Study On The Preparation, Quality Control Method And Pharmacokinetics Of Gushudan Granules

Gushudan is a Chinese compound formulation composed of Herba Epimedii,Fructus Cnidii, Rhizoma Drynariae and Radix Salviae Miltiorrhizae.We studied the preparation and quality control of Gushudan granules,the validated method was successfully applied to the pharmacokinetic studies of neoeriocitrin and naringin in rats after oral administration of the chinese compound formulation.1.Preparation of Gushudan granulesWith hydroscopicity and shaping as the index,five commonly used auxiliary materials were tested by single and double auxiliaries method respectively.Orthogonal test was used to choose the Gushudan granules preparation technique.That is,the ratio extract powder to vehicle is 2:1, the ratio lactose to dextrine is,the concentration of ethanol is 85%.the fluidity and moisture of absorption of Gushudan granule was detected with angle of repose and CRH.2.Study on the quality control of Gushudan granulesTLC methods have been developed for identification of icariin,osthole and naringin in Gushudan granules.A high performance liquid chromatography（HPLC）method has been developed for the determination of neoeriocitrin,icariin,osthole,naringin,imperatorin in Gushudan granule.The HPLC assay was performed on a Diamondsil ^TMC₁₈（250.0 mm×4.6 mm,5μm）column with a mobile phase of acetonitrile-water（21:79,v/v）at a flow-rate of 1.0 mL·min^-1.The UV detector was set at 285 nm.A good linearity was found over the range 5.32-160 mg.L^-1,4.90-147 mg·L^-1,10.2-307 mg·L^-1,1.58-47.3 mg·L^-1and 8.02-241 mg·L^-1,and calibration equations was Y= 1.01×10⁴x + 11.0,Y= 4.63 x 10⁴x + 65.7,Y= 2.26×10⁴ x -17.7,Y= 2.80x10⁴x + 3.11 and Y= 2.56×10⁴x - 8.36 for neoeriocitrin,icariin,osthole,naringin, imperatorin,respectively.The recoveries were 95.8%,97.0%,97.1%,98.3%and 98.6%for neoeriocitrin,icariin,osthole,naringin,imperatorin,respectively（n = 5）.The validated method has the advantages of simplicity,precision and reliability,allowing quality control of Gushudan granule.3.Pharmacokinetics of neoeriocitrin and naringin of Gushudan in ratA sensitive,specific method has been developed for simultaneous determination of neoeriocitrin and naringin in rat plasma using liquid-chromatography tandem mass spectrometry （LC-MS/MS）.With hesperidin as the internal standard（IS）,plasma samples were prepared by protein precipitation with methanol.The analysis was carried out on an ACQUITY UPLC^TM BEH C₁₈column using acetonitrile-water（20:80,v/v）as the mobile phase.The detection was performed by means of electrospray ionization mass spectrometry in negative ion mode with multiple reaction monitoring（MRM）.Linear calibration curves of neoeriocitrin and naringin were obtained over the concentration ranges of 15.0 - 960 ng/ml and 12.0 - 1200 ng/ml respectively.The intra- and inter-day precisions（RSD）were within 9.7%and 7.6%for neoeriocitrin and 7.8%and 12.9%for naringin.The accuracy（RE）was from -4.3 to 0.43%for neoeriocitrin and from -3.8 to 3.0%for naringin.The recoveries were all above 90%.The validated method was successfully applied to the pharmacokinetic studies of neoeriocitrin and naringin in rats after oral administration of Gushudan.The C_maxof neoeriocitrin and naringin were 236.5 ng·mL^-1and 332.5 ng·mL^-1,respectively, the T_maxwere both 0.20 h,the t_1/2were 0.47 h and 0.41 h,and the AUC_0～∞were 204.5 ng·mL^-1·h and 386.2 ng·mL^-1·h,respectively.Neoeriocitrin and naringin appeared to be absorbed and eliminated fast in vivo.