Studies On Chemical Constituents Of The Tender Leaves Of Eucommia Ulmoides Oliv.

Eucommia Leaf,the dried leaf of Eucommia ulmoides Oliv.,has been used more than2000 years as a traditional Chinese medicine in China.Phytochemical investigations on Eucommia folium reported the isolation of a series of lignans,iridoids,phenylpropanoids,flavonoids,triterpenoids and so on.Its wide bioactivities include anti-hypertensive,anti-hyperglycemic,preventive osteoporosis,blood lipid regulation,immunoregulation,anti-inflammatory,antifatigue,anti-oxidant,antibacterial,antiviral and other pharmacological effects.Many previous studies and literature investigations on Eucommiae folium have reported the difference in chemical composition and application between tender leaves and leaves that were collected in summer and autumn when there was extensive branching and growth of the foliage.However,active ingredients and mechanisms of tender leaves of Eucommia ulmoides Oliv.are not clear.Thus,we make a research on the chemical composition of Eucommiae folium in order to obtain the lead compounds with novel structure and significant activity which can enrich its chemical constituents and comprehensively elaborate the material basis of the leaves of Eucommia ulmoides Oliv.on pharmacological effects.This paper illustrates the research of the leaves of E.ulmoides extracted with 75%ethanol.The extract was suspended in water,then partitioned with petroleum ether and ethyl acetate respectively.By using DPPH method to study the antioxidant activity of the total extract and its extract parts,it was decided to give priority to the study of ethyl acetate and water parts.And the anti-inflammatory activity of the extracts was studied with RAW264.7 cells.Then monomers were separated and purified through LH-20 dextran gel,silica gel and ODS reverse phase column chromatography,middle pressure liquid chromatography,preparative high performance liquid chromatography（pre-HPLC）,preparative thin layer chromatography and other proper means,and the UV,IR,NMR,CD spectra and MS data were determined to identify the structures for them.Finally,the new compounds were screened for their antioxidant activity by DPPH assay and anti-inflammatory effect on RAW264.7 cells.53 monomers were isolated from water and ethyl acetate sections,there were 12 iridoids（1-12）:Eucommiate A（1）,eucommiol-2-hydroxy-propanoate（2）,asperuloside（3）,6′-acetyl asperuloside（4）,deacetylasperuloside（5）,asperulosidic acid（6）,deacetylasperulosidic acid（7）,11-demethoxy-11-ethoxydaphylloside（8）,eucommiol（9）,geniposidic acid（10）,aucubin（11）,artselaenin C（12）;21 phenolic compounds（13-33）:2-{[3-（3,4-dihydroxyphenyl）propanoyl]oxy}propanoic acid（13）,2-{[3-（3,4-dihydroxyphenyl）propanoyl]oxy}propanoic acid methyl（14）,（±）3-{2-[1-（3′,4′-dihydroxy-phenyl）ethyl]-4,5-dihydroxyphenyl}propanoic acid methyl（15）,（±）3-{2-[1-（3′,4′-dihydroxy-phenyl）ethyl]-4,5-dihydroxyphenyl}propanoic acid（16）,methyl 3-（3,4-dihydroxyphenyl）-propanoate（17）,dihydrocaffeic acid（18）,caffeic acid（19）,caffeic acid ethyl ester（20）,catechol（21）,1,3-trans-5,6-dihydroxy-3-（3’,4’-dihydroxyphenyl）indan-1-methyl（22）,protocatechuic acid（23）,m-hydroxybenzoic acid（24）,p-hydroxybenzoic acid（25）,p-coumaric acid（26）,3,4-bis（4-hydroxyphenyl）cyclobutane-1,2-dicarboxylic acid（27）,1-[（7S）-7-hydroxy-1′-（3′,4′-dihydroxyphenyl）methanol]-3-hydroxybenzaldehyde（28）,chlorogenic acid methyl ester（29）,methyl 4,5-dicaffeoylquinate（30）,ethyl 4,5-dicaffeoylquinate（31）,3,5-dicaffeoyl quinic acid（32）,3,5-dicaffeoylquinic acid ethyl ester（33）;9 flavonoids（34-42）:quercitin（34）,isoquercitrin（35）,rutin（36）,quercetin 3-O-（6’’-O-acetyl）-β-D-glucopyranoside（37）,quercetin 3-O-α-L-arabinopyranosyl-（1→2）-β-D-glucopyranoside（38）,kaempferol（39）,kaempferol-3-O-rutinoside（40）,astragalin（41）,kaempferol 3,7-di-O-β-D-glucopyranoside（42）;4 lignanoids（43-46）:olivil（43）,cycloolivil（44）,medioresinol（45）,pinoresinol（46）;7 other compounds（47-53）:3,4-dihydroxycyclohexane-1-carboxylic acid（47）,uridine（48）,adenosine（49）,aeculetin（50）,dibutylphthalate（51）,palmitic acid（52）,α-linolenic acid（53）.6 new compounds（1、2、13、14、15、16）,and the absolute configurations of the compound 1 and 2 was determined by CD spectrum.17 compounds were isolated from Eucommiaceae for the first time.In addition,the antioxidant and anti-inflammatory activities of the six new compounds were screened,respectively.The DPPH.radical scavenging activity of the six new compounds was determined by DPPH assay.Compared with the positive control Vc,compound 16 showed a stronger DPPH.radical scavenging activity with EC₅₀ value of60.44?M,Compounds 13,14 and 15 showed weaker DPPH.radical scavenging activity than Vc with EC₅₀ value of 203.5?M,185.4?M and 222.2?M,respectively.Two new iridoids 1 and 2 were showed with EC₅₀ value of>500?M.On the other hand,the release of NO was taken as the index to evaluate the anti-inflammatory activity of the compound through the LPS-induced RAW264.7 macrophage model.Four new phenolic compounds（13,14,15,16）were shown moderate inhibitory effect with IC₅₀ value of 56.3?（44）,10.6?（44）（11）25.7?M and 29.9?M,respectively.Two new iridoids 1 and 2 were found to exhibit negligible inhibitory effect with IC₅₀ value of>100?M.In this study,on the basis of the screening of antioxidant and anti-inflammatory activities,the chemical constituents of the tender leaves of Eucommia ulmoides Oliv.were studied.53 monomers were isolated,including 6 new compounds,17 compounds were isolated from Eucommiaceae for the first time,and the activities of the new compounds were determined.The results of the research enriched the chemical information of the leaves of Eucommia ulmoides Oliv.,laid a foundation for the further improvement of its quality standard,and provided a reference for the basic research of its pharmacodynamic substances.