EVALUATION OF ANTI ARTHRITIC ACTIVITY OF THE ROOT EXTRACT OF ACALYPHA INDICA LINN. USING IN VITRO TECHNIQUES

Acalypha indica L., belonging to family Euphorbiaceae, is an herb used in traditional medicine for the treatment of bronchitis, asthma, pneumonia, rheumatism, earache, syphilitic ulcers, bacterial and renal diseases. Acalypha indica crude root powder was extracted with solvents of increasing polarity viz. petroleum ether, chloroform and methanol successively by continuous hot percolation method. Acalypha indica methanol extract was evaluated using three different in vitro models to explore antiarthritic potential such as inhibition of protein denaturation, proteinase inhibitory action and anti-hyaluronidase activity. The concentrations of 10 to 200 µg/ml of Acalypha indica methanol extract were prepared using DMSO. Diclofenac was used as the positive control. All in vitro determinations were done in triplicate. A dose dependent increase in percentage inhibition was observed for all the three models. The inhibitory concentration (IC 50 ) was found to be 52 µg/ml for protein denaturation assay, 37 µg/ml in proteinase inhibitory action and 18 µg/ml for anti-hyaluronidase activity. Diclofenac offered protective activity at even much lower concentrations compared to Acalypha indica methanol extract producing IC 50 values of 40 and 13 µg/ml for protein denaturation and proteinase inhibitory assays. Acalypha indica exhibited a very good anti-arthritic activity in all the methods checked confirming its traditional use. Further in vivo studies are to be carried out to confirm their activity and explore the mechanism by which this plant acts in protecting from autoimmune disease and rationalize their use.

IJPP VOL 2 ISSUE 6 headache and applied as a local application in syphilitic ulcers. The leaf of this plant is used as an antiparasiticide and applied externally with common salt or quicklime or lime juice 4 . Acalypha indica is reported to possess anthelmintic 5 , antibacterial 6 cardioprotective 7 , antidiabetic 8 and antioxidant 9 activities. The main active phytoconstituents present in the root extract of Acalypha indica were alkaloids acalyphus, acalyphine, quinine, amides such as acalyphamide and sterols, stigmasterol and a flavonol kaempherol and cyanogenic glycoside 10 . Therefore the present study was undertaken to evaluate the anti-arthritic activity of the root extract of Acalypha indica using three different in vitro models.

Drugs and chemicals:
Bovine serum albumin, bovine hyaluronidase, casein and diclofenac were procured from Sigma Aldrich, USA, phosphate buffer, tris-hydrochloride buffer, acetate buffer, dimethyl sulfoxide and dimethyl amino benzaldehyde were purchased from SD Fine Chem. Limited, Mumbai and sodium sodium hyaluronate (Hyalgan) was obtained from Fidia Farmaceutici S.P.A Abano Terme (PD), Italy and imported by Lupin Ltd, India. All other reagents used were of analytical grade and were purchased from local drug suppliers.

Plant collection and authentication:
The root of Acalypha indica have been collected from Coimbatore district, Tamil Nadu, and dried under shade. The root was identified and certified by Dr. C. Kunhikannan, Scientist-D, Biodiversity division, Institute of forest genetics & tree breeding, Coimbatore. The voucher specimen is preserved in the herbarium file of our department.

Preparation of root powder for extraction:
The roots of Acalypha indica plant were separated, cleaned and well dried at room temperature to avoid the degradation of phytoconstituents. The dried root part of the crude drug was ground well for getting semi coarse powder.

Process of extraction:
About 1000 gram of Acalypha indica crude drug root powder was extracted separately with 1000 ml of solvents of increasing polarity viz. petroleum ether, chloroform and methanol successively by continuous hot percolation method using Soxhlet extraction apparatus at a constant temperature of 30-45°C. The crude powder was extracted with each solvent for three consecutive days. After extraction, the extracts were collected and dried under air at room temperature to get a well dried extracts. Then the dried extracts were weighed and the percentage yield of each solvent extract was calculated from the weighed powder of each plant. The extract was reconstituted with the respective solvents for further analysis.

Preparation of reagents
5% Bovine serum albumin (BSA): Dissolved 5 g of BSA in 100 ml of water. Phosphate buffer saline pH 6.3: Dissolved 8 g of sodium chloride (NaCl), 0.2 g of potassium chloride (KCl), 1.44 g of disodium hydrogen phosphate (Na 2 HPO 4 ), 0.24 g of potassium dihydrogen phosphate (KH 2 PO 4 ) in 800 ml distilled water. The pH was adjusted to 6.3 using 1N HCl and make up the volume to 1000 ml with distilled water. 25mM of tris-HCl buffer pH 7.4: Dissolved 3.94 g in 800 ml of deionized water and the pH was adjusted to 7.4 using 1M hydrochloric acid (HCl) and made up to 1000 ml with deionized water. 0.8% (w/v) casein: Dissolved 0.8 g in 100 ml of distilled water. Dimethyl amino benzaldehyde solution: Dissolved 4.0 g of para-dimethyl amino benzaldehyde in 350 ml of 100% acetic acid and 50 ml of 10 N hydrochloric acid)

Inhibition of protein denaturation:
The reaction mixture consisted of 0.5 ml of 5% aqueous solution of bovine serum albumin and 0.05 ml of various concentrations (25-100 µg/ml) of the methanol extract of Acalypha indica. The pH of the reaction mixture was adjusted to 6.3 using 1 N hydrochloric acid (HCl) and it was then incubated at 37°C for 20 minutes and then heated at 57°C for 3 minutes. The reaction mixture was allowed to cool and added 2.5 ml of phosphate buffer saline. Turbidity was measured at 340 nm 11 . In control 0.05 ml distilled water was used instead of test extract while product control lacked bovine serum albumin and diclofenac sodium was used as the standard. The percentage inhibition of protein denaturation was calculated. The control represents 100% protein denaturation. All determinations were done in triplicate. perchloric acid was added and the cloudy solution was centrifuged at 2500 rpm for 5 minutes. Diclofenac was used as the standard 12 . Absorbance of the supernatant was measured at 217 nm keeping buffer as blank. The percentage inhibition of proteinase was calculated. All determinations were done in triplicate.

Anti-hyaluronidase activity:
Hyaluronidase activity was determined spectrophotmetrically by measuring the amount of N-acetyl glucosamine formed from sodium hyaluronate. 50 µL of bovine hyaluronidase (7900 units/ml) dissolved in 0.1M acetate buffer (pH 3.5) was mixed with 100 µL of a designated concentration of sample (total MeOH extract) dissolved in 5% dimethyl sulfoxide and then incubated in a water bath at 37°C for 20 minutes. The control group was treated with 100 µL of 5% DMSO instead of the sample. To this 100 µL of 12.5mM CaCl 2 was added to the reaction mixture and then the mixture was incubated in a water bath at 37°C for 20 minutes. The Ca 2+ activated hyaluronidase was treated with 250 µL of sodium hyaluronate (1.2 mg/ml) dissolved in 0.1M acetate buffer (pH 3.5) and then incubated in a water bath at 37°C for 40 minutes. About 100 µL of 0.4N NaOH and 100 µL of 0.4N potassium bromate (K 3 BO 3 ) were added to the reaction mixture and then incubated in a boiling water bath for 3 minutes. After cooling to room temperature, 3 ml of dimethyl amino benzaldehyde solution was added to the reaction mixture which was again incubated in a water bath at 37°C for 20 minutes. Optical density was measured at 404 nm for the reaction mixture using a spectrophotometer. All determinations of the assay were done in triplicate for reproducibility 13 .

Results and Discussion
Inflammatory arthritis is a synovial disease characterized by chronic inflammation of the joints and can result in disability owing to joint destruction. In vitro anti-arthritic activity was performed using most popular methods such as inhibition of protein denaturation, proteinase inhibitory action and antihyaluronidase activity. Concentrations ranging from 10-200 µg/mL were tested to find out the percentage inhibition and 50% inhibitory concentrations. In inhibition of protein denaturation assay it was found that the methanol extract of Acalypha indica at concentration of 25 µg/ml it offered an percentage inhibition of 31.18 and in 50µg/ml it has increased further to 48% and likewise at 75µg/ml it was 70% and in 100µg/ml it could offer 80.36% activity. A dose dependent increase in the percentage inhibition was observed for all the concentrations tested with a dose dependent increase in anti-arthritic activity ( Table 1). The IC 50 values of the Acalypha indica methanol extract was found to be 52 µg/ml. In this method diclofenac was used as standard for comparing its anti-arhtritic activity and when compared to our extract it was found that diclofenac could offer anti-arthritc potential at much lower concentrations with an IC 50 value of 40 µg/ml. In case of arthritis autoantigens were produced due to protein denaturation. A decrease in the absorbance reflects the inhibition of the protein denaturation, which further reveals that the compound has the capability to control the production of autoantigens by inhibiting denaturation of proteins in rheumatic disease 14 . Proteinase inhibitory action was also done for the Acalypha indica methanol extract and the extract could produce 21% inhibitory action at 10 µg/ml and a dose dependant activity was produced with a greater inhibitory percentage of 79 at 100 µg/ml and with an effective IC 50 value of 37 µg/ml. However studies done with standard drug diclofenac exhibited remarkable activity in all the concentrations tested with a maximum percentage inhibition of 92.68 at 100 µg/ml dose with an IC 50 of 13 µg/ml. In assay conducted with the enzyme hyaluronidase present mainly in extracellular matrix a greater activity could be observed in all the tested concentrations. Methanol extract of Acalypha indica produced 29.3% inhibition at 12.5 µg/ml and this gradually increased to 83.81% at 100 µg/ml and to 83.93 at 200 µg/ml. However the % inhibition decreased when concentration was increased to above 200 µg/ml and dose dependent effect could be noted up to 200 µg/ml. The IC 50 score was found to be 18 µg/ml. Hyaluronidases are the family of enzymes that degrade hyaluronic acid. By catalyzing the hydrolysis of hyaluronan, a constituent of the extracellular matrix, hyaluronidase lowers the viscosity of hyaluronan, thereby increasing tissue permeability. In our experiment we have observed that the root extract of Acalypha indica have the potential activity to inhibit a key enzyme involved in RA, hyaluronidase, an enzyme that destroys the hyaluronic acid backbone of cartilage matrix. It is indeed IJPP VOL 2 ISSUE 6 found that the elevated serum level of hyaluronic acid is a reliable biomarker of arthritis progression 15 . Hence, it can be confirmed that our plant Acalypha indica root extract has the potential to inhibit the enzymes involved in disease progression of RA.

Conclusion
The plant extract obtained from Acalypha indica used commonly in traditional Indian system of medicine produced an excellent in vitro anti-arthritic activity when tested using standard methods. However, treatment with plant extracts although may be have some unpredictability in the effectiveness; being non-toxic, side effect less alternative, purified plant extracts and their isolated phytoconstituents can be very useful against rheumatoid arthritis. Further to corroborate the in vitro anti-arthritic activity, studies are to be carried out in vivo to confirm their activity and also the active principles has to be identified and isolated to explore the possible mechanism by which this plant acts in protecting from autoimmune disease, rheumatoid arthritis.