Study On The Extraction And Purification Process Of Punicalin And The Procucts Activity

Punicalin, one important ellagitannin in pomegranate husk, has alreadyshown its potential in health effects including antioxidant activities, multitargeted therapy of cancer, inhibitory activity on HIV-1reverse transcriptase,etc. However, due to its low concentration in pomegranate husk and lowproduct purity, its physiological activities were not systematically studied ascompared to punicalagin, which has a higher content in pomegranate husk.Thus, in order to perform studies evaluating the biological effects of punicalinin animal or clinical trials, it is necessary to obtain reasonably large quantitiesof highly purified compounds for experimental purposes.In this paper we studied the separation and purification method ofpunicalin. We established the optimum extraction conditions for both sulfuricacid hydrolysis extraction and flash extraction. We employed H-103resin-based column chromatograph and Medium Pressure LiquidChromatography (MPLC) to obtain punicalin of high purity. What’s more, weinvestigated the combination of punicalin and BSA and tried to explain the action mechanism. We also preliminarily studied punicalin’s antioxidantactivity, sodium nitrite scavenging ability and NDMA interdiction ability.1The extraction condition established:①sulfuric acid hydrolysisextraction: Sulfuric acid concentration (3%), temperature (100℃), time (5h),and solid-to-solvent volume ratio (1:12). The yield of punicalin could reach8.91%.②flash extraction:voltage (81V), extraction time (1.83min),solid-to-solvent volume ratio (1:21.4). The yield of punicalin could reach6.64%.2The optimum condition for H-103resin-based column chromatograph:900ml sample liquid with a concentration of4mg/ml and pH of4.0was loadedonto the column filled with150ml H-103resin at a flow rate of0.6BV/h.Then the column was eluted with5BV50%methanol in water at a flow rate of1BV/h. The fraction was collected and dried. After the separation process, wecould obtain punicalin with the purity of37.7%and recovery of83%.3Purification of punicalin using MPLC: Pure punicalin could beobtained by MPLC using a mobile phase consisting of5%methanol in water.The folw rate was80ml/min and the loading amount was1.0g. We can obtainpunicalin with the purity and recovery of95.89%and90.04%, respectively.Also, we could obtain gallic acid with the purity of78.04%as a by-product.4Study on the interaction of punicalin and protein: Punicalin was foundto have a BSA flocculating property, and it turned out to be a balance ofhydrophobic interactions and hydrogen bonding. There was no synergistic or antagonism interaction between punicalin and punicalagin except a weekantagonism interaction under the ratio of24.4:1. Both punicalin andpunicalagin can be used as clarifers in beer. In comparison, it was punicalaginwho had a more favorable effect on protein flocculating property in beer.5Investigate the activity of punicalin: The results showed that punicalinhad excellent anti-oxidant activity, sodium nitrite scavenging ability andNDMA interdiction ability. According to the Maximum Dose of crude product(punicalin), the maximum daily dose of punicalin should not exceed4.0g/kg.