Biotransformation and Metabolic Profile of Limonin in Rat Liver

Sep 27, 2018 - Limonin is a triterpenoid in citrus seeds, which has significant biological activities. However, the metabolic profile of limonin has n...
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Biotransformation and Metabolic Profile of Limonin in Rat Liver Microsomes, Bile, and Urine by High-Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry Shijia Liu,† Guoliang Dai,† Luning Sun,† Bingting Sun,‡ Du Chen,§ Lei Zhu,† Yao Wang,† Li Zhang,‡ Peidong Chen,*,‡ Dong Zhou,*,∥ and Wenzheng Ju*,† Downloaded via UNIV OF LOUISIANA AT LAFAYETTE on September 27, 2018 at 15:30:31 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210029, People’s Republic of China Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210016, People’s Republic of China § State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang Road, Nanjing, Jiangsu 210009, People’s Republic of China ∥ Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, United States ‡

S Supporting Information *

ABSTRACT: Limonin is a triterpenoid in citrus seeds, which has significant biological activities. However, the metabolic profile of limonin has not been fully understood. To expound its metabolism in vivo and in vitro, the metabolites of limonin was studied by rat liver microsomes, urine, and bile. High-performance liquid chromatography/quadrupole time-of-flight mass spectrometry was used for identification. Among the metabolites, the structures of M1 and M3 were confirmed by chemical synthesis and nuclear magnetic resonance spectra analysis. Our results indicated that reduction and hydrolysis were the two major pathways during limonin metabolism in vivo and in vitro. The results from this work are valuable and important for understanding the metabolic process of limonin. KEYWORDS: limonin, metabolism, reduction, hydrolysis

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INTRODUCTION Limonin is a triterpenoid in citrus seeds, which contains a furanolactone core structure.1 Extensive studies have been conducted on the biological activities of limonin, such as antioxidant,2,3 anti-inflammatory,4 anti-human immunodeficiency virus (HIV), and anticancer.5 It was reported that limonin has strong activity to inhibit the rat hepatocarcinogenesis.6 Other studies have demonstrated that limonin has the effects on inhibiting proliferation of human colon cancer.7,8 The method to evaluate limonin in rat plasma has been reported,9 and previous experiments have elucidated the pharmacokinetic parameters of limonin in dog plasma and human urine.10,11 Limonin could produce many metabolites by hydroxylation and glycine reduction in liver microsomes,12 and the metabolic processes have also been demonstrated in zebrafish compared to other limonoid constituents recently.13 Bile excretion is an important route for the metabolism of drugs through the liver,14 and many compounds from traditional medicine have been studied for their metabolites and possible biotransformation in rat bile.15 However, the metabolic pathways and possible metabolites of limonin in vivo have not been fully understood, and this evidence is important for the exploration of the activity of limonin and its clinical therapeutic effect. Thus, a more accurate investigation is necessary to explore the metabolic characteristics of limonin for further understanding of its biological activity. Therefore, the purpose of the current study © XXXX American Chemical Society

is to identify limonin metabolites using high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (HPLC−Q−TOF/MS) in rat liver microsomes, urine, and bile. The results of our study may provide useful evidence for further metabolism and action mechanism evaluation on limonin.

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MATERIALS AND METHODS

Materials and Reagents. Limonin (purity of >96.8%) was obtained from Zhejiang Nanyang Pharmaceutical Group. Methanol and formic acid were purchased from Merck (Darmstadt, Germany), and acetonitrile was purchased from Tedia (Fairfield, OH, U.S.A.). All other chemicals and solvents were of analytical grade. Ultrapure water was purified by a Milli-Q system (Millipore, Bedford, MA, U.S.A.). Animals and Preparation of Limonin Samples for Oral Administration. Animal experiments were approved by the guidelines for animal care in Nanjing University of Chinese Medicine and based on the internationally accepted guidelines for the use and care of laboratory animals. Eight Sprague Dawley (SD) rats (180−220 g) were purchased from the Experimental Animal Center of China Pharmaceutical University (four females and four males). At 12 h before the experiment, the rats were kept at a fasting state but allowed access to water. The method for preparation of rat bile and urine Received: May 4, 2018 Revised: September 6, 2018 Accepted: September 12, 2018

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DOI: 10.1021/acs.jafc.8b02057 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry samples was in accordance with a previously published report.16 Blank samples (without drug substance) and drug-containing samples were frozen before the test. Biological Sample Collection and Pretreatment. Urine samples (n = 6) were collected in metabolic cages at 6, 12, and 24 h after oral administration of limonin (80 mg/kg). Each urine sample (0.2 mL) was extracted with ethyl acetate (1 mL) and immediately centrifuged at 12000g for 10 min. Supernatants were separated and removed the solvent by a nitrogen evaporator. The residue was dissolved in methanol and stored at −80 °C until analysis. Under light anesthesia, bile samples (n = 6) were obtained from the bile duct by a polyethylene tube at 6, 12, and 24 h after oral administration of limonin (80 mg/kg). Bile samples were centrifuged at 12000g for 10 min, and the separated supernatants were stored at −80 °C for the test. Blood samples were collected from the suborbital vein at 6, 12, and 24 h after oral administration of limonin (80 mg/kg). An aliquot of 200 μL of plasma was transferred to a 1.5 mL centrifuge tube, vortexmixed for 30 s, added to 1.0 mL of acetonitrile, and then vortex-mixed for another 3 min. The organic phase was separated to a polypropylene tube and centrifugated at 3500 rpm for 10 min. The supernatant fraction was evaporated to dryness at 50 °C for 40 min using a centrifugal thickener (CentriVap console, Labconco Corp., Kansas City, MO, U.S.A.). The residue was dissolved with 100 μL of mobile phase, vortexed for 2 min, and centrifuged at 12000g for 10 min at 4 °C. The supernatant fraction was collected, and 10 μL of the samples was injected for HPLC−Q−TOF/MS analysis.17,18 In Vitro Samples. Limonin was incubated with rat liver microsomes. The method for incubation was chosen based on the linear rate of formation of the metabolite. A typical incubation mixture consisted of 1 mg of microsomal protein/mL and other compositions as previously reported.18 Rat liver microsomes were prepared from adult SD rats in half genders. Briefly, rat livers were removed immediately after euthanasia. After the connective tissue around the liver was removed under aseptic conditions, the liver was washed repeatedly with 0.15 mol/L potassium chloride solution for 3 min. Liver scrap was homogenized at 9000 g/min for 10 min, and the supernatant was centrifuged at 100000g/min for another 60 min. The pink precipitate was collected, and the liver microsomes were suspended in phosphate-buffered saline (PBS), which contained 30% glycerol and was stored at −80 °C until use. Rat liver microsomes (1 mg/mL), MgCl2 (10 mmol/L), PBS (0.1 mol/L), and NADPH (1 mmol/L) were mixed. The mixture was preincubated at 37 °C for 60 min. The reaction was terminated at 4 °C by cold acetonitrile. A total of 200 μg of limonin was dissolved in 1 mL of methanol, and organic solvent was added to the reaction system with the volume less than 1% of the incubation reaction solution. After incubation in water bath for 12 h, the reaction was terminated by adding cool acetonitrile. The incubation mixtures were shaken intensely and centrifugated at 18000g for 5 min to remove precipitated protein. The supernatant was extracted again with ether, and 5 mL of the solution was dried by nitrogen at 45 °C. The residues were dissolved in 100 μL of mobile phase and centrifuged at 12000g for 10 min to obtain 10 μL of supernatants for HPLC−Q−TOF/MS analysis. Synthesis. To identify the structure of limonin metabolites, we synthesized M1 according to the method of Ruberto et al.19 M3 was synthesized according to the method of English and Williams.20 The synthesized products were identified by means of 1H and 13C nuclear magnetic resonance (NMR), heteronuclear single-quantum correlation (HSQC), and heteronuclear multiple-bond correlation (HMBC) with a Bruker Avance 500 (Bruker BioSpin, Rheinstetten, Germany). A total of 200 mg of limonin was dissolved in methanol (8 mL) and refluxed continuously for 2 h with 0.8 g of NaBH4 to obtain white powder (142 mg). The powder was purified by a silica gel column (12 g, 200−300 mesh) and eluted in a mixed solution made of chloroform and methanol with gradient elution. The eluent was divided into 32 fractions. Fractions 14 and 15 were collected and purified by chloroform/methanol (15:l) to obtain three fractions. The second

fraction produced a white crystal (M1, 102 mg) by crystallization with ether acetate and was identified as limonol.21 Limonin (200 mg) was dissolved in methanol (5 mL) and refluxed for 5 h with 0.6 g of LiOH to produce a white powder (142 mg). A silica gel column (12 g, 200−300 mesh) was used to purify the powder with a mixed solution made of chloroform and methanol with gradient elution. The eluent was divided into 45 fractions. Fractions 22−25 were collected and eluted with chloroform/methanol (9:l) to obtain four fractions. The last fraction was crystallized to yield a white crystal (86 mg) with ether acetate. The crystal was identified as limonoate A-ring lactone.22 The NMR spectra data of M1 and M3 were shown in Table 1.

Table 1. Chemical Shifts and J Value Data for M1 and M3 M1

M3

number

δH (J = Hz)

δC

δH (J = Hz)

δC

1 2

3.99, s H2a: 2.66, d (16.4) H2b: 2.57, d (16.4)

78.8 35.6

4.10, s H2a: 2.80, d (16.4) H2b: 2.64, d (16.4)

78.4 35.6

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

2.02, d (14.3) H6a: 1.64, d (11.1) H6b: 1.53, d (15.4) 3.71, d (4.6) 2.28, d (11.4, 5.9) H11a: 1.66, m H11b: 1.99, m H12a: 1.58, m H12b: 1.19, m

4.42, s 5.53, s 0.97, s H19a: 4.65, d (6.0) H19b: 4.77, d (6.6) 7.70, s 6.48, s 7.66, s 0.83, s H25a: 1.17, s H25b: 1.20, s

170.2 79.6 56.5 27.8 76.3 42.9 44.6 45.1 16.8 25.4 38.4 65.2 55.0 168.0 77.4 18.2 65.2 120.4 141.7 110.2 143.3 13.5 30.3 21.4

2.42, d (11.3) H6a: 2.29, d (9.6) H6b: 3.18, d (16.2)

2.54, d (11.1) H11a: 1.79, m H11b: 1.88, m H12a: 1.72, m H12b: 1.23, m

4.10, s 5.47, s 1.02, s H19a: 4.51, d (11.9) H19b: 4.94, d (11.9) 7.76, s 6.50, s 7.72, s 1.00, s H25a: 1.11, s H25b: 1.18, s

170.1 79.4 58.0 37.6 207.9 50.2 46.4 45.2 19.6 29.7 38.6 64.8 53.6 167.2 77.4 17.5 65.8 120.1 141.6 110.1 143.3 13.5 29.7 21.4

a

s, singlet hydrogen proton signal; d, doublet hydrogen proton signal; and m, multiplet hydrogen proton signal.

Chromatography and Mass Spectrometry (MS) Conditions. An Agilent 1200 high-performance liquid chromatography (HPLC) system equipped with a binary pump was used for the chromatographic analysis. The experiments were performed using an Agilent Zorbax SB-C18 column (100 × 3.0 mm). The mixed mobile phase consisted of solvent A (0.1% formic acid in water containing 2 mmol/ L ammonium formate) and solvent B (methanol). Separation was carried out by gradient elution. The gradient was as follows: 0−9 min, B increased linearly from 5 to 95%; 9−14.5 min, B was constant at 95%; 14.4−14.6 min, B decreased linearly from 95 to 5%; and 14.6− 21 min, B was constant at 5%. Other parameters were set as follows: flow rate, 0.35 mL/min; autosampler temperature, 15 °C; and column temperature, 35 °C. B

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Journal of Agricultural and Food Chemistry Table 2. Mass Spectral Data for Limonin and Its Metabolitesa metabolite

tR (min)

(m/z) [M + H]+

formula

error (ppm)

species/matrix

limonin M1 M2 M3 M4 M5

11.52 11.25 10.89 11.47 11.0 12.4

471.1981 473.2145 487.1937 489.2314 457.2207 455.2038

C26H30O8 C26H32O8 C26H30O9 C26H32O9 C26H32O7 C26H30O7

2.3 1.0 1.0 3.7 3.2 3.4

RB, RU, and RLM RB, RU, and RLM RU and RLM RB, RU, and RLM RB and RU RB and RU

fragment ions (m/z) 161.0594, 161.0588, 95.0490, 95.0151, 95.0490, 161.0604,

339.1931, 413.1947, 267.0933, 161.0609, 227.1781, 281.1157,

367.1884, 427.2102, 423.1802, 410.2041, 369.2050, 349.1777,

and and and and and and

425.1933 455.2043 441.1902 472.2042 421.2002 353.1730

a The [M + H]+ (m/z) values were accurate-mass-detected. The error (ppm) is the difference between the calculated and observed m/z values. These are tentative identifications based on HPLC−Q−TOF/MS. RU, rat urine; RB, rat bile; and RLM, rat liver microsomes.

Figure 1. Limonin metabolites in rat urine measured by LC−ion trap (IT)−TOF/MS: (A and B) extract ion and second-stage mass spectra of M1, (C and D) extract ion and second-stage mass spectra of M2, (E and F) extract ion and second-stage mass spectra of M3, (G and H) extract ion and second-stage mass spectra of M4, and (I and J) extract ion and second-stage mass spectra of M5. The MS experiment was performed on TripleTOF 5600 MS (AB SCIEX). A hybrid triple Q−TOF mass spectrometer equipped with an electrospray ionization (ESI) source in positive-ion mode was used to detect ion fragments. The conditions of TOF−MS were set as follows: curtain gas, 30 psi; gas 1, 55 psi; gas 2, 55 psi; TOF mass range, m/z 80−1000; temperature, 550 °C; declustering potential, 100 V; collision energy, 10 eV; and ion spray voltage, 5500 V.

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Identification of Metabolites. General. Five metabolites were identified in rat liver microsomes, urine, and bile as shown in S7 of the Supporting Information. To confirm the structure of the metabolites, we synthesized M1 and M3, identified their structure by NMR, and compared the synthesized product HPLC/MS spectra to the metabolites. Metabolite M1. The peak eluting of M1 was observed at approximately 11.2 min, and the positive ion of M1 was observed at m/z 473.2145, which was 2 Da higher than limonin, as shown in Figure 1B. On the basis of the data of fragment ions at m/z 455.2043, we propose that M1 may miss the hydroxyl group in the B ring. The peak products at m/z 427.2102 and 413.1947 indicated the missing ester group in lactonic rings A and D. Additionally, a fragment, which was observed at m/z161 [M + H]+ in M1, indicated the decomposition of rings C and D in the metabolites. The possible fragmentation of M1 was shown in S2 of the Supporting Information. We synthesized M1 from the parent compound limonin by NaBH4. The major different characteristic of the reaction product from limonin in 13C NMR was the missing peak of the carbonyl group, as shown in panels A and

RESULTS AND DISCUSSION

Identification of Limonin. The data of ion fragments of limonin and its five metabolites were summarized in Table 2. The limonin eluting peak was observed at 11.82 min, and the tandem mass spectrometry (MS/MS) spectra of collision dissociation of limonin ions were detected at m/z 471.1981. The typical positive ions at m/z 453.1893, 425.1933, 367.1884, and 339.1931 were joined to the loss of [H2O1 (18 Da)], [C1O4 (44 Da)], [C3H8O5 (104 Da)], and [C4H12O5 (132 Da)], respectively. Additionally, m/z 161.0594 and 95.0141 were also the product ions. The possible fragment ions of limonin were shown in S1 of the Supporting Information. C

DOI: 10.1021/acs.jafc.8b02057 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry B of Figure 2. However, an additional carbon signal at δ 76.3 ppm indicated the 7-hydroxyl group in the reaction product.

retention time and NMR experiments, M3 was identified as limonoate A-ring lactone.20 Metabolite M4. The peak eluting of M4 was observed at 11.5 min. The positive ion was detected at m/z 457.2207. The fragment, which was 14 Da lower than the parent drug, indicated the missing carbonyl group in ring B. Other fragment ions were observed at m/z 351.1944, 369.2050, and 421.2002. The fragment ion observed at m/z 227.1781 indicated that an open furan ring was connected to ring A. The proposed fragmentation of M4 was shown in S5 of the Supporting Information. Metabolite M5. The peak eluting M5 was at approximately 11.2 min. The positive ion of M5 was observed at m/z 455.2038, which was 16 Da lower than limonin. In comparison to M4, the fragment ions at m/z 281.1157, 349.1777, 353.1730, and 395.1843 indicated a double band derivative site in ring A of the metabolite. The proposed fragmentation of M5 was shown in S6 of the Supporting Information. Additionally, the fragment ions at m/z 161 and 95, which were similar to those observed in limonin and other metabolites, indicated that the existence of a furan ring. The structure of M5 was identified as deoxylimonin.24 A number of Citrus species have been used in clinics in China, and identifying the metabolites of the constituents in this phytomedicine is very important to understand their benefits and risks.25 Animal urine and blood are very important research objects for us to understand the absorption and metabolism of compounds in the body.26 In the present study, the comparative metabolism of limonin was established in rat liver microsomes, urine, and bile. Five metabolite profiles of limonin were identified. Among them, M1 and M3 were identified on the basis of NMR data. Five phase I metabolites were detected in rat urine; four of them (excluding M2) were detected in rat bile; and 3 metabolites were detected in rat liver microsomes (M1−M3). No phase II metabolite was found in the three samples. The primary metabolic pathways include the hydrolysis of lactone,27 carbonyl reduction,28 and the decomposition of the epoxy group.29 In our experiment, MS2 data of limonin, observed at m/z 453.1893, 425.1933, 367.1884, and 339.1931, and the positive ion at m/z 473 were consistent with the previous report.13 Different from the literature, the reduction site, which was located on the carbonyl group of ring A, was confirmed by NMR data. Additionally, although the metabolites appeared to be primarily metabolized via various pathways, the metabolite MS2 data of limonin observed at m/z 161 in our experiment were the same as described in the reported literature.12 It was reported that citrus seeds contained limonoate hydrogenase, which can convert limonin to 17-dehydrolimonoate A-ring lactone (M2) and limonoate A-ring lactone (M3).30 Furthermore, previous research has demonstrated that the enzyme of Pseudomonas sp. has the ability to produce limonin A- and D-ring hydrolysates.31 Under the catalysis of limonoate dehydrogenase, limonin was converted into different metabolites.32 In our study, M2, which was one of the authentic degradation products of limonin, was detected in the samples other than in rat urine, which may be due to the different metabolic pathways. Among the five metabolites, M1, M2, M3, and M5 were natural products in a previous study. The results from this work are valuable and important for understanding the metabolic process of limonin and provided a reference for

Figure 2. 13C NMR spectra of limonin and metabolites: (A) limonin, (B) limonol (M1), and (C) limonoate A-ring lactone (M3).

The HPLC retention time and production spectra of the synthetic constituent were similar to M1. On the basis of these experiments and data comparison, M1 was identified as limonol, which is the natural product isolated from Citrus paradisi.21 Metabolite M2. M2 had a retention time of 10.8 min. The protonated molecular ion [M + H]+ of M2 was detected at m/ z 487.1937, as shown in panels C and D of Figure 1. The protonated molecular ion of M2 was 16 Da higher than limonin, indicating that there was an additional oxygen group in M2. The MS2 spectra of m/z 487 observed a series of fragment ions at m/z 423.1802, 441.1902, and 267.0933, as shown in Table 2. The structure of M2 was identified as 17dehydrolimonoate A-ring lactone, which was a natural product isolated from Citrus fruits based on the ion data.23 The possible fragmentation and product ions of M2 were shown in S3 of the Supporting Information. Metabolite M3. The peak eluting of M3 was observed at approximately 11.4 min, as shown in panels E and F of Figure 1. The positive ion was at m/z 489.2314, which was 18 Da higher than limonin, indicating the lactone hydrolysis in ring D. The MS2 spectra showed fragment ions at m/z 436.1811, 426.1988, 410.2041, and 368.1949. The product ions of M3 were shown in S4 of the Supporting Information. We also observed the fragment ions at m/z 161.0609 and 95.0151, which were similarly to those detected in limonin and other metabolites. The result indicated that a furan ring was located in ring D. Limonin was treated with LiOH to yield a white crystal, which has the same NMR characteristics as limonoate A-ring lactone (Figure 2C). The signals of the proton in the furan ring at C-17 were 7.76, 6.50, and 7.72 ppm. The higher chemical shift compared to those of limonin indicated that there was an open ring D.21 On the basis of the HPLC D

DOI: 10.1021/acs.jafc.8b02057 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

replication on infected human mononuclear cells. Planta Med. 2003, 69, 910−913. (6) Langeswaran, K.; Jagadeesan, A. J.; Revathy, R.; Balasubramanian, M. P. Chemotherapeutic efficacy of limonin against Aflatoxin B1 induced primary hepatocarcinogenesis in Wistar albino rats. Biomed. Aging Pathol. 2012, 2, 206−211. (7) Chidambara Murthy, K. N.; Jayaprakasha, G. K.; Patil, B. S. Citrus limonoids and curcumin additively inhibit human colon cancer cells. Food Funct. 2013, 4, 803−810. (8) Tanaka, T.; Maeda, M.; Kohno, H.; Murakami, M.; Kagami, S.; Miyake, M.; Wada, K. Inhibition of azoxymethane-induced colon carcinogenesis in male F344 rats by the citrus limonoids obacunoneand limonin. Carcinogenesis 2001, 22, 193−198. (9) Liang, Y.; Xie, L.; Liu, X. D.; Lu, T.; Wang, G. J.; Hu, Y. Z. Determination of limonin in rat plasma by liquid chromatography− electrospray mass spectrometry. J. Pharm. Biomed. Anal. 2005, 39, 1031−1035. (10) Liu, S. J.; Zhou, L.; Zhang, J.; Yu, B. Y.; Li, C. Y.; Liu, Z. X.; Ju, W. Z. Determination of limonin in dog plasma by liquid chromatography-tandem mass spectrometry and its application to a pharmacokinetic study. Biomed. Chromatogr. 2013, 27, 515−519. (11) Liu, S.; Zhang, J.; Zhou, L.; Yu, B.; Li, C.; Liu, Z.; Ju, W. Quantification of limonin in human urine using solid-phase extraction by LC-MS/MS. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2012, 907, 163−167. (12) Ren, W.; Xin, S. K.; Han, L. Y.; Zuo, R.; Li, Y.; Gong, M. X.; Wei, X. L.; Zhou, Y. Y.; He, J.; Wang, H. J.; Si, N.; Zhao, H. Y.; Yang, J.; Bian, B. L. Comparative metabolism of four limonoids in human liver microsomes using ultra-high-performance liquid chromatography coupled with high-resolution LTQ-Orbitrap mass spectrometry. Rapid Commun. Mass Spectrom. 2015, 29, 2045−2056. (13) Ren, W.; Li, Y.; Zuo, R.; Wang, H. J.; Si, N.; Zhao, H. Y.; Han, L. Y.; Yang, J.; Bian, B. L. Species-related difference between limonin and obacunone among five liver microsomes and zebrafish using ultrahigh-performance liquid chromatography coupled with a LTQOrbitrap mass spectrometer. Rapid Commun. Mass Spectrom. 2014, 28, 2292−2300. (14) Wu, C.; Benet, L. Predicting drug disposition via application of BCS: Transport/absorption/ elimination interplay and development of a biopharmaceutics drug disposition classification system. Pharm. Res. 2005, 22, 11−23. (15) Du, L.; Qian, D.; Shang, E.; Liu, P.; Jiang, S.; Guo, J.; Su, S.; Duan, J.; Xu, J.; Zhao, M. UPLC−Q−TOF/MS-based screening and identification of the main flavonoids and their metabolites in rat bile, urine and feces after oral administration of Scutellaria baicalensis extract. J. Ethnopharmacol. 2015, 169, 156−162. (16) Tong, Z.; Chandrasekaran, A.; DeMaio, W.; Espina, R.; Lu, W.; Jordan, R.; Scatina, J. Metabolism of vabicaserin in mice, rats, dogs, monkeys, and humans. Drug Metab. Dispos. 2010, 38, 2266−2277. (17) Jin, Y.; Wu, L.; Tang, Y.; Cao, Y.; Li, S.; Shen, J.; Yue, S.; Qu, C.; Shan, C.; Cui, X.; Zhang, L.; Duan, J. A. UFLC−Q−TOF/MS based screening and identification of the metabolites in plasma, bile, urine and feces of normal and blood stasis rats after oral administration of hydroxysafflor yellow A. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2016, 1012−1013, 124−129. (18) Ren, Q.; Wang, Y. L.; Wang, M. L.; Wang, H. Y. Screening and identification of the metabolites in rat urine and feces after oral administration of Lycopus lucidus Turcz extract by UHPLC−Q− TOF−MS mass spectrometry. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2016, 1027, 64−73. (19) Ruberto, G.; Renda, A.; Tringali, C.; Napoli, E. M.; Simmonds, M. S. J. Citrus limonoids and their semisynthetic derivatives as antifeedant agents against Spodoptera f rugiperda larvae. A structure− activity relationship study. J. Agric. Food Chem. 2002, 50, 6766−6774. (20) English, B. J.; Williams, R. M. Synthesis of (±)-oleocanthal via a tandem intramolecular michael cyclization-HWE olefination. Tetrahedron Lett. 2009, 50, 2713. (21) Bennett, R. D.; Hasegawa, S. 7α-Oxygenated limonoids from the rutaceae. Phytochemistry 1982, 21, 2349−2354.

the related mechanistic research as well as further development of new drugs.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b02057.

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Possible fragment ions of limonin and its metabolites (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Telephone: +86-25-86552301. Fax: +86-25-86515447. Email: [email protected]. *Telephone: +01-4126489508. Fax: +01-4126481916. E-mail: [email protected]. *Telephone: +86-25-86617141. Fax: +86-25-86555033. Email: [email protected]. ORCID

Shijia Liu: 0000-0002-5060-6145 Funding

This work is financially supported by the National Natural Science Foundation of China (81403174 and 81774096), the Natural Science Foundation of Jiangsu Province (BK20161610 and BK20151602), the Jiangsu Provincial Medical Youth Talent (QNRC2016642), the Chinese Talented Person Training Project for China Association of Chinese Medicine (QNRC2-B04), the Science and Technology Projects of Jiangsu Province Hospital of Traditional Chinese Medicine (Y18010), and the Top Six Talent Project of Jiangsu Province 2016 (WSN-051). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED HPLC−Q−TOF/MS, high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry; NMR, nuclear magnetic resonance; HIV, human immunodeficiency virus; SD, Sprague Dawley; HSQC, heteronuclear singlequantum correlation; HMBC, heteronuclear multiple-bond correlation; HPLC, high-performance liquid chromatography; ESI, electrospray ionization

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REFERENCES

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DOI: 10.1021/acs.jafc.8b02057 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jafc.8b02057 J. Agric. Food Chem. XXXX, XXX, XXX−XXX