Preparation Process And Bioactivities Of Salvianolic Acid A From The Roots Of Salvia Miltiorrhiza Bunge F.alba Wu And Li

Salvia miltiorrhiza Bunge f.alba Wu and Li,the white variant of Salvia miltiorrhiza Bunge,is native to mountainous areas of Laiwu and Zhangqiu in Shandong province.The roots of S.miltiorrhiza Bunge.f.alba are often used as Danshen,a traditional Chinese medicine,for the treatment of cardiovascular and cerebrovascular diseases such as coronary heart disease,angina,hyperlipidemia,hypertension and so on,as a substitute of the roots of S.miltiorrhiza in local clinical practice.And it was also used for the treatment of thromboangiitis obliterans(TAO).Modern studies indicated that the main chemical constituents of the roots of S.miltiorrhiza Bge.f.alba are tanshinones and phenolic acids,roughly the same as that of the roots of S.miltiorrhiza.Salvianolic acid A is one of the important phenolic acids from the roots of S.miltiorrhiza Bunge f.alba.The content of salvianolic acid A is very low in the root of S.miltiorrhiza Bge.f.alba,but it has good pharmacological activities.We have investigated a method for converting salvianolic acid B into salvianolic acid A in the plant tissues of the roots of S.miltiorrhiza Bunge f.alba.Based on it,we extracted salvianolic acid A from the treated roots of S.miltiorrhiza Bge.f.alba by using ultrasonic with ethanol.After acidification and liquid-liquid extraction,we got the extract of salvianolic acid A.Then,we studied the optimal separation and purification conditions of salvianolic acid A with polyamide.The results were as follows:the loaded sample concentration was 11 g/L with the flow rate 1.0 mL/min and the sample solution 150 mL,eluted with 5 retention volumes by 20%ethanol to remove impurities,and then eluted with 10 retention volumes by 50%ethanol.The content of purified salvianolic acid A extract was 40.36%.After that,we purified the crude product by MCI gel column.The 7 g of the crude product was loaded in the 250 mL MCI gel column,eluted with 20 retention volumes by 30%methanol to remove impurities,and then eluted by 40%methanol.During the eluted 9th to 12th retention volume,the purified salvianolic acid A extract was obtained and its purity was about 65%.Finally,after purified using Sephadex LH-20 and silica gel C18 columns,the purity of salvianolic acid A was more than 97%.Afterwards,a larger mass of salvianolic acid A were prepared using above methods for next work.In order to increase the solubility of salvianolic acid A in water,it was made into salvianolic acid A ammonium salt.The conditions,such as pH vaules of ammonia solution and reaction solution,were investigated.The results indicated that the content of salvianolic acid A ammonium salt was more than 95%,while the pH values of ammonia solution and reaction solution were 9.5 and 7.5,respectively.In addition,the water solubility of salvianolic acid A ammonium salt was tested,and it was more than 40 mg/mL.We evaluated the anti-inflammatory activity of the ethanol extract from the treated roots of S.miltiorrhiza Bunge f.alba.The result showed that the ethanol extract significantly inhibited the overexpression of inflammatory cytokines IL-8 and IL-1? in LPS-induced THP-1 cells and had moderate anti-inflammatory activity.Moreover,the antioxidant activity of the extract purified by polyamide was investigated by DPPH scavenging assay,determination of reducing power,and hydroxyl radical scavenging assay.The results displayed that the salvianolic acid A extract purified by polyamide had significant antioxidant activity.In this study,we also investigated the anti-hepatic fibrosis of salvianolic acid A ammonium salt,using CCl4-induced hepatic fibrosis in rats by tail vein injection,while fufangbiejiaruanganpian was used as positive control by intragastric administration.We observed the changes of physical signs,liver pathologic sections,a-SMA immunohistochemical analysis,and the contents of AST,ALT,and LN in serum and SOD,MDA in liver tissue.The results showed that the salvianolic acid A ammonium salt improved conditions of hepatic fibrosis rats,and reduced the degree of fibrosis of liver tissue.Meanwhile,it inhibited the overexpression of a-SMA,and remarkably decreased contents of AST,ALT,and LN in serum.Moreover,it also increased SOD and reduced MDA levels.In conclusion,salvianolic acid A ammonium salt showed significant therapeutic effects on hepatic fibrosis rats.The results provide good references for the development of salvianolic acid A.