ISSN - 0975-7058 Vol 10, Special Issue 1, 2018 ISSN - 0975-7058 ANTITHROMBOTIC ACTIVITY OF TAMARINDUS INDICA L. IN MICE FADLINA CHANY SAPUTRI 1 *, CHAVELLA AVATARA 2 , DIAN RACHMAWATI 1 1 Department of Pharmacology, Faculty of Pharmacy, Universitas Indonesia, Depok 16424, Indonesia. 2 Drug Development Laboratory, Faculty of Pharmacy, Universitas Indonesia, Depok 16424, Indonesia. Email: fadlina.chany@farmasi.ui.ac.id Received: 05 May 2018, Revised: 20 September 2018, Accepted: 15 November 2018 ABSTRACT Objective: This study aimed to investigate the antithrombotic activity of Tamarindus indica L. extract (TIE) in mouse models (in vivo). Methods: TIE was orally administered to mice at three different doses for 7 days. TIE-treated mice were used in two experiments of antithrombotic activity: An examination of bleeding time following tail cutting and an examination of survival rate after collagen-epinephrine-induced thromboembolism. The TIE groups were observed after 7 days of treatment and compared to an aspirin-treated group and a control group. Results: Treatment with TIE led to a significant increase in bleeding time compared with that in the control group. TIE treatment also protected mice from thromboembolic death, significantly increasing survival rates in a dose-dependent manner. Conclusion: TIE has the potential as an antithrombotic agent against platelet thromboembolism. Keywords: Antithrombotic, Bleeding time, Survival rate, Tamarindus indica L. INTRODUCTION Thrombosis, as one of the risk factors of cardiovascular disease, is the formation of a blood clot in an artery or a vein which starts with platelet aggregation [1,2]. Deep vein thrombosis (DVT) refers to the formation of one or more blood clots in one of the major blood vessels of the body, most commonly in the lower limbs. The most serious complication that can arise from DVT is a pulmonary embolism (PE), which occurs in more than one-third of patients with DVT and often causes sudden death [3-5]. Acetylsalicylic acid (ASA), commonly known as aspirin, is often used as a platelet aggregation inhibitor agent. ASA inhibits platelet aggregation by inhibiting the cyclo-oxygenase (COX) enzyme within the COX pathway, thus preventing thrombus formation [6]. In addition to chemical drugs, herbal medicines have been studied for their potentials as the antithrombotic agent. Tamarind (Tamarindus indica L.), a flowering plant within the family Fabaceae, is a potential antithrombotic agent due to its active compound dotriacontanoic acid [7]. This compound is one component of D-003, a natural compound comprising triacontanoic, dotriacontanoic, and tetradecanoic acid [8,9]. Compound D-003 has been tested in vitro on a venous thrombosis model; a dose of 200 mg/kg has shown to increase the number of prostacyclin molecules (prostaglandin I 2 [PgI 2 ]) and reduce thromboxane A2 (TxA 2 ). In vivo experiments with D-003 have demonstrated that this compound can reduce the formation of venous thrombus at a dose of 400 mg/kg in collagen-induced and epinephrine-induced mice [8]. In this study, the effects of T. indica L. extract (TIE) were tested in vivo on mice using bleeding time and survival rate as metrics of antithrombotic activity. MATERIALS AND METHODS Materials Ethanolic TIE was obtained from the Indonesian Spice and Medicinal Crops Research Institute, Bogor. This study used aspirin (Medifarma, Indonesia) as a positive control. Carboxymethyl cellulose (CMC) (0.5%; Brataco, Indonesia) and saline (Euro-Med, Indonesia) were used as a vehicle group (normal and negative control group). Voucher specimens were deposited at the Center for Plant Conservation Botanic Gardens (No. B-1693/IPH.3./KS/VI/2017). Total flavonoid and phenolic content assays Total flavonoid content was measured using the aluminum chloride colorimetry method, with absorbance measured at λ=510 nm [10]. The total phenolic compound content was measured using the Folin– Ciocalteu method, with absorbance measured at λ=725 nm [11]. Animals Male mice (Mus musculus, ddY strain) weighing between 20 and 30 g were obtained from the Institute Pertanian Bogor. The use of animals in this study was approved by the Ethics Committee of Faculty of Medicine, Universitas Indonesia (Number. 232/UN2.F1/ETIK/2017). Antithrombotic activity tests Bleeding time test The treatment of bleeding time was determined according to protocols described by Saputri et al. (2017) with the following modifications. Mice were divided into the treatment groups shown in Table 1 and acclimatized for 1 week. Experimental treatment doses were given to mice through oral administration for 7 days. The dose of aspirin applied was 80 mg/day [12-14]. Experimental TIE doses were 14, 28, and 56 mg/20 g mice. Antithrombotic activity was evaluated by a bleeding time of 5 h after experimental day 7. The animals were anesthetized with ether by inhalation and placed in a horizontal position. An approximately 10-mm segment of the tail was amputated from each animal using a scalpel. The end of each severed tail was immediately immersed in a 15 mL Falcon tube containing isotonic saline. The remaining portion of the tail was vertically positioned with the tip horizontally placed approximately 2 cm below the body. Bleeding observations were conducted for 20 min [12]. Differences in bleeding time with TIE treatment were obtained by comparing each experimental dosage group to the vehicle group. Survival rate test Mice used for survival rate tests were divided into treatment groups as shown in Table 2. The survival rate tests were performed 24 h after experimental day 7. To induce pulmonary thrombosis, experimental mice were injected with a mixture of collagen (700 µg) and epinephrine (42 µg) Research Article © 2018 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ijap.2018.v10s1.08 The 2 nd Physics and Technologies in Medicine and Dentistry Symposium (PTMDS), Universitas Indonesia. Depok, Indonesia