Immunomodulatory Effects and Toxicity

of pudica, the Sensitive

by

Cheng Yuk Kwan，Anna

Thesis submitted as partial fulfilment for

the degree of Master of Philosophy

June, 1993 气、• \ Division of Biology

Graduate School , ./ - ：："

The Chinese University of Hong Kong U.L^

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~UNIVERSITY /M// ^^gSilBRARY system/^ Thesis Committee:

Dr. Paul P.H. But (Advisor)

Dr. Vincent E.G. Ooi (Advisor)

Prof. C.F. Chen (External Examiner)

Dr. K.W. Chiu Acknowledgements

With great pleasure I acknowledge my gratitude to my supervisors, Dr. Paul

P.H. But and Dr. Vincent E.G. Ooi. Their enthusiasm, patience, continuing encouragement and sincere supervision are memorable.

I am especially indebted to Prof. C.F. Chen, who serves as the external examiner, and Dr. K.W. Chin, who sits in the thesis committee, for their invaluable advice for the preparation of this dissertation.

I would like to express my grateful thanks to Mr. H.M. Xu and Mr. C.P. Lau for their training of techniques.

Finally, may I also thank my co-workers in the laboratory and all the staffs in the Department of Biology and the Chinese Medicinal Material Research Centre,

The Chinese University of Hong Kong, for their kind co-operation. TABLE OF CONTENTS

Acknowledgements

Table of Contents i Abbreviations iv Abstract vi List of figures ix List of tables xi

Chapter One: Introduction 1.1 Objective and scope of the project 1

1.2 Literature review of Mimosa pudica 1.2.1 Morphology of Mimosa pudica 3 1.2.2 Chemistry of Mimosa pudica 5 1.2.3 Uses in traditional medicine 5 1.2.4 Clinical and pharmacological studies of Mimosa pudica 6 1.2.5 Toxicology of Mimosa pudica 8 1.2.6 Characteristics and toxicology of 9

1.3 Immunomodulation 1.3.1 Overview of the immune system 11 1.3.2 Strategies on the study of immunomodulation of Mimosa pudica 13

1.4 Toxicology 1.4.1 Principles of the toxicological assays 1.4.1.1 LD50 1.4.1.2 Enzyme assays 18 1.4.1.3 Subacute toxicity test 24 1.4.1.4 Reproductive toxicity test 25

i Chapter Two: Materials and methods 2.1 Materials 2.1.1 Mimosa pudica 27 2.1.2 Animals 27 2.1.3 Chemicals 28

2.2 Methods 2.2.1 Extraction of Mimosa pudica 32

2.2.2 Assays for the immunomodulatory effects of Mimosa pudica 2.2.2.1 Cell preparation a) Splenocytes 35 b) Thymocytes 35 c) Macrophages 36 2.2.2.2 Splenocyte proliferation 37 2.2.2.3 Thymocyte proliferation 38 2.2.2.4 Phagocytic activity of macrophages 39 2.2.2.5 Release of IL-1 by macrophages 40 2.2.2.6 Plaque forming cells 41 2.2.2.7 Restoration on splenocyte blastogenesis of old mice 42

2.2.3 Assays for the toxicity of Mimosa pudica 2.2.3.1 LD50 43 2.2.3.2 Enzyme assays 43 2.2.3.3 Subacute toxicity 43

2.2.3.4 Reproductive toxicity 44

2.2.4 Statistical analysis 44

Chapter Three: Results 3.1 Immunomodulatory effects of Mimosa pudica 3.1.1 In vitro study on the lymphocyte proliferation

ii 3.1.1.1 Splenocyte proliferation 45 3.1.1.2 Thymocyte proliferation 50 3.1.2 In vivo study on the lymphocyte proliferation 53 3.1.3 Phagocytic activity of macrophages 58 3.1.4 Release of IL-1 by macrophages 64 3.1.5 Plaque forming cells 67 3.1.6 Restoration on splenocyte blastogenesis of old mice 69

3.2 Toxicity of Mimosa pudica 3.2.1 LD50 72 3.2.2 Enzyme assays 75 3.2.3 Subacute toxicity 80 3.2.4 Reproductive toxicity 85

Chapter Four: General discussion on the immunomodulatory effects and toxicity of Mimosa pudica 4.1 Immunomodulatory effects of Mimosa pudica 88 4.2 Toxicity of Mimosa pudica 95

Chapter Five: Concluding remarks 99

References 1 以 Appendix 113

iii Abbreviations

ADP adenosine diphosphate ALP alkaline phosphatase ALT alanine aminotransferase APC antigen-presenting cell AST aspartate aminotransferase ATP adenosine triphosphate B cell bum-derived cell CK creatine kinase Con A concanavalin A cpm counts per minute DWE dry weight equivalent FBS fetal bovine serum G-6-P glucose-6-phosphate G-6-PDH glucose-6-phosphate dehydrogenase GPT glutamic pyruvic transaminase GOT glutamic oxalacetic transaminase ^H-TdR tritiated thymidine HC hydrocortisone i.g. intragastrical Ig immunoglobulin i.p. intraperitoneal IL-1 interleukin 1 K cell antibody-dependent cytotoxic cell LDH lactate dehydrogenase LPS lipopolysaccharide NAD nicotinamide adenine dinucleotide NADH reduced nicotinamide adenine dinucleotide NADPH reduced nicotinamide adenine dinucleotide phosphate NK cell natural killer cell O.D. optical density

iv PBS phosphate-buffered-saline PDi day-1 of pregnancy

PDio day-10 of pregnancy PDi6 day-16 of pregnancy PEC peritoneal exudate cell PFC plaque forming cell PHA phytohaemagglutinin P.O. per OS (L) by mouth RBC red blood cell SF U/ml Sigma-Frankel Units/millilitre SRBC sheep red blood cell T cell thymus-derived cell Tc cell cytotoxic T cell

Th cell helper T cell Ts cell suppressor T cell TNF tumour necrosis factor U/L Units/litre WBC white blood cell

V Abstract

Mimosa pudica is a pan-tropical weed. It is used in Chinese medicine for analgesic and anti-inflammatory purposes. In Hong Kong the herb is often used to treat osteophyte. However, little effort has been made to evaluate its pharmacological effects. This project is designed to investigate its immunomodulatory values.

Moreover, as the plant was recorded to have a mild toxicity, its toxicity was also assessed to ensure the safe use of this herb. If this plant is shown to have pharmacological value, this may help turn a weed to human beneficial use.

The Mimosa extract used in this study was the water extract of the whole plant. The dry plant material contained in each gram of extract, or dried weight equivalent (DWE) was 5.3. From the in vitro immunological studies, millipore- filtered fraction of the water extract of M. pudica was found to have modulatory effect on both cellular and humoral immunities. It showed a significant stimulatory effect on splenocyte proliferation and a co-mitogenic effect with lipopolysaccharide

(LPS) but not with concanavalin A (Con A). However, it did not show prominent effect on thymocyte proliferation. Moreover, the extract inhibited the thymocyte proliferation in the presence of Con A. Some of these findings could also be observed in the in vivo system. In mice fed with the extract for 10 days, the splenocyte proliferation was enhanced but the thymocyte proliferation was suppressed.

Moreover, the mitogenic responses of splenocytes to both LPS and Con A and of thymocytes to Con A were enhanced. From the immunorestoration sturdy on 20- week-old mice, it was noted the blastogenic response of the mice could be partially

vi restored by Mimosa extract. Besides the modulatory effects on lymphocytes, the extract could increase the phagocytosis of macrophages and reverse the inhibition caused by hydrocortisone. However, this effect could only be observed when the macrophages were cultured in the presence of the extract, but not for the macrophages cultured from the mice after daily oral administration of the extract for

10 days. Besides, Mimosa extract inhibited the release of IL-1 by macrophages but enhanced the release by LPS-stimulated macrophages. The extract also activated the primary humoral immune response by increasing the release of haemolysin from antibody-forming-cells •

For the toxicological studies, the results of acute and subacute toxicity tests indicated the water extract of M. pudica was practically non-toxic when taken orally.

No mortality was observed in LD50 study by i.g. administration even at 15.0 g/kg

(DWE = 79.5 g/kg). Similarly, no toxic symptom was observed in the subacute study at 10.0 g/kg/day (DWE = 53.0 g/kg/day) for 30 days. It also did not have any influence on early pregnancy at 3.75 g/rat/day (DWE = 19.9 g/rat/day) when administered orally for 10 days. However, when the extract was injected intraperitoneally, toxic symptoms of hindlimb paralysis and elevation of serum enzyme levels such as LDH, CK, GOT and GPT were observed. LD50 by i.p. injection for male and female mice were found to be 3.3土0.8 and 3.4±0.9 g/kg, respectively (DWE 二 17.5±4.2 and 18.0土4.8 g/kg). Apparently, the toxicity of

Mimosa extract was greatly reduced when given through the gastro-intestinal tract.

As a conclusion, the results of our present studies showed that Mimosa pudica

vii had immunostimulatory effects. Although the potency was not high when the extract was given orally, its stimulatory effects may play a role in some of the traditional uses of this herb.

viii List of Figures

Figure 1.1: A diagram showing a flowering branch of Mimosa pudica L. 4

Figure 2.1: Flow chart showing the extraction of Mimosa pudica 34

Figure 3.1: In vitro concentration-response curve of Mimosa pudica on the splenocyte proliferation 47

Figure 3.2: In vitro concentration-response curve of Mimosa pudica on the splenocyte proliferation in the presence of 10 ^g/ml LPS 48

Figure 3.3: In vitro concentration-response curve of Mimosa pudica on the splenocyte proliferation in the presence of 1 jug/ml Con A 49

Figure 3.4: In vitro concentration-response curve of Mimosa pudica on the thymocyte proliferation 51

Figure 3.5: In vitro concentration-response curve of Mimosa pudica on the thymocyte proliferation in the presence of Con A 52

Figure 3.6: In vitro effect of Mimosa pudica on the phagocytosis of macrophages 60

Figure 3.7: In vitro effect of Mimosa pudica on the phagocytosis of macrophages in the presence of hydrocortisone 61

Figure 3.8: In vivo effect of Mimosa pudica on the phagocytosis of macrophages 62

Figure 3.9: In vivo effect of Mimosa pudica on the phagocytosis of macrophages 63

Figure 3.10: In vitro effect of Mimosa pudica on the release of IL-1 from macrophages 65

Figure 3.11: In vitro effect of Mimosa pudica on the release of IL-1 from LPS-stimulated macrophages 66

Figure 3.12: Primary humoral antibody response to SRBC in mice treated with Mimosa pudica 68

Figure 3.13: Dose-lethal toxicity curve of Mimosa pudica on mice 73

ix Figure 3.14: Dose-response curve of Mimosa pudica (i.p.) on the serum level of lactate dehydrogenase (LDH) of rats 76

Figure 3.15: Dose-response curve of Mimosa pudica (i.p.) on the serum level of creatine kinase (CK) of rats 77

Figure 3.16: Dose-response curve of Mimosa pudica (i.p.) on the serum level of glutamic oxalacetic transaminase (GOT) of rats 78

Figure 3.17: Dose-response curve of Mimosa pudica (i.p.) on the serum level of glutamic pyruvic transaminase (GPT) of rats 79

Figure 3.18: Effect of Mimosa pudica (p.o.) on the body weight of mice after subacute treatment 81

Figure Al: Calibration curve showing O.D. value of the amount of neutral red 113

Figure A2: Calibration curves of glutamic oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) activities 119

Figure A3: Serum level of lactate dehydrogenase (LDH) at different time after i.p. injection of Mimosa extract to rats 121

Figure A4: Serum level of creatine kinase (CK) at different time after i.p. injection of Mimosa extract to rats 122

Figure A5: Serum level of glutamic oxalacetic transaminase (GOT) at different time after i.p. injection of Mimosa extract to rats 123

Figure A6: Serum level of glutamic pyruvic transaminase (GPT) at different time after i.p. injection of Mimosa extract to rats 124

V List of Tables

Table 1.1: Classification of toxicity 18

Table 3.1: In vivo effect of Mimosa pudica on the mice splenocyte proliferation 55

Table 3.2: In vivo effect of Mimosa pudica on the mice thymocyte proliferation 56

Table 3.3: In vivo effect of Mimosa pudica on the lymphoid organ weights 57

Table 3.4: In vivo effect of Mimosa pudica on splenocyte blastogenesis of old mice 70

Table 3.5: In vivo effect of Mimosa pudica on the lymphoid organ weights of old mice 71

Table 3.6: Death time of female mice after i.p. injection of Mimosa pudica 74

Table 3.7: Death time of male mice after i.p. injection of Mimosa pudica 74

Table 3.8: Effect of subacute oral administration of Mimosa pudica on the serum enzymatic activities of mice 82

Table 3.9: Effect of subacute oral administration of Mimosa pudica on the blood cell counts of mice 83

Table 3.10: Effect of subacute oral administration of Mimosa pudica on the vital organ weights of mice 84

Table 3.11: Effect of Mimosa pudica (p.o.) on the percentage of pregnancy in rats 86

Table 3.12: Effect of Mimosa pudica (p.o.) on the fetus implantation in rats 86

Table 3.13: Effect of Mimosa pudica (p.o.) on the fetus formation in rats 87

Table 3.14: Effect of Mimosa pudica (p.o.) on corpora lutea formation- in rats 幻

xi Chapter One

Introduction

1.1 Objective and Scope of the Project

Recently, many pharmaceutical research institutes are interested in higher as a source for new drugs (Hamburger and Hostettmann, 1991; Malone, 1983;

Phillips, 1992). Plants used in traditional medicine are considered likely to yield pharmacologically active compounds. The World Health Organization (WHO) is also aware of the importance of herbal medicines for the health of many people throughout the world. Herbal medicines have been recognized as a valuable and readily available resource for primary health care, and WHO has endorsed their safe and effective use

(WHO, 1993). Since the use of most herbal medicines is empirically, few of them have been subjected to scientific study. Therefore, scientific research on evaluating the efficacy and safety of herbal medicines should be encouraged (WHO, 1993).

Mimosa pudica is a wide-spread plant and an inexpensive Chinese medicine.

It is used as a herbal medicine in southern provinces of China. It was first recorded as a medicinal plant in Qing dynasty in Sheng Cao Yao Xin Bei Yao (He, 1711), according to which the plant had analgesic and anti-inflammatory effects. According to the Encyclopedia of Chinese Materia Medica (Anonymous, 1977), Mimosa pudica was recorded to have the value of cleaning the evil-heat, tranquillizing； relieving dyspepsia, and removing toxic material. It is used to treat enteritis, gastritis,

1 insomnia, infantile malnutrition, redness and swelling of eye, deep abscess and herpes zoster. In Hong Kong, many people take it to treat osteophyte (personal observation, unpublished). Although this plant is used in Chinese medicine, little effort has been made to evaluate its pharmacological effects. Traditionally, the guiding principle of

Chinese medicine is to maintain the natural balance of the human body or to restore it to a balanced state during periods of illness, rather than a direct assault on the disease. Based on this concept, some people believe that many traditional medicines exert their effects by improving our immune system (Chihara, 1992; Labadie et al.,

1988). Therefore, this project is designed to investigate whether Mimosa pudica has any immunomodulatory effect which, if present, may help offer rationale for the use of this herb in Chinese medicine. Moreover, Villarreal et al. (1991) reported that three different extracts of related species in the same genus, Mimosa tenuiflora, could modify the growth rate of normal embryonic fibroblasts and KB cells from a nasopharyngeal carcinoma in tissue culture conditions. It seems that Mimosa pudica may probably contain some components which also have immunomodulatory effects.

On the other hand, as this plant was recorded to have a mild toxicity (Anonymous,

1983)，its toxic effects are also studied in order to ensure the safe use of this herb.

2 1.2 Literature Review of Mimosa pudica

1.2.1 Morphology of Mimosa pudica

Mimosa pudica L.(含羞草；sensitive plant) belongs to the family

Leguminosae, subfamily (Elias，1981). It is a scrambling shrubby plant growing to one meter high with branching stem (Figure 1.1). The plant is hairy and spiny. Leaves are alternate, bipinnatedly compound, and would fold and droop upon disturbance. Pinnae are usually four on each leaf, 4-7 cm long, digitately arranged at the apex of the main , bearing 30-40 leaflets. Leaflets are linear-oblong, sessile, 8-13 mm long, short and sharp at the tip, with scattered spiny hair.

Inflorescence is a globular capitulum with long stipe, single or 2 to 3 arising from the leaf axil, 1 cm in diameter. Flowers are pink and numerous. Calyx and corolla are minute; are 4 in number, 3-5 mm long, exerted beyond the corolla. is a loment, flattened, slight curved, 1-2 cm long, composed of 3-5 one-seeded segments which disarticulate and fall apart when mature, leaving only the spinose- bristly margins of the loment. Flowering season is in August but may be observed from June to September (Hou, 1956).

M. pudica originally grows in tropical America but is now wide-spread as a pan-tropical weed (Good, 1964). It grows on the hillside, roadside, lawns and damp fertile pasture. It is found in eastern, southern and southwestern China. It is cultivated in temperate areas (eg. Inner Mongolia) as an ornamental plant

(Anonymous, 1977).

3 •••••^••••••hiZ^IHBIBI^^^HSEHIHBiHBIIII^HBBSlSSIHI

Figure 1.1: A diagram showing a flowering branch of Mimosa pudica L.

4 1.2.2 Chemistry of Mimosa pudica

The major chemical constituents of the whole plant of Mimosa pudica are

flavonoid glycosides, phenols, amino acids and organic acids. It also contains mimosine (Notation and Spenser, 1964) and mimosine-o-B-D-glucoside (Murakoshi

广 al., 1971). The leaves contain a kind of myosin-like contractile protein (Biswas and Bose, 1968)，which is involved in the movement of the leaves. Seeds were found to contain 7.4% of moisture, 25.7% of crude protein, 12.3% of pentosan, and 6.5% of hexane extractable oil. The defatted seed powder contained a which is a neutral polysaccharide (galactomannan). The mucilage contains 35.5% of galactan and negligible amount of pentoses (Farooqi et al., 1977). It is also found to have approximately 17% of oil. The fatty acids of the oil include 0.4% of linolenic acid,

51% of linoleic acid, 31% of oleic acid, 8.7% of palmitic acid, and 8.9% of stearic acid. The seeds also contain 2.5% of unsaponifiable matter which contained two sterols (Aggarwal and Karimullah, 1945). Roots contain , lactone substances and flavonoid glycoside (Anonymous, 1977). Mimosine present in the plant is a toxic amino acid. Its toxicity will be discussed in another section (section: 1.2.6).

1.2.3 Uses in traditional medicine

According to the Encyclopedia of Chinese Materia Medica (Anonymous,

1977), Mimosa pudica tastes sweet and has cold properties. The whole plant has medicinal uses for cleaning away evil-heat, tranquillizing, relieving dyspepsia, and treating enteritis, gastritis, insomnia, redness and swelling of eye, infantile

5 malnutrition and inflammation. The root can be used as an expectorant and

antitussive agent. Other literatures recorded that Mimosa pudica is used to treat

neurasthenia, high fever in children, acute conjunctivitis, urolithiasis and

rheumatalgia. It also has analgesic and diuretic effects. It can be used topically in

traumatic injury, pyoderma and herpes zoster (Cheung and Li, 1981; Zhong Cao Yao

Xue, 1976).

In Southeast Asia, the water extract of roots is used to treat malaria,

dysmenorrhoea and asthma, and to induce vomiting. Leaves are used in the treatment

of swollen sore and pain while seeds are used in throat diseases (Cheung and Li,

1981; Cheung, 1963). In Hong Kong, many people claim that the whole plant or root

of M. pudica is effective in the treatment of osteophyte (personal observation,

unpublished).

1,2.4 Clinical and pharmacological studies of Mimosa pudica

Although there are many uses of Mimosa pudica, very little pharmacological

and clinical information can be found. Only one clinical study had been conducted

on the treatment of 30 cases of chronic bronchitis of the aged people by using the root

extract of the herb. After three courses of treatment (10 days for one course of

treatment), 24 cases were satisfactorily controlled and 3 cases showed significant

improvement (Anonymous, 1972). A compound prescription containing Mimosa pudica and Rubia cordifolia was also used to treat 86 patients of chronic bronchitis

of the aged people; the results indicated a 95.4% efficiency after the first course of

6 treatment (Anonymous, 1972).

Besides the clinical study, there were a few pharmacological studies on the water extract of the herb. They are summarized here:

a) Expectorant and antitussive effects

A study showed that the extract of the root of M. pudica had antitussive effect in mice; the whole plant and leaves had expectorant effect. The assay of using isolated intestinal segment indicated M. pudica had no spasmolytic effect

(Anonymous, 1971).

b) Effect on smooth muscle

It was reported that the root extract did not show any dilation effect on isolated bronchus of guinea pig by perfusion study, but had significant anti- acetylcholine effect on isolated ileum of rabbit (Anonymous, 1977).

c) Effect on cardiovascular system

In experiment using anaesthetized cat, it was found that intravenous injection of the root extract could steadily increase the blood pressure by 10-20 mmHg. It then returned to normal after 1 hour (Anonymous, 1971).

d) Antibacterial effect

A compound prescription containing the roots of Mimosa pudica and Rubia cordifolia showed a strong inhibitory effect on Diplococcus pneumoniae,

7 Streptococcus alpha, Staphylococcus aureus and influenza (Anonymous, 1971).

e) Effect on urolithiasis

Joyamma et al (1990) investigated the effect of M. pudica on treatment of urolithiasis. A model of urolithiasis was established by the implantation of zinc discs in urinary bladder of albino rats. It was found that the herb was not effective in either preventing stone deposition or dissolving preformed stones.

Based on the traditional medicinal uses of Mimosa pudica, such as treating rheumatalgia and some inflammatory diseases, this herb seems to affect the immune system. Therefore, its immunomodulatory effects were examined in this study. As the crude extract usually contains only a small amount of the active constituents, highly sensitivity bioassays should be used (Hamburger and Hostettmann, 1991). In order to meet this requirement, immunological assays are chosen in this project.

1.2.5 Toxicology of Mimosa pudica

A toxicity study was reported on the water extract of the root of Mimosa pudica in a concentration of 0.5g plant material per ml (Anonymous, 1971). The result showed that the highest tolerance dose of the root extract in mice was 0.8 ml/mice by intravenous injection. On the other hand, the highest tolerance dose of

300% of the water extract of the whole plant was found to be 0.5 ml/mice by oral administration. The major toxicity symptom was the inhibition on nervous system.

Oral administration of large doses could induce diarrhoea. Another study reported

8 that oral administration of 200 g/kg of the root extract to five mice, all mice showed reduction in activities and one had diarrhoea. No death was observed within 24 hours. When the dosage was increased to 250 g/kg, the mice had diarrhoea and a significant reduction of activities. Two of the five mice died (Anonymous, 1977).

As little toxicity information on the whole plant of M. pudica, this project will study the general acute and subacute toxicity of this herb. Its effect on fertility and pregnancy of rats was also investigated as the herb is said to be contraindicated in pregnancy (Anonymous, 1976, 1983; Cheung and Li, 1981).

1.2.6 Characteristics and toxicology of mimosine

In contrast to the few toxicity studies on Mimosa pudica, many studies had been carried out on mimosine, which is a toxic amino acid found in the legume genera Mimosa and Leucaena. The structure of mimosine is shown below:

NH „ I Cli ,C11C00I1 I

八I

0

Mimosine forms dl-form crystal from water with melting point at 235-236

It is slightly soluble in water (Ig in 500 ml at 25°C) (Lin and Ling, 1961a). It is much less soluble in methanol and ethanol, practically insoluble in the higher alcohols such as dioxane, ethylacetate, ether, benzene, chloroform, glacial acetic acid, pyridine

9 and cellosolve，but soluble in diluted acids or bases. The maximum UV absorption is at 282 nm (Budavari, 1989).

Animals fed with a diet containing 0.5-1.0% of mimosine would result in growth retardation, shorten life span, alopecia (Lin and Ling, 1961)，cataract (Yang et aL, 1968; Von Sallmann and Grimes, 1959), hair loss (Crounse et al, 1962), infertility (Hylin and Lichton, 1965) and deformity of fetus (Dewreede and Way man,

1970). However, Lin et al. (1964) demonstrated that addition of 1% of DL- phenylalanine to the diet containing 0.5% of mimosine could reverse 37% of the growth inhibition caused by mimosine. Moreover, the growth retardation could be completely reversed by feeding the animals with 1% of L-tyrosine. Lin and Ling

(1961b) found that protein and amino acids were present in the urine of the animals fed with the diet containing 0.5% of mimosine. Three possible mechanisms were suggested on the toxicity of mimosine: (1) antimetabolic effects on amino acid metabolism or protein biosynthesis as mimosine is structurally similar to phenylalanine and tyrosine (Lin et al.，1967; Lin et al.，1964); (2) mimosine forming a complex with pyridoxal phosphate which would then inhibit the activities of Bg- requiring enzymes (Lin et al” 1962); and (3) mimosine acting as a chelating agent which would inhibit some metal-containing enzymes (Chang, 1960; Tsai, 1961; Lin and Tung, 1964).

10 1.3 Immunomodulation

1.3.1 An overview of the immune system

Our immune system defends ourselves against invasion by infectious agents,

such as bacteria, viruses, fungi and other parasites, as well as spontaneously arising

neoplasms. However, it is also responsible for hypersensitivity diseases and

autoimmune diseases when there are defects in the immune system. Moreover, it

plays a role in the rejection problems when organs or tissues are transplanted.

The immune system is composed of several cell types (eg. phagocytes,

lymphocytes, natural killer cells and mast cells) and many different kinds of soluble

factors (eg. complements, lymphokines and interferons). All the cellular components

of the system arise from haemopoietic stem cells, which are a unique group of

unspecialized cells. The haemopoietic stem cells are present in the fetal liver or adult

bone marrow. During development, the haemopoietic stem cells differentiate into

different cell types: erythroids (erythrocytes), megakaryocytoids (platelets), myeloids

(granulocytes and monocytes) and lymphoids (lymphocytes). The latter two, namely

myeloids and lymphoids, are particularly important in the immune system. There are

two types of lymphocytes, namely B cells and T cells. The maturation of most cells

occur in bone marrow while T lymphocytes mature in the thymus. These organs are

referred to as primary lymphoid organs. After maturation, the cells enter the

circulation and recirculate through the secondary lymphoid tissues, including spleen,

.lymph nodes, tonsils and gut-associated lymphoid tissues (Roitt et al, 1989),

11 The protective mechanisms of the immune system may be divided into two groups: the innate and adaptive immunities. The innate immunity includes mechanical barrier, soluble factors (eg. lysosomes and complements), and inflammatory cells (eg. phagocytes and natural killer cells). This innate resistance is nonspecific and indiscriminate. In contrast to the innate immunity, the adaptive immunity is specific and discriminate. The adaptive resistance can be improved by immunization. This immunity is carried out by the cooperation and interaction of the two immune responses: humoral- and cellular-mediated immune responses (Sell,

1987).

The humoral immunity involves the production of antibodies by B lymphocytes. After antigens are recognized by certain B lymphocytes, the B cells would begin to proliferate and differentiate into plasma cells which in turn produce a large amount of antibodies. These antibodies protect the host by binding to the antigens and hence facilitate the phagocytes to engulf the antibody-antigen complex.

Moreover, the binding can activate the complement system and some other antibody- dependent cellular cytotoxicity to destroy the antigens (Dean et al., 1989).

The cellular immunity primarily involves T lymphocytes, where the helper T cells (Th) play the central role. After antigens are recognized and processed by antigen-presenting cells (APC) (eg. macrophages), the antigens are presented to the helper T cells which in turn help B cells to produce antibodies. Also, helper T cells can modulate the other effector cells, including cytotoxic T cells (Tc), natural killer cells (NK), macrophages, granulocytes, antibody-dependent cytotoxic cells (K) and

12 suppressor cells (Ts), by releasing different kinds of lymphokines. Besides, cytokines

(eg. interleukins and tumor necrosis factor) released by macrophages also take an important part in these actions (Roitt et al., 1989).

1-3.2 Strategies on the study of immunomodulation of Mimosa pudica

Immunomodulation can be defined as any type of therapeutic intervention aimed at restoring the normal function of the immune system. Immunomodulators are drugs that directly modify a specific immune function or have a net positive effect

(immunostimulation) or a net negative effect (immunosuppression) on the activity of the immune system. The potential uses of immunomodulators in clinical medicine include the reconstitution of immune deficiency and the suppression of normal or excessive immune functions such as in the treatment of graft rejection or autoimmune diseases (Goust et al., 1990; Jaffe and Sherwin, 1991).

It is a worldwide interest in searching for potential drugs which have immunostimulatory or immunomodulatory effects (Labadie et al., 1988; Chihara,

1992). Although the indication 'immunodeficiency' cannot be found in traditional medicine, it is obvious that traditional medicines contain a large amount of drugs which from ancient times had been used successfully against infections, cancer, and various chronic diseases. Therefore, it is reasonable to select those plant drugs which have antimicrobial, antiviral and antitumour activities for studying their immunomodulatory activities (Wagner, 1985; Wagner and Jurcic, 1991). From folkloric records and current experimental studies (Anonymous, 1977), it appeared

13 that Mimosa pudica had some antibacterial, antiviral and anti-inflammatory effects, and it was therefore selected in this study to determine if it had immunomodulatory effects. Some assays were selected to measure its influence on the phagocytic system, the T- and B-lymphocytes, and the secretion of some soluble factors. These assays include lymphocyte proliferation, phagocytic capability of macrophages, secretion of interleukin 1 (IL-1) by macrophages and secretion of immunoglobulin M

(IgM) by B cells. The principles of these assays are summarized as follows:

a) Lymphocyte proliferation

Lymphocyte proliferation or transformation is the process in which T or B lymphocytes undergo new DNA synthesis and cell division in response to a given stimulus. The commonly used stimulants are mitogens such as phytohaemagglutinin

(PHA), concanavalin A (Con A) and lipopolysaccharide (LPS), which are non-specific polyclonal activators. Mitogen-induced proliferation, unlike specific antigens, does not require previous exposure, and can be used to assess the responsiveness of T or

B lymphocyte populations as a whole. Mitogens activate lymphocytes by binding to surface receptors which are present on almost all lymphocytes of a given type.

Different mitogens can selectively activate T or B cell populations (Ashman, 1984;

Miller et al, 1991; Greaves and Janossy, 1972).

Lymphocyte proliferation is performed by in vitro culturing of a lymphocyte population in the presence or absence of a selected stimulus for a period of time.

During incubation, the stimulated lymphocytes are transformed into large： blast-like cells that undergo DNA synthesis and mitosis. Evaluation of lymphocyte

14 transformation can be quantitated by determination of the increased DNA synthesis,

which in turn can be detected by the incorporation of a radiolabeled DNA precursor,

eg. ^H-thymidine, and expressed in counts per minute (cpm) (Miller et al., 1991).

b) Phagocytic activity of macrophages

Acute inflammation in the peritoneal cavity can be induced by the i.p. injection of thioglycollate. After 72 hours, peritoneal cellular exudates which are predominantly composed of macrophages can be obtained (Gervais et al，1984). The phagocytosis may be analyzed quantitatively by measuring the accumulation of particles within the macrophages (Southewick and Stossel, 1986).

Glucocorticoids are potent immunosuppressive agents. They have inhibitory effect on the phagocytosis of macrophages (Grasso et al., 1981; Wiener and

Marmary, 1969; Parrillo and Fauci, 1979). Therefore, hydrocortisone is used as a positive control in this assay to study the effect of Mimosa pudica on the phagocytic activity of macrophages in the presence of hydrocortisone.

c) Release of interleukin 1 (IL-1) by macrophages

IL-1 plays an important role in inflammation and many immune responses.

It is produced by a range of cell types, predominantly monocytes and macrophages

(Ward et al., 1988) which are induced to released IL-1 following the uptake of antigen-antibody complexes or LPS (Pignol et al., 1989). Since IL-1 can promote the growth of T lymphocytes, thymocyte proliferation can be used as an index for IL-

1 release by macrophages (Yang and Li, 1989; Ward et al., 1988; Snyder and

15 Unanue, 1982).

d) Plaque forming cell (PFC)

Antibody production is one of the major lymphocyte effector functions. The antibody-producing cells are measured by plaque forming cell assay. By modifying the plaque forming cell assay, it is possible to separatively identify different classes of immunoglobulins, as well as to identify the total number of antibody-producing cells (Steward and Male, 1989). Antibody-forming cells can be measured quantitatively by mixing the test population with antigen sensitized red cells. The red cells that surrounding the specific antibody-secreting cells would be coated with the haemolysin and then lysed by complement. Therefore, the amount of antibody- secreting cells and the amount of antibodies that released can be determined by the degree of haemolysis of red cells (Simpson and Gozzo, 1978).

e) Immunorestoration on splenocyte blastogenesis of old mice

It has been known that decay of the immune system is accompanied with the aging process. The high susceptibility of aged people to infectious diseases is closely related to the decline of immune response (Antonaci et al.，1987; Kim et al.，1985).

If Mimosa pudica can enhance the immune system, it is interesting to see whether it can also restore the declined immune response in the old animals. As a preliminary study, the assay of lymphocyte proliferation was chosen to investigate the effect of the Mimosa extract on the cellular immunity of the old animals.

16 1.4 Toxicology

Toxicology involves the study of the adverse effects of chemicals on living systems in order to predict chemical hazard to man. This study helps in defining possible toxicity from short-term use, but is particularly important in detecting toxicity that may occur either after prolonged exposure or years after the exposure has been discontinued (Anderson and Conning, 1988; WHO, 1993).

1.4.1 Principles of the toxicological assays

1.4.1.1 LD50

Acute toxicity has been defined by the Organization for Economic Cooperation and Development (OECD) as "the adverse effects occurring within a short time of administration of a single dose of a substance or multiple doses given within 24 hours". The determination of the median lethal dose (LD50) of a compound is the most frequently used acute toxicity test. LD50 is the dose of a compound that causes

50% mortality in a population. OECD panel of experts defined LD50 as the

"statistically derived single dose of a substance that can be expected to cause death in 50% of the animals" (Chan and Hayes, 1989). Although many critics criticize that

LD50 has limited significance in predicting safety or toxicity in man, preliminary lethality data are required to plan a more extensive toxicity study. Moreover, LD50 can provide a measure of relative toxicity which allows the tested substance, especially new compounds, to be evaluated and allocated to a particular class of

17 toxicity (Table 1.1) (Pascoe, 1983; Gad and Chengelis, 1988).

Table 1.1: Classification of toxicity (Pascoe, 1983)

LD50 (mg/kg) Classification

< 5 super-toxic 5-50 extremely toxic 50-500 very toxic 500-5000 moderately toxic 5000-15000 slightly toxic > 15000 practically non-toxic

1.4.1.2 Enzyme assays

Enzymes are proteins that act as biological catalysts, which are always highly specific for particular types of biochemical reactions, by altering the rate and providing a means of regulation of metabolism. Their activities are subject to the influence of many variables including the temperature and pH of their environment, the supply of reacting materials, the removal of reaction products, and the presence of activators or inhibitors (Walmsley and White, 1988; Wilkinson, 1976).

Although a great diversity of enzymes are found in the tissues and fluids of the body, only a few are routinely studied for clinical diagnosis. Since enzymes are primarily intracellular, the presence of increased amounts of an enzyme in serum or plasma is generally a consequence of cell injury that allows the escape of intracellular molecules. Therefore, abnormal elevation of enzymes in serum can be used as an

18 evidence of cell or organ damage. Enzymes released into the blood circulation have

no physiological function and are gradually removed by normal routes of excretion

(Sacher et al, 1991).

Some most commonly used enzymes have a widespread distribution in most

. body tissues; an increased activity of these enzymes would result in a number of

diseases. On the other hand, some enzymes are organ-specific or occur in different

forms eg. isoenzymes, according to the tissue from which they are derived. The

measurement of such appropriate plasma enzyme activities can yield organ-specific

diagnostic information. Although many commonly used enzymes are non-specific,

the pattern of their increase may give some information in diagnosis, or combine with

clinical findings to indicate the organs involved (Walmsley and White, 1988;

McLauchlan, 1988).

Five of the commonly used enzymes are selected here to study the general

toxicity of Mimosa pudica. Their properties are summarized as follows:

a) Lactate dehydrogenase (LDH)

Lactate dehydrogenase (LDH) is a hydrogen transfer enzyme which catalyses

the reversible oxidation of L-lactate to pyruvate with the mediation of L-NAD as a

hydrogen acceptor.

LDH CH3.CHOH.COO- + NAD+ < > CH3.CO.COO- + NADH + H+ lactate pyruvate

LDH is a zinc-containing enzyme, present in almost all the tissues of the body

19 and located only in the cytoplasm of the cell. Intracellular enzyme level is many

folds higher than in the serum. Therefore, a significant elevation of LDH in serum level can be observed even though there is only a small extent of tissue damage

(Kachmar and Moss, 1976). The principal sources of LDH are heart, liver, skeletal musde，kidney, erythrocytes, pancreas and lung. Thus, LDH activity may be significantly elevated in some diseases such as myocardial diseases, liver diseases, skeletal muscle diseases, renal diseases and haematological disorders (Walmsley and

White, 1988).

The principle for the determination of LDH activity is based on the fact that, during reduction of pyruvate, an equimolar amount of NADH is oxidized to NAD.

The oxidation of NADH is monitored continuously by measuring the rate of absorbance decrease at 340 nm in a spectrophotometer. The rate of decrease is directly proportional to LDH activity in the sample (McLauchlan, 1988). The detailed procedure is referred to Appendix I.

b) Creatine kinase (CK)

Creatine kinase (CK), also referred to as creatine phosphokinase or creatine phosphotransferase, catalyses the reversible phosphorylation of creatine by adenosine triphosphate (ATP).

adenosine triphosphate (ATP) CK adenosine diphosphate (ADP) + < > +

creatine creatine phosphate

Mg2+ is an obligate activating ion which exists in the form of its -salts with

ADP and ATP. CK is found in highest activity in skeletal muscle, heart muscle and

20 brain tissue. The determination of CK activity has proved to be much more sensitive

than many other laboratory tests in the investigation of skeletal muscle diseases, and

is also useful in the diagnosis of myocardial infarction and cerebrovascular accidents

(Kachmar and Moss, 1976).

The colorimetric method used to determine the CK activity is recommended by the German Society for Clinical Chemistry (1977). The series of linked reactions involved in the assay procedure are as follows:

CK creatine phosphate + ADP > creatine + ATP

hexokinase ATP + glucose > ADP + glucose-6-phosphate

G-6-PDH G-6-P + NADP > 6-phosphogluconate + NADPH + H+

ATP formed from creatine phosphate and ADP by the action of CK reacts with glucose in the presence of hexokinase to form glucose-6-phosphate (G-6-P).

Subsequently, G-6-P is oxidized by NADP in the presence of glucose-6-phosphate dehydrogenase (G-6-PDH) to form 6-phosphogluconate and NADPH. The rate of increase in absorbance at 340 nm due to the formation of NADPH is directly proportional to CK activity as CK catalysed reaction is the rate-limiting step. The detailed procedure for the determination of CK activity is referred to Appendix II..

c) Aspartate aminotransferase (AST) / Glutamic oxalacetic transaminase (GOT) and

Alanine aminotransferase (ALT) / Glutamic pyruvic transaminase (GPT)

Aminotransferases or transaminases constitute a group of enzymes which catalyse the process of transferring an amino group from an a-keto acid. As a result,

21 a different a-amino acid and a different a-keto acid are formed. Transamination

plays a key role in intermediate metabolism as it takes part in the synthesis and

degradation of amino acids in living cells. The three amino acids, glutamate,

aspartate and alanine, are converted into corresponding ce-keto acids which are the

components of the tricarboxylic acid cycle. Thus, they are oxidized to provide a

source of energy. Moreover, transamination is also involved in providing aspartate

for the urea cycle (Wilkinson, 1976). Aminotransferases require pyridoxal-5,- phosphate (vitamin Bg) as a co-factor which mediates amino group transfer and forms pyridoxamine derivatives (Sacher et al,, 1991). Two clinically important aminotransferases are aspartate aminotransferase (AST) and alanine aminotransferase

(ALT). These two enzymes are also known as GOT and GPT, respectively. They catalyse the following reactions:

GOT aspartic acid + a-ketoglutaric acid > oxalacetic acid + glutamic acid

GPT alanine + a-ketoglutaric acid > + glutamic acid

GOT is widely distributed in body tissues, heart, liver, skeletal muscle and

kidney being the richest sources. Smaller amounts are also present in pancreas,

spleen, lung and erythrocytes. On the other hand, liver is the major source of GPT

while heart, skeletal muscle and kidney are the minor sources. As the two

aminotransferases can be found in large amounts in the liver, they have been widely

used for diagnostic purposes of liver damage. GOT is present in both the

mitochondria and cytosol of liver cells, whilst GPT is found in the cytosoL Damaged

hepatocytes release these enzymes into extracellular fluid and result in elevation of

22 serum levels of aminotransferase activity. Usually, GPT level is greater than that of

GOT in hepatitis. However, if GOT is found to be greater than that of GPT, it suggests that there is a widespread of hepatitic necrosis as the mitochondrial enzymes are released. Thus, the relative activities of these two enzymes can provide some information on the underlying pathology and severity of the disease processes

(Walmsley and White, 1988).

The method for the determination of GOT and GPT is generally based on those described by Reitman and Frankel (1957). The oxalacetic or pyruvic acid formed in the GOT or GPT catalysed reactions can react with 2,4- dinitrophenylhydrazine to form a coloured product of phenylhydrazones. The colour intensity of this product is proportional to the transaminase activity. Although the a- ketoglutaric acid in the substrate mixture, as well as the keto acids produced by transamination, would form coloured hydrazones, the hydrazones formed from the reaction products of oxalacetic or pyruvic acid are more chromogenic and possess different spectral absorption maxima than that of of-ketoglutaric acid. Therefore, this colorimetric measurement of the enzyme activity is still feasible and being commonly used. The detailed procedures of GOT and GPT assays are referred to Appendix III and IV respectively.

d) Alkaline phosphatase (ALP)

Alkaline phosphatases (ALP) are a group of relatively non-specific enzymes which hydrolyse a variety of ester orthophosphates under alkaline conditions. Mg2+ and Zn2+ are the activators of ALP; an optimal Mg2+/Zn2+ ratio is required for its

23 maximum activity (McLauchlan, 1988).

The enzyme is present in almost all tissues of the body, the richest sources being intestinal mucosa, osteoblasts of bone, bile canaliculi of liver, proximal tubules of kidney, placenta and lactating mammary glands. In all these sites, ALP activity is localized at the cell membrane. Therefore, ALP appears to be involved in the transport mechanisms of phosphate as active transport is the important function in these tissues. ALP activity is useful in diagnosing hepatobiliary diseases and bone disorders associated with increased osteoblastic activity (McLanchlan, 1988).

The principle for the determination of ALP activity is according to its ability to catalyse the following reaction:

ALP p-Nitrophenyl phosphate + HjO > p-Nitrophenol + phosphate

Para-nitrophenylphosphate (PNPP) is colourless. ALP splits off the phosphate group of PNPP to form free p-nitrophenol, which is converted to the p-nitrophenoxide ion in alkaline conditions and shows an intense yellow colour with an absorbance maximum at 405 nm. Thus, the rate of increase in absorbance is directly proportional to ALP activity in the serum (Bowers and McComb, 1966). The detailed procedure is referred to Appendix V.

1.4.1.3 Subacute toxicity test

Subacute toxicity studies are designed to examine the adverse effects on an

24 experimental animal resulting from repeated exposure to the test substance over a period of time. A compound shown to be non-toxic in acute toxicity test may be toxic after prolonged exposure, even at low doses, due to accumulation, changes in enzyme levels, and disruption of physiological and biochemical homeostasis.

Therefore, subacute studies can provide information on the cumulative toxicity of a substance on target organs and on physiological and metabolic tolerance of a compound after prolonged exposure. It also aids in selecting doses for chronic, reproductive, and carcinogenicity studies (Mosberg and Hayes, 1989). Some of the physical examinations (eg. body weight and organ weights), biochemical examinations

(eg. enzyme assays), and pathological examinations (eg. microscopic histology) can be conducted to assess the effects of a test substance on the animals (Loomis, 1978).

1.4.1.4 Reproductive toxicity test

The mammalian reproductive process is long and complex, and thus susceptible to influence by exogenous agents. It is known that many foreign compounds can induce developmental abnormalities in the embryo or fetus. Some investigations are designed for evaluating chemical injury to reproductive function in experimental animals. The approach commonly adopted is the use of a three segment scheme of studies. The，Segment 1，study is designed to assess the toxic effect of a substance on gonadal function and mating behaviour in both male and female animals.

In addition, the effects on conception rate, the early and late stages of gestation, parturition, lactation, and the development of offspring are assessed. The: 'Segment

2，study is conducted to evaluate the teratogenic potential of a test compound.

25 Female animals only are treated with the test compound post-mating and during the period of major organogenesis. The 'Segment 3' study is used to assess the effects of a substance on the peri- and post-natal development of fetus (Gangolli and Phillips,

1988). Since Mimosa pudica is recorded to be contraindicated in pregnancy (Cheung and Li, 1981),，Segment 2’ study is adopted to evaluate its effects on implantation and pregnancy. The employed assay is a preliminary test aimed at determining if the

Mimosa extract has any effect on the first 10 days of pregnancy in the rat so as to find out if there is any influence on the process of implantation or disruption of early pregnancy.

26 Chapter Two

Materials and Methods

2.1 Materials

2.1.1 Mimosa pudica

Whole plants of Mimosa pudica, including flowers and seeds, were collected in Hong Kong (the campus of The Chinese University of Hong Kong) from June to

September, 1991. The botanical identity of the plant material was verified by Dr.

Paul P. H. But and the voucher specimens (Cheng 1-4) were deposited in the

Herbarium, Biology Department, CUHK. Samples were cut into small pieces and dried for storage.

2.1.2 Animals

BALB/c An (6-8-week-old), C57BL/6N (6-8-week-old) and ICR (4-6-week- old) male or female mice, Sprague-Dawley male or female rats (180-220g), Sprague-

Dawley pregnant rats (220 ± lOg) and Dunkin-Hartley male guinea pigs (3 month-old) were supplied from the Animal House, The Chinese University of Hong Kong. They were fed with animal diet (Rodent Laboratory Diet 5001 for mice and rats, Guinea

Pig Diet 5025 for guinea pigs, Purina Mills，U.S.A.) ad libitum with free access to

water. They were kept under the conditions of 21 -24 ° C and a 12-hours light-dark cycle.

27 2.1.3 Chemicals

Saline

Normal saline was prepared by dissolving 0.9g sodium chloride (NaCl) (Ajax,

Australia, analytical grade) in 100 ml distilled water.

Phosphate-Buffered-Saline rPRS^

PBS was prepared by dissolving 1.48g disodium hydrogen phosphate

(Na2HP04) (Ajax, , analytical grade), 0.43g potassium dihydrogen phosphate

(KH2PO4) (Ajax, Australia, analytical grade) and 6.8g NaCl in IL of double-distilled water. The solution was adjusted to pH 7.2 by IM sodium hydroxide (NaOH) or IM hydrochloric acid (HCl), and was sterilized by autoclaving at 121 °C for 20 minutes.

Penicillin-Streptomycin (PS)

Penicillin-Streptomycin was purchased from GIBCO (U.S.A.). The antibiotics solution was separated into 5 ml aliquots and stored at -20°C.

Fetal Bovine Serum (TBS)

FBS was purchased from GIBCO (U.S.A.). The serum was separated into 20 ml aliquots and stored at -20°C.

RPMI culture medium

RPMI 1640 medium (GIBCO, U.S.A.) was prepared by dissolving 1 litre- powder package and 0.87g sodium hydrogen bicarbonate (NaHCO�）(Ajax Australia, ,

28 analytical grade) in IL double-distilled water. The solution was adjusted to pH 7.2 by IM NaOH or IM HCl. It was sterilized by 0.2 /^m millipore filtration. The medium was supplemented with 100 units/ml of penicillin G sodium, 100 /xg/ml streptomycin sulfate (GIBCO, U.S.A.) and 10% fetal bovine serum (GIBCO,

U.S.A.), hereafter referred to as "complete culture medium".

Mitogens

Stock solutions of concanavalin A (Con A) and lipopolysaccharide (LPS)

(Sigma, U.S.A.) were prepared by dissolving the chemicals into PBS to make the concentration of 100 jixg/100 /al. They were sterilized by 0.2 fxm millipore filtration and stored at -20

Trypan blue solution

0.2% solution of trypan blue (Sigma, U.S.A.) was prepared by dissolving the powder in PBS solution.

Neutral red solution

0.1% solution of neutral red (Sigma, U.S.A.) was prepared by dissolving the powder in saline solution.

Methyl-^H Thymidine r^H-TdR)

0.5 mCi/ml solution of ^H-thymidine (2 Ci/mmol, Amersham, U.K.) was prepared by diluting the stock solution with RPMI 1640 medium.

29 Thioglvcollate

3% thioglycollate (DIFCO, U.S.A.) in PBS was prepared and sterilized by autoclaving at 121°C for 20 minutes.

Sheep Red Blood Cell rSRBn

SRBC was purchased from SEROTEC (U.K.) and was washed by PBS for several times before used.

Hydrocortisone (UC)

Stock solution of lO'-'^M of hydrocortisone (Sigma, U.S.A.) in RPMI 1640 medium was prepared and sterilized by 0.2 ixm millipore filtration. Different molarities were made by RPMI dilution.

Mimosine

Mimosine was purchased from Sigma (U.S.A.) and was suspended in normal saline when administered to mice.

Scintillation fluid Scintillation fluid was prepared by dissolving 15g of 2,5-diphenyloxazole

(PPO) and 0.125g of dimethyl-1,4-bis (2-(5-phenyloxazol)) benzene (POPOP), both from Sigma (U.S.A.), in 2.5L of toluene (Merck, Germany, scintillation grade) and stirred overnight to dissolve the compounds completely.

30 Lytic solution

Lytic solution was prepared by mixing glacial acetic acid (Peking, China, analytical grade) and absolute alcohol (Merck, Germany, GR grade) in 1:1 ratio.

Guinea pig complement

Blood from adult guinea pig was obtained by heart puncture and serum was isolated for performing the plaque-forming-cells assay. Before the assay, 1 ml of undiluted serum was mixed with 0.2 ml packed SRBC at 4。C for 30 minutes in order to remove any non-specific complement-mediated haemolysin. The absorbed complement was recovered by centrifuging at 3,000 rpm for 10 minutes and made a

1:20 dilution by PBS.

Enzyme Kits

Kits of lactate dehydrogenase (DG1340-K), creatine kinase (DG147-K), aspartate aminotransferase/alanine aminotransferase (505-OP) and alkaline phosphatase (DG1245-K) were purchased from Sigma (U.S.A.) for performing the enzyme assays.

31 2.2 Methods

2.2.1 Extraction of Mimosa pudica

In every extraction, 50g whole plant dry material were cut into small pieces

and soaked in 2 litres of distilled water for 1 hour, and then boiled for 1 hour. After

boiling，the extract was filtered by a gauze to separate it from the plant material. The

extract was saved and the plant material was added with another 2 litres of distilled

water and boiled for 1 hour. The second extract was also obtained by filtering

through a gauze. The two water extracts were pooled together and concentrated to

a smaller volume («200 ml) by evaporation. Afterwards, it was centrifuged at 3,000

rpm for 10 minutes to remove the large particles and impurities. Finally, the water

extract was lyophilized into dry powder for storage in desiccator (Figure 2.1). A

total of 3 kg dry plant material was extracted to obtain a total yield of 567.9g dry

powder (Dry Weight Equivalent (DWE) = 5.3g dry plant material per g of extract).

The extract is hereafter referred to as the Mimosa extract.

a) Preparation of the extract for cell culture:

A stock solution was prepared by dissolving the powder extract of Mimosa pudica in RPMI medium to make a concentration of 20 mg powder/ml and was stored

at -20°C. It was defrost and made a 5-fold dilution by RPMI medium. Then it was

sterilized by 0.45 /xm millipore filtration and was ready for cell culture.

32 b) Preparation of the extract for oral administration or intraperitoneal injection:

The powder extract was dissolved in saline or distilled water according to different dosages in the experiment.

33 50g dry plant material was soaked in 2L of distilled water for 1 hr

Boiled for 1 hr

Filtered by a gauze

Residue was added with Extract 1 another 2L of distilled water

•4/ Boiled for 1 hr

�f

Filtered by a gauze

、‘ ^

Discarded residue Extract 2 、梦

Pooled two extracts together

Concentrated the extract to «200 ml by evaporation

•y Centrifugation at 3,000 rpm for 10 minutes

Supernatant Discarded precipitate lyophilization Powder of water extract stored in desiccator

Figure 2.1: Flow chart showing the extraction of Mimosa pudica.

34 2.2.2 Assays for the immunomodulatory effects of Mimosa pudica

2.2.2.1 Cell preparation

a) Splenocytes

Four to Five mice were sacrificed by cervical dislocation. Their spleens were removed aseptically and pooled together in RPMI 1640 medium. Isolated cells were obtained by teasing the spleens through a sterile sieve in RPMI medium with a plunger of plastic syringe. The splenocytes were washed 2 times with RPMI medium by centrifuging at 1,500 rpm for 10 minutes. The cell pellet was resuspended in 20%

FBS. The cell number was counted by preparing a 10-fold dilution of 1 % acetic acid which was used to lyse the RBC. After a few minutes, the cells were counted by using a haemocytometer. The viability of the cell suspension was over 95% by trypan blue exclusion test (Hudson and Hay, 1976). The cell suspension was adjusted to a concentration of 5X10^ cells/ml by adding 20% FBS and was ready for cell culture.

b) Thymocytes

Preparation of thymocyte cell suspension was similar to that of splenocytes

(section: 2.2.2.1a). Mice were sacrificed and their thymuses were removed. Isolated thymocytes were obtained and washed 2 times by RPMI medium. The cell pellet was resuspended in 20% FBS. The cell number was counted by preparing a 10-fold dilution of 1% acetic acid. The viability of the cell suspension was over 95% by trypan blue exclusion test (Hudson and Hay, 1976). The thymocyte concentration

35 was adjusted to 1X10? cells/ml by 20% FBS and was ready for cell culture,

c) Macrophages

The method from Gu et al, (1990) was followed with some modifications.

Five to six mice were administered with 3 ml of 3% thioglycollate per mouse by intraperitoneal injection to induce the peritoneal exudate cells (PEC). After 3 days, the mice were sacrificed by cervical dislocation and their abdominal skins were removed aseptically. The peritoneal cavity was lavaged by injecting 5-6 ml of PBS through a 19-G needle. After lavaging for about 1 minute, the peritoneal fluid was collected by the needle, and the lavaging process was repeated once. All the peritoneal fluids from the mice were pooled together and washed by centrifuging at

1,500 rpm for 5 minutes. The cell pellet was resuspended in 10% FBS. The cell number was counted and adjusted to 2X10^ cells/ml. The viability of the cell suspension was over 95 % when checked by trypan blue exclusion test (Hudson and

Hay, 1976). Then 0.1 ml of the cell suspension was added into each well of 96-well flat-bottomed microtitre plate (Nunc, Denmark). The plate was incubated at 37°C in a humidified atmosphere containing 5% CO2 in air for 2 hours. After incubation, the non-adherent cells and the medium were discarded. The macrophages adhered at the bottom of the plate was washed by warm PBS for 2 times. Finally, 0.1 ml

20% FBS was added to each well and was ready for cell culture.

36 2.2.2.2 Splenocyte proliferation

a) In vitro study:

The method from Leung et al. (1986) was followed with some modifications.

Splenocyte suspension from C57BL/6N mice was prepared and adjusted to 5X10^ cells/ml (section: 2.2.2. la). 100 of the suspension was added to each well of a 96- well microtitre plate. Different concentrations of Mimosa extract and/or mitogens were added to make a final volume of 0.2 ml/well. Two mitogens, LPS and Con A were used in their sub-optimal concentrations of 10 /xg/ml and 1 /zg/ml，respectively.

The plate was incubated at 37°C in a humidified atmosphere containing 5% CO2 in air for 48 hours. After 42 hours of incubation, 50 /xl of 0.5 /xCi ^H-thymidine was added to each well and further incubated for 6 hours. Then the cells were harvested onto a glass fibre filter (Flow Lab, U.K.) by a Titertek cell multiharvester (Flow Lab,

U.K.). The radioactivity incorporated into the cells was counted by a liquid scintillation counter (Beckman LSI801，U.S. A.). The DNA synthesis was determined by ^H-TdR incorporation and was expressed in counts per minute (cpm). Usually, each sample was done in quadruplication.

b) In vivo study:

BALB/c An male mice in groups of four were fed with different dosages of the Mimosa extract (2.5，5.0 or 10.0 g/kg/day; DWE =13.3，26.5 or 53.0 g/kg/day) in saline by stomach tube. The control group was fed with the same volume of saline. After feeding continuously for 10 days, the mice were sacrificed and the splenocyte suspensions were prepared as mentioned in section 2.2.2.1a. Then the in

37 v/w study of the mitogenic effect of Mimosa extract was determined as the in vitro assay except no further Mimosa extract was added to the culture. Also, the weight of the spleens was measured.

2.2.2.3 Thymocyte proliferation ^

a) In vitro study:

The method from Ward et al (1988) was followed with slight modifications.

Thymocytes from C57BL/6N mice was prepared and cultured in RPMI complete culture medium which containing 20% of FBS in a 96-well microtitre plate with 100 jul per well (section: 2.2.2.1b). Different concentrations of Mimosa extract and/or mitogen Con A were added to make a final volume of 200 per well. The plate was incubated at 37°C in a humidified atmosphere containing 5% CO2 in air for 72 hours.

The culture was pulsed with 50 yil of 0.5 /xCi/well of ^H-thymidine 8 hours before cell harvesting with a Titertek cell multiharvester (Flow Lab, U.K.). The cell proliferation was determined by measuring the amount of incorporated ^H-TdR by liquid scintillation and expressed in cpm. Usually, each sample was done in quadruplication.

b) In vivo study:

Thymocyte preparation and the in vivo assay were the same as that in splenocytes (section: 2.2.2.1b and 2.2.2.2). Then the thymocyte culture for determining the mitogenic effect of Mimosa extract was the same as the in vitro assay except no further Mimosa extract was added. Also, the weight of the thymuses was

38 measured.

2.2.2.4 Phagocytic activity of macrophages

a) In vitro study:

The phagocytosis was measured by using spectrophotometrical method as mentioned by Wang et al (1987) and Gu et al. (1990). Macrophages from

C57BL/6N mice was prepared (section: 2.2.2.1c) and each well of a 96-well microtitre plate contained 100 pX of the cells. Different concentrations of Mimosa extract and/or hydrocortisone were added to make a final volume of 200 yiX per well.

The plate was incubated at SVC in a humidified atmosphere containing 5% CO2 in air for 24 hours. After incubation, the drugs were discarded and the cells were washed by warm PBS for 2 times. Then 0.1% of prewarmed neutral red solution was added with 50 /xl/well and the incubation was continued for 30 minutes. After that, excess neutral red solution was discarded and the cells were washed by warm PBS for 2 times. The plate was dried in a 60°C oven for 1 hour. 200 /xl of lytic solution was added to each well and the plate was left overnight. The macrophages were lysed to release the engulfed neutral red solution which was measured by microplate reader at O.D. 540 nm (BIORAD 3550, U.S.A.). The amount of neutral red uptake by macrophages was determined from a standard curve which was constructed by different concentrations of neutral red solution (Appendix, Figure A1). Usually, each sample was done in quadruplication.

39 b) In vivo study:

BALB/c An male mice in groups of four were administered by intragastrical administration with different dosages of Mimosa extract (2.5，5.0 or 10.0 g/kg/day;

DWE = 13.3, 26.5 or 53.0 g/kg/day) in saline for 10 days. Control group was fed with the same volume of saline. 3 days prior to the end of drug administration, 3 ml of 3% thioglycollate was injected intraperitoneally to each mice. After feeding continuously for 10 days, the mice were sacrificed and the macrophages were prepared as mentioned in section 2.2.2.1c. The phagocytic assay was performed as the in vitro study except no further Mimosa extract was needed.

2.2.2.5 Release of IL-1 by macrophages

a) Preparation of macrophages:

The method from Kurakata et al. (1991) was followed with slight modifications. Peritoneal exudate cells (PEC) were obtained from C57BL/6N mice and macrophages were purified in the same way as mentioned before

(section:2.2.2.1c) except that the PEC were adjusted to 1.5X10^ cells/ml and added at 1.0 ml per well to a 24-well flat-bottomed plate (Dune, Denmark). After macrophages purification, non-adherent cells were discarded and 0.9 ml complete culture medium containing 10% FBS was added. Different concentrations of Mimosa extract in the presence or absence of 10 jL^g/ml LPS were added and incubated at

37°C in a humidified atmosphere containing 5% CO2 in air for 24 hours. Then, the supernatants were collected and centrifuged at 2,000 rpm for 10 minutes to remove any non-adherent cells. The cell-free supernatants containing IL-1 were distributed

40 into aliquots and stored frozen at -20°C.

b) IL-1 Assay:

Supernatants of macrophage culture were determined for their IL-1 activities by using the mouse thymocyte proliferation assay (section: 2.2.2.3). Mouse thymocytes were obtained from C57BL/6N mice and cultured at IXIO? cells/ml with three dilutions of supernatants (1:40，1:80 and 1:160) in the presence of 1 /xg/ml of

Con A.

2.2.2.6 Plaque forming cells (PFC)

The method from Simpson and Gozzo (1978) was followed with some modifications. BALB/c An male mice in groups of four were intragastrically administered with two different concentrations of Mimosa extract (5.0 or 10.0 g/kg/day; DWE = 26.5 or 53.0 g/kg/day) in saline continuously for 10 days. The control group was given the saline. On the sixth day, the mice were immunized by intraperitoneal injection of 2% SRBC in saline at 0.5 ml/mouse. After drug administration for 10 days, the mice were sacrificed by cervical dislocation. Their spleens were removed and teased through a sieve in PBS. The splenocytes were washed 2 times with PBS by centrifuging at 1,500 rpm for 5 minutes. The cell pellet was resuspended in PBS and adjusted to 5X10^ cells/ml. In each 75 mm X 10 mm culture tube, 1 ml of the splenocyte suspension was incubated with 1 ml of a 0.2%

SRBC suspension in PBS and 1 ml of a 1:20 dilution of guinea pig complement. At the same time, a blank was prepared by 1 ml of PBS instead of splenocytes used in

41 the mixture. The mixtures were incubated at 37。C for 1 hour. Then they were

centrifuged at 3,000 rpm for 5 minutes and the supernatant haemolysis was

determined at O.D. 413 nm. Usually, each sample was done in quadruplication.

2.2.2.7 Restoration on splenocyte blastogenesis of old mice

BALB/c An male mice (20-week-old) in groups of four were administered with Mimosa extract by intragastrical administration at the dose level of 5.0 g/kg/day

(DWE = 26.5 g/kg/day). Two control groups, group 1 (6-week-old) and group 2

(20-week-old), were given the same volume of distilled water. After feeding for 10 days, all mice were sacrificed and their splenocyte suspensions were prepared for the proliferation assay. The assay procedure was the same as that of the splenocyte proliferation (section: 2.2.2.2). The weights of their lymphoid organs, namely the spleen and thymus, were measured.

42 2.2.3 Assays for the toxicity of Mimosa pudica

2.2.3.1 LD50

ICR male or female mice were randomly divided in groups of fifteen. After

fasting for 4 hours, each animal was given a single dose of different concentrations

of the Mimosa extract by either intragastrical or intraperitoneal administration. The

mice in the control group were given the vehicle. The animals were observed for

general symptoms of toxicity and mortality for one week (Chan and Hayes, 1989).

2.2.3.2 Enzyme assays

Male or female Sprague-Dawley rats (200±20g) were randomly allotted to

different test and control groups. After overnight fasting, the test groups were intraperitoneally injected with different dosages of Mimosa extract. The control

group was injected with saline. After 10 hours, the rats were sacrificed. Their blood

was collected by heart puncture and the serum was isolated for analyzing the enzyme levels of lactate dehydrogenase (LDH), creatine kinase (CK), glutamic oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT). Procedures for the enzyme assays were referred to appendix I-V.

2.2.3.3 Subacute toxicity

The method was modified from Mosberg and Hayes (1989) and Qureshi et al

(1992). ICR female mice (15±lg) were randomly assigned into different test and

control groups. Each group contained ten mice. The mice in the experimental

groups were intragastrically given Mimosa extract at 2.5, 5.0, or 10.0 g/kg/day

43 (DWE = 13.3，26.5 or 53.0 g/kg/day). The mice in the control group was given the vehicle. The treatment was continued for 1 month. Animals were observed for external general symptoms of toxicity, change of body weight and mortality. After the completion of the drug administration, the animals were sacrificed. Their blood was collected for red blood cell (RBC) and total white blood cell (WBC) count and their serum was isolated for the enzyme assays: LDH, CK, GOT, GPT and ALP.

The animals were subjected to a gross pathology examination and their vital organ weights were recorded.

2.2.3.4 Reproductive toxicity

The method from Kong et al. (1989) was followed. Female Sprague-Dawley rats (220±10g) were supplied by the Animal House, CUHK in the first morning when the presence of postcoital plug was observed. It was designated as Day-1 of pregnancy (PDj). They were randomly assigned into two groups with ten rats per group. The rats in the experimental group were orally administered with the Mimosa extract by stomach tube. The extract was dissolved in distilled water and was given daily for 10 days at the highest possible dose of 3.75 g/rat/day (DWE = 19.9 g/rat/day) from PDi to PDjo- The rats in the control group were given the vehicle.

The animals were autopsied at PDjg and the numbers of pregnant animals, implantation sites, normal fetuses and corpora lutea of pregnancy were recorded.

2.2.4 Statistical analysis

All results were expressed as the arithmetic mean 士 standard error. .Student's t test was used to determine the confidence limits in group comparisons.

44 Chapter Three

Results

3.1 Assays for the Immunomodulatory Effects of Mimosa pudica

3.1.1 In vitro study on the lymphocyte proliferation

3.1.1.1 Splenocyte proliferation

Splenocytes from C57BL/6N mice were used to study the mitogenic effect of

Mimosa extract on the proliferation of T cells and B cells. Splenocytes were cultured with different concentrations of Mimosa extract in the presence or absence of the B cell mitogen LPS or the T cell mitogen Con A at their sub-optimal concentrations (10 jug/ml of LPS and 1 ixg/ml of Con A). From trypan blue exclusion test, the non- cytotoxic concentrations of the extract was found to be 100 /xg/ml or below. Within this concentration, the extract enhanced the proliferation of the splenocytes in a dose- dependent manner. At the optimal concentration 100 /ig/ml, the proliferation rate was five times of the control (Figure 3.1). However, when the concentration was greater than 100 ixg/ml, the proliferation rate decreased.

When the splenocytes were cultured in the presence of the B cell mitogen

LPS, the addition of the extract enhanced the cell proliferation at-, the low concentrations (1-25 /zg/ml), but the proliferation decreased when the concentration

45 was greater than 25 /xg/ml (Figure 3.2). However, when the splenocytes were

cultured in the presence of the T cell mitogen Con A, the addition of the extract did

not show any stimulatory effect at the non-cytotoxic concentrations (<100 /xg/ml)

(Figure 3.3). These results suggested that low concentrations of Mimosa extract had a strong stimulatory effect on splenocyte proliferation and a significant co-mitogenic activities with LPS but not with Con A.

46 2400 ~~— 2200 — 了

5 2000 — /I\ 眷 control O Z \ O Mimosa extract --1800 - (Y \

I 1600 - j \〇 I 誦-J \ 6 1200 -X \ 呂 1000 ^ K 800 j V 600 I 〇 co^ 400

200 - Q 1 I I 1 1 I 1 I I 0 100 200 300 400 500

Final concentration of Mimosa extract (yL6g/ml)

Figure 3.1: /n vitro concentration-response curve of Mimosa pudica on the splenocyte proliferation.

C57BL/6N splenocytes were cultured with different concentrations of the water extract of Mimosa pudica for 48 hours. Pulse labelling with 0.5 jLcCi/well of tritiated thymidine was performed for 6 hours.

Data are expressed as mean 士 S.E.

47 50

^ ^U W ^ 鲁 control 0I 40 V： h \ O Mimosa extract

1 g 30 Q \\ I 25 -

20- \ •IS 15- \ ^ \

“ 5 - • \〇 Q I I I I I I I 1 1 0 100 200 300 400 500

Final concentration of Mimosa extract (/xg/ml)

Figure 3.2: In vitro concentration-response curve of Mimosa pudica on the splenocyte proliferation in the presence of 10 /xg/ml LPS.

C57BL/6N splenocytes were cultured with different concentrations of the water extract of Mimosa pudica for 48 hours. Pulse labelling with 0.5 )uCi/well of tritiated thymidine was performed for 6 hours.

Data are expressed as mean 士 S.E.

48 70 —

3 60 - • control ^ X O Mimosa extract

讓5。丨 ^ \ 二 40 一 \ .2 〇\

^ 3〇一

謹20 - \〇

^ 10 -

CO Q I I 1 I I I I I 1 0 100 200 300 400 500

Final concentration of Mimosa extract (yczg/ml)

Figure 3.3: In vitro concentration-response curve of Mimosa pudica on the splenocyte proliferation in the presence of 1 /xg/ml Con A.

C57BL/6N splenocytes were cultured with different concentrations of the water extract of Mimosa pudica for 48 hours. Pulse labelling with 0.5 /iCi/well of tritiated thymidine was performed for 6 hours.

Data are expressed as mean 士 S.E.

49 3.1.1.2 Thymocyte proliferation

In contrast to splenocytes which contain a mixture of T and B cell populations, thymocytes contain only T cells at different stages of maturation. Thymocyte culture was used to study the effect of Mimosa extract on the proliferation of these different populations of T cells and their mitogenic response to Con A. It was found that non- cytotoxic concentrations (<100 //g/ml) of the extract could only cause a mild stimulatory effect on the thymocyte proliferation. At the concentration of 100 /xg/ml, it caused only an increase of 40% when compared with the control (Figure 3.4).

When the thymocytes were cultured in the presence of Con A at three different concentrations of 1.0，1.5 or 3.0 /zg/ml, the addition of Mimosa extract inhibited the mitogenic response of thymocytes to Con A at all three concentrations. The concentration of the extract at 12.5 �ug/m indicatel d the greatest inhibitory rate; further increase of the concentration did not show any further suppression (Figure

3.5). The results showed that Mimosa extract exhibited only a weak stimulatory effect on thymocyte proliferation, but, the extract caused a suppressive effect on the mitogenic response of thymocytes to Con A.

50 160

？ 140 - ^^ P. rS一一 3 120 - /

〇 一- 80 - 〇 0 .S 60 - P：： 40 — • control 1 ,丨 I O Mimosa extract

CO 20 —

Q I I I I I I I I I 1 1 1 0 20 40 60 80 100 120

Final concentration of Mimosa extract {ixg/mX)

Figure 3.4: In vitro concentration-response curve of Mimosa pudica on the thymocyte proliferation.

C57BL/6N thymocytes were cultured with different concentrations of the water extract of Mimosa pudica for 72 hours. Pulse labelling with 0.5 /xCi/well of tritiated thymidine was performed for 8 hours.

Data are expressed as mean 士 S.E.

51 ^ s I] a 4 o 7 -X

^2 6 \

5- 一 \ i"c^ 4 - \ fe \ 3 - •—• Q 口 ConA 3/^g/ml

E-h 1 # • ConA 1.5/zg/ml

^ iQ O —O O ConA Wird 0 I I I 1 I I 0 20 40 60 80 100 120 140

Final concentration of Mimosa pudica (ycxg/ml)

Figure 3.5: In vitro concentration-response curve of Mimosa pudica on the thymocyte proliferation in the presence of Con A.

C57BL/6N thymocytes were cultured with different concentrations of the water extract of Mimosa pudica for 72 hours. Pulse labelling with 0.5 /xCi/well of tritiated thymidine was performed for 8 hours.

Data are expressed as mean 士 S.E.

52 3.1.2 In vivo study on the lymphocyte proliferation

BALB/c An mice in groups of four were administered with Mimosa extract by intragastrical administration at one of the three different dose levels, 2.5, 5.0 or

10.0 g/kg/day (DWE = 13.3，26.5 or 53.0 g/kg/day), for 10 days. The control group was given equal volume of saline. Splenocytes and thymocytes from these groups were then assayed for their mitogenic-induced lymphocyte proliferation.

Table 3.1 showed that splenocytes from the groups fed with Mimosa extract had a higher proliferation rate than the control group. The greatest stimulatory effect was found at the dose of 5.0 g/kg/day at which the proliferation rate was four times greater than that in the control group. The stimulatory effect was also observed in the mitogenic response of splenocytes to LPS and Con A. It caused an increase of

50-60% at the dose of 5.0 g/kg/day in the proliferation induced by either LPS or Con

A. However, the mitogenic response to Con A was not in a dose-dependent manner as all three groups administered with the corresponding doses of Mimosa extract showed nearly the same degree of stimulation.

However, thymocytes from the mice given the same dosages of Mimosa extract as those for the splenocyte assays did not show any enhancement in the cell proliferation when compared with the control group. Instead of stimulation, all three dose levels of the extract induced a reduction on the thymocyte proliferation by nearly

50% (Table 3.2). Nevertheless, the suppression of the thymocytes could be reversed when mitogen Con A was added in the cell culture. At the dose of 5.0 g/kg/day, the thymocytes exhibited 85% enhancement in response to 2 figlmX of Con A.

53 Spleen and thymus weights were measured and the results did not show any significant change on the spleen weight after the administration of Mimosa extract.

However, the treated-mice showed a slight decrease on thymus weight. There were decreases of 20% or 30% after the mice were fed with 5.0 or 10.0 g/kg/day of the extract, respectively (Table 3.3).

54 Table 3.1: In vivo effect of Mimosa pudica on the mice splenocyte proliferation.

Treatment RPMI LPS(10 辟/ml) Con A(1 ixg/ml) saline 503.17 40939.83 121150.00 士 32.33 ±1080.56 土5673.900

Mimosa extract 2.5 g/kg/day 1514.50* 58706.25* 188351.67* ±95.97 ±2829.81 士 3998.38

5.0 g/kg/day 2652.00* 61041.25* 187151.67* ±185.85 ±2593.88 士4650.86

10.0 g/kg/day 1214.88* 51904.50* 183396.67* ±99.53 ±1275.10 ±4572.26

BALB/c An mice in groups of four were administered with either saline (control group) or one of the three dose levels of the water extract of Mimosa pudica (2.5, 5.0 or 10.0 g/kg/day; DWE = 13.3，26.5 or 53.0 g/kg/day) by intragastrical administration for 10 days. Splenocytes were assayed for mitogenic response in vitro.

Data are expressed as mean 士 S.E.

* Significantly different when compared with the saline control group, PCO.Ol.

55 Table 3.2: In vivo effect of Mimosa pudica on the mice thymocyte proliferation.

Treatment RPMI Con A(1 ；xg/ml) Con A (2 /xg/ml) saline 136.13 9708.04 57042.00 ±12.18 士751.66 ±3891.93

Mimosa extract 2.5 g/kg/day 69.75* 10384.50 95870.83* ±4.56 ±826.06 ±5898.70

5.0 g/kg/day 64.50* 14897.99* 105529.17* 土 3.59 ±503.58 土 6441.21

10.0 g/kg/day 77.50* 5896.91* 80244.44* ±2.81 土 328.04 士 6025.38

BALB/c An mice in groups of four were administered with either saline (control group) or one of the three dose levels of the water extract of Mimosa pudica (2.5，5.0 or 10.0 g/kg/day; DWE = 13.3, 26.5 or 53.0 g/kg/day) by intragastrical administration for 10 days. Thymocytes were assayed for mitogenic response in vitro.

Data are expressed as mean 士 S.E.

* Significantly different when compared with the saline control group, P<0.01.

56 Table 3.3: In vivo effect of Mimosa pudica on the lymphoid organ weights.

Organ weight (g) per lOOg body weight Treatment Body weight (g) spleen thymus saline 24.17 0.45 0.29 ±0.51 ±0.03 ±0.01

Mimosa extract 2.5 g/kg/day 23.65 0.42 0.30 ±0.31 ±0.01 ±0.02

5.0 g/kg/day 23.40 0.41 0.23* ±0.97 土 0.03 土 0.02

10.0 g/kg/day 23.04 0.42 0.21* ±0.59 土 0.01 ±0.02

BALB/c An mice in groups of four were administered with either saline (control group) or one of the three dose levels of the water extract of Mimosa pudica (2.5, 5.0 or 10.0 g/kg/day; DWE = 13.3, 26.5 or 53.0 g/kg/day) by intragastrical administration for 10 days.

Data are expressed as mean ± S.E.

* Significantly different when compared with the saline control group, P<0.05.

57 3.1.3 Phagocytic activity of macrophages

In vitro study:

Mouse peritoneal macrophages were incubated with different non-cytotoxic concentrations (confirmed by trypan blue exclusion test) of Mimosa extract (1-1000

/Ag/ml). After incubation for 24 hours, neutral red which acted as a foreign particle was added to check if the phagocytic activities of extract-treated macrophages were enhanced. The uptake of neutral red was measured at O.D. 540 nm and the actual amount was read from a standard curve (Appendix, Figure Al). Figure 3.6 showed that Mimosa extract enhanced the uptake of neutral red and the effect was in a dose- dependent manner. At the optimal dose of 800 /xg/ml, the uptake was three times that of the control. Besides, the stimulatory effect of the extract was also observed in the presence of hydrocortisone and was also in a dose-dependent manner (Figure 3.7).

The suppressive effects caused by hydrocortisone at lO'^M and lO'^M were reversed by Mimosa extract at the dose levels of 200 /xg/ml and 400 /xg/ml, respectively.

In vivo study:

BALB/c An mice in groups of four were administered with Mimosa extract by intragastrical administration at one of the three different dose levels of 2.5, 5.0 or

10.0 g/kg/day (DWE 二 13.3，26.5 or 53.0 g/kg/day) for 10 days. The control group was given the same volume of saline. Peritoneal macrophages were obtained to study whether the treated-macrophages had any change on their phagocytosis activities. It was found that the macrophages did not show any change in the uptake of neutral red in all the three groups administered with different dosages of the extract when

58 compared with the control group (Figure 3.8). The same negative result was also observed when the macrophages were cultured with hydrocortisone (Figure 3.9).

59 0.6

ClO 丁 3 0.5 - ^

CD _ /

^ 0-4 — /

§ 0.2 杂 ， <； 0 r • control 6 "“ 0- Mimosa extract 0.1 -

• 〇〇 I I I I I I I I 1 I 1 “〇 200 400 600 800 1000 1200

Final concentration of Mimosa extract (yUg/ml)

Figure 3.6: In vitro effect of Mimosa pudica on the phagocytosis of macrophages.

C57BL/6N macrophages were cultured with different concentrations of the water extract of Mimosa pudica for 24 hours. 0.1% neutral red was added and incubated for another 30 minutes. The amount of uptake was measured at O.D. 540 nm.

Data are expressed as mean 士 S.E.

60 0.6 —

3 〇.5 - � f ‘ 口 ^ 0.4 — L I _ y^/^f 广/V z

a _：// O HC 0.1 參 HC 10 M O z y -9 C^ • HC 10 M 7 〇.〇(^—I 1 1 ‘ 1 〇 200 400 600 800 1000 1200

Final concentration of Mimosa extract (/xg/ml)

Figure 3.7: In vitro effect of Mimosa pudica on the phagocytosis of macrophages in the presence of hydrocortisone.

C57BL/6N macrophages were cultured with different concentrations of the water extract of Mimosa pudica for 24 hours in the presence of hydrocortisone (HC). 0.1% neutral red was added and incubated for another 30 minutes. The amount of uptake was measured at O.D. 540 nm.

Data are expressed as mean 士 S.E.

61 .2.2 2.0 - tiO ：！ 1.8 - � T T ^ 1.6- T ^^ ri

“ ————

^ 1.4 —

1.2 — (D ^口 1.0 -

^ 0.8 —

o 0.6 — S C 0.4 -

0.2 - 0.0 saline 2.5 5.0 10.0 Dosages of Mimosa extract (g/kg)

Figure 3.8: In vivo effect of Mimosa pudica on the phagocytosis of macrophages.

BALB/c An mice in groups of four were administered with either saline (control group) or one of the three different dose levels of the water extract of Mimosa pudica (2.5, 5.0 or 10.0 g/kg/day; DWE 二 13.3，26.5 or 53.0 g/kg/day) by intragastrical administration for 10 days. Macrophages were cultured at 37。C for 24 hours. 0.1% neutral red was added and incubated for another 30 minutes. The amount of uptake was measured at O.D. 540 nm.

Data are expressed as mean 士 S.E.

62 I I HC—； [Z3 HC~®M ； •

^ 2 0-

saline 2.5 5,0 10.0

Dosages of Mimosa extract (g/kg)

Figure 3.9: In vivo effect of Mimosa pudica on the phagocytosis of macrophages.

BALB/c An mice in groups of four were administered with either saline (control group) or one of the three different dose levels of the water extract of Mimosa pudica (2.5, 5.0 or 10.0 g/kg/day; DWE = 13.3，26.5 or 53.0 g/kg/day) by intragastrical administration for 10 days. Macrophages were cultured with different concentrations of hydrocortisone (HC) at 37°C for 24 hours. 0.1% neutral red was added and incubated for another 30 minutes. The amount of uptake was measured at O.D. 540 nm.

Data are expressed as mean 士 S.E.

63 3.1.4 Release of IL-1 by macrophages

The effect of Mimosa extract on the synthesis of IL-1 was examined by culturing macrophages with different concentrations of the extract in the absence or presence of LPS. The quantity of IL-1 released was then assayed by Con A- stimulated thymocyte proliferation. As the supernatant from macrophage culture contained some other factors such as thymine and prostaglandins besides IL-1, high concentration of the supernatant was found to inhibit thymocyte proliferation.

However, the inhibitory effect of these factors could be eliminated when the supernatant was diluted by at least 40-fold (Otterness et al, 1988; Shang et al.,

1986). Therefore, three dilutions of the supernatant at 1:40，1:80 and 1:160 were made. The results showed that IL-1 released was suppressed by the extract when the concentration was 50 /xg/ml or greater (Figure 3.10). However, for LPS-stimulated macrophages the extract could enhance IL-1 released at the low concentrations up to

200 jLtg/ml (Figure 3.11). At the concentration of 100 /xg/ml, the amount of IL-1 released was 60% higher than that of the corresponding LPS control group when the supernatant was diluted at 1:40.

64 4000 "1 I I 1:40 ； [Z3 1:80 ; • 1:160

3500 - *

？ T 工 a -r I I

^ 3000 - T f 工 ^

^ 2500 - I - - 「- * ^ 々 氺木 I 2000 \ : *

； ^^ 1500 - I ；/ / / p *f * * 丄 1000 - g / 5 / : / */ /

：ii —丨」_i J i—

blank RPMI 1 25 50 200 300 400 Final concentration of Mimosa extract (yUg/ml) Figure 3.10: In vitro effect of Mimosa pudica on the release of IL-1 from macrophages.

C57BL/6N macrophages were cultured with different concentrations of Mimosa extract for 24 hours. Supernatants were collected and made into three dilutions (1:40, 1:80 and 1:160). The IL-1 activity was determined using the thymocyte proliferation assay. The blank was the thymocyte culture without any supernatant.

The data are expressed as mean 士 S.E.

* Significantly different when compared with the corresponding control group (RPMI), P<0.05.

65 3500 -|

I I 1:40 ； [Z3 1:80 ; • 1:160

-腳〇- ** *T 日 T r^JI Oh t Z T ^ T * Z 7 * 2500 - T * z T/ a TP= T^ 「-/ 己 o I / 厂； - 5 2000 - ^ J z, / / / K ；r 6 I - - - - 产- - a 1500 一 ¥ F^z / / / / - fz 节 々 / z / / / , z / ^ 1000 - X / z / / / : / /

500 -1 / : : : : ； ： ；

0 圓 Ikl Ikl Ikl Ikl ItJ Irl I PI blank LPS l 25 50 100 200 300 400 Final concentration of Mimosa extract (/zg/ml)

Figure 3.11: In vitro effect of Mimosa pudica on the release of IL-1 from LPS- stimulated macrophages.

C57BL/6N macrophages were cultured with different concentrations of Mimosa extract in the presence of 10 /xg/ml LPS for 24 hours. Supernatants were collected and made into three dilutions (1:40，1:80 and 1:160). The IL- 1 activity was determined using the thymocyte proliferation assay. The blank was the thymocyte culture without any supernatant.

The data are expressed as mean 土 S.E.

* Significantly different when compared with the corresponding control group (RPMI), P<0.05.

66 3.1.5 Plaque forming cell (PFC) assay

As Mimosa extract was found to have mitogenic effect on B cells, plaque forming cell (PFC) assay was carried out to investigate if the extract could also enhance the primary humoral immune response. BALB/c An mice in groups of four were intragastrically given Mimosa extract at the dose of either 5.0 or 10.0 g/kg/day

(DWE = 26.5 or 53.0 g/kg/day) for 10 days. The control group was administered with the same volume of saline. Then the splenocytes were prepared for the PFC assay four days after SRBC antigen sensitization. The amount of SRBC lysed was measured at O.D. 413 nm. Figure 3.12 showed that the O.D. value of the two test groups were increased. At the dose group of 10 g/kg/day, the increase was three times of the control group. This result indicated that the amount of SRBC lysed was increased.

67 0.20

^ 0.15 - * -H

CO 寸 q 0.10 - 6

氺

0.05 — “

J nz

0.00 saline 5.0 10.0

Dosages of Mimosa extract (g/kg)

Figure 3.12: Primary humoral antibody response to SRBC in mice treated with Mimosa pudica.

BALB/c An mice in groups of four were administered with either saline (control group) or one of the two dose levels of the water extract of Mimosa pudica (5.0 or 10.0 g/kg/day; DWE = 26.5 or 53.0 g/kg/day) by intragastrical administration for 10 days. Splenocytes were incubated with SRBC in the presence of guinea pig complement for 1 hour. The amount of lysis of SRBC were determined at O.D. 413 nm.

The data are expressed as mean 士 S.E.

* Significantly different when compared with the corresponding control group (RPMI), P<0.05.

68 3.1.6 Restoration on splenocyte blastogenesis of old mice

BALB/c An mice 20-week-old, in groups of four, were given Mimosa extract by intragastrical administration at the dose level of 5.0 g/kg/day (DWE = 26.5 g/kg/day) for 10 days. Two control groups, group 1 (6-week-old) and group 2 (20- week-old), were administered with the same volume of distilled water. Splenocytes were then assayed for their blastogenic responses in the presence or absence of the mitogens LPS and Con A. Table 3.4 showed that the mitogenic response of the control group 2 (20-week-old) decreased to nearly one half of the control group 1 (6- week-old). From the test group fed with Mimosa extract, it indicated that the extract could partially restore the blastogenic response of the test group. A similar reversed effect could be observed in the presence of mitogens LPS or Con A. The effect was much more prominent in the presence of Con A as the proliferation rate of the test group (20-week-old) was restored nearly to the same level of the group 1 (6-week- old).

However, Mimosa extract did not show any significant change on the spleen weight and the thymus weight of the 20-week-old mice (Table 3.5). Both groups with

20-week-olcl mice indicated a decrease on these lymphoid organs when compared with the 6-week-old mice.

69 Table 3.4: In vivo effect of Mimosa pudica on splenocyte blastogenesis of old mice.

Mice with RPMI LPS(10 爬/ml) Con A(1 /xg/ml) treatment

6-week-mice 300.25 5903.91 8657.96 distilled water ±11.04 士 197.62 ±505.17

20-week-mice 152.88* 2280.75* 5433.03* distilled water 士7.05 ±39.92 士243.03

20-week-mice 205.63*# 3242.：38*# 7848.19# Mimosa extract ±10.08 ±40.11 土271.16

Young (6-week-old) or old (20-week-old) BALB/c An mice in groups of four were administered with either distilled water (control group) or Mimosa extract at the dose of 5.0 g/kg/day (DWE = 26.5 g/kg/day) by intragastrical administration for 10 days. Splenocytes were assayed for mitogenic response in vitro. Data are expressed as mean 士 S.E.

* Significantly different when compared with the young mice (6-week-old) distilled water control group, P<0.05.

# Significantly different when compared with the old mice (20-week-old) distilled water control group, P<0.05.

70 Table 3.5: In vivo effect of Mimosa pudica on the lymphoid organ weights of old mice.

Mice with Organ weight (g) per lOOg body weight treatment Body weight (g) spleen thymus

6-week-old 24.83 0.47 0.22 distilled water ±0.46 ±0.02 士0.02

20-week-olci 29.47* 0.34* o.lO* distilled water ±0.52 ±0.01 士0.01

20-week-old 30.40* 0.36* 0.13* Mimosa extract ±0.65 ±0.01 ±0.02

Young (6-week-old) or old (20-week-old) BALB/c An mice in groups of four were administered with either distilled water (control group) or Mimosa extract at the dose of 5.0 g/kg/day (DWE = 26.5 g/kg/day) by intragastrical administration for 10 days.

Data are expressed as mean 士 S.E.

* Significantly different when compared with the young mice (6-week-old) distilled water control group, P<0.05.

71 3.2 Results for the Toxicity of Mimosa pudica

3.2.1 LD50

ICR male mice (20±2g) were randomly divided into two groups of ten animals each. One group of mice was given Mimosa extract by intragastrical administration at the limit dose of 15.0 g/kg of the powder extract (DWE = 79.5 g/kg). The control group was given the same volume of saline. The results showed that no mortality was observed after one week and the animals did not show any abnormal behaviour.

ICR male or female mice in groups of fifteen were administered with Mimosa extract by intraperitoneal injection at different dose levels of 1.0-6.0 g/kg (DWE =

5.3-31.8 g/kg). The number of dead animals was recorded within one week. By using the statistical graphic method of Litchfield and Qilcoxon (1947), the LD50 with

95% confidence limit of male and female mice were found to be 3.3土0.8 g/kg (DWE

=17.5土4.2 g/kg) and 3.4±0.9 g/kg (DWE == 18.0土4.8 g/kg), respectively (Figure

3.13). The death time of the mice after drug administration was recorded (Tables

3.6-3.7). It indicated that over one-third of the dead mice died after 24 hours.

Hindlimb paralysis was observed in nearly half of the mice before death.

72 100

90 — // ^ • male S 80 - 〇 female D/Z 1 - X ：60 - Z I - 1 40 — Xo z ？ 30 - /

1 1 1 1 0.0 0.2 0.4 0.6 0.8 1.0

Log dose of Mimosa extract

Figure 3.13: Dose-lethal toxicity curve of Mimosa pudica on mice.

ICR mice in groups of fifteen were injected a single dose of different dose levels of Mimosa extract (1.0-6.0 g/kg; DWE = 5.3-31.8 g/kg) by intraperitoneal injection. LD50 in male and female mice were found to 3.3土0.8 and 3.4士0.9 g/kg, respectively.

73 Table 3.6: Death time of female mice after i.p. injection of Mimosa pudica.

Treatment of Death time after drug administration (day) "^Mimosa extract (g/kg) 1 2 3 4 5

1 0 0 0 0 0 2 0 4 0 0 0 3 3 2 10 0 4 8 10 0 0 5 9 10 0 0 6 11 0 0 0 0

ICR mice in groups of fifteen were injected a single dose of different dose levels of Mimosa extract by intraperitoneal injection. *DWE = 5.3g dry plant material per g of extract

Table 3.7: Death time of male mice after i.p. injection of Mimosa pudica.

Treatment of Death time after drug administration (day) "^Mimosa extract (g/kg) 1 2 3 4 ^

1 0 0 0 0 0 2 112 0 0 3 2 3 1 0 0 4 4 0 2 1 1 5 10 1 0 0 0 6 12 0 0 0 0

ICR mice in groups of fifteen were injected a single dose of different dose levels of Mimosa extract by intraperitoneal injection. *DWE = 5.3g dry plant material per g of extract

74 3.2.2 Enzyme assays

Male or female Sprague-Dawley rats (200±20g) in groups of six were

administered with one of the different dose levels of 0.5-3.0 g/kg (DWE = 2.7-15.9

g/kg) of Mimosa extract by intraperitoneal injection. The control group was injected

with the same volume of saline. After 10 hours, the rats were sacrificed and their

serum was obtained for the determination of the enzyme activities of LDH，CK, GOT and GPT. Preliminary study on the time course using the dose level of 1.5 g/kg

(DWE = 8.0 g/kg) was conducted. This dosage was used because animal death was observed when the dose was greater than 1.5 g/kg. The results on the time course study indicated that all four enzyme activities could reach an optimal level 10 hours after the drug administration. Some of the enzyme activities, such as GOT, CK and

LDH, started to return to normal after 12 hours (Appendix, Figures A3-A6). Since the animals may start to recover or the released enzymes are gradually removed from the circulation, the serum level of the enzyme activities start to decrease. Thus, 10- hour time period was chosen for studying the dose-response relationship of different enzyme activities.

The results showed that all the four enzymes, LDH, CK, GOT and GPT

(Figures 3.14-3.17)，exhibited a dose-dependent relationship when Mimosa extract was given to rats by intraperitoneal injection. CK and GOT had a significant increase at the dose level of 1.0 g/kg or greater, while LDH had a significant elevation even at the lower dose level of 0.5 g/kg. However, an elevation of GPT could only be observed when the dose level of 2.5 g/kg was reached. Besides, some of the rats also showed a hindlimb paralysis when the dosage greater than 2.0 g/kg was given.

75 1200

1000 -

§ T * — 800 - 氺 丁

600 - 人Z Ct /V l/^ Q / 口 400 - IX / 200 - / 0 0 1 1 1 1 0.0 0.5 1.0 1.5 2.0 2.5 Dosages of Mimosa extract (g/kg)

Figure 3.14: Dose-response curve of Mimosa pudica (i.p.) on the serum level of lactate dehydrogenase (LDH) of rats.

Sprague-Dawley rats were given a single dose of either saline or Mimosa extract by intraperitoneal injection. After 10 hours, serum was collected for the enzyme measurement. Data are expressed as mean 士 S.E.

DWE = 5.3g dry plant material per g of extract

* Significantly different when compared with the correspondingcontro l group (RPMI), P<0.05.

76 1200

1000 一 氺

\ B 800 - I /宁 I _ — / 丄 M * /

。400 - f y

200 — 丄 . 0 1 1 1 1 0.0 0.5 1.0 1 .5 2.0 2.5 Dosages of Mimosa extract (g/kg)

Figure 3.15: Dose-response curve of Mimosa pudica (i.p.) on the serum level of creatine kinase (CK) of rats.

Sprague-Dawley rats were given a single dose of either saline or Mimosa extract by intraperitoneal injection. After 10 hours, serum was collected for the enzyme measurement. Data are expressed as mean 土 S.E.

DWE = 5.3g dry plant material per g of extract

* Significantly different when compared with thecorrespondin gcontro l group (RPMI), P<0.05.

77 240 — ^ * * T*^ >I 200 - 丁 .(V^ 二 XI

a _ * 丄

fe 160 - *

.- - T 120- 丄 ^ 丄 O 80 -

I I I I I I 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Dosages of Mimosa extract (g/kg)

Figure 3.16: Dose-response curve of Mimosa pudica (i.p.) on the serum level of glutamic oxalacetic transaminase (GOT) of rats.

Sprague-Dawley rats were given a single dose of either saline or Mimosa extract by intraperitoneal injection. After 10 hours, serum was collected for the enzyme measurement. Data are expressed as mean 土 S.E.

DWE = 5.3g dry plant material per g of extract

* Significantly different when compared with thecorrespondin gcontro l group (RPMI), P<0.05.

78 80

一 70 - g * > 60 - * T

-+-> 丁 c A ：=> 50 - ^^

⑷ /T --

各別— K __1/ 5 40〇- /I ^ \ * Y

I 2。-\口7

10 -

Q I I ！ I I I 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

Dosages of Mimosa extract(g/kg)

Figure 3.17: Dose-response curve of Mimosa pudica (i.p.) on the serum level of glutamic pyruvic transaminase (GPT) of rats.

Sprague-Dawley rats were given a single dose of either saline or Mimosa extract by intraperitoneal injection. After 10 hours, serum was collected for the enzyme measurement. Data are expressed as mean 土 S.E.

DWE = 5.3g dry plant material per g of extract

* Significantly different when compared with the corresponding control group (RPMI), P<0.05.

79 3.2.3 Subacute toxicity test

ICR female mice (15 土 Ig) in groups of ten were administered with Mimosa extract at one of the three different dose levels, 2.5, 5.0 and 10.0 g/kg/day (DWE =

13.3，26.5 and 53.0 g/kg/day) for 30 days, by intragastrical administration. The control group was given the same volume of saline. From the physical observation, there was no behavioral change in all three test groups; the food and water intake were normal when compared with the control group. The growth rate was also normal (Figure 3.18). After one month, the mice were sacrificed and their serum was collected for biochemical tests. Five enzyme assays, LDH, CK, GOT, GPT and

ALP, were measured, but none was affected. Haematological studies included red blood cell (RBC) count and total white blood cell (WBC) count. It was found that all five enzymes did not increase in serum level when compared with those in the control (Table 3.8). The RBC count and total WBC count did not show any significant change (Table 3.9). All the animals were subjected to a gross pathological examination and the result did not show any abnormality. Among the vital organ weights recorded (Table 3.10)，only the weight of liver showed a significant decrease in the dose groups of 2.5 and 5.0 g/kg/day, but no increase in the liver enzymes

LDH, ALP, GOT or GPT was observed.

80 34.00 —

32.00 —

30.00 — 二：

—28.00 - Jc^f^私時•.壬 I

^ 26.00 - T'f^i:.:.....土

, 24.00 — ,办/ 士

^ 22.00 - Mr 。 o 〇 丨〖ne PQ _ _ y # Mimosa exlracL 2.5 g/kg/day 20.00 — _/ ^r� .''•\7 � ,, �'•. �/IL • Mimosa extract 5.0 g/kg/day Z®/ T/ T Mimosa extract 10.0 g/kg/day

18.。。-J^^

16.00善…至 1 4.00 ‘ I I 1 1 I I I I I I I I 1 1 1 1_ ‘ 0 4 8 12 16 20 24 28 32 Days

Figure 3.18: Effect of Mimosa pudica (p.o.) on the body weight of mice after subacute treatment.

ICR mice in groups of ten were orally given with either saline (control group) or one of the three different dose levels of Mimosa extract (2.5, 5.0 or 10.0 g/kg/day; DWE = 13.3, 26.5 or 53.0 g/kg/day) for one month. Data are expressed as mean 士 S.E.

81 Table 3.8: Effect of subacute oral administration of Mimosa pudica on the serum enzymatic activities of mice.

Enzyme activities Treatment

LDH CK GOT GPT ALP (U/L) (U/L) (SF U/ml) (SF U/ml) (U/L)

saline 342.84 25.39 81.39 38.88 173.95 ±36.20 ±6.22 ±21.72 土 3.09 ±19.89 oo Mimosa extract ~ 2.5 g/kg/day 367.89 45.91 84.25 47.50 179.39 ±55.10 ±16.27 土 18.68 ±4.93 士 10.90

5.0 g/kg/day 469.59 41.59 98.50 47.75 191.80 ±55.09 ±7.87 ±3.45 ±3.45 ±15.08

10.0 g/kg/day 379.36 32.92 48.25 44.50 154.26 ±47.75 土 6.18 ±6.63 ±3.02 ±6.31

ICR mice in groups of ten were orally given with either saline (control group) or one of the three dose levels of Mimosa extract (2.5, 5.0 or 10.0 g/kg/day; DWE = 13.3，26.5 or 53.0 g/kg/day) for one month. Serum was collected for the enzyme measurements. Data are expressed as mean 士 S.E. P>0.05 (Student's t test) _ Table 3.9: Effect of subacute oral administration of Mimosa pudica on the blood cell counts of mice.

Treatment RBC counts (XlOVml) Total WBC counts (XlOVml)

s业ne 817.13 士 60.85 112.50 土 30.06

Mimosa extract

2.5 g/kg/day 753.14 土 44.04 96.44 土 31.50

5.0 g/kg/day 723.14 土 56.35 105.38 士 39.50

10.0 g/kg/day 869.75 土 50.02 101.56 ±11.13 ICR mice in groups of ten were orally given with either saline (control group) or one of the three different dose levels of Mimosa extract (2.5, 5.0 or 10.0 g/kg/day; DWE =13.3, 26.5 or 53.0 g/kg/day) for one month. Data are expressed as mean 土 S.E.

P>0.05 (Student's t test)

83 Table 3.10: Effect of subacute oral administration of Mimosa pudica on the vital organ weights of mice.

Body weight (g) Organ weight (g) per lOOg body weight Treatment

before treatment after treatment spleen liver heart left kidney right kidney

saline 16.9 士 0.3 30.8±1.0 0.457 士 0.018 5.601 ±0.290 0.529±0.015 1.697±0.018 0.714±0.016

Mimosa extract 2.5 g/kg/day 16.5土0.2 28.8土0.9 0.432±0.018 4.350±0.140* 0.546±0.012 0.674土0.022 0.696土0.024

(X

5.0 g/kg/day 16.1 土0.3 28.3土0.08 0.439±0.025 4.443±0.240* 0.551±0.019 0.697土0.024 0.723士0.033

10.0 g/kg/day 16.1±0.4 28.4±0.7 0.435±0.023 5.068±0.210 0.523±0.013 0.697±0.021 0.727±0.029

ICR mice in groups of ten were orally given with either saline (control group) or one of the three different dose levels of Mimosa extract (2.5，5.0 or 10.0 g/kg/day; DWE = 13.3，26.5 or 53.0 g/kg/day) for one month. Data are expressed as mean 士 S.E. .，. * Signifi,cantly different when compared with the saline control group, P<0.05. 3.2.4 Reproductive toxicity

Female Sprague-Dawley rats (220土 lOg) after successful coitus were subjected to drug administration. They were divided into two groups with ten animals each.

One group was given Mimosa extract at the highest possible daily dose of 3.75 g/rat/day (DWE = 19.9 g/rat/day) from PD, to PD,o by intragastrical administration.

The control group was given the same volume of distilled water. The results were shown in Tables 3.11-3.14. It was found that Mimosa extract did not affect the percentage of pregnancy. It also did not show any reduction on the number of implantation sites, the number of normal fetuses and the number of corpora lutea per pregnant animal when compared with the control.

85 Table 3.11: Effect of Mimosa pudica (p.o.) on the percentage of pregnancy in rats.

Treatment No. of animal pregnant Percentage of pregnancy per no. of animal dosed distilled water 10/10 100

Mimosa extract 10/10 100

Sprague-Dawley rats in groups of ten were orally administered with either distilled water (control) or Mimosa extract at the dose of 3.75 g/rat/day (DWE = 19.9 g/rat/day) from PD^ to PDjo- Rats were autopsied at PDi6.

Table 3.12: Effect of Mimosa pudica (p.o.) on the fetus implantation in rats.

Treatment No. of implantation sites Percentage of reduction

per pregnant animal distilled water 14 • 5 土 1.9 --

Mimosa extract 14.6 土 1.5 0 Sprague-Dawley rats in groups of ten were orally administered with either distilled water (control) or Mimosa extract at the dose of 3.75 g/rat/day (DWE = 19.9 g/rat/day) from PDi to PDio- Rats were autopsied at PDi^. Data are expressed as mean 士 S.E.

86 Table 3.13: Effect of Mimosa pudica (p.o.) on the fetus formation in rats.

Treatment No. of normal fetuses Percentage of normal per pregnant animal distilled water 12.7土2.75 -

Mimosa extract 13.7±2.63 107.9

Sprague-Dawley rats in groups of ten were orally administered with either distilled water (control) or Mimosa extract at the dose of 3.75 g/rat/day (DWE = 19.9 g/rat/day) from PD! to PD,o. Rats were autopsied at PDig. Data are expressed as mean 士 S.E.

Table 3.14: Effect of Mimosa pudica (p.o.) on corpora lutea formation in rats.

Treatment No. of corpora lutea Percentage of reduction per pregnant animal distilled water 14.7土 1.8 --

Mimosa extract 15.1 ± 1.0 0

Sprague-Dawley rats in groups of ten were orally administered with either distilled water (control) or Mimosa extract at the dose of 3.75 g/rat/day (DWE = 19.9 g/rat/day) from PDj to PDjo. Rats were autopsied at PDi^. Data are expressed as mean 土 S.E.

87 Chapter Four

General Discussion on the Immunomodulatory Effects and Toxicity of Mimosa pudica

4.1 Immunomodulatory Effects of Mimosa pudica

a) Effect of Mimosa pudica on lymphocyte proliferation

Splenocytes contain a mixed population of B and T lymphocytes while thymocytes contain different sub-populations of T cells. By using different mitogens for B cells and T cells, the effect of a drug or chemical on these two cell types can be studied. Moreover, by comparing the mitogenic response of T cells of splenocytes and of thymocytes to Con A, the collaboration of T cells and B cells can be determined (Andersson et al., 1972).

In in vitro assays, the non-cytotoxic concentrations (1-100 jLtg/ml) of Mimosa extract showed a strong stimulatory effect on splenocyte proliferation (Figure 3.1).

This stimulatory effect was also be observed when the splenocytes cultured in the presence of LPS (Figure 3.2)，but not in the presence of Con A (Figure 3.3).

However, the non-cytotoxic concentrations of the extract did not enhance thymocyte proliferation (Figure 3.4). Moreover, it inhibited the mitogenic response of the thymocytes to Con A (Figure 3.5). As splenocytes contained a mixture of B and T

88 lymphocytes while thymocytes contained only T lymphocytes, the mitogenic effect of

Mi麵“ extract might only act on B cells but not on T cells. Since the activation of

B cells can be stimulated either by T-dependent antigens which require the collaboration of T-helper cells or by T-independent antigens which stimulate the proliferation and differentiation of B cells directly (Lanier, 1991; Kumazawa et al.,

1982)，there is a possibility that Mimosa extract contains some components which act as antigens for B cell activation.

By the use of trypan blue exclusion test (Hudson and Hay, 1976), it was found that the concentration of Mimosa extract greater than 100 jxglml had a slight cytotoxic effect to the lymphocytes. Therefore, the decrease of the splenocyte proliferation in the presence or absence of LPS or Con A at the concentrations of Mimosa extract greater than 100 /xg/ml might be due to cytotoxicity (Figures 3.1-3.3). However, the inhibition on mitogenic response of thymocytes to Con A was not due to the cytotoxic effect since the concentration used was within the non-cytotoxic dose range (Figure

3.5). From the in vitro study, it may be proposed that components in Mimosa extract activate B cells but suppress T cells.

The in vivo study was consistent with the in vitro study that Mimosa extract could enhance splenocyte proliferation and its mitogenic response to LPS (Table 3.1).

Instead of suppression, the in vivo study found that the extract could also strengthen the mitogenic response of splenocytes and thymocytes to Con A (Tables 3.1-3.2).

However, it showed an inhibition on thymocyte proliferation in the absence of the

89 mitogen. As immune response is the outcome of collaboration of different cell populations, results observed in the in vivo system could be different from the isolated

/n vitro system. The degree of lymphocyte activation is also a function of cellular regulation for the outcome of immune response. T cells are heterogenous in nature and comprised of different sub-populations. Therefore, there is the possibility that

Mimosa extract might have greater effect on certain sub-population of T cells such as suppressor T cells.

b) Effect of Mimosa pudica on phagocytosis of macrophages

The engulfing and killing of infectious agents by macrophages represent a major host defense mechanism. Therefore, strengthening the phagocytic capability of macrophages can reduce the susceptibility to infection. From the in vitro study

(Figures 3.6-3.7), it was noted that Mimosa extract could strongly enhance the phagocytic activities of peritoneal macrophages. The same stimulatory effect was also observed even in the presence of hydrocortisone. Hydrocortisone is a kind of glucocorticoids which are potent immunosuppressive agents. They have multiple effects on many stages in inflammatory and immune processes. The major cell targets are mononuclear leukocytes, particular lymphocytes and cells of the monocyte/macrophage lineage (Fauci, 1978; Goulding and Guyre, 1993). Werb et al (1978) suggested that some of these effects were mediated through direct interaction of glucocorticoids with macrophages by specific receptor binding on macrophages. Grasso et al (1982) found that these agents might contribute to the decrease in host resistance by directly inhibiting the phagocytic capability of

90 macrophages. As our findings indicated that Mimosa extract could restore the

suppressive effect on the phagocytic capability of macrophages caused by

hydrocortisone, it might be suggested that the extract could strengthen the. host

defense system.

On the other hand, the in vivo studies indicated that Mimosa extract did not

show any effect on the phagocytic capability of macrophages in both the absence or

presence of hydrocortisone in the culture (Figures 3.8-3.9). Similar to the findings

of hydrocortisone, Wiener et al (1972) found that in vitro hydrocortisone decreased

phagocytosis in mouse peritoneal macrophages, but Van Zwet et al (1975) found that

in vivo hydrocortisone produced no effect on the phagocytosis in mouse peritoneal

macrophages. Although the reasons for the inconsistent results of some in vitro and

in vivo studies are unknown, some hypotheses have been made that the stimulatory

effects of some drugs are reversible or not persistent so that the macrophages of the

in vivo study do not show any enhancement in phagocytosis once the administration

of the drug is stopped (Grasso et al,, 1982). Therefore, the different responses of

macrophages in the in vitro and in vivo studies to Mimosa extract might be explained

.to have different mechanisms and further studies are required.

c) Effect of Mimosa pudica on IL-1 release by macrophages

Macrophages release many cytokines to regulate the immune response. IL-1

is one of the major cytokines responsible for the immunoregulation. It can stimulate

the proliferation and differentiation of T and B cells and potentiate their responses to

91 other lymphokines. It also activates natural killer cells and macrophages, either

directly or indirectly, via other lymphokines (Feldmann and Male, 1989). As IL-1 is a stimulator responsible for T cell activation, the suppression on the release of IL-1 caused by Mimosa extract (Figure 3.10) agreed with the results of the thymocyte proliferation studies (Figure 3.5). A possibility might be suggested that the inhibitory effect of Mimosa extract on thymocyte proliferation could be caused either by suppressing the proliferation of T cells directly or by inhibiting the release of IL-1 by macrophages or by both mechanisms. Since the concentration of the extract was within the non-cytotoxic dose range, the decrease in the IL-1 released was probably due to the inhibitory effect of the extract but not a cytotoxic effect.

However, there was an opposite effect of the extract on the IL-1 released by the LPS-stimulated macrophages (Figure 3.11). Low concentrations of the extract could slightly enhance the release of IL-1. This synergistic effect with LPS to induce the IL-1 release agreed with the results in splenocyte proliferation. As IL-1 can activate B cells, there might be the possibility that the B cell activation induced by the extract was via both the direct interaction of the extract components on B cells and indirectly by the co-stimulating effect of IL-1. It is known that LPS mainly stimulates the level of intracellular IL-1, the amount of IL-1 released by macrophages is low. Since this assay can only determine the extracellular IL-1, the level of intracellular IL-1 cannot be measured (Gery et al。1981; Shang et al., 1986).

Therefore, it is not clear whether Mimosa extract can also increase the intracellular

IL-1 or not. This can be further investigated by using the other reagents such as

92 silica or glucocerebroside which can act synergistically with LPS on macrophages to increase the release of intracellular IL-1 (Gery et al., 1981).

d) Effect of Mimosa pudica on primary humoral iiiinuiiic response

After activation, B lymphocytes will proliferate and differentiate into plasma cells to secrete antibodies. The direct plaque forming assay can reflect the productivity of antigen-specific IgM which are capable of causing the coniplement- mediated lysis of the T-dependent antigen SRBC (Simpson and Gozzo, 1978; Steward and Male, 1989). This assay is used to study the later stage of the B cell response to an antigen, the enhancement of the lysis of SRBC may be clue to the increase of the number of plaque forming cell and/or the increase of the release of IgM per cell

(Yang and Zhang, 1990). Thus, it was suggested that the enhancement of the lysis of SRBC observed in our results (Figure 3.12) might be due to one or both of these mechanisms and further studies are needed.

e) Effect of Mimosa pudica on the restoration of splenocyte blastogenesis of

old mice

According to the studies on the lymphocytes and macrophages, the results showed that Mimosa extract had immunostimulatory effect. It was therefore interesting to see whether the extract could restore the impaired immunity as there was clinical study showing M. pudica could cure chronic bronchitis of the aged people (Anonymous, 1972). Lymphocyte proliferation of the old mice was used as the assay model in this preliminary study. From the previous studies on the

93 lymphocyte proliferation, the extract at the dose of 5.0 g/kg/day (DWE = 26.5

g/kg/day) exhibited the greatest enhancement effect. Therefore, this dose level was

chosen in this restoration study. The results indicated that the extract could partially

restore the blastogenic response of the lymphocytes, especially on the mitogenic response to the T cell mitogen Con A (Table 3.4). Although the extract could enhance the blastogenesis of the lymphocytes, the lymphoid organs spleen and thymus did not show any significant change (Table 3.5); this might be due to the short dosing period. As the aging process is very complicated and involves multiple factors, more extensive studies should be performed to investigate if Mimosa pudica can be used to treat immunodeficiency.

94 4.2 Toxicity of Mimosa pudica

a) LD50

All chemicals can produce toxicity at an unreasonably high dose. It is therefore misleading to demonstrate acute toxicity at such conditions which may be irrelevant to the test substances themselves (Chan and Hayes, 1989). In the present acute toxicity study, the test limit dose of 15.0 g/kg was administered orally to mice in a single dose and neither mortality nor abnormal behaviour was seen. This dose level is equivalent to 79.5g dry weight of the herb per kg body weight. Thus, it can be suggested that Mimosa extract is practically non-toxic or non-lethal after an acute oral exposure (Table 1.1).

However, if the extract was applied to the mice by intraperitoneal injection, mortality was observed when the dosages were at 2.0 g/kg (DWE = 10.6 g/kg) or greater (Figure 3.13). Among all the dead animals, nearly one third of the mice died after 24 hours. Death happened after the first 24 hours may be due to delayed toxic effects which can be direct or indirect (WHO, 1978). As the symptom of hindlimb paralysis was observed in nearly half of the dying mice, there is the possibility that intraperitoneal injection of the extract may exert its toxic effect on specific systems, such as muscular system, and eventually lead to death.

b) Enzyme assays

From the result of LD50, it was found that the toxicity for oral administration

95 of Mimosa extract was too low for carrying out the enzymatic assays. Therefore, enzyme activity changes were checked only for intraperitoneal injection of Mimosa extract. Although the enzymes that we used in this study are not organ-specific and widely distributed in the body tissues, we still can use them to narrow down the possible target organs by combining the pattern of the enzymatic assays and some intoxicated symptoms (Walmsley and White, 1988). The results of the enzymatic assays showed that all the four test enzymes, LDH, CK, GOT and GPT, were increased in serum level when the extract was injected at the sublethal dosages

(Figures 3.14-3.17). Since liver, heart, skeletal muscle and kidney are the principal sources of LDH and GOT, the elevation of these two enzymes suggests that these organs may be involved in the Mimosa intoxication. Although GPT is also rich in heart, skeletal muscle and kidney, liver contains much greater concentration than the other organs. A significant increase of GPT could only be observed at a much higher dose level (2.5 g/kg) than that of the other enzyme activities, at which the dose level as low as 0.5-1.0 g/kg would be sufficient to cause a significant elevation of the enzyme activities, it may suggest that liver is not the primary target organ of Mimosa intoxication. Skeletal muscle is the major source of CK, the elevation of CK activity at the dose level of 1.0 g/kg suggests that skeletal muscle may be the primary target organ. This result was consistent with the findings that the symptom of hindlimb paralysis was observed in some rats of this study and also in mice during the study of LD50. Thus, further studies can be conducted by using some organ-specific isozymes such as carbonic anhydrase III (V^n^en et al, 1988) to verify that the skeletal muscle is involved in the Mimosa intoxication. Besides, a more direct

96 histological study of the vital organs can also be carried out in addition to enzyme assays.

c) Subacute toxicity

Overall speaking, from the results of physical, biochemical and pathological examinations in the subacute toxicity test, Mimosa extract did not show any significant toxic effect. Although there was a slight decrease on the weight of liver, the decrease could only be observed at the two dose groups of 2.5 and 5.0 g/kg/day

(DWE = 13.3 and 26.5 g/kg/day) but not at the higher dose group of 10.0 g/kg/day

(DWE = 53.0 g/kg/day) (Table 3.10). Moreover, LDH, ALP, GOT and GPT are present abundantly in liver, but the absence of significant increase in these enzyme activities might indicate that the liver was not intoxicated (Table 3.8). It is realized that normal biological variations will occasionally show up as statistically significant changes (Mosberg and Hayes, 1989). Therefore, the slight decrease of the liver weight might be probably due to the biological variation as a great variation could also be observed in the enzyme assays.

Mimosa pudica is known to contain a toxic amino acid: mimosine (Notation and Spenser, 1964). Previous toxicity studies on mimosine demonstrated adverse biological effects when animals were fed with an animal diet containing 0.5-1.0% of mimosine (approximately equivalent to 0.8-1.6 g/kg) (Lin and Ling, 1961; Yang et al，1968; Von Sallmann and Grimes, 1959; Crounse et al.，1962; Hylin and Lichton，

1965; Dewreede and Way man, 1970). In our preliminary study, mice intragastrically

97 fed with 0.5 g/kg/day of mimosine for 1 month, showed a growth retardation by 70-

80%. However, the Mimosa extract did not show any toxic effect when it was administered orally. Therefore, the mimosine content present in the Mimosa extract might be negligible or at least lower than the toxic dose used in this study. Further chemical analysis are required to determine the mimosine content in the extract quantitatively.

d) Reproductive toxicity

As the amount of Mimosa extract was administered at the highest possible dose of 3.75 g/rat/day (DWE = 19.9 g/rat/day) in the reproductivity test, no abnormal change on the number of fetus implantation, normal fetuses and corpora lutea could suggest that Mimosa extract did not cause any adverse effect on early pregnancy

(Tables 3.11-3.14). However, whether longer exposure to the extract can cause abnormal development of embryo required further investigation.

98 Chapter Five

Concluding Remarks

From the results of the immunomodulatory studies on the water extract of

Mimosa pudica, it can be concluded that Mimosa extract has a modulatory effect on

both cellular and humoral immunities.

In in vitro studies, Mimosa extract showed a significant stimulatory effect on

splenocyte proliferation and a co-mitogenic effect with LPS but not with Con A.

However, it did not show stimulation on thymocyte proliferation. Moreover, it inhibited the thymocyte proliferation in the presence of Con A. Therefore, there is

the possibility that the extract exerts its stimulatory effect on B cells but suppressive effect on T cells. Further studies can be conducted to verify this stimulatory effect on B cells by depleting T cell populations in the splenocytes to obtain a B cell population culture. The method can be done by treating spleen cells with appropriate monoclonal antibody plus complement (Kumazawa et al., 1982).

The immunomodulatory action of Mimosa extract is not only effective on the in vitro system but also on the in vivo system. The extract can enhance the splenocyte proliferation and its mitogenic response to both LPS and Con A.

However, the extract inhibits the thymocyte proliferation in the absence of mitogens

.but enhances thymocyte proliferation in the presence of Con A. As a conclusion for

99 the in vitro and in vivo splenocyte and thymocyte proliferation studies, it might be

suggested that B cells and T cells were the target cells of Mimosa extract. However,

it is unclear whether Mimosa extract exerts its effects directly on B and T cells or

indirectly on some other accessory cells such as macrophages which are known to

play an essential role in the induction of specific immune responses. As the different

sub-populations of T cells, such as suppressor T cells and helper T cells, B cells and

mononuclear cells are all capable of modifying the degree of proliferation response,

the exact mechanism or the precise cellular targets for Mimosa extract have not yet

been identified and some other experiments may be carried to verify these findings.

Besides the modulatory effect on lymphocytes, in vitro administration of

Mimosa extract can also enhance the phagocytosis of macrophages and reverse the

inhibition caused by in vitro addition of hydrocortisone to the cell culture. Since

phagocytosis is only one of the process of macrophages to protect the host from

infection, the effect of Mimosa extract on the ability of degranulation, oxygen

metabolism, and bactericidal activity of macrophages may also be carried out to

ensure its effect on strengthening the host defense system against infection.

Mimosa extract inhibits the release of IL-1 by macrophages but enhances the

‘ release in the presence of LPS. Further experiments are required to work out the

exact mechanism. As Mimosa extract can affect the release of cytokine IL-1 by

macrophages, whether it can affect the release of tumour necrosis factor" (TNF) is

worthy of studying. TNF is another important cytokine released by macrophages and

100 have many effects in common with IL-1; there is the possibility that the release of

TNF may also be affected by Mimosa extract. If the extract can induce the release of TNF, a series of studies can be carried out on its anti tumour activity.

Besides the effect on the release of IL-1, Mimosa extract also enhances the primary humoral immune response by increasing the release of IgM from antibody- forming-cells. Since IgM is only one of the five classes of immunoglobulins produced by plasma cells, other assays can be carried out to see if the production of other classes of immunoglobulins is also induced by the extract.

Generally, immunopotentiating agents can augment the immune responses or normalize a depressed response, they can be used to combat cancer, reverse iatrogenic immunosuppression, treat immunodeficiency and chronic infection (Goust et aL, 1990). In conclusion for the immunomodulatory effect of Mimosa extract, it shows some stimulatory effect on both the cellular and humoral immunities.

Moreover, the study on the blastogenic response of the 20-week-old mice to Mimosa extract showed that the extract could partially restore the impaired immunity.

Therefore, these results may partially support the traditional use of this herb in treating some inflammatory diseases or chronic infection.

It would be relevant to note that immunomodulatory properties were also found in another species of Mimosa, namely M. tenuiflora. Villarreal et 'al (1991) reported that three different extracts of Mimosa tenuiflora had different patterns of

101 immune responses even on the same cell line. They found that the ethylacetate and

butanol extracts inhibited cell growth, but the petroleum ether extract significantly

enhanced proliferation in both human embryonic fibroblasts W138 and KB cells.

There is a possibility that the aqueous extract of Mimosa pudica may also contain

various components that exhibit diverse and sometimes opposite effects on cellular

proliferation in in vivo and in vitro animal models.

Besides the study on the immunomodulation, Mimosa pudica is also subjected

to the toxicological studies in order to ensure the safe use of the herb. From the

results of acute and subacute toxicity tests, it can be concluded that Mimosa extract

is practically non-toxic when taken orally, causing no mortality, no change in body

weight, no change in serum enzyme level and no gross anatomical change in vital

organs. Moreover, the extract also cannot affect the early pregnancy when it is given

orally. However, when the extract was injected intraperitoneally, mortality and toxic

symptoms such as the increase in serum enzyme levels and hindlimb paralysis were observed. It is known that different route of drug administration can significantly affect the toxic effect of a compound by the differences on local effects; absorption and distribution; and metabolism (Gad and Chengelis, 1988). Thus, the toxicity of

Mimosa extract is probably due to less the bioavailability as eliminated via passing through the gastro-intestinal tract or first pass effect.

As a conclusion, the results of our present studies showed that Mimosa pudica had immunostimulatory effects. Although the potency was not high when the extract

102 was given orally, its stimulatory effects may play a role in some of the traditional uses of this herb.

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112 Appendix

3.0 p 7

2.5 - 2.0 - / / SI 1.5- / 〇 Q/ 1.0 - /

O.OC^I——-J ‘ 1 1 1 ‘ 01 2 3 4 5 6 7 8

Amount of neutral red (XZ/ug)

Figure Al: Calibration curve showing O.D. value of the amount of neutral red.

113 Appendix I: Procedure on determining the activity of lactate dehydrogenase

(LDH)

1. Prepare the sample start reagent by mixing 10 ml reagent A, which consists of

0.194 mmol/L NADH and 54 mmol/L phosphate buffer of pH 7.5，with 0.4

ml reagent B which consists of 16.2 mmol/L pyruvate.

2. Set the spectrophotometer wavelength at 340 nm, absorbance reading to zero with

water as reference and temperature of the cuvet compartment to 25°C.

3. Add 2.5 ml of the prewarmed sample start reagent and 0.1 ml of sample to the

cuvet and mix immediately by inversion.

4. Incubate for 30 seconds.

5. Read and record absorbance at 340 nm versus water as reference and start a

stopwatch at the same time.

6. Continue incubation at 25and record absorbance exactly 1，2 and 3 minutes,

following initial absorbance reading.

7. Determine the mean absorbance change per minute (i^^A/min) and calculate LDH

activity by multiplying c^jA/min with the factor 4180.

114 Appendix II: Procedure on determining the activity of creatine kinase (CK)

1. Prepare the sample start reagent by combining 10 ml reagent A, which consists

of 2.28 mmol/L ADP, 5.7 mmol/L adenosine monophosphate, 2.28 mmol/L

NADP, 22.8 mmol/L N-acetyl-L-cysteine, 2850 U/L HK (yeast), 1710 U/L

G-6-PDH (Lm), 11.4 mmol/L magnesium ions, 22.8 mmol/L D-glucose, 11.4

/zmol/L Di(adenosine 5,)pentaphosphate and 2.28 mmol/L EDTA, with 1 ml

reagent B which consists of 342 mmol/L creatine phosphate and 1.14 mol/L

imidazole buffer of pH 6.7.

2. Set the spectrophotometer wavelength at 340 nm, absorbance reading to zero with

water as reference and temperature of the cuvet compartment to 25°C.

3. Add 2.5 ml of the prewarmed sample start reagent and 0.1 ml of sample to cuvet

and mix immediately by inversion.

4. Incubate for 3 minutes.

5. Read and record absorbance at 340 nm versus water as reference and start a

stopwatch at the same time.

6. Continue incubation at 25°C and record absorbance exactly 1, 2 and 3 minutes,

following initial absorbance reading.

7. Determine the mean absorbance change per minute ((^A/min) and calculate CK

activity by multiplying (^A/min with the factor 4180.

115 Appendix III: Procedure on determining the activity of aspartate

aminotransferase / glutamic oxalacetic transaminase (AST/GOT)

1. Add 0.5 ml substrate solution, which consists of 0.2 mol/L DL-aspartate and 1.8

mmol/L a-ketoglutaric acid in phosphate buffer of pH 7.5, into a test tube and

prewarm it in a 37°C water bath.

2. Add 0.1 ml sample and mix it gently.

3. Incubate for 1 hour at the water bath.

4. Add 0.5 ml color reagent, which contains 20 mg/dL 2,4-dinitrophenylhydrazine

(DNP) in IN hydrochloric acid, and shake gently to stop the reaction. Leave

it at room temperature.

5. After 20 minutes, add 5 ml 0.4N sodium hydroxide solution and mix by

inversion.

6. Wait at least 5 minutes.

7. Read and record absorbance at wavelength 505 nm using water as reference.

8. Determine GOT activity from the calibration curve (Figure A2).

116 Appendix IV: Procedure on determining the activity of alanine

aminotransferase/glutamic pyruvic transaminase (ALT/GPT)

1. Add 0.5 ml substrate solution, which consists of 0.2 mol/L DL-alanine and 1.8

mmol/L Qf-ketoglutaric acid in phosphate buffer of pH 7.5, into a test tube and

prewarm it in a 37°C water bath.

2. Add 0.1 ml sample and mix it gently.

3. Incubate for 30 minutes at the water bath.

4. Add 0.5 ml color reagent, which contains 20 ing/dL 2,4-dinitrophenylhydrazine

(DNP) in IN hydrochloric acid, and shake gently to stop the reaction. Leave

it at room temperature.

5. After 20 minutes, add 5 ml 0.4N sodium hydroxide solution and mix by

inversion.

6. Wait at least 5 minutes.

7. Read and record absorbance at wavelength 505 nm using water as reference.

8. Determine GPT activity from the calibration curve (Figure A2).

117 Procedure for preparing a calibration curve for the measurement of GOT or

GPT activities

1. Add different volumes of the solutions indicated in the following table into test

tubes:

tube *calibration substrate water serum GOT serum GPT no. standard solution (ml) activity activity solution (ml) (ml) (SF Units/ml) (SF Units/ml)

1 0.0 0.5 0.1 0 0 2 0.05 0.45 0.1 20 23 3 0.1 0.4 0.1 55 50 4 0.15 0.35 0.1 95 83 5 0.2 0.3 0.1 148 125 6 0.25 0.25 0.1 216 -

* Calibration standard solution consists of 1.5 mmol/L sodium pyruvate in phosphate buffer of pH 7.5.

2. Add 0.5 ml color reagent to each tube. Shake gently and leave at room

temperature.

3. After 20 minutes, add 5 ml 0.4 N sodium hydroxide solution to each tube and mix

by inversion.

4. Wait at least 5 minutes.

5. Read and record the absorbance at wavelength 505 nm using water as reference.

6. Plot GOT and GPT calibration curves of absorbance values versus the

corresponding units of GOT and GPT respectively (Figure A2).

118 0.8 p

0.7 - ！ 0.6 _

g 0.5 - I�.[^^^ ^ “ 參 GPT

4 0.2 — 〇 GOT

0.1 -

0.0 1 1 ‘ ‘ 〇 50 100 150 200 250 GOT and GPT activity (SF Units/ml)

Figure A2: Calibration curves of glutamic oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) activities.

119 Appendix V: Procedure on determining the activity of alkaline phosphatase

(ALP)

1. Prepare the sample start reagent by combining 25 ml reagent A, which consists

of 0.607 mmol/L magnesium ions and 1.214 mol/L diethanolamine buffer of

pH 9.8, with 5 ml reagent B which contains 60.8 mmol/L p-nitrophenyl

phosphate.

2. Set the spectrophotometer wavelength at 405 nm, temperature of cuvet

compartment to 30°C and absorbance reading to zero with water as reference.

3. Add 3 ml of the prewarmed sample start reagent and 0.05 ml of sample to the

cuvet. Mix immediately by inversion.

4. Read absorbance at 405 nm versus water as reference and start a stopwatch at the

same time.

5. Continue incubation at SC'C and record absorbances after exactly 1，2 and 3

minutes, following the initial absorbance reading.

6. Determine mean absorbance change per minute (t^A/min) and calculate ALP

activity by multiplying t^A/min by factor 3306.

120 1200

• saline ^^^ 1 000 * 〇 Mimosa extract -J 丁

y 800 — X\ 奢 \ ^ 600 一 \ 4 \ *

二 \ 3 40〇- \T

2。。_ • \o

Q I I I I I I I I I I 〇 5 10 15 20 25 30 35 40 45 50 Time (hours)

Figure A3: Serum level of lactate dehydrogenase (LDH) at different time after i.p. injection of Mimosa extract to rats.

Sprague-Dawley rats were given a single dose of either saline or Mimosa extract of 1.5 g/kg (DWE = 8.0 g/kg) by intraperitoneal injection. After different period of time, serum was collected for the enzyme measurement. Data are expressed as mean 土 S.E.

* Significantly different when compared with the saline control group, P<0.05.

121 500

450 - * • saline ^^ 400 - 0 Mimosa extract hJ 丁 ^ 350 — 0 ^ 300 - \ •r-l \ I 250 - \ ^ 200 - \ 。 \ 150 - \ T \T 100 - I

50- ^^^

Q I I I I I I I I I 0 5 10 15 20 25 30 35 40 45 50

Time (hours)

Figure A4: Serum level of creatine kinase (CK) at different time after i.p. injection of Mimosa extract to rats.

Sprague-Dawley rats were given a single dose of either saline or Mimosa extract of 1.5 g/kg (DWE = 8.0 g/kg) by intraperitoneal injection. After different period of time, serum was collected for the enzyme measurement. Data are expressed as mean 土 S.E.

* Significantly different when compared with the saline control group, P<0.05.

122 240 - ^ • saline ^ _ � g * T 〇 extract > 200 - T i

Iw 160 -- * t - / K

O 120 - J *

S 1 • 80 - 參

I I I I I I 1 I I 〇 5 10 15 20 25 30 35 40 45 50 Time (hours)

Figure A5: Serum level of glutamic oxalacetic transaminase (GOT) at different time after i.p. injection of Mimosa extract to rats.

Sprague-Dawley rats were given a single dose of either saline or Mimosa extract of 1.5 g/kg (DWE 二 8.0 g/kg) by intraperitoneal injection. After different period of time, serum was collected for the enzyme measurement. Data are expressed as mean 士 S.E.

* Significantly different when compared with the saline control group, P<0.05.

123 80 p

一 70 - • saline 71I 〇 Mimosa extract E >-+- ) 60 - T "3 氺 氺 二 50 - 丁 T

巴 Cr^ O

I -- /

2。-‘ 、 i 10 - 0 1 i 1 1 〇 10 20 30 40 50

Time (hours)

Figure A6: Serum level of glutamic pyruvic transaminase (GPT) at different time after i.p. injection of Mimosa extract to rats.

Sprague-Dawley rats were given a single dose of either saline or Mimosa extract of 1.5 g/kg (DWE == 8.0 g/kg) by intraperitoneal injection. After different period of time, serum was collected for the enzyme measurement. Data are expressed as mean 土 S.E.

* Significantly different when compared with the saline control group, P<0.05.

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