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

22 3.3.3 Preparation of solutions from crude CPH extract and derived fractions for evaluation of phenolic content, antioxidation potential and antimicrobial activity .... LIST OF ABBREVI

MINISTRY OF EDUCATION AND TRAINING

NHA TRANG UNIVERSITY

MINISTRY OF EDUCATION AND TRAINING

NHA TRANG UNIVERSITY

Topic allocation decision 192/QD-DHNT on 03/03/2020 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 declare that the thesis entitled “Extraction and assessment of antioxidant and

antimicrobial activities of phenolics 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

3rd September, 2020

Uwihaye Festus

ACKNOWLEDGEMENTS

Many thanks and praises to God Almighty that he has been with me throughout

my study period in Vietnam

Firstly, I would highly acknowledge Dr Nguyen Van Tang, my principal supervisor, and Dr Tran Thi My Hanh, my co-supervisor for their help and guidance, they gave me an amazing experience I also would like to thank 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”

Secondly, I also wish to appreciate my country, Rwanda through the Ministry of Labour as it accepted me to join in Master program at the Nha Trang University, Vietnam

Thirdly, I am grateful to the supports from VLIR, Graduate Studies Department, and Faculty of Food Technology, Nha Trang University At large, I can not forget the Nha Trang University’s entire teaching staff and my classmates for providing me with

a good and favorable working environment

Lastly but not least, my special acknowledgment goes to my wife, Ugwaneza Grace and my children, Jayden, Joanna and Jordan for their undeniable love, encouragement, patience and understanding

3rd September, 2020

Uwihaye Festus

TABLE OF CONTENTS

UNDERTAKING iii

ACKNOWLEDGEMENTS iv

TABLE OF CONTENTS v

LIST OF ABBREVIATIONS ix

LIST OF TABLES x

LIST OF FIGURES xi

CHAPTER 1 INTRODUCTION 1

1.1 PROBLEM STATEMENT 3

1.2 RESEARCH SIGNIFICANCE 4

1.3 RESEARCH AIMS 4

1.3.1 Overall aim 4

1.3.2 Specific aims 4

CHAPTER 2 REVIEW OF THE LITERATURE 5

2.1 COCOA AND COCOA BY-PRODUCTS AS WASTES AND VALUE ADDITION 5

2.1.1 Husk of cocoa pod 5

2.1.2 Cocoa bioactive compounds with emphasis on phenolics 6

2.1.3 CPH applications and phenolic compounds 9

2.2 IMPACT OF PROCESSING METHODS ON PHENOLIC COMPOUNDS AND ANTIOXIDATION POTENTIAL OF CPH 12

2.3 FRACTIONATION, PURIFICATION, CHARACTERIZATION AND QUANTIFICATION OF PHENOLIC COMPOUNDS 16

2.4 EVALUATION OF ANTIOXIDANTION POTENTIAL OF PHENOLICS 17

2.5 COCOA PHENOLIC COMPOUNDS AND BIOLOGICAL ACTIVITIES 18

CHAPTER 3 MATERIALS AND METHODS 20

3.1 EXPERIMENTAL PROCEDURE 20

3.2 MATERIAL AND CHEMICALS 21

3.2.1 Fresh cocoa pod husk 21

3.2.2 Analytical reagents 21

3.3 EXPERIMENTAL METHODS 21

3.3.1 Preparation of powder from crude CPH extract 21

3.3.2 Fractionation of crude extract from CPH by column chromatography 22

3.3.3 Preparation of solutions from crude CPH extract and derived fractions for evaluation of phenolic content, antioxidation potential and antimicrobial activity 23

3.3.4 Phenolic compounds identification in CPH extract 23

3.3.5 Analysis of physicochemical properties of powdered extract from CPH 25

3.3.6 Analysis of bioactive compounds 26

3.3.7 Evaluation of biological activities 27

3.3.8 Statistical analysis 29

CHAPTER 4 RESULTS AND DISCUSSIONS 30

4.1 PHYSICOCHEMICAL PROPERTIES OF CRUDE CPH POWDER 30

4.2 IDENTIFICATION OF PHENOLIC COMPOUNDS BY THIN-LAYER CHROMATOGRAPHY 32

4.3 IDENTIFICATION OF PHENOLIC COMPOUNDS BY FTIR SPECTROSCOPY 33

4.4 IDENTIFICATION OF PHENOLIC COMPOUNDS BY HPLC 35

4.5 BIOACTIVE COMPOUNDS OF DERIVED CPH FRACTIONS 42

4.5.1 Total phenolic content (TPC) 42

4.5.2 Total flavonoids content (TFC) 44

4.5.3 Saponins 45

4.6 ANTIOXIDANT ACTIVITIES OF CPH EXTRACT AND DERIVED FRACTIONS 46

4.6.1 DPPH radical scavenging capacity (DRSC) 46

4.6.2 Cupric reducing antioxidant capacity (CUPRAC) 47

4.6.3 Ferric reducing antioxidant power (FRAP) 48

4.7 ANTIMICROBIAL ACTIVITY OF CRUDE CPH EXTRACT 49

CHAPTER 5 CONCLUSIONS AND FUTURE PERSPECTIVES 52

5.1 CONCLUSIONS 52

5.2 FUTURE PERSPECTIVES 52 REFERENCES I

LIST OF ABBREVIATIONS

CBS : Cocoa bean shells

CE : Catechin equivalents

CUPRAC : Cupric reducing antioxidant capacity DPPH : 2,2-diphenyl-1-picrylhydrazyl

DRSC : DPPH radical scavenging capacity

EE : Escin equivalents

FRAP : Ferric reducing antioxidant power

FTIR : Fourier-transform infrared spectroscopy GAE : Gallic acid equivalents

HPLC : High performance liquid chromatography MAE : Microwave assisted extraction

MeOH : Methanol

MIC : Minimum inhibition concentration

ORAC : Oxygen radical absorbance capacity

RE : Rutin equivalents

SFE : Supercritical fluid extraction

TAA : Total antioxidant activity

TE : Trolox equivalents

TFC : Total flavonoid content

TLC : Thin layer chromatography

TPC : Total phenolic content

WSI : Water soluble index

LIST OF TABLES

Table 2.1 Total phenolic content (TPC) of cocoa by-products 10

Table 2.2 MAE characteristics 15

Table 4.1 Physiochemical properties of crude CPH powder 31

Table 4.2 Bioactive compounds of crude extract and fractions from CPH 46

Table 4.3 Antioxidant capacity of crude extract and fractions from CPH 49

Table 4.4 Antimicrobial activity of phenolic-enriched powder from CPH 51

LIST OF FIGURES

Figure 2.1 Classification of dietary phenolic compounds in foods and their general

chemical structures 7

Figure 2.2 The cocoa fruit structures1and wastes2 Source: 11

Figure 2.3 Scheme of MAE apparatus 15

Figure 2.4 Strategic steps for treating material from plant by targeting phenolics 17

Figure 3.1 Overall experimental procedure for extraction and assessment of biological activity of phenolics from CPH 20

Figure 4.1 TLC of phenolic-enriched extract from cocoa pod husk using 30% methanol as solvent 33

Figure 4.2 FTIR spectrum of phenolic-enriched extract from CPH 34

Figure 4.5 HPLC chromatograms at 272 nm of mixed standards (A and B) and 8 fractions (C to J) 39

Figure 4.6 HPLC chromatograms at 272 nm of 6 re-fractionated fractions (A to F) 41

ABSTRACT

The main objectives of this research were to extract phenolic compounds from the CPH using already optimized conditions as well as evaluate bioactive compounds and biological functions of obtained extracts and fractions Microwave-assisted extraction (MAE) was used for extraction under optimum condition (50 mL/g, 600 W,

30 min, and 5 s/min) and water as a solvent The CPH extract fractionation was done

by column chromatography (CC) Qualitative analysis of phenolic compounds was done using TLC, FTIR, and HPLC Quantitative determination for total phenolics (TPC), total flavonoids (TFC) and total saponins (SC) shown in milligram of gallic acid, catechin and escin equivalents per gram of dried CPH, respectively, together with antioxidant abilities were analyzed by colorimetric assays coupled with UV spectrophotometry The results showed that there was a significant difference (p<0.05), with higher bioactive compounds in crude CPH extract, 14.96  0.86 for TPC, 119.17  18.63 for TFC and 451.58  44.09 for SC, compared to the content of CPH extract fractions The antioxidation analysis results showed that there was a significant difference (p<0.05), with CPH extract having higher antioxidation capacities, DRSC, CUPRAC and FRAP with values of 54.12  0.19, 66.12  1.94 and 76.17  5.00 mg TE/g dry extract, respectively, than values in CPH extract fractions FTIR and HPLC analysis indicated that the fractions from the crude CPH extract contained some major phytochemical compounds including theobromine, theophylline, and (-)-epigallocatechin gallate The crude CPH extract antimicrobial

activity was evaluated on two bacterial strains (Bacillus subtilis and Escherichia coli) and one fungal strain (Candida albicans) by use of agar diffusion method The extract

showed some traces and 1 mm diameter of inhibition zones against test bacterial strains and fungal strain, respectively Hence, this study underline the probable exploitation of CPH extract as natural source of antioxidant and antibacterial which could have considerable and valuable use in different domains including food, cosmetic sector, and pharmacy, to mention few

Keywords: Cocoa pod husk, microwave-assisted extraction, bioactive

compounds, phenolic compounds, biological activities, antioxidant, antimicrobial

CHAPTER 1 INTRODUCTION

The cocoa tree (Theobroma cacao L.) is said to be have been first found in

Amazon region of South America, and now its farming has been expanded and vast plantations are localized in both tropical and subtropical regions Africa high percentage

of cocoa production share (68%), followed by Asia (17%) and Latin America (15%) [1]

In 2013, 3.9 million tons of cocoa bean, worth around $12 billion, global production was achieved [2,3] In general, cocoa market place increase by 3% each year and 40 to 50 million people worldwide benefit from cocoa farming opportunities [1] It was reported that not more than 450g of chocolate are obtained from 400 cocoa beans [1], which means that 10 tons of wet cocoa pod husk (CPH) come from 1 ton of dry beans [4]

Cocoa producing countries generate a lot of by-products considered as waste [3] , and they are not used but discarded Considering fresh weight, a great percentage

of fresh cocoa pod is considered to be waste [2,5] That portion covers cocoa shell (8.15 ± 0.78%), cocoa pod husk (70.15 ± 0.67%) and cocoa bean pulp (21.70 ± 0.20%) [6] Not only the bad smell, but also cocoa pod husk left in the field of cocoa plantation is associated with environmental problems and disease propagation like black pod rot [4] and with ches' bloom [7] Around 6.4 (1.5 times cocoa beans) [2] and

55 million tons (13 times cocoa beans) [8] of cocoa pod shell and cocoa pod husk are world annual production, respectively There is a need to exploit those cocoa by-products The interest in cocoa by-products comes from their availability and high phenolic content [1] Phenolics from cocoa, the same as those of other plants, have been part of various studies as bioactive compounds [9], and were proven to have properties which are important to the health like antioxidants, [10]

In the current trend, the health benefits of phenolic compounds has been topic

of discussion and research [11] Epidemiological studies have reported that phenolics

are probable candidates for prevention of chronic diseases [5] Farahmandfar et al.[12]

reported that the phenomenon through which antioxidant ability happens is by redox reactions that inhibit highly reactive molecules effects As it was reported, Abdul

Karim et al.[13] has shown that phenolic compounds delayed skin aging and protected

it from UV radiations Grillo et al.[14] showed that because of high fiber and

antioxidants content, the CPH could be used in food and beverages

Cocoa bioactive compounds are in various categories which include phenolics, flavonoids, saponins, and many others, and they affect some of the bioactivities [9] It was reported that the minimization of waste from cocoa industry by value addition should be strategic [10] By use of supercritical fluid extraction of cocoa hulls, a value

of 1.8% TPC was achieved [9], and another process at the optimum conditions using MeOH for the extraction, 0.52 % yield was obtained [5]

Exploitation of cocoa by-products considered as waste can reduce environmental pollution and improve the value for both cocoa farming and processing

industry [2] Valadez-Carmona et al.[5] reported that cocoa waste have a considerable

phenolic content which make them attractive to production of value-added products Polyphenol content of 1.09g/100g dry weight was achieved from cocoa shell [6] and 4.6% soluble phenolics in CPH extraction [4] Extraction of phenolics is very important and the conditions during process can affect bioactive compounds and their

activities [15] Md Yusof et al.[10] indicated that its crucial to properly choose an

extraction method as different techniques give different outcomes of bioactive compounds Various solvents extracted polyphenolics in the CPH, 8.48 mg GAE/g total phenolic as the maximum value was obtained by water extraction [6] The choice

of solvent is important as its physicochemical characteristics impact on amount and type of phenolic compound [15] More polyphenolic content, 2-3 times more, were realized in cocoa bean husk by use of aqueous solvents, than using pure ones [16] The process of extracting of polyphenolics has been implemented traditionally way by use

of conventional methods, currently there is a need to adapt novel methods and one of

them is microwave-assisted extraction Abbas Delazar et al [17] reported that method relies on microwaves heating during extraction

By referring to the previous studies, extraction of phenols in the CPH by microwave-assisted extraction (MAE) and water serving as extraction medium on optimized conditions has not been reported

There are a number of advantages associated with the use of MAE including does not take long to get the yield, just little quantity of extraction medium is required,

very effective and cheap [17], and using water during extraction process is safe and environmental friendly due to that it can produce clean and safe extracts [18]

In most cases, a traditional technique yields low amount of phenolics when compared

to modern technology [10]

The research main intentions are: (a) extracting and recovering of phenolic compounds in the CPH using already optimized conditions, and (b) evaluating bioactive functions of the extracted phenolics from the CPH

1.1 PROBLEM STATEMENT

So many researches have been conducted and identified natural bioactive compounds with different biological activities (prevention of oxidation, aging, cancer, ) in different edible plants Nevertheless, the use of normal foods to generate other food components is seen as unprofessional which could also affect food prices, and consumers will be required to incur more cost [15] By referring to the trending demand for maximum valorization of food waste [14], there was an involvement and willingness to isolate natural bioactive compounds from unused and ignored plant materials such as peels, leaves and pulps, and worldwide many industries have embarked in drafting and implementing measures which can minimize process by-products considered as waste [14] Additionally, the use of synthetic additives has been quite long under criticism due to probable undesirable side effects to human health [19]

Grillo et al.[14] also reported that despite the publication number of patents and

research papers related to food waste value addition strategies, the targeted objective is still far from being full achieved

In the cocoa industry, chocolate production requires the use of fresh cocoa beans, which are fermented and dried, while cocoa pod husk and shell account for high percentage of cocoa fruit are discarded to the landfill Every year, there is an increase

in production and processing of cocoa beans which correlate with an increase in waste reading to the disposal of million tons of cacoa pod husks [4] Chocolate market was projected to increase at a 2.3% annual rate, and wastes generated by chocolate industry were estimated at a 3.1% increase annual rate between 2014 and 2019 [14]

Despite being considered to be waste, research has proven some of potential

uses and detection of bioactive compounds Vriesmann et al.[4] has showed that a

4.6% soluble phenolics was found in cocoa pod husk, and 45.6-46.4 mg GAE of

soluble phenolics were reported by Abdul Karim et al.[20] It was reported that cocoa

pod husk contains phenolic acid including caffeic acid [15] It was also indicated that cocoa pod husk has anti-caries activity [16]

The extraction and valorization of phenolic compounds from cocoa pod husk which is renewable, sustainable and its availability is in huge quantity source and is of great importance as it has many applications in different domains (food industry, cosmetics, pharmacy, ) The proper usage of the cocoa pod husk could decrease their environmental impact and problems, and contribute to the economic advantages [4,16,21] It is required to apply appropriate techniques, which can achieve the intention of waste value addition, and this needs a shift from conventional methods These techniques are associated with so many drawbacks including being hard to apply, a lot of time of getting for high yield, huge quantity of solvents, and sometimes degradation of bioactive compounds and vaporization of other important components, especially aromas and flavours Non-conventional techniques are able to overcome the mentioned limitations, hence fit for positive outcomes in terms of projected bioactive compound quantity and quality

1.2 RESEARCH SIGNIFICANCE

The outcomes of this research could probably be significant for the potential different beneficial uses in food industry, pharmacy, cosmetics, etc In addition, the information obtained could be the base for further researches It would also strengthen valorization of the CPH, which will further reduce the environmental problems and cocoa plantation diseases spread caused by its discard

1.3 RESEARCH AIMS

1.3.1 Overall aim

The general intention of the research was to extract phenolic compounds in the CPH

using already optimized conditions and evaluate their biological functions in vitro

CHAPTER 2 REVIEW OF THE LITERATURE

There was a focus of some topics which included cocoa and cocoa wastes, mainly the CPH, bioactive compounds with emphasis on phenolics, MAE as one of methods for phenolics extraction, phenolics purification, and then phenolic biological activities (antioxidant, antibacterial, )

2.1 COCOA AND COCOA BY-PRODUCTS AS WASTES AND VALUE ADDITION

2.1.1 Husk of cocoa pod

The fresh fruits of cocoa tree are in various shapes and colours, the tree is fixed either on main branches or those of aside Once cocoa is broken down and inner content known as cocoa beans are removed, the remaining material consist of three main parts which are cocoa mucilage, cocoa bean shell and cocoa pod husk (Figure 2.2) [22]

Since the CPH has been considered as one of the most important chocolate processing cocoa pod derivative products ignored [3,4] The CPH pose a serious disposal problem, as per the estimations quantity of CPH related to the production of 1 ton of dry beans, are 10 times higher that of dry beans [4,14] In fact, the CPH is not used properly and could be handled as unwanted material to be discarded In normal case, the activity of removing cocoa seeds from cocoa pods is done directly in the field and the CPH is not collected, so it decomposes and creates inappropriate conditions to the environment

Beyond production of bad smells, the CPH under decomposition in the field could also spread cocoa tree diseases, and the most common known is black pod rot [4,20,23,24] The estimated production loss per year caused by black pod rot could be

of 30 up to 90%, whereas at the global level it ranges between 20 to 30% [25]

Almost 75% of fresh cocoa pod is covered by CPH mass [25], meaning also that around 750 kg will be considered as material to discard [26,27], and it contains 11.88 g/kg tannins [27] Even though, there still some issues on value addition of the CPH, different ways of exploitation have been implemented and are targeting to minimize the discard of cocoa by-products and this lead to the production of valuable

products, used in different domains such as food antioxidants, dietary fibers, animal feed, etc

Bioactive compounds occur in some foods in little amount and are beyond the

normal nutritional role [28], whereas Rachmawaty et al.[29] defines bioactive

compounds just as food components in little quantity which could be vitally nutritional

or not, and could give important properties to life Phenolic compounds in plants (around 8000 types) [30,31] are secondary metabolites, often produced in plant subsequent phase to growth Shikimic acid and malonic acid pathways are channels for phenolic compounds generation and this induce to define the terms phenolic and polyphenol as all secondary natural metabolites which are end-products to shikimate phenylpropanoids-flavonoids pathways by biogenetic [32]

For Vriesmann and de Oliveira Petkowicz, and Dias et al.[32,33], "phenolic"

or "polyphenol" as a chemical definition, is a chemical material which the hydroxyl replacements attached to thebenzene ring are one of esters, methyl ethers, glycosides and others, and is called a phenol when one hydroxyl is replaced and polyphenols

when more than one are replaced Lecumberri et al.[34] defines phenols as a big

group of chemicals that their hydroxyl moieties are attached on aromatic ring, and numerous categories of these compounds exist More than 4000 known phenolic compounds are flavonoids, one of the main phenolics found in plants Flavonoids chemical structure consist of C15 which in positions of 6-3-6 carbon atoms The main classification of flavonoids comes from different substituents to C3 in the middle of two aromatic rings [33] (Figure 2.1)

Figure 2.1 Classification of dietary phenolic compounds in foods and their

general chemical structures Source: Andrea and Cano[27]

Generally, the main objectives of the techniques applied when extracting compounds with bioactivities among many others are: (a) to obtaining bioactive compounds of target function in a plant under study, (b) increasing the efficacy of techniques used in analysis, (c) boosting bioassay sensitivity by the increase the interested compound concentration, (d) obtaining converted bioactive compounds which are much applicable due to they are easy to sense and extract, and (e) providing

a stable technique which is not affected by changes in plant under study [35] Cocoa has been related to several health and technological benefits due to phenolic fraction in its composition Of those, phenolic compounds and procyanidins have been ranked the most important subgroup under flavonoids classification [30]

Cocoa bioactive composition is made of various group of compounds and those include phenolic acids, flavonoids, saponins and their derivatives and other small

fractions constituents [9] Among many phenolic compounds identified in cocoa products, procyanidins emerge as the most abundant and its content is functional of where it orginated and production status [36] Cyanidin-3-galactoside as well as cyanidin-3-arabinoside as anthocyanins at a concentration of 0.02 to 0.4% dry mater together with epicatechin and catechin as polyphenols at a level of 2 to 4% dry mater, these compounds were detected in cocoa bean flour, which its fat content was removed The procyanidins content level obtained after extraction is various dependent

by-of some intrinsic and extrinsic factors during extraction process Its probable to work under maximum conditions which give higher percentage of end-product, and methanol-water (8:2 v/v) was proposed to be suitable mixture in extracting coloring chemicals in cocoa pod shell samples [2] In comparative study done by Azila Abdul

Karim et al.[13] in Malaysia on antioxidant properties of cocoa pod husk and shell, it

was noticed that between them, cocoa pod shell was lower in antioxidation by DPPH scavenging ability, but both were below antioxidation shown by gallic acid, a phenolic used as standard, and the CPH was much higher in antioxidation by measured by FRAP method than cocoa pod shell

Different plants have shown to have extracts with bioactive functions For

example, extracted compounds from P amarus were possibly active in healing some

of the health ailment including hepatitis, plasmodia, inflammation, malaria, diabetic Those functional properties observed are much related to the biochemical

constituents in extracts [37] Another plant, the artichoke (Cynara scolymus L.) has

phenolic compounds which have shown bioactivity through their antioxidation, antifungal, and antimicrobial activities [38] The presence of phenolic compounds with

biological activities, in Oxalis corniculta L was also confirmed [39] Cocoa and its

by-products have high content of phenolic compounds and many studies have been conducted so far due to some properties of their phenolic content like antioxidant and antiradical activities Cocoa bean hull has pigments and bioactive compounds with distinct biological activities, and this has made cocoa bean hull to be a more attractive material for extraction By use of the most common method for total phenolic content evaluation using Folin Ciocalteu reagent, a 1.8% TPC in average was realized [9]

In general, it was reported that the extracts with high total phenolic contents would be expected to exhibit much of oxidation and microbial inhibitions hence their

effects will be profound when used as food components [19] In a study of evaluating

antioxidantion abilities of cocoa and its by-products, Martínez et al.[24] reported a

strong correlation (R2 > 0.95) was observed between TPC and all antioxidation activity assays (DPPH, ABTS and FRAP) A study conducted on cocoa liquors to evaluate its phenolic compounds level and their structure and antioxidation capability has realized that antioxidation obtained was much related to the quantity of phenolics (coefficient

of determination was greater than 0.9), this allowed the suggestion that the dominant constituents which contributed to the antioxidantion achieved were having a characteristic of polyphenolic structure [31] Tiburcio [40] also confirmed a very

corresponding ratio of phenolics level to antioxidation effect was observed Sotelo et

al.[31] has reported that alteration of the quantity and the quality of the polyphenols in

the food matrix is a result of modulation of the polyphenolics production synthesis of phenolic compounds The unique chemical structure will determine specific antioxidant capacity of each type of substance, and those alterations in the type of phenolic compounds could change the ability of inhibiting oxidation for the particular food being analyzed

2.1.3 CPH applications and phenolic compounds

The CPH value has many fields of applications It has been reported to be the main ingredient in many of value-added products (animal feed, soap ) Other biotechnological opportunities for CPH value addition are its use to make fuels and as functional ingredient in food [22]

The good proximate composition (protein around 6%, crude fat up to 10%, fiber around 4%) of CPH has attracted the attempt to consider it as an important ingredient

to be incorporated in feed of animals [22], but theobromine presented at high concentration has a detrimental effect on animals [3] Though, the chemical composition of the CPH is complex but one of chemicals contained is phenolic compounds [41] Due to the availability of cocoa pod husk, its important to exploit them and generate compounds with potential functional activities to be used in food industry and pharmacy [4]

Table 2.1 Total phenolic content (TPC) of cocoa by-products

Cocoa by-products shell of

bean (%)

husks of pod (%)

Pulp (g/100g DM)

As per reports from Lu et al., and Rachmawaty et al.[25,29], phenolic acids

content in the CPH was between 0.46 and 0.69 g GAE/g, whereas the TPC of fresh CPH reported was around minimum of 2 and maximum of 3 mg GAE/g [25], and

45.6-46.4 mg GAE/g of soluble phenolics were reported by Abdul Karim et al.[20] For Martínez et al.[24], a range of 2.07 and 3.65 mg GAE/g was established as the

TPC value of the CPH, whereas range of 16.40-23.01 mg GAE /g was realized by Sotelo, Alvis and Arrázola [42]

Phenolic compounds were detected during the study of rheological properties of pectins in the CPH determined under various treatments [43] It was indicated that the proportion of cocoa bean and cocoa pulp of fresh cocoa pod fruit was 23% and 26% and that of the CPH was the more than the double of the two above [44] In general, in plants, polyphenols are found in most external parts [4,14,45] The value addition of cocoa by-products which are discarded, has been discovered by referencing on cocoa beans [14] (Table 2.1)

A study conducted on polyphenolic compounds and their antioxidation in the extracts from some plants has established higher concentration of polyphenols in fruit peels than pulp, reaching up to twice the amount found in pulps of bananas and tangerines, which also reflected in higher oxidation inhibition power [31] The flesh of kiwifruit was less in polyphenolics and flavonoids content than its pericarp, also the content of peels and reflected biological functions including antioxidation and bacterial inhibition were much more than that of the flesh [46] Cocoa pod has got three successive parts, the one out known as epicarp, the intermediate part known as mesocarp, and the one inside known as endocarp [22] Depending on the clones, at the ripening, there is variation in thickness and color, green to red at the ripening stage Those changes could reflect the maturity stages and be associated with accumulation

of bioactive compounds at different levels [47]

Figure 2.2 The cocoa fruit structures 1 and wastes 2 Source: Campos-Vega et al.[47]

A number of conditions can impact on cocoa phenolic compound composition and those include environmental associated factors like where the cocoa originated, growth stage, factors in production process like high or low temperature, use of chemicals, and storage associated factors [22] Procyanidins have been identified as the most abundant polyphenol group in cocoa [48] A variation in polyphenolic compounds of cocoa derived products has been reported Cocoa powder had a higher range of TPC (0.33-6.5%) than dark chocolate (0.17-3.65%) [45], whereas in the cocoa pod shell, range of polyphenolics was 1.3-1.8%, which include some of flavan-3-ols, little quantity of anthocyanins and flavonols [14]

Yapo et al.[23] reported that during the CPH compounds extraction, the four

step sequential method was used whereby fractions in hot aqueous ethanol, cold aqueous acetone and luke warm water were considered as soluble phenolics, and fractions in hot acidified butanol as insoluble portion From the study results, TPC from husk of “fresh” cocoa pod has been realized to be > 6.9%, higher than the range 2.0 to 3.0% of both cocoa bean hull and kernel products under study, and which have undergone fermentation and roasting The above results suggest that the CPH was rich

in proanthocyanidins compared to other by-products from fermentation and roasting treatments It was found that condensed tannins were lower in the CPH samples than the other two cocoa by-products, and it could have been the results of chemical changes, the most important being phenolics converted into insoluble products, when

kernel was fermented Also, the insoluble portion was increased by end-products of Maillard reaction, which occurred in the presence of heat Discovery for the outcomes

of oxidation inhibition ability evaluation that active radicals were scavenged by soluble phenolics from the CPH compounds at the level of more than 85% compared

to the maximum scavenging ability of 68.0% realized by both kernel and hull products This showed that antioxidation measured by scavenging ability was higher in the CPH products than in kernel and hull products Additionally, the calculated EC50

of kernel and hull products with a maximum value of 55.0 g/g was two times higher than that of CPH products The same trend was also obtained by scavenging ability

evaluation by ABTS method Abdul Karim et al.[20] reported that in two different

studies, TPC in the CPH varied between 45.6-46.4 mg GAE of soluble phenolics and 55.93 - 57.07 mg GAE per g

2.2 IMPACT OF PROCESSING METHODS ON PHENOLIC COMPOUNDS AND ANTIOXIDATION POTENTIAL OF CPH

Among some of extraction processes having an impact on the CPH phenolics

content and antioxidants activity have been published Though, Abdul Karim et al.[20]

reported that the CPH total phenolic content with a maximum value of 57 mg GAE/g dry mater but minimum value of TPC of around 4 mg GAE/g dry mater was realized

in function of sample origin and type of solvent Campos-Vega, Nieto-Figueroa and Oomah [47] found that much of TPC (3.5 mg GAE/g) was obtained in the CPH samples from Cone region (Ecuador) when extracting the CPH by MeOH-acetone, compared to 2.0 mg GAE/g realized by ethanolic extraction The same trend was also observed for its antioxidation capacity The CPH extract was gained by two different solvents by maceration method, in which 70% acetone was found to have extracted phenolics content almost double times (around 95 mg GAE/g) as compared to ethanol-

water (7:3 v/v) at 50 mg GAE/g) [41] In addition, the milling method has been proven

to probably impact on the CPH phenolic content When the CPH powder under oven drying of less than one millimeter was treated by dry-milling, around 5% of phenolic content by dry weight was achieved, but for those of less than one point seven millimeter, this gave a 7% phenolic content when extracted [22]

The use of microwave as a novel method may protect phenolic content and increase the extraction yield, but it should be noted that each method has its effect

Some of processing methods, which affected phenolics and antioxidants of CPH were reported It was found that in comparison to hot air and freeze-drying methods, drying

of CPH using microwave achieved higher TPC and other phenolic compounds For example, during the CPH extraction at the power of 595 W for 11.5 min, 3.4 higher times TPC content was achieved

Drying by use of microwave is recommended as it can improve on the extraction of bioactive constituents [47] Though, generally freeze drying has been performed well than drying by air in terms of affecting extraction of phenolic compounds, but its implementation should be done careful to avoid negative impacts

on composition of extracts, which could lead to the reduction of phenolic content [49]

In another report, it was shown that there was a considerable increase in TPC when the CPH treated with acetic acid was sun-dried, compared to those were not treated Acid treatment was suggested to be important in inhibiting oxidation caused by PPO [50]

Furthermore, Daniel Oduro et al.[42] indicated that higher phenolic extracted by

ultrasound with maximum TPC of 23 mg GAE/g compared to the maximum TPC of

19 mg GAE/g obtained when extraction was done by agitation process

The TPC of dry CPH extracted by ethanol solvent (5:5 v/v) reduced by 42% when samples were autoclaved at 120oC but a 37% more TPC was achieved when the

CPH samples were fermented by a fungus strain (Rhizopus stolonifera NRRL 28169)

It is believed that fermentation has caused conversion of some molecules into new products, which contributed to the increase of phenolic content [51] Shalini [52] found the same way that antioxidation ability was increased when the CPH was treated

by a different fungus strain (Rhizopus stolonifera LAU 07) However, Peralta-Jiménez

and Cañizares-Macías [51] found that there was much of phenolic content in the extract from the CPH without any treatment than those found in the CPH, which has undergone fermentation and the latter has higher antioxidation ability measured by scavenging capacity of DPPH and reduction of ORAC The observed outcome is assumed to be molecular changes of polyphenolics

A study, where the CPH was treated by a fungus strain, more than 11 and 89% decrease in phenolic content and tannin content was observed, respectively The decrease could have been the results of some compounds decomposition which were then used by fungus [53] Observed rise in phenolics of the CPH after fermentations is due to the phenolics migration induced from the inner parts of cocoa pod, which is the

usual store of phenolic compounds to the outer part (CPH) During the CPH pectin extraction by hot water at 50oC and 100oC for 90 min, and 1:25 w/v, pectin extracts was co-extracted along with phenolics It was found that higher phenolic content (0.098 GAE/g) was realized by pectin extraction at low temperature than the one achieved at high temperature (0.083 GAE/g) [4]

It was reported that there is no single technique which could be effective in process of extracting of all phenolic compounds as the difference in the natural composition of plant phenolic content brings variation in extraction results [49] Many factors could affect extraction of phytochemicals Among others, there is chemical nature of compounds, extraction method applied, particle size of the sample, etc [54] The traditional methods for bioactive compounds extraction though being associated with many drawbacks but are still somehow relied on A number of limitations have been recorded during conventional extraction and these include among others: required longer time, need for pure solvent which is expensive, a lot of solvent to be evaporated, minimal extraction selectivity, and thermal decomposition of heat sensitive compounds [35] Since then, organic solvents extraction methods of bioactive compounds from cocoa pod husk have been trending The variation in chemical characteristics of organic solvents has big impact to the outcome of extraction [22] Nowadays, many new (modern or greener) methods have been developed along with conventional methods, and they present many advantages than those traditional methods (for example: higher yield, shorter time, easier to apply )

During extraction by microwave-assisted technique, solvents and plants tissues are heated by use of microwaves energy, which activate the extraction kinetic [17] By the fact that the microwave-assisted extraction does not require the use of much solvent, it does not affect environment, contrary to most of common organic solvents

in extraction methods Its application is associated with a lot of benefits which include boosting of extraction efficacy, minimal heat effect of bioactive compound, cheap to

be applied, does not require much solvents, microwave energy deep penetration and does not take long to get yield [17,55,56]

The current available microwave instruments work with the power which can

go up to 700 W [28,57] Microwave as a source of energy for heating during extraction

is build on its effect on polarity of compounds to be extracted [58] (Figure 2.3)

Transparent solvent used in microwave allows heat from microwave radiations

to reach plant material and the residual moisture content in the solid heats up Generated heat induces moisture evaporation and high pressure vapor builds up which cause the substrate cell wall to break and its content get released into solvent, and at the end the phytoconstituents yield becomes higher [59] Generally, some factors which can impact to the microwave-assisted technique, which are type and quantity of solvent to be used, energy output range of microwave instrument, length of extraction, composition of material under extraction, and heating intensity [17]

Figure 2.3 Scheme of MAE apparatus Source: Mashuni et al.[60]

Table 2.2 MAE characteristics

Source: Zhang et al.[55]

In a research of the optimization on MAE of CPH polyphenolics Microwave energy was the greatest to impact TPC and antioxidation ability of cocoa pod husk, and other parameters were ranked as follows: irradiation time, solvent and material

proportions ratio (w/v), then lastly the length of extraction [61] Research work done

by Kim et al.[16] on MAE of cocoa bean hull, the solvent effect was demonstrated as

the polyphenolic compounds to be double to triple by organic extraction with water dilution (4-6 v/v) than process using pure ones The dielectric constant of water (high value) had impacted on the extraction efficiency observed when water was added in solvents (Table 2.2)

Valadez-Carmona et al.[5] reported that polyphenols are medium polar

compounds For the necessity of extracting much as efficiently as possible, the optimization of various factors is necessary Comparison with other methods applied

in extraction of rutin and quercetin in plants, MAE emerged the most rapid with only 6 min extraction time, minimum solvent consumption and a maximized yield [17] In the study when apple pomace was extracted for TPC, MAE took short time (15 min), compared to 180 min by soxhlet, and 1,440 min by maceration [62]

2.3 FRACTIONATION, PURIFICATION, CHARACTERIZATION AND QUANTIFICATION OF PHENOLIC COMPOUNDS

Separation of bioactive compounds for their structure complete elucidation and description has been a problem as those phytochemicals are sometimes presented in complex form combined with other molecular materials This can reduce the bioactive content [63] Strategies relying on polar as well as acidic compounds were applied to concentrate and obtain fractions rich in polyphenol prior to analysis For the purpose of isolation, various methods have been implemented with the intention of reaching pure material from crude [63] From experience, column chromatography gives higher quantity of fractions and has been used to fractionate phenolic extracts [49] (Figure 2.4)

The choice of method of analysis of phenolic compounds is dictated by objective

of the research, sample material origin and specific bioactive compound in question Various methods have been applied but were not 100% effective [64] The Folin-Ciocalteu method looked different and was chosen to be the best of all methods [65]

Generally, conventional methods are not hard and quick to apply but they have limitations due to they are linked to the structural nature of phenolic compounds in

plants, which create various reactions behaviour and this make the use of a broad spectrum of methods, hence the results obtained from those methods are not similar and cannot be compared Additionally, as consequence, those methods have many interferences and result could be over or underestimation of the contents [49]

Instrumental analysis coupled with modern high-performance chromatographic techniques is promising move for phenolic compounds profiling and quantification HPLC is trending as an effective and reliable method for the study of phenolics [64]

Figure 2.4 Strategic steps for treating material from plant by targeting phenolics

Source: Cseke et al.[49]

2.4 EVALUATION OF ANTIOXIDANTION POTENTIAL OF PHENOLICS

Many assays used to measure antioxidants in plant sources, and they differ in their principle of application [48] Antioxidation evaluation methods were grouped into two categories First category includes those methods, which involve in transferring hydrogen atoms to active molecules, and the second category is made of

methods where antioxidant compounds free their electrons to active molecules Those methods of analysis are used to assess the intensity of antioxidation capacity of bioactive compounds and the reduction is observed by color changes There is proportionality between color level and antioxidant power [49]

In general, the natural structure of bioactive compounds in plants may be very complex The antioxidation evaluation can not rely on one single method It is recommended for better practice to combine methods The use of more than one methods together gives better results and is easy to be interpreted [23]

2.5 COCOA PHENOLIC COMPOUNDS AND BIOLOGICAL ACTIVITIES

It was reported that phenolics in their nature have a number of health beneficial physiological activities and that include among others, prevention and/or inhibition of allergy, inflammations, microbial growth, oxidation, thrombosis, atherogenesis, heart disease, and vasodilatation adverse effects [33] Colored phenolic pigment have been extracted in cocoa bean hull by supercritical fluid extraction method, and they were found to have important properties which are antioxidation and antiradical [2]

Various researches have published important physiological activities of phenolic compounds from cocoa and different plants [9]

Antioxidation property of phenolic compounds is beneficial and useful in many systems It involves a process where phenolics acting like antioxidants release hydrogen atoms and transfer to free radicals or reduce and scavenge free radicals [12] The potentiality of having oxidation inhibition ability makes phenolic compounds to

be good candidates in food systems [32] Polyphenols in nature have a various range

of biological functions By chemical interpretation, polyphenols are capable of reacting with one-electron oxidants, and this in turn prevents formation of free radicals

in biological system Phenolic compounds bioactivity is strengthened by the fact that they can interact with metal ions such as Fe2+, which is capable of generating free

radicals [66] Dias et al and Tiburcio [33,40] reported that the antioxidation capacity

of polyphenolics is related to their structural composition

In vitro, it was shown that for flavonoids and their metabolites, their functional

groups arrangement, determine their antioxidant activity [40,64] The study has

evaluated biological function of cocoa phenolic compounds in living organism, and it

was found that there was a proportion correlation between the rate at which the organism uptake the phenolic compounds and the resulted function [9]

Many studies have reported that the plant polyphenolics present an established relationship to their antoxidation capacity and microbial inhibition properties [67] Campos-Vega, Nieto-Figueroa and Oomah [47] found the effectiveness of CPH extracts as an antibacterial agent It was revealed that the extract was effective against two major bacteria strains with higher minimum inhibitory concentration for Gram-positive bacteria (0.0025 g/mL) than Gram-negative bacteria (0.001 g/mL)

Martínez et al.[24] indicated that antioxidant property of polyphenols can be

resulted from a single compound or complex interaction of many compounds, and this could affect the intensity of antioxidation capacity A crude husk extract fermented without starter culture was found to have an antimicrobial activity on two different strains, and the extract concentration was less than 9 mg/mL showing a positive action

at a 5.0 mg/mL MIC Furthermore, fermented crude husk extract sub-fractions had a

wide variation in antimicrobial activity with a MIC of 0.001 g/mL for S choleraesuis and 0.0025 g/mL for S epidermidis [7] Lu et al.[25] reported the use of CPH extract

in cosmetic creams as it has shown properties of inhibiting development of winkles

and skin dryness during the experiment carried out on human being skin

CHAPTER 3 MATERIALS AND METHODS

3.1 EXPERIMENTAL PROCEDURE

The overall experimental procedure is described in Figure 3.1 below:

Figure 3.1 Overall experimental procedure for extraction and assessment of

biological activity of phenolics from CPH

Fresh cacao pod husk (CPH)

Microwave drying:720W, 60 min

Grinding using hammer mill

MAE: 600 W, 30 min, 5 s/min

Centrifugation:4000 rpm, 15 min

Crude extract

Column chromatography fractionation

Analysis of bioactive compounds (TPC, TFC and SC), and evaluation of biological activities (Antioxidation and antimicrobial)

Dried CPH

Dried CPH powder

Identification of phenolic compounds by FTIR, TLC and HPLC

3.2 MATERIAL AND CHEMICALS

3.2.1 Fresh cocoa pod husk

Fresh CPHs were obtained from Thanh Trieu commune, Chau Thanh district, Ben Tre province, Vietnam in 12th October, 2019 and were covered by iced box to avoid oxidation (browning), deterioration and spoilage during transportation to Nha Trang University (NTU) After collection, the CPHs were quickly transported to the NTU laboratories for necessary treatments before extraction and any analysis Freshly corrected CPHs were chopped into small slices (4-5 mm in size) and packed into sealed polyamide bags, which were then kept in a freezer at -18oC until required

3.2.2 Analytical reagents

Utilized reagents in various analysis were graded of analytical quality include: (+)-catechin, (-)-epicatechin, (-)-epigallocatechin, (-)-epigallocatechin gallate, theobromine, theophyllin, caffeine and gallic acid were purchased from TCI (Tokyo, Japan) Potassium persulfate, trolox, methanol, ethanol, copper (II) chloride, iron (III) chloride, sodium acetate, TPTZ (2,4,6-tripyridyl-s-triazine), ammonium acetate, aluminium trichloride (anhydrous), neocuproine, sodium hydroxide, sodium carbonate (anhydrous), vanillin, sulfuric acid, hydrochloric acid, reagent of Folin-Ciocalteu, acetonitrile (HPLC grade), acetic acid and DPPH (2,2-Diphenyl-1-Picrylhydrazyl) were obtained from Merck (Darmstadt, Germany)

3.3 EXPERIMENTAL METHODS

3.3.1 Preparation of powder from crude CPH extract

The CPH slices kept in freezer at -18oC were removed from their packages, defrosted completely at room temperature, then put on rotating glass plate inside the microwave, and were heat-dried between 40-60 min at 600-720 W to constant weight (to avoid high temperature inside the oven, it was opened 5s for each min as drying was going on) Dried cocoa pod husk was ground into very tiny particles (powder)

The extracts of CPH were obtained by applying procedures published in Nguyen et

al [68] For extraction process, 20 g CPH powder was soaked into 1000 mL of

distilled water, kept at room temperature (RT), then the mixture was put inside the microwave oven (Panasonic NN-ST651M, Shanghai) set at 600 W for half an hour period and irradiated at the rate of 5 s/min The mixture was dipped into a water

(cold) bath to cool it down to RT and then it was centrifuged by use of the centrifuge (HERMLE Z323) set at 4000 rpm for 15 min and supernatant separated from solid material A portion of centrifuged CPH extract was condensed using rotary vacuum evaporator (LABOTA 4001 OB Heidolph, Germany) at 65-80C, and a final concentration of around 40-45% was achieved The condensed extract was dried into powder in a freeze drier (Telstar, RD-18, Spain) at -40C and 0.014 mbar for a period

of 52 h Finally, the crude phenolic-enriched CPH powder obtained was kept in desiccator to avoid moisture uptake until it was used for further analysis

3.3.2 Fractionation of crude extract from CPH by column chromatography

A sample of reconstituted crude CPH extract was filtered twice through 0.45 and 0.22 m nylon syringe filters, respectively 500-1000 L of crude CPH extracts were injected using a pippette into the column repeatedly one after another and different fractions were collected for each injection The technique consisted of a glass column graduated for different compound fractions, then filled with stationary phase (Silica gel: Silica 60A) and the sample (crude CPH extract) was added to the top of the eluting column to be separated A mobile phase (30% MeOH) was added above injected sample and solvent was allowed to flow continuously through the column Compounds including phenolics were separated on the basis of their differential migration through the column and the affinity to stationary phase (polar and apolar) Eluted compounds were collected into different tubes (8 fractions), then condensed under vacuum rotary evaporator (LABOTA 4001 OB Heidolph, Germany) at 60-65oC, and analyzed for phenolic content by HPLC In order to elute almost pure compounds, re-fractionation was carried out whereby collected and condensed fractions from the CPH extract were re-injected (500 L/injection), and 6 fractions were collected from each individual of the 8 fractions The fractions of the same number of elution location were then mixed (e.g: 1.1; 1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8) to form one fraction which was condensed and analyzed for phenolic content by HPLC

3.3.3 Preparation of solutions from crude CPH extract and derived fractions for evaluation of phenolic content, antioxidation potential and antimicrobial activity

3.3.3.1 Preparation of the crude extract solution

In order to achieve the concentration of 200 ppm and 500 ppm, 0.1 g and 0.025

g of freeze-dried CPH powder were dissolved into 50 ml distilled water The solutions were completely dissolved by use of vortex mixer (HWASHIN Technology co, 250

VM, Korea), then they were kept in the freezer at -18oC for the analysis of bioactive compounds and antioxidant capacity For antimicrobial activity, various CPH extract solutions were prepared At first, 0.01 g, 0.05 g and 0.15 g of freeze-dried CPH powder were dissolved into 5 mL distilled water to form 103, 104, and 3*104 g/mL solutions, respectively The solutions were thoroughly mixed using voltex mixer, kept

in a water bath at 37oC for 15 min to completely dissolve the solids into the solutions, then they were filtered by 0.45 and 0.22 m filters and kept in the freezer at -18oC till the use for further analysis

3.3.3.2 Preparation of fractions solutions

2 and 4 ml of condensed fractions were put in the crucibles, which were then completely evaporated in a dessicator for a period of 68 and 89 h, respectively Evaporated fractions were dissolved into methanol, which its volume was depended on evaporated fraction weight in order to give 500 and 2000 ppm concentrations, respectively The mixtures were dissolved using vortex mixer, and kept in freezer at -

18oC till the next use in analysis

3.3.4 Phenolic compounds identification in CPH extract

Some techniques were used which include FTIR, TLC and HPLC as follows:

3.3.4.1 Thin layer chromatography (TLC)

Around 2 μL CPH extract (200 ppm) and standard compounds solutions (100 ppm) were deposited on a silica gel plate using glass capillary spotter after filtration though 0.45 μm nylon syringe filter The samples were applied at 15 mm from the base

of plates A volume of 30 ml of different acetonitrile and methanol concentrations (30,

50, 80 and 100% v/v) was put in the TLC separation glass chamber, silica gel plates having extract were then immersed in the chamber After development of the chromatogram, the plates were dried under the fan at room temperature and the

detection was mainly carried out using a UV lamp at 254 nm, and separated visible bands corresponding to phenolic compounds were observed The best solvent for TLC separation of phenolic compounds from the crude extract was selected for further use

in column chromatography

3.3.4.2 Fourier-transform infrared spectroscopy (FTIR)

The spectral features of CPH extract and fractions derived from CPH extract

were obtained by FTIR analysis following the method reported by Batista et al.[69]

using a ALPHA FTIR spectrometer (Bruker Optics Inc, Billerica, MA, USA) 2 mL CPH extract was frozen for 24 h at −20°C Then, the frozen sample was subjected to lyophilizing step for 30 h A solid sample was ground with IR transparent potassium bromide (KBr) and pressed into a pellet FTIR spectra of CPH extract was recorded on

a Digilab Excalibur coupled to an attenuated total reflectance (ATR) accessory equipped with a ZnSe reflection crystal The spectra was acquired at room temperature with 32 scans/sample in the range of 4400 to 600 cm−1 at a resolution of 4 cm−1 using Opus 7.5 software

3.3.4.3 High performance liquid chromatography (HPLC) analysis of CPH phenolics

Identification of phenolic compounds of fractions derived from the CPH extract was done by help of HPLC system (Chromaster, HITACHI, Japan) as per procedure

described by Maity et al.[70] and published in Nguyen et al.[68] Solutions of

authentic standards were produced using distilled water as solvent to the level of 400

g/mL, and from prepared solutions, 300 L each solution was collected and put together in order to form a solution of mixed authentic standards Impurities were removed from authentic standards and CPH derived fraction solutions by use of 0.45 and 0.22 μm nylon syringe filters, then 20 μL each filtered solution was separately inserted by an automatic injection in the column, which was kept at 35°C Mobile phases included 0.2% phosphoric acid (A) and 100% acetonitrile (B) served as the eluents moving in the column at a flow rate of 1 mL/min The change in the flow eluent concentration was like the: 0 to 5 min - 0% B; 5 to 15 min - 8% B; 15 to 30 min

- 15% B; 30 to 50 min - 20% B; 50 to 55 min - 15% B; 55 to 60 min - 8% B; and 60 to

65 min - 0% B Detection of phenolics was done at 272 nm using a UV–VIS detector Phenolics identification was done by checking similarity of retention times (Rt) in the

CPH extract and fractions to the ones of authentic standards (gallic acid, theobromine, theophylline and caffeine, (+)-catechin, (-)-epicatechin, (-)-epigallocatechin and (-)-epigallocatechingallate)

3.3.5 Analysis of physicochemical properties of powdered extract from CPH

The residual moisture was analyzed according to AOAC official method (AOAC1998) as reported by Nguyen, Ueng and Tsai [71] with some modifications Briefly, 0.2 g of freeze-dried CPH extract was put into drying crucibles which were earlier dried in a hot-air oven (Memmert, Schwabach, Germany), then crucibles were placed dried in oven at 105oC for a 5 h time period until a constant weight (weight of the sample did not change) To lower temperature to room ambient very quickly, crucibles removed from oven were put in a desiccator, the sample was then reweighed after cooling Content of moisture was estimated as per formula below:

3.3.5.3 pH determination

pH measurement was done as per procedure reported by Taylor et al.[7] To

make a solution, 0.5 g dried CPH powder was reconstituted by dissolving in 10 ml distilled water, then mixed thoroughly using a vortex mixer (HWASHIN Technology

co, 250 VM, Korea) The obtained solution was poured in beaker and pH was then measured at room temperature at 25oC using a pH meter (HORIBA Advance Techno Co., Ltd, LAQUA-PH1100, Japan) which was calibrated by standards pH 4 and 7 buffer solutions

3.3.5.4 Water solubility index (WSI)

WSI of CPH freeze-dried extract was determined as per procedure of Anderson

et al.[73] and reported by Taylor et al.[72] 0.5 g dried CPH powder was put in plastic

test tube, and distilled water was filled up to 10 mL mark The mixture was vigorously mixed using vortex mixer, incubated at 37oC for 30 min in a water bath, after that centrifuged for 20 min in centrifuge (HERMLE Z323, Labnet International, USA) The supernatant was carefully collected in a dried crucible, which were put in a hot-air oven at 103±2oC for a 7 h period The estimation of WSI (%) was from the percentage ratio related to the weight of dried supernatant and that of freeze-dried CPH powder

3.3.5.5 Bulk density

Bulk density (g/mL) of CPH powder was measured as per procedure reported

by Taylor et al [72] Briefly, 0.3 g CPH powder was put inside a 10 mL glass cylinder

by avoiding touching sides, and it was shaken vigorously using a vortex mixer for 60

s Weight of CPH sample in cylinder over the volume occupied by particles during shaking was estimate of bulk density

3.3.6 Analysis of bioactive compounds

Crude phenolic-enriched powder and fractions from crude CPH extract obtained by column chromatography were analyzed for bioactive compounds content

3.3.6.1 Total phenolic content (TPC)

To analyze TPC in the extract of CPH and its derivatives, the procedure of

Vuong et al.[74] and published by Nguyen et al.[68] was referred with some

adjustments 0.5 mL CPH extract or fractions and 2.5 mL prepared Folin-Ciocalteu solution (10% v/v) were throughly mixed in test tubes and the mixture was left for 6 min at room temperature After the waiting time, 2 mL Na2CO3 solution (7.5% by mass) was added in test tubes covering by aluminium foil to avoid light The mixture was left for 1 h and the absorbance was recorded at 765 nm by using a UV-VIS spectrophotometer (Biochrom Libra S50, Cambridge, England) For the control and standard purposes, water and gallic acid were served, respectively Results of TPC were shown in mg GAE/g

3.3.6.2 Total flavonoid content (TFC)

Evaluation of TFC in the extract from CPH and its derived fractions was performed based on the method reported by Vuong et al.[74] and Nguyen et al.[68] In

test tubes, 0.5 mL CPH extract or fractions, 0.15 mL Na2CO3 (5% w/v) and 2 mL distilled water were vigorously mixed The blend was tightly covered to avoid light and incubated for 6 min in the dark at room temperature 0.15 mL AlCl3 solution (10% w/v) was added in the mixture and kept for more 6 min After that, both 2 mL NaOH solution (4% by mass) and 0.7 mL distilled water were added in the mixture and the blend was kept for 15 min The absorbance was read at 510 nm by using a UV-VIS spectrophotometer Water and catechin were used as the control and standard, respectively Results of TFC were presented as mg CE/g

3.3.6.3 Saponin content (SC)

The SC of the CPH extract and its derived fractions was done following

procedures of Vuong et al.[74] and reported by Nguyen et al.[6], with some

adjustments 0.5 mL CPH extract or fractions and 0.5 mL vanillin (8% by mass) in methanol were mixed together 5 mL H2SO4 (72% v/v) was then poured in the mixture and left at 70oC for 15 min After that, it was quickly dipped in a cold water bath to reach room temperature The absorbance was checked at 560 nm by a UV-VIS spectrophotometer For the control and standard purposes, water and escin were served, respectively Results of SC were reported as mg EE/g

3.3.7 Evaluation of biological activities

The crude CPH extract and fractions were assessed for their bioactive functions including antioxidation for both, and antimicrobial capacity for the crude CPH extract

3.3.7.1 DPPH radical scavenging capacity (DRSC)

Antioxidant analysis of CPH extract and fractions based on DRSC was

performed follow procedures published by Vuong et al [74] and Nguyen et al [68] In

short, DPPH stock solution (0.024% by mass) in methanol was prepared and maintained in freezer at-18oC 1 mL DPPH stock solution was mixed with 4.5 mL methanol to make a working solution, which was then adjusted the absorbance to 1.08-1.12 at 515 nm To induce hydrogen transfer to DPPH, 0.15 mL CPH extract or fractions was vigorously mixed with 2.85 mL DPPH working solution, the blend was

then covered to avoid light and left for 3 h at room temperature in the dark The absorbance was read at 515 nm using a UV-VIS Water and trolox were used as the control and standard, respectively Results of DRSC were shown in mg TE/g

3.3.7.2 Cupric reducing antioxidant capacity (CUPRAC)

Antioxidant assay based on CUPRAC of CPH extract and fractions were relied

on procedure reported by Vuong et al [74] and Nguyen et al [68] At first, 1 mL

CuCl2 (10 mM), 1 mL neocuproine (7.5 mM) and 1 mL AcONH4 solution (7.7% w/v) were blended together, then 1.1 mL CPH extract or fractions was added in the mixture The blend was covered to avoid light and incubated at ambient temperature for 1.5 h before checking the absorbance at 450 nm using a UV-VIS spectrophotometer For the control and standard purposes, water and trolox were used, respectively Results of CUPRAC were indicated in mg TE/g

3.3.7.3 Ferric reducing antioxidant power (FRAP)

The antioxidant determination of CPH extract and fractions by FRAP assay was

done in compliance to Kamonwannasit et al [75] method and published by Nguyen et

al [68] with some adjustments Briefly, A, B and C reagents as the order of mixing at

a ratio of 10 : 1 : 1, (A) being a solution of acetate buffer (300 mM, pH 3.6); (B) is 10

mM TPTZ solution prepared by mixing with in 40 mM HCl and (C) is a solution of 20

mM FeCl3.6H2O To induce ferric reduction, 0.15 mL pipetted from CPH extract or fractions together with 2.85 mL picked from the blend of A, B, and C were mixed, covered to avoid light and left for 60 min at ambient temperature The absorbance of mixtrure was then detected at 593 nm using a UV-VIS spectrophotometer For the control and standard purposes, water and trolox were served, respectively Results of FRAP were described as mg TE/g

3.3.7.4 Antimicrobial assay

a Microbial culture

As per Bhuyan et al [76] with some modifications, two bacterial strains: a negative bacteria Escherichia coli (ATCC 8739), a Gram-positive bacteria Bacillus

Gram-subtilis (ATCC 6633), and a yeast strain Candida albicans (ATCC10231) were used

in this study The microbial cultures were obtained from Thermo Scientific™ Oxoid™ in the form of Culti-Loops® The stock bacterial and fungal cultures were