INTRODUCTION:

Inflammation is a normal protective response to tissue injury and it involves a complex array of enzyme activation, mediater release, fluid extravasations, cell migration, tissue breakdown and repair¹. It is a complex process, which is frequently associated with pain and involves occurrences such as: the increase in vascular permeability, increase of protein denaturation and membrane alterations². Harmful stimuli including pathogens, irritants or damaged cells initiate response of vascular tissue as inflammation. Inflammation is a protective attempt by the organism to remove injurious stimuli as well as initiate the healing process for the tissue³. However, if inflammation is not treated it leads to onset of diseases like vasomotor rhinorrhoea, rheumatoid arthritis and atherosclerosis⁴. It is believed that current drugs available such as opoids and non-steroidal anti-inflammatory drugs (NSAIDS) are not useful in all cases of inflammatory disorders, because of their side effects and potency⁵. Now a days much interest has arisen in the search of medicinal plants with anti-inflammatory activity which may lead to the discovery of new therapeutic agents without too much side effects.

Drynaria quercifolia (Asvakatri) belongs to Family of Polypodiaceae is found throughout India, especially in the plains or very low down in the mountains, on trees or rocks^6,7.

The whole plant of Drynaria quercifolia is anthelmintic, pectoral, expectorant and tonic, and is used to treat skin diseases and loss of appetide⁸. The rhizomes of the plant have antibacterial properties and are used traditionally for the treatment of cough, tuberculosis and typhoid fever^9,10. It is also used to treat chronic jaundice. The rhizome is also reported to have antifertility¹¹, anti inflammatory¹² and analgesic¹³, antipyretic¹⁴, antibacterial¹⁵ and antiulcer¹⁶ properties. In the present study, in vitro anti-inflammatory activity of methanolic rhizome extract of Drynaria quercifolia was analysed by HRBC membrane stabilization method.

MATERIALS AND METHODS:

Collection and Processing of Plant material:

The rhizome of Drynaria quercifolia Linn were collected from Kollimalai, Namakkal District, Tamil Nadu, India. The collected samples were carefully kept in polythene bags. These plant samples were authenticated by Dr. S. Johnbritto, The Director, The Rabinet Herbarium Centre for Molecular Systematic, St. Joseph’s College, Tiruchirappalli and a voucher specimen (Voucher No:001) was deposited in the Department of Biochemistry, S.T.E.T Women’s College, Mannargudi, Thiruvarur, Tamil Nadu. The rhizome is covered with small brown coloured hair like structures. They were removed using sterile scalpel and washed with sterile distilled water. They were cut into small pieces and dried in shade and made into fine powder, using blender, and stored in air tight containers until further studies.

Extraction of Plant Material:

20g of powder of rhizome of Drynaria quercifolia was weighed and macerated in methanol in the ratio of 1:6. They were kept at room temperature for 72 h. The mixture was stirred every 24 h using a sterile glass rod. Then it was filtered through the Whatman No:1 filter paper. Extraction procedure was done further twice for complete extraction of bioactive compounds. The obtained filtrate was combined together and concentrated in vacuum using rotary evaporated. The dried residue was used for this study.

HRBC Membrane stabilization Method:

The HRBC membrane stabilization has been used as a method to study the anti-inflammatory activity¹⁷. Blood was collected from healthy volunteers who had not taken any NSAIDs for 2 weeks prior to the experiment. The collected blood was mixed with equal volume of sterilized Alsever solution (2% dextrose, 0.8% sodium citrate, 0.05% citric acid and 0.42% sodium chloride in water). The blood was centrifuged at 3000 rpm and packed cells washed with isosaline (0.85%, Ph 7.2) and a 10% v/v suspension was made with isosaline. The assay mixer contains the drug (at various concentrations), 1 ml phosphate buffer (0.155 m, Ph 7.4), 2 ml of hyposaline (0.36%) and 0.5 ml of HRBC suspension. Ibuprofen was used as the reference drug. Instead of hyposaline 2ml of distilled water was used in the control. All the assay mixtures were incubated at 37ºc for 30 min and centrifuged. The haemoglobin content in the supernatant solution was estimated using spectrophotometer at 560nm. The percentage haemolysis was calculated by assuming the haemolysis produced in the presence of distilled water as 100%. The percentage of HRBC membrane stabilization or protection was calculated by using the formula,

100 – Optical density of drug treated sample

% Protection = --------------------------------------------------------× 100

Optical density of control

RESULTS AND DISCUSSION:

Methanolic extract of Drynaria quercifolia rhizome was studied for in-vitro anti-inflammatory activity by HRBC membrane stabilization method. Various concentration of methanolic extracts of Drynaria quercifolia (100, 200, and 300mcg/ml) were used for this experiment and the results were represented in Table 1 and Fig 1.

Table 1: Effect of Drynaria quercifolia rhizome on HRBC membrane stabilization

Concentration(µg/ml)	Methanolic extract of Drynaria quercifoila	Standard Diclofenac
100	33.64±4.34	35.52±0.57
200	34.14±4.24	39.09±2.00
300	34.96±5.01	41.62±1.27

Values are expressed as mean± SEM. n=3 in each groups

Among the three concentrations, maximum protection was observed at 300mcg of plant extract which showed 34.94% protection of HRBC in hypotonic solution followed by 200mcg (34.14%) and 100mcg (33.64%). Standard drug diclofenac were also performed and their protection percentage were 35.52%, 39.09% and 41.62% for 100, 200 and 300mcg respectively.

Fig 1: Effect of Drynaria quercifolia rhizome on HRBC membrane stabilization

The results revealed the antiinflammatory activity of Drynaria quercifolia increased markedly with the increase in the concentration of extract that is dose dependant response were observed. The main action of anti-inflammatory agents is the inhibition of cyclooxygenase enzyme which is responsible for conversion of arachidonic acid to prostaglandins (PG). The extract exhibited membrane stabilization effects by inhibiting hypotonicity induced lysis of erythrocyte membrane¹⁸. The erythrocyte membrane is analogues to the lysosomal membrane and its stabilization implies that the extract may well stabilize lysosomal membranes. Stabilization of lysosomal membrane is important in limiting the inflammatory responses by preventing the release of lysosomal constituents of activated neutrophil such as bactercidal enzymes and proteases, which further tissue inflammation and damage up on extra cellular release¹⁹. Some of the NSAIDs are known to exhibit membrane stabilization due to osmotic loss of intracellular electrolyte and fluid components²⁰. In the present study, the methanolic plant extract may inhibit the processes, which may stimulate or enhance the efflux of these intracellular components, thereby it is used as a anti-inflammatory agent. The activity may be due to one or more phytochemical constituents present in it.

ACKNOWLEDGEMENT:

Authors are thankful to our Managing Trustee, Correspondent, and Principal of S.T.E.T. Women’s College, Mannargudi, Thiruvarur, Tamil Nadu for their moral support, encouragement and guidance during research work.

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Received on 28.07.2014 Modified on 20.08.2014

Res. J. Pharmacognosy & Phytochem. 7(1): Jan.-Mar. 2015; Page 06-08

DOI: 10.5958/0975-4385.2015.00002.3