Final Report of the UGC Minor Research Project

Screening of phytochemical and biological properties of selected plants used for the

treatment of arthritis

Submitted to UNIVERSITY GRANTS COMMISION

Submitted by

PRINCIPAL INVESTIGATOR : Dr. FRANCIS MATHEW

CO-INVESTIGATOR : Mrs. CHRISTY K. JOSE, M. Pharm

DEPARTMENT OF BOTANY

St. THOMAS COLLEGE RANNY, PATHANAMTHITTA-689673, KERALA

Phone:(office) 0473-5226238

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Declaration

We hereby declare that this minor project entitled “Screening of

phytochemical and biological properties of selected plants used for the

treatment of arthritis ” is an authentic report of work carried out by us at

the Dept. of Botany, St. Thomas College, Ranni under UGC- Minor

Research Project (Plan) for the year 2013-2014.

Dr. FRANCIS MATHEW

PRINCIPAL INVESTIGATOR :

Mrs. CHRISTY K. JOSE, M. Pharm

CO-INVESTIGATOR :

Ranni

30.03.2017

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CONTENTS

Page No. Title of the Research project 4

Brief objective of the project 4

Summary of the work done 4

Details of the work done and results

obtained 5 - 22

Introduction 5 - 11

Review of literature 12-13

Materials and methods 14 - 17

Results, Discussion & Conclusion 18 -22

References 23-24

Achievements 25

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Title of the Research project “Screening of phytochemical and biological properties of selected plants used for the treatment of arthritis ”

Brief objective of the project

• Selection of plants with anti-arthritic activity based on their ethnomedical uses

• In-vitro anti-arthritic and antioxidant studies on the extracts of selected plants

• Isolation and characterization of compounds from extract which shows higher

activity

• In-vivo anti-arthritic and antioxidant studies on the isolated compounds

Summary of the work done

A list of medicinal plants commonly used for the treatment of arthritis was

prepared based on the survey. From this the list three plants namely Naravalia

zeylanica, Strobilanthus ciliates and Samadera indica were selected for the

phytochemical analysis and invivo screening of anti-arthritic studies. Plant specimens

were collected from different parts of Kerala State, identified and authenticated . The

collected plant parts were used for the studies. Phytochemical analysis was conducted

by qualitative studies and extraction by using standard solvents like petroleum ether,

Chloroform, Ethanol and water. These extractives were used for invivo screening for

arthritis. Anti-inflammatory and anti-arthritic screening was done by model studies.

The Chloroform and ethanolic extracts of these plants showed more or less the same

anti-inflammatory activity compared to the standard drug used. In the case of anti-

arthritic activity the ethanolic extract showed the better performance which is

significant. Among the three plants analysed, Naravalia zeylanica showed the highest

performance both in anti-inflammatory and anti-arthritic activity.

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CHAPTER 1

INTRODUCTION Arthritis

Arthritis is one of the oldest diseases. It is a systemic inflammatory disease, affecting

mainly joints. Arthritis is classified into rheumatoid arthritis and osteoarthritis. Gout is also a

type of inflammatory disease, caused by the pathogenic deposition of uric acid crystals in

joints and tissues.

Rheumatoid arthritis (RA) It is a chronic multisystem disease characterized by hyperactivity of certain immune

reactions, persistent synovitis with diffuse proliferation. The typical features of RA are

bilateral symmetric inflammatory polyarthritis involving small and large joints in both upper

and lower extremities with sparing of axial skeleton except the cervical spine. In most of the

cases, deposition of autoantibodies to immunoglobulins known as rheumatoid factor (RF). In

severe cases, the synovial inflammation leads to articular cartilage damage, bone erosion and

subsequent changes in joint integrity. Usually peripheral joints are involved. The prevalence

of RA is about 0.8 %; women are affected more often than men. The cause of RA is almost

unknown.

Osteoarthritis (AO) OA is a joint failure often initiated by joint injury. The pathological changes are

hyaline articular cartilage loss, increased thickness and hardening of the subchondrial bony

plate, outgrowth of osteophytes at the joint margin, stretching of the articular capsule and

mild synovitis in many affected joints and weakness of muscles bridging . OA is the most

common type of arthritis with high rate of disability and high prevalence in the elderly. It is

the most common cause of chronic knee pain in persons above 40 years of age. The

symptoms are pain and disability; bursitis occurs commonly around knee and hip. Likely

sources of pain include effusions, marrow edema and synovial inflammation. The loss of

function is a consequence of weakness across the joint and of laxity and instability. As

disease progresses the pain becomes continuous. Stiffness for a short time (less than 30 min in

the morning) of the affected joint may be prominent.

In osteoarthritis (OA) synovitis is mild and not accompanied by conspicuous

proliferation of cells. The hyper activity of certain immune reactions occurring in RA are

absent in AO and RF is not present. OA commonly affects the cervical and lumbosacral joint,

hip, knee and first metatarsal phalangeal joint; in the hands the distal and proximal inter

phalangeal joints and the base of the thumb are often affected. Usually spared are the wrist,

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elbow and ankle. OA is uncommon in adults under 60 years of age and more common in

women than in man. Age is the most potent risk factor for AO and the joint vulnerability

increases with age due to weakening of joint protective mechanisms against loading and

minor injuries. Joint vulnerability and loading are the two major factors contributing to the

development of AO.

Gout Gout is a metabolic disease typically characterized by episodic acute or chronic

arthritis or periarthritis which is caused by deposition of monosodium urate (MSU) crystals in

joints and connective tissue tophi. It is the result of an increased pool of urate with

hyperuricemia. Most often, it affects middle aged to elderly men and postmenopausal women.

Acute arthritis is the most frequent early clinical anifestation of gout. Usually only one joint is

affected initially, but poly articular acute gout can occur subsequently. The metatarso

phalangeal joint of the toe is often involved, but tarsal joints, ankles and knees are also

commonly affected.

Medicines used to control RA in conventional medicine The available therapies are not curative and are aimed at reducing the symptoms pain,

inflammation, damage to articular structure, functional impairment and systemic involvement.

Some of the therapies employed are directed at non-specific suppression of the inflammatory

or immunological process (Lipsky, 2008). The therapies can be classified into 5 groups.

(i) Non steroidal anti-inflammatory drugs (NSAIDs) and simple analgesics: NSAIDs

include ibuprofen, nabumetone, naproxen, salsalate, piroxican, ketorolak, ketoprofen, etc.

Although these drugs reduce inflammation and pain, they exert minimal effects on the

progression of the disease. Further, the major side effect of NSADs is gastro-duodenal

ulceration which may range from mild dyspepsia to ulceration.

(ii) Oral glucocorticoids: Low dose of oral glucocorticoids are used as additional second line

of therapy to suppress signs and symptoms of inflammation. Low dose may also retard the

progression of bone erosions.

.

(iii) Disease modifying antirheumatic drugs (DMARDs): These drugs show clinical

improvement in a majority of cases and decrease elevated levels of acute-phase reactants in

treated patients. Therefore, these are thought to modify the inflammatory component of RA.

These drugs include methotrexate, sulfasalazine, hydroxychloroquine, gold salts, D-

penicillamine, etc. Most of them exert minimal direct anti-inflammatory and analgesic effects

and therefore, NSAID may be continued with them. Toxicity of DMARDs includes

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gastrointestinal upset, oral ulceration, liver function abnormalities and drug induced

pneumonitis.

(iv) Biologics: These include TNF-α neutralizing agents such as infliximab, etanercept and

adalimumab, IL-1 neutralizing agents (anakinra), those that deplete B cells (rituximab) and

those that interfere with T cell activation. These agents improve signs and symptoms of RA

and decrease disability. The major side effects of these agents include potential for an

increase in the risk of serious infections such as tuberculosis; increase in the risk of

lymphoma and other malignancies.

(v) Immune suppressive and cytotoxic drugs: These include leflunomide, cyclosporine,

azathioprine and cyclophosphamide. These may ameliorate the disease process in some

patients and exerts the therapeutic effects somewhat similar to those of DMARDs. But they

cause a variety of toxic side effects. For example, leflunomide alters liver function enzymes

(Lipsky, 2008).

Gout Treatment: Acute attack is generally treated with anti-inflammatory drugs such as

NSAIDs , colchicines or glucocorticoids. Both NSAIDs and colchicines may show adverse

side effects; they may be dangerous in the elderly and in the presence of renal insufficiency

and gastrointestinal disorders. Intra articular glucocorticoid injections may be preferable and

effective in some cases. Adreno-carticotrophic hormone may be effective in patients with

acute poly articular refractory gout. Oral administration of colchicine is a traditional and

effective treatment, if used early in the attack. High doses of colchicines are extremely toxic

and lethal. Hypouricemic therapy should be considered, as in most patients, when

hyperuricemia cannot be corrected by control of body weight, low purine diet, increase in

liquid intake, limitation of ethanol use, etc. Urate lowering agents such as probenecid can be

used to patients with good renal function, but under excrete uric acid. Probenecid is not

generally effective in patients with high serum creatine levels (above 177 µmol/L).

Plant based ayurvedic treatment for arthritis Many plants are used in the Ayurvedic medicines used to treat arthritis and related

disease conditions. In an excellent review, most of the important plant based therapies used in

Ayurveda and the known scientific basis of their actions are given (Mishra, 2003). Arthritis

and its various manifestations are described in Ayurveda in light of ayurvedic thought and

philosophy. Ayurvedic treatment for any disease including arthritis differs from the treatment

in modern medicine.

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According to Ayurveda, over eating of foods that are too salty, sour, alkaline, fatty,

improperly cooked, meat of the animals or birds of marshy and desert regions which have

been soaked in water, excessive drinking of sugarcane juice, exposure to cold winds, sleeping

in the daytime and not in the night, travelling long distances at a stretch, etc. cause gout in

susceptible persons ( Ashtangahrudayam).

As per ayurveda, arthritis results by the accumulation of toxic waste materials in the

joints, so efforts are made to digest these materials and to reduce the accumulation of it. For

digestion of toxic materials various herbal combinations such as Hinguvachadichoornam,

Vaishwanarachoornam and Amrithotharam Kashayam are used. Then, external as well as

internal applications of herbs in oil are followed. Medicated oils like Dhanwantharam,

Mahanarayanam, Sahacharadi and Pindathailam are used for external massaging. Internal

medications with ghee and oil medium are also used. This is followed by cleansing the body

from accumulated toxic materials (Pancha karma). Numerous kashayams are used to treat

rheumatic diseases and osteoarthritis. However, these Kashayas are used in the treatment of

many other diseases also. Kashayams containing plant ingredients and used mainly for

arthritis

Plants with anti-arthritis or/and anti-inflammatory properties Numerous plants are used to treat arthritic conditions in traditional medicine which

include local health traditions and traditional systems of medicine such as Ayurveda and

Sidha. Based on the traditional information many of the plant extracts or/and active fractions

were tested in experimental animal models of arthritis and inflammation.

Mechanism of action of plant based antiarthritic drugs Some of the anti-inflammatory and/or antiarthritis plants were studied for their

mechanisms of actions. However, most of the studies were not carried out systematically and

fully with a therapeutic approach. Plant extracts and active fractions are likely to have more

than one mechanisms of action. The mechanisms emerged from the studies include

suppression of certain hyper immune reactions occurring in the case of RA, exerting anti-

inflammatory action by one or more mechanisms such as inhibition of phospholipase A2,

phospholipase D, Cyclooxygenases, lipoxgenases, etc. Another important mechanism is

immune modulation by influencing the levels of specific cytokines and lymphokines. Release

of arachidonic acid by the action of phospholipase A2 may result in production of excess

eicosanoids which in turn can result in inflammation. Some plant constituents are known to

activate phospholipase A2. Flavonoids from the bark of the Samoan anti-inflammatory plant,

Erythrina variegata L. (Legumenosae) inhibit Phospholipase A2. Zanhasaponin A and B

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(triterpene saponins) and cyclitol pinitol isolated from the root bark of Zanha africana

(Sapindaceae) are also inhibitors of phospholipase A2 (Shah Biren et al., 2006). Two new

triterpene saponins (anti-inflammatory phytochemicals) from the leaves of Myrsine australis

inhibited PMA (phorbal-12-myristate-13-acetate) as well as fMLP (Nformyl- methionyl-

leucyl-phenylalanine) stimulated phospholipase D activation (Shah Biren et al. 2006). The

anti-arthritic rhizome of Z. officinale (ginger) is an inhibitor of both prostaglandin and

leucotrine bio-synthesis (Srivastava and Mustafa, 1992). Anti-inflammatory biflavonoids

from the rhizome of Sacrophyte piriei inhibits prostaglandin synthesis and platelet activation

factor induced exocytosis. Anti-inflammatory coumarins isolated and purified from Santolina

oblongifolia (Compositae) inhibit eicosanoid release from ionophore stimulated macrophages

(Shah Biren et al. 2006). One of the important traditional antiarthritic medicinal plants is

Boswellia serrata. Boswellic acids (active principles) present in the oleogum of this plant

inhibit 5-lipoxygenase, a key enzyme involved in the synthesis of leukotrienes. This could be

at least one of the mechanisms of its anti-arthritic activity (Shah Biren et al. 2006). Further,

acetyl-boswellic acids inhibit lipopolysaccharide mediated TNF-α induction in monocytes by

direct interaction with IκB kinases (Syrovets et al., 2005). The oleoresin fraction of

Commiphora mukul possesses significant anti-arthritic activity

Methods of screening plant extracts and active fractions/compounds for anti-arthritic action

Rheumatoid arthritis

The commonly used important in vivo methods to test anti- RA agents (drug

candidates) are adjuvant-induced arthritis, collagen-induced arthritis, collagen anti-body-

induced arthritis, zymogen-induced arthritis, antigen-induced arthritis, streptococcal cell wall-

induced arthritis and spontaneous transgenic models of arthritis (Asquith et al. 2009; Bendele,

2001; Oliver and Brahn, 1996). Agents currently in clinical use or trials that are active in

these models include corticosteroids, methotrexate, NSAIDs, cyclosporin A, leflunomide,

interleukin-1 receptor antagonists and soluble TNF-α receptors (Mishtra 2003). Other

common methods include formaldehyde induced arthritis in rats and cotton pellet induced

granuloma. The other methods such as carrageenan-induced paw edema are widely used to

measure anti-inflammatory activity.

Adjuvant-induced arthritis in rats This method is widely used for preclinical testing of many antiarthritic agents

(Bendele, 2001). This model shows reliable onset and progression of the disease, easily

measureable, polyarticular inflammation, bone resorption, etc. However, in this model

cartilage destruction is mild in comparison to the inflammation and bone destruction. Male

Lewis rats (165-200 grams) are generally used in studies of adjuvant arthritis. In females, the

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disease is much more variable in onset and severity (Henson and Brunson, 1970). Induction

of adjuvant disease can be done with either Freunds complete (FCA) supplemented with

mycobacterium or by injection of the synthetic adjuvant N, N-dioctadecyl- N', N'-bis(2-

hydroxyethyl) propanediamine (LA) (Chang, 1980). Adjuvant can be injected at the base of

the tail or in one of the foot pads. If injection is into the footpad, it allows study of the acute

inflammatory reactions in that local area as well as the immunological reaction that develops

approximately 9 days later in the paw and various organs. Hind paw welling is monitored

from day 9 (onset of disease) to 15 or greater depending on duration desired (Bendele, 2001).

To assess disease progression, measurements of ankle joint width or volume using a

plethysmometer are done prior to the onset of arthritis, and then every other day until the

study is terminated on day 15 post injection of the adjuvant. Treatments are initiated on day 0

(prophylactic model) or day 8 (therapeutic model). At termination, the tibiotarsal joint is

transected and weighed. Paws are then collected into formalin for histopathological

evaluation for beneficial effects on arthritis parameters and also for evaluation of potential

deleterious effects of treatment on bone marrow (review, Bendele, 2001). Ankle joints (with

digits removed) are decalcified and transected in the longitudinal plane to give approximately

equal halves. Then, joints are processed for paraffin embedding, sectioned and stained with

hematoxylin and eosin for general evaluation and stained with toluidine blue for specific

evaluation of cartilage changes.

Collagen-induced arthritis in rats or mice Collagen-induced arthritis (CIA) shares many similarities with human RA and is a

useful model (Asquith et al., 2009). CIA was first described in rats; it is inducible in

susceptible strains of mice, following inoculation with type II heterologous collagen

in complete Freund’s adjuvant (Trenthan et al., 1999). DBA/1 mice are most widely used in

CIA model.

Development of polyherbal drugs and pure chemical entity combination drugs for arthritis

More than 110 plants are used in various polyherbal preparations to treat arthritis and

related diseases in Ayurveda. Many of these plant drugs are not scientifically evaluated for

their possible anti-inflammatory and/or antiarthritic activities. Further, many traditional

medicinal plants used to treat arthritis in local health traditions in remote villages and tribal

pockets remain to be studied. Although there are numerous studies on anti-inflammatory

and/or arthritis traditional medicinal plants and isolated pure anti-inflammatory chemical

entities, these studies do not suggest the use of any of them as such (extract or active fraction

or isolated pure compound) as a satisfactory medicine to treat RA or OA. Most of the studies

were not focused on possible drug development. Some of the traditionally used anti-arthritis

plants may be potential as anti- inflammatory agents.

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However, there are anti-arthritis medicinal plants such as Boswellia serrata,

Commiphora mukul, Withania somnifera, Curcuma longa, Tinosphora cordifolia, Zingiber

officinale and Ncytanthes arbortristis with not only anti-inflammatory activity but also with

other beneficial pharmacological properties. Other important plants include Aloe vera,

Camellia sinensis and Cyperus rotundus. These plant active fractions or principles are to be

subjected to detailed investigations including mechanism of action studies. Mecchanism

action studies are required, among other things, for their use in rational poly herbal

formulation development.

Significance of the study: Although a number of drugs (non-steroidal or steroidal anti-inflammatory

agents and immunosuppressants) being used in the treatment of rheumatoid arthritis,

there is still an urgent need for more effective drugs with lower side effects (Badger

and Lee, 1997). The plant kingdom is abundant in species that act as anti-

inflammatory agents to animal tissue. The steroidal anti-inflammatory drugs were

developed from plant material and are still largely synthesized from saponins such as

diosgenin from the Mexican Yam: Dioscorea floribunda (Tyler, 1981). Herbalists

around the world know many plants with inflammation inhibiting and anti-arthritic

properties. In a well known study, 163 species of plants and fungi were tested to

determine their anti-inflammatory activity. Of the species tested, 17 exhibited

between 30/39% inhibition of inflammation, 21 between 40/49%, 15 between

50/59%, 4 between 60/69%, and 2 gave greater than 70 % inhibition (Benoit., 1976).

In many examinations, the herb or its components are compared to a commonly used

anti-inflammatory drug such as aspirin or phenylbutazone. Results are compared to

see if the effect of the herb is statistically significant. Systematic studies considering

all the phytochemical and clinical aspects of plant products for the treatment of

arthritis are limited. In this context it is necessary to have a detailed investigation on

plants commonly used for arthritis and not yet screened for this specific activity.

(iii) Objectives: • Selection of plants with anti-arthritic activity based on their ethnomedical uses

• In-vitro anti-arthritic and antioxidant studies on the extracts of selected plants

• Isolation and characterization of compounds from extract which shows higher

activity

• In-vivo anti-arthritic and antioxidant studies on the isolated compounds

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CHAPTER 2

REVIEW OF LITERATURE

Herbal drugs constitute a major part in all the traditional systems of

medicine. Plants, above all other agents, have been used for medicine form time

immemorial because they have fitted the immediate need; they are easily accessible

and inexpensive. Herbal drugs can make a dent in international markets, which are

looking towards alternative medicine for the cure of ailments to which even modern

system has no answer. These ailments include metabolic or degenerative disorders

like arthritis, lifestyle induced problems of heart, diabetes, cancer, etc.

Phytochemistry deals with the analysis of plant chemicals called natural

products, and with changes occurring in such chemicals due to alterations in

environmental conditions.

Antioxidants are now known to play a major role in the resolution of

inflammatory conditions (Sakai et al., 1999) and several anti-inflammatory agents

from plant sources have been found to exhibit antioxidant properties

(Narendhirakannan et al., 2005; Vijayalakshmi et al., 1997). The antioxidant activities

of some of these plants have been ascribed to their phenolic constituents (Ozgova et

al., 2003).

One of the widely used models for studying, the anti-nflammatory/ anti-

rheumatic properties of compounds is Complete Freund’s Adjuvant (CFA) induced

arthritis in rats (Jiang JY, Xu Q). It is an experimental immunopathy that is thought to

share many features with human rheumatoid arthritis (Billingham MEJ, Davies EG).

Sakai et al. (1999) studied the role of antioxidants in the resolution of inflammatory

conditions. Several anti-inflammatory agents from plant sources have been found to

exhibit antioxidant properties (Narendhirakannan et al., 2005; Vijayalakshmi et al.,

1997). The antioxidant activities of some of these plants have been ascribed to their

phenolic constituents (Ozgova et al., 2003).

In a study with 163 species of plants and fungi to determine their anti-

inflammatory activity, 17 exhibited between 30/39% inhibition of inflammation, 21

13

between 40/49%, 15 between 50/59%, 4 between 60/69%, and 2 gave greater than 70

% inhibition (Benoit., 1976). Steroidal anti-inflammatory drugs were developed from

plant material and are still largely synthesized from saponins such as diosgenin from

the Mexican Yam: Dioscorea floribunda (Tyler., 1981). Systematic studies

considering all the phytochemical and clinical aspects of plant products for the

treatment of arthritis are limited.

Anti-inflammatory and anti-arthritic activity:

The secondary metabolites in plants contribute significantly towards the

biological activities such as anti-inflammatory anti-oxidant, anti-osteoartritic,

analgesic activities, anti-diabetic, anti-microbial and hepatoprotective (Brahma et al.,

2011; Brijyog, et al 2014) . Strobilanthes ciliatus of Acanthaceae family is a highly

potential medicinal plant in ayurveda in the treatment of inflammatory disorders

(Warrier et al. 1994; Thomas et al, 2000)The root and stem paste of Naravelia

zeylanica is used to treat rheumatism, itches, scabies, allergies, headache and back

pain (Arun Vijayan et al., 2007; Ramachandran et al., 2009). Ashoka Shenoy M et al

(2009) evaluated the effect of lyophilized aqueous extract of Naravelia zeylanica

leaves in various in vitro and in vivo inflammatory models. Sutharsingh R et

al.,(2011) evaluated the chloroform and ethanolic extracts of aerial parts of Naravelia

zeylanica (200mg/kg) for anti-inflammatory activity by Carrageenan induced paw

edema method in wister albino rats

.

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CHAPTER 3

MATERIALS & METHODS Selection of plants

A survey was conducted among registered ayurvedic medical practitioners,

local ayurvedic practitioners and the tribal populations in Pathanamthitta district of

Kerala state. The survey was made by personal interviews and a list of plants used for

arthritis was prepared according to the interviews. From this list the most commonly

used and less studied (based on literature survey) plants – Naravalia zeylanica DC,

Strobilanthus ciliates and Samadera indica were selected.

Plant Descriptions

Naravelia zeylanica (Linn.) DC (Figure 3.1) is a woody stout climbing perennial

shrub with tuberous roots and long tendrils belonging to the family ranunculaceae. It

is distributed throughout india mainly in warm regions It is a climbing shrub with

long tendrils, stem is serrate, leaves are pinnately compound opposite, entire margin,

trifoliate, terminal leaflet is modified into a three branched tendril. Leaflets are ovate-

lanceolate serrate. Inflorescence is panicules, flowers are yellow with fragrance ,

Samadera indica (Figure 3.2) - Synonym: Quassia indica Niepa Bark Tree belongs

to the family Simaroubaceae, is an evergreen tree or shrub up to 10 m tall. Leaves are

elliptic-oblong, somewhat pointed-rounded at base, pointed to tapering at tip, leathery,

hairless, shining, netveined. Leaf-stalks are 1-2 cm long, stout. Flowers are 20 or

more in umbel-like hairless or finely velvet-hairy clusters. Fruits are 1-4 together, flat,

smooth, glandular and netveined. Niepa Bark Tree is found in India, Myanmar and Sri

Lanka. Flowering: All year.

Strobilanthes ciliatus Wall. ex Nee(Figure 3.3) - Synonym - Nilgirianthus ciliatus

(Wall. ex Nees) Bremek.- The plant is a shrub growing in southern India. It has a

weak stem and in most cases a prostrate mode of growth habit is exhibited.

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Collection of Plant Material

Naravalia zeylanica DC was collected from Agastyamala,

Thiruvananthapuram, Kerala State, Strobilanthus ciliaturs from Nedumkandam,

Idukki district, Kerala, and Samadera indica from Ranni, Pathanamthitta district,

Kerala. The plant was identified and authenticated by Dr. Vinodkumar T.G., Dept. of

Botany, St. Thomas College, Ranni, Kerala. The identification was confirmed by

comparing the standard herbarium sheets of these specimen maintained in the college

Herbarium collection. A voucher specimen has been maintained in the college for

future reference. The aerial part of the plants were collected in the case of Naravalia,

roots of Strobilanthes and levaes of Samadera.. The extracted plants parts were

washed in running water and dried in the shade. .

Extraction of Phytochemicals

Coarse powder of the dried plant parts was prepared by grinding it in a mixer

grinder and fine mesh sieving. Plant extractives were made by cold maceration

method using successive solvents such as petroleum ether, chloroform and ethanol in

increasing polarity for 48 hours each. The extracts were concentrated by distillation

the solvent and dried under reduced pressure.

Preliminary Phytochemical Tests

Petroleum ether, chloroform, ethanol and aqueous extracts were subjected to

phytochemical chemical tests to identify the phyto-constituents using standard

qualitative tests (Kokate, 2005; Khandelwal , 2006; Gibbs, 1974; Harborne, 1984;

Trease and Evans, 1989) . From the phytochemical test, presence of triterpenoids,

carbohydrates, phenols, polyphenolases, proteins, steroids, tannins alkaloids,

flavonoids and saponins were observed in chloroform and ethanolic extracts.

Phytochemical tests : Phytochemical analysis for various chemical constituents was

carried out using standard methods, and detailed protocol is given below (Harborne,

1973;, Ramaan, 2006).

Test for alkaloids- (Wagner’s Test): Small aliquots of methanolic extract were

stirred separately with few drops of dilute HCl and filtered. The presence of alkaloids

was detected by treating the filtrate with Wagner’s reagent to confirm the presence of

alkaloids in the sample with reddish-brown precipitate.

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Test for saponins (Foam test) : 2 ml of filtrate is diluted with 5 ml distilled water.

The suspension is shaken in a graduated cylinder for 15 min. A two cm layer of foam

indicates the presence of saponins.

Test for terpenoids (Salkowski test) : 1 ml of extract, to this 2 ml of chloroform is

added, and the content is shaken well. Now, an equal volume of concentrated

sulphuric acid is added to the test tube. Yellow to brick red color indicates the

presence of terpenoids.

Test for flavonoids (Alkaline reagent test) : The extract is treated with 10%

ammonium hydroxide solution. Yellow fluorescence indicates the presence of

flavonoids.

Test for phenolic compounds (Ferric chloride test) : 2 ml extract, to this few drops

of neutral 5% ferric chloride solution is added. A dark green color indicates the

presence of phenolic compounds.

Invivo studies The animal studies were carried at the Pushpagiri College of Pharmacy,

Thiruvalla, Kerala, after getting approval from the institutional animal ethical

committee. The animals were collected from Govt. Veternery college, Mannuthy,

Thrichur, Kerala.

Acute toxicity study An acute toxicity study was carried out by up and down method. Drugs were

administered orally to overnight fasted animals. The rats were observed continuously

for 2h for behavioral, neurological and autonomic profiles and after 24h and 72h for

any lethality. None of the animals died even at a dose of 3000mg/kg b.w. of each

extract. Hence one tenth (1/10th of LD50) cut off dose (i.e. 300mg/kg) was selected

for the subsequent study.

Anti inflammatory studies Anti inflammatory studies was carried out by Carrageenan induced oedema

method in rats (Winter et,al 1962). Healthy young adult winstar albino rats,

weighing about 150- 170gms of either sex were divided into four groups each of five

animals. The four groups were treated with ethanol and chloroform extracts

(200mg/kg), indomethacin (10mg/kg) and control vehicle orally. After 30 minutes,

the rats were challenged with subcutaneous injection of 0.1ml of 1%w/v solution of

17

carrageenan into the sub planar region of left paw. The paw volume was measured

using mercury plethysmometer. at 0,1,2,3 and 4 hr after carrageenan injection The

difference between initial and subsequent reading gave the actual edema volume.

Anti arthritic activity screening

Experimental design Male Wistar rats weighing between 150-200gm were selected for the

experiment. They were grouped in a group of six animals each in to five group. The

treatment schedules of rats belonging to the different groups are shown below

Group 1: Normal (Normal saline )

Group 2: Control (Complete Freund.s adjuvant 0.1ml)

Group 3: Indomethacin (10mg/kg p.o)

Group 4: Ethanol extract (300mg/kg p.o)

Group 5: Aqueous extract (300mg/kg p.o)

On the 0th day, the basal paw volume of left hind paw of each animal was

measured using mercury plethysmometer. On the 1st day all the animals except

normal group were once anaesthetized, they were injected in to the ankle joint of left

hind paw with 0.1 ml of Complete Freund.s adjuvant (Sigma Aldrich, USA)

containing 0.1 mg of heat killed Mycobacterium

tuberculosis cells in liquid paraffin and were allowed to recover to serve as control.

Dosing with standard drug Indomethacin and extracts was started on the same day i.e.

1st day and continued for 21st day. Normal and arthritic control groups rats receives

normal saline through out study while the rest experimental groups animals receives

respective treatment once daily by

oral route. The gum acacia 2%w/w was used as vehicle for suspended the extracts.

Paw volume of injected paw was measured on 4th, 8th, 14th and 21st day of study

period.

The body weight of the animals was measured by digital balance to access the

course of the disease at the initial day before induction and at the end of 21st day.

Statistical Analysis

The experimental results were expressed as mean ± S.E.M. Data were assessed

by the method of analysis of Oneway ANOVA followed by Dunnett’s test. P value,

p<0.01 & p<0.05was considered as statistically significant.

18

CHAPTER 4

RESULTS, DISCUSSION & CONCLUSION

Selection of plants The survey on medicinal plants used for the treatment of arthritis in local area

reveals that there are more than 100 plants being used as single or in combination

with other plants. The most commonly using c plants are listed in table 4.1. Based on

this list three plants namely Naravelia zeylanica, Strobilanthes ciliates and Samadera

indica were selected for the present study Table 4.1. Plants commonly used for the treatment of arthritis in in Kerala Sl # Botanical Name Family Plant parts used 1 Madhuca longifolia L. Sapotaceae Seed 2 Rubia cordifolia Rubiaceae Roots 3 Semecarpu sanacardium Linn Anacardiaceae Nut 4 Vitex Negundo Linn . Lamiaceae Leaves 5 Ficus bengalensis Linn Moraceae Bark 6 Sida rhombifolia Linn. Malvaceae Stems and roots 7 Naravelia zeylanica Linn. Acanthaceae Leaves & Roots 8 Strobilanthes ciliates Acanthaceae Leaves & roots 9 Vitex negundo Linn. Verbenaceae Leaves 10 Samadera indica Simarubae Leaves

Extraction of Phytochemicals

The coarse powder of plant parts of Naravalia zeylanica, Strobilanthes ciliates and Samadera indica extracted with various solvents successively by cold maceration method and the percentage yield were presented in table 4.2 . Table 4.2 Yield (w/w) of extractives received by using different solvents Sl Plant Petroleum

ether Chloroform Ethanol Aqueous

1 Naravalia zeylanica 6.5 % 10.8 % 12.7% 19.4 % 2 Strobilanthes ciliates 8.4 % 12.2 % 13.1% 24.6% 3 Samadera indica 6.1% 10.5% 11.3% 18.6% Preliminary Phytochemical Tests

The results of preliminary phytochemical test conducted in Naravalia zeylanica, Strobilanthes ciliates and Samadera indica were presented in table 4.3

19

Table 4.3 Results of phytochemical tests Sl Plant Alk Triterp Sap Phen Flav 1 Naravalia

zeylanica +

(Chl & Ethan)

+

( Chl & Ethan)

+

(Ethan & Aqu)

+

(Ethan & Aqu)

+

( PetE, Chl & Ethan)

2 Strobilanthes ciliates

+

(Chl & Ethan)

+

(Chl & Ethan)

+

(Ethan & Aqu)

+

( PetE, Chl & Ethan)

+

( PetE, Chl & Ethan)

3 Samadera indica

+

( PetE, Chl & Ethan)

+

(Chl & Ethan)

- +

( PetE, Chl & Ethan)

+

( PetE, Chl & Ethan)

Acute toxicity studies

In Acute oral toxicity screening after administration of single oral dose of

2000 mg/kg, of three plant extractives, there was no death and other complications

reported with in the 14 days.

Anti inflammatory studies

The results of the anti-inflammatory action of phytochmicals from the three

plants is given in table 4.4. The chloroform extract of aerial part of Naravalia

zeylanica DC possessed significant anti-inflammatory activity that may be due to its

ability to prevent the production of some pro-inflammatory mediators.

Table 4.4: Anti-inflammatory action of chlrofom and ethanolic extract of three plants in carrageenan induced rat paw edema (percentage of reduction of edema after 4 hrs) Sl Plant Indomethacin

(standard)

Chloroform Ethanol control

1 Naravalia zeylanica (22.04 ± 3.03%)

19.60 ± 2.73%

23.43 ± 2.45%)

40.34 ± 2.34%)

2 Strobilanthes ciliates (22.04 ± 3.03%)

21.43 ± 2.64%

23.57 ± 2.33%)

40.34 ± 2.34%)

3 Samadera indica (22.04 ± 3.03%)

20.60 ± 2.73%

25.43 ± 2.37%)

40.34 ± 2.34%)

Values are expressed as Mean percentage reduction of edema ±S.E.M. p*<0.05, p**<0.01 considered significant (n=6) when compared to control. The results were statistically significant (p<0.01).

20

Anti arthritic activity screening

From the Freud's adjuvant induced arthritis models, the percentage increase in

paw volume 7days and 21 days after the drug administration were noted. Mean

changes in paw volume in the experimental animals were presented in Table 4.5

(Naravelia zeylanica), Table 4.6 (Strobilanthes ciliates) and Table 4.7 (Samadera

indica). In the anti arthritic activity screening both chloroform and ethanolic extract

showed significant activity when compared with the standard and control (Figure

1&2). The cordial signs of the chronic inflammatory reactions like redness, swelling,

arthalgia and immobility of affected joints were significantly less in the drug treated

animal than those of the control. The activity may due to the presence of triterpenoids

and poly phenolic constituents

From the results observed in the current investigation, it may be concluded

that the ethanolic extracts of the three plants displays a significant anti-arthritic

activity compared to the standard drug. This h may due to the presence of

phytoconstituents such as alkaloids, steroids, and glycosides. Several studies indicate

that these phytoconstituents possess significant anti-arthritic activity (Rajendran and

Krishnakumar 2010)

Table 4.5: Mean changes in paw volume at different intervals in Adjuvant-induced arthritis in rats (Plant extract used – Naravelia zeylanica). Treatment groups

Mean changes in paw volume ±SEM4th day 8th day 14th day 21st day

Control 4.657±0.0233 4.783±0.0600 4.850±0.0562 4.990±0.1100

Standard

Indomethacin

(10 mg/kg)

3.822±0.0307* 3.643±0.0223** 1.853±0.0307** 1.155±0.1444**

Ethanol extract

300 mg/kg)

3.844±0.0130* 3.142±0.0157** 2.356±0.0146** 1.253±0.0521**

Aqueous extract

(300 mg/kg)

4.1273±0.0327 . 3.754±0.0210* 2.652±0.0223** 1.685±0.0354**

n=6, values are expressed as mean ± SEM, .Non significant (P>0.05), *Significant(P<0.05), **More significant(P<0.01) ,when compared to control

21

Table 4.6: Mean changes in paw volume at different intervals in Adjuvant-induced arthritis in rats (Plant extract used – Strobilanthes ciliates). Treatment groups

Mean changes in paw volume ±SEM4th day 8th day 14th day 21st day

Control 4.742±0.0233 4.855±0.0600 4.897±0.0562 4.952±0.144

Standard

Indomethacin

(10 mg/kg)

3.817±0.0307* 3.425±0.0223** 2.983±0.0307** 1.2480±0.1400**

Ethanol extract

300 mg/kg)

3.756±0.0130* 3.145±0.0157** 2.178±0.0146** 1.315±0.0500**

Aqueous extract

(300 mg/kg)

4.258±0.0333 . 3.441±0.0210* 2.254±0.0223** 1.425±0.0365**

n=6, values are expressed as mean ± SEM, .Non significant (P>0.05), *Significant(P<0.05), **More significant(P<0.01) ,when compared to control Table 4.7: Mean changes in paw volume at different intervals in Adjuvant-induced arthritis in rats(Plant extract used – Samadera indica). Treatment groups

Mean changes in paw volume ±SEM4th day 8th day 14th day 21st day

Control 4.744±0.0233 4.790±0.0600 4.825±0.0562 4.915±0.1100

Standard

Indomethacin

(10 mg/kg)

3.645±0.0307* 3.548±0.0223** 2.983±0.0307** 1.159±0.1400**

Ethanol extract

300 mg/kg)

3.849±0.0130* 3.245±0.0157** 2.373±0.0146** 1.381±0.0500**

Aqueous extract

(300 mg/kg)

4.276±0.0333 . 3.482±0.0210* 2.450±0.0223** 1.548±0.0365**

n=6, values are expressed as mean ± SEM, .Non significant (P>0.05), *Significant(P<0.05), **More significant(P<0.01) ,when compared to control Table 4.8: Percentage inhibition of paw volume in Adjuvant-induced arthritis in rats. Plant extract used – Naravelia zeylanica). Treatment groups

% inhibition of paw volume4th day 8th day 14th day 21st day

Control -- -- -- 0 Standard 13.87 32.33 57.38 78.89

Ethanol extract 10.54 30.61 49.23 74.88 Aqueous extract 8.43 27.51 46.35 70.82

22

Table 4.8: Percentage inhibition of paw volume in Adjuvant-induced arthritis in rats. Plant extract used – Strobilanthes ciliates). Treatment groups

% inhibition of paw volume4th day 8th day 14th day 21st day

Control -- -- -- 0 Standard 13.24 31.47 59.41 79.16 Ethanol extract 11.23 32.69 51.24 76.81 Aqueous extract 10.28 24.11 44.59 71.41 Table 4.8: Percentage inhibition of paw volume in Adjuvant-induced arthritis in rats. Plant extract used – Samadera indica). Treatment groups

% inhibition of paw volume4th day 8th day 14th day 21st day

Control -- -- -- 0 Standard 13.75 30.39 59.38 80.16 Ethanol extract 11.57 32.81 53.27 76.43

Aqueous extract 9.48 29.51 44.18 68.42 Conclusion

Phytochemicals offers good source of drugs for the treatment of many

diseases in man and animals. The secondary metabolites in plants has the potentiality

for the use as drugs. Many of the systems of treatment like Ayurveda, Sidha, Unani,

and even Homeopathic use plant derived products for treatment. There are more than

100 plants at present used as anti inflammatory and anti arthritic agents. These plants

may used as single or in combination with other plants. The present study anlyses the

potentialities of three plants Naravalia zeylanica, Strobilanthes ciliates and

Samadera indica to be used in the treatment of arthritis. This study reveals that the

ethanolic extract of these plants showed more or less the same activity compared to

the standard drug used. Of the three plants studies, Naravalia zeylanica showed better

performance than the other. As a future step, for the effective utilization of these

plants, further investigation is required by isolating the active components from these

plants and thorough screening.

23

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25

Results Achieved a) 5 UG Botany students were trained for the extraction of phytochemicals from

plant parts and three B.Pharm students were trained to conduct in vivo studies using animal models.

b) One paper presented in an International Seminar.

Francis Mathew & Christy J Jose (2016) Anti-inflamatory and anti-arthritic activities of leaves of Naravalia zeylanica L(DC). Proceedings of the International Seminar on Phytochemistry and Pharmacogonocy. Pushapagiri College of Pharmacy, Thiruvalla, Kerala, Jan 11 & 12, 2016. P. 41-44

c) One research paper is communicated to International Journal.

Francis Mathew & Christy J Jose . Anti-inflammatory and anti-arthritic

activities of selected for arthritis in traditional medicine. International

Journal of Research in Pharmacy & Chemistry (communicated)