Separation,Identification And Biological Activity Of Flavonoids From Physalis Pubescence L.

Physalis pubescens L.is a characteristic berry in the Northeast.It is not only a good source of dietary nutrient supplements,but also has health benefits such as anti-inflammatory,detoxification,anti-oxidation and anti-tumor,which has been widely circulated among the people.But it lacks basic material research and modern scientific evidence.researches are currently focused on the anti-tumor effect of steroid monomers.A large number of chemical substances,especially flavonoids and their contribution to bioactivity,are very limited in their efficacy.Based on aforementioned,the extraction and purification process of flavonoids from fruits and calyxes was explored in this paper.The antioxidant activity and hepatic cell bioactivity of two sourses of flavonoids from Physalis pubescens L.were compared.Meanwhile,the relationship between the cellular ROS changes and the cellular biological activity was explored.The main"efficiency group"on cellular bioactivity was further screened out,which composition of the compound was determined.The material basis of the inhibitory activity on hepatic cells was establishe.The mechanism of inhibition on Hep G2cells was studied by using network pharmacology,bioinformatics and molecular biology techniques.This paper aims to provide basic data and reference for the future deep processing of Physalis pubescens L.,and provide modern scientific support for the traditional liver and detoxification effect of Physalis pubescens L.The specific methods and conclusions are as follows:?1?The ultrasonic assisted extraction?UAE?,used for fruits-derived total flavonoids from P.pubescens?FTFP?L.,and ultrasonic assisted water bath shaking extraction?UAWBSE?,used for calyxes-derived total flavonoids from P.pubescen?CTFP?,was optimized by using Box-Behnken experimental design.The effects of various parameters on the antioxidant activity of FTFP were investigated.The calyx was degreased with petroleum ether,and its fatty acids and volatile components were analyzed by GC-MS.After optimization,the FTFP and CTFP yields was to 3.39±0.17 mg/g DW and 8.71±0.11 mg/g DW,respectively.The yield of fatty acids and volatile components in the saponin was3.85447%.48 volatile compounds were identified.Fatty acids accounted for 71.1%.The volatile components such as methyl eugenol and endogenous borneol were firstly identified in this plant.?2?The antioxidant activity and the cellular biological activity of TFP on Malhavu line were evaluated.Meanwhile,The correlation between the cellular biological activity and the changes of intracellular ROS after TFP treatment was also investigated.The results showed that CTFP inhibited Malhavu proliferation in a time-and dose-dependent manner with IC₅₀?24 h?=267.4?g/m L,?R²=0.7446?and IC₅₀?48h?=114.6?g/m L?R²=0.9752?.After CTFP treatment,the fluorescence intensity of ROS in Malhavu cells was significantly increased?p<0.05?.Oxidative stress induced cells apoptosis with apoptotic rate in a dose-dependent manner,which suggested that CTFP has the potential to inhibit hepatoma cells.On the contrary,FTFP did not affect the proliferation rate of Malhavu cells?p>0.05?,and the fluorescence intensity of ROS in the low dose group was significantly increased.There was no significant difference between the high dose group and the control group?p<0.05?.The antioxidative activity of TFP was detected by chemical method and CAA method.The oxidative-stress cell model was constructed by ROS activator ABAP combined with DCFH-DA fluorescent probe.It was found that both FTFP and CTFP had the property of scavenging ROS,and FTFP activity was higher than that of CTFP,but with the results of the chemical evaluation method are reversed.The experimental results show that FTFP has low cytotoxicity and has the potential to reduce oxidative stress damage.The CAA method is more suitable for the evaluation of the antioxidant properties of the compound.?3?FTFP and CTFP were enriched and purified using macroporous adsorptive resins,which parameters was optimized.The purity of FTFP,purified with AB-8 under optimizd condition increased from 3.30±0.05%to 35.02±3.42%.The purity of CTFP,purified with D-101 under optimizd condition increased,increased from 7.82±1.00%to 55.23±2.67%.The antioxidant activity,including reducing power,·OH,·O₂^-and DPPH scavenging activity,of puried FTFP was higher than that of the un-purified FTFP and the control group of VC.?4?Furthermore,the CTFP was isolated and the effects of different fractions of CTFP on the proliferation and apoptosis of Malhavu cells were evaluated.EAFPC,the isolated component of CTFP,was identified as the main"efficiency group"of CTFP for hepatocyte protection,which IC₅₀?24h?=37.15?g/m L,R²=.9771,IC₅?48h?=12.52?g/m L,R²=0.9868.16compounds with characteristic absorption under UV spectrum in EAFPC was identified by using HPLC/Q-TOF-MS/MS combined with infrared spectroscopy,including of 9 flavonoids,5 botulinum lactones,1 sterol and 1 species.Three flavonoids of which,luteolin-7-O-?-D-glucoside,quercetin-3,7-di-O-?-D-glucoside and luteolin-7,3'-Di-O-?-D-glucoside were firstly discovered in this plant.?5?The inhibitory activity and cell pathway of EAFPC on hepatoma cell lines was analyzed using network pharmacology technology combined with bioinformatics technology.Through analysis,115 target targets and 229 synergistic relationships of EAFPC were obtained,and quercetin-3-O-?-D-glucoside,3,7-di-O-methyl quercetin and?-sitosterol in EAFPC were determined the most import constituents mainly contributed to the anti-hepatocarcinogenic effect with a synergistic manner.TCGA and text mining tools were utilized to screen the HCC target genes significantly associated with survival and liver cancer.There are 172 key genes to be identified by establishing and analyzing the PPI relationship network between component-target and liver cancer-related disease targets.Finally,Clue GO was used to perform GO annotated analysis and pathway enrichment analysis on these key targets.The results show that EAFPC mainly antagonizes liver cancer by regulating cell cycle,apoptotic biological processes,oxidative stress and inflammatory signaling pathways.?6?In this part,the effect of EAFPC on proliferation,apoptosis and cell cycle arrest of hepatoma cell lines Hep G2 were studied.RT-PCR and WB assays were used to detect relative levels expression of related m RNA and proteins in mitochondrial apoptosis pathway,Keap1-Nrf2/HO-1 pathway?anti-oxidative stress pathway?and cell cycle regulatory factors.The dose-effect of EAFPC on the proteins expression of P53 and Cyvlin D1 was examined.The correlation of the inhibitory effect of EAFPC on Hep G2 cell lines with these abiological processes and signaling pathways were explored.The results showed that EAFPC had a significant inhibitory effect on Hep G2,and its IC₅₀value reached to 121.4?g/m L at 24h.EAFPC induced G0/G1 cell arrest and cell apoptosis with dose dependtent manner,up-and down-regulated the relative proteins expression of P53 and Cyclin D1 in dose-and time-manner,significantly down-regulated the relative expression of m RNA and proteins for Bcl-2and Bcl-2/Bax on the mitochondrial apoptotic pathway,and up-regulated the expression of Bax m RNA and protein with dose and time dependtent effect.The inhibitory effect of EAFPC on Hep G2 hepatocellular carcinoma cell line was closely related to the down-regulation of the antioxidative stress signaling pathway of Keap1-Nrf2/HO-1.At 24h,Keap1 m RNA expression was consistent with Nrf2 m RNA in each treatgment group.At 48h,Keap1 m RNA was significantly down-regulated,and Nrf2 m RNA level was not significantly different contrast to the control group?p>0.05?.The related protein expression was further detected.At48h,the relative protein levels of Keap1 in cytoplasmand was consistent with Nrf2 in cytoplasm,which significantly descreased in time and dose effects?p<0.05?.With the resultant downregulation of Keap1 and Nrf2,HO-1 m RNA and proteins,the downstream antioxidant enzyme or two-phase detoxification enzyme,desceased evidently,which indicates that Keap1 accelerats the degradation of Nrf2 in nucleus.Therefore,EAFPC inhibits the overactivation of Keap1/Nrf2 pathway,down-regulates the expression of its'downstream enzyme HO-1,and reduces cells self-repairing effect gainst the oxidative-stress injury,thereby chemically preventing or blocking HCC process and exerting the protective effect on liver cells.In conclusion,EAFPC can regulate the expression of genes and proteins in Bcl2and Bax on mitochondrial apoptotic pathway,up-regulate the expression of P53,down-regulate the expression of Cyclin D1,promote cell G0/G1 arrest,induce cell apoptosis and inhibit the proliferation of Hep G2 cells;EAFPC can also down-regulate the activation of Keap1-Nrf2/HO-1 antioxidant stress pathway,promote cell oxidative stress,and induce hepatocellular carcinoma cells to develop apoptosis,thereby prevent or block the process of HCC chemically and exert the protective effect on hepatocytes.