| Objective:Separating and identifying the common peaks in the fingerprint spectrum through the established HPLC fingerprint of Rheum lhasaense,establishing a high performance liquid chromatography method that can simultaneously determine the content of multiple compounds in Rheum lhasaense and formulating and revising the quality standard of Rheum lhasaenseb Provide theoretical basis.Methods:(1)Preparing extract from rhubarb crude powder by alcohol impregnation method.(2)Separating Rheum lhasaense extract according to the different nature of the adsorption capacity of different polar substances fixed in the chromatographic column,normal phase silica gel is used as the stationary phase,and the chloroform-methanol system is a stepwise enrichment for the mobile phase.(3)A medium-high pressure preparative chromatograph was used to reversely separate and purify Rheum lhasaense extract by different ratios of acetonitrile-water flow to obtain a single compound.After structural identification,the fingerprints of Rheum lhasaense were identified.peak.(4)Determing the contents of the abtained ompounds 1,2,4,7,8,and 10 by high performance liquid chromatography using external standard method.Chromatography column is C18 column(250mm × 4.6mm,5?m);mobile phase is acetonitrile-0.1%phosphoric acid aqueous solution,gradient elution;flow rate is 1.0mL/min;column temperature is 30?;detection wavelength is 319nm;injection volume is 10?L.(5)Establishing an in vitro LPS-induced inflammation model of Raw264.7 monocytes/macrophages,treat cells with different concentrations of compounds,and detect compounds 1,4,6,and 7 by the single solution cell prolifera-tion method(MTS),8,10 on cell viability,and according to the results to determine the follow-up test concentration,and finally by enzyme-linked immunosorbent assay(Elisa)to detect these six compounds on LPS induced secrete IL-6.Results:(1)Compound 1(Piceatannol),Compound 2(Resveratrol),Compound 3(New Compound),Compound 4(Desoxyrhapontigenin),Compound 5(s-Viniferin),Compound were isolated and identified from the ethanol extract of Rhubarb 6(Ampelopsm E),compound 7(Rhapontigenin 3'-O-?-D-glucopyranoside),compound 8(new compound),compound 9(Piceatannol-4'-O-?-D-(6"-Op-coumaroyl)-Glucopyranoside),compound 10(Desoxyrhaponticin 6 "-O-gallate)was separated from ethanol extract of Rheum lhasaense and 5 common peaks was identified by the established Rheum lhasaense HPLC fingerprint which are compound 1,4,7,8,10.(2)There is a good linear relationship between compounds 1 between 0.0399?19.96?g/ml,compounds 2 between 0.0080?19.98?g/ml,compounds 4 between 0.0080?20?g/ml,compounds 7 between 0.0399?19.97?g/ml,compounds 8 between0.0400?20?g/ml,compounds 10 between 0.0392?19.96?g/ml and the peak area which is determined by the high-performance liquid chromatography method which can mensurate multiple compounds simultaneously.The average recoveries are 97.82%(RSD=1.15%),N=9),98.61%(RSD=1.75%,n=9),98.43%(RSD=0.81%,n=9),100.20%(RSD=0.72%,n=9),99.17%(RSD=1.73%,n=9)and 102.41%(RSD=0.65%,n=9).(3)Cytotoxicity and anti-inflammatory tests showed that these 6 compounds have less cytotoxicity and better IL-6 inhibition Compared with the pomalidomide control group,the compound 4,6,10 concentration groups showed better IL-6 inhibition rate.Conclusions:(1)There are 5 common HPLC fingerprint peaks in the 10 compounds obtained from the ethanol extract of rhubarb by column chromatography.(2)The high-performance liquid chromatography established can be used to determine the content of multiple compounds in rhubarb.(3)There is good anti-inflammatory effects in the 6 compounds measured in the in vitro pharmacological test. |
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