Absorption and Metabolism of Piceatannol in Rats - Journal of

Article pubs.acs.org/JAFC

Absorption and Metabolism of Piceatannol in Rats Yuko Setoguchi,*,† Yukihiro Oritani,‡ Ryouichi Ito,† Hiroyuki Inagaki,† Hiroko Maruki-Uchida,‡ Takashi Ichiyanagi,§ and Tatsuhiko Ito‡ †

Research Institute, Morinaga & CO., Ltd., 2-1-16 Sachiura, Kanazawa-ku, Yokohama 236-0003, Japan Research Institute, Morinaga & CO., Ltd., 2-1-1 Shimosueyoshi, Tsurumi-ku, Yokohama 230-8504, Japan § Faculty of Environmental Science, Niigata Institute of Technology, 1719 Fujihashi, Kashiwazaki, Niigata 945-1195, Japan ‡

S Supporting Information *

ABSTRACT: Piceatannol (trans-3,3′,4,5′-tetrahydroxystilbene), a natural analogue of resveratrol, has multiple biological functions. Nevertheless, piceatannol’s biological fate is yet to be determined. In this study, we evaluated the absorption and metabolism of piceatannol in rats. Furthermore, the area under the plasma concentration curves (AUC) and metabolic pathway of piceatannol were compared with those of resveratrol. We determined the plasma concentrations of piceatannol, resveratrol, and their respective metabolites following their intragastric administration. Resveratrol metabolites were only conjugates, whereas piceatannol metabolites were piceatannol conjugates, O-methyl piceatannol, and its conjugates. The AUC for piceatannol, resveratrol, and their metabolites increased in a dose-dependent manner (90−360 μmol/kg). The AUC for total piceatannol was less than that for total resveratrol, whereas the AUC for piceatannol (8.6 μmol·h/L) after piceatannol and resveratrol coadministration was 2.1 times greater than that for resveratrol (4.1 μmol·h/L). The greater AUC for piceatannol was a result of its higher metabolic stability. KEYWORDS: piceatannol, resveratrol, absorption, metabolism, passion fruit, stilbene

■

INTRODUCTION Piceatannol (trans-3,3′,4,5′-tetrahydroxystilbene; Figure 1) is a naturally occurring analogue of resveratrol (trans-3,4′,5trihydroxystilbene) that is present in foods such as peanuts (Arachis hypogaea), Vitis amurensis, and Vaccinium berries.1 We found previously that passion fruit (Passif lora edulis) seeds are a useful dietary source because they contain large amounts of piceatannol.2 Piceatannol has beneficial effects that are similar to those of resveratrol,3 including antioxidative, anticancer, antiinflammatory, and SIRT1-activation effects.1,4 In addition, we demonstrated that piceatannol also upregulates endothelial nitric oxide synthase expression,5 exhibits vasorelaxant activity,6 increases collagen synthesis,2 protects the skin from UVB irradiation,7 and inhibits melanogenesis.2

The biological behavior of piceatannol has not been fully investigated.8−10 Piceatannol administered intravenously in the rat was metabolized into a glucuronide conjugate and excreted in urine, whereas its plasma concentration was maintained at a level higher than that of its glucuronide conjugate.8 However, absorption and metabolism of piceatannol following intragastric administration are yet to be examined. Compared to piceatannol, resveratrol has been investigated for its biological behavior in many studies.11−19 These studies indicate that resveratrol is rapidly absorbed, metabolized, and eliminated through feces and urine. Major resveratrol metabolites identified in plasma are various glucuronide and sulfate conjugates. No difference in type of metabolites has been observed between plasma and urine in human,12 and those in rats and pigs were similar.17,19 Dihydroresveratrol, a microbiota-derived metabolite of resveratrol, was the major metabolite found in pig and rat colon contents.17,20 It was also detected, along with its conjugates, in human and pig plasma or urine.11−13,17 The bioavailability of resveratrol was assessed by comparing plasma levels measured following intravenous (15 mg/kg) and oral (50 mg/kg) administration of resveratrol to rats and determined to be 38%.15 The area under the plasma concentration curve (AUC) values for resveratrol were 1/7 and 1/46 of those for glucuronide conjugates after intravenous and oral administration, respectively. The difference in AUC indicates that the small intestine plays an important role in Received: Revised: Accepted: Published:

Figure 1. The chemical structures of stilbenes. © 2014 American Chemical Society

2541

October 17, 2013 February 28, 2014 March 3, 2014 March 13, 2014 dx.doi.org/10.1021/jf404694y | J. Agric. Food Chem. 2014, 62, 2541−2548

Journal of Agricultural and Food Chemistry

Article

administration. Plasma and urine samples were stored at −30 °C until analysis. In the coadministration study, 6 cannulated rats were given a mixture of piceatannol and resveratrol (each at 180 μmol/kg, 44.0 mg/ kg for piceatannol and 41.1 mg/kg for resveratrol) through intragastric administration. Blood samples were collected and plasma samples were stored as described above. Enzymatic Hydrolysis of Conjugated Metabolites in Biological Samples. We hydrolyzed the plasma samples to determine the total amount of piceatannol and resveratrol. The samples were hydrolyzed according to the method of Maeda-Yamamoto et al.,21 but with slight modification. Briefly, 5 μL of β-glucuronidase (15.6 units) and 5 μL of sulfatase (1.3 units) were added to 50 μL of plasma samples diluted with 90 μL of sodium phosphate buffer (final concentration of 27 mmol/L; pH 6.5) and incubated at 37 °C for 45 min. The reaction was stopped with 300 μL of ice-cold acetonitrile (ACN) as described below. Plasma Preparation for High-Performance Liquid Chromatography (HPLC) Analysis. Plasma aliquot samples (50 μL) were extracted with 100 μL of ice-cold ACN and centrifuged at 10000g for 10 min at 4 °C. The supernatant was transferred to another microcentrifuge tube, and the precipitate was washed with 100 μL of ice-cold ACN and then centrifuged. Thereafter, the supernatant was combined with the previous supernatant and evaporated to dryness in vacuo. The residue was redissolved in 50 μL of the initial mobile phase and used for HPLC analysis. Appropriate dilutions were performed when concentrations fell outside the analytical range (0.2−10 μmol/ L). The mean recovery (at 0.2, 0.5, 1, 5, 10, and 50 μmol/L; n = 3) for piceatannol and resveratrol was 95.9% and 95.0%, respectively. HPLC Analysis of Stilbenes. The stilbenes were analyzed using a Shimadzu (Kyoto, Japan) Prominence HPLC system equipped with an SPD-M20A photodiode array detector. The data were processed using LabSolutions chromatography workstation software (version 1.21; Shimadzu). Chromatographic separations were performed on a Mightysil RP-18 GP ODS column (150 × 4.6 mm i.d., 5 μm; Kanto Chemical) equipped with a Mightysil RP-18 GP guard column (5 × 4.6 mm i.d., 5 μm; Kanto Chemical) at 40 °C, with 0.1% (v/v) phosphoric acid/water as mobile phase A, and 0.1% (v/v) phosphoric acid/ACN as mobile phase B at a flow rate of 1 mL/min. Sample aliquots (10 μL) were injected into the HPLC system and were eluted with the following gradient conditions: 0−2 min, 14% B; 2−30 min, 14−30% B. The eluate was monitored at 320 nm by the detector. Calibration curves were prepared by supplementation with known concentrations (0.2, 0.5, 1, 2, 5, and 10 μmol/L) of piceatannol, resveratrol, isorhapontigenin, and rhapontigenin in the initial mobile phase. The correlation coefficients evaluated by linear regression analysis were 0.999 or above. The limit of detection (LOD) and the limit of quantitation (LOQ) were defined as the concentrations of the stilbenes spiked into blank plasma and prepared according to the method described in the previous section that produced a signal-tonoise ratio of at least 3 and 10, respectively. The LOD and LOQ for stilbenes were 0.1 μmol/L and 0.2 μmol/L, respectively. The intraday and interday precision of assay were assessed with two concentrations as quality control samples (0.5 and 10 μmol/L stilbenes in blank plasma) in triplicate, with precision estimates ranging from 1.0 to 9.4%. Identification of Stilbene Metabolites. Major metabolites of piceatannol and resveratrol were fractionated from the rat urine samples using the same HPLC system and column described above. Urine aliquot samples (5 μL) were prepared according to the method used for the plasma samples injected into the HPLC system. Elution was carried out with a mixture of 0.1 mmol/L acetic acid/water and ACN at a flow rate of 1 mL/min at 40 °C. The gradient conditions were as follows: (a) 0−2 min, 10% ACN, and (b) 2−50 min, 10−30% ACN. Each peak was monitored at 320 nm and collected individually. The collected peaks were evaporated to dryness in vacuo and dissolved in 50 μL of 0.1% (v/v) formic acid/10% ACN/water. The fractionated peak aliquots (10 μL) were injected into a Shimadzu Prominence ultrafast liquid chromatography (UFLC) system equipped with a Kinetex 2.6 μm C18 100 Å ODS column

the glucuronidation of resveratrol. Hence, it is important to evaluate the first-pass effect for stilbenes. In this study, we investigated the absorption and metabolism of piceatannol in rats. We then determined the plasma concentration of piceatannol and identified its metabolites following intragastric administration. Furthermore, the AUC values for intact compounds and their respective metabolites were determined following administration of piceatannol and resveratrol, and the metabolic pathways were compared.

■

MATERIALS AND METHODS

Chemicals. Piceatannol (>98.0% purity), isorhapontigenin (>95.0% purity), and rhapontigenin (>98.0% purity) were obtained from Tokyo Chemical Industry (Tokyo, Japan). Resveratrol (>98.0% purity) was obtained from Cayman Chemical (Ann Arbor, MI). Sulfatase Type VIII from abalone entrails (EC 3.1.6.1) and βglucuronidase Type X-A from Escherichia coli (EC 3.2.1.31) were obtained from Sigma (St. Louis, MO). All other reagents were purchased from Wako (Osaka, Japan) and Kanto Chemical (Tokyo, Japan), and were used without any other purification. Animals and Treatments. Eight-week-old male Sprague−Dawley rats (Japan SLC, Shizuoka, Japan) were used in the study. The rats were housed individually in plastic cages in a room with controlled temperature (21−25 °C) and humidity (40−60%). The rats were maintained on a 12 h dark/light cycle with artificial lighting and received AIN-93G (Oriental Yeast, Tokyo, Japan) and water ad libitum. Following a conditioning period that lasted two or three days, the rats were anesthetized with pentobarbital and the right jugular vein was catheterized with silicone tubing (0.5 mm inner diameter (i.d.), 1.0 mm outer diameter). After the catheterization procedure, the rats were allowed a 1 week postoperative recovery period during which they were fed AIN-93G. Following the recovery period, the rats were fasted overnight and were then administered stilbenes. Thereafter, blood samples were collected from the rats. All animal experiments were carried out with strict compliance with the Guidelines for Proper Conduct of Animal Experiments by the Science Council of Japan. The experimental protocols and procedures were approved by the Animal Experimental Committee of Morinaga & CO., Ltd. Experimental Design. Thirty-six cannulated rats were randomly assigned to six equal groups (n = 6) and transferred to plastic cages with stainless wire mesh floor mats. Each group was administered a different dose of piceatannol or resveratrol to evaluate the dose dependency of their absorption. Stilbene doses were based on the previous study by Marier et al.,15 which examined resveratrol bioavailability and estimated a maximum concentration (Cmax) of 6.57 ± 1.55 μmol/L and AUC of 7.1 ± 2.0 μmol·h/L following oral administration of 50 mg/kg (219 μmol/kg). The rats received piceatannol or resveratrol prepared with 50% (v/v) polyethylene glycol 400/saline through direct stomach intubation at doses of 90, 180, or 360 μmol/kg (22.0, 44.0, or 87.9 mg/kg for piceatannol and 20.5, 41.1, or 82.2 mg/kg for resveratrol; 2 mL/kg). Blood samples (0.4 mL) were collected from each rat before piceatannol or resveratrol administration, and at 0.25, 0.5, 1, 2, 4, 8, and 24 h after administration. The samples were collected into heparinized tubes directly from the jugular vein catheter. The rats were transferred to metabolic cages after the 8 h blood collection, where they received AIN-93G. The samples were centrifuged (4 °C, 10 min, 5000g), and 1/10 volume of a stabilizer (0.4% ascorbic acid/0.02% EDTA disodium/80 mmol/L sodium phosphate buffer, pH 3.6) was added to the plasma samples. Urine samples were collected using pipettes when urination was observed during the 8 h blood collection period, and 1/10 volume of the same stabilizer used for plasma samples was added to the urine samples. The urine samples were combined every four hours. After blood collection for 8 h, the rats were placed in metabolic cages and urine samples were collected in plastic tubes containing the stabilizer once between 8 and 12 h, once between 12 and 24 h, and once between 24 and 48 h following stilbene 2542

dx.doi.org/10.1021/jf404694y | J. Agric. Food Chem. 2014, 62, 2541−2548


Article

Figure 2. Representative high-performance liquid chromatography (HPLC) chromatograms of rat plasma samples before and 0.5 h after intragastric administration of 360 μmol of stilbene per kg of body weight: (A) before administration; (B) administration of piceatannol; (C) administration of piceatannol, hydrolyzed with enzymes; (D) administration of resveratrol; (E) administration of resveratrol, hydrolyzed with enzymes. (100 × 2.1 mm i.d.; Phenomenex, Torrance, CA). Elution was carried out at 40 °C, with 0.1% (v/v) formic acid/water as mobile phase A, and 0.1% (v/v) formic acid/ACN as mobile phase B; both were carried out with a flow rate of 0.2 mL/min. The gradient conditions were as follows: (a) 0−2 min, 10% B, and (b) 2−40 min, 10−25% B. The eluate was monitored at 320 nm by using a Shimadzu SPD-M20A. Mass spectrometry analysis was performed using a 3200 QTRAP system (AB SCIEX, Framingham, MA) with an electrospray ionization source. Electrospray ionization tandem mass spectrometry was performed in negative polarity mode with the following settings: curtain gas, 40 psi; nebulizer gas, 50 psi; turbo gas, 80 psi; capillary temperature, 600 °C; ion spray voltage, −4.5 kV; declustering potential, −40 V; and collision energy, −30 V. The full-scan mass spectrum to obtain product ion of the metabolite was collected in the mass range from m/z 100 to 1000. Data Analysis. Experimental values are expressed as the mean ± SEM. The AUC was calculated using the linear trapezoidal method.

The ratio of intact (unmetabolized) compound to total detected signal was calculated by dividing the AUC corresponding to the peak of the intact compound by the total AUC (sum of intact and all metabolites). Linear regression analysis was used to assess the association between the dose and plasma AUC. Statistical significance of differences in mean values between AUC values for piceatannol and resveratrol was evaluated using 2-way analysis of variance (ANOVA) in the individual administration study, and was evaluated by paired sample t-test in the coadministration study. Repeated-measures ANOVA was used to evaluate the time-dependent variation in plasma concentrations between piceatannol and resveratrol for the coadministration study. Student’s t-test was used to evaluate the differences in mean AUC values between individual administration and coadministration. Differences were considered statistically significant when p < 0.05. All statistical analyses were performed using SPSS version 13.0 J for Windows (SPSS, Tokyo, Japan). 2543



■

Article

RESULTS Identification of Piceatannol Metabolites. Figure 2 shows representative HPLC chromatograms of rat plasma samples before and after intragastric administration of piceatannol or resveratrol. No remarkable peak was observed in the plasma before administration (Figure 2A), whereas piceatannol (P5) and several metabolite peaks were detected following piceatannol administration (Figure 2B). Piceatannol and P9 markedly increased together, whereas several peaks (P1, P2, P3, P4, P6, and P7) disappeared when the plasma was hydrolyzed with enzymes (Figure 2C). Moreover, P10 was newly produced after plasma hydrolysis. Resveratrol was undetected in the plasma of any rat that received piceatannol. Following resveratrol administration, a resveratrol peak (R3) was detected in rat plasma with two remarkable hydrophilic peaks (R1 and R2; Figure 2D). When the plasma was hydrolyzed with enzymes, R1 disappeared and R2 only slightly decreased, but resveratrol increased dramatically (Figure 2E). Piceatannol, isorhapontigenin, and rhapontigenin were undetected in the plasma of rats that received resveratrol. The peaks indicating the metabolites found in rat plasma (P1−P10, R1−R3) were also observed in rat urine (Figure S1 in the Supporting Information); thus, we fractionated the metabolites from urine and identified their mass spectra (Figure S2 in the Supporting Information). Table 1 summarizes the

piceatannol. Based on the retention time and the mass spectrum, P9 was identified as isorhapontigenin (3,4′,5trihydroxy-3′-methoxystilbene; Figure 1) and P10 was identified as rhapontigenin (3,3′,5-trihydroxy-4′-methoxystilbene). In addition to the nine metabolites, other peaks speculated to be piceatannol metabolites were observed in small amounts, but the mass spectra were unidentified. The mass spectra of R1 and R2 fractionated from rat urine after resveratrol administration confirmed that R1 and R2 were conjugate metabolites of resveratrol. Plasma Concentration Profiles of Stilbenes. Figure 3 shows the time-dependent plasma concentration profiles for piceatannol and its metabolites following piceatannol administration. Piceatannol and isorhapontigenin were detected in rat plasma, whereas rhapontigenin was undetected at any dose (Figure 3A). Piceatannol reached the Cmax within 15 min of administration, with the measured Cmax of 3.3 μmol/L following the 90 μmol/kg dose, 7.5 μmol/L following 180 μmol/kg, and 8.1 μmol/L following 360 μmol/kg. Piceatannol concentration for any dose subsequently declined over time to an undetectable level at 24 h. Isorhapontigenin showed a profile similar to that of piceatannol until 1 h after administration, but at lower concentrations. Isorhapontigenin concentration was almost constant between 1 and 8 h, and trace amounts of isorhapontigenin were detected at 24 h only following 360 μmol/kg administration. The plasma concentration of piceatannol and isorhapontigenin were increased with elevating doses, except the Cmax of piceatannol between the doses of 180 and 360 μmol/kg. Conjugate metabolites of intact and O-methyl piceatannol were detected in rat plasma after piceatannol administration (Figure 3B). Since P8 was not hydrolyzed, its levels were quantified using the calibration curve for piceatannol and are shown as O-methyl piceatannol-monosulfate in Figure 3B. Piceatannol conjugates were the dominant metabolites in plasma up to 2 h after 90 and 180 μmol/kg administration and up to 4 h after 360 μmol/kg administration, but could not be detected at 24 h. Conversely, O-methyl piceatannol conjugates were still detectable at 24 h, except for isorhapontigenin conjugates following 90 μmol/kg administration, and O-methyl piceatannol-monosulfate following 90 and 180 μmol/kg administration. The plasma concentration of conjugates of intact and O-methyl piceatannol was increased with elevating doses. Plasma concentration reached the Cmax within 15 min of resveratrol administration, with the Cmax determined to be 3.8, 3.5, and 5.0 μmol/L following administration of 90, 180, and 360 μmol/kg, respectively (Figure S3 in the Supporting Information). Although the concentration of resveratrol declined within 2 h, secondary peaks appeared at 4 h and were notable following 360 μmol/kg administration, and very small following 180 μmol/kg administration. The plasma concentrations of resveratrol conjugates, including that of R2 quantified using the calibration curve for resveratrol, were much higher than the concentrations of the intact resveratrol. In the analysis of conjugates, notable secondary peaks appeared following 360 μmol/kg resveratrol administration, and, with lower peak AUC, following 180 μmol/kg administration. The concentration of resveratrol and its conjugates tended to increase with higher doses of resveratrol. The AUC for piceatannol and resveratrol increased in a dosedependent manner (Table 2). The correlation between AUC and the administered dose was well described by a linear

Table 1. Mass Spectra of Metabolites of Piceatannol and Resveratrol peak

tRa

P1 P2 P3

5.1 6.9 8.9

P4 P5 P6

11.0 14.5 15.2

P7

17.5

P8

22.5

P9 P10 R1 R2 R3

24.2 26.3 11.8 18.1 20.1

a

compound piceatannol-diglucuronide piceatannol-monoglucuronide O-methyl piceatannolmonoglucuronide piceatannol-monoglucuronide piceatannol O-methyl piceatannolmonoglucuronide O-methyl piceatannolmonoglucuronide O-methyl piceatannolmonosulfate isorhapontigenin rhapontigenin resveratrol-monoglucuronide resveratrol-monosulfate resveratrol

parent and product ion pairs (m/z) 595/419/243 419/243 433/257 419/243 243 433/257 433/257 337/257 257 257 403/227 307/227 227

HPLC retention time.

mass spectra for the piceatannol and resveratrol metabolites fractionated from rat urine. As the table indicates, various piceatannol conjugate metabolites were identified. Based on the mass spectra and the disappearance of the peaks after hydrolysis, P1 (m/z 595/419/243) was identified as a diglucuronide metabolite of piceatannol, and both P2 (m/z 419/243) and P4 (m/z 419/243) were identified as monoglucuronide metabolites of piceatannol. Likewise, P3 (m/z 433/257), P6 (m/z 433/257), and P7 (m/z 433/257) were identified as O-methyl piceatannol-monoglucuronide metabolites. Although P8 was not hydrolyzed by sulfatase, P8 was considered an O-methyl piceatannol-monosulfate metabolite based on the mass spectrum (m/z 337/257). The mass spectra for P9 and P10 were similar to that for O-methyl 2544



Article

Figure 3. Plasma concentration profiles for piceatannol and its metabolites after intragastric administration of 90−360 μmol of piceatannol per kg of body weight: (A) aglycon and (B) conjugates. Values are expressed as mean ± SEM (n = 6).

regression model, with the coefficients of determination (R2) for piceatannol and resveratrol estimated as 0.770 and 0.907, respectively. Some correlation between the administered dose and AUC of piceatannol conjugates was detected (R2 = 0.486), with the AUC increasing with higher doses. The relationship of the AUC with dose for O-methyl piceatannol metabolites, resveratrol conjugates, and total piceatannol and resveratrol was good, with R2 ranging from 0.688 to 0.945. The AUC values for piceatannol were 2.1- to 2.6-fold higher than those for resveratrol, with statistically significant differences found in all comparisons, except for administration of 90 μmol/kg. The total AUC calculated following piceatannol administration was significantly lesser than the AUC calculated for resveratrol. The ratio of intact compound to the total AUC for piceatannol was 3.7- to 4.3-fold higher than the ratio for resveratrol (p < 0.05). Comparison between Piceatannol and Resveratrol Plasma Concentrations after Coadministration. Plasma concentration was compared between piceatannol and resveratrol after administering a mixture of these two stilbenes (each at 180 μmol/kg). The Cmax of piceatannol was 4.2 μmol/ L at 15 min, whereas the Cmax of resveratrol was 1.6 μmol/L at 30 min (Figure 4A). Piceatannol concentration was maintained at a level higher than that of resveratrol for up to 8 h following administration. Furthermore, the AUC for piceatannol was 2.1fold greater than that for resveratrol (p < 0.05; Table 3). In contrast, the total concentration corresponding to piceatannol was less than that associated with resveratrol administration, with statistically significant differences detected at 0.25, 0.5, and 2 h after administration (Figure 4B). Furthermore, the AUC for total piceatannol was significantly less than that for resveratrol. The intact ratio for piceatannol was 3.0-fold higher than that for resveratrol (p < 0.05). The statistically significant differences in AUC values between individual administration and coadminis-

tration were detected only for conjugates of isorhapontigenin and rhapontigenin, whereas no significant differences in AUC values of intact compounds or of other metabolites of piceatannol and resveratrol were observed.

■

DISCUSSION The focus of our research was to evaluate absorption and metabolism of piceatannol following intragastric administration in rats. Roupe et al. described that only a glucuronide metabolite was evident in rat plasma after intravenous administration of piceatannol.8 In our study, a monoglucuronide metabolite (P2) was the most abundant metabolite following intragastric administration of piceatannol. However, a number of additional metabolites were also observed in plasma. The difference in the diversity of metabolites identified between the study of Roupe et al. and the current study may be due to differences in the administration routes or, more likely, due to different analytical methods used. In addition to three conjugate metabolites (P1, P2, and P4), four O-methyl piceatannol conjugates (P3, P6, P7, and P8) and isorhapontigenin (P9) were identified by liquid chromatography−tandem mass spectrometry (Table 1). In contrast to piceatannol, methylated metabolites were not found with resveratrol. The occurrence of methylation only in piceatannol indicates that the catechol ring of piceatannol was metabolized by catechol-Omethyltransferase, as in the case of flavonoids.22−24 The disappearance of O-methyl piceatannol conjugates with plasma hydrolysis was accompanied by an increase in isorhapontigenin and the appearance of rhapontigenin. Therefore, the O-methyl piceatannol conjugates (P3, P6, P7, and P8) were considered conjugates of isorhapontigenin and rhapontigenin. The nine piceatannol metabolites shown in Table 1 indicate the major metabolic pathway for piceatannol, involving glucuronidation, 2545



Article

Table 2. Cumulative Plasma AUCs after Intragastric Administration of Piceatannol and Resveratrol to Rats AUC0−8hb (μmol·h/L) dose (μmol/kg)

piceatannol

resveratrol

intact

90 180 360

4.3 ± 0.3 12.3 ± 1.8 20.6 ± 2.2

conjugates

90 180 360

isorhapontigenin

coefficient of determinationd

statistical significance between stilbenese

piceatannol

resveratrol

main effect

simple main effect

2.0 ± 0.2 4.7 ± 0.4 8.6 ± 0.4

0.770

0.907

− + +

11.3 ± 1.1 19.5 ± 1.7 30.2 ± 5.8

37.6 ± 1.6 79.6 ± 4.9 118.8 ± 4.1

0.486

0.938

+ + +

90 180 360

1.7 ± 0.2 4.2 ± 0.3 7.4 ± 0.6

ndc nd nd

0.858

isorhapontigenin conjugates

90 180 360

1.9 ± 0.2 4.3 ± 0.3 8.3 ± 1.3

nd nd nd

0.688

rhapontigenin

90 180 360

nd nd nd

nd nd nd

rhapontigenin conjugates

90 180 360

3.3 ± 0.3 8.1 ± 0.6 12.4 ± 1.3

nd nd nd

0.796

O-methyl piceatannol-monosulfate

90 180 360

1.0 ± 0.1 3.0 ± 0.1 4.7 ± 0.4

nd nd nd

0.885

total

90 180 360

23.5 ± 1.6 51.4 ± 1.4 83.5 ± 9.6

39.6 ± 1.7 84.3 ± 5.0 127.4 ± 4.1

0.788

0.945

+ + +

intact ratioa (%)

90 180 360

18.6 ± 1.3 23.9 ± 3.3 25.1 ± 1.4

5.1 ± 0.5 5.6 ± 0.5 6.8 ± 0.4

+

a

Intact ratio was calculated by dividing the AUC corresponding to the peak of the intact compound by the total AUC, and expressed as a percentage. Values are expressed as mean ± SEM (n = 6). cNot detected. dLinear regression analysis was used to assess the association between the stilbene dose and plasma AUC. The analysis demonstrated a statistically significant correlation between the dose and AUC for all compounds (p < 0.05). e Statistical significance of differences in mean values between AUC values for stilbenes was assessed using 2-way ANOVA. When a significant interaction was detected (p < 0.05), simple main effect was analyzed for each dose. +, significant difference; −, no significant difference. b

Figure 4. Plasma concentration profiles of piceatannol and resveratrol after intragastric coadministration at 180 μmol per kg of body weight: (A) intact stilbene and (B) total stilbene. Values are expressed as mean ± SEM (n = 6). *Statistically significant differences (p < 0.05) between stilbenes were evaluated by using repeated-measures ANOVA.

2546



Article

vivo and in vitro conditions. Similar disparities were observed with apigenin metabolism between in vivo and in vitro studies.31 Therefore, we conclude that the piceatannol measured in rat plasma in the present study was from the administered dose, and not a resveratrol metabolite. Our second concern was that, in some cases, the bioavailability of polyphenols was affected by other compounds ingested simultaneously, and the AUCs for these polyphenols were higher than expected.32−34 However, in our study, the AUC values for intact and total piceatannol or resveratrol in the coadministration study were similar to those in the individual administration study, and no statistically significant difference was observed between the administration methods. These results indicate that coadministration of piceatannol and resveratrol did not affect the absorption and metabolism of either stilbene. Hence, piceatannol was absorbed at levels that were 2-fold higher than those for resveratrol in the intact form. This finding predicts strong biological activity for piceatannol. In contrast, the total amount of piceatannol absorbed through the gastrointestinal tract into blood circulation (AUC for total) was significantly lower than that for resveratrol. Therefore, these findings indicate that piceatannol is more stable than resveratrol in metabolism. In conclusion, we determined that the metabolic pathway for piceatannol is more complicated than that for resveratrol; piceatannol metabolism involves conjugation, methylation, or both. Furthermore, we showed that the AUC for intact piceatannol was higher than that of intact resveratrol. A greater AUC for intact piceatannol likely contributes to its important biological functions. Further studies examining the biological fate of piceatannol are required to determine its efficacy, and to determine its biological activity following administration.

Table 3. Cumulative Plasma AUCs after Intragastric Coadministration of Piceatannol and Resveratrol to Rats AUC0−8hb (μmol h/L) intact conjugates isorhapontigenin isorhapontigenin conjugates rhapontigenin rhapontigenin conjugates O-methyl piceatannol-monosulfate total intact ratioa (%)

piceatannol

resveratrol

8.6 ± 1.3 a 18.8 ± 1.8 a 4.7 ± 0.6 6.4 ± 0.8 nd 10.2 ± 0.7 3.0 ± 0.4 51.7 ± 4.4 a 16.3 ± 1.7 a

4.1 ± 1.0 b 70.2 ± 5.8 b ndc nd nd nd nd 74.3 ± 6.0 b 5.5 ± 1.3 b

a

Intact ratio was calculated by dividing the AUC corresponding to the peak of the intact compound by the total AUC, and expressed as a percentage. bValues are expressed as mean ± SEM (n = 6). Different letters (a, b) in the same row indicate significant differences (p < 0.05) determined using paired sample t-test. cNot detected.

sulfation, and methylation. Compared to piceatannol, only two conjugate metabolites (R1 and R2) were detected in the plasma of the rats that received resveratrol. In recent studies, dihydroresveratrol and its conjugates, which are microbiotaderived metabolites, were detected in rat liver, adipose tissue, skeletal muscle, and the colon content following repeated oral administration of 30 and 60 mg/kg resveratrol,18,20 but not in the plasma.20 We acquired HPLC chromatograms at 276 nm, the maximal absorbance for quantification of dihydroresveratrol, but no noticeable peak corresponded to a dihydro metabolite of resveratrol. It was not possible at this point to explore the possible presence or absence of the dihydroresveratrol metabolite in rat plasma, because dihydroresveratrol standard was not available. Even though unidentified metabolites of resveratrol were present in the plasma in our study, piceatannol could conceivably have a more complicated metabolic pathway due to the presence of a catechol ring, which enables methylation and increases the number of pathways available for its metabolism. Further studies are required to elucidate piceatannol metabolism. A remarkable finding was that isorhapontigenin was detected in the intact form as a piceatannol metabolite in rat plasma, whereas, similar to flavonoids,22,23 rhapontigenin was found only in conjugated forms. Isorhapontigenin has antioxidant, antiarteriosclerosis, and anticancer effects.25−27 In particular, isorhapontigenin promotes inhibition of cyclooxygenase-1 and cyclooxygenase-2, whereas piceatannol does not.27 Therefore, piceatannol can exhibit additional biological functions when it is metabolized to isorhapontigenin. The AUCs for piceatannol and resveratrol increased in a dose-dependent manner after their intragastric administration (Table 2). These results indicate that stilbene absorption was not saturated with doses that ranged between 90 and 360 μmol/kg. Therefore, piceatannol and resveratrol were simultaneously administered at 180 μmol/kg. We had two concerns with the coadministration study. First, previous studies that examined resveratrol metabolism in vitro or in athymic mice found that resveratrol was metabolized to piceatannol.28−30 In the present study, we did not detect piceatannol in rat plasma or urine after resveratrol administration. The reasons for the disagreement between our results and those of the previous study are unclear. We suspect the disparity might result from the differences between rats and athymic mice, or between in

■

ASSOCIATED CONTENT

S Supporting Information *

Figure S1: Representative HPLC chromatograms of rat urine samples after intragastric administration of 360 μmol/kg stilbene. Figure S2: Representative liquid chromatography− tandem mass spectrometry chromatograms and full scan product ion mass spectra of fractionated rat urine samples after intragastric administration of 360 μmol/kg stilbene. Figure S3: Plasma concentration profiles for resveratrol and its metabolites after intragastric administration of 90−360 μmol/ kg resveratrol. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Tel: +81-45-791-7670. Fax: +81-45-791-7675. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors thank Professor Yukihiro Nakabou of Kawasaki University of Medical Welfare and Professor Yasuhiro Kido of Kyoto Prefectural University for their technical guidance with catheterization, and Dr. Eisaku Nishimura and Masahiko Sai of Morinaga CO., Ltd., for comments on this paper. 2547



■

Article

García-Conesa, M. T.; Espín, J. C. Metabolites and tissue distribution of resveratrol in the pig. Mol. Nutr. Food Res. 2011, 55, 1154−1168. (18) Andres-Lacueva, C.; Macarulla, M. T.; Rotches-Ribalta, M.; Boto-Ordóñez, M.; Urpi-Sarda, M.; Rodríguez, V. M.; Portillo, M. P. Distribution of resveratrol metabolites in liver, adipose tissue, and skeletal muscle in rats fed different doses of this polyphenol. J. Agric. Food Chem. 2012, 60, 4833−4840. (19) Wenzel, E.; Soldo, T.; Erbersdobler, H.; Somoza, V. Bioactivity and metabolism of trans-resveratrol orally administered to Wistar rats. Mol. Nutr. Food Res. 2005, 49, 482−494. (20) Alfaras, I.; Juan, M. E.; Planas, J. M. trans-Resveratrol reduces precancerous colonic lesions in dimethylhydrazine-treated rats. J. Agric. Food Chem. 2010, 58, 8104−8110. (21) Maeda-Yamamoto, M.; Ema, K.; Tokuda, Y.; Monobe, M.; Tachibana, H.; Sameshima, Y.; Kuriyama, S. Effect of green tea powder (Camellia sinensis L. cv. Benifuuki) particle size on O-methylated EGCG absorption in rats; The Kakegawa Study. Cytotechnology 2011, 63, 171−179. (22) Piskula, M. K.; Terao, J. Accumulation of (-)-epicatechin metabolites in rat plasma after oral administration and distribution of conjugation enzymes in rat tissues. J. Nutr. 1998, 128, 1172−1178. (23) Graf, B. A.; Mullen, W.; Caldwell, S. T.; Hartley, R. C.; Duthie, G. G.; Lean, M. E. J.; Crozier, A.; Edwards, C. A. Disposition and metabolism of [2−14C]quercetin-4′-glucoside in rats. Drug Metab. Dispos. 2005, 33, 1036−1043. (24) Chen, Z.; Chen, M.; Pan, H.; Sun, S.; Li, L.; Zeng, S.; Jiang, H. Role of catechol-O-methyltransferase in the disposition of luteolin in rats. Drug Metab. Dispos. 2011, 39, 667−674. (25) Wang, Q. L.; Lin, M.; Liu, G. T. Antioxidative activity of natural isorhapontigenin. Jpn. J. Pharmacol. 2001, 87, 61−66. (26) Liu, Y.; Liu, G. Isorhapontigenin and resveratrol suppress oxLDL-induced proliferation and activation of ERK1/2 mitogenactivated protein kinases of bovine aortic smooth muscle cells. Biochem. Pharmacol. 2004, 67, 777−785. (27) Lee, D.; Cuender, M.; Vigo, J. S.; Graham, J. G.; Cabieses, F.; Fong, H. H. S.; Pezzuto, J. M.; Kinghorn, A. D. A novel cyclooxygenase-inhibitory stilbenolignan from the seeds of Aiphanes aculeata. Org. Lett. 2001, 3, 2169−2171. (28) Niles, R. M.; Cook, C. P.; Meadows, G. G.; Fu, Y. M.; McLaughlin, J. L.; Rankin, G. O. Resveratrol is rapidly metabolized in athymic (Nu/Nu) mice and does not inhibit human melanoma xenograft tumor growth. J. Nutr. 2006, 136, 2542−2546. (29) Potter, G. A.; Patterson, L. H.; Wanogho, E.; Perry, P. J.; Butler, P. C.; Ijaz, T.; Ruparelia, K. C.; Lamb, J. H.; Farmer, P. B.; Stanley, L. A.; Burke, M. D. The cancer preventative agent resveratrol is converted to the anticancer agent piceatannol by the cytochrome P450 enzyme CYP1B1. Br. J. Cancer 2002, 86, 774−778. (30) Zhu, Y.; Chiang, H.; Zhou, J.; Xie, F.; Kissinger, P. T. In vitro metabolism study of resveratrol and identification and determination of its main metabolite piceatannol by LC/EC and LC/MS/MS. Asian J. Drug Metab. Pharmacokinet. 2005, 5, 49−54. (31) Gradolatto, A.; Basly, J. P.; Berges, R.; Teyssier, C.; Chagnon, M. C.; Siess, M. H.; Canivenc-Lavier, M. C. Pharmacokinetics and metabolism of apigenin in female and male rats after a single oral administration. Drug Metab. Dispos. 2005, 33, 49−54. (32) Johnson, J. J.; Nihal, M.; Siddiqui, I. A.; Scarlett, C. O.; Bailey, H. H.; Mukhtar, H.; Ahmad, N. Enhancing the bioavailability of resveratrol by combining it with piperine. Mol. Nutr. Food Res. 2011, 55, 1−8. (33) Matsumoto, H.; Ito, K.; Yonekura, K.; Tsuda, T.; Ichiyanagi, T.; Hirayama, M.; Konishi, T. Enhanced absorption of anthocyanins after oral administration of phytic acid in rats and humans. J. Agric. Food Chem. 2007, 55, 2489−2496. (34) Chen, L.; Lee, M. J.; Li, H.; Yang, C. S. Absorption, distribution, and elimination of tea polyphenols in rats. Drug Metab. Dispos. 1997, 25, 1045−1050.

ABBREVIATIONS USED AUC, area under the plasma concentration curve; i.d., inner diameter; Cmax, maximum concentration; ACN, acetonitrile; ANOVA, analysis of variance; HPLC, high-performance liquid chromatography; LOD, limit of detection; LOQ, limit of quantitation; R2, coefficient of determination; UFLC, ultrafast liquid chromatography

■

REFERENCES

(1) Piotrowska, H.; Kucinska, M.; Murias, M. Biological activity of piceatannol: Leaving the shadow of resveratrol. Mutat. Res. 2012, 750, 60−82. (2) Matsui, Y.; Sugiyama, K.; Kamei, M.; Takahashi, T.; Suzuki, T.; Katagata, Y.; Ito, T. Extract of passion fruit (Passif lora edulis) seed containing high amounts of piceatannol inhibits melanogenesis and promotes collagen synthesis. J. Agric. Food Chem. 2010, 58, 11112− 11118. (3) Smoliga, J. M.; Baur, J. A.; Hausenblas, H. A. Resveratrol and healthA comprehensive review of human clinical trials. Mol. Nutr. Food Res. 2011, 55, 1129−1141. (4) Howitz, K. T.; Bitterman, K. J.; Cohen, H. Y.; Lamming, D. W.; Lavu, S.; Wood, J. G.; Zipkin, R. E.; Chung, P.; Kisielewski, A.; Zhang, L. L.; Scherer, B.; Sinclair, D. A. Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature 2003, 425, 191−196. (5) Kinoshita, Y.; Kawakami, S.; Yanae, K.; Sano, S.; Uchida, H.; Inagaki, H.; Ito, T. Effect of long-term piceatannol treatment on eNOS levels in cultured endothelial cells. Biochem. Biophys. Res. Commun. 2013, 430, 1164−1168. (6) Sano, S.; Sugiyama, K.; Ito, T.; Katano, Y.; Ishihata, A. Identification of the strong vasorelaxing substance scirpusin B, a dimer of piceatannol, from passion fruit (Passiflora edulis) seeds. J. Agric. Food Chem. 2011, 59, 6209−6213. (7) Maruki-Uchida, H.; Kurita, I.; Sugiyama, K.; Sai, M.; Maeda, K.; Ito, T. The protective effects of piceatannol from passion fruit (Passif lora edulis) seeds in UVB-irradiated keratinocytes. Biol. Pharm. Bull. 2013, 36, 845−849. (8) Roupe, K. A.; Yáñez, J. A.; Teng, X. W.; Davies, N. M. Pharmacokinetics of selected stilbenes: rhapontigenin, piceatannol and pinosylvin in rats. J. Pharm. Pharmacol. 2006, 58, 1443−1450. (9) Miksits, M.; Maier-Salamon, A.; Vo, T. P. N.; Sulyok, M.; Schuhmacher, R.; Szekeres, T.; Jäger, W. Glucuronidation of piceatannol by human liver microsomes: major role of UGT1A1, UGT1A8 and UGT1A10. J. Pharm. Pharmacol. 2010, 62, 47−54. (10) Miksits, M.; Sulyok, M.; Schuhmacher, R.; Szekeres, T.; Jäger, W. In-vitro sulfation of piceatannol by human liver cytosol and recombinant sulfotransferases. J. Pharm. Pharmacol. 2009, 61, 185− 191. (11) Wenzel, E.; Somoza, V. Metabolism and bioavailability of transresveratrol. Mol. Nutr. Food Res. 2005, 49, 472−481. (12) Cottart, C. H.; Nivet-Antoine, V.; Laguillier-Morizot, C.; Beaudeux, J. L. Resveratrol bioavailability and toxicity in humans. Mol. Nutr. Food Res. 2010, 54, 7−16. (13) Walle, T. Bioavailability of resveratrol. Ann. N.Y. Acad. Sci. 2011, 1215, 9−15. (14) Amri, A.; Chaumeil, J. C.; Sfar, S.; Charrueau, C. Administration of resveratrol: What formulation solutions to bioavailability limitations? J. Controlled Release 2012, 158, 182−193. (15) Marier, J. F.; Vachon, P.; Gritsas, A.; Zhang, J.; Moreau, J. P.; Ducharme, M. P. Metabolism and disposition of resveratrol in rats: Extent of absorption, glucuronidation, and enterohepatic recirculation evidenced by a linked-rat model. J. Pharmacol. Exp. Ther. 2002, 302, 369−373. (16) Cottart, C. H.; Nivet-Antoine, V.; Beaudeux, J. L. Review of recent data on the metabolism, biological effects, and toxicity of resveratrol in humans. Mol. Nutr. Food Res. 2014, 58, 7−21. (17) Azorín-Ortuño, M.; Yáñez-Gascón, M. J.; Vallejo, F.; Pallarés, F. J.; Larrosa, M.; Lucas, R.; Morales, J. C.; Tomás-Barberán, F. A.; 2548


Absorption and Metabolism of Piceatannol in Rats - Journal of

Recommend Documents