UNIVERSITI PUTRA MALAYSIA

SCREENING OF MALAYSIAN MEDICINAL PLANTS FOR IMMUNOMODULATORY AND HAEMOLYTIC ACTIVITIES

NOR AZURA BT. SAMAT

FPSK (M) 2003 6

SCREENING OF MALAYSIAN MEDIONAL PLANTS FOR IMMUNOMODULATORY AND HAEMOLYTIC ACTlVmES

By

NOR AZURA BT. SAMAT

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for the Degree of Master of

Science

September 2003

DEDICATION

This thesis is specially dedicated to my beloved husband, Molm. Zaki, parents Samat and Zamzimar, and daughters, Nur Nadia Najiha and Nur Nadia Nazreell.

Thank you for all the understanding, support, advice and opinion during the course of completing of this thesis.

For Najiha and Nazreeu . . . . . . . . . . .

Thanks for bringing me such joy and laughter

Thanks for everything.

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Abstract of thesis presented to the Senate ofUniversiti Putra Malaysia in fulfilment of the requirements for the degree of Master of Science

SCREENING OF MALAYSIAN MEDICINAL PLANTS FOR IMMUNOMODVLATORY AND HAEMOLYTIC ACTIVITIES

By

NOR AZURA BT. SAMAT

September 2003

Chairman Associate Professor Daud Ahmad Israf Ali, Ph.D.

Faculty Medicine and Health Science

Twenty-six water-soluble extracts from eighteen local medicinal plant specirs

and fourteen methanol extracts from ten medicinal plants were screened in

order to find new immunomodulating substances from those medicinal plants.

Effects on lymphocyte proliferation and macrophage function in host defense

mechanism including the nitric oxide (NO) prodUction, phagocytic activity and

intracellular killing ability were tested. Lymphocyte or macrophage was treated

with those extracts at the concentration of 2.5 - 50J..lg/ ml. Four of the water-

soluble extracts shows mitogenicity where it causes highest mitogenicity at

different concentrations. Both methanol and water-soluble extracts failed to

alter or improve macrophage function neither of its phagocytic activity nor its

intracellular killing ability. The water extracts of S. torvum (stem bark), C.

asiatica, S. grandiflora, U. lobata (stem bark) and G. vulgaris (leaves) produce NO

significantly. Apart from that, haemolyti c activity was also determined. Both

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significantly. Apart from that, haemolytic activity was also determined. Both

methanol and water-soluble extracts causes less than 32% haemolysis of SRBC

as com pared to hdemolysis caused by saponin except for methanol extracts of E.

guainensis leaf which causes 68.2% haemolysis. Overall results indicate that the

extracts effect on all parameter were influenced by a few factor such as dose,

cells involved, solvent used for extraction and constituents of each extracts.

IV

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master 5ains

PENYARINGAN EKSTRAK TUMBUHAN UBATAN MALAYSIA BAGI AKTIVITI IMMUNOMODULA TORI DAN HEMOLISIS

Oleh

NOR AZURA BT. SAMAT

September 2003

Pengerusi Profesor Madya Daud Ahmad Israf Ali, Ph.D.

Fakulti Perubatan dan Sains Kesihatan

Dua puluh enam ekstrak larutan air daripada lapan belas spesis tumbuhan

ubatan tempatan dan empat belas ekstrak methanol daripada sepuluh

tumbuhan ubatan tempatan telah disaring bagi menges an ekstrak yang

berpotensi dalam meningkatkan sistem imun. Kesan ekstrak terhadap

proliferasi limfosit, fungsi-fungsi makrofaj dalam penghasilan nitrik oksida

(NO), aktiviti fagositosis dan keupayaannya membunuh secara intraselular

merupakan parameter yang dilihat dalam ujikaji ini. Ekstrak-ekstrak tersebut

telah di inkubasi Limfosit dan makrofaj telah dieram bersama ekstrak-ekstrak

tersebut di mana kepekatan ekstrak yang digunakan adalah di antara 2.5 -

50f.lg/ ml. Empat daripada ekstrak larutan air telah menunjukkan peningkatan

di dalam aktiviti proliferasi sel limfosit pada kepekatan yang berbeza. Walau

bagaimanapun, kesemua ekstrak yang diuji tidak berupaya meningkatkan

kadar fagositosis dan keupayaan makrofaj untuk membunuh secara

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intraselular. Ekstrak larutan air bagi S. torvum (kulit pokok), C. asiatica, S.

grandiflora, U. lobata (kulit pokok) dan G. vulgaris (daun) menunjukkan

peningkatan yang signifikan dalam penghasilan nitrik oksida (NO). Selain itu,

kadar hemolisis SRBC juga dikaji bagi memastikan ekstrak tidak toksik.

Kesemua ekstrak menyebabkan hemolisis kurang daripada 32% jika

dibandingkan dengan hemolisis yang disebabkan oleh saponin. Hanya ekstrak

methanol bagi daun E. guainensis sahaja yang mengakibatkan kadar hemolisis

yang agak tinggi iaitu 68.2%. Kesimpulannya, kesan ekstrak terhadap semua

parameter yang diuji bergantung kepada beberapa faktor seperti kadar

kepekatan ekstrak yang digunakan, sel-sel yang terlibat, jenis pelarut yang

digunakan semasa proses ekstraksi dan juga kandungan setiap ekstrak yang

diuji

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ACKNOWLEDGEMENT

I would like to thank the entire supervisory committee member, Assoc. Prof. Daud Ahmad Israf Ali, Prof Nordin Lajis and Assoc. Prof Abdul Rahman Omar for their kind supervision, guidance and advice through out the project.

Thank you to all the staff members of Faculty of Veterinary and Department of Chemistry, Universiti Putra Malaysia for their cooperations.

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I certify that an Examination Committee met on 24 September 2003 to conduct the final examination of Nor Azura Bt. Samat on her Master of Science thesis entitled IIScreening of Malaysian Medicinal Plants for Immunomodulatory and Haemolytic Activities" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulation 1981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows:

Muhammad Nazrul Hakim, Ph.D. Associate Professor Faculty of Medicine and Health Science Universiti Putra Malaysia (Chairman)

Daud Ahmad Israf Ali, Ph.D. Associate Professor Faculty of Medicine and Health Science Universiti Putra Malaysia (Member)

Dr. Nordin Hj. Lajis, Ph.D. Professor Institute of Bioscience Universiti Putra Malaysia (Member)

Abdul Rahman Omar, Ph.D. Associate Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member)

GULAM RUS Professor/Deputy, ean School of Gradua e Studies Universiti Putra Malaysia

Date: IS j"AN �4

viii

This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Master of Science. The members of the Supervisory Committee are as follows:

Daud Ahmad Israf Ali, Ph.D. Associate Professor Faculty of Medicine and Health Science Universiti Putra Malaysia (Chairman)

Dr. Nordin Hj. Lajis, Ph.D. Professor Institute of Bioscience Universiti Putra Malaysia (Member)

Abdul Rahman Omar, Ph.D. Associate Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member)

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AINI IDERIS, Ph.D. Professor jDean School of Graduate Studies Universiti Putra Malaysia

Date: 0 3 FEB 2004

DEClARATION

I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

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TABLE OF CONTENTS

Page

DEDICATION ........ ............ ...... ......... . .. ............ ........... ........ . ... ii ABSTRACT ........................................................................... ... iii ABSTRAK ...... .......................... ........ ....................................... v ACKNOWLEDGEMENTS .. ......... . . ............... . ...... . ..................... vii APPROV AL SlIEET ........... . . . ............................... ........... .... .. . ... viii DECLARATION ....................................................................... x LIST OF TABLES ...................................................................... xiii LIST OF FIGURES ..................................................................... xiv LIST OF PLATES ...................................................................... xv

CHAPTER

1

2

3

4

INTRODUCTION 1

LITERATURE REVIEW 4

2. 0 The Mammalian Immune System 4 2.1 Immunomodulation 2.2 Plant-derived Immunomodulatory Substance 12 2.3 Low Molecular Weight Compounds 13

2.3.1 Alkaloids 13 2.3.2 Terpenoids 14 2.3.3 Phenolics and Polyphenols 17

2.4 High Molecular Weight Compounds 18 2. 4. 1 Polysaccharides 18 2. 4.2 Lectins 20

GENERAL MATERIALS AND METHODS

3. 1 Plant extraction 3.2 Preparation of extracts for assay 3.3 Statistical analysis

MITOGENIC ACTIVITY OF PLANT EXTRACTS

4. 0 Introduction 4. 1 Materials and methods 4.2 Results 4.3 Discussion

xi

22

22 23 2 4

2 8

28 30 33 39

5 PHAGOCYTOSIS AND INTRACELLULAR KILLING 41

5.0 Introduction 41 5.1 Materials and methods 44 5.2 Results 48 5.3 Discussion 53

6 NITRIC OXIDE PRODUCTION 56

6.0 Introduction 56 6.1 Materials and methods 59 6.2 Results 61 6.3 Discussion 66

7 HAEMOL YfIC ACTNITY 69

7.0 Introduction 69 7.1 Materials and methods 71 7.2 Results 73 7.3 Discussion 78

8 GENERAL DISCUSSION AND CONCLUSION 81

REFERENCES 88

APPENDICES 95

BIODATA OF THE AU1HOR 99

xii

Table

21

2.2

3.1

4.1

4.2

5.1

5.2

5.3

6.1

6.2

7.1

7.2

8.1

LIST OF TABLES

Comparison of innate and adaptive immunity

Immunostimulating substances and extracts from higher plants and fungi

Plant used in this studies

Stimulation index of water-soluble extracts screened

Stimulation index of methanol soluble extracts screened

Toxic components of mammalian macrophages and neutrophils that mediate killing and digestion

Effects of water extracts on macrophages phagocytic and intracellular killing

Effects of methanol extracts on macrophages phagocytic and intracellular killing.

Nitric oxide production by peritoneal macrophage treated with water-soluble extracts

Nitric oxide production by peritoneal macrophage treated with methanol soluble extracts

Haemolysis percentage of SRBC treated with water-soluble extracts

Haemolysis percentage of SRBC treated with methanol soluble extracts

List of extracts with immunomodulatory effect

Xlll

Page

6

12

25

36

38

42

51

52

64

65

76

77

87

Figure

4.1

6.1

LIST OF FIGURES

Lymphocyte proliferation by ConA shows stimulation where the maximum activity was at 25f.!g/ ml

Standard curve for nitric oxide determination using sodium nitrate

xiv

Page

35

63

Plate

1

2

LIST OF PLATES

Use of acridine orange assay to determine

phagocytosis and intracellular killing ability

by peritoneal macrophages.

The haemolysis of SRBC cause by extracts

xv

Page

50

75

CHAPTERl

INTRODUCTION

Plants play an important role both as providers of foods in the form of

carbohydrates, fibres, proteins and oils, and a source of natural products that

can be used as traditional medicines. Preparations from leaves, seeds, stem bark

and roots of plants have been widely used in traditional medicines. Although

the use of plants for medicinal purposes has been practiced over thousands of

years, it is only relatively recent time that scientific evaluation of plant extracts

and decoctions have been assessed. Most studies described antiviraL

antihelminthic, antitumoral, anti-inflammatory and antirheumatic activities

(Nores, 1997). However, studies on effects upon the immune system are

generally lacking.

Manipulation of the immune system is very important where it involves

immunomodulation process. Immunomodulation can either suppress or

enhance the immune system. It is also essential to improve protective immunity

and reduce immune-mediated disease such as allergy and autoimmunity.

Previous studies showed that plants may consist of several compounds, which

are immunomodulatory. Plant are rich in a wide variety of secondary

metabolites such as tannins, terpenoids, alkaloids and flavonoids, which have

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been found in vitro to have anti-microbial properties (Cowan, 1999). Plant

constituents such as saikosaponin-d extracted from roots of Bupleurum radix,

modifies immune response of T and B cells, and activates immunological

function of the macrophage (Ushio, 1 991). Apart from that, plants also may

contain substances that cause haemolysis such as saponin from Quillaja saponaria

(bark).

Traditional healers have long used plants to prevent or cure infectious

conditions. Herbal medicines play an important and integral part in different

communities and ethnic groups that make up the Malaysian society. Malaysian

herbal medicine has its grass roots in the beliefs, religion, philosophical and

practical aspects of health and diseases as defined by the Malays, Chinese,

Indians and indigenous groups in Malaysia. However, scientific studies on

these medicinal plants are very limited.

In MalaYSia, the use of traditional medicine has been a part of life of multi­

ethnic society for a long time (Khalilur, 1981). Traditional medication is

common especially in rural areas. In urban areas, though modern medicines are

widely used, traditional medicine is not discarded altogether. Very often, we

find urbanities seek traditional medicine as an alternative when modern

medicines does not bring desired results. Plant materials as herbal remedies are

commonly practiced among different communities in Malaysia. Two common

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local medicinal plants are Eurycoma IOllgifolia (tongkat Ali) and Celltella asiatica

(pegaga). E. IOllgifolia is consumed as an aphrodisiac and tonic whilst C. asiatica

is widely used in health food and cosmetic industries. Research is actively

pursued in order to characterize their bioactive compounds. Standardization of

toxicity and efficacy evaluation is important to obtained herbal medicines,

which are of high quality, safe and efficacious. Other popular herbs used are

Morinda citrofolia (mengkudu), Labisia pumila (kacip Fatimal:t) and Andrographis

paniculata (hempedu bumi). These products are being manufactured and sold

regardless of the fact that concrete scientific data on the efficacy and even

toxicity of the herbs used are inadequate or lacking.

Therefore, the objective of this study is to screen crude aqueous and organic

extracts of selected indigenous plants for immunomodulatory and haemolytic

activity.

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CHAPTER 2

LITERATURE REVIEW

2.0 The Mammalian Immune System

The immune system provides the defense mechanisms to protect from hostile

forces in the environment, among which are toxic substances and

microorganisms. Its primary function is to discriminate between self and

nonself. The mammalian immune system involves the action of cellular and

soluble components of the blood. The cellular components are white blood cell

or leukocytes, which consist of neutrophil, basophiL eosinophiL lymphocytes

and monocyte-macrophages but it depends primarily on 3 major cell types:

macrophage, thymus-derived lymphocyte (T cells) and bone-marrow derived

lymphocyte (B cells). The mammalian immune system provides a mechanism

for specific response to the invasion of particular pathogenic microorganisms

and other substances. It is largely this specific physiological response that

protects us against disease.

There are two types of immunity: innate and adaptive (refer Table 2.1). The

important difference between these is that adaptive immune response is highly

specific for a particular pathogen. Innate immunity is a non-specific response

(not antigen specific), where it does not distinguish between self and non-self. It

4

is sometimes called natural immunity where it is present from birth and

includes numerous nonspecific elements. Body surfaces, especially skin, form

the first line defense against penetration by microorganisms. One of the

important group of leukocytes is the phagocytic cells such as monocytes,

macrophage and polymorphonuclear neutrophils. These cells bind to

microorganisms, internalize them and then kill them (Riott et. aI., 1996). In non­

specific immunity, macrophage plays an essential role by ingesting and killing

invading microorganism and by releaSing many soluble factors that contribute

to host defense and to inflammation.

The second type of immunity is called adaptive or acquired immunity. This

system retains a memory of all the invaders it has faced. It is a specific response.

The adaptive immune response is triggered by the presence of a foreign agents

that escape early elimination by the innate immune system. It involves memory

cell formation and antibody production by B cells. Adaptive immunity is

classified into two responses: humoral response and cell-mediated response.

Humoral response acts principally against extracellular phases of infection, that

is, through the secretion of proteins called antibodies or immunoglobulins.

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Table 2.1: Comparison of innate and adaptive immunity

Property Innate Immunity Adaptive Immunity

Physical barriers Skin and mucous membranes None

Soluble factors Enzymes (eg. lysozyme and Antibodies complement) Lymphokines Acute-phase proteins (eg. CRP) a and f3 interferons

Cells Macrophages, T and B lymphocytes Polymorphonuclear neutrophils (PMN), NK cells, eosinophils

Self-nonself Yes Yes discrimination

Specificity No Yes

Memory No Yes

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In contrast, the cell-mediated response is effective in eliminating infections by

pathogenic microorganism that develop within host cells, which involves direct

interaction between lymphocytes and the pathogens. It depends on the actions

of T lymphocytes that divide and differentiate into cytotoxic T cells (killer T

cells). Both lymphocytes are responsible for the defense mechanisms of the

body.

T-Iymphocytes are involved in cell-mediated immunity where it does not

involve antibody production. It only recognizes antigen when following

association with major histocompatibilty (MHC) molecules, which are on the

cell surface. Two classes of MHC namely MHC Class I and Class II are involved

in the elimination of virus infected cells and non-viral infected cells respectively.

The recognition of MHC Class I-antigen will activate cytotoxic T cells to

diminish the infected cells directly whereas MHC Class II-antigen activates

effector T helper cells (Th). Th cells induce cytokine production that will bind to

pathogens and activate cells in secreting proteolytic enzymes, which causes

destruction of the pathogen.

On the other hand, B cells play an important role in the humoral response where

they recognize non-self antigen directly. The development of B cells involves 4

stages; stem cell, pre-B cell, B cell and plasma cells. It enters the circulation and

migrates to spleen and other peripheral lymphoid tissue. When antigen that is

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complementary to surface immunoglobulin receptors and with the help of

appropriate T cells, the B cell will differentiate into antibody secreting plasma

and memory cells. There are five types of imunoglobulin: IgA, IgG, IgM, IgE

and IgD. Each immunoglobulin has specific functions in the immune systems.

Antibodies can also neutralize bacterial toxin and viruses. Apart from antibody

production, the B cell also facilitates the engulfment of opsonized or

agglutinated bacteria by phagocytes and initiates their destruction through

activation of the complement system.

Involvement of soluble mediators in immune response is important. A wide

variety of molecules are involved in the development of immune responses.

These includes antibody as mentioned before and cytokines. During infections,

these proteins concentration in serum increases rapidly. Cytokines are proteins

or pep tides. A number of cytokines produces by the immune cells are

interferons, interleukins and tumor necrosis factors. Each cytokines plays a

different role for example, interferons is important in limiting the spread of

certain viral infections where else interleukins which consist of IL-l to IL15

produced mainly by T cells involved in directing other cells to divide and

differentiate.

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2.1 Immunomodulation

Immunomodulation is a modification process of immune response, which is

generally used to describe the pharmacological manipulation of immune

system. It can either increase the magnitude of immune response;

immunostimulation or decrease it; immunosuppression. This

immunomodulation process can either be a specific immunomodulation which

depends on a particular antigenic stimulus (e.g vaccination) or a non-specific

where the immune system is altered by a wide range of antigenic stimuli or

immunocompromised. The primary targets for this process include T cells, B

cells and monocytes-macrophage.

The mechanisms involve in modifying the immune response are alteration of

responsiveness either at cellular level or intracellular nucleotide level. The

cellular level involves changes in membrane surface and metabolism of

phospholipids, fatty acids and prostaglandins. In the intracellular level, the

alteration of cyclic AMP (cAMP) and cyclic GMP (cGMP) in lymphocyte occur.

High level of cAMP will inhibit the effector function of lymphocyte where as the

increase in cGMP level will promote lymphocyte activity and thus have

immunopotentiating properties. Substances that can increase cAMP are

glucocorticoids. Plant lectins and lipopolysaccharides act in cGMP agonists.

The effect of cyclic nucleotides on lymphocyte indicate that cGMP stimulates

9