Screening Of Plant Polyphenols With Gastric Mucosal Repair And Melanoma Inhibitory Activity Based On WNT Pathway

Abnormal WNT signaling is closely related to various diseases such as gastric ulcer and melanoma,so looking for WNT pathway regulators has become an important means of treating diseases related to WNT signaling.Plant polyphenols have various physiological activities such as anti-inflammatory,anti-oxidation,bacteriostatic,anti-tumor and immunity enhancement.Polyphenols have become a research hotspot for the development of WNT signal-regulating small molecule compounds.In this thesis,two kinds of plant materials,Citrus aurantium L.and Cinnamomum cassia Presl,with good WNT signal inhibitory activity were selected from26 kinds of plant materials.Citrus aurantium L.is a health food raw material approved by the State Food and Drug Administration of China,and Cinnamomum cassia Presl is a spice commonly used in daily diet and rich in polyphenols.The crude fraction of plant polyphenols is extracted from(Alpinia officinarum Hance,Poria cocos(Schw.)Wolf,Dolicho Lablab L.,Astragalus propinquus Schischkin,Euodia rutaecarpa(Juss.)Benth,Amomum cardamon,Cinnamomum cassia Presl,Curcuma rcenyujin Y,Angelica dahurica,Citrus aurantium L.,Atractylodes macrocephala Koidz.,Citrus medica L.var.sarcodactylis Swingle,Pinellia ternata(Thunb)Breit.,Dendrobium nobile Lindl,Pogostemon cablin(Blanco)Benth.,Glycyrrhiza uralensis Fisch,Panax ginseng C.A.Mey,Angelica sinensis,Ophiopogon japonicus(Linn.f.)Ker-Gawl.,Common Vladimiria Root,Bupleurum chinense,Amomum villosum Lour.,Citri Reticulatae Pericarpium,Codonopsis pilosula,Paeonia lactiflora Pall,and Zingiber oj-jicinale Rosc.)plant raw materials with a volume fraction of 75%ethanol.Determine the maximum non-toxic concentration of different plant polyphenols on HEK293-TOPflash cells by MTT method In this thesis,the HEK293-TOPflash cell model was used to add cells stimulated with WNT3A conditioned medium,and the luciferase reaction intensity value was 123.33±21.72;however,WNT3A conditioned medium was added for stimulation,while adding 5.00?g/m L Citrus aurantium L.and 25.00?g/m L Cinnamomum cassia Presl polyphenol extracts,the luciferase reaction intensity values were 39.56±14.42(p<0.01)and 2.41±1.03(p<0.001),indicating that Citrus aurantium L.and Cinnamomum cassia Presl polyphenol extracts have a good WNT signal inhibition activity.However,the oether 24 plant polyphenol extracts had no significant WNT signal inhibitory activity.The rats were administrated with ethanol in order to construct a gastric ulcer rat model.The visual images of the stomach and the results of HE staining showed that compared with the normal group,the gastric mucosa of the rats with ethanol treatment was severely damaged,indicating that WNT The pathway activated gastric mucosa injury rat model was successfully constructed.By comparing the visual images of the stomach,it is found that the pretreatment of Citrus aurantium L.and Cinnamomum cassia Presl polyphenol extract has a good protective effect on gastric ulcers,and the specific manifestation is that the bleeding point is reduced,and there is no significant difference from the positive control drug treatment effect(p>0.05),indicating that The extracts of Citrus aurantium L.and Cinnamomum cassia Presl polyphenols have a good protective effect in the rat model of gastric ulcer induced by absolute ethanol.The statistics of the injury inhibition rate of the stomach HE staining diagram showed that the positive control,Citrus aurantium L.and Cinnamomum cassia Presl polyphenol extracts could increase the injury inhibition rate of rats by 35.29±22.01%(p<0.05),23.53±35.29%(P<0.05)and 15.77±26.64%(p<0.05).The HE staining results of gastric tissues also indicated that the pretreatment of Citrus aurantium L.and Cinnamomum cassia Presl polyphenols can protect the gastric mucosa injury induced by absolute ethanol,but protective effect of Citrus aurantium L.polyphenols is better than that of Cinnamomum cassia Presl polyphenols.B16 cells were injected subcutaneously into the groin and melanoma grew in situ at the injection site one week later,and the normal control mice did not receive B16 cells had no tumor.This indicates that the WNT pathway activated melanoma mouse model was successfully constructed.By comparing the tumor size of mice in different groups,the average tumor size of the model group mice was 1614.89±170.08 mm~3,and the average tumor size of the mice in the dacarbazine injection group was 977.92±18.61 mm~3(p<0.05).The mice in the Citrus aurantium L.?group were able to achieve similar results as daily injections of dacarbazine,with an average tumor size of 881.52±35.94 mm~3(p<0.05),and the average tumor size of the mice in the Cinnamomum cassia Presl?group was 1147.41±66.60mm~3(p>0.05),and the tumor size data also showed that the extract of Citrus aurantium L polyphenols was superior to the Cinnamomum cassia Presl polyphenols in inhibiting tumors.Tumor HE staining results showed that:Citrus aurantium L polyphenol extract combined with dacarbazine as an anti-melanoma adjuvant agent can inhibit the growth of melanoma,specifically manifested by nuclear shrinkage of tumor cells and melanin deposition.In summary,combined with tumor size and HE staining results of liver,kidney and tumor tissues,it shows that the administration of Citrus aurantium L.polyphenol extract combined with dacarbazine has a significant inhibitory effect on the tumor,compared with the positive drug.However,the inhibitory effect of Cinnamomum cassia Presl polyphenol extract combined with dacarbazine on tumor is not significant,indicating that in the B16 melanoma mouse model,Citrus aurantium L.polyphenol extract has better in vivo biological activity.By comparing the static adsorption and desorption effects of four macroporous resins on Citrus aurantium L.and Cinnamomum cassia Presl polyphenols,the weakly polar resin AB-8 with good adsorption and desorption effects was selected.The extraction parameters of Citrus aurantium L.polyphenols were selected as follows:material-liquid ratio 1:10,ethanol volume fraction 75%,the extraction time is 90 min and the extraction temperature is 35°C.When purifying citrus aurantium polyphenols by column chromatography,elute with 40%and 60%ethanol to obtain the fraction ZS-1 and ZS-2,respectively.125.00?g/m L 40%ethanol elution of Citrus aurantium L.polyphenol component can reduce the proliferation rate of HCT116 cells from 73.99±8.06%to 44.21±11.15%(p<0.01).Through UHPLC-Q-TOF/MS analysis,68compounds were identified from the Citrus aurantium L.polyphenol fraction eluted with 40%ethanol,of which 6 compounds named Unkown1-6 were unknown compounds that were not found in the literature and in the database..In summary,the Citrus aurantium L.and Cinnamomum cassia Presl polyphenols screened from 26 plant materials can inhibit WNT signal activated by WNT3A conditioned medium in vitro;then through ethanol-induced gastric ulcer rats and B16 melanoma mice.Two animal models of WNT signaling pathway activation evaluated the inhibitory activity of Citrus aurantium L.and Cinnamomum cassia Presl polyphenols in vivo.It was found that the protective effect and antitumor effect of Citrus aurantium L.polyphenols on gastric mucosa were superior to Cinnamomum cassia Presl polyphenols.The optimal condition parameters and macroporous resin types of purified polyphenols were screened through a single factor experiment.To investigate the WNT signal inhibitory activity and the inhibitory effect on the proliferation of WNT-dependent HCT116 tumor cells in vitro by purified Citrus aurantium L.and Cinnamomum cassia Presl polyphenols.The purified Citrus aurantium L.polyphenols have better tumor proliferation inhibitory activity than the purified Cinnamomum cassia Presl polyphenols,and then the Citrus aurantium L.polyphenols component ZS-1 was analyzed by UHPLC-Q-TOF/MS.The above results reveal that Citrus aurantium L.polyphenols,due to its good WNT signal inhibitory activity in vivo and in vitro,is expected to be used as a gastric mucosal protection and adjvant agent of melanoma protection or special medical food ingredient,and has good application prospects in the protection of WNT signal pathway related diseases.