Thrombolysis Potential of Methanol Extracts from the Five Medicinal Plants Leaf, Available in Bangladesh

INTRODUCTION

Thrombosis is the formation of an atypical mass inside the vascular system of a living animal¹. It is a serious happening in the arterial diseases linked with myocardial infarction and stroke and venous thromboembolic disorder report for substantial morbidity and mortality. In addition, venous thrombosis is the second foremost cause of death in patients with cancer².

Clinical thrombolytic agents such as urokinase (UK), streptokinase (SK) or tissue plasminogen activators (t-PA) are broadly indicated in the coronary artery thrombosis for the management of severe or massive deep venous thrombosis, pulmonary embolism, myocardial infarction and occluded intravenous or dialysis cannulas³. Due to lesser cost, UK and SK are commonly used in India, Bangladesh and other developing countries⁴ as compared to other thrombolytic drugs, but the use was combined with elevated risk of bleeding intracranial hemorrhage, severe anaphylactic reaction and lacks specificity^5,6. The therapy for thrombolysis with recombinant t-PA was effective in acute myocardial infarction, however, the treatment was inadequate by a fairly sluggish reperfusion rate and frequent early reocclusions. Moreover, the platelet-rich thrombi are extremely resistant to lysis by t-PA⁷. Further, fucoidan as a thrombolytic agent, a branched sulfated fucan extracted from brown seaweeds with anticoagulant and antithrombotic effects mediated via straight thrombin inhibition has been reported newly³. On the other hand, herbal drugs are safe because it has been coming from natural sources. The acceptability of traditional herbal drugs increased day by day due to their barely credible pharmacological activities, cost-effective viability and less side effects in different health care management system^8,9.

Jacquemontia paniculata (Burm.f.) Hallier f. belongs to family Convolvulaceae. Jacquemontia is one of the superior genera with approximately 120 species¹⁰. Psychotria adenophylla Wall. (Family: Rubiaceae). Traditionally, roots used in mouth sore and rheumatism¹¹. According to the plant list and Wikipedia, Cheilocostus speciosus (J.Konig) C.Specht or (syn. Costus speciosus) was probably the top identified species in the genus Cheilocostus (family: Costaceae). The hexane extract of C. speciosus rhizome have anti-hyperglycemic, hypolipidemic activity, hepatic antioxidant enzyme activity, affects neurotransmitters and monoamine oxidase activity, anti-inflammatory, antipyretic properties and hepatoprotective activity¹². Primula sikkimensis Hook. (Family: Primulaceae) is an endemic species in the Himalaya-Hengduan Mountains (HMM) region and is the only species in Primula sect¹³. Dioscorea pentaphylla Linn (Family: Dioscoreaceae), potentially used as vegetables, tubers are boiled and eaten, leaf paste mixed with mustard oil was rubbed on the effective part to treat rheumatism^14-17.

Nowadays, natural medicines from plant sources have been shown versatile pharmacological activities in various disease model. The pharmacological activities due to the different phytochemicals that are available in different parts of plants. Considering the thrombosis model, the aim of the present study was to investigate in vitro thrombolysis activity of five Bangladeshi medicinal herbs such as J. paniculata, P. adenophylla, C. speciosus, P. sikkimensis and D. pentaphylla.

MATERIALS AND METHODS

Plant collection and identification: The leaves of the plant of J. paniculata, P. adenophylla, C. speciosus, P. sikkimensis and D. pentaphylla were collected from different parts of Chittagong region, Bangladesh. All plants were identified by Prof. Dr. Shaikh Bokhtear Uddin, Department of Botany, University of Chittagong. All experiments and collection of plant specimens during October, 2015 to April, 2016.

Preparation of crude extract: The fresh leaves of all herbs were separated, washed and air dried at room temperature (24±2°C) for about 10 days. Then the leaves were grounded into coarse powder (250 g) with a mechanical grinder (Moulinex three-in-one grinder, China). The coarse powder emerged using methanol and shaking by rotary shaker apparatus for 7 days. The tincture was collected using Buchner funnel. Methanol was evaporated at a temperature below 45°C then 25 g of the concentrated crude extract was weighed and stored at 4°C temperatures.

Streptokinase (SK): Thrombolysis activities of all plant extracts were tested by a method using streptokinase (SK: 15,00,000 I.U.) as a reference standard. Five milliliters sterile distilled water was added to streptokinase vial and mixed properly. From this suspension, 100 μL (30,000 I.U) was used for in vitro thrombolysis¹⁸.

Sample preparation: A hundred milligrams each of the extracts was suspended in 10 mL distilled water and the suspension was shaken vigorously by using a vortex mixer. Then, the suspension was reserved during the night and decanted to remove the soluble supernatant, which was filtered.

Blood sample: For this test, blood samples (Five mL) were drawn from healthy human volunteers (n = 10) with maintaining aseptic circumstance devoid of a history of oral contraceptive or anticoagulant therapy. The study protocol was accepted by the Planning and Development Committee (Grant No. Pharmacy P&D 68/09-15), Department of Pharmacy, International Islamic University Chittagong, Bangladesh.

Thrombolysis activity: An in vitro thrombolytic model used to investigate the thrombolysis activity¹⁸. Venous blood was drawn from every volunteer which were taken in ten different pre-weighed sterile microcentrifuge tubes and permitted to incubate at 37°C for 45 min. Later than clot formation, fluid was wholly removed from all microcentrifuge tubes and determined clot weight by subtracting the weight of clot containing tube from the weight of tube alone. In addition, 100 μL of streptokinase (SK) used as a positive control but 100 μL of distilled water as a negative control. Moreover, 100 μL of every sample also independently added to the microcentrifuge tubes. All the tubes were followed by incubated at 37°C for 90 min and observed for clot lysis. Later than incubation, the released fluid was discarded and tubes were again weighed to observe the difference in weight after clot disruption. The percentage of clot lysis was calculated as following⁶:

Statistical analysis: Data were expressed as the mean±standard error of mean (SEM). Statistical comparisons were performed using one-way ANOVA followed by Tukey/Tukey-Kramer (equal/unequal observations). The values obtained were compared with the control group and considered statistically significant when p<0.001, p<0.01 and p<0.05. All statistical analysis was performed using MaxStat Lite 3.60 version software (MaxStat Software, Oliver Wurl Grüner Weg 1726441 Jever-OT Cleverns, Germany).