- Email: [email protected]
Tissue distribution of naringin and derived metabolites in rats after a single oral administration
Journal of Chromatography B 1136 (2020) 121846
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/jchromb
Short communication
Tissue distribution of naringin and derived metabolites in rats after a single oral administration
T
Xuan Zenga, Hongliang Yaoa,b, Yuying Zhenga, Yudong Hea, Yan Hea, Hongyu Raoa, Peibo Lia, ⁎ Weiwei Sua, a
Guangdong Engineering & Technology Research Center for Quality and Efficacy Reevaluation of Post-Market Traditional Chinese Medicine, Guangdong Provincial Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, 510275 Guangzhou, People’s Republic of China b Guangdong Key Laboratory of Animal Conservation and Resource Utilization, Guangdong Public Laboratory of Wild Animal Conservation and Utilization, Drug Synthesis and Evaluation Center, Guangdong Institute of Applied Biological Resources, 510260 Guangzhou, People’s Republic of China
ARTICLE INFO
ABSTRACT
Keywords: Naringin Metabolites Rat Tissue distribution UFLC-Q-TOF-MS/MS
Naringin has been documented to possess multiple pharmacological activities. Reported pharmacokinetic studies revealed that oral bioavailability of naringin was low, in contrast to its significant pharmacological effects. The in vivo distribution of naringin and derived metabolites might partly explain this discrepancy. In this study, an ultra-fast liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry system (UFLC-Q-TOFMS/MS) was used for profiling the distribution of naringin and its metabolites in rat plasma and fourteen tissues after oral administration. Naringin was widely distributed and its concentrations in certain tissues were much higher than that in plasma, especially in trachea and lung. Moreover, a total of 23 flavonoid metabolites and 15 phenolic catabolites were screened. Naringenin glucuronides were principal metabolites in plasma, while free naringenin and naringenin-7-O-sulfate were the major molecular forms in most tissues. Meanwhile, phenolic catabolites derived from naringin were found to be abundant in liver and kidney. These pharmacokinetic results would be useful to explain the pharmacodynamics of naringin.
1. Introduction
time-of-flight tandem mass spectrometry system (UFLC-Q-TOF-MS/MS) was used to analyze plasma and fourteen tissues collected in different time points after a single oral administration, to illustrate the tissue distribution profile of naringin and its metabolites in rats. Obtained results would be essential in the interpretation of pharmacodynamics for naringin.
Naringin, chemically called 5,7,4′-trihydroxy-flavanone-7-O-rhamnoglucoside, is a typical phytopharmaceutical which has been experimentally documented to relieve multiple diseases, including inflammation, neurodegeneration, cardiovascular disorders, metabolic syndrome, and respiratory diseases [1,2]. Thanks to its multiple bioactivities, naringin has been involved in many pharmacokinetic studies [3–6]. The bioavailability of naringin was found to be low, which contradicted to its remarkable pharmacological effects. For example, its oral bioavailability in human was only 5–9% [7]. To date, several studies have been carried out to explain the discrepancy [8–10]. However, these studies generally focused on naringin and its aglycon naringenin obtained after the incubation with glucuronidase and/or sulfatase, while the molecular forms of derived metabolites were still unknown. Hence, it is necessary to systematically investigate the distribution of derived metabolites in main tissues after oral administration of naringin. In the present work, ultra-fast liquid chromatography-quadrupole-
2. Materials and methods 2.1. Chemicals and materials Naringin (purity: 94.7%), apigenin (purity: 99.6%), and hippuric acid (purity: 99.9%) were acquired from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Naringenin (purity: 99.5%), hesperetin (purity: 95.0%), 4-hydroxybenzonic acid (purity: 99.0%), 3-(4′-hydroxyphenyl)propionic acid (purity: 98.0%), and MS grade formic acid were purchased from SigmaAldrich (St. Louis, USA). Eriodictyol (purity: 97.0%) was obtained from Sinova (Shenzhen, China). Hesperetin-7-O-glucuronide (purity: 95.0%)
Abbreviations:UFLC-Q-TOF-MS/MS, ultra-fast liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry ⁎ Corresponding author at: No. 135, Xingang Xi Road, 510275 Guangzhou, People’s Republic of China. E-mail address: [email protected] (W. Su). https://doi.org/10.1016/j.jchromb.2019.121846 Received 16 April 2019; Received in revised form 29 September 2019; Accepted 20 October 2019 Available online 31 October 2019 1570-0232/ © 2019 Published by Elsevier B.V.
Journal of Chromatography B 1136 (2020) 121846
X. Zeng, et al.
Fig. 1. A heat map of the AUC (shown in logarithm of 10) of naringin and derived metabolites in rat tissues after oral administration.
and hesperetin-7-O-sulfate (purity: 95.0%) were purchased from Toronto Research Chemicals (Toronto, Canada). Naringenin-7-O-glucuronide (purity: 97.0%) was obtained from Cayman Chemical Company (Ann Arbor, USA), while naringenin-4′-O-glucuronide (purity: 96.0%) was supplied from Shanghai ZZBIO Co., Ltd (Shanghai, China). The stable isotope labeled internal standard (IS) [2′,3′,5′,6′-D4]4,6,4′-trihydroxydihydroaurone (purity: 94.5%) was obtained from Artis-chem Co. Ltd. (Shanghai, China). LC-MS grade methanol was obtained from Fisher Scientific Inc. (Fair Lawn, USA), while HPLC grade acetonitrile and ethyl acetate were purchased from Honeywell B&J Chemicals Inc. (New Jersey, USA). Milli-Q deionized water (specific electric conductivity = 18.2 MΩ) was filtered through a 0.22 μm membrane filter before use. For oral administration, naringin was extracted from Exocarpium Citri Grandis with a purity of 98.8%, which was determined by HPLC method with external standard [3].
2.2. Animals Male and female Sprague-Dawley rats (600–800 g) were purchased from Chengdu Dossy Experimental Animals Co. Ltd. (Chengdu, China, Certificate No. SCXK2015-030). Rats were housed in controlled environmental conditions (20–25 °C, 55 ± 15% relative humidity, 12 h light/dark cycles), and fasted overnight with water available ad libitum before experiment. All experimental procedures were approved by the Animal Ethics Committee of the School of Life Sciences in Sun Yat-sen University. 2.3. Administration and sampling Briefly speaking, 96 rats (half male and half female) were randomly assigned into 8 groups (corresponding to the corresponding collection time points, i.e. 0.25, 1, 3, 6, 8, 10, 15, and 24 h post dose) and each orally administrated with 42 mg/kg naringin. The dose and route of 2
Journal of Chromatography B 1136 (2020) 121846
X. Zeng, et al.
administration were the same as our preliminary studies [3,4,9]. After blood collection, the organs (including stomach, duodenum, jejunum, ileum, colon, liver, kidney, heart, lung, trachea, spleen, brain, fat, and skeletal muscle) were dissected, washed with saline, blotted dry, accurately weighed, and then homogenized in 0.9% saline (1 g tissue/ 20 mL for trachea, while 1 g tissue/5 mL for other organs) on ice with an IKA T10 basic homogenizer (Staufen, Germany) to afford tissue extract. Obtained biological samples were stored at −70 °C until analysis.
few naringin-O-glucuronide and naringin-O-sulfate were detected in jejunum, ileum, and colon. Although gut microbiota-mediated hydrolysis was important in the absorption [11], naringin was widely distributed in tissues outside gastrointestinal tract, and the concentrations of naringin in certain tissues were much higher than that in plasma. Apart from gastrointestinal tract, free naringin was detected in trachea, lung, kidney, fat, liver, muscle, heart, plasma, brain, and spleen in a descending order of distributed amounts within 24 h. Findings of tissue distribution of naringin provided a partial explanation for the discrepancy between low bioavailability of naringin and its pharmacological effects. Naringin extensively distributed in trachea and lung, provided evidence for its remarkable activities of relieve cough, phlegm, and asthma [12]. What’s more, distribution of naringin in fat, heart, and brain probably associated with its antihypercholesterolemic, cardioprotective and neuroprotective effects [1]. Gastrointestinal tracts were main region for the first-pass metabolism of flavonoids [13]. Mediated by lactase-phlorizin hydrolase and gut microbiota, naringin was hydrolyzed into naringenin. Generated naringenin was absorbed through both passive diffusion as well as active transport [14], and subsequently engaged in in vivo phase I and phase II metabolism, giving rise to an array of metabolites. Free naringenin (M3), naringenin-7-O-sulfate (M10), and naringenin-O-glucuronide-O-sulfate (M11) were found to be predominant metabolites in gastrointestinal tracts. Naringenin-7-O-glucuronide (M5) and naringenin-4′-O-glucuronide (M6) were relatively abundant in jejunum, while naringenin-4′-O-sulfate (M9) was solely detected in jejunum and ileum. Meanwhile, phase I metabolites were abundant in duodenum, jejunum, ileum, and colon, probably associated with the abundance of gut microbiota in these regions [15]. Through the portal vein, generated metabolites were transferred from gastrointestinal tract to liver to be further processed. The major metabolites in liver were naringenin (M3) and naringenin-7-O-sulfate (M10), followed by free naringin, naringenin-4′-O-glucuronide (M6), and apiferol (M14). After the hepatic processes, naringin and derived metabolites were released into systemic circulation and then distributed to other tissues. Aligned with reported studies [3], the major metabolites of naringin in most tissues were identified as glucuronide and sulfate conjugates of naringenin. However, the dominant existing forms were found to be different in multiple tissues, implying tissue distribution specificity and binding site specificity of phase II metabolic enzymes in rats. In plasma, naringenin glucuronides were predominant, including naringenin-7-Oglucuronide (M5), naringenin-4′-O-glucuronide (M6), and naringeninO-glucuronide-O-sulfate (M11). While in gastrointestinal tracts, liver, kidney, lung, trachea, heart, spleen, muscle, free naringenin (M3) and naringenin-7-O-sulfate (M10) were the major forms. Therefore, it can be assumed that when naringenin glucuronides transferred from circulation into these tissues, they were hydrolyzed by tissue b-glucuronidase to free naringenin, crossed lipophilic cell membrane, and then sulfated by sulfotransferases. As naringenin-7-O-sulfate was the principal metabolite in most tissues, its bioactivities warrant more investigations. Except for these flavonoid metabolites, 15 phenolic catabolites, such as phenylpropenoic acid, phenylpropionic acid, benzoic acid, and benzoylglycine derivatives, including free phenolics and phase II sulfate conjugates were detected. Phenolic catabolites mainly flowed from the microbiota-mediated ring fission of unabsorbed flavonoids [16], as well as the metabolites excreted by enterohepatic circulation. Reported studies have revealed that 3-(4′-hydroxyphenyl)propionic acid (HPPA) and 4′-hydroxybenzoic acid (HBA), which were the phenolic catabolites of naringenin, could effectively improve the cholesterol and antioxidant metabolism [17]. In this study, 3-(phenyl)-2-propenoic acid-4′-O-sulfate (C3), HPPA (C9), HPPA-4′-O-sulfate (C11), and HBA-4-O-sulfate (C13) were found to be abundant in liver and kidney, which were the main metabolic and excretive tissues, suggesting the relation to mentioned bioactivities. Thus, microbial-derived phenolic catabolites could be an important part to understand the pharmacodynamics of naringin.
2.4. Metabolite profiling For metabolites profiling, tissue extract (100 μL) were deproteinized with 2-fold acetonitrile (containing IS), vortex-mixed for 3 min, and centrifuged at 15,000 × g for 30 min at 25 °C. Finally, 10 μL supernatant was analyzed with Shimadzu UFLC XR system tandem a hybrid quadrupole time-of-flight mass spectrometer system (Triple TOFTM 5600 plus; Sciex, Foster City, USA), which equipped with an ESI source in negative-ionization mode. The accuracy of the instrument for m/z measurement was up to 1 ppm. Chromatographic separation was conducted using a Kinetex C18 column (3.0 × 150 mm, 2.6 μm, 100 Å; Phenomenex, Torrance, USA) at 40 °C. A gradient elution program was conducted for chromatographic separation, with the mobile phase A (0.1% formic acid*water) and mobile phase B (0.1% formic acid–methanol) as follows: 0–10 min (5–65% B), 10–20 min (65–82.5% B), maintained at 100% B for 4 min (20.1–24.1 min), and then decreased to 5% B to equilibrate for 5 min (25.0–30.0 min). The flow rate was 0.3 mL/min. The optimal MS parameters were set as follows: TOF-MS scan range m/z 100 to 1500, product ion scan range m/z 50 to 1500, ion source gas 55 psi, curtain gas 35 psi, ion source temperature 550 °C, ion spray voltage −4,500 V, collision energy 35 eV, collision energy spread 25 eV, declustering potential 80 V. Nitrogen was used as nebulizer and auxiliary gas. Data acquisition was performed with Analyst® TF 1.6 software (Sciex, Foster City, USA) in information-dependent acquisition (IDA) mode. 2.5. Data analysis Metabolites were screened by the software (PeakView, Version 1.6; MetabolitePilot, Version 1.0; Sciex, Foster City, USA) but identified manually. If the IS-standardized response of a chromatographic peak in tissue homogenate is five times higher than that in blank sample, it can be considered as the peak of a potential metabolite. The metabolites profiling was carried out based on following two points: (I) reported results concerning the metabolism of naringin and other flavonoids, (II) full understanding of the MS/MS fragmentation patterns of the parent compound and authentic standards. TOF-MS chromatographic peak areas of identified metabolites, as well as that of IS, were obtained with MultiQuant software (Version 2.1; Sciex, Foster City, USA) and then used to calculate the peak area ratios. Furthermore, area under curve (AUC) were acquired from the mean peak area ratio-time curves to reflect cumulative exposure of identified metabolites. Obtained AUC values were shown in logarithm of 10 and plotted in Fig. 1 with GraphPad Prism (Version 7.0). 3. Results and discussion In this study, high resolution UFLC-Q-TOF-MS/MS based metabolites profiling was performed on plasma as well as fourteen other tissues, including stomach, duodenum, jejunum, ileum, colon, liver, kidney, heart, lung, trachea, spleen, brain, fat, and skeletal muscle. Finally, a total of 23 flavonoid metabolites and 15 phenolic catabolites were identified. Detailed information of identified metabolites was presented in Table 1. The AUC (shown in logarithm of 10) of naringin and derived metabolites were presented in a heat map (Fig. 1). Administered by gavage, naringin was in direct contact with gastrointestinal tract and found to be abundant in these organs. Moreover, 3
4
Naringenin-4′-O-sulfate
Naringenin-7-O-sulfate
Naringenin-O-glucuronide-Osulfate
Naringenin-O-glucoside-O-sulfate
Methylnaringenin-O-glucuronide
Apiferol
Apigenin
Apigenin-O-glucuronide
Apigenin-O-glucuronide
Apigenin-O-sulfate
M9
M10
M11
M12
M13
M14
M15
M16
M17
M18
Eriodictyol-O-sulfate
M20
Hesperetin
Hesperetin-7-O-glucuronide
M21
M22
b
Eriodictyol
M19
b
Naringenin-5,7-O-di-glucuronide
M8
b
Naringenin-4′,7-O-di-glucuronide
M7
b
C22H22O12
C16H14O6
C15H12O9S
C15H12O6
C15H10O8S
C21H18O11
C21H18O11
C15H10O5
C15H14O5
11.8
13.8
12.3
12.7
13.4
12.3
11.1
15.0
13.8
13.5
9.4
C21H22O13S C22H22O11
10.1
12.1
C21H20O14S
C15H12O8S
10.0
9.4
C27H28O17 C15H12O8S
8.7
11.7
11.5
C27H28O17
C21H20O11
Naringenin-4′-O-glucuronide
M6
C21H20O11
Naringenin-7-O-glucuronide
M5
10.7
C21H20O11
b
Naringenin-5-O-glucuronide
M4
13.7
10.2
9.7
11.4
RT (min)
C15H12O5
b
Naringenin
M3
b
Naringin-O-sulfate
C27H32O17S
C27H32O14
M2
b
Formula
C33H40O20
Naringin
Identified metabolites
Flavonoid metabolites M1 Naringin-O-glucuronide
Parent drug
No.
513.0691 (-3.3) 461.1082 (-1.6) 273.0765 (-1.4) 269.0447 (-3.0) 445.0781 (1.0) 445.0780 (0.9) 349.0016 (-2.2) 287.0569 (2.9) 367.0133 (1.0) 301.0709 (-2.9) 477.1033 (-1.3)
527.0496 (-0.9)
623.1270 (2.6) 623.1257 (0.6) 351.0178 (-0.5) 351.0178 (-0.5)
447.0924 (-2.1) 447.0929 (-1.0) 447.0934 (0.1)
755.2064 (3.2) 659.1272 (-2.4) 271.0618 (2.2)
579.1717 (-0.4)
[M−H]− (Error, ppm)
301.0727[M-H-GlcUA]−, 175.0233[M-H-HE]−, 151.0046[M-H-GlcUA-C9H10O2]−, 113.0265[M-H-HE-CO2-H2O]−, 95.0156
(continued on next page)
plasma, stomach, duodenum, jejunum, ileum, liver, kidney duodenum, jejunum, ileum, colon, liver, kidney, lung plasma
348.9218[M-H-H2O]−, 287.0583[M-H-SO3]−, 151.0029[M-H-SO3-C8H8O2]−, 135.0441[M-H-SO3-C7H4O4]−, 107.0148[M-H-SO3-C8H8O2-CO2]− 286.0466[M-H-CH3]−, 151.0004[M-H-C9H10O2]−, 107.0159[M-H-C9H10O2-CO2]−
241.1767, 151.0031[M-H-C8H8O2] , 135.0469[M-H-C7H4O4]
plasma, stomach, duodenum, jejunum, ileum, colon, liver, kidney, lung, trachea, heart, spleen colon, liver
269.0442[M-H-SO3]−, 225.0522, 117.0366[M-H-SO3-C7H4O4]− −
liver
399.0513, 269.0462[M-H-GlcUA]−, 113.0255[M-H-AE-CO2-H2O]−
−
stomach, duodenum, jejunum, ileum, colon, liver, kidney, lung, trachea, heart plasma
jejunum, ileum, colon, liver
plasma
liver
plasma, stomach, jejunum, ileum, liver, kidney, lung plasma, stomach, duodenum, jejunum, ileum, colon, liver, kidney, lung, trachea, heart, spleen, muscle, fat plasma, stomach, duodenum, jejunum, ileum, liver, kidney, lung, trachea, heart, fat
plasma, kidney
plasma, stomach, jejunum, ileum, liver, kidney
plasma, stomach, duodenum, jejunum, ileum, liver, kidney, lung, heart, muscle, fat plasma, stomach, duodenum, jejunum, ileum, liver, kidney, lung, trachea, heart, muscle, fat
229.0875, 179.0360, 167.0334, 153.0062[M-H-C8H8O]−, 123.0460, 119.0506[M-H-C7H6O4]−, 107.0133[M-H-C8H8O-CO-H2O]−, 93.0338[M-H-C9H8O4]− 225.0557, 151.0058[M-H-C8H6O]−, 117.0334[M-H-C7H4O4]−, 107.0227[M-H-C8H6O-CO2]− 269.0468[M-H-GlcUA]−, 175.0269[M-H-AE]−, 113.0269[M-H-AE-CO2-H2O]−, 97.9613
447.0962[M-H-GlcUA]−, 271.0632[M-H-2GlcUA]−, 175.0238[M-H-NE-GlcUA]−, 113.0232[M-H-NE-GlcUA-CO2-H2O]− 271.0612[M-H-SO3]−, 151.0027[M-H-SO3-C8H8O]−, 119.0492[M-H-SO3-C7H4O4]−, 107.0146[M-H-SO3-C8H8O-CO2]−, 93.0389[M-H-SO3-C9H6O4]− 271.0609[M-H-SO3]−, 177.0188[M-H-SO3-C6H6O]−, 151.0033[M-H-SO3-C8H8O]−, 119.0514[M-H-SO3-C7H4O4]−, 107.0169[M-H-SO3-C8H8O-CO2]−, 93.0362[M-H-SO3-C9H6O4]− 447.0953[M-H-SO3]−, 351.0203[M-H-GlcUA]−, 271.0624[M-H-SO3-GlcUA]−, 254.9827, 175.0251[M-H-SO3-NE]−, 151.0028[M-H-SO3-GlcUA-C8H8O]−, 113.0253[M-H-SO3-NE –CO2-H2O]− 467.2386, 433.1195[M-H-SO3]−, 313.0580[M-H-SO3-C4H8O4]−, 271.0648[M-H-SO3-Glc]−, 240.9983, 151.0056[M-H-SO3-Glc-C8H8O]− 285.0758[M-H-GlcUA]−, 175.0215[M-H-MNE]−, 113.0250[M-H-MNE-CO2-H2O]−
271.0613[M-H-GlcUA]−, 175.0206[M-H-NE]−, 151.0031[M-H-GlcUA-C8H8O]−, 119.0493[M-H-GlcUA-C7H4O4]−, 113.0199[M-H-NE-CO2-H2O]− 271.0616[M-H-GlcUA]−, 227.0702, 175.0244[M-H-NE]−, 151.0040[M-H-GlcUA-C8H8O]−, 119.0507[M-H-GlcUA-C7H4O4]−, 113.0251[M-H-NE-CO2-H2O]− 313.0730[M-H-C4H6O5]−, 271.0618[M-H-GlcUA]−, 227.0730, 177.0205[M-H-GlcUA-C6H6O]−, 175.0252[M-H-NE]−, 151.0038[M-H-GlcUA-C8H8O]−, 119.0501[M-H-GlcUA-C7H4O4]−, 113.0256[M-H-NE-CO2-H2O]− 447.0939[M-H-GlcUA]−, 271.0626[M-H-2GlcUA]−, 175.0242[M-H-NE-GlcUA]−
colon, liver
579.1711[M-H-SO3]−, 527.2455, 459.1262[M-H-SO3-C8H8O]−, 351.0248[M-H-Rha-Glc]−, 313.0700[M-H-SO3-C8H8O-Rha]−, 271.0612[M-H-SO3-Rha-Glc]− 225.0697, 177.0166[M-H-C6H6O]−, 151.0029[M-H-C8H8O]−, 119.0501[M-H-C7H4O4]−, 107.0152[M-H-C8H8O-CO2]−, 93.0364[M-H-C9H6O4]−
plasma, stomach, duodenum, jejunum, ileum, colon, liver, kidney, lung, trachea, heart, spleen, muscle, fat plasma, jejunum, kidney
jejunum, ileum, colon
579.1693[M-H-GlcUA]−, 429.0959, 271.0612[M-H-GlcUA-Rha-Glc]−, 175.0269[M-H-NG]−
Source plasma, stomach, duodenum, jejunum, ileum, colon, liver, kidney, lung, trachea, heart, spleen, brain, muscle, fat
a
459.1175[M-H-C8H8O]−, 339.0710, 313.0725[M-H-C8H8O-Rha]−, 271.0626[M-H-Rha-Glc]−, 151.0044[M-H-Rha-Glc-C8H8O]−, 119.0492[M-H-Rha-Glc-C7H4O4]−
Fragment ions in negative mode
Table 1 UFLC-Q-TOF-MS/MS based identifications of flavonoid metabolites and phenolic catabolites in rat tissues collected 0–24 h after a single oral administration of naringin.
X. Zeng, et al.
Journal of Chromatography B 1136 (2020) 121846
5
b
a
Hippuric acid
b
7.9 7.0 8.0 10.2 10.8 8.3
C9H10O4 C9H10O7S C9H10O7S C9H10O3 C9H10O3 C9H10O6S
C9H9NO3
8.2
C10H12O7S
8.9
273.0076 (0.4) 163.0410 (5.9) 242.9971 (0.8) 242.9972 (1.3) 275.0236 (2.0) 181.0512 (3.4) 261.0074 (-0.2) 261.0073 (-0.5) 165.0564 (4.3) 165.0564 (4.4) 245.0130 (1.8) 245.0127 (0.5) 216.9815 (1.1) 216.9817 (2.0) 178.0518 (4.8)
381.0275 (-2.9)
[M−H]− (Error, ppm)
a
−
160.0415[M-H-H2O]−, 134.0610[M-H-CO2]−, 77.0426
146.0830, 137.0241[M-H-SO3] , 93.0379[M-H-SO3-CO2]
−
−
165.0528[M-H-SO3]−, 121.0659[M-H-SO3-CO2]−, 119.0503[M-H-SO3-HCOOH]−, 106.0425[M-H-SO3-CO2-CH3]− 146.0824, 137.0245[M-H-SO3]−, 116.0722, 93.0353[M-H-SO3-CO2]−, 88.0424
165.0556[M-H-SO3]−, 121.0673[M-H-SO3-CO2]−, 93.0362[M-H-SO3-CO2-C2H4]−
147.9822, 121.0647[M-H-CO2] , 119.0506[M-H-HCOOH] , 106.0429[M-H-CO2-CH3]
plasma, stomach, liver, kidney, lung, trachea, heart, spleen, brain, muscle, fat
kidney
liver, kidney
plasma, stomach, colon, liver, kidney, lung, heart, muscle, fat plasma, colon, kidney
plasma, colon, kidney, brain
plasma, colon, liver, kidney, lung, heart, muscle −
147.9836, 137.0251[M-H-CO]−, 121.0655[M-H-CO2]−, 93.0361[M-H-CO2-C2H4]− −
kidney, brain
217.0353[M-H-CO2]−, 203.0812[M-H-CO2-CH2]−, 181.0481[M-H-SO3]−, 131.0163
−
plasma, stomach, ileum, liver, lung, trachea, heart, spleen, brain, muscle, fat plasma, kidney, brain
plasma, kidney
plasma, stomach, jejunum, ileum, colon, liver, kidney, lung, trachea, heart, spleen, muscle, fat plasma, liver, kidney
plasma, liver, kidney
plasma
duodenum, jejunum, ileum, liver, kidney
Source
230.9960[M-H-CO2]−, 195.0656[M-H-SO3]−, 177.0563[M-H-SO3-H2O]−, 133.0645[M-H-SO3-H2O-CO2]− 163.0387[M-H-H2O]−, 136.9842[M-H-CO2]−, 135.0453[M-H-H2O-CO]−, 119.0505[M-H-H2O-CO2]−, 107.0492[M-H-H2O-2CO]−, 92.9940 216.9980[M-H-CO2]−, 181.0487[M-H-SO3]−, 137.0596[M-H-SO3-CO2]−, 95.0493
163.0393[M-H-SO3]−, 130.9664, 119.0497[M-H-SO3-CO2]−
163.0404[M-H-SO3] , 119.0510[M-H-SO3-CO2] , 96.9609
−
228.8822[M-H-CO2]−, 193.0496[M-H-SO3]−, 178.0285[M-H-SO3-CH3]−, 134.0367[M-H-SO3-CH3-CO2]− 119.0509[M-H-CO2]−, 93.0354[M-H-CO2-2CH]−
301.0714[M-H-SO3]−, 286.0463[M-H-SO3-CH3]−, 151.0029[M-H-SO3-C9H10O2]−
Fragment ions in negative mode
The losses are: Rha = rhamnose moiety, Glc = glucose moiety, GlcUA = glucuronyl moiety, NG = naringin, NE = naringenin, AE = apigenin, HE = hesperetin. Confirmation in comparison with authentic standards.
C15
9.4
C9H8O6S
6.9
8.6
C9H8O6S
C7H6O6S
Benzoic acid-3-O-sulfate
10.9
C9H8O3
C14
8.8
C10H10O7S
6.4
12.3
C7H6O6S
C16H14O9S
8.6
b
RT (min)
C9H10O6S
Hesperetin-7-O-sulfate
M23
Formula
Phenolic catabolites C1 3-(4′-Methoxyphenyl)-2propenoic acid-3′-O-sulfate C2 3-(4′-Hydroxyphenyl)-2propenoic acid C3 3-(Phenyl)-2-propenoic acid-4′-Osulfate C4 3-(Phenyl)-2-propenoic acid-3′-Osulfate C5 3-(4′-Methoxyphenyl)propionic acid-3′-O-sulfate C6 3-(3′,4′-Di-hydroxyphenyl) propionic acid C7 3-(3′-Hydroxyphenyl)propionic acid-4′-O-sulfate C8 3-(4′-Hydroxyphenyl)propionic acid-3′-O-sulfate C9 3-(4′-Hydroxyphenyl)propionic acid b C10 3-(3′-Hydroxyphenyl)propionic acid C11 3-(Phenyl)propionic acid-4′-Osulfate C12 3-(Phenyl)propionic acid-3′-Osulfate C13 Benzoic acid-4-O-sulfate
Identified metabolites
No.
Table 1 (continued)
X. Zeng, et al.
Journal of Chromatography B 1136 (2020) 121846
Journal of Chromatography B 1136 (2020) 121846
X. Zeng, et al.
4. Conclusion [3]
In this work, distribution of naringin and derived metabolites in rat plasma and fourteen tissues were investigated with UFLC-Q-TOF-MS/ MS system. After a single oral administration, naringin was found to be widely distributed and its concentrations in certain tissues were much higher than that in plasma, especially in trachea and lung. This at least partially explains the naringin’s effects on respiratory disease. A total of 23 flavonoid metabolites and 15 phenolic catabolites were detected. Naringenin glucuronides were predominant metabolites in plasma, while free naringenin and naringenin-7-O-sulfate were the major molecular forms in most tissues, implying deglucuronidation and subsequent sulfation in the tissue distribution of naringenin glucuronides. Besides, phenolic catabolites derived from naringin, which also possessed certain bioactivities, were abundant in liver and kidney. In summary, naringin as well as derived metabolites were more widely distributed in tissues than in plasma after oral administration. These pharmacokinetic results would be helpful in the interpretation of pharmacodynamics for naringin.
[4] [5] [6] [7] [8]
[9] [10]
Declaration of Competing Interest
[11]
All the authors declare that there are no known conflicts of interest with this publication.
[12]
Acknowledgements
[13] [14]
This work was financially supported by National Natural Science Foundation of China(No. 31571830), Applied Science and Technology R &D Special Fund Project of Guangdong Province (No. 2016B020239003), and Guangdong Academic of Sciences Special Project of Science and Technology Development (No. 2016GDASRC-0104).
[15]
[16]
References
[17]
[1] S. Bharti, N. Rani, B. Krishnamurthy, D.S. Arya, Preclinical evidence for the pharmacological actions of naringin: a review, Planta Med 80 (2014) 437–451. [2] R. Shi, Z.T. Xiao, Y.J. Zheng, Y.L. Zhang, J.W. Xu, J.H. Huang, W.L. Zhou, P.B. Li,
6
W.W. Su, Naringenin regulates CFTR activation and expression in airway epithelial cells, Cell. Physiol. Biochem. 44 (2017) 1146–1160. M. Liu, W. Zou, C. Yang, W. Peng, W. Su, Metabolism and excretion studies of oral administered naringin, a putative antitussive, in rats and dogs, Biopharm. Drug Dispos. 33 (2012) 123–134. X. Zeng, W. Su, Y. Zheng, Y. He, Y. He, H. Rao, W. Peng, H. Yao, Pharmacokinetics, tissue distribution, metabolism, and excretion of naringin in aged rats, Front. Pharmacol. 10 (2019) 34. M.L. Matabilbao, C. Andréslacueva, E. Roura, O. Jáuregui, E. Escribano, C. Torre, R.M. Lamuelaraventós, Absorption and pharmacokinetics of grapefruit flavanones in beagles, Br. J. Nutr. 98 (2007) 86–92. X. Zeng, Y. Bai, W. Peng, W. Su, Identification of naringin metabolites in human urine and feces, Eur. J. Drug Metab. Pharmacokinet. 42 (2017) 647–656. C. Felgines, O. Texier, C. Morand, C. Manach, A. Scalbert, F. Régerat, C. Rémésy, Bioavailability of the flavanone naringenin and its glycosides in rats, Am. J. Physiol. Gastrointest. Liver Physiol. 279 (2000) G1148–G1154. M.A.E. Mohsen, J. Marks, G. Kuhnle, C. Rice-Evans, K. Moore, G. Gibson, E. Debnam, S.K. Srai, The differential tissue distribution of the citrus flavanone naringenin following gastric instillation, Free Rad. Res. Commun. 38 (2004) 1329–1340. W. Zou, C. Yang, M. Liu, W. Su, Tissue distribution study of naringin in rats by liquid chromatography-tandem mass spectrometry, Arzneimittelforschung 62 (2012) 181–186. S. Lin, Y. Hou, S. Tsai, M. Wang, P. Chao, Tissue distribution of naringenin conjugated metabolites following repeated dosing of naringin to rats, BioMedicine 4 (2014) 16. X. Zeng, W. Su, Y. Bai, T. Chen, Z. Yan, J. Wang, M. Su, Y. Zheng, W. Peng, H. Yao, Urinary metabolite profiling of flavonoids in Chinese volunteers after consumption of orange juice by UFLC-Q-TOF-MS/MS, J. Chromatogr. B 1061–1062 (2017) 79–88. P. Li, Y. Wang, Z. Wu, W. Peng, C. Yang, Y. Nie, M. Liu, Y. Luo, W. Zou, Y. Liu, The pre-clinical studies of naringin, an innovative drug, derived from Citri Grandis Exocarpium (Huajuhong), Acta Scient. Naturalium Univ. Sunyatseni 54 (2015) 1–5. P.I. Oteiza, C.G. Fraga, D.A. Mills, D.H. Taft, Flavonoids and the gastrointestinal tract: local and systemic effects, Mol. Aspects Med. 61 (2018) 41–49. L. Zhang, L. Song, P. Zhang, T. Liu, L. Zhou, G. Yang, R. Lin, J. Zhang, Solubilities of naringin and naringenin in different solvents and dissociation constants of naringenin, J. Chem. Eng. Data 60 (2015) 932–940. T. Chen, W. Su, Z. Yan, H. Wu, X. Zeng, W. Peng, L. Gan, Y. Zhang, H. Yao, Identification of naringin metabolites mediated by human intestinal microbes with stable isotope-labeling method and UFLC-Q-TOF-MS/MS, J. Pharm. Biomed. Anal. 161 (2018) 262–272. W. Zou, Y. Luo, M. Liu, S. Chen, S. Wang, Y. Nie, G. Cheng, W. Su, K. Zhang, Human intestinal microbial metabolism of naringin, Europ. J. Drug Metab. Pharmacokinet. 40 (2015) 363–367. S.-M. Jeon, H.K. Kim, H.-J. Kim, G.-M. Do, T.-S. Jeong, Y.B. Park, M.-S. Choi, Hypocholesterolemic and antioxidative effects of naringenin and its two metabolites in high-cholesterol fed rats, Translat. Res. 149 (2007) 15–21.
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/jchromb
Short communication
Tissue distribution of naringin and derived metabolites in rats after a single oral administration
T
Xuan Zenga, Hongliang Yaoa,b, Yuying Zhenga, Yudong Hea, Yan Hea, Hongyu Raoa, Peibo Lia, ⁎ Weiwei Sua, a
Guangdong Engineering & Technology Research Center for Quality and Efficacy Reevaluation of Post-Market Traditional Chinese Medicine, Guangdong Provincial Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, 510275 Guangzhou, People’s Republic of China b Guangdong Key Laboratory of Animal Conservation and Resource Utilization, Guangdong Public Laboratory of Wild Animal Conservation and Utilization, Drug Synthesis and Evaluation Center, Guangdong Institute of Applied Biological Resources, 510260 Guangzhou, People’s Republic of China
ARTICLE INFO
ABSTRACT
Keywords: Naringin Metabolites Rat Tissue distribution UFLC-Q-TOF-MS/MS
Naringin has been documented to possess multiple pharmacological activities. Reported pharmacokinetic studies revealed that oral bioavailability of naringin was low, in contrast to its significant pharmacological effects. The in vivo distribution of naringin and derived metabolites might partly explain this discrepancy. In this study, an ultra-fast liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry system (UFLC-Q-TOFMS/MS) was used for profiling the distribution of naringin and its metabolites in rat plasma and fourteen tissues after oral administration. Naringin was widely distributed and its concentrations in certain tissues were much higher than that in plasma, especially in trachea and lung. Moreover, a total of 23 flavonoid metabolites and 15 phenolic catabolites were screened. Naringenin glucuronides were principal metabolites in plasma, while free naringenin and naringenin-7-O-sulfate were the major molecular forms in most tissues. Meanwhile, phenolic catabolites derived from naringin were found to be abundant in liver and kidney. These pharmacokinetic results would be useful to explain the pharmacodynamics of naringin.
1. Introduction
time-of-flight tandem mass spectrometry system (UFLC-Q-TOF-MS/MS) was used to analyze plasma and fourteen tissues collected in different time points after a single oral administration, to illustrate the tissue distribution profile of naringin and its metabolites in rats. Obtained results would be essential in the interpretation of pharmacodynamics for naringin.
Naringin, chemically called 5,7,4′-trihydroxy-flavanone-7-O-rhamnoglucoside, is a typical phytopharmaceutical which has been experimentally documented to relieve multiple diseases, including inflammation, neurodegeneration, cardiovascular disorders, metabolic syndrome, and respiratory diseases [1,2]. Thanks to its multiple bioactivities, naringin has been involved in many pharmacokinetic studies [3–6]. The bioavailability of naringin was found to be low, which contradicted to its remarkable pharmacological effects. For example, its oral bioavailability in human was only 5–9% [7]. To date, several studies have been carried out to explain the discrepancy [8–10]. However, these studies generally focused on naringin and its aglycon naringenin obtained after the incubation with glucuronidase and/or sulfatase, while the molecular forms of derived metabolites were still unknown. Hence, it is necessary to systematically investigate the distribution of derived metabolites in main tissues after oral administration of naringin. In the present work, ultra-fast liquid chromatography-quadrupole-
2. Materials and methods 2.1. Chemicals and materials Naringin (purity: 94.7%), apigenin (purity: 99.6%), and hippuric acid (purity: 99.9%) were acquired from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Naringenin (purity: 99.5%), hesperetin (purity: 95.0%), 4-hydroxybenzonic acid (purity: 99.0%), 3-(4′-hydroxyphenyl)propionic acid (purity: 98.0%), and MS grade formic acid were purchased from SigmaAldrich (St. Louis, USA). Eriodictyol (purity: 97.0%) was obtained from Sinova (Shenzhen, China). Hesperetin-7-O-glucuronide (purity: 95.0%)
Abbreviations:UFLC-Q-TOF-MS/MS, ultra-fast liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry ⁎ Corresponding author at: No. 135, Xingang Xi Road, 510275 Guangzhou, People’s Republic of China. E-mail address: [email protected] (W. Su). https://doi.org/10.1016/j.jchromb.2019.121846 Received 16 April 2019; Received in revised form 29 September 2019; Accepted 20 October 2019 Available online 31 October 2019 1570-0232/ © 2019 Published by Elsevier B.V.
Journal of Chromatography B 1136 (2020) 121846
X. Zeng, et al.
Fig. 1. A heat map of the AUC (shown in logarithm of 10) of naringin and derived metabolites in rat tissues after oral administration.
and hesperetin-7-O-sulfate (purity: 95.0%) were purchased from Toronto Research Chemicals (Toronto, Canada). Naringenin-7-O-glucuronide (purity: 97.0%) was obtained from Cayman Chemical Company (Ann Arbor, USA), while naringenin-4′-O-glucuronide (purity: 96.0%) was supplied from Shanghai ZZBIO Co., Ltd (Shanghai, China). The stable isotope labeled internal standard (IS) [2′,3′,5′,6′-D4]4,6,4′-trihydroxydihydroaurone (purity: 94.5%) was obtained from Artis-chem Co. Ltd. (Shanghai, China). LC-MS grade methanol was obtained from Fisher Scientific Inc. (Fair Lawn, USA), while HPLC grade acetonitrile and ethyl acetate were purchased from Honeywell B&J Chemicals Inc. (New Jersey, USA). Milli-Q deionized water (specific electric conductivity = 18.2 MΩ) was filtered through a 0.22 μm membrane filter before use. For oral administration, naringin was extracted from Exocarpium Citri Grandis with a purity of 98.8%, which was determined by HPLC method with external standard [3].
2.2. Animals Male and female Sprague-Dawley rats (600–800 g) were purchased from Chengdu Dossy Experimental Animals Co. Ltd. (Chengdu, China, Certificate No. SCXK2015-030). Rats were housed in controlled environmental conditions (20–25 °C, 55 ± 15% relative humidity, 12 h light/dark cycles), and fasted overnight with water available ad libitum before experiment. All experimental procedures were approved by the Animal Ethics Committee of the School of Life Sciences in Sun Yat-sen University. 2.3. Administration and sampling Briefly speaking, 96 rats (half male and half female) were randomly assigned into 8 groups (corresponding to the corresponding collection time points, i.e. 0.25, 1, 3, 6, 8, 10, 15, and 24 h post dose) and each orally administrated with 42 mg/kg naringin. The dose and route of 2
Journal of Chromatography B 1136 (2020) 121846
X. Zeng, et al.
administration were the same as our preliminary studies [3,4,9]. After blood collection, the organs (including stomach, duodenum, jejunum, ileum, colon, liver, kidney, heart, lung, trachea, spleen, brain, fat, and skeletal muscle) were dissected, washed with saline, blotted dry, accurately weighed, and then homogenized in 0.9% saline (1 g tissue/ 20 mL for trachea, while 1 g tissue/5 mL for other organs) on ice with an IKA T10 basic homogenizer (Staufen, Germany) to afford tissue extract. Obtained biological samples were stored at −70 °C until analysis.
few naringin-O-glucuronide and naringin-O-sulfate were detected in jejunum, ileum, and colon. Although gut microbiota-mediated hydrolysis was important in the absorption [11], naringin was widely distributed in tissues outside gastrointestinal tract, and the concentrations of naringin in certain tissues were much higher than that in plasma. Apart from gastrointestinal tract, free naringin was detected in trachea, lung, kidney, fat, liver, muscle, heart, plasma, brain, and spleen in a descending order of distributed amounts within 24 h. Findings of tissue distribution of naringin provided a partial explanation for the discrepancy between low bioavailability of naringin and its pharmacological effects. Naringin extensively distributed in trachea and lung, provided evidence for its remarkable activities of relieve cough, phlegm, and asthma [12]. What’s more, distribution of naringin in fat, heart, and brain probably associated with its antihypercholesterolemic, cardioprotective and neuroprotective effects [1]. Gastrointestinal tracts were main region for the first-pass metabolism of flavonoids [13]. Mediated by lactase-phlorizin hydrolase and gut microbiota, naringin was hydrolyzed into naringenin. Generated naringenin was absorbed through both passive diffusion as well as active transport [14], and subsequently engaged in in vivo phase I and phase II metabolism, giving rise to an array of metabolites. Free naringenin (M3), naringenin-7-O-sulfate (M10), and naringenin-O-glucuronide-O-sulfate (M11) were found to be predominant metabolites in gastrointestinal tracts. Naringenin-7-O-glucuronide (M5) and naringenin-4′-O-glucuronide (M6) were relatively abundant in jejunum, while naringenin-4′-O-sulfate (M9) was solely detected in jejunum and ileum. Meanwhile, phase I metabolites were abundant in duodenum, jejunum, ileum, and colon, probably associated with the abundance of gut microbiota in these regions [15]. Through the portal vein, generated metabolites were transferred from gastrointestinal tract to liver to be further processed. The major metabolites in liver were naringenin (M3) and naringenin-7-O-sulfate (M10), followed by free naringin, naringenin-4′-O-glucuronide (M6), and apiferol (M14). After the hepatic processes, naringin and derived metabolites were released into systemic circulation and then distributed to other tissues. Aligned with reported studies [3], the major metabolites of naringin in most tissues were identified as glucuronide and sulfate conjugates of naringenin. However, the dominant existing forms were found to be different in multiple tissues, implying tissue distribution specificity and binding site specificity of phase II metabolic enzymes in rats. In plasma, naringenin glucuronides were predominant, including naringenin-7-Oglucuronide (M5), naringenin-4′-O-glucuronide (M6), and naringeninO-glucuronide-O-sulfate (M11). While in gastrointestinal tracts, liver, kidney, lung, trachea, heart, spleen, muscle, free naringenin (M3) and naringenin-7-O-sulfate (M10) were the major forms. Therefore, it can be assumed that when naringenin glucuronides transferred from circulation into these tissues, they were hydrolyzed by tissue b-glucuronidase to free naringenin, crossed lipophilic cell membrane, and then sulfated by sulfotransferases. As naringenin-7-O-sulfate was the principal metabolite in most tissues, its bioactivities warrant more investigations. Except for these flavonoid metabolites, 15 phenolic catabolites, such as phenylpropenoic acid, phenylpropionic acid, benzoic acid, and benzoylglycine derivatives, including free phenolics and phase II sulfate conjugates were detected. Phenolic catabolites mainly flowed from the microbiota-mediated ring fission of unabsorbed flavonoids [16], as well as the metabolites excreted by enterohepatic circulation. Reported studies have revealed that 3-(4′-hydroxyphenyl)propionic acid (HPPA) and 4′-hydroxybenzoic acid (HBA), which were the phenolic catabolites of naringenin, could effectively improve the cholesterol and antioxidant metabolism [17]. In this study, 3-(phenyl)-2-propenoic acid-4′-O-sulfate (C3), HPPA (C9), HPPA-4′-O-sulfate (C11), and HBA-4-O-sulfate (C13) were found to be abundant in liver and kidney, which were the main metabolic and excretive tissues, suggesting the relation to mentioned bioactivities. Thus, microbial-derived phenolic catabolites could be an important part to understand the pharmacodynamics of naringin.
2.4. Metabolite profiling For metabolites profiling, tissue extract (100 μL) were deproteinized with 2-fold acetonitrile (containing IS), vortex-mixed for 3 min, and centrifuged at 15,000 × g for 30 min at 25 °C. Finally, 10 μL supernatant was analyzed with Shimadzu UFLC XR system tandem a hybrid quadrupole time-of-flight mass spectrometer system (Triple TOFTM 5600 plus; Sciex, Foster City, USA), which equipped with an ESI source in negative-ionization mode. The accuracy of the instrument for m/z measurement was up to 1 ppm. Chromatographic separation was conducted using a Kinetex C18 column (3.0 × 150 mm, 2.6 μm, 100 Å; Phenomenex, Torrance, USA) at 40 °C. A gradient elution program was conducted for chromatographic separation, with the mobile phase A (0.1% formic acid*water) and mobile phase B (0.1% formic acid–methanol) as follows: 0–10 min (5–65% B), 10–20 min (65–82.5% B), maintained at 100% B for 4 min (20.1–24.1 min), and then decreased to 5% B to equilibrate for 5 min (25.0–30.0 min). The flow rate was 0.3 mL/min. The optimal MS parameters were set as follows: TOF-MS scan range m/z 100 to 1500, product ion scan range m/z 50 to 1500, ion source gas 55 psi, curtain gas 35 psi, ion source temperature 550 °C, ion spray voltage −4,500 V, collision energy 35 eV, collision energy spread 25 eV, declustering potential 80 V. Nitrogen was used as nebulizer and auxiliary gas. Data acquisition was performed with Analyst® TF 1.6 software (Sciex, Foster City, USA) in information-dependent acquisition (IDA) mode. 2.5. Data analysis Metabolites were screened by the software (PeakView, Version 1.6; MetabolitePilot, Version 1.0; Sciex, Foster City, USA) but identified manually. If the IS-standardized response of a chromatographic peak in tissue homogenate is five times higher than that in blank sample, it can be considered as the peak of a potential metabolite. The metabolites profiling was carried out based on following two points: (I) reported results concerning the metabolism of naringin and other flavonoids, (II) full understanding of the MS/MS fragmentation patterns of the parent compound and authentic standards. TOF-MS chromatographic peak areas of identified metabolites, as well as that of IS, were obtained with MultiQuant software (Version 2.1; Sciex, Foster City, USA) and then used to calculate the peak area ratios. Furthermore, area under curve (AUC) were acquired from the mean peak area ratio-time curves to reflect cumulative exposure of identified metabolites. Obtained AUC values were shown in logarithm of 10 and plotted in Fig. 1 with GraphPad Prism (Version 7.0). 3. Results and discussion In this study, high resolution UFLC-Q-TOF-MS/MS based metabolites profiling was performed on plasma as well as fourteen other tissues, including stomach, duodenum, jejunum, ileum, colon, liver, kidney, heart, lung, trachea, spleen, brain, fat, and skeletal muscle. Finally, a total of 23 flavonoid metabolites and 15 phenolic catabolites were identified. Detailed information of identified metabolites was presented in Table 1. The AUC (shown in logarithm of 10) of naringin and derived metabolites were presented in a heat map (Fig. 1). Administered by gavage, naringin was in direct contact with gastrointestinal tract and found to be abundant in these organs. Moreover, 3
4
Naringenin-4′-O-sulfate
Naringenin-7-O-sulfate
Naringenin-O-glucuronide-Osulfate
Naringenin-O-glucoside-O-sulfate
Methylnaringenin-O-glucuronide
Apiferol
Apigenin
Apigenin-O-glucuronide
Apigenin-O-glucuronide
Apigenin-O-sulfate
M9
M10
M11
M12
M13
M14
M15
M16
M17
M18
Eriodictyol-O-sulfate
M20
Hesperetin
Hesperetin-7-O-glucuronide
M21
M22
b
Eriodictyol
M19
b
Naringenin-5,7-O-di-glucuronide
M8
b
Naringenin-4′,7-O-di-glucuronide
M7
b
C22H22O12
C16H14O6
C15H12O9S
C15H12O6
C15H10O8S
C21H18O11
C21H18O11
C15H10O5
C15H14O5
11.8
13.8
12.3
12.7
13.4
12.3
11.1
15.0
13.8
13.5
9.4
C21H22O13S C22H22O11
10.1
12.1
C21H20O14S
C15H12O8S
10.0
9.4
C27H28O17 C15H12O8S
8.7
11.7
11.5
C27H28O17
C21H20O11
Naringenin-4′-O-glucuronide
M6
C21H20O11
Naringenin-7-O-glucuronide
M5
10.7
C21H20O11
b
Naringenin-5-O-glucuronide
M4
13.7
10.2
9.7
11.4
RT (min)
C15H12O5
b
Naringenin
M3
b
Naringin-O-sulfate
C27H32O17S
C27H32O14
M2
b
Formula
C33H40O20
Naringin
Identified metabolites
Flavonoid metabolites M1 Naringin-O-glucuronide
Parent drug
No.
513.0691 (-3.3) 461.1082 (-1.6) 273.0765 (-1.4) 269.0447 (-3.0) 445.0781 (1.0) 445.0780 (0.9) 349.0016 (-2.2) 287.0569 (2.9) 367.0133 (1.0) 301.0709 (-2.9) 477.1033 (-1.3)
527.0496 (-0.9)
623.1270 (2.6) 623.1257 (0.6) 351.0178 (-0.5) 351.0178 (-0.5)
447.0924 (-2.1) 447.0929 (-1.0) 447.0934 (0.1)
755.2064 (3.2) 659.1272 (-2.4) 271.0618 (2.2)
579.1717 (-0.4)
[M−H]− (Error, ppm)
301.0727[M-H-GlcUA]−, 175.0233[M-H-HE]−, 151.0046[M-H-GlcUA-C9H10O2]−, 113.0265[M-H-HE-CO2-H2O]−, 95.0156
(continued on next page)
plasma, stomach, duodenum, jejunum, ileum, liver, kidney duodenum, jejunum, ileum, colon, liver, kidney, lung plasma
348.9218[M-H-H2O]−, 287.0583[M-H-SO3]−, 151.0029[M-H-SO3-C8H8O2]−, 135.0441[M-H-SO3-C7H4O4]−, 107.0148[M-H-SO3-C8H8O2-CO2]− 286.0466[M-H-CH3]−, 151.0004[M-H-C9H10O2]−, 107.0159[M-H-C9H10O2-CO2]−
241.1767, 151.0031[M-H-C8H8O2] , 135.0469[M-H-C7H4O4]
plasma, stomach, duodenum, jejunum, ileum, colon, liver, kidney, lung, trachea, heart, spleen colon, liver
269.0442[M-H-SO3]−, 225.0522, 117.0366[M-H-SO3-C7H4O4]− −
liver
399.0513, 269.0462[M-H-GlcUA]−, 113.0255[M-H-AE-CO2-H2O]−
−
stomach, duodenum, jejunum, ileum, colon, liver, kidney, lung, trachea, heart plasma
jejunum, ileum, colon, liver
plasma
liver
plasma, stomach, jejunum, ileum, liver, kidney, lung plasma, stomach, duodenum, jejunum, ileum, colon, liver, kidney, lung, trachea, heart, spleen, muscle, fat plasma, stomach, duodenum, jejunum, ileum, liver, kidney, lung, trachea, heart, fat
plasma, kidney
plasma, stomach, jejunum, ileum, liver, kidney
plasma, stomach, duodenum, jejunum, ileum, liver, kidney, lung, heart, muscle, fat plasma, stomach, duodenum, jejunum, ileum, liver, kidney, lung, trachea, heart, muscle, fat
229.0875, 179.0360, 167.0334, 153.0062[M-H-C8H8O]−, 123.0460, 119.0506[M-H-C7H6O4]−, 107.0133[M-H-C8H8O-CO-H2O]−, 93.0338[M-H-C9H8O4]− 225.0557, 151.0058[M-H-C8H6O]−, 117.0334[M-H-C7H4O4]−, 107.0227[M-H-C8H6O-CO2]− 269.0468[M-H-GlcUA]−, 175.0269[M-H-AE]−, 113.0269[M-H-AE-CO2-H2O]−, 97.9613
447.0962[M-H-GlcUA]−, 271.0632[M-H-2GlcUA]−, 175.0238[M-H-NE-GlcUA]−, 113.0232[M-H-NE-GlcUA-CO2-H2O]− 271.0612[M-H-SO3]−, 151.0027[M-H-SO3-C8H8O]−, 119.0492[M-H-SO3-C7H4O4]−, 107.0146[M-H-SO3-C8H8O-CO2]−, 93.0389[M-H-SO3-C9H6O4]− 271.0609[M-H-SO3]−, 177.0188[M-H-SO3-C6H6O]−, 151.0033[M-H-SO3-C8H8O]−, 119.0514[M-H-SO3-C7H4O4]−, 107.0169[M-H-SO3-C8H8O-CO2]−, 93.0362[M-H-SO3-C9H6O4]− 447.0953[M-H-SO3]−, 351.0203[M-H-GlcUA]−, 271.0624[M-H-SO3-GlcUA]−, 254.9827, 175.0251[M-H-SO3-NE]−, 151.0028[M-H-SO3-GlcUA-C8H8O]−, 113.0253[M-H-SO3-NE –CO2-H2O]− 467.2386, 433.1195[M-H-SO3]−, 313.0580[M-H-SO3-C4H8O4]−, 271.0648[M-H-SO3-Glc]−, 240.9983, 151.0056[M-H-SO3-Glc-C8H8O]− 285.0758[M-H-GlcUA]−, 175.0215[M-H-MNE]−, 113.0250[M-H-MNE-CO2-H2O]−
271.0613[M-H-GlcUA]−, 175.0206[M-H-NE]−, 151.0031[M-H-GlcUA-C8H8O]−, 119.0493[M-H-GlcUA-C7H4O4]−, 113.0199[M-H-NE-CO2-H2O]− 271.0616[M-H-GlcUA]−, 227.0702, 175.0244[M-H-NE]−, 151.0040[M-H-GlcUA-C8H8O]−, 119.0507[M-H-GlcUA-C7H4O4]−, 113.0251[M-H-NE-CO2-H2O]− 313.0730[M-H-C4H6O5]−, 271.0618[M-H-GlcUA]−, 227.0730, 177.0205[M-H-GlcUA-C6H6O]−, 175.0252[M-H-NE]−, 151.0038[M-H-GlcUA-C8H8O]−, 119.0501[M-H-GlcUA-C7H4O4]−, 113.0256[M-H-NE-CO2-H2O]− 447.0939[M-H-GlcUA]−, 271.0626[M-H-2GlcUA]−, 175.0242[M-H-NE-GlcUA]−
colon, liver
579.1711[M-H-SO3]−, 527.2455, 459.1262[M-H-SO3-C8H8O]−, 351.0248[M-H-Rha-Glc]−, 313.0700[M-H-SO3-C8H8O-Rha]−, 271.0612[M-H-SO3-Rha-Glc]− 225.0697, 177.0166[M-H-C6H6O]−, 151.0029[M-H-C8H8O]−, 119.0501[M-H-C7H4O4]−, 107.0152[M-H-C8H8O-CO2]−, 93.0364[M-H-C9H6O4]−
plasma, stomach, duodenum, jejunum, ileum, colon, liver, kidney, lung, trachea, heart, spleen, muscle, fat plasma, jejunum, kidney
jejunum, ileum, colon
579.1693[M-H-GlcUA]−, 429.0959, 271.0612[M-H-GlcUA-Rha-Glc]−, 175.0269[M-H-NG]−
Source plasma, stomach, duodenum, jejunum, ileum, colon, liver, kidney, lung, trachea, heart, spleen, brain, muscle, fat
a
459.1175[M-H-C8H8O]−, 339.0710, 313.0725[M-H-C8H8O-Rha]−, 271.0626[M-H-Rha-Glc]−, 151.0044[M-H-Rha-Glc-C8H8O]−, 119.0492[M-H-Rha-Glc-C7H4O4]−
Fragment ions in negative mode
Table 1 UFLC-Q-TOF-MS/MS based identifications of flavonoid metabolites and phenolic catabolites in rat tissues collected 0–24 h after a single oral administration of naringin.
X. Zeng, et al.
Journal of Chromatography B 1136 (2020) 121846
5
b
a
Hippuric acid
b
7.9 7.0 8.0 10.2 10.8 8.3
C9H10O4 C9H10O7S C9H10O7S C9H10O3 C9H10O3 C9H10O6S
C9H9NO3
8.2
C10H12O7S
8.9
273.0076 (0.4) 163.0410 (5.9) 242.9971 (0.8) 242.9972 (1.3) 275.0236 (2.0) 181.0512 (3.4) 261.0074 (-0.2) 261.0073 (-0.5) 165.0564 (4.3) 165.0564 (4.4) 245.0130 (1.8) 245.0127 (0.5) 216.9815 (1.1) 216.9817 (2.0) 178.0518 (4.8)
381.0275 (-2.9)
[M−H]− (Error, ppm)
a
−
160.0415[M-H-H2O]−, 134.0610[M-H-CO2]−, 77.0426
146.0830, 137.0241[M-H-SO3] , 93.0379[M-H-SO3-CO2]
−
−
165.0528[M-H-SO3]−, 121.0659[M-H-SO3-CO2]−, 119.0503[M-H-SO3-HCOOH]−, 106.0425[M-H-SO3-CO2-CH3]− 146.0824, 137.0245[M-H-SO3]−, 116.0722, 93.0353[M-H-SO3-CO2]−, 88.0424
165.0556[M-H-SO3]−, 121.0673[M-H-SO3-CO2]−, 93.0362[M-H-SO3-CO2-C2H4]−
147.9822, 121.0647[M-H-CO2] , 119.0506[M-H-HCOOH] , 106.0429[M-H-CO2-CH3]
plasma, stomach, liver, kidney, lung, trachea, heart, spleen, brain, muscle, fat
kidney
liver, kidney
plasma, stomach, colon, liver, kidney, lung, heart, muscle, fat plasma, colon, kidney
plasma, colon, kidney, brain
plasma, colon, liver, kidney, lung, heart, muscle −
147.9836, 137.0251[M-H-CO]−, 121.0655[M-H-CO2]−, 93.0361[M-H-CO2-C2H4]− −
kidney, brain
217.0353[M-H-CO2]−, 203.0812[M-H-CO2-CH2]−, 181.0481[M-H-SO3]−, 131.0163
−
plasma, stomach, ileum, liver, lung, trachea, heart, spleen, brain, muscle, fat plasma, kidney, brain
plasma, kidney
plasma, stomach, jejunum, ileum, colon, liver, kidney, lung, trachea, heart, spleen, muscle, fat plasma, liver, kidney
plasma, liver, kidney
plasma
duodenum, jejunum, ileum, liver, kidney
Source
230.9960[M-H-CO2]−, 195.0656[M-H-SO3]−, 177.0563[M-H-SO3-H2O]−, 133.0645[M-H-SO3-H2O-CO2]− 163.0387[M-H-H2O]−, 136.9842[M-H-CO2]−, 135.0453[M-H-H2O-CO]−, 119.0505[M-H-H2O-CO2]−, 107.0492[M-H-H2O-2CO]−, 92.9940 216.9980[M-H-CO2]−, 181.0487[M-H-SO3]−, 137.0596[M-H-SO3-CO2]−, 95.0493
163.0393[M-H-SO3]−, 130.9664, 119.0497[M-H-SO3-CO2]−
163.0404[M-H-SO3] , 119.0510[M-H-SO3-CO2] , 96.9609
−
228.8822[M-H-CO2]−, 193.0496[M-H-SO3]−, 178.0285[M-H-SO3-CH3]−, 134.0367[M-H-SO3-CH3-CO2]− 119.0509[M-H-CO2]−, 93.0354[M-H-CO2-2CH]−
301.0714[M-H-SO3]−, 286.0463[M-H-SO3-CH3]−, 151.0029[M-H-SO3-C9H10O2]−
Fragment ions in negative mode
The losses are: Rha = rhamnose moiety, Glc = glucose moiety, GlcUA = glucuronyl moiety, NG = naringin, NE = naringenin, AE = apigenin, HE = hesperetin. Confirmation in comparison with authentic standards.
C15
9.4
C9H8O6S
6.9
8.6
C9H8O6S
C7H6O6S
Benzoic acid-3-O-sulfate
10.9
C9H8O3
C14
8.8
C10H10O7S
6.4
12.3
C7H6O6S
C16H14O9S
8.6
b
RT (min)
C9H10O6S
Hesperetin-7-O-sulfate
M23
Formula
Phenolic catabolites C1 3-(4′-Methoxyphenyl)-2propenoic acid-3′-O-sulfate C2 3-(4′-Hydroxyphenyl)-2propenoic acid C3 3-(Phenyl)-2-propenoic acid-4′-Osulfate C4 3-(Phenyl)-2-propenoic acid-3′-Osulfate C5 3-(4′-Methoxyphenyl)propionic acid-3′-O-sulfate C6 3-(3′,4′-Di-hydroxyphenyl) propionic acid C7 3-(3′-Hydroxyphenyl)propionic acid-4′-O-sulfate C8 3-(4′-Hydroxyphenyl)propionic acid-3′-O-sulfate C9 3-(4′-Hydroxyphenyl)propionic acid b C10 3-(3′-Hydroxyphenyl)propionic acid C11 3-(Phenyl)propionic acid-4′-Osulfate C12 3-(Phenyl)propionic acid-3′-Osulfate C13 Benzoic acid-4-O-sulfate
Identified metabolites
No.
Table 1 (continued)
X. Zeng, et al.
Journal of Chromatography B 1136 (2020) 121846
Journal of Chromatography B 1136 (2020) 121846
X. Zeng, et al.
4. Conclusion [3]
In this work, distribution of naringin and derived metabolites in rat plasma and fourteen tissues were investigated with UFLC-Q-TOF-MS/ MS system. After a single oral administration, naringin was found to be widely distributed and its concentrations in certain tissues were much higher than that in plasma, especially in trachea and lung. This at least partially explains the naringin’s effects on respiratory disease. A total of 23 flavonoid metabolites and 15 phenolic catabolites were detected. Naringenin glucuronides were predominant metabolites in plasma, while free naringenin and naringenin-7-O-sulfate were the major molecular forms in most tissues, implying deglucuronidation and subsequent sulfation in the tissue distribution of naringenin glucuronides. Besides, phenolic catabolites derived from naringin, which also possessed certain bioactivities, were abundant in liver and kidney. In summary, naringin as well as derived metabolites were more widely distributed in tissues than in plasma after oral administration. These pharmacokinetic results would be helpful in the interpretation of pharmacodynamics for naringin.
[4] [5] [6] [7] [8]
[9] [10]
Declaration of Competing Interest
[11]
All the authors declare that there are no known conflicts of interest with this publication.
[12]
Acknowledgements
[13] [14]
This work was financially supported by National Natural Science Foundation of China(No. 31571830), Applied Science and Technology R &D Special Fund Project of Guangdong Province (No. 2016B020239003), and Guangdong Academic of Sciences Special Project of Science and Technology Development (No. 2016GDASRC-0104).
[15]
[16]
References
[17]
[1] S. Bharti, N. Rani, B. Krishnamurthy, D.S. Arya, Preclinical evidence for the pharmacological actions of naringin: a review, Planta Med 80 (2014) 437–451. [2] R. Shi, Z.T. Xiao, Y.J. Zheng, Y.L. Zhang, J.W. Xu, J.H. Huang, W.L. Zhou, P.B. Li,
6
W.W. Su, Naringenin regulates CFTR activation and expression in airway epithelial cells, Cell. Physiol. Biochem. 44 (2017) 1146–1160. M. Liu, W. Zou, C. Yang, W. Peng, W. Su, Metabolism and excretion studies of oral administered naringin, a putative antitussive, in rats and dogs, Biopharm. Drug Dispos. 33 (2012) 123–134. X. Zeng, W. Su, Y. Zheng, Y. He, Y. He, H. Rao, W. Peng, H. Yao, Pharmacokinetics, tissue distribution, metabolism, and excretion of naringin in aged rats, Front. Pharmacol. 10 (2019) 34. M.L. Matabilbao, C. Andréslacueva, E. Roura, O. Jáuregui, E. Escribano, C. Torre, R.M. Lamuelaraventós, Absorption and pharmacokinetics of grapefruit flavanones in beagles, Br. J. Nutr. 98 (2007) 86–92. X. Zeng, Y. Bai, W. Peng, W. Su, Identification of naringin metabolites in human urine and feces, Eur. J. Drug Metab. Pharmacokinet. 42 (2017) 647–656. C. Felgines, O. Texier, C. Morand, C. Manach, A. Scalbert, F. Régerat, C. Rémésy, Bioavailability of the flavanone naringenin and its glycosides in rats, Am. J. Physiol. Gastrointest. Liver Physiol. 279 (2000) G1148–G1154. M.A.E. Mohsen, J. Marks, G. Kuhnle, C. Rice-Evans, K. Moore, G. Gibson, E. Debnam, S.K. Srai, The differential tissue distribution of the citrus flavanone naringenin following gastric instillation, Free Rad. Res. Commun. 38 (2004) 1329–1340. W. Zou, C. Yang, M. Liu, W. Su, Tissue distribution study of naringin in rats by liquid chromatography-tandem mass spectrometry, Arzneimittelforschung 62 (2012) 181–186. S. Lin, Y. Hou, S. Tsai, M. Wang, P. Chao, Tissue distribution of naringenin conjugated metabolites following repeated dosing of naringin to rats, BioMedicine 4 (2014) 16. X. Zeng, W. Su, Y. Bai, T. Chen, Z. Yan, J. Wang, M. Su, Y. Zheng, W. Peng, H. Yao, Urinary metabolite profiling of flavonoids in Chinese volunteers after consumption of orange juice by UFLC-Q-TOF-MS/MS, J. Chromatogr. B 1061–1062 (2017) 79–88. P. Li, Y. Wang, Z. Wu, W. Peng, C. Yang, Y. Nie, M. Liu, Y. Luo, W. Zou, Y. Liu, The pre-clinical studies of naringin, an innovative drug, derived from Citri Grandis Exocarpium (Huajuhong), Acta Scient. Naturalium Univ. Sunyatseni 54 (2015) 1–5. P.I. Oteiza, C.G. Fraga, D.A. Mills, D.H. Taft, Flavonoids and the gastrointestinal tract: local and systemic effects, Mol. Aspects Med. 61 (2018) 41–49. L. Zhang, L. Song, P. Zhang, T. Liu, L. Zhou, G. Yang, R. Lin, J. Zhang, Solubilities of naringin and naringenin in different solvents and dissociation constants of naringenin, J. Chem. Eng. Data 60 (2015) 932–940. T. Chen, W. Su, Z. Yan, H. Wu, X. Zeng, W. Peng, L. Gan, Y. Zhang, H. Yao, Identification of naringin metabolites mediated by human intestinal microbes with stable isotope-labeling method and UFLC-Q-TOF-MS/MS, J. Pharm. Biomed. Anal. 161 (2018) 262–272. W. Zou, Y. Luo, M. Liu, S. Chen, S. Wang, Y. Nie, G. Cheng, W. Su, K. Zhang, Human intestinal microbial metabolism of naringin, Europ. J. Drug Metab. Pharmacokinet. 40 (2015) 363–367. S.-M. Jeon, H.K. Kim, H.-J. Kim, G.-M. Do, T.-S. Jeong, Y.B. Park, M.-S. Choi, Hypocholesterolemic and antioxidative effects of naringenin and its two metabolites in high-cholesterol fed rats, Translat. Res. 149 (2007) 15–21.