Extraction and assessment of antioxidant and antimicrobial activities of saponins from cocoa pod husk (theobroma cacao l )

MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY ABOSEDE, FUNMILOLA FANIFOSI EXTRACTION AND ASSESSMENT OF ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF SAPONINS FROM COCOA POD

MINISTRY OF EDUCATION AND TRAINING

NHA TRANG UNIVERSITY

ABOSEDE, FUNMILOLA FANIFOSI

EXTRACTION AND ASSESSMENT OF ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF SAPONINS FROM

COCOA POD HUSK (Theobroma Cacao L.)

MASTER THESIS

KHANH HOA - 2020

MINISTRY OF EDUCATION AND TRAINING

NHA TRANG UNIVERSITY

ABOSEDE, FUNMILOLA FANIFOSI

EXTRACTION AND ASSESSMENT OF ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF SAPONINS FROM

COCOA POD HUSK (Theobroma Cacao L.)

MASTER THESIS

Decision in establishing the Committee 899/QD-DHNT on 04/09/2020

1 Dr Nguyen Van Tang (Principal supervisor)

2 Dr Tran Thi My Hanh (Co-supervisor)

Chairman

Assoc Prof Dr Huynh Nguyen Duy Bao

Department of Graduate Studies

KHANH HOA – 2020

UNDERTAKING

I undertake that the thesis entitled ―Extraction and assessment of antioxidant

and antimicrobial activities of saponins from cocoa pod husk (Theobroma cacao

L.)” is my own work The work has not been presented elsewhere for assessment until

the time this thesis is submitted

20th August 2020

Fanifosi Abosede Funmilola

ACKNOWLEDGMENTS

Firstly, I want to express my sincere gratitude to God Almighty for protection, provision and help through this program I also would like to express my gratitude to the following organizations and persons for their invaluable support, help and encouragement to me in conducting this study: Faculty of Food Technology, Nha Trang University in VLIR-UOS program for giving me the opportunity to study and conduct this Master thesis on ―Extraction and assessment of antioxidant and

antimicrobial activities of saponins from cocoa pod husk (Theobroma cacao L.)‖

Secondly, I would like to express special appreciation to my supervisors Dr Nguyen Van Tang and Dr Tran Thi My Hanh whom I have learned a lot from their guidance, useful advices and valuable comments throughout the period of study My special thank is given to the financial support for my Master thesis through the research project funded by the Ministry of Education and Training, Vietnam entitled

―Extraction of some bioactive compounds from cocoa pod husk for potential application in the functional foods‖

Finally, I would like to express my sincere gratitude to my parent (Engr Olaniyi and Mrs Kikelomo Fanifosi), my uncle and aunt (Mr Olufemi and Mrs Opeyemi Fanifosi), my bachelor‘s degree thesis supervisor (Dr Olugbenga Awolu), friends, classmates and colleagues for their love, encouragement and help

Nha Trang, 20th August 2020

Fanifosi Abosede Funmilola

TABLE OF CONTENTS

UNDERTAKING iii

ACKNOWLEDGMENTS iv

TABLE OF CONTENTS v

LIST OF ABBREVIATIONS viii

LIST OF TABLES ix

LIST OF FIGURES x

ABSTRACT xii

CHAPTER 1 : INTRODUCTION 1

1.1 Aims of the research 3

1.1.1 Overall aim 3

1.1.2 Specific aims 3

1.2 Contents of research 3

CHAPTER 2 : LITERATURE REVIEW 4

2.1 Cocoa 4

2.1.1 Cocoa pod husk 4

2.1.2 Composition (chemical) of CPH 5

2.2 Bioactive compounds 5

2.2.1 Polyphenols 6

2.2.2 Flavonoids 7

2.2.3 Saponin 8

2.2.4 Alkaloids 9

2.2.5 Extraction method 10

2.2.6 Conventional extraction method 10

2.2.7 Pressurized liquid extraction (PLE) 11

2.2.8 Ultrasound-assisted extraction (UAE) 11

2.2.9 Enzyme-assisted extraction (EAE) 11

2.2.10 Microwave-assisted extraction (MAE) 12

2.2.11 Freeze drying 12

2.2.12 Fractionation and identification methods 14

2.2.13 Column chromatography (CC) 14

2.2.14 Fourier-transform infrared spectroscopy (FTIR) 15

2.2.15 Thin layer chromatography (TLC) 16

2.2.16 High performance liquid chromatography (HPLC) 16

2.2.17 Anti-oxidation 17

2.2.18 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPH) 18

2.2.19 Cupric reducing antioxidant capacity (CUPRAC) 18

2.2.20 FRAP (ferric reducing antioxidant power) 18

2.2.21 Antimicrobial activity 19

CHAPTER 3 : MATERIALS AND METHODS 21

3.1 Overall experimental design 21

3.2 Study area 21

3.3 Preparation of dried sample for extraction 21

3.4 Extraction 24

3.5 Preparation of crude saponin-enriched powder 26

3.6 Identification of saponin-enriched extract and powder 28

3.6.1 Fourier-transform infrared spectroscopy (FTIR) 28

3.6.2 Thin-layer chromatography (TLC) 28

3.6.3 High performance liquid chromatography (HPLC) 30

3.7 Purification/fractionation of crude saponin-enriched extract 31

3.7.1 Preparation of column 31

3.7.2 Preparation of sample 31

3.7.3 Sample fractionation 32

3.8 Analytical methods 32

3.8.1 Determination of total saponin content 32

3.8.2 Total phenolic content (TPC) determination 33

3.8.3 Determination of total flavonoid content (TFC) 33

3.8.4 DPPH radical scavenging capacity (DRSC) determination 33

3.8.5 Ferric reducing antioxidant power (FRAP) determination 34

3.8.6 Determination of cupric reducing antioxidant capacity assay (CUPRAC) 34

3.8.7 Determination of antimicrobial activity 34

3.9 Statistical Analysis 35

CHAPTER 4 : RESULTS AND DISCUSSION 36

4.1 Physicochemical properties of crude saponin extract from cocoa pod husk 36

4.2 Bioactive compounds of crude saponin extract and fractions from CPH 37

4.2.1 Total phenol content (TPC) of crude saponin extracts and fractions of CPH 37

4.2.2 Total flavonoid content (TFC) of crude saponin extract and fractions from CPH 38

4.2.3 Saponin content (SC) of crude saponin extract and fractions from CPH 39

4.3 Antioxidant activity of crude saponin extract and fractions from CPH 39

4.3.1 DPPH radical scavenging capacity (DRSC) of crude saponin extract and fractions from CPH 39

4.3.2 Ferric reducing antioxidant power (FRAP) of crude saponin extract and fractions from CPH 41

4.3.3 Cupric reducing antioxidant capacity (CUPRAC) of crude saponin extract and fractions from CPH 41

4.4 Identification of phytochemicals in extract and fractions from CPH 42

4.4.1 TLC spectrum of crude saponin-enriched extract 42

4.4.2 FTIR spectrum of crude saponin-enriched powder 43

4.4.3 HPLC results of mixed standards and saponin fractions 46

4.4.4 HPLC result of re-fractionated fractions from fractions 1 and 2 52

4.5 Antimicrobial activity of saponin-enriched powder from CPH 55

CHAPTER 5 : CONCLUSIONS AND RECOMMENDATION 57

5.1 Conclusions 57

5.2 Recommendations 57 APPENDICES I

LIST OF ABBREVIATIONS

CUPRAC : Cupric reducing antioxidant capacity

DPPH : 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl DRSC : DPPH radical scavenging capacity

FRAP : Ferric reducing antioxidant capacity

HPLC : High performance liquid chromatography MAE : Microwave-assisted extraction

TFC : Total flavonoid content

TPC : Total phenolic content

LIST OF TABLES

Table 2.1: Composition of cocoa pods based on structure 6Table 4.1: Physicochemical properties of crude saponin extract from CPH 36Table 4.2: Bioactive compounds of crude saponin extract and fractions from CPH 37Table 4.3: Antioxidant capacity of crude saponin extract and fractions from CPH 40Table 4.4: FTIR peaks of saponin-enriched extract from CPH 45Table 4.5 Antimicrobial activity of saponin-enriched powder from cocoa pod husk 56

LIST OF FIGURES

Figure 2.1: Schematic diagram of a simple chromatographic system 15

Figure 3.1: Fresh Cocoa pod husk stored in Styrofoam boxes 21

Figure 3.2: Preparation, purification and assessment of biological activity of saponins from cocoa pod husk (CPH) 22

Figure 3.3: Cutting Cocoa pod husk into thin slices 23

Figure 3.4: Dried cocoa pod husk 23

Figure 3.5: Milled cocoa pod husk 24

Figure 3.6: MAE set-up 25

Figure 3.7: Crude saponin extract 25

Figure 3.8: Filtering crude saponin extract 26

Figure 3.9: Condensed crude saponin extract 27

Figure 3.10: Sample prepared for freeze drying 27

Figure 3.11: Crude saponin-enriched powder after freeze drying 28

Figure 3.12: TLC plate undergoing 29

Figure 3.13: TLC plate under UV light 30

Figure 3.14: Colum set up for fractionation 32

Figure 4.1: TLC of saponin-enriched extract from cocoa pod husk using 80% methanol as solvent 43

Figure 4.2: FTIR spectrum of standard (-)-epigallocatechin gallate 43

Figure 4.3: FTIR spectrum of standard theophylline, theobromine and caffeine 44

Figure 4.4: FTIR spectrum of saponin-enriched extract from CPH 45

Figure 4.5: HPLC chromatogram of standards (4, 6, 7 and 8) acquired at 272 nm 47

Figure 4.6: HPLC chromatogram of standards (1, 2, 3 and 5) acquired at 272 nm 47

Figure 4.7: HPLC chromatogram of fraction 1 acquired at 272 nm 48

Figure 4.8: HPLC chromatogram of fraction 2 acquired at 272 nm 49

Figure 4.9: HPLC chromatogram of fraction 3 acquired at 272 nm 49

Figure 4.10: HPLC chromatogram of fraction 4 acquired at 210-280 nm 50

Figure 4.11: HPLC chromatogram of fraction 5 acquired at 272 nm 50

Figure 4.12: HPLC chromatogram of fraction 6 acquired at 210-280 nm 51

Figure 4.13: HPLC chromatogram of fraction 7 acquired at 272 nm 51

Figure 4.14: HPLC chromatogram of fraction 8 acquired at 272 nm 52

Figure 4.15: HPLC chromatogram of sub-fraction 1.1 acquired at 272 nm 53

Figure 4.16: HPLC chromatogram of sub-fraction 1.2 acquired at 272 nm 53

Figure 4.17: HPLC chromatogram of sub-fraction 1.3 acquired at 272 nm 54

Figure 4.18: HPLC chromatogram of sub-fraction 1.4 acquired at 272 nm 54

Figure 4.19: HPLC chromatogram of sub-fraction 1.5 acquired at 272nm 55

Figure 4.20: HPLC chromatogram of sub-fraction 1.6 acquired at 272 nm 55

Cacoa (Theobroma cacao L.) is a foison which has a great economic

importance worldwide, and the main base material for manufacturing chocolate, the pod is said to be a material with a naturally coated surface, the pod husk is trashed and bound to cause problems to the environment when disposed carelessly in areas where processing takes place Saponins are said to be one of the popular varying categories

of natural compounds that are discovered in plants and aquatic organisms Previous studies show that saponins have proven to contain a number of biological activities and they are therefore important in both food and medical fields This study aimed to research how to prepare, purify and assess the biological activity (antioxidant, antimicrobial) of saponins from cocoa pod husk

In this study, saponins were extracted from cocoa pod husk through assisted extraction, fractionation of the extract was done using column chromatography The saponin, flavonoid and phenolic contents, and antioxidant activity of extracts and fractions were carried out using UV-VIS spectrophotometer The bioactive compounds identification in the extracts and fractions were done using

microwave-TLC, FTIR and HPLC Two bacterial strains: Escherichia coli (ATCC 8739) and

Bacillus subtilis (ATCC 6633) and Candida albicans (ATCC 10231) belonging to the

yeast strain were used to determine the antimicrobial activities

The results showed that there were different levels of SC, TPC, TFC, DRSC, CUPRAC, and FRAP in the crude saponin extract as well as the fractions Antimicrobial assay indicated that inhibition zones for 10 mg/mL of extract was 2 ± 0 mm and for 30 mg/mL of extract was3 ± 0.5 mm, both these values were recorded for Bacillus subtilis (ATCC 6633) The extract did not show any effect on the tested Escherichia coli (ATCC 8739) nor Candida albicans (ATCC 10231) at every concentrations Saponin-

enriched extract and fractions from cocoa pod husk have shown potent antioxidant activity, and they also contained some key compounds (theobromine, theophylline, (+)-catechin, (-)-epicatechin, (-)-epigallocatechin and (-)-epigallocatechingallate), it could therefore be used and incorporated into diets as functional foods or food supplements

CHAPTER 1 : INTRODUCTION

Cocoa (Theobroma cacao L.) is a foison which has a great economic

importance worldwide, and the main base material for manufacturing chocolate

(Krähmer et al., 2015) Cocoa is one of the main agricultural commodity exported by

most countries that produce it, these countries are situated in the Central and West

Africa (Nigeria, Cote d‘Ivoire, Ghana and Cameroon) (Afoakwa et al., 2011a) The

potential of Vietnam becoming a large cocoa producer is high as the country experiences climatic conditions which favors the growth of the crop, but to see to this large production genetic methods are required to identify and conserve promising

cacao cultivars (Everaert et al., 2020) The genus of Cocoa; Theobroma consists of twenty two species and having Theobroma cacao L as the very economically

relevant because of its seeds value (CacaoNet, 2012) Cocoa seeds which are generally called cocoa beans are obtained from cocoa pods, cocoa pods are oval in shape, contains around 35 to 45 beans wrapped in a pulp (depending on the size of pod) which approximately is about 40% of the fresh mass of bean and finally cocoa

pods has a length between 12 and 30 cm (Lima et al., 2011)

The pod is said to be a material with a naturally coated surface, the pod consists

of three layers with obvious differences; endocarp, mesocarp and epicarp (inner pericarp, middle and outer pericarp, respectively) Campos-Vega et al., (2018) analyzed the composition (chemical) of these layers and also did a comparison with whole cocoa pod husk (CPH) after incorporating them separately as broiler chick feed Camps-Vega et al., (2018) also found out in the CPH that high proportion of ash (47%), minerals (Ca, P and K) (41 to 66%) and hemicellulose (50%) were pre-dominated within the outer pericarp; cellulose (53%) and fiber (NDF, ADF and crude 44-48%) were found in the middle pericarp; and, crude fat (50%), pectin (59%) and protein (50%) were major compositions in the inner pericarp

Once the cocoa beans are removed, the husk is mostly trashed on the farm which can be used as a fertilizer, this puts in natural pesticide-free matter to the soil and sees to the replenishing of nutrients to the soil after decomposition (Aboyeji, 2016) However, untreated cocoa pod husk carelessly sprawled on the soil surface

may create room for inoculum which causes some plant diseases such as black pod

rot if Phytophthora spp is present (Vriesmann et al., 2011b) 20% to 30% of annual

yield loss are caused by black pod rot worldwide, while the yield of individual farms annually are reduced by 30% to 90% (Lu et al., 2018)

The food industry is at the moment searching for novel origin of non-artificial antioxidants, and it is found that the cocoa pod husk (CPH) happens to be a good carrier of phenols and other bioactive compounds which can work as antioxidants and also have a place as functional foods ingredients Nevertheless, due to their moisture content being high, fresh CPH tend to spoil easily and so they have to be dried to increase the shelf-life (Valadez-Carmona et al., 2017) Some studies on the use of cocoa wastes have been carried out, one of such experiments is the use of supercritical

CO2 to extract some pigmented phenolic compound fractions from cocoa hulls with

bioactive and antiradical properties (Arlorio et al 2005)

Saponins are said to be one of the popular varying categories of non-artificial compounds discovered in plants and aquatic organisms, it has opposing molecules which includes a steroid aglycone or triterpene alongside one or more chains of sugar (Osbourn, 2011) From previous studies, saponins are discovered to inhibit a number of organic activity, like antitumor, anti-cholesterol, anti-oxidant, anti-obesity, anti-diabetic, antiinflammatory, anti-hepatitis B and C and anti-cancer properties Oxidation process is directly linked to free radicals and active oxygen (Naumovski, 2014), and this may distort biological macromolecules and damage cells, which leads to aging of cell and long-term ailments, like diabetics, cardio-vascular diseases, cancer and others Distortion of biological macromolecules can be controlled by averting or putting a stop

to the oxidative radical chain reactions involved in the process, this can be achieved by the use of antioxidants gotten from non-artificial materials, thereby holding back the process of oxidation (Nimse, 2015)

Recently, due to the evolution of Fourier transform infrared spectroscopic (FTIR) arrangement, the implementation of this method is now rampant in the study of fats and edible oils (Guillen & Cabo, 2000) The relevance of infrared spectroscopy for spotting molecular structures is as a result of a great number of information gotten and

also the feasibility to put some absorption bands, here majority of the peaks in oils and fats are attributed to certain functional groups (Bendini et al., 2007)

Microwave-assisted extraction (MAE) has been found out in previous studies as

a novel means for extracting phyto-chemicals from various natural sources, having the minimum time of extraction with minimum consumption of energy, and methanol being the best solvent found for saponin extraction from these sources (Nguyen, 2016b) Optimization of saponin extract has been done in a previous study but there has been no analysis carried out on the extract Therefore, this study aims to research how to prepare, purify and assess the biological activity (antioxidant, antimicrobial) of saponins from cocoa pod husk

1.1 Aims of the research

1.1.1 Overall aim

The overall aim of this experiment is to extract, purify and assess the biology activity of saponin-enriched extract from cocoa pod husk (CPH)

1.1.2 Specific aims

(1) To prepare saponins-enriched extract from the CPH

(2) To purify pure saponins from saponins-enriched extract obtained from the CPH

(3) To assess the biological activity in vitro of saponins-enriched extract,

saponin fractions and pure saponins obtained from the CPH

1.2 Contents of research

(1) Preparation of saponins-enriched extract from CPH

(2) Purification of saponin fractions and pure saponins from saponins-enriched extract obtained from the CPH

(3) Assessment of the biological activity in vitro of saponins-enriched extract,

saponin fractions and pure saponins obtained from the CPH

CHAPTER 2 : LITERATURE REVIEW 2.1 Cocoa

Cocoa tree is a tree that buds after two years, it‘s about 8-15m high (Fowler, 1999) and grows best in moist, hot state, the tree is in no way resistant to lengthened drought circumstances and the fruit from it varies in size, appearance, shape and

external colour depending on the specie Cocoa (Theobroma cacao L) gives

significant economic importance in countries that are still developing, cocoa beans production was valued to be 4.7 million tones worldwide between 2016 and 2017 (Lu

et al., 2018) It is the main agricultural commodity of export for various countries which produce in Central and West Africa like Nigeria, Cote D‘Ivoire, Ghana and Cameroon (Afoakwa et al., 2011a) 67% of the global production of cocoa beans comes from Ghana, Cote D‘Ivoire and Indonesia while 70% is carried out in the central region of West Africa Cocoa genus consists of twenty two species and

Theobroma cacao L is the most relevant because of its seed value (CacaoNet, 2012)

because it is the only specie cultivated for business purposes and most well known in markets (World Agriculture, 2011)

Cocoa by-products include pulp and cocoa pod husk (CPH) produced in generous amounts but under-used, these products are gotten by separating the pods from the beans (FAO, 2015)

2.1.1 Cocoa pod husk

The pod is said to be a material with a naturally coated surface, the pod consists

of three layers with obvious differences; endocarp, mesocarp and epicarp (inner pericarp, middle and outer pericarp, respectively) The inner pericarp is a soft tissue closest to the bean and protecting the beans in a properly greased inner part; the middle pericarp is hard and therefore able to grip the cocoa beans in place when in contact with high impact (Babatope, 2005) After cocoa beans are removed from the husk, the cocoa pod husk accounts for about 52-77% of the total weight of cocoa (Vriesmann, Teófilo, & Lúcia de Oliveira Petkowicz, 2012), the pod husk are trashed and likely lead to environmental issues when left carelessly in areas of processing acting as a an inoculum source for black pod rot plant disease (Vriesmann et al., 2011) but it can function as a fertilizer that puts in non-artificial matter to the soil which then

see to the replenishing of nutrients when properly managed (Aboyeji, Olofintoye, Olaleye, Olugbemi, & Adetula, 2016) CPH is an under-utilized sustainable material that has various compounds (polyphenols), and consumable fiber (Lu et al., 2018)

2.1.2 Composition (chemical) of CPH

The CPH is made up of 67–76% of cocoa fruit in terms of weight, farmers have used it in formulation of feeds for their poultry and livestock because it has some amount of minerals, crude fat (1.2–10%), protein (5.9–9.1%) and fiber (22.6–35.7%) among others (Oddoye, Agyente-Badu && GyeduAkpto, 2013) The fiber fraction consists of 80.7% neutral fibers, 74.6% acid detergent fibers, 35.3% hemicellulose, and 38.8% lignin (Laconi & Jayanegara, 2015)

2.2 Bioactive compounds

Essential or nonessential compounds given freely by nature or formulated during food processing or processing of plants are known as bioactive compounds (Ortega & Campos, 2019), these compounds are presented in small quantities in foods and are mainly found in fruits, vegetables and whole grains, they are said to provide benefits to health beyond the base nutrient value (Gokmen, 2016) Essential compounds carry out

an important organic function in y of humans and when there is a deficiency of this compound in the body, it can cause diseases development, and no important biological function is achieved by nonessential compounds in humans, therefore not having these cannot adversely have an impact on health (Biesalski et al., 2013)

In plants, typical bioactive compounds is produced as secondary metabolite; the metabolites formed in a pre-growth stage are called secondary metabolites, have no role to play in growth and have uncommon chemical structure which are normally made as blends of firmly related individuals from a synthetic family (Azmir et al., 2013), therefore bioactive mixes in plants are characterized as auxiliary plant metabolite which have pharmacological or toxicological impacts in people and in creatures (Magnúsdóttir, 2002)

Classifying bioactive mixes into various classifications is as yet conflicting because the categories depend on the intention of that particular classification, phytochemicals are the major bioactive mixes and are characterized by how their

synthetic structure is framed and this classification is given as : terpenoids and steroidal glycosides (saponins), alkaloids, organosulfur compounds and polyphenols

(Somani et al., 2015)

Table 2.1: Composition of cocoa pods based on structure (Sobamiwa and Longe (1994)

oxidative corruption and consequently improving the nutritional value and quality of foods has increased the interest for its study in food industries (Manach et al., 2004) Polyphenols are cancer prevention agents and have different natural properties, for example, anticancer (Odongo, 2017), antiasthmatic (Shaw, 2016), cholesterol lowering, antihypertensive (Tenore, 2017), anti-inflammatory (Sajid, 2017), anti-diabetic and antiviral properties (Omodanisi, 2017) They can go about as chain breakers or radical scroungers dependent on their compound structures (Rice-Evans, 2001), in light of their structure they show a wide scope of properties which incorporate yellow, red, blue and orange shades

The test for polyphenol group in plants is known as total phenolic content, many literatures have been written on the test of total phenol content one of which is cell reinforcement action and complete phenolic substance of Iranian Ocimum increases, the total content of phenols was carried out by Javanmardi et al., (2003) using the Folin-Ciocalteu reagent, a method developed by Spanos & Wrolstad (1990), and re-modeled by Lister & Wilson (2001)

2.2.2 Flavonoids

Flavonoids occur only in plants mainly as glycosides, like other types of polyphenols, they are generally present in leaves, tissues of blossoming plants and

woody pieces of plants, for example, barks and stems (Kahkonen et al., 1999) They

consist of a central three ring structure with pro-anthocyanidins being its oligomer, all the compounds of a flavonoid contain phenol groups that are effective as general antioxidant Stilbenes are found in not many amounts in human eating routine, they are found mostly in grapes, peanuts and red wine but resveratrol is the most well-known compound among stilbenes group

Total flavonoid content of cocoa was evaluated by Crozier et al., (2011), while establishing cocoa as a great fruit while comparing it with other products, fruits and powders, the flavonoid substance was resolved colorimetrically at 640 nm with DMAC (4-dimethylaminocinnamaldehyde), the method used is as determined by Payne et al., (2010)

2.2.3 Saponin

Glycosides consist of various classes of secondary metabolites which is attached

to either a mono-or oligosaccharide or to uronic corrosive The part of the uronic acid

is termed glycone while the other part is termed aglycone Glycosides have five main groups which are glucosinolates, anthraquinone glycosides, cardiac glycosides,

cyanogenic glycosides and saponins (Magnúsdóttir, 2002)

Saponin is a collective name given to triterpenoids and steroidal glycosides, they are bioactive compounds found in many plant-derived foods especially in legumes (Rao & Gurfinkel 2000), and they are also present in many medicinal plants such as ginseng Saponins have been found to serve as nutraceuticals and also known for its

hypocholesterolemic and anti-coagulant activity both in animals and in vitro models

(Rao & Gurfinkel, 2000)

2.2.3.1 Prevention of cardiovascular diseases

Cardiovascular infection is the most prominent reason for death around the world

as it is a big social and economic problem (Pagliaro et al., 2015) Antherosclerosis is a main risk causing factor during the progressive development of acute myocardial infraction and this is the main cause of CVD (Golia et al., 2014) It has been found out that a few dynamic mixes found in food plants have properties that ensures against cardiovascular infection, some of these active compounds include phytochemicals such as resveratrol (Riccioni et al., 2015) and lycopene (Liu, 2013) The main biological activities that prevents CVD also lowers lipid, are anti-inflammatory and anti-thrombotic antioxidants

2.2.3.2 Prevention of chronic diseases

Chronic diseases are known to be diseases which occur for long terms, they exist

in human body long before they are clinically discovered Example of some of these diseases are cancer, diabetes and Alzheimer‘s disease (Martin, 2007) Diabetes is a disease characterized by its resistance to insulin, reduced insulin secretion, glucose intolerance, chronic hyperglycemia and chronic pro-inflammatory position (Al-Goblan

et al., 2014), some major health risks factors linked with diabetes are obesity, genetics

and too much ingestion of fats and sugars (Wu et al., 2014) Studies on epidemiology showed us that serum levels of lycopene, α-carotene, β-carotene, β-cryptoxanthin and zeaxanthin help to improve glucose prejudice just as diabetes mellitus type II (Coyne

et al., 2005)

Around the world, cancer is a generally known problem that affects health and it

is one of the diseases accountable for highest deaths rates (Stewart & Wild, 2014) Known as an infection that affects the genes, it has a relationship with exposure to smoke from cigarette, viruses, hormones, manufactured cancer-causing substances, drugs, liquor, bright radiation and hefty metals (Blackadar, 2016) Some phytochemicals such as lycopene, resveratrol and curcumin have been found to have anticancer potential and show efficient healing effect (Dhillon et al., 2008)

2.2.4 Alkaloids

Alkaloids are compounds which contain nitrogen, they happen normally in plants and furthermore in microorganisms, aquatic animals and organisms, they are in most occasions utilized as medications or natural tests for physiological examinations (Kuramoto, Arimoto, & Uemura, 2004) They are structurally the most diverse class of secondary metabolites and range from simple structure to complex ones (Bandaranayake, 2002), the use of alkaloids in chemotherapy increased the importance

of bioactive alkaloids Although, alkaloids are made up of various groups with diverse clinical properties, they are of restricted dissemination in the plant realm, below are a list of five groups of alkaloids (Bernhoft, 2010)

 Methylxanthine alkaloids: the main source of this group of alkaloids are

Coffea arabica (coffee) and Theobroma cacao (cacao), methylxanthine is bound to

adenosine receptor and causes neurological effects in human and animals which when taken at low or moderate doses is regarded as stimulating

 Tropane alkaloids: this group of alkaloid compounds have anticholinergic activity and they are used in medicine to reduce smooth muscle spasm, pain and

hypersecretion Sources are: Datura spp (thorn apples), Hyoscyamus niger (henbane) and Atropa belladonna (deadly nightshade)

 Isoquinoline alkaloids: isoquinoline alkaloids are products from Papaveraceae (poppy family) and Berberidaceae (barberry family), they are used widely in medicine due

to their biochemical effect in inhibition of varying conditions such as pain, stimulation of bone marrow leucocytes and also inhibition of cancer cells

 Pyrrolizidine alkaloids: Found particularly in Senecio spp (Ragworts) and

Boraginaceae (borage family) They could have a hepatotoxic adverse effect after

bioactivation

2.2.5 Extraction method

The subjective and quantitative investigations of bioactive mixes from plant materials predominantly rely upon the decision of extraction technique (Sasidharan et al., 2011) So many factors influence the extraction cycle and output of bioactive mixes in plant however the most common ones are solvent used in extraction, extraction time, method of extraction, temperature of extraction, pressure and matrix properties of the plant (Hernandez, 2009) Bioactive compound extraction from plant sources can be done either by conventional of non-conventional methods, the non-conventional method include microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), enzyme-assisted extraction (EAE), Pressurized Liquid Extraction (PLE), pulse-electric field extraction (PEF) and supercritical fluid extraction (SFE)

2.2.6 Conventional extraction method

Soxhlet extraction, maceration and hydrodistillation are the techniques employed

in conventional method of extracting bioactive compounds from plants, these procedures depend on removing intensity of different solvents utilized and use of warmth or potentially blending (Azmir et al., 2013) In an ideal conventional extraction, the desired compound should be highly soluble in the solvent used while other compound shouldn‘t be soluble in that solvent during extraction (Pronyk & Mazza, 2009) but this is usually not the case and this led to research on other factors that will help in conventional method of extraction other than type of solvent used Other optimizing conditions include: particle size, pressure, flow rate, temperature and solvent to sample ratio (Wijngaard et al., 2012) Conventional method of extracting bioactive compounds have been used in the past but there are new technologies which aid the extraction of these bioactive compounds in order to greater yields, one of such

is the microwave-assisted extraction which will be used in this thesis

2.2.7 Pressurized liquid extraction (PLE)

For pressurized fluid extraction, pressure is applied in this way permitting the utilization of temperature over the breaking points of solvents (Mendiola et al., 2007), having a higher temperature of extraction will expand the mass exchange and extraction rates on the grounds that having an increased temperature will bring about

an expansion in limit of solvents to dissolvable solutes, a decrease in the viscosity of the solvent, an expansion in dissemination rate and an abatement in surface strain (Ramos, Kristenson, & Brinkman, 2002) The applied high weight extending from 4 to

20 MPa guarantees that the dissolvable stays in its fluid state at the temperature applied and this is the motivation behind why pressure is utilized

2.2.8 Ultrasound-assisted extraction (UAE)

Ultrasound is a kind of sound wave that is above human hearing, it is typically between 20 kHz and 100 MHz The mechanism of extraction using ultrasound includes two fundamental sorts of physical wonders which are: the dispersion over the cell divider and washing the substance of cell in the wake of breaking the divider The fundamental preferred position of UAE is seen in strong plant tests when ultrasound vitality encourages natural and inorganic compound filtering from plant framework (Herrera and Luque de Castro, 2005), other advantages include reduction in time of extraction, facilitation of effective mixing, reduced thermal gradient, reduced use of energy and use of solvent, quicker reaction to deal with extraction control and quicker transfer of energy (Chemat et al., 2008) Ghafoor et al., (2011) separated anthocyanins and phenolic mixes from grape strip employing the use of UAE and optimization of the extraction measure was finished concerning dissolvable, extraction time, and temperature

2.2.9 Enzyme-assisted extraction (EAE)

A few phytochemicals in frameworks of plants are scattered in cell cytoplasm and some different mixes are found in the polysaccharide-lignin arrange by hydrophobic or hydrogen holding and these cannot be accessed by solvents in a solvent based extraction process The use of enzymes in pre-treatment of these plants

is being found as a novel and effective approach to deliver limited compounds and increment in general yield by breaking the cell divider and hydrolysis of the basic

polysaccharides, this is achieved by expansion of explicit compounds like α-amylase, pectinase and cellulose while extracting (Azmir et al., 2013)

2.2.10 Microwave-assisted extraction (MAE)

Electromagnetic waves, normally worked at a recurrence of 2.45 GHz are said to

be microwaves, microwaves have the ability to access organic frameworks and also cooperate with some polar particles, for example, water The rise in temperature prompts upgraded extraction proficiency (Wang & Weller, 2006) In a research paper written by Chen et al., (2007), microwave-assisted extraction was used for isolating a

kind of saponin known as total triterpenoid from Ganoderma atrum using ethanol as

solvent The microwave-assisted extraction was done alongside other extraction methods so as to determine the best method for isolating a kind of saponin known as

total triterpenoid from Ganoderma atrum, these other methods of extraction include;

ultrasonic extraction, shaking extraction, heat flux and supercritical fluid extraction (SFE) Extraction using microwave was influenced by some factors which includes: type of solvent, material to solvent ratio, extraction temperature and time Ideal states

of MAE of saponins (triterpenoid) from Ganoderma atrum was tested and given to be

95% ethanol as the best solvent, proportion of dissolvable to material given as 25 mL/g, term of microwave radiation of 5 min and temperature of extraction as 90ºC The impact of microwave vitality and treatment time on the extraction of phenolic mixes from citrus mandarin strip and pomace was depicted in the investigation of Hayat et al (2010) In all microwave-helped extraction was appeared to give the least extraction time in minutes and higher yield while it took hours for extraction using other extraction methods

2.2.11 Freeze drying

In preservation and processing of foods removal of water is essential to ensure extension of shelf life is extended, ease of handling, sanitation and quality enhancement A drying cycle in which the dissolvable (typically water) as well as the suspension medium is solidified at a low temperature and from there on sublimated from the strong state straightforwardly into the fume stage is known as freeze drying (lyophilization) (Liu et al., 2008), it is the most effective method of removal of water from materials that are highly heat sensitive, freeze drying helps in getting products

with higher worth compared to either oven drying or other methods of food drying (Taylor et al., 2012), protects food materials against chemical decomposition and ensures easy rehydration (Tao et al., 2005) Low temperature for processing and depletion of oxygen in the process are factors that help in maintaining high quality during freeze drying process (Strumillo & Adamiec, 1996) The main disadvantages associated with freeze drying include high operating (capital and energy) cost, lengthy dehydration process (Litvin et al., 1998) Freeze drying has been applied to different food products (herbs, tea, vegetables and fruits) (Chan et al., 2009), flowers, medical devices, cosmetics, microorganisms, pharmaceuticals, enzymes, and pigments (Ciurzyńska & Lenart, 2011)

Freeze drying process/cycle as given by Tang & Pikal, (2004) is consisted of four steps: Freezing, annealing, primary drying and secondary drying

 Step 1: Freezing

In this step, the samples are made solid by exposing them to a low temperature, this changes the physical properties of the sample and is caused by ice crystal formation (Wang, 2000) In order to have a well formulated solid sample, some parameters have to be optimized this include the eutectic temperature (Teu) and glass transition temperature (Tg) (Morais et al., 2016)

 Step 2: Annealing

During the annealing step, the product is held for a definite time and temperature above glass transition temperature (Tg) but below eutectic temperature (Teu) of the bulking agent, this will ensure effective crystallization of the glasslike building operator (Morais et al., 2016)

 Step 3: Primary drying

The essential drying is the longest advance in freeze drying measure, this progression permits sublimation of ice utilizing low temperatures and pressures (Tang

& Pikal, 2004) The ice formed in the first step (freezing step) will be sublimed and the sublimation process begins from the outer part of the sample by diffusion through porous layers and goes gradually to the bottom Pressure and collapse temperature (Tc) are two important parameters that should be monitored in this step, lower chamber pressure results in faster sublimation of ice (Ingvarsson et al., 2011), also keeping

collapse temperature (Tc) above sample temperature will help to retain the structure of the sample (Levi & Karel, 1995)

 Step 4: Secondary drying

Decrease of water to an ideal level for security is the principle point of this step, the balance in adsorption-desorption balance of dampness and permeable medium is a significant factor in this progression along these lines there temperature and dampness content must be controlled in order to get a last item with worthy item quality (Wang

et al., 2012)

2.2.12 Fractionation and identification methods

Extracts from plants are usually made up of different types and groups of bioactive compounds which may include saponins, phenolic groups and some other phytochemicals These compounds have different polarities and this has posed as a big challenge in separation and identification of bioactive compounds into their different groups or individual compounds For the purpose of isolation, separation and purification of bioactive compounds, different techniques have been used over the years some of which are thin layer chromatography (TLC), flash chromatography, sephadex chromatography, column chromatography, phytochemical screening assay, high performance liquid chromatography (HPLC) and Fourier-transform infrared spectroscopy (FTIR) (Tradit et al., 2007)

2.2.13 Column chromatography (CC)

Chromatography is a technique scientists use for separating organic and inorganic compounds in a sample to make analysis easier, in the real sense chromatography is a term that means ―color writing‖ (Kondeti, Mulpuri, & Meruga, 2014) A chromatography system is made up of various components including a fluid delivery system (pump), a column with chromatographic gel, a buffer reservoir or reservoirs, a means of detecting molecules eluting from column and finally a collecting system (Burden & Whitney, 1995) Column chromatography itself is a technique used in cleaning of individual mixes from a blend of various mixes, in chemistry it is used for preparative applications on scales ranging from micrograms to kilograms (Kondeti, Mulpuri, & Meruga, 2014) To achieve separation by chromatography, substances within a mixture are distributed between two phases that

is a moving phase and a stationary phase by so doing, substances which interact more strongly with the stationary phase will remain in the chromatographic system much longer than substances which interact with the mobile phase (Scott, 1983)

Figure 2.1Schematic diagram of a simple chromatographic system

(Burden & Whitney, 1995)

2.2.14 Fourier-transform infrared spectroscopy (FTIR)

Fourier-transform infrared spectroscopy (FTIR) instrumentation application, has become popular since its development and has been used in the identification of various extracts (Guillen & Cabo, 2000), its use is in the mapping out of cellular components that is: carbohydrates, proteins and lipids, the mapping out is done to help

in pointing out and characterizing abnormal cells (Levin & Bhargava, 2005) The principle of operation is that FTIR tests the properties of vibration of amino acids and co-factors that are known to have high sensitivity to very tiny structural changes (Berthomieu & Hienerwadel, 2009), Rhoman et al., (2010) told us about the significance of infrared spectroscopy in ID of atomic structure, in his report he expressed that the enormous data content acquired from the result and the ease of assigning absorption bands with peaks that could be attributed to certain functional group has seen to the increased use of infrared spectroscopy In an experiment carried out and reported by Freitas, (2013), FTIR was used in characterization of purified polysaccharide of cashew acquired from trees of A occidentale by liquor precipitation,

in the outcome acquired the spectra from FTIR indicated O-H, C-O-C and C-O bunches available in the structure of the cashew polysaccharide

2.2.15 Thin layer chromatography (TLC)

Thin-layer chromatography (TLC) is a simple, quick, and relatively affordable chromatographic procedure that helps a researcher to know the different components

in a mixture (Tradit et al., 2007), TLC has a long history in its use in analysis of compound‘s fractions through column chromatography (Altemimi et al., 2017) It has found its way through different disciplines and has been applied by various specialists

in their day to day research, the common areas where it has been applied include food science, chemistry, toxicology, pharmacy and environmental sciences (Gorman & Jiang, 2004) The continuous and frequent use of TLC has been attributed to its convenience, cost-effectiveness, speed of separation, high sensitivity, availability in a number of stationary phases and because it has a feature complementary to HPLC (Zhang et al., 2005) In TLC, the extract is applied as a small dot or a streak on a marked origin of the TLC plate making use of capillary tubes, the plate is then placed

in a shallow pool of mobile phase in a glass container and sample solvent is allowed to evaporate from the plate (Gorman & Jiang, 2004) One of the application of TLC in chemistry is found in the research of Bober, Bȩ benek, & Boryczka, (2019) where TLC was used for determining the lipophilicity of new synthesized esters of Betulin derivatives

2.2.16 High performance liquid chromatography (HPLC)

High performance liquid chromatography (HPLC) is a flexible, strong, and broadly utilized strategy for the segregation and recognizable proof of regular items (Cannell, 1998) It is a form of column chromatography with the principle of pumping

a sample (analyte) dissolved in a mobile phase (solvent) at elevated pressure along a column with a stationary phase (an immobilized packaging material) (Yeo, Wang, & Hammond, 2017) Chemical separation has been accomplished over the years using HPLC by applying the different migration rates, each compound has depended on the mobile phase and column The degree or level of partition is generally dictated by the decision of fixed stage and portable stage (Tradit et al., 2007) HPLC has been used widely in food technology and food sciences for identification, analysis and purification of compounds in food and beverages, ranging from the determination of food additives (antioxidants, sweeteners, dyes), monitoring of food irradiation, study

of food aroma, product purity specification, etc (Yashin & Yashin, 2004) Gordon et al., (2011) did a comprehensive characterization of phenolic constituent in the edible part of the four Amazonian fruits using high performance liquid chromatography, Lee

et al., (2010) determined the quality and quantity of crystal violet and malachite green

in processed fish products using liquid chromatography

on biological systems through various mechanisms including metal ion chelation, co-antioxidants, electron donation or gene expression regulation (Lobo et al., 2010)

Antioxidants naturally found in plant products, especially in vegetables and fruits

is gaining more grounds and attracting the interest of consumers as well as food scientists and technologists this is because studies have shown that repeated intake of non-artifical antioxidants helps to achieve a decreased chance of cancer and diseases

of the heart (Temple, 2000) Three main groups have an effect on how antioxidants serve as defense against cardiovascular diseases, these include vitamins, phenolics and carotenoids (Thaipong et al., 2006) The functional and nutritional properties of plant based phenolic compounds which include antioxidant and antimicrobial activity are of immense benefit (Bubonja-Sonje et al., 2011) and cocoa bean (seed of cocoa) is said to

be rich in polyphenols such as flavanol monomers and procyanidin oligomers (Wollgast & Anklam, 2000)

With regards to free radical scavenging, many methods have been formulated to calculate the potential of antioxidant of a food or substance The ABTS/TEAC (2,20-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid/trolox equivalent antioxidant

Fresh cocoa pod husk

Drying in microwave oven

Grinding Dried

Dried CPH Extraction (using methanol)

Analysis of bioactive

activities (antioxidant and

Pure saponin compounds Analysis of bioactives, and

Analyzed Purified Analyzed Crude

Saponin Extract

capacity) (Re et al., 1999), ORAC (oxygen radical absorbance capacity) (Cao et al., 1995), DPPH (2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl) (Sánchez-Moreno et al., 1998), FRAP (ferric reducing antioxidant power) (Benzie & Strain, 1996), and CUPRAC (Cupric reducing antioxidant capacity) (Apak et al., 2004)

various plant extracts, fruits and vegetable juices (Sendra et al., 2006)

The DPPH antioxidant assay has its basis on transfer of electron that gives a violet colored solution when mixed with ethanol (Huang, Ou, & Prior, 2005), the free radical known to be stable at room temperature is reduced when it comes in contact with an antioxidant molecule and thus changing the violet colored solution to a colorless ethanol solution (Garcia et al., 2012) Evaluation of antioxidant activities of extracts and sample solutions by spectrophotometry has been made easy using DPPH assay (Huang, Ou, & Prior, 2005)

2.2.19 Cupric reducing antioxidant capacity (CUPRAC)

The CUPRAC method of antioxidant test is also a simple assay useful for polyphenols (flavonoids, phenolic acids, and carotenoids), vitamins C, Vitamins E and synthetic antioxidant (Apak et al., 2004), the method uses neocuproine (NC) as the chelating agent and has been stated as suitable for assessment of the antioxidant power of biological and non-biological samples (Karaman et al., 2010 & Apak et al., 2005), but another report by Da Cruz, (2003) showed that bathocuproinedisulfonic acid disodium salt (BCS) can also be used as a chelating agent

2.2.20 FRAP (ferric reducing antioxidant power)

The FRAP measured the antioxidants‘ power to lower ferric iron in a sample/product, the principle is based on lowering of 2,3,5-triphenyl-1,3,4-triaza-2-

azoniacyclopenta-1,4-diene chloride (TPTZ) and ferric iron to ferrous at low pH (Alam, Bristi, & Rafiquzzaman, 2013), the FRAP test has been carried out on various plant extract with varying reports based on the type and composition of the extract

El Jemli et al., (2016) carried out a ferric reduction test on aqueous extracts from

leaf of Juniperus oxycedrus, Juniperus thurifera, Tetraclinis articulate and Juniperus

Phoenicea, and from Morocco, it was aimed to compare the reducing activity of all

four extracts, the result showed that extracts of Juniperus oxycedrus have the best

performance reducing power activity

2.2.21 Antimicrobial activity

An antimicrobial is any substance that can restrain development of microorganisms or reduce bacterial loads in foods and material surfaces, an antimicrobial may be an antibiotics used for human and animals, chemicals used for cleaning surfaces or may be freely found in some foods (Verraes et al., 2013) Spoilage of food is characterized as an adjustment in the nature of food that makes it unsuitable for utilization due to spoilage, the spoilage indicators for food include offensive odor, off-flavors, slime growth, changes in surface and appearance (Lianou, Panagou, and Nychas, 2016; Nychas, and Panagou, 2011), the most well-known reason for food deterioration are microbes and they are found everywhere, but because they very small size and cannot be seen without the use of microscope, they may remain unnoticed in exposed food except for molds which can be seen without the use

of microscope (Odeyemi et al., 2020)

Reported by (Lorenzo et al., 2018), microbes such as bacteria, molds, and yeasts are said to be used in the production of some foods like beer, wine and baked products,

it should also be noted that yeasts are fungi that grow as solitary cells that reproduce

by budding and can be classified either as Ascomycetes or Basidiomycetes (Johnson,

2013a) and any microorganism that has the ability to multiply in foods to a high level, also has the ability to cause foods to spoil In the case of beneficial microorganisms, they convert substance piece of crude materials during aging which at that point improves the healthy benefit of some foods (Tamang, Watanabe, & Holzapfel, 2016)

* Sources of natural antimicrobial

González-Fandos et al., (1994), Kim et al., (2004) and Quinto et al.,( 2019) all gave reports about the antimicrobial activities of onions and garlics, the juice from

onions were said to inhibit growth of some bacteria ( Escherichia coli, Clostridium

Saccharomyces, Candida, etc.) Some coffee processing industries‘ by-products

include coffee pulp, peel and husks (Esquivel & Jiménez, 2012), products gotten from extraction procedure of these by-products are tested and said to possess reasonable amount of phenolic compounds which are known to be potential natural preservatives for food (Gyawali & Ibrahim, 2014)

In a bid to test for activity of antimicrobes of some extracts, Sagdic et al., (2011) did an antimicrobial test on beef patties using grape pomace extracts as an antimicrobial at different concentration (1-10%) and time (12, 24, 48 hrs), the result

showed that spoilage microorganisms and Enterobacteriaceae were inhibited at 10%

concentration across all storage time period Friedman et al., (2013) also carried out a test to show the inhibitory property of olive pomace extract against the development of

S aureus, L monocytogenes, Enterobacter spp., E coli and Salmonella spp

CHAPTER 3 : MATERIALS AND METHODS

3.1 Overall experimental design

Overall experimental design of the study is shown in Figure 3.1

3.2 Study area

This study is conducted at the Nha Trang University, Nha Trang city, latitude prolonger from 12º14' N to 12º19.6' N and longitude prolonger from 109º11' E to 109º48.2' E, this area is within the Khanh Hoa province

Figure 3.1 Fresh Cocoa pod husk stored in Styrofoam boxes

3.3 Preparation of dried sample for extraction

Fresh cocoa pods (Theobroma cacao trinitario) was removed from the husk,

cut into tiny bits about 1 mm in size and dried at 720W in a microwave oven to get the best extraction yield according to the description given by Nguyen et al (2015b) The irradiation used by microwave breaks the shell of the pod thereby releasing bioactive compounds The dried cocoa pod husk was loaded in plastic bags, sealed and kept in freezer till it was needed, this is to preserve the bioactivity of the husk Before the dried cocoa pod husk was used for all experiments and analysis, the dried samples were ground to powder form with the use of a hammer mill after which a stainless-steel mesh sieve (200 mm) was used for screening to achieve very fine particle size

Figure 3.2: Preparation, purification and assessment of biological activity of

saponins from cocoa pod husk (CPH)

Fresh cocoa pod husk (CPH)

Drying in microwave oven at 720 W

Grinding with hammer mill

85% methanol extraction for 40min

Purification using column chromatography with silica gel

Figure 3.3: Cutting Cocoa pod husk into thin slices

Figure 3.4: Dried cocoa pod husk

Figure 3.5: Milled cocoa pod husk 3.4 Extraction

According to the results found by Nguyen et al., (2016b) on extraction and solvent method, the crude saponins from cocoa pod husk were extracted by using diluted methanol (85%) for 30 min at room temperature, wrapped up by microwave-assisted extraction (MAE) using a microwave oven at a power of 600 W, irradiation time of 6 s/min meaning the microwave was allowed to run for only 6s and left to rest the remaining 54s in a minute, extraction time of 40 min and ratio of solvent to sample

of 50 mL/g The flask was connected to a condenser to further help in regulation of the temperature, after extraction the flask was covered with foil to stop release of vapor and then quickly cooled to room temperature utilizing a water bath, lastly sifted utilizing vacuum filter through a 55 mm breadth Whatman filter paper 1 to acquire the extract for additional trials

Figure 3.6: MAE set-up

Figure 3.7: Crude saponin extract

Figure 3.8: Filtering crude saponin extract 3.5 Preparation of crude saponin-enriched powder

After filtration, the crude saponin-enriched extract was concentrated in a 500 mL flask until thick but not completely dried to increase the concentration of the sample, concentrate was gotten using a condenser rotary evaporator unit with vacuum pump set

at 50ºC (temperature lower than boiling point of methanol) 10 mL each of the extract was pipetted into 5 closed glass containers and prepared for freeze-drying into powders

Figure 3.9: Condensed crude saponin extract

Figure 3.10: Sample prepared for freeze drying

Figure 3.11: Crude saponin-enriched powder after freeze drying

3.6 Identification of saponin-enriched extract and powder

The identification was done using different methods which include:

3.6.1 Fourier-transform infrared spectroscopy (FTIR)

FTIR analysis of saponin-enriched powder was analyzed using the description of Sakugawa et al., (2004) with little modifications FTIR spectra of saponin-enriched powder was recorded in absorbance with 64-128 scans at a resolution of 4 cm-1 using a Nicolet Magna 860 FTIR spectrometer

3.6.2 Thin-layer chromatography (TLC)

Two milliliters of saponin-enriched extract was further identified by means of commercially prepared thin layer chromatography plates according to the method of Chatterjee et al., (2011) using 100% acetonitrile; 30% acetonitrile:70% distilled water; 50% acetonitrile:50% distilled water; 70% acetonitrile:30% distilled water; 100% methanol; 20% methanol:80% distilled water; 50% methanol:50% distilled water; and 80% methanol:20% distilled water as a mobile phase The sample was prepared by filtering the saponin-enriched extract using 0.45 and 0.22 µm nylon Phenex syringe membrane filters

The thin plates were divided into four (4) equal parts so as to fit into the TLC glass, one part of the thin plate was used for each experiment A line 1.5 cm from the base of the plate was drawn so as to mark the point where sample was injected, two