| Anthocyanins are widely distributed in the plant kingdom, responsible for red and blue pigmentation in fruits, berries, and petals. They are good antioxidant and have positive effect on human health. Structurally, anthocyanins are glycosides of anthocyanidins. Dp, Cy, Pt, Pn, Mv, Ma, and Mc are considered as the most important compounds for the quality and color of Vitis vinifera grapes and wine.It is reported that there are more than 500 kinds of anthocyanins, but most of them are not commercial. The anthocyanin standards which can be bought are expensive. In many researches, anthocyanins were only semi-quantitified by Mv. Standard anthocyanins prove to be very useful for biochemical studies, because they provide quick identification of these compounds in the field of anthocyanins biosynthesis and food quality determination.In the present work, the isolation method of seven anthocyanins monomers from raw grape extracts was introduced, including extraction, purification and isolation. Anthocyanins were extracted with ethanol, purified by XAD-7HP and then directly fractionated by Xbridge Prep C18 column on Preparative HPLC (auto-purity system, waters). This work prepared two kinds of purified acylated anthocyanins successfully, named Ma and Mc, whose commercial standards are not available up to now. The stability of the seven isolated anthocyanins to light, heat, and pH was also examined.(1) Optimal extraction of seven key anthocyanins from grape skinThe rapid extraction method of Dp, Cy, Pt, Pn, Mv, Ma, and Mc from the wine pomace was optimized by using high HPLC. The ANOVA of factors and orthogonal experimental design showed that it was a rapid approach to extract these main anthocyanins in following conditions: 4 liquid-solid ratio (mL/g), 0.1% hydrochloric acid (v/v) in ethanol, 40℃, 30 min and twice extraction, ultrasonic treatment for 2 min.(2) Purification and stabilities of anthocyanins from grape skinPurification and stability of anthocyanins from Yan73 grape were studied. HPLC was used to analyze the residual anthocyanins in the stability test. According to the experiment results, XAD-7HP exhibited the excellent characteristics for anthocyanins purification. The purity of grape anthocyanins was raised from 48.16 % to 94.93 %, the colour value was improved to 291.6 and the recovery rate of anthocyanins was 85.7%.(3) Isolation of seven anthocyanins from Yan73 grapeAnthocyanin monomers, including Dp, Cy, Pt, Pn, Mv, Ma, and Mc were successfully isolated with Xbridge Prep C18 column on a preparative scale. Pigments Dp, Cy, Ma, and Mc were yielded in a one-step separation by preparative HPLC, with purities up to 99.9%, 99.9%, 91.7% and 94.5%, respectively. Pigments Pt, Pn, and Mv were further purified with another elution method and their purities were all improved up to 99.9 %.(4) Stabilities study on the seven anthocyanin monomersThe stabilities of the seven anthocyanin monomers to light, heat, and pH were different, but the trend in changes was similar. The higher light intensity, temperature, and pH values, the more unstable the anthocyanins were. Among the seven anthocyanins, Dp, Ma and Mc were more sensitive to light heat and pH than Cy Pt, Pn, and Mv. When stored at pH 3, 4℃and in dark, the residual anthocyanins kept upon 95% in one week, so the situation was suitable for anthocyanins short storage.(5) Varieties and viny regions analysis of ChangYu red winesDp, Cy, Pt, Pn, Mv, Ma, and Mc were the main anthocyanins of Cabernet Sauvignon wine, Cabernet Gernischt wine, Merlot wine and Yan wine. In four wines, the content of Mv is highest in Cabernet Sauvignon, followed by Ma or Pn. Anthocyanin fingerprints of red wine were used to categorize wine to different varieties and different viny regions. That helps to appraisal of red wines. |
