Isolation and Characterisation of Biopolymers from Anti-cancer Medicinal Herbs

210 Pages·2012·1.81 MB·English

Isolation and Characterisation of Biopolymers from Anti-cancer
Medicinal Herbs

By

Lin Zhang

A thesis submitted to fulfil the requirements for the degree of
Master of Science (Honours)

School of Science and Health
University Of Western Sydney, Australia

March 2012
Table of Contents
Contents i
Declaration vii
Acknowledgements viii
List of Tables ix
List of Figures xi
Publications and Paper submitted xiv
Abstract xv
Chapter 1: Introduction……………………………………………………………………….1
1.1 Introduction……………………………………………………………………………2
1.2 Previous research in this area………………………………………………………….5
1.3 Thesis plan……………………………………………………………………………..7

Chapter 2: Antioxidant activity of selected Traditional Chinese Medicinal herbal extracts...9
2.1 Introduction…………………………………………………………………….…....10
2.1.1 Anti-oxidants and Traditional Chinese medicine…………………………..12
2.1.2 Anti-oxidant constituents in medicinal plants……………………………..16
2.1.2.1 Polyphenols in medicinal herbs………………………………...16
2.1.2.2 Trace metals in medicinal herbs………………………………....17
i
2.1.3 Methods of measurement of antioxidant activity of medicinal herbs…….18
2.1.4 Principles of DPPH free radical scavenging activity test………………….19
2.1.5 Principles of the Yeast Oxidative stress model……………………………21
2.2 Materials and methods……………………………………………………………....21
2.2.1 Plant material………………………………………………………………..21
2.2.2 Water extracts……………………………………………………………….22
2.2.3 Ethanol extracts……………………………………………………………..22
2.2.4 Determination of total phenolic compounds……………………………….22
2.2.5 Determination of total flavonoids…………………………………………..24
2.2.6 Sample digestion and mineral analysis by ICP-MS………………………..25
2.2.7 DPPH free radical scavenging assay………………………………………..26
2.2.8 The high throughput Saccharomyces cerevisiae-based biological assay…..29
2.3 Results………………………………………………………………………………..30
2.3.1 Total phenolics and flavonoids content in the selected plants…………......32
2.3.2 Anti-oxidant activities of selected plant extracts……………………………34
2.3.3 Correlation of antioxidant activities of selected plants in terms of their total
phenolics and flavonoid content…………………………………………….36
2.3.4 Total phenolic and flavonoid content of twenty nine plants……………….39
2.3.5 Trace metal quantification of the selected 29 medicinal herbs……………..43
2.3.6 Anti-oxidant activities of 29 herbs………………………………………….45
2.4 Discussion…………………………………………………………………………...47
Chapter 3: Anti-inflammatory activities of selected medicinal plants……………………..53
3.1 Introduction……………………………………………………………………..…...54
3.1.1 Inflammatory molecular…………………………………………………….55
ii
3.1.2 Role of antioxidants in controlling inflammatory disorders…………………57
3.1.3 Anti inflammatory properties of traditional medicinal herbs………………58
3.1.4 Evaluation methods for anti-inflammatory activity of medicinal herbs……..60
3.1.4.1 Determination of nitric oxide using the Griess reagent Test…….60
3.1.4.2 ELISA Test………………………………………………………..61
3.1.4.3 Cytotoxicity tests………………………………………………….62
3.2 Materials and methods………………………………………………………………64
3.2.1 Acquisition of plant material and preparation of herbal extracts………….65
3.2.2 Maintenance, preparation and activation of RAW 264.7 macrophages…...65
3.2.3 Determination of nitric oxide production by Griess assay…………………66
3.2.4 Determination of cell viability by Alamar Blue assay……………………...66
3.2.5 TNF-α determination by ELISA…………………………………………….67
3.3 Results………………………………………………………………………………..67
3.3.1 Anti-inflammatory properties of selected medicinal herbs against NO
production……………………………………………………………………67
3.3.2 Anti-inflammatory properties of selected medicinal herbs against TNF-α
production……………………………………………………………………68
3.4 Discussion…………………………………………………………………………...72

Chapter 4: Anti-fungal and anti-bacterial activities of ethanol extracts of medicinal
plants…………………………………………………………………………….79
4.1 Introduction…………………………………………………………………………..80
4.2 Materials and methods……………………………………………………………….82
4.2.1 Collection of medicinal plants……………………………………………….82
4.2.2 Preparation of the ethanol extract………………………………………….86
iii
4.2.3 Test microorganisms………………………………………………………….86
4.2.4 Anti-bacterial assay on A. baumannii………………………………………..86
4.2.5 Anti-infective assay on S. aureus……………………………………………..88
4.2.6 Anti-infective assay on P. aeruginosa……………………………………….89
4.2.7 Anti-infective assay on C. albicans…………………………………………..89
4.2.8 Anti-infective assay on Aspergillus fumigates………………………………..90
4.2.9 Statistical analysis……………………………………………………………91
4.3 Results………………………………………………………………………………..92
4.3.1 Anti-fungal activity of selected plant extracts………………………………92
4.3.2 Antibacterial activity of selected plant extracts against
gram-negative bacteria......................………………………………………..93
4.3.3 Anti-bacterial activity of selected plant extracts against
gram-positive bacteria……………………….………………………………96
4.4 Discussion……………………………………………………………………………97
4.5 Conclusion……………………………………………………………………….…101

Chapter 5: Anti-cancer properties of selected medicinal plants…………………………..103
5.1 Introduction…………………………………………………………………………104
5.1.1 The main anti-cancer agents found in medicinal herbs…………………….106
5.1.2 Free radicals as cancer causing agents…………………………………….107
5.1.3 Principles and methods of Herbal Treatment for cancer………………….108
5.2 Materials and methods……………………………………………………………..113
5.2.1 Collection of medicinal plants and preparation of their extracts…………113
5.2.2 Tumour cell lines……………………………………………………………114
iv
5.2.3 Antitumor assay…………………………………………………………….115
5.2.4 Data presentation and analysis……………………………………………..116
5.3 Results……………………………………………………………………………….116
5.3.1 Anti-proliferative effects of selected plant extracts against MCF7 cells…..117
5.3.2 Anti-proliferative effects of selected plant extracts against A549 cells……120
5.3.3 Anti-proliferative effects of selected plant extracts against HL60 cells…...121
5.3.4 Anti-proliferative effects of selected plant extracts against HepG2 cells….121
5.3.5 Anti-proliferative effects of selected plant extracts against HT29 cells…...122
5.3.6 Anti-proliferative effects of selected plant extracts against Fa2N4 cells….123
5.4 Discussion…………………………………………………………………………..124

Chapter 6: Isolation and Characterisation of Polysaccharidess from Taxillus chinensis and
Sanguisorba officinalis…………………………………………………………133
6.1 Introduction…………………………………………………………………………134
6.1.1 Inmmunostimulatory activities of plant polysaccharides…………………134
6.1.2 Antioxidant activities of plant polysaccharides……………………………136
6.2 Materials and Methods……………………………………………………………...140
6.2.1 Plants………………………………………………………………………..140
6.2.2 Chemicals…………………………………………………………………...140
6.2.3 Fractionation and purification of Polysaccharidess from Taxillus chinensis and
Sanguisorba officinalis……………………………………………………...141
6.2.4 Analysis of Fractions………………………………………………………..143
6.2.4.1 Protein content of polysaccharides…………………………………143
v
6.2.4.2 Sugar content of isolated polysaccharides…………………………144
6.2.4.3 Sugar composition analysis………………………………………...145
6.2.4.4 Amino acid analysis………………………………………………..147
6.2.5 Bioactivity tests……………………………………………………………. 148
6.2.5.1 Antioxidant activity………………………………………………..148
6.2.5.2 Measurement of NO production……………………………………149
6.2.5.3 Assay of TNF- α production…………………………………….....151
6.2.5.4 Determination of cell viability by Alamar Blue assay…………….152
6.2.6 Statistical analysis…………………………………………………………...152
6.3 Results………………………………………………………………………………152
6.3.1 Fractionation and purification of Polysaccharidess from T. chinensis and S.
officinalis……………………………………………………………………152
6.3.2 Anti-oxidant activities of selected plant extracts………………………….159
6.3.3 Effect of polysaccharides derived from Taxillus chinensis and Sanguisorba
officinalis on macrophage TNF-α and NO production……………………161

6.4 Discussion…………………………………………………………………………..166

Chapter 7: Conclusion and Future Research Possibilities…………………………………169
References………………………………………………………………………………….176

vi
Declaration

This is to certify that the work presented in this thesis is original, except where
acknowledgment is made in the text, and has not been submitted to any other University or
Institution for a higher degree.

Signed…………………………………
Lin Zhang

vii
Acknowledgements
I am sincerely greatful to my supervisors: Dr. Paul Smith, Dr. Narsimha Reddy and Dr.
Sunder Rao from School of Science and Health, University of Western Sydney, for their
valuable guidance and advice in this research.
I also would like to thank Dr. Jeong, and Dr. Ming Wu, Kirubukaran Shanmugam, for
helping me in biological experiments.
I would like to thank the technical staff of School of Science and health, for their support
during my research.
Last but not least, I wish thank my parents for encouraging me to pursue higher education
and their priceless support.
I specially thank Dr. Narsimha Reddy, Dr. Sunder Rao and Dr. Paul Smith for their valuable
guidance and giving me an opportunity to pursue research career.

viii
List of Tables
Table 1.1: Traditional Chinese Medicinal plants that have been used to study in this
research………………………………………………………………………...4
Table 2.1: Antioxidant activities of selected 14 Chinese medicinal herbs along with their
total phenolics and flavonoid contents………………………………………31
Table 2.2: Antioxidant activities of selected 29 Chinese medicinal herbs along with their
total phenolics and flavonoid contents (Scientific names of the 29 plants
labeled as 1 to 29 are given in Table 1.1 in Chapter 1)……………………..41

Table 2.3: The trace metal content of twenty nine selected medicinal plants (Scientific
names of the 29 plants labeled as 1 to 29 are given in Table 1.1 in Chapter 1)
……………………………………………44

Table 3.1: Anti-inflammatory activities of water extracts of selected medicinal herbs
against NO and TNF-α production and associated cell viability……………70
Table 3.2: The anti-inflammatory properties (Inhibitory activities of NO and TNF-α
excess production) of some of the selected plants with respect to their
antioxidant contents (Total phenolics and flavonoids, trace metals including
Cu, Zn, Mn, Mg, Se and Mo)………………………………………………...75

Table 3.3: Some anti-inflammatory activity that are used by Chinese medical
practitioners and in clinical studies…………………………………………77
Table 4.1: List of plants screened for anti-microbial activities in this study…………….84

Table 4.2: Anti-fungal activities of medicinal plant extracts towards A.fumigatus and C.
albicans……………………………………………………………………… 94
Table 4.3: Anti-bacterial activities of medicinal plant extracts towards A.baumannii and
P. Aeruginosa (gram-negative bacteria)…………………………………….. 95
Table 4.4: Antibacterial activities of medicinal plant extracts towards S. aureus (gram
positive bacteria)……………………………………………………………..96
Table 4.5: Summary of important findings of this study providing correlation of anti-
microbial activity of the active plant extracts and their flavonoid contents
along with their traditional use………………………………………………98
Table 5.1: List of the Traditional Chinese Medicinal (TCM) plants that were used in the
study………………………………………………………………………...111
Table 5.2: Some anti-cancer formulations that are used by Chinese medical practitioners
and also in clinical studies……………………………………………….…112
Table 5.3: In vitro cytotoxicity (% of inhibition at 1 mg/ml) of aqueous and ethanol
extracts of the plants on five cancer cell lines measured by the MTT
assay………………………………………………………………………………………119
ix