Interpretation
Der Songtext
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Abstract
The aim of this study was to identify the metabolites of kinsenoside in vivo and to study the pharmacokinetic properties of kinsenoside and syringin in rats. After a single oral administration of kinsenoside (2, 4 or 8 mg/kg), the plasma, urine, feces, bile, and different tissues were isolated, and the sample pretreatment procedures including protein precipitation and liquid–liquid extraction were optimized. The detection of kinsenoside and syringin was performed using a high-performance liquid chromatography–mass spectrometry method. The pharmacokinetic profiles of kinsenoside and syringin were studied in rats by intravenous or oral administration. The concentration of kinsenoside and syringin in plasma and various tissues was determined by high-performance liquid chromatography–mass spectrometry. Kinsenoside could be detected in the plasma, liver, lung, kidney, heart, and brain after oral administration of kinsenoside at 2, 4, or 8 mg/kg. Syringin could be detected in the plasma, liver, lung, kidney, heart, and brain after oral administration of syringin at 2, 4, or 8 mg/kg. The pharmacokinetic behaviors of kinsenoside and syringin in rats were compared with each other. The area under the concentration–time curve values of syringin were approximately 1.99-fold to 4.53-fold greater than those of kinsenoside in all tissues and plasma. The half-life and time to reach maximum concentration (Tmax) of kinsenoside were longer than those of syringin. In conclusion, syringin was more easily absorbed, distributed, and eliminated than kinsenoside in vivo. The results of this study might provide useful information for the further study of pharmacokinetics and pharmacological action of kinsenoside.
Keywords: Pharmacokinetics, kinsenoside, syringin, HPLC-MS
How to cite this article:
Zhang L, Zhang Q, Jiang Q. Metabolite Identification and Pharmacokinetics of Kinsenoside after Oral Administration in Rats. Int J Med Sci 2014; 11(12):1214-1220. doi:10.7150/ijms.10013. Available from http://www.medsci.org/v11p1214.htm
Abstract
Kinsenoside (Ginsenoside F1, KSD) is an important bioactive ingredient in Anoectochilus roxburghii (Wall.) Lindl., a traditional Chinese medicine (TCM). The present study aims to identify the metabolites of kinsenoside in vivo and investigate its pharmacokinetics. The urine, feces, plasma, and various tissues were isolated, and the sample pretreatment procedures including protein precipitation and liquid-liquid extraction were optimized. The concentrations of kinsenoside and its metabolite syringin in the plasma and various tissues were determined by HPLC-MS. The pharmacokinetics of kinsenoside and syringin were investigated after oral administration of kinsenoside in rats. The results showed that kinsenoside and its metabolite syringin could be detected in the plasma, liver, lung, kidney, heart, and brain after oral administration of kinsenoside at the doses of 2, 4, or 8 mg/kg. The AUC0-∞ values of syringin were approximately 1.99- to 4.53-fold higher than those of kinsenoside in all tissues and plasma. The half-life (t1/2) and time to reach maximum concentration (Tmax) values of kinsenoside were longer than those of syringin. In conclusion, syringin was more easily absorbed, distributed, and eliminated than kinsenoside in vivo. The results of this study provide useful information for further investigations on the pharmacokinetics and pharmacological actions of kinsenoside.
Keywords: kinsenoside, syringin, metabolism, pharmacokinetics
Introduction
Anoectochilus roxburghii (Wall.) Lindl. (Orchidaceae) is a traditional Chinese medicinal plant widely used in China and Southeast Asia for the treatment of various diseases, including cancer [1-6]. Kinsenoside (KSD), also called ginsenoside F1, is a major bioactive compound in A. roxburghii [7]. Previous studies have shown that KSD has various pharmacological activities, including anti-inflammatory [8], immuno-enhancement [9], anti-oxidant [10], antiviral [11], and anti-cancer effects [12]. However, little is known about the absorption, distribution, metabolism, and excretion of KSD in vivo.
In the present study, we investigated the metabolites of KSD and their pharmacokinetics, which may provide a basis for the study of the mechanism of action of KSD and its clinical application.
Materials and Methods
Chemicals and reagents
KSD (purity > 98%) was purchased from the Chengdu Mansite Biological Technology Co., Ltd. (Chengdu, China). Syringin (purity > 98%) was purchased from the National Institutes for Food and Drug Control (Beijing, China). Acetonitrile and methanol of HPLC grade were purchased from Fisher Scientific (USA). Ammonium acetate was purchased from Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). Formic acid of HPLC grade was purchased from Sigma-Aldrich (USA). Water was purified using a Milli-Q water purification system (Millipore, Bedford, USA). The other reagents were of analytical grade, and all the solvents used in the experiment were of ultrapure grade.
Apparatus
The analysis was performed using an Agilent 1260 liquid chromatography system (Agilent Technologies, USA) equipped with a quaternary pump, an autosampler, a column oven and a diode array detector (DAD). The separation of the analytes was performed on a ZORBAX SB-C18 column (250 mm × 4.6 mm, 5 μm, Agilent Technologies, USA). The mobile phase consisted of acetonitrile-water (55:45, v/v) containing 0.1% formic acid, and was delivered at a constant flow rate of 1.0 mL/min. The column temperature was set at 30°C.
Animals
Male Sprague-Dawley rats (weighing 180-220 g) were purchased from the Experimental Animal Center of Zhejiang Medicine Co., Ltd (Huzhou, China, Certificate No. SCXK (Zhejiang) 2008-0010). The rats were kept in a temperature- and humidity-controlled environment (20 ± 2°C and 50% ± 5%) with a 12-h light/dark cycle, and were supplied with water and food ad libitum. The animal experiments were approved by the Animal Ethics Committee of the Zhejiang University of Traditional Chinese Medicine (Hangzhou, China) and were conducted according to the Guidelines for the Care and Use of Laboratory Animals.
Sample preparation and extraction
Stock solutions of KSD (1 mg/mL) and syringin (1 mg/mL) were prepared in methanol. Working solutions were prepared by appropriate dilutions of the stock solutions with methanol. The calibration curves of KSD and syringin were obtained by analysis of standard solutions prepared at different concentrations. The calibration curves of KSD and syringin were linear in the range of 0.05-10 μg/mL and 0.02-10 μg/mL, respectively.
To obtain pharmacokinetic data for KSD and syringin alone, rats (n = 6) were administered KSD (2, 4, and 8 mg/kg, respectively) via a single tail vein injection. Blood (0.3 mL) was collected from each rat at 0.083, 0.25, 0.5, 0.75, 1, 2, 4, 6 and 8 h after administration, and the plasma samples were obtained by centrifugation at 4000 rpm for 10 min. The organs (liver, lung, kidney, heart and brain) were harvested at 0.5 and 2 h after administration. Urine and feces samples were collected over 24 h after administration. All of the samples were stored at -80°C until analysis.
Sample preparation for plasma and tissue samples
A 100-μL aliquot of plasma or tissue homogenate was mixed with 10 μL of internal standard solution (50 μg/mL of ginsenoside Rb1 in methanol) in a 1.5 mL centrifuge tube, and then 1 mL of methanol and 10 μL of formic acid were added. The mixture was vortex-mixed for 1 min and sonicated for 10 min in an ice bath, and then centrifuged at 12000 rpm for 10 min. The supernatant was filtered through a 0.22 μm polytetrafluoroethylene syringe filter prior to HPLC analysis.
Sample preparation for urine and feces samples
Urine (0.5 mL) or feces (0.2 g) was mixed with 10 μL of internal standard solution in a 1.5 mL centrifuge tube, and then 1 mL of methanol and 10 μL of formic acid were added. The mixture was vortex-mixed for 1 min and sonicated for 10 min in an ice bath, and then centrifuged at 12000 rpm for 10 min. The supernatant was filtered through a 0.22 μm polytetrafluoroethylene syringe filter prior to HPLC analysis.
HPLC analysis
The HPLC system was equipped with a quaternary pump, an autosampler, a column oven, and a diode array detector (DAD). Chromatographic separation was performed on a ZORBAX SB-C18 column (250 mm × 4.6 mm, 5 μm, Agilent Technologies, USA) maintained at 30°C. The mobile phase consisted of acetonitrile-water (55:45, v/v) containing 0.1% formic acid, and was delivered at a flow rate of 1.0 mL/min. The injection volume was 10 μL.
The detection wavelength was set at 203 nm for KSD and syringin, and 203 nm for ginsenoside Rb1. KSD, syringin and ginsenoside Rb1 were eluted at 3.8, 4.7 and 11.9 min, respectively. The method was validated over a concentration range of 0.05-10.0 μg/mL for KSD and 0.02-10.0 μg/mL for syringin. The calibration curve was constructed by plotting the peak area ratio (analyte/internal standard) against the nominal concentration.
Pharmacokinetic analysis
Pharmacokinetic parameters were calculated using non-compartmental analysis with DAS 2.1.1 software (Chinese Pharmacological Society) and expressed as the mean ± standard deviation (SD). The peak concentration (Cmax) and the time to reach maximum concentration (Tmax) were directly obtained from the plasma concentration-time data. The area under the plasma concentration