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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
2
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
3
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
4
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.
5
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,
6
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
7
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.
8
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
9
(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
10
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.
11
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
12
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
.
14
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.
15
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.
16
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)