Tissue distribution and excretion of resveratrol in rat after oral administration of Polygonum cuspidatum extract (PCE).
Purpose: Polygonum cuspidatum extract as a traditional Chinese medicine is extracted from the dried rhizome and root of Polygonum cuspidatum Sieb.et Zucc. Resveratrol is one of its active components. Studies were performed in rats to define the tissue distribution and excretion of resveratrol in urine and bile, and to characterize (if possible) any metabolites of resveratrol observed in tissues after ig 20 mg/kg Polygonum cuspidatum extract.
Method: For tissue distribution studies, tissues (300 mg) were homogenized and centrifuged with methanol, and metabolites found in selected tissue extract were identified by LC/MS/MS. For urinary and biliary excretion experiments, urine and bile samples were cleaned up by using solid-phase extraction (SPE) with polyamide cartridges. All the concentrations of resveratrol in these biological samples were determined by HPLC with UV detection.
Result: After a single oral dose of 20 mg/kg PCE in rats, resveratrol was mainly distributed in stomach, duodenum, liver and kidney with detectable metabolites resveratrol monoglucuronide and resveratrol monosulfate. The majority of the resveratrol was excreted as metabolites, only 0.59% and 0.027% of the dosage were excreted in urine and bile respectively as unchanged drug within 24 h.
[c] 2008 Elsevier GmbH. All rights reserved.
Keywords: Polygonum cuspidatum extract; Resveratrol; Tissue Distribution; Excretion
Polygonum cuspidatum extract (PCE) is made from the dried rhizome and root of Polygonum cuspidatum Sieb.et Zucc. As a traditional Chinese medicine it has been used in the treatment of anti-inflammatory, atherosclerosis and other therapeutic purposes (Zhang et al., 2003; Jie and Hu, 2000), it has also been recorded in China Pharmacopoeia (CP). Resveratrol (3, 4', 5-trihy-drohydroxystilbene) is one of its active components.
Resveratrol is a phenolic compound found in many families of plants such as peanuts (Ingham, 1976), grapes (Landcake and Price, 1976), wine (Siemann and Creasy, 1992) and Polygonum cuspidatum (Vastano et al., 2000). The content of resveratrol in Polygonum cuspidatum was much higher than in grape and other plants. It was reported that resveratrol has many protecting properties to human health such as an antioxidant (Frankel et al., 1993), modulator of lipoprotein metabolism (Soleas et al., 1997; Goldberg et al., 1995), inhibitor of platelet aggregation (Bertelli et al., 1995; Pace-Asciak et al., 1995) and vasorelaxing agent (Chen and Pace-Asciak, 1996; Jager and Nguyen-Duong, 1999). Above all the most beneficial effect of resveratrol is its cancer chemopreventive activity for it is involved in the inhibition of tumor initiation, promotion and progression (Jang et al., 1997).
Paramount to the development of a drug for certain disease treatment is an understanding of its distribution, metabolism and excretion after oral administration to preclinical species. Over recent years, many studies on resveratrol including its tissue distribution (Vitrac et al., 2003) and metabolism (Kuhnle et al., 2000; Yu et al., 2002; Wang et al., 2005) have been reported. Although PCE has been recorded in China Pharmacopoeia (CP), to our knowledge, there are few reports on bioavailability, disposition and metabolism of its main components. Here we tentatively do some research work on tissue distribution and excretion of resveratrol in rat after oral administration of PCE.
Materials and methods
Chemicals and reagents
Trans-resveratrol and naproxen (internal standard) were purchased from Sigma (St. Louis, MO, USA), and both were 99% pure. HPLC-grade methanol and acetonitrile were from Merck KGaA (Darmstadt, Germany). Water was deionized distilled water. The polyamide cartridge (180 mg, particle size 40 [micro]m) was prepared in our laboratory.
The PCE was prepared in our laboratory through multiple extraction procedures. The herb, which was pulverized into powder form (less than 0.9 mm), was initially extracted with 95% ethanol by the refluxing method; the yield of extract was 23.6%. Then the dry product mentioned above was again extracted with ethyl acetate-petroleum ether (3:7), and back extracted with 4% sodium bicarbonate. After the pH was adjusted to between 6 and 6.5, the aimed compounds were again extracted into ethyl acetate-petroleum ether (3:7). During the final extraction, the yield of resveratrol was 96.5%, and the content of resveratrol was over 50.0%.
Animals and dosing
Male Sprague-Dawley rats weighing 200-230 g were obtained from the Breeding Laboratories in China Pharmaceutical University. Rats were housed in metabolic cages with water and a solid diet freely available, and maintained at 22 [+ or -] 3[degrees] C with 40-70% relative humidity. Rats were acclimatized under these conditions for at least 2 days before dosing. The research using rats adhered to the "Principles of Laboratory Animal Care" (NIH publication # 85-23, revised in 1985).
For tissue distribution study, rats were divided in three groups (n = 5 per group), and a dose of 20 mg/kg of Polygonum cuspidatum extract dissolved in a hydro-alcoholic solution (approx. 4.0% v/v ethanol in water) was orally administered by gastric intubation to overnight fasted rats. The rats in the four groups were sacrificed by decapitation at 0, 10, 30, 60 min postdosing, respectively. The tissues or organs, including brain, heart, lungs, liver, spleen, stomach, small intestine and kidneys, were excised, trimmed of extraneous fat, residual muscle and connective tissue, thoroughly rinsed of residual blood or contents with physiological saline solution, and blotted dry.
For urinary excretion experiments, eight rats each was administered a single dose of 20 mg/kg of PCE by the same way as described above, and were then placed in four stainless-steel metabolic cages (n = 2 per cage) that allowed separate collection of urine and feces. They were provided with standard food and water ad libitum throughout the experiment. Urine samples were collected from rats at-12 to 0 h predose and at intervals of 0-2, 2-4, 4-8, 8-12 and 12-24, postdosing. The volume of each collected urine sample was recorded separately and stored at approximately-20 [degrees]C before the sample preparation using polyamide solid-phase extraction was performed.
For biliary excretion study, five male Sprague-Dawley rats (BW 200 [+ or -] 50 g) were anesthetized by intraperitoneal injection with 200 g/1 urethane (1.0 g/kg). Following a midline abdominal incision, the common bile duct was exposed and the distal end ligated with a silk suture. The bile duct was cannulated with a small length of PE-10 tubing for collection of bile samples, and closed with surgical clips. Then the rats were administered a single dose of PCE by the same way as described above. Bile samples of each rat were collected at 0-2, 2-4, 4-6 and 6-8 postdosing, and the volume of each collected sample was recorded separately.
For tissue distribution study, small slices of tissues (300 mg) were individually homogenized with methanol (4.0 ml), vortexed and centrifuged (GS-15R centrifuge; Beckman Instruments, Palo Alto, CA) at 16,000g for 15 min at 4 [degrees]C. The supernatant was separated and evaporated under [N.sub.2] gas, and the residue was reconstituted in 200 [micro]l methanol before HPLC analysis. The internal standard (20.3 mg/1), 100 [micro]l, was added in tissue analyte samples before homogenization.
Rat urine samples were thawed in a water bath at room temperature and then centrifuged at approximately 2000g for 5 min. Polyamide SPE cartridges (180 mg) were conditioned with 5 ml of methanol followed by 10 ml of water. Aliquots of 1 ml urine samples were loaded onto the cartridges, which were then rinsed with 10 ml of water and 5 ml of methanol/water (15:85, v/v) in that order. The analytes were finally eluted from the cartridges by 4 ml methanol. The eluate was evaporated to dryness under [N.sub.2] gas at 40 [degrees]C and was redissolved in 200 [micro]l of 80% methanol. A volume of 20 [micro]l was applied to the HPLC system. The bile samples were processed by the same way as that of urine.
The analyses were carried out in Agilent 1100 Series (USA) liquid chromatograph equipped with two pumps (model G1312A), an autosampler (Model G1313A) and a VWD detector (model G13I4A). A Nucleosil 100 [C.sub.18] reverse-phase column (150 x 4.6 mm; particle size, 5 [micro]m; Knauer, Berlin, Germany) protected by a precolumn was used.
Tissue distribution studies: We used acetonitrile/water (10/90, v/v) as solvent A, and acetonitrile/water (80/20, v/v) as solvent B, at a flow rate of 1.0 ml/min with the following gradient: the initial composition of the mobile phase was 10/90 of acetonitrile/water. Within 5 min the concentration of organic solvent (acetonitrile) increased to 30% (linear increase). From 5.1 to 14 min, the concentration of acetonitrile increased to 41%. From 14.1 to 22 min, the concentration of acetonitrile increased to 80%, after which the concentration of acetonitrile declined to the initial value of 10% (linear decline in 5 min). For the purpose of equilibration this concentration (10% acetonitrile) was kept constant for l0 min. Samples were filtered (0.45 [micro]m, Millipore) and 20 [micro]l was directly injected. Chromatograms were monitored at 306 nm using the UV detector.
Urinary and biliary excretion studies: The HPLC instrument was the same as described above. Separation was carried out by using a [C.sub.18] reverse-phase column (100 x 3 mm; particle size, 5 [micro]m; Shimadzu, Japan) with isocratic conditions (26% acetonitrile) at a flow rate of 1.0 ml/min. Column temperature was set to 28[degrees]C, and the detector wavelength was set to 306 nm.
Tentative identification of metabolites in selected tissue extract was achieved by LC/MS/MS. The LC/MS/MS analyses were performed using a system consisting of a Finnigan autosampler (Thermo Electron Corporation, USA), a Finnigan LC pump, a Finnigan TSQ Quantum Ultra equipped with an electrospray ion source and operated by XCalibur software.
The separating conditions were the same as described above in tissue distribution studies, but only l0 [micro]l was directly injected to the LC/MS/MS system.
The mass spectral analysis was performed in a negative electrospray ionization mode. The capillary and orifice voltages were set at-3.7 kV and-65 V, respectively. The nebulizer gas was set at 50 psi. The nitrogen auxiliary was adjusted to a constant flow rate of 21/min. The turbo-ionspray temperature was set at 400 [degrees]C. Collision-induced dissociation (CID) studies were performed using a collision energy of 30 eV. The ionspray interface and mass spectrometric parameters were optimized to obtain maximum sensitivity at unit resolution.
For determination of resveratrol in selected tissues and excreta samples (urine and bile), the analytical procedures were validated with respect to linearity, precision, limit of detection and recovery. For tissue distribution studies, calibration curves were derived from peak area ratios (analyte/internal standard) using a least-squares regression of the ratio versus the nominal concentration of the calibration curve standard. The results showed good relationships ([r.sup.2] > 0.98) and the linear range of the assays was found to be 71.5-57332 ng/mg for the entire matrix. The RSD of precision (within-day precision and between-day precision) were both less than 10%. The limit of detection was 35.5 ng/mg and the average recovery of resveratrol was over 10%.
In urinary and biliary excretion studies, samples were treated with the method of polyamide solid-phase extraction, and then detected by HPLC with isocratic conditions. The assay was found to be linear in the range 43-8600 ng/ml ([r.sub.2] > 0.99). The lower limit of quantitation was set to 35.5 ng/ml. The recovery and RSD (precision) were over 89.3% and under 8.1%, respectively.
Tissue distribution studies
Fig. 1 shows a representative chromatogram of rat tissues corresponding to liver extracted 10 min after oral administration of PCE extract. Four major peaks corresponding to trans-resveratrol (Tr = 9.83 min), naproxen (internal standard, Tr = 14.4) and two unidentified peaks (metabolites M1, M2), were detected. As can be seen, the extraction is very selective with no interference at the retention times of trans-resveratrol and internal standard.
Tissue distribution of resveratrol
Tissue distribution of resveratrol was investigated following a single PCE administration to rats at 20 mg/kg dosage. The results (Table 1) indicated that the resveratrol underwent a rapid and wide distribution in the tissues/organs throughout the whole body within the time course examined. Following 10 min of PCE administration, most of the tissues analyzed contained a significant amount of resveratrol. Except in the brain resveratrol showed substantial disposition in heart, lungs, liver, spleen, stomach, small intestine and kidneys, which is similar to the previous report (Vitrac et al., 2003). The highest levels (48,236.7 [+ or -] 48,156.2 and 3768.5 [+ or -] 2343.9 ng/mg) were detected in stomach and small intestine, respectively, followed by liver (2433.6[+ or -]291.5 ng/mg) and kidneys (1317.7[+ or -]3.97 ng/mg).
[FIGURE 1 OMITTED]
Table 1. Concentration of resveratrol in rats at 10, 30 and 60 min after ig 20 mg/kg PCE (ng/mg, n = 5) Tissue 10 min 30 min 60 min Heart 196.2 [+ or -] 743.4 [+ or -] 5.0 [+ or -] 12.8 45.77 12.9 Liver 2433.6 [+ or -] 1581.5 [+ or -] 2126.4 [+ or -] 291.5 1154.3 50.7 Spleen 611.6 [+ or -] 497.8 [+ or -] 267.8 [+ or -] 315.6 78.2 43.9 Lung 167.7 [+ or -] 281.4 [+ or -] 2859.3 [+ or -] 23.0 55.5 1040.9 Kidney 360.2 [+ or -] 1317.7 [+ or -] 826.8 [+ or -] 101.7 3.97 225.1 Brain nd nd nd Stomach 46,184.5 [+ or -] 46,321.9 [+ or -] 48,236.7 [+ or -] 1261.0 5781.8 48.156.2 Intestine 20,404.4 [+ or -] 12,615.0 [+ or -] 3768.5 [+ or -] 8171.9 5217.9 2343.9 nd: not detectable.
For the clearance organs, liver and kidneys, liver initially absorbed much more resveratrol than kidney did. The concentration in liver peaked early (10 min postdosing) and decreased slowly, while resveratrol in kidneys reached the maximum until 30 min.
Resveratrol: As listed in Table 2, the negative electrospray mass spectrum of resveratrol showed a [[M-H].sup.-] ion at m/z 227. The CID spectrum of m/z 227 generated a series of fragment ions at 185, 143, 117 and 119. The fragment ion at m/z 185 was generated from the loss of 42 ([C.sub.2] [H.sub.2]O) from the ion at m/z 227. The m/z 185 further fragmented to m/z 143 after loss of 42 ([C.sub.2] [H.sub.2]O). The m/z 119 and 117 were formed after the losses of 108 ([C.sub.6] [H.sub.4][O.sub.2]) and 110 ([C.sub.6] [H.sub.6][O.sub.2]), respectively. This result is consistent with that of the report by Yu et al. (2002). The fragmentation pathway may be explained according to Fig. 2.
Metabolite Ml: The mass spectra of M1 are listed in Table 2. It gave an [[M-H].sup.-] ion at m/z 403, 176 Da higher than that of resveratrol. The CID spectrum of Ml showed a series of fragment ions at m/z 227, 185, 175, 117 and 113. m/z 227, 185 and 117 corresponded to the characteristic fragment ions of resveratrol. m/z 175 and 113 can be considered characteristic of the presence of a glucuronic acid moiety (Stecher et al., 2001; Debrauwer et al., 2001; Manini et al., 1998). The ion at m/z 113 was formed by further dissociation of the fragment ion of m/z 175 and is common in the negative ion mass spectra of glucuronide metabolites. Therefore, M1 was identified as resveratrol monoglucuronide. The fragmentation pathway may be explained in terms of Fig. 3.
[FIGURE 2 OMITTED]
Metabolite M2: The [[M-H].sup.-] ion of m/z 307 of M2 fragmented to form a product ion of m/z 227 that corresponded to resveratrol itself after the loss of sulfate. The m/z 227 further fragmented to m/z 185. Therefore, M2 was identified as resveratrol monosulfate (MW = 307). Only one resveratrol monosulfate and no disulfate metabolites were detected. The fragmentation pathway may be explained according to Fig. 4.
[FIGURE 4 OMITTED]
Table 2. Mass spectral data for resveratrol and its metabolites in rats Metabolite [[M-H].sup.-]ion m/z CID spectra data Resveratrol 227 185, 143, 117, 119 Ml 403 227, 185, 175, 117, 113 M2 307 227, 185
HPLC chromatograms of samples before and after polyamide solid extraction
In order to eliminate the endogenesis interferer, rat urine samples were cleaned up by using solid-phase extraction (SPE) with polyamide cartridges. The extraction and HPLC assay resulted in symmetrical peak shape and good baseline resolution of resveratrol. Urine matrix components did not interfere with the analysis. Using this system, the retention time for resveratrol (peak 1) was 15 min.
Fig. 5 illustrates typical chromatograms of urine samples before and after SPE. (1) and (3) show chromatograms of blank urine sample without or with SPE treatment, respectively. (4) is a chromatogram from SPE-treated urine sample of rat after oral administration of 20 mg/kg b.wt. PCE extract.
[FIGURE 5 OMITTED]
Urinary and biliary excretion
Fig. 6 and Table 3 show the urinary excretion time profile of resvertrol after oral administration of 20 mg/kg PCE. It shows that the excretion of resveratrol peaked during 4-8 h post administration, and the mean cumulative excretion of resveratrol was 0.59% of the dosage within 24 h.
The biliary excretion data of resveratrol after a single oral administration of 20 mg/kg PCE to intact and bile duct cannulated rats are listed in Table 4. Only 0.027% of the dosed resveratrol was excreted unchanged drug into bile up to 8 h postdosing. These findings suggested the presence of the entero-hepatic circulation of resveratrol and its metabolites.
[FIGURE 6 OMITTED]
Table 3. Urine excretion of resveratrol in rat after ig dose of 20 mg/kgPCE Time (h) Amount excreted ([mu]g) Group I Group II Group III Mean 0-2 3.08 2.16 1.31 2.18 2-4 0.54 0.19 0.84 0.52 4-8 21.97 23.06 9.82 18.28 8-12 10.00 7.00 1.88 6.29 12-24 2.37 2.14 3.17 2.56 0-24 37.96 33.55 17.04 29.52 Total dose ([mu]g) 4740 4860 6020 5206.67 Excretion (%) 0.80 0.69 0.28 0.59
The results from the current study indicate that after oral administration, resveratrol had a rapid distribution to various organs. The highest concentrations were found in stomach and small intestine; the reasons may be mainly attributed to the residual drug content and to entero-hepatic circulation. On the contrary, decreasing concentrations of resveratrol in kidney over time indicates that renal excretion might be one of the major ways of elimination of resveratrol, observations also supported by the relatively high concentrations found in urine.
HPLC analysis of resveratrol in most of selected tissue extract showed the presence of resveratrol, together with a relatively high concentration of metabolites (M1, M2). M1 and M2 were thereafter identified as resveratrol monoglucuronide and resveratrol monosulfate by LC/MS/MS. However, we could not find another two metabolites (dihydroresveratrol monosulfate and dihydroresveratrol), which were found in our previous experiment (Wang et al., 2005); this may be due to their lower concentration.
The oldest and most basic sample preparation method is extraction, in which the analyst aims to separate the analyst of interest from a sample matrix using a solvent with an optimum yield and selectivity, so that as few potential interfering species as possible are carried through to the analytical separation stage. Solid-phase extraction technique (SPE) is one of the methods that usually used, and is often applied in the extraction of drugs and their metabolites from body fluids. In our experiment, we applied polyamide cartridges, mainly used for the separation of compound containing hydroxyl or carbonyl group, to the pretreatment of urine and bile samples, and found it was a very effective tool in offering specificity, sample concentration and matrix reduction.
After oral administration to rats, resveratrol was excreted mainly in the forms of metabolites; this was confirmed by our experiment and other reports (Yu et al., 2002; Wang et al., 2005). In the present study on excretion, the data showed very low urinary and biliary excretion of resveratrol (0.59% and 0.027% of the dose, respectively) after oral administration of PCE to rats.
Table 4. Bile excretion of resveratrol in rat after ig dose of 20 mg/kg PCE extract Time (h) Amount excreted ([mu]g) 1 2 3 4 Mean 0-2 0.41 0.50 0.20 0.28 0.34 2-4 0.28 0.29 0.14 0.36 0.27 4-6 0.13 0.01 0.19 0.14 0.12 6-8 0.01 nd 0.01 0.08 0.03 0-8 0.84 0.79 0.53 0.86 0.75 Total dose ([mu]g) 2240 3110 2816 2816 2745 Excretion (%) 0.04 0.025 0.019 0.03 0.027 nd: not detectable.
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* Corresponding author.
E-mail address: email@example.com (D. Wang).
0944-7113/$-see front matter (C) 2008 Elsevier GmbH. All rights reserved.
doi:10. 1016/j.phymed. 2008.02.009
Donggeng Wang (a),*, Yuerong Xu (b), Wenying Liu (c)
(a) Nan Tong University, Nan Tong 226001, China
(b) Hospital of Hunan Agriculture University, Changsha, Hunan 410128, China
(c) Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, China
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|Author:||Wang, Donggeng; Xu, Yuerong; Liu, Wenying|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Oct 1, 2008|
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