| Euphorbiae Semen is the dry and mature seeds of Euphorbia Lathyris L.,which is the plant of Euphorbia Linn of Euphorbiaceae,and its main function is to remove water,swelling,blood and tumor.Diterpene esters are the main active ingredients in Euphorbiae Semen,and have anti-tumor,anti-tumor multi-drug resistance,freckle whitening,diarrhea and other pharmacological effects.So far,there are many literatures on the extraction and separation of diterpene alcohol ester chemical constituents in Euphorbiae Semen,but there are still many blanks in the determination of the content of diterpene ester compounds in Euphorbiae Semen and the research on the pharmacological effects of the chemical constituents of Euphorbiae Semen.In order to deeply study the medicinal substance basis of Euphorbiae Semen and improve its quality standards,and then develop its new medical applications,in this paper,the diterpene monoesters compounds were separated from the Euphorbiae Semen,and their chemical structures were identified.Based on this,the new content determination methods of diterpene esters was studied.Firstly,by using the silica gel column chromatography,ODS column chromatography,and preparative chromatography and other separation methods,thirteen diterpene monoesters were isolated from the Euphorbiae Semen,and their chemical structures were identified by NMR,MS and other spectroscopy methods.The results showed that one of them was new compounds and was identified as 5,7,15-triacetyl-3-benzoyl-7-hydroxylathyrol,and the other twelve known compounds were identified as 5,15-diacetoxy-3-nicotinoyloxy-6,17-epoxylathyra-12-en-14-one,jolkinol-5?,6?-oxide-3-nicotinoyl-15-acetylester,(12E,2S,3S,4R,5R,6R,9S,11S,15R)-15-acetoxy-5,6-epoxylathyra-12-en-3-ol-14-one,Euphorbia factor L7b,L11,L12,L15,L18,L23,L24,L25 and L29,respectivelyOn the basis of the above chemical composition research,the matrix solid phase dispersion-high performance liquid chromatography was used in this work for first time to determine the contents of five diterpene esters in Euphorbiae Semen,namely Euphorbia factors L7b,Ls,L11,L15 and L23.Through single factor investigation,the optimal experimental conditions of this method were determined and described as follow:neutral alumina was used as the solid phase dispersant,and the amount of neutral alumina was 1.0 g;methanol was used as the eluent,and the volume of methanol was 20 mL.Under the appropriate optimal conditions,the linear response ranged from 0.002 to 0.496 mg/mL for Euphorbia factors L7b,Ls,L11,L15,L23,respectively.The detection limits of the five diterpene ester compounds were in the range from 0.10 to 0.32 ng,and the relative standard deviations(RSDs)of precision,repeatability and stability were all less than 3.0%(n=6).The sample recovery rates were in the range from 96.5%to 104.9%with the relative standard deviations(RSDs)were less than 3.0%(n=6),indicating that this method can effectively overcome the interference of fatty oil on target components extraction,and has the characteristics of high extraction rate,high sensitivity,good accuracy,high precision,etc.The experimental results showed that the contents of Euphorbia factor L7b,Ls,L11,L15 and L23 in Euphorbiae Semen are 0.940?1.305 mg/g,0.312?0.394 mg/g,0.261?1.120 mg/g,0.051?0.120 mg/g and 0.113?0.131 mg/g,respectively,and the sum of the contents of the five diterpene ester compounds are 1.677?3.070 mg/gFinally,the contents of six diterpene esters in Zijin Ding Tablets,a compound preparation of Euphorbiae Semen as one of the prescription medicinal materials,were for the first time determined using dispersive liquid-liquid microextraction combined with high performance liquid chromatography.Through single-factor investigation,the optimal experimental conditions of the method were determined as follows:the sample dosage is 5.0 g,trichloromethane was used as the extraction solvent with the volume of 600 ?L;200 ?L anhydrous ethanol were used as the dispersant;the PH was 7;the ultrasonic time was 4min;the centrifugation time was 8min(5000 r/min).Under the appropriate optimal conditions,the linear response ranged from 0.002 to 0.496 ?g/mL for Euphorbia factors Li,L2,L3,L7b,L8 and L11.The limits of detection(LODs)of six diterpene esters were in the range from 0.19 to 0.65 ?g/mL,the relative standard deviations(RSDs)of intraday and interday precisions were in the range from 0.4 to 2.1%,the relative standard deviations(RSDs)of repeatability and stability were all less than 4.0%(n=6),the sample recovery rates were in the range from 94.7%to 106.6%with the relative standard deviations(RSDs)were in the range from 3.2%to 4.2%(n=6).The results showed that the method can effectively enrich the diterpene ester compounds from the Zijin Ding tablet which has a complex chemical composition,and has the characteristics of high extraction efficiency,fastness,high sensitivity,good precision and high accuracy,etc.The experimental results showed the contents of Euphorbia factor Li,L2,L3,L7b,Ls and L11 in Zijin DingTablets are 161.52 ?g/g,24.83 ?g/g,126.60 ?g/g,34.68 ?g/g,7.32 ?g/g and 50.05 ?g/g,respectively,and the sum of the contents is 405.00 ?g/g.In summary,a total of 13 diterpene ester compounds,including a new diterpene ester compound,were isolated and identified from Euphorbiae Semen.The matrix solid phase dispersion-high performance liquid chromatography was established in this work for first time to determine the contents of five diterpene ester compounds in Euphorbiae Semen simultaneously.In addition,the dispersive liquid-liquid microextraction combined with high performance liquid chromatography was developed in this work for first time to determine contents of six diterpene ester compounds in Zijin Ding.The research results not only enriched the species and quantity of diterpene esters in Euphorbiae Semen,but also provides the new scientific basis for the improvement of the quality standards of Euphorbiae Semen and its compound preparations and for the intensive study of its pharmacodynamic substance basis. |
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