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2 nd International Non-Wood Products Symposium 8-10 September 2011 - Isparta/TURKEY , total phenolics and radical scavenging activity of metanolic extract of defatted seed from B. orphanidis growing wild in Mediterranean region of Turkey. 2. MATERIAL AND METHOD 2.1.Plant Material The aerial parts (calyx) of B. orphanidis were collected from C3 Gazipasa Maha Yaylasi Antalya district in Mediterranean region of Turkey, on Cedrus libani and Abies cilicica forest, about 1850 m above the sea level at the end of June 2010 (Fig. 1). Seeds of B. orphanidis were used as materials in present study. Firstly, all seeds samples were air-dried at room temperature in the dark and stored in a deep fridge at -20 o C. Before the analyses, the samples were oven-dried at 105 o C. A voucher specimen is deposited at Herbarium of the Forest Botany Department of Suleyman Demirel University (ISPO) as Fakir 4857. 2.2.Determination of oil yield Dried seed were crushed in a grinder for two min, but at 15 s intervals the process was stopped for 15 s to avoid heating the sample. Oil content was determined according to AOCS method (AOAC, 1990). The powdered grape seeds were extracted in a Soxhlet extractor (Büchi Universal Extraction System B-811, Germany) for 6 h with 150 mL of hexane at 60 o C. Data presented are average of three measurements. The oil was stored in a deep fridge at -20 o C until use. 2.3.Determination of phenolic yield After defatting from dried and crushed seed, seeds were dried again at 105 o C to evaporate hexane. And then the defatted seed powder was reextracted in a Soxhlet apparatus for 6 h with 200 ml of methanol at 60 o C. The extracts were concentrated by rotary evaporation under vacuum at 70 o C to get crude extracts. Data presented are average of three measurements. The oil was stored in a deep fridge at -20 o C until use. a fused silica capillary column (MN FFAP (50 m x 0.32 mm i.d.; film thickness 0.25 μm). It was operated under the following conditions: oven temperature program, 120 o C for 1 min. raised to 240 o C at a rate of 6 o C/min and then kept at 240 o C for 15 min; injector and detector temperatures, 250 and 260 o C; respectively, carrier gas, helium at flow rate of 15 cm/s; split ratio, 1/20 mL/min. Fatty acids were identified by comparing retention times with standard compounds. Five fatty acids were considered in this study. These were palmitic (C16:0), stearic (C18:0), oleic (C18:1, n:9), oleic (C18:1, n:7) and linoleic (C18:2) acids expressed as percentages of fatty acids. Data presented are average of three measurements. 2.5.Determination of tocopherols In the tocopherol analyses, the HPLC method of Lampi et al. (1999) was modified. Tocopherols (α, β, γ and δ-tocopherol) were evaluated by highperformance liquid chromatography with direct injection of an oil extract in a mixture of heptane:tetrahydrofuran (THF) (95:5) solution. Detection and quantification was carried out with a SCL-10Avp System controller, SIL–10ADvp Autosampler, LC-10ADvp pump, CTO-10 Avp column heater and fluorescence detector with wavelengths set at 295 nm for excitation and 330 nm for emission. The 150cm x 4,6 mm i.d. column used was filled with Supelcosil Luna, 5 (Supelco, Inc. Bellefonte, PA). The mobile phase consisted of heptane/THF (95/5) (v/v) at a flow rate of 1.2 mL/min and the injection volume 10L. The data were integrated and analyzed using the Shimadzu Class-VP Chromatography Laboratory Automated Software system. Standard samples of α, β, γ and δ isomers of tocopherol (Sigma Chemical Co., St. Louis, Mo., USA) were dissolved in hexane and used for identification and quantification of peaks. The amount of tocopherols in the extracts was calculated as mg tocopherols in kg oil extract using external calibration curves (r=0.999), which were obtained for each tocopherol standard. 2.4.Determination of fatty acids composition The fatty acid composition of the oil extracts was determined by gas chromatography (GC). Fatty acid composition was performed using a method as given by Marquard (1987). The chromatographic separation was performed in a Perkin Elmer Auto System XL gas chromatograph equipped with a flame ionizing detector (FID), and 97
2 nd International Non-Wood Products Symposium 8-10 September 2011 - Isparta/TURKEY 2.6.Determination of total phenolic content Total phenolic contents of the extracts were determined spectrophotometrically according to the Folin-Ciocalteu colorimetric method (Singleton & Rossi, 1965), calibrating against gallic acid standards and expressing the results as mg gallic acid equivalents (GAE) /g extract. Data presented are average of three measurements. Fig. 1. A: Habitat, B: Ripe fruits, C: Infrutescence, D: Habitus and inflorescence, E: Root, F: Coffee of fruit, G: Calyxes used as herbal tea (Photographs by FAKIR). 98
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