Article pubs.acs.org/JAFC
Tissue Distribution, Excretion, and Metabolic Profile of Dihydromyricetin, a Flavonoid from Vine Tea (Ampelopsis grossedentata) after Oral Administration in Rats Li Fan,†,⊥ Qing Tong,†,⊥ Weiwei Dong,§ Guangjie Yang,† Xiaolong Hou,† Wei Xiong,† Chunyang Shi,† Jianguo Fang,† and Wenqing Wang*,† †
Department of Pharmacy, Tongji Hospital Affiliated with Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China § Wuhan Institute for Drug and Medical Device Control, Wuhan 430075, China S Supporting Information *
ABSTRACT: Dihydromyricetin (DMY), a flavanonol compound found as the most abundant and bioactive constituent in vine tea (Ampelopsis grossedentata), possesses numerous biological activities. In the present study, an HPLC-MS/MS method for the determination of DMY in tissues, urine, and feces was developed and applied to the tissue distribution and excretion study after oral administration in rats, and the metabolic profile of DMY was further investigated using UPLC-QTOF-MS. The results indicated that DMY could be distributed rapidly in various tissues and highly in the gastrointestinal tract. The elimination of DMY occurred rapidly as well, and most unconverted forms were excreted in feces. A total of eight metabolites were identified in urine and feces, while metabolites were barely found in plasma. The predicted metabolic pathways including reduction, dehydroxylation, methylation, glucuronidation, and sulfation were proposed. The present findings may provide the theoretical basis for evaluating the biological activities of DMY and will be helpful for its future development and application. KEYWORDS: dihydromyricetin, tissue distribution, excretion, metabolic profile
■
in rats,21 and the gastrointestinal stability of DMY in vitro was investigated as well.22 These previous results showed that DMY was poorly absorbed into bloodstream after oral administration and was instable under the intestinal environment, suggesting that DMY might be metabolized and eliminated in the intestinal tract. Also the high apparent volume of distribution of DMY indicated that it could be widely distributed in various tissues, which should be further clarified in the tissue distribution and excretion studies. In addition, although several metabolites of DMY were previously reported,23 the metabolic profile in vivo of DMY has not been systematically illustrated. Therefore, it is crucial to investigate the tissue distribution, excretion, and metabolic profile in vivo of DMY after oral administration. Hence in the present study, an HPLC-MS/MS method for the determination of DMY in rat tissues, urine, and feces was developed and validated, and the method was successfully applied to the first tissue distribution and excretion study of DMY after oral administration in rats. Moreover, the metabolic profile of DMY in rat urine, feces, and plasma was investigated by ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS), a dominant technique for the metabolite identification by enabling chromatographic separations and providing elemental compositions with accurate mass from MS and MS2 spectra.
INTRODUCTION Ampelopsis grossedentata (family: Vitaceae; genus: Ampelopsis Michx.), a traditional Chinese medicinal and edible herb that is also known as vine tea, is widely distributed in southern China.1 Its tender stem and leaves have been consumed as a healthy tea beverage and an herbal medicine for hundreds of years to clear away heat, promote diuresis and bloodflow, and remove channel obstructions.2,3 Dihydromyricetin (DMY) is found as the most abundant (with a content of more than 30%) and bioactive flavanonol compound in Ampelopsis grossedentata.3 The numerous biological activities of DMY have long been demonstrated, such as antioxidant,4 anticancer,5,6 anti-inflammation,7 antialcoholic intoxication,8 hepatoprotective, and cardioprotective effects,9−11 as well as improvement of memory impairment,12 physical performance,13 and insulin resistance.14,15 Presently, DMY-containing products are sold in the U.S. as a nutraceutical supplement to prevent alcohol hangovers.16 Also a recent randomized controlled clinical trial proved that DMY could improve glucose and lipid metabolism and exert anti-inflammatory effects in nonalcoholic fatty liver disease.17 The pharmacokinetic characteristics including absorption, distribution, metabolism, and excretion of active ingredients in plants or foods contribute to the understanding of their interactions with the human body, which are vital to the evaluation of their efficacy and in vivo toxicity.18−20 In our previous studies, a first high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the determination of DMY in rat plasma was developed and applied to the pharmacokinetic study after oral administration © 2017 American Chemical Society
Received: Revised: Accepted: Published: 4597
March 14, 2017 May 18, 2017 May 23, 2017 May 23, 2017 DOI: 10.1021/acs.jafc.7b01155 J. Agric. Food Chem. 2017, 65, 4597−4604
Article
Journal of Agricultural and Food Chemistry Table 1. MRM Conditions for Determination of DMY in Rat Tissues, Urine, and Feces by HPLC-MS/MS
a
analyte
ionization modea
precursor ion (m/z)
product ion (m/z)
DP (V)
CE (eV)
EP (V)
CXP (V)
dwell time (ms)
DMY IS
− −
319.1 301.1
192.8 150.9
−82 −91
−15 −28
−10 −10
−13 −13
100 100
−, Negative ionization mode. for the purpose of maintaining stability of DMY. Then 100 μL tissue homogenate was added with 50 μL IS working solution (blank tissue homogenate was added with 50 μL methanol), and 350 μL acetonitrile containing 1% formic acid was added. After being vortexed for 5 min and centrifuged at 15 616g for 10 min, 100 μL supernatant was collected for HPLC-MS/MS analysis. Urine and Feces Samples for Excretion Study. The feces samples were homogenized in ice-cold methanol/water (1:1, v/v) with a w/v ratio of 1:9, and the feces homogenate or urine sample was pretreated with methanol/water (1:1, v/v, containing 3% formic acid and 100 mM ascorbic acid) at a volume ratio of 25:1. Then 100 μL feces homogenate or urine sample was added with 50 μL IS working solution (blank feces homogenate and urine sample was added with 50 μL methanol), and 100 μL water (containing 1% formic acid) and 1 mL ethyl acetate were added. After being vortexed for 5 min and centrifuging at 15 616g for 5 min, 800 μL supernatant was dried under nitrogen gas flow, and redissolved in 200 μL acetonitrile containing 1% formic acid, followed by being centrifuged at 15 616g for 2 min. Finally, 100 μL supernatant was collected for HPLC-MS/MS analysis. Urine and Feces Samples for Metabolite Identification Study. The feces samples were homogenized in ice-cold methanol/water (1:1, v/v) with the w/v ratio of 1:9, and the feces homogenates or urine samples collected from six rats at the same time point were pooled. Then 200 μL pooled sample was added with 100 μL water and 1 mL ethyl acetate. After being vortexed for 5 min and centrifuged at 15 616g for 5 min, 800 μL supernatant was dried under nitrogen gas flow and redissolved in 200 μL methanol, followed by being centrifuged at 15 616g for 5 min. Finally, 200 μL supernatant was collected for UPLC-QTOF-MS analysis. Determination of DMY by HPLC-MS/MS. The HPLC-MS/MS method for the determination of DMY in rat tissues, urine, and feces was optimized on the basis of our previous pharmacokinetic study21 and validated in accordance with the principles of Guideline on Bioanalytical Method Validation from the European Medicines Agency,24 including specificity, sensitivity, linearity, carry over, precision, accuracy, dilution integrity, recovery, matrix effect, and stability. The chromatographic separation was performed on a Prominence LC-20A HPLC system (Shimadzu, Kyoto, Japan) with a Welch Ultimate XB-C18 HPLC column (50 × 2.1 mm, 5 μm). The mobile phase consisted of water (mobile phase A) and acetonitrile (mobile phase B), and the gradient elution program at the flow rate of 0.8 mL/ min was as follows: mobile phase B from 10% to 90% (0−2.00 min), maintained at 90% (2.00−3.00 min), from 90% to 10% (3.00−3.10 min) and maintained at 10% (3.10−3.60 min). The temperature of column and autosampler was controlled at 40 and 4 °C, respectively. The injection volume was 5 μL. An API 4000 Triple Quadrupole mass spectrometer (AB SCIEX, Concord, Canada) equipped with turbo ionspray source (TIS) was operated in negative ionization mode with multiple reaction monitoring (MRM). The MS parameters were controlled as follows: TIS temperature 550 °C, ionspray voltage −4500 V, curtain gas 25, nebulizing gas 50, and TIS gas 50. The MRM transitions, declustering potential (DP), collision energy (CE), entrance potential (EP), collsion cell exit potential (CXP), and dwell time were optimized as shown in Table 1. Identification of Metabolites by UPLC-QTOF-MS. The chromatographic separation was performed on an Acquity UPLC system (Waters, Milford, U.S.A.) with a Welch Ultimate XB-C18 UPLC column (100 × 2.1 mm, 1.8 μm). The mobile phase consisted of water (mobile phase A) and acetonitrile (mobile phase B), and the gradient elution program at a flow rate of 0.3 mL/min was as follows: mobile phase B from 10% to 30% (0−10.00 min), from 30% to 80%
The predicted metabolic pathways of DMY were proposed as well.
■
MATERIALS AND METHODS
Chemicals and Reagents. DMY was prepared in-house and the purity was above 99% (detailed preparation procedures are shown in the Supporting Information, SI). Quercetin was purchased from the National Institutes for Food and Drug Control (Beijing, China). HPLC-grade acetonitrile and methanol were purchased from Fisher Scientific (Fairlawn, NJ, U.S.A.). The deionized water was produced with a Milli-Q water purification system (Milford, MA, U.S.A.). Formic acid and ethyl acetate were purchased from Sinopharm Chemistry Reagent Co., Ltd. (Shanghai, China). K2-EDTA and ascorbic acid were purchased from Ruite Biotechnology Co., Ltd. (Guangzhou, China). Animals and Drug Administration. Sprague−Dawley rats (220−300 g, 7−8 weeks) were obtained from Hubei Provincial Center for Disease Control and Prevention (Wuhan, China), and had free access to food and water in a controlled environment (temperature: 22 ± 2 °C, relative humidity: 50 ± 10%) for 1 week. The animal experiment was conducted in accordance with the Guidelines for Animal Experimentation of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China), which was approved by the Animal Ethics Committee of the institution. Before the experiments, the rats were fasted overnight with access to water. The blank feces and urine samples were collected in the metabolism cages. DMY suspended in 0.5% sodium carboxymethylcellulose (CMC-Na) solution was oral administered to rats at a dose of 100 mg/kg (equivalent to a human dose of 15.9 mg/ kg according to the conversion method of body surface area). Sample Collection. Plasma Samples for Metabolite Identification Study. Six rats were used for identification of DMY metabolites in plasma. 200 μL whole blood samples were obtained from tail vein at predosing, 2 h, 4 h, 6 h, and 8 h after dosing, and treated with K2EDTA. Then each whole blood sample was centrifuged at 1328g for 10 min to obtain the plasma sample. All of the plasma samples were properly stored at −80 °C until sample preparation and analysis. Tissue Samples for Tissue Distribution Study. Rats were randomly divided into five groups (six rats per group) for tissue distribution study. Tissue samples including heart, liver, spleen, lung, kidney, brain, stomach, and small intestine were collected at 15 min, 45 min, 2 h, 6 h, and 12 h after dosing. After rinsing with saline to remove the blood and content, tissue samples were blotted on filter paper, and then weighed and stored at −80 °C until sample preparation and analysis. Urine and Feces Samples for Excretion and Metabolite Identification Study. Six rats were used for the excretion and metabolite identification study. Urine and feces were collected at 0−2, 2−4, 4−6, 6−8, 8−12, and 12−24 h after dosing in metabolic cages. Food and water were replaced at 2 h postdosing. After measuring the volume of urine samples and dry weight of feces samples, all the urine and feces samples were stored at −80 °C until sample preparation and analysis. Sample Preparation. Plasma Samples. Plasma samples collected from six rats at the same time point were pooled. Then 100 μL pooled sample was added to 600 μL acetonitrile. After being vortexed for 5 min and centrifuged at 15 616g for 10 min, 500 μL supernatant was dried under nitrogen gas flow and redissolved in 200 μL methanol, followed by being centrifuged at 15 616g for 5 min. Finally, 200 μL supernatant was collected for UPLC-QTOF-MS analysis. Tissue Samples. Each weighed tissue was homogenized in ice-cold methanol/water (1:1, v/v) with the w/v ratio of 1:9, and the tissue homogenate was pretreated with methanol/water (1:1, v/v, containing 3% formic acid and 100 mM ascorbic acid) at a volume ratio of 25:1 4598
DOI: 10.1021/acs.jafc.7b01155 J. Agric. Food Chem. 2017, 65, 4597−4604
Article
Journal of Agricultural and Food Chemistry
Table 2. Distribution of DMY in Rat Tissues (ng/g of tissue) at Different Time Points after Oral Administration at a Dose of 100 mg/kg (n = 6, Mean ± SD)a tissues heart liver spleen lung kidney brain stomach small intestine a
15 min 3847 1729 1523 5468 1574 2685 84350 34950
± ± ± ± ± ± ± ±
1436 1048 376.5 1896 432.0 977.8 31506 8980
45 min 491.3 968.7 332.7 750.8 571.0 439.8 85150 2060
± ± ± ± ± ± ± ±
2h
166.6 599.9 101.7 107.0 180.5 135.6 36935 946.0
140.3 132.9 37.07 241.3 209.2 37.98 520.0 246.3
± ± ± ± ± ± ± ±
6h 45.61 37.04 6.058 26.85 69.47 16.14 167.3 93.12
100.1 70.82 − 315.4 62.15 42.73 522.3 24.93
12 h
± 52.10 ± 19.98 ± ± ± ± ±
354.5 23.41 13.62 275.1 5.558
96.57 251.8 − 789.0 92.05 169.2 440.5 38.73
± 27.47 ± 70.6 ± ± ± ± ±
236.5 10.07 127.0 96.64 9.128
−, undetected.
(10.00−15.00 min), from 80% to 10% (15.00−17.00 min), and maintained at 10% (17.00−20.00 min). The temperature of column and autosampler was controlled at 30 and 4 °C, respectively. The injection volume was 5 μL. A Xevo G2-XS QTOF mass spectrometer (Waters, Milford, U.S.A.) equipped with electro-spray ionization (ESI) source was operated in negative ionization mode. The mass spectrometer parameters were as follows: capillary voltage 2.00 kV, sampling cone 15 V, extraction cone 4.0 V, source temperature 120 °C, desolvation temperature 300 °C, cone gas flow rate 50 L/h, and desolvation gas flow rate 600 L/h. Leucine-enkephalin was used as the lock mass, generating an [M − H]− ion (m/z 554.2615) to ensure accuracy during the MS analysis. The date was collected in centroid mode by MSE acquisition. The MSE experiment in two scan functions was carried out as follows: function 1 (low energy): mass-scan range 100−1000 Da, scan time 0.2 s, interscan time 0.05 s, collision energy 2 eV; function 2 (high energy): mass-scan range 100−1000 Da, scan time 0.2 s, interscan time 0.05 s, collision energy ramp 20−30 eV. The data acquisition and processing were conducted by MetabolynxXS software (Waters, Milford, U.S.A.). All of the DMY-containing samples were analyzed together with blank solvent or matrix samples to eliminate the matrix interferences.
■
RESULTS
Method Development and Validation. The present HPLC-MS/MS method for the determination of DMY in rat tissues, urine, and feces was developed based on our previous HPLC-MS/MS method for the determination of DMY in rat plasma.21 Quercetin was chosen as internal standard (IS) because of its similar chemical structure as DMY, which would result in similar behavior during the sample preparation and HPLC-MS/MS analysis. The tissue samples were prepared using the protein precipitation approach, of which the procedure optimization was described in our previous study.21 The urine and feces samples were prepared using a common liquid−liquid extraction approach with the extraction solvent of ethyl acetate, for the purpose of better eliminating the endogenous interferences from the urine and feces matrix. The results of method validation including specificity, sensitivity, linearity, carry-over, precision, accuracy, dilution integrity, recovery, matrix effect, and stability are shown in SI Tables S1 and S2. The UPLC-QTOF-MS method for the identification of DMY metabolites in rat urine, feces, and plasma was developed and optimized. Different gradient elution programs with mobile phases of acetonitrile and water were investigated to achieve satisfactory chromatographic separation and peak shape. For the optimization of QTOF-MS conditions, the negative and positive ionization modes were both evaluated, and the negative ionization mode was selected for higher MS responses. It was noteworthy that the in-source collision-induced dissociation of DMY could easily occur because of its instability, which
Figure 1. Urinary and fecal cumulative excretion ratio of DMY in rats after oral administration at a dose of 100 mg/kg (n = 6, Mean ± SD).
resulted in the interferences of data processing. Thus, various sampling cone voltage (8, 10, 15, 20, 25, 30, and 40 V), desolvation temperature (250, 300, 325, and 350 °C), and source temperature (100, 115, 120, and 125 °C) were tested to reduce the in-source collision-induced dissociation and acquire the proper sensitivity. Finally, the sampling cone voltage was set at 15 V, the desolvation temperature was maintained at 300 °C, and the source temperature was 120 °C. Tissue Distribution Study. The distribution of DMY in rat tissues at different time points was investigated and the results were shown in Table 2. After oral administration at a dose of 100 mg/kg, the peak concentrations of DMY in different tissues were observed at 15 or 45 min, and the peak concentrations in 4599
DOI: 10.1021/acs.jafc.7b01155 J. Agric. Food Chem. 2017, 65, 4597−4604
Article
Journal of Agricultural and Food Chemistry
Figure 2. (A) MS2 spectrum of DMY and (B) the proposed fragmentation pathways of DMY.
stomach (85150 ± 36935 ng/g) and small intestine (34950 ± 8980 ng/g) were much higher than those in heart (3847 ± 1436 ng/g), liver (1729 ± 1048 ng/g), spleen (1523 ± 376.5 ng/g), lung (5468 ± 1896 ng/g), kidney (1574 ± 432.0 ng/g), and brain (2685 ± 977.8 ng/g). After 45 min, the concentrations of DMY in tissues underwent a downward trend over time, which were reduced by approximately 90% of peak levels at 12 h. These results demonstrated that DMY could be distributed rapidly in various tissues and was able to cross the blood−brain barrier. Compared with other tissues, DMY could be highly distributed in the gastrointestinal tract after oral administration. Urinary and Fecal Excretion Study. The concentrations of DMY in rat urine and feces at different time periods after oral administration at a dose of 100 mg/kg were determined, and the cumulative excretion ratios were calculated as shown in Figure 1. The urinary and fecal cumulative excretion ratios of DMY reached a plateau after 12 h, meaning that the elimination of DMY could be completed within 12 h after dosing, which was in accordance with the findings in tissue distribution study. The final urinary cumulative excretion ratio was only 0.0145 ± 0.0069%, while the final fecal cumulative excretion ratio could
reach 18.95 ± 6.87%, indicating that most unconverted forms of DMY were excreted in feces. Taken together, DMY was mainly excreted as metabolites. Fragmentation of DMY Standard. Since metabolites might share the similar skeleton structure and retain the basic substructure of the parent compound, a detailed MS 2 fragmentation of DMY standard was performed as the first step using UPLC-QTOF-MS with Mass Fragment software (Waters, Milford, U.S.A.), which was important for further identification of DMY metabolites. The representative MS2 spectrum and proposed fragmentation pathways of DMY are shown in Figure 2. As a result, the fragmentation of the molecular ion of DMY at m/z 319.0442 led to the predominant product ions at m/z 193.0135 and 125.0228 by loss of B-ring, because the bond between C2−C1′ could be easily fractured because of the hydroxy group at the C-ring. The ion at m/z 301.0312 could be produced by loss of a water molecular. As we know, the Retro Diels−Alder (RDA) fragmentation and loss of neutral fragments were the basic fragmentation pathways of flavonoids, so the ion at m/z 151.0042 was inferred to be formed because of RDA fragmentation. In addition, the ions at m/z 165.0170 and 153.0204 could be generated because of the 4600
DOI: 10.1021/acs.jafc.7b01155 J. Agric. Food Chem. 2017, 65, 4597−4604
Article
plasma
− − − − − − − −
feces
− + + − + + + +
bond cleavage between C2−C3 and C9−O1, respectively. The ion at m/z 165.0170 produced the ions at m/z 137.0209 and 109.0296 by loss of the carbanyl group, and the ion at m/z 151.0042 could generate an ion at m/z 109.0296 because of the bond cleavage between C4−C10 and C9−O1. Identification of DMY Metabolites. The metabolites of DMY in urine, feces, and plasma were investigated by UPLCQTOF-MS with MetabolynxXS software (Waters, Milford, U.S.A.). The mass errors between the measured and calculated values were in the range of ±2 mDa, demonstrating the high level of mass accuracy for the identification of metabolites. A total of eight metabolites (M1-M8) were identified, and the representative MS2 spectra are shown in SI Figure S2. The retention time (tR), metabolic pathways, measured and calculated mass, mass errors, double bond equivalents (DBE), neutral formulas, and sources of the speculated metabolites are summarized in Table 3. M1 (tR = 2.67 min) was identified with an accurate [M − H]− ion at m/z 495.0782 (0.7 mDa) in urine, which was 176 Da higher than that of the parent compound, implying the glucuronidation of DMY. QTOF MS2 fragmentation generated product ions at m/z 193.0152, 301.0374, and 319.0437. The diagnostic ion at m/z 319.0437 was produced by loss of the glucuronic acid group. The product ion at m/z 301.0374 could be produced by loss of a water molecular and the ion at 193.0152 generated because of the bond cleavage between C2− C1′, which were identical to the MS2 fragmentation patterns of DMY. Thus, M1 was characterized as the glucuronidation metabolite. M2 (tR = 3.33 min) was identified with an accurate [M − H]− ion at m/z 321.0608 (−0.2 mDa) in feces, which was 2 Da higher than that of the parent compound, implying the reduction of DMY. QTOF MS2 fragmentation generated the product ions at m/z 125.0240, 195.0285, and 303.0463. The product ion at m/z 303.0463 could be produced by loss of a water molecular. And the bond between C2−C1′ could be easily fractured, generating ions at m/z 193.0152 and 125.0240. Thus, M2 was identified as the reduction metabolite. M3 (tR = 3.83 min) was identified with an accurate [M − H]− ion at m/z 303.0505 (0.0 mDa) in feces, which was 16 Da less than that of the parent compound, implying the dehydroxylation of DMY. As the same fragmentation pathway of DMY, the RDA fragmentation generated the product ion at m/z 151.0067. The cleavage of bonds between C3−C4 and C9−O1 produced ion at m/z 137.0251, and those between C4−C10 and C2−O1 generated ion at m/z 125.0255. The bond between C2−C1′ could be easily fractured owing to the hydroxy group at the C-ring. However, no other fragments caused by the bond cleavage between C2−C1′ were observed, indicating that the dehydroxylation was more likely to occur at the C-ring. According to the present findings and previous study,23 M3 was tentatively identified as the 3-dehydroxylation metabolite. M4 (tR = 3.92 min) was identified with an accurate [M − H]− ion at m/z 399.0032 (1.0 mDa) in urine, which was 80 Da higher than that of the parent compound, implying the sulfation of DMY. QTOF MS2 fragmentation generated the product ions at m/z 193.0136, 301.0352, and 319.0474. The diagnostic ion at m/z 319.0474 was produced by loss of the sulfuric acid group. The product ions at m/z 301.0352 and 193.0136 were identical to the fragment ions of DMY. Thus, M4 was characterized as the sulfation metabolite.
319.0437 303.0463 151.0067 319.0474 179.0277, 181.0159, 207.0296, 207.0305, 301.0374, 195.0285, 137.0251, 301.0352, 151.0135, 153.0185, 165.0250, 165.0222, C21H20O14 C15H14O8 C15H12O7 C15H11O11S C15H12O7 C15H14O9 C16H13O8 C16H13O8 12.5 9.5 10.5 10.5 10.5 9.5 10.5 10.5
DBE error (mDa) calculated [M − H] (m/z) measured [M − H] (m/z)
495.0775 321.0610 303.0505 399.0022 303.0505 305.0661 333.0610 333.0610
−
a
+, detected. b−, undetected.
495.0782 321.0608 303.0505 399.0032 303.0509 305.0663 333.0619 333.0611 2.67 3.33 3.83 3.92 3.98 4.01 4.05 4.20
glucuronidation reduction dehydroxylation sulfation dehydroxylation reduction dehydroxylation methylation methylation
tR (min) no.
M1 M2 M3 M4 M5 M6 M7 M8
metabolic pathway
−
Table 3. MS Data and Identification of DMY Metabolites in Rat Urine, Feces, and Plasmaa,b
0.7 −0.2 0.0 1.0 0.4 0.2 0.9 0.1
formula (neutral)
193.0152, 125.0240, 125.0255, 193.0136, 125.0243, 125.0246, 125.0242, 125.0243,
fragment ions
287.0557 287.0560 315.0518 315.0485
+ − − + − − + +
urine
source
Journal of Agricultural and Food Chemistry
4601
DOI: 10.1021/acs.jafc.7b01155 J. Agric. Food Chem. 2017, 65, 4597−4604
Article
Journal of Agricultural and Food Chemistry
Figure 3. Proposed metabolite structures and predicted metabolic pathways of DMY: (a) glucuronidation, (b) sulfation, (c) reduction, (d) dehydroxylation, and (e) methylation.
■
M5 (tR = 3.98 min) was identified with an accurate [M − H]− ion at m/z 303.0509 (0.4 mDa,) in feces, which was 16 Da less than that of the parent compound, implying the dehydroxylation of DMY. As the same fragmentation pathway of DMY, the product ion at m/z 151.0135 was generated because of the RDA fragmentation, and the ion at m/z 287.0557 was produced by loss of a water molecular. The bond cleavage between C4−C10 and C2−O1 produced the product ions at m/z 125.0243 and 179.0277. According to the present findings and previous study,23 M5 was tentatively identified as the 4′-dehydroxylation metabolite. M6 (tR = 4.01 min) was identified with an accurate [M − H]− ion at m/z 305.0663 (0.2 mDa) in feces. QTOF MS2 fragmentation generated the fragment ions at m/z 125.0246, 153.0185, 181.0159, and 287.0560. The product ion at m/z 287.0560 was produced by loss of a water molecular, and the product ion at m/z 153.0185 could be generated because of the RDA fragmentation. The bond cleavage between C4−C10 and C2−O1 produced ions at m/z 125.0246 and m/z 181.0159. Thus, M6 was identified as the reduction and dehydroxylation metabolite. M7 and M8 (tR = 4.05 and 4.20 min) were identified with accurate [M − H]− ions at m/z 333.0619 (0.9 mDa) and m/z 333.0611 (0.1 mDa), respectively. M7 and M8 could be both detected in urine and feces. QTOF MS2 fragmentation of M7 generated the product ions at m/z 125.0242, 165.0250, 207.0296, and 315.0518. The product ion at m/z 315.0518 was produced by loss of a water molecular, and the ion at m/z 165.0250 could be generated because of the RDA fragmentation. The bond between C2−C1′ could be easily fractured, producing ions at m/z 125.0242 and 207.0296. QTOF MS2 fragmentation of M8 generated the product ions at m/z 125.0243, 165.0222, 207.0305, and 315.0485, which was identical to the MS2 fragmentation patterns of M7. As a result, M7 and M8 were stuctually identifed as the 5- and 7methylation metabolites.
DISCUSSION In our previous study, a first HPLC-MS/MS method for the determination of DMY in rat plasma was developed and applied to a pharmacokinetic study after oral administration in rats.21 The results preliminarily indicated that DMY was poorly absorbed into bloodstream. Recently, another pharmacokinetic study of DMY after oral and intravenous administration was reported, and the results showed that the absolute bioavailability of DMY was only 4.02%.25 In the present study, an HPLC-MS/MS method for the determination of DMY in rat tissues, urine, and feces was developed based on our previous method, and the tissue distribution and excretion of DMY after oral administration were studied for the first time. It was found that the unconverted DMY could be rapidly distributed in various tissues and was able to cross the blood−brain barrier, which confirmed the previous reports that DMY possessed numerous biological activities in vivo including antialcohol intoxication, hepatoprotective and cardioprotective effects, and improvement of memory impairment, physical performance, and insulin resistance.8−15 The elimination of DMY was rapid as well, which could almost be completed within 12 h, proving that no long-term accumulation issue occurred. Notably, DMY could be highly distributed in the gastrointestinal tract after oral administration, and the unconverted forms of DMY were mainly excreted in feces rather than urine. A large amount of DMY was excreted in the forms of metabolites, which needed to be identified in the metabolism study. Thus, this work further investigated the metabolic profile of DMY in urine, feces, and plasma using UPLC-QTOF-MS. Consequently, a total of eight metabolites were detected and identified in urine and feces. Three metabolites (M4, M7, and M8) were reported for the first time. Six phase I and II metabolites formed by reduction (M2), dehydroxylation (M3 and M5), reduction and dehydroxylation (M6), and methylation (M7 and M8) were mainly detected in the feces samples, while four phase II metabolites derived from glucuronidation (M1), sulfation (M4), and methylation (M7 and M8) could be 4602
DOI: 10.1021/acs.jafc.7b01155 J. Agric. Food Chem. 2017, 65, 4597−4604
Journal of Agricultural and Food Chemistry
■
detected in the urine samples. However, the metabolites of DMY were hardly found in the plasma samples. The proposed metabolite structures and predicted metabolic pathways of DMY are illustrated in Figure 3. Similar to other phenolics that are structurally related with DMY such as querceitin and gallocatechin, there might be several major factors influencing the bioavailability and pharmacokinetic characteristics of DMY including the oxidative degradation, gastrointestinal instability, metabolic transformations, poor permeability, and multidrug resistance-associated protein (MRP) efflux.26−28 Thus, one possible explanation for the present findings was that DMY could be rapidly metabolized by phase I modification and phase II conjugation in the intestinal tract after oral administration, resulting in the significant plasma concentration reduction of the parent compound. Meanwhile, DMY underwent the oxidative degradation and was instability under the alkaline conditions in the intestinal tract.22 The intestinal efflux of the parent compound and metabolites might take place as well. Then the metabolites were directly excreted in feces. However, DMY and metabolites arriving in the bloodstream were further metabolized by phase II conjugating enzymes, and these metabolites could be either excreted in urine or directed to biliary excretion. Additionally, renal phase II conjugation of DMY might also occur. In order to confirm the above hypothesis, further metabolism studies of DMY using in vitro incubation method and in vivo germ-free rat model should be carried out in the future.
■
REFERENCES
(1) Ye, L.; Wang, H.; Duncan, S. E.; Eigel, W. N.; O’Keefe, S. F. Antioxidant activities of Vine Tea (Ampelopsis grossedentata) extract and its major component dihydromyricetin in soybean oil and cooked ground beef. Food Chem. 2015, 172, 416−422. (2) Gao, Q.; Ma, R.; Chen, L.; Shi, S.; Cai, P.; Zhang, S.; Xiang, H. Antioxidant profiling of vinetea (Ampelopsis grossedentata): off-line coupling heart-cutting HSCCC with HPLC-DAD-QTOF-MS/MS. Food Chem. 2017, 225, 55−61. (3) Hou, X. L.; Tong, Q.; Wang, W. Q.; Shi, C. Y.; Xiong, W.; Chen, J.; Liu, X.; Fang, J. G. Suppression of inflammatory responses by dihydromyricetin, a flavonoid from Ampelopsis grossedentata, via inhibiting the activation of NF-κB and MAPK signaling pathways. J. Nat. Prod. 2015, 78, 1689−1696. (4) Hou, X. L.; Tong, Q.; Wang, W. Q.; Xiong, W.; Shi, C. Y.; Fang, J. G. Dihydromyricetin protects endothelial cells from hydrogen peroxide-induced oxidative stress damage by regulating mitochondrial pathways. Life Sci. 2015, 130, 38−46. (5) Zhang, Q.; Liu, J.; Liu, B.; Xia, J.; Chen, N.; Chen, X.; Cao, Y.; Zhang, C.; Lu, C.; Li, M.; Zhu, R. Dihydromyricetin promotes hepatocellular carcinoma regression via a p53 activation-dependent mechanism. Sci. Rep. 2015, 4, 4628. (6) Fan, T. F.; Wu, T. F.; Bu, L. L.; Ma, S. R.; Li, Y. C.; Mao, L.; Sun, Z. J.; Zhang, W. F. Dihydromyricetin promotes autophagy and apoptosis through ROS-STAT3 signaling in head and neck squamous cell carcinoma. Oncotarget. 2016, 7, 59691−59703. (7) Qi, S.; Xin, Y.; Guo, Y.; Diao, Y.; Kou, X.; Luo, L.; Yin, Z. Ampelopsin reduces endotoxic inflammation via repressing ROSmediated activation of PI3K/Akt/NF-κB signaling pathways. Int. Immunopharmacol. 2012, 12, 278−287. (8) Shen, Y.; Lindemeyer, A. K.; Gonzalez, C.; Shao, X. M.; Spigelman, I.; Olsen, R. W.; Liang, J. Dihydromyricetin as a novel antialcohol intoxication medication. J. Neurosci. 2012, 32, 390−401. (9) Murakami, T.; Miyakoshi, M.; Araho, D.; Mizutani, K.; Kambara, T.; Ikeda, T.; Chou, W. H.; Inukai, M.; Takenaka, A.; Igarashi, K. Hepatoprotective activity of tocha, the stems and leaves of Ampeolopsis grossedentata, and ampelopsin. BioFactors 2004, 21, 175−178. (10) Chen, Y.; Lv, L.; Pi, H.; Qin, W.; Chen, J.; Guo, D.; Lin, J.; Chi, X.; Jiang, Z.; Yang, H.; Jiang, Y. Dihydromyricetin protects against liver ischemia/reperfusion induced apoptosis via activation of FOXO3amediated autophagy. Oncotarget. 2016, 7, 76508−76522. (11) Liu, S.; Ai, Q.; Feng, K.; Li, Y.; Liu, X. The cardioprotective effect of dihydromyricetin prevents ischemia−reperfusion-induced apoptosis in vivo and in vitro via the PI3K/Akt and HIF-1α signaling pathways. Apoptosis 2016, 21, 1366−1385. (12) Liu, P.; Zou, D.; Chen, K.; Zhou, Q.; Gao, Y.; Huang, Y.; Zhu, J.; Zhang, Q.; Mi, M. Dihydromyricetin improves hypobaric hypoxiainduced memory impairment via modulation of SIRT3 signaling. Mol. Neurobiol. 2016, 53, 7200−7212. (13) Zou, D.; Chen, K.; Liu, P.; Chang, H.; Zhu, J.; Mi, M. Dihydromyricetin improves physical performance under simulated high altitude. Med. Sci. Sports Exercise 2014, 46, 2077−2084. (14) Liu, L.; Wan, J.; Lang, H.; Si, M.; Zhu, J.; Zhou, Y.; Mi, M. Dihydromyricetin delays the onset of hyperglycemia and ameliorates insulin resistance without excessive weight gain in Zucker diabetic fatty rats. Mol. Cell. Endocrinol. 2017, 439, 105−115. (15) Le, L.; Jiang, B.; Wan, W.; Zhai, W.; Xu, L.; Hu, K.; Xiao, P. Metabolomics reveals the protective of dihydromyricetin on glucose homeostasis by enhancing insulin sensitivity. Sci. Rep. 2016, 6, 36184. (16) Wang, C.; Xiong, W.; Perumalla, S. R.; Fang, J.; Sun, C. C. Solidstate characterization of optically pure (+)dihydromyricetin extracted from Ampelopsis grossedentata leaves. Int. J. Pharm. 2016, 511, 245− 252. (17) Chen, S.; Zhao, X.; Wan, J.; Ran, L.; Qin, Y.; Wang, X.; Gao, Y.; Shu, F.; Zhang, Y.; Liu, P.; Zhang, Q.; Zhu, J.; Mi, M. Dihydromyricetin improves glucose and lipid metabolism and exerts anti-inflammatory effects in nonalcoholic fatty liver disease: A randomized controlled trial. Pharmacol. Res. 2015, 99, 74−81.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b01155. Preparation of DMY from Ampelopsis grossedentata, preparation of stock solutions, working solutions, calibration standards and quality control samples, HPLC-MS/MS method validation, and MS2 spectra of DMY metabolites by UPLC-QTOF-MS (PDF)
■
Article
AUTHOR INFORMATION
Corresponding Author
*Tel: +86-027-83649095. E-mail:
[email protected] (W.W.). ORCID
Wenqing Wang: 0000-0003-0703-9787 Author Contributions ⊥
L.F. and Q.T. contributed equally to this work.
Funding
The present study was financially supported by the National Natural Science Foundation of China (81503013). Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS The authors are very grateful for the theoretical guidance from Dr. Zhongzhe Cheng, Dr. Chang Chen, Dr. Guiying Chen, and Prof. Dr. Hongliang Jiang from Tongji School of Pharmacy, and the experimental platform in Wuhan Institute for Drug and Medical Device Control. 4603
DOI: 10.1021/acs.jafc.7b01155 J. Agric. Food Chem. 2017, 65, 4597−4604
Article
Journal of Agricultural and Food Chemistry (18) Zhang, L.; Wang, Y.; Li, D.; Ho, C.-T.; Li, J.; Wan, X. The absorption, distribution, metabolism and excretion of procyanidins. Food Funct. 2016, 7, 1273−1281. (19) Chen, L.; Liu, Y.; Jia, D.; Yang, J.; Zhao, J.; Chen, C.; Liu, H.; Liang, X. Pharmacokinetics and biodistribution of aurantiamide and aurantiamide acetate in rats after oral administration of Portulaca oleracea L. extracts. J. Agric. Food Chem. 2016, 64, 3445−3455. (20) Jiang, P.; Ma, Y.; Gao, Y.; Li, Z.; Lian, S.; Xu, Z.; Jiang, W.; Tian, X.; Huang, C. Comprehensive evaluation of the metabolism of genipin-1-β-d-gentiobioside in vitro and in vivo by using HPLC-QTOF. J. Agric. Food Chem. 2016, 64, 5490−5498. (21) Tong, Q.; Hou, X.; Fang, J.; Wang, W.; Xiong, W.; Liu, X.; Xie, X.; Shi, C. Determination of dihydromyricetin in rat plasma by LCMS/MS and its application to a pharmacokinetic study. J. Pharm. Biomed. Anal. 2015, 114, 455−461. (22) Xiang, D.; Wang, C.-g.; Wang, W.-q.; Shi, C.-y.; Xiong, W.; Wang, M.-d.; Fang, J.-g. Gastrointestinal stability of dihydromyricetin, myricetin, and myricitrin: an in vitro investigation. Int. J. Food Sci. Nutr. 2017, 1−11. (23) Zhang, Y.; Que, S.; Yang, X.; Wang, B.; Qiao, L.; Zhao, Y. Isolation and identification of metabolites from dihydromyricetin. Magn. Reson. Chem. 2007, 45, 909−916. (24) European Medicines Agency (EMA). Committee for Medicinal Products for Human Use (CHMP), Guideline on Bioanalytical Method Validation, 2011. (25) Liu, L.; Yin, X. L.; Wang, X.; Li, X. H. Determination of dihydromyricetin in rat plasma by LC-MS/MS and its application to a pharmacokinetic study. Pharm. Biol. 2017, 55, 657−662. (26) Guo, Y.; Bruno, R. S. Endogenous and exogenous mediators of quercetin bioavailability. J. Nutr. Biochem. 2015, 26, 201−210. (27) D’Andrea, G. Quercetin: a flavonol with multifaceted therapeutic applications? Fitoterapia 2015, 106, 256−271. (28) Bansal, S.; Vyas, S.; Bhattacharya, S.; Sharma, M. Catechin prodrugs and analogs: a new array of chemical entities with improved pharmacological and pharmacokinetic properties. Nat. Prod. Rep. 2013, 30, 1438−1454.
4604
DOI: 10.1021/acs.jafc.7b01155 J. Agric. Food Chem. 2017, 65, 4597−4604
