Phenolic Acid Quantitative Analysis In Rapeseed Oil And Its Inhibition Of Lipotoxicity In HepG2 Cells

Rapeseed oil is one of the major consumer vegetable oils in the world.Compared to other vegetable oils,enzyme-assisted-aqueous extracted rapeseed oil(EAO)contains high levels of phenolic compounds,especially phenolic acids and has significant antioxidant activity.Although there have been studies on the anti-inflammatory,anti-oxidation,anti-tumor and antiapoptotic effects of polyphenols in rapeseed and its products from the pharmacological point of view,compared with phenolic substances such as flavonoids,studies on phenolic acids activity in rapeseed oil are still relatively rare.A large number of studies have shown that some phenolic acids can significantly improve lipotoxicity,but as an important dietary polyphenol source in edible oils,the effect of phenolic acids in rapeseed oil on lipotoxicity has not been reported.Therefore,the phenolic acid of rapeseed oil was quantitatively identified by Ultra Performance Liquid Chromatography-tandem Quadrupole Mass Spectrometry(UPLC-MS/MS).Based on the lipoapoptosis model established by Oleic acid,the regulation of phenolic acids in rapeseed oil on HepG2 lipoapoptosis was explored in HepG2 cells in order to provide a theoretical basis for the study of its biological activity.(1)The phenolic substances in the aqueous enzymatic rapeseed oil were extracted and determined by Folin-Ciocalteu colorimetric method.The results showed that the total phenolic content of the aqueous enzymatic rapeseed oil was 253.47 � 2.15 ?mol GAE/g;UPLC-MS/MS technology was used to quantitatively analyze the phenolic extracts by internal standard method.The results showed that the highest content of phenolic substances in rapeseed oil was 2,6-Dimethoxy-4-ethenylphenol(Canolol,CA),and the content of the CA is 174.76 � 8.02 ?mol / g,accounting for more than 99% of the detection of phenolic acid,followed by Sinapic acid(SA),salicylic acid and syringic acid,with the contents of 0.96 � 0.03,0.16 � 0.00 and 0.17 � 0.10 ?mol/g respectively;1,1-diphenyl-2-tricrylhydrazyl(DPPH)was used to test the antioxidant capacity of the phenolic acids in vitro.The results showed that the antioxidant capacity of each phenolic acid was as follows: sinapic acid > Canolol > syringic acid > salicylic acid,and the half-inhibitory concentrations were 8.45,8.80,11.62 and 14.24 ?mol/L respectively..(2)Establish a lipoapoptosis model of HepG2 induced by Oleic acid(OA).After induction of HepG2 cells with different concentrations of OA for 24 h,it was found that when HepG2 cells were stimulated with 0.7 mmol/L OA for 24 h,cell viability decreased significantly,LDH cytotoxicity was at a high level;lipid deposition was significant,and intracellular triglyceride content was significant.At the same time,oxidative stress indicators Alanine Aminotransferase(ALT)and aspartate Aminotransferase(AST)release,lipid peroxide malondialdehyde(MDA)content increased significantly,superoxide dismutase(Superoxide Dismutase,SOD)activity decreased significantly;cell cycle was inhibited in G1-S phase,apoptosis was significant,total apoptotic rate increased significantly,and the model was established successfully.(3)Although the content of phenolic substances in the aqueous enzymatic rapeseed oil is high,it is still a trace substance.In order to study the effects and molecular mechanisms of phenolic acids in rapeseed oil on lipid metabolism and apoptosis induced by OA,SA and CA with the highest content and the strongest antioxidant capacity were selected for the lipoapoptosis model group.Intervene.Firstly,the safe concentration of the two phenolic acids was determined to be 500 ?mol/L by CCK-8 and LDH cytotoxicity experiments,and the model group cells were intervened at a safe concentration.The results showed that both SA and CA can reverse the decrease of cell viability caused by OA to different degrees,and significantly inhibit OA-induced cytotoxicity.In terms of lipid metabolism: from the biochemical indicators,different concentrations of SA and CA can promote intracellular lipid accumulation,increase intracellular TG content;in order to further explore the SA and CA on Oleic acid lipid metabolism from the genetic level Effects: The results showed that 10 ?mol/L SA treatment down-regulated the expression of SREBF-1 compared with the model group;100 ?mol/L SA treatment of ACOX2 and PPAR? and SCD-1 expression did not change,while down-regulating SREBF-1 expression,300 ?mol/L SA treatment up-regulated the expression of ACOX2 and PPAR? and SREBF-1 and SCD-1;10 ?mol/L CA treatment up-regulated the expression of SREBF-1 and SCD-1,and 100 ?mol/L CA treatment down-regulated the expression of ACOX2,PPAR? and SREBF-1,The expression of SCD-1 was up-regulated.300 ?mol/L CA treatment up-regulated the expression of ACOX2 and PPAR? and SCD-1,and down-regulated the expression of SREBF-1.This result indicates that both SA and CA can promote lipid deposition in HepG2 cells to varying degrees.In terms of oxidative stress induced by Oleic acid: different concentrations of SA and CA can reduce the cytotoxicity caused by Oleic acid,reduce the reactive oxygen species ROS induced by Oleic acid,and reduce the formation of lipid peroxide MDA.Increase the activity of SOD enzymes.In terms of apoptosis induced by oxidative stress: different concentrations of SA and CA can accelerate the cell cycle and reduce the total apoptosis rate.Further,the effects of SA and CA on oleate lipoapoptosis were further investigated.Compared with the model group,10,100,and 300 ?mol/L SA significantly down-regulated the expression of Bax and caspase-3 and upregulated the expression of Bcl-2.Bcl-2/Bax was significantly elevated,but the opposite result was observed for their upstream gene p53;in contrast,10 and 100 ?mol/L CA down-regulated Bax expression;meanwhile,10,100 and 300 ?mol/L CA can up-regulate Bcl-2 expression;10 and 100 ?mol/L CA can significantly increase Bcl-2/Bax;100?mol/L CA can significantly down-regulate p53 expression,but 300 ?mol/L CA significantly up-regulates p53 gene expression;10,100 ?mol/L CA significantly down-regulated the expression of caspase-3,while 300 ?mol/L CA significantly up-regulated the expression of caspase-3.The results indicated that SA may regulate Bax,Bcl-2 and caspase-3 through other pathways,thereby inhibiting cell apoptosis;low concentration of CA may inhibit apoptosis through p53-Bax-caspase-3 pathway.The high concentration of CA has a limited inhibitory effect on lipotoxic apoptosis induced by high Oleic acid.