| Shengyangsanhuo Soft-extract, which is composed of ten herbs:Radix Bupleuri, Rhizoma Cimicifugae, Radix Puerariae, Rhizoma Notopterygii, Radix Saposhnikoviae, Radix Angelicae Pubescentis, Radix Glycyrrhizae Preparata, Licorice Root, Radix Ginseng and Radix Paeoniae Alba, is an effective prescription from the book of spleen and stomach written by LiDong-yuan. In the clinical practice of traditional Chinese medical science, the soft-extract has used to treat spleen and stomach debility and reduce internal heat of haslet. The modern doctors use it to treat toothache, congestive eyes, chronic pharyngitis, chronic oral ulcer, chronic tonsillitis, et al.This study is to optimize the process of extracting effective constituents from Sheng yangsanhuo powdered extract by central composite design and response surface methodology. Independent variables were ethanol concentration, boiling time and solvent fold. Dependent variables were contents of Puerarin, Ferulic acid, Isoferulic acid, Osthol, Isoimperatorin and recovery of extract. Dependent variables were transformed into desirability mathematically by Hassan’s method. Datas of overall desirability were fitted to a second order polynomial equation, through which three dimensional response surface graphs were produced. Optimum experimental conditions were selected from the stationary point of the response surfaces. Regression coefficients of binomial fitting complex model was as high as 0.995 3, the optimum condition of extraction process was 160 min for boiling time,16 fold water and 1 time for extraction. Ethanol concentration was 75% ethanol. Bias between observed and predicted values was-6.7%. Central composite design and response surface methodology is useful for the optimization of the process of extracting effective constituents from Shengyangsanhuo Soft-extract. The estimation of the established model is good.RP-HPLC methods were established to determine the contents of puerarin, ferulic acid, isoferulic acid, osthol, isoimperatorin, cimicifugoside, liquiritin,4’-O-β-D-glucosyl-5-O-methylvisamminol and paeoniflorin in Shengyangsanhuo powdered extract. The determination could be accomplished by different mobile phase system in DiamonsilTM C18(200 mm×4.6 mm,5μm)columns. The linear ranges were 15.6-156μg·mL-1(r= 0.999 5),0.66-6.60μg·mL-1(r= 0.999 7),8.96-89.6μg·mL-1(r= 0.999 5),6.30-63.0μg·mL-1(r= 0.999 3),1.32-13.2μg·mL-1(r= 0.999 6),3.02-30.2μg·mL-1(r= 0.999 8),17.9-179μg·mL-1(r= 0.999 8),3.80-38.0μg·mL-1(r= 0.999 5) and 9.52-95.2μg·mL-1(r= 0.9993), respectively. The recoveries were 99.7%(RSD= 0.9%),100.6%(RSD= 1.0%),100.2% (RSD= 1.1%),100.6%(RSD= 0.7%),99.8%(RSD= 0.9%),101.1%(RSD= 0.9%),100.9% (RSD= 1.1%),100.5%(RSD= 1.3%) and 100.4%(RSD= 0.6%), respectively. The assay methods are simple, rapid and reproducible, which provide a quantitive basis for the quality assessment of Shengyangsanhuo powered extract.In order to investigate the pharmacokinetics of Isoferulic acid in rats after oral administration of Shengyangsanhuo powdered extract, an HPLC method was established to determine concentrations of Isoferulic acid in rat plasma. With Hyperoside as internal standard, the analytes were separated through a DiamonsilTM C18 (200 mm×4.6 mm,5um) column at 35℃. The mobile phase consisted of a mixture of 0.1% phosphate acid in water-methanol-acetonitrile (120:25:45, v:v) at a flow rate of 1.0 mL·min-1. The detection wave length was set at 320 nm. The linear range of concentrations for Isoferulic acid in rat plasma was 0.05-20.0μg·mL-1(r= 0.997 8). The extracting recoveries at the concentrations of 0.10,5.00,16.0μg·mL-1 were 55.0%,58.3%,57.7%, respectively. Within-day RSD and between-day RSD were both less than 15%. The lower limit of quantization was 0.05μg·mL-1. The method was proved to be accurate and simple. And after oral administration of Shengyangsanhuo powdered extract to rats, the Pharmacokinetics parameters were achieved as follows:Cmax was 3311.6±1829.5 ng·mL-1, Tmax was 7.667±3.141 min, t1/2 was 54.75±14.10 min, AUC0→t was 161419.6±58739.6 ng·min·mL-1, AUC0→∞was 161419.6±58739.6 ng·min·mL-1. The method is simple, special, accurate and reproducible, which was suitable for the preclinical pharmacokinetics study of Isoferulic acid in rats.
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