Phytochemical investigation and arthritic activity seeds

Phytochemical investigation and arthritic activity seeds of mesua ferraa linn

By Yuvaraj D Mandavkar

Under the guidance of

Dr S S Jalapure Dept of Pharmaceutical Phytochemistry

KLES College of Pharmacy Belgaum May 2010

ABSTRACT

Background and objectives

Rheumatoid arthritis has afflicted humankind since antiquity and can persist, with serious

medical consequences, throughout a patient’s lifetime. In spite of tremendous advances in the

field of medicine, there is no truly satisfactory drug for the treatment of rheumatoid arthritis. In

traditional system of medicine, seeds of Mesua ferrea linn. is used to treat wound, microbes,

pain and rheumatism (swelling). Hence, in the present study, seeds of Mesua ferrea have been

randomly selected to study their anti-arthritic properties in Complete Freund’s adjuvant (CFA)

and formaldehyde induced arthritic rats.

Materials and methods

The seeds of Mesua ferrea were dried in shade, powdered and then extracted successively

with pet. Ether, ethyl acetate and alcohol. The extracts were subjected to preliminary

phytochemical analysis. Acute toxicity study was carried out using “Up and Down” method.

Male Wistar albino rats were used for assessment of anti-arthritic activity. Arthritis was induced

by injecting 0.1ml of CFA in sub-plantar region of left hind paw on day 1. Pet. Ether, eth. Acet.E

and AlcE of seed (300 mg/kg b.w., p.o.) were administered from 1st day till 14th day. Diclofenac

sodium (13.5 mg/kg b.w., p.o.) was used as a standard drug. The assessment of anti-arthritic

activity was done by measuring the mean changes in paw edema on 4th,8th, 14th and 21st day

after induction. The changes in paw volume were recorded and % inhibition of paw edema was

calculated.

Arthritis is also induced by injecting formaldehyde 0.1 ml in sub plantar region of left hind paw

on 1st and 3rd day. The extract were administered from 1 to 10 days and paw edema was

measured daily

Results and discussion

Preliminary phytochemical investigation revealed the presence of fixed oil, Coumarins,

carbohydrates and Saponins in Pet. Ether(kernels and coat), eth aceE and AlcE of Mesua ferrea.

LD50 cut-off dose was found to be 3000 mg/kg b.w. for all extracts. The pet. etherE of coat and

kernels of Mesua ferrrea significantly reduced (P<0.01) the CFA induced paw edema on 21st

day as compared to standard drug (Diclofenac sodium).

Conclusion

The presented data indicate that administration of pet. etherE and eth aceE of Mesua

ferrea to rats with CFA-induced arthritis and formaldehyde-induced paw edema significantly

inhibited the edema compared to the standard drug, supporting folk information regarding anti-

inflammatory activity of Mesua ferrea. The inhibitory effect of the seed may possibly be due to

the phytoconstituents (coumarins and fixed oil) present in it.

Keywords: Arthritis; CFA; Mesua ferrea; formaldehyde. Anti-inflammatory

CONTENTS

SL. NO.

TITLE PAGE NO.

1. INTRODUCTION 1-14

2. RESEARCH ENVISAGED 15-19

3. REVIEW OF LITERATURE 20-32

4. PHYTOCHEMICAL INVESTIGATION 33-51

5. PHARMACOLOGICAL INVESTIGATION 52-72

6. RESULTS 73-89

7. DISCUSSION 90-92

8. SUMMARY AND CONCLUSION 93

9. BIBLIOGRAPHY 94-101

10. ANNEXURE 101-108

LIST OF TABLES

Table no. Name of table

Pg. no.

1. Drugs derived from plants, with their Ethnomedical correlation & sources.

10

2. Macroscopic Characteristic of seeds of Mesua ferrea (Linn.). 73

3. Microscopic Characteristic of seeds of Mesua ferrea (Linn.). 73

4. Physical Constants for seed of Mesua ferrea (Linn.) 74

5. Percent yield of Pet. ether, ethanol and aqueous extracts of Mesua ferrea linn.

75

6. Results of Qualitative Chemical Investigation of Mesua ferrea (Linn.)

76

7. Characterization of seed oil of Mesua ferrea linn 79 8.

Paper Chromatography of carbohydrates 79

9. TLC of Isolated coumarins 80 10. The UV absorption maxima (λmax) were found at nm for compound 1

and 2 81

11. FTIR absorption bands of isolated compounds 81 12. Acute oral toxicity study of Pet. ether, ethyl acetate and ethanol extracts

of M. ferrea Linn

82

13. Percentage inhibitions of Freund’s adjuvant-induced paw edema by seed extracts and standard drug in injected (left) hind paw.

83

14. Effect on haematological parameters in adjuvant induced arthritis in rats 84 15. Change in body weight in adjuvant induced arthritis in rats 87 16. Percentage inhibition of paw volume IN Formaldehyde induced arthritis 89

SL NO.

NAME OF FIGURE PAGE NO.

1 Explanatory model for molecular Pharmacognosy

9

2 Documented species distribution

22

LIST OF FIGURES

3 Diagram showing the differences between a normal healthy joint, a joint affected by osteoarthritis, and one affected by rheumatoid arthritis.

55

4 The pathogenesis of rheumatoid arthritis 59 5 CFA induced paw edema on 21st day in (a) control group and

(b) Pet.ether extract (Mesua ferrea) treated group.

87

Graph-1

Column statistic of various extracts on 0th to 21st day in injected hind paw.

84

Graph-2

Column statistic of various extracts on haematological parameters

86

LIST OF ABBREVIATIONS

AlcE - Alcoholic extract

ANOVA - Analysis of variance

Approx. - Approximate

b.w. - Body weight

CFA - Complete freund’s adjuvant

conc. - Concentrated

Eth aceE - Ethyl acetate extract

FTIR - Fourier transform infra-red

g - Grams

GHS - Global harmonization system

h - Hour

HPTLC - High performance thin layer

chromatography

I.P. - Indian pharmacopoeia

Kg - Kilogram

LD50 - 50 % of the lethal dose

mg/dl - Milligram per deciliter

mg/g - Milligram per gram

min - Minutes

N - Normality

p.o. - Per oral

Pet.Ether - Petroleum ether (40-600C)

pH - Hydrogen ion concentration

q.s. - Quantity sufficient

Rf - Retardation factor

s.c. - Sub-cutaneous

SEM - Standard error of mean

T.S. - Transverse section

TLC - Thin layer chromatography

UV - Ultra-violet

INTRODUCTION CHAPTER-1

Dept. of Pharmacognosy and Phytochemistry, klecop, belgaum 1

History and Introduction to Herbals.

Historically, plants have provided a source of inspiration for novel drug

compounds, as plant derived medicines have made large contributions to human health

and well-being. Their role is two fold in the development of new drugs:

(1) They may become the base for the development of a medicine, a natural

Blue print for the development of new drugs, or;

(2) A Phytomedicine to be used for the treatment of disease1

Medicinal plants had been in use since 5000 B.C. Oldest known herbal is Pen-

t’sao written by emperor Shen-Nung around 3000 B.C. It contains 365 drugs, one for

each day of the year. Indians worked meticulously to examine and classify the herbs.

Charaka made 50 groups of 10 herbs, each of which would suffice an ordinary

physician's need1. Similarly, Sushruta arranged 760 herbs in 7 distinct sets based on to

some of their common properties. Charaka says, “There is no substance in the world that

has no medicinal value, provided you know how to use it”2.

It is very surprising to know, that according to the literature, around 60,000-

75,000 species of higher plants exists on earth and around 15,000 to 20,000 have been

reported to have medicinal value but nearly 1500 plants have been currently used in

various Ayurvedic preparations, where as only 750 to 840 have been evaluated for its

therapeutic value the form of extract.3



Herbal Drugs:

Man’s existence on this earth has been made possible only because of the vital

role played by plant kingdom. Nature always stands as golden mark to amplify the

outstanding phenomenon of symbiosis.1 Medicinal plants existing even before human

being made their appearance on the earth.4

Practically every country develops its own medical system, which includes the

ancient civilization of China, Egypt and India. Thus, the Indian Medical System-

Ayurveda came into existence. The raw materials for Ayurvedic medicines were mostly

obtained from plant sources in the form of crude drugs such as dried herbal powders or

their extracts or mixture of products.5 Also, Siddha, Unani and Tibetan are traditional

health care systems have been flourishing for many centuries. Apart from these systems

there has been a rich heritage of ethnobotanical usage of herbs by various colorful tribal

communities in the country.6

If we do well for a moment on our hoary past, Rigveda, one of our oldest

repositories of human knowledge written between 4,500-1,500 B.C. mentions the use of

67 plants for the therapeutic purposes and Yajurveda enlists 81 plants whereas

Atharvaveda written somewhere 1,200 B.C. describes 290 plants.

India unquestionably occupies the top position in the use of herbal drugs. It is one

of the foremost countries exporting plant drugs or their derivatives and excels in home

consumption too. According to Indian mythology, when the illness and diseases got



rampant on the earth, the sages learnt the science of healing from Lord Indra and

recorded them in scriptures.7

It has been estimated that about 75,000 species of higher plants exist on the earth.

A reasonable estimate of about 10% has been used in traditional medicine. However,

perhaps only about 1% of these are acknowledged through scientific studies to have

therapeutic value when used in extract form by human.3

Natural products have been derived from higher plants, microbes or animals and

those can be of either terrestrial or marine or aquatic origin. The medicinal preparations

based on these raw materials were in the form of crude drugs. With the advent of

scientific methods, many of these reputed medicinal plants came under chemical

investigation leading to the isolation of active principles. Beginning with 1,800 A.D.,

there was continuous activity in this area and many of the well known medicinal plants

were chemically analyzed and their active principles characterized. Subsequently, such

compounds became part of pharmacopoeias of several countries. This is where herbal

medicine and modem medicine have a common link7.

Indian Herbs: 8

Now-a-days natural products are an integral part of human health care system,

because there is popular concern over toxicity and resistance of modern drugs. India is

one of the 12 leading biodiversity centers with presence of over 45,000 different plant

species, 15000-18000 flowering plants, 23,000 fungi, 16,000 lichens, 18,000 bryophytes

and 13 million marine organisms. From this flora, 15,000 to 20,000 have good medicinal



value. Among those only about 7,000 plants are used in Ayurveda, 600 in Siddha, 700 in

Unani and 30 in modern medicines.

Distribution of medicinal plants

(Modified by Anonymous 2004, 2005).

Country or region

Total number of

native species in

flora

No of medicinal

plant species reported

% of medicinal

plants

Source

World 297000 52885 10 Schippmann et al., 2002

India 17000 7500 44 Shiva, 1996

Indian Himalays

800 1748 22 Samant

Pharmacognosy: Base of Phytopharmaceuticals 1

As a result of rapid development of phytochemistry and pharmacological testing

methods in recent years, new plant drugs are finding their way into medicine as purified

phytochemical. Pharmacognosy is the infrastructure on which depends evolution of novel

medicine, as it is a source of therapeutically significant substance that cannot be

synthesized economically. Further, the crude drugs also provide essential intermediates

for final synthesis of active compounds.

Prospects of Pharmacognosy9:



There is a worldwide ‘green’ revolution, which is reflected in the belief that

herbal remedies are safer and less damaging to human body, furthermore, underlying this

upsurge of interest in plants is the fact that many important drugs in use today were

derived from plants or from starting molecules of plant origin. Hence, plants seem to

have served as models in drug development.

Herbal Drug Market8:

The global herbal products market is worth of US $32 billion and is growing at a

rate of about 9-15%. The average turnover of Indian herbal medicine industry is about

2,300 crore rupees. However, to achieve the goal of major exporter of herbal remedies,

several steps need to be taken.

• Systematic study of world market demand and short-listing of medicinal herbs with

good potential.

• Systematic cultivation of medicinal herbs on a large scale.

• Encouragement for agro-based phytochemical and pharmaceutical industries to

manufacture value added herbal products.

• Strict legislation to control quality and purity.

• Up gradation of cultivation and collection process.

• Documentation of research work and standardization for quality.



Herbal Drugs Promotion10:

Phytochemistry or natural product chemistry research is the backbone of herbal

industry. For promoting use of herbals in modern medicine, Phytochemistry should be

envisaged for:

• Isolation, purification and characterization of new phytoconstituents.

• Use of newly isolated phytoconstituents as “lead” compound for the synthetic design

of analogues with either improved therapeutic activity or reduced toxicity.

• Conservation of lead phytoconstituents into medicinally important drugs.

Ethnopharmacological Approach to Herbal Drugs10:

The term ethno-pharmacology refers the interdisciplinary scientific observation,

description and experimental investigation of indigenous drugs and biological activities.

There are 119 drugs of known structure that are still extracted from higher plants and

used globally in allopathic medicine.

Practical Aspects of Herbal Drug Discovery10:

The following scheme represents a summary of the stages involved in the

development of pure drug from a plant source.

• Collection and identification of the plant and deposition of voucher sample in

herbaria.

• Literature survey on the plant species selected for studies.



• Extraction with solvent and preparation of non-polar and polar extracts for initial

biological testing.

• Evaluation of plant extract against a panel of biological test methods, as exemplified

by receptor binding, enzyme inhibition and /or cytotoxicity assays.

• Activity guided fractionation on the extract showing activity, by monitoring each

chromatographic fraction with bioassay chosen from the panel available to the

investigation.

• Structure elucidations of pure active isolate (s) using spectroscopic techniques and

chemical methods, if necessary.

• Test each active compound (whether of novel or known chemical structure) in all in

vitro and in vivo biological test methods available, in order to determine potency and

selectivity of the drug.

• Molecular modeling studies and preparation of derivatives of active compound.

• Large-scale isolation of interesting active compounds for toxicological,

pharmacological and for mutation studies, when total synthesis is not practical.

• Clinical trials (Phase I – III).



Molecular Pharmacognosy

The subject of Pharmacognosy deals with natural products used as drugs or for

the production and discovery of drugs. The poisonous or healing properties of plants

were discovery by man in his search for food11. Ayurvedic knowledge supported by

modern science is necessary to isolate, characterized and standardize the active

constituents from herbal sources12.

“Pharmacognosy is a molecular science that explores naturally occurring

structure- activity relationships with a drug potential” The term “molecular” here has a

broader meaning than in “ Molecular Biology” and other sciences which have included

molecular as a part of their name to imply a focuses on enzymes and genes. In the

definition of molecular Pharmacognosy “molecular” simply means that it is sciences

which focus on molecules and involves isolation and determination of the structure of

pharmacologically active molecules as well as the study of their biosynthesis, including

the enzymes and genes which are involved. Also the term “structure-activity

relationships” is used here in a broader sense than is commonly the case in medicinal

chemistry. The definition also restricts the research activity to molecules which could be

used as drug, thereby stressing that Molecular Pharmacognosy is a pharmaceutical

research subject also suggest the following explanatory model.

The starting point is an organism (Upper left corner) which displays some kind of

biological activity (upper right corner). Bioassay direct separation from the complex

biomass will lead to the identification of a chemical structure (central) which can be

correlated to the observed biological activity. The chemical structure can than be used for



Organism

development of a drug (lower left corner) or as a model, precursor or tool in the drug

research (lower right corner) as shown in figure.2.11.

Observation

Possible Applications

Fig 1: Explanatory model for molecular Pharmacognosy

Model Precursor Tool

Drug Herbal Drug

Chemical Structure

Biological Activity



Table 1: Drugs derived from plants, with their ethnomedical correlation & sources.

Sr.No. Drug Action or Clinical use Plant source

1 Ajmalicine Circulatory disorder Rauwolfia serpentina (L.) Benth ex.

Kurz.

2 Aesculetin Antidysentry Fraxeinus rhynchophylla

3 Arecoline Anthelmintic Areca catechu L.

4 Atropine Anticholinergic Atropa belladonna L.

5 Bromelain Anti-inflammatory;

proteolytic agent

Ananas comosus (L.)Merrill.

6 Berberine Bacillary dysentry Berberies vulgaris

7 Caffeine CNS stimulant Camellia sinensis (L.)Kuntze.

8 Curcumin Choleretic Curcuma longa L

9 Cocaine Local anaesthetic Erythroxylium coca Lam K.

10 Colchicine Antitumour agent Colchicum autumnale

11 Digoxin Cardiotonic Digitalis lanata

12 Emetine Amoebicide Cephaelis ipecacuanha

13 Glycyrrhizin Sweetener Glycyrrhiza glabra L

14 Hyoscamine Anticholinergic Hyoscamus niger L



Plants as a Source of Antibiotics11:

The antimicrobial agents have grown rapidly in the field of medicine and enabled

the eradication of most microbial infections. Numerous advances in medical technology

have brought rapid and accurate analysis of specific invading organisms, the

determination of status of blood and immunological systems and levels of drugs to

administer to patients. Thus, there is need to provide necessary pharmacological tools for

the discovery of more appropriate and effective drugs.

Plants have developed an arsenal of weapons to survive attacks by microbial

invasion. Since the advent of antibiotics in the 1950s, the use of plant derivatives as

antimicrobials has been virtually non-existent but that pace is rapidly on the increase as

we begin to realize the need for new and effective treatments. The worldwide spending

on finding new anti-infective agents is expected to increase 60% from 1993 and plant

sources are especially being investigated.

Literally, thousands of phytochemical with inhibitory effects on microorganisms

have been found to be active in vitro. One may argue that these compounds have not been

tested in vivo and therefore activity cannot be claimed but one must take into

consideration that many, if not all, of these plants have been used for centuries by various

cultures in the treatment of disease. Another argument could possibly be that at very high

concentrations, any compound is likely to inhibit the growth of microorganisms.



Anti-inflammatory and Antiarthritic herbal drugs13,14

Inflammatory diseases including different types these of rheumatic diseases are

very common throughout the world. This has been called the “King of Human Miseries”.

Although rheumatism is one of the oldest known disease of mankind and affects the large

population of the world, no substantial progress has been made in achieving a permanent

cure till the synthesis of aspirin in 1899 by the German company Bayer, the hint of which

also obtained from a plant, willow bark (Salix Alba) used worldwide in folk medicine for

the relief of aches, fever and rheumatic pain.

In India, many Ayurvedic practitioners are using various indigenous plants for the

treatment of different types of arthritic conditions. Although the applications of theses

medicaments have a sound tradition and a rational background according to the Indian

system of medicines, perhaps it is essential to investigate the rationality of their use in

modern scientific terms. The presently available drugs provide only symptomatic relief

and not free from side effects. The target should be to discover a newer drugs from plant

kingdom, which may provide therapeutic cure and would be free from undesirable effects

as well as economical, which would be accepted by the developing nations like India.

A systematic study of anti-inflammatory effects of Indian medicinal plants by

Gujral and his associates in 1956 and they screened a no. of plants for their antiarthritic

effects. Subsequently various workers from different laboratories in India have made

significant contribution. For arthritis Freund’s complete adjuvant models are used

workers in different laboratories tested their drugs. The greatest disadvantage in the

presently available potent synthetic drug lies in their toxicity and reappearance of the



symptoms after discontinuation. Therefore, the search for screening and development of

drugs for their anti-inflammatory activity is an unending problem.

Aqueous extract of roots of Maringa olifera shows antiarthritic activity in rats. In which

polar constituents are present which are responsible for antiarthritic activity.

Curcumine, a constituent of Curcuma longa chemically known as diferuloyl, has been

shown to be an effective anti-inflammatory agent. It is as potent as phenyl butazone in the

carrageenan edema test. Coumarins and fixed oil from Occimum sanctum, Ochrocarpus

longifolius, Arnebia hispidissima, Rhus undulata, Dalbergia volubilis etc. are found to be

active as anti-inflammatory and antiarthritic agent.13,14,15.

Current Status of Herbal Drugs10:

In recent years, newer diseases are posing threat to humanity. Despite this, World

Health Organization (WHO) had taken the vouch of providing ‘Health for all’ by 2,000

A.D.

Inspite of tremendous advances made by modem medicine, drugs for viral

diseases like AIDS, certain type of cancers, arthritis, Parkinsonism are yet to come. The

newer concepts about herbal drugs have immunomodulators and adaptogens gaining

importance and are recognized for prophylactic and preventive therapy.

Surprisingly, a recent survey revealed that more than 50% of all prescription

drugs are either directly derived from the natural sources or synthesized from the natural

models as the sole ingredient or as one of the several ingredients. It seems certain that the



continued scientific study of medicinal plants afford a plethora of novel, structurally

diverse and bioactive compounds. Multidisciplinary research on plants has lead to many

new drugs as well as prototype active molecules and biological tools.

Future Prospects in Herbal Medicines

At the moment, scientific research on medicinal plants is being carried out most

intensely in research institutes, universities and pharmaceutical laboratories as well as in

the clinics of many developed countries. This research is oriented mainly in two

directions. Firstly, the active ingredients of plants that have long been known for their

healing properties are investigated. The second sphere of basic research is directed

towards the discovery of new kinds of medicinal plants and new drugs from the more

remote regions of the world, which have not been explored so far.

Drugs of each and every traditional medicine, like Ayurveda, Unani and Siddha

need to be tested and validated scientifically. Council for Scientific and Industrial

Research (CSIR), New Delhi, is already involved in this field and validated about 350

formulations for different activities. This is a welcome trend since it attempts too many

traditional practices with modern knowledge for the betterment of health.16

WHO has emphasized the need to ensure the quality control of herbs and herbal

formulations by using modern techniques? Several countries have herbal pharmacopoeias

and lay down monographs to maintain their quality. Ayurvedic Pharmacopoeia of India

recommends basic quality parameters for 80 common herbal drugs17.

RESEARCH ENVISAGED CHAPTER-2


RESEARCH ENVISAGED

2. RESEARCH ENVISAGED

Rheumatoid arthritis (RA) is a chronic inflammatory disease of unknown etiology and

complex multifactorial pathogenesis, affecting joints and other tissues18. The number of

population affected are staggering. About 2% of the world’s population suffers from RA

and one of every six people have some form of arthritis in India, in a female/male ratio of

2.5:1 resulting in symptoms that range from mild annoyance to crippling disability. The

disease can occur at any age, but it is most commonly among those aged 40-70 years, its

incidence increasing with age19.

The natural history of RA is poorly defined; its clinical course is fluctuating and the

prognosis is unpredictable. RA is characterized by progressive and irreversible damage of

the synovial lined joints causing loss of joint space of bone and of function, as well as

deformity. Extra-cellular matrix degradation is a hallmark of RA, which is responsible for

the typical destruction of cartilage, ligaments, tendons and bones. Mediators responsible

for the joint destruction includes TNF-α, IL-1, IL-6, IL-15 and interferon produced locally

by macrophages, play central role18.

The activation of a cellular immune response in the genetically susceptible host

marks the beginning of the rheumatoid arthritis. The cause may be due to a single virus or

several viruses that generate an immune response or cross-react with host tissues. The

ensuing proliferation of the polyclonal B lymphocytes is centered in a proliferative

synovitis. Cytokines drive the proliferation of synovial cells, which ultimately amass to

invade and destroy articular cartilage20.



We have underestimated the morbidity and mortality of rheumatoid arthritis. Our

goals must be to intervene with focused but less toxic drugs as early as feasible in the

disease process20.

2.1 Need for study

Drug therapy for RA is based on two principal approaches: symptomatic treatment

with non-steroidal anti-inflammatory drugs (NSAIDs) and disease modifying anti-

rheumatic drugs (DMARDs). However most of the currently available drugs are primarily

directed towards the control of pain and/or the inflammation associated with joint

synovitis, but do little to interfere with the underlying immuno-inflammatory events, and

consequently also do little to block the disease progression and reduce cartilage and bone

destruction of joints. A systematic review of randomized placebo-controlled trials

conducted recently demonstrates that the published evidences supports only the efficacies

of nine agents in Western medicine, i.e., Infliximab, Cyclosporine, Sulphasalazine,

Leflunomide, Methotrexate, Parenteral Gold, Corticosteroids, Auranofin And Il-1 receptor

antagonist, in decreasing radiological progression in RA. Nevertheless, a variety of

problems exist with drugs. For example, the use of Methotrexate and Leflunomide is

impeded by their long term side effects and toxicity, while cytokines antagonists, despite

substantial efficacy and clinical improvements, entail high cost and hypersensivity to

medication and infections. Consequently, there is dramatically growing interest in herbal

medicines among persons with RA and RA research community21.

RA is an autoimmune disease which is chronic affecting the people of all ethnic

groups worldwide even though various categories like immunosuppressant, NSAIDS,

steroidal anti-inflammatory drugs are being used till now, the development of new



antiarthritic drugs is aimed towards the discovery of safe, potent drugs with minimal side

effects22.

In fact, herbal medicine is being widely used virtually around the world for

treatment of rheumatic and arthritic diseases, and in recent decades considerable advances

have been made in both clinical and basic research on the treatment of RA. Thus herbal

medicines constitute a potentially important avenue leading to novel therapeutic agents for

RA that may not only prevent structural damage of arthritic joints caused by tissue and

bone breakdown, but also be safe, relatively inexpensive, highly tolerated and convenient

for many patients21.

Their modes of action appear to be generalized in nature and doubt has been cast

on their ability to influence the long term course of disease. Furthermore, long term

studies have shown significant morbidity and mortality in up to 90% of treated

rheumatoid arthritis patient. So there is much hope of finding antirheumatic drug from

indigenous plants. Future attempts for developing anti inflammatory drugs from plants are

likely to yield fruitful results23.

Fruits of M. ferrea are used as astringent, gastric troubles; seeds used in

rheumatism and cutaneous affections; flowers astringent, stomachic and expectorant;

powder mixed with ghee applied externally in bleeding piles; buds useful in dysentery;

leaves as poultice applied on forehead in severe colds; bark astringent, decoction with

ginger used as sudorific24.

However the literature survey reveals that the seeds of Mesua ferrea Linn is used

traditionally as antiarthritc25,26,27,28. but it has not been scientifically investigated for anti-

arthritic activity.

Natural drugs possessing anti arthritic property are Aloe vera29 (Linn.) and

Barringtonia racemosa30 (L.) R.Br. contains steroid and Coumarin and fixed oil

derivatives. Since Mesua ferrea Linn also contains fixed oil and coumarin derivatives.



Due to similar chemical constituents Mesua ferrea Linn may also shows anti arthritic

activity

Hence, in the present study, the petroleum ether, ethyl acetate, and ethanol

extracts of the seeds of Mesua ferrea have been selected for phytochemical investigation

and anti-arthritic activity on experimentally induced arthritis in rats.

2.2 Objectives of study

The overall aim of proposed study is to explore the application of traditional

medicinal plants of India. The specific objectives aimed in the present work are as

follows:

To explore the possibilities of traditional uses of the plants with proper chemical

and pharmacological profiles.

To conduct systematic chemical investigation of seeds of Mesua ferrea Linn.

Screening of the Petroleum ether, Ethyl acetate and Ethanol extract of seeds of

Mesua ferrea Linn for in-vivo anti-arthritic activity on experimentally induced arthritis in

rats.

However, for analyzing and correlating the data obtained, correctly and more

precisely, the present study is designed and carried out in different steps, which are

schematically represented as follows:

Phytochemical investigations

• Collection and authentication

• Extraction

• Preliminary qualitative chemical analysis



• Separation and isolation of active phytoconstituents

• Characterization of active phytoconstituents

Pharmacological screening

• Acute toxicity studies.

• Evaluation of anti-arthritic activity.

a) CFA induced arthritis model.

b) Formaldehyde induced arthritis model.

REVIEW OF LITURATURE CHAPTER-3


Mesua ferrea Linn.

Mesua ferrea (Ceylon ironwood, Indian rose chestnut, Cobra's saffron or locally,

Penaga Lilin, Na (Sinhalese) or Nahar/Nahor is a species in the family Clusiaceae. The

plant is named after the heaviness of its timber and cultivated in tropical climates for its

form, foliage, and fragrant flowers. It is native to tropical Sri Lanka but also cultivated in

Assam, southern Nepal, Indochina, and the Malay Peninsula.

The National Ironwood Forest is a 96 ha (238 acre) forest in Sri Lanka where

Mesua ferrea trees dominate the vegetation. It is said that during King Dappula IV's

period (8th century AD) this forest was created and the remaining trees are the shoots of

it. Hence it is considered the oldest man made forest in Sri Lanka. According to botanists

this is the only ironwood forest in the dry zone with wet zone vegetation31

Biological source:

It is obtained from dried plant of Mesua ferrea. It is belonging to family

Guttiferae, Clusiaceae26

Synonym27:

Sanskrit: Nagkeshara;

Hindi: Nagkeshar;

English: Cobra’s saffron;

Marathi: Nagchampa;



kannada: Nagkesara, nagsampige;

Telagu: Nagkesara;

MULTILINGUAL MULTISCRIPT PLANT NAME DATABASE32

Sorting Mesua names

( A = names approved by most authorities, s = approved as synonyms) :

• Mesua coromandelina Wight -> Mesua ferrea L. A

• Mesua ferrea L. A

• Mesua ferruginea (Pierre) Kosterm.

• Mesua nagassarium (Burm. f.) Kosterm. -> Mesua ferrea L. A

• Mesua nervosa Planch. & Triana

• Mesua pedunculata Wight -> Mesua ferrea L. A

• Mesua roxburghii Wight -> Mesua ferrea L. A

• Mesua sclerophylla Thw. -> Mesua ferrea L. A

• Mesua speciosa Choisy -> Mesua ferrea L. A

Taxonomy33

Domain: Eukaryota

Kingdom: Plantae

Subkingdom: Viridaeplantae

Phylum: Tracheophyta

Subphylum: Euphyllophytina

Infraphylum: Radiatopses



Class: Magnoliopsida

Subclass: Dilleniidae

Superorder: Theanae

Order: Hypericales

Family: Clusiaceae

Subfamily: Kielmeyeroideae

Tribe: Calophylleae

Genus: Mesua

Specific epithet: ferrea

Botanical name: - Mesua ferrea

• HABITAT31:

The plant grows all over India, in evergreen forests upto 1500 meters altitude

found throughout the eastern Himalayas, Assam, Andaman’s; Burma and Bangladesh.

• BIOPHYSICAL LIMITS

• Altitude: up to 2,300 m

• Soil type: M. ferrea requires a fairly rich, well drained soil.

• DOCUMENTED SPECIES DISTRIBUTION

Native range: Cambodia, India, Malaysia, M Native: Myanmar, Philippines,

Singapore, Sri Lanka, Thailand, Vietnam



• FIG: 2 : Documented Species Distribution

BIOLOGY

It flowers during the dry season and flushes of new leaves are produced just after

flowering at the start of the rainy season. The bisexual flowers open for one day, between

3 and 4 a.m. and closing around sunset.

ECOLOGY

M. ferrea is a canopy component in lowland forest, but commonly features as an

understorey tree in montane evergreen or semi-evergreen forest. In Borneo, the species is

associated with dipterocarps.

BIOPHYSICAL LIMITS

• Altitude: up to 2,300 m

• Soil type: M. ferrea requires a fairly rich, well drained soil.



Plant Description

It is the National tree of Sri Lanka. The wood is very heavy, Hard & Strong.

Weight is about 72 lbs per cubic foot & density is 1.12 kg/m3.Caller is deep dark red.

Refractory in sawing & mechanics moderately well. It is used for railroad ties and heavy

structural timber31.

• The plant grows all over India, in evergreen forests upto 1500 meters altitude.

• It is a medium sized beautiful tree with glabrous, shiny, lush green leaves.

• The leaves are opposite, lanceolate, 5-15 cm long and 4-5 cm broad. Their lower

surface is covered with a waxy bloom and looks rather pale and red when young25.



Flowers terminal or axillary, bisexual, solitary or in an up to 9-flowered open

panicle, pedicel with small paired bracts. Sepals 4 decussate, suborbicular, persistent and

variously enlarged and thickened in fruit. Petals 4, white or pink. Stamens numerous, free

or connate only at the base, ovary superior (1-2 celled) each cell with 1-2 axillary ovules.

Style slender with a peltate to lobed stigma31. Flowers are 4-7.5 cm diameter with white

petals and center of numerous yellow stamens29a-34.

• The fruits, ovoid, 2.5-5 cm in length, 1-4 dark brown seeds within24

• The bark is grayish or reddish brown, exfoliating in large thin flakes, wood

extremely hard.



• Seeds are 1-4, dark brown, up to 2.5cm diameter.

Stamen

• stamen consist of anther, connective and filament; coppery or golden brown;

• Filament united at base forming a fleshy ring; 0.8-1.0cm long slender, filiform,

more or less twisted, soft to touch, quite brittle;

• Connective not visible with naked eye; odor fragment; task astringent.

• Each stamen 0.9-1.9cm. Long;

• Anther about 0.5cm. Long, linear, basifixed, containing pollen grains;



Chemical constituents30,31,37:

• From the plant, xanthones, a number of 4- phenylcoumarin derivatives, friedelin

and triterpenes have been isolated.

• Xanthones are isolated from the heartwood young fruits contain an oleo resin

which yields an essential oil,

• Octadecatrienoic and hexadecanoic acids identified in seed oil.

• new 4-phenylcoumarin-mesuagin- viz; mesuone, mammeigin, Mesugin and

mesuol isolated

• Another coumarin named Mammeisin was isolated from seeds

• The presence of acetaldehyde, n-hexaldehyde and glyoxal in the seed oil has been

reported

• Fatty acids viz. palmitic oleic, linoleic and arachidic are reported from the seed

oil. Mesuol and mesuaferrol are also isolated. Mammeisin isolated from seeds;

mesuagin – isolated from seed oil and characterized; mammeigin and mesuol

isolated from seed oil.

• Mammeisin is isolated from the seeds and stamens afford two novel biflavanones

designated as mesuaferrone.

• Lipids: The kernels contain about 75% of a yellowish oil.

• A new biflavone – measuaferrone A – isolated from stamens and characterized



structure elucidation of another biflavone mesuaferrone B – isolated from stamens.

• Canophyllal, canophyllol and canophyllic acid from the leaves. Leaves are

reported to contain unnamed alkaloid.

Mammeisin Canophyllal

Mesuaferrol



Pharmacological properties:

Uses of specific part of M. ferrea,25,26,27

Flowers

Flowers used for cough, stomachic and astringent, stimulant, carminative,

expectorant, powder mixed with ghee (butter fat) applied externally in bleeding

pile,

The dried flowers are given in vomiting, thirst, irritability of the stomach,

excessive perspiration.

The fragrant flowers are used to stuff pillows and cushions and in cosmetic

products. Buds useful in dysentery.

Stamens are used in treatment of anthelminitic.

Decoction of the flowers is drunk by women after childbirth

Tannin or dyestuff: present in the flowers are used in dyeing for fixed colours.

Seeds

Seeds yield fatty oil which may be bleached and used for soap making, also used

for skin troubles and as an embrocating in rheumatism and cutaneous affections

such as sores, scabies, and wounds.

Phenolic constituent of seed oil revealed a potent antiasthmatic effect.

Decorticated seed kernel meal can be incorporated up to the 10% level to replace

maize in the feed of poultry without adverse effects on their performance.

Seed meal is a good source of protein and energy, and its used as a feed ingredient

for cattle is proposed.



Seed meal contains 12.8% digestible crude protein and 87.3% total digestible

nitrogen on a DM basis.

Fruits:

Astringent, useful in gastric troubles,

It is stimulating and alterative; it is given in disease of the genitourinary organs as

a substitute for cubebs.

Bark

It is bitter, aromatic and mildly astringent.

It is combined with ginger used as sudorific.

It is given in the form of decoction, or infusion is a bitter tonic and is very useful

in gastritis and bronchitis.

Leaves: as poilitis applied on forehead in severe colds.

Wood

Used for railway sleepers, bridges and posts, beams, and other construction work.

Also suitable for electric poles and currier’s cutting blocks, boat building, tool-

handles, golf club head and walking stick.

Heavy wood used for gun-stock, musical instruments, and cabinet work.



Rewiew of Literature:

Gautam Das and Niranjan Kara (2009) An epoxidized vegetable oil of Mesua

ferrea L. seed was prepared and used as a reactive diluent for commercial BPA-based

epoxy resin at different compositions for the first time38.

Nunthaun Uawonggul et al,(2006), studied aqueous extracts of 64 plant species

were screened for their activity against fibroblast cell lysis after Heterometrous laoticus

scorpion venom treatment39.

Md. Taufiq Hassan et al(2006), studied n-hexene, ethyl acetate, and methanol

fractions for analgesic activity of Mesua ferrea linn in acetic acid induced rats40.

Dutta et al(2006), synthesized A series of polyurethane resins with varying

NCO/OH ratios (0.8–2.0) from the monoglyceride of Mesua Ferrea L. seed oil41

Nurdin, Rudy et al(2006), studied, A microwave extraction system (MES) was

developed for the extraction of essential oils from Mesua ferrea L. leaves and Jasminum

sambac flowers42.

M. Abu Sayeed et al(2004); Studied Physico-chemical Characteristics of Mesua Ferrea

Seed Oil and Nutritional Composition of Its Seed and Leaves43

Verotta L et al(2004),Extracted 4-Alkyl- and 4-phenylcoumarins from Mesua

ferrea blossoms by using supercritical co2 as promising multidrug resistant antibacterials44

FI Sohel, MST Sarmina Yasmin(2004) Studied antibacterial and antifungal

activities of various solvent extracts were tested against fourteen pathogenic bacteria

tested and six fungi respectively45.



Rupa Mujumdar et al (2004), studied antibacterial efficacy of the methanol extract

of whole flowers of Mesua ferrea against various strains of bacteria and the extract

significantly reduce the viable count of strain46

US Patent 6267996 - Pharmaceutical and cosmetic formulations with

antimicrobial activity US Patent Issued on July 31, 200147

Europian patent. Pharmaceutical And Cosmetic Antiacne Formulations

Containing Plant Extracts (Krameria Triandra Or Mesua Ferrea)48

Dolon Konwer, et al(1989), Extracted Liquid Fuels from Mesua ferrea L. Seed

Oil49.

C. Gopalakrishnan,et al,(1980) Studied anti inflammatory and CNS depressant

activities of xanthones from calophyllum inophyllum and Mesua ferrea, where All the

xanthones produced varying degrees of C.N.S. depression characterised by sedation,

decreased spontaneous motor activity, loss of muscle tone, potentiation of pentobarbitone

sleeping time and ether anaerthesia in mice and rats50.

T. R. Govindachari et al(1967), Isolated Two new yellow pigments,

Mesuaxanthone A and Mesuaxanthone B, and the known Euxanthone have been isolated

from the heartwood extracts of Mesua ferrea L. Evidence is presented to show that

Mesuaxanthone A is 1,5-dihydroxy-3-methoxy-xanthone and Mesuaxanthone B is 1,5,6-

trihydroxyxanthone. Mesuaxanthone A has been synthesized51.

STANDARDIZATION AND PHYTOCHEMICAL INVESTIGATIOn CHAPTER-4

Dept. of Pharmacognosy and Phytochemistry, klecop, belgaum. 33

STANDARDIZATION AND PHYTOCHEMICAL INVESTIGATION

OF SEEDS OF MESUA FERREA LINN.

PHARMACOGNOSTIC INVESTIGATION53,54,55

• Collection & Authentification of seeds of Mesua ferrea (Linn.)

• Analysis of powder characteristics.

• Determination of physical constants.

1. Ash Value.

2. Loss on Drying.

3. Fluorescence analysis of drug.

4. Extractive Values.

a) Petroleum ether soluble extractive of seed kernels.

b) Ethyl acetate soluble extractive.

c) Alcohol soluble extractive.

d) Petroleum ether soluble extractive of seed coat.

5. Chemical evaluation:

Acid value.

Iodine value.

Ester value.

Saponification value.

Unsaponificable matter.



4.1. Collection & Authentification of Seeds of Mesua ferrea (Linn.)

The seeds of the Mesua ferrea (Linn.) were collected in the month of May

from Botanical garden of Karnataka University, Dharwar. The seeds are authenticated by

Mr. Harsha Hegde, Research Officer, Regional Medical Research Centre, ICMR, Nehru

Nagar, Belgaum-590 010. The certificate is given in Annexure A-1.

4.2. Preliminary Pharmacognostic Characteristics:

In present study, the seed of Mesua ferrea (Linn.) was investigated for its

macroscopic characteristics and microscopic characteristics. Results are given in table

no-2

4.3. Microscopic Characteristics56:

Powder Characteristic:-

In present study, the dried seeds of Mesua ferrea (Linn.) was pulverized into fine

powder separately. The powder was investigated for their microscopic characteristic.

Procedure:

The pulverized powder of seeds was boiled separately with chloral hydrate

solution in small quantity. Remove cleaved powder in three separate watch glass

respectively and stain with one drop each of phloroglucinol and concentrated

hydrochloric acid.

Mount a little of the treated powder in dilute glycerin and observed the slide under

microscope at low power. Table no.-3



4.4. DETERMINATION OF ASH VALUES54

1) Total Ash Value:

Method:-

Weigh accurately 2 to 3 gm of air–dried seeds of Mesua ferrea separately in a

tarred platinum or silica dish and incinerate at a temperature not exceeding 4500C until

free from carbon, cool and weigh. If a carbon free ash cannot be obtained in this way

exhaust the charred mass with hot water, collect the residue on an ash less filter paper,

incinerate the residue and filter paper until the ash is white or nearly so, add the filtrate,

evaporate to dryness and ignite at a temperature not exceeding 4500C. Calculate the

percentage of ash with reference to the air–dried drug.

2) Determination of Acid Insoluble Ash:

Method: -

Boil the ash with 25 ml of 2M Hydrochloric acid for 5min, collect the insoluble

matter in a Gooch crucible or on an ashless filter paper, wash with hot water, ignite, cool

in a desiccator and weigh. Calculate the percentage of acid insoluble ash with reference

to the air–dried drug.

3) Determination of Water Soluble Ash:

Method:-

Boil the ash for 5 min. with 25 ml of water, collect the insoluble matter in a Gooch

crucible or on an ashless filter paper, wash with hot water, and ignite for 15 min at a

temperature not exceeding 4500C. Subtract the weight of insoluble matter from the



weight of the ash, the difference in weight represent the water-soluble ash. Calculate the

percentage of water-soluble ash with reference to the air-dried drug.

The results were given in (Table no. 4)

4.5. FLUORESCENCE ANALYSIS OF THE DRUG55

Many crude drugs show the fluorescence when the sample is exposed to

ultraviolet radiation. Evaluation of crude drugs based on fluorescence in daylight is not

much used, as it is usually unreliable due to the weakness of the fluorescence effect

(umbelliferone test used for galbanum and asafoetida is, however, an exception).

Fluorescence lamps are fitted with suitable filters, which eliminate visible radiation from

the lamp and transmit ultraviolet radiation of definite wavelength. Several crude drugs

show characteristic fluorescence useful for their evaluation.

Objective

To examine the crude drugs under ultraviolet radiation and report their

authenticity.

Materials

Ultraviolet lamp (200-400 nm), crude drugs (entire and powder).

The results are given in (Table no.4)



4.6. LOSS ON DRYING55

Loss on drying of the air-dried seeds of Mesua ferrea (Linn.). was analyzed.

Weigh about 1.5 gm of air-dried seed powder of Mesua ferrea (Linn.) into a

weighed flat and thin porcelain dish. Dry it in the oven at 1000C or 1050. Cool in a

desiccator and observe. The loss in weight is usually recorded as moisture and the results

are given in (Table no.4).

4.7. EXTRACTIVE VALUES56

The extractive values for various solvents of air-dried sample were evaluated.

1) Petroleum ether soluble extractive.

2) Ethyl acetate soluble extractive.

3) Alcohol soluble extractive.

1) Petroleum ether soluble extractive value:

5 grams of air-dried seed coat powder of Mesua ferrea (Linn.) was macerated with

100 ml of petroleum ether in a closed flask, shaking frequently during the first 6 hours

and allowed to stand for 18 hours separately. Thereafter, it was filtered rapidly taking

precaution against loss of petroleum ether. Evaporated 25 ml of filtrate to dryness in a

tared flat bottom shallow dish dried at 1050C and weighed. Percentage petroleum ether

soluble extractive was calculated with reference to the air-dried seed.



2) Petroleum ether soluble extractive value:

5 grams of air-dried seed kernel powder of Mesua ferrea (Linn.) was macerated

with 100 ml of petroleum ether in a closed flask, shaking frequently during the first 6

hours and allowed to stand for 18 hours separately. Thereafter, it was filtered rapidly

taking precaution against loss of petroleum ether. Evaporated 25 ml of filtrate to dryness

in a tarred flat bottom shallow dish dried at 1050C and weighed. Percentage petroleum

ether soluble extractive was calculated with reference to the air-dried seed.

3) Ethyl acetate soluble extractive value:

5 grams of air-dried seed powder of Mesua ferrea (Linn.) was macerated with 100

ml of petroleum ether in a closed flask, shaking frequently during the first 6 hours and

allowed to stand for 18 hours separately. Thereafter, it was filtered rapidly taking

precaution against loss of ethyl acetate. Evaporated 25 ml of filtrate to dryness in a tarred

flat bottom shallow dish dried at 1050C and weighed. Percentage petroleum ether soluble

extractive was calculated with reference to the air-dried seed.

4) Alcohol soluble extractive:

5 grams of air-dried seed powder of Mesua ferrea (Linn.) macerated with 100 ml

of alcohol in a closed flask, shaking frequently during the first 6 hours and allowed to

stand for 18 hours separately. Thereafter, it was filtered rapidly taking precaution against

loss of alcohol. Evaporated 25 ml of filtrate to dryness in a tarred flat bottom shallow

dish dried at 1050C and weighed. Percentage alcohol soluble extractive values was

calculated with reference to the air-dried seeds.

Results are given in table no-4



4.8. PHYTOCHEMICAL INVESTIGATION:

• Extraction of pulverized seeds of Mesua ferrea (Linn.). with Petroleum ether (40-

600C) followed by ethyl acetate and Alcohol solvents.

• Qualitative chemical tests.

• Chromatographic studies

Paper Chromatography

Thin layer Chromatography

High Performance Thin Layer Chromatography (HPTLC).

• Spectroscopic studies

U.V. Spectroscopy

I.R Spectroscopy.

Extraction: 57

The air-dried seeds of Mesua ferrea (Linn.) were reduced to fine powder of

required particle size and around 200 gm of powder was subjected to successive hot

continuous extraction (soxhlet) with petroleum ether (40-600C), ethyl acetate and finally

alcohol. After the effective extraction, the solvents were distilled off. The extract was

then concentrated on water bath and finally reduced to dryness. After drying, the

respective extracts were weighed and yields were recorded in Table No.5

All the extracts were subjected to chemical investigation and anti-arthritic activity.



4.9. Qualitative Chemical Investigation:58

The Petroleum ether (40-600C), ethyl acetate and Ethanol extracts were subjected

to qualitative chemical investigation.

Details of various tests:

The following procedures were adopted to test for the presence of various chemical

constituents in the extracts.

1. Test for Sterols:

a) Salkowski test: When a few drops of conc. sulphuric acid is added to the test solution,

shaken and allowed to stand, lower layer turns red indicating the presence of sterols.

b) Liebermann Burchardt test: The test solution treated with few drops of acetic

anhydride and mixed well. When conc. sulphuric acid is added from the sides of the test

tube, it shows a brown ring at the junction of the two layers and the upper layer turns

green.

c) Sulphur test: Sulphur when added into the test solution, it sinks in it.

2. Test for Triterpenoids:

a) Salkowski test: When a few drops of conc. sulphuric acid is added to the test solution,

shaken and allowed to stand, lower layer turns yellow.

b) Liebermann Burchardt test: The test solution treated with acetic anhydride, mixed

well and conc. sulphuric acid is added from the sides of the test tube. Deep red colour

forms.



3. Test for Glycosides:

a) Baljet's test: The test solution treated with sodium picrate gives yellow to orange

colour.

b) Keller-Killiani test: The test solution with few drops glacial acetic acid in 2ml of

ferric chloride solution and conc. sulphuric acid is added from the sides of test tube which

shows the separation between two layers, lower layer shows reddish brown and upper

layer turns bluish green.

c) Raymond's test: Test solution treated with dinitrobenzene in hot methanolic alkali

gives violet colour.

d) Bromine water test: Test solution dissolved in Bromine water gives yellow

precipitate.

e) Legal's test: Test solution when treated with pyridine (made alkaline by adding

sodium Nitroprusside solution) gives pink to red colour.

4. Test for Saponins:

a) Foam test: Saponins when mixed with water and shaken shows the formation of

foam which is stable at least for 15 minutes.

b) Haemolysis test: 2 ml of 18% sodium chloride in two test tubes was taken. To one

test tube distilled water is added and to the other test tube 2ml of filtrate and then few

drops of blood is added to both the test tubes. Mixed and observed for haemolysis under

microscope.



5. Test for Carbohydrates:

a) Molisch's test: Test solution with few drops of Molisch's reagent and 2ml of conc.

sulphuric acid is added slowly from the sides of the test tube shows a purple ring at the

junction of two liquids.

b) Barfoed's test: Test solution treated with Barfoed’s reagent on boiling on a water

bath shows brick red precipitate.

c) Benedict's test: Test solution treated with Benedict's reagent and boiling on a water

bath shows reddish brown precipitate.

6. Tests for Alkaloids:

a) Mayer's test: Test solution treated with Mayer's reagent (Potassium mercuric iodide)

gives cream coloured precipitate.

b) Wagner's test: The acidic solution treated with Wagner's reagent (Iodine in potassium

iodide) gives brown precipitate.

c) Hager's test: The acidic solution with Hager's reagent (Saturated picric acid solution)

gives yellow precipitate.

d) Dragendorff's test: The acidic solution with Dragendorff's reagent (potassium

bismuth iodide) shows reddish brown precipitate.

7. Test for Flavonoids:

a) Ferric chloride test: Test solution with few drops of ferric chloride solution shows

intense green colour.

b) Shinoda test: Test solution with few fragments of magnesium ribbon and conc.

hydrochloric acid, shows pink to magenta red colour.



c) Zinc – Hydrochloric acid-reduction test: Test solution with zinc dust and few drops

of HCl shows magenta red colour.

d) Alkaline reagent test: Test solution when treated with sodium hydroxide solution

shows increase in the intensity of yellow colour which becomes colourless on addition of

few drops of dilute acid.

e) Lead acetate solution test: Test solution with few drops of lead acetate solution

(10%) gives yellow precipitate.

8. Tests for Tannins:

a. Ferric-chloride test: Test solution with few drops of ferric chloride solution gives

dark colour.

b. Gelatin test: Test solution treated with gelatin solution gives white precipitate.

9. Tests for Proteins:

a) Million's test: Test solution treated with million's reagent and heated on a water bath,

protein is stained yellow on warming.

b) Xanthoprotein test: Test solution treated with conc. nitric acid and on boiling gives

yellow precipitate.

c) Biuret test: Test solution treated with 40% sodium hydroxide and dilute copper

sulphate solution gives blue colour.

d) Ninhydrin test: Test solution treated with ninhydrin reagent gives blue colour.

10. Test for Free Amino Acids:

Ninhydrin test: Test solution treated with ninhydrin reagent gives blue colour.

11. Test for Fats:



a) Solubility test: Oils are soluble in ether, benzene and chloroform, but insoluble in

90% ethanol and in water. (Exception- Castor oil, soluble in alcohol).

b) Filter paper test: Filter paper gets permanently stained with oils.

12. Test for Volatile Oils:

a) Odour test: Volatile oils have characteristic odour.

b) Filter paper test: Filter paper is not permanently stained with volatile oil

Results are given in table no-6

4.10. Characterization of seed oil of Mesua ferrea linn59.

A) Acid value:

2g oil was weighed accurately by transfer method into a 250ml

conical flask neutral ethanol(20ml) was added by means of pippate and the

flask heated on a steam bath for 3 min. then the flask was cooled and the

contents were titrated with 0.1 M alcoholic KOH solution using

phenolphthalein as an indicator. A blank titration was also conducted side

by side

Acid value= 5.61X n

W

Where n = the number of ml of 0.1 M KOH required

W= the weight (in g) of the substance

B) Iodine value

2 gm oil was weighed accurately by transfer method into a 250 ml iodine flask

and dissolved in chloroform (2o ml). Wijs reagent (Iodine monochloride, 20 ml ) was

added by means of pipette. The flask was stoppered and kept in darkness for 1 hour with

intermittent shaking. Then 15 % of KI solution (10 ml) and 15 ml of DW were added to



the flask and mixture was shaken well. The liberated iodine was titrated with 0.1 M

sodium thiosulphate solution using fresh starch solution as a indicator. A blank titration

was also conducted side by side.

Iodine value = 1.269 X (B – S ) W

Where, B = the number of ml of 0.1 M sodium thiosulphate required for blank

S =t he number of ml of 0.1 M sodium thiosulphate required for substance

W= the weight (in g ) of the substance

C) Saponification value

2 gm oil was weighed accurately by transfer method into a 250 ml round bottom

flask. Freshly prepared 0.5 M alcoholic potassium hydroxide solution (25) ml was added

to the sample by means of pipette and the mixture gently refluxed on a steam bath using

an air condenser for 1 hour. Then the flask was cooled to about 60 – 70 0 c, the condenser

tip washed with little distilled water and the contents were titrated with 0.5 M HCL

Solution using phenolphthalein as indicator. A blank titration was carried out

simultaneously.

Saponification value = 28.05 X (B-S)

W

Where B= the number of ml of 0.5 M HCL required for blank,

S= the number of ml of 0.5 M HCL required for the substance,

W= the weight (in g) of the substance.

D) Unsaponifiable matter:

After the titration of the sample for Saponification value was completed, the

contents of the flask were made alkaline and extracted with light petroleum ether and

diethyl ether. The combined ethereal solution was washed thoroughly with distilled



water, dried over anhydrous sodium sulfate, evaporated the solvent and the residue was

weighed. It was dissolved in neutral alcohol and the free acid was titrated with 0.02N

alcoholic potassium hydroxide solution using phenolphthalein as indicator.

Unsaponifiable matter = 100 x [W1-(0.282 x N x V)] W Where, W1=the weight (in g) of the residue,

V= No. of ml of 0.02N KOH Solution,

N= Normality of the titrant

W= The weight in gm of the substance.

E) Ester value:

The ester value is the number of milligrams of potassium hydroxide required to

saponify the ester present in 1 gram of the substance.

Ester value= Saponification value – acid value Results are given in table no-8

CHROMATOGRAPHIC STUDIES:

The Paper Chromatography and Thin Layer Chromatography studies of

alcoholic extracts of Hemidesmus indicus roots were carried out to confirm the presence

of phytoconstituents.

Paper Chromatography for Carbohydrates60

Method for Paper Chromatography of Carbohydrates:-

Whatman’s chromatography paper no. 42 (55x45 cm) was used for quantitative

separation of amino acids in ascending manner.



Solvent System:-

N-butanol : glacial acetic acid : water (50 :10 :40 v/v) was used.

The paper was spotted with ethanolic extract of seeds of Mesua ferrea. After

development of chromatogram, the spots were visualized by using Phenolic-sulphuric

acid. The Rf values were determined and given in Table no.9

THIN LAYER CHROMATOGRAPHY:

Thin Layer Chromatography studies were carried out for various extracts to

confirm the presence of different phytoconstituents in these extract. TLC is a mode of

liquid chromatography, in which, the extract is applied as a small spot or band at the

origin of thin sorbent layer supported on a glass/plastic/metal plate. The mobile phase

migrates through the stationary phase by capillary action. The separation of solutes takes

place due to their differential absorption/ partition coefficient with respect to both mobile

and stationary phases. Each separated component has same migration time but different

migration distance.

The mobile phase consists of a single solvent or a mixture of solvents.

Although, a number of sorbent like silica gel, cellulose, polyamide, alumina, chemically

modified silica gel etc. are used, silica gel(type 60) is most commonly used sorbent.

Handmade plates are prepared by using techniques like pouring, dipping or spraying.

Now-a-days, readymade precoated plates are also available. The plates need to be

activated at 1100c for 1h. this removes water/ moisture loosely bound to silica gel surface.



The retardation factor (Rf) is calculated using following formula,

Rf = Distance traveled by sample from base line

Distance traveled by solvent from base line

Thin Layer Chromatography 62,63

The pet. ether extract were subjected to thin layer chromatography for the presence of

phytoconstituents.

In this technique, the Silica gel-GF254 (for TLC) was used as an adsorbent and

plates were prepared by spreading technique, then air dried for an over-night and

activated for one hour at 1100C and used.

TLC of coumarins

Stationary phase : Silica gel GF-254

Mobile Phase : Benzene: Glacial acetic acid: water

Proportion : 10:7:3

Detection : UV -356

Rf : 0.36

Results are in table no-7



Isolation of Phytoconstituents:

Preparative Thin Layer Chromatography: 63

A thick layer of silica gel GF-254 was coated on the square shaped plate and

activated at 1100C for one hour. The broad band (2 mm width) of extracted sample was

applied on the plate.

The details of plate were as follows:-

Plate dimension : 10 x 20 cm

Adsorbent : Silica gel GF254

Activation : 1100 C for 1 hr

Band parameter : Width – 2 mm

Length – 8 cm

The plate was developed in a saturated chamber having desired solvent system.

After developing the plate was dried and if the band gives fluorescence then it can be

easily scraped. Otherwise a small portion of the band was sprayed with detecting agent,

by taking care to avoid the exposure of remaining plate to spray reagent. Then the band is

scraped by measuring the height of sprayed band.

The scraped band was then suspended in desired solvent and filtered on Whatman

filter paper no.1 and washed several times with same solvent. The filtrates were

combined and concentrated and reduced to dryness. This procedure was followed for

several scrapings.



Then the resulted compound was run with original sample to confirm the isolation

and subjected to U.V. absorption and I.R. spectroscopy studies for identification and

partial characterization.

Characterization of Isolated Compound:

From the separated bands, the substance of interest was scrapped from the plate

and it dissolved in methanol. The mixture was filtered and the filtrate was evaporated to

dryness. The isolated compound was then subjected for further studies.

Fig. No. 6: Showing TLC Profile of extract and isolated Coumarins

Spectral Characterization of Isolated Coumarins

• UV Spectrum of coumarin64

UV spectrum was recorded in JASCO UV530 Spectrophotometer for isolated

coumarin in AR grade methanol, which gave a sharp peak at 223 nm.

Results are mentioned in the Annexure A-2.

• IR of Coumarin64

IR spectrum was recorded in “Themro Nicolet” IR-200 spectrometer for isolated

Coumarin using IR grade KBr.

Results are mentioned in the Annexure A-3.



HPTLC of Extracts and Isolated Compound65:

Pet. ether extract of seed and isolated compounds were subjected to High

Performance Thin Layer Chromatography.

The details of HPTLC were as follows:-

Plate : Aluminium plate precoated with Silica gel GF254

Thickness : 0.2 mm

Plate size : 10 x 10 cms

Sample application : 10 μl

Solvent system : Benzene: Glacial acetic acid: Water (10:7:3)

Detection : U.V. (200, 254, 366 nm)

Instrument : CAMAG TLC Scanner 3 & LINOMAT-V

CAMAG TLC Scanner 3 & LINOMAT-V densitometric evaluation system with

WINCAT software was used for scanning of thin layer chromatogram objects in

reflectance or transmission mode by absorbance or by fluorescence at 254 and 366 nm

respectively.

The fingerprint of HPTLC profile of ethanolic extracts and isolated compound

were taken using computer. Rf value of various samples was evaluated using following

formula:

Distance traveled by sample from base line Rf = Distance traveled by solvent from base line

Results are mentioned in the Annexure A-3

PHARMACOLOGICAL INVESTIGATIONS CHAPTER-5

Dept. of Pharmacognosy and Phytochemistry, klecop, belgaum. Page 53

PHARMACOLOGICAL INVESTIGATIONS

5.1 Introduction to Arthritis:

Arthritis is a term used to describe a number of painful conditions of the joints

and bones. Two of the main types of arthritis are osteoarthritis and rheumatoid arthritis.

Osteoarthritis

Osteoarthritis is the most common form of arthritis. Cartilage (connective tissue)

between the bones gradually wastes away (degenerates), and this can lead to painful

rubbing of bone on bone in the joints. It may also cause joints to fall out of their natural

positions (misalignment). The most frequently affected joints are in the hands, spine,

knees and hips.

Rheumatoid arthritis

Also known as inflammatory arthritis, rheumatoid arthritis is a more severe, but

less common condition. The body's immune system attacks and destroys the joint,

causing pain and swelling. It can lead to reduction of movement, and the breakdown of

bone and cartilage66.

5.2 Rheumatoid arthritis

5.2.1 Introduction

Rheumatoid arthritis (RA) is a chronic systemic inflammatory disorder that may

affect many tissues and organs-skin, blood vessels, heart, lungs and muscles. But

principally attacks the joints producing a non-suppurative, proliferative synovitis that

often progresses to destruction of the articular cartilage and ankylosis of the joints67.



Fig. No. 3: Diagram showing the differences between a normal healthy joint, a joint

affected by osteoarthritis, and one affected by rheumatoid arthritis.

RA is characterized by progressive and irreversible damage of the synovial lined

joints causing loss of joint space of bone and of function, as well as deformity. Extra-

cellular matrix degradation is a hallmark of RA, which is responsible for the typical

destruction of cartilage, ligaments, tendons and bone68.

RA is characteristically a symmetric arthritis. Articular and periarticular

manifestations include joint swelling and tenderness to palpation, with morning stiffness

and severe motion impairment in the involved joints. Extra-articular signs can involve

pulmonary, cardiovascular, nervous and reticuloendothelial systems. The clinical

presentation of RA varies, but an insidious onset of pain with symmetric swelling of the

small joints is the most frequent finding. Onset of RA is acute or subacute in about 25%

of patients, but its patterns of presentation also include palindromic onset, monoarticular



presentation (both slow and acute form), extra-articular synovitis (tenosynovitis, bursitis),

polymyalgic-like onset, and general symptoms (malaise, fatigue, weight loss, fever).

The palindromic onset is characterized by recurrent episodes of oligoarthritis

with no residual radiologic damage, while the polymyalgic-like onset may be clinically

indistinguishable from polymyalgia rheumatica in elderly subjects.

Early RA is characterized by symmetric polyarthritis involving the small joints

of the hands and feet with no radiologic changes. RA most frequently affects the

metacarpophalangeal, proximal interphalangeal and wrist joints. Although any joint,

including the cricoarytenoid joint, can be affected, the distal interphalangeal, the

sacroiliac and the lumbar spine joints are rarely involved, which is peculiar because these

are some of the most typical targets of seronegative spondylarthropathies, such as

psoriatic arthritis and aknylosing spondylitis69.

As in many autoimmune diseases a clear differences in RA susceptibility exists

between females and males, the former being 2-3 times likely of developing the disease.

A difference in RA susceptibility is maintained at all ages but is more pronounced before

the age of 50. These observations can be interpreted as females being more susceptible to

RA and having a lower threshold for developing the disease69.

The disease can occur at any age (No age is immune), but it is most common

among those aged 40-70 years, its incidence increasing with age. RA is the most common

form of rheumatic diseases, and has a substantial societal effect in terms of cost,

disability, and lost productivity70.



The geographic distribution of RA is worldwide, with a notably low prevalence

in rural Africa and high prevalence in specific tribes of Native Americans. There is no

clear association between prevalence of RA and socioeconomic status70.

5.2.2 Clinical Features

The range of presentations of RA is broad, but disease onset is insidious in most

cases, and several months can elapse before a firm diagnosis can be ascertained. The

predominant symptoms are pain, stiffness, and swelling of peripheral joints69.

Clinical Features of RA

Symptoms: -

• Joint swelling

• Pain/ stiffness (Commonly in morning and lasting > 1hr)

• Weakness

• Deformity

• Fatigue

• Malaise

• Fever

• Weight loss

• Depression

Articular Characteristics: -

• Palpation tenderness

• Synovial thickening

• Effusion (early on)

• Erythema (early on)



• Decreased range of motion (later on)

• Ankylosis (later on)

• Subluxation (later on)

Distribution: -

• Symmetrical (especially later on)

• Distal more commonly than proximal

• Proximal interphalangeal joint, metacarpophalangeal joint/ metatarsophalangeal

joint, wrist/ ankle more commonly than elbow/ knee, shoulder/ hip.

5.2.3 Etiology

Possible Primary Causes:

The cause of rheumatoid arthritis is unknown. Indeed, it is possible that many

different arthritogenic stimuli activate the immune response in the immunogenetically

susceptible host. Nevertheless, current research is focusing on exogenous infectious

candidates as the causative agent or agents, as well as endogenous substances, such as

connective-tissue proteins (e.g. collagen and proteoglycans) and altered

immunoglobulins71.



Fig no:4- The pathogenesis of rheumatoid arthritis

Infectious Agents:

At various times in recent years, agents such as human T-cell lymphotropic virus

Type I, rubella virus, cytomegalovirus, herpesvirus, and mycoplasma, have been

proposed as the etiologic agent of rheumatoid arthritis, but none have received sustained

scientific support71.

Epstein-Barr virus has been linked to rheumatoid arthritis for more than 10

years because of the following observations. Eighty percent of patients with rheumatoid

arthritis have a circulating antibody directed against antigens specific for Epstein-Barr

virus and the autoantibody response in rheumatoid arthritis enhances the response to

these antigens. Patients with rheumatoid arthritis shed more Epstein-Barr virus in throat

washings than do control subjects. They also have increased numbers of circulating B

cells infected with the virus and a diminished cytotoxic T-cell response to immune

responses induced by Epstein-Barr virus71



The relation of mycobacteria to rheumatoid arthritis is also enjoying a

resurgence of interest because these bacteria express heat-shock proteins, which are the

arthritogenic factors of adjuvant arthritis in rats. Heat-shock proteins appear on cell

surfaces in response to various kinds of stress. Animal and bacterial heat-shock proteins

have much homology with human heat-shock proteins and are believed to have a role in

inflammation. Patients with rheumatoid arthritis have elevated levels of antibodies to

heat-shock proteins from recombinant mycobacteria. It is of particular interest that in

synovial fluid from patients with rheumatoid arthritis there are relatively large number of

"double-negative" T-lymphocytes (lymphocytes without CD4 or CD8 surface markers)

with a distinct CD3-associated T-cell receptor composed of γ and δ chains. These are the

cells that proliferate in response to mycobacterial antigens71.

Autoimmunity:

Although the cause of RA remains unknown, autoimmunity plays a pivotal

role in its chronicity and progression. There is little question among most investigators

that autoimmunity has a major role in the progression of rheumatoid arthritis, but data

supporting autoimmunity as the initial cause of rheumatoid arthritis are less firm.

Collagen and IgG are the endogenous proteins most often implicated in these hypotheses.

Collagen is a direct cause of arthritis in rodents and higher forms of animals

(including monkeys), and collagen-induced arthritis is often considered to be a relevant

model for rheumatoid arthritis. Most data from studies in humans are consistent with the

hypothesis that rheumatoid arthritis is not caused by the development of antibodies to the

collagen (Type II) found in articular cartilage, but rather that synovitis and perhaps the

centripetal polarization of destructive arthritis are amplified by anticollagen antibodies.



Elevated titers of antibody to both native and denatured forms of Type II collagen are

found in the serum of patients with rheumatoid arthritis; however, there are data showing

that antibodies against native Type II collagen do not precede the clinical onset of

rheumatoid arthritis. It is probable that as cartilage is invaded and destroyed by the

proliferative synovitis, an immune response is mounted against epitopes on degraded

portions of collagen and that collagen antigen-antibody complexes, along with

rheumatoid factor-IgG complexes, precipitate within superficial layers of cartilage and

serve as a chemo attractant for the invasive tissue67,71.

Genetic Susceptibility67:

The results of several studies have shown a higher disease concordance

among monozygotic twins (12-15%) than dizygotic twins (4%), implying the influence of

genetic factors. Heritability analysis of these studies suggests that about 60% of a

population's predisposition to rheumatoid arthritis can be accounted for by genetic

factors, although on analysis of twin pairs concordant for rheumatoid arthritis, striking

diversity in disease severity was noted.

Analysis of genetic markers has revealed an association between

development of rheumatoid arthritis and the presence of a shared epitope on small

regions of the DRB1 *0401 and *0404 alleles. These analysis have also suggested that

certain HLA alleles correlate with features of worse disease such as rheumatoid factor,

nodules, and erosion; rapid advances in genetic methods also hold promise for

identification of non-HLA disease-association genes.

Histological Changes67:



An inflamed synovium is central to the pathophysiology of rheumatoid

arthritis. It is histologically striking, showing pronounced angiogenesis; cellular

hyperplasia; an influx of inflammatory leucocytes; and changes in the expression of cell-

surface adhesion molecules, proteinases, proteinase inhibitors, and many cytokines.

Synovial changes in rheumatoid arthritis vary with disease progression. In the first weeks

of the disease, tissue oedema and fibrin deposition are prominent and can manifest

clinically as joint swelling and pain. Within a short period, the synovial lining becomes

hyperplasic, commonly becoming ten or more cells deep and consisting of type A

(macrophage-like) and type B (fibroblast-like) synoviocytes. The sublining also

undergoes striking alterations in cellular number and content, with prominent infiltration

of mononuclear cells including T-cells, B-cells, macrophages, and plasma cells.

Synovial-vessel endothelial cells transform into high endothelial venules early in the

course of the disease. High endothelial venules are specialized post-capillary venules

found in secondary lymphoid tissue or inflamed non-lymphoid tissues; they facilitate the

transit of leucocytes from the bloodstream into tissues.

The formation of locally invasive synovial tissue pannus – is a characteristic

feature of rheumatoid arthritis. This tissue is involved in the joint erosions seen in

rheumatoid arthritis. Pannus is histologically distinct from other regions of the synovium

and shows phases of progression. Initially, there is penetration of the cartilage by

synovial pannus composed of mononuclear cells and fibroblasts with high-level

expression of matrix metalloproteinases by synovial lining cells. In later phases of the

disease, cellular pannus can be replaced by fibrous pannus comprised of a minimally

vascularised layer of pannus cells and collagen overlying cartilage. The tissue derivation



of pannus cells has not been fully elucidated, although they are thought to arise from

fibroblast-like cells (type B synoviocytes). In-vitro work shows that these fibroblast-like

synoviocytes have anchorage-independent proliferation and loss of contact inhibition,

which are phenotypes shown by transformed cells. However, the molecular pathogenic

mechanisms driving pannus formation remain poorly understood.

T-cells67:

Several lines of evidence implicate the participation of T-cells in the

pathogenesis of rheumatoid arthritis. T-cells account for part of the mononuclear

infiltrate in the synovial sublining, and with slight differences, these lymphocytes can

organize into aggregates similar to those found in lymph nodes and Peyer's patches. The

genetic evidence implicating HLA-DR (MHC class II) alleles also suggests a role for T-

lymphocytes. CD4 T-cell specificity is mediated by interaction of a specific T-cell

receptor with a peptide presented by an MHC class II molecule. Thus, the predilection

for HLA-DR alleles in rheumatoid arthritis suggests a pathogenic process either at the

level of antigen presentation by the MHC molecule or at the level of MHC plus antigen

recognition by CD4 T-cells. To delineate further the restricting elements defining

potential auto-reactive T-cell clones; precise use of T-cell receptor chains in rheumatoid

arthritis has been defined in synovial and peripheral T-cells. Although these studies

suggest over-representation of certain receptor chains, they also document heterogeneous

receptor populations in the inflamed synovium, arguing against a single pathogenic T-cell

receptor allele. Further analysis of peripheral T-cell homeostasis in populations of

patients with rheumatoid arthritis shows decreased general diversity of T-cell-receptor

use, specific changes in receptor selection, and clonal outgrowth of subsets of CD4 cells.



Thus, one hypothesis for rheumatoid arthritis pathogenesis is aberrant systemic selection

or activation of T-cells via MHC class II alleles interacting with several T-cell receptors

of limited diversity. The molecular bases underlying the synovial predilection for disease

activity remain unknown.

Despite the evidence implicating T-cells in the pathogenesis of rheumatoid

arthritis, other evidence suggests that T-cells do not directly cause synovitis in the joint

microenvironment. Analysis of synovial infiltrating T-cells does not show much

proliferation of this population. Furthermore, comparison of CD45 isoforms in synovial

T-cells with peripheral blood T-cells reveals an enrichment of cells expressing CD45

isoforms characteristic of memory T-cells in the rheumatoid arthritis synovium. This

phenotype suggests recruitment of previously stimulated and mature T-cells as opposed

to in-situ maturation of these cells. Finally by contrast with known T-cell dependent

inflammatory processes, analysis of synovial T-cell lymphokine production has shown a

small T-cell contribution on cytokine profiles. Thus, although T-cells probably play a

part in the systemic initiation of the process in rheumatoid arthritis, their direct role in

synovitis and joint destruction is unclear.

5.2.4 Treatment for rheumatoid arthritis72

There is no known cure for rheumatoid arthritis. To date, the goal of treatment

in rheumatoid arthritis is to reduce joint inflammation and pain, maximize joint function,

and prevent joint destruction and deformity. Early medical intervention has been shown

to be important in improving outcomes. Aggressive management can improve function,

stop damage to joints as seen on X-rays, and prevent work disability. Optimal treatment

for the disease involves a combination of medications, rest, joint-strengthening exercises,



joint protection, and patient (and family) education. Treatment is customized according to

many factors such as disease activity, types of joints involved, general health, age, and

patient occupation. Treatment is most successful when there is close cooperation between

the doctor, patient, and family members.

5.2.4.1 Drugs

Two classes of medications are used in treating rheumatoid arthritis: fast-acting

"first-line drugs" and slow-acting "second-line drugs" (also referred to as disease-

modifying antirheumatic drugs or DMARDs).

The first-line drugs, such as aspirin and cortisone (corticosteroids), are used to

reduce pain and inflammation.

The slow-acting second-line drugs, such as gold (Solganal), Methotrexate

(Rheumatrex, Trexall), and hydroxychloroquine (Plaquenil) promote disease remission

and prevent progressive joint destruction, but they are not antiinflammatory agents. Some

newer "second-line" drugs for the treatment of rheumatoid arthritis include leflunomide

(Arava) and the "biologic" medications Etanercept (Enbrel), Infliximab (Remicade),

Anakinra (Kineret), Adalimumab (Humira), Rituximab (Rituxan), and Abatacept

(Orencia).

5.2.4.2 Rest and exercise



A balance of rest and exercise is important in treating rheumatoid arthritis.

During flare-ups (worsening of joint inflammation), it is best to rest the joints that are

inflamed. When joint inflammation is decreased, guided exercise programs are necessary

to maintain flexibility of the joints and to strengthen the muscles that surround the joints.

• Range-of-motion exercises should be done regularly to maintain joint mobility.

• Swimming is particularly helpful because it allows exercise with minimal stress

on the joints.

• Physical and occupational therapists are trained to provide specific exercise

instructions and can offer splinting supports. For example, wrist and finger splints

can be helpful in reducing inflammation and maintaining joint alignment.

• Devices, such as canes, toilet seat raisers, and jar grippers can assist daily living.

• Heat and cold applications can ease symptoms before and after exercise.

5.2.4.3 Surgery

Surgery may be recommended to restore joint mobility or repair damaged joints.

Doctors who specialize in joint surgery are orthopedic surgeons. The types of joint

surgery range from arthroscopy (insertion of a tube-like instrument into the joint to see

and repair abnormal tissues) to partial and complete replacement of the joint. Total joint

replacement is a surgical procedure whereby a destroyed joint is replaced with artificial

materials. For example, the small joints of the hand can be replaced with plastic material.

Large joints, such as the hips or knees, are replaced with metals.

5.2.4.4 Diet and other treatments



There is no special diet for rheumatoid arthritis. Fish oil may have anti-

inflammatory beneficial effects, but so far this has only been shown in laboratory

experiments studying inflammatory cells. Likewise, the benefits of cartilage preparations

remain unproven. Symptomatic pain relief can often be achieved with oral

acetaminophen (Tylenol and others) or over-the-counter (OTC) topical preparations,

which are rubbed into the skin.

5.3ANTI-ARTHRITIC ACTIVITY

5.3.1 Acute oral toxicity73:

The acute oral toxicity was carried out as per the guidelines set by Organization

for Economic Co-operation and Development (OECD), revised draft guidelines 423,

received from Committee for the Purpose of Control and Supervision of Experiments on

Animals, Ministry of Social Justice and Empowerment, Government of India.

It is the principle that is based on a stepwise procedure with the use of a

minimum number of animals per step to obtain sufficient information is obtained on the

acute toxicity of the test substance to enable its classification. The substance is

administered orally to a group of experimental animals at one of the defined doses. The

substance is tested using a stepwise procedure, each step using three animals of either

sex. Absence or presence of compound related mortality of the animals dosed at the step

will determine the next step of;

- No further testing is required

- Dosing of three additional animals with the same dose



- Dosing of 3 animals at the next higher or the next lower dose level

The method enabled judgment with respect to classifying the test substance to

one of the series of toxicity classes defined by fixed LD50 cut off values.

Description of the methods:

1) Selection of animal species:

Healthy young mice of either sex weighing 25-30 gms were used for acute

toxicity study to determine LD50 of test compounds. Each group contained 3 animals.

2) Housing and feeding condition:

The temperature in the experimental room was maintained around 25°C.

Lighting was natural; sequence being 12 hours dark, 12 hours light cycle. The

conventional laboratory diet was fed with adequate supply of drinking water.

3) Preparation of Animals:

The animals were randomly selected, marked to permit individual identification

and kept in polypropylene cages for one week prior to dosing to allow acclimatization of

them to laboratory conditions.

4) Preparation of doses:

The test compounds (P.E., alcoholic and aqueous extracts) were prepared as a

suspension by triturating with water and 2% tween 80.

5) Administration of doses:



The test substances were administered in a single dose by using a mice oral

feeding needle. Prior to dosing, animals were kept for 12 hours fasting. Then animals

were weighed and test substance was administered. After the administration of dose, food

was withheld for a further 3-4 hours.

6) Number of animals and dose levels:

In each step three animals were used in each group. Study begun at 50mg/kg

body weight and 3000-mg/kg-body weight.

Observations:

Animals were observed initially after dosing at least once during the first 30

minutes, periodically during the first 24 hours, with special attention given during the

first 4 hours. In above case, death was observed within first 24 hours. Additional

observations like changes in skin, fur, eyes, mucous membranes, respiratory, circulatory,

autonomic, central nervous system, somatomotor activity and behavior pattern were also

noted. Attention was also given to observation of tremors and convulsions.

5.3.2 Evaluation of anti-arthritic activity74,75,

Animals:

Male Albino rats (Wistar strain) weighing 150-200gm, procured from Sri

Venkateshwara Enterprises, Bangalore, were used for the study.

Housing of the Animals:



Animals were kept for one week to acclimatize to laboratory conditions before

starting the experiment. They were given free access to water and standard rat feed. 12

hrs prior to an experiment, the rats were deprived of food but not water.

Chemicals and drugs:

1) Freund’s Complete Adjuvant (Sigma, St. Louis, USA).

2) Formaldehyde.

3) Diclofenac sodium (collected from local market)

4) Tween 80 (Himedia Mumbai)

5) Sterile water for injection (Core Health Care Ltd., Mumbai).

Dose selection

The doses of 300 mg/kg b.w of petroleum ether, ethanol and ethyl acetate extract of

Mesua ferrea were chosen for Freund’s Complete Adjuvant induced arthritic in rats.

Diclofenac sodium 13.7mg/Kg body weight was used as standard drug.

Preparation of Mesua ferrea dose:

Dose of extracts of Mesua ferrea were prepared as a suspension by triturating extract,

with water and 2% Tween 80.

Preparation of Diclofenac sodium dose:

Diclofenac sodium dosage was prepared 30 min. before administration, as a suspension

by triturating with water and 1 % Tween 80.

Procedure:



Male Wistar albino rats weighing between 180 and 210 g were randomly selected.

Animals were divided into 6 groups containing six in each group. Arthritis was induced

in all animals by injecting 0.1 ml of CFA in sub-plantar region of left hind paw. Control

group was treated with vehicle and the standard group was treated with Diclofenac

Sodium (13.5mg/kg BW). The other groups were treated with petroleum ether(seed coat

and kernels), ethanol and aqueous extracts of Mesua ferrea at the dose of 300mg/kg BW.

On 0th day, the left hind paw volume of all animals was measured using

plethysmometer. The dosing was started on 1st day and continued till 14th day. The

assessment of anti-arthritic activity was done by measuring the mean changes in paw

edema on 4th,8th,14th and 21st day after induction. The changes in paw volume were

recorded and % inhibition of paw edema was calculated.

5.3.3 Observations and Calculations:

The paw edema was measured on 4th,8th,14th and 21st day after induction of CFA

using digital plethysmometer. The mean changes in injected paw edema, with respect to

initial paw volume, were calculated on respective days and percentage inhibition of paw

edema with respect to untreated group was calculated using following formula.



The changes in body weight were recorded daily, on the day 21, blood was withdrawn

from the each animal by retro-orbital vein puncture by anesthetizing each animal by

using anesthetic ether. The blood was collected into vials containing EDTA for assessing

hematological parameters.

Parameter measured29,76,77:

The following parameters were studied during the course of experiment.

1. Change in paw edema 2. Body weight changes 3. Haematological parameters • Haemoglobin (Hb %) • RBC count • WBC count • ESR count

b) Evaluation of formaldehyde induced arthritis78

Male Wistar rats weighing between 150-200 g. will be randomly selected. They will be

grouped in a group of 6 animals each into 4 groups. On the 0th day, the basal paw volume

of left hind paw of each animal will measure using Plethysmometer. On day 1 and day 3,

they will be injected into the sub-plantar region of the left hind paw with 0.1 ml of 2 %

v/v formaldehyde in normal saline. Dosing with standard drug, Diclofenac sodium and

extracts will be started on same day and continued for 10 days.

Group I - Arthritis control

Group II - Diclofenac Sodium (standard drug) treated.

Group III - Petroleum ether extract treated

Group IV - Ethyl acetate extract treated.

Group V - Alcohol extract treated.

Arthritic control rats received normal saline throughout the study. While the rest

experimental group animals receive respective treatments once daily by oral route. Paw

volume of injected paw will be measured daily. The mean changes in injected paw

edema, with respect to initial paw volume, will be calculated on respective days and

percentage rate inhibition of paw edema with respect to untreated group will be



calculated.

The percentage inhibition of paw edema will be calculated as compared to control on 10th

day.

Statistical analysis79

All the data obtained from the various parameters were statistically evaluated by one way

analysis of variance test (ANOVA) followed by Dunnet’s t post test. Significance level

was p<0.05.

RESULTS CHAPTER-6


RESULTS

7.1.1 Authentication of plant

The plant was authenticated by Mr. Harsha Hegade, research scientist, RCMR,

ICMR, Belgaum.

CODE

NAME OF PLANT

RMRC-472 Mesua ferrea linn

7.1.2 Preliminary Pharmacognostic Characteristics22, 41:

In present study, the seeds of Mesua ferrea (Linn.) was investigated for its

macroscopic characteristics and microscopic characteristics. Given in table

Table No.2

Macroscopic Characteristic of seeds of Mesua ferrea (Linn.).

Sl. No. Parameters Observation of Seed

1. Colour Brown

2. Odour Odourless

3. Taste Bitter

4. Size 1.2- 2.0 cm in diameter

5. Shape Oval

RESULTS CHAPTER-6


7.1.3 Microscopic Characteristics:

Microscopic characters of seeds of Mesua ferrea are given in table

Table No. 3

Microscopic Characteristic of seeds of Mesua ferrea (Linn.).

Sr. No. Observation of Seed

1. Oil globules

2. Lignified xylem

3. Covering trichomes

4. Parenchymatous cells

Table No. 4

Physical Constants for seeds of Mesua ferrea (Linn.)

Sl. No. Physical Constants Result

1. Ash Value (% w/w)

• Total Ash

• Acid Insoluble Ash

• Water Soluble Ash

14.4

1.25

6.87

2. Fluorescence Analysis

• At 254 nm

Fluorescence

RESULTS CHAPTER-6


• At 366 nm Fluorescence

3. Loss on Drying (% w/w) 13.2

4. Extractive Values (% w/w)

Pet. ether soluble extractive value (kernels)

Ethyl acetate soluble extractive value

Alcohol soluble extractive value

Pet.ether soluble extractive value of seed

coat.

42

31

9

1.4

7.1.4 Percent yield

The percent yield of Pet. ether, ethyl acetate and ethanol extracts of Mesua ferrea is

given in Table

Table No. 5: Percent yield of Pet. ether, ethyl acetate and ethanol extracts of

Mesua ferrea linn.

Extract Colour Consistency Yield (% w/w)

Mesua ferrea linn

Pet. Ether ( kernels) Light red Oil 22.12

Pet.ether( coat) Yellowish Solid 1.53

Ethyl acetate Reddish brown Oil 20.32

Ethanol Dark black Semisolid 8.23

RESULTS CHAPTER-6


7.1.5 Preliminary qualitative test:

Results of Qualitative Chemical Investigation of Mesua ferrea (Linn).

Table-6

Sl. No

Chemical tests Petroleum Ether Extract(coat)

Petroleum Ether Extract(kernels)

Ethyl acetate extract

Ethanol Extract

Tests for Carbohydrates Molish's test (General test)

+++

++

++

++

A Tests for reducing sugars a)Fehling's test b)Benedicts test

+++ +++

+++ +++

++ ++

++ ++

B Test for Monosaccharides a) Barfoeds test

-

-

-

C Test for Pentose sugars

- - -

D Tests for Hexose sugars a)Selwinoff’s test b)Cobalt chloride test

- -

- -

- -

1

E Tests for Non-reducing polysaccharides (starch) a)Iodine test b)Tannic acid test for starch

- -

- -

- -

RESULTS CHAPTER-6


2 Tests for Proteins: a)Biuret test b)Millions test c)Precipitation test

++ ++ ++

- - -

+ - +

+ +

3.

Tests for Amino acids: a)Ninhydrin test b)Test for tyrosine c)Test of tryptophan d)Test for cysteine

- -- -

- - - -

- - -

4.

Tests for Fats and oils: a)Solubility test b)Saponification test c)Paper staining test

+++ +++ +++

+++ +++ +++

+++ +++ +++

+ + +

5.

Tests for Steroids: a)Salkowski reaction b)Liebermann–Burchard reaction c)Liebermann reaction

- - -

- - -

- - -

6.

Tests for Triterpenoids: a)Salkowski reaction b)Liebermann– Burchard test

- -

- -

- -

Tests for Glycosides: A Tests for Cardiac

Glycosides: a) Baljet test b) Legal test c) Keller-Killani test d) Liebermann's test

- - - -

- - - -

- - - -

B Tests for Anthraqu- inone glycosides : a) Borntraggers test b) Modified Borntragger's test

- -

- -

- -

7.

C Tests for Saponin glycosides : a) Foam test b) Haemolysis test c)Bromine water test

+++ +++

+

+

++ ++

+ +

RESULTS CHAPTER-6


Note: "+" Present, "-" Absent.

The table indicated that the extracts contain mainly carbohydrates, coumarins,

Phenolic compound and fats and oil compound etc.

Pet. Ether extract showed the presence of carbohydrates, fats and oil, amino

acids, Coumarin, Saponins and Proteins. Ethanol extract showed the presence of

carbohydrates, fats and oil, amino acids, Coumarin, phenolic compound and Proteins.

D Tests for Coumarin glycosides : a) Alkaline reagent test b)NaOH Soaked paper test

+++

+++

+++

+++

++

++

+ +

8 Tests for Flavanoids: a. Ferric Chloride test b. Shinoda test c. Alkaline reagent test d. Lead acetate test

- -

- -

- -

9.

Tests for Alkaloids: a) Dragendroff's test b) Mayers test c) Hagers test d) Wagners test e)Murexide test for purine alkaloids

- - -

- - -

- - - - -

10

Tests for Tannins and Phenolic compounds: a) 5% FeCl3 solution b) Lead acetate solution c)Gelatin solution d)Bromine water e)Acetic acid solution f) Dilute iodine solution g) Dilute HNO3 h)Dilute potassium permagnate solution

++ ++

++ + +

- -

- +

-

- + - -

- +

- + - + - -

+ +

+ + + - -

RESULTS CHAPTER-6


Ethyl acetate extract showed the presence of carbohydrates, fats and oil, amino acids,

Coumarin and Proteins.

7.1.6 Characterization of seed oil of Mesua ferrea linn

TABLE NO-7

Extract Oil characteristic Result

Acid value 10.15

Iodine value 91.23

Saponifiable Value 204.07

Unsaponificable Matter 1.38

Pet. Ether

Ester value 192.54

7.1.7 Chromatographic studies:

Results of paper chromatography:

Table no. 8

Paper Chromatography of carbohydrates

Extracts No .of spots

observed.

Colour Rf – values

Pet. Ether 2 Black 0.43, 0.77

The qualitative TLC analysis resulted in separation of different phytoconstituents in

different solvent systems and they were identified by their characteristic coloured bands

with the corresponding visualizing reagent (Table No. 8).

RESULTS CHAPTER-6


Table No. 9 Qualitative TLC analysis for separation of different

phytoconstituents

HPTLC:

HPTLC of pet ether extract of seeds was performed using CAMAG -TLC Scanner 3

and LENOMAT- V

The Detail profiles Of HPTLC is Given in Annexure-4

7.1.8 Spectroscopic study

UV spectroscopy

The UV absorption maxima (λmax) were found at 284nm for compound 1

(Annexure A-.3)

Table-10

Phytoconstituents Absorbance

Compound 284

Phytoconstituents Solvent System Visualizing Reagent

Colour Rf Value

Compound 1 Benzene: Glacial acetic acid: Water (10:7:3)

UV-366 Orange 0.37

RESULTS CHAPTER-6


FTIR spectroscopy

The FTIR spectrum of isolated compound has shown characteristic peaks as listed in

Table. (Annexure A-5).

Table No. 11: FTIR absorption bands of isolated compounds

Compound 1 Compound 2

Wave

number(cm-1)

Mode of Vibration

Wave

number

(cm-1)

Mode of

Vibration

RESULTS CHAPTER-6


7.2 Pharmacological investigations

7.2.1 Acute toxicity study

Acute toxicity study was carried out according to OECD guidelines. The extracts did

not show any toxic symptoms and mortality in animals at various dose levels tested. The

LD50 cut-off doses obtained for various extracts are enlisted in Table No.10

Table No. 12 Acute oral toxicity study of Pet. ether, ethyl acetate and

ethanol extracts of M. ferrea Linn

Extract LD50 cut-off dose ED50 Effective dose

Mesua ferrea linn

Pet. Ether(kernels) 3000 mg/kg b.w. 300mg/kg

Pet. Ether(coat) 3000 mg/kg b.w. 300mg/kg

Ethyl acetate 3000 mg/kg b.w. 300mg/kg

Ethanol 3000 mg/kg b.w. 300mg/kg

7.2.2 Anti-arthritic activity

The anti-arthritic activity was evaluated by using CFA induced arthritis model in

Wistar albino rats. The assessment made on the 21st day showed that, treatment with all

extract of Mesua ferrea Linn significantly reduced (P<0.01) the swelling in the injected

left hind paw (Fig. No. 5) as compared to Diclofenac sodium treated group. On the 21st

day the % inhibition of paw edema exhibited by pet ether(kernels), pet ether(coat), ethyl

acetate and ethanolic extract were 71.90 68.21 68.48 and 58.12% respectively; while

Diclofenac sodium treated animals showed maximum % of inhibition of paw edema

79.36% on 21st day. The results are shown in table no (Table No.13, Graph No.1)

RESULTS CHAPTER-6


RESULTS CHAPTER-6


TABLE NO-13

Percentage inhibitions of Freund’s adjuvant-induced paw edema by seed extracts and standard drug in injected (left) hind paw.

Change in paw edema

Extracts

0th day 4th day 8th day 14th day 21st day

Inhibition

Normal control 0.831±0.023 0.833±0.056 0.835±0.203 0.837±0.013 0.840±0.023

Arthritic control 0.836±0.0133 1.585±0.099 1.652±0.0195 1.738±0.0186 1.975±0.056

Diclofenac sod. (13.5mg/kg BW)

0.888±0.0268 1.412±0.017** 1.242±0.017** 1.167±0.0194** 1.110±0.015** 79.36

Pet. Ether(kernels)

0.850±0.0278 1.520±0.010** 1.352±0.013** 1.393±0.0311** 1.177±0.014** 71.90

Pet. Ether (Seed coat)

0.8450±0.0168 1.533±0.008* 1.464±0.010** 1.460±0.0673** 1.207±0.033** 68.21

Ethyl acetate 0.8717±0.0401 1.538±0.010* 1.527±0.008** 1.525±0.0633** 1.230±0.033** 68.48

Mesuaferrea

Ethanol 0.865±0.0321 1.548±0.011NS 1.595±0.0078* 1.538±0.0388* 1.342±0.044** 58.12

RESULTS CHAPTER-6


Graph No.1 Column statistic of various extracts on 0TH to 21st day in injected

(left) hind paw.

HAEMATOLOGICAL PARAMETERS:

1. White blood cell count

WBC count seems to be raised in arthritis condition there is a mild to

moderate rise in WBC count due to release IL-1β inflammatory response. IL-1β

increases the production of both granulocytes and macrophages colony stimulating

factor.

It appears from our study that, pet. Ether treated group significantly reduces the

WBS count may be due to inhibiting the release of IL-1β in arthritis condition.

2. Red blood cell count

RESULTS CHAPTER-6


Results indicating the total average RBC count in the entire test materials

treated group raised marginally, in arthritis there is a mild anaemia, which may be due to

iron deficiency or low serum iron with normal iron store. The results indicated that, the

pet ether treated groups are significantly (p<0.01) increases the RBC count

3. Haemoglobin count:

The results indicated that the haemoglobin count in severe arthritis condition is

decreased than normal. Diclofenac sodium and Pet ether (kernels and coat) extract

treated rats showed significant (p<0.01) increase in the haemoglobin count compared to

arthritis control rats.

4. Erythrocyte sedimentation rate:

The results indicated that the ESR count in severe arthritis condition was

marginally increased than normal. Diclofenac sodium and Pet ether (kernels and coat)

extract treated rats showed significant (p<0.01) reduction in the ESR rate compared to

arthritis control rats. Results are given in table no-14

5. Body weight changes:

Arthritis control group rats exhibited mean weight loss of 3.65±0.50 gm over a

period of 21 days. Diclofenac sodium and extract treated rats appeared to normalize the

body growth by exhibiting mean change in body weight nearly equivalent to normal rats.

However, the differences in the mean body weight exhibited by Diclofenac sodium and

extract treated rats were significant (p<0.01) compare to arthritis control rats.

Results are shown in table no-15

RESULTS CHAPTER-6


Table no-14

Effect on haematological parameters in adjuvant induced arthritis in rats

Group Changes in haematological parameters (Mean ±SEM)

WBC (x103 cells/mm3)

RBC count(x106/mm3)

Hb (%)

ESR

Normal(-ve control)

7.00± 0.10 5.88± 0.11 14.08 ±0.103 12.95±0.06

+ve Control 8.017±0.060 5.717±0.060 12.61±0.123 14.47±0.054

Standard 7.10±0.013** 7.03±0.113** 14.64±0.103** 10.61±0.073**

Pet. Ether kernel

7.145±0.015** 6.748±0.0956** 14.60±0.033** 11.95±0.077**

Pet.ether coat 7.812±0.0407NS 6.183±0.1337* 13.52±0.1655** 13.71±0.2341**

Ethyl acetate 7.812±0.040** 6.038±0.031* 13.20±0.099* 14.17±0.007NS

Alcohol 7.908±0.071NS 5.805±0.068* 12.34±0.051 NS 13.93±0.1450*

Graph No.2 Column statistic of various extracts on Haematological parameters

RESULTS CHAPTER-6


TABLE NO-15

Change in body weight in adjuvant induced arthritis in rats

Groups Mean body weight in gm before injection

Mean body weight in gm on 21 day

Mean body weight in gm (±SEM)

Normal 184.43±0.56 213.8±0.46 29.36±9.90

Control 197.45±0.42 193.8±0.87 - 3.650±0.495

Standard 182.45±1.545 204.90±3.25** 22.45±2.500

Pet. Ether (kernels)

185.60±1.540 203.00±1.32** 18.60±0.650

Pet ether (coat) 185.78±1.052 198.87±1.56** 13.09±0.458

Ethyl acetate 188.34±1.656 197.90±0.374* 8.12±0.754

Alcohol 183.34±1.323 188.70±1.256NS 5.36±0.640

(A)

RESULTS CHAPTER-6


(B)

Fig. No.5: CFA induced paw edema on 21st day in (A) Control group and (B) Pet.ether extract (Mesua ferrea) treated group.

7.2.3. Formaldehyde induced arthritis

The anti-arthritic activity was also evaluated by using formaldehyde induced

arthritis model in Wistar albino rats. The assessment made on the 10th day showed that,

treatment with all extract of Mesua ferrea more significantly reduced (P<0.01) the

swelling in the injected (left) hind paw as compared to Diclofenac sodium treated group.

On the 10th day the % inhibition of paw edema exhibited by pet ether, ethyl acetate and

alcoholic extract were 76.58%, 66.11, and 65.84% respectively; while Diclofenac

sodium treated animals showed maximum % of inhibition of paw edema 81.37 on 21st

day. The results are shown in table no-16.

RESULTS CHAPTER-6


TABLE NO-16

Percentage inhibition of paw volume

Paw volume(mean ± SEM) Group

0TH DAY 10TH Day

% INHIBITION

(10TH DAY)

Control 0.9133±0.0284 2.002±0.0104

Standard 0.9217±0.0265 1.105±0.0224** 80.65

Pet. ether extract

0.9300±0.0713 1.180±0.0388** 76.58

Ethyl acetate extract

0.8417±0.0392 1.210±0.0535** 66.11

Alcohol extract 0.9250±0.0453 1.297±0.0170** 65.84

DISCUSSION CHAPTER-7

DEPT. OF PHARMACOGNOSY AND PHYTOCHEMISTRY, KLES, COP, BELGAUM 90

DISCUSSION

In the present study, the seeds of Mesua ferrea were dried in shade, powdered

and then extracted successively with Pet. Ether, Ethyl acetate and alcohol. The extracts

were subjected to preliminary phytochemical analysis. The preliminary phytochemical

study showed the presence of coumarin, saponins, protein, phenols, tannins and

carbohydrates in Mesua ferrea, by qualitative TLC, U.V and FTIR analysis, compound in

Mesua ferrea, may be coumarin. However, detailed study is necessary to elucidate the

chemical structure of Phytoconstituents.

In this study, acute oral toxicity was carried out using Wistar albino mice of either

sex weighing between 25 and 30 g; while, male Wistar albino rats weighing between 180

and 210 g were employed for assessment of anti-arthritic activity. Acute toxicity study by

“Up and Down” method showed the LD50 cut-off doses at 3000 mg/kg body weight,

indicating that the drug to be much safer. Hence, 300mg/kg b.w. dose were selected for

anti-arthritic activity.

Arthritis was induced in left hind limb of rats using CFA. The most objective

measurement that can be made to assess the anti-arthritic activity is the determination of

magnitude of swelling of hind paws. The left injected hind paw is used to assess the acute

inflammatory response to the injection of the adjuvant27. In CFA induced arthritis model,

rats develop a chronic swelling in multiple joints with influence on inflammatory cells,

erosion of joint cartilage, bone destruction and remodeling. These inflammatory changes

ultimately result in the complete destruction of joint integrity and function in the affected

animal27,80



Chronic inflammation involves the release of number of mediators like cytokines

[interleukin-1β(IL-Iβ) and tumour necrosis factor (TNF-α)], granulocyte-macrophage

colony stimulating factor (GM-CSF), interferon and platelet derived growth factor

(PGDF). These mediators are responsible for the pain, destruction of bone and cartilage

that can lead to severe disability27.

Other miscellaneous information related to the pathology of arthritis that has been

obtained during this study includes haematological parameters and body weight changes.

In the PEE- and Eth aceE-treated groups there was restoration of the body weights of the

rats. A report suggests that the decrease in the body weight during inflammation is due to

deficient absorption of nutrients through the intestine and that treatment with anti-

inflammatory drugs normalizes the process of absorption. The evident restoration of the

body weight of rats in the PEE- and Eth aceE -treated groups may involve improvement

of intestinal absorption of the nutrients and a reduction in the distress caused by the

severity of the arthritis.

It has been reported that a moderate rise in the WBC count occurs in arthritic

conditions due to an IL-1B-mediated rise in the respective colony-stimulating factors.

The present study reveals that PEE- and Eth aceE treatments tend to normalize the WBC

count. In addition to this, other characteristic haematological alterations such as the

decreased Hb count and increased erythrocyte sedimentation rate(ESR) were also restored

by the PEE- and Eth aceE treatments. It is proposed that the reduction in the Hb count

during arthritis results from reduced erythropoietin levels, a decreased response of the

bone marrow erythropoietin and premature destruction of red blood cells. Similarly, an



increase in the ESR is attributed to the accelerated formation of endogenous proteins such

as fibrinogen and /β globulin, and such a rise in the ESR indicates an active but obscure

disease process. Thus, the reduction in the ESR and increase in the Hb count brought

about by PEE- and Eth aceE treatment further support its anti-arthritic effect.

It appears from our findings, the PEE(kernels and coat) and eth. aceE of Mesua

ferrea significantly reduced (P<0.01) the CFA induced paw edema on 21st day as

compared to standard drug (Diclofenac sodium), which may be due to inhibiting the

release of above mediators or inhibiting the response of inflammatory cells or protecting

erosion of joint cartilage and bone destruction in chronic arthritis model.

SUMMARY AND CONCLUSION CHAPTER-8

93

SUMMARY AND CONCLUSION

In the present study, the seeds of Mesua ferrea were dried in shade, powdered and

then extracted successively with pet. Ether, ethyl acetate and alcohol. The extracts were

subjected to preliminary phytochemical analysis. The preliminary phytochemical study

showed the presence of Coumarin, Saponins, Protein, Phenols, Tannins and Carbohydrates

in Mesua ferrea were partially confirmed by qualitative TLC, UV and FTIR analysis.

However, detailed study is necessary to elucidate the chemical structure of

phytoconstituents.

The pharmacological screening included evaluation of anti-arthritic activity of

petroleum ether, ethanol and ethyl acetate extracts of Mesua ferrea at the dose of

300mg/kg BW in rats with CFA-induced arthritis. In injected (left) paw Pet. ether Extract

exhibited 71.69% % inhibition and eth,aceE of Mesua ferrea exhibited 68.48% inhibition

against CFA induced paw edema on 21st day as compared to Diclofenac sodium (79.36%).

Where as Pet. ether Extract exhibited 76.58% inhibition and eth,aceE exhibited 66.11%

inhibition against formaldehyde induced paw edema on 10th day as compared to

Diclofenac sodium (80.65%).

In conclusion, the increased protection against CFA induced arthritis paw edema

exhibited by PEE and eth. aceE of Mesua ferrea concludes the prominent anti-arthritic

activity of the seeds. However, further research on detailed pharmacological screening,

isolation of active phytoconstituents possessing the therapeutic activity and clinical study

for evaluation of safety and efficacy of the drug needs to be assessed.

Dept. of Pharmacognosy and Phytochemistry, klecop, belgaum

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Annexure- 1

Animal ethical committee letter


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ANNEXURE –3: U.V. Absorption spectrum

U.V. absorption spectra of Compound


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Annexure-4

HPTLC spectra of Compound 1

3D View of all tracks at 366


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Annexure-5


Documents

Phytochemical investigation and arthritic activity seeds