Study On Se-enriched Danshen Acid Soluble Protein Extraction Method And Antioxidant Activity Of Se-enriched Danshen

Danshen（Salvia Miltiorrhiza Bge.）is a kind of lamiaceae sage plants,which is one of the traditional Chinese medicines for promoting blood circulation and removing blood stasis.It has a therapeutic effect on cardiovascular,tumors,cancers and other diseases.Selenium is one of the essential trace elements in humans and animals,which is mainly used as the active center of glutathione peroxidase in the body,participates in the antioxidant function in the body,and removes toxic substances such as peroxides.Both Danshen and selenium have antioxidant activity,but the mechanism of action is different.The antioxidant activity of Danshen mainly stem from tanshinones,phenolic acids and flavonoids react with active oxygen directly.However Selenium exerts endogenous antioxidant activity by binding to enzymes in the human body.The combined effect of active ingredients of Danshen and selenoenzyme in human body may produce synergistic effects in the treatment of diseases.Zhongjiang is located in the selenium deficiency areas of China’s soil.Se-enriched cultivation can effectively increase the selenium content of Danshen.The extraction method of the roots protein and selenoprotein of Danshen was optimized.In order to evaluate the effect of se-enriched on the antioxidant activity of Danshen,this article were used the in vitro and in vivo experiments to evaluate the antioxidant activity of se-enriched Danshen.Box-Behnken test design was used to explore the effects of different p H values,different extraction times and different liquid-solid ratios on the extraction of protein and selenoprotein content in roots of Danshen.Experimental results the optimal extraction conditions of protein and selenoproteins in roots of Danshen were p H=6.25,repeated extraction 3 times,and the liquid-solid ratio was 35 m L/g.Verification of the difference in the protein content of 1.26 g/100 g between the Danshen and the model was 2.4%,and found that acidic buffer solution can not extract selenoprotein.Se-enriched cultivation was conducted by spraying sodium selenite and sodium selenite solution in the field,sodium selenite and sodium selenate divided the low,medium,and high dose groups（Se concentrations 0.060,0.120,and 0.240 g/L,respectively）,as well as blank control group without selenium spraying,all of the dose 3 times in parallel.Danshen is classified as roots,stems and leaves after harvest.The hydrophilicity and lipophilic components of roots,stems and leaves of Danshen were extracted with different volume（32%,42%,90%）fractions of ethanol by ultrasonic extraction,potassium ferricyanide method,o-phenanthroline-Fe²⁺method,pyrogallol autoxidation method,DPPH method and ABTS method were used to determine the in vitro antioxidative activity of hydrophilicity and lipophilic components of Danshen plant and to evaluate differences of antioxidant activities of different selenate and the enrichment of different doses in Danshen.Five in vitro antioxidant activity evaluation experiments show that differences between the control group and between all dose groups rich in selenium Danshen roots are not significant.The antioxidation activity of the ethanol extracts of Danshen stem and leaves in the low-dose sodium selenite group is higher than that in the control group（P<0.05）.The antioxidant activity of stem and leaf of the other se-enriched groups is higher than that of the control group,but the data in the field parallel experimental group fluctuate greatly,and these groups not show significant differences.By comparing the contents of total phenolic acids,total flavonoids and total tanshinones in Danshen plant,the in vitro antioxidant activity of Danshen ethanol extract is positively correlating with the content of secondary metabolites in Danshen,therefore in vitro antioxidant activity experiments reflect the antioxidant activity of secondary metabolites of Danshen.In order to overall assessment the antioxidant activity of the secondary metabolites and selenium in Danshen,the antioxidant activity of Danshen root extract was determined by myocardial ischemia-reperfusion injury model in SD rats.Using a volume fraction of 32%,42%,90%ethanol solution and 0.05 mol/L Na OH continuous extraction of secondary metabolites and selenium compounds in Danshen made extracts,each gram of extract equivalent to the original drug 1 gram.The sham operation group,myocardial ischemia model group,Danshen group,Danshen+Na₂Se O₃solution group,Danshen+Na₂Se O₄solution group,Se（IV）-enrich Danshen group,and Se（VI）-enrich Danshen group were set up,10 rats in each group.After continuity intragastric administration 7 day according to kilograms per kilogrambody weight 5 g of extract,the hemodynamic parameters such as left ventricular systolic pressure（LVSP）,left ventricular developed pressure（LVDP）,the maximal change rate of left ventricular pressure(dp/dt _Max)and the minimum change rate of left ventricular pressure(dp/dt _Min)were measured.After 4 h of myocardial ischemia and reperfusion in rats determination of serum levels activity or content of creatine kinase isoenzymes（CK-MB）,lactate dehydrogenase（LDH）,superoxide dismutase（SOD）,glutathione peroxidase（GSH-Px）,malondialdehyde（MDA）,nitric oxide（NO）,to evaluate the differences in antioxidant activity in rats of extracts of Danshen with inorganic selenium and Danshen roots extract enriched with different doses and different selenate.It was found that rats continuity intragastric administration with Danshen have elevated LVSP,dp/dt _Max,dp/dt _Minlevels,and reduced LVDP levels compared to non-irrigated rats,indicating that Danshen can significantly improve cardiac function in rats.Se（IV）-enrich Danshen group,Se（VI）-enrich Danshen group and Danshen+Na₂Se O₄solution group compared to myocardial ischemia model group and Danshen group,SOD,GSH-Px activity and NO content increased,CK-MB,LDH activity and MDA content reduced.But Danshen+Na₂Se O₃solution group compared to myocardial ischemia model group and Danshen group,SOD,GSH-Px activity were reduced,CK-MB,LDH activity and NO,MDA content were increased.The inhibitory effects of different Danshen extracts on myocardial ischemia injury in rats were as follows:Se（IV）-enrich Danshen group>Se（VI）-enrich Danshen grou>Danshen+Na₂Se O₄group>Danshen group>myocardial ischemia model group～Danshen+Na₂Se O₃group.It is conclude that se-enriched secondary metabolites and selenium compounds work together in rats and show synergistic effects in myocardial ischemia and antioxidation.