Identification of anthocyanins and their stability and antioxidant activity in wax apple

Abstract: 【Objective】Although the coloration of wax apple fruit is determined by the composition andcontents of anthocyanins, the mechanism is still unclear. Therefore, the analysis of content and composi-tionof the anthocyanins from wax apple, and investigation on their stability and antioxidant activities, would pave a way to further understanding the colors formation of wax apple fruit, and finally applyingin food industry.【Methods】'Black pearl'fruits with pink peel and'Tub Ting Jiang'fruits with dark redpeel were chosen in this study. Sixty ripe fruit samples were harvested from three trees (twenty samplesfrom each plant) and were used for chromatism analysis and anthocyanin extraction. The pericarp sepa-rated from forty fruits was immediately frozen in the liquid nitrogen and kept at -80℃ for anthocyaninextraction. Pericarp color measurement was performed by a HP-200 precision colorimeter. The RGBvalues were then converted to CIELAB parameter (L, a, b, C, h) . Anthocyanins Extraction:5 g of tissuewere incubated with 50 mL of MeOH-HCl (pH=3) , kept in dark for 12 h, and then centrifuged at 4 000 r·min^-1 (RT) . Supernatant was collected and dried in a vacuum (40 ℃) and then dissolved in 10 mL 0.01% (v/v) HCl. The product was washed with 10 mL ethyl acetate for three times, and then the aqueousphase was collected. After using AB-8 macroporous resin adsorbed, the product was washed with 0.01% (v/v) HCl, and target components were eluted with 0.01% (v/v) MeOH-HCl. Finally, the product wasconcentrated to 5 mL for HPLC-ESI-MS/MS analysis as well as testing for stability and antioxidant ac-tivities. The HPLC-MS analysis was carried out using an Agilent 1100 LC/MSD Trap VL detector. Thechromatographic separation was performed on a C18 column (Luna, 5 µm, 4.6 mm×250 mm) . The injec-tion sling was 10 μL. Elution was performed using mobile phase A (aqueous 10% formic acid solution) and mobile B (methanol) . The detection was at 520 nm, and the column oven temperature was set at35 ℃. The flow rate was 1 mL· min^-1. The gradient program is described as follows: 0-20 min, 5%-60%B; 20-25 min, 60%-100% B; 25-30 min, 100% B. The mass spectrometry conditions were as follows:electrospray ion source; positive ion mode; capillary voltage, 3 500 V; nebulizer, 45 psi; dry gas: nitro-gen; cone gas flow, 12 L· min-1; dry temperature, 300 ℃; ion trap, scan from m/z 200 to 1300. The an-thocyanins were quantified by external calibration using Peonidin-3-O-glucoside standard (Extrasyn-these, France) . Stability Determination: (1) 1 mL of extract was diluted with a set of solutions (9 mL) with different pH (1-8) , kept at room temperature for 2 h, and then measured absorption spectrum by aUV spectrophotometer (PerkinElmer Lambda 25) at 300-700 nm. (2) 1 mL of extract was diluted with 4 mL of ddH2 O, kept at 4 ℃, 20 ℃, 30 ℃ and 50 ℃ for 1.5 h, 3 h, 5 h, 7 h and 9 h. The absorbance at530 nm and 600 nm was measured to calculate the residual rate.【Results】Four kinds of anthocyaninswere identified: cyanidin-3, 5-glucoside (Cy3 Gu5 Gu) , peonidin-3, 5-glucoside (Pe3 Gu5 Gu) , cyaniding-3-glucoside (Cy3 Gu) and peonidin-3-O-glucoside (Pe3 Gu) . The anthocyanin types, contents and propor-tion were different between two wax apple varieties. Pe3 Gu and Cy3 Gu were the major components of'Black pearl', and the contents of Pe3 Gu and Cy3 Gu were 15.94±1.90 mg· mL^-1 and 2.42±0.79 mg· mL^-1.The content of Cy3 Gu (97.40±11.22) mg· mL^-1 was the highest in'Tub Ting Jiang'. The differences inthe colorimetric parameters and the Cy3 Gu contents between two varieties were extremely significant.Correlation analysis shown that the content of Cy3 Gu was significantly positively correlated with colori-metric parameter a and b, significantly negatively correlated with L and h°. Therefore, the fruits of twovarieties with different colors was related to the content of Cy3 Gu. The anthocyanins of wax apple werestable under acidic conditions, and its stability decreased when the pH increased. The radical rate of an-thocyanins decreased significantly with the time at 70 ℃, but not so at 4-50 ℃. The residual rate of an-thocyanins decreased with increasing temperature. Except for 1.5 h treatment, the residual rate of antho-cyanins at 70 ℃was significantly lower than other temperature treatments. The scavenging ability of hy-droxyl radicals and DPPH radicals increased with the concentration.【Conclusion】The main anthocya-nins in wax apple fruits were cyanidin and peonidin, and different varieties contained various compo-nents and contents. The anthocyanins of wax apple were highly stable under acidic conditions and at 4-50 ℃. The strong antioxidant activity would be a new and worthy resource to be developed into func-tional ingredients and applied products with anthocyanin pigments.