Isolation And Qualitative And Quantitative Analysis Of Anti-Complement Active Components Of Italian Medicine Stellera

Objective:To study the anti-complementary component and set up the preliminary quality standard of Anchusa italica Retz.which may be help to establish the substance basis of looking for the natural complement inhibitor and provide a scientific basic for further development and utilization.Methods:Through in-vitro hemolysis test,CH50 as the index to observe the anti-complementary activity of different fraction of ethanol extracts in Anchusa italica Retz.,to confirm the active parts.The chemical components of the different action fraction were isolated and purified by various chromatographic methods,including silica gel,Sephadex LH-20,ODS and semi-preparative HPLC.Structural elucidation of compounds was carried out according to the physical and chemical properties and spectroscopic data.Regarding the anti-complementary activities of classical pathway(CH50)and alternative pathway(AP50)as index,to determine the active momomeric compounds.UPLC-PDA was employed,and the analysis was performed on a ACQUITY UPLC BEH C18(100mm × 2.1 mm,1.7 μm)with methanol(A)-0.4%formic acid solution(B)as the mobile phases with gradient elution(0-3 min,5-31%A;3-10 min,31%-37%A;10-12 min,37%-40%A;12-30 min,40%-40%A).The column temperature was set at 30℃,and the UV detection wavelength was 366 nm,to determine the content of rutin and kaempferol in Anchusa italica Retz.Results:1)Through in-vitro hemolysis test,the ethanol extract of Anchusa italica Retz.showed strong anti-complement activity.Then,Anchusa italica Retz.was fractioned with petroleum ether,ethyl acetate and n-butanol.The ethyl acetate and n-butanol showed good anti-complement activity,and the anti-complement activity of petroleum ether does not good,while the aqueous extract showed no activity.The anti-complment test of all extract showed that the ethyl acetate and n-butanol extract possessed strong activity,so these two extracts were chosen as the target fraction.2)Through anti-complement activity-directed isolation,10 compounds were isolated and purified from the EtOAc extract of Anchusa italica Retz.by various chromatographic separation methods and the spectral analysis techniques.Among of them,8 compounds were elucidated on the base of spectral data.3)Through in-vitro hemolysis test,kaempferol,rutin,2,6,10,14-tetramethyl-18-butanecarboxymethylene-henecos-12-en-17p-of and nonyl benzoate exhibited anti-complementary activities through the classical pathway and alternative pathway(CH50 0.085～1.23 mg/mL;AP50 0.45～0.96 mg/mL),while dibutly phthalate,phthalic acid bis-(2-methyl-propyl)ester and O-phthalic acid bis-(2-ethyl decyl)-ester showed weak inhibitory effect;2-furoic acid showed no inhibitory effect.4)The contents of rutin and kaempferol in Anchusa italica Retz.were in the range of 1.25～3.34 mg/g and 2.86～6.05 mg/g,respectively.The average recovery different concentration were 100.09%and 97.78%,respectively.The RSD of precision tests,stability tests and repeatability tests were less than 2.0%.Conclusions:As expected,we isolated and purified 10 compounds and 8 compounds were determined.O-phthalic acid bis-(2-ethyl decyl)-ester was isolated from this plant for the first time.2-furoic acid,2,6,10,14-tetramethyl-18-butanecarboxymethylene-henecos-12-en-17β-ol and nonyl benzoate were isolated from the plant in this genus for the first time.Among these compounds,2,6,10,14-tetramethyl-18-butanecarboxymethylene-henecos-12-en-17β-ol possessed strong anti-complement activity,equal to positive drug heparin sodium.Two flavonoids:kaempferol and rutin also had good activity.This may be help to establish the substance basis of looking for the natural complement inhibitor.There was no significant difference in the contents of kaempferol and rutin in Anchusa italica Retz.from the same regions,but had differences in the different regions.The results of the study lay the foundation of improvement of the quality standard of Anchusa italica Retz.