SELECTED PHYTOCHEMICAL, NUTRITIONAL and ANTIOXIDANT PROPERTIES of FRESH, OVEN and FREEZE-DRIED Streblus Asper LEAVES

SELECTED PHYTOCHEMICAL, NUTRITIONAL AND ANTIOXIDANT PROPERTIES OF FRESH, OVEN AND FREEZE-DRIED Streblus asper LEAVES

NOR MAWARTI BINTI IBRAHIM

Thesis submitted in fulfillment of the requirements for the degree of Master of Science

FEBRUARY 2014

ACKNOWLEDGEMENTS

Bismillahirrahmanirrahim. In the name of Allah, The Most Gracious, Most Merciful.

Shalawat and remembrance for the Holy Prophet, Muhammad S.A.W.

Alhamdulillah, with the Allah help and His amazing grace, I’m finally completing this journey successfully after going through the two years of challenging. Nevertheless, this period is so meaningful because it gives me an opportunity to step foot into the realm of the true research work. This research has been developed and implemented with assistance of several generous individuals. I would like to take this opportunity to express my profound gratitude to a number of individuals who have been inspirational to my study.

First and foremost, my deepest appreciation goes to my supervisor and academic mother; Dr Ruzita Ahmad for her continuous support of this research project. She shows me different ways to approach a research problem and the need to be persistent to accomplish any goal. Dr Ruzita, May Allah make all your ways straight, successful and bless you abundantly.

I am also indebted to my co-supervisors; Prof Ishak Mat and Dr Lim

Vuanghao for guidance, advice, valuable discussions, encouragement, assistance and support throughout the period of my study.

I am appreciative of the valuable assistance to all the staff of the Integrative

Medicine Lab, Advanced Medical & Dental Institute (AMDI). I would like to thank my friends, especially to Dr Hazwani Ahmad and Rabiatul Basria for her discussions.

Finally, I would like to express my indebtedness and offer my special thanks to my mother; Sharipah Zainab Syed Ali and families for their endless supports, encouragement and understanding. To my lovely hubby, Muhammad Nazrin Md

Yunos who always welcomed me with a smile no matter my troubles, I say may

Allah grant all your heart desires and continue to bless more people through you.

Thank you, thank you, and thank you.

TABLE OF CONTENTS

Acknowledgements II

Table of Contents IV

List of Tables X

List of Figures XI

List of Abbreviations XV

List of Publications XVII

Abstrak XVIII

Abstract XX

CHAPTER 1 INTRODUCTION

1.1 Background 2

1.2 Problem Statements 4

1.3 Significance of Research 4

1.4 Research Objectives 5

CHAPTER 2 LITERATURE REVIEWS

2.1 Morphological descriptions of S. asper 6

2.2 Traditional Uses and Medicinal Properties of S. asper 8

2.3 Antioxidant 9

2.3.1 1,1-diphenyl-1- picrylhydrazyl (DPPH) Radical Scavenging 11

2.4 Secondary Metabolites 12

2.5 Phytochemicals 13

2.5.1 Classification of Phytochemicals 14

2.5.2 Potential Sources of Phytochemicals 14 5

2.6 Phenolic 15

2.6.1 Total Phenolic Content 17

2.6.2 Total Flavonoid Content 18

2.7 Chemical and Physical Analysis 19

2.7.1 Chemical Analysis 19

2.7.1.1 Proximate 19

2.7.1.2 Minerals 20

2.7.1.3 Chlorophyll 22

2.7.2 Phytochemical Screening 25

2.7.2.1 Alkaloid, Tannin, Saponin,Terpenoids, Steroids and 26 Cardiac Glycosides

2.7.2.2 Screening of Volatiles by Gas Chromatography 31 Mass Spectrometry (GCMS)

2.7.3 Physical Analysis 33

2.7.3.1 Colour Attributes 33

2.8 Drying techniques; oven drying and freeze drying 36

2.8.1 Effect of Drying on the Proximate Composition, Minerals, 38 Chlorophyll and Phenolics

2.9 Cytotoxicity Study 39

2.9.1 HT29 Cell Lines 41

2.9.2 MTS Assay Principles 42

CHAPTER 3 MATERIALS AND METHOD

3.1 Study Outline 44

3.2 Sample Preparation 46

3.3 Sample Extraction 47

3.4 Visual and Odour Observation of the Extracts 47

3.5 Chemicals Analysis 48

3.5.1 Proximate 48

3.5.1.1 Moisture 48

3.5.1.2 Crude Fat 49

3.5.1.3 Crude Fibre 50

3.5.1.4 Crude Protein 51

3.5.1.5 Ash 53

3.5.1.6 Nitrogen Free Extract 54

3.5.2 Caloric Value 54

3.5.3 Minerals 55

3.5.4 Chlorophyll 56

3.5.5 Preliminary Phytochemical Screening 57

3.5.5.1 Qualitative Screening of Alkaloid, Steroids, Terpenoids 57 Tannins, Saponin and Cardiac Glycoside

3.5.5.1.1 Alkaloids 57

3.5.5.1.2 Steroids 58

3.5.5.1.3 Terpenoids 58

3.5.5.1.4 Tannins 58

3.5.5.1.5 Saponin 58

3.5.5.1.6 Cardiac Glycosides 59

3.5.5.2 Screening of Volatile Compounds by GCMS 59

3.6 Physical Analysis 60

3.6.1 Colour Measurement 60

3.7 Extract Yield Determination 61

3.8 Antioxidant Activity 61

3.9 Analysis of Phenolics 62

3.9.1 Total Phenolic Content 62

3.9.1.1 Folin-Ciocalteu Method 62

3.9.1.2 HPLC 64

3.9.2 Total Flavonoids Content 65

3.9.2.1 Colorimetric Method 65

3.9.2.2 HPLC 66

3.10 Cytotoxicity Analysis 67

3.10.1 Materials and Reagents 67

3.10.2 Sample Extractions 67

3.10.3 Preparation of Medium 68

3.10.4 Subculture of HT 29 68

3.10.5 Enumeration of Cells 69

3.10.6 Sample Preparation 70

3.10.7 MTS Assay 70

3.11 Statistical Analysis 71

CHAPTER 4 RESULTS AND DISCUSSION

4.1 Visual and Odour Observation of Extract 72

4.2 Chemicals Analysis 74

4.2.1 Proximate 74

4.2.2 Determination of Mineral Content 77

4.2.3 Chlorophyll 82

4.2.4 Phytochemical Analysis 84

4.2.4.1 Preliminary Phytochemical Screening of S. asper leaves 84

4.2.4.2 Screening of Volatile Compound using GCMS 90

4.2.4.2.1 Effect of drying methods on the Screening of 98 Volatile Compound using GCMS

4.3 Physical Analysis 103

4.3.1 Colour Measurement 103

4.4 Percentage Yield of S. asper Leaves Extract 106

4.5 Antioxidant Activity 108

4.6 Analysis of Phenolics 117

4.6.1 Total Phenolic Content 117

4.6.2 Total Flanovoid Content 123

4.6.3 Gallic acid and Quercetin by HPLC 127

4.7 Comparison of the efficiency of different solvents on the 141 antioxidant activity from S. asper leaves extracts

4.8 Cytotoxicity activity of S. asper leaves extract against HT29 cell lines 145 in vitro

CHAPTER 5 CONCLUSIONS AND RECOMMENDATIONS

5.1 General Conclusions 150

5.2 Recommendations for future works 152

REFERENCES 153

APPENDICES

Appendix A: Voucher specimens of S. asper

Appendix B: Calibration Curves for Minerals Analysis

Appendix C: CIE L*, a*, b* Colour Space

Appendix D: Calibration curve for linearity standard of Gallic acid and Quercetin using HPLC 9

Appendix E: Percentages of cell viability at 24, 48 and 72 h of incubation

LIST OF TABLES

Page

Table 2.1 Scientific classification of S. asper plant 7

Table 2.2 Phytochemical constituents of different plant parts of S. asper 21

Table 2.3 Cytotoxicity studies of different plant parts of S. asper 34

Table 3.1 Oven condition of GCMS 54

Table 3.2 The experimental condition of HPLC 58

Table 4.1 The extraction yield obtained by the different solvents 87 from fresh, oven-dried and freeze-dried leaves

Table 4.2 Phytochemicals screening of S.asper leaves 89

Table 4.3 Volatile screening of the chemical composition (%) in 30% 91 EtOH extracts from fresh, oven dried and freeze dried samples

Table 4.4 Volatile screening of the chemical composition (%) in 50% 93 EtOH extracts from fresh, oven dried and freeze dried samples

Table 4.5 Volatile screening of the chemical composition (%) in 70% 97 EtOH extracts from fresh, oven dried and freeze dried samples

Table 4.6 Volatile screening of the chemical composition (%) in aqueous 100 extracts from fresh, oven drying and freeze drying

Table 4.7 Pearson correlation coefficients between TPC, TFC and DPPH 114 assay in the S. asper leaf extract

Table 4.8 Total phenolic constituents of S. asper leaves aqueous and 118 ethanol extracts from fresh leaf (FL), oven-dried (OD) and freezedried (FD) samples

Table 4.9 Total flavonoid content of S. asper leaves aqueous and ethanol 123 extracts from fresh leaf (FL), oven-dried (OD) and freeze-dried (FD) samples

Table 4.10 Concentration of gallic acid from different extracts and drying 127 treatments of S. asper leaves

Table 4.11 Concentration of quercetin from different extracts and drying 128 treatments of S. asper leaves

LIST OF FIGURES

Page

Figure 2.1 Streblus asper leaf 7

Figure 2.2 Common simple phenol and flavonoids in plants 16

Figure 2.3 The basic unit of flavonoids 19

Figure 2.4 Chlorophyll a and chlorophyll b 23

Figure 2.5 The structure of alkaloids 26

Figure 2.6 The structure of tannins 27

Figure 2.7 The structure of saponins 28

Figure 2.8 The structure of terpenoids 29

Figure 2.9 The structure of steroids 30

Figure 2.10 The structure of cardiac glycosides 31

Figure 2.11 a*, b* Chromaticity Diagram 34

Figure 2.12 Flow chart on standardization and evaluation of plant herbal 35

Figure 2.13 The structures of HT 29 cell lines 42 Figure 3.1 Flow chart of the study 45 Figure 4.1 Comparative percentage proximate composition of S. asper leaves by different drying methods 76

Figure 4.2 Macronutrients composition of S. asper leaves by different drying methods 79

Figure 4.3 Effects of different drying methods on Ca, Cb and Tc of S. asper leaves 84

Figure 4.4(a) GCMS chromatogram of 30% EtOH extract of S. asper 89 fresh leaves

Figure 4.4(b) GCMS chromatogram of 30% EtOH extract of S. asper 89 leaves from oven dried S. asper leaves

Figure 4.4(c) GCMS chromatogram of 30% EtOH extract of S. asper 90 leaves from freeze-dried S. asper leaves

Figure 4.5(a) GCMS chromatogram of 50% EtOH extract of S. asper 92 fresh leaves

Figure 4.5(b) GCMS chromatogram of 50% EtOH extract of S. asper 92 leaves from oven dried leaves

Figure 4.5(c) GCMS chromatogram of 50% EtOH extract of S. asper 93 leaves from freeze-dried S. asper leaves

Figure 4.6(a) GCMS chromatogram of 70% EtOH extract of S. asper 95 fresh leaves

Figure 4.6(b) GCMS chromatogram of 70% EtOH extract of S. asper 95 leaves from oven dried leaves

Figure 4.6(c) GCMS chromatogram of 70% EtOH extract of S. asper 96 leaves from freeze-dried S. asper leaves

Figure 4.7(a) GCMS chromatogram of S. asper aqueous fresh leaves 98 extract

Figure 4.7(b) GCMS chromatogram of S. asper aqueous leaves extract 98 from oven dried leaves

Figure 4.7(c) GCMS chromatogram of S. asper aqueous leaves extract 99 from freeze-dried S. asper leaves

Figure 4.8 Effects of different drying methods on L*, a*, b*,C* and h 105 Values of S. asper leaves

Figure 4.9(a) DPPH free radical scavenging activity (%) of the aqueous and 110 ethanol S. asper fresh leaf extract and BHA

Figure 4.9(b) DPPH free radical scavenging activity (%) of the aqueous and 110 ethanol S. asper oven-dried leaf extract and BHA

Figure 4.9(c) DPPH free radical scavenging activity (%) of the aqueous and 112 ethanol S. asper freeze-dried leaf extract and BHA.

Figure 4.10 Gallic acid calibration curve for determination of total phenolic 119 using Folin–Ciocalteu colorimetric assay

Figure 4.11 Quercetin calibration curve for determination of total flavonoid 124 using colorimetric assay

Figure 4.12(a) HPLC chromatogram of 30% EtOH extract of S. asper 134 fresh leaves

Figure 4.12(b) HPLC chromatogram of 30% EtOH extract of S. asper 134 leaves from oven-dried leaves

Figure 4.12(c) HPLC chromatogram of 30% EtOH extract of S. asper 135 leaves from freeze dry

Figure 4.13(a) HPLC chromatogram of 50% EtOH extract of S. asper 135 fresh leaves

Figure 4.13(b) HPLC chromatogram of 50% EtOH extract of S. asper 136 leaves from oven-dried leaves

Figure 4.13(c) HPLC chromatogram of 50% EtOH extract of S. asper 136 leaves from freeze-dried leaves

Figure 4.14(a) HPLC chromatogram of 70% EtOH extract of S. asper 137 fresh leaves

Figure 4.14(b) HPLC chromatogram of 70% EtOH extract of S. asper 137 leaves from oven-dried leaves

Figure 4.14(c) HPLC chromatogram of 70% EtOH extract of S. asper 138 leaves from freeze-dried leaves

Figure 4.15(a) HPLC chromatogram of aqueous extract of S. asper 138 fresh leaves

Figure 4.15(b) HPLC chromatogram of aqueous extract of S. asper 139 leaves from oven-dried leaves

Figure 4.15(c) HPLC chromatogram of aqueous extract of S. asper 139 leaves from freeze-dried leaves

Figure 4.16(a) Effects of different drying methods on total phenolic contents 141 of S. asper leaves.

Figure 4.16(b) Effects of different drying methods on total flavonoid contents 142 of S. asper leaves

Figure 4.17(a) Effects of different solvents for extraction of total phenolic 145 content of S. asper leaves.

Figure 4.17(b) Effects of different solvents for extraction of flavonoid 146 contents of S. asper leaves

Figure 4.18(a) Cytotoxic effect of aqueous extract of S. asper leaves 145 14

at 24 h, 48 h and 72 h incubation

Figure 4.18(b) Cytotoxic effect of 70% EtOH extract of S. asper 148 at 24h, 48h and 72h incubation

LIST OF ABBREVIATIONS

% percent ˚C Celsius µl microlitre 30% EtOH 30% of ethanol 50% EtOH 50% of ethanol 70% EtOH 70% of ethanol AAS Atomic Absorption Spectrometry AOAC Association of Official Analytical Chemists BHA Butylated hydroxyanisole BHT Butylated hydroxytolune Ca Calcium cm2 centimeter square

CO2 carbon dioxide dH20 distilled water DPPH 1, 1-diphenyl-2-picrylhydrazyl

FeCl3 Iron (III) chloride g gram GCMS Gas Chromatography Mass Spectrometry GAE Gallic acid equivalent h hour

H2O2 Hydrogen peroxide

H2SO4 Sulfuric acid HBV Hepatitis B Virus HCl Hydrochloric acid Hg Mercury

HNO3 Nitric acid HPLC High Performance Liquid Chromatography HT 29 Colon carcinoma cell lines

IC50 Inhibition concentration K Potassium L liter 16

LC Liquid Chromatography Mg Magnesium mg milligram min minute ml milliliter MTS CellTiter 96® Aqueous One Solution Cell Proliferation Assay N Nitrogen Na Sodium

Na2CO3 Sodium carbonate NaOH Sodium hydroxide nm nanometer

O2 Oxygen P Phosphorus PBS Phosphate Buffered Saline QE Quercetin equivalent rpm revolutions per minute S. asper Streblus asper UV Ultra Violet v/v volume per volume VOCs Volatile Organic Compounds w/v weight per volume

LIST OF PUBLICATIONS

Journal of Antioxidant (International Open Access Journal)

Antioxidant Activity and Phenolic Content of Streblus asper Leaves Ethanol and Aqueous Extracts from Various Drying Methods

Oral Presentation

1. Antioxidant Activity and Phytochemical Screening of the Streblus asper (kesinai) leaf ethanol extracts

Poster Presentation

1. Screening of volatiles in Streblus asper leaves by Gas Chromatography-Mass Spectrometry

2. Viability of Lactobacillus and Chemical Composition of Cultured Jackfruit Dadih

3. Phytochemicals screening and antioxidant activity of Streblus asper (kesinai) leaf in different solvent extracts

4. Antioxidant Activity and Phytochemical Screening of the Streblus asper (kesinai) leaf aqueous extracts

5. Cytotoxicity Effect of Cigarette Extract On Fibroblast 3T3 Cell Line and Effect of Pereskia bleo on HEp-2 Human Laryngeal Carcinoma Cells

6. Effect of different drying methods on the Proximate Composition, Chlorophyll Contents and Colour of Streblus asper (casino) leaves

MEMILIH FITOKIMIA, NUTRISI DAN CIRI-CIRI ANTIOKSIDA DAUN

SEGAR, PENGERINGAN KETUHAR DAN SEJUKBEKU Streblus asper

ABSTRAK

Streblus asper dari famili Moraceae dikenali dengan nama tempatan sebagai kesinai di Malaysia. Herba berubat ini didapati di wilayah utara semenanjung Malaysia seperti Kedah dan Perlis. Kebanyakan kajian berkaitan S. asper menggunakan bahagian akar dan kulit pokok. Namun, terdapat dokumentasi tentang kegunaan tradisional daun kesinai sebagai rawatan pembengkakan saluran kencing, penyakit keputihan, keradangan kencing, cirit-birit, melancar pengeluaran susu ibu, dan sebagai agen diuretik. Dalam kerja ini, ciri-ciri fitokimia daun S. asper dari pelbagai kaedah pengeringan telah dikaji untuk memperolehi prosedur yang paling sesuai untuk memelihara dan mengekalkan kualiti tinggi tumbuhan perubatan ini tanpa mengurangkan nilai perubatan dan nutrisinya. Daun S. asper juga disaring untuk sebatian organik meruap menggunakan GCMS dan penaksiran jumlah asid galik dan kuersetin dilakukan dengan HPLC. Tambahan, kesan pengeringan dan pelarut terhadap sampel terhadap aktiviti antioksidan dalam ekstrak tumbuhan S. asper juga ditentukan. Kajian ini menyediakan data asas tentang nilai fizikokimia, nutrisi dan ciri-ciri antioksidan daun S. asper daripada pelbagai kaedah pengeringan dan pelarut yang berbeza. Secara umumnya rawatan pengeringan mempengaruhi secara signifikan (p < 0.05) kebanyakkan komposisi kimia. Keputusan mencadangkan pengeringan sejukbeku adalah lebih efisien dan disyorkan untuk digunakan dalam penyediaan tumbuhan ubatan ini. Dari penyaringan fitokimia, sebatian utama yang telah dikenalpasti dalam S. asper adalah fitol, asid lemak, flavonoid, fenolik,

alkaloid, saponin, tanin, terpenoid, glikosida kardiak, klorofil dan juga jumlah ketara mineral, asid galik dan kuersetin. Beberapa unsur lain seperti protin, serat, karbohidrat, asid palmitik, asid linoleik, 12,15-oktadekatrien-1-ol, asid n- heksdekanoik, β-tokoferolerol, vitamin E total telah dikesan dari daun S. asper.

Kajian ini menunjukkan daun S. asper mempunyai aktiviti antioksidan yang baik.

Satu pertalian kuat antara fenolik total dan aktiviti penyingkiran radikal telah diperhatikan. Bagaimanapun, ekstrak mentah air dan etanol dari daun S. asper tidak menunjukkan kesan kesitotoksikan terhadap sel HT29 secara in vitro. Secara amnya, ekstrak pelarut EtOH 70% menunjukkan prestasi lebih baik dari segi aktiviti penyingkiran radikal, kandungan fenolik dan juga kandungan flavonoid berbanding dengan ekstrak air.

SELECTED PHYTOCHEMICAL, NUTRITIONAL AND ANTIOXIDANT

PROPERTIES OF FRESH, OVEN AND FREEZE-DRIED Streblus asper

LEAVES

ABSTRACT

Streblus asper from the family Moraceae is known locally as kesinai in

Malaysia. This medicinal herb is found in the northern region of peninsular Malaysia especially in Kedah and Perlis. Most studies on S. asper concentrate on the root part and stem bark. However, there is documentation on the traditional use of kesinai leaf for the treatment of urinary tract swelling, candidiasis, dysuria, diarrhoea, increase breast milk supply and as diuretic agent. In this research, phytochemical properties of

S. asper leaves from various drying treatments were studied to get a suitable procedure to preserve and maintain a high quality medicinal plant without reducing its medicinal and nutritive value. The S. asper leaves were also screened for VOCs using GCMS and quantification of gallic acid and quercetin were done using HPLC.

Additionally, the effects of drying and solvent used for extraction on the level of antioxidant in S. asper plant extracts were determined. This study provided preliminary data on the physicochemical, and nutritional values as well as the antioxidant properties of S. asper leaves from various drying treatments and solvent extracts. Generally, the drying treatment had significant (p < 0.05) effect on most of the chemical constituents. The result suggested that freeze drying was more efficient and is recommended to be used in preparation of this medicinal plant. The compounds identified in S. asper is mainly phytol, fatty acids, flavonoids, phenolics, alkaloids, saponin, tannins, terpenoids, cardiac glycosides, chlorophyll; S. asper also

contains appreciable amounts of minerals, gallic acid and quercetin. Some of the other phytoconstituents detected in S. asper leaves were proteins, fibres, carbohydrates, palmitic acid, linoleic acid, 12,15-octadecatrien-1-ol, n-hexdecanoic acid, β-tocopherol and vitamin E. The study showed that S. asper leaves possessed antioxidant activity. A strong correlation between total phenolics and radical scavenging activity was observed. However, crude aqueous and ethanol extracts of S. asper leaves did not show any cytotoxicity activity against HT29 cell lines in vitro.

Generally, 70% EtOH solvent extracts gave better performance and possessed greater radical scavenging activity had higher amounts of phenolic and flavonoid contents compared to aqueous extracts.

CHAPTER 1

INTRODUCTION

1.1 Background

Medicinal plants continue to attract increasing attention because of their potential benefits especially in the field of medicine and pharmacology. Medicinal plants have been recognized for their therapeutic benefits for centuries. Polyphenolic compounds are proven to be potent antioxidants and contain important biological, pharmacological and medicinal properties. The biological activities are antioxidant, anti-proliferative, antibacterial, antifungal, antiviral, anti-diabetic, antihypertensive and anti-inflammatory (Muktar et al., 2005; Arif et al., 2009: Huang et al., 2010).

Recently, people have started to look for high-quality dried herbal products that are closely associated with the quality of common raw herbal materials. Several factors contribute to the quality of herbs which are color and drying method. The final color of a dried plant product is a strong factor for marketing.

Drying of herbal plant is done either naturally or by machine. Natural drying is the standard practice that is currently used by most of the Malaysian herbal producers. Machine drying provides higher drying rate and is more hygienic as compared to natural method since it uses heat and operated in a closed chamber. The main purpose of drying is to extend product shelf life (Hamrouni-Sellami et al.,

2011) by slowing microorganism growth and preventing certain biochemical reactions that might alter the organoleptic characteristics (Diaz-Maroto et al., 2003;

Hamrouni-Sellami et al., 2011).

Recently, most people have started to look into local traditions and natural sources of medicinal plant that may provide potent and safe medicines. They are now starting to be concerned about the side effects of synthetic antioxidants to our health due to enhanced public awareness of the health issues. Natural antioxidants are preferable by consumers and occur naturally from plants, animals, microbial sources, and processed food products. The volume of natural products is increasing day by day and they are available in the market in the forms pills, capsules, liquids and creams; however, the recent trend was ‘‘tea infusion’’ as herbal supplements

(Mellgren, 2001). Hence, extensive researches on the use of natural based supplement used are imperative nowadays to identify their biologically active compounds.

Streblus asper Lour (S. asper), is an herbal plant known locally as kesinai. S. asper (family Moraceae) is found in tropical countries such as Malaysia, Thailand and India. In Thailand and India, this plant has been extensively used in popular folk and Ayurvedic medicine for centuries. However, in Malaysia, there is still limited study and evidence on their typical mechanism of actions and the potential uses of S. asper leaf as herbal supplements. This plant is found mainly in surrounding villages and open areas in the northern region of Malaysia. S. asper has been used in Malay traditional medicine as decoction and pastes for wound infections. Previously, it has been described and its uses have been identified in Thailand, but only from the bark, stem and root. Thus far, very little is known about on the complete phytochemical and nutritional properties of the leaves. S. asper plant has been reported to possess anticancer activity, and to be useful in the treatment of wounds, skin disease, filariasis, leprosy, toothache, diarrhoea, dysentery and especially in the oral cavity 24

(Rastogi et al., 2006; Taweechaisupapong et al., 2006). It also possesses antibacterial and anti inflammatory activities (Wongkham et al., 2001). Leaf extracts may contain many nutrients that are beneficial for our body and health.

1.2 Problem Statements

There is much information regarding S. asper cited in the literature but these studies mainly focus on its stem, root and bark. However, information regarding the specific properties of S. asper leaves as affected by various drying methods is not available. Studies on the physichochemical characteristics of S. asper leaves as a potential herbal supplement are still lacking. Thus, this study was conducted to find the most suitable drying procedure to preserve S. asper leaves with minimal damage to their properties

1.3 Significance of Research

This research was to explored a new potential natural antioxidant with the goal to provide nature based alternative health supplement to the public. In general, when we consume natural and fresh plant leaves, it may contain rich phytochemical and nutrient which is claimed to overcome degenerative diseases that affect the human body (Alothman et al., 2009). About 80% of the world populations presently use herbal medicines for some aspects of primary health care (WHO, 1993). The results obtained from the present research will be useful to provide information on the phytochemical and nutrient contents of the S. asper leaves. Most studies in other

countries were limited to their locally grown cultivar; additionally, drying treatments and extraction solvents data may be used for herbal product standardization.

1.4. Research Objectives

The purpose of this study was to compare the effects of different drying treatments on the physicochemical properties of S. asper leaves in order to explore the potential uses of S. asper extracts as an alternative form of food supplement.

The specific objectives of this study were:

1. To analyse the physical (colour), chemical (preliminary

phytochemical screening, chlorophylls, minerals), phenolics and

proximate compositions of S. asper leaves.

2. To evaluate the efficiency of different solvents on the antioxidant

activity of S. asper leaves extract.

3. To determine the cytotoxicity effect of S. asper leaves extract against

HT29 cell lines in vitro.

CHAPTER 2

LITERATURE REVIEW

2.1 Morphological descriptions of Streblus asper

Streblus asper Lour (S. asper) is also known by several common names, including kesinai, siamese rough bush, koi, serut, berrikha, sheora, shaokota, rudi and toothbrush tree. It is a medium-sized tree native to dry regions in Asian countries such as Malaysia, Thailand, India, China and Vietnam. Table 2.1 shows the scientific classification of S. asper plant. It is also commonly made into a bonsai.

Table 2.1: Scientific classification of S. asper plant.

Scientific classification Kingdom Plantae Order Rosales Family Moraceae Genus Streblus Species S. asper

S. asper (Fig. 2.1), a medicinal plant, is a bushy, small, evergreen tree with milky juice, large shrub with crooked stem. The bark is light grey or greenish with faint ridges and is laticiferous. The leaves are alternate, 5-10 cm long, rigid, oval- shaped, acute or shortly acuminate, cuneate at base, denticulate, irregularly toothed, and borne on small petioles.

Fig. 2.1: Streblus asper leaf.

The leaves are also very rough on both surfaces, petiole very short. The flowers are unisexual, axillary; male flowers in globose pedunculate heads; stamens

4, inflexed in bud, anthers reinform; female flowers are solitary, inconspicuous long peduncles (Madhavan et al., 2009). The fruits are seeded berry, 5 mm diam., yellow when ripe.

2.2 Traditional Uses and Medicinal Properties of S. asper

S. asper (family Moraceae), is an ethno-medicinal plant used in most of the

Asian country in the treatment for diarrhoea, dysentery, stomach ache and wounds.

Many of the compounds in S. asper exhibited a wide range of biological activities, including antioxidative properties. In Malaysia the crude leaf extract has been reported, as an antibacterial agent (Nurul Ain, 2009), to be used as oral health care

(Hazizul, 2009). S. asper contains milk coagulating protease which could be used as a rennet substitute (Manap et al., 1992). The root extract has antifungal properties

(Nurbaiti, 2009) and cytotoxicity activity (Ilaina, 2009). In other Asian countries such as China, they used S. asper as medicinal herb in the treatment for Hepatitis B virus (HBV) infection, fever, dysentery, toothache, gingivitis, inflammation and antibacterial agent (Jun et al., 2012; Casaroto and Lara, 2010; Li et al., 2011). Hong et al., 2012 also reported that heartwood, barks, and roots of S. asper exhibited good anti-HBV activities. In India, it is used traditionally in Ayurveda in the treatment for leprosy, piles, diarrhoea, dysentery and cancer (Chopra et al., 1956; Bhakuni et al.,

1969). A decoction of the bark was used for treatment in fever, dysentery, and diarrhoea (Burkil, 1966). The powdered root is also used for dysentery, applied to unhealthy ulcers, epilepsy and inflammatory swellings (Kirtikar and Basu, 1933).

The latex of the S. asper is used as an astringent and antiseptic; it is also used as a sedative and applied to sore heels, chapped hands, glandular swellings (Kirtikar and

Basu, 1933). The seeds are beneficial in epistaxes, piles, diarrhoea and applied as a paste in leucoderma (Nadkarni, 1976). The juice was used as astringent, antiseptic, swells of cheek and applied to chapped hands and sore feet (Chopra et al., 1956; Jain,

1991). In Vietnam, the bark, root, stem and leaves of this plants are used as medicine

in the treatment for dental decay, sore throat, fever, cough, leprosy, piles, diarrhoea, dysentery and elephantiasis; it also possesses anticancer activity (Vo, 1997).

Other uses have been reported by Gaitonde et al. (1964); Lewis (1980);

Zhang et al. (1985); Rastogi et al. (2006); where the bark extract was used for relief of fever, dysentery, toothache and gingivitis. The branch part has been used as a toothbrush for strengthening teeth and gums, while the leaf extract has demonstrated insecticidal activity towards mosquito larvae, antibacterial action, and inhibitory effect on oral and dental diseases.

2.3 Antioxidant

Antioxidants are compounds that help in delaying, inhibiting lipid oxidation, minimise rancidity, retard the formation of toxic oxidation products, help to maintain nutritional quality and increase shelf life in the foods (Fukumoto and Mazza, 2000).

There are two main types of antioxidants; primary (chain breaking, free radical scavengers) and secondary (preventive). Gordon (1990) who explained that secondary antioxidant mechanisms include regeneration of primary antioxidants, deactivation of metals, singlet oxygen quenching and inhibit the breakdown of lipid hydroperoxides to unwanted volatile products. The oxidative damage created by free radicals is referred as oxidative stress that has been associated with several degenerative diseases such as cardiovascular, inflammatory diseases, cancer, aging, stroke (Machlin and Bendich 1987; Uddin and Ahmad 1995), antioxidant defence systems, altered calcium homeostasis, changes in gene expression and induction of

abnormal proteins which may contribute significantly to human disease (Bagchi,

2000; Bouhamidi et al., 1998).

Natural antioxidants from phytochemical are not only used as food additive but it has also been used to improve the quality of the food and to extend the shelf life of herbal product. Nowadays, researchers are starting to look into phytochemicals because of their natural antioxidative properties that can be used as food or dietary supplements. For health and safety reasons, there is growing need to identify new alternatives for safer sources of food antioxidants. The growing interest in the substitution of synthetic food antioxidants by natural ones has led to research on plant medicinal sources and the screening of raw materials to identify potential antioxidants.

Previous studies have demonstrated on the antioxidant activity of medicinal plants such as Centella asiatica, Polygonum minus, Peperomia pellucid, Withania somnifera, Croton argyratus, Phyllanthus amarus extracts (Lim and Murtijaya,

2007), Moringa oleifera leaves, Mentha spicata leaves (Arabshahi et al., 2007),

Azadirachta indica, Acacia nilotica, Eugenia jambolana, Terminalia arjuna, and

Aloe barbadensis leaves (Bushra et al., 2009), Euphorbia hirta (Abu et al., 2011), leaf extracts of Morus alba and Morus nigra (Radojkovic et al., 2012). The data suggest that all these medicinal plants have strong antioxidant potential. Various antioxidant activity methods have been used to monitor and compare the antioxidant activity, such as DPPH radical assay, thiobarbituric acid-reactive-substances

(TBARS), 2,2-azinobis, 3-ethylbenzothiazoline-6-sulfonic acid (ABTS), oxygen radical absorbance capacity (ORAC), Fe2+ chelating activity, inhibition of lipid 31

peroxidation, superoxide anion radical, ferric reducing antioxidant power (FRAP) assays, β–carotene bleaching (BCB) and the hydroxyl radical (OH) assays. The

DPPH test is a simple, rapid and inexpensive method which is based on the exchange of hydrogen atoms between the antioxidant. The determination of scavenging stable

DPPH is a very fast sensitive method, with ease of use and convenient (Arulpriya et al., 2010). TBARS method can be time consuming because it depends on the oxidation of a substrate which is influenced by temperature, pressure and may not be practical when large numbers of samples are involved.

2.3.1 1,1-diphenyl-1- picrylhydrazyl (DPPH) Radical Scavenging

Phytochemicals such as ascorbic acid, carotenoids, phenolics, and flavonoids with antioxidant potentials have been characterised by the free radical scavenging assay (Saxena et al., 2009). In this case, DPPH assay was used to measure antioxidant activity of S. asper leaf extracts using a spectrophotometer. DPPH is widely used to test the ability of compounds to act as free radical scavengers or hydrogen donors. It has also been used to quantify antioxidants in complex biological systems in recent years. It is very reproducible and does not require special instrumentation (Koleva et al., 2002). The DPPH method can be used for solid or liquid samples and is not specific to any particular antioxidant component, but applies to the overall antioxidant capacity of the sample. A measure of total antioxidant capacity helps understand the functional properties of foods. Basically, the reaction of antioxidant compounds present in S. asper leaves with DPPH radical decolourised the visible deep purple colour by measuring the changes of the degree of discolouration in absorbance at 517 nm in the presence of a hydrogen donating 32

antioxidant (AH) due to the formation of the non-radical form DPPH-H by the reaction donation (Verma et al., 2009):

DPPH + AHDPPH-H + A

The more antioxidant presence in the extract, the more the DPPH reduction will occur. High reduction of DPPH is related to the high scavenging activity performed in the sample (Abu et al., 2011). The antioxidant activity of some herbs such as

Centella asiatica, Eichhornia crassipes (Lalitha and Jayanthi, 2012), Azadirachta indica, Acacia nilotica, Eugenia jambolana, Terminalia arjuna, and Aloe barbadensis leaves were studied using DPPH assay (Bushra et al., 2009).

2.4 Secondary Metabolites

Phytochemicals are divided into two groups, which are primary and secondary constituents; according to their functions in plant metabolism. Primary constituents comprise common sugars, amino acids, proteins and chlorophyll while secondary constituents consists of alkaloids, terpenoids, tannins, phenolic compounds and flavonoids. Secondary metabolites are organic compounds that are not directly involved in the normal growth, development, or reproduction of an organism. It is plays an important role in plant defense against herbivory. Secondary metabolite is crucial for plant defenses (e.g. as an antioxidant or antimicrobial agent) which has enabled plants to survive. Generally, humans use secondary metabolites as medicines, flavourings, and recreational drugs.

2.5 Phytochemicals

Phytochemicals are plant chemicals that protect plant cells from environmental hazards such as pollution, stress, drought, UV exposure and pathogenic attack. These compounds are known as secondary metabolites that have important biological activities such as antioxidant, cytotoxcity, antimicrobial, antibacterial and etc. that act as human health protector against diseases. Study by

Rastogi et al. (2006); Prakash et al. (1992); Barua et al. (1968) showed the presence of secondary metabolites such as cardiac glycosides, pregnane glycoside named sioraside from the roots and stem barks, α-amyrin acetate, lupeol acetate, β-sitosterol,

α-amyrin and lupoel that have been isolated from S. asper. Other literatures by

Phutdhawong et al. (2004); Chaturvedi (2006); Malika (2007); Li et al. (2008); Lu et al. (2009); Tripahti et al. (2011) also reported that chemical constituent characterised from S. asper were steroids, cardiac glycosides, lignans, flavonoids, triterpenoids, protease, oleanolic acid, α-farnesene, phytol, trans-farnesyl acetate, caryophyllene and trans-trans-α-farnesene.

Research was carried out on the medicinal plants such as Murraya koenigii leaves, Eurycoma longifolia, Andrographis paniculata, Labisia pumila, Spondias mombin (Nijoku and Akumefula, 2007), Schotia latifolia (Mbaebio et al., 2012),

Cadaba trifoliata, Syzygium cumini, Anchomanes difformis, Anisopus mannii,

Pavetta crassipes, Stachytarpheta angustifolia and Vernonia blumeoides (Aliyu et al., 2008) and Aframomum melegueta and Garcinia Kola Heckel (Okwu, 2005).

Phytochemical screening of the extracts showed the presence of tannins, steroids, alkaloids, terpenoids, saponin, glycosides and flavonoids.

2.5.1 Classification of Phytochemicals

Phytochemical evaluation is one of the tools for the quality assessment in producing herbal products which include preliminary phytochemical screening, chemo-profiling and marker compound analysis using modern analytical techniques in order to maintain the high quality of the herbal product. Phytochemicals are bioactive substances found in the plants which have been associated with the protection of human health against chronic degenerative diseases. Bao and Fenwick

(2004); Shahidi and Ho (2005) also described that phytochemicals are important sources of natural food antioxidants where some of them have been developed and processed for popular functional beverages and dietary supplements.

Phenolic phytochemicals are the largest category that is widely distributed in the plant kingdom. According to a study by Shahidi (2004), phytochemical mainly phenolic compounds can act as antioxidants, deactivate procarcinogenes, maintain

DNA repair and inhibit formation of N-nitrosamines. Soetan and Oyewole (2009) reported that flavonoids, flavones, glucosides and other secondary plant metabolites such as tannins, alkaloid, terpenoid, steroids, saponin are some of the phytochemicals can be widely seen in plants and have pharmacologically active agents.

2.5.2 Potential Sources of Phytochemicals

Study by Altiok (2010) reported that the processing of plant foods results in the production of by-products that are rich sources of bioactive compounds, including phenolic compounds. By-products of plant food processing represent a

major disposal problem for the industry concerned, but they are also promising sources of compounds which may be used because of their favourable properties.

2.6 Phenolic

Phenolic compounds are responsible for major organoleptic characteristics of plant derived foods and beverages, particularly colour and taste (Cheynier, 2005).

Phenolic compounds belong to the category of natural antioxidants and found most abundant of antioxidants in the diet (Boskou et al., 2006). The term ‘phenolic’ refers to any compounds with a phenol type structure (Vaquero et al., 2007) that consists of three most important groups; phenolic acids, flavonoids and polyphenols (Fig. 2.2).

Phenolic compounds such as quercetin and ellagic acid are known as a good antioxidants that able to protect the body cells from injuries which are caused by reactive oxygen and nitrogen species (Sroka and Cisowski, 2003). The antioxidant capacity is attributed largely to its phenolic compounds (Huang et al., 2009) which have beneficial effects on the body (Heim et al., 2002). Study by Chang et al. (2001) revealed that the phenolic content in the plants is correlated well with their antioxidant activities which probably due to their redox properties that as reducing agents, hydrogen donors, and singlet oxygen quenchers. Singleton and Rossi (1965) have described those three classes of phenolics from relatively simple to the complex which are; non – flavonoids (hydroxybenzoic acid and hydrocinnamic acid), flavonoids (flavones, flavonols, flavanones, flavononols and flavans) and tannins.

Tepe et al. (2006); Luthria et al. (2006) stated that the phenolic contents depending on the genetic, environmental factors, storage conditions as well as post – harvest processing.

The biosynthesis of phytochemicals; flavonoids are a defensive response of plants to the environment that functions as protectors from ultraviolet sunlight and lipid peroxidation (Mariani et al. 2008). Flavonoids are characterised as C6-C3-C6 structure with free hydroxyl groups that attached to aromatic rings. Flavonoids have known to possess strong and effective antioxidant properties capable of scavenging oxygen radicals and break the chain mechanism (Kandaswami and Middleton, 1997;

Yanishlieva, 2001). Other literatures also described that flavonoid has been proven to display a wide range of pharmacological and biochemical actions such as antimicrobial, antithrombotic, anti-mutagenic, anti-inflammatory, anti-allergic, antihypertensive and anti-carcinogenic activities (Cook and Samman, 1996;

Kandaswami and Middleton, 1996; Sahu and Gray, 1997).

Fig. 2.2: Common simple phenol and flavonoids in plants (Evren, 2010).

Dinelli et al. (2006); Elizabeth et al (2007) who explained that flavonoids are representing a large family of low molecular weight phenolics and divided into

several chemical classes such as flavones, flavanones, isoflavones, isoflavans, pterocarpans, coumestans, anthocyanins, flavanols (or catechins) and flavonols according to their molecular structure. Flavonoids and other classes of phenolic compounds such as tocopherols, carotenoids and ascorbic acids are important for health benefits which derived from consuming high levels of fruits and vegetables

(Hertog et al., 1993; Parr and Bolwell, 2000; Johnson, 2001; Meyers et al., 2003).

2.6.1 Total Phenolic Content

Phenolics are secondary metabolites and known as radical scavengers, metal

chelators, reducing agents, hydrogen donors that present in edible plants (Saleem et

al., 2002). It has been known their potential as antioxidant activity. Other literatures

found that phenolics have been claimed as medicinal values in the management for

diabetes, anti-allergic, anti-oxidative activities, anti-carcinogenic, anti-inflammatory

antimicrobial, anti-mutagenic, anti-atherosclerosis and cardiovascular protector

(Han, 2007; Arts and Hollman, 2005; Scalbert et al., 2005). Phenolic acids can be

divided into two classes; derivatives of benzoic acid (gallic acid) and derivatives of

cinnamic acid (coumaric, caffeic and ferulic acid). They also have activity either as

a single compound or as synergistically by two or more compounds. Singleton

(1999); Shakirin et al. (2010) who described that phenolic compound undergo a

complex redox reaction with the phosphotungstic and phospho-molybdic acids

present in the Folin Ciocalteu reagent used for the colorimetric assay of phenolic

and polyphenolic antioxidants. The principle of Folin Ciocalteu is based on the

reduced ability of phenol functional group, where the oxidation and reduction

reaction of phenolat ion takes place on base condition. The reduction of this reagent 38

by phenolat ion will change its colour to be blue (Prior et al., 2005). Therefore, the reduction will increase when the extract contains more phenolic compounds indicating the colour will be darker and the observance will be higher.

2.6.2 Total Flavonoid Content

Gheldof and Engeseth (2002) described that flavonoids (secondary metabolites) are natural antioxidants derived from plants and found most in the foods, fruits, vegetables and medicinal plant leaves with the ability to inhibit lipid oxidation through both metal chelating and free radical scavenging mechanisms that defend against oxidative stress, reduce heart disease, cardiovascular disease, prevent cancer, and slowing the aging processes in cells responsible for degenerative diseases (Hollman and Katan 1997). Flavonoids (Fig. 2.3) are divided into several subclasses which are flavanols, flavanones, flavones, isoflavones, anthocyanidins, and flavonols (Hollman and Katan 1997). The presence of hydroxy groups, 2,3- double bond and orthodiphenolic structure can donate electrons through resonance to stabilise the free radicals (Machlin and Bendich 1987) and enhance antioxidative and antiradical activity of flavonoids (Tapas et al., 2008).

Fig. 2.3: The basic unit of flavonoids (Tapas et al., 2008).

2.7 Chemical and Physical Analysis

2.7.1 Chemical Analysis

2.7.1.1 Proximate

Medicinal plants play a crucial role in giving good benefits to the health significantly (Taiga et al., 2008; Kochhar et al, 2006; Pandey et al., 2006). Plants are known to have high amounts of essential nutrients, minerals, fatty acids and fibre.

WHO (2007) emphasised on the needs and the importance in determining proximate and micronutrient analysis for herbal drug standardisation. Carbohydrates, fats, proteins, vitamins, minerals and water are important constituents in the diet

(Indrayan et al., 2005). Plant materials form a major portion of the diet so that it is important for us to know their nutritive value. In this study, the determination of proximate composition has been carried out to determine the amount of moisture, fat,

ash, fibre, protein and nitrogen free extract in S. asper leaves. Every component of the composition has an important role in the health. Similar studies have been carried out on the medicinal plants leaves such as Tylophora glauca (Ajibade and Fagbohun,

2010), Azadirachta indica (Atangwho et al., 2009; Onyimonyi et al., 2009), Morus alba (Shahid et al., 2012), Moringa oleifera and Piper sarmentosum. Research by

Ajibade and Fagbohun (2010) found that the proximate composition of Tylophora glauca was (7.11%), ash (6.66%), crude protein (20.03%), fat (14.01%), fibre

(7.19%) and carbohydrate (45.00%). The result of the proximate analysis of dried leaves of Moringa oleifera revealed that the early stage has the highest carbohydrate content (55.14%) while mild stage recorded the highest moisture (6.3%) and the late stage has the highest protein content (28.08%), with crude fibre (10.11%), ash

(9.25%), fat (2.5) and pH (6.27). Onyimonyi et al. (2009) reported that the

Azadirachta indica leaf meal has a proximate composition of 3.5% moisture; 24.06% crude protein; 12.00% crude fibre; 6.00% ash and 51.94% Nitrogen free extract. The moisture, ash, fibre and protein contents of the fresh Morus alba leaves were 5.3% ±

0.2, 8.91% ± 0.51, 10.11% ± 0.37 and 18.41% ± 1.36 (Shahid et al., 2012) respectively.

2.7.1.2 Minerals

Generally, the knowledge of proximate, micronutrients and phytochemical composition is fundamental in understanding the mechanisms and modes of action in medicinal plants. Many medicinal plants are studied for their impact on health, yet little research has examined on the mineral content. Every constituent plays an important role and deficiency of the constituent may lead to abnormal developments 41

in the body. Mineral analysis the has been carried out to determine the concentration levels of minerals such as Phosphorus (P), Sodium (Na), Potassium (K), Calcium

(Ca) and Magnesium (Mg) in S. asper leaf using atomic absorption spectrometry

(AAS). The mineral composition is not only depending on the species or varieties, but also on the growing conditions, such as soil and geographical conditions. Mineral determination was carried out on medicinal plants like Morus alba (Omidiran et al.,

2012), Froriepia subpinnata, Eryngium caucasicum leaves (Ebrahimzadeh et al.,

2009), rosemary leaves (Arslan and Ozcan, 2008), Murraya koenigii, Mentha piperitae, Ocimum sanctum and Aegle marmelos (Narendhirakannan et al., 2005).

The study showed that these medicinal plants contain an appreciable amount of minerals (Narendhirakannan et al., 2005; Ebrahimzadeh et al., 2009). Study by

Arslan and Ozcan (2008) found that fresh and dried rosemary leaves had high amounts of K, Ca, Na, Mg and P minerals, where Al, B, Ba, Cu, Fe and P values of the fresh and dried samples were not statistically significant. The mineral compositions of dried rosemary leaves were higher because of the increased dry matter content. The mineral compositions of Morus alba leaves showed the presence of Ca (1.56 mg/100g), Zn (3.60 mg/100g), K (31.20 mg/100g), P (7.66 mg/100g) and

Mg (1.64 mg/100g) (Omidiran et al., 2012). They indicated that the mineral compositions of the plants were low which correlate with the low ash contents of the plants. Study by Ogungbenle (2003) reported that some differences were found when comparing the quinoa mineral contents. These differences may be linked to the fact that the quinoa samples were from different cultivation areas. Thus, the mineral content may vary depending on several factors such as ripeness, variety, soil type, the use of fertilizers, intensity and exposure time to sunlight, temperature and rain.

2.7.1.3 Chlorophyll

Chlorophyll is a natural, green pigment, antioxidant compound which is stored in the chloroplast of green leafy plants and a major agent that capable of absorbing light energy and transmitting into the carbohydrates synthesised during photosynthesis to carry out the various important of biological functions. Chlorophyll degradation is an integral part of leaf senescence. It has been found in green leaves, stems, flowers and roots. Chlorophyll is an antioxidant which is usually found in nature especially in green leafy plants (Buavaroon et al., 2011) and is a widely distributed plant pigment. Chlorophyll is a major of supplement source in the production of nutraceutical products (Higdon, 2004; Suzuki and Shioi 2003); where it has been used as an indicator for quality of herbal product. Chlorophyll can reveal plant ageing (Merzlyak et al., 1999), plant nutrition (Moran et al., 2000) and also useful in the study the symptoms of senescence in attempts to maintain the green colour for producing high quality herbal products. Study by Monica (2005) also reported the uses of chlorophyll in helping to absorb and to build up blood and fight anaemia, supports liver function and detoxification of the body. It could cure or ease acute infection of the respiratory tract and sinuses, chronic ulcers, and bad breath and also accelerates wound healing (Monica, 2005). Those benefits influence us to study the importance of chlorophyll in this research

Chlorophyll is not a single molecule but a family of related molecules, designated as chlorophyll a, b, c and d (only present in red algae). The chlorophylls; chlorophyll a (Ca) and b (Cb) are virtually essential pigments for the conversion of light energy to store chemical energy. The amount of solar radiation absorbed by a

leaf is a function of the photosynthetic pigment content (Monteith 1972, Foyer et al.

1982). Pan Dong (1995) described that chlorophyll a and b are present in the leaves of higher plants; the main pigments of photosynthesis in the chloroplasts. They have important functions in the absorption and exploitation of light energy, thereby influence photosynthetic efficiency. Chlorophyll a is the primary and the pigment that related to the photosynthesis and produces the energy for plant chlorophyll b

(accessory pigment). The main function of chlorophyll b is to gather the light energy that working together with chlorophyll a and carotenoids. Chlorophylls a and b have a side phytol chain. The two chlorophylls are always accompanied by yellow pigments, carotene and xanthophyll. Chlorophyll b differs from chlorophyll a by having an aldehyde group (-CHO) on carbon atom 3 instead of a methyl group (Fig.

2.4).

C32H30ON4Mg COOCH3

COOC20H30 chlorophyll a

C32H28O2N4Mg COOCH3

COOC20H39 chlorophyll b

Fig. 2.4: Chlorophyll a and chlorophyll b.

Several studies have been carried out on the medicinal plants leaves such as

Mentha spicata, Podophyllum peltutum, Ricinus communis (Nouri et al., 1954),

Apium graveolens, Averrhoa bilimbi, Hydroctyle asiatica, Mentha arvensis, Psidium guajava, Sauropus androgynous, Solanum nigrum and Polygonum minus (Mahanom

et al., 1999) to maintain the appearance of green in dried herb leaves. Study by

Arathi and Suneetha (2011) showed that the chlorophyll content of Azadirachta indica L, Murraya koenigii L, Ocimum tenuiflorum L and Mentha arvensis L by using spectrophotometric methods using 80% acetone as the solvent, showed that

Azadirachta indica L has the highest chlorophyll content among the four followed by

Murraya koenigii L and thus these leaves can be utilized to avail the health benefits of chlorophyll compound along with their other medicinal benefits which provide a natural source of chlorophyll readily without using any chemical supplements.

Chlorophyll has been seen to help neutralize free radicals that do damage to healthy cells. It is also useful in treating infected wound naturally and calcium oxalate stone ailments, possesses anti-atherogenic, antimutagenic and anticarcinogenic properties which is helpful in protecting your body against toxins and in reducing drug side effects (Arathi and Suneetha, 2011).

2.7.1.4 pH

The pH indicators (acid-base indicator or neutralisation indicator) are substances which the colour change due to the changes in pH. They are usually weak acids or bases, but their conjugate base or acid forms have different colours due to differences in the absorption spectra. Das et al. (2010) reported that the variation may occur due to the pH of the solvent. The general formula for the indicator is HIn for acidic indicators and InOH for basic indicator.

Factors such as temperature and pH of the media, antioxidant concentration, processing treatment and storage strongly influence the antioxidant activity (Gazzani 45

et al., 1998). The pH test was done on some medicinal plants such as Moringa oleifera leaves and Mentha spicata leaves (Arabshahi et al., 2007). Study by

Arabshahi et al. (2007) showed that the antioxidant activity of Mentha spicata leaves extract was higher at pH 9 than pH 4, while Moringa oleifera leaves extract remained the same under both pH conditions. These extracts stored in the dark at 5 and 25°C after a 15 day period did not show any significant change (p < 0.05) in their antioxidant activity. These data indicate that selected plant extracts are potential sources of dietary antioxidants.

2.7.2 Phytochemical Screening

Phytochemicals are bioactive substances of plants found in plants, such as

vegetables, fruits, medicinal plants, flowers, leaves and roots that work with

nutrients and fibers that have been associated with the protection of human health

against chronic degenerative diseases and have high antioxidant activities (Moure et

al., 2001). For this reason, they are widely used as food or dietary health

supplements. The phytochemical screening assay is a simple, quick, and

inexpensive procedure that gives a quick answer to the various types of

phytochemicals and it is an important tool in bioactive compound analyses. Hence,

phytochemicals screening serves as the initial step in predicting the types of

potential active compounds from plants.

2.7.2.1 Alkaloid, Tannin, Saponin, Terpenoids, Steroids and Cardiac Glycosides

In this study, phytochemical compounds such as alkaloids, tannins, saponin, terpenoids, steroid and cardiac glycosides were screened. Alkaloid is a group of nitrogen containing bases, usually a small, heavily derivatized amino acid, crystalline, odourless, colourless, bitter taste and non-volatile compounds which are found to be rich in barks, leaves and fruits. Alkaloids (Fig. 2.5) are produced by a large variety of organisms, including bacteria, fungi, plants, and animals, and are part of the group of natural products. Alkaloids have diverse and important physiological effects on humans and other animals where most are poisonous in nature; some are used medicinally as analgesics (pain relievers) or anaesthetics.

Fig. 2.5: The structure of alkaloids.

Tannins (Fig. 2.6) are naturally occurring plant polyphenols located mainly in the vacuoles or surface wax of the plants. They are composed of a very diverse group of oligomers and polymers which are relatively high molecular weight compounds that have ability to form complexes with metal ions and macro-molecules such as proteins and polysaccharides (De-Bruyne et al., 1999; Dei et al., 2007) and constitute the third important group of phenolics; it may be subdivided into hydrolysable and condensed tannins (Porter, 1989). Their main characteristic is to bind and precipitate proteins. Tannins has high potential in treating intestinal disorders such as diarrhoea and dysentery and also aids in wound healing.

Fig. 2.6: The structure of tannins.

Saponins (Fig. 2.7) are glycosides, which include steroid saponins with a

distinctive foaming characteristic and triterpenoid saponins (Dei et al., 2007) widely

distributed in the plant kingdom. It is traditionally used as medicine preparations, as

dietary supplements and nutraceuticals (Xu et al., 1996) which are characterized by

their structure containing a triterpene or steroid aglycone and one or more sugar

chains. Saponin are natural surfactants, or detergents, found in many plants,

especially certain desert plants. Saponins have been known to have strong biological

activity. They consist of a polycyclic aglycone that is either a choline steroid or

triterpenoid attached via C3 and an ether bond to a sugar side chain. The antifungal

and antibacterial properties of saponins are important in cosmetic applications, in

addition to their emollient effects. Saponin-protein complex formation can reduce

protein digestibility. It is also traditionally used to cleanse and purify blood because

one of saponins medicinal uses is as a gentle blood cleanser.

Fig. 2.7: The structure of saponins.

Terpenoids (Fig. 2.8) are called isoprenoids, are a large and diverse class of

naturally occurring organic chemicals similar to terpenes, derived from five carbon

isoprene units assembled. Most are multicyclic structures that differ from one

another not only in functional groups but also in their basic carbon skeletons. These

lipids can be found in all classes of living things, and are the largest group of natural

products. Plant terpenoids are used extensively for their aromatic qualities. They

play a role in traditional herbal remedies in nutrition and human health such as a

rejuvenating agent because it has been found to be a very useful remedy for anti-

aging and overall beauty, enhancement anti-malarial, anticancer drugs, commercial

flavour and also as fragrance compounds (Vincent, 2003). Terpenoid also is a heart-

friendly phytochemical constituent. It is shown that terpenoids strengthen the skin,

increase the concentration of antioxidants in wounds, and restore inflamed tissues

by increasing blood supply.

Fig. 2.8: The structure of terpenoids.

Steroids (Fig. 2.9) are a class of organic compounds with a chemical structure that contains a characteristic arrangement of four cycloalkane rings that are joined to each other. Steroids are frequently used signalling molecules for examples; the steroid hormones. Steroids with phospholipids function as components of cell membranes. Steroids such as cholesterol decrease membrane fluidity. It is also used to increase protein synthesis, promoting growth of muscles and bones. They reduce the recovery time needed between training sessions and enable athletes to train more intensively for longer periods.

Fig. 2.9: The structure of steroids.

Cardiac glycosides (Fig. 2.10) are compounds containing a carbohydrate

(sugar) and non carbohydrate residue in the same molecule and includes well-

known drugs such as ouabain and digoxin. An acetyl linkage at carbon atom 1

attaches the carbohydrate residue to a noncarbohydrate or non sugar residue. The

nonsugar component is known as the aglycone and the sugar component is glycone.

Glycosides play numerous important roles in living organisms. Many plants store

chemicals in the form of inactive glycosides. It is used widely used in traditional

medicine in the treatment for congestive heart failure and cardiac arrhythmia.

Fig. 2.10: The structure of cardiac glycosides.

2.7.2.2 Screening of Volatiles by Gas Chromatography Mass Spectrometry (GCMS)

Volatile constituents are the most sensitive components in the process of

plant herbal drying. Study by Paula and Manuel (2001) reported that the percentage

composition and characteristics of Volatile Organic Compounds (VOCs) provide an

important parameter for the characterisation of plant. Other literatures by Edris

(2007) also reported that the interest in plant volatiles is focused on their biological

activity potential such as antioxidant, antibacterial, anti-diabetic, anti-platelet,

antifungal effect and inhibition of prostaglandin. It is also important in cosmetics,

food and pharmaceutical industries due to their flavours and fragrances. Table 2.2

shows the phytochemical constituents of different plant parts of S. asper that has

been characterised. In this study, GCMS was used to screen VOCs in S. asper leaf.

The effect of drying method on the volatile compounds and on the structural

integrity and sensory characteristics of the spice spearmint (Mentha spicata L.) was

studied by Diaz-Maroto et al. (2003).

Table 2.2: Phytochemical constituents of different plant parts of S. asper.

Plant part Phytochemical constituents References Aerial n-triacontane, tetraiacontan-3-one, β- Chawla et al., 1990 sitosterol, stigmasterol, betulin and oleanolic acid Bark Cardio active glycosides, strebloside and Ghani, 2003 asperosid, pregnane glycoside, sioraside Root Kamaloside, asperoside, indroside, Rastogi and luknoside, along with amorphous Mehrotra, 1990 glycosides G, G´ and H. Cannodimethoside, strophalloside, glucogitodimethoside, strophanolloside, glucokamaloside, sarmethoside and glucostrebloside Cardiac glycosides; kamloside, asperoside, Reichstein et al., strebloside, indroside, cannodimemoside, strophalloside, strophanolloside, 16-O- acetylglucogitomethoside, glucogitodimethoside, glucokamloside, sarmethoside and glucostrebloside. β-sitosterol-3-O-β-D-arabinofuranosyl- Chaturvedi and O-α-l-rhamnopyranosyl-O-β-D- Saxena, 1984 glucopyranoside Lupanol-3-O-β-D-glucopyranosyl-[1-5]-O- Chaturvedi and β-D-xylofuranoside Saxena, 1985 Vijaloside; periplogenin-3-O-β-D- Saxena and glucopyranosyl-[1-5]-O-β-D- Chaturvedi,1985 xylopyranoside Stem β-amyrin acetate, lupeol Barua et al., 1968 acetate, β-sitosterol, β-amyrin, lupeol and diol Strebloside and mansonin Fiebig et al., 1985

Sioraside Prakash et al., 1992 Essential oil Phytol, α-farnesene, trans-farnesyl acetate, Phutdhawong et al., 53

of fresh caryophyllene, trans-trans-α-farnesene,α- 2004 leaves copaene, β-elemene, caryophyllene, geranyl,acetone, germacrene, δ-cadinene, caryophyllene oxide and 8-heptadecene.

2.7.3 Physical Analysis

2.7.3.1 Colour Attributes

Colour measurement is one of the important organoleptic properties that provides an objective index of the quality of herbal product which has to be analysed and evaluated (Fig. 2.12). Colour has significant impact on consumer perception and influences other important quality factors such as flavour and aroma. The colour of herbal products must be measured and standardised in order to obtain and maintain high quality of herbal products as well as the raw materials. In this study, colour changes were determined by measuring colour attributes (L*, a*, b*, C and hue angle) value. Colour analysis was based on the L a b Hunter colour scale. The lightness (L*), greenness (a*) and yellowness (b*) of dried leaves were compared with the fresh sample. The a* and b* are the chromaticity coordinates (Fig. 2.11).

The center is achromatic. As a* and b* values increase and the point moves out from the center, the saturation of the colour increases. C* is chroma and h is the hue angle which expresses the colour tonality from 0/360° (magenta-red), 90° (yellow), 180°

(bluish-green), to 270° (blue) (Minolta, 1998; Chaovanalikit et al., 2012).

Chroma, C*=√(a*)2 + (b*)2

-1 Hue angle, hab= tan b* a* + b (yellow)

hue angle

(green) -a + a (red)

- b (blue)

Fig. 2.11: a*, b* Chromaticity Diagram

The latter aspect is not considered in most research concerning drying and there are limited reports for colour characteristics of the dried plant (Arslan and

Ozcan, 2008; Yousif et al., 1999). Colour is an indication of ripeness, spoilage and also indicates problems with the processing or packaging. For instance, the colour changes from browning to blackish indicate either enzymatic or non-enzymatic reactions. Scientists are more interested in researching the Maillard reaction (major non-enzymatic reaction) which is the dominant browning reaction. Enzymatic browning is due to the enzymatic catalysed oxidation of the phenolic compounds which is naturally occurring pigments that plays a role in food colouring. Medicinal plants such as Thymys daenensis (Rahimmalek and Goli, 2013) and rosemary leaves

(Arslan and Ozcan, 2008) were analysed for the colour. It is important to maintain

the green colour for producing higher quality medicinal plant products. Food products are sensitive to drying temperature, which can induce degradation for example loss of colour, loss of texture and nutritional–functional properties. There is a direct relationship between the colour parameter and the drying temperature

(Miranda et al., 2009). Study by Genin and Rene (1995) reported that freeze drying is the most suitable drying method for maintaining the colour quality of dried chilli where freeze-dried sample gave more bright-red colour. They also found that drying samples at 80 and 90°C showed the highest total colour variation compared to the fresh sample. It is due to the effect of temperature on heat-sensitive compounds such as carbohydrates, proteins, and vitamins, which cause colour degradation in fresh food.

Qualitative Quantitative – HPTLC Finger Printing, 2nd Metabolites, DNA Finger Printing Chromatography Heavy Metal Pesticide Residue Mycotoxin • Moisture Content • Extraction Values Physical • Ash Values Chemical • Fluorescence Analysis

Standardisation of herbal drug • Microbial Contamination Biological • Toxicological Pharmacological •Other specific activities

• Colour • Odour Botanical Organoleptic • Taste • Texture •Macroscopic – Shape, External, Marking •Microscopic – Qualitative, Quantitative, SEM Studies, Powder Studies

Fig. 2.12: Flow chart on standardization and evaluation of herbal drug (Neeli et al., 2008). 56

2.8 Drying techniques: oven drying and freeze drying

Medicinal plants are perishable in the fresh state and may deteriorate after a few days. One way to preserve plant products is by drying where which helps to conserve desirable qualities, reduces storage volume and also extend the shelf life of herbal products. Drying can inactivate the enzymes polyphenol oxidases. It can be performed by traditional sun drying, microwave drying, oven drying and freeze drying. However, enzymatic and/or non-enzymatic processes that occur while drying leads to significant changes in the composition of phytochemicals (Capecka,

Mareczeek and Leja, 2005) which might affect on the final product. Drying process also inhibits microbial growth but at the same time, it can give rise to other alterations that affect the quality of herbal products. Drying caused shrinkage and made the plant material more brittle, thus making it easier to grind for extraction.

Oven drying is commonly used in herbal production but consumes longer drying time which usually results in inferior product quality. Plants that were dried in an oven at 40°C for 72 h for potential bioactive compounds (Das et al., 2010;

Ncube et al., 2008). This temperature is acceptable as recommended by Jackman et al. (1987) who reported that conventional extraction was conducted at temperatures ranging from 20 to 50°C. The lower temperatures were more suitable because temperature over 60°C will degrade the phenolic compounds rapidly (Havlikova and

Mikova, 1985). However, there are some disadvantages in oven drying method. It can cause a decline in density and water absorbance capacity from the internal part of the drying material to the surface due to longer drying period and high temperature

(Maskan, 2001). 57

Another alternative is by freeze drying method. Freeze drying is based on the dehydration by sublimation of the frozen product. Due to the absence of the liquid water and the low temperature required for the process, most deterioration and microbiological reaction are stopped which gives an excellent quality of the final product. Freeze drying would give the highest product quality but it incurs high production cost (Ratti, 2001). Genin and Rene (1995) and Irzyniec et al., (1995) also found that freeze drying is the best method of water removal giving final products of highest quality compared to other methods of food drying. The variations in different extraction methods could cause or affect the quantity and secondary metabolite composition of an extract which depends on the type of extraction, time of extraction, temperature, solvent, the concentration and the polarity of the solvent used.

Research was carried out on the effects of various drying methods of some medicinal plants such as Felicia muricata, Orthosiphon stamineus, Mentha spicata L.

(Diaz-Maroto et al., 2003), fresh and dried Phyllanthus amarus (Lim and Murtijaya,

2007), rosemary, oregano, marjoram, age, basil and thyme (Hossain et al., 2010), olive leaf (Martin and Molina, 2008) and Angelica sinensis. Results revealed that different drying treatments and conditions affect all phytochemicals analysed. Herbal preparation developed using oven drying was found to have inferior phytochemical content compared to freeze drying (Mahanom et al., 1999). The drying of Mentha spicata is an effective method that increases the shelf life of the final product by slowing the growth of microorganisms and preventing certain biochemical reactions that may alter the organoleptic characteristics (Diaz-Maroto et al., 2003).

2.8.1 Effect of Drying on the Proximate Composition, Minerals, Chlorophyll and Phenolics

Study by Hossain et al. (2010) reported that total phenols, and antioxidant capacity of six Lamiaceae herbs (rosemary, oregano, marjoram, age, basil and thyme) after three drying treatments (air drying, freeze drying and vacuum oven drying) stored for 60 days at 20°C compared to fresh samples was significantly (p <

0.05) different. Air-dried samples had significantly (p < 0.05) higher total phenols and antioxidant capacity than freeze-dried and vacuum oven-dried samples throughout the storage period. Fresh samples had the lowest values for the parameters tested. general from these works; oven drying methods might cause some variations in the amount and proportions of the components tested in the S. asper powdered leaves. From study by Martin and Molina (2008) also reported that drying tends to reduce the content of the analysed chemical compounds such as dry matter, organic matter, fiber and total nitrogen. Furthermore, heat treatment caused changes in the final product quality in terms of physical, chemical, organoleptic and nutritional (Vega et al., 2009). Abascal et al. (2005); Pinela et al. (2011) found that freeze drying gave better quality to medicinal plants during processing. Genin and

Rene (1995); Irzyniec et al. (1995) found that freeze drying is the best method of water removal as it gives a final product of the highest quality without heat compared to other methods of food drying.

Study by Nouri et al. (1954) reported that more total chlorophyll was obtained from the freeze-dried leaves of Mentha spicata, Podophyllum peltutum,

Ricinus communis than from the oven-dried leaves. They found that less chlorophyll

was retained in the oven-dried samples than in the freeze-dried samples; so that enzymatic activity was retarded to a greater extent in the freeze-dried samples than in the oven-dried ones, hence the less destruction and breakdown of chlorophyll in the freeze-dried samples. Study by Abascal et al. (2005) reported that plant medicinal from freeze drying extract such as Apium graveolens, Averrhoa bilimbi, Centella asiatica, Mentha arvensis, Psidium guajava, Sauropus androgynous, Solanum nigrum, Polygonum minus preserved more chlorophyll and Vitamin C compare than oven drying at 50 °C for 9 h or at 70 °C for 1 h. Their study showed freeze-dried asparagus and hyacinth leaves had higher antioxidant activity compared with oven- dried at 40°C method.

2.9 Cytotoxicity Study

Cancer is a class of diseases in which a group of cells display uncontrolled growth, invasion, and metastasis. Cancer may affect people at all ages, the risk increases with the age and it causes about 13% of all human deaths because of the abnormalities in the genetic material of the transformed cells. These abnormalities may be due to the effects of carcinogens (tobacco smoke, radiation, chemicals, or infectious agents) and errors in DNA replication, or inherited. A diet that rich in plant foods with a variety of secondary metabolites may protect the body against cancer. Secondary metabolites are known to have a wide range of protective functions in the human body including boosting the immune system, protect the body from free radicals, exhibit anti-carcinogenic and anticancer activity as well. Study by

Jang et al. (1997) reported that phytochemicals such as flavonoids and phenols are

known to have chemopreventive responses. Since S. asper was found to contain high amounts of phenolic composition, its cytotoxic properties need to be studied.

Table 2.3 shows the cytotoxicity studies of different plant parts of S. asper that has been characterised. Cytotoxicity screening models are the preliminary methods for selection of active plant extracts against cancer. Various cytotoxicty methods have been used to monitor to determine the anticancer effects of all plant extracts against a panel of cancer cell lines, such as MTS assay, Brine Shrimps lethality bioassay and Sulforhodamine B assay. The MTS cytotoxic assay has been most considered a valuable tool in studying cytotoxicity induced by chemical agents, for many years. It uses a tetrazolium salt, which is reduced to a coloured formazan product by mitochondrial dehydrogenases in living cells. It is frequently used and is reported to be suitable for in vitro drug screening tests (Maznah et al., 2012). Brine

Shrimps lethality bioassay is one of an excellent method for preliminary investigation of toxicity for screening medicinal plant (Gupta et al. 1996). This technique is inexpensive, utilize little amount of material and easy to perform, and is a useful tool for preliminary assessment of toxicity, for the detection of fungal toxins and plant extract toxicity (Kumar et al., 2011).

Previous studies have demonstrated on the antioxidant activity of Malaysian medicinal plants such as Casearia capitellata, Baccaurea motleyana, Phyllanthus pulcher and Strobilanthus crispus for their in vitro anticancer properties, using the

MTS assay, on four human cancer cell lines: colon (HT-29), breast (MCF-7), prostate (DU-145) and lung (H460) cancers. The results showed that anticancer activity was observed for the ethyl acetate extract of Casearia capitellata leaves on 61

MCF-7 cell lines with IC50 2.0 μg/mL and its methanolic extract showed an outstanding activity against H460 cell lines. Phyllanthus pulcher dichloromethane extract from aerial parts showed the highest anticancer activity against DU-145 cell lines, while significant activity was exhibited by dichloromethane extract of

Phyllanthus pulcher roots on HT 29 cell lines with IC50 value of 8.1 μg/mL (Maznah et al., 2012).

Table 2.3: Cytotoxicity studies of different plant parts of S. asper.

Plant part Cytotoxicity studies Method References Leaves Found that methanol extract of S. Brine Kumar et al., 2011 (methanol asper was weakly toxic Shrimps and (LC50≥500≤1,000 μg/mL) may be lethality petroleum due to the presence of cardiac bioassay ether glycosides and bioactive extracts) compounds, however petroleum ether extract of S. asper was non toxic ((LC50>1,000 μg/mL). Volatile Showed significant anticancer MTS assay Phutdhawong et oil leaves activity (ED50 << 30 μg/ml) from al., 2004 extract cytotoxicity primary screening tests with P388 (mouse lymphocytic leukaemia) cells

2.9.1 HT 29 Cell Lines

HT 29 (Fig. 2.13) cell line was derived in 1964 from the tumour of a 44-year- old woman with colon adenocarcinoma in 1964. HT 29 cells are human intestinal epithelial cells which produce the secretory component of Immunoglobulin A (IgA) and carcino embryonic antigen (CEA). The HT29 line is designated heterotransplantable, forming well-differentiated grade I tumours. The structure of

HT29 cells includes microvilli, microfilaments, mitochondria, smooth and rough

endoplasmic reticulum with free ribosomes, lipid droplets, limited primary lysosomes and many secondary lysosomes. The cells express urokinase receptors, but do not have detectable plasminogen activator activity. HT29 cells have been shown to be negative for CD4, but there is an expression of galactose ceramide.

HT29 cells are used for tumourigenicity studies. The line is considered a useful model system to study epithelial differentiation in vitro.

Fig. 2.13: The structures of HT 29 cell lines.

2.9.2 MTS Assay Principles

The Promega CellTiter 96® AQueous One Solution Cell Proliferation Assay

Boyd (2007) is a very easy to use, simple solution, colorimetric method used to determine the number of viable cells in proliferation or cytotoxicity assays. The one solution assay system contains a tetrazolium compound (MTS) and an electron

coupling reagent (PBS). PBS combines with MTS and confers enhanced chemical stability and allows the formation of a stable solution.

The principle of the assay is that the MTS tetrazolium compound is reduced in cells into a coloured formazan compound which is soluble in tissue culture media.

This conversion occurs via dehydrogenase enzymes which are present in live cells.

Cells are plated in a 96-well cell culture plate and allowed to grow over a pre- determined period of time, typically 48-72 hrs, in 100 µl of culture medium. At the end of the incubation period, the CellTiter 96® AQueous One Solution is thawed and

20 µl is added to each well. The absorbance is then read at 490 nm with a microplate spectrophotometer. The quantity of formazan produced when measured by absorbance at 490 nm is directly proportional to the number of viable cells in culture.

Typically, this reagent was used to determine the toxicity or anti-proliferative characteristics of a chemical entity. It should be noted that the reagent would also function well to determine the effect of a growth factor which increases growth. It is possible to determine the IC50 or ED50 of such an entity by plotting the concentration of the chemical entity against the absorbance at 490 nm.

CHAPTER 3

MATERIALS AND METHOD

3.1 Study outline

In this research, Streblus asper leaf (fresh, oven-dried and freeze-dried) were subjected to extraction using two different kinds of solvents (ethanol and water) to obtain the ethanol and aqueous crude extract. Both the crude extracts were then analysed for physicochemical and antioxidant, and cytotoxicity properties.

Phytochemical screening was carried out using standard phytochemical methods and the phenolic content was determined by analysing the TFC and TPC.

The details of the study outline are described by the following flowchart

(Figure 3.1).

S. asper leaves

Sample pre treatments

Fresh leaves sample Oven-dried powdered sample Freeze-dried powdered sample

Extractions

30% EtOH 50% EtOH 70% EtOH Aqueous

Analysis

Chemical Physical Antioxidant Phytochemicals Phenolics MTS Analysis Analysis Analysis Screening Analysis Assay

DPPH Color TFC TPC Proximate Assay GCMS (moisture, fat, protein, Minerals Chlorophyll saponin, alkaloid, steroid, tannin, Colorimetric ash & fiber) (Mg, Ca, K, (a,b & total Folin- chlorophyll) terpenoids, Ciocalteu P & Na) HPLC glycosides

Fig. 3.1: Flow chart of the study

3.2 Sample Preparation

Fresh leaves of S. asper were collected at Sungai Petani, Kedah. The samplings were started in January 2011 until Mac 2012. Botanical preparation of these species was carried out by a plant botanist, Mr. Shanmugan. Voucher specimen: no 11248 (Appendix A) was deposited in the herbarium unit of the School of Biological Sciences, Universiti Sains Malaysia (USM).

The fresh leaves of S. asper was weighed, cut into small pieces and washed several times under running tap water and finally rinsed with distilled water (dH2O).

The samples were fresh leaves, powdered oven-dried leaves and powdered freeze- dried leaves. For fresh S. asper leaves, extraction was carried out immediately. Oven and freeze drying were applied in this experiment to compare the effects of each drying process on antioxidant activity and phenolic properties of S. asper leaves.For freeze drying, S. asper leaves were dried with a freeze dryer at -50˚C under vacuum

(1.6 mm Hg) with a pressure of 1.1×10-2 mB for 72 h and then ground with a dry grinder to obtain fine powdered leaves. For oven drying, S. asper leaves were dried in an oven (Memmert, Germany) at 40˚C for 72 h (Tambunan et al., 2001) and then ground using the same procedure as that in the freeze-dried powdered leaves.The dried powdered leaves were sealed properly and stored in a freezer at -20˚C until used for further analysis (Amin and Tang, 2002; Satpathy et al., 2011).

3.3 Sample Extraction

All chemicals used in the following analysis were of analytical grade except for GCMS and HPLC whereby chemicals of LC and HPLC grades were used respectively.

The S. asper fresh leaves and dried powdered leaves were extracted with either water and ethanol - water; [(30:70, v/v), (50:50,v/v) and (70:30,v/v)] using a water bath at 40˚C, 60 rpm for 24 h. Each aqueous and ethanol extract was filtered through muslin cloth and centrifuged (Shimadzu LC-200 ) at 1500 rpm for 20 min.

The aqueous and ethanol extracts were then freeze-dried at -50˚C under vacuum (1.6 mm Hg) with a pressure of 1.1×10-2 mB for 3 days and then evaporated using a rotary evaporator at 40˚C until dry. The crude extracts were stored at -20˚C for further analysis (Amin & Tan, 2002; Satpathy et al., 2011).

3.4 Visual and Odour Observation of the Extracts

The powdered and fresh leaves extracts of S. asper was examined for colour, texture and odour in respective solvents for wet as well as dry conditions. According to the method by Nitin et al. (2010) organoleptic examination refers to the evaluation by means of the organ of sense; touch, vision and its odour.

3.5 Chemical Analysis

The chemical analysis which were determined include pH, proximate

(moisture, protein, crude fat, crude fibre, ash and total carbohydrate) and caloric value, minerals (calcium, phosphorus, sodium, potassium and magnesium), and chlorophyll (chlorophyll a, b and total chlorophyll).

Proximate and mineral analyses were part of the nutritional value of the leaf extract, while chlorophyll is significant as a food supplement in the production of nutraceutical products. pH measurement ensures optimal conditions of extracts. For colour, it is an important quality attributes of a plant product and in most herbal products the colour values are related to chlorophyll contents.

3.5.1 Proximate

3.5.1.1 Moisture

The method was carried out according to AOAC, 2005; Reference No.

935.29 – Oven Drying. The apparatus used were moisture dishes and cover, desiccator, oven 105˚C (Memmert UL40, Germany) and Analytical balance (Mettle, model: AE 200, Switzerland).

Firstly, a clean moisture dish and cover were weighed. Then, the fresh and dried powdered S. asper leaves were weighed (5 – 10 g) and spread evenly in the moisture dish. After that, the uncovered moisture dish was put in the oven at 105˚C.

The samples were dried overnight until a constant weight was obtained. Moisture dish containing dried samples were then covered and cooled in desiccators before reweighing.

The percentage (%) of moisture in the sample was calculated using the equation below:

% moisture = (Weight of wet sample – Weight of dried sample) × 100% Eq. 1.1 Weight of wet sample

Dried (moisture free) samples obtained were kept for crude fat analysis.

3.5.1.2 Crude Fat

The method was carried out according to AOAC, 2005; Reference No.

2003.06 – Soxhlet Extraction Method . The apparatus used were soxhlet extractor, extraction thimble, extraction flask, cotton (fat-free), heater, condenser and filter paper. Petroleum ether (boiling point 40˚C - 60˚C) was used as the solvent.

First, the fresh and dried powder of S. asper leaves were weighed (5 – 10 g) in the extraction thimble and covered with fat-free cotton. The extraction thimble was then placed in the soxhlet extractor. Extraction flask was weighed. Petroleum ether was poured into the soxhlet extractor until it siphoned into the boiling flask

(three times). Extraction flask, soxhlet extractor, condenser and heater were assembled. The extraction (reflux process) was allowed for 4 h. The endpoint of the extraction was confirmed by dripping the solvent onto a filter paper (no trace of oil).

The remaining petroleum ether in the boiling flask was evaporated (prevent from burning). The boiling flask was placed in the oven (105˚C). After an hour, the boiling flask was cooled in a desiccator. The samples were then weighed.

The percentage of crude fat was calculated using the equation below:

% Crude Fat (dry basis) = Weight of fat × 100 Eq. 1.2 Weight of dried sample

Dried and defatted samples obtained were kept for crude fibre analysis.

3.5.1.3 Crude Fibre

The method was carried out according to AOAC,2005; Method Reference

No. 978.10 – Neutralization Method. The apparatus used were 500 ml reflux flask, glass ball, ashless filter paper (Whatman No. 541), condenser, litmus paper, silica ashing dish, muffle furnace and hot plate (Gerhardt). The reagents were H2SO4 solution 5% (v/v), NaOH solution 40% (w/v), NaOH solution 25% (w/v), HCl solution 1% (v/v), blue litmus paper, methyl spirit and antifoam.

The dried and defatted samples were weighed (1 - 2 g) in the 500 ml reflux flask. 150 ml water, 50 ml of 5 % H2SO4, and 3 - 4 glass balls were added into the flask. The flask was attached to the condenser and refluxed for 30 min (count when the solution started to boil). 40 % of NaOH solution was added into the flask until the acid was neutralised (using blue litmus paper). Then, 10 ml of 25 % NaOH was

added and the mixture was refluxed for another 30 min (when the solution started to boil). After that, the mixtures were filtered through weighed the ashless filter paper.

The residue was washed using 1 % of HCl followed by hot water to wash away the acid using litmus paper. Then, the residues were washed using methylated spirit. The filter paper containing the residues was then transferred into silica ashing dish and were dried in an oven at 105˚C until constant weight. The silica ashing dish was then weighed after being cooled in the desiccators. It was later burned using a Bunsen burner until no fumes can be seen. Finally, the silica ashing dish was transferred into the muffle furnace until the absence of black particles can be seen. The weight of the crucible containing ash was weighed after being cooled in the desiccator. Percentage

(%) of crude fibre was calculated using the equation below:

% Crude Fibre = (S – K) – A × 100 Eq. 1.3 W where,

S = weight of ashing dish + ashless filter paper + residue (g)

K = weight of ashless filter paper (g)

A = weight of ashing dish + ashless filter paper + ash (g)

W = weight of dried and defatted sample (g)

3.5.1.4 Crude Protein

The method was carried out according to AOAC, 2005; Reference No.

976.05 – Micro-Kjeldahl Method. The apparatus used were digestion system PS

(Tecator), distillation Unit (Kjeltec System 1002, Tecator), micro-kjehdahl tube, 250 ml conical flask, litmus paper and filter paper nitrogen-free (Whatman No. 41). The reagents were copper sulphate (CuSO4), sulfuric acid (H2SO4) concentrated 95-98 % nitrogen free, 50 % of sodium hydroxide (NaOH) (w/v), methyl red-methyl blue indicator, 2 % of boric acid with methyl red-methyl blue receiver solution and standard HCl solution 0.02M.

The procedure consists of three steps;

(a). Digestion

About 0.3 g of fresh and dried powdered leaves were weighed on nitrogen- free filter paper and placed on micro-Kjehdahl tube. 2 ml of concentrated H2SO4 and

5 mg of CuSO4 were added into the tube. The tube was placed in the digestion system and the samples were digested until it becomes a clear solution. Then, the tube was taken out from the digestion system and cooled at room temperature.

(b). Distillation

The digested samples were transferred into the distillation unit with very minimum water. 10 ml boric acid was poured into a 250 ml conical flask. The tip of the condenser tube was immersed under the surface of boric acid solution. The distillation process was started and 50 % of NaOH was added slowly through a dispenser attached to the distillation unit. Distillation was continued until the colour of the solution changed from clear to dark brown. Finally, the clear blue solution

obtained was checked with red litmus paper. The tip of the condenser was then rinsed with dH2O.

(c). Titration

Distillate in the conical flask was titrated with standard 0.02M HCl. The distillate was titrated until the colour changed to purple.

*An empty nitrogen-free filter paper was used for blank and the same procedure was followed.

Percentage (%) of the crude protein was calculated using the equation below;

% (w/w) nitrogen :

ml HCl for sample - ml HCl for blank) × molarity HCl ×14 × 100% Eq. 1.4 Weight of sample (g)

% Crude protein (wet basis) = % nitrogen × factor 6.25

% Crude protein (dry basis);

100 × 100% crude protein (wet basis) Eq. 1.5 (100 - % moisture content)

3.5.1.5 Ash

The method was carried out according to AOAC 2005; Reference No.

900.02A – Drying Ashing Method. The apparatus used were electric muffle furnace

500˚C - 600˚C (Thermolyne Sybran, model: 6000, USA) and silica ashing dishes.

The silica ashing dish was heated using a Bunsen burner (~ 20 min). After that, the silica ashing dish was cooled and weighed together with its cover. 5 – 10 g of fresh and dried powdered samples were weighed in silica ashing dish and then it was burned using a Bunsen burner until no fumes can be seen. Then, the silica ashing dishes containing the residue were placed in a muffle furnace at 550˚C until no black particles can be seen. After that, the silica ashing dish was taken out from muffle furnace and cooled in the desiccator before being weighed. The percentage (%) of ash content was calculated as follows:

% Ash (wet basis) = Weight of ash × 100% Eq. 1.6 Weight of sample

% Ash (dry basis) = % ash (wet basis) × 100% Eq. 1.7 100 - % moisture content

3.5.1.6 Nitrogen Free Extract (NFE)

The amount of total carbohydrate ( percentage ) was calculated using the formula; expressed as the nitrogen free extract (NFE):

100 – [Ash (%) + Moisture (%) + Fat (%) + Protein (%) + Fibre (%)] Eq. 1.8

3.5.2 Caloric Value

Nutritive value was finally determined using the following formula and expressed in kilocalories (kcal) (Onyeike and Ikru, 1998; Imran et al., 2008).

[4 × Protein (%)] + [9 × Fat (%)] + [4 × Carbohydrate (%)] Eq. 1.9

3.5.3 Minerals

The method was carried out according to AOAC, 2005; Reference number

956.52 – Atomic Absorption Spectroscopy. The apparatus used were AA-

Spectrophotometer flame Perkin Elmer, hollow cathodes lamp and volumetric flask.

The reagents were concentrated HNO3, 30% H2O2 and standard stock solution of P,

Na, K, Ca and Mg.

The sample extractions were carried out according to the method of Shahidi et al. (1999) with slight modification. 10 g of ground samples of fresh and dried powdered leaves were subjected to dry ashing in a well-cleaned porcelain crucible at

550 °C in a muffle furnace until there was no evolution of smoke. The ash was dissolved in 5 ml of HNO3 /H2O2 (1:1) and heated gently on a hot plate until brown fumes disappeared. To the remaining material in each crucible, 5 ml of deionised water was added and heated until a colourless solution was obtained. The mineral solution in each crucible was transferred into a 100 ml volumetric flask by filtration through a Whatman filter paper and the volume was made to mark with deionised water. This solution was used for elemental analysis by AAS (AAS Flame Perkin-

Elmer, Model 3110).

Standard solution of each element was prepared and calibration curves

(Appendix B) were plotted for each element. The mineral content was expressed as mg per 100 g sample.

The concentration of minerals was calculated using the equation below:

Minerals (mg/100g) = Concentration of the mineral × final volume × dilution Weight of sample Eq. 1.10

3.5.4 Chlorophyll

Chlorophyll a, b and total chlorophyll were determined by spectophotometric method as described by Gao et al. (2000) with slight modification. The apparatus used were UV- Vis spectrophotometer, centrifuges, and filter paper (Whatman No.1).

The reagent used was 80% acetone.

100 mg of fresh and dried powdered leaves of S. asper was suspended in 10 ml 80% acetone until well mixed and kept in a refrigerator at 4˚C overnight in the dark. The mixtures were then filtered using filter paper and centrifuged at 4600 rpm for 4 minutes. The absorbance of the chlorophyll extracted was recorded at 645 and

663 nm for chlorophyll a, chlorophyll b and total chlorophyll.

The results were calculated by Arnon’s formula (Arnon, 1949) as below:

Chlorophyll a (mg/g) = 12.7 (D663) - 2.69 (D645) × V Eq. 1.11 1000 x W

Chlorophyll b (mg/g) = 22.9 (D645) - 4.68 (D663) × V Eq. 1.12 1000 x W

Total chlorophyll (mg/g) = 20.2 (D645) + 8.02 (D663) × V Eq. 1.13 1000 x W

where,

D = optical density

V = final volume of 80 % acetone (ml)

W = dry weight of sample taken (g)

3.5.5 Preliminary Phytochemical Screening

3.5.5.1 Qualitative Screening of Alkaloid, Steroids, Terpenoids, Tannins,

Saponin and Cardiac Glycoside

A qualitative phytochemical test was used to detect the presence of alkaloid, steroids, terpenoids, tannins, saponin and cardiac glycoside according to standard methods as described by Harborne (1998) and Aguinaldo et al. (2005). The apparatus used were water bath and filter paper (Whatman No.1) and the reagents used were methanol, 1% HCl, Mayer’s reagent, chloroform, concentrated H2SO4, acetic anhydride, 0.1% FeCl3.

3.5.5.1.1 Alkaloids

About 20 mg of leaf extract was added to 10 ml methanol and placed in a sonic bath to dissolve. The extracts were then filtered using a Whatman No.1 filter paper, 2 ml of filtrate was taken and mixed with 1% HCl. To 1 ml of this mixture, 6 drops of Mayer’s reagent were added. Within a few mins, the presence of alkaloids indicates a yellow-creamish precipitate colour.

3.5.5.1.2 Steroids

About 20 mg of leaf samples were mixed with 1 ml methanol and filtrated.

Then, 1 ml chloroform and 1 ml concentrated H2SO4 were added into the filtrate in which a yellow green fluorescent indicates the presence of steroids.

3.5.5.1.3 Terpenoids

About 10 ml chloroform was added into 20 mg of leaf samples. This was followed by the filtration process, whereby 2 ml acetic anhydride and concentrated

H2SO4 were added into the filtrate. The presence of terpenoids is indicated by a blue green ring which appears on top of the mixture.

3.5.5.1.4 Tannins

About 0.5 g of leaf sample was boiled in 20 ml of distilled water in a test tube and then filtered. 0.1% FeCl3 was added to the filtered samples and observed for brownish green or a blue black colouration, which shows the presence of tannins.

3.5.5.1.5 Saponin

About 2 g of the leaf samples was boiled in 20 ml of distilled water in a water bath and filtered. 10 ml of the filtrate was mixed with 5 ml of distilled water and

shaken vigorously for a stable persistent froth. The frothing was shaken vigorously and then observed in the formation of emulsions.

3.5.5.1.6 Cardiac Glycosides

About 100 mg of the extract was dissolved in 1 ml of glacial acetic acid containing one drop of ferric chloride solution. This was then underlayer with 1 ml of concentrated sulphuric acid. A brown ring obtained at the interface indicated the presence of a de-oxy sugar characteristic of cardenolides.

3.5.5.2 Screening of Volatile Compounds by GCMS

The method was adapted from Medini et al. (2009); Kumar et al. (2010);

Rahimmalek and Goli (2012) with modifications. The apparatus used was GCMS

(Agilent 7890A/ 5975CGC System). The reagent was methanol LC grade.

Chromatography analysis of S. asper extracts was carried out using GCMS

(Agilent 7890A/5975C GCMS System) equipped with capillary HP-5-MS column, auto sampler/autoinjector (HP-7683B) using helium gas. For gas chromatography conditions, inlet temperature was set to 250°C with splitless mode. 1 µl of the sample was injected into the GC/MS system. Flow was set to 1.0 ml/min and held for 5 min.

The initial oven temperature was set at 40°C, increased to 150°C and kept increasing to 280°C and then maintained for 5 min. Total run time was 53 min (Table 3.1).

MSD Transfer Line Heater was set to 260°C. For mass spectrometer conditions, scan

parameters was set from 35.00 to 800.00 a.m.u. Solvent delay was set for 3.00 min.

MS source temperature was set at 230°C and the MS quadrupole temperature was set at 150°C. Chromatograms that have been obtained from the analysis were analysed using MSD Chemstation Data Analysis software using NIST02 library. Each mass spectrum that has been obtained was compared to NIST02 library. Each mass spectrum was given the most matching compounds contained in the NIST library

(NIST, 2005; Medini et al., 2009).

Table 3.1: Oven condition of GCMS.

Rate Value ˚C Hold time Run time ˚C/min (min) (min) 40 0 0 5 150 0 22 5 280 5 53

3.6 Physical Analysis

3.6.1 Colour Measurement

The apparatus used was Minolta Spectrophotometer (CM-3500d Model).

First, the spectrophotometer was calibrated with an empty dish (CM-A120) followed by white calibration using white opaque dish. Then, the colours of the samples were measured. The colour of the fresh leaves, oven and freeze-dried powdered of S. asper leaves were determined by using a colorimeter. The colour of the powdered oven-dried, freeze-dried and fresh leaves of S. asper was compared. L*, a* and b* values were obtained from the colorimeter.

CIE (Appendix C) is a standard illuminant to determine CIE colour space coordinates; L*, a*, b*, C* and h values. Lightness value, L* measures the whiteness value of a colour and ranges from black at 0 to white at 100. The chromaticity coordinates a* measured greenness/redness (varying from −60 to + 60); where red and green were positive and negative respectively. The chromaticity coordinates b* measured the grade of blueness/yellowness (also varying from −60 to + 60) where yellow when positive and blue when negative. The chromatic (C*) measured perpendicular distance from the lightness axis (more distance being more chroma).

The hue angle (h) is expressed in degrees, with 0° being a location on the +a* axis, then continuing to 90° for the +b* axis, 180° for -a*, 270° for -b*, and back to 360°

= 0°. (Doymaz et al., 2006; Soysal, 2004).

3.7 Extract Yield Determination

The extracts of aqueous, 30% EtOH, 50% EtOH and 70% EtOH of S. asper leaves obtained were evaporated to dryness by rotary evaporator (Allerod, Denmark) with vacuum at 40°C. The extract yield was defined as the amount of dried extract

(g) obtained from 50 g of DW of S. asper leaves.

3.8 Antioxidant Activity

UV-Vis spectrophotometer and the reagent 1, 1-diphenyl-2-picrylhydrazyl

(DPPH) and 70% Ethanol were used for scavenging activity.

About 5 ml of DPPH in 70% ethanol was prepared and added into 0.2 ml of each sample extract. The extracts were mixed well and kept in the dark for 45 min.

The decrease in the absorbance was monitored at 517 nm (A517 extract). The absorbance of a control (distilled water instead of sample extract) was recorded after

1 min at the same wavelength (A517 control) (Kwon et al., 2006). The percentage of activity was calculated using the formula as below: (Yen and Duh, 1994).

% Activity = A517 (control) - A517 (extract) × 100 Eq. 1.14 A517 (control)

The inhibition concentration of sample required to scavenge DPPH radical by

50% (IC50) was obtained from the calculated graph plotting between % inhibition versus concentration. BHA was used as standard. The DPPH solution was prepared freshly during analysis which was stored and kept in a flask covered with aluminium foil at 4°C between the measurements in the dark. All determinations were performed in triplicate. A stock solution of BHA was prepared by dissolving 10 mg of BHA in ethanol and making up the volume to 10 ml with methanol to get the final concentration of 1mg/ml.

3.9 Analysis of Phenolics

3.9.1 Total Phenolics Content

3.9.1.1 Folin-Ciocalteu Method

The total phenolic content (TPC) of extracts was measured using the Folin -

Ciocalteu method by Chew et al. (2011) with slight modifications. The apparatus used was UV-Vis spectrophotometer and the reagent was 10 % Folin-Ciocalteu’s phenol reagent (Sigma) and 7.5 % Na2CO3.

About 1.5 mL Folin-Ciocalteu’s phenol reagent (Sigma) (10% v/v) and 1.2 mL 7.5% w/v Na2CO3 were added to 0.3 mL sample extracts. The extracts were oxidised with Folin-Ciocalteu reagent, and the reaction was neutralised with Na2CO3.

The reaction mixtures were thoroughly mixed and incubated in the dark for 30 min before the absorbance was measured at 725 nm. The mean (±SD) results of triplicate analyses were expressed as mg of gallic acid equivalents per gram of dry extract (mg

GAE/g). The calibration equation for gallic acid was y = 0.116x (R2 = 0.992), where x was the gallic acid concentration in mg/ml and y was the absorbance reading at 725 nm. The total content of phenolic compounds. Gallic Acid Equivalents (GAE) was calculated by using the formula below:

T = C: V Eq. 1.15 M

where;

T= total content of phenolic compounds, milligram per gram of plant extract, in

GAE; C= the concentration of gallic acid established from the calibration curve, mg/ml; V= the volume of extract, ml; M= the weight of plant extract, g (Adesegun et al., 2007). A stock solution of gallic acid was prepared by dissolving 10 mg of gallic

acid in 70% EtOH and making up the volume to 10 ml with 70% EtOH to get the final concentration of 1mg/ml.

3.9.1.2 HPLC

HPLC Agilent Technologies 1200 Autosampler series was equipped with a

DAD 1100 photodiode array detector. The reagent used were acetic acid, acetonitrile and gallic acid. All solvents used during the HPLC study were HPLC grade having a purity of > 99.5%.

The extracts (5 ml) were filtered through a 0.20 µm filter. A volume of 20 µl of samples was injected using an Agilent Technologies 1200 Autosampler series

HPLC equipped with a DAD 1100 photodiode array detector. The solvents used for gradient elution were (A) 100% methanol and (B) water-acetic acid 25:1, v/v. The methanol concentration was increased to 50% for the first 5 min and gradually to

80% for the next 10 min (total run time, 11 min) (Table 3.2). The analytical column used was Agilent (250 X 4.6 mm) with packing silica material of 5 µm particle size; the flow rate was 1ml/min at room temperature. During each run the absorbance was recorded at 260 nm and 280 nm. The chromatogram was integrated using Agilent

Chemstation enhanced integrator. Pure standards of gallic acid in 100 % methanol were used to calibrate the standard curves and retention times (modified method from Weerasak and Suwanna, 2007).

Table 3.2: The experimental condition of HPLC.

Time (min) A B Flow (ml/min) 0 100 100 1.000 5 50 50 1.000 10 80 20 1.000

For the quantitative analysis of 30 % EtOH, 50 % EtOH, 70 % EtOH and aqueous from S. asper leaf extracts, the calibration curves for gallic acid was prepared by plotting peak area vs. concentrations as attached in Appendix D [Fig.

5.5(a)].

3.9.2 Total Flavonoids Content

3.9.2.1 Colorimetric Method

Total flavonoids content of S. asper extracts were determined by using the colorimetric method as described by Sakanaka et al. (2005). The apparatus used was

UV-Vis spectrophotometer and the reagent used were 5 % sodium nitrite solution,

10% aluminium chloride, and NaOH.

The extract of 0.25 ml was mixed with 1.25 ml of distilled water in a test tube, followed by the addition of 75 μl of a 5 % sodium nitrite solution. After 6 min,

150 μl of a 10 % aluminium chloride solution was added and the mixture allowed to stand for a further 5 min before the addition of 0.5 ml of NaOH. The mixture was made up to 2.5 ml with distilled water and mixed well. The absorbance was measured immediately at 510 nm. The mean (±SD) results of triplicate analyses were

expressed as mg of quercetin equivalents per gram of dry extract (mg QE/g). The calibration equation for quercetin was y = 0.043x + 0.024 (R2 = 0.980), where x was the quercetin concentration in mg/ml and y was the absorbance reading at 510 nm.

The total content of flavonoid compounds in the extract in Quercetin Equivalents

(QE) was calculated using the formula below:

T = C: V Eq. 1.16 M

where;

T=total flavonoid contents, milligram per gram of plant extract, in QE; C= the concentration of quercetin established from the calibration curve, mg/ml; V= the volume of extract, ml; M= the weight of plant extract, g (Adesegun et al., 2007). A stock solution of quercetin was prepared by dissolving 10 mg of quercetin in 95%

EtOH and making up the volume to 10 ml with 95% EtOH to get the final concentration of 1mg/ml.

3.9.2.2 HPLC

Method as described in section 3.9.1.2.

For the quantitative analysis of 30 % EtOH, 50 % EtOH, 70 % EtOH and aqueous from S. asper leaf extracts, the calibration curves for quercetin was prepared by plotting peak area vs. concentrations as attached in Appendix D [Fig. 5.6(a)].

3.10 Cytotoxicity Analysis

3.10.1 Materials and Reagents

Established colon carcinoma cell lines (HT 29) were obtained from

Advanced Medical and Dental Institute (AMDI) lab, Universiti Sains Malaysia,

Penang. Reagents and materials used were RPMI 1640, HCl, L-glutamine, phosphate buffered saline (PBS), penincillin, tryspin xpress, fetal bovine serum (FBS), and cellTiter 96® aqueous one solution cell proliferation assay (MTS) kit (Promega). All reagents used were in analytical grades.

3.10.2 Sample Extractions

The fresh leaves of S. asper was weighed, cut into small pieces and washed under running tap water and dried in the oven under 40˚C. The dried powdered of leaves were blended where 50g were macerated with 590 ml of pure water and 70%

EtOH. The dried powdered were extracted with aqueous and 70% EtOH using a water bath at 40˚C, 60 rpm. After 24 h, the mixture was centrifuged at 1500 rpm for

20 min. The supernatants were freeze-dried at -50˚C under vacuum (1.6 mm Hg) with a pressure of 1.1×10-2 mB for 3 days for aqueous extract and evaporated at 40˚C until completely dry for 70% EtOH. The crude powder extracts obtained were stored in the fridge at -20˚C for further analysis (Hazwani et al., 2010).

3.10.3 Preparation of Medium

For the preparation of medium, 1L powder of RPM-1 1640 and 2 g of sodium bicarbonate was dissolved in 900 ml ultra pure water. The pH of the solution was adjusted to 7.1 using 1 N hydrochloric acid and 1 N sodium hydroxide, monitored by a pH meter followed by a top up of ultra pure water to make the final volume of solution to 1000ml. The solution was filtered with 1L disposable filter unit with vacuum pump. Filtration step was performed in a sterile condition using a Biohazard safety cabinet class II. 5 ml of the filtered solution was aliquoted in a 25cm2 culture flask and kept in a CO2 incubator for 24 h. The remaining was kept in a refrigerator at 4°C. After 24 h of incubation, the solution was checked for any sign of contamination under a microscope with transmitted light illumination. Signs of contamination are cloudy solution using naked eyes and poor background vision under microscope. Then, contamination-free solution was added with 10% of fetal bovine serum, 1% penicillin and 1% of L-glutamine. The final solutions were filtered and mixed well, forming a complete RPMI 1640 medium. In order to maintain its quality, the complete medium was stored in the refrigerator at 4°C.

3.10.4 Subculture of HT 29

HT 29 cell lines were washed several times with PBS. The pellet containing cells was mixed with complete medium by re-suspension and then sub-cultured in

125 cm2 flasks containing 15 ml of complete medium. The cells were incubated in a

CO2 incubator at 37°C in a humidified atmosphere 5% CO2/95% O2. Cells condition was continuously checked for the presence of any contamination. Ninety percent confluent cells were sub-cultured into a new flask on a regular basis. The cells were counted using haemocytometer and the concentration of the cells used was 1 x 105 cells/ml.

3.10.5 Enumeration of Cells

HT 29 cells were detached from cell culture flask using trypsin Xpress solution and incubated several minutes in a CO2 incubator. Then, the cells were centrifuged at 1000 rpm for 7 min. Supernatant was discarded and pellet containing cells were resuspended with a complete medium. Cells were added with trypan blue and counted using a haemocytometer. Trypan Blue is a vital dye. Live, healthy cells unstained by trypan blue were counted. The reactivity of trypan blue is based on the fact that the chromopore is negatively charged and does not interact with the cell unless the membrane is damaged. All the cells which exclude the dye are viable.

Therefore, the cell concentration per ml (and the total number of cells) was determined using the following calculations.

Cells per ml = the average count per square x the dilution factor x 104 (count

10 squares) Eq. 1.17

3.10.6 Sample Preparation

The stock solution of 70% EtOH extract was prepared by dissolving 200 mg of S. asper in 1 ml 70% EtOH as for aqueous extract, preparation was done by dissolving 20 mg and 5 mg of S. asper leaves in 1 ml of ultra pure water.

3.10.7 MTS Assay

The method was modified from Padma et al. (2006). Complete medium was added into respective wells of 96 well-plates for 24 h. The cell line was grown and maintained in RPM-1 1640 culture medium. Cytotoxic analysis of the cells was determined using the MTS assay. The range of the concentration of sample extracts used was 15.63, 31.25, 62.5, 125, 250, 500 and 1000 µg/ml for triplicate assay. Cells were treated with the extracts for 24 h, 48 h, 72 h and cytotoxic effects were determined by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-

(4-sulfophenyl)-2H-tetrazolium (Sigma Chemical Co.) based on colorimetric assay.

This method depends on the cleavage of tetrazolium salt to purple formazan crystals by mitochondrial enzymes of metabolically active cells. Briefly; 4 h before the end of each incubation period, the medium of the cells was removed and the wells were washed by pre-warmed PBS to remove any trace of extracts and to prevent colour interference while optical density determination. The MTS stock solution was diluted at a 1:20 ratio into complete culture media, 20 μl of MTS dilution was added into each well and incubated. Cell viability was measured using a microplate reader with

490 mm test wavelength. The results were represented as percentage viability and calculated using the following formula:

Cell viability (%) = (Absorbance of treated cell / Absorbance of untreated cell) × 100

Eq. 1.18

The IC50 which inhibits cell proliferation by 50% was then being calculated graphically.

3.11 Statistical Analysis

All measurements were carried out in triplicates (n=3). Statistical analyses were performed using a one-way analysis of variance ANOVA, and the significant differences between means were determined by Duncan’s multiple range tests.

Differences at p < 0.05 were considered statistically significant. The results were presented as mean values ± SD (standard deviations).

CHAPTER 4

RESULTS AND DISCUSSION

4.1 Visual and Odour Observation of Extract

The colour, texture and odour of the plant extracts in different solvents in both wet and dried conditions were characterized in Table 1. Dried extracts obtained generally appeared darker and more turbid than the wet extracts.

Table 1: Visual and Odour Observation of S. asper leaves

Plant name Plant Wet extract Dried extract Method Solvent Code (Latin) part Colour Texture Odour Colour Texture Odour

Macerate, Murky, pale Slightly foamy Golden Sticky, Unpleasant Water AE Leafy smell 24h orange when shaken brown resinous smell

Light Shiny, Macerate, 70% 70% Clear, light Water-like Strong Unpleasant green, slightly 24h Ethanol EtOH green consistency alcoholic smell Leaf part glistening waxy S. asper Dark Macerate, 50% 50% Shiny, Unpleasant Clear, dark Not foamy Alcoholic yellowish 24h Ethanol EtOH thick smell green green

Macerate, 30% 30% Pale Less Yellowish Unpleasant Viscous Sticky 24h Ethanol EtOH brownish alcoholic brown smell

4.2 Chemical Analysis

4.2.1 Proximate

The proximate composition of S. asper leaves produced by freeze drying and oven drying and fresh leaves are presented in figure 4.1. There were significant differences (p < 0.05) for all the proximate components except for crude fat and crude fibre between the samples. Overall, the S. asper fresh leaves had higher concentrations of most of the proximate parameters compared to oven-dried and freeze-dried powdered leaves. Moisture was highest in fresh leaf of S. asper with

5.2% followed by oven-dried and freeze-dried S. asper leaves with 0.75% and 0.44% respectively. There were significant differences (p < 0.05) in moisture content of the treated samples. Dried powder from freeze-dried samples had the lower moisture content compared to oven-dried samples. Crude fat content of the S. asper fresh leaves recorded the highest value (2.78%) compared to oven-dried (2.13%) and freeze-dried powdered leaves (1.94%).

For protein, results showed that fresh S .asper (13.83%) had the highest concentration of protein as compared to rest of the leaves. Freeze-dried and oven- dried powdered leaves contained 12.50% and 11.40% protein respectively. Ash content, which is an index of mineral contents showing that there were significant (p

< 0.05) differences in the ash contents among the fresh sample which recorded the highest amount (8.38%) compared to freeze-dried (7.65%) and oven-dried (7.19%) ones. Nitrogen free extract of oven-dried powdered leaves was significantly (p <

0.05) higher (56.23%) compared to freeze-dried powdered leaves (52.07 %) and fresh leaves (44.37%).

Fig. 4.1: Comparative percentage proximate composition of S. asper leaves by different drying methods. The reported values are the means ± S.D. (n=3). FLa: fresh leaves as is basis; FLdw: fresh leaves dry weight; FDa: freeze drying as is basis; FDdw: freeze drying dry weight; ODa: oven drying as is basis; ODdw: oven drying dry weight; NFE: nitrogen free extract.

The highest proximate component of S. asper leaves were carbohydrate. This agrees with other publication which showed that herbs are rich in total carbohydrate

(Manay, 1987). The carbohydrates are the main source of energy store that enhance substances for biological synthesis of many compounds. Caloric value of oven-dried powdered leaves was highest (378.81 kcal) followed by freeze-dried powdered leaves (377.34 kcal). The fresh leaves were found to contain less calories (367.58 kcal). Thus, S. asper leaves could be considered as a rich natural source of valuable

nutrients such as carbohydrates, fibres and proteins. This suggests that the leaves can be used as a nutraceutical because the leaves have some nutritional value when taken as food or potent medicinal properties when used as a food supplement.

The result is comparable to Krishnurthy et al. (2010) who reported that the proximate content of oven-dried Withania domnifera, a medicinal plant were; moisture 7.30%, ash 3.17%, fat 1.13%, fibre 5.00%, protein 0.61% and carbohydrate

24.34%. The value obtained for the moisture, protein, fibre, ash and NFE are also comparable with values reported for oven-dried Azadirachta indica (neem) leaves which were 3.5%, 13.42%, 20.11%, 6.00% and 51.94% (Atangwho et al., 2009;

Onyimonyi et al., 2009) respectively. Study by Shahid et al. (2012) confirmed the moisture content of Morus alba, M. nigra and M. rubra leaves with a lower range from 4.5 – 6.7% may contribute towards the roughness of the leaves since S. asper leaves also were very rough on both surfaces. The low moisture contents may reduce the chance of microbial growth and improved shelf life (Egharevba et al., 2010).

S. asper leaves also contain low fat; according to Antia et al. (2006), fat in sweet potato leaves play a role in retaining and absorbing flavours thus increasing its palatability. As the amount of protein from fresh and freeze-dried S. asper leaves were more than 12%, it can be considered as a good source of protein as reported in the literature by Pearson (1976). Adequate intake of fibre was shown to be beneficial in reducing cholesterol level, diabetes, risk of coronary heart disease, hypertension, colon and breast cancer (Rao and Newmark, 1998; Ishida et al., 2000) and improve

digestion and absorption processes (Ogungbenle, 2003). Study by Shahid et al.

(2012) found that fibre is known as an anti-nutrient, as it has the potential to obstruct the utilization of minerals. Similarly finding by Betancur et al. (2004) confirm that chemical substances in plants including protein, carbohydrate, vitamin and fibre also contribute to the antioxidant capacity.

According to the result, the ash content for all treatment, even in the fresh leaves was about 7.19-8.38% which implies that the S. asper leaves have a good organic or low inorganic and mineral constituent components. The value of ash and protein obtained in this study is agreeable with the earlier research by Chumpawadee and Pimpa (2009) who noted that the ash values of Siamese rough bush (S. asper) leaves were 10.7 % and 12.0 % respectively. The ash content of S. asper leaves contained nutritionally important mineral elements. There was a significant (p >

0.05) increase in ash content between fresh, oven-dried and freeze-dried leaves.

Mineral elements are essential in daily requirement and tissue functioning in our body, therefore it is essential to determine the mineral content of the S. asper leaves.

4.2.2 Determination of Mineral Content

The fresh and dried powdered S. asper leaves from different drying treatments are summarized in figure 4.2. Results revealed that fresh leaves had significant (p < 0.05) different compared with treated leaves. For oven-dried and freeze-dried powder of S. asper leaves extract the result found that there was no

significant different. The fresh leaves of S. asper was found to be rich in P

(85.5±5.29 mg/100g) compared to oven-dried and freeze-dried powdered leaves with

1.9 mg/100g. Study by Roberson et al. (2007) found that the timing and extent of plant freezing and drying would influence the potential for P losses in alfalfa. For fresh leaves of S. asper, the concentration of various elements analysed in the present work decreases in the order of ; P > Na > K > Ca > Mg.

Fig. 4.2: Macronutrient composition of S. asper leaves by different drying methods. Bars with different letters are significantly different (p < 0.05). The reported values are the means ± S.D. (n=3). FL: fresh leaves; FD: freeze drying; OD: oven drying; P:phosphorus; Na:sodium; K:potassium; Mg:magnesium; Ca:calcium.

The level of Na had higher in freeze-dried leaves (20.8±1.53 mg/100g) followed by oven-dried (20.0±1.53) and fresh leaves of S. asper (15.8±1.53). The

concentration of K decreased significantly (p < 0.05) in the following order; oven- dried (20.0±0.58) < freeze-dried (17.9±1.00) < fresh leaves (7.6±1.37). Freeze-dried

S. asper leaves had the highest concentration of Mg (2.5±0.58) while oven-dried and fresh leaves had 2.3±1.00 and 1.2±1.00 of Mg respectively. For Ca content, the order decrease as follows: oven-dried powdered leaves, 25.3±0.58 mg/100g > freeze-dried powdered leaves, 24.3±0.58 > fresh leaves, 7.3±2.08. These results are in agreement with the studies done by Ozcan et al. (2005) that Ca content in oven-dried basil herb was higher than fresh basil herb because of the increase of dry matter content. Joshi and Mehta (2010) also showed that the Ca content of M. oleifira leaf had higher in oven-dried followed by sun-dried and fresh leaf samples.

Generally, the mineral content of fresh leaves were lower compared to treated leaves since the minerals in dried samples were in concentrated forms after the removal of water. The high ash content is a reflection of the mineral contents found in the plant materials. A higher deposit of mineral elements is found in medicinal leaves (Antia et al., 2006). The results of the mineral composition clearly indicate that S. asper leaves contain a rich source of mineral elements in preventing several diseases. This becomes important when the usefulness of such minerals like phosphorus (P), sodium (Na), potassium (K), magnesium (Mg) and calcium (Ca) in the body is considered. Minerals are an important part of the diet, even though it comprised only 4–6% of the human body (Ozcan, 2004). Major minerals such as Ca,

P, Mg, Na and K are required in amounts greater than 100 mg per day (Ozcan, 2004) which serve as structural components of tissues, in cellular function, basal

metabolism, water and acid-base balance (Macrae et al., 1993). The intakes of elements such as Na, K, Mg and Ca could reduce individual risk factors such as cardiovascular disease.

Among the minerals of S. asper fresh leaves, P was the highest, while Mg was the lowest. Oven-dried and freeze-dried powder of S. asper leaves contained highest amount of Ca and the lowest amount of P, with significant (p < 0.05) differences on Ca between different drying treatments (p < 0.05). Higher amounts of

K and Ca in oven-dried extracts were found in S. asper leaves. This result was confirmed by Abascal et al. (2005) who found that oven drying resulted in the greatest nutritional loss but preserved the highest mineral content. The biological roles for K and Ca are essential in preventing and controlling diseases, therefore it may contribute to the traditional medicinal influences of the plants (Aliyu et al.,

2008). K is essential as an activator for enzymes involved in the synthesis of certain peptide bonds (Alabi and Alausa, 2006); important for diuretic, maintain tissue excitability and ionic balance in the human body (Tapan, 2011). While Na plays an important role in the transport of metabolites (Tapan, 2011). The ratio of K/Na is an important factor in the prevention of hypertension and arteriosclerosis with K depressing and Na enhancing blood pressure (Saupi et al., 2009). This is strongly supported by the study of Gaitonde et al. (1964) who reported that S. asper leaves have an activity on anti-hypertension.

Ca was found to be the most abundant mineral in oven-dried powder of S. asper leaves. P and Ca have been reported to be good for blood clotting, muscle contraction, strong bone, and for infants in the development of bones and teeth formation (Ogungbenle, 2003). The results indicated that there was a variation in concentration among the samples in terms of minerals. These results were supported by Derya and Musa (2008); Zielisnki and Koslowska (2000); Miranda et al. (2009) that the changes in the concentrations of minerals were dependent on the method of treatment, the drying temperature, and exposure time to sunlight and rain.

Furthermore, no significant (p < 0.05) differences were encountered for the P and Mg compositions between oven-dried and freeze-dried S. asper leaves. Mg was found least in all samples. Lack of Mg may cause abnormal irritability of muscle.

Thus, Mg is required in enzyme-catalysed reactions, plasma and extracellular fluid which help in maintaining osmotic equilibrium in the human body (Indrayan et al.,

2005). Even though mineral contents of S. asper leaves were found in small amount but it may be very important to our health. Therefore, such amounts obtained from minerals showed that S. asper leaves could provide the variable secondary metabolites that may help in preventing illness. No other works in the literature have been found regarding mineral composition of S. asper leaves. S. asper leaves could serve as a good booster in body and immune system.

4.2.3 Chlorophyll

The estimated chlorophyll contents of S. asper leaves revealed that fresh leaves had higher chlorophyll a (Ca), chlorophyll b (Cb) and total chlorophyll (Tc) compared to the oven-dried and freeze-dried powdered leaves (Fig. 4.3). For fresh leaves of S. asper, the Ca, Cb and Tc were 0.52±0.03, 0.23±0.04 and 0.72±0.01 mg/100g respectively. The values obtained after drying treatments ranged from 0.27-

0.41±0.16, 0.10-0.18±0.05 and 0.37 - 0.68 mg/100g for Ca, Cb and Tc. In this case, oven drying treatments had significant (p < 0.05) effect on the chlorophyll content, whereby the chlorophyll contents of fresh leaves and freeze-dried powdered leaves were found to be very close to each other, with no significant ( p>0.05) differences.

As chlorophyll is an important component, comparisons on chlorophyll a, b and total chlorophyll in S. asper leaves have been made to observe differences between fresh leaves and dried leaves as illustrated in figure 4.3. Results indicated that there was significant (p < 0.05) effect on chlorophyll content as a result of the drying treatments. There was no significant difference in chlorophyll content between the fresh and freeze-dried powdered leaves of S. asper. However, there was a significant (p < 0.05) decrease in the chlorophyll content in oven-dried powdered leaves compared to fresh and freeze-dried samples. These findings correlated with the results reported by Venskutonis (1997) that freeze drying treatment has been found to retain the features that are closer to the characteristic appearance of the fresh one. Therefore, freeze drying proved to preserve more chlorophyll contents

(Chan et al., 2009; Pinela et al. 2011) than oven drying. Similarly, degradation of chlorophyll has been reported to occur because of the heat treatment (Rahimmalek

and Goli, 2012; Weemaes et al., 1999). Other findings by Nouri et al. (1954) also indicated that more total chlorophyll was obtained from the freeze-dried leaves of

Mentha spicata. Freeze-drying facilitated total chlorophyll extraction.

Fig. 4.3: Effects of different drying methods on Ca, Cb and Tc of S. asper leaves. Bars with different letters are significantly different (p < 0.05). The reported values are the means ± S.D. (n=3). FL: fresh leaves; FD: freeze drying; OD: oven drying; Ca: chlorophyll a; Cb: chlorophyll b; Tc: total chlorophyll.

Leaf chlorophyll content will provide valuable information about the physiological status of the plants itself. Chlorophyll seems to have an important role in health in which it can bring the essential nutrients to human haemoglobin, the human cell, decrease blood sugar, indigestion, excretion and lowering the allergens

(Buavaroon et al., 2011) helps in protecting gastric mucosa cells, in detoxification in the liver and kidney, lower blood sugar (Kimura et al., 2007) and also helps in increasing body immune system. Another finding also indicated that the chlorophyll

consumed by human oral intakes will increase the level of heme production and chlorophyll can be absorbed in the small intestine totally (Buavaroon et al., 2011).

In addition, chlorophyll also gives an indirect estimation of the nutrient values; where leaf nitrogen is much incorporated into chlorophyll molecules (Filella et al., 1995). The importance of chlorophyll a and b are derived from their part in the green colour; but, it is easy to degrade during processing, storage and also depends on the temperature (Monreal et al, 1999; Heaton and Marangoni, 1996). Similarly, heat treatment might cause the loss of green colour by chlorophyll degradation in the leaf cells (Arslan and Ozcan, 2011). Therefore, the colour of the leaves needs to be analysed in order to maintain their chlorophyll values. So far, there is no information about S. asper leaves chlorophyll contents effect from various drying treatments found in the literature.

4.2.4 Phytochemical Analysis

4.2.4.1 Preliminary Phytochemical Screening of S. asper Leaves

The preliminary phytochemical analysis of fresh, oven-dried and freeze-dried powder of S. asper leaves showed the presence of alkaloids, saponin, tannins,

terpenoids and cardiac glycosides. The results are summarised in Table 4.2. The dried powder and fresh leaf of S. asper was proven to contain alkaloids, saponins, tannins, terpenoids and cardiac glycosides. No steroid was detected.

Phytochemical screening provides a preliminary guide to the possible use of medications of plants. Recently, more attention has been focused on the standardisation of medicinal plant materials for their therapeutic potentials such as anticancer, antioxidant, antimicrobial and anti-inflammatory activities. They are known to exhibit medicinal activity as well as physiological activity (Sofowora,

1993). These phytochemicals may be present in trace amounts and are important to health. Hence in this study, the major groups of phytochemicals such as alkaloids, saponins, tannins, terpenoids and cardiac glycosides from S. asper plant leaf was studied qualitatively.

Table 4.2: Preliminary phytochemicals screening of S.asper leaves.

Assay Chemical groups Fresh Oven-dried Freeze-dried powdered powdered Alkaloids + + + Saponins + + + Steroids - - - Tannins + + + Terpenoids + + + Cardiac glycosides + + + Phytochemical groups of S. asper contributed to the antioxidant activity in the present study. Alkaloids compound which is important in medicine and constitute most of the valuable drugs (Edeoga and Eriata, 2001). Saponins, which are glycosides commonly present in higher plants and used as dietary supplements,

antioxidant, hyperchlolestrolaemia, nutriceuticals, hemolytic, hyperglycaemia, anticancer, anti-inflammatory, antifungal and weight loss agent (Aiyelaagbe and

Paul, 2009). Tannins have potential value as cytotoxic, astringent, anti-neoplastic

(Aguinaldo et al., 2005), antifungal, antimicrobial agent, antioxidant, remedy for intestinal disorders such as diarrhoea and dysentery (Akinpelu and Onakoya, 2006) and also in repairing for skin wounds (Shyamala and Vasantha, 2010). The antioxidant activity present in S. asper leaves could be attributed to the presence of tannins, as the tannins have shown antioxidant and protein-precipitating properties

(Ruch et al., 1989).

Apart from saponin and tannin, terpenoids which were found in S. asper is widely used in herbal medicine (Hayashi et al., 1993) for wound and scar healing

(Hawkins and Ehrlich, 2006). From clinical studies by Krishnaiah et al. (2009), it was shown that terpenoids strengthen the skin, increase the concentration of antioxidants in wounds, and restore inflamed tissues by increasing blood supply.

Cardiac glycosides shown to be effective as an antioxidant (Imaga et al. 2010), in the treatment of congestive heart failure and regulation of heart beat (Leverin and

McMatron, 1999; Taiwo et al., 2009). Therefore, the presence of alkaloids, saponins, tannins, terpenoids and cardiac glycosides in extracts supported the antioxidant activity.

4.2.4.2 Screening of Volatile Compound using Gas Chromatograph Mass Spectrometry (GCMS)

The results indicated that leaf extracts from freeze-dried contained more volatile compounds compared to oven-dried and fresh leaf extracts. The percentage of total area varied with the drying methods where freeze-dried samples showed the highest total area from all extracts tested. The screening of volatile compounds using

GCMS from 30% EtOH, 50% EtOH, 70% EtOH and aqueous extracts of fresh, oven- dried and freeze-dried S. asper leaves were conducted. The compound identification was based on NIST library with resemblance percentage above 90% (Zaibunnisa et al., 2009). Figure 4.4(a) - (c) are shown the chromatogram and in Table 4.3 shows the identification of compounds in 30% EtOH leaves extract. Acetic acid, n- hexadecanoic acid, 1,2-benzenediol and 2-methoxy-4-vinylphenol were detected, having the total area of 7.18% , 4.19% and 27.01% from 30% EtOH of fresh, oven- dried and freeze-dried S. asper leaves extracts respectively.

Table 4.3: Volatile screening of the chemical composition (%) in 30% EtOH extracts from fresh, oven-dried and freeze-dried samples.

Compounds FL OD FD Acetic acid 6.40 23.78 n-hexadecanoic acid 0.78 1,2-benzenediol 1.79 1.91 2-methoxy-4-vinylphenol 2.40 1.32 Total area (%) 7.18 4.19 27.01

The reported values are the means ± S.D. (n=3). FL: fresh leaves; FD: freeze drying; OD: oven drying.

Fig. 4.4(a): GCMS chromatogram of 30% EtOH extract of S. asper fresh leaves.

Fig. 4.4(b): GCMS chromatogram of 30% EtOH extract of S. asper leaves from oven-dried S. asper leaves.

Fig. 4.4(c): GCMS chromatogram of 30% EtOH extract of S. asper leaves from freeze-dried S. asper leaves.

For 50% leaves extracts, freeze-dried sample shows seven volatile compounds (Fig. 4.5c) compared to oven-dried and fresh leaf extracts which showed five peaks (Fig. 4.5b) and only one peak (Fig. 4.5a). The main compounds found in these extracts were listed in Table 4.4. For fresh leaves, acetic acid (31.59%) was found while for oven-dried extracts, there were five compounds identified; acetic acid (15.44%), 2-methoxy-4-vinylphenol (1.98%), phytol (33.13%), 9,12,15- octadecatrien-1-ol (2.29%) and 9,12,15-octadecatrienoic acid, ethyl ester (2.04%).

The results revealed that acetic acid (33.29%) and phytol (13.51%) were the major component in 50% EtOH extract from freeze drying whereas the five minor compounds were 1,2-benzenediol (1.13%), 2-methoxy-4-vinylphenol (1.24%), n- hexadecanoic acid (1.48%), 9,12,15-octadecatrienoic acid, methyl ester (5.03%) and

9,12,15-octadecatrienoic acid, ethyl ester (1.15%). The total area of respective extracts from fresh, oven-dried and freeze-dried were 31.59%, 54.88% and 57.01%.

Table 4.4: Volatile screening of the chemical composition (%) in 50% EtOH extracts from fresh, oven-dried and freeze-dried samples.

Compounds FL OD FD Phytol 33.13 13.51 Acetic acid 31.59 15.44 33.29 n-hexadecanoic acid 1.48 1,2-benzenediol 1.31 2-methoxy-4-vinylphenol 1.98 1.24 9,12,15-octadecatrien-1-ol 2.09 9,12,15-octadecatrienoic 2.04 1.15 acid, ethyl ester 9,12,15-octadecatrienoic 5.03 acid, methyl ester Total area (%) 31.59 54.68 57.01

The reported values are the means ± S.D. (n=3). FL: fresh leaves; FD: freeze drying; OD: oven drying.

Fig. 4.5(a): GCMS chromatogram of 50% EtOH extract of S. asper fresh leaves.

Fig. 4.5(b): GCMS chromatogram of 50% EtOH extract of S. asper leaves from oven-dried leaves. 18

Fig. 4.5(c): GCMS chromatogram of 50% EtOH extract of S. asper leaves from freeze-dried samples.

Five compositions were identified (Table 4.5) from 70% EtOH of fresh leaves with total area (78.80%) and the major peak exhibited by acetic acid (30.48% and phytol (40.23%). The other chemical compositions as illustrated in figure 4.6(a) were n-hexadecanoic acid (2.23%), 9, 12, 15-octadecatrien-1-ol (4.05%) and

9,12,15-octadecatrienoic acid, ethyl ester (1.81%). Table 4.5 lists the identified compositions in 70% EtOH extract from oven drying and their areas relative to the total areas (78.61%). Twelve compositions were identified from this sample as shown in figure 4.6(b) which mainly shown by phytol (37.23%), acetic acid

(13.56%) and 9, 12,15-octadecatrien-1-ol (7.20%). The other identified compositions were vitamin E (4.91%), γ-sitosterol (4.16%), n-hexadecanoic acid (3.88%), 9,12- octadecadienoic acid (2.40%), 9,12,15-octadecatrienoic acid, ethyl ester (1.76%), nonanoic acid (1.02%), 2-methoxy-4-vinylphenol (0.78%), β-tocopherol (0.86%) and hexadecanoic acid (0.85%).

Table 4.5: Volatile screening of the chemical composition (%) in 70% EtOH extracts from fresh, oven-dried and freeze-dried samples.

Compounds FL OD FD Phytol 40.23 37.23 33.75 Acetic acid 30.48 13.56 8.26 n-hexadecanoic acid 2.23 3.88 4.77 2-methoxy-4-vinylphenol 0.78 0.56 9,12,15-octadecatrien-1-ol 4.05 7.20 0.48 9,12,15-octadecatrienoic 1.81 1.76 2.20 acid, ethyl ester 9,12-octadecadienoic acid 2.40 hexadecanoic acid, 0.85 0.47 2-hydroxy-1- Nonanoic acid 1.02 β-tocopherol 0.86 1.21 Vitamin E 4.91 8.21 γ-sitosterol 4.16 4.76 2-hexadecene 0.37 Hexadecanoic acid, ethyl ester 0.69 Tetracosahexaene 1.60 Octadecanoic acid 1.26 9,12,15-octadecatrienoic acid 11.09 Total area (%) 78.80 78.61 79.68

The reported values are the means ± S.D. (n=3). FL: fresh leaves; FD: freeze drying; OD: oven drying

Fig. 4.6(a): GCMS chromatogram of 70% EtOH extract of S. asper fresh leaves.

Fig. 4.6(b): GCMS chromatogram of 70% EtOH extract of S. asper leaves from oven-dried leaves.

Fig. 4.6(c): GCMS chromatogram of 70% EtOH extract of S. asper leaves from freeze-dried S. asper leaves.

The result of the GCMS analysis identified various compounds present in

70% EtOH from freeze drying method with total area of 79.68%. Figure 4.6(c) shows the chromatogram of the extract. A total of fifteen volatile compounds were identified. Phytol (37.88%) was the major compositions followed by 9,12,15- octadecatrienoic acid (11.09%), acetic acid (8.26%) and vitamin E (8.21%) (Table

4.5). The other compositions were n-hexadecanoic acid (4.77%), tetracosahexaene

(1.6xxxxxxxxxx0%), β-tocopherol (1.21%), hexadecanoic acid, ethyl ester (0.69%),

2-methoxy-4-vinylphenol (0.56%), 9,12,15-octadecatrien-1-ol (0.48%), hexadecanoic acid, 2-hydroxy-1- (0.47%) and 2-hexadecene (0.37%).

Table 4.6: Volatile screening of the chemical compositions (%) in aqueous extract from fresh, oven-dried and freeze-dried samples.

Compounds FL OD FD Acetic acid 100 48.56 43.77 n-Hexadecanoic acid 2.10 2,3-Butanediol 3.12 2-Methoxy-4-vinylphenol 1.97 1.32 Total area (%) 100 55.75 45.09

The reported values are the means ± S.D. (n=3). FL: fresh leaves; FD: freeze drying; OD: oven drying

Acetic acid was detected as the main compound from fresh, oven-dried and freeze-dried with 100%, 48.59% and 43.77% of aqueous extract as listed in Table

4.6. Figure 4.7(a) - (c) shows an aqueous extract from fresh, oven-dried and freeze- dried extracts. The other minor identified compositions were 2,3-butanediol (3.12%), n-hexadecanoic acid (2.10%) and 2-methoxy-4-vinylphenol (1.97%) from oven drying. The freeze-dried aqueous extract was shown to exhibit lesser compositions compared to than the ethanol extract.

Fig. 4.7(a): GCMS chromatogram of S. asper aqueous fresh leaves extract.

Fig. 4.7(b): GCMS chromatogram of S. asper aqueous leaves extract from oven- dried leaves.

Fig. 4.7(c): GCMS chromatogram of S. asper aqueous leaves extract from freeze- dried samples Most of the studies focused on essential oils from different parts of the plant, however none reported from leaves. Knowledge of the phytochemical is very essential to investigate the actual effectiveness and benefit of the plant in medicine and human body. Research on the phytochemistry of S. asper leaves extracts from various drying treatments using GCMS has not been reported.

Nineteen compositions were identified in the 30% EtOH, 50% EtOH and

70% EtOH extracts of S. asper leaf with different drying methods. In order to study the effects of drying method on the chemical composition of S. asper leaves extracts, percentages of volatile compounds of fresh, oven-dried and freeze-dried extracts were compared. The relative percentage varied with the drying methods. In 30%

EtOH leaves extract from oven-dried, fresh and freeze-dried the total relative area

increased significantly (p < 0.05) from 4.19% < 7.18% < 27.01% respectively. For

50% EtOH leaves extracts, freeze-dried (57.01%) was significantly (p < 0.05) higher compared to oven-dried (54.68%) and fresh samples (31.59%). Whereas for the 70%

EtOH leaves extracts, oven-dried samples had a lower amount (78.61%) followed by fresh ones (78.80%) and freeze-dried (79.68%).

Different extracts exhibited different volatile profiles. Freeze-dried exhibited more compounds and possessed a higher percentage of the total relative area. The main factor could be the temperature. Venskutonis (1997) reported that the loss of volatile constituents in herbs and spices depends on the drying methods and biological characteristics of the plants. The temperatures might cause the biological structure of the oil glands and the epithelial cells in the dried samples to collapse

(Venskutonis, 1997); Hamrouni et al., 2011). Freeze-drying significantly decreased losses of several flavour and aroma compounds in blueberries (Vaccinium spp.) and volatile components of Antriscus sylvestris (L.) compared to fresh plant (Feng et al.,

1999); Bos et al., 2002). However, in aqueous leaf extracts, fresh leaves had a significantly (p < 0.05) higher total relative area as compared to oven-dried (55.75%) and freeze-dried (45.09%) samples.

Ethanol extracts exhibited more volatile compounds compared to aqueous extracts. Different techniques and solvents lead to a variation of extraction. In this case, 70% EtOH had more percentage of total relative area compared to 50% and

30% EtOH extracts. Ethanol has been proven as better solvent compared to aqueous

extraction. As the polarity of the solvents increases, extraction efficiency and volatile components would be increased which shows distinct compounds affinities (Taveira et al., 2009). Different phytoconstituents possessed different degrees of solubility in various types of solvents depending on their polarity (El-Mahmood and Doughari,

2008).

Major compounds identified in S. asper leaf extracts were acetic acid and phytol. This is consistent with data reported by Wei et al. (2011) and Sathish and

Maneemegalai (2008) which showed phytol as the major compound found in dried medicinal plants such as Peperomia pellucida and Lantana camara leaf methanol extract. 70% EtOH exhibited more volatile compositions compared to others such as

9,12,15-Octadecatrien-1-ol, n-Hexadecanoic acid, 9,12,15-octadecatrienoic acid, β- tocopherol and vitamin E. Extract from fresh leaf exhibited higher amount of acetic acid and phytol compared to freeze-dried and oven-dried samples. Phytol has been reported to possess antibacterial activities against Staphylococcus aureus (Sathish

Kumar and Maneemegalai, 2008), anti-diabetic activity in type-11 diabetic

(McCarty, 2001), antioxidant property (Jyotsna et al., 2007), anti-inflammatory, antimicrobial and anticancer activities (Kumar et al., 2010; Rani et al., 2011). The presence of this compound strongly supports the use of S. asper leaves extracts as antibacterial, antimicrobial and anticancer activities (Sowiseth et al., 1991;

Leungpailin and Leenasirimakul, 1992; Wongkham et al., 1996). Phytol has been found to be one part of the chlorophyll that is important in plant biosynthesis and

also be used in cosmetics, fine fragrances, shampoos, toilet soaps and other toiletries as well as in non-cosmetic (McGinty et al., 2010).

Acetic acid present mainly in all samples. In comparison to all samples, freeze-dried samples possessed higher content of acetic acid in 30% and 50% EtOH extracts. However, acetic acid in fresh leaf extracts was found to be higher in 70%

EtOH and aqueous extracts. Acetic acid (ethanoic acid) is the most important organic acid and used in plastics, chemical, pharmaceutical industries, as a food additive

(UK FSA, 2011 and US FDA, 2011) and natural latex coagulant (Ernstgard et al.,

2006). Other commercial uses include the manufacture of vitamins, antibiotics, hormones, and organic chemicals (Peter, 1994). Acetic acid is a common constituent for the formation of food substances, metabolism for animal and plant tissues. Acetic acid has been reported to reduce weight, lower blood pressure (Kondo et al., 2001) and is an anti-diabetic (Carol et al., 2004). This acid helps to keep the skin healthy, prevent early aging, and prevent the formation of cholesterol in arteries

(http://wiki.answers.com). Therefore, this indication may support compounds in S. asper leaves as an antioxidant agent.

GCMS analysis showed phytochemicals which contributed to the medicinal activities of the plant (Tables 4.4 and 4.5). The detected compounds of S. asper leaf extracts were 9, 12, 15-octadecatrien-1-ol, 2-methoxy-4-vinylphenol, 9, 12- octadecadienoic acid, n-hexadecanoic acid, β-tocopherol, vitamin E, hexadecanoic acid, ethyl ester and 9, 12, 15-octadecatrienoic acid methyl ester. Vitamin E is lipid-

soluble substance and functions as a chain-breaking antioxidant that prevents the propagation of a free radical reaction (Packer, 1994; Kamal-Eldin A and Appelqvist,

1996). β-Tocopherol, a minor constituent of vitamin E possess antioxidant activity

(Regina and Maret, 1999). Study by Grassman (2005) found that vitamin E and phytol are responsible in antioxidant activity. Vitamin E is an example of powerful phenolic antioxidant which can destroy free radicals to keep tissues healthy and reduces risk of incidence of cardiovascular (Lee and Shibamoto, 2001; Sang et al.,

2002; Sen et al., 2007). It is also used as anti-diabetic, anti-leukemic, anti-coronary and antitumour agent (Kumar et al., 2010; Rani et al., 2011). The presence of this component may further support the present result of the antioxidant activity, total phenolic and flavonoid as well.

Meanwhile, polyunsaturated fatty acid 9, 12,15-octadecadienoic acid is a conjugated linoleic acid which has been known as an antioxidant, has anticancer, hypocholesterolemic, anti-acne (Ha et al., 1990; Kumar et al., 2010), anti- inflammatory and anti-arthritic property(Jones, 2002), and believed to lower type 2 diabetes (Salmeron et al., 2001) and coronary heart disease (Willett, 2007). Study by

Bodoprost and Rosemeyer (2007) described that hexadecanoic acid act as antioxidant and have antimicrobial activities. It was also reported that flavonoids, palmitic acid

(hexadecanoic acid, ethyl ester and n-hexadecaonoic acid), unsaturated fatty acid and linolenic (docosatetraenoic acid and octadecatrienoic acid) act as antimicrobial, anti- inflammatory, antioxidant, hypocholesterolemic, cancer preventive, hepatoprotective, antiarthritic, antihistimic, antieczemic and anticoronary

aagents(Kumar et al., 2010). Naturally occurring phenolic compound, 2-methoxy-4- vinylphenol was used as flavouring agent (Jeong and Jeong, 2010) and food additives and it is safe for use in food (EFSA, 2008). The presence of 2-methoxy-4- vinylphenol in S. asper leaf extracts confirmed the leaves as an alternative food preservative and flavour.

4.3 Physical Analysis

4.3.1 Colour Measurement

Results obtained for the colour from fresh, powdered oven-dried and freeze- dried of S. asper leaves were illustrated in figure 4.8. L*, a*, b*, C* and h values showed significant (p < 0.05) differences among the samples. The L* (lightness) values of the S. asper leaves decreased during drying indicating a darker colour. The

L* value of oven-dried leaves (47.30) was the lowest, followed by freeze-dried leaves (49.76), and fresh leaves (53.99). Fresh leaves had the highest a* value (-

5.77±0.11) which is more greenish as compared to freeze-dried (-3.11±0.22) and oven-dried (-0.12±0.03) leaves. The b* (yellow) values increased significantly in the following order; fresh leaves (12.92±0.06) < freeze-dried (13.33±0.22) < oven-dried

(15.97±0.26). Oven drying results in a loss of the green colour of the S. asper leaves and a considerable decrease in the colour quality. The chroma (C* value) of oven- dried leaves were the highest (most intense) followed by freeze-dried and fresh leaves. The hue angle (h) is an indicator of the colour tone. The h-value of all the samples falls within the yellow-green quadrant. The colour of fresh leaves was more

green (114.07˚) as compared to freeze-dried (103.13˚) and oven-dried (90.43˚) leaves. A significant (p < 0.05) difference was observed in the hue of all S. asper leaves.

Fig. 4.8: Effects of different drying methods on L*, a*,b*,C* and h values of S. asper powdered leaves. Bars with different letters are significantly different (p < 0.05). The reported values are the means ± S.D. (n=3). FL: fresh leaves; FD: freeze drying; OD: oven drying; L*: lightness; a*: greenness; b*: yellowness; C*: chroma; h: hue angle

Colour is one of the most important quality parameters in a plant medicate in order to preserve their appearance of green in dried herb leaves. Study by Arslan and

Ozcan (2008) reported that high temperature and long drying are the most effective factors in colour damage during drying. It is clearly indicated that h values have significant variation among the samples. In this study, heat treatment caused colour degradation in oven-dried powdered leaves as C* values were shown to be more saturated towards the bright yellow colour. Oven-dried powdered leaves also showed 31

the highest b* values in comparison to others indicating that this sample had the highest yellow colour while powdered fresh leaf had the lowest yellow colour (b* value). The highest intensity of yellow colour was observed in oven drying followed by freeze drying while fresh samples resulted in a very light green colour. Therefore, oven-dried powdered of the S. asper leaves were significantly (p < 0.05) darker in colour when compared to the freeze-dried powdered leaf samples. This study is in agreement with Soysal (2004) who showed that oven drying resulted in some darkening of the leaf colour compared to the fresh herb. For a* value (greenness) the fresh one had the highest value (more negative) compared to oven-dried and freeze- dried leaves. Oven drying caused a significant increment of a* values than freeze drying treatment, which suggests that the loss of green colour in the final product was greater. This result was supported by other literature that oven drying method was known as the least desirable drying method regarding to final colour of dried products (Arslan and Ozcan, 2011). Also, Arabhosseini et al. (2007) found that hue angle, lightness and saturation in tarragon leaves would decrease due to drying temperature. It can be concluded that drying methods gave varying effects on the colour contents of S. asper leaves. The possible colour changes in this plant leaves would influence their organoleptic properties and limit their potential applications in producing products (Femenia et al., 2003).

A decrease in L* value correlated well with an increase in brown coloration of oven-dried samples. Cemeroglu and Acar (1986) also found that the colour alteration might be due to the chemistry of the pigments from the chlorophylls

(chlorophyll a and b) being converted to brown pheophytins and some other compounds during the thermal processing and storage. This result confirmed previous reports in the literature and supports the advantages of freeze drying over oven drying (Salunkhe, 1974). Freeze drying allows preservation of the greenish colour of fresh leaves and enhance their luminosity. This indicated that different drying treatment contributed varied colours to the S. asper leaves. The adverse changes in colour may alienate potential customer due to suggestion of possibility of poorly controlled processing where this must be prevented or minimized (Ramana et al., 1988).

This is the first evaluation of the effects of various drying on some properties of S. asper leaves, such as proximate composition, chlorophyll, colour and the mineral contents. The drying process allows preservation, improvement or preservation of nutritive and functional values of the leaves. Raw medicinal plants are often dried and stored for a long time before it is used to produce various types of herbal products (Lin et al., 2011).

4.4 Percentage Yield of S. asper Leaves Extract

The yields of the extracts obtained by different solvents from fresh, oven- dried and freeze-dried are given in Table 4.1. The yield of solid residue was obtained after extraction and evaporation from 50 g leaves. Among all the plant extracts, aqueous extract was found to have a higher yield followed by 70% EtOH, 50%

EtOH, 30% EtOH extracts. This result was consistent with the study reported by Tien

et al. (2011) in Angelica sinensis leaves extract. Amirah et al. (2012) indicates that the variation of solvent polarities (ethanol to water) will increase the yield of extraction. Extraction yield was effective with increasing yield using freeze-dried followed by oven-dried and fresh leaf.

Table 4.1: The extraction yield obtained by different solvents from fresh, oven- dried and freeze-dried leaves.

Solvent Yield of extract (%) FL OD FD AE 6.31+0.41 8.08+0.11 11.21+0.06 70% EtOH 2.12+0.28 3.51+0.02 4.01+0.24 50% EtOH 1.29+0.10 1.68+0.10 2.15+0.08 30% EtOH 1.04+0.03 1.42+0.01 2.10+0.01

FL: fresh leaves; FD: freeze drying; OD: oven drying AE = aqueous extract, 70% EtOH = 70% ethanol extract, 50% EtOH =50% ethanol extract, 30% EtOH = 30% ethanol extract. The reported values are the means ± S.D. (n=3).

Generally, drying the plant material resulted either increased or decreased in the yield depending on the time of the drying and temperature (Hamrouni., 2011).

Freeze-dried samples showed the highest yield compared to oven-dried samples which is similarly found by Rahimmalek and Goli (2012). Higher temperature may decrease the extraction yield. This statement is supported by Buggle et al. (1999);

Braga et al. (2005); Khangholi and Rezaeinodehi (2008) where temperatures range from 30–90˚C caused the decrease in extraction percentage in Cymbopogon citrates,

Piper hispidinervium and Artemisia annua. From the result demonstrated that the aqueous extract of S. asper leaves had lower pH ranged 4.8 - 5(acidic) value, were exhibited higher yield of extraction. These results are also confirmed by a study by

Liang and Xu (2001) who found that the yield of tea leaf extract increased when

extracted at lower pH. Therefore, the yield of extraction depends on the type of solvents with varying polarities, extraction time, temperature, sample-to-solvent ratio as well as on the chemical composition and physical characteristics of the samples.

4.5 Antioxidant Activity

From the results obtained, fresh leaf extracts (Fig. 4.9a) radical scavenging activity increased in the following order: 30% EtOH; 66.31% ± 0.01 < 50% EtOH;

72.52% ± 0.01 < 70% EtOH; 84.70% ± 0.01 < aqueous; 82.07% ± 0.03. Figure

4.11(b) and 4.11(c) show the antioxidant activity by the DPPH radical scavenging method at various drying treatments of S. asper leaves extracts. Extraction from freeze-dried (Fig. 4.9c) samples had significantly (p < 0.05) higher antioxidant activity in comparison to oven-dried samples where 70% EtOH (89.25% ± 0.01) contained higher antioxidant activity followed by aqueous extract (84.88% ± 0.01),

50% EtOH (78.10% ± 0.03) and 30% EtOH (69.48% ± 0.03).

Fig. 4.9(a): DPPH free radical scavenging activity (%) of the aqueous and ethanol S. asper fresh leaf extract and BHA. Mean ± standard deviation (n = 3). AE = aqueous extract, 70% EtOH = 70% ethanol extract, 50% EtOH = 50 % ethanol extract, 30% EtOH = 30% ethanol extract. b

Fig. 4.9(b): DPPH free radical scavenging activity (%) of the aqueous and ethanol S. asper oven-dried leaf extract and BHA. Mean ± standard deviation (n = 3). AE = aqueous extract, 70% EtOH = 70% ethanol extract, 50% EtOH = 50% ethanol extract, 30% EtOH = 30% ethanol extract. 36

On the other hand, for oven-dried (Fig. 4.9b) extracts scavenging effects of

70% EtOH extract indicates the highest scavenging activity with 86.68% ± 0.01 as compared to the aqueous extract of S. asper leaves which recorded a value of 82.88%

± 0.02. For 50% and 30% EtOH extracts the antioxidant activities were 77.2% ± 0.01 and 68.56% ± 0.01 respectively. The results were comparable to a study done by

Qader et al. (2011) the antioxidant activity of dried Polygonum minus leaf aqueous and ethanol extracts were 81.88% ± 0.98 and 89.5% ± 1.07 respectively.

The 50 % inhibition of DPPH radical (IC50) by different treatments of S. asper leaves were determined graphically. From fresh leaves, the lowest IC50 was obtained from aqueous and 70% EtOH (0.14 mg/ml) whereas 30% EtOH (0.77 mg/ml) extracts was found to have higher IC50 value followed by 50% EtOH (0.17 mg/ml). The IC50 values ranged from 0.14 to 0.75 mg/ml. Moreover, aqueous and

70% EtOH extracts from oven-dried reported a lower IC50 value of 0.14 mg/ml compared to other extracts with a value of 0.17 mg/ml (50% EtOH) and 0.77 mg/ml

(30% EtOH).

Fig. 4.9(C): DPPH free radical scavenging activity (%) of the aqueous and ethanol S. asper freeze-dried leaf extract and BHA. Mean ± standard deviation (n = 3). AE = aqueous extract, 70% EtOH = 70% ethanol extract, 50% EtOH = 50 % ethanol extract, 30% EtOH = 30% ethanol extract.

For freeze-dried samples, the IC50 values from aqueous, 70% EtOH, 50%

EtOH and 30% EtOH extracts values increased in the following order; 0.13 < 0.13 <

0.16 < 0.56 mg/ml respectively. The data indicated that 70% EtOH and aqueous

extracts have lower IC50 values as compared to other extracts while IC50 values of

BHA was found to be active at 0.1 mg/ml.

In this study, the potential of S. asper leaf extracts as a new antioxidant agent

was explored. Undesirable side effects of commercial synthetic antioxidants such as

BHT and BHA which has been reported toxic to man (Lobo et al., 2010) and

carcinogenic have led to a continuous search in finding for a new natural bioactive

compounds with antioxidant activities that have significant roles in humans for the 38

prevention of chronic diseases. The antioxidant activities of the aqueous and ethanol extracts of S. asper leaves were investigated by the ability of the extract to scavenge hydroxyl radicals. This is the first report on the antioxidant activity of this plant leaves extracts from various drying treatments. Overall, 70% EtOH extract exhibited significantly (p < 0.05) higher DPPH radical scavenging activities compared to aqueous extract. Thus, the highest antioxidant activity among different drying methods were shown by 70% EtOH extracts from freeze-dried (89.25% ± 0.01) followed by the other; 70% EtOH extracts oven-dried (86.68% ± 0.01) and 70%

EtOH extracts from fresh leaves (84.70% ± 0.01). The antioxidant activity was significantly (p < 0.05) different among fresh leaves, oven-dried and freeze-dried extracts. Whereas for BHA (standard) was found to be 99.5%±0.03 at the same concentration.

Antioxidant activity of aqueous, 30 % EtOH, 50 % EtOH and 70 % EtOH of

S. asper leaves extract from fresh, oven-dried, freeze-dried and BHA in terms of free radical scavenging ability was evaluated using DPPH free radical assay; as expressed as percentages of antioxidant activity at 1.0mg/ml. The scavenging activity of all samples on the DPPH radical was found to be strongly dependent on concentration which showed that the antioxidant activity increased with the increase of the concentration of all extracts. The extracts of all tested S. asper leaves possessed high radical scavenging abilities. According to the results, oven-dried and freeze-dried extracts of S. asper leaves might possess more antioxidant properties than the fresh ones. Consistent with the results described by Hossain et al. (2010) and Suhaj (2006),

the fresh samples might lose antioxidant compounds due to the enzymatic degradation during processing as the enzymes were still active in fresh samples.

Thus, it tends to lower the percentage of antioxidant in the fresh sample extracts.

Suhaj (2006) also reported that the use of dried samples might avoid the loss of enzymes action, as the enzymes were inactivated due to decreased water activity and retained high antioxidant capacity and total phenols in the dried extracts. Moreover, in comparison between fresh, oven-dried and freeze-dried leaves aqueous extracts, freeze-dried aqueous extract had significant (p < 0.05) higher values than others.

This result was confirmed by Bodo et al. (2004), whereby freeze-dried water hyacinth leaves had a higher antioxidant activity in vitro than oven-dried at 40°C. In this case also, 70% EtOH from freeze-dried samples had a significantly (p < 0.05) higher antioxidant activity comparing to oven-dried and fresh leaf extracts. The result is in an agreement with Katsube et al. (2009) who reported that freeze-dried

Morus alba leaves showed relatively high levels of polyphenolic compounds. Lim and Murtijaya (2007) also reported that oven drying led to significant reduction in the DPPH scavenging ability of Phyllanthus amarus.

According to the result obtained, different solvent extraction would result in different antioxidant capacity. Ethanol extraction particularly, 70% EtOH extract had significantly (p <0.05) higher values compared to 30% and 50% EtOH extracts.

Finding by Chew et al. (2011) reported that the antioxidant capacities of crude extracted were found to be sensitive to ethanol concentration where antioxidant capacities (ABTS and DPPH) of crude extract increased which was associated with

the increase of ethanol concentration. Study by Othman et al. (2007) also reported that the solvent significantly influences the measurement of antioxidant properties of the extract.

The potential of S. asper leaves as a strong antioxidant was studied using

DPPH radical scavenging methods. All the extracts exhibit high antioxidant activities, which make them potent antioxidant source. The DPPH antioxidant assay is a stable free radical based on the ability of DPPH by the degree of discoloration of

DPPH (Arulpyria et al., 2010) in which decolorise the deep purple colour into yellow in the presence of antioxidants. DPPH assay was used in this study because it is one of the most effective, reactive, reliable, simple and reproducible in vitro method for evaluating important activity of compounds as well as plant extracts (Koleva et al.,

2002; Balestrin et al., 2008) and also can accommodate many samples in a short period and it is sensitive enough to detect active ingredients at low concentrations

(Sanchez, 2002).

Free radicals are molecules, usually of oxygen, that have lost an electron that contribute to a variety of health problems such as cancer, diabetes, hypertension, heart disease and aging (Stauth, 2007) which was generated during body metabolism continuously. This study also found that the radical scavenging activity of both extracts increased with concentrations. It shows that 70% EtOH leaf extracts have the strongest potential as a source of antioxidant activity, which is above 80%. It can be concluded that the extracts obtained using high polarity solvents (ethanol) were

considerably more effective radical scavengers than using less polar solvents

(aqueous), indicating that antioxidant or active compounds of different polarity could be present (Ghasemzadeh et al., 2011) in the leaves. This data may indicate that 70%

EtOH extracts was the most effective DPPH radical scavengers.

The IC50 value is a widely used parameter to measure the free radical scavenging activity. A lower IC50 indicates a higher antioxidant activity

(Maisuthisakul et al., 2007). IC50 values for aqueous and ethanol extract ranged from

0.13 – 0.77 mg/ml. IC50 values of the investigated extracts slightly differ depending on the solvent applied (IC50 values of ethanol extracts are higher than IC50 of aqueous extract).

Table 4.7: Pearson correlation coefficients between TPC, TFC and DPPH assay in the S. asper leaf extract.

TPC TFC DPPH TPC 1 TFC 0.592 1 DPPH 0.929 0.602 1

TPC = total phenolic content; TFC = total flavonoid content; DPPH = 1, 1-diphenyl- 2-picrylhydrazyl radical, (n=24, p < 0.01).

Correlations among the antioxidant assays were also carried out in this study in order to obtain the relationship between the phenolic contents and antioxidant capacities of crude extract. The antioxidant activities of S. asper leaves correlated significantly (p < 0.05) with the DPPH radical scavenging activity and total phenolic compounds with r2 = 0.929, p < 0.01 as shown in Table 4.7. The results obtained in

the present study are comparable to those reported by Katsube et al. (2009) where

DPPH radical scavenging activities and the levels of polyphenolic compounds correlated well (r2 = 0.940) in the Morus alba leaves air-dried at various temperatures and freeze-dried. In the present study, total phenolic content seems to correlate with the antioxidant capacity of the extracts as their correlation coefficients are greater than 0.8. A good correlation was found between antioxidant activity and phenolic contents. This likely indicates that the antioxidant activity of S. asper leaf extracts might originate from the phenolic compounds. The result may suggest that

92.9% of the plant antioxidant activity results from activity of phenolic compounds.

It can also be concluded that the antioxidant activity is not only limited to the phenolics itself but may come about 7.1% from the presence of the other secondary metabolites such as vitamin and other volatile compounds. The results are consistent with those recently obtained by Yan and Asmah (2010); Nsimba et al. (2008);

Zielisnki and Koslowska (2000); and Oktay et al. (2003) who presented a strong relationship between antioxidant activity and the phenolic content, in which phenolic compounds might contribute to the antioxidant activity. Similarly, free radical scavenging activity of antioxidant is very much associated with total phenolic and total flavonoids content (Ghasemzadeh et al., 2010). According to Cao et al. (1997);

Prior et al. (1998) found that the antioxidant capacity was mainly due to the phenolic compounds, so it is reasonable to expect that the total phenolic content should be highly correlated with the antioxidant capacity (Cao et al., 1996; Ou et al., 2002).

However, the correlation of total flavonoid contents and antioxidant activity was found moderate (r2 = 0.602), and low correlation was found between flavonoid content with the total amount of phenolics (r2 = 0.592). This indicates that flavonoids had a lesser antioxidant activity than phenolic compounds. Similar findings have been made while assessing the antioxidant activities in medicinal plant reported that there were apparent linear relationships between antioxidant capacity and total phenolics (Pietta et al., 1998). Therefore, the extent of antioxidant capacities was correlated with the contents of total phenolics and flavonoids. Kahkonen et al.

(1999); Lagouri and Boskou, (1996) also described that phenolic acids and flavonoids might play the major role in responsible for antioxidant capacity among the plant phenolics.

4.6 Analysis of Phenolics

4.6.1 Total Phenolic Content (TPC)

Table 4.8 presents the results of the total phenolic content of fresh and dried

S. asper plant leaves extracted using different solvents, namely aqueous and ethanol;

30% EtOH, 50% EtOH and 70% EtOH from different drying methods using regression equation y = 0.116x, R2 = 0.9924 (Fig. 4.10). There was a variation in the amount of the total phenolics in the treated leaves from 117.37 ± 0.04 to 302.85 ±

0.03 and 196.43 ± 0.01 to 226.80 ± 0.03 mgGAE/g for ethanol and aqueous extracts respectively.

Fig. 4.10: Gallic acid calibration curve for determination of total phenolic using Folin–Ciocalteu colorimetric assay.

When compared between different solvents, the highest total phenolic content of aqueous extracts was found in freeze-dried extracts (226.80 ± 0.03 mgGAE/g) and the lowest in the fresh leaf extracts (196.43 ± 0.01 mgGAE/g). Whereas from ethanol extracts, 70 % EtOH extracts from freeze-dried had higher total phenolic (302.85 ±

0.03 mgGAE/g) compared to the lowest was 30 % EtOH from fresh leaf (117.37 ±

0.04 mgGAE/g) extracts. In the present study, it was observed that the total phenolic content decreased significantly during drying. All the sample extracts from freeze drying showed significantly (p < 0.05) higher total phenolic content as compared to oven drying and fresh leaf extracts. The amount of total phenolic content of the S. asper leaf extracts can be arranged in descending order from freeze-dried and oven-

dried; 70% EtOH < aqueous < 50% EtOH < 30% EtOH. Thus, drying treatments had significant (p < 0.05) effects on the phenolic content of the S. asper extracts.

Table 4.8: Total phenolic constituents of S. asper leaves aqueous and ethanol extracts from fresh leaf (FL), oven-dried (OD) and freeze-dried (FD) samples.

Extraction Total phenolic content (mgGAE/g) solvent (ml) *FL *OD *FD AE 196.43±0.01a 223.95±0.05b 226.80±0.03c 70% EtOH 239.07±0.03a 296.37±0.04b 302.85±0.03c 50% EtOH 126.95±0.03a 155.66±0.04b 163.17±0.05c 30% EtOH 117.37±0.04a 146.64±0.08b 153.09±0.03c

AE = aqueous extract, 70% EtOH = 70% ethanol extract, 50% EtOH =50% ethanol extract, 30% EtOH = 30% ethanol extract. The reported values are the means ± S.D. (n=3). Data bearing different letters in the same row for each different drying method is significantly different (p < 0.05).

Extractions from oven-dried samples showed that the total phenolic was significantly (p < 0.05) higher in 70% EtOH extract (296.37 ± 0.04 mgGAE/g) and lowest in 30% EtOH (146.6 4± 0.08). For aqueous and 50% EtOH extracts, the phenolic contents were 223.95 ± 0.05 mgGAE/g and 155.66 ± 0.04 mgGAE/g respectively. The total phenolic content from freeze-dried extracts were decreased significantly in the following order; 302.85 ± 0.03 mgGAE/g (70% EtOH), 226.80 ±

0.03 mgGAE/g (aqueous extract), 163.17 ± 0.01 mgGAE/g (50% EtOH) and 153.09

± 0.02 mgGAE/g (30% EtOH). In this case, gallic acid was used as standard. Table

4.8 shows the phenolic concentration in the leaf extracts, expressed as milligram of gallic acid equivalents (GAEs) per gram of extract.

In this study, total phenolic of the leaf extracts was determined because plant phenolics constitute one of the major compound groups which act as primary antioxidants and it has been reported that leafy parts are rich in phenolic compounds

(Wong and Kitts, 2006). Phenolic compounds are major constituents that play an important role in the nutritional values, organoleptic properties, commercial properties, stabilized lipid peroxidation and direct antioxidative activity due to their scavenging abilities from their hydroxyl group (Paul et al., 2011).

Based on the results obtained, there were significant (p < 0.05) differences between all the extracts. The data showed that the range of total phenolic contents from S. asper leaf extracts from aqueous, 30% EtOH, 50% EtOH and 70% EtOH extracts were from 117.37–302.85 mgGAE/g. The result closely agreed with finding by Eric (2012); the antioxidant activity of Morus alba, Cassia angustifolia and Orthosiphon aristatus aqueous leaf extracts ranged from 120.5 ± 35 – 327.1 ±

288 mgGAE/g. For aqueous extracts of S. asper leaves, freeze-dried leaves had significantly (p < 0.05) higher values than oven-dried and fresh leaf extracts. The data also clearly showed that 70% EtOH extracts possessed a higher amount of total phenolic than aqueous extracts. The results were in agreement with that of Miranda et al. (2010) who showed that the influence of temperature on total phenolic content for fresh and dehydrated quinoa is significantly different (p < 0.05). Study by Yu et al. (2005) also indicated that in extracting phenolic compounds from peanut skin, ethanol was the most efficient extraction solvent than aqueous. However, when comparing among ethanol extractions, 70% EtOH freeze-dried leaf had significantly

(p < 0.05) higher values than others. It can be concluded the phenolic content increase with the increase of the solvent polarity. Lapornik et al. (2005); Turkmen et al. (2006); Rodtjer et al. (2006) reported that a high content of phenolic compounds showed that the extraction yield of phenolic compounds is greatly dependent on the solvent polarity. Yilmaz and Toledo (2006); Pinelo et al. (2005) also agreed that ethanol containing different volumes of water (10–20–30–40–50–60%) have revealed to be more efficient than the mono-component solvent system in extracting phenolic constituents. In this study, it was also found that 50% and 30% EtOH of S. asper freeze-dried extracts were significantly (p < 0.05) higher than oven-dried and fresh leaves ethanol extracts. The result obtained was supported a study by Nourhene et al. (2009) who stated that the phenolic content of fresh leaves was less than the dried leaves. Different drying treatments were shown to reduce the phenolic contents of the ethanol and aqueous extracts significantly, with oven drying causes the highest decrease in phenolic content.

There could be a few explanations for the decrease in phenolic contents. Loss of phenolic content after oven drying may be caused by enzymatic processes where heat treatments not only deactivate the enzymes, but also was able to degrade the phytochemicals, therefore, they are able to degrade phenolic compounds before the plant materials are completely dry. Comparing different methods of drying found that freeze drying caused the smallest loss of phenolic contents than oven drying.

Chan et al. (2009) reported the losses of phenolic compound due to thermal degradation. Freeze drying showed the highest antioxidant phenolic content among

the samples tested. This statement was supported by the finding by Abascal and

Ganora (2005) who described that freeze drying retains higher levels of phenolic content in plant samples than oven-drying. Spigno et al. (2007) confirmed that freeze-drying did not lead to a reduction in phenol content. Yousif et al. (1999) also reported that freeze drying showed a less pronounced damaging effect on the tissue structure than oven drying.

The results showed that S. asper leaves possess a high amount of antioxidant, a majority that may come from the phenolic compounds. This is supported by the finding of Rahman and Moon (2007) who reported that the antioxidant effect is mainly due to radical scavenging activity of phenolic, flavonoids and polyphenol compounds. Study by Lim and Murtijaya (2007) also described that a reduction in total phenolic values due to various drying treatments was accompanied by the respective decrease in antioxidant activities. Findings from this study also showed that the antioxidant activity is well correlated with the amount of phenolics found in the extracts. Consumption of foods with high phenolic, which acts as antioxidants, may reduce the risk of heart disease by slowing the progression of atherosclerosis

(Kaur and Kapoor, 2002). These results could be considered helpful for promoting biological activity of total phenolic that can prolong the use of dried S. asper leaves in food, cosmetic or therapeutic applications.

4.6.2 Total Flavonoid Content (TFC)

The total flavonoid content was reported as quercetin equivalents by reference to standard curve; y = 0.043x + 0.024, R2 = 0.980 (Fig. 4.11). The amounts of total flavonoids in the aqueous and ethanol extracts of S. asper leaves are shown in Table 4.9. For fresh leaves, low total phenolic contents were found in all extracts when compared to oven drying and freeze drying extracts. When comparing among different solvent extracts from S. asper fresh leaves, aqueous extract (3.97% ± 0.13) was a bit significantly (p < 0.05) higher than 70% EtOH extract (3.57% ± 0.06) followed by 50% EtOH and 30% EtOH with 1.84% ± 0.03, 1.81% ± 0.02 respectively.

Fig. 4.11: Quercetin calibration curve for determination of total flavonoid using colorimetric assay.

Among the different techniques applied, freeze drying showed higher values of total flavonoid as compared to oven drying. There was a significant (p < 0.05)

effects on the drying treatment of the flavonoid content. For oven-dried extract, it was found that the 70% EtOH (22.28 ± 0.03 mgQE/g) had the highest phenolic contents followed by 50% EtOH (19.16 ± 0.03 mgQE/g), aqueous extract (10.26 ±

0.04 mgQE/g) and 30% EtOH (8.87 ± 0.02 mgQE/g). For freeze-dried extract, total flavonoid can be ranked as follows; 70% EtOH (22.70 ± 0.02 mgQE/g) > 50% EtOH

(19.74 ± 0.05 mgQE/g) > aqueous extract (15.38 ± 0.05 mgQE/g) > 30% EtOH (9.92

± 0.05 mgQE/g). Comparison of extracts between different drying treatments showed that 70% EtOH extraction of S. asper leaves from freeze-dried samples had the highest levels of total flavonoids content.

Table 4.9: Total flavonoid content of S. asper leaves aqueous and ethanol extracts from fresh leaf (FL), oven-dried (OD) and freeze-dried (FD) samples.

Extraction Total flavonoid content (mgQE/g) solvent (ml) *FL *OD *FD AE 3.97±0.13 a 10.26±0.04b 15.38±0.01c 70% EtOH 3.57±0.06a 22.28±0.02b 22.70±0.04c 50% EtOH 1.84±0.03a 19.16±0.01b 19.74±0.02c 30% EtOH 1.81±0.02a 8.87±0.02b 9.92±0.02c

The total flavonoids content of the crude plant extracts as determined by the colorimetric method are reported as quercetin equivalents (Table 4.9). This data indicated that the range of total flavonoid contents from S. asper leaf extracts from aqueous, 30% EtOH, 50% EtOH and 70% EtOH extracts were 1.81–22.70 mgQE/g. 51

The amount of total flavonids at 70% EtOH extracts ranged 3.57 ± 0.06 to 22.70 ±

0.04 magic/g. The result is comparable to Radojkovic et al. (2012) who showed that the amount of total flavonoids in bonsai species such as Morus alba leaves 70%

EtOH and Morus nigra was ranged 0.894 to 67.369 mg RE/g. There were significant

(p < 0.05) differences between fresh, oven-dried and freeze-dried samples of S. asper leaves extracts. The flavonoid contents of S. asper fresh leaves were found to be significantly lower compared to oven-dried and freeze-dried extracts. In addition, the amount of total flavonoid content in S. asper leaf extract decreased significantly (p <

0.05) in oven-dried samples compared to freeze-dried samples. This result was supported by study from Schieber et al. (2001) who reported that the loss of macromolecules like flavonoid during heat treatment might be due to the drying condition particularly the temperature and duration used.

Among different solvent used, ethanol extract shows more flavonoid extracted. The results also showed that among different ethanol extracts, 70% EtOH had higher amount of flavonoids compared to 50% and 30% EtOH extracts. It can be concluded that total flavonoids content of S. asper leaf extracts increased with the increase of solvent polarity. Finding by Bazykina et al. (2002) described that ethanol is a polar solvent that is effective in extracting flavonoids from raw plant.

Similarly, report by Lapornik et al. (2005) and Turkmen et al. (2006) showed that the extraction yield of phenolic and flavonoid content is greatly dependent on the solvent polarity.

Flavonoids and other phenolic compounds are potent water soluble antioxidants and free radical scavengers, which prevent oxidative cell damage and have strong anticancer activity (Salah et al., 1995; Del-Rio et al., 1997; Okwu, 2004).

Flavonoids have a beneficial effect on human health such as hypertension and diabetes (Mbaebie et al., 2012), anti-aging, natural antioxidant, antimicrobial, anti- carcinogenic activities (Andrade et al., 2009; Ghasemzadeh et al., 2011) and also have been shown to possess anti-inflammatory activities and effective against diarrhoea (Schuier et al., 2005; Manga et al., 2004).

Flavonoids are subgroups of phenolic compounds; they are a broad class of low molecular weight compounds with chelating properties which is highly effective as antioxidants (Yanishlieva, 2001) and less toxic (Naczk, 2004). Flavonoids intake has a protective role of diet in the prevention of coronary heart disease (Hertog et al.,

1993; Peluso, 2006). The antioxidant effect demonstrated in this study might probably be due in part to the presence of flavonoids and tannins in S. asper leaf extracts. Antioxidants neutralize highly unstable and extremely reactive molecules, called free radicals, which attack the cells of human body every day (Stauth, 2007).

The antioxidant activity comes from phenolic compounds mainly flavonoids, polyphenols, tannins, and phenolic terpenes (Rahman and Moon, 2007). Here, can conclude that the higher the total phenolic content, the higher is the total flavonoid contents. These works were supported by study from Luximon et al. (2002);

Maisuthisakul et al. (2007) who described that the plant extracts with higher levels of

phenolic contains higher level of flavonoid and also exhibit greater free radical scavenging as well (Yingming et al., 2004; Ghasemzadeh et al., 2010).

4.6.3 Gallic acid and Quercetin by HPLC

According to the results, the range of gallic acid is between 0.0034 g/g –

0.0161 g/g in all extracts. The analysis of gallic acid content in S. asper leaf extracts illustrated in Table 4.10 from the linear calibration curve of gallic acid (y = 6789.2x

– 1076.3, R2 = 0.9736). The highest amount of gallic acid was observed in freeze– dried extracts compared to fresh leaf extracts (lowest). The highest amount of gallic acid was observed in aqueous extract from freeze-dried (0.0161 g/g) and the lowest was fresh leaves of 30% EtOH (0.0034 g/g) extract. For 30% EtOH extract, the concentration of gallic acid decreased as follows; freeze-dried (0.0075) > oven-dried

(0.0043 g/g) > fresh leaf (0.0034 g/g) while in 50% EtOH extract, gallic acid showing decreasing in freeze-dried (0.0043 g/g) > oven-dried (0.0039 g/g) > fresh leaf (0.0036 g/g). Freeze-dried extracts of 70% EtOH showed higher amounts of gallic acid (0.0070 g/g) followed by oven-dried and fresh leaf extracts with 0.0053 g/g and 0.0035 g/g respectively. In aqueous extracts, the freeze-dried showed higher content of gallic acid (0.0161 g/g) followed by oven-dried and fresh leaf extract with

0.0132 g/g and 0.0035 g/g respectively.

Table 4.10: Concentration of gallic acid from different extracts and drying treatments of S. asper leaves.

Samples Drying Total Phenolics of extract (g/g)

30% EtOH FL 0.0034a OD 0.0043b FD 0.0075c 50% EtOH FL 0.0036a OD 0.0039b FD 0.0043c 70% EtOH FL 0.0038a OD 0.0053b FD 0.0070c AE FL 0.0035a OD 0.0132b FD 0.0161c

FL: fresh leaves; FD: freeze drying; OD: oven drying. a-c Different letter from different drying treatment indicates significant differences (p<0.05). Mean ± standard deviation (n = 3). AE = aqueous extract, 70% EtOH = 70% ethanol extract, 50% EtOH = 50 % ethanol extract, 30% EtOH = 30% ethanol extract.

Table 4.11 showed the amount of quercetin in S. asper leaf extracts from the regression equation (R) for the calibration curve of standard quercetin, y = 2580.5x -

481.8; R2 = 0.9815. The average amount of quercetin in S. asper leaf extracts was comparatively low (0.0037 g/g – 0.0041 g/g) where 30% EtOH extracts from fresh leaf and oven drying exhibited higher amount of quercetin with 0.0041 g/g whereas the aqueous extract from fresh leaf possessed the lowest amount of quercetin (0.0037 g/g). No significant difference was shown for an amount of quercetin for all extracts and different drying treatments. The analysis demonstrated that the amount of quercetin in 50% EtOH extracts from fresh leaf and oven drying, 70% EtOH extract from oven drying, and aqueous extracts from oven drying and freeze drying was

0.0038 g/g. The quercetin content for freeze-dried of 30% and 50% EtOH was

0.0039 g/g while in fresh leaf and freeze-dried extracts of 70% EtOH was 0.0040 g/g.

Table 4.11: Concentration of quercetin from different extracts and drying treatments of S. asper leaves.

Samples Drying Total Flavanoids of extract (g/g) 30% EtOH FL 0.0041a OD 0.0041a FD 0.0039b 50% EtOH FL 0.0038a OD 0.0038a FD 0.0039a 70% EtOH FL 0.0040a OD 0.0038b FD 0.0040a AE FL 0.0037a OD 0.0038b FD 0.0038b

In this study, the analysis of phenolic and flavonoids in S. asper leaves extract from various drying treatments was done by using HPLC. The presence of gallic acid and quercetin in S. asper leaves supported the claim as natural antioxidants for nutritional as health supplements and therapeutic purposes.

Representative chromatograms of different solvent extracts from the leaves of S. asper was shown in figure 4.12(a) – figure 4.15(c). HPLC is reliable for quantify the chemical constituents in a plant and it has been reported that the analysis of gallic acid and quercetin shown for their stability, linearity, accuracy and precision

(Weerasak and Suwanna, 2007). Gradient elution was carried out for separation using 100 % methanol: water-acetic acid 25:1, v/v as the mobile phase with an analysis time of 10 min. 56

A wavelength at 280 nm for gallic acid and quercetin was set to confirm the existence in crude extracts. Gallic acid and quercetin were detected at retention time of 1.510 min and 9.808 min respectively in Appendix D [Fig. 5.5(b) and Fig. 5.6(b)].

The content of phenolic acid (gallic acid) and flavonoids (quercetin) was calculated from the corresponding calibration curves and presented in Table 4.10 and Table

4.11. The chromatogram of gallic acid and quercetin from various solvent extracts and drying methods are illustrated in figure 4.12(a) – figure. 4.15(c). The differences in amount of gallic acid content might be due to the methods of extraction. It was found that drying treatment showed significantly (p < 0.05) to the concentration of gallic acid. The finding obtained from this study is in agreement with Schieber et al.

(2001). They showed higher amounts of phenolic compounds in freeze-dried extracts of apple pomace. The amount of gallic acid decreased when the temperature (35-

55°C) was raised (Amirah et al., 2012). Temperature higher may affect physical properties, including surface tension, solubility, viscosity and diffusivity (Yang et al.,

2008). In conclusion, gallic acid is easily degraded when exposed to high temperature (Amirah et al., 2012).

The amount of gallic acid in S. asper leaf extract ranged from 0.0034 g/g to

0.0161 g/g. The amount of gallic acid increased as the solvent increased up to 70%.

The amount of gallic acid varied with different concentrations of ethanol extracts indicating the content of gallic acid is strongly dependent on the type of the solvents and concentrations (Turkmen et al., 2006; Lapornik et al., 2005). Aqueous extract

exhibited fairly high content of gallic acid as compared to ethanol extract. The same result was also obtained by Kwon (2007) in green tea leaves aqueous extracts.

Studies by Del-Rio et al. (1997) and Okwu (2004) reported that phenols are potent water soluble antioxidants which prevent oxidative cell damage which act as antiseptics, anticancer, anti-inflammatory effects and having mild anti-hypertensive.

Gallic acid is a natural phenolic compound that widely found in the plant kingdom (Rajalakshmi et al., 1996). Gallic acid is usually found in nature as esters and rarely as glycosides or in free form or as a part of tannin molecule. Gallic acid has been reported to possess anti-inflammatory, antimutagenic, anticancer, antioxidant (Nakatani, 1992; Huang et al., 1995), antimicrobial, antidiabetes (Martin et al., 2006), antifungal and antiviral properties (Elvira et al., 2006). A similar report was demonstrated by Inoue et al. (1994); Sohi et al. (2003) showed that gallic acid acted as a free radical scavenger, as an inducer of differentiation and apoptosis in leukaemia, lung cancer, and colon adenocarcinoma cell lines, as well as in normal lymphocyte cells. Gallic acid has also been used in the prevention of malignant transformation and cancer development which has the same properties of quercetin

(Ali et al., 2010).

Flavonoid content was found to be lower than the phenolic content in S. asper leaves extracts. Results from the study revealed that different solvent extraction and drying treatments did not affect the amount of quercetin in S. asper leaves extracts.

Study by Abascal et al. (2005) described that drying did not change the composition

from Euphorbia esula. As in comparison among the extractions, 30% and 70% EtOH showed higher amounts of quercetin compared to 50% EtOH and aqueous extracts.

30% and 70% EtOH from freeze-dried samples have proven to be the most efficient in extracting flavonoids from S. asper leaves (Table 4.7). This is in agreement with a study done by Keinanen and Julkunen (1996) content of quercetin glycosides were higher reported in freeze-dried compared to samples dried at 40˚C.

The present study showed that drying methods and solvents (pure or mixed) of varying polarities significantly (p < 0.05) while aqueous extract and freeze drying method performed better. However, no significant difference was encountered in quercetin contents of S. asper leaves from various drying treatments and solvents.

Here, gallic acids were the predominant phenolic compounds that might contribute highly to the high antioxidant activities of S. asper leaves extracts. The presence of these compounds may play an important role in S. asper leaves.

Fig. 4.12(a): HPLC chromatogram of 30% EtOH extract of S. asper fresh leaves.

Fig. 4.12(b): HPLC chromatogram of 30% EtOH extract of S. asper leaves by oven- dried leaves.

Fig. 4.12(c): HPLC chromatogram of 30% EtOH extract of S. asper leaves by freeze- dried leaves.

Fig. 4.13(a): HPLC chromatogram of 50% EtOH extract of S. asper fresh leaves.

Fig. 4.13(b): HPLC chromatogram of 50% EtOH extract of S. asper leaves by oven – dried leaves.

Fig. 4.13(c): HPLC chromatogram of 50% EtOH extract of S. asper leaves by freeze- dried leaves.

Fig. 4.14(a): HPLC chromatogram of 70%8 EtOH extract of S. asper fresh leaves.

Fig. 4.14(b): HPLC chromatogram of 70% EtOH extract of S. asper leaves by oven- dried leaves.

Fig. 4.14(c): HPLC chromatogram of 70% EtOH extract of S. asper leaves by freeze- dried leaves.

Fig. 4.15(a): HPLC chromatogram of aqueous extract of S. asper fresh leaves.

Fig. 4.15(b): HPLC chromatogram of aqueous extract of S. asper leaves by oven- dried leaves.

Fig. 4.15(c): HPLC chromatogram of aqueous extract of S. asper leaves by freeze- dried leaves.

The extraction of bioactive compounds from plant materials is the first step in the utilisation of phytochemicals in the preparation of dietary supplements, nutraceuticals, food ingredients, pharmaceutical, and cosmetic products. Pre- treatment is a crucial step to improve the quality of herbal products and its efficiency

(Jha and Prasad, 1996) without degrading the chemical compounds. Phenolics can be extracted from fresh, frozen or dried plant. Since most of the herbs are used as dried form, drying process may affect their phenolics content and antioxidant activity. It is important to evaluate the effect of different drying methods to maintain the antioxidant activity, total phenolics and flavonoid contents of S. asper leaves extracts as this information is not reported. In this work, oven drying and freeze drying were employed for sample treatment. Different drying treatments and solvents influenced the total phenolic and flavonoid content, as well as antioxidant activity of S. asper leaf extracts. Reduction in total phenolic and flavonoid contents from oven drying treatment would cause the decrease in antioxidant activity due to long drying period and temperature (Lin Tein et al., 1998; Maskan, 2001).

Drying resulted a significant decline in the phytochemical compositions of aqueous and ethanol S. asper leaves extract. Oven drying showed significantly (p <

0.05) lower values compared to freeze-dried extracts [Fig. 4.16(a) and 4.16(b)].

These results agree with study done by Ciou et al. (2008) where some compounds are degraded by Maillard reaction or enzymatic browning. This reduces the contents of flavonoid and phenolic contents. Phenolic compounds are easily hydrolysed and oxidised. Therefore, long extraction time and higher temperature increase the of

oxidation of phenolics. This decreases the yield of phenolics in the extracts (Lim and

Murtijaya, 2007).

From the analysis, freeze-drying process shown to enhance and preserve the

functional values of S. asper leaves. Freeze drying treatment is preferred for of S.

asper leaves. Others may degrade phytochemicals, inactivate polyphenols oxides,

enzymes and also some phenolic compounds decompose rapidly when it dries at

elevated temperature (Mueller-Harvey, 2001).

Fig. 4.16(a): Effects of different drying methods on total phenolic content of S. asper leaves. Bars with different letters are significantly different (p < 0.05). AE = aqueous extract, 70% EtOH = 70% ethanol extract, 50% EtOH = 50 % ethanol extract, 30% EtOH = 30% ethanol extract.

Fig. 4.16(b): Effects of different drying methods on total flavonoid contents of S. asper leaves. Bars with different letters are significantly different (p < 0.05). AE = aqueous extract, 70% EtOH = 70% ethanol extract, 50% EtOH = 50 % ethanol extract, 30% EtOH = 30% ethanol extract.

4.7 Comparison of the efficiency of different solvents on the antioxidant

activity from S. asper leaves extracts

Another factor to be considered is the type of solvent used for extraction. The

selection of extraction solvents is critical for plant samples as it will determine the

amount and type of phenolic compounds being extracted. Aqueous, alcohols

particularly acetone, ethanol and methanol are most commonly employed in phenolic

extraction of botanical materials (Naczk and Shahidi, 2004; Hayouni et al., 2007). In

this study, ethanol/water (70% EtOH, 50% EtOH, 30% EtOH) and water (Fig. 4.17a

and 4.17b) were chosen as extraction solvents because ethanol/water and water

formulations are relatively safe for human consumption as compared with other

organic solvents, such as acetone or methanol (Wendakoon et al., 2012). 68

Ethanol/water extraction of S. asper leaves showed a better performance compared to aqueous extracts. Findings by Suzuki et al. (2002) found that ethanol or methanol solutions containing some water, particularly those ranging from 40% to 80% ethanol, are more efficient in the extraction of polyphenolic compounds than pure water, ethanol or methanol itself.

Differences in extraction efficiency can be explained by the different polarity of the solvents used; in this case, ethanol extracted the most polar antioxidants. The same finding was reported by Marinova and Yanishlieva (1997) who suggested that the antioxidant activity of the extracts is strongly dependent on the types of solvent used due to compounds with different polarity exhibiting differing rates of antioxidant potential. Jung et al. (2006) also found out that the ethanol extracts contained higher amounts of total phenolics and flavonoids than aqueous from wild ginseng leaves. Thus, it can be assumed that S. asper leaves extracts possesses different antioxidant potential in different extraction mediums.

Solvents with different polarity had significant effects on polyphenol content and antioxidant activity where higher content was found in more polar solvents

(Siddhuraju et al., 2003; Thurkmen et al., 2006; Sultana et al., 2007). Similar results also have been reported by Amensour et al. (2009) and Liu et al. (2008) for other medicinal plants, where the more polar was the solvent used, the higher the amount of total phenolic and flavonoid compounds were extracted out. The difference in the antioxidant activity of each sample in different extracts implies that the extracting solvent used would affect the radical scavenging potency. Duffy and Power (2001)

also observed that different samples in different solvents would result in different antioxidant potentials. Study by Amensour et al. (2009) was reported in previous studies that ethanol extracts of licorice samples displayed high antioxidant potential compared to water extracts. This finding supports the present antioxidant results.

The data clearly indicated that 70% EtOH of S. asper leaf extract had higher amount of antioxidant activity, phenolic and flavonoid compounds as ethanol is an organic and volatile solvent as compared to aqueous extracts. 70% EtOH extract was slightly significantly (p < 0.05) higher than aqueous extract. Similarly, result from

Narender et al. (2012) using 70% EtOH Mesua ferrea leaves showed better activity.

These results were also strongly supported in a study by Bimakr (2010) who reported that the highest concentrations of more bioactive compounds were detected with ethanol 70% due to its higher polarity than pure ethanol. Ethanol alone or acetone alone was not effective as a solvent for extraction of phenolic compounds

(Wendakoon et al., 2012). Apart from that also, these results were in agreement with

Normala and Mardhiah (2010) who reported that phenolic and flavonoid compounds were not extracted completely in distilled water. This is because the solvent only extract the hydrophilic compound such as quercetin, gallic acids, free and bound cuticular phenolics (Claudina et al., 2004).

Volatile solvent is more efficient in plant cell wall degradation, therefore, it has the ability to extract a greater amount of endocellular materials than water. Most medicinal herbs are popularly prepared using an aqueous extract as extraction solvent

in seeking a reduction on the use of organic solvents as well as the cost of extraction

(Cacace and Mazza, 2002), yet ethanol is one of the best solvents for polyphenol

extraction and safe for human consumption (Shi et al., 2005). However, ethanol is

more preferable solvent because of its nontoxic, environmentally safe and

inexpensive features. Thus, ethanol and aqueous extracts are the most widely

employed solvents due to their more hygienic characteristics (Moure et al., 2001). a )

Fig. 4.17(a): Effects of different solvents for extraction of total phenolic content of S. asper leaves. Bars with different letters are significantly different (p < 0.05). FL: fresh leaves; FD: freeze drying; OD: oven drying.

Fig. 4.17(b): Effects of different solvents for extraction of flavonoid contents of S. asper leaves. Bars with different letters are significantly different (p < 0.05). FL: fresh leaves; FD: freeze drying; OD: oven drying. 4.8 Cytotoxicity activity of S. asper leaves extract against HT29 cell lines in

vitro

In figure 4.18 (a), the extract has significantly (p < 0.05) inhibited the

proliferation of HT29 cells in time-dependent manner at concentrations above 125

µg/ml. At 24 h, the extract has induced the proliferation of the cells at concentrations

ranging from 15.6 to 62.5 µg/ml. On the other hand, the growth inhibition was

observed at concentrations from 125 to 1000 µg/ml. The inhibition of cell

proliferation was consistently observed in following incubation times with significant

differences in concentrations from 125 to 1000 µg/ml. These findings are in

agreement with previous report where most plant extracts depend on dose and time to

demonstrate their cytotoxicity effects (Shahin et al., 2008).

Interestingly, a different pattern of the cell proliferation was observed in response to the ethanolic extract, as shown in figure 4.18(b). The result indicated that the maximal cell growth inhibition was observed in the lower concentration treatment which was 15.63 µg/ml at all incubation times. This result suggested that the extract at low concentration, particularly 15.63 µg/ml induced higher cytotoxicity effect while the higher concentrations above 15.63 µg/ml induced the proliferation of the cells. Furthermore, the cell treatment at 24 h produced highest inhibition of the cell growth, followed by 48 and 72 h. This indicates that the compounds in the ethanolic extract might initiate cell recovery throughout the incubation times. This data suggested that the HT29 cells were not sensitive to the ethanolic extract with more than 70% of cell viability.

Fig. 4.18(a): Cytotoxic effect of aqueous extract of S. asper leaves at 24, 48 and 72 h of incubation. The reported values are the means ± S.D. (n=3).

The MTS test is a simple bioassay used for the primary screening of crude plant extracts and isolated compounds. So far, no studies on the anticancer potential of crude extracts from S. asper leaves have been undertaken on human cancer cell lines such as HT29. Therefore, a preliminary cytotoxicity study of crude extracts of aqueous and 70% EtOH of S. asper leaves were evaluated on HT29 colorectal cancer cell line. The HT29 cells were treated with various concentrations in a double dilution manner which were 15.6, 31.3, 62.5, 125.0, 250.0, 500.0 and 1000.0 µg/ml and incubated at 24, 48 and 72 h. Overall, as depicted in figure 4.20(a), and 4.20(b), the results revealed that aqueous and ethanolic extracts inhibited the proliferation of

HT29 cells, in comparison with control. However, there were no IC50 values exhibited by both extracts, indicating that the extracts do not have active compounds.

According to American National Cancer Institute (US NCI), a crude extract is generally considered to have in vitro cytotoxic activity if the IC50 value in carcinoma cells, following incubation between 48 and 72 h is less than 20 µg/ml (Boik, 2001).

Cancer is known as uncontrolled cell proliferation which is caused by abnormalities in the genetic material of the transformed cells. These abnormalities may be due to the effects of carcinogens, such as air pollution, tobacco smoke, UV radiation, chemicals, or infectious agents.

The preliminary cytotoxicity study demonstrated different patterns of cell proliferation in response towards the aqueous and ethanolic extracts of S. aper leaves. Even though no IC50 values were observed upon various concentrations up to

1000 µg/ml, it has been shown that the aqueous extract has extracted active

compounds that produced higher toxicity effects as compared to the ethanolic extract. Similar finding by Qader et al., (2011) also found that crude ethanol and aqueous extract of kesum leaf extract did not show any inhibition percentage of cell viability against Hs888Lu cell lines. Study by Evren (2010) reported that they also did not find any inhibitory activity of the crude extract of olive leaf on the PC3 and

MCF7 cell lines. This result agrees with many previous researches that reported most plant extracts which were reported on having other activities related to cancer preparation and had no cytotoxic activity, such as Smilax glabra, Derris scandens and Strychnos nux-vomica which showed anti-inflammatory activities and analgesic effects (Jiang et al., 1997; Yin et al., 2003). The discrepancy of this finding with

Maznah et al. (2012) findings might be due to different extraction process and the sensitivity of the cell line to the anticancer compounds in the extract or tissue specific response. Rosskopf and co-workers (1992) also reported that some anticancer agents might exhibit their antitumor activity in vivo but not in vitro cytotoxic activity and this may be due to immune modulation by the compound which could lead to antitumor activity in vivo.

Fig. 4.18(b): Cytotoxic effect of 70% EtOH extract (200 mg/ml) of S. asper at 24h, 48h and 72h incubation. The reported values are the means ± S.D. (n=3).

CHAPTER 5

CONCLUSIONS AND RECOMMENDATIONS 76

5.1 General Conclusions

The present study has provided some comparative data on the physical, chemical, nutritive and phytochemical properties of S. asper leaves from various extracts and drying treatments. S. asper leaves, fresh and treated with various drying treatments (oven-drying and freeze-drying) and extracted with different solvents

(aqueous and ethanol) are good sources of nutrients and phytochemicals. S. asper leaves contains a substantial amount of protein, fibre, fat, carbohydrate, minerals, chlorophyll, gallic acid and quercetin which can contribute to the nutrient requirements of a human body and it is suitable as a health supplement. S. asper leaves was found to be a potential source of bioactive compounds such as phenolic, flavonoids, tannins, saponin, alkaloid, terpenoids and cardiac glycosides which have favourable properties such as antioxidant, antimicrobial activities, anti-diabetic, anti- inflammatory and cytotoxic agent needed to combat various kinds of chronic diseases in human beings. Apart from that, S. asper aqueous and ethanol leaves extract also exhibited high antioxidant activity, total phenolic and total flavonoid contents thus it can be used as a good natural antioxidant agent. Generally, 70%

EtOH extract of S. asper leaves gave better performance, possessed greater radical scavenging activity, phenolic and flavonoid contents compared to aqueous extracts where 70% EtOH extract was slightly significant (p < 0.05) higher than in aqueous extract. A strong correlation was found between antioxidant activity and phenolic contents. Phytol and acetic acid are the major compounds detected while other compounds such as n-Hexadecanoic acid, Hexadecanoic acid, 9,12,15-

Octadecatrienoic acid, β-Tocopherol and Vitamin E were detected during the screening of VOCs of S. asper all extracts and fresh leaf. However, crude aqueous and ethanol extracts did not show any inhibition activity on HT 29 cell lines in vitro.

IC50 > 20 µg/ml suggested that S. asper crude extracts are not potent as an anticancer therapeutic (colon cancer). The mode of action for cytotoxicity of S. asper leaves may be different on different human cancer cell lines.

Drying treatment in general, had significant (p < 0.05) effect on most of the chemical constituents. Different drying treatment and extraction solvent of varying polarities differ significantly (p < 0.05) in which oven drying caused significantly (p

< 0.05) decrease compared to freeze-dried extracts. In this study, freeze drying treatment seemed to be the preferred choice of drying in the preparation and processing of S. asper since it resulted in appreciable yield, phytochemical and nutritive composition and better colour, and it is less time consuming and simple treatment. In fact, through this study, herbal preparation developed using oven drying was found to be inferior on most phytochemical content and physicochemical properties compared freeze-dried products. However, the herbal preparation developed using oven drying treatments still retain an appreciable amount of phytochemicals and physico-chemical of S. asper leaves, and thus have potential for commercial purposes.

5.2 Recommendations for future works

Further studies should be performed to isolate and characterise the active components and also evaluate the most potent fraction of S. asper leaves crude extracts to test its cytotoxicity. Future efforts should also concentrate more on the in vivo studies of the isolated active component and also in clinical trials in order to confirm its efficacy and understand its mechanism of action to be used for medicinal purposes, particularly in Malaysia. In this study, HT29 cell lines were used to study the cytotoxicity effect of S. asper extracts. Further studies could be conducted on other cell lines. The effectiveness of the herbal preparation in reducing risks for chronic diseases such as cancer, coronary heart disease, and diabetes particularly from the leafy part of S. asper could also be tested in vivo.

REFERENCES

Abascal, K., Ganora, L. and Yarnell, E., 2005. The Effect of Freeze drying and Its Implications for Botanical Medicine. A review Phytotherapy Research,19,pp.655-660.

Abu Arra Basma1, Zuraini Zakaria1, Lacimanan Yoga Latha2， Sreenivasan Sasidharan., 2011. Antioxidant activity and phytochemical screening of the methanol extracts of Euphorbia hirta L. Asian Pacific Journal of Tropical Medicine, pp.386-390

Adesegun, S.A., Fajana, A., Orabueze, C.I. and Coker, H.A.B., 2007. Evaluation of Antioxidant Properties of Phaulopsis Fascisepala CBCl. (Acanthaceae). Oxford Journal,6,pp.227-213. Aherne, S.A. and O’Brien N.M., 2002. Dietary Flavonols: Chemistry, Food Content, and Metabolism. Nutrition,18,pp.75-81. Aiyelaagbe, O.O. and Paul M. Osamudiamen., 2009. Phytochemical Screening for Active Compounds in Mangifera indica Leaves from Ibadan, Oyo State. Plant Sciences Research,2(1),pp.11-13. Ajibade, V. A. and Fagbohun, E. D., 2010. Phytochemical, Proximate Analysis and Antimicrobial Activities of Methanolic Crude Extract of Tylophora glauca (Bullock). J. Pharm. Biomed. Sci., 1(1),pp.7- 12 Ali Ghasemzadeh, Hawa Z. E., Jaafar and Asmah Rahmat., 2011. Effects of Solvent Type on Phenolics and Flavonoids Content and Antioxidant Activities in Two Varieties of Young Ginger (Zingiber Officinale Roscoe) Extracts. Journal of Medicinal Plants Research,5(7),pp.1147-1154.

Aliyu, A.B., Musa, A.M., Oshanimi, J.A., Ibrahim H.A., and Oyewale, A.O., 2008. Phytochemical Analyses and Mineral Elements Composition of Some Medicinal Plants of Northern Nigeria. Journ. Pharm. Sci., 7(1),pp.119– 125. Alok Saxena, Bawa, A.S. and Raju, P.S., 2009. Phytochemical Changes in Fresh- Cut Jackfruit (Artocarpus Heterophyllus L.) Bulbs during Modified Atmosphere Storage. Food Chemistry,115,pp.1443–1449.

Amensour, M., Sendra, E., Abrini, J., Bouhdid, S., Perez-Alvarez, J.A. and Fernandez-Lopez, J., 2009. Total Phenolic Content and Antioxidant Activity of Myrtle (Myrtus Communis) Extracts. Nat Pro Comm,4,pp.819–824.

Amin, I., and Tan, S. H., 2002. Antioxidant Activity of Selected Commercial Seaweeds. Mal J Nutr,8(2),pp.167-177. Amirah, D.M., Reddy Prasad. and Maksudur, R. Khan., 2012. Comparison of Extraction Techniques on Extraction of Gallic Acid from Stem Bark of Jatropha curcas. Journal of Applied Sciences,12,pp.1106-1111.

Amitabye Luximon-Ramma, Theeshan Bahorun, Mohammed A., Soobrattee. and Okezie I. Aruoma., 2002. Antioxidant Activities of Phenolic, Proanthocyanidin, and Flavonoid Components in Extracts of Cassia fistula. Journal Agricultural Food Chemistry,50,pp.5042-5047.

Amokaha, R.A., Ubwa, S.T., Otokpa, M. and Shenga, G., 2002. Phytochemical Screening of Danta Stramo niumlinn (Silanacease) Seeds. Journal of Chemical, 27,pp.105-106. Arnon, D.I., 1949. Copper Enzymes in Isolated Chloroplasts Polyphenoloxidase in Beta vulgaris. Plant Physiology,24,pp.1–15. Andrade, D., Gil, C., Breitenfeld, L., Domingues, F. and Duarte, A.P., 2009. Bioactive Extracts from Cistus ladanifer and Arbutus unedo L. Ind Crops Prod,30,pp.165–167. Antia, B.S., Akpan, E.J., Okon , P.A. and Umoren, I.U., 2006. Nutritive and Anti-Nutritive Evaluation of Sweet Potatoes (Ipomoea batatas) Leaves. Pak. J. Nutr.,5,pp.166-168. AOAC., 2005. Official Methods of Analysis. Association of Official Analytical Chemist. EUA. Arabhosseini, A., Huisman, W., van Boxtel, A. and Muller, J., 2007. Long Term Effects of Drying Conditions on The Essential Oil and Color of Tarragon Leaves During Storage. Journal of Food Engineering,79,pp.561–566.

Arabshahi, D.S. and Urooj, A., 2007. Antioxidant Properties of Various Solvent Extracts of Mulberry (Morus indica L.) leaves. Food Chemistry, 102,pp.1233– 1240.

Arabshahi, D.S., Devi, D.V. and Urooj, A., 2007. Evaluation of Antioxidant Activity of Some Plant Extracts and Their Heat, pH and Storage Stability. Food Chemistry,100,pp.1100–1105.

Arathi, K. and Suneetha, V., 2011. Estimation of chlorophyll content in common household medicinal leaves and their utilization to avail health benefits of chlorophyll. Journal of Pharmacy Research,4(5),pp.1412-1413.

Arslan, D. and Ozcan, M.M., 2008. Evaluation of Drying Methods with Respect to Drying Kinetics, Mineral Content and Color Characteristics of Rosemary Leaves. Energy Convers. Manage.,49,pp.1258–1264.

Arslan, D. and Ozcan, M.M., 2011. Evaluation of Drying Methods With Respect to Drying Kinetics, Mineral Content, And Color Characteristics of Savory Leaves. Food Bioprocess Technology,5(3),pp.983-991.

Arts, I.C. and Hollman, P.C., 2005. Polyhenols and Disease Risk in Epidemiological Studies. Am J Clin Nutr,81,pp.317-325.

Arulpriya, P., Lalitha, P. and Hemalatha, S., 2010. Antioxidant Activities of the Extracts f the Aerial Roots of Pothos aurea. Der Chemica Sinica,1(2).pp.73-79. Atangwho, I. J., Ebong, P. E.,Eyong, E. U., Williams, I. O., Eteng, M. U. and Egbung, G. E., 2009. Comparative chemical composition of leaves of some antidiabetic medicinal plants: Azadirachta indica, Vernonia amygdalina and Gongronema latifolium. African Journal of Biotechnology, 8(18),pp.4685-4689. Ayitey-Smith, E. and Addae-Mensah I.W., 1977. Preliminary Phytochemical Analysis of some Plant Seeds. Afr J Pharmacol Drug Res 4,pp.7-8.

Azizah, A. H., Nik Ruslawati, N. M., and SweeTee, T., 1999. Extraction and Characterization of Antioxidants from Cocoa By-Products. Food Chemistry,64,pp.199–202.

Bahorun, T., Trotin, F., Pommery, J., Vasseur, J. and Pinkas, M., 1994. Antioxidant Activities of Crataegus monogyna Extracts. Planta Med.,60,pp.323- 328.

Balestrin, L., Dias, J.F.G., Miguel, O.G., Dall’Stella, D.S.G. and Miguel, M.D., 2008. Contribuição ao estudo Fitoquímico deDorstenia Multiformis Miquel (Moraceae) Comabordagem em Atividade Antioxidante. Rev Bras Farmacogn,18,pp.230-235. Barua A.K., Pal S.K. and Basu K.K., 1968. Chemical Examination of Streblus asper. Journal Indian Chemical Society,45,pp.7.

Bazykina, N. I., Nikolaevskii, A. N., Filippenko, T. A. and Kaloerova, V. G., 2002. Optimization of conditions for The Extraction of Natural Antioxidants from Raw Plant Materials. Pharmaceutical Chemistry Journal,36(2),pp.100–103.

Betancur-Ancona, D., Peraza-Mercado, G., Moguel-Ordoñez, Y. and Fuertes- Blanco, S., 2004. Physico-chemical characterization of lima bean (Phaseolus lunatus) and Jack bean (Canavalia ensiformis) fibrous residues. Journal Food Chem,84,pp. 287–29.

Bhakuni, D.S., Dhar, M.L., Dhar, M.M., Dhawan, B.N. and Mehrotra, B.N., 1969. Screening of Indian Plants for Biological Activity. Indian J Exp Biol,7,pp.250–62.

Bhargava, A., Shukla, S. and Ohri, D., 2006. Chenopodium quinoa—An Indian perspective. Ind. Crop. Prod.,23,pp.73–87. Bhattacharya, S., Kamat, J.P., Bandyopadhyay, S.K. and Chattopadhyay, S., 2009. Comparative Inhibitory Properties of Some Indian Medicinal Plant Extracts Against Photosensitization-induced Lipid Damage. Food Chemistry,113,pp.975-979. Bimakr, M., 2010. Comparison of Different Extraction Methods for The Extraction of Major Bioactive Flavonoid Compounds from Spearmint (Mentha spicata L.) Leaves. Food Bioproduction Process,pp.1-6. Bodo, R., Azzouz, A. and Hausler, R., 2004. Antioxidative Activity of Water Hyacinth Components. Plant Sci,166,pp.893–899.

Bodoprost, J. and Rosemeyer, H., 2007. Analysis of Phenacylester Derivatives Fatty Acids From Human Skin Surface Sebum By Reversed-Phase HPTLC:Chromatographic Mobility as A Function of Physicochemical Properties. Int J Mol Sci,8,pp.1111-1124.

Boik J., 2001. Natural Compounds in Cancer Therapy. Oregon Medical Press, Minnesota, USA, p. 25.

Bombardelli, E., Cristoni, A. and Morazzoni, P., 2000. Ginkgo biloba, in: T.A. van Beek (Ed.), Medicinal and Aromatic Plants Industrial Profiles,12,pp. 475.

Boots, A.W. et al., 2008. In vitro and Ex Vivo Anti-Inflammatory Activity of Quercetin in Healthy Volunteers. Nutrition,24,pp.703-710. Bos, R., Koulman, A. and Woerdenbag, H.J., et al., 2002. Volatile components from Antriscus sylvestris (L.) Hoffm. J Chromatography,966:pp.233–238.

Bouktaib, M., et al. 2002. Regio- and Stereoselective Synthesis of the Major Metabolite of Quercetin, Quercetin-3-O-b-D-glucuronide. Tetrahedron Letters,43,pp. 6263-6266. Boyd Scott., 2007. CellTiter 96® Aqueous One Solution Cell Proliferation Assay From Promega.

Braga, N.P., Cremasco, M.A. and Valle, R.C.C.R., 2005. The Effects of Fixed- bed Drying on The Yield and Composition of Essential Oil from Long Pepper (Piper hispidinervium CDC) Leaves. Brazil Journal Chemical Engineering,22,pp.257–262.

Bravo L., 1998. Polyphenols: Chemistry, Dietary sources, Metabolism and Nutritional Significance. Nutrition Reviews,56,pp.317-333. Buavaroon, Srichaikul., Ronchai, Bunsang., Supachai, Samappito., Luchai, Butkhup. and Gordon, Bakker., 2011. Comparative Study of Chlorophyll Content in Leaves of Thai Morus alba Linn. Species. Plant Sciences Research,3(2),pp.17- 20 Buggle, V., Ming, L.C., Furtado, E.L., Rocha, S.F.R. and Marques, M.O.M., 1999. Influence of Different Drying Temperatures on The Amount of Essential Oils and Citral Content in Cymbopogon citrates (DC) Stapf.-Poaceae. Acta Hortic.,500,pp.71–74. Burkill, I.H. 1966. A Dictionary of the Economic Products of the Malay Peninsula. Ministry of Agriculture and Cooperatives, Kuala Lumpur. Cannell, R.J.P., 1998. Natural Products Isolation. Human Press Inc. New Jersey, pp. 165-208. Bushra, S., Farooq, A. and Muhammad A,. 2009. Effect of Extraction Solvent/Technique on the Antioxidant Activity of Selected Medicinal Plant Extracts. Molecules, 14, pp. 2167-2180 Carol, S.J., Cindy, M., Kim, M.S., Amanda, J. and Buller, M.S., 2004. Vinegar Improves Insulin Sensitivity to a High-Carbohydrate Meal in Subjects With Insulin Resistance or Type 2 Diabetes. Diabetes Care"

Cemeroglu, B. and Acar, J., 1986. Fruit and Vegetable Processing Technology. Food Technology Association,6,pp.501-507.

Chan, E.W.C., Lim, Y.Y., Wong, S.K., Lim, K.K., Tan, S.P., Lianto, F.S. and Yong, M.Y., 2009. Effects of Different Drying Methods on The Antioxidant Properties of Leaves and Tea of Ginger Species. Food Chemistry,113,pp.166– 172. Chaovanalikit, A., Mingmuang, A., Kitbunluewit, T., Choldumrongkool, N., Sondee, J. and Chupratum, S., 2012. Anthocyanin and Total Phenolics Content of Mangosteen and Effect of Processing on The Quality of Mangosteen Products, International Food Research Journal,19(3),pp.1047-1053.

Chaturvedi, S.K. and Saxena, V.K., 1984. β-sitosterol-3-O-β -D- arabinofuranosyl-O-α-L-rhamnopyranosyl-O-β-D-glucopyranoside from Roots of Streblus asper Lour. Acta Cienc Indica (Ser) Chem,10,pp.22–23.

Chaturvedi, S.K. and Saxena, V.K., 1985. A New Saponin Lupanol-3-O-β-D- Glucopyranosyl (1-5)-O-β-D xylofuranoside from The Roots of Streblus asper. Indian Journal Chemistry,24,pp.562.

Chaturvedi, S.K., 2006. Chemical analysis of Streblus asper seeds. Asian Journal Chemical,32(4),pp.351–352.

Chawla AS, Kapoor VK, Mukhopadhyay R, Singh M. Constituents of Streblus asper. Fitoterapia 1990;61:186.

Chew, K. K., Khoo, M. Z., Ng, S. Y., Thoo, Y. Y., Wan Aida, W. M. and Ho, C. W., 2011. Effect of Ethanol Concentration, Extraction Time and Extraction Temperature on The Recovery of Phenolic Compounds and Antioxidant Capacity of Orthosiphon stamineus Extracts. International Food Research Journal,18(4),pp.1427-1435

Chew, Y.L., Goh, J.K. and Lim, Y.Y., 2009. Assessment of In Vitro Antioxidant Capacity and Polyphenolic Composition of Selected Medicinal Herbs from Leguminosae family in Peninsular Malaysia. Food Chemistry,119,pp.373-378.

Chopra RN, Nayar SL, Chopra IC., 1956. Glossary of Indian Medicinal Plants,1st edn, New Delhi: NISCOM,pp.235.

Chumpawadee, S. and Pimpa, O., 2009. Effect of Burma Padauk (Plerocarpus indicus), Rain Tree (Samanea saman (Jacg.) Merr.) and Siamese Rough Bush (Streblus asper) Leaves as Fiber Sources in Total Mixed Ration on in vitro Fermentation. Asian Journal of Animal and Veterinary Advances, 4,pp.1-8. Ciou, J.Y., Wang, C.C.R., Chen, J. and Chiang, P.Y., 2008. Total Phenolics Content and Antioxidant Activities of Extracts from Dried Water Caltrop (Trapa taiwanensis Nakai) hulls. Journal Food Drug Analysis,16,pp.41–47.

Cristina Wasowski MM., 2012. Flavonoids as GABAA receptor ligands: The whole story. J Exp Pharmacol,4,pp.9-24.

Das, K., Tiwari, R.K.S. and Shrivastava, D.K., 2010. Techniques for Evaluation of Medicinal Plant Products as Antimicrobial Agent: Current Methods and Future Trends. Journal of Medicinal Plants Research,4(2),pp.104-111.

De-Bruyne, T,, - Pieters, L., Deelstra, H. and Ulietinck, A., 1999. Condensed Vegetable Tannins: Biodiversity in Structure and Biological Activities. In Biochemical Systematic and Ecology,27,pp.445-459.

Dehpour, A.A., Ebrahimzadeh, M.A. and Nabavi, S.F., 2009. Antioxidant Activity of Methanol Extract of Ferula assafoetida and Its Essential Oil Composition. Grasas Aceites,60(4),pp.405-412. Dei, H.K, Rose, S.P. and Mackenzie, A.M., 2007. Shea nut (Vitellaria paradoxa) Meal as A Feed Ingredient for Poultry. In World’s Poultry Science Journal,63,4,pp. 611-624.

Del Bano, M.J, Lorente, J., Castillo, J., Benavente-Garcia, O., Marin, M.P., Del Rio, J.A., Ortuno, A. and Ibarra, I., 2004. Flavonoid Distribution During The Development of Leaves, Flowers, Stems, and Roots of Rosmarinus officinalis. Postulation of a biosynthetic pathway. Journal Agricultural Food Chemistry,52,pp.4987-4992. Del-Rio, A., Obdululio, B.G., Casfillo, J., Marin, F.G. and Ortuno, A., 1997. Uses and Properties of Citrus Flavonoids. Journal Agricultural Food Chemistry,45,pp.4505-4515. Depkevicius, A., Van Beek, T.A., Lelyveld, G.P., Van Veldhuizen, A., De Groot, A. and Linssen J.P., 2002. Isolation and Structural Elucidation of Radical Scavengers from Thymus vulgaris Leaves. Journal of Natural Products,65,pp.892–896. De-whalley C, Rankin S M, Houct J R S, Jessup W, and Leake D S,1990. Flavonoids Inhibit the Oxidative Modification of Low Density Lipoproteins by Macrophages. Biochemistry Pharmacology,39,pp.1743-1750.

Diaz-Maroto, M.C., Perez-Coello, M.S., Vinas, M.A.G. and Cabezudo, M. D., 2003. Influence of Drying on The Flavour Quality of Spearmint (Mentha spicata L.). Journal of Agricultural and Food Chemistry,51,pp.1265-1269.

Doymaz, I., Tugrul, N,, Pala, M., (2006). Drying characteristics of dill and parsley leaves. Journal Food Engineering,77(3),pp.559–565.

Dr. Parul Lakhanpal, M.D. and Dr. Deepak Kumar Rai, M.D., 2007. Quercetin: A Versatile Flavonoid. Internet Journal of Medical Update,2,pp.2.

Duffy, C.F. and Power, R.F., 2001. Antioxidant and Antimicrobial Properties of Some Chinese Plant Extracts. International Journal of Antiomicrobial Agents,17,pp.527-529. Ebrahimzadeh, M.A., Nabavi, S.F. and Nabavi, S.M., 2009. Antioxidant Activities of Methanol Extract of Sambucus ebulus L. Flower. Journal Biology Science,12(5),pp.447-450. Edeoga, H.O. and Eriata, D.O.,2001. Alkaloid, Tannin and Saponin Contents of Some Nigeria Medicinal Plants. Journal Medical Aromatic Plant Science,23,pp.344-349. Edris, A.E., 2007. Pharmaceutical and Therapeutic Potencials of Essential Oils and Their Individual Volatile Constituents: A Review, Phytotherapy Research, 21,pp. 308–323.

EFSA (European Food Safety Authority)., 2008. Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with

Food. Flavouring Group Evaluation 88, (FGE.88)[1] - Consideration of Phenol and Phenol Derivatives. The EFSA Journal,857,pp.1-18.

EFSA (European Food Safety Authority)., 2012. Scientific Opinion on the safety and efficacy of phenol derivatives containing ring-alkyl, ring-alkoxy and side- chains with an oxygenated functional group (chemical group 25) when used as flavourings for all species. The EFSA Journal,10(2),pp.2573-2592.

Elegbede, J.A.,1998. Legumes. In: Nutritional quality of plant foods. Osagie, A. U. Eka, O. U. (Eds), Post harvest research Unit, University of Benin,pp.53-83.

Ellen, D.G., Ellen, L, Danny, G. and Guy, S., 2007. Novel Advances with Plant Saponins as Natural Insecticides to Control Pest Insects. Pest Technology,1(2),pp.96-105. El-Mahmood, A.M. and Doughari, J.H., 2008. Phytochemical Screening and Antibacterial Evaluation of The Leaf and Root Extracts of Cassia alata Linn, African Journal of Pharmacy and Pharmacology,2(7),pp.124-129.

Elvira, G.M., Chandra, S., Marco Vinicio, R.M. and Wenyi, W., 2006. Food Chemistry Toxicology,44,pp.1191.

Eric Wei Chiang Chan, Suit Ying Eng, Yuen Ping Tan, Zhiew Cheng Wong, Phui Yan Lye and Lea Ngar Tan. (2012). Antioxidant and Sensory Properties of Thai Herbal Teas with Emphasis on Thunbergia laurifolia Lindl. Chiang Mai Journal Science,39(4),pp.599-609.

Evans, W.C., 1996. Trease and Evans’ Pharmacognosy. 14th ed. WB Saunders Company, London; pp.224-228.

Evren Altiok, 2010. Recovery Of Phytochemicals (Having Antimicrobial And Antioxidant Characteristics) From Local Plants. Ph.D thesis, İzmir Institute of Technology.

Fan, X.H., Cheng, Y.Y., Ye, Z.L., Lin, R.C. and Qian, Z.Z., 2006. Multiple Chromatographic Fingerprinting and Its Application To The Quality Control of Herbal Medicines. Anal. Chim. Acta,555,pp.217-224. Femenia, A., Garcia-Pascual, P. G., Simal, S. and Rossello, C., 2003. Effects of Heat and Dehydration on Bioactive Polysaccharide Acemannan and Cell Wall Polymers from Aloe barbadensis Miller. Carbohydrate Polymers,51,pp.397–405.

Feng, H., Tang, J., Mattinson, D.S. and Fellman, J.K., 1999. Microwave and Spouted Bed Drying of Frozen Blueberries: The Effect of Drying and Pretreatment Methods on Physical Properties and Retention of Flavor Volatiles. J Food Proc Preserv,23,pp.463–479.

Fiebig, M., Duh, C.Y., Pezzuto, J.M., Kinghorn, A.D. and Farnsworth, N.R., 1985. Plant Anticancer Agents, XLI. Cardiac glycosides from Streblus asper. Journal Natural Product,48,pp.981–85.

Filella, I., Serrano, I., Serra, J. and Penuelas. J., 1995. Evaluating Wheat Nitrogen Status with Canopy Reflectance Indices and Discriminant Analysis. Crop Sci. ,35,pp.1400-1405. Fukumoto, L.R., and Mazza, G., 2000. Assessing Antioxidant and Prooxidant Activities and Phenolic Compounds. Journal of Agricultural and Food Chemistry, 48,pp.3597–3604. Gaitonde, B.B., Vaz, A.X. and Patel, J.R., 1964. Chemical and Pharmacological Study of Root Bark of Streblus asper Linn. Indian Journal Medical Science,18,pp.191–9. Gallaher, R.N., 2004. Methods for Analysis of Dry Tea Material and Herbal Infusions. Personal communication, University of Florida. Gao, X., Ohlander, M. J.N., Bjork, L. and Trajkovski, V. 2000. Changes of antioxidant effects and their relationnship to phytonutrients in fruits of sea buckthorn (Hippophae rhamnoides L.) during maturation. Journal Agricultural Food Chemistry,48,pp.1485–90. Gartner, C., Stahl, W. and Sies, H., 1997. Lycopene is More Bioavailable from Tomato Paste than from Fresh Tomato. Am. Journal Clinical Nutrition,66,pp.116-122. Gazzani, G., Papetti, A., Massolini, G., & Daglia, M. (1998). Anti- and prooxidant activity of water soluble components of some common diet vegetables and the effect of thermal treatment. Journal of Agricultural and Food Chemistry, 46, 4118–4122.

Genin, N. and Rene, F. 1995. Analyse du role de la transition vitreuse dans les procedes de conservation agroalimentaires. Journal of Food Engineering,26,pp.391-408.

Ghasemzadeh, A., Jaafar, H.Z.E. and Rahmat, A., 2010. Antioxidant Activities, Total Phenolics and Flavonoids Content in Two Varieties of Malaysia Young Ginger (Zingiber officinale Roscoe). Molecules,15,pp.4324-4333.

Giorgia, S., Lorenza T. and Dante M.D.F., 2007. Effects of Extraction Time, Temperature and Solvent on Concentration and Antioxidant Activity of Grape Marc Phenolics, Journal of Food Engineering,81,pp.200–208.

Gouri, S., Yogesh K.T. and Rajinder K.G., 2011. Preliminary Evaluation of Nutraceutical and Therapeutic Potential of Raw Spondias pinnata K., An Exotic Fruit of India. Food Research International,44,pp.2076–2087. 88

Gowthami, M., Tamil, S.S., Senthil, K.G. and Panneerselvam, A., 2012. Phytochemical Analysis and Antibacterial Properties of Leaf Extract of Azima tetracantha (Lam.). Asian Journal of Plant Science and Research,2 (2),pp.110- 114

Grassman. J., (2005). Terpenoids as plant antioxidant. Vitam Horn,72,pp.505-535 Guardia, T., Rotelli, A.E., Juarez, A.O., Pelzer, L.E., 2001. Anti-inflammatory properties of plant flavonoids. Effects of Rutin, Quercetin and Hesperidin on Adjuvant Arthritis in Rat. Farmaco.,56(9),pp.683-687.

Ha, Y.L., Storkson, J. and Pariza, M.W., 1990. Inhibition of Benzo (a) Pyrene- Induced Mouse Forestomach Neoplasia by Conjugated Dienoic Derivatives of Linoleic Acid. Cancer Research,50,pp.1097-1124.

Hamrouni-Sellami, I., Wannes, W.A., Bettaieb, I., Berrima, S., Chahed, T., Marzouk, B. and Limam, F., 2011. Qualitative and Quantitative Changes in The Essential Oil of Laurus Nobilis L. Leaves as Affected by Different Drying Methods. Food Chemistry,126,pp.691–769. Han, X., Shen, T., Lou, H., 2007. ''Dietary Polyphenols and Their Biological Significance''. International Journal of Molecular Science 8, pp 950-988. Hatano, T.,1995. Constituents of Natural Medicines With Screening Effects on Active Oxygen Species Tannins and Related Polyphenols. Nature Medicine,49,pp.357-363.

Havlikova, L. and Mikova, K., 1985. Heat Stability of Anthocyanins. Z. Lebensm.-Unters. Forsch.,181,pp.427-432.

Hawkins, E.B. and Ehrlich, S.D., 2006. Gotu Kola. University of Maryland Medical Center. Baltimore. USA. Hayashi, T., Okamuka, K., Kawasaki, M. and Morita, N., 1993. Production of Diterpenoids by Cultured Cells from Two Chemotypes Scoparia dulcis. Phytochemistry,35(2),pp.353-356. Hayouni, E.A., Abedrabba, M., Bouix, M. and Hamdi, M., 2007. The Effects of Solvents and Extraction Method on The Phenolic Contents and Biological Activities In Vitro of Tunisian Quercus coccifera L. and Juniperus phoenicea L. Fruit Extracts. Food Chemistry,105,pp.1126-1134. Hazrizul A.Z., Rabia S.S. and Siti A.H., 2009. Antibacterial Effects of Streblus asper Lour Leaves Crude in Ethanol Extract on Oral Bacteria. Archives of Orofacial Sciences,4(2),pp.47-64.

Hertog, M.G., Feskens, E.J. and Hollman, PC., 1993. Dietary Antioxidant Flavonoids and Risk of Coronary Heart Disease. The Zutphen Elderly Study, Lancet,342,pp.1007-1011.

Hollman, P.C.H. and Katan M.B., 1997. Absorption, Metabolism and Health Effects of Dietary Flavonoids in Man. Biomed. and Pharmacother.,51,pp.305- 310. Hong, C., Jun, L., Qiang, W., Xiao-Tao Niu, et al., 2012. Anti-HBV activities of Streblus asper and Constituents of Its Roots. Fitoterapia, doi:10.1016/j.fitote.2012.01.009.

Hossain, M., Barry-Ryan, C., Martin-Diana, A. and Brunton, N., 2010. Effect of Drying Method on the Antioxidant Capacity of Six Larniaceae Herbs. Food Chemistry,123(1),pp.85-91. How, Y.L. and Yau, Y.L., 2011. Antioxidant Properties of Some Malaysian Ferns. 3rd International Conference on Chemical, Biological and Environmental Engineering. IPCBEE vol.20. Quo Shen Shu, 2011. [Online], [Accessed 11th June 2011]. Available from World Wide Web; http://www.stuartxchange.org/Kalios.html. Hutadilok, T.N. Chaiyamutti, P. , Panthong, K., Mahabusarakam, W. and Rukachaisirikul. V., 2006. Antioxidant and Free Radical Scavenging Activities of Some Plants Used In Thai Folk Medicine. Pharm. Biol.,44,pp.221-228. Huxley, R.R. and Neil, H., 2003. The Relation Between Dietary Flavonol Intake and Coronary Heart Disease Mortality: A Meta-Analysis of Prospective Cohort Studies. European Journal Of Clinical Nutrition,57,pp.904-908. Ibrahim, M. H., Daud, W. R. W. and Talib, M. Z. M., 1997. Drying characteristics of oil palm kernels. Drying Technology,15,pp.1103-1117.

Ilaina, S, Rabia, S.S., Azizah, Y. and Azman S., 2009. Immunomodulating effects of Stichopus hermanni and Streblus asper Lour. Archives of Orofacial Sciences,4(2),pp.47-64.

Indrayan, A.K., Sharma, S., Durgapal, D., Kumar, N. and Kumar, M., 2005. Determination of Nutritive Value and Analysis of Mineral Elements for Some Medicinally Valued Plants from Uttaranchal. Current Sci,89,pp.1252-1255. Inoue, M., Suzuki, R., Koide, T., Sakaguchi, N., Ogihara, Y. and Yabu, Y., 1994. Antioxidant, gallic acid, Induces Apoptosis in HL-60RG Cells. Biochem. Biophys. Res. Commun.,204,pp.898–904.

Irzyniec, Z., Klimezak, J. and Michalowski, S., 1995. Freeze-drying of The Black Currant Juice. Drying Technology,13,pp.417-412.

Jackman, R.L., Yada, R.Y., Tung, M.A. and Speers, R. A., 1987. Anthocyanins as Food Colorants - A Review. Journal Food Biochemistry,11,pp.201-247.

Jain, S.K., 1991.Dictionary of Indian Folk Medicine and Ethnobotany, New Delhi: Deep Publications. pp. 172.

Jha, S.N. and Prasad, S., 1996. Determination of Processing Conditions of Gorgon Nut (Euryale Ferox). Journal of Agricultural Engineering Research,63,pp.103-112. Jiang, J.Y., Wu, F.H., Lu, J.F., Lu, Z.H., and Xu, Q.N. 1997. Anti-infammatory activities of aqueous extract from rhizome Smilax glabra. Pharmaco. Res., 36,pp.309-314

Jin, B,J. and Hyung, J.J., 2010. 2-Methoxy-4-vinylphenol can induce cell cycle arrest by blocking the hyper-phosphorylation of retinoblastoma protein in benzo[a]pyrene-treated NIH3T3 cells. Biochemical and Biophysical Research Communications, 400,pp.752–757.

Jones, P.J., 2002. Functional foods more than just nutrition. CMAJ,166,pp.1555- 1563.

Joshi, P. and Mehta, D., 2010. Effect of dehydration on the nutritive value of drumstick leaves. Journal of Metabolomics and Systems Biology, 1(1), pp. 5 -9.

Jun, L., Yan H., Xin L.G., Jian, L., Sheng, P. D., et al., 2012. Anti-hepatitis B virus Constituents from The Stem Bark of Streblus asper. Phytochemistry,82,pp.100–109.

Jung, C.H., Seog, H.M., Choi, I.W., Park, M.W. and Cho, H.Y., 2006. Antioxidant Properties of Various Solvent Extracts from Wild Ginseng Leaves. Food Science Thechnology,39,pp.266–274.

Juntachote, T. and Berghofer. E., 2005. Antioxidative, Properties and Stability Of Ethanolic Extracts of Holy Basil and Galangal. Food Chemistry,92,pp.193-202. Jyostna, M., Srivastava, R.K., Shukla, S.V. and Raghav, C.S., 2007. Antioxidants in Aromatic and Medical plants. Science Tech Entrepreneur; Fragrance and Falvour Development Centre, Kannauj.

Kahkonen, M. P., Hopia, A. I., Vourela, H. J., Rauha, J.P., Pihlaja,K., Kujala, T.S., et al., 1999. Antioxidant Activity of Plant Extracts Containing Phenolic Compounds. Journal of Agricultural and Food Chemistry,47,pp.3954–3962. Kamal-Eldin, A. and Appelqvist, L.A., 1996. The Chemistry and Antioxidant Properties of Tocopherols and Tocotrienols. Lipids,31,pp.671–701.

Kaur, C. and Kapoor, H.C., 2002. Antioxidant Activity and Total Phenolic Content of Some Asian Vegetables. International Journal Food Science Technology,37,pp.153-161.

Keinanen, M. and Julkunen-Tiitto, R., 1996. Effect of Sample Preparation Method on Birch (Betula Pendula Roth) Leaf Phenolics. Journal of Agricultural and Food Chemistry,44,pp.2724–2727. Khangholi, S. and Rezaeinodehi, A., 2008. Effect of Drying Temperature on Essential Oil Yield and Composition of Sweet Wormwood (Artemisia annua) Growing Wild in Iran. Pakistan Journal Biology Science,11,pp.934–937. Kimura, Nakagawa, T.K., Kubota, H., Kojima, Y. and Goto, Y., 2007. Food- grade Mulberry Powder Enriched with 1-deoxynojirimycin Suppresses The Elevation of Postprandial Blood Glucose in Humans. Journal Agricultural Food Chemistry,55,pp.5869-5874. Kirtikar, K.R. and Basu, B.D., 1933. Indian Medicinal Plants, Allahabad: Lalit Mohan Basu Publications,3,pp.2291.

Koleva, I.I., Van Beek, T.A., Linssen, J.P.I.I., deGroot, A. and Evstatieva, L.N., 2002. Screening of Plant Extracts for Antioxidant Activity: A Comparative Study on Three Testing Methods. Phytochemistry Analysis,13,pp.8-17. Kondo, S., Tayama, K., Tsukamoto, Y., Ikeda, K. and Yamori, Y., 2001. Antihypertensive Effects of Acetic Acid and Vinegar on Spontaneously Hypertensive Rats. Biosci Biotechnol Biochem.,65(12),pp.2690-2694.

Konishi, Y., Hirano, S., Tsuboi, H. and Wada, M., 2004. Distribution of Minerals in Quinoa (Chenopodium quinoa Willd.) Seeds. Biosci. Biotechnol. Biochem.,68,pp.231–234. Krishnaiah, D., Sarbatly, R. and Bono, A., 2007. Phytochemical Antioxidants for Health and Medicine: A Move Towards Nature. Biotechnol. Mol. Biol. Rev.,1(4),pp.097-104. Krishnamuthy, K.R. and Sarala, P., 2010. Proximate Nutritive Values and Mineral Components of Withania Somnifera (Linn.) Dunal, J. of Chem., 7(3),pp.985-996.

Kumar, P.P., Kumaravel, S. and Lalitha, C., 2010. Screening of Antioxidant Activity, Total Phenolics and GCMS Study of Vitex negundo. African J Biochem Res,4(7),191-196. Kumar, R.B.S., Puratchikodi, A., Prasanna, A., Dolai, N., Majumder, P., Mazumder, U.K., Haldar, P.K., 2011. Pre clinical studies of Streblus asper Lour in terms of behavioural safety and toxicity. Orient Pharm Exp Med,11,pp.243– 249. Kwon, Y.I, Vattem, D.V. and Shetty, K., 2006. Evaluation of Clonal Herb of Lamiaceae Species for Management of Diabetes and Hypertension. Asia Pacific Journal Clinical Nutrition,15,pp.107–18.

Kwon, Young-In., 2007. Food Ingredient Design Strategies For Chemoprevention of disease using phenolic phytochemicals.

Lagouri, V. and Boskou, D., 1996. Nutrient Antioxidants in Oregano. International Journal of Food Sciences and Nutrition,47,pp.493–497. Lai, L.S., Chou, S.T. and Chao, W.W., 2001. Studies on The Antioxidative Activities of Hsian-Tsao (Mesona procumbens hemls) Leaf Gum. Journal Agricultural Food Chemistry,49,pp.963-968. Lamson, D.W. and Brignall, M.S., 2000. Antioxidants and Cancer III: Quercetin. Alt Med Rev,5(3),pp.196-208.

Lapornik, B., Prosek, M. and Wondra, A.G., 2005. Comparison of Extracts Prepared from Plant By-Products Using Different Solvents And Extraction Time. Journal Food Engineering,71,pp.214-222.

Lee, K.G. and Shibamoto, T., 2001. Antioxidant Activities of Volatile Components Isolated from Eucalyptus Sp. Journal of the Science of Food and Agriculture,81,pp.1573–1579.

Lee, S.W., Wendy,W., Julius, Y.F.S. and Desy, F.S., 2011. Characterization of Anticancer, Antimicrobial, Antioxidant Properties and Chemical Compositions of Peperomia pellucida Leaf Extract; Acta Medica Iranica,49(10),pp.670-674.

Lena, E., Anders, I., Bengt, S. and Gunnar, J., 2006. Acute Effects of Exposure to Vapours of Acetic Acid in Humans, Toxicology Letters, 165,pp.22–30.

Leverin, G. and McMatron, H., 1999. Alkaloids and glycosides. Clinical Microbiology Reviews,11,pp.156-250.

Lewis, W.H., 1980. Plants Used as Chewing Sticks. J Prev Dent,6,pp.71–73.

Li, J., Zhang, Y.J., Jin, B.F., Su, X.J., Tao, Y.W., She, Z.G., et al., 2008. 1H and 13C NMR Assignments for Two Lignans from The Heartwood of Streblus asper. Magn Reson Chem,46,pp.497–500.

Liang, Y.R. and Xu, Y.R., 2001. Effect of pH on Cream Particle Formation and Solids Extraction Yield of Black Tea. Food Chemistry,74,pp.155–160.

Lim, Y.Y. and Murtijaya, J., 2007. Antioxidant Properties of Phyllanthus amarus Extracts as Affected by Different Drying Methods. LWT, 40,pp.1664–1669 Limam, B.F., 2011. Qualitative and Quantitative Changes in The Essential Oil of Laurus Nobilis L. Leaves as Affected By Different Drying Methods. Food Chemistry, 126,pp.691–769.

Limsong, J., Benjavongkulchai, E. and Kuvatanasuchati, J., 2004. Inhibitory Effect of Some Herbal Extracts on Adherence of Streptococcus mutans. Journal of Ethnopharmacology,92,pp.281–289. Lin Tein, M., Durance, T.D., and Scaman, C.H., 1998. Characterization of Vacuum Microwave, Air and Freeze Dried Carrot Slices. Food Research International,3,pp.111-117. Liu, H., 1970. Catalysis of Lipid Peroxidation in Meats I. Linoleate peroxidation Catalyzed by Metmb of Fe (II)-EDTA. Journal of Food Science,35,pp.590–595.

Liu, H., Qiu, N., Ding, H. and Yao, R., 2008. Polyphenols Contents and Antioxidant Capacity of 68 Chinese Herbals Suitable for Medical Food Uses. Food Research International,41,pp.363–370.

Lobo, V., Patil, A., Phatak, A. and Chandra N., 2010. Free Radicals, Antioxidants and Functional Foods: Impact on Human Health. Pharmacogn Rev,4(8),pp.2968-2972. Lopez, V., Akerreta, S., Casanova, E., Garcia-Mina, J., Cavero, M. and Calvo, M.I., 2007. In Vitro Antioxidant and Anti-Rhizopus Activities of Lamiaceae Herbal Extracts. Plant Foods For Human Nutrition,62(4),pp.151−155. Lu, X.W., Li, J., Huang, C.P., Meng, A.P. and Zhu, S.J., 2009. Chemical Constituents in Leaves of Streblus asper. Guangxi Shifan Daxue Xuebao (Ziran Kexueban),27(4),pp.61–64.

Machlin, L.J. and Bendich, A., 1987. Free radical tissue damage: protective role of Antioxidant Nutrients. The FASEB Journal,1,pp.441-445. Macrae, R., Robinson, R. K. and Sadler, M. J., 1993. Encyclopaedia of Food Science, Food Technology And Nutrition, vol. 5. San Diego, CA: Academic Press INC. Madhavan, V., Pravinkumar, P.Z., Gurudeva, M.R. and Yoganarasimhan, S.N., 2009. Pharmacognostical evaluation of Root Bark of Streblus asper Lour. Indian Journal of Traditional Knowledge,8(2),pp.176-180.

Maestri, D.M., Nepote, V., Lamarque, A.L., Zygadlo, J.A., 2006. Natural products as antioxidants. In: Filippo Imperato (Ed.), Phytochemistry: Advances in Research. Research Signpost, pp. 105–135. Mahanom, H. , Azizah, AH and Dzulkifly, M.H., 1999. Effect of different drying methods on concentrations of several phytochemicals in herbal preparation of 8 medicinal plants leaves. Mal J Nutr, 5,pp.47-54 Maisuthisakul, P., Suttajit, M. and Pongsawatmanit, R., 2007. Assessment of Phenolic Content and Free Radical-Scavenging Capacity of Some Thai Indigenous Plants. Food Chemistry,100,pp.1409–1418. 94

Malika, I.O., 2007. Recent Advances in Oxasteroids Chemistry. Steroids,72:,pp.475–508.

Manay, N.N., 1987. Spices. Edn. In: Foods, facts and principle. London: Wiley, pp.300-334.

Manga, H.M., Brkic, D, Marie, D.E.P and Quetin-Leclercq J., 2004. In Vivo Anti-Inflammatory Activity of Alchonear cordifolia. Journal Ethnopharmacology,92,pp.209-214.

Manzetti, A.R. and Reichstein, T., 1964. Die Glykoside von Streblus asper Lour. 3. Mitteilung Untersuchung der stark wasserloslichen Anteile. Helv Chem Acta,47,pp.2303-2309 Marcos, T., Paula, G.D.P., Rui, F., Gonçalves, P.B., Andrade, P.V., 2009. Determination of Eighty-one Volatile Organic Compounds in Dietary Rumex induratus Leaves by GC/IT-MS, Using Different Extractive Techniques, Microchemical Journal,93,pp.67–72.

Margarita, Miranda., Antonio, Vega-Galvez., Jessica, lopez., et al., 2010. Impact of Air-Drying Temperature on Nutritional Properties, Total Phenolic Content and Antioxidant Capacity of Quinoa Seeds (Chenopodium quinoa Wild.). Industrial Crops and Products,32,pp.258–263. Maria jancy Rani, P. Kannan, P.S.M. and Kumaravel, S., 2011. Screening of Antioxidant Activity, Total Phenolics and Gas Chromatograph and Mass Spectrometer (GCMS) Study of Delonix regia. African Journal of Biochemistry Research,5(12),pp.341-347.

Mariani, C., et al., 2008. Flavonoid Characterization and In Vitro Antioxidant Activity of Aconitum anthora L. (Ranunculaceae). Phytochemistry,69,pp.1220- 1226. Marija, M., Radojkovic, Zoran. P., Zekovic, Senka, S., Vidovic, et al., 2012. Free Radical Scavenging Activity and Total Phenolic and Flavonoid Contents of Mulberry (Morus spp. L., Moraceae) Extracts. Hem. ind.,66(4),pp.547–552.

Marinova, E.M. and Yanishlieva, N.V., 1997. Antioxidative Activity of Extracts from Selected Species of the Family Lamiaceae in Sunflower Oil. Food Chemistry,58,pp. 245-248.

Martin-Cabrejas, M.A., Aguilera, Y., Pedrosa, M., Cuadrado, C., et al., 2009. The Impact of Dehydration Process on Antinutrients and Protein Digestibility of Some Legume Flours. Food Chemistry,114,pp.1063–1068.

Martin-Garcia A.I., Molina-Alcaide E., 2008. Effect of different drying procedures on the nutritive value of olive (Olea europaea var. europaea) leaves for ruminants. Animal Feed Science and Technology, 142,pp.317–329 Martin-Gil, J., Ramos-Sanchez, M.C., Martin-Gil, F.J. and Jose Yacaman, M., 2006. Chemical Composition of a Fountain Pen Ink. Journal Chemical Education,83,pp.1476.

Maskan, M., 2001. Drying, Shrinkage and Rehydration Characteristics of Kiwifruits During Hot Air and Microwave Drying. Journal of Food Engineering,48,pp.177-182.

Maznah Ismail, Gururaj Bagalkotkar, Shahid Iqbal and Hadiza Altine Adamu., 2012. Anticancer Properties and Phenolic Contents of Sequentially Prepared Extracts from Different Parts of Selected Medicinal Plants Indigenous to Malaysia. Molecules,17,pp.5745-5756.

Mbaebie, B.O, Edeoga, H.O and Afolayan, A.J., 2012. Phytochemical Analysis and Antioxidants Activities of Aqueous Stem Bark Extract of Schotia latifolia Jacq. Asian Pacific Journal of Tropical Biomedicine,pp.118-124.

McCarty MF., 2001. The Chlorophyll Metabolite Phytanic Acid is A Natural Rexinoid-Potential for Treatment and Prevention of Diabetes. Medical Hypothesis,56,pp.217-219.

McGinty, D., Letizia, C.S. and Api, A.M., 2010. Fragrance Material Review on Phytol. Food and Chemical Toxicology,48,pp.59–63.

Md. Shahadat Hossan, Abu Hanif, Bipasha Agarwala, Md. Shahnawaz Sarwar, Masud Karim, et al., 2010. Traditional Use of Medicinal Plants in Bangladesh to Treat Urinary Tract Infections and Sexually Transmitted Diseases. Ethnobotany Research & Applications,8,pp.061-074.

Mellgren, J., 2001. Healthy teas. The Gourmet Retailer,22(11),pp.58–63.

Minguez-Mosquera, M.I., Jaren-Galan, M. and Garrido-Fernandez, J., 1994. Carotenoid Metabolism during the Slow Drying of Pepper Fruits of The Agridulce Variety. Journal of Agricultural and Food Chemistry,42,pp.2260- 2264.

Minolta., 1998. Precise Color Communication, Minolta Co.Ltd. Japan. Miranda, M., Maureira, H., Rodriguez, K., Vega-Galvez, A., 2009. Influence of Temperature on the Drying Kinetic, Physicochemical Properties, and Antioxidant Capacity of Aloe Vera (Aloe Barbadensis Miller) gel. Journal Food Engineering,91,pp.297–304.

Moller, J.K.S., Madsen, H.L., Aaltonen, T. and Skibsted, L.H., 1999. Dittany (Origanum dictamnus) as a Source of Water Extractable Antioxidants. Food Chemistry,64,pp. 215-219. Monica Marcu., 2005. The Miracle Tree. 1st Edt., KOS Health Publications.

Monjoy K.C., Venkatraman, S. and Lokesh U., 2012. Phytochemical Analysis and Peripheral Glucose Utilization Activity Determination of Steblus asper. Asian Pacific Journal of Tropical Biomedicine,pp.656-661.

Moure, A., Cruz, J.M., Franco, D., Dominguez, J.M., et al., 2001. Review: Natural Antioxidants from Residual Sources. Food Chemistry,72,pp.145-171. Mueller-Harvey, I., 2001. Analysis of Hydrosable Tannins. Animal Feed Science. Technology,91,pp.3-20. Murota, K. and Terao, J., 2003. Antioxidative Flavonoid Quercetin: Implications of Its Intenstinal Absorption and Metabolism. Archives of Biochemistry and Biophysics,417,pp.12-17. Naczk M., 2004. Extraction and Analysis of Phenolics in Food. Journal Chromatography A,1054,pp.95-11. Naczk, M. and Shahidi, F., 2004. Extraction and Analysis of Phenolics In Food. Journal of Chromatography,1054,pp.95–111.

Nadkarni, K.M., 1976. Indian Materia Medica. Bombay: The Karnataka Printing Press and The Popular Press Ltd, p.1171.

Naga, V.K.A, Venkata, R.B., Kasetti, R.B., Cjippada, A., 2012. Antioxidant Activity and GCMS Analysis of Phragmytes allatoria Leaf Ethanolic Extract. International Research Journal of Pharmacy,3(3),pp.252-254.

Narendhirakannan, Subramanian, and Kandaswamy., 2005. Mineral Content of Some Medicinal Plants Used in the Treatment of Diabetes Mellitus. Biological Trace Element Research,103,pp.109-115.

Narender, P.D, Ganga Rao, B., Sambasiva Rao, E., Mallikarjuna Rao, T. and Praneeth D., 2012. Quantification of Phytochemical Constituents and In-Vitro Antioxidant Activity of Mesua ferrea leaves, Asian Pacific Journal of Tropical Biomedicine,pp.539-542.

Ncube, N.S., Afolayan, A.J. and Okoh, A.I., 2008. Assessment Techniques of Antimicrobial Properties of Natural Compounds of Plant Origin: Current Methods and Future Trends. African Journal of Biotechnology,7(12),pp.1797- 1806.

Neeli, R.E., Kamta, P.N. and Pradeep, K.S., 2008. Standardization Strategies for Herbal Gruds-An Overview. Research Journal Pharma and Tech.,1(4),pp.310- 312.

Ngozi, A.I., George, O.G., Veronica, I., Okochi, S.A., et al., 2010. Phytochemical and Antioxidant Nutrient Constituents of Carica papaya and Parquetina nigrescens extracts. Scientific Research and Essays, 5(16),pp. 2201-2205. Nindo, C., Sun, T., Wang, S.W. and Tang, J., 2003. Evaluation of Drying Technologies for Retention of Physical Quality and Antioxidants in Asparagus. Lebensmittel Wissenschaft Technology,36,pp.507–516.

Nitin, K.B., Pankaj, K., Devender, R., Reena, D. and Manohar, L., 2010. Pharmacognostic and Phytochemical Investigations of Bark of Tecomella Undulata Seem. Int. J. of Pharma, Research & Development ,2(7),pp.0974-9446. Nor Hazwani Ahmad, Rohanizah Abdul Rahim and Ishak Mat., 2010. Catharanthus roseus Aqueous Extract is Cytotoxic to Jurkat Leukaemic T-cells but Induces the Proliferation of Normal Peripheral Blood Mononuclear Cells. Tropical Life Sciences Research,21(2),pp.101–113. Normala Halimoon and Mardhiah Hayati Abdul Hasan ., 2010. Determination and Evaluation of Antioxidative Activity in Red Dragon Fruit (Hylocereus undatus) and Green Kiwi Fruit (Actinidia deliciosa). American Journal of Applied Sciences,7(11),pp.1432-1438.

Nouri, Y.M., Bernard, V.C. and Jack, L.B., 1954. of Freeze-Drying on Chlorophyll in the Leaves of Some Selected Drug Plants. Journal of the American Pharmaceutical Association, 43(9),pp.554-557.

Nsimba, R.Y, Kikuzaki, H. and Konishi, Y., 2008. Antioxidant Activity of Various Extracts Fractions of Chenopodium quinoa and Amaranthus spp. Seeds. Food Chemistry,106,pp.760–766. Nurbaiti Ridzuan, Ban Tawfeek Shareef, Habsah Hassan, Azman Seeni, Rabia Sultanam Siddiqui.,2009. In Vitro Antifungal Analysis of Streblus asper Root- Extract. Archives of Orofacial Sciences,4(2),pp.47-64.

Nurul Ain Ahmad Jamallullail, Azizah Yusof, Rabia Sultana Siddiqui, Azman Seeni, Siti Asma’ Hassan.,2009. In vitro study of Antibacterial Activity of Streblus asper Lour Root and Gamodulin (Stichopus hermanii) Extract. Archives of Orofacial Sciences,4(2),pp.47-64.

Ogungbenle, H.N., 2003. Nutritional Evaluation and Functional Properties of Quinoa (Chenopodium quinoa) flour. International Journal Food Science Nutrition,54,pp.153–158.

Oktay, M., Gulcin, I. and Kufrevioglu, O.I., 2003. Determination of In Vitro Antioxidant Activity of Fennel (Foeniculum Vulgare) Seed Extracts. Lebens- Wiss Technology,36,pp.263-271. Okuda, T., 1995. Tannins, A New Family of Bioactive Natural Organic Compounds. Yakugaku Zasschi,115,pp.81-100.

Okwu, D.E., 2004. Phytochemicals and Vitamin Content of Indigenous Spices of Southeastern Nigeria. Journal Sustain Agricultural Environment,6(1),pp.30-37. Olaleye, M.T., 2007.Cytotoxicity and Antibacterial Activity of Methanolic extract of Hibiscus sabdariffa. Journal Medical Plants Research,1(1),pp.009- 013. Oliver, M.F., 1997. It is More Important to Increase the Intake of Unsaturated Fats than to Decrease the Intake of Saturated Fats: Evidence from Clinical Trials Relating to Ischemic Heart Disease. Am J Clin Nutr,66,pp.980-986.

Omidiran, M.O., Baiyewu, I.T., Ademola, O.C., Fakorede, O.C., et al., 2012. Phytochemical Analysis, Nutritional Composition and Antimicrobial Activities of Morus alba (White Mulberry). Pakistan Journal of Nutrition,11(5), pp.456- 460.

Omotade Oloyede, Jeferson Franco, Daniel Roos, Joao Rocha, Magareth Athayde, Aline Boligon., 2012. Antioxidative Properties of Ethyl Acetate Fraction of Unripe Pulp of Carica Papaya In Mice, Journal of Microbiology, Biotechnology and Food Sciences,1 (3),pp.409-425.

Onyimonyi, A.E., Adeyemi, O. and Okeke, G.C. 2009. Performance and Economic Characteristics of Broilers Fed Varying Dietary Levels of Neem Leaf Meal (Azadirachta indica). International Journal of Poultry Science,8(3),pp.256- 259. Othman, A., Ismail, A., Abdul Ghani, N. and Adenan, I., 2007. Antioxidant Capacity and Phenolic Content of Cocoa Beans. Food Chemistry,100,pp.1523- 1530.

Ow, Y.Y. and Stupans, I., 2003. Gallic acid and Gallic Acid Derivatives: Effects on Drug Metabolizing Enzymes. Curr Drug Metab.,4(3),pp.241-8.

Ozcan, M., 2004. Mineral Contents of Some Plants Used as Condiments in Turkey. Food Chemistry,84,pp.437–440. Packer, L., 1994. Vitamin E is Nature’s Master Antioxidant. Sci. Am. Sci. Med.1,pp.54–63.

Pan, R. and Dong. Y., 1995. Photosynthesis and Respiration-In: Pan R, Y Dong, Plant Physiology 3rd Ed. Higher educational press, Beijing.,pp.67-143. 99

Pandey, R.M. and Rekha Singh., 2010. Genetic Studies for Biochemical and Quantitative Characters in Grain Amaranth (Amaranthus hypochondriacus L.). Plant Omics Journal,3(4),pp.129-134 Patel, V.R., Patel, P.R. and Kajal, S.S., 2010. Antioxidant Activity of Some Selected Medicinal Plants in Western Region of India. Adv Biol Res.,4,pp.23–26.

Pathan Mohd Arif Khan and Mazahar Farooqui.,2011. Analytical Applications of Plant Extract as Natural pH Indicator: A Review; Adv Scient Res.,2(4),pp.20-27.

Paul Raj, K., Irudayaraj, V., Johnson, M. and Patric Raja, D., 2011. Phytochemical and Antibacterial Activity of Epidermal Glands Extract of Christella parasitica (L.) H. Lev. Asian Pacific Journal Tropical Biomedicine,1(1),pp.8-11. Paula, C.S.G. and Manuel, F.F., 2001. Organ and season dependent variation in the essential oil composition of Salvia officinalis L. cultivated at two different sites. Journal of Agricultural and Food Chemistry,49,pp.2908.

Peluso, M.R., 2006. Flavonoids Attenuate Cardiovascular Disease, Inhibit Phosphodiesterase, and Modulate Lipid Homeostasis in Adipose Tissue and Liver. Exp Biol Med,231,pp.1287-1299. Peter C. Sherertz, 1994. [Online]. [Accessed 1th June 1994]. Available from World Wide Web; http://www.vdh.virginia.gov/epidemiology/DEE/PublicHealthToxicology/docum ents/pdf/aceticacid.PDF

Peterson, T.A., Blackmer, T.M., Francis, D.D., Scheppers, J.S., 1993. Using a Chlorophyll Meter to Improve N Management. A Web guide in Soil Resource Management: D-13 Fertility. Cooperative Extension, Institute of Agriculture and Natural Resources, University of Nebraska, Lincoln, NE, USA. Phutdhawong, W., Donchai, A., Korth, J., Pyne, S.G., Picha, P., Ngamkham, J. and Buddhasukh, D., 2004. The Components and Anticancer Activity of The Volatile Oil from Streblus asper. Flavour Fragrance Journal,19,pp.445–447.

Pietta, P., Simonetti, P. and Mauri, P., 1998. Antioxidant Activity of Selected Medicinal Plants. Journal Agricultural Food Chemistry,46,pp.4487-4490.

Pinelo, M., Del Fabbro, P., Marzocco, L., Nunez, M. J. and Vicoli, M.C., 2005. Optimization of Continuous Phenol Extraction from Vitis vinifera Byproducts. Food Chemistry,92,pp.109–117.

Prakash, K., Deepak, D., Khare, A. and Khare, M.P., 1992. A Pregnane Glycoside from Streblus asper. Phytochemistry,31,pp.1056.

100

Praveen Kumar, P.. Kumaravel, S. and Lalitha. C., 2010. Screening of Antioxidant Activity, Total Phenolics and GCMS Study of Vitex negundo. African Journal of Biochemistry Research,4(7),pp.191-195.

Prior, R.L., et.al., 2005. Standardized Method for Determination of Antioxidant Capacity and Phenolic in Food and Dietary Supplement”, Journal Agricultural Food Chemistry,53,pp.4290-4302.

Qader, S.W., Abdulla, M.A., Chua, L.S., Najim, N., Zain, M.M. and Hamdan, S., 2011. Antioxidant, Total Phenolic Content and Cytotoxicity Evaluation of Selected Malaysian Plants. Molecules,16,pp.3433-3443.

Qu, W., Pan, Z., Ma, H., 2010. Extraction Modeling and Activities of Antioxidants from Pomegranate marc. Journal Food Engineering,99,pp.16–23. Quercetin., 2007. Royal Pharmaceutical Society of Great Britain,pp.272-275. Raghuveer, C. and Tandon, R.V., 2009. Consumption of Functional Food and Our Health Concerns. Pak J Physiol,5(1),pp.76-83. Rahimmalek, M. and Goli, S.A.H., 2012. Evaluation of Six Drying Treatments With Respect to Essential Oil Yield, Composition and Color Characteristics of Thymys daenensis subsp. daenensis. Celak leaves. Industrial Crops and Products,42,pp.613– 619. Rahman, M.A.A. and Moon, S.S., 2007. Antioxidant Polyphenol Glycosides from the Plant Draba Nemorosa. Bull. Korean Chemistry Society,28(5),pp.827- 831. Rajalakshmi, D., Naraimban, S., Madhavi, D.I., Deshpande, S.S and Salunkhe, D.K., 1996. Food antioxidants:sources and methods of evaluation. Food Antioxidants,65,pp.157.

Ramana, S.V., Jayaraman, K.S. and Mohan Kumar, B.L.,1988. Studies on the Color of some Dehydrated Green Leafy Vegetables. Indian Food Packer,pp.19- 23.

Roberson, T., Bundy, L.G., and Andraski, T.W., 2007. Freezing and drying effects on potential plant contributions to phosphorus in runoff. J Environ Quality,1;36(2):532-539

Rodrıguez, H., Landete, J. M., de las Rivas, B. and Munoz, R., 2008. Metabolism of Food Phenolic Acids by Lactobacillus plantarum CECT 748T. Food Chemistry, 107,pp.1393–1398.

Ruch RJ, Cheng SJ, Klaunig JE.,1989. Prevention of Cytotoxicity and Inhibition of Intracellular Communication by Antioxidant Catechins Isolated from Chinese Green Tea. Carcinogen,10,pp.1003-1008.

101

Runrawee, T., 1999. Samunpaithainaru. Bangkok: Ummarin printing and publishing. Rupasinghe, H.P., Jackson, C.J., Poysa, V., Di Berado, J., Bewley, J.D. and Jenkinson, J., 2003. Journal of Agricultural and Food Chemistry,51,pp.5888– 5894. Sakanaka, S., Tachibana, Y. and Okada, Y., 2005. Preparation and Antioxidant Properties of Extracts of Japanese Persimmon Leaf Tea (kakinoha-cha). Food Chemistry,89,pp.569–575. Salah, N., Miller, N.J., Pagange, G., Tijburg, L., Bolwell, G.P., Rice, E. and Evans ,C., 1995. Polyphenolic Flavonols as Scavengers of Aqueous Phase Radicals as Chain Breaking Antioxidant. Arch. Biochem. Biophys,2,pp.339-346. Sale, J.F. and Maji, J.O., 2006. The Phytochemical and Antomicrobial Screening of Honey Based (Ilesha Wonder Drug). Proceedings of the 1st National Conference of the Faculty of Natural Sciences, University of Abuja. Salmeron J, Hu FB, Manson JE, Stampfer MJ, Colditz GA, Rimm EB, Willett WC.,2001. Dietary fat intake and risk of type 2 diabetes in women. Am J Clin Nutr.,73,pp.1019–1026.

Sam-Long H, Ping-Hsiao S, and Gow-Chin Y., 2012. Neuroprotective Effects of Citrus Flavonoids. Journal Agricultural Food Chemistry,60(4),pp.877-885.

Sanchez-Castillo, C. P., Dewey, P. J. S., Aguirre, A., Lara, J. J., et al.,1998. The Mineral Content of Mexican Fruits and Vegetables. Journal of Food Composition and Analysis,11,pp.340–356. Sanchez-Moreno C.,2002. Methods used to Evaluate The Free Radical Scavenging Activity in Foods And Biological Systems. Food Science Technology International,8,pp.121–137.

Sang, S., Cheng, X., Stark, R.E., Rosen, R.T., Yang, C.S. and Ho, C.T., 2002. Chemical Studies on Antioxidant Mechanism of Tea Catechins: Analysis of Radical Reaction Products of Catechin and Epicatechin with 2, 2-diphenyl-1- picrylhydrazyl. Bioorganic and Medicinal Chemistry,10,pp.2233–2237. Sathish-Kumar, M. and Maneemegalai, S., 2008. Evaluation of Larvicidal Effect of Lantana Camara Linn Against Mosquito Species Aedes aegypti and Culex quinquefasciatus. Advances in Biological Research,2(3-4),pp.39-43. Saupi, N., Zakaria, M.H. and Bujang, J.S., 2009. Analytic Chemical Composition and Mineral Content of Yellow Velvetleaf (Limnocharis flava L. Buchenau)’s edible parts. Journal Applied Science,9,pp.2969-2974. Saxena, V.K. and Chaturvedi, S.K., 1985. Cardiac Glycosides from The Roots of Streblus asper. Planta Medical,4,pp.343. 102

Scalbert A, Manach C, Morand C, Remesy C, Jimenez L.,2005. Dietary Poly Phenols and The Prevention of Diseases. Crit Rev Food Sci Nutrition,45,pp.287- 306.

Schieber, A., Keller, P. and Carle, R., 2001. Determination of Phenolic Acids and Flavonoids of Apple and Pear by High Performance Liquid Chromatography. Journal of Chromatography A,910,pp.265-273.. Schuier, M., Sies, H., Billek, B. and Fischer, H., 2005. Cocoa-Related Flavonoids Inhibit CFTR-Mediated Chloride Transport across T84 Human Colon Epithelia. Journal Nutrition,135(10),pp.2320-2325. Sen CK, Khanna S, Rink C, Roy S., 2007. Tocotrienols: the emerging face of natural vitamin E. Vitam Horm.,76,pp.203–261.

Shahid Iqbal, Umer Younas, Kim Wei Chan, Muhammad Zia-Ul-Haq and Maznah Ismail.,2012. Chemical Composition of Artemisia annua L. Leaves and Antioxidant Potential of Extracts as a Function of Extraction Solvents. Molecule, 17,pp.6020-6032.

Shahid Iqbal, Umer Younas, Sirajuddin, Kim Wei Chan, Raja Adil Sarfraz and Md. Kamal Uddin.,2012. Proximate Composition and Antioxidant Potential of Leaves from Three Varieties of Mulberry (Morus sp.): A Comparative Study. International Journal Molecular Science,13,pp.6651-6664.

Shahin, S.A., Naresh, K., Abhinav, L., Angad, S., Hallihosur, S., Abhishek, S and Utpal, B., 2008. Food Research International,41(1),pp.1-15.

Shakirin, F.H., Prasad, K.N., Ismail, A., Yuon, L.C. and Azlan, A., 2010. Antioxidant capacity of underutilized Malaysian Canarium odontophyllum (dabai) Miq., Fruit. J Food Compos Anal,23,pp.777-781. Shi, J., Nawaz, H., Pohorly, J., Mittal, G., Kakuda, Y. and Jiang, Y., 2005. Extraction of Polyphenolics from Plant Material for Functional Foods- Engineering and Technology. Food Reviews International,21,pp.139-166.

Shyamala, G. S. and Vasantha, K.., 2010. Phytochemical Screening and Antibacterial Activity of Syzygium cumini (L.) (Myrtaceae) Leaves Extracts. International Journal of PharmTech Research,2(2),pp.1569-1573 Siddhuraju, P. and Becker, K., 2003. Antioxidant Properties of Various Extracts of Total Phenolic Constituents from Three Different Agroclimatic Origins of Drumstick Tree (Moringa oleifera lam.) Leaves. Journal Agricultural Food Chemistry,51,pp. 2144-2155.

Singh, V. and Jain, D.K., 2006. Nootan biology. Nageen Prakashan Pvt Ltd, Meerut, India, Ed 7th. 103

Singleton, Vernon L., Orthofer, Rudolf., Lamuela-Raventos. and Rosa M.,1999. Analysis of Total Phenols and Other Oxidation Substrates and Antioxidants By Means of Folin Ciocalteu Reagent,299,pp.152.

Skerget, M., Kotnik, P., Hadolin, M., Hras, A.R., Simonic, M. and Knez, Z., 2005. Phenols, Proanthocyanidins, Flavones and Flavonols in Some Plant Materials and Their Antioxidant Activities. Food Chemistry,89,pp.191–198. Sodipo, O.A., Akiniyi, J.A. and Ogunbamosu, J. A.,2000. Studies on certain Characteristics of Extracts of Bark of Pansinystalia macruceras (K schemp) pierre Exbeille. Global J. Pure Appl. Sci.,6,83-87. Soetan, K.O. and Oyewole, O.E., 2009. The need for adequate processing to Reduce thr Anti-Nutritional Factors In Animal Feeds: A review. In African Journal of Food Science,3(9),pp.223-232.

Sofowora, L.A., 1993. Medicinal Plants and Traditional Medicine in Africa. Spectrum Books Ltd, Ibaban, pp.55-71. Sohi, K.K., Mittal, N., Hundal, M.K. and Khanduja, K.L., 2003. Gallic acid, an Antioxidant, Exhibits Anti Apoptotic Potential in Normal Human Lymphocytes: a Bcl-2 Independent Mechanism. J. Nutr. Sci. Vitaminol.,49,pp.221–227.

Soong, Y.Y. and Barlow, P.J., 2006. Quantification of Gallic Acid And Ellagic Acid from Longan (Dimocarpus longan Lour.) Seed and Mango (Mangifera indica L.) Kernel and Their Effects on Antioxidant Activity. Food Chemistry,97,pp.524–530.

Soysal, Y., 2004. Microwave drying characteristics of parsley. Biosystems Engineering, 89,pp.167–73.

Sparg, S.G., Light, M.E. and van Staden, J., 2004. Biological Activities and Distribution of Plant Saponins. Journal of Ethnopharmacolog,94,(2–3),pp.219– 243.

Stauth, D. Oregon State University. Studies Force New View On Biology of Flavonoids. Available online: http://www.sciencedaily.com/releases/2007/03/070305140834.htm (accessed on 12th March 2012). Suhaj, M.,2006. Spice Antioxidants Isolation and Their Antiradical Activity: A Review. Journal of Food Composition and Analysis,19, pp.531–537. Sultana, B., Anwar, F. and Pirzybyiski, R., 2007. Antioxidant Activity of Phenolic Components Present in Barks of Azadirachta indica, Terminalia arjuna, Acacia nilotica and Euginea jambolana Lam. Trees. Food Chemistry,104,pp.1106-1114.

104

Sultana, B., Anwar, F. and Przybylski, R., 2007. Antioxidant Activity of Phenolic Components Present In Barks of Barks of Azadirachta indica, Terminalia arjuna, Acacia nilotica, and Eugenia jambolana Lam. trees. Food Chem., 104: 1106- 1114.

Surya Prakash D.V., Sree Satya Nandam and Meena Vangalapati., 2012. Optimization of Physico-Chemical Parameters for the Extraction of Phenolic Components from Terminalia chebula Species. Research in Pharmacy,2(5),pp.01-08.

Suzuki, M., Watanabe, T., Miura, A., Harashima, E., Nakagawa, Y. and Tsuji, K., 2002. An Extraction Solvent Optimum for Analyzing Polyphenol Contents by Folin–Denis Assay. Nippon Shokuhin Kagaku Kaishi,49,pp.507–511.

Taiwo A, Abidemi C, Oyedepo J, Adebayo B, Oluwadare I and Agboto D,., 2009. Nutrient Content and Anti Nutritional Factor in Shea butter (Butryospermum parkii) Leaves, African Journal of Biotechnology,8(21),pp.5888-5890. Takuya Katsube, Yoko Tsurunaga, Mari Sugiyama, Toshimichi Furuno, Yukikazu Yamasaki., 2009. Effect of Air-Drying Temperature on Antioxidant Capacity and Stability of Polyphenolic Compounds in Mulberry (Morus alba L.) leaves. Food Chemistry,113,pp.964–969.

Tambunan, A.H., Yudistira, K. and Hernani., 2001. Freeze drying Characteristics of Medicinal Herbs. Drying Techology,19,pp.325-331. Tanaka, T., 1999. Structure, property and function of plant polyphenols. Food Ingred. J. Jpn.,180,pp.64.

Taweechaisupapong, P., Klanrit, P., Singhara, S., Pitiphat, W. and Wongklam, S., 2006. Inhibitory Effect of Streblus asper Leaf Extract on Adhesion of Candida albicans to Dentene Acrylic. Journal of Ethnopharmacology,106,pp.414–417.

Taweechaisupapong, S., Wongkham, S., Chareonsuk, S., Suparee, S., Srilalai, P., Chaiyarak, S., 2000. Selective Activity of Streblus asper on Mutans streptococci. Journal of Ethnopharmacology,70,pp.73–79. Taylor, R.S.L., Hudson, J.B., Manandhar, N.P. and Towers, G.H.N., 1996. Antiviral Activities of Medicinal Plants of Southern Nepal. Journal of Ethnopharmacology ,53,pp.97-104. Tien-Tso Wu, Sy-Yu Shiau and Rei-Chu Chang, 2011. Effects of Drying Temperature and Extraction Solvent on The Antioxidant, Color and Sensory Properties of Angelica Sinensis Leaves. Journal of Food Processing and Preservation,pp.1745-4549.

105

Tomaino, A., Cimino, F., Zimbalatti, V., Venuti, V., Sulfaro, V. and De Pasquale, A., 2005. Influence of Heating on Antioxidant Activity and The Chemical Composition of Some Spice Essential Oils. Food Chemistry,89,pp.549–554.

Tripathi, P., Tomar, R. and Jagannadham, M.V., 2011. Purification and Biochemical Characterisation of a Novel Protease Streblin. Food Chemistry,125,pp.1005–1012.

Turkmen, N. Sari, F. and Velioglu, Y.S., 2006. Effect of Extraction Solvents on Concentration and Antioxidant Activity of Black and Black Mate Polyphenols Determined by Ferrous Tartrate and Folin-Ciocalteu Methods. Food Chemistry,99,pp.838-841.

Uddin, S. and Ahmad, S., 1995. Dietary Antioxidants Protection Against Oxidative Stress. Biochemical Education,23,pp.2-7.

UK Food Standards Agency, 2011. Current EU Approved Additives and Their E Numbers".

United State National Nutritional Data, 2010. United State Document on Nutrition. US Food and Drug Administration, 2011. Listing of Food Additives Status Part I.

Vega-Galvez, A., Di Scala, K., Rodriguez, K., Lemus-Mondaca, R., Miranda, M., Lopez, J. and Perez-Won, M., 2009. Effects of Air-Drying Temperature on Physicochemical Properties, Antioxidant Capacity and Total Phenolic Content of Red Pepper (Capsicum annuum, L. var. Hungarian). Food Chemistry,117,pp.647–653. Velioglu, Y.S., Mazza, G., Gao, L. and Oomah, B.D., 1998. Antioxidant Activity and Total Phenolics in Selected Fruits, Vegetables, and Grain Products. Journal of Agriculture and Food Chemistry,46,pp.4113–4117. Venskutonis, P.R., 1997. Effect of Drying on The Volatile Constituents of Thyme (Thymus vulgaris L.) and Sage (Salvia officinalis L.). Food Chemistry,59,pp.219–227. Verma, A.R., Vijayakumar, M., Mathela, C.S. and Rao, C.V., 2009. In vitro and In Vivo Antioxidant Properties of Different Fractions of Moringa oleifera Leaves. Food and Chemical Toxicology,47,pp.21196–21201.

Veronique Cheynier, 2005. Polyphenols in foods are more complex than often thought. Am J Clin Nutr;81,pp.223–229.

106

Vincent JJ Martin, Douglas J Pitera , Sydnor T Withers, Jack D Newman and Jay D Keasling., 2003. Engineering a Mevalonate Pathway In Escherichia Coli for Production of Terpenoids. Nature Biotechnology,21,pp.7.

Vinson, J.A., Dabbagh, Y.A., Serry, M.M. and Jang, J. 1995. Plant Flavonoids, especially Tea Flavonoids, are Powerful Antioxidants Using In Vitro Oxidation Model for Heart Disease. Journal of Agricultural and Food Chemistry,43,pp. 2800–2802.

Vo Van Chi, 1997. A dictionary of medicinal plants of Vietnam, Hanoi. Medicinal Publishing House,pp.419.

Waliullah, G.U., Abdur, R., Bina, S.S., Taj, U.R., Sadiq, A. and Muhammad Qaisar., 2011. Chemical Constituents and Biological Screening of Grewia optiva drummond ex Burret Whole Plant. American-Eurasian J Agric & Environ Sceince,11(4),pp.542-546.

Weemaes, C.A., Ooms, V., Van Loey, A.M. and Hendrickx, M.E., 1999. Kinetics of Chlorophyll Degradation and Color Loss in Heated Broccoli Juice. Journal Agricultural Food Chemistry,47,pp.2404–2409. Weerasak Samee and Suwanna Vorarat, 2007. Simultaneous Determination of Gallic acid, Catechin, Rutin, Ellagic acid and Quercetin in Flower Extracts of Michelia alba, Caesalpinia pulcherrima and Nelumbo nucifera by HPLC. Thai Pharm Health Sci J,2(2),pp.131-137. Wendakoon, Chitra, Peter Calderon and Daniel Gagnon. 2012. Evaluation of Selected Medicinal Plants Extracted in Different Ethanol Concentrations for Antibacterial Activity against Human Pathogens. Journal of Medicinally Active Plants,1(2),pp.60-68. Why does the human body need acetic acid, 2012. [Online], [Acecssed 2th Nov 2012]. Available from World Wide Web: http://wiki.answers.com/Q/.

Willett, W.C.,2007. The Role of Dietary N-6 Fatty Acids in The Prevention of Cardiovascular Disease. J Cardiovasc Med (Hagerstown),8(1),pp.42–45.

Wong, P.Y.Y. and Kitts, D.D., 2006. Studies on the Dual Antioxidant and Antibacterial Properties of Parsley (Petroselinum crispum) and Cilantro (Coriandrum sativum) Extracts. Food Chemistry,97(3),pp.505–515.

Wongkham, S., Laupattarakasaem, P., Pienthaweechai, K., Areejitranusorn, P., et al., 2001. Antimicrobial of Streblus asper Leaf Extract. Phytotherapy Research,15,pp.119–121.

107

Wongkham, S., Pienthaweechai, K., Laupattarakasaem, P., Areejitranusorn, P., et al., 1996. Bactericidal activity of Streblus asper. In: Second Thai–French Symposium on Plant Molecular Biology. Bordeaux, France, pp.363–374. Xu, R., Zhao, W., Xu, J., Shao, B. and Qin, G., 1996. Studies on Bioactive Saponins from Chinese Medicinal Plants. Advances in Experimental Medicine and Biology, 404,pp.371–82.

Yan, S.W. and Asmah, R., 2010. Comparison of Total Phenolic Contents and Antioxidant Activities of Turmeric Leaf, Pandan Leaf and Torch Ginger Flower. International Food Research Journal,17,pp.417-423. Yen, G.C. and Duh, P.D., 1994. Scavenging of Methanolic Extracts of Peanut Hulls on Free Radical and Active Oxygen Species. Journal Agricultural Food Chemistry,42,pp.629-632. Yilmaz, Y. and Toledo, R., 2006. Oxygen Radical Absorbance Capacities of Grape/Wine Industry Byproducts and Effect of Solvent Type on Extraction of Grape Seed Polyphenols. Journal of Food Composition and Analysis,19,pp.41- 48.

Yin, W., Wang, T.S., Yin, F.Z., and Cai, B.C. 2003. Analgesic and anti- inflammatory properties of brucine and brucine N-oxide extracted from seeds of Strychnos nux-vomica. J. Ethnopharm.,88,pp.204-205.

Yingming, P., Ping, L., Hengshan, W. and Min, L., 2004. Antioxidant Activities of Several Chinese Medicinal Herbs. Food Chemistry,88,pp.347–350.

Yousif, A.L., Scaman, C.H., Durance, T.D. and Girard, B.,1999. Flavor Volatiles and Physical Properties of Vacuum-Microwave And Air-dried Sweet Basil (Ocimum basilicum L.). Journal of Agricultural and Food Chemistry,47,pp. 4777–4781. Yu, J., Ahmedna, M. and Goktepe, I., 2005. Effects of Processing Methods and Extraction Solvents on Concentration and Antioxidant Activity of Peanut Skin Phenolics. Food Chemistry,90,pp.199-206.

Zaibunnisa, A.H., Norashikin, S., Mamot, S. and Osman, H., 2009. An Experimental Design Approach for The Extraction of Volatile Compounds From Turmeric Leaves (Curcuma Domestica) Using Pressurised Liquid Extraction (PLE). Food Science and Technology,42,pp.233–238.

Zhang, X.S., Wu, Z.Y. and Zao, Z.Y., 1985. The Plant Index of China, Beijing: Scientific Press,23,pp.38.

Zhongbing, L., Guangjun, N., Peter, S., et al., 2006. Structure Activity Relationship Analysis of Antioxidant Ability and Neuroprotective Effect of Gallic Acid Derivatives. Neurochemistry International,48,pp.263–274. 108

Zielisnki, H., Koslowska, H., 2000. Antioxidant activity and Total Phenolics in Selected Cereal Frains and Their Different Morphological Fractions. Journal Agricultural Food Chemistry,48,pp.2008–2016.

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APPENDICES

Appendix A: Voucher Specimen of S. asper

FLORA OF MALAYSIA

Universiti Sains Malaysia Herbarium

Voucher Specimen No.: No.11248 Family : Moraceae Name : Streblus asper Date : 20 July 2011 Locality & Habitat: Can be found at the corner roadside. Located opposite towards T junction of the Eureka Complex entrance at USM Health Campus Penang. Found in seasonal climates and is absent from rain forest. It is found surrounding of the villages, open areas and secondary forest. Notes: Is evergreen with unisexual flowers in the same plant of different plants shrub or small tree that grow up to 15 m tall. The crown resembles an umbrella with twiggy, drooping and straggling branches. The twigs and leaves are rough and hairy with copious white latex. The leaves are arranged alternately, elliptical to reverse egg-shaped with size 1.2-13 cm X 0.6-6.5 cm. The base is partially heart shaped to partially triangular acute, margin is serrate, dentate or has small teeth and is rough to touch on both sides. The stalk measures 1-3(-5) mm long and hairy. The inflorescence is arising from the axils. The male flowers are small and have stalk heads which are 4-10 mm in diameters. There are 5-15 flowers and 4 stamens while female are solitary or several together with long pedicel. The ovary is with prominent bifid stigmatic arms. The fruits are spherical drupe with measures 6-8mm long and in yellow to orange color. They are at first enclosed by enlarged segments of floral leaves, measures 5-8mm long but exposed at maturity and the segments of the floral leaves are abruptly bent. The spherical seed is 4-5 mm in diameter.

Collector&No: Identified: Nor Mawarti Ibrahim Mr Shanmugan PIPM0007/11(R)

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Appendix B: Calibration Curves for Minerals Analysis

Fig. 5.1(a): Calibration curve for Phosphorus (P): Absorbance vs P concentration (ppm) Graph.

Fig. 5.1(b): Calibration curve for Potassium (K): Absorbance vs K concentration (ppm) Graph.

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Fig. 5.1(c): Calibration curve for Sodium (Na): Absorbance vs Na concentration (ppm) Graph.

Fig. 5.1(d): Calibration curve for Calcium (Ca): Absorbance vs Ca concentration (ppm) Graph

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Fig. 5.1(e): Calibration curve for Magnesium (Mg): Absorbance vs Mg concentration (ppm) Graph.

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Appendix C: CIE L*, a*, b* Color Space

Fig. 5.2(a): a*, b* chromaticity diagram.

2 2 -1 Chroma, C*=√(a*) + (b*) ; Hue angle, hab=tan (b*/a*)

Fig. 5.2(b): Portion of a*, b* chromaticity diagram of Figure 5.1(a).

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Fig. 5.3: Representation of color solid for L* a* b* color space (Minolta, Precise color communication, Minolta, Co., Ltd., Osaka, Japan, 1998; 9242-4830-92 IHCAJ).

Fig. 5.4: Chroma and Lightness.

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Appendix D: Calibration curve for linearity standard of Gallic acid and Quercetin using HPLC

Fig. 5.5(a): Calibration curve for linearity standard of Gallic acid (GA): Area vs GA concentration (mg/ml) Graph.

Fig. 5.5(b): Chromatogram for gallic acid standard at Rt=1.510.

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Fig. 5.6(a): Calibration curve for linearity standard of Quercetin (Q): Area vs Q concentration (mg/ml) Graph.

Fig. 5.6(b): Chromatogram for Quercetin standard at Rt=9.808.

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Appendix E: Percentages of cell viability at 24, 48 and 72 h of incubation

Table 4.18(a): Percentages of cell viability of aqueous extract of S. asper leaves.

Concentration 24 48 72 (µg/ml) (100%) (100%) (100%) 0 100.0 100.0 100.0 15.6 98.3 96.9 92.2 31.3 94.9 94.2 90.8 62.5 92.8 92.8 92.5 125.0 86.3 88.0 83.4 250.0 87.2 91.4 76.3 500.0 80.6 86.1 75.4 1000.0 79.1 77.9 65.7

Figure 5.7(a): Mean OD of aqueous extract of S. asper leaves.

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Table 4.18(b): Percentages of cell viability of 70% EtOH extract of S. asper leaves.

Concentration 24 48 72 (µg/ml) (100%) (100%) (100%) 0 100.0 100.0 100.0 15.6 71.1 76.3 80.9 31.3 72.4 85.1 89.9 62.5 80.9 88.7 87.6 125.0 77.4 95.6 90.6 250.0 78.2 96.1 97.8 500.0 82.7 79.9 92.2 1000.0 81.7 89.5 92.2

Figure 5.7(b): Mean OD of 70% EtOH extract of S. asper leaves.

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