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(Ordu) in Turkey on the Effects of Tyrosinase Journal of Pharmaceutical Research International 25(6): 1-10, 2018; Article no.JPRI.47707 ISSN: 2456-9119 (Past name: British Journal of Pharmaceutical Research, Past ISSN: 2231-2919, NLM ID: 101631759) Biological Screening of Traditional Medicinal Plants from Villages of Akkuş (Ordu) in Turkey on the Effects of Tyrosinase Merve Badem1, Nuriye Korkmaz1*, Sıla Ozlem Sener2, Seyda Kanbolat1, Ufuk Ozgen2, Serhat Sevgi3, Rezzan Aliyazicioglu1 and Maksut Coskun4 1Department of Biochemistry, Faculty of Pharmacy, Karadeniz Technical University, 61080 Trabzon, Turkey. 2Department of Pharmacognosy, Faculty of Pharmacy, Karadeniz Technical University, 61080 Trabzon, Turkey. 3Department of Pharmacology, Faculty of Pharmacy, Karadeniz Technical University, 61080 Trabzon, Turkey. 4Department of Pharmaceutical Botany, Faculty of Pharmacy, Ankara University, 06100 Ankara, Turkey. Authors’ contributions This work was carried out in collaboration between all authors. Authors UO, MB, NK, SÖS, SK, SS and RA designed and performed the study, and wrote the first draft of the manuscript. Author MC helped to deposite the herbariums of the plants. All authors read and approved the final manuscript. Article Information DOI: 10.9734/JPRI/2018/v25i630118 Editor(s): (1) Dr. R. Deveswaran, Associate Professor & Head, Drug Design and Development Centre, Faculty of Pharmacy, M. S. Ramaiah University of Applied Sciences, India. Reviewers: (1) Bogumil E. Brycki, Adam Mickiewicz University, Poland. (2) Esther Van Der Werf, Taylor’s University, Malaysia. (3) Mustafa Sevindik, Akdeniz University, Biology, Turkey. Complete Peer review History: http://www.sdiarticle3.com/review-history/47707 Received 17 November 2018 Original Research Article Accepted 26 February 2019 Published 11 March 2019 ABSTRACT In the present study, ethnomedicinal uses of the plants naturally growing in 35 villages of Akkuş District have been determined. Informations about the uses of the plants have been obtained from villagers using a questionnaire. At the end of the identification of the plants collected from villages, 58 taxa belong to 32 families have been determined. Furthermore, tyrosinase enzyme activity studies of methanolic extracts of all the species carried out spectroscopicallly. Eventually, the _____________________________________________________________________________________________________ *Corresponding author: E-mail: [email protected]; Badem et al.; JPRI, 25(6): 1-10, 2018; Article no.JPRI.47707 plants has been demonstrated to be used as folk medicine, food animal feed, ornamental, stuff, dyeing, odour, insecticide and fishing. According to the biological screening studies, some species have been indicated to have inhibitory effect, but the others have activator effect on tyrosinase enzyme. Keywords: Akkuş; biological screning; ethnomedicinal; folk medicine; tyrosinase enzyme. 1. INTRODUCTION Irano-Turanian, Euro-Siberian and has a rich flora [8]. Turkey is a leading country in terms of Melanin is known to be one of the major medicinal and endemic plants in the World [9]. It pigments for skin and hair color of mammals. is known that about 10000 flowering and fern Melanin have been synthesized in differentiated plant species has naturally grown in Turkey, and cells such as melanocytes in the skin, retinal 30% of them are endemic [10-12]. epithelium, and central nervous system in mammals [1-3]. Besides, our country which hosted many civilizations has a rich cultural heritage and a Tyrosinase, being copper-containing in active wealth of ethnobotanical datas [13]. The site, is known to be a member of the polyphenol traditional knowledge on plants and their uses oxidase enzyme family, which is an key enzyme has been disappearing in recent years because in charge of melanin biosynthesis. The of urbanization, increased migration to urban tyrosinase enzyme inhibitors from natural areas, developments in technology, and health resources have been investigated by many services easier accessibility [14-16] Therefore, researchers but there is not been enough studies any information about uses of the plant in our on the tyrosinase enzyme activators [3]. daily life should be recorded as soon as possible. Defect of melanin synthesis have been Ordu province has been located (40°18’-41°08’ associated with pigmentation disorders. N, 36°52’-38°12’ E) in Euro-Siberian Decreasing of melanin level have been caused phytogeographic region [17]. However There are various skin diseases suh as psoriasis, vitiligo, the plenty of studies on the folk medicine in the because of hypopigmentation [4]. Using the Black Sea region, some of which is agents having tyrosinase activator effect is an ethnobotanical research about Ordu, any study in efficient approach for treatment of Akkus district has not been found [9,14,18-26]. hypopigmentation disorders. Hyperpigmentation Also, the effects of tyrosinase enzyme of the knowed increasing production of melanin have plants naturally growing in 35 villages of Akkuş been induced some disorders like actinic District and used as folk medicine have been damage, melasma, freckle and age-related explored for the first time in this study. stains The tyrosinase inhibitors can be used as a remedy for these diseases [5]. 2. MATERIALS AND METHODS Also, Tyrosinase inhibitors have been believed to be used to cure ailments related to neurotoxicity 2.1 Field Trips like Parkinson. Tyrosinase have been give rise to accumulating of oxide-dopamine derivatizations The materials of our research includes plant due to neuronal damage [6]. samples collected from 35 villages of Akkus District of Ordu province in Turkey. Plant Ethnobotany is described as “the use of plants by samples were obtained by visiting the research the local population” by John W. Harsberger and area twice between July and September 2016. A etnobotanical studies are important to determine questionnaire form (name, surname, age, the relationship between ethnobotany and plants telephone numbers, local names of plants, parts [7]. In Turkey, the interest in ethnobotanical used, preparation methods and the purpose of studies beginning in the early 19th century has using plants) was prepared for the participants. been observed to be higher in recent years [8]. The participants were visited at their homes and asked to them to fill out the questionnaire form. Turkey, located at the junction of three different After each interview, plant samples were phytogeographic regions, namely Mediterranean, collected with assistance from the participants. 2 Badem et al.; JPRI, 25(6): 1-10, 2018; Article no.JPRI.47707 2.2 Study Area spectrophotometric method in a microplate reader. The percentage of tyrosinase inhibitory Akkus is located in the inner parts of the Middle activity was calculated using the formula follows: Black Sea in Turkey. The height of the sea is 1340 meters and has a rough land (Fig. 1). The % Inhibition = [[(A-B)-(C-D)] / (A-B)] x 100 Akkus region includes 6 towns, 35 villages and 15 neighborhoods. Continental climate prevails in The results were given as IC50 levels. the region [27]. 2.4.4 Tyrosinase enzyme activation 2.3 Identification of Plants Tyrosinase Enzyme Activation was determined The plant samples collected from the research employing various concentrations of 8- area were identified using references, i.e. Flora Methoxsalen (8-MOP) solutions as standard [31]. of Turkey and the East Aegean Islands Tyrosinase solution (46 U/mL), methonolic plant [11,28,29]. Identifications were performed by two extract (500, 100, 50 and 25 μg/mL) were of the authors (MB and UO). The dried plant prepared. 120 μL of 0.2 M phosphate buffer (pH samples are deposited at the Herbarium of the 7.0), and 40 μL tyrosinase solution for A wells; Faculty of Pharmacy of Ankara University. 160 μL of 0.2 M phosphate buffer (pH 6.8) for B wells; 80 μL of 0.2 M phosphate buffer (pH 6.8), 2.4 Studies on the Tyrosinase Enzyme 40 μL tyrosinase solution and 40 μL sample solution for C wells; 120 μL of 0.2 M phosphate 2.4.1 Chemical materials used in experiments buffer (pH 7.0) and 40 μL sample solution for D wells were added and mixed in a 96-well plate Methanol (CH3OH-Merck, 106009), Tyrosinase and incubated for 10 min at 23°C. Then, 2.5 mM (Sigma, T3824-25KU), kojic acid (Sigma, K3125- L-DOPA solution (40 μL) was added to all wells 5G), potassium dihydrogen phosphate and incubated for 10 min at 23°C. The (NaH2PO4-Sigma Aldrich, 7558-80-7), disodium absorbance of the reaction mixture was hydrogen phosphate anhydride (Na2HPO4-Sigma determined at 490 nm using the Aldrich, 10039-32-4), L-DOPA (Sigma, D9628- spectrophotometric method in a microplate 5G) and 8-Methoxsalen (Roth, 5497.2) were reader. The percentage of tyrosinase enzyme used in the experiments. activation was calculated using the formula follows: 2.4.2 Preparation of the extract % Activation = [[(A-B)-(C-D)] / (A-B)] x 100 Methanol extract (10 mg/mL) of each species from Akkus was prepared. Diluted samples were The results were given as AC50 levels. obtained from methanol extract with potassium phosphate buffer (pH 6.8) at concentrations of 3. RESULTS AND DISCUSSION 25, 50, 100 and 500 µg / mL. 3.1 The Results of Ethnobotanical 2.4.3 Tyrosinase enzyme inhibition Studies Tyrosinase inhibitory activity was determined The plants naturally growing in 35 villages of employing various concentrations of kojic acid Akkuş District has been showed to be used as solutions as standard (30). Tyrosinase solution folk medicine (30 taxa), food (21 taxa), animal (46 U/mL), methonolic
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