Study On The Structure Identification, Stability And Biological Effect Of Black Carrot Anthocyanin

Black carrots(Daucus carota ssp. sativus var. atrorubens Alef.), a variant of carrot originate from Turkey, Afghanistan, Egypt, Pakistan, India and Far East, where they have been cultivated for at least 3000 years. Black carrots have atropurpureus root and contain high amount of anthocyanins, which can contribute some health benefits, such as antioxidation effects, antiproliferation activity and so on. On the basis of structure identification of black carrot anthocyanins, the binding interaction of crude extract and monomer of anthocyanin with bovine serum albumin, stability and antioxidant activity of black carrot anthocyanin were investigated in order to expand the application of black carrot anthocyanin in the food and pharmaceutical industries. The results obtained are summarized as follows:1.The total anthocyanin content of black carrot was 5.42%. Cyanidin 3-xylosyl-(glucosyl)galactoside, cyanidin 3-xylosyl-galactoside, cyanidin 3-xylosyl(sinapoylglucosyl)galactoside, cyanidin 3-xylosyl(feruloylglucosyl)galactoside which content had risen to above 50%, cyanidin 3- xylosyl(coumaroylglucosyl)galactoside and pelargonidin 3-xylosyl(feruloylglucosyl)- galactoside were detected as major anthocyanins by high-performance liquid chromatography with diode array detection(HPLC-DAD) and with electrospray ionization multiple mass spectrametry(HPLC-ESI-MSn) analysis. The purity of cyanidin 3-xylosyl(feruloylglucosyl)galacto- side obtained by semi-preparative liquid chromatography was 98.92%.2.The pK of black carrot anthocyanin was about 4.8, which was the reason of unstablility of black carrot anthocyanin at p H 5. The degration rate of black carrot anthocyanin was fast at 70-90℃. In addition, black carrot anthocyanin was sensitive to hydrogen peroxide and ascorbic acid.The storage stability of black carrot anthocyanin was not effected by Na+ and K+, but it was increased by high concentration of Ca2+, Mg2+, Zn2+ and Al3+ and reduced by Cu2+ and Fe3+.3.The interactions between black carrot anthocyanin and cyanidin 3-xylosyl(feruloyl-glucosyl)galactoside and bovine serum albumin were investigated systematically by fluorescence, synchronous fluoscence, three-dimensional fluorescence, circular dichroism spectroscopy and microscale thermophoresis technology. The results indicated that the fluorescence intensity of bovine serum albumin was quenched by black carrot anthocyanin and cyanidin 3-xylosyl(feruloyl-glucosyl)galactoside. The fluorescence quenching observed is attributed to the formation of a complex by static quenching. The hydrogen bonds and van de Waals forces played major roles in stabilizing the black carrot anthocyanin-BSA and cyanidin 3-xylosyl(feruloyl- glucosyl)galactoside-BSA complex. The binding distance was 4.010 nm and 3.813 nm, respectively. During this process the polarity of microenvironment of tryptophan and tryosine of bovine serum albumin was not changed, but secondary structure was changed, in which the content of α-helix was mainly decreased. When the molar ratio of black carrot anthocyanin and bovine serum albumin was 2.5:1, the content of α-helix was decreasced from 52.21% to 46.84%. When the molar ratio of cyanidin 3-xylosyl(feruloyl-glucosyl)galactoside and bovine serum albumin was 2.5:1, the content of α-helix was decreasced from 52.21% to 51.35%. According to data fitting by microscale thermophoresis, the Ka of cyanidin 3-xylosyl(feruloyl-glucosyl)galac- toside binding to bovine serum albumin was 5208L/mol, which was consitent with the results of fluorescence spectroscopy. The fluorescence intensity of bovine serum albumin was littlely descreased by different ions. The quenching mechanism, number of binding site and conformationl changed by black carrot anthocyanin and cyanidin 3-xylosyl(feruloyl- glucosyl)galactoside interacted with bovine serum albumin were not changed in the prescence of different concentration ions. 4.The antioxidant capacity of black carrot anthocyanin was observed by scavenging DPPH and ABTS free radicals, capacity of total reducing power and ferric reducing antioxidant capacity. The results showed that the antioxidant capacity of black carrot anthocyanin improved with concentration increasing and was about half of ascorbic acid. The IC50 of scavenging DPPH and ABTS was 26.51mg/L and 167.49mg/L respectively.