| Daphne genkwa Sieb.et Zucc., a well-known medicinal plant,is widely used for treatment of codein,removing the phlegm , antibacterial and inflammation etc. So far, there are many researches in chemical ingredient and pharmacological function of Daphne genkwa Sieb.et Zucc. For the sake of the better use of this herb medicine, this paper describes the extraction, isolation and identification. Moreover, the method has been established for content determination of flavonoid tiliroside [kaempferol 3-O-β-D-( 6"-p-coumaroyl) glucopyranoside] in the Daphne genkwa Sieb.et Zucc in this paper. This method is accurate, reliable and reproducible to determine tiliroside in the Daphne genkwa Sieb.et Zucc. Extraction and SeparationDried floweres (2kg) were extracted by heat reflux in 95% Ethanol (solvent volume were 15, 12 and 10 L respecti- vely ) for three times (extraction time were 2,1.5 and 1h resp- ectively),the ethanol was evaporated then decompressed and concentrated to cream. The extraction was subjected to silica gel columnchromatography using ethyl acetate– methanol– water (ratio:25:1:0.5) as mobile phase to afford compounds YA (250mg),YB (90mg),YC (350mg),YD (300mg),YE (37mg),YF(6mg) and YG(18mg)。Compounds YA, YB, YC, YD, YE,YF and YG were identifyed respectively by UV, IR,MS, 1DNMR and 2DNMR as genkwanin, 3′-hydroxygenkwanin, apigenin, kaempferol-3-O-β-D-(6"-p- coumaroyl)-glucopyranoside, genkwain-5-O-β-D-glucoside,3′-hydroxygenkwanin-3′-O-β-D-glucoside and kaempferol-3-O-β-D-glucoside. Compounds YE, YF and YG were isolated the first time from Daphne genkwa Sieb.et Zucc.Content determination of tiliroside in the Daphne genkwa Sieb. et ZuccIn this paper, we present a more sensitive and accu-rate quantification method by HPLC for determining the major flavonoids (compound YD-tiliroside) in Daphne genkwa Sieb. et Zucc. HPLC was performed on a ZORBAX Eclipse XDB-C18 column(250 mm×4.6 mm,5μm)at 25℃with the rate of 1.0 ml/min,using the mixture of acetonitrile and water/acetic acid (1000/1 v/v) as mobile phase (gradient elution: 0-18min, 26:74; 18-33min, 80:20).Conclusions:ⅰThe linear range of tiliroside is 0.1μg～5.0μg. Y=23017.47X+23.55 r=0.9999ⅱTiliroside solution was injected into HPLC for six times, the RSD of precision tests is 1.32 % ( n=6).ⅲThe repeatability tests for six samples showed RSD is 0.98 % ( n=6).ⅳWhen the concentration of tiliroside solution descrease to 0.0001mg/ml, S/N=3.6 after determined by HPLC, So the detectability is 0.001μg (1ng)。ⅴThe test of stability showed its RSD is 3.00%, so the tiliroside solution is stable in eight hours.ⅵThe average recovery is 101.5%, its RSD is 0.92 %( n=6).ⅶNine batches samples of Daphne genkwa Sieb.et Zucc. have been measured by the above method, the results showed that their contents were variety in different regions. The content of tiliroside in Daphne genkwa Sieb.et Zucc. from AnHui province is higher than in Jilin province and HeBei province. The contents were showed as: 0.177%, 0.161%, 0.158%, 0.173%and 0.169% respectively from Anhui province; 0.141% from Jilin province; 0.149%,0.154% and 0.138% respectively from Hebei province. The results showed that the content of tiliroside in Daphne genkwa Sieb.et Zucc. should be above 0.12% in all the samples of Daphne genkwa Sieb.et Zucc.The results of this paper can provide with a new item on content determination for establishment of quality standards of Daphne genkwa Sieb.et Zucc., and also can offer science foundation for the development and utilization of this medicine plant.
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