Biological activities and structural characterisation of anticancer Herbal Polysaccharides
Lin Zhang
A thesis submitted in fulfilment of the requirement of Doctor of Philosophy
National Institute of Complementary Medicine School of Science and Health Western Sydney University, Australia
Supervisors
Dr. Narsimha Reddy
Dr. Sundar Rao Koyyalamudi
Dr. Cheang Khoo
March 2017 Signed Statement of Authentication
I, Lin Zhang, declare that this thesis contains no material that has been accepted for the award of any other degree or diploma and that, to the best of my knowledge and belief, this thesis contains no material previously published or written by another person, except when due reference has been made in the text of this thesis.
Signed ...... 张璘......
i Abstract
In recent years, medicinal plants have become popular for the treatment of several diseases due to their efficacy and cost effectiveness. Plant derived therapeutic agents are increasingly sought out as pharmaceuticals for the treatment of life-threatening illnesses. Important contributions have already been made in the recent years to the drug market by the compounds isolated from natural sources or from their derivatives. There is no doubt that, novel lifesaving drugs could be discovered by a systematic evaluation of ethno medicinal information using modern scientific tools.
This thesis attempts to systematically combine ethnomedicinal knowledge together with the
contemporary scientific methods to evaluate immunomodulatory and anticancer properties of
sixteen traditional medicinal herbs with an aim to discover anticancer formulations. The
major objective of this project is to isolate and characterise potent anticancer
polysaccharides from Traditional Chinese Medicinal (TCM) herbs.
In order to achieve these objectives, sixteen Chinese Medicinal herbs have been successfully
screened for their antioxidant, immunomodulatory and anticancer activities. Based on
their immunomodulatory and anticancer activities, three traditional anticancer TCM
herbs, namely, Amauroderma rugosum, Lobelia chinensis and Artemisia annua have been
selected for further studies. Isolation and characterisation of polysaccharides from these three medicinal herbs has been carried out. The structural characterization
employing FT-IR and NMR spectroscopy has been carried out and presented in Chapters
5 to 7.
ii A total of eight potent immuno-therepuritic polysaccharides have been discovered for the first time from the three important anticancer TCM herbs (A. annua, L. chinensis and A. rugosum).
The structure of three of them have been determined. The MRI contrast potentials of Gd(III) complexes of three of the herbal polysaccharides have also been studied. Low molecular weight polysacchro-proteins were found to be T1 agents and high molecular weight polysacchro-proteins were T2 agents.
Findings of this thesis have contributed significant scientific knowledge that has offered some suggestions for designing new immuno-therepeutic formulations based on the eight polysaccharides discovered in this thesis. However, further research is required in order to design such formulations.
iii Acknowledgements
I am sincerely greatful to my supervisors: Dr. Narsimha Reddy, Dr. Sundar Rao and Dr.
Cheang Khoo 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. Christopher E Jones, 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. Sundar Rao and Dr. Cheang Khoo for their valuable guidance and giving me an opportunity to pursue research career.
iv Table of contents
Abstract ii
Acknowledgements iv
Table of contents v
List of Figures xv
List of Tables xviii
Publications and Papers Submitted
Chapter One: Introduction
1.1. Introduction 1
1.2. Thesis plan 2
1.3. References 5
Chapter Two: Literature review
2.1. Introduction 6
2.2. Traditional Chinese Medicine and Cancer treatment 7
2.3. Molecular basis of cancer formation 9
2.3.1. Free radicals and cancer 9
2.3.2. Chemokines and cancer 12 2.3.2.1. Immunosurveillance and immunoediting of cancer: Role of botanic polysaccharides 12
2.3.2.2. Antioxidant and immunomodulatory activities of polysaccharides from medicinal plants/mushroom 13
2.4. Review of literature on anticancer TCM herbs with special emphasis on bioactive polysaccharides 14
2.4.1. Anticancer organic molecules isolated from TCM herbs 16
2.4.2. Anti-tumor polysaccharides isolated from medicinal mushrooms 17
2.5. An overview of the factors influencing the mechanism of action of plant polysaccharides as anticancer agents 23
v 2.6. Structural characterization of polysaccharides 24
2.7. Basis for the selection of medicinal herbs used in this project 29
2.8. Aims and objectives 34
2.9. References 36
Chapter Three: Evaluation of Immunomodulatory and anticancer potentials of sixteen traditional anticancer herbs - selection of best herbs for further study
Abstract 50
3.1. Introduction 51
3.2. Materials and Methods 55
3.2.1. Procurement of medicinal plants associated with this research 55
3.2.2. Chemicals and reagents/enzymes 55
3.2.2.1. Chemicals 55
3.2.2.2. Reagents/enzymes 56
3.2.3. Extraction of crude polysaccharides from sixteen medicinal plants 56
3.2.4. Analysis of chemical composition 59
3.2.5. Bio-Assays 60
3.2.5.1. Antioxidant activities 60
3.2.5.1.1. Scavenging activity against DPPH● radicals 60
3.2.5.1.2. ABTS●+radical scavenging assay 60
3.2.5.1.3. Ions (Fe2+) Chelating assay 61
3.2.5.1.4. Ferric ions (Fe3+) reducing antioxidant power 61
3.2.5.2. Immunomodulatory activity assays 62
3.2.5.2.1. Production of IL-6 62
3.2.5.2.2. Production of TNF- α 63
3.2.5.2.3. Toxicity test 63
3.2.5.3. In vitro anticancer assays against various cancer cell lines 64
vi 3.2.6. Statistical analysis 65
3.3. Results and Discussion 65
3.3.1. Chemical composition of polysaccharides 65
3.3.2. Antioxidant activities of selected herbal polysaccharides 67
3.3.2.1. Scavenging abilities against DPPH● and ABTS●+ radicals 67
3.3.2.2. Ions (Fe2+) Chelating assay 69
3.3.2.3. Fe3+ reducing abilities 69
3.3.3. Immunomodulatory activities of crude polysaccharides 70
3.3.3.1. Effect of herbal polysaccharides to activate mouse macrophages
and produce TNF-α and IL-6 70
3.3.3.2. Toxicities of polysaccharides 76
3.3.4. Anticancer activities of crude polysaccharides 76
3.4. Conclusions 83
3.5. References 84
Chapter Four: Evaluation of Immunomodulatory and anticancer potentials of ethanol soluble organics from sixteen traditional anticancer herbs Abstract 96
4.1. Introduction 97
4.2. Materials and Methods 100
4.2.1. Collection of medicinal herbs associated with this research 100
4.2.2. Chemicals and reagents 101
4.2.3. Ethanol soluble water extraction preparations for bioactivity studies 101
4.2.4. Determination of total phenolic compounds 103
4.2.5. Determination of total flavonoids 104
4.2.6. Bioactivity tests 104
4.2.6.1. DPPH● radical scavenging assay 104
4.2.6.2. ABTS●+radical scavenging assay 104
vii 4.2.6.3. Immunomodulatory activities assays 105
4.2.6.3.1. Culture RAW 264.7 macrophages 105
4.2.6.3.2. NO production 105
4.2.6.3.3. TNF- α production 105
4.2.6.3.5. Determination of cell viability by MTT assay 105
4.2.6.4. Anticancer assays against various cancer cell lines 106
4.2.7. Statistical analysis 107
4.3. Results and Discussion
4.3.1. Chemical composition 107
4.3.2. Antioxidant activities 108 4.3.2.1. DPPH● and ABTS●+ scavenging activities 108
4.3.2.2. Fe3+ reducing power 110
4.3.3. Anti-inflammatory activities 114
4.3.3.1. Cell viability 116
4.3.4. Anticancer activities 118
4.4. Conclusions 123
4.5. References 126
Chapter Five: Biological and structural characterisation of polysaccharides fractions isolated from Amauroderma rugosum Trrond
Abstract 137
5.1. Introduction 138
5.1.1. Traditional use of Coriolus versicolor 139
5.1.1.1. Clinical studies of anti-tumor polysaccharides from C. versicolor 139
5.1.1.2. Anti-tumor mechanism of PSP and PSK isolated from C. versicolor 141
5.1.2. Anticancer polysaccharides from Ganodermataceae family of mushroom 143
5.1.2.1. Polysaccharides from Amauroderma genus 143
5.2. Materials and Methods 144
5.2.1. Materials 144
viii 5.2.2. Extraction and fractionation of polysaccharides from
Amauroderma rugosum 145
5.2.3. Determination of molecular weights of polysaccharide fractions 146
5.2.4. Analysis of mono-saccharides 147
5.2.5. Fourier transforms infrared (FT-IR) spectroscopy 147
5.2.6. NMR analysis 148
5.2.7. Bioactivity tests 148
5.2.7.1 . Antioxidant activity 148
5.2.7.2. Assay for the measurement of IL-6 production 149
5.2.7.3. Assay for the measurement of TNF- α production 150
5.2.7.4 . Determination of cell viability by MTT assay 150
5.2.8. Statistical analysis 151
5.3 Results and Discussion 151
5.3.1. Fractionation and purification of polysaccharides from A. rugosum 151
5.3.2. FT-IR spectroscopic characterisation of active polysaccharides 154
5.3.3. NMR spectroscopic study 156
5.3.4. Antioxidant activities ARPs 164
5.3.5. Immunomodulatory effects of polysaccharides from A. rugosum 166
5.3.6. Mechanism of action of ARPs 169
5.3.7. Cell viability 170
5.4 Conclusions 170
5.5 References 172
Chapter Six: Biological and structural characterisation of polysaccharides fractions isolated from Lobelia chinensis Lour
Abstract 183
6.1. Instruction 184
6.2. Materials and methods 187
ix 6.2.1. Material 187
6.2.2. Chemicals 187
6.2.3. Extraction and fractionation of polysaccharides from Lobelia chinensis 187
6.2.4. Determination of average molecular mass 188
6.2.5. Determination of chemical composition 188
6.2.6. FT-IR analysis 188
6.2.7. NMR analysis 188
6.2.8. Bioactivity tests 189
6.2.8.1. DPPH● scavenging assay 189
6.2.8.2. ABTS●+radical scavenging assay 189
6.2.8.3. OH● scavenging assay 189
6.2.8.4. Immunostimulatory activity assays 189
6.2.8.4.1. Culturing of macrophage cells 189
6.2.8.4.2. IL-6 production 190
6.2.8.4.3. TNF- α production 190
6.2.8.5. Determination of toxicity by MTT test 190
6.2.9. Statistical analysis 190
6.3 Results and Discussion 191
6.3.1. Extraction and fractionation of polysaccharides from L. chinensis 191
6.3.2. Chemical compositions of the fractions 192 6.3.3. FT-IR spectroscopy analysis 194
6.3.4. NMR spectroscopy analysis 196
6.3.5. Radical scavenging activities 201
6.3.6. Immunostimulatory activities of L. chinensis polysaccharides 204
6.3.7. Cell viability 206
6.4 Conclusions 207
6.5 References 209
x Chapter Seven: Biological and structural characterisation of polysaccharides
isolated from Artemisia annua L
Abstract 216
7.1. Introduction 217
7.1.1. Ethnopharmacology of the plants from Artemisia genus and
their photochemistry 219
7.1.2. Important polysaccharides from Artemisia genus 221
7.2 Materials and methods 223
7.2.1. Material 223
7.2.2. Chemicals 223
7.2.3. Extraction and fractionation of polysaccharides from A. annua 223
7.2.4. Determination of molecular weights of polysaccharide fractions 224
7.2.5. Analysis of mono-saccharides 224
7.2.6. Bioactivity tests 224
7.2.6.1. DPPH● scavenging assay 224
7.2.6.2. ABTS●+radical scavenging assay 225
7.2.6.3. OH● radical scavenging assay 225
7.2.6.4. Immunomodulatory activity assays 225
7.2.6.4.1. Maintenance, preparation and activation of RAW 264.7 macrophages 225
7.2.6.4.2. Assay for the measurement of IL-6 production 225
7.2.6.4.3. Assay for the measurement of TNF- α production 226 7.2.6.5. Determination of cell viability by MTT assay 226
7.2.7. Fourier transform infrared (FT-IR) spectroscopy 226
7.2.8. Statistical analysis 226
7.3 Results and Discussion 226
xi 7.3.1. Fractionation and purification of polysaccharides from A. annua L 227
7.3.2. FT-IR spectroscopic characterisation of active polysaccharides 232
7.3.3. Antioxidant activities AAPs 235
7.3.4. Immunomodulatory effects of polysaccharides from A. annua L 237
7.3.5. Cell viability 242
7.4. Conclusions 242
7.5. References 244
Chapter Eight: Evaluation of MRI contrast potential of Gd(III) complexes with
bioactive herbal polysaccharides
Abstract 253
8.1. Introduction 254
8.1.1. Principles of NMR spectroscopy 256
8.1.1.1. NMR relaxation phenomenon 257
8.1.1.2. Spin-lattice relaxation 258
8.1.1.3. Spin-spin relaxation 258
8.1.1.4. Factors influencing relaxation rates 259
8.1.1.5. Relaxation of water protons in biological tissues 260
8.1.2. Influence of paramagnetic contrast agents on water proton relaxivities 261
8.1.2.1. Mechanism of paramagnetic relaxation of water molecules 262
8.1.3. Basic principles of magnetic resonance imaging (MRI) 264
8.2. Experimental Methodology 265
8.2.1. NMR relaxation experiments 265
8.2.2. MRI experiments 268
8.2.3. T2-weighted images 268
8.2.4. T1-weighted images 269
xii 8.2.5. Acquisition of MR images 271
8.2.6. Construction of phantom for the evaluation of performance of
T1 and T2 agents 272
8.2.7. Inversion recovery spin-echo MRI experiments
(T1 contrast images) 273
8.2.8. Spin-echo / CPMG pulse sequence based MRI experiments
(T2 contrast images) 274 8.3. Results and Discussion 274
8.3.1. NMR relaxivity Results 274
8.3.1.1. Results on T1 agents 275
8.3.1.2. Relaxation values of [Gd-Dextran-10] at different
concentrations 275
8.3.1.3. Relaxation values of [Gd-LCP-2] at different concentrations 276
8.3.1.4. Relaxation values of [Gd-AAP-1] at different concentrations 279
8.3.2. MRI Results 283
8.3.2.1. Performance evaluation of T1 contrast agents 283
8.3.2.2. MRI performance evaluation of T1 agents 283
8.3.2.2.1. Evaluation of MRI performance of [Gd- Dextran-10]
complex: a T1 agent 283
8.3.2.2.2. Evaluation of MRI performance of [Gd-LCP-2]
complex: a T1 agent 284
8.3.2.3. Performance evaluation of T2 contrast agents 285
8.3.2.4. MRI performance evaluation of T2 agent 285
8.3.2.4.1. Evaluation of MRI performance of [Gd-AAP-1] complex:
a T2 agent 286
xiii 8.4. Conclusion 286
8.5. References 288
Chapter Nine: Conclusions and Future Research 292
xiv List of Tables
Chapter Two
Table 2.1. Some anticancer polysaccharides from medicinal mushrooms and
their biological activities 22
Table 2.2. Important anti-cancer herbs that are used by Chinese medical practitioners
and in clinical studies 34
Chapter Three
Table 3.1. The Chemical composition and monosaccharide content of crude polysaccharides extract from dried plant material 66
Table 3.2. Antioxidant activities of crude polysaccharides extracted from 68
sixteen medicinal plants
Table 3.3. Ferric ion reducing power of polysaccharides extracted from
sixteen medicinal plants 72
Table 3.4. Immunomodulatory activities of polysaccharides extracted from
selected medicinal herbs 74
Table 3.5. In vitro cytotoxicity (IC50) of crude polysaccharides isolated from herbs
against five cancer cell lines 78
Chapter Four
Table 4.1. Antioxidant activities of the extracts from sixteen Chinese Medicinal herbs along with their Total phenolic and flavonoid contents 109 Table 4.2. Ferric ions reducing power of the extracts isolated from sixteen medicinal herbs 111 Table 4.3. Anti-inflammatory activities of the extracts from selected medicinal herbs 115
Table 4.4. In vitro cytotoxicity (IC50) of the extracts from herbs against
xv five cancer cell lines 120
Table 4.5. Important anticancer herbal extracts identified in this research
together with their polyphenol contents 125
Chapter Five
Table 5.1. Chemical composition (sugar contents) of ARPs 153
Table 5.2. Chemical shift (ppm) of 1H NMR signal for ARP-2 163
Table 5.3: Antioxidant activities of polysaccharide fractions of A. rugosum
along with their molecular weights 165
Chapter Six
Table 6.1. The Chemical composition and monosaccharide contents of LCPs 193
Table 6.2. 1H and 13C chemical shifts (ppm) of LCP-2 from L. chinensis 198
Table 6.3. Proton and carbon correlations from 2D-NMR spectra data
of LCP-2 from L. chinensis 198
Table 6.4. Radical scavenging activities of LCPs along with their
average molecular mass 202
Chapter Seven
Table 7.1. Chemical composition (sugar contents) of polysacharide
fractions isolated from A. annua L 229
Table 7.2. Antioxidant activities of polysaccharide fractions of A. annua
along with their molecular weights 231
Table 7.3. Antioxidant activities of polysaccharide fractions of A. annua 236
xvi Chapter Eight
Table 8.1. Relaxation data for Gd3+ complex with Dextan-10 which is a
standard α-glucan (Average molecular mass =10 kDa) 277
Table 8.2. Relaxation data for Gd3+ complex with LCP-2 which is a
β-D-(2→1)-fructan discovery in this research 278
Table 8.3. Relaxation data for Gd3+ complex with AAP-1 which is a complex
polysaccharides (containing arabinose, galactose and glucose units)
discovered in this research 280
Table 8.4. Classification of type of Gd (III) based MRI contrast agents
formed with bio-macromolecules with different molecular masses
studied in Authors’ Laboratory (unpublished data) 282
xvii List of Figures
Chapter Two
Fig. 2.1. Simplified diagram showing free radical damage of biopolymers that leads to carcinogenic response 11
Fig. 2.2. Various anticancer effects of components 15 Fig. 2.3. Flow chart showing project summary 35
Chapter Three
Fig.3.1. Flow chart for the extraction of polysaccharides from selected herbs 58 Fig. 3.2. Concentration dependant immunomodulatory activities of most active polysaccharide extracts 75
Fig. 3.3. Anticancer activities (IC50) of polysaccharide extracts against four different cancer cell lines 79 Fig. 3.4. Dose dependant variation of anticancer activities of the polysaccharides from the three most active herbs 80 Fig. 3.5. A diagram developed to visualise the relationship of anticancer
activities and monosaccharide composition 81
Chapter Four
Fig. 4.1. Schematic diagram for the separation of ethanol soluble organics from hot water extracts of medicinal herbs 103 Fig. 4.2. Correlation between antioxidant activity and the total
phenolic and flavonoid contents in the extracts 113
Fig. 4.3. Concentration dependant immunomodulatory activities of most
active extracts from the selected herbs 117
Fig. 4.4. Anticancer activities (IC50) of the extracts against
four different cancer cell lines 121
Fig. 4.5. Dose dependant variation of anticancer activities of the extracts
from the three most active herbs 122
xviii Chapter Five
Fig. 5.1. Flow chart for the extraction of polysaccharides from A. rugosum 146
Fig. 5.2. Gel filtration chromatograms of polysaccharide fractions from A. rugos um 152
Fig. 5.3. Calibration curve for the determination of molecular weights of polysaccharides A. rugosum L based on the elution volume and the molecular mass of standard dextran series of T2000 (2,000 kDa), T450 (450 kDa), T150 (150 kDa), T70 (70 kDa), T40 (40 kDa), T10 (10 kDa) and Glucose (180 Da) (Note: Kav = (Ve – Vo) / (Vt – Vo), Vo is void volume, Vt is total volume, Ve is elution volume) 153
Fig 5.4. The FT-IR spectra of the three fractions from A. rugosum 155
Fig. 5.5. 1H NMR spectra of A. rugosum polysaccharides 157
Fig. 5.6. 13C NMR spectra of A. rugosum polysaccharide 159
Fig. 5.7. (a) g-COSY and (b) HSQC spectra of ARP-2 160
Fig. 5.8. DEPT spectra of ARP-2 161
Fig. 5.9. Structure of ARP-2 162
Fig. 5.10. Concentration dependant radical scavenging activities of two polysaccharide fractions (LCP-1 and LCP-2) 165
Fig. 5.11. Effects of A. rugosum polysaccharides on murine RAW 264.7 macrophages 168
Fig. 5.12. Cell viabilities of isolated polysaccharide fractions from A. rugosum at different concentrations 170
Chapter Six
Fig. 6.1. Isolation and purification of LCPs using Sepharose LC-6B column 191
xix Fig. 6.2. FTIR spectrum of LCP-1 and LCP-2 195
Fig. 6.3. NMR spectra of LCP-2 isolated from L. chinensis 199
Fig. 6.4. Structure of LCP-2 200
Fig. 6.5. Concentration dependant radical scavenging activities of two polysaccharide fractions (LCP-1 and LCP-2) 202
205 Fig. 6.6. Effects of L. chinensis polysaccharides on murine RAW 264.7 macrophages 207 Fig. 6.7. Cell viabilities of isolated polysaccharides from L. chinensis
Chapter Seven
Fig. 7.1. Size-exclusion chromatogram representing polysaccharide and
protein profiles of A. annua 227
Fig. 7.2. Calibration curve for the determination of molecular weights of polysaccharides of A. annua L based on the elution volume and the molecular mass of standard dextran series of T2000 (2,000 kDa), T450 (450 kDa), T150 (150 kDa), T70 (70 kDa), T40 (40 kDa), T10 (10 kDa) and Glucose (180 Da) (Note: Kav = (Ve – Vo) / (Vt – Vo), Vo is void volume, Vt is total volume, Ve is elution volume) 230
Fig. 7.3. The FT-IR spectra of the three fractions from A. annua 234
Fig. 7.4. Concentration dependant radical scavenging activities of two polysaccharide fractions 236
Fig. 7.5. Effects of A. annua polysaccharides on murine RAW 264.7 macrophages 239
Fig. 7.6. Schematic representation of mechanism of immunostimulatory activity F induced by inulin-type fructans (β-D-(2→1)-fructan) of longer chain length (Note: Fructans with shorter chain leads to decreased production of cytokines) 241
xx Fig. 7.7. Cell viabilities of isolated polysaccharide fractions from A. annua at different concentration 242
Chapter Eight
Fig 8.1. Spin-echo MRI pulse sequence 268
Fig 8.2. Inversion recovery spin-echo MRI pulse sequence 270
Fig 8.3. Phantom for T1 and T2- weighted MR imaging 273
Fig. 8.4. Relaxivity graphs plotted against concentration: (A) T1 relaxivity, and (B) T2 relaxivity (Results confirm that this is a T1 agent with r2/r1=1.2) 277
Fig. 8.5. Relaxivity graphs plotted against concentration: (A) T1 relaxivity, and (B) T2 relaxivity (Results confirm that this is a T1 agent with r2/r1=1.17) 278
Fig. 8.6. Relaxivity graphs plotted against concentration: (A) T1 relaxivity, and (B) T2
relaxivity (Results confirm that this is a T2 agent with r2/r1=9.66) 280
Fig. 8.7. T1-weighted images from the phantom made with [Gd-Dextran-10] samples with three different concentrations (using Bruker FLASH pulse sequence). The concentration used were: Tube-1) 2.88 mM, Tube-2) 1.63 mM and Tube-3) 1.15 mM. The imaging parameters employed were: TE=1.8 ms, TR=5.6 ms 284
Fig. 8.8. T1-weighted images from the phantom made with [Gd-LCP-2] samples with three different concentrations (using Bruker FLASH pulse sequence). Theconcentration used were: Tube-1) 3.57mM, Tube-2) 2.6mM and Tube-3) 1.8mM. The imaging parameters employed were: TE=1.9 ms, TR=5.6 ms 285
Fig. 8.9. T2-weighted image from the phantom made with [Gd-AAP-1] samples with three different concentrations (using Bruker CPMG pulse sequence). The concentration used were: Tube-1) 0.3 mM, Tube-2) 0.9 mM and Tube-3) 2.5 mM. The imaging parameters employed were: TE=18 ms, TR=3 s 286
xxi Publications and Papers Submitted during PhD research
1. Zhang, L., Li CG, Liang H, Reddy N (2017) Bioactive Mushroom Polysaccharides:
Immunoceuticals to Anticancer Agents. J Nutraceuticals Food Sci. 2(2), 1.
2. Zhang, L., Koyyalamudi, S. R., Khoo, C. S., Reddy, N., 2017. Antioxidant, anti-
inflammatory and anticancer activities of ethanol soluble organics from water extracts
of selected Medicinal Herbs and their relation with flavonoid and phenolic contents.
Pharmacologia 8 (2): 59-72
3. Zhang, L., Reddy, N. & Koyyalamudi, S.R. (2014). “Chapter 5: Isolation,
characterization and biological activities of polysaccharides from medicinal plants and
mushrooms”. In: Studies in Natural Products Chemistry. Atta-ur-Rahman (Ed.),
Elsevier Science Publishers, Oxford, UK. (ISBN 978-0-444-63281-4). 42, 117-151.
4. Zhang, L., Koyyalamudi, S. R., Jones, C. E., Khoo, C. S., Reddy, N., 2017.
Antioxidant and immunomodulatory activities and structural characterization of
polysaccharides isolated from Lobelia chinensis Lour. Int. J. Mol. Sci (submitted)
5. Zhang, L., Koyyalamudi, S. R., Jones, C. E., Khoo, C. S., Reddy, N., 2017. Biological
activities and structural characterization of polysaccharides isolated from
Amauroderma rugosum. Int. J. Mol. Sci (submitted)
xxii Chapter One
Introduction 1.1.Introduction
Cancer is one of the leading causes of death in the modern world (Zhang et al., 2014). The occurrence of cancer is increasing because of the aging of the population as well as an increasing prevalence of established risk factors such as lifestyle factors. Based on
GLOBOCAN estimates, about 14.1 million new cancer cases and 8.2 million deaths occurred in 2012 worldwide (Torre et al., 2015).
A closer look into Traditional Chinese Medicine (TCM) for anticancer herbal formulations could be of great importance in the discovery of new and effective anticancer agents based on herbal polysaccharides.In recent decades, botanical polysaccharides have attracted a great deal of attention in biomedical research due to their significant immunomodulatory and anti- proliferation capacities (Vetvicka and Vetvickova, 2012, Schepetkin and Quinn, 2006,
Friedman, 2016, Sugiyama, 2016 and Zhang et al., 2014). It is important to note that novel and lifesaving therapeutic agents could be discovered by a systematic evaluation of TCM herbs/mushrooms using modern scientific techniques .
In order to discover new and effective polysaccharide based cancer therapeutic agents from medicinal herbs, it is essential to:
• Extract and screen the herbal polysaccharides for their biological activities (e.g. antioxidant, anti-inflammatory, anti-cancer) from a well-chosen set of TCM based anticancer herbs with a view to select a few of the best herbs in this chosen set
• Purify and study the biological activities of the polysaccharides isolated from the selected herbs based on the studies carried out in the preceeding step
• Undertake detailed scientific investigations on the active herbs to identify the chemical structures and possible mechanism of action of the purified herbal polysaccharides.
• Develop formulations by combining the most active polysaccharides to design effective therapeutic agents. Such a design of effective formulations must take advantage of synergistic effects. It should be noted that the synergy effects of a
1
formulation can be maximised by including the componetnts that act by differing mechanisms.
The first three of the above investigations have been successfully undertaken in this study with the aim to discovering anticancer polysaccharides. The results presented in Chapters 3 to 7 are extremely promising. The last dot point above is the subject of future research.
Sixteen herbs selected for initial screening in this research are presented in Table 3.1 of
Chapter 3. Table 3.1 gives the list of TCM anticancer herbs that have been chosen and studied in this project. Selection of these plants was on the basis of their TCM knowledge and therapeutic properties repoted in the literature (Huang et al., 2008; Wei et al., 2010).
Previous research on these selected sixteen polysaccharides is reviewed in Chapter 2.
Based on the initial screening results (Chapter 3), three of the most active herbs have been selected for further detailed study of their polysaccharides. These herbs are: Artemisia annua
L, Lobelia chinensisLour and Amauroderma rugosum. The detailed results on the pure polysaccharides isolated from these three herbs are presented in Chapters 5 to 7. Excellent results have been obtained from these studies and this research has produced about ten pure polysaccharides with very high immunomodulatory and antioxidant potential indicating that they are potent candidates for immuno-chemotherapy. MRI contrast potential of four of the discovered herbal polysaccharides have also been evaluated and presented in Chapter 8.
1.2. Thesis plan
Encompassing all the above mentioned objectives, this thesis has been divided into nine chapters as described below.
Chapter 1 provides an introduction to anticancer plants with special reference to TCM herbs and the significance of their polysaccharides. The importance of modern scientific research to
2 develop effective anticancer formulations with their polysaccharides is also presented. This
Chapter also gives the plan of this thesis.
Chapter 2 presents a thorough literature review on TCM herbs and their polysaccharides. A review of literature on botanical polysaccharides is also included in this chapter.The basis of the selection of sixteen anticancer herbs is also provided in this chapter.
Chapter 3 presents the results on anticancer, immunomodulatory and antioxidant activities of crude polysaccharides extracted from the sixteen selected herbs.The results are thoroughly analysed in this chapter and the relationship of anticancer activities with mono-saccharide composition is are discussed. Based on these results, three of the best herbs are chosen for further detailed study. They are, Artemisia annua L, Lobelia chinensisLour and
Amauroderma rugosum.
Chapter 4 presents the results on anticancer, anti-inflammatory and antioxidant activities of small organics extracted from the sixteen selected herbs with hot water with a view to obtaining a comprehensive biological picture of all the hot water extractable constituents.
The results are analysed in detail in this chapter and the relationship of anticancer activities with flavonoid contents are discussed. The results presented in this chapter together with the results of Chapter 3 provide a comprehensive picture of most of the bioactive constituents
(polysaccharides and flavonoids) present in the hot water extracts of the selected sixteenherbs.
Based on these results, three of the best herbs were chosen for further detailed study.
Chapter 5 describes the isolation of polysaccharides from Amauroderma rugosumare discribed . The structural characterisation of the pure polysaccharides has been carried out
3 using GC, FT-IR and NMR techniques and the results are presented. Antioxidant and
immunomodulatory activities of polysaccharides from the anticancer mushroom
A. rugosumare also presented and discussed.
Chapter 6 presents the isolation, characterisation and bioactivity results of polysaccharides
from Lobelia chinensis. Structural characterisation including sugar composition, FTIR spectral features and NMR spectroscopic analysis have been carried out and presented in this
chapter. The antioxidant and immunomodulatory activities of these polysaccharides are also
presented and discussed.
Chapter 7 presents the results on isolation and characterisation of polysaccharides from
Artemisia annua. GC and FT-IR have been employed in order to obtain structural information
on the polysaccharides and the results are present in this chapter.
Chapter 8 povides the results on the evaluation of the bioactive herbal polysaccharides as
potential MRI contrast agents discovered in this study (described in Chapters 5-7). Three of
the bioactive polysaccharides: LCP-2, AAP-1 and AAP-2 have been used for the studies carried out in this Chapter. These bio-polymers have been complexed with Gd(III) ions and their relaxivities determined at 300 MHz. These results together with MRI performance evaluation results are presented.
The overall conclusions of the study and future directions of anticancer herbal polysaccharide
research is presented in Chapter 9.
4 1.3. References
Zhang, L., Koyyalamudi, S. R., Reddy, N., (2014). Isolation, characterization, and biological
activities of polysaccharides from medicinal plants and mushrooms, in ? Atta-ur-Rahman,
F.R.S (Ed), studies in Natural Products Chemistry, 1st ed.; UK
Torre, L. A., Bray, F., Siegel, R. L., Ferlay, J., Lortet‐Tieulent, J., and Jemal, A. (2015).
Global cancer statistics, 2012. CA: a cancer journal for clinicians, 65(2), 87-108.
Bubb, W. A. (2003). NMR spectroscopy in the study of carbohydrates: Characterizing the structural complexity. Concepts in Magnetic Resonance Part A, 19, 1-19.
Vetvicka, V., Vetvickova, J., (2012). Combination of glucan, resveratrol and vitamin C demonstrates strong anti-tumor potential. Anticancer Res. 32 (1), 81-87.
Schepetkin, I. A., and Quinn, M. T. (2006). Botanical polysaccharides: macrophage immunomodulation and therapeutic potential. International immunopharmacology, 6, 317-
333.
Friedman, M. (2016). Mushroom Polysaccharides: Chemistry and Antiobesity, Antidiabetes,
Anticancer, and Antibiotic Properties in Cells, Rodents, and Humans. Foods, 5, 80.
Sugiyama, Y., (2016). Polysaccharides. In Yamaguchi, Y (Ed), Immunotherapy of Cancer,
Springer: pp 37-50.
Huang, H. B., Li K. X., Liu, T., Zeng, C. Q., Lin, J., Qiu, M., (2008). Kang Zhong Liu Zhong
Yao Lin Chuang Ying Yong Yu Tu Pu (Clinical application of anti-tumor Chinese medicine).
1st ed. Guang Zhou: Guangdong Science and Technology Press.
Wei C. Z., Qu L., Kou T. Q., (2010) Kang Zhong Liu Zhang Yao Ji Jin (Views of Anticancer
Chinese Medicinal Herbs), 1st ed, Zhong Yao guji Press, Bei Jing
5
Chapter Two
Literature Review
2.1 Introduction
The occurrence of cancer is increasing because of the aging of the population as well as an
increasing prevalence of established risk factors such as smoking, overweight, physical
inactivity, and changing reproductive patterns associated with urbanization and economic
development. Lung cancer is the leading cause of cancer death among males in both more
and less developed countries, and has surpassed breast cancer as the leading cause of cancer
death among females in more developed countries; breast cancer remains the leading cause of cancer death among females in less developed countries (Torre et al., 2015). Other leading causes of cancer death in more developed countries include colorectal cancer among males and females and prostate cancer among males (Torre et al., 2015). In less developed
countries, liver and stomach cancer among males and cervical cancer among females are also
leading causes of cancer death (Torre et al., 2015). Although incidence rates for all cancers
combined are nearly twice as high in more developed than in less developed countries in both
males and females, mortality rates are only 8% to 15% higher in more developed countries
(Torre et al., 2015). This disparity reflects regional differences in the mix of cancers, which is affected by risk factors and detection practices, and/or the availability of treatment. Risk factors associated with the leading causes of cancer death include tobacco use (lung, colorectal, stomach, and liver cancer), overweight/obesity and physical inactivity (breast and
colorectal cancer), and infection (liver, stomach, and cervical cancer) (Torre et al., 2015).
Conventionally, surgical procedures, chemotherapy and radiotherapy are the major treatments
for cancer. However, these procedures have drawbacks which include traumatic side effects,
expensive diagnosis and treatment (Cho 2010). Medicinal herbs have therefore received a
significant interest in anti-cancer therapy as they alleviate some of these drawbacks.
6
2.2. Traditional Chinese Medicine and cancer treatment
In traditional Chinese medicine (TCM), the pathological condition of the patient is
established by using four basic diagnostic methods (Cho, 2010). The origin of cancer is
considered to be mainly due to lack of “Zheng Qi” (immune system deficiency) and
accumulation of “Xie Qi” (pathogenic factors) (Cho, 2010). After an accurate diagnosis of
cancer is established, five TCM principles are then employed to design an appropriate herbal
formulation for the treatment. These five principles organize the formulation of many herbal
prescriptions for the treatment of cancer: supplement the ‘qi’ and blood to strengthen host
resistance; activate circulation to dispel blood stasis and ecchymosis; relieve pain; eliminate
heat and toxins; and soften lumps and dissolve masses. In simpler terms, a herbal treatment
principle involves two main aspects, namely, (i) to improve blood circulation and (ii) to
strengthen the immune system of the patient (Fuzheng) while simultaneously regenerating
and repairing the body (Guben) (Ravipati et al., 2012 ;Cho, 2010). Chinese medicinal herbal
formulations to treat cancer may also be thought of as strengthening healthy ‘qi’ to eliminate
pathogens.
The treatment aims to supplement the “qi” and the blood to improve the immune system of
the patient and to activate circulation. According to TCM theory, “Xie qi” is recognised as
the main factor in the early stage of cancer, whereas the deficiency of “Zheng qi” is
considered to be the key factor in the middle and late stages of cancer. Blood stasis is also
recognised as one of the factors in the development of tumour (Ravipati et al., 2012 ;Cho,
2010). Therefore in CMH therapy for cancer in early stages, the prescription must address
clearing the excess of “Xie qi”, but it should also address protection of the “Zheng qi” to maximize the cancer patient’s immunity (Ravipati et al., 2012; Cho, 2010). According to
TCM theory, the early stages of tumour development is mainly caused by ‘qi’ stagnation
7
and blood stasis caused by heat, cold and phlegm. Thus the CMH prescription for tumours in their early stages should primarily focus on promoting circulation of ‘qi’ and blood, though it optimally should include herbs that clear heat and phlegm, reduce phlegm, and resolve masses in accordance with the individual patient’s status based on the four diagnostics examination.
In treating tumours in the later stages of the disease process, the cancer patient’s ‘Zheng qi’ is the primary factor to be addressed in the CMH prescription so as to maximize their immune status. This is essential to assist the body to attack the tumour and to achieve optimal clinical results. Studies of Fuzheng therapy in the United States and China have demonstrated its value in treating a wide range of immune-compromised conditions, including cancer and leukemia. In a study of 76 patients with Stage II primary liver cancer, 29 of the 46 people receiving Fuzheng therapy in combination with radiation or chemotherapy survived for a year, and 10 survived for 3 years. Only 6 of the 30 patients who received radiation or chemotherapy alone survived 1 year, and all died by the third year (Cho, 2010).
It should be noted that there is a similar concept of balance between antioxidants and oxidants in modern medicine (Zhang et al., 2014; Cho, 2010). Imbalance between the level of antioxidant defence system and the production of free radicals and other oxygen-derived
●- ● species (ODS) such as superoxide radicals (O2 ), hydroxyl radicals (HO ), nitric oxide and
hydrogen peroxide (H2O2) are the main factors causing oxidative stress (Zhu et al., 2004).
These highly reactive species can cause DNA damage and also modify proteins (Zhu et al.,
2004). Such damage to the structures of biopolymers causes pro-carcinogenic response and
alters the cellular antioxidant defence system (Zhang et al., 2014; Zhu et al., 2004). Many
studies have shown that free radicals that produce oxidative stress in the body contribute to
8
both initiation and promotion of multistage carcinogenesis (Klaunig and Kamendulis, 2004;
Zhu et al., 2004). Recent literature also supports that inflammatory pathways activated by
oxidative stress lead to the process of cancer formation (Grivennikov, 2010). In this context
botanical polysaccharides have been shown to be of great importance to fight against
oxidative stress and to improve antioxidant defence of biological systems (Zhang et al., 2012;
Zhang et al., 2013; Zhang et al., 2014).
2.3. Molecular basis of cancer formation
It is known that the process of cancer formation is very complex. Generally, oxidative stress
has been proven to be a significant factor leading to the formation of cancer (Reuter et al.,
2010). It has been shown in the literature that excessive oxidative stress in the body for
extended periods of time activates inflammatory pathways which cause the transformation of
normal cells into cancer cells, support the survival of cancer cells, and finally leading to cancer cell proliferation (Ravipati et al., 2013; Zhang et al., 2011; Zhang et al., 2014).
2.3.1. Free radicals and cancer
The molecular basis of cancer formation is an important aspect and some of the details are discussed in this section. First, it is of interest to make a comparison between traditional and modern knowledge on pathology of cancer formation. In TCM, the common pathogenesis of cancer is considered to be the fatal imbalance of “yin-yang” energies due to deficiency of “qi’ and blood (Cho, 2010). There is a similar concept of balance between antioxidants and oxidants in modern medicine (Ravipati et al., 2013). Over-production of free radicals and
●- ● radical oxygen species (ROS) such as superoxide radicals (O2 ), hydroxyl radicals (HO ),
nitric oxide (NO) and hydrogen peroxide (H2O2) can cause oxidative stress which might lead
9
to DNA damage by converting guanine into 8-hydroxyguanine and also by protein modification (Zhang et al., 2014; Zhu et al., 2004). Such damage to the structures of biopolymers causes pro-carcinogenic response and alters the cellular antioxidant defence system (Zhang et al., 2014; Zhu et al., 2004). Many studies have shown that free radicals that produce oxidative stress in the body contribute to both initiation and promotion of multistage carcinogenesis (Zhu et al., 2004). This is illustrated in the Figure 2.1 (Zhang et al, 2014).
It should be mentioned here that, oxidative stress/inflammatory pathway is one of the causes
of cancer formation. Other pathways such as genetic factors can also lead to cancer formation
(Dunn et al., 2002; Dunn et al., 2004; Dunn et al., 2006) which is beyond the scope of this study .
10
Fig. 2.1. Simplified diagram showing free radical damage of biopolymers that leads to carcinogenic response (Adapted from Zhang et al, 2014; Zhu et al., 2004;Klaunig and Kamendulis, 2004)
11
2.3.2. Chemokines and cancer
Recent literature suggests that inflammatory cells can be activated by oxidative stress leading
to chronic infections. The associated inflammation may be further enhanced contributing to
the process of cancer formation (Grivennikov et al., 2010; Zhang et al., 2014). Inflammatory
cytokines such as tumour necrosis factor (TNF), interleukin-1 (IL-1), IL-6 and chemokines such as IL-8, CXC, chemokine receptor 4 (CXCR4) are the important products produced by inflammatory cells (Reuter et al., 2010). Evidence from the literature shows that chemokines, chemokine receptors and the epidermal growth factor receptor molecules such as human epidermal growth factor receptor (HER) family of molecules play a critical role in the formation and growth of tumour (Grivennikov et al., 2010). The effect of chemokines and chemokine receptors on tumour cells is very complex (Zhang et al., 2014). A detailed
discussion of this topic is beyond the scope of this research.
2.3.2.1. Immunosurveillance and immunoediting of cancer: Role of botanic polysaccharides
According to immunosurveillance theory, the immune system recognizes the cancer cells and destroys them. However, cancer immunosurveillance alone is not adequate to prevent cancer formation (Dunn et al., 2002). Literature has revealed that the cancer cells are capable of escaping immune recognition and survive (Dunn et al., 2004; Dunn et al., 2002; Dunn et al.,
2006), which is the basis of the new concept known as cancer immunoediting (Zhang et al.,
2014). The three phases of cancer immunoediting consist of elimination, equilibrium, and escape (Dunn et al., 2004).
This new concept of immunoediting emphasises that the weak immune system of the host is one of the key factors responsible for the formation of cancer. Hence, keeping the immune system healthy is an important way to initiate timely production of appropriate chemokines and cytokines that recognize and destroy cancer cells and prevent cancer formation (Dunn et
12
al., 2004). In this context, immunomodulatory plant/mushroom polysaccharides are very
important for the prevention and cure for cancer. Discovery of immunomodulatory
polysaccharides is one of the main aims of this project.
2.3.2.2. Antioxidant and immunomodulatory activities of polysaccharides from medicinal
plants/mushroom
As pointed out before, the defence system in humans is complex and involves many cell
types with distinct and overlapping roles (Janeway and Medzhitov, 2002). The cells relevant
for initial immune response are phagocytes such as neutrophils and monocytes and
macrophages, which are key participants in the innate immune response (Janeway and
Medzhitov, 2002, Uthaisangsook et al., 2002). Macrophages are the first line of host defence.
In addition, macrophages can interact with T lymphocytes to modulate the adaptive immune
response (Uthaisangsook et al., 2002).
Many studies have demonstrated that polysaccharides derived from higher plants possess
high antioxidant and immunomodulatory properties (Jeong et al., 2004; Jeong et al., 2012;
Zhang et al., 2012; Zhang et al., 2013). These compounds have been shown to produce a
range of responses by interacting with immune cells. Such responses include: (i) an increase
in macrophage cytotoxic activity against tumour cells and microorganisms, and (ii) activate
phagocytic activity, and enhance secretion of cytokines and chemokines, such as tumour
necrosis factor (TNF-α), interleukin (IL)-1β, IL-6, IL-8, IL-12, IFN-γ and IFN-β (Schepetkin and Quinn, 2006).
13
2.4. Review of literature on anticancer TCM herbs with special emphasis on bioactive
polysaccharides
It is well-known that cancer is a complex and dynamic disease and its treatment requires
chemo-therapeutics that possesses several biological activities (Cho, 2010). To be effective
for cancer treatment, a chemo-therapeutic agent must display a variety of anticancer effects summarised in figure below (Fig 2.2).
14
Various anticancer effects of components that is suitable for cancer treatment
Inhibition of Inhibition of Anti- Induction of Antioxidant Immunomodulatory proteinkinases topoisomerase angiogenesis apoptosis activity activity
Fig. 2.2. Various anticancer effects of components (Ravishankar et al., 2013, Sghaier et al., 2011; Boulikas and Tsogas, 2008;Dumontet and Jordan, 2010;Shah et al., 2013)
15
2.4.1. Anticancer organic molecules isolated from TCM herbs
Various bioactive compounds have been isolated from TCM herbs and clinically used as anticancer agents (Shah et al., 2013). Vinca alkaloids such as vinblastine, vinorelbine, vincristine and vindesine are the earliest and important anticancer agents in clinical use
(Boulikas and Tsogas, 2008, Dumontet and Jordan, 2010, Shah et al., 2013). Many scientific investigations has demonstrated that vinca alkaloids can inhibit cancer cell proliferation by binding to the tubulin during the cell mitotic stage and inhibition of cell mitosis as well as induce cell apoptosis (Dumontet and Jordan, 2010, Botta et al., 2009,
Govind, 2011). However, it should be noted that vinca alkaloids can also bind to the tubulin in the normal cell and lead to various negative effects such as reducing the number of white blood cells and bone marrow depression (Gragg and Newman, 2005, Lee et al., 2015). These risks limited the progression of vinca alkaloids in clinical use.
Flavonoids are the other class of anticancer therapeutic agents in clinical use/clinical trial
(Talhouk et al., 2007, Ravishankar et al., 2013, Sghaier et al., 2011). Scientific investigations demonstrate that flavonoids prevent cancer formation via (i) inhibition of protein kinases production, (ii) blocking the cell cycle, (iii) inducing apoptosis, and (iv) inhibiting angiogenesis (Rusak et al., 2005;Ravishankar et al., 2013). For instance, flavone and flavopiridol isolated from Dysoxylumbinectariferum Hook can prevent cancer formation by inhibition of several protein kinases such as cyclin-dependent kinases and tyrosine kinases
(Shah et al., 2013, Ravishankar et al., 2013). In addition, plant flavonoids such as quercetin, genistein, daidzein prevent cancer formation by their antioxidant and immunomodulatory activities (Hämäläinen et al., 2007; Fantini et al., 2015; Rusak et al., 2005; Ravishankar et al.,
2013). However, literature indicates that flavonoids exhibit significant multidrug resistance
(MDR) modulatory activity (Ravishankar et al., 2013), that causes a major problem of flavonoid use in anticancer clinical chemotherapy.
16 2.4.2. Anti-tumour polysaccharides isolated from medicinal mushrooms
In recent decades, botanical polysaccharides have attracted a great deal of attention in biomedical research due to their significant immunomodulatory and anti-proliferation capacities (Vetvicka and Vetvickova, 2012, Schepetkin and Quinn, 2006, Friedman, 2016,
Sugiyama, 2016 and Zhang et al., 2014). Several bioactive polysaccharides isolated from mushrooms have been clinically used as anticancer agents, these polysaccharides include: lentinan derived from Lentinula edodes (Sugiyama, 2016; Daba et al., 2003; Ina et al., 2013).
Polysaccharide Krestin (PSK) derived from Coriolus versicolor (Friedman, 2016, Sugiyama,
2016, Hattori et al., 2004 and Torisu et al., 1990), Polysaccharopeptide (PSP) isolated from
Coriolus versicolor (Ng, 1998, Cui and Chisti, 2003; Cheng and Leung, 2008), and schizophyllan from Schizophyllum commune (Sugiyama, 2016;Daba et al., 2003). These mushroom polysaccharides and their anticancer mechanisms are briefly discussed below.
Polysaccharide-Krestin (PSK) and polysaccharopeptide (PSP) are two famous anti- tumour polysaccharides isolated from medicinal mushroom Coriolus versicolor
(Polyporaceae) (Vetvicka and Vetvickova, 2012; Schepetkin and Quinn, 2006; Friedman,
2016; Sugiyama, 2016; Zhang et al., 2014). Clinical studies demonstrated that PSP and PSK, when used as immunochemotherapeutic agents in combination with chemotherapeutic agents such as FOLFOX4 (5-FU/folinic acid/oxaliplatin), can significantly reduce the risk of recurrence and increase the survival rates in patients with gastric and colorectal cancer
(Ohwada et al., 2006; Shibata et al., 2011; Sakai et al., 2008). Clinical studies also showed that PSP and PSK can inhibit tumour cell proliferation through the induction of apoptosis and cell cycle arrest (Zhang e al., 2014; Hirahara et al., 2011; Jiménez-Medina et al., 2008). More
17 details of these polysaccharide and their antitumour mechanism of action are provided in section 5.1.1.1 (Chapter 5).
Lentinan derived from a commonly used food mushroom Lentinula edodes (Berk) Pegler
(Polyporaceae),is an important anti-tumour polysaccharide which has been clinically used for the treatment of gastric cancer in Japan (Sugiyama, 2016, Daba et al., 2003and Ina et al.,
2013). Clinical studies indicate that lentinan used in chemo-immunotherapy in combination with chemotherapeutic agents (oral fluoropyrimidine) can significantly prolong the survival of patients with advanced gastric cancer, as compared to chemotherapy alone (Ina et al.,
2013). In addition, an in vivo study clearly demonstrated that lentinan can enhance the anti- tumour ability of trastuzumab, and significantly suppress tumour growth (Cheung et al.,
2002). However, without using lentinan, trastuzumabhas limited anti-tumour effect (Ina et al., 2013).
Anti-tumour mechanism oflentinan
Several studies have indicated that the significant immunomodulatory activities and cell signal modulation capacity of lentinan make it a suitable candidate for anticancer therapy
(Chen et al., 2007, Brown and Gordon, 2003, Ina et al., 2013). The molecular mechanisms of action of lentinan may be described as:
• Lentinan can bind with Dectin-1 receptor and activate several signalling pathways to
promote innate immune response. For example there is activation of phagocytosis and
production of anti-tumour cytokines such as IL-12 and TNF-α (Ina et al., 2013,
Brown and Gordon, 2003).
18
• Complement receptor type 3 (CR3) or scavenger receptors can recognise lentinan and
activate anti-tumour T-cell function as well as NK cells (natural killer cells) (Chen et
al., 2007).
• Granulocytes suppress the antitumour activities of lymphocytes (Ina et al., 2013).
Lentinan can reduce the ratio of granulocytes/ lymphocytes, and improve anti-tumour
effect of lymphocytes.
Schizophyllan (SPG) is another important mushroom polysaccharide isolated from functional food mushroom Schizophyllum commune Fr (Schizophyllaceae) (Zhang et al.,
2013, Daba et al., 2003), which has been clinically used for the treatment of cancer in Japan
(Zhang et al., 2013, Daba et al., 2003). Komatsu et al. (1969) have discovered SPG and demonstrated that this polysaccharide displays host-mediated antitumour activity against
Sarcoma 180. Clinical studies indicated that the antitumour capacity of SPG can be attributed to its significant immunostimulatory activities (Zhang et al., 2013). SPG stimulates the immune system and activates NK cells, spleen cells, lymphoid cells as well as bone marrow cells, thereby enhancing the production of antitumour cytokines such as interleukines 1,2 and
3 (Tsuchiya et al., 1989). Clinical studies have also indicated that SPG used in chemo- immunotherapy in combination with chemotherapeutic agents (tegafur or mitomycin and 5- fluorouracil) can significantly prolong the survival of patients with stage II cervical cancer, as compared to chemotherapy without SPG (Zhang et al., 2013).
Anti-tumour mechanism of Schizophyllan
The current understanding of the antitumour and immunomodulating effects of mushroom polysaccharides are: (i) prevention of oncogenesis by oral consumption of mushrooms or their preparations; (ii) direct antitumour activity against various allogeneic and syngeneic
19 tumours; (iii) immunopotentiation activity against tumours in combination with chemotherapy; (iv) preventive effect on tumour metastasis (Wasser, 2002; Zhang et al., 2007;
Zhang et al., 2014). Schizophyllan is similar to lentinan in biological activity, and its mechanism of immunomodulation and antitumour action appears to be quite similar (Zhang et al., 2013). Therefore, the antitumour activity of schizophyllan is due mainly to host- mediated immune responses (Zhang et al., 2013). Some anticancer polysaccharides from medicinal mushrooms and their biological activities are summarised in Table 2.1 below.
20
Table 2.1.Some anticancer polysaccharides from medicinal mushrooms and their biological activities
Medincal Types of Biological activities References Mushroom polysaccharides Agaricusblazei Glucan Immunomodulatory and anticancer activities Wasser, 2002, Akramiene et al., 2006 murrill Glucan Anti-tumour Armillaria mellea Galactoglucan Immunomodulatory and antioxidant activities Muszynska et al., 2011 Glucan-peptide complex Immunomodulatory and antioxidant activities Antrodia (1,3)-β-D-glucan Enhancing immunity, and anticancer Liu et al., 2004 camphaorate Auriculariaauricul (1,4)-D-glucan Induced apoptosis of cancer cell Ma et al., 2010 a-judae (1,3)-β-D-glucan Anticancer (1,6)-β-D-glucan Induced apoptosis of cancer cell Mannoglucan Immunomodulating Cordyceps Yu et al., 2007, Lee et al., 2010 militaris Galactoglucan Treatment of hypoglycemic, anticancer Poly-N- Antifibrotic acetylhexosamine (1,3)-, (1,4)- and (1,6)-β- Anticancer via inducing cell-cycle arrest and apoptosis; D-glucan immunomodulating Glucuronoglucan Treatment of hyperglycemia Ganoderma Zhang et al., 2010, Gao et al., 2003, Stachowiak lucidum Mannoglucan Immunomodulatory and antioxidant activities and Regula 2012 Heteroglucan Immunomodulatory and antioxidant activities Proteoglucan Immunomodulatory and antioxidant activities Lentinan Anticancer Mannoglucan Immunomodulating Ooi and Liu, 2000, Bisen et al., 2010, Hobbs et Lentinuse dodes Heteroglucan Antiviral al., 2000 Mannan-protein Hepatoprotective and antioxidant Heteroglucan-protein Hepatoprotectiveand antioxidant
21
Table 2.1.Some anticancer polysaccharides from medicinal mushrooms and their biological activities (Continued)
Types of Medincal Mushroom Biological activities References polysaccharides β-pachyman β-glucan Promoted the immune respones and increasing expression of Zhang et al., 2006, Huang and Zhang, Poria cocos (1,3)-α-D-glucan cytokines 2005 (1,3)-β-D-glucan Schizophyllum Schizaphyllan Anticancer and immunomodulating El Enshasy and Hatti-Kaul, 2013 commune Sclerotinia SSG Improveing the development of Th1 cells Ohno and Yamamoto, 1987 sclerotiorum Enhancing hematopoiesis and promoting the production of Sparassi scrispa SCG Tada et al., 2007 cytokine Trametes versicolor PSK Anticancer antioxidant and immunomodulating
PSP Anticancer antioxidant and immunomodulating Cui and Chisti, 2003
Heteroglucan Anticancer antioxidant and immunomodulating
Acidic Tremella fuciformis Inducing human monocytes to express interleukines Wasser, 2002 glucuronoxylomannan
22
2.5. An overview of the factors influencing the mechanism of action of plant
polysaccharides as anticancer agents
As discussed before, botanical polysaccharides (PS) display a variety of pharmacological
activities that include antitumour activity and immune regulation (Jeong et al., 2010, Jeong et al., 2012; Schepetkin and Quinn, 2006). Literature demonstrates that the differences in bioactivities of plant polysaccharides originate from the differences in their structures
(Lerouxel et al, 2006; Sheehan, 2008;Zhang et al., 2014). A review of important publications on plant / fungal polysaccharides (Jiang et al., 2010, Nie and Xie, 2011), revealed that certain structures such as β-glucans, acetylated glucomannans, sulfated PS, and arabinogalactans are responsible for their action (Jiang et al., 2010). A variety of factorssuch as monosaccharide composition, the main chain structure, branching, and functional groups impact on the anticancer activity of polysaccharides.(Zhang et al., 2007), these include:
• Literature strongly indicates that the antitumour properties of polysaccharides
isolated from medicinal plants/mushrooms are associated with their glucan
structures (Zhang et al., 2014).
• The type of glycosidic linkage in polysaccharides is the dominant factor for their
anticancer capacity. Many studies have indicated that most antitumour
polysaccharides contain β-(13)- and/or β-(16)-glycosidic linkages (Vetvicka
and Vetvickova, 2012; Zhang et al., 2007; Jiang et al., 2010; Zhang et al., 2014).
• High molecular mass is favourable for increasing immunomodulatory and
anticancer effects (Zhang et al., 2014, Jeong et al., 2010, Jeong et al., 2012, Mei
et al., 2016). For instance, polyporus polysaccharides (PPS) isolated from
Polyporusumbellatus with high molecular mass (1600kDa) possesses significant
immunomodulatory and anticancer activities (Zong et al., 2012).
23
• Degree of branching in the range of 0.2 to 0.33 is favourable for anticancer and
immunomodulatory activities (Zhang et al., 2014). For example, the degree of
branching of the anticancer agent schizophyllan is 0.33, and PSK and PSP are 0.2
(Zhang et al., 2014).
2.6. Structural characterization of polysaccharides
As discussed above, the structure of polysaccharides is the most important feature that
determines their function. The chemical structures of polysaccharides, such as the mono-
sugar composition, type of glycosidic linkage and their branching can be characterized by spectroscopic analysis, chemical analysis and chromatography techniques. Multi-dimensional
solution NMR spectroscopic techniques have also proven to be the most appropriate means to
determine the precise tertiary structures of the polysaccharides in addition to confirming
structural details including glycosidic linkages and branching (Zhang et al., 2014). Solution
NMR spectroscopy is therefore an indispensable tool for a clear understanding of the structure-function relationship of polysaccharides. Various techniques that are useful for structure determination of polysaccharides are discussed below.
Analysis of mono-saccharide composition
In generally, mono-saccharide composition in botanical polysaccharides is analysed by gas chromatography (GC) (Zhang et al., 2014). The main procedure for GC sample preparation includes hydrolysis, reduction, acetylation and methylation. The detailed information for GC analysis is described by Zhang et al (2014).
24
FTIR spectroscopy
FTIR spectroscopy is used to investigate the vibrational modes of molecules and is used to
study functional groups. The IR spectrum can be divided into three main regions: the far-IR
(< 400 cm-1), the mid-IR (4000 – 400 cm-1), and the near-IR (13,000 – 4,000 cm-1) (Stuart,
2005). Generally, many IR applications employ the mid-IR region, and it includes four
regions: (i) X-H stretching region (4000–2500 cm-1), (ii) triple-bond region (2500 – 2000 cm-
1), (iii) double-bond region (2000 – 1500 cm-1), and (iv) fingerprint region (1500 – 600 cm-1)
(Stuart, 2005). Important structural details of polysaccharides, such as the type of glycosidic
bonds and other functional groups can easily be identified using FTIR spectroscopy (Zhang et
al., 2014; Yang, and Zhang 2009) in the fingerprint region. For example, three strong IR absorption peaks result from pyranosides and two peaks from furanosides in the range of
1100 – 1010 cm-1 (Yang, and Zhang 2009; Zhang et al., 2014). Polysaccharides containing
mono-sugar units with functionally important β-structures have different IR stretching
frequencies when compared to those with generally inactive α-structures. For example,
polysaccharides with β-D-Glucose units give their stretching bands at 905 – 876 cm-1 and
those with α-D-Glucose units appear at 855 – 833 cm-1. Similar differences are observed in the stretching frequencies of other pyranoses with α- and β-structures(Yang, and Zhang 2009;
Zhang et al., 2014).
NMR spectroscopy
Liquid state NMR spectroscopy has played an important role in the structural studies of polysaccharides (Bubb, 2003; Yang, and Zhang 2009; Zhang et al., 2014). This is an excellent technique for the detailed structure determination of polysaccharides as long as they are soluble in a suitable solvent. Deuterated water and DMSO-d6 are common solvents used to run the 1 D- and 2D-NMR spectra in liquid state (Bubb, 2003).
25
Literature indicates that the 1HNMR signals of polysaccharides display severe overlap in the
chemical shift range of 3.5–5.5 ppm and it is difficult to assign them using one dimensional
1H NMR spectrum alone (Bubb, 2003; Yang, and Zhang 2009; Zhang et al., 2014). In a recent study by Leeuwen et al, (2008), the primary structural characterization of α-D-glucans
has been analysed using 1H NMR spectrum with some difficulty. The range of 13C chemical
shifts of polysaccharides is much wider than that of their 1H chemical shifts. Carbon chemical
shifts of polysaccharides range from 60 to 110 ppm (Bubb, 2003; Yang, and Zhang 2009;
Zhang et al., 2014).
A suite of two-dimensional (2D) NMR techniques that include DQF-COSY (double quantum
filtered-COSY), TOCSY (total correlation spectroscopy), HMQC (heteronuclear multiple-
quantum coherence spectroscopy) have been used for proton and carbon resonance
assignments of polysaccharides. These in turn (proton and carbon chemical shifts) have been
used for the analysis of polysaccharide structures, configurations and types of glycosidic
linkages (Bubb, 2003; Yang, and Zhang 2009; Zhang et al., 2014)
The salient features, of multidimensional NMR spectral analysis of polysaccharides, are
summarized here.
Homonuclear correlation (1H – 1H correlation) techniques, such as DQF-COSY and TOCSY
together with 1H-NMR are employed to assign proton chemical shifts of polysaccharides
(Bubb, 2003; Yang, and Zhang 2009; Zhang et al., 2014; Dunn, 2000; Ning, 1998). Once the
proton assignments are done, the strategy is then to use heteronuclear direct correlation (1H –
13C correlation) techniques, such as HMQC or HSQC to assign carbons that are directly
bonded to protons (Bubb, 2003; Yang, and Zhang 2009; Zhang et al., 2014; Duus, 2000;). In many situations, it is not possible to assign all the protons with homouclear 2D-NMR spectra
26
due to severe resonance overlap. In such a situation, one can begin assignments from known
13C resonances. It should be noted that the quaternary carbons and other non-protonated carbons cannot be assigned using HMQC or HSQC techniques. Such carbons are assigned by employing heteronuclear long range correlation methods, such as HMBC (heteronuclear multiple bond correlation spectroscopy) technique. Once all the proton and carbon chemical shifts of polysaccharides are assigned, the next step is to employ 2D-NOESY (nuclear
Overhauser enhancement spectroscopy) and / or 2D-ROESY (rotating frame Overhauser enhancement spectroscopy) to determine the three-dimensional structures (conformations) of polysaccharides (Bubb, 2003; Yang, and Zhang 2009; Zhang et al., 2014; Duus, 2000). Some details about practical approach to analyze solution NMR spectra of polysaccharides are provided below for a ready reference (Bubb, 2003; Yang, and Zhang 2009; Zhang et al.,
2014; Duus, 2000).
• The anomeric carbon resonances of polysaccharides (chemical shift range of 90–110
ppm) can be assigned by using 13C NMR spectra as per the method suggested by
Zhang, (1994)and hence the saccharide residues are determined. With anomeric
carbon assignments in hand, the assignment of anomeric proton resonances (chemical
shift range of 4.4–5.5 ppm) may easily be accomplished by using heteronuclear direct
correlation experiments, such as HMQC or HSQC.
• The assignment of individual monosaccharide residues of polysaccharides is carried
out starting from the known anomeric proton assignments (Duus et al., 2000; Zhang et
al., 2014). H2, H3, H4, H5 and H6 protons of each monosaccharide unit can be
recognized step by step based on the cross peaks starting from the anomeric proton in
homonuclear DQF-COSY or TOCSY spectra. It is then straight forward to assign C2,
27
C3, C4, C5 and C6 of individual monosaccharides by employing heteronuclear
HMQC (or HSQC) and HMBC spectra(Duus et al., 2000, Zhang et al., 2014).
• The long range heteronuclear correlation between the anomeric proton on one
monosacchride unit and the carbon on the adjacent monosaccharide can be established
using HMBC spectrum and hence the glycosidic linkage and the monosaccharide
sequence of polysaccharide can be obtained. Such sequential structure can be
confirmed from the through space correlations observed in NOESY spectra (Zhang et
al., 2014).
• The anomeric configuration can be obtained by recognizing the fact that the 13C
resonances of β-anomeric configuration always appears in the downfield region when
compared to that of α-anomeric configuration (Zhang et al., 2014; Zhang et al., 1994).
The range of chemical shifts of the β-anomeric 13C resonances is from 103 to105 ppm,
while the chemical shifts range of α-anomeric 13C is from 97 to 101 ppm (Zhang et al.,
2014; Bubb, 2003). In addition, the homonuclear and heteronuclear coupling
constants help to assign the anomeric configuration of saccharides (Zhang et al.,
2014).
• Positions of substituted groups (e.g. methyl, acetyl, sulfate substitutions on hydroxyl
groups of polysaccharides) can be distinguished by chemical shifts of corresponding
protons and carbon. For example, according to the study by Duus et al.,(2000), the
chemical shifts of substituted saccharides normally downfield shift about 0.2 to 0.5
ppm for protons and 6 to 7 ppm for 13C when compared to the unsubstituted
saccharides.
High molecular weight polysaccharides, which pose the problem of solubility, can be analyzed to some extent by using solid state 13C NMR techniques (Zhang et al., 2014).
28
Normally, magic-angle-spinning (MAS) experiments with cross polarization from proton to
13C nuclei are employed to obtain high resolution solid state NMR spectra with good
sensitivity to enable the structural analysis of insoluble polysaccharides (Zhang et al., 2014).
2.7. Basis for the selection of medicinal herbs used in this project
As mentioned above, TCM is considered as one of the oldest system used for the treatment of cancer. Three recently published monographs (Zhou et al., 2007; Huang et al., 2008; Wei et
al., 2010) summarized more than 800 species of TCM herbsassociated with cancer
treatment. Most of the documented anticancer herbal medicines and the relevant prescriptions
have been verified in clinical reports (Zhou et al., 2007; Huang et al., 2008; Wei et al., 2010).
From these published monographs, sixteen medicinal herbs (Table 2.2) were chosen in this study for the discovery of polysaccharides with anti-cancer properties. Most of these plants are important constituents in TCM anticancer formulations (Huang et al., 2008; Cho, 2010;
Wei et al., 2010) and hence are chosen for this study. Some details of these TCM anticancer herbs are given below.
Akebiaquinata (Lardizabalaceae) is one of the popular plants widely distributed in the south-
eastern provinces of China used in the treatment of various types of cancer such as lung, liver
and rectal cancers (Huang et al., 2008). In support of this, scientific investigations revealed
that the water extract of this herb can significantly inhibit the growth of JTC26 (inhibition rate
about 50%-70%), S180 and P38(Huang et al. 2008).
The stems of ArtemisiaannuaL. (Asteraceae) is used in traditional anticancer formulations
for the treatment of various cancers such as lung cancer, breast cancer, liver cancer and
29
stomach cancer (Huang et al., 2008). Several bioactive compounds were isolated from
A. annua, which include sesquiterpenoids, flavonoids, triterpenoids, steroids, (Bhakuni et
al., 2001, Bilia et al., 2006, Bora and Sharma, 2011). Several studies reported that,
artemisin isolated from A.annua can prevent cancer formation by blocking the cell cycle
to induce apoptosis, modulation of signalling pathway, inhibition of angiogenesis and
prevention of metastasis (Ghantous et al., 2010, Thoppil and Bishayee, 2011). A very recent
study indicated that polyphenols isolated from A.annua displayed significant anticancer
activity through the inhibition of highly metastatic breast cancer cells MDA-MB-231(Ko et
al., 2016). To the best our knowledge there is no literature on polysaccharides isolated from
A.annua, and this herb has been chosen for detailed study in this thesis (Chapter 7)
Artemisia vulgaris has been used byTCM practitioners for the treatment of cancer in addition
to other ailments (Zhou et al., 2007). TCM literature demonstrates that A. vulgaris is widely used in traditional anticancer formulations for the treatment of pancreatic cancer, lung cancer, and intraspinal cancer (Zhou et al., 2007). Several bioactive components have been isolated from this plant that include coumarins, and many types of terpenes (Govindaraj et al., 2013).
A recent study indicated that the methanol extract from the dry leaves of A. vulgaris
displayed anticancer activity against the human hepatocellular carcinoma cell line HepG2 by
induced cancer cell apoptosis (Sharmila and Padma, 2013). Aqueous extracts from A.
vulgaris were shown to induce apoptosis in prostate, breast and colon cancer cell lines
(Nawab et al., 2011). It is reported in the literature that the essential oil from A. vulgaris
displayed significant anticancer properties by mitochondria-dependent apoptosis (Saleh et al.,
2014). To the best of our knowledge there is no literature on polysaccharides isolated from A.
vulgaris.
30 Rabdosiarubescens (Lamiaceae), a plant native to southern China, has been used in TCM for
the treatment of esophagus, cardia, mammary, liver and prostate cancers (Bai et al., 2010).
R. rubescens is rich in ent-kauranediterpenoids, which have been verified as the
main biologically active constituent (Bai et al., 2010). Other compounds were also
isolated including flavonoids, such as cirsiliol and pedalitin (Bai et al., 2010). A
recent study indicated that flavonoids isolated from R. rubescens inhibit the growth of
human leukemia HL-60 cells (Bai et al., 2010). Polysaccharides isolated from R. rubescens
showed significant anticancer activity against Sarcoma 180 in vivo test (Wang et al., 2001).
Lobelia chinensis Lour (Campanulaceae) is an important anticancer herb used in several
traditional anticancer formulations to treat gastric cancer, lung cancer, colorectal cancer and
liver cancer (Wei et al., 2010; Li et al., 2016). Studies have revealed that L.chinensis contains several important classes of bioactive compounds such as piperidine alkaloids, flavonoids,
terpenoids and coumarins(Kuo et al.,2011; Yang et al., 2014).The hot water extracts
(decoction) from L.chinensis have significant immunostimulatory and anticancer activity
against liver cancer (H22) and gastric cancer (BC-38) (Wei et al., 2010; Chen et al., 2014; Liu and Zhang, 2009; Shao and Zhang, 2010). Recently, an immunostimulatory α-glucan was isolated from L. chinesis (Li et al., 2016), and its structure determined. It should be noted that such an activity has not been found in the literature for α-glucans. Also, water soluble polysaccharides isolated in this study from this herb did not contain any α-glucans (Chapter
6).
Amaurodermarugosum has been chosen for this study because this mushroom belongs to the
famous family of Ganodermataceae (Chan et al., 2013). Traditionally, A. rugosum is used as
31 an anticancer, anti-inflammatory anti-epilepsy and anti-diuretic agent (Dai and Yang,
2008;Chan et al., 2015). To the best of our knowledge, there are very limited scientific
investigations on bioactive compounds from this mushroom (Chan et al., 2015). A very
recent study reported that, methanol extracts from A. rugosum displayed significant immuno-
suppressing activity (Chan et al., 2015). Sixteen anticancer herbs selected for this research are summarized in Table 2.2.
32
Table 2.2. Important anti-cancer herbs used by Chinese medical practitioners and in clinical studies
Plant name and number Chinese Name Family names Traditional Uses and Scientific study References Treatment of rheumatism, allergies 1. Akebia quinata (Houtt.) Decne. Ba yuezha Lardizabalaceae (Kang et al., 2010) diabetics, and anticancer (Samarghandian et al., 2. Alpinae officinarum Hance Gao liangjiang Zingiberaceae Anticancer and anti-allergic 2014) 3. Artemisia annua L Qing gao Asteraceae treat malaria and cancer (Ferreira et al., 2010) 4. Artemisia scopariaWaldst. & Kit. Yinchanhao Asteraceae Antioxidant, treat malaria and cancer (Cha et al., 2005) Anticancer; Inhibition growth of HL-60 5. Artemisia vulgaris L Ai ye Asteraceae leukemic cell line by mitochondria- (Saleh et al., 2014) dependent apoptosis Anticancer; Inhibition growth of Human 6. Citrus reticulataBlanco Ju ye Rutaceae (Xiu et al., 2015) Gastric Cancer Cells SNU-668 Anti-tumour; Inhibtion growth of lung 7. Curcuma aromaticaSalisb Yu jin Zingiberaceae (Ma et al., 2016) carcinoma cells 8. Cynanchum paniculatum L Xu changqing Apocynaceae Anticancer (Kim et el., 2013) antidiabetic, anti-obese, anti-platelet, 9. Cyperus rotundusL Xiang fu Cyperaceae anti-allergic, anti-inflammatory, and (Pirzada et al., 2015) anticancer Antioxidant activity, Anti-mutagenic 10. Lobelia chinensisLour Ban bianlian Campanulaceae activity and Anti-microbial activity and (Li et al., 2016) anticancer, Anticancer; inhibition growth of human 11. Polygonum cuspidatumSieb Hu zhang Polygonacae (Lee et al., 2015) skin melanoma cells Anticancer; apoptosis in human laryngeal 12. Rabdosia rubescens (Hamst.)Wuet. Dong ling cao Labiatae (Kang et al., 2015) cancer cells Antitumour, anti-inflammatory, 13. Rheum palmatum L Da huang Polygonaceae (You et al., 2013) antimicrobial and hemostatic properties 14. Spatholobus suberectus Dunn. Ji xieteng Leguminosae Anticancer (Wang et al., 2011) Anticancer; anti-inflammatory responses via the inhibition of nuclear factor-κB 15. Xanthium sibiricum L Chang ErZi Asteraceae (NF-κB) and signal transducer and (Ju et al., 2015) activator of transcription 3 (STAT3) in murine macrophages 16. Amauroderma rugosum(Blume & T. Jiazhi Ganodermataceae Anticancer (Chan et al., 2015) Nees)
33 2.8. Aims and objectives
The aim of this project was to investigate the antioxidant, immunomodulatory and anti-cancer
activities of polysaccharides from several medicinal plants and mushrooms, and to undertake
structural characterisation of the active polysaccharides in order to establish their structure-
function relationship. Initial screening involved evaluation of crude polysaccharides from 16
Chinese medicinal plants for their biological activities with the aim to select 3
herbs/mushrooms for further systematic studies. The selection was alsobased on their
traditional application in addition to the scientific investigation carried out in this project.
The extracts of herbs/mushrooms identified in the initial screening (Chapter 3) are
further, purified, screened for bioactivities and structures of the molecules determined.
Discovery of novel polysaccharides with anti-cancer therapeutic properties, which is the major objective of this project, is expected to lead the way towards new clinical trials in the
field of oncology. Concerted and collaborative scientific/medical advancements in these
fields of research can lead ultimately to more options for cancer treatment.An overview of the research undertaken in this project is summarised in the flow chart given below.
34 Selecting16 medicinal plants/mushroom Ethanol treated supernatant to isolate organic molecules
Hot water extraction Crude polysaccharides (PSs) Screening for bioactivities
(Chapter 3)
Selecting 3 most active plants based on initial screening
Separation and purification of PSs employing chromatographic methods
Conducting bioactivity tests on purified compounds
Structural characterisation using GC, FT-IR and NMR spectroscopy
Fig. 2.3. Flow chart showing project summary
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49 Chapter Three Evaluation of Immunomodulatory and anticancer potentials of sixteen traditional anticancer herbs _ selection of best herbs for further study Abstract
Plant-derived polysaccharides are known to possess significant antioxidant,
immunomodulatory and anticancer activities. In this research, six polysaccharides
formulations were designed and their biological activities studied. The antioxidant activities
were examined using 1,1-diphenyl-2-picrylhydrazyl (DPPH●) scavenging, 2,2'-azino-bis(3-
ethylbenzothiazoline-6-sulphonic acid) (ABTS●+) scavenging and OH● scavenging assays.
The immunomodulatory properties of the polysaccharides were determined by evaluating
their capacity to activate mouse macrophages (RAW 264.7) to produce the cytokines
interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α). The anticancer activities of the polysaccharides were determined against five human cancer cell lines. Cell viabilities were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in order to assess the toxicities of these polysaccharides. The total sugar content and monosaccharide composition of the polysaccharide extracts were determined with a view to correlate their bioactivities with chemical constituents. The polysaccharides isolated from
Artemisia annua L, Lobelia chinensis Lour, Amauroderma rugosum (Blume & T. Nees),
Artemisia scoparia Waldst. & Kit,Artemisia vulgaris L, Curcuma aromatica Salisb,
Rheum palmatum L and Cyperus rotundus Blanco show significant anticancer, immunomodulatory and antioxidant activities with low toxicity. The results suggest that the polysaccharides extracted from some of the selected TCM herbs have strong therapeutic potential for the treatment of cancer. These herbs include A. annua, L. chinensis, A. rugosum and C. rotundus. An analysis of the results indicate that the mannose, glucose and galactose contents of these polysaccharides correlate well with their immunomodulatory and anticancer activities.
50
3.1. Introduction
Traditional Chinese Medicinal (TCM) plants have thousands of years of history of successfully treating various life-threatening diseases including cancer (Cho, et al., 2010,
Ravipati et al., 2012, Ravipati et al., 2013, Lee et al., 2005, Zhang et al., 2012, Zhang et al.,
2013, Jeong et al., 2016, Palaniyandi, et al., 2016). Numerous scientific studies involving isolation of bioactive compounds from TCM plants exist in the literature (Ravipati et al.,
2013, Zhang et al., 2012; Zhang et al., 2013, Jeong et al., 2016; Palaniyandi, et al., 2016,
Huang et al., 2008; Ravipati et al., 2012). Such studies have led to the discovery of several important chemotherapeutic compounds and many of these agents are in an advanced stage of clinical usage/trials (Vetvicka and Vetvickova, 2012, Ooi et al., 1999, Schepetkin and Quinn,
2006, Butler et al., 2014, Shah et al., 2013, Ghorbani and Hosseini, 2015, Friedman, 2016 and Sugiyama, 2016). In this context plant/mushroom polysaccharides are of great interest in order to discover novel therapeutic agents with minimal side effects (Schepetkin and Quinn,
2006, Jeong et al., 2004, Jeong et al., 2010, Jeong et al., 2012; Zhang et al., 2014). During the past several years, botanical polysaccharides with immunomodulatory and anti-proliferative properties have been of enormous interest for the discovery of chemotherapeutic agents and significant progress has been achieved in this field (Vetvicka and Vetvickova, 2012,
Schepetkin and Quinn, 2006, Friedman, 2016, Sugiyama, 2016, Zhang et al., 2014). Anti- tumour activities of mushroom polysaccharides are well known, for example lentinan derived from Lentinula edodes (Sugiyama, 2016, Chihara, 1970, Chihara et al., 1987, Hamuro et al.,
1980, Taguchi, 1986, Daba et al., 2003, Ina et al., 2013). Polysaccharide Krestin (PSK) derived from Coriolus versicolor (Friedman, 2016; Sugiyama, 2016; Hattori et al., 2004,
Torisu et al., 1990), Polysaccharopeptide (PSP) isolated from Coriolus versicolor (Ng, 1998,
Cui and Chisti, 2003, Cheng and Leung, 2008), and schizophyllan from Schizophyllum commune (Sugiyama, 2016, Daba et al., 2003) are important anticancer agents.
51 All of these mushroom polysaccharides have been used as anticancer agents for many years
in Japan following excellent results in several clinical trials and government approval have
been given for their use (Friedman, 2016, Sugiyama, 2016, Chihara, 1970, Chihara et al.,
1987, Hamuro et al., 1980, Taguchi, 1986, Cheng and Leung, 2008, Cui and Chisti, 2003 and
Daba et al., 2003). Recent scientific investigations (Han et al., 2016 and Ayeka et al., 2016)
reveal that the polysaccharides isolated from Glycyrrhiza uralensis Fisch and Undaria
pinnatifida (Harvey) Suringar showed significant inhibition of cancer cell growth invivo (Han
et al., 2016) and invitro (Ayeka et al., 2016). Much more important advancements are
possible in this area in the near future with the availability of modern state of the art
techniques for characterisation of bioactive polysaccharides.
A program of research has been initiated in our laboratory for the discovery of novel
anticancer polysaccharides from TCM herbs. In this investigation, sixteen traditional
anticancer herbs have been carefully selected and studied with a view to identify a few
potential TCM plants/mushroom for further detailed study to isolate, screen and characterise
anticancer polysaccharides. The selected traditional anticancer plants/mushrooms used in this
study are given in Table 2.2. The Table also provides information on their traditional use and
published scientific literature on their immunomodulatory, anticancer and other ethno-
pharmacological properties (Cho et al., 2010, Vetvicka and Vetvickova, 2012, Wei et al.,
2010, Zhou et al., 2007, Kim et al., 2005, Xiu et al., 2015, Pirzada et al., 2015, Ma et al.,
2015, Kim et al., 2013, Li et al., 2016, Kang et al., 2010, You et al., 2013, Wang et al., 2011 and Ju et al., 2015). It should be noted that there is very limited literature available on polysaccharides from these sixteen herbs/mushrooms. To the best of our knowledge there are only a few scientific studies on polysaccharides isolated from two of these sixteen herbs (Li et al., 2016 and Wang et al., 2001). Immunomodulatory α-glucan has been isolated from L.
52 chinesis (Li et al., 2016), and its structure determined. The rest of the scientific studies on these plants/herbs involve the organic extracts (of small organic molecules) and their biological activities. A brief review of these studies is provided here. Akebia quinata is one of the important medicinal herbs used in traditional anticancer formulations (Zhao et al.,
2007 and Kang et al., 2010). Scientific studies have indicated that bioactive constituents isolated from A. quinata display significant anticancer properties and anti-inflammatory
activity (Kang et al., 2010 and Jung et al., 2004). Alpinia officinarum is another important
anticancer herb used in traditional formulations (Wei et al., 2010 and Samarghandian et al.,
2014), and which is also used externally for skin infection and skin cancer (Yasukawa et al.,
2008). Literature reports that the ethanol extracts of A. officinarum possesses significant
antitumor activity (Samarghandian et al., 2014). A. annua is yet another important TCM herb
used to treat cancer (Huang et al., 2008). Literature indicates that artemisinin isolated from
Artemisia annua showed significant anticancer activities invitro and invivo (Ferreira et al.,
2010). Lobelia chinensis Lour. (ban bian lian) has been widely used in anticancer formulations, as a venom antidote, and to treat liver cirrhosis and haemostat in TCM
(Shibano et al., 2001 and Yong and Lei, 2006). Literature reports that the extracts of L. chinesis display significant anticancer properties (Li et al., 2016, Shao and Zhang, 2010).
Amauroderma rugosum (Blume & T. Nees) has been traditionally used as anticancer, anti- inflammatory and anti-diuretic agents (Dai and Yang, 2008 and Chan et al., 2015). A recent study on the ethanol extracts of A. rugosum showed significant immunomodulatory activities
(Chan et al., 2015). Scientific investigations carried out on the bioactive components extracted from Artemisia scoparia, Artemisia vulgaris, Citrus reticulate, Cynanchum paniculatum, Cyperus rotundus, polygonum cuspidatum, Rabdosia rubescens,
Rheum palmatum, Spatholobus suberectus, Curcuma aromatica Salisb and Xanthium sibiricum revealed significant anticancer properties (Kim et al., 2005, Xiu et al., 2015,
53 Pirzada et al., 2015, Ma et al., 2015, Kim et al., 2013, Kang et al., 2015, You et al., 2013,
Wang et al., 2011, Ju et al., 2015, Cha et al., 2005, Saleh et al., 2014, Ma et al., 2016 and Lee et al., 2015). These studies and the associated traditional knowledge demonstrate great potential of TCM herbs for the discovery of anticancer agents.
In this chapter, polysaccharides have been extracted from the selected sixteen herbs and their
biological activities evaluated. It is well known that botanical polysaccharides displaying
significant antioxidant and immunomodulatory effects are likely to exhibit anticancer
properties (Schepetkin and Quinn, 2006 and Zhang et al., 2014). Therefore this chapter aims
to evaluate antioxidant, immunomodulatory and anticancer properties of the polysaccharides
isolated from these selected herbs.
In order to evaluate antioxidant activities, a few rapid and efficient screening methods have
been employed. These include diphenylpicrylhydrazyl radical (DPPH●) scavenging, 2,2'-
azino-bis(3-ethylbenzothiazoline-6-sulphonic acidradical (ABTS●+) scavenging, iron (Fe2+)
chelating and Fe3+ reducing assays (Lee et al., 2015, Alam et al., 2013, Rajendran et al.,
2016, Li et al., 2011, Umamaheswari and Chatterjee, 2008, Halliwell et al., 1987, Chen et al.,
2005 and Wang et al., 2008). The immunomodulatory properties of the polysaccharides were
determined by evaluating their capacity to activate mouse macrophages (RAW 264.7) to
generate interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) (Zhang et al., 2012 and
Zhang et al., 2013). Cell viabilities were evaluated by MTT test to assess the toxicities of these polysaccharides (Thambiraj et al., 2015). Anticancer activities of polysaccharide extracts were measured against five different cancer cell lines, which include HT29 (colon
carcinoma), MCF7 (breast carcinoma), A549 (lung carcinoma), HepG2
(Hepatocites carcinoma), MiaPAca2 (pancreatic cancer), using MTT assay. This study also
54 aims to evaluate the monosaccharide content of these polysaccharides with a view to
understanding the correlation between their activities and mono-sugar content.
3.2. Materials and Methods
3.2.1. Procurement of medicinal plants associated with this research
The herbal plant materials have been purchased from a Chinese herbal medical centre known
as Bei Jing Tong Ren Tang located in Sydney (Australia). Sample specimens of all the herbs
are stored in our research laboratory. This company has branches all over the world and
is well known for their best practice in TCM. The herbs traded in the Sydney centre
have approvals from both Australian and Chinese governments. The company undertakes
stringent authentication and quality control procedures for all the herbal materials supplied by
them. The details of these selected herbs are presented in Table 2.2. All herbal samples
were powdered and subjected to an extraction procedure.
3.2.2. Chemicals and reagents/enzymes 3.2.2.1. Chemicals
The DPPH●, ABTS●+, dimethyl sulfoxide (DMSO), Folin–Ciocalteu reagent (F-C reagent),
sodium carbonate, 95% ethanol, ascorbic acid, Trypan blue 0.4%, tetra methyl benzidine,
sulfanilamide, N-(1-1-napthyl) ethylenediamine dihydrochloride, lipopolysaccharide (LPS) were purchased from Sigma (Australia) and Lomb Scientific Pty Ltd (Australia). The Foetal bovine serum (FBS), antibiotics, and Dulbecco’s modified Eagle’s medium (DMEM) with
gluMax were purchased from BD bioscience. The tumour necrosis factor-α (TNF-α) and
interleukin (IL-6) (mouse) ELISA standards and antibodies were purchased from BD
Bioscience (USA).
55 3.2.2.2. Reagents/enzymes
Mouse macrophage cells (RAW 264.7) have been used in this study to test for immunostimulatory activities of polysaccharides from these traditional anticancer herbs and mushroom. Lipopolysaccharide (LPS) is used as positive control. The capacity of polysaccharides to produce IL-6 and TNF-α was measured by using suitable ELISA kits.
DPPH● and ABTS●+ radical scavenging assays, iron (Fe2+) chelating and ferric ion reducing power assay were used for the measurement of antioxidant activity. Anticancer activities of polysaccharides are determined using five different tumour cell lines, which are MCF7
(ATCC HTB-22 Breast carcinoma) HT29 (ATCC HTB-38 Colon carcinoma), A549 (ATCC
CCL-185 lung carcinoma), HepG2 (ATCC HB-8065 hepatocytes carcinoma), and MiaPAca2
(ATCC CRL-1420 pancreatic cancer).
3.2.3. Extraction of crude polysaccharides from medicinal plants
30 g of dried medicinal plant were ground to powder form and mixed well. The powdered material was subjected to hot water extraction using an autoclave method (at 121oC for 2 hours) and then cooled to laboratory temperature and the supernatant separated by filtration.
The supernatant (extract) was then treated with 95% ethanol (extract:ethanol = 1:4 volume ratio) for 24 hours at 4.1oC. The resulting precipitate consisting of biopolymers
(polysaccharides and proteins) was centrifuged (10,000 rpm for about 20 minutes) to obtain the sample in the form of a pellet which was re-dissolved in deionised water. The solution was subjected to filtration using 0.45 µm Whatman filter paper. The solution was then freeze dried (Zhang et al., 2012, Zhang et al., 2013, Jeong et al., 2004, Jeong et al., 2010, Jeong et al., 2012 and Thambiraj et al., 2015). The entire extraction process is summarised in Fig. 3.1.
The freeze dried polysaccharide extracts were re-dissolved in deionised water (10 mg/mL),
56 and mixed with 1/4 volume mixture of the Sevag reagent (n-butanol:chloroform = 1:4 volume ratio) for removing the free protein (Zhang et al., 2014). De-proteinated polysaccharide extracts were stored at -20oC until use.
57 Powdered plant/mushroom
material • Hot water extraction (autoclaving) (121 for 2 h)
• Centrifugation (10,000 rpm for 20 min)℃
Marc Water Supernatant
• Precipitation using 95% EtOH(volume : volume =1:4 for 24 h) • Centrifuge (10,000 rpm for 20 min) • Freeze dry
Crude biopolymer
Crude biopolymer dissolved in water
• Sevag reagent • Votex 20 min • Contrifuge (5000 rpm for 15 min)
Precipitation using 95% EtOH (Volume:Volume = 1:4)
• Centrifuge (10,000 rpm for 20 min)
• Freeze dry
Crude polysaccharide
Fig.3.1. Flow chart for the extraction of polysaccharides from selected herbs
58 3.2.4. Analysis of chemical composition
The method, phenol-sulfuric acid method developed originally by Dubios et al., (Dubios et
al., 1956) was employed to measure the total sugar content. In this method, the concentrated
sulfuric acid breaks down any polysaccharides, oligosaccharides, and disaccharides to
monosaccharides. Pentoses (5-carbon compounds) are then dehydrated to furfural, and
hexoses (6-carbon compounds) to hydroxymethyl furfural. These compounds then reacted
with phenol to produce a yellow-gold colour. Glucose was used to construct a standard
curve: y = 0.0018x + 0.0374 (R2 = 0.9964). The method of Lowry et al., (Lowry et al., 1951)
was employed to measure the total bound protein in the polysaccharide samples.
Gas chromatographic analysis was performed to measure the mono-sugar content of the polysaccharides. Analysis was performed using a Hewlett Packard 7890B gas chromatograph with a flame ionisation detector (FID) detector and a capillary medium polarity column
(HP-5 column). The method developed by Jones and Albersheim (Jones and Albersheim
1972 and Zhang et al., 2014) was used to prepare the polysaccharide samples and for GC analysis. Fucose, arbinose, ribose, xylose, rhamnose, fructose, galactose and glucose sugars were used as standards.
Instrumentation and analytical conditions for the GC-FID system
GC chromatograph Hewlett Packard 7890
HP-5 capillary column (30m long, 0.32mm i.d., 0.25µm film thickness; SGE Analytical Science Pty Ltd., Ringwood, VIC, Australia)
GC Inlet 240°C, split ratio 90:10
Carrier gas Nitrogen
Oven temperature 90 °C (3 min), 4 °C/min to 240°C(5 min)
Detector FID
Injection volume 5µL
59 3.2.5. Bio-Assays
3.2.5.1. Antioxidant activities
3.2.5.1.1. Scavenging activity against DPPH● radicals
The Blois method (Blois, 1958) was employed to determine the DPPH● scavenging ability of
the polysaccharide samples. DPPH● solution was prepared using the procedure outlined in a
previous publication (Zhang et al., 2012, Zhang et al., 2013 and Alam et al., 2013). 150 μL of
DPPH● solution (62.5 μM) was added to 50 μL of polysaccharide sample and incubated for
about 30 minutes in a 96 well microtiter plate. The absorbance values of the incubated
samples were then determined using a UV spectrophotometer at 492 nm (Multiskan 141 EX,
Thermo Electron, USA). Ascorbic acid (a known standard) was employed as positive control
and deionised water was used as blank. A standard curve was built using different
concentrations of ascorbic acid solutions (in 60% methanol) in the range of 0 to 200 µM. The
regression of the standard curve gave a linear equation (y = -0.0026x + 0.5578 with R² =
0.9715). DPPH● scavenging potential of the polysaccharides was determined as the ascorbic acid equivalence using the above equation.
3.2.5.1.2. ABTS●+radical scavenging assay
A stock solution of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) was
prepared at a concentration of 7 mM using PBS buffer (pH 7.4). ABTS stock solution was
mixed with potassium persulfate (2.45 mM) to initiate the formation of radical cations
(ABTS●+). The mixture was then kept in the dark overnight to make sure that the radical
formation iscomplete (Li et al., 2011 and Alam et al., 2013). Absorbance of the ABTS●+
radical solution was adjusted to about 0.74 using PBS buffer (pH 7.4) to dilute the solution.
200 μL of ABTS●+ solution was added to 20μL of polysaccharide sample and incubated for
about 30 minutes in a 96 well microtiter plate. The absorbance value of the incubated
60 samples was then determined using a UV spectrophotometer at 734 nm (Multiskan 141 EX,
Thermo Electron, USA). Ascorbic acid was employed as positive control and PBS buffer (pH
7.4) was used as blank. A standard curve was built using different concentrations of ascorbic acid solutions (in 60% methanol) in the range of 0 to 400 µM. The regression of the standard curve gave a linear equation (y = -0.0019x + 0.7274 with R² = 0.9852). The radical scavenging capacities of the polysaccharides were determined as the ascorbic acid equivalence using the above equation.
3.2.5.1.3. Ions (Fe2+) chelating assay
The affinity of polysaccharides to complex with Fe2+ were determined by measuring the
absorbance of the complex formed in the presence of ferrozine (Jeong et al., 2016 and Li et al., 2011). First, 0.1mL of polysaccharide sample was mixed with 0.5 mL of FeCl2 (0.2 mM) to form the Fe-polysaccharide complex. To this complex, 0.2mL of ferrozine (5 mM) was added and thoroughly mixed to trigger the competition between polysaccharide and ferrozine for Fe2+. The mixture was incubated for about 10 min. The absorbance of the red coloured
ferrozine-Fe2+ complex was determined using a UV spectrophotometer at 562 nm (Genesys
10S UV-Vis spectrophotometer, Thermo Fisher Scientific, Australia) (Li et al., 2011).
Ethylenediaminetetraacetic acid (EDTA) was employed as positive control and deionised
water was used as blank. A standard curve was built using different concentrations of EDTA
solution in the range of 0 to 855 µM. The regression of the standard curve gave a linear
equation (y = -0.002x + 1.7779 with R² = 0.9726). The chelating activities of the
polysaccharide samples were calculated using the above equation.
3.2.5.1.4. Ferric ions (Fe3+) reducing antioxidant power
Polysaccharide samples were prepared at different concentrations in the range of 0-1000
µg/mL. 100µL of the sample was added with phosphate buffer (250 μL, 0.2 mol/L, pH 6.6)
61 and then mixed with K3Fe(CN)6 (250μL, 1% w/v). The solution was vortexed and incubated
at about 50 ºC for 25 min. 250μL of 10% trichloroacetic acid (w/v) was then added to the
incubated samples and the supernatant collected by centrifuging at 3500 rpm for about 10
min. The supernatant was then added with equivalent volumes of distilled water and FeCl3
(0.1% w/v) and placed immediately into a spectrophotometer to measure the absorbance
values 700 nm. The samples were analysed in groups of three, and when the analysis of one
group has finished, the next group of three samples were mixed with FeCl3 to avoid oxidation by air. Reducing power of ascorbic acid (standard) was also measured for comparison purposes (Lee et al., 2015, Alam et al., 2013, Wang et al., 2008).
3.2.5.2. Immunomodulatory activity assays
3.2.5.2.1. Production of IL-6
Mouse macrophages (RAW 264.7) are first added to DMEM (culture medium containing 1%
antibiotic and 5% FBS) and incubated for 4 days at 37°C in 5% CO2 in air. Cells were then
diluted with the medium to achieve a density of 2x105 cells/mL. 200µL of diluted cells were
transferred into a 96-well microtiter plate and then for a further 20 hours at 37 °C in 5% CO2
in air. The old medium was then removed from all the wells and 180 µL of new medium added to each well containingthe incubated cells. Immediately following this, to each well
was added either 20µL of polysaccharide samples at different concentrations (0-1 mg/mL:
final concentration) or 20 µL of LPS (100 ng/mL: final concentration). Then the microtiter plate was incubated overnight at 37°C in 5% CO2 (Jeong et al., 2004, Jeong et al., 2010,
Jeong et al., 2012 and Ni et al., 2016). The supernatant (100 µL) from each well was then
carefully transferred into a new multi-well plate. ELISA kit (IL-6, BD Biosciences, San Jose,
CA, USA) was then used to measure the concentration of IL-6 as per the procedure provided
in the manufacturer’s manual. Triplicate measurements were made .
62 The regression of the standard curve gave a linear equation (y = 0.0018x + 0.0294with R² =
0.9887). The concentration of IL-6 produced by the polysaccharide extracts were calculated using the above equation.
3.2.5.2.2. Production of TNF- α
The initial cell macrophage culturing and sample preparation were carried out as described in section 3.2.5.2.1 above. The supernatant (100 µL) from each well was carefully transferred
into a new multi-well plate. ELISA kit (TNF-α, BD Biosciences, San Jose, CA, USA) was
then used to measure the concentration of TNF-α as per the procedure provided in the
manufacturer’s manual (Zhang et al., 2012 and Zhang et al., 2013). Triplicate measurements
were made .
The regression of the standard curve gave a linear equation (y = 0.0015x + 0.0734 with R² =
0.9879). The concentration of TNF-α produced by the polysaccharide extracts were calculated
using the above equation.
3.2.5.2.3. Toxicity test
Viability of macrophage cells (RAW 264.7) were measured employing 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Thambiraj et al., 2015).
Briefly, mouse macrophages were treated by herbal polysaccharides, and incubated at 37 °C for 18 hours. After that, the supernatant was removed and 100 µL of MTT solution (0.2 mg/mL, dissolved in DMEM medium) added to each well and incubated at 37 oC for 4 hours.
The supernatant was then discarded and 50 µL DMSO was added to each well to solubilise
the crystalline formazan. The absorbance was measured at 595 nm.
(%) = 100%
𝑂𝑂𝑂𝑂 𝑜𝑜𝑜𝑜 𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣 𝑋𝑋 Where, positive control is mouse macrophages𝑂𝑂𝑂𝑂 𝑜𝑜𝑜𝑜treated𝑝𝑝𝑝𝑝𝑝𝑝 𝑐𝑐𝑜𝑜with𝑛𝑛𝑛𝑛𝑛𝑛 𝑛𝑛𝑛𝑛 DMEM medium (without LPS)
63 3.2.5.3. In vitro anticancer assays against various cancer cell lines
The cancer cell lines were cultured and incubated according to the procedure outlined in
previous publications (Royo et al., 2003, Tormo et al., 2005). All the cancer cell lines studied
in this research were purchased from the American Type Culture Collection (ATCC,
Manassas, VA, USA). 1) HT29 cells (human colon carcinoma HTB-38), were cultured in
McCoy`s 5A medium modified 10 % FBS and 2 mM glutamine. 2) A549 monolayer cells
(human lung carcinoma, CCL-185) were grown in Ham`s F12 medium containing 10% FBS,
2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. 3) MiaPAca2 cells
(pancreatic cancer CRL-1420) were cultured in ATCC-formulated DMEM with 10 %
qualified FBS. 4) Hep_G2 cells (human liver carcinoma, CCL-8065) were grown in ATCC-
formulated Eagle´s M essential medium (MEM) with 10 % qualified FBS, 2 mM L-
glutamine, 1 mM sodium pyruvate, and 100 µM MEM non-essential amino acids. 5) MCF7
cells (human breast HTB-22) were preserved in MEM containing with 0.01 mg/mL bovine
insulin. All cell cultures were incubated at 37 ºC under 5% CO2.
Five cancer cell lines were treated by herbal polysaccharides, and kept at 37 °C overnight.
After that, the supernatant was removed and treated with 100 µL MTT solution (0.2 mg/mL,
dissolved in DMEM medium) added to each well and kept at 37 oC for 4 hours. The supernatant was discarded and 50µL DMSO added to each well to dissolve the crystalline formazan. The rate of decrease of MTT is a measure of cytotoxicity. The cell density per well was adjusted to 200,000 for MCF7, 200,000 for A549, 100,000 for MiaPAca2, 150,000 for
HepG2, and 150,000 for HT29 by diluting with medium. Polysaccharide samples (3-50
µg/mL) were then added to each well and kept at 37 ºC under 5% CO2 for 24 hours. Optical
density was then determined at 570 nm using a spectrofluorometer.
64 % = 100 sample pos contr OD − OD 𝑖𝑖𝑖𝑖ℎ𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 𝑁𝑁𝑁𝑁𝑁𝑁 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑝𝑝𝑝𝑝𝑝𝑝 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑋𝑋 Where, ODNeg Contr represents the optical𝑂𝑂 𝐷𝐷density of− the 𝑂𝑂𝐷𝐷 negative control
ODPos Contr represents the optical density of the positive control
The culture medium containing DMSO (1%) was used negative control and the medium
with 2mM MMS was used as positive control.
3.2.6. Statistical analysis
All data were measured in triplicate and the mean ± SD calculated. The group mean was
compared using a one-way analysis of variance (ANOVA) and Duncan’s multiple range tests.
Statistical calculations were done using IBM SPSS, OriginPro 8.5 and Excel 2016. The
data were considered as statistically significant if p < 0.05.
3.3. Results and Discussion
3.3.1. Chemical composition of polysaccharides
The total sugar content of polysaccharides extracted from the selected herbs was measured
using phenol-sulfuric acid method (Dubois et al., 1956) and the results are given in Table 3.2.
It is clear that most polysaccharides contained a significant quantity of total sugar content
(>50%) except for R. rubescens. Table 3.1 also shows the monosaccharide composition of all these polysaccharides (monosaccharide standards used included: rhamnose, frucose, ribose,
arabinose, xylose, fructose, galactose and glucose). It is interesting to note that glucose is the
main constituent in all polysaccharides except in C. aromatica which contained 98.7%
galactose. These other monosaccharide (glucose, mannose and arabinose) concentrations are
appreciable. Ribose is absent in all polysaccharides.
65 Table 3.1. The Chemical composition and monosaccharide content of crude polysaccharides extract from dried plant material
Total Plant Rhamnose Ribose Fucose Arabinose Xylose Mannose Glucose Galactose Name of the plants carbohydrate No content (%)* (%) (%) (%) (%) (%) (%) (%) (%) 1 Akebia quinata (Houtt.) Decne. 86 ± 6 1.21 0.89 97.36 0.54 2 Alpinae officinarum Hance 90 ± 4 1.29 1.25 0.74 95.84 0.88 3 Artemisia annua L 53 ± 2 5.32 24.24 4.24 9.88 35.79 20.53 4 Artemisia scoparia Waldst. & Kit. 79 ± 1 13 16.75 13.08 12.1 19.05 26.02 5 Artemisia vulgaris L 53 ± 1 6.68 14.64 6.00 7.62 47.05 18.01 6 Citrus reticulata 77 ± 5 2.23 9.82 3.5 77.46 6.99 7 Curcuma aromatica Salisb 99 ± 3 1.26 98.74 8 Cynanchum paniculatum L 83 ± 2 2.62 7.4 1.35 2.83 76.96 8.84 9 Cyperus rotundus L 95 ± 7 3.8 18.03 2.29 62.34 13.54 10 Lobelia chinensis Lour 94 ± 1 3.54 10.01 1.45 11.04 65.09 8.84 11 Polygonum cuspidatum Sieb. et Zucc 76 ± 1 4.72 2.06 86.33 6.89 12 Rabdosia rubescens (Hamst.)Wuet. 37 ± 6 4.34 19.29 3.27 2.14 60.02 10.94 13 Rheum palmatum L 92 ± 4 2.64 12.02 75.62 9.72 14 Spatholobus suberectus Dunn. 66 ± 1 9.12 24.1 40.03 26.75 15 Xanthium sibiricum L 79 ± 4 8.89 36.00 10.63 24.91 19.57 16 Amauroderma rugosum 83 ± 4 2.96 18.03 2.68 5.73 45.93 24.67 * These polysaccharides have combined protein. The numbers represent percentage carbohydrate content and the remaining portion is protein content
66 3.3.2. Antioxidant activities of selected herbal polysaccharides
3.3.2.1. Scavenging abilities against DPPH● and ABTS●+radicals
DPPH● and ABTS●+scavenging abilities of the polysaccharides are provided in Table 3.2. It is
clear from the results that the majority of the polysaccharides effectively scavenged DPPH●
radicals and these activities ranged from 109 µM to 178 µM ascorbate equivalent/g. Their
ABTS●+scavenging activity ranged from 158 µM to 296 µM ascorbate equiv/g. Highly
effective radical scavenging abilities were displayed by polysaccharides isolated from
Artemisia annua, Artemisia scoparia, Artemisia vulgaris, Rabdosia rubescens, Polygonum cuspidatum,Spatholobus suberectus and Amauroderma rugosum (Blume & T. Nees) (Table
3.2). However, polysaccharides extracted from C. aromatica and C. rotundus did not show
any scavenging activity. This may be due to the fact that these two polysaccharides did not
fully dissolve in water and formed starchy/turbid solutions.
67 Table 3.2. Antioxidant activities of crude polysaccharides extracted from medicinal plants.
#DPPH scavenging activity #ABTS scavenging activity &Chelating activity (EDTA Plant.No Name of the plants (Ascorbate equivalent (µM)) (Ascorbate equivalent (µM)) equivalent (µM))
1 Akebia quinata (Houtt.) Decne. 124.06 ± 1.88 258.38 ± 0.25 395.45 ± 0.01 2 Alpinae officinarum Hance 146.77 ± 1.57 231.86 ± 0.66 306.95 ± 0.02 3 Artemisia annua L 154.48 ± 2.53 274.61 ± 0.43 606.28 ± 1.53 4 Artemisia scoparia Waldst. & Kit. 167.4 ± 1.44 296.78 ± 0.87 597.28 ± 0.29 5 Artemisia vulgaris L 154.27 ± 0.72 256.59 ± 0.18 562.62 ± 0.76 6 Citrus reticulata 147.6 ± 2.19 261.57 ± 0.43 306.95 ± 0.02 * 7 Curcuma aromatica Salisb NA NA 306.95 ± 0.03 8 Cynanchum paniculatum L 110.73 ± 1.05 210.26 ± 1.15 405.95 ± 0.50 9 Cyperus rotundus L NA NA 306.95 ± 0.01 10 Lobelia chinensis Lour 109.9 ± 1.57 158.96 ± 1.15 602.28 ± 0.29 11 Polygonum cuspidatum Sieb. et Zucc 158.85 ± 1.3 276.35 ± 1.57 601.78 ± 1.26 12 Rabdosia rubescens (Hamst.)Wuet. 178.44 ± 1.88 287.07 ± 0.66 446.28 ± 1.61 13 Rheum palmatum L 133.85 ± 2.82 237.80 ± 1.00 414.12 ± 0.29 14 Spatholobus suberectus Dunn. 161.35 ± 2.01 282.87 ± 0.43 391.95 ± 0.50 15 Xanthium sibiricum L 109.69 ± 1.88 197.94 ± 0.66 612.78 ± 0.29 16 Amauroderma rugosum 109.9 ± 3.15 214.46 ± 0.66 615.62 ± 0.76
# DPPH, ABTS free radical scavenging activity was expressed as equivalent of ascorbic acid. & chelating activity was measured with equivalent of EDTA * NA: no activity Values are: mean ± standard deviation (n = 3)
68 Interestingly, the results presented in Tables 3.1 and 3.2 indicate that the most active polysaccharides contain large quantities of galactose and glucose, and average quantities of mannose. This suggests that these monosaccharides may be responsible for the observed radical scavenging activities (Chen et al., 2005 and Chen et al., 2014). Numerous studies have demonstrated that galactose, glucose and mannose indeed contribute to the radical
scavenging abilities of plant based polysaccharides (Zhang et al., 2014, Thambiraj et al., 2015,
Chen et al., 2015, Xu et al., 2013 and Deng et al., 2016). Chen et al., (Chen et al., 2014)
demonstrated that polysaccharides isolated from Elaegnus angustifolia L display significant radical scavenging activity and contained large quantities of galactose, glucose and mannose.
Thambiraj et al., (Thambiraj et al., 2015) showed that the polysaccharides extracted and
purified from Lupinusangustifolius displayed high antioxidant activities and contained large
quantities of glucose, and siginificant quantities of mannose, galactose and rhamnose/frucose.
3.3.2.2. Ions (Fe2+) chelating assay The results for the chelating activity of polysaccharides are presented in Table 3.2. The Fe2+ chelating ability of the polysaccharide extracts were monitored by spectrophotometric method. The absorbance of the red coloured complex formed in the reaction decreases as the concentration of the polysaccharide-Fe(II) complex increases. Among the polysaccharides studied, those from A. annua, A. scoparia, A. vulgaris L. chinensis,P. cuspidatum, X. sibiricum, and A. rugosum showed significant Fe2+ chelating capacity. It is clear from Tables
3.1 and 3.2 that galactose and glucose are most likely candidates for the chelating ability of these polysaccharides (Thambiraj et al., 2015).
Literature reports indicate that the antioxidant capacities of natural products are determined by their combined abilities to scavenge radicals and chelate with Fe2+ (Thambiraj et al., 2015,
69 Raza et al., 2017). Most of the herbal polysaccharides studied in this research displayed
significant radical scavenging abilities as well as Fe2+-chelating potential. Hence, it is
expected that the herbal polysaccharides considered in this study will have high antioxidant
potential.
3.3.2.3. Fe3+ reducing abilities
The results of the Fe3+ reducing abilities of polysaccharides is presented in Table 3.3.The
reducing ability was measured by monitoring Fe3+→Fe2+ conversion by herbal
polysaccharides. The formation of Fe2+ is determined from the absorbance values at 700 nm
(Umamaheswari and Chatterjee, 2008 andMeir et al., 1995) at five different concentrations. It can be seen that most of the polysaccharides exhibited good reducing power. Polysaccharides
from S. suberectus, A. vulgaris, R. rubescens, P. cuspidatumn and A. annua have showed
highly significant reducing ability.
These activities of herbal polysaccharides may be linked to the presence of reducing sugars
such as galactose, glucose and xylose (Table 3.1) (Selvendran and Isherwood, 1967).
It should be noted that all the active polysaccharides studied here contain large quantities
of galactose and glucose.
3.3.3. Immunomodulatory activities of crude polysaccharides
3.3.3.1. Effect of herbal polysaccharides to activate mouse macrophages and produce TNF-α
and IL-6
Strong evidence exists in the literature that the botanical polysaccharides activate the immune
system to produce various cytokines (Schepetkin and Quinn, 2006, Zhang et al., 2014 and Li
70 et al., 2016). Treatment of RAW 264.7 cells with polysaccharides from these medicinal herbs showed concentration-dependent enhancement in the production of TNF-α and IL-6 (Table
3.4 and Fig. 3.2).
71 Table 3.3. Ferric ion reducing power of polysaccharides extracted from medicinal plants
Absorbance at 700 nm Plants/standard 1000 µg/mL 500 µg/mL 250 µg/mL 125 µg/mL 62.5 µg/mL Vit C 1.707±0.096 0.706±0.001 0.331±0.001 0.193±0.001 0.127±0.001 Akebia quinata 0.163±0.022 0.088±0.001 0.048±0.002 0.029±0.001 0.014±0.002 Alpinae officinarum 0.103±0.002 0.049±0.001 0.03±0.001 0.017±0.002 0.006±0.002 Artemisia annua 0.409±0.001 0.228±0.002 0.115±0.001 0.056±0.003 0.037±0.001 Artemisia scoparia 0.302±0.003 0.173±0.002 0.086±0.002 0.047±0.002 0.014±0.001 Artemisia vulgaris 1.035±0.001 0.605±0.002 0.325±0.002 0.179±0.001 0.078±0.001 Citrus reticulata 0.218±0.002 0.115±0.003 0.053±0.003 0.026±0.002 0.014±0.003 Curcuma aromatica NA NA NA NA NA Cynanchum paniculatum 0.257±0.001 0.126±0.002 0.063±0.002 0.039±0.001 0.036±0.047 Cyperus rotundus 0.026±0.003 0.013±0.002 NA NA NA Lobelia chinensis 0.067±0.002 0.049±0.001 0.026±0.002 0.015±0.003 0.009±0.001 Polygonum cuspidatum 0.467±0.002 0.215±0.001 0.086±0.001 0.035±0.001 0.014±0.002 Rabdosia rubescens 0.693±0.002 0.385±0.001 0.217±0.001 0.114±0.003 0.053±0.006 Rheum palmatum 0.169±0.001 0.087±0.001 0.045±0.001 0.023±0.002 0.014±0.002 Spatholobus suberectus 1.581±0.172 0.952±0.001 0.523±0.001 0.267±0.001 0.112±0.001 Xanthium sibiricum 0.064±0.001 0.044±0.001 0.019±0.001 0.009±0.001 0.007±0.001 Amauroderma rugosum 0.176±0.002 0.098±0.001 0.045±0.001 0.026±0.002 0.013±0.002 * NA: no activity # Values given in this table are the absorbance values measured at 700 nm. The values are: mean ± STD (n = 3)
72 The immunomodulatory activities were measured at different concentrations 0 to 1 mg/mL and expressed in term of EC50 values (Table 3.4). Toxicities for various polysaccharides are also given in Table 3.5. The results indicat e that most of the isolated polysaccharides from the 16 herbs display significant immuno-stimulatory activity by increasing the production of
TNF-α and IL-6. A. annua , A. rugosum, L. chinensis, C. reticulata and S. suberectus showed
high immunostimulatory activities as evidenced by the production of TNF-α and IL-6 (EC50 values less 120 µg/m) L . Polysaccharides from the remaining herbs have also displayed
significant immunostimulatory activities with EC50 values in the range of 130 µg/mL to 300
µg/mL.
Concentration dependant immunostimulatory activities of most active herbs are shown in Fig.
3.2. Polysaccharides from L. chinensis and S. suberectus display extremely high activity as indicated by TNF-α production. The other two herbs A. rugosum and A. annua have also
displayed significant activity. With respect to the production of IL-6, the polysaccharides
from A. rugosum and A. annua are the best. L. chinensis and S. suberectus have also induced
the production of significant quantities of IL-6.
73 Table 3.4. Immunomodulatory activities of polysaccharides extracted from selected medicinal herbs
EC for the IL-6 50 EC for TNF-a production Cell viability (% 50 Cell viability (% Plant.No Name of the plants # production # (µg/mL)* of cell survival) of cell survival) (µg/mL)*
1 Akebia quinata (Houtt.) Decne. 266.59 ± 4.00 99.17 ± 3.55 324.6 ± 3.37 107.97 ± 0.50 2 Alpinae officinarum Hance 322.78 ± 4.78 94.8 ± 6.82 334.94 ± 4.59 105.17 ± 1.53 3 Artemisia annua L 107.78 ± 4.94 90.90 ± 6.20 107.35 ± 5.26 74.97 ± 2.52 4 Artemisia scoparia Waldst. & Kit. 337.82 ± 1.40 96.07 ± 7.83 347.75 ± 3.10 94.67 ± 3.51 5 Artemisia vulgaris L 159.95 ± 2.00 97.77 ± 1.17 203.82 ± 4.50 86.67 ± 4.49 6 Citrus reticulata 94.23 ± 3.40 97.87 ± 5.04 79.56 ± 4.52 95.07 ± 4.24 7 Curcuma aromatica Salisb 337.37 ± 1.46 86.5 ± 5.63 308.14 ± 4.52 68.50 ± 0.50 8 Cynanchum paniculatum L 160.37 ± 4.52 98.53 ± 6.69 104.18 ± 5.48 76.2 ± 2.55 9 Cyperus rotundus L 113.52 ± 5.31 94.87 ± 4.76 193.09 ± 2.29 65.73 ± 2.97 10 Lobelia chinensis Lour 93.23 ± 4.22 93.7 ± 3.94 61.74 ± 1.19 87.33 ± 2.08 11 Polygonum cuspidatum Sieb. et Zucc 62.17 ± 2.99 93.23 ± 10.51 174.91 ± 2.89 76.53 ± 5.71 12 Rabdosia rubescens (Hamst.)Wuet. 233.79 ± 5.22 93.5 ± 7.13 130.93 ± 3.9 78.13 ± 4.92 13 Rheum palmatum L 175.13 ± 5.05 88.43 ± 0.45 118.24 ± 5.56 96.5 ± 5.07 14 Spatholobus suberectus Dunn. 59.28 ± 0.51 98 ± 14.29 62.36 ± 2.3 75.3 ± 5.98 15 Xanthium sibiricum L 113.52 ± 5.31 91.5 ± 0.01 263.13 ± 2.02 96.4 ± 4.61 16 Amauroderma rugosum 62.5 ± 2.43 94.87 ± 1.79 62.09 ± 4.27 96.43 ± 1.40 * IL-6 and TNF-α production was expressed in terms of EC50 values # Cell viabilities are measured at the EC50 values corresponding to the production of 50% IL-6 and TNF-α
74 A B
Fig. 3.2. Concentration dependant immunomodulatory activities of most active polysaccharide extracts: (A). TNF-α production and (B) IL-6 production. Results are given as mean ± SD (n = 3); p < 0.05 is considered to be statistically significant.
75 It is therefore expected that the herbal polysaccharides from A. rugosum, A. annua , L. chinensis and S. suberectus have huge potential to be used as immunostimulators and this property makes them highly desirable candidates as anticancer agents (Schepetkin and Quinn,
2006 and Zhang et al., 2014). It is important to note from the literature that plant derived immunostimulatory polysaccharides are potential agents for cancer therapy (Schepetkin and
Quinn, 2006, Zhang et al., 2014, Zhang et al., 2013). For instance, Lentinan is one of the well-known immune-enhancing mushroom polysaccharide that has been successfully used in chemo-immunotherapy in combination with fluoropyrimidine to improve survival rates of patients with gastric cancer (Ina et al., 2013). It is therefore expected that the polysaccharides extracted from A. rugosum, A. annua, L. chinensis and S. suberectus are promising candidates for the formulation of immunotherapeutic agents.
3.3.3.2. Toxicities of polysaccharides
The toxicities of the polysaccharide extracted from selected medicinal plants are given in
Table 3.4. Cell viabilities were measured at the concentrations corresponding to the EC50
values of IL-6 and TNF-α production. The results demonstrate that the polysaccharides
studied here display good cell viabilities (Table 3.5) indicating low toxicity. These results are
in agreement with literature findings (Schepetkin and Quinn, 2006, Zhang et al., 2012 and
Zhang et al., 2014).
3.3.4. Anticancer activities of crude polysaccharides
The anticancer activities of the polysaccharides isolated from 16 Chinese medicinal plants
were measured against several cancer cell lines, namely, A549 (lung carcinoma), MCF7
76 (breast carcinoma), HT29 (colon carcinoma), HepG2 (hepatocites carcinoma) and MiaPAca2
(pancreatic carcinoma) and the results are presented in Table 3.5 and Figures 3.3 and 3.4.
The polysaccharides from A. annua, A. rugosum , C. rotundus and L. chinensis display
significant anticancer activities against two or more cancer cell lines (Table 3.5 and Fig. 3.3).
It is interesting to note from Figure 3.3 that the polysaccharides extracted from A. annua
displayed extremely high anticancer activity against A529 (lung carcinoma) and MiaPAca2
(pancreatic carcinoma). A. annua was therefore been chosen as an important candidate for detailed study. Similarly, L. chinensis and A. rugosum also exhibited significant anticancer activities against two cancer cell lines (Fig. 3.3). These two herbs were also chosen for
detailed study. It can be seen from Figure 3.4 that three of the selected herbs have highly
significant activities in a dose dependant manner against cancer cell lines.
77 Table 3.5. In vitro cytotoxicity (IC50) of crude polysaccharides isolated from herbs against five cancer cell lines
# IC50 (µg/mL) P.No Name of the plants A549 MCF7 HT29 MiaPAca2 HepG2* P.1 Akebia quinata (Houtt.) Decne. P.2 Alpinae officinarum Hance P.3 Artemisia annua L 11.97 ± 2.10 18.98 ± 1.49 7.51 ± 2.82 P.4 Artemisia scoparia Waldst. & Kit. 13.15 ± 2.48 P.5 Artemisia vulgaris L 10.58 ± 3.95 P.6 Citrus reticulata P.7 Curcuma aromatica Salisb 17.78 ± 1.18 P.8 Cynanchum paniculatum L P.9 Cyperus rotundus L 15.02 ± 0.4 20.49 ± 1.58 P.10 Lobelia chinensis Lour 15.6 ± 1.38 18.18 ± 1.95 P.11 Polygonum cuspidatum Sieb. et Zucc P.12 Rabdosia rubescens (Hamst.)Wuet. P.13 Rheum palmatum L 18.02 ± 1.85 P.14 Spatholobus suberectus Dunn. P.15 Xanthium sibiricum L P.16 Amauroderma rugosum 13.48 ± 2.32 11.82 ± 1.35 * None of the polysaccharide extracts were active against HepG2 cell lines # Smaller IC50 value indicates high activity
78 Fig. 3.3.Anticancer activities (IC50) of polysaccharide extracts against four different cancer cell lines.
79 Fig. 3.4. Dose dependant variation of anticancer activities of the polysaccharides from the three most active herbs.
80 A diagrammatic representation of the correlation of anticancer activities of the polysaccharides from 16 herbs and their monosaccharide contents are presented in Fig. 3.5.
Fig. 3.5. A diagram developed to visualise the relationship of anticancer activities and monosaccharide composition. (A) Plants P3, P4, P5 and P10 showed anticancer activity and contain significant quantities of four monosaccharides (mannose, galactose, glucose and arabinose); (B) Plants P9, P13 and P16 showed anticancer activity and contain significant quantities of three monosaccharides (galactose, glucose and arabinose/mannose); (C) Plant P7 showed anticancer activity and only contains high glucose content; (D) Plants P1, P2, P6, P8, P11 and P12 showed no anticancer activity and only contains high galactose content; (E) Plants P14 and P15 showed no anticancer activity and did not contain any Arabinose or Mannose contents (Note: Refer to Table 3.1 for the details of plant names and numbers)
An examination of the activities (Table 3.5) and the monosaccharide contents (Table 3.1) indicates that galactose, glucose, mannose and arabinose are likely to contribute towards the anticancer activities of these polysaccharides. The pie-chart given in Figure 3.5 indeed shows a good correlation of the activities with these four monosaccharides. For example, the plants
Artemisia annua, Artemisia scoparia, Artemisia vulgaris and Lobelia chinensis showed good anticancer activity also contain significant quantities of galactose, glucose, mannose, and arabinose (Tables 3.1 and 3.5); The plants Cyperus rotundus, Rheum palmatum and
Amauroderma rugosum showed anticancer activity also contain significant quantities of the monosaccharides galactose and glucose, and arabinose/mannose (Tables 3.1 and 3.5). The
81 plant Curcuma aromatica Salisb showed anticancer activity and only contains a high galactose content (Tables 3.1 and 3.5) The plants Akebia quinata, Alpinae officinarum, Citrus reticulata, Cynanchum paniculatum , and Polygonum cuspidatum did not show anticancer activity and only contain high glucose content. The plants Spatholobus suberectus and
Xanthium sibiricum did not show anticancer activity also did not contain any arabinose or mannose . Rabdosia rubescens is the only plant that deviated from this correlation diagram.
This exception may be due to the fact that the polysaccharides from this plant contain significantly low total sugar content (38.62%).
It is pertinent to further discuss the findings on immunostimulatory and anticancer effects of these polysaccharides in the light of published literature. PSP and PSK are the well-known anticancer polysaccharides that have been successfully used to treat several types of cancers in Japan (Vetvicka and Vetvickova, 2012, Friedman, 2016, Sugiyama, 2016, Ina et al., 2013,
Cheng and Leung, 2008). The most important benefit of PSP and PSK in treating cancer patients is their simultaneous immunomodulatory and anticancer activities (Zhang et al., 2014 and Cheng and Leung, 2008). Another example of anticancer polysaccharides with immuno- enhancing capability is schizophyllan which is a mushroom polysaccharide that increases cellular immunity by activating killer-cells to normal levels (Zhang et al., 2013). It is important to activate this immune function as killer-cells become dormant in cancer patients.
It is also demonstrated in the literature that several plant derived polysaccharides are efficient immunostimulators (Ina et al., 2013, Zhang et al., 2013, Wang et al., 2017 and He et al.,
2016). Lentinan is one of the well-known immune-enhancing mushroom polysaccharide that has been successfully used in chemo-immunotherapy in combination with fluoropyrimidine to improve survival rates of patients with gastric cancer (Ina et al., 2013).
It is important to examine the findings of this Chapter in the light of the above literature
(Friedman, 2016, Sugiyama, 2016, Ina et al., 2013, Cheng and Leung, 2008, Zhang et al.,
82 2013). Three of the herbal polysaccharides studied in this research (namely, A. annua, L.
chinesis, and A. rugosum) exhibited significant immunostimulatory as well as anticancer activities. Hence, they are potential candidates for the discovery of anti-tumour polysaccharides. Also, several herbal polysaccharides studied in this research displayed highly significant immunostimulatory activities (namely, A. annua, A. rugosumL. chinensis,C. reticulata and S. suberectus) which are likely candidates as chemo- immunotherapeutic agents in combination with known chemotherapeutic agents.
3.5. Conclusion
In this study, polysaccharides from selected TCM herbs/mushroom have been investigated for
their antioxidant, immunomodulatory and anticancer activities. Polysaccharides from A.
annua, A. rugosum, L. chinensis,S. suberectus, A. scoparia, P. cuspidatum and X. sibiricum
showed significant radical scavenging and iron chelating activities indicating that these
polysaccharide extracts have great antioxidant potential. In addition, the immunomodulatory
activities revealed that crude polysaccharides from A. annua, A. rugosum L. chinensis,C.
reticulata and S. suberectus exhibited significant stimulation of mouse macrophages to
produce TNF-α and IL-6 (Table 3.4, Figure 3.2). It is interesting to note that monosaccharides
such as galactose, glucose, mannose and arabinose are most likely to be responsible for the
antioxidant and immunomodulatory activities. Amongst these polysaccharides studied, four of
them from A. annua, A. rugosum,L. chinensis, and C. rotundus, showed significant anticancer activities against two or more cancer cell lines. Three of these most active herbs (A. annua, A. rugosum,and L. chinensis) have been chosen for further study. The polysaccharides
from these three herbs also showed significant antioxidant and immunomodulatory activities.
Hence, these herbs have great potential for the isolation of anticancer polysaccharides with
immune enhancing capabilities.
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95 Chapter Four Evaluation of Immunomodulatory and anticancer potentials of ethanol soluble organics from sixteen traditional anticancer herbs Abstract
Medicinal herbs offer an important traditional way to prevent and cure several diseases such as
chronic inflammation and cancer as they contain bioactive compounds including those with
antioxidant, immunomodulatory and anticancer activities. In this study, sixteen herbs were
selected based on their traditional medicinal uses and their hot water extracts obtained. Ethanol
soluble organic molecules have been separated from these extracts and their antioxidant, immunomodulatory and anticancer activities assessed. Antioxidant activities were evaluated by using DPPH●, ABTS●+scavenging methods, and ferric ion reducing assay. Total phenolic and total
flavonoid contents of these extracts were estimated based on the Folin-Ciocalteau and aluminium
chloride methods. The immunomodulatory properties of the herbal extracts were determined on
the basis of their ability to inhibit NO and TNF-α production in LPS induced RAW 264.7
macrophages. Cell viabilities were determined using MTT assay. Anticancer activity was
measured against five human cancer cell lines. Organic molecules extracted from Alpinae
officinarum, Artemisia annua, Cynanchum paniculatum, Lobilia chinesis, Spatholobus suberectus,
Xanthium sibiricum and Amauroderma rugosum exhibited significant antioxidant activities and
greatly inhibited the production of NO and TNF-α. Seven herbal extracts out of the sixteen herbs
studied showed highly significant anticancer activity against MCF7 (breast cancer cell line). The
extract from Rabdosia rubescens displayed significant anticancer activity against three cancer cell
lines. Observed biological activities of the extracts showed good correlation with their flavonoid
contents. Extracts from A. quinata, A. officinarum, A. scoparia, L. chinesis, S. suberectus and A.
rugosum exhibited significant biological activities with large quantities of polyphenols. These
herbs are potential candidates for the isolation of novel anticancer agents.
96 4.1. Introduction
It is well-known that medicinal herbs have been an important source of novel drugs and can be lead compounds for the discovery of new drugs (Shah et al., 2013, Lee et al., 2005, Palaniyandi et al., 2016). Herbs continue to contribute to the development of important new classes of therapeutics including anticancer drugs and hence it is beneficial to investigate their medicinal value using modern scientific tools (Shah et al., 2013). Several medicinal herbs have been used traditionally to treat different types of cancer (Huang et al., 2008, Shah et al., 2013). However the large majority of these are yet to be investigated comprehensively.
Traditional Chinese Medicinal (TCM) herbs have been used for treatment of different types of cancers for thousands of years in Asian countries (Ravipati et al., 2013, Lee et al., 2005,
Zhang et al., 2013, Jeong et al., 2016, Palaniyandi et al., 2016). Many bioactive compounds isolated from medicinal herbs are in clinical use as anticancer agents. Literature on some of these molecules and their anticancer potentials are briefly discussed here. One such class of anticancer agents are the vinca alkaloids such as vinblastine, vinorelbine, vincristine and vindesine (Boulikas and Tsogas, 2008, Dumontet and Jordan, 2010, Shah et al., 2013). Numerous investigations demonstrated that vinca alkaloids can induce cancer cell apoptosis by inhibition of cell mitosis
(Jordan et al., 1998, Gragg and Newman, 2005, Nobili et al., 2009, Dumontet and Jordan, 2010,
Botta et al., 2009, Govind, 2011, Lee et al., 2015). However, it should be noted that vinca alkaloids can also reduce the number of white blood cells (Gragg and Newman, 2005, Lee et al.,
2015) leading to various side effects which include bone marrow depression, nausea, vomiting, fatigue, headaches, dizziness, peripheral neuropathy, hoarseness, ataxia, dysphagia, urinary retention, constipation, diarrhea (Lee et al., 2015). These risks have limited the progression of vinca alkaloids into clinical use. Another class of important therapeutic agents consist of flavonoids that display anticancer activities (Talhouk et al., 2007, Ravishankar et al., 2013,
97 Sghaier et al., 2011). For example, investigations demonstrate that flavone and flavopiridol
isolated from Dysoxylum binectariferum Hook can prevent cancer formation by inhibition of several protein kinases such as cyclin-dependent kinases and tyrosine kinases (Shah et al., 2013,
Ravishankar et al., 2013). Curcumin from Curcuma longa L has been used in anticancer clinical
trials due to its significant immunomodulatory properties (Aggarwal et al., 2003, Julie and
Jurenka, 2009) as well as protein kinase inhibition activities with minimal toxicity (Aggarwal et
al., 2003, Li and Zhang, 2014). Genistein isolated from soybeans displays antiangiogenic effects
by regulating the expression of vascular endothelial growth factor (Ravishankar et al., 2013,
Farina et al., 2006, Guo et al., 2007, Su et al., 2005). In addition, plant flavonoids such as
quercetin, genistein, daidzein prevent cancer formation by their antioxidant and
immunomodulatory activities (Hämäläinen et al., 2007,Fantini et al., 2015, Rusak et al., 2005,
García-Lafuenteet al., 2009, Ravishankar et al., 2013). Some of these compounds are in advanced
phases of clinical trials for several types of cancers (Bulter, 2008, Ravishankar et al., 2013). There
is abundant literature to indicate the existence of several prenylated flavonoids which exhibit a
broad spectrum of properties relevant to anticancer activity (Venturelli et al., 2016). However,
this important class of molecules have not fully been exploited to unravel their cancer-preventative properties and their therapeutic potential to treat cancer (Venturelli et al., 2016). It is therefore important to undertake a detailed study on the anticancer behaviour of flavonoids from herbal medicine.
As part of the research program initiated in our laboratory to discover anticancer agents from
TCM herbs, sixteen traditionally known anticancer herbs (Table 3.1 in Chapter 3) have been
carefully selected and investigated. Table 3.1 provides their traditional uses and biological
activities. Available scientific studies and the TCM knowledge demonstrate that these sixteen
medicinal herbs exhibit significant therapeutic properties such as immunomodulatory, anticancer
98 and other pharmacological potential. The literature on some of the selected herbs is reviewed in the following paragraphs.
Artimisia annua is a famous traditional herb that is widely used for the treatment of malaria and cancer (Huang et al., 2008, Ferreira et al., 2010). Scientific studies have indicated that artemisinin
from A. annuacan prevent cancer formation by blocking the cell cycle to induce apoptosis,
modulation of signalling pathway, inhibition of angiogenesis and metastasis (Ghantous et al.,
2010, Huang et al 2012, Thoppil and Bishayee, 2011). Chrysosplenetin isolated from A. annua
showed significant anticancer activities against three different cancer cell lines which are A549,
HL60 and U87 (Chu et al., 2014).A very recent study indicated that polyphenolic compounds
isolated from A. annua displayed significant anticancer activity through inhibition adhesion and
epithelial-mesenchymal transition of highly metastatic breast cancer cells MDA-MB-231(Ko et
al., 2016),
Artemisia vulgaris is traditionally used for treatment of fever, malaria, and cancer (Huang et al.,
2008, Saleh et al., 2010). A recent study indicated that the methanol extract from the dry leaves of
A. vulgaris displayed anticancer activity against human hepatocellular carcinoma cell line HepG2
by induced cancer cell apoptosis (Sharmila & Padma, 2013). In addition, the essential oil of A.
vulgaris displayed significant anticancer properties by mitochondria-dependent apoptosis (Saleh et al., 2010).
Artemisia scoparia is an important traditional Chinese medicinal herb for the treatment of
inflammation and cancer (Abad et al., 2012). Several pharmacological compounds such as
flavones (Lin et al., 2004), coumarins (Ali et al., 2003), and various volatile oils (Singh et al.,
2009, Singh et al., 2008) have been isolated from A.scoparia. A study indicated that methanol
extracts from A.scoparia display significant anticancer activity against human breast cancer (Choi
et al., 2013).
99 The outcomes of the above investigations and the traditional knowledge of the selected herbs
warrant further study on their organic extracts. It is common practice in the TCM to use hot
water extracts for cancer and other treatments (Cho, 2010). Systematic studies involving hot
water extractable therapeutic agents from the selected sixteen TCM herbs is very limited (Cai et
al., 2004, Zhang et al., 2011, Ravipati et al., 2012). The objectives of this study are to isolate
ethanol soluble organic molecules from hot water extracts of the selected herbs and to determine
their antioxidant, anti-inflammatory and anticancer properties. It also aims to correlate the bioactivities of these extracts with the total flavonoid and phenolic content.
In this study, antioxidant activities of the selected herbal extracts were evaluated by DPPH●
scavenging (diphenylpicrylhydrazyl), ABTS●+ scavenging (2,2'-azino-bis(3-ethylbenzothiazoline-
6-sulphonic acid), and ferric ions reducing assays (Li et al., 2011; Umamaheswari et al., 2008,
Alam et al., 2013). Anti-inflammatory properties of the extracts from these herbs were evaluated
by measuring their ability to inhibit the production of NO and TNF-α in LPS-induced RAW 264.7 macrophages (Zhang et al., 2011, Ravipati et al., 2012). The anticancer activities were evaluated against five different cancer cell lines which included A549 (lung carcinoma), MCF7 (breast
carcinoma), HT29 (colon carcinoma), HepG2 (hepatocites carcinoma), MiaPAca2 (pancreatic cancer) (Ravipati et al., 2013). Toxicities of the herbal extracts were determined by MTT assay by measuring cell viability (Zhang et al., 2011, Ravipati et al., 2012).
4.2 Materials and methods
4.2.1. Collection of medicinal herbs associated with this research
The herbal plant materials were purchased from a Chinese herbal medical centre known as Bei
Jing Tong Ren Tang located in Sydney (Australia). Sample specimen of all the herbs is stored in our research laboratory. This company has branches all over the world and is well known for their
100 best practice in TCM. The herbs traded in Sydney centre have approvals from both Australian and
Chinese Governments.
The details of these selected herbs are presented in Table 2.2 (Chapter 2). All herbal samples were powdered and subjected to an extraction procedure.
4.2.2. Chemicals and reagents
The gallic acid, quercetin, sodium nitrate aluminium chloride, DPPH●, ABTS●+, DMSO, F-C reagent, sodium carbonate, 95% ethanol, ascorbic acid, Trypan blue 0.4%, tetra methyl benzidine, sulfanilamide, N-(1-1-napthyl) ethylenediamine dihydrochloride, lipopolysaccharide (LPS) were purchased from Sigma (Australia) and Lomb Scientific Pty Ltd (Australia). The Foetal bovine serum (FBS), antibiotics, and Dulbecco’s modified Eagle’s medium (DMEM) with gluMax were purchased from BD Bioscience. The tumour necrosis factor-α (TNF-α) – ELISA standards and antibodies were purchased from BD Bioscience (USA).
In this study, mouse leukaemia monocyte macrophage cell line (RAW 264.7 macrophages) was used to test the immunomodulatory properties of herbal extracts from sixteen traditional anticancer herbal and mushroom (Refs). Lipopolysaccharide (LPS) is used as positive control. The abilities of herbal extracts to inhibit production of NO and TNF-α were measured by Griess assay and ELISA tests. DPPH● radical scavenging assay, ABTS●+ radical scavenging assay, Iron (Fe2+) chelating and ferric ions reducing power assays were used for the analysis of antioxidant activity.
Anticancer activities of herbal extracts are determined using five different tumour cell lines which are MCF7 (breast carcinoma) HT29 (colon carcinoma), A549 (lung carcinoma), HepG2
(hepatocytes carcinoma), MiaPAca2 (pancreatic cancer).
101 4.2.3. Ethanol soluble water extraction preparations for bioactivity studies
About 30g of dried herbs were ground to powder form and mixed well. The powdered plant material was subjected to hot water extraction using the autoclave method (at 121 oC for 2 hours) and then cooled to laboratory temperature and the supernatant was separated by filtration. The supernatant (extract) was then treated with 95% ethanol (extract: ethanol = 1:4 volume ratio) for
24 hours at 4.1 oC.The ethanol supernatant was then collected and filtered through a 0.45 µm
Whatman filter paper. The solution was then freeze dried and kept in -20 °C until use (Jeong et al., 2016). The entire process of extraction from ethanol supernatant is summarised in Figure 4.1.
102 Powdered plant/mushroom material • Hot water extraction (autoclaving) (121 for 2 h) • Centrifugation (10,000 rmp for 20 min)℃
Marc Supernatant
• EtOH precipitation (95%) (Supernatant: EtOH =1:4 for 24 h, 4°C)
Ethanol supernatant
• Filtration • Freeze drying
Small organic molecules
Fig. 4.1. Schematic diagram for the separation of ethanol soluble organics from hot water extract of medicinal herbs
4.2.4. Determination of total phenolic compounds
The Folin-Coicalteu (F-C) reagent method was employed for determination of total phenolic
content (Cicco et al., 2009). The procedure followed for the assay is similar to the one published
previously (Cicco et al., 2009, Zhang et al., 2011, Ravipati et al., 2012). A standard curve was
built using different concentrations of gallicacid (0-1000 µg/Lm) (Cai et al., 2004). The regression of the standard curve gave a linear equation (y = 0.004x + 0.0496, R2 = 0.9961). The samples were
analysed in triplicate. The total phenolics in the ethanol soluble water extracts were calculated
using the above equation.
103 4.2.5. Determination of total flavonoids
The aluminium chloride method was employed using for determination of total flavonoids
(Zhishen et al., 1999). The procedure followed for the assay is based on the method published
before (Zhishen et al., 1999, Zhang et al., 2011, Ravipati et al., 2012). A standard curve was built
using different concentrations of quercetin (0-1000 µg/mL) (Cai et al., 2004). The regression of
the standard curve gave a linear equation (y = 0.0006x - 0.0033, R2 = 0.9942). The samples were
analysed in triplicate.
4.2.6. Bioactivity tests
4.2.6.1. DPPH● radical scavenging assay
The Blois method (Blois, 1958, Zhang et al., 2011, Ravipati et al., 2012) was employed to determine the DPPH● radical scavenging ability of the herbal extracts . The methodology
employed for this assay is similar to that used in the Chapter 3. A standard curve was built using
different concentrations of ascorbic acid solutions (in 60% methanol) in the range of 0 to 200 µM.
The regression of the standard curve gave a linear equation (y = -0.0016x + 0.3515 with R² =
0.9648). The free radical scavenging activity was calculated as the ascorbic acid equivalent using
the above equation.
4.2.6.2. ABTS●+radical scavenging assay
ABTS●+ was employed in order to determine the radical scavenging ability of the herbal extracts
(Li et al., 2011, Alam et al., 2013).The methodology employed is similar to the method used in the Chapter 3. The regression of the standard curve gave a linear equation (y = -0.0023x + 0.6996 with R² = 0.9852). The free radical scavenging activity was calculated as the ascorbic acid equivalent using the above equation.
104 4.2.6.3. Immunomodulatory activities assays
4.2.6.3.1. Culture RAW 264.7 macrophages
Mouse macrophages (RAW 264.7) are first added to DMEM (culture medium containing 1%
antibiotic and 5%FBS) and incubated for 4 days at 37 °C in 5% CO2 (Ni et al., 2016, Zhang et
al., 2012, Zhang et al., 2013, Thambiraj et al., 2015, Jeong et al., 2012). The methodology
employed for this assay was similar that used in the Chapter 3.
4.2.6.3.2. NO production
The supernatant (100 µL) from each well is then carefully transferred into a new multi-well plate.
50µL of suffanilamide (1% w/v, dissolved in 5% H3PO4) was then added to the supernatant and kept for 5 min at room temperature, and 50 µL naphthyl ethylenediamine (0.1% w/v) was added to measure the concentration of NO as per the described procedure (Jeong et al., 2012, Zhang et al., 2013, Zhang et al., 2012, Thambiraj et al., 2015). Triplicate measurements were made .
Sodium nitrate was used as standard. The regression of the standard curve gave a linear equation
(y = 0.0011x + 0.3975 with R² = 0.9757). The immunomodulatory activities of the herbal extracts
were calculated using the above equation.
4.2.6.3.3. TNF- α production
The supernatant (100 µL) from each well was transferred into a new multi -well plate (Jeong et al.,
2012, Zhang et al., 2013, Zhang et al., 2012). The methodology employed for this assay is similar to that used in the Chapter 3.Triplicate measurements were made . The regression of the standard
curve gave a linear equation (y = 0.001x + 0.1069 with R² = 0.9879). The immunomodulatory
activities of the herbal extracts were calculated using the above equation.
4.2.6.3.5. Determination of cell viability by MTT assay
105 Viability of macrophage cells (RAW 264.7) were measured employing 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Zhang et al., 2013, Zhang et al., 2012,
Thambiraj et al., 2015). The methodology employed for this assay was similar to that used in the
Chapter 3. The absorbance values were the measured at 595 nm.
(%) = 100%
𝑂𝑂𝑂𝑂 𝑜𝑜𝑜𝑜 𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣 𝑋𝑋 Where, positive control is mouse macrophages𝑂𝑂𝑂𝑂 treated𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝 w𝑝𝑝ith𝑐𝑐𝑜𝑜 𝑛𝑛DMEM𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛 Medium (without LPS)
4.2.6.4. Anticancer assays against various cancer cell lines
The cancer cell lines were cultured and incubated according to procedure out-lined in a previous
publication (Royo et al., 2003; Tormo et al., 2005;Ravipati et al., 2013, Prodo et al., 2013). The
method employed for this assay was similar to that used in the Chapter 3.
Optical density was determined at 570 nm using a spectrofluorometer (Victor2TM Wallac
spectrofluorometer). The percentage inhibition against various cancer cells was calculated using
the following equation.
% = 100 pos contro ODsample− OD 𝑖𝑖𝑖𝑖ℎ𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 𝑁𝑁𝑁𝑁𝑁𝑁 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑝𝑝𝑝𝑝𝑝𝑝 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑋𝑋 Where, ODNeg Contr is the optical density of𝑂𝑂 𝐷𝐷the negative− 𝑂𝑂control𝐷𝐷
ODPos Contr is the optical density of the positive control
The culture medium containing DMSO (1%) was used as a negative control and the medium
with 2mM MMS was used as positive control.
106 4.2.5. Statistical analysis
All data were measured in triplicate and the mean ± SD was calculated. The group mean was
compared using a one-way analysis of variance (ANOVA) and Duncan’s multiple range tests.
Statistical calculations were done using IBM SPSS, OriginPro 8.5 and Excel 2016. The data is
considered to be statistically significant if p < 0.05.
4.3. Results and discussion
4.3.1. Chemical composition
Total flavonoid and phenolic content of ethanol soluble organics in the water extracts of the selected sixteen herbs was measured using Folin-Coicalteu and aluminium chloride assays
respectively and the results are presented in Table 4.2. The phenolic and flavonoid contents were expressed in gallic acid equivalent (GAE mg/g) and quercetin equivalent (QE mg/g) of the extract per gram of the dried plant material. The total phenolic content of the selected plant extracts ranged
from 0.12 to 14.33 (mg/g). The highest phenolic content was observed in the extracts of P.
cuspidatum (14.33 ± 0.14 mg/g). The flavonoid content was relatively large in all the extracts
compared to their phenolic contents (Table 4.2). Highly significant flavonoid contentwas found in
P. cuspidatum (24.86 ± 4.19 mg/g), X. sibiricum (20.61 ± 1.67 mg/g), A. quinata (20.46 ± 1.67
mg/g), A. vulgaris (19.09 ± 0.96 mg/g), and A. rugosum (17.85 ± 1.67 mg/g). The herbs A. officinarum, A. annua, A. scoparia and S. suberectus also had significant levels of flavonoid
content.
107 4.3.2. Antioxidant activities
4.3.2.1. DPPH● and ABTS●+ scavenging activities
In this study, the antioxidant activities of the extracts from sixteen selected medicinal herbs were evaluated using DPPH● and ABTS●+radical scavenging activity. The results of free radical
scavenging capacity of the extracts are presented in Table 4.2. Most of the plant extracts showed
significant scavenging activity. The values of DPPH● scavenging activity ranged from 112.4 µmol
to 198.9 µmol ascorbic acid equivalent. The ABTS●+scavenging activity of the extracts ranged
from 121 µmol to 303.74 µmol ascorbic acid equivalents. Highly significant DPPH● scavenging activities are shown by the extracts of R. rubescens, P. cuspidatum, A. officinarum , A. quinata, A. scoparia, A. vulgaris, A. annua,S. suberectus, X. sibiricum and A. rugosum for which the activities were greater than 180 µM ascorbic acid equivalent. High ABTS●+ scavenging activities were shown by the extracts of R. rubescens, A. quinata, A. officinarum, A. scoparia, A. annua, A.
vulgaris, P. cuspidatum, and A. rugosum which were greater than 270 µM ascorbic acid
equivalent.
108 Table 4.1. Antioxidant activities of the ethanol soluble organics from water extracts from sixteen Chinese medicinal herbs along with their total phenolic and flavonoid content
DPPH scavenging ABTS scavenging Phenolic content Flavonoid content Name of herbs * activity (Ascorbate activity (Ascorbate (GAE mg/g) (QE mg/g)* # # S. No equivalent (µM) equivalent (µM) 1 Akebia quinata (Houtt.) Decne. 8.87 ± 0.14 20.46 ± 1.67 188.65 ± 0.36 302.43 ± 0.01 2 Alpinae officinarum Hance 5.2 ± 0.14 14.38 ± 1.67 190.1.1 ± 0.36 283.59 ± 0.25 3 Artemisia annua L 7.19 ± 0.29 12.98 ± 4.41 185.31 ± 0.63 296.35 ± 0.04 4 Artemisia scoparia Waldst. & Kit. 3.77 ± 0.25 11.51 ± 0.96 182.69 ± 0.63 263.74 ± 0.03 5 Artemisia vulgaris L 6.09 ± 0.52 19.09 ± 0.96 185.06 ± 0.36 294.17 ± 0.01 6 Citrus reticulata 4.68 ± 0.75 4.39 ± 1.67 145.31 ± 0.63 203.16 ± 3.70 7 Curcuma aromatica Salisb 1.82 ± 0.29 3.24 ± 0.47 125.31 ± 0.72 142.43 ± 0.25 8 Cynanchum paniculatum L 0.12 ± 0.63 1.36 ± 0.96 112.4 ± 2.89 133.16 ± 0.25 9 Cyperus rotundus L 3.41 ± 0.29 4.66 ± 0.02 154.06 ± 0.63 192.43 ± 0.01 10 Lobelia chinensis Lour 2.45 ± 0.14 8.13 ± 1.92 174.31 ± 1.08 208.43 ± 0.02 11 Polygonum cuspidatum Sieb. et Zucc 14.33 ± 0.29 24.86 ± 4.19 189.69 ± 0.72 298.81 ± 0.25 12 Rabdosia rubescens (Hamst.)Wuet. 8.4 ± 0.66 9.51 ± 3.47 198.9 ± 2.25 303.74 ± 0 13 Rheum palmatum L 0.16 ± 0.29 2.16 ± 1.92 118.44 ± 0.63 121.86 ± 0 14 Spatholobus suberectus Dunn. 5.62 ± 1.28 15.35 ± 0.96 187.19 ± 1.65 228.67 ± 0.25 15 Xanthium sibiricum L 6.22 ± 0.14 20.61 ± 1.67 184.69 ± 1.25 238.81 ± 0.5 16 Amauroderma rugosum 8.18 ± 0.38 17.85 ± 1.67 176.56 ± 2.86 275.19 ± 5.02 *Total phenolic and flavonoid content were expressed in gallic acid and quercetin equiv/mg respectively. #DPPH, ABTS free radical scavenging activity was expressed as equivalent of ascorbic acid (µM). All values are mean of triplicate determination ± standard deviation.
109 4.3.2.2. Fe3+ reducing power
Fe3+ reducing power of the extracts were also measured as part of evaluating the antioxidant potential of the herbal extracts. The results of concentration dependant reducing power of the extracts are presented in Table 4.2. The extracts from A. officinarum, A. vulgaris, R. palmatum and
S. suberectus showed significant reducing ability.
Antioxidants eliminate oxidative stress by scavenging free radicals that cause damage to DNA and other biopolymers. Natural antioxidants from TCM herbs are attractive alternatives to synthetic antioxidants (Zhu et al., 2004). Extracts from several selected herbs studied in this research have exhibited significant radical scavenging as well as Fe3+ reducing ability. The more potent extracts include A. officinarum, A. vulgaris, P. cuspidatum, R. rubescens, S. suberectus and X. sibiricum. It is therefore expected that these herbs are potential candidates for the isolation of antioxidant compounds. A correlation of antioxidant activities of the selected herbal extracts and their polyphenol contents is presented in Table 4.2 below.
110 Table 4.2. Ferric ions reducing power of the ethanol soluble water extracts isolated from sixteen medicinal herbs
Absorbance at 700 nm S. No Herbs/standard 1000 µg/mL 500 µg/mL 250 µg/mL 125 µg/mL 62.5 µg/mL Vit C 1.707±0.006 0.706±0.001 0.331±0.001 0.193±0.001 0.127±0.001 1 Akebia quinata (Houtt.) Decne. 0.285±0.001 0.136±0.002 0.074±0.002 0.035±0.003 0.014±0.004 2 Alpinae officinarum Hance 1.138±0.001 0.741±0.005 0.345±0.001 0.214±0.002 0.106±0.002 3 Artemisia annua L 0.37±0.001 0.257±0.001 0.133±0.002 0.066±0.002 0.033±0.002 4 Artemisia scoparia Waldst. & Kit. 0.349±0.001 0.234±0.024 0.121±0.004 0.05±0.001 0.027±0.001 5 Artemisia vulgaris L 1.157±0.003 0.734±0.001 0.316±0.002 0.184±0.001 0.086±0.003 6 Citrus reticulata Blanco 0.269±0.001 0.153±0.003 0.075±0.001 0.052±0.001 0.048±0.001 7 Curcuma aromatica Salisb NA* NA NA NA NA 8 Cynanchum paniculatum L 0.444±0.003 0.254±0.002 0.153±0.004 0.077±0.002 0.035±0.001 9 Cyperus rotundus L 0.274±0.002 0.137±0.002 0.042±0.002 0.024±0 NA 10 Lobelia chinensis Lour NA NA NA NA NA 11 Polygonum cuspidatum Sieb. et Zucc 0.876±0.002 0.518±0.002 0.281±0.007 0.097±0.001 0.017±0.003 12 Rabdosia rubescens (Hamst.)Wuet. 0.726±0.002 0.412±0.003 0.208±0.001 0.108±0.001 0.058±0.001 13 Rheum palmatum L 1.091±0.001 0.709±0.002 0.407±0.001 0.216±0 0.122±0.001 14 Spatholobus suberectus Dunn. 1.962±0.045 0.976±0.003 0.447±0.001 0.285±0.001 0.192±0.005 15 Xanthium sibiricum L 0.504±0.003 0.247±0.002 0.127±0.003 0.076±0.002 0.017±0.001 16 Amauroderma rugosum(Blume & T. Nees) Torrend 0.296±0.002 0.204±0.001 0.088±0.002 0.038±0.002 0.014±0.002 *NA: no activity; P < 0.05
111 Correlation plots were developed in order to reveal the relationship between the antioxidant activity and polyphenol content of the extracts (total phenolics and flavonoids) (Fig.4.2). DPPH● and ABTS●+ scavenging activities of the extracts showed significant correlation (R2 >0.55) with total phenolic content (Fig.4.2A and 4.2C) and also with the total flavonoid content (Fig. 4.2B and
4.2D). The results indicate that the total phenolic and flavonoid contents are important contributors to the antioxidant activities of the ethanol soluble water extracts of the herbal medicine and this observation is in agreement with the literature (Cai et al., 2004, Chan et al.,
2014, Joeng et al., 2016, Zhang et al., 2011, Ravipati et al., 2012). The observed correlations of radical scavenging activities of the extract from A. rogosum with total phenolics is in agreement with those reported in the literature (Chan et al., 2014). It should be noted that some of the herbal extracts significantly deviated from the linear correlation. For instance, C. reticulata and C. rotundus were found to have high antioxidant activities but low levels of phenolic/flavonoid content (Table 4.2). It is therefore reasonable to conclude that in addition to phenolics and flavonoids there are other antioxidant components that are likely to be present in the ethanol soluble water extracts, which may also contribute to their antioxidant activities.
112 250 250 A B 200 200 150 150 R² = 0.641 R² = 0.5563 100 100 50 50 0 0 in ethanol extracts (µM) ethanol extracts in
in ethanol extracts (µM) ethanol extracts in 0 5 10 15 20 0 10 20 30 DPPH Scavegning activity activity DPPH Scavegning DPPH Scavegning activity activity DPPH Scavegning Phenolics content in water extracts (mg/g) Flavonoids content in water extracts (mg/g)
400 350 D
350 C 300 300 250 250 R² = 0.6869 R² = 0.6485 200 200 150 150 100 100 50 50
ethanol extracts (µM) extracts ethanol 0 0 in ethanol extracts (µM) extracts ethanol in ABTS Scavegning activity in in activity Scavegning ABTS 0 5 10 15 20 ABTS Scavegning activity 0 10 20 30 Phenolics content in water extracts (mg/g) Flavonoids content in water extracts (mg/g)
Fig. 4.2. Correlation between antioxidant activity and the total phenolic and flavonoid contents in the extracts: (A) DPPH● and phenolics, (B): DPPH● and flavonoids, (C): ABTS●+ and phenolics, (D): ABTS●+ and flavonoids.
113 4.3.3. Anti-inflammatory activities
Evidence from the literature indicates that increased production of NO and TNF-α can cause the development of inflammation (Durga et al., 2014, Kiemer et al., 2002). This study investigated the ability of the herbal extracts to inhibit the production of NO and TNF-α in LPS-induced RAW
264.7 macrophages. The inhibition activity is expressed in terms of IC50 values and the results are presented in Table 4.4. It can be seen that many herbal extracts showed inhibitory activity against the production of nitric oxide (NO). The extracts from A. annua, A. rugosum, A. vulgaris, A.
rugosum, L. chinesis, S. suberectus, and X. sibiricum significantly down regulated the NO
production with IC50 values that are less than 0.23 mg/mL. There was no inhibition activity in the
extracts from Curcuma aromatic, Cynanchum paniculatum and Rheum palmatum .
Results presented in Table 4.4 indicate that the extracts from A. annua, A. rugosum, A. vulgaris,
Cyperus rotundus and L. chinesis showed significant inhibitory activity against TNF-α production
with IC50 values less than 330 µg/mL. Interestingly, the extracts from C. aromatica, C.
paniculatum and R. palmatum did not show inhibitory activities against both NO and TNF-α
production.
114 Table 4.3. Anti-inflammatory activities of the extracts from selected medicinal herbs
IC for the IC50 for the inhibition 50 Cell viability (% Cell viability (% S. No Name of herbs inhibition of NO & of TNF-α production & of cell survival) of cell survival) production (µg/mL)* (µg/mL) 1 Akebia quinata. 687.12 ± 5.22 94.4 ± 1.47 484.64 ± 2.42 89.17 ± 3.55 2 Alpinae officinarum 229.31 ± 4.61 96.8 ± 1.73 348.67 ± 2.43 91.47 ± 10.09 3 Artemisia annua 47.42 ± 0.78 106.43 ± 4.04 156.67 ± 1.78 87.57 ± 8.99 4 Artemisia scoparia. 245.13 ± 3.99 94.5 ± 3.5 388.49 ± 1.92 86.07 ± 2.29 5 Artemisia vulgaris 108.15 ± 6.21 97.77 ± 1.17 128.42 ±0.48 95.43 ± 0.93 6 Citrus reticulata 333.92 ± 1.92 92.73 ± 3.31 343.08 ± 4.90 74.53 ± 3.52 7 Curcuma aromatica NA NA 8 Cynanchum paniculatum NA NA 9 Cyperus rotundus 217.72 ± 2.54 65.73 ± 2.97 271.58 ± 4.96 78.2 ± 1.54 10 Lobelia chinensis 159.95 ± 2 87.33 ± 2.08 143.76 ± 5.34 77.03 ± 1.96 11 Polygonum cuspidatum 265.14 ± 4.95 76.53 ± 5.71 330.37 ± 5 88.43 ± 0.45 12 Rabdosia rubescens. 253.42 ± 2.88 96.5 ± 5.07 430.83 ± 3.44 93.5 ± 7.13 13 Rheum palmatum NA NA 14 Spatholobus suberectus 200.94 ± 3.35 88.63 ± 0.32 301.69 ± 1.17 57 ± 2.58 15 Xanthium sibiricum 148.13 ± 3.01 79.73 ± 10.71 313.91 ± 5.02 68.5 ± 0.5 16 Amauroderma rugosum 81.31 ± 4.47 88.77 ± 2.87 327.38 ± 3.67 94.87 ± 1.79
* Inhibition of NO and TNF- α production was expressed in terms of IC50 values; P < 0.05 & Cell viabilities were measured at appropriate IC50 values corresponding to the inhibition of NO and TNF-α production. NA: no activity
115 Concentration dependant anti-inflammatory activities of most active herbal extracts are shown in
Figures 4.3A and 4.3B. Extracts from A. annua and A. vulgaris,displayed high activity against
TNF-α production. The extracts of C. rotundus and L. chinesis also displayed significant activity
against TNF-α production (Fig.4.3B). The extracts from A. annua,A. vulgaris,A. rugosum and X.
sibiricum show concentration dependant inhibition of NO production (Fig.4.3A).
4.3.3.1. Cell viability
The effect of the extract from the sixteen medicinal herbs on the viability of mouse macrophages
is given in Table 4.4. Cell viability was measured at appropriate IC50 values corresponding to the production of NO and TNF-α. It is clear from these results that all of the extracts showed significant cell viabilities (> 57% ). These results indicate that the extracts of chosen herbs exhibit
low toxicity and this is consistent with literature reports (Zhang et al., 2011, Ravipati et al., 2012,
Ravipati et al., 2013).
116 A B
Fig. 4.3. Concentration dependant immunomodulatory activities of most active extracts from the selected herbs: (A). NO production, and (B). TNF-α production. Values are expressed as mean ± SD (n=3), P < 0.05.
117 It is interesting to note from the results that there is a good correlation between the anti-
inflammatory activities and total phenolic and flavonoid content. For instance, A. officinarum, A.
annua, X. sibiricum,S. suberectus and A. rugosum which inhibited the production of NO/TNF-α
(low IC50 values) and also had significant levels of total phenolics and flavonoid content. Other
herbs such as C. aromatic, C. paniculatum and R. palmatum which were found to contain low
levels of phenolic and flavonoid content also did not display anti-inflammatory activity (Table
4.4). These results are in agreement with previous studies that report that phenolics and
flavonoids influence anti-inflammatory activities (da Silva Oliveira et al., 2011). Literature reports
indicates that polyphenols isolated from medicinal herbs display strong anti-inflammatory activities. For instance the polyphenolic compounds galangin and 5-hydroxy-7-(4″-hydroxy-3″- methoxyphenyl)-1-phenyl-3-heptanone, isolated from A. officinarum significantly inhibited the production of pro-inflammatory factor (COX-2) (Honmore et al., 2016). On the other hand, the anti-inflammatory activities of some of the herbs are not consistent with their total phenolic and flavonoid content. For instance, C. reticulata, and C. rotunduswere found to have significant anti- inflammatory activities (inhibition of production of both NO and TNF-α), but contained low levels of phenolics and flavonoids. Hence, it is concluded that chemical constituents other than phenolics and flavonoids may also be responsible for the anti-inflammatory properties of such plants (Zhang et al., 2011, Ravipati et al., 2013).
4.3.4. Anticancer activities
The anticancer activities of these extracts from Chinese medicinal herbs were evaluated against
five human cancer cell lines which included A549 (lung carcinoma), MCF7 (breast
carcinoma), HT29 (colon carcinoma), HepG2 (hepatocites carcinoma), MiaPAca2 (pancreatic
cancer). These results are expressed in terms of IC50 values and presented in Table 4.4.
118 A review of the literature (Zhu et al., 2004, Zhang et al., 2014) offers strong evidence that prolonged oxidative stress leads to inflammation and tissue damage that can potentially cause cancer . Therefore, the agents that simultaneously possess antioxidant, anti-inflammatory and anticancer properties are of great importance for the prevention and treatment of cancer
(Ravishankar et al., 2013, Sghaier et al., 2011). Many of the herbal extracts studied in this research exhibited these important biological activities and hence are highly suitable for the isolation of anticancer agents. Polyphenols in general and flavonoids in particular are known to be potential anticancer agents (Durga et al., 2014, Ravishankar et al., 2013, Sghaier et al., 2011). Correlation of observed anticancer activities of herbal extracts with their polyphenol contents is discussed below.
119
Table 4.4. In vitro cytotoxicity (IC50) of the extracts from herbs against five cancer cell lines
IC50 (µg/mL)* No Name of Herbs A549 MCF7 HT29 MiaPAca2# Hep_G2 1 Akebia quinata. 14.65 ± 0.94 2 Alpinae officinarum 14.64 ± 1.34 3 Artemisia annua 4 Artemisia scoparia 6.86 ± 2.60 5 Artemisia vulgaris 6 Citrus reticulata 7 Curcuma aromatica 36.23 ± 2.53 8 Cynanchum paniculatum 9 Cyperus rotundus 10 Lobelia chinensis 11.75 ± 3.91 11 Polygonum cuspidatum 12 Rabdosia rubescens . 15.33 ± 0.22 22.00 ± 8.64 10.75 ± 2.91 13 Rheum palmatum 14 Spatholobus suberectus 10.65 ± 1.3 15 Xanthium sibiricum 16 Amauroderma rugosum 11.75 ± 5.64 # None of the extracts were active against Hep_G2 cell lines *Smaller IC50 value indicate high activity
120 Fig. 4.4. Anticancer activities (IC50) of the extracts against four different cancer cell lines: (A).The extracts from Akebia quinata, Alpinae officinarum, Artemisia scoparia, Curcuma aromatica, Lobelia chinensis, Spatholobus suberectus and Amauroderma rugosum against breast cancer cell line (MCF-7) and (B).The extracts from Rabdosia rubescens against three different cancer cell lines (A529, HT29 and Hep_G2).
121 Fig. 4.5.Dose dependant variation of anticancer activities of the extracts from the three most active herbs. (a) the extracts from Rabdosia rubescens against three different cancer cell lines (A529, HT29 and Hep_G2), (b) the extracts from Artemisia scoparia, against breast cancer cell line (MCF-7), and (c) the extracts from Spatholobus suberectus against breast cancer cell line (MCF-7).
122 Many of the herbal extracts studied in this research showed significant correlation of anticancer
activities with their phenolic and flavonoid content. For example, A. quinata, A. officinarum, A.
scoparia, L. chinensis, R. rubescens, S. suberectus and A. rugosum contained medium to high quantities of polyphenols and exhibited significant anticancer activities. This observation is consistent with the literature reports that flavonoids possess significant anticancer activities
(Ravishankar et al., 2013, Sghaier et al., 2011). It should be noted that some of the herbal extracts
studied here contain polyphenols (Table 4.2), but did not display any anticancer activity (e.g. A.
annua, A. vulgaris, C. reticulate, C. rotundus, P. cuspidatum and X. sibiricum). This may be due
to the fact that the flavonoids present in these extracts may not be structurally relevant to
anticancer activities (Ravishankar et al., 2013, Silici et al., 2007, Chaubal et al., 2005). Evidence
from the literature indicates the existence of a relationship between the chemical structure of
flavonoids and their anticancer properties (Ravishankar et al., 2013, Scotti et al., 2012, Dai and
Mumper, 2010). For example, the anticancer activities of flavonoids depend on the number and
position of hydroxyl groups, methoxy groups and presence of C-C double bond in ring-B
(Ravishankar et al., 2013, Casagrande and Darbon, 2001,Scotti et al., 2012).
4.4. Conclusions
A summary of the spectrum of biological activities of herbal extracts studied in this research are
presented in Table 4.5. In this Table, the notation “+++” is used to represent very high activity or
large quantity of total polyphenols in the extracts. The notation “++” is used to represent
significant activity or significant quantity of polyphenols. “+” means average activity or average
quantity of polyphenols in the extracts. It can be seen from Table 4.6 that the extracts from A.
quinata, A. officinarum, A. scoparia, L. chinensis, S. suberectus and A. rugosum exhibited highly
significant activities and also contain large quantities of polyphenols. It is therefore concluded that
these six herbs are very important candidates for the discovery of novel anticancer agents.
123 Findings of this research strongly support the traditional use of these herbs. It is interesting to
note that the six important herbs short-listed by this research (Table 4.6) are extensively used by
TCM practitioners in anticancer formulations.
124 Table 4.6. Important anticancer herbal extracts identified in this research together with their polyphenol content
Plant Total quantity Antioxidant Anti-inflammatory Anticancer No Name of the herbs of polyphenol activity activity activity Comment 1 Akebia quinata +++ +++ + +++ High potential candidate* 2 Alpinae officinarum ++ +++ ++ +++ High Potential candidate 3 Artemisia annua ++ +++ +++ 4 Artemisia scoparia ++ ++ ++ +++ Potential candidate 5 Artemisia vulgaris +++ +++ +++ 6 Citrus reticulata + + 7 Curcuma aromatica + 8 Cynanchum paniculatum 9 Cyperus rotundus ++ +++ 10 Lobelia chinensis + ++ +++ +++ Potential candidate 11 Polygonum cuspidatum +++ +++ ++ 12 Rabdosia rubescens + +++ ++ 13 Rheum palmatum 14 Spatholobus suberectus ++ ++ +++ +++ High Potential candidate 15 Xanthium sibiricum +++ ++ +++ 16 Amauroderma rugosum +++ +++ +++ +++ High potential candidate “+++” represents extremely high activity or large quantity of polyphenols “++” represents significant activity or significant quantity of polyphenols “+” represents average activity or average quantity of polyphenols *Potential candidates for anticancer flavonoids
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Zhang, L., Ravipati, A.S., Koyyalamudi, S.R., Jeong, S.C., Reddy, N., Smith, P.T., Bartlett, J., Shanmugam, K., Münch, G., and Wu, M.J. (2011). Antioxidant and anti-inflammatory activities of selected medicinal plants containing phenolic and flavonoid compounds. J Agric Food Chem 59, 12361-12367.
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136 Chapter Five
Biological and structural characterisation of polysaccharides fractions isolated from Amauroderma rugosum Abstract
Amauroderma rugosum is an important medicinal mushroom used for the treatment of cancer in Chinese medicine. To examine the biological activities of polysaccharides from A. rugosum, water-soluble polysaccharides were isolated and their pharmacological and anticancer activities determined. A.rugosum polysaccharides (ARPs) were extracted and purified using size-exclusion chromatography to obtain three main polysaccharides, ARP-1,
ARP-2 and ARP-5, having molecular masses of 1498 kDa, 450 kDa and 7 kDa respectively.
The antioxidant potentials of the isolated polysaccharides were evaluated by measuring radical scavenging activities against DPPH● (2,2-diphenyl-1-picrylhydrazyl radical), ABTS●+
(2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical), and OH● (hydroxyl radical).
Immunostimulatory activities of ARP-1, ARP-2 and ARP-5 were measured by using mouse macrophages. The three polysaccharide fractions displayed significant antioxidant activities and stimulate the production of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6), where the ARP-1 and ARP-2 were found to be the most active . A detailed structural characterisation of these ARPs was carried out by gas chromatography (GC) and FT-IR techniques and NMR spectroscopic methods. ARP-1 and ARP-2 consist of β- (1→3)-D- glucans with β-(1→6)-D-glucopyranosyl branches. The results also suggest that the polysaccharides from A. rugosum are potential candidates for immunotherapeutic agents.
137 5.1 Introduction
Generally mushrooms are used as functional foods or dietary supplements for their
nutritional and medicinal values (Chang and Miles, 2004). The bioactivities of mushrooms
have been confirmed by extensive studies (Jeong et al., 2004, Cui and Chisti, 2003 and
Cheng and Leung, 2008, Jeong et al., 2010, Jeong et al., 2012). Several medicinal
mushrooms were identified and used to treat cancer in traditional Chinese medicine (TCM)
for many centuries (Cheng and Leung, 2008). For instance, Corolus versicolor is one such
mushroom that has been well investigated and is currently used in China and Japan in
combination with chemotherapy (Cheng and Leung, 2008). Several decades ago Lucas
(1957) demonstrated that the biopolymers isolated from a food mushroom (Boletus edulis)
exhibited significant anticancer activity against Sarcoma 180 tumour cells (Lucas, 1957).
Subsequently, numerous antitumour polysaccharides have been extracted from a variety of
medicinal mushrooms (Jeong et al., 2004, Cui and Chisti, 2003, Cheng and Leung, 2008,
Jeong et al., 2010, Jeong et al., 2012, Zhang et al., 2007, Vetvicka and Vetvickova, 2012,
Schepetkin and Quinn, 2006, Friedman, 2016, Sugiyama, 2016, Zhang et al., 2014). For
instance, β-glucans isolated from medicinal mushroom (such as Corolus versicolor,
Schizophyllum commune), have proven to possess pharmacological effects such as immunomodulatory, anticancer, anti-inflammatory and antioxidant activities (Rout and
Banerjee, 2007, Moradali et al., 2007, Schepetkin and Quinn, 2006, Lu et al., 2010, Mau et
al., 2001). In particular, several polysaccharides and polysaccharoproteins have been isolated
from medicinal mushrooms and clinically used for anticancer therapy (Schepetkin and Quinn,
2006, Zhang et al., 2014, Friedman, 2016, Zhang et al., 2007, Vetvicka and Vetvickova,
2012; Sugiyama, 2016, Cheng and Leung, 2008). For example lentinan derived from
Lentinula edodes (Sugiyama, 2016, Chihara, 1970, Chihara et al., 1986, Daba et al., 2003 and
Ina et al., 2013; Cheng and Leung, 2008), polysaccharide Krestin (PSK) derived from
138 Coriolus versicolor (Friedman, 2016, Sugiyama, 2016, Hattori et al., 2004; Cheng and
Leung, 2008 and Torisu et al., 1990); polysaccharopeptide (PSP) isolated from Coriolus
versicolor (Ng, 1998, Cui and Chisti, 2003 and Cheng and Leung, 2008); and schizophyllan
from Schizophyllum commune (Sugiyama, 2016 and Daba et al., 2003) are some of the
important immune enhancing and anticancer polysaccharides. All of these mushroom
polysaccharides have been used as anticancer agents for many years in Japan following good
results in several clinical trials (Friedman, 2016, Sugiyama, 2016, Chihara, 1970, Chihara et
al., 1987, Cheng and Leung, 2008, Cui and Chisti, 2003, Daba et al., 2003). A brief review of
literature on these mushrooms and their polysaccharides is provided below.
5.1.1. Traditional use of Coriolus versicolor
Coriolus versicolor belongs to the Basidiomycetes class and Polyporaceae family (Hobbs,
1995, Ng, 1998, Huang et al., 2008, Chu et al., 2007, Cui and Chisti, 2003). According to the
theory of TCM, C. versicolor has a slightly sweet taste and ‘cold’ property, exerting its
effects via the liver and spleen (Huang et al., 2008, Cho, 2010). TCM literature reports that
C. versicolour dispells heat, removes toxins, strengthens physique, increases energy and spirit, and enhances the host’s immune function (Chu et al., 2007, Cho, 2010, Huang et al.,
2008). In the clinical practice of TCM, C. versicolor is often indicated for the treatment of
various types of cancers such as uterine cancer and liver cancer (Huang et al., 2008, Cho,
2010).
5.1.1.1. Clinical studies of anti-tumorpolysaccharides from C. versicolor
Polysaccharide-Krestin (PSK) and polysaccharopeptide (PSP) are the most important polysaccharides isolated from C. versicolor, and both of them are clinically used for the treatment of cancer (Zhang et al., 2007, Vetvicka and Vetvickova, 2012, Schepetkin and
139 Quinn, 2006, Friedman, 2016, Sugiyama, 2016, Zhang et al., 2014). PSK was first isolated in
Japan and developed as a cancer therapeutic agent after an extensive study (Zhang et al.,
2007, Vetvicka and Vetvickova, 2012, Schepetkin and Quinn, 2006, Friedman, 2016,
Sugiyama, 2016 and Zhang et al., 2014). PSK has also been used clinically as a
immunochemotherapeutic agent in combination with one of the chemotherapeutic agents
such as fluoropyrimidines (Sakai et al., 2008), UFT (tegafur/ uracil) (Ohwada et al., 2006;
Yoshitani and Takashima, 2009), S-1 (tegafur/gimeracil/oteracil) (Kono et al., 2008) and
FOLFOX4 (5-FU/folinic acid/oxaliplatin) to treat gastric and colorectal cancer (Shibata et al.,
2011). This treatment protocol can improve long-term prognosis (Sakai et al., 2008), reduce
the risk of recurrence and increase the survival rates in patients with gastric and colorectal
cancer (Ohwada et al., 2006, Shibata et al., 2011). In addition, in vivo studies have shown that
PSK can also enhance the cytotoxicity of such chemotherapeutic drugs (Katoh and Ooshiro,
2007; Kinoshita et al., 2010). For example, PSK could enhance the anti-tumor efficacy of
trastuzumab, and significantly suppressed tumour growth through its immunostimulatory
activities by binding with toll-like receptor (TLR-2) and inhibition of the TGF-β pathway
(Lu et al., 2011, Ono et al., 2012). PSK also showed direct anti-tumour effects by inhibiting the proliferation of various tumour cell lines via the arrest of cell cycle and the induction of apoptosis (Hirahara et al., 2010, Hirahara et al., 2011, Jiménez-Medina et al., 2008).
PSP is another important polysaccharide isolated from C. versicolor in China and was
developed as an anticancer agent after extensive scientific and clinical studies (Cheng and
Leung, 2008). It has been demonstrated that PSP inhibits tumour cell proliferation through
the induction of apoptosis and cell cycle arrest (Hsieh et al., 2006). PSP enhances the
cytotoxicity of certain S-phase targeted chemotherapeutics, such as doxorubicin, etoposide,
camptothecin and cyclophosphamide against human cancer cells (Wan, Sit, and Louie, 2008,
140 Wan et al., 2010). It has been shown that PSP kills cancer cells in the S-phase (Hui et al.,
2005, Wan et al., 2008) and potentiates the efficacy of camptothecin (Wan et al., 2010). In addition, PSP has shown chemo-preventive effect on prostate cancer by targeting prostate cancer stem cell-like populations (Luk et al., 2011). Clinical studies have demonstrated that
PSP in combination with chemotherapeutic agents can reduce chemotherapy-induced side effects and increase survival rate and quality of life of patients (Cheng and Leung, 2008). A new formulation composed of PSP and Astragalus polysaccharide (APS) has shown enhanced immunomodulatory effects and can restore the immunological effects against
Adriamycin induced immunosuppression. These results indicate that the new formulation has better effects than using PSP alone (Jin et al., 2008).
5.1.1.2. Anti-tumour mechanism of PSP and PSK isolated from C. versicolor
Two important mechanisms have been thought to be responsible for the anticancer action of these polysaccharides: they are: (i) indirect anticancer effect though immunomodulatory activity and (ii) direct anticancer effect (Maehare et al., 2012, Jiang et al., 2010, Zhang et al.,
2007).
The indirect anticancer activity of PSP and PSK occurs mainly through their immunomodulatory activities by activation of B lymphocytes, T lymphocytes, macrophages and monocytes, bone marrow cells, natural killer cells, and lymphocyte-activation killer cells
(Cui andChisti, 2003, Zhang et al., 2007, Lu et al., 2011, Zhang et al., 2014, Meng et al.,
2016). This activation of immune cells can induce the production of various cytokines and antibodies such as interferons, interleukin-2 and interleukin-6 (IL-6), tumour necrosis factor
(TNF-α), and immunoglobulin-G (Cui and Chisti, 2003, Zhang et al., 2014, Meng et al.,
2016, Zhang et al., 2017). The immuno-regulation effects of PSK are attributed to the prevention of the apoptosis of circulating T cells, restoration of immunosuppression resulting
141 directly from cancer as well as from the treatment (Maehara et al., 2012) and bone marrow
suppression induced by chemotherapy (Kono et al., 2008, Shibata et al., 2011).
PSP and PSK exhibit direct antitumour effects by: (i) inducing apoptosis and preventing
angiogenesis via the production of glutathione peroxidase and superoxide dismutase
(Maehara et al., 2012), and (ii) reducing the expression of cancer cell cycle related proteins such as cyclin D1, cyclin E and Bcl-2 and up-regulation of p12 (Maehara et al., 2012).
The polysaccharide portion of PSP and PSK has β-(1→3)-glucan structure with branching at
4 and 6 positions. This structure has been suggested to be responsible for the immuno- regulatory and anticancer activities of these polysaccharides (Zhang et al., 2007, Zhang et al.,
2014).
As discussed in the previous sections, there is an abundance of literature on anticancer and
immunomodulatory activities of polysaccharides isolated from medicinal mushrooms. Some of these are listed in Table 2.4 (Chapter 2) along with their specific activities. The biological activities of the polysaccharides given in Table 2.4 have been investigated either by in vivo or in vitro methods.
This chapter aims to investigate immunomodulatory activities of polysaccharides from
Amauroderma rugosum which is a well know medicinal mushroom (Chan et al., 2015). This
mushroom belongs to the Ganodermataceae family. A brief description of traditional use and
previous research on Ganodermataceae polysaccharides is provided below.
142 5.1.2. Anticancer polysaccharides from the Ganodermataceae family of mushroom
Ganodermataceae family (known as “Lingzhi” in China) is an important family of medicinal mushrooms and are widely used for treatment of heart disease, hypertension, hepatitis, diabetes, neurasthenia and cancer in China and other countries (Zhou et al., 2007, Huang et al., 2008). Ganodermataceae mushrooms contain a numbers of bioactive natural constituents such as polysaccharides, ganoderic acids, ergosterols, proteins, unsaturated fatty acids, vitamins and minerals (Niuet al., 2002). However, polysaccharides are the most important constituents of the Lingzhi family of mushrooms. Studies involving polysaccharides and protein-conjugated polysaccharides indicated that a majority of bioactive polysaccharides isolated from the Ganoderma species contain β-(1, 3)-D-glucans with β-(1, 6)-D-
glucopyranosyl branches (Zhou et al., 2007).
5.1.2.1. Polysaccharides from Amauroderma genus
Amauroderma genus belongs to Ganodermataceae family. Mushrooms of this genus are widespread in Malaysia and other tropical areas and comprises about 30 species (Chan
et al., 2015). There is very limited literature on the bioactive constituents from the
Amauroderma species (Pen et al., 2015, Chan et al., 2015). A polysaccharide isolated from
Amauroderma rude exhibited significant immunostimulatory and anticancer activities (Pan
et al., 2015).
Amauroderma rugosum (Blume and T. Nees) Torrendis a traditional anticancer, anti- inflammatory anti-epilepsy and anti-diuretic agent (Dai and Yang, 2008, Chan et al., 2015). A recent study involving the ethanol extracts of A. rugosum showed significant anti- inflammatory activities (Chan et al., 2015). To the best of our knowledge, there is no report on polysaccharides from A. rugosum. Preliminary studies in the authors’ laboratory on water soluble crude polysaccharides from A. rugosum revealed significant immunostimulatory and
143 anticancer activities (Chapter 3). Therefore, this chapter describes the purification of the
polysaccharides from A. rugosum and the investigation of their immunomodulatory and antioxidant properties. It is important to recognise at this point that, suitable modulation of the immune system and addressing the oxidative stress are the two key aspects to be considered while formulating the treatment protocols for cancer (Cui and Chisti, 2003,
Schepetkin and Quinn, 2006, Jeong et al., 2010, Jeong et al., 2012, Zhang et al., 2013, Zhang
et al., 2014, Thambiraj et al., 2015, Yuan et al., 2015, Friedman, 2016, Sugiyama, 2016,
Cheng and Leung, 2008). In addition, structural characterisation of these polysaccharides
using FT-IR and NMR is also described with a view to understanding the structure -activity
relationships.
5.2. Materials and Methods
5.2.1. Materials
500 g of the dried roots of Amauroderma rugosum (known as “JiaZhi” in TCM terminology)
was obtained from Herbal Life Chinese Herbal Medicine shop, Sydney, Australia. A voucher specimen of the plant has been deposited in the laboratory. The plant materials was ground to a fine powder in a kitchen grinder (Inalsa Mixer Grinder) before extraction.
In this study, mouse leukaemia monocyte macrophage cell line (RAW 264.7 macrophages)
was used to test the immunodulatory properties of polysaccharide fractions extracted from
Amaurodermarugosum. RAW 264.7 cells were treated with polysaccharide fractions extracted
from Lobelia chinensisto induce the production of IL-6 and TNF-α, lipopolysaccharides
(LPS) was used as positive control. The ability of polysaccharides to produce IL-6 and TNF-
α were measured by ELISA tests. DPPH ABTS and NO radical scavenging assays were used
as measure of antioxidant activity.
144 5.2.2. Extraction and fractionation of polysaccharides from Amauroderma rugosum
The whole plant of Amaurodermarugosum was powdered, and then homogenized. The sample was then autoclaved for 2 hours at 121 oC, followed by cooling to room temperature before filtration. The procedure followed for the crude polysaccharides preparation is described in Chpater 3 (Staub, 1965, Jeong et al., 2004, Jeong et al., 2006, Jeong et al., 2007, Zhang et al., 2012) A crude polysaccharide extract with a yield of 4g/kg was obtained.
The crude polysaccharide extract was dissolved in 3 mL distilled water at a concentration of
10 mg/mL and then fractionated by size-exclusion chromatography using a Sepharose CL-
6B column (2.4 x 99 cm) equilibrated with distilled water and eluted with distilled water at a
flow rate of 0.51 mL/min. The polysaccharide elution profile was determined by the phenol–
sulfuric acid method and Lowry’s method (Lowry et al., 1951; Dubios et al., 1956; Jeong et
al., 2006; Jeong et al., 2012). The relevant fractions were collected, pooled, and concentrated
by freeze-drying. The entire extraction, de-proteination and purification processes are
illustrated Figure 5.1.
145 Powdered plant material
Hot water extraction (autoclaving) (121 for 2 h) Centrifugation (10,000 rpm for 20 min) ℃
Marc Water Supernatant
Precipitation using 95% EtOH (v/v =1:4 for 24 h)
Centrifuge (10,000 rpm for 20 min)
Freeze Dry
Crude biopolymer
Sevag reagent (1:4 v/v) Votex 20 mins Contrifuge (5000 rpm for 15 mins)
Precipitation using 95% EtOH (Volume:Volume = 1:4)
Centrifuge (10,000 rpm for 20 min)
Freeze Dry
Crude polysaccharides
Fractionation using CL-6B size
ARPs fractions
Fig. 5.1:Flow chart for the extraction of polysaccharides from A. rugosum
5.2.3. Determination of molecular weight of polysaccharide fractions
Estimation of molecular weights of the purified polysaccharide fractions was done on the basis of the elution volume and the molecular weight using a standard dextran series that
146 included T2000 (2000 KDa), T450 (450 KDa), T150 (150 KDa), T70 (70 KDa), T40 (40
KDa) T10 (10 KDa) and glucose at a concentration of 10 mg/mL each for calibration of the
Sepharose CL-6B column (Jeong et al., 2004, Jeong et al., 2012, Zhang et al., 2012).
The linear regression of the data has provided the standard equation, y = -0.2291x + 1.5495 with a high correlation coefficient (R² = 0.9716). This standard curve was used to determine the molecular weight of the extracted polysaccharide fractions.
5.2.4. Analysis of mono-saccharides
The total sugar content was determined by the phenol–sulfuric acid method (Dubios et al.,
1956) using glucose as a reference standard. Total protein content of polysaccharides was determined by the method of Lowry et al., (Lowry et al., 1951). Bovine serum albumin
(BSA) was used as a standard to measure the protein content (Jeong et al., 2004). The monosaccharide composition was analysed by a Hewlett Packard 7890gas chromatograph
(Agilent Technologies, Palo Alto, CA, USA) equipped with a flame-ionization detector using a HP-5 capillary column (30m long, 0.32mm i.d., 0.25 µm film thickness; SGE Analytical
Science Pty Ltd., Ringwood, VIC, Australia). Mono-sugars, produced from the polysaccharide fractions by hydrolysis were acetylated and injected into the GC-FID (Jones andAlbersheim, 1972). Fructose, glucose, xylose, arbinose, galactose, ribose, rhamnose and fucose sugars were used as standards.
5.2.5. Fourier transforms infrared (FT-IR) spectroscopy
Three fractions of A. rugosumwere characterized by FTIR spectroscopy on a TENSOR II
FTIR Spectrometer (BRUKER) at room temperature (25 °C), and spectra were scanned between 4000 and 450 cm−1 with a resolution of 4 cm−1.
147 5.2.6. NMRanalysis
1H, 13C, g-COSY and HSQC spectral data were acquired using a Bruker Avance 400MHz
NMRspectrometer equipped with an inverse detection probe with pulsed field gradient capabilities. ARP-2 (25 mg) was dissolved in 600 µL of D2O (99.9%) containing 0.15%
trimethylsilylpropanoic acid (TSP) (v/v ratio). TSP was used to reference 1H and 13C chemical shifts. All NMR experiments were performed at 25°C.
5.2.7. Bioactivity tests
5.2.7.1 . Antioxidant activity
The DPPH● free radical scavenging assay was conducted using the Blois method. Each fraction (50 μL) was added to a 150 μL of 62.5 μM DPPH●. After 30 min incubation, the
absorbance of the reaction mixture was measured at 492 nm using a microplate reader
(Multiskan 141 EX, Thermo Electron, USA). Ascorbate (vitamin C), an antioxidant, was
used as a positive control. The blank control was distilled water instead of polysaccharides.
The standard concentrations (0, 10, 20, 40, 60, 80, 100 and 200 µM) were prepared in 60%
methanol. The free radical scavenging activity of the polysaccharides was calculated as the ascorbic acid equivalent against the calibration standard curve. The linear regression equation of y = -0.0026x + 0.5578 was obtained with a correlation coefficient (R² = 0.9715).
ABTS●+ free radical reagent was dissolved in water to a 7 mM concentration. ABTS radical
cation (ABTS●+) was produced by reacting ABTS stock solution with 2.45 mM potassium
persulfate (final concentration) and allowing the mixture to stand in the dark at room
temperature for 12–16 hours before use (Li et al., 2011, Alam et al., 2013). For the study of
plant extracts, the ABTS•+solution was diluted with PBS (pH 7.4), to an absorbance of
0.70±0.02 to 0.75±0.025 at 734 nm and equilibrated at 30°C. Ascorbate (vitamin C), an
148 antioxidant, was used as a positive control. The blank control was distilled water instead of
polysaccharides. The standard concentrations (0, 10, 20, 40, 60, 80, 100, 200 and 400 µM)
were prepared in 60% methanol. The free radical scavenging activity of the polysaccharides
was calculated as the ascorbic acid equivalent against the calibration standard curve. The
linear regression equation of y = -0.0019x + 0.7274 was obtained with a good correlation
coefficient (R² = 0.9852).
The OH● scavenging assay was modified on the basis of a method described by de Avellar et
al (2004). Peptide samples or glutathione (50 μL at a final concentration of 1mg/mL in 0.1 M
sodium phosphate buffer, pH 7.4) were first added to a 96-well macroplate followed by the of
addition 50 μL 3 mM 1,10-phenanthroline (in phosphate buffer) and 50 μL of 3 mM
FeSO 4 (in water). To initiate the reaction, 50 μL of 0.01% aqueous H2O2 was added, and
the reaction mixture was covered and incubated at 37°C for 1 hour with shaking. The absorbance was measured at 536 nm using a spectrophotometer. The absorbance was also determined for blank (without polysaccharides and H2O2) and a control (without polysaccharides). The OH• scavenging activity was calculated as below:
● OH scavenging activity (%) = X100% 𝑂𝑂𝑂𝑂 𝑜𝑜𝑓𝑓 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠−𝑂𝑂𝑂𝑂 𝑜𝑜𝑜𝑜 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑂𝑂𝑂𝑂 𝑜𝑜𝑜𝑜 𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏−𝑂𝑂𝑂𝑂 𝑜𝑜𝑜𝑜 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐
5.2.7.2. Assay for the measurement of IL-6 production
Mouse macrophages (RAW 264.7) was first added to DMEM (culture medium containing
1% antibiotic and 5% FBS) and incubated for ≈ 4 days at 37°C in 5% CO2. Cells were then
diluted with the medium to achieve a density of 2x105 cells/mL. The procedure is based on a
published method (Jeong et al., 2004; Yao et al., 2015; Yao et al., 2016; Jeong et al.,
2012;Ni, Wang, Zhang, Guo, and Shi, 2016).
149 The respective intercept and slope values were used in the measurement of IL-6 production
capacity of polysaccharides. The linear regression equation of y = 0.002x + 0.1482 was
obtained with a good correlation coefficient (R² = 0.9935).
5.2.7.3. Assay for the measurement of TNF- α production
RAW 264.7 cells were incubated for 40 hours in culture medium with or without various
doses of polysaccharide fractions (20 µL) or LPS as a positive control in 96-well flat-bottom microtiter plates at a density of 4 x 105 cells per well. TNF-α secreted in the culture
supernatants were quantified using enzyme-linked immunosorbent assay kits (BD
Biosciences, San Jose, CA, USA). Cytokine concentrations were determined by extrapolation from a TNF-α standard curve, according to the manufacturer’s protocol (Joeng et al., 2012).
All measurements are performed in triplicate.
The respective intercept and slope values were used in the measurement of TNF-α production
capacity of polysaccharides. The linear regression equation of y = 0.0021x + 0.1634 was
obtained with a significant correlation coefficient (R² = 0.9921).
5.2.7.4 . Determination of cell viability by MTT assay
Viability of macrophage cells (RAW 264.7) were measured employing a MTT assay
described in the recent publications (Dore et al., 2013, Thambiraj et al., 2015). Briefly, mouse
macrophages were treated by herbal polysaccharides, and incubated at 37 °C for 12 hours.
After that, the supernatant was removed and treated with100 µL of MTT solution (0.2 mg/mL, dissolved in DMEM medium) added to each well and incubated at 37oC for 4 hours.
150 Then, the supernatant was removed and 50 µL of DMSO was added to each well to solubilise the crystalline formazan. The absorbance values were the measured at 595nm.
(%) = 100%
𝑂𝑂𝑂𝑂 𝑜𝑜𝑜𝑜 𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣 𝑋𝑋 𝑂𝑂𝑂𝑂 𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐
5.2.8. Statistical analysis
All data were measured in triplicate and mean ± SD was determined. A one-way analysis of variance (ANOVA) and Duncan’s multiple range tests were used for data analysis . Statistical calculations were performed using OriginPro 8.5 and Excel 2016. The data were considered to be statistically significant if p < 0.05.
5.3. Results and discussion
5.3.1. Fractionation and purification of polysaccharides from A. rugosum
Polysaccharides were extracted from A. rugosum (ARPs) as described in Chapter 2 and were fractionated by Sepharose CL-6B size-exclusion chromatography. Five fractions were selected on the basis of the total carbohydrate elution profiles obtained by phenol-sulfuric acid test. These crude polysaccharide fractions are designated as ARP-1, ARP-2, ARP-3,
ARP-4 and ARP-5 (Fig. 5.2). The results for total carbohydrate contents in each of the fractions is presented in Table 5.1. Based on the calibration curve obtained from analysis of dextran molecular weight standards (Fig. 5.3), the average molecular masses of the various fractions have been determined. The molecular mass of the fraction ARP-1 was found to be relatively high, with an estimated average molecular mass of 1498 kDa (Fig. 5.2).
151 Fig. 5.2: Gel filtration chromatograms of polysaccharide fractions from A. rugosum
This was followed by ARP-2 and ARP-3 which contained polysaccharides with average molecular masses of 450 and 152 kDa respectively (Fig. 5.3), and ARP-5 showed a minimal average molecular weight of 7 kDa. Table 5.1 shows total sugar and protein content of the various polysaccharide fractions. It can be seen from these results that ARP-1 only consisted of glucose (100%). The main mono-sugar present in ARP-2 is glucose (95.04%) with small quantities of mannose (2.47%) and galatcose (2.48%). The monosaccharides in ARP-5 were glucose (75.34%), and galactose (17.15%), with small amount of fucose and mannose (Table
5.1). It is interesting to note that ARP-1 and ARP-2 consisted mainly of glucose indicating that these are possibly glucans. The fractions ARP-3 and ARP-4 yielded only a small quantity in SEC purification and were not sufficient to undertake further biological and structural studies.
152 Fig. 5.3: Calibration curve for the determination of molecular weights of polysaccharides of A. rugosumL based on the elution volume and the molecular mass of standard dextran series of T2000 (2,000 kDa), T450 (450 kDa), T150 (150 kDa), T70 (70 kDa), T40 (40 kDa), T10 (10 kDa) and Glucose (180 Da) (Note: Kav = (Ve – Vo) / (Vt – Vo), Vo is void volume, Vt is total volume, Ve is elution volume).
Table 5.1 Chemical composition (sugar contents) of ARPs
ARP-1 ARP-2 ARP-3* ARP-4* ARP-5 Total Protein (%) 21.57 14.95 6.95 1.31 0.32
Total Carbohydrate (%) 78.43 85.05 93.05 98.69 99.68
Monosaccharide (%)
Rhamnose (%) Ribose (%) Fucose (%) 3.58 3.21 3.3 Arabinose (%) Xylose (%) Mannose (%) 2.47 4.65 6.48 4.21 Glucose (%) 100% 95.04 51.12 50.76 75.34 Galactose (%) 2.48 40.65 39.55 17.15 Unknown (%) *ARP-3 and ARP-4 were minor fractions and yielded very small quantity. Hence, these fractions were not considered for further study
153 5.3.2. FT-IR spectroscopic characterisation of active polysaccharides
Fig. 5.4 presents FT-IR spectra of A. rugosum polysaccharides (ARP-1, ARP-2 and ARP-5).
ARP-1 showed peaks corresponding to β-glycosidic linkage (891 cm-1) (Fig. 5.4a) (Zhang et
al., 2014, Yang and Zhang, 2009, Thambiraj et al., 2015, Pawar and Lalitha, 2014). The
spectrum also showed three strong absorption bands at 995 cm-1, 1061 cm-1 and 1033 cm-1
(corresponding to C-O stretching vibrations related to glycosidic linkage) indicating the
presence of pyranose sugar in ARP-1 (Zhang et al., 2014, Yang and Zhang, 2009, Thambiraj
et al., 2015, Pawar and Lalitha, 2014). The rest of the vibrational bands conform to a
polysaccharide structure. The broad band centred on 3316 cm−1 corresponds to the hydroxyl
stretching vibrations of the polysaccharide and the peak at 2919 cm−1 belongs to C-H
stretching vibrations (Zhang et al., 2014, Yang and Zhang, 2009). These observations lead to
the conclusion that ARP-1 contains pyranose sugars with β-glycosidic linkages. These
spectral feature together with GC findings strongly indicate that ARP-1 is a β-glucan.
ARP-2 (Fig. 5.4b) has peaks at 891 cm−1 indicating the presence of β-glycosidic linkage
(Zhang et al., 2014, Yang and Zhang, 2009, Thambiraj et al., 2015, Pawar and Lalitha, 2014).
The three strong absorption peaks in the range of 1066 cm−1 – 1000 cm−1 (corresponding to
C-O stretching vibrations related to glycosidic linkage) indicating the presence of pyranose sugars in ARP-2 (Zhang et al., 2014, Yang and Zhang, 2009). The rest of the FT-IR bands conform to a polysaccharide structure. These observations confirm that ARP-2 also contains pyranose sugars with β-glycosidic linkages. These findings together with the results presented in section 5.3.1 indicate that ARP-2 may also be a β-glucan. Results presented in sections
5.3.4 and 5.3.5 demonstrates that ARP-1 and ARP-2 are the most active polysaccharides from
A. rugosum. Therefore, a detailed structural analysis of ARP-1 and ARP-2 were carried out
employing NMR spectroscopy (section 5.3.3).
154 a: ARP-1
b: ARP-2
c: ARP-5
Fig 5.4: The FT-IR spectra of the three fractions from A. Rugosum. (a: ARP-1, b: ARP-2, and c: ARP-5)
155 ARP-5 (Fig. 5.4c) has peaks at 893 cm-1 indicating the presence of β-glycosidic linkage
(Zhang et al., 2014, Yang and Zhang, 2009, Thambiraj et al., 2015, PawarandLalitha, 2014).
The spectrum showed three strong absorption peaks in the range of 1061cm−1 – 1008 cm−1
(corresponding to C-O stretching vibrations related to glycosidic linkage) indicating the presence of furanose sugars in ARP-5 (Zhang et al., 2014, Yang and Zhang, 2009). Rest of
the FT-IR bands are consistent with polysaccharide structure.
5.3.3. NMR spectroscopic study
Results presented in sections 5.3.5 and 5.3.6 demonstrate that ARP-1 and ARP-2 are the most
active polysaccharides isolated from A. rugosum. Therefore, a detailed structural analysis of
ARP-1 and ARP-2 was carried out using NMR spectroscopy. 1H and 13C NMR spectra of
ARP-1 and ARP-2 are given in Figures 5.5 and 5.6. From Fig. 5.5, the sugar proton region (3
to 4.6 ppm) has superimposable spectral features for these two polysaccharides (expect that
ARP-1 gave sharper proton resonances than ARP-2). Proton chemical shift of both fractions
are very similar. Figure 5.5 also shows that these polysaccharides are protein conjugates
(region between 0.5 to 2.6 ppm) and this is expected in most bioactive mushroom
polysaccharides reported in the literature (Ng, 1998, Cui and Chisti, 2003, Cheng and Leung,
2008, Friedman, 2016, Sugiyama, 2016, Hattori et al., 2004).
156 Fig. 5.5: 1H NMR spectra of A. rugosum polysaccharides: (A) ARP-1 and (B) ARP-2. Parameter used: number of scans = 128; relaxation delay ≈ 4 sec; data points = 64 k (zero filled to 128 k before Fourier transform).
157 13C NMR spectra of ARP-1 and ARP-2 also display identical spectral features with minor
variations in chemical shift (Fig. 5.6). The detailed 2D-NMR assignments are presented for one of these active polysaccharides (ARP-2). Fig. 5.7 shows g-COSY and HSQC spectra of
ARP-2. Standard assignment protocols were used to obtain proton and carbon chemical shift assignments from 1D- and 2D-NMR spectra (Tables 5.2). These proton and carbon assignments are consistent with those reported in the literature for β-glucans from medicinal mushrooms (Saito et al., 1977; Bubb, 2003; Zhang et al., 2007; Sutivisedsak et al,. 2013; Liu et al., 2014). The DEPT spectrum of ARP-2 (Fig. 5.8) further confirmed the assignments of
C6 carbons (CH2-carbons). Three distinct -CH2 signals were identified at 62.98 ppm (C6 of
D-ring), 63.48 ppm (C6 of A and C rings), and 71.6 ppm (C6 of B-ring) (Table 5.2 and Fig.
5.9). These assignments are consistent with HSQC (Table 5.2 and Fig. 5.5b). NMR findings together with GC and FT-IR results strongly indicate that ARP-2 consists of β- (1→3)-D- glucan with β-(1→6)-D-glucopyranosyl branches (Fig. 5.9).
Very similar 1H and 13C NMR chemical shifts of ARP-1 and ARP-2 (Figs. 5.5 and 5.6) indicate that ARP-1 is also a β- (1→3)-D-glucan with very similar structure (Fig. 5.9).
158 Fig. 5.6. 13C NMR spectra of A. rugosum polysaccharide: (A) ARP-1 and (B) ARP-2. Parameter used: number of scans = 2048; relaxation delay ≈ 3.36 sec; data points = 64 k (zero filled to 128 k before Fourier transform).
159 Fig. 5.7. (a) g-COSY and (b) HSQC spectra of ARP-2. Parameter used for g-COSY: Number of scans = 48; relaxation delay ≈ 1.68 sec; data points: 2048 in t2 dimension and 256 in t1 dimension (zero filled to 4 k and 1 k before Fourier transform in t2 and t1 dimensions respectively). Parameter used for HSQC: Number of scans = 40; relaxation delay ≈ 1.6 sec; data points: 1024 in t2 dimension and 256 in t1 dimension (zero filled to 4 k and 1 k before Fourier transform in t2 and t1 dimensions respectively). PS: sugar rings A, B, C and D are represented in Fig. 5.9.
160 Fig. 5.8. DEPT spectra of ARP-2. Parameter used: Number of scans = 3000; relaxation delay ≈ 3.36 sec; data points = 64 k (zero filled to 128k before Fourier transform); DEPT pulse angel = 135º. PS: sugar rings A, B, C and D are represented in Fig. 5.9.
161 OH
OH OH D ring H O
OH 1 H O β (1, 6) OH OH 6 O O O 5 H OH OH OH CH3 H O O 3 1 3 O 1 OH OH OH OH β (1,3) H β (1,3) C ring A ring B ring H
n
Fig. 5.9. Structure of ARP-2
162 Table 5.2. Chemical shift (ppm) of NMR signal for ARP-2
H1/C1 (ppm) H2/C2 (ppm) H3/C3 (ppm) H4/C4 (ppm) H5/C5 (ppm) H6/C6 (ppm)
A* 4.75 105.47 3.32 75.89 3.75 87.09 3.50 70.91 3.49 78.33 3.89 63.49
B* 4.75 105.47 3.52 75.89 3.75 87.09 3.50 70.91 3.76 78.65 3.85/4.2 71.61
C* 4.75 105.47 3.32 75.89 3.75 87.09 3.50 70.91 3.49 78.33 3.89 63.49
D* 4.53 105.33 3.40 72.37 3.64 77.64 3.50 70.91 3.49 78.33 3.74 62.99
*sugar rings A, B, C and D are represented in Fig. 5.7.
163 Summary of findings of the three structural characterisation techniques:
• GC analysis (section 5.3.1) revealed that ARP-1 and ARP-2 contains mainly glucose
as the building block.
• FT-IR results (section 5.3.2) indicated that ARP-1 and ARP-2 contains pyranose
sugars with β-glycosidic linkages
• NMR results confirm that ARP-1 and ARP-2 have a β-D-(1→3)-glucopyranosyl units
in the backbone with β-(1→6)-glucopyranosyl branches in their structure (Fig. 5.9)
The structures of ARP-1 and ARP-2 determined in this Chapter are similar to that of
schizophyllan isolated from Schizophyllum commune (Saito et al., 1977, Zhang et al., 2007,
Sutivisedsak et al,. 2013).
5.3.4. Antioxidant activities ARPs
The results of free radical scavenging capacity of these major polysaccharide fractions (ARP-
1, ARP-2 and ARP-5) are presented in Table 5.3. All the polysaccharide fractions exhibited
significant DPPH● and ABTS●+scavenging capacity that ranged from 128 µM to 150 µM
ascorbate equiv/g and 266 µM to 308 µM ascorbate equiv/g, respectively. Very high
antioxidant activity was displayed by polysaccharide fractions ARP-1, with DPPH● and
ABTS●+ scavenging capacities more than 60% and 70% respectively (Fig. 5.10 A and B).
164 Table 5.3: Antioxidant activities of polysaccharide fractions of A. rugosum along with their molecular weights.
S.NO Molecular Mass DPPH scavenging activity(Ascorbate ABTS scavenging activity (Ascorbate %OH● (1mg/ml) (kDa) equivalent µM)# equivalent µM)# scavenging ARP-1 1498 150.50 ± 4.07 308.53 ± 4.59 88.02 ARP -2 450 146.27 ± 3.29 289.31 ± 5.65 67.63 ARP-5 7 128.83 ± 2.56 266.96 ± 8.49 53.72 # ABTS andDPPH free radical scavenging activity was measured as equivalent of ascorbic acid. * the Percentage of inhibition of NO production after treatment with polysaccharides. All the values are mean of triplicate determination ± standard deviation (SD).
Fig. 5.10. Concentration dependant radical scavenging activities of two polysaccharide fractions (LCP-1 and LCP-2): (A). DPPH● scavenging activity, (B) ABTS●+ scavenging activity, and (C) OH● scavenging activity. p < 0.05 is considered to be statistically significant (n=3). (% activity has been calculated with respect to negative control)
165 Antioxidant activities of polysaccharide fractions of A. rugosum were also measured by
hydroxyl (OH●) radical scavenging assay and the results are presented in Table 5.3.
Fractions ARP-1 shows extremely high OH● scavenging activity (more than 70 %), followed
by ARP-2 andARP-5 (Fig. 5.10C).
Literature reports indicate that various factors can influence the antioxidant capacity of
botanical polysaccharides (Wang et al., 2013, Kong et al., 2010, Thambiraj et al., 2015, Wang
et al., 2016), which includes: (i) higher average molecular mass (>90 kDa) of polysaccharides
display higher antioxidant activities (Wang et al., 2013), (ii) β-glycosidic linkages present in
the polysaccharide structure is favourable for antioxidant activity (Wang et al., 2013, Kong et
al., 2010), (iii) chemical composition of plant polysaccharides such as glucose and galactose
can improve antioxidant capacity of the polysaccharides (Wang et al., 2013, Thambiraj et al.,
2015, Wang et al., 2016), and (iv) protein conjugated polysaccharides increase radical
scavenging abilities. For instance, PSP and PSK exhibit significant superoxide radical and
hydroxyl radical scavenging activities (Wang et al., 2016, Zhang et al., 2014).
The observed results for ARP-1 are in good agreement with those reported in the literature.
ARP-1 with significant antioxidant activities has (i) large molecular weight (1498 kDa)
(Table 5.2 and Fig. 5.2), (ii) contain β-glycosidic linkages (Fig. 5.3), (iii) binds with protein
(20% protein content) (iv) is mainly composed of glucose (Table 5.1).
5.3.5. Immunomodulatory effects of polysaccharides from A. rugosum
From the results presented in Figure 5.11 it is evident that all the polysaccharides isolated from A. rugosum display significant immunomodulatory activity by increasing the IL-6 and
166 TNF-α production in a dose-dependent manner (Fig. 5.11 A to D). Results presented in
Figures 5.11A and B indicate that ARP-1 and ARP-2 exhibit significantly superior activities than LPS (a well-known positive control) with respect to the production of both TNF-α and
IL-6. As can be seen from Figures 5.11 C and D, the immunostimulatory activities of ARP-1,
ARP-2 and ARP-5 increase sharply when the polysaccharide concentration is greater than 10
µg/mL. The excellent immunostimulatory activities are exemplified by the fact the ARP-1
showed: (i) over 16-fold increase in the production of TNF-α (at 100 µg/mL) (Fig. 5.11C),
and (ii) more than 17-fold increase in the production of IL-6 as compared to the negative
control (untreated macrophages) (Fig. 5.11D). Significant immunostimulatory activities of
ARP-2 at 100µg/mL are exemplified by the fact that: (i) over 13-fold increase in the
production of TNF-α (Fig. 5.11C), and (ii) nearly 15-fold increase in the production of IL-6
compared to the negative control (untreated macrophages) (Fig. 5.11D). ARP-5 showed the least activity amongst the three fractions (however the activities are very significant when compared to polysaccharides from other anticancer herbs). These observations are highly significant and demonstrate that ARP-1, ARP-2 and ARP-5 are prime candidates for stimulating the immune system.
167 Fig. 5.11. Effects of A. rugosum polysaccharides on murine RAW 264.7 macrophages. (A and C): represent Interleukin 6 (IL-6) production, and (B and D): represent tumor necrosis factor-α (TNF-α) production. Statistical difference for the positive control (LPS treated group) and the samples was significant, n = 3, p < 0.05
168 5.3.6. Mechanism of action of ARPs
Information from the literature indicates that β-glucans exhibit immunostimulatory activity
by their interaction with immune cell receptors (Jiang et al., 2010, Cleary et al., 1999,
Batbayar et al., 2012, Brown and Gordon, 2003, Volman et al., 2008, El Enshasy and Hatti-
Kaul, 2013). This mechanism of action stimulates innate as well as adaptive immune
response and leads to the production of various cytokines such as IL-6, IL-1 and TNF-α
(Jiang et al., 2010, Cleary et al., 1999, Batbayar et al., 2012, Brown and Gordon, 2003,
Volman et al., 2008, El Enshasy and Hatti-Kaul, 2013). This property of β-D-(1→3)-glucans
is responsible for their indirect anticancer action via the activation of immune system.
β-glucans are known to interact with two types of immune receptors: (i) Dectin-1 receptor
(also known as β-glucan receptor) which is the most important receptor that can recognise β-
(1→3)-glucans, thereby activating macrophages as well as dendritic cells (DCs) (Jiang et al.,
2010, Cleary et al., 1999, Batbayar et al., 2012, Brown and Gordon, 2003, Volman et al.,
2008, El Enshasy and Hatti-Kaul, 2013), and (ii) Toll like receptors (TLR), especially TLR-2
and TLR-4 which can also recognise β-(1→3)-glucans and activate macrophages to produce
cytokine secretion (Jiang et al., 2010, El Enshasy and Hatti-Kaul, 2013). It is also known that
the molecular mass of β-glucans is one of the important factors in this mechanism of action
and this is consistent with the high immunomodulatory activity determined for ARP-1 which
has largest molecular weight (Cleary et al., 1999, Batbayar et al., 2012, Brown and Gordon,
2003, Volman et al., 2008, El Enshasy and Hatti-Kaul, 2013). The structures of ARP-1 and
ARP-2 (section 5.3.2 and 5.3.3) have been identified in this chapter as β-(1→3)-glucans
branched with β-(1→6) linked side chains, which is favourable for immunomodulatory
activity, is another factor that determines immunostimulatory activities (Cleary et al., 1999,
169 Batbayar et al., 2012, Brown and Gordon, 2003, Volman et al., 2008, El Enshasy and Hatti-
Kaul, 2013).
5.3.7. Cell viability
The effect of ARP-1, ARP-2 and ARP-5 on the viability of mouse macrophage cells is given
in Figure 6. It is clear from these results that, from the polysaccharides from A. rugosum
show significant cell viabilities even at the highest concentration (100 µg/mL) used in this
study (Fig. 5.12) indicating that they are essentially non-toxic. These results are consistent
with literature reports (Jeong et al., 2004, SchepetkinandQuinn, 2006, Jeong et al., 2010,
Jeong et al., 2012) that botanical polysaccharides have lowertoxicity.
120
100
80
60 ARP-1 ARP-2 40 ARP-5 Cell viabilities (%) Cell viabilities 20
0 0 10 25 50 100 Concentration of polysaccharides (µg/mL)
Fig. 5.12. Cell viabilities of isolated polysaccharide fractions from A. rugosum at different concentrations.
5.4. Conclusion
In this study, three polysaccharides were isolated from A. rugosum (ARP-1, ARP-2 and ARP-
5). Two novel immunostimulatory polysaccharides (ARP-1 and ARP-2) having a β-D-
(1→3)-glucan backbone with β-D-(1→6) branched structures have been isolated for the first
time from A. rugosum. These polysaccharides also displayed high antioxidant activities and
170 lowtoxicity demonstrating that they are potentially powerful immunotherapeutic agents.
These results suggest the potential of A. rugosum polysaccharides as natural immuno-
enhancing agents. Findings of this chapter together with the anticancer properties of the crude
polysaccharides from A. rugosum (Chapter 3) strongly demonstrate the potential of ARPs to be used in anticancer formulations.
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182 Chapter Six
Biological and structural characterisation of polysaccharides fractions isolated from Lobelia chinensis Lour Abstract
Lobelia chinensis Lour is an important anticancer herb used in traditional Chinese medicine.
The molecular basis underpinning the anticancer activity is not well understood, but polysaccharides, broadly recognised as having immunomodulatory, antioxidant and anticancer activity, are potentially key active agents. To examine the function of polysaccharides in L. chinensis were isolated and the water-soluble polysaccharides characterised and their activity in biological assays relevant to anticancer function was determined. Water-soluble L.chinensis polysaccharides (LCPs) were extracted and purified using size-exclusion chromatography to obtain two dominant polysaccharides, LCP-1 and
LCP-2, having molecular masses of 1899 kDa and 5.3 kDa respectively. The antioxidant potentials of the isolated polysaccharides were evaluated by measuring radical scavenging activities against DPPH● (2,2-diphenyl-1-picrylhydrazyl radical), ABTS●+ (2,2'-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid) radical), and OH● (hydroxyl radical).
Immunostimulatory activities of LCP-1 and LCP-2 were measured using mouse macrophages. The two polysaccharide fractions displayed significant antioxidant activities and stimulate the production of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6), although LCP-2 is more effective in all assays. Preliminary structural characterisation of both
LCPs was carried out by gas chromatography (GC) and FT-IR techniques. A detailed structural characterisation by NMR was undertaken for the most active fraction (LCP-2) showing that LCP-2 is inulin-type of fructan. The results suggest that another polysaccharide may be viewed as a potential candidate for immunotherapy and treatment of cancer.
183 6.1. Introduction
Lobelia chinensis Lour (Campanulaceae) is an important anticancer herb which has been widely used as diuretic, hemostat, antidote and anticancer agents in traditional Chinese medicine (TCM) for decades (Wei, Qu, and Kou 2010, Li et al., 2016, Shibano et al., 2001).
This herb is used in several traditional anticancer formulations to treat gastric cancer, lung cancer, colorectal cancer and liver cancer (Wei et al., 2010, Li et al., 2016). Modern studies revealed that L. chinensis contains several important classes of bioactive compounds such as piperidine alkaloids, flavonoids, terpenoids andcoumarins (Kuo et al., 2011, Chen et al.,
2014, Yang et al., 2014).Several studies demonstrated that extracts from this herb displayed antibacterial, anti-venom and anticancer activities (Kuo et al., 2011, Chen et al., 2014, Yang et al., 2014). The hot water extracts (decoction) from L. chinensis have significant immunostimulatory and anticancer properties against liver cancer (H22) and gastric cancer
(BC-38) (Wei et al., 2010, Chen et al., 2014, Liu and Zhang, 2009, Shao and Zhang, 2010).
Preliminary scientific studies from authors’ laboratory indicated that the aqueous extracts from L. chinensis have high antioxidant and immunostimulatory activities (Ravipati et al.,
2012). Also, water soluble crude polysaccharides from this herb have shown significant immunomodulatory and anticancer activities (Chapter 3). However literature on the biological activities of purified polysaccharides from L. chinensis and their characterisation is very limited (Li et al., 2016).
Literature reports indicate that botanical polysaccharides exhibit a variety of pharmacological activities that include anticancer, immune regulation and antioxidant activities (Cui and Chisti, 2003, Schepetkin and Quinn, 2006, Jeong et al., 2010, Jeong et al.,
2012, Zhang et al., 2013, Zhang, Koyyalamudi and Reddy, 2014, Thambiraj et al., 2015,
Yuan et al., 2015, Friedman, 2016, Sugiyama, 2016, Cheng
184 and Leung, 2008, Yao, Zhu and Ren, 2016). Over recent decades, herbal polysaccharides are
proving to be ideal candidates for anticancer agents due to their relevant biological activities
with minimal side effects (Schepetkin and Quinn, 2006, Zhang et al., 2014, Zhu et al., 2016,
Kowalczewska et al., 2016, Shan et al., 2017, Hu et al., 2016, Li et al., 2016, Seedevi et al.,
2016, Liu et al., 2017). It is important to recognise that, there are two strategies to develop
suitable polysaccharides for cancer treatment.
(i). Anticancer polysaccharides: All the anticancer polysaccharides known in the literature
have significant immunomodulatory activities (Friedman, 2016, Sugiyama, 2016, Zhang et al., 2014, Schepetkin and Quinn, 2006, Cheng and Leung, 2008, Cui and Chisti, 2003, Daba
and Ezeronye, 2003). For example, polysaccharide Krestin (PSK) and polysaccharopeptide
(PSP) isolated from medicinal mushrooms are clinical anticancer polysaccharides and possess
excellent immuno-regulatory abilities (Friedman, 2016, Sugiyama, 2016, Zhang et al., 2014,
Schepetkin and Quinn, 2006, Cheng and Leung, 2008, Cui and Chisti, 2003, Daba and
Ezeronye, 2003). Therefore, screening for immunomodulatory polysaccharides is an
important step towards the discovery of new anticancer agents.
(ii). Immunomodulatory polysaccharides: It is known from the literature that the most
effective strategy for cancer treatment is a combination therapy (also known as immuno-
chemotherapy) which involves the use of both immuno-regulatory agents and anticancer agents (Friedman, 2016, Sugiyama, 2016, Zhang et al., 2014). Lentinan and schizophyllan are the immuno-regulatory agents used in combination anticancer therapy (Friedman, 2016,
Sugiyama, 2016, Zhang et al., 2014). Therefore, the discovery of polysaccharides with good
185 immuno-regulatory abilities is an alternate strategy to develop immunotherapy agents for
cancer treatment.
Both of these strategies lead to the conclusion that the discovery of immunomodulatory
polysaccharides is important. It is therefore of great interest to investigate herbal
polysaccharides with immunomodulatory potential to develop novel therapeutics for the
treatment of cancer. To the best of our knowledge, there is only one publication involving
isolation of α-Glucan from L. chinensis that displayed significant immunostimulatory activity
(Li et al., 2016).
Hence, the objectives of this study are to isolate polysaccharides from L. chinensis, and evaluate their antioxidant and immunostimulatory activities. It is important to recognise that
suitable modulation of the immune system and addressing the oxidative stress are the two key
aspects to be considered while formulating the treatment protocols for cancer (Cui and Chisti,
2003, Schepetkin and Quinn, 2006; Jeong et al., 2010, Jeong et al., 2012, Zhang et al., 2013,
Zhang et al., 2014, Thambiraj et al., 2015, Yuan et al., 2015, Friedman, 2016, Sugiyama,
2016, Cheng and Leung, 2008, Yao et al., 2016). Hence we sought to evaluate the immunomodualty and antioxidant behaviour of the polysaccharides from L. chinensis. It is also important to carry out structural characterisation of L. chinensis polysaccharides using
FT-IR and NMR spectroscopic techniques with a view to gaining a more detailed
understanding of the structure-activity relationship.
186 6.2. Materials and Methods
6.2.1. Materials
Lobelia chinensis Lour was purchased from Bei Jing Tong Ren Tang, a Chinese Herbal
Medical Centre located in Sydney (Australia). This company has branches all over the world and is well known for their best practice in TCM. The herbs traded in Sydney centre have approvals from both Australian and Chinese Governments.
6.2.2. Chemicals
● ●+ The DPPH , ABTS , bovine serum albumin (BSA), 1,10-phenanthroline, H2O2, dimethyl sulfoxide (DMSO), 95% ethanol, ascorbic acid, Trypan blue 0.4%, sulfanilamide, N-(1-1- napthyl) ethylenediaminedihydrochloride, lipopolysaccharide (LPS) were purchased from
Lomb Scientific Pty Ltd (Australia) and Sigma-Aldrich (Australia). The foetal bovine serum
(FBS), Dulbecco’s modified Eagle’s medium (DMEM) with gluMax, antibiotic, tumour necrosis factor-α (TNF-α) and interleukin (IL-6) (mouse) – ELISA standards and antibodies were purchased from BD Bioscience (USA).
6.2.3. Extraction and fractionation of polysaccharides from Lobelia chinensis
To extract water-soluble compounds, 25 g of Lobelia chinensis Lour was powdered then autoclaved (121°C, 2 hours). Details of the procedure followed for the extraction and purification of polysaccharides is similar to that published previously and is described in the
Chapter 5 (Fig. 5.2.2). (Lowry et al., 1951, Dubois et al., 1956, Zhang et al., 2012, Zhang et al., 2013, Zhang et al., 2014, Jeong et al., 2004, Jeong et al., 2010, Jeong et al., 2012,
Thambiraj et al., 2015). These fractions were collected and concentrated by freeze-drying, then stocked at -20°C for further studies.
187 6.2.4. Determination of average molecular mass
Molecular weights of these fractions were determined by calibrating the Sepharose CL-6B
gel filtration column. Standard dextrans with average molecular weights (from 2000 kDa to 1
kDa) were used to obtain a calibration curve (Jeong et al., 2004, Jeong et al., 2012,
Thambiraj et al., 2015, Zhang et al., 2012, Zhang et al.,2013, Zhang et al.,2014). The
regression of the data for dextran standards produced the calibration curve (y = -0.2291x +
1.5495, with R² = 0.9716). This equation was used to estimate the average molecular weight
of polysaccharides obtained from L. chinensis (LCPs).
6.2.5. Determination of chemical composition
The monosaccharide composition was analysed employing Hewlett Packard 7890B gas
chromatograph Mono-sugars were prepared from polysaccharide fractions and analysis mothed were described in Chapter 3 (Jones and Albersheim, 1972, Zhang et al., 2014).
Fructose, glucose, galactose, xylose, arabinose, rhamnose, fucose and ribose were used as
monosaccharide standards. The detailed information was described in Chapter 5 (Section
5.2.4).
6.2.6. FT-IR analysis
The procedure employed for this assay was similar to that described in Chapter 5 (Section
5.2.5).
6.2.7. NMR analysis
1H, 13C, g-COSY and HSQC spectral data were acquired using a Bruker Advance 400MHz
NMR spectrometer equipped with an inverse detection probe with pulsed field gradient
188 capabilities. LCP-2 (25 mg) was dissolved in 600 µL D2O (99.9%) containing 0.15% TSP
(v/v ratio) and all NMR experiments were performed at 40°C.
6.2.8. Bioactivity tests
6.2.8.1. DPPH● scavenging assay
Blois method (Blois, 1958) was employed in order to determine the DPPH● scavenging abilities of polysaccharides. The procedure employed for this assay was similar to the method in Chapter 5 (Section 5.2.7.1) (Zhang et al., 2012; Zhang et al., 2013; Jeong et al., 2012).
6.2.8.2. ABTS●+radical scavenging assay
ABTS●+ scavenging abilities of polysaccharides were determined using a method published
in the literature (Li et al., 2011; Alam et al., 2013; Thambiraj et al., 2015; Jeong et al., 2016).
The procedure employed for this assay was similar to the method described in Chapter 5
(Section 5.2.7.1).
6.2.8.3. OH● scavenging assay
The OH● scavenging assay was modified on the basis of a method described by de Avellar et al (2004) with minor modification. Detailed procedure for this assay is described in chapter 5
(Section 5.2.7.1).
6.2.8.4. Immunostimulatory activity assays
6.2.8.4.1. Culturing of macrophage cells
Mouse macrophages (RAW 264.7) are first added to DMEM (culture medium containing 1% antibiotic and 5% FBS) and incubated for ≈ 4 days at 37°C in 5% CO2. The approach
followed to implement this procedure is based on published literature (Jeong et al., 2004; Yao
189 et al., 2015; Yao et al., 2016; Jeong et al., 2012; Ni et al., 2016). Details of this assay are discussed in section 5.2.7.2 (Chapter 5).
6.2.8.4.1. IL-6 production
The procedure employed for this assay was similar to that described in Chapter 5 (Section
5.2.7.2). The regression of the standard curve gave a linear equation (y = 0.0019x + 0.0248 with R² = 0.992). The concentrations of IL-6 produced by the polysaccharides were
calculated using the above equation.
6.2.8.4.2. TNF- α production
The procedure employed for this assay was similar to that described in Chapter 5 (Section
5.2.7.3). The regression of the standard curve gave a linear equation (y = 0.0017x + 0.0706
with R² = 0.9875). The concentration of TNF- α produced by the polysaccharide extracts
were calculated using the above equation.
6.2.8.5. Determination of toxicity by MTT test
The procedure employed for this assay was similar to that described in Chapter 5 (Section
5.2.7.4). The absorbance values were the measured at 595nm.
(%) = 100%
𝑂𝑂𝑂𝑂 𝑜𝑜𝑜𝑜 𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣 𝑋𝑋 𝑂𝑂𝑂𝑂 𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝 𝑐𝑐𝑜𝑜𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛
6.2.9. Statistical analysis
All data were measured in triplicate and mean ± SD were determined. A one-way analysis of variance (ANOVA) and Duncan’s multiple range tests were used for analysis of data.
190 Statistical calculations were performed using OriginPro 8.5 and Excel 2016. The data were
considered to be statistically significant if p < 0.05.
6.3. Results and Discussion
6.3.1. Extraction and fractionation of polysaccharides from L. chinensis
Two polysaccharide fractions, namely, LCP-1 and LCP-2, were isolated from L. chinensis after water extraction under high temperature/pressure and purification on a Sepharose CL-
6B column (Fig. 6.1). Calibration of the column with dextran standards (Section 2.4) revealed that the average molecular masses of LCP-1 and LCP-2 were 1899kDa and 5.3kDa respectively (Fig. 6.1).
Fig. 6.1. Isolation and purification of LCPs using Sepharose LC-6B column (two fractions: LCP- 1and LCP-2 were separated). Average molecular masses of LCP-1 and LCP-2 were determined using dextran standards.
191 6.3.2. Chemical compositions of the fractions
Total carbohydrate and total protein composition of isolated fractions (LCP-1 and
LCP-2) were analysed and these results presented in Table 6.1. The carbohydrates are the chief constituents of each fraction (94% of LCP-1 and 96% of LCP-2).
192 Table 6.1. The Chemical composition and monosaccharide contents of LCPs LCP-1 LCP-2* Total Protein (%) 5.95 3.57 Total Carbohydrate (%) 94.05 96.43 Monosaccharide (% ratio) Rhamnose (%) 14.39 Fucose (%) Ribose (%) Arabinose (%) 24.39 Xylose (%) 1.87 Mannose (%) 3.31 43.83* Glucose (%) 18.87 56.17* Galactose (%) 33.80 Unknown (%) 3.46 *As discussed in section 3.2 and 3.3, fructose is the major monosaccharide present in LCP-2. It should be noted that, during the reduction step of GC sample preparation, fructose gets reduced to mannnitol and glucitol. Therefore, mannose and glucose are seen in the GC results instead of fructose.
193 Table 6.1 also presents the monosaccharide compositions of the isolated fractions. It can be
seen from these results that LCP-1 consisted mainly of rhamnose (14.39%), arabinose
(24.39%), galatcose (33.80%), and glucose (18.87%). However, the main monosaccharides from LCP-2 were mannose (43.83%) and glucose (56.17%) (Table 6.1). It is important to note from the literature that reduction of fructose during GC sample preparation yields mannnitol and glucitol (Xu et al., 2005, Fan et al.,2014, Li et al., 2017). It is therefore
expected that LCP-2 may possibly contain glucomannan and/or fructan. Further
characterisation is necessary to understand the structure of LCP-2. FT-IR and NMR
spectroscopic investigations provided further structural details of LCP-2 (section 6.3.3 and
6.3.4).
6.3.3. FT-IR spectroscopy analysis
Figure 6.2 presents FT-IR spectra of L. chinensis polysaccharides LCP-1 and LCP-2. LCP-1
showed peaks consistent with α-glycosidic linkage (755.6 cm-1) and β-glycosidic linkage
(893 cm-1 and 914 cm-1) (Fig. 6.2A) (Zhang et al., 2014, Yang and Zhang, 2009, Thambiraj et
al., 2015, Pawar and Lalitha, 2014). The spectrum also showed three strong absorption bands
at 1014 cm-1, 1073 cm-1 and 1097 cm-1 (corresponding to C-O stretching vibrations related to
glycosidic bonds) indicating the presence of a pyranose sugar in LCP-1 (Zhang et al., 2014,
Yang and Zhang, 2009, Thambiraj et al., 2015, Pawar and Lalitha, 2014). The broad band
centred at 3294 cm−1 corresponds to hydroxyl stretching vibrations of the polysaccharide and
the peaks at 2935 cm−1 are due to C-H stretching vibrations (Zhang et al., 2014, Yang and
Zhang, 2009). The remaining peaks are consistent with a polysaccharide structure.
194 Fig. 6.2. FTIR spectrum of LCP-1 and LCP-2
195 These observations lead to the conclusion that LCP-1 contains pyranose sugars with α- and β-
glycosidic linkages. LCP-2 (Fig. 6.2B) has peaks at 908 cm-1 and 853 cm−1 indicating the
presence of β-glycosidic linkage (Zhang et al., 2014, Yang and Zhang, 2009, Thambiraj et al.,
2015, Pawar and Lalitha, 2014). The two strong absorption peaks in the range of 1011 cm−1 –
1053 cm−1 (corresponding to C-O stretching vibrations related to glycosidic bonds) indicate
the presence of furanose sugars in LCP-2 (Zhang et al., 2014, Yang and Zhang, 2009). As can
be seen from the vibrational bonds in the range 1010 cm-1 to 1100 cm-1 of the FT-IR
spectrum (Fig. 6.2B), there are no pyranose type sugars in LCP-2 (Zhang et al., 2014, Yang and Zhang et al., 2009). The presence of only furanose sugars indicates that LCP-2 might be a fructan (Fan et al., 2014, Li et al., 2017). The broad band centred on 3264cm−1 corresponds
to the hydroxyl stretching vibrations of the polysaccharide and the peaks at 2930.72 cm−1 and
2887cm−1 belongs to C-H stretching vibrations. These observations confirm that LCP-2
contains furanose sugars with β-glycosidic linkages.
6.3.4. NMR spectroscopy analysis
Results presented in this section (section 6.3.5 and section 6.3.6) demonstrate that LCP-2 is
the most active polysaccharide fraction isolated from L. chinensis. Therefore, a detailed
structural analysis of LCP-2 was carried out by NMR spectroscopy. 1H, 13C, g-COSY, and
HSQC spectra of LCP-2 are given in Fig. 6.3. Standard assignment protocols were used to obtain proton and carbon chemical shift assignments (Table 6.2) using 1D- and 2D-NMR
spectra. Absence of intense proton resonances in 4.4-5.5 ppm range (Fig. 6.3A) indicates that
there are no anomeric protons in LCP-2. In addition, LCP-2 showed six intense carbon peaks
indicating that it contains a single monosaccharide unit. These observations together with GC
and FT-IR results strongly indicate that LCP-2 is a fructan with fructose units in the backbone. The weak anomeric doublet at 5.42 ppm in the proton spectrum (Fig. 6.3A) is
196 likely due to the presence of a chain terminating glucose residue indicating a fructan. A set
of weak resonances between 3.3-3.6 ppm confirms the terminal glucose residue.
The fructan backbone structure of LCP-2 can be confirmed from the proton and carbon connectivities in g-COSY and HSQC spectra. The g-COSY spectrum showed several proton connectivities as shown in Table 3. Clearly, the protons H3 to H6 within the fructofuranosyl ring (Fig. 6.3) displayed correlations in the g-COSY spectrum. Also, the two allylic protons
H1ꞌ and H1" attached to C1 showed intense cross peak in the g-COSY spectrum (Table 6.3 and Fig. 6.3C). Five intense cross peaks are clearly observed for the five protonated carbons in the HSQC spectrum (Table 6.3 and Fig. 6.3D). The anomeric carbon (C2) at 105.91 ppm
(Table 6.2 and Fig. 6.3B) did not show any cross peak as there are no protons attached to this carbon (Table 6.3 and Fig. 6.3). Literature shows that β-anomeric 13C resonances are
commonly located between 103 and 105 ppm (Zhang et al., 2014, Yang and Zhang, 2009,
Bubb, 2003). Therefore, the chemical shift value (105.91 ppm) observed for the anomeric
carbon demonstrates that LCP-2 likely has a β-glycosidic linkage (Yang and Zhang, 2009,
Bubb, 2003, Zhang et al., 2014). Proton and carbon chemical shifts of LCP-2 (Table 6.2) are
consistent with the correlations observed in the 2D-NMR spectra. These assignments indicate that LCP-2 contains the following fructan backbone with glucose in the chain terminating position (Fig. 6.4).
197 Table 6.2.1H and 13C chemical shifts (ppm) of LCP-2 from L. chinensis
H1'/H1" C1 H2 C2 H3 C3 H4 C4 H5 C5 H6'/H6" C6
3.7/3.9 63.66 - 105.91 4.24 79.78 4.08 77.08 3.85 83.78 3.77/3.82 64.83
Table 6.3. Proton and carbon correlations from 2D-NMR spectra data of LCP-2 from L. chinensis
1H-1H connectivities from g-COSY (Fig. 4C) 1H-13C connectivities from HSQC (Fig. 4C)
H1ꞌ ↔ H1" C1 ↔ H1
H3 ↔ H4 C2 ↔ No cross peak*
H4 ↔ H5 C3 ↔ H3
H5 ↔ H6 C4 ↔ H4
C5 ↔ H5
C6 ↔ H6
*There is no proton on C2 (no H2) in fructan
198 Fig. 6.3. NMR spectra of LCP-2 isolated from L. chinensis: (A) 1H-NMR, (B) 13C-NMR, (C) g-COSY and (D) HSQC. Parameter used for 1H: Number of scans =128; relaxation delay ≈ 4 sec; data points = 64 k (zero filled to 128k before Fourier transform) Parameter used for 13C: Number of scans = 2048; relaxation delay ≈ 3.36 sec; data points = 64 k (zero filled to 128k before Fourier transform) Parameter used for g-COSY: Number of scans = 8; relaxation delay ≈ 2.2 sec; data points: 2048 in t2 dimension and 1024 in t1 dimension (zero filled to 4 k and 1 k before Fourier transform in t2 and t1 dimensions respectively). Parameter used for HSQC: Number of scans = 16; relaxation delay ≈ 1.6 sec; data points: 1024 in t2 dimension and 512 in t1 dimension (zero filled to 4 k and 1 k before Fourier transform in t2 and t1 dimensions respectively).
199 β (2, 1)
α (1, 2)
Fig. 6.4. Structure of LCP-2: α-D-glucopyranosyl-(1→2)- [β-D-fructofuranosyl -(2, 1)-β-D- fructofuranosyl]n-(2→1)- β-D-fructofuranosyl
200 The chemical shifts of LCP-2 presented in Table 6.2 match well with the inulin type β-
fructans isolated from Saussureacostus (Fan et al., 2014), Artemisia japonica (Li et al.,
2017), Matrisiamaritima (Cérantola et al., 2004) and Ophiopogon japonicas (Xu et al., 2005).
It is interesting to know that, consistent with LCP-2, all the herbal fructans reported in the
literature are low molecular weight polysaccharides (Fan et al., 2014, Li et al., 2017,
Cérantola et al., 2004).
Conclusions from the findings of the three characterisation techniques:
• GC analysis (section 6.3.2) revealed that LCP-2 may contain galactomannan and/or
fructan.
• FT-IR results (section 6.3.3) indicated that LCP-2 contains only furanose sugars with
β-glycosidic linkage (and no pyranose sugars are present)
• NMR results confirm that LCP-2 is a β-D-(2→1) fructofuranoside
Overall finding from these results confirm β-D-(2→1)-fructofuranosyl units in the backbone
with one chain terminating glucosyl residue in the structure of LCP-2 (Fig. 6.4).
6.3.5. Radical scavenging activities
Polysaccharides are often able to act as antioxidants, therefore we wanted to test whether the
L. Chinensis polysaccharides LCP-1 and LCP-2 had this activity. The results of free radical scavenging activities of LCPs (against three different radicals) are presented in Table 6.4.
201 Table 6.4.Radical scavenging activities of LCPs along with their average molecular mass.
Sample Molecular ABTS●+ scavenging activity DPPH● scavenging activity % OH radical (1mg/Lm) mass (kDa) (Ascorbic acid equivalent µM) # (Ascorbic acid equivalent µM) # scavenging * LCP-1 1899 206.12 ± 0.29 145.56 ± 2.32 48.21 ± 1.04 LCP-2 5.3 155.78 ± 0.37 103.76 ± 2.31 44.08 ± 0.86 # ABTS and DPPH free radical scavenging activity was expressed as equivalent of ascorbic acid. *the percentage of inhibition of OH production after treatment with polysaccharides. Values: mean ± standard deviation (n=3)
60 80 C 70 A 50 60 40 50 activity (%) activity
40 30 LCP-1 LCP-1 30 LPC-2 20 LCP-2 20 scavenging 10 10 OH DPPH scavenging activity (%) activity DPPH scavenging 0 0 1000 500 250 125 63 31 16 0 1000 500 250 125 62 Concentration of polysaccharide fractions (µg/ml Concentration of polysaccharide fractions (µg/ml)
Fig. 6.5. Concentration dependant radical scavenging activities of two polysaccharide fractions (LCP-1 and LCP-2): (A). DPPH● scavenging activity, (B) ABTS●+ scavenging activity, and (C) OH● scavenging activity. p< 0.05 is considered to be statistically significant (n=3).
202 The concentration dependant variation of radical scavenging activities of LCP-1 and LCP-2 is
shown in Figure 6.5. As can be seen from these results, both LCP-1 and LCP-2 displayed
significant radical scavenging activities against the three radicals tested. It is clear from these
results that at the higher concentrations tested LCP-1 exhibited better scavenging activity
than LCP-2 (Table 6.4 and Fig. 6.5). This may be due to various factors suggested in the
literature (Wang et al., 2013, Kong et al., 2010, Thambiraj et al., 2015):
• High molecular weight (>90 kDa) is favourable for antioxidant activity
• Presence of β-glycosidic linkages in the main chain/side chain increase antioxidant
activities
• Presence of monosaccharides such as galactose, rhamnose and arabinose increase the
activity
The observed results for LCP-1 are in good agreement with those reported in the literature.
For instance, LCP-1 with high antioxidant activity has large molecular weight (1899 kDa)
(Table 6.4 and Fig. 6.1), and contains β-glycosidic linkages (section 6.3.6) and is composed
of glucose, rhamnose and arabinose in addition to galactose.
It should be noted that the antioxidant activities of LCP-2 are also significant (slightly lower
than LCP-1) (Table 6.4 and Fig. 6.5). An interesting point to be noted here is that the major monosaccharide present in LCP-2 is fructose (section 6.3.3 and 6.3.4). The antioxidant activities of LCP-2 observed in this research are consistent with the literature showing that fructans have good antioxidant activities (Peshev and Van den Ende, 2014). Overall, it may be concluded that the antioxidant activities of LCPs correlate well with fructose, glucose, galactose, arabinose, galactose and rhamnose contents.
203 6.3.6. Immunostimulatory activities of L. chinensis polysaccharides
Immunostimulatory activities of LCP-1 and LCP-2 were measured by treating RAW 264.7
cells with purified fractions. It can be seen from the results that LCP-1 and LCP-2 exhibited immunostimulatory effects as demonstrated by an increase in the production of TNF-α and
IL-6 as a function of polysaccharide concentration (Fig. 6.6). Results presented in Figure
6.6A indicate that LCP-2 exhibits better activity than LPS (positive control) with respect to
the production of TNF-α. From Figures 6.6C and 6.6D, the immunostimulatory activities of
LCP-1 and LCP-2 increase sharply when the polysaccharide concentration is >30 µg/Lm.
Excellent immunostimulatory activities have been observed for LCP-2 at 125 µg/mL as
indicated by (i) over 12-fold increase in the production of TNF-α as compared to the negative control (untreated macrophages) (Fig. 6.6C), and (ii) nearly 8-fold increase in the production of IL-6 (Fig. 6.6D). These observations are very significant and demonstrate that LCP-2 is a highly suitable candidate to stimulate the immune system. Literature reports indicate that fructans can stimulate immune cells by binding to toll-like receptor (TLR) (Peshev and Van den Ende, 2014). It is of interest to note that the high immunostimulatory activities of the
LCPs correlate well with their fructose, glucose, arabinose, galactose and rhamnose contents.
204 1400 1200 C 1000 800 600 LCP-1 LCP-2
- α Production (%) 400 200 TNF 0 0 15.6 31.25 62.5 125 Concentration of biopolymer fractions (µg/ml)
1000 D 800 600
400 LCP-1
- 6 Production (%) LCP-2
IL 200 0 0 15.6 31.25 62.5 125 Concentration of biopolymer fractions (µg/ml)
Fig. 6.6. Effects of L. chinensis polysaccharides on murine RAW 264.7 macrophages. A and C: represent the production of tumour necrosis factor-α (TNF-α), and B and D: represent the production of interleukin 6 (IL-6). LPS was the positive control (100 ng/mL). ELISA assay was used for the quantification of IL-6 and TNF-α production. * Statistical difference for the positive control (LPS treated group) and the samples was significant (p < 0.02, n = 3) ** Statistical difference for the positive control (LPS treated group) and the samples was significant (p < 0.01, n = 3)
205 These observations are consistent with the literature findings that plant polysaccharides
containing the above mono-sugars display immunomodulatory activities (Jiang et al., 2010,
Thambiraj et al., 2015, Zhang et al., 2007).
The structure of LCP-2 (section 6.3.2) has been identified as a β-(2→1)-fructan with an average molecular mass of 5.3 kDa. Fructans of this size are known in the literature to possess significant immunostimulatory activities (Vogt et al., 2013, Franco-Robles and
Lopez, 2015). β-(2→1)-linked fructans with large chain length (consisting of 11 - 60 fructose
units) can directly interact with toll-like receptors (TLRs) present on DCs or macrophages,
and stimulate immune response and produce cytokines (such as IL-6, IL-1 and TNF-α) (Vogt
et al., 2013; Franco-robles and Lopez, 2015; Jiang et al., 2010). It has also been recognised
that the β-fructan chain length is an important factor in this mechanism of action with long
chain length favouring greater immunostimulatory activity (Vogt et al., 2013; Franco-Robles and Lopez, 2015).
It is important to note from the literature that immunomodulatory activity plays an important
role in anticancer activity (Zhang et al., 2014; Schepetkin andQuinn, 2006; Bafna and Mishra,
2009, Jeong et al., 2012, Zhang et al., 2012, He et al., 2016, Zhang et al., 2013, Ayeka, 2016,
Wang et al., 2016, Yu et al., 2017). Hence, L. chinensis polysaccharides isolated in this study are the potential candidates for developing new anticancer agents.
6.3.7. Cell viability
The effect of LCP-1 and LCP-2 on cell viabilities are given in Fig. 6.7. Results demonstrate
that the polysaccharides from L. chinensis showed low toxicities even at the highest
concentrations (125 µg/mL) studied in this research (Fig. 6.7). These findings are in
206 agreement with those reported in the literature (Zhang et al., 2014, Thambiraj et al., 2015, Li
et al., 2016, Kowalczewska et al., 2016, Shan et al., 2017, Hu et al., 2016) that plant
polysaccharides display less toxicity.
110 100 90 80 70
(%) 60 50 LCP-1 40 LCP-2 30 20
Cell viability viability Cell 10 0 15.625 31.25 62.5 125 Concentration of polysaccharides fractions (µg/mL)
Fig. 6.7. Cell viabilities of isolated polysaccharides from L. chinensis
6.4. Conclusion
In this study, two polysaccharide fractions were isolated from L. chinensis (LCP-1 and LCP-
2). Furthermore, a novel immunostimulatory polysaccharide (LCP-2) with a β-D-(2→1)- fructofuranoside structure has been isolated for the first time from L. chinensis. GC, FT-IR and NMR sepectroscopic techniques have been used to unambiguously determine the structure of LCP-2. LCP-1 and LCP-2 showed highly significant immunostimulatory and antioxidant activities. LCP-2 particularly displayed very high immunostimulatory activity as well as low toxicity demonstrating that it is has great potential as an immunotherapeutic agent. These results suggest that the L. chinensis polysaccharides are most likely candidates to be used as natural immuno-enhancing agents. Findings reported in this chapter together
207 with the anticancer properties reported in Chapter 3 strongly demonstrate the potential of
LCP-s to be used in anticancer formulations.
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215 Chapter Seven
Biological and structural characterisation of polysaccharides isolated from Artemisia annua L Abstract
Arimisia annua L is an important anticancer herb used in traditional Chinese medicine. The
molecular basis underpinning the anticancer activity is not fully understood, but polysaccharides, broadly recognised as having immunomodulatory, antioxidant and anticancer activities, are potentially key active agents. To examine the function of polysaccharides in A. annua,water-soluble polysaccharides were extracted from this herb
(AAPs) and purified using size-exclusion chromatography to obtain three dominant
polysaccharides, AAP-1, AAP-2 and AAP-3 having molecular masses of 1685 kDa, 445 kDa
and 5.9 kDa respectively. The antioxidant potentials of the isolated polysaccharides were evaluated by measuring radical scavenging activities against DPPH● (2,2-diphenyl-1-
picrylhydrazyl radical), ABTS●+ (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)
radical), and OH● (hydroxyl radical). Immunostimulatory activities of AAP-1, AAP-2 and
AAP-3 were measured using mouse macrophages. The three polysaccharide fractions display
significant antioxidant activities and stimulate the production of tumour necrosis factor-α
(TNF-α) and interleukin-6 (IL-6), Preliminary structural characterisation of both AAPs were
carried out employing gas chromatography (GC) and FT-IR techniques. The results suggest
that these polysaccharides are potential candidates for immunotherapy and treatment of
cancer.
216 7.1. Introduction
Natural products derived from plants have been used to improve human health for a long time
due to their significant pharmacological properties such as immunomodulatory, anti-
microbial, anti-diabetic, anti-hypertensive and anticancer activities (Huang et al., 2008; Wei
et al., 2007; Zhou et al., 2010). Several types of bioactive compounds have been isolated
from plants which include polysaccharides, anthraquinone glycosides, polyphenols, tannins,
terpenoids, diterpenoids, resins, lignans, alkaloids, protein and peptides (Shah et al., 2013;
Ravishankar et al., 2013; Sghaier et al., 2011; Zhang et al., 2011, Ravipati et al., 2012, Zhang et al., 2012, Zhang et al., 2013, Zhang et al., 2014).
Traditionally, medicinal plants have been used for thousands of years for the treatment of a
wide variety of diseases such as inflammation, malaria, fever, cough, cold and cancer in
China, Japan, Korea, India, and South East Asian countries (Cho, 2010, Huang et al., 2008,
Wei et al., 2007, Zhou et al., 2010, Zhang et al., 2011, Ravipati et al., 2012, Cai et al, 2004).
In this arena, plant based Traditional Chinese Medicinal (TCM) herbs are of enormous interest. Over the years, traditional Chinese practitioners have also developed suitable herbal formulations for the treatment of several diseases (Huang et al., 2008, Wei et al., 2007, Zhou et al., 2010, Cho, 2010). These developments have occurred with the transfer of traditional knowledge and experiences through multiple generations over a long period (Cho, 2010,
Huang et al., 2008; Wei et al., 2007; Zhou et al., 2010) and are available to current
researchers . Corroboration of the wealth of traditional knowledge with modern scientific
techniques has resulted in the discovery of several therapeutic agents (Cho, 2010). Many such
therapeutics derived from traditional medicinal plants are currently in clinical use for the
treatment of life-threatening diseases (Cheng and Leung, 2008, Cui and Chisti, 2003, Daba,
217 and Ezeronye, 2003, Cho, 2010), and some are in clinical trials (Shah et al., 2013) owing to
theirbiological activities such as immunomodulatory, antioxidant, anticancer, antibacterial
and anti-microbial activities (Friedman, 2016, Sugiyama, 2016, Cheng and Leung, 2008,
Cho, 2010, Boulikas and Tsogas, 2008, Dumontet and Jordan, 2010, Shah et al., 2013). For
instance, vinca alkaloids (vinblastine and vincristin), polysaccharide-Krestin (PSK), lentinan,
polysaccharopeptide (PSP) and schizophyllan (SPG) that have been isolated from medicinal
plants/mushrooms are in clinical use for the treatment of various cancers (Shah et al., 2013,
Friedman, 2016, Sugiyama, 2016, Cheng and Leung, 2008). This chapter aims to isolate
polysaccharides from Artmesiaannua L which belongs to the Asteraceae family and
Artemisia genus. A brief introduction to the medicinal plants of this genus is provided in the
following paragraphs.
The Artemisia L (Asteraceae) is one of the largest and diverse genus of plants, which consist of more than 500 species and mainly found in Asia, Europe and North America (Abad et al.,
2012). Many of these plants are widely used for medicinal purposes (Bora and Sharma,
2011, Abad et al., 2012), such as treatment of cancer, malaria, hepatitis, inflammation and infections caused by fungi, bacteria and viruses (Abad et al., 2012; Bora and Sharma, 2011;
Willcox et al., 2009). For instance, Artemisiaannua L, Artemisia scoparia and Artemisia vulgaris have been used as anticancer, anti-inflammatory, anti-microbial and anti-malaria agents (Huang et al., 2008, Abad et al., 2012, Ferreira et al., 2010, Saleh et al., 2014).
Several classes of bioactive compounds such as terpenoids, flavonoids, coumarins, sterols,
acetylenes, volatile components such as essential oils and sesquiterpene lactones have been
isolated from Artemisia genus plants (Bora and Sharma, 2011). Pharmacological activities
218 and important phyto-constituents isolated from this genus are summarised in the following
section.
7.1.1. Ethnopharmacology of the plants from Artemisia genus and their photochemistry
Traditionally, ArtemisiaannuaL. (Asteraceae) is a perennial plant found in the northern parts
of China. The stems of A. annua were traditionally used in Chinese medicine for preventing
malaria, and enhancing immunity in patients(Elfawal et al., 2012). A. annua is also used in
traditional anticancer formulations for the treatment of lung cancer, breast cancer, liver
cancer and stomach cancer (Huang et al., 2008). Several bioactive compounds were isolated
from A. annua, which include sesquiterpenoids, flavonoids, coumarins, triterpenoids,
steroids, phenolics purines, and lipids (Bhakuni et al., 2001, Bilia et al., 2006, Bora and
Sharma, 2011). Among these bioactive components, artemisinin is considered the main active constituent (Bilia et al., 2006, Salminen et al., 2008, Ghantous et al., 2010, Bora and
Sharma, 2011). Artemisinin from A. annua L is wieldy used for treatment of malaria in
China, Vietnam and other countries (Bore and Sharma, 2011, Salminen et al., 2008,
Ghamtous et al., 2010). Several studies reported that artemisinin can also prevent cancer formation by blocking the cell cycle to induce apoptosis, modulation of signalling pathway, inhibition of angiogenesis and prevention of metastasis (Ghantous et al., 2010, Huang et al
2012, Thoppil and Bishayee, 2011). A recent study indicated that polyphenols isolated from
A. annua display significant anticancer activity through inhibition of highly metastatic breast
cancer cells MDA-MB-231 (Ko et al., 2016). To the best our knowledge there is no literature
on polysaccharides isolated from A. annua.
Seeds of Artemisia sphaerocephala Krasch was traditionally used in noodles and other
traditional Chinese foods to improve sensory qualities such as elasticity and chewing quality
219 in northwest China (Ren et al., 2017). According to practitioners of TCM powdered seeds from A. sphaerocephala are useful for the treatment of diabetes and other diseases such as parotitis, tonsillitis, scabies and ileuses (Ren et al., 2017). Phytochemical studies indicated that A. sphaerocephala contains polyphenols (Sun et al., 2016) and triterpenoids (Sun et al.,
2016). Recent reports indicate that a polysaccharide extracted from A. sphaerocephala seeds exhibits biological activities, such as alleviating hyperglycemia, hyperlipidemia and insulin resistance (Ren et al., 2014). A recent study indicated that polysaccharides from A. sphaerocephala displayed immune enhancing abilities by activating RAW 264.7 macrophages and production of cytotoxic molecules (NO) and the cytokines (TNF-α, INF-β, and IL-6) (Ren et al., 2017).
Artemisia argyi (Asteraceae) is a perennial herbaceous plant widely distributed in China, and has been used in TCM for the treatment of ailments such as coughs, colds, headaches, microbial infections, inflammatory diseases, diarrhoea, hepatitis, malaria, cancer, and circulatory disorders (Adams et al., 2006, Khan et al., 2011, Bora and Sharma, 2011).
Phytochemical studies have revealed that A. argyi contains coumarins, glycosides, flavonoids, polyacetylenes, monoterpenes, sterols, triterpenes, sesquiterpene lactones and essential oils (Bao et al., 2013). Some of these compounds displayed biological activities, such as antiulcer (Yoon et al., 2011), antidiabetic (Adams et al., 2012), antioxidant (Ferreira et al., 2010), antimutagenic (Nakasugi et al., 2000), anti-inflammatory (Cai, 2001) and anticancer properties (Adams et al., 2006). Recently a polysaccharide (consisting of N-acetyl- d-glucosamine, glucose, mannose, galactose, rhamnose, arabinose, xylose and ribose) was isolated from A. argyi that displayed anticancer activity against mice Sarcoma 180 (S180) tumour by immunostimulation in a combination therapy along with 5-FU, combination therapy along with 5-FU, resulting in enhanced e survival rates in mice (Bao et al., 2013).
220 Artemisia japonica Thunb is a traditional medicinal herb used for the treatment of fever, headache, malaria, hypertension and tuberculosis in Japan, China, Korea and Vietnam (Giang et al., 2014). Phytochemical studies have shown that A. japonica contains acetylenic , sesquiterpenes and polyphenol compounds (Kwon and Lee, 2001). Pharmacological studies have revealed that the ethanol extracts of A. japonica display anti-malarial activity (Valecha et al, 1994), methanol extracts from A. japonica display significant anticancer activity against the human breast cancer estrogen receptor-α positive T47D (Choi et al., 2013). Recently, an inulin-type fructan (ASKP-1) was isolated from A. japonica that displayed significant anti- arthritic effects (Li et al., 2017).
Artemisia asiatica Nakai has been used in traditional medicine for the treatment of cancer, inflammation, infections and ulcerogenic diseases (Bora and Sharma, 2011). Phytochemical studies revealed that several active components are found in A. asiatica such as flavonoids, sesquiterpenoids, and essential oils such as eupatilin, artemisolide, 1,8-cineole, and terpinen-
4-ol (Kalemba et al., 2002; Kim et al., 2004; Reddy et al., 2006). Eupatilin (DA-9601
(Stillen™)) isolated from the ethanol extract of A. asiatica, is the main active constituent, which has been used clinically to treat gastric mucosal ulcers and inflammation in South
Korea (Jeong et al., 2014). Literature also indicate that eupatilin can inhibit 5-lipoxygenase activity in mastocytoma cells and induce apoptotic death of cultured human promyelocytic leukaemia (HL-60) cells (Koshihara et al., 1983, Seo et al., 2002).
221 7.1.2. Important polysaccharides from the Artemisia genus
Polysaccharides are the ideal candidates to develop novel therapeutic agents for the treatment of cancer due to their excellent biological activities such as immunomodulatory, antioxidant an anticancer activities (Jeong et al., 2004; Schepetkin and Quinn, 2006; Jeong et al., 2010;
Jeong et al., 2012; Zhang et al., 2013; Zhang et al., 2014; Thambiraj et al., 2015). As described in Chapter 2, several polysaccharides purified from medicinal mushrooms have been clinically used for the treatment of various cancers due to their significant anticancer and immunomodulatory activities (Friedman, 2016, Sugiyama, 2016; Daba and Ezeronye 2003;
Ina et al., 2013).
Artemisia L genus plants display significant anticancer and immunomodulatory properties.
Especially, A. annua has been used in anticancer formulations for treating liver cancer, breast cancer, lung cancer and stomach cancer (Huang et al., 2008, Wei et al., 2010, Zhou et al.,
2007). Limited scientific literature exists on the polysaccharides isolated from genus
Artemisia (Chen et al., 2014, Bao et al., 2013, Ren et al., 2017, Li et al., 2017). The polysaccharides isolated from A. apiacea displayed significant immunomodulatory and anticancer activities (Chen et al., 2014), polysaccharide (ASKP-1) isolated from A. sphaerocephala exhibit immune echancing capacity (Ren et al., 2017), an inulin-type fructan from A. japonica displayed significant anti-arthritic effects (Li et al., 2017). To the best of our knowledge, there is no study involving polysaccharides from A. annua which is one of the herbs chosen for a detailed study in this research.
Preliminary studies carried out in Chapter 3 strongly indicate that A. annua has good potential for isolating immunomodulatory and anticancer polysaccharides. The aim of thischapter is to describe the purification of the polysaccharides from A. annua and to evaluate their antioxidant and immunomodulatory activities. It is important to recognise that
222 suitable modulation of the immune system and addressing oxidative stress are the key
aspects to be considered while formulating the treatment protocols for cancer (Cui & Chisti,
2003; Schepetkin & Quinn, 2006; Jeong et al., 2010; Jeong et al., 2012; Zhang et al., 2013;
Zhang et al., 2014; Thambiraj et al.,2015; Friedman, 2016; Sugiyama, 2016, Cheng & Leung,
2008; Yao et al., 2016). Hence, we aim to evaluate immunomodualty, antioxidant potentials
as well as structural characterisation of the polysaccharides from A. annua to understand
structure-activity relationship and the mechanism of action.
7.2. Materials and Methods
7.2.1. Material
Artemisia annuaL(Qing Hao, full plant) have been purchased from Herbal life Chinese
Herbal Medicine shop, Sydney, Australia. The herbs traded in Sydney centre have approvals from both Australian and Chinese Governments.
7.2.2. Chemicals
● ●+ The DPPH , ABTS , 1,10-phenanthroline, H2O2, Dimethyl sulfoxide (DMSO), 95%
ethanol, ascorbic acid, Trypan blue 0.4%, sulfanilamide, N-(1-1-napthyl)
ethylenediaminedihydrochloride, and lipopolysaccharide (LPS) were purchased from Sigma
(Australia) and Lomb Scientific Pty Ltd (Australia). The foetal bovine serum (FBS),
Antibiotics, and Dulbecco’s modified Eagle’s medium (DMEM) with gluMax were purchased from BD Bioscience. The tumour necrosis factor-α (TNF-α) and interleukin (IL-6)
(mouse) – ELISA standards and antibodies were purchased from BD Bioscience (USA).
7.2.3. Extraction and fractionation of polysaccharides from A. annua
To extract water-soluble compounds, 25 g of A. annua was powdered then autoclaved
(121°C, 2 hours). Details of the procedure followed for the extraction and purification of
223 polysaccharides is similar to that published previously and is described in the Chapter 5 (Fig.
5.1). (Zhang et al., 2012, Zhang et al., 2013, Zhang et al., 2014, Jeong et al., 2004, Jeong et al., 2010, Jeong et al., 2012, Thambiraj et al., 2015). These fractions were collected and
concentrated by freeze-drying, then stored at -20°C for further studies.
7.2.4. Determination of molecular weights of polysaccharide fractions
Estimation of molecular weights of the purified polysaccharide fractions was done on the
basis of the elution volume and molecular weight using a standard dextran series that
included T2000 (2000 kDa), T450 (450 kDa), T150 (150 kDa), T70 (70 kDa), T40 (40 kDa)
T10 (10 kDa) and glucose at a concentration of 10 mg/mL each for calibration of the
Sepharose CL-6B column (Jeong et al., 2004, Jeong et al., 2012, Zhang et al., 2012, Zhang et
al.,2013, Zhang et al.,2014).
Linear regression of the data provided the standard equation, y = -0.2291x + 1.5495 with R²
= 0.9716. This standard curve was used to determine the molecular weights of extracted polysaccharide fractions.
7.2.5. Analysis of mono-saccharides
The monosaccharide composition was analysed employing a Hewlett Packard 7890B gas
chromatograph Mono-sugars were prepared from polysaccharide fractions and analysis
method are described in Chapter 3 (Jones and Albersheim, 1972, Zhang et al., 2014).
Fructose, glucose, galactose, xylose, arabinose, rhamnose, fucose and ribose were used as
monosaccharide standards.
7.2.6. Bioactivity tests
7.2.6.1. DPPH● scavenging assay
224 The Blois method (Blois, 1958) was employed to determine the DPPH● scavenging ability of
polysaccharides. The procedure employed for this assay is similar to the method in Chapter 5
(Section 5.2.7.1) (Zhang et al., 2012, Zhang et al., 2013, Jeong et al., 2012).
7.2.6.2. ABTS●+radical scavenging assay
ABTS●+ scavenging abilities of polysaccharides were determined using a published method
(Alam et al., 2013, Thambiraj et al., 2015, Jeong et al., 2016). The procedure employed is
similar to the method described in Chapter 5 (Section 5.2.7.1).
7.2.6.3. OH● radical scavenging assay
The OH● scavenging assay was modified on the basis of a method described by de Avellar et
al (2004) with minor modifications. A detailed procedure for this assay is described in chapter 5 (Section 5.2.7.1).
7.2.6.4. Immunomodulatory activity assays
7.2.6.4.1. Maintenance, preparation and activation of RAW 264.7 macrophages
Mouse macrophages (RAW 264.7) is first added to DMEM (culture medium containing
1% antibiotic and 5% FBS) and incubated for 4 days at 37°C in 5% CO2. Cells were then diluted with the medium to achieve a density of 2x105 cells/mL. The approach
followed to implement this procedure is based on published literature (Jeong et al., 2004,
Jeong et al., 2010, Jeong et al., 2012, Ni et al., 2016).
7.2.6.4.1. Assay for the measurement of IL-6 production
The procedure employed for this assay is similar to that described in Chapter 5 (Section
5.2.7.2).The regression of the standard curve gave a linear equation (y = 0.0019x + 0.0248
225 with R² = 0.992). The concentrations of IL-6 produced by the polysaccharides were
calculated using the above equation.
7.2.6.4.2. Assay for the measurement of TNF- α production
The procedure employed for this assay is similar to that described in Chapter 5 (Section
5.2.7.3).The regression of the standard curve gave a linear equation (y = 0.0017x + 0.0706
with R² = 0.9875). The concentration of TNF-α produced by the polysaccharide extracts was calculated using the above equation.
7.2.6.5. Determination of cell viability by MTT assay
The procedure employed for this assay is similar to that described in Chapter 5 (Section
5.2.7.4).The absorbance values were the measured at 595nm.
(%) = 100%
𝑂𝑂𝑂𝑂 𝑜𝑜𝑜𝑜 𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣 𝑋𝑋 𝑂𝑂𝑂𝑂 𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝 𝑐𝑐𝑜𝑜𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛
7.2.7. Fourier transform infrared (FT-IR) spectroscopy
The procedure employed for this assay is similar to that described in Chapter 5 (Section
5.2.5).
7.2.8. Statistical analysis
Data is expressed as mean ± standard deviation (SD) values. The group mean was compared
using a one-way analysis of variance (AOVAN) and Duncan’s multiple range tests. The
statistical difference was considered significant if p < 0.05. All statistical analyses was
performed using OriginPro 8.5 and Excel 2016.
7.3. Results and discussion
226 7.3.1. Fractionation and purification of polysaccharides from A. annua L
Polysaccharides were extracted from A. annua L (AAPs) as described in Chapter 2and fractionated by Sepharose CL-6B size-exclusion chromatography. Three fractions were selected on the basis of the total carbohydrate elution profile (Fig. 7.1) obtained by the phenol-sulfuric acid method. These crude polysaccharide fractions were designated as AAP-
1, AAP-2, and AAP-3 (Fig. 7.1). Figure 7.1 also gives the protein profile of the fractions.
Fig. 7.1. Size-exclusion chromatogram representing polysaccharide and protein profiles of A. annua
The results for total carbohydrate and protein content in each of the fractions are presented in
Table 7.1. Based on the calibration curve obtained from the analysis of dextran molecular weight standards (Fig. 7.2 and Table 7.2), the average molecular masses of the various fractions were determined. The molecular mass of the fraction AAP-1 was found to be relatively large, with an estimated average molecular mass of 1685 kDa (Fig. 7.2 and Table
7.2). This was followed by AAP-2 and AAP-3 which had average molecular masses of 449 and 5.9 KDa respectively (Table 7.2 and Fig. 7.2). Table 7.1 shows the total sugar and protein
227 contents of the various polysaccharide fractions. Table 7.1 also provides the results of
monosaccharides content of each polysaccharide fraction. It can be seen that AAP-1 consists
mainly of arabinose (33.35%), glucose (8.69%), and galatcose (30.92%). The main mono-
sugar content in AAP-2 are arabinose (36.02%), glucose (22.16%), mannose (18.17%) and
galatcose (16.07%). The monosaccharides detected in AAP-3 are mannose (53.08%),
glucose (46.92%) (Table 7.1). It should be noted that reduction of fructose yields mannnitol
and glucitol during GC sample preparation (Fan, Liu, Bligh, Shi, & Wang, 2014; Li et al.,
2017). Therefore AAP-3 might be 100% fructose . This aspect is discussed along with the
FT-IR results. All of the fractions consisted primarily of glucose, galatcose, mannose, and arbinose.
228 Table 7.1. Chemical composition (sugar content) of polysacharide fractions isolated from A. annua L
AAP-1 AAP-2 AAP-3 Total Protein (%) 29.57 10.95 9.32 Total Carbohydrate (%) 70.43 89.05 90.68 Monosaccharide (% ratio) Rhamnose (%) 9.67 Ribose (%) Fucose (%) Arabinose (%) 33.35 36.02 Xylose (%) 7.67 Mannose (%) 1.44 18.17 53.09 Galactose (%) 30.92 16.07 Glucose (%) 8.69 22.16 46.92 Unknown (%) 8.26 7.58
229 AAP-3 (5.9kDa)
AAP-2 (445kDa) AAP-1
(1685kDa)
Fig. 7.2. Calibration curve for the determination of molecular weights of polysaccharides of A. annua L based on the elution volume and the molecular mass of standard dextran series of T2000 (2,000 kDa), T450 (450 kDa), T150 (150 kDa), T70 (70 kDa), T40 (40 kDa), T10 (10 kDa) and Glucose (180 Da) (Note: Kav = (Ve – Vo) / (Vt – Vo), Vo is void volume, Vt is total volume, Ve is elution volume).
230 Table 7.2. Antioxidant activities of polysaccharide fractions of U. rhyncophylla and T. chinensis along with their molecular weights.
S.NO Molecular ABTS scavenging activity of water DPPH scavenging activity of water ● # # %OH scavenging (1mg/Lm) Mass (kDa) extracts (Ascorbate equivalent µM) extracts (Ascorbate equivalent µM) AAP-1 1685 302.56 ± 3.26 120.35 ± 0.04 78.09 ± 3.49 AAP-2 445 301.00 ± 2.10 118.96 ± 4.31 49.17 ± 2.28 AAP-3 5.9 150.67 ± 4.32 100.46 ± 2.32 47.65 ± 1.80 # ABTS andDPPH free radical scavenging activity was measured as equivalent of ascorbic acid. * the percentage of inhibition of NO production after treatment with polysaccharides. All the values are mean of triplicate determination ± standard deviation (SD).
231 7.3.2. FT-IR spectroscopic characterisation of active polysaccharides
Figure 7.3 presents FT-IR spectra of A. annua polysaccharides (AAP-1, AAP-2 and AAP-2).
AAP-1 showed peaks corresponding to β-glycosidic linkage (914 – 891 cm-1) (Fig. 7.3a)
(Zhang et al., 2014, Yang and Zhang, 2009, Thambiraj et al., 2015). The spectrum of AAP-1
also showed three strong absorption bands at 1018 cm-1, 1047 cm-1 and 1074 cm-1
(corresponding to C-O stretching vibrations related to glycosidic linkage) indicating the presence of pyranose sugar in AAP-1 (Zhang et al., 2014; Yang and Zhang, 2009; Thambiraj et al., 2015; Pawar&Lalitha, 2014). The rest of the vibrational bonds conform to a
polysaccharide structure. The broad band centred on 3394 cm−1 corresponds to the hydroxyl
stretching vibrations of the polysaccharide and the peak at 2934 cm−1 belongs to C-H
stretching vibrations (Zhang et al., 2014; Yang and Zhang, 2009). These observations lead to
the conclusion that AAP-1 contains pyranose sugars with β-glycosidic linkages.
AAP-2 (Fig. 7.3b) has a peak at 914 cm−1 indicating the presence of β-glycosidic linkage
(Zhang et al., 2014, Yang and Zhang, 2009, Thambiraj et al., 2015, Pawar and Lalitha, 2014).
The three strong absorption peaks in the range of 1025 cm−1 – 1072 cm−1 (corresponding to
C-O stretching vibrations related to glycosidic linkage) indicate the presence of pyranose
sugar in AAP-2 (Zhang et al., 2014, Yang and Zhang, 2009). The broad band centred on 3339
cm−1 corresponds to the hydroxyl stretching vibrations of the polysaccharide and the peak at
2944 cm−1 belongs to C-H stretching vibrations. These observations confirm that AAP-2
contains pyranose sugars with β-glycosidic linkages.
AAP-3 (Fig. 7.3c) showed a distinctly different FT-IR spectral feature compared to AAP-1
and AAP-2. Especially the region between 815 to 1025 cm-1 shows different structural
features for AAP-3. The peaks at 873 cm-1 and 815 cm−1 indicate the presence of α- as well as
β-glycosidic linkages (Zhang et al., 2014, Yang and Zhang, 2009, Thambiraj et al., 2015).
Two strong absorption peaks in the range of 1000 cm−1 – 1100 cm−1 (corresponding to C-O
232 stretching vibrations related to glycosidic linkage) indicate the presence of furanose sugars in
AAP-3 (Zhang et al., 2014, Yang and Zhang, 2009). It is important to note the absence of
pyranose sugars in AAP-3 (as there are only two strong absorption bands in the range of 1000
– 1100 cm-1). These spectral feature together with GC findings strongly point to the fact that
AAP-3 is a fructan (Fig. 7.3c) (Zhang et al., 2014; Yang and Zhang et al., 2009; Fan et al.,
2014; Li et al., 2017). The broad band centred on 3280.6 cm−1 corresponds to the hydroxyl stretching vibrations of the polysaccharide and the peaks at 2929 cm−1 and 2889 cm−1 belongs
to C-H stretching vibrations. These observations indicate that AAP-3 mainly contains
furanose sugars with α- and β-glycosidic linkages. These findings together with the results
presented in Chapter 6 for LCP-2 indicates that AAP-3 may be a β- fructofuranoside. It will
be interesting to study the detailed structure of AAP-3 using NMR spectroscopy. This was not possible in this study as the size exclusion separation gave a very small quantity of this
fraction.
233 Fig 7.3. The FT-IR spectra of the three fractions from A. annua. (a: AAP-1, b: AAP-2, and c: AAP-3)
234 7.3.3. Antioxidant activities AAPs
The results of free radical scavenging capacity of these polysaccharide fractions are presented in Tables 7.2 and 7.3. All the polysaccharide fractions exhibited significant DPPH● and
ABTS●+scavenging capacity that ranged from 150 µMto 302 µM ascorbate equiv/g and 100
µM to 120 µM ascorbate equiv/g, respectively. Very high radical scavenging activity was displayed by polysaccharide fraction AAP-1, with respect to DPPH● and ABTS●+ radicals
(more than 60% and 70% respectively) (Table 7.3 and Fig. 7.4A and B).
Antioxidant activities of polysaccharide fractions of A. annua were also measured by hydroxyl (OH●) radical scavenging abilities and the results are presented in Table 7.2 and
Figure 7.4. These results indicate that AAP-1 shows very high OH● scavenging activity (
>70 %), followed by AAP-2 andAAP-3 (Fig. 7.4C). Literature reports indicate that various factors can influence the antioxidant capacities of botanical polysaccharides (Wang et al.,
2013, Kong et al., 2010, Thambiraj et al., 2015, Wang et al., 2016): (i) higher average molecular weight (more than 90 kDa) is favourable for antioxidant (Wang et al., 2013), (ii) presence of β-glycosidic linkages can enhance the activity (Wang et al., 2013; Kong et al.,
2010), (iii) presence of large quantities of glucose, galactose, rhamnose and arabinose can improve the activities (Wang et al., 2013, Thambiraj et al., 2015, Wang et al., 2016); (iv) protein or peptide conjugation will increase the radical scavenging abilities of polysaccharides (Wang et al., 2016, Zhang et al., 2014).
235 Table 7.3. Antioxidant activities of polysaccharide fractions of A. annua.
S.NO ABTS scavenging activity (%) DPPH scavenging activity (%) (1mg/ml) AAP-1 73.37 ± 0.03 68.24 ± 0.98 AAP-2 70.39 ± 0.05 59.99 ± 2.24 AAP-3 59.15 ± 0.08 47.93 ± 0.59
Fig. 7.4. Concentration dependant radical scavenging activities of two polysaccharide fractions (LCP-1 and LCP-2): (A). DPPH● scavenging activity, (B) ABTS●+ scavenging activity, and (C) OH● scavenging activity. p < 0.05 is considered to be statistically significant (n = 3).
236 The results for AAP-1 are in good agreement with those reported in the literature (Wang et
al., 2013; Kong et al., 2010; Zhang et al., 2014; Kong et al., 2010; Thambiraj et al., 2015;
Wang et al., 2016). AAP-1 with significant antioxidant activities has (i) high molecular
weight (1899 kDa) (Table 7.2 and Fig. 7.2), (ii) contains β-glycosidic linkages (Fig. 7.3 in
section 7.3.2), (iii) AAp-1 has protein conjugation (29% protein content), and (iv) AAP-1
contains glucose, rhamnose and arabinose in addition to galactose (Table 7.1).
7.3.4. Immunomodulatory effects of polysaccharides fromA. annuaL
Immunomodulatory activities of the three isolated polysaccharide fractions were determined
by the treatment of RAW 264.7 macrophages with AAPs and the results on the production of
TNF-α and IL-6 are presented in Figure 7.5.
The results from Figure 7.5) show that AAPs has significant immunomodulatory activity as
indicated by increasing IL-6 and TNF-α production in a dose-dependent manner (Figs. 7.5).
As can be seen from Figures 7.5C and D, the immunostimulatory activities of AAP-1, AAP-2 and AAP-3 increases sharply when the polysaccharide concentration was greater than 15
µg/mL. Excellent immunostimulatory activities are observed for AAP-1 at 125 µg/mL as
indicated by: (i) over 14-fold increase in the production of IL-6 compared to the control
(untreated macrophages) (Fig. 7.7C), and (ii) nearly 18-fold increase in the production of
TNF-α (Fig. 7.7D). Immunostimulatory activities of AAP-2 at 125 µg/mL was: (i) had nearly
8-fold increase in the production of IL-6 compared to the control (untreated macrophages)
(Fig. 7.7C), and (ii) nearly 10-fold increase in the production of TNF-α (Fig. 7.7D).
Immunostimulatory activities was observed for AAP-3 at 125 µg/mL as indicated by: (i) over 15-fold increase in the production of IL-6 compared to the control (untreated
237 macrophages) (Fig. 7.7C), and (ii) a more than 12-fold increase in the production of TNF-α
(Fig. 7.7D). These observations are very significant and demonstrate that AAP-1, AAP-2 and
AAP-3 are highly suitable candidates to stimulate the immune system.
Information from literature indicates that toll like receptors (TLR) can recognise and bind
with various types of polysaccharides such as protein-polysaccharide complexes, inulins and
glucans and activate macrophages to promote cytokine secretion (Jiang et al., 2010, Peshev and Van de Ende, 2014). For instance, high molecular weight polysaccharide-protein
complex isolated from Lentinusedodes displayed significant immunomodulatory activities
(Jiang et al., 2010; Franco-robles and Lopez, 2015).
These observations are consistent with literature findings that the plant polysaccharides
display immunomodulatory activities (Jiang et al., 2010; Thambiraj et al., 2015; Zhang et al.,
2007; Vogt et al., 2013; Franco-robles and Lopez, 2015).
238 Fig. 7.5. Effects of A. annua polysaccharides on murine RAW 264.7 macrophages. (A and C): represent Interleukin 6 (IL-6) production, and (B and D): represent tumour necrosis factor-α (TNF-α) production. * Statistical difference for the positive control (LPS treated group) and the samples was significant, n = 3, P < 0.05. ** Statistical difference for the positive control (LPS treated group) and the samples was significant, n = 3, P < 0.03.
239 The structure of AAP-3 (section 7.3.2) has been identified as a β-(2→1)-fructan with an average molecular mass of 5.9 kDa. Fructans of this size are known to possess significant immunostimulatory activities (Vogt et al., 2013, Franco-Robles and Lopez, 2015). β-(2→1)-
linked fructans with large chain length (consisting of 11 - 60 fructose units) can directly
interact with dendritic cells (DCs) (Fig. 7.6). The toll-like receptors (TLRs) present on DCs
and macrophages recognise β-(2→1)-linked fructans (inulin-type fructans) can activate TLR-
2 to stimulate immune response and produce cytokines (such as IL-6, IL-1 and TNF-α) (Vogt
et al., 2013, Franco-robles and Lopez, 2015, Jiang et al., 2010). Fructans can mainly activate
TLR-2, but also TLR-4 and other TLRs to a lesser extent (Vogt et al., 2013, Franco-Robles
and Lopez, 2015). It has also been recognised that the β-fructan chain length is an important
factor in this mechanism of action (Fig. 7.5) with large chain length favouring better
immunostimulatory activity (Vogt et al., 2013; Franco-Robles and Lopez, 2015).
It is important to note that two β-(2→1)-linked fructans (inulin type immunomodulatory
fructans) have been discovered in this research: (i) LCP-2 with an average molecular mass of
5.3 kDa (Chapter 6), and (ii) AAP-3 with an average molecular mass of 5.9 kDa. Consistent
with the literature (Vogt et al., 2013, Franco-robles and Lopez, 2015), AAP-3 with a larger
molecular mass showed significantly higher immunostimulatory activity than LCP-2 (Section
7.3.4; Section 6.3.6 of Chapter 6).
240 Fig. 7.6. Schematic representation of mechanism of immunostimulatory activity induced by inulin-type fructans (β-D-(2→1)-fructan) of longer chain length (Note: Fructans with shorter chain leads to decreased production of cytokines) (Vogt et al., 2013; Franco-Robles & Lopez, 2015).
It is important to note from the literature that (Zhang et al., 2014, Schepetkin and Quinn,
2006, Jeong et al., 2012, Zhang et al., 2012, Zhang et al., 2013, Ayeka, 2016, Wang, 2016,
Yu et al., 2017) the immunomodulatory activity plays an important role in cancer treatment.
Hence, A. annua polysaccharides isolated in this study are a potential candidate for developing anticancer agents.
241 7.3.5. Cell viability
The effect of AAP-1, AAP-2 and AAP-3 on the viability of mouse macrophage cells is given
in Figure 7.7. The results that the polysaccharides from A. annua show significant cell viabilities even at the highest concentration (125 µg/mL) used in this study (Fig. 7.7) indicating that they are less toxic. These results are consistent with literature reports (Jeong et al., 2004; Schepetkin&Quinn, 2006;Jeong et al., 2010; Jeong et al., 2012) that plant polysaccharides are non-toxic.
Fig. 7.7. Cell viabilities of isolated polysaccharide fractions from A. annua at different concentrations.
7.4. Conclusion
Three polysaccharide fractions were isolated from A. annua (AAP-1, AAP-2 and AAP-3).
FT-IR results indicated that AAP-1 and AAP-2 are pyranose containing polysaccharides with
β-linkages. GC and FT-IR findings lead to the conclusion that AAP-3 is a β-fructofuranoside.
It is pertinent to further compare the biological and structural results of LCP-2 (Chapter 6) with those of AAP-3 (this Chapter). Both polysaccharides gave very similar mono-sugar ratios, similar FT-IR spectral features and very comparable biological activities. These results
242 strongly indicate that AAP-3 is a β-fructan. Unfortunately, the fraction corresponding to
AAP-3 yielded low quantity and was not sufficient to undertake a detailed structural study
by NMR spectroscopy which is the subject of future study.
AAPs isolated from A. annua has highly significant immunostimulatory capacity and
antioxidant activities. Especially, AAP-1 has displayed very high immunostimulatory
activity and low toxicity demonstrating that it has high potential as an immune-enhancing
agent. These results suggest the potential of A. annua polysaccharides as a natural immuno-
enhancing agent. The findings of this chapter together with the anticancer properties of crude polysaccharides from A. annua (Chapter 3) strongly demonstrate the potential of AAPs to be
used in anticancer formulations.
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252 Chapter Eight
Evaluation of MRI contrast potential of Gd(III) complexes with bioactive herbal polysaccharides Abstract
Results presented in Chapter 3 demonstrate that the herbal polysaccharides display significant
metal-chelation abilities. To examine this aspect further and to evaluate the MRI contrast
potential of the purified bioactive herbal polysaccharides, NMR relaxivities of three of the
bioactive polysaccharides (LCP-2, AAP-1 and AAP-2) have been studied in this Chapter. The results indicated that the three herbal polysaccharides (LCP-2, AAP-1 and AAP-2)
discovered in this thesis (Chapters 6 and 7) formed stable complexes with Gd(III) ions and
each of these complex has significantly influenced the water relaxation parameters (T1 and
T2). The relaxivity results also indicated that LCP-2 (5.3 kDa) was a T1 contrast agent and the
other two herbal polysaccharides (AAP-1 and AAP-2) with high molecular mass displayed the relaxation behaviour consistent with T2 contrast agents. The relaxivity results on standard
Dextrans (linear α-glucans) with different molecular weights showed that these linear α-
glucans are T1 agents at low as well as high molecular weights (even the highest molecular
weight Dextran with 2000 kDa showed T1 behaviour). However, this is in contrast to the
results on the high molecular weight herbal polysaccharides (AAP-1 and AAP-2) with
attached protein which showed T2 relaxivity behaviour. This is a novel finding that has not
been explored in the literature. The results on MRI performance evaluation using the
phantom samples of these herbal polysaccharides have demonstrated bright contrast (with
LCP-2) and dark contrast (with AAP-1) which are consistent with T1 and T2 agents respectively.
253 8.1. Introduction
Magnetic Resonance Imaging (MRI) is a non-invasive diagnostic imaging technique that is widely used in modern medicine. MRI provides images of the whole human body without exposure to harmful and high energy electromagnetic radiation; rather the technique uses low energy radiofrequency (RF) waves. MRI is based on the principles of nuclear magnetic resonance (NMR) and the object to be imaged must contain atoms with non-zero nuclear spin
(e.g. 1H, 13C, 31P). 1H is the most commonly used nuclear spin in many medical imaging
applications due to its large natural abundance and also due to the presence of large quantity
of water in living systems.
Good resolution, soft tissue contrast and anatomical details can easily be obtained with
modern MRI techniques (Reddy and Smith, 2012). As discussed in Chapter 2, simultaneous
application of external static and varying magnetic fields together with RF pulses provides spatial encoding necessary for MR imaging (Reddy and Smith, 2012; Morris and Liberman,
2005). Rich information content of these images is due to the fact that the MR signal intensity/contrast depends on a variety of physical variables such as proton density and relaxation parameters (T1 and T2) that characterise biological tissue (Moser, 2009). Also, MRI
is very sensitive to soft tissue and fluid flow in addition to its ability to visualise hard tissues
(joints/bones) (Li and Poon, 1988; Morris and Liberman, 2005). Due to these advantages
MRI has become extremely important tool in modern clinical diagnostics (Morris and
Liberman, 2005; Yao et al., 1996; Koh and Collins, 2012; Bookheimer, 1996).
The NMR signal arising from the nuclei present in the imaging object is spatially encoded and imaged by applying linear magnetic field gradients (Mansfield, 1982) (Section 8.1.3).
The intensity of MR images of biological tissue depends on proton density, the relaxation
254 times T1 and T2, and the pulse sequence parameters (Reddy and Smith, 2012; Callaghan,
1991). It is well known that, biological tissues are characterised by large variations in their proton density and the relaxation parameters T1 and T2 (Callaghan, 1991). It is also important to note that the relaxation parameters change dramatically when changes occur in the pathological state of the tissue (Callaghan, 1991). Thus, variations in the nature of biological tissue and their pathological state greatly influence the intensity and the contrast of MR images. It is therefore clear that MR imaging has inbuilt ability to provide contrast between different tissue types and different organs. Especially, such a contrast is prominent if there is underlying pathology in the tissue being imaged (e.g. presence of cancer) (Weishaupt et al.,
2008; Hennig et al., 2003; Smith et al., 2001). Therefore, MRI provides natural image contrast even without the use of any contrast agents and this is the unique characteristics of this imaging modality (Callaghan, 1991). In addition, the opportunity of using paramagnetic metal complexes to improve image contrast makes this technique an extremely valuable and sensitive diagnostic method to determine the underlying pathological state of soft tissue in human body (Caravan, 1999). Furthermore, the non-invasive nature of MRI makes it a versatile and indispensable clinical tool.
As discussed above, the proton NMR relaxation parameters (T1 and T2) are extremely
important to determine tissue contrast in MRI. Hence, section 8.1.1 provides general
principles of NMR, relaxation phenomenon and also a description of water proton relaxation
in biological tissue. A description of the influence of paramagnetic contrast agents on water
proton relaxivities is then provided (section 8.1.2), before providing a brief description of the
methods to measure NMR relaxation parameters (T1 and T2). A brief discussion on MRI principles is then provided in section 8.1.3, followed by a short sketch of MRI pulse sequences employed in this chapter to evaluate the performance of Gd (III) complexes of bioactive herbal polysaccharides discovered in this thesis (Chapters 5 to 7). A description of
255 the instrumentation used for realaxivity measurements and the micro-imaging system used
for MRI experiments is provided in section 8.1.4.Section 8.2 deals with the experimental
methodology and the protocols employed to evaluate the performance of herbal
polysaccharide based macromolecular Gd (III) contrast agents. Finally, the results on
relaxivities and MRI performance evaluation are presented and discussed in section 8.3,
before providing concluding remarks.
8.1.1. Principles of NMR spectroscopy
Nuclei with non-zero spin act as tiny magnets that are in random orientation in an initial state
with no applied magnetic field. When a steady magnetic field (B0) is applied, the nuclear
magnetic moments tend to either align with or align against the magnetic field. The aligned
spins are associated with lower energy compared to those opposing the magnetic field.
According to classical description, for a spin I=½ nucleus (e.g. 1H), the vector sum of these
oriented nuclear magnetic moments is known as the net magnetisation (M0) (Derome, 1987).
The net nuclear magnetisation (M0) interacts with applied magnetic field (B0) leading to the
classical precessional motion of nuclear spins with respect to the applied magnetic field
(usually Z-axis). The angular frequency of precession is given by ω0 = γB0; with ω0 = 2πv0,
where v0 is the frequency expressed in cycles per second (Hz) (James, 1998). It is important
to note that the classical and quantum mechanical descriptions of nuclear spins, in a magnetic
field, lead to the identical results in the case of non-interacting isolated spins (Slichter , 1990;
Slitcher, 1978; Derome, 1987). However, the classical mechanics fails to accurately describe
the spin system for bulk samples involving several interacting spins (Slitcher, 1978). In
modern NMR spectroscopy, radiofrequency (RF) field is applied in the form of narrow pulses
of a few microseconds duration. The field generated by RF pulses is called the B1 magnetic field (alternating magnetic field). The B1 field is always applied in a direction perpendicular
256 to the direction of the main magnetic field (e.g. X-axis). Application of RF pulses cause
rotations of magnetisation by a well-defined angle (as viewed from a fictitious rotating
frame). For example, a 90o pulse applied along X-axis rotates the magnetisation vector clockwise by 90o about the X-axis (down to the Y-axis) and a 180o pulse rotates the
magnetisation vector by 180o (down to negative Z-direction). Such RF pulses provide the
desired responses from an NMR sample and all the modern NMR/MRI experiments are based
on suitably designed RF pulse sequences. For example, a single RF pulse yields an FID (free
induction decay) signal and a two pulse sequence yields a spin-echo signal. A detailed
discussion of modern pulsed NMR spectroscopy is beyond the scope of this thesis and
excellent text books are available for such purposes (Slichter , 1990).
8.1.1.1. NMR relaxation phenomenon
Application of a resonant RF pulse disturbs the magnetisation (M0) from its thermal
equilibrium (Abraham et al., 1988). Subsequently, a process known as relaxation will restore
the equilibrium magnetisation. The process by which the Z-magnetisation (Mz) returns to
thermal equilibrium is known as spin-lattice relaxation (also known as longitudinal
relaxation) and is characterised by the time constant T1. Concurrent to spin-lattice relaxation, another process known as spin-spin relaxation (also known as transverse relaxation) takes place, when a spin system is disturbed from thermal equilibrium. Spin-spin relaxation process
is characterised by the time constant T2 and involves a dephasing process amongst the spins.
An understanding of spin-lattice and spin-spin relaxation processes is very important to gain
insight into the NMR and MRI experiments. A brief description of these processes is given
below.
257 8.1.1.2. Spin-lattice relaxation
After the application of a RF pulse, the Z-magnetisation (Mz) returns to thermal equilibrium
by spin-lattice relaxation process. This process can be described by the following differential
equation (Callaghan, 1991):
dM M0 Mz z = Eq 8.1 d − 𝑡𝑡 1 𝑇𝑇
Where, M0 is the Z-magnetisation at thermal equilibrium and T1 is the time constant that
describes the return of Z-magnetisation (Mz) towards the equilibrium. This time constant is known as spin-lattice relaxation time and the solution of Equation 8.1 is given by:
Eq 8.2 Mz( ) = M0(1 2 ) 𝑡𝑡 − �𝑇𝑇 1 𝑡𝑡 − 𝑒𝑒 As the name suggests, the process of spin-lattice relaxation involves an exchange of spin
energy to surrounding thermal reservoir, known as lattice. The dominant sources of
interactions responsible for spin-lattice relaxation are magnetic dipolar couplings that are
modulated by molecular motions.
8.1.1.3. Spin-spin relaxation
After the application of a RF pulse (e.g. 90ᵒ RF pulse), the net magnetisation in the X-Y-plane
undergoes transverse relaxation. During this process, the spins in the transverse plane
exchange their energy with other spins to reach a thermal equilibrium among them. This
process is known as spin-spin relaxation and the characteristic time constant that governs the
process is denoted by T2. Spin-spin relaxation is described by the following
phenomenological equation (Mansfield, 1982):
, = , Eq 8.3 𝑑𝑑𝑴𝑴𝑥𝑥 𝑦𝑦 𝑴𝑴𝑥𝑥 𝑦𝑦 𝑑𝑑𝑑𝑑 − 𝑇𝑇2
258 Where, Mx,y denotes the transverse magnetisation along X- or Y-axis. A simple exponential solution of Equation 8.3 is given by (Abraham et al., 1988):
Mx,y( ) = Mx,y(0) Eq 8.4 −𝑡𝑡 �𝑇𝑇2 𝑡𝑡 𝑒𝑒 T2 values of water protons in biological tissue can be determined from NMR experiments
employing Carr-Purcel-Meiboom-Gill pulse sequence (Bodenhausen, 1983) and an
exponential regression of the data with Equation 8.4.
8.1.1.4. Factors influencing relaxation rates
It is important to note that the relaxation rates are dependent on many factors such as
molecular structure, size, solution viscosity and temperature. Relaxation process can be
visualised as transitions between the spin energy levels caused by randomly fluctuating
magnetic fields produced by molecular motions (Derome, 1987).
Such randomly fluctuating fields generated by interactions within the spins and also with the
environment. These fields become time dependant by molecular rotations (tumbling) and
other motions (Geraldes, 1999; Botta, 2000).
Important factors that influence the relaxation rate are: (i) strength of the interaction
responsible for relaxation which depends on the detailed knowledge of the origin of the
random fields and often it is related to geometry (Derome, 1987), and (ii) spectral density
which has a large effect on relaxation rates and is an important factor to consider when
discussing T1 and T2 mechanisms (Derome, 1987). Spectral density is a measure of the intensity of fluctuating field created by random molecular motions (characterised by molecular rotational correlation time, τc) (Derome, 1987).
259 8.1.1.5. Relaxation of water protons in biological tissues
1H nuclei are present in the biological tissues in the form of water and other bio-molecules.
Water is the most abundant amongst these various molecular species and exists in two states
in biological tissue (free and bound). The 1H (proton) NMR signal from water is the strongest
and is most commonly used for in-vivo MRI (Reddy and Smith, 2012). As discussed before,
dipolar interactions between the water protons and the random molecular motions produce
fluctuating magnetic fields that cause nuclear relaxivities (Reddy and Smith, 2012;
Callaghan, 1991). Due to the dependence of nuclear spin relaxation on molecular motion the
proton NMR signal (and hence MRI) is very sensitive to the physical environment of the host
molecule (e.g. water molecules in different biological tissue) (Reddy and Smith, 2012). This
sensitivity is reflected in the variation of MRI signal intensity from one type of tissue to
another. Therefore, nuclear spin relaxation is an important property that can be used to
improve contrast, which is crucial for diagnostic MR images (Reddy and Smith, 2012).
These interactions fluctuate as the water molecules undergo rotational reorientation and the
resulting fluctuating fields induce transitions responsible for relaxation. For free water the
rotational reorientation is fast and the value of T1 is large (2.72 s) (Reddy and Smith, 2012;
Callaghan, 1991). The water molecules in different states (bound and free states) exchange with one another and this exchange correlation also contributes to relaxation. Another important contribution to spin relaxation in tissue arises from the fast exchange of labile protons on biopolymers with water protons. Water molecules and water protons therefore undergo complex dynamics that varies significantly from one type of tissue to another.
Variations in water dynamics in different types of tissue cause variation of fluctuating dipolar interaction responsible for spin relaxation. These significant differences in proton relaxation behaviour (T1 and T2) of water molecules in different tissues arise because of the variation in
260 water dynamics (Reddy and Smith, 2012). In addition, the dynamics of water molecules also
changes with pathological state of the tissues (such as cancer) resulting in variation in
relaxivities.
8.1.2. Influence of paramagnetic contrast agents on water proton relaxivities
Addition of agents with electronic spins (such as paramagnetic agents) introduces further
pathways of relaxivities of water protons in biological tissue. These pathways are caused by
electronic spin-nuclear spin interactions, which are extremely strong compared to nuclear
spin-spin interactions. Paramagnetic agents therefore improve relaxivities of water in
biological systems by many folds.
Larger electron magnetic moment of paramagnetic metals (e.g. Gd3+ in contrast agents) leads
to larger strength of the local magnetic field fluctuations at the site of the protons and is much
more effective in promoting nuclear spin relaxation when compared to the nuclear-nuclear
dipolar interaction. Hence, the paramagnetic contrast agents provide very efficient nuclear
relaxation (Jame, 1998).
The strength of the interaction between paramagnetic metal centre and the water hydrogen nuclei depends on several factors and a relation was first described by Bloembergen et al.,
(Bloembergen, 1957; Bloembergen and Morgan, 1961) and subsequently modified by others
(Runge e al., 1983).
= 𝟐𝟐 𝟐𝟐 𝟐𝟐 Eq 8.5 𝟏𝟏 𝟏𝟏𝟏𝟏𝝅𝝅 𝜸𝜸 𝝐𝝐𝝁𝝁 𝑵𝑵 �𝑻𝑻𝟏𝟏� 𝟓𝟓𝟓𝟓𝟓𝟓 Where µ is the effective magnetic moment of the paramagnetic ions, ϵ is the viscosity of the
solvent, k is Boltzman’s constant, T is the absolute temperature, γ is the gyromagnetic ratio
(for the hydrogen nucleus), and N is the number of paramagnetic ions per unit volume
261 (concentration) (Runge e al., 1983). From this equation, it is apparent that the spin-lattice
relaxation rate (r1) (and also, spin-spin relaxation rate, r2) is directly dependent on: (i) the
concentration of the paramagnetic agent (N) and (ii) the square of the effective magnetic
moment (µ2) of the paramagnetic ions. By appropriate selection of these two factors, one can achieve a greater decrease of the T1 and T2 parameters (faster relaxation rate). It is also important to remember while developing contrast agents that the paramagnetic effect falls off rapidly with the sixth power of the distance between the paramagnetic centre and the resonating nucleus (1/r6) (Runge e al., 1983). Therefore, facile access of water molecules
closer to coordination sites increases paramagnetic relaxation efficiency. The value of “r”
which is the distance between the paramagnetic metal centre and bound water protons mainly
depends on water coordination strength and the exchange rate of water molecules between
the inner coordination sphere and the bulk. Also, the effect of a paramagnetic contrast agent
increases with the number of unpaired electrons as the magnetic moment (µ) increases. For
example, Gd3+ seven unpaired electrons have a large electron paramagnetic moment and is an
ideal metal ion to design contrast agents (Chen et al., 1984).
8.1.2.1. Mechanism of paramagnetic relaxation of water molecules
The paramagnetic relaxation of water molecules in the presence of paramagnetic metals is
characterised by two mechanisms: inner sphere (IS) and outer sphere (OS) mechanisms. The
inner sphere relaxation relies on the exchange of water molecules between the inner
coordination sphere of the metal ion and the bulk solvent. This exchange helps to propagate
the influence of paramagnetic effect to the totality of the bulk water (Burtea et al., 2008).
This exchange process directly influences the coordination sphere residence time (τm) of
water molecules and has pronounced effect on paramagnetic relaxivity (Mercier, 1998).46
A strong metal-water coordination means relatively longer τm which will directly contribute
262 to faster relaxivity as long as the system is in the fast exchange limit (Mercier, 1998). It
should be noted that fast exchange limit is important in order to allow the propagation of
paramagnetic effects to the bulk. This means that τm should not be too long.
The contribution of the inner sphere mechanism to relaxivity is given by (Burtea et al., 2008;
Chan et al., 2007):
= Eq 8.6 𝑰𝑰𝑰𝑰 𝟏𝟏 𝑹𝑹𝟏𝟏 𝑪𝑪𝑪𝑪 𝑻𝑻𝟏𝟏𝟏𝟏+𝝉𝝉𝑴𝑴 Where:
C = the concentration of the paramagnetic metal complex
q = the number of water molecules in the first coordination sphere
τm = the residence life time of water molecules in the IS
The outer sphere contribution to the paramagnetic relaxation is due to the long distance dipolar interaction between the paramagnetic metal centre and the hydrogen atoms of water molecules. This mechanism is governed by the relative translational diffusion (D) of the paramagnetic centre and the bulk water (Burtea et al., 2008).
Some of the paramagnetic contrast agents (e.g. small molecular contrast agents) produce similar improvements in both T1 and T2 relaxivities. These are known as T1-contrast agents that produce brighter MR images. However, certain class of paramagnetic agents (e.g. macrocyclic contrast agents) lead to much larger improvement in T2 relaxivities when
compared to T1 relaxivities. These are known as T2-contrast agents that produce darker MR
images. Both types of paramagnetic contrast agents are extremely useful in diagnostic MRI
and the focus of this chapter has been to evaluate the potential of Gd (III) complexes of
bioactive polysaccharides as T1 and T2 agents.
263 8.1.3. Basic principles of magnetic resonance imaging (MRI)
In MRI, linearly varying magnetic fields (known as ‘field gradients’) are applied to the
sample in addition to the static B0 field. Under the influence of such field gradients, the
Larmor frequencies of the spins display linear spatial dependence (Callaghan, 1991). This
linear relationship between the spatial coordinates of the spins and the Larmor frequencies forms the basis of MRI (Reddy and Smith, 2012). When a single field gradient is applied, a mathematical expression for such an encoding is given by,
( ) = Eq. 8.7 𝛄𝛄 𝐁𝐁𝐨𝐨+𝐆𝐆𝐗𝐗 𝛎𝛎𝐩𝐩 𝟐𝟐𝟐𝟐
Where νp is the larmor frequency in Hz,γ is the gyromagnetic ratio of proton, B0is the static
magnetic field strength, Gx is the magnetic field gradient applied along X-direction.
The application of a selective RF pulse (pulse with narrow excitation bandwidth) then excites only a specific region of the sample. In the case of a single X-gradient, NMR signal is obtained from the spins in YZ-plane. This is equivalent to obtaining a projection of the object perpendicular to X-axis (plane sensitive image) (Bovey and Mirau, 1996).
Simultaneous application of double or triple orthogonal gradients will result in a line sensitive or point sensitive images respectively from the object to be imaged. For example, with a single gradient field, simply changing the direction of the gradient field can be produced several two-dimensional projections of the object (Bovey and Mirau, 1996). These
projections can then be used to reconstruct the image of the original object by means of
‘projection reconstruction techniques’ (Bovey and Mirau, 1996). It should be noted that the
original method of Lauterbur (Bovey and Mirau, 1996) was based on the reconstruction of
objects from the projections taken from rotating an object around an axis perpendicular to the
linear gradient direction (or vice versa), then applying the projection reconstruction algorithm
264 to generate a matrix representation of the image in question. This method is referred to as
‘plane sensitive’ projection reconstruction technique. While this is conceptually a simple
method, it takes a long time to obtain reasonable MR images. Another disadvantage with
projection reconstruction based MRI is the relatively poor image quality (Reddy and Smith,
2012; Callaghan, 1991).
The most commonly used method in modern MRI instruments is based on Fourier imaging
(Kumar et al., 1975). This technique eliminates most of the disadvantages of the projection
reconstruction method. The Fourier MRI method involves the application of a sequence of
pulsed orthogonal linear magnetic field gradients to an object during the FID or a spin echo
signal (Kumar et al., 1975). Detailed description of the Fourier MRI method can be found in
the literature (Reddy and Smith, 2012). MRI pulse sequences/techniques used in this research
include, (i) two-dimensional spin-echo based Fourier imaging, (ii) two-dimensional inversion
recovery spin-echo Fourier imaging and (iii) CPMG pulse sequence based Fourier imaging
techniques are used in this research. Detailed descriptions of these methods can be found in
the literature (Reddy and Smith, 2012).
8.2. Experimental Methodology
Varian Unity plus 300 MHz NMR spectrometer was used for the measurement of NMR
relaxation parameters reported in this chapter. Bruker Avance 600 MHz microimaging
system was employed for performing the MRI experiments reported in this chapter.
8.2.1. NMR relaxation experiments
Materials
Gadolinium(III) chloride (AR grade) used in these experiments were purchased from Sigma
Aldrich. Precision NMR tubes (5 mm x 7” L) were purchased from Wilmad. Pure de-ionised
265 water (resistivity 1 – 10 MΩ.cm and conductivity 1.0 – 0.1 µS/cm) was used to prepare
solutions of paramagnetic metal complexes with the herbal polysaccharides discovered in this
research (Chapters 6 and 7). A concentrated stock solution of each paramagnetic metal
complex in pure water was serial diluted to make the required concentrations for the NMR
relaxation measurements.
1 o H NMR relaxation times T1 and T2 (25 C) of the water protons (H2O samples) were measured at 300 MHz on a Varian Unity plus 300 MHz NMR spectrometer equipped with a wide bore magnet and a micro-imaging probe. T1 and T2 measurements have been conducted
at various concentrations of paramagnetic metal complexes of bioactive polysaccharides. The
T1 and T2 measurements were conducted at different concentrations as follows: (i) 2.88 mM,
1.63 mM and 1.15 mM (Dextran-10), (ii) 3.57 mM, 2.6 mM, 1.8 mM (LCP-2), and (iii) 2.5
mM, 0.9mM and 0.3 mM (AAP-1). These concentrations are close to the concentrations used
to test the MRI contrast performances of the Gd-metal complexes of bioactive
polysaccharides.
Probe was tuned with every sample before conducting T1 and T2 experiments and the RF
pulses have been calibrated to ensure the accuracy of results. The spin-lattice relaxation
time(T1)was determined by using a standard inversion recovery pulse sequence (180° – τ –
90°) followed by signal detection (Vold et al., 1968;Waugh et al., 1968).Recovery of the
inverted signal was recorded as a function of the inversion delay (Ti) between the pulses. The
resulting data was fitted to the exponential function (Eq 8.8) to determine T1 values:
S(τ) = S(0) [1 – 2exp(-Ti/T1)] Eq 8.8
Where, S(0) is the signal intensity when Ti = 0, and S(τ) is the signal intensity for different values of Ti.. All T1 experiments were conducted with a minimum array size of 15 Ti values
266 (at least 15 increments). Four scans (or multiples of 4 scans) were used for each increment in
order to be consistent with CYCLOPS phase cycling procedure. Array dimension and the
increment size were carefully determined for each concentration of paramagnetic agents
keeping in view of the possibility of extremely small T1 values.
Pulse sequence repetition time was suitably adjusted to more than 5 x T1 value to enable the
magnetisation to fully recover between the scans and also between the increments. Care was
taken to make an initial quick estimation of T1 values, which were then employed to
determine relaxation delays for the final measurements. All measurements were conducted in
duplicate in order to achieve reliability.
The Carr–Purcell–Meiboom–Gill (CPMG) pulse sequence (Meiboom and Gill, 1958) was
employed to determine the spin-spin relaxation time (T2). In this sequence, an initial 90°
pulse was followed by a burst of 180° pulses with suitable delays (τ). Decay of the resulting
spin-echo signal was monitored and an exponential regression of the data (using the
following equation) yielded values for T2.
S(τ) = S(0) exp (-τ/T2) Eq 8.9
Where, S(0) is the spin-echo signal intensity when τ = 0, and S(τ) is the spin-echo signal
intensity for different values of τ. The parameter τ is related to “bt” in Varian CPMG pulse sequence program.
All T2 experiments were conducted with a minimum array size of 15 τ values (at least 15
increments). All other necessary precautions have been exercised in order to ensure the
accuracy of the results (similar to T1 experiments described above).
267 T1 and T2 data was processed using Varian VNMR 6.1C software on Sun Microsystems
(ULTRA 10) host computer system. The standard Varian relaxation analysis protocols have
been employed for data processing.
8.2.2. MRI experiments
Bruker Avance 600 MHz microimaging system was employed for performing the MRI
experiments reported in this chapter.
8.2.3. T2-weighted images
Spin-echo MRI pulse sequence (Bovey and Mirau, 1996) is given in the Fig 8.1 below. This
pulse Spin-echo sequence was employed for generating non-selective MR images and also
for producing T2-weighted images (a CPMG variant of this pulse sequence was actually used
for producing T2-weighted images). However, a simple interpretation for T2-weighted image formation is provided below using this pulse sequence.
Fig 8.1. Spin-echo MRI pulse sequence (Bovey and Mirau, 1996).
268 The response of spin-echo pulse sequence is given by (Reddy, 2006),
( ) = ( )/ / Eq. 8.10 𝑻𝑻𝑻𝑻−𝑻𝑻𝑻𝑻 𝑻𝑻𝟏𝟏 −𝑻𝑻𝑻𝑻 𝑻𝑻𝟐𝟐 𝑰𝑰 𝑺𝑺𝑺𝑺 𝑴𝑴𝒐𝒐�𝟏𝟏 −𝒆𝒆 �𝒆𝒆 Where I(SE) is the maximum spin-echo signal intensity, Mo is the nuclear spin concentration
that determines the transverse (Iy) magnetisation generated by spin-echo sequence, TR is the pulse sequence repetition time, TE is the echo time (see Fig 8.1), T1 is the spin-lattice relaxation time and T2 is the spin-spin relaxation time of water protons. It should be noted that the values of T1 and T2 would be extremely small in the presence of paramagnetic contrast agents.
An examination of spin-echo pulse sequence (Fig 8.1) reveals that an increase of TE (echo
time delay) leads to the decrease of spin-echo signal intensity due to the loss of signal due to spin-spin relaxation and hence produces darker image. It can also be seen that the spin-echo signal intensity decreases dramatically in the presence of a contrast agent with extremely short T2 values (T2 contrast agents). Hence, T2-agents produce dark contrast (also known as negative contrast) (Caravan, 1999). If a study phantom contains several samples with different T2 values, the sample with shortest T2 gives darkest image and the sample with longest T2 gives brightest image. This sequence therefore produces T2-weighted images and can be used to test the performance of T2 contrast agents. In this chapter, Bruker CPMG pulse sequence has been used to test the performance of T2 contrast agents.
8.2.4. T1-weighted images
Inversion recovery based spin-echo MRI pulse sequence is shown in the following figure. This pulse sequence is employed for generating T1-weighted images.
269 Fig 8.2. Inversion recovery spin-echo MRI pulse sequence (Reddy and smith, 2012; Reddy,
2006).
Taking relaxation into consideration, the response of inversion recovery pulse sequence is
given by (Reddy, 2006),
( ) = ( )/ / / Eq. 8.11 𝑻𝑻𝑻𝑻+𝑻𝑻𝑻𝑻−𝑻𝑻𝑻𝑻 𝑻𝑻𝟏𝟏 −𝑻𝑻𝑻𝑻 𝑻𝑻𝟏𝟏 −𝑻𝑻𝑻𝑻 𝑻𝑻𝟐𝟐 𝑰𝑰 𝑰𝑰𝑰𝑰 𝑴𝑴𝒐𝒐�𝟏𝟏 −�𝟐𝟐 − 𝒆𝒆 �𝒆𝒆 �𝒆𝒆 Where I(IR) is the intensity of the spin-echo signal formed after the initial inversion pulse
(Fig 8.2), Mois the initial nuclear spin magnetisation that determines the transverse
magnetisation generated by IR spin-echo sequence, TR is the pulse sequence repetition time,
TE is the echo time (Fig 8.1), T1 is the spin-lattice relaxation time and T2 is the spin-spin relaxation time of water protons. It should be noted that the values of T1 and T2 would be extremely small in the presence of paramagnetic contrast agents.
It can easily be seen from the IR pulse sequence and Eq 8.11 that, the inverted magnetisation
recovers towards thermal equilibrium during TI period at a rate dictated by T1 value of the sample/tissue. The recovered magnetisation is proportional to the signal intensity in IR
270 imaging sequence (Fig 8.2). If a phantom containing several samples (each with different T1 values) is studied by this sequence, the sample with shortest T1 (recovers fastest) gives highest signal. Hence, the signal intensity in an IR pulse sequence decreases as T1 increases. The sample with shortest T1 gives brightest image and the sample with longest T1 gives weakest image. This sequence therefore produces T1-weighted images and can be used to test the performance of T1 contrast agents. Bruker FLASH imaging pulse sequence has been used in this research for the evaluation of the performance of T1 contrast agents. Extremely small TR value (5.6 ms) was used in this pulse sequence to obtain T1 contrast.
8.2.5. Acquisition of MR images
Standard Bruker operating procedure was used to acquire all the images. RF and gradient pulse calibrations were performed to set up the imaging system in the beginning of each series of imaging experiments. Probe tuning and shimming were performed before acquiring images from each phantom sample.
All Gd3+ metal complexes were produced by titrating with the herbal polysaccharides
discovered in this thesis (Chapters 6 and 7). Pure de-ionised water was used to prepare
solutions of paramagnetic metal complexes. Precision NMR tubes (5 mm x 7” L) were
purchased from Wilmad.
All MRI experiments were conducted on phantom samples specifically constructed for
imaging studies. Separate phantom was constructed for experiments with each paramagnetic
metal complex studied. Phantoms for the performance evaluation of T1 and T2 agents
consisted of three (3) 5 mm NMR tubes with four different concentrations. A brief procedure
for the construction of these phantoms is given below.
271 8.2.6. Construction of phantom for the evaluation of performance of T1 and T2 agents
A large glass tube with 3cm internal diameter and 10 cm long was used for this purpose.
This tube has been fitted with a foam lid with three equally spaced holes (spacer). A 25 mM solution of the desired Gd3+-complex was used to prepare three samples with three different
concentrations by dilution with deionised water. Concentrations used for each imaging
experiment was varied slightly and the exact values are given under each performance
evaluation MR images (Figs. 8.7 to 8.9)
These solutions with varying paramagnetic metal concentration were transferred into three clean 5 mm NMR tubes and these tubes were inserted into the large glass tube through the
holes of the foam lid. The holes in the foam lid on the large tube were equally spaced that
acted as a spacer to hold 5 mm sample tubes in position. A cross-section of the phantom with
three tubes for acquiring T1 and T2 contrast images is shown in Fig 8.3 below. The samples
were arranged in the phantom such that the concentrations decreased from tube-1 to tube-3 in
T1 contrast experiments (tube-1 being highest concentration). Whereas, the concentration of
samples increased from tube-1 to tube-3 in T2 contrast experiments (tube-3 being highest
concentration).
The phantom was inserted up-right into the vertical bore of the micro-imaging magnet system
for MRI studies.
272 2
3 1
Fig 8.3. Phantom for T1 and T2- weighted MR imaging. (Concentrations in the tube-1, tube-2 and tube-3 were suitably adjusted as discussed in section 8.1.4.5).
8.2.7.Inversion recovery spin-echo MRI experiments (T1 contrast images)
T1-weighted images have been acquired using standard inversion recovery spin-echo MRI pulse sequence. All images were transverse sections of the phantom object. In this experiment, the inversion recovery time (TI), echo time (TE) and the repetition time (TR) are
the parameters, which control the timing of the sequence.
TI was varied in the range of 50 to 80 ms and the TI value that gave optimum contrast
between the four concentrations was taken as the final TI. TR, which controls the repetition
time of the pulse sequence, was set according to the pre-determined spin-lattice relaxation
time T1 for the sample with lowest concentration within the phantom (section 8.3). The value
of TR was set to at least five times that of T1 value. Following imaging parameters were used
for T1-contrast experiments. TR was 1.0 s; TE was varied in the range of 9.6 to 20 ms
(generally a TE value in the range of 11-15 ms was found to be optimum for T1-contrast experiments; slice thickness was 1 mm; field of view parameter was set to 3.5 x 3.5 cm. Total acquisition time for each transverse section was about 9 - 12 minutes.
273 8.2.8. Spin-echo / CPMG pulse sequence based MRI experiments (T2 contrast images)
In these experiments, the echo time (TE) and the repetition time (TR) are the parameters,
which control the timing of the sequence. TE was determined from the range of T2 valuesof
the paramagnetic complex with different concentrations. TR, which controls the repetition
time of the pulse sequence, was set according to the pre-determined spin-lattice relaxation
time T1 for the sample with lowest concentration within the phantom (section 5.3). The value of TR was set to at least five times T1 value of the sample with lowest concentration in the
phantom. Following imaging parameters were used for T2-contrast experiments: TR was 1.0 s; TE was in the range of 9.6 to 20 ms; slice thickness was 1 mm; field of view parameter was set to 3.5 x 3.5 cm. Total acquisition time for each transverse section was about 9 - 12 minutes.
8.3. Results and Discussion
In this section, the results of experimentally determined relaxation parameters (T1 and T2), as
a function of the concentration of Gd-herbal polysaccharide complexes, are presented for
each of the three herbal polysaccharides used for MRI studies (and also for one of the
Dextran standard studied for comparison purposes). The relevant relaxivities (r1 and r2) have also have calculated by graphical method for each of the Gd-herbal polysaccharide complex studied and these results are presented in section 8.3.1 below. The MRI performance evaluation results are presented in section 8.3.2.
8.3.1. NMR relaxivity Results
T1 and T2 relaxation parameters of water protons were measured at various concentrations of
the Gd3+ complexes of three herbal polysaccharides discovered in this research and also for
274 the Gd3+ complexes of Dextran (a polysaccharide standard). These results are presented and
discussed in this section.
8.3.1.1. Results on T1 agents
Relaxation measurements of water protons at different concentrations of complexes were performed for [Gd-Dextran-10] and [Gd-LCP-2] and the results are presented below (Tables
8.1 and 8.2). From the measured relaxation parameters (T1 and T2), relaxation rates (R1 =
1/T1 and R2 = 1/T2) have been computed for these complexes (Tables 8.1 and 8.2). The plots
(Figures 8.4 and 8.5) of relaxation rates as a function of concentration yielded relaxivities of individual complexes (gradient of the plots gives relaxivities r1 and r2). Longitudinal (r1) and
transverse (r2) relaxivity values determined for individual complexes are also presented in
Tables 8.1 and 8.2. It should be noted that the magnitude of ratio of relaxivities (r2/r1)
classifies paramagnetic contrast agents into T1 or T2 agents (Caravan et al, 1999). T1 agents, that produce bright contrast, usually have r2/r1 ratio of 1-2 and the T2 agents, that produce
dark contrast, have an extremely large r2/r1 ratio of about 8 or more (Caravan et al, 1999).
8.3.1.2. Relaxation values of [Gd-Dextan-10] at different concentrations
The results present in Table 8.1 and Figure 8.4 clearly demonstrate that the [Gd-Dextan-10]
complex is T1 agent. Indeed, the MRI r esults present in section 8.3.2.2 prove that this agent
performs as a bright contrast agent. It should be noted that we have tested t he relaxivities of
four Dextran samples in the molecular weight range of 10 to 2000 kDa .All of them showed results consistent with T1 agents (Table 8.4). Iit s surprising tot note tha , even the Dextran
with the largest mass formed T1 agent. Results presented in table 8.4 indicate that protein
standard BSA with 66.5 kDa formed a T2 agent. This is an interesting result in the sense that
the biopolymers (polysaccharides and proteins) studied in this research behaved differently as
far as the type of MRI agents are concerned. Table 8.4 also highlights that low molecular
275 weight proteins are T1 agents whereas the high molecular weight proteins/peptides form T2
agents (~ 10 kDa or more). However, the Dextrans formed T1 agents independent of
molecular mass (Table 8.4). This may be due to the fact that the Dextrans studied in this
research are linear α-glucans. It will be interesting / necessary to study the MRI contrast
behaviour of the Gd-complexes with branched polysaccharides and also those with
conformational structures.
8.3.1.3. Relaxation values of [Gd-LCP-2] at different concentrations
The results present in Table 8.2 and Fig. 8.5 clearly demonstrate that the [Gd-LCP-2] complex is T1 agent. Indeed, the MRI results presented in section 8.3.2.2 prove that this agent performs as a bright contrast agent. This observation is consistent with the results presented
in Table 8.4. LCP-2 is low molecular weight linear β-fructan (5.3 kDa). As expected low
molecular weight polysaccharides form T1 agents (Table 8.4).
276 Table 8.1. Relaxation data for Gd3+ complex with Dextan-10 which is a standard α-glucan (Average molecular mass =10 kDa)
Concentration of -1 -1 -1 -1 Gd-D10 T1 (ms) T2 (ms) R1 (1/T1 s ) R2 (1/T2 s ) r1 (mM s ) r2 (mM s ) r2/r1 complex(mM) 1.15 111 ± 0.05 86.1 ± 2.6 9.01 11.61 1.2 1.63 79.7 ± 0.08 62.8 ± 2.2 12.55 15.92 7.45 8.95 (T1 agent) 2.88 45.7 ± 0.04 36.9 ± 1.8 21.88 27.1
Dextran-10kDa A Dextran-10kDa B 30.00 30.00 y = 8.9494x + 1.3283
) ) 1 20.00 y = 7.4463x + 0.4305 2 20.00 (1/T (1/T
1 10.00 2 R 10.00 R
0.00 0.00 0 1 2 3 4 0 1 2 3 4 Concentration (mM) Concentration (mM)
Fig. 8.4. Relaxivity graphs plotted against concentration: (A) T1 relaxivity, and (B) T2 relaxivity (Results confirm that this is a T1 agent with r2/r1=1.2)
277 Table 8.2. Relaxation data for Gd3+ complex with LCP-2 which is a β-D-(2→1)-fructan discovered in this research (Chapter 6)
Concentration of T (ms) T (ms) R (1/T s-1) R (1/T s-1) r (mM s-1) r (mM s-1) r /r Gd-LCP-2 complex(mM) 1 2 1 1 2 2 1 2 2 1 1.8 112 ± 0.2 92.5 ± 3.9 8.93 10.81 2.6 72.3 ± 0.2 58.2 ± 1.8 13.83 17.18 1.17 4.66 5.44 3.57 55.5 ± 0.2 45.2 ± 2.5 18.02 22.12 (T1 agent) 6.82 30.5 ± 0.01 25.7 ± 1.1 32.79 38.91
LCP-2 A LCP-2 B 35.00 50.00 y = 4.6638x + 1.1467 30.00 40.00 y = 5.4427x + 2.1326
) 25.00 ) 1 20.00 2 30.00 (1/T (1/T
1 15.00 2 20.00 R 10.00 R 5.00 10.00 0.00 0.00 0 2 4 6 8 0 2 4 6 8 Concentration (mM) Concentration (mM)
Fig. 8.5. Relaxivity graphs plotted against concentration: (A) T1 relaxivity, and (B) T2 relaxivity (Results confirm that this is a T1 agent with r2/r1=1.17)
278 8.3.1.4. Relaxation values of [Gd-AAP-1] at different concentrations
Relaxation measurements of water protons at different concentrations of complex [Gd-AAP-
1] were performed and the results are presented below (Table 8.3). From the measured
relaxation parameters (T1 and T2), relaxation rates (R1 = 1/T1 and R2 = 1/T2) have been
computed (Table 8.3). The plots (Figure 8.6) of relaxation rates as a function of
concentration yielded relaxivities of [Gd-AAP-1] (gradient of the plots gives relaxivities r1
and r2). Longitudinal (r1) and transverse (r2) relaxivity values determined for [Gd-AAP-1]
complex are also presented in Table 8.3.
The r2/r1 ratio for this Gd-complex with AAP-1 (1685 kDa) was 9.66 indicating that this
herbal polysaccharide (AAP-1) forms a T2 agent with Gd(III). However, it should be noted
that even the high molecular weight Dextran (2000 kDa) formed a T1 agent (Table 8.4). As
can be seen from the results present in Table 8.4 for Dextran standards, the complex formed
by AAP-1 (1685 kDa) is expected to be a T1 agent if it is a pure polysaccharide without any
protein conjugation. The results presented in Chapter 7 indicated that AAP-1 is a
polysaccharo-protein (with about 20 % protein). These results suggest that protein plays an
important role in determining the agent type with biomacromolecular ligands
(polysaccharides and proteins). This leads to the conclusion that pure polysaccharides with no
protein conjugation form T1 agents independent of molecular weight (Table 8.4). However, in
the case of polysaccharo-proteins or pure proteins as ligands lead to the formation of T2
agents at high molecular mass (~ 10 kDa or more). The change from T1 to T2 agents occurred at a molecular mass of about 10 kDa for pure proteins (Table 8.4). These are extremely important results observed with macromolecular complexes with Gd 3+.
279 Table 8.3. Relaxation data for Gd3+ complex with AAP-1 which is a complex polysaccharide (containing arabinose, galactose and glucose units) discovered in this research (Chapter 7) Concentration of -1 -1 -1 -1 AAP-1 complex T1 (ms) T2 (ms) R1 (1/T1 s ) R2 (1/T2 s ) r1 (mM s ) r2 (mM s ) r2/r1 (mM) 0.14 425 ± 3.2 54.5 ± 1.0 2.35 18.35 0.3 222 ± 1.2 27.7 ± 0.9 4.5 36.1 9.66 12.71 122.84 0.9 72 ± 0.03 8.45 ± 0.35 13.89 118.34 (T2 agent) 2.5 30.8 ± 0.06 3.25 ± 0.11 32.47 307.69
B AAP-1 A AAP-1 35 350 y = 12.711x + 1.1004 y = 122.84x + 2.1956 30 300
25 250 ) ) 2 1 20 200 (1/T (1/T
2 1 15 150 R R 10 100 5 50 0 0 0 0.5 1 1.5 2 2.5 3 0 0.5 1 1.5 2 2.5 3 Concentration (mM) Concentration (mM)
Fig. 8.6. Relaxivity graphs plotted against concentration: (A) T1 relaxivity, and (B) T2 relaxivity (Results confirm that this is a T2 agent with r2/r1=9.66)
280 To the best of our knowledge, there are no reports in the literature demonstrating a systematic
switch occurring from T1 to T2 type agents when the biomolecular mass is increased. In this
study, we observed this switch to occur for peptides at about 10 kDa in the case of pure
protein/peptide based contrast agents. However, the exact molecular weight required for such
a switch could not be determined for polysaccharo-proteins due to non-availability of such
samples with different molecular masses. Polysaccharo-proteins with all the available
molecular masses were tested (Table 8.4). Such a switch has been observed at about 46 kDa
in the case of polysaccharo-protein isolated in Authors’ laboratory (Table 8.4). However,
further research is required to determine exact molecular mass at which the switchoccurs.
It should be noted that, very few Gd-complexes of T2 type exist in the literature (Botta,
2000). A few macrocyclic ligands have been designed by previous authors’ in our research group (Chalmers, 2014).
281 Table 8.4. Classification of type of Gd (III) based MRI contrast agents formed with bio-macromolecules with different molecular masses studied in Authors’ Laboratory (unpublished data)
Biopolymer Type of agents Dextran-10 (10 kDa) T1 agent Dextran-40 (40 kDa) T1 agent Polysaccharide standards Dextran-70 (70 kDa) T1 agent Dextran-2000 (2000 kDa) T1 agent
* Polysaccharides LCP-2 (5.3 kDa) T1 agent * AAP-2 (445 kDa) T2 agent * Bioactive polysaccharo-proteins from AAP-1 (1658 kDa) T2 agent # Authors' laboratory YLP-3 (4 kDa) T1 agent # BLP-5 (46 kDa) T2 agent # WLP-1 (1162 kDa) T2 agent
Protein standard BSA (66.5 kDa) T2 agent
** Protein/peptides Fl-4h (< 2kDa) T1 agent Bioactive peptides from Authors' ** Fl-2h (< 10kDa) T2agent laboratory ** Pep-3h (< 10kDa) T2 agent *Details of bioactive polysaccharide LCP-2, AAP-1 and AAP-2 are presented in Chapter 6 and Chapter 7 of this Thesis.
# Thambiraj, S. (2017), “Biological activities and Structural characterisation of Three Australia Sweet Lupin Species”, PhD Thesis, Submitted to Western Sydney University.
** Kamran, F. (2017), “Enzymatic hydrolysis of Lupin protein: Isolation and characterisation of bioactive peptides”, PhD Thesis, Submitted to Western Sydney University.
282 8.3.2. MRI Results
In this section, the results of MRI experiments designed to evaluate the performances of
paramagnetic contrast agents prepared by complexing herbal polysaccharides with Gd3+ ions
are presented (and also with one of the Dextran standard studied for comparison purposes).
Measured relaxivities of these paramagnetic contrast agents (section 8.3.1) have been used to
devise and implement suitable MRI protocols to test their efficacy as contrast agents.
Experimentally determined relaxivities (section 8.3.1) have also assisted in the explanation of
the observed MRI contrast efficiencies of these agents developed in this research.
8.3.2.1. Performance evaluation of T1 contrast agents
Gd3+ complexes with low molecular weight herbal polysaccharides and the Gd3+ complex
with Dextran standard were found to be T1 agents (section 8.3.1). Results on T1 contrast performances of these agents are presented and discussed in this sub-section.
8.3.2.2. MRI performance evaluation of T1 agents
3+ Two Gd complexes formed with [Gd-Dextan-10] and [Gd-LCP-2] have been found to be T1 agent in this research. As can be seen from the relaxivity studies (section 8.3.1.1), both these
Gd3+ complexes formed by a low molecular weight polysaccharide LCP-2 (5.3 kDa) and
medium molecular weight Dextran standard (10 kDa) formed T1 agents (Table 8.4). These
two complexes have been studied to evaluate their efficacies as T1 contrast agents and the
results are presented in this section below.
8.3.2.2.1. Evaluation of MRI performance of [Gd-Dextran-10] complex: T1 agent
Results presented in Figure 8.7 indicated that [Gd-Dextran-10] complex is a T1 agent.
Clearly, the sample with highest concentration gave brightest image (Tube 1 in the image).
The sample with lowest concentration gave darkest image (Tube 3 in the image). Hence, this
283 complex showed a bright contrast in MR image (Fig. 8.7), consistent with the results
expected for a T1 agents.
Fig. 8.7. T1-weighted images from the phantom made with [Gd-Dextran-10] samples with three different concentrations (using Bruker FLASH pulse sequence). The concentrations used were: Tube-1) 2.88 mM, Tube-2) 1.63 mM and Tube-3) 1.15 mM. The imaging parameters employed were: TE=1.8 ms, TR=5.6 ms.
8.3.2.2.2. Evaluation of MRI performance of [Gd-LCP-2] complex: T1 agent
Results presented in Figure 8.8 indicated that [Gd-LCP-2] complexT is a 1 agent. Clearly, the sample with highest concentration gave brightest image (Tube 1 in the image). The sample
with lowest concentration gave darkest image (Tube 3 in the image). Hence, this complex
showed a bright contrast in MR image (Fig. 8.8), consistent with the results expected for a T1 agent.
284 Fig. 8.8. T1-weighted images from the phantom made with [Gd-LCP-2] samples with three different concentrations (using Bruker FLASH pulse sequence). Theconcentrations used were: Tube-1) 3.57 mM, Tube-2) 2.6 mM and Tube-3) 1.8 mM. The imaging parameters employed were: TE=1.9 ms, TR=5.6 ms.
8.3.2.3. Performance evaluation of T2 contrast agents
Gd3+ complex with one of the high molecular weight herbal polysaccharide was found to be a
T2 agent in this research (section 8.3.1). Results on T2 contrast performance of this agent are
presented and discussed in this sub-section.
8.3.2.4. MRI performance evaluation of T2 agent
Only one Gd3+ complex formed with [Gd-AAP-1] has been studied in this research as this
was the only herbal polysaccharide that had T2 type behaviour (Table 8.3). As can be seen
from the relaxivity s tudies (Table 8.3), this polysaccharo-protein hadr2/r1 ratio of about 9.66
indicating that this is a T2 agent.
285 8.3.2.4.1. Evaluation of MRI performance of [Gd-AAP-1] complex: T2 agent
Results present in Fig. 8.9 indicate that [Gd-AAP-1] complex showed MRI performance
consistent with that of a T2 agent. Clearly, the sample with lowest concentration gave
brightest image (Tube 1 in the image). The sample with highest concentration gave darkest
image (Tube 3 in the image). Hence, this complex showed a dark contrast in MR image (Fig.
8.9), and this is consistent with the results expected for a T2 agent.
Fig. 8.9. T2-weighted image from the phantom made with [Gd-AAP-1] samples with three different concentrations (using Bruker CPMG pulse sequence). The concentrations used were: Tube-1) 0.3 mM, Tube-2) 0.9 mM and Tube-3) 2.5 mM. The imaging parameters employed were: TE=18 ms, TR=3 s.
8.4. Conclusion
In this chapter, relaxation behaviour of three herbal polysaccharide based Gd complexes and
their MRI performances have been evaluated. Relaxation results on several other
286 macromolecular-Gd complexes (both polysaccharides and peptides) have also been presented in this Chapter for a more detailed understanding of macromolecular-Gd complexes (Table
8.4). The results presented in Table 8.4 are the unpublished data from other co-workers in authors’ laboratory. However, it should be noted that only three of the complexes listed in
Table 8.4 are from this thesis, which are, ([Gd-Dextan-10], [Gd-LCP-2] and [Gd-AAP-1]).
Results presented in this Chapter clearly demonstrate that the Gd-macromolecular complexes formed by polysaccharo-proteins display T2 behaviour at higher molecular weight (about 46 kDa or more). However, the Gd-macromolecular complexes formed by linear polysaccharides displayed only T1 behaviour at low as well as high molecular weights (even
at 2000 kDa). This is a very interesting result and needs further investigations in order to
develop a clear understanding of such behaviour which is expected to depend on molecular
mass as well as protein content. This behaviour may partially be explained by rotational
dynamics of these macromolecular complexes. Proteins are well-structured molecules and the complexes of Gd(III) formed with such ligands are expected to form rigid structures and hence display slow tumbling.
287 8.5. References
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288 Chalmers, G. (2014). “Design and evaluation of novel Gd(III) complexes as MRI contrast agents”. PhD thesis, submitted to Western Sydney Unversity.
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291 Chapter Nine
Conclusions and Future Work 9. Conclusion and Future Research Possibilities
Systematic studies involving pharmacological activities and establishing structure – function relationships of bioactive consituents derived from herbal medicine are only emerging in recent years. Traditional medicine therefore has great future potential for the discovery of new and effective medications not only to treat cancer but also other life-threatening ailments.
The main objectives of this thesis were to discover potent immuno-therapeutic and anticancer polysaccharides, with minimal side effects. In order to fulfil these objectives, the crude polysaccharides from sixteen carefully chosen traditional anticancer Chinese medicinal herbs have been screened for their antioxidant, immunomodulatory and anticancer properties.
Quantification of bioactive components (total sugar, and protein contents) has also been carried out in order to establish a relationship between the crude polysaccharides and bioactive properties of these sixteen herbal polysaccharides. Many polysaccharides that have been studied in this thesis displayed significant antioxidant, immunomodulatory, and anti- cancer properties (Chapter 3). Crude polysaccharides from three of these herbs performed extremely well in terms of their immunomodulatory and anticancer properties (Chapter 3).
These herbs are: A. annua, L. chinensis, and A. rugosum which have been selected for turther detailed study.
In order to obtain a comprehensive picture of the activities of all hot water extractable constituents from the sixteen selected herbs, ethanol soluble organics have been isolated and screening for their antioxidant, anti-inflammatory and anticancer activities (Chapter 4). This result together with the results of polysaccharides (Chapter 3) lead to the conclusion that the crude polysacchairdes from A. annua, L. chinensis, and A. rugosum are the best candidates for further purification and detailed study.
292 The detail results of these three herbal polysaccharides are presented in Chapter 5 to 7. From the results presented in Chapter 5, two novel immunostimulatory polysaccharides (ARP-1
and ARP-2) having β-D-(1→3)-glucan backbone with β-D-(1→6) branched structures have
been identified for the first time in the literature from A. rugosum which is the well-known
traditional anticancer medicinal mushroom. These results suggest the potential of A. rugosum
polysaccharides as natural immuno-enhancing agents. Findings of this chapter together with
the anticancer properties of crude polysaccharides from A. rugosum (Chapter 3) strongly demonstrate the potential of ARPs to be used in anticancer formulations. It will be interesting to determine anticancer activities of these two A. rugosum polysaccharides (ARP-1 and ARP-
2). Another interesting future direction of A. rugosum polysaccharides is to undertake animal and human studies to assess their immuno-therapeutic efficacies. These are the subjects of future studies. Such future research is expected to provide cues to design effective immuno- therepurtic and anti-cancer formulations.
Results presented in Chapter 6 leads to the conclusion that, two pure immunostimulatory
polysaccharides were identified from L. chinensis (LCP-1 and LCP-2). Detailed structural studies on one of these immunostimulatory polysaccharides (LCP-2) showed a β-D-(2→1)-
fructofuranoside structure. A plausible mechanism of action of this β-fructan has also been proposed in Chapter 6. LCP-2 has displayed extremely high immunostimulatory activity and was less toxic demonstrating that it is a highly potent immuno-therapeutic agent. These
results suggest that the L. chinensis polysaccharides are most likely candidates to be used as
natural immuno-enhancing agents. Findings of this chapter together with the anticancer
properties reported in Chapter 3 strongly demonstrate the potential of L. chinensis
polysaccharides to be used in anticancer formulations. It will be interesting to determine anticancer activities of L. chinensis polysaccharides (LCP-2). Another interesting future
293 direction of L. chinensis polysaccharides is to undertake animal and human studies to assess their immuno-therapurtic effecacies. These are the subjects of future studies. Such future
research is expected to provide cues to design effective immuno-therepurtic and anti-cancer formulations.
Results presented in Chapter 7 lead to the conclusion that, three pure immunostimulatory
polysaccharides were identified from A. annua (AAP-1, AAP-2 and AAP-3). Structural
studies on one of these immunostimulatory polysaccharides (AAP-3) showed that it is
possibly a β-D-(2→1)-fructofuranoside. A plausible mechanism of action of this β-fructan
has been provided in Chapter 7. AAP-1 and AAP-3 have displayed extremely high
immunostimulatory activity and were less toxic demonstrating that they are highly potent
immuno-therapeutic agents. These results suggest that the A. annua polysaccharides are most
likely candidates to be used as natural immuno-enhancing agents. Findings of this chapter
together with the anticancer properties reported in Chapter 3 strongly demonstrate the
potential of A. annua polysaccharides to be used in anticancer formulations. It will be
interesting to determine anticancer activities of A. annua polysaccharides (AAP-1, AAP-2
and AAP-3). Another interesting future direction of A. annua polysaccharides is to undertake
animal and human studies to assess their immuno-therapeutic efficacies. These are the
subjects of future studies. Such future investigations are expected to provide cues to design
effective immuno-therepurtic and anti-cancer formulations.
Overall, the reseach carried out as part of this PhD thesis has led to the discovery of eight
potent immuno-therepuritic agents for the first time from three important anticancer TCM
herbs (A. annua, L. chinensis and A. rugosum).
294 Results presented in Chapter 8 clearly demonstrated that the Gd-macromolecular complexes formed by polysaccharo-proteins display T2 behaviour at higher molecular weight (at about
46 kDa or more) (Table 8.4). However, the Gd-macromolecular complexes formed by linear
polysaccharides displayed T1 behaviour at low as well as high molecular weights (even at
2000 kDa) (Table 8.4). This is a very interesting result and needs further investigations in
order to describe such behaviour. This behaviour may partially be explained by rotational
dynamics of these macromolecular complexes. Proteins are well-structured molecules and
complexes formed with such ligands are expected to form rigid structures with least internal mortion and hence display slow tumbling. Further research in this area is required in order to make firm conclusions.
295