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PHYTOCHEMICAL AND PHARMACOLOGICAL INVESTIGATION ON TEPHROSIA PURPUREA,
FICUS GLOMERATA, FICUS RELIGIOSA IN ANIMAL MODELS
THESIS SUBMITTED TO
The Tamilnadu Dr. M.G.R. Medical University, Guindy, Chennai-600032, Tamilnadu, India.
As a partial fulfillment of the requirement for the award of the degree of
DOCTOR OF PHILOSOPHY (Faculty of Pharmacy)
Submitted By
Vishal S. Gulecha
Under the guidance of
DR. T. SIVAKUMAR M.Pharm., Ph.D. Professor and Principal
Nandha College of Pharmacy, Erode, Tamilnadu, India.
2011-2012
DECLARATION
I here by declare that the thesis entitled “Phytochemical and
pharmacological investigation on Tephrosia purpurea, Ficus
glomerata, Ficus religiosa in animal models” submitted to the
Tamilnadu Dr. M.G.R. Medical University, Chennai as partial fulfilment
of the requirements for the award of degree of DOCTOR OF
PHILOSOPHY (Faculty of pharmacy) was completely carried out by me
during the period of 2008-2011 under the guidance of Prof. Dr. T.
Sivakumar, M.Pharm., Ph.D., Professor and Principal Nandha College
of Pharmacy, Erode, Tamilnadu, India. This work is original and has not
formed the basis for the award of any Degree, Diploma, Associateship,
Fellowship or other similar title.
Place: Erode
Vishal S. Gulecha
AACCKKNNOOWWLLEEDDGGEEMMEENNTT
I wish to express my profound gratitude and indebtness to my esteemed guide Dr. T. Sivakumar, Professor and Principal Nandha College of Pharmacy, Erode, Tamilnadu, India, for his expert guidance and scholarly supervision, endless motivation, encouragements and freedom to carry research work. His appreciation and encouragement always boosted me during the investigation.
I owe sincere gratitude to Dr. C. D. Upasani, Principal, SSDJ College of Pharmacy, chandwad for their encouragement and moral support.
A Special vote of thanks to Dr. Nitin Desai, Dean, Faculty of Sciences, Dr. D.Y Patil Deemed University, Sector15, CBD Belapur, Navi Mumbai From whom I learnt the concept and techniques of anticancer activity
I deeply acknowledge my warmest thanks for the assistance rendered by my friends Dr. Aman, Manoj, Rakesh, Santosh, Hemant,Mayur and every other person who has directly or indirectly been responsible in extending help to successfully complete the research work.
The support, patience and encouragement rendered by my brother Dr. Vaibbhav & Rahul have enabled me to overcome the obstacles in the path of progress.I have no words to express gratitude to my parents Mr.Subhasnchand Gulecha & Mrs.Tarabai S Gulecha for their unstinted care and love. I am indeed grateful to my life companion, Vaishali and Son Veedant for their encouragement, confidence and forbearance.
I will remain forever indebted to the lives of the animals, sacrificed, during the experiment, which was done with the noble purpose of widening of horizons of science.
I apologize for not being able to accommodate many more names that have contributed little bit in this endeavor.
Vishal S. Gulecha
IINNDDEEXX Title Page
Abbreviation
Contents
List of tables
List of figures
Chapter 1 Introduction 1-34
Chapter 2 Literature review 35-44
Chapter 3 Plant profile 45-56
Chapter 4 Aim and Objective, Scope and plan of work 57-60
Chapter 5 Materials and Method 61-88
Chapter 6 Results 89-120
Chapter 7 Discussion 121-
Chapter 8 Summary and Conclusion 130-132
Reference
Appendix
Publications
CONTENTS
Title Page
CHAPTER-1 INTRODUCTION 1
1.1 Plant-Derived Natural Products 3
1.2 Natural Product Research And Development 6
1.3 Inflammation 8
1.3.1 Mechanism of inflammation 11
1.3.2 Chemical Mediators of Acute Inflammation 14
1.3.2.1 Chemical mediators released from cells 14
1.3.2.2 Complement system 15
1.3.2.3 Kinin system 16
1.3.2.4 Coagulation system 16
1.3.2.5 Fibrinolytic system 17
1.3.2.6 Eicosanoids 17
1.3.2.7 Interleukin 19
1.3.2.8 Nitric Oxide 19
1.3.2.9 Cytokines 19
1.3.2.10 Tumor Necrosis Factor-alpha 20
1.3.2.11 Endotoxin 20
1.4 Types Of Inflammation 22
1.4.1 Acute Inflammation 22
1.4.1.1 Hereditary defects that impair the acute inflammatory response
23
1.4.2 Sub-Acute Inflammation 23
1.4.3 Chronic Inflammation 23
1.4.3.1 Nonspecific Chronic Inflammation 24
1.4.3.2 Granulomatous Lesions 25
1.5 Rheumatoid arthritis 27
1.6 Comprehensive list of plants with anti-inflammatory action 32
1.7 Selection of Plants 34
1.7.1 Criteria for selection of plants 34
CHAPTER 2 LITERATURE REVIEW 35
CHAPTER 3 PLANT PROFILE 45
Tephrosia purpurea 45
Ficus glomerata 48
Ficus religiosa 52
CHAPTER 4 AIM AND OBJECTIVE, SCOPE AND PLAN OF WORK 35-36
CHAPTER 5 MATERIALS AND METHOD 61
5.1 Plant material 61
5.1.1 Identification and Authentication of Plant Material 61
5.2 Chemicals 61
5.3 Drugs 62 5.4 Method 62
5.4.1 Extraction Procedure of plant materials 62
5.4.1.1 Petroleum ether extract 61
5.4.1.2 Chloroform extract 62
5.4.1.3 Methanolic extract 62 5.4.1.4 Aqueous extract 62
5.4.2 Phytochemical analysis of extracts 62 5.4.2.1 Test for Carbohydrates 62 5.4.2.2 Test for proteins 64 5.4.2.3 Test for amino acids 64 5.4.2.4 Test for tannins and phenols 65 5.4.2.5 Test for glycosides 65 5.4.2.6 Test for saponins 65 5.4.2.7 Test for flavonoids 66 5.4.2.8
Test for Alkaloids 66
5.4.2.9 Test for Steroids 66 5.4.3.1 Preparation of dosage form 67 5.4.3.2 Animals 67 5.4.3.3 Approval of Protocol 68
5.4.3.4 Acute toxicity study 68 5.4.4 Fractionation 69
5.4.4.1 Fractionation of Pet ether extract of T. purpurea 69
5.4.4.1.1 Chromatographic separation of acetone soluble part 69
5.4.4.1.2 Chromatographic separation of acetone insoluble part 71
5.4.4.2 Fractionation of Pet ether extract of F. religiosa and F.glomerata 71
5.4.4.3 Fractionation of Methanol extract of F. religiosa and F.glomerata 71
5.4.5 Thin layer chromatography 72 5.4.6 Pharmacological activity 73
5.4.6.1 Evaluation of biological activity 74 5.4.6.1.1 Screening of In-vitro Anticancer Activity 74
5.4.6.2 Evaluation Of Analgesic Activity 75 5.4.6.2.1 Evaluation of Tail flick latency period in rats 75 5.4.6.2.2 Evaluation of Acetic acid induced writhing in mice 75
5.4.6.3 Evaluation of anti-inflammatory activity induced by Carrageenan hind paw edema
76
5.4.6.3.2 Evaluation of anti-inflammatory activity induced by Serotonin and histamine induced paw edema 77
5.4.6.3.3 Evaluation of Cotton pellet granuloma formation in rats 78 5.4.6.4 Evaluation of anti-arthritis activity by adjuvant induced
arthritis in rats 78
5.4.6.4.1 Evaluation of anti-arthritis activity by formalin induced arthritis in rats 79
5.4.6.4.2 Evaluation of Antihyperlipidemic activity 80 5.4.6.5 Preparation of normal and high-fat diet 80
5.4.6.5.1 Biochemical analysis of T. purpurea, F. religiosa and F. glomerata 81
5.4.6.5.2 Statistical method 88 CHAPTER-6 RESULT AND ANALYSIS
6.1 Results 89 6.2 Extractive value 89 6.2 Phytochemical investigation of T.purpurea, F.religiosa and F.
glomerata leaves 89
6.3 Acute toxicity assessment 90 6.4 Thin layer chromatography 90
6.5 Screening of In-vitro Anticancer Activity 90 6.6 Evaluation of Tail flick latency period in rats and evaluation
of acetic acid induced writhing in mice 91
6.7 Evaluation of anti-inflammatory activity induced by Carrageenan hind paw edema 92
6.8 Evaluation of anti-inflammatory activity induced by Serotonin and histamine induced paw edema 92
6.9 Evaluation of Cotton pellet granuloma formation in rats 93 6.10 Evaluation of anti-arthritis activity by adjuvant induced
arthritis in rats 93
6.11 Evaluation of anti-arthritis activity by formalin induced arthritis in rats 93
6.12 Evaluation of Antihyperlipidemic activity 94 6.12.1 Effect of t. purpurea, F.religiosa, and F.glomerata on serum
lipid profile 94
6.12.2 Effect of t. purpurea, F.religiosa, and F.glomerata on biochemical parameters 95
CHAPTER-7 DISCUSSION 121 CHAPTER-8
SUMMARY AND CONCLUSION 132
REFERENCE
APPENDIX
PUBLICATIONS
LIST OF TABLES
Table No.
Title Page
1 Summary of Mediators of Acute Inflammation 21
2 Some clinically used NSAIDs and their therapeutic scope 30
3 Extractive values (% w/w yield) of plant material with different solvents
97
4 Phytochemical analysis of different extracts T. purpurea 98
5 Phytochemical analysis of different extracts F. religiosa 99
6 Phytochemical analysis of different extracts F. glomerata 100
7 Effect of different fractions of T. purpurea, F. religiosa and F. glomerata on Tail flick latency period in rats
104
8 Effect of different fractions of T. purpurea, F. religiosa and F. glomerata on acetic acid induced writhing in mice
106
9 Effect of different fractions of T. purpurea, F. religiosa and F. glomerata in Carrageenan induced rat paw edema
108
10 Effect of different fractions of T. purpurea, F. religiosa and F. glomerata on serotonin induced rat paw edema
109
11 Effect of different fractions of T. purpurea, F. religiosa and F. glomerata on histamine induced rat paw edema
110
12 Effect of different fractions of T. purpurea, F. religiosa and F. glomerata on Cotton pellet granuloma formation in rats
111
13 Effect of different fractions of T. purpurea on adjuvant induced arthritis in rat
113
14 Effect of different fractions of F. religiosa on adjuvant induced arthritis in rat
114
15 Effect of different fractions of F. glomerata on adjuvant induced arthritis in rat
115
16 Effect of different fractions of T. purpurea on formalin induced arthritis in rat
116
17 Effect of different fractions of F. religiosa on formalin induced arthritis in rat
117
18 Effect of different fractions of F. glomerata on formalin 118
induced arthritis in rat
19 Effect of different fractions of T. purpurea, F. religiosa and F. glomerata on serum lipid profile in hyperlipidemia-induced rats.
119
20 Effect of different fractions of T. purpurea, F. religiosa and F. glomerata on biochemical parameters in hyperlipidemia-induced rats
120
LIST OF FIGURES
Figure No.
Title Page
1 Schematic diagram representing the key events of the inflammatory response
10
2 Vascular phase of acute inflammation 12
3 Prostaglandin, thromboxane and leukotriene biosynthesis 5-HPETE-hydroperoxy eicosa tetraenoic acid
18
4 Effect of varying concentration of TPI on Trypan blue Exclusion test of cell viability
101
5 Effect of varying concentration of TPIII on Trypan blue Exclusion test of cell viability
101
6 Effect of varying concentration of FRI on Trypan blue Exclusion test of cell viability
102
7 Effect of varying concentration of FRIII on Trypan blue Exclusion test of cell viability
102
8 Effect of varying concentration of FGI on Trypan blue Exclusion test of cell viability
103
9 Effect of varying concentration of FGIII on Trypan blue Exclusion test of cell viability
103
10 Effect of different fractions of T. purpurea, F. religiosa and F. glomerata on Tail flick latency period in rats
105
11 Effect of different fractions of T. purpurea, F. religiosa and F. glomerata on acetic acid induced writhing in mice
107
12 Effect of different fractions of T. purpurea, F. religiosa and F. glomerata on Cotton pellet granuloma formation in rats
112
5-HT 5-Hydroxy tryptamine
AA Arachidonic acid
BK Bradykinin
C5a Complement 5a
CGRP Calcitonin gene-related peptide
COX Cyclooxygenase
FG Ficus glomerata
FR Ficus religiosa
GABA Gamma-aminobutyric acid
i.p. Intraperitoneal
LTB4 Leukotriene B4
LTC4 Leukotriene C4
LTD4 Leukotriene D4
NA Noradrenaline
NF Nuclear factor
NSAIDs Non-steroidal anti-inflammatory
drugs
NO Nitric oxide
OTC Over the counter
p.o. Per oral
PAF Platelet activating factor
PDES Phosphodiesterases
PGD2 Prostaglandin D2
PGI2 Prostaglandin I2, Prostacyclin
PGJ2 Prostaglandin J2
PGs Prostaglandins
SP Substance P
SRS-A Slow releasing substance of
anaphylaxis
TP Tephrosia purpurea
TNFα Tumour necrosis factor alpha
TXA2 Thromboxane A2
1
1. INTRODUCTION:
Since antiquity, there has been a search by mankind for
alleviating inflammation, pain and arthritis which has been associated
with numerous ailments. In this endeavour a number of natural
products have been found to be useful for relief and cure. Systematic
knowledge of these materials can be fathomed in earliest literature of
indigenous system in different civilizations such as Chinese, Indian,
Arabian and Egyptian. A considerable amount of information has been
passed about the usage of flora and fauna by the tribals inhabiting
different parts of the world. This knowledge has led to the
development of materia medica and later into the pharmacopoeias of
different systems of medicine such as Ayurvedic, Unani, Tibb.
Natural products are generally either of prebiotic origin or
originate from microbes, plants, or animal sources. As chemicals,
natural products include such classes of compounds as terpenoids,
flavonoids, polyketides, amino acids, peptides, proteins,
carbohydrates, lipids, nucleic acid bases, ribonucleic acid (RNA),
deoxyribonucleic acid (DNA), and so forth. Natural products are not
just accidents or products of convenience of nature. More than likely
they area natural expression of the increase in complexity of
organisms1 Interest in natural sources to provide treatments for pain,
palliatives, or curatives for a variety of maladies or recreational use
reaches back to the earliest points of history.
Discovery of a new drug is time consuming and laborious
process. Natural products have been a thriving source for discovery of
natural drugs due to their chemical diversity and ability to act on
various biological targets. The phytochemical exploration of a
indigenous flora has been part of our lifestyle since ages and classical
texts like Ayurveda and Charak Samhita have served as materia
medica for this purpose. Natural products remain a prolific source for
the discovery of new drugs and drug leads even from Vedic period.
2
Recent data suggest that 80% drug molecule were natural products or
natural compound inspired2.
Studies on source of new drugs from 1981 to 2007 reveal that
almost half of the drugs approved since 1994 are based on natural
product3. Indian natural product, particularly those from traditional
medicinal plants which are reported in the classic texts like Ayurveda,
Charka Samhita have contributed toward this bloom in drug
discovery. The rich biodiversity of India has remained untouched as
far as discovery of new chemical entities is concerned. The traditional
Indian system of medicine has a very long term history of a usage in
number of diseases and disorders, but as it lacking in recording of the
safety and efficacy data. However the main cause for their scientific
neglect is due to multi-constituent mainstay and the mechanism of
action being unclear. But recently, it has been suggested that drug
discovery should not always limited to discovery of single molecule
and current belief one diseases one drug approach may be untenable
in future and that rationally designed polyherbal formulation could
also be investigated as an alternative in multitarget therapeutics and
prophylaxis4.
The role of individual drugs either single or in combination with
other for the relief of symptoms has to be critically studied and
understood so that a rational treatment is evolved. Inspite of having a
number of anti-inflammatory drugs which will be useful in the
treatment of intractable inflammation and severe pain. There is also
urgent need to develop drugs of low toxicity for long duration
treatment.
Natural products isolated from higher plants arid
microorganisms have been providing novel, clinically active drugs. The
key to the success of discovering naturally occurring therapeutic
agents rests on bioassay-guided fractionation and purification
procedures.
3
1.1 PLANT-DERIVED NATURAL PRODUCTS
Plants produce a huge array of natural products (secondary
metabolites). These compounds have important ecological functions,
providing protection against attack by herbivores and microbes and
serving as attractants for pollinators and seed-dispersing agents. They
may also contribute to competition and invasiveness by suppressing
the growth of neighbouring plant species (a phenomenon known as
allelopathy). Humans exploit natural products as sources of drugs,
flavouring agents, fragrances and for a wide range of other
applications. Rapid progress has been made in recent years in
understanding natural product synthesis, regulation and function and
the evolution of metabolic diversity. It is timely to bring this
information together with contemporary advances in chemistry, plant
biology, ecology, agronomy and human health to provide a
comprehensive guide to plant-derived natural products5.
The use of natural products as medicinal agents presumably
predates the earliest recorded history as the earliest humans used
various, but specific plants to treat illness. Records from as early as
2700 B.C. from China, traced to the Emperor Shennung, indicate the
usefulness of plants for treating disease, and the Ebers papyrus,
written in about 1550 B.C., includes many of the plants used in
Egyptian medicine. Theophrastus (370-285 BC) began the scientific
classification of plants, and Dioscorides De Materia Medica (77 AD)
reported the uses, medicinal and otherwise, of over 600 plants. Ibn al-
Baitar (1197-1248) listed over 1400 drugs and medicinal plants in his
Corpus of Simples. In Europe, after the tenth century, much of the
medicinal lore was based in the church, particularly the monastic
orders, but by the 1500’s, after the invention of the printing press,
herbals available to the general public were popular, particularly in
England. By the late 1700’s, studies like William Withering’s An
Account of the Foxglove and its Medicinal Uses (1785) began to
4
appear. These were based on case histories and described specific
doses and gave administration instructions for herbal remedies. In the
United States, before the advent of specific pharmaceuticals, herbal
medicine was relied upon to treat many illnesses. Development of
drugs based on natural products has had a long history in the United
States, and in 1991, almost half of the best selling drugs were natural
products or derivatives of natural products. There has recently been a
resurgence of interest in herbal remedies, and a Reuters/Zogby poll in
2000 showed that 40 % of people in the U.S. had tried herbal
remedies. In 1998, the U.S. market for natural supplements was over
$12 billion in sales and increasing by as much as 10 % per year.
Herbs such as St. John’s Wort, ginkgo, echinacea, and ginseng are
among the most popular herbs. In 1999, echinacea was reported to
make up 38 % of the U.S. market, with ginkgo a close second at 34%.
The efficacy of these herbs is being investigated in many laboratories,
and efforts are also being made to isolate and identify any active
constituents6.Natural products, as the term implies, are those
chemical compounds derived from living organisms, plants, animals,
insects, and the study of natural products is the investigation of their
structure, formation, use, and purpose in the organism. Drugs derived
from natural products are usually secondary metabolites and their
derivatives, and today those must be pure and highly characterized
compounds. Until the late 1800's, organic chemistry was almost
exclusively the study and use of natural products. The purpose of
these compounds in the organisms and their formation was little
understood or investigated, primarily due to the lack of appropriate
techniques and structural theory. The natural products that were
studied and used tended to be the compounds that occurred in the
largest amounts, mostly in plants, and were most easily isolated in a
pure, or sometimes not very pure, form by techniques such as simple
distillation, steam distillation, or extraction with acid or base.
5
Originally teas or decoctions (aqueous extracts) or tinctures or elixirs
(alcoholic extracts) were used to prepare and administer herbal
remedies - these were usually the starting points for isolation work.
We now employ different solvents, e.g., ethanol to extract, hexane to
concentrate non-polar constituents, methanol to concentrate polar
constituents, and modern isolation techniques include all types of
chromatography, often guided by bioassays, to isolate the active
compounds. Up until the 1950’s, the structures of natural products,
when determined, were determined by degradative techniques, and a
structure was not proven until the compound had been synthesized in
an unambiguous manner. Stereochemistry was not often determined.
Now, structures are elucidated primarily by spectroscopic techniques,
and the stereochemistry is an important feature of the structure7.
The treatment of diseases with pure pharmaceutical agents is a
relatively modern phenomenon. However, as European explorers and
merchants spread out to the Western and Eastern parts of the world,
some of the benefits they would bring back were newly discovered
pharmaceutical preparations of natural origin. One of the earliest
success stories in developing a drug from a natural product was
aspirin. The Ebers papyrus indicates the use of willow leaves as an
anitpyretic treatment, and early English herbals also recommend the
use of teas made from willow bark for this use. Following on these folk
treatments, chemists and pharmacists began to isolate the
compounds responsible for the remedy. Among the earliest pure
compounds discovered was salicin, isolated from the bark of the white
willow, Salix alba, in 1825-26. It was subsequently converted to
salicylic acid via hydrolysis and oxidation, and proved so successful
as an antipyretic (fever reducing) that it was actively manufactured
and used worldwide. The use of salicylic acid, however, often led to
severe gastrointestinal toxicity. This was overcome when Felix
Hoffmann of Bayer Company converted salicylic acid into
6
acetylsalicylic acid (ASA) via acetylation. Bayer then began marketing
ASA under the trade name aspirin in 1899. Today, aspirin is still the
most widely used analgesic and antipyretic drug in the world. By definition, the word natural is an adjective referring to
something that is present in or produced by nature and not artificial
or man-made. When the word natural is used in verbiage or written,
many times it is assumed that the definition is something good or
pure. However, many effective poisons are natural products. The term
natural products today is quite commonly understood to refer to herbs,
herbal concoctions, dietary supplements, traditional Chinese
medicine, or alternative medicine. That will not be the case in this
chapter. The information presented here will be restricted to the
discovery and development of modern drugs that have been isolated or
derived from natural sources. While in some cases, such discovery
and development may have been based on herbs, folklore, or
traditional or alternative medicine, the research and discovery of,
along with the development of, herbal remedies or dietary
supplements typically present different challenges with different goals.
So while the stories of herbs and drugs are very much intertwined, it
needs to be fully appreciated that the use of herbs as natural product
therapy is different than the use of herbs as a platform for drug
discovery and further development 8.
1.2 NATURAL PRODUCT RESEARCH AND DEVELOPMENT
The World Health Organization estimates that approximately 80
percent of the world’s population relies primarily on traditional
medicines as sources for their primary health care. Over 100 chemical
substances that are considered to be important drugs that are either
currently in use or have been widely used in one or more countries in
the world have been derived from a little under 100 different plants.
Approximately 75 percent of these substances were discovered as a
direct result of chemical studies focused on the isolation of active
7
substances from plants used in traditional medicine .The number of
medicinal herbs used in China in 1979 has been estimated to be
numbered at 5267.
Experience has persistently and repeatedly demonstrated that
nature has evolved over thousands of years a diverse chemical library
of compounds that are not accessible by commonly recognized and
frequently used synthetic approaches. Natural products have revealed
the ways to new therapeutic approaches, contributed to the
understanding of numerous biochemical pathways and have
established their worth as valuable tools in biological chemistry and
molecular and cellular biology. Just a few examples of some natural
products that are currently being evaluated as potential drugs are
(natural product, source, target, indication, status): manoalide,
marine sponge, phospholipage- A2 Ca2+-release, anti-inflammatory,
clincial trials; dolastatin 10, sea hare,microtubules, antineoplastic,
nonclinical; staurosporine, streptomyces, protein kinase C,
antineoplastic, clinical trials; epothilone, myxobacterium,
microtubules, antineoplastic, research; calanolide A, B, tree, DNA
polymerase action on reverse transcriptase, acquired
immunodeficiency syndrome (AIDS), clinical trials; huperzine A, moss,
cholinesterase, alzheimer’s disease, clinical trials9 .
About 80% of the world’s population uses folk medicine in
traditional medicine states World Health Organisation. Since ancient
time plants as sources of medicinal compounds have continued to
play a dominant role in the maintenance of human health10. India is
one of the richest countries in the world with regard to diversity of
medicinal plants. For centuries, plants have been used throughout the
world as drugs and remedies for various diseases since they have
great potential for producing new drugs of great benefit to mankind.
There are many approaches to search for new biologically active
principles in higher plants. Even though pharmacological industries
8
have produced a number of antibiotics in last three decades,
resistance to these drugs by micro organism has been increased. The
problem of microbial resistance is growing and the outlook for the use
of antimicrobial drugs in the future is still uncertain. It is expected
that plant extract showing target sites other then the dose used by
antibiotics will be active against drug resistant microbial pathogens.
Despite the advances made in orthodox medicine, there has
been global resurgence of interest in traditional system of medicine.
The reasons for these are many, but the most common would be
disillusionment with the result of or the lack of faith in the orthodox
medicine therapy and apprehension concerning the toxicity and safety
of modern drugs. All of the world culture contained element of
traditional medicine in which both drug therapy and drugless therapy
are used and considerable emphasis is placed on the moral and
spiritual aspects of life11 World health organization (WHO) has been
defined a traditional system of medicine as “the sum total of all the
knowledge and practices, whether explicable or not; used in diagnosis,
preventions and elimination of physical, mental or social imbalance and
relying exclusively on practical experience and observation handed
down by generation to generation whether verbally or in writing”. In
other words traditional system of medicine might also be considered
as a solid amalgamation of dynamic medical know-how and
experience12.
1.3 INFLAMMATION:
Inflammation is the means by which the body deals with insult
and injury. Insult may be caused: mechanically (e.g., by pressure or
foreign bodies), chemically (e.g., by toxins, acidity, alkalinity),
physically (e.g., by temperature), by internal processes (e.g., uremia),
and by microorganisms (e.g., bacteria, virus, parasites). Inflammation
is a complicated and not fully understood communication between
cellular and humoral elements.
9
The same exogenous and endogenous stimuli that cause cell
injury also elicit a complex reaction in vascularized connective tissues
called inflammation. Reduced to its simplest terms, inflammation is a
protective response intended to eliminate the initial cause of cell
injury as well as the necrotic cells and tissues resulting from the
original insult. Inflammation accomplishes its protective mission by
diluting, destroying, or otherwise neutralizing harmful agents (e.g.,
microbes or toxins). It then sets into motion the events that eventually
heal and reconstitute the sites of injury. Thus, inflammation is also
intimately interwoven with repair processes whereby damaged tissue
is replaced by the regeneration of parenchymal cells, and/or by filling
of any residual defect with fibrous scar tissue. Although inflammation
helps clear infections and, along with repair, makes wound healing
possible, both inflammation and repair have considerable potential to
cause harm.
Thus, inflammatory responses are the basis of life-threatening
anaphylactic reactions to insect bites or drugs, as well as of certain
chronic diseases such as rheumatoid arthritis and atherosclerosis.
Other harmful examples include inflammation in the peritoneum
leading to fibrous bands that cause intestinal obstruction, or
pericardial inflammation resulting in dense encasing scar that impairs
cardiac function.
Inflammation or phlogosis is pathological response of living
tissue to injuries that leads to the local accumulation of plasmatic
fluid and blood cells. Although it is defense mechanism, the complex
events and mediators involved in inflammatory reaction can be
induced, maintain or aggravate many diseases. It is a complex
phenomenon, comprising of biochemical as well as immunological
factors. Inflammation is recognized by Rubor (redness), Tumor
(Swelling), Calor (heat), Dolor (pain) and Functio laesa (Loss of
functions) 13.
10
The cardinal signs develop irrespective of stimuli, but the actual
expression of these processes depends on the site of inflammation e.g.
lung inflammation may manifest only with loss of function, while skin
abscess may display all cardinal signs. In these cardinal signs,
redness is caused by vasodilatation. The swelling results mainly from
the accumulation of fluid exudates consequent to increased vascular
permeability, with smaller contributions from the cellular infiltrations
of the affected tissues and the engorgement of their blood vessels.
However, sensation of heat is attributable to the rapid flow of relatively
warm blood through dilated vessels in inflamed area. Various factors
contributing to pain include distension of tissue particularly when
there is little room for expansion, kinins, histamine and metabolites,
which are liberated or activated by injured cells14.
Figure1: Schematic diagram representing the key events of the
inflammatory response.
11
A: intracellular kinase activation by cytokines; B: activation of NF-κB;
C: adhesion molecule expression; D: migration (of specific
leukocytesubsets); E: stimulation of inflammatory phagocytes,
including changes in cytosolic Ca2+ and plasma membrane NADPH
oxidase; F: opsonisation involving activation of the complement
cascade; G: release and activation of MMPs within the extracellular
matrix; H: breakdown of the extracellular matrix by proteinases e.g.,
degradation of cartilage by a MMPs and aggrecanase.
1.3.1 Mechanism of inflammation:
Inflammatory process is a continuous process operating
through many mechanisms. The histopathological and biochemical
studies of inflammation indicate that it involves two distinct events: A) Vascular events B) Cellular events (leucocytic infiltration)
A) Vascular events: It contains: - a) Changes in vascular flow and
caliber b) Vascular leakage
a) Changes in vascular flow and caliber:
It begins early after injury and proceeds at different rates
depending on severity of injury.
Changes occur in the following manner:
After an inconstant and transient vasoconstriction of arterioles,
lasting few seconds, vasodilation occurs. Vasodilation first involves
the arterioles and then results in opening of new capillary beds in the
area. Along with this increased blood flow, this is the cause of heat
and redness. Different mediators are involved in this process of
vasodilation such as vasoactive amines15, kinins and
prostaglandins16. Slowing of circulation is brought about by increased
permeability of microvasculature, with outpouring of protein rich fluid
into extravascular tissues. Stasis develops due to loss of fluid, which
results in concentration of red cells in small vessels and increased
12
viscosity of the blood. After this, there occurs peripheral orientation of
leukocytes, a process called leukocytic migration.
b) Vascular Leakage:
Increased vascular permeability leading to the escape of protein
rich fluid is the hallmark of acute inflammation. In normal conditions,
fluid loss through the vascular bed is governed by the balance
between hydrostatic and oncotic pressures in the plasma and tissues
with fluid tending to be lost at the arterial end and reabsorbed at the
venular end. Increase in capillary hydrostatic pressure resulting from
local vasodilation after an inflammatory injury drives fluid out of the
vascular compartment and extravascular spaces at the rate that is too
rapid for reabsorption by lymphatics to prevent the resulting edema
formation and tissue swelling17. Three phases of vascular
permeability are described by Mohan, 2000:
I) Immediate-transient phase: This lasts about 30 min. affects
venules and is largely mediated by histamine.
II) Immediate – prolonged phase: The exudation starts immediately
but persists for days. It appears to be due to direct damage to vessels.
III) Delayed prolonged phase: Both capillaries and venules are affected
by the direct injury of the agent (e.g. heat) and by chemical mediators.
Figure 2: Vascular phase of acute inflammation. (A) Normal capillary
bed. (B) Acute inflammation with vascular dilation causing increased
redness (erythema) and heat (calor), movement of fluid into the
13
interstitial spaces (swelling), extravasation of plasma proteins into the
extracellular spaces (exudate), and emigration of leukocytes.
B) Cellular Events:
A critical function of inflammation is the delivery of leukocytes
to the site of injury. Leukocytes ingest offending agents, kill bacteria
and other microbes and degrade necrotic tissue & foreign antigens.
Leukocytes may also prolong inflammation and induce tissue damage
by releasing enzymes, chemical mediators and toxic oxygen
radicals.The migration of leukocytes at the site of inflammation takes
place as follows14,17.
a) Sticking of Neutrophils:
Neutrophils adhere to walls of venules and get pushed by the
blood cells slowly. As they come in contact with endothelium of
venules in injured tissue, the cells adhere to the endothelium and the
margin of cell in contact with endothelium becomes flattened. Some
of these neutrophils appear to move on the inner surface of the
endothelium by ameboid motion. This is mediated by chemo
attractants and certain cytokines.
b) Escape of neutrophils:
Once a neutrophil becomes closely opposed to the endothelium
of venules, it extrudes pseudopod preparatory to migrating. Neutrophil
that manages to emigrate between the endothelial cells come against a
second barrier, the basement membrane with which periendothelial
cells and connective tissue, collectively forms the periendothelial
sheath. This barrier hinders the continued migration of neutrophil so
that its pseudo pod usually changes course to take up a position
between an endothelial cell and its basement membrane. Eventually,
the neutrophil penetrates the periendothelial sheath and emerges into
the connective tissue around the venule. Thus, the migration of
neutrophil takes place without leaving breach in the endothelium.
14
The escape of neutrophil is sometimes followed by a trickle of
erythrocytes from the same site in endothelium. This process,
diapedesis of red cells, appears to be passive.Tissue damage initiates
or activates the local release of various chemotactic factors that
provokes directly or indirectly the appearance of mediators of pain and
inflammation 18,19.
1.3.2 Chemical Mediators of Acute Inflammation
The spread of the inflammatory response following injury to a
small area of tissue suggests that chemical substances are released
from injured tissues, spreading outwards into uninjured areas. These
chemicals, called endogenous chemical mediators, cause
vasodilatation, emigration of neutrophils, chemo taxis and increased
vascular permeability.
1.3.2.1 Chemical mediators released from cells
a) Histamine:
This is the best-known chemical mediator in acute
inflammation. It causes vascular dilatation and the immediate
transient phase of increased vascular permeability. It is stored in mast
cells, basophil and eosinophil leukocytes, and platelets. Histamine
release from those sites (for example, mast cell degranulation) is
stimulated by complement components C3a and C5a, and by
Iysosomal proteins released from neutrophils 20.
b) Lysosomal compounds:
These are released from neutrophils and include cationic
proteins, which may increase vascular permeability, and neutral
proteases, which may activate complement.
c) Prostaglandins:
These are a group of long-chain fatty acids derived from
arachidonic acid and synthesised by many cell types. Some
prostaglandins potentiate the increase in vascular permeability
caused by other compounds. Others include platelet aggregation
15
(Prostaglandin I is inhibitory while prostaglandin A2 is stimulatory).
Part of the anti-inflammatory activity of drugs such as aspirin and the
non-steroidal anti-inflammatory drugs is attributable to inhibition of
one of the enzymes involved in prostaglandin synthesis 21,22.
d) Leukotrienes:
These are also synthesised from arachidonic acid, especially in
neutrophils, and appear to have vasoactive properties. SRS-A (slow
reacting substance of anaphylaxis), involved in type I hypersensitivity,
is a mixture of leukotrienes 23,24.
e) 5-hydroxytryptamine (serotonin):
This is present in high concentration in mast cells and platelets.
It is a potent vasoconstrictor. It also may play a role in mediating
inflammation, but their antagonists ameliorate only certain types of
inflammatory responses 25
f) Lymphokines:
This family of chemical messengers is released by lymphocytes.
Apart from their major role in type IV hypersensitivity, lymphokines
may also have vasoactive or chemotactic properties.
g) Plasma factors:
The plasma contains four enzymatic cascade systems
complement, the kinins, the coagulation factors and the fibrinolytic
system which is inter-related and produce various inflammatory
mediators 26.
1.3.2.2 Complement system:
The complement system is a cascade system of enzymatic
proteins. It can be activated during the acute inflammatory reaction in
various ways:
-In tissue necrosis, enzymes capable of activating complement are
released from dying cells.
16
During infection, the formation of antigen-antibody complexes can
activate complement via the classical pathway, while the endotoxins of
Gram-negative bacteria activate complement via the alternative
pathway.
Products of the kinins, coagulation and fibrinolytic systems can
activate complement. The products of complement activation most
important in acute inflammation include:
• C5a: chemotactic for neutrophils; increases vascular permeability;
releases histamine from mast cells
• C3a: similar properties to those of C5a, but less active
• C567: chemotactic for neutrophils
• C56789: cytolytic activity
• C4b, 2a, 3b: opsonisation of bacteria (facilitates phagocytosis by
macrophages) 27.
1.3.2.3 Kinin system:
The kinins are peptides of 9-11 amino acids; the most important
vascular permeability factor is bradykinin. The kinin system is
activated by coagulation factor XII. Bradykinin is also a chemical
mediator of the pain, which is a cardinal feature of acute
inflammation 26.
1.3.2.4 Coagulation system:
The coagulation system is responsible for the conversion of
soluble fibrinogen into fibrin, a major component of the acute
inflammatory exudates 27. The coagulation system is responsible for
the conversion of soluble fibrinogen into fibrin, a major component of
the acute inflammatory exudates. Coagulation factor XII (the Hageman
factor), once activated by contact with extracellular materials such as
basal lamina, and various proteolytic enzymes of bacterial origin, can
activate the coagulation, kinin and fibrinolytic systems 28,29 .
17
a) Coagulation factor XII (Hageman factor):
Once activated by contact with extracellular materials such as
basal lamina, and various proteolytic enzymes of bacterial origin, can
activate the coagulation, kinin and fibrinolytic systems.
b) Platelet Activating Factors:
Newly defined class of biologically active lipids, which can
produce effects at low concentrations. PAF has actions on the variety
of different target cells and is believed to an important mediator in
both acute and persisting allergic and inflammatory phenomena 30, 31.
1.3.2.5 Fibrinolytic system:
Plasmin is responsible for the lysis of fibrin into fibrin
degradation products, which may have local effects on vascular
permeability 32
1.3.2.6 Eicosanoids:
They are generated from phospholipids in response to a wide
range of different stimuli, and their presence has been detected in
every tissue in the body. The control of many physiological process
and are the most important mediators and modulators of the
inflammatory action33, 34
18
Figure 3: Prostaglandin, thromboxane and leukotriene biosynthesis
5-HPETE-hydroperoxy eicosa tetraenoic acid (PGs+ TXA)
Arachidonic Acid Metabolism involves cell membrane
phospholipid which under the influence of phospholipase A2 will
release the arachidonic acid.Two pathways: 1.Lipoxygenase – give rise
to various leukotrienes. Responsible for vasoconstriction,
bronchospasm, increased permeability e.g. Leukotrienes B4, C4, D4,
and E4 Cycloxygenase inhibitors prevent prostaglandin synthesis.
Thromboxane effects are opposite of prostacyclin effects. e.g. TXA2,
PGI2 (Prostacyclin), PGD2, and PGF2α.
19
1.3.2.7 Interleukin-1 (IL-1)
Macrophages and monocytes are the main source of this
cytokine. IL-1 has both Paracrine effects on cells in the vicinity 35-37.
1. Causing them to produce tissue factor and thus triggering the
blood-clotting cascade. 2. Stimulating the synthesis and secretion of a
variety of other interleukins.
3. Helping to activate T cells and thus initiate an adaptive immune
response.
4. Hormonal effects as it is carried in the blood throughout the body.
5. Decreasing blood pressure.
6. Inducing fever. IL-1 causes fever by stimulating the release of
prostaglandin’s, which act on the temperature control center of the
hypothalamus.
1.3.2.8 Nitric Oxide:
Formed from the amino acid arginine by nitric oxide syntheses
present in endothelium (constitutive) and macrophages (inducible).
Many of the long-lived actions of nitric oxide in vivo appear to be
caused by stable S-nitroso compounds (R-SNO) Actions of nitric oxide
are vasodilator, anti-platelet aggregation, and cytotoxic/antimicrobial 38.
1.3.2.9 Cytokines
Cytokines are proteins that are secreted by various types of
immune cells and serve as signaling chemicals22. The central role of
cytokines is to control the direction, amplitude, and duration of the
inflammatory response.
There are two main groups of cytokines:
a) Pro-inflammatory Cytokines: Pro-inflammatory cytokines are
produced predominantly by activated immune cells such as microglia
and are involved in the amplification of inflammatory
reactions.
20
b) Anti-inflammatory Cytokines:
Anti-inflammatory cytokines are involved in the reduction of
inflammatory reactions.
1.3.2.10 Tumor Necrosis Factor-alpha (TNF-α)
Large amounts of TNF-α are quickly released by stimulated
mast cells. All the cells involved in inflammation have receptors for
TNF-α, and are activated by it to synthesize more on their own. This
positive feedback quickly amplifies the response 36-37.
1.3.2.11 Endotoxin:
Bacterial products and toxins can act as exogenous mediators
of inflammation i.e. endotoxin or lipopolysachharides of Gram-
negative bacteria. The immune system of higher organisms has
probably evolved in a veritable sea of endotoxin, so it is perhaps not
surprising that this substance evokes powerful responses19. For
example, endotoxin can trigger complement activation, resulting in the
formation of anaphylatoxins C3a and C5a, which cause vasodilation
and increase vascular permeability. Endotoxin also activates the
Hageman factor, leading to activation of the coagulation and
fibrinolytic pathways as well as the kinin system. In addition,
endotoxins elicit T cell proliferation, and have been described as super
antigen for T cells 39.
21
Table 1: Summary of Mediators of Acute Inflammation40
Mediators Source Evoked Responses
Histamine and Serotonin
Mast cells, platelets
Vascular leakage.
Bradykinin Plasma substrate Pain, Vascular leakage. C3a, C5a. Plasma protein via
liver, macrophages Vascular leakage opsonic fragment, leukocyte adhesion.
Prostaglandins Mast cells, from membrane phospholipids
Potentiation of other mediators, vasodilatation, pain, fever.
Leukotriene B4 Leukocytes Leukocytes adhesion, activation.
Leukotriene C4, D4, E4.
Leukocytes, Mast cells.
Bronchoconstriction, Vasoconstriction.
Oxygen metabolites Hydroxyl radical Hydrogenperoxide Hypochloride Superoxide radicals
Leukocytes Vascular leakage, Chemotaxis, Endothelial damage, Tissue damage.
PAF Leukocytes, mast cells.
Vascular leakage, Chemotaxis, Bronchoconstriction Leukocyte priming.
IL-1 & TNF Macrophages Chemotaxis, acute phase reaction, endothelial activation.
IL-8 Macrophages endothelium.
Leukocyte activation, chemo taxis.
Nitric oxide Macrophages, endothelium.
Vasodilation, cytotoxicity.
Integrins selectins immunoglobulin
Cell surface endothelial cells
Vascular leakage.
So, in brief, the sequence of early events in inflammation may be
summarized as
• Initial injury which causes the release of inflammatory mediators
22
• Vasodilatation
• Increased vascular permeability, resulting into cellular infiltration
• Migration of phagocytic cells to the inflamed area, resulting into
release of lytic enzymes due to rupturing of cellular lysosomal
membranes.
1.4 TYPES OF INFLAMMATION:
Depending upon the defense capacity of the host and duration
of response, mainly three types of inflammation are recognized or
inflammatory response occurs in two distinct phases, each apparently
mediated by different mechanisms. Based on the course and duration,
the inflammation can be called as: -
1.4.1 Acute Inflammation:
Inflammation is the response of living tissue to damage. The
acute inflammatory response has 3 main functions.
• The affected area is occupied by a transient material called the
acute inflammatory exudate. The exudate carries proteins, fluid and
cells from local blood vessels into the damaged area to mediate local
defenses.
• If an infective causative agent (e.g. bacteria) is present in the
damaged area, it can be destroyed and eliminated by components
of the exudate.
• The damaged tissue can be broken down and partially liquefied,
and the debris removed from the site of damage.
The cause of acute inflammation may be due to physical damage,
chemical substances, micro-organisms or other agents. The
inflammatory response consists of changes in blood flow, increased
permeability of blood vessels and escape of cells from the blood into
the tissues. It is characterized by local vasodilation and increased
capillary permeability. It is immediate and early responses to
injurious agents have three major components 1) Increase in blood
flow 2) Structural changes that leads to plasma protein and
23
leukocytes into circulation 3) Accumulation of leukocyte in focus of
injury.
1.4.1.1 Hereditary Defects that Impair the Acute Inflammatory
Response
Deficiency of Complement Components: Increased susceptibility to
infection, Deficiency of factors C2, C3, and C5
Defects in Neutrophil
a.) Chronic granulomatous disease of childhood
• X-linked disorder
• deficient activity of NADPH oxidase
b.) Myeloperoxidase deficiency
• sometimes associated with recurrent infections but is often of little
clinical consequence
c.) Chediak-Higashi syndrome
• autosomal recessive disorder
• neutropenia, albinism, cranial and peripheral neuropathy, & a
tendency to repeated infections
• presence of abnormal WBC 40
1.4.2 Sub-Acute Inflammation:
The inflammation lasts for 1 to 6 weeks or more. The type that
is neither acute nor chronic is termed as sub-acute inflammation. It
lasts longer as compared to acute inflammation. Microscopically
vascular, exudative as well as proliferative changes of acute and
chronic inflammation are present. Exudate chiefly consists of
eosinophils, lymphocytes, plasma cells, histocytes and fibroblasts.
1.4.3 Chronic Inflammation:
Chronic inflammation can evolve from acute inflammation or
occur without an acute phase. Histologically, chronic inflammation
has two main features: The presence of granulation tissue and
mononuclear predominance. The combination of new blood vessels,
fibroblasts, and extracellular matrix is termed “granulation tissue.”
24
Mononuclear predominance can also be seen in the latter part of
acute inflammation as mononuclear phagocytes or macrophages. In
comparison to ordinary loose connective tissue, granulation tissue is
more cellular and contains neutrophils, inflammatory cells, and
fibroblasts. Granulation tissue is more vascular and has “leaky”
capillaries. The formation of granulation tissue is the response of
connective tissue and vessels to irritation.
In some forms of chronic inflammation, other cell types appear.
This suggests the development of immunologic reactions that may
include lymphocytes, eosinophils, and plasma cells. In other forms,
where no immune response is present, the mononuclear cells are
almost entirely macrophages. When inflammation is chronic, the
vascular component, vasodilation, and exudation is minimal, and,
therefore, manifests clinically with little (possibly no) redness and heat 41. Most chronic inflammation without bacterial invasion is pointless
and may even prove to be harmful. For example, edema raises tissue
tension and causes pain, impeding movements that are important for
normal joint function and homeostasis. Pressure from edema to
vascular tissues can result in poor drainage of toxins (ibid).
1.4.3.1 Nonspecific Chronic Inflammation:
Nonspecific chronic inflammation involves a diffuse
accumulation of macrophages and lymphocytes at the site of injury.
Ongoing chemotaxis causes macrophages to infiltrate the inflamed
site, where they accumulate because of prolonged survival and
immobilization. These mechanisms lead to fibroblast proliferation,
with subsequent scar formation that in many cases replaces the
normal connective tissue or the functional parenchymal tissues of the
involved structures. For example, scar tissue resulting from chronic
inflammation of the bowel causes narrowing of the bowel lumen.
25
1.4.3.2 Granulomatous Lesions:
A granulomatous lesion results from chronic inflammation. A
granuloma typically is a small, 1 to 2 mm lesion in which there is a
massing of macrophages surrounded by lymphocytes. These modified
macrophages resemble epithelial cells and sometimes are called
epithelioid cells. Like other macrophages, these epithelioid cells are
derived originally from blood monocytes. Granulomatous
inflammation is associated with foreign bodies such as splinters,
sutures, silica, and asbestos and with microorganisms that cause
tuberculosis, syphilis, sarcoidosis, deep fungal infections, and
brucellosis. These types of agents have one thing in common: they are
poorly digested and usually are not easily controlled by other
inflammatory mechanisms. The epithelioid cells in granulomatous
inflammation may clump in a mass (granuloma) or coalesce, forming a
large, multinucleated giant cell that attempts to surround the foreign
agent. A dense membrane of connective tissue eventually
encapsulates the lesion and isolates it.
The nonsteroidal anti-inflammatory drugs (NSAIDs) e.g.
ibuprofen, aspirin, phenylbutazone, diclofenac, etc. are associated
with analgesic and antipyretic activity. Considerable evidence has now
been accumulated to show that prostaglandins are thebasic cause
behind pain and inflammatory conditions. They have the ability to
sensitize the pain receptors to mechanical and chemical stimulations.
The biosynthesis of prostaglandins is catalysed by lysosomal enzymes
present in almost all mammalian cell type except erythrocytes.
Prostaglandins potentiate the early inflammatory response causing
vasodilation42, increased permeability43, facilitating celluar infiltration
and sensitizing pain receptors. The NSAIDs do not act centrally to
intervene in the perception of pain44 but act peripherally to inhibit the
biosynthesis and release of prostaglandins 45. People who have taken
non-steroidal anti-inflammatory drugs (NSAIDs), are at reduced risk of
26
colon cancer 46,47. This may also be true for cancers of the oesophagus,
stomach, and rectum, and in rodents experimental bladder, breast,
and colon cancer is reduced when NSAIDs are administered
concurrently with carcinogens48.NSAIDs inhibit cyclooxygenase
enzymes and angiogenesis. Cyclooxygenase-2 is induced by cytokines
and expressed in both inflammatory disease and cancer.
The functional relationship between inflammation and
cancer is not new. In 1863, Virchow hypothesized that the origin of
cancer was at sites of chronic inflammation, in part based on his
hypothesis that some classes of irritants, together with the tissue
injury and ensuing inflammation they cause, enhance cell
proliferation49 Although it is now clear that proliferation of cells alone
does not cause cancer, sustained cell proliferation in an environment
rich in inflammatory cells, growth factors, activated stroma, and DNA-
damage-promoting agents, certainly potentiates and/or promotes
neoplastic risk. During tissue injury associated with wounding, cell
proliferation is enhanced while the tissue regenerates; proliferation
and inflammation subside after the assaulting agent is removed or the
repair completed. In contrast, proliferating cells that sustain DNA
damage and/or mutagenic assault (for example, initiated cells)
continue to proliferate in microenvironments rich in inflammatory
cells and growth/survival factors that support their growth. In a
sense, tumours act as wounds that fail to heal50
Today, the causal relationship between inflammation, innate
immunity and cancer is more widely accepted; however, many of the
molecular and cellular mechanisms mediating this relationship
remain unresolved. Furthermore, tumour cells may usurp key
mechanisms by which inflammation interfaces with cancers, to
further their colonization of the host. Although the acquired immune
response to cancer is intimately related to the inflammatory response 51,52.
27
Inflammation is a major symptom of various connective tissue
diseases. These include:
a) Rheumatic fever which is characterized by arthritis, swelling,
immobility of joints and fever
b) Rheumatoid arthritis, ankylosing spondylitis and osteoarthritis
c) Systematic lupus erythematosus and polyarteritis nodosa.
1.5 Rheumatoid arthritis (RA)
RA is an inflammatory autoimmune disease- a type of condition
in which the immune system, which normally protects the body by
fighting infections and diseases, instead targets the body. RA is
different from other types of arthritis such as osteoarthritis, a wear-
and-tear condition that commonly occurs as people age. In RA, the
immune system attacks the tissues that line the joints, causing pain,
swelling, and stiffness in the joints and affecting their ability to work
properly. Over time, RA may damage bone and cartilage within the
joints, weaken muscles and tendons that support the joints, and lead
to joint destruction53.
The NSAIDs are clinically employed in the treatment of all above
conditions. They form the mainstay of drug therapy of inflammation.
Due to their analgesic, anti-inflammatory, smooth muscle spasm
relieving, platelet aggregation inhibitory actions, NSAIDs are
increasingly used in field of medicine. They provide relief in moderate
dysmenorrohea, biliary and renal colic, migrane, moderate post
operative pain and febrile conditions 54. Since the list of NSAIDs has
been ever increasing, we have a long list of NSAIDs with dissimilar
chemical structures, which nevertheless share certain therapeutic
actions and side effects55. Some of clinically used NSAIDs and their
therapeutic scope listed in Table 2
NSAIDs produce adverse effects which can be categorized in two
classes:
a) Related to inhibition of prostaglandin synthesis
28
b) Unrelated to the inhibition of prostaglandin synthesis
The adverse effects related to inhibition of PG synthesis include
erosive gastritis, peptic ulceration, gastrointestinal blood loss,
prolonged bleeding time,fluid retention, azotemia, hyperkalemia,
oliguria, rhinitis, delayed parturition etc.56
The adverse reaction unrelated to the inhibition of
prostaglandin synthesis include hepatitis, hepatic nerosis, cholestatic
jaundice, photosensitivites, epidermal nerosis, headache, dizziness,
tinnitus, deafness, confusion, nervousness, increased sweating,
retinal disturbances56. The other clinically used anti-inflammatory
drugs are glucocorticoids. They are powerful anti-inflammatory and
immunosuppressant agents. They inhibit both early and late
manifestations of inflammation i.e. not only the redness, heat, pain,
and swelling but also the later stages of wound healing and repair and
the proliferative reactions seen in chronic inflammation.
In early events glucocorticoids reduce the acute inflammatory
response causing: vasoconstriction, reduce exudation, decrease in
number and activity of leucocytes and decrease in inflammatory
mediators. In late events they cause decreased in number and activity
of mononuclear cells and fibroblasts, decreased proliferation of blood
vessels and less fibrosis thus decreased chronic inflammation but also
decreased healing. They affect all types of inflammatory reactions
whether caused by invading pathogen, by chemical or physical stimuli
or by inappropriate deployed immune responses e.g. hypersentivity.
The anti-inflammatory effects are produced by
a) Decreased production of inflammatory mediators and cells
b) Generation of anti-inflammatory mediators like lipocortin.
Glucocorticoids reduce:
a) Synthesis of both eicosanoids and platelet activating factor
b) Histamine release from basophils
c) Concentration of complement components
29
d) Release of tissue dimensions toxic oxygen metabolites
e) Synthesis of lymphokines from macrophages
f) Accumulation of mononuclear leucocytes
g) Activity of fibroblast thereby reducing collagen synthesis
h) Activity of osteoblast
However they carry the hazard that they can suppress the
necessary protective responses to infection and cause decrease
essential healing process. The other major adverse effects include
leucocytosis, lymphopenia, peptic ulceration, pancreatitis,
hyperglycemia, protein wasting, obesity, growth failure, glaucoma,
depression, psychosis. Patients become more susceptible to bacterial
infections as well as fungal infections after chronic treatment with
glucocorticoids. Further, they provide only a palliative relief as the
underlying cause remains. Corticosteroids mask the valuable
symptoms which are essential for the diagnosis e.g. an infection may
continue while patient superficially appears to improve56.
30
Table 2: Some clinically used NSAIDs and their therapeutic scope
Drugs Rheumatoid arthritis
Ankylosing spondylitis
Gout Juvenile arthritis
Salicylates Aspirin Diflunisal Benorylate
+ + +
+ + +
Propionic acid derivatives Ibuprofen Naproxen Ketoprofen Fenoprofen Flurbiprofen
+ + + + +
+ + + + +
+
+ +
Indole derivatives Indomethacin Sulindac Tolmetin
+ + +
+ +
+
+
Phenyl acetic acid derivatives Diclofenac Fenclofenac
+ +
Pyrazoles derivatives Phenylbutazone Oxyphenbutazone Azapropazone
+
+ +
+ + +
Fenamates Mefenamic acid Flufenamic acid
+ +
Oxicams Piroxicam
+
+
+
+
31
Herbal medicine is still the mainstay of about 75–80% of the
world population, mainly in the developing countries, for primary
health care because of better cultural acceptability, better
compatibility with the human body and lesser side effects. However,
the last few years have seen a major increase in their use in the
developed world. In Germany and France, many herbs and herbal
extracts are used as prescription drugs and their sales in the
countries of European Union were around $ 6 billion in 1991 and may
be over $ 20 billion now. In USA, herbal drugs are currently sold in
health food stores with a turnover of about $ 4 billion in 1996 which
is anticipated to double by the turn of the century. In India, the herbal
drug market is about $ one billion and the export of plant-based crude
drugs is around $ 80 million. Herbal medicines also find market as
nutraceuticals (health foods) whose current market is estimated at
about $ 80–250 billion in USA and also in Europe57.
The World Health Organization (WHO) has recently defined
traditional medicine (including herbal drugs) as comprising
therapeutic practices that have been in existence, often for hundreds
of years, before the development and spread of modern medicine and
are still in use today58 Or say, traditional medicine is the synthesis of
therapeutic experience of generations of practicing physicians of
indigenous systems of medicine. The traditional preparations
comprise medicinal plants, minerals, organic matter, etc. Herbal
drugs constitute only those traditional medicines which primarily use
medicinal plant preparations for therapy. The earliest recorded
evidence of their use in Indian, Chinese, Egyptian, Greek, Roman and
Syrian texts dates back to about 5000 years. The classical Indian
texts include Rigveda, Atherveda, Charak Samhita and Sushruta
Samhita. The herbal medicines/traditional medicaments have,
32
therefore, been derived from rich traditions of ancient civilizations and
scientific heritage.
Recent comprehensive list of such plants as follows59-109
Comprehensive list of plants with anti-inflammatory action
NAME SPECIES FAMILY
Aparajita Clitonia ternatea Linn Papilionaceae
Apamarga Achyranthes aspera Linn Amaranthaceae
Arkapuspi Holostemma adekodier Asetepiadaceae
Ashoka Saraca asoca Roxb Caesalpiniaceae
Aragvadha Cassia fistula Linn Caesalpiniaceae
Eranda Ricinus communis Linn. Euphorbtaceae
Kapitta Limonia acidissima Linn. Rutaceae
Kakodumbarika Ficus hispida Linn Moraceae
Kancanara Bauhiia variegate Linn. Caesalpiniaceae
Kukundara Blumea lacera Burm Compositae
Kumari Aloe barbadensis Mill. Liliaceae
Kulattha Dolichos biflorus Linn Pipilionaceae
Gambhari Gmelina arborea Linn. Verbenaceae
Goksura Tribulus terrestris Linn Zygophyllaceae
Jatamansi Nardostachys grandiflora Valerianaceae
Jatiphala Myristica fragrans Houtt Myristicaeae
Tankari Physalis peruviana Linn Solanaceae
Tambula Piper betle Linn. Piperaceae
Tinduka Diospyros peregrine Gurke Ebenaceae
Trivrita Operculina turpethum Linn. Convolvulaceae
33
Devadaru Cedrus deodara Roxb Pinaceae
Dronapuspi Leucas cerphalotus Spreng Labiateae
Nimba Azadirachta indica Juss Meliaceae
Nirgundi Vitex negundo Linn. Verbenaceae
Puskarmula Inula racemosa Hook. Compositae
Bobbula Acacia nilotica Benth Mimosaceae
Bibhitaka Terminalia bellirica Roxb. Combretaceae
Bilva Aegle marmelos Corr. Rutaceae
Brahmi Bacopa monnieri Linn. Scrophulariaceae
Brhati Solnum ferox Linn Solanaceae
Bharangi Clerodendrum serratum Linn Verbenaceae
Bhumyamalaki Phyllanthus fraternus Hook Euphorbiaceae
Mandukaparni Centella asiatica Linn Umbelliferae
Methika Trigonella foenum-graecum Linn. Fabaceae
Yavani Trachyspermum ammi Linn. Apiaceae
Rasna Pluchea lanceolata Clarke. Compositae
Rohitaka Tecomella undulate Seem. Bignoniaceae
Latakaranja Caesalpinia bonduce Linn. Caesalpiniaceae
Lodhra Symplocos cochinchinensis Lour. Symplocaceae
Vaca Acorus colamus Linn. Araceae
Vatsanabha Aconitum ferox Wall. Ranunculaceae
Vasa Justicia adhatoda Linn. Acanthaceae
34
The above information indicates that a considerable interest
have been taken in investigating anti-inflammatory action of plant
products. It is felt that this venture should be followed by further work
so as to enhance our knowledge regarding this activity and would lead
to further finding of potential new drugs.
The plants selected for studying anti-inflammatory, analgesic,
anticancer, antihyperlipidemic and anti arthritis effects in the present
work are Tephrosia purpurea, ficus religiosa, and ficus glomerata.
These plants have been mentioned in the literature and this
selection is based on there usage and included in some marketed
preparation such as Sarapunkha svarasa, Sarapunkha lepa,
Sarapunkha ghanavati, Pancha, Valkaladi, Tailum, Pancha
ValkalaKashaya.
1.6 SELECTION OF PLANTS
1.6.1Criteria for selection of plants:
The plants should be recommended for the anti-inflammatory
activity in the traditional Indian system of medicine and should be
used in the medicinal preparation for the treatment of
inflammatory disorders.
Plant should be recommended individually for the treatment of
inflammation and related disorders
Literature review of scientific work may be suggestive of effects on
inflammation but should not be proved for activity under
investigation.
Literature on pharmacological investigation done so far should be
indicative that the plants are devoid of toxicity.
Phytochemical investigations done so far should suggest that plant
dose not contain any harmful or toxic compound.
Availability of selected plant should be much easier.
35
2. LITERATURE REVIEW
Saxena et al., 1997,reported rutin (2.5 % in leaf and root);
quercetin (0.29 % in leaf); rotenoids (leaves 1.7 %; root 1.2 %; stem 0.6
%) - mainly deguelin, rotenone, tephosin, neoflavonoid glycoside, and
lupeol124.
Ram et al., 1985, reported that it contains β-sitosterol (leaves),
delphinidin chloride (flowers), and cyaniding chloride (flowers), caffeic
acid (seeds). Pyranoflavone; candidin (seeds), flavone; pongachin with
pongachalcone-1 and dehydrodeguelin (seeds), flavonol; candidol (seeds).
β-hydroxyl chalcone; purpurenone (roots) with purpurene,
dehydroisoderricin and maackiain. Also having pongamol,
flemichapparins B and C, spinasterol and ursolic acid. On the other
hand, it contains gum, albumin, colouring matter and brown resin.122
Gupta et al., 1980, reported the presence of flavones and
flavanones123.
Pelter et al., 1981 reported the presence of 8-Substituted
flavonoids and 30-substituted 7-oxygenated chalcones125.
Sinha et al., 1982, Ventakata et al., 1984, Chang et al., 2000
reported the presence of prenylated flavonoids126-128
Khatri et al., 2009, shows that the aerial parts of Tephosia
purpurea (L.) pers. (Fabaceae) and stem bark of Tecomella undulata seem.
(Bignoniaceae) are used for liver disorders in the traditional system of
medicine. Maximum hepatoprotective activity was observed at 500mg/kg
dose level of Tephosia purpurea (aerial parts), which was comparable to
that of silymarin. Extract of Tephosia purpurea was found to be more
potent than the extract of Tecomella undulata 129
Lodhi et al., 2006, reported wound healing potential of ethanolic
extract of Tephosia purpurea (aerial part) in the form of simple ointment
using thee types of wound models in rats as incision wound, excision
wound and dead space wound. This extract effectively stimulates wound
36
contraction; increase tensile strength of incision and dead space wounds
as compared to control group 130.
Chinniah et al., 2009, reported the methanolic extract showed
promising activity against clinical isolates and standard strains of
Helicobacter pylori, including metronidazole-resistant strains.
Fractionation of the extract revealed the n-hexane and chloroform
fractions to possess marked activity. The extract and the less polar
fractions remained functionally active in acidic condition similar to
stomach environment, exhibited consistent bacteriostatic activity during
repeated exposure, and demonstrated synergism, complete or partial,
even with antibiotic-resistant strains 131.
Damre et al., 2003, reported the flavonoid fraction of Tephosia
purpurea (FFTP) was studied for its effect on cellular and humoral
functions and on macrophage phagocytosis in mice. Oral administration
of FFTP (10–40 mgykg) to modulate both the cell-mediated and the
humoral components of the immune system132.
Mohammad et al., 2001, reported the effect of topical application of
Tephosia purpurea on TPA-mediated depletion in the level of enzymatic
and non-enzymatic molecules in skin was also evaluated and it was
observed that topical application of Tephosia purpurea prior to TPA
resulted in the significant recovery of TPA-mediated depletion in the level
of these molecules, namely glutathione, glutathione S-transferase,
glutathione reductase and catalase. From these data we suggest that
Tephosia purpurea can abrogate the tumor-promoting effect of croton oil
(phorbol ester) in murine skin133.
Deshpande et al., 2003, reported the aqueous extract of Tephosia
purpurea possesses significant antiulcer property, which could be due to
cytoprotective action of drug or by strengthening of gastric and duodenal
mucosa and thus enhancing mucosal defense134.
37
Bouthaina et al., 2008, reported the study of the oligosaccharides
extracted from Tephosia purpurea seeds was undertaken using the
instant controlled pressure drop (DIC) as a pre-treatment prior to
conventional solvent extraction. This DIC procedure provided structural
modification in terms of expansion, higher porosity and improvement of
specific surface area; diffusion of solvent inside such seeds and
availability of oligosaccharides increase notably135
Soni et al., 2006, reported T. Purpurea exhibits antioxidant activity
in vivo and the ethyl acetate soluble fraction has improved antioxidant
potential than the extract136.
Jain et al., 2006, reported ethanol extract of leaves and flavonoid
(isolated from leaves extract) from Tephosia purpurea were evaluated for
hepatoprotective activity in rats by inducing hepatotoxicity with carbon
tetrachloride. These fractions were administered orally at a dose of 100
mg/kg/day. Serum level of transaminases, alkaline phosphate, and total
bilirubin were used as biochemical markers of hepatotoxicity.
Histopathological changes in the liver were also studied. The results of
the study indicated that the hepatoprotective activity was more in
ethanolic extract of leaves than isolated flavonoid137.
Suresh et al., 1979 reported the stem bark of Ficus racemosa
contains tannin, wax, saponin gluanol acetate, β-sitosterol,
leucocyanidin- 3 – O – β – D - glucopyrancoside, leucopelargonidin – 3 –
O – β – D - glucopyranoside, leucopelargonidin – 3 – O – α – L -
rhamnopyranoside, lupeol, ceryl behenate, lupeol acetate, α-amyrin
acetate, leucoanthocyanidin, and leucoanthocyanin from trunk
bark,lupeol, β-sitosterol and stigmasterol were isolated20. Fruit contains
glauanol, hentriacontane,β sitosterol, glauanolacetate, glucose, tiglic
acid, esters of taraxasterol, lupeolacetate, friedelin, higherhydrocarbons
and other phytosterol145.
38
Deva et al., 2008 reported a new tetra triterpene glauanol acetate
which is characterized as 13α, 14β, 17βH, 20 α H-lanosta-8, 22-diene-
3βacetate and racemosic acid were isolated from the leaves. An unusual
thermostable aspartic protease was isolated from latex of the plant. The
stem bark and fruit showed the presence of glauanol acetate 146.
Ahmed et al., 2010, reported methanol extract of Ficus racemosa
stem bark were studied using the model of hepatotoxicity induced by
carbon tetrachloride (CCl4) in rats. CCl4 administration induced a
significant increase in total bilirubin associated with a marked elevation
in the activities of aspartate aminotransferase (AST), alanine
aminotransferase (ALT) and alkaline phosphatase (ALP) as compared to
control rats. Pretreatment with methanol extract resulted in significant
decreases in the activities of AST, ALT and ALP, compared to CCl4-
treated rats. The results indicate that F. racemosa possesses potent
hepatoprotective effects against CCl4-induced hepatic damage in rats 147.
Veerapur et al., 2009, reported ethanol extract (FRE) and water
extract (FRW) of Ficus racemosa were subjected to free radical scavenging
both by steady state and time resolved methods such as nanosecond
pulse radiolysis and stopped-flow spectrophotometric analyses. FRE
exhibited significantly higher steady state antioxidant activity than FRW.
FRE exhibited concentration dependent DPPH, ABTS, hydroxyl radical
and superoxide radical scavenging and inhibition of lipid peroxidation
with IC50 comparable with tested standard compounds. In vitro radio
protective potential of FRE was studied using micronucleus assay in
irradiated Chinese hamster lung fibroblast cells (V79). Maximum
radioprotection was observed at 20 μg/ml of FRE. The cytokinesis-block
proliferative index indicated that FRE does not alter radiation induced
cell cycle delay. Based on these results it is evident that the ethanol
extracts of F. racemosa acts as a potent antioxidant and a probable
radioprotector 148.
39
Chandrashekhar et al., 2008, reported the bark extract were
evaluated for anthelmintic activity using adult earthworms, which
exhibited a spontaneous motility (paralysis) With 50 mg/mL of aqueous
extract the effects were compared with 3% piperazine citrate. There was
no final recovery in the case of worms treated with aqueous extract in
contrast to piperazine citrate, the worms recovered completely within 5 h.
This result shows the anthelmintic nature of the extract 149.
Khan et al., 2005, reported Ficus racemosa extract at a dose of 200
and 400 mg/kg when given orally a significant decrease in lipid
peroxidation, xanthine oxidase, γ-glutamyl transpeptidase and hydrogen
peroxide (H2O2) generation with reduction in renal glutathione content
and antioxidant enzymes generated by Potassium bromate (KBrO3), a
potent nephotoxic agent that induces renal carcinogenesis in rats. There
was significant recovery of renal glutathione content and antioxidant
enzymes. There was also reversal in the enhancement of renal ornithine
decarboxylase activity, DNA synthesis, blood urea nitrogen and serum
creatinine. This result suggests that Ficus racemosa extract is a potent
chemopreventive agent and suppresses KBrO3-mediated nephotoxicity in
rats 150.
Kar et al., 2003, reported the ethanol extract (250mg/kg/day)
lowered blood glucose level within 2 weeks in the alloxan diabetic albino
rats confirming its hypoglycemic activity Βsistosterol isolated from the
stem bark was found to posses potent hypoglycemic activity when
compared to other isolated compound 151.
Biswas et al., 2003, ethanol extracts of stem bark show a potent
wound healing in excised and incised wound model in rat 152.
Ratnasooriya et al., 2003, reported the decoction (D) of the bark of
Ficus racemosa at a dose of 250, 500 or 1000 mg/kg induced
antidiuresis, had a rapid onset (within 1 h), peaked at 3 h and lasted
thoughout the study period (5 h). However, antidiuretic potential of D
40
was about 50 % lower than that of ADH. The D was well tolerated even
with subchonic administration. The D caused a reduction in urinary Na+
level and Na+/K+ ratio, and an increase in urinary osmolarity indicating
multiple mechanisms of action. This proves its efficacy as antidiuretic
agent 153.
Vonshak et al., 2003, reported the 50 methylene chloride in hexane
flash column fraction of the extract of the leaves of Ficus racemosa was
found to have antifungal activity. The extract inhibited the growth of
several plant pathogens (Curvularia sp, Colletotrichum gloeosporioides,
Alternaria sp, Corynespora cassiicola and Fusarium sp). Psoralen was
identified as the active compound and was shown to be biodegradable,
having the potential to be developed as a fungicide against pathogens
causing diseases on crops of economic importance 154.
Mandal et al., 2000, reported different extracts of leaves were
tested for antibacterial potential against Escherichia coli, Bacilus pumitis,
Bacillus subtilis, Pseudomonas aureus. Out of all extracts tested,
petroleum ether extract was the most effective extract against the tested
microorganism 155.
Mukherjee et al., 1998, reported ethanol extract of stem bark has
shown significant inhibitory activity against castor oil induced diarrhoea
and PEG2 induced enteropooling in rats and also showed a significant
reduction in gastro intestinal motility in charcoal meal test in rats which
proves its efficacy as antidiarrhoel agent 156.
Agarwal et al., 1988, reported fruits when fed to rats in diet
induced hypocholesterolemic effect, as it increased faecal excretion of
cholesterol 157.
Niranjan et al., 2007 reported the leaves of F. religiosa contain a
high amount of l-cystine, llysine, l-arginine, dl-serine, dl-aspartic acid,
glycine, dl-theonine, dl-∞-alanine, l-proline, tryptophan, l-tyrosine, dl-
methionine, dlvaline, dl-isoleucine and l-leucine. Fibers like, acid
41
detergent fiber (ADF), neutral detergent fiber (NDF), acid detergent lignin
(ADL) have been identified in the leaves of F. religiosa170.
Singh et al., 1978, Panda et al., 1983 reported the leaves contain around
1.5% of total tannin content, which comprises tannic acid and
condensed tannins 171-172.
Desai et al., 1980 Williamson et al., 2002 reported the leaves are
rich in minerals like, calcium, phosphorous, iron, copper, manganese,
magnesium, zinc, potassium and sodium 173,174.
Bhadauria et al., 2002, Bamikole et al., 2003, reported variety of
proteins and carbohydrates are present in the leaves, making them a
good fodder 175,176.
Behari et al., 1984, reported Phytosterols like, campesterol,
stigmasterol Sitosterol, isofucosterol, triterpene alcohols like, amyrin,
lupeol have been isolated from the non-saponifiable fraction of light
petroleum leaf extract of F. religiosa. Along with phytosterols and
triterpene, long-chain hydrocarbons [n-nonacosane, n-hentriacontane],
aliphatic alcohols [nhexacosanol, n-octacosanol] have also been isolated
from the same fraction 177.
Osima et al., 1939, reported the bark of F. religiosa comprises total
tannin content 178.
Swami et al., 1989, the vitamin K1, n-octacosanol, methyl oleonate,
lupen-3-one, have been isolated from the petroleum ether extract of the
bark 179.
Swami et al., 1996, carbocyclic polyol, “Inositol” has been isolated
from the alcoholic bark extract180.
Mali et al., 2003, Phenolics, fibre, alkaloids, saponins, and
cyanogenic glycosides have been identified in the inner bark of F.
religiosa 181.
Pandit et al., 2010, recently the antidiabetic effect of the aqueous
bark extract of F. religiosa in streptozotocin -induced diabetic rats has
42
been investigated 182. The extract was prepared by maceration of the
powdered bark with distilled water for 48 h at room temperature, filtered
and air dried (yield: 2%, w/w). The extract was administered (25, 50 and
100 mg/kg; p.o.) to normal, glucose loaded and streptozotocin (55
mg/kg; i.p.) diabetic rats. In all the cases treatment with the extract
resulted in a dose-dependent decrease in the blood glucose level.
Moreover, the extract treatment increased the level of serum insulin,
body weight, and glycogen content of the liver and skeletal muscle and
reduced the level of serum triglyceride and total cholesterol of the
streptozotocin diabetic rats. Anti-lipid peroxidative effect on the pancreas
of streptozotocin diabetic rats was also observed after the extract
treatment. In the study glibenclamide at a dose of 10 mg/kg served as
standard. The study proved the effectiveness of the bark in type 1
diabetes.
Kaur et al., 2010, reported the antiamnesic effect of the methanolic
fruit extract was studied using scopolamine-induced anterograde and
retrograde amnesia model in mice 183. Elevated plus-maze and modified
passive avoidance paradigm served as behavioral models to study the
effect on learning and memory of the animal. Scopolamine (1 mg/kg; i.p.)
was administered before training for the induction of anterograde
amnesia and before retrieval for the induction of retrograde amnesia.
Piracetam at 200 mg/kg; i.p. served as standard. Treatment with the
extract (25, 50, and 100 mg/kg; i.p.) attenuated the scopolamine-
induced anterograde and retrograde amnesia in a dose-dependent
manner. In order to investigate the antiamnesic mechanism, the extract
was administered to the animals pretreated with 4mg/kg; i.p. of
cyproheptadine (non-selective 5-HT1/2 blocker). Reversal of the
antiamnesic effect of the extract by cyproheptadine pretreatment
substantiated the involvement of serotonergic mechanisms. Further
studies are required to investigate its precise downstream signaling.
43
Roy et al., 2009, reported wound healing activity of the hydro-
alcoholic leaf extract of F.religiosa has been investigated. 184. Leaf powder
was extracted with 70 % hydro-alcoholic solvent, dried under reduced
pressure to get a semisolid extract (yield 32.5 %, w/w). Phytochemical
screening showed the presence of glycosides and tannins in the extract.
The activity of the extract was determined using excision and incision rat
wound models. Treatment with 5 and 10 % extract ointment promoted
the healing of wound in a dose-dependent manner, indicated by
increased rate of wound contraction, decrease in the period for
epithelialisation and high skin breaking strength.
Vyawahare et al., 2007, reported based on the Ayurvedic reports,
the effect of F. religiosa leaves was investigated in the pentylenetetrazol
(PTZ)-induced convulsion model. The animals pretreated with the leaf
extract 30 min prior to PTZ (60 mg/kg; i.p.) exhibited 80–100 % seizures
protection 185.
Uma et al., 2009, investigated the effect of different solvent extracts
(aqueous, methanol, chloroform, petroleum ether and hexane) of the bark
of F. religiosa on the growth of thee enteroxigenic E. coli, isolated from the
patients suffering from diarrhoea. The aqueous, methanol and
chloroform extracts of the bark inhibited the growth of all the thee tested
enteroxigenic E. coli, showing zone of inhibition of 8, 12 and 10 mm
respectively. However, the petroleum ether and hexane extracts of the
bark were found to be ineffective. The study validated the traditional use
of the bark in diarrhoea, dysentery and menorrhagia186.
Kusumoto et al., 1995, studied the human immunodeficiency
virus-I protease (HIV-1 PR) inhibitory activity of the aqueous and
methanolic bark extracts of F. religiosa. HIV-1 PR from bacterial cells of
JM105 E. coli expressing the DNA sequence for HIV-1 PR was employed
in the study. Acetylpepstatin served as an enzyme inhibitory control. The
activity was assessed by calculating the ratio of the substrate peak the
45
3. PLANT PROFILE
TEPHOSIA PURPUREA
Kingdom: Plantae
Division: Magnoliophyta
Class: Magnoliopsida
Order: Fabales
Family: Fabaceae
Tribe: Millettieae
Genus: Tephosia
Species: T. purpurea
46
Botanical Description: Tephosia purpurea (Family--- Fabaceae). The plant is a
copiously branched herbaceous perennial plant distributed thoughout
the tropics and commonly known as Sarponkha (Hind.), jhila (San.),
Thila (Guj.), purple tephosia (Eng.)110 In the traditional system of
medicine the plant is used for different types of diseases and health
problems.
Pharmacognostical Characteristic:
Macroscopy
Plant is copiously branched suberect herbaceous perennial;
leaves 5-10 cm long, short petioles 6-12 cm long, leaflets13 to 21,
narrowly oblanceolate, green and glabrous above, obscurely silky
beneath, flowers in racemes, flowers fascicled, pedicels short,
bracteoles minute, pod long, glabrescent, slightly recurved, 6-8
seeded111.
Microscopy
TS of leaflet show isobilateral nature. Bicellular, uniseriate
covering trichomes are present on both the surfaces; mesophyll
consist of 3-5 layers of upper palisade cells and 2-3 layers of lower
palisade cells with parenchyma in between. One or two monoclinic
prisms of calcium oxalate crystals are present in the cells of
mesophyll. In the midrib region palisade layers are not continuous
above and below the vascular bundle and are replaced by
parenchyma; vascular bundle is large, more or less surrounded by
cells containing prisms of calcium oxalate crystals; xylem and phloem
capped externally by group of sclerenchymatous fibres. TS of mature
stem shows a well developed periderm comprising 8 to 11 layers of
tangentially elongated cork cells, 3 to 5 layers of phelloderm, cortex
with parenchymatous cells, groups of thick-walled pericyclic fibres,
groups of lignified phloem fibres; prisms of calcium oxalate crystals
are present in phloem parenchyma; abundant starch grains are
present in all the parenchymatous cells 112,113
47
Physical constant
Foreign matter: Not more than 2 %. Total ash: Not more than 7
%, Acid-insoluble ash: Not more than 1.2 %, Ethanol-soluble
extractive: Not less than 7 %, Water-soluble extractive: Not less than
12 %, Loss on drying: Not more than 7 %113
Traditional uses:
According to Ayurveda literature this plant has also given the
name of “Sarwa wranvishapaka” which means that it has the property
of healing all types of wounds114. It is an important component of
some preparations such as Tepholi and Yakrifit used for liver
disorders 115,116. In Ayurvedic system of medicine various parts of this
plant are used as remedy for impotency, asthma, diarrhoea,
gonorrhoea, rheumatism, ulcer and urinary disorders. The plant has
been claimed to cure diseases of kidney, liver spleen, heart and
blood111. The dried herb is effective as tonic laxative, diuretics and
deobstruents. It is also used in the treatment of bronchitis, bilious
febrile attack, boils, pimples and bleeding piles. The roots and seeds
are reported to have insecticidal and piscicidal properties and also
used as vermifuge. The roots are also reported to be effective in
leprous wound and their juice, to the eruption on skin. An extract of
pods is effective for pain, inflammation and their decoction is used in
vomiting117. Ethanolic extracts of aerial plant parts have medicinal
properties including anticancer activity against a human
nasopharyngeal epidermoid tumor cell line (KB)117. An aqueous seed
extract has significant in-vivo hypoglycaemic activity in diabetic
rabbits118. The ethanolic extracts of Tephosia purpurea possessed
potential antibacterial activity. The flavanoids were found to have
antimicrobial activity 119. The dried herb is effective as a tonic,
laxative, and diuretic. It is also used in the treatment of bronchitis,
bilious febrile attack, boils, pimples, and bleeding piles. The roots and
seeds are reported to have insecticidal, piscicidal, and vermifugal
properties120. The roots are effective in leprous wounds and root juice
48
to skin eruptions. Some important Ayurvedic marketed formulations
are: Sarapunkha svarasa, Sarapunkha lepa, Sarapunkha ghanavati 121.
FICUS GLOMERATA
Kingdom : Plantae
Division : Magnoliophyta
Class : Magnolipsida
Order : Urticales
Family : Moraceae
Genus : Ficus
Species : F. glomerata
49
Botanical Description: Ficus racemosa Linn syn. Ficus glomerata Family — Moraceae).
The plant is a large deciduous tree distributed all over India from outer
Himalayan ranges, Punjab, Khasia mountain, Chota Nagpur, Bihar,
Orissa, West Bengal, Rajasthan Deccan and common in south india It
is commonly known as Gular fig, Cluster fig in English, Gular in Hindi
and Udumbara Sanskrit. This plant is an evergreen, moderate to large
sized spreading, lactiferous, deciduous tree, without much prominent
aerial roots found thoughout greater part of India in moist localities
and is often cultivated in villages for its edible fruit. The tree is up to
18m high, leaves ovate, ovate-lanceolate or elliptic, sub acute, entire
and petiolate. Leaves are shed by December and replenished by
January and April, when the tree becomes bare for short period. Figs
subglobose or pyriform, red when ripe, borne in large clusters, on
short, leafless branches emerging from the trunk and the main
branches 118.
Pharmacognostical Characteristic:
Macroscopy The tree is medium tall with quite rich green foliage that
provides good shade. The leaves are dark green, 7.5-10 cm long, ovate
or elliptic. The fruit receptacles 2-5 cm in diameter, pyriform, in large
clusters, arising from main trunk or large branches. The fruits
resemble the figs and are green when raw, turning orange, dull
reddish or dark crimson on ripening. The seeds tiny, innumerable,
grain like, the outer surface of the bark consists of easily removable
translucent flakes grayish to rusty brown, uniformly hard and non-
brittle. Bark grayish green, soft surface and uneven 0.5-1.8 cm thick,
on rubbing white papery flakes come out from the outer surface, inner
surface light brown, fracture fibrous, taste mucilaginous without any
characteristics odour 138,139.
Microscopy
The cork is made up of polygonal or rectangular cells. The
.
50
phellogen is made up of 1-2 layers of thin walled cells. Phelloderm is
well marked compact tissue consisting of mainly parenchymatous cells
with isolated or small groups of sclereids, particularly in inner region.
Sclereids are lignified with simple pits. Several parenchymatous cells
contain single prism of calcium oxalate or some brownish content. The
cortex is wide with numerous sclereids and some cortical cells contain
resinous mass. Prismatic crystals of calcium oxalate are present in
some of the cells. Sclereids are rectangular or isodiametric and pitted
thick walled Phloem composed of sieve tubes companion cells, phloem
parenchyma sclereids, phloem fibres and medullary rays. Sclereids
have lignified walls with simple pits like those of cortex. Phloem fibres
are non-lignified, having narrow lumen without any septa. Prismatic
crystals of calcium oxalate and few clustered crystals are also present.
Starch grains are ovoid to spherical. Laticiferous vessels with a light
brown granular material are present in the phloem region. Cambium
when present 2-3 layered of tangentially elongated thin walled cells 140,141.
Physical constant
Foreign matter about 2 %, total ash 14 %, acid soluble ash 1 %,
alcohol soluble extractive 7 % and water soluble extractive 9 % 142.
Traditional uses:
The roots, bark-skin, fruits, latex and leaves have great
medicinal value. It is a one of the herbs mentioned in all ancient
scriptures of Ayurveda. Udumbara is considered sacred to God
Dattaguru. It is otherwise called Udumbara. Udumbara has various
synonyms like yajnanga, yajniya, yajnayoga, yajnyasara etc.
suggesting its use in ritual sacrifice. It is one of the ksiri viksa – on
cutting or plucking the leaf, lates oozes out. It is one of the plants
from a group, called pancavalkala, meaning the thick bark skins of
five herbs, viz. udumbara, vata, asvattha, parisa and plaksa. The
decoction of pancavalkala is used internally or for giving enema in
bleeding per rectum and vagina (Raja Nighantu). Maharishi Charka
51
has categorized udumbara as mutra sangrahaniya anti-udumbara as
mutra sangrahaniya – antidiuretic herb. Susruta has described the
properties of the plant, like astringent, promotes callus healing in
fractures (bhagna sandhaniya), alleviates Rakta pitta, burning
sensation and obesity, and useful in vaginal disorders. The roots,
bark-skin, fruits, lates and leaves of udumbara have great medicinal
value. Udumbara is used both, internally as well externally,
externally; the latex is applied on chonic infected wounds to alleviate
edema, pain and to promote the healing. The tender leaf buds are
applied on the skin, in the form of paste, to improve the complexion;
the decoction of leaves is salutary in washing the wounds for better
cleansing and healing. The decoction of its bark-skin is an effective
gargle in stomatitis and sore thoat. Application of latex alleviates the
edema in adenitis, parotitis, orchitis, traumatic swelling and
toothache. Internally, udumbara is used in vast range of maladies.
The decoction of bark skin is extremely useful in diarrhea, dysentery
and ulcerative colitis. In children, the latex is given along with sugar
to combat diarrhea and dysentery. The cold infusion of ripened fruits
mixed with sugar, is salutary in Rakta pitta is effectively controlled
with the decoction of bark-skin. In diabetes, the ripe fruits or bark-
skin decoction is beneficial, as it works well as anti-diuretic. The
decoction of leaves is an effective remedy in glandular swelling,
abscess, chonic wounds, cervical adenitis etc. In uterine bleeding,
abortion, leucorrhea and vaginal bleeding the decoction of its bark-
skin is given orally, as well as in a form of basti (enema). The latex
mixed with sugar is benevolent in sexual debility in males. The juice of
its fruit is a panacea for hiccup. The powder of the bark-skin works
well as an anorexient, hence, beneficial in gyperphagia-bhasmaka.
According to Ayurveda, roots are useful in hydrophobia whereas bark
is acrid, cooling, galactagogue and good for gynaecological disorders.
Fruits are astringent to bowels, styptic, tonic and useful in the
treatment of leucorrhoea, blood disorders, burning sensation, fatigue,
52
urinary discharges, leprosy, menorrhagia, epistaxis and intestinal
worms. According to Unani system of medicine, leaves are astringent
to bowels and good in case of bronchitis whereas fruits are useful in
treatment of dry cough, loss of voice, diseases of kidney and spleen.
Bark is useful in Asthma and piles. Latex is applied externally on
chonic infected wounds to alleviate edema, pain and to promote the
healing. The tender leaf buds are applied on the skin, in the form of
paste, to improve the complexion 143,144.
FICUS RELIGIOSA
Kingdom : Plantae
Division : Magnoliophyta
Class : Magnolipsida
Order : Urticales
Family : Moraceae
Genus : Ficus
Species : F. religiosa
53
Botanical Description: Ficus religiosa L. is the most popular member of the genus
Ficus, and is known English: Peepul tree, Pipal tree, Sacred fig Hindi:
Asvattha, Pipal Sanskrit: Achyutavas, Ashvatha, Bodhidru,
Bodhidruma, Marathi: Ashvatha, Pimpala. It is native of the sub-
Himalayan tract, Bengal and central India. It has been extensively
distributed worldwide though cultivation 158,159. F. religiosa tree begin
its life epiphytically and then strangle the host by its far-growing roots
that extend to the ground, establishing it as an independent tree. It is
found in the areas up to 1500melevation having an annual rainfall
varying from 50 to 500cm during the monsoon season and tolerates a
wide variation in temperature (below 0°C and above 40°C) 160. It is the
most sacred tree of South Asia, to both Hindus and Buddhists. The
specific epithet “religiosa” and synonym “bodhi tree” alludes to the
religious significance attached to this tree 161,162. Since antiquity, F.
religiosa has got mythological, religious and medicinal importance in
Indian culture. It is the oldest portrayed tree in India. Atharvaveda
(sacred text of Hinduism) links it with the third heaven and discusses
its medicinal properties along with Soma and Kustha (holy medicinal
herbs). References to F. religiosa are found in several ancient holy
texts like, Arthasastra, Puranas, Upanisads, Ramayana,
Mahabharata, Bhagavadgita, Buddhistic literature, etc.163. The
therapeutic utilities of F. religiosa have been indicated in traditional
systems of medicine like, Ayurveda, Unani, etc. It has been used to
cure the disorders of the central nervous system (epilepsy, migraine,
etc.), endocrine system (diabetes, etc.), gastrointestinal tract (vomiting,
ulcers, stomatitis, constipation, liver diseases, etc.), reproductive
system (menstrual irregularities, etc.), respiratory system (asthma,
cough, etc.) and infectious diseases (chickenpox, elephantiasis,
leprosy, tuberculosis, gonorrhea, scabies, etc.).
54
Pharmacognostical Characteristic:
Macroscopy F. religiosa is a large deciduous tree with few or no aerial roots.
It is often epiphytic with the drooping branches bearing long petioled,
ovate, cordate shiny leaves. Leaves are bright green, the apex
produced into a linear-lanceolate tail about half as long as the main
portion of the blade. The receptacles occurring in pairs and are
axillary, depressed globose, smooth and purplish when ripe. The bark
is flat or slightly curved, varying from 5 to 8 mm in thickness, outer
surface is grey or ash with thin or membranous flakes and is often
covered with crustose lichen brown or ash coloured, surface has
shallow irregular vertical fissures and uneven due to exfoliation of
cork, inner surface smooth, yellowish to orange brown and fibrous.
Microscopy Bark differentiated into outer thick periderm and inner
secondary phloem. Periderm is differentiated into phellem and
phelloderm. Phellem zone is 360 mm thick and it is wavy and uneven
in transection. Phellem cells are organized into thin tangential
membranous layers and the older layers exfoliate in the form of thin
membranes. The phelloderm zone is broad and distinct. Phelloderm
cells are turned into lignified sclereids. Secondary phloem
differentiated into inner narrow non-collapsed zone and outer broad
collapsed zone. Non-collapsed zone consists of radial files of sieve tube
members, axial parenchyma, and gelatinous fibres. Outer collapsed
phloem has dilated rays, crushed obliterated sieve tube members,
thick walled and lignified fibres, and abundant tannin filled
parenchyma cells. Laticifers are fairly abundant in the outer
secondary phloem zone. Phloem rays are both uniseriate and
multiseriate. Multiseriate rays are homocellular and uniseriate rays
are either homocellular or heterocellular.
.
55
Physical constant
Foreign matter about 2 % w/w, total ash 7.86 % w/w, acid
soluble ash 0.41 % w/w, alcohol soluble extractive 7.21 % w/w and
water soluble extractive 15.76 % w/w
Traditional Uses:
F. religiosa has been extensively used in traditional medicine for
a wide range of ailments. Its bark, fruits, leaves, adventitious roots,
latex and seeds are medicinally used in different forms, sometime in
combination with other herbs. The bark forms an important ingredient
of many Ayurvedic formulations, like “Pancha Valkaladi Tailum” (oil
containing F. religiosa, Ficus benghalensis L., Ficus glomerata Roxb.,
Ficus infectoria Willd., Azadirachta indica A. Curcuma longa L. and
Hemidesmus indicus R. Br.) and “Pancha Valkala Kashaya” (decoction
containing F. religiosa, F. benghalensis, F. glomerata., F. infectoria and
A. indica) 164,165. The Ayurvedic properties of F. religiosa include Rasa:
kashaya(astringent), Guna: guru (heavy), ruksha (dry), Veerya: shita
(cold)and Vipaka: katu (pungent)166. It has coloring or pigmenting
(varnya) action, ability to arrestpain (vedana sthaapana), remove
edematous swellings (shothahara) and conserves blood (rakta
samgrahaka167. The central concept of Ayurvedic medicine is a theory
that, health exists when there is an overall balance in the thee
organizing principles of Dosha called Vata, Pitta and Kapha,
imbalance in any of these results in diseased state. Vata express the
entire locomotor system of the body. Pitta (heat and metabolism)
represent all the metabolic activities, biochemical reactions and the
process of energy exchange like, digestion, exocrine and endocrine
glands functions, metabolic functions, etc. Kapha govern the structure
and cohesion of the organism, it is responsible for the biological
strength, natural tissue resistance and body structure. The use of
specific combinations in traditional medicine produces synergistic
effect and minimizes side effect. In case of infectious diseases,
combined therapy expands the antimicrobial spectrum and prevents
56
the emergence of resistance 168. In Ayurvedic terminology, drug
vehicles are known as “Anupaan”. The anupaan accelerates
circulation, absorption and assimilation of the drug into the body. For
example, the leaves of F. religiosa are applied on the inflammatory
ulcers using butter fat, such a type of vehicle helps in deep
penetration of the medicament by causing swelling of the skin and
invigorating the internal tissue 169.
57
4. AIMS AND OBJECTIVE
The costs of drug discovery and drug development continue to
increase at astronomical rates, yet despite these expenditures, there is
a decrease in the number of new medicines introduced into the world
market. Despite the successes that have been achieved over the years
with natural products, the interest in natural products as a platform
for drug discovery has waxed and waned in popularity with various
pharmaceutical companies. Natural products today are most likely
going to continue to exist and grow to become even more valuable as
sources of new drug leads. This is because the degree of chemical
diversity found in natural products is broader than that from any
other source, and the degree of novelty of molecular structure found
in natural products is greater than that determined from any other
source. Medicinal plant, which forms the back bone of traditional
medicine, have in the last few decades been the subject of very intense
pharmacological studies, this has been brought about by the
acknowledgement of the value of the value of medicinal plant as
potential source of new compounds in the drug development. In
developing countries, it is estimated that about 80 % of the population
rely on traditional medicines for their primary health care. Cancer
continues to represent a largest source of mortality in the world and
claims over 6 millions lives every year.
An extremely promising strategy for cancer prevention today is
chemoprevention, which defines as the use of synthetic or natural
agent alone or combination to block the development of cancer in
humans.
There is growing interest in the pharmacological evaluation of
various plants used in Indian traditional system of medicines. There
arises a need therefore to screening medicinal plant for bioactive
compounds as a basis for further pharmacological studies and also
isolated the active plant constituent which is responsible for
pharmacological activity and characterization, structural elucidation
58
of that compound by using newly developed and available resources.
The plants selected for anticancer activity was based on its easy
availability, degree of research work which is not done and folklore
claiming its therapeutic activity without any side effects. These plants
are also known as an anti-tumor agent in ancient system of
medicines. Hence, these plants having wide scope for detailed
pharmacological and phytochemical structural elucidation and
investigation of Analgesic activity, Anti-inflammatory activity,
Antihyperlipidemic activity, cytotoxicity scientifically explored for the
common cause of Indian economically connected to poor people.
59
SCOPE
Man’s existence on this earth has been made possible only
because of the vital role played by the plant kingdom in sustaining his
life. In recent years, there has been a phenomenal rise in the interest
of scientific community to explore the pharmacological actions or to
confirm the veracity of claims made about herbs in the official book of
the Ayurveda. Plant remedies contain active principles that are
constantly being screened for their possible pharmacological value. It
is necessary to screen plants for new activities. To meet this objective,
judicious efforts are required in selection of plant material, time for its
collection, method of processing and pharmacological screening for
proper therapeutic effects.
Survey of Ayurvedic literature including Ayurvedic preparations,
books based on folklore medicines and medicinal plants used in India
suggest that in Indian system of medicine, about 2000 plant were
used for the treatment of inflammatory disorders. Very few of them
were investigated scientifically. Most frequently used plants for the
treatment of inflammation, cancer, arthritis, and hyperlipidemic
disorders. There is paucity of scientific data about the inflammation
and related activity of T. purpurea, F. religiosa, and F. glomerata.
From the literature review it is reviews that these plants are
Not exploded to pharmacological activity and hence following
the plan of work as under
60
PLAN OF WORK
PHASE – 1
Selection of plants: -
Criteria for the selection of plant shall be set and following these
criteria extensive review of literature based on traditional system of
medicine and scientific work shall be carried out.
Collection, Identification and Authentication: -
Selected plant material shall be collected and authenticated
from authorized persons.
PHASE – 2
Preparation of extracts: -
Various extracts shall be prepared using solvents of different
polarity.
Preliminary Phytochemical evaluation of extracts: -
All extracts shall be investigated for the presence of chemical
constituents in it.
Acute toxicity study: -
All the extracts shall be investigated for its acute toxicity.
PHASE – 3
Fractionation: -
The extract showing promising activity shall be subjected to
fractionation by column chromatography.
Pharmacological screening of fractions: -
Fractions showing chemical composition shall be pooled and
then screened for analgesic, anti-inflammtory, anti arthritis, anti
cancer and antihyperlipidemic activity using standard animal models.
44
hydrolysate peak area using HPLC. The aqueous and methanolic extracts
of the bark exhibited excellent HIV-1 PR inhibitory activity to the extent
of 54.6 ± 0.5 and 42.9± 2.7 % respectively187.
Agarwal et al., 1988, reported treatment with the fruit fiber diet
showed significant hypolipidemic effect, indicated by reduced level of
serum cholesterol and phospholipids, and liver total lipids and
cholesterol. Moreover, its treatment showed increased fecal excretion of
cholesterol and bile salts 188.
Preethi et al., 2010, reported the bark, roots and fruits,the
antioxidant effect of the aqueous, methanolic and ethanolic leaf extracts
at 35–36g/100mg concentration, in the in vitro DPPH assay has been
reported 189.
Mallurwar and Pathak, 2008 investigated the immune stimulant
activity of the ethanolic bark extract of F. religiosa. Pyrogallol-induced
immune suppression model was employed to induce immune
suppression in mice. Sheep red blood cells were injected as an antigenic
material to sensitize the mice. Vitamin E suspension at a dose of 150
mg/kg; p.o. served as standard. The investigators found that the extract
at 100 mg/kg; p.o. dose stimulates the humoral and cell mediated
immune response190.
Patel and Patel, 2000, reported the study proved the
bronchodilator effect of the bark and validated its traditional use in
asthma. Invented a composition made from the powdered interior bark of
F. religiosa, admixed with a rice pudding containing milk, sugar, rice and
cardamom. The composition claimed to suppress all the symptoms of
asthma in a human191.
61
5. MATERIALS AND METHODS 5.1 Plant material:
The leaves of Tephrosia purpurea (family- Fabaceae), Ficus religiosa
(family- Moraceae), Ficus glomerata (family- Moraceae) were collected
from Nasik district in the month of July.
5.1.1 Identification and Authentication of Plant Material:
Confirmation of identity and authentication, on the basis of
organoleptic characters, exomorphology and pharmacognostic study of
all plant materials were carried out by Prof. P.G.Diwakar, Botanical
survey of India, Pune and the Voucher No. BSI/WC/Tech/08/340. They
were placed in a cool and dry place. The certificate for the authentication
was obtained.
5. 2 Chemicals:
Carrageenan, Freund’s complete adjuvant, serotonin, histamine
were purchased from Sigma Chemicals Co., St Louis, MO, USA. Gum
acacia was purchased from Research Lab. Mumbai. Borosil glass column
(height, 60 cm; diameter, 3 cm) (J-Sil, Mumbai), TLC grade silica gel were
purchased from Research Lab, Mumbai.
All the solvents such as petroleum ether (60-80°C), chloroform,
methanol, benzene, ethyl acetate, methanol and acetone were procured
from Orchid Scientifics, Mumbai, India.
5.3 Drugs:
Ibuprofen (Brufen tablet, Abbott India Ltd, Goa) water for injection
was purchased from medical stores.
5.4 METHOD 5.4.1Extraction Procedure of plant materials:
5.4.1.1 Petroleum ether extract:-
Coarsely powdered plant materials (500 g) were extracted with
2000 ml of petroleum ether (60-80°C) for 48 h using Soxhlet apparatus.
Residue was filtered through filter paper to obtain a clear extract.
62
Residues were pooled, transferred to previously weighed petri dish and
evaporated to dryness at room temperature (35-40°C) so as to obtain
dried extracts. After completion of drying the petri dish was weighed
again. The yield of extract was calculated by subtracting initial weight of
petri dish from final weight. The yield was represented as percent yield.
5.4.1.2 Chloroform extract:-
The residue remained after petroleum ether extraction, was dried
in air. The dried residue was extracted following same procedure as
above by using chloroform as solvent, to obtain dried chloroform extract.
5.4.1.3 Methanolic extract:-
The residue remain after chloroform extraction, was dried in air.
The dried residue was extracted following same procedure as above by
using methanol as solvent, to obtain dried methanolic extract.
5.4.1.4 Aqueous extract:-
Residue of methanolic extraction was dried in air. The dried extract
was then extracted following same procedure as in petroleum ether
extraction by using distilled water as solvent. The aqueous extracts were
evaporated to dryness at 55°C (Percent yield by different solvent
extraction is shown in following table).
5.4.2 Phytochemical analysis of extracts
The phytochemical analysis of extracts was carried out by the
method according to standard procedure 192
5.4.2.1 Test for Carbohydrates:
Preparation of extract solutions:-
Test solutions of petroleum ether, chloroform, methanol and water
extract were prepared in petroleum ether, chloroform, methanol and
water respectively in concentration 100 mg/ml.
Molish’s test:-
Few drops (2-3) of α-naphthol solution in alcohol was added to 2-3
ml of test solution, shaken for few min and then 0.5 ml of conc. H2SO4
63
was added from the side of test tube. The formation of violate ring at the
junction of two solutions indicated presence of carbohydrates.
Test for reducing sugar
Fehling’s test:-
Fehling’s A and B solutions (1 ml each) were added to the test tube
and boiled for 1 min. To this 2 ml of test solution was added and heated
in boiling water bath for 5-10 min. Appearance of yellow and then brick
red precipitate indicated the presence of reducing sugars.
Benedict’s test:-
Benedict’s reagent (1 ml) and test solution(1ml) was mixed in test
tube and heated in boiling water bath for 5-10 min. Change in color to
yellow, green or red indicated the presence of reducing sugar.
Test for monosaccharide
Barfoed’s test:-
Barfoed’s reagent (1 ml) and test solution (1 ml) was mixed in test
tube, heated in boiling water bath for 1-2 min. and then cooled. The
appearance of red precipitate indicated the presence of monosaccharides.
Test for pentose sugar
Bial’s test:-
To the boiling Bial’s reagent (2 ml) test solution (4 drops) was
added. The appearance of green or purple color indicated the presence of
pentose sugar.
Test for hexose sugar
Selwinoff’s test (for fructose):-
Selwinoff’s reagent (3 ml) and test solution (1 ml) was heated on
water bath for 1-2 min. The change in color to red indicated presence of
hexose sugar.
Cobalt chloride test:-
Test solution (3 ml) and cobalt chloride solution (2 ml) was mixed
in test tube, boiled for 2 min and cooled. NaOH solution (2-5 drops) was
64
added to test tube. The change in color of solution to greenish blue,
purpulish or upper layer greenish blue and lower layer purpulish
indicated presence of glucose, fructose or mixture of glucose and fructose
respectively.
5.4.2.2 Test for proteins:
Biuret test:
To the test solution (3 ml), 4 % NaOH (2-5 drops) and 1% CuSO4
(2-5 drops) was added.The change in color of solution to violet or pink
indicated presence of proteins.
Million’s test:
Test solution(3 ml) and Million’s reagent (5 ml) were mixed in test
tube. The appearance of white precipitate changing to brick red or
dissolved and gave red color to solution on heating indicated presence of
proteins.
Test for proteins containing tyrosine and tryptophan
Xanthoprotein test:
To the test tube containing test solution (3 ml), 1 ml of conc.
H2SO4 was added. Appearance of white precipitate which turns to yellow
on boiling and orange on addition of NH4OH indicated presence of tyrosin
and/or tryptophan containing proteins.
Test for protein containing sulphur
Test solution(5 ml), 40 % NaOH (2 ml) and 2 drops of lead acetate
solution was mixed in test tube and boiled for 5 min. Change in color to
brownish or black indicated presence of sulphur containing protein.
5.4.2.3 Test for amino acids:
Ninhydrin test:
Test solution(3 ml) and 3 drops of 5 % lead acetate solution were
boiled on water bath for 10 min. Change in color of solution to purple or
blue indicated presence of amino acids.
65
5.4.2.4 Test for tannins and phenols:
Following reagent (3-5 drops) were added to the 2-3 ml of test
solution. Change in color to blue-black, appearance of white precipitate
or decoloration of solution indicated presence of tannins and phenols.
5% Ferric chloride: blue- black color.
Lead acetate: white precipitate.
Potassium permanganate: decoloration.
5.4.2.5 Test for glycosides:
Cardiac glycosides
Legal’s test:
To the extract, 1 ml of pyridine and 1 ml of sodium nitroprusside
was added. Change in color to pink or red indicated presence of cardiac
glycosides.
Keller-Killiani test:
Glacial acetic acid (3-5 drops), one drop of 5 % FeCl3 and conc.
H2SO4 was added to the test tube containing 2 ml of test solution.
Appearance of reddish-brown color at the junction of two layers and
bluish green in the upper layer indicated presence of cardiac glycosides.
Anthraquinone glycosides
Borntrager’s test:
Dilute H2SO4 was added to 2 ml of solution of extract, boiled for
few minutes and filtered. To the filtrate 2 ml of benzene or chloroform
was added and shaken well. Separated the organic layer and ammonia
was added. The Change in color of ammonical layer to pink-red indicated
presence of anthraquinone glycosides.
5.4.2.6 Test for saponins:
Foam test:
Extract (10-20 mg) was shack vigorously with water (1 ml).
Development of persistent foam indicated presence of saponins.
66
5.4.2.7 Test for flavonoids:
Shinoda test:
To dry extract (10-20 mg), 5 ml of ethanol (95 %), 2-3 drops of HCl
and 0.5 g magnesium turnings were added. Change of solution color to
pink indicated presence of flavonoids.
5.4.2.8 Test for Alkaloids:
To the dry extract (10-20 mg) dilute HCl (1-2 ml) was added,
shaken well and filtered. With filtrate following test was performed.
Mayer’s test:
To 2-3 ml of filtrate 2-3 drops of Mayer’s reagent was added.
Appearance of precipitate indicated presence of alkaloids.
Wagner’s test:
To 2-3 ml of filtrate Wagner’s (3-5 drops) reagent was added.
Appearance of reddish brown precipitate indicated presence of alkaloids.
Hager’s test:
To 2-3 ml of filtrate 4-5 drops of Hager’s reagent was added.
Appearance of yellow precipitate indicated presence of alkaloids.
5.4.2.9 Test for Steroids:
Salkowski reaction:
Chloroform (2 ml) and of H2SO4 (2 ml) was added to 2 ml of test
solution and Shaken well. Change in color of chloroform layer to red and
acid layer to greenish yellow fluorescence indicated presence of steroids.
Liebermann-Burchard reaction:
Test solution 2 ml was mixed with chloroform (2 ml). To the
solution, 1-2 ml of acetic anhydride and 2 drops of conc. H2SO4 from the
side of test tube was added. It gives colored change to red, then blue and
finally green indicated presence of steroids.
67
5.4.3.1 Preparation of dosage form:
Dosage forms of individual extracts were prepared as per the
following procedures.
Petroleum ether extract:
Emulsion of petroleum ether extracts of T.purpurea, F.religiosa and
F.glomerata were prepared by triturating extract with Gum acacia (0.5%)
in glass mortar with gradual addition of water to make the volume.
Chloroform extract:
Emulsion of chloroform extracts of T.purpurea, F.religiosa and
F.glomerata were prepared by triturating extract with Gum acacia (0.5%)
in glass mortar with gradual addition of water to make the volume.
Methanol extract:
Emulsion of methanol extracts of T.purpurea, F.religiosa and
F.glomerata prepared by triturating extract with Gum acacia (0.5%) in
glass mortar with gradual addition of water to make volume.
Aqueous extract:
Solution of aqueous extract T.purpurea, F.religiosa and F.glomerata
were prepared in water.
Vehicles:
Respective vehicles were prepared by the same procedure without
addition of extracts.
5.4.3.2 Animals
Albino Wister rats (150 ± 200 g) of either sex were obtained from
the animal house of Yash Institute of India Ltd, Pune. On arrival animals
were placed randomly in polypropylene cages (six per cage) with paddy
husk as bedding. Standard laboratory conditions of temperature 24 ± 2
°C, relative humidity 55 ± 5 % and 12:12 h light dark cycle were
maintained throughout all the experiments.
Animals had free access of water filtered through Aquaguard® and
68
standard pellet animal diet (Chaken oil Mill, Pune; India) ad libitum. On
the day of experiments animals were food deprived at 06:00 h and placed
in the experimental room at 08:00 h.
In case of behavioral study, before start of the session, the
apparatus was cleaned with hydrogen peroxide to avoid possible bias due
to odor trails left by previous animal.
5.4.3.3 APPROVAL OF PROTOCOL
All the experimental procedures and protocols used in this study
were reviewed and approved by the Institutional Animal Ethical
Committee (IAEC) of SSDJ college of Pharmacy, Chandwad (Nasik)
constituted under Committee for Purpose of Control and Supervision of
Experiments on Animals (CPCSEA), Ministry of Environment and
Forests, Government of India. Ethical guidelines were strictly followed
during all the experiments.
5.4.3.4 Acute toxicity study
Doses of T. purpurea, F.religiosa and F. glomerata 30, 100, 300,
1000 and 2000 mg/kg of all above extracts were administered
intraperitonial to mice. TP was administered 30, 100, 300, 1000 and
2000 mg/kg in case of oral toxicity study. Mice were then observed for
incidence of mortality or any sign of toxicity up to 24 h after injection.
The dosing schedule as per the OECD (guidelines 425)193 followed.
1st animal received a dose of 30 mg/kg intraperitonial or per oral
route. Animal was observed for 3 h after injection for any toxicity signs,
survival, or death. If the 1st animal died or appeared moribund, the 2nd
animal received lower dose (10 mg/kg).
The dose progression or reduction factor was 3.2 times of previous
dose. If no mortality was observed in 1st animal then 2nd animal received
higher dose (100 mg/kg). Dosing of next animal was continued
depending on outcome of previously dosed animal for fixed time interval
(3h). The test was stopped when one of stopping criteria was met.
69
- 5 reversals occur in any 6 consecutive animal tested
- 3 consecutive animal died at one dose level
Survived animals were observed for outcomes for period of 24 h.
5.4.4 Fractionation
The pet ether extract of T. purpurea, F. religiosa and F.glomerata
and Methanol extract of F. religiosa and F.glomerata, these extracts were
subjected to further fractionation.
5.4.4.1 Fractionation of Pet ether extract of T. purpurea
The pet ether extract was allowed to evaporate slowly in shallow
dish and resinous mass was discarded. The remaining material was
further fractionated into acetone soluble and acetone insoluble portions.
These two portions were dissolved in benzene and subjected to column
chromatographic separation as detailed below:
5.4.4.1.1 Chromatographic separation of acetone soluble part:
Slurry of activated silica G was prepared and then the column was
packed with slurry. The sample was loaded on the packed silica gel. After
stabilization column was eluted with mobile phase. Fractions were
collected and analyzed by TLC.
Activation of silica:-
Column grade silica G was kept in oven at 150°C for 3 h to remove
the all moisture content present in it.
Weighed quantity of activated silica was added to the beaker containing
mobile phase and stirred with glass rod to prepare the slurry.
Preparation of mobile phase:-
The solvents Benzene and ethyl acetate were distilled and then
used for the preparation of mobile phase. The composition of mobile
phase was Benzene: ethyl acetate (7:3).
Packing of column:-
A clean and dry borosil glass column (60 cm, height; 3 cm,
70
diameter) was aligned in a vertical position with the help of clamps
attached to metal stand. A piece of cotton soaked in mobile phase was
placed in the bottom of the column and gently tamped down with a glass
rod. Column was then filled about 1/3 volume by the mobile phase.
The column was slowly and evenly filled about 5/6 volumes full
with gradual addition of silica gel slurry. Stopcock was opened to allow
excess mobile phase to drain into the beaker. Side of the
chromatographic column was gently tapped with a cork during the
packing process to make the silica gel compact. Meanwhile the stopcock
was opened to allow excess mobile phase to run out. When the packing
was finished the excess mobile phase was drained until it just reaches
top level of silica.
Application of sample:-
Weighed quantity of the sample was mixed with 1-2 g of activated
silica gel and 3-4 ml of mobile phase to prepare slurry. The slurry of
sample was added to top of the packed silica in column. Stopcock was
opened to drain excess mobile phase until it reaches top level of sample.
A thin disc (column diameter) of cotton soaked in mobile phase
was placed on top of the bed to prevent disturbing the sample layer after
addition of mobile phase. Column was filled to the top with the mobile
phase and allowed to stand for overnight (~24 hours) to develop a
chromatogram.
Elution:-
Elution was carried out by the gravity at the flow rate of 1 ml/min.
Mobile phase was added to the top of the column and fractions were
collected in amber colored bottle. Fractions were concentrated by
evaporating at room temperature until volume reduced to ¼ of the total
volume. TLC of concentrated fractions was carried out to detect similarity
between the chromatograms of different fractions 194.
71
5.4.4.1.2 Chromatographic separation of acetone insoluble part:
The separation of acetone insoluble part was carried out as
described above.
5.4.4.2 Fractionation of Pet ether extract of F. religiosa and
F.glomerata
The pet ether extracts of both the above plants were subjected to
column chromatography using silica gel. The solvents such as benzene
and ethyl acetate were used for separation. The procedure employed is as
described earlier. Only benzene fractions were used for the study.
5.4.4.3 Fractionation of Methanol extract of F. religiosa and
F.glomerata
The dried methanolic extract was separated into water soluble and
water insoluble portion Water soluble portion was shaken vigorously with
absolute alcohol yielded a gelatinous precipitate. The water insoluble
part was dissolved in minimum volume of absolute alcohol and column
chromatography was carried out with benzene and ethyl acetate.
72
Source Extract Fraction Abbreviation
T. purpurea Pet ether Acetone soluble
Benzene fraction TPI
Acetone insoluble
Benzene fraction TPIII
F.religiosa Pet ether Benzene fraction FRI
Alcoholic Water soluble Precipitate FRIII
F.glomerata Pet ether Benzene fraction FGI
Alcoholic Water soluble Precipitate FGIII
5.4.5 Thin Layer Chromatography (TLC): The slurry was prepared by suspending silica gel G in distilled
water (1:2). Measured amount of slurry was put on the clean and dry
glass plate, which was kept on a level surface. The plate was then tipped
back and forth to spread the slurry uniformly over the surface. The
plates were dried in air for 30 min and then in oven at 110°C for another
60 min for the activation of adsorbent layer.
A spot of sample was applied on the starting line, which was
parallel and about 10 mm above the lower edge, with help of glass
capillary. Sample spots were allowed to dry at room temperature.
Sufficient quantity (3-4 ml) of mobile phase was poured into the
chamber. To achieve saturation, chamber was closed and allowed to
stand for 15-20 min. The plate was placed as nearly vertical as possible
into the chamber, ensuring that the points of application were above the
surface of the mobile phase. Chamber was closed and mobile phase was
73
allowed to ascend to specified distance. Plate was removed, position of
mobile phase front was marked and mobile phase was allowed to
evaporate at room temperature.
Plate was observed in the daylight, under UV light and then in
iodine chamber. After each observation the central points of spots
appeared on chromatogram were marked with needle. Retention factor
(Rf) was calculated by following formula:-
Rf = A/B
A = distance between point of application and central point of spot
of material being examined.
B = distance between point of application and the mobile phase
front 194.
5.4.6 PHARMACOLOGICAL ACTIVITY OF T. PURPUREA, F.
RELIGIOSA AND F. GLOMERATA:
5.4.6.1 Evaluation of biological activity
5.4.6.1.1. Screening of In-vitro Anticancer Activity
5.4.6.2 EVALUATION OF ANALGESIC ACTIVITY:
5.4.6.2.1 Tail flick latency period in rats
5.4.6.2.2 Acetic acid induced writhing in mice
5.4.6.3 EVALUATION OF ANTI-INFLAMMATORY ACTIVITY
5.4.6.3.1carrageenan induced paw edema method ,
5.4.6.3.2Serotonin and Histamine induced paw edema,
5.4.6.3.3Cotton pellet granuloma formation in rats.
5.4.6.4 EVALUATION OF ANTI-ARTHRITIS ACTIVITY
5.4.6.4.1 Adjuvant induced arthritis in rats
5.4.6.4.2 Formalin induced arthritis in rats
74
5.4.6.5 EVALUATION OF ANTIHYPERLIPIDEMIC ACTIVITY
5.4.6.5.1 Preparation of normal and high-fat diet
5.4.6.5.2 Biochemical analysis of T. purpurea, F. religiosa and F.
glomerata
A) Serum Total Cholesterol B) Serum Triglycerides
C) Serum High density lipoproteins D) Very low density lipoproteins
E) Low density lipoprotein F) Serum SGOT
G) Serum SGPT
5.4.6.6 STATISTICAL ANALYSIS
5.4.6.1 IN-VITRO ANTICANCER ACTIVITY:
5.4.6.1.1 Trypan Blue Exclusion Test of Cell Viability
The dye exclusion test is used to determine the number of viable
cells present in a cell suspension. It is based on the principle that live
cells possess intact cell membranes that exclude certain dyes, such as
trypan blue, Eosin, or propidium, whereas dead cells do not. In this test,
a cell suspension is simply mixed with dye and then visually examined to
determine whether cells take up or exclude dye. In the protocol presented
here, a viable cell will have a clear cytoplasm whereas a nonviable cell
will have a blue cytoplasm. In brief an aliquot of MCF -7 cell suspension
was centrifuged for 5 minute at 100 rpm and supernatant was discarded.
The pellet formed thus resuspended in 1 ml of PBS which serves as cell
suspension. 1 part of 0.4 % trypan blue was mixed with 1 part cell
suspension and supplemented with TPI, TPIII, FRI, FRIII and FGI, FGIII
fractions (20, 40, 60, 80, 100, 120, 140, 160, 180 and 200 μg/ml)
allowed to incubate for 3 min. at room temperature. A drop of trypan
blue/cell mixture was applied to a haemocytometer and placed on the
stage of an inverted microscope and focus on the cells. The unstained
(viable) and stained (nonviable) cells were counted separately to obtain
the total number of viable cells per ml of aliquot 195196
75
5.4.6.2 EVALUATION OF ANALGESIC ACTIVITY:
5.4.6.2.1 Evaluation of Tail flick latency period in rats:
The fraction obtained from T. purpurea, (TPI, TPIII), F.religiosa (FRI,
FRIII) and F. glomerata (FGI, FGIII) were evaluated for analgesic activity.
Male rats of 125-150 g. rats were divided into fourteen groups containing
five animals in each group. A tail flick response was evoked by placing
each rat tail over the wire heated electrically, using Analgesiometer
(Space Scientific, Nashik, India). The intensity of heat was adjusted so
that baseline tail flick latency averaged 3-4 sec in all animals. Cut off
time was 15 sec in order to avoid injury to tail. All groups received
respective doses 1 h prior to the test 197.
The percentage analgesia was computed using the formula
% analgesia = Test latency- control latency X 100
15- control latency
Group I served as control and received vehicle.
Group II, III, received the fractions TPI (20 and 40mg/kg, p.o)
Group IV and V received the fractions TPIII (20 and 40mg/kg, p.o)
Group VI, VII, received the fractions FRI (20 and 40mg/kg, p.o)
Group VIII, IX received the fractions FRIII (20 and 40mg/kg, p.o)
Group X, XI, received the fractions FGI (20 and 40mg/kg, p.o)
Group XII and XIII received the fractions FGIII (20 and 40mg/kg, p.o)
Group XIV received the reference standard i.e. ibuprofen (40mg/kg, p.o)
5.4.6.2.2 Evaluation of Acetic acid induced writhing in mice:
The fraction obtained from T. purpurea, (TPI, TPIII), F.religiosa (FRI,
FRIII) and F. glomerata (FGI, FGIII) were evaluated for acetic acid induced
writhing. Male albino mice of 20-25 g. were divided into fourteen groups
containing five animals in each group. The writhing syndrome was
elicited by intraperitonial injection of acetic acid (0.1ml of 0.6% solution)
and numbers of writhes displayed from 5 to 20min were recorded 198. All
groups received respective doses 30 min prior to the test.
76
Group I served as control and received vehicle. The percentage analgesia
was computed using the formula
% inhibition of writhes= S-T/S X100
Where S is the number of writhes in control group and T is the number
of writhes in treated group
Group II, III, received the fractions TPI (20 and 40mg/kg, p.o)
Group IV and V received the fractions TPIII (20 and 40mg/kg, p.o)
Group VI, VII, received the fractions FRI (20 and 40mg/kg, p.o)
Group VIII, IX received the fractions FRIII (20 and 40mg/kg, p.o) whereas
Group X, XI, received the fractions FGI (20 and 40mg/kg, p.o)
Group XII and XIII received the fractions FGIII (20 and 40mg/kg, p.o)
Group XIV received the reference standard i.e. ibuprofen (40mg/kg, p.o)
5.4.6.3.1 Evaluation of anti-inflammatory activity induced by
Carrageenan hind paw edema
The fraction obtained from T. purpurea, (TPI, TPIII), F.religiosa (FRI,
FRIII) and F. glomerata (FGI, FGIII) were evaluated for anti-inflammatory
activity. The anti-inflammatory activity using carrageenan induced hind
paw edema was carried out as described by Winter et al., (1962)199.
Male rats of 125-150 g were used. Rats were divided into fourteen groups
containing five animals in each group.
Group I served as control group and received distilled water (DW), orally.
Group II received Ibuprofen (40 mg/kg, p.o.) as standard.
Group III and IV animals received fraction at a dose of TPI 20, and 40
mg/kg p.o.
Group V and VI animals received the fractions of TPIII at a dose of 20,
and mg/kg p.o.
Group VII and VIII, animals received fraction of FRI at a dose of 20, and
40 mg/kg p.o.
Group IX and X received fraction of FRIII at a dose of 20, and 40 mg/kg
p.o.
77
Group XI and XII fraction of FGI at a dose of 20, and 40 mg/kg p.o.
Group XIII and XIV received fraction of FGIII at a dose of 20, and 40
mg/kg p.o.
After 1 h, 0.1 ml of 1% w/v Carrageenan suspension was
injected subcutaneously in to the plantar surface of the right hind paw.
The paw volume was measured using a Digital Plethysmometer (UGO
Basile, Italy. Model.7130) at 0, 1, 2 and 3 h after carrageenan injection.
The percentage inhibition of paw edema was calculated at 3 h using the
formula:
% inhibition= (Vt-Vc/Vc) X100, where, Vt- volume of paw for
treated group, Vc- volume of paw for control group
5.4.6.3.2 Evaluation of anti-inflammatory activity induced by
serotonin and histamine paw edema 200,201
The fraction obtained from T. purpurea, (TPI, TPIII), F.religiosa (FRI,
FRIII) and F. glomerata (FGI, FGIII) were evaluated for serotonin and
histamine induced paw edema. Male rats of 125-150 g were used. Rats
were divided into eight groups containing five animals in each group.
Group I served as control group and received distilled water (DW), orally.
Group II received Ibuprofen (40 mg/kg, p.o.) as standard.
Group III and IV animals received fraction at a dose of TPI 20, and TPIII
40 mg/kg p.o. while Group
V and VI animals received the fractions of FRI 20 mg/kg, p.o. and FR III
40mg/kg;
Group VII and VIII, animals received fraction of FGI 20 mg/kg, p.o., and
FGIII40 mg/kg p.o.
Edema was induced into the hind paw by injecting 0.1 ml of 0.1 %
w/v serotonin and also edema was induced into the hind paw by
injecting 0.1 ml of 0.1 % w/v histamine. The paw volume was measured
using a Digital Plethysmometer (UGO Basile, Italy.Model.7130)
immediately and 1 h after serotonin and histamine injection
78
5.4.6.3.3 Evaluation of Cotton pellet granuloma formation in rats
The fraction obtained from T. purpurea, (TPI, TPIII), F.religiosa (FRI,
FRIII) and F. glomerata (FGI, FGIII) were evaluated for Cotton pellet
granuloma formation in rats. Male rats of 125-150 g. were divided into
eight groups containing five animals in each group. The cotton pellet
weighing 50±2 mg was sterilized in an autoclave (Lab hosp, Mumbai,
India) handled with sterile instrument. The pellet was inserted in each
animal on the back. All the fractions were administered for consecutive
six days 202,203. The animals were sacrificed on seventh day and cotton
pellet along with granuloma mass were collected, it was weighed and
dried at 60°C. % inhibition of dry weight of granuloma formation by
using the formula:
100 (A-B)/A, where, A= gain in dry weight of control pellet (mg), B=
gain in dry weight of drug treated (mg).
Group I served as control and received vehicle.
Group II received the reference standard i.e. ibuprofen (40mg/kg, p.o)
Group III, IV, received the fractions TPI and TPIII (40mg/kg, p.o)
Group V and VI received the fractions FRI and FRIII (40mg/kg, p.o)
Group VII, VIII, received the fractions FGI and FGIII (40mg/kg, p.o)
5.4.6.4 Evaluation of anti-arthritis activity by adjuvant induced
arthritis in rats
The fraction obtained from T. purpurea, (TPI, TPIII), F.religiosa (FRI,
FRIII) and F. glomerata (FGI, FGIII) were evaluated for Adjuvant induced
arthritis in rats. Male rats of 125-150 g. were divided into fourteen
groups containing five animals in each group. Arthritis was induced in
rats by injecting 0.1 ml of Freund’s complete adjuvant in to sub plantar
region of the right hind paw. The above fractions were administered
orally from the 13th day to the 40th day. The volume of edema was
79
measured daily using a Digital Plethysmometer (UGO Basile, Italy.
Model.7130)204.
The volume of edema was compared with the vehicle treated group. The
percentage inhibition was determined using the formula
% inhibition= 1-(A-X)/(B-Y)x 100
Where A- the volume of paw on ‘nth day, in the treated group
X- the volume of paw on first day before adjuvant, in the same
group
B- the volume of paw on ‘nth day, in the control group
Y- the volume of paw on first day before adjuvant, in the same
group
Group I served as control group and received distilled water (DW), orally.
Group II received Ibuprofen (40 mg/kg, p.o.) as standard.
Group III and IV animals received fraction at a dose of TPI 20, and TPIII
40 mg/kg p.o.
V and VI animals received the fractions of FRI 20 mg/kg, p.o. and FR III
40 mg/kg p.o.
Group VII and VIII, animals received fraction of FGI 20 mg/kg, p.o., and
FGIII40 mg/kg p.o.
5.4.6.4.2 Evaluation of anti-arthritis activity by formalin induced
arthritis in rats
The fraction obtained from T. purpurea, (TPI, TPIII), F.religiosa (FRI,
FRIII) and F. glomerata (FGI, FGIII) were evaluated for formalin induced
arthritis in rats. Male rats of 125-150 g. were divided into fourteen
groups containing five animals in each group. Arthritis was induced in
rats by injecting 0.1 ml of 2% formalin in to sub plantar region of the
right hind paw on first and third day of experiment at 11.00 h. The
volume of edema was measured using a Digital Plethysmometer (UGO
Basile, Italy. Model.7130) before the injection of the irritant and once
daily at 15 h for 10 days. The fractions were administered orally from the
80
day first to day ten of the experiment. The mean increase in the paw
volume of each group over a period of ten days was calculated and
compare with the control 205,206.
The percentage inhibition was determined using the formula
% inhibition=% inhibition= (Vt-Vc/Vc) X100, where, Vt- volume of
paw for treated group, Vc- volume of paw for control group
Group I served as control group and received distilled water (DW), orally.
Group II received Ibuprofen (40 mg/kg, p.o.) as standard.
Group III and IV animals received fraction at a dose of TPI 20, and TPIII
40 mg/kg p.o.
Group V and VI animals received the fractions of FRI 20 mg/kg, p.o. and
FR III 40mg/kg p.o.
Group VII and VIII, animals received fraction of FGI 20 mg/kg, p.o., and
FGIII 40 mg/kg p.o.
5.4.6.5 EVALUATION OF ANTIHYPERLIPIDEMIC ACTIVITY
The method described by Ram et al., (1996) 207 was employed in
the study. The fraction obtained from T. purpurea, (TPI, TPIII), F.religiosa
(FRI, FRIII) and F. glomerata (FGI, FGIII) were evaluated for
antihyperlipidemic activity. Male Albino rats were divided into eight
groups each comprising five rats.
5.4.6.5.1 Preparation of normal and high-fat diet
The compositions of both normal and high-fat diets were as
follows:
The normal diet contained whole wheat (67.5 g), yellow corn (62.5
g), barley (37.5 g), anik spray (37.5 g), bone meal (2.5 g), calcium chloride
(2.5 g), salt (2.5 g), oil (37.5 g) and 1 tablet of VitaminB12.
The high-fat diet were prepared by mixing calculated amounts of
whole wheat (50.0 g), yellow corn (50.0 g), barley (25.0 g), anik spray
(37.5 g), bone meal (2.5 g), calcium chloride (2.5 g), salt (2.5 g), oil (25.0
g), butter (25.0 g), 1 tablet of Vitamin B12 and cholesterol
81
(200mg/kg/day). Twelve grams of diet of above composition was supplied
to each animal everyday.
Group I: Normal Control, Rats were fed on normal diet consist of
standard laboratory feed through out the whole experimental period (i.e.
0-30 days)
Group II: positive control, Rats were fed with high fat diet for initial 15
days. After that high fat diet were withdrawn and for remaining period
(i.e. 16-30 days). Rats were kept only on normal diet and Atorvastatin
suspension prepared with Tween 80 (10mg/kg; p.o.).
For the initially period of 15 days all remaining six groups (III-VIII)
were kept on same diet as mentioned for positive control i.e high fat diet
after which high fat diet withdrawn from their diet, normal diet
continued and drug therapy started with respective fractions.
Group III: Treated group: 20mg/kg/day of TP I was given for 15 days
Group IV: Treated group: 40mg/kg/day of TP III was given for 15 days
Group V: Treated group: 20mg/kg/day of FR I was given for 15 days
Group VI: Treated group: 40mg/kg/day of FR III was given for 15 days
Group VII: Treated group: 20mg/kg/day of FG I was given for 15 days
Group VIII: Treated group: 40mg/kg/day of FG III was given for 15 days
5.4.6.5.2 Biochemical analysis of antihyperlipidemic activity
3-4 ml of blood was collected from all rats with the help of
disposable syringes on 0th day, after 30 days on feeding high-fat diet and
finally after 15 and 30 days of normal diet and drug treatment in both
control and experimental groups. The samples were transferred to
centrifuge tubes and allowed to clot at 37°C. The serum was separated
by centrifugation for 10 minutes.
82
These serum samples were used to determine total cholesterol,
HDL- cholesterol, LDL- cholesterol, triglycerides and total lipids as
described in the instruction sheets provided with the reagent kits of
Reckon Diagnostics Pvt. Ltd.
A) Determination of total cholesterol
Total cholesterol was determined by reagents kits of Reckon
Diagnostics Pvt. Ltd., Baroda.
Method:
CHOD-PAP method has been described by Allain et al., 1974 208. It
is a highly specific, enzymatic, colorimetric test for measurements in
visible range which is distinguished by its high flexibility.
Test principle
The cholesterol esters are hydrolysed to free cholesterol by
cholesterol esterase (CE). The free cholesterol is then oxidized by
cholesterol oxidase (CO) to cholesten 4-en-3-one with the simultaneous
production of hydrogen peroxide. The hydrogen peroxide reacts with 4-
aminoantipyrine (AAP) and phenolic compound in the presence of
peroxidase to yield a coloured complex which is read at 505nm (500-540
nm, GREEN filter). The intensity of colour produced is directly
proportional to the concentration of total cholesterol in the sample.
Cholesterol esters + H2O CE Cholesterol + RCOOH
(Fatty acids)
Cholesterol + O2 CO Cholesterol - 4-en- one + H2O2
2H2O2 + 4- AAP + Phenol POD Quinoneimine dye+4 H2O
Sample material
Serum
83
Procedure:
3 tubes are taken and labelled as blank, standard and test. 0.01ml
of standard and serum are added to their respective tubes. 1 ml of
Cholesterol reagent solution was added to all tubes i.e. blank, standard
and test. Mix well and incubate for 10 minutes at 37°C. Read absorbance
of test and standard at 505 nm (500-540 nm) or with Green filter against
reagent blank.
Calculations:
Cholesterol (mg/dl) = Absorbance of Test x 200
Absorbance of Std.
B) Determination of Triglycerides
Triglyceride was determined by reagents kits of Reckon Diagnostics
Pvt. Ltd., Baroda.
Method:
High performance enzymatic GPO-PAP method modified according
to Fossati and Principe, 1982; McGowan et al., 1983 209,210
Test principle
Lipase hydrolyses triglycerides sequentially to Di & Monoglycerides
and finally to Glycerol. Glycerol kinase (GK) using ATP as PO4 source
converts Glycerol liberated to Glycerol-3-phosphate (G-3-Phosphate). G-
3-phosphate Oxidase (GPO) oxidise G-3- phosphate formed to Dihydroxy
acetone phosphate and hydrogen peroxide is formed. Peroxidase (POD)
uses the hydrogen peroxide formed, to oxidise 4 aminoantipyrine and
chlorophenol to a pink coloured complex. The absorbance of the coloured
complex is measured at 520nm (500-550nm or with green filter) which is
proportional to Triglycerides concentration.
Triglyceride + H2O Lipase Glycerol + Fatty acids
Glycerol+ ATP GK Glycerol-3-phosphate+ADP
84
Glycerol-3-phosphate+O2 GPO Dihydroxyacetonephosphate+ H2O2
H2O2 +4-aminoantipyrine+Chlorophenol POD Colored complex+H2O
Sample material
Serum
Procedure:
0.05 ml of serum, standard and distilled water were placed in the
tubes marked as test, standard and blank respectively. Then 1 ml of
working solution was added to each tube, mixed well and incubates at
for 20 minutes at 37°C.
After incubation period 1.5ml of distilled water was added to each tube,
mixed well. Read absorbance of test and standard against the blank at
520 nm (500-550 nm)
Calculations:
Triglyceride (mg/dl) = Absorbance of Test x 200
Absorbance of Std.
C) Determination of HDL- cholesterol
HDL- cholesterol was determined by reagents kits of Reckon
Diagnostics Pvt. Ltd., Baroda.
Method:
High performance enzymatic PTA (Phosphotungstic acid) method
according to Burstein et al., 1980 211
Test principle
High density lipoproteins (HDL) are separated from other
lipoprotein fractions by treating serum with phosphotungstic acid and
magnesium chloride. HDL remains in solution while all other lipoprotein
fractions are precipitated. Cholesterol content of which is estimated by
enzymatic method.
Serum+ PTA reagent Supernatant (HDL) + Precipitates (other
fraction)
85
Procedure:
3 tubes are taken and labelled as blank, standard and test. 0.1ml
of standard and serum are added to their respective tubes. 1 ml of
Cholesterol reagent solution was added to all tubes i.e. blank, standard
and test. Mix well and incubate for 20 minutes at 37°C. After incubation
period 2 ml of distilled water was added to each tube, mixed well. Read
absorbance of test and standard against the blank at 505 nm (500-540
nm) or with Green filter against reagent blank.
Calculations:
Serum HDL- cholesterol (mg/dl) = Absorbance of Test x 100
Absorbance of Std.
D) Determination of VLDL- cholesterol
VLDL cholesterol was calculated using Friedewald et al., (1972)212
formulas as TG/5
E) Determination of LDL- cholesterol
LDL cholesterol was estimated using Friedewald et al., (1972) 212
formula as follows:
LDL (mg/dl) = TC − (HDL + VLDL)
F) Determination of SGOT
SGOT was determined by reagents kits of Crest Biosystems, Goa.
Method:
Serum glutamate oxalate transaminase (SGOT) were estimated
according to the method described by Reitman and Frankel (1957)213
Test principle
SGOT converts L-asparate and α ketoglutarate to oxaloacetate and
glutamate. The oxaloacetate formed reacts with 2, 4, dinitrophenyl
86
hydrazine (DNPH) to produce a hydrazone derivative, which in an
alkaline medium produces a brown coloured complex whose intensity is
measured. The reaction does not obey Beers law and hence calibration
curve is plotted using Pyruvate standard. The activity of SGOT is read off
this calibration curve.
L-asparate+ α ketoglutarate SGOT oxaloacetate+ L-Glutamate
pH 7.4
Oxaloacetate+ 2, 4, DNPH Alkaline DNPH
Medium (Brown coloured complex)
Procedure:
SGOT activity in the serum was measured using standard
calibration curve method. 5 test tubes were labelled for increasing
enzyme activity (U/ml) as blank (0), 24, 61,114,190 with the addition of
substrate reagent as 0.5, 0.45, 0.40, 0.35, and 0.30 respectively.
Pyruvate standard was added in test tubes 2 -5 in increasing manner as
0.05, 0.1, 0.15, and 0.20 respectively. 0.1ml distilled water was added in
each test tubes. At last 0.5ml DNPH reagent was added in each test tube.
Content of each test tubes were mixed well and allow to stand for 20
min. at room temperature. Finally 5 ml of working NaOH regent was
added in each test tube and mixed well. It was kept aside for 10 min. at
room temperature. The absorbance was measured against blank and a
graph of absorbance versus enzyme activity (U/ml) was plotted for
standard.
For measuring enzyme activity in test 2 more test tubes were
labelled as blank and test. 0.5ml substrate reagent was added in the
both the test tubes and incubated at 37°C for 3 min. in the test 0.1ml
serum sample was added and again incubated for 60 min at 37°C. Then
in both test tubes, 0.5ml DNPH reagent was added mixed well and kept
aside for 20 min at room temperature. 0.1ml distilled water was added to
blank only. Finally 5 ml of working NaOH reagent was added in both test
87
tubes mix well kept aside for 10 min at room temperature. The
absorbance was measured against blank and the activity was read from
the calibration curve plotted earlier.
G) Determination of SGPT
SGPT was determined by reagents kits of Crest Biosystems, Goa.
Method:
Serum glutamate pyruvate transaminase (SGPT) were estimated
according to the method described by Reitman and Frankel (1957) 213
Test principle
SGPT converts L-alanine and α ketoglutarate to Pyruvate and
Glutamate. The Pyruvate formed reacts with 2, 4, dinitrophenyl
hydrazine (DNPH) to produce a hydrazone derivative, which in an
alkaline medium produces a brown coloured complex intensity is
measured. The reaction does not obey Beers law and hence calibration
curve is plotted using Pyruvate standard. The activity of SGOT is read off
this calibration curve.
L-Alanine+ α ketoglutarate SGPT Pyruvate + L-Glutamate
pH 7.4
Pyruvate + 2, 4, DNPH Alkaline DNPH
Medium (Brown coloured complex)
Procedure:
SGPT activity in the serum was measured using standard
calibration curve method. 5 test tubes were labelled for increasing
enzyme activity (U/ml) as blank (0), 28, 57, 97,150 with the addition of
substrate reagent as 0.5, 0.45, 0.40, 0.35, and 0.30 respectively.
Pyruvate standard was added in test tubes 2 -5 in increasing manner as
0.05, 0.1, 0.15, and 0.20 respectively. 0.1ml distilled water was added in
each test tubes. At last 0.5ml DNPH reagent was added in each test tube.
88
Content of each test tubes were mixed well and allow to stand for 20
min. at room temperature. Finally 5 ml of working NaOH regent was
added in each test tube and mixed well. It was kept aside for 10 min. at
room temperature. The absorbance was measured against blank and a
graph of absorbance versus enzyme activity (U/ml) was plotted for
standard.
For measuring enzyme activity in test 2 more test tubes were
labelled as blank and test. 0.5ml substrate reagent was added in the
both the test tubes and incubated at 37°C for 3 min. in the test 0.1ml
serum sample was added and again incubated for 30 min at 37°C. Then
in both test tubes, 0.5ml DNPH reagent was added mixed well and kept
aside for 20 min at room temperature. 0.1ml distilled water was added to
blank only. Finally 5 ml of working NaOH reagent was added in both test
tubes mix well kept aside for 10 min at RT. The absorbance was
measured against blank and the activity was read from the calibration
curve plotted earlier.
5.4.6.6 STATISTICAL ANALYSIS
Results of all the above estimations have been indicated in terms of
mean ± SEM. Difference between the groups was statistically determined
by analysis of variance (ANOVA) with Dunnett’s test multiple
comparisons test using GraphPad InStat version 5.00, GraphPad
Software, CA, USA. The level of significance was set at P < 0.05.
89
89
6. RESULTS:
6.1 Extractive value:
Percent yield by different solvent extraction was shown in
following Table No. 3
6.2 Phytochemical investigation of T. purpurea, F.religiosa and F.
glomerata leaves:
Preliminary Phytochemical analysis revealed the presence of
different phytochemicals in different extracts of T.purpurea, F.religiosa,
and F.glomerata.
T.purpurea mainly showed the presence of saponins and steroids
as major constituents of petroleum ether extract whereas chloroform
extract was rich in flavonoids. Most of the phytoconstituents of
T.purpurea were found to be present in methanolic extract including
glycosides, saponins, flavonoids and steroids. Aqueous extract also
showed positive tests for tannins, glycosides and saponins. Apart from
these constituents each of the extracts showed presence of proteins and
amino acids. (Table No. 4)
F. religiosa extracts were also tested for different
phytoconstituents. Tests revealed the presence of saponins and steroids
in the petroleium ether extract. Chloroform extract of F. religiosa
possess saponins and flavonoids while methanolic extract showed
positive tests for all the constituents except alkaloids. Aqueous extract
was also rich in tannins, glycosides, saponins, flavonoids. On the other
hand all these extracts showed presence of proteins and amino acids.
(Table 5)
A result obtained with phytochemical investigation of F.glomerata
extracts was somewhat similar to that of F. religiosa. Petroleum ether
extract was positive for saponins and steroids. Chloroform extract was
rich in saponins and flavonoids. Methanolic extract showed presence of
carbohydrates, proteins, amino acids, tannins, saponins, flavonoids
and steroids. Aqueous extract was found positive for the presence of
carbohydrates, proteins, amino acids, tannins, saponins, and
flavonoids. (Table No. 6)
90
6. 3 Acute toxicity assessment: Acute toxicity studies reveled that non toxic nature of extracts.
There were no lethality or toxic reactions found at any stage of the
study period. All the animals were alive, healthy and active during the
observation study for the given dose so the doses were fixed for
pharmacological study.
6. 4 Thin layer chromatography: Column chromatographic separation of the T. purpurea,
F.religiosa and F.glomerata gave fractions of 20 ml each. TLC of each
fraction was carried out during column chromatography and on the
basis of similarity in the Rf and appearance of color in daylight, UV
(254 and 366 mn) and after exposure to iodine vapors, the fractions
were combined to get pooled fraction TP I, TPIII, FR I,FR III, FG I , FG
III. Fraction was obtained by the eluting column with mobile phase
Benzene: ethyl acetate (7:3).The Rf value for TPI (0.72), TP III(0.91), FR I
(0.181),FR III(0.218), FG I(0.363) , FG III(0.45).
6.5 Screening of In-vitro Anticancer Activity: Anticancer activities of T. purpurea and F.religiosa were carried
out using human MCF 7 cell line. The exposure of above fractions with
cell line showed good anticancer activity of both the fractions. Results
showed sharp inhibition in the cancer cell lines with no changes in log
cell count. It can be interpreted as the drug scaffold having inhibitory
activity against the breast cancer cell lines but in case of normal cell
line drug is ineffective. The drug neither allows the cell growth nor does
it kill the existing cell. The IC50 value for TPI was found to be 152.4 µM,
(Fig 4) whereas the IC50 value for TPIII was found to be 158.71 µM.(Fig
5)
Similarly fractions of F. religiosa showed inhibition in the cancer
cell lines with no changes in log cell count. This shows the signs of
cytotoxicity with the normal epithelial cell. The IC50 value for FRI was
found to be 160.3 µM, (Fig 6) whereas the IC50 value for FRIII was
found to be 222.7 µM. (Fig7) same result was obtained with the fraction
91
of F.glomerata. The IC50 value for FGI was found to be 120.42 µM,( Fig
8)while the IC50 value for FGIII was found to be 212.23µM.( Fig 9)
6.6 Evaluation of Tail flick latency period in rats and evaluation of
Acetic acid induced writhing in mice:
Treatment of TPI and TPIII (20 mg/kg, p.o.) significantly inhibited
nociception in rats by 17.60 % and 20.02 % respectively. Whereas, TPI
and TPIII (40 mg/kg, p.o), significantly inhibited pain perception by
16.58 % and 12.53 % respectively. Ibuprofen treatment (40 mg/kg, p.o)
significantly inhibited pain perception by 27.92 %. (Table No. 7)
The effect of different fractions of T.purpurea against acid induced
writhing in mice. It was observed that mice treated with TPI 20
(39.86%) and TPIII 20 (34.88 %) shows significant (P < 0.01) protection
compared to control group, however TPI 40 (49.16 %) and TPIII 40
(48.50%) was found to be more significant (P < 0.01) in protecting acetic
acid induced writhing compared to control group. (Table No. 8)
Treatment of FRI and FRIII (20 mg/kg, p.o.) significantly
inhibited nociception in rats by 19.26 % and 20.92 % respectively.
Whereas, FR I and FRIII (40 mg/kg, p.o) significantly inhibited pain
perception by 17.5 % and 15.29 % respectively. Ibuprofen treatment
(40mg/kg, p.o) significantly inhibited pain perception by 27.92 %.
(Table No. 7) The effect of different fractions of F.religiosa against acid induced
writhing in mice. It was observed that mice treated with FRI 20 (31.89
%) and FRIII 20 (36.21 %) shows significant (P < 0.01) protection
compared to control group, however FRI 40 (44.18 %) and FRIII 40
(46.51 %) was found to be more significant (P < 0.01) in protecting
acetic acid induced writhing compared to control group. (Table No. 8)
Treatment of FGI and FGIII (20 mg/kg, p.o.) significantly
inhibited nociception in rats by 20.22 % and 20.96 % respectively.
Whereas, FG I and FGIII (40 mg/kg, p.o) significantly inhibited pain
perception by 18.20% and 16.40% respectively. (Table No. 7) The effect of different fractions of F.glomerata against acid
induced writhing in mice. It was observed that mice treated with FGI 20
92
(36.21 %) and FGIII 20 (34.21 %) shows significant (P < 0.01) protection
compared to control group, however FGI 40 (47.50 %) and FGIII 40
(51.16 %) was found to be more significant (P < 0.01) in protecting
acetic acid induced writhing compared to control group. Ibuprofen
showed 56.14 % protection against acetic acid induced writhing in
mice. (Table No. 8)
6.7 Evaluation of anti-inflammatory activity induced by
Carrageenan hind paw edema:
Treatment with T. purpurea fraction TPI and TPIII (20 and 40
mg/kg, p.o.) showed a significant (p<0.01) inhibition of paw volume
after 1h, 2 h, and 3 h. Maximum inhibition was observed at a dose of
40 mg/kg as 63.75 %.Treatment with the F,religiosa fractions FRI and
FRIII (20 and40 mg/kg, p.o.) showed significant (p<0.01) inhibition of
carrageenan induced rat paw edema. Maximum inhibition was
observed at 40 mg/kg dose as 48.12 % compared to the
control.F.glomerata fraction FGI and FGIII (20 and40 mg/kg, p.o.)
showed a significant (p<0.01) inhibition of paw volume. Maximum
inhibition was observed at 40 mg/kg dose as 44.15 % compared to the
control. (Table No. 9)
6.8 Evaluation of anti-inflammatory activity induced by serotonin
and histamine paw edema:
In the serotonin induced paw edema, the fraction TPI inhibited
serotonin induced paw edema by 39.32 % and the fraction TPIII
inhibited paw edema by 46%. The fraction FRI and FRIII inhibited paw
edema by 32.31 % and 41 % whereas the fraction FGI and FGIII
inhibited paw edema by 30 % and 42 %.The reference standard
ibuprofen inhibited paw edema by 56.05 %. (Table10). In histamine
induced rat paw edema the fraction TPI and TPIII inhibited paw edema
34.39 % and 50 % respectively. The fraction FRI and FRIII inhibited
paw edema by 29.28 % and 35 % whereas the fraction FGI and FGIII
inhibited paw edema by 26 % and 38 %.Whereas the reference
standard ibuprofen inhibited paw edema by 55.20 %. (Table No. 11)
93
6.9 Evaluation of Cotton pellet granuloma formation in rats: The fraction obtained from T. purpurea, F.religiosa and F.
glomerata produced a dose-dependent inhibition of which was
comparable with known anti-inflammatory drugs. The percent
inhibition for ibuprofen as a standard was found to be 48.28 %. The
percent inhibition for the fraction TPI and TPIII was 24.84%, 21.25 %
respectively. The percent inhibition for the fraction FRI and FRIII was
38.75 %, 40.31 % respectively and the percent inhibition for fraction
FGI and FGIII was 40.20 %, 42.45 % respectively. (Table No. 12)
6.10 Evaluation of anti-arthritis activity by adjuvant induced
arthritis in rats:
The fraction obtained from T. purpurea, F.religiosa and F.
glomerata produced a dose-dependent inhibition of which was
comparable with known anti-inflammatory drugs. The percent
inhibition for ibuprofen as a standard was found to be 47%. The
percent inhibition for the fraction TPI and TPIII was 61%, 49%
respectively (Table13). The percent inhibition for the fraction FRI and
FRIII was 50, 57% respectively (Table14) and the percent inhibition for
fraction FGI and FGIII was 56%, 59% respectively. (Table No. 15)
6.11 Evaluation of anti-arthritis activity by formalin induced
arthritis in rats: The effect of different fractions of T. purpurea, F.religiosa and F.
glomerata against formalin induced arthritis in rats. It was observed
that rats treated with TPI 40(66.19 %) and TPIII 40(73.70 %) shows
significant (P < 0.01) protection compared to control group,(Table 16)
similarly FRI 40 (74.64 %) and FRIII 40 (71.83 %) shows significant (P <
0.01) protection compared to control group,( Table No. 17 ) however FGI
40 (72.30 %) and FGIII 40 (65.25 %) was found to be more significant (P
< 0.01) in protecting formalin induced arthritis compared to control
group. (Table No. 18) Ibuprofen showed 75.58 % protection against
formalin induced arthritis in rats.
94
6.12 Evaluation of Antihyperlipidemic activity: Antihyperlipidemic activities of T. purpurea, F.religiosa and
F.glomerata were carried out using high fat diet induced hyperlipidemia
in rat. Treatment with T. purpurea fractions [TPI (20 mg/kg, p.o), TPIII
(40 mg/kg, p.o.) F.religiosa fractions [FRI (20 mg/kg, p.o), FRIII (40
mg/kg, p.o.) and F.glomerata [FGI (20 mg/kg, p.o), FGIII (40 mg/kg,
p.o.) showed significant lipid lowering activity which was confirmed by
evaluating serum lipid profile and marker enzymes such as SGOT and
SGPT.
6.12.1 Effect of T. purpurea, F.religiosa, and F.glomerata on
serum lipid profile:
The rats when fed high-fat diet showed marked hyperlipidemia.
For the whole group, there was a significant (P < 0.0001) increase in TC
(93.4±7.84 to 294±6.00 mg/dl), LDL cholesterol (37.2±3.68 to
201.52±5.88 mg/dl), HDL cholesterol (43.4±9.45 to 55.2±7.98 mg/dl)
and TG (64±6.05 to 239.6±11.89 mg/dl). These rats were then divided
into a control group and experimental groups each containing 6 rats.
By the replacement of high-fat diet with normal diet and by
continuation of treatment up to 30th day, the lipid levels were
significantly reduced. At the 30th day, most significant (P < 0.0001)
reduction in lipid levels in the TP treated (40 mg/kg; p.o.) groups as
compared to the rats fed with high-fat diet at the initial day were: TC
273.4±11.80 mg/dl vs. 242.2±14.08mg/dl, LDL cholesterol
178.64±9.31mg/dl vs. 142.84±11.82mg/dl, TG 208.8±11.04 mg/dl vs.
188.8±9.90 mg/dl, VLDL cholesterol 41.76±2.2 mg/dl vs. 37.76±1.98
mg/dl (P < 0.0001). Conversely, HDL cholesterol levels were
significantly (P < 0.0001) increased from 53±7.24 to 61.6±7.30 mg/dl in
the TP treated groups at the 30th day of treatment.
FR (40 mg/kg; p.o.) groups as compared to the rats fed with
high-fat diet at the initial day were: TC 279.8±15.1 mg/dl vs.
217.2±8.57mg/dl, LDL cholesterol 179.68±12.6mg/dl vs.
106.32±8.51mg/dl, TG 239.6±11.89 mg/dl vs. 178.4±2.76 mg/dl, VLDL
cholesterol 47.92±2.38 mg/dl vs. 35.68±0.55 mg/dl (P < 0.0001).
95
Conversely, HDL cholesterol levels were significantly (P < 0.0001)
increased from 52.2±5.02 to 75±5.38 mg/dl in the FR treated groups at
the 30th day of treatment.
whereas FG (40 mg/kg; p.o.) groups as compared to the rats fed
with high-fat diet at the initial day were: TC 269.6±15.93 mg/dl vs.
227.8±13.3mg/dl, LDL cholesterol 173.84±14.3mg/dl vs.
115.36±11.5mg/dl, TG 240.8±9.8 mg/dl vs. 171.4±11.07 mg/dl, VLDL
cholesterol 48.16±1.96 mg/dl vs. 34.28±2.2 mg/dl (P < 0.0001).
Conversely, HDL cholesterol levels were significantly (P < 0.0001)
increased from 47.6±2.42 to 78.2±2.51 mg/dl in the FG treated groups
at the 30th day of treatment.
Atorvastatin (10mg/kg; p.o.) markedly exerted the most significant (P
< 0.0001) effects as: TC 294±6.00 mg/dl vs. 179.6±4.94 mg/dl, LDL
cholesterol 201.52±5.88mg/dl vs. 72.24±4.49mg/dl, TG 228.4±4.88
mg/dl vs. 133.8±3.94 mg/dl, VLDL cholesterol 45.68±0.97 vs.
26.76±0.78 and HDL cholesterol 46.8±5.89 vs.80.6±3.01. (Table No. 19)
6.12.2 Effect of T. purpurea, F.religiosa, and F.glomerata on
biochemical parameters:
The TP (40 mg/kg/day), FR (40 mg/kg/day) and FG(40
mg/kg/day), as well as Atorvastatin (10mg/kg) after high fat diet
feeding, SGOT levels (units/ml) increased substantially from
29.88±1.99 to 48.75±3.77 (units/ml), 29.88±1.99 to 46.97±1.42
(units/ml) and 29.88±1.99 to 45.88±4.62 (units/ml) (P < 0.0001).
Interestingly, these levels were decreased from 48.75±3.77 to
33.88±2.5units/ml, 46.97±1.42 to 35.91±1 units/ml and 45.88±4.62 to
33.18±4.42 units/ml in the T. purpurea, F.religiosa, and F.glomerata
treated groups (P < 0.0001).Similarly, the levels of SGPT have been
substantially increased from 39.26±0.91 to 65.45±3.29 units/ml,
39.26±0.91 to 56.6±6.88 units/ml, 39.26±0.91to 52.73±3.34 units/ml
and significantly (P < 0.0001) decreased to 45.57±3.50 units/ml,
36.99±3.50 units/ml and 35.19±3.21 units/ml by the administration of
T. purpurea, F.religiosa, and F.glomerata in the experimental groups.
Atorvastatin also showed less significant (P < 0.05) reduction of SGOT
96
levels from 45.24±2.26 to 31.23±2.94 and significant reduction of SGPT
levels (P < 0.001)from 62.74±4.52 to 46.82±4.01. (Table No. 20)
97
Table No. 3: Extractive values (% w/w yield) of plant material with
different solvents
Solvent T.purpurea F.religiosa F.glomerata
Petrolum ether 2.96 4.2 4.8
Chloroform 7.52 8.26 7.56
Methanol 11.26 13.2 14
Aqueous 2.7 4.9 5.86
98
Table No. 4: Phytochemical analysis of different extracts T.
purpurea
Test Pet ether Chloroform Methanol Aqueous
Carbohydrates Molish’s test - - + +
Proteins Biuret test + + + +
Amino acids Ninhydrin test
+ + + +
Tannins
and Phenols
FeCl3 tests - - - +
Lead Acetate - - - +
Glycosides Borntrager's test
- - + +
Saponins Foam test + + + +
Flavonoids Shinoda tests
- + + +
Alkaloids
Mayer’s test - - - -
Wagner’s test
- - - -
Hager’s Test - - - -
Steroids
Salkowski reaction
+ - + -
Liebermann-Burchard reaction
+ - + -
+ indicates Positive test
- indicates Negative test
99
Table No. 5: Phytochemical analysis of different extracts F.
religiosa
Test Pet ether Chloroform Methanol Aqueous
Carbohydrates Molish’s test - - + +
Proteins Biuret test + + + +
Amino acids Ninhydrin test
+ + + +
Tannins
and Phenols
FeCl3 tests - - + +
Lead Acetate - - + +
Glycosides Borntrager's test
- - + +
Saponins Foam test + + + +
Flavonoids Shinoda tests
- + + +
Alkaloids
Mayer’s test - - - -
Wagner’s test
- - - -
Hager’s Test - - - -
Steroids
Salkowski reaction
+ - + -
Liebermann-Burchard reaction
+ - + -
+ indicates Positive test
- indicates Negative test
100
Table No. 6: Phytochemical analysis of different extracts
F.glomerata
Test Pet ether Chloroform Methanol Aqueous
Carbohydrates Molish’s test - - + +
Proteins Biuret test + + + +
Amino acids Ninhydrin test + + + +
Tannins
and Phenols
FeCl3 tests - - + +
Lead Acetate - - + +
Glycosides Borntrager's test
- - - -
Saponins Foam test + + + +
Flavanoids Shinoda tests - + + +
Alkaloids
Mayer’s test - - - -
Wagner’s test - - - -
Hager’s Test - - - -
Steroids
Salkowski reaction
+ - + -
Liebermann-Burchard reaction
+ - + -
+ indicates Positive test
- indicates Negative test
101
Fig 4:Effect of varying concentration of TPI on Trypan blue Exclusion test of cell viability
Fig 5:Effect of varying concentration of TPIII on Trypan blue Exclusion test of cell viability
102
Fig 6:Effect of varying concentration of FRI on Trypan blue Exclusion test of cell viability
Fig 7:Effect of varying concentration of FRIII on Trypan blue Exclusion test of cell viability
103
Fig 8:Effect of varying concentration of FGI on Trypan blue Exclusion test of cell viability
Fig 9:Effect of varying concentration of FGIII on Trypan blue Exclusion test of cell viability
104
Table No. 7: Effect of different fractions of T. purpurea, F.
religiosa and F. glomerata on Tail flick latency period in rats
Treatment Mg/kg Tail flick latency in sec
% Analgesia
Control 4.15 ± 0.44 --
TPI (20) 6.06 ± 0.29* 17.60
TPI (40) 5.95 ± 0.33* 16.58
TPIII (20) 6.54±0.24** 22.02
TPIII (40) 5.51±0.22* 12.53
FRI (20) 6.24 ± 0.021* 19.26
FRI (40) 6.0 ± 0.035* 17.05
FRIII (20) 6.42 ± 0.041** 20.92
FRIII (40) 5.81 ± 0.01* 15.29
FGI (20) 6.35 ± 0.04* 20.22
FGI (40) 6.13 ± 0.04* 18.20
FGIII (20) 6.43±0.04** 20.96
FGIII (40) 5.93 ± 0. 10* 16.40
Ibuprofen (40) 7.18 ± 0.88** 27.92
n= 5, treatment, mg/kg, data were analyzed using ANOVA and expressed as
Mean ± SEM followed by Dunnett’s test and differences between means were
regarded significant at * (P<0.05), ** (P<0.01)
105
Fig 10: Effect of different fractions of T. purpurea, F. religiosa and F.
glomerata on tail flick latency period in rats
106
Table No. 8: Effect of different fractions of T. purpurea, F.
religiosa and F. glomerata on acetic acid induced writhing in mice
Treatment
Mg/kg
Number of writhing % Inhibition
Control 60.2±2.49 --
TPI (20) 36.2±1.93** 39.86
TPI (40) 30.6±2.27** 49.16
TPIII (20) 39.2±2.15** 34.88
TPIII (40) 31±1.41** 48.50
FRI (20) 41±1.14** 31.89
FRI (40) 33.6±2.92** 44.18
FRIII (20) 38.4±1.13** 36.21
FRIII (40) 32.2±1.96** 46.51
FGI (20) 38.4±3.82** 36.21
FGI (40) 31.6±2.37** 47.50
FGIII (20) 39.6±2.92** 34.21
FGIII (40) 29.4±3.41** 51.16
Ibuprofen (40) 26.4±0.92** 56.14
n= 5, treatment, mg/kg, data were analyzed using ANOVA and expressed as
Mean ± SEM followed by Dunnett’s test and differences between means were
regarded significant at ** (P<0.01)
107
Fig 11: Effect of different fractions of T. purpurea, F. religiosa and F.
glomerata on acetic acid induced writhing in mice
108
Table No.9: Effect of different fractions of T. purpurea, F. religiosa
and F. glomerata in Carrageenan induced rat paw edema
n= 5, treatment, mg/kg, data were analyzed using ANOVA and expressed as
Mean ± SEM followed by Dunnett’s test and differences between means were
regarded significant at * (P<0.05), ** (P<0.01)
Treatment
(mg/kg)
Mean increase in paw volume (mL) % Decrease in paw
volume at 3 h
0 h 1 h 2 h 3 h
Control 0.91 ±0.007 1.56 ±0.007 1.95 ± 0.01 2.51 ± 0.011 -
IBU (40) 0.91 ±0.008 1.00 ±0.01** 130 ± 0.003** 1.62 ± 0.001** 52.34
TPI (20) 0.85 ±0.002 1.4 ±0.001** 1.64± 0.026** 1.80 ± 0.012** 40.62
TPI (40) 0.92 ±0.015 1.34 ±0.007** 1.39 ± 0.01** 1.5 ± 0.013** 63.75
TPIII (20) 0.84± 0.007 1.50 ± 0.01** 1.62 ± 0.007** 1.85 ± 0.007* 36.87
TPIII (40) 0.91± 0.007 1.32 ± 0.007** 1.59 ± 0.007** 1.71 ± 0.014** 50.00
FRI (20) 0.89±0.012 1.42 ±0.015** 1.65 ± 0.02** 1.99 ± 0.027** 31.25
FRI (40) 0.92 ±0.018 1.23 ±0.022** 1.51 ± 0.037** 1.83 ± 0.028** 43.12
FRIII (20) 0.88± 0.008 1.28 ± 0.013** 1.50 ± 0.021** 1.87± 0.009** 38.12
FRIII (40) 0.90± 0.003 1.11 ± 0.033** 1.42 ± 0.021** 1.73 ± 0.02** 48.12
FGI (20) 0.90±0.026 1.37 ±0.012** 1.67 ± 0.04** 1.95 ± 0.027** 34.37
FGI (40) 0.87 ±0.036 1.30 ±0.028** 1.49 ± 0.016** 1.86 ± 0.023** 38.12
FGIII (20) 0.90± 0.042 1.32 ± 0.033** 1.52 ± 0.025** 1.91± 0.042** 36.87
FGIII (40) 0.97± 0.018 1.22 ± 0.051** 1.44 ± 0.029** 1.86 ± 0.046** 44.15
109
Table No.10: Effect of different fractions of T. purpurea, F. religiosa
and F. glomerata on serotonin induced rat paw edema
n= 5, treatment, mg/kg, data were analyzed using ANOVA and expressed as
Mean ± SEM followed by Dunnett’s test and differences between means were
regarded significant at * (P<0.05), ** (P<0.01)
Treatment
(mg/kg)
Mean increase in paw volume (mL) % Decrease in paw volume at
3 h 0 h 1 h
Control 0.91 ± 0.076 1.80 ± 0.075 -
IBU(40) 0.93 ±0.077 1.33 ± 0.066** 56.05
TPI (40) 0.86 ± 0.074 1.4 ± 0.085** 39.32
TPIII (40) 0.88 ± 0.071 1.36 ± 0.07** 46.00
FRI(40) 0.85 ± 0.057 1.46 ± 0.07 * 32.31
FRIII(40) 0.89 ± 0.077 1.4 ± 0.064** 41.00
FGI(40) 0.81 ± 0.061 1.47± 0.08* 30.00
FGIII(40) 0.90 ± 0.074 1.39 ± 0.07** 42.00
110
Table No.11: Effect of different fractions of T. purpurea, F. religiosa
and F. glomerata on histamine induced rat paw edema
n= 5, treatment, mg/kg, data were analyzed using ANOVA and expressed as
Mean ± SEM followed by Dunnett’s test and differences between means were
regarded significant at * (P<0.05), ** (P<0.01)
Treatment
(mg/kg)
Mean increase in paw volume (mL) % Decrease in paw volume at
3 h 0 h 1 h
Control 0.87 ± 0.03 1.83 ± 0.1 -
IBU(40) 0.92 ±0.032 1.35 ± 0.085** 55.20
TPI (40) 0.87 ± 0.037 1.5 ± 0.085* 34.39
TPIII (40) 0.90 ± 0.047 1.38 ± 0.087 ** 50.00
FRI(40) 0.89 ± 0.038 1.41 ± 0.098* 29.28
FRIII(40) 0.90 ± 0.07 1.49 ± 0.088* 35.00
FGI(40) 0.85 ± 0.041 1.33 ± 0.09* 26.00
FGIII(40) 0.89 ± 0.074 1.52 ± 0.088* 38.00
111
Table No.12: Effect of different fractions of T. purpurea, F.
religiosa and F. glomerata on Cotton pellet granuloma formation
in rats
Treatment
Mg/kg
Average weight of
cotton pellet
Average weight of cotton pellet with
granuloma
% Inhibition
Control 50±0.01 128±4.99
Ibuprofen (40) 50±0.01 66.2±6.87** 48.28
TPI (40) 50±0.01 96.2±2.47** 24.84
TPIII (40) 50±0.01 100.8±3.20** 21.25
FRI (40) 50±0.01 78.4±0.92** 38.75
FRIII (40) 50±0.01 76.4±4.26** 40.31
FGI (40) 50±0.01 74.2±3.98** 42.20
FGIII (40) 50±0.01 77.4±5.94** 42.45
n= 5, treatment, mg/kg, data were analyzed using ANOVA and expressed as
Mean ± SEM followed by Dunnett’s test and differences between means were
regarded significant at * (P<0.05), ** (P<0.01)
112
Fig 12: Effect of different fractions of T. purpurea, F. religiosa and F.
glomerata on cotton pellet granuloma formation in rats
113
Table No.13: Effect of different fractions of T. purpurea on
adjuvant induced arthritis in rat
Day Control IBU TPI TPIII 0 1.002±0.04 0.86±0.024 0.98±0.032 1.004±0.048 14 1.65±0.072 1.40±0.04 1.18±0.02 1.33±0.044 15 1.79±0.068 1.48±0.051 1.29±0.026 1.42±0.048 16 1.67±0.083 1.37±0.035 1.34±0.035 1.49±0.036 17 1.82±0.099 1.29±0.028 1.30±0.044 1.43±0.041 18 1.83±0.086 1.42±0.029 1.46±0.037 1.70±0.029 19 1.92±0.069 1.49±0.042 1.54±0.022 1.96±0.038 20 1.95±0.098 1.68±0.026 1.59±0.024 1.83±0.019 21 1.93±0.018 1.55±0.021 1.63±0.026 1.94±0.025 22 2.05±0.048 1.54±0.021 1.58±0.022 1.97±0.033 23 2.11±0.058 1.56±0.027 1.55±0.022 1.93±0.018 24 2.18±0.059 1.76±0.017 1.55±0.023 2.14±0.02 25 2.12±0.035 1.65±0.019 1.66±0.02 1.94±0.018 26 2.11±0.031 1.52±0.02 1.63±0.019 1.93±0.017 27 2.19±0.027 1.75±0.022 1.55±0.02 1.85±0.017 28 2.18±0.023 1.65±0.021 1.54±0.02 1.92±0.018 29 2.32±0.019 1.54±0.018 1.54±0.02 2.04±0.019 30 2.33±0.019 1.66±0.018 1.56±0.013 1.93±0.017 31 2.31±0.025 1.74±0.016 1.47±0.016 2.05±0.017 32 2.35±0.016 1.64±0.018 1.53±0.017 2.17±0.069 33 2.51±0.02 1.52±0.021 1.47±0.015 2.04±0.022 34 2.66±0.018 1.65±0.019 1.61±0.021 2.04±0.022 35 2.85±0.015 1.75±0.017 1.69±0.019 2.08±0.016 36 3.04±0.02 1.93±0.017 1.63±0.02 2.32±0.024 37 3.08±0.012 1.93±0.017 1.76±0.015 2.04±0.02 38 3.05±0.017 1.74±0.019 1.90±0.017 2.06±0.04 39 3.09±0.032 2.05±0.022 1.87±0.016 2.15±0.015 40 3.10±0.032 1.98±0.013 1.81±0.023 2.09±0.017
n= 5, treatment, mg/kg, data were analyzed using ANOVA and expressed as
Mean ± SEM followed by Dunnett’s test and differences between means were
regarded significant at * (P<0.05), ** (P<0.01)
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Table No. 14: Effect of different fractions of F. religiosa adjuvant induced arthritis in rat
Day Control IBU FRI FRIII 0 1.002±0.04 0.86±0.024 0.87±0.029 0.83±0.034 14 1.65±0.072 1.40±0.04 1.19±0.018 1.27±0.014 15 1.79±0.068 1.48±0.051 1.27±0.025 1.36±0.02 16 1.67±0.083 1.37±0.035 1.39±0.043 1.45±0.041 17 1.82±0.099 1.29±0.028 1.35±0.029 1.46±0.018 18 1.83±0.086 1.42±0.029 1.41±0.026 1.43±0.024 19 1.92±0.069 1.49±0.042 1.40±0.024 1.43±0.027 20 1.95±0.098 1.68±0.026 1.49±0.022 1.47±0.023 21 1.93±0.018 1.55±0.021 1.54±0.022 1.43±0.018 22 2.05±0.048 1.54±0.021 1.57±0.021 1.38±0.02 23 2.11±0.058 1.56±0.027 1.56±0.017 1.48±0.022 24 2.18±0.059 1.76±0.017 1.53±0.019 1.52±0.018 25 2.12±0.035 1.65±0.019 1.60±0.027 1.55±0.017 26 2.11±0.031 1.52±0.02 1.66±0.018 1.54±0.021 27 2.19±0.027 1.75±0.022 1.65±0.018 1.46±0.025 28 2.18±0.023 1.65±0.021 1.54±0.019 1.55±0.025 29 2.32±0.019 1.54±0.018 1.57±0.021 1.66±0.018 30 2.33±0.019 1.66±0.018 1.58±0.023 1.63±0.02 31 2.31±0.025 1.74±0.016 1.64±0.019 1.70±0.017 32 2.35±0.016 1.64±0.018 1.71±0.02 1.77±0.021 33 2.51±0.02 1.52±0.021 1.74±0.015 1.88±0.016 34 2.66±0.018 1.65±0.019 1.83±0.015 1.89±0.019 35 2.85±0.015 1.75±0.017 1.80±0.015 2.03±0.02 36 3.04±0.02 1.93±0.017 1.87±0.019 1.93±0.022 37 3.08±0.012 1.93±0.017 1.96±0.017 1.82±0.014 38 3.05±0.017 1.74±0.019 1.94±0.018 1.84±0.017 39 3.09±0.032 2.05±0.022 1.89±0.016 1.83±0.017 40 3.10±0.032 1.98±0.013 1.95±0.018 1.74±0.016
n= 5, treatment, mg/kg, data were analyzed using ANOVA and expressed as
Mean ± SEM followed by Dunnett’s test and differences between means were
regarded significant at * (P<0.05), ** (P<0.01)
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Table No.15: Effect of different fractions of F. glomerata adjuvant induced arthritis in rat
Day Control IBU FGI FGIII 0 1.002±0.04 0.86±0.024 0.87±0.04 0.79±0.031 14 1.65±0.072 1.40±0.04 1.23±0.02 1.29±0.015 15 1.79±0.068 1.48±0.051 1.34±0.032 1.37±0.05 16 1.67±0.083 1.37±0.035 1.38±0.045 1.42±0.033 17 1.82±0.099 1.29±0.028 1.32±0.016 1.39±0.025 18 1.83±0.086 1.42±0.029 1.39±0.029 1.44±0.039 19 1.92±0.069 1.49±0.042 1.38±0.038 1.41±0.042 20 1.95±0.098 1.68±0.026 1.54±0.025 1.52±0.029 21 1.93±0.018 1.55±0.021 1.58±0.029 1.46±0.026 22 2.05±0.048 1.54±0.021 1.57±0.021 1.43±0.025 23 2.11±0.058 1.56±0.027 1.55±0.018 1.40±0.022 24 2.18±0.059 1.76±0.017 1.51±0.015 1.47±0.019 25 2.12±0.035 1.65±0.019 1.57±0.017 1.55±0.018 26 2.11±0.031 1.52±0.02 1.63±0.025 1.54±0.021 27 2.19±0.027 1.75±0.022 1.60±0.027 1.51±0.022 28 2.18±0.023 1.65±0.021 1.57±0.017 1.45±0.025 29 2.32±0.019 1.54±0.018 1.61±0.021 1.49±0.031 30 2.33±0.019 1.66±0.018 1.6±0.0250 1.44±0.02 31 2.31±0.025 1.74±0.016 1.63±0.017 1.53±0.019 32 2.35±0.016 1.64±0.018 1.67±0.023 1.63±0.019 33 2.51±0.02 1.52±0.021 1.71±0.015 1.70±0.016 34 2.66±0.018 1.65±0.019 1.75±0.017 1.76±0.026 35 2.85±0.015 1.75±0.017 1.72±0.021 1.77±0.02 36 3.04±0.02 1.93±0.017 1.75±0.019 1.71±0.025 37 3.08±0.012 1.93±0.017 1.75±0.017 1.65±0.02 38 3.05±0.017 1.74±0.019 1.71±0.015 1.64±0.02 39 3.09±0.032 2.05±0.022 1.71±0.022 1.65±0.019 40 3.10±0.032 1.98±0.013 1.78±0.019 1.66±0.016
n= 5, treatment, mg/kg, data were analyzed using ANOVA and expressed as
Mean ± SEM followed by Dunnett’s test and differences between means were
regarded significant at * (P<0.05), ** (P<0.01)
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Table No.16: Effect of different fractions of T. purpurea on formalin induced arthritis in rat
Day Control IBU TPI TPIII
1 0.71±0.08 0.70±0.04 0.71±0.05 0.74±0.03
2 1.84±0.16 1.44±0.04 1.78±0.09 1.67±0.06
3 1.98±0.17 2.03±0.066 1.84±0.1 1.98±0.12
4 2.70±0.17 2.11±0.09 2.27±0.077 2.35±0.076
5 2.58±0.15 2.05±0.07 2.13±0.11 2.20±0.09
6 2.65±0.03 1.69±0.02 1.80±0.03 1.58±0.04
7 2.79±0.04 1.52±0.03 1.67±0.03 1.38±0.03
8 3.01±0.05 1.44±0.03 1.64±0.03 1.38±0.03
9 2.89±0.04 1.30±0.05 1.49±0.04 1.32±0.04
10 2.84±0.03 1.22±0.07 1.43±0.05 1.30±0.04
n= 5, treatment, mg/kg, data were analyzed using ANOVA and expressed as
Mean ± SEM followed by Dunnett’s test and differences between means were
regarded significant at * (P<0.05), ** (P<0.01)
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Table No.17: Effect of different fractions of F. religiosa on formalin induced arthritis in rat
Day Control IBU FRI FRIII
1 0.71±0.08 0.70±0.04 0.70±0.06 0.72±0.04
2 1.84±0.16 1.44±0.04 1.71±0.07 1.64±0.03
3 1.98±0.17 2.03±0.066 2.05±0.09 1.72±0.07
4 2.70±0.17 2.11±0.09 2.42±0.077 2.28±0.059
5 2.58±0.15 2.05±0.07 2.35±0.058 2.10±0.06
6 2.65±0.03 1.69±0.02 1.87±0.05 1.79±0.02
7 2.79±0.04 1.52±0.03 1.49±0.06 1.68±0.02
8 3.01±0.05 1.44±0.03 1.36±0.07 1.57±0.03
9 2.89±0.04 1.30±0.05 1.30±0.07 1.47±0.04
10 2.84±0.03 1.22±0.07 1.24±0.08 1.32±0.06
n= 5, treatment, mg/kg, data were analyzed using ANOVA and expressed as
Mean ± SEM followed by Dunnett’s test and differences between means were
regarded significant at * (P<0.05), ** (P<0.01)
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Table18: Effect of different fractions of F. glomerata on formalin induced arthritis in rat
Day Control IBU FGI FGIII
1 0.71±0.08 0.70±0.04 0.75±0.026 0.73±0.01
2 1.84±0.16 1.44±0.04 1.69±0.02 1.58±0.01
3 1.98±0.17 2.03±0.066 1.81±0.03 1.72±0.02
4 2.70±0.17 2.11±0.09 2.18±0.05 1.98±0.06
5 2.58±0.15 2.05±0.07 2.12±0.03 1.93±0.04
6 2.65±0.03 1.69±0.02 1.90±0.03 1.83±0.02
7 2.79±0.04 1.52±0.03 1.71±0.04 1.74±0.02
8 3.01±0.05 1.44±0.03 1.51±0.04 1.65±0.01
9 2.89±0.04 1.30±0.05 1.42±0.02 1.55±0.03
10 2.84±0.03 1.22±0.07 1.34±0.02 1.47±0.05
n= 5, treatment, mg/kg, data were analyzed using ANOVA and expressed as
Mean ± SEM followed by Dunnett’s test and differences between means were
regarded significant at * (P<0.05), ** (P<0.01)
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Table 19: Effect of different fractions of T. purpurea, F. religiosa and F. glomerata on serum lipid profile in hyperlipidemia-induced rats.
Lipid profile
Control Treatment Serum lipid levels (mg/dl) on day(s)
0th day X 15th day XX 30th day XX TC 93.4±7.84 TPI 20 284±19.39a 274±19.89N 262±19.21N TPIII 40 273.4±11.80a 249.6±14.35N 242.2±14.08N
FRI 20 293.2±5.36a 268 ±8.99d 233±7.53a FRIII 40 279.8±15.18a 260.4±10.80N 217±8.57b FGI 20 289.2±11.44a 270.8±6.31d 239±6.7a
FGIII 40 269.6±15.93a 255.8±16.04N 227.8±13.3b AT 10 294±6.00a 214.8±3.15a 179.6±4.94a
TG 64±6.05 TPI 20 218.2±6.42a 209.2±6.53N 203.2±6.55N TPIII 40 208.8±11.04a 197.6±10.61N 188.8±9.90N
FRI 20 234.2±13.38a 223.4±11.72N 186±10.48c FRIII 40 239.6±11.89a 205±3.4c 178.4±2.76c FGI 20 237.6±9.9 a 225±9.83 N 188.6±7.7c
FG III40 240.8±9.8 a 211.2±8.83c 171.4±11.07c AT 10 228.4±4.88a 180.8±3.62a 133.8±3.94a
HDL 43.4±9.45 TPI 20 49.4±6.42N 53±1.94N 55.6±1.88N TP III40 53±7.24N 56.6±2.94N 61.6±7.30N
FRI 20 55.2±7.98N 63.8±2.95N 70.6±4.93b FRIII 40 52.2±5.02N 66.8±3.12d 75±5.38a FG I20 53±4.6N 59.4±2.8N 72.6±2.31b
FGIII 40 47.6±2.42N 61.4±3.42d 78.2±2.51a AT 10 46.8±5.89N 71.8±2.55a 80.6±3.01a
LDL 37.2±3.68 TPI 20 190.6±19.67a 189.16±20.5N 165.76±19.96N TPIII 40 178.64±9.31a 153.48±12.3N 142.84±11.82d
FRI 20 191.16±6.46a 159.52±8.41c 125.6±8.01b FRIII 40 179.68±12.6a 152.68±10.4N 106.32±8.51b FGI 20 188.68±11.3a 166.4±5.86c 127.97±8.39 b
FG III40 173.84±14.3a 152.16±15.8N 115.36±11.5 b AT 10 201.52±5.88a 106.84±3.51a 72.24±4.49a
VLDL 12.8±1.21 TPI 20 43.64±1.28a 41.84±1.3N 40.64±1.31a TPIII 40 41.76±2.2a 39.52±2.12N 37.76±1.98N
FRI 20 46.84±2.67a 44.68±2.34N 37.2±2.09c FRIII 40 47.92±2.38a 40.92±0.74c 35.68±0.55b FGI 20 47.52±1.98a 45±1.96N 37.72±1.41c
FGIII 40 48.16±1.96a 42.24±1.76c 34.28±2.2b AT 10 45.68±0.97a 36.16±0.72a 26.76±0.78a
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Table 20: Effect of different fractions of T. purpurea, F. religiosa and F. glomerata on biochemical parameters in hyperlipidemia-induced rats
Parameters Control Treatment Effects during experimental period
0th dayX 15th dayXX 30th dayXX
SGOT 29.31±1.99 TPI 20 56.31±6.47b 51.95±6.52N 48.41±6.84N
TPIII 40 48.75±3.77b 40.94±3.06N 33.88±2.56c
FRI 20 52.91±3.40b 46.98±3.63N 38.70±4.24c
FRIII 40 46.97±1.42a 40.76±0.86b 35.91±1.00a
FGI 20 50.46±2.95 b 44.57±2.14N 35.43±1.81c
FGIII 40 45.88±4.62a 40.65±4.58 b 33.18±4.42a
AT 10 45.24±2.66b 39.05±2.98N 31.23±2.94b
SGPT 39.26±0.91 TPI 20 58.33±4.93b 54.0 ±4.59N 50.0 ±4.96N
TPIII 40 65.45±3.29a 58.29±2.90N 45.57±3.50c
FRI 20 57.67±4.79b 50.46±4.94N 41.35±4.29c
FRIII 40 56.6±6.88c 46.79±6.04N 36.99±6.69c
FGI 20 56.7±3.34 b 49.02±2.73N 40.98±3.19c
FGIII 40 52.73±5.06c 43.90±3.48N 35.19±3.21c
AT 10 62.74±4.52a 54.77±5.27N 46.82±4.01c
ATV: Atorvastatin; Values are expressed as mg/dl± S.D. (n = 5). Values
are statistically significant at a P < 0.0001 and bP < 0.001, c P < 0.01,
dP < 0.05, N—non significant (P > 0.05).X Results of 0th day treatment
(hyperlipidemic control) are compared with normal control. XX Results
of 15th and 30th day treatment (treated groups) are compared with 0th
day treatment (hyperlipidemic control) (unpaired two-tailed student t-
test).
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7. DISCUSSION
Inflammation is a necessary function of the human immune
system but may become problematic and potentially lethal when acute
inflammation becomes chronic inflammation. As opposed to acute
inflammation, chronic inflammation develops over time and can last
months to years supported by extensive research and clinical
evidence. Chronic inflammation promotes disease development and
progression by impairing endothelial function, vascular lining,
increasing platelet activation, clotting, depleting intrinsic antioxidants,
generating free radicals, amplifying oxidative stress, delaying wound
healing and tissue regeneration, promoting cell aging and premature
cell death, suppressing or amplifying immune responses.
Inflammatory abnormalities are a large group of disorders which
underlie a vast variety of human diseases and clinical conditions like
arthritis, cancer and vascular diseases. There has been an alarming
increase in the inflammation linked diseases which are the leading
cause of morbidity and interference of socioeconomic life of
individuals.
Chronic inflammation promotes progression of atherosclerosis
and destabilizes fatty plaque in coronary and carotid arteries, leading
to heart attack and stroke. Chronic inflammation also destroys nerve
cells in the brains of Alzheimer's victims and facilitates the
development and progression of cancer. It has been widely accepted
that chronic inflammation may be the engine that drives many of the
most feared illnesses of middle and old age.
A number of pharmaceutical compounds with anti-inflammatory
and analgesic properties, designated as nonsteroidal anti-
inflammatory drugs (NSAIDs). NSAIDs are one of the most frequently
used classes of medicines in the world, accounting for nearly 5 % of
all prescribed medications214 share the inhibition of cyclooxygenase
enzymes (COX) as their main mechanism of action. These enzymes
participate in the metabolism of arachidonic acid, resulting in the
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production of potent inflammatory mediators such as prostaglandins
and thromboxanes. Two isoenzymes of COX, COX-1 (constitutive form)
and COX-2 (inducible form) have been identified. The classical NSAIDs
inhibit both isoenzymes and their use is often accompanied by
gastrointestinal intolerance due to a decreased production of
protective prostaglandin E2 in the stomach.
New drugs that inhibit selectively COX-2 exhibit a better gastric
tolerance profile, although their introduction into clinical practice has
been associated with severe cardiovascular adverse events that led to
the recommendation for careful utilization in patients with previous
vascular diseases215. Analgesic and anti-inflammatory medications are
widely used in all age groups for the treatment of pain, inflammation
and fevers of diverse etiologies. A large proportion of the population is
exposed to these drugs, making them the second cause of untoward
reactions.
Therefore it is necessary to find the alternative therapy for
treating inflammation and related disorders with minimum or no
adverse effects. The answer probably lies in the natural remedies.
Natural or alternative, medicine is often thought of as a
phenomenon of as the so-called new age; in reality much of it is older
than human history. Every society has herbal cure and folk remedies,
many of which have been incorporated into orthodox medicine. In fact,
it is esteemed that many of the modern drugs originated with natural
plant sources. The ancient Ayurveda and Chinese medicine offer a
safer alternative solution to controlling inflammation. Many botanical
medicinal herbs, used in the Traditional Chinese Medicine and
Ayurvedic Medicine for thousands of years, have anti-inflammatory
properties. These anti-inflammatory herbs have been carefully
examined and a large number of valuable information becomes
available.
Tephrosia purpurea (TP) L. (Fabaceae) is the plant with many
biological activities but very few of them were explored scientifically.
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No, scientific reports is observed till date regarding the effects of T.
purpurea fractions on analgesic and anti inflammatory activity. Whole
plant has been used to cure tumors, ulcers, leprosy, allergic and
inflammatory conditions such as rheumatism, asthma and
bronchitis112. The aqueous extract of Tephrosia purpurea seeds has
shown significant in vivo hypoglycemic activity in diabetic rabbits and
the ethanolic extracts possess potential antibacterial activity. The
flavanoids isolated from the plant has been reported to have
antimicrobial activity120.It has also been reported to acquire
hepatoprotective, mast cell stabilizing and erythrocyte membrane
integrity enhancing effect in various animal models. The plant was
chosen in the present investigation as it shows diverse constituents
responsible for pharmacological activity including glycosides,
rotenoids, isoflavones, flavanones, chalcones, flavanols, flavones and
sterols125.
Ficus religiosa (FR) L. (Moraceae) commonly known as ‘Peepal’. It
is reported to have numerous therapeutic uses in folkmedicine viz.:
leaf juice has been used for the treatment of asthma, cough, sexual
disorders, diarrhea, haematuria, ear-ache and toothache, migraine,
eye troubles, gastric problems and scabies; leaf decoction has been
used as an analgesic for toothache; fruits for the treatment of asthma,
other respiratory disorders and scabies; stem bark is used in
gonorrhea, bleeding, paralysis, diabetes, diarrhea, bone fracture,
antiseptic, astringent and antidote. It is claimed to possess
anticonvulsant activity, acetyl cholinesterase inhibitory activity and
antianxiety activity185. Fruits of this plant contain numerous amino
acids whereas figs of this plant has been reported to contain highest
amount of serotonin [5-HT] as compare to figs of other Ficus
species187.
Ficus glomerata (Moraceae), commonly known as Cluster fig is
most frequently used plant in the traditional medicines. The bark,
fruits and latex are used to treat anemia and gastrointestinal
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disorders. The alcoholic extract of the fruit also possessed anti-filarial
activity. Fruits of F. glomerata contain glauanol, glauanol acetate, b-
sitosterol, lupeol acetate. The aerial part of plant contains b-sitosterol,
lupeol and quercetin as major active constituent149. Fruits of F.
glomerata showed significant gastroprotective activity on physically
and chemically induced gastric ulceration in rats147.
Rheumatoid Arthritis is one of the leading cause of disability for
many. It affects mainly middle aged women, who complain generally of
early morning stiffness, pain and swelling in their smaller joints of
fingers and writs joints. The stiffness can sometimes felt in the whole
body and goes away slowly as the sun goes up later in the afternoon.
These kind of symptoms are seen in very mild and initial stages of
rheumatism. Later on the pain and swelling becomes persistant and
starts to cripple the smaller as well as bigger joints. The joints gets
eroded and there is significant destruction of the joints in serious
cases. Sometimes the problem can arise acutely involving all the joints
simultaenously particularly in young patients. This is called juvenile
rheumatoid arthritis.
The selection of Ficus glomerata for the present investigation
was on the basis of utilization in Ayurvedic preparations and
recommendation of this plant for treatment of inflammation.
Authentication of the collected plant material confirms the
identity of plant whereas study of physicochemical characters as per
WHO guidelines approved the purity of samples.
From the phytochemical tests of plant extracts used in this
study it was revealed the presence of different chemical constituents
including steroids, flavonoids and tannins are being promising along
with the other minor constituents. All the crude test extracts as well
as the fractions pooled from them were found biologically active with
slight difference in their potency. The decision regarding the inclusion
of the extract or the fraction in main screening procedure was made
from the preliminary studies conducted with the crude extract and
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their possible fractions.
Phytochemical valuation of all plants was found to consist
similar constituents isolated earlier by the various authors, which
further support the authenticity of plant materials and validity of
method.
Cardiovascular diseases remain by far the number one cause of
death for both men and women of all ethnic backgrounds. Although
many causative factors of these diseases are recognized (smoking,
high blood pressure, genetic background, diabetes mellitus and
obesity) high serum LDL-C and elevated total cholesterol levels are the
most prevalent indicators for susceptibility to atherosclerotic heart
disease. Atherosclerosis is a disorder of the arterial wall characterized
by accumulation of cholesterol ester in cells derived from the
monocyte-macrophage line, smooth muscle cell proliferation and
fibrosis, and results in narrowing the blood vessel. An association of
dietary cholesterol with cardiac and cerebral vascular diseases is
based on several lines of evidence, including studies in animal models
and epidemiological data in humans. There has been an increasing
demand from patients for the use of natural products with Anti-
hyperlipidemic activity. The undesirable side effects and
contraindications of synthetic. There are many classes of lipid
lowering agents available, these drugs have different mechanisms of
action and variable efficacy depending on the lipid profile of an
individual. In spite of their lipid-lowering effect, these drugs have
many side effects. Thus, research is still pursuing to find out novel
agents that are more effective and safe.In the present work, the
selected plants were tested for their anti-inflammatory, analgesic,
anti-arthritic, anti-hyperlipidemic and anti-cancer activities using well
validated and universally accepted animal models and in vitro
techniques.
The anti-inflammatory activity was carried out using well
established animal models like carrageenan induced rat paw edema,
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serotonin induced rat paw edema, histamine induced rat paw edema
and cotton pellet induced granuloma formation.
The fractions TPIII, FRIII, and FGIII inhibited serotonin induced
edema by 46%, 41% and 42% respectively. All these fractions were
less potent than ibuprofen. The fractions shows that fractions TPIII,
FRIII, and FGIII inhibited histamine induced edema by 50%, 35% and
38% respectively whereas ibuprofen inhibited histamine induced
edema by 55.20%.The effectiveness of these fractions at 1 and 3 h in
carrageenan induced paw edema indicates their antagonistic effect at
serotonin, histamine, bradykinin and prostaglandin. Because
carrageenan induced rat paw edema has been a popular inflammatory
model to investigate nonsteroidal anti-inflammatory effect of
compounds216. Serotonin, histamine, bradykinin and prostaglandins
have been identified as mediators for carrageenan induced rat paw
edema 217. The first phase is due to release of histamine and
serotonin (5-HT) (1 h), plateau phase is maintained by kinin like
substance (2 h) and second accelerating phase of swelling is attributed
to PG release (3 h.) 218.
It has been reported that the second phase edema is sensitive to
most clinically effective steroidal and non-steroidal anti-inflammatory
agents 216, while a variety of clinically ineffective agents are capable of
reducing 1 h initial phase.
The initial phase of carrageenan paw edema is mediated by
histamine and serotonin, while the mediators in the later phase were
suggested to be arachidonate metabolites (prostaglandins,
leukotrienes) producing an edema dependent on mobilisation of
neutrophils. In our experiments the edematous response was only
significantly suppressed in rats pre-treated with the test extracts and
their fractions on the first phase of the edema, suggesting an
inhibitory effect on the release of histamine and/or serotonin. On the
second and third phase of edema, suggesting an inhibition of 5-
lipoxygenase and/or cyclooxygenase, both enzymes involved in the
127
formation of prostaglandins and leukotrienes. This edematous
response was also significantly reduced in rats pre-treated with
ibuprofen known to be cyclooxygenase inhibitors219
These effects were obtained due to presences of diversity
phytochemicals like flavonoids, tannins, steroids etc. Flavonoids
isolated from some medicinal plants have been proven to posses
antinociceptive and/ or anti-inflammatory effects 220. It has been
shown by Meli et al. 221, Dicarlo et al. 222 that flavonoids also inhibit
gastric motility in a dose - dependent, manner. It is therefore possible
that the inhibitory effects on anti-inflammatory effects observed in
these fractions may be attributed in part to its flavonoid, triterpenoid
and steroid content. Flavonoids also inhibit the phosphodiesterases
involved in cell activation222. Much of this effect is upon the
biosynthesis of protein cytokines that mediates adhesion of circulating
leukocytes to sites of injury. Flavonoids inhibit biosynthesis of
prostaglandins, which are involved in various immunologic responses
and are the end products of the cyclooxygenase and lipoxygenase
parthways 223. Protein Kinases are another class of regulatory
enzymes affected by flavonoids. Inhibition of these enzymes provides
the mechanism by which flavonoids inhibit inflammatory processes 224.
Hence it can safely interpreted that series of anti-inflammatory
plants mostly contains sterol and flavonoid moieties which perhaps
suppress inflammation by inhibiting synthesis of eicosanoids and
PAF.
The fractions thus obtained from above plants were tested in
models of subacute and chronic inflammation of arthritis. Formalin
induced arthritis and cotton pellet granuloma were used as subacute
models whereas adjuvant induced arthritis was used as chronic model
of inflammation. The results of formalin induced arthritis method
show that the fractions inhibited inflammation by TPI (66.19%), TPIII
(73.70%), FRI (74.64%) FRIII 40 (71.83%) and FGI 40 (72.30%) and
128
FGIII 40 (65.25%) was found to be more significant (P < 0.01) in
protecting formalin induced arthritis compared to control group.There
is stimulation of fibroblast development in formalin induced arthritis
which is better inhibited by NSAIDs whereas cotton pellet granuloma
is known as foreign body granuloma and is model of non-immunologic
type of inflammation mediated mostly by kinins and the inflammation
is more sensitive to steroidal drugs than the NSAID.202
In cotton pellet granuloma method TPI and TPIII inhibited
granuloma formation by 24.84%, 21.25% respectively whereas fraction
FRI and FRIII inhibited granuloma formation by 38.75%, 40.31%. The
percent inhibition for the fraction FRI and FRIII was 38.75%,
40.31%.The standard drug ibuprofen was found to be more effective in
preventing granuloma formation.
The adjuvant induced arthritis is a syndrome more akin to
rheumatoid arthritis than any other method it was observed that the
fractions TPI and TPIII inhibited edema to the extent of 61%, 49% and
the percent inhibition for the fraction FRI, FRIII, FGI and FGIII
inhibited edema by 50, 57%,56%, 59% respectively and ibuprofen
inhibited edema by 48.28%.
Since inflammation is also associated with pain, cancer,
ischemic heart diseases and liver disorders. So it is necessary to
evaluate the potential of these fractions in attenuation of disorder
realted to inflammation as mentioned above. In the tail flick test TPI
and TPIII was found least potent with percentage analgesia of 20.02%,
16.58% whereas the fractions FRI, FRIII, FGI and FGIII were most
potent and showed 20.92%, 17.5%, 20.96%,18.20% analgesia.
The anticancer activity was carried out by using trypan blue
exclusion test of cell viability. The principle involved in this method is
human breast cancer cell line such as MCF-7 cells. MCF-7 cells is
estrogen receptor (ER)-dependent and carries the wild type tumour
129
suppressor p53 gene 256 is tested by using trypan blue exclusion
method which is based on decrease in viable tumor cell count and
increased non-viable tumor cell count. The Ic 50 value for the
fractions TPI and TPIII was found to be 152.4 µM and 158.71 µM.
however the Ic 50 value for the fractions FRI, FRIII, FGI and FGIII
160.3 µM, 222.7 µM, 120.42 µM,212.23 µM.
Antihyperlipidemic activities of T. purpurea, F.religiosa and
F.glomerata were carried out using high fat diet induced
hyperlipidemia in rat. The results reveal that the fractions
TPI,TPIII,FRI,FRIII,FGI and FGIII when administered initially to the
hyperlipidemic rats causes a sharper and more significant decrease in
the serum TC and LDL cholesterol and TG level. Interestingly, these
fractions showed significantly increase in the HDL cholesterol levels of
the experimental groups at both 15th and 30th day of treatment.
Atorvastatin (10mg/kg; p.o.) has also been included in the study in
order to understand how far fractions activity is comparable to that of
a standard drug.
Several studies reveal that an increase in HDL cholesterol and
decrease in TC, LDL cholesterol and TG is associated with a decrease
in the risk of ischemic heart diseases226. Most of the
antihyperlipidemic drugs are causing significant reduction in both TC
and HDL cholesterol levels227. In the present study, significant
decrease of cholesterol in the fractions treated groups is manifested in
all the lipoprotein fractions. TPI, TPIII, FRI, FRIII, FGI and FGIII
significantly increased the levels of HDL cholesterol and decreased the
TC and TG levels. This is an important advantage in treatment of
hypercholesterolemia particularly among Indians where low HDL
cholesterol is the most prevalent lipoprotein abnormality228. High
130
levels of TC and most importantly, LDL cholesterol are the predictors
of atherosclerosis229. TPI, TPIII, FRI, FRIII, FGI and FGIII significantly
reduced both the TC and LDL cholesterol. Recent studies also show
that triglycerides are directly or indirectly related to coronary heart
diseases. In the present study, fractions markedly decreased the
triglycerides level. This is an important advantage in treatment of
ischemic heart diseases where the predictors are triglyceride and LDL
cholesterol.230
Additionally, the biochemical parameters studied did not show
any of the adverse effect of fractions on experimental animals. Liver
enzymes such as SGOT and SGPT are considered to be biochemical
markers for assessing liver function. Hepatotoxicity is evidenced by an
elevation of the serum marker enzymes. Fractions treatment reduced
these liver enzyme levels significantly in experimental animals
showing that TP, FR and FG fractions have hepatoprotective action.
During the experimentation, albino rats did not show any mortality or
any other adverse effects when the rats fed orally with at the doses of
20-40mg/kg/day. It is indicating that the leaves of the plants have a
definite antihyperlipidemic and hence can be screen for
cardioprotective and anti atherosclerotic potential.
Thus these plant medicines, T.purpurea, F.religiosa and
F.glomerata appears to be highly beneficial not only inflammation but
also in the related disorders. These plants medicines contains mostly
flavonoids and triterpinoids compound, they have high potential of
being good substitutes to NSAIDs and steroidal anti-inflammatory
agents.
The work presented here was attempted to evaluate the herbal
drugs. This is significant contribution of scientifically utilizing these
131
natural products. Further work on dosage form, clinical trials, toxicity
etc. will have to be carried out before introducing into modern
medicines. It is hoped that such type studies will reclaim our faith in
herbal products and traditional medicines so it will add novel drugs in
our armoury to fight various diseases and pain. The WHO dream of
providing health for all by the turn of century would only be realized
by such systematic knowledge of traditional medicines and tribal
medicines with modern medicines.
132
8. SUMMARY AND CONCLUSION:
In technical terms, the inflammatory response directs immune
system components to the site of injury or infection and is manifest by
increased blood supply and vascular permeability which, in technical
terms, allows chemotactic peptides, neutrophils, and mononuclear cells
to leave the intravascular compartment. Microorganisms are engulfed by
phagocytic cells (e.g., neutrophils and macrophages) in an attempt to
contain the infection in a small-tissue space. The response includes
attraction of phagocytes in a chemotactic gradient of microbial products,
movement of the phagocyte to the inflammatory site and contact with the
organism, phagocytosis (ingestion) of the organism, development of an
oxidative burst directed toward the organism, fusion of the phagosome
and lysosome with degranulation of lysosomal contents, and death and
degradation of the organism. When quantitative or qualitative defects in
neutrophil function result in infection, the infection usually is prolonged
and recurrent and responds slowly to antimicrobial agents.
Staphylococci, gram-negative organisms, and fungi are the usual
pathogens responsible for these infections. Purpose of In- vivo Models of
Inflammation is to provide the biomedical researcher in both the
pharmaceutical industry and academia with a description of the state of
the art animal model systems used to emulate diseases with components
of inflammation.
Moreover, the cell migration within the injured tissue is an
important step of the inflammatory process. Thus, using carrageenan as
stimulus it was possible to produce an acute inflammatory response
inside of the peritoneal cavity of rats 4 h later, with a large number of
polimorphonuclear cells in the exudate.cells in the exudate.
Edema formation, leukocyte infiltration and granuloma formation
represent such components of inflammation. Edema formation in the
133
paw is the result of a synergism between various inflammatory mediators
that increase vascular permeability and/or the mediators that increase
blood flow. Several experimental models of paw oedema have been
described. Carrageenan-induced paw oedema is widely used for
determining the acute phase of inflamemation. Histamine, 5-
hydroxytryptamine and bradykinin are the first detectable mediators in
the early phase of carrageenan-induced inflammation whereas
prostaglandins are detectable in the late phase of inflammation.
Maximum inhibition was observed at 40 mg/kg dose as 48.12%
compared to the control. F.glomerata fraction FGI and FGIII (20 and40
mg/kg, p.o.) showed a significant (p<0.01) inhibition of paw volume.
Maximum inhibition was observed at 40 mg/kg dose as 44.15%
compared to the control.
The cotton-pellet granuloma is widely used to evaluate the
transudative and proliferative components of the chronic inflammation.
The moist weight of the pellets correlates with transude, the dry weight of
the pellet correlates with the amount of granulumatous tissues. Chronic
inflammation occurs by means of the development of proliferate cells.
These cells can be either spread or in granuloma form. Non-steroidal
anti-inflammatory drugs decrease the size of granuloma which results
from cellular reaction by inhibiting granulocyte infiltration, preventing
generation of collagen fibers and suppressing mucopolysaccharides. The
present study showed significant anti-inflammatory activity in cotton
pellet induced granuloma and thus found to be effective in chronic
inflammatory conditions, which reflected its efficacy in inhibiting the
increase in the number of fibroblasts and synthesis of collagen and
mucopolysaccharides during granuloma tissue formation. The percent
inhibition for ibuprofen as a standard was found to be 48.28%. The
percent inhibition of the extracts T.Purpurea F.religiosa and F. glomerata
of the extracts for the fraction TPI and TPIII was 24.84%, 21.25%
134
respectively. The percent inhibition for the fraction FRI and FRIII was
38.75%, 40.31% respectively and the percent inhibition for fraction FGI
and FGIII was 40.20%, 42.45% respectively.
There has been an increasing demand from patients for the use of
natural products with Anti-hyperlipidemic activity. The undesirable side
effects and contraindications of synthetic drugs, does not appear without
risk and the fact that they are not suitable for use during pregnancy,
have made scientists look towards natural products with anti-
hyperlipidemic activity. Lipids play an important role in the pathogenesis
of complications involved with diabetes mellitus, where elevated level of
serum cholesterol and reduced level of serum HDL cholesterol, possese
to be a risk factor for developing microvascular complications leading to
atherosclerosis and further leads to cardiovascular diseases like coronary
heart disease drugs, does not appear without risk and the fact that they
are not suitable for use during pregnancy, have made scientists look
towards natural products with anti-hyperlipidemic activity. Lipids play
an important role in the pathogenesis of complications involved with
diabetes mellitus, where elevated level of serum cholesterol and reduced
level of serum HDL cholesterol, possese to be a risk factor for developing
microvascular complications leading to atherosclerosis and further leads
to cardiovascular diseases like coronary heart disease.
In conclusion, the present study reveals that T.Purpurea,
F.religiosa and F. glomerata and their fractions showed the presences of
diverse phytochemicals which are responsible for the different
pharmacological activities of the drugs. In the present study T.Purpurea,
F.religiosa and F. glomerata showed better analgesic, anti-inflammatory
activity however T.Purpurea F.religiosa and F. glomerata showed good
activity in arthritis, cancer and hyperlipidemic model. The extracts have
the potential safety and efficacy advantages for anti-cancer
chemoprevention as well as utility for treating malignant disease if
135
combined with chemotherapy. However, much work remains to be done
in order to achieve definitive conclusions about their potential
usefulness. Further study need for the isolation and toxicity studies.
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List of chemicals used for phytochemical analysis.
Name Manufacturer
α-naphthol Ranbaxy Fine Chemicals Ltd, New Delhi.
Acetic anhydride Poona Chemical Laboratories, Pune.
Ammonia Ranbaxy Fine Chemicals Ltd, New Delhi.
Ammonium hydroxide Sisco Research Lab, Mumbai
Benzene
Ranbaxy Fine Chemicals Ltd, New Delhi.
Chloroform
Cobalt chloride
Conc. Sulfuric acid
Copper sulphate
Ethanol Space Scientifics, Nasik
Ferric chloride
Ranbaxy Fine Chemicals Ltd, New Delhi.
Glacial acetic acid
Hydrochloric acid
Iodine
Lead acetate
Magnesium turnings Space Scientifics, Nasik
Mercuric chloride
Ranbaxy Fine Chemicals Ltd, New Delhi
Mercury
Ninhydrine
Nitric acid
Potassium iodide
Potassium permagnet
Potassium sodium tartarate
Pyridine
Sodium hydroxide Space Scientifics, Nasik
Sodium nitroprusside Ranbaxy Fine Chemicals Ltd, New Delhi
List of reagents used for phytochemical analysis
Name Preparation method or manufacturer Fehling’s solution
Copper sulphate (34.66 g) was dissolved in distilled water volume made upto 500 ml to prepare solution A (Fehling’s A). Potassium sodium tartarate (50 g) and sodium hydroxide was dissolved in distilled water and volume made upto 500 ml to prepare solution B (Fehling’s B).
Benedict’s reagent
Ranbaxy Fine Chemicals Ltd, New Delhi
Barfoed’s reagent
Space Scientifics, Nasik
Bial’s reagent S. d Fine chemicals, Mumbai Selwinoff’s reagent
Poona Chemical Laboratories, Pune
Millon’s test Mercury (1g) was dissolved in fuming nitric acid (9 ml) after cooling equal volume of distilled water was added.
Mayer’s reagent Mercury chloride (1.36 g) was dissolved in 60 ml distilled water. This solution of mercury chloride was added to another solution containing potassium iodide (5 g) dissolved in 20 ml distilled water. Volum was made upto 100 ml with distilled water.
Wagner’s reagent
Iodine (1.27 g) and potassium iodide (2 g) were dissolved in 5 ml water and volume made upto 100 ml with distilled water.
Hager’s reagent Space Scientifics, Nasik
• Vishal Gulecha, Thangavel Sivakumar, et al.,
Screening of Ficus religiosa leaves fractions for
analgesic and anti-inflammatory activities. Indian
journal of Pharmacology (In press)