Comparison of polysaccharides from two species of...
Transcript of Comparison of polysaccharides from two species of...
Comparison of polysaccharides from two species of Ganoderma
by
Xie Jing
Master of Science
2012
Institute of Chinese Medical Sciences
University of Macau
Comparison of polysaccharides from two species of Ganoderma
by
Xie Jing
A thesis submitted in partial fulfillment of the
requirements for the degree of
Master of Science
Institute of Chinese Medical Sciences
University of Macau
2012
Approved by __________________________________________________
Supervisor
__________________________________________________
__________________________________________________
__________________________________________________
Date __________________________________________________________
碩士學位論文
兩種靈芝多糖類成分比較研究
研究生姓名: 謝靜
導 師: 李紹平 教授
專 業: 中 藥 學
日 期: 2012.07
澳門大學中華醫藥研究院
In presenting this thesis in partial fulfillment of the requirements for a Master's
degree at the University of Macau, I agree that the Library and the Institute of
Chinese Medical Sciences shall make its copies freely available for inspection.
However, reproduction of this thesis for any purposes or by any means shall
not be allowed without my written permission. Authorization is sought by
contacting the author at
Address:
Telephone:
Fax:
E-mail:
Signature ______________________
Date __________________________
Master Thesis, University of Macau
I
Acknowledgement
My study will soon come to an end and at the completion of my graduation thesis
I wish to express my sincere appreciation to all those who have offered me invaluable
help during my undergraduate study here at the Institute of Chinese Medical Sciences,
University of Macao.
Firstly, I would like to express my deepest gratitude to my supervisor, Professor
Shaoping Li, for his constant encouragement and guidance. I am deeply encouraged
by his rigorous scholarship and positive working attitude. I learned a lot about life
from him. I am also very grateful to Professor Yitao Wang for he offers me the
opportunity to study here and providing valuable resources to me. I would like to
express my heartfelt gratitude to Dr. Jing Zhao, Dr. Ying Zheng, Dr. Qingwen Zhang,
Dr. Simon Lee, Dr. Ru Yan and other teachers in our institute. I benefit a lot from
their lectures.
I am also deeply indebted to all the staffs in our institute: Leon LAI, Sio Kio
KUONG, Sandy LAO, Wing LEONG, Hattie U, Ada WONG, etc. for their direct and
indirect help to me throughout the project.
Lastly, my thanks would go to my beloved family, friends and classmates, for
their loving considerations and great confidence in me all through these years.
Master Thesis, University of Macau
II
Table of contents
Acknowledgement ........................................................................................................ I
Table of contents ......................................................................................................... II
Abstract ....................................................................................................................... IV
摘要 ............................................................................................................................. VI
List of Figures ........................................................................................................... VII
List of Tables .............................................................................................................. IX
Abbreviation ................................................................................................................ X
Chapter1: Research Progress on Polysaccharides of Ganoderma ........................... 1
1.1 Background ....................................................................................................... 1
1.2 Chemistry Researches of Polysaccharides from Lingzhi .................................. 3
1.3 Pharmacological Actions of Polysaccharides from Lingzhi ............................ 11
1.3.1 Immuno-modulating Effects .................................................................. 11
1.3.2 Anti-tumor Activities ............................................................................. 14
1.3.3 Anti-oxidative Property ......................................................................... 15
1.3.4 Other Effects .......................................................................................... 16
1.4 Quality Control of Ganoderma Polysaccharides ............................................ 18
1.4.1 Determination of Purity and Molecular Weight .................................... 18
1.4.2 Monosaccharide Composition and Linkage Analysis ........................... 19
1.5 Conclusions ..................................................................................................... 20
Chapter 2 Comparison of Polysaccharides from Two Species of Ganoderma
using HPTLC .............................................................................................................. 21
2.1 Materials and Methods .................................................................................... 21
2.1.1 Herbal Materials and Chemicals ............................................................ 21
2.1.2 Preparation of Solutions ........................................................................ 22
2.1.3 Preparation of Polysaccharides .............................................................. 22
2.1.4 Acid Hydrolysis for Crude Polysaccharides .......................................... 24
2.1.5 HPTLC Analysis.................................................................................... 24
2.2 Results and Discussion .................................................................................... 25
2.2.1 Optimization of TFA Hydrolysis Condition .......................................... 25
Master Thesis, University of Macau
III
2.2.2 HPTLC Chromatograms of Monosaccharides and Protein Ingredients in
Polysaccharides .............................................................................................. 27
2.2.3 TLC Fingerprint Profiling of Acid Hydrolyzates of Polysaccharides
from Lingzhi ................................................................................................... 29
2.3 Conclusions ..................................................................................................... 31
Chapter 3 Comparison of Polysaccharides from Two Species of Ganoderma
Using Enzymatic Digestion........................................................................................ 32
3.1 Materials and Methods .................................................................................... 32
3.1.1 Herbal Materials and Chemicals ............................................................ 32
3.1.2 Extraction of Polysaccharides from Ganderma ..................................... 33
3.1.3 HPSEC-ELSD Analysis ........................................................................ 33
3.1.4 HPSEC-MALLS-RI Analysis................................................................ 34
3.1.5 Enzymatic Digestion.............................................................................. 34
3.2 Results and Discussions .................................................................................. 35
3.2.1 HPSEC-ELSD Profiles and Molecular Weights of Polysaccharides from
Lingzhi ............................................................................................................ 35
3.2.2 Investigation on Enzymatic Digestion of Polysaccharides from Lingzhi
........................................................................................................................ 38
3.3 Conclusions ..................................................................................................... 41
Chapter 4 Separation and Purification of Polysaccharides from Ganoderma
Lucidum ....................................................................................................................... 42
4.1 Materials and Methods .................................................................................... 42
4.1.1 Materials and Chemicals ....................................................................... 42
4.1.2 Extraction Procedure ............................................................................. 42
4.1.3 Fractionation and Purification ............................................................... 44
4.1.4 HPSEC-ELSD Analysis ........................................................................ 44
4.2 Results and Discussions .................................................................................. 45
4.2.1 Optimization of Ethanol Concentration for Alcohol Precipitation ........ 45
4.2.2 Purification with Ion-exchange Chromatography ................................. 46
4.2.3 HPSEC-ELSD Analysis of Different Fractions ..................................... 47
4.3 Conclusions ..................................................................................................... 49
Summary ..................................................................................................................... 50
References ................................................................................................................... 51
Appendix ..................................................................................................................... 62
Publications ................................................................................................................ 88
Master Thesis, University of Macau
IV
University of Macau
Abstract
COMPARISON OF POLYSACCHARIDES FROM
TWO SPECIES OF GANODERMA
by
XIE Jing
Supervisor: Prof. Shaoping Li
Chinese medicinal science
Ganoderma lucidum and Ganoderma sinense, known as Lingzhi in Chinese, are
commonly used Chinese medicines with excellent beneficial health effects.
Triterpenes and polysaccharides are usually considered as their main active
components. However, the content of triterpenes differs significantly between the two
species of Ganoderma. Polysaccharides in Lingzhi have attracted much attention in
biomedical research because of their significant pharmacological activity and
relatively low toxicity, for example, antitumor, immuno-modulating, anti-oxidative,
hypoglycemic, and platelet aggregation inhibiting activities. To date, a careful
comparison of polysaccharides from the two species of Ganoderma has not been
performed. Therefore, more attention should be paid on the quality control of
polysaccharides in Lingzhi.
In this study, polysaccharides from fruiting bodies of two species of Lingzhi
collected from different regions of China were analyzed and compared based on
Master Thesis, University of Macau
V
HPSEC-ELSD and HPSEC-MALLS-RI analyses, as well as enzymatic digestion and
HPTLC of acid hydrolysates. Then water soluble polysaccharides from Ganoderma
lucidum were extracted and purified. The results indicated that HPTLC profiles of
their TFA hydrolysates colored with different reagents and their monosaccharides
composition were similar between different samples, but their protein ingredients are
significantly different. Both HPSEC-ELSD profiles and molecular weights of the
polysaccharides for these two species were also similar. Enzymatic digestion showed
that polyshaccharides from all samples of Lingzhi could be hydrolyzed by pectinase
and dextranase.
Key words: Ganoderma lucidum, Ganoderma sinense, polysaccharides, quality
control
Master Thesis, University of Macau
VI
澳門大學
摘要
兩種靈芝多糖的比較研究
謝靜
指導老師:李紹平 教授
中藥學
靈芝是一種名貴的中藥材,因其獨特的保健作用,被廣泛使用。赤芝和紫芝
均為《中華人民共和國藥典》的收載品種,其化學成份和生物活性上的差異引起
人們的廣泛關注。三萜和多糖是靈芝中的主要活性成份,然而靈芝三萜在兩種靈
芝中的差別很大。藥理學研究表明靈芝多糖具有抗腫瘤、免疫調節、抗氧化、活
血化瘀、降糖保肝等作用,且毒性較低,越來越受到人們的重視。目前為止,有
關赤芝和紫芝多糖的比較研究較少,引起了我們的關注。
本研究採用高效薄層色譜法對來自不同產地的紫芝和紫芝多糖的酸水解產
物進行了比較分析,同時用 HPSEC-ELSD和 HPSEC-MALLS-RI的方法,對這不同產
地的靈芝多糖及其酶解產物進行了分析,最後對赤芝多糖進行了分離純化。HPTLC
結果表明,兩種靈芝多糖的單糖組成相似,氨基酸含量具有一定差異。HPSEC-ELSD
色譜以及分子量分布差別很小。酶解結果顯示,赤芝和紫芝多糖均能被果膠酶和
葡聚糖酶水解,兩者沒有特徵性差異。
關鍵詞: 赤芝,紫芝,多糖,質量控制
Master Thesis, University of Macau
VII
List of Figures
Figure 1.1 Raw materials of G. sinense (A) and G. lucidum (B) ........................... 2
Figure 2.1 Effects of (A) concentration of TFA for 2 h, (B) hydrolysis time treated
with 5 mol/L, and (C) optimum conditions (5 mol/L for 6 h) on acid
hydrolysis of polysaccharides from Ganoderma lucidum (GL07). ... 26
Figure2.2 HPTLC profiles, colored with aniline-diphenylamine-phosphoric acid
(Left) and ninhydrin (Right) solutions, of acid hydrolysates of
polysaccharides from Linzhi. ......................................................... 28
Figure 2.3 HPTLC profiles of G. lucidum samples and their module plot .......... 30
Figure 2.4 HPTLC profiles of G. sinense samples and their module plot ........... 30
Figure 2.5 Module plots of G. lucidum and G. sinense ....................................... 31
Figure 3.1 HPSEC-ELSD profiles of crude polysaccharides from Lingzhi. ........ 36
Figure 3.2 HPSEC-RI profiles with molecular weight distribution of
polysaccharides from typical samples of G. lucidum (GL02) and G.
sinense (GS12) ..................................................................................... 37
Figure 3.3 HPSEC-ELSD profiles of polysaccharides from G. lucidum (GL02) and
G. sinense (GS10) treated with (P+E) or without (P) selected enzyme
(E). ....................................................................................................... 39
Figure 4.1 Scheme for the extraction of polysaccharides from G. lucidum. ........ 43
Figure 4.2 HPSEC-ELSD profiles of different ethanol precipitate fractions of
polysaccharides from G. lucidum ........................................................ 46
Master Thesis, University of Macau
VIII
Figure 4.3 Elution profile of GLP30 on DEAE-cellulose column ....................... 47
Figure 4.4 HPSEC-ELSD profiles of different fractions of water extracted
polysaccharides from G. lucidum eluded on DEAD-cellulose column
.......................................................................................................... 48
Master Thesis, University of Macau
IX
List of Tables
Table 1.1 Polysaccharides purified from fruit bodies of Ganoderma .................... 7
Table 2.1 Summary of Lingzhi Samples ............................................................... 23
Table 3.1 Digestion buffers for various enzyme digestion modified from the
operation manual. .................................................................................. 35
Table 3.2 Responses of polysaccharides from Lingzhi to enzymatic digestion. .. 40
Master Thesis, University of Macau
X
Abbreviation
Abreviations Full Name
DAD diode array detector
DEAE diethylaminoethyl cellulose
ELSD evaporative light scattering detector
GC gas chromatography
HPLC high performance liquid chromatography
HPSEC high performance size exclusion chromatography
HPTLC high performance thin layer chromatography
IR infrared spectrum
MS mass spectrometry
MW molecular weight
MALLS multi-angle laser light scattering photometer
NMR nuclear magnetic resonance
N/A not available
RI refractive index detector
TFA trifluoroacetic acid