Study On Separation,Structure Identification And Preliminary Mechanism Of Anti-inflammatory Compounds From Equisetum Palustre L. And Their Purification Process

Equisetum palustre L.belongs to the Equisetum genus of Equisetaceae family,and is a perennial fern plant.In folk,it is traditionally used to treat inflammation-related diseases,such as prostatitis,cystitis,peptic ulcers,hemorrhoids,rheumatism et ai.However,the reports on the chemical constituents and pharnacological activity ofE.palustre L.are very few.In the present study,the isolation,purification and structure identification of chemical constituents from E,palustre L.were guided by anti-inflammatory activity for the first time to clarify the material basis of its efficacy;A HPLC method vas developed for quality control analysis of active ingredients in E.palustre L.;A highly efficient,high-yield and high-purity purification process for flavonoid aglycones with anti-inflamaatory activity was innovatively developed.The present research provided scientific basis and technical support for the use of E.palustre L.resource,and the material basis for the development of natural anti-inflammatory agents.Sixteen compounds were obtained from the active fraction of E.palustre L.extracts by using a variety of chromatographic techiques such as macroporous absorption resin,normal or reverse phase silica gel,Sephadex LH-20 and semi-prepare HPLC,et al.On the basis of chetical and spectroscopic evidences including IR,MS,1H-NMR,13C-NMR,2D-NMR and X-ray,fourteen compounds were identified as follows:genkwanin(1),apigenin(2),luteolin(3),4-O-(p-coumaroyl)shikimic acid(4),equisetumone(5),quercitrin(6),isoquercitrin(7),genkwanin-5-O-?-D-glucoside(8),kaempferol-3,7-di-O-?-D-glucoside(10),kaempferol-3-O-?-D-rutinoside?7-O-?-D-glucoside(11),apigenin-6,8-C-di-?-D-glucopyranoside(12),]uteolin-7-O-?-D-glucoside(13),rutin(14),apigenin-5-O-?-D-glucoside(15).Among them,compound 5 is a new sesquiterpene,which is named as equisetumone.Compounds 1-4,6-8 and 12-15 were isolated from E.palustre L.for the first time.The structure of other two compounds(9 and 16)is identifying.2.The anti-inflammatory activity of purified compounds was evaluated in terms of NO.The results showed that three flavonoid aglycones genkwanin(1),apigenin(2)and luteolin(3)and two flavonoid glycosides kaempferol-3,7-di-O-p-D-glucoside(10)and luteolin-7-O-?-D-glucoside(13)possessed strong anti-inflammatory activity in vitro.3.A HPLC method for quality control analysis was developed to sinultaneously separate and determine active ingredients in E.palustre L.Chromatographic conditions:Chromatographic column:Luna C18(5 ?m,250 mm × 4.6 mm i.d.);Mobile phase consisted of 0.1%formic acid aqueous solution(A)and 0.1%formic acid in methanol(B);Gradient elution:0-A min,23%B;4-35 min,23-35%B;35-47 min,35%B;47-68 min,35-45%B;68-80 min,45-65%B;80-81 min,65-76%B;81-90 min,76-90%B;Flow rate:1.0 mL/min;Injection volume:5 ?L;Column temperature:25?.Under the conditions above,thirteen active ingredients in E.palustre L.were separated and determined for the first time.The developed method was sensitive and accurate,and was suitble for the quality control analysis of active ingredients in E.palustre L.,which provided scientific basis for the further study on active ingredients in E.palustre L.,and was significant for the development and use of E,palustre L.resource.4.The inhibition of three flavonoid aglycones for inflammation and the prelimixiary mechanism were compared and investigated.The results showed that luteolin,apigenin and genkwanin could inhibit the release of TNF-??IL-1? and IL-6 of RAW264.7 cell induced by LPS,and the inhibition of luteolin and apigenin was more prominent.The further investigation confirmed that the mechanism was related to their inhibition for the phosphorylation of p38 protein of RAW264.7 cell induced by LPS.5.A highly efficient,high-yield and high-purity purification process for flavonoid aglycones with anti.inflammatory activity was developed by gel resin flash chromatography combined with macroporous resin.(1)Determining the process conditions of macroporous resin enrichment for luteolin,apigenin and genkwanin:Resin type:D101 resin;Sample concentration:luteolin 0.1999 mg/mL,apigenin 0.0835 mg/mL,genkwanin 0.0482 mg/mL;Processing volume:3 BV;Adsorption flow rate:1 BV/h;Desorption solution and volume:7/3 BV 40%ethanol and 4 BV 90%ethanol;Desorption flow rate:3 BV/h.After treated by D101 resin under the above optinum conditions,the contents of luteolin,apigenin and genkwanin in the processing were 13.3-,12.5-and 12.9-fold increased with the recovery yields of 83.6%,78.8%and 81.2%,respectively.(2)Determining the process condition of the separation and purification of luteolin,apigenin and genkwanin in enriched sample by Toyopearl HW-40S gel resin flash chromatography:Gradient elution:0-20 min,80%aqueous methanol;20-40 min,92%aqueous methanol;40-55 min,pure methanol;55-70 min,methanol/acetone(3:1,v/v);70-80 min,pure acetone;Flow rate:30 mL/min;Processing volume:3/40 BV.After purified by Toyopearl HW-40S gel resin flash chromatography under the above optimum conditions and recrystallization,the purities of luteolin,apigenin and genkwanin were more than 97%,and the recovery yields of them were more than 90%.The developed procedure boasts easily reusable solvents,production of high-purity product as well as high recoveries,and allows an easy scale.up.Compared with silica gel flash chromatography,Toyopearl HW-40S gel resin flash chromatography was more suitable for fast purification of luteolin,apigenin and genkwanin from E.palustre L.