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NorCal Open Access Publications NORCAL Journal of Allied Pharmaceutical OPEN ACCESS PUBLICATION Sciences Volume 1; Issue 1 Karakaya S et al. Research Article Identification and Quantification of Coumarins in Four Ferulago Species (Apiaceae) Growing in Turkey by HPLC-DAD Songül Karakaya1*, Hilal Özbek1, Zühal Güvenalp1, Hayri Duman2, Cavit Kazaz3 and Ceyda Sibel Kılıç4 1Department of Pharmacognosy, Faculty of Pharmacy, Atatürk University, Erzurum 25240, Turkey 2Department of Biological Sciences, Faculty of Art and Sciences, Gazi University, Ankara 06500, Turkey 3Department of Chemistry, Faculty of Science, Atatürk University, Erzurum 25240, Turkey 4Department of Pharmaceutical Botany, Faculty of Pharmacy, Ankara University, Ankara 06100, Turkey *Corresponding author: Songül Karakaya, Department of Pharmacognosy, Faculty of Pharmacy, Atatürk University, Erzurum 25240, Turkey, Tel: +90 4422315318; E-mail: [email protected] Received Date: 08 August, 2017; Accepted Date: 12 September, 2017; Published Date: 29 September, 2017 Abstract Turkey 19 of which are endemic. Therefore, Anatolia is considered to be the gene center of this genus [1]. F. blancheana, F. pachyloba Ferulago species have been used since ancient times for the treatment and F. bracteata are endemic perennial species, growing only in of intestinal worms, hemorrhoids and as tonic, digestive, Kayseri, Nigde and Gaziantep, Turkey, respectively [2,3]. aphrodisiac and sedative. They have also been used as salad or spice due to their special odors. Samples were analyzed on a ACE Ferulago species have been used in folk medicine for their 5 CN S/N-A21532 column (250 x 4.6 mm, 10 μm) with hexane aphrodisiac, digestive, tonic, sedative, antiseptic, carminative and and ethyl acetate as mobile phases at a flow rate of 1.0 mL x min-1 vermifuge properties as well as for the treatment of hemorrhoids, and the method was successfully validated according to ICH ulcers, snake bites, spleen diseases and headache [4,5]. Except guideline acceptance criteria for selectivity, linearity, precision, medicinal usage, they have been consumed as salad or spice due and accuracy. The structures of isolated compounds were to their special odor, also as food for goats and deer’s [6]. elucidated by detailed analyses of 1D and 2D NMR and Previous phytochemical studies indicated that Coumarins are the HR-ESI-MS data. Osthol (1), prantschimgin (2), felamidin (3) and most common metabolites on Ferulago species [7]. Coumarins umbelliferone (4) contents of roots and aerial parts from F. blancheana, have various biological activities such as anticancer [8,9], F. pachyloba, F. trachycarpa F. bracteata were found to be 0.39, antiinflammatory [10,11], anticoagulant [12,13], antiadipogenic 2.76, 1.26, 0.20, 0.10, 1.07, 0.26, 0.05; 0.14, 2.83, 0.56, 0.44, 0.23, [14], antitubercular [15], antihyperglycemic [16,17], antiviral [18], 0.59, 0.26, 0.11; 0.11, 2.45, 1.29, 0.24, 0.27, 0.72, 0.43, 0.26 and antifungal [19,20], antibacterial [21], antihypertensive [22,23], 0.30, 2.28, 0.48, 0.21, 0.40, 0.83, 0.78, 0.05 %, respectively. A anticonvulsant [24], antioxidant [25,26], neuroprotective [27] new Coumarin, peucedanol-2'-benzoate (5) was only found to be and antidiabetic [28,29]. from roots of F. bracteata 1.12 %. This study aims to give first report to identify and establish the Keywords content of several Coumarins like osthol, prantschimgin, felamidin, umbelliferone in F. blancheana, F. pachyloba, F. trachycarpa Apiaceae; Coumarin; Ferulago; Peucedanol-2′-benzoate; and F. bracteata by using HPLC-DAD. Also, a new Coumarin, Validation; HPLC peucedanol-2′-benzoate was isolated from roots of F. bracteata and established the content of in all species. Furthermore, the method Introduction was validated on the basis of selectivity, linearity, precision, and accuracy. To the best of our knowledge, this is the first report on Ferulago W Koch, belonging to Apiaceae family, is represented the HPLC analysis and isolation of Coumarins from F. blancheana, by approximately 50 taxa throughout the world and 34 taxa in F. pachyloba, F. trachycarpa and F. bracteata. NorCal Open Access Publications .01. Citation: Karakaya S, Özbek H, Güvenalp Z, Duman H, Kazaz C, et al. (2017) Identification and Quantification of Coumarins in Four Ferulago Species(Apiaceae ) Growing in Turkey by HPLC-DAD. J Allied Pharm Sci 2017: 35-42. DOI: https ://doi.org/10.29199/JAPS.101016. Material and Methods respectively and identified by Prof. Dr. Hayri Duman, a plant taxonomist in the Department of Biological Sciences, Faculty of Plant material Art and Sciences, Gazi University. The voucher specimens are kept in the Herbarium of Ankara University, Faculty of Pharmacy The aerial parts and roots of Ferulago blancheana, F. pachyloba, F. (Herbarium numbers are AEF 26673, AEF 26674, AEF 26677 and trachycarpa and F. bracteata were collected during flowering time AEF 26676, respectively). Collected locations of these species in 2014 from Kayseri, Niğde, Antalya and Gaziantep (Turkey), are given in table 1. Species Collected Location Voucher Numbers F. blancheana B6 Kayseri: Between Pınarbası-Sarız, 4 Km to Sarız, rocky slopes, 1633 m, 13.07.2014. AEF 26673 F. pachyloba C5 Nigde: Camardı Demirkazık village, Cimbar channel, rocky slopes, 1696 m, 13.07.2014. AEF 26674 F. trachycarpa C3 Antalya: Akseki-Seydisehir road 35. Km, calcareous slopes, 1785 m, 12.07.2014. AEF 26677 F. bracteata C6 Gaziantep: Nurdagı Antep road 22. Km, calcareous cliff slopes, 1642 m, 14.07.2014. AEF 26676 Table 1: Collected locations of Ferulago species. Reagents and chemicals TLC analyses were carried out on silica gel F 254 TLC plates aluminum sheets (Merck). Compounds were detected by UV Hexane and ethyl acetate were HPLC-grade (Sigma, Germany). fluorescence and spraying 1% 2H SO4 reagent, followed by heating Analytical grade solvents of dichloromethane, ethyl acetate at 105°C for 1-2 min. (Merck, Darmstadt, Germany), n-butanol (Analar NORMAPUR, Fontenay, France), methanol (Sigma, France), sulphuric acid Extraction, fractionation and isolation (Merck, Germany), and also silica gel (Merck-737, Germany) were used. Osthol, prantschimgin, felamidin and umbelliferone Extracts were obtained from the aerial parts and roots of Ferulago were isolated from the dichloromethane extracts of F. blancheana, blancheana, F. pachyloba, F. trachycarpa and F. bracteata with F. pachyloba, F. trachycarpa and F. bracteata roots using silica gel Methanol (MeOH). Aerial parts and roots were grounded and macerated (Heidolph MR3001, Germany) for 8 hours/3 days with columns. NMR spectra were recorded on a Varian Mercury Plus at 1 13 methanol in a water bath not exceeding 45°C using a Heidolph 400 MHz for H NMR and 100 MHz for C NMR by using TMS mechanical mixer (300 rpm). The extracts, filtered and concentrated as the internal standard. The chemical shifts were measured in till dryness using a Heidolph rotary evaporator (Heidolph VV2000, CDCl3. Mass spectra were recorded on a Waters Micromass ZQ Germany), were dispersed in methanol-water (1:9) and fractionated Mass Spectrophotometer ESI and Agilent 6530 Accurate- Mass three times with 400 mL of dichloromethane, ethyl acetate, n-butano Q-TOF LC/MS. UV spectra were measured using Thermo Scientific l and aqueous residue in a separatory funnel, respectively. Each fraction Multiskan GO microplate and cuvette spectrophotometer. IR was then concentrated to dryness. Amounts of the powdered plants spectra were run on a Bruker VERTEX 70v FT-IR Spectrophotometer. and obtained extracts are given in table 2. Species Used parts Powdered MeOH CHCl EtOAc n-BuOH Aqueous (g) (g) (g) (g) (g) residue (g) F. blancheana Root 750 86.62 28.52 2.32 12.24 23.35 Aerial part 50 3.22 1.89 0.46 0.57 0.39 F. pachyloba Root 600 83.25 23.63 1.53 13.13 21.29 Aerial part 50 3.32 1.78 0.45 0.59 0.45 F. trachycarpa Root 450 86.77 26.29 2.41 13.55 22.08 Aerial part 50 3.41 1.67 0.5 0.61 0.55 F. bracteata Root 450 60.94 17.96 2.44 14.98 13.98 Aerial part 50 3.65 1.55 0.61 0.59 0.61 Table 2: Amounts of the powdered plants and obtained extracts. As a result of the previous bioguided fractionation study [30], (Fr. A-E) were obtained. Repetitive silica gel column chromatography the effective dichloromethane extract was first submitted to a with n-hexane-ethyl acetate (90:10 and 95:5) solvent system on silica gel column (Merck 737, 70-230 Mesh) and eluted with Fr. A gave compound 1 (220 mg). Eluting with n-hexane-ethyl a gradient of n-hexane:ethyl acetate (100:0 → 0:100, v/v) and acetate (80:20) over silica gel column of Fr. B gave compound 2 ethyl acetate:methanol (100:0 → 0:100, v/v), and five fractions (420 mg). Fr. C was fractioned by column chromatography over J Allied Pharm Sci 2017: 35-42. .02. Citation: Karakaya S, Özbek H, Güvenalp Z, Duman H, Kazaz C, et al. (2017) Identification and Quantification of Coumarins in Four Ferulago Species (Apiaceae) Growing in Turkey by HPLC-DAD. J Allied Pharm Sci 2017: 35-42. silica gel using n-hexane:ethyl acetate mixtures (70:30 and 90:10) and the calibration curve was established by plotting peak areas consecutively and compound 3 was obtained (330 mg). Fr. D was against the curve equation. The extract was transferred into submitted on a silica gel column using n-hexane:ethyl acetate a 10 mL volumetric flask which was made up to its volume (65:35) and the resulting fraction was chromatographed on silica with ethyl acetate and filtered through a 0.45 µm Econofilter gel column using n-hexane:ethyl acetate (90:10) to give compound (Agilent Technologies, USA) prior to injection into the HPLC 4.