Study On Extraction, Purification, Identification And Antioxidant In Vitro Of Anthocyanins From Wild Berries

Anthocyanins are the water-soluble pigments in plants which contribute to the brilliant color of blue, red, mauve in flowers, fruits and leaves, belong to polyphenol compound. The harmless natural edible pigment was conspicuous topic in the phyto-chemistry and the food scientific research. The anthocyanins are important natural organic compound, besides taking the edible pigment; it also has the vital significance to treatments and the prevention disease, such as tumor, senile, cardiovascular of humanity. In order to establish definite academic foundation of healthy food and medicine, the extraction, purification, identification and antioxidant activity of the anthocyanins from three northeast special wild berries, such as Vaccinium uliginosum, Rubus idaeus, Ribes nigrum L., for affording the theoretically basis for industrial production and the betterment of these anthocyanins were mainly studied.The extractions of anthocyanins were performed method of acidic ethanol according to the procedures described by other authors who had studied the same materials. Through experiments, the optimum extraction conditions of ethanol exrtaetion for three anthocyanins were obtained. The optimum conditions of Vaccinium uliginosum anthocyanins were extraction solvent ethanol/TFA/water= 49.5:0.5:50 (v/v/v), the proportion of stuff and liquid=1:10, centrifugal extraction at the condition of 4000rpm,15min. The optimum conditions of Rubus idaeus anthocyanins were that extraction solvent ethanol/TFA/water= 49.5:0.5:50 (v/v/v), pH 2, the ratio of solid to liquid(1:10), extraction 60min at 70℃. The optimum extraction conditions of Ribes nigrum L. anthocyanins as follows:extraction at 48℃for 120min with 1:7 ratio of marc to 75% ethanol. The total anthocyanin content of these three berries were determined from the crude extracts by the pH differential method. Ribes nigrum L. was the richest regarding the total amount of anthocyanins (217.81±8.56mg/100g fresh weight (fw)). Correspondingly, the total content of anthocyanins was 90.6±1.1mg/100g fw in Vaccinium uliginosum and 76.09±0.8mg/100g fw in Rubus idaeus. This anthocyanin concentration of each berry is higher than that of fruits with substantial anthocyanin pigment. These facts suggest that these three berries anthocyanins could be good source of pigment.The anthocyanin was purified by macroporous adsorption resin in this thesis. The optimum purifieation conditions of the Vaccinium uliginosum anthocyanins by X-5 macroporous resin adsorption were studied. The adsorbed Vaccinium uliginosum anthocyanin was good to eluted with 70% ethanol. The optimum conditions of dynamic adsorption were pH 1,6 times column volume, 1mL/min as flow rate. The color value of purified anthocyanins was 21.2 times higher than that of the beginning. Compared with four kinds of resin, the best one for Rubus idaeus anthocyanins was AB-8, the concentration of desorption ethanol was 60%, pH value was 2,2mL/min was flow rate,6 times column volume. The color value of purified anthocyanins was 17.7 times higher than that of the beginning. The optimum purification conditions for Ribes nigrum L. anthocyanins were as follows:X-5 macroporous resin,70% ethanol, flow rate 1mL/min,5 times column volume, pH value was 2. The color value of purified anthocyanin was 20.9 times higher than that of the beginning.The anthocyanin composition of the purified extract from three berries was determined by means of HPLC analysis. The major anthocyanins were further identified by HPLC-ESI/MS using full scan, precursor ion scan, and product ion scan experiments.11 anthocyanins and 2 non-anthocyanins were characterized in Vaccinium uliginosum berry, the major anthocyanins of them as petunidin-3-glucoside and malvidin-3-glucoside. There were also 9 anthocyanins detected in the Rubus idaeus berry and 10 anthocyanins in the Ribes nigrum L. berry, respectively.The antioxidant of anthocyanins extracted from each berry of three was determined using hydroxyl free radicals, superoxide anion free radicals, DPPH free radicals, reducing force and lipidosome antioxidation reaction system. Results showed that all of the three anthocyanins had strong antioxidant activity. There exists obvious concentration-response relation between the antioxidant activity and its concentration of anthocyanins, as increasing the concentration, the antioxidant activity being stronger. Compared with other methods, lipidosome antioxidation reaction system, hydroxyl free radicals and superoxide anion free radicals had higher relativity with other methods for measure antioxidant activity of anthocyanins.