Xenobiotica the fate of foreign compounds in biological systems

ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20

Pharmacokinetic properties of trifolirhizin, (–)-maackiain, (–)-sophoranone and 2-(2,4dihydroxyphenyl)-5,6-methylenedioxybenzofuran after intravenous and oral administration of Sophora tonkinensis extract in rats Soo Min Jang, Soo Hyeon Bae, Woong-Kee Choi, Jung Bae Park, Doyun Kim, Jee Sun Min, Hunseung Yoo, Minseok Kang, Keun Ho Ryu & Soo Kyung Bae To cite this article: Soo Min Jang, Soo Hyeon Bae, Woong-Kee Choi, Jung Bae Park, Doyun Kim, Jee Sun Min, Hunseung Yoo, Minseok Kang, Keun Ho Ryu & Soo Kyung Bae (2015) Pharmacokinetic properties of trifolirhizin, (–)-maackiain, (–)-sophoranone and 2-(2,4-dihydroxyphenyl)-5,6-methylenedioxybenzofuran after intravenous and oral administration of Sophora tonkinensis extract in rats, Xenobiotica, 45:12, 1092-1104, DOI: 10.3109/00498254.2015.1041181 To link to this article: http://dx.doi.org/10.3109/00498254.2015.1041181

Published online: 11 Jun 2015.

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Date: 27 October 2015, At: 02:12

http://informahealthcare.com/xen ISSN: 0049-8254 (print), 1366-5928 (electronic) Xenobiotica, 2015; 45(12): 1092–1104 ! 2015 Informa UK Ltd. DOI: 10.3109/00498254.2015.1041181

RESEARCH ARTICLE

Pharmacokinetic properties of trifolirhizin, (–)-maackiain, (–)-sophoranone and 2-(2,4-dihydroxyphenyl)-5,6-methylenedioxybenzofuran after intravenous and oral administration of Sophora tonkinensis extract in rats

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Soo Min Jang1*, Soo Hyeon Bae2*, Woong-Kee Choi1, Jung Bae Park1, Doyun Kim1, Jee Sun Min1, Hunseung Yoo3,4, Minseok Kang4, Keun Ho Ryu5, and Soo Kyung Bae1 1

College of Pharmacy and Integrated Research Institute of Pharmaceutical Sciences, The Catholic University of Korea, Bucheon, Republic of Korea, Department of Pharmacology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea, 3College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Republic of Korea, 4New Drug Preclinical & Analytical Team, and 5New Drug Team 1, Life Science R&D Center, SK Chemicals, Sungnam, Republic of Korea

2

Abstract

Keywords

1. SKI3301, a standardized dried 50% ethanolic extracts of Sophora tonkinensis, contains four marker compounds (trifolirhizin, TF; (–)-maackiain, Maack; (–)-sophoranone, SPN, and (2-(2,4dihydroxyphenyl)-5,6-methylenedioxybenzofuran, ABF), is being developed as an herbal medicine for the treatment of asthma in Korea. This study investigates the pharmacokinetic properties of SKI3301 extract in rats. 2. The dose-proportional AUCs suggest linear pharmacokinetics of TF, Maack, SPN and ABF in the SKI3301 extract intravenous dose range of 5–20 mg/kg. After the oral administration of 200–1000 mg/kg of the extract, TF and Maack exhibited non-linearity due to the saturation of gastrointestinal absorption. However, linear pharmacokinetics of SPN and ABF were observed. 3. The absorptions of TF, Maack, SPN and ABF in the extract were increased relative to those of the respective pure forms due to the increased solubility and/or the decreased metabolism by other components in the SKI3301 extract. 4. No accumulation was observed after multiple dosing, and the steady-state pharmacokinetics of TF, Maack, SPN and ABF were not significantly different from those after a single oral administration of the extract. 5. The pharmacokinetics of TF, SPN and ABF were not significantly different between male and female rats after oral administration of the extract, but a significant gender difference in the pharmacokinetics of Maack in rats was observed. 6. Our findings may help to comprehensively elucidate the pharmacokinetic characteristics of TF, Maack, SPN and ABF and provide useful information for the clinical application of SKI3301 extract.

Pharmacokinetics, rats, SKI3301, (–)-maackiain, (–)-sophoranone, Sophora tonkinensis, trifolirhizin, 2-(2,4-dihydroxyphenyl)-5, 6-methylenedioxybenzofuran

Introduction The dried roots and rhizomes of Sophora tonkinensis are traditionally used in herbal medicines in Korea and China under the name Shan-dou-gen for the treatment of asthma, allergic dermatitis, gastrointestinal hemorrhage, chronic bronchitis, acute pharyngolaryngeal infections and sore throat (He et al., 2013; Wu, 2005; Xiao, 1993). They are *These authors contributed equally to this work. Address for correspondence: Soo Kyung Bae, College of Pharmacy and Integrated Research Institute of Pharmaceutical Sciences, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon 420-743, Republic of Korea. Tel: +82 2 2164 4054. Fax: +82 2 2164 4096. E-mail: [email protected]

History Received 25 March 2015 Revised 10 April 2015 Accepted 12 April 2015 Published online 11 June 2015

known to contain mainly isoprenylated flavonoids, quinolizidine alkaloids, and triterpenoids with various pharmacological activities (Ding & Chen, 2006; Ding et al., 2006; Li et al., 2008a, 2008b; Xiao et al., 1999). The individual components or extracts of S. tonkinensis have also been found to possess anti-inflammatory (Lee et al., 2015; Li et al., 2012; Yoo et al., 2014a), anti-cancer (Chui et al., 2005; Liu et al., 2006), and antipyretic functions as well as hepato-protective effects (Chai et al., 2012; Cho et al., 1986; Long et al., 2004). The roots and rhizomes of S. tonkinensis are being developed as an herbal medicine for the treatment of asthma by SK Chemicals Ltd, a Korean pharmaceutical company (Suwon, Republic of Korea). SKI3301, a standardized extract obtained from dried 50% ethanolic extracts of

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Figure 1. Chemical structures of TF (A), Maack (B), SPN (C), and ABF (D).

S. tonkinensis, contains four marker compounds, three flavonoids (trifolirhizin, TF; (–)-maackiain, Maack; and (–)-sophoranone, SPN) and one 2-arylbenzofuran derivative (2-(2,4-dihydroxyphenyl)-5,6-methylenedioxybenzofuran, ABF) (Figure 1). After the oral administration of SKI3301 extract at a dose of 200 mg/kg twice a day for two weeks, it demonstrated an anti-allergic property on mast cell-mediated allergic response in an ovalbumin-induced asthma mouse model (data not shown). Of the four marker compounds, TF has been proposed for use in the treatment of asthma by significantly inhibiting the acetylcholine-induced airway smooth muscle contraction (Yang et al., 2013). Maack and SPN inhibit 5-lipoxygenase, which plays a potent pathophysiological role in asthma development, with IC50 values of 7.9 and 1.6 mM, respectively, in vitro (data not shown). Thus, these three flavonoids (TF, Maack and SPN) likely account for the spectrum of medicinal properties of SKI3301 extract. Bioactive constituents with favorable pharmacokinetic properties present in adequate amounts in medicinal herbs most likely to account for the pharmacological effects and form the basis of their therapeutic efficacy (Liu et al., 2009). However, the pharmacokinetic properties of constituents contained in an herbal medicine can be quite different compared with those of respective pure constituent due to its multi-components. Recently, we have published an analytical LC-MS/MS method for the simultaneous quantification of the four marker compounds (TF, Maack, SPN and ABF) in SKI3301 in rat plasma (Yoo et al., 2014b). Using this bioanalytical method, we could obtain the pharmacokinetic properties of TF, Maack, SPN and ABF after a single oral administration of SKI3301 at a dose of 400 mg/kg to rats. As a continuation of our previous research, we carried out this work to study the pharmacokinetic properties of TF, Maack, SPN and ABF after the intravenous or oral administration of SKI3301 extract in male rats at various doses and to assess the dose-proportionality of the four marker compounds. We also compared the pharmacokinetic properties

following oral administration of equivalent doses of the individual four marker compounds (TF, Maack, SPN and ABF) and following oral administration of SKI3301 extract in male rats. Additionally, we performed steady-state pharmacokinetic studies on TF, Maack, SPN and ABF after multiple oral dosing of the SKI3301 extract to assess whether accumulations of TF, Maack, SPN and ABF occurred in the body. The pharmacokinetic properties of TF, Maack, SPN and ABF after oral administration of SKI3301 extract in both female and male rats were also studied. This work provides significant evidence to contribute to a comprehensive understanding of the pharmacokinetic properties of SKI3301 extract in vivo.

Materials and methods Chemicals and reagents SKI3301 extract (Lot No. SOTB07-10-07), trifolirhizin (98.0% purity; TF), (–)-macckiain (98.4% purity; Maack), (–)-sophoranone (98.0% purity; SPN) and 2-(2,4-dihydroxyphenyl)-5,6-methylenedioxybenzofuran (99.3% purity; ABF) were supplied by SK Chemicals Ltd. (Suwon, Gyeonggi-do, Republic of Korea). The respective contents of TF, Maack, SPN and ABF in the SKI3301 extract (Lot No. SOTB07-1007) were 4.76, 1.84, 3.17, and 0.25%, respectively. Ammonium acetate, chlorpropamide, dimethylsulfoxide, formic acid, methyl cellulose with a viscosity of 4000 and polyethylene glycol 400 were products of Sigma-Aldrich (St. Louis, MO). All solvents were of high-performance liquid chromatography (HPLC) grade and were obtained from Burdick & Jackson Company (Morristown, NJ), and other chemicals were of the highest quality available. Animals Male and female Sprague–Dawley rats (7–9 weeks, 250–310 g) were purchased from Orient Bio (Sungnam,

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Gyeonggi-do, Republic of Korea). The protocol of this animal study was approved by the Department of Laboratory Animals, Institutional Animal Care and Use Committee on the Sungsim Campus of the Catholic University of Korea. The rats were maintained under controlled environmental conditions (temperature 20 ± 2  C; relative humidity 55 ± 5%; 12-h light/dark cycle). Rats were used for pharmacokinetic studies after 1-week acclimatization with free access to water and feed. During the experiments, each rat was housed in a metabolic cage (Tecniplast, Varese, Italy).

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Single intravenous administration of SKI3301 extract at doses of 5, 10 and 20 mg/kg to male rats To assess whether the four marker compounds of SKI3301 (namely TF, Maack, SPN and ABF) followed dose-proportional pharmacokinetics, the extract was administered intravenously at various doses to rats. The procedures used for anesthesia and animal handling have been reported previously (Bae et al., 2014). The carotid artery (for blood sampling) and the jugular vein (for drug administration) of each rat were cannulated with polyethylene tube 50 (Clay Adams, Parsippany, NJ). SKI3301 extract (dissolved in dimethylsulfoxide: polyethylene glycol 400: distilled water ¼ 5:50:45 (v/v/ v)) at doses of 5, 10, and 20 mg (2 mL)/kg (n ¼ 9, 9, and 9, respectively) was infused over 1 min via the jugular vein. Blood samples (0.12 mL, each) were collected via the carotid artery at 0 (pre-dose), 1 (at the end of the infusion), 5, 15, 30, 45, 60, 90, 120, 180, 240, 360, and 480 min after the intravenous administration of SKI3301 extract. Then, 0.3 mL of a heparinized 0.9% NaCl-injectable solution (20 units/mL) was used to flush the cannula immediately after each blood sampling to prevent blood clotting. The blood samples were immediately centrifuged and a 50-mL aliquot of each plasma sample were stored at 80  C until further use in the LC-MS/ MS analysis. At the end of 24 h, each metabolic cage was rinsed with 20 mL of distilled water and the rinsing was combined with the 24-h urine sample. After measuring the exact volume of the combined urine sample, two 50-mL aliquots of the combined urine sample were stored at 80  C until further use in the LC-MS/MS analysis. At the same time (24 h), each rat was euthanized with CO2, the abdomen was operated, and the entire gastrointestinal tract (including its contents and feces) of each rat was excised, transferred into a beaker containing 50 mL of methanol (to facilitate the extraction of TF, Maack, SPN and ABF), and cut into small pieces using scissors. After sonication for 20 min, duplicate aliquots (50 mL) of the supernatant were collected from each beaker and stored at 80  C until further use in the LC-MS/ MS analysis.

Xenobiotica, 2015; 45(12): 1092–1104

30, 60, 90, 120, 180, 240, 360 and 480 min after the oral administration of SKI3301 extract. The other procedures for the oral study were similar to those of the intravenous study. Pharmacokinetic studies of TF, Maack, SPN and ABF after oral administration of their pure forms at a dose equivalent to 400 mg/kg of SKI3301 extract to male rats SKI3301 extract contains 4.76% TF, 1.84% Maack, 3.17% SPN and 0.25% ABF, respectively. Considering their purities, an oral dose of pure TF, Maack, SPN, and ABF containing 19.4, 7.48, 12.9, and 1.00 mg/kg (n ¼ 9, 9, 9 and 9), respectively, which is equivalent to 400 mg/kg of SKI3301 extract, was administered to the rats. Pure TF, Maack, SPN and ABF were suspended in the same solution as used in the oral studies. Blood samples were collected at 0, 5, 15, 30, 60, 90, 120, 180, 240, 360 and 480 min after oral administration of TF, Maack, SPN and ABF. The other procedures were similar to those used for the oral study. Multiple oral administration of SKI3301 extract at a dose of 400 mg/kg twice a day for six days to male rats The male rats were grouped randomly into two groups (n ¼ 8 per group). One group was orally dosed with 400 mg (10 mL)/ kg of SKI3301 extract (suspended in 0.5% methyl cellulose with a viscosity of 4000) twice daily for six days via gastric gavage to reach a steady state, and the other group was orally dosed with 0.5% methyl cellulose with a viscosity of 4000 (10 mL/kg) twice daily for six days via gastric gavage. On the seventh day morning, SKI3301 (the same solution used in the oral study) at a dose of 400 mg (10 mL)/kg was administered orally using a gastric gavage tube to rats in each group. Blood samples were collected at 0, 5, 15, 30, 60, 90, 120, 180, 240, 360 and 480 min after the oral administration of SKI3301 extract. The other procedures were similar to those used for the oral study. Gender differences in the pharmacokinetics of TF, Maack, SPN and ABF after oral administration of SKI3301 to rats SKI3301 extract (the same solution used in the oral study) at a dose of 400 mg (10 mL)/kg was administered orally using a gastric gavage tube to male (n ¼ 8) and female (n ¼ 10) rats. Blood samples were collected at 0, 5, 15, 30, 60, 90, 120, 180, 240, 360 and 480 min after oral administration of SKI3301. The other procedures were similar to those used for the oral study. LC-MS/MS analysis

Single oral administration of SKI3301 extract at doses of 200, 400 and 1000 mg/kg to male rats For oral administration, the carotid arteries of each rat were cannulated with polyethylene tubing for blood sampling. SKI3301 extract (suspended in 0.5% methyl cellulose with viscosity 4000) at doses of 200, 400 and 1000 mg (10 mL)/kg (n ¼ 12, 11 and 10, respectively) was administered orally using a gastric gavage tube to rats after overnight fasting with free access to water. Blood samples were collected at 0, 5, 15,

In our lab, we have developed and validated LC-MS/MS methods for the simultaneous determination of TF, Maack, SPN and ABF in rat plasma (Yoo et al., 2014b). The LC-MS/ MS system used was an API 5500 Q-Trap mass spectrometer (AB SCIEX, Foster City, CA) equipped with a 1260 HPLC system (Agilent Technologies, Wilmington, DE) in electrospray ionization mode to generate both positive [M + H]+ and negative [M  H]– ions simultaneously. In brief, a 150-mL aliquot of acetonitrile containing 100 ng/mL chlorpropamide

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Figure 2. Mean arterial plasma concentrations of TF (A), Maack (B), SPN (C), and ABF (D) after intravenous administration of SKI3301 extract at doses of 5 mg/kg (*, n ¼ 9), 10 mg/kg ( , n ¼ 9), and 20 mg/kg (g, n ¼ 9) to male rats. Vertical bars indicate standard deviation.



(internal standard, IS) was added to a 50-mL aliquot of rat plasma sample and mixed by vortexing for 2 min. After the mixture was centrifuged at 13 000 rpm for 15 min at 4  C, the supernatant fraction was separated, transferred into a 1.5-mL Eppendorf tube and evaporated under a gentle stream of nitrogen gas at 40  C. The residue was reconstituted with a 100-mL aliquot of the mobile phase (7.5 mM ammonium acetate:acetonitrile, 50:50, v/v), and a 5-mL aliquot was injected directly into the LC-MS/MS system. All prepared samples were kept in an autosampler at 4  C until injection. Four marker compounds were separated on a C18 reversedphase column (Acclaim RSLC120, 100  2.1 mm i.d., 2.2 mm particle size; Thermo Fisher, Sunnyvale, CA) with a gradient mobile phase consisting of 7.5 mM ammonium acetate (A) and acetonitrile with 0.1% formic acid (B). The gradient program was as follows: 0–2.5 min, 50% (A, v/v); 2.5– 3.0 min, from 50 to 2% (A); 3.0–5.25 min, 2% (A); 5.25– 5.4 min, from 2 to 50% (A), and 5.4–7 min, continuing 50% (A) for equilibration. The column and autosampler

temperature were maintained at 40 and 4  C, respectively. The mobile phase was consistently eluted at 0.4 mL/min, and the run time for each sample was 7 min. The optimized ion spray voltage was set at 5500 V for positive and 4500 V for negative, and the temperature was 600  C. The nitrogen gas used as the nebulizer gas, curtain gas, and collision-activated dissociation gas was set at 60, 20 and 60 psi for positive mode and 40, 20 and 60 psi for negative mode, respectively. Detection and quantification of target compounds were performed using a mass spectrometer in selected reactionmonitoring mode with positive electrospray ionization at m/z 447.1 ! 285.1 for TF and m/z 277.0 ! 192.1 for IS and with negative electrospray ionization at m/z 283.1 ! 255.1 for Maack, m/z 459.3 ! 255.1 for SPN, m/z 269.3 ! 241.1 for ABF, and m/z 275.1 ! 190.0 for the IS. TF, Maack, SPN, ABF, and the IS were eluted at 0.85, 1.91, 4.66, 1.87, and 1.96 min, respectively. The calibration ranges of TF, Maack, SPN, and ABF in rat plasma were 50–5000 ng/mL, 25– 2500 ng/mL, 5–250 ng/mL and 1–250 ng/mL, respectively.

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Table 1. Pharmacokinetic parameters of TF, Maack, SPN, and ABF after single intravenous administration of SKI3301 extract at doses of 5 mg/kg, 10 mg/kg, and 20 mg/kg to male rats.

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Parameters TF AUCt (mg min/mL)a Dose-normalized AUCtb t1/2 (min) CL (mL/min/kg) MRT (min) Vdss (mL/kg) GI24 h (% of dose)c Ae24 h (% of dose)d Maack AUCt (mg min/mL)a Dose-normalized AUCtb t1/2 (min) CL (mL/min/kg) MRT (min) Vdss (mL/kg) GI24 h (% of dose)c Ae24 h (% of dose)d SPN AUCt (mg min/mL)a Dose-normalized AUCtb t1/2 (min) CL (mL/min/kg) MRT (min) Vdss (mL/kg) GI24 h (% of dose)c Ae24 h (% of dose)d ABF AUCt (mg min/mL)a Dose-normalized AUCtb t1/2 (min) CL (mL/min/kg) MRT (min) Vdss (mL/kg) GI24 h (% of dose)c Ae24 h (% of dose)d

5 mg/kg (n ¼ 9)

10 mg/kg (n ¼ 9)

20 mg/kg (n ¼ 9)

7.90 ± 0.981 7.90 ± 0.981 3.26 ± 0.228 28.9 ± 3.72 4.25 ± 0.430 124 ± 25.2 NDe NDe

18.8 ± 1.44 9.39 ± 0.720 6.61 ± 0.483 24.5 ± 1.68 6.32 ± 0.584 155 ± 21.8 NDe NDe

42.0 ± 5.31 10.5 ± 1.33 10.6 ± 0.895 22.4 ± 2.42 8.33 ± 1.22 187 ± 32.1 NDe NDe

2.62 ± 0.507 2.62 ± 0.507 20.7 ± 5.20 26.4 ± 4.56 25.5 ± 5.78 657 ± 123 NDe NDe

8.62 ± 0.901 4.31 ± 0.451 26.1 ± 8.06 18.5 ± 1.95 31.3 ± 6.98 575 ± 114 NDe NDe

16.8 ± 2.67 4.21 ± 0.668 31.8 ± 4.67 20.4 ± 2.39 34.7 ± 4.06 709 ± 126 NDe NDe

7.43 ± 1.53 7.43 ± 1.53 33.7 ± 10.1 21.1 ± 4.60 15.5 ± 3.43 324 ± 103 NDe NDe

17.8 ± 5.75 8.88 ± 2.87 47.6 ± 14.8 18.8 ± 5.12 17.8 ± 8.74 362 ± 287 NDe NDe

43.4 ± 7.60 10.8 ± 1.90 83.5 ± 11.9* 14.8 ± 2.73 17.9 ± 2.48 269 ± 83.8 NDe NDe

0.0659 ± 0.0186 0.0659 ± 0.0186 4.16 ± 1.18 175 ± 30.4 5.68 ± 2.12 1040 ± 499 NDe NDe

0.208 ± 0.0263 0.104 ± 0.0132 13.4 ± 6.03 107 ± 14.5 11.9 ± 5.41 1250 ± 4.39 NDe NDe

0.471 ± 0.0856 0.118 ± 0.0214 14.6 ± 2.47 102 ± 14.1 12.2 ± 3.78 1250 ± 386 NDe NDe

Data are presented as mean ± SD. At 5 mg/kg, plasma concentrations of TF, Maack, SPN and ABF obtained from only three blood sampling points. Thus, the statistical analysis was performed except for all pharmacokinetic parameters of 5 mg/kg groups. a Total area under the plasma concentration–time curve from time zero to time last sampling time. b The values were based on 5 mg/kg of SKI3301. c Percentages of the doses of TF, Maack, SPN and ABF calculated from their contents in SKI3301 extract recovered from the gastrointestinal tract (including its contents and feces) at 24 h. d Percentages of the doses of TF, Maack, SPN and ABF calculated from their contents in SKI3301 extract excreted in the 24-h urine. e Not detected. *The group of 20 mg/kg was significantly different (p50.05) from groups of 10 mg/kg.

The mean intra- and inter-day coefficients of variation (CVs) of the analysis on five consecutive days were 13.1% and 13.0%, and the corresponding assay accuracies ranged over 91.8–111% and 87.9–110%. All analytes were stable under various storage and handling conditions, and no relevant cross-talk or matrix effects were observed. This method was fully validated according to the Guidance for IndustryBioanalytical Method Validation, recommended by the U.S. Food and Drug Administration (US FDA, 2013). In this study, a dilution integrity test was performed to ensure that the plasma samples could be diluted with blank plasma without affecting the final concentration. The dilution QC samples (50,000 ng/mL for TF, 50 000 ng/mL for Maack, and 10 000 ng/mL for SPN, and 10 000 ng/mL for ABF) were diluted 50-fold with blank rat plasma prior to extraction in six replicates and analyzed. The dilution

integrity data showed that the accuracies for dilution integrity (50-fold) were in the range of 92.3–107.8%, and the precisions were less than 7.19%, which met the acceptance criteria. These results support plasma sample dilution up to 50-fold for analysis. In the rat urine and gastrointestinal samples, the calibration ranges of TF, Maack, SPN and ABF were 0.5–50 mg/mL, 0.25–25 mg/mL, 0.5–50 mg/mL and 0.1–10 mg/mL, respectively. In brief, a 150-mL aliquot of acetonitrile containing 1 mg/mL chlorpropamide (IS) was added to a 50-mL aliquot of sample and mixed by vortexing for 2 min. After centrifuging, the supernatant fraction was transferred into a 1.5-mL Eppendorf tube and evaporated under a gentle stream of nitrogen gas at 40  C. The residue was reconstituted with a 1-mL aliquot of the mobile phase, and a 5-mL aliquot was injected into the LC-MS/MS system. The other treatment

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Figure 3. Mean arterial plasma concentrations of TF (A), Maack (B), SPN (C), and ABF (D) after oral administration of SKI3301 extract at doses of 200 mg/kg (*, n ¼ 9), 400 mg/kg ( , n ¼ 9), and 1000 mg/kg (g, n ¼ 9) to male rats. Vertical bars indicate standard deviation.



procedures for the urine and gastrointestinal samples were the same as those for the plasma samples.

SPN and ABF after oral administration of SKI3301 extract was calculated as: F ¼ ðAUCoral =AUCiv Þ  ðdoseiv =doseoral Þ  100%

Pharmacokinetic analysis Standard methods were used to calculate the following pharmacokinetic parameters using a non-compartmental analysis (WinNonlinÕ , ver. 5.2; Pharsight Corporation, Mountain View, CA): the total area under the plasma concentration–time curve from time zero to time t (AUCt), time-averaged total body clearance (CL), terminal half-life (t1/2), mean residence time (MRT), apparent volume of distribution at steady state (Vdss), peak plasma concentration (Cmax), time to reach Cmax (Tmax), and oral bioavailability (F) (Bae et al., 2009). The oral bioavailability (F) of TF,

where, AUCoral is the AUCt after oral administration of SKI3301 at a dose of 400 mg/kg, and AUCiv is the AUCt after intravenous administration of SKI3301 at a dose of 10 mg/kg. The dose-normalised AUCs or Cmax of TF, Maack, SPN and ABF were obtained by dividing the AUC by the respective dose of SKI3301 extract. Statistical analysis A p value 50.05 was deemed to be statistically significant using a t-test between the two means for the unpaired data, or a Duncan’s multiple range test of the Statistical Package for

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Table 2. Pharmacokinetic parameters of TF, Maack, SPN, and ABF after single oral administration of SKI3301 extract at doses of 200 mg/kg, 400 mg/kg, and 1000 mg/kg to male rats.

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Parameters TF AUCt (mg min/mL)a Dose-normalized AUCtb t1/2 (min) Cmax (mg/mL) Dose-normalized Cmaxb Tmax (min)c GI24 h (% of dose)d Ae24 h (% of dose)e F (%) Maack AUCt (mg min/mL)a Dose-normalized AUCtb t1/2 (min) Cmax (mg/mL) Dose-normalized Cmaxb Tmax (min)c GI24 h (% of dose)d Ae24 h (% of dose)e SPN AUCt (mg min/mL)a Dose-normalized AUCtb t1/2 (min) Cmax (mg/mL) Dose-normalized Cmaxb Tmax (min)c GI24 h (% of dose)d Ae24 h (% of dose)e F (%) ABF AUCt (mg min/mL)a Dose-normalized AUCtb t1/2 (min) Cmax (mg/mL) Dose-normalized Cmaxb Tmax (min)c GI24 h (% of dose)d Ae24 h (% of dose)e F (%)

200 mg/kg (n ¼ 12)

400 mg/kg (n ¼ 11)

1000 mg/kg (n ¼ 10)

78.2 ± 17.8 78.2 ± 17.8 132 ± 80.0 0.858 ± 0.302 0.858 ± 0.302 30 (30–30) 0.871 ± 0.188 0.356 ± 0.157

162 ± 41.3 81.0 ± 20.7 132 ± 77.7 1.23 ± 0.461 0.616 ± 0.230 30 (30–120) 1.19 ± 0.503 0.273 ± 0.147 19.2%

203 ± 52.5 40.5 ± 10.5* 162 ± 120 1.774 ± 0.568 0.355 ± 0.114* 30 (30–30) 11.4 ± 8.99* 0.253 ± 0.0786

29.6 ± 5.51 29.6 ± 5.51 99.4 ± 47.4 0.212 ± 0.0548 0.212 ± 0.0548 60 (30–60) 2.79 ± 1.04 7.11 ± 3.09

53.7 ± 13.5 26.9 ± 6.75 109 ± 40.0 0.293 ± 0.0757 0.147 ± 0.0379 60 (30–120) 2.98 ± 1.37 5.21 ± 2.73

69.0 ± 17.3 13.8 ± 3.47* 118 ± 29.9 0.375 ± 0.107 0.0749 ± 0.0214x 60 (30–120) 3.31 ± 3.25 5.03 ± 1.80

2.02 ± 1.30 2.02 ± 1.30 63.8 ± 61.0 0.0379 ± 0.0198 0.0379 ± 0.0198 30 (5–30) 6.32 ± 2.56 0.187 ± 0.157

4.69 ± 3.27 2.34 ± 1.64 69.4 ± 42.4 0.0540 ± 0.0351 0.0270 ± 0.0175 30 (30–60) 5.60 ± 1.61 0.223 ± 0.219 0.540%

5.69 ± 3.29 1.14 ± 0.658 80.6 ± 73.2 0.0860 ± 0.0350 0.0172 ± 0.00700y 30 (30–0) 6.56 ± 3.74 0.194 ± 0.100

0.223 ± 0.0155 0.223 ± 0.0155 40.9 ± 17.4 0.00472 ± 0.00338 0.00472 ± 0.00338 30 (5–30) NDf NDf

0.642 ± 0.377 0.321 ± 0.189 63.3 ± 67.6 0.0112 ± 0.00797 0.00561 ± 0.00399 30 (15–60) NDf NDf 6.82%

1.044 ± 1.04 0.209 ± 0.207 42.7 ± 33.8 0.0208 ± 0.0190 0.00416 ± 0.00380 15 (15–0) NDf NDf

Data are presented as mean ± SD. Total area under the plasma concentration–time curve from time zero to time last sampling time. The values were based on 200 mg/kg of SKI3301. c Time to reach Cmax; Median (ranges). d Percentages of the doses of TF, Maack, SPN and ABF calculated from their contents in SKI3301 extract recovered from the gastrointestinal tract (including its contents and feces) at 24 h. e Percentages of the doses of TF, Maack, SPN and ABF calculated from their contents in SKI3301 extract excreted in the 24-h urine. f Not detected. *The group of 1000 mg/kg was significantly different (p50.05) from groups of 200 mg/kg and 400 mg/kg. xThree groups were significantly different (p50.05). yThe group of 1000 mg/kg was significantly different (p50.05) from groups of 200 mg/kg. a

b

the Social Sciences (SPSS) a posteriori analysis of variance among the four means for the unpaired data. All results are expressed as means ± standard deviation (SD), except for the medians (ranges) for Tmax.

Results and discussion Pharmacokinetics of TF, Maack, SPN, and ABF after intravenous administration of SKI3301 extract at doses of 5, 10 and 20 mg/kg to male rats The mean arterial plasma concentration–time profiles of TF, Maack, SPN and ABF after intravenous administration

of SKI3301 at doses of 5, 10 and 20 mg/kg are shown in Figure 2(A–D). The relevant pharmacokinetic parameters are listed in Table 1. After intravenous administration of SKI3301 extract, the mean arterial plasma levels of TF and ABF declined rapidly (Figure 2A and D), with mean values of terminal half-life of TF and ABF of 3.26–10.6 min and 4.16– 14.6 min (Table 1), respectively, with detection only up to 45 min (Figure 2A and D). However, the levels of Maack and SPN declined slowly compared with those of Maack and SPN (Figure 2B and C) with mean values of terminal half-life (t1/2) of Maack and SPN of 20.7–31.8 min and 33.7–83.5 min, respectively (Table 1). As shown in Figure 2(D), the plasma

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Figure 4. (A) Mean plasma concentrations of TF ( ) and Maack (*) after oral administration of TF in its pure form at a dose of 19.4 mg/kg to male rats. (B) Mean plasma concentrations of SPN ( , n ¼ 4) after oral administration of SPN in its pure form at a dose of 12.9 mg/kg in male rats. Vertical bars indicate standard deviation.



concentrations of ABF were the lowest among the four marker compounds of SKI3301 due to their having the lowest contents. At the lowest dose of SKI3301 (5 mg/kg), plasma concentrations of TF, Maack, SPN and ABF obtained from only four or five blood sampling points. Thus, the pharmacokinetic parameters of TF, Maack, SPN, and ABF could not be compared with the other groups, and the statistical analysis was performed on all except for the 5 mg/kg group. Note that the dose-normalized (based on 5 mg/ kg of SKI3301) AUCt values of TF were comparable (not significantly different) for the two SKI3301 intravenous doses studied, with values of 9.39 ± 0.720 mg min/mL and 10.5 ± 1.33 mg min/mL for 10 and 20 mg/kg, respectively (Table 1). The corresponding values of Maack, SPN and ABF did not show a significant difference between the two dose groups (Table 1). Moreover, the other pharmacokinetic parameters of TF, Maack, SPN and ABF listed in Table 1 were also dose-independent, except for the terminal half-life (t1/2) of SPN (Table 1), for which the 20 mg/kg dose had a significantly longer duration than the 10 mg/kg group. This could have been due to the assay sensitivity. These data indicate that TF, Maack, SPN and ABF exhibited linear pharmacokinetic characteristics after intravenous administration of SKI3301 over the dose ranges studied. Note that after the intravenous administration of SKI3301 extract, the excreted amounts of TF, Maack, SPN and ABF as unchanged drugs in urine over 24 h (Ae24 h) or in the gastrointestinal tract including the feces over 24 h (GI24 h) were below the detection limit for all three doses studied (Table 1). These findings suggest that almost all of the TF, Maack, SPN and ABF after intravenous dosing of SKI3301 were metabolized and the metabolites were excreted into bile and/or urine.

Pharmacokinetics of TF, Maack, SPN, and ABF after oral administration of SKI3301 extract at doses of 200, 400 and 1000 mg/kg to male rats The minimum effective oral dose of SKI3301 in rats was 200 mg/kg twice daily (our unpublished data). Thus, in this study, we examined the pharmacokinetic properties of TF, Maack, SPN, and ABF after oral administration of SKI3301 extract at doses of 200 mg/kg, 400 mg/kg and 1000 mg/kg to rats. The mean arterial plasma concentration–time profiles of TF, Maack, SPN, and ABF after oral administration of SKI3301 at doses of 200, 400, and 1000 mg/kg are shown in Figure 3(A–D). The relevant pharmacokinetic parameters are listed in Table 2. After oral administration of SKI3301, TF, SPN and ABF were detected in the plasma from the first blood sampling time point (5 min) and reached Cmax rapidly (30–60 min; Table 2), suggesting that the absorption of TF, SPN, and ABF from the rat gastrointestinal tract is rapid. After oral administration of SKI3301 at a dose of 1000 mg/ kg, the dose-normalized (based on 200 mg/kg SKI3301) AUCt values of TF were significantly smaller than those at doses of 200 and 400 mg/kg; the values were 78.2 ± 17.8 mg min/mL, 81.0 ± 20.7 mg min/mL, and 40.5 ± 10.5 mg min/mL for 200, 400 and 1000 mg/kg, respectively (Table 2). Similar patterns were observed in the dose-normalized (based on 200 mg/kg SKI3301) Cmax values of TF (Table 2), indicating that TF exhibited non-linear pharmacokinetic properties. The recovered amounts of TF from the rat gastrointestinal tract including the feces over 24 h (GI24 h) after oral dosing for 200, 400 and 1000 mg/kg were found to be 0.871 ± 0.188, 1.19 ± 0.503, and 11.4 ± 8.99% of the dose of TF calculated from their contents in the SKI3301 extract, respectively (Table 2). Note that the GI24 h at 1000 mg/kg was significantly larger than those at 200 mg/kg and 400 mg/kg of SKI3301. Thus, the non-linearity in the oral pharmacokinetics of TF in

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Table 3. Pharmacokinetic parameters of TF, Maack, SPN, and ABF after oral administration of their pure forms of TF (19.4 mg/kg), Maack (7.48 mg/kg), SPN (12.9 mg/kg), and ABF (1.00 mg/kg) as their pure compounds, respectively, to rats. TF (n ¼ 8) a

AUCt (mg min/mL) Cmax (mg/mL)b Tmax (min)c GI24 h (% of dose)d Ae24 h (% of dose)e

25.6 ± 14.3 0.210 ± 0.0809 15 (15–240) 4.64 ± 0.887 NDf

Maack (n ¼ 8) f

ND NDf NDf 1.06 ± 0.589 5.12 ± 2.20

SPN (n ¼ 8)

ABF (n ¼ 8)

0.922 ± 0.373 0.0131 ± 0.00416 60 (30–90) 47.4 ± 16.8 0.819 ± 0.548

NDf

Formed Maack from TF AUCt (mg min/mL)a Cmax (mg/mL)b Tmax (min)c

10.1 ± 6.67 0.0626 ± 0.0210 105 (60–240)

a

Total area under the plasma concentration–time curve from time zero to time last sampling time. Peak plasma concentration. c Time to reach Cmax; Median (ranges). f Not detected.

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b



Figure 5. Mean arterial plasma concentrations of TF (A), Maack (B), SPN (C), and ABF (D) after single ( , n ¼ 8) and multiple (*, n ¼ 8) oral administration of SKI3301 extract at a dose of 400 mg/kg to male rats. Vertical bars indicate standard deviation.

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Table 4. Pharmacokinetic parameters of TF, Maack, SPN and ABF after single and multiple (twice a day for six days) oral administration of SKI3301 extract at a dose of 400 mg/kg to male rats.

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Parameters TF AUCt (mg min/mL)a t1/2 (min) Cmax (mg/mL) Tmax (min)b GI24 h (% of dose)c Ae24 h (% of dose)d Maack AUCt (mg min/mL)a t1/2 (min) Cmax (mg/mL) Tmax (min)b GI24 h (% of dose)c Ae24 h (% of dose)d SPN AUCt (mg min/mL)a t1/2 (min) Cmax (mg/mL) Tmax (min)b GI24 h (% of dose)c Ae24 h (% of dose)d ABF AUCt (mg min/mL)a t1/2 (min) Cmax (mg/mL) Tmax (min)b GI24 h (% of dose)c Ae24 h (% of dose)d

Single (n ¼ 8)

Multiple (n ¼ 8)

197 ± 22.7 69.8 ± 20.9 1.34 ± 0.283 30 (30–90) 1.87 ± 1.93 0.235 ± 0.176

169 ± 43.2 70.6 ± 35.0 1.27 ± 0.831 75 (30–120) 2.34 ± 1.70 0.188 ± 0.155

80.2 ± 16.3 94.9 ± 25.1 0.390 ± 0.0746 75 (30–120) 4.12 ± 2.33 3.35 ± 1.59

66.2 ± 13.5 95.2 ± 28.3 0.385 ± 0.159 90 (15–120) 4.79 ± 3.14 2.47 ± 2.38

7.77 ± 3.56 51.9 ± 13.6 0.0827 ± 0.0277 30 (30–90) 4.46 ± 2.37 0.565 ± 0.389

4.66 ± 3.32 60.6 ± 23.3 0.0530 ± 0.0485 60 (30–90) 5.12 ± 2.19 0.853 ± 0.477

1.16 ± 0.194 58.1 ± 27.6 0.0126 ± 0.00425 22.5 (15–60) NDe NDe

0.891 ± 0.395 51.6 ± 17.4 0.00974 ± 0.00657 45 (15–120) NDe NDe

Data are presented as mean ± SD. a Total area under the plasma concentration–time curve from time zero to time last sampling time. b Time to reach Cmax; Median (ranges). c Percentages of the doses of TF, Maack, SPN and ABF calculated from their contents in SKI3301 extract recovered from the gastrointestinal tract (including its contents and feces) at 24 h. d Percentages of the doses of TF, Maack, SPN, and ABF calculated from their contents in SKI3301 extract excreted in the 24-h urine. e Not detected. *The female group was significantly different (p50.05) from that of the male group.

SKI3301 might be a result of saturation of the absorption in the gastrointestinal tract. Similarly, after oral administration of SKI3301 at 1000 mg/ kg, the dose-normalized (based on 200 mg/kg SKI3301) AUCt values of Maack were significantly smaller than those at doses of 200 and 400 mg/kg (Table 2). The dose-normalized Cmax values of Maack were significantly different among the three SKI3301 doses, with smaller than dose-proportional increases observed (Table 2). However, this non-linear pharmacokinetics may not be attributable to absorption saturation. The amounts of Maack recovered from the GI24 h values were comparable (not significantly different) among the three SKI3301 oral doses studied, with 2.79 ± 1.04, 2.98 ± 1.37, and 3.31 ± 3.25% of the dose of Maack calculated from their contents in SKI3301 extract, respectively (Table 2). In addition, note that Maack was not detected in all plasma samples after oral administration of Maack as a pure compound, indicating the poor absorption from the gastrointestinal tract and/or the rapid metabolism of Maack in the gastrointestinal tract and liver (see Section PKs of the pure forms). However, after oral administration of TF as a pure compound, Maack was detected in the plasma although

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lower levels were detected compared to those of TF (see Section PKs of the pure forms). TF (maackiain-3-O-glucoside) is hydrolyzed in vivo by b-glucosidase into Maack. Moreover, after oral administration of SKI3301 extract, the conversion rates of TF into Maack, expressed as AUCt, Maack/AUCt, TF, were comparable (not significantly different) among the three oral doses studied, with values of 37.9, 33.1 and 34.0% at doses of 200 mg/kg, 400 mg/kg, and 1000 mg/kg, respectively (Table 2). Based on our results, the non-linear pharmacokinetics of Maack might be attributable to that of TF, as its saturation of absorption with respect to the dose was similar to that of TF. However, the dose-normalized AUCt and Cmax values of SPN and ABF were comparable (not significantly different) among the three oral doses studied and appeared to show linear pharmacokinetic properties from oral doses of 200 to 1000 mg/kg of SKI3301 extract (Table 2). As shown in Figure 3(C), a double-peak feature of SPN in rats was found (one presenting at 45 min and another at 240 min), except at a dose of 200 mg/kg. However, this double-peak feature may not be attributable to enterohepatic circulation, multi-site absorption, transformation from other ingredients of SKI3301 or other reasons. The considerably high plasma levels of SPN were observed in three rats of total nine rats up to 360 min, whose plasma profiles showed a plateau in the time interval (240 and 360 min) corresponding to the second peak. Representative plasma profiles of three rats at doses 400 and 1000 mg/kg who presented double peaks in the plasma are presented in Figure 3(C). Moreover, this feature was not observed in multiple-dosing and gender-difference experiments. The oral bioavailabilities (F) of TF, SPN, and ABF, based on an oral dose of 400 mg/kg and an intravenous dose of 10 m/kg, were relatively low (19.3, 0.540, and 6.82%, respectively), which might be partly because of insufficient absorption from the gastrointestinal tract and extensive firstpass metabolism in the liver. Pharmacokinetic properties of TF, Maack, SPN and ABF after oral administration of their pure forms equivalent to 400 mg/kg of SKI3301 The mean arterial plasma concentration–time curves after oral administration of the pure forms of TF (19.4 mg/kg), Maack (7.48 mg/kg), SPN (12.9 mg/kg), and ABF (1.00 mg/kg), which is equivalent to administration of 400 mg/kg of SKI3301 extract, are shown in Figure 4(A) and (B), and the relevant pharmacokinetic parameters are shown in Table 3. However, after oral administration of Maack and ABF in their pure forms, they were not detectable (below LLOQ) in plasma samples. It was reported that flavonoids show poor oral bioavailability (51%) (Manach & Donovan, 2004; Manach et al., 2005; Walle et al., 2004, 2005). Various studies have demonstrated that phase II metabolism, such as glucuronidation and sulfation in both the small intestine and the liver, is the major barrier to their bioavailability (Gao & Hu, 2010; Gao et al., 2011; Zhang et al., 2007). It has been reported (Gao et al., 2011) that after intravenous administration of Maack (as a pure compound) in mice, it is extensively metabolized in vivo, and thus, maackiain-sulfate and maackiain-glucuronide were detected in plasma as the major metabolites. It is generally accepted that UGT’s expression

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Figure 6. Mean arterial plasma concentrations of TF (A), Maack (B), SPN (C), and ABF (D) after single and multiple oral administration of SKI3301 extract at a dose of 400 mg/kg to male ( , n ¼ 8) and female (*, n ¼ 10) rats. Vertical bars indicate standard deviation.



in the intestine is much lower than that in the liver, but due to the location, the small intestine is the first organ that encounters the absorbed flavonoids that are present at relatively high concentrations. Therefore, the contribution of the intestine in limiting the oral flavonoids’ bioavailability should not be underestimated. For example, Cermak et al. (2003) found that the swine portal blood contained only quercetin metabolites and no parent compound, suggesting that quercetin was totally biotransformed in the gastrointestinal tract before reaching the liver. Thus, the absence of detectable Maack in the plasma after oral administration of Maack might be due to its extensive and rapid glucuronidation or sulfation in both the intestine and liver. Maack is expected to be present in the plasma as only glucuronide or sulfateconjugated forms. However, after oral administration of TF in its pure form, Maack (aglycone of TF) was detected in plasma, although at lower levels than those of TF (Figure 4A). This result suggests that TF (maackiain-3-O-glucoside) is hydrolyzed by

b-glucosidase into Maack after TF absorption from the gastrointestinal tract, indicating that TF (maackiain-3-Oglucoside) is better absorbed in the intestine than its aglycone, Maack. Similar results have been previously reported (Hollman et al., 1995, 1996, 1997) that quercetin glycosides exhibit higher bioavailability than their aglycones. Note that the conversion rate of TF into Maack, expressed as AUCt, Maack/AUCt, TF, was approximately 39.5% after oral administration of TF, comparable to the values of 33.1%–37.9% achieved with the oral administration of SKI3301 extract (Table 2), suggesting that the conversion rate was the same for TF in the SKI3301 extract and TF alone. As shown in Figure 4(A), a bimodal distribution (second peak phenomenon) appeared in rats after oral administration of TF, which might be due to enterohepatic circulation, multi-site absorption, transformation from other ingredients or other reasons. After oral administration of TF in its pure form, the Cmax (0.210 ± 0.0809 mg/mL) and AUCt (25.6 ± 14.3 mg min/mL) of TF at the dosage of 19.4 mg/kg were significantly smaller

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than the values of Cmax (1.23 ± 0.461 mg/mL, Table 2) and AUCt (162 ± 41.3 mg min/mL, Table 2) of TF in 400 mg/kg SKI3301 extract. Larger amounts of TF (4.64 ± 0.887%) were recovered from the gastrointestinal tract over 24 h after oral dosing in its pure form compared with after oral dosing of 400 mg SKI3301 (1.19 ± 0.503%). After oral administration of SPN in its pure form, the concentration of SPN in plasma was so low that it was difficult to measure. In only four of the nine rats, the level of SPN could be detected. Furthermore, the plasma levels of SPN at the dosage of 12.9 mg/kg were also significantly smaller than those of SKI3301 at the dosage of 400 mg/kg. After oral dosing of SPN in its pure form to rats, considerable amounts (GI24 h, 47.4 ± 16.8%) of SPN were recovered from the gastrointestinal tract over 24 h (Table 3). However, a smaller amount of SPN was found in the gastrointestinal tract over 24 h (5.60 ± 1.61%) after oral administration of SKI3301 extract at a dose of 400 mg/kg. It has been shown that the herbal extract SKI3301 increases the bioavailability of TF and SPN in rats comparing with the respective pure compounds. It may be explained through the increased solubility or the decreased metabolism of TF, Maack, SPN and ABF in both the intestine and liver, which can improve absorption due to the synergistic effect of other components in the SKI3301 extract. Multiple oral administration of SKI3301 extract at a dose of 400 mg/kg twice a day for six days to male rats The pharmacokinetic profiles of TF, Maack, SPN and ABF after a single or multiple oral doses are shown in Figure 5(A–D), and the pharmacokinetic parameters are shown in Table 4. There are no significant differences (p > 0.05) in all pharmacokinetic parameters of TF, Maack, SPN and ABF between the single and multiple oral administration groups (Table 4). The conversion rates of TF into Maack, expressed as AUCt, Maack/AUCt, TF, were also not changed (38.2% versus 40.2%) after multiple dosing compared with that after single dosing. This result suggests that no time-dependent auto-induction or auto-inhibition occurs after multiple dosing of SKI3301. Gender differences in the pharmacokinetics of TF, Maack, SPN, and ABF after oral administration of SKI3301 to rats The mean arterial plasma concentration–time curves of TF, Maack, SPN and ABF in male and female rats after oral administration of SKI3301 at a dose of 400 mg/kg are shown in Figure 6(A–D), and the relevant pharmacokinetic parameters are shown in Table 5. The plasma concentrations and pharmacokinetic parameters of TF, except the t1/2, were not significantly different between male and female rats, but the AUCt and Cmax of Maack in female rats were significantly lower than those in male rats (Table 5). Thus, in female rats, the conversion rate of TF into Maack, expressed as AUCt, Maack/AUCt, TF, was significantly lower (32.0 versus 22.5%) than in male rats. Gender-dependent pharmacokinetics results in individual differences in drug efficacy and toxicity (Anderson, 2008; Floridia et al., 2008). One of the major reasons for gender-based pharmacokinetics

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Table 5. Pharmacokinetic parameters of TF, Maack, SPN, and ABF after single oral administration of SKI3301 extract at a dose of 400 mg/kg to male and female rats. Parameters TF AUCt (mg min/mL)a t1/2 (min) Cmax (mg/mL) Tmax (min)b GI24 h (% of dose)c Ae24 h (% of dose)d Maack AUCt (mg min/mL)a t1/2 (min) Cmax (mg/mL) Tmax (min)b GI24 h (% of dose)c Ae24 h (% of dose)d SPN AUCt (mg min/mL)a t1/2 (min) Cmax (mg/mL) Tmax (min)b GI24 h (% of dose)c Ae24 h (% of dose)d ABF AUCt (mg min/mL)a t1/2 (min) Cmax (mg/mL) Tmax (min)b GI24 h (% of dose)c Ae24 h (% of dose)d

Male rats (n ¼ 8)

Female rats (n ¼ 10)

182 ± 61.7 65.3 ± 7.50 1.47 ± 0.589 30 (30–30) 3.87 ± 2.93 0.470 ± 0.276

179 ± 38.0 90.2 ± 24.7* 1.58 ± 0.437 30 (15–30) 3.35 ± 2.70 0.362 ± 0.277

58.3 ± 18.7 84.0 ± 12.0 0.343 ± 0.117 60 (60–120) 4.97 ± 3.03 2.12 ± 1.19

40.3 ± 9.29* 118 ± 31.7* 0.245 ± 0.0680* 60 (30–60) 5.34 ± 3.60 2.38 ± 1.60

5.24 ± 2.87 60.5 ± 28.3 0.0579 ± 0.0278 30 (30–60) 8.19 ± 4.25 0.418 ± 0.187

3.45 ± 1.42 43.4 ± 12.0 0.0592 ± 0.0172 30 (30–60) 9.20 ± 6.07 0.290 ± 0.146

0.665 ± 0.331 34.9 ± 6.43 0.0110 ± 0.00583 30 (30–30) NDe NDe

0.759 ± 0.322 29.7 ± 9.74 0.0162 ± 0.00623 30 (15–30) NDe NDe

Data are presented as mean ± SD. a Total area under the plasma concentration–time curve from time zero to time last sampling time. b Time to reach Cmax; Median (ranges). c Percentages of the doses of TF, Maack, SPN, and ABF calculated from their contents in SKI3301 extract recovered from the gastrointestinal tract (including its contents and feces) at 24 h. d Percentages of the doses of TF, Maack, SPN, and ABF calculated from their contents in SKI3301 extract excreted in the 24-h urine. e Not detected. *The female group was significantly different (p50.05) from that of the male group.

is sex-dependent metabolism. Although the exact reason is unclear, gender-dependent differences in the activity or expression level of b-glucosidase might be one of the determining factors in these results. It was reported that a marked sexual difference in b-glucosidase activity was found in the adult rat kidney and that the activity in adult male rats was 1.4–2.1 times higher than that in adult female rats (Hatakeyama et al., 1980). However, the other pharmacokinetic profiles of SPN and ABF in both male and female rats were comparable.

Conclusions An understanding of the pharmacokinetic profiles of bioactive constituents can be extremely beneficial to an assessment of the drug responses of a medicinal herb or a natural product. This is the first study to characterize the detailed pharmacokinetic properties of four marker components of S. tonkinensis, namely TF, Maack, SPN, and ABF, after intravenous and oral administration of SKI3301, a dried 50% ethanolic extract of S. tonkinensis, to rats. These findings may help us to

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comprehensively understand the pharmacokinetic characteristics of TF, Maack, SPN, and ABF and provide useful information for the further nonclinical and/or clinical study of SKI3301 extract for the treatment of asthma.

Declaration of interest This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation funded by the Ministry of Science, ICT & Future Planning (No. 2013M3A9B5075838) and the Ministry of Trade, Industry and Energy R&D program of Korea (No. 10039320). None of the authors declares any conflict of interest regarding this manuscript.

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