EVALUATION OF VARIOUS

PHARMACOLOGICAL PROPERTIES OF

THREE INDIAN MEDICINAL PLANTS

A THESIS

Submitted by

VINOTHAPOOSHAN G

In partial fulfillment for the award of the degree

Of

DOCTOR OF PHILOSOPHY

DEPARTMENT OF BIOTECHNOLOGY

KALASALINGAM UNIVERSITY

(Kalasalingam Academy of Research And Education)

ANAND NAGAR, KRISHNANKOIL - 626 126

OCTOBER 2013

ii 

 

KALASALINGAM UNIVERSITY

KRISHNANKOIL - 626 126

BONAFIDE CERTIFICATE

Certified that this thesis titled “EVALUATION OF VARIOUS

PHARMACOLOGICAL PROPERTIES OF THREE INDIAN

MEDICINAL PLANTS” is the bonafide work of Mr. G. Vinothapooshan,

who carried out the research under my supervision. Certified further, that to

the best of my knowledge the work reported herein does not form part of any

other thesis or dissertation on the basis of which a degree or award was

conferred on an earlier occasion on this or any other scholar.

Place: Krishnankoil Dr. K. Sundar

Date: 18.10.13 SUPERVISOR

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ABSTRACT

Natural products isolated from higher plants and microorganisms have been a

source of novel and clinically active drugs. The success of discovering naturally

occurring therapeutic agents rests on bioassay-guided fractionation and

purification procedures. In the present study, immature plant leaves of Mimosa

pudica, Artabotrys hexapetalus and Adhatoda vasica were collected from

Courtallum and Thaniparai Hills in the state of Tamilnadu, India during early

winter season. The leaves of the above plants were shade-dried and made into

coarse powder which was passed through a 40-mesh sieve to get a uniform

particle size and then used for extraction. 500 g of the powder was subjected to

continuous hot extraction in Soxhlet apparatus with methanol, chloroform and

diethyl ether and the residual marc was collected.

The extracts were subjected to qualitative tests for identifying various plant

constituents. The acute toxicity study was performed for various extracts of M.

pudica, A. hexapetalus and A. Vasica according to the acute toxic classic method

as per Organization for Economic Co-operation and Development (OECD)

guidelines. Then the extracts were subjected to various pharmacological activities

such as immunomodulatory, hepatoprotective, anti-ulcer and wound healing

activities. The extracts were also subjected to antimicrobial and antioxidant

studies. The extracts were also separated using Thin Layer Chromatography

iv 

 

(TLC) and High performance liquid chromatography (HPLC); the purified

compounds were analyzed by Fourier transform infrared spectroscopy (FTIR).

The extracts of M. pudica, A. hexapetalus and A. vasica administered orally on

albino rats showed significant increase in adhesion of neutrophils to nylon fibers

which correlates to the process of margination of cells in blood vessels. The

neutrophil adhesion was found to increase significantly with increasing in

concentration. The methanolic extracts of all the three plants exhibited a strong

immmunomodulatory activity, when compared to other extracts and untreated

control. The diethyl ether extracts of all three plants did not exhibit any influence

in modulating the immune response, with values being 43.29%, 34.95% and

63.31% for M. pudica, A. hexapetalus and A. vasica, respectively.

The delayed type hypersensitivity reaction has been widely used as one of the

parameters to measure cell-mediated immune response in a rat model. The

reaction was measured by the percent increase in the paw volume over the

control. A significant increase in paw volume was observed with diethyl ether

extracts of all the three plants, the values being 23.8%, 24.2% and 20.1%

respectively. These results were comparable to that of the positive control

(30.44%). The increase in DTH reaction in rats suggests the stimulatory effect of

v 

 

the antigen on T-lymphocytes and accessory cell types required for the

expression of the reaction.

The hepatoprotective ability of the plant extracts was assessed by their ability to

protect the liver from carbon tetrachloride (CCl4) injury. Four marker enzymes

SGPT, SGOT, ALP and TBL were used for assessing hepatoprotective ability.

All three extracts of all the three test plants were found to be protective against

CCl4 injury. The animals were found to be markedly recovering from CCl4 effect

as noted from the activity of the enzymes. The enzyme activity was found to

decrease about one-half the injured liver and was almost equivalent to control.

The histopathological examination of liver sections of the control group showed

normal cellular architecture with distinct hepatic cells, sinusoidal spaces, and

central vein. The disarrangement of normal hepatic cells with necrosis and

vacuolization were observed in carbon tetrachloride intoxicated liver. The liver

sections of the albino rat treated with 200mg/kg body weight p.o. of methanolic,

chloroform and diethyl ether extracts of the selected plant, followed by carbon

tetrachloride intoxication, showed less vacuole formation and absence of

necrosis. The overall less visible changes observed were comparable with the

standard silymarin, supplementing the protective effect of ether extract of

selected plant and the standard hepatoprotective drug.

vi 

 

All the three plant extracts exhibited anti-ulcer activity in all three models tested

(aspirin induced, alcohol induced and pylorus ligation), when treated with two

different concentrations viz. 100 mg/kg and 200 mg/kg, the methanolic extracts

of the plants exhibited a stronger anti-ulcer activity than other organic solvents.

The ulcer index was considerably reduced in albino rats treated with methanolic

extracts when compared to other solvents. The reduction in ulcer index was

found to be between 60-75% treated with methanol extracts of all the three

plants. The reduction in ulcer index was statistically significant and comparable

to that of the standard drug Ranitidine (20 mg/kg).

All three plant extracts exhibited significant wound healing activity, the activity

of different extracts varied from 65.46% to 93.87%. A lower wound healing

activity was exhibited by the diethyl ether extract whereas the activity was found

to be higher with methanolic extracts. The methanolic extract of M. pudica

exhibited a higher activity of 93.87% whereas the methanolic extracts of A.

hexapetalus and A. vasica exhibited 78.61% and 87.46% respectively.

The increasing in failure of existing chemotherapeutic agents and the rise in

antibiotic resistance of pathogenic microorganisms led to the search for newer

anti-microbial agents particularly from the plant kingdom. In the present study,

all the three extracts were found to exhibit a very strong and broad spectrum

vii 

 

antibacterial activity. The effect was found to be pronounced against gram-

positive bacteria (Micrococcus luteus, Staphylococcus aureus and Bacilluccerus)

than against Gram-negative bacteria (Klebsiellapneumonieae, Salmonella

typhimurium and Salmonella paratyphimurium). Antimicrobial activity of the

plant extracts was analyzed by Gram- positive and Gram- negative organisms by

the well-diffusion assay using ciprofloxacin as standard.

As many plant metabolites exhibit potential anti-oxidant activity, the metabolites

of the plants under study were also assessed for anti-oxidant activity using DPPH

assay, FRAP assay and reducing power measurement methods. The TLC-

purified methanolic extracts of all the three plants exhibited potent anti-oxidant

activity as measured by potassium ferricyanide, FRAP and DPPH assays. The

activities of all three plants were comparable in all these assays.

In conclusion, the methanolic extracts exhibited remarkable ulcer-protective

properties when compared to other two extracts. Similarly methanolic extracts of

all the three extracts exhibited higher antimicrobial activity against Gram-

positive and Gram-negative pathogens and the methanolic extracts of all the three

plants showed better anti-oxidant activity. The methanolic extracts of M. pudica

was found to exhibit better wound healing activity compared to other extracts. In

contrast to this the ether extracts of selected plants (200 mg/kg) were found to

viii 

 

possess significant hepatoprotective activity against carbon tetrachloride induced

hepatotoxification. Interestingly the methanolic extracts of M. pudica, A.

hexapetalus and A. vasica were found to have compounds mimopudine,

artobotrycinol and vasicine. Further investigations on the solvent extracts with

potential pharmacological activity could result in potential therapeutic agents

from these plants.

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ACKNOWLEDGEMENT

I am very much thankful to my mentor, Dr. K. Sundar, Professor and Head,

Department of Biotechnology, Kalasalingam University, Krishnankoil for

suggesting this problem, his expert guidance, constructive criticism, keen interest

and his enthusiastic support shown throughout my project work.

I express my sincere and respectful regards to “Kalvivallal” Thiru

T. Kalasalingam, Chairman, A.K. group of Institutions and ‘Ilayavallal’

Mr. K. Sridharan, Chancellor, Kalasalingam University for providing necessary

facilities in carrying out this work.

I am thankful to Dr. S. Saravanasankar, Vice Chancellor, Kalasalingam

University for permitting me to carry out this work at the Department of

Biotechnology.

I express my sincere thanks to Dr. H. Nellaiah, Dr. K. Palanichelvam, Dr. T.

Kathiresan and Dr. A. Muthukumaran for their critical comments and enthusiastic

support during the preparation of the thesis.

Dr. B. Balamurugan, Ms. V. Anbini, Ms. B. Vinobiah, Ms. G. Nadana Raja

Vadivu, Ms. V. ArunaJanani and Ms. J. Christina Rosy of the Department of

Biotechnology, Kalasalingam University are acknowledged for their helpful

suggestions during the course of this work.

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The enthusiastic help and suggestions of Dr. A. Manohar, Associate Professor,

Department of Chemistry, Kalasalingam University in IR studies is gratefully

acknowledged.

I express my sincere thanks to Dr. S. Balamurali, Professor and Head,

Department of Computer Applications, Kalasalingam University for their critical

comments and enthusiastic support during the preparation of the thesis.

The kind support and suggestions given by Mr. R. Haribalaganesh, Mr. M.

Manikandan and Mr. V. Deepak, Research Scholars of the department during the

course of the study and in the final preparation of the thesis is greatly

appreciated.

My regards are due for Dr. P. Bharathidasan, Professor and Head, Dept. of

Pharmaceutics, Mr. N. R. Livingston Raja, Assistant Professor, Dept. of

Pharmacology, Arulmigu Kalasalingam college of Pharmacy, Krishnankoil for

their support and help in carrying out the Pharmacological work in their

institution.

I express my sincere thanks to Mr. R. Kalirajan, M.Pharm. Professor, Dept. of

Pharmaceutical Chemistry, J.S.S College of Pharmacy, Ooty, for his valuable

help and suggestions offered during the IR studies.

I take this opportunity to express my sincere thanks to all my batch mates, Mr. R.

Kasimani, Mrs. L. Muthulakshmi, Mr. S. RamkumarPandian, Mrs. L. Harini,

xi 

 

Mr. B. Karthikeyan, Ms. M. Ajitha and Mr. C. Mariappan, Research Scholars of

the Department of Biotechnology, Kalasalingam University for making this

experience a memorable one.

I express my sincere thanks to Mr. P. Bharath and Mr. B. Ramar, Administrative

Staff of the Department of Biotechnology, Kalasalingam University for their help

during the course of the study.

The constant support of my family is recognized with gratitude.

Lastly, but not the least, the sacrifice made by the animals during the course of

my study will not be a waste as this study will be helpful in uplifting the

mankind.

Signature

(Vinothapooshan. G)

xii 

 

TABLE OF CONTENTS CHAPTER

NO TITLE PAGE

NO ABSTRACT iii

LIST OF TABLES xvi

LIST OF FIGURES xvii

LIST OF SYMBOLS AND ABBREVIATIONS xix

1. INTRODUCTION 1.1 Natural Products 2

1.2 Sources of Natural Products 3

1.3 Plant Sources 6 2. REVIEW OF LITERATURE 2.1 Inflammation and Inflammatory Diseases 13

2.2 Inflammation and Cancer 14

2.3 Importance of plant extracts for treatment 16

2.4 Inflammation and Liver 17

2.5 Ulcer- inflammation 21

2.6 Inflammation-ROS-anti-oxidative system 24

2.7 Cancer- Immunomodulation 27

2.8 Plants as anti-microbial 28 3. MATERIALS AND METHODS 3.1 Collection of Plant Materials 29

3.2 Phytochemical Studies 29

3.2.1 Preparation of Plant Extracts 29

3.2.2 Qualitative Chemical Evaluation 29

3.3 Pharmacological study 31

3.3.1 Screening for Immunomodulatory activity 31

3.3.1.1 Neutrophil adhesion test in rats 31

3.3.1.2 Delayed type hypersensitivity (DTH) 33

3.3.2 Hepatoprotective Activity Screening 34

xiii 

 

3.3.3 Anti-ulcer activity screening 37

3.3.4 Wound Healing Activity Screening 40

3.4 Isolation and Identification of Bioactive Compounds

42

3.5 Screening of anti-microbial Activity 45

3.5.1 Test microorganisms 46

3.6 Assay of anti-oxidant activity of the plant extracts

47

3.6.1 Quantitative assay for DPPH free-radical scavenging activity

47

3.6.2 Determination of Reducing Power 47

3.6.3 Determination of antioxidant activity by FRAP Assay

47

4. RESULTS

4.1 Analysis of Phytochemicals present in the plants

49

4.1.1 Extraction of phytochemicals 49

4.2 Chromatographic analysis of plant extracts 50

4.2.1 Isolation of active principles 50

4.2.1.1 Fractionation of compounds from Mimosa pudica using Column Chromatography

50

4.2.2 Isolation of compounds from Mimosa pudica using thin layer chromatography

52

4.3 Fractionation of compounds from Artabotrys hexapetalus using Column Chromatography

53

4.3.1 Isolation of compounds from Artabotrys hexapetalus using thin layer chromatography

55

4.4 Fractionation of compounds from Adhatoda vasica using Column Chromatography

56

4.5 Isolation of compounds from Adhatoda vasica using thin layer chromatography

58

4.6 Pharmacological Evaluation of Plant Extracts

59

4.6.1 Immunomodulatory activity 59

xiv 

 

4.6.2 Delayed type hypersensitivity test 62

4.7 Hepatoprotective Activity Screening 63

4.7.1 Acute toxicity studies 63

4.7.2 Hepatoprotective activity 64

4.8 Histopathological section of liver 66

4.9 Anti-ulcer activity 68

4.9.1 Aspirin induced ulcer 69

4.9.2 Alcohol induced ulcer 69

4.9.3 Pylorus ligation induced ulcer 70

4.10 Wound healing Activity 75

4.10.1 M. pudica (MP) 75

4.10.2 A. hexapetalus (AH) 76

4.10.3 A. vasica (AV) 77

4.11 Wound Healing Activity of Fractions of Extracts

81

4.12 HPLC analysis 85

4.12.1 HPLC analysis of methanolic extract of Mimosa pudica

85

4.12.2 HPLC analysis of methanolic extract of Artabotrys hexapetalus

87

4.12.3 HPLC analysis of methanolic extract of Adhatoda vasica

88

4.13 FTIR Spectra of Compounds 89

4.13.1 FTIR Spectral Analysis 89

4.13.2 IR Spectral Studies 89

4.14 Microbiological Analysis 96

4.14.1 Anti-bacterial activity 96

4.15 Anti-oxidant activity of MP, AH and AV 99

4.15.1 Potassium ferricyanide assay 99

4.15.2 Fluorescence Recovery after Photobleaching(FRAP) Assay

100

xv 

 

4.15.3 DPPH Assay 102 5. DISCUSSION 5.1 Phytochemical studies 104

5.2 Evaluation of Immunomodulatory Activity (neutrophil adhesion assay)

104

5.2.1 Delayed type hypersensitivity activity 106

5.3 Hepatoprotective activity 107

5.3.1 Histopathological section of liver 109

5.4 Anti-ulcer activity 110

5.5 Wound healing activity 112

5.6 Anti-bacterial activity 115

5.7 Separation of active principles using HPLC 116

5.8 Infrared Spectral Studies 116

5.8.1 IR studies on Artabotrycinol 116

5.8.2 IR Studies on Mimopudine 117

5.8.3 IR Studies on Vasicine 117

5.9 Anti-oxidant activity 118 6. CONCLUSION 120 7. REFERENCES 121 PUBLICATIONS CURRICULUM VITAE

xvi 

 

S.NO LIST OF TABLES PAGE NO

1. List of cancer and the related chronic immunological conditions 15 2. Summary of major functions of liver 17 3. Formulation of Ointment 40 4. Extraction of phytochemicals from medicinal plants 49 5. Qualitative analysis of plant extracts 50 6. Methanolic extract of Mimosa pudica 50 7. Chloroform extract of Mimosa pudica 51 8. Diethyl ether extract of Mimosa pudica 51

9. Thin layer chromatographic analysis of methanol extract of Mimosa pudica 52

10. TLC analysis of chloroform extract of Mimosa pudica 52 11. TLC analysis of diethyl ether extract of Mimosa pudica 53 12.1 Methanol extract of Artabotrys hexapetalus 53 12.2 Chloroform extract of Artabotrys hexapetalus 54 12.3 Diethyl ether extract of Artabotrys hexapetalus 54 13.1 TLC analysis of methanolic extract of Artabotrys hexapetalus 55 13.2 TLC analysis of chloroform extract of Artabotrys hexapetalus 55 13.3 TLC analysis of diethyl ether extract of Artabotrys hexapetalus 56 14.1 Methanol extract of Adhatoda vasica 56 14.2 Chloroform extract of Adhatoda vasica 57 14.3 Diethyl ether extract of Adhatoda vasica 57 15.1 TLC analysis of methanolic extract of Adhatoda vasica 58 15.2 TLC analysis of chloroform extract of Adhatoda vasica 58 15.3 TLC analysis of diethyl ether extract of Adhatoda vasica 58

16. Effect of MP, AH and AV extracts on delayed type hypersensitivity footpad thickness 62

17. Effect of MP, AH and AV extracts on carbon tetrachloride induced hepatotoxicity 66

18. Effect of various plant extracts on aspirin and alcohol induced gastric ulcer in rats 71

19. Effect of plant extracts of MP, AH and AV against pylorus ligation induced gastric ulcer in rats 73

20. Effect of methanolic, chloroform and diethyl ether extract ointments of MP, AH and AV on excision wound model 78

21. Effect of methanolic extract ointments of Mimosa pudica fractions and their wound healing activity on excision wound model

82

22. Evaluation of anti-microbial activities of plant extracts 96

xvii 

 

S.NO LIST OF FIGURES PAGE NO

1. Major factors that lead to the inflammation of liver which finally leads to cancer 19

2. Possible pathways which might trigger ROS and DNA damage which later leads to hepatocarcinoma 20

3. Involvement of H. pylori in the inflammation induced at gastric epithelial cell 21

4. Role of H. pylori in the induction of various factors leading to gastric cancer 22

5. Possible involvement of various factors that induce COX-2 as a factor that induces epithelial neoplasms which may end in cancer 23

6. Possible mutation sites for the ROS to exert its effects in leading to cancer 25

7. Both extrinsic and intrinsic pathways can induce ROS production where the anti-oxidant system can block the production of ROS 26

8. Use of immune modulators in the treatment of prostate cancer, where the T cells are activated by either threshold reduction or by enhancing the life cycle of T effector cells

27

9. Analysis of haemotological parameters in rats treated with the extracts of MP, AH and AV 60

10. Effect of Leaf Extracts of MP, AH and AV on Neutrophil counts in rats 61

11. Effect of Leaf Extracts of MP, AH and AV on Neutrophil Adhesion 61

12. Effect of MP, AH and AV extracts on delayed type hypersensitivity footpad thickness 63

13. Effect of M. pudica on Carbon tetrachloride induced hepatotoxicity in rats 64

14. Effect of A. hexapetalus on carbon tetrachloride induced hepatotoxicity in rats

65

15. Effect of A. vasica on carbon tetrachloride induced hepatotoxicity in rats 65

16. Resolution of CCl4 induced toxicity in liver of rats by extracts of various plants, Effect of Mimosa pudica on Carbon tetrachloride induced hepatotoxicity in rat’s enzymes

67

17. Effect of various extracts of A. hexapetalus against aspirin, alcohol and pylorus ligation induced gastric ulcer in rats 74

18. Effect of various extracts of MP, AH and AV against Anti-ulcer activity in rats 75

19. Effect of methanolic, chloroform and diethyl ether extract 79

xviii 

 

ointments of M. pudica on excision wound model

20. Effect of methanolic, chloroform and diethyl ether extract ointments of Artabotrys hexapetalus on excision wound model 80

21. Effect of methanolic, chloroform and diethyl ether extract ointments of Adhatoda vasica on excision wound model 80

22. Effect of methanolic, chloroform and diethyl ether extract ointments of MP, AH and AV on excision wound model 81

23. Effect of methanolic extract ointments of Mimosa pudica fractions and their wound healing activity on excision wound model

84

24. Effect of methanolic extract ointments of Mimosa pudica fractions and their wound healing activity on excision wound model

85

25.1 Standard Mimopudine 86 25.2 HPLC results of methanolic extract of Mimosa pudica 86 26.1 Standard Artabotrycinol 87 26.2 HPLC results of methanolic extract of Artabotrys hexapetalus 87 27.1 Standard Vasicine 88 27.2 HPLC results of methanolic extract of Adhatoda vasica 88 28. FTIR Analysis of Mimopudine 90 29. FTIR analysis of methanolic extract of M. pudica 91 30. FTIR Analysis of Artabotrycinol 92 31. FTIR Analysis of Methanolic Extract of A. hexapetalus 93 32. FTIR Analysis of Vasicine 94 33. FTIR Analysis of Methanolic Extract of A. vasica 95 34. The antibacterial activity of Plant Extracts 98 35.1 Anti-oxidant activity (Potassium ferricyanide assay) 99 35.2 Pottasium ferricyanide assay fractions after TLC 100 36.1 Fluorescence Recovery After Photobleaching (FRAP) Assay 101 36.2 FRAP assay fractions after TLC 101 37.1 1,1-Diphenyl-2-Picryl Hydrazyl(DPPH)Assay 102 37.2 DPPH assay fractions after TLC 103

xix 

 

LIST OF SYMBOLS AND ABBREVIATIONS

ALP - Alkaline Phosphatase

CCL4 - Carbon tetrachloride

CO2 - Carbon-dioxide

CuSO4 - Copper sulphate

dl - Deci liter

DLC - Differential leucocyte count

DMF - Dimethyl formaide

DTH - Delayed type hypersensitivity

ED - Effective dose

ES - Eosin stain

FeCl2 - Ferric chloride

FPT - Food pad thickness

g - gram

H2So4 - Sulphuric acid

Hb - Hemoglobin

HCL - Hydrochloric acid

HNO3 - Nitric acid

HPLC - High performance liquid chromatography

I.P - Intra peritoneal

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IAEC - Institutional Animal Ethics Committee

IC - Inhibition concentration

ie. - that is

IR - Infrared spectrophotometer

K - Potassium

KCL - Potassium chloride

Kg - Kilogram

L - Liter

LD - Lethal dose

M - Molarity

Meq/l - Milli-equlence per liter

Mg/dl - Milligram/deciliter

MIC - Minimum inhibition concentration

NAD - Nicotine adenine dinucleotide

NFTB - Nylon-fiber treated blood

NSAID’s - Non-steroidal anti-inflammatory drugs

OECD - Organisation for Economic Co-operation and

Development

P.O - Per oral

PBS - Phosphate buffer

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pH - Hydronium ion concentration

ppm - Parts per million

PSI - Pressure per square inch

PUD - Peptic ulcer disease

RBC - Red blood cells

Rf - Relative front

rpm - Revolutions per minute

SEM - Standard error mean

SGOT - Serum glutamic oxaloacetic transaminase

SGPT - Serum glutamic pyruvic transaminase

SRBC - Sheep red blood cells

TB - Total Bilirubin

TLC - Total leucocyte count

TLC - Thin layer Chromatography

U/L - Unit/liter

UB - Untreated blood

W.B.C - White blood cells

 

1. Introduction

Interest in herbal remedies has been revived recently with a new zeal. Around the

world, research has been carried out to explore the hidden truths and to utilize the

healing property of herbs. Previously, information on the healing power of herbs

in traditional systems of medicine was considered as un-codified data. But the

recent scientific validation of herbs has changed the view of the scientists on the

miraculous effects of natural products. Production of drugs without proper quality

control measures would be harmful to both traditional systems of medicine and

human welfare. Hence, the World Health Organization (WHO), in 1991, brought

out guidelines for the assessment of herbal medicines with the objective of

defining basic criteria for the evaluation of quality, safety and efficacy of herbal

medicines. The assessment includes evaluating the effect of the crude raw drugs,

their preparation, and the finished product; these apart, stability and biological

activity studies also form part of the evaluation (Kamboj, 2000). Such studies help

the development process and also propagate these traditional systems of medicine.

Plants are the richest resource of drugs used in both the traditional and modern

medicinal systems; they are being used in folk medicines, as nutraceuticals,

pharmaceutical intermediates, food supplements and also provide chemical entities

for semi-synthetic drugs (Hammer et al., 1999). Plants and their products might

have been used as medicines right from the beginning of human civilizations. The

uses of plants for medicinal purpose have been practiced for centuries in the

Indian subcontinent. The “Aushadhisuktha” in the Rigveda, which is said to have

been written between 4500 - 1600 B.C., is the oldest document available on

medicinal plants (Shwetha et al., 2012). It briefly describes the morphological

characteristics of medicinal plants, their habitats and therapeutic classification, and

their uses in various ailments.

2  

Medicinal plants are a source of great economic value all over the world. Nature

has conferred human a very rich and diverse kinds of plants present throughout the

world. In India, herbal medicine is still being used by huge population, where the

major portion of traditional therapy utilizes plant extracts and the active

constituents present in it (Akerele and Heywood, 1991). Plants have been used in

traditional medicines to treat a wide range of diseases in India (Kritikar and

Basu,1993). Approximately 3000 plant species in India are known to have

medicinal properties (Prakasha et al., 2010). The traditional Indian systems of

medicine viz., Ayurveda, Siddha and Unani, describes the use of plant products for

enhancing immunity and healing (Jain et al., 2006).

The Western Ghats (10°10′N 77°04′E), is one of the ‘Hotspots of Biodiversity’

identified in the world (Myers et al., 2000). About 5,000 species of an estimated

17,000 species of flowering plants of India are found in the Western Ghats and

almost all have at least one medicinal property (Nayar, 1996). A huge amount of

the plant types found here viz., 54 genera, 1720 species and 135 infra-specific taxa

are found to be endemic (Shetty and Kaveriappa, 1991).

1.1 Natural Products

By definition, the word ‘natural’ is an adjective referring to something that is

present in or produced by nature and not artificial or man-made. Today many

natural products are quite commonly understood to refer herbs, herbal

concoctions, dietary supplements, traditional or alternative medicines. But the use

of herbs as natural-product therapies is different from their use as a platform for

drug discovery process. The development of medicinal plants into therapeutic

drugs is a process that is time consuming and capital-intensive; the risks are also

high with low success rate. Despite all this, natural product drug discovery

programs are still in existence all over the world, mainly because of:

3  

• The higher chemical diversity in natural products as compared to synthetics

and the largely unexplored potential of these products.

• The large number of terrestrial and marine species yet uninvestigated and the

back to nature syndrome.

• Modern technology and advancements made in this field in the last few years

that have made such programmes attractive.

• High-throughput screens and sensitive instrumentation for structure

elucidation that have greatly reduced the amount of time (and also the

amount of sample) required for the first stage of investigation (Lang et al.,

2001).

1.2 Sources of Natural Products

Natural products isolated from higher plants and microorganisms have been

providing novel and clinically active drugs. Screening of natural products has

resulted in a wide array of bioactive agents. For example, there are about 50

commercially available anticancer drugs (excluding endocrines) which have

been approved till date by the USFDA; and significantly, one-third of them are

based on natural products. The most recent addition is taxol, a natural product

derived from the Pacific yew tree, Taxus brevifolia, which is used for the

treatment of ovarian and breast cancers (Kharwar et al., 2011).

The sources of natural products vary from plants and animals to microorganisms

like bacteria, fungi and algae. Historical evidence indicates that certain

Neanderthal remains have been found to contain remnants of medicinal herbs.

One of the earliest collections about health sciences dates back to the 13th

century B.C. which is called as The Nei Ching. But the use of natural products

in medicines recorded dates back to 2600 B.C. which were in cuneiform in

Mesopotamia. Interestingly, these agents have still being used one or the other

4  

way in the treatment of influenza, cough, inflammation and parasitic infestations

(Holt and Chandra, 2002).

There were several references for the use of the herbs in the medicines,

including ayurvedic hymns describing use of various herbs. Theophrastus, a

philosopher and natural scientist circa 300 B.C. wrote a History of Plants in

which he addressed the medicinal qualities of herbs and the ability to cultivate

them. The Greek botanist, Pedanius Dioscorides, circa 100 A.D. produced a

work entitled De Materia Medica, a very well-known European document, on

the use of herbs in medicine. Monks in monasteries in the Middle Ages copied

manuscripts about herbs and their uses. However, Arabs are the ones who

maintained most of the documentations of the Roman and Greeks knowledge of

medicinal plants and the natural products along with the information of their

knowledge of Chinese and Indian herbal medicine (Kroll, 2001). The first semi-

synthetic drug based on a natural product, aspirin was introduced by Bayer in

1899.

Peptic ulcer disease (PUD) was recognized through ages and civilizations. In

fact, peptic-ulcer has attracted most attention among gastro-intestinal diseases

by both the patients and clinicians (Naik and Dhiman, 1993). Dyspepsia in its

variable forms has been a companion to human ever since the advent of bad

cooking, over-indulgence and anxiety (Goodman and Gilman, 1991). The term

“peptic ulcer” is used to refer a group of ulcerative disorders of the upper

gastrointestinal tract which appear to have a common role to play in the

participation of acid-pepsin in their pathogenesis (Jain and Santani, 1994). There

are many causative agents for PUD including stress, hyperacidity, food habits,

NSAIDs and the mucosal barriers are to name a few.

5  

Recent information suggests that the prevalence and changing patterns of the

disease are mainly due to a Gram-negative bacterium, Helicobacter pylori,

which colonize the gastric mucosa, particularly the antral region. About 60% of

patients with gastric ulcers were reported to have H. pylori infection (Jain and

Santani, 1994). Allopathic treatment of PUD has undergone a remarkable degree

of transformation. The therapeutic management includes antacids, anti-

cholinergic and anti-spasmodic drugs, H2-receptor antagonists such as

cimetidine, ranitidine, famotidine and proton-pump inhibitors viz., omeprazole,

lansprazole etc. Previously, since the discovery of the association of H. pylori

with PUD, many antibiotics have been used in combination including ampicillin,

tetracycline, clariothromycin and amoxicillin etc, for killing the bacteria and for

histological remedies. Apart from being highly expensive, these 3-4 drug

regimes produce many side-effects viz. constipation, osteomalacia,

encephalopathy, osteodystrophy and mild diarrhea and CNS depression in case

of non-systemic antacids (Romano and Cuomo, 2004).

Systemic antacids results in side-effects such as occasional risk of gastric

perforation by sodium bicarbonate, systemic alkalosis and edema due to sodium

retention. In case of anti-cholinergic drugs, dry mouth and blurred vision are the

main side effects. H2- receptor blockers mainly cause skin rashes, diarrhea,

muscle pain, hepatotoxicity, gynecomastia, sexual impotence, granulocytopenia

and reversible confusion (Fisher and Lecouteur, 2001). Proton-pump inhibitors

such as omeprazole and lansprezole cause hyper-gastrinaemia due to prolonged

achlorhydria. Other miscellaneous agents like bismuth salts, amylopectin sulfate,

gafarnate and sucralfate cause constipation. Anti-protozoal drugs like tinidazole

and metronidazole produce nausea and a metallic taste; these drugs have also

been found to be carcinogenic in rats (Laine et al., 2000).

6  

As many conventional allopathic medicines for treating various ulcer conditions

with special reference to peptic ulcer are found to have toxic effects on chronic

administration, there is an urgent need for finding alternative herbal remedies for

PUD (Goodman and Gilman, 1991).

1.3 Plant Sources

Natural products, once served mankind as source of all drugs, were mostly

provided by higher plants. Even today, higher plant-derived natural products

represent about 50% of natural products available for clinical use. The WHO

estimates that 80% of people in developing countries rely on traditional

medicine for their primary healthcare and about 85% of traditional medicine

involves the use of plant extracts. This shows that about 3.5-4 billion people

depend upon the plants and their products as source of drugs. About 39% of

newly approved drugs were of natural origin including original natural products,

products derived semi-synthetically from natural products and synthetic

products based on natural product models (Jarvis, 2000).

The use of biodiversity as a source of medicine is an ancient and well proven

concept. At the start of the 21st century, an estimated 75% of the world’s

population continued to depend on traditional plant based medicines for primary

healthcare (Mann, 2002), and among the newly developed chemical entities for

the cancer treatment from 1940’s, over 70% were obtained from natural

products (Johnston, 1998). But the real exploration for the novel natural

products has not been seriously initiated since 1960s, where the modern and safe

equipments for the diving have been discovered (Kim et al., 1995) along with

safe unmanned submerged vehicles a decade later (Bhattaram et al., 2002).

7  

All kinds of animals irrespective of their positions in the phylogenetic tree, their

dwelling place can be a good source of natural products. Unicellular organisms

like bacteria, yeasts and molds, which are considered as primitive life can

produce compounds or provide basic blue print for the production of new

compounds which might be potential therapeutic agents. The use of a natural

product as a therapeutic agent requires that the particular characteristic of the

compound should match with a disease. Developing natural products for therapy

needs to have knowledge of the therapeutic target and thorough understanding

of the pathophysiology of the disease, where the presence of a particular

character in the natural product may suggest the use in the particular condition.

Although the choice of the natural products for therapeutics is a trial and error

one, the search yielded many natural products at serendipity (Hogg, 1971).

The investigation of micro-organisms as sources of potential therapeutic

compounds has much shorter history than compounds from plant as a source of

human medicines. Secondary metabolites secreted by micro-organisms are the

natural substances which may not have any important role in the growth of the

organism which produces it. These metabolites might be secreted because of the

interactions between the various organisms present in the environment (Demain,

1983).

Although almost 20,000 microbial metabolites and approximately 100,000 plant

products have been described so far, secondary metabolites still appear to be an

inexhaustible source of lead structures for new antimicrobials, anti-virals, anti-

tumour drugs, agricultural and pharmacological agents. Later various secondary

metabolites like benzylpenicillin, erythromycin, strobilurin and cephalosporin

etc, were used as lead structures upon which numerous synthetic and semi-

8  

synthetic compounds were derived with improved pharmacological properties

(Vicente et al., 2003).

Plant products have been used in different sectors like medical, industrial,

veterinary and diagnostic applications. Although several medicinal plant extracts

have been used for the treatment for centuries, only 1-10% of the estimated

250,000 to 500,000 species only have been exploited for the purpose (Borris,

1996). Plant products are relatively inexpensive source of biological products

which contains a wide spectrum of primary and secondary metabolites. Modern

medicine is increasingly expecting plant derivatives for the use of antimicrobial

and other drugs, since the traditions antibiotics are becoming ineffective.

Moreover the other reason for the growing interest on plant antimicrobials is the

extinction of rare plants (Lewis and Lewis, 1995). The scientific discipline,

Ethnobotany, utilizes the impressive array of facts gathered by indigenous

peoples about the plant and animal products they have used to maintain health

(Georges and Pandelai, 1949). Lastly, the emergence of new virus entities such

as human immunodeficiency virus (HIV) has spurred intensive investigations

into plant derivatives which may be effective, especially for use in developing

nations.

Various natural products have been already reported in the literature for the

treatment of leukemia, virus infection, thrombosis and coagulopathy, anemia,

malaria and bone marrow diseases. Extracts of Trichothecium roseum (fungus),

Cucumaria japonica (the sea cucumber), Amorpha fruitcosa (legume),

Magnolia officinalis (tree), etc. may be highly useful in treatment of Epstein-

Barr virus. Extracts from Mycena pura, Nidula candida and basidiomycetes, are

useful in the treatment of leukemia and compounds extracted from Streptomyces

platensis may be useful in the thrombocytopenia treatment (Miles et al., 1998).

9  

Compounds obtained from the marine sponge, Aplysina archeri, have been

reported to inhibit the growth of the feline leukemia virus. A number of blood-

sucking invertebrates have small, low-molecular-weight proteins in their salivas

that interfere with the clotting of blood and therefore might be of value as

potential anticoagulants (Zhu et al., 1997). Streptomyces hygroscopicus var

ascomyceticus produces a macrolide that has been reported to have

immunosuppressive activity and may prove to be beneficial in preventing

transplant rejection in humans. It is quite possible that the plant compounds and

the other biological compounds offer a wide range of biological activity,

adequate structural diversity and difference in the mechanism of action.

Therefore a new, safer and more efficient drugs for the treatment of blood-based

disorders could well arise from this family (Sehgal, 2003).

There are several natural products which were claimed to possess the

immunosuppressive function, but often it is associated with cytotoxicity (Mann,

2002). Right from the first heart transplant which occurred in late 1960s,

modern medicine has travelled to a point where organ transplants have become

rather a routine procedure. The survival of the patients with transplants is due to

Cyclosporin A, a fungal metabolite discovered in 1970, which is being used for

immunosuppression since 1978 (Lechler et al., 2005). Apart from

immunosuppression, currently cyclosporine A is being investigated for the

treatment of Rheumatoid arthritis, Crohn’s disease and systemic lupus

erythematosus (Karampetsou et al., 2010).

Apart from cyclosporine A, a methyl analog of oligomycin F, which was

originally isolated from Streptomyces ostreogriseus, was reported to quite

efficiently suppress the activation of B-cell and T-cell in the presence of

mitogens at treatment concentrations equivalent to that of cyclosporine A.

10  

Concanamycin F which was first isolated from the fungus Streptomyces

diastatochromogenes in 1992, has been reported to possess a wide spectrum of

biological activities, including antiviral and immunosuppressive activities

(Mann, 2001). The experimental immunosuppressant (+)-discodermolide,

isolated from the marine sponge Discodermia dissolute, exhibits relatively

nonspecific immunosuppression, causing the cell-cycle to be arrested during the

G2 and M phases. Current the compound is being investigated as a potential

neoplastic agent since it has been found to stabilize the microtubules and thwarts

the depolymerization effectively resulting in the cell cycle arrest in between

metaphase to anaphase transition (Goyal et al., 2010). The same mode of

activity is seen in taxol (Paclitaxel), epothilones, eleutherobin and sarcodictyins.

The cyclic peptide didemnins, first isolated from a marine tunicate,

Trididemnum solidum was found to exhibit immunosuppressive activity. It

involved the induction of cytotoxicity through inhibition of the cell cycle

progression through G1 phase but the mechanism was unknown (Janin, 2003).

The trichopolyns I to V produced by Trichoderma polysporum (fungus) are

lipopeptides which was reported to suppress the lymphocyte proliferation in a

murine allogeneic model (Mann, 2001). Triptolide a product from Tripterygium

winfordii (plant) exhibits immunosuppressive activity through the inhibition of

expression of IL-2 receptor and the subsequent signal transduction (Mann,

2002).

Anti-cancer drug discovery is one of the hottest fields of science where natural

product based anti-cancer drug remain as an active area of research throughout

the world. The tumor incidences, frequency and the type of tumor differ from

country to country (Shu, 1998). The most common positions in the body where

the frequency to develop cancer more is prostrate, breast, colon, rectum, breast,

11  

cervix, uterus, liver, lung, stomach, esophagus kidney, urinary bladder, oral

cavity, blood and ovary (Bostwick and Brawer 1987). A variety of plant and

their derivatives based chemicals are used for the chemotherapeutic treatment of

the aforesaid cancers. They fall into drug classes like the lignans, taxanes, vinca

alkaloids, stilbenes, cephalotaxanes, flavones and camptothecins (Da Rocha et

al., 2001).

Although the occurrence of cancer is wide spread in the human body in different

organs with different functions, yet there remain basic similarities in the

pathogenesis of cancer. When more details about the molecular mechanism in

cancer get revealed, there is every chance of getting more targets for the

possible potential interventions in the growth and development of cancer. A

relatively new approach called cancer chemoprevention which either prevents or

delays or reverses the carcinogenesis (Mehta and Pezzuto, 2002).

Natural products, besides revealing new therapeutic approach had also played a

vital role in the understanding of various biochemical pathways. It also has

proved its volubility by acting as a tool in understanding biological chemistry,

molecular and cellular biology. Some more natural products which have been

used as potential drugs include staurosporine from Streptomyces, huperzine A

from moss and manoalide from marine sponge (Grabley and Sattler, 2003).

There is a steep increase in the costs of drug discovery and development

whereas there is also a decrease in the number of drugs which comes to the

market after all evaluations. Although there is huge amount of success with the

natural products in the drug discovery process, yet natural products have waxed

and waned in pharmaceutical industries. Since there is a large chemical diversity

in natural products, they are most likely to continue to exist and grow to become

12  

even more valuable as sources of new drug leads. This is also because of the

novel molecular structures present in natural products that are much greater in

number and diversity than the other sources (Dahanukar et al., 2000).

There is a major concern today to improve the tools to develop new drugs and

pace by which new products are discovered and developed in the pharmaceutical

industries. This can be successfully achieved when the knowledge about the

procedures of drug-target elucidation followed by the optimization of the

procedures for the lead compound identification and optimization. Human

genome analysis will also help in developing innumerable potential targets

which may also need to be evaluated (Grabley and Sattler, 2003).

The objective of this study is to evaluate the pharmacologic potential of three

Indian medicinal plants viz. Mimosa pudica, Artabotrys hexapetalus and

Adhatoda vasica available in the Western Ghats, for their immunomodulatory,

hepatoprotective, anti-ulcer, wound healing, antimicrobial and anti-oxidant

activities. These three plantschosen are widely distributed, commonly used as a

part of herbal medicine and cultivated in gardens throughout India (Kritikar,

1993).

13  

2. REVIEW OF LITERATURE 2.1 Inflammation and Inflammatory Diseases

Inflammation is considered as the most potent defense in the immune system

(Mogensen et al., 2009). It is a part of complex biological response by the vascular

tissues to harmful stimuli, such as pathogens, tissue injury or irritants (Eming et al.,

2007). A set of events that follows the wound or invasion of a pathogen, which may

result in a specific immune response for the clearance of the invasion or the invader

by the innate immune system is called the inflammatory response (Kindt and Kuby,

2007). It can be recognized based on symptoms like swelling, pain, heat and redness

in the affected tissue. It may occur around a skin infection like a boil or within a

tendon (tendinitis), a joint (arthritis) or a vital organ. Inflammation is mediated by

immune cells by releasing specific mediators which control local circulation and

cell activities. It can also occur when the host fights infection. It is a protective

attempt by the organism to remove the injurious stimuli and to initiate the healing

process.

Inflammation can be as either acute or chronic. The inflammatory response in the

former one is short-lived but in the latter the response stays relatively much longer.

Acute inflammation usually is highly helpful in isolating the damaged tissue and

healing the affected region. It is the initial response of the body to the harmful

stimuli and is achieved by the increased movement of plasma and leucocytes from

the blood into the injured tissues. This is followed by a cascade of biochemical

events that proceeds with the inflammatory response. On the other hand during

chronic inflammation, there will be prolonged secretion of various inflammatory

factors. Although chronic inflammation seems to be advantageous, the prolonged

effect has its own consequences of leading to various kinds of disorders such as hay

fever, atherosclerosis, rheumatoid arthritis and cancer. Balkwill and Mantovani

(2001) rightly mentioned that cancer is a fire lighted by the genetic mutations but

the inflammatory response may be the fuel for the flames of the cancer.

14  

2.2 Inflammation and Cancer

In 1863, Virchow (Balkwill et al., 2001) in his hypothesis stated that some of the

irritants have potential for inducing cancer through inflammation. Later this was

found to be due to the irritants along with the tissue injury, which are for the wound

healing resulting in enhanced cell proliferation. As the wound gets healed these

inflammatory factors recedes from the site. But due to the chronic inflammation and

the presence of inflammatory factors along with various agents including DNA

damaging agents, there is a chance that some cells undergo mutations and continue

to proliferate in the nutrient rich microenvironment resulting in cancer (Coussens

and Werb, 2002). There are many factors which triggers the cancer via

inflammation which include autoimmune disorder (colon cancer–inflammatory

bowel disease), microbial factors (gastric cancer-Helicobacter pylori infections) and

miscellaneous factors (prostate cancer- prostitis) (Table: 1).

Peyton Reus reported that many factors including viral infections result in the sub

threshold neoplastic states (Rous, 1910). This part is referred to as “initiation” step

of cancer. During cancer the first step is always followed by secondary signals,

including irritants and chemicals like phorbol esters and chemicals produced at the

site of wound healing, organ resection etc. This step is referred to as “promotion”

step. This step is where the cells which have the mutations, in the presence of

various inflammatory factors continue to proliferate and at later stage results in a

tumor (Cossens and Werb, 2002).

The host leucocytes including macrophages, dendritic cells and lymphocytes are

present in the inflammatory microenvironment both in the supporting stroma and

the tumor (Lu et al., 2006). Tissue mast cells have also shown to play a major role

in inflammation. All these factors prepare a provisional extracellular matrix where

the endothelial and fibroblast cells grow and produce an environment where the

15  

“promoted” cells grow. These conditions prevail during the wound healing of

injured tissues also. During tissue injury, platelet aggregation results in release of

thrombin which initiates the blood clotting preventing the loss of nutrients. Apart

from this, the platelet aggregation also induces various inflammatory processes by

secreting various proteins and α-granules to the affected site thus initiating

inflammation. During chronic inflammation, the process continuously goes on

resulting in possible mutations and suitable microenvironment for the cancer cells to

grow, thus resulting in tumor (Cossens and werb, 2002). Based on this Dovorak

(1986) called tumors as wounds that do not heal.

There are many inflammatory disorders. Some of them are not harmful to the body.

But some inflammatory bowel disorders like ulcerative colitis and Crohn’s disease

have strongest association with the tumor development in colon. Apart from these

schistosomiasis also plays a major role in colon carcinoma whereas the chronic

infection by H. pyroli is the leading cause for the development of stomach cancer.

The Gram-negative bacterium was proved to be the causative agent for gastric

cancer, where the mechanism is believed to be the DNA damage arising as a result

of chronic inflammation. Hepatatis C infection in liver also has strong influence

over the development of hepatocarcinoma. Here in this thesis, the effect of various

plant extracts on the possible inflammatory damage sites like liver (hepato

protection), stomach (ulcer) and external wounds (wound healing) with respect to

immune modulation was checked. Apart from this various extracts has been checked

for the potential anti-oxidative properties too.

Table 1: List of cancer and the related chronic immunological conditions

(Balkwill and Manowani, 2012)

Malignancy Inflammatory stimulus/condition

Bladder Schistosomiasis Cervical Papillomavirus

16  

Ovarian Pelvic inflammatory disease/talc/tissue remodeling

Gastric H. pylori induced gastritis MALT lymphoma H. pylori Oesophageal Barrett’s metaplasia Colorectal Inflammatory bowel disease Hepatocellular Hepatitis virus (B and C) Bronchial Silica, asbestos, cigarette smoke Mesothelioma Asbestos Kaposi’s sarcoma Human herpesvirus type 8

2.3 Importance of plant extracts for treatment

Extracts from plants contains compounds which are used for curing various

disorders. Ayurveda is the Indian traditional medicine which utilizes the plant and

plant derived compounds for the treatment of various disorders including cancer.

Based on Ayurveda, cancer can be developed from both inflammation and non-

inflammatory disorders. But for development of tumors, inflammation plays a major

role (Garodia et al., 2007). But the use of plant extracts for the treatment is now

limited to use in a particular region. First one is the variations and the number of

herbs used for the preparation of the extracts. This might in turn result in its effect in

the levels of the alkaloids present in the extracts. Among all plants, active principle

has been defined only for certain plants and for only some the chemical structures

are known. Therefore it cannot be completely ascertained that the cure/side effect is

because of a particular compound or multiple compounds. The second reason

attributed is the lack of complete clinical studies. Therefore safety of the plant

extracts was the concern of the scientists (liver herbal products). Therefore more

studies have to be performed in order to find new plant sources and new compounds

for the treatment of various disorders.

17  

2.4 Inflammation and Liver

Liver is an important organ in the human body. It controls the major part of the

human internal environment through various biochemical pathways. Some of the

major functions of the liver include protein and lipid metabolism, detoxification etc.

A summary of the functions of the liver is shown in the Table. 2. But the liver is

always subjected to huge amount of stress because of the pesticide contamination in

the food, alcoholism etc. which leads to increased oxidative stress in the liver.

Although liver has the capacity to regenerate itself, but if the contamination goes

beyond threshold limit, there will be change in the metabolism in the liver. Due to

this oxidative stress various potential disorders may raise in the liver including

inflammation.

Table 2: Summary of major functions of liver (table adopted from Treadway,

1998)

Carbohydrate Metabolism

Produces and stores glycogen (glycogenesis), produces glucose from liver glycogen and other molecules (gluconeogenesis) and releases it into the blood

Lipid Metabolism Oxidizes fatty acids to acetyl-CoA for energy production, synthesizes cholesterol, phospholipids, and bile salts, and excretes cholesterol in bile

Protein Metabolism Deamination of amino acids and produces urea, albumin, plasma transport proteins, and clotting factors Forms the intermediate product in the synthesis of active vitamin D hormone Stores iron as ferritin, and stores large amounts of vitamins A, D, and B12, and smaller amounts of other

Formation and Storage of Vitamins and Minerals

B-complex vitamins and vitamin K. Conjugates and excretes steroid hormones.

Detoxification of Blood

Biotransforms endogenous and exogenous compounds via Phase I and Phase II pathways of detoxif ication (glucuronidation, etc.)

18  

Hepatic fibrosis is the response to the wounds formed in the liver due to chronic

hepatic injury (Curcumin inflammation). Hepatic injuries may arise due to hepatitis,

fatty liver, cirrhosis, biliary cirrhosis and alcoholic liver disease (Treadway, 1998).

This condition is characterized by the abnormal formation of extracellular matrix

(ECM) in the wounded site. Research showed that the hepatic stellate cells and

kupffer cells secrete various factors including PDGF-β during this condition. The

key event in HSC activation followed by hepatic fibrosis is the inflammation caused

due to oxidative stress. Carbon tetrachloride (CCl4) induced hepatic fibrosis has

been used as an experimental model system. The reponse to chronic administration

of CCl4 by the liver tissue in rats is similar to human cirrhosis (Tamayo, 1983).

CCl4 induces lipid peroxidation and production of free radicals in the liver (Basu,

2003) which leads to necrosis in the hepatocytic cells, inflammation and finally

mimics the conditions of hepatic fibrinogenesis (Curcumin inflammation).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

19  

 

 

 

 

 

Figure 1: Image showing the major factors that lead to the inflammation of liver

which finally leads to cancer (Image adopted from

http://www.in.gov/isdh/17438.htm).

20  

Figure 2: Possible pathways which might trigger ROS and DNA damage which

later leads to hepatocarcinoma (Image modified and adopted from Sun and Karin,

2012).

21  

2.5 Ulcer- inflammation

Now it has been proven through various studies that there is a strong relationship

between H. pylori infection in stomach and adenocarcinoma. There is a hypothesis

porposed that H. pylori infection causes gastric inflammation which may lead to

atrophic gastritis and this finally may lead to gastric cancer. Moreover the

organisms infection is also related to the gastric and peptic ulcerations, as described

by many studies which have shown the role of H. pylori in idiopathic peptic ulcer.

But most of the infections which may lead to chronic gastric inflammation remain

clinically silent (Blazer et al., 1995).

Figure 3: Image showing the involvement of H. pylori in the inflammation induced

at gastric epithelial cell (Adapted from Smith et al., 2006).

22  

Figure 4: Role of H. pylori in the induction of various factors leading to gastric

cancer (Image adopted from Kwiecien et al., 2002).

IL-1β is the prime most cytokine activated by the H. pylori infection. The infection

not only induces inflammation but also the neoplastic stage of the intestine. IL-1β is

a proinflammatory cytokine and inhibits the secretion of acid in the stomach.

Therefore when the bacterial virulence factors and the inflammatory factors

combine, the effect will be multi-factorial and may result in cancer (Smith et al.,

2006). Besides, during inflammation Tumor necrosis factor-α is also secreted which

along with IL-1β act as proinflammatory factors leading to various disorders.

23  

Figure 5: Possible involvement of various factors that induce COX-2 as a factor

that induces epithelial neoplasms which may end in cancer (Image adopted from

Kwiecien et al., 2002).

These inflammatory factors invite various cells to the affected site including

neutrophils. Neutrophils secrete various reactive oxygen species including super

oxide (O2-). This superoxide radical reacts with various lipids present in the cell

forming lipid peroxides. Therefore chronic inflammation enhances the production of

ROS and further may result in cancer (Kwiechen et al., 2002). Apart from

cytokines, Cyclooxygenase -2 (COX-2) was also found to play a major role in the

healing of ulcer. Various Non-steroidal anti-inflammatory agents (NSAIDs) have

been used for the treatment of gastric and peptic ulcers. When inhibitors for COX-1

were administered, the ulcer healed very slowly than the non-specific inhibitors for

the COX-2. Therefore, COX-2 was considered to be one of the candidates which are

targeted for treating gastric and peptic ulcer inflammation (Halter et al., 2001).

24  

2.6 Inflammation – ROS – antioxidative system

Reactive Oxygen Species (ROS) is a name given to particular set of ions and

radicals which include super oxide (O2), peroxyl (RO2), hydroxyl (OH), alkoxyl

(RO) group. Apart from these there are certain other members like HOCl, Ozone

(O3), singlet oxygen (O2) and H2O2 which can be easily converted into radicals also

fall in to this category. Mutations induced by ROS can lead to cancer and can occur

in the following three ways.

1. Alterations in base pairs – ROS induced alternations in DNA may result in

mutations in proto-oncogenes and tumor suppressor genes which may lead to

cancer.

2. Affect the cytoplasmic signal pathways – enhanced H2O2 production may lead to

loss of the inhibitory segment in NF-κB which continuously result in

transcriptional activation.

3. Modulation of the stress related genes – H2O2 can activate c-jun and MAP kinase

(Wiseman and Halliwell, 1996).

25  

 

Figure 6: Possible mutation sites for the ROS to exert its effects in leading to

cancer (Image modified and adopted from Nelson and Montgomery, 2003).

Oxidative stress is a major factor that triggers hepatic fibrosis, which in turn has

been shown to enhance the possibility of cancer (Curcumin inflammation liver).

Similarly H. pylori and other agents are shown to increase the ROS in the stomach

which in turn may result in cancer (Smith et al., 2006). Therefore, in both the cases

of liver and stomach, ROS plays a major role in inflammation and cancer. Therefore

inhibition of enhanced ROS production may be a possible strategy for the treatment

of the disorders in liver and stomach.

26  

Figure 7: Both extrinsic (eg. radiations) and intrinsic pathways can induce ROS

production where the antioxidant system can block the production of ROS (Image

modified and adapted from Perera and Bardeesy, 2011).

27  

2.7 Cancer – Immunomodulation

Many chemotherapy agents induce their effect through anti-proliferative and

cytotoxic effects. But the same agents had the capacity for immunosuppression as

the drug reduces the proliferation of immune cells which multiplies at a fast rate.

Some agents which exhibit the property of immunomodulation augmented the

treatment of cancer through the modulation of the immune system (Ehrke and Jane,

2003). Cyclophosphamide is a very good example for the immunomodulatory drug.

The drug has different role at higher concentrations but when the concentration is

lowered, administered alone or with some other agents exhibited anti-cancer and

immunomodulatory properties and cured cancer in mouse models. Apart from

cyclophosphamide, several other agents are shown to modulate the immune system,

nitrosoreus compounds such as adriamycin, arabinosylcitrosine etc. have been

shown to have the same potential (Ehrke et al., 1996).

 

Figure 8: Use of immune modulators in the treatment of prostate cancer, where the

T cells are activated by either threshold reduction or by enhancing the life cycle of T

effector cells (Image adopted from Kwek et al., 2012).

28  

Immunomodulation is used as a method in the treatment various cancers including

prostate cancer where, by inhibiting the activity of an immune checkpoint protein,

Cytotoxic T Lymphocyte-associated antigen 4 (CTLA-4), a crucial impedance can

be removed. This is followed by activation of T cells by lowering the threshold or

by eliminating the inhibitory signals which attenuate the effector T cells (Kwek et

al., 2012).

2.8 Plants as anti-microbials

Apart from H. pylori in causing inflammatory effect, humans in day to day life have

been infected by various microorganisms, which range from infections which are

easily controlled by the host to serious infections resulting in severe morbidity and

mortality. This prompted many scientists to look for new anti-microbial agents

from various sources including plants. Since time immemorial, human have used

plants and their extracts to treat various infectious diseases. Some plants like

cranberry (Vaccinium macrocarpon) was used to treat urinary infections, garlic

(Allium sativum), lemon (Melissa officinalis) etc have been used as antimicrobial

agents. The compounds can be used either directly as phytomedicine for a particular

disease or used as a base compound from which new compounds can be derived

(Iwu et al., 1999).

The search for newer drugs from plant products continues every day since the

scientists predicted that the average effective of every antibiotic is limited, which

kindles the way to produce new antibiotics for the use of mankind.

29  

3. MATERIALS AND METHODS

3.1 Collection of Plant Materials

The plant materials M. pudica, A. hexapetalus and A. vasica were collected from the

foot hills of Western Ghats in and around Courtallum and Thaniparari Hills, Tamil

Nadu, India during early winter season.

3.2 Phytochemical Studies

3.2.1 Preparation of Plant Extracts

The leaf extracts of all the three plants were prepared as described by Kokate,

(1991). The leaves of all three plants (M. pudica, A. hexapetalus and A. vasica) were

shade-dried and made into a coarse powder which was passed through a 40-mesh

sieve to get uniform particle size. A weighed quantity (500 g) of the powder was

then subjected to continuous hot extraction in Soxhlet apparatus individually with

methanol, chloroform and diethyl ether and the residual marc was collected. The

extract was filtered through a cotton plug, followed by Whatman filter paper (No.

1). The extract was evaporated under reduced pressure using a rotary evaporator

until all the solvent had been removed to obtain methanol, chloroform and diethyl

ether extracts.

3.2.2 Qualitative Chemical Evaluation

The chemical composition of the plant extracts was evaluated as described by

Harbone, (1984). The obtained extracts were tested for the presence of various plant

constituents such as alkaloids, flavanoids, tannins, saponins, glycosides, steroids,

steroidal terpenes, phenolic compounds, gums & muciages and carbohydrates.

Alkaloids: The extracts were dissolved in 1 ml of dilute H2SO4 and filtered using

Whatman No.1 filter paper and the filtrate was treated with Mayer’s, Dragendrof’s,

Hager’s and Wagner’s reagents separately. The appearance of cream, orange brown,

30  

yellow and reddish brown precipitates in response to the above reagents respectively

indicates the presence of alkaloids.

Flavonoids: The extracts were mixed with 1-2 ml of alcohol and heated with 1-2

mgs of magnesium and then concentrated HCl was added under cooling. The

appearance of pink colour indicates the presence of flavonoids.

Test for Tannins: The extracts were dissolved in 10 ml of distilled water and

allowed to settle and filtered. To the filtrate 1-2 ml of 5% ferric chloride was added.

The appearance of deep green color indicates the presence of tannin. Another

portion of the filtrate was treated with 1-2 ml of iodine solution and a faint bluish

color confirmed the presence of tannin.

Saponins: About 1 ml of the test extract was dissolved in 20 ml of distilled water

and shaken in a graduated cylinder for 15 minutes. Formation of 1 cm layer of foam

indicates the presence of saponins.

Test for Glycosides: The extracts were dissolved in 10 ml of distilled water under

boiling conditions. This was filtered and 2 ml of the filtrate was hydrolyzed with a

few drops of concentrated HCl and the solution was rendered alkaline with 1-2

drops of ammonia solution. Five drops of this solution was added to 2 ml of

Bennedict’s qualitative reagent and boiled. A reddish–brown precipitate showed the

presence of glycosides.

Test for Steroids: The extractswere dissolved in 2 ml of chloroform. To this 2 ml

of concentrated sulphuric acid was carefully added to form a lower layer. A reddish

brown color at the interface indicated the presence of steroids.

31  

Test for Steroidal Terpenes: The extractswere dissolved in 2 ml of chloroform and

1 ml of acetic anhydride. To this solution 2 drops of concentrated sulphuric acid

were added. A pink colour which changes to bluish green on standing indicated the

presence of steroidal terpenes.

Tannins and Phenols: The extractswere dissolved in 10 ml of water and ferric

chloride solution (5%) or gelatin solution (1%) or lead acetate solution (10%). The

appearance of blue colour with ferric chloride or precipitation with other reagents

indicates the presence of tannins and phenols.

Gums and Mucilages: About 10 ml of test extract was slowly added to 25 ml of

absolute alcohol under constant stirring. The precipitation indicates the presence of

gums and mucilages.

Carbohydrates: The extracts were dissolved in 2 ml of distilled water and then

filtered. The filtrate was treated with concentrated sulphuric acid then Molisch’s

reagent was added. The appearance of pink to violet color indicates the presence of

carbohydrates. The filtrate was boiled with Fehling’s or Benedict solutions. The

formation of brick red precipitate in Fehling’s and Benedict’s solution indicates the

presence of reducing sugars and non-reducing sugars respectively.

3.3 Pharmacological study

3.3.1 Screening for Immunomodulatory activity

3.3.1.1 Neutrophil adhesion test in rats

Adult male Wistar rats were weighing about 150-200gms were divided into

11groups of each 5 animals. The dosage of drugs was administered to the different

groups were as follows:

Group-1: Control (normal saline 10 ml/kg) - used common for all

32  

Group -2: Cedrus deodara wood oil 100 mg/kg (Standard) - used common for all

Group -3: Methanolic extract of M. pudica 200 mg/kg & 400 mg/kg

Group -4: Chloroform extracts of M. pudica 200 mg/kg & 400 mg/kg

Group -5: Diethyl ether extracts of M. pudica 200 mg/kg & 400 mg/kg

Group -6: Methanolic extract of A. hexapetalus 200 mg/kg & 400 mg/kg

Group -7: Chloroform extract of A. hexapetalus 200 mg/kg & 400 mg/kg

Group -8: Diethyl ether extract of A. hexapetalus 200 mg/kg & 400 mg/kg

Group -9: Methanolic extract of A. vasica 200 mg/kg & 400 mg/kg

Group -10: Chloroform extract of A. vasica 200 mg/kg & 400 mg/kg

Group -11: Diethyl ether extract of A. vasica 200 mg/kg & 400 mg/kg

The neutrophil adhesion test was performed according to the methodology of

Wilkinson et al., (1978). The rats were divided into 11 groups, each group

consisting of 5 animals. First group was administrated with normal saline at

concentration of 10 ml/kg (negative control), the second group with C. deodara

wood oil by oral route (positive control) and the third to eleventh groups with

methanolic, chloroform and diethyl ether extracts to all three plants (M. pudica, A.

hexapetalus and A. vasica ) at a dose of 200mg/kg and 400mg /kg/day for 8 days.

On the 8th day blood samples were collected from the retro-orbital plexus in

heparinized vials and analyzed for total leukocyte count (TLC) using Erma PC-607

cell counter (Transasia Ltd., Mumbai, India). The differential leukocyte count

(DLC) was performed by fixing the blood smear and staining with leucofine and

neutrophils percentage in each sample were determined. After the initial counts,

blood samples were incubated with 80mg/ml of nylon fibers for 10 minutes at 370C.

The incubated blood samples were again analyzed for TLC, DLC and neutrophils

percent and neutrophil index was calculated. The neutrophil adhesion percent was

calculated from the following formula;

33  

Neutrophil adhesion % = NIu—NIt NIu

Where,

NIu - Neutrophil index of untreated blood sample

NIt - Neutrophil index of the treated blood sample.

3.3.1.2 Delayed type hypersensitivity (DTH)

The hypersensitivity reaction to Sheep red blood cells (SRBC) was induced in mice

as per the method described by Ray et al., (1996). The sheep erythrocytes were

washed with pyrogen-free sterile normal saline and adjusted to a concentration of

1×108 cells/ml and used for sensitization and challenge. The control group was

administered with an equal volume of PBS (pH 7.4) orally and positive control

group with standard Levamisole 50mg/kg. The negative control group was treated

with Normal saline (10ml/kg) and the test groups with methanolic, chloroform and

diethyl ether extracts ofthree plants (M. pudica, A. hexapetalus and A. vasica) at the

dose of 400mg /kg/day for 9 days. On 9th day, all the groups were challenged with

1×108 SRBC cells, administered intradermally into the left footpad of each mouse,

and the increase in footpad thickness (FPT) was measured 24 h after the SRBC

challenge by volume differential meter.

Group -1: Negative control (normal saline 10ml/kg) - used common for all

Group -2: Positive control PBS (pH 7.4) +Levamisole 50mg/kg - used common for all

Group -3: Methanolic extract of M. pudica 400 mg/kg

Group -4: Chloroform extract of M. pudica 400 mg/kg

Group -5: Diethyl ether extract of M. pudica 400 mg/kg

Group -6: Methanolic extract of A. hexapetalus 400 mg/kg

Group -7: Chloroform extract of A. hexapetalus 400 mg/kg

Group -8: Diethyl ether extract of A. hexapetalus 400 mg/kg

34  

Group -9: Methanolic extract of A. vasica 400 mg/kg

Group -10: Chloroform extract of A. vasica 400 mg/kg

Group -11: Diethyl ether extract of A. vasica 400 mg/kg

3.3.2 Hepatoprotective Activity Screening

Animals: Male albino rats weighing 150-200g maintained under standard

husbandary conditions (temp 23±2oC, relative humidity 55±10% and 12 hours light

dark cycle) were used for the screening. Animals were fed with standard laboratory

food and ad libitum during the entire period of study. All the experimental protocols

were conducted at the Arulmigu Kalasalingam College of Pharmacy, Krishnankoil,

Tamil Nadu, India and were approved by the Institutional Animal Ethics Committee

at Arulmigu Kalasalingam College of Pharmacy, Krishnankoil, India (Reg.

No.509/02/C/CPCSEA/2002).

Toxicity studies: The acute toxicity study was performed for various extracts of

three plants (M. pudica, A. hexapetalusand A. vasica) according to the acute toxic

classic method as per OECD guidelines (Ecobichon, 1997). The male albino rats

were used for acute toxicity study. The animals were kept fasting for overnight

providing only water, after which various extracts were administered orally at the

dose of 300mg/kg and observed for 14 days. If mortality was observed in two

animals out of three animals, then the dose administered was assigned as toxic dose.

If the mortality was observed in one animal, then the same dose was repeated to

confirm the toxic dose. If mortality was not observed, the procedure was repeated

for further higher doses such 400, 500 & 2000mg/kg body weight. The animals

were observed for toxic symptoms for 72 h.

Carbon tetrachloride induced hepatotoxicity: Male albinorats were divided into

12 groups of 6 animals in each group. Group I served as a control, which was

administrated normal saline (3 ml/kg, p.o.). Group II received CCl4 (0.5 ml/kg, i.p.),

35  

Group III received CCl4 (0.5 ml/kg, i.p.) with Silymarin (100 mg/kg, p.o), Group IV,

V and VI received CCl4 (0.5 ml/kg, i.p.) with methanolic extract of three plants

namely, M. pudica, A. hexapetalusand A. vasica (200 mg/kg, p.o.), Group VII,VIII

and IX received CCl4 (0.5 ml/kg, i.p) with chloroform extract of three plants (M.

pudica, A. hexapetalus and A. vasica) (200 mg/kg, p.o.), Group X, XI and XII

received CCl4 (0.5 ml/kg, i.p) with diethyl ether extract of 3 plants namely M.

pudica, A. hexapetalusand A. vasica (200 mg/kg p.o.) separately for 7 days. After 7th

days of treatment and overnight fasting of rats blood samples were collected from

retro-orbital plexus region under mild ether anesthesia and the serum was separated

and to determine the various biochemical parameters (Rao and Mishra, 1997).

Group-I: Normal saline (3ml/kg, p.o.) - Solvent control

Group -II: CCl4 (0.5ml/kg, i.p.) - Hepatic control

Group -III: CCl4 (0.5ml/kg, i.p.) + Silymarin (100mg/kg p.o.)

Group-IV: CCl4 (0.5ml/kg, i.p.)+Methanolic extract of M.pudica (200mg/kg, p.o.)

Group-V: CCl4 (0.5ml/kg, i.p.)+Chloroform extract of M pudica (200mg/kg, p.o.)

Group-VI: CCl4 (0.5ml/kg, i.p.)+Diethyl ether extract of M pudica (200 mg/kg, p.o.)

Group-VII: CCl4 (0.5ml/kg, i.p.)+ Methanolic extract of A.Hexapetalus (200 mg/kg, p.o.)

Group-VIII: CCl4 (0.5ml/kg, i.p.)+Chloroform extract of A.Hexapetalus (200 mg/kg, p.o.)

Group-IX: CCl4 (0.5ml/kg, i.p.)+Diethylether extract of A Hexapetalus (200 mg/kg, p.o.)

Group-X: CCl4 (0.5ml/kg, i.p.) + Methanolic extract of A. vasica (200 mg/kg, p.o.)

Group-XI: CCl4 (0.5ml/kg, i.p.) + Chloroform extract of A. vasica (200 mg/kg, p.o.)

Group-XII: CCl4 (0.5ml/kg, i.p.)+Diethylether extract of A.vasica (200 mg/kg, p.o.)

Assessment of liver function: Blood was collected from all the groups by

puncturing the retro-orbital plexus and was allowed to clot at room temperature.

Serum was separated by centrifugation at 2500 rpm for 10 min for the estimation of

biochemical parameters and to determine the functional state of the liver. The

Serum Glutamic Oxaloacetic Transaminase (SGOT) and Serum Glutamic Pyruvic

36  

Transaminase (SGPT) were estimated by a UV kinetic method based on the

reference method of International federation of Clinical Chemistry in which both

SGOT and SGPT were assayed based on enzyme-coupled system, where keto acid

formed by the aminotransferase reacts with NADH. The coenzyme is oxidized to

NAD and the decrease in absorbance at 340 nm is measured. For SGOT malated

dehydrogenase is used to reduce oxaloacetate to malate where as for SGPT the

pyruvate formed during the reaction is converted to lactate by lactate dehydrogenase

(Raitman and Frankel, 1957).

Alkaline phosphatase (ALP) was estimated as described by Comb and Bowers,

(1972). The total Bilirubin (TBL) was estimated by the method of Jendrassik and

Grof, (1938) which involves the reaction of bilirubin with diazotized sulphanilic

acid to form an azo compound and the colour was measured at 546 nm.

Histopathological studies: The abdomen of the animal was cut open and the liver

was removed. The liver was fixed in Boucin’s solution (mixture of 75 ml of

saturated picric acid, 25 ml of 40 % formaldehyde and 5 ml of glacial acetic acid)

for 12h, and then embedded in paraffin using conventional methods (Galighor and

Kozloff, 1976) and cut into 5μm thick sections and stained with haematoxylin-eosin

dye and finally mounted in di-phenyl xylene. The sections were then observed under

microscope for histopathological changes in liver architecture and their

photomicrographs were well documented.

Statistical analysis: The values of mean ± SEM were calculated for each of the

parameters. For determining the significant inter group difference in each

parameters were analysed separately. The analysis of variance one-way (Gennaro,

1995) was carried out and their individual comparisons of the each group mean

values were performed using Dunnet’s test (Dunnet, 1964).

37  

3.3.3 Anti-ulcer activity screening

Animals: Male albino rats weighing 150-200 g were obtained from Madurai and

maintained under standard husbandry conditions (temp 23±2oC, relative humidity

55±10% and 12 hours light dark cycle) were used for the screening. The animals

were fed with standard laboratory diet ad libitum during the entire period of study.

The experimental protocol has been approved by institutional animal ethics

committee, Arulmigu Kalasalingam College of Pharmacy, Krishnankoil (Regd.

No.509/02/C/CPCSEA/2002.) India.

Toxicity studies: The acute toxicity study was performed using the extracts of

selected plant according to the acute toxic classic method as per OECD guidelines

(Ecobichon, 1997). The albino rats of both sexes were used for acute toxicity study.

The animals were kept fasting for overnight providing only water, after which the

various extracts were administered orally at the dose of 300 mg/kg and observed up

to 14 days. If mortality was observed in two animals out of three animals, then the

dose administered was assigned as toxic dose. If the mortality was observed in one

animal, then the same dose was repeated to confirm the toxic dose. If mortality was

not observed, the procedure was repeated for further higher doses such as 400, 500

and 2000 mg/kg body weight. The animals were observed for toxic symptoms for

72h.

Aspirin-induced gastric ulcer: In aspirin induced ulcer experiments, 11 groups of

male albino rats (100–200 g), with each group consisting of 5 animals were used.

The first group that served as a control administrated with normal saline (2 ml/kg),

the second group which served as positive control with ranitidine (20 mg/kg) orally

and the 3 to 11 groups were served as the test extracts (100mg/kg and 200 mg/kg)

groups for 8 days. The ulcer was produced by administration of aqueous suspension

of aspirin (a dose of 200 mg/kg orally) for eight days. After 8 days of treatment,

38  

animals were allowed to fast for 24 h. On the 8thday the animals were sacrificed 4 h

later of the drug treatment and stomach was surgically opened to calculate the ulcer

index (Kunchandy et al., 1985).

Alcohol-induced gastric ulcer: The male rats were randomly divided into 11

groups each group consisting of 5 animals and fasted for 24 h with free access to

water. The animals were given methanolic, chloroform and diethyl ether extracts of

the 3plants (M. pudica, A. hexapetalus and A. vasica) at a dose of 100mg/kg and

200 mg/kg and Ranitidine (20 mg/kg) orally for seven days. One hour later, 1 ml of

80% ethanol was administered orally to each animal for 7th days. On the seventh day

animals were sacrificed by cervical dislocation, one hour after ethanol

administration, stomachs were surgically cut open along the greater curvature and

pinned on a soft board. The length of each gastric lesion was measured and the

lesion index was expressed as sum of the length of the entire lesion in mm

(Kunchandy et al., 1985).

Pylorus-ligation induced gastric ulcer: The male albino rats weighing 150-200 g

were selected for pyloric ligation ulcer model. The rats were divided into 11 groups,

each group consisting of 5 animals. First group administrated normal saline 2 ml/kg

(negative control), the second group with Ranitidine 20 mg/kg by oral route

(positive control) and the third to eleventh groups with methanolic, chloroform and

diethyl ether extracts ofthree plants(M. pudica, A. hexapetalus and A. vasica)

(100mg/kg and 200 mg/kg) by oral route for seven days. After one hour of the last

dosing, pylorus ligation was made under ether anesthesia. The animals were

returned to the observation chamber for 4h. After 4h, the animals were sacrificed by

decapitation, the abdomen of each animal was surgically opened and the stomach

was isolated after suturing the lower esophageal end. The gastric juice was collected

and the mucosal layer was washed with 1 ml distilled water. The ulcer scoring was

39  

performed in the stomach of each animal. The total volume of gastric content was

also measured. The gastric contents were centrifuged at 1000 rpm for 10 min. One

ml of the supernatant liquid was pipetted out and diluted with 10 ml with distilled

water. The solution was titrated against 0.01 N NaOH using Topfer’s reagent as

indicator, to the endpoint when the solution turned to orange colour. The volume of

NaOH needed was taken as corresponding to the free acidity. The titration was

further continued till the solution regained pink colour (Shay et al., 1945).

The volume of NaOH required was noted and was taken as corresponding to

the total acidity. Acidity was expressed as:

Acidity = Volume of NaOH x Normality x 100 mEq/1

0.1

Statistical analysis: The values mean ± SEM are calculated for each parameter. For

determining the significant inter group differences, each parameter was analysed

separately and one-way analysis of variance (Gennaro, 1995) was carried out and

the individual comparisons of the group mean values were done using Dunnet’s test

(Dunnet, 1964).

Group-1: Normal saline (2 ml/kg, p.o.) - solvent control

Group -2: Ranitidine (20 mg/kg, p.o.) - standard

Group -3: Methanolic extract of M. pudica (100 mg/kg &200 mg/kg, p.o.)

Group -4: Chloroform extract of M. pudica (100 mg/kg &200 mg/kg, p.o.)

Group -5: Diethyl ether extract of M. pudica (100 mg/kg &200 mg/kg, p.o.)

Group -6: Methanolic extract of A. hexapetalus (100 mg/kg &200 mg/kg, p.o.)

Group -7: Chloroform extract of A. hexapetalus (100 mg/kg &200 mg/kg, p.o.)

Group -8: Diethyl ether extract of A. hexapetalus (100 mg/kg &200 mg/kg, p.o.)

Group -9: Methanolic extract of A. vasica (100 mg/kg &200 mg/kg, p.o.)

Group -10: Chloroform extract of A. vasica (100 mg/kg &200 mg/kg, p.o.)

40  

Group -11: Diethyl ether extract of A. vasica (100 mg/kg &200 mg/kg, p.o.)

3.3.4 Wound Healing Activity Screening

Experimental Animals: Male albino rats (150-200 gm) were provided with a

standard diet (Pranav Agro, India) and water ad libitum and maintained under

standard laboratory conditions in the institutional animal house facility.

Table 3: Formulation of Ointment

Type: water miscible base

S. No. Ingredients Official Formula (gm) Working Formula (gm)

1. 2. 3.

Emulsifying wax White soft paraffin Liquid paraffin

30 50 20

3 5 2

Method of Preparation: The ingredients were mixed, heated gently with

continuous stirring until a homogenous mixture was formed. The above contents

were cooled at room temperature. The10% concentration of ointment was

prepared.In case of the plant extracts, 1 gm of suitable extract was mixed with 10

gms of ointment base (10%); then it was stirred well until a homogenous ointment

was obtained.

Types of ointment prepared: Eleven types of ointments were prepared as indicated

below:

a) Simple ointment base - Control

b) 0.2% w/w of Nitrofurazone ointment -Standard

c) Base + Methanolic Extract of M. pudica (10% w/w)

d) Base + Chloroform Extract of M. pudica (10% w/w)

e) Base + Diethyl Ether Extract of M. pudica (10% w/w)

f) Base + Methanolic Extract of A. hexapetalus (10% w/w)

41  

a) Base + Chloroform Extract of A. hexapetalus (10% w/w)

b) Base + Diethyl Ether Extract of A. hexapetalus (10% w/w)

c) Base + Methanolic Extract of A. vasica (10% w/w)

d) Base + Chloroform Extract of A. vasica (10% w/w)

e) Base + Diethyl Ether Extract of A. vasica (10% w/w)

Excision Wound Model: Male Albino rats (150-200gms) were selected and divided

into 11 groups of 5 animals for each of these experiments. The animals were housed

in the experimental room which was maintained as per IAEC guide lines. The

experimental animals were anaesthetized using lignocaine 2% injections, over the

local selected region. The rats were depilated and an excision wound was created by

cutting away 500 mm square thickness of skin from the predetermine area, the

wound was left open then the drugs, reference standard (0.2 % w/w Nitrofurazone

ointment), control (simple ointment base B.P) and methanol, chloroform and diethyl

ether extracts of 3 plants ointment (M. pudica, A. hexapetalus and A. vasica) were

applied until the wound was healed. This model was used to monitor the wound

contraction and wound closer time. The wound contraction was calculated as

percentage reduction in wound area. The progressive change in wound area is

monitored by calculating the decreasing area (Muppayavarmath and Patil,

1999).RWH = Size of Wound in surface area (mm2) at Day 16 / Size of Wound in

surface area (mm2) at Day 1 X 100

% Reduction in Healing = 100 – RWH

Fractionation of Mimosa pudica Extract: Liquid–liquid extraction, also known as

solvent extraction and partitioning, is a method to separate compounds based on

their relative solubility in two different immiscible liquids, usually water and an

organic solvent. It is an extraction of a substance from one liquid phase into another

liquid phase. Methanolic extract of M pudica was subjected to solvent fractionation

between the following immiscible solvent mixtures in 1:1 ratio (purified water:

42  

hexane, purified water: ethyl acetate, purified water: chloroform and purified water:

n-butanol).

Protocol: Two grams of methanolic extract of M. pudica was dissolved in 10

volumes of solvent purified water and equal volume of hexane was added in a first

separating funnel. This was shaken for 10 min, then allowed to settle or centrifuged

at a low speed for 15 minutes. The lower phase and the upper phase were collected

in different containers. Evaporate the two liquid phases separately. Repeat the

procedure using different solvent mixtures such as purified water: ethyl acetate,

purified water: chloroform and purified water: N-butanol. The combined aqueous

extracts and different organic extracts were used for evaluation of wound healing

activity (McCraken and Chaikin, 1974).

The ointments were prepared with different fractions of M. pudica as per the protocols described earlier.

a) Simple ointment base - Control

b) 0.2% w/w of Nitrofurazone ointment - Standard

c) Base + Aqueous fraction of M. pudica (10% w/w)

d) Base + Hexane fraction of M. pudica (10% w/w)

e) Base + Ethyl acetate fraction of M. pudica (10% w/w)

f) Base + Chloroform fraction of M. pudica (10% w/w)

g) Base + N-butanol fraction of M. pudica (10% w/w)

10% concentration of ointment was prepared.

3.4 Isolation and Identification of Bioactive Compounds

Column Chromatography: Column Chromatography was used for the separation

of extracted compounds. The method described by Wagner and Blatt (1996) was

used. The separation of the compounds was achieved by either increasing or

decreasing the polarity of the solvent system.

43  

Sample Preparation: The methanol, chloroform and diethyl ether extracts (2 gm)

were dissolved in 1 ml of chloroform and this was loaded into the column for the

separation of metabolites.

Preparation of Column: The wet packing of column chromatography was adopted

for the preparation of the column. The column matrix, silica gel, was mixed with

petroleum ether and poured gently from the top of the column to a desired length.

Then the same solvent was run through the column for 2-3 times to prevent air

entrapment. The solvent was maintained up to 10cm above the column bed. The

sample mixture was applied on top of the column with the aid of a funnel. The

column was allowed to stand over-night undisturbed. On the next day, column was

eluted with different solvents viz. petroleum ether, benzene, chloroform, ethyl

acetate and methanol with gradual increasing in polarity. The flow rate of solvent

was adjusted between 16 and 20 drops per minute. 20 ml fractions were collected

and evaporated at room temperature. The extracted material was further analyzed

using Thin Layer Chromatography (TLC).

Thin Layer Chromatography: Glass plates coated with silica gel-G as stationery

phase were used to perform TLC. Plates were developed using iodine, and the spots

were identified and their Rf values determined by using the following formula;

Rf value = Distance traveled by solute / Distance traveled by solvent

The spots identified at the same Rf value were pooled and evaporated to dryness by

using vacuum drier and proceded for further studies (Beckett and Stenlake, 1986).

HPLC analysis: HPLC experiments were carried out using a Shimadzu HPLC - A

HT 2010, made in Japan. The stationary phase Phenomnex C18 column (100Å5µ,

150X4.6mm) and Princeton SPHER-100, C18 colum (100Å5µ, 150X4.6mm) were

used for the analysis of the isolated compounds. Commercially available

44  

mimopudine, artabotricinol and vasicine were used as standards (Natural Remedies

Pvt. Ltd., Bangalore). The mimopudine andartabotricinol were detected at 254nm

and vasicine was detected at 300nm.

Chromatographic conditions for mimopudine standard

Stationary Phase : Phenomnex C18 100Å5µ, 150X4.6mm

Mobile Phase : Methanol: Water (pH 3.4) (60:40)

Detection Wave Length : 254nm

Flow Rate : 1.0 ml/min

Column Washing : Water: Methanol (50:50)

Injection Volume : 20µL/inj

Injection Type : Rheodyne injector

Chromatographic conditions for artabotrycinol

Stationary Phase : Phenomenex C18 100Å5µ, 150X4.6 mm

Mobile Phase : Methanol: Water (90:10)

Detection Wave Length : 254nm

Flow Rate : 1.0 ml/min

Column Washing : Water: Methanol (50:50)

Injection Volume : 20µL/inj

Inject ion Type : Rheodyne injector

Chromatographic conditions for vasicine

Stationary Phase : Princeton SPHER-100, C18 100Å5µ, 150X4.6mm

Mobile Phase : Acetonitrile–0.1M Phosphate buffer–Acetic acid

(15:85:1, v/v/v)

Detection Wave Length : 300nm

Flow Rate : 1.0 ml/min

45  

Column Washing : Water: Methanol (50:50)

Injection Volume : 20µL/inj

Injection Type : Rheodyne injector

Instruments Used

Shimadzu, Detector UV 2487 Dual wave length, Pump 1515

Preparation of standard stock solution: Accurately weighed 5mg of standards viz.

mimopudine, artabotrycinol and vasicine were transferred in to separate tubes and

the contents were dissolved in a few ml of methanol and the volume was made up to

5 ml (1000µg/ml).

Preparation of extracts

1. Accurately weighed 10mg of methanolic extract of M. pudica, A. hexapetalus

and A. vasica weretransferred to separate tubes, the contents dissolved with

few ml of methanol and the volume was made up to 5 ml (1000µg/ml)

(Sharma et al.,1992; Srivastava et al., 1999).

2. 1 ml stock was made up to 10 ml with mobile phase.

3. The sample solution was injected and analysed.

FTIR Spectral Analysis: Methanolic extracts of three plants and their chemical

structure were analyzed by Fourier Transformer - Infrared Spectrophotometer

(Shimadzu – 8400) by KBr pellet method as described by Narmato, (1997).

3.5 Screening of anti-microbial Activity

Preparation of Mueller – Hinton Agar medium

Beef extract - 300 gm

Peptone - 17.5 gm

46  

Starch - 1.5 gm

Agar - 17 gm

Cold distilled water - up to 1000ml

All the ingredients were weighed and suspended in 1000 ml of cold distilled water

and heated to boiling. The pH of the media was adjusted to 7.4 with 5 M sodium

hydroxide solution. Then 5 – 20 ml of this agar medium was transferred into each

boiling tube and plugged with non-absorbent cotton. The tube containing agar

medium was sterilized by pressure controlled heat sterilization technique using an

autoclave at 15 lbs at 121°C for 20 minutes. After sterilization the agar medium

was melted and cooled. A well was prepared in the plates with help of a cork-borer

(0.85 cm) and 100 µl of the test compound was introduced into the well. The plates

were incubated overnight at 370 C. The microbial growth was determined by

measuring the diameter of zone of inhibition. For each bacterial strain controls were

maintained where pure solvents were used instead of the extract. The result was

obtained by measuring the zone diameter (Mukerjee, 1996).

3.5.1 Test microorganisms

Known microbial strains were obtained from the National Chemical Laboratory

(NCL), Pune, India. They were three Gram-positive bacteria viz. Micrococcus

luteus, Staphylococcus aureus, Bacillus cerus and three Gram-negative bacteria viz.

Klebsiella pneumonieae, Salmonella typhimurium and Salmonella

paratyphimurium. The disc diffusion assay was used for testing the antibacterial

activity. The ciprofloxacin (100µg/ml) was used as a positive control along with the

extracts of three plants, M. pudica, A. hexapetalusand A. vasic (200 µg/ml)

incubated at 37° C for 24 hours (Adeniyi et al., 1996; Bauer et al., 1966).

47  

3.6 Assay of anti-oxidant activity of the plant extracts

3.6.1 Quantitative assay for DPPH free-radical scavenging activity

The scavenging activity for DPPH free radicals assay was performed as per the

method of Zhao et al., (2006). One milliliter of 0.1 mM DPPH solution in ethanol

and 0.5 ml of each of the test extracts was mixed. The reaction mixture was shaken

vigorously and allowed to reach a steady state at 37°C for 30 min. Decolorization of

DPPH was determined by measuring the decrease in absorbance at 517 nm. The

DPPH radical scavenging effect was calculated according to the following equation:

 

Where,

A0 - Absorbance of the control

A1 - Absorbance of extract

A2 - Absorbance without DPPH.

3.6.2 Determination of Reducing Power

The reducing power of the extracts was determined as described by Chang et al.,

(2002). An aliquot test extracts (0.5 ml) were added to 0.1 ml of 1% (w/v)

potassium ferricyanide. After incubating the mixture at 50°C for 30 min,

supplemented with 0.1 ml of 1% (w/v) trichloroacetic acid and 0.1% (w/v) FeCl3

left for 20 min. The absorbance was read at 700 nm. The increase in absorbance of

the reaction mixture indicates higher reducing power of the sample.

3.6.3 Determination of antioxidant activity by FRAP Assay

48  

The FRAP assay was carried out according to Othman et al., (2007). The FRAP

reagent was prepared by mixing 300 mM acetate buffer (pH 3.6), 10 mM TPTZ and

20 mM ferric chloride in the ratio of 10:1:1. For FRAP assay, the reaction mixture

containing 2 ml of FRAP reagent, 0.5 ml of test extracts and 1 ml of distilled water

was incubated for 10 min. The absorbance was measured at 593 nm. The

antioxidant potential of the sample was compared with the activity of 0.5 ml stock

solution of 1 mg/ml FeSO4.

49  

4. RESULTS

4.1 Analysis of Phytochemicals present in the plants

4.1.1 Extraction of phytochemicals

Mimosa pudica leaf extracts were prepared using three solvents viz. methanol,

chloroform and diethyl ether; the percentage yields of the extracts were found to be

6.4, 8 and 5.2 respectively. Similar results were obtained when extraction was

carried out with the other two plants, with A. hexapetalusthe yield was found to be

7, 6 and 5 with methanol, chloroform and diethyl ether respectively and extraction

of A. vasica powder with methanol, chloroform and diethyl ether yielded 7, 6.2 and

5.2 respectively (Table 4).

Table 4: Extraction of phytochemicals from medicinal plants

Weight of drug

Method of Extraction Solvent used Weight of

extract (gm) Yield (%)

Methanol 16 6.4

Chloroform 20 8

500gms Mimosa pudica powder

Soxhlet apparatus

Diethyl Ether 13 5.2

Methanol 15 7 Chloroform 13.5 6

Artabotrys hexapetalus powder (500gms)

Soxhlet apparatus

Diethyl Ether 12 5 Methanol 16 7

Chloroform 14 6.2

500gms Adhatoda vasica powder

Soxhlet apparatus

Diethyl Ether 12 5.2

50  

Qualitative analysis of the extracts for the presence of phytochemicals

The plant extracts were analyzed for the presence of various phytochemicals using

standard analytical methods. The extracts of all three plants showed the presence of

alkaloids, flavanoids, tannins, steroids and phenolic compounds (Table 5).

Table 5: Qualitative analysis of plant extracts

Phytochemicals Extracts of Mimosa pudica

Extracts of Artabotrys hexapetalus

Extracts of Adhatoda vasica

CHLO DEE METH CHLO DEE METH CHLO DEE METHAlkaloid + ‐ +  + ‐ +  + ‐ +  Flavanoid ‐ + + ‐ + + ‐ + + Tannins ‐  +  +  ‐  +  +  ‐  +  + Saponins ‐ + + ‐ + + ‐ + + Glycosides ‐ + + ‐ + + ‐ + + Steroids + + + + + + + + + Steroidal terpenes ‐  +  +  ‐  +  +  ‐  +  + Phenolic compounds

‐ ‐ + ‐ ‐ + ‐ ‐ +

Gums&mucilages ‐  ‐  +  ‐  ‐  +  ‐  ‐  + Carbohydrate ‐ ‐ + ‐ ‐ + ‐ ‐ +

“+” indicates the presence of phytochemicals and “-“indicates absence of

phytochemicals

4.2 Chromatographic analysis of plant extracts

Various constituents of all the extracts were separated by column chromatography

using silica gel as a matrix. They were separated using various solvents and a

different ratio. The solvents and the ratio used are presented in Table 6, 7 and 8.

4.2.1 Isolation of active principles

4.2.1.1 Fractionation of compounds from Mimosa pudica using Column Chromatography

Table 6: Methanolic extract of Mimosa pudica

51  

S. No. Solvent Ratio Nature of Residue 1 Petroleum Ether: Benzene 20:80 Colorless Residue 2 Petroleum Ether: Benzene 50:50 Colorless Residue 3 Benzene 100 Light Green Residue 4 Benzene: Chloroform 70:30 Light Green Residue 5 Benzene: Chloroform 20:80 Green Residue 6 Chloroform 100 Green Residue

7 Chloroform: Ethyl Acetate 80:20 Greenish Yellow Residue

8 Chloroform: Ethyl Acetate 20:80 Greenish Yellow Residue

9 Ethyl Acetate 100 Light Brown Residue 10 Ethyl Acetate: Methanol 60:40 Light Brown Residue 11 Ethyl Acetate: Methanol 20:80 Dark Brown Residue 12 Methanol 100 Brownish Residue

When all the fractions were analyzed by TLC for the presence of compounds, the

fraction obtained using ethyl acetate and methanol at a ratio of 20:80, yielded

identifiable compound. Hence, this fraction was used for further analysis.

Table 7: Chloroform extract of Mimosa pudica

S. No. Solvent Ratio Nature of Residue 1 Petroleum Ether: Benzene 50:50 Colorless Residue 2 Petroleum Ether: Benzene 20:80 Colorless Residue 3 Benzene 100 Light Green Residue 4 Benzene: Chloroform 40:60 Light Green Residue 5 Benzene: Chloroform 20:80 Green Residue 6 Chloroform 100 Green Residue 7 Chloroform: Ethyl Acetate 70:30 Greenish Yellow Residue 8 Chloroform: Ethyl Acetate 10:90 Greenish Yellow Residue 9 Ethyl Acetate 100 Light Brown Residue 10 Ethyl Acetate: Methanol 70:30 Light Brown Residue 11 Ethyl Acetate: Methanol 20:80 Dark Brown Residue 12 Methanol 100 Brownish Residue

TLC analysis does not yield any significant band from these fractions.

Table 8: Diethyl ether extract of Mimosa pudica

S. No. Solvent Ratio Nature of Residue 1 Petroleum Ether: Benzene 40:60 Colorless Residue

52  

2 Petroleum Ether: Benzene 10:90 Light Green Residue 3 Benzene 100 Light Green Residue 4 Benzene: Chloroform 30:70 Green Residue 5 Benzene: Chloroform 10:90 Green Residue 6 Chloroform 100 Green Residue 7 Chloroform: Ethyl Acetate 80:20 Greenish Yellow Residue 8 Chloroform: Ethyl Acetate 20:80 Greenish Yellow Residue 9 Ethyl Acetate 100 Light Brown Residue 10 Ethyl Acetate: Methanol 60:40 Light Brown Residue 11 Ethyl Acetate: Methanol 10:90 Dark Brown Residue 12 Methanol 100 Brownish Residue

TLC analysis does not yield any significant band from these fractions.

4.2.2 Isolation of compounds from Mimosa pudica using thin layer chromatography

The compounds present in the extracted fractions were resolved by TLC using ethyl

acetate and methanol at different ratios. The results are presented in Table 9, 10 and

11.

Table 9: Thin layer chromatographic analysis of methanol extract of Mimosa pudica

Detecting Reagent

Distance Run by Solvent

Number of spots

Distance Run by

solute (cm)

Rf Value Color of the spot

8 0.5333 Greenish Black 8.5 0.5666 Light Green 9.3 0.6200 Light Green 10.4 0.6933 Light Green

Iodine 15cm 5

12.8 0.8533 Light Brown

Note: Solvent system Ethyl Acetate: Methanol (20:80)

The spot at Rf value 0.62 matches with that of the standard marker Mimopudine

was collected and utilized for further HPLC and FTIR studies.

Table 10: TLC analysis of chloroform extract of Mimosa pudica

Detecting Reagent

Distance Run by Solvent

Number of spots

Distance Run by

solute (cm)

Rf Value Color spot

53  

8.4 0.6461 Light Green 9 0.6923 Light Green

9.5 0.7307 Light Brown 10 0.7692 Dark Brown

Iodine 13cm 5

10.4 0.8 Dark Brown Note: Solvent system (Ethyl Acetate: Methanol (20:80)

None of the spots were matching with that of the standard marker Mimopudine.

Table 11: TLC analysis of diethyl ether extract of Mimosa pudica

Detecting Reagent

Distance Run by Solvent

Number of spots

Distance Run by solute

Value Color spot

7.5 0.5357 Light Brown 8 0.5714 Light Green

9.4 0.6714 Dark green

10.5 0.7500 Brownish Orange

Iodine 14cm 5

11.2 0.8000 Yellow Note: Solvent system (Ethyl Acetate: Methanol (10:90)

None of the spots were matching with that of the standard marker Mimopudine.

4.3 Fractionation of compounds from Artabotrys hexapetalus using Column Chromatography

Table 12.1: Methanolic extract of Artabotrys hexapetalus S. No. Solvent Ratio Nature of Residue

1 Petroleum Ether: Benzene 20:80 Colorless Residue 2 Petroleum Ether: Benzene 30:70 Colorless Residue 3 Benzene 100 Light Green Residue 4 Benzene: Chloroform 50:50 Green Residue 5 Benzene: Chloroform 20:80 Green Residue 6 Chloroform 100 Greenish Yellow Residue 7 Chloroform: Ethyl Acetate 70:30 Greenish Yellow Residue 8 Chloroform: Ethyl Acetate 20:80 Greenish Yellow Residue 9 Ethyl Acetate 100 Light Brown Residue

10 Ethyl Acetate: Methanol 50:50 Light Brown Residue 11 Ethyl Acetate: Methanol 10:90 Dark Brown Residue 12 Methanol 100 Dark Brown Residue

54  

The fraction obtained from elution using Ethyl Acetate: Methanol (10:90) yielded a

partially pure marker compound. This fraction was subjected to TLC studies for

further purification.

Table 12.2: Chloroform extracts of Artabotrys hexapetalus

S. No. Solvent Ratio Nature of Residue 1 Petroleum Ether: Benzene 30:70 Colorless Residue 2 Petroleum Ether: Benzene 20:80 Colorless Residue 3 Benzene 100 Light Green Residue 4 Benzene: Chloroform 40:60 Light Green Residue 5 Benzene: Chloroform 10:90 Light Green Residue 6 Chloroform 100 Greenish Yellow Residue 7 Chloroform: Ethyl Acetate 60:40 Greenish Yellow Residue 8 Chloroform: Ethyl Acetate 10:90 Greenish Yellow Residue 9 Ethyl Acetate 100 Light Brown Residue

10 Ethyl Acetate: Methanol 70:30 Light Brown Residue 11 Ethyl Acetate: Methanol 20:80 Dark Brown Residue 12 Methanol 100 Brownish Residue

TLC analysis does not yield any significant band from these fractions.

Table 12.3: Diethyl ether extract of Artabotrys hexapetalus

S. No. Solvent Ratio Nature of Residue

1 Petroleum Ether: Benzene 60:40 Colorless Residue 2 Petroleum Ether: Benzene 20:80 Light Green Residue 3 Benzene 100 Light Green Residue 4 Benzene: Chloroform 50:50 Green Residue 5 Benzene: Chloroform 20:80 Green Residue 6 Chloroform 100 Green Residue 7 Chloroform: Ethyl Acetate 80:20 Greenish Yellow Residue 8 Chloroform: Ethyl Acetate 20:80 Greenish Yellow Residue 9 Ethyl Acetate 100 Light Brown Residue 10 Ethyl Acetate: Methanol 50:50 Light Brown Residue 11 Ethyl Acetate: Methanol 10:90 Dark Brown Residue

55  

12 Methanol 100 Brownish Residue TLC analysis does not yield any significant band from these fractions.

4.3.1 Isolation of compounds from Artabotrys hexapetalus using thin layer chromatography

The compounds present in the extracted fractions were resolved by TLC using ethyl

acetate and methanol at different ratios. The results are presented in Table 13.1, 13.2

and 13.3.

Table 13.1: TLC analysis of methanolic extract of Artabotrys hexapetalus

Detecting Reagent

Distance Run by Solvent

Number of spots

Distance Run by

solute (cm)

Rf Value Color spot

9.4 0.9400 Greenish Black 8.6 0.8600 Greenish Black 8.2 0.8200 Green 8.0 0.8000 Green

Iodine 10cm 5

7.6 0.7600 Brown Note: Solvent system (Ethyl Acetate: Methanol (10:90)

The spot at Rf value 0.80 matches with that of the standard marker Artabotrycinol

was collected and utilized for further HPLC and FTIR analyses.

Table 13.2: TLC analysis of chloroform extract of Artabotrys hexapetalus

Detecting Reagent

Distance Run by Solvent

Number of spots

Distance Run by

solute (cm)

Rf Value Color spot

9.8 0.8909 Light Green 9.2 0.8363 Green 8.6 0.7818 Green 7.8 0.7090 Dark Brown

Iodine 11cm 5

7.2 0.6545 Dark Brown

56  

Note: Solvent system (Ethyl Acetate: Methanol (20:80). The Rf value of the

compounds separated were not found to be similar to already known compound

Artabotrycinol.

Table 13.3: TLC analysis of Diethyl ether extract of Artabotrys hexapetalus

Detecting Reagent

Distance Run by Solvent

Number of spots

Distance Run by solute

(cm)

Rf Value Color spot

10.0 0.9523 Light Green 9.6 0.9142 Dark Green 9.2 0.8761 Dark Brown 8.4 0.8000 Brownish Orange

Iodine 10.5cm 5

7.2 0.6857 Yellow

Note: Solvent system (Ethyl Acetate: Methanol (10:90), none of the spots were

matching with that of the standard marker Artabotrycinol.

4.4 Fractionation of compounds from Adhatoda vasica using Column Chromatography

Table 14.1: Methanolic extract of Adhatoda vasica

S. No. Solvent Ratio Nature of Residue 1 Petroleum Ether: Benzene 30:70 Colorless Residue 2 Petroleum Ether: Benzene 40:60 Colorless Residue 3 Benzene 100 Light Green Residue 4 Benzene: Chloroform 60:40 Green Residue 5 Benzene: Chloroform 10:90 Green Residue 6 Chloroform 100 Greenish Yellow Residue 7 Chloroform: Ethyl Acetate 70:30 Greenish Yellow Residue 8 Chloroform: Ethyl Acetate 20:80 Light Brown Residue 9 Ethyl Acetate 100 Light Brown Residue

10 Ethyl Acetate: Methanol 50:50 Light Brown Residue 11 Ethyl Acetate: Methanol 10:90 Dark Brown Residue 12 Methanol 100 Dark BrownResidue

57  

The fraction obtained from elution using Ethyl Acetate: Methanol (50:50) yielded a

partially pure marker compound. This fraction was utilized for TLC analyses for

further purification.

Table 14.2: Chloroform extract of Adhatoda vasica

S.No. Solvent Ratio Nature of Residue 1 Petroleum Ether: Benzene 40:60 Colorless Residue 2 Petroleum Ether: Benzene 20:80 Colorless Residue 3 Benzene 100 Green Residue 4 Benzene: Chloroform 50:50 Green Residue 5 Benzene: Chloroform 10:90 Green Residue 6 Chloroform 100 Greenish Yellow Residue 7 Chloroform: Ethyl Acetate 60:40 Greenish Yellow Residue 8 Chloroform: Ethyl Acetate 10:90 Greenish Yellow Residue 9 Ethyl Acetate 100 Light Brown Residue 10 Ethyl Acetate: Methanol 70:30 Light Brown Residue 11 Ethyl Acetate: Methanol 10:90 Dark Brown Residue 12 Methanol 100 Brownish Residue

The Rf value of the compounds separated were not found to be similar to already

known compound Adhatoda vasica.

Table 14.3: Diethyl ether extract of Adhatoda vasica

S. No. Solvent Ratio Nature of Residue 1 Petroleum Ether: Benzene 60:40 Colorless Residue 2 Petroleum Ether: Benzene 10:90 Light Green Residue 3 Benzene 100 Light Green Residue 4 Benzene: Chloroform 50:50 Green Residue 5 Benzene: Chloroform 10:90 Green Residue 6 Chloroform 100 Green Residue 7 Chloroform: Ethyl Acetate 80:20 Greenish Yellow Residue 8 Chloroform: Ethyl Acetate 20:80 Greenish Yellow Residue 9 Ethyl Acetate 100 Light Brown Residue

58  

10 Ethyl Acetate: Methanol 50:50 Light Brown Residue 11 Ethyl Acetate: Methanol 10:90 Dark Brown Residue 12 Methanol 100 Brownish Residue

The Rf value of the compounds separated were not found to be similar to already

known compound Adhatoda vasica.

4.5 Isolation of compounds from Adhatoda vasica using thin layer chromatography

The compounds present in the extracted fractions were resolved by TLC using ethyl

acetate and methanol at different ratios. The results are presented in Table 15.1, 15.2

and 15.3.

Table 15.1: TLC analysis of methanolic extract of Adhatoda vasica

Detecting Reagent

Distance Run by Solvent

Number of spots

Distance Run by

solute (cm) Rf Value Color spot

10.5 0.8750 Greenish Black 9.4 0.7833 Greenish Black 9 0.7500 Light Green

8.2 0.6833 Green

Iodine 12cm 5

7.4 0.6166 Brown Note: Solvent system Ethyl Acetate: Methanol (10:90)

The spot at RF value 0.6833 matches with that of the standard marker Vasicine was

collected and utilized for further HPLC and FTIR analyses.

Table 15.2: TLC analysis of chloroform extract of Adhatoda vasica

Detecting Reagent L Number

of spots Distance Run by solute (cm)

Rf Value Color spot

8 0.8000 Light Green 8.2 0.8200 Green 7.5 0.7500 Light Brown 7.2 0.7200 Dark Brown

Iodine 10cm 5

6.8 0.6800 Dark Brown

Note: Solvent system (ethyl acetate: methanol (10:90))

59  

None of the spots were matching with that of the standard marker Vasicine.

Table 15.3: TLC analysis of diethyl ether extract of Adhatoda vasica

Detecting Reagent

Distance Run by Solvent

Number of spots

Distance Run by solute

(cm)

Rf Value Color spot

10.2 0.9272 Light Green 9.6 0.8727 Light Green 9.2 0.8363 Dark Brown

8.4 0.7636 Brownish Orange

Iodine 11cm 5

8 0.7272 Yellow

Note: Solvent system: ethyl acetate: methanol (10:90)

None of the spots were matching with that of the standard marker Vasicine.

4.6. Pharmacological Evaluation of Plant Extracts

4.6.1 Immunomodulatory activity

Chloroform extracts of M. pudica, A. hexapetalus and A. vasica were found to be

modulating the innate immune response by increasing the neutrophil counts as

assayed by the neutrophil adhesion assay. The percentage of activity was found to

be 64.99, 54.12 and 84.30 for M. pudica, A. hexapetalus and A. vasica, respectively.

Methanolic extracts of all three plants were found to positively modulate the

immune response. Their values were found to be 78.73%, 71.80% and 94.72% for

M. pudica, A. hexapetalus and A. vasica, respectively. The diethyl ether extracts

were not found to positively influence the immune response, with values of 43.29%,

34.95% and 63.31% respectively (Figure 9).

Various extracts of Mimosa pudica, Artabotrys hexapetalus and Adhatoda vasica

administered at a dose of 200 and 400mg/kg/ per oral for 8 days significantly

inhibited the adhesion of neutrophils to nylon a fibre which stimulates the process of

margination of cells in the blood vessels. The methanol extracts of the plants to

produce maximum activity in the study. At the site of inflammation the chloroform

60  

and ether extracts of selected plant to reduced the number of neutrophils and thus

decreased their phagocytosis action and the release of various enzymes and

mediators that make inflammation (Figure 10).

Percentages of neutrophil adhesion observed were on 8th day after treatment with

both the plant extracts. The percentage increase in neutrophil adhesion after

Mimosa pudica, Artabotrys hexapetalus and Adhatoda vasica

methanolextractsshowed a dose dependent activity. The methanol extract of Mimosa

pudica, Artabotrys hexapetalus and Adhatoda vasica at a dose of 400mg/kg showed

the highly significant activity in the study (Figure 11).

Figure 9: Analysis of haemotological parameters in rats treated with the extracts of

MP, AH and AV

UB - Untreated blood; NFTB – Nylon fibre treated blood; MP- M. pudica; AH- A. hexapetalus; AV- A. vasica

61  

Figure 10: Effect of Leaf Extracts of MP, AH and AV onNeutrophil counts in rats

UB - Untreated blood; NFTB – Nylon fibre treated blood; MP- M. pudica; AH- A. hexapetalus; AV- A. vasica

MP- M. pudica AH- A. hexapetalus AV- A. vasica

Figure 11: Effect of Leaf Extracts of MP, AH and AV onNeutrophil Adhesion

62  

4.6.2 Delayed type hypersensitivity test

Delayed type hypersensitivity reaction has been widely used as one of the

parameters to measure cell-mediated immune response of the animal (Shwetha et

al., 2012). The reaction was measured by the percent increase in the paw volume

over the control animal. A significant percent increase in paw volume was observed

with diethyl ether extracts of all the three plants, the values being 23.80%, 24.20%

and 20.10% for M. pudica, A. hexapetalus and A.vasica respectively. A significantly

present in paw volume was observed with methanol extracts of all the three plants,

the values being 15.20%, 15.80% and 11.56% for M. pudica, A. hexapetalus and A.

vasica respectively. A significantly present in paw volume was observed with

chloroform extracts of all the three plants, the values being 19.40%, 19.60% and

15.60% for M. pudica, A. hexapetalus and A. vasica respectively (Table 16). These

results were comparable to that of the positive control (30.44%) (Figure12).

Increase in DTH reaction in rats in response to thymus-dependent antigen suggests

that the stimulatory effect of NAEE on T-lymphocytes and accessory cell types is

required for the expression of the reaction (Luster et al., 1982).

Table 16: Effect of MP, AH and AV extracts on delayed type hypersensitivity footpad thickness

S. No. Group (Treatment) Dose (mg/Kg p.o)

Paw volume at 24 hrs (%)

1. Negative control (normal saline) 10ml/Kg 10.22±3.68 2. Positive control

(PBS + Levamisole 50mg/kg) 10ml/Kg 30.44 ± 5.24

3. MPME 400 mg/kg 15.20 ± 4.38* (50.06)

4. MPCE 400 mg/kg 19.40 ± 4.34* (36.44)

5. MPDEEE 400 mg/kg 23.80 ± 4.30* (21.81)

6. AHME 400 mg/kg 15.80 ± 3.38* (48.09)

63  

7. AHCE 400 mg/kg 19.60 ± 3.26* (35.61)

8. AHDEEE 400 mg/kg 24.20 ± 3.12* (20.49)

9. AVME 400 mg/kg 11.56 ± 4.37* (62.02)

10. AVCE 400 mg/kg 15.60 ± 4.30* (48.75)

11. AVDEEE 400 mg/kg 20.10 ± 4.22* (33.96)

Results are expressed as mean ± SEM from five observations as compared to control group by students “t” test, n=5; *P<0.05; In brackets inhibition % is reported.

MP- M. pudica AH- A. hexapetalus AV- A. vasica

Figure 12: Effect of MP, AH and AV extracts on delayed type hypersensitivity

footpad thickness

4.7 Hepatoprotective Activity Screening

4.7.1 Acute toxicity studies

In acute toxicity study, it was found that the animals were safe up to a

maximumdose of 2000mg/kg body weight in rats, there were no changes in normal

behavioral pattern and no signs and symptoms of toxicity and mortality were

64  

observed and hence the extract was considered to be safe and non-toxic for further

pharmacological screening.

4.7.2 Hepatoprotective activity

The hepatoprotective ability of the plant extracts was assessed by their ability to

protect the liver from CCl4 injury. Four marker enzymes SGPT, SGOT, ALP and

TBL were used for assessing hepatoprotective ability. All three extracts of plants

tested (M. pudica, A. hexapetalus and A. vasica) were found to be protective against

CCl4 injury. The animals were found to be markedly recovering from CCl4 effect as

noted from the activity of the marker enzymes (Figure 13, 14 and 15). The enzyme

activity was found to decrease about one-half the injured liver and was almost

equivalent to control (Farooq et al., 1997).

Figure 13: Effect of M. pudica on CCl4 induced hepatotoxicity in rats

65  

Figure 14: Effect of A. hexapetalus on CCl4 induced hepatotoxicity in rats

Figure 15: Effect of A. vasica on CCl4 induced hepatotoxicity in rats

66  

4.8 Histopathological section of liver

Histopathological liver sections of the Control group showed normal cellular

architecture with distinct hepatic cells, sinusoidal spaces, and central vein.

Disarrangement of normal hepatic cells with necrosis and vacuolization were

observed in Carbon tetrachloride intoxicated liver. The liver sections of the rat

treated with 200mg/kg bodyweight p.o. of methanolic, chloroform and diethyl ether

extracts of the selected plant, followed by Carbon tetrachloride intoxication, showed

less vacuole formation and absence of necrosis. Overall the less visible changes

observed were comparable with the standard silymarin (Mitra et al., 1998) (Table

17) (Figure 16).

Table 17: Effect of MP, AH and AV extracts on carbon tetrachloride induced hepatotoxicity

Group Dose SGPT(U/L) SGOT(U/L) ALP(U/L) TBL(mg/dl)

I-Control 3ml/kg p.o. 127.16±5.12 106.40±2.60 200.80±5.80 1.18±0.6

II-Ccl4 0.5ml/kg i.p. 285.10±35.60a

244.30±38.20a

428.60±47.60a 2.98±0.38a

III-Silymarin

100mg/kg p.o

124.10±11.60b 104.60±6.60b 194.60±7.20b 1.38±0.32

b

IV-MPME 200mg/kg p.o

132.20±15.16b 119.80±9.30b 205.60±49.80

b 1.52±0.34b

V-MPCE 200mg/kg p.o 156.62±8.14b 141.62±16.2

0b 220.80±32.60b

1.68±0.12b

VI-MPDEE 200mg/kg p.o 174.14±6.22b 172.24±1.60b 250.62±16.20

b 1.86±2.40b

VII-AHME 200mg/kg p.o

140.12±12.40b 125.12±6.40b 240.26±10.12

b 1.69±0.62b

VIII-AHCE 200mg/kg p.o 156.28±6.12b 139.81±8.12b 268.62±9.18b 1.92±0.84

b IX-AHDEEE

200mg/kg p.o 178.28±6.12b 162.74±6.40b 299.14±7.62b 2.34±0.14

b

X-AVME 200mg/kg p.o

130.62±14.12b 114.62±8.14b 203.12±44.62

b 1.49±0.62b

67  

XI-AVCE 200mg/kg p.o

144.48±10.26b

132.62±14.40b

218.64±36.14b

1.62±0.24b

XII-AVDEEE

200mg/kg p.o 165.82±7.16b 158.46±2.62b 240.38±18.14

b 1.74±0.26b

Values are expressed as mean SEM; n=6 in each group, aP<0.01 Vs control group, bP<0.01 Vs CCl4 – treated group (ANOVA followed by Dunnet’s t- test).

a) b) C)

Control CCl4: central vein is damaged Standard

d) e) f)

MP-Methanolic extract: Central vein MP-Chloroform extract: Central vein and MP-Diethyl ether extract : and surrounding hepatocytes are seen. surrounding hepatocytes are seen without necrosis. There is no evidence of necrosis.

68  

g) h) i)

AH - methanolic extract : damaged AH-Chloroform extract : damaged AH-Diethyl ether extract :damaged hepatocytes are not seen hepatocytes are not seen hepatocytes andnecrosis is not found.

j) k) l)

AV- methanolic extract: radiating AV-chloroform extract: central vein AV-diethyl ether extract: Central vein with columns of hepatocytes are seen with radiating columns of hepatocytes radiating columns of hepatocytes are seen are seen Figure 16: Resolution of CCl4 induced toxicity in liver of rats by extracts of various

plants. Effect of MP, AH and AV extractson CCl4 induced hepatotoxicity in rat’s

enzymes.

4.9 Anti-ulcer activity

All the three plant-extracts exhibited anti-ulcer activity in all three models tested

(aspirin induced, alcohol induced and pylorus ligation). When treated with two

different concentrations viz. 100 mg/kg and 200 mg/kg, the methanolic extracts of

the plants exhibited a stronger anti-ulcer activity than other organic solvents. The

ulcer index was considerably reduced in animals treated with methanolic extracts

when compared to other solvents. The reduction in ulcer index was found in animals

69  

treated with methanol extracts of all three plants. The reduction in ulcer index was

statistically significant and comparable to that of the standard drug Ranitidine (20

mg/kg).

4.9.1 Aspirin induced ulcer

Aspirin-induced gastric ulceration in rats was another model used to study the effect

of extracts. The methanolic extract was found to possess remarkable ulcer-

protective properties at 100 and 200 mg/kg when compare to other two extracts. The

ulcer protection was found to be 70.46%, 57.84% and 46.46% at 200 mg/kg

respectively for methanolic, chloroform and diethyl ether extracts of M. pudica. The

ulcer protection was 66.15%, 54.76% and 47.07% (Figure 17c) at 200 mg/kg for

methanolic, chloroform and diethyl ether extracts of A. hexapetalus and 73.23%,

56.00% and 49.23% were produced at 200 mg/kg for methanolic, chloroform and

diethyl ether extracts of A. vasica; the standard drug, Ranitidine at 20 mg/kg gave

81.53% of ulcer protection (Table 18: Figure 18).

4.9.2Alcohol induced ulcer

The results obtained with the alcohol-inducedgastric ulceration model in rats were

comparable to that of the aspirin induced ulcer. Here also the methanolic extract was

found to exhibit good ulcer-protective properties at 100 and 200 mg/kg when

compare to other two extracts. The ulcer protection was found to be 69.53%,

58.76% and 47.38% at 200 mg/kg for methanolic, chloroform and diethyl ether

extracts of M. pudica; the protection was 67.07%, 56.61% and 48.30%(Figure 17d)

at 200 mg/kg for methanolic, chloroform and diethyl ether extracts of A.

hexapetalus and 74.46%, 57.84% and 49.84% were produced at 200 mg/kg for

methanolic, chloroform and diethyl ether extracts of A. vasica and the standard drug

(Ranitidine 20 mg/kg) gave 81.53% of ulcer protection. Pre-treatment of rats with

M. pudica extracts produced a dose dependent protection in the ethanol induced

70  

ulceration model as compared to control group (Table 18). However the protection

was statistically significant reduced the severity of ulcer and caused a significant

reduction of ulcer index in this model. Ranitidine produced significant gastric ulcer

protection as compared to control group (Figure 18).

4.9.3 Pylorus ligation induced ulcer

The results obtained in the experimental model of Pylorus ligation induced gastric

ulceration in rats. The methanolic extract was found to possess remarkable ulcer-

protective properties at 100 and 200 mg/kg when compare to other two extracts. The

methanolic extract was found to possess remarkable ulcer-protective properties at

200 mg/kg when compare to other two extracts. The maximum effect of ulcer

protection (70.10%), (58.69%) and (46.73%) were produced at 200 mg/kg for

methanolic, chloroform and diethyl ether extracts of M. pudica, (67.39%), (56.52%)

and (48.36%) (Figure 17e)were produced at 200 mg/kg for methanolic, chloroform

and diethyl ether extracts of A. hexapetalus and (73.91%), (57.06%) and (50.00%)

were produced at 200 mg/kg for methanolic, chloroform and diethyl ether extracts

of A. vasica and the standard drug (Ranitidine 20 mg/kg) gave 80.70% of ulcer

protection (Table 19; Figure 18).

The methanolic, chloroform and diethyl ether extracts of the M. pudica in the doses

of 200 mg/kg produced a reduction in the ulcer index, gastric volume, free acidity,

total acidity and raised gastric pH significantly in comparison with control group.

Ranitidine reference drug produced significant reduction of gastric ulcer and total

acid output as compared to control group. The present study indicates that the

methanolic extract of selected M. pudica significantly reduces the total volume of

gastric juice, free and total acidity of gastric secretion and also has activity against

gastric ulcers in rats when compare to other two extracts. The control animals had

71  

ulcers and haemorrhagic streaks. Where as in animals administered with the extracts

of M. pudica there was significant reduction in ulcer index.

Preliminary phytochemical screening revealed the presence of Alkaloids, Steroids,

polyphenolic constituents like flavonoids, Saponins, glycosides, tannins, gums and

mucilages. Acute toxicity studies of the various extracts of the M. pudica did not

exhibit any signs of toxicity up to 2 g/kg body weight. Since there was no mortality

of the animals found at high dose. Hence 100 and 200 mg/kg dose of the extract

selected for evaluation of anti-ulcer activity.

Table 18: Effect of various plant extracts on aspirin and alcohol induced gastric ulcer in rats

Aspirin Alcohol Treatment

Dose (mg/kg)

p.o. Ulcer Index

% of ulcer protection

Ulcer Index

% of ulcer protection

Control (Normal saline)

2ml/kg 6.5± 0.50 _ 6.5± 0.50 _

Standard (Ranitidine) 20mg/kg 1.20± 0.24 81.53 1.20± 0.24 81.53

100mg/kg 4.14±0.24 36.30 4.10±0.22 36.92 MPME 200mg/kg 1.92± 0.32 70.46∗∗∗ 1.98± 0.38 69.53∗∗∗ 100mg/kg 4.68±0.28 28.00 4.62±0.25 28.92 MPCE 200mg/kg 2.74± 0.36 57.84∗ 2.68± 0.33 58.76∗ 100mg/kg 4.86±0.29 25.23 4.82±0.26 25.84 MPDEEE 200mg/kg 3.48± 0.33 46.46 3.42± 0.29 47.38 100mg/kg 4.22±0.28 35.07 4.18±0.26 35.69 AHME 200mg/kg 2.20± 0.30 66.15∗∗ 2.14± 0.32 67.07∗∗ 100mg/kg 4.74±0.24 27.07 4.60±0.28 29.23 AHCE 200mg/kg 2.94± 0.34 54.76∗ 2.82± 0.30 56.61∗ 100mg/kg 5.14±0.22 20.92 5.06±0.22 22.15 AHDEEE 200mg/kg 3.44± 0.36 47.07 3.36± 0.32 48.30 100mg/kg 4.20±0.27 35.38 4.10±0.24 36.92 AVME 200mg/kg 1.74± 0.36 73.23∗∗∗ 1.66± 0.34 74.46∗∗∗

72  

100mg/kg 4.60±0.24 29.23 4.44±0.27 31.69 AVCE 200mg/kg 2.86± 0.34 56.00∗ 2.74± 0.31 57.84∗ 100mg/kg 4.92±0.26 24.30 4.82±0.28 25.84 AVDEEE 200mg/kg 3.30± 0.35 49.23 3.26± 0.26 49.84

Results are mean ± S.E.M. (n=5); statistical comparison was performed by using ANOVA coupled with student’s t-test.*P<0.05, **P<0.01, ***P<0.001 were consider statistically significant when compared to control group.

73  Table 19: Effect of plant extracts of MP, AH and AV against pylorus ligation induced gastric ulcer in rats

Treatment Dose (mg/kg) p.o

Volume of gastric juice (ml/4h) PH Free Acidity

(mEq/L) Total Acidity

(mEq/L) Ulcer Index %Inhibition of ulcer

Control (Normal saline) 2ml/kg 4.02± 0.11 1.84± 0.14 26.84±

0.08 70.16± 0.30 3.68± 0.56 _

Standard (Ranitidine) 20mg/kg 1.94± 0.06 4.96± 0.18 10.42± 0.02 22.24± 0.18 0.71± 0.14 80.70

100mg/kg 3.66± 0.16 3.12± 0.14 21.18± 0.05 52.14± 0.38 2.34± 0.24 36.41 MPME 200mg/kg 2.40± 0.14 4.56± 0.18 11.76± 0.06 30.62± 0.26 1.10± 0.29 70.10*** 100mg/kg 3.79± 0.16 3.20± 0.14 22.96± 0.08 60.48± 0.24 2.62± 0.36 28.80 MPCE 200mg/kg 3.28± 0.21 3.88± 0.16 13.68± 0.02 35.45± 0.33 1.52± 0.44 58.69* 100mg/kg 3.86± 0.14 2.86± 0.14 25.54± 0.04 64.16± 0.19 2.76± 0.49 25.00 MPDEEE 200mg/kg 3.68± 0.12 3.24± 0.16 16.62± 0.06 39.52± 0.32 1.96± 0.56 46.73 100mg/kg 3.66± 0.12 3.12± 0.14 20.14± 0.02 54.62± 0.68 2.42± 0.24 34.23 AHME 200mg/kg 2.40± 0.14 4.48± 0.12 11.64± 0.03 33.62± 0.42 1.20± 0.28 67.39∗∗∗ 100mg/kg 3.78± 0.16 3.22± 0.18 18.46± 0.05 64.96± 0.62 2.66± 0.44 27.71 AHCE 200mg/kg 3.14± 0.12 3.82± 0.14 14.56± 0.03 39.76± 0.46 1.60± 0.48 56.52∗∗ 100mg/kg 3.98± 0.15 2.80± 0.18 23.86± 0.08 69.12± 0.12 2.88± 0.52 21.73 AHDEEE 200mg/kg 3.67± 0.16 3.20± 0.12 18.42± 0.02 43.42± 0.42 1.90± 0.56 48.36 100mg/kg 3.62± 0.14 3.14± 0.15 22.16± 0.03 56.34± 3.16 2.32± 0.20 36.95 AVME 200mg/kg 2.42± 0.18 4.52± 0.14 12.86± 0.04 32.46± 0.20 0.96± 0.22 73.91∗∗∗ 100mg/kg 3.74± 0.18 3.18± 0.16 20.84± 0.04 62.84± 0.42 2.50± 0.42 32.06 AVCE 200mg/kg 3.16± 0.16 3.86± 0.12 15.46± 0.02 38.54± 0.32 1.58± 0.46 57.06∗∗ 100mg/kg 3.92± 0.17 2.84± 0.16 24.84± 0.06 68.64± 0.39 2.84± 0.54 23.36 AVDEEE 200mg/kg 3.64± 0.14 3.18± 0.10 18.86± 0.02 42.58± 0.34 1.84± 0.52 50.00∗

Results are mean ± S.E.M.(n =5). Statistical comparison was performed by using ANOVA coupled with student’s t-test.* P<0.05, ** P<0.01, *** P<0.001 were consider statistically significant when compared to control group.

74  

a) b)

Control Standard

 

c) d) e)

AH-aspirin induced AH-alcohol induced AH-pyloric ligation induced gastric ulcer in rat gastric ulcer in rat gastric ulcer in rat Figure 17: Effect of various extracts of A. hexapetalus against aspirin, alcohol and

pylorus ligation induced gastric ulcer in rats (Ulcer in rats was induced as discussed

earlier; the animals were sacrificed on 8th day and the intestine was photographed)

 

75  

MP- M. pudica AH- A. hexapetalus AV- A. vasica

Figure 18: Effect of various extracts of MP, AH and AV against Anti-ulcer activity in

rats

4.10 Wound healing Activity

All three plant extracts exhibited wound healing activity. A lower wound healing

ability was observed with the diethyl extract whereas the activity was higher in

methanolic extracts. The methanolic extract of M. pudica exhibited a higher activity of

93.87% whereas the methanolic extracts of A. hexapetalus and A. vasica exhibited

78.61% and 87.46% respectively.

4.10.1 M. pudica (MP)

The wound healing activity was studied by using five groups; Group I negative control

simple ointment, In Group II positive control Nitrofurazone ointment (0.2% w/w),

Group III MPME, Group IV MPCE and Group V MPDEE. The size of the wound in

 

76  

surface area, On the Day 1 (50.24) (50.36) (51.26) (50.54) (50.42). On the Day 4

(48.24) (28.26) (38.46) (38.36) (48.46). On the Day 8 (44.20) (12.56) (28.26) (30.26)

(40.32). On the Day 12 (40.46) (3.14) (12.56) (20.54) (36.16). On the Day 16 (35.46)

(0.758) (3.14) (18.76) (20.45). The mean percentage closure of excision wound model

on Day 16 (40.45) (98.44) (93.87) (80.72) (70.76) (Table 20; Figure 19 and 22).

4.10.2 A. hexapetalus(AH)

The wound healing activity was studied by using five groups; Group I negative control

base ointment, In Group II positive control Nitrofurazone ointment (0.2% w/w), Group

III AHME, Group IV AHCE and Group V AHDEE. The size of the wound was

determined by measuring the surface area. The area of wound on Day 1 was found to

be 48 to 50.24sq.mm. After 16 days, the size of the wound, in animals treated with A.

vasica extracts, were found to be reduced to 6.42 sq.mm, 9.80 sq.mm and 15.60 sq.mm

for methanol, chloroform and diethyl ether extracts (Table 20; Figure 20 and 22).

Contraction of the excision wound was observed from Day 4 and it progressed till Day

16. The percent wound contraction after 16 days was found to be 98.44, 78.61, 70.82

and 65.46 respectively in nitrofurazone, methanol, chloroform and diethyl ether extract

treated groups. Significant wound contraction was observed on 16th day for all treated

groups (p<0.001 for standard and methanolic extracts; p<0.01 for chloroform and

diethyl ether extracts), in comparison with the control group. Time for complete

epithelization was significantly short in drug and standard treated groups.

The epithelization of wound in case of rat treated with extracts was found to be quite

earlier than control. It is also comparable with the marketed preparation. It suggests

that the leaves extracts of Artabotrys hexapetalus promoted wound healing activity.

The excision wound model showed excellent wound healing property in methanolic

leaf extract which was well compared with standard drug.

 

77  

4.10.3 A. vasica(AV)

The wound healing activity was studied by using five groups; Group I negative control

simple ointment, In Group II positive control Nitrofurazone ointment (0.2% w/w),

Group III AVME, Group IV AVCE and Group V AVDEE. The size of the wound in

surface area, On the Day 1 (50.24) (50.36) (51.16) (50.62) (49.84). On the Day 4

(48.24) (28.26) (38.14) (36.90) (37.10). On the Day 8 (44.20) (12.56) (27.84) (27.52)

(29.34). On the Day 12 (40.46) (3.14) (16.12) (18.42) (21.64). On the Day 16 (35.46)

(0.758) (6.42) (9.80) (15.60). The mean percentage closure of excision wound model

on Day 16 (40.45) (98.44) (87.46) (80.65) (68.70) (Table 20; Figure 21 and 22).

Contraction of the excision wound was promoted from day 1 to day 16. In excision

wounds, wound contraction was 98.44, 87.46, 80.65 and 68.70% respectively on 16th

day for nitrofurazone, methanolic, chloroform and diethyl ether extract treated groups.

Significant wound contraction was also observed on 16th day for all treated groups

(p<0.001 for standard and methanolic extracts; p<0.01for chloroform and diethyl ether

extract), in comparison with the control group. Time for complete epithelization was

significantly short in drug and standard treated groups.

The epithelization of wound in case of rat treated with extracts was found to be quite

earlier than control. It is also comparable with the marketed preparation. It suggests

that the leaves extracts of Adhatoda vasica promoted woundhealing activity. The

excision wound model showed excellent wound healing property in methanolic leaf

extract which was well compared with standard drug.

 

78  

Table 20: Effect of methanolic, chloroform and diethyl ether extract ointments of MP, AH and AV on excision wound model

Size of wound surface area (mm 2) Group

Avg. wt of

animal Drug /extract Day

0 Day

1 Day

4 Day

8 Day 12

Day 16

% wound healing

I Control 50.24 50.24 48.24 44.20 40.46 35.46 40.45

II

Nitrofurazone ointment (0.2% w/w)

50.36 50.36 28.26 12.56 3.14 0.758 98.44

III

MPME (10%w/w) 51.26 51.26 38.46 28.26 12.56 3.14 93.87

**

IV MPCE(10%w/w) 50.54 50.54 28.36 30.26 20.54 18.76 80.72 **

V

150-200 gm

MPDEEE (10%w/w) 50.42 50.42 48.46 40.32 36.16 20.45 70.76**

VI AHME (10%w/w) 48.60 48.60 36.20 27.12 18.14 10.40 78.61**

VII AHCE (10%w/w) 49.20 49.20 35.22 28.84 21.20 14.36 70.82**

VIII AHDEEE (10%w/w) 50.42 50.42 38.96 32.14 24.20 17.42 65.46*

IX AVME (10%w/w) 51.16 51.16 38.14 27.84 16.12 6.42 87.46**

X AVCE (10%w/w) 50.62 50.62 36.90 27.52 18.42 9.80 80.65**

XI

150-200 gm

AVDEEE 49.84 49.84 37.10 29.34 21.64 15.60 68.70*

Values are mean ± SEM of 5 animals in each group. Numbers in Parenthesis indicate

percentage of wound contraction. * P<0.01, **P<0.001Vs respective control by

students t- test.

 

79  

a) b)

Control 0 day Standard 0 day

c) d)

Control 16 day Standard 16 day

e) f) g)

Mimosa pudica Mimosa pudica Mimosa pudica (Diethyl ether Extract10%W/W) (Chloroform Extract10 %W/W) (Methanol Extract 10%W/W)

Figure 19: Effect of methanolic, chloroform and diethyl ether extract ointments of

M.pudica on excision wound model.

 

80  

a) b) c)

        Artabotrys hexapetalus Artabotrys hexapetalus Artabotrys hexapetalus (Diethyl ether Extract10%W/W) (Chloroform Extract10 %W/W) (Methanol Extract 10%W/W)

Figure 20: Effect of methanolic, chloroform and diethyl ether extract ointments of

Artabotrys hexapetalus on excision wound model.

a) b) c)

          Adhatoda vasica Adhatoda vasica Adhatoda vasica (Diethyl ether Extract10%W/W) (Chloroform Extract10 %W/W) (Methanol Extract 10%W/W) Figure 21: Effect of methanolic, chloroform and diethyl ether extract ointments of

Adhatoda vasica on excision wound model.

 

81  

MP- Mimosa pudica AH- Artabotrys hexapetalus AV- Adhatoda vasica

Figure 22: Effect of methanolic, chloroform and diethyl ether extract ointments of MP,

AH and AV on excision wound model.

4.11. Wound Healing Activity of Fractions of Extracts

Among the different fractions of M. pudica extract that were tested (aqueous, Hexane,

n-butanol, chloroform and ethyl acetate fractions) for wound healing activity the n-

butanol fraction exhibited a higher wound healing activity (71.70%); this was

comparable to that of the standard nitrofurazone ointment (0.2% w/w).

Contraction of the excision wound was promoted from day 1 of the treatment and

healing was observed on day 16. The epithelization of wound in case of rat treated with

fraction of extracts was found to be quite earlier than control. It is also comparable with

the commercial product available in the market. It suggests that the leaves extracts of

M. pudica promoted wound healing activity. The excision wound model showed

 

82  

excellent wound healing property in methanol leaf extract which was well compared

with standard drug.

The results are given control (31.24%), standard (89.94%), hexane fraction (51.36%)

(Figure 23c), aqueous fraction-(67.56%) (Figure 23d), Ethyl acetate fraction-(61.81%)

(Figure 23e), chloroform fraction (57.52%) (Figure 23f) and n-butanol fraction

(71.70%) (Figure 23g). Overall n-butanol and aqueous fractions showed higher wound

healing activity compared to other fractions (Table 21; Figure 24).

Fractions and their wound healing activity:

1. Aqueous fraction

2. Hexane fraction

3. Chloroform fraction

4. N-butanol fraction

5. Ethyl acetate fraction

Table 21: Effect of methanolic extract ointments of Mimosa pudica fractions and their wound healing activityon excision wound model

Size of wound surface area (mm2) Group

Avg. wt of

animal

Drug /extract Day

0 Day

1 Day

4 Day

8 Day 12

Day 16

Percentage of wound healing

I Control 51.48 51.48 48.10 43.12 40.56 35.40 31.24

II

Nitrofurazone ointment

(0.2% w/w) 51.66 51.66 40.62 32.10 17.84 5.20 89.94

III

150-200 gm.

Aqueous fraction

(10%w/w) 50.80 50.80 45.40 38.64 30.60 16.48 67.56

 

83  

IV

Hexane fraction

(10%w/w) 48.64 48.64 45.24 39.84 34.30 23.66 51.36

V

Chloroform fraction

(10%w/w) 49.10 49.10 44.20 37.64 28.94 20.86 57.52

VI N-butanol fraction

(10%w/w) 50.38 50.38 44.20 36.46 29.82 14.26 71.70

VII Ethyl acetate

fraction (10%w/w)

50.22 50.22 45.24 37.94 30.24 19.18 61.81

Values are mean ± SEM. Statistical comparison was performed by using ANOVA and

student’s t- test. Numbers in Parenthesis indicate percentage of wound contraction.

P<0.001 respective control.

 

84  

a) b)

Control Standard c) d) e)

Hexane fraction Aqueous fraction Ethyl acetate fraction f) g)

Chloroform fraction n-Butanol fraction Figure 23: Effect of methanolic extract ointments of Mimosa pudica fractions and their

wound healing activityon excision wound model.

 

85  

Figure 24: Effect of methanolic extract ointments of Mimosa pudica fractions and their

wound healing activity on excision wound model.

4.12 HPLC analysis

4.12.1 HPLC analysis of methanolic extract of Mimosa pudica

HPLC analysis of partially purified methanolic extract of Mimosa pudica presented a

distinct peak at a retention time of 9.141 which is similar to standard mimopudine

(retention time 9.439). Few more peaks with varying retention times were also

observed in this fraction.

 

86  

AU

0.00

0.10

0.20

0.30

0.40

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

9.43

9

Figure 25.1: HPLC profile of Mimopudine standard.

AU

0.00

1.00

2.00

3.00

4.00

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00

9.14

1

Figure 25.2: HPLC profile of methanolic extract of Mimosa pudica. A peak similar to

Mimopudine with a retention time of 9.14 was observed.

 

87  

4.12.2 HPLC analysis of methanolic extract of Artabotrys hexapetalus

HPLC analysis of partially purified methanolic extract of A. hexapetalus gave a peak

with a retention time of 11.304 which is similar to the artabotrycinol standard

(retention time 10.788). Few more peaks with varying retention times were also

observed in this fraction.

0.0 2.5 5.0 7.5 10.0 12.5 min

-0.25

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

mAU(x10)254nm,4nm (1.00)

/8.539/5127

Artabotrycinol/10.788/532607

Figure 26.1: HPLC profile of standard Artabotrycinol

0.0 2.5 5.0 7.5 10.0 12.5 min

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

mAU(x1,000)254nm4nm (1.00)

/3.428/15987

/3.853/21710

/4.134/10800

/4.501/6361

/4.770/5704

/5.223/4007

/5.653/22886942

Artaboteycinol/11.304/3571572

Figure 26.2: HPLC profile of methanolic extract of Artabotrys hexapetalus. A peak

similar to Artabotrycinol with a retention time of 11.304 was observed.

 

88  

4.12.3 HPLC analysis of methanolic extract of Adhatoda vasica

HPLC analysis of partially purified methanolic extract of A. vasica gave a peak with a

retention time of 6.433 which is similar to the vasicine standard (retention time 6.22).

Few more peaks with varying retention times were also observed in this fraction.

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min

-1.0

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

mAU(x10)302nm,4nm (1.00)

vasicine/6.266/1125785

Figure 27.1: HPLC profile of standard Vasicine

0.0 2.5 5.0 7.5 10.0 min

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

mAU302nm4nm (1.00)

vasicine/6.443/8879

Figure 27.2: HPLC profile of methanolic extract of Adhatoda vasica. A peak similar to

Vasicine with a retention time of 6.433 was observed.

 

89  

4.13 FTIR SPECTRA OF COMPOUNDS

4.13.1 FTIR Spectral Analysis

The structure of isolated compound was elucidated by Shimadzu – 8400 Series Fourier

Transformer - Infrared Spectrophotometer using KBr pallet method. IR results are

shown below:

4.13.2 IR Spectral Studies

FT-IR spectra of the methanolic extracts of Mimosa pudica. Artabotrys hexapetalus

and Adhatoda vasica confirm the existence of mimopudine, artabotrycinol and vasicine

respectively. Interestingly, the characteristic stretching frequencies of the functional

group (الO-H, الN-H, الC-H, الC-N, الC=O, الC=C), presentin the IR spectra of the methanolic

extracts match with the stretching frequencies obtained for the commercial samples. A

few characteristics peaks were shifted by 1-2cm-1 wave number, which may be due to

the presence of other organic moieties as impurities and solvent effects. The absorption

by the impurities is masked by the strong signals associated with mimopudine,

artabotrycinol and vasicine molecules. These trends confirm the presence of

mimopudine, artabotrycinol and vasicine of the plants (Figure 28-33).

 

90  

Figure 28: FTIR analysis of standard Mimopudine: The absorption frequencies in the

IR spectrum of commercial mimopudine such as γc=0 (conjugated ketone), γN-H

(stretching), γN-H (bending) and γO-H (bending) are identified correctly.

 

91  

Figure 29: FTIR Analysis of Methanolic Extract of Mimosa pudica: A characteristic

peak for the conjugated ketonic group (C=O) is observed at 1683cm-1. The N-H

stretching and N-H bending peaks are observed at 3416 and 1595cm-1 respectively.

TheγO-H (bending) frequencies appear in the range 1350-1250cm-1

 

92  

Figure 30: FTIR analysis of standard Artabotrycinol: The group frequencies for H-

bonded OH stretching, in-plane bending OH, alkenyl (-C=C-) stretching and C-H

stretching are identified correctly from the IR spectrum of commercial Artabotrycinol.

 

93  

Figure 31: FTIR Analysis of Methanolic Extract of A. hexapetalus: A hydrogen

bonded broad band due to OH groups is observed in the range of 3570-3200cm-1.

Intrestingly γO-H frequency portions to -CH2OH group shows a strong band at

1290cm-1 due to in-plane bending vibrations. A characteristic alkenyl (-C=C-)

stretching frequency is observed at 1643cm-1.

 

94  

Figure 32: FTIR analysis of standard Vasicine: The ring frequencies associated with

the aromatic system and group frequencies of imino group (C=N-) and hydroxyl group

are observed correctly from the IR spectrum of commercial Vasicine.

 

95  

Figure 33: FTIR Analysis of Methanolic Extract of Adhatoda vasica: Prominent and

strong band is observed at 1641cm-1 for imino group (C=N-). A broad band is observed

in the range of 3550-3250cm-1 due to inter molecular association related with hydroxy

groups.

 

96  

4.14 Microbiological Analysis

4.14.1 Antibacterial activity

The increasing failure of existing chemotherapeutic agents and the rise in antibiotic

resistance of pathogenic microorganisms have led to the screening of newer anti-

microbial agents; several medicinal plants are being explored for their potential

antimicrobial activity (Scazzocchio et al., 2001). All three methanoic extracts were

found to exhibit a very strong broad spectrum antibacterial activity. The effect was

found to be pronounced against Gram-positive bacteria (Micrococcus luteus,

Staphylococcus aureus and Bacilluc cerus) than against Gram-negative bacteria

(Klebsiella pneumonieae, Salmonella typhimurium and Salmonella paratyphimurium)

(Table 22; Figure 34).

Antimicrobial screening

B –20 % DMF used as blank; Standard: S1 – Ciprofloxacin 0.1mg/ml;

S2 – Ciprofloxacin 1mg/ml; Anti-bacterial Activity of Plant Extracts

Test Organisms: Gram-positive bacteria: Micrococcus luteus, Staphylococcus aureus and Bacilluc cerus

Gram-negativebacteria: Klebsiella pneumonieae, Salmonella typhimurium and

Salmonella paratyphimurium.

Table 22: Evaluation of Antimicrobial activities of Plant Extracts

Zone of clearance in diameter (mm) Organism used

A A1 A2 B B1 B2 C C1 C2

Bacillus cereus 26 25 23 22 23 24 23 23 27

Klesiella pneumoniae 22 19 17 19 17 19 19 15 19

 

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(109)

Salmonella typhimurium B 19 18 18 16 15 17 14 17 17

Salmonella paratyphimurium 23 23 23 19 23 24 23 18 20

Micrococcus luteus 38 38 34 30 35 38 32 33 36

Staphylococcus aureus 28 25 21 24 23 25 24 22 25

Salmonella typhimurium (1251) 25 22 19 18 18 20 18 18 19

A, A1, A2= Methanolic extract of AH, MP, AV; B, B1, B2= Chloroform extract of

AH, MP, AV; C, C1, C2= Diethyl ether extract of AH, MP, AV

AH- Artabotrys hexapetalus MP- Mimosa pudica AV- Adhatoda vasica

 

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Figure 34: Anti bacterial activity of plant extracts treated with Staphylococcus aureus a. A, A1, A2= Methanolic extract of AH, MP, AV; AH- Artabotrys hexapetalus; MP- Mimosa pudica; AV- Adhatoda vasica. b. B, B1, B2 = Chloroform extract of AH, MP, AV; AH- Artabotrys hexapetalus; MP- Mimosa pudica; AV- Adhatoda vasica. c. C, C1, C2= Diethyl ether extract; C, C1, C2= Diethyl ether extract

a. b.

   

Control CHO-Chloroform DEE-Diethyl ether MET-Methanol DMF-Dimethyl formamide

A, A1, A2= Methanolic extract of AH, MP, AV. AH- Artabotrys hexapetalus; MP- Mimosa pudica; AV- Adhatoda vasica

c. d.

B, B1, B2 = Chloroform Extract of AH, MP, AV. AH- Artabotrys hexapetalus; MP- Mimosa pudica; AV- Adhatoda vasica

C, C1, C2= Diethyl ether Extract; C, C1, C2= Diethyl ether Extract of AH, MP, AV. AH- Artabotrys hexapetalus; MP- Mimosa pudica; AV- Adhatoda vasica

 

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of AH, MP, AV. d. Control CHO-Chloroform DEE-Diethyl ether MET-Methanol DMF-Dimethyl formamide. AH-Artabotrys hexapetalus; MP- Mimosa pudica; AV- Adhatoda vasica. 4.15 ANTI-OXIDANT ACTIVITY OF MP, AH AND AV

Crude extracts and TLC- purified methanolic extracts of all three plants exhibited a

potent anti-oxidant activity as measured by Potassium ferricyanide, FRAP and DPPH

assays. The activities of all three plants were comparable in all three assays.

4.15.1 Potassium ferricyanide assay

All the three plant extracts (crude and purified) were tested for potassium ferricyanide

reduction. Among the crude extracts of all the three plants, AV methanolic extracts

shown the higher reduction compare with other two plants methanolic extracts. Further

the TLC purified methanolic samples of all three palnts were tested again. In the

purified form sample A1 gives maximal reduction of potassium ferricyanide (Figure

35.1 and 35.2).

 

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Figure 35.1: The reduction of potasium ferricyanide, sample A2 gives more reduction

compared with other crude samples. Sample A and A1 gives followed level of the

reduction respectively.

Figure 35.2: TLC purified samples were tested potasium ferricyanide reduction,

sample A1 gives the maximal reduction when compared with crude samples.

A –AH Methanolic extract, A1-MP Methanolic extract, A2-AV Methanolic extract, B-

AH Chloroform extract, B1-MP Chloroform extract, B2-AV Chloroform extract, C-AH

Diethyl ether extract, C1-MP Diethyl ether extract, C2-AV Diethyl ether extract

AH- Artabotrys hexapetalus MP- Mimosa pudica AV- Adhatoda vasica

4.15.2 Fluorescence Recovery after Photobleaching (FRAP) Assay

All the three plants extracts of crude and purified were tested for the ferric ion

reduction. In the crude extracts of all the three plants, AV methanolic extracts shown

the higher reduction compare with other two plants methanolic extracts. Further,

 

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purified AV methanolic samples were tested again. In the purified form sample AV-A2

gives maximal reduction of potassium ferricyanide (Figure 36.1 and 36.2).

Figure 36.1: Crude extracts of all the three plants samples were tested forferric ion

reduction. The AV-A2 samples reduce the maximal level and indicate that higher anti-

oxidative property compared with other samples.

Figure 36.2: The purified AV-A2 samples were tested again forferric ion reduction,

The AV-A2 extracts shown the maximal reduction and anti-oxidant activity. A –AH

Methanolic extract,A1-MP Methanolic extract, A2-AV Methanolic extract, B-AH

Chloroform extract, B1-MP Chloroform extract, B2-AV Chloroform extract, C-AH

Diethyl ether extract, C1-MP Diethyl ether extract, C2-AV Diethyl ether extract.

 

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AH- Artabotrys hexapetalus MP- Mimosa pudica AV- Adhatoda vasica

4.15.3 DPPH

The stock solution of plant extracts at a concentration of 100µl/ml of all the extracts

was assayed for antioxidant activity. The maximum inhibition for DPPH radical was

A2 (5.80) for followed by A1 (5.10), A (5.04), B1 (3.04), C (2.90), B (2.66), B2 (2.28),

C1 (2.19) and C2 (2.19). The reducing potential of 2.0, 1.1, 1.04, and 1.02 was

observed in sample B1, B, C, B2, C1 and C2, respectively. The A2-AV methanolic

extracts shows the maximal reduction in crude and purified state (Figure 37.1 and

37.2).

Figure 37.1: The observation of ferric ion reduction, sample A, A1 and A2 shows the

maximal level of absorbance indicates the anti-oxidant activity compare with other

extracts.

 

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Figure 37.2: Purified A, A1 and A2 samples were shown the maximum absorbance

level indicates that the anti-oxidant activity compared with crude extracts.

A –AH Methanolic extract, A1-MP Methanolic extract, A2-AV Methanolic extract, B-

AH Chloroform extract, B1-MP Chloroform extract, B2-AV Chloroform extract, C-AH

Diethyl ether extract, C1-MP Diethyl ether extract, C2-AV Diethyl ether extract

AH- Artabotrys hexapetalus MP- Mimosa pudica AV- Adhatoda vasica

 

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5. DISCUSSION

Natural products have been the source of most of the active ingredients that are used in

modern medicine (Harvey, 2008). More than 80% of the substances used to make drugs

are natural products or inspired by natural compounds (Sneader, 1996) and half of the

drugs approved since 1994 are based on natural products (Newman and Cragg, 2007;

Butler, 2008). Natural products and /or compounds derived from natural products

continue to play a major role in drug development process (Newman et al., 2003). The

natural products include compounds from plants, microbes and animals and synthetic

or semi-synthetic compounds based on natural drugs. They have been used to treat a

variety of disease conditions such as cancer, infections, diabetic and other metabolic

diseases.

In the present study, leaf extracts of three plants viz. M. pudia, A. hexapetalus and A.

vasica were evaluated for pharmacological activity in animal models.

5.1 Phytochemical studies

The preliminary phytochemical analysis of all three extracts (viz. methanol, chloroform

and diethyl ether) of M. pudica, A. hexapetalus and A. vasica indicate the presence of

alkaloids, flavanoids, tannins, saponins, glycosides, steroids, steroidal terpenes,

phenolic compounds, gums and muciages and carbohydrates.

5.2 Evaluation of Immunomodulatory Activity (neutrophil adhesion assay)

The immunomodulatory agents obtained from plant and animal origin, activate and

enhance the immune response of the host against the invading pathogens (Desai et al.,

1966). Inflammation is one of the conditions where in neutrophils, a part of innate

immunity, play a major role. The present study reveals the immunomodulatory activity

 

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of methanolic extracts of the selected plant extracts. Many plant extracts are known to

exhibit anti-inflammatory activities. However, more studies have to be performed to

understand the underlying mechanism of these medicinal plants regard to their

therapeutic activities (Fulzele et al., 2003).

The methanol extracts of all three plants, when administered orally, exhibited

significant immune modulatory activity as measured by two assays: an increase in

adhesion of neutrophils to nylon fibers which correlates to the process of margination

of cells in blood vessels. The neutrophil adhesion was found to be significantly

increased in animals fed with the test extracts compared to untreated control.

Neutrophils circulate in the vasculature in a passive state and become more adhesive

upon stimulation at sites of inflammation; this would be followed by margination to the

vessel wall and subsequent transmigration and phagocytosis. The plant extracts

significantly increased the neutrophil chemotactic movement as indicated by the

increase in number of cells reaching the lower surface of the filter; therefore, the

extracts act as chemo attractants. These results are in comparison to that of a compound

isolated from R. communis leaf extract which significantly increased the neutrophil

chemotactic movement (Konig et al., 1987). Natural products derived antibiotics like

azithromycin are known to exhibit anti-inflammtory activities by suppressing the

abundance of neutrophils (Ivetic Tkalcevic et al., 2006). Another natural product

derived drug telithromycin reduces inflammation through reduction in cytokine

production (Lotter et al., 2006).

At the time of pathogens invasion, macrophages detect the invasion in the tissues and

recruit neutrophils to the affected site from the reserve pool in order to assist them in

eliminating the invaders (Hoffstein et al., 1981). Therefore, macrophages are supported

by neutrophils, which supply the effective antimicrobial neutrophil granules. This

 

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would enhance the capacity of the macrophages which do not have these granules in

them (Silva et al., 1989).

5.2.1 Delayed type hypersensitivity activity

Delayed type hypersensitivity reaction is a type IV hypersensitivity reaction

characterized by large influxes of non-specific inflammatory cells. Generally these

cells are of TH1 subpopulation although rarely cytotoxic T-cells (TC) are also involved.

Activation of TDTHcells results in the secretion of various cytokines that includes

interleukin-2, interferon-γ, macrophage migration inhibition factor and tumor necrosis

factor-β (Askenase and Van Loveren, 1983). These cytokines recruit macrophages into

the area of inflammation and activate them. There are several evidences to postulate

that DTH is an important phenomenon to eliminate the parasites and bacteria which can

survive and grow intracellularly.

In the present study, it was observed that the diethyl ether extracts of all three plants

exhibited a strong T-cell immune response as measured by DTH. The interaction of

activated T-cells with the presented antigen is associated with the release of soluble

mediators like histamine which are products of arachidonic acid metabolism (Griswold

et al., 1987) and eventually interferon-gamma leading to DTH. Therefore, the

inhibitory action could be due to an influence of fraction on the biological mediators.

Prasad et al., (2006) reported the immunomodulatory activity of Momordica charantia

extracts using delayed type-hypersensitivity assay on rats. The authors observed a

significant increase in paw volume in animals administrated with the drug at a

concentration of 350 mg/kg/day. Similar observations were made by others in animals

administered with herbal drugs (Fulzel et al., 2003; Hafeez et al., 2003; Pradhan et al.,

2009). Dashputre and Naikwade (2010), while studying Abutilon indicum, attributed

 

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these activities to the presence of flavonoids (quercetin), alkaloids, tannins, saponin,

glycosides and phenolic compounds.

Treatment of ether extract of selected plant enhanced DTH reaction, which is reflected

from the increased footpad thickness compared to control group suggesting heightened

infiltration of macrophages to the inflammatory site. This study may be supporting a

possible role of ether extract ofselected plantin assisting cell-mediated immune

response. T-sesquiterpene lactones, a group of compounds isolated from the Tridax,

have been reported to induce delayed type hypersensitivity (Picman, 1986). So it might

be possible that these sesquiterpene lactones may be present in the ether extract of the

plant extracts that was tested.

5.3 Hepatoprotective activity

Liver plays a major role in the detoxification and excretion of many endogenous and

exogenous compounds and any injury or impairment of its function may lead to several

complications. Management of liver diseases is still a major challenge to modern

medicine. Conventional drugs used in the treatment of liver diseases are often

inadequate. It is therefore necessary to search for alternative drugs for the treatment of

liver diseases that could replace the existing drugs.

Liver damage induced by CCl4 is a commonly used model for the screening of

hepatoprotective drugs. The hepatic cytochrome P-450 converts CCl4 into a reactive

halogenated free-radical which covalently binds with the membranes (both cell and

organelle) resulting in lipid peroxidation and tissue injury (Recknage et al., 1989). The

prophylactic administration of plant extracts offered significant protection against

CCl4-induced liver injury, as evident by the reduction in serum levels of liver enzymes,

 

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SGPT, SGOT, ALP, and total bilirubin in rats. All three plant extracts (M. pudica, A.

hexapetalus and A. vasica) were found to be protective against CCl4 injury. The

animals were found to be markedly recovering from CCl4 effect as noted from the

activity of the marker enzymes. Incidentally, unlike immunomodulation, the ether

extracts of all three plants exhibited a higher hepato-protective activity compared to the

other extracts. This protective effect could possibly be due to the presence of tannins

and flavonoids (Brattin et al., 1985). The changes associated with CCl4 induced liver

damage of the present study appeared similar to the acute viral hepatitis (Venukaumar

and Latha, 2002).

Animals received CCl4 significantly lost their body weight and showed reduced food

consumption as compared to control group. Whereas animals of other groups treated

with CCl4 and test extracts / standard drug Silymarin showed a significant increase in

body weight and food consumption when compared to CCl4 administrated group

animals. These findings suggest that the extracts administered has significantly

neutralized the toxic effects of CCl4 resulting in the regeneration of hepatocytes

(Farooq et al., 1997).

Estimating the activities of serum marker enzymes, such as SGPT, SGOT, ALP can

make the assessment of liver function when liver cell plasma membrane is damaged, a

variety of enzyme normally located in the cytosol are released into the blood stream.

Estimation of these factors is now used as markers to determine the extent and type of

liver damage (Mitra et al., 1998). The tendency of these enzymes to return to near

normal level in extract administered group is a clear manifestation of anti-hepatotoxic

effects.

 

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Reduction in the levels of SGPT and SGOT towards the normal value is an indication

of regeneration process. Reduction in ALP levels with concurrent depletion of raised

bilirubin levels suggests that the stability of the biliary function during injury with

CCl4. This hepato protective effect exhibited by the methanolic, chloroform and diethyl

ether extracts of selected plant at the dose level of 200mg/kg body weight was

comparable with the standard drug, Silymarin.

The methanolic extract of aerial parts of Plumbago zeylanica administered

prophylactically exhibited significant protection against CCl4-induced liver injury as

manifested by the reduction in toxin mediated increase in serum level of SGPT, SGOT,

ALP and total bilirubin in rats. The phytochemical analyses of the plant extracts

indicated the presence of carbohydrate, terpenes, steroids, tannins and flavonoid which

could play a role in protection against liver damage (Brattin et al., 1985). The ether

extracts of plants found to have significant hepatoprotective activity. This may

probably due to the higher content of the terpenes, tannins and flavonoids.

The potential usefulness of the plant extracts in clinical conditions associated with liver

damage is still need to be demonstrated. Further investigations on the isolation of the

active principle responsible for hepatoprotective activity are needed.

5.3.1 Histopathological section of liver

Histopathological liver sections of the Control group showed normal cellular

architecture with distinct hepatic cells, sinusoidal spaces and central vein.

Disarrangement of normal hepatic cells with necrosis and vacuolization were observed

in CCl4 intoxicated liver. The liver sections of the rat treated with 200mg/kg

bodyweight p.o. of methanolic, chloroform and diethyl ether extracts of the selected

 

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plant, followed by CCl4 intoxication, showed less vacuole formation and absence of

necrosis. Overall the less visible changes observed were comparable with the standard

Silymarin (Mitra et al., 1998).

Histopathological liver sections of the animals also revealed that the normal liver

architecture was disturbed by hepatotoxin in CCl4 group, whereas in the liver sections

of rats treated with the methanolic, chloroform and diethyl ether extracts and

intoxicated with carbon tetrachloride, the normal cellular architecture was retained and

it was comparable with the standard Silymarin group, hence confirming the significant

hepato-protective effect of the selected plant extracts. The inhibitory effect of the plant

extractscan be attributed to chemical substances such as gallic acid and ethyle gallate that

are present in these plants (Krishna Mohan et al., 2007).

Based on the earlier observations the presence of phytoconstituents such as tannins,

flavonoid, saponin, alkaloid and glycosides which are present in the ether extracts of

plants tested could be contributing to the significant hepatoprotective activity. The

ether extracts of these plants exhibited a significant hepatoprotective effect against

carbon tetrachloride induced hepatotoxicity when compared to other two extracts.

5.4 Antiulcer activity

The proper cause of developing peptic ulcer is still unknown where ulcer may be

induced because of stress, alcoholism, long-time use of anti-inflammatory drugs and

many more (Barocelli, 1997). But, it is believed that gastric ulcers develop because of

the imbalance between the factors affecting and the maintenance of the mucosal

integrity by the host defence mechanisms (Szabo et al., 1987; Piper and stiel, 1986).

Prostaglandin (PG) induces formations of excess gastric acids which not only increases

the mucosal resistance but also decrease in the aggressive factors that induces the ulcer

 

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(Aly and Scand, 1987). Therefore continuous aspirin intake inhibits PG synthesis

which results in the damage to the cells lining the mucosal layer (Rainsford, 1984). To

regain the balance, different therapeutic agents including plant extracts may be used.

There are various animal models available to study ulcer such as by employing asprin,

alcohol and pylorus ligation ulcer models. The main causative factor for gastric ulcer

pyloric ligation was found to be stress-induced escalation in the secretion and/stasis of

hydrochloric acid in the stomach. Here volume of HCl secreted is also a problem as it

might affect the unprotected lumen in the stomach (Raju, 2009). Pylorus ligation

induced ulcers are caused by the gastric mucosa autodigestion and subsequent

interruption of the gastric mucosal barrier (Wagnar, 1990). Ulceration in gastric

mucosa can also be induced by ethanol. When ethanol is metabolized, superoxide and

hydroperoxy radicals are released which cause the ulceration (Pihan et al., 1987; Jude

and paul, 2009). Ethanol also results in the gastric damage which is mainly due to the

stasis in the blood flow (Guth et al., 1984).

The anti-ulcer activity of the selected plant extracts were evaluated by employing

asprin, alcohol and pylorus ligation ulcer models. These models represent some of the

most common causes of gastric ulcer in humans. Many factors and mechanisms are

implicated in the ulcerogenesis and gastric mucosal damage induced by different

models employed. In the present study, methanolic extracts of the selected plants were

significantly effective in protecting gastric mucosa against aspirin, alcohol and pylorus

ligation-induced ulcers at all the dose levels examined.

The protection of ulceration by these plant extracts is evident by reduced values of

lesion index was compared to control group suggesting its potent cytoprotective effect.

Since the pyloric ligation is caused by the accumulation of gastric juice and meddling

of blood circulation, the antiulcer activity of selected plant extracts in pylorus ligation

 

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model is evident from its significant reduction in gastric volume, total acidity, free

acidity, ulcer index and increase in pH of gastric juice. The animals treated with the

methanolic extracts not only inhibited the formation of pylorus ulcer but also reduced

the gastric volume, acid concentration and increased the pH values in the stomach. This

study suggests that methanolic extracts of selected plant can suppress gastric damage

induced by aggressive factors.

The antiulcer property of A. indicum in pylorus ligation model is evident from its

significant reduction in free acidity, total acidity, number of ulcers and ulcer index. In

the treated animals, formation of ulcer has been inhibited and also resulted in increase

in pH. Therefore it was suggested that A. indicum has the capacity to suppress the

gastric damage induced by the aggressive factors. The significant increase in the

antiulcer activity of A. indicum could be attributed to the presence of flavonoids

(quercetin), alkaloids, tannins, saponin, glycosides and phenolic compounds.

Flavonoids are among the cytoprotective materials for which anti-ulcerogenic efficacy

has been extensively confirmed. It was suggested that the compounds present in the

extract may accelerate mucous formation, bicarbonate secretion and also increase the

prostaglandin secretion. They also might counteract the effects of the reactive oxidants

present and causes ulcer in the gastrointestinal lumen (Sakat and Juvekar, 2009). So

the antiulcer activity of methanolic extract of selected plantmay be attributed to its

flavonoids content. The results of the present study suggest that the methanolic extract

of selected plant may be beneficial in the treatment of gastric lesions.

5.5 Wound healing activity

Wound healing is a complex multi-stage process with many different stages such as

contraction, epithelization, granulation and collagenation. It normally involves an

initial inflammatory phase followed by fibroblast proliferation, formation of collagen

 

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fibres and shrinking, occurring concurrently but independent of one another. Several

plants are known to have wound healing potential. Flavonoids, glycosides and tannins

present in plant extracts are known to promote the wound healing process mainly by

their astringent and antimicrobial property (Nayak et al., 2007). Flavonoids are also

known to reduce lipid peroxidation not only by preventing or slowing onset of cell

necrosis, but also by improving vascularity. Lipid peroxidation is an important process

in several types of injuries like burns, infected wounds and skin ulcers. Hence all the

drugs which inhibit the lipid peroxidation are believed to enhance the strength of the

collagen fibres by preventing the cell damage or by increasing the circulation in the

tissue and by promoting the DNA synthesis.

Preliminary phytochemical analysis of the leaves of the three selected plant extracts

revealed the presence of alkaloids, glycosides, flavonoid, tannins and phenolic

compounds; the presence of these compounds may contribute to the wound healing

activity. The results of the present investigation indicate significant wound healing

activity by the methanolic, chloroform and diethyl ether extract ointment (10 % w/w)

ofM. Pudica when compared to the other extracts. Methanolic extract ointments (10 %

w/w) of the other two plants also showed a significant effect when compared to the

standard drug.

On the basis of the results obtained in the present investigation it is possible to

conclude that the methanolic, chloroform and diethyl ether extract ointment (10% w/w)

of M. pudica has significant wound healing activity. In both extract ointment, the

methanolic extract of mimosa pudica ointment (10% w/w) showed significant effect

when compare to standard drug (0.2% w/w of Nitrofurazone ointment) and other two

extract in excision wound model.

 

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Minimization of the tissue damage, proper nutrition, providing necessary oxygen in the

wounded tissue and moist environment at the wound region are some of the basic

criteria needed for the wound healing which would reestablish the anatomical

continuity and the function in the wounded region. It can be found that there is no

significant increase in the contraction of the wound in the first four days when

compared with the control group. The results of the 8th day indicate that there is

significant increase in the percentage wound contraction in the group treated with

standard drug (nitrofurazone) and methanolic extract of Mimosa pudica ointment,

revealing that the extract has ability to induce cellular proliferation. Hydroxyproline is

an amino acid which is required for synthesis of protein collagen and it hydroxyproline

is a major component of the protein collagen. Hydroxyproline content has been used as

an indicator to determine the collagen synthesis.

Chemically, M. pudica is a rich source of steroidal and triterpenoidal saponin. The

constituents like steroids viz β -sitosterol and triterpenoides viz Lupeol seem to have

major role in pharmacological activities. The proliferative phase is characterized by

granulation tissue proliferation formed mainly by fibroblast and the angiogenesis

process. In the proliferative phase, angiogenesis is essential for the provision of oxygen

and metabolites to tissues. It is already reported that, β -sitosterol has therapeutic

angiogenic effect on damaged blood vessels (Choi et al., 2002). β - Sitosterol also

exhibited a M. pudica anti-inflammatory, anti-pyretic, anti-arthritic and anti-ulcer

activities (Patra et al., 2010). Lupeol shows activities like anti-protozoal, anti-

inflammatory and anti-microbial which are also supporting the wound healing process.

Lupeol is also used as utraceautical/chemopreventive agent (Gallo and Sarachine,

2009).

 

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The results indicate that the plant extracts have significantly promoted collagen

synthesis as compared to that of control. Use of single model is inadequate and there is

no reference standard which can collectively represent the various components of

wound healing as drugs which, influence one phase may not necessarily influence

another (Sharma and Sikarwar, 2008).

5.6 Anti-bacterial activity

Natural products were considered as “silver bullets” in treating infectious diseases

(Baker et al., 2007). The tested plant extracts also exhibited anti-bacterial activity

against various clinical isolates. Methanolic extracts of all the three plants exhibited

broad-spectrum inhibitory activity against both Gram-positive and Gram-negative

clinical isolates. Recent reports suggest that tannins and propylgallate could be

inhibitory to food-borne, water-borne and off-flavor producing microorganisms (Neeraj

and Sharma, 2007). The tested plant extracts are found to have tannins, saponin and

terpenes in methanolic extracts which could be responsible for the significant anti-

bacterial activity.

The plant extracts showed significant antibacterial activity against almost all the

microorganisms that were tested. Particularly significant activity was found against

Micrococcus luteus. However, the ether extracts of selected plants exhibited lesser

antimicrobial activity. Whereas significant antimicrobial activity was observed in

methanolic extracts of selected plant. Amongst the test organisms used, Micrococcus

luteus was found to be most sensitive followed by Staphylococcus aureus and Bacilluc

cerus, Salmonella typhimurium, Klebsiella pneumonieae and Salmonella

paratyphimurium. The effect was found to be pronounced against Gram-positive

bacteria (Micrococcus luteus, Staphylococcus aureus and Bacilluc cerus) than against

 

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Gram-negative bacteria (Klebsiella pneumonieae, Salmonella typhimurium and

Salmonella paratyphimurium).

These studies were in corroboration with that of C. pubescens, wherein the presence of

terpenes was found to be responsible for the antimicrobial activity (Toyota and

Asakawa, 1999). The inhibitory effect of the plant extractscan also be attributed to the

other chemical substances such as gallic acid and ethyle gallate that are present (Chung

et al., 1998). 5.7 Separation of active principles using HPLC

HPLC analysis of the partially purified methanolic extracts revealed the presence of

compound similar to mimopudine, atrabotrycinol and vasicine as determined by

retention time.

5.8 Infrared Spectral Studies

FT-IR spectra of the methanolic extracts are confirming the existence of artabotrycinol,

mimopudine and vasicine compounds in the respective extracts. IR spectra were

recorded by Shimadzu – 8400 Series Fourier Transformer - Infrared Spectrophotometer

using KBr pallet method.

5.8.1 IR studies on Artabotrycinol

The IR spectrum of methanolic extract of artabotrycinol shows the observed broad

band in the range of 3570-3200cm-1 is probably due to the dominant functions of

hydroxyl group present in the artabotrycinol structure. The observation shows the

hydroxyl group does not exist in isolation and a high degree of association may be

experienced as a result of extensive hydrogen bonding with other hydroxyl groups. A

characteristic absorption at 1290cm-1 is due to in-plane bending vibrations of γO-H

 

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pertains to –CH2OH group. A strong band observed at 1643cm-1 is associated with the

possible existence of alkenyl -C=C- stretching in the structure. The γC-Hstretching

appears at 2962cm-1. The ring frequencies for the aromatics are also observed from IR

spectrum.

5.8.2 IR Studies on Mimopudine

The IR spectrum of methanolic extract of mimopudine shows an intense band at 1683

cm-1, which is due to the presence of conjugated ketonic group in the

structure. The absorption frequencies observed at 3416 and 1595cm-1 are due to the

characteristic N-H stretching and N-H bending associated with the aliphatic primary

amine group respectively. In plane bending γO-H vibrations are observed in the range

1350-1250 cm-1.

C=O

5.8.3 IR Studies on Vasicine

The IR spectrum of methonolic extracts of vasicine shows the vibrational modes at

1450 and 1504cm-1 are associated with the aromatic system present in the compound.

Prominent and strong band observed at 1641cm-1 is due to presence of double banded

nitrogen containing imino group. A broad band observed in the range 3550

- 3250cm-1 due to γO-H group indicates a probable intermolecular association in the

structure. The γC-H frequency appears at 2922 cm-1.

C=N

The characteristic absorptions of the functional groups present in purified extracts are

matching with the absorption frequencies obtained for the commercial samples.

However, the absorption frequency values are shifted by 1-2 cm-1 for a few

characteristic peaks. This trend may be due to the presence of other organic moieties as

impurities and solvent effect. The absorptions by the impurities are masked by the

strong signals associated with artabotrycinol and vasicine molecules. The overall trend

 

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based on IR spectral studies confirms the presence of artabotrycinol, mimopudine and

vasicine organic compounds in the respective methanolic extracts.

5.9 Anti-oxidant activity

Antioxidants are known to play a pivotal role in conferring protection against several

diseases. Epidemiological studies have demonstrated that higher intake of antioxidants

results in reduced risk of heart disease and many other diseases. Because of this there

are lot of interests in natural antioxidants and to study their role in human health and

nutrition. Several medicinal plants, spices, vegetables, fruits and fungi have been

researched as sources of potentially safe natural antioxidants. Various compounds have

been isolated and many of these are polyphenols. Recently, various fungi, endophytes

and mushrooms have been reported to produce antioxidant activity. They are known to

produce several novel metabolites possessing antioxidant activity and are equally

potent as synthetic antioxidants and phytochemicals (Chandra and Arora, 2009).

Natural antioxidants that are present in herbs and spices are responsible for inhibiting

or preventing the deleterious consequences of oxidative stress. Spices and herbs

contain free radical scavengers like polyphenols, flavonoids and phenolic compounds.

In the present study, the free radical scavenger activity of methanolic extracts of

selected plants was evaluated. It was interesting to find that although methanolic

extract exhibited potent antioxidant activity, it was less effective in reducing power.

The ability of the compound to reduce (by addition of H+) is regarded as an indicator of

the potential antioxidant activity (Meir et al., 1995). Besides several other mechanisms

also has been proposed for the various antioxidants like binding to the transition metal

ion, decomposition of the peroxides formed, prevention of the H+ removal, prevention

of chain initiation and radical scavenging (Diplock et al., 1997). Hence we can suggest

that there is always no linear correlation between total antioxidant activity and reducing

 

119  

power activity. Thus, although methanolic extract has low reducing power, it could

have high total antioxidant activity. The present study suggests that the methanolic

extract of selected plant might be a potential source of natural antioxidant. The

phytochemical analysis indicated the presence of alkaloids, glycosides, tannins and

flavonoids in the crude methanolic extract. Several of such compounds are known to

possess potent antioxidant activity (Lee et al., 2004).

Penicillium roquefortti produces various secondary metabolites like phenolic acid

derivatives, terpenoids, benzoic acid, rutin with antioxidant activity and also a wide

range of other biological activities such as antibacterial, antiviral, anti-mutagenic and

immunomodulatory activities (Huang et al., 2007). The results obtained from various

analyses indicate that the methanolic extracts of the plants exhibit a good antioxidant

activity. The activity was higher than many other already reported fungi, plants and

mushrooms (Bounatirou et al., 2007).

Phytochemical screening of extracts of all the three plants showed the presence of

alkaloids, flavanoids, tannins, steroids, Steroidal terpenes, Phenolic compounds,

Carbohydrate, Gums and mucilages. Methanolic extracts of all the three plants exhibits

a strong immuno-modulatory activity, where as the ether extracts exhibit a stronger

activity in delayed type hypersensitivity. Ether extracts of all three plants exhibited a

higher activity in hepato-protective as compare with other extracts. Methanolic extract

of M. pudica exhibited a higher level of wound healing activity than the others. In

solvent fractionation, n-butanol fraction showed well-mannered wound healing

activity. Methanolic extracts of all three plants exhibited comparably strong anti-ulcer

activity. The extracts of three plants were screened for antibacterial and anti-oxidant

activity, the methanolic extracts of all plants exhibit antibacterial and anti-oxidant

activity.

 

120  

6. CONCLUSION

The extracts of leaves of three plants viz. Mimosa pudica, Artabotrys hexapetalus and

Adhatoda vasica were subjected to preliminary chemical characterization followed by

evaluation of pharmacological activity. Methanolic extracts of all the three plants

exhibited a strong immuno-modulatory activity as assayed by NAT assay whereas the

ether extracts exhibited a stronger activity in delayed type hypersensitivity assay. Ether

extracts of all three plants exhibited a higher hepato-protective activity than the other

extracts. Methanolic extracts of all the three plants exhibited comparably stronger anti-

ulcer activity. The methanolic extract of M. pudica exhibited a higher level of wound

healing activity than the others. In solvent fractionation, the n-butanol fraction showed

very good wound healing activity. The methanolic extracts of all the plants showed

significant antibacterial activity; similarly methanolic extracts of all the plants showed

significant anti-oxidant activity implying that they could be used as potential anti-

inflammatory agents.

The bioactive compounds were separated by TLC and HPLC and IR studies were

carried out using the purified compounds. The HPLC and IR analysis indicated that the

compounds isolated from M. pudica, A. hexapetalus and A. vasica were similar to

standard mimopudine, artabotrycinol and vasicine respectively.

 

121  

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PUBLICATIONS

LIST OF PUBLICATIONS IN JOURNALS:

1. Vinothapooshan G and Sundar K, (2011), “Immunomodulatory Activity of Various

extracts of Adhatoda vasica Linn. In experimental rats” African Journal of

Pharmacy and Pharmacology, Vol.5 (3), pp. 306-310.

2. Vinothapooshan G and Sundar K, (2010), “Wound Healing effect of various

extracts of Mimosa pudica” Pharmacologyonline,Vol. 1, pp. 307-315.

3. Vinothapooshan G and Sundar K, (2010), “Hepatoprotective activity of Adhatoda

vasica leaves against carbontetrachloride induced toxicity” Pharmacologyonline,

Vol. 2, pp. 551-558.

4. Vinothapooshan G and Sundar K, (2010), “Wound Healing effect of various

extracts of Adhatoda vasica” International Journal of Pharma and Bio

Sciences,Vol. 1(4), pp. 530-536.

5. Vinothapooshan G and Sundar K, (2010), “Anti-ulcer activity of Mimosa pudica

leaves against gastric ulcer in rats” Research journal of Pharmaceutical, Biological

and chemical Sciences, Vol. 1(4),pp. 606-614.

 

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Curriculum Vitae

Mr. G. Vinothapooshan is currently working as an Assistant professor at Arulmigu

Kalasalingam College of Pharmacy, Krishnankoil, India. Mr. Vinothapooshan obtained

his Bachelor of Pharmacy degree from Arulmigu Kalasalingam College of

Pharmacyaffiliated to Tamilnadu Dr. M.G.R Medical University, Chennai and Master

of Pharmacy in Pharmaceutics from J.S.S. College of Pharmacy, Ooty affiliated to

Tamilnadu Dr. M.G.R Medical University, Chennai. He has authored 15 peer-reviewed

research articles and presented more than 20 research papers in National and

International conferences. His research interests include exploration of natural products

for various pharmacological activities.