Effects of organic extracts and their different fractions of five Bangladeshi plants on in vitro thrombolysis

Leaf of T. orientalis (Accession No. 30816), leaf of B. monnieri (Accession No. 32216), fruits of C. frutescens (Accession No. 33018), fruits of B. oleracea (Accession No. 30072) and leaf of U. sinuata (Accession No. 36155) were collected from different parts of Chittagong region (Chakaria Upazila, University of Chittagong campus and Hill tracts area of Chittagong, Bangladesh). The plants were identified by Bangladesh Forest Research Institute (BFRI), Chittagong-4211, Bangladesh. The sample specimens of the identified plants have been preserved in the national herbarium with the mentioned accession numbers.

Plant materials were dried and ground (Moulinex Blender CK-243, Moulinex, France) into powder (50–80 mesh, 450 g approx.) to soak in 2.5 L of methanol for 14 days 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 45°C using rotary evaporator (Buchi Rotavapor R-200, Germany). The solvent was completely removed by using rotary evaporator (Buchi Rotavapor R-200, Germany) and obtained T. orientalis 7.74 g (yield 3.87% w/w), B. monnieri 18 g (yield 3.60% w/w), C. frutescens 23 g (yield 5.50% w/w), B. oleracea 48 g (yield 6.40% w/w) and U. sinuata 16 g (yield 3.4% w/w) of dried crude extracts. All of the extracts were placed in glass Petri dishes (90 × 15 mm, Pyrex, Germany).

Crude extracts were undertaken for solvent-solvent partitioning by using the protocol designed by Kupchan and Tsou [17] and modified version of Wagenen et al. [17]. The crude extract (5 g) was triturated by dissolving in 10% aqueous methanol (methanol: water; 9:1 v/v) to make the mother solution which was successively partitioned by four solvents such as n-hexane, chloroform, ethyl acetate and hydro-methanol in order of increasing polarity by using separating funnel (Figure 1). Physical appearances of the fractions and their quantity after partitioning are shown in Table 1. Resulting fractions of each plant extract were dried by evaporating respective solvent using rotary evaporator. All extracts were stored at 4°C in air tight containers till further analysis [18]. 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 [13].

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 previously [19]. Briefly, 4 mL venous blood drawn from the healthy volunteers was distributed in eight different pre-weighed sterile microcentrifuge tubes (0.5 mL/tube) and incubated at 37°C for 45 minutes. 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 different organic extracts of the five plants (T. orientalis, B. monnieri, C. frutescens, B. oleracea and U. sinuata) 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 minutes and observed for clot lysis. After incubation, released fluid 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 twenty (20) healthy volunteers.

The significance between % clot lysis by SK and plant extracts was tested by the paired t-test analysis using the software SPSS, version 18.0 (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 values < 0.05 and 0.001.