Purification,Characterization And Biological Effects Of Proteins From Angelica Sinensis

Angelica sinensis is one of the famous Chinese traditional medicines.Modern research has confirmed that all of the polysaccharides and small molecules of Angelica sinensis have pharmacological effects,while little is known about the variety and biological effects of Angelica sinensis proteins.Two proteins ASPR-F(Pathogenesis-related proteins from the fresh Angelica sinensis roots)of highest content with similar molecular weights and named as ASPR-F-1 and ASPR-F-2 were purified from the fresh root of Angelica sinensis by 80%ammonium sulfate precipitation,Sephadex G-50 gel filtration chromatography,and DEAE-Sepharose anion exchange chromateography.The molecular weights of ASPR-F-1 and ASPR-F-2 on SDS-PAGE were 16.66 kDa and 16.46 kDa,respectively.Both ASPR-F-1 and ASPR-F-2 are glycoproteins with glycosyl content of 1.8%and 3.4%,respectively.They were mainly monomeric in solution,but partially formed dimers.ASPR-F-1's N-terminal sequence of first 15 amino acid residues was determined as GIQKTEVEAPSTVS,and showed that it related to pathogenesis-related(PR)class 10 protein.ASPR-F-2 was also identified to be member of PR-10 family by mass spectrometry.They both have ribonuclease(RNase)activities,and the specific RNase activity of ASPR-F-2(128.85 U/mg)was higher than that of ASPR-F-1(68.87 U/mg).Two isoforms have the different optimal pH of pH 5.0(ASPR-F-1)and 6.0(ASPR-F-2),respectively.But they share the same optimum temperature(50 ?)and both exhibit the greatest thermal stability at 50?.Similarly,ASPR-C-1 and ASPR-C-2,two proteins with the molecular weights of 17.33 kDa and 17.1 8 kDa were purified from the crude drug of Angelica sinensis.The glycosyl content of two isoforms were 2.6%(ASPR-C-1)and 8.2%(ASPR-C-2),respectively.Their specific RNase activities were 73.60 U/mg and 146.76 U/mg,respectively.They have the different optimal tempe-rature of 50?(ASPR-C-1)and 60?(ASPR-C-2),respectively.But they share the same optimum pH(pH 5.6)and both exhibit the greatest thermal stability at 60 ?.However,ASPR-C-2 exhibited better thermal stability than ASPR-C-1.The latter rapidly inactivated and retained only about 20%residual activity while the former still maintained about 80%of its original activity at a higher treatment temperature(80-100 ?).Furthermore,the total ASPR proteins were prepared from the fresh root,crude drug and prepared slices of Angelica sinensis(named as ASPR-F,ASPR-C and ASPR-S)to investigate the biological activities of the total ASPR proteins in different processing stages.The results showed that all proteins can promote the proliferation of normal human hepatic cells L 02 and prevent carbon tetrachloride CCl4-induced acute hepatic injury in mice.ASPR-S protein presented most hepato-protective effect among ASPR proteins.The cell viability of L 02 cells was up to 263.14%with the pretreatment of 0.5 mg/mL ASPR-S protein.In addition,ASPR-S(at the doses of 1.25 mg/mL)pretreatment could significantly reduce hepatic MDA level of by 44.27%and significantly increase the CAT,GST and T-AOC activities by 113%,54%,and 87.63%,respectively.At the same time,the vitality of serum ALT and AST were significantly decreased by 98%and 77.53%,respectively,which almost were reversed to the normal level.In this study,we not only first purified PR-10 proteins from the fresh root and the crude drug of Angelica sinensis,revealed their excellently hepatoprotective activities,but also verified their hepatotective activities could be enhanced by processing.These findings not only enrich the current knowledge of poorly annotated Angelica sinensis proteins,but provided an evidence for the necessity of processing of Chinese traditional medicines.