Isolation,Structure Identification,and Biological Activity Of The Anthocyanin In Fruit Of Lycium Ruthenicum Murray

Lycium ruthenicum Murr(LRM)is a typical eremophytes and long-lived perennial shrub in member of Lycium genus in the family of Solanaceae,mostly located in the dry and saline-alkali land in the northwestern of China.LRM was recorded as a Tibetan medicine in medical classic "Jing Zhu Ben Cao" and "Si Bu Yao Dian",and it was used to treat heat disease,heart disease,irregular menstruation,menopause hypertension,endocrine disordersand and other diseases.Modern research have shown the fruit of LRM is abundant of anthocyanin,alkaloid,polysaccharide,essential fatty acids,amino acid,Vc,tocopherol and other nutrients and active ingredients,which possesses a variety of biological activities such as anti-oxidant,neuroprotection,bacteriostasis,lowering blood sugar and preventing cardiovascular disease.Compared with the traditional wolfberry,LRM contains more anthocyanins active substances,becoming a research focus recently.However,there is a limit progress in the study of LRM at present.In this paper we have carried out a systematic research on the optimization of extraction process,stability,separation and purification,structure identification,antioxidation and immune activity of anthyanins in LRM.1.Research on the extraction process and stability of anthocyanin from LRM.In this study,we experimented on the four single factors:ratio of water to material,extraction time,ethanol concentration.extraction temperature and determined the center of RMS experimental factors.Through the regression analysis of experimental data,we concluded the optimum extraction process as follows:ratio of water to material was 40 mL/g,the extraction time was 3.07 h,the concentration of ethanol was 71.36%.We finally modified the programme as 40 mL/g,3 h and 70%.The study on the stability of anthocyanins from LRM showed that the stability of anthocyanin could be protected by acid condition,avoiding heat and light,metal ion such as A13+,Ca2+,Mg2+,Zn2+ and glucose.2.Isolation,purification and identification of anthocyanins from LRM.In this study,we purified the component 1 with high purity(97%)through the AB-8 macroporous resin and AKTA semi preparative chromatography.The anthocyanin content of component 1 was measured as 845.6±43.9 mg C3G/100g DW by pH differential method.HPLC,HPLC-MS,and NMR methods were used to analyze the structure of component 1.Combined with the experimental data of mass,NMR spectrometry and the related reports,componentl was identified as petunidin 3-0-[6-0-(4-0-(trans-p-coumaroyl)-a-L-rhamnopyranosyl)-b-D-glucopyranoside]-5-0-[b-Dglucopyranoside](compound 1).3.Study on biological activity of anthocyanins from LRM.Through the vitro antioxidant chemistry experiment,it was proved that the anthocyanin and purified 1 of LRM had strong DPPH,superoxide anion,ABTS radical scavenging ability and general total antioxidant capacity.The PC 12 rat adrenal chromaffin tumor neural cell model have demonstrated the 1 in a range of concentrations could inhibit H2O2 induced cell oxidative damage,protect the cell membrane integrity,reduce the secretion of LDH and MDA in cell lipid peroxidation reaction,enhance the activity of antioxidant enzymes such as SOD,CAT,GSH-Px and anti-apoptosis.Through mouse peritoneal macrophages(RAW264.7)cell model,it was found that the 1 of LRM could improve the cell proliferation ability and neutral red phagocytosis,increased intracellular acid phosphatase activity and stimulated NO secretion of RAW264.7 cell.