Purification And Activity Study Of Polyphenols Extract From Porphyra Haitanensis

Porphyra haitanensis belongs to the genus of red algae seaweed with high production.Because of its high nutritional value and the protection of blood vessels,as well as some other effects such as antioxidant activity,hypolipidemic effect,anti-tumor effect,and chemical defense effect,it has been very attractive and popular in this field.Using Porphyra haitanensis as material,we investigate the antioxidant activity and anti-tumor effects of these crude polyphenolsDry Porphyra haitanensis was crushed into powder and then it was introduced to study the effects of kinds and concentration of solvents,solid-liquid ratio on the yield of polyphenols.Based on the single factor experiment and orthogonal test,the optimal extraction conditions were ascertained.Under the optimal conditions which was described as follows:70%ethanol,1/8(g solid/ml liquid),60?,1.5 hours and operation twice,the extraction yield of polyphenols would be 6.52 mg/g dry raw materia.The component B was separated from crude extract A by silica gel column chromatography in the condition of ethyl acetate/methanol and methanol/water gradient elution.The component C1 and C2 was separated subsequently from component B by high performance liquid chromatography via acetonitrile/water gradient elution.The constituents of component C1 and C2 were analized by means of HPLC-MS,1H-NMR,13C-NMR and other modern spectroscopy technololies.The antioxidant activities of component A were evaluated by DPPH free radical elimination method,ferric-reducing antioxidant power(FRAP)method,and 3 carotene-linoleate bleaching assays,linoleic acid peroxidation method.The results showed that component A has high antioxidative activity and it is better on elimination of DPPH free radical.Polyphenols from Porphyra haitanensis exerted protective effects against H2O2-induced injury in human vascular endothelial cells(HAEC),which may act through its anti-oxidative effects and the inhibition of apoptotic of HAEC cells.We investigated the function of component B in cancer resistant through MTT assay.Four cell lines(HepG2,MCF-7,A375,HeLa)can be inhibited by the polyphenols.The inhibition of A375 cell survival was the most significant one,with the IC50 values for 48 h is 7.55 mg/ml.Then we selected A375 for the study in component C1 and C2,with the IC50 values for 48 h were 1.10,1.58 mg/ml?The PI staining and Annexin V/PI staining in the flow cytometry showed that the ratio of apoptotic A375 cells is related to the dosage of component B,C1 and C2 but not significant.Then it showed that component C1 can also positively increase the expression level of melanin and the activity of total tyrosinase in A375 cells.To detect the ultrastructural changes of A375 cells,we used transmission electron microscopy to observe the ultrastructural structure of A375 cells.The results showed that the proportion of nucleus decreased obviously and the proportion of cytoplasm increased.The quantities of cytoplasmic organelles,such as mitochondria,endoplasmic reticulum,Golgi apparatus,etc,were enriched significantly.These results suggested that component C1 can induce the normal differentiation of A375 cells.This study on the antioxidant and anti-tumor aspects of polyphenols from Porphyra haitanensis was executed in this work.We believe these studies will serve as a theoretical and experimental underpinning for food and health care in human life.