Studies On The Isolation, Structure Elucidation And Bioactivity Of Anthocyanins From Waxberry And Mulberry

Anthocyanins are the water-soluble pigments in plants which contribute to the brilliant color of blue, red, scarlet in leaves, flowers and fruits, belong to flavonoid-polyphenols. It is reported that they have many bioactivities, such as anti-oxidation, anti-mutation, anti-cancer and anti-inflammation. Waxberry and mulberry are rich of anthocyanins. In the present study, the anthocyanins in waxberry and mulberry were extracted, separated and identified, and the antioxidant and anti-allergic activities of four anthocyanin monomers and total anthocyanins extracts of waxberry (TAW) and mulberry (TAM) were assayed.Waxberry and mulberry fruits were extracted with methanol acidified with 5% formic acid, respectively. The extraction solutions were evaporated in vaccum to remove organic solvents. The residues were dissolved into water to be subjected to cation exchange column Chromatography to yield TAW and TAM. TAW was separated by high speed counter-current chromatography (HSCCC) using a biphasic solvent system composed of methyl tert-butyl ether-n-butanol-acetonitrile-water-trifluoroacetic acid (1.5:2: 1: 5:0.001,Ⅴ/Ⅴ) to yield two anthocyaninsⅠandⅡwhile TAW was separated by HSCCC using biphasic solvent systems composed of methyl tert-butyl ether-n-butanol-acetonitrile-water-trifluoroacetic acid (1:3:1:5:0.001,Ⅴ/Ⅴ) and (1:4:1:5:0.001,Ⅴ/Ⅴ) to yield six anthocyaninsⅢ,Ⅳ,Ⅴ,Ⅵ,ⅦandⅧ.Based on the ESI-MS, 1H-NMR and 13 C-NMR data, anthocyaninⅠandⅦwere identified as cyanidin-3-O-β-D-glucopyranoside (Cy3G); and anthocyaninⅣ,ⅥandⅧwere identified as cyanidin-3-O-(6"-O-α-rhamnopyranoside-β-D-glucopyranoside) (Cy3RG), cyanidin-7-O-β-D-glucopyranoside (Cy7G) and cyanidin-3-O-β-D-galacopyranoside (Cy3Ga), respectively. AnthocyaninⅡandⅤwere speculated as delphinidin-3-glucoside and cyanidin-3-O-(6"-O-α-hhamnopyranoside-β-D-galacopyranoside)(Cy3RGa), respectively.The antioxidant activity of TAW, TAM, Cy3G, Cy3Ga, Cy7G and Cy3RG was investigatedby 1,1-diphenyl-2-picrylhydrazyl (DPPHo) free radical scavenging method. The results showed that TAW, TAM, Cy3G, Cy3Ga, Cy7G and Cy3RG have higher scavenging ability on DPPH·than V_c at concentration more than 25.0μg/mL. Cy3G, Cy3Ga and Cy7G with one sugar group showed similar antioxidant activity, while Cy3RG that possesses two sugar groups showed lower antioxidant activity than those with one sugar.Anti-allergic activity of TAW, TAM, Cy3G, Cy3Ga, Cy7G and Cy3RG was investigated by measuring their inhibition effects on hyaluronidase from bovine testis in vitro. TAW and TAM showed higher inhibition activity to hyaluronidase while Cy3G, Cy3Ga, Cy3RG and Cy7G showed lower. inhibition activity at a concentration of 1.0 mg/mL. The result implies that TAW and TAM contain the anti-allergic compounds.