Isolation,Identification And Action Mechanism Of Chemical Constituents With Anti-Inflammatory Activity Stems From Dendrobium Huoshanense

Dendrobium huoshanense,commonly known as “mihu”,is an edible medicinale plant,which has a great value for development and utilization.At present,the research on D.huoshanense mainly focuses on biological macromolecules such as polysaccharides,while the research on small molecule compounds is relatively less.In this paper,the small molecular compounds from the stem of D.huoshanense were isolated and identified,and their anti-inflammatory activity and mechanism were further investigated in LPS-induced RAW264.7 macrophages.These results would be used to clarify the anti-inflammatory substance basis of D.huoshanense,and provide theoretical guidance for the development and utilization of D.huoshanense.The research contents and results of this paper are as follows.(1)Screening of the anti-inflammatory fraction from stem of D.huoshanense.The dry stem powder was extracted with cold 95% ethanol,and D101 macroporous resin was used to enrich the anti-inflammatory active components.Gradient elution was performed with water,30%,50%,70% and 95% ethanol successively,and the corresponding fraction with different polarity were obtained,which were recorded as HA,HB,HC,HD and HE.Based on the activity evaluation of LPS-induced RAW264.7macrophage inflammation model,HC and HD showed significant anti-inflammatory activities,and the activity of HD was better than that of HC.(2)Isolation,purification and structure identification of monomer compounds.Fourteen monomer compounds were isolated by silica gel column,MCI column,ODS column,gel column and pre-HPLC.The structure of monomer compounds was identified by modern spectral techniques including NMR and MS.The results showed that compound 1(3-hydroxy-5-methoxybibenzyl),compound 2(3-hydroxy-5,4’-dime thoxybibenzyl),compound 3(dendrocandin U),compound 4(dendrocandin B),compound 5(3,3’-dihydroxy-5-methoxybibenzyl),compound 6(batatasin)III)and compound 7(3,4’-dihydroxy-5-methoxybibenzyl)were bibenzyl compounds,compounds 8(dihydroconiferyl dihydro-p-coumarate),compound 9(syringaresinol)and compound 10(ethyl 3-(4-hydroxyphenyl)propanoate)were phenylpropion compounds,and compound 11(R)-1-(3-ethyl)phenylpropanoatePhenyl)ethane-1,2-diol,compound 12(4-hydroxybenzaldehyde),compound 13(2(5H)-furanone,5-hydroxy-3,4-dimethyl-5-pentyl)and compound 14((+)-Loliolide)were other compounds.(3)Screening of anti-inflammatory activity of monomeric compounds in vitro.The evaluation of RAW264.7 macrophage inflammation model induced by LPS showed that compound 1,2,3,5,6,7 could significantly inhibit the production of NO RT-PCR analysis showed that compounds 1,2,3,5,6,7 can significantly inhibit mRNA expression of inflammatory cytokines iNOS,TNF-α,and IL-1β.These results proved that compounds 1,2,3,5,6,7 has significant anti-inflammatory activity,and they may be the material basis for the anti-inflammatory effect of D.huoshanese.(4)Study on the anti-inflammatory mechanism of active compounds in stem of D.huoshanese.The effects of compounds 3 and 5 on the inflammatory signaling of NF-κB,MAPK and Akt was detected by Western-Blot,and results suggested compounds 3 and5 exerted an anti-inflammatory function by inhibiting the phosphorylation of LPS-induced IκB,p65,p38,ERK,JNK and Akt,which resulted in the inhibition of NF-κB,MAPK and Akt signaling pathwaysas well as the downregulation of inflammatory cytokine expression and secretion in macrophages.