Article Cite This: J. Agric. Food Chem. 2018, 66, 12227−12235
pubs.acs.org/JAFC
Absorption, Metabolism, and Pharmacokinetics Profiles of Norathyriol, an Aglycone of Mangiferin, in Rats by HPLC-MS/MS Xiaozhen Guo,†,‡ Mingcang Cheng,† Pei Hu,† Zhangpeng Shi,† Shuoji Chen,† Huan Liu,†,‡ Haoyun Shi,†,‡ Zhou Xu,† Xiaoting Tian,*,†,‡ and Chenggang Huang*,†,‡ †
Shanghai Institute of Material Medica, Chinese Academy of Sciences, Shanghai 201203, China University of Chinese Academy of Sciences, Beijing 100049, China
Downloaded via UNIV OF NORTH DAKOTA on November 21, 2018 at 09:23:53 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
‡
ABSTRACT: Norathyriol, an aglycone of mangiferin, is a bioactive tetrahydroxyxanthone present in mangosteen and many medicinal plants. However, the biological fate of norathyriol in vivo remains unclear. In this study, the absorption and metabolism of norathyriol in rats were evaluated through HPLC-MS/MS. Results showed that norathyriol was well absorbed, as indicated by its absolute bioavailability of 30.4%. Besides, a total of 21 metabolites of norathyriol were identified in rats, including methylated, glucuronidated, sulfated and glycosylated conjugates, which suggested norathyriol underwent extensive phase II metabolism. Among those metabolites, 15 metabolites were also identified in hepatocytes incubated with norathyriol, indicating the presence of hepatic metabolism. Furthermore, glucuronide and sulfate conjugates, rather than their parent compound, were found to be the main forms existing in vivo after administration of norathyriol, as implicated by the great increase of exposure of norathyriol determined after hydrolysis with β-glucuronidase and sulfatase. The information obtained from this study contributes to better understanding of the pharmacological mechanism of norathyriol. KEYWORDS: norathyriol, pharmacokinetics, bioavailability, metabolism, HPLC-MS/MS
■
(about 1.2%28). Thus, given its promising pharmacological effects and as the potentially bioactive metabolite responsible for the antidiabetic and hypolipidemic activities of mangiferin, norathyriol is considered a valuable lead for therapeutic development. The efficacy and safety of drugs or food supplements are closely related to their disposition in vivo. Knowledge of the fate of norathyriol in vivo is important because the abovementioned medicinal plants are extensively used in traditional Chinese medicine, and mangosteen is widely consumed in daily life. Our previous research on the pharmacokinetics profile of norathyriol in rats reported that the exposure to norathyriol after oral administration of mangiferin accounted for 3%−11% of that of mangiferin.29,30 Nevertheless, the metabolism and pharmacokinetics profiles of norathyriol after direct oral administration remain unclear. Moreover, knowledge on the bioavailability of norathyriol and its form in the blood circulation after oral administration might contribute to better understanding of its pharmacological mechanism and facilitates its further development as therapeutic agent. Hence, in the present study, biological samples in metabolism studies in vivo and in vitro were collected and prepared for HPLC-QTOF-MS/MS analysis to obtain the comprehensive metabolic pattern of norathyriol in rats. To evaluate the absolute bioavailability and pharmacokinetics characteristics of norathyriol, the previously validated HPLC-MS/MS method with acetonitrile containing 3% acetic acid as protein precipitation
INTRODUCTION Throughout history, natural products from fruits, plants, and herbs, have been valuable sources of medicines or drugs.1,2 Norathyriol (Figure 1) is a tetrahydroxyxanthone of natural
Figure 1. Chemical structure of norathyriol.
origin, and can be found in the fruit hulls and heartwood of Garcinia mangostana (mangosteen)3,4 and medicinal plants belonging to the families of Guttiferae and Gentianaceae, such as Hypericum perforatum (St. John’s Wort) and Canscora decussata.5−7 Over the past few years, a wide range of pharmacological activities of norathyriol have been reported,8−16 such as antitumor,8−11 anti-inflammatory,12,13 and cardiovascular protective effects, including vasorelaxation,14 antiplatelet,15 and antihemostatic activities.16 Our previous studies showed that norathyriol is the primary metabolite of mangiferin (C-glucoside of 1,3,6,7-tetrahydroxyxanthone),17−19 a major constituent of mango fruit with great potential for treatment of metabolic disorders and cancers.20,21 Increasing number of research demonstrated that norathyriol is likely to be responsible for the pharmacological actions of mangiferin against diabetes mellitus,22−24 hyperlipidemia25,26 and hyperuricemia;27 this finding could partly explain the discrepancy between the remarkable biological effects of mangiferin and its extremely poor systemic bioavailability © 2018 American Chemical Society
Received: Revised: Accepted: Published: 12227
July 15, 2018 October 7, 2018 October 9, 2018 October 9, 2018 DOI: 10.1021/acs.jafc.8b03763 J. Agric. Food Chem. 2018, 66, 12227−12235
Article
Journal of Agricultural and Food Chemistry
administration of norathyriol. The rats were accustomed to the environment for a week. Pharmacokinetics Study. Male Wistar rats received norathyriol either by intragastric [i.g., 150 mg/kg; vehicle, 0.5% carboxymethyl cellulose sodium salt (CMC-Na)] or intravenous (i.v., 1 mg/kg; vehicle, mixture of physiological saline and tween-80) administration. The rats were anaesthetized with isoflurane and then blood (0.5 mL) were collected from the angular vein into 1.5 mL sodium heparinized tubes at 0.167, 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h (i.g. administration) or 0.083, 0.25, 0.42, 0.67, 1, 2, 4, and 6 h (i.v. injection) after dosing. Plasma samples were obtained by centrifugation at 12 000 rpm and 4 °C for 10 min and kept at −80 °C until analysis. In Vivo Metabolism Studies. The animals were randomly separated into 15 groups, with three rats each. Two groups were placed in the metabolism cages, and urine and fecal samples were collected into tubes from 0 to 24 h after oral administration of norathyriol (150 mg/kg) or 0.5% CMC-Na vehicle. Another two groups, in which rats were orally given with norathyriol or 0.5% CMC-Na vehicle, were anaesthetized with 20% urethane through intraperitoneal injection. The bile ducts were inserted with micropolyethylene tubing. Bile samples were obtained with 1.5 mL centrifuge tubes over the intervals of 0.25−2, 2−4, 4−8, 8−24 h. Seven groups were anaesthetized as mentioned above and sacrificed to collect the blood from the abdominal artery, liver, kidney, heart, lung, and spleen at 0.5, 1, 2, 4, 8, 12, and 24 h after oral administration of norathyriol. The four control groups were sacrificed to collect blood, liver, kidney, heart, lung, and spleen at 1, 4, 8, and 24 h after oral administration of 0.5% CMC-Na vehicle. The plasma samples were separated from blood by centrifugation at 12 000 rpm and 4 °C for 10 min. All the biological samples were stored at −80 °C until analysis. In Vitro Metabolism Studies. The primary hepatocytes were freshly isolated from Wistar rats according to a previously reported method.32 The hepatocytes were suspended at a density of 0.5 × 106 per well in six-well plates and spiked with known amounts of norathyriol to yield a concentration of 2 μM. The samples were then incubated at 37 °C for 3 h under shaking. Then, 300 μL of cell suspensions were collected at 0.5, 1, and 3 h after incubation and added with 900 μL of ice-cold methanol to terminate the reaction. After vortexing and centrifugation, the resultant supernatant was evaporated under vacuum to dryness at 37 °C. The resultant residue was reconstituted with 10% acetonitrile and subjected to vortexing and centrifugation. The supernatant (5 μL) was used for HPLC-QTOF-MS/MS analysis. Sample Pretreatment. Pharmacokinetics Study. The concentrations of the intact and conjugated (glucuronidated or sulfated) metabolites of norathyriol were determined without and with enzyme hydrolysis. For quantification of glucuronidates/sulfates, 100 μL of the plasma sample was mixed with enzyme buffer solution containing 1850 units glucuronidase and 63 units sulfatase in 0.1 M ammonium acetate buffer (pH 5.0) and 10 μL of ascorbic acid (125 mg/mL) and coincubated at 37 °C for 3 h. After hydrolysis, the plasma was mixed with 3-fold volume of acetonitrile containing 3% acetic acid and 10 μL of 500 ng/mL internal standard, according to our previously validated method.29 The mixture was vortexed for 5 min and centrifuged at 4 °C and 12 000 rpm for 10 min. The supernatant was then evaporated under vacuum at 40 °C to dryness and reconstituted with 100 μL of 8% acetonitrile. For determination of free norathyriol, 100 μL of the plasma samples were processed in the same manner, except that enzyme-free buffer was not added and incubation was not performed. Calibrator samples were then prepared. In brief, 100 μL of blank plasma was added with 10 μL of the appropriate norathyriol working solution and treated with the same procedure used for determination of the free form of norathyriol. Appropriate dilutions were carried out when the concentration was outside the linear range (1−3000 ng/ mL). Metabolism Study. Aliquots of plasma were extracted with three times the volume of acetonitrile containing 3% acetic acid, and centrifuged at 12 000 rpm and 4 °C for 10 min. The supernatant was separated and evaporated under vacuum at 40 °C. The resultant residue was added with 100 μL of 5% acetonitrile, and a 5 μL aliquot
solution was employed for determination of norathyriol in rat plasma.29
■
MATERIALS AND METHODS
Chemicals and Reagents. Norathyriol for reference (purity >98%, HPLC) and dosing (purity >95%, HPLC) was prepared through deglycosylation of mangiferin and identified by MS and NMR.31 β-glucuronidase (type H-2, Helix pomatia, > 85000 units/ mL), sulfatase (type H-1, Helix pomatia, > 53570 units/g), and HPLC-grade formic acid were products of Sigma-Aldrich (St. Louis, MO). Deionized water was purified by a Milli-Q system (Millipore Corporation, Milford, MA). Standard puerarin as internal standard (IS) (purity >98%), HPLC-grade acetonitrile, and other analyticalgrade reagents were products of the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China), Fisher Scientific (Loughborough, U.K.), and Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), respectively. Instrumentations. HPLC-Q-TOF-MS/MS. Norathyriol metabolites in various biological samples were identified using an HPLC-MS/MS system that consist of an Agilent 1260 HPLC and Agilent 6530 QTOF mass spectrometer (Agilent Technologies, CA, U.S.A.). The mass spectrometers were equipped with a dual AJS electrospray ionization source and Masshunter software. Samples (7 μL) were loaded onto an Agilent Poroshell 120 EC-C18 column (100 × 2.1 mm2, 2.7 μm) at 35 °C. The mobile phase consists of solvent A (water containing 0.1% formic acid) and solvent B (acetonitrile containing 0.1% formic acid) at a flow rate of 0.35 mL/min. Gradient separation was achieved by changing the proportion of the mobile phase as follows: 0−30 min, 5% B-50% B; 30−45 min, 50% B-90% B; 45−47 min, 90% B; 47−48 min, 90% B-5% B; and 48−53 min, 5% B. The mass spectrometer was operated in negative Auto MS/MS mode and the parameters were set as follows: temperature of drying and sheath gas, 350 and 300 °C; capillary voltage, 3500 V; skimmer, 65 V; nozzle voltage, 1000 V; fragmentor, 150 V; collision energy, 37 eV; pressure of nebulizer, 35 psi; and flow rate of the drying and sheath gas, 8 and 11.0 L/min, respectively. The Q-TOF mass spectra were obtained in high-resolution mode. The range of mass-to-charge ratio (m/z) scanning was set between 100 and 1200 by using the extended dynamic range. HPLC-QQQ-MS/MS. In our laboratory, an HPLC-QQQ-MS/MS bioanalytical method was established and validated for quantitation of norathyriol in rat plasma.29 Samples (10 μL) were injected to an Agilent 1260 HPLC coupled with Agilent 6460 QQQ mass spectrometer. The mobile phase consists of water containing 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent B) at a flow rate of 0.35 mL/min. The gradient separation was achieved through the following program: 8% B for 0−3.0 min; 25% B−75% B for 3.4−3.8 min; 75% B−95% B for 5−5.5 min; 95% B−8% B for 6.0−6.1 min, and 8% B was maintained until 8.5 min. The operating parameters of the mass spectrometer were optimized as follows: gas temperature, 350 °C; gas flow rate, 10 L/min; nebulizer, 45 psi; capillary, 3500 V; nozzle voltage, 1000 V; sheath gas temperature, 400 °C, and sheath gas flow rate, 12 L/min. The negative multiple reaction monitoring (MRM) mode was employed to quantify compounds. The precursor-to-product ion pairs were m/z 258.9 → 150.8 for norathyriol (the fragmentor voltage and collision energy were 138 V and 42 eV, respectively) and 415.1 → 294.9 for puerarin (151 V and 21 eV, respectively). The qualitative ion pairs were m/z 258.9 → 94.9 (138 V and 50 eV, respectively) for norathyriol and 415.1 → 266.9 (151 V and 34 eV, respectively) for puerarin. Animal Studies. All animal experiments were conducted with procedures approved by the Institutional Animal Care and Use Committee of Shanghai Institute of Materia Medica, China Academic Science. Male Wistar rats (220 ± 20 g) were acquired from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). The rats were housed in an environment under controlled humidity (45%−55%) and temperature (20 °C−24 °C) and given free access to water and standard diet, except in the overnight fasting period before 12228
DOI: 10.1021/acs.jafc.8b03763 J. Agric. Food Chem. 2018, 66, 12227−12235
Article
Journal of Agricultural and Food Chemistry
Figure 2. (A) Extracted ion chromatogram, (B) product ion spectrum, and (C) proposed fragmentation pattern of norathyriol.
Figure 3. Extracted ion chromatogram of norathyriol and its metabolites. was injected into the HPLC-Q-TOF-MS/MS system after vortexing and centrifugation. The tissue samples were homogenized with three times the volume of physiological saline water (0.9%) to prepare tissue homogenates. Urine, bile and tissue homogenates were pretreated with the same method used for plasma samples. Fecal samples (0.6 g) were initially prepared by supersonic extraction with five times the volume of methanol (twice, 0.5 h for each time. The suspension was centrifuged and diluted with 10 times the volume of 5% acetonitrile. About 5 μL of the resultant aliquot was injected into the HPLC-Q-TOF-MS/MS system. The blank biological samples as control were treated with the same method used for norathyriolcontaining samples. Data Analysis. Pharmacokinetics analysis was conducted using Winnonlin software (Pharsight 6.2, NC, U.S.A.). Kinetic parameters were determined via noncompartmental model analysis. These parameters include the time to maximum concentration (Tmax), maximum concentration (Cmax), area under the concentration- time curve (AUC), half elimination time (T1/2), and mean residence time (MRT). Total plasma clearance (CL) and apparent distribution
volume (Vd) were also calculated for i.v. dosing. The absolute oral bioavailability (F) of norathyriol was calculated by the equation F(%) = (AUC0−t, i.g. × dosei.v.)/ (AUC0−t, i.v. × dosei.g.) × 100, where AUC0−t, i.g. and AUC0−t, i.v. represent the AUC of norathyriol after intragrastic and intravenous administration, respectively; and dosei.g. and dosei.v. stand for the dose used for intragrastic and intravenous administration of norathyriol, respectively. All data are presented as mean ± standard deviation (SD).
■
RESULTS Mass Fragmentation Pattern of Norathyriol. This study employed MS/MS fragmentation-based HPLC-MS method by comparison of molecular masses and fragmentation pattern of the metabolites with those of the parent drug to identify the metabolites of norathyriol. In this regard, the first step of the study was to identify the chromatographic and mass spectrometric characteristics of the norathyriol standard. Negative-ion detection mode was adopted to acquire better 12229
DOI: 10.1021/acs.jafc.8b03763 J. Agric. Food Chem. 2018, 66, 12227−12235
Article
Journal of Agricultural and Food Chemistry
Figure 4. Proposed metabolic pathway of norathyriol in vivo.
drug-containing biological samples and their corresponding mass spectra data, 21 metabolites were found and tentatively characterized. The extracted ion chromatograms of these metabolites and the proposed metabolic pathway of norathyriol are shown in Figures 3 and 4, respectively. The results suggested that norathyriol underwent extensive phase II metabolism, including glucuronidation, sulfation, methylation and glycosylation. A summary of the retention times, the mass spectral data, and their designations is presented in Table 1. Parent Compound (M0). M0, eluted at 15.60 min, displayed a deprotonated molecular ion at m/z 259.0242, suggesting an elemental composition of C13H7O6− (2.32 ppm). The retention time, precursor ion, and product ions of M0 were identical to those of norathyriol standard. Thus, M0 was designated as norathyriol. Metabolites M8, M11, and M4. M8 and M11 were detected with the retention time of 10.08 and 12.37, respectively. Their MS spectra displayed the same [M−H]− ions at m/z 435.0557 (2.76 ppm, C19H15O12−) in negative-ion ESI mode. In their MS/MS spectra, apart from the characteristic loss of the glucuronic acid moiety (176 Da), the two ions experienced the same fragmentation pathways to yield the common product ions at m/z 231.0270, 215.0342, and 187.0387, which were the same as the characteristic product ions of norathyriol. Hence, M8 and M11 were a pair of isomers and identified as the monoglucuronidation metabolites of norathyriol. Likewise, M4 at 9.14 min showed the [M−H]− ion at m/z 611.0875 (2.45 ppm, C25H23O18−), 176 Da greater than that of M8 or M11. The product ion scanning spectrum of M4 showed ions at m/z 435.0564 and 259.0244, because of the continuous loss of the glucuronic acid moiety (176 Da). The ions were identical to the diagnostic ions of norathyriol at m/z 230.0192, 215.0318, and 187.0056. Therefore, M4 was tentatively proposed to be the diglucuronidation metabolite of norathyriol.
responses of norathyriol and other related components in the MS spectra. As shown in Figure 2A, norathyriol was eluted at 15.60 min in the extracted ion chromatogram (EIC) and displayed a deprotonated molecular ion [M−H]− at m/z 259.0242 (2.32 ppm, elemental composition C13H7O6−) in the full-scan mass spectrum. In the MS/MS spectrum (Figure 2B), the product ions at m/z 231.0306 (C12H7O5−, −3.03 ppm), 230.0221 (C12H6O5−, 0 ppm), and 215.0340 (C12H7O4−, 4.65 ppm) were derived from the loss of CO (28 Da), •CHO (29 Da), and CO2 (44 Da) from [M−H]−, respectively. The other ions at m/z 231.0306 and 215.0340 further eliminated CO (28 Da) to obtain ions at m/z 203.0333 (C11H7O4−, 8.37 ppm) and 187.0398 (C11H7O3−, 1.60 ppm), respectively. The ion at m/z 169.0298(C11H5O2−, −1.77 ppm) was due to the neutral loss of H2O from the ion at m/z 187.0398. Norathyriol also underwent the Retro Diels−Alder (RDA) rearrangement via the different retrocyclization fragments shown in Figure 2C, where the superscripts on the left of the A or B ring indicate the bonds that have been broken. Accordingly, the most abundant ions at m/z 107.0134 (C6H3O2−, 4.67 ppm) were attributed to 1,2B+ or 0,3A+ fragment and those at m/z 151.0035 (C7H3O4−, 1.32 ppm) were designated as 1,2A+ or 0,3 + B RDA ion. The proposed fragmentation pattern of norathyriol in negative mode is shown in Figure 2C. Thus, the ions at m/z 231.0306 (C12H7O5−), 230.0221 (C12H6O5−), 215.0340 (C12H7O4−), 203.0333 (C11H7O4−), 187.0398 (C 1 1 H 7 O 3 − ), 151.0035 (C 7 H 3 O 4 − ), and 107.0134 (C6H3O2−) were considered as the diagnostic ions of norathyriol. This finding provided a basis for characterization of the metabolites of norathyriol. Identification of Metabolites of Norathyriol. The metabolites of norathyriol in rat plasma, urine, bile, feces, tissues, and hepatocytes were analyzed using HPLC-Q-TOFMS/MS. After the analysis of the chromatography patterns of 12230
DOI: 10.1021/acs.jafc.8b03763 J. Agric. Food Chem. 2018, 66, 12227−12235
Article
Journal of Agricultural and Food Chemistry Table 1. Summary of the Mass Spectral Data of Norathyriol and its Metabolites Detected in Rats [M − H]− no.
description
tR (min)
measured
calculated
error (ppm)
15.60
259.0242
259.0248
2.32
C13H8O6
formula (neutral)
M0
Norathyriol
M1
Norathyriol sulfate glucuronide glucoside
6.22
677.0653
677.0665
1.77
C25H26O20S
M2 M3
Norathyriol disulfate Norathyriol sulfate glucoside
7.15 7.69
418.9381 501.0332
418.9384 501.0344
0.72 2.40
C13H8O12S2 C19H18O14S
M4
Norathyriol diglucuronide
9.14
611.0875
611.0890
2.45
C25H24O18
M5 M6
9.19 9.67
597.1087 529.0289
597.1097 529.0294
1.67 0.95
C25H26O17 C20H18O15S
M7
Norathyriol glucuronide glucoside Norathyriol methyl ether glucuronide sulfate Norathyriol glucuronide sulfate
9.80
515.0121
515.0137
3.11
C19H16O15S
M8
Norathyriol glucuronide
10.08
435.0557
435.0569
2.76
C19H16O12
M9
Norathyriol methyl ether diglucuronide
10.71
625.1048
625.1046
−0.32
C26H26O18
M10
Norathyriol glucuronide sulfate
10.86
515.0121
515.0137
3.11
C19H16O15S
M11
Norathyriol glucuronide
12.37
435.0557
435.0569
2.76
C19H16O12
M12
Norathyriol methyl ether glucuronide
14.02
449.0714
449.0725
2.45
C20H18O12
M13
Norathyriol methyl ether glucuronide
14.88
449.0714
449.0725
2.45
C20H18O12
M14
Norathyriol sulfate
15.03
338.9813
338.9816
0.89
C13H8O9S
M15
Norathyriol methyl ether sulfate
16.64
352.9962
352.9973
3.12
C14H10O9S
M16
Norathyriol methyl ether sulfate
17.65
352.9962
352.9973
3.12
C14H10O9S
M17 M18
Norathyriol methyl ether Norathyriol dimethyl ether glucuronide
18.32 19.30
273.0401 463.0877
273.0405 463.0882
1.46 1.08
C14H10O6 C21H20O12
M19 M20
Norathyriol methyl ether Norathyriol methyl ether disulfate
19.54 21.89
273.0410 432.9533
273.0405 432.9541
−1.83 1.85
M21
Norathyriol methyl ether
22.53
273.0396
273.0405
3.30
Metabolites M14, M2, M7, and M10. Metabolite M14, observed at 15.03 min, showed the deprotonated molecular ion at m/z 338.9813, suggesting the molecular formula of C13H7O9S−(0.89 ppm). The MS/MS spectrum exhibited the characteristic neutral loss of SO3 (80 Da) and the identical product ions at m/z 259.0258, 231.0314, 215.0350, 187.0406, and 107.0146, similar to those of norathyriol. M14 was thus assumed to be a monosulfate conjugate of norathyriol. Similarly, the [M−H]− ion of M2 (0.72 ppm, C13H7O12S2−) at 7.15 min was 80 Da greater than that of M14. The fragment ions at m/z 338.9788 and 259.0254 corresponded to the successive losses of SO3, and the diagnostical ions of norathyriol were shown in the product ion spectrum of M2. Thus, M2 was tentatively assumed to be the disulfation metabolite of norathyriol. M7 and M10 at 9.80 and 10.71 min showed the same [M−H]− ion at m/z 515.0121 (3.11 ppm, C19H15O15S−), 80 Da higher than that of M8 or M11. Their further decomposition paralleled that of the molecule ions of M8 or M11, indicating that M7 and M10 might be a pair of isomers as the glucuronidation and sulfation metabolites of norathyriol. Metabolites M17, M19, M21, M20, M6, M12, M13, M15, M16, M9, and M18. Metabolites M17, M19, and M21 were
C14H10O6 C14H10O12S2 C14H10O6
MS/MS fragment m/z (% base peak) 231.0306(10),230.0221(10),215.0340(91) ,203.0333(22),187.0398(65),169.0298(33) ,151.0035(85),107.0134(100) 597.1061(35),435.0564(22),421.0764(98) ,259.0238(100),215.0336(1) 338.9788(5),259.0254(100), 168.9855(22) 421.0790(10),259.0279(100),230.0247(6) ,215.0412(3),187.0477(2),106.9872(1) 435.0564(4),259.0244(100),230.0192(1) ,215.0318(1),187.0056(1) 435.0559(100),259.0249(94),215.0272(1) 449.0802(1),273.0421(93),258.0189(100) ,230.0245(4),150.9729(1) 435.0545(1),259.0241(100),231.0299(1) ,215.0370(2),203.0371(1),187.0361(1) 259.0247(100),231.0270(4),215.0342(14) ,187.0387(7) 449.0754(2),273.0404(100),258.0167(40) ,230.0177(2) 435.0545(1),259.0241(100),231.0299(1) ,215.0370(2),203.0371(1),187.0361(1) 259.0247(100),231.0270(4),215.0342(15) ,187.0387(7) 273.0433(11),258.0185(100),230.0232(64) ,186.0323(3) 273.0433(11),258.0185(100),230.0232(64) ,186.0323(3) 259.0258(100),231.0314(12),215.0350(52) ,187.0406(26),107.0146(10) 273.0366(7),258.0152(100),230.0208(79) ,201.0179(10),186.0310(16) 273.0366(7),258.0152(100),230.0208(79) ,201.0179(10),186.0310(16) 258.0158(100),230.0193(66),185.0219(24) 287.0543(14),257.0096(100),229.0141(63) ,187.0885(30) 258.0158(100),230.0193(66),185.0219(24) 353.0276(22),273.0406(100),258.0151(53) ,229.0181(18),215.0445(10) 258.0158(100),230.0193(66),185.0219(24)
separated at 18.32, 19.54, and 22.53 min, respectively, and their [M−H]− ions yielded the same elemental composition of C14H9O6− with error less than 5 ppm. In their MS/MS spectra, following the loss of •CH3 (15 Da), the product ions observed at m/z 230.0193 and 185.0219 were identical to those of norathyriol. Hence, M17, M19, and M21 were suggested to be isomers and the methylated metabolites of norathyriol. Among the four hydroxyl groups in the structure of norathyriol, methylation occurred preferentially at the hydroxyls at C-3, C-6, and C-7, not at C-1 due to the intramolecular hydrogen bond between the hydroxyl at C-1 and an ortho carbonyl. The ClogP values of 3-, 6-, and 7-Omethyl-norathyriol calculated by Chemdraw (version 14.0, CambridgeSoft Corporation) were 2.49, 2.17 and 2.17, respectively. Thus, according to their retention times, M17 and M19 were considered to be 7- or 6-O-methyl norathyriol and M21 was tentatively designated as 3-O-methyl norathyriol. Likewise, M20 at 21.89 min showed the [M−H]− ion at m/z 432.9533 (1.85 ppm, C14H10O11S2−), which is 15 Da higher than that of M2, and its fragment ions at m/z 353.0276 and 273.0406 were 15 Da higher than the corresponding product ions of M2 at m/z 338.9788 and 259.0254. Combined with the diagnostic ions of norathyriol shown in the product ion 12231
DOI: 10.1021/acs.jafc.8b03763 J. Agric. Food Chem. 2018, 66, 12227−12235
Article
Journal of Agricultural and Food Chemistry
Figure 5. Plasma concentration−time curves of norathyriol after (A) intragastric and (B) intravenous administration of norathyriol to rats (each point represents mean ± SD, n = 6).
Table 2. Distribution of Norathyriol and its Metabolites in Rat Urine, Fece, Plasma, Heart, Liver, Spleen, Lung, and Kidney after Oral Administration of Norathyriol and in Hepatocytes no.
M0
urine plasma feces bile heart liver spleen lung kidney hepatocytes
+ + + + + + + + + +
M1
+
M2
M3
M4
M5
M6
M7
M8
M9
M10
M11
M12
M13
M14
M15
M16
M17
M18
M19
M20
M21
+
+
+ +
+ +
+ +
+
+ +
+ +
+ +
+
+ +
+
+ +
+
+
+
+ + + +
+ +
+ + +
+
+
+
+
+
+
+
+
+
+
+
+ + +
+
+ + +
+ + + + + + + + + +
+ +
+ + +
+ + + + + + + + + +
+ + +
+ + +
+ + + + + + + + + +
+ + +
+ + +
+ + +
+ + +
+
+
+
+ + +
+
+
+ +
+
+ +
Da); both ions were 176 Da higher than the products ions of M3 at m/z 421.0790 and 259.0279, which is due to the corresponding neutral losses. Thus, M1 was postulated as the glucuronidation metabolite of M3, namely, the diglucuronidation conjugate of norathyriol. Pharmacokinetics Studies of Norathyriol after Intragastric and Intravenous Administration. After the rats had been administrated with norathyriol orally (150 mg/kg) or intravenously (1 mg/kg), the time course of the mean plasma concentration of norathyriol determined with and without enzyme hydrolysis are displayed in Figure 5. The relevant pharmacokinetics parameters are tabulated in Table 3. Following i.v. administration, the concentration of norathyriol exhibited a biexponential decline with an initial steep phase over the first hours and an intermediate T1/2 of 2.3 h for norathyriol to be eliminated from systemic circulation. The mean Vd of norathyriol (7.1 L/kg) was far in excess of the total amount of rat body water (0.67 L/kg), which indicates that norathyriol could be widely distributed to the tissues. The mean CL of norathyriol (2.6 L/h/kg) was close to the rat hepatic blood flow (3.4 L/h/kg).33 Following i.v. administration, the AUC of norathyriol determined after enzymatic hydrolysis (2743.9 h·ng/mL) was twice that determined without hydrolysis (449.4 h·ng/mL), thereby indicating the extensive glucuronidation and sulfation metabolism of norathyriol. Following oral administration, norathyriol peaked at 3.6 h and was cleared at a moderate rate from the body, as indicated by its mean T1/2 of 5.7 h. Plasma levels of norathyriol after oral administration were more variable than those observed after i.v. injection. By the comparing the AUCs of norathyriol observed after i.v. and i.g. administration, the absolute bioavailability of norathyriol was calculated to be 30.4%.
spectrum of M20, M20 was tentatively identified as the methylation metabolite of M2, namely, the disulfation and methylation metabolite of norathyriol. M6 (the deprotonated ion at m/z 529.0289 with the corresponding elemental composition C20H17O15S−, 0.95 ppm) at 9.67 min, M12 or M13 (m/z 449.0714, C20H17O12−, 2.45 ppm) at 14.02 or 14.88 min, M15 or M16 (m/z 352.9962, C14H9O9S−, 3.12 ppm) at 16.64 or 17.65 min, M9 (m/z 625.1048, C26H25O18−, 0.01 ppm) at 10.71 min and M18 (m/z 463.0877, C21H19O12−, 1.08 ppm) at 19.30 min were tentatively characterized as the methylation metabolites of M7 (or M10), M8 (or M11), M14, M4, and M12 (or M13), respectively. Metabolites M3 and M5. M3 at 7.69 min displayed the [MH]− ion at m/z 501.0332 (C19H17O14S−, 2.40 ppm), 162 Da higher than that of M14. The fragment ions at m/z 421.0790 and 259.0279 were due to successive losses of SO3 (80 Da) and a glucose moiety (162 Da). Further decomposition paralleled that of the molecular ions of M14. Thus, M3 was postulated to be the glycosylation product of M14. Likewise, M5 at 9.19 min showed a precursor ion at m/z 597.1087 (1.67 ppm, C25H25O17−), 162 Da greater than that of M8 or M11. In the product ion spectrum of M5, the fragment ion at m/z 435.0559 is due to the characteristic loss of a glucose moiety (162 Da), and the other ions at m/z 259.0249 and 215.0272 were identical to the [M−H]− and product ions of M8 (or M11), respectively. Hence, M5 was identified as the glycosylated conjugate of M8 or M11. Metabolite M1. M1 was eluted at 6.22 min with the deprotonated molecular ion at m/z 677.0653 (C25H25O20S−, 1.77 ppm,), which was 176 Da greater than that of M3. In its product ion spectrum, the fragment ions at m/z 597.1061 and 435.0564 were the products obtained after consecutive losses of SO3 (80 Da) and a glucose moiety (162 12232
DOI: 10.1021/acs.jafc.8b03763 J. Agric. Food Chem. 2018, 66, 12227−12235
Article
Journal of Agricultural and Food Chemistry
Table 3. Pharmacokinetic Parameters of Norathyriol in Male Wistar Rats Following Intragastric (i.g.) and Intravenous (i.v.) Administration of Norathyriol (Mean ± SD, n = 6) PK parameters Tmax(h) Cmax(ng/mL) AUC0‑t(h·ng/mL) T1/2(h) CL (L/h/kg) MRT(h) Vd (L/kg) F (%)
i.v.- without enzymolysis
449.4 2.3 2.6 1.3 7.1
± ± ± ± ±
183.7 1.3 1.4 0.6 2.9
i.g.- without enzymolysis 3.6 2262.8 20513.3 5.7
± ± ± ±
2.3 592.0 8630.8 0.9
8.2 ± 2.2
i.g.- with enzymolysis 4.5 16461.9 233750.8 8.9
2743.9 ± 411.5 1.3 ± 0.2 1.2 ± 0.06
± ± ± ±
1.91 2309.5 39920.7 3.4
9.0 ± 1.1
30.4
After oral administration of norathyriol, the respective Cmax and AUC of norathyriol determined with enzyme hydrolysis were 7.3- and 11-fold those determined without hydrolysis, which indicates that its glucuronidation and sulfation metabolites are the predominant forms in which norathyriol exists. Comparisons between norathyriol determined with and without enzymatic hydrolysis also suggested that the extent of glucuronidation and sulfation depends on route of dosing.
■
i.v.- with enzymolysis
dosing appears to have a significant impact on the extent of its glucuronidation and sulfation. The respective AUCs of norathyriol determined with enzyme hydrolysis after i.g. and i.v. administration were 11- and 2-fold those determined without hydrolysis (Table 3). Such extensive metabolism partly limits the systemic exposure of norathyriol, resulting in its intermediate bioavailability of approximately 30.4%. The distribution of norathyriol was widespread, as indicated by its detection in various tissues and Vd (7.1 L/kg), which was far in excess of the total amount of rat body water (0.67 L/kg)33 and rat plasma volume (0.031 L/kg).33 Norathyriol exhibited an intermediate rate of clearance after either intravenous or oral administration, as indicated by its T1/2 of 2.3 and 5.7 h, respectively. Given its great potential activity against diseases such as diabetes, hyperuricemia, and cancer, and its favorable pharmacokinetics properties, norathyriol may be considered a promising candidate worthy of further development into a multipotency drug. To better understand the mechanism of the pharmacological action of norathyriol, information on the major form of norathyriol circulating in vivo among the identified metabolites must be collected. However, due to the lack of a reliable reference for norathyriol metabolites, comparisons of the exposure of norathyriol between samples treated with and without enzyme hydrolysis were used instead to indirectly determine the main form for norathyriol in vivo. As shown in Figure 5B and Table 3, the AUC of norathyriol determined after oral administration with enzymolysis was 11-fold that determined without enzymolysis. This result indicates that its glucuronidation and sulfation metabolites, instead of the parent compound, are the predominant forms in which norathyriol exists. Screening for potential interactions with norathyriol as perpetrator or as victim is important to evaluate its safety for therapeutic applications. Norathyriol has been reported to exhibit inhibitory effects on UGT1A3, UGT1A7, and UGT1A9 with the corresponding IC50 values of 8.2, 4.4, and 12.3 μM, respectively,34 which pointed toward potential interactions with other molecules being substrates for the identified UGT isoforms. In summary, while norathyriol undergoes extensive phase II metabolism and predominantly exists as glucuronidation and sulfation metabolites in vivo, the aglycone exhibits favorable pharmacokinetics properties, as partly indicated by its intermediate bioavailability. The results suggest a potential interaction between norathyriol and other molecules undergoing UGT- or SULT- mediated metabolism.
DISCUSSION
In this study, the comprehensive metabolism in vivo and in vitro, bioavailability, and pharmacokinetics profile of norathyriol were evaluated. With the application of HPLC-Q-TOFMS/MS, and analysis of the mass fragmentation pattern of norathyriol which was characterized with neutral losses of CO, H2O, and CO2, and RDA fragmentation (Figure 2C), a total of 21 metabolites were tentatively characterized, including methylated, sulfated, glucuronidated, and glycosylated conjugates. However, the exact position of substitution could not be determined because of lack of the standards of the metabolites. As shown in the Table 2, 20, 6, 15, 16, 6, 15, 3, 10, and 12 metabolites were identified in rat urine, feces, plasma, bile, heart, liver, spleen, lung, and kidney, respectively. Notably, the parent compound, M10, M11, and M12 were detected in all the tested biological samples of rats, suggesting the extensive tissue distribution of norathyriol and these metabolites. In addition, M2 and M20 were only identified in rat urine, M1 was only identified in bile, and M18 was only identified in bile and urine. Such limited distribution partly indicates that these metabolites are minor products and that their concentration in other matrices may be lower than the limit of detection of the mass spectrometer used in this work. Among the metabolites identified in vivo, 15 metabolites (M4, M6−17, M19, and M21) were also identified in vitro in primary hepatocytes incubated with norathyriol; this finding reveals the significant hepatic metabolism of norathyriol and highlights the liver as the place for the generation of these metabolites. The metabolic pathway of norathyriol in rat is proposed as shown in Figure 4, which reveals that norathyriol undergoes extensive phase II metabolism in rat and that the parent compound and other metabolites (M10, M11, and M12) exhibit widespread distribution. The expected dehydroxylated metabolites, including 1,3,7-trihydroxyxanthone, 1,7-dihydroxanthone, and their conjugated metabolites, were not found in rats orally administrated with norathyriol, in line with our previous metabolism study of mangiferin,17 wherein these metabolites were not screened in conventional rats but found in pseudogerm-free rats. The extensive conjugation metabolism of norathyriol was confirmed by pharmacokinetics studies, and the route of
■
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (X.T.). 12233
DOI: 10.1021/acs.jafc.8b03763 J. Agric. Food Chem. 2018, 66, 12227−12235
Article
Journal of Agricultural and Food Chemistry *E-mail:
[email protected] (C.H.).
(9) Fu, W.-m.; Zhang, J.-f.; Wang, H.; Tan, H.-s.; Wang, W.-m.; Chen, S.-C.; Zhu, X.; Chan, T.-m.; Tse, C.-m.; Leung, K.-s.; Lu, G.; Xu, H.-x.; Kung, H.-f. Apoptosis induced by 1,3,6,7-tetrahydroxyxanthone in Hepatocellular carcinoma and proteomic analysis. Apoptosis 2012, 17 (8), 842−851. (10) Li, J.; Malakhova, M.; Mottamal, M.; Reddy, K.; Kurinov, I.; Carper, A.; Langfald, A.; Oi, N.; Kim, M. O.; Zhu, F.; Sosa, C. P.; Zhou, K.; Bode, A. M.; Dong, Z. Norathyriol suppresses skin cancers induced by solar ultraviolet radiation by targeting ERK kinases. Cancer Res. 2012, 72 (1), 260−70. (11) Li, J.; Li, X.; He, Z.; Zhou, K. Norathyriol suppresses transformation in JB6 P+ cells by the inhibition of Akt. J. Cancer Res. Ther. 2012, 8 (4), 561−4. (12) Hsu, M.-F.; Lin, C.-N.; Lu, M.-C.; Wang, J.-P. Inhibition of the arachidonic acid cascade by norathyriol via blockade of cyclooxygenase and lipoxygenase activity in neutrophils. NaunynSchmiedeberg's Arch. Pharmacol. 2004, 369 (5), 507−15. (13) Chen, Y.-c.; Chen, K.-t. Novel selective inhibitors of hydroxyxanthone derivatives for human cyclooxygenase-2. Acta Pharmacol. Sin. 2007, 28 (12), 2027−2032. (14) Ko, F. N.; Lin, C. N.; Liou, S. S.; Huang, T. F.; Teng, C. M. Vasorelaxation of rat thoracic aorta caused by norathyriol isolated from Gentianaceae. Eur. J. Pharmacol. 1991, 192 (1), 133−139. (15) Teng, C. M.; Lin, C. N.; Ko, F. N.; Cheng, K. L.; Huang, T. F. Novel inhibitory actions on platelet thromboxane and inositolphosphate formation by xanthones and their glycosides. Biochem. Pharmacol. 1989, 38 (21), 3791−3795. (16) Teng, C. M.; Ko, F. N.; Wang, J. P.; Lin, C. N.; Wu, T. S.; Chen, C. C.; Huang, T. F. Antihemostatic and antithrombotic effect of some antiplatelet agents isolated from Chinese herbs. J. Pharm. Pharmacol. 1991, 43 (9), 667−9. (17) Liu, H. H.; Wu, B.; Pan, G. Y.; He, L.; Li, Z. X.; Fan, M. S.; Jian, L. H.; Chen, M. C.; Wang, K.; Huang, C. G. Metabolism and Pharmacokinetics of Mangiferin in Conventional Rats, Pseudo-GermFree Rats, and Streptozotocin-Induced Diabetic Rats. Drug Metab. Dispos. 2012, 40 (11), 2109−2118. (18) Liu, H. H.; Wang, K.; Tang, Y. H.; Sun, Z. L.; Jian, L. H.; Li, Z. X.; Wu, B.; Huang, C. G. Structure elucidation of in vivo and in vitro metabolites of mangiferin. J. Pharm. Biomed. Anal. 2011, 55 (5), 1075−1082. (19) Wang, H.; Ye, G.; Ma, C. H.; Tang, Y. H.; Fan, M. S.; Li, Z. X.; Huang, C. G. Identification and determination of four metabolites of mangiferin in rat urine. J. Pharm. Biomed. Anal. 2007, 45 (5), 793−8. (20) Nunez Selles, A. J.; Daglia, M.; Rastrelli, L. The potential role of mangiferin in cancer treatment through its immunomodulatory, antiangiogenic, apoptopic, and gene regulatory effects. BioFactors 2016, 42 (5), 475−491. (21) Fomenko, E. V.; Chi, Y. Mangiferin modulation of metabolism and metabolic syndrome. BioFactors 2016, 42 (5), 492−503. (22) Wang, F.; Yan, J.; Niu, Y.; Li, Y.; Lin, H.; Liu, X.; Liu, J.; Li, L. Mangiferin and its aglycone, norathyriol, improve glucose metabolism by activation of AMP-activated protein kinase. Pharm. Biol. 2014, 52 (1), 68−73. (23) Ding, H.; Zhang, Y.; Xu, C.; Hou, D.; Li, J.; Zhang, Y.; Peng, W.; Zen, K.; Zhang, C. Y.; Jiang, X. Norathyriol reverses obesity- and high-fat-diet-induced insulin resistance in mice through inhibition of PTP1B. Diabetologia 2014, 57 (10), 2145−54. (24) Shi, Z. L.; Liu, Y. D.; Yuan, Y. Y.; Song, D.; Qi, M. F.; Yang, X. J.; Wang, P.; Li, X. Y.; Shang, J. H.; Yang, Z. X. In Vitro and In Vivo Effects of Norathyriol and Mangiferin on alpha-Glucosidase. Biochem. Res. Int. 2017, 2017, 1−7. (25) Li, J.; Liu, M. Y.; Yu, H. Y.; Wang, W.; Han, L. F.; Chen, Q.; Ruan, J. Y.; Wen, S. S.; Zhang, Y.; Wang, T. Mangiferin Improves Hepatic Lipid Metabolism Mainly Through Its Metabolite-Norathyriol by Modulating SIRT-1/AMPK/SREBP-1c Signaling. Front. Pharmacol. 2018, 9, 13. (26) Wilkinson, A. S.; Monteith, G. R.; Shaw, P. N.; Lin, C.-N.; Gidley, M. J.; Roberts-Thomson, S. J. Effects of the mango components mangiferin and quercetin and the putative mangiferin
ORCID
Chenggang Huang: 0000-0001-9209-7484 Funding
This work was funded by research grants from The National Natural Science Foundation of China (No. 81030065), and National Natural Science Foundation for Young Scientists of China (No. 81603280), Strategic Priority Scientific and Technological Project of Chinese Academy of Science (No. XDA12040203), National Science and Technology Major Project of the Ministry of Science and Technology of China (No. 2018ZX09731016-003 and No. 2018ZX09201001-001008). Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS The authors are grateful to Le Wang (Shanghai Institute of Material Medica, Chinese Academy of Science) for his kind help in the preparation of the rat primary hepatocytes for the in vitro metabolism study of norathyriol.
■
ABBREVIATIONS USED HPLC-MS/MS, high-preformance liquid chromatography tandem mass spectrometry; Q-TOF, quadrupole time-of flight; QQQ, triple quadrupole; MRM, multiple reaction monitoring mode; IS, internal standard; AUC, area under concentration versus time curve; Cmax, maximum concentration; Tmax, time to reach peak concentration; T1/2, elimination half-life; MRT, mean retention time; CL, total plasma clearance; Vd, the apparent distribution volume; UGT, UDP-glucuronosyltransferase; SULT, sulfotransferase.
■
REFERENCES
(1) Koehn, F. E.; Carter, G. T. The evolving role of natural products in drug discovery. Nat. Rev. Drug Discovery 2005, 4 (3), 206−20. (2) Carter, G. T. Natural products and Pharma 2011: strategic changes spur new opportunities. Nat. Prod. Rep. 2011, 28 (11), 1783−9. (3) Holloway, D. M.; Scheinmann, F. Extractives from Guttiferae. 30. Phenolic compounds from the heartwood of Garcinia mangostana. Phytochemistry 1975, 14 (11), 2517−18. (4) Jiang, H. Z.; Quan, X. F.; Tian, W. X.; Hu, J. M.; Wang, P. C.; Huang, S. Z.; Cheng, Z. Q.; Liang, W. J.; Zhou, J.; Ma, X. F.; Zhao, Y. X. Fatty acid synthase inhibitors of phenolic constituents isolated from Garcinia mangostana. Bioorg. Med. Chem. Lett. 2010, 20 (20), 6045−6047. (5) Tusevski, O.; Stanoeva, J. P.; Stefova, M.; Kungulovski, D.; Pancevska, N. A.; Sekulovski, N.; Panov, S.; Simic, S. G. Hairy roots of Hypericum perforatum L.: a promising system for xanthone production. Cent. Eur. J. Biol. 2013, 8 (10), 1010−1022. (6) Tusevski, O.; Vinterhalter, B.; Krstic Milosevic, D.; Sokovic, M.; Ciric, A.; Vinterhalter, D.; Zdravkovic Korac, S.; Petreska Stanoeva, J.; Stefova, M.; Gadzovska Simic, S. Production of phenolic compounds, antioxidant and antimicrobial activities in hairy root and shoot cultures of Hypericum perforatum L. Plant Cell. Plant Cell, Tissue Organ Cult.. 2017, Ahead of Print.128 (3), 589−605. (7) Ghosal, S.; Chaudhuri, R. K. Chemical constituents of the Gentianaceae. VI. New tetraoxygenated xanthones of Canscora decussata. Phytochemistry 1973, 12 (8), 2035−8. (8) Han, B.; Yang, X.; Liao, X.; Gao, C.; Wang, F.; Jiang, R.; Yang, B.; Zhao, Y. Host-guest inclusion system of norathyriol with βcyclodextrin and its derivatives: preparation, characterization, and anticancer activity. J. Biosci. Bioeng. 2014, 117 (6), 775−9. 12234
DOI: 10.1021/acs.jafc.8b03763 J. Agric. Food Chem. 2018, 66, 12227−12235
Article
Journal of Agricultural and Food Chemistry metabolite norathyriol on the transactivation of peroxisome proliferator-activated receptor isoforms. J. Agric. Food Chem. 2008, 56 (9), 3037−42. (27) Niu, Y.; Liu, J.; Liu, H.-Y.; Gao, L.-H.; Feng, G.-H.; Liu, X.; Li, L. Hypouricaemic action of mangiferin results from metabolite norathyriol via inhibiting xanthine oxidase activity. Pharm. Biol. (London, U. K.) 2016, 54 (9), 1680−1686. (28) Han, D.; Chen, C.; Zhang, C.; Zhang, Y.; Tang, X. Determination of mangiferin in rat plasma by liquid-liquid extraction with UPLC-MS/MS. J. Pharm. Biomed. Anal. 2010, 51 (1), 260−3. (29) Tian, X.; Gao, Y.; Xu, Z.; Lian, S.; Ma, Y.; Guo, X.; Hu, P.; Li, Z.; Huang, C. Pharmacokinetics of mangiferin and its metaboliteNorathyriol, Part 1: Systemic evaluation of hepatic first-pass effect in vitro and in vivo. BioFactors 2016, 42 (5), 533−544. (30) Tian, X.; Xu, Z.; Li, Z.; Ma, Y.; Lian, S.; Guo, X.; Hu, P.; Gao, Y.; Huang, C. Pharmacokinetics of mangiferin and its metabolitenorathyriol, Part 2: Influence of UGT, CYP450, P-gp, and enterobacteria and the potential interaction in Rhizoma Anemarrhenae decoction with timosaponin B2 as the major contributor. BioFactors 2016, 42 (5), 545−555. (31) Guo, J.; Xu, C.; Xue, R.; Jiang, W.; Wu, B.; Huang, C. Cytotoxic activities of chemical constituents from rhizomes of Anemarrhena asphodeloides and their analogues. Arch. Pharmacal Res. 2015, 38 (5), 598−603. (32) Shen, L.; Hillebrand, A.; Wang, D. Q. H.; Liu, M. Isolation and primary culture of rat hepatic cells. J. Visualized Exp. 2012, 64, E39171−E3917-4. (33) Davies, B.; Morris, T. Physiological parameters in laboratory animals and humans. Pharm. Res. 1993, 10 (7), 1093−5. (34) Sun, D.; Zhang, C. Z.; Ran, R. X.; Cao, Y. F.; Du, Z.; Fu, Z. W.; Huang, C. T.; Zhao, Z. Y.; Zhang, W. H.; Fang, Z. Z. In Vitro Comparative Study of the Inhibitory Effects of Mangiferin and Its Aglycone Norathyriol towards UDP-Glucuronosyl Transferase (UGT) Isoforms. Molecules 2017, 22 (6), 1008.
12235
DOI: 10.1021/acs.jafc.8b03763 J. Agric. Food Chem. 2018, 66, 12227−12235
