Determination Of The Active Constituents In Gushudan Decoction And Pharmacokinetics Study In Rat

Gushudan decoction is formed according to "The Basic Conception of Establishing the Technical Operation Flat Stage for the Fined Selective System of Small Decoction of Chinese Herbs" and "The Database Management System of PUJIFANG". This decoction is composed of Herba Epimedii, Fructus Cnidii, Rhizoma Drynariae and Radix Salviae Miltiorrhizae, and commonly used for the treatment of osteoporosis. Its activity stimulating osteoblastic bone formation was investigated previously in our laboratory.In this study five compounds were isolated from the n-butanol fraction of Herba Epimedii extract, and two compounds, naringin and neoeriocitrin, were isolated from the n-butanol fraction Rhizoma Drynariae extract. Their structures were identified by UV,IR,NMR and MS.A high performance liquid chromatography (HPLC) method has been developed for the determination of icariin, epimedin B and epimedin C in Herba epimedii. The HPLC assay was performed on a Hypersil ODS 2 column using acetonitrile - 0.4% acetate acid (25: 75, v/v) as the mobile phase at a flow-rate of 1.0 mL·min^-1, The UV detector was set at 260 nm, and trimethoprim as internal standard. A good linearity between peak area ratio and the concentration was found over the range 0.21-172μg·mL^-1,0.18-144μg·mL^-1 and 0.16-132μg·mL^-1 for icariin,epimedin B and epimedin C, respectively. The recoveries were 97.2 %,98.0 % and 98.3 %, with RSD of 2.0%,2.1% and 1.5% for icariin,epimedin B and epimedin C, respectively (n = 9). The contents of icariin, epimedin B, epimedin C in the species of Herba epimedii from 16 sources were determined. An HPLC method for determining naringin and neoeriocitrin in Rhizoma Drynariae has been also developed and the contents of naringin and neoeriocitrin in Rhizoma Drynariae from 7 sources were determined. The HPLC assay was performed on a Hypersil ODS2 column using acetonitrile-0.4% acetate acid (20: 80, v/v) as the mobile phase at a flow-rate of 1.0 mL.min^-1, and the UV detector at 283 nm. A good linearity between peak and the concentration was found over the range 10-400μg·mL^-1 and 12-480μg·mL^-1 for naringin and neoeriocitrin, respectively. The recoveries were 100.0% and 99.4%, with RSD of 1.4% and 2.0% for naringin and neoeriocitrin, respectively(n=9).The optimal extraction process of Gushudan decoction was studied by using orthogonal design with extraction yield and content of icarrin, osthole, naringin and tanshinoneⅡ_A as the indices. Factors including the solvent ammount, concentration of ethanol and extraction time were investigated. The optimal condition was 75% ethanol consumed 12 times of the amount of raw material, triplicate extraction and 90 minutes for each time.A study on the quality control methods of Gushudan decoction was carried out. The contents of icarrin, osthole, naringin and tanshinoneⅡ_A in Gushudan decoction were determined by RP-HPLC, separately. The methods were simple, rapid and accurate, and provided a qualitative and quantitative basis for the quality assessment of Gushudan decoction.RP-HPLC methods have been developed for the determination of epimedin C and osthole in plasma of rats after oral administration of Gushudan decoction. Afar addition of carbamaze- pine or fluocinonide as internal standard, plasma samples were extracted with ethyl acetate and diethyl ether, respectively. The linear ranges for epimedin C and osthole were 0.05-4μg·mL^-1 (r=0.999 0) and 0.052-5.2μg·mL^-1 (r=0.9952), respectively. The LOQs were 0.05μg·mL^-1 and 0.052μg·mL^-1, respectively. Both accuracy and precision of all the assays were satisfactory. The plasma concentration-time curves of epimedin C and osthole were plotted and the pharmacokin- etic parameters were calculated.The C_max, t_max, t_1／2 and AUC_0-∞ of epimedin C in rats receiving Herba Epimedii extract were 0.862μg·mL^-1, 2.5 h, 8.3 h and 7.90μg·mL^-1·h,, respectively. While those parameters in rats having Gushudan extract were 0.806μg·mL^-1, 3.0 h, 8.0 h and 9.35μg·mL^-1·h. The C_max, t_max, t_1／2 and AUC_0-∞ of osthole in rat plasma after oral administration of Fructus Cnidii extract were 0.776μg·mL^-1, 1.0 h, 3.6 h and 3.55μg·mL^-1·h,, respectively. While those parameters from Gushudan decoction were 1.18μg·mL^-1, 1.5 h, 4.0 h, 5.11μg·mL^-1·h respective ly. The parameter of C_max were significantly different (P＜0.05).In conclusion, under the direction of theory and clinical practice of TCM, The extraction technology was optimized and quality assessment methods for Gushudan decoction were developed. Pharmacokinetic study of epimedin C and osthole were carded out in rats to take a limited view of pharmacokinetic profiles of Gushudan decoction. This research provided an exploration for the modernization of TCM.