Study On Extraction,Purification And Antioxidant Activity Of Flavonoids From Mesona Blume

This experiment used Mesona blume of Fujian as raw materials to study optimiz extraction conditions of flavonoid compounds by the response surface method in Mesona blume.In this study,isolation and purification of flavonoids from Mesona blume by macroporous resin and Sephadex gel column chromatography were investigated,and the flavonoids were identified,at the same time,study of the antioxidant activity and mechanism of flavonoids from Mesona blume.Further,it provides a theoretical reference for the study of Mesona blume.The main experimental results are as follows:Flavonoids was extracted by ethanol extraction methods.The effects of factors such as ethanol concentration,extraction time,and ratio of liquid to material on the yield of Mesona blume flavonoids were investigate.The optimum conditions for extraction of flavonoids were obtained by response surface optimization experiments:ethanol 45%,extraction time 2 h,liquid to solid ratio 15:1,Under the optimal condition,the maximum yield of flavonoids was 13.41%.Macroporous resin and Sephadex gel column chromatography were employed to separate and purify the flavonoids extracts from Mesona blume.The results indicated that HPD-100macroporous resin was optimum for isolation of flavonoids,and the optimal the concentration30 mg/m L,injection volume 3 BV,sample flow rate 3 BV/h elution reagent 60%ethanol,elution flow rate 6 BV/h.Sephadex LH-20 column was applied to further separate the flavonoids of Mesona blume?CMBF?.The separation conditions were as follows:the concentration 10 mg/mL,injection volume 2 mL,elution flow rate 0.5 ml/min,30%,50%,70%,and 90%methanol gradient elution each 100 mL,the CMBF separation of two components:MBF1 and MBF2.Then,the flavonoids of Mesona blume were identified by HPLC,NMR analysis.The results showed MBF1 may be rosmarinic acid and MBF2 was5,7,4'-trihydroxy-flavonoid-3-O-?-D-glucoside.The flavonoids of Mesona blume CMBF showed obvious antioxidative activity in vitro.And scavenging DPPH free radicals,hydroxyl free radicals and superoxide anion free radicals were determination.The IC₅₀ values 14.63?g/m L,0.28 mg/m L and 1 mg/m L,respectively.At the cellular level,the Mesona blume flavonoids could have protective the LO₂ cells from H₂O₂oxidative damage from 5?g/m L-250?g/mL.Compared with the model group,CMBF?5?g/m L?could weaken H₂O₂ induced decrease in LO₂ cell viability,LDH activity and MDA levels in the supernatant of the cells were reduced 1.1-fold,1.3-fold,?P<0.01?significantly,and CAT activity remarkably increased 56.5%.CMBF highly observably increased 64.3%the intracellular content of GPx?250?g/mL??P<0.01?,CMBF could significantly increased SOD?5?g/m L??P<0.05?.The expression levels of antioxidant gene HO-1,Nrf2,GPx,SOD and CAT were determined by RT-PCR.Preliminary study on the anti-oxidation mechanism of Mesona blume flavonoids.The results showed oxidative stress is produced under the induction of H₂O₂,which could upregulated the expression of HO-1 and Nrf2 genes,and GPx,SOD,and CAT mRNA expression levels rised in LO₂ cells.Compared with the model group,when the concentration ofCMBF 50?g/mL and 250?g/m L,the expression of HO-1 and Nrf2 genes in LO₂ cellsincreased significantly?P<0.01?;CMBF?250?g/mL?resulted GPx and CAT genes expression in a marked increase 1.31-fold,1.89-flod?P<0.01?;CMBF?5?g/mL?significantly enhance SOD mRNA expression?P<0.01?.Under the effect of flavonoids of Mesona blume,it have strongly inducted the HO-1 and Nrf2 mRNA levels on LO₂ cells damaged by H₂O₂,and by improving the antioxidant enzymes GPx,CAT,SOD activity to strengthen antioxidant capacity.