Anti-inflammatory activity of Erythrina variegata a member of Papilonaceace Family was tested as possible anti-arthritic effects of ethanol (EEEV) and aqueous (AEEV) extracts. Herein we show that both EEEV and AEEV extract have potent anti arthritic effect on Freund's Complete adjuvant (FCA) induced arthritis model. Both the extracts at two different concentrations (250, 500 mg/kg body weight, per oral, once a day for 21 days) marked reduced swelling of the paw and body weight during the secondary lesions although the effect of EEEV was significantly higher. Hematological parameters like Hemoglobin content, total WBC count, total RBC count and ESR showed that both the extracts prevented arthritis induced animal was increase in WBC count. Together for the first time our studies have established the anti-arthritic potential of Erythrina variegata leaves extracts.
Keywords; - Erythrina variegate, Adjuvant induced arthritis, Hematological parameters
Rheumatoid arthritis is a chronic proliferative disease affecting connective tissues with thickening of soft tissues of the joint, erosion of articular cartilages and extension of synovial tissue over articular cartilages. The joints become stiff, swellon and eroded. There will be disability and deformities. Its affects nearly 1% of the population (Thabrew, et al. 2001). The disease is common in women and obese peoples.
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Current drugs include NSAIDs, Glucocorticoids, Gold salts and immunosuppressant's and Drugs modifying anti-rheumatoid arthritis. The most common toxicities associated with these drugs include ulcers, gastrointestinal hemorrhage, osteoporosis, nephritis and dermatitis etc.
Though many drugs are available in the allopathic system, they provide only symptomatic relief and not a permanent cure for the underlying pathological process. Traditional medicinal uses various plants as drugs to treat different diseases are an integral part of the culture identity of rural India. However the enormous wealth of information about the medicinal values of the different medicinal herbs in India is on the decline. Non documentation and lack of knowledge about the valuable active ingredients are the primary cause. In an effective better define medicinal value of one such plant.
The paribhadra is an acrid with anthelminthic, carminative, diuretic, galactogogue, expectorant, febrifuge, laxative, emmenegogue and anti-bilious properties (The Wealth of India, 2003). The alkaloids showed weak spasmolytic and relaxant action on smooth muscles of guinea pig ileum and rat uterus and curarimimetic action on skeletal muscles by in vitro and in vivo tests. They also produced CNS depression in rats.
In siddha, the bark, leaves, flower and seeds are used for the treatment of stomatitis, dysentery, diabetes, sterility, vomiting, arthritis and eye diseases (Yohanarashiman, 2001; Madhava chetty, et al. 2008; The useful plants of India, 2000). The juices made from leaves are used in blood dysentry, earache, and toothache. Juice of bark is used in fever and juices made from roots are used to induce menstrual flow when periods are absent (Rahman, et al. 2008). Liver trouble (Selvam, 2008). In Ayurveda, the bark and leaves are used in worm infestations, ear diseases, leprosy, dyspnoea and alleviation of Kapha, vata, anorexia and cough (Kapoor, 2001).
Although the medicinal value of E. variegata is well documented only very few pharmacological activities have been scientifically evaluated for the plant. The higher dose of the extract showed a sharp fall in blood pressure and cardiac arrest. It also produced a muscle contraction of isolated guinea pig ileum. The fall in blood pressure and contraction of ileum were completely blocked by an anti-histaminic agent diphenhydramine. The aqueous extract did not show any CNS activity (Chatterjee, et al. 1981), but it is used in Leucorrhoea and Menorrhagia (Hemadri and Rao, 1983) and also has Anti-microbial activity (Bhale and Bokadia, 1979). However the anti-inflammatory effect of E. variegata has never been reported in Freund's complete adjuvant induced arthritis. Therefore, by considering the nutritive value of E. variegata as well as its various haematological activities, we investigated the effect of E. variegata on adjuvant-induced arthritis in rats, a well-established model for rheumatoid arthritis.
2. MATERIALS AND METHODS
Colony inbred strains of wistar rats of both sex weighing (180-200 g) and mice (20-25g) were used for the pharmacological studies. The animals were kept under standard conditions (day/night rhythm) 8.00 a.m to 8.00 p.m, 22±2°C room temperature, standard pelleted diet (Hindustan Lever, Bangalore) and water ad libitum. The animals were housed for one week in polypropylene cages prior to the experiments to acclimatize to laboratory conditions. Rats and mice were divided into groups. Each groups containing six animals and were kept in different cages. Animals were selected at random and both sexes were used. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC).
2.2. Plant collection and identification
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The fresh leaves of E. variegata, were collected from the forest in Courtallum, Tirunelvelli dist, during the month of July to August-2006. The plant was identified and authenticated by Dr. S. Jayaraman, Botanist, Plant anatomy Research Centre, West Tambaram, Chennai-600045. A voucher specimen was deposited in the Department of Pharmacology, C. L. Baid Metha College of pharmacy, Chennai, Tamil Nadu. (Voucher no-207/ 2006).
2.3. Preparation of extract
Leaves were collected, dried under shade, coarsely powdered and extracted with ethanol aqueous (EEEV) and (AEEV) using soxhlet extractor. Extracts were dried under reduced pressure using a rotary flash evaporator and stored between 0-4°C protected from sunlight. The percentage yield of ethanol and aqueous extract were found to be 19%w/w and 21%w/w. The extracts were used for the pharmacological studies by suspended in 4 %v/v Tween 80.
2.4. Chemicals required
Complete Freund's adjuvant was supplied by Sigma Chemicals Company.
3. ANIMAL STUDIES
3.1. Acute toxicity studies
Acute oral toxicity study was performed as per OECD-423 guidelines (acute toxic class method), (Ecobichon, 1997). Wistar rats (n=6) of either sex selected by random sampling technique were used for acute toxicity study. The animals were kept fasting for overnight providing only water, after which the extracts were administered orally at the dose level of 5 mg/kg body weight by gastric intubation and observed for 14 days. If mortality was observed in 2 out of 3 animals, then the dose administered was assigned as toxic dose. If mortality was observed in 1 animal, then the same dose was repeated again to confirm the toxic dose. If mortality was not observed, the procedure was repeated for further higher doses such as 50, 300, and 2000 mg/kg body weight.
The adjuvant induced arthritis assay procedure were carried out according to the method is (Newbould, 1963). Animals were divided into seven groups of six animals each and arthritic syndrome was induced by subcutaneous injection of 0.1 ml of Complete Freund's Adjuvant (10 mg of heat killed mycobacterium tuberculosis per ml of liquid paraffin) into the sub-plantar area of the left hind paw. Group I served as normal control rats receiving 5 ml/kg of 4% Tween 80 per oral as vehicle, Group II arthritic rats served as induced-untreated, Group III and Group IV (EEEV 250 and 500 mg/kg b.wt, p.o), Group V and Group VI (AEEV 250 and 500 mg/kg b.wt, p.o) and Group VII Standard drug (Indomethacin 20 mg/kg b.wt, p.o). The different dose levels of E. variegata were dosed suspending in 4% Tween 80 (5ml/kg b.wt, p.o) orally to animals from 14th day of adjuvant induction and were terminated on day 28. The mean changes in paw volume was measured weekly by dipping the left hind paw of all rats into mercury column up to the ankle joint and noticing the mercury displacement by using plethysmograph (Ugo Basile, Italy).
Body weight and paw volume changes were also recorded at every week throughout the duration of study. Animals were sacrificed on the day 29 by cervical decapitation; blood was collected to determine the following haematological parameters like Haemogloin, total WBC count, total RBC count and ESR (Ghai, 1995). The Liver, kidneys and spleen were dissected out, washed and transferred to an ice-cold saline solution. The organs were weighed.
3.2. STATISTICAL ANALYSIS;
The data represents mean ± SEM. results were analysed statistically by one way analysis of variance (ANOVA) followed by Dunnet s t- test. The difference was considered significant when p<0.05.
The mean changes in paw swelling were about 0.62 ± 0.01 in the complete Freund's adjuvant induced control group. E. variegata significantly (p<0.001) and (p<0.01) reduced the mean percentage change in paw swelling on 28th day evaluation and the percentage protection of EEEV was 38.7% and 51.6% in different dose levels at 250, 500mg/kg, b.wt, p.o. respectively. The percentage protection of AEEV was 35.9% and 48.4% in different dose levels at 250, 500mg/kg, b.wt, p.o, respectively. However, standard drug Indomethacin exhibited 56.5% (p<0.001) protection compared with the group-II animals. Results were shown Table 1.
In adjuvant-induced arthritis model, rats developed a chronic swelling in multiple joints with influence of inflammatory cells, erosion of joint cartilage, bone destruction and remodeling. These inflammatory changes ultimately result in the complete destruction of joint integrity and functions in the affected animal (Carl, 1963).
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AIA rats showed only a mild increase in body weight during the observation period against control rats. Compared with Group-II animal all the Groups of E.variegata treated rats showed a gradually increased the body weight in a dose-dependent manner after 14 days from the day of adjuvant injection. Further, there was no significant change observed in the liver and kidney weights of arthritic animals, rather a significant increase (p<0.01) in spleen weight was observed. Upon E. variegata administration, a significant decrease (p<0.01) in the spleen weight was observed in all treated animals when compared to adjuvant-induced arthritis rats. Results were shown Table 2 and 3.
The EEEV and AEEV extracts and standard drug have shown an increase in Haemoglobin content compared to control. The total WBC counts were remarkably increased in adjuvant-induced rats. However, the E. variegata leaves extracts and standard drug administered group significantly decreased (p<0.01) in total WBC and RBC count. ESR was drastically increased in arthritic control group, it has been remarkably counteracted by the standard and extracts, restoring it back to normal, which justify its significant role in arthritic conditions. Results were shown Table 4.
Results of the present study revealed the potential inhibition of arthritic effect of E. variegata at 400 mg/kg, b.wt, as evidenced by (a) a significant increase in body weight, (b) a significant reduction in paw volume, (c) a significant decrease in spleen weight, (d) a significant protection in haematological parameters in E. variegata treated AIA rats.
FCA is an inflammatory agent is frequently used for induction of rheumatoid arthritis was used as a model for this disease (Choi, et al. 2002; Giffen, et al. 2003). RA affects approximately 1% of the adult population worldwide (Gabriel, 2001). In the present study, rats were selected to induce arthritis because rats develop a chronic swelling in multiple joints with influence of inflammatory cells, erosion of joint cartilage and bone destruction. It has close similarities to human rheumatoid disease (Singh and Majumdar, 1996).
The measurement of paw swelling is apparently simple, sensitive and quick procedure for evaluating the degree of inflammation and the therapeutic effects of test drugs. Chronic inflammation involves the release of number of inflammatory mediators like cytokines, Interleukin-IB and Tumor Necrosis Factor-α (IL-IB and TNF-α), GM-CSF, interferon's and PGDF. These mediators are responsible for the pain, destruction of bone and articular cartilages that can lead to severe disability (Eric and Lawrence, 1996). However, the extracts and standard drug significantly suppressed the paw swelling.
Changes in body weight have also been used to assess the course of the disease and the response to therapy of anti-inflammatory drugs (Winder, et al. 1969). As the incidence and severity of arthritis increased, the changes in the body weights of the rats also occurred during the course of the experimental period. The loss of the body weight during arthritic condition was also supported by earlier observation on alterations in the metabolic activities of diseased rats (Walz, et al. 1971). During the treatment with anti-inflammatory drugs, the decrease in absorption was nullified (Somasundaran, et al. 1983b). It shows that the anti-inflammatory drugs decrease the absorption capacity of intestine during inflammated conditions. The increased body weight during treatment of extracts and standard drug may be due to the restoration of absorption capacity of intestine.
Further, the spleen weights were increased in adjuvant induced arthritis animals have been reported to be associated with an enlarged spleen (spleenomegaly) and can increase the risk of inflammation. The spleen weight was decreased significantly during the administration of extracts and standard drug.
In arthritis condition there is a mild to moderate rise in WBC count due to release of IL-IB inflammatory response. IL-IB increases the production of both granulocyte and macrophages colony stimulating factor (Eric and Lawrence, 1996; William, 1996). In the present study, the migration of leucocytes into the inflamed area is significantly suppressed by the extracts and standard drug as seen from the significant decrease in total WBC count.
Erythrocyte sedimentation rate is determined the suspension stability of RBC's in plasma. It is related to the number and size of the red cells and to the relative concentration of plasma proteins, especially fibrinogen, α and β globulins. Increase in the rate is 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 like injection, injury, and surgery and tissue necrosis. The ESR count, which drastically increased in arthritic control group, has been remarkably counteracted by the standard, extracts and brought back to normal thus justifying its significant role in arthritic conditions (William, 1996).
The reports showed as ethanol extract of E.variegata are potential suppression of rheumatoid arthritis when compared with other extract and it does not produce any toxicity in haematological parameters. The mechanism of the effects may depend on the formation of several inflammatory mediators.
In conclusion, the study demonstrates that ethanol and aqueous extracts of E. variegata have marked anti-rheumatoid arthritis activities. Based on the acute toxicity study, EEEV and AEEV could be considered as almost non-toxic to tested experimental animals and further studies are in progress for isolation and characterization of the active principles of the extract.