Phytochemicals and Antioxidative Properties of Coffea Liberica Green Beans and Comparison with That of Coffea Arabica and Coffea Robusta

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PHYTOCHEMICALS AND ANTIOXIDATIVE PROPERTIES OF COFFEA LIBERICA GREEN BEANS AND COMPARISON WITH THAT OF COFFEA ARABICA AND COFFEA ROBUSTA

TEO HUI MIN

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

December 2016 ACKNOWLEDGEMENT

First of all, I would like to express my deepest gratitude towards my supervisor, Dr. Lai Choon Sheen, for giving me her guidance, valuable suggestions and continuous encouragement throughout the course of this research project and thesis writing. She never fails to do her best in introducing me to many innovative ideas of conducting research and studying as well as directing me to a right path.

Thank you for being such a dedicated mentor. I would also like to extend my gratitude to my co-supervisor, Dr. Yam Wan Sinn for her help and advices throughout my study.

I would like to thank the Ministry of Higher Education (MOHE) (My Master) and the Research Creativity and Management Office (RCMO) of USM (grant:

1001/PKIMIA/811187) for their financial support in the completion of this thesis. I thank Centre for Drug Research (CDR) for providing me with the facilities and a comfortable environment to conduct the experiments.

I have been fortunate enough to have many people who had helped me along the way. Without them, I could not have completed my project smoothly. I thank Mr

Zahari and Mr Nizam from the School of Chemical Sciences, USM for helping me to carry out the NMR analysis. Also, I would like to thank Mr Azizo who had assisted me in performing the FTIR analysis. I wish to thank all the support staffs in CDR especially Mr. Hilman, Mr. Asokan and Mr Razak for their assistance in GC-MS and

HPLC analysis. I thank Mr. Zamri, Mr Rahim and all other lab and admistrative staffs who had helped me in one way or another. Special thanks to John Dolan from the Separation Science website, who had shared the knowledge of the

ii chromatography with me. Many thanks to my colleagues, Nelson, Thenmoly, Gaik

Ling, Sutha, Yasodha and Jimmy for lending their hands and moral support during my hard time. I greatly appreciate it.

I thank my best friends, Hsin Chi, Gui San, Chian Fang, Hui Teng, Chiew

Ling, Si Ning, Yi Ying, Jia Min, Joshua and Seow Lay who shared my ups and downs and are always supportive.

Last but not least, I wish to thank my dearest family members especially my parents, aunties and brothers who have never stopped believing in me, and for their love, support, understanding and encouragement that gave me the strength to complete my studies.

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TABLE OF CONTENTS

ACKNOWLEDGEMENT ii

TABLE OF CONTENTS iv

LIST OF TABLES ix

LIST OF FIGURES xi

LIST OF SYMBOLS AND ABBREVIATIONS xx

ABSTRAK xxv

ABSTRACT xxvii

CHAPTER 1 INTRODUCTION 1

CHAPTER 2 LITERATURE REVIEW 4

2.1 Antioxidant as chemopreventive and therapeutic agent 4

2.1.1 Natural antioxidants 5

2.1.2 Synthetic antioxidants 8

2.2 Rubiaceace family 9

2.3 Genus of Coffea 9

2.4 Coffee as a source of medicine and beverage 13

2.5 Green beans 13

2.5.1 Phytochemicals in green coffee beans 15

2.5.1(a) Lipids 15

2.5.1(b) Volatile compounds 18

2.5.1(c) Alkaloids 20

2.5.1(d) Polyphenols 22

2.5.1(e) Carbohydrates 25

2.6 Pharmacological activities of coffee 26

2.7 Roasted beans 28

2.8 Green coffee beans extract as a supplement 29

2.9 Chromatographic analysis of phenolic acids and caffeine 30

2.10 Coffea liberica 33

2.10.1 Geographical distribution and botanical description 33

2.10.2 Economical importance and uses 36

2.10.3 Phytochemicals and pharmacological activities 36

CHAPTER 3 METHODOLOGY 41

3.1 Materials 41

3.1.1 Chemicals, reagents and materials 41

3.1.2 Equipment and instrumentation 43

3.2 Plant materials 43

3.3 Experimental methods 44

3.3.1 Extraction of plant materials 44

3.3.2 Chromatography 44

3.3.2(a) Isolation of compounds 1 and 2 from the 44 precipitate obtained in Coffea liberica MeOH extract

3.3.2(b) Partitioning of the chemical constituents in 46 Coffea liberica MeOH extract

3.3.2(c) Chromatography of EtOAc fraction 46

3.3.2(d) Isolation of compound 3 from EF3 47

3.3.2(e) Isolation of chemical constituents from EF7 48

3.3.3 Identification and structure elucidation of compounds 55

3.3.3(a) Gas chromatography-mass spectrometry 55

3.3.3(b) Mass spectrometry 55

3.3.3(c) Melting point analysis 55

3.3.3(d) Ultraviolet-visible spectroscopy 56

3.3.3(e) Fourier Transformed Infrared Spectroscopy 56 (FTIR)

3.3.3(f) Nuclear magnetic resonance spectroscopy 57

3.3.4 Detection of compounds on TLC by post-chromatographic 57 treatment

3.3.4(a) Anisaldehyde 57

3.3.4(b) Ferric (III) chloride 58

3.3.4(c) DPPH 58

3.3.5 Quantitative analysis of phytochemcial contents in MeOH 59 extract of Coffea liberica, Coffea arabica and Coffea robusta by HPLC

3.3.5(a) Standard and sample preparation 59

3.3.5(b) Instrumentation and chromatographic condition 60

3.3.5(c) Method validation 61

3.3.5(c)(i) Linearity 61

3.3.5(c)(ii) Sensitivity 62

3.3.5(c)(iii) Accuracy 62

3.3.5(c)(iv) Precision 63

3.3.6 Antioxidant assays 63

3.3.6(a) DPPH free radical scavenging assay 63

3.3.6(b) Ferric reducing antioxidant power (FRAP) assay 64

3.3.7 Determination of total phenolic content 64

3.3.8 Determination of total sugar content 65

3.3.9 Protective effects of the MeOH extract of three Coffea 66 species on hydrogen peroxide-induced oxidative stress in human skin fibroblasts

3.3.9(a) Test sample preparation 66

3.3.9(b) Cell culture 66

3.3.9(c) Cell treatment 66

3.3.10 Statistical analysis 68

CHAPTER 4 RESULTS AND DISCUSSION 69

4.1 Extraction yield 69

4.1.1 Antioxidant activities of the extracts of Coffea liberica 70

4.1.2 Total phenolic content of the extracts of Coffea liberica 72

4.1.3 Antioxidant activities and total phenolic content of MeOH 73 PPT

4.2 Comparison of the chemical composition of MeOH PPT versus 74 MeOH extract

4.3 Bioactivity-guided isolation of antioxidative compounds from Coffea 76 liberica MeOH extract

4.3.1 Antioxidant activities of EtOAc and water fractions 76

4.3.2 Total phenolic content of EtOAc and water fractions 78

4.3.3 Total sugar content of EtOAc and water fractions 79

4.3.4 Comparison of the phytochemicals of EtOAc and MeOH 81 extract

4.3.5 Subfractionation of active EtOAc fraction 83

4.3.5(a) Antioxidant activities of subfractions (EF1 EF7) 84

4.3.5(b) Total phenolic content of subfractions (EF1 86 EF7)

4.3.5(c) Phytochemical evaluation of active subfractions 87

4.4 Bioactive phenolic compounds obtained from the selected 89 subfractions

4.5 Structure elucidation of isolated compounds 93

4.5.1 Compound 1 93

4.5.2 Compound 2 107

4.5.3 Compound 3 114

4.5.4 Compound 4 122

4.5.5 Compound 5 131

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4.5.6 Compound 6 141

4.5.7 Compound 7 151

4.5.8 Compound 8 164

4.5.9 Compound 9 176

4.6 DPPH radical scavenging activity of the isolated compounds 187

4.7 Comparison of the chemical profiles and antioxidant activities of 191 Coffea liberica, Coffea arabica and Coffea robusta MeOH extracts

4.7.1 HPLC method validation 191

4.7.1(a) Selectivity 193

4.7.1(b) Calibration curve, limit of detection and limit of 197 quantification

4.7.1(c) Precision 198

4.7.1(d) Accuracy 199

4.7.2 Quantification of the marker compounds in the MeOH 203 extract of three Coffea species

4.7.3 Antioxidant activities and total phenolic content of the 206 MeOH extract of three Coffea species

4.7.4 Protective effect of the MeOH extracts of the three Coffea 208 species against hydrogen peroxide-induced oxidative stress in human skin fibroblasts

CHAPTER 5 CONCLUSION 211

REFERENCES 214

APPENDICES

LIST OF PUBLICATIONS

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LIST OF TABLES

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Table 2.1 The main classes of phenolic compounds. 6

Table 2.2 Characteristic differences between Coffea arabica, Coffea 12 robusta and Coffea liberica.

Table 2.3 Caffeine content in green coffee beans, expressed in % 21 (gram per gram of coffee beans, w/w dry matter basis).

Table 2.4 Chlorogenic acids content in green coffee beans, 24 expressed in % (gram per gram of coffee beans, w/w dry matter basis).

Table 2.5 Chlorogenic acids and caffeine content (mg per gram of 29 coffee beans, w/w dry matter basis) (Mean SD, n =3) in Ethiopian green coffee beans.

Table 2.6 Summary of the chromatograpic separation of analytes in 31 32 various coffee samples.

Table 3.1 List of chemicals used, their abbreviations and 41 42 manufacturing country.

Table 3.2 List of equipment and instrumentation. 43

Table 3.3 Solvent system used for eluting the EtOAc fraction. 47

Table 3.4 Solvent system used for the fractionation of EF7. 48

Table 3.5 Mobile phase for the separation of EF7/SF1 to EF7/SF4 49 using HPLC.

Table 3.6 Solvent system used for the separation of EF7/SF1. 50

Table 3.7 Chromatographic conditions used for the scaled-up 51 separation of EF7/SF2-EF7/SF4.

Table 3.8 Concentration of the individual compounds for each 59 standard mixture.

Table 3.9 Gradient elution program used in the mobile phase 61 system for HPLC analysis of Coffea liberica, Coffea arabica and Coffea robusta using Xselect HSS T3 XP column.

Table 4.1 DPPH radical scavenging activity of the extracts of 71 Coffea liberica.

Table 4.2 Total sugar content of MeOH extract, EtOAc and water 79 fractions.

Table 4.3 1D and 2D NMR data of compound 1. 99

Table 4.4 1H and 13C NMR data of compound 2. 111

Table 4.5 1D and 2D NMR data of compound 3. 119

Table 4.6 1D and 2D NMR data of compound 4. 127

Table 4.7 1D and 2D NMR data of compound 5. 136

Table 4.8 1D and 2D NMR data of compound 6. 146

Table 4.9 1D and 2D NMR data of compound 7. 157

Table 4.10 1D and 2D NMR data of compound 8. 170

Table 4.11 1D and 2D NMR data of compound 9. 181

Table 4.12 The linear regression data, limit of detection (LOD) and 197 limit of quantification of compounds 1 5 and 7 9 (n = 3).

Table 4.13 Results of precision study on compound 1 5 and 7 9 at 198 three concentration levels (low, medium, high) (n = 3).

Table 4.14 Method accuracy for the analysis of compounds 1 5 and 200 7 9 in the MeOH extract of Coffea liberica (n = 3).

Table 4.15 Method accuracy for the analysis of compounds 1 5 and 201 7 9 in the MeOH extract of Coffea arabica (n = 3).

Table 4.16 Method accuracy for the analysis of compounds 1 5 and 201 7 9 in the MeOH extract of Coffea robusta (n = 3).

LIST OF FIGURES

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Figure 2.1 Chemical structures of ascorbic acid, α-tocopherol and 5 β-carotene.

Figure 2.2 The chemical structures of caffeic acid, ferulic acid, p- 7 coumaric acid and sinapic acid.

Figure 2.3 Chemical structure of BHT (butylated 8 hydroxytoulene), BHA (butylated hydroxyanisole), THBP (2,4,5-trihydroxybutyrophenone), propyl gallate and octyl gallate.

Figure 2.4 World coffee production. 10

Figure 2.5 The structure of coffee fruit. 14

Figure 2.6 The dried green beans of Coffea arabica, Coffea 14 robusta and Coffea liberica.

Figure 2.7 Chemical structures of Nβ-arachidoyl-5- 16 hydroxytryptamine, Nβ-behenoyl-5-hydroxytryptamine and Nβ-lignoceroyl-5-hydroxytryptamine.

Figure 2.8 Chemical structures of major free fatty acids. 17

Figure 2.9 Chemical structures of major volatile compounds. 19

Figure 2.10 Chemical structures of caffeine (1,3,7- 20 trimethylxanthine), theophylline (1,3- dimethylxanthine), theobromine (3,7- dimethylxanthine) and trigonelline.

Figure 2.11 Chemical structures of quinic acid, trans- 23 hydroxycinnamic acids and chlorogenic acids.

Figure 2.12 Chemical structures of the backbone of 25 galactomannans, the backbone of cellulose and sucrose.

Figure 2.13 The leaves of Coffea liberica. 34

Figure 2.14 The ripe berries of Coffea liberica. 34

Figure 2.15 Coffea liberica. a. A cluster of three calycesn with the 35 calyculate upper bracteoles covering the short pedicels, the middle calyx shown after the fall of its corolla, the right-hand one with a fully expanded corolla, and the left-hand one with young unexpanded corolla; b. Vertical section of the ovary and epigynous disk by central plane perpendicular to the septum; c. A pyrene as from the face, natural size; d. Lateral view of the same; natural size; dorsal view of a bean (natural size) cut away rather obliquely towards the base so as to expose the embryo with its inferior radicle. Drawn by D. Blair.

Figure 2.16 Chemical structures of the fatty acids present in Coffea 37 liberica green beans.

Figure 2.17 Chlorogenic acids in Coffea liberica. 39 40

Figure 3.1 Semi-preparative HPLC chromatogram of EF7/SF2 52 detected at 325 nm.

Figure 3.2 Semi-preparative HPLC chromatogram of EF7/SF3 52 detected at 325 nm.

Figure 3.3 A schematic diagram of compound isolation from 54 Coffea liberica.

Figure 4.1 Extraction yield of hexane, DCM, MeOH extracts of 70 Coffea liberica. Results are expressed as mean SD (n = 3).

Figure 4.2 Ferric reducing antioxidant activity of the extracts of 71 Coffea liberica. Results are expressed as mean SD (n = 3 2) in mg of ascorbic acid equivalent per 1g of dry extract. Extracts marked with different superscripts are significantly different (p < 0.05) by Tukey’s test.

Figure 4.3 Total phenolic content of the extracts of Coffea 73 liberica. Results are expressed as mean SD (n = 3 2) in mg of gallic acid equivalent per 1g of dry extract. Extracts marked with different superscripts are significantly different (p < 0.05) by Tukey’s test.

Figure 4.4 Reversed phase TLC of MeOH extract (left) and 75 MeOH extract’s precipitate (MeOH PPT) (right) under A) short wavelength (254 nm) B) long wavelength (365 nm), separated with the mobile phase MeOH (1% A.A):1% A.A. The circles indicated the location of compound 1 and 2.

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Figure 4.5 DPPH radical scavenging activity of MeOH extract, 77 EtOAc and water fractions. Results are expressed as mean SD (n = 3 2). Samples marked with different superscripts are significantly different (p < 0.05) by Tukey’s test.

Figure 4.6 Ferric reducing activity of MeOH extract, EtOAc and 77 water fractions. Results are expressed as mean SD (n = 3 2) in mg of ascorbic acid equivalent per 1 g of dry extract. Samples marked with different superscripts are significantly different (p < 0.05) by Tukey’s test.

Figure 4.7 Total phenolic content of EtOAc and water fractions. 78 Results are expressed as mean SD (n = 3 2) in mg of gallic acid equivalent per 1 g of dry extract.

Figure 4.8 IR spectrum of water fraction from Coffea liberica 81 MeOH extract.

Figure 4.9 Normal phase TLC of MeOH extract and EtOAc 82 fraction A) under short wavelength (254 nm) B) under long wavelength (365nm) C) stained with anisaldehyde, under visible light D) sprayed with FeCl3, under visible light after separated with the mobile phase EtOAc:MeOH (1% acetic acid):1% acetic acid (7:2:1).

Figure 4.10 Percentage of yield of subfractions EF1-EF7. 83

Figure 4.11 DPPH radical scavenging activity of EF2-EF7. Results 85 were expressed in mean SD (n = 3 2). (IC50 of EF 1 not achieved at the concentration of 500µg/mL). Subfractions marked with different superscripts are significantly different (p < 0.05) by Tukey’s test.

Figure 4.12 Ferric reducing activity of EF1-EF7. Results are 85 expressed as mean SD (n = 3 2) in mg of ascorbic acid equivalent per 1g of dry extract. Subfractions marked with different superscripts are significantly different (p < 0.05) by Tukey’s test.

Figure 4.13 Total phenolic content of EF1-EF7. Results are 86 expressed as mean SD (n = 3 2) in mg of gallic acid equivalent per 1g of dry extract. Subfractions marked with different superscripts are significantly different (p < 0.05) by Tukey’s test.

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Figure 4.14 Normal phase TLC of EtOAc fraction, EF3, EF6 and 88 EF7 A) under long wavelength (365nm) B) stained with anisaldehyde C) sprayed with FeCl3 D) sprayed with 2.0 mM DPPH reagent, separated with the mobile phase EtOAc: MeOH (1% acetic acid): 1% acetic acid. The circles indicated the phenolic compounds that exhibited DPPH scavenging activity.

Figure 4.15 The HPLC chromatogram of A) EF3 B) compound 3 89 on a Waters Sunfire C18 (4.6 250 mm, 5 µm) detected at 325 nm.

Figure 4.16 Normal phase TLC of EF7 and subfractions EF7/SF1- 91 EF7/SF4 A) under long wavelength (365nm) B) sprayed with 2.0 mM DPPH reagent, developed with the mobile phase EtOAc: MeOH (1% acetic acid): 1% acetic acid.

Figure 4.17 The HPLC chromatogram of A) EF7/SF1 B) EF7/SF2 92 C) EF7/SF3 D) EF7/SF4 on a Waters Sunfire C18 column (250 4.6 mm i.d., 5 µm), detected at 325 nm.

Figure 4.18 Chemical structure of compound 1 (5-caffeoylquinic 93 acid).

Figure 4.19 ESI-MS (negative mode) of compound 1. 94

Figure 4.20 UV spectrum of compound 1 (MeOH). 94

Figure 4.21 FT-IR spectrum of compound 1 (ATR). 96

1 Figure 4.22 H NMR spectrum of compound 1 (D2O, 500 MHz). 100

Figure 4.23 HMBC spectrum of compound 1 (D2O, 500MHz). 101 102

Figure 4.24 COSY spectrum of compound 1 (D2O, 500MHz). 103

13 Figure 4.25 C NMR spectrum of compound 1 (D2O, 125 MHz). 104

Figure 4.26 A) 13C DEPT 90 B) 13C DEPT 135 spectrum of 105 compound 1 (D2O, 125 MHz).

Figure 4.27 HSQC spectrum of compound 1 (D2O, 500MHz). 106

Figure 4.28 The chemical structure of compound 2 [caffeine 107 (1,3,7-trimethylxanthine)].

Figure 4.29 EI-MS of A) compound 2 B) Caffeine standard. 108

Figure 4.30 UV spectrum of compound 2 (MeOH). 109

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Figure 4.31 FT-IR spectrum of compound 2 (ATR). 110

13 Figure 4.32 C NMR spectrum of compound 2 (CDCl3, 125 112 MHz).

1 Figure 4.33 H NMR spectrum of compound 2 (CDCl3, 500 MHz). 113

Figure 4.34 Chemical structure of compound 3 (caffeic acid; 3,4- 114 dihydroxycinnamic acid).

Figure 4.35 ESI-MS (negative mode) of compound 3. 115

Figure 4.36 UV spectrum of compound 3 (MeOH). 115

Figure 4.37 FT-IR spectrum of compound 3 (ATR). 117

1 Figure 4.38 H NMR spectrum of compound 3 (CD3OD, 500 120 MHz).

13 Figure 4.39 C NMR spectrum of compound 3 (CD3OD, 125 121 MHz).

Figure 4.40 Chemical structure of compound 4 (3-caffeoylquinic 122 acid).

Figure 4.41 ESI-MS (negative mode) of compound 4. 123

Figure 4.42 UV spectrum of compound 4 (MeOH). 123

Figure 4.43 FT-IR spectrum of compound 4 (ATR). 125

1 Figure 4.44 H NMR spectrum of compound 4 (D2O, 500 MHz). 128

13 Figure 4.45 C NMR spectrum of compound 4 (D2O, 125 MHz). 129

Figure 4.46 HMBC spectrum of compound 4 (D2O, 500MHz). 130

Figure 4.47 Chemical structure of compound 5 (4-caffeoylquinic 131 acid).

Figure 4.48 ESI-MS (negative mode) of compound 5. 132

Figure 4.49 UV spectrum of compound 5 (MeOH). 132

Figure 4.50 FT-IR spectrum of compound 5 (ATR). 134

1 Figure 4.51 H NMR spectrum of compound 5 (D2O, 500 MHz). 137

13 Figure 4.52 C NMR spectrum of compound 5 (D2O, 125 MHz). 138

Figure 4.53 COSY spectrum of compound 5 (D2O, 500MHz). 139

Figure 4.54 HMBC spectrum of compound 5 (D2O, 500MHz). 140

Figure 4.55 Proposed chemical structure of compound 6 (5- 141 feruloylquinic acid).

Figure 4.56 ESI-MS (negative mode) of compound 6. 142

Figure 4.57 UV spectrum of compound 6 (MeOH). 142

Figure 4.58 FT-IR spectrum of compound 6 (ATR). 144

1 Figure 4.59 H NMR spectrum of compound 6 (CD3OD, 500 147 MHz).

Figure 4.60 HMBC spectrum of compound 6 (CD3OD, 500 MHz). 148

13 Figure 4.61 C NMR spectrum of compound 6 (CD3OD, 125 149 MHz).

Figure 4.62 COSY spectrum of compound 6 (CD3OD, 500 MHz). 150

Figure 4.63 Chemical structure of compound 7 (4,5- 151 dicaffeoylquinic acid).

Figure 4.64 ESI-MS (negative mode) of compound 7. 152

Figure 4.65 UV spectrum of compound 7 (MeOH). 152

Figure 4.66 FT-IR spectrum of compound 7 (ATR). 154

1 Figure 4.67 H NMR spectrum of compound 7 (DMSO-d6, 500 158 MHz).

Figure 4.68 COSY spectrum of compound 7 (DMSO-d6, 159 500MHz).

13 Figure 4.69 C NMR spectrum of compound 7 (DMSO-d6, 125 160 MHz).

Figure 4.70 A) 13C DEPT 90 B) 13C DEPT 135 NMR spectrum of 161 compound 7 (DMSO-d6, 125 MHz).

Figure 4.71 HSQC spectrum of compound 7 (DMSO-d6, 162 500MHz).

Figure 4.72 HMBC spectrum of compound 7 (DMSO-d6, 163 500MHz).

Figure 4.73 Chemical structure of compound 8 (3,4- 164 dicaffeoylquinic acid).

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Figure 4.74 ESI-MS (negative mode) of compound 8. 165

Figure 4.75 UV spectrum of compound 8 (MeOH). 165

Figure 4.76 FT-IR spectrum of compound 8 (ATR). 167

1 Figure 4.77 H NMR spectrum of compound 8 (DMSO-d6, 500 171 MHz).

Figure 4.78 COSY spectrum of compound 8 (DMSO-d6, 172 500MHz).

13 Figure 4.79 C NMR spectrum of compound 8 (DMSO-d6, 125 173 MHz).

Figure 4.80 HSQC spectrum of compound 8 (DMSO-d6, 174 500MHz).

Figure 4.81 HMBC spectrum of compound 8 (DMSO-d6, 175 500MHz).

Figure 4.82 Chemical structure of compound 9 (3,5- 176 dicaffeoylquinic acid).

Figure 4.83 ESI-MS (negative mode) of compound 9. 177

Figure 4.84 UV spectrum of compound 9 (MeOH). 177

Figure 4.85 FT-IR spectrum of compound 9 (ATR). 179

1 Figure 4.86 H NMR spectrum of compound 9 (DMSO-d6, 500 182 MHz).

Figure 4.87 COSY spectrum of compound 9 (DMSO-d6, 183 500MHz).

13 Figure 4.88 C NMR spectrum of compound 9 (DMSO-d6, 125 184 MHz).

Figure 4.89 HSQC spectrum of compound 9 (DMSO-d6, 185 500MHz).

Figure 4.90 HMBC spectrum of compound 9 (DMSO-d6, 186 500MHz).

Figure 4.91 DPPH radical scavenging activity (IC50 in µM) of 190 compounds 1, 3 9 that were isolated from C.liberica. Results are expressed as the mean ± SD (n 3). Compounds marked with different superscripts are significantly different (p < 0.05) by Tukey’s test.

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Figure 4.92 Representative chromatograms of Coffea liberica 192 MeOH extract (2 mg/mL) obtained on A) Phenomenex Kinetex PFP (250 4.6 mm, 5 μm) B) Waters Sunfire C18 (250 4.6 mm, 5 μm) and Agilent Zorbax SB C18 (250 4.6 mm, 5 μm) columns at 280 nm.

Figure 4.93 UV spectrum of: 5-caffeoylquinic acid (1), caffeine 194 (2), caffeic acid (3), 3-caffeoylquinic acid (4), 4- caffeoylquinic acid (5), 4,5-caffeoylquinic acid (7), 3,4-caffeoylquinic acid (8), 3,5-caffeoylquinic acid (9). Each subfigure shown an overlay of five spectra (represented by blue, red, green, pink and yellow colour lines) obtained at different point of the peak.

Figure 4.94 Representative HPLC chromatograms of A) Coffea 195 liberica MeOH extract (2 mg/mL) B) Coffea arabica MeOH extract (2 mg/mL) C) Coffea robusta MeOH extract (2 mg/mL) D) standard mixture consisting: 5- caffeoylquinic acid (1), caffeine (2), caffeic acid (3), 3- caffeoylquinic acid (4), 4-caffeoylquinic acid (5), 4,5- caffeoylquinic acid (7), 3,4-caffeoylquinic acid (8) and 3,5-caffeoylquinic acid (9) detected at 280 nm.

Figure 4.95 Representative HPLC chromatograms of A) Coffea 196 liberica MeOH extract (2 mg/mL) B) Coffea arabica MeOH extract (2 mg/mL) C) Coffea robusta MeOH extract (2 mg/mL) D) standard mixture consisting: 5- caffeoylquinic acid (1), caffeine (2), caffeic acid (3), 3- caffeoylquinic acid (4), 4-caffeoylquinic acid (5), 4,5- caffeoylquinic acid (7), 3,4-caffeoylquinic acid (8) and 3,5-caffeoylquinic acid (9) detected at 325 nm.

Figure 4.96 The composition of compounds 1-5 and 7-9 in the 205 MeOH extract of Coffea liberica, Coffea arabica and Coffea robusta (n = 3 2) expressed in mg/g of dry extract (mean SD). The statistical differences (p < 0.05) among the content of individual compounds in three species were indicated with different superscripts by Tukey’s test.

Figure 4.97 DPPH radical scavenging activity of Coffea liberica, 206 Coffea arabica and Coffea robusta MeOH extracts. Results are expressed as mean SD (n = 3 2).

Figure 4.98 Ferric reducing activity of Coffea liberica, Coffea 207 arabica and Coffea robusta MeOH extracts. Results are expressed as mean SD (n = 3 2) in mg of ascorbic acid equivalent per 1g of dry extract.

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Figure 4.99 Total phenolic content of Coffea liberica, Coffea 207 arabica and Coffea robusta MeOH extracts. Results are expressed as mean SD (n = 3 2) in mg of ascorbic acid equivalent per 1g of dry extract.

Figure 4.100 A) pre-treatment and B) post-treatment effect of the 210 MeOH extracts of Coffea liberica, Coffea arabica and Coffea robusta on cell viability following H2O2 oxidative stress. Results are presented as mean SEM (n = 3 2). a indicates p < 0.05 against control; b, p < 0.01 and c, p < 0.001 against H2O2-treated cells by Dunnett’s test.

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LIST OF SYMBOLS AND ABBREVIATIONS

% Percentage

α Alpha

β Beta

® Registered

°C Degree celcius

x Mean

ε Molar absorptivity c Centi

Standard deviation

S Slope

µL Microliters g Gram

J Coupling constant p Para-substituted r2 Coefficient of determination

λmax Maximum absorption m Meter

µm Micrometer

µg Microgram dm3 Cubic desimeter cm Centimeter mm Millimeter nm Nanometer mg Milligram mM Millimolar

µM Micromolar mg AAE/g Milligram ascorbic acid equivalent per gram mg GAE/g Milligram gallic acid equivalent per gram mg Glc/g Milligram glucose equivalent per gram mL Mililiters mL/min Milliliters per minute kHz Kilo hertz

ACN Acetonitrile

1D One dimension

2D Two dimension

A.A Acetic acid

AR Analytical grade

ATR Attenuated Total Reflection

ANOVA One way analysis of variance

BHT Butylated hyroxytoulene

CQA Caffeoylquinic acid

COSY Homonuclear Correlation spectroscopy

CD3OD Deuterated chloroform

CHCl3 Chloroform

CDCl3 Deuterated chloroform

C-5-HT Nβ-alkanoyl-5-hydroxytryptamine

DCQA Disubstituted caffeoylquinic acid/ dicaffeoylquinic acid

DCM Dichloromethane

D2O Deuterium oxide

DAD Dioade array

DEPT Distortionless enhancement by polarization transfer

DMSO Dimethyl sulfoxide

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DPPH 2,2-α-diphenyl-1-picrylhydrazil et al. and other

ESI Electrospray ionization

EtOAc Ethyl acetate ft Feet

F.A Formic acid

FC Folin-ciocalteu

FeCl3 Ferric (III) chloride

FeCl3•6H2O Ferric (III) chloride hexahydrate

FRAP Ferric reducing antioxidant power

FTIR Fourier Transformed Infrared spectroscopy g/g Gram per gram

HSQC Heteronuclear Single Quantum Correlation spectroscopy

HMBC Heteromultiple Bond Correlation spectroscopy

HPLC High Performance Liquid Chromatography

H2O Water

HCl Hydrochloric acid

IC50 Half maximal inhibitory concentration

ICH International Conference on Harmonisation i.d. Internal diameter i.e. That is

IM/oa-TOF- Ion mobility/orthogonal acceleration time-of-flight mass MS spectrometry

LOD Limit of detection

LOQ Limit of quantification lit. Literature min Minute

xxii ms Mass spectrometry

MeOH Methanol

MCT Monocarboxylic acid transporter

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NaCO3 Sodium carbonate

NIST National Institute of Standards and Technology

NMR Nuclear Magnetic Resonance no. Number ppm Parts per million

PPT Precipitate

PFP Penfluorophenyl

PDA Photodiode array

PTLC Preparative thin layer chromatography

Rf Retention factor

Rt Retention time

RSD Relative standard deviation

ROS Reactive oxygen species

RP C18 Reversed phase carbon 18

Semi-prep Semi preparative

TPTZ 2,4,6-tris(2-prydyl)-s-triazine

TLC Thin layer chromatography

THBP 2,4,5-trihydroxybutyrophenone

T Temperature temp. Temperature

TFA Trifluoroacetic acid

UV Ultraviolet

UV-Vis Ultraviolet-visible

xxiii

USDA United States Department of Agriculture w/w Weight per weight w/v Weight per volume

xxiv

FITOKIMIA DAN SIFAT ANTIOKSIDAN BIJI KOPI MENTAH COFFEA

LIBERICA DAN PERBANDINGAN DENGAN COFFEA ARABICA DAN

COFFEA ROBUSTA

ABSTRAK

Coffea arabica, Coffea robusta dan Coffea liberica merupakan tiga spesies kopi utama yang ditanam untuk kegunaan komersial. Walaupun C. liberica merupakan spesies yang paling jarang diperdagangkan, ia merupakan suatu komoditi yang penting di Malaysia. Penyelidikan terhadap spesies ini adalah sangat terhad terutamanya terhadap biji mentahnya. Projek penyelidikan ini telah dijalankan untuk mengenalpasti juzuk kimia yang bertanggungjawab terhadap aktiviti antioksidan biji mentah C. liberica dan juga kandungan fitokimia serta aktiviti antioksidan berbanding dengan kedua-dua spesies yang lebih popular. Dengan menggunakan pendekatan pemencilan sebatian kimia berpandukan bioaktiviti, kafein (2), asid kafeik (3), dan satu siri asid klorogenik termasuk asid 5-kafeoilkuinik (1), asid 3- kafeoilkuinik (4), asid 4- kafeoilkuinik (5), asid 5- kafeoilkuinik (6), asid 4,5-di kafeoilkuinik (7), asid 3,4-di kafeoilkuinik (8) dan asid 3,5-di kafeoilkuinik (9) telah dipencilkan daripada biji mentah C. liberica. Aktiviti pemerangkapan radikal bebas bagi sebatian-sebatian ini yang ditentukan melalui cerakin DPPH adalah antara IC50

12.33 39.76 µM. Secara amnya, asid kafeoilkuinik dwitukarganti dan asid kafeik menunjukkan aktiviti pemerangkapan radikal DPPH yang lebih tinggi (IC50 = 12.33

22.99 µM) berbanding dengan asid kafeoilkuinik mono-tukarganti (IC50 = 29.90

39.76 µM) dan asid 5-feruloikuinik (IC50 = 84.99 µM). Aktiviti bagi asid kafeoilkuinik mono- dan dwitukarganti serta asid kaffeik adalah setanding dengan aktiviti asid askorbik (IC50 = 21.27 µM) dan trolox (IC50 = 26.72 µM) manakala

xxv aktiviti bagi asid 5-feruloylquinik adalah setanding dengan aktiviti BHT (IC50 =

78.80 µM). Hasil kajian ini menunjukkan bahawa ektrak metanol C. liberica mempunyai profil kimia yang serupa dengan kedua-dua spesies Coffea yang lain dengan menggunakan kaedah kromatografi cecair berprestasi tinggi dengan pengesan tatasusun diod (HPLC-DAD) yang telah dibangunkan dan disahkan. Kuantiti asid kafeoilkuinik mono-tukarganti dan kafein yang terdapat dalam C. liberica adalah di antara kuantiti yang didapati dalam C. arabica dan C. robusta manakala kuantiti asid kafeoilkuinik dwitukarganti adalah lebih kurang daripada yang terdapat dalam kedua-dua spesies yang lain. Namun begitu, perbezaan dalam kandungan sebatian- sebatian ini tidak menyebabkan banyak perbezaan dalam aktiviti-aktiviti antioksidan antara spesies ini. Ketiga-tiga ekstrak kopi tersebut juga didapati mempunyai keupayaan yang serupa dalam sifat perlindungan mereka ke atas fibroblas kulit manusia diaruh tekanan oksidatif daripada hidrogen peroksida. Kajian ini menunjukkan bahawa biji mentah C. liberica mempunyai fitokimia dan potensi antioksidan yang setara dengan C. arabica dan C. robusta. Dengan itu, spesies ini mungkin berfungsi sebagai satu alternatif yang sesuai kepada C. arabica dan C. robusta sebagai sumber yang kaya dengan antioksidan.

xxvi

PHYTOCHEMICALS AND ANTIOXIDATIVE PROPERTIES OF COFFEA

LIBERICA GREEN BEANS AND COMPARISON WITH THAT OF COFFEA

ARABICA AND COFFEA ROBUSTA

ABSTRACT

Coffea arabica, Coffea robusta and Coffea liberica are three main coffee species cultivated for commercial consumption. C. liberica, being the least traded species among the three, is an important commodity to Malaysia. However, limited research has been carried out on this species, in particular on its green beans. The present work was carried out to identify the chemical constitutents responsible for the antioxidant activity of C. liberica green beans, as well as to compare its phytochemical content and antioxidant activities with the other two more popular species. By using an bioactivity-guided approach, caffeine (2), caffeic acid (3) and a series of chlorogenic acids, namely 5-caffeoylquinic acid (1), 3-caffeoylquinic acid

(4), 4-caffeoylquinic acid (5), 5-feruloylquinic acid (6), 4,5-dicaffeoylquinic acid (7),

3,4-dicaffeoylquinic acid (8) and 3,5-dicaffeoylquinic acid (9) were isolated from the green beans of C. liberica. The free radical scavenging activity of these compounds as determined by the DPPH assay was between IC50 12.33 39.76 µM. Generally, the di-substituted caffeoylquinic acids and caffeic acid exhibited stronger DPPH radical scavenging activity (IC50 = 12.33 22.99 µM) than the mono-substituted caffeoylquinic acids (IC50 = 29.90 39.76 µM) and 5-feruloylquinic acid (IC50 = 84.99

µM). The activity of the mono- and di- substituted caffeoylquinic acids as well as caffeic acid was comparable with that of the ascorbic acid (IC50 = 21.27 µM) and trolox (IC50 = 26.72 µM) while the activity of 5-feruloylquinic acid was comparable with that of BHT (IC50 = 78.80 µM). The study revealed that C. liberica methanol

xxvii extract has a similar chemical profile as those of the other two Coffea species by means of high performance liquid chromatography method with diode array detection (HPLC-DAD) that was developed and validated in-house. The quantity of the mono-substituted caffeoylquinic acids and caffeine in C. liberica was in between those found in C. robusta and C. arabica, while the quantity of the di-substituted caffeoylquinic acids was slightly lower than that in the other two species.

Nevertheless, the differences in the content of these compounds did not result in much difference in the antioxidant activities among these species. All three coffee extracts demonstrated similar capacity in their protective properties towards human skin fibroblasts against hydrogen peroxide-induced oxidative stress. The present study indicated that C. liberica green beans have similar constituents and antioxidative potential as those of C. arabica and C. robusta. The species may serve as a good alternative to C. arabica and C. robusta for their rich source of antioxidants.

xxviii

CHAPTER 1

INTRODUCTION

Coffee, being the most consumed beverage in the world, is well known for its antioxidative properties. There are three main coffee species cultivated for commercial consumption, namely Coffea arabica, Coffea robusta and Coffea liberica, among which C. arabica and C. robusta are more popular. Another species,

C. liberica, is less traded because its cultivation is limited to small scale in few places; hence, it is commercially less successful. The total production of C. liberica only accounts for less than 1% of the world’s coffee production (Davis et al., 2006).

Even so, this species is rather important to Malaysia as it accounts for approximately

95% of the total coffee production of this country (Wallengren, 2002).

Throughout centuries, coffee is brewed from roasted beans. The roasting process changes the flavour of green coffee beans and brings out its delightful aroma and taste. However, during the roasting process, many natural products present originally in the green coffee beans are lost due to degradation and/or transformation

(Perrone et al., 2008; Moon et al., 2009; Moreira et al., 2013). Green coffee beans are rich in chlorogenic acids such as caffeoylquinic acids, feruloylquinic acids, p- coumarouylquinic acids, small amount of sinapoylquinic acids and other types of mixed esters (Clifford et al., 2003; Jaiswal et al., 2010). The loss of chlorogenic acids due to roasting directly influences the antioxidant capacity of green coffee and hence the many natural benefits of coffee are lost (Daglia et al., 2000; Castillo et al., 2002;

Somporn et al., 2011; Pino-García et al., 2012). Green coffee beans extract was also found to have better anti-inflammatory activity than that of the roasted beans of C. arabica (Moreira et al., 2013). Its consumption may also prevent various chronic diseases such as cancer, cardiovascular disease and diabetes (Kozuma et al., 2005).

For many years, tremendous amount of research have been done on coffee but the focus was mainly on the roasted beans and their products. There have been renewed interests of late, concerning the beneficial effects of green coffee beans towards human health. However, due to the popularity of C. arabica and C. robusta, most of the research mainly revolved around these two species but not C. liberica

(Naidu et al., 2008; Ludwig et al., 2012; Baeza et al., 2014). Phytochemical investigations of the C. liberica green beans and their antioxidant activities in comparison with those of C. arabica and C. robusta are still lacking up to these days.

Hence, the present study with the following objectives was carried out:

1. To evaluate the antioxidant activities of the green bean extracts of C. liberica

through several in vitro assays.

2. To isolate the phytochemicals responsible for the antioxidant activities of C.

liberica using a bioactivity-guided approach.

3. To determine the antioxidant activities of the isolated compounds.

4. To compare the antioxidant activities by using chemical and cell-based

models as well as the content of the major phytochemicals of C. liberica with

those of its more popular counterparts – C. arabica and C. robusta green

beans by HPLC.

Coffea liberica MeOH extracts (Coffea arabica & Coffea robusta) Extraction

Hexane DCM MeOH

Liquid2 extraction

EtOAc H2O Precipitate VLC RP C18 column chromatography EF1 EF2 EF3 EF4 EF5 EF6 EF7 RP C18 column Compound 1 Sephadex LH-20 chromatography Compound 2

SF1 SF2 SF3 SF4

RP C18 column Semi-prep HPLC Semi-prep HPLC PTLC Compound 3 chromatography Compound 4 Compound 1 Compound 8 Compound 7 Compound 5 Compound 9 Compound 6

Method validation

DPPH radical scavenging activity Quantitative analysis of phytochemical Antioxidant activity (DPPH, FRAP), content (HPLC) (except compound 6) TPC and protective effect against H2O2- induced oxidative stress in human skin fibroblasts (Hs27)

CHAPTER 2

LITERATURE REVIEW

2.1 Antioxidant as chemopreventive and therapeutic agent

- Reactive oxygen species (ROS) such as superoxide anion (O2• ), hydroxyl (•OH), peroxyl (ROO•), alkoxyl (RO•) and hydroperoxyl (HO2•) radicals are continuously generated as the byproducts of normal cellular metabolism in human body. The body then regulates these free radicals with its natural antioxidant defense system which comprises of the endogenous enzymes such as superoxide dismutase (SOD), catalase and glutathione peroxidase (Halliwell, 1991; Valko et al., 2007). However, air pollution, ultraviolet radiation, unhealthy lifestyle such as smoking and many more can cause an overproduction of ROS. Thus, a depletion of the antioxidant defenses in the endogenous protective system occurs. This phenomenon is known as oxidative stress and can cause the damage in cellular lipids, proteins or DNA which in turn gives rise to various types of chronic diseases such as cancer, diabetes mellitus, cardiovascular diseases, rheumatoid arthritis, ageing and neurodegerative disorder

(Halliwell, 1997; Valko et al., 2007; Ziech et al., 2010; Baeza et al., 2014). In order to help reverse the effects caused by the overwhelming presence of ROS, exogenous antioxidants of natural or synthetic origin may be recruited (Wootton-Beard & Ryan,

2011).

2.1.1 Natural antioxidants

Natural antioxidants may be categorized into hydrophilic or lipophilic, depending on their solubility. They consist of polyphenols, carotenoids, vitamins and minerals which are mostly found in fruits, vegetables and beverages (Bravo, 1998; Wootton-

Beard & Ryan, 2011). Vitamin C, E and β-carotene are amongst the most established antioxidants found in dietary sources. Vitamin C, often known as ascorbic acid, is a hydrophilic antioxidant, while vitamin E (mainly presents as α-tocopherol) and β- carotene are lipophilic antioxidants (Niki et al., 1995) (Figure 2.1).

Ascorbic acid (vitamin C)

α-tocopherol (vitamin E)

β-carotene

Figure 2.1: Chemical structures of ascorbic acid, α-tocopherol and β-carotene.

Polyphenols, the largest class of dietary antioxidants, are another group of hydrophilic compounds present abundantly in fruits, vegetables and beverages

(Ratnam et al., 2006). These types of compounds are synthesized by plant as secondary metabolites to protect themselves against ultraviolet radiation and pathogenic invasion (Manach et al., 2004). Polyphenols can be divided into two groups: non-flavonoid and flavonoid compounds, with different classes and subclasses according to the number of phenol rings and the type of substituents attached to the core structure (Table 2.1). Phenolic compounds present in nature ranges from molecules as simple as phenolic acids, flavonols, flavones, flavanones, flavanols and isoflavones to highly polymerized structures like tannins (Bravo, 1998;

Manach et al., 2004).

Table 2.1: The main classes of phenolic compounds (Source: Manach et al., 2004 & Wootton-Beard & Ryan, 2011). Class Subclass Examples Non-flavonoid compounds Phenolic acids Benzoic acids Gallic acid, protocatechuic acid, p-hydroxybenzoic acid Hydroxycinnamic acids p-coumaric acid, caffeic acid, ferulic acid, sinapic acid Tannins Hydrolyzable tannins Pentagalloyloylglucose, punicalagins, ellagitannins Stilbenes Resveratrol Lignans Secoisolariciresinol, matairesinol, lariciresinol, pinoresinol Flavonoid compounds Flavonols Kampferol, quercetin, myricetin Flavones Apigenin, luteolin Flavanones Narigenin, hesperetin Flavanols Catechins, gallocatechins Tannins Condensed tannins Proanthocyanidins Isoflavones Daidzein, genistein, glycitein

Hydroxycinnamic acids (HCA) are ubiquitous constituents in the plant kingdom and well known for their antioxidant activities, particularly the radical scavenging ability. These phytochemicals possess a phenylpropanoic structure with

C6-C3 as their basic skeleton (Bravo, 1998; Esteves et al., 2008; Razzaghi-Asl et al.,

2013). The para hydroxy group on the benzene ring and the α, β unsaturated bond of

HCA (highlighted in Figure 2.2) are the requisite factors of the radical scavenging capacity of HCA. The benzene structure which bears the hydroxy group is capable of forming phenoxy radical intermediate that are responsible for free radical chain termination, whereas the α, β unsaturated bond favours the stability of the phenoxy radical by increasing its electron delocalization, at the same time it acts as an additional reaction site for ROS (Graf, 1992; Barone et al., 2009). Caffeic acid, ferulic acid, p-coumaric acid and sinapic acid are the most typical examples of HCA

(Figure 2.2) (Razzaghi-Asl et al, 2013).

R = OH, R = H caffeic acid 1 2 R1=OCH3, R2 =H ferulic acid R1= H, R2 =H p-coumaric acid R1=OCH3, R2 = OCH3 sinapic acid

Figure 2.2: The chemical structures of caffeic acid, ferulic acid, p-coumaric acid and sinapic acid.

2.1.2 Synthetic antioxidants

Synthetic antioxidants are used as food additives or preservatives to help prevent food rancidity due to oxidation process. Some of the examples of these antioxidants which are widely used in the food industry include BHT (butylated hydroxytoulene),

BHA (butylated hydroxyanisole), THBP (2,4,5-trihydroxybutyrophenone), propyl gallate and octyl gallate (Xiu-Qin et al., 2009; Yehye et al., 2015) (Figure 2.3).

However, studies have shown that some of these synthetic antioxidants may have adverse effect on human health (Yehye et al., 2015). Therefore, attention has now been switched to the use of naturally occuring antioxidants as safer substitutes.

BHT (butylated hydroxytoulene) BHA (butylated hydroxyanisole)

THBP (2,4,5-trihydroxybutyrophenone) Propyl gallate

Octyl gallate

Figure 2.3: Chemical structures of BHT (butylated hydroxytoulene), BHA (butylated hydroxyanisole), THBP (2,4,5-trihydroxybutyrophenone), propyl gallate and octyl gallate.

2.2 Rubiaceae family

Rubiaceae family is one of the largest flowering plant in the plant kingdom with around 600 genera and 13000 species commomly found in the tropical region.

Rubiaceae is subdivided into four subfamilies, which are Cinchonoideae, Ixoroideae,

Antirheoideae and Rubioideae. Their species vary from small trees, shrubs, herbs to even large woody plant (Bremer, 1996; Davis et al., 2009). Some of the species in this family which are valuable to mankind includes Cinchona officinalis (medicinal plant), Rubia tinctoria (madder; dye plant), Neolamarckia chinensis (timber trees),

Gardenia jasminoides (ornamental plant) and several Coffea species which are by far the most economically important species due to its popularity as a beverage (Kew

Royal Botanic Gardens, n.d.).

2.3 Genus of Coffea

The genus Coffea is native to Africa and is widely distributed across the tropical countries for centuries (Clarke & Macrae, 1985) (Figure 2.4). It is classified under the Ixoroideae subfamily which is divided into two subgenus, Coffea subgenus

Coffea and Coffea subgenus Baracoffea based on their morphological characteristics

(Davis et al., 2006; Bremer & Eriksson, 2009). There are 103 species identified in this genus, of which 95 species are grouped under Coffea subgenus Coffea including the three main coffee species that are traded in the market, namely, Coffea arabica,

Coffea robusta (C. canephora) and Coffea liberica, whilst another eight species are under Coffea subgenus Baracoffea (Davis et al., 2006).

Figure 2.4: World coffee production (Source: Jha et al., 2014).

The spread of coffee cultivation is mainly based on the economical importance of a species and geographical aspects such as adaptation to soils, climate and altitude (Wellman, 1961). Among the three coffee species sold for coffee beverages, C. arabica and C. robusta are more widely traded, while C. liberica is less sought after (Davis et al., 2006; USDA, 2014). The main producers for C. arabica are countries like Brazil, Central America and Africa, where their contribution of coffee production was 41.4%, 15.4% and 10.9%, respectively. C. robusta is mainly produced in Vietnam, Brazil, Indonesia and Africa accounting for

42.1%, 25.3%, 11.0% and 10.4%, respectively (USDA, 2014). On the other hand, C. liberica is mainly produced in Malaysia, some places in the Philipines and parts of

Africa such as Liberia (Wellman, 1961; Davis et al., 2006).

C. canephora is more known as C. robusta due to its resistance towards

Hemileia rust (Wellman, 1961; Davis et al., 2006). There are few interesting differences among the trees of these three coffee species (Table 2.2).

C. arabica is self-pollinating, has little blossoms and small flowers while the flowers of C. robusta and C. liberica are often bigger and rely on cross-pollination by means of pollination agents like wind and insects. Under cultivation, C. liberica is grown as the tallest tree and C. arabica being the shortest. Unlike C. robusta and C. liberica, C. arabica has the smallest leaves. The leaves of C. liberica are leathery and the largest among three species. Apart from that, the cherries of C. liberica are large and thick- skinned with big seeds/beans. For C. arabica, the cherries and beans are often medium-sized while the cherries and beans of C. robusta are the smallest. The beans of C. liberica and C. arabica are oval in shape except for C. robusta, which are round. Among the three species, C. arabica is more vunerable to rust, disease and harsh climates (Wellman, 1961). C. liberica is well adapted to various elevations,

11 soils, climates and sun expose cultivations. Therefore, it is probably more suited to grow at lowland and is also the most tolerant species towards heat amongst the three.

Table 2.2: Characteristic differences between Coffea arabica, Coffea robusta and Coffea liberica (Wellman, 1961; Clarke & Macrae, 1985; Davis et al. 2006; Ismail et al., 2014). Species Characteristic C. arabica C. robusta C. liberica

Pollination Self-fertile Self-sterile Self-sterile

Tree height in 26-33 ft 6.5-16 ft 18-36 ft wild

Relative leaf size Smallest; dark Largest; light green Medium; dark green, green leathery

Relative flower Small Medium Large size

Cherries/fruits Medium; red Small; red Large; red to red- brownish

Bean size (no. of 1200 1600 800 beans per pound)

Bean shape Oval Round Oval

Optimum altitude 2500-5000 ft 600-2400 ft Sea level-1800 ft for growth

Ecological niche Humid, evergreen Humid, evergreeen Humid, evergreen forest forest, sometimes in forest, or seasonally seasonally dry dry, evergreen humid forest, rarely forest, sometimes in in gallery forest seasonally dry mixed evergreen- deciduous forest, gallery forest

2.4 Coffee as a source of medicine and beverage

In the early days, coffee was used as medicine in places like Arabia, Europe and

America. Coffee infusion is believed to soothe the mind, relieve pains in the head, lethargy and cough. Besides, it is also effective in treating rheumatism, gout and intermittent fever. Before roasted coffee beans were introduced, dried young coffee leaves, cherry pulp or whole cherry were infused like tea in order to make a refreshing drink. The coffee leaf is also used traditionally for preparing drinks in some parts of Malaysia, Jawa and Sumatra (Wellman, 1961). This refreshing and stimulating effect of coffee was most probably due to the presence of caffeine.

2.5 Green beans

A coffee fruit consists of the skin, pulp, mucilage, pachment, silverskin and the bean

(Figure 2.5). Green coffee beans are produced by removal of pulp including mucilage and pachment of the ripe coffee berries. After depulping the berries, the green beans (with or whithout the silverskin) are sundried, separated and graded accordingly (Sivaram, 1980; Esquivel & Jiménez, 2012). A picture of the green coffee bean of C. arabica, C. robusta and C. liberica, respectively, is shown in

Figure 2.6.

Figure 2.5: The structure of coffee fruit (Source: Esquivel & Jiménez, 2012).

Coffea arabica Coffea robusta Coffea liberica

Figure 2.6: The dried green beans of Coffea arabica, Coffea robusta and Coffea liberica.

2.5.1 Phytochemicals in green coffee beans

The phytochemicals of coffee have been studied for more than a century. There are tremendous amount of compounds present in coffee beans and more than a thousand of compounds have been identified in roasted beans to date (Hoffman & Gerber,

2012). Green coffee beans are rich in polyphenols particularly 5-caffeoylquinic acid, caffeine other than lipids, volatile compounds and carbohydrates (Redgwell &

Fischer, 2006; Speer & Kölling-Speer, 2006; Alonso-Salces et al., 2009; Wagemaker et al., 2011). Most of the studies on the phytochemicals were done on C. arabica and

C. robusta but there is scant information about the phytoconstituents of C. liberica

(Fischer et al., 2001; Oosterveld et al., 2003; Moon et al., 2009; Perrone et al., 2012).

In this section, only important and major components present in green coffee bean especially in C. arabica and C. robusta were discussed.

2.5.1(a) Lipids

The lipid fraction of coffee beans consists of a minute amount of coffee wax extracted from the outer layer of the beans and coffee oils. Three of the Nβ-alkanoyl-

5-hydroxytryptamine (C-5-HT), Nβ-arachidoyl-5-hydroxytryptamine, Nβ-behenoyl-5- hydroxytryptamine and Nβ-lignoceroyl-5-hydroxytryptamine were found as the predominent components present in the coffee wax (Figure 2.7) (Folstar et al., 1979).

The coffee oils is constituted of free fatty acids, triglycerols, sterols, diterpenes and other lipid components found in the endosperm (Speer & Kölling-Speer, 2006)

(Figure 2.8). Among the fatty acids present are palmitic (C16:0), stearic (C18:0), oleic (C18:1), linoleic (C18:2), linolenic (C18:3) and arachidic ((C20:0) acids in the form of free fatty acids, with palmitic and linoleic acids being the most predominat

15 free fatty acids present in the green beans of 10 coffee species (Martin et al., 2001;

Wagemaker et al., 2011). However, most of the fatty acids present in nature are either esterified with glycerol or diterpenes to form triacylglycerols or diterpene esters whilst small amounts are esterified with sterol as sterol esters (Nikolova-

Damyanova et al., 1998). The diterpenes, cafestol and kahweol are the predominant unsaponifiable matter of coffee oil which is seldom found in free form and present as diterpene esters (Scharnhop & Winterhalter, 2009). The main sterols in green coffee are β-sitosterol, followed by stigmasterol and then campesterol (Carrera et al., 1998).

β R = CH2OH; n = 18 N -arachidoyl-5-hydroxytryptamine β R = CH2OH; n = 20 N -behenoyl-5-hydroxytryptamine β R = CH3; n = 22 N -lignoceroyl-5-hydroxytryptamine

Figure 2.7: Chemical structures of Nβ-arachidoyl-5-hydroxytryptamine, Nβ- behenoyl-5-hydroxytryptamine and Nβ-lignoceroyl-5-hydroxytryptamine.

palmitic acid

cafestol kahweol

R= H; free diterpene R’= fatty acid; diterpene ester

stigmasterol β-sitosteol

campesterol

Figure 2.8: Chemical structures of major free fatty acids, diterpenes and sterols.

2.5.1(b) Volatile compounds

Approximately 300 or more volatile compounds were identified in green coffee.

These compounds mainly consisted of hydrocarbons, alcohols, pyrazines, ketone, furans, aldehydes, phenols, esters and some sulfur compounds (Flament & Bessière-

Thomas, 2002). Among these volatile components, there are a few major compounds which give rise to the odour of green coffee beans (Figure 2.9). Pentanal or known as veleraldehyde was identified in green beans and is believed in giving penetrating odour to the green coffee beans (Zlatkis & Sivetz, 1960; Flament & Bessiere-Thomas,

2002). The ‘green’ smell of green coffee was possibly caused by the presence of compounds like 2-isobutyl-3-methoxypyrazine and two pairs of unsaturated aldehydes, (E,E)-2,4- and (2E,4Z)-2,4-nonadienal; (E,E)-2,4- and (2E,4Z)-2,4- decadienal. These two pairs of unsaturated aldehyde were identified in the

Colombian green coffee (Boosfeld & Vitzthum, 1995). Other than these, p- vinylguaiacol was also found to be a major compound present in green coffee that gives its strong, spicy, clove-like odour (Flament & Bessiere-Thomas, 2002; Saw et al., 2015).

Pentanal 2-isobutyl-3-methoxypyrazine

(E,E)-2,4-nonadienal (2E,4Z)-2,4-nonadienal

(E,E)-2,4-decadienal (2E,4Z)-2,4-decadienal

p-vinylguaiacol

Figure 2.9: Chemical structures of major volatile compounds.

2.5.1(c) Alkaloids

Caffeine (1,3,7-trimethylxanthine), theophylline (1,3-dimethylxanthine), theobromine (3,7-dimethylxanthine) and trigonelline are the alkaloids found in green coffee beans (Clifford & Kazi, 1987; Alonso-Salces et al., 2009) (Figure 2.10).

The content of caffeine, a major alkaloid in coffee beans is higher in C. robusta than

C. arabica. The content of caffeine also varies depending on its geographical origin

(Table 2.3). Theophylline is only present in C. robusta and is often used as a chemical marker for this species. Trigonelline is the second most abundant alkaloid after caffeine, while only trace amount of theobromine is observed in green coffee beans (Clifford & Kazi, 1987; Ky et al., 2001; Alonso-Salces et al., 2009).

caffeine theophylline theobromine

trigonelline

Figure 2.10: Chemical structures of caffeine (1,3,7-trimethylxanthine), theophylline (1,3-dimethylxanthine), theobromine (3,7-dimethylxanthine) and trigonelline.