phytochemicals and antioxidative properties of …
TRANSCRIPT
PHYTOCHEMICALS AND ANTIOXIDATIVE
PROPERTIES OF COFFEA LIBERICA GREEN
BEANS AND COMPARISON WITH THAT OF
COFFEA ARABICA AND COFFEA ROBUSTA
by
TEO HUI MIN
Thesis submitted in fulfillment of the requirements
for the degree of
Master of Science
December 2016
ii
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
iii
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.
iv
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
v
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
precipitate obtained in Coffea liberica MeOH
extract
44
3.3.2(b) Partitioning of the chemical constituents in
Coffea liberica MeOH extract
46
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
vi
3.3.3(e) Fourier Transformed Infrared Spectroscopy
(FTIR)
56
3.3.3(f) Nuclear magnetic resonance spectroscopy 57
3.3.4 Detection of compounds on TLC by post-chromatographic
treatment
57
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
extract of Coffea liberica, Coffea arabica and Coffea robusta
by HPLC
59
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
species on hydrogen peroxide-induced oxidative stress in
human skin fibroblasts
66
3.3.9(a) Test sample preparation 66
3.3.9(b) Cell culture 66
3.3.9(c) Cell treatment 66
vii
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
PPT
73
4.2 Comparison of the chemical composition of MeOH PPT versus
MeOH extract
74
4.3 Bioactivity-guided isolation of antioxidative compounds from Coffea
liberica MeOH extract
76
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
extract
81
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 EF7)
86
4.3.5(c) Phytochemical evaluation of active subfractions 87
4.4 Bioactive phenolic compounds obtained from the selected
subfractions
89
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
Coffea liberica, Coffea arabica and Coffea robusta MeOH extracts
191
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
quantification
197
4.7.1(c) Precision 198
4.7.1(d) Accuracy 199
4.7.2 Quantification of the marker compounds in the MeOH
extract of three Coffea species
203
4.7.3 Antioxidant activities and total phenolic content of the
MeOH extract of three Coffea species
206
4.7.4 Protective effect of the MeOH extracts of the three Coffea
species against hydrogen peroxide-induced oxidative stress
in human skin fibroblasts
208
CHAPTER 5 CONCLUSION 211
REFERENCES 214
APPENDICES
LIST OF PUBLICATIONS
ix
LIST OF TABLES
Page
Table 2.1 The main classes of phenolic compounds. 6
Table 2.2 Characteristic differences between Coffea arabica, Coffea
robusta and Coffea liberica.
12
Table 2.3 Caffeine content in green coffee beans, expressed in %
(gram per gram of coffee beans, w/w dry matter basis).
21
Table 2.4 Chlorogenic acids content in green coffee beans,
expressed in % (gram per gram of coffee beans, w/w dry
matter basis).
24
Table 2.5 Chlorogenic acids and caffeine content (mg per gram of
coffee beans, w/w dry matter basis) (Mean SD, n =3) in
Ethiopian green coffee beans.
29
Table 2.6 Summary of the chromatograpic separation of analytes in
various coffee samples. 31 32
Table 3.1 List of chemicals used, their abbreviations and
manufacturing country. 41 42
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
using HPLC.
49
Table 3.6 Solvent system used for the separation of EF7/SF1. 50
Table 3.7 Chromatographic conditions used for the scaled-up
separation of EF7/SF2-EF7/SF4.
51
Table 3.8 Concentration of the individual compounds for each
standard mixture.
59
Table 3.9 Gradient elution program used in the mobile phase
system for HPLC analysis of Coffea liberica, Coffea
arabica and Coffea robusta using Xselect HSS T3 XP
column.
61
Table 4.1
DPPH radical scavenging activity of the extracts of
Coffea liberica.
71
x
Table 4.2 Total sugar content of MeOH extract, EtOAc and water
fractions.
79
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
limit of quantification of compounds 1 5 and 7 9 (n = 3).
197
Table 4.13 Results of precision study on compound 1 5 and 7 9 at
three concentration levels (low, medium, high) (n = 3).
198
Table 4.14 Method accuracy for the analysis of compounds 1 5 and
7 9 in the MeOH extract of Coffea liberica (n = 3).
200
Table 4.15 Method accuracy for the analysis of compounds 1 5 and
7 9 in the MeOH extract of Coffea arabica (n = 3).
201
Table 4.16 Method accuracy for the analysis of compounds 1 5 and
7 9 in the MeOH extract of Coffea robusta (n = 3).
201
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LIST OF FIGURES
Page
Figure 2.1 Chemical structures of ascorbic acid, α-tocopherol and
β-carotene.
5
Figure 2.2 The chemical structures of caffeic acid, ferulic acid, p-
coumaric acid and sinapic acid.
7
Figure 2.3 Chemical structure of BHT (butylated
hydroxytoulene), BHA (butylated hydroxyanisole),
THBP (2,4,5-trihydroxybutyrophenone), propyl gallate
and octyl gallate.
8
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
robusta and Coffea liberica.
14
Figure 2.7 Chemical structures of Nβ-arachidoyl-5-
hydroxytryptamine, Nβ-behenoyl-5-hydroxytryptamine
and Nβ-lignoceroyl-5-hydroxytryptamine.
16
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-
trimethylxanthine), theophylline (1,3-
dimethylxanthine), theobromine (3,7-
dimethylxanthine) and trigonelline.
20
Figure 2.11 Chemical structures of quinic acid, trans-
hydroxycinnamic acids and chlorogenic acids.
23
Figure 2.12 Chemical structures of the backbone of
galactomannans, the backbone of cellulose and
sucrose.
25
Figure 2.13 The leaves of Coffea liberica. 34
Figure 2.14 The ripe berries of Coffea liberica.
34
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Figure 2.15 Coffea liberica. a. A cluster of three calycesn with the
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.
35
Figure 2.16 Chemical structures of the fatty acids present in Coffea
liberica green beans.
37
Figure 2.17 Chlorogenic acids in Coffea liberica. 39 40
Figure 3.1 Semi-preparative HPLC chromatogram of EF7/SF2
detected at 325 nm.
52
Figure 3.2 Semi-preparative HPLC chromatogram of EF7/SF3
detected at 325 nm.
52
Figure 3.3 A schematic diagram of compound isolation from
Coffea liberica.
54
Figure 4.1 Extraction yield of hexane, DCM, MeOH extracts of
Coffea liberica. Results are expressed as mean SD
(n = 3).
70
Figure 4.2 Ferric reducing antioxidant activity of the extracts of
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.
71
Figure 4.3 Total phenolic content of the extracts of Coffea
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.
73
Figure 4.4 Reversed phase TLC of MeOH extract (left) and
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.
75
xiii
Figure 4.5 DPPH radical scavenging activity of MeOH extract,
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.
77
Figure 4.6 Ferric reducing activity of MeOH extract, EtOAc and
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.
77
Figure 4.7 Total phenolic content of EtOAc and water fractions.
Results are expressed as mean SD (n = 3 2) in mg
of gallic acid equivalent per 1 g of dry extract.
78
Figure 4.8 IR spectrum of water fraction from Coffea liberica
MeOH extract.
81
Figure 4.9 Normal phase TLC of MeOH extract and EtOAc
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).
82
Figure 4.10 Percentage of yield of subfractions EF1-EF7. 83
Figure 4.11 DPPH radical scavenging activity of EF2-EF7. Results
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.
85
Figure 4.12 Ferric reducing activity of EF1-EF7. Results are
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.
85
Figure 4.13 Total phenolic content of EF1-EF7. Results are
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.
86
xiv
Figure 4.14 Normal phase TLC of EtOAc fraction, EF3, EF6 and
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.
88
Figure 4.15 The HPLC chromatogram of A) EF3 B) compound 3
on a Waters Sunfire C18 (4.6 250 mm, 5 µm)
detected at 325 nm.
89
Figure 4.16 Normal phase TLC of EF7 and subfractions EF7/SF1-
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.
91
Figure 4.17 The HPLC chromatogram of A) EF7/SF1 B) EF7/SF2
C) EF7/SF3 D) EF7/SF4 on a Waters Sunfire C18
column (250 4.6 mm i.d., 5 µm), detected at 325 nm.
92
Figure 4.18 Chemical structure of compound 1 (5-caffeoylquinic
acid).
93
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
Figure 4.22 1H 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
Figure 4.25 13
C NMR spectrum of compound 1 (D2O, 125 MHz). 104
Figure 4.26 A) 13
C DEPT 90 B) 13
C DEPT 135 spectrum of
compound 1 (D2O, 125 MHz).
105
Figure 4.27 HSQC spectrum of compound 1 (D2O, 500MHz). 106
Figure 4.28 The chemical structure of compound 2 [caffeine
(1,3,7-trimethylxanthine)].
107
Figure 4.29 EI-MS of A) compound 2 B) Caffeine standard. 108
Figure 4.30 UV spectrum of compound 2 (MeOH). 109
xv
Figure 4.31 FT-IR spectrum of compound 2 (ATR). 110
Figure 4.32 13
C NMR spectrum of compound 2 (CDCl3, 125
MHz). 112
Figure 4.33 1H NMR spectrum of compound 2 (CDCl3, 500 MHz). 113
Figure 4.34 Chemical structure of compound 3 (caffeic acid; 3,4-
dihydroxycinnamic acid).
114
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
Figure 4.38 1H NMR spectrum of compound 3 (CD3OD, 500
MHz).
120
Figure 4.39 13
C NMR spectrum of compound 3 (CD3OD, 125
MHz).
121
Figure 4.40 Chemical structure of compound 4 (3-caffeoylquinic
acid).
122
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
Figure 4.44 1H NMR spectrum of compound 4 (D2O, 500 MHz). 128
Figure 4.45 13
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
acid).
131
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
Figure 4.51 1H NMR spectrum of compound 5 (D2O, 500 MHz). 137
Figure 4.52 13
C NMR spectrum of compound 5 (D2O, 125 MHz). 138
Figure 4.53 COSY spectrum of compound 5 (D2O, 500MHz). 139
xvi
Figure 4.54 HMBC spectrum of compound 5 (D2O, 500MHz). 140
Figure 4.55 Proposed chemical structure of compound 6 (5-
feruloylquinic acid).
141
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
Figure 4.59 1H NMR spectrum of compound 6 (CD3OD, 500
MHz).
147
Figure 4.60 HMBC spectrum of compound 6 (CD3OD, 500 MHz). 148
Figure 4.61 13
C NMR spectrum of compound 6 (CD3OD, 125
MHz).
149
Figure 4.62 COSY spectrum of compound 6 (CD3OD, 500 MHz). 150
Figure 4.63 Chemical structure of compound 7 (4,5-
dicaffeoylquinic acid).
151
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
Figure 4.67 1H NMR spectrum of compound 7 (DMSO-d6, 500
MHz).
158
Figure 4.68 COSY spectrum of compound 7 (DMSO-d6,
500MHz).
159
Figure 4.69 13
C NMR spectrum of compound 7 (DMSO-d6, 125
MHz).
160
Figure 4.70 A) 13
C DEPT 90 B) 13
C DEPT 135 NMR spectrum of
compound 7 (DMSO-d6, 125 MHz).
161
Figure 4.71 HSQC spectrum of compound 7 (DMSO-d6,
500MHz).
162
Figure 4.72 HMBC spectrum of compound 7 (DMSO-d6,
500MHz).
163
Figure 4.73 Chemical structure of compound 8 (3,4-
dicaffeoylquinic acid).
164
xvii
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
Figure 4.77 1H NMR spectrum of compound 8 (DMSO-d6, 500
MHz).
171
Figure 4.78 COSY spectrum of compound 8 (DMSO-d6,
500MHz).
172
Figure 4.79 13
C NMR spectrum of compound 8 (DMSO-d6, 125
MHz).
173
Figure 4.80 HSQC spectrum of compound 8 (DMSO-d6,
500MHz).
174
Figure 4.81 HMBC spectrum of compound 8 (DMSO-d6,
500MHz).
175
Figure 4.82 Chemical structure of compound 9 (3,5-
dicaffeoylquinic acid).
176
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
Figure 4.86 1H NMR spectrum of compound 9 (DMSO-d6, 500
MHz).
182
Figure 4.87 COSY spectrum of compound 9 (DMSO-d6,
500MHz).
183
Figure 4.88 13
C NMR spectrum of compound 9 (DMSO-d6, 125
MHz).
184
Figure 4.89 HSQC spectrum of compound 9 (DMSO-d6,
500MHz).
185
Figure 4.90 HMBC spectrum of compound 9 (DMSO-d6,
500MHz).
186
Figure 4.91 DPPH radical scavenging activity (IC50 in µM) of
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.
190
xviii
Figure 4.92 Representative chromatograms of Coffea liberica
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.
192
Figure 4.93 UV spectrum of: 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), 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.
194
Figure 4.94 Representative HPLC chromatograms of A) Coffea
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.
195
Figure 4.95 Representative HPLC chromatograms of A) Coffea
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.
196
Figure 4.96 The composition of compounds 1-5 and 7-9 in the
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.
205
Figure 4.97 DPPH radical scavenging activity of Coffea liberica,
Coffea arabica and Coffea robusta MeOH extracts.
Results are expressed as mean SD (n = 3 2).
206
Figure 4.98 Ferric reducing activity of Coffea liberica, Coffea
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.
207
xix
Figure 4.99 Total phenolic content of Coffea liberica, Coffea
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.
207
Figure 4.100 A) pre-treatment and B) post-treatment effect of the
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.
210
xx
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
xxi
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
xxii
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-
MS
Ion mobility/orthogonal acceleration time-of-flight mass
spectrometry
LOD Limit of detection
LOQ Limit of quantification
lit. Literature
min Minute
xxiii
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
xxiv
USDA United States Department of Agriculture
w/w Weight per weight
w/v Weight per volume
xxv
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
xxvi
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.
xxvii
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
xxviii
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.
1
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;
2
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.
3
Precipitate EtOAc H2O
Compound 3
Compound 1
Compound 2
Coffea liberica
EF1 EF2 EF3 EF4 EF5 EF6 EF7
Extraction
Liquid2 extraction
VLC
Sephadex LH-20
RP C18 column
chromatography
RP C18 column
chromatography
MeOH extracts (Coffea arabica & Coffea robusta)
SF1 SF4 SF2 SF3
Compound 4 Compound 1
Compound 5
Compound 6
Compound 8
Compound 9 Compound 7
RP C18 column
chromatography PTLC Semi-prep HPLC
Semi-prep HPLC
MeOH Hexane DCM
DPPH radical scavenging activity Quantitative analysis of phytochemical
content (HPLC) (except compound 6)
Method validation
Antioxidant activity (DPPH, FRAP),
TPC and protective effect against H2O2-
induced oxidative stress in human skin
fibroblasts (Hs27)
4
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).
5
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).
Figure 2.1: Chemical structures of ascorbic acid, α-tocopherol and β-carotene.
Ascorbic acid (vitamin C)
α-tocopherol (vitamin E)
β-carotene
6
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
7
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).
Figure 2.2: The chemical structures of caffeic acid, ferulic acid, p-coumaric acid and
sinapic acid.
R1= OH, R2 = H caffeic acid
R1=OCH3, R2 =H ferulic acid
R1= H, R2 =H p-coumaric acid
R1=OCH3, R2 = OCH3 sinapic acid
8
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.
Figure 2.3: Chemical structures of BHT (butylated hydroxytoulene), BHA
(butylated hydroxyanisole), THBP (2,4,5-trihydroxybutyrophenone), propyl gallate
and octyl gallate.
BHA (butylated hydroxyanisole) BHT (butylated hydroxytoulene)
Propyl gallate THBP (2,4,5-trihydroxybutyrophenone)
Octyl gallate
9
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).
10
Figure 2.4: World coffee production (Source: Jha et al., 2014).
11
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,
12
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).
Characteristic Species
C. arabica C. robusta C. liberica
Pollination Self-fertile Self-sterile Self-sterile
Tree height in
wild
26-33 ft 6.5-16 ft 18-36 ft
Relative leaf size Smallest; dark
green
Largest; light green Medium; dark green,
leathery
Relative flower
size
Small Medium Large
Cherries/fruits Medium; red Small; red Large; red to red-
brownish
Bean size (no. of
beans per pound)
1200 1600 800
Bean shape Oval Round Oval
Optimum altitude
for growth
2500-5000 ft 600-2400 ft Sea level-1800 ft
Ecological niche Humid, evergreen
forest
Humid, evergreeen
forest, sometimes in
seasonally dry
humid forest, rarely
in gallery forest
Humid, evergreen
forest, or seasonally
dry, evergreen
forest, sometimes in
seasonally dry
mixed evergreen-
deciduous forest,
gallery forest
13
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.
14
Figure 2.5: The structure of coffee fruit (Source: Esquivel & Jiménez, 2012).
Figure 2.6: The dried green beans of Coffea arabica, Coffea robusta and Coffea
liberica.
Coffea arabica Coffea robusta Coffea liberica
15
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, N
β-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
16
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).
Figure 2.7: Chemical structures of Nβ-arachidoyl-5-hydroxytryptamine, N
β-
behenoyl-5-hydroxytryptamine and Nβ-lignoceroyl-5-hydroxytryptamine.
R = CH2OH; n = 18 Nβ-arachidoyl-5-hydroxytryptamine
R = CH2OH; n = 20 Nβ-behenoyl-5-hydroxytryptamine
R = CH3; n = 22 Nβ-lignoceroyl-5-hydroxytryptamine
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Figure 2.8: Chemical structures of major free fatty acids, diterpenes and sterols.
cafestol kahweol
palmitic acid
5
β-sitosteol stigmasterol
campesterol
R= H; free diterpene
R’= fatty acid; diterpene ester
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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).
19
Figure 2.9: Chemical structures of major volatile compounds.
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
20
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).
Figure 2.10: Chemical structures of caffeine (1,3,7-trimethylxanthine), theophylline
(1,3-dimethylxanthine), theobromine (3,7-dimethylxanthine) and trigonelline.
theobromine theophylline caffeine
trigonelline