Study On The Chemical Constituents And Pharmacological Activities Of Rehmannia Glutinosa

Background:The fruit of Vernonia anthelmintica(L.)Wild.(Family of Asteraceae),V anthelmintica,is representative of Traditional Uygur Medicine(TUM).With function of activating tyrosinase,improving melanin synthesis,inhibiting tumor growth and regulating immune,it has widely used in clinical treatment,including vitiligo,damp and cold inflammation,and intestinal parasites.In recent years,domestic academic research of Vernonia anthelmintica mainly focused on clinical application,but few on the active ingredient and pharmacological action mechanism.Firstly,we systematically studied the chemical constituents of Vernonia anthelmintica,and evaluated the activity of tyrosinase,antioxidant and anti-tumor of monomer compounds.Then,we found that isorhamnetin and luteolin have strong inhibitory effect on PANC-1 cells growth.However,the molecular mechanism of the anti-pancreatic cancer of isorhamnetin has not been reported in the literature.Objective:In order to lay the foundation for future study of Vernonia anthelmintica,the chemical composition of Vernonia anthelmintica have been studied through the extraction and separation technology and HPLC-MS technology.At the same time,quantitative analysis,pharmacological activities and molecular mechanism of monomer compounds isolated from Vernonia anthelmintica also have been studied.Methods:Part 1:The study of the chemical constituents from the fruit of Vernonia anthelmintica.Methods of silica gel,Sephadex LH-20 and preparative HPLC column chromatography were applied to make a systemic chemical investigation of the ethyl acetate extract of Vernonia anthelmintica.The structures of separated compounds were identified by HPLC,MS and NMR.Then,we analyze fragmentation pathways of flavonoids and caffeoylquinic acids by UHPLC-LTQ-Orbitrap-MS/MS,and rapidly identify phenolic constituents of ethanol extract of Vernonia anthelwmintica based on the fragmentation patterns we summarized before.Part 2:Quantitative analysis of the fruit of Vernonia anthel,mintica by HPLC.An HPLC method was used for simultaneous determination of eriodictyol,butein,butin and isorhamnetin.The analysis was performed on a ZORBAX SB-C18 column(4.6 mm×250 mm,5?m)with the mobile phase consisted of mobile phase A(acetonitrile)and mobile phase B(0.2%phosphoric acid solution)in a programme of gradient elution.The detection wavelength was 310 nm,the flow rate was 1.0 m L·min-1 and the column temperature was 25?.Part 3:Evaluation of pharmacological activity of polyphenols from the fruit of Vernonia anthelmintica.The activation functions of mushroom tyrosinase of seven flavonoids from Vernonia anthelmintica were estimated by measuring the oxidation rate of L-dopa.The antioxidant activity in vitro of nine polyphenolic compounds from Vernonia anthelmintica were evaluated by DPPH method.The proliferation inhibition activities of 9 polyphenolic compounds on human pancreatic cancer cell line(PANC-1)and colorectal cancer cell line(LoVo)were screened using CCK-8 assay.Part 4:The molecular mechanism of isorhamnetin against pancreatic cancerHoechst 33258 and Annexin V-FITC/PI were used to detect cell apoptosis.Flow cytometry was used to analyze the distribution of tumor cell in cell cycle phases.Western blot technology was used to detect the protein expression of cell cycle and RAS/MAPK pathway.qRT-PCR technology was used to detect mRNA expression of the genes involved in cell cycle control.Scratch test was used to observe cell migration ability.Results:Part 1:Fourteen.compounds were isolated from the ethyl acetate extract of Vernonia anthelmintica,including seven flavonoids,four phenolic acids,two indole alkaloids,and one sterol.The structures were identified as N-acetylindole-3-carboxaldehyde(1),1-ethylindole-3-carboxylicacid(2),stigmasterol(3),eriodictyol(4),apigenin(5),butein(6),butin(7),isorhamnetin(8),sulphuretin(9),luteolin(10),caffeicacid(11),protocatechuicacid(12),3,5-O-dicaffeoylquinicacidmethylester(13),3,4-O-dicaffeoylquinicacidmethylester(14).Compounds 12?14 were isolated from the plant of Veronia anthelmintica for the first time,compounds 1?2?9 were isolated from this genus for the first time.In particular,alkaloids were firstly found in this plant.Using UHPLC-LTQ-Orbitrap-MS/MS technology,forty-three phenolic compounds were identified from the ethanol extract of Vernonia anthelmintica,including thirteen phenolic acids and thirty flavonoids,and twenty-six compounds were firstly reported from this plant.Part 2:An HPLC method was firstly established for simultaneous determination of four flavonoids in the fruits of Vernonia anthelmintica through investigating extraction methods,detection wavelength and proportion of mobile phase of HPLC.Results showed the linear ranges of eriodictyol,butein,butin and isorhamnetin were 13.44?107.52,0.24?241.92,9.60?76.80,6.25?49.97 ng.The average recovery rates(n = 9)for the sample preparation of the markers were in the range of 99.70%?100.56%and RSD was less than 2.0%.This method is simple,rapid and replicable,,which can be used for the quality control of Vernonia anthelmintica Willd.Part 3:The results of the oxidation rate of L-dopa showed that seven flavonoid compounds isolated from Vernonia anthelmintica have no stimulating effect on tyrosinase activities.Oppositely,they can significantly inhibit tyrosinase activities.It indicated that these flavonoids can't promote the synthesis of melanin by activating tyrosinase activities.The results of DPPH assay showed that there was a significant difference in the antioxidant capacity of nine polyphenolic compounds isolated from Vernonia anthelmintica.,which may be related to their own structure.Among of them,3,5-O-dicaffeoylquinicacidmethylester,3,4-O-dicaffeoylquinicacidmethylester,luteolin,isorhamnetin and butein had a stronger antioxidant activity than Vc,and the two caffeoylquinic acids had a strongest antioxidant activity.The results of CCK-8 assay showed that only isorhamnetin and luteolin had the strong antiproliferative activity against PANC-1 cells among the nine polyphenolic compounds isolated from Vernonia anthelmintica.,in a dose-dependent fashion,with IC50 values of 19.6 ± 4.1 and 18.1 ± 3.8 ?M,respectively.Part 4:The results of Hoechst 33258 showed that isorhamnetin didn't induce morphological alterations of cell nuclei.The results of Annexin V-FITC/PI double staining showed that isorhamnetin remarkably induced early apoptosis,but mildly induced late apoptosis.The results of cell cycle assay showed that isorhamnetin can noticeably enhance S-phase cell cycle arrest in PANC-1 cell through increasing the expression of Cyclin E/CDK2 complexes and decreasing the expression of Cyclin A.At the same time,isorhamnetin decreased cyclin A protein level by down-regulating transcription,but regulation of Cdk2 and cyclin E protein is not through transcriptional alteration.The results of western blotting showed isorhamnetin remarkably inhibited the phosphorylation level of MEK in a dose-dependent manner,but had only marginal effect on ERK phosphorylation.The results of scratch test showed that the migratory potential of PANC-1 cells was remarkably inhibited by isorhamnetin.And the migration activity was almost completely inhibited at high concentration of isorhamnetin.Conclusion:By the systematic separation and analysis of the chemical constituents of Vernonia anthelmintica Willd.,a method for the rapid identification of polyphenolics was established,which could lay the foundation for basic research of pharmacological function of Vernonia anthelmintica.Moreover,a method for the content determination of Vernonia anthelmintica was established through the selections of indicators,which could provide the quality standard for the Vernonia anthelmintica medicine.Isorhamnetin separated from Vernonia anthelmintica can significantly inhibit the proliferation of PANC-1 cells in a dose dependent manner,and the mainly mechanism are to induce cell apoptosis,enhance S-phase cell cycle arrest by regulating the expression of cell cycle protein and inhibit the activation of RAS/MAPK signaling pathway.