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

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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

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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