Article pubs.acs.org/JAFC
Liquid Chromatography−Tandem Mass Spectrometry Determination and Pharmacokinetic Analysis of Amentoflavone and Its Conjugated Metabolites in Rats Sha Liao,† Qiuxia Ren,‡ Cuiping Yang,† Tianhong Zhang,† Jinglai Li,† Xiaoying Wang,† Xinyan Qu,‡ Xiaojuan Zhang,‡,§ Zhe Zhou,‡ Zhenqing Zhang,*,† and Shengqi Wang*,‡ †
Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Haidian District, Beijing, 100850 P. R. China Beijing Institute of Radiation Medicine, 27 Taiping Road, Haidian District, Beijing 100850, P. R. China § Department of Preventive Medicine, Qinghai University School of Medicine, 6 Kunlunlu, Xining 810001, P. R. China ‡
S Supporting Information *
ABSTRACT: Amentoflavone (AMF) is a biflavone found in many herbal dietary supplements. To investigate the pharmacokinetic profile of AMF in rats, a sensitive, simple, and accurate liquid chromatography−tandem mass spectrometry (LC−MS/MS) method was developed and used to monitor AMF and its conjugated metabolites in plasma. AMF was administered to rats by oral gavage (po), or by intravenous (iv) or intraperitoneal (ip) injection. Plasma samples (with apiolin as an internal standard) were liquid/liquid extracted after hydrolysis with β-glucuronidase/sulfatase in vitro. Following chromatographic separation on a C18 column with a methanol:water:formic acid (70:30:0.1, v/v/v) mobile phase, AMF and internal standard were determined by electrospray ionization in negative ion mode and their precursor−product ion pairs (m/z 537.1 → 374.9 and m/z 269.2 → 224.9, respectively) were used for measurement. This bioanalytical method was fully validated and showed good linearity (r2 > 0.99), wide dynamic range (0.93−930 nmol/L), and favorable accuracy and precision. After iv or ip AMF (10 mg/kg) injection, 73.2% ± 6.29% and 70.2% ± 5.18% of the total AMF detected in plasma was present as conjugated metabolites. Furthermore, AMF and AMF conjugates showed similar time courses with no significant differences in the time to reach the maximum plasma concentration (tmax) and terminal half-life (t1/2) (p > 0.05). Following po AMF administration (300 mg/kg), 90.7% ± 8.3% of the total AMF was circulating as conjugated metabolites. When compared with iv administration (with dose correction), the bioavailability of po AMF was very low (0.04% ± 0.01% for free AMF; 0.16% ± 0.04% for conjugated AMF). Collectively, these data provided a preliminary pharmacokinetic profile for AMF that should inform further evaluations of its biological efficacy and preclinical development. KEYWORDS: amentoflavone, pharmacokinetics, β-glucuronidase/sulfatase, liquid-phase extraction
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INTRODUCTION
of which are based on high-performance liquid chromatography (HPLC) with detection by UV spectrometry,4 Fourier transform infrared (FTIR) spectrometry,5 or mass spectrometry.6 These methods were developed to quantify flavonoids and their derivatives in raw herbal extracts, where flavonoid concentrations are often very high. Consequently, these methods are not sufficiently sensitive or selective to enable the detection of low concentrations of AMF in plasma. Recently, Michler et al.7and Wang et al.8 reported the detection of free AMF and AMF conjugates in rat or human blood following administration of extracts from St. John’s wort and the Traditional Chinese Medicine (TCM), Shixiao San, both of which contain AMF. However, because flavonoids are reported to be substrates of uptake and efflux transporters such as sodium dependent glucose transporter 1 (SGLT1), monocarboxylate, multidrug resistance-associated proteins 2 (MRP2), and other MRP2 isoforms,9−13 as well as potent
Biflavonoids are polyphenolic compounds that are widely distributed and abundant in plants. These compounds are known to have antioxidant activities that can be beneficial for treating various diseases associated with oxidative stress, such as cancer, cardiovascular, and neurodegenerative diseases. Amentoflavone (AMF, Figure 1A), a biflavonoid active constituent of Biophytum sensitivum and some other plants, is commonly used in oriental medicines. AMF has been reported to have multiple biological activities, including anti-inflammatory1 and antioxidant effects, inhibition of nitric oxide synthase in macrophages,2 inhibition of nonenzymatic lipid peroxidation, superoxide scavenging, and inhibition of cytosolic fraction (IκBα) degradation and prevention of nuclear factor κB (NF-κB) translocation to the nucleus.3 Despite these promising activities, the pharmacokinetics of AMF has not been well characterized, beyond establishing its poor oral bioavailability. An improved understanding of the pharmacokinetics of AMF would help to clarify its in vivo mechanisms of action and could facilitate its use as a human therapeutic agent. Several methods for the qualitative and quantitative characterization of AMF have been published, most © XXXX American Chemical Society
Received: April 26, 2014 Revised: November 19, 2014 Accepted: November 21, 2014
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(HPLC grade) was purchased from Sigma-Aldrich (St. Louis, MO). βGlucuronidase (type H-1 from Helix pomatia, which contained 300 units of β-glucuronidase and 15.3 units of sulfatase/mg), was purchased from Sigma-Aldrich. Formic acid was purchased from J.T. Baker. Wahaha deionized water (Hangzhou, China) was used throughout the study. Ethyl acetate and other chemicals were of analytical grade and obtained from Beijing Chemical Co. (Beijing, China). Animal Treatment and Pharmacokinetic Analysis. Male Sprague−Dawley (SD) rats (190−210 g) were supplied by Beijing Vital River Laboratories Animal Technology Co. Ltd. All the experimental protocols were approved by the Animal Ethics Committee of Beijing Institute of Pharmacology and Toxicology (No. 11400700031602). Fifteen SD rats were randomly divided into three groups and were fasted overnight with free access to water prior to AMF administration. Animals in the first group received a single 10 mg/kg intravenous (iv) dose of AMF via the tail vein. Animals in the second group received a single 10 mg/kg intraperitoneal (ip) dose of AMF suspended in 0.1% Tween 80. Animals in the third group received a single 300 mg/kg dose of AMF suspended in 1% Tween 80 by oral gavage (po). Blood samples were obtained via retro-orbital puncture with a glass capillary at 0, 0.033, 0.083, 0.167, 0.25, 0.5, 1, 2, 4, 6, 9, 12, and 24 h after iv and ip administration and at 0, 0.017, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, and 24 h after po administration. The blood samples were collected into heparinized plastic tubes, and the plasma was separated by centrifugation at 4000 rpm at 4 °C for 10 min. The resulting plasma samples were stored frozen (−30 °C) until analyzed. LC−MS/MS Method. The LC method employed a Thermo Syncronis C18 column (2.1 mm × 50 mm, 5 μm) equipped with an online filter and maintained at room temperature. Using a Finnigan Surveyor HPLC system, peaks were separated using a mobile phase of methanol, water, and formic acid (70:30:0.1, v/v/v) at a flow rate of 0.2 mL/min. The injection volume was 5 μL, and the run time was 3 min. A triple-stage quadrupole mass spectrometer (TSQ Quantum Discovery, ThermoElectron, San Jose, CA) equipped with an electrospray ionization (ESI) source was used for AMF detection. Xcalibur version 1.4 software (Thermo-Fisher, USA) controlled the HPLC system, mass spectrometer, and autosampler, and data analyses were completed using LCQuan software (Thermo Finnigan, USA). The mass spectrometer was operated in the negative ion detection mode, and quantification was performed using selected reaction monitoring (SRM) of the transitions of m/z 537.1 → m/z 374.9 for AMF (Figure 2A) and of m/z 269.2 → m/z 224.9 for apiolin (as an internal standard, Figure 2B) with a scan time of 0.1 s per transition. The optimum ion source parameters were as follows: ESI source temperature, 300 °C; spray voltage, 3500 V; sheath gas pressure, 43 psi; auxiliary gas pressure, 20 psi. Standard and Quality Control (QC) Sample Preparation. Stock solutions of AMF and the internal standard were prepared individually by dissolving 10 mg of AMF or 20 mg of apiolin in 100 mL of methanol. Ten working standard solutions containing 9.3, 18.6, 37.2, 93.0, 186, 465, 930, 1860, 4650, and 9300 nmol/L of AMF were prepared by serial dilution of the stock solution using appropriate volumes of methanol. A working solution of the internal standard was prepared by diluting the stock solution with ethyl acetate by a factor of 1000 to a final concentration of 700 nmol/L. All stock solutions were stored at 4 °C, and all working solutions were freshly prepared each week. Plasma calibration standards were prepared by spiking 45 μL of fresh rat plasma with 5 μL of the appropriate working solution to achieve AMF concentrations of 0.93, 1.86, 3.72, 9.30, 18.6, 46.5, 93, 186, 465, and 930 nmol/L. QC samples containing three concentrations of AMF (low, 2.79 nmol/L; medium, 37.2 nmol/L; and high, 744 nmol/L) were prepared similarly. Sample Preparation. The utilization of a mixture of βglucuronidase/sulfatase to hydrolyze glucuronidated/sulfated conjugates has been successfully applied for the quantification of drugs in plasma.19−23 In brief, prior to the determination of the total AMF (including free AMF plus its conjugated metabolites), plasma samples were treated with β-glucuronidase/sulfatase (type H-1) in vitro,
Figure 1. Chemical structure of amentoflavone AMF (A) and the internal standard (B).
inhibitors of cytochrome (CYP) 2C9, CYP 3A4, and especially CYP 1A2/1A1,14−16 the other flavones present in the herbal extracts used in these studies could potentially alter the metabolism and disposition of AMF. On the other hand, because of simultaneous quantitation of multiple free and total biflavone components, including AMF, in rat plasma and human urine, respectively, the short cycle time and limit of detection were sacrificed for sufficient separation and accurate quantification of multicomponents in both methods. To improve understanding of the bioactivity and role of AMF beyond that provided by previous pharmacodynamics studies,17,18 we developed a new specific, sensitive, and rapid liquid chromatography−tandem mass spectrometry (LC−MS/MS) method to detect the total content of AMF following βglucuronidase/sulfatase hydrolysis. The present method had the advantages of a shorter detection time and lower limit of detection than the two published methods.7,8 We used this method to generate a full pharmacokinetic profile for AMF in rats by administering a single dose by various routes, and then fully analyzing the conjugate metabolites of AMF, including its glucuronide/sulfate forms. Further, the pharmacokinetic differences between AMF administered to rats via alternative routes and the bioavailability of the compound were investigated. The resulting data are very useful for developing an improved understanding of the pharmacological profile of AMF in vivo, which will be beneficial for preclinical research and clinical use of AMF.
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MATERIALS AND METHODS
Chemicals. AMF (98.0%) and apiolin (internal standard, 99.0%) were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Methanol B
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Figure 2. Product ion mass spectra of deprotonated molecules of amentoflavone (AMF, A) and the internal standard (B). whereas no enzyme treatment was used before determination of free AMF. However, extensive efforts were made to optimize the conditions used in this first enzymatic step prior to liquid−liquid extraction (Supporting Information). Based on these results, we established the following optimal enzymolysis conditions: For enzymolysis, 50 μL plasma samples were mixed with 50 μL of βglucuronidase/sulfatase solution in 0.1 M acetate buffer, pH 5 (500/ 16.6 units/mL for samples from rats dosed po and 5,000/166 units/ mL for samples from rats dosed iv and ip), and 10 μL of ascorbic acid (200 mg/mL), followed by incubation at 37 °C for 12 h under anaerobic conditions in the dark with gentle shaking. After hydrolysis, the plasma was partitioned using 1 mL of ethyl acetate containing 700 nmol/L of the internal standard. The mixture was vortexed for 3 min and centrifuged at 4000g for 10 min at 4 °C. The ethyl acetate layer was then evaporated to dryness under N2 gas and reconstituted with 50 μL of methanol. For determination of free AMF, 50 μL plasma samples were processed in the same manner, except that no enzyme was included in the 0.1 M acetate buffer. To prepare calibrator samples, 45 μL of plasma was mixed with 5 μL of the appropriate AMF working solution. The mixture was added to 50 μL of 0.1 M acetate buffer (pH 5) and processed in the same way as the other samples. Method Validation. The method was validated for selectivity, linearity, precision, accuracy, recovery, matrix effect, and stability. The validation assays were carried out in accordance with currently
accepted U.S. Food and Drug Administration (FDA) guidelines for use in industry.24 Specificity. The specificity of the LC−MS/MS method was investigated by comparing blank rat plasma from six different rats with the corresponding rat plasma samples spiked with AMF at the lower limit of quantification (LLOQ, 0.93 nmol/L). This enabled evaluation of the influences of endogenous substances in rat plasma on AMF quantification. Recovery and Matrix Effect. The extraction recovery of AMF from rat plasma was determined at the LLOQ (0.93 nmol/L) and at 2.79, 37.2, and 744 nmol/L for QC samples. Extraction recovery was defined as the ratio of the analyte peak area from extracted QC plasma to the mean peak area from extracted blank plasma spiked with AMF. Matrix effects were quantified as the ratio of the analyte peak area from an extracted plasma blank spiked with AMF to the mean peak area observed during analysis of the standard AMF solution, at the same concentration, prepared in methanol. Linearity and LLOQ. Calibration curves were calculated by linear regression analysis of the peak area ratio of AMF to internal standard plotted against the normalized concentration of AMF, with a weighting factor of 1/x2. Curve fits were not forced to intersect the origin. The LLOQ of the assay was defined as the lowest concentration of AMF detected with precision and accuracy lower than 20% (n = 6). Precision and Accuracy. QC samples were processed (n = 6) at the LLOQ and at 2.79, 37.2, and 744 nmol/L in rat plasma for each run C
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Figure 3. Representative selected reaction monitoring (SRM) chromatograms of amentoflavone (AMF, I) and internal standard (II) in rat plasma: (A) blank plasma sample; (B) blank plasma sample spiked with AMF at the lowest limit of quantification (0.93 nmol/L) and the internal standard (700 nmol/L); (C) plasma sample obtained from a rat 3 h after oral administration of AMF, when the concentration of AMF was found to be 0.16 nmol/mL. over 3 days. The concentrations in the QC and the unknown samples were calculated from the equation of the calibration curve. Accuracy
was measured by the deviation or bias (%) of the mean calculated concentration from the actual concentration. Precision was defined as D
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Journal of Agricultural and Food Chemistry the coefficient of variation (CV, %). Precision and accuracy were determined within each day for the LLOQ and three concentrations of QCs, with six replicates and one calibration set prepared each day. Precision and accuracy between different days were evaluated for each QC concentration and for the LLOQ using 18 replicates obtained over three different days. Stability and Dilution. The stability of AMF in rat plasma at the LLOQ and at the three QC concentrations was determined at room temperature (24 °C for 6 h), after three freeze−thaw cycles (−30 °C to room temperature), at the storage temperature (14 days at −30 °C), and in the autosampler at 4 °C. Five aliquots were analyzed for each condition, and the stability was calculated according to a published procedure.25 The analyte was considered stable when the accuracy bias was within ±15% of the nominal concentration. Notably, some samples obtained from animals receiving iv AMF were observed to have AMF levels exceeding the highest concentration of the calibration curve. Therefore, to accurately evaluate the precision and accuracy for these samples, 25- and 5-fold more concentrated QC samples were diluted into blank matrix and analyzed in the same way as the other samples.26 For these samples, accuracy was within ±15% and precision was ≤20%. Carry-Over. To evaluate carry-over between samples, a blank sample was tested immediately after testing an AMF standard at the upper limit of quantitation (ULOQ).27 Data Evaluation and Statistics. The concentrations of the conjugate metabolites of AMF in plasma were calculated by comparing samples treated with β-glucuronidase/sulfatase to samples with no enzyme treatment, as follows:
z 374.9) and the internal standard (m/z 224.9) are shown in Figure 2. We found that changing the methanol:water ratio from 90:10 to 50:50 (v/v) improved the MS response area and peak symmetry and also reduced the run time for the target ions. For chromatographic separation, we found that a mobile phase of 70% methanol at a flow rate of 0.2 mL/min gave an ideal retention time of less than 3 min, which enabled rapid analysis of multiple samples. We also observed that, compared to lower percentages, 70% methanol enhanced the peak signal, most likely by enhancing ionization. Like most biflavonoids, AMF contains a carboxylic acid group, making the pH of the solvent critical for mass response. When 0.1% formic acid was included in the mobile phase, the peaks of both AMF and the internal standard were symmetrical and sharp. The sample preparation method was also optimized to ensure reproducibility, minimal matrix effects, and a low cost. Apiolin was chosen as the internal standard because it has similar chemical structure, physicochemical properties, and mass spectrometric behavior to those of AMF. A single-step liquid−liquid extraction procedure was used to enrich and clean the target compound from plasma. Although several organic solvents, including ethyl acetate, acetonitrile, methanol, and diethyl ether, were investigated as extraction solvents, ethyl acetate produced the highest recovery and cleanest samples. Method Validation. Selectivity. The SRM chromatograms of blank rat plasma, blank plasma spiked with AMF at the LLOQ (0.93 nmol/L) and with the internal standard (700 nmol/L), and plasma samples obtained 3 h after po administration of 300 mg/kg AMF are shown in Figure 3. No endogenous interference was observed in the chromatograms of control blank rat plasma (n = 6) over the period when AMF and the internal standard eluted, indicating that the analysis was highly selective. Linearity and Sensitivity. The LLOQ for this method was 0.93 nmol/L for AMF in rat plasma. The standard curve was obtained by plotting the ratio of AMF to internal standard peak area versus the concentration of AMF added (0.93−930 nmol/ L). Linear regression to 1/x2 yielded a correlation coefficient ≥0.9955, and the slope of each regression line was reproducible, with a relative standard deviation of 0.05).
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DISCUSSION Herein, we report the development and validation of a method for the accurate determination of plasma concentrations of the free and conjugate metabolized forms of AMF, an important bioactive biflavonoid derived from various herbal sources. Since no authentic standards exist for the glucuronide or sulfate metabolites of AMF, the method included in vitro treatment with β-glucuronidase/sulfatase, liquid−liquid extraction, centrifugation, and analysis by LC−MS/MS. Compared with the previously reported methods,7,8 the present assay showed excellent performance in terms of sensitivity (increased by about 4-fold) and throughput (3 min per sample versus 6−10 min per sample). Using this method, we could accurately detect both the conjugated and nonconjugated forms of AMF. The high sensitivity and accuracy, as well as the rapid sample analysis, make this method suitable for routine evaluation of AMF pharmacokinetics. We used the method to compare pharmacokinetic behavior following administration by three different routes and to study the bioavailability of AMF in rats. Although encouraging efficacy data has been reported for AMF in rats,17,18there have been no careful analyses of the drug metabolism and pharmacokinetic (DMPK) properties of AMF. Previous analyses of related compounds demonstrated that the biflavonoid glycoside moiety can undergo extensive biotransformation in vivo,29,31 and that the glucuronide and sulfate conjugates are actually the predominant forms found in the bloodstream, urine, and bile. Hence, it is reasonable to assume that the conjugates are the bioactive forms.30 Our preliminary investigations of the biotransformation of AMF after po dosing in rats revealed that glucuronide, sulfate, and sulfate/ glucuronide conjugates predominated over the parent compound in the urine and feces, and an in situ perfused rat intestine−liver model also confirmed this result (unpublished data). Thus, like other biflavonoids, the conjugated form of AMF is most likely to be the bioactive form. The dose of AMF used in this study was based on previously reported levels, as well as on our recent pharmacodynamics studies of efficacy. We have previously observed that AMF (20, 10, and 5 mg/kg iv and ip, and 300 mg/kg po) protected from the sperm damage induced by microwave radiation in rats (unpublished data). There are at least two published papers
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ASSOCIATED CONTENT
S Supporting Information *
Figure depicting time course of AMF release by βglucuronidase/sulfatase at different concentrations in plasma samples obtained from rats after iv, ip, and po administration. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*(Z.Z.) Tel/fax: +86 10 66931632. E-mail: zqzhang55@126. com;
[email protected]. H
DOI: 10.1021/jf5019615 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry *(S.W.) Tel/fax: +86 10 66932211. E-mail:
[email protected]. ac.cn.
caffeine N3-demethylation by naturally occurring flavonoids. Biochem. Pharmacol. 1998, 55, 1369−1375. (16) Zhai, S.; Dai, R.; Friedman, F. K.; Vestal, R. E. Comparative inhibition of human cytochromes P450 1A1 and 1A2 by flavonoids. Drug Metab. Dispos. 1998, 26, 989−992. (17) Kim, H. K.; Son, K. H.; Chang, H. W.; Kang, S. S.; Kim, H. P. Amentoflavone, a plant biflavone: a new potential anti-inflammatory agent. Arch. Pharm. Res. 1998, 21, 406−410. (18) Sakthivel, K. M.; Guruvayoorappan, C. Amentoflavone inhibits iNOS, COX-2 expression and modulates cytokine profile, NF-kappaB signal transduction pathways in rats with ulcerative colitis. Int. Immunopharmacol. 2013, 17, 907−916. (19) Azorin-Ortuno, M.; Yanez-Gascon, M. J.; Pallares, F. J.; Vallejo, F.; Larrosa, M.; Garcia-Conesa, M. T.; Tomas-Barberan, F.; Espin, J. C. Pharmacokinetic study of trans-resveratrol in adult pigs. J. Agric. Food. Chem. 2010, 58, 11165−11171. (20) Azuma, K.; Ippoushi, K.; Nakayama, M.; Ito, H.; Higashio, H.; Terao, J. Absorption of chlorogenic acid and caffeic acid in rats after oral administration. J. Agric. Food. Chem. 2000, 48, 5496−5500. (21) Jan, K. C.; Ho, C. T.; Hwang, L. S. Bioavailability and tissue distribution of sesamol in rat. J. Agric. Food. Chem. 2008, 56, 7032− 7037. (22) Konishi, Y.; Hitomi, Y.; Yoshida, M.; Yoshioka, E. Pharmacokinetic study of caffeic and rosmarinic acids in rats after oral administration. J. Agric. Food. Chem. 2005, 53, 4740−4746. (23) Zhao, G.; Zou, L.; Wang, Z.; Hu, H.; Hu, Y.; Peng, L. Pharmacokinetic profile of total quercetin after single oral dose of tartary buckwheat extracts in rats. J. Agric. Food. Chem. 2011, 59, 4435−4441. (24) Center for Drug Evaluation and Research of the U.S. Department of Healthand Human Services Food and Drug Administration, Guidance for indus-try: Bioanalytical method validation, 2001. http://www.fda.gov/Drugs/ GuidanceComplianceRegulatoryInformation/Guidances/ucm064964. htm (accessed 10.12.12). (25) Timm, U.; Wall, M.; Dell, D. A new approach for dealing with the stability of drugs in biological fluids. J. Pharm. Sci. 1985, 74, 972− 977. (26) Amoako, A. A.; Marczylo, T. H.; Lam, P. M.; Willets, J. M.; Derry, A.; Elson, J.; Konje, J. C. Quantitative analysis of anandamide and related acylethanolamides in human seminal plasma by ultra performance liquid chromatography tandem mass spectrometry. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2010, 878, 3231−3237. (27) Ghassabian, S.; Moosavi, S. M.; Valero, Y. G.; Shekar, K.; Fraser, J. F.; Smith, M. T. High-throughput assay for simultaneous quantification of the plasma concentrations of morphine, fentanyl, midazolam and their major metabolites using automated SPE coupled to LC-MS/MS. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2012, 903, 126−133. (28) Guruvayoorappan, C.; Kuttan, G. Amentoflavone inhibits experimental tumor metastasis through a regulatory mechanism involving MMP-2, MMP-9, prolyl hydroxylase, lysyl oxidase, VEGF, ERK-1, ERK-2, STAT-1, NM23 and cytokines in lung tissues of C57BL/6 mice. Immunopharmacol. Immunotoxicol. 2008, 30, 711−727. (29) Hollman, P. C.; de Vries, J. H.; van Leeuwen, S. D.; Mengelers, M. J.; Katan, M. B. Absorption of dietary quercetin glycosides and quercetin in healthy ileostomy volunteers. Am. J. Clin. Nutr. 1995, 62, 1276−1282. (30) Akao, T.; Kawabata, K.; Yanagisawa, E.; Ishihara, K.; Mizuhara, Y.; Wakui, Y.; Sakashita, Y.; Kobashi, K. Baicalin, the predominant flavone glucuronide of scutellariae radix, is absorbed from the rat gastrointestinal tract as the aglycone and restored to its original form. J. Pharm. Pharmacol. 2000, 52, 1563−1568. (31) Williamson, G.; Day, A. J.; Plumb, G. W.; Couteau, D. Human metabolic pathways of dietary flavonoids and cinnamates. Biochem. Soc. Trans. 2000, 28, 16−22. (32) Osorio, E.; Londono, J.; Bastida, J. Low-density lipoprotein (LDL)-antioxidant biflavonoids from Garcinia madruno. Molecules 2013, 18, 6092−6100.
Funding
This work was supported by two grants from the National Natural Science Foundation of China (No. 81172981, No. 81001469 and No. 81373472) and the National Science and Technology Major Projects for Major New Drugs Innovation and Development of China (2012ZX09301003-001-007). Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Kim, H. K.; Son, K. H.; Chang, H. W.; Kang, S. S.; Kim, H. P. Amentoflavone, a plant biflavone: a new potential anti-inflammatory agent. Arch. Pharm. Res. 1998, 21, 406−410. (2) Woo, E. R.; Lee, J. Y.; Cho, I. J.; Kim, S. G.; Kang, K. W. Amentoflavone inhibits the induction of nitric oxide synthase by inhibiting NF-kappaB activation in macrophages. Pharmacol. Res. 2005, 51, 539−546. (3) Banerjee, T.; Valacchi, G.; Ziboh, V. A.; van der Vliet, A. Inhibition of TNFalpha-induced cyclooxygenase-2 expression by amentoflavone through suppression of NF-kappaB activation in A549 cells. Mol. Cell. Biochem. 2002, 238, 105−110. (4) Simmen, U.; Higelin, J.; Berger-Buter, K.; Schaffner, W.; Lundstrom, K. Neurochemical studies with St. John’s wort in vitro. Pharmacopsychiatry 2001, 34 (Suppl. 1), S137−142. (5) Rager, I.; Roos, G.; Schmidt, P. C.; Kovar, K. A. Rapid quantification of constituents in St. John’s wort extracts by NIR spectroscopy. J. Pharm. Biomed. Anal. 2002, 28, 439−446. (6) Tatsis, E. C.; Boeren, S.; Exarchou, V.; Troganis, A. N.; Vervoort, J.; Gerothanassis, I. P. Identification of the major constituents of Hypericum perforatum by LC/SPE/NMR and/or LC/MS. Phytochemistry 2007, 68, 383−393. (7) Michler, H.; Laakmann, G.; Wagner, H. Development of an LCMS method for simultaneous quantitation of amentoflavone and biapigenin, the minor and major biflavones from Hypericum perforatum L., in human plasma and its application to real blood. Phytochem. Anal. 2011, 22, 42−50. (8) Wang, X.; Zhao, X.; Gu, L.; Lv, C.; He, B.; Liu, Z.; Hou, P.; Bi, K.; Chen, X. Simultaneous determination of five free and total flavonoids in rat plasma by ultra HPLC-MS/MS and its application to a comparative pharmacokinetic study in normal and hyperlipidemic rats. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2014, 953−954, 1−10. (9) Walgren, R. A.; Lin, J. T.; Kinne, R. K.; Walle, T. Cellular uptake of dietary flavonoid quercetin 4′-beta-glucoside by sodium-dependent glucose transporter SGLT1. J. Pharmacol. Exp. Ther. 2000, 294, 837− 843. (10) Wolffram, S.; Block, M.; Ader, P. Quercetin-3-glucoside is transported by the glucose carrier SGLT1 across the brush border membrane of rat small intestine. J. Nutr. 2002, 132, 630−635. (11) Walgren, R. A.; Lin, J. T.; Kinne, R. K.; Walle, T. Cellular uptake of dietary flavonoid quercetin 4′-beta-glucoside by sodium-dependent glucose transporter SGLT1. J. Pharmacol. Exp. Ther. 2000, 294, 837− 843. (12) Vaidyanathan, J. B.; Walle, T. Cellular uptake and efflux of the tea flavonoid (−)epicatechin-3-gallate in the human intestinal cell line Caco-2. J. Pharmacol. Exp. Ther. 2003, 307, 745−752. (13) Walle, T.; Walle, U. K. The beta-D-glucoside and sodiumdependent glucose transporter 1 (SGLT1)-inhibitor phloridzin is transported by both SGLT1 and multidrug resistance-associated proteins 1/2. Drug Metab. Dispos. 2003, 31, 1288−1291. (14) Tsyrlov, I. B.; Mikhailenko, V. M.; Gelboin, H. V. Isozyme- and species-specific susceptibility of cDNA-expressed CYP1A P-450s to different flavonoids. Biochim. Biophys. Acta 1994, 1205, 325−335. (15) Lee, H.; Yeom, H.; Kim, Y. G.; Yoon, C. N.; Jin, C.; Choi, J. S.; Kim, B. R.; Kim, D. H. Structure-related inhibition of human hepatic I
DOI: 10.1021/jf5019615 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry (33) Piskula, M. K.; Terao, J. Accumulation of (−)-epicatechin metabolites in rat plasma after oral administration and distribution of conjugation enzymes in rat tissues. J. Nutr. 1998, 128, 1172−1178. (34) Rondini, L.; Peyrat-Maillard, M. N.; Marsset-Baglieri, A.; Berset, C. Sulfated ferulic acid is the main in vivo metabolite found after shortterm ingestion of free ferulic acid in rats. J. Agric. Food. Chem. 2002, 50, 3037−3041. (35) Spencer, J. P.; Chowrimootoo, G.; Choudhury, R.; Debnam, E. S.; Srai, S. K.; Rice-Evans, C. The small intestine can both absorb and glucuronidate luminal flavonoids. FEBS Lett. 1999, 458, 224−230. (36) Griffiths, L. A.; Barrow, A. Metabolism of flavonoid compounds in germ-free rats. Biochem. J. 1972, 130, 1161−1162. (37) Bokkenheuser, V. D.; Shackleton, C. H.; Winter, J. Hydrolysis of dietary flavonoid glycosides by strains of intestinal Bacteroides from humans. Biochem. J. 1987, 248, 953−956.
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