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Anticoagulant Potential and Total Phenolic Content of Six Species of the Genus Ficus from Azad Kashmir, Pakistan

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Anticoagulant Potential and Total Phenolic Content of Six Species of the Genus Ficus from Azad Kashmir, Pakistan Ambreen et al Tropical Journal of Pharmaceutical Research June 2019; 18 (6): 1245-1251 ISSN: 1596-5996 (print); 1596-9827 (electronic) © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. Available online at http://www.tjpr.org http://dx.doi.org/10.4314/tjpr.v18i6.14 Original Research Article Anticoagulant potential and total phenolic content of six species of the genus Ficus from Azad Kashmir, Pakistan Sumaira Ambreen1, Muhammad Tariq1*, Muhammad S Masoud2, Imran Ali1, Muhammad Qasim2, Aamar Mushtaq1, Maqsood Ahmed1, Rehana Asghar1 1Department of Biotechnology, Mirpur University of Science and Technology Mirpur 10250, AJK Pakistan, 2Department of Bioinformatics and Biotechnology, Government College University, Faisalabad, Pakistan *For correspondence: Email: [email protected]; Tel: +92 5827 961999 Sent for review: 10 October 2019 Revised accepted: 17 May 2019 Abstract Purpose: To investigate the total phenolic and flavonoid contents of Ficus benghalensis, Ficus elasticaa, Ficus palmata, Ficus religiosa, Ficus semicordata and Ficus auriculata, and to determine their anticoagulant potential. Methods: Crude methanol extracts were prepared from the plant leaves, and fractionated using liquid- liquid partition with n-hexane, chloroform and ethyl acetate. The total flavonoid and total phenolic contents of the extracts and their fractions were determined. The anticoagulant potential of the six Ficus species were evaluated in healthy human plasma, using activated partial thromboplastin time (APTT) and prothrombin time (PT) methods. Results: Phytochemical analysis showed the presence of considerable amounts of flavonoids ranging from 5.3 ± 0.7 to 11.8 ± 0.3 mg rutin equivalents (RE)/g, and phenolic compounds ranging from 8.0 ± 0.7 to 86.5 ± 1.5 mg gallic acid equivalents (GAE)/g in each fraction of the six species. Results from in vitro anticoagulant potential assays showed significant anticoagulant properties, with prothrombin time (PT) ranging from 17.7 ± 0.7 to 26.7 ± 2.2 s, and activated partial thromboplastin time (APTT) varying from 47.7 ± 3.3 to 72.3 ± 5.4 s. Conclusion: The results indicate that F. semicordata and F. Religiosa have higher anticoagulant potential than the other Ficus species studied. Keywords: Medicinal plants, Anticoagulant, Polyphenols, Flavonoids, Ficus This is an Open Access article that uses a fund-ing model which does not charge readers or their institutions for access and distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0) and the Budapest Open Access Initiative (http://www.budapestopenaccessinitiative.org/read), which permit unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Tropical Journal of Pharmaceutical Research is indexed by Science Citation Index (SciSearch), Scopus, International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index Copernicus, EBSCO, African Index Medicus, JournalSeek, Journal Citation Reports/Science Edition, Directory of Open Access Journals (DOAJ), African Journal Online, Bioline International, Open-J-Gate and Pharmacy Abstracts INTRODUCTION Abnormal coagulation leads to the formation of thrombus in blood vessels resulting in ischemia Cardiovascular diseases (CVDs) are associated [1]. In atherosclerotic arteries, platelet activation with poor health and high incidence of death induces arterial thrombosis [2]. Therefore, it is worldwide [1]. The major cause of death in CVD important to regulate platelet function in order to patients is thrombus-induced myocardial prevent thrombotic events. However, the infarction which also contributes to the currently available antithrombotic and antiplatelet pathogenesis of atherosclerotic CVDs [1]. drugs produce unsatisfactory results, and often ----------------------------------------------------------------------------------------------------------------------------------------------------- © 2019 The authors. This work is licensed under the Creative CommonsTrop Attribution J Pharm 4.0Res, International June 2019; License18(6): 1245 Ambreen et al lead to vascular relapses. Indeed, most thrombo- auriculata were collected from Azad Kashmir, embolic processes require anticoagulant therapy. Pakistan at coordinates 34.22°N 73.28°E, in Therefore, there is need for development of August 2016. The material was identified by Dr. novel, more effective and less toxic bioactive Ajaib of the Department of Botany, Mirpur anticoagulant and antithrombotic drugs with University of Science and Technology, Mirpur, different mechanisms of action. Anticoagulants Azad Jammu and Kashmir, Pakistan. The arrest coagulation after the initial platelet voucher specimens (no. MUST722 – MUST727) aggregation with blood clotting factors [3]. were deposited at the Herbarium of the Generally, anticoagulation therapy reduces the Department of Biotechnology. The leaves were severity of CVDs, and prevents existing clots shade-dried at room temperature for 2 weeks, from growing larger [4]. Anticoagulants reduce triturated with a blender and kept in sealed the formation of blood clots in other vital organs containers away from light and humidity until such as lungs and brain [3]. Moreover, they used. diminish the risks of developing other diseases such as atrial fibrillation, deep vein thrombosis, Extract preparation myocardial infarction, stroke and pulmonary embolism [3,4]. Although anticoagulants such as The dried leaf powder was soaked in methanol at heparin, aspirin and warfarin are often used in a plant material: methanol ratio of 1:5 (w: v) for 6 clinical practice, some patients may develop days. The soaked samples were occasionally aspirin resistance. Therefore, natural sources of shaken to ensure proper mixing. The mixture anticoagulants (plants/herbs) may provide was then filtered using a muslin cloth and alternative and safer therapy for thrombotic Whatman filter paper. The filtrate was further disorders [3]. Herbal drugs have shown great concentrated using a rotary evaporator, to obtain efficacy in the treatment of many diseases. a semi-solid material i.e. crude methanol extract According to the World Health Organization (MCE). A portion of the crude extract was (WHO), about 25% of drugs used in the world dissolved in phosphate buffered saline (PBS) are plant-based [5]. Indeed, in many parts of the and used for biological screening. world, there is a significant preference for herbs over orthodox drugs for primary health care. The Fractionation of MCEs high cost of synthetic medicines and their limited supply to rural areas in many developing The MCEs were subjected to liquid–liquid countries have driven the continuous partitioning using solvents of increasing polarity dependence on traditional herbal medicines [6]. to obtain n-hexane, chloroform and ethyl acetate Medicinal plant-based products are used as raw fractions. The fractions were dried through material in several multibillion-dollar industries all solvent evaporation in a rotary evaporator under over the world [7]. reduced pressure. The genus Ficus of the Moraceae family has Phytochemical analysis about 800 species of woody trees, shrubs and vines [8]. These species are found mainly in Determination of total phenolic content (TPC) tropical areas, and are used in medical practice worldwide [8,9]. The medicinal plants of genus The TPCs of the plant extracts were determined Ficus contain triterpenes, polyphenols, using the Folin-Ciocalteau method as reported by flavonoids, sterols, alkaloids, coumarins and Harborne [12]. The principle involved in this other secondary metabolites [10]. These assay is that polyphenols are reduced by Folin- phytochemicals are used as anti-rheumatic, Ciocalteau reagent to produce a blue-coloured antidiabetic, anti-helminthic, digestive, mild- complex, the intensity of which is proportional to laxative and anti-dysentery agents [11]. In the the amount of polyphenol present. The present study, six species of genus Ficus, concentrations of polyphenols in the plant namely, Ficus benghalensis, Ficus elasticaa, extracts were calculated from a gallic acid Ficus palmata, Ficus religiosa, Ficus semicordata standard calibration curve. For the calibration and Ficus auriculata were screened for their curve, different concentrations of gallic acid anticoagulant potential. (12.5, 25, 50, 100, 200, and 400 μg/mL) were prepared. Then, 0.5 mL of each solution was EXPERIMENTAL added to 2.5 mL of 10 % Folin-Ciocalteau reagent and 2.5 mL of 7.5 % sodium carbonate Plant materials solution, followed by incubation at room temperature for 1 h. Thereafter, the absorbance Leaves of F. benghalensis, F. elastica, F. of the solution was read at 765 nm in a UV- palmata, F. religiosa, F. semicordata, and F. visible spectrophotometer. The readings were Trop J Pharm Res, June 2019; 18(6): 1246 Ambreen et al taken in triplicate for each sample and the mean Evaluation of anticoagulant potential value was calculated. The same procedure was used for plant extracts. The TPC content was The anticoagulant potential of each plant expressed as mg GAE/g of plant extract as sample was tested on human blood sample. shown in Equation 1: For this purpose, six healthy human donors were selected. Care was taken to ensure that TPC = (C x V) ………………. (1) the blood donors had not taken any drugs for m at least seven days prior to sampling period. The blood samples were collected in where C is the concentration of gallic acid Eppendorf tubes containing
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