Original Research Paper

Evaluation of thrombolytic potential of three medicinal available in , as a potent source of thrombolytic compounds

Md. Ramjan Ali1*, Marjan Hossain1, Jannatul Ferdous Runa1, Md. Hasanuzzaman1, Md. Mahmodul Islam1

1Department of Pharmacy, Noakhali Science and Technology University, Sonapur, Noakhali- 3814, Bangladesh

Article history: Abstract Received: Jan 30, 2014 Objective: The present study is aimed to investigate in vitro Received in revised form: May 5, 2014 thrombolytic activity of three Bangladeshi medicinal plants Accepted: May 27, 2014 bilimbi (Oxalidiaceae), Clerodendrum viscosum Vol. 4, No. 6, Nov-Dec 2014, (Verbanaceae) and Drynaria quercifolia (Polypodiaceae). 430-436. Materials and methods: Each the was extracted with methanol at room temperature and the concentrated methanolic * Corresponding Author: extracts (MEF) were fractionated by the modified Kupchan Tel: +8801732522854‎ partitioning method to render pet-ether soluble fraction (PESF), [email protected] carbon tetrachloride soluble fraction (CTSF), chloroform soluble fraction (CSF) and aqueous soluble fraction (AQSF). To observe Keywords: their thrombolytic potential, a prompt and swift method was In vitro involved where streptokinase and water were used as positive and Streptokinase thrombolytic negative control, respectively. activity Result: Among the three plants, AQSF and PESF of D. % Clot lysis quercifolia with CTSF of C. viscosum exhibited highest

thrombolytic activity by clot lysis of 34.38%, 34.27% and 28.64%, respectively. Among other extracts A. bilimbi, C. viscosun and D.quercifolia showed significant percentage (%) of clot lysis compared to standard streptokinase (41.05%) while the negative control water revealed 3.31 % lysis of clot. Conclusion: From our findings it is observed that all the plants revealed remarkable thrombolytic activity. Therefore, steps should be taken to observe in vivo clot dissolving potential and to isolate active component(s) of these extracts. Please cite this paper as: Ali R, Hossain M, Runa JF, Hasanuzzaman M, Islam M‎. Evaluation of thrombolytic potential of three medicinal plants available in Bangladesh, as a potent source of thrombolytic compounds. Avicenna J Phytomed, 2014; 4 (6): 430-436.

Introduction of the tissue in that area. Thrombin formed Recently blood clot formation has been blood clot from fibrinogen and is lysed by a severe problem of blood circulation. plasmin, which is activated from Thrombus or embolus hinders the blood plasminogen by tissue plasminogen flow by blocking the blood vessel therefore activator (tPA). The purpose of a depriving tissues of normal blood flow and fibrinolytic drug is to dissolve thrombin in oxygen. These consequence yield necrosis acutely occluded coronary arteries thereby

AJP, Vol. 4, No. 6, Nov-Dec 2014 430 Evaluation of thrombolytic potential of three medicinal plants to restore blood supply to ischemic hairy leaves with a disagreeable odor myocardium, to limit necrosis and to (Kirtikar et al., 2001). The shrub is about 2- improve prognosis (Laurence et al., 1992). 4 feet in height. Various parts of the plant For the treatment of myocardial infarction, are used by tribes for the treatment of colic, many thrombolytic agents are used. Among scorpion sting and snake bite, tumors and them, streptokinase is remarkable and certain skin diseases, while the leaves are widely used. Moreover, Tissue-type somewhat bitter, cure inflammation, skin Plasminogen activator is more effective and diseases and good in small pox (Santanuet safer than either urokinase or streptokinase al., 2009). type activators. It is noted that all available Drynaria quercifolia J. Smith belongs to thrombolytic agents still have significant the family of Polypodiaceae, locally known deficiencies, including the necessity of as Gurar, is a parasitic fern (Bhattacharya large doses to be maximally effective, 1990; Kirtikar et al., 1994) that is generally limited fibrin specificity and a significant distributed in Bangladesh, and associated bleeding tendency. Therefore, Thailand. The rhizomes of the plant have steps are taken to develop improved antibacterial properties and are used recombinant variants of these drugs in traditionally for management of cough, order to minimize deficiencies of the tuberculosis and typhoid fever. ASEAN available thrombolytic drugs (Adams et al., Centre for Biodiversity stated in their 1991; Nicolini et al., 1992; Lijnen et al., Checklist of Medicinal Plant in Southeast 1991; Marder 1993; Wu et al., 2006). that rhizome decoction or drink of D. Averrhoa bilimbi Linn. belongs to the quercifolia rhizome uses as antipyretic family of having some local preparation (Ramesh et al., 2001). name including belembu, belemburi; In English, this is also familiar with- bilimbi, cucumber tree, tree sorrel. The plant has a Materials and Methods short trunk soon dividing into a number of Collection and identification upright branches; attractive, long-lived Different parts of Averrho abilimbi tropical tree, reaches 16 to 33 ft. (5-10 m) (fruits), Clerodendrum viscosum (leaves) in height. Probably, A. bilimbi is inborn of and Drynaria quercifolia (leaves) were the Moluccas in . This plant is collected from Noakhali, Bangladesh also available in Brazil, Cuba, , during the month of July 2013 and voucher , Bangladesh, (Burma) specimens for each of the collections and (Hasanuzzaman et al., 2013). (DACB 37752, 35979 and 37654 A. bilimbi is medicinally used as a folk respectively) have been deposited in therapy for many purposes including Bangladesh National Herbarium (BNH) for antibacterial, antiscorbutic, astringent; future references. Different parts of the postpartum protective medicine. It is also plants were washed with clean water to used for the management of fever, mumps, discard dirt materials, dried under shade pimples, inflammation of the rectum and with casual sun drying, ground into coarse diabetes, itches, boils, rheumatism, powder and preserved in a closed container syphilis, bilious colic, whooping cough, at 25°C for further use. hypertension, stomach ache, aphthous ulcer and as a cooling drink (Roy et al., 2011). Preparation, extraction and Clerodendrum viscosum Linn. belongs fractionation of plant material to the family of Verbanaceae, commonly The extraction was performed using cold known as Bhat in Hindi and Ghentu, in maceration technique. Powder portions of Bengali, is a terrestrial shrub having different plants (500 g) were soaked in square, blackish stem and simple, opposite, 2500 ml of methanol for about 10 days at decussate, petiolate, exstipulate, coriacious, room temperature with occasional stirring.

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The solution was filtered through filter consent was obtained from each volunteer cloth followed by Whatman’s‎ filter‎ paper prior to collect blood sample. and the filtrate thus obtained was concentrated designated as MEF. This was Thrombolytic activity done by evaporation method under ceiling 5 ml of venous blood were drawn from fan and in a water bath below 40°C each volunteers which were taken in five temperature. The concentrated MEF was different pre weighed sterile micro partially separately by modified Kupchan centrifuge tube and allowed to incubate at method (Vanwagenen et al., 1994) using 37 °C for 45 minutes. After clot formation, chloroform, carbon tetrachloride and pet- fluid was completely released from each ether to yield CSF, CTSF and PESF. microcentrifuge tubes and determined clot Aqueous portion was retained as AQSF. weight by subtracting weight of clot Four organic solvent soluble fractions were containing tube from weight of tube alone. dried for 7 days at room temperature by As‎ a‎ positive‎ control,‎ 100‎ μl‎ of‎ evaporation method. streptokinase (SK) and as a negative non thrombolytic‎ control,‎ 100‎ μl‎ of‎ distilled‎

water along‎ with‎ 100‎ μl‎ of‎ each‎ samples Streptokinase (SK) Streptokinase (15, 00,000 I.U.,) used as were separately added to the a standard which was collected from microcentrifuge tubes. All the tubes were Beacon pharmaceutical Ltd, Bangladesh. 5 then incubated at 37 °C for 90 minutes and ml sterile distilled water was added to observed for clot lysis. After incubation, streptokinase vial and mixed properly. the released fluid was discarded and tubes From‎ this‎ suspension100μl‎ (30,000‎ I.U)‎ were again weighed to observe the was used for in vitro thrombolysis (Prasad difference in weight after clot disruption. et al., 2007). Finally percentage of clot lysis was determined as followings: % of clot lysis = (wt of released clot /clot Preparation of sample The thrombolytic activities of all plant wt) × 100 extracts were evaluated by a method using streptokinase (SK) as a reference standard. Statistical analysis 100 mg of MEF, CSF, CTSF, PTSF, and Data obtained were analyzed using AQSF of different plants were dissolved SPSS version 16.0 (SPSS Inc. Chicago, IL. respectively in 10 ml of methanol, USA). All values are expressed as chloroform, carbon tetrachloride, pet-ether, mean±SEM for three replicates. Data were distilled water and were kept overnight. analyzed by one-way ANOVA and the statistical significance differences were Then the soluble supernatant was decanted and filtered. analyzed using paired t- test. p<0.05 was considered statistically significant. Blood sample Blood samples were collected from healthy human volunteers (n=5) by Results maintaining aseptic condition without a The thrombolytic activity of A.bilimbi, C. history of oral contraceptive or viscosum and D. quercifolia extracts were anticoagulant therapy. 1ml of blood was determined as a part of exploration of transferred to the previously weighed cardio protective drugs from plants microcentrifuge tubes to form clots. Study reserves, and the conclusion are conferred protocol was approved by ethical in Table 1. It presents 41.05 % lysis of clot committee of Pharmacy department, in a process of subsequent incubation for Noakhali Science and Technology 90‎ minutes‎ at‎ 37°C,‎ where‎ 100μl‎ SK‎ was‎ University, Noakhali, Bangladesh. Written added as a positive control (30,000 I.U.).

AJP, Vol. 4, No. 6, Nov-Dec 2014 432 Evaluation of thrombolytic potential of three medicinal plants

On the other side, it showed negligible CSF fraction of A. bilimbi (23.94% and percentages of lysis of clot (3.31 %), when 23.51%). we use distilled water as a negative control. Besides, MEF and CTSF of D. In this observation, highest thrombolytic quercifolia, exhibit 23.46% and 20.33% of activity (34.38%) is demonstrated by the clot lysis respectively while AQSF of A. AQSF of D. quercifolia. Besides, bilimbi exhibit 17.06% of clot lysis. On the convincing thrombolytic activity were also other hand, MEF and CSF of C. viscosum exibited by PESF of D. quercifolia along with CSF of D. quercifolia showed (34.27%), CTSF of C. viscosum (28.64%) negligible amount of clot lysis. Figure 1 and A. bilimbi (27.72%), PESF of A. represents comparative thrombolytic bilimbi (27.5%) and C. viscosum (25.29%), activity of A. bilimbi, C. viscosum and D. AQSF of C. viscosum (24.39%), MEF and quercifolia for each extract.

Table-Thrombolytic activity (in terms of % of clot lysis) of A. bilimbi, C. viscosum and D. quercifolia

Treatment % of clot lysis

A.bilimbi C.viscosum D.quercifolia MEF 23.94 ± 7.58** 11.25± 2.32*** 23.46± 5.53** PESF 27.5 ± 6.89** 25.29± 6.80** 34.27± 1.34* CSF 23.51 ± 4.92** 4.1± 6.76*** 6.16± 7.45*** CTSF 27.72 ± 4.92** 28.64± 3.76* 20.33± 4.81** AQSF 17.06± 6.99** 24.39± 4.98** 34.38± 5.33*

Results represented in means ± SD (n = 3); Level of Significance *** p<0.001, ** p<0.01, * p<0.05 comparing with standard Streptokinase (41.05%). Here, MEF-methanolic extract fraction, PESF- pet-ether soluble fraction, CSF-chloroform soluble fraction, CTSF- carbon tetrachloride soluble fraction, AQSF-aqueous soluble fraction

Figure-Comparative thrombolytic activity of A. bilimbi, C. viscosum and D. quercifolia for each extract. Significant at **p<0.01 and for A. bilimbi, ###p<0.001, ##p<0.01 and #p<0.05 for C. viscosum, and ap<0.001, bp<0.01 and cp<0.05 for D. quercifolia when compared to standard streptokinase. Here, MEF-methanolic extract fraction, PESF- pet-ether soluble fraction, CSF-chloroform soluble fraction, CTSF- carbon tetrachloride soluble fraction, AQSF-aqueous soluble fraction.

Discussion treatment of many diseases, Nowadays From the beginning of civilization, phytopharmacological investigation has human are dependable on plants for the created a new field to discovery plant

AJP, Vol. 4, No. 6, Nov-Dec 2014 433 Ali et al. derivative drugs, which are effective in The present study was carried out to remedial of certain diseases, and renewed investigate thrombolytic activity of three the attention in herbal medicines. It is plants available in Bangladesh. estimated that about 30% of the Streptokinase (SK), a known thrombolytic pharmaceuticals are prepared from plants drug is used as a positive control (Prasad et derivatives (Leta et al., 2002; Gillman et al., 2007). Water, on the other hand, was al., 1995). A number of research works designated as a negative control. The have been conducted to discover the plants comparison of positive control with and natural food sources and their negative control clearly confirmed that clot supplements having antithrombotic dissolution does not occur when water was (anticoagulant and antiplatelet) effect and added to the clot. By comparing with this there is indication that consuming such positive & negative control, a significant food leads to prevention of coronary events and stroke (Ratnasooriyaet al., 2008; thrombolytic activity was observed after Joshipura et al., 1999; Liu et al., 2000; treating the clots with A.bilimbi, C. Bazzano et al., 2002). Although there are viscosum & D. quercifolia extracts. several thrombolytic drugs with those In conclusion from our recorded data, it obtained by recombinant DNA technology, can be demonstrated that our findings may but side effects related to some of these have significant implications in drugs that lead to further difficulties have cardiovascular health. In addition, this been reported (Baruah et al., 2006; Gallus finding may indicate the possibility of et al., 1998; Wardlaw et al,. 2004; Capstick developing novel thrombolytic compounds et al,. 2005). from A. bilimbi, C. viscosum, and D. Platelets play a significant role in the quercifolia extracts. Further studies are development of atherothrombosis as well as ongoing to isolate and characterize the damage the regions of endothelial surface compounds responsible for thrombolytic (produced by reactive oxygen species). The activity stimulated platelets form platelets to platelets bonds, binds also to leucocytes Acknowledgement carrying them into an intricate process of The authors are grateful to Beacon plaque development and progression pharmaceutical Ltd., Bangladesh for their (Prenticeet al., 1999). Plasmin, a natural generous donation of S-Kinase TM fibrinolytic agent, lyses clot by breaking (Streptokinase), and BNH to identify the down the fibrinogen and fibrin contained in plant/. The authors are also grateful to all a clot. Streptokinase forms a 1:1 the teachers and staffs of the Department of stoichiometric complex with plasminogen Pharmacy, Noakhali Science and that can convert additional plasminogen to Technology University for their support plasmin (Banerjee et al., 2004). Moreover, and co-operation. phlorotannin, isolated from marine brown algae, have a unique property in promotion Conflict of interest of dissolution of intravascular blood clot The authors declare no conflict of via antiplasmin inhibition (Prasad et al., interest with this study. 2007). Several studies reveal that A. bilimbi, C. viscosum and D. quercifolia possesses tannin, alkaloid saponin References (Hasanuzzaman et al., 2013; Runa et al., Adams DS, Griffi LA, Nachajko WR, Reddy 2013; De et al., 2013) which could be VB, Wei CM. 1991.A synthetic DNA participated for its clot lysis activity (Ali et encoding a modified human urokinase al., 2013) resistant to inhibition by serum plasminogen

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