| The perennial herbaceous plant Eupatorium lindleyanum DC.(Compositae),which showed antihistamine and antibacterial activities,is indigenous to China.Its dried aerial part is called"Ye-Ma-Zhui" by local residents.It is used for the treatment of chronic bronchitis,tracheobronchitis,cough,hypertension.E.lindleyanum contains a variety of chemical components,such as sesquiteipenes,flavonoids,volatile oil and coumarins.Up to now,we haven't defined the specific constituent which played the effect of anti-inflammatory activities on account of lacking systematic research.Therefore,the systematic research of chemical ingredients,anti-inflammatory activities and preparation technology had been carried out in order to find novel bioactive component of this herb,meanwhile provided a scientific foundation for the rational and efficient application of E.lincdleyanum.This paper takes the E.lindleyanum as the research object,and the done work is as follows:1.Screening for anti-inflammatory activity fraction from E.lindleyanum:After preliminary separation,we used xylene-induced mouse ear edema and the carrageenan-induced mouse paw edema model to screen the anti-inflammatory fraction of E.lindleyanum.Result of experiment indicated that the water extract and the 85%ethanol extract on AB-8 macroporous resin significantly decreased xylene-induced mouse ear edema and attenuated carrageenan-induced mouse paw edema,85%ethanol part was the major part of anti-inflammatory activity fraction.2.Isolation and identification of compounds:Macroporous resin column chromatography,silica medium pressure column chromatography,ODS-C18 medium pressure column chromatography and PHPLC were used to isolate constituents from E.lindleyanum,and 18 compounds were obtained.Subsequently,the structures of those compounds were elucidated by UV,IR,MS,NMR spectral analysis.Finally,the compounds were identified as eupalinolide L(1),eupalinolide M(2),eupalinolide A(3),eupalinolide B(4),eupalinolide C(5),3?-acetoxy-8?-(4'-hydroxytigloyloxy)-14-hydroxycostunolide(6),eupalinolide K(7),2a-hydroxyeupatolide(8),eupalinolide H(9),zhebeiresinol(10),medioresinol(11),salicifoliol(12),5-hydroxy-3,4-dimethy-5-pentyl-2(5H)-furanone(13),3-(2-hydroxy-4-methylbutyl)-4-methoxyacetophenone(14),coniferyl alcohol(15),p-hydroxybenzaldehyde(16),3-(hydroxymethyl)-1,14,15-trihydroxy-7;11,15-trimethyl-2,6,10-hexadecatrien-13-acetate(17),3-(hydroxymethyl)-1,13,15-trihydroxy-7,11,15-trimethyl-2,6,10-hexadecatrien-14-acetate(18).Among them,compounds 1?2 were new sesquiterpene lactones,and compounds 10?16 were isolated from genus Eupatorium for the first time.3.Anti-inflammatory activity of compounds isolated from E.lindleyanum:The in vitro anti-inflammatory activity of E.lindleyanum compounds was evaluated with the lipopolysaccharide(LPS)-induced murine macrophage RAW264.7 cell line.Then using ELISI assay to explore anti-inflammatory activity of different chemical constituents,from the expression of TNF-a and IL-6.In the concentration range of safety,compounds 1?9(sesquiterpene lactones)and compounds 10?12(bisepoxylignans)could suppress the production of TNF-a and IL-6 in varying degrees,and the efficacy showed a concentration-dependent effect.Among them,compound 1(eupalinolide L)had the strongest inhibitory effect on TNF-?,compound 5(eupalinolide C)had the strongest inhibitory effect on IL-6.4.An efficient protocol for production of sesquiterpene lactones from Eupatorium lindleyanum DC.by combination of macroporous adsorption resin and CO2 supercritical fluid extraction:First of all,adopting the method of orthogonal experiment to optimize the extraction methods and conditions of sesquiterpene lactones of E.lindleyanum with eupalinolide A(EA)and eupalinolide B(EB)as index.The optimum technological conditions of reflux extraction were as follows:ethanol concentration 60%,ratio of material to liquid was 1:14 and 1:10,extraction time was 1.5 hours and 1 hours,extract 2 times.Secondly,investigating the optimal separation method of macropourous resin through the static,dynamic adsorption and desorption experiment:macropourous resin type was AB-8,ratio of drug to macropourous was 4.2:1(g/g),eluent was 70%ethanol,the volumn of eluent was 4 BV.In the end,establishing a method for CO2 supercritical fluid extraction(SFE)of sesquiterpene lactones part purified from the resin process extraction.The result showed that the optimal condition paremeters were as follows:extraction pressure 35 MPa,temperature 45?,dynamic extraction time 2 hours,30%ethanol was chosen as cosolvent with the flow rate of 40 mL/min.Under the optimized conditions,the purity of EA and EB was increased from 0.5 8%to 39.07%.The combination of macroporous resin and SFE can improve the internal quality of the product as well as shorten the production cycle,thus deserving to be popularized. |
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