Objective: The present study is aimed to evaluate the leaf extracts of Calotropis gigantea Linn. for acclaimed anti-arthritic activity using albino rats.
Anti-Arthritic Activity Of Leaves Of Calotropis Gigantea Linn.
Objective: The present study is aimed to evaluate the leaf extracts of Calotropis gigantea Linn. for acclaimed anti-arthritic activity using albino rats. Materials and Methods: The arthritic action of leaves of C.gigantea was studied in Freund's adjuvant induced arthritis in rats. Diclofenac sodium was used as a standard drug. Results: The study revealed that the petroleum ether (40—60) extract and aqueous extract of C.gigantea possessed significant anti-arthritic activity in all parameters of the study compared to control group. Conclusion: The more potent anti-arthritic activity of leaves of C.gigantea may be due to nature of steroids, triterpenoids and saponin glycosides. This claim demands further detailed phytochemical profile to identify the active constituent responsible for the anti-arthritic activity.
Keywords: Calotropis gigantea Linn.; Anti-arthritic activity; Freund's adjuvant; Diclofenac sodium.
Adjuvant induced arthritis is a chronic crippling, skeletomuscular disorder having nearest approximation to human rheumatoid arthritis for which no medicine is available effecting a permanent cure in the recent times. The modern drugs both steroidal and non-steroidal anti-inflammatory drugs are used for the amelioration of the symptoms of the disease, however they offer only temporary relief and also produce severe side effects. Therefore, increasing efforts are being directed towards the development of drugs with long acting anti-inflammatory effects with the minimum side effects1. In view of these, at present a search has been made from the traditional remedies to find a drug that will be effective and will offer a long time relief in arthritis.
C.gigantea belonging to family Asclepiadaceae is a shrub or small tree 8-10 feet high, bearing unscented, pale purple or white flowers with spreading corolla lobes. This species is common throughout India2. The dried whole plant is a good tonic, expectorant, depurative and anthelmintic. The leaves are useful in the treatment of paryalysis, arthralgia, swellings and intermittent fevers3. In traditional medicine, the leaves of Calotropis gigantea are used for rheumatism4,5.
2. MATERIALS AND METHODS
2.1. Plant Material
Fresh leaves were collected from the local areas of Belgaum in November 2004 and botanically identified by Mr.R.S.Goudar, R.L. Science Institute, Belgaum.
2.2. Preparation of Extracts:
The leaves were shade dried at room temperature and powdered until able to pass through sieve no.40. Powdered leaves were subjected to hot continuous extraction (soxhlet)6 with petroleum ether (40-60). After complete extraction, the solvent was distilled off and concentrated on a water bath to a dry residue. The marc was dried completely at 50C and again loaded in the extractor and further extracted successively with ethyl acetate and alcohol.
Finally, the marc was macerated with chloroform water to obtain the aqueous extract. Each extract is concentrated by distilling off the solvent and then evaporating to dryness on water bath. The different extracts were subjected to qualitative chemical investigation and were taken for pharmacological studies.
Female wistar albino rats weighing between 130-200 gm were selected between 130-200 gm were selected for the pharmacological study. The animals were kept in standard polypropylene cages. The bedding material of cages were changed everyday. The temperature of the experimental animals room was maintained at 223c and lighting was kept artificial. The study protocol was approved from Animal Ethical Committee of the Institution.
The rats were divided into seven groups each consisting 6 rats. The first group represented normal rats. Second group that was treated as arthritic control. These two groups were received saline orally. The third group received the standard drug Diclofenac sodium at a dose of 10mg/kg7. 1% Tween 80 was used as a vehicle to suspend the various extracts. The fourth, fifth, sixth and seventh groups received petroleum ether, ethyl acetate, alcohol and aqueous extracts at a dose of 300 mg/kg, 200mg/kg, 30mg/kg and 300mg/kg8 respectively by oral route.
2.5. Anti-arthritic Activity 1,9,10,11
Six groups, except normal group, were made arthritic by injecting 0.1 ml Freund's complete adjuvant (Sigma, Germany) into the subplantar region of left hind paw on day '0'. This adjuvant consists of dead mycobacterium tuberculosis bacteria suspended in heavy paraffin oil to give final concentration of 0.5 mg/ml.
Saline or extracts or Diclofenac sodium were administered orally once daily, from the initial day i.e. from the day of adjuvant injection (0 day), 30 minutes before adjuvant injection, and continued till 21st day.
The anti-arthritic effect of the extracts as well as Diclofenac sodium was evaluated by measuring paw volume of injected paw on 4th, 8th, 14th and 21st day of study by using Digital Plethysmometer (UGO Basile, Italy). The mean changes in injected paw volume with respect to initial paw volume were calculated on respective days and % inhibition of paw volume with respect to control group was calculated. The changes in body weight were recorded daily. On the day 22nd blood was withdrawn from the each animal through retroorbital vein puncture by anaesthetizing the animals with anaesthetic ether. The blood was collected into vials containing EDTA and the biochemical parameters like haemoglobin content, total WBC count, differential WBC count, ESR and RBC were analysed.
2.6. Statistical Analysis 12
The results were analysed by using one way analysis of variance (ANOVA) followed by Dunnet's 't' test to determine the statistical significance.
3. RESULTS AND DISCUSSION
In adjuvant induced arthritis model, rats develop a chronic swelling in multiple joints with influence of inflammatory cells, erosion of joint cartilage and bone destruction and remodeling. These inflammatory changes ultimately result in the complete destruction of joint integrity and function in the affected animals13. Chronic inflammation involves the release of number of mediators like cytokines (IL-I and TNF-), GM-CSF, interferons and PGDF. These mediators are responsible for the pain, destruction of bone and cartilage that can lead to severe disability14. It appears from our findings that the petroleum ether extract treated groups significantly reduced paw swelling may be due to inhibiting the release of above mediators or inhibiting the response of inflammatory cells or protecting the release of joint cartilage and bone destruction in chronic arthritic model.
WBC count seems to be raised in control group, but in petroleum ether extract, aqueous extract and Diclofenac sodium treated groups seems to lower (p<0.01). In arthritis condition there is a mild to moderate rise in WBC count due to release of IL-I inflammatory response. IL-I increases the production of both granulocyte and macrophages colony stimulating factor15. T-lymphocytes have been reported to play a central role in the pathogenesis of rheumatoid arthritis. These cells comprise the majority of the lymphoid cells found in the rheumatoid synovium. In arthritic condition there is a moderate elevation in lymphocyte count7. In all treated groups the lymphocyte count was suppressed significantly (p<0.01) as compared to control. The results indicated that the total average RBC count in the entire test materials treated group raised marginally as compared to control group. However, the changes in RBC count were found to be statistically non-significant (p>0.05), which may be due to iron deficiency or low serum iron with normal iron store15. ESR is an estimate of the suspension stability of RBC's in plasma, related to the number of size of red cells and to the relative concentration of plasma proteins especially fibrinogen and the and globulins. Increases are an indication of active but obscure disease processes. The acute phase proteins in ESR and C-reactive protein share the property of showing elevations in the concentration in response to stress or inflammation that occurs like infection, injury, surgery and tissue necrosis. So in arthritic condition, ESR is elevated. It appears from the study that decrease in ESR denotes the anti-arthritic activity15.
Changes in the body weight have also been used to assess the course of the disease and the response to the therapy of anti-inflammatory drugs. During the course of the experimental period, as the incidence and severity of the arthritis is increased, the changes in body weight of the routs also occur. The loss of body weight during arthritis condition was also supported by earlier observation on alterations in the metabolic activities of diseased rats. Earlier findings suggests the absorption of 14C-glucose and 14C-leucine in rats intestine was reduced in the case of inflamed rats. But on the treatment with anti-inflammatory drugs, the decrease in the absorption was nullified and this shows that the anti-inflammatory drugs correct the decreased/ deranged absorption capacity of intestine during inflammation. The significant increase in the body weight during treatment of Diclofenac sodium, petroleum ether extract and aqueous extract when compare to control was may be de to the restoration of absorption capacity of intestine1.
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