Article pubs.acs.org/JAFC
Pharmacokinetics and Safety of Resveratrol Derivatives in Humans after Oral Administration of Melinjo (Gnetum gnemon L.) Seed Extract Powder Hiroko Tani,*,† Susumu Hikami,† Sanae Iizuna,† Maiko Yoshimatsu,† Takashi Asama,† Hidetaka Ota,§ Yuka Kimura,† Tomoki Tatefuji,† Ken Hashimoto,† and Kazutaka Higaki# †
Institute for Bee Products and Health Science, Yamada Bee Company, Inc., 194 Ichiba, Kagamino-cho, Tomata-gun 708-0393, Japan Department of Geriatric Medicine, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan # Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Okayama University, Kita-ku, Okayama 700-8530, Japan §
ABSTRACT: Fruits and seeds of melinjo (Gnetum gnemon L.) are resveratrol derivative-rich materials. Pharmacokinetics of resveratrol derivatives in healthy volunteers after oral administration of 1000 mg of melinjo seed extract (MSE) powder were assessed and compared with those after oral dosing of trans-resveratrol (tRV) powder containing 4.8 mg of tRV only, equivalent to the content in 1000 mg MSE powder. Plasma tRV concentrations with enzymatic hydrolysis were maintained over 24 h, with a tmax of 12 h and a mean residence time (MRT) of 14 h, 5 and 2 times higher than those for tRV powder intake, respectively. Gnetin C, a resveratrol dimer, with hydrolysis was maintained in plasma for >96 h with a 36 h MRT. With repeated doses once daily for 28 days, plasma tRV and gnetin C concentrations with hydrolysis were in good agreement with the theoretical curves. MSE powder was well tolerated up to the oral dosing of 5000 mg with no serious adverse events. KEYWORDS: melinjo seed extract, resveratrol derivatives, human, plasma, metabolites
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metabolized to sulfates and glucuronides of tRV in humans.15 Although MSE contains a variety of resveratrol derivatives, the pharmacokinetics of tRV and its monomer derivatives such as piceid and isorhapontigenin from melinjo seed has not been reported yet. Furthermore, the most abundant resveratrol derivatives in melinjo seed are resveratrol dimers, and these dimers could play an important role in biological activity. However, there has been no report on the pharmacokinetics of resveratrol dimer derivatives including gnetin C, gnetin L, and glucosides of gnetin C (gnemonosides A, C, and D). Therefore, we investigated the plasma concentration profile of tRV, dihydroresveratrol, isorhapontigenin, gnetin C, and gnetin L after a single oral administration of MSE powder and compared it with administration of tRV powder. In addition, we also examined the persistence of plasma concentrations of tRV and gnetin C after repeated oral administration, which were described by a one-compartment model, and we assessed the safety of MSE powder by blood chemical analysis and examination by a physician. This is the first report on the pharmacokinetics and metabolism of resveratrol monomer and dimer derivatives from melinjo seed in human subjects.
INTRODUCTION The use of plants as a traditional folk medicine has been well recognized from ancient times. A medium-size tree, Gnetum gnemon, which is distributed from Southeast Asia to the Western Pacific region, is commonly called melinjo in Indonesia, and its fruits and seeds are used as ordinary food in Indonesia. Melinjo seed extract (MSE) has been reported to show several pharmacological activities, such as antimicrobial activity,1 inhibition of lipase and amylase,1 modulation of cytokine production,2 antiangiogenesis,3 antimetabolic syndrome,4 and prevention of endothelial senescence.5 Recently, we have also reported that MSE would decrease serum uric acid levels in nonobese Japanese males.6 MSE contains trans-resveratrol (tRV); isorhapontigenin, which is a resveratrol monomer derivative; gnetin C, gnemonosides A, C, and D, and gnetin L, which are resveratrol dimer derivatives; and other derivatives.1,2,7 Some resveratrol derivatives have been reported to have biological activity, including antioxidant activity,1,7 antimicrobial activity,1 inhibition of lipase and amylase,1 modulation of cytokine production,2 antiangiogenesis,3 inhibition of tyrosinase, and melanin biosynthesis.8 Many pharmacokinetic studies, including in vitro, ex vivo, and in vivo studies, have demonstrated that tRV is well absorbed, but quickly eliminated, mainly as sulfates and glucuronides of tRV in the small intestinal lumen and liver.9−13 Furthermore, it is known that tRV is also subject to hydroxylation and/or hydrogenation in humans.13 Dihydroresveratrol is, for example, one of hydrogenated metabolites of tRV and is formed in the intestinal lumen by hydrogenation of a double bond by microflora.11,14 In addition, piceid (tRV 3-O-D-glucoside), a major glycoside form of resveratrol in plants, is absorbed after deglycosylation and is © 2014 American Chemical Society
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MATERIALS AND METHODS
Materials. We obtained tRV (≥99%), trans-piceid (≥97%), sulfatase type H-1 from Helix pomatia (≥10000 units/g, including ≥30000 units/ g β-glucuronidase), β-glucuronidase type H-2 from H. pomatia (85000 Received: Revised: Accepted: Published: 1999
October 27, 2013 January 29, 2014 February 3, 2014 February 3, 2014 dx.doi.org/10.1021/jf4048435 | J. Agric. Food Chem. 2014, 62, 1999−2007
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units/mL, including ≥7500 units/mL sulfatase), S9 mix, NADP, UDPglucuronic acid (UDPGA), Tris-dithiothreitol, MgCl2, and bovine serum albumin from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Isorhapontigenin was purchased from TCI (Tokyo, Japan). The compound cis-resveratrol (≥98%) was obtained from Cayman Chemical Co., Inc. (Ann Arbor, MI, USA). Dihydroresveratrol (≥98%) was purchased from LKT Laboratories, Inc. (St. Paul, MN, USA). The compounds tRV 4′-O-glucuronide, tRV 3-O-glucuronide, and tRV 3sulfate sodium salt were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). β-Glucosidase from almonds (≥2000 units/g) was obtained from Shin Nihon Chemical Industries, Inc. (Aichi, Japan). tRV powder, 720 mg (3.2 mmol) tRV/g, was obtained by extracting tRV from capsules of dietary supplements of resveratrol (Douglas Laboratories, Pittsburgh, PA, USA) with a 10-fold amount of ethanol three times (25 mL × 3). The MSE powder (YMP-M-100109) containing 91% MSE and 9% dextrin was obtained from Yamada Bee Co., Inc. (Kagamino, Japan). The preparation of solutions and all experiments using resveratrol derivatives were performed in dim LED light to avoid the photochemical isomerization of the trans form to the cis form. Contents of Resveratrol Derivatives in tRV and MSE Powder. Analysis of resveratrol derivatives in tRV and MSE powder with and without hydrolysis was performed by the Prominence high-performance liquid chromatography (HPLC) system (Shimadzu) with a Cosmosil 5C18-ARII column (Nacalai Tesque, 4.6 × 250 mm), following the analysis conditions reported by Mark et al.16 with minor modifications. A gradient solvent system of MeOH (20%, 5 min hold; 20−70%, 45 min linear gradient; 100%, 10 min hold; and 20%, 10 min hold) in 0.1% trifluoroacetic acid was used at a flow rate of 1.0 mL/min, and detection was at 320 nm. Enzymatic Hydrolysis of Glucosides in MSE Powder. Hydrolysis of glucosides of resveratrol derivatives in MSE powder was performed following the method reported by La Torre et al.17 with minor modifications. Briefly, a total of 100 mg of β-glucosidase in 1 mL of 100 mM acetic acid was added to MSE powder (10 mg) in 1 mL of 100 mM acetic acid and incubated at 55 °C for 2 h. A volume of 3 mL of acetonitrile was added to the reaction mixture, and after stirring, was centrifuged at 13000g for 15 min at 4 °C. The residue was washed twice with 1 mL of acetonitrile and centrifuged at 13000g for 15 min at 4 °C. The supernatant was collected and evaporated with nitrogen gas and dissolved in 10 mL of 30% aqueous acetonitrile containing 1% acetic acid for HPLC analysis. Extraction and Isolation. The extraction of melinjo was performed as previously described by Kato et al.1 with slight modifications. In brief, the dried endosperms (250 g) were powdered and soaked in 55% ethanol (750 mL) at room temperature for 3 days. The filtrate was evaporated in vacuo to obtain the MSE (23 g). The extract was then purified as previously described1 to obtain gnetin C (≥99%), gnemonoside A (≥99%), gnemonoside C (≥99%), gnemonoside D (≥99%), and gnetin L (≥99%). Gnetin C monoglucuronide was obtained by following the method reported by Otake et al.18 with minor modifications. First, gnetin C (24 mg) was incubated with incubation mixtures containing 250 μL of S9 mix (5 μg of protein), 72 mg of UDPGA, 95 mg of NADP, 570 mg of MgCl2, 185 mg of dithiothreitol, and 480 mg of bovine serum albumin in 480 mL of 50 mM Tris buffer (pH 7.4). After incubation at 37 °C for 24 h, the reactions were terminated by the addition of 480 mL of acetonitrile. The samples were centrifuged at 13000g for 15 min at 4 °C, and supernatants were evaporated. After 6.7 g of residue was dissolved in 36 mL of water, 3 mL of the solution was loaded onto the column (Strata C18-E 55 μm, 70A, 100 mg/3 mL, Shimadzu GLC Ltd., Tokyo, Japan), which was preconditioned with 3 mL of methanol and 3 mL of water. After a washing with 9 mL of methanol, all of the eluates were collected and evaporated. The crude glucuronides (71 mg) were dissolved in 470 mL of methanol and loaded onto a preparative C18 column (Cosmosil 5C-18-AR-II, 20 × 250 mm). The semipreparative HPLC Waters 600E multisolvent delivery system was used with a 200 μL loop (Waters, Milford, MA, USA). The mobile phase consisting of water/trifluoroacetic acid (0.1%) and methanol in the ratio of (45:55, v/v) was
delivered at 7.5 mL/min. The injection volume was 40 μL. The eluent was monitored at 320 nm and the fraction at 23 min was collected. The methanol was evaporated, and the aqueous layer was lyophilized to obtain the glucuronide as a white solid (2.9 mg ≥96%). The structure was confirmed by liquid chromatography−tandem mass spectrometry (LC-MS/MS). Subjects and Study Design. The study design was approved by the Yamada Bee Co. Ethics Committee. All of the subjects gave their written informed consent. Single-Dose Study. Ten healthy volunteers (23−34 years; six men and four women) were divided into two groups: a tRV dose group and an MSE dose group. Two days before the study and during the study, the volunteers were given a diet that was low in polyphenols. Grape and peanut products were excluded. Subjects in the tRV dose group were provided a capsule containing 1.36 mg of tRV powder, 149 mg of dextrin, 29 mg of cellulose, 9 mg of sucrose esters of fatty acids, and 64 mg of gelatin. Subjects in the tRV dose group consumed five capsules after breakfast. Subjects in the MSE dose group were provided a capsule containing 250 mg of MSE powder, 29 mg of cellulose, 9 mg of sucrose esters of fatty acids, and 64 mg of gelatin. Subjects in the MSE dose group consumed four capsules after breakfast. Blood samples were collected at 1, 2, 4, 6, 8, 12, 24, 48, 72, and 96 h after intake of the capsules. The collected blood samples were centrifuged at 1200g for 10 min at ambient temperature. The obtained plasma samples were stored at −80 °C until UPLC-MS/MS analysis. Repeated-Dose Study. In a placebo-controlled study, 44 healthy volunteers (32−49 years; 22 men and 22 women) were divided into four groups: placebo and 1000, 2000, or 5000 mg of MSE powder. In the placebo capsule, 250 mg of dextrin was used instead of 250 mg of MSE powder. All subjects consumed 20 capsules every morning for 28 days in the proportion of placebo and melinjo capsule, 20:0 (placebo group), 16:4 (1000 mg MSE dose group), 12:8 (2000 mg MSE dose group), and 0:20 (5000 mg MSE dose group). During the study, the volunteers were given a diet low in polyphenols. Grape and peanut products were excluded. The portion of plasma samples obtained at 14 and 28 days after the intake of 1000 mg of MSE powder was used for measuring blood levels of resveratrol derivatives. In addition, before and 28 days after the intake of placebo, 1000, 2000, or 5000 mg of MSE powder, clinical laboratory tests, including blood pressure, pulse, body mass index, and blood and urine biochemical parameters, were performed for the volunteers. Sample Preparation for Plasma. For UPLC-MS/MS analysis, 250 μL of human plasma was mixed with 750 μL of cold acetonitrile and centrifuged at 13000g for 15 min at 4 °C. The supernatant was evaporated with nitrogen gas and dissolved in 750 μL of 0.5% citric acid/ acetonitrile (1:3) solution. The solution was loaded onto an SPE column (Superco HybridSPE PPT/30 mg, Sigma-Aldrich, St. Louis, MO, USA), preconditioned with 400 μL of 0.5% citric acid/acetonitrile (1:3) solution. After washing with 0.5% citric acid/acetonitrile solution, the eluate was combined and concentrated by evaporation with nitrogen gas. The pellet was resuspended in 250 μL of 30% aqueous acetonitrile before filtering with a 0.2 μm filter (DISMIC-13HP, Advantec, Tokyo, Japan), and 4 μL of the filtrate was injected onto UPLC-MS/MS. Enzymatic Hydrolysis of Glucuronate and Sulfate Conjugates. The enzymatic cleavage of glucuronate and sulfate conjugate of plasma metabolites was performed as follows. A volume of 250 μL of human plasma was mixed with 750 μL of cold acetonitrile and centrifuged at 13000g for 15 min at 4 °C. The supernatant was evaporated with nitrogen gas and dissolved in 250 μL of water. The solution was mixed with 100 μL of sulfatase type H-1 from H. pomatia (≥10000 units/g, including ≥30000 units/g β-glucuronidase) solution in 0.1 M sodium acetate buffer (pH 4.7). The mixture was incubated at 37 °C in a heating block for 2 h. The reaction was terminated by adding 750 μL of 0.5% citric acid/acetonitrile (1:3) solution. The reaction mixture was centrifuged at 2000g for 5 min at ambient temperature. The supernatant was treated in the same way as that for samples with no hydrolysis, and UPLC-MS/MS analysis was performed. UPLC-MS/MS Analysis. UPLC analysis was performed using an Acquity UPLC (Waters) system. Separation of metabolites was performed at 40 °C using a reversed-phase column (Acquity UPLC 2000
dx.doi.org/10.1021/jf4048435 | J. Agric. Food Chem. 2014, 62, 1999−2007
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Table 1. Mass Parameters for Melinjo Constituents and Their Metabolites mode
precursor (m/z)
cone (V)
product (m/z)
collision (eV)
RT (min)
tRV
compound
ES−
227.3
36.0
143.0 159.0 185.2
25.0 19.0 18.0
7.7
7.9/26
cis-resveratrol
ES−
227.3
36.0
143.0 159.0 185.2
25.0 19.0 18.0
9.2
26/88
dihydroresveratrol
ES−
229.2
30.0
81.0 106.0 123.0
28.0 18.0 16.0
8.1
26/87
piceid
ES−
389.0
20.0
143.0 185.0 227.0
45.0 40.0 22.0
6.2
3.6/12
isorhapontigenin
ES−
257.0
30.0
224.0 241.3
20.0 20.0
8.2
19/65
gnetin C
ES+
455.1
42.0
329.1 361.2 437.2
329.1 361.2 437.2
12.4
1.7/5.7
cis-gnetin C
ES+
455.1
42.0
329.1 361.2 437.2
329.1 361.2 437.2
14.5
gnetin C monoglucuronide
ES+
455.1
42.0
329.1 361.2 437.2
329.1 361.2 437.2
10.1
1.4/16
gnetin L
ES+
485.7
30.0
257.2 361.2 467.4
24.0 20.0 20.0
13.2
0.50/1.7
gnemonoside A
ES−
777.5
50.0
411.3 452.7 615.2
44.0 36.0 24.0
6.3
4.2/14
gnemonoside D
ES−
615.5
50.0
315.5 241.3 453.3
46.0 20.0 24.0
7.9
19/65
BEH C18, 2.1 × 100 mm, 1.7 μm). Injections were carried out with an autosampler maintained at 20 °C. The mobile phase consisted of water and acetonitrile and was pumped at a flow rate of 0.3 mL/min. The gradient system was as follows: 5% acetonitrile (0−2 min), 5−15% acetonitrile (2−7 min), 15−50% acetonitrile (7−27 min), 50−100% acetonitrile (27−31 min), and linear gradient, 100% acetonitrile (31−35 min). There was a 5 min reequilibration period with the initial solvent mixture between analyses. MS/MS was performed on a Q-Premier XE triple-quadrupole mass spectrometer (Waters) equipped with a turbo electrospray ion source (ESI) in the positive and negative modes and a heated curtain gas interface to the high-vacuum region of the mass analyzer. Mass detection and quantification of analytes were accomplished in the multiple reaction monitoring (MRM) mode, and spectra were analyzed by MassLynx Software (Waters). The ESI source was operated in the positive/negative ionization mode at 120 °C, with a desolvation temperature of 400 °C, an 800 L/h desolvation gas flow rate, and a capillary voltage set at 3.34 kV. The extractor and radiofrequency voltage were fixed to 3.0 and 0.1 V, respectively. The cone voltage varied from 20 to 50 V depending on the compound investigated. Argon was
LOD/LOQ (nM)
used as the collision gas at a flow rate of 0.25 mL/min, with collision energies of 16−46 eV. Quantification of tRV, cis-resveratrol, dihydroresveratrol, piceid, isorhapontigenin, gnetin C, gnetin C monoglucuronide, gnetin L, and gnemonosides A, C, and D was performed using the plasma matrix standard curve. The limits of detection (LOD) and limits of quantitation (LOQ) were determined as the analyte concentration that gave a signal-to-noise ratio of 3 and 10, respectively. Optimal conditions, LOD, and LOQ for each compound are shown in Table 1. The retention time of cis-gnetin C was confirmed using gnetin C solutions irradiated with artificial UV light for 0.5 h. Pharmacokinetic Analysis. Maximum plasma concentration (Cmax) and the time to reach Cmax (tmax) were taken directly from observed plasma concentration−time profiles. The area under the plasma concentration time curve (AUC0−96, nM·h) and the area under the first moment curve (AUMC0−96, nM·h2) were calculated by the trapezoidal rule to the last time point, 96 h.19 The mean residence time (MRT, h) was calculated from the following formula:19
MRT = AUMC/AUC 2001
dx.doi.org/10.1021/jf4048435 | J. Agric. Food Chem. 2014, 62, 1999−2007
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Figure 1. Liquid chromatography−mass spectrometry chromatogram of resveratrol derivatives and their major metabolites in plasma: tRV 4′-Oglucuronide (M1), isorhapontigenin monoglucuronide (M2), tRV 3-O-glucuronide (M3), tRV 3-sulfate (M4), isorhapontigenin monosulfate (M5), resveratrol monosulfate (M6), gnetin C monoglucuronides (M7, M8), cis-gnetin C (M9), dihydroresveratrol (M10), and cis-resveratrol (M11) without hydrolysis (A) and with hydrolysis (B) in a subject 8 h after a single oral administration of MSE powder (1000 mg). Plasma concentration (Cp) time profiles of tRV and gnetin C with hydrolysis after a single dose of MSE were analyzed by a onecompartment model with first-order absorption. A curve-fitting study was performed with the least-squares regression program MULTI20 by using the equation19
Cp =
kaFD (e−kelt − e−kat ) Vd(ka − kel)
where ka, kel, F, D, and Vd are the absorption rate constant, elimination rate constant, bioavailability, dose, and distribution volume, respectively. Plasma concentrations after multiple doses of MSE were simulated by using the parameters obtained with the fitting study for the single dose of MSE. Statistical Analysis. Statistical analyses were carried out using JMP for Windows 5 (SAS Institute Japan, Tokyo, Japan). In all of the tests, the criterion for statistical significance was p < 0.05.
Figure 2. Plasma concentration−time curves of tRV with enzymatic hydrolysis in healthy human subjects who received a single dose of MSE powder (1000 mg) or tRV powder (n = 5). Results are expressed as the mean, and the bars indicate the SD.
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RESULTS AND DISCUSSION Identification of Metabolites for Resveratrol Monomer and Dimer Derivatives from MSE Powder. The contents of resveratrol monomer and dimer derivatives in MSE powder were as follows: resveratrol monomers, 1.2 mg/g (5.26 μmol/g) tRV, 5 mg/g (12.8 μmol/g) trans-piceid, 2.0 mg/g (7.75 μmol/g) isorhapontigenin; and dimers, 28 mg/g (61.7 μmol/g) gnetin C, 102 mg/g (131 μmol/g) gnemonoside A, 18 mg/g (29.2 μmol/ g) gnemonoside C, 47 mg/g (76.3 μmol/g) gnemonoside D, 2.4 mg/L (4.95 μmol/g) gnetin L. After enzymatic hydrolysis of glucosides, the content of tRV was 5.4 mg/g (23.7 μmol/g) and those of isorhapontigenin, gnetin C, and gnetin L were 5.6, 150, and 12 mg/g (21.7, 350, and 24.8 μmol/g), respectively.
Blood collected up to 96 h from volunteers who received 1000 mg of MSE powder was used for identification of metabolites of resveratrol monomer and dimer derivatives. Samples were prepared from plasma with and without enzymatic hydrolysis of glucuronide and sulfate. Samples were analyzed by UPLCMS/MS. Figure 1A shows the representative metabolites in plasma after intake of MSE powder. Five conjugates of resveratrol were found as two monoglucuronides (403/227) and two monosulfates (307/227) by MRM. The conjugate pattern of tRV was the same as that after intake of tRV powder intake (data not shown), which was also in good agreement with previous studies.21,22 Two 2002
dx.doi.org/10.1021/jf4048435 | J. Agric. Food Chem. 2014, 62, 1999−2007
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Table 2. Cmax, tmax, AUC, and MRT of Resveratrol Derivatives after Hydrolysis in Plasma Volunteers after a Single Oral Dose of MSE Powdera
a
sample
conjugated metabolite
Cmax (nM)
tmax (h)
AUC1−96h (nM h−1)
MRT (h)
tRV MSE
tRV tRV dihydroresveratrol isorhapontigenin gnetin C gnetin C monoglucuronide gnetin L
473 ± 117 462 ± 314 124 ± 228 262 ± 155 143 ± 84 77.9 ± 44.4 13.1 ± 8.7
2.4 ± 0.9 11.6 ± 7.3* 18.0 ± 10.4 6.7 ± 1.2 16.6 ± 7.5 10.4 ± 8.3 6.8 ± 5.0
3326 ± 470 7401 ± 5774 1371 ± 1770 2962 ± 2010 6977 ± 4097 2511 ± 990 372 ± 412
6.6 ± 1.4 13.7 ± 7.7 6.7 ± 1.2 11.3 ± 7.0 36.3 ± 8.1 36.5 ± 5.8 6.6 ± 1.4
Results are expressed as the mean ± SD of experiments (n = 5). *, P < 0.05, Welch’s test.
the following MRM transitions: 433.0 → 257.0 (loss of a glucuronide from isorhapontigenin) and 337.0 → 257.0 (loss of a sulfate from isorhapontigenin). Moreover, two monoglucuronides of gnetin C were found by the following MRM transitions: 631.0 → 455.1 (loss of glucuronic acid from gnetin C). In this research, the glucosides of gnetin C, such as gnemonosides A, C, and D, were not detected in plasma, suggesting that resveratrol dimers are absorbed after deglucosylation, as is the case in the intake of piceid.15 After enzymatic hydrolysis, most of these conjugates disappeared and tRV, cis-resveratrol, dihydroresveratrol, isorhapontigenin, and gnetin C were released (Figure 1B). However, two peaks of gnetin C glucuronides were not hydrolyzed, but the peak area of gnetin C was increased (Figure 1B). This finding indicated that several metabolites of gnetin C, other than monoglucuronide, remain to be identified. Among the detected conjugates, two monoglucuronides and one sulfate of resveratrol were identified as tRV 4′-Oglucuronide, tRV 3-O-glucuronide, and tRV 3-sulfate by comparing them with their authentic samples. The conjugates of isorhapontigenin and gnetin C were characteristic metabolites of MSE, and their structures need to be elucidated in the future. On the other hand, the dihydro derivatives of gnetin C were not observed after enzymatic hydrolysis, suggesting that gnetin C is not susceptible to hydrogenation by colonic bacteria. Pharmacokinetics of tRV and Its Derivative after Oral Dosing of MSE Powder. Without enzymatic hydrolysis, tRV, piceid, isorhapontigenin, and gnemonosides A, C, and D were not detectable in any of the plasma samples. Among tRV dimer derivatives, gnetin C was only detected (96 h might be due to slow absorption of their glucosides, because they would be absorbed as aglycone after release from glucosides by bacterial microflora as is the case
of tRV. Furthermore, their elimination from plasma might also be mediated via several transporters as described above, and a possible inhibition by metabolites and conjugates of gnetin C and its glucoside might be responsible for their long residence in plasma. However, further studies are required to clarify the reason for this finding. Plasma Persistence of Metabolites and Safety Assessment. In the repeated-dose administration experiment, 1000 mg of MSE powder was administered to subjects once daily after breakfast for 28 days. On days 14 and 28, blood samples were collected at 24 h after taking the dose. In this study, we focused on tRV and gnetin C, because gnetin C was found to be the most abundant dimer in plasma after enzymatic hydrolysis (Figure 3b,c). Plasma concentrations of tRV and gnetin C that were determined after enzymatic hydrolysis are shown with those after a single administration in Figure 4a,b. Following the repeated dosing of MSE powder, a cumulative increase in plasma concentrations of tRV with hydrolysis was not observed (Figure 4a). Although the ratios of plasma tRV concentrations on days 14 and 28 to those at 24 h after the first dose were under unity, the tRV value at 24 h had a large variability, and observed concentrations on days 14 and 28 were not contradictory to the simulation curve calculated on the basis of a onecompartment model with first-order absorption (Figure 4a). The theoretical accumulation ratio calculated with ka and kel values was 1.28, suggesting that during multiple doses of MSE powder, tRV with hydrolysis follows pharmacokinetics similar to those after a single oral administration. We observed that plasma gnetin C concentrations after hydrolysis were increased by multiple dosing of MSE powder, which were in good agreement with the theoretically calculated simulation curve (Figure 4b). However, the accumulation ratio was moderate (2.8), which is also consistent with the theoretical value of 2.33 calculated by ka and kel values. This suggests that plasma gnetin C with hydrolysis during multiple doses follows similar pharmacokinetics to that after a single dose of MSE powder. After intake of MSE powder, the variability in plasma concentrations of tRV and its derivatives with hydrolysis was considerably high in subjects (Figures 2 and 3; Table 2). This finding could be due to the interindividual differences of metabolism and pharmacokinetics of resveratrol derivatives. 2004
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Table 3. Blood Biochemistry Parameters in Healthy Subjects (n = 11) before and after Repeated Oral Administration of Melinjo for 28 Daysa dose of MSE powder days
placebo
1000 mg
2000 mg
5000 mg
ALT (U/L)
0 28
19.3 ± 11 19.7 ± 10.5
22.8 ± 12.4 19.3 ± 10.6
15.5 ± 4.6 17.4 ± 6.7
13.9 ± 6.0 15.0 ± 5.3
γ-GTP (U/L)
0 28
26.9 ± 19.4 26.8 ± 20.1
31.5 ± 17.8 27.8 ± 14.4
17.4 ± 4.9 19.5 ± 8.8
15.5 ± 4.2 14.6 ± 3.5
blood glucose (mg/dL)
0 28
89.8 ± 7.9 93.8 ± 6.3
91.9 ± 8.23 93.4 ± 8.2
92.2 ± 7.9 93.3 ± 8.3
91.9 ± 9.2 91.2 ± 8.5
total protein (g/dL)
0 28
7.42 ± 0.35 7.23 ± 0.34
7.35 ± 0.56 7.35 ± 0.52
7.38 ± 0.28 7.28 ± 0.3
7.37 ± 0.43 7.05 ± 0.42
albumin (g/dL)
0 28
4.52 ± 0.17 4.36 ± 0.17
4.41 ± 0.37 4.37 ± 0.38
4.56 ± 0.16 4.45 ± 0.14
4.45 ± 0.25 4.2 ± 0.28
A/G ratio
0 28
1.56 1.53
1.54 1.49
1.64 1.49
1.54 1.58
LDH (U/L)
0 28
153 ± 27 148 ± 24
148 ± 27 155 ± 26
164 ± 30 158 ± 25
157 ± 22 154 ± 19
total bilirubin (mg/dL)
0 28
0.79 ± 0.36 0.66 ± 0.18
0.77 ± 0.31 0.82 ± 0.34
0.68 ± 0.31 0.78 ± 0.35
0.53 ± 0.19 0.59 ± 0.27
creatinine (g/L)
0 28
1.51 ± 0.82 1.34 ± 0.66
1.28 ± 0.65 1.85 ± 1.09
1.42 ± 0.83 1.88 ± 1.04
1.68 ± 1.30 1.50 ± 1.09
sodium (g/L)
0 28
2.96 ± 1.18 3.22 ± 1.45
3.15 ± 1.48 2.68 ± 1.01
3.28 ± 1.21 3.09 ± 0.91
2.27 ± 1.06 2.72 ± 1.32
potassium (g/L)
0 28
2.66 ± 1.63 1.97 ± 0.75
2.00 ± 1.13 2.97 ± 1.51
2.59 ± 0.96 3.33 ± 1.36
2.15 ± 1.36 2.28 ± 1.15
HS-CRP (mg/dL)
0 28
0.052 ± 0.088 0.044 ± 0.086
0.10 ± 0.28 0.13 ± 0.37
0.022 ± 0.034 0.098 ± 0.17
0.061 ± 0.155 0.021 ± 0.019
chloride (g/L)
0 28
6.14 ± 2.58 5.86 ± 2.65
5.58 ± 2.31 5.48 ± 1.67
6.49 ± 2.14 7.18 ± 1.96
4.42 ± 2.66 5.07 ± 1.99
a Results are expressed as the mean ± SD. ALT, alanine aminotransferase; γ-GTP, γ-glutamyl transpeptidase; AG ratio, albumin/globulin ratio; LDH, lactate dehydrogenase; HS-CRP, high-sensitivity C-reactive protein.
For the safety assessment of MSE powder, in addition to the 1000 mg dosing study, repeated-dose administration experiments were also performed at 0 (placebo), 2000, and 5000 mg for 28 days. Before and after 28 day multiple dosings, subjects were monitored by a physician and evaluated for body weight, heart rate, and blood pressure. Throughout the single and repeated administration experiments, no clinically noteworthy abnormalities were observed. Additionally, blood biochemistry parameters were not significantly different (p > 0.05) by Dunnet’s test for parametric parameters, between before and after each oral administration of 1000, 2000, and 5000 mg of MSE powder (Table 3). This finding suggests that MSE is safe, at least in the short term, such as 1 month, and in an amount consumed in a usual manner. Most studies have focused on tRV in the in vitro experiments, but it has been reported that cis-resveratrol, dihydroresveratrol, resveratrol conjugates, and other metabolites, such as piceatan-
nol, a hydroxylated metabolite of resveratrol, have some biological activity.26−31 Furthermore, pterostilbene, a methoxy derivative of resveratrol, is better absorbed and more bioactive once it is absorbed than tRV in the rat.32 Recently, we reported that gnetin C reduces cell viability of tube-forming human umbilical vein epithelial cells more potently than tRV.3 We also found that a monoglucuronide of gnetin C did not lose xanthine oxidase inhibitory activity.6 In animal experiments, it has been reported that resveratrol metabolites and conjugated resveratrol metabolites were distributed into several tissues and that these metabolites might provide a pool for local or systemic regulation of resveratrol metabolites.33,34 Therefore, the metabolism and distribution of resveratrol derivatives from MSE in several tissues need to be clarified. Further studies are now in progress to elucidate the details of metabolism of resveratrol derivatives from MSE. 2005
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In conclusion, the repeated oral administration of a daily dose of MSE powder was well-tolerated up to 5000 mg/day. Clinical usefulness of MSE powder is likely because of the persistence of resveratrol monomer and dimer conjugates in plasma. However, the relationships between plasma concentrations of these conjugates and biological activity remain unclear.
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AUTHOR INFORMATION
Corresponding Author
*(H.T.) E-mail:
[email protected]. Phone: +81-868-543825. Fax: +81-868-54-3211. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We express gratitude to Dr. E. Kato in Hosoda SHC Co. Ltd. and the Institute of World Health Development of Mukogawa Women’s University for helpful discussions.
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NOTE ADDED AFTER ASAP PUBLICATION There was an error in the fifth paragraph of the Pharmacokinetics of tRV and Its Derivative after Oral Dosing of MSE Powder section in the version of this paper published February 12, 2014. The correct version published February 13, 2014.
2007
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