| Tusizi is the seed of Cuscuta chinensis Lam, 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 constituents and pharmacological function of Cuscuta chinensis Lam. For the sake of the better use of this herb medicine, this paper describes the extraction, isolation and identification. Moreover, the determination method has been established for content determination of hyperin and kaempferol-3-O-β-D- glucoside in the Cuscuta chinensis Lam in this paper. This determination method was accurate, reliable and reproducible to determine hyperin and kaempferol-3-O-β-D- glucoside in the Cuscuta chinensis Lam.Extraction and SeparationDried seeds (3kg) were extracted by heat reflux in 85% Ethanol (solvent volume were 15, 12 and 10 L respectively ) for three times (extraction time were 2,1.5 and 1h respectively), the ethanol was evaporated in vacuum and concentrated to cream. The cream was subjected to silica gel column chromatography using ethyl acetate– methanol– water (8:1:0.5) as mobile phase to afford compounds A (50mg),B (500mg),C(300mg), D(50mg), E(200mg), F(30mg), G(50mg).All the compounds were identifyed respectively by UV, IR, MS, 1DNMR and 2DNMR as kaempferol(A), hyperin(B), kaempferol-3-O-β-D- glucoside, quercetin-3-O-β-D-apiofuranosyl-(1→2)-β-D-galactoside(C),. Content determination of hyperin, kaempferol-3-O-β-D- glucoside(D), 6-O-(E)-P-coumaroyl)-β-D-fructofuranosyl-(2→1)-α-D-glucopyranoside(E), daucosterol(F) andβ-sitosterol(G).Compound E has been isolated from this plant for the first time.In this paper, we present a more sensitive and accurate quanti-fication method by HPLC for determining the major flavonoids (compound C-hyperin and compound D-kaempferol-3-O-β-D- glucoside) in the Cuscuta chinensis Lam. HPLC was performed on Agilent 1200 instrument and a ZORBAX Eclipse XDB-C18 column(250 mm×4.6 mm,5μm)at 25℃with the rate of 1.0 mL·min-1,using the mixture of acetonitrile and water/acetic acid (1000/1 v/v) as mobile phase (gradient elution: 0-24 min, 15:85; 24-27 min, 18:82).Resultsⅰboth hyperin and kaempferol-3-O-β-D- glucoside gave the same linear range ,viz.0.1μg～5.0μg. y=13411.58x-41.45 r=0.9999 y=19556.42x-31.24 r=0.9999ⅱHyperin solution was injected into HPLC for six times, the RSD of precision tests was 1.11 % ( n=6). The solution of kaempferol-3-O-β-D- glucoside was injected into HPLC for six times, the RSD of precision tests was 0.87 % ( n=6).ⅲThe repeatability tests for six samples showed RSD of hyperin and kaempferol-3-O-β-D-glucoside were 1.23%, 1.47% ( n=6 ) respectively.ⅳThe concentration of solution of hyperin and kaempferol- 3-O-β-D- glucoside diluted to 0.0005mg/mL and determined by HPLC, it showed the values of S/N were 4.2 and 6.0 respectively, So their detectabilities were 0.005μg (5ng)。ⅴThe test of stability showed its RSD were 1.16%,1.90%, so the solution of hyperin and kaempferol-3-O-β-D- glucoside was stable in ten hours.ⅵThe average recovery of hyperin and ikaempferol-3-O-β-D- glucoside were 98.5%( RSD=1.23 ), 97.6%( RSD=1.47 )( n=6) respectively。ⅶFour batches samples of Cuscuta chinensis Lam. have been measured by the above method, the results showed that their contents were variety in different regions. The content of hyperin in Cuscuta chinensis Lam from NeiMeng province is higher than from LiaoNing province, JiLin province and ShanXi province. The average contents were showed as 0.43%, 0.091%, 0.20%, 0.32% respectively; The content of kaempferol-3-O-β-D- glucoside in Cuscuta chinensis Lam from JiLin province is higher than from LiaoNing province, NeiMeng province and ShanXi province. The average contents were showed as: 0.25%, 0.086%, 0.0%, 0.15%respectively; The results showed that the contents of hyperin and kaempferol-3-O-β-D- glucoside in Cuscuta chinensis Lam. from JiLin province is higher than from other provinceThe results of this paper can provide a new index of content determination for establishment of quality standards of Daphne genkw Cuscuta chinensis Lam., and also can offer science foundation for the development and utilization of this medicine plant...
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