| Polygonum Orientale.L,as a plant of traditional Chinese medicine is mainly distributed in Heilongjiang , Neimenggu, Jilin, Liaoning, Hebei, Shandong, and Jiangsu provinces etc. in China. The seeds of Polygonum Orientale.L can be used to treat bad stomach digest, intumescene, inflammation and stomach-ache etc.. So, it has been inscrolled in Chinese pharmacopoeia from the edition of 1977.According to the literature , vitexin, orietin, quercetin and other flavonoids were found in the Polygonum Orientale.L .In order to find a kind of chemical constituent as a representational compound with a high content in Polygonum Orientale.L , we studied chemical constituent of Polygonum Orientale.L and measured the content of the bioactivity compound in the seeds of the Polygonum Orientale.L. The extraction and isolation were accomplished with heat reflux and column chromatographical method, 5 compounds were obtained and identified by IR,UV, NMR and MS analysis methods as Taxifolin, 3, 5, 7 -trihydrochromone, quercetin,β-sitosterol and daucosterol. Among the 5 compounds, daucosterol was obtained the first time from the seeds of Polygounum Orientale.L..All the five compounds mentioned Above, Taxifolin, not only is in relative high content, but also has important bio-activity. Therefore, the analysis method has been established for determining the content of Taxifolin in seeds of Polygonum Orientale L. with HPLC. A series of methodical experiments proved that this method has the advantages of accuracy, precision, sensitivity, convenience etc. Extraction and SeparationDried leaves (3kg) were extracted by heat reflux in ethanol (95%) (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-ether-ethylacetate-ethanol as mobile. Three partitions were obtained as Fr.1, Fr.2 and Fr.3 parts.Fr.1 and Fr.3 were subjected to silica gel colum chromatography using petroleum-chloroform-methanol as mobile phase. Monomer compounds S5(12mg), S2 (203mg), S3 (110mg) and S4 (10mg) were obtained. The part of Fr.2 was recrystallized by petroleum ether–ethyl acetate to gain monomer compound S1 (32mg).Compounds S1, S2, S3, S4 and S5 were identified by UV, IR, MS, 1H-NMR, 13C-NMR etc. as 3, 5, 7-trihydrochromone, taxifolin, quercetin,β-sitoserol and daucosterol respectively.HPLC determination of TaxifolinIn this paper we presented a more sensitive quantification method by HPLC for measuring the main flavonoid (taxifolin).HPLC was performed on a ZORBAX XDB C18 column (250 mm×4.6 mm , 5μm) with the mixture of 30% methanol-70% acetic acid solution as mobile phase in gradient elution: 0-22min, 70% methanol; 22-32min, 100% methanol and the flow rate is 1.0ml/min at 25℃.ⅰThe linear range of taxifolin is 0.5μg～10μg. Y=1232.96X+10.12, r=0.9999.ⅱTaxifolin solution was injected into HPLC for six times, the result of precision tests is RSD<1%.ⅲThe reprodurity tests for six samples showed RSD<2%.ⅳWhen the concentration of taxifolin solution descrease to 0.001mg/ml, S/N=3.8>3 after determined by HPLC, So the detectability is 0.01μg。ⅴThe stability of taxifolin solution is good in ten hours because the results is RSD<1%.ⅵThe results of recovery is 100.9% and RSD is 1.7%.ⅶThree samples of the seeds Polygonum Orientale L. have been measured, the results show the content of taxifolin is 0.61%,0.60% and 0.59% respectively,the average is 0.60%。The results of this paper can offer a new item of content determination for establishment of quality standard of Polygonum Orientale.L ,also, can offer science foundation for the development from this medicine plant.
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