Study On Extraction And Purification Of Polyphenols From Water Hemp Leaves And Their Antioxidant And Anti-cancer Activities

Debregeasia edulis is a wild medicinal and edible plant,mainly distributed in eastern Asia and northern Africa,including China,Bhutan,northeastern India,Nepal,Taiwan and Japan.Chinese people use the leaves,stems and roots of debregeasia edulis to make Chinese medicines,which have a function to clearing the damp and swell in human body.Debregeasia edulis fruit can be ate and used to brewed wine.However,debregeasia edulis have not get the attention it deserve,which is not conducive to develop and utilize this wild plant resources.The leaves and fruits of Debregeasia edulis are rich in functional ingredients,of which the highest content is polyphenols,with strong antibacterial,anti-oxidation and anticancer activity.However,the mechanism of the antioxidant and anticancer activity is not clear.Therefore,the polyphenols of Debregeasia edulis leaves were extracted,purified and analyzed,then measured the antioxidant activity and anticancer activity of the crude extracts and purified products of Debregeasia edulis leaves and further clarify the molecular mechanism of its antioxidant and anticancer activity.In this study,ultrasound-assisted extraction was used to extract the polyphenols from Debregeasia edulis leaves and optimized the extraction parameters.Macroporous resin was selected to purify the Debregeasia edulis leaves polyphenols and optimized the related parameters.The components of the extract were analysis by high performance liquid chromatography(HPLC),and the total reducing power and the ability to scavenge OH·,DPPH· and ABTS-were determined by spectrophotometry.Debregeasia edulis leaf polyphenols were used to treated human non-small lung cancer cell A549 and human cervical cancer cell Hela.The proliferation of A549 cells and Hela cells were observed by MTT assay,the inverted fluorescence microscope and flow cytometer(FCM)were used to test the changes of the cellular morphology,cell cycle,apoptosis,reactive oxygen species(ROS)and mitochondrial transmembrane potential(??m).The expression of related apoptotic proteins p53,Bcl-2/Bax,Cyt-c,caspase-3,caspase-9 and PARP were measured by Western blot.The main results are as follows:(1)The results of orthogonal experiments show that,the optimum extracting process of Debregeasia edulis leaves polyphenols were as follows:the methanol concentration was 70%,the ultrasonic power was 100w,the extraction temperature was 70 ?,the extraction time was 50min.The rate of Debregeasia edulis leaves polyphenols extracted under this optimal extraction process was 10.88%.(2)10 monomer phenols in DELPs were analysed by high performance liquid chromatography.Which may be the following substances:1,Gallic acid;2,Protocatechuic acid;3,Epigallocatechin;4,(+)-Catechins;5,Caffeine;6,Chlorogenic acid;7,Epigallocatechin gallate;8,Epicatechin;9,Gallocatechin gallate;10,Epicatechin gallate.Among the ten phenolic acids,Chlorogenic acid,Caffeine,Protocatechuic acid,Epigallocatechin and Epicatechin were the highest content components,which were 22.67,15.80,10.13,7.60 and 6.04 mg/100 mg respectively.(3)The results showed that the optimized adsorption and desorption conditions were as follows:LSA-5B resin were chose and the concentration of polyphenols in feeding solution is 1.5 mg/mL;processing volume is 3.6 BV,pH value is 3.5,flow rate is 3 BV/h(1.5 mL/min),and temperature is 25 ?.The elution conditions were as follows:7 BV ethanol-water solution(70:30,v/v)with its flow rate 3 BV/h.The total polyphenols content of the final purified products is 75.36 mg/100mg.(4)The results of antioxidant assay show that DELPs has strong antioxidant activity,the total reducing power,DPPH radical scavenging ability and ABTS radical scavenging capacity of the crude extract and purified product of debregeasia edulis leaves were stronger than those of vitamin C.(5)This study show that,DELPs could inhibit the proliferation of A549 cells and Hela cells significantly.Inducing the apoptosis of A549 cells and Hela cells by arrest the cell cycle in G2/M phase and S phase respectively.Besides,the cells morphology,ROS and ??m were changed significantly,and the expression of related apoptotic proteins P53,Bax/Bcl-2,Cyt-c,Caspase3,Caspase9 and PARP in A549 cells was significantly increased.The apoptosis process of A549 cells and Hela cells was involved in the intracellular mitochondrial apoptosis pathway.This paper had studied the extraction,purification,composition,antioxidant activity,anticancer activity and its mechanism of polyphenols from Debregeasia edulis leaves,which is of great significance for fully exploiting the medicinal and health care value of Debregeasia edulis leaves polyphenols.It provides the theoretical basis and technical support for the further research and development and utilization of Debregeasia edulis.