مکانیسم فعالیت آنتی‌اکسیدانی اسید جنتیسیک در پایداری اکسیداتیو روغن‌های زیتون و سویا

Introduction: Lipid oxidation is the oxidative deterioration of unsaturated fatty acids via an autocatalytic radical chain process that leads to the deterioration both the sensory and nutritional quality of lipids. Lipid oxidation has adversely consequences for the quality of lipid and human health by producing a wide range of harmful reactive radicals. Using antioxidants is the most important defense means to delay or slow rate of lipids oxidation by scavenge chain-propagating peroxyl radicals. Recently, the interest in natural antioxidants has been increased due to the reported toxicologically negative effects of the synthetic antioxidants.
The most powerful types of natural antioxidants are the phenolic acids, which are widely found as secondary metabolites in plant kingdom acids. A relationship between the antioxidant activities and the structures of the phenolic acids were the object of a number of investigations. The number of hydroxyl groups and the position of these groups in the phenolic ring have a significant influence in antioxidant activity of phenolic acids. The activity increases in the following order: monohydroxyo< dihydroxy< trihydroxy. Gentisic acid (2,5-dihydroxybenzoic acid) is one of the interesting subset of dihydroxybenzoic acids, present in many natural sources such as citrus fruits, grapes, olive, peanuts and herbs. The antioxidant activity of the Gentisic acid was the object of lot investigations. Gentisic acid has been reported to has anxiolytic, antirheumatic, anticarcinogenetic, anti-inflammatory and antimutagenic properties. The capacity of Gentisic acid to prevent lipid oxidation is related to its mechanism of antioxidant activity. The relative positions of the two hydroxyl groups in Gentisic acid phenolic ring have significantly influence in antioxidant property.
Material and methods: Purifed triacylglycerols of sunflower and olive oils were obtained by removing all pro- and anti- oxidants by adsorption chromatography: 130 g of oils were purified twice by passing in a glass column (25 × 2.5 cm i.d), packed with 70 g of aluminum oxide 60 activated at 200 °C for 3 h in bottom and, 15 g of silica gel activated at 160 C for 3 h in upper layer. Triacylglycerols were drawn in dark through the column by suction without solvent. Then soybean and olive oil samples, containing different concentrations of the Gentisic acid (0.02, 0.04, 0.08, 0.16 and 0.32 wt. %) were prepared by adding aliquots of its solutions in purified acetone. The solvent was removed under nitrogen. 1-mm layer of the prepared oils samples (4 g) were oxidized in a Petri dish having a diameter of 9 cm Under these conditions, the process took place in a kinetic regime, i.e., at a sufficiently high oxygen concentration which the diffusion rate does not influence the oxidation rate. Oxidation was performed in dark at 60, 80, 100 and 120 °C.
The oxidation process was followed by withdrawing samples at certain time intervals and subjecting them to spectrophotometric determination of the peroxide value (PV) as primary oxidation products and different kinetic parameters i.e. stabilizing factor (F), oxidation rate ratio (ORR), and antioxidant activity (A). In order to PV measurement, the vegetable oil samples (≤0.01–0.30 g) were added to a glass tube containing 9.8mL chloroform–methanol (7:3 v/v) and were vortexed for 2–4 s. 50mL of ammonium thiocyanate solution (30 % w/v) was added the sample was mixed on a vortex mixer for 2–4s. Then iron (II) chloride solution [50mL, (0.4g barium chloride dehydrate dissolved in 50mL H2O) + (0.5 g FeSO4·7H2O dissolved in 50mL H2O) + 2mL 10 M HCl], with the precipitate, barium sulfate, filtered off to produce a clear solution]) were added, and the sample was vortexed for 2-4 s. After 5min incubation at room temperature, the absorbance of the sample was read against a blank sample (containing all the reagents except the sample) at 500nm (UV-VIS spectrophotometer, Model 160A Shimadzu, Kyoto, Japan). Results were reported as milliequivalents of oxygen per kilogram of oil. Kinetic curves of peroxide accumulation were plotted. The x-coordinate of intersection point of two straight lines fitted on the initiation and propagation stages of the oil oxidation was calculated as induction period (IP). Mechanism of antioxidative action was determined according to kinetic parameters and the based on the participation of Gentisic acid molecules and radicals in a series of reactions).
Results and discussion: Gentisic acid as a phenolic antioxidant was effectiveness during autoxidation in both lipid systems (i.e. olive and soybean oil) without reactive free radicals production. The dependence of the parameter F on antioxidant concentration is linear only in the case of Gentisic acid-inhibited sunflower oil oxidation that showed the participation of antioxidant molecules mainly in reaction 7. The absence of F linearity in other treatments (α-Resorcylic acid in both olive and sunflower oils oxidation and Gentisic acid in olive oil oxidation) is due to the participation of the inhibitors molecules in the side reactions other than the main reaction of chain termination 7, namelyreaction 11 or/and 12. The mean rate of antioxidant consumption increased as the unsaturation degree of the soybean oil increased. Despite having the more appropriate performance in the olive oil sample, which normally contains higher amounts of oleic acid. The overall performance of Gentisic acid was attributed to the main reaction of chain termination (ROO• + InH ® ROOH + In•) as competed with the main oxidation reaction of chain propagation (ROO• + RH ® ROOH + R•). Due to the higher percentage of non-saturated fatty acids in soybean oil, antioxidant activity of gentisic acid promoted with increased concentrations, while in olive oil because higher content of monosaturated fatty acid (e.g. oleic acid), Gentisic acid participate in side oxidation reactions (InH + ROOH ® In • + R • + H₂O) as prooxidant in high concentration.
Conclusion:The present study was performed to elucidate the mechanism of antioxidative action of Gentisic acid. It was concluded that Gentisic acid as a phenolic antioxidant was effectiveness during autoxidation in both lipid systems (i.e. olive and soybean oil) without reactive free radicals production. The mean rate of antioxidant consumption increased as the unsaturation degree of the oil system increased. Despite having the more appropriate performance in the olive oil sample, which normally contains higher amounts of oleic acid.