| Background:Brachybotrys paridiformis Maxim.ex Oliv.(B.paridiformis),belonging to Boraginaceae family,is widely distributed in Northeast China and has strong reproductive ability and low requirements for growth environment.B.paridiformis has good research value and development potential in food,health products and medicine because it is rich in amino acids,trace elements and antioxidants and has significant anti-inflammatory activity.In this study,we report compounds from B.paridiformis and their anti-hepatitis B virus(HBV)activities.Objective:1.Establish scientific experimental method and reasonable data model to determine the extraction conditions of B.paridiformis.2.Extract,isolate,and purify monomeric compounds from B.paridiformis;identify their structures;and summary major compound type.Aim to discover undescribed compounds.3.Explore the anti-HBV activities of eluting fractions and monomeric compounds from B.paridiformis;screen out the optimally active compounds;and summarize the structure-activity relationship.Methods:1.Study on the extraction process of B.paridiformis: on the basis of literature review,rosmarinic acid was determined as standard and High Performance Liquid Chromatography as test method.Four factors(extraction time,extraction times,ethanol concentration,and solid-liquid ratio)were investigated by Single-factor experiment combined with Response surface methodology,using the principle of box-benhnken experimental design and taking the extraction rate of rosmarinic acid as response value.2.Extraction,isolation,purification and structure elucidation of phytochemicals from B.paridiformis.Air-dried aerial parts of B.paridiformis were extracted with52% ethanol(liquid-solid ratio 18:1)at room temperature for 3 times,each time for 3days according to the extraction process obtained above.The extracts were combined and dried under reduced pressure,dissolved in distilled water,filtrated under reduced pressure,and separated through D101 macroporous resin preliminarily.30% and 50%ethanol eluting solutions were collected and further separated through silica gel column chromatography and semi preparative HPLC to obtain monomeric compounds.The compounds were identified using chemical and spectroscopic data(1D and 2D NMR,as well as HR-ESI-MS,GC,Optical rotation,and CD).3.Anti-HBV activities of eluting factions and monomeric compounds from B.paridiformis: Hep G 2.2.15 cell cytotoxicities of 30%,50% eluting factions and monomeric compounds were detected by MTT method,and inhibitory effects on the secretion of HBs Ag and HBe Ag by ELISA method.The inhibitory activities of the tested compounds on HBV were evaluated to select the optimally active compound.And the inhibition rates of the best compound on HBV DNA,HBs m RNA,HBe m RNA and HBV pg RNA were detected by Quantitative Real-time PCR.Results:1.The experimental results of single factor experiment and response surface methodology were comprehensively analyzed and the extraction conditions of ethanol concentration(52%),extraction time(3 days),extraction times(3 times)and liquid-solid ratio(18:1)were determined.2.50 monomers were obtained from the dried aerial parts of B.paridiformis.Among them,47 compounds were obtained from B.paridiformis for the first time and5,19,21,24,26,27,30,38,41,48,and 49 were undescribed compounds.The structures of 11 undescribed compounds were as follows:brachnan A: 2R-3-(3,4-dihdroxyphenyl)-2-[(3-methoxy-4-hydroxy-phenyl)-6,7-dihydroxy-3-naphthoyl-oxy]-propanoic acid(5);brach acid A:(2R)-2-[[(2E)-3-(3,4-dihydroxyphenyl)-1-oxo-2-propenyl]]oxy]-3-(2-formyl-4,5-dihydroxyphenyl)propanoic acid(24);Brach acid B: 2-hydroxy-5-O-β-D-[6-(E)-2-methyl-2-butenoate-glucopyranosyl]benzoic acid(38);brachoside A: 3-O-feruloyl-6′-O-(4-O-α-L-rhamnopyranosylcoumaroyl)sucrose(27);brachoside B:(7R,8S,7′E)-balanophonin-4,9-di-O-β-D-glucopyranoside(41);brachoside C: eugenol 4-O-β-D-glucopyranosyl-(2→1)-β-D-glucopyranoside(30);brachoside D:(7S,8R,7′E)-7,9-dihydroxy-3,3′-dimethoxy-8-O-4′-neolignan-7′-formylvinyl-4-O-β-D-glucopyranoside(26);brachin A: 3f→2a:6f→3a-[(3S,4R)-4′,7-dihydroxy-3′,6-dimethoxy-4,10-cycloligna-1-en-2a,3a-dicarbonyl] sucrose(21);brachin B: 3f→2a:6f→3a-[(3S,4R)-4′,7-dihydroxy-3′,6-dimethoxy-4,10-cycloligna-1-en-2a,3a-dicarbonyl]-6g-(4′′-(1r-α-L-rhamnopyranosyl-p-courmaroyl))sucrose(48);brachin C: 3f→2a-[(3S,4R)-4′,7-dihydroxy-3′,6-dimethoxy-4,10-cycloligna-1-en-2a,3a-dicarbonyl]-6g-(4′′-(1r-α-L-rhamnopyranosyl-p-courmaroyl))sucrose(49);brachin D: 3f→2a:6g→3a-[(3R,4R)-4′,7-dihydroxy-3′,5,6-trimethoxy-4,10-cycloligna-1-en-2a,3a-dicarbonyl] sucrose(19).3.With the exception of 6 active compounds against HBV that have been reported in articles,30% and 50% eluting fractions and seven compounds from B.paridiformis were proved to have significant anti-HBV activities,among which caffeic anhydride had the strongest activity.Caffeic anhydride can significantly inhibit HBs Ag and HBe Ag secretion and HBV-DNA replication in a dose-dependent manner.The HBs Ag inhibition rate of 76 μM caffeic anhydride at 9 days was 55.06%,which was higher than that of lamivudine(11.38%);the inhibition rate of HBe Ag was22.80%,which was higher than that of lamivudine(3.53%).The 6-day HBV-DNA inhibition rate of 38 μM caffeic anhydride was 58.12%,which was slightly lower than that of lamivudine(67.23%).38 μM caffeic anhydride down-regulated the relative expression level of HBV pg RNA and HBs m RNA(P <0.05),but had no effect on HBe m RNA.The results showed that the anti HBV activity of caffeic anhydride might be related to the inhibitions of HBV pg RNA and HBs m RNA expressions.Conclusion:1.Response surface methodology was used to optimize the extraction process of B.paridiformis.The best extraction process conditions can be used for subsequent experiments and guiding practice.2.50 compounds were obtained from the aerial parts of B.paridiformis,including 11 new compounds.It was found that most of these compounds were phenylpropanoids and its derivatives,which provided a theoretical basis for the study of B.paridiformis.3.The in vitro anti-HBV test showed that 30% and 50% eluting fractions had significant inhibitory effects on HBV antigen and the potential to be developed into anti HBV drugs.All of the compounds showing anti HBV activities contained one or more 3,4-dihydroxybenzene groups except for(7R,8S)-balanophonin.Thus,3,4-dihydroxybenzene group may be the key group against HBV.Among eight active compounds,caffeic anhydride had the strongest anti HBV activity which might be related to the inhibition of HBV pgRNA and HBs m RNA expressions. |
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