Plant material: Fresh leaves of B. platyphylla were collected from Bandarban, Chittagong, Bangladesh in the month of March 2015. It was authenticated by Dr. Shaikh Bokhtear Uddin, Professor, Department of Botany, University of Chittagong, Chittagong-4331, Bangladesh.
Preparation of Extract: The leaves were dried for a period of 10 days under shade and ground. The ground leaves (450 gm) were soaked in sufficient amount of ethanol for one week at room temperature with occasional shaking and stirring then the whole mixture was filtered and the filtrate thus obtained was concentrated using a water bath to get a viscous mass. The viscous mass was kept at room temperature under a ceiling fan to get a dried extract (yield value, 5.3%). The extract prepared was for pharmacological screening.
Chemicals and equipment: To the commercially available lyophilized streptokinase (SK) vial (Square Pharmaceuticals Ltd. Dimethyl sulfoxide) of 1500000 I.U., 5mL sterile distilled water was added and mixed properly. This suspension was used as a stock from which 100 L (30,000 I.U.) was used for in vitro thrombolysis. Methanol was purchased from Merck (Germany). Dimethyl sulfoxide (DMSO) and Vincristine sulfate (2mg/vial; Techno Drugs Limited Bangladesh). Kanamycin (30µg/disc, Oxoid, England) was used as a standard antibiotic disc.
Brine Shrimp Lethality Bioassay: Brine shrimp lethality bioassay was carried out with the method as described by Meyer et al. to investigate the cytotoxicity of methanol extract of B. platyphylla leaves. The dried extract preparations were redissolved in DMSO to obtain a solution of 10 mg/ml which was subjected to serial dilution to get the concentrations between 12.5 μg/ml- 400 μg/ml. Standard drug Vincristine Sulphate (VS) was used as positive control at concentrations of 5 μg/ml - 0.312 μg/ml. A 5.0 ml of artificial sea water was added into all the test tubes. Simple zoological organism (Artemia salina) was used as a convenient monitor for cytotoxic screening. The eggs of the brine shrimps were collected from local aquarium shop (Dhaka, Bangladesh) and hatched in artificial seawater (Prepared by using sea salt 38 g/L and adjusted to pH 8.5 using 1N NaOH) under constant aeration for 24 h under the light. The hatched shrimps were allowed to grow by 48 h to get shrimp larvae called nauplii. After 48 h, active nauplii were attracted to one side in a glass petri dish by using a micropipette. The nauplii were then separated from the eggs by aliquoting them in another glass petri dish containing artificial sea water and used for the assay. Suspension containing 10 nauplii was added into each test tube and was incubated at room temperature (25±1°C) for 12 h under the light. The tubes were then examined after 24 h and the number of surviving larvae in each tube was counted with the aid of a 3X magnifying glass. Experiments were conducted along with VS in a set of three tubes per dose. The concentration that would kill 50% of the nauplii (LC50) was determined from a linear regression equation using the software “Microsoft excels 2007”.
In vitro Thrombolytic activity Blood specimen: Whole blood (1.5 ml) was drawn from healthy human volunteers (n = 12) without a history of oral contraceptive or anticoagulant therapy. A new consent, approved by Mohammed Abu Sayeed, Assistant professor & Head of Department of Pharmacy, International Islamic University Chittagong, Bangladesh, for collection of blood samples from Human volunteers. Blood collection were conducted by Md. Shariful Islam (Lab technician, Department of Pharmacy, IIUC) and preservation were conducted by Abdul Karim (Lab technician, Department of Pharmacy, IIUC), who stored the clot containing Eppendorf tube in the refrigerator in Microbiology lab, Department of Pharmacy, IIUC. A 500 μl of blood was transferred to each of the three previously weighed Eppendorf tube tubes to form clots.
Statement on informed consent of the donors: The volunteer donors were supplied a consent form which informed the title of the research project, name and detail contact of investigators as well as purpose of the research. Description of the research mentioning step-by-step brief of the proposed research, inclusion and exclusion criteria of the donors, whether donors will receive any therapy or not, volume of blood to be taken, possible discomfort of the puncture sites, time required for the blood sampling. Benefits of the volunteer described. It was indicated to the consent form that the volunteers might refuse to donate blood at any time. Donor, whether could withdraw his sample data, was disclosed. The sample was restricted for that individual study not for future research projects was presented in the consent form. Potential harm, injuries, discomforts or inconvenience associated with donors in this study was added as an informed consent statement. If there was known harm to the donors, the potential harm, current knowledge regarding the probability of the occurrence of the harm, clinical importance of the harm; and any relevant knowledge regarding the probability of reversibility. Treatment alternative and possibility of the research was described. Confidentiality statement was included in the consent form in the way that “confidentiality will be respected and no information that discloses the identity of the participant will be released or published without consent unless required by law of states. Finally identification of investigators was provided in case of further query. The consent form was concluded with major questions on above disclosures in Yes/NO form followed by the signature (with date) of the donor.
In Vitro Thrombolytic Study procedure: Experiments for clot lysis were carried as reported earlier [17-19]. Briefly, 1.5 ml venous blood drawn from the healthy volunteers was distributed in three different pre-weighed sterile Eppendorf 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 Eppendorf tube containing preweighed clot, 100 μl of methanol extract of B. platyphylla leaves were added separately. As a positive control, 100 μl of SK and as a negative nonthrombolytic control, 100 μl of distilled water were separately added to the control tubes numbered. 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 obtained in weight taken before and after clot lysis was expressed as percentage of clot lysis. The experiment was repeated with the blood samples of the 12 volunteers.