Plant materials: The leaves of M. sylvatica were collected from Chittagong hill tracts area specifically from the area of Department of Forestry, University of Chittagong in May, 2014 and it was authenticated by Dr. Shaikh Bokhtear Uddin, Associate Professor, Department of Botany, University of Chittagong, Chittagong-4331, Bangladesh.
Preparation of extracts: The leaves of the plant were air dried at room temperature before grinding them to powdered form with the help of mechanical grinder (NOWAKE, Japan). Dried and powdered leaves were soaked separately in different solvents having different polarity (i.e., benzene, hexane, petroleum ether (40-60°C), chloroform, methanol, ethanol and ethyl acetate). Each extract was first filtered through muslin cloth to clarify and then through a Whatman No. 1 filter paper. The filtrate was evaporated under reduced pressure in vacuum evaporator. The dried crude extracts were sterilized overnight by UV radiation and then stored at room temperature in amber color glass vials until used for test of thrombolytic activity.
Reagents and chemicals: All the chemicals and reagents (i.e., benzene, hexane, petroleum ether, chloroform, ethanol and ethyl acetate) were of analytical grade and were provided by the Department of Pharmacy, International Islamic University Chittagong. Streptokinase (1.5 million unit/vial; Sanofi-aventis Bangladesh Limited) were used as positive control and water as negative control for in vitro thrombolytic test.
Ethical consideration: The study protocol was approved by the P and D Committee (pharmacy and drug committee-institutional ethics committee), Department of Pharmacy, International Islamic University Chittagong, Bangladesh. Blood samples were collected from the students of the Department of Pharmacy, International Islamic University Chittagong. A written consent was taken from all the volunteers.
Phytochemical evaluation: The leaves powder was dissolved in distilled water and screened for the presence of various chemical constituents like alkaloids, carbohydrates, proteins, cardiac glycosides, steroids, saponins, flavonoids, terpenoid, tannins and phenols (Chowdhury et al., 2014; Musa et al., 2009; Kokate, 1997; Ugochukwu et al., 2013; Yadav and Agarwala, 2011).
Preparation of test solution: Hundred milligram extract was suspended in 10 mL distilled water and the suspension was shaken vigorously on a vortex mixer. The suspension was kept overnight and decanted to remove the soluble supernatant. Hundred microliter of this aqueous preparation of herbs was added to the microcentrifuge tubes containing the clots to check thrombolytic activity.
In vitro thrombolytic test: The thrombolytic activity was evaluated by the method prescribed earlier (Prasad et al., 2006). In our study, 100 µL of each fractional extract was used as experimental drug. Five milliliter of blood samples were collected from volunteer and distributed as 0.5 mL of blood into seven separate pre-weighed (W1) sterile microcentrifuge tubes. The blood samples were collected from ten healthy different volunteers, who did not have contraceptive or anticoagulant therapy for the last 7 days. The blood specimen was incubated for 45 min at 37°C. After clotting of blood, serum was decanted and removed. Then weight of clotted blood (ΔW) was taken by subtracting the pre-weight (W1) from the weight of clot containing tube (W2) as, ΔW = W2-W1. The equation for calculating weight of clot is given below:
Clot weight = Weight of tube containing clot-weight of empty tube
Then 100 µL extracts of each fraction of M. sylvatica were added to the clot containing tubes. Similarly, 100 µL of streptokinase was added to clot of standard tube and 100 µL of water was added to the clot of blank tube, those were used as positive and negative control, respectively. Then all the tubes were incubated at 37°C for 90 min and weighed again for getting the weight variation among the pre weight and final weight that was achieved for clot lyses (thrombolysis). The experiment was repeated three times with same blood sample of 10 volunteers. The percentage of clot lysis was calculated using the following equation:
Clot lysis % = Weight of lysis / Weight of clot before lysis x 100
Statistical analysis: The significance between percentage of clot lysis by streptokinase and herbal extracts by means of weight difference was tested by one-way ANOVA followed by Dunnett’s test analysis. Data is expressed as Mean±SE with 95% confidence interval. The statistical analysis was carried out by GraphPad Prism ver. 5.04.