Studies On The Chemical Constituents And Anti-inflammatory Of Yao Medicine Pothos Chinensis(Raf.) Merr

ObjectivePothos chinensis(Raf.)Merr,an annual herb plant of the genus Pothos,Araceae family,was used to treat various rheumatoid arthritis by Yao Medicine was the Huluzuan of Shibazuan.It was reported that P.Chinensis was focus on antitumor and antioxidant,etc,but it has less reported on anti-infla-mmatory than antitumor and anti-hypoglycemic,and has not reported about anti-inflammatory activity of the different extracts or effective components.The topic screened effective parts in vitro cell mode to find pathways and targets on anti-inflammatory.Advanced separation methods was used to isolate the effective compound that contain anti-inflamatory activity and the mechanism could be illustated.The compounds that contain anti-inflamatory activity were isolated by advanced separation methods and its mechanisms of action were investigated.These findings might be establish fundament for rational developing of P.· Chinensis.Methods1.obtaining different polar extraction of P.ChinensisFirst,extracted with methyl alcohol,then the crude extract was removed the solvent by reduced pressure distillation.After the dry extract was dispersion by water,with petroleum ether,chloroform,ethyl acetate,n-butanol and water extracted successively.finally,Five different extraction was obtained.2.Anti-inflammatory activity screening of the effective partsFive different extraction was screened whether have anti-inflammatory,according to LPS-stimulated-RAW 264.7 cells.MTT assay was performed to determine the viability of RAW 264.7 cells,RT-qPCR was performed to determine mRNA expression of iNOS,COX-2 in RAW 264.7 cells,and Western blot was performed to determine protein expression of iNOS,COX-2 in RAW 264.7 cells.3.isolated and purified the chemical components of the effective partsSystematic separation method was adopted,and compound of Antiinflammatory activity were obtained using silica gel column chromatography,reverse phase silica gel column chromatography,MCI,Sephadex LH-20,preparation HPLC,purifi-cation and so on.And then the structures of the isolated compounds were determined by spectroscopic methods,including ESI-MS,EI-MS,1H-NMR,13C-NMR and their physicochemical Properties.4.Anti-inflammatory activity screening of chemical components separate from the effective partsThat chemical components were separated from the effective parts was screened according to LPS-stimulated-RAW 264.7 cells whether have anti-inflammatory.MTT assay was performed to determine the viability of RAW 264.7 cells,fireflyluciferase was performed to determine bioluminescence expressing according to LPS-stimulated-NF ?B+/+ RAW 264.7 cells and Griess was performed to determine NO expressing according to LPS-stimulated-RAW 264.7 cells.Finnally,That the chemical components were separated from the effective parts was quick screened.Results1.Petroleum ether,chloroform,ethylacetate,n-butanol and water extracted was obtained from herb plant of P.Chinensis.2.Petroleum ether extract,chloroform extract,ethylacetate extract,n-butanol extract,and water extraction part of the safe range of site were less than 25.00,6.25,12.50,25.00 and 25.00 ?g/mL in RAW 264.7 cells;When the concentration of LPS was 0.5 ?g/mL that the mRNA and the protein expressions of iNOS could be decreased by petroleum ether extract(25 ?g/mL);The mRNA and the protein of COX-2 could be decreased by chloroform extract(6.25 ?g/mL).3.16 compounds were isolated and puried from the effective parts of P.Chinensis,respectively Aurantiamide Acetate,N-trans-p-coumaroyltyramine,N-trans-feruloyltyramine,Erucylamide,E-4-hydroxyhex-2-enoic acid,Azelaic Acid,Sebacic acid,9(S),12(S),13(S)-Trihydroxyoctadeca-10(E),15(Z)-dienoic acid,Syringate,vanillic acid,19 ?-hydroxyoleanolic acid,ilexgenin A,(9E)-8,11,12-trihydroxyoctadecenoic acid methyl ester,Monomethyl fumarate,Stigmasterol and p-Hydroxybenzaldehyde.4.When the concentration was suitable that the NF-? B expressing could be inhibited and the NO releaseing could be decreased by Aurantiamide Acetate.ConclusionAccording to quick screened in vitro cell mode,petroleum ether extract and chloroform extract could be inhibite inflammatory by LPS-stimulated-RAW 264.7 cells-producting,and the protecting mechanism could be relate to inhibiting the mRNA and the protein expressions of iNOS and COX-2.16 compounds were separated and identified from the chloroform extract part of Pothos chinensis(Raf.)Merr.,and Aurantiamide Acetate could be regulate the pathway of NF-? B that inhibiting transcription factors activated,thus reduced NO releaseing.