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Pharmacokinetics, tissue distribution and excretion study of trans-resveratrol-3-O-glucoside and its two metabolites in rats
Phytomedicine 58 (2019) 152882
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Original Article
Pharmacokinetics, tissue distribution and excretion study of transresveratrol-3-O-glucoside and its two metabolites in rats Meiying Sua, Chao Donga, Jiyun Wana, Maojin Zhoub,c,
T
⁎
a
Taian Central Hospital of Shandong Province, 29 Longtan Road, Taian 271000, PR China Laboratory of Drug Metabolism and Pharmacokinetics, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China c HQ Bioscience Co. Ltd., 11/F, Building D, 398 Ruoshui Road, Suzhou Industrial Park, Suzhou 2151123, PR China b
ARTICLE INFO
ABSTRACT
Keywords: Trans-resveratrol-3-O-glucoside Trans-resveratrol-3-O-glucuronide Trans-resveratrol Pharmacokinetics Distribution Excretion
Background: Trans-resveratrol-3-O-glucoside (TRG), isolated from the Chinese traditional herbal medicine Huzhang, has been shown to have a wide range of pharmacological benefits. Purpose: The aim of this study is to investigate the pharmacokinetics, tissue distribution and excretion of TRG and its metabolites, (TRN (trans-resveratrol-3-O-glucuronide) and TR (trans-resveratrol)), following a single intragastric (i.g.) administration of TRG in rats. Study design: To evaluate the pharmacokinetic properties of TRG, TRN and TR, groups of rats were administrated a single i.g. dose of either 75, 150 or 300 mg/kg TRG. The absolute bioavailability of TRG was estimated from the ratios of AUC0-∞ values for oral and intravenous administration. Tissue distributions of TRG, TRN and TR in rats were investigated following a single i.g. administration to four groups at 150 mg/kg dosage of TRG. For urinary, fecal and biliary excretion study, TRG, TRN and TR excretions were recovered from a group of rats administered a single i.g. dose of 150 mg/kg TRG. Methods: The levels of TRG, TRN and TR in plasma, tissues, bile, urine and feces were measured by a rapid and sensitive LC-UV method. The precision was below 10.0%, and the accuracy was within ± 9.9% for TRG, TRN and TR. Results: The concentrations of TRN were markedly higher than those of TRG and TR in plasma, urine and bile. TRG, TRN and TR showed linear dynamics in dose range of 75–300 mg/kg TRG. TRG had poor absolute bioavailability in rats. The major distribution tissues of TRG, TRN, and TR in rats were in the digestive tract. TRG, TRN and TR were all eliminated from tissues quickly. TRG was mostly excreted via the renal route in the form of TRN, which accounted for 52.8% of the administered dose up to 72 h. Conclusion: Following a single i.g. administration to rats TRG was easily absorbed and rapidly converted to the metabolites TR and TRN. These metabolites were found to be mainly excreted by the kidneys.
Introduction Trans-resveratrol-3-O-glucoside (TRG, also named polydatin, Fig. 1), a glycoside of trans-resveratrol (TR), is an active monomer isolated from the dried roots of Polygonum cuspidatium Sieb.et Zucc., a traditional Chinese medicine commonly known as Huzhang. TRG is also found in grapes, peanuts, hop cones and pellets, red wines, cocoa/ chocolate products and other daily diets (Du et al., 2013). In recent years, numerous pharmacological studies have indicated TRG has numerous physiological benefits, including cardiovascular effects, hepato-
protective effects, neuroprotective activity, lung protective effects, antiarteriosclerosis, anti-inflammatory activity, anti-shock effects, antitumor activity, anti-oxidative activity, inhibition of melanogenesis, anti-microbial activity, inhibition of thrombus formation, immuneregulatory effects (Du et al., 2013; Jin et al., 2016; Wang et al., 2015; Zhang et al., 2016), protection against kidney injury (Meng et al., 2016), induction of bone marrow stromal cell migration (Chen et al., 2016), and anti-osteoporotic activity (Zhou et al., 2016). Although the pharmacological efficacy of TRG after oral administration is known to depend on its absorption, tissue distribution,
Abbreviations: ANOVA, a one-way analysis of variance; AUC, the area under the plasma concentration−time curve; HPLC, high-performance liquid chromatography; LLOQ, lower limit of quantification; TR, trans-resveratrol; TRG, trans-resveratrol-3-O-glucoside; TRN, trans-resveratrol-3-O-glucuronide; UV, ultraviolet detection ⁎ Corresponding author at: HQ Bioscience Co. Ltd., 11/F, Building D, 398 Ruoshui Road, Suzhou Industrial Park, Suzhou 2151123, PR China. E-mail address: [email protected] (M. Zhou). https://doi.org/10.1016/j.phymed.2019.152882 Received 14 November 2016; Received in revised form 28 February 2019; Accepted 9 March 2019 0944-7113/ © 2019 Published by Elsevier GmbH.
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Fig. 1. Chemical structures of TRG, its metabolites (A) and baicalin (internal standard) (B).
metabolism and excretion, few studies have included TRG pharmacokinetics (Ding et al., 2014; Gao et al., 2006; He et al., 2007; Lv et al., 2006; Zhou et al., 2009), tissue distribution (Gao et al., 2006; Lv et al., 2006; Zhang et al., 2008) and excretion (Gao et al., 2006) in rats. The limited studies available mainly focus on the disposition of the unchanged TRG; only two reports address TRG and its metabolites in rat plasma (Zhou et al., 2009) or tissues (Zhang et al., 2008). To our knowledge, specific disposition of TRG and its metabolites TRN and TR in rats has not been reported. Therefore, the aim of our study is to analyze the pharmacokinetics, tissue distributions and excretions of TRG, TRN and TR following oral administration of TRG in rats.
TRG (99.3% purity) was obtained courtesy of Liaoning Institute of Medical Industrial Research (Shenyang, China). TR (99.5% purity) and baicalin (internal standard, 99.0% purity) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). TRN (98.5% purity by HPLC) was isolated and prepared from rat urine in our laboratory (Zhou et al., 2007) and identified by MSn, 1H NMR, 13C NMR (Learmonth, DA., 2003) and HMBC. Acetonitrile and methanol (Yuwang Chemical, Shandong, China) were of HPLC grade. Other chemicals were of analytical grade. Distilled water, prepared from demineralized water, was used throughout the study.
To study the tissue distribution of orally administered TRG, sixteen male and sixteen female rats were divided into four groups at random and each rat was administered a single dose of TRG at 150 mg/kg. Tissue samples were collected from heart, liver, spleen, lung, kidney, stomach, duodenum, jejunum, ileum, caecum, colon, pancreas, brain, fat, muscle, ovary, uterus and testis at time points 0.33, 2.0, 6.0 and 12.0 h post TRG delivery. Time points to collect tissues were designed according to the concentration-time curves. One additional rat was sacrificed pre-dose to provide control tissues for analysis. Tissue samples were weighed rapidly, rinsed with physiological saline to remove the blood or content, blotted on filter paper, and stored at −20ºC until analysis. For study of urinary and fecal excretion, three female and three male rats were orally administered a single dose of TRG at 150 mg/kg. After dosing, the rats were housed in individual stainless steel metabolism cages designed for the separation and collection of urine and feces. Urine and feces were collected between –18 and 0 h pre-dosing and during 0−4, 4−8, 8−12, 12−24, 24−36, 36−48, 48−72 h postdosing. Collected feces were dried at room temperature. Following measurement of feces dry weight and urine volume for each collection period, samples were stored at −20 ºC. For study of biliary excretion, three female and three male rats were anesthetized and cannulas were surgically inserted into the bile duct to collect bile. A single 150 mg/kg dose of TRG was orally administered to each rat. Bile samples were collected before dosing and during 0−4, 4−8, 8−12, 12−24, 24−36, 36−48 h after dosing. Samples were stored at −20 °C until analysis.
Animals, drug administration and sampling
Sample processing
Wistar rats (male and female, ∼200−240 g, Accreditation No.: [Liao] 008, Shenyang) were obtained from the Department of Experimental Animals, Shenyang Pharmaceutical University (Shenyang, China). All animals were acclimatized to 1aboratory conditions and starved overnight prior to drug administration, yet retained free access to drinking water at all times. All animal experiments were approved by the institutional ethics committee prior to the study. For pharmacokinetic study, sixteen male and sixteen female rats were randomly divided into four groups (n = 8 per group). TRG, dissolved in a saline solution (0.9% NaCl) of 20% 2-hydroxypropyl-β-cyclodextrin (Marier et al., 2002), was administered intragastrically at a single dose of 75, 150 and 300 mg/kg to groups 1–3, respectively. The remaining group was administered intravenously a single dose of TRG at 75 mg/kg. Blood samples were collected at 0, 0.17, 0.33, 0.67, 1.0, 2.0, 4.0, 6.0, 8.0, 12.0 and 24.0 h following oral administration and at 0.08, 0.17, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 12.0 and 24.0 h after intravenous administration. Plasma was obtained by centrifugation at 10,000 rpm for 10 min and stored at −20ºC. Due to the generally high ratio of trans-to cis-isomers all samples and standards were handled with no exposure to light.
Plasma, Bile and Urine: A 100 μl aliquot of each sample was combined with 100 μl of the internal standard, 100 μl of formic acid in water (pH 3.5) and 300 μl of acetonitrile. The samples were vortexed for 1 min, and centrifuged at 10,000 rpm for 10 min. The supernatant was transferred to a new tube and evaporated to dryness at 40 ºC under a gentle stream of nitrogen. The residue was reconstituted in 100 μl of the mobile phase and 50 μl of the solution was injected into the HPLC system. Tissue samples were thawed and homogenized in methanol (1:2, w/ v). 100 μl of tissue homogenate was processed as described above for plasma samples. Fecal samples were pulverized with a mortar and pestle and homogenized in methanol (1:2, w/v). 100 μl of fecal homogenate was processed as described above for plasma samples.
Materials and methods Materials
Analysis of TRG, TRN and TR in rat biological samples The concentrations of TRG, TRN and TR in rat biological samples were analyzed using an LC-UV method previously developed by our laboratory (Zhou et al., 2007) with some modifications. Chromatography was performed on a Diamonsil C18 column (200 mm × 4.6 mm 2
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Fig. 2. HPLC-UV chromatograms of a blank liver homogenate sample (A); a blank liver homogenate sample spiked with TRG (0.04 μg/ml), TRN (0.04 μg/ml), TR (0.04 μg/ml) and IS (20 μg/ml) (B); a liver homogenate sample at 2 h after oral administration of 150 mg/kg TRG to a rat (C). Peak I, TRG (tR = 4.7 min); Peak II, TRN (tR = 6.2 min); Peak III, IS (tR = 11.7 min); Peak IV, TR (tR = 14.4 min).
I.D., particle 5 μm, Dikma, Beijing, China). The isocratic mobile phase consisted of 25% acetonitrile and 75% formic acid in water at pH 3.5. The flow-rate was 1.0 ml/min. Ultraviolet detection was set at 320 nm and the column temperature was kept at 25 °C. The LC-UV method developed previously was only validated for rat plasma samples. In the present study, the method was further validated with respect to selectivity, linearity, lower limit of quantification, precision and accuracy, absolute recovery and stability for plasma, tissue, bile, urine and fecal samples. Blank rat plasma, tissue homogenate, bile, urine and feces were used to establish and validate a standard protocol.
Results Method validation Fig. 2 shows representative chromatograms of blank rat liver homogenate (A), blank rat liver homogenate spiked with TRG, TRN and TR (B), and rat liver homogenate sample after i.g. administration of TRG (C). TRG, TRN, TR and internal standard were eluted at approximately 4.7, 6.2, 14.4 and 11.7 min, respectively. No detectable interfering peaks were found with the retention time close to that of TRG, TRN, TR and internal standard. For quantification of TRG, TRN and TR in plasma, urine, bile, tissue and fecal homogenate samples, standard curves of seven relevant concentrations for each standard substance were performed. The curves were characterized by regression coefficients of r = 0.99 or higher. The coefficient of variation of a sixfold replicate was less than 5.0%. The LLOQs of TRG, TRN and TR, defined as the lowest concentration at which both precision and accuracy were less than or equal to 20.0%, were 0.04 μg/ml, 0.10 μg/ml, 0.10 μg/ml, 0.04 μg/g, and 0.10 μg/g in 100 μl rat plasma, urine, bile, tissue and fecal homogenate, respectively. Absolute recoveries of TRG, TRN and TR in rat biological samples were more than 90.0%. The precision, expressed as the intra-day and inter-day relative standard deviation (RSD%), was below 10.0%, and the accuracy, expressed as the relative error (RE%), was within ± 9.9% for TRG, TRN and TR. The results of stability experiments showed that all the biological samples were stable for 12 h at room temperature, after three freeze-thaw cycles and for 30 days under −20 ºC, with a reduction of less than 15.0%.
Pharmacokinetic and statistical analysis All TRG, TRN and TR concentrations in plasma are expressed as TRG, TRN and TR mass equivalents. The Cmax is the highest concentration observed, and Tmax is time at which Cmax occurred. The pharmacokinetic parameters were calculated by non-compartmental analyses using the Topfit software package (version 2.0, Thomae GmbH, Germany). Terminal elimination rate constant (Ke) was determined by linear regression of the terminal portion of plasma concentration-time data, and the elimination half-life (t1/2) was calculated as 0.693/Ke. The area under the plasma concentration−time curve (AUC) from time zero to the last quantifiable concentration (Clast) was determined using the linear trapezoidal method. AUC0-∞ was calculated by adding Clast/λz to AUC0-t, where λz represents the terminal phase of the plasma concentration profile. The total body clearance (CL) was calculated as the quotient of the dose (D) and AUC0−∞. The absolute bioavailability of TRG in rats was estimated from the ratios of AUC0−∞ values for oral and intravenous administration. Dose-proportionality after a single i.g. administration of three dosages was determined by comparison of the dose-normalized AUC0−∞ across dosage levels using the SPSS 13.0 one-way ANOVA analysis and linear regression analysis. All the data were expressed as mean ± S.D. and a p value < 0.05 was deemed to be statistically significant.
Pharmacokinetic studies The HPLC method was successfully applied for investigation of the pharmacokinetics of TRG, TRN and TR following a single i.g. administration of either 75, 150 or 300 mg/kg TRG and a single i.v. TRG administration of 75 mg/kg. The corresponding pharmacokinetic parameters of TRG, TRN and TR, calculated using non-compartmental analysis are listed in Tables 1–3. Mean plasma concentration-time
Table 1 Pharmacokinetic parameters of TRG in rats following i.g. administration of TRG at a single dose of 75, 150 and 300 mg/kg, and following i.v. administration TRG at a single dose of 150 mg/kg, respectively. Parameters
i.g. dose (mg/kg) 75
150
300
i.v. dose (mg/kg) 75
p value
t1/2 (h) AUC0-∞(μg•h/ml) CL (ml/min) Vz (L)
1.61 ± 0.72 1.25 ± 0.51 1.18 ± 0.59 88.5 ± 69.5
1.29 ± 0.66 3.72 ± 1.43 0.75 ± 0.22 83.5 ± 57.7
1.31 ± 0.17 7.62 ± 2.23 0.72 ± 0.26 82.8 ± 35.0
1.51 ± 0.25 112 ± 42.2 12.5 ± 4.19 1.59 ± 0.69
>0.05 >0.05 >0.05 >0.05
Data are expressed as mean ± S.D. (n = 8). t1/2, terminal half-life; AUC0−∞, area under the analyte concentrations vs.time curve from time 0 to infinity; Vz, terminal volume of distribution; CL, total body clearance. p values are obtained by evaluating the pharmacokinetic parameters across the three i.g. administration of TRG dosage groups using one-way ANOVA. AUC0−∞ was normalized by the corresponding dosages when conducting comparison among the three i.g. 3
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Table 2 Pharmacokinetic parameters of TRN in rats following i.g. administration of TRG at a single dose of 75, 150 and 300 mg/kg, and following i.v. administration TRG at a single dose of 150 mg/kg, respectively. Parameters
i.g. dose (mg/kg) 75
150
300
i.v. dose (mg/kg) 75
P value
t1/2 (h) AUC0-∞(μg•h/ml) CL (ml/min) Vz (L)
3.23 ± 0.92 104 ± 39.2 13.4 ± 4.76 3.88 ± 1.86
2.79 ± 0.75 191 ± 46.9 13.7 ± 3.04 3.31 ± 1.22
3.02 ± 0.48 313 ± 112.2 18.1 ± 7.06 4.01 ± 2.21
2.80 ± 0.91 25.6 ± 7.17 53.2 ± 16.4 13.5 ± 8.49
>0.05 >0.05 >0.05 >0.05
Data are expressed as mean ± S.D. (n = 8). t1/2, terminal half-life; AUC0−∞, area under the analyte concentrations vs. time curve from time 0 to infinity; Vz, terminal volume of distribution; CL, total body clearance. P values are obtained by evaluating the pharmacokinetic parameters across the three i.g. administration of TRG dosage groups using one-way ANOVA. AUC0−∞ was normalized by the corresponding dosages when conducting comparison among the three i.g. administration of TRG groups. Table 3 Pharmacokinetic parameters of TR in rats following i.g. administration of TRG at a single dose of 75, 150 and 300 mg/kg, respectively. Parameters
i.g. dose (mg/kg) 75 150
300
t1/2 (h) AUC0-∞(μg•h/ml) CL(ml/min) Vz (L)
2.23 ± 0.34 1.85 ± 0.33 0.70 ± 0.14 0.13 ± 0.03
2.72 ± 0.32 8.21 ± 1.82 0.64 ± 0.14 0.15 ± 0.03
2.87 ± 0.68 3.53 ± 0.77 0.74 ± 0.18 0.18 ± 0.05
P value >0.05 >0.05 >0.05 >0.05
Data are expressed as mean ± S.D. (n = 8). t1/2, terminal half-life; AUC0−∞, area under the analyte concentrations vs. time curve from time 0 to infinity; Vz, terminal volume of distribution; CL, total body clearance. P values are obtained by evaluating the pharmacokinetic parameters across the three i.g. administration of TRG dosage groups using one-way ANOVA. AUC0−∞ was normalized by the corresponding dosages when conducting comparison among the three i.g. administration of TRG groups.
Fig. 4. Mean plasma concentration-time curves of TRN in rats (mean ± S.D., n = 8) following i.g. administration at a single dose of 75, 150 and 300 mg/kg and i.v. administration of 75 mg/kg TRG to rats.
Fig. 5. Mean plasma concentration-time curves of TR in rats (mean ± S.D., n = 8) following i.g. administration at a single dose of 75, 150 and 300 mg/kg TRG to rats.
Fig. 3. Mean plasma concentration-time curves of TRG in rats (mean ± S.D., n = 8) following i.g. administration at a single dose of 75, 150 and 300 mg/kg and i.v. administration of 75 mg/kg TRG to rats.
Tissue distribution studies
curves (n = 8) are presented in Figs. 3–5, demonstrating rapid elimination of TRG from the plasma. This elimination correlates with TR and TRN formation. The AUC0−∞ values of TRG, TRN and TR of the three dosages of TRG indicated an apparent dose-proportionality. The increases in AUC0−∞ of TRG, TRN and TR were roughly dose proportional at 75, 150 and 300 mg/kg dose range (1.25 μg•h/ml vs. 3.72 μg•h/ml vs. 7.62 μg•h/ml for TRG; 104.5 μg•h/ml vs. 191.2 μg•h/ml vs. 312.6 μg•h/ml for TRN; 1.85 μg•h/ml vs.3.53 μg•h/ml vs. 8.21 μg•h/ml for TR). There were no significant differences for other parameters including CL, Vz and t1/2 among the three dosages analyzed by ANOVA (p > 0.05). The absolute bioavailability of TRG in rats was estimated to be 1.11%, 1.66% and 1.70% after a single oral dose of 75, 150 and 300 mg/kg TRG, respectively.
Tissue distributions of TRG and its metabolites were investigated following a single i.g. administration of 150 mg/kg in rats. The concentrations of TRG, TRN and TR in tissues, following a single oral administration, are shown in Figs. 6–8. These data indicate that TRG, TRN and TR underwent a rapid and widespread distribution with no longterm accumulation in any of the tissues tested. At 0.33 h after dosing, the highest level of TRG was found in stomach, amounting to 80.0 µg/g, then in duodenum and jejunum. The concentrations of TRG in these tissues were higher than those in other tissues at any of the time points tested. Subsequently, the concentrations of TRG in all tested tissues decreased rapidly. At 12.0 h after dosing TRG was not detectable in most tissues except for stomach, duodenum, ileum and colon. At all time points, concentrations of TRG in tested tissues were higher than 4
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Fig. 6. Mean concentrations (mean ± S.D., n = 8) of TRG in tissues at different time points after i.g. administration of 150 mg/kg TRG to rats (μg/g or μg/ml).
Fig. 7. Mean concentrations (mean ± S.D., n = 8) of TRN in tissues at different time points after i.g. administration of 150 mg/kg TRG to rats (μg/g or μg/ml).
those in plasma. After TRG was administered orally to rats, the concentrations of TRN in ileum, kidney, jejunum, liver, and duodenum were higher than in other tissues at each time point. The amount of TRN in ileum reached a peak level of 20.0 µg/g at 6.0 h after dosing. Notably, the concentrations of TRN in tested tissues were lower than those in plasma at each time point. TR was mainly distributed into the jejunum, ileum, caecum, and colon, and the concentration in caecum reached maximum value at 6.0 h post-dosing, which was 77.0 µg/g.
and 0.35% up to 72 h in feces. With regard to TRG, only 0.59% of the dose was recovered up to 72 h in urine, 0.08% up to 48 h in bile. Discussion In recent years, an increasing body of work has focused on Chinese herbal medicine, particularly isolated monomers. Although the biological activity of TRG has been shown, pharmacokinetic knowledge of TRG, especially its metabolism and disposition in vivo, is lacking. This type of in depth analysis is very important for better understanding the pharmacological effects of TRG. Our lab has previously reported both TRN and TR are metabolites of TRG, of which TRN is a main metabolite following i.g. administration of TRG in rats (Zhou et al., 2007). With this study, we present the first findings of the pharmacokinetics, tissue distribution and excretion of TRG and its metabolites after i.g. administration of TRG in rats. A simple, specific and sensitive LC-UV method previously developed by our laboratory has been further validated and
Excretion studies The urinary, fecal and biliary excretions of TRG, TRN and TR in rats following a single oral administration of TRG are summarized in Fig. 9. After oral administration of TRG at dose of 150 mg/kg, TRN was the predominant metabolite collected, accounting for 52.8% of the dose up to 72 h in urine and 8.07% up to 48 h in bile. The cumulative excretion ratio of TR was low, amounting to 1.37% of the dose up to 72 h in urine 5
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Fig. 8. Mean concentrations (mean ± S.D., n = 8) of TR in tissues at different time points after i.g. administration of 150 mg/kg TRG to rats (μg/g or μg/ml).
successfully applied to quantification of TRG, TRN and TR in a wide range of rat biological samples. Our pharmacokinetic analysis, following a single i.g. administration in rats, shows TRG was rapidly absorbed and eliminated from rat plasma with an average t1/2 of 1.40 h. TRN was the predominant compound in rat plasma, eliminated more slowly with an average t1/2 of 3.01 h. Intravenous administration of TRG in rats showed a much higher concentration of TRG than TRN, while TR was almost undetectable in plasma. The mean molar ratios of AUC0−∞ of TRN/TRG and TR/TRG were 56.0 and 1.98 at three oral doses, respectively; while the mean molar ratio of AUC0−∞ of TRN/TRG was 0.221 at single i.v. dose (much lower than that at oral dose). This result indicates a certain amount of TRG absorbed into the body was hydrolyzed to form TR by the intestines and/or liver first pass effects (Henry-Vitrac et al., 2006); subsequently the TR was mainly in the form of TRN (a glucuronic acid
conjugate of TR) after entering the blood circulation. Besides, the extent of glucuronidation of TR in intestine was much higher than that in liver. The mean absolute bioavailability of TRG in rats was estimated to be 1.49% after a single oral three doses. Similar findings for low absolute bioavailability (2.9%) of TRG have been previously reported (Ding et al., 2014). It was inferred that rapid and extensive metabolism of TRG in rats might be the main cause of low bioavailability of TRG after oral administration. In addition, the linear pharmacokinetic behavior of TRG across the investigated dosage range (75–300 mg/kg) was verified by other investigators (Zhou et al., 2009). In our analysis of the tissue distribution of TRG and its metabolites, concentrations of TRG, TRN and TR increased following i.g. administration of TRG and decreased with time. We found TRG was distributed into a variety of tissues. The concentration of TRG in the gastrointestinal tract was highest at the tested time points; consistent with
Fig. 9. Urinary (A), fecal (B) and biliary (C) cumulative excretions of TRG and its metabolites in rats (mean ± S.D., n = 6) following i.g. administration at a single dose of 150 mg/kg TRG. 6
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previous reports (Lv et al., 2006; Zhang et al., 2008). The metabolite TRN was also widely distributed, but the concentrations of TRN in tested tissues were significantly lower than those in plasma at corresponding time points. Interestingly, levels of TRG, in all tested tissues, were higher than those in plasma at the corresponding time points. Taken together, these data imply TRN is more easily cleared from rat tissues compared with TRG. Additionally, TR was mainly distributed into the jejunum, ileum, caecum and colon, with these concentrations of TR being significantly higher than in other tissues. This result further supports the conclusion that TRG is principally metabolized into TR in the intestine. At 12 h after dosing, concentrations of TRG, TRN and TR were low or undetectable in all tested tissues. TRG, TRN and TR were poorly distributed into brain, indicating that they cannot effectively cross the blood-brain barrier. In our analysis of TRG, TRN and TR excretion following i.g. administration of TRG in rats, we detected and quantified levels of TRG, TRN and TR in urine, TR in feces and TRG and TRN in bile. Overall, the urinary, fecal and biliary excretions of TRG and TR were very low yet the excretions of TRN were very high, accounting for 52.8% of the dose in urine, 8.07% in bile; suggesting that TRG underwent deglycosylation followed by glucuronidation before being excreted in rat urine and bile. After TRG was administered orally to rats the unchanged TRG in feces was undetectable, whereas TR was the principal metabolite detected. Therefore, it is inferred that TRG reaching the cecum is extensively hydrolyzed by enzymes of rat microflora (Wang et al., 2011), and that the resulting TR is excreted via feces. TR, acetylated TR (prodrug for TR) and TRG are stilbenoid compounds that have aroused interest due to their potential health benefits (Hogg et al., 2015). Following a single i.g. 100 mg/kg TR administration and a single i.g. (77.5, 155 and 310 mg/kg acetylated TR) administration to rats, TR was eliminated rapidly from the plasma with an average t1/2 of approximately 1.97 h, which was slightly longer than the average t1/2 of TRG (1.40 h), yet acetylated TR was eliminated much more slowly in the form of TR with mean t1/2 of approximately 6.58 h (Liang et al., 2013). The increases in AUC0−∞ of acetylated TR (Liang et al., 2013) and TRG were roughly dose proportional over three doses in rats. The major distribution tissues of TR or acetylated TR in rats were in liver, spleen, heart and lung (Liang et al., 2013), while TRG was mainly distributed into gastrointestinal tract. There was no longterm accumulation of TR (Liang et al., 2013) and TRG in rat tissues. Whether rats were i.g. administrated at a single dose of 155 mg/kg TR or 100 mg/kg acetylated TR, total recoveries of TR in urine and feces within 36 h were low (0.99% or 0.07% in urine and 1.69% or 0.15% in feces), while acetylated TR was undetectable (Liang et al., 2013; Wang et al., 2008), indicating that TR and acetylated TR as same as TRG had poor bioavailability being rapidly and extensively metabolized, accordingly that their biological activities were deprived (Calamini et al., 2010; Hoshino et al., 2010; Miksits et al., 2009). Additionally, after healthy volunteers were orally administered TRG, grape extract tablets, red wine, respectively, the glucuronic acid and sulfate metabolites of TR or TRG were principal compounds in plasma and urine, while level of TRG or TR was very low (Burkon and Somoza, 2008; Rotches-Ribalta et al., 2012). These results indicate the disposition of TRG in humans is similar to that found in rats. Therefore, there is a need to modify structure of TRG to improve its bioavailability in the future.
dynamics in dose range of 75–300 mg/kg. The major distribution tissues of TRG, TRN, and TR in rats were found in the digestive tract. There was no long-term accumulation of TRG, TRN and TR in rat tissues. TRG, TRN and TR were poorly distributed into brain, indicating that they were unable to cross the blood-brain barrier. TRG was mainly excreted in the form of TRN via the renal route. We conclude that after i.g. administration to rats, TRG is easily absorbed by the body and subsequently rapidly converted to TR and TRN. These metabolites are then mainly excreted by the kidneys. The present disposition studies of TRG in rats will provide helpful information for the further investigation and development of TRG. Conflict of interest The authors declare no conflict of interest. Acknowledgments This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References Burkon, A., Somoza, V., 2008. Quantification of free and protein-bound trans-resveratrol metabolites and identification of trans-resveratrol-C/O-conjugated diglucuronidestwo novel resveratrol metabolites in human plasma. Mol. Nut. Food Res. 52, 549–557. Calamini, B., Ratia, K., Malkowski, MG., Cuendet, M., Pezzuto, JM., Santarsiero, BD., Mesecar, AD., 2010. Pleiotropic mechanisms facilitated by resveratrol and its metabolites. Biochem. J. 429, 273–282. Chen, Z., Wei, Q., Hong, G., Chen, D., Liang, J., He, W., Chen, MH., 2016. Polydatin induces bone marrow stromal cells migration by activation of ERK1/2. Biomed. Pharmaco. 82, 49–53. Ding, XY., Hou, XL., Gao, SH., Sun, MX., Lin, FW., Cai, GC., Xiao, K., 2014. Pharmacokinetics and bioavailability study of polydatin in rat plasma by using a LC–MS/MS method. Pak. J. Pharmaceut. Sci. 27, 1931–1937. Du, QH., Peng, C., Zhang, H., 2013. Polydatin: a review of pharmacology and pharmacokinetics. Pharm. Biol. 51, 1347–1354. Gao, SH., Fan, GR., Hong, ZY., Yin, XP., Yang, SL., Wu, YT., 2006. HPLC determination of polydatin in rat biological matrices: application to pharmacokinetic studies. J. Pharm. Biomed. Anal. 41, 240–245. He, H., Zhao, Y., Chen, XJ., Zheng, YT., Wu, XL., Wang, R., Li, TT., Yu, QL., Jing, J., Ma, L., Ren, WC., Han, D., Wang, GJ., 2007. Quantitative determination of trans-polydatin, a natural strong anti-oxidative compound, in rat plasma and cellular environment of a human colon adenocarcinoma cell line for pharmacokinetic studies. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 855, 145–151. Henry-Vitrac, C., Desmoulie`re, A., Girard, D., Merillon, JM., Krisa, S., 2006. Transport, deglycosylation, and metabolism of trans-piceid by small intestinal epithelial cells. Eur. J. Nutr. 45, 376–382. Hogg, S.J., Chitcholtan, K., Hassan, W., Sykes, P.H., Garrill, A., 2015. Resveratrol, acetylresveratrol, and polydatin exhibit antigrowth activity against 3D cell aggregates of the SKOV-3 and OVCAR-8 ovarian cancer cell lines. Obs. Gyn. Int. 5, 1–14. Hoshino, J., Park, E.J., Kondratyuk, T.P., Marler, L., Pezzuto, J.M., van Breemen, R.B., Mo, S., Li, Y., Cushman, M., 2010. Selective synthesis and biological evaluation of sulfate-conjugated resveratrol metabolites. J. Med. Chem. 53, 5033–5043. Jin, J., Li, Y., Zhang, XL., Chen, TS., Wang, YF., Wang, ZP., 2016. Evaluation of both free radical scavenging capacity and antioxidative damage effect of polydatin. Adv. Exp. Med. Biol. 923, 57–62. Learmonth, D.A., 2003. A concise synthesis of the 3-O-beta-D- and 4′-O-beta-D-glucuronide conjugates of trans-resveratrol. Bioconjug. Chem. 14, 262–267. Liang, L., Liu, X.Y., Wang, Q.W., Cheng, S.K., Zhang, S.Y., Zhang, M., 2013. Pharmacokinetics, tissue distribution and excretion study of resveratrol and its prodrug 3,5, 4′,-tri-O-acetylresveratrol in rats. Phytomedicine 20, 558–563. Lv, C.Y., Zhang, L.T., Wang, Q., Liu, W.N., Wang, C.Y., Jing, X.J., Liu, Y., 2006. Determination of piceid in rat plasma and tissues by high-performance liquid chromatographic method with UV detection. Biomed. Chromatogr. 20, 1260–1266. Marier, J.F., Vachon, P., Gritsas, A., Zhang, J., Moreau, J.P., Ducharme, M.P., 2002. Metabolism and disposition of resveratrol in rats: extent of absorption, glucuronidation, and enterohepatic recirculation evidenced by a linked-rat model. J. Pharmacol. Exp. Ther. 302, 369–373. Meng, Q.H., Liu, H.B., Wang, J.B., 2016. Polydatin ameliorates renal ischemia/reperfusion injury by decreasing apoptosis and oxidative stress through activating sonic hedgehog signaling pathway. Food Chem. Toxicol. 96, 215–225. Miksits, M., Wlcek, K., Svoboda, M., Kunert, O., Haslinger, E., Thalhammer, T., Szekeres, T., Jäger, W., 2009. Antitumor activity of resveratrol and its sulfated metabolites against human breast cancer cells. Planta Med. 75, 1227–1230. Rotches-Ribalta, M., Andres-Lacueva, C., Estruchc, R., Escribano, E., Urpi-Sardaa, M., 2012. Pharmacokinetics of resveratrol metabolic profile in healthy humans after moderate consumption of red wine and grape extract tablets. Pharmacol. Res. 66,
Conclusion To our knowledge, we present the first comprehensive pharmacokinetics, tissue distribution and excretion study of TRG and its metabolites in rats after i.g. administration. A rapid, reliable and sensitive LC-UV method was used for simultaneous analysis of TRG and its metabolites in rat biological samples including plasma, urine, feces, bile and tissue. The absolute bioavailability of TRG in rats was very low due to extensive metabolism, resulting in only trace amounts of unchanged TRG in the systemic circulation. TRG, TRN, and TR manifested linear 7
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M. Su, et al. 375–382. Wang, D.G., Xu, Y.R., Liu, W.Y., 2008. Tissue distribution and excretion of resveratrol in rat after oral administration of polygonum cuspidatum extract (PCE). Phytomedicine 15, 859–866. Wang, D.G., Zhang, Z.W., Ju, J.F., Wang, X.Y., Qiu, W.J., 2011. Investigation of piceid metabolites in rat by liquid chromatography tandem mass spectrometry. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 879, 69–74. Wang, H.L., Gao, J.P., Han, Y.L., Xu, X., Wu, R., Gao, Y., Cui, X.H., 2015. Comparative studies of polydatin and resveratrol on mutual transformation and antioxidative effect in vivo. Phytomedicine 22, 553–559. Zhang, M.M., Zhao, Z.J., Shen, M., Zhang, Y.M., Duan, J.H., Guo, Y.J., Zhang, D.W., Hu, J.Q., Lin, J., Man, W.R., Hou, L.C., Wang, H.C., Sun, D.D., 2016. Polydatin protects cardiomyocytes against myocardial infarction injury by activating Sirt3. Biochimica et Biophysica Acta-Mol. Basis Dis. 16, 30221–30226.
Zhang, W.T., Li, Q., Zhu, M., Huang, Q.W., Jia, Y., Bi, K.S., 2008. Direct determination of polydatin and its metabolite in rat excrement samples by high-performance liquid chromatography. Chem. Pharmaceut. Bull. 56, 1592–1595. Zhou, M.J., Chen, X.Y., Zhong, D.F., 2007. Simultaneous determination of trans-resveratrol-3-O- glucoside and its two metabolites in rat plasma using liquid chromatography with ultraviolet detection. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 854, 219–223. Zhou, Q.L., Qin, R.Z., Yang, Y.X., Huang, K.B., Yang, X.W., 2016. Polydatin possesses notable anti-osteoporotic activity via regulation of OPG, RANKL and β-catenin. Mol. Med. Rep. 14, 1865–1869. Zhou, S.Y., Yang, R.T., Teng, Z.H., Zhang, B.L., Hu, Y.Z., Yang, Z.F., Huan, M.G., Zhang, X., Mei, Q.B., 2009. Dose-dependent absorption and metabolism of trans-polydatin in rats. J. Agric. Food Chem. 57, 4572–4579.
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Original Article
Pharmacokinetics, tissue distribution and excretion study of transresveratrol-3-O-glucoside and its two metabolites in rats Meiying Sua, Chao Donga, Jiyun Wana, Maojin Zhoub,c,
T
⁎
a
Taian Central Hospital of Shandong Province, 29 Longtan Road, Taian 271000, PR China Laboratory of Drug Metabolism and Pharmacokinetics, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China c HQ Bioscience Co. Ltd., 11/F, Building D, 398 Ruoshui Road, Suzhou Industrial Park, Suzhou 2151123, PR China b
ARTICLE INFO
ABSTRACT
Keywords: Trans-resveratrol-3-O-glucoside Trans-resveratrol-3-O-glucuronide Trans-resveratrol Pharmacokinetics Distribution Excretion
Background: Trans-resveratrol-3-O-glucoside (TRG), isolated from the Chinese traditional herbal medicine Huzhang, has been shown to have a wide range of pharmacological benefits. Purpose: The aim of this study is to investigate the pharmacokinetics, tissue distribution and excretion of TRG and its metabolites, (TRN (trans-resveratrol-3-O-glucuronide) and TR (trans-resveratrol)), following a single intragastric (i.g.) administration of TRG in rats. Study design: To evaluate the pharmacokinetic properties of TRG, TRN and TR, groups of rats were administrated a single i.g. dose of either 75, 150 or 300 mg/kg TRG. The absolute bioavailability of TRG was estimated from the ratios of AUC0-∞ values for oral and intravenous administration. Tissue distributions of TRG, TRN and TR in rats were investigated following a single i.g. administration to four groups at 150 mg/kg dosage of TRG. For urinary, fecal and biliary excretion study, TRG, TRN and TR excretions were recovered from a group of rats administered a single i.g. dose of 150 mg/kg TRG. Methods: The levels of TRG, TRN and TR in plasma, tissues, bile, urine and feces were measured by a rapid and sensitive LC-UV method. The precision was below 10.0%, and the accuracy was within ± 9.9% for TRG, TRN and TR. Results: The concentrations of TRN were markedly higher than those of TRG and TR in plasma, urine and bile. TRG, TRN and TR showed linear dynamics in dose range of 75–300 mg/kg TRG. TRG had poor absolute bioavailability in rats. The major distribution tissues of TRG, TRN, and TR in rats were in the digestive tract. TRG, TRN and TR were all eliminated from tissues quickly. TRG was mostly excreted via the renal route in the form of TRN, which accounted for 52.8% of the administered dose up to 72 h. Conclusion: Following a single i.g. administration to rats TRG was easily absorbed and rapidly converted to the metabolites TR and TRN. These metabolites were found to be mainly excreted by the kidneys.
Introduction Trans-resveratrol-3-O-glucoside (TRG, also named polydatin, Fig. 1), a glycoside of trans-resveratrol (TR), is an active monomer isolated from the dried roots of Polygonum cuspidatium Sieb.et Zucc., a traditional Chinese medicine commonly known as Huzhang. TRG is also found in grapes, peanuts, hop cones and pellets, red wines, cocoa/ chocolate products and other daily diets (Du et al., 2013). In recent years, numerous pharmacological studies have indicated TRG has numerous physiological benefits, including cardiovascular effects, hepato-
protective effects, neuroprotective activity, lung protective effects, antiarteriosclerosis, anti-inflammatory activity, anti-shock effects, antitumor activity, anti-oxidative activity, inhibition of melanogenesis, anti-microbial activity, inhibition of thrombus formation, immuneregulatory effects (Du et al., 2013; Jin et al., 2016; Wang et al., 2015; Zhang et al., 2016), protection against kidney injury (Meng et al., 2016), induction of bone marrow stromal cell migration (Chen et al., 2016), and anti-osteoporotic activity (Zhou et al., 2016). Although the pharmacological efficacy of TRG after oral administration is known to depend on its absorption, tissue distribution,
Abbreviations: ANOVA, a one-way analysis of variance; AUC, the area under the plasma concentration−time curve; HPLC, high-performance liquid chromatography; LLOQ, lower limit of quantification; TR, trans-resveratrol; TRG, trans-resveratrol-3-O-glucoside; TRN, trans-resveratrol-3-O-glucuronide; UV, ultraviolet detection ⁎ Corresponding author at: HQ Bioscience Co. Ltd., 11/F, Building D, 398 Ruoshui Road, Suzhou Industrial Park, Suzhou 2151123, PR China. E-mail address: [email protected] (M. Zhou). https://doi.org/10.1016/j.phymed.2019.152882 Received 14 November 2016; Received in revised form 28 February 2019; Accepted 9 March 2019 0944-7113/ © 2019 Published by Elsevier GmbH.
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Fig. 1. Chemical structures of TRG, its metabolites (A) and baicalin (internal standard) (B).
metabolism and excretion, few studies have included TRG pharmacokinetics (Ding et al., 2014; Gao et al., 2006; He et al., 2007; Lv et al., 2006; Zhou et al., 2009), tissue distribution (Gao et al., 2006; Lv et al., 2006; Zhang et al., 2008) and excretion (Gao et al., 2006) in rats. The limited studies available mainly focus on the disposition of the unchanged TRG; only two reports address TRG and its metabolites in rat plasma (Zhou et al., 2009) or tissues (Zhang et al., 2008). To our knowledge, specific disposition of TRG and its metabolites TRN and TR in rats has not been reported. Therefore, the aim of our study is to analyze the pharmacokinetics, tissue distributions and excretions of TRG, TRN and TR following oral administration of TRG in rats.
TRG (99.3% purity) was obtained courtesy of Liaoning Institute of Medical Industrial Research (Shenyang, China). TR (99.5% purity) and baicalin (internal standard, 99.0% purity) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). TRN (98.5% purity by HPLC) was isolated and prepared from rat urine in our laboratory (Zhou et al., 2007) and identified by MSn, 1H NMR, 13C NMR (Learmonth, DA., 2003) and HMBC. Acetonitrile and methanol (Yuwang Chemical, Shandong, China) were of HPLC grade. Other chemicals were of analytical grade. Distilled water, prepared from demineralized water, was used throughout the study.
To study the tissue distribution of orally administered TRG, sixteen male and sixteen female rats were divided into four groups at random and each rat was administered a single dose of TRG at 150 mg/kg. Tissue samples were collected from heart, liver, spleen, lung, kidney, stomach, duodenum, jejunum, ileum, caecum, colon, pancreas, brain, fat, muscle, ovary, uterus and testis at time points 0.33, 2.0, 6.0 and 12.0 h post TRG delivery. Time points to collect tissues were designed according to the concentration-time curves. One additional rat was sacrificed pre-dose to provide control tissues for analysis. Tissue samples were weighed rapidly, rinsed with physiological saline to remove the blood or content, blotted on filter paper, and stored at −20ºC until analysis. For study of urinary and fecal excretion, three female and three male rats were orally administered a single dose of TRG at 150 mg/kg. After dosing, the rats were housed in individual stainless steel metabolism cages designed for the separation and collection of urine and feces. Urine and feces were collected between –18 and 0 h pre-dosing and during 0−4, 4−8, 8−12, 12−24, 24−36, 36−48, 48−72 h postdosing. Collected feces were dried at room temperature. Following measurement of feces dry weight and urine volume for each collection period, samples were stored at −20 ºC. For study of biliary excretion, three female and three male rats were anesthetized and cannulas were surgically inserted into the bile duct to collect bile. A single 150 mg/kg dose of TRG was orally administered to each rat. Bile samples were collected before dosing and during 0−4, 4−8, 8−12, 12−24, 24−36, 36−48 h after dosing. Samples were stored at −20 °C until analysis.
Animals, drug administration and sampling
Sample processing
Wistar rats (male and female, ∼200−240 g, Accreditation No.: [Liao] 008, Shenyang) were obtained from the Department of Experimental Animals, Shenyang Pharmaceutical University (Shenyang, China). All animals were acclimatized to 1aboratory conditions and starved overnight prior to drug administration, yet retained free access to drinking water at all times. All animal experiments were approved by the institutional ethics committee prior to the study. For pharmacokinetic study, sixteen male and sixteen female rats were randomly divided into four groups (n = 8 per group). TRG, dissolved in a saline solution (0.9% NaCl) of 20% 2-hydroxypropyl-β-cyclodextrin (Marier et al., 2002), was administered intragastrically at a single dose of 75, 150 and 300 mg/kg to groups 1–3, respectively. The remaining group was administered intravenously a single dose of TRG at 75 mg/kg. Blood samples were collected at 0, 0.17, 0.33, 0.67, 1.0, 2.0, 4.0, 6.0, 8.0, 12.0 and 24.0 h following oral administration and at 0.08, 0.17, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 12.0 and 24.0 h after intravenous administration. Plasma was obtained by centrifugation at 10,000 rpm for 10 min and stored at −20ºC. Due to the generally high ratio of trans-to cis-isomers all samples and standards were handled with no exposure to light.
Plasma, Bile and Urine: A 100 μl aliquot of each sample was combined with 100 μl of the internal standard, 100 μl of formic acid in water (pH 3.5) and 300 μl of acetonitrile. The samples were vortexed for 1 min, and centrifuged at 10,000 rpm for 10 min. The supernatant was transferred to a new tube and evaporated to dryness at 40 ºC under a gentle stream of nitrogen. The residue was reconstituted in 100 μl of the mobile phase and 50 μl of the solution was injected into the HPLC system. Tissue samples were thawed and homogenized in methanol (1:2, w/ v). 100 μl of tissue homogenate was processed as described above for plasma samples. Fecal samples were pulverized with a mortar and pestle and homogenized in methanol (1:2, w/v). 100 μl of fecal homogenate was processed as described above for plasma samples.
Materials and methods Materials
Analysis of TRG, TRN and TR in rat biological samples The concentrations of TRG, TRN and TR in rat biological samples were analyzed using an LC-UV method previously developed by our laboratory (Zhou et al., 2007) with some modifications. Chromatography was performed on a Diamonsil C18 column (200 mm × 4.6 mm 2
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Fig. 2. HPLC-UV chromatograms of a blank liver homogenate sample (A); a blank liver homogenate sample spiked with TRG (0.04 μg/ml), TRN (0.04 μg/ml), TR (0.04 μg/ml) and IS (20 μg/ml) (B); a liver homogenate sample at 2 h after oral administration of 150 mg/kg TRG to a rat (C). Peak I, TRG (tR = 4.7 min); Peak II, TRN (tR = 6.2 min); Peak III, IS (tR = 11.7 min); Peak IV, TR (tR = 14.4 min).
I.D., particle 5 μm, Dikma, Beijing, China). The isocratic mobile phase consisted of 25% acetonitrile and 75% formic acid in water at pH 3.5. The flow-rate was 1.0 ml/min. Ultraviolet detection was set at 320 nm and the column temperature was kept at 25 °C. The LC-UV method developed previously was only validated for rat plasma samples. In the present study, the method was further validated with respect to selectivity, linearity, lower limit of quantification, precision and accuracy, absolute recovery and stability for plasma, tissue, bile, urine and fecal samples. Blank rat plasma, tissue homogenate, bile, urine and feces were used to establish and validate a standard protocol.
Results Method validation Fig. 2 shows representative chromatograms of blank rat liver homogenate (A), blank rat liver homogenate spiked with TRG, TRN and TR (B), and rat liver homogenate sample after i.g. administration of TRG (C). TRG, TRN, TR and internal standard were eluted at approximately 4.7, 6.2, 14.4 and 11.7 min, respectively. No detectable interfering peaks were found with the retention time close to that of TRG, TRN, TR and internal standard. For quantification of TRG, TRN and TR in plasma, urine, bile, tissue and fecal homogenate samples, standard curves of seven relevant concentrations for each standard substance were performed. The curves were characterized by regression coefficients of r = 0.99 or higher. The coefficient of variation of a sixfold replicate was less than 5.0%. The LLOQs of TRG, TRN and TR, defined as the lowest concentration at which both precision and accuracy were less than or equal to 20.0%, were 0.04 μg/ml, 0.10 μg/ml, 0.10 μg/ml, 0.04 μg/g, and 0.10 μg/g in 100 μl rat plasma, urine, bile, tissue and fecal homogenate, respectively. Absolute recoveries of TRG, TRN and TR in rat biological samples were more than 90.0%. The precision, expressed as the intra-day and inter-day relative standard deviation (RSD%), was below 10.0%, and the accuracy, expressed as the relative error (RE%), was within ± 9.9% for TRG, TRN and TR. The results of stability experiments showed that all the biological samples were stable for 12 h at room temperature, after three freeze-thaw cycles and for 30 days under −20 ºC, with a reduction of less than 15.0%.
Pharmacokinetic and statistical analysis All TRG, TRN and TR concentrations in plasma are expressed as TRG, TRN and TR mass equivalents. The Cmax is the highest concentration observed, and Tmax is time at which Cmax occurred. The pharmacokinetic parameters were calculated by non-compartmental analyses using the Topfit software package (version 2.0, Thomae GmbH, Germany). Terminal elimination rate constant (Ke) was determined by linear regression of the terminal portion of plasma concentration-time data, and the elimination half-life (t1/2) was calculated as 0.693/Ke. The area under the plasma concentration−time curve (AUC) from time zero to the last quantifiable concentration (Clast) was determined using the linear trapezoidal method. AUC0-∞ was calculated by adding Clast/λz to AUC0-t, where λz represents the terminal phase of the plasma concentration profile. The total body clearance (CL) was calculated as the quotient of the dose (D) and AUC0−∞. The absolute bioavailability of TRG in rats was estimated from the ratios of AUC0−∞ values for oral and intravenous administration. Dose-proportionality after a single i.g. administration of three dosages was determined by comparison of the dose-normalized AUC0−∞ across dosage levels using the SPSS 13.0 one-way ANOVA analysis and linear regression analysis. All the data were expressed as mean ± S.D. and a p value < 0.05 was deemed to be statistically significant.
Pharmacokinetic studies The HPLC method was successfully applied for investigation of the pharmacokinetics of TRG, TRN and TR following a single i.g. administration of either 75, 150 or 300 mg/kg TRG and a single i.v. TRG administration of 75 mg/kg. The corresponding pharmacokinetic parameters of TRG, TRN and TR, calculated using non-compartmental analysis are listed in Tables 1–3. Mean plasma concentration-time
Table 1 Pharmacokinetic parameters of TRG in rats following i.g. administration of TRG at a single dose of 75, 150 and 300 mg/kg, and following i.v. administration TRG at a single dose of 150 mg/kg, respectively. Parameters
i.g. dose (mg/kg) 75
150
300
i.v. dose (mg/kg) 75
p value
t1/2 (h) AUC0-∞(μg•h/ml) CL (ml/min) Vz (L)
1.61 ± 0.72 1.25 ± 0.51 1.18 ± 0.59 88.5 ± 69.5
1.29 ± 0.66 3.72 ± 1.43 0.75 ± 0.22 83.5 ± 57.7
1.31 ± 0.17 7.62 ± 2.23 0.72 ± 0.26 82.8 ± 35.0
1.51 ± 0.25 112 ± 42.2 12.5 ± 4.19 1.59 ± 0.69
>0.05 >0.05 >0.05 >0.05
Data are expressed as mean ± S.D. (n = 8). t1/2, terminal half-life; AUC0−∞, area under the analyte concentrations vs.time curve from time 0 to infinity; Vz, terminal volume of distribution; CL, total body clearance. p values are obtained by evaluating the pharmacokinetic parameters across the three i.g. administration of TRG dosage groups using one-way ANOVA. AUC0−∞ was normalized by the corresponding dosages when conducting comparison among the three i.g. 3
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Table 2 Pharmacokinetic parameters of TRN in rats following i.g. administration of TRG at a single dose of 75, 150 and 300 mg/kg, and following i.v. administration TRG at a single dose of 150 mg/kg, respectively. Parameters
i.g. dose (mg/kg) 75
150
300
i.v. dose (mg/kg) 75
P value
t1/2 (h) AUC0-∞(μg•h/ml) CL (ml/min) Vz (L)
3.23 ± 0.92 104 ± 39.2 13.4 ± 4.76 3.88 ± 1.86
2.79 ± 0.75 191 ± 46.9 13.7 ± 3.04 3.31 ± 1.22
3.02 ± 0.48 313 ± 112.2 18.1 ± 7.06 4.01 ± 2.21
2.80 ± 0.91 25.6 ± 7.17 53.2 ± 16.4 13.5 ± 8.49
>0.05 >0.05 >0.05 >0.05
Data are expressed as mean ± S.D. (n = 8). t1/2, terminal half-life; AUC0−∞, area under the analyte concentrations vs. time curve from time 0 to infinity; Vz, terminal volume of distribution; CL, total body clearance. P values are obtained by evaluating the pharmacokinetic parameters across the three i.g. administration of TRG dosage groups using one-way ANOVA. AUC0−∞ was normalized by the corresponding dosages when conducting comparison among the three i.g. administration of TRG groups. Table 3 Pharmacokinetic parameters of TR in rats following i.g. administration of TRG at a single dose of 75, 150 and 300 mg/kg, respectively. Parameters
i.g. dose (mg/kg) 75 150
300
t1/2 (h) AUC0-∞(μg•h/ml) CL(ml/min) Vz (L)
2.23 ± 0.34 1.85 ± 0.33 0.70 ± 0.14 0.13 ± 0.03
2.72 ± 0.32 8.21 ± 1.82 0.64 ± 0.14 0.15 ± 0.03
2.87 ± 0.68 3.53 ± 0.77 0.74 ± 0.18 0.18 ± 0.05
P value >0.05 >0.05 >0.05 >0.05
Data are expressed as mean ± S.D. (n = 8). t1/2, terminal half-life; AUC0−∞, area under the analyte concentrations vs. time curve from time 0 to infinity; Vz, terminal volume of distribution; CL, total body clearance. P values are obtained by evaluating the pharmacokinetic parameters across the three i.g. administration of TRG dosage groups using one-way ANOVA. AUC0−∞ was normalized by the corresponding dosages when conducting comparison among the three i.g. administration of TRG groups.
Fig. 4. Mean plasma concentration-time curves of TRN in rats (mean ± S.D., n = 8) following i.g. administration at a single dose of 75, 150 and 300 mg/kg and i.v. administration of 75 mg/kg TRG to rats.
Fig. 5. Mean plasma concentration-time curves of TR in rats (mean ± S.D., n = 8) following i.g. administration at a single dose of 75, 150 and 300 mg/kg TRG to rats.
Fig. 3. Mean plasma concentration-time curves of TRG in rats (mean ± S.D., n = 8) following i.g. administration at a single dose of 75, 150 and 300 mg/kg and i.v. administration of 75 mg/kg TRG to rats.
Tissue distribution studies
curves (n = 8) are presented in Figs. 3–5, demonstrating rapid elimination of TRG from the plasma. This elimination correlates with TR and TRN formation. The AUC0−∞ values of TRG, TRN and TR of the three dosages of TRG indicated an apparent dose-proportionality. The increases in AUC0−∞ of TRG, TRN and TR were roughly dose proportional at 75, 150 and 300 mg/kg dose range (1.25 μg•h/ml vs. 3.72 μg•h/ml vs. 7.62 μg•h/ml for TRG; 104.5 μg•h/ml vs. 191.2 μg•h/ml vs. 312.6 μg•h/ml for TRN; 1.85 μg•h/ml vs.3.53 μg•h/ml vs. 8.21 μg•h/ml for TR). There were no significant differences for other parameters including CL, Vz and t1/2 among the three dosages analyzed by ANOVA (p > 0.05). The absolute bioavailability of TRG in rats was estimated to be 1.11%, 1.66% and 1.70% after a single oral dose of 75, 150 and 300 mg/kg TRG, respectively.
Tissue distributions of TRG and its metabolites were investigated following a single i.g. administration of 150 mg/kg in rats. The concentrations of TRG, TRN and TR in tissues, following a single oral administration, are shown in Figs. 6–8. These data indicate that TRG, TRN and TR underwent a rapid and widespread distribution with no longterm accumulation in any of the tissues tested. At 0.33 h after dosing, the highest level of TRG was found in stomach, amounting to 80.0 µg/g, then in duodenum and jejunum. The concentrations of TRG in these tissues were higher than those in other tissues at any of the time points tested. Subsequently, the concentrations of TRG in all tested tissues decreased rapidly. At 12.0 h after dosing TRG was not detectable in most tissues except for stomach, duodenum, ileum and colon. At all time points, concentrations of TRG in tested tissues were higher than 4
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Fig. 6. Mean concentrations (mean ± S.D., n = 8) of TRG in tissues at different time points after i.g. administration of 150 mg/kg TRG to rats (μg/g or μg/ml).
Fig. 7. Mean concentrations (mean ± S.D., n = 8) of TRN in tissues at different time points after i.g. administration of 150 mg/kg TRG to rats (μg/g or μg/ml).
those in plasma. After TRG was administered orally to rats, the concentrations of TRN in ileum, kidney, jejunum, liver, and duodenum were higher than in other tissues at each time point. The amount of TRN in ileum reached a peak level of 20.0 µg/g at 6.0 h after dosing. Notably, the concentrations of TRN in tested tissues were lower than those in plasma at each time point. TR was mainly distributed into the jejunum, ileum, caecum, and colon, and the concentration in caecum reached maximum value at 6.0 h post-dosing, which was 77.0 µg/g.
and 0.35% up to 72 h in feces. With regard to TRG, only 0.59% of the dose was recovered up to 72 h in urine, 0.08% up to 48 h in bile. Discussion In recent years, an increasing body of work has focused on Chinese herbal medicine, particularly isolated monomers. Although the biological activity of TRG has been shown, pharmacokinetic knowledge of TRG, especially its metabolism and disposition in vivo, is lacking. This type of in depth analysis is very important for better understanding the pharmacological effects of TRG. Our lab has previously reported both TRN and TR are metabolites of TRG, of which TRN is a main metabolite following i.g. administration of TRG in rats (Zhou et al., 2007). With this study, we present the first findings of the pharmacokinetics, tissue distribution and excretion of TRG and its metabolites after i.g. administration of TRG in rats. A simple, specific and sensitive LC-UV method previously developed by our laboratory has been further validated and
Excretion studies The urinary, fecal and biliary excretions of TRG, TRN and TR in rats following a single oral administration of TRG are summarized in Fig. 9. After oral administration of TRG at dose of 150 mg/kg, TRN was the predominant metabolite collected, accounting for 52.8% of the dose up to 72 h in urine and 8.07% up to 48 h in bile. The cumulative excretion ratio of TR was low, amounting to 1.37% of the dose up to 72 h in urine 5
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Fig. 8. Mean concentrations (mean ± S.D., n = 8) of TR in tissues at different time points after i.g. administration of 150 mg/kg TRG to rats (μg/g or μg/ml).
successfully applied to quantification of TRG, TRN and TR in a wide range of rat biological samples. Our pharmacokinetic analysis, following a single i.g. administration in rats, shows TRG was rapidly absorbed and eliminated from rat plasma with an average t1/2 of 1.40 h. TRN was the predominant compound in rat plasma, eliminated more slowly with an average t1/2 of 3.01 h. Intravenous administration of TRG in rats showed a much higher concentration of TRG than TRN, while TR was almost undetectable in plasma. The mean molar ratios of AUC0−∞ of TRN/TRG and TR/TRG were 56.0 and 1.98 at three oral doses, respectively; while the mean molar ratio of AUC0−∞ of TRN/TRG was 0.221 at single i.v. dose (much lower than that at oral dose). This result indicates a certain amount of TRG absorbed into the body was hydrolyzed to form TR by the intestines and/or liver first pass effects (Henry-Vitrac et al., 2006); subsequently the TR was mainly in the form of TRN (a glucuronic acid
conjugate of TR) after entering the blood circulation. Besides, the extent of glucuronidation of TR in intestine was much higher than that in liver. The mean absolute bioavailability of TRG in rats was estimated to be 1.49% after a single oral three doses. Similar findings for low absolute bioavailability (2.9%) of TRG have been previously reported (Ding et al., 2014). It was inferred that rapid and extensive metabolism of TRG in rats might be the main cause of low bioavailability of TRG after oral administration. In addition, the linear pharmacokinetic behavior of TRG across the investigated dosage range (75–300 mg/kg) was verified by other investigators (Zhou et al., 2009). In our analysis of the tissue distribution of TRG and its metabolites, concentrations of TRG, TRN and TR increased following i.g. administration of TRG and decreased with time. We found TRG was distributed into a variety of tissues. The concentration of TRG in the gastrointestinal tract was highest at the tested time points; consistent with
Fig. 9. Urinary (A), fecal (B) and biliary (C) cumulative excretions of TRG and its metabolites in rats (mean ± S.D., n = 6) following i.g. administration at a single dose of 150 mg/kg TRG. 6
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previous reports (Lv et al., 2006; Zhang et al., 2008). The metabolite TRN was also widely distributed, but the concentrations of TRN in tested tissues were significantly lower than those in plasma at corresponding time points. Interestingly, levels of TRG, in all tested tissues, were higher than those in plasma at the corresponding time points. Taken together, these data imply TRN is more easily cleared from rat tissues compared with TRG. Additionally, TR was mainly distributed into the jejunum, ileum, caecum and colon, with these concentrations of TR being significantly higher than in other tissues. This result further supports the conclusion that TRG is principally metabolized into TR in the intestine. At 12 h after dosing, concentrations of TRG, TRN and TR were low or undetectable in all tested tissues. TRG, TRN and TR were poorly distributed into brain, indicating that they cannot effectively cross the blood-brain barrier. In our analysis of TRG, TRN and TR excretion following i.g. administration of TRG in rats, we detected and quantified levels of TRG, TRN and TR in urine, TR in feces and TRG and TRN in bile. Overall, the urinary, fecal and biliary excretions of TRG and TR were very low yet the excretions of TRN were very high, accounting for 52.8% of the dose in urine, 8.07% in bile; suggesting that TRG underwent deglycosylation followed by glucuronidation before being excreted in rat urine and bile. After TRG was administered orally to rats the unchanged TRG in feces was undetectable, whereas TR was the principal metabolite detected. Therefore, it is inferred that TRG reaching the cecum is extensively hydrolyzed by enzymes of rat microflora (Wang et al., 2011), and that the resulting TR is excreted via feces. TR, acetylated TR (prodrug for TR) and TRG are stilbenoid compounds that have aroused interest due to their potential health benefits (Hogg et al., 2015). Following a single i.g. 100 mg/kg TR administration and a single i.g. (77.5, 155 and 310 mg/kg acetylated TR) administration to rats, TR was eliminated rapidly from the plasma with an average t1/2 of approximately 1.97 h, which was slightly longer than the average t1/2 of TRG (1.40 h), yet acetylated TR was eliminated much more slowly in the form of TR with mean t1/2 of approximately 6.58 h (Liang et al., 2013). The increases in AUC0−∞ of acetylated TR (Liang et al., 2013) and TRG were roughly dose proportional over three doses in rats. The major distribution tissues of TR or acetylated TR in rats were in liver, spleen, heart and lung (Liang et al., 2013), while TRG was mainly distributed into gastrointestinal tract. There was no longterm accumulation of TR (Liang et al., 2013) and TRG in rat tissues. Whether rats were i.g. administrated at a single dose of 155 mg/kg TR or 100 mg/kg acetylated TR, total recoveries of TR in urine and feces within 36 h were low (0.99% or 0.07% in urine and 1.69% or 0.15% in feces), while acetylated TR was undetectable (Liang et al., 2013; Wang et al., 2008), indicating that TR and acetylated TR as same as TRG had poor bioavailability being rapidly and extensively metabolized, accordingly that their biological activities were deprived (Calamini et al., 2010; Hoshino et al., 2010; Miksits et al., 2009). Additionally, after healthy volunteers were orally administered TRG, grape extract tablets, red wine, respectively, the glucuronic acid and sulfate metabolites of TR or TRG were principal compounds in plasma and urine, while level of TRG or TR was very low (Burkon and Somoza, 2008; Rotches-Ribalta et al., 2012). These results indicate the disposition of TRG in humans is similar to that found in rats. Therefore, there is a need to modify structure of TRG to improve its bioavailability in the future.
dynamics in dose range of 75–300 mg/kg. The major distribution tissues of TRG, TRN, and TR in rats were found in the digestive tract. There was no long-term accumulation of TRG, TRN and TR in rat tissues. TRG, TRN and TR were poorly distributed into brain, indicating that they were unable to cross the blood-brain barrier. TRG was mainly excreted in the form of TRN via the renal route. We conclude that after i.g. administration to rats, TRG is easily absorbed by the body and subsequently rapidly converted to TR and TRN. These metabolites are then mainly excreted by the kidneys. The present disposition studies of TRG in rats will provide helpful information for the further investigation and development of TRG. Conflict of interest The authors declare no conflict of interest. Acknowledgments This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References Burkon, A., Somoza, V., 2008. Quantification of free and protein-bound trans-resveratrol metabolites and identification of trans-resveratrol-C/O-conjugated diglucuronidestwo novel resveratrol metabolites in human plasma. Mol. Nut. Food Res. 52, 549–557. Calamini, B., Ratia, K., Malkowski, MG., Cuendet, M., Pezzuto, JM., Santarsiero, BD., Mesecar, AD., 2010. Pleiotropic mechanisms facilitated by resveratrol and its metabolites. Biochem. J. 429, 273–282. Chen, Z., Wei, Q., Hong, G., Chen, D., Liang, J., He, W., Chen, MH., 2016. Polydatin induces bone marrow stromal cells migration by activation of ERK1/2. Biomed. Pharmaco. 82, 49–53. Ding, XY., Hou, XL., Gao, SH., Sun, MX., Lin, FW., Cai, GC., Xiao, K., 2014. Pharmacokinetics and bioavailability study of polydatin in rat plasma by using a LC–MS/MS method. Pak. J. Pharmaceut. Sci. 27, 1931–1937. Du, QH., Peng, C., Zhang, H., 2013. Polydatin: a review of pharmacology and pharmacokinetics. Pharm. Biol. 51, 1347–1354. Gao, SH., Fan, GR., Hong, ZY., Yin, XP., Yang, SL., Wu, YT., 2006. HPLC determination of polydatin in rat biological matrices: application to pharmacokinetic studies. J. Pharm. Biomed. Anal. 41, 240–245. He, H., Zhao, Y., Chen, XJ., Zheng, YT., Wu, XL., Wang, R., Li, TT., Yu, QL., Jing, J., Ma, L., Ren, WC., Han, D., Wang, GJ., 2007. Quantitative determination of trans-polydatin, a natural strong anti-oxidative compound, in rat plasma and cellular environment of a human colon adenocarcinoma cell line for pharmacokinetic studies. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 855, 145–151. Henry-Vitrac, C., Desmoulie`re, A., Girard, D., Merillon, JM., Krisa, S., 2006. Transport, deglycosylation, and metabolism of trans-piceid by small intestinal epithelial cells. Eur. J. Nutr. 45, 376–382. Hogg, S.J., Chitcholtan, K., Hassan, W., Sykes, P.H., Garrill, A., 2015. Resveratrol, acetylresveratrol, and polydatin exhibit antigrowth activity against 3D cell aggregates of the SKOV-3 and OVCAR-8 ovarian cancer cell lines. Obs. Gyn. Int. 5, 1–14. Hoshino, J., Park, E.J., Kondratyuk, T.P., Marler, L., Pezzuto, J.M., van Breemen, R.B., Mo, S., Li, Y., Cushman, M., 2010. Selective synthesis and biological evaluation of sulfate-conjugated resveratrol metabolites. J. Med. Chem. 53, 5033–5043. Jin, J., Li, Y., Zhang, XL., Chen, TS., Wang, YF., Wang, ZP., 2016. Evaluation of both free radical scavenging capacity and antioxidative damage effect of polydatin. Adv. Exp. Med. Biol. 923, 57–62. Learmonth, D.A., 2003. A concise synthesis of the 3-O-beta-D- and 4′-O-beta-D-glucuronide conjugates of trans-resveratrol. Bioconjug. Chem. 14, 262–267. Liang, L., Liu, X.Y., Wang, Q.W., Cheng, S.K., Zhang, S.Y., Zhang, M., 2013. Pharmacokinetics, tissue distribution and excretion study of resveratrol and its prodrug 3,5, 4′,-tri-O-acetylresveratrol in rats. Phytomedicine 20, 558–563. Lv, C.Y., Zhang, L.T., Wang, Q., Liu, W.N., Wang, C.Y., Jing, X.J., Liu, Y., 2006. Determination of piceid in rat plasma and tissues by high-performance liquid chromatographic method with UV detection. Biomed. Chromatogr. 20, 1260–1266. Marier, J.F., Vachon, P., Gritsas, A., Zhang, J., Moreau, J.P., Ducharme, M.P., 2002. Metabolism and disposition of resveratrol in rats: extent of absorption, glucuronidation, and enterohepatic recirculation evidenced by a linked-rat model. J. Pharmacol. Exp. Ther. 302, 369–373. Meng, Q.H., Liu, H.B., Wang, J.B., 2016. Polydatin ameliorates renal ischemia/reperfusion injury by decreasing apoptosis and oxidative stress through activating sonic hedgehog signaling pathway. Food Chem. Toxicol. 96, 215–225. Miksits, M., Wlcek, K., Svoboda, M., Kunert, O., Haslinger, E., Thalhammer, T., Szekeres, T., Jäger, W., 2009. Antitumor activity of resveratrol and its sulfated metabolites against human breast cancer cells. Planta Med. 75, 1227–1230. Rotches-Ribalta, M., Andres-Lacueva, C., Estruchc, R., Escribano, E., Urpi-Sardaa, M., 2012. Pharmacokinetics of resveratrol metabolic profile in healthy humans after moderate consumption of red wine and grape extract tablets. Pharmacol. Res. 66,
Conclusion To our knowledge, we present the first comprehensive pharmacokinetics, tissue distribution and excretion study of TRG and its metabolites in rats after i.g. administration. A rapid, reliable and sensitive LC-UV method was used for simultaneous analysis of TRG and its metabolites in rat biological samples including plasma, urine, feces, bile and tissue. The absolute bioavailability of TRG in rats was very low due to extensive metabolism, resulting in only trace amounts of unchanged TRG in the systemic circulation. TRG, TRN, and TR manifested linear 7
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M. Su, et al. 375–382. Wang, D.G., Xu, Y.R., Liu, W.Y., 2008. Tissue distribution and excretion of resveratrol in rat after oral administration of polygonum cuspidatum extract (PCE). Phytomedicine 15, 859–866. Wang, D.G., Zhang, Z.W., Ju, J.F., Wang, X.Y., Qiu, W.J., 2011. Investigation of piceid metabolites in rat by liquid chromatography tandem mass spectrometry. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 879, 69–74. Wang, H.L., Gao, J.P., Han, Y.L., Xu, X., Wu, R., Gao, Y., Cui, X.H., 2015. Comparative studies of polydatin and resveratrol on mutual transformation and antioxidative effect in vivo. Phytomedicine 22, 553–559. Zhang, M.M., Zhao, Z.J., Shen, M., Zhang, Y.M., Duan, J.H., Guo, Y.J., Zhang, D.W., Hu, J.Q., Lin, J., Man, W.R., Hou, L.C., Wang, H.C., Sun, D.D., 2016. Polydatin protects cardiomyocytes against myocardial infarction injury by activating Sirt3. Biochimica et Biophysica Acta-Mol. Basis Dis. 16, 30221–30226.
Zhang, W.T., Li, Q., Zhu, M., Huang, Q.W., Jia, Y., Bi, K.S., 2008. Direct determination of polydatin and its metabolite in rat excrement samples by high-performance liquid chromatography. Chem. Pharmaceut. Bull. 56, 1592–1595. Zhou, M.J., Chen, X.Y., Zhong, D.F., 2007. Simultaneous determination of trans-resveratrol-3-O- glucoside and its two metabolites in rat plasma using liquid chromatography with ultraviolet detection. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 854, 219–223. Zhou, Q.L., Qin, R.Z., Yang, Y.X., Huang, K.B., Yang, X.W., 2016. Polydatin possesses notable anti-osteoporotic activity via regulation of OPG, RANKL and β-catenin. Mol. Med. Rep. 14, 1865–1869. Zhou, S.Y., Yang, R.T., Teng, Z.H., Zhang, B.L., Hu, Y.Z., Yang, Z.F., Huan, M.G., Zhang, X., Mei, Q.B., 2009. Dose-dependent absorption and metabolism of trans-polydatin in rats. J. Agric. Food Chem. 57, 4572–4579.
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