| Euphorbia ebracteolata Hayata, as one of the original plant of traditional Chinese medicine Langdu, is mainly distributed in Anhui, Shandong, Henan, Hubei, and Jiangsu provinces etc. in China. The plant is used for the treatment insecticide, abdominal distension, lymphnodes and chronic bronchitis etc.. Also, it can be used to treat of pulmonary ruberculosis and chronic tracheitis in clinical.According to the literature, tannins, flavonoids, hypnones, terpenoids, sterols and other biological active compounds were found in the Euphorbia ebracteolata Hayata. In order to find a kind of triterpenoids chemical constituent as a representational compound with a high content in Euphorbia ebracteolata Hayata, we studied chemical constituent in the root of Euphorbia ebracteolata Hayata and determined the content of the bioactivity compound in it.The extraction and isolation were accomplished with heat reflux and column chromatographical method, seven compounds were obtained and identified by IR, 1H NMR and 13C NMR spectra analysis asβ-amyrin acetate,24-methylene cycl- oanol,3,3′-diacetyl-4,4′-dimethoxy-2,2′,6,6′-tetrahydrxydiphenylmethane, jolkinolide B, 2,4-dihydroxy-6-methoxy-3-methy-1-acetophenone, ebractedatanolide A andβ-sitosterol.Among the seven compounds mentioned above,β-amyrin acetate and 24- methylene cycloartanol, not only are in relative high content, but also have important bioactivity. Therefore, the method of quantitative analysis has been established for determining the contents of the two compounds in Euphorbia ebracteolata Hayata with HPLC.A series of methodical experiments proved that this method has the advantages of accuracy, precision, sensitivity, convenience etc. Extraction and Separation Dried leaves (3kg) were extracted by heat reflux in ethanol (85%) (30L, 24L, 18L respectively) for three times (2h, 1.5h, 1h respectively). The extraction was obtained after ethanol had been evaporated.The extraction was subjected to silica gel column chromatography using petroleum etherethyl acetate as mobile phase. Seven partitions were obtained as A, B, C, D , E ,F, G and H parts.E, F, G and H were subjected to silica gel and ODS colum chromatography repeatedly. Monomer compounds Y2 (200mg), Y3 (90mg), Y4 (120mg), Y5 (90mg), Y6(20mg) and Y7(50mg) were obtained. The part B was recrystallized by petroleum etherethyl acetate to gain monomer compound Y1 (120mg).Compounds Y1, Y2, Y3, Y4, Y5, Y6 and Y7 were identified by IR, 1H NMR, 13C NMR etc. asβ-amyrin acetate,24-methylene cycloartanol,3,3′-diacetyl-4,4′- dimethoxy-2,2′,6,6′-tetrahydroxydiphenylmethane, jolkinolide B, 2,4-dihyd- roxy-6-methoxy-3-methy-1-acetophenone, ebractedatanolide A andβ-sitosterol resp- ectively.HPLC determination of triterpenoids (Y1 and Y2)In this paper we presented a more sensitive quantification method by HPLC for determining the main triterpenoids.HPLC was performed on a Agilent 1100 HPLC instrument with a Agilent HC-C8 column (150 mm×4.6 mm,5μm) with the mixture of CH3CN-H2O(85:15) solution as mobile phase and the flow rate is 1.0mL·min-1 at 25℃. ResultsⅰThe linear range ofβ-amyrin acetate was 0.1μg～5.0μg. Y=7.511×103X+10.00 r=0.9998 The linear range of 24-methylene cycloartanol was 0.1μg～5.0μg. Y=13.403×103X+21.81 r=0.9999.ⅱThe solution of compound Y1 and Y2 were injected into HPLC for six times, the result of precision tests was RSD<1%.ⅲThe reprodurity tests for six samples showed RSD<2%(n=6).ⅳWhen the concentration of compound Y1 and Y2 solution was descreased to 0.0005 mg·mL-1 and determined by HPLC, S/N≥3, so the detectabilities were 1.0ng.ⅴThe solution of compound Y1 and Y2 were stable in tweleve hours because of it's RSD<1%.ⅵThe average recoveries were 99.27%(compound Y1) and 96.92% (compound Y2) respectively.ⅶThe samples of Euphorbia ebracteolata Hayata have been determined fromdifferent place, the results showed that the average contens of compound Y1 and Y2 were 0.049% and 0.173%(from Anguo in Hebei), 0.062% and 0.266%(from southward), 0.000% and 0.138%(from northward).The results of this paper can offer a new index of content determination for establishment of quality standard of Euphorbia ebracteolata Hayata, also, can offer science foundation for the development of new traditional medicine from this plant.
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