Isolation And Purification Of Polyphenol From Hemerocallis Citrina Baroni And The Mechanism Of Inhibiting Hepatoma Cell Proliferation

Primary hepatocellularcarcinoma(HCC)usually refers to non-benign neoplasms present in the liver.Up to now,the treatment methods for HCC mainly include surgery,radiotherapy,chemotherapy,and immunotherapy.However,surgical trauma and the killing of normal cells by radiotherapy and chemotherapy can lead to low immunity.In recent years,researchers have extracted a variety of clinical anti-liver cancer drugs from natural products.Hemerocallis citrina Baroni is an angiosperm of the genus Hemerocallis in the lily family.Studies have shown that there are a wide range of active components in daylily,including flavonoids,alkaloids,polyphenols,carotenoids,and trace elements.Among them,polyphenols have extensive pharmacological effects.In this study,Datong daylily was used as raw material to extract polyphenols.Polyphenols were further purified using macroporous adsorption resin.Purified polyphenols with strong anti-liver cancer and antioxidant activities were screened and analyzed in vitro.The molecular mechanism of active polyphenols from daylily against hepatoma was investigated.The main research contents are divided into the following three parts:Part I: Extraction,purification and antioxidant activity of polyphenols from daylily.The crude polyphenols(HcBPP)from Hemerocallis citrina Baroni were extracted in an anhydrous ethanol water bath,followed by centrifuging,concentrating by rotary evaporation,and vacuum freeze-drying.The contents of HcBPP were determined by folinol colorimetry.MTT assay was used to identify the antitumor activity of HcBPP,and it was found that HcBPP reduced the viability of Bel-7402 cells in a dose dependent manner.AB-8 resin was determined to be the optimal resin for purifying HcBPP.After optimizing the static adsorption-desorption conditions of AB-8 resin,it was found that when the sample concentration was controlled at 3.172 mg/m L,the adsorption amount of AB-8 resin reached the maximum;The pure polyphenols HcBPP1 and HcBPP2 from daylily were obtained by elution with 20% and 40% ethanol,respectively.In vitro antioxidant and cell survival experiments showed that HcBPP2 had the strongest antioxidant and anti-tumor activities.Infrared spectrum analysis found that HcBPP2 contains hydroxy O-H,C-H bonds,carbonyl-C=0 bonds,aromatic C=C skeleton,C-O-C bonds,and other polyphenol structures.HPLC-LTQ-Orbitrap MS/MS identified that HcBPP2 contains 17 polyphenolic component: chlorogenic acid,caffeic acid,coumaric acid,eugenoic acid,neochlorogenic acid,catechin,epicatechin,orientin,p-coumaric acid,isoorientin,rutin,erucic acid,ferulic acid,gallic acid,p-hydroxycinnamic acid,quercetin,and protocatechin.Part II: HcBPP2 induces apoptosis and inhibits proliferation of hepatocellular carcinoma.MTT assay and cell cloning experiments showed that HcBPP2 inhibited the proliferation of Hep G2 and Bel-7402 cells.DAPI staining and flow cytometry detection showed that HcBPP2 could induce apoptosis in liver cancer cells.Western blotting results showed that cytochrome C and proapoptotic protein Bax significantly increased,while the expression of anti apoptotic protein Bcl-2 significantly decreased..At the same time,the expression of activated caspase-9 and caspase-3 significantly increased.The above results indicated that HcBPP2 induced apoptosis in Hep G2 and Bel-7402 cells.Part III: HcBPP2 induced apoptosis in liver cancer cells via Wnt/β-catenin signaling.Western blotting was used to detect the effect of HcBPP2 treatment on Wnt/β-catenin signaling in Hep G2 and Bel-7402 cells.It was found that the total β-catenin,nucleusβ-catenin and cytoplasm β-catenin protein levels decreased in a concentration dependent manner after HcBPP2 treatment.Wnt/β-catenin signaling pathway activator Li Cl treatment reversed HcBPP2-induced downregulation of Bcl-2 protein expression and upregulation of Bax and cytochrome C protein levels.In addition,the upregulation of activated-caspase-3 and caspase-9 induced by HcBPP2 was also reversed with Li CI treatment.Flow cytometry analysis showed that the apoptosis rate of cells treated with Li Cl and HcBPP2 was significantly lower than that treated with HcBPP2 alone;MTT results showed that Li Cl treatment also reversed the decrease in Hep G2 and Bel-7402 cell viability induced by HcBPP2.These results indicated that HcBPP2 induced apoptosis in liver cancer cells through Wnt/β-catenin signaling.To sum up,polyphenol HcBPP2 is a extracted and purified from Hemerocallis citrina Baroni.HcBPP2 has good antioxidant activity and anti-liver cancer cell proliferation in vitro,and it contains 17 polyphenol components.Furthermore,HcBPP2 induced apoptosis in hepatocellular carcinoma cells via Wnt/β-catenin signaling.