the study of pharmacognostical, phytochemical and...

THE STUDY OF PHARMACOGNOSTICAL,

PHYTOCHEMICAL AND BIOLOGICAL ACTIVITIES

OF RUBUS RACEMOSUS FAMILY ROSACEAE (ROXB)

Thesis submitted to

THE TAMILNADU DR.M.G.R. MEDICAL UNIVERSITY

CHENNAI-600032

For the award of degree of

DOCTOR OF PHILOSOPHY

By

P. R. KUMAR, M.Pharm.,

Under the guidance of

Prof. V.VAIDHYALINGAM, M.Pharm,Ph.D.

C.L.BAID METHA

COLLEGE OF PHARMACY

CHENNAI – 600 097 APRIL 2009

CERTIFICATE

I certify that the thesis entitled “THE STUDY

OF PHARMACOGNOSTICAL, PHYTOCHEMICAL AND

BIOLOGICAL ACTIVITIES OF RUBUS RACEMOSUS FAMILY

ROSACEAE (ROXB)” submitted for the award of degree of Doctor of

Philosophy by Mr.P.R.Kumar is the record of research work carried out

by him during the period of 2005 – 2009 under my guidance and

supervision and this work has not formed the basis for the award to the

candidate of any degree, diploma, associateship, fellowship or other titles

in this or any other University or Institution of Higher Learning.

Date: Place:

(V.VAIDHYALINGAM)

DECLARATION I declare that the thesis entitled “THE STUDY

OF PHARMACOGNOSTICAL, PHYTOCHEMICAL AND

BIOLOGICAL ACTIVITIES OF RUBUS RACEMOSUS FAMILY

ROSACEAE (ROXB)” submitted by me for the award of degree of

Doctor of Philosophy is the record work carried out by me during the

period from 2005-2009 under the guidance of

Prof.V.VAIDHYALINGAM and has not formed the basis for the award

of any degree, diploma, associateship, fellowship or other titles in this or

any other University or Institution of Higher Learning.

Date: Place:

(P.R.KUMAR)

ACKNOWLEDGEMENT

I express my deep sense of gratitude and most respectful regards to my

guide Prof.V.Vaidhyalingam M.Pharm., Ph.D., Director, K.K.College of

Pharmacy, Chennai, for his excellent guidance, encouragement and continuous

inspiration throughout my dissertation work.

I am extremely grateful and express my heartful thanks to

Prof. Mrs. Grace Ratnam, M.Pharm, (PhD), Head of the Department,

Department of Pharmaceutics and Director of C.L.Baid Metha College of

Pharmacy, for her valuable contribution to my dissertation work.

I express my sincere thanks and gratitude to Dr.A.Shantha Arcot,

Principal, C.L.Baid Metha College of Pharmacy, Chennai for all the

encouragement and useful suggestions given by her throughout my research

work.

I am greatly indebted to my co-guide Prof.P.Muthusamy Ph.D. for his

valuable guidance.

I express my heartfelt thanks to Prof.P.Muralidharan, Head of the

Department, Pharmacology, C.L.Baid Metha College of Pharmacy, Chennai for

his valuable support in pharmacological studies.

I am very much thankful to Dr.S.Rajan, Field Botanist, Survey of

Medicinal Plants & Collection Unit, (Central Council for Research in

Homoeopathy), Department of AYUSH, Ministry of Health & Family Welfare,

Govt. of India, 112, Government Arts College Campus, Udhagamandalam,

Pin – 643 002 for his valuable suggestions about the plant.

I am highly thankful to Dr.S.Ravi, Senior Scientist, Centre for Medical

Plants Research, Kottakkal for his valuable suggestions for my thesis work.

It is my privilege to express my heartful sense of gratitude and immense

respect to Prof.V.Jayasingh, Professor, Jamia Salafia College of Pharmacy,

Kerala for his valuable suggestions and constant encouragement in my thesis

work.

I am extremely thankful to Prof.P.Jayaraman, Director, Plant Anatomy

Research Centre, Chennai- 45 for the helpful suggestions given during the

progress of my work.

I express my heartfelt thanks to Dr.Padma Venkat and

Mr.Chandrasekhar, Foundation for Revitalization of Local Health Traditions

(FRLHT), Bangalore for their help to collect the literature survey.

I owe my gratitude to Mrs.Banumathi, Librarian, C.L.Baid Metha

College of Pharmacy, Chennai., Mrs.Pratima Mathur, Drug Information

Centre for their help to collect the literature.

I take the privilege to thank Mr.Srinivasaraghavan, Mrs.Usha,

Mrs.Kalpakam, Mrs.Valli and Mrs.Radha the administration staff, C.L.Baid

Metha College of Pharmacy for their help.

Words are inadequate to express my gratitude and indebtedness to my

parents Mr.P.S.Ramachandran and Mrs.K.Padma.

I acknowledge deep sense of gratitude to my wife Mrs.V.Andal and my

beloved Daughters Harini and Deeptha for their abundant support.

Above all my humble thanks and prayers to Almighty who gave me

strength, confidence and capacity to complete my work.

Dedicated

to my

beloved parents

CONTENTS

CHAPTER TITLE PAGE NO. NO.

I INTRODUCTION 1 1.1 AIM AND OBJECTIVES OF THE STUDY 17

II REVIEW OF LITERATURE 18

III MATERIALS AND METHODS 26

3.1 GLASSWARE AND CHEMICALS 26

3.2 PLANT PROFILE 27

3.3 PLAN OF WORK 28

3.4 ANATOMICAL STUDIES 32

3.5 POWDER ANALYSIS 34

3.6 QUANTITATIVE MICROSCOPY 35

3.7 DETERMINATION OF LEAF

CONSTANTS 37

3.8 DETERMINATION OF

PHYSIOCHEMICAL CONSTANTS 40

3.9 PLANT MATERIAL AND EXTRACTION 44


3.10 PRELIMINARY PHYTOCHEMICAL

SCREENING 46

3.11 THIN LAYER CHROMATOGRAPHY 50

3.12 ISOLATION OF CONSTITUENTS BY

COLUMN CHROMATOGRAPHY 51

3.13 HPTLC STUDIES 53

3.14 EXPERIMENTAL ANIMALS 54

3.15 TOXICOLOGICAL EVALUATION 55

3.16 ANTIDIABETIC ACTIVITY 60

3.17 BIOCHEMICAL ESTIMATIONS 65

3.18 ESTIMATION OF ANTIOXIDANT

ENZYME LEVELS IN VARIOUS TISSUES 68

3.19 ANTIOXIDANT STUDIES 73

3.20 ANTIEPILEPTIC ACTIVITY 76

3.21 ANTIMICROBIAL METHODS 80


IV RESULTS 84

4.1 PHARMACOGNOSTICAL STUDIES 84

4.1.1 Macroscopy 84

4.1.2 Microscopy 85

4.1.3 Powder analysis 88

4.1.4 Fluorescence analysis 89

4.1.5 Quantitative microscopy 90

4.1.6 Determination of Leaf constants 91

4.1.7 Determination of physiochemical

constant 92

4.2 PHYTOCHEMICAL STUDIES 92

4.2.1 Extractive Values of different extracts 93 4.2.2 Preliminary Phytochemical Screening 93

4.2.3 Fluorescence analysis of different

extracts 96

4.2.4 Thin layer chromatography 96


4.2.5 Isolation of constituents by column

Chromatography 98

4.2.6 Spectral studies 100

4.2.6.1 Compound – I 100

4.2.6.2 Compound – II 103

4.2.7 HPTLC Studies 106

4.3 PHARMACOLOGICAL STUDIES 107

4.3.1 Acute oral toxicity studies 107

4.3.2 Sub acute toxicity studies 107

4.3.3 Haematological Parameters 108

4.3.4 Antidiabetic activity 108

4.3.4.1 Effect of MERR on blood glucose level in normal rats 108

4.3.4.2 Effect of MERR on blood glucose level on glucose fed hyperglycemic rats 108

4.3.4.3 Effect of acute treatment of MERR on blood glucose level in streptozotocin induced diabetic rats 109


4.3.4.4 Effect of sub-acute treatment of

MERR on blood glucose level in

streptozotocin induced diabetic rats 109

4.3.4.5 Biochemical Estimation 110

4.3.4.5.1 Total bilirubin 110

4.3.4.5.2 Serum glutamate

oxalocetate transminase 110

4.3.4.5.3 Serum glutamate

pyruvate transminase 110

4.3.4.5.4 Serum total protein 111

4.3.4.5.5 Alkaline phosphatase 111

4.3.4.5.6 Serum total cholesterol 111

4.3.4.5.7 Serum HDL cholesterol 112

4.3.4.5.8 Serum triglyceride 112

4.3.4.5.9 Serum LDL-Cholesterol 112

4.3.4.6 Antioxidant enzymes in liver

Homogenate 113

4.3.4.6.1 Superoxide dismutase 113

4.3.4.6.2 Catalase 113

4.3.4.6.3 Glutathione peroxidase 113


4.3.4.6.4 Glutathione reductase 114

4.3.4.6.5 Lipid peroxidation 114

4.3.5 In vitro anti-oxidant studies 115

4.3.5.1 Free radical scavenging activity

by DPPH reduction 115

4.3.5.2 Nitric oxide scavenging activity 115

4.3.5.3 Hydroxyl radicals scavenging activity 115

4.3.5.4 Determination of reducing power 116

4.3.5.5 Determination of total phenolic

compounds 116

4.3.6 Antiepileptic activity 117

4.3.6.1 Maximal electroshock induced

convulsion 117

4.3.6.2 Effect of MERR on neurotransmitter

levels in MES induced rats 117

4.3.6.2.1 Serotonin 117

4.3.6.2.2 Nor-adrenaline 117

4.3.6.2.3 Dopomine 118


4.4 ANTIMICROBIAL STUDIES 119

4.4.1 Antibacterial Activity 119

4.4.2 Antifungal Activity 120

V DISCUSSION 161

VI SUMMARY AND CONCLUSION 173

VII REFERENCES 176

LIST OF TABLES

TABLE TITLE NO.

1. Fluorescence analysis

2. Preliminary phytochemical screening

3. Fluorscence analysis of different extracts

4. TLC for flavanoids

5. TLC for glycoside

6. TLC for tannins

7. Examination of eluates

8. TLC for isolated compound

9. Spectral studies – compound II

10. HPTLC studies

11. Acute toxicity class method OECD guidelines 423

12. Haematological parameters of MERR

treatment on sub-acute toxicity study

TABLE TITLE NO.

13. Histopathology report of various organs on

MERR treatment on sub-acute toxicity study

14. Effect of Rubus Racemosus treatment on blood

glucose level in normoglycaemic rats

15. Effect of Rubus Racemosus on blood

glucose fed hyperglycemic rats

16. Effect of acute treatment of Rubus Racemosus

on blood glucose in STZ induced diabetic rats

17. Effect of sub-acute treatment of Rubus Racemosus


18. Effect of Methanolic Extract of Rubus Racemosus on

Serum Total Bilirubin in STZ induced diabetic rats

19. Effect of Methanolic Extract of Rubus Racemosus

on SGOT in STZ induced diabetic rats

TABLE TITLE NO.


on SGPT in STZ induced diabetic rats


on Serum Total Protein in STZ induced diabetic rats


on Serum Alkaline phosphatase in STZ induced

diabetic rats


on Serum Total Cholesterol in STZ induced

diabetic rats


on Serum HDL- Cholesterol in STZ induced

diabetic rats


on Serum Triglyceride in STZ induced

diabetic rats

TABLE TITLE NO.


on Serum LDL- Cholesterol in STZ induced

diabetic rats

27. Effect of Methanolic Extract on Superoxide

dismutase in STZ induced diabetic rats

28. Effect of Methanolic Extract on Catalase in STZ

induced diabetic rats

29. Effect of Methanolic Extract on Glutathione

Peroxidase in STZ induced diabetic rats


reductase in STZ induced diabetic rats

31. Effect of Methanolic Extract on Lipid

Peroxidation in STZ induced diabetic rats

TABLE TITLE NO.

32. Histopathological studies of the Liver

33. Histopathological Studies of the Pancreas

34. Free radical scavenging activity of MERR

by DPPH reduction

35. Nitric oxide scavenging activity of MERR

36. Hydroxyl radical scavenging activity of MERR

37. Effect of MERR and BHT on reducing power

38. Effect of MERR on MES induced epilepsy

39. Effect of MERR on Serotonin levels in MES

induced epilepsy

40. Effect of MERR on Non adrenaline levels in

MES induced epilepsy

41. Effect of MERR on Dopamine levels in MES induced epilepsy

TABLE TITLE NO.

42. In vitro evaluation of Anti-microbial activity

of different extracts of Rubus racemosus on

Staphylococcus aureus



Staphylococcus epidermidis



Bacillus cereus



Micrococcus luteus



Klebsiella pneumoniae

TABLE TITLE NO.



Pseudomonos aeruginosa



Escherichia coli

49. In vitro evaluation of Anti-fungal activity


Aspergillus Niger



Aspergillus fumigatus

LIST OF FIGURES

FIGURE TITLE NO.

1. Rubus racemosus - Habitat

1.1 Flower of Rubus Racemosus

1.2 Leaves of the plant

2. OECD guideline

3. Cross Sectional View of young folded Leaf

4. Anatomy of the Leaf

4.1 Transverse Section of Leaf through midrib with lamina

4.2 Transverse Section of Midrib

4.3 Transverse Section of Lamina

5. Anatomy of the midrib and Trichomes

5.1 Transverse section of young leaf showing midrib

and lateral vein

5.2 Glandular and Non-glandular trichomes in sectional view

5.3 Glandular and Non-glandular trichomes enlarged

FIGURE TITLE NO.

6. Anatomy of the Petiole

6.1 Transverse Section of petiole entire view [Distal region]

6.2 Transverse Section of petiole ground Plane [Proximal region]

7. Structure of the Petiole – Proximal region

8. Anatomy of the Young Stem

8.1 Transverse Section of Stem under low magnification

8.2 Transverse Section of Stem under high magnification

9. Glandular trichome of the Stem

9.1 Transverse Section of Stem showing a glandular trichome

9.2 Glandular trichome enlarged

10. Trichome Morphology

10.1 Non-glandular trichome stained with Toluidine Blue

10.2 Non-glandular trichome stained with Safranin

11. Histopathology of various organs in toxicity studies

FIGURE TITLE NO.


glucose level in normoglycaemic rats


glucose fed hyperglycemic rats

14. Effect of acute treatment of Rubus Racemosus

on blood glucose level in STZ induced diabetic rats

15. Effect of sub-acute treatment of Rubus Racemosus


16. Effect of Methanolic Extract of Rubus Racemosus on

Serum Total Bilirubin in STZ induced diabetic rats


on SGOT in STZ induced diabetic rats


on SGPT in STZ induced diabetic rats

FIGURE TITLE NO.


on Serum Total Protein in STZ induced diabetic rats


on Serum Alkaline phosphatase in STZ induced

diabetic rats


on Serum Total Cholesterol in STZ induced

diabetic rats


on Serum HDL- Cholesterol in STZ induced

diabetic rats


on Serum Triglyceride in STZ induced diabetic rats

FIGURE TITLE NO.


on Serum LDL- Cholesterol in STZ induced

diabetic rats

25. Effect of Methanolic Extract on Superoxide

dismutase in STZ induced diabetic rats

26. Effect of Methanolic Extract on Catalase

in STZ induced diabetic rats

27. Effect of Methanolic Extract on Glutathione Peroxidase in STZ induced diabetic rats


reductase in STZ induced diabetic rats

29. Effect of Methanolic Extract on Lipid Peroxidation

in STZ induced diabetic rats

FIGURE TITLE NO.

30. Histopathology of the liver section

31. Histopathology of the pancreatic section

32. Free radical scavenging activity of MERR

by DPPH reduction

33. Nitric oxide scavenging activity of MERR

34. Hydroxyl radical scavenging activity of MERR

35. Effect of MERR and BHT on reducing power

36. Effect of MERR on MES induced epilepsy

51. Effect of MERR on Serotonin levels in MES

induced epilepsy

52. Effect of MERR on Non adrenaline levels in

MES induced epilepsy

53. Effect of MERR on Dopamine levels in MES

induced epilepsy

FIGURE TITLE NO.



Staphylococcus aureus






Bacillus cereus



Micrococcus luteus



Klebsiella pneumoniae

FIGURE TITLE NO.



Pseudomonos aeruginosa



Escherichia coli



Aspergillus Niger




LIST OF SPECTRA

SPECTRA TITLE NO.

1. Compound I

1.1 I.R. Spectrum

1.2 1H NMR Spectrum

1.3 13C NMR Spectrum

1.4 MASS Spectrum

2. Compound II

2.1 I.R. Spectrum

2.2 I.R. Spectrum

2.3 1H NMR Spectrum

2.4 13C NMR Spectrum

2.5 MASS Spectrum

3. HPTLC Studies

3.1 Spectrum scan parameters

3.2 Spectrum for extract

3.3 Spectrum for isolated compound

LIST OF ABBREVIATIONS USED

Ab - Accessary bundle

AbE - Abaxial Epidermis

Abs - Abaxial side

AdG - Adaxial groove

Ads - Adaxial side

ALP - Alkaline Phosphatase

ANOVA - Analysis of Variance

AR - Analytical Reagent

B - Body of the trichome

beta - β cell

BHT - Butylated Hydroxyl Toluene

b.w - body weight

CAT - Catalase

cfu - colony forming unit

Chl - Chlorenchyma

Co - Cortex

Col - Colenchyma

CPCSEA - Committee for the Purpose of Control

and Supervision of Experiments on Animals

Cr - Crystal

DA - Dopamine

DNA - Deoxyribonucleic acid

DPPH - 1,1-diphenyl-2-picrylhydrazyl

EDTA - Ethylene Diamine Tetra Acetic acid

Ep - Epidermis

ESR - Erythrocyte Sedimentation Rate

GABA - Gamma-Aminobutyric Acid

GHS - Globalised Harmonized System

GPx - Glutathione Peroxidase

GSH - Reduced Glutathione

GSH-Rase - Glutathione Reductase

GSSG - Glutathione oxidized

GST - Glutathione –s-transferase

GT - Glandular Trichome

H - Head

HCl - Hydrochloric acid

H and E - Hematoxylin and Eosin stain

Hb - Haemoglobin

HDL - High-Density Lipoprotein

HLTE - Hind Limb Tonic Extension

HPTLC - High Performance Thin Layer

Chromatography

H2O2 - Hydrogen peroxide

Hy - Hypoderm

IAEC - Institutional Animal Ethics Committee

ICDRA - International Conference of Drug Regulatory

Authorities

i.p - intra peritoneal

IDDM - Insulin Dependent Diabetes Mellitus

La - Lamina

LB - Lateral Bundle

LC50 - Median Inhibition Concentration LD50 - Lethal Dose 50%

LDL - Low-Density Lipoprotein

LPO - Lipid Peroxidation

Lv - Lateral vein

IU/dl - International Units per deciliter

MB - Median Bundle

Mc - Mucilaginous cell

MDA - Malondialdehyde

MERR - Methanolic Extract of Rubus Racemosus

MES - Maximal electroshock

mg - milligram

mg/dl - milligram/deciliter

MIC - Minimal Inhibitory Concentration

min - minutes

ml - milliliter

mM - milimoles

MR - Midrib

NA - Noradrenaline

NaCl - Sodium chloride

NADP - Nicotinamide adenine Dinucleotide

Phosphate Reduced

NADPH - Nicotinamide Adenosine Dinucleotide

Phosphate

NGT - Non glandular trichome

Nm - Nanometer

NO - Nitric oxide

Ns - Non significant

OD - Optical Density

OECD - Organisation for Economic Co operation

and Development

OGTT - Oral Glucose Tolerance Test

OPT - O-phthalaldehyde

P - Parenchyma

Pa - Palisade tissue

PCV - Packed Cell Volume

Pf - Pericyclic fibre

Ph - Phloem

PHT - Phenytoin

Pi - Pith

PM - Palisade Mesophyll

po - per oral

RBC - Red Blood Cells

Rf - Relative factor

ROS - Reactive Oxygen Species

rpm - revolutions per minute

Sc - Sclerenchyma cells

SCMC - Sodium Carboxymethyl- Cellulose

SEM - Standard Error Mean

SGOT - Serum Glutamic-Oxaloacetic Transaminase

SGPT - Serum Glutamic-Pyruvate Transaminase

SI - Stomatal Index

SM - Spongy Mesophyll

SOD - Superoxide Dismutase

Sp - Spongy tissue

sq.mm - square millimeter

ST - Stalk cells

St - Stoma

STZ - Streptozotocin

T1DM - Type 1 Diabetes Mellitus

T2DM - Type 2 Diabetes Mellitus

TBA - Thio Barbituric Acid

TCA - Trichloro Acetic acid

TBARS - Thio Barbituric Acid Reactive Substances

TLC - Thin Layer Chromatography

Tr - Trichome

TRIS-HCL - Tris Hydrochloride

UV - Ultraviolet-Visible

V - Vessel

Vb - Vascular bundle

Vi - Vein islet

Vs - Vascular strand

Vt - Vein termination

WBC - White Blood Cells

W/V - Weight/Volume

WHO - World Health Organisation

Wi - Wing

Xy - Xylem

% - Percentage

µg - Microgram

µl - Microlitre

INTERNATIONALLY APPROVED NUMBER FOR STRAINS

NAME OF THE STRAIN

MICROBIAL TYPE OF CULTURE COLLECTION

AMERICAN TYPE OF CULTURE COLLECTION

Staphylococcus aureus 737 6538 p

Staphylococcus epidermides 435 155

Bacillus cereus 430 11778

Micrococcus luteus 106 4698

Klebsiella pneumoniae 618 29665

Pseudomonos aeruginosa 1688 9027

Escherichia coli 1687 8739

Aspergillus niger 1344 16404

Aspergillus fumigate 2550 13073

INTRODUCTION

CHAPTER - I

INTRODUCTION

Herbal Medicine sometimes referred as Herbalism or Botanical

Medicine is the use of herbs for their therapeutic or medicinal value. A herb is

a whole plant or plant part valued for its medicinal, aromatic or savory

qualities. Herbal plants contain a variety of chemical substances that act upon

the body.

Herbal medicine is a major component in all indigenous traditional

system of medicine and a common element in Ayurvedic, homeopathic,

naturopathic, traditional oriental and Native American Indian medicine. WHO

notes that of 119 plant-derived pharmaceutical medicines, about 74 percent are

used in modern medicine in ways that correlated directly with their traditional

uses as plant medicines by native cultures.

The World Health Organization (WHO) estimates that 4 billion people,

i.e. 80 percent of the world population, presently use herbal medicine for some

aspect of primary health care1.

In 20th century much of the pharmacopoeia of scientific medicine was

derived from the herbal folklore of native peoples. Many drugs commonly used

today are of the herbal origin. Indeed, about 25 percent of the prescribed drugs

dispensed in the United States contain at least one active ingredient derived

from plant material. Some are made from plant extracts, others are synthesized

to mimic a natural plant compound.

The use of medicinal plants for healing purpose predates human history

and forms the origin of much modern medicines. Many conventional drugs

originate from plant sources. A century ago most of the few effective drugs

were plants based. Examples include aspirin (from willow bark), Digoxin (from

fox glove), Quinine (from cinchona bark) and Morphine (from the opium

poppy).

The leads of CNS active medicinal plants which have emerged besides

Rawolfia serpentina, Mucuna pruriens for Parkinson’s disease, Ocim santum as

an antistress agent, Withania somnifera as anxiolytic. The study related to

epilepsy is focused towards the traditional medicine. The recent trends in the

pharmacological studies are based on the biochemical and molecular

mechanism which leads to the development of CNS active principles from the

herbal drugs2.

Traditional medicine3 has been practiced for millennia. Resulting in a

particularly long and rich heritage that continues to influence growing

acceptance of the efficacy and clinical use of traditional herbal medicines.

Practices of traditional medicine can be categorized into two sample groups.

One category constitutes highly evolved and disciplined applications of both

medical and pharmaceutical methodologies. Among some of the most

developed practices are Traditional Chinese Medicine and Traditional Indian

Medicine (such as Ayurveda and Sidda). Which engender distinct,

comprehensive and systematic principles as the foundation of their specific

therapeutic practices? Traditional Chinese Medicine has been widely adopted

in many neighbouring regions of China, where it has been customarily

modified by incorporating indigenous ingredients and/ or local practices; a few

specific examples include, Kampo Medicine (Japan), Han Medicine (Korea),

and North Medicine (Vietnam). Ayurveda is also popularly exercised

worldwide, largely advocated and exercised by Indian scholars and migrants.

Distinctly, Folk Medicine comprises another category of traditional

medicine varies considerably around the world. In Japan, the system of

traditional (Kampo) medicine was officially integrated into the healthcare

system in1976. Today many Kampo herbal medicines are commonly available

as OTC or prescription drugs. Japan’s pharmaceutical industry accounts for

sales of approximately 6.2 trillion yen annually, of which Kampo medicines

constitute around 2%, according to 1995 statistics. Nearly 65-70% of Japanese

medical doctors utilize Kampo medicines in clinical practice, either exclusively

or in combination with modern drugs.

The nation’s medical school curriculum is now being revised, to better

educate physicians on the theories and uses of Kampo medicines, while until

now Japanese doctors had been trained only in the applications of modern

drugs.

In Germany, herbal remedies are particularly prevalent, both as self-

medications and prescription drugs, constituting nearly 5.4% of all medical

prescriptions and 10% of the entire drug market. In the US, however the food

and drug administration has conventionally prohibited the lawful use of herbal

medicines. Nevertheless, the growing interest in herbal medicines throughout

the world can be ascribed to benefits of cost effectiveness, efficacy and

generally reduced side-effects, of many of these treatments. The US

government has recently classified herbal medicines and the products of

medicinal plants as dietary supplements. However, this half-step measure lacks

the regulation necessary to prevent the misuse of herbal medicines (designated

as dietary supplements) which can exhibit adverse effects, and chronic or acute

toxicity if over-ingested.

More recently, though, the US has substantially increased its funding

allocated to Complimentary and Alternative Medicine by millions of dollars,

while further establishing. The office of alternative medicine, in order to better

facilitate research and clinical trials. These are welcome measures towards the

development of more practical and effective regulatory policies concerning the

use of traditional herbal remedies and phytomedicines.

In the midst of the present boom in natural products and herbal

medicines, and intends to introduce some of the most current research on

traditional herbal medicines, and to depict potential and functional applications

of these agents in our modern healthcare systems, which continue to be

practiced most widely in Asia. The contributing studies also characterize state-

of –the-art pharmacological mechanisms and develop more effective

formulations of traditional herbal remedies, in concert with their potential roles

in our modern healthcare systems. The editors believe much more basic

pharmacological research into traditional herbal medicines is essential, and that

will assist continued research as an important reference.

During the past decade, traditional systems4 of medicine have become a

topic of global importance. Current estimates suggest that, in many developing

countries, a large proportion of the population relies heavily on traditional

practitioners and medicinal plants to meet primary health care needs. Although

modern medicine may be available in these countries, herbal medicines

(phytomedicines) have often maintained popularity for historical and cultural

reasons. Concurrently, many people in developed countries have begun to turn

to alternative or complementary therapies, including medicinal herbs.

Few plant species that provide medicinal herbs have been scientifically

evaluated for their possible medical application. Safety and efficacy data are

available for even fewer plants, their extracts and active ingredients, and the

preparations containing them. Furthermore, in most countries the herbal

medicines market is poorly regulated, and herbal products are often neither

registered nor controlled. Assurance of the safety, quality, and efficacy of

medicinal plants and herbal products has now become a key issue in

industrialized and in developing countries. Both the general consumer and

health-Care professionals need Up-to-date, authoritative information on the

safety and efficacy of medicinal plants.

During the fourth International Conference of Drug Regulatory

Authorities (ICDRA) held in Tokyo in 1986, WHO was requested to compile a

list of medicinal plants and to establish international specifications for the most

widely used medicinal plants and simple preparations. Guidelines for the

assessment of herbal medicines were subsequently prepared by WHO and

adopted by the sixth ICDRA in Ottawa, Canada, in 1991. As a result of

ICDRA’s recommendations and in response to requests from WHO’s member

states for assistance in providing safe and effective herbal medicines for use in

national health-care systems, WHO is now publishing this first volume of 28

monographs on selected medicinal plants; a second volume is in preparation.

Preparation of the monographs

The medicinal plants featured in this volume were selected by an

advisory group in Beijing in 1994. The plants selected are widely used and

important in all WHO regions, and for sufficient scientific information seemed

available to substantiate safety and efficacy. The monographs were drafted by

the WHO Collaborating Centre for Traditional Medicine at the University of

Illinois at Chicago, United States of America. The content was obtained by a

systematic review of scientific literature from 1975 until the end of 1995:

review articles: bibliographies in review articles; many pharmacopoeias-the

International, African, British, Chinese, Dutch, European, French, German,

Hungarian, Indian, and Japanese; as well as many other reference books.

Draft monographs were widely distributed, and some 100 experts in

more than 40 countries commented on them. Experts included members of

WHO’s expert Advisory panels on Traditional Medicine, on the International

pharmacopoeia and pharmaceutical preparations, and on Drug Evolution and

National Drug policies; and the drug regulatory authorities of 16 countries.

A WHO Consultation on selected Medicinal plants was held in Munich,

Germany, in 1996. Sixteen experts and drug regulatory authorities from

Member states participated. Following extensive discussion, 28 of 31 draft

monographs were approved. The monographs on one medicinal plant was

rejected because of the plant’s potential toxicity. Two others will be

reconsidered when more definitive data are available. At the subsequent eighth

ICDRA in Bahrain later in 1996, the 28 model monographs were further

reviewed and endorsed, and Member states requested WHO to prepare

additional model monographs.

Purpose and content of the monographs

Provide scientific information on the safety, efficacy, and quality

control/quality assurance of widely used medicinal plants, in order to facilitate

their appropriate use in Member states;

Provide models to assist member states in developing their own

monographs or formularies for these or other herbal medicines; and facilitate

information exchange among member states.

Readers will include members of regulatory authorities, practitioners of

orthodox and of traditional medicine, pharmacists, other health professionals,

manufactures of herbal products, and research scientists.

Each monograph contains two parts. The first part consists of

pharmacopoeial summaries for quality assurance: botanical features,

distribution, identity tests, purity requirements, chemical assays, and active or

major chemical constituents. The second part summarizes clinical applications,

pharmacology, contraindications, warnings, precautions, potential adverse

reactions, and posology.

In each pharmacopoeial summary, the Definition section provides the

Latin binomial pharmacopoeial name, the most important criterion in quality

assurance. Latin pharmacopoeial synonyms and vernacular names, listed in the

sections Synonyms and selected vernacular names, are those names used in

commerce or by local consumers. The monographs place outdated botanical

nomenclature in the synonyms category, based on the International Rules of

Nomenclature.

For example, Aloe barbadensis Mill is actually Aloe Vera (L.) Burm.

Cassia acutifolia Delile and Cassia angustifolia Vahl., often treated in separate

monographs, are now believed to be the same species, Cassia senna L.

Matricaria chamomile L., M.recutita L., and M.suaveolens L. have been used

for many years as the botanical name for chamomile. However, it is now

agreed that the name Chamomilla recutita (L.) Rauschert is the legitimate

name.

The vernacular names listed are a selection of names from individual

countries worldwide, in particular from areas where the medicinal plants in

common use. The lists are not complete, but reflect the names appearing in the

official monographs and reference books consulted during preparation of the

WHO monographs and in the Natural products Alert (NAPRALERT) database

(a database of literature from around the world on ethnomedical, biological and

chemical information on medicinal plants, fungi and marine organisms, located

at the WHO Collaborating Centre for Traditional Medicine at University of

Lillinois at Chicago).

A detailed botanical description (under Description) is intended for

quality assurance at the stages of production and collection, whereas the

detailed description of the drug material (under Plant material of interest) is for

the same purpose at the manufacturing and commerce stages. Geographical

distribution is not normally found in official compendia, but it is included here

to provide additional quality assurance information.

General identity tests, Purity tests, and chemical assays are all normal

compendial components included under those headings in these monographs.

Where purity tests do not specify accepted limits, those limits should be set in

accordance with national requirements by the appropriate Member State

authorities.

Each medicinal plant and the specific plant part used (the drug) contain

active or major chemical constituents with a characteristic profile that can be

used for chemical quality control and quality assurance. These constituents are

described in the section major chemical constituents.

The second part of each monograph begins with a list of Dosage forms

and of Medicinal uses categorized as those uses supported by clinical data,

those uses described in pharmacopoeias and in traditional systems of medicine,

and those uses described in folk medicine, not yet supported by experimental or

clinical data.

The first category includes medical indications that are well established

in some countries and that have been validated by clinical studies documented

in the world’s scientific literature. The clinical trials may have been controlled,

randomized, double-blind studies, open trials, or well-documented observations

of therapeutic applications. Experts at the Munich Consultation agreed to

include Folium and Fructus Sennae, Aloe, Rhizoma Rhei, and herba Ephedrae

in this category because they are widely used and their efficacy is well

documented in the standard medical literature.

The second category includes medicinal uses that are well established in

many countries and are included in official pharmacopoeias or national

monographs. Well-established uses having a plausible pharmacological basis

and supported by older studies that clearly need to be repeated are also

included. The references cited provide additional information useful in

evaluating specific herbal preparations. The uses described should be reviewed

by local experts and health workers for their applicability in the local situation.

The third category refers to indications described in unofficial

pharmacopoeias and other literature, and to traditional uses. The

appropriateness of these uses could not be assessed, owing to a lack of

scientific data to support the claims. The possible use of these remedies must

be carefully considered in the light of therapeutic alternatives.

The final sections of each monograph cover Pharmacology (both

experimental and clinical); Contraindications such as sensitivity or allergy;

Warnings; Precautions, including discussion of drug interactions,

carcinogenicity, teratogenicity and special groups such as children and nursing

mothers; Adverse reactions; and Posology.

Man has been using herbs and plants products for combating diseases

since times immemorial.

The Indian subcontinent is enriched by a variety of flora – both aromatic

and medicinal plants. This is due to the wide diversity of climate conditions in

India ranging from deserts to swamplands. Numerous types of herbs have been

well recognized and catalogued by botanists from the high ranges of the

Himalayan tract up to the sea-shore of Kanyakumari. This extensive flora has

been greatly utilized as a source of many drugs in the Indian traditional system

of medicine.

In India, the earliest mention of the use of medicinal plants is to be

found in the Rigveda which was written between 4500-1600 BC. A detailed

account of the world’s first symposium on medicinal plants is given in the first

chapter of Vrihat Samhita and since 1600 BC the amount of literature on this

subject is boundless. The traditional system of medicine is so engrained in our

culture that, even now 75% of the Indian population depend on this indigenous

system for relief. With such a huge section of an ever-increasing population

relying on herbal remedies, it is imperative that the plant products which have

been in use for such a long time be scientifically supported for their efficacy.

The World Health Organisation is now actively encouraging developing

countries to use herbal medicine which they have been traditionally used for

centuries. They have identified 3000 plants from the forests of India and other

tropical countries which can be used as medicine. The active ingredients from

these plants are worth nearly Rs.2000 crores of rupees for the US market alone

and nearly 8 times that for the world market. Only with the scientific

advancements in the fields of pharmacology and toxicology in the western

hemisphere, has drug development based on natural products gained intensity

in Europe and USA. The importance of such an investigation, in India was

realized long back and the first systematic study with these aims was started by

Sir. Ramanath Chopra at Calcutta about 45 years back.

In the early stages, the science of medicine developed around those

plants which had curative properties. A continued search for medicinal plants

during the last several centuries has given rise to a long list of plants which are

of great use in the treatment of diseases, and for promoting health. It can be

stated, more or less truthfully, that every disease has a cure in a plant growing

in nature. Recently, Moose has described a number of vegetable drugs that can

be used as single drug remedies.

Drug used in medicine today, are either obtained from nature or are of

synthetic origin. Natural drugs are those obtained from plants, animals,

microbes or minerals. Those obtained from plants and animals are called drugs

of biological origin and are produced in the living cells of plants or animals.

Until now, only 6000 plant constituents have been isolated and studied.

The flora on this earth, representing and inexhaustible source of medicinal

plants, remains incompletely explored. This unexplored world provides the

most challenging aspects of pharmaceutical and medical science to scientists in

search of new and more potent drugs with marked therapeutic virtues and

negligible side effects. During the last few decades, tremendous progress has

been made in the study of phytochemicals.

Natural products, as a basis for new drugs, have great promise and it is

gratifying to note that the World Health Organization have shown an abiding

interest in plant-derived medicines, described in the folklore of various

countries.

Plants have been one of the important sources of medicines since the

dawn of human civilization. For instance, the Chinese drug Mahung was in use

for over 5000 years for the treatment of different types of fever and respiratory

disorders. Cinchona sp was in use in Peru even in 1825, primarily for

controlling malaria. In spite of the tremendous development in the field of

synthetic drugs and antibiotics during the 21st century, plants still contribute

one of the major sources of drugs in modern as well as traditional medicine

throughout the world. One-third of the world’s population treat themselves

with traditional medicines. Some of the compounds now commonly used in

medicine were isolated from plant sources and used as early as in the 19th

century. Examples are morphine (1803), quinine (1812), atropine (1831),

papaverine (1848), cocaine (1860), digitoxin (1865), and pilocarpine (1875).

Examples of some important compounds isolated in the 20th century include

ergotamine (1518), labeline (1921), digoxin (1930), reserpine (1931),

tubocuraine (1935) diosganin, vincristine (1961) and vinblastine (1963).

Plants are the only economic source of a number of well-established and

important drugs. In addition, they are also the source of chemical intermediates

needed for the production of some drugs.

As stated before, about 75% of the Indian population relies heavily on

the use of herbal drugs for the treatment of diseases. The factory responsible

for the continued and extensive use of herbal remedies in India are their

effectiveness, easy availability, low cost, comparatively less toxic effect and

the shortage of practitioners of modern medicine in rural areas. There is a

growing appreciation in India, as in many other developing countries, of the

need to make greater use of traditional remedies in order to be able to provide

medicine for primary health care.

Although use of traditional remedies is advantageous, it does suffer

some limitations. The main limitation is the lack of standardization of raw

materials, of processing methods and of the final products, dosage formulation,

and the non-existence of criteria for quality control.

Research has to be directed to the use of modern scientific methodology

and techniques to standardize all these steps and for quality control.

Before Independence, the production of plant-based drugs in India was

confined mainly to cinchona and opium alkaloids, galenicals (i.e. medicine

extracted from plants) and tinctures. In the last three decades, bulk production

of plant drugs has become an important aspect of the India pharmaceutical

industry. Some of the drugs which are manufactured today include morphine,

codeine, papaverine, the baine, emetine, quinine, quinidine, digoxin, caffeine,

hyoscine, hyocyamine, atropine, xanthotoxin, sennosides, colchicines,

berberine, vinblastine, vincristine and ergot alkaloids, papaine, nicotine,

strychnine, brucine and pyrethroids.

In India, there are about 20 well-recognised manufacturers of herbal

drugs, 140 medium or small-scale manufacturers, and about 1200 licensed

small manufacturers on record, in addition to many vaidyas having small

manufacturing facilities. The estimated current annual production of herbal

drugs is around Rs. 100 crores. The demand for herbal remedies is ever-

increasing. Herbal medicines represent an estimated $60-billion a year global

market, about 20 percent of the overall drug market, according to the United

Nations agency. There are 1650 herbal formulations in the Indian market and

540 major plants involved in their formulations.

During the last two decades, over 3000 plants have been screened in

India for their biological activities. As a result, a number of new drugs have

been introduced in clinical practice and some are in the advance stages of

clinical development.

There are well-documented scientific data on a good number of

medicinal plants that have been investigated. In spite of all these efforts, very

few drugs of plant origin would reach stage I of a clinical trial or gain enough

creditability for clinical use by practitioners of modern medicine.

This is because herbal drugs are sometimes considered dubious and its

practitioners considered as quakes. The reasons are many. Doubts have been

raised on the use of herbal drugs for the following reasons:

1. Herbal of different origin are after known by the same popular name.

2. Plants growing in different climatic and seasonal conditions do not have

identical chemical constituents or therapeutic effect.

3. The process of collection (fresh, shade or sun-dried), extraction,

processing and storage of herbal medicines cause variation in potency

and safety.

4. The lack of specific standards for herbal medicines in suitable dosage

form creates difficulty in administration.

These shortcomings have delayed the integration of some of the better

known Ayurvedic and Unani principles with the modern system of medicine.

But things are looking up with the gradual acceptance of Ayurvedic

medicine. Further, detailed investigation on the mode of action of herbal drugs

has revealed that they are involved in enzymatic, endocrine and

immunomodulation functions. These have helped to widen their profile of

activity and opened new vistas of therapeutic applications.

1.1 AIMS AND OBJECTIVES OF THE STUDY

The Plant selected for the project work is Rubus racemosus family

Rosaceae.

Rubus species are known to provide extracts which have been used in

traditional medicine as astringent, emmenagogne, abortfacient, anti-microbial,

anti-convulsant, anti-diabetic, muscle relaxant, free radical scavenging agents5.

Decoction of the root is useful for relaxed bowel and dysentery6. Infusion of

leaves was administered to stop diarrhoea and some bleeding. Family Rosaceae

is known as a source of folk medicine for treatment to nervous disorders7.

Preliminary phytochemical screening of the plant Rubus racemosus

revealed the presence of flavanoids and phenolic compounds and tannins.

Flavanoids have been reported to exert multiple biological effects due to their

antidiabetic, antioxidant and free radical scavenging activity8.

But the literature review revealed no documentation of scientific work

on the aerial parts of Rubus racemosus. This prompted us to take up this

project.

In the present study, an attempt was made to isolate individual phyto-

constituent and extracts were subjected to anti-diabetic, anti-oxidant, anti-

convulsant activity and antimicrobial studies.

REVIEW OF

LITERATURE

CHAPTER - II

REVIEW OF LITERATURE

Rubus (Rosaceae)

Sharma.B.B. and Varshney M.D. et al (1986) screened the antifertility

activity of the plant Rubus Niveus on early and late pregnancy in albino rats9.

Bhakuni.R.S. et al (1987) reported chemical examination of the roots

of Rubus ellipticus10.

Rana.A.C. and Saluja.A.K. et al (1990) undertook pharmacological

screening of the alcoholic extract of the leaves of Rubus ellipticus and reported

the acute toxicity studies, anticonvulsant, analgesic, anti-inflammatory and

hypnotic activities11.

Pal.R. et al (1991) studied chemical examination of Rubus ellipticus

and reported the presence of saponins and glucosides12.

Costantino.L. et al (1992) reported antilipoperoxidant activity of

polyphenolic crude extracts of Rubus idaeus, Rubus fruticosus and Rubus

occidentalis fruits13.

Xiao-Hong Zhou et al (1992) reported the phytoconstituents of Rubus

species and reported the presence of holeanane and ursane glucosides14.

Emile M. Gaydou (1995) studied phytoconstituents and reported the

presence of long chain epoxide from stem of Rubus thibetanus15.

Robertson.G.W. et al (1995) observed changes in the chemical

composition of volatiles released by the flowers and fruits of the red raspberry

(Rubus idaeus) cultivar glen prosen16.

Durham et al (1996) reported the isolation of phytoconstituents from

the roots of Rubus pinfaensis17.

Wang.B.G. et al (1997) reported the isolation of phytoconstituents from

the aerial parts of Rubus pileatus18.

Nogueira.E. et al (1998) studied involvement of GABAA-

benzodiazepine receptor in the anxiolytic effect induced by hexanic fraction of

Rubus brasiliensis7.

Vassilieff.V.S. et al (1998) reported the anxiolytic effect of Rubus

brasilensis in rats and mice19.

Zhong-Jian Jia et al (1998) conducted chemical investigations of

Rubus Pungens camb. Var Oldham II and reported the presence of triterpenes

and triterpene glycosyl ester20.

Lien et al (1999) reported the isolation of phytoconstituents from

ethanolic and butanolic extracts of the leaves of Rubus cochinchinensis21.

Kim.T.G. et al (1999) studied the inhibitory effects of Rubus coreanus

on hepatitis B virus replication in Hep G2 2.2.15 cells22.

Kim S.Y. et al (1999) reported the phytoconstituents from the roots of

Rubus parvifoluis and evaluated anti-inflammatory activity using invivo mouse

ear edema test23.

Lemus.I. et al (1999) reported the hypoglycaemic activity in petroleum

ether extract of Rubus Ulmifolius24.

Shepherd.T et al (1999) studied the epicuticular wax ester and

triacylglycerol composition in relation to aphid infestation and resistance in red

raspberry25 (Rubus idaeus L.).

Gunter Adam et al (1999) reported the phytoconstituents of Rubus

colchinchinensis and reported the presence of triterpenes26.

Tom Shepherd et al (1999) studied epicuticular wax composition in

relation to aphid infestation and resistance in red raspberry27 (Rubus idaeus L.).

Yunes.R.A. et al (1999) identified phytoconstituents and evaluated

antinociceptic activity from the aerial parts of Rubus imperialis28.

Deighton.N. et al (2000) studied antioxidant properties from fruits of

Rubus occidentalis, Rubus idaeus and Rubus strigosus29.

Dhanabal.S.P. et al (2000) validated alcoholic extracts of leaves of

various species of Rubus, Rubus ellipticus, Rubus niveus, Rubus Racemosus

and Rubus rugosus (Rosaceae). They were tested for antifertility activity in

female Wistar albino rats. The results indicate decreased implantation sites and

increased resorption sites, which denote anti implantation and early

abortifacient activities of Rubus species. The results are in agreement with the

traditional use of this plant as abortifacient by the tribals of Nilgiris6.

Kim.N.D. et al (2000) studied the activity of Crude extract of Rubus

roots as crataegifolius roots as a potent apoptosis inducer and DNA

topoisomerase I inhibitor30.

Lin.H.S. et al (2000) has studied antioxidant activity in fruits and leaves

of blackberry, raspberry, and strawberry varies with cultivar and developmental

stage31.

Vassilieff.V.S. et al (2000) studied the hypnotic, anticonvulsant and

muscle relaxant effects of Rubus brasiliensis and involvement of GABAA-

System32.

Wang.B.G. et al (2000) reported the phytoconstituents from an ethanol

extract of the aerial parts of Rubus Pungens33.

Wynne Griffiths.D. et al (2000) made a comparitive study of the

composition of epicuticular wax from red raspberry (Rubus idaeus L.) and

hawthorn (Crataefus monogyna Jacq.) flowers34.

Derek Stewart et al (2001) made studies on ripening related changes in

raspberry cell wall composition and structure35.

Dugo.P. et al (2001) identified the anthocyanins in berries by narrow-

bore high-performance liquid chromatography with electrospray ionization

detection36.

Seeram.N.P. et al (2001) reported the cyclooxygenase inhibitory and

antioxidant cyanidin glycosides in cherries and berries37.

Wang.S.Y et al (2001) reported the changes in oxygen-scavenging

systems and membrance lipid peroxidation during maturation and ripening in

blackberry38.

Catalano.S. et al (2002) has studied the phytoconstituents and exhibited

the in vitro antimicrobial activity of Rubus ulmifolius39.

Cho.S.M. et al (2002) studied the phytoconstituents and evaluated

inhibitory effects in B16 mouse melanoma cells from the fruits of Rubus

Coreanum40.

Corao.G.M. et al (2002) reported hyaluronidase inhibitory activity from

the polyphenols in the fruit of Rubus fruticosus41.

Cui.C.B. et al (2002) identified phytoconstituents and evaluated new

cell-cycle inhibitors from air dried roots of Rubus aleaefolius42.

De Coroa et al (2002) reported antiviral activity from crude extract of

Rubus fruticosus43.

Gabriela Maria Konig et al (2002) reported that the methanolic extract

of Rubus rigidus inhibited the activity of both enzymes HIV1 reverse

transcriptase (HIV1-RT) and TKP5644.

Mohamed Eddouks et al (2002) reported the hypoglycaemic effect of

Rubus fructicosis L. and Globularia alypum L. in normal and streptozotocin-

induced diabetic rats45.

Mohamed Eddouks et al (2002) reported that the fruits of blackberry

exhibited significant hyaluronidase inhibitory activity from polyphenols46.

Moyer.R.A. and Wrolstad.R.E. et al (2002) reported the anthocyanins,

phenolics, and antioxidant capacity in diverse small fruits: Vaccinium and

Rubus47.

Nakatani.K. et al (2002) reported the inhibitions of histamine release

and prostaglandin E2 synthesis from aqueous of Rubus suavissimus48.

Patel.A.V. et al (2002) reported the uterine relaxant activity from

methanolic extract of Rubus idaeus49.

Wada.L. et al (2002) reported the antioxidant activity and phenolic

content from Rubus occidentalis50.

Alan Crozier et al (2003) analysed ellagitannins and conjugates of

ellagic acid and quercetin in raspberry fruits51.

Brian E. Ellis et al (2003) has studied the family of polyketide synthase

genes expressed in ripening from the fruits of Rubus idaeus52.

Hamill.F.A. et al (2003) has studied the physical characterization of

bioactive alkanols from Rubus apetalus53.

Mahmoud A M Nawwar et al (2003) has studied the phytoconstituents

of Rubus sanctus and reported the presence of caffeoyl sugar esters and

ellagitannin5.

Yesilada et al (2003) reported the anti-inflammatory and

antinociceptive activity assessment of plants used as remedy in ethanolic and

aqueous extracts of Rubus hirtus and Rubus sanctus54.

Meckes.M. et al (2004) reported the carrageenan induced rat paw

edema activity of methanolic extract of Rubus Coriifolius55.

Moon P.D. et al (2004) studied the inhibition of mast cell-mediated

anaphylactic- like reaction and tumor necrosis factor-alpha-secretion from the

methanolic extract of Rubus croceacanthus56.

Thiem.B. et al (2004) reported antibacterial activity of Rubus

chamaemorus leaf butanolic fraction of the methanolic extract was evaluated

against gram positive and gram negative bacteria57.

Ivanova.D. et al (2005) studied the polyphenols and antioxidant

capacity from the leaves of Rubus sp. Diversa58.

Liu.Z. et al (2005) reported the antiangiogenic activity in a novel

human tissue based in vitro fibrin clot angiogenesis assay from Rubus

occidentalis59.

Tomezyk.M. et al (2005) reported three phenolic compounds from

Rubus saxatilis60.

Brisht.A. et al (2006) made a review on ethanobotanical studies on

Rubus ellipticus and Rubus pedunuculosus61.

Venskutonis.P.R. et al (2007) reported that the radical scavenging

activity and composition of raspberry (Rubus idaeus) leaves from different

locations in Lithuania62.

Elizabeth Barbosa et al (2007) reported in vivo antigiardial activity of

three flavonoids isolated of some medicinal plants used in Rubus Coriifolius

for the treatment of diarrhea8.

A Perusal literature review on Rubus racemosus revealed that only one

pharmacological activity has been reported on the plant and no phytoconstiuent

has been isolated and characterized.

MATERIALS AND

METHODS

CHAPTER – III

MATERIAL AND METHODS

3.1 GLASSWARE AND CHEMICALS

For the entire project, Borosil glasswares were used. They were soaked

in chromic acid for 3 days, washed with tap water, rinsed with distilled water

and dried over hot air oven.

Analytical grade chemicals supplied by S.D.Fine Chemicals, Sigma

Chemicals Co and Qualigens Fine Chemicals were used. All chemical

solvents, enzyme kits used for this research work were of analytical reagent

grade.

3.2 PLANT PROFILE

The plant seen in Rubus racemosus

Vernacular Name: Tamil - Cheethi

Botanical information

Distribution:

The straggling shrub Rubus Racemosus belongs to the family Rosaceae.

It is indigenous to India, distributed in the Southern Western ghats, the Nilgiri

and Palani hills at an altitude of 1,880 metre.

Description

It occurs as decidious shrub; Subshrub; tender parts glandular; prickles

recurved. Leaves odd-pinnate, to 12(16) x 8 (10) cm, chartaceous; margin

serrate; petiole to 5 (7) cm; stipules adnate to petiole, to 6 mm, persistent;

terminal leaflet ovate, acute, to 8 x 6 cm, often sublobulate; laterals ovate-

lanceolate, 7 x 3.5 cm. Inflorescence axillary, a few-flowered; peduncle 2 cm.

Flowers 1 cm wide; pedicel to 1 cm; bracts subulate, 6mm. calyx-tube

shallowly cup-shaped, with glandular hairs; lobes 5, ovate-acuminate. Petals 5,

red, longer than sepals. Stamens α.Ovary glabrous; ovule 1. Fruits globose,

1 cm wide, purple.

Fig. 1 RUBUS RACEMOSUS - HABITAT

Fig. 1.1 Flower of Rubus Racemosus

Fig.1.2 Leaves of the plant

3.3 PLAN OF WORK

I. Pharmacognostical studies of the leaf

a) Macroscopy

b) Microscopy

c) Powder analysis

d) Fluorescence analysis

e) Quantitative microscopy

f) Determination of leaf constants

g) Determination of physiochemical constants

II. Phytochemical studies

a) Extractive values of different extracts

b) Preliminary phytochemical screening

c) Fluorescence analysis of extracts

d) Thin layer chromatography

e) Isolation of constituents by column chromatography

f) Spectral studies

g) HPTLC studies

III. Pharmacological and Biochemical Studies

a) Acute oral toxicity studies

b) Sub acute toxicity studies

c) Haematological Parameters

d) Antidiabetic activity

i. Effect of MERR on blood glucose level in normal rats

ii. Effect of MERR on blood glucose level on glucose fed

hyperglycemic rats

iii. Effect of acute treatment of MERR on blood glucose level

in streptozotocin induced diabetic rats

iv. Effect of sub-acute treatment of MERR on blood glucose

level in streptozotocin induced diabetic rats

v. Biochemical Estimation

a. Total bilirubin

b. Serum glutamate oxalocetate transminase

c. Serum glutamate pyruvate trasaminase

d. Serum total protein

e. Alkaline phosphatase

f. Serum total cholesterol

g. Serum HDL cholesterol

h. Serum triglyceride

i. Serum LDL-Cholesterol

vi. Antioxidant enzymes in liver homogenate

1. Superoxide dismutase

2. Catalase

3. Glutathione peroxidase

4. Glutathione reductase

5. Lipid peroxidation

vii. Histopathological studies on Liver and pancreas

e) In vitro anti-oxidant studies

i. Free radical scavenging activity by DPPH

ii. Nitric oxide scavenging activity

iii. Hydroxyl radicals scavenging activity

iv. Determination of reducing power

v. Determination of total phenolic compounds

f) Antiepileptic activity

i. Maximal electroshock induced convulstion

ii. Effect of MERR on neurotransmitter levels in MES

induced rats

1) Determination of the effect of Rubus racemosus and

standard on neurotransmitter concentrations in rat brain after

induction of epilepsy

IV. Antimicrobial Studies

a) Antibacterial Activity

b) Antifungal Activity

3.4 ANATOMICAL STUDIES

Collection of specimens

The plant specimens for the proposed study were collected from Nilgiri

Hills. Care was taken to select healthy plants and normal organs. The required

samples of different organs were cut and removed from the plant and fixed in

FAA (Formalin-5ml + Acetic acid – 5ml + 70% Ethyl alcohol – 90ml). After

24 hrs of fixing, the specimens were dehydrated with graded series of tertiary-

Butyl alcohol as per the schedule given by Sass, 1940. Infiltration of the

specimens was carried out by gradual addition of paraffin wax (melting point

58-600 C) until TBA solution attained supersaturation. The specimens were cast

into paraffin blocks63.

Sectioning

The paraffin embedded specimens were sectioned off with the help of

Rotary Microtome. The thickness of the section was 10-12 µm. Dewaxing the

sections was achieved by customary procedure64. The sections were stained

with Toluidine blue as per the method published by O’Brien et al., 1964.

Since Toluidine blue is a polychromatic stain, the staining results were

remarkably good and some Cytochemical reactions were also obtained. The

dye rendered pink colour to the cellulose walls, blue to the lignified cells, dark

green to suberin, violet to the mucilage, blue to the protein bodies etc.

wherever necessary sections were also stained with safranin and Fast-green

and I+KI for Starch65.

Photomicrographs

Microscopic descriptions of tissues are supplemented with micrographs

wherever necessary. Photographs of different magnifications were taken with

Nikon Labphot 2 Microscopic Unit. For normal observations bright field was

used. For the study of crystals, starch grains and lignified cells, polarized

light was employed. Since these structures have birefringent property, under

polarized light they appear bright against dark back ground, Magnifications of

the figures are indicated by the Scale-bars.

Descriptive terms of the anatomical features are as given in the standard

Anatomy books66.

3.5 POWDER ANALYSIS

The Powdered aerial parts of Rubus Racemosus was passed through

sieve no.100 and subjected to microscopical features67

i. The powder was mounted in glycerine water and observed for

calcium oxalate crystals.

ii. The powder was stained with Iodine solution and observed for

starch grains.

iii. The powder was cleared with chloral hydrate and stained with

phloroglucinol and concentrated HCl and observed in a self-

illuminating compound microscope.

3.6 QUANTITATIVE MICROSCOPY68

Measurement of length and width of trichomes in powdered leaf of

Rubus racemosus

Powder of the leaf was observed under low power magnification.

Trichomes of simple unicellular, uniseriate type were observed and hence their

dimensions were determined.

The first step involved is the calibration of the eyepiece micrometer

using stage micrometer. To determine this calibration factor, the eyepiece was

replaced by eyepiece micrometer in the ridge. The stage micrometer was then

placed on the stage of the microscope and focused under high power with the

eyepiece scale. The calibration factor was calculated by applying the formula

given below after noting down the coincidence of micrometer division with

eyepiece.

No of division of stage micrometer Each division of eyepiece micrometer ________________________________ x 10 No. of divisions of eyepiece micrometer

The stage micrometer was replaced with the slide containing the

powdered drug. For the preparation of the slide a little quantity of powder was

first boiled with chloral hydrate solution. The cleared powder was taken in a

watch glass and stained with one drop each of phloroglucinol and concentrated

hydrochloric acid.

A little of this powder was then placed on a slide mounted in dilute

glycerin and observed under low power. The length and width of the trichomes

were measured by focusing them on the lines of the eyepiece micrometer.

The dimensions for about 40 trichomes were measured and multiplied

with the calibration factor to give the dimensions of the trichomes in microns.

Calibration factor

Seventh division of eyepiece coincides with tenth division of the stage

micrometer.

One smallest divisions of stage = 0.01mm (or) 10µm

No. of divisions of stage micrometer x 10 Each divisions of eyepiece micrometer = No. of divisions of eyepiece micrometer

= 10 x 10 7

= 14.4 µm

3.7 DETERMINATION OF LEAF CONSTANTS69

a) Determination of vein islet and vein termination numbers

Vein islet number is defined as the number of vein islets present per

square millimeter of the leaf surface midway between the mid rib and the

margin. It is constant and characteristic feature for a given species of the plant

and for used for differention from allied species.

Vein let termination number is defined as the number of vein let

termination present per square millimeter of the leaf surface midway between

the mid rib and the margin. A vein termination is the ultimate free termination

of vein let.

Procedure

Few leaves were boiled in chloral hydrate solution in a test tube placed in a

boiling water bath. The preparation was mounted in glycerin water. The camera

lucida was set up and the black board was divided into squares of 0.5 sq mm by

means of the stage micrometer. The stage micrometer was replaced by the

cleared leaf preparation and the veins were traced in sixteen continuous

squares. The vein islets and vein let terminations were traced by looking

through the microscope when a superimposed image of the leaf portion and

paper were seen at the same time. The number of vein islets and vein let

termination present within the square were counted by taking into consideration

incomplete vein islet on any two adjacent sides of the square. The value for 1

sq mm was calculated. 16 sets of such counts were taken. The observations

were recorded in the form of range and mean values.

b) Determination of stomatal number and stomatal index

Stomata is a minute epidermal opening covered by two kidney shaped

guard cells in dicot leaves. Those guard cells in turn are surrounded by

epidermal (subsidiary) cells. Stomata performs the functions of gaseous

exchange and transpiration in plants. The nature of the stomata as well as

stomatal index and stomatal number are important diagnostic characteristics of

dicot leaves.

Stomatal number is defined as the number of stomata present per sq mm

of epidermis of the leaf. The actual number of stomata per sq mm may vary for

the leaves of the same plant grown in different environments or under different

climatic conditions. It is however shown that the ratio of the number of

epidermal cells in a given area of epidermis is fairly constant for any age of the

plant under different climatic conditions.

Stomatal index is a percentage in which the number of stomata forms

the total number of epidermal cells, each stomata being counted as one cell.

Stomatal index can be calculated by using the following equation

S.I = S x 100 E+S

Where,

S.I. = Stomatal index

S = Number of stomata per unit area

E = Number of epidermal cells in the same unit area

It is employed for the differentiation of closely related species of the

same genus in air dried as well as fresh condition.

Procedure

Fragments of the leaves from the middle of the lamina were cleared by

boiling with chloral hydrate solution. The upper and the lower epidermis were

peeled out separately by means of forceps. The mounts of the upper and lower

epidermis were separately prepared in glycerin water. A square of known

dimension was drawn by means of stage micrometer and camera lucida on

black board drawing paper. The stage micrometer was replaced by the cleared

leaf preparation focused under the same magnification and the epidermal cells

and stomata were traced by looking through the microscope when a super

imposed image of the leaf is seen at the same time. The number of epidermal

cells and stomata within the square were counted, a cell being counted if at

least half of its area lies within the square provided, two adjacent sides are

counted for the purpose of calculation. Successive adjacent fields were

examined until about hundred cells were counted and the stomatal number and

stomatal index were calculated.

3.8 DETERMINATION OF PHYSIOCHEMICAL

CONSTANTS

1. Determination of ash values70

Procedure

The ash content of a crude drug is generally taken to be the residue

remaining after incineration. It usually represents the inorganic salts naturally

occurring in the drug and adhering to it, but it may also involve the inorganic

matter added for the purpose of adulteration. There is a considerable difference

which varies within narrow limits in the case of some individual drug. Hence

an ash determination furnishes a basis for judging the identity and cleanliness

of a drug and gives information related to its adulteration with inorganic

matter. Ash standards have been established for a number of official drugs.

Usually these standards set a maximum limit on the total ash or on the acid

insoluble ash permitted, the total ash is the residue remaining after incineration.

The acid insoluble ash is a part of the total ash, which is insoluble in dilute

hydrochloric acid.

The ash or residue yielded by an organic chemical compound is a rule to

measure the amount of inorganic matter which is present as impurity. In most

cases the inorganic matter is present in small amounts which are difficult to

remove in the purification process and which are not objectionable if only

traces are present. Ash values are helpful in determining the quality and purity

of the crude drug in powdered form.

1a. Determination of total ash

About 3g of accurately weighed powdered drug was taken in tarred

silica cruicible previously ignited and weighed, the powdered drug was

scattered in a fine even layer at the bottom of the crucible and incinerated

gradually by increasing the temperature not exceeding dull red heat until free

from carbon, cooled and weighed. As, a carbon free form was not obtained the

charred mass was extracted with hot water and the residue was collected on an

ashless filter paper. The residue and the filter paper were incinerated and the

filtrate was added evaporated to dryness and ignited at low temperature. The

percentage of ash with reference to air-dried drug was calculated.

1b. Determination of acid insoluble ash

The above procedure was repeated to collect the total ash and was boiled

for 5 minutes with 25ml of conc. hydrochloric acid (AR grade) and the

insoluble matter was collected on an ashless filter paper wet with hot water

ignited and weighed. The percentage of acid insoluble ash was calculated with

reference to air dried drug.

1c. Determination of sulphated ash

The total ash was moistened with conc. sulphuric acid (AR grade)

ignited gently again moistened with sulphuric acid reignited cooled and

weighed. The percentage of sulphated ash was calculated with reference to air

dried drug.

1d. Determination of water soluble ash

The total ash was boiled for 5 minutes with 25ml of water and filtered.

The insoluble matter was collected on an ashless filter paper wet with hot water

and ignited to constant weight at a low temperature. The weight of insoluble

matter was subtracted from the weight of the ash. The difference in weight

represents the water soluble ash. The percentage of water soluble ash was

calculated with reference to air dried drug.

2. Determination of extractive values

Procedure

Extractive values of crude drugs are useful for their evaluation

especially when the constituents of the drug cannot be readily estimated by any

other means. Further, these values indicate the nature of the constituents

present in a crude drug.

2a. Alcohol soluble extractive

Macerated 5 g of dried coarse powder of Rubus racemosus with 100 ml

of 90% ethanol in a closed flask for 24 hours, shaking frequently during 6

hours and allowing to stand for 18 hours.

It was filtered immediately taking precaution against loss of alcohol and

25ml of filtrate was evaporated to dryness in a tarred flat bottomed shallow

dish and dried at 105o C and weighed. The percentage of alcohol soluble

extractive was calculated with reference to air dried drug.

2b. Water Soluble Extractive

Rubus racemosus weighing 5 g of powder was added to 50ml of water at

80oC in a stoppered flask. It was shaken well and allowed to stand for 10

minutes. It was Cooled to 15oC, 2g of kieselghur was added into it and filtered.

Transferred 5ml of the filterate to a tarred evaporating basin and evaporated on

a water bath and the residue was weighed. The percentage of water soluble

extractive was calculated with reference to air dried drug.

3. Determination of loss on drying

The loss on drying is the loss of weight in percentage w/w resulting

from water and volatile matter of any kind that can be driven of under specified

conditions. The test was carried out on well-mixed sample of the substance.

Glass stoppered shallow bottle was weighed that had been dried in the

same conditions to be employed in the determination. Transferred 1.0g of the

sample powder to the bottle. The loaded bottle was placed in a drying chamber.

The sample was dried at a temperature 1050 C to a constant weight. The drying

chamber was opened and bottle was allowed to cool. The bottle and contents

were weighed. The process was repeated until the successive weights differed

not more than 0.5mg.

3.9 PLANT MATERIAL AND EXTRACTION

The aerial parts of Rubus racemosus were collected from Nilgiri Hills in

the month of August in the year 2006. The plant was authenticated by

Dr.S.Rajan, Field Botanist, Survey of Medicinal Plants & Collection Unit,

(Central Council for Research in Homoeopathy), Department of AYUSH,

Ministry of Health & Family Welfare, Govt. of India, 112, Government Arts

College Campus, Udhagamandalam – 643 002.

The aerial parts were shade dried for seven days and then powdered by

means of a grinder and the powder was passed through the sieve no.60. Fine

powder was used for microscopical analysis and coarse powder was used for

phytochemical work. Powdered material was extracted successively with

petroleum ether (60-800), ethyl acetate (770C), chloroform (640C), methanol

(700C) and water. The residues were collected by evaporation of solvent under

reduced pressure by rotary evaporator.

a. Purification of Solvents

1. Petroleum ether

The petroleum ether was distilled and the fraction boiling between

60o-80oc was collected and used for extraction and chromatographic purposes.

2. Chloroform

The chloroform was shaken well with equal volume of distilled water

twice to remove water soluble impurities and separated using a separating

funnel. It was dried over anhydrous calcium chloride for 24 hours, filtered and

dried again over anhydrous potassium carbonate for 24 hours. This was

decanted, distilled and the fraction boiling at 64oc was collected and stored in a

dark brown bottle. Absolute alcohol of 1ml was added as preservative.

3. Ethyl acetate

Ethyl acetate was refluxed for 4 hours and distilled. The distillate was

shaken with sufficient amount of anhydrous potassium carbonate, filtered and

redistilled. The fraction boiling at 770C was collected and used.

4. Methanol

Methanol was lime distilled and used for extraction and

chromatographic purposes.

3.10 PRELIMINARY PHYTOCHEMICAL SCREENING71

Various extracts of the aerial parts of Rubus racemosus were subjected

to preliminary phytochemical screening.

1. Test for alkaloids

All the extracts were treated with dilute hydrochloric acid and filtered.

The filtrate was treated with alkaloidal reagents like Mayer’s reagent,

Dragendroff’s reagent, Hager’s reagent and Wagner’s reagent. There was no

characteristic colour change indicating the absence of alkaloids.

2. Test for carbohydrate (Reducing and Non Reducing Sugar)

a) The extracts on treatment with Molisch’s reagent showed violet ring at

the junction of two liquids suggesting the presence of the carbohydrate.

b) The extracts were treated with Fehling’s solution A and B and heated. A

reddish brown precipitate is formed indicating presence of reducing

sugar.

c) The extracts on treatment with Benedict’s reagent gave reddish orange

colour indicating the presence of reducing sugar.

d) The extracts, when treated with Barford’s reagent gave no characteristic

reaction confirming the presence of non-reducing sugar.

3. Test for steroids and sterols

a) Libermann Burchard test: The extracts were treated with concentrated

sulphuric acid, glacial acetic acid and acetic anhydride which did not

show green colour. This confirms the absence of steroids.

b) When the extracts treated with 5% potassium hydroxide, they did not

give pink colour which indicates the absence of sterols.

4. Test for Proteins

a) Biuret test: The extracts were treated with copper sulphate and sodium

hydroxide they did not show violet colour confirming the absence of

proteins.

b) Millon’s reagent: The extracts were treated with Millon’s reagent it did

not show pink colour which confirms of the absence of proteins.

5. Test for phenols

a) The extracts on treatment with ferric chloride produced violet colour

confirming the presence of phenols.

b) The extracts were treated with 10% sodium chloride solution and

showed cream colour. This confirms the presence of phenols.

6. Test for tannins

When the extracts treated with 10% lead acetate, 10% sodium chloride

and aqueous bromine solutions separately, produced white precipitate

indicating the presence of tannins.

7. Test for flavanoids

The extracts on treatment with amyl alcohol followed by sodium acetate

and ferric chloride produced a pink or blood red colour which shows the

presence of flavanoids.

8. Test for gums and mucilages

The extracts were treated with 25ml of absolute alcohol and filtered. The

filterate was examined for its swelling properties. No swelling was observed.

This shows the absence of gums and mucilage.

9. Test for glycosides

A pinch of the substance was dissolved in glacial acetic acid and few

drops of ferric choloride was added followed by concentrated sulphuric acid. A

red ring formed at the junction of two liquids. It shows the presence of

glycosides.

10. Test for Saponins

Foam test: All the dry extracts weighing 1g were treated with distilled

water and shaken well in a test tube. Formation of foam was observed in the

upper part of the test tube due to the presence of saponins.

11. Test for terpenes

Extracts were treated with tin and thionyl chloride produced pink colour.

Confirming the presence of terpenes.

3.11 THIN LAYER CHROMATOGRAPHY72

Preparation of the plate

About 40 gm of silica gel G was shaken to a homogenous suspension

with 100 ml of distilled water to form a slurry. This suspension was poured

onto a TLC plate of 0.25mm thickness of 20x5 cm. The plates were kept for air

drying until the transparency of the layer disappeared, dried in hot air oven at

121oC for 30 minutes for activation and stored in a dry atmosphere.

Application of the substance mixture for separation

The substance mixture was taken in a capillary tube and it was spotted

on TLC plate, 2cm above its bottom end. Most solutions for application were

between 0.1-1% strength. The start points were equally sized as far as possible

and a diameter ranging from 2-5mm was seen.

Development of chromatogram

The plates were developed in a chromatographic tank by using a range

of solvents from non polar to polar as mobile phase. The raise of the mobile

phase was allowed upto 3/4 the length and then removed. The solvent front

was marked immediately and the plates were allowed to dry in a dryer. The

spots were identified and their Rf values determined after spray reagents. The

results are furnished in Table no.4 to 6.

3.12 ISOLATION OF CONSTITUENTS BY COLUMN

CHROMATOGRAPHY73

As many chemical constituents show their presence in the methanolic

extract and due to its quantitative abundance, this extract had been chosen for

the isolation of the individual phytochemicals by means of column

chromatography.

A suitable column of 2.5 cm in diameter and 60cm in length was

selected, washed thoroughly with water, dried and rinsed with acetone and

dried completely. A little pure cotton was placed in to column with help of a

big glass rod upto neck to avoid the leakage of smaller particles of the

adsorbent. A piece of filter paper with suitable size was placed over the cotton.

The column was packed with silica gel 100-200 mesh upto 1/3 of its length by

carefully pouring the slurry through the funnel to prevent the formation of air

bubbles in the column. The sides of the column was tapped slowly in order to

facilitate the packing of the adsorbent. The prepared column was thoroughly

washed with petroleum ether and the liquid level was always kept above the

surface of the column to prevent the cracking. A round filter paper of suitable

size was placed above the packed column.

Package of the sample in the column

About 2 gms of the concentrated methanolic extract was mixed with

suitable quantity of silica gel (100-200 mesh) to ensure the free flow of the

extract along with adsorbent it was packed in the column through the funnel,

then petroleum ether was added through the column and kept aside over night.

The column was eluted with different organic solvents in increasing order of

polarity

1. Petroleum ether

2. Chloroform

3. Ethyl acetate

4. Ethyl acetate and isopropanol at different ratios

5. Isopropanol and ethyl alcohol at different ratios

6. Ethyl alcohol

7. Ethyl alcohol and methyl alcohol at different ratios

8. Methyl alcohol

The fraction 100ml each of the eluate from the column was collected

into series of 500ml glass beakers. The eluate was concentrated by evaporating

the solvent and the residues if any were identified by Thin Layer

Chromatography.

3.13 HPTLC STUDIES

Materials and Methods

Name of the instrument : Shimadzu

Plate material : HPTLC precoated plates silica gel MERCK -

60F254

Solvent : Ethylacetate: Hexane (4:6)

Detecting agent : Fluorescence Mode

Chloroform extract and isolated pure compound were applied on

silicagel in 0.2mm layer thickness precoated on aluminium sheets using

linomet IV sample applicator and determined their Rf values.

The mobile phase used for developing the plate under study is given

above. The plate was scanned using camag densitometer scanner equipped with

cats v 3.20 software. The chromatogram is furnished in spectrum no.3.2 and

3.3.

3.14 EXPERIMENTAL ANIMALS

Inbred adult wistar rats 150-200 gm of either sex were obtained from the

animal house of C.L.Baid Metha College of Pharmacy. The animals were

maintained in well ventilated rooms with 12:12 light/dark cycle in

polypropylene cages. Standard pelleted feed and drinking water were provided

ad libitum throughout the experimental period. Animals were acclimatized to

the laboratory conditions one week prior to the initiation of the project work.

The project has got the ethical committee clearance from IAEC of CPCSEA.

IAEC Ref: no: IAEC/XIII/17/CLBMCP/2007-2008 dated on 20.04.2007

3.15 TOXICOLOGICAL EVALUATION

Acute oral toxicity studies74

The procedure was followed according to the OECD guidelines 423

(Acute toxic class method). The acute toxic class method is a stepwise

procedure with 3 animals of single sex per group. Depending on the mortality

and or moribund status of the animals, on an average 2-4 steps may be

necessary to allow judgment on the acute toxicity of the testing substance.

According to this procedure minimum number of animals were to be used for

acceptable data band scientific conclusion. The method uses defined doses

(5, 50, 300, 2000 mg/kg body weight) and the results allow a substance to be

ranked and classified according to the globally harmonized system (GHS) for

the classification of chemical which cause acute toxicity.

Adult Male Wistar rats weighing between 150-200 g were used for the

study. The starting dose of Rubus racemosus was 2000 mg/kg body wt. as most

of the crude extracts possess LD50 value more than 2000 mg/kg body weight.

The dose was administered to over night fasted rats and food was withheld for

a further 3-4 hours after administration of the drug and observed for signs of

toxicity.

Body weight of the rats before and after treatment were noted and any

changes in the skin, eyes and mucous membranes, salivation, nasal discharge

urination, and behavioral (sedation, depression), neuromuscular (tremors,

convulsions), cardiovascular lethargy and sleep and coma were noted. The

onset of toxicity and signs of toxicity was also noted. The animals were kept

under observation for 14 days.

Sub acute toxicity studies

Adult male albino rats (120 – 150 g) were used for sub acute toxicity.

These animals were maintained in polypropylene cages under identical animal

house conditions and provided with standard pellet and water and libitum.

Six groups of rats were used in sub acute toxicity study of methanolic

extract consists of 6 rats each at the dose of 400 mg/kg were given orally for 28

days.

Animals were observed for signs and symptoms, alteration of

behaviours, food and water intake and body weight changes. Blood samples

were collected after 24hrs of the last dose of methanolic extracts for

haematological studies. A portion of liver, brain, kidney, spleen, heart, lungs,

testis and ovary were dissected out and kept in 10% formalin for

histopathalogical studies.

In these samples haematological parameters such as Hb, RBC and total

WBC and differential WBC were determined using routine methods.

Figure: 2

Haematological Parameters75

Hb (haemoglobin), RBC (red blood cell) count, total WBC (white blood

cell) differential count, ESR and PCV were determined in the blood.

Estimation of Haemoglobin

The haemoglobin content was estimated by the standard method using

Sahli’s haemoglobinometer.

Apparatus and reagents

1. Comparator

2. Haemoglobinometer

3. Haemoglobin tube

4. Haemoglobin pipette

5. 0.1N HCI

Procedure

A clean and dry Sahli’s tube was filled with 0.1N HCL up to the mark.

Blood was drawn in to the pipette up to the mark and pushed into Sahli’s tube

containing HCL solution. Contents were mixed well and compared after 10

min. Distilled water was added until the colour matches the standard. The

reading was taken from the upper meniscus. Values are expressed as g/dl.

Estimation of total red blood cell count

The total red cell count was determined by the standard method using

haemocytometer.


1. Haemocytometer

2. RBC pipette

3. Hayem’s fluid

4. Microscope

Procedure

Blood was taken up to the 0.5 ml in the RBC pipette followed by

Hayem’s fluid to the mark 101, thus achieving 1: 200 dilution of blood sample.

The counting chamber was charged with the fluid and cells were counted with

the aid of microscope.

Calculations

Total RBC count = cells counted x 5 (1/5 sq. cm) x 10 (depth) x 200 (dilution factor)

Values are expressed as million-cells/cu.mm of blood.

Estimation of total white blood cell count

The total and differential white cell count was estimated by the standard

method using haemocytometer


1. Haemocytometer

2. WBC pipette

3. Turk’s fluid

Procedure

Blood was taken up to the 0.5 ml in the RBC pipette followed by Turk’s

fluid to the mark 11, thus achieving 1: 20 dilution of blood sample. The

counting chamber was charged with the fluid and cells were counted with the

aid of microscope.

Calculations

Cells counted x 10 (depth) x 20 (dilution factor) Total WBC count = _______________________________________

4 (sq.mm counted)

Values are expressed as thousand cells/ cu.mm.

Differential WBC count


1. Microscope

2. Leishman’s stain

3. Pasteur pipette

Procedure

A thin film of blood was prepared on a clear glass slide and stained by

using Leishman’s stain for 2 min. The slide was washed with distilled water

and allowed to stand for 6 min. Then it was observed under microscope and

different WBC cells were identified and counted. Values are expressed as % of

cells.

3.16 ANTIDIABETIC ACTIVITY

Induction of diabetes mellitus in experimental rats

Adult inbred wistar albino rats [42 numbers] of either sex were over

night fasted, and subjected to a single intrapertoneal injection of freshly

prepared streptozotocin [50 mg/kg] dissolved in ice-cold citrate buffer and PH

4.5. After injection, the animal had free access to food and water and were

given 5 % glucose solution to drink overnight to counter the hypoglycemic

shock76.

The development of diabetes was confirmed after 48 hr of the

streptozotocin injection. The animals with fasting blood glucose level more

than 200mg/dl were selected for the experimentation. Out of the 42 animals

subjected for diabetes induction, 6 animals died before grouping and four

animals were omitted from the study, because of mild hyperglycemia. Of the

remaining 32 diabetic animals, four groups of eight animals each were formed

and used for the experimentation. In the present study, glibenclamide (0.5

mg/kg b.w) was used as the standard drug.

Collection of blood sample and blood glucose determination

Blood samples were collected by end tail vein cutting method and blood

glucose level was determined by one touch electronic glucometer using glucose

test strips. This method permits the measurement of blood glucose levels with

minimal injury to the animal and was previously validated by comparison with

glucose oxidase method.

Blood glucose in fasting rats

Effect of MERR treatment on blood glucose level in normo glycemic rats

The animals were divided into three groups of six rats each,

GROUP 1 Animals received normal control [1% SCMC 1ml/100gm/po/b.w

of rat].

GROUP 2 Animal received MERR [200mg/kg/po/b.w of rat in 1% SCMC].

GROUP 3 Animal received MERR [400mg/kg/po/b.w of rat in 1% SCMC].

In this study, the entire group of animals was overnight fasted prior to

the experimentation and administered with the respective drugs as per the

above mentioned dosage schedule. Blood samples were collected before

administration of the drugs and at 30, 60, 90 and 120th min after drug

administration to determine the blood glucose levels by using electronic

glucometer77.

*MERR – Methanolic Extract of Rubus Racemosus

Induced blood glucose level

Effect of MERR on blood glucose level on glucose fed hyperglycemic rats

Oral glucose tolerance test – [OGTT]

The animals were divided into four groups of six rats each.

GROUP 1 Animals received glucose solution at a dose of 2gm/kg/p.o.

GROUP 2 Animals received glibenclamide 0.5mg/kg and glucose solution at a

dose of 2 gm/kg/p.o

GROUP 3 Animals received MERR* 200mg/kg/b.w and glucose solution at a

dose of 2 gm/kg/oral.

GROUP 4 Animals received MERR 400mg/kg/b.w and glucose solution at a

dose of 2 gm/kg/p.o.

In this study, the entire group of animals was fasted and treated with

above dosage schedule only. The MERR and glibenclamide were administered

half an hour before administration of glucose solution. Blood samples were

collected before glucose administration and at 30,60,90 and 120th min after

glucose administration to determine the blood glucose level by using electronic

glucometer78.

*MERR – Methanolic Extract of Rubus Racemosus

Effect of acute treatment of MERR on blood glucose level in STZ induced

diabetic rats

The animals were divided into five groups. Group 1 consisted of 6

normal animals. The remaining 4 groups consisted of 6 STZ* induced diabetic

rats.

GROUP 1 Normal control animals received 1% SCMC 2ml/kg/p.o

GROUP 2 Streptozotocin [50 mg/kg/b.w] induced diabetic animals received

1% scmc 2ml/kg/p.o

GROUP 3 Streptozotocin [50mg/kg/b.w] induced diabetic animals received

glibenclamide 0.5 mg/kg/p.o


MERR 200mg/kg/p.o


MERR 400mg/kg/p.o

In the single day acute study all the surviving diabetic animals and

normal animals were fasted overnight. Blood samples were collected from the

fasted animals prior to the treatment with above dosage schedule and after drug

administration at 1st, 3 rd and 5th hour to determine the blood glucose level by

using electronic glucometer79.

* STZ - Steptozotocin

Effect of sub acute treatment of MERR on blood glucose level in STZ


GROUP 1 Normal control animals received 1% SCMC 2ml/kg/p.o for 28 days.

GROUP 2 Streptozotocin [50 mg/kg/b.w] induced diabetic animals received 1% scmc 2ml/kg/p.o for 28 days

GROUP 3 Streptozotocin [50mg/kg/b.w] induced diabetic animals received glibenclamide 0.5 mg/kg/p.o for 28 days.

GROUP 4 Streptozotocin [50 mg/kg/b.w] induced diabetic animals received MERR 200mg/kg/p.o for 28 days.

GROUP 5 Streptozotocin [50 mg/kg/b.w] induced diabetic animals received MERR 400mg/kg/p.o for 28 days.

The above mentioned treatment schedule was followed for the

respective group of animals for 10 days. Blood samples were collected from

overnight fasted animals, in morning one hour after drug administration on the

1st, 7th, 14th, 21th and 28th day and the blood glucose levels were estimated using

electronic glucometer80.

At the end of the study, all the surviving animals of the respective

groups were anaesthetised by anaesthetic ether. Blood was collected by

bleeding carotid artery and serum was separated to study the biochemical

parameters. After exsanguination of the animals, the liver and pancreas were

removed immediately and washed with ice-cold saline. The liver and

pancreatic tissues were preserved in bovine fluid for histopathological studies.

3.17 BIOCHEMICAL ESTIMATIONS

All the animals were sacrificed at the end of the experiment under light

ether anesthesia, the blood samples were collected separately by carotid

bleeding into sterile dry centrifuge tubes and allowed to coagulate for 30 min at

370C the clear serum was separated at 2500 rpm for 10 min and biochemical

investigations were carried out to assess liver function Viz.,

• Total bilirubin

• Serum glutamate oxalocetate transaminase (SGOT)

• Serum glutamate pyruvate transaminase (SGPT)

• Total protein

• Alkaline phosphatase

• Total cholesterol

• HDL cholesterol

• Triglyceride

• LDL-Cholesterol

All the enzyme assays were carried out at using shimadzu

spectrophotometer, UV – 1601 model81.

Estimation of total bilirubin

The total bilirubin of the serum was estimated using the test kit based on

the Vadeberg’s method82 .

Estimation of glutamate oxaloacetate transaminase (GOT)

The enzyme activity was estimated based on test kit by Reitman and

Frankel method83.

Estimation of Glutamate pyruvate transaminase (GPT)

The enzyme activity was estimated in the serum by using GPT test kit83.

Estimation of Total protein

Total protein was estimated using total protein kit based on biuret

method84.

Estimation of Alkaline phosphatase (ALP)

Alkaline Phosphatase was estimated using Alkaline Phosphatase kit.

Based on para nitro phenyl phosphate method85.

Estimation of Total cholesterol

Total cholesterol was estimated in the serum by using test kit86.

Estimation of HDL-Cholesterol

HDL cholesterol was estimated in the serum by using test kit86.

Estimation of Triglycerides

Triglyceride levels were estimated by using test kit86.

Estimation of LDL-Cholesterol

LDL-cholesterol was calculated by using the formula

LDL-cholesterol = Total cholesterol – [HDL-C + (triglycerides/5)

3.18 ESTIMATION OF ANTIOXIDANT ENZYME LEVELS

IN VARIOUS TISSUES

Preparation of liver homogenate

Immediately after the sacrifice, the liver was excised devoid of blood

and washed in icecold physiological saline. Small pieces of liver was collected

in 10% formalin solution for histopathological examination. The remaining

portion of the liver was weighed and homogenate prepared in Tris HCI buffer

(0.5 M pH 7.4) at 40C. The homogenate was centrifuged and the supernatant

was used for the assay of total protein and cytoprotective enzymes namely

glutathione peroxidase (GPX), superoxide dismutase (SOD), Catalase (CAT),

glutathione reductase (GR), lipid peroxidation (LPO). All the enzymatic assays

were carried out using Shimadzu Spectrometer UV 16.1 model87.

Superoxide dismutase (SOD)

Superoxide dismutase activity was measured by inhibition of pyrogallol

autoxidation at 420 nm for 10 min88

Reagents

1. Tris-HCI buffer

2. Pyrogallol

3. HCL

Procedure

The assay mixture consisted of 1.8 ml of 50mM Tris-HCL buffer

(containing 10mM EDTA), 0.1 ml of 6.0 mM pyrogallol and the diluted tissue

supernatant to a final volume of 2 ml. The reaction was stopped by adding

0.05ml of 1N HC1. The enzyme activity was expressed as Unit/mg protein,

where one unit of superoxide dismutase is the amount of enzyme required to

bring about 50% inhibition of autoxidation of pyrogallol.

Estimation of catalase activity

Catalase activity was determined89.

Reagents

1. Phosphate buffer (pH 7.0)

2. H2O2 freshly prepared

Procedure

The incubation mixture consisted of 0.05 ml of 10% liver homogenate,

1.95 ml of 50 mM phosphate buffer (pH 7.0), to this 1 ml of freshly prepared

30 mM H2O2 was added. The rate of decomposition of H2O2 was measured

spectrophotometrically at 240 nm for 1 min. The result for catalase activity was

expressed as n moles of H2O2. The catalase activity is expressed as n moles of

H2O2 utilized /min/mg protein in tissue homogenate.

Glutathione peroxidase

The activity of glutathione peroxidase was determined90.

Reagents:

1. Phosphate buffer

2. Glutathione

3. Glutathione reductase

4. EDTA

5. NADPH

6. H2O2

Procedure

The assay mixture consisted of 2.0 ml of 10% liver homogenate, 2 ml of

75 mM Phosphate buffer (pH 7.0), 50 ml of 60 mM glutathione, 0.1 ml of 30

units/ml glutathione reductase, 0.1ml of 15mM EDTA, 0.1 ml of 3mM

NADPH and made up a final volume of 3 ml. The reaction was started by the

addition of 0.1 ml of 7.5 mM H2O2. The rate of change of absorbance during

the conversion of NADPH to NADP+ was recorded spectrophotometrically at

340 nm for 3 min. GPx activity for tissues was expressed as µM of NADPH

oxidised to NADP+/min/mg/protein.

Glutathione reductase

Glutathione reductase activity was assayed by measuring the decrease in

absorbance at 340 nm91.

Reagents

1. Sodium phosphate buffer pH 7.5)

2. EDTA

3. GSSG

4. NADPH

Procedure

The reaction mixture containing 50mM sodium phosphate buffer

(pH 7.5), 10 mM EDTA, 0.67 nM glutathione oxidized, and 0.1 mM NADPH

was made upto 3 ml with water. The change in the optical density was

monitored after adding 0.1ml liver homogenate at 340 nm for 3 minutes at 30

seconds interval. The enzyme activity is expressed as n moles of GSSG

utilized/ min/mg protein in tissue homogenate.

Lipid peroxidation

Lipid peroxidation was assayed by the measurement of malondialdehyde

(MDA) levels on the basis of reaction with thiobarbituric acid92.

Reagent:

1. Sodium dodecyl Sulphate AR

2. Acetic acid AR

3. TBA

4. n-butanol AR

Procedure

The incubation mixture consisted of 0.2 ml of 10% liver homogenate, to

this added 0.2 ml of 8.1% Sodium dodecyl sulphate, 1.5 ml of 20% acetic acid

(pH 3.5) and 1.5 ml of 0.8% TBA. The mixture was made upto 4.0 ml with

distilled water and heated in a water bath at 900C for 60 min. After cooling

with tap water, 1.0 ml of distilled water and 5.0 ml of n – butanol were added

and shaken vigorously and centrifuged at 4000 rpm for 10 min. The upper

butanol layer was taken and its absorbance at 532 nm was measured. The lipid

peroxide concentration was expressed as n moles of MDA liberated/min/mg

protein in liver homogenate.

Histopathology

After draining the blood, liver samples were excised, washed with

normal saline and proceeded separately for histological observations. The

materials were fixed in 10% buffered neutral formalin for 48 hrs. Paraffins

sections were taken at 5µm thickness, processed in alcohol-xylene series and

were stained with haematoxylin-eosin dye. The sections were examined

microscopically for histopathological changes93.

Statistical analysis

The statistical analysis was carried out using analysis of variance

(ANOVA) followed by Dunnet’s ‘t’ test. p values <0.05 were considered as

significant.

3.19 ANTIOXIDANT STUDIES

IN VITRO EXPERIMENTS

DPPH radical scavenging activity: 94

To a methanolic solutions of DPPH (2.95 ml of 100 µM), 0.05 ml of the

test compounds dissolved in methanol were added at different concentrations

(25 µg/ml to 1000 µg/ml). Equal amount of methanol was added to the control.

Absorbance was recorded at 517 nm at regular intervals of 30 sec for 5 min.

% Inhibition = [(control – test)] x 100 Control

Nitric oxide scavenging activity: 95

Nitric oxide scavenging activity was measured by spectrophotometry.

Sodium nitroprusside (4ml of 5 mM) in phosphate buffered saline was mixed

with different concentrations of the extracts (25µg/ml to 1000 µg/ml) dissolved

in 10ml of methanol and incubated at 250 C for 30 min along with a control.

After 30 min, 1.5 ml of the incubated solution was diluted with 1.5 ml of Greiss

Reagent (1% sulphanilamide, 2% phosphoric acid and 1% N-1-

napthylethylene diamine hydrochloride in water) and mixed well. The

absorbance of the chromophore formed during diazotization of the

sulphanilamide and subsequent coupling with N-1 napthylethylene diamine

was measured at 546 nm.


Scavenging of hydroxyl radicals92

The reaction mixture contained deoxyribose (2.8 mM), FeCl3 (0.1 mM),

EDTA (0.1 mM) H2O2 (1 mM), ascorbic acid (0.1 mM), KH2 PO4-KOH buffer

(20 mM, pH 7.4) and various concentrations of the extracts (25 µg/ml to 1000

µg/ml) to a final volume of 1 ml, was incubated for 1 hr. at 370C. Deoxyribose

degradation was measured as TBARS. The percentage inhibition was

determined by comparing the results of test and control.


Determination of reducing power97

The reducing power of Rubus racemosus was determined according to

the following method. Mixed 10 mg of methanolic extract of Rubus racemosus

in 1 ml of distilled water with phosphate buffer (PH 6.8) and potassium

ferricyanide [K3 Fe (CN)6] (2.5 ml, 1 %). The mixture was incubated at 500 C

for 20 min. A portion (2.5 ml) of trichloroacetic acid (15%) was added to the

mixture, which was then centrifuged at 3000 rpm for 10 min. The upper layer

of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and ferric

chloride (0.5 ml, 0.1%), and the absorbance was measured at 700nm. Increased

absorbance of the reaction mixture indicates increased reducing power.

Determination of total phenolic compounds98

Total phenolic compounds in the methanolic extract of Rubus racemosus

were determined with folin-ciotalteu reagent according to the method of

Slinkard using pyrocatechol as a standard. To 0.1 ml of extract solution

containing 1000 µg of drug extract in a 50ml volumetric flask diluted with

distilled water. Added 1 ml of folin-ciotalteu reagent and the contents of the

flask mixed thoroughly. After 3 min, 3 ml of 2% Sodium carbonate was added,

then the mixture was allowed to stand for 2 hrs with intermittent shaking and

made up to the volume with distilled water. The absorbance was measured at

760 nm. The phenolic compound in Rubus racemosus was determined as

catechol by interpolation from the calibration curve. The equation is given

below:

Absorbance = 0.001 x pyrocathecol (µg) + 0.0033

3.20 ANTIEPILEPTIC ACTIVITY

a.Maximal electroshock induced convulsion99

Procedure

Seizures are induced to all the groups by using an

electroconvulsiometer. Maximal electroshock seizures were elicited by a 60 Hz

alternating current of 150 mA intensity for 0.2 sec. A drop of electrolyte

solution (0.9% NaCl) with lignocaine was applied to the corneal electrodes

prior to application to the rats. This increased the contact and reduced the

incidence of facilities. Different doses of the MERR were administered for 14

days before induction of seizures. The duration of various phases of epilepsy

were observed. The percentage protection was estimated by observing the

number of animals showing abolition of Hind Limb Tonic Extension100

(HLTE).

Group I Animals treated with 1% SCMC,1ml/100g

Group II Animals treated with phenytion (25mg/kg) suspended in 1% w/v

SCMC

Group III Animals treated with MERR (200mg/kg) suspended in 1% w/v

SCMC

Group IV Animals treated with MERR (400mg/kg) suspended in 1% w/v

SCMC

b.Determination of the effect of Rubus racemosus and on neurotransmitter

concentrations in rat brain after induction of epilepsy

The various biogenic amines in discrete regions of the rat brain were

estimated by spectroflurorimetric method.

Reagents

1) HCl-Butanol – 0.85ml of 37% HCl was added to one liter of

butanol to get HCl-butanol solution

2) Heptane

3) 0.4M HCl

4) EDTA (pH 6.9)

5) 0.1 M Iodine

6) Sodum thiosulphate solution

7) 5 M Sodium hydroxide

8) 10 M acetic acid

9) Dopamine Standard

10) Nor-adrenaline Standard

11) 0.1M HCl

12) o-pthaldialdehyde reagent

PROCEDURE

Preparation of Tissue Extracts100

Dissected frozen rat brains were first cut on a cooled microtome (-200)

in to frontal slices (about 1mm thick) at pre determined antero posterior levels.

The frontal slices were subsequently placed on the cooled stage (-200) of a

punching apparatus where cylindrical tissue samples (usually 1 mm in

diameter, same thickness as the slice) were punched out of selected brain areas

with a glass tube. The x and y co-ordinates of the center of the area were

adjusted is a stereo microscope, ocular of which contained crossline that were

concentric with the center of the glass tube. For weight determination the tissue

pieces were transferred immediately to pre-cooled microhomogenizers which

were closed with glass stored at –250.

Extraction

The tissue was homogenized in 0.1ml HCl-Butanol for 1 minute in a

glass homogenizer made from a small centrifuge tube (vol. 1.5ml) the total

volume was considered to give 0.105ml, taking account of the tissue volume

(1mg = 0.001ml) the sample was then centrifuged for 10 min at 2000 rpm.An

aliquot of the supernatant phase (0.8ml) was added to an eppendroff reagent

tube containing 0.2ml heptane (for spectroscopy) and 0.025ml HCl 0.1M. After

10 min of vigorous shaking, the tube was centrifuged under the same condition

as above in order to separate the two phases and the overlaying organic phase

was discarded, the aqueous phase (0.02ml) was then taken either for a 5-HT or

NA and DA assay. All steps carried at 00C.

Serotonin Assay101

As mentioned earlier some modifications in reagent concentration

became necessary together with changes in proportions of the solvent, in order

to obtain in a good fluorescence yield with reduced volume for 5-HT

determination, when o-pthaldialdehyde (OPT) method was employed. Added

0.025ml of OPT reagent to 0.02ml of the HCl extract. The fluorophore was

developed by heating to 100oC for 10min. After the samples reached

equilibrium with the ambient temperature, excitation and emission spectra of

intensity reading at 360 and 470nm were recorded.

Nor-Adrenaline and Dopamine assay100

To 0.02ml of HCl phase, 0.005ml 0.4M HCl and 0.01ml EDTA/Sodium

acetatebuffer (pH 6.9) were added, followed by 0.01ml iodine solution for

oxidation. The reaction was stopped after two minutes by the addition of

0.01ml sodium thiosulphate in 5 M sodium hydroxide and 10 M acetic acid was

added 1.5 minutes latter. The solution was then heated to 1000C for 6 minutes.

When the sample again reached room temperature, excitation and emission

spectra were read (330 and 375 nm for Dopamine and 395 – 485 nm for Nor-

adrenaline) in a spectrofluorimeter compared the tissue values (fluorescence of

tissue extract minus fluorescence of tissue blank) with an internal reagent

standard (fluorescence of internal reagent standard minus fluorescence of

internal reagent blank). Tissue blanks for the assay were prepared by adding

the reagents of the oxidation step in reversed order (sodium thiosulphate before

iodine). Internal reagent standards were obtained by adding 0.005ml distilled

water and 0.1 ml HCl Butanol to 20 ng of dopamine and Nor-adrenaline

standard.

3.21 ANTIMICROBIAL METHODS

DETERMINATION OF MINIMUM INHIBITORY CONCENTRATION

Agar streak dilution method102

MIC of the extract was determined by agar streak dilution method. A

stock solution of the extract (100 µg mL-1) in respective solvent was prepared

and incorporated in specified quantity of molten sterile agar (nutrient agar for

anti-bacterial activity and sabourand dextrose agar medium for anti-fungal

activity). A specified quantity of the medium (40 – 500C) containing the extract

was poured into a petridish to give a depth of 3-4 mm and allowed to solidify.

Suspension of the microorganism were prepared to contain approximately 105

cfu mL-1 (colony forming unit per milliliter) and applied to plates with serially

diluted extracts in respective solvent and incubated at 370C for 24 hr and 48 hr

for bacteria and fungi, respectively. The MIC was considered to be the lowest

concentration of the test substance exhibiting no visible growth of bacteria of

fungi on the plate.

a. Antibacterial Activity103

The antibacterial activity of different extracts were studied by Disc

Diffusion method against the following organisms.

• Staphylococcus aureus (gram positive)

• Staphylococcus epidermides (gram positive)

• Bacillus cereus (gram positive)

• Micrococcus luteus (gram positive)

• Klebsiella pneumoniae (gram negative)

• Pseudomonos aeruginosa (gram negative)

• Escherichia coli (gram negative)

Extracts were used in the concentrations of 25, 50 and 100 µl, using

their respective solvents comparing with Ciproflaxacin (5 mcg/disc) as

standard. The disc diffusion method was employed for the screening of

antibacterial activity.

Media Used

Soyabean casein digest agar media gm/lit

Casein enzymic hydrolysate 15.0

Papain digest of soyabean meal 5.0

Sodium chloride 5.0

Agar 15.0

Final pH at 250C 7.3 + 0.2

Disc Diffusion Method104

A suspension of Micrococcus luteus was added to sterile soyabean

casein digest agar media at 450C. The mixture was transferred to sterile petri

dishes and allowed to solidify. Sterile discs of 5mm in diameter (made from

whatmann filter paper previously sterilized by U.V.lamp) dipped in solutions of

the different extracts, standard and a blank were placed on the surface of agar

plates.

The plates were allowed to stand for 1 hour at room temperature as a

period of preincubation diffusion to minimize the effects of variation in time

between the application of the different solutions. Then the plates were

incubated at 370C for 18 hours and observed for antibacterial activity. The

diameters of the zones of inhibition was observed and measured.

The average area of zone of inhibition was calculated and compared

with that of the standards.

A similar procedure was carried out for the study of antibacterial activity

of other extracts against Staphylococcus aureus, Staphylococcus epidermides,

Bacillus cereus, Kl.pneumoniae, Pseudomonos aeruginosa and E.coli.

b. Antifungal activity

The anti fungal activity of the extracts were studied by disc diffusion

method against the following organisms.

1. Aspergillus niger

2. Aspergillus fumigates

Extracts were used in the concentrations of 25, 50 and 100 µ/l using

their respective solvents. The standard used was ketaconazole (50 mcg/disc)

against both the organisms. The disc diffustion method was employed for the

screening of anti fungal activity.

Media used

Sabouraud Dextrose Agar Medium gm/lit.

Mycological peptone 10.0

Dextrose 40.0

Agar 15.0

Final pH at 250C 5.6 + 0.2

Disc Diffusion Method

A suspension of Aspergillus niger was added to Sabouraud Dextrose

Agar Media at 450C. The mixture was transferred to sterile petridishes and

allowed to solidify. Sterile discs 5mm in diameter (made from whatmann filter

paper previously sterilized in U.V.lamp) dipped in solutions of the different

extracts, standard and a blank were placed on the surface of agar plates.

The plates were allowed to stand for 1 hour at room temperature as a

period of preincubation diffusion to minimize the effects of variation in time

between the application of the different solutions.

Then the plates were incubated at 370C for 18 hours and observed for

antifungal activity. The diameters of the zones of inhibition was measured for

the plates in which the zone of inhibition was observed. The average area of

zone of inhibition was calculated and compared with that of the standards.

A similar procedure was carried out for the study of antifungal activity

of other extracts against Aspergillus fumigates.

RESULTS

CHAPTER – IV

RESULTS

4.1 PHARMACOGNOSTICAL STUDIES

4.1.1 Macroscopy

Colour : Green

Odour : Characteristic

Taste : Characteristic

Size : 3 – 5 cm in length

1.5 – 2.5 cm in width

Shape : Oval

Petiole : A small petiole present

Margin : Serrate

Apex : Acute

Base : Symmetrical

Veins : 6 – 8 veins on each side

Fig: 3 Cross Sectional View of young folded Leaf

[GT – Glandular trichome; La – Lamina; LV – Lateral vein;

MR – Midrib; NGT – non glandular trichome; VB – Vascular bundle].

Fig:4 Anatomy of the Leaf

4.1 Transverse Section of Leaf through midrib with lamina

4.2 & 4.3 Transverse Section of Midrib & Lamina

[AbE – Abaxial epidermis ; Abs – Abaxial side; Ads – Adaxial side;

Ep – Epidermis; GT – Ground tissue; La - Lamina; LV – Lateral vein;

MR – Midrib; Ph – Phloem; PM – Palisade mesophyll; SM – Spongy

mesophyll; VB – Vascular bundle; X - Xylem].

Fig:5 Anatomy of the midrib and Trichomes

5.1 Transverse section of young leaf showing midrib and lateral vein

5.2 Glandular and Non-glandular trichomes in sectional view

5.3 Glandular and Non-glandular trichomes enlarged

[B – Body of the trichome; Ep – Epidermis; GT – Ground tissue;

LV – Lateral vein; MR – Midrib; NGT – Non-glandular trichome; Ph –

Phloem; St – Stalk cells; X - Xylem].

Fig :6 Anatomy of the Petiole

6.1 Transverse Section of petiole entire view [Distal region]

6.2 Transverse Section of petiole ground Plane [Proximal region]

[AdG – Adaxial groove; Col – Collenchyma; Ep – Epidermis; LB – Lateral

bundle; AB – Abaxial bundle; MB – Median bundle; Pa – Parenchyma cells;

Ph – Phloem; Sc – Sclerenchyma cells; Tr – Trichome; X – Xylem].

Fig :7 Structure of the Petiole – Proximal region

[AB – Adaxial bundle; AdG – Adaxial groove; Col – Collenchyma;

Ep – Epidermis; LB – Lateral bundle; MB – Median bundle; Pa – Parenchyma

cells; Ph – Phloem; Sc – Sclerenchyma cells; X – Xylem].

Fig :8 Anatomy of the Young Stem

8.1 Transverse Section of Stem under low magnification

8.2 Transverse Section of Stem under high magnification

[Co – Cortex; Col –Collenchyma cells; Ep – Epidermis;

MR – Medullary – ray; Ph – Phloem; Pi – Pith; Sc – Sclerenchyma; St – Stele;

X - Xylem].

Fig: 9 Glandular trichome of the Stem

9.1 Transverse Section of Stem showing a glandular trichome

9.2 Glandular trichome enlarged

[Co – Cortex; Col – Collenchyma; H – Head; Pi – Pith; St – Stalk].

Fig: 10 Trichome Morphology

10.1 Non-glandular trichome stained with Toluidine Blue

10.2 Non-glandular trichome stained with Safranin

[NGT – Non-glandular trichome].

Anatomy of the leaf

The leaf has prominent midrib and lateral veins and their lamina (Fig.3).

The leaf has dense epidermal trichomes on the adaxial side and smooth on the

abaxial side. Young leaves are plicate longitudinally and the mature leaves are

flat (Fig.4.1).

4.1.2 Microscopy

Midrib (Fig.4.1, 4): The midrib has concave adaxial side and broad and thick

abaxial part. It has the length of 1.6mm vertically and 1.75 mm horizontally.

The epidermal layer is thin, made up of small, thick walled cells. Two or three

layers inner to the epidermis are collenchymatous, rest of the ground tissue has

thin walled, circular, compact parenchyma cells. The vascular bundle is single,

broad and bowl shaped. It consists of several long, radial rows of xylem

elements and several small groups of phloem elements.

Lamina (Fig.4.3): The lamina is 120 µm thick. The adaxial side is even and

smooth; the abaxial side is undulate and hairy. The adaxial epidermis is thick

with large squarish cells and thick cuticle; the cells are 20 µm thick. The

abaxial epidermis is thin comprising of narrow, rectangular cells. The

mesophyll tissue consists of two layers of short, thin palisade cells and 6-8

layers of small, lobed spongy parenchyma cells. The vascular bundles of the

veinlets are small, surrounded by a layer of dilated hyaline bundle-sheath cells

and narrow adaxial and abaxial extensions.

Trichomes: The leaf has dense glandular and nonglandular trichomes. The non

glandular trichomes are unicellular, unbranched, thick walled and pointed at the

tip (Fig5.2,3). They arise from a pedestal of a group of cells raised above the

level of the epidermis (Fig.5.2). The non glandular trichomes are 250-650 µm

long.

The glandular trichomes are more complex in structure. They occur on

the leaf (Fig.5.1 – 3) and the petiole or stem (Fig.9.1,2).The glandular

trichomes have a long filamentous stalk and prominent, spherical head. The

stalk consists of a single vertical row of cells or multiseriate elongated cells

(Fig.9). The head of the gland has darkly staining compact mass of cells. The

gland varies in length from 120 µm to 1.3 mm. The head is 50-120 µm thick.

Petiole: The petiole is circular in sectional outline and has shallow adaxial

groove (Fig.6.1, 2). It is 1.75mm in diameter. The upper part (distal part) of the

petiole has a broad main, medianly placed vascular bundle and three accessory

lateral bundles on either side, which diminish in size towards adaxial part

(Fig.6.1). The lower part (proximal part) has wider median lervelle, two

prominent lateral bundles and two smaller less prominent bundles (one set on

either side of the adaxial part) (Fig.6.2,7). All the bundles are collateral with

thick band of sclerenchyma cells abutting the phloem tissue. The petiole has

their epidermis with small thick walled cells. Inner to the epidermis is a narrow

zone of two or three layers of collenchyma cells. The remaining ground tissue

is homogeneous and parenchymatous (Fig.7).

Stem (Fig.8.1.2): The stem is uneven in outline with ridges and furrows due to

the presence of thick epidermal trichomes. The stem has a thin layer of

epidermis comprising of small thick walled cells. Epidermis is followed by

about five layers of collenchyma cells. Inner to the collenchyma zone is a

narrow cortical zone of parenchyma cells, some of them having chloroplasts.

The vascular cylinder is thin and continuous and consisting of several

wedge-shaped vascular bundles placed close to each other. The vascular

bundles are collateral and have thick mass of sclerenchyma caps, wide zone of

phloem and radial files of xylem. The sclerenchyma cells are thin walled and

wide lumened. Xylem elements are circular and thick walled. The pith is wide

and parenchymatous.

Trichome Morphology (Fig.10)

The powdered sample of the leaf shows the trichomes and epidermal

fragments. The non glandular trichomes are seen randomly distributed on the

fragments of the epidermis. They are long, whiplike, thickwalled with smooth

surface; they are tapering at the ends. The glandular trichomes are long stalked

with spherical head. The cells of the head portion are darkly staining.

4.1.3 Powder analysis

Sensory Characters

Appearance Coarse

Colour Green

Odour Characteristic

Taste Characteristic

Microscopical Characters

Trichomes Unicellular, Non glandular trichomes

Stomata Anisocytic

Epidermal cells Cells are thin and wavy in nature in lower surface

and straight walls in upper surface

Mesophyll Palisade and spongy parenchyma with epidermis

Vessels They are spiral in nature

Calcium oxalate Prismatic type

Crystals

4.1.4 Flourescence analysis

The powder of Rubus racemosus was treated with various reagents and

visualized under ultra violet radiations. The observations are furnished in Table

No.1.

Table: 1

Drug Fluorescent colours

Powder Green

Powder + Water Green

Powder + Conc.H2SO4 Dark green

Powder + Conc.HNO3 Pale green

Powder +Conc.HCl Dark green

Powder + dil.H2SO4 Light green

Powder + dil.HNO3 Light green

Powder + dil.HCl Green

Powder +1N NaOH Blackish green

Powder + Ac.anhydride Emerald green

Powder + Acetone Dark green

4.1.5 Quantitative microscopy

The length of trichomes of Rubus racemosus is

Range 165µ 320.18µ 622.5µ

Minimum Average Maximum

The width of trichomes of Rubus racemosus is

Range 12.7µ 34.82µ 41.4µ


4.1.6 Determination of Leaf Constants

The range for the vein islet number of Rubus racemosus is

Range 2 3 4


The vein let termination number of Rubus racemosus is

Range 5 7 9


The range for the stomatal number of Rubus racemosus is adaxial surface

Range 4 6 8


The stomatal index of of Rubus racemosus in adaxial surface

Range 6.7 11.2 14


The range of stomatal number of Rubus racemosus in abaxial surface

Range 2.5 3.75 5


The range for stomatal index of of Rubus racemosus in abaxial surface

Range 5 3.7 2.2


4.1.7 Determination of physiochemical constant

The ash values of Rubus racemosus is

1. Total ash - 2.97% w/w

2. Acid insoluble ash - 1.80% w/w

3. Sulphated ash - 2.65% w/w

4. Water soluble ash - 0.18% w/w

The extractive values of Rubus racemosus is

1. Alcohol soluble extractive value is 8.35% w/w

2. Water soluble extractive value is 4.21% w/w

The loss on drying of Rubus racemosus at 1050C is 7.84% w/w

4.2 PHYTOCHEMICAL STUDIES

4.2.1 Extractive Values of different extracts

The successive solvent extractive values of the aerial parts of Rubus

racemosus

Petroleum ether : 12.87% w/w

Ethyl acetate : 10.22% w/w

Chloroform : 12.52% w/w

Methanol : 23.76% w/w

Aqueous : 14.32% w/w

4.2.2 Preliminary phytochemical screening

Preliminary phytochemical analysis of methanolic extract of Rubus

racemosus

The result of preliminary phytochemical analysis of Rubus Racemosus

extracts is shown in table 2. All the extracts showed the presence of various

phytochemical constituents like terpenes, tannins, flavanoids, saponins,

carbohydrates, glycosides and phenols. However the extracts showed negative

results for proteins, steroid, sterols, alkaloids, gum and mucilages.

Table: 2

Phytoconstituents Petroleum

Ether Ethyl

AcetateChloroform Methanol Aqueous

Alkaloids

Mayer’s - - - - -

Hager’s - - - - -

Wagner’s - - - - -

Dragendroff’s - - - - -

Carbohydrates

Molisch + + + + +

Fehling’s + + + + +

Benedict’s + + + + +

Barford’s + + + + +

Steroids

Liberman - - - - -

Burchard Test - - - - -

Sterols

5% potassium hydroxide

- - - - -

Proteins

Biuret - - - - -

Millon’s - - - - -

Phenols

Ferric chloride + + + + +

10% sodium Chloride

+ + + + +

Tannins

10% lead acetate + + + + +

Phytoconstituents Petroleum

Ether Ethyl

AcetateChloroform Methanol Aqueous

!0 % sodium chloride

+ + + + +

Aqueous bromine Solution

+ + + + +

Flavanoids

Amylalcohol + Sodium acetate + Ferric chloride

+ + + + +

Gums and Mucilage

- - - - -

Glycosides

Glacial acetic acid + ferric chloride + conc. H2SO4

+

+

+

+

+

Saponins

Foam test + + + + +

Terpenes

Tin + Thionyl Chloride

+ + + + +

+ = Showed colour reactions

– = Did not show colour reaction

4.2.3 Flourscence analysis of different extracts

The different extracts were observed under ultra violet light and the

findings are furnished in Table no.3.

Table: 3

Extracts Fluorescent colours

Petroleum ether Dark green

Ethyl acetate Pale green

Chloroform Green

Methanol Emerald green

Aqueous Dark green

4.2.4 Thin layer chromatography

Petroleum ether, Ethyl acetate, Chloroform, Methanol and Aqueous

extracts of Rubus Racemosus were subjected to thin layer chromatography for

flavanoids, glycoside and tannins with respective solvent system. Their Rf

values were determined and furnished in Table 4,5 and 6.

TLC for flavanoids

Table: 4

Extracts Solvent System Detecting agent Rf Petroleum ether UV radiation 0.35 Ethyl acetate 0.48 Chloroform 0.56 Methanol 0.59 Aqueous

Chloroform: Ethyl acetate 60:40

0.35

TLC for glycoside

Table: 5

Extracts Solvent System Detecting agent Rf

Petroleum ether UV radiation 0.52

Ethyl acetate 0.62

Chloroform 0.51

Methanol 0.32

Aqueous

Chloroform: Methanol: Water 30:25:10

0.31

TLC for tannins

Table: 6

Extracts Solvent System Detecting agent Rf

Petroleum ether UV radiation 0.55

Ethyl acetate 0.65

Chloroform 0.34

Methanol 0.45

Aqueous

n-butanol: Glacial acetic acid: Water 14:1:5

-

4.2.5 Isolation of Constituents by column Chromatography

Examination of eluates

Table: 7

Eluent % of solvent Fractions Compound

Petroleum ether 100% 1-100 Wax

Chloroform 100% 101-149

Ethyl acetate 100% 150-154

Ethyl acetate : Isopropanol 90:10 155-159



Ethyl acetate : Isopropanol 60:40 170-174 Compound I

Ethyl acetate : Isopropanol 50:50 175-179 Compound I





Isopropanol 100% 200-204

Isopropanol : Ethyl alcohol 90:10 205-209


Isopropanol: Ethyl alcohol 70:30 215-219







Ethyl alcohol 100% 250-254 Compound II

Ethyl alcohol : Methyl alcohol 90:10 255-259 Compound II

Ethyl alcohol : Methyl alcohol 80:20 260-264

Eluent % of solvent Fractions Compound








Methyl alcohol 100% 300-304

Eluates (170-179), (250-259) showed residues with similar Rf values

with respect to thin layer chromatography. Hence all the residues were mixed

together and recrystallized from methanol: water (1:1). The substance was

examined for further process. The isolated compounds are yellow in colour and

highly hygroscopic in nature.

TLC for isolated compound

Table: 8

Compound Solvent System Detecting agent Rf

Compound I Compound II

Ethyl acetate : Isopropanol Ethyl alcohol : Methyl alcohol

UV radiation 0.62 0.59

4.2.6 Spectral studies

4.2.6.1 Compound I

The IR spectrum of the isolated compound I from Rubus racemosus

shows characteristic absorption bands at 3411cm-1 for –OH group and 1651 cm-

1 for α, β – unsaturated carbonyl group.

The 1HNMR spectrum revealed three distinct patterns of proton

resonances.

a) The first is typical for esterified p-hydrozy benzoic acid and contained

two signals at δ6.85 (d, 2H) and δ7.11 (d, 2H).

b) The second pattern is characteristic for sugar protons and contained four

anomeric resonances at δ5.3, δ5.2, δ4.91 and δ4.92. The chemical shift

values of these doublet signals indicated esterification of the anomoric

hydroxyl group and four sugar units were present. Other sugar protons

resonate at chemical shift values between δ3.04 to δ3.84.

c) The third pattern of H-NMR signals is indicative of a long chain epoxide

and contained signals at δ0.84 (t, 3H, CH3), a strong singlet at δ1.22 for

long chain methylene protons, two multiplets at δ2.83 and δ2.69 for

epoxide hydrogens and a signal at δ1.57 for the α – methylene protons to

the epoxide ring.

The above datas were strongly supported by the 13C-NMR spectral

datas. It exhibited a signal at δ174.99 for ester carbonyl group and at 156.46

(C-1), 143.36 (C-4), 114 (C-2, C-6) and 127.89 (C-3, C-5) for aromatic ring

carbons. The downfield signal at δ156.46 is due to the substitution with a –OH

group in that position.

The spectrum also indicated the presence of four anomeric carbon

signals at δ102.44, δ98.49, δ97.35 and δ92.68. The other carbon signals of the

sugar moiety appeared between δ63.38 to δ82.35. Further the 13C-NMR

spectral data exhibited the signals at δ14.39 (CH3), δ61.69 and δ63.38 (epoxide

ring carbons), 26.00 and 27.00 for α – carbons to the epoxide ring.

The presence of fragment ions at m/z 309 and m/z 153 arising from

fragmentations adjacent to the oxygen bearing carbons suggest that the epoxide

was located on C-20 and C-21 of the chain. All the other fragments are closely

related to the fragments of the compound II suggesting the same moiety may be

present in compound I.

On alkaline hydrolysis, compound II gave D-glucose as the sugar

component which was identified by direct comparison with an authentic

sample. Considering the above datas into consideration the following structure

is proposed for the compound I (1-[20,21 – dodeacylnanone] – α-1→6-D-

glucotetraose-6’’’(P-hydroxy) benzoate).

Spectrum 1.1

Specturm 1.2

Spectrum 1.3

Spectrum 1.4

Compound I

Considering the above facts the structure is arrived as follows.

OHO

HO OHO

1

2

3

45

6

G

OH1

2

3

C

O

O4

5

6

OHO

HO OH O

6'

G'1'

OHO

HO OH O

6"

G"1''

OHO

HO OH O

6"'

G'''1'''

O29

22

21

20

191

2

m/z 309

3 5 7

4.2.6.2 Compound II

The molecular formulae of the isolated compound II is found to be

C29 H58 O from the E1 mass spectrum [MH]+ m/z 423. The ‘H and 13C NMR

spectra indicated that II was a linear hydrocarbon with an epoxide ring (δH,

2.90; δc, 58.19). The 200MHz 1H-NMR data further showed two very close

methyl resonances at δ0.84 and δ 0.88. The methylene protons ά to the epoxide

ring resonated at δ1.47 and gave the chemical shifts of the corresponding

carbons at δc 27.42 and 28.65. The strong singlet at δH 2.25 and δc 30.52

corresponds to the long chain methylene protons and carbons respectively. The

epoxide position in the aliphatic chain was determined using mass

spectrometry. The presence of two fragments at m/z 155(10%) and m/z 309

(5%) arising from fragmentations adjacent to the oxygen bearing carbons,

suggested that the epoxide was located on C-9 and C-10 of the chain.

A search in the literature reveals that the compound was earlier isolated

from Rubus thibetanus peak by peak comparison was made. Comparison of 1H-

NMR and 13C-NMR data of compound II with the values reported in the

literature are furnished in Table no.9.

Spectrum 2.1

Spectrum 2.2

Spectrum 2.3

Spectrum 2.4

Spectrum 2.5

Compound II

Considering the above facts the structure is arrived as follows.

Om/z 155

m/z 309m/z 144m/z57

m/z43

29

10 9 1

O

10 9 1

29

Comparision of the spectra of isolated compound II with the reported are

furnished in Table no.9

Table: 9

Proton δH of II Literature value

Carbon position δc of II Literature

value

1-CH3 29-CH3

8&11-CH2

9&10, CH CH2

0.84 0.88 1.47 2.90

2.25

0.860 0.861 1.48 2.88

-

1&29 9,10

8 11

CH2

14.92 58.19 27.42 27.92 30.52

32.75 23.50

14.2 57.4 26.70 28.65 29.50 29.30 29.80 29.70 32.00 22.80

The compound II may be 9,10 epoxynonacosane.

4.2.7. HPTLC studies

HPTLC Studies of the extract of Rubus racemosus

The Chloroform extract was subjected to HPTLC studies. The sample showed 8 peaks scanning 254 nm and their Rf values were furnished in Table no.10 and Spectrum no. 3.1.

Table: 10

Track Peak Rf values Area Percentage 1 0.07 11.45%

2 0.08 24.33%

3 0.27 6.15%

4 0.39 13.63%

5 0.54 9.48%

6 0.59 4.99%

7 0.67 26.02%

8 0.81 3.94%

Specturm 3.1

Spectrum 3.2

HPTLC fingerprint of the isolated compound showed single peak with

Rf value 0.39.

Spectrum 3.3

4.3 PHARMACOLOGICAL STUDIES

4.3.1 Acute oral toxicity studies

The acute oral toxicity study was carried out according to the OECD

guidelines 423 (acute toxic class method) as represented in Table 11. The

starting dose of 2000 mg/kg body weight / p.o of the one methanolic extract of

Rubus racemosus was administered to 3 male rats and observed for three days.

There was no considerable weight change in body weight before and after

treatment and no signs of toxicity was observed. When the experiment was

repeated again with the same dose level 2000 mg/kg body weight / p.o of the

methanolic extract of Rubus racemous for three more days and observed for

fourteen days no change was observed from the first set of experiments. LD50

cut off mg/kg was observed as class X (unclassified) and Globally Harmonized

system (GHS).

4.3.2 Sub Acute toxicity Studies

The Methanolic extract of Rubus Racemosus at a dose of 400 g/kg b.w

p.o was administered for 28 days. The changes in body weight, food and water

intake was observed for entire study. No significant decrease in body weight

was observed. The MERR treated rats did not show any significant changes in

haematological parameters like Hb, RBC, WBC, neutrophils, monocytes,

eosinophil and lymphocyte, ECR, PCV values when compared with normal

control animal. Histopathological examination of internal organs like liver,

kidney, spleen, heart, lungs, testis, ovary and brain showed no change in their

normal architecture suggesting no damage caused by MERR treatment.

4.3.3 Haematological Parameters

The methanolic extract of Rubus racemosus did not show any

remarkable changes in Haemoglobin, RBC, WBC, ESR, PCV and Neutrophils.

The Results are shown in Table.12.

4.3.4 Antidiabetic Activity

4.3.4.1 Effect of MERR on blood glucose level in normal rats

The MERR at a dose level of 200mg/kg p.o did not exhibit any

significant hypoglycemic effect in the fasted normal rats after 60 min and 120

min of oral administration, when compared with the initial blood glucose levels

before administration of the extracts. A higher dose of 400 mg/kg, also did not

exhibit significant hypoglycemic action at the end of 60,90 and 120 min after

oral administration in the normal fasted rats. Results are shown in table 14 and

figure 12.

4.3.4.2 Effect of MERR on blood glucose level on glucose fed

hyperglycemic rats

The MERR at a dose level of 200mg/kg and high dose 400mg/kg/p.o

reduced the raised blood glucose level [hyperglycemic due to glucose load

2 gm/kg/p.o at 4 ml/kg] significantly [p<0.001] after 120 min of oral

administration when compared to control group. This standard drug

glibenclamide also produced significant [p<0.001] drop in blood glucose level.

Results are shown in table 15 and figure 13.

4.3.4.3 Effect of acute treatment of MERR on blood glucose level in STZ


The effect of MERR was evaluated at a double dose administration of

MERR [low dose 200 mg/kg/p.o and high dose of 400mg/kg/p.o] a double dose

of extracts did not produce significant reduction in the blood glucose levels in

STZ induced diabetic rats.

Treatment with glibenclamide also did not reduce the blood glucose

levels significantly. Results are shown in table 16 and figure 14.

4.3.4.4 Effect of sub acute treatment of MERR on blood glucose level in

STZ induced diabetic rats

In the sub acute study STZ induced diabetic rats were treated with

MERR 200 mg/kg/p.o and 400mg/kg/p.o for duration of 28 days. Treatment

with MERR significantly [p<0.001] decreased the blood glucose level after 21

days and concomitantly after that in diabetic rats. Treatment with MERR

produced a significant drop in blood glucose level from 21st day onwards upto

28 days. Treatment with gilbenclamide produced significant [p<0.001]

decrease in blood glucose level steadily after the 14th day or oral administration

and thereafter. Results are shown in table 17 and figure 15.

4.3.4.5 Biochemical Estimation

4.3.4.5.1 Total bilirubin

The diabetic control animals showed significant [p<0.001] increase in

serum total bilirubin level when compared to control animals. The serum total

bilirubin levels in MERR (200mg and 400 mg/kg b.wt) treated diabetic rats

showed significant increase [p<0.001] and glibenclamide (0.5 mg/kg b.w/p.o)

treatment showed significant [p<0.001] decrease when compared to STZ

induced diabetic animals. Results are shown in Table 18 and figure 16.

4.3.4.5.2 Serum glutamate oxalocetate transminase (SGOT)

The SGOT level was significantly [p<0.01] increased in STZ induced

diabetic rats when compared to control animals. SGOT levels of diabetic rats

treated with MERR (200mg and 400 mg/kg b.wt) showed significant decrease

[p<0.01] and [p<0.001] respectively. However glibenclamide (0.5 mg/kg

b.w/p.o) treatment showed significant [p<0.001] decrease when compared to

STZ induced diabetic rats. Results are shown in Table 19 and figure 17.

4.3.4.5.3 Serum glutamate pyruvate trasaminase (SGPT)

The SGPT level was significantly [p<0.01] increased in STZ induced

diabetic rats when compared to control animals. SGPT levels of diabetic rats

treated with MERR (400 mg/kg b.w p.o) and glibenclamide (0.5 mg/kg

b.w/p.o) showed significant (p<0.01) decrease when compared to STZ induced

diabetic rats. Results are shown in Table 20 and figure 18.

4.3.4.5.4 Serum total protein

The diabetic control animals showed significant [p<0.001] decrease in

serum total protein level when compared to control animals. The serum total

protein levels in MERR (200mg and 400 mg/kg b.wt) treated diabetic rats

showed significant increase [p<0.001]. Glibenclamide (0.5 mg/kg/p.o) showed

significant increase [p<0.001] when compared to STZ induced diabetic rats.

Results are shown in table 21 and figure 19.

4.3.4.5.5 Alkaline phosphatase (ALP)

The ALP level in serum was significantly [p<0.001] increased in STZ

induced diabetic rats when compared to control. ALP levels of diabetic rats

treated with MERR (400 mg/kg b.w p.o) and glibenclamide (0.5 mg/kg

b.w/p.o) showed significant (p<0.001 and p<0.001, respectively) decrease

when compared to STZ induced diabetic rats. Results are shown in Table 22

and figure 20.

4.3.4.5.6 Serum total cholesterol

The total cholesterol level significantly [p<0.001] increased in STZ

induced diabetic rats when compared to control rats. Serum total cholesterol

levels of diabetic animals treated with MERR (200 and 400 mg/kg b.w/p.o)

showed significant [p<0.01] decrease in cholesterol level when compared to

STZ induced diabetic animals. Results are shown in Table 23 and figure 21.

4.3.4.5.7 Serum HDL-Cholesterol

The serum HDL-cholesterol level was significantly decreased [p<0.001]

in STZ induced diabetic rats when compared to control rats. HDL-cholesterol

level of diabetic rats treated with MERR (200 and 400 mg/kg b.wt/p.o) showed

significant increase [p<0.001] and glibenclamide (0.5 mg/kg b.w/p.o) treatment

showed significant [p<0.001] increase HDL-cholesterol when compared to

STZ induced diabetic animals. Results are shown in Table 24 and figure 22.

4.3.4.5.8 Serum triglycerides

The serum triglyceride level was significantly [p<0.001] increased in

STZ induced diabetic rats when compared to control rats. Triglyceride level of

diabetic rats treated with MERR (200 and 400mg/kg b.wt/p.o) showed

significant decrease [p<0.001] and glibenclamide (0.5 mg/kg b.w/p.o)



4.3.4.5.9 Serum LDL-Cholestrol

The serum LDL-cholesterol level was significantly [p<0.001] increased

in STZ induced diabetic rats when compared to control rats. LDL-cholesterol

level of diabetic rats treated with MERR (200 and 400 mg/kg b.wt/p.o) was

showed significant decrease [p<0.001] and glibenclamide (0.5 mg/kg b.w/p.o)



4.3.4.6 Antioxidant enzymes in liver Homogenate

4.3.4.6.1 Superoxide Dismutase

A significant [p<0.01] decrease in the liver SOD was observed in STZ

induced diabetic animals when compared to control animals. The liver SOD

levels of diabetic animals treated with double dose of MERR and

glibenclamide showed significant [p<0.01] increase when compared to STZ

induced diabetic animals. Results are shown in table 27 and figure 25.

4.3.4.6.2 Catalase

A significant [p<0.001] decrease in the liver CAT was observed in STZ

induced diabetic animals when compared to control animals. The liver CAT

level of diabetic rats treated with double dose of MERR was significantly

[p<0.001] increased. Glibenclamide treatment showed slight [p<0.01] increase

in CAT when compared to STZ induced diabetic rats. Results are shown in

table 28 and figure 26.

4.3.4.6.3 Glutathione Peroxidase

The GSH PX lelvel in lever was significantly [p<0.001] decreased in

STZ induced diabetic animals when compared to control animals. Double dose

of MERR significantly [p<0.001] increased the GSH-PX level when compared

to STZ induced diabetic animals. Glibenclamide treatment showed [p<0.001]

increase in GSH-PX when compared to STZ induced diabetic rats. Results are

shown in table 29 and figure 27.

4.3.4.6.4 Glutathione Reductase

The GSH Rase level in and liver was significantly [p<0.001] decreased

in STZ induced diabetic rats when compared to control. The liver GSH Rase

level of diabetic rats treated with double dose of MERR significantly [p<0.01]

increased and glibenclamide also showed significant [p<0.05] increase when

compared to STZ induced diabetic animals. Results are shown in table 30 and

figure 28.

4.3.4.6.5 Lipid Peroxidation (LPO)

A significant [p<0.001] increase in the liver lipid peroxidation was

observed in STZ induced diabetic animals when compared to control animals.

Lipid peroxidation level of animals treated with double dose of MERR showed

significant [p<0.001] decrease and glibenclamide treatment showed significant

[p<0.001] decrease when compared to STZ induced diabetic animals. Results

are shown in table 31 and figure 29.

4.3.5 In vitro Antioxidants Studies

4.3.5.1 Free radical scavenging activity by DPPH reduction

Reduction of the DPPH radicals can be observed by the decrease in the

absorbance at 517 nm. The scavenging capacity of the MERR was found to be

86.81% with IC50 being 348.87 µg/ml. Results are shown in Table 34 and

figure 32.

4.3.5.2 Nitric oxide scavenging activity

The scavenging of nitric oxide by MERR was concentration dependent

and the IC50 value was found to be 219.53µg/ml. The percentage scavenging of

Nitric oxide was as high as 84.26% at the concentration of 1000 µg/ml. Results

are shown in Table 35 and figure 33.

4.3.5.3 Hydroxyl radical scavenging activity

The MERR scavenged the hydroxyl radicals generated by the

EDTA/H2O2 system, when compared with that of control. The percentage

scavenging of the Hydroxyl radicals by MERR increased in a dose dependent

manner and was found to the 83.42% at 1000 µg/ml concentration. The IC50

value was found to be 520.32 µg/ml. Results are shown in Table 36 and figure

34.

4.3.5.4 Determination of reducing power

The reducing power of MERR with increasing concentration of MERR

showed significant increase in activities than control. Results were comparable

with the standard (BHT). Results are shown in Table 37 and figure 35.

4.3.5.5 Determination of total phenolic compounds

Phenols are very important plant constituents because of their

scavenging ability due to their hydroxyl groups. In the MERR (1mg),

137±0.078 µg pyrocatechol equivalent of phenols was detected.

4.3.6 Antiepileptic activity

4.3.6.1 Maximal electroshock induced convulsion

Effects of MERR on MES induced Epilepsy

Phenytoin treated animals have shown 100% protection against MES

induced seizures where as MERR 200mg/kg and 400 mg/kg have shown

54.35% and 67.31% protection respectively against MES induced seizures.

The MERR at both doses and standard treated rats did not show any

significant change in duration of tonic flexion and clonic convulsions.

MERR 200 mg/kg and 400 mg/kg had shown a significant decrease in

the duration of tonic extensor phase and comparable p<0.001 with the standard.

Results are shown in Table 38 and figure 36.

4.3.6.2 Effect of MERR on neurotransmitter levels in MES induced rats

4.3.6.2.1 Serotonin

A significant p<0.001 & p<0.001 decrease in brain Serotonin levels was

observed in forebrain of epileptic control animals. MERR 200mg/kg and 400

mg/kg treated rats have shown a significant p<0.001, p<0.001 increase in

Serotonin levels in forebrain. The results are shown in Table 39 and figure 37.

4.3.6.2.2 Noradrenaline

A significant p<0.001 decrease is observed in forebrain in epileptic

control animals. MERR 200mg/kg and 400 mg/kg and PHT treated animals

showed a significant p<0.05 & p<0.001 increase in Noradrenaline levels in

forebrain of MERR treated animals. Results are shown in Table 40 and

figure 38.

4.3.6.2.3 Dopamine

A significant p<0.001 decrease in the dopamine levels is observed in

forebrain in epileptic control animals and a significant p<0.001 increase is

observed in forebrain on MERR 200mg/kg and 400 mg/kg treated rats, PHT

treated animals showed a significant p<0.001 increase forebrain. Results are

shown in Table 41 and figure 39.

4.4 ANTIMICROBIAL STUDIES

4.4.1 Antibacterial Activity

Methanol, Chloroform, Petroleum ether and Aqueous extracts exhibited

significant activity at the concentration of 100µg/ml whereas it showed

moderate activity at the concentration of 25µg/ml and 50µg/ml against

Staphylococcus aureus. The Results were shown in Table 42 and figure 40.




Staphylococcus epidermidis. The Results were shown in Table 43 and figure

41.



moderate activity at the concentration of 25µg/ml and 50µg/ml against Bacillus

cereus. The Results were shown in Table 44 and figure 42.




Micrococcus luteus. The Results were shown in Table 45 and figure 43.




Klebsiella pneumoniae. The Results were shown in Table 46 and figure 44.




Pseudomonos aeruginosa. The Results were shown in Table 47 and figure 45.




Escherichia coli. The Results were shown in Table 48 and figure 46.

4.4.2 Antifungal Activity




Aspergillus Niger and Aspergillus fumigatus. The Results were shown in

Tables 49 , 50 and figures 47 and 48.

The minimum inhibitory concentration values of all the extracts were

determined.

Acute toxicity class method OECD guidelines 423

Table: 11

Weight Of Animal Group

S. No

Group Dose Before Test

(on 1st day)

After Test

(on 4th day)

Signs Of toxicity

Onset of toxicity

Reversible Of

Irreversible Duration

1

MERR

2000mg

158

155

No sign of

toxicity

Nil

Nil

14 days

2

MERR

2000mg

162

159

No sign of

toxicity

Nil

Nil

14 days

3

MERR

2000mg

160

159

No sign of

toxicity

Nil

Nil

14 days

As no toxicity or death was observed for these dose levels the same dose level was tried again

1

MERR

2000mg

160

157

No sign of

toxicity

Nil

Nil

14 days

2

MERR

2000mg

163

161

No sign of

toxicity

Nil

Nil

14 days

3

MERR

2000mg

162

159

No sign of

toxicity

Nil

Nil

14 days

MERR: Methanolic Extract of Rubus Racemosus

121

Haematological parameters of MERR treatment on sub-acute toxicity study

Table: 12

Groups Hb% RBC

X106/mm3

WBC

Cells/mm3 ESR PCV

%

Neutrophil

%

Eosonophil

%

Lympocytes

%

Monocytes

Control 13.86±.76 4.7±.58 8.3±.1 3.4 ±1 40.2 ± 2.83 24±.8 4.38±.7 75.2±.4 2.15±.4

MERR 13.52±.68ns 4.57±.52ns 7.92±.26ns 3.2 ±.6 45.25 ± 3.56 21.1±.7ns 3.21±.92ns 69.1±1.6ns 1.3±.4ns

Values are expressed in mean ± SEM. Each group consists of 6 rats.

Statistical significance test for comparison was done by Student ‘t’ test.

ns – non significant

Histopathology report of various organs on MERR

treatment on sub-acute toxicity study

Table: 13

Report

S. No

Organ Control I Group II

1.

Liver

Shows normal liver with central vein with cords of hepatocytes

Shows normal liver with central vein with cords of hepatocytes

2.

Kidney

Normal kidney with glomeruli and tubules

Normal kidney with glomeruli and tubules

3.

Heart

Normal cardiac fibre

Normal cardiac fibre

4.

Testis

Shows normal testicular tubules with normal spermatogenesis

Shows normal testicular tubules with normal spermatogenesis

5.

Lung

Shows normal lung tissue with bronchi and alveoli

Shows normal lung tissue with bronchi and alvcoli

6.

Brain

Shows normal brain tissues with astrocytes and nerve fibres.

Shows normal brain tissues with astrocytes and nerve fibres.

7.

Spleen

Spleen with follicles and hyaline septa

Spleen with follicles and hyaline septa

8.

Ovary

Ovary with maturing follicles

Ovary with maturing follicles

Group I – Rats received 1% SCMC (2ml/kg) orally for 28 days.

Group II – rats received MERR (400mg/kg) orally for 28 days.

Figure.11

Histopathology of various organs in toxicity studies

ANTIDIABETIC ACTIVITY

Effect of Rubus Racemosus treatment on blood glucose level in normoglycaemic rats

Table: 14

Groups Initial 30min 60min 90min 120min

I 75.56 ± 0.87 87.85 ± 0.59 112.05 ± 1.07 105.03 ± 0.37 97.16 ±0.71

II 76.1 ± 0.47 111.8 ± 0.76 117.16 ± 0.78 85.33 ± 0.77 72.73 ± 1.49

III 76.08 ± 0.41 95.16 ± 1.05 104 ± 0.93 91.66 ± 0.64 80.8 ± 0.36

The values are expressed as mean ± SEM. Each groups having six animals.

Statistical significant test for comparison was done by ANOVA, followed by

Dunnet’s “t” test. The blood glucose values of group II and III are compared

with control animal values.

Figure.12

Effect of Rubus Racemosus of blood glucose level in normoglycaemic rats

0

20

40

60

80

100

120

140

Int ial 30min 60min 90min 120min

mg/

dl I

II

III

Effect of Rubus Racemosus on blood glucose fed hyperglycaemic rats

Table: 15

Groups Initial 30min 60min 90min 120min I 76.28 ± 0.7 74.8 ± 0.64 70.71 ± 1.00 69.66 ± 0.79 72.08 ± 0.69 II 79.71 ± 1.24a*** 126.5 ± 0.84a*** 129 ± 0.52a*** 113.46 ± 1.27a*** 80.33 ± 0.52a*** III 77.81 ± 0.79b*** 130 ± 0.93b*** 117.5 ± 1.05b*** 95.66 ± 0.91b*** 82.33 ± 0.60b*** IV 80 ± 0.78b*** 138.1 ± 0.52b*** 114.98 ± 0.65b*** 98.7 ± 0.64b*** 88.56 ± 0.8b***

The values are expressed as mean + SEM. Each group having six animals.

Statistical significant test for comparison done by ANOVA, followed by

Dunnet’s “t” test.

(a) Group II is compared with group I values

(b) Group III and IV are compared with group II values.

The 60th, 90th and 120th min values are compared with initial values: non

significant.

***p<0.001

Figure.13

Effect of Rubus Racemosus on blood glucose fed hyperglycaemic rats

0

20

40

60

80

100

120

140

160

Intial 30min 60min 90min 120min

mg/

dl

I

II

III

IV

Effect of acute treatment of Rubus Racemosus on

blood glucose in STZ induced diabetic rats

Table: 16

Groups Initial 1hr 3hr 5hr

I 86.28 ± 0.4 95.8 ± 0.53 95 ± 0.66 86.05 ± 0.57

II 274.33 ± 2.26ns 286 ± 1.07ns 297.83 ± 0.9ns 294.1 ± 0.61ns

III 270.16 ± 1.66ns 279 ± 2.18 ns 273.7 ± 1.94 ns 256.8 ±0.52ns

IV 245.83 ± 0.52ns 277.67 ± 2.07ns 250.5 ± 1.50ns 236.7 ± 0.52ns

V 234.33 ± 0.74ns 247.33 ± 1.38ns 256 ± 1.30ns 230 ± 3.34ns

The values are expressed as mean + SEM. Each groups having six animals.

Statistical significant test for comparison done by ANOVA, followed by

Dunnet’s “t” test. The values 1st, 3rd and 5rd hour are compared with initial

values. ns – non significant

Figure.14

Effect of acute treatment of Rubus Racemosus on blood glucose in STZ induced diabetic rats

0

50

100

150

200

250

300

350

Intial 1hr 3hr 5hr

mg/

dl

I

II

III

IV

V

Effect of sub-acute treatment of Rubus Racemosus on Blood glucose in STZ induced diabetic rats

Table: 17

Groups Initial 1 day 7 day 14 day 21 day 28 day

I 67.66 ± 0.53 75.33 ± 0.84 67.9 ± 0.54 71.5 ± 0.52 67.8 ± 0.37 69.33 ± 1.36

II 257.5 ± 0.96 277.66 ± 1.05 a*** 297.73 ± 0.63 a*** 334.5 ± 4.06a*** 356.33 ± 1.84 a*** 365.16 ± 1.20a***

III 255.16 ± 0.9 276.66 ± 0.39b** 278.83 ± 1.35b** 242.5 ± 3.28b** 218.16 ± 1.94b*** 191.66 ± 2.64b***

IV 248.66 ± 1.38 267.66 ± 0.90b** 271.26 ± 1.50b** 255.3 ± 1.12b** 228.5 ± 1.86b*** 163.16 ± 8.31b***

V 248.66 ± 1.08 264.83 ± 1.37 b** 268.66 ± 3.56 b** 191.66 ± 1.12b*** 168.33 ± 2.02b*** 135.3 ± 1.2b***


Statistical significant test for comparison was done by ANOVA, followed by

Dunnet’s “t” test.


(b) Group III, IV and V are compared with group II values.

***p<0.001;** p<0.01

Figure.15

Effect of Sub-acute treatment on Rubus Racemosus on blood glucose in STZ induced diabetic rats

0

50

100

150

200

250

300

350

400

1 2 3 4 5 6

mg/

dl

Series1Series2Series3Series4Series5

BIOCHEMICAL PARAMETERS

Effect of Methanolic extract of Rubus Racemosus on Serum Total Bilirubin in STZ induced diabetic rats

Table: 18

Group Treatment Total Bilirubin (mg/dl)

I (n=6) Control 0.17 ± 0.006

II (n=6) Diabetic Control 0.38 ± 0.007a***

III (n=6) Gilbenclamide 0.25 ± 0.01b***

IV (n=6) MERR 200mg 0.19 ± 0.006b***

V (n=6) MERR 400mg 0.23 ± 0.004b***


n= number of animals in each group. Statistical significant test for comparison

was done by ANOVA, followed by Dunnet’s “t” test.

(a) Group II is compared with group I values (b) Group III, IV and V are compared with group II values. ***p<0.001

Figure.16

Effect of Rubus Racemosus on serum Total bilirubin of STZ induced diabetic rats

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

I II III IV V

mg/

dl

Effect of Methanolic extract of Rubus Racemosus on SGOT in STZ induced diabetic rats

Table: 19

Group Treatment SGOT (IU/dl)

I (n=6) Control 36.9 ± 0.46

II (n=6) Diabetic Control 92.57±0.22a**

III (n=6) Gilbenclamide 63.46±0.23b**

IV (n=6) MERR 200mg 42.01±0.75b**

V (n=6) MERR 400mg 45.62±0.06b*** The values are expressed as mean ± SEM. Each groups having six animals.



(a) Group II is compared with group I values (b) Group III, IV and V are compared with group II values. ***p<0.001; ** p<0.01

Figure.17

Effect of Rubus Racemosus on serum SGOT of STZ induced diabetic rats

0

20

40

60

80

100

I II III IV V

Groups

IU/d

l

Effect of Methanolic extract of Rubus Racemosus on SGPT in STZ induced diabetic rats

Table: 20

Group Treatment SGPT (IU/dl)

I (n=6) Control 42.8 ± 0.55

II (n=6) Diabetic Control 95.36±1.1a**

III (n=6) Gilbenclamide 64.18±0.51b**

IV (n=6) MERR 200mg 55.58±0.24b**

V (n=6) MERR 400mg 58.35±0.22b**

The values are expressed as mean ± SEM. Each groups having six animals. n= number of animals in each group. Statistical significant test for comparison was done by ANOVA, followed by Dunnet’s “t” test. (a) Group II is compared with group I values (b) Group III, IV and V are compared with group II values. **p<0.01

Figure.18

Effect of Rubus Racemosus on serum SGPT of STZ induced diabetic rats

0

20

40

60

80

100

120

I II III IV V

Groups

IU/d

l

Effect of Methanolic extract of Rubus Racemosus on Serum Total Protein in STZ induced diabetic rats

Table: 21

Group Treatment Total protein (mg/dl)

I (n=6) Control 7.3 ± 0.11



IV (n=6) MERR 200mg 6.65 ± 0.07b***

V (n=6) MERR 400mg 6.16 ±0.03b***




(a) Group II is compared with group I values (b) Group III, IVand V are compared with group II values. ***p<0.001

Figure.19

Effect of Rubus Racemosus on serum Total Protein of STZ induced diabetic rats

0

1

2

3

4

5

6

7

8

I II III IV V

Groups

mg/

dl

Effect of Methanolic extract of Rubus Racemosus on Serum Alkaline Phosphatase in STZ induced diabetic rats

Table: 22

Group Treatment Alkaline

Phosphatase (mg/dl)

I (n=6) Control 68.1 ± 0.31



IV (n=6) MERR 200mg 96.1 ± 0.71b***

V (n=6) MERR 400mg 104.8 ± 0.34b***




(a) Group II is compared with group I values (b) Group III, IVand V are compared with group II values. ***p<0.001

Figure.20

Effect of Rubus Racemosus on serum Alkaline phosphatase of STZ induced diabetic rats

0

20

40

60

80

100

120

140

160

180

I II III IV V

Groups

mg/

dl

Effect of Methanolic extract of Rubus Racemosus on Serum Total Cholesterol in STZ induced diabetic rats

Table: 23

Group Treatment Total Cholesterol

(mg/dl)

I (n=6) Control 64.46 ± 0.15



IV (n=6) MERR 200mg 74.75 ± 0.28b***

V (n=6) MERR 400mg 68.5 ± 0.32b***





(b) Group III, IVand V are compared with group II values.

***p<0.001

Figure.21

Effect of Rubus Racemosus on serum Total Cholesterol of STZ induced diabetic rats

0

20

40

60

80

100

120

140

I II III IV V

Groups

mg/

dl

Effect of Methanolic extract of Rubus Racemosus on Serum HDL-Cholesterol in STZ induced diabetic rats

Table: 24

Group Treatment HDL- Cholesterol

( mg/dl)

I (n=6) Control 48.13 ± 0.26



IV (n=6) MERR 200mg 45.2 ± 0.3b***

V (n=6) MERR 400mg 46.13 ± 0.13b***


n= number of animals in each group.Statistical significant test for comparison




***p<0.001

Figure.22

Effect of Rubus Racemosus on serum HDL-Cholesterol of STZ induced diabetic rats

0

10

20

30

40

50

60

I II III IV V

Groups

mg/

dl

Effect of Methanolic extract of Rubus Racemosus on Serum Triglyceride in STZ induced diabetic rats

Table: 25

Group Treatment Triglyceride (mg/dl)

I (n=6) Control 43.93 ± 0.26 II (n=6) Diabetic Control 64.9 ± 0.13a*** III (n=6) Gilbenclamide 56.9 ± 0.18b*** IV (n=6) MERR 200mg 58.1 ± 0.12b***

V (n=6) MERR 400mg 55.6 ± 0.11b***






***p<0.001

Figure.23

Effect of Rubus Racemosus on serum Triglyceride of STZ induced diabetic rats

0

10

20

30

40

50

60

70

I II III IV V

Groups

mg/

dl

Effect of Methanolic extract of Rubus Racemosus on Serum LDL-Cholesterol in STZ induced diabetic rats

Table: 26

Group Treatment LDL- Cholesterol ( mg/dl)

I (n=6) Control 18.6 ± 0.40 II (n=6) Diabetic Control 40.13 ± 0.78a*** III (n=6) Gilbenclamide 16.35 ± 0.19b*** IV (n=6) MERR 200 mg 14.55 ± 0.07b***

V (n=6) MERR 400 mg 13.92 ± 0.09b***






***p<0.001

Figure.24

Effect of Rubus Racemosus on serum LDL-Cholesterol of STZ induced diabetic rats

0

5

10

15

20

25

30

35

40

45

I II III IV V

Groups

mg/

dl

INVIVO – ANTIOXIDANT STUDIES

Effect of Methanolic Extract on Superoxide dimutase in STZ induced diabetic rats

Table: 27

Groups Treatment SOD (mg/dl)

I (n=6) Control 7.48 ± 0.11 II (n=6) Diabetic Control 4.47 ± 0.17a** III (n=6) Gilbenclamide 5.98 ± 0.096b** IV (n=6) MERR 200 mg 6.8 ± 0.06 b**

V (n=6) MERR 400 mg 7.58 ± 0.16b**




(a) Group II is compared with group I values (b) Group III, IV and V are compared with group II values. **p<0.01 SOD Units – Units/mg protein 1Units = The amount of enzyme required bring about 50% of inhibition of auto oxidation of pyrogallol

Figure.25

Effect of Rubus Racemosus extract on SOD levels in STZ induced diabetic rats

0

1

2

3

4

5

6

7

8

9

I II III IV V

Groups

U/m

g of

pro

tein

Effect of Methanolic Extract on Catalase in STZ induced diabetic rats

Table: 28

Groups Treatment Catalase (mg/dl)

I (n=6) Control 85.31 ± 0.34

II (n=6) Diabetic Control 76.4 ± 0.39 a***

III (n=6) Gilbenclamide 78.5 ± 0.56b**

IV (n=6) MERR 200 mg 80.8 ± 0.28b***

V (n=6) MERR 400 mg 82.8 ± 0.26b***






***p<0.001;**p<0.01

Figure.26

Effect of Rubus Racemosus extract on Catalase in STZ induced diabetic rats

70

72

74

76

78

80

82

84

86

88

I II III IV V

Groups

n m

oles

of h

ydro

gen

pero

xide

de

com

pose

d/m

in/m

g pr

otei

n

Effect of Methanolic Extract on Glutathione Peroxidase in STZ induced diabetic rats

Table: 29

Group Treatment Glutathione Peroxidase (mg/dl)

I (n=6) Control 9.18 ± 0.17 II (n=6) Diabetic Control 4.7 ± 0.18a*** III (n=6) Gilbenclamide 5.7 ± 0.27b*** IV (n=6) MERR 200 mg 7.4 ± 0.14b***

V (n=6) MERR 400 mg 8.6 ± 0.13b***

The values are expressed as mean ± SEM. Each groups having six animals. n= number of animals in each group. Statistical significant test for comparison was done by ANOVA, followed by Dunnet’s “t” test.



***p<0.001

Units – n moles of hydrogen peroxide decomposed/min/mg protein.

Figure.27

Effect of Rubus Racemosus extract on Glutathione Peroxidase in STZ induced diabetic rats

0

1

2

3

4

5

6

7

8

9

10

I II III IV V

Groups

mol

es/m

in/m

g of

pro

tein

Effect of Methanolic Extract on Glutathione Reductase in STZ induced diabetic rats

Table: 30

Group Treatment Glutathione Reductase (mg/dl)

I (n=6) Control 1.29 ± 0.05 II (n=6) Diabetic Control 0.68 ± 0.01a*** III (n=6) Gilbenclamide 0.81 ± 0.01b* IV (n=6) MERR 200 mg 0.87 ± 0.02b** V (n=6) MERR 400 mg 1.04 ± 0.06b***





(b) Group III, IV, V are compared with group II values.

***p<0.001; **p<0.01; *p<0.05

Units – n moles of GSSG utilized/min/mg protein.

Figure.28

Effect of Rubus Racemosus extract on Glutathione Reductase in STZ induced diabetic rats

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

I II III IV V

Groups

n m

oles

of G

SSG

util

ized

m

in/m

g pr

otei

n

Effect of Methanolic Extract on Lipid Peroxidation in STZ induced diabetic rats

Table: 31

Groups Treatment Lipid Peroxidation

(mg/dl) I (n=6) Control 0.58 ± 0.01 II (n=6) Diabetic Control 0.92 ± 0.02a*** III (n=6) Gilbenclamide 0.83 ± 0.01b*** IV (n=6) MERR 200 mg 0.75 ± 0.01b*** V (n=6) MERR 400 mg 0.66 ± 0.02b***






***p<0.001

Figure.29

Effect of Rubus Racemosus extract on Lipid Peroxidation in STZ induced diabetic rats

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

I II III IV V

Groups

n m

oles

of M

DA

libe

rate

/min

/mg

prot

ein

Histopathological studies of the Liver

Table: 32

Sl.No.

Group

Observation

1.

Group I

H & E section shows normal liver with central vein surrounded by hepatocytes

2.

Group II

Liver shows vacuolated hepatocytes

3.

Group III

Liver shows almost normal hepatocytes (minimum vacuolation)

4.

Group IV

Liver shows almost normal liver parenchyma with minimal vacuolation

Figure.30

Histopathology of the liver section

Histopathological Studies of the Pancreas

Table: 33

Sl.No.

Group

Observation

1.

Group I

The pancreas section showed normal acini with islet of β-cells

2.

Group II

Pancreas shows atrophic acini and reduction islet β-cell size.

3.

Group III

Pancreas shows markedly proliferative (hyperplastic) islets β-cells

4.

Group IV

Pancreas shows preserved islet cells.

Figure.31

Histopathology of the pancreatic section

Free radical scavenging activity of MERR by DPPH reduction

Table: 34

SL. NO.

Concentration in MERR (µg/ml)

% Inhibition

IC50 value

(µg/ml) 1. 25 10.35±0.19

2. 50 29.42±2.01 3. 100 39.14±0.22 4. 200 54.54±0.20

5. 400 60.65±0.29 6. 800 71.13±1.19 7. 1000 86.81±0.19

348.87

Values are mean ± SEM of 6 parallel measurements.

MERR: Methanolic extract of Rubus Racemosus

Figure.32

0

20

40

60

80

100

25 50 100 200 400 800 1000

Concentration in mcg/ml

% in

hibi

tion

Nitric oxide scavenging activity of MERR

Table: 35

SL. NO. Concentration

of MERR (µg/ml)

% Inhibition IC50 value (µg/ml)

1. 25 22.01 ± 0.28

2. 50 32.28 ± 0.51

3. 100 53.87 ± 0.59

4. 200 53.78 ± 0.71

5. 400 77.03 ± 0.39

6. 800 82.22 ± 0.37

7. 1000 84.26 ± 0.40

219.53

Values are mean ± SEM of 6parallel measurements.


Figure.33

0

20

40

60

80

100

25 50 100 200 400 800 1000


% in

hibi

tion

Hydroxyl radical scavenging activity of MERR

Table: 36

SL. NO. Concentration

of MERR (µg/ml)% Inhibition IC50 value

(µg/ml)

1. 25 52.53±0.41

2. 50 62.82±0.15

3. 100 67.92±0.26

4. 200 74.65±0.31

5. 400 82.57±0.22

6. 800 84.15±0.20

7. 1000 83.42±0.32

520.32

Values are mean ± SEM of parallel measurements.


Figure.34

0

20

40

60

80

100

25 50 100 200 400 800 1000


% in

hibi

tion

Effect of MERR and BHT on reducing power

Table: 37

SL.NO. Concentration

of MERR (µg/ml)

Absorbance (OD) of MERR

Absorbance (OD) of BHT

1. Control 0.094

2. 25 0.086 ± 0.002 0.268±0.004

3. 50 0.108 ± 0.003 0.287±0.004

4. 100 0.194 ± 0.005 0.316±0.002

5. 200 0.213 ± 0.002 0.340±0.002

6. 400 0.967 ± 0.003 0.361±0.002

7. 800 1.016 ± 0.002 0.382±0.003

8. 1000 1.130±0.002 0.389±0.098

Values are mean ± SEM of 6 parallel measurements.


Figure.35

0

0.2

0.4

0.6

0.8

1

1.2

25 50 100 200 400 800 1000


Abs

orba

nce

MERRBHT

Effect of MERR on MES induced epilepsy Table: 38

Groups %

Protection Flexion Extensor Clonus Stupor Recovery

I. Control (CMC)

0 6.87±0.41 13.89±0.81 9.89±0.52 16±0.32 190.82

II. PHT 100 5.27±0.38 0 9±0.32** 12.17±0.58*** 109.34

III. MERR

200 51.69 6.18±0.35ns 6.28±0.61*** 10±0.43ns 15.98±0.47ns 147.35

IV.MERR 400

61 5.87±0.37*** 5.07±0.37*** 8.78±0.55ns 14.68±0.57ns 122.81

Values are expressed as mean± SEM of six observations. Comparison between Group I Vs Group II, Group II Vs Group II & Group IV. Statistical significant test for comparison was done by ANOVA, followed by Dunnet’s ‘t’ test *p<0.05; ** p<0.01; ***p<0.001; ns-non significant. Values expressed in seconds.

Figure.36

0

2.5

5

7.5

10

12.5

15

17.5

20

Flexion Extensor Clonus Stupor

Tim

e in

sec

onds Control (CMC)

PHTMERR 200MERR 400

Effect of MERR on Serotonin levels in MES induced epilepsy

Table: 39

Groups Drug used Serotonin (ng/mg)

I Control 181.33 ± 1.84

II MES 77.83 ± 1.34a***

III PHT 98.16 ± 0.9b***

IV MERR 200 84.5 ± 1.73b***

V MERR 400 88.83 ± 1.02b***

Values are expressed as mean ± SEM of six observations. Comparison between

Group I Vs Group II, Group III Vs Group IV & Group V. Statistical significant

test for comparison was done by ANOVA, followed by Dunnet’s ‘t’ test

*p<0.05; ** p<0.01; ***p<0.001; ns-non significant. Units = ng/mg of wet

tissue

Figure.37

Effect of MERR on Serotonin levels in MES induced epilepsy

0

20

40

60

80

100

120

140

160

180

200

I II III IV V

ng/m

g w

et ti

ssue I

IIIIIIVV

Effect of MERR on Non adrenaline levels in MES induced epilepsy

Table: 40

Groups Drug used Nor adrenaline (ng/mg)

I Control 718.3 ± 2.50

II MES 440.16 ± 1.02a***

III PHT 597.66 ± 1.90b***

IV MERR 200 490.83 ± 3.17b***

V MERR 400 520.3 ± 2.52b***

Values are expressed as mean± SEM of six observations. Comparison between

Group I Vs Group II, Group III Vs Group IV & Group V. Statistical significant

test for comparison was done by ANOVA, followed by Dunnet’s ‘t’ test.

*p<0.05; ** p<0.01; ***p<0.001; ns-non significant. Units = ng/mg of wet

tissue

Figure.38

Effect of MERR on Non adrenaline levels in MES induced epilepsy

0

100

200

300

400

500

600

700

800

I II III IV V

ng/mg wet tissue IIIIIIIVV

Effect of MERR on Dopamine levels in MES induced epilepsy

Table: 41

Groups Drug used Dopamine

(ng/mg)

I Control 813.3 ± 0.84

II MES 478.16 ± 1.29a***

III PHT 810 ± 1.12b***

IV MERR 200 626.66 ± 3.45b***

V MERR 400 710.3 ± 2.71b***

Values are expressed as mean± SEM of six observations. Comparison between

Group I Vs Group II, Group III Vs Group IV & Group V. Statistical

significant test for comparison was done by ANOVA, followed by Dunnet’s ‘t’

test. *p<0.05; ** p<0.01; ***p<0.001; ns-non significant. Units = ng/mg of wet

tissue.

Figure.39

Effect of MERR on Dopamine levels in MES induced epilepsy

0

100

200

300

400

500

600

700

800

900

I II III IV V

ng/m

g w

et ti

ssue I

IIIIIIVV

RESULTS OF ANTIMICROBIAL STUDIES

In Vitro evaluation of Anti-microbial activity of different

extracts of Rubus Racemosus on Staphylococcus aureus

Table: 42

Zone of Inhibition (mm) Extract Micro-

Organism Std. Control 25 (µl)

50 (µl)

100 (µl)

MIC Values

(µl)

Methanol 30 0 16 19 25 15

Chloroform 30 0 17 24 27 14

Petroleum ether 30 0 18 20 24 13

Aqueous

Staphylococcus

aureus

29 0 17 19 22 14

Figure.40

[S.aureus - Staphylococcus aureus; STD – Standard; C – Control; M.ex –

Methanol Extract; P.E – Petroleum ether Extract; Chlor. – Chloroform Extract;

Aque. – Aqueous Extract]

25, 50, 100 – Concentration of the extracts in mcg/ml.

STD Used – Ciprofloxacin → 5mcg/disc

In vitro evaluation of Anti-microbial activity of different

extracts of Rubus racemosus on Staphylococcus epidermidis

Table: 43



50 (µl)

100 (µl)

MIC Values

(µl)

Methanol 31 0 18 21 24 17

Chloroform 30 0 17 19 21 16


Aqueous


30 0 15 17 20 14

Figure.41

[S.epi - Staphylococcus epidermidis; STD – Standard; CON – Control; Me.ext

– Methanol Extract; Pe.ex. – Petroleum ether Extract; Ch. ex. – Chloroform

Extract; AQ. ex. – Aqueous Extract]


STD Used – Ciprofloxacin → 5 mcg/disc


extracts of Rubus racemosus on Bacillus cereus

Table: 44



50 (µl)

100 (µl)

MIC Values

(µl)

Methanol 31 0 16 19 26 15

Chloroform 30 0 17 20 27 14


Aqueous

Bacillus cereus

30 0 14 18 22 13

Figure.42

[B.Cereus – Bacillus cereus; STD – Standard; CON – Control; Met.ext –

Methanol Extract; Pe.ext. – Petroleum ether Extract; Ch. ext. – Chloroform



STD Used – Ciprofloxacin → 5 mcg /disc


extracts of Rubus racemosus on Micrococcus luteus

Table: 45

Zone of Inhibition (mm) Extract

Micro-

Organism Std. Control 25

(µl) 50

(µl) 100 (µl)

MIC Values

(µl)

Methanol 30 0 17 22 25 16

Chloroform 31 0 15 21 24 14


Aqueous

Micrococcus luteus

30 0 17 21 24 16

Figure.43

[M.luteus – Micrococcus luteus; STD – Standard; C – Control; Me.ex. –

Methanol Extract; P.E. – Petroleum ether Extract; Ch. ex. – Chloroform



STD Used – Ciprofloxacin → 5mcg/disc


extracts of Rubus racemosus on Klebsiella pneumoniae

Table: 46



50 (µl)

100 (µl)

MIC Values

(µl)

Methanol 30 0 17 19 22 14

Chloroform 29 0 16 20 23 15


Aqueous

Klebsiella

pneumoniae

28 0 14 16 21 12

Figure.44

[Kl.Pne – Klebsiella pneumonia; STD – Standard; C – Control; Me.ex. –

Methanol Extract; Pe.ex. – Petroleum ether Extract; Ch. ex. – Chloroform





extracts of Rubus racemosus on Pseudomonos aeruginosa

Table: 47



50 (µl)

100 (µl)

MIC Values

(µl)

Methanol 31 0 18 20 22 16

Chloroform 30 0 18 20 23 15


Aqueous

Pseudomonos

aeruginosa

32 0 14 19 23 12

Figure.45

[Ps.aeru – Pseudomonos aeruginosa; STD – Standard; CON – Control; Me.ex.

– Methanol Extract; Pe.ext. – Petroleum ether Extract; Ch. ex. – Chloroform



STD Used – Ciprofloxacin → 5 mcg /disc


extracts of Rubus racemosus on Escherichia coli

Table: 48


Organism Std. Control 25 (µl) 50 (µl) 100

(µl)

MIC Values

(µl)

Methanol 30 0 15 19 23 14

Chloroform 32 0 18 22 26 17


Aqueous

Escherichia coli

30 0 15 19 24 14

Figure.46

[E.coli – Escherichia coli; STD – Standard; CON – Control; Me.ex. – Methanol

Extract; Pe.ext. – Petroleum ether Extract; Ch. ex. – Chloroform Extract; AQ.

ex. – Aqueous Extract]



In Vitro evaluation of Anti-fungal activity of different

extracts of Rubus Racemosus on Aspergillus niger

Table: 49


Organism Std Control 25 (µl)

50 (µl)

100 (µl)

MIC Values

(µl)

Methanol 31 0 15 19 22 14

Chloroform 30 0 14 18 23 13


Aqueous

Aspergillus

niger

30 0 13 17 21 12

Figure.47

[A.niger – Aspergillus Niger ; STD – Standard; C – Control; Me.ex. –

Methanol Extract; P.E.. – Petroleum ether Extract; Ch. ex. – Chloroform



STD Used – Ketoconazole → 50 mcg /disc

In Vitro evaluation of Anti-fungal activity of different

extracts of Rubus Racemosus on Aspergillus fumigatus

Table: 50


Organism Std Control 25 (µl)

50 (µl)

100 (µl)

MIC Values

(µl)

Methanol 31 0 17 20 23 16

Chloroform 30 0 16 21 24 15


Aqueous


32 0 15 18 21 13

Figure.48

[A.fumigatus – Aspergillus fumigatus; STD – Standard; CON – Control;

Me.ex. – Methanol Extract; Pe.ext. – Petroleum ether Extract; Ch. ex. –

Chloroform Extract; AQ. ex. – Aqueous Extract]


STD Used – Ketoconazole → 50 mcg /disc

DISCUSSION

CHAPTER - V

DISCUSSION

The aerial parts of Rubus racemosus were identified, collected,

authenticated and were subjected to organoleptic, microscopical and physical

studies.

Plant anatomical studies may have a crucial role in plant identification

and standardization.

In the midrib region, inner epidermis are collenchymatous and the rest

of the ground tissue parenchyma. The vascular bundle consists of radial rows

of xylem and phloem elements.

In the lamina region adaxial epidermis is thick with large squarish cells

and thick cuticle whereas abaxial epidermis shows narrow rectangular cells.

The mesophyll tissue consists of two layers of short, thin palisade cells and 6-8

layers of small, lobed spongy parenchyma cells.

The leaf has dense glandular and non-glandular trichomes. The non

glandular trichomes are unicellular, unbranched, thick walled and pointed at the

tip. They arise from a pedestal of a group of cells raised above the level of the

epidermis.

The upper part (distal) of the petiole has three accessory lateral bundles,

whereas lower part (proximal) has 2 prominent lateral bundles. All the bundles

are collateral with thick band of sclerenchyma cells abutting the phloem tissue.

Next to the epidermis, 2 to 3 layers of collenchyma cells are seen.

The Stem has a thin layer of epidermis comprising of small thick walled

cells. The vascular cylinder is thin and continuous consisting of several wedge

shaped vascular bundles. The vascular bundles are collateral. The

sclerenchyma cells are thin walled and wide lumened. Xylem elements are

circular thick walled. The pith is parenchymatous.

The powdered sample of the leaf shows non glandular trichomes, they

are long whip like thick walled with smooth surface. The glandular trichomes

are long stalked with spherical head.

Powder microscopical character of the plant Rubus racemosus showed

unicellular, non-glandular trichomes, anisocytic type of stomata and prismatic

type of calcium oxalate crystals.

Powdered material was extracted successively with petroleum ether

ethyl acetate, chloroform, methanol and water. The extracts were subjected to

preliminary phytochemical screening to find the chemical constituents present.

It was found that flavonoids were present in petroleum ether, ethyl acetate,

chloroform, methanol and aqueous extracts of hot percolation type which was

confirmed by treatment of extracts with different respective reagents.

Alkaloids, Sterols and Proteins were absent in Petroleum ether, ethylacetate,

chloroform, methanol and aqueous extracts of hot percolation. Phenols,

tannins, carbohydrates, glycosides, Saponins and terpenes were observed in

pertoleum ether, ethyl acetate, chloroform, methanol and aqueous extracts of

hot percolation and confirmed by their respective reagents. Gums and mucilage

is absent in all the extracts.

All the extracts were subjected to thin layer chromatography and their Rf

values were determined.

The compounds isolated from methanolic extract of Rubus

racemosus was found to be Compound I - 1-[20, 21 – dodeacylnanone] – α-

1→6-D-glucotetraose-6’’’(P-hydroxy) benzoate. Compound II-9, 10

epoxynonacosane. The Rf values of the isolated compounds were found to be

0.62, 0.59.

The chloroform extract of Rubus racemosus subjected to HPTLC

studies. It showed 8 peaks with Rf values 0.07, 0.08, 0.27, 0.39, 0.54, 0.59, 0.67

and 0.81. HPTLC fingerprint of the isolated compound showed single peak

with Rf value 0.39.

Acute toxicity studies revealed that MERR is relatively nontoxic up to

2000 mg/kg b.w. p.o. indirectly pronouncing the safety profile of the extract.

The subacute toxicity also supports that the MERR is nontoxic, predicted by

there is no remarkable changes in the both haematological and biochemical

parameters in the blood and histopathological parameters of tissues.

The MERR of a dose of (200 mg/kg/b.w/p.o) did not significantly

suppress blood glucose levels in over night fast normal animals. The same

effect was observed at a higher dose level of 400 mg/kg/b.w/p.o of the MERR

in over night fasted normal animals after 60, 90, 120 min of oral

administration, when compared with control group of animals.

MERR showed significant improvement in glucose tolerance in glucose

fed hyperglycemic rats. Such an effect may be accounted for, in part, by a

decrease in the rates of intestinal glucose absorption, achieved by an extra

pancreatic action including the stimulation of peripheral glucose utilization or

enhancing glycolytic and glycogenic process with concomitant decrease in

glycogenolysis and gluconeogenesis105. However the effect was less significant

when compared standard drug glibenclimide.

Sterptozotocin is the most commonly employed agent for the induction

of experimental diabetic animal models of human insulin dependent diabetes

mellitus106. There is increasing evidence that streptozotocin causes diabetes by

rapid depletion of β cells, by DNA alkylation and accumulation of cytotoxic

free radicals that is suggested to result from initial islet inflammation, followed

by infiltration of activated macrophages and lymphocyte in the inflammatory

focus. It leads to a reduction in insulin release thereby a drastic reduction in

plasma insulin concentration leading to stable hyperglycemic states. In this

study significant hyperglycemia was achieved within 48 hours after

streptozotocin (50 mg/kg/b.w/i.p) injection. Streptozotocin induced diabetic

rats with more 200 mg/dl of blood glucose were considered to the diabetic and

used for the study.

A double dose of MERR did not bring about any hypoglycemic action.

In the sub-acute study, glibenclamide treatment brought down the blood sugar

levels from the 14th day of treatment MERR (high dose) treatment produced

and a steady decrease in blood glucose levels from 21st day of treatment and a

steady decrease was observed there after. At the end of the study, a marked anti

hyperglycemic effect was observed in the plant extract treatment. The activity

may be due to the presence of phytochemicals like flavanoids, glycoside,

phenolics etc.

Sulphonylureas produces a hypoglycemic effect by stimulating

endogenous insulin secretion from β cells after binding to their receptors on the

plasma membrane and enhancing tissue sensitivity to insulin. The other

mechanism is extra pancreatic, action mainly upon liver, muscle and adipose

tissue.

Histopathological studies showed prominent islet cell hyperplasia and

regeneration of islet cells shows a proof for the possible antidiabetic property

of MERR.

Lipids play an important role in the pathogenesis of diabetes mellitus.

One level of serum lipids is usually raised in diabetic condition and such an

elevation posses to be a risk factor for cardiovascular diseases like coronary

heart disease and a two to four fold risk for the atherosclerosis which

constitutes the main cause of morbidity and mortality in diabetes mellitus. The

abnormal high concentration of serum lipids in diabetes is mainly due to

increased mobilization of free fatty acids from the peripheral depots, since

insulin inhibits the hormone sensitive lipase107. There hyperlipemia in diabetic

state may be regarded as a consequence of uninhibited actions of lipytic

hormones on fat depots108. In the present study, streptozotocin diabetic rats

clear cut abnormalities in the lipid profile were evidence by elevated serum

total cholesterol and reduced serum HDL cholesterol. The glibenclamide

treatment MERR in diabetic animals produced beneficial improvement in the

lipid profile by a reduction in the total cholesterol levels and increase in HDL-

Cholestrol level, which may not only be due to better glycemic control but

could also be due to the drug’s direct action on lipid metabolic pathways. Such

a biochemical state is desirable for preventing the progression of diabetes

related cardiovascular problems.

Free radicals can be defined as chemical species possessing an unpaired

electron, which is formed by homolytic cleavage of a covalent bond of a

molecule, by the loss of a single electron from a normal molecule or by

addition of a single electron to a normal molecule. Cells are equipped with both

enzymatic and non-enzymatic defence mechanisms to minimize cellular

damage resulting form interaction between cellular constituents and ROS. The

enzymatic antioxidant defence mechanism contains various forms of

superoxide dismutases, catalase, glutathione peroxides as well as enzymes

involved in the recycling of oxidized gluthione such as glutathione reductase

and glutathione-S-tranferases. Despite the presence of such delicate cellular

antioxidant systems, an over production of ROS in both intra and extracellular

spaces often occur upon exposure of cells to certain chemicals like

streptozotocin that yields to the pathogenesis of diabetes mellitus in

experimental animals. In the present study, we have examined the oxidative

stress pathway markers in streptozotocin diabetic rats. Superoxide dismutase

and catalase are the most important enzymes that scavenge toxic free radicals

and forms the superoxide anion, which initiates peroxidation of unsaturated

lipids.

In the biological system, it reduces the potential for hydroxyl radical

generation by catalyzing the reduction of superoxide radical to form hydrogen

peroxide, thereby diminishing the toxic effect of the free radicals. Catalase a

heme protein is the major determinant of hepatic antioxidant status. The

enzyme is localized in the cellular peroxisomes and micro-peroxisomes. It

catalyses the decomposition of hydrogen peroxide to water and oxygen, thus

protecting the cell from oxidative damage109. Superoxide dismutase and

catalase activity in the diabetic control animals was significantly low due to

increased oxidative stress when compared to the normal animals. However a

significant increase in the enzyme activity was observed in the glibenclamide

and MERR.

GSH is mainly involved in the synthesis of important macrolecules and

in protection against ROS. It is also essential for the maintenance of thiols of

proteins and components of antioxidants like ascorbates Tocopherol110. GSH-

Px is a selenium containing enzyme, that is active in the reduce form. This

enzyme catalyses the oxidation of GSH to GSSG at the expense of hydrogen

peroxide. A marked decrease in the hepatic GSH was observed in

streptozotocin diabetic rats. Such a decrease contributes to the pathogenesis of

complications associated with chronic diabetic state109. Glibenclamide, MERR

treated animals showed a marked increase in the hepatic glutathione peroxidase

antioxidant level significantly. This indicates that both the extracts have potent

properties to inhibit oxidative damage to tissues.

Impairment of enzymatic antioxidant system due to reduce levels of

insulin in diabetic state increases fatty acyl-coA oxidase and initates β

oxidation of fatty acids favours accumulation of free radials resulting in lipid

peroxidation. Increased lipid peroxidation impairs membrane function by

decreasing the membrane fluidity and changing the activity of membrane

bound enzymes and receptors111.

An elevated TBARS level in diabetic rats suggests the extent of

peroxidative injury, indicative of the development of diabetic complications. In

the present study, it was found that streptozotocin induction caused a

significant increase in TBARS in the hepatic tissue. But glibenclamide, MERR

exerted a protective effect against peroxide damage to the tissues.

Free radical scavenging activity of the extracts was measured in an

invitro chemical system by DPPH radical scavenging activity, Nitric oxide

scavenging activity, scavenging of Hydroxyl radical, Determination of

reducing power, Determination of total phenolic compounds methods while for

the antiperoxidative activity, a biological system comprising of hepatic tissue

homogenates were employed.

MERR is tested by their ability to bleach the stable radical DPPH. This

assay provided information on the reactivity of the compounds with a stable

free radical. Because of the odd electron, DPPH shows a strong absorption

band at 517nm in visible spectroscopy (deep violet color) as this electron

becomes paired off in the presence of a free radical scavenger, the absorption

vanishes and the resulting decolourisation is stoichiometric with respect to the

number of electrons taken up.

Nitric oxide is an important chemical mediator generated by the

endothelial cells, marophages, neurons, etc and involved in the regulation of

various physiological processes. Oxygen reacts with excess nitric oxide to

generate nirite and peroxynitrite anions, which act as free radicals. In this study

the extracts competes with oxygen to react with nitric oxide and thus inhibits

the generation of anions.

The hydroxyl radical scavenging activity in MERR is to inhibit hydroxyl

radical mediated deoxyribose degradation in a reaction mixture. The relative

extents of inhibition of deoxyribose degradation will give an induction of

scavenging and in the presence of EDTA, and mannitol, a classical hydroxyl

scavenger, significantly inhibited deoxyribose degradation in a concentrated

dependent manner112.

Incubation of sodium nitroprusside in phosphate buffer saline at 250C

for 2 hrs resulted in the time dependent nitric production which was reduced by

MERR.

The reducing capacity of a compound may serve as a significant

inductor of its potential antioxidant activity113. However the antioxidant activity

of putative antioxidants have been attributed to various mechanisms, among

which are prevention of chain initiation, binding of transition metal ion

catalysis, decomposition of peroxides, prevention of continuous hydrogen

abstraction and radical scavenging114&115. The reducing power of MERR

increased with increasing amount of the sample.

Phenols are very important plant constituents because of their

scavenging ability due to their hydroxyl groups116. The phenolic compounds

may contribute directly to antioxidative action. It is suggested that

polyphenolic compounds have inhibitory effects on necrosis in humans, this

made us to investigate the amount of total phenolic compound present in

MERR. In the MERR (1 mg), 129 ± 0.998 µg pyrocatechol equivalent of

phenols was detected.

In epilepsy, normal pattern of neuronal activity becomes disturbed

briefly when the nerves in the brain “Fire” spontaneously causing strange

sensations, emotions, behaviours and often times seizures with muscle spasms

as well as loss of consciousness117.

It has been reported to increase the brain levels of Dopamine and

Noradrenaline which causes an inhibition of seizure activity118.

MES induced epilepsy was altering the levels of monoamines like

noradrenaline, serotonin, dopamine119.

It is found that treatment with MERR on rats significantly reduces in

tonic hind limb extensor stage in MES induced epilepsy. Methanolic Extract of

Rubus Racemosus markedly protects epilepsy induced by MES which are

mediated by levels of monoamines.


extract of Rubus Racemosus were subjected to antibacterial and antifungal

against following organisms. Staphylococcus aureus, Staphylococcus

epidermides, Bacillus cereus, Micrococcus luteus, Kl.pneumoniae,

Pseudomonos aeruginosa, E.coli, Aspergillus fumigates, Aspergillus niger

respectively.

Petroleum ether extract exhibited significant activity against all the

tested organisms at the concentration of 100 µg/ml whereas it showed moderate

activity at the concentration of 25µg/ml and 50µg/ml when compared with

standard drug.

Methanolic extract exhibited significant activity against all the tested

organisms at the concentration of 100 µg/ml whereas it showed moderate


standard drug.

Chloroform extract exhibited significant activity against all the tested



standard drug.

Aqueous extract exhibited significant activity against all the tested



standard drug.

The Minimum Inhibitory concentration values of all the extracts

were determined.

From the above studies it reveals that all the extracts were active against

tested bacterial and fungal organisms.

SUMMARY AND

CONCLUSION

CHAPTER - VI

SUMMARY & CONCLUSION

The plant Rubus racemosus belongs to the family Rosaceae was

selected. A detailed pharmacognostical, toxicological and pharmacological was

studied on the plant. The aerial parts of the plant Rubus racemosus were

anatomically studied and reported first time. A detailed anatomical description

of the anatomical structures were studied. The midrib contains 2-3 layers of

epidermis, vascular bundle. Lamina contains mesophyll tissue consists of

palisade cells and spongy parenchyma. The vascular bundles are surrounded by

dilated hyaline bundle sheath cells. The type of trichomes present is glandular

and non-glandular trichomes. All the bundles in petiole are collateral with thick

band of sclerenchyma cells abutting the phloem tissues. Stem contains wedge

shaped vascular bundles. Sclerenchyma cells are thin walled and wide

lumened. The pith is parenchymatous.

The powder microscopy of the plant shows unicellular, non-glandular,

trichomes, anisocytic type of stomata, parenchyma cells and prismatic type of

calcium oxalate crystals. Length and Width of trichomes, leaf constants, physio

chemical constants, loss on drying and extractive values were determined.

Preliminary phytoconstituent screening of the plant Rubus Racemosus

showed the presence of various phytochemical constituents like flavanoids,

glycosides, phenols, carbohydrates, terpenes, tannins, saponins and the

compounds were isolated from the methanolic extract of Rubus racemosus. All

the extracts were subjected to thin layer chromatography, HPTLC and their Rf

values were determined. Both acute and Sub acute toxicity studies revealed that

MERR did not produce any mortality or sign of toxicity at the dose of

200mg/kg/b.w/p.o in the experimental rats. Treatment of MERR did not exhibit

any significant hypoglycemic effect on normal animals.

Significant improvement in glucose tolerance was observed with

methanolic extract treatment on glucose fed hyperglycemic rats. Experimental

diabetes in rats was induced by STZ (50 mg/kg/i.p) and animals with blood

glucose level more than 200 ml/dl were considered as diabetic and used for this

study. No significant reduction of blood glucose level was observed on the

acute treatment of MERR in STZ induced diabetic rats. In the sub acute study a

steady decrease in blood glucose level was observed on MERR treatment, in

STZ induced diabetic rats. The treatment of MERR showed marked increase in

the Total protein and HDL Cholesterol in serum of STZ induced diabetic

animals.

At the same time significant decrease in Total bilirubin, SGOT, SGPT,

ALP, Total Cholesterol, Triglycerides, LDL Cholesterol levels in serum of

diabetic animals were observed. The hepatic antioxidant enzyme levels

superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase

are significantly decreased in STZ induced diabetic animals with a high degree

of lipid peroxidation. The enzyme levels increased significantly on treatment

with MERR. Further, the antioxidant activity is confirmed by free radical

scavenging activity by DPPH, Nitric oxide, Reducing power, Hydroxyl radical

activity by MERR.

The antiepileptic activity of MERR was assessed by MES induced

convulsion.

In MES induced epilepsy, MERR at both 200 mg/kg and 400 mg/kg exhibits

significant antiepileptic activity particularly tonic hind limb extensor stage.

Neurochemical estimations of dopamine, nonadrenaline, and serotonin

in forbrain of MES induced rats reveals that MERR significantly increases

dopamine levels in forebrain. A significant increase in nonadrenaline levels

was observed in forebrain and also serotonin levels are shown to rise in

forebrain of MERR treated rats.

From the observations of the studies performed it could be predicted that

the MERR at both 200 mg/kg and 400 mg/kg exhibited significant anti-

epileptic activity.


extracts of Rubus racemosus have showed significant and moderate activity

against anti-bacterial and anti-fungal organisms.

Based on the results obtained and observation we can infer that the plant

under study, Rubus racemosus could be used for the supportive treatment of

diabetes mellitus, as the plant also offers effective protection against the attack

of free radicals that forms the basis for the development of diabetic

complications. And it also showed significant anti-epileptic, anti-bacterial and

anti-fungal activity.

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