Antioxidant Activity Of Main Pyrazine Compounds In Fujian Monascus Aged Vinegar

As a traditional Chinese fermented food,vinegar not only has unique flavor,but also has the functions of scavenging free radicals,anti-lipid peroxidation injury,lowering blood sugar,lowering cholesterol,lowering blood pressure,lowering blood pressure,bacteriostasis and sterilization,Osteoporosis and other physiological functions.Studies have found that pyrazines----the important products of Maillard reaction in food,is one kind of important flavor compounds in Chinese vinegar and Chinese liquor----the traditional Chinese fermentation products.Pyrazines and pyrazine derivatives are widely used in food(flavors and fragrances),medicine(pyrazinamide(2-methyl pyrazine as raw material)anti-tuberculosis drugs),and chemical industry.Among them,2,3,5,6-tetramethylpyrazine(TMP)has a good effect in the prevention and treatment of ischemic cerebrovascular disease,and is widely used in the treatment of ischemic cardiovascular and cerebrovascular diseases such as atherosclerosis,high blood pressure and stroke.The researches of TMP in the removal of reactive oxygen species in cells,protecting cells from injury are many.However,there are few studies on the antioxidation of other pyrazines(such as 2,5-dimethylpyrazine and 2,3,5-trimethylpyrazine)derived from vinegar sources.We would like to use Fujian Monascus aged vinegar as a carrier to study the antioxidant effect of its major pyrazines,TMP can be used as a control to compare the antioxidant activity of other pyrazines.It is expected to find out the antioxidation mechanism of the main pyrazine compounds in Fujian Monascus aged vinegar.The selection of topics can also lay a theoretical foundation for the development of novel functional foods riched in pyrazines.Headspace solid-phase microextraction combined GC-MS was used to qualitatively and quantitatively determine the volatile components of Fujian Monascus aged vinegar:Yongchun aged vinegar(eight years)and Yongchun aged vinegar(Gold Medal).2,3,5-trimethylpyrazine and TMP were the main pyrazine compounds in Yongchun aged vinegar(eight years)and Yongchun aged vinegar(Gold Medal),with the content(peak area percentage)of 0.13±0.09%,0.74±0.78%and 0.69±0.52%,0.33±0.17%,respectively.Alcohols,esters,ketones,acids,aldehydes,phenols,etc.are also detected.HepG2 cells were grown in growth medium(DMEM supplemented with 10%FBS,0,5,10,20,30,40,60,80 μg/mL 2,3,5-trimethylpyrazine and TMP)by inhibition 24、48、72 h,respectively.Using MTT assay to select optimum time and test concentration by cell inhibition rate.The optimum time and test concentration of 2,3,5-trimethylpyrazine and TMP were 24 h and 0～10 μg/mL,24 h and 0～20μg/mL,respectively.H2O2-induced HepG2 oxidative damage model was needed to investigate the antioxidant activity of 2,3,5-trimethylpyrazine and TMP.HepG2 cells were grown in growth medium(DMEM supplemented with 10%FBS,0,200,400,600,800,1000 μmol/L H2O2)by inhibition 4、8、24 h,respectively.The inhibition rates of cells,GPT activity,GOT activity,LDH activity,and CAT activity as indexes to screen out the optimum concentration and time of H2O2 needed for H2O2-induced oxidative damage model.800μmol/L H2O2 after 8 h incubation,the model was established.The antioxidantion of 2,3,5-trimethylpyrazine and TMP was studied as follows:a.Blank group:0 μg/mL 2,3,5-trimethylpyrazine and TMP+0μmol/L H2O2;b.H2O2 treament group:0 μg/mL 2,3,5-trimethylpyrazine and TMP+800 μmol/L H2O2;c.Drug group 1:5 μg/mL TMP+800 μmol/L H2O2;d.Drug group 2:10 μg/mL TMP+800 μmol/L H2O2;e.Drug group 3:5 μg/mL 2,3,5-trimethylpyrazine+800 μmol/L H2O2;f.Drug group 4:10 μg/mL 2,3,5-trimethylpyrazine+800 μmol/L H2O2;The activities of GPT,GOT,LDH,CAT in culture supernatants and intracellular MDA content,SOD and GSH-Px activity were determined.We found TMP and 2,3,5-trimethylpyrazine in the concentration range of the test could significantly reduced the oxidative damage of HepG2 cells induced by H2O2,and was positively correlated with the dosage of TMP and 2,3,5-Trimethylpyrazine.TMP and 2,3,5-trimethylpyrazine can inhibited the oxidative damage of HepG2 cells induced by H2O2.