Evaluation of in vitro anti-inflammatory activity of the spadix of Colocasia affinis

Contact us: sciencevision@outlook.com The methanolic extract of C. affinis was evaluated for its in-vitro anti-inflammatory activity by bovine serum albumin denaturation method, egg albumin denaturation method and protease inhibition method at different concentrations. Diclofenac sodium was used as the reference drug. The extract exhibits anti-inflammatory activity in a concentration-dependent manner. In bovine serum albumin denaturation method, the extract at concentrations of 100, 200, 400, 600, 800, 1000 μg/ml showed 25.49, 28.43, 31.37, 37.25, 41.17, 48.03% inhibition respectively. In egg albumin denaturation method, the concentrations of 50, 100, 200, 300, 400, 500 μg/ml showed 5.3, 9.89, 28.12, 32.8, 43.2, 52.8% inhibition respectively. In protease inhibition method the extract at concentration of 100, 200, 300, 400, 500 μg/ml showed 8.87, 19.32, 28.56, 43.96, 59.92% inhibition respectively. Therefore, from the results it can be concluded that the methanolic extract of C. affinis possesses anti-inflammatory activity.

were confirmed to have antioxidant properties. 5 This study is carried out to screen the in vitro antiinflammatory activity of C. affinis.
Inflammation is a protective response of the body to tissue injury, microbial infection, allergy or chemical irritants. It is an important immune response which acts to remove harmful stimuli as well as initiates the healing of damaged tissue. The inflammatory response is characterized by redness, pain, swelling and heat. 6 Non-steroidal and steroidal drugs were used for the treatment of inflammatory diseases. The non-steroidal anti-inflammatory drugs (NSAIDs) are the main drugs used for reducing the untoward consequences of inflammation and inhibit early steps in the biosynthesis pathway of prostaglandins by inhibition of COX enzymes. 7 But NSAIDs cause unwanted effects like gastric injury and ulceration, renal injury and bronchospasm due to their non-selective inhibition of both isoforms of COX enzymes. 8 Steroidal drugs also possess multiple side effects and their used as anti-inflammatory drugs is becoming highly controversial. 9 Therefore,

Introduction
Plants have a long history of improving human health and for curing various diseases. Plants play a major role in traditional systems of medicine for thousands of years and continue providing new remedies to mankind. 1 Therefore, the research on plant is rising all over the world and there is a huge evidence showing massive potential of medicinal plants used in various traditional systems. 2 Colocasia affinis, belonging to the family Araceae, is a broad leave and perennial herb with slightly pink petiole. It grows in an altitude below 900 m. Flowers and fruits are mainly produced during June-August. It is found in Bangladesh, Tripura and Assam. In Mizoram, it is mainly found in new jhum land. In Mizo tradition, it is mainly consumed as dish after boiling with water. Traditionally, juice of the plant is used externally for snake bite. The leaf is also used for catching land leech from the body. 3 The methanolic rhizome extract was reported to possess remarkable analgesic activity. 4 The leaves and stolon of the plant

Collection of plant material
Spadix of C. affinis was collected from the local market of Aizawl, Mizoram. It was dried under shade to remove moisture. It was ground to coarse powder. The plant specimen was authenticated at the Botanical Survey of India, Shillong (AP/ RIPANS/04).

Extraction of plant material
The dried and powdered materials of spadix of C. affinis were defatted with petroleum ether (60-80 o C) and then extracted with chloroform and methanol in a soxhlet apparatus. The extraction was carried out exhaustively and the solvents were recovered by simple distillation. The concentrated extracts were kept in refrigerator at 4 o C for further use.

Phytochemical studies
The methanolic extract of C. affinis was screened for the presence of alkaloids, glycosides, fats and fixed oils, proteins, amino acids, saponins, tannins and flavonoids and aqueous extract for carbohydrates. 10

Evaluation of in vitro anti-inflammatory activity
The in vitro anti-inflammatory activity was studied using bovine serum albumin denaturation method, egg albumin denaturation method and protease inhibition method.
Test control solution (0.5 ml) consist of 0.45 ml of BSA (5% w/v aqueous solution) and 0.05 ml of distilled water. Product control solution (0.5 ml) consists of 0.45 ml of distilled water and 0.05 ml of test solution. Standard solution (0.5 ml) consist of 0.45ml of BSA (5% w/v aqueous solution) and 0.05 ml of diclofenac sodium (100, 200, 400, 600, 800, 1000 µg/ml). All the above solutions were adjusted to pH 6.3 using 1N Hydrochloric acid. The samples were incubated at 37°C for 20 minutes and the temperature was increased to keep the samples at 57°C for 3 minutes. After cooling, 2.5 ml of phosphate-buffered saline was added to the above solutions. The absorbance was measured using UVvisible spectrophotometer at 416 nm. The percentage inhibition of protein denaturation was calculated by the formula: 11 Percentage inhibition (%) = 100-Absorbance of control-Absorbance of sample x 100 Absorbance of control

Egg albumin denaturation method
The reaction mixture (5 ml) consist of 0.2 ml of egg albumin (from fresh hen's egg), 2.8 ml phosphate buffered saline (pH 6.4) and 2 ml of varying concentration of plant extracts. Similar volume of double distilled water served as control. Then the mixtures were incubated at 37±2ºC in an incubator for 15 minutes and then heated at 70ºC for 5 minutes. After cooling, their absorbance was measured at 660 nm by using vehicle as blank. Diclofenac was used as reference drug and treated similarly for determination of absorbance. The Percentage inhibition of protein denaturation was calculated as follows: 12 Percentage inhibition (%) = 100-Absorbance of control-Absorbance of sample x 100 Absorbance of control

Protease inhibition assay
The reaction mixture (2 ml) was containing 0.06 mg trypsin, 1 ml 20 mM Tris buffer (pH 7.4) and 1 ml test sample of different concentration (100-500 µg/ml). The mixture was incubated at 37 o C for 5 min and then 1 ml of 0.8% (w/v) casein was added. The mixture was incubated for an additional 20 min. 2 ml of 70% perchloric acid was added to arrest the reaction. Cloudy suspension was centrifuged and the absorbance of the supernatant was read at 210 nm against buffer as blank. The percentage inhibition of protease inhibitory activity was calculated: 13 Percentage inhibition (%) = 100-Absorbance of control-Absorbance of sample x 100 Absorbance of control

Phytochemical screening
The phytochemical screening of methanolic extract of C. affinis showed the presence of flavonoids and tannins and aqueous extract showed the presence of carbohydrates as shown in Table 1.

Discussion
The preliminary phytochemical screening showed the presence of flavonoids and tannins in methanolic extract and carbohydrates in aqueous extract. In the in vitro anti-inflammatory activity screening, it was observed that the methanolic extract of C. affinis showed significant activity. The activity was shown in concentration-dependent manner, i.e. with the increase in concentration the activity was also increased. Denaturation of protein is one of the causes of inflammation. The production of auto antigens in inflammation disease may be due to in vivo denaturation of proteins. The mechanism of denaturation possibly involves alteration in electrostatic, hydrogen, hydrophobic and disulphide bonding. 14 A number of anti-inflammatory drugs have shown the ability to inhibit thermally induced denaturation. 17 Inhibition of denaturation of protein by the extract of C. affinis is possibly a contributing factor for its anti-inflammatory activity. Neutrophils are the group of white blood cells known to be a rich source of proteinase enzyme which carries in their lysosomal granules or vesicles contain many serine proteinases. It was reported that leukocytes proteinase plays a major role in the development of tissue damage during inflammatory reactions and efficient level of protection was provided by proteinase inhibitors. 15 The methanolic extract exhibit anti-proteinase activity at various concentrations as shown in Table 4. The antiinflammatory activity of C. affinis may be due to the presence of flavonoids. Flavonoids are reported to inhibit prostaglandin synthase specifically endoperoxidase and able to produce antiinflammatory effects. 16 The therapeutic application of flavonoids on inflammation have been reported. 18,19

Conclusion
The result of the in-vitro studies showed that the extract possesses anti-inflammatory activity which may be due to the presence of flavonoids. However further studies involving the isolation and purification of the chemical constituents of the plant and the investigations in the biochemical pathways may result in the development of a potent antiinflammatory agent with a low toxicity.