SUPPLEMENTARY MATERIAL Evaluating the Potential of Natural Curcumin for Oxidative Stability of Soybean Oil NaeemeEshghiª, Maryam Asnaashariᵇ1, Mohammad Hossein Haddad Khodaparastᵇ, Fereshteh Hosseini. ͣ Iranian Applied Research Center for Public Health and Sustainable Development (IRCPHD), North Khorasan University of Medical Sciences, Bojnurd, Iran; ᵇ Food Science and Technology Department, College of Agriculture, Ferdowsi University, Mashhad, Iran; ᶜ Department of Food Additives, Iranian Academic Centre for Education Culture and Research (ACECR)- Mashhad branch, Iran The active substance of turmeric rhizome (curcumin) was extracted and identified with TLC and NMR. To evaluate the antioxidant efficiency of different concentrations of curcumin (120, 160 and 200 ppm) in comparison with TBHQ (120 ppm), α-tocopherol (200 ppm) in two temperatures (25 and 55 ºC) under dark and light conditions during 90 days, the soybean oil with no added antioxidant was used as a food model and its fatty acids profile was determined by GC. Results showed increasing curcumin concentration leads to decreaseoxidation rate significantly. The soybean oil containing curcumin at 25˚C in darkness which the lowest rate of increased of PVs indicated the same antioxidant activity with αtocopherol and TBHQ. Moreover, the results of acid value (AV) and iodine value (IV) showed samples with curcumin were more effective to avoid oxidation at both temperatures in comparison to control. However, lightness did not have has not any significant effect. Keywords: Curcumin; Natural antioxidant; Soybean oil; Turmeric 1. Materials Soybean oil with no added components was supplied from Damoon (Fariman, Iran). Rhizome of turmeric provided by local market. α-tocopherol and TBHQ as the control antioxidants and standard curcumin purchased from sigma company. All chemicals and solvents used in this study were of analytical reagent grade and purchased from Merck and Sigma Chemical Companies. 2. Methods 1 Corresponding author. Email: email@example.com 2.1. Extraction of curcumin The rhizomes of turmeric (Curcuma longa L.) were peeled, powdered and sieved then 500 g dried powder was steeped for 48 hours in hexane in a shaker with 750 rpm. The combined hexane solution was filtered and evaporated at a reduced pressure to produce turmeric oil in the form of a yellow oil. To increase the extraction yield, this operation was done twice. The powder from the hexane extraction was further steeped overnight in 95% ethanol in a shaker (750 rpm) and this process was repeated. The combined alcoholic extract was concentrated in a rotary evaporator. Then concentrated mixture was poured in dish to eliminate remained solvent in the room temperature in darkness. And then 1:5 mixture of water: ethanol added to sediment. After filtration and drying at room temperature, curcumin powder was obtained (Apisarlyakul et al. 1995). 2.2. Identification of the curcumin A dichloromethane solution (5%) of the extracted curcumin and its standard (50 mg/plate) was streaked using a thin-layer chromatography (TLC) applicator on a precoated silica gel plate 2×10 cm which had been activated for 15 min at 100°C. then the plate was developed in the ascending direction for 10 cm with the solvent system dichloromethane: methane (9.9: 0.1, by volume) ( Almeida et al. 2005). The NMR spectra were recorded by an NMR instrument (Bruker AC-80) at 300 MHz at room temperature, using methanol as a solvent. A solution was prepared by dissolving 10 mg of curcumin in 0.5 mL of methanol to NMR tube. The samples were prepared freshly and carefully protected from light during the NMR experiments. 2.3. Fatty acid composition of soybean oil Fatty acid composition of the oil samples was determined by gas-liquid chromatography and was reported in relative area percentages. Fatty acid composition of the soybean oils were transesterified into their corresponding FAME by vigorous shaking of a solution of oil in hexane (0.3 g in 7 mL) with 2 mL 7 N methanolic potassium hydroxide at 50-55 °C for 10 min. After shaking, the solution was allowed to settle for 5 min. The upper layer was collected for GC analysis after mixing with anhydrous natrium sulphate and filtering. The FAME were identified using HP-5890 chromatograph (Hewlett-Packard, CA, USA) equipped with a CP-SIL 88 (Supelco, Bellefonte, PA, USA) capillary column of fused silica, 60 m _ 0.32 mm i.d., 0.2 mm film thickness, and a flame ionization detector (FID). Helium was used as carrier gas with a flow rate of 1 ml/min. The oven temperature was maintained at 195 °C, and that of the injector and the detector at 210 °C (Metcalf et al. 1996). 2.4. Preparation of oil samples The soybean oil was mixed for 15 min separately with 120, 160 and 200 ppm of the curcumin and 200 ppm of α-tocopherol and 120 ppm of TBHQ as control antioxidants and then devided in dark and light glasses. The glasses were washed thoroughly with distilled water and 0.5% EDTA and dried in 180˚C before usage. After preparation of oil samples containing antioxidant, two set of dark and light glasses stored in 25˚C and 55˚C in incubator equipped with the optical system. progress of oxidation was monitored by determination of the peroxide value (PV), acid value (AV) and iodine value (IV) in determined interval for 90 days. 2.5. Calculated oxidisability value The calculated oxidisability (Cox) value of the oils was calculated by the percentage of unsaturated C18 fatty acids, applying the following equation proposed by Fatemi and Hammond (1980): Cox = [1(18:1%) + 10.3(18:2%) + 21.6(18:3%)] / 100 2.6. Peroxide value Determination of PV was made following the AOCS official methods Cd 8-53 (AOCS 1998). In this method, a solution of oil (5± 0.05 g) in 30 Ml acetic acid – chloroform with 0.5 ml saturated potassium iodide solution and titration with 0.1 N sodium thiosulfate. 2.7. Iodine value The iodine value (IV) was determined according to the AOAC Official Method 920.158 (Hanus method). Iodine chloride was used for double- bond saturation analysis and the consumed iodine was measured by titration with 0.1 M standard sodium thiosulfate solution (AOAC 2005). 2.8. Acid value The acid value (AV) was determined according to the AOCS (1993) Official Method Cd 3d-63. In this method, for determination of FFA, 15±0.01 gr of each oil sample was placed into a 250 ML Erlenmeyer flask and dissolved in 70 ml reagent grade alcohol containing phenolphthalein indicator and then each oil solution was subsequently titrated with the potassium hydroxide solution. 2.9. Statistical analysis All determinations were carried out in triplicate, and data were subjected to analysis of variance (ANOVA). ANOVA analyses were performed according to SAS software. Significant differences between means were determined by Duncan’s multiple range tests; p values less than 0.05 were considered statistically significant. References Almeida LP, Cherubino APF, Alves RJ, Dufosse L, Gloria MBA. 2005. 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