Chemical and pharmacological evaluations of panax notoginseng and swietenia macrophylla

CHEMICAL AND PHARMACOLOGICAL EVALUATIONS
OF PANAX NOTOGINSENG AND
SWIETENIA MACROPHYLLA








TOH DING FUNG

(B.Sc. (Pharm.) (Hons.), NUS)







A THESIS SUBMITTED
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
DEPARTMENT OF PHARMACY
NATIONAL UNIVERSITY OF SINGAPORE
2010

ii

ACKNOWLEDGEMENTS

I would like to extend my sincere gratitude to my thesis supervisor, Prof Koh Hwee Ling,
for her patient guidance, suggestions and advice throughout the whole course of this project and
thesis write-up. I would also like to thank Dr Eric Chan Chun-Yong and Miss New Lee Sun, for
their helpful guidance and advice in the metabolomics work on P. notoginseng. I would like to
thank Dr Neo Soek-Ying and Dr Alvin Teo for their advice and guidance in the cancer cell work.
I would like to express my heartfelt gratitude Mr Li Lin for his help in LC-MS and NMR and Mr
Johannes Murti Jaya for his help in preliminary work on isolation of limonoids. I am also grateful
to the financial support of a research scholarship from the National University of Singapore
research scholarship. The technical assistance from the laboratory officers in the Department of
Pharmacy, especially Ms Ng Sek Eng is greatly appreciated. I also wish to thank everyone in the
department who has helped me in one way or another, especially my laboratory mates (namely,
Li Lin, Jianhong, Dhaval and Peiling) for their help and enjoyable times in the laboratory.
Special thanks also go to all my fellow friends in the department for their moral support, helpful
discussions, and for sharing all the woes and wonderful times together during my postgraduate
years. Last but not least, I would like to thank my family for their understanding and continuous
support.







iii

LIST OF PUBLICATIONS AND CONFERENCE PRESENTATIONS

Publications

1. Toh DF, New LS, Koh HL, Chan EC. Ultra-high performance liquid chromatography/time-

of-flight mass spectrometry (UHPLC/TOFMS) for time-dependent profiling of raw and
steamed Panax notoginseng. J Pharm Biomed Anal 2010; 52: 43-50.

2. Toh DF, Patel DN, Chan EC, Teo A, Neo S-Y, Koh HL. Antiproliferative effects of raw
and steamed extracts of Panax notoginseng and its ginsenoside constituents on human liver
cancer cells. Chinese medicine 2011, In press.

3. Lau AJ, Toh DF, Chua TK, Pang YK, Woo SO, Koh HL. Antiplatelet and anticoagulant
effects of Panax notoginseng: comparison of raw and steamed Panax notoginseng with
Panax ginseng and Panax quinquefolium. J Ethnopharmacol 2009; 125: 380-386.

4. Chan EC, Yap SL, Lau AJ, Leow PC, Toh DF, Koh HL. Ultra-performance liquid
chromatography/time-of-flight mass spectrometry based metabolomics of raw and steamed
Panax notoginseng. Rapid Commun Mass Spectrom 2007; 21: 519-528.

5. Toh DF, New LS, Chan EC, Koh HL. Ultra-performance Liquid Chromatography - Time
of flight Mass Spectrometry (UPLC-TOFMS) metabolite profiling of raw and steamed
Panax notoginseng: Application on commercial products. (In preparation)



Conference presentations

1. Toh DF, Koh HL. A review of the traditional uses, phytochemical constituents and
biological activities of Swietenia macrophylla, 3rd Medicinal Chemistry Symposium, 28
July 2008, National University Singapore, Singapore.

2. Toh DF, Koh HL. Medicinal plants in Drug Discovery: A focus on Swietenia macrophylla,
Medicinal Chemistry Symposium, 23 January 2008, National University Singapore,
Singapore.

3. Toh DF, Koh HL. A literature review of the traditional uses, phytochemical constituents
and biological activities of the medicinal plant Swietenia macrophylla, The Inaugural
Singapore-Taiwan-Hong Kong (CU) Meeting of Pharmacologists, 28-29 May 2007,
National University Singapore, Singapore.

4. Ching JH, Toh DF, Tan CH, Koh HL. Antiplatelet activities of Ardisia elliptica and
Swietenia macrophylla, Congress of the Asian-Pacific Society on Thrombosis and
Haemostasis, 18-20 September 2008, Grand Copthorne Waterfront Hotel.

5. Low WL, Toh DF, Liesbet Tan, Belinda Tan, Low MY, Chan CL, Koh HL. Safety of
botanical health supplements and other complementary medicine, Research Awareness
Symposium 2009, 1 April 2009, National University Singapore, Singapore.


iv
6. Tan CJ, Ching JH, Toh DF, Neo SY, Koh HL. Effects of Ardisia elliptica and Strobilanthes
crispus on hepatocellular carcinoma cell proliferation. Research Awareness Symposium
2009, 1 April 2009, National University Singapore, Singapore.

7. Patel DN, Toh DF, New LS, Chan EC, Koh HL. Quality control of Panax notoginseng
using metabolomics Approach, 4
th
PharmSci@Asia 2009 Symposium, 27 May 2009, China
Pharmaceutical University, China.

8. Patel DN, Toh DF, New LS, Chan EC, Koh HL. Effects of steaming on the types and
concentrations of ginsenosides in Panax notoginseng, Conference on Recent Development
in Chinese Herbal Medicine jointly with the Consortium for Globalization of Chinese
Medicine (CGCM), 25-26 January 2010, Nanyang Technological University, Singapore.

v


TABLE OF CONTENTS

Title page
i
Acknowledgements

ii
List of publications and conference presentations
iii
Table of contents
v
Summary
ix
List of tables

xi
List of figures
xiii
List of abbreviations
xvi
Chapter 1. Introduction

1.1

Herbal medicine



1.1.1

Importance of herbal medicine

1

1.1.2

Quality control of herbal medicine




1.1.2.1

Importance of quality control

4



1.1.2.2

Traditional methods of quality control of herbal medicine

6



1.1.2.3

Modern methods of quality control of herbal medicine

8

1.1.2.4

Metabolomics

13

1.2

Medicinal plants and herbal medicine as potential sources of novel therapeutics

23


1.2.1

Cance
r

25


1.2.2

Cardiovascular diseases

26


1.2.3

Medicinal plants in drug discovery




1.2.3.1

Cancer

27



1.2.3.2


Cardiovascular diseases

31

1.3

Panax notoginseng



1.3.1

Introduction

36


1.3.2

Processing of
P. notoginseng

37


1.3.3

Chemical cons
tituents of
P. notoginseng

39


1.3.4

Quality control of
P. notoginseng

41


1.3.5

Pharmacological studies of
P. notoginseng

48

1.4

Swietenia macrophylla



1.4.1

Introduction

50


1.4.2

Botanical description

51


1.4.3

Identification of
Swietenia

specie
s

51


1.4.4

Traditional uses and biological activities

53


1.4.5

Phytochemical constituents


61

Chapter 2.

Hypothesis and Objectives


66

Chapter 3. Ultra-high pressure liquid chromatography / time-of-flight mass
spectrometry (UHPLC/TOFMS) analysis of raw and steamed Panax

notoginseng and application on commercial products


3.1

Development of metabolomic platform for time
-
dependent profiling



3.1.1

Introduction

69


3.1.2

Objective

75


3.1.3

Experimental

vi


3.1.3.1

Materials and reagents

75



3.
1.3.2

Steaming of raw
P. notoginseng

herb

75



3.1.3.3

Extraction and sample preparation

76

3.1.3.4

Instrumentation

76



3.1.3.5

Method validation

77



3.1.3.6

Chemometric/Multivariate data analysis

78



3.1.3.7

Statistical analysis

79


3.1.4

Resu
lts and Discussion




3.1.4.1

Ultra
-
high performance liquid chromatography time
-
of
-
flight
mass spectrometry (UHPLC/TOFMS) 79


3.1.4.2

Method validation

83

3.1.4.3

PCA & PLS
-
DA analysis

84



3.1.4.4

Loadings plot

89



3.1.4.5

Univariate data analyse
s of therapeutically important
ginsenosides
97



3.1.4.6

Differences of raw and steamed
P. notoginseng

in claimed
therapeutic effects and scientific evidence
97

3.2

Application of method developed on commercial products



3.2.1

Introduction

101


3.2.2

Objective

101


3.2.3

Experimental

3.2.3.1

Materials and reagents

101



3.2.3.2

Sample preparation

101



3.2.3.3

UHPLC/TOFMS method

104



3.2.3.4

Chemometric/Multivariate data analysis

104


3.2.4

Results and Discussion




3.2.4.1

Optimization an
d processing of raw, 2h and 4h steamed
samples
104



3.2.4.2

Application on four pairs of raw and steamed commercial
products (1R,S to 4R,S)
108



3.2.4.3

Application on thirteen commercial products labeled as “raw”
(5R to 17R)
116

3.3

Conclusion


126

Chapter 4. Chemical analysis of Panax notoginseng and Investigation of its
antiproliferative activities against liver cancer cells


4.1

Introduction



4.1.1

Liver cancer

127


4.1.2

P. notoginseng

as a potential source of anticancer therapeutics

130

4.2

Objective

133

4.3

Experimental

134


4.3.1

LC
-
LTQ Orbitrap FTMS Analysis




4.3.1.1

Sample preparation

134



4.3.1.2

Standards preparation

134



4.3.1.3

LC
-
LTQ Orbitrap FTMS method

135



4.3.1.4

Method validation

136

4.3.1.5

Data analysis

136


4.3.2

Cell culture

137


4.3.3

Cell proliferation analysis using WST
-
1 assay
–

Cell proliferation
screening
137


4.3.4

Statistical analysis of results

138

4.4

Results and discussion



4.4.1

UHPLC/TOFMS



vii


4.4.1.1

UHPLC/TOFMS metabolite profiles of r
aw and steamed
extracts of P. notoginseng
139



4.4.1.2

Use of Chemometrics in the Identification of important
ginsenosides responsible for the differences between raw and
steamed P. notoginseng
141


4.4.2

LC
-
LTQ Orbitrap FTMS




4.4.2.1

Method validati
on

144



4.4.2.2

Effects of steaming on the ginsenoside compositions of
P.
notoginseng

147


4.4.3

Effects of raw and steamed
P. notoginseng

extracts on human liver
cancer cells
150


4.4.4

Effects of ginsenosides on the proliferation of human liver cance
r cells

154


4.4.5

Changes of the important ginsenosides upon steaming

160


4.4.6

Dose
-
response effects of selected ginsenosides on SNU449 cell line

164

4.5

Conclusion


166

Chapter 5. Chemical and pharmacological evaluations of Swietenia macrophylla

5.1

Isolation and identification of chemical components



5.1.1

Introduction

167


5.1.2

Objective

168


5.1.3

Experimental




5.1.3.1

Materials and reagents

168



5.1.3.2

Extraction

168



5.1.3.3

Isolation and purification of compounds from kernel e
xtract

169



5.1.3.4

Isolation and purification of compounds from seed coat extract

170



5.1.3.5

Identification

171


5.1.4

Results and Discussion

172


5.1.5

Conclusion

191

5.2

Screening for antiproliferative activities against liver cancer cells



5.2.1

Introduction

191


5.2.2

Objective

193


5.2.3

Experimental

5.2.3.1

Materials and reagents

193



5.2.3.2

Cell culture

193



5.2.3.3

Sample preparation

193



5.2.3.4

WST
-

1 Assay: extracts, fractions, compounds

194


5.2.4

Results and Discussio
n




5.2.4.1

Effects of
Swietenia macrophylla

extracts on human liver
cancer cells in vitro
194



5.2.4.2

Effects of compounds isolated from ether extract of seed coat
against human liver cancer cells in vitro
198

5.2.4.3

Effects of compounds isolated

from ether extract of kernel
against human liver cancer cells in vitro
201



5.2.4.4

Dose
-
response effects of compounds on SNU449, SNU182
and HepG2 cell lines
204


5.2.5

Conclusion

207

5.3

Screening for antiplatelet and anticoagulant activities

5.
3.1

Introduction

208


5.3.2

Objective

209


5.3.3

Experimental




5.3.3.1

Materials and reagents

209

5.3.3.2

Sample preparation

210



5.3.3.3

In vitro

platelet aggregation assay

210



5.3.3.4

In vitro

plasma coagulation assay

212

viii


5.3.3.5

Statist
ical analysis

213


5.3.4

Results and Discussion




5.3.4.1

Effects of
Swietenia macrophylla

extracts on platelet
aggregation
214



5.3.4.2

Effects of compounds isolated from ether extract of
Swietenia
macrophylla on platelet aggregation
216



5.3.4.3

E
ffects of
Swietenia macrophylla

extracts on plasma
coagulation
220



5.3.4.4

Effects of compounds isolated from ether extract of
Swietenia
macrophylla on plasma coagulation
222


5.3.5

Conclusion

226

5.4

Conclusion

226

Chapter 6. Conclusions and future prospects
227

Bibliography
234

Appendices


Appendix I Typical chromatograms of standard mixtures of
ginsenosides (A and B) and P. notoginseng (raw and 15h
steamed) samples
261


Appendix II

Schematic equations for the chemical conversions of the
ginsenosides
263

Appendix III
13
C and
1
H NMR spectra of compounds isolated from
Swietenia macrophylla
264














ix

SUMMARY
The overall objectives of this work are to chemically and pharmacologically evaluate
medicinal plants, namely, to develop a metabolomic platform for the quality control of raw and
steamed P. notoginseng and study the effects of steaming of raw herb in terms of chemical and
biological activity and to investigate the chemical components and biological activity of the
seeds of S. macrophylla.

Metabolomic platform using ultra-high pressure liquid chromatography / time-of-flight
mass spectrometry (UHPLC/TOFMS) and chemometrics has been successfully developed and
validated for the time-dependent profiling of raw and differentially steamed P. notoginseng. It
has also been successfully applied to commercial products of raw and steamed P. notoginseng.
Important ginsenosides of the raw and steamed form were identified. Two commercial products
were found to have been subjected to heat treatment despite being labeled as the “raw” form.
The raw and steamed P. notoginseng were also evaluated for their antiproliferative
activities against three liver cancer cell lines, namely, SNU449, SNU182 and HepG2. The
antiproliferative activities of P. notoginseng increased with progressive steaming up to 24h as
this process enriched the bioactive components such as ginsenosides Rh2, Rk1, Rk3 and 20S-
Rg3. These antiproliferative ginsenosides were identified in the complex multi-ingredient
mixture in P. notoginseng using the metabolomics method developed by correlating the changes
of antiproliferative activity of P. notoginseng with different steaming durations. The platform
facilitates drug discovery from P. notoginseng. Ginsenoside Rh2 was found to be the most potent
antiproliferative component with an IC
50
of 56 µM as compared to the well-known anticancer
agent 20S-Rg3 of 199 µM against the same cell line SNU449. This is the first report of the
antiproliferative activity of ginsenoside Rk3.
Besides Chinese herbs, locally grown medicinal plants are also important resources. In
this work, the seeds of S. macrophylla are investigated for their chemical components and
pharmacological activities, namely, antiproliferative and antithrombotic (antiplatelet and

x
anticoagulant) activities. Two pure compounds and five partially purified compounds were
isolated from the seed coat while five partially purified compounds were isolated from the kernel.
The two pure compounds were determined to be the limonoids swietenolide and swietenine. The
compounds isolated are potentially bioactive compounds responsible for the various
pharmacological activities in S. macrophylla. Some extracts and partially purified compounds
showed promising antiproliferative and antiplatelet for the first time. Some of the partially

purified compounds had IC
50
values comparable to that of aspirin in antiplatelet activity. Hence,
they could be potential leads for antiplatelet drugs.
In conclusion, a metabolomics platform has been successfully developed for the quality
control of P. notoginseng and its products, and facilitates drug discovery. The chemical and
pharmacological investigations on S. macrophylla show that it is a useful medicinal plant with
promising anticancer and antiplatelet activity. Further work needs to be carried out to further
develop the biologically active components from both P. notoginseng and S. macrophylla into
useful therapeutics. This work is a step towards the discovery of potential anticancer and
antithrombotic drug leads.










xi

LIST OF TABLES
Page no.
Table 1.1

Botanical features of three
Swietenia

species, namely
S.
macrophylla, S. mahogany and S. humilis

54

Table 1.2

Traditional uses

of
S. macrophylla


55

Table 1.3

List of reported biological activities of
S. macrophylla


57

Table 1.4

List of reported biological activities of
S. mahagoni

59

Table 1.5

Reported biological activities of
S. humilis


60

Table 1.6

Structural classifica
tions of the limonoids present in the seeds of
S.
macrophylla

63

Table 3.1

List of important components in
P. notoginseng

used in
differentiating raw and steamed form
95

Table 3.2

List of
P. notoginseng

CPMs samples (raw and steamed products)

102

Tabl
e 4.1

Important saponins found in raw and steamed
P. notoginseng

and
their respective Variable importance plot (VIP) values.
145

Table 4.2

Linear calibration curve, concentration range, limit of detection
(LOD) and limit of quantification (LOQ) of the twelve saponins (n =
6).

146

Table 4.3

Concentrations of ginsenoside (in mg/g) in raw and steamed
P.
notoginseng samples (n = 6).

148

Table 4.4

Inhibition of proliferation of SNU449, SNU182 and HepG2 human
liver cancer cells by raw and steamed P. notoginseng extracts (n =
6).

153

Table 4.5

Inhibitory concentrations of different ginsenosides on proliferation
of SNU449 human liver cancer cells (n = 3).

165

Table 5.1

The extraction yields of the extracts of seed coat and kernel of
S.
macrophylla (n = 3).

177

Table 5.2

List of isolated compounds with their molecular formula, accurate
mass and proposed identity.

180

Table 5.3

1
H
-
NMR chemical shifts (δ, ppm) of samples sce1, sce2, sce3, sce4,
sce5 and sce6, obtained in CDCl
3
at 300 MHz.

182

Table 5.
4

13
C
-
NMR chemical shifts (δ, ppm) of samples sce1, sce2, sce3, sce4,
sce5 and sce6, obtained in CDCl
3
at 300 MHz.

184

Table 5.5

1
H
-
NMR chemical shifts (δ, ppm) of samples eks1 and eks6,
188


xii
obtained in CDCl
3
at 300 MHz.


Table 5.6

13
C
-
NMR chemical shif
ts (δ, ppm) of samples eks1 and eks6,
obtained in CDCl
3
at 300 MHz.

189

Table 5.7

Inhibitory concentrations of the ether, methanol and water extracts
of the seed coat and kernel of the seeds of S. macrophylla on

proliferation of SNU449, SNU182 and HepG2 human liver cancer
cells (n = 3).

197

Table 5.8

Inhibitory concentrations of the partially purified compounds
isolated from the ether extract of the seed coat of the seeds of S.
macrophylla on proliferation of SNU449, SNU182 and HepG2
human liver cancer cells (n = 3).

205

Table 5.9

Inhibitory concentrations of the ether extracts and partially purified
compounds of S. macrophylla and aspirin (n = 3).

219

xiii
LIST OF FIGURES
Page no.
Figure 1.1


The root of
P. notoginseng

can be in the form of whole root (coated and
uncoated) and slices.

37

Figure 1.2

Chinese proprietary medicines (CPMs) of
P. notoginseng

available in raw
and steamed form.

40

Figure 1.3

Chemical structures of some saponins in
P. notoginseng
.


42

Figure 1.4

The fruits and seeds of
S. macrophylla
.


52

Figure 1.5

Chemical structures of some limonoids in
S. macrophylla
.


64

Figure 3.1

Representative total ion chro
matograms (TICs) of raw and steamed
P.
notoginseng extracted samples (2h and 15h) obtained from
UHPLC/TOFMS.

80

Figure 3.2

PCA score plot of raw and steamed
P. notoginseng

samples (1, 2, 4, 6, 9,
12, 15 and 24 h).

86

Figure 3.3

(A) Three dimensional P
LS
-
DA score plot of batch 1 raw and steamed
P.
notoginseng samples (1, 2, 4, 6, 9, 12, 15 and 24h);
(B) Three dimensional PLS-DA score plot of batch 1 model set and batch
2 prediction set samples comprising raw, 2, 6, 9, 12 and 15h steamed
samples.

88

F
igure 3.4

Plot of PLS
-
DA cross
-
validated predicted Y scores of raw and steamed
P.
notoginseng samples (2, 6, 9, 12 and 15h) belonging to the batch 1 model
and batch 2 prediction set.

90

Figure 3.5

Two dimensional PLS
-

DA score plot of raw and steamed
P.
notoginseng
.

91

Figure 3.6

Loadings plot of PLS
-
DA model of raw and steamed
P. notoginseng
.


92

Figure 3.7

An enlarged view of the loadings plot of PLS
-
DA model of raw and
steamed P. notoginseng.
93

Figure 3.8

Chemical structures of some saponins pre
sent in raw and steamed

P.
notoginseng.

96

Figure 3.9

Plots of integrated peak area of the [M+HCOO]
-

ions of (A) 20S
-
Rg3 and
(B) Rh2 against the duration of steaming process (hours) for the batch 1
samples.
98

Figure 3.10

Typical total ion chromatogram
s (TICs) of raw and steamed
P.
notoginseng extracted samples (2h and 4h) obtained from
UHPLC/TOFMS.

105

Figure 3.11

PCA score plot of raw and steamed (2hr and 4hr steamed)

P.notoginseng
.


107

Figure 3.12

PLS
-
DA score plot of raw and steamed (2hr and 4
hr steamed)
P.notoginseng.
109


xiv
Figure 3.13

PCA score plot of raw and steamed (2hr and 4hr steamed)
P.notoginseng

in comparison to the commercial products (pairs 1R/1S to 4R/4S).
110

Figure 3.14

PLS
-
DA score plot of raw and steamed (2hr and 4hr steamed)

P.notoginseng in comparison to the commercial products (pairs 1R/1S to
4R/4S).
112

Figure 3.15

UHPLC/TOFMS chromatograms of commercial sample 3S and 4h
steamed P. notoginseng extracted samples.

113

Figure 3.16

PCA score plot of raw and steamed (2hr an
d 4hr steamed)
P.notoginseng

in comparison to the commercial products labeled as “steamed” (1S to
4S).
115

Figure 3.17

PCA score plot of raw and steamed (2hr and 4hr steamed)
P.notoginseng

in comparison to the commercial products (5R to 17R).
117

Figur
e 3.18

PCA score plot of raw
P.notoginseng

in comparison to the commercial
products 11R, 12R, 13R and 16R.

119

Figure 3.19

PLS
-
DA score plot of raw
P.notoginseng

in comparison to the
commercial products 11R, 12R, 13R and 16R.

121

Figure 3.20

Loading
s plot of PLS
-
DA model of raw
P.notoginseng

and commercial
products 11R, 12R, 13R and 16R.
122

Figure 3.21

UHPLC/TOFMS chromatograms of commercial samples 11R and 12R.


123

Figure 3.22

UHPLC/TOFMS chromatograms of commercial samples 13R and 16R
and raw P. notoginseng extracted samples.

125

Figure 4.1

Representative total ion chromatograms (TICs) of (A) raw and (B)
steamed P. notoginseng (15h) extracted samples obtained from
UHPLC/TOFMS.

140

Figure 4.2

PLS

-
DA score plot of raw and steamed
P. notogin
seng

samples
(2, 6, 9,
15 and 24 h).
142

Figure 4.3

Loadings plot of PLS
-
DA model of raw and steamed
P. notoginseng

samples (2, 6, 9, 12 and 15 h).
143

Figure 4.4

The saponins content in the (A) raw and differentially (B) steamed
P.
notoginseng samples.
149

Figure 4.5

In vitro

antiproliferative effects of raw and steamed
Panax notoginseng

extracts in SNU449, SNU182 and HepG2 human liver cancer cells.
151

Figure 4.6

The relationship between the duration of steaming of
P. notoginseng

and
its antiproliferative activity on the SNU449, SNU182 and HepG2 human
liver cancer cells.
155

Figure 4.7

In vitro

antiproliferative effects of saponins in the (A) raw and (B)
steamed Panax notoginseng extracts in SNU449, SNU182 and HepG2
human liver cancer cells.
157

Figure 4.8

Plot of concentrations of ginsenosides (A) Rk3, (B) 20S
-

Rg3, (C) Rk1
161


xv
and (D) Rh2 in the raw and steamed
P. notoginseng

samples.

Figure 4.9

The changes of the concentration of individual ginsenosides Rk1, Rk3,
Rh2 and 20S-Rg3 in the raw and steamed P. notoginseng extracts and the
changes in the antiproliferative activity (in terms of inhibitory
concentration IC
50
on cell proliferation of SNU449, SNU182 and HepG2.

163

Figure 5.1

Typical HPLC chromatograms of different extr
acts of seed coat.


174

Figure 5.2

Typical HPLC chromatograms of different extracts of kernel.


175

Figure 5.3

Typical HPLC chromatograms of ether extract of (A) seed coat and (B)
kernel of S. macrophylla.

178

Figure 5.4

Structures of limonoids in
S.
macrophylla
.


179

Figure 5.5

In vitro

antiproliferative effects of extracts of (A) seed coat and (B) kernel
in SNU449, SNU182 and HepG2 human liver cancer cells.
195

Figure 5.6

In vitro

antiproliferative effects of isolated compounds from ether extract

of seed coat of S. macrophylla in SNU449, SNU182 and HepG2 human
liver cancer cells.
199

Figure 5.7

In vitro

antiproliferative effects of isolated compounds from ether extract
of kernel of S. macrophylla in SNU449, SNU182 and HepG2 human liver
cancer cells.
202

Figure 5.8

Effects of extracts of (A) seed coat and (B) kernel of
S. macrophylla

on
platelet aggregation.

215

Figure 5.9

Effects of compounds isolated from the ether extract of seed coat of
S.
macrophylla on platelet aggregation.

216

Figur
e 5.10

Effects of compounds isolated from the ether extract of kernel of
S.
macrophylla on platelet aggregation.

218

Figure 5.11

Effects of extracts of seed coat and kernel of
S. macrophylla

on
prothrombim time (PT).

221

Figure 5.12

Effects of extrac

ts of seed coat and kernel of
S. macrophylla

on activated
partial thromboplastin time (aPTT).

221

Figure 5.13

Effects of compounds isolated from the ether extract of seed coat of
S.
macrophylla on prothrombin time (PT).

223

Figure 5.14

Effects of com
pounds isolated from the ether extract of seed coat of
S.
macrophylla on activated partial thromboplastin time (aPTT).

223

Figure 5.15

Effects of compounds isolated from the ether extract of kernel of
S.
macrophylla on prothrombin time (PT).

225

Figu
re 5.16

Effects of compounds isolated from the ether extract of kernel of
S.
macrophylla on activated partial thromboplastin time (aPTT).
225



xvi

LIST OF ABBREVIATIONS
δ

chemical shifts

µM

micro molar

µg/ml

micro gram per milliliter

ACS

American Cancer

Society

ADP

Adenosine diphosphate

ANOVA

Analysis of variance

AP
-
PCR

Arbitrarily
-
primed polymerase chain reaction

aPTT

activated partial thromboplastin time

Ara(f)

Arabinose in furanose form

Ara(p)

Arabinose in pyranose form

CAM

Complementary and Al
ternative Medicine

cAMP

cyclic adenosine monophosphate

CE

Capillary electrophoresis

CNS

Central nervous system

CO
2

Carbon dioxide

CPM

Chinese Proprietary Medicine

DAD

Diode array detector

DMSO

Dimethyl sulfoxide

DNA

Deoxyribonucleic acid

DPPH

1,1
-
diphenyl
-
2
-
picrylhydrozyl

DRE

Dynamic range enhancement

EBV
-
EA

Epstein
-

Barr virus
-
early antigen

e.g.

For example

ELISA

Enzyme
-
linked immunosorbent assay

ELSD

Evaporative light scattering detection

EMEA

European Medicines Agency

ESI

Electrospray io
nization

et al.

and others

etc.

and so forth

FBS

Fetal bovine serum

FDA

Food and Drug Administration

FTMS

Fourier Transform Mass Spectrometry

GC

Gas Chromatography

GC
-
MS

Gas Chromatography Mass Spectrometry

Glc

Glucose

GPIIb
-
IIIa

Glycoprotein IIb
-
IIIa

HBV

Hepatitis B virus

HCA

Hierarchical cluster analysis

HCC

Hepatocellular carcinoma

HCV

Hepatitis C virus

HIT

Heparin
-
induced thrombocytopenia

HIV

Human immunodeficiency virus

HMWK

High molecular weight kininogen

HPLC

High
-
performance liqui
d chromatography

IC
50

50% inhibitory concentration

i.e.

That is

IR


Infrared

KNN

K
-
nearest neighbours

LC


Liquid Chromatography


xvii
LMWHs

Low molecular weight heparins

LOD

Limit of detection

LOQ

Limit of quantification

LTQ

Linear ion trap

LV

Laten
t variable

MEKC

Micellar Electrokinetic Chromatography

mg/ml

milligram per milliliter

MS

Mass Spectrometry

MTT

3
-
[4,5
-
dimethylthiazol
-
2
-
yl)
-
2,5
-

diphenyltetrazolium bromide

m/z

mass over charge ratio

NAD(P)H

Nicotinamide adenine dinucleotide (phospha
te) (reduced form)

NCCAM

National Center for Complementary and Alternative Medicine

NCE

New chemical entities

NCI

National Cancer Institute

NIR

Near infrared


NMR

Nuclear Magnetic Resonance

PAF


Platelet
-
activating factor

PBS

Phosphate buffered sal
ine

PC

Principal component

PCA

Principal component analysis

PCR

Polymerase chain reaction

PLS
-
DA

Partial least square
-

discriminant analysis

PNS

P. notoginseng

saponins

PPARγ

Peroxisome proliferator
-
activated receptor γ

ppm

parts per million

PT

Prothrombin time

QCAR

Quantitative composition
-
activity relationship

R


Raw

R
2

correlation factor

RAPD

Random amplified polymorphic DNA

RFLP

Restriction fragment length polymorphism

Rha

Rhamnose

rpm

revolutions per minute

R.S.D.

Relative standard

deviation

RT

Retention time

S

Steamed

SFDA

State Food and Drug Administration

S/N

Signal
-
to
-
noise ratio

TCM


Traditional Chinese Medicine

TICs

Total ion chromatograms

TLC

Thin layer chromatography

TMS

Tetramethylsilane

TOFMS

Time
-
of
-
flight Mass S
pectrometry

TPA

12
-
O
-
tetradecanoylphorbol
-
13
-
acetate

UHPLC

Ultra
-
high Pressure Liquid Chromatography

UHPLC/TOFMS

Ultra
-
high Pressure Liquid Chromatography/Time
-
of
-
flight Mass Spectrometry

UPLC

Ultra Performance Liquid Chromatography

UV

Ultraviolet

V
IP

Variable importance plot

vWf

von Willebrand factor

WHO

World Health Organization

WST
-
1

4
-
[3
-
(4
-
iodophenyl)
-
2
-
(4
-
nitrophenyl)
-
2H
-
5
-

tetrazolio]
-
1,3
-
benzene disulfonate

XTT

2,3
-
bis[2
-
methoxy
-
4
-
nitro
-
5
-
sulfophenyl]
-
2H
-
tetrazolium
-
5
-
carboxanilide

Xyl

X
ylose


1
CHAPTER 1
INTRODUCTION
1.1 Herbal medicine
1.1.1 Importance of herbal medicine
According to the World Health Organization (WHO), traditional medicine refers
to the knowledge, skills and practices based on the theories, beliefs and experiences
indigenous to different cultures, used in the maintenance of health and in the prevention,
diagnosis, improvement or treatment of physical and mental illness (WHO, 2008).
Traditional medicine covers a wide variety of therapies and practices, which vary from
country to country and region to region. In some countries, it is referred to as
complementary and alternative medicine (CAM). According to National Center for
Complementary and Alternative Medicine (NCCAM), CAM is a group of diverse
medical and health care systems, practices and products that are not generally considered
part of conventional medicine (NCCAM, 2010). Herbal medicine (also known as
botanical medicine, phytomedicine, natural products) is part of traditional medicine or
CAM, will include herbs, herbal materials, herbal preparations and finished herbal
products that contain as active ingredients parts of plants, or other plant materials, or
combinations (WHO, 2000). Herbal medicine is also defined as naturally occurring,
plant-derived substances with minimal or no industrial processing that have been used
for medicinal purposes (Tilburt & Kaptchuk, 2008).
Traditional medicine has been used for thousands of years with great
contributions made by practitioners to human health, particularly as primary health care
providers at the community level (WHO, 2010). Traditional medicine or CAM has

maintained its popularity worldwide. Besides their long history of use, herbal medicine is
getting significant attention in global healthcare due to their great medicinal and

2
economic importance. In some countries, herbal medicine is still a central part of the
medical system, such as Ayurverdic medicine in India and Traditional Chinese Medicine
(TCM) in China. In Europe, North America and other industrialized regions, herbal
medicinal products are becoming increasingly popular, and the worldwide annual market
for these products approaches US$ 60 billion (WHO, 2002; WHO, 2008). According to
WHO, its use has surged in many developed and developing countries since 1990s
(WHO, 2008). In some Asian and African countries, 80% of the population depends on
traditional medicine for primary health care. In many developed countries, 70% to 80%
of the population has used some form of alternative or complementary medicine (e.g.
acupuncture) (WHO, 2008). Herbal treatments are the most popular form of traditional
medicine, and are highly lucrative in the international marketplace. Annual revenues in
Western Europe reached US$ 5 billion in 2003-2004, sales of products totaled US$ 14
billion in 2005 in China and herbal medicine revenue in Brazil was US$ 160 million in
2007 (WHO, 2008). These statistics show the growing worldwide importance of herbal
medicine in both developing and industrialized countries. In developing countries, the
broad use of herbal medicine is often attributed to its accessibility, affordability and their
cultural beliefs. While in many developed countries, the increasing use of herbal
medicine is often fuelled by concerns regarding adverse effects and unsatisfactory
treatments from modern western drugs, the need for apparently milder treatments for
chronic debilitating diseases and greater public access to health information.
Although herbal medicine has a long history of use, there are challenges to ensure
its safety, quality and efficacy (Rousseaux & Schachter, 2003). The most important one
is that the application of herbal medicine is often based on long-term empirical and
traditional uses rather than on scientific evidences (Bardia et al., 2007). Scientific
evidence from tests done to evaluate the safety and efficacy of traditional medicine

3
products and practices is limited (WHO, 2008). Scientific evidences are still far from
sufficient to meet the criteria needed to support its worldwide use (Zhao & Chan, 2003).
This has highlighted the need for up-to-date scientific information on herbal medicine to
assure their quality, safety and efficacy. Requirements and methods for research and
evaluation are complex. For example, it can be difficult to assess the quality of finished
herbal products. The safety, efficacy and quality of finished herbal medicine products
depend on the quality of their source materials (which can include hundreds of natural
constituents), and how materials are handled through production processes (WHO, 2008).
The chemical constituents bring about the effects of herbal medicine. Thus, the chemical
analysis of herbal medicine is especially important because it enables profiling and
identification of biologically active chemical components in the herbal medicine and to
establish scientific and rational quality control methods. Many people believe that
because remedies are herbal (natural) or traditional they are safe (or carry no risk for
harm). However, traditional medicine and practices can cause harmful, adverse reactions
if the product or therapy is of poor quality, or it is used inappropriately or in conjunction
with other incompatible medicine (WHO, 2008). Hence, rigorous scientific approach is
required to realize evidence-based medicine in these herbal medicine.
Fortunately, research on herbal medicine has been increasing over the years and
these can be seen by the increase in funding and establishment of CAM research and
research units in both developed and developing countries (National Center for
Complementary and Alternative Medicine (NCCAM), 2004; WHO, 2008). Over the past
decade, advanced chemical, pharmacological and biological technologies have facilitated
an increasing number of research in the search for possible ways to explore the potential
healthcare benefits of herbal medicine (Xie & Leung, 2009). Furthermore, due to an
increasing interest among public in herbal medicine as an alternative therapeutic

4
modality, many scientific studies have been conducted on its various aspects, including
taxonomy, authentication, isolation and elucidation of chemical constituents,

pharmacology, toxicology, bioactivity screening, and others (Xie & Leung, 2009).
Parallel to the increased popularity of herbal medicine, clinical pharmacological interest
in the quality, safety and efficacy of herbal remedies in general have also grown. One of
the aims of WHO and its Member States collaboration is to ensure the use of safe,
effective and quality products and practices, based on available evidence (WHO, 2008).

1.1.2 Quality control of herbal medicine
1.1.2.1 Importance of quality control
Systematic research on herbal medicine such as TCM has centered on
identification of chemical components, pharmaceutical activity, processing methods and
quality control. The accumulated knowledge about TCM makes their use reasonable and
safe, but many problems are not resolved. In the process of “modernization” and
“globalization” of TCM, a key issue is the consistency and quality control of TCM. The
quality and safety of TCM attract more attention especially after the toxicological cases
of aristolochic acids and pyrrolizidine alkaloids (Xiao & Liu, 2004). Quality control
plays an important role in the application and development of herbal medicine. The
WHO recognized this in a document entitled “General Guidelines for Methodologies on
Research and Evaluation of Traditional Medicines” (WHO, 2000). Species
authentication and quality consistency of the herbal material used is of the utmost
importance regardless of whether one’s interest is in research or manufacture of herbal
medicine. This is because results based on unauthenticated or inconsistent materials are
irreproducible, rendering the research efforts in vain or the manufactured products
ineffective. Currently, pursuing stringent quality control and keeping consistent quality
of herbal medicine should be placed first. It is necessary to carry out scientific quality

5
control in order to ensure the quality of TCM, as well as its safety, effectiveness, stability
and good manufacturing practice.
An estimated number of 11,145 medicine were developed from medicinal plants
are known in China (Huang et al., 2004). Their efficacy and toxicity are mostly based on

historical, long-term clinical experience instead of modern scientific evaluation. A
challenge is the variation in chemical contents caused by the variation of growth during
the day-night rhythm, seasonal change, age and polymorphisms within one species (Zhu,
1998; Chang et al., 2006). The variation in chemical contents contributes to varying
observations of potential toxicity of herbal medicine (Wang et al., 2009b). Herbal
remedies are complex mixtures, containing a myriad of compounds. However,
biologically active compounds form just a minute part of herbs being diluted with a large
amount of proteins, sugars or tannins, which, in some cases, does not contribute to the
pharmaceutical effect but they make the quality control of crude drugs and their medical
preparations extremely difficult (Drasar & Moravcova, 2004). Quality control is a major
issue of herbal medicine that is related to adverse reactions, such as contamination
(pesticide, heavy metals, microbes etc.), adulteration, misidentification, improper
processing and preparation (Ko, 2004; Chan, 2005; Maxion-Bergemann et al., 2006). It
is necessary to develop a type of quality assessment system that adequately meets the
complex characteristics of TCM.
It is well known that the holistic system of herbal medicine such as TCM is
featured by the integrity of the ingredients contained in the Chinese herbal medicine.
Unlike the single chemical entity that forms the basis of modern pharmacology and drug
development, the paradigm of traditional Chinese herbal medicine views the multi-
compound, multi-ingredients preparations typical of herbal medicine as representing the
activity of the herbal drug (Xie et al., 2006). Selection of individual analytical

6
compounds for determining either efficacy or quality is contrary to TCM principles (Xie
et al., 2006). This creates a challenge in establishing quality control standards for raw
materials and the standardization of finished herbal drugs because no single component
can contribute to the total efficacy. This difficulty has been acknowledged in the
publication of ‘Regional strategy for traditional medicine in the Western Pacific Region”
by WHO (WHO, 2001). The characteristics of multi-target and synergistic action of
TCM come from their multiple constituents. Thus, a comprehensive method that could

reflect the variation of most constituents in the crude drugs is necessary, especially the
variation correlating with pharmacological and clinical efficacy.

1.1.2.2 Traditional methods of quality control of herbal medicine
Traditionally, quality control of herbal medicine is performed via morphological
identification method and microscopical identification method, physical and chemical
identification methods and one or two markers’ thin layer chromatography (TLC)
identification and/or content determination. Most of the traditional physical and chemical
identifications belong to qualitative analysis. Morphological identification method is
determined via physical features such as the shape, colour, smell, the surface texture,
cross section, water entry, burning and other features, which is dependent upon the
appearance of TCM to determine its quality standards (Lombard & Wadley, 2007).
Microscopic identification includes the observations of transverse or longitudinal section,
the powder, the surface, the dissociated tissue, and the polarized light microscopy
(Zhong et al., 2009). Physical and chemical identification is the method which certain
chemical reactions or physical properties of chemical constituents in herbal medicine are
introduced to carry out qualitative and quantitative analysis (Cheng et al., 2007). For
example, the identification of the physical constants (density, rotation, refractive index,
hardness, viscosity, boiling point, freezing point, and melting point), colour reaction,

7
precipitation reaction (Zhong et al., 2009). They generally apply to TCM with different
chemical composition, or similar traits, but without a clear characteristic of microscopic
identification.
These traditional methods have their own disadvantages. Morphological and
microscopic identification methods require experts who have a wealth of practical
experience, so human factors play an important role on the result. Physical and chemical
identification method only considers the individual “active ingredient” of the many
complex components, neglects to some degree the synergism of the complex ingredients
in TCM (Zhong et al., 2009). The use of one or two markers or pharmacologically active

components in herbs and/or herbal mixtures employed for evaluating the quality and
authenticity of herbal medicine does not give a complete picture of herbal product,
because multiple constituents are usually responsible for its therapeutic effects. These
multiple constituents may work ‘synergistically’ and could hardly be separated into
active parts. As many substances used in traditional Chinese herbal medicine contain the
same compounds such an approach fails to be able to even confirm the identity of a
specific plant, let alone make any determination regarding its quality (Liang et al., 2004).
Moreover, the chemical constituents in component herbs in the herbal medicine products
may vary depending on harvest seasons, plant origins, drying processes and other factors
(Liang et al., 2004). These methods do not provide a complete profile of the drug, so it
cannot distinguish drugs with similar appearance and/or similar main chemical
constitution. For example, chlorogenic acid cannot differentiate the flowers of Lonicera
japonica and Chrysanthemi indici, or oleanolic acid cannot identify the roots of
Ligustrum lucidum, Achyranthes bidentata or Clemantis chinensis (The State
Pharmacopoeia Commission of People's Republic of China, 2005). Simple quantitative

8
analysis of one or several active components in herbal medicine does not represent its
quality because its efficacy results from multiple components at multiple targets.
Traditional methods for measuring the quality control of herbal medicine are not
sufficient for applying to herbal medicine. As a consequence of the lack of efficient
quality control tools, large variations of herbal medicine are encountered in the markets.
Since the chemical composition of herbal medicine depends on the plant growth
environment, the season of collection and preparation methods used such as during
processes and extraction procedures, it is vital that quality control of the herbal medicine
takes these factors into consideration. Revision of the techniques currently used is clearly
necessary in order to meet the increasing demands for accuracy and consistency of
products. It is therefore desirable to establish an analytical method able to profile whole
plant extracts in a holistic manner and thus provide a means for standardising and
controlling the quality of herbal medicine based on the entire biochemical composition

of the preparation without reference to ‘active’ molecules. Thus, it is necessary to
develop a type of quality assessment system that adequately meets the complex
characteristics of traditional Chinese herbal medicine.

1.1.2.3 Modern Methods of quality control of herbal medicine
Modern methodologies for quality control of herbal medicine include DNA
methods and chemical fingerprinting.
DNA methods
Authentication is one of the most important steps for ensuring the quality and
therapeutic effectiveness of herbal medicine. DNA analysis is one of the most reliable
methods of identification for medicinal plants or TCM since genetic composition is
unique for each individual. DNA methods are less affected by age, physiological
conditions, environmental factors, harvest, storage and processing methods (Yip et al.,