Studies On Fractionation And Bioactivity Of Polyphenols From Pomegranate Pericarp

Plant polyphenol is a general term of polyhydroxy phenolic compounds that has variousphysiological activities, such as anti-oxidation, anti-inflammatory, anti-aging, anti-bacterial,anti-tumor, preventing a variety of cardiovascular diseases, which are widely used in food andcosmetic industries. China has rich vegetable resources such as pomegranate, butpomegranate pericarp (20%~30%of fruit weight) which is rich in polyphenols has the lowutility ratio of resources. To exploit rationally pomegranate pericarp resources and to promotepomegranate processing industry development in China, the studies on fractionation andbioactivity of polyphenols from pomegranate pericarp is of significance.In this paper, pomegranate pericarp was used to extract polyphenols by ultrasound-assist.Microfiltration and ultrafiltration membrane technology were used to separate and purifypolyphenols from pomegranate pericarp, then the components with different molecular weightwere subdivided by ultrafiltration with different aperture. Biological activities of the productswere analyzed by the antioxidant tests in vitro as well as their apoptotic regulation mechanismin PC-3cells. The main research contents were as follows:Firstly, ultrasonic assisted extraction was used to extract polyphenols from pomegranatepericarp. The results showed that the extractive rate was182.8±3.4mg/g at the optimumconditions of ethanol concentration60%(v/v), solid to liquid ratio of1:35(m/v, g/mL),ultrasonic-assist extraction time30min, mesh beads100.Secondly, pomegranate pericarp polyphenol extracts were purified by microfiltration andultrafiltration membrane technology. The conditions of microfiltration process were asfollows: membrane pore size0.2μm, operating pressure0.18MPa, the penetration rate ofpolyphenol was93.4%, the intercept rate of protein, total sugar and solids were18.45%,4.21%and10.59%respectively. The conditions of ultrafiltration process were the membranepore size8000Da, operating pressure18bar, the penetration rate of polyphenol79.03%, theintercept rate of protein, total sugar and solids were80.59%,52.32%and61.28%respectively.The purity of pomegranate pericarp polyphenol from23.75%increased to39.53%, the finalyield is73.84%.Thirdly, the purified polyphenols form pomegranate pericarp were subdivided byultrafiltration with different aperture (3000Da and1000Da), and three different fractions(PTP1, PTP2and PTP3) which made from polyphenols in different proportions were gained.The bioactivities of the products such as PTP, PTP0, PTP1, PTP2and PTP3were evaluatedby their antioxidant capacity in vitro and inhibiting rate of PC-3cell. The results showed thatPTP0had stronger antioxidant and cell inhibition than PTP; among three fractions, PTP2hadthe strongest antioxidant capacity and inhibiting rate of PC-3cell.Finally, the apoptotic mechanism was studied by observing PC-3cell morphology,detecting cell apoptosis, mitochondrial membrane potential (MMP) and the expression ofrelated proteins and enzymes. The result of PC-3cells with morphology showed PTP2hadan ability to promote the PC-3cell apoptosis, which was confirmed by Flow Cytometry.With the increase of PTP2concentrations, MMP increased and PC-3cell apoptosisenhanced.To regulate the expression of key proteins and enzyme, PTP2inhibited the expression of anti-apoptotic protein Bcl-2, promoted the expression of apoptotic proteinBax and release of Cyt C, meanwhile, the key apoptotic enzyme Caspase-3was activated,which leaded to cell apoptosis irreversibly. Thus, PTP2regulated PC-3cell apoptosis bymediating Caspase dependent cell apoptosis via the mitochondrial pathway.