Study On The Species And In Vitro Antioxidant Activity Of Se In Se-enriched Salvia Miltiorrhiza

As an essential trace element for human beings and animals,Selenium（Se）functions as active center for glutathione peroxidase in physiological activities,and shortage of it will cause some diseases.The Se of human body come mainly from plant.Se-fertilization of crops can enrich Se content in food chain,improve quality and increase yields as well as enhance crop’s resistance.Salviae Miltiorrhizae（SM）,a kind of Chinese traditional herbal medicine with diverse important physiological activities that Se shared.The functions SM and Se can aggregate into Selenium-enriched SM to enhance the quality of general SM,so the absorption and transformation of Se in Selenium-enriched plants is such an important course that attracted lots of scientists’interests.We studied the main occurring state of Se in Selenium-enriched SM roots and the antioxidant activity in vitro of main occurrence compounds to learn the biotransformation efficiency of Se and Se-contained compounds’in vitro antioxidant activities.Se-binding proteins,polysaccharides and nucleic acids could be extracted from Se-enriched SM with corresponding solvents.And the contents of them and the concentration of Se in them could be detected with spectrophotometry,Hydride generation atomic fluorescence spectrometry and Graphite furnace atomic absorption spectrometry.Bulk polysaccharides,can be obtained with sequentially 0.1mol/L NaCl solvents extraction of Salvia Miltiorrhiza Polysaccharides（SMP）,Sevage de-proteinization,dialysis,concentration and ethanol precipitation,and it showed that of which the content increases with the increase of Se content in SM,so does the content of polysaccharides combined Se,possibly attribute to Se’s affection of SMP’s metabolism and promotion of the production of Se-contained polysaccharide（SM-SeP）.The SM-SeP was applied to DEAE-52 cellulose column chromatography（DEAE-52 CCC）,and four different SePs were gained.According to the IR data showed SMP from control experiment and four isolated SePs were all polysaccharides withβ-pyran ring,and Se exists mainly as selenate ester and some Se-H either.The extraction of SM-protein（SMPr）was carried out with sequential extraction by two 0.03 mol/L different pH phosphate buffer solution,deionized water,75%ethanol,0.5 mol/L NaCl and 0.1 mol/L NaOH,and the extract were applied to following saturated（NH₄）₂SO₄ precipitatation,dialysis and concentration to give bulk SMPr,the content analysis results showed that SMPr content increased with Se content increasing and the obtained 6 SMPrs positively correlated with Se in the term of content（P<0.007）,implied that Se can accelerate the synthesis of SMPr.Applying SMPr to DEAE-52 CCC,it can release two selenium proteins（SePrs）and a selenium-free protein.IR spectrum showed that SMPrs from control experiment and the handled three SePrs wereβ-sheet structured and Se mainly exists as selenate ester.SephadexG-75 gel column chromatography（Sephadex G-75 GCC）was used for further separation of the above three proteins,all component fractions appeared at the outer volume of water;identical results were gained with Sephadex G-200 GCC,indicating that the molecular weight of SMPr is larger than the 600000 Da—the separation limitation of Sephadex G-200.2 wt%of cetyl trimethyl ammonium bromide（CTAB）buffer solution was used to extract selenium-binding nucleic acid,and phenol-chloroform-isoamyl alcohol was used to extract protein,isopropyl alcohol-70%alcohol to remove impurities like polysaccharide and small inorganic molecules.The content of nucleic acid,Se was determined to be 0.61%,and Se was tested to take up 2.3%of Selenium-binding nucleic acids.We selectively studied the content of SePrs,SePs and SeNAs within high-dosage groups of Se-enriched SM.The results showed that in ratio to total amount of Selenium of tested SM,SePrs take up 74.21%,SePs take up 39.48%,while SeNAs take up 2.3%.The existence of Glycoprotein and Protein-Nucleic Acid allowed the appearance of protein in both polysaccharide and nucleic acid,which made the Se content outweigh 100%.In the complexes obtained within sequential water,NaCl solution,75%ethane and NaOH solution extraction,water-soluble proteins were tested to be the most Se-binder with a ratio of 46.0%Se/Se.In the complexes obtained within two 0.03 mol/L different pH phosphate buffer solution extraction,weeak base-soluble proteins were tested to be the most Se-binder with a ratio of51.2%Se/Se.Being extracted by the weak acid and alkali phosphoric acid buffer solutions,SePrs are more concentrated in the alkali buffer solution,taking up 51.19percent of the total amount.In Se-enriched SM,Se exists mainly in the forms of Selenium-binding proteins and Selenium-binding saccharides.Cultivation of Se-enriched SM can lead the biotransformation of inorganic Selenium compounds into organic compounds efficiently.We also carried out the·OH-scavenging and Fe³⁺-reduction experiments,to study the antioxidant activity of protein components from SM and Se-enriched SM.Results showed that SePs and SePrs from Se-enriched SM have better·OH-scavenging and Fe³⁺-reduction abilities over that of polysaccharides and proteins from SM.And within same-contents of polysaccharides and proteins,·OH-scavenging and Fe³⁺-reduction abilities related to Se-content in a certain dose-effect manner,namely the higher the selenium content,the stronger the in vitro antioxidant activities of SM-SePs and SM-SePrs.Na₂SeO₄ complexes with SMPs and SMPrs have better·OH-scavenging and Fe³⁺-reduction abilities over simple addition of that of Na₂SeO₄,SMPs and SMPrs with attributing to synergistic effects.Predictably,SePs and SePrs from SeSM will have good antioxidant activities in vivo to human body.