08 12.83 ± 0.09
	Paullinic acid (C20.1)	6.50 ± 0.12 6.19 ± 0.12 13.51 ± 0.11 5.19 ± 0.09	2.41 ± 0.10
	Unknown 1	10.66 ± 0.09 10.14 ± 0.09 7.81 ± 0.10 15.14 ± 0.09 15.31 ± 0.10
	Unknown 2	9.55 ± 0.11	9.96 ± 0.10 6.00 ± 0.10	4.96 ± 0.10	8.69 ± 0.11
	Unknown 3	11.79 ± 0.12 8.83 ± 0.11	9.07 ± 0.09	7.98 ± 0.11 12.32 ± 0.11

  . Dengue fever treatment with Carica papaya leaves extracts. Asian Pacific Journal of Tropical Disease, 1, 330-333. Ali, M. A., Sayeed, M. A., Reza, M. S., Yeasmin, M. S., & Khan, A. M. (2008). Characteristics of seed oils and nutritional compositions of seeds from different varieties of Momordica charantia Linn. cultivated in Bangladesh. Czech Journal of Food Science, 26(4), 275-283.

2.2.2 The M. charantia seed oil extraction method

Aedes aegypti Aedes albopictus

Ces résultats ont également montré que la composition de l'huile différait en fonction des différents rapports d'enzymes X7. Dans M4, la distribution en pourcentage de l'acide élaïdique, de l'acide vaccinique, de l'acide palmitique, de l'acide stéaridonique et de l'acide paullinique augmentait avec l'augmentation du rapport X7. Les résultats suggèrent que le cocktail d'enzymes X7 favorise la réaction du cocktail d'enzymes HEL1 pour hydrolyser les structures cellulaires des graines, car chaque cocktail d'enzymes est constitué de différents types de mélange d'enzymes.En outre, la composition biochimique du pétrole extrait diffère également des études précédentes. Ali et son collègue ont extrait l'huile de graines avec les compositions suivantes : acide palmatique (4.84 ± 0.13 %), acide stéarique (20.21 ± 0.40 %), acide linoléique (6.37 ± 0.46 %) et acide éléostéarique (53.22 ± 0.36 %)[START_REF] Ali | Characteristics of seed oils and nutritional compositions of seeds from different varieties of Momordica charantia Linn. cultivated in Bangladesh[END_REF]. Tandis que les huiles de graines extraites à l'aide de l'AEE contiennent une quantité plus importante d'acide palmitique. En outre, l'acide gras saturé, l'acide stéarique, n'a été détecté que dans le M2. En outre, l'acide gras oméga-3 détecté par Ali et son collègue était l'acide éléostéarique, tandis que l'huile extraite à l'aide de l'AEE était l'acide stéaridonique hautement insaturé. Cette découverte était en accord avec les conclusions de chercheurs antérieurs qui avaient montré que l'extraction assistée par des enzymes peut entraîner une augmentation du rendement en huile ainsi qu'une légère modification des propriétés biochimiques de l'huile extraite(Zuorro

CHAPTER 4 RESULT AND DISCUSSION

4.1

The studies of M. charantia seeds oil and lignocellulosic biomass

The M. charantia seeds oil were extracted using aqueous enzyme extraction (AEE) method. The seed oil composition was determined using GC-MS. The seeds lignocellulosic biomass composition were identified in terms of lignin, soluble sugars, uronic acid, and hemicelullose content.

Aqueous enzyme extraction (AEE) of M. charantia seed oil

The idea of using enzyme in aqueous extraction process was to facilitate the breakdown of cellular and subcellular structures of the seed and increased the dissolution of the soluble compound presence in the seed into the water matrix as well as promote oil released (Ricochon & Muniglia, 2010). Theoretically, this method based on the insolubility of oil rather than on dissolution of oil [START_REF] Johnson | Comparison of alternative solvents for oils extraction[END_REF][START_REF] Rosenthal | Aqueous and enzymatic processes for edible oil extraction[END_REF][START_REF] Kalia | Using Enzymes for Oil Recovery from Edible Seeds[END_REF]. Table 4.1 shows the amount of enzyme cocktail introduced in the reaction process and the percentages of seed oil recovered by aqueous enzymatic extraction method using combination of different enzyme cocktail ratios.

141

In the 1 H NMR spectrum (Figure 4.23) of compound 90b, the -CH signal of H-3 was observed at δH 5.45 indicating the formation of ring as there are no signal observed at δH 5.45-5.00 in compound 86 and 89b [START_REF] Ali | Discovery of novel methanone derivatives acting as antimycobacterial agents[END_REF]. Besides, two high intensity singlet signals were observed at δH 3.82 and 3.85 corresponding to the four methoxy -OCH3 moieties of only two chemical shifts were observed indicating that the signal were overlapped as the nuclei are magnetically equivalent to each other [START_REF] Pavia | Introduction to spectroscopy[END_REF]. The signal integrals showed a proximate number of proton, 7 and 5 which similar the number of proton presence in the H-7', H-7'', H-8'' and H-9''.

The multiplet can be observed at the downfield region of δH 7.48-7.64 was assigned as the H-aromatic of benzene ring. The two doublet located at δH 6.96 (J=8.5 Hz) and δH 7.88 (J=10.0 Hz) with a triplet located at δH 7.39 (J= 5.0 Hz) were assigned to aromatic proton as the signal appeared in the aromatic region (Lampman et al., 2010). It is hard to assign the proton presence in a benzene or multiple aromatic in this case compound 90b. Three benzene rings in compound 90b increased the deshielded effect by creating its own ring current when placed in the magnetic field.

In addition, the carbons with sp 2 bond in the benzene ring holds charge closer to the nucleus, thus easier to be deshielded (Balci, 2005a).

The two low intensity signal were observed at δH 2.78 and 3.09 of H-4. The 1 H-1 H COSY NMR spectrum (Figure 4.24) showed a cross peak between the neighbouring proton H-11 at δH 3.96. The signal appears as two singlet signals instead of doublet suggested due to the identification of each hydrogen as it is equivalent to each other [START_REF] Balci | Chapter 12: Chemical Shift[END_REF]. The spectrum revealed a cross peak of H-3 and H-11 at δH 5.45 and 3.96 respectively, indicating that both signal are correlated with each other.

This figure highlighted the binding interaction of the original ligand and positive docked ligand confirmation with the protease active site, which were in an almost identical conformation with each other. The similar conformation indicated that programs used were able to reproduce the experimentally-known complex. The docking parameters obtained was utilized in the ligand-DENV-2 NS2B/NS3 protease docking studies using the synthesized compounds.

The ligand-DENV-2 NS2B/NS3 protease docking studies

The DENV-2 NS2B/NS3 protease was built up by a single-stranded RNA of positive polarity with 3391 amino acids residue comprising three structural and seven non-structural proteins, arranged in the order of C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5 [START_REF] Irie | Sequence analysis of cloned dengue virus type 2 genome (New Guinea-C strain)[END_REF]. The NS2B/NS3 protease played an essential role in virus replication [START_REF] Othman | Docking of Noncompetitive Inhibitors into Dengue Virus Type 2 Protease: Understanding the Interactions with Allosteric Binding Sites[END_REF][START_REF] Phong | Dengue protease activity: The structural integrity and interaction of NS2B with NS3 protease and its potential as a drug target[END_REF]. Thus, it been selected as the anti-dengue target.

The targeted amino acid residue possesses in the NS2B/NS3 protease were identified as three catalytic triad amino acid residues that located in NS3 serine protease, namely His51, Asp75 and Ser135. Whilst, the NS2B protease acted as a NS3 serine protease co-factor for an optimal catalytic activity and reported to be responsible for cleaving the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 junctions, preferentially with adjacent basic residues [START_REF] Arias | Dengue 2 virus ns2b and ns3 form a stable complex that can cleave ns3 within the helicase domain[END_REF][START_REF] Preugschat | In vitro processing of dengue virus type 2 nonstructural proteins NS2A, NS2B, and NS3[END_REF][START_REF] Cahour | Cleavage of the dengue virus polyprotein at the NS3/NS4A and NS4B/NS5 junctions is mediated by viral protease NS2B-NS3, whereas NS4A/NS4B may be processed by a cellular protease[END_REF][START_REF] Falgout | Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins[END_REF].

Altogether, 20 final synthesized compounds [90a-t], and gallic acid were docked excluding, the positive control peptide inhibitor. The ligand-protein docking results based on the free energy of binding (FEB) scores are tabulated in Table 4.12.

All the docked ligand showed positive interaction with the DENV-2 NS2B/NS3 protease with the binding energy less than -5.0 kcal/mol. The synthesized compounds displayed good binding interaction with the FEB less than the gallic acid and the peptide inhibitor. Five synthesized compounds displayed the FEB less than -7.55 kcal/mol with the synthesized compound 90p showed the lowest FEB of -8.12 kcal/mol and the binding interaction of synthesized compound 90p was selected to be discussed further. (--hydrogen bond; --π-π stacking; --π-alkyl)

ABSTRACT

There are neither curative nor preventive therapies of dengue fever (DF). Only supplemental medical care and fluid therapy are available to fight DF. Some patients opt for prevention and treatment measure used by the ancient people or folk remedy to treat sickness. One of folk remedy used among patients with dengue fever in the Malaysia hospital is consumption of frog meat soup with bitter melon (Momordica charantia). The fruits flesh and seeds of Momordica charantia (M. charantia) were utilized in this study. The oil of M. charantia seeds were extracted via aqueous enzymatic extraction (AEE) and the oil yield increased with optimum condition to extract the seed oil was to use HEL1:X7 (5:1.25). The seeds oil as well as the lignin, hemicelullose, soluble sugars, and uronic acids content were different accordance to the type of enzyme cocktail ratio. On the other hand, the M. charantia fruits flesh ethyl acetate extract was found to inhibit 38.11 ± 1.06 % of the DENV-2 NS2B/NS3 protease. Based on the GC-MS analysis the trace of gallic acid was found and was selected the basic structural frameworks for synthesis of the DENV-2 NS2B/NS3 protease inhibitor. The pyrazolyl substituted indanone derivatives, 90a-t were synthesized and characterized using FTIR, NMR, and LC-MS analysis. An appropriate percentage of yield of the compounds were obtained. The compounds 90a-t underwent in silico study using AutoDock4.2 to identify the binding mode and conformation with the Wichapong homology protein crystal structure. Five compounds were found to obtained free energy of binding (FEB) less than -7.55 kcal/mol. Compound 90p showed eight hydrogen bond interactions, one π-π stacking interaction, and one π-alkyl interaction with the amino acid residue in the DENV-2 NS2B/NS3 protease. Lastly, the in vitro studies of top five docked compoundtowards DENV-2 NS2B/NS3 protease by using fluorogenic peptide substrate Boc-Gly-Arg-7-amino-4-methylcoumarin were conducted. The panduratin A was used as the positive control. Compound 90p showed a high DENV-2 NS2B/NS3 protease inhibitor activity (65.61 ± 0.98 %), while panduratin A (47.59 ± 1.03 %), and gallic acid (25.11 ± 1.11 %). The IC50 value for compound 90p was observed to be 78.87 µM which is lower compared to the panduratin A, 290.27 µM. The compound 90p is concluded to be a potent hit-to-led compound in the development of DENV-2 NS2B/NS3 protease inhibitor.