Collection and proper identification of plant Macaranga denticulata was collected from Chittagong Hill Tracts area, Chittagong. The sample was identified by Dr. Shaikh Bokhtear Uddin, Associate Professor, Department of Botany, University of Chittagong.
Preparation of extract Plant extract was dried and ground (Moulinex Blender AK241, Moulinex, France) into powder (40 to 80 mesh, 500 g) and soaked for 7 days with 2 to 3 days interval both in 2.0 L of methanol and ethanol at room temperature (23 ± 0.5 °C). Filtrate obtained through cheese cloth and Whatman filter paper No. 1 was concentrated under reduced pressure at a temperature below 50 °C using a rotary evaporator (RE 200, Sterling, UK). The extracts (yield 4.4 to 5.6% W/W) were all placed in glass petri dishes (90 × 15 mm, Pyrex, Germany). A 100 mg each of the extracts was suspended in 10 ml distilled water and the suspension was shaken vigorously by using a vortex mixer. In this way, the concentration (10 mg/ml) of both methanolic and ethanolic extracts were prepared for screening the anti-arthritc, thrombolytic and cytotoxic properties. The crude methanol extract was suspended with distilled water (150 ml) and partitioned with ethanol and chloroform. The resultant partitionates i.e. ethanol was used for the biological screenings.
Chemicals and reagents The chemicals used were bovine serum albumin (BSA), diclofenac sodium, absolute methanol and ethanol (99.50%) and vincristine sulfate (VS) were purchased from SigmaAldrich, Munich, Germay. Lyophilized Streptokinase vial (Durakinase, Dongkook Pharma. Co. Ltd, South Korea) of 15 00000 I.U., 5 ml sterile distilled water was added and mixed properly. This suspension was used as a stock from which 100 μl (30,000 I.U) was used for in vitro thrombolysis. All chemicals in this investigation were of analytical reagent grade.
Inhibition of protein denaturation For the evaluation in vitro anti-arthritic activity of M. denticulata, the method used was “inhibition of protein denaturation” with diclofenac sodium as a standard. The test solution (0.5 ml) consists of 0.45 ml. of bovine serum albumin (5% w/v aqueous solution) and 0.05 ml of methanol and ethanol extract of M. denticulata. The control solution (0.5 ml) consists of 0.45 ml of bovine serum albumin (5% w/v aqueous solution) and 0.05 ml of distilled water. Product control (0.5 ml) consists of 0.45 ml of distilled water and 0.05 ml of methanolic and ethanolic extract of M. denticulata. Standard solution (0.5 ml) consists of 0.45 ml of bovine serum albumin (5% w/v aqueous solution) and 0.05 ml of diclofenac sodium. Various concentrations (31.25, 62.5, 125, 250, 500, and 1000 µg/ml) of both methanol and ethanol extracts of Macaranga denticulata (MD) and diclofenac sodium (standard) were used respectively. All the solutions were adjusted to pH 6.3 using 1 N HCL. Samples were kept in incubator at 37 °C for 20 min and the temperature was increased to keep the samples at 57 for 3 min. 2.5 ml of phosphate buffer was added to the previous solutions after cooling. The absorbance was determined by UV-Visible spectrophotometer at 416 nm. The control represents 100% protein denaturation. The results were compared with diclofenac sodium used as a standard.
The percentage inhibition of protein denaturation can be calculated as:
% inhibition = [100 - (OD of test solution - OD of product control)] ×100
Where OD = optical density. The control represents 100% protein denaturation. The results were compared with diclofenac sodium.
Thrombolytic activity Blood specimen Total blood (2 ml) was drawn from healthy human volunteers (n = 5) without a history of oral contraceptive or anticoagulant therapy. A 500 μl of blood was transferred to each of the three previously weighed microcentrifuge tubes to form clots.
Clot lysis Experiments for clot lysis were carried as reported earlier. Briefly, 2 ml venous blood drawn from the healthy volunteers was distributed in three different pre weighed sterile microcentrifuge tube (0.5 ml/tube) and incubated at 37 °C for 45 min. After clot formation, serum was completely removed without disturbing the clot and each tube having clot was again weighed to determine the clot weight (clot weight = weight of clot containing tube – weight of tube alone). To each microcentrifuge tube containing pre-weighed clot, 100 μl of methanol and ethanol extracts of M. denticulata were added separately. 100 μl of streptokinase as positive control and 100 μl of distilled water as negative control were used and separately added to the marked control tubes. All the tubes were then incubated at 37 °C for 90 min and observed for clot lysis. After incubation, fluid released was removed and tubes were again weighed to observe the difference in weight after clot disruption. Difference in weight before and after clot lysis was expressed as percentage of clot lysis as shown below:
Percent (%) of clot lysis = (Weight of released clot /clot weight) ×100
The experiment was repeated with the blood samples of the 5 volunteers