The Formation Of 9,10-Epoxystearic Acid Generated From Oils During Frying Process And Its Effect On HepG2 Cells

Oils are susceptible to thermal oxidation during high temperature frying process and thus produce a series of lipid oxidation products.9,10-Epoxystearic acid(ESA)is produced by the oxidation of oleic acid during frying process.However,the formation mechanism of ESA during high temperature thermal oxidation and the effects of ESA on cells remain unclear.The generation of ESA and its distribution in frying oils as well as its effect on body health were investigated in the present thesis.Firstly,the conditions of separation and preconcentration based on solid phase extraction(SPE)of ESA were optimized,and the gas chromatographic detection method was evaluated.Secondly,the formation mechanism of ESA and its content in oils with different fatty acid composition during high temperature thermal oxidation process were determined.Lastly,the effects of ESA on the cytotoxicity,oxidative damage and lipid metabolism of HepG2 cells were investigated.The expression of genes involved in apoptosis and lipid metabolism was further studied to elucidate its effects on apoptosis and lipid metabolism in cells.The present study provides novel insights into the formation of ESA over deep-frying process and its effects on toxicity and lipid metabolism in vitro.The main research contents and results were listed as follows:(1)In order to obtain good extraction efficiency and high analytical sensitivity,the factors affecting SPE sampling(SPE type,sorbent amount,eluent type and volume)of frying oils for ESA were optimized.In summary,an optimized SPE procedure was established for the extraction and concentration of epoxystearates in frying oils.Epoxystearates in samples were extracted by the silica SPE cartridge filled with 1g of sorbent and were eluted with 15 mL diethyl ether.At the same time,the TR-FAME 260M154 P column(60 m × 0.25 mm × 0.25 ?m)was applied in gas chromatographic and the method validation of quantitative analysis of ESA by GC was also evaluated.A good repeatability and accuracy were displayed,where the standard recovery of the method is above 89 %,intraday precision ranges from 0.93 %-2.35 % and interday precision ranges from 2.44 %-11.68 %.Thus,the improved sample preparation and detection method for ESA analysis could be highly accepted in the following studies.Further analysis on the ESA showed that ESA was not found in unfried oils,and the content of ESA in palm oil(PO)and sunflower oil(SO)increased as the frying time prolonged.Compared to the oil samples fried for 5 h,the ESA content in PO and SO fried for 50 h was increased by 94.70 % and 97.02 %,respectively,and the content of ESA in PO was always higher than that in SO.Besides,cis-ESA content in PO fried from 0 h to 15 h and SO samples was higher than trans-ESA,and the main ESA in PO samples after frying for 15 h was stable trans-ESA.It was also found that the loss rate of oleic acid in PO and SO fried for 10 h was similar,while after that the oleic acid loss in SO was higher than that in PO.And ESA content in PO was higher than that in SO when the oleic acid loss was same.The above results indicated that the oxidative degree and fatty acid composition of frying oils could affect the formation of ESA.(2)The ESA in frying oils was produced by thermal oxidation of oleic acid(OA).OA was applied in successive high temperature heating process.The formation of ESA during thermal oxidation process was studied using the electron spin resonance(ESR)spectra.Meanwhile,changes of free radicals and oxidative volatile in oxidized OA were also investigated.Results showed that the content of free radicals,volatile compounds and ESA in oxidized OA increased with the heating time prolonged,while the OA content showed the opposite trend simultaneously,and the OA content in 50 h heating OA samples decreased by 63.74 % compared to unheated OA.Alkyl radical was the mainly identified free radical in oxidized OA,and small amount of alkoxy radical was also exist.During the early stage of thermal oxidation in OA(0 h-30 h),the free radical content gradually increased and was relatively low,after that it increased significantly.In addition,the free radical content increased first and then decreased,and finally maintained at a certain level within the 28 min-detection time of ESR.The detected volatiles including octanal,nonanal,(E)-2-decenal,2-undecenal were produced by degradation of hydroperoxide formed by oxidized OA,and(E,E)-2,4-decadienal and 2,4-nonadienal were also identified in oxidized OA.ESA was not found in unheated OA.The ESA content was low in oxidized OA heated in the first 25 hours,then it significantly increased,and the trans-ESA content was higher than cis-ESA.Pearson correlation coefficient analysis was applied to analyze the free radicals and oxidation products of OA and to estimate the formation of ESA.It was concluded that OA lost hydrogen radicals to form alkyl radicals which could react rapidly with oxygen to form hydroperoxides,then the alkoxyl radicals were formed by the break of OO bonds of hydroperoxides and abstracted hydrogen from another OA to form ESA.(3)Effects of OA and ESA on cytotoxicity and oxidative damage of HepG2 cells were investigated in the present thesis.The results showed that although the cell viability decreased after treated with high concentration of OA,it remained above 57 %,indicating that OA showed little toxic effect on cells.While viability of cells treated with ESA decreased significantly with the increase of the concentration and treating time of ESA.The viability of cell treated with 500 ?M ESA for 48 h was only 24.41 %,indicating that ESA had toxic effects on HepG2 cells.Results of cell cycle experiments showed that ESA inhibited cell proliferation in the G0/G1 phase by increasing cells in the G0/G1 phase and decreasing cells in the S phase.Apoptosis experiments further elucidated the cytotoxic effect of ESA.Results revealed that cells treated with ESA for 12 h mainly induced the early apoptosis,while cells treated with ESA for 24 h and 48 h mainly caused the late apoptosis.Besides,the effects of OA and ESA on the level of intracellular reactive oxygen species(ROS)were also investigated.It was found that OA could not cause the increase of ROS levels in cells,while the intracellular ROS content induced by ESA presented a dose-and time-dependent increase.Notably,the intracellular ROS content induced by ESA for 48 h increased compared to the control group,but was much lower than the cells treated with the same concentration of ESA for 12 h and 24 h.Results of intracellular lipid peroxidation(MDA)and antioxidant capacity(SOD,CAT and GSH-Px)showed that OA had little effect on intracellular lipid peroxidation and antioxidant system,while MDA content in cells treated by ESA 12 h and 24 h increased significantly with the increasing concentration of ESA,the antioxidant enzyme content including SOD,CAT and GSH-Px decreased remarkably.The transcription study of genes involved in apoptosis showed that ESA could inhibit the expression of Bcl-2 gene and promote the expression of Bax gene as well as Cyt C,Caspase-9 and Caspase-3 gene.The above results showed that ESA reduced the antioxidant capacity and induced oxidative damage of HepG2 cells,and apoptosis induced by ESA maybe through a mitochondria-mediated endogenous pathway.(4)Effects of OA and ESA on lipid metabolism in HepG2 cells were investigated.Intracellular lipid droplet accumulation,triacylglycerol(TAG),total cholesterol(TC),and fatty acid composition were analyzed.The results showed that low concentration of OA(10 ?M-20 ?M)could not cause intracellular lipid accumulation.A small amount of lipid droplets formed in the cells as the OA concentration increased,meanwhile TAG and TC levels of cells treated by OA showed little difference with the control group.While the number of lipid droplets,TAG and TC content in ESA-induced cells presented a dose-and time-dependent increase.The content of palmitoleic acid and OA in cells treated by OA increased with the increasing concentration,while the content of palmitic acid,stearic acid and other unsaturated fatty acids decreased.Similar trends were also observed in cells treated with ESA for 12 h and 24 h,but the influences of ESA on intracellular fatty acid profile were much greater than OA-induced cells,and the ESA content in cells increased in a dose-and time-dependent manner.The intracellular lipid profile and the transcription level of genes involved in lipid metabolism showed that low concentrations of OA could not cause changes in the expression levels of genes involved in fatty acid metabolism in cells,while high concentration of OA slightly increased the expression Srebp-1c and Scd1 gene,and decreased the expression of PPAR?,Cpt1? and Acox1 gene in HepG2 cells.ESA significantly promoted the expression of Srebp-1c and Scd1 gene,and significantly inhibited the expression of PPAR?,Cpt1? and Acox1 gene.Our results indicated that ESA facilitated the fatty acid synthesis and suppressed fatty acid oxidation on peroxisomes and mitochondria.