74.3 % ~ 89.1 % 1.08 h

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1 31 Traditional Chinese Drug Research & Clinical Pharmacology 11 May Vol.22 No.3 /μg ml Figure 2 Concentration-time curve of the puerarin in compound puerarin or puerarin injection [2] P > t / min 3P9 t t 1/2α t 1/2β V c Cl s AUC AUC - AUC -t MRT - MRT -t P >.5 [1]. [J].. [J]. [3]. [J] [4]. [J] Beagle [5] [J] Agilent 1 Zorbax SB-C mm 4.6 mm 5 μm.1 % - 1. ml / min 1 μl 2 nm 9 mg/kg WinNolin.9 ~. μg/ml r= % ~ 89.1 %.4 μg/ml 1.8 h 3.6 μg/ml R285.5 A ma96912@umac.mo yzheng@umac.mo RG85/9-1S/ZY/ICMS

2 Danshensu Pharmacokinetics in Beagle Dogs Plasma by HPLC-UV Method After Oral Administration YOU Chang SUN Jiabei SUN Shaoping ZHENG Ying Institute of Chinese Medical Science University of Macau Macau SAR 9998 China Abstract: Objective To establish a method to determine the plasma concentration of danshensu in beagle dog using HPLC with DAD detector and to calculate the pharmacokinetic parameters of danshensu in beagle dog after oral administration. Methods Caffeic acid was used as the internal standard and drugs in plasma were extracted by ethyl acetate. Agilent 1 HPLC system with Zorbax SB-C 18 column 25 mm 4.6 mm 5 μm was used. The mobile phase conditions were as follows:.1 % formic acid-acetonitrile with gradient elution flow rate of 1. ml/min injection volume of 1 μl and wavelength of DAD detection being 2 nm. After danshensu pellets were orally administered to beagle dogs at 9 mg/kg single dose plasma samples were collected at predetermined time. Concentrations of drugs in plasma were quantified by HPLC and pharmacokinetic parameters were calculated by non -compartment model with WinNolin software. Results The linear range of calibration curve of danshensu was.9 ~. μg/ml with good linearity r= The drug recovery was 4.3 %~89.1 % and the lowest quantitative concentration was.4 μg/ml. After oral administration in beagle dogs the average time for danshensu to reach the peak was 1.8 h with the mean maximum concentration C max of 3.6 μg/ml. Conclusion This method is simple convenient and accurate for pharmacokinetic study of danshensu in beagle dogs. Keywords: Danshensu Beagle dogs HPLC Pharmacokinetics lent Type Salvia miltiorrhiza Bge N-EVAP/OA-SYS Organomation Associates MilliQ-water Milli- [1] pore Danshensu [2] 1.2 Sodium Danshensu 98 % [3] [4] [5] [6] [] Caffeic acid 98 % [8] Merck 1.3 [9] - 1 kg~12 kg [1] - 12 h [11] mg/ ml - 1 % μg/ml μg/ml 1 1. mg/ml 1.1 Agilent 1 Agilent Agilent Agi- - 5 μg/ml

3 312 Traditional Chinese Drug Research & Clinical Pharmacology 11 May Vol.22 No.3 6 μl 5 μl+ 5 % 1 %.16 μg/ml 1.34 μg/ml 5.38 μg/ HCl 1 μl 15 ml ml 3 5 % 1 % 5 μg/ml 5 μl 5 HCl 1 μl 2.2 ml 1 min 4 5 r/min 5 1 min 3 d 3 15 μl.2 % 1 % 13 r/ min 5min HPLC 2. 4 μl μl 2.3 Agilent Zorbax SB-C mm 4.6 mm 5 μm Agilent Zorbax SB-C mm 4.6 mm 5 μm.1 % μg/ml h 5% 24 h % HCl 1 μl h mg/kg μl 4.25 h.5 h.5 h 1. h 1.5 h 2. h μg/ml h 3. h 4. h 5. h 6. h 12. h 5 % 1 % HCl 2 ml 5 r/min 1 μl min 5 μl 1 5 % 1 % HCl 1 μl - 14 d 14 d.16 μg/ml 1.34 μg/ml 5.38 μg/ ml 3 5% 1 1 % HCl 1 μl ml/min 1 μl 2 nm A 1 μl 25 1 % 15 μl 1.1 % - A 2 Table 1 Procedure of formic acid.1 % -acetonitrile gradient R=A 1 / A 2 1 % 5 elution μl /min % /% /% μl.16 μg/ml 1.34 μg/ml 5.38 μg/ml 3 5 % 1 %HCl 1 μl d μl

4 LOQ.4 μg/ml WinNolin Mountain View Pharsight CA ±11. % 4.3±3.9 % 4.4±3.8 %.5 min 13.3 min 1 2 Table 2 Intra- and inter-day precisions and accuracies of HPLC method for determination of danshensu in beagle dog plasma /μg ml / % RSD / % min a min c..5 h Table 3 3 Stability test of HPLC method for determination of danshensu in beagle dog plasma /μg ml -1 /μg ml % /μg ml /% min /μg ml b /% HPLC Figure 1 Representative chromatograms of danshensu in beagle dog plasma determined by HPLC-UV method 5 ml ml 5 ml ph.31~.42 Y= HCl ph.5x -.36 r=.9999 n= ~. μg/ml [9] [12] S/N =1

5 314 Traditional Chinese Drug Research & Clinical Pharmacology 11 May Vol.22 No.3 /μg ml -1 /μg ml t / h a. 1 h C max = 3.6 ±.64 μg/ml T 1/2 =.65 ±.32 h HPLC-DAD [1]. [J] [2]. - [J] [3] Ding M Ye TX Zhao GR et al. Aqueous extract of Salvia miltiorrhiza attenuates increased endothelial permeability induced by tumor necrosis factor -α [J]. International Immunopharmacology 5 5 t / h b. - [4] Zhang LJ Chen L Lu Y et al. Danshensu has anti-tumor activity 2 HPLC-UV in B16F1 melanoma by inhibiting angiogenesis and tumor cell - n=3 invasion [J]. European Journal of Pharmacology Figure 2 Plasma concentration-time profile of danshensu determined 1. by HPLC-UV method after oral administration of danshensu to beagle dog [5]. H + ~ATP [J] n=3 [6] Zhou LM Zuo Z Chow SS. Danshen an overview of its chemistry Table 4 Pharmacokinetics parameters of danshensu after oral pharmacology pharmacokinetics and clinical use [J]. Journal of administration of danshensu powder or pellets in beagle dog Clinical Pharmacology []. LDL ±SD ±SD [J] C max/μg ml -1 T max / h 3.6± ± ± ± [8]. [J] AUC -t/μg h ml ± ± [ 9] Tong Lu Junling Yang Chuan Li. Plasma and Urinary Tanshinol AUC -i/μg h ml -1 MRT/h T 1/2/h Kc/h-1 4.6± ±.43.65± ± ± ± ±1.6.62± from Salvia miltiorrhiza Danshen Can Be Used as Pharmacokinetic Markers for Cardiotonic Pills a Cardiovascular Herbal Medicine [J]. Drug Metabolism and Disposition [1] Luo XJ Bi KS Zhou SY et al. Determination of Danshensu a major active compound of Radix Salvia miltiorrhiza in dog plasma by HPLC with fluorescence detection [J]. Bimedical Chromatography [11]. [J] [12] [J] [13]. [J] [14]. [J] [13-14].

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