Plant collection and identification
Whole plants of A. conyzoides (Accession No. 36073), root of C. suffruticosa (Accession No. 32909), leaf of L. indica (Accession No. 36078), whole plants of L. aspera (Accession No. 36070), leaf of S. sophera (Accession No. 36072) and fruits of S. torvum (Accession No. 36071) were collected from different parts of Chittagong region, Bangladesh. The plants were identified by Dr. Shaikh Bokhtear Uddin, Taxonomist and Associate Professor, Department of Botany, University of Chittagong. The sample specimens of the identified plants have been preserved in the national herbarium with the mentioned accession numbers.
Chemicals and reagents
To the commercially available lyophilized Streptokinase (SK) vial (Durakinase, Dongkook Phama. 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. Absolute ethanol (99.50%) and vincristine sulfate (VS) were purchased from Sigma-Aldrich, Munich, Germany.
Each of the plant materials was dried and ground (Moulinex Blender AK-241, Moulinex, France) into powder (40-80mesh, 500 g) and soaked for 7 days with 2–3 days interval in 2.0 L of ethanol at room temperature (23 ± 0.5°C). Filtrate obtained through cheesecloth and Whatman filter paper No. 1 was concentrated under reduced pressure at the temperature below 50°C using rotary evaporator (RE 200, Sterling, UK). The extracts (yield 4.4–5.6% W/W) were all placed in glass Petri dishes (90 X 15 mm, Pyrex, Germany). A 100 mg each of the extracts 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, which was filtered through a 0.22-μm syringe filter. A 100 μl of this aqueous preparation was added to the microcentrifuge tubes containing the clots to check thrombolytic activity. The same concentration (10 mg/ml) of extracts was prepared for screening the cytotoxic properties.
Whole blood (4 ml) was drawn from healthy human volunteers (n = 20) without a history of oral contraceptive or anticoagulant therapy using a protocol approved by the Institutional Ethics Committee of Chittagong University, faculty of medicine. An earlier consent, approval number HET-CU2011/1, was taken from the faculty of medicine, University of Chittagong, for collection of blood samples from Human volunteers. Blood collection and preservation were conducted by Dr. M Rafiqur Rahman (Pathologist, faculty of Medicine, University of Chittagong). A 500 μl of blood was transferred to each of the eight previously weighed microcentrifuge 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. Explanation was made on if future use of the research data beyond the current study is anticipated, whether this is a focus group if so the Principal Investigator should put a procedure in place in which the researchers caution people about the limit on confidentiality. Access to Research Information regarding who would have access to the collected sample, information regarding retention of sample and schedules for their disposal were also detailed. 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 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; for example the possibility of bruising or swelling while giving blood, or some other discomforts at the site where blood is drawn and that there might be minimal chance of infection, and that these discomforts were brief and transient were also added. Potential benefits of the donors, not directly, but the society in general or individuals with a similar condition might benefit from the results of this study was explained. 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. The legal obligation includes a number of circumstances, such as suspected child abuse and infectious disease, expression of suicidal ideas where research documents are ordered to be produced by a court of law and where researchers are obliged to report to the appropriate authorities. In those rare instances where it will not be possible to assure complete confidentiality”, the limits on this obligation were carefully explained. Reimbursement issue was also mentioned whether the donors or their parents may be offered money for reasonable out-of-pocket expenses for example, transportation costs, meals, etc. Finally detail contact (Name, area code and phone number) of investigators was provided in case of any questions of the donors about this study. The consent form was concluded with major questions on above disclosures in Yes/NO form followed by the signature (with date) of the donor.
Experiments for clot lysis were carried as reported earlier . Briefly, four ml venous blood drawn from the healthy volunteers was distributed in eight 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 organic extracts of the six plants (A. conyzoides, C. suffruticosa, L. indica, L. aspera, S. sophera and S. torvum) were added separately. As a positive control, 100 μl of SK and as a negative non-thrombolytic 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 20 volunteers.
Brine shrimp bioassay was carried out with the method as described by Meyer et al. (1982)  to investigate the cytotoxicity of the extracts. The dried extract preparations were re-dissolved in DMSO to obtain a solution of 10 mg/ml which was subjected to serial dilution to get the concentrations between 20 μg/ml- 800 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 the Institute of Marine Science and Fisheries, University of Chittagong, 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 20 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 “BioStat-2009”.
The significance between % clot lysis by SK and plant extracts, LC50 values by VS and extracts was tested by the paired t-test analysis using the software SPSS, version 19.0 (SPSS for Windows, Version 18.0, IBM Corporation, New York, USA). Data are expressed as mean ± standard deviation. The mean difference between positive and negative control was considered significant at p < 0.05.