Pharmacokinetics and Metabolism of Naringin and Active Metabolite Naringenin in Rats, Dogs, Humans, and the Differences Between Species

Background Pharmacokinetics provides a scientific basis for drug product design, dosage regimen planning, understanding the body's action on the drug, and relating the time course of the drug in the body to its pharmacodynamics and/or toxic effects. Recently, naringin, a natural flavonoid, was approved for clinical trials as a first-class new drug product by the China Food and Drug Administration, owing to its nonclinical efficacy in relieving cough, reducing sputum, and low toxicity. Previous reports focused on the pharmacokinetic studies of naringin or its active metabolite naringenin in rats, which were scattered and insufficient because naringin was coadministered with mixtures such as herbs, fruits, and other traditional medicines. The purpose of this study was to evaluate the pharmacokinetics and metabolism of naringin and naringenin, following oral and intravenous administration of naringin in rats, dogs, and humans, which can be beneficial for new drug development. Methods Separate bioanalytical methods were developed and validated to determine the concentrations of naringin and its active metabolite naringenin in biological samples obtained from rats, dogs, and humans. Comprehensive nonclinical and clinical data were used to estimate the pharmacokinetic parameters of naringin and naringenin. Experiments included single-dose studies (oral and intravenous administration), multiple-dose studies, and an assessment of food-effects. Furthermore, the metabolism of naringin and naringenin was studied in rat and human liver and kidney microsomes. All biological samples were analyzed using liquid chromatography-tandem mass spectrometry. Results The pharmacokinetic parameters of naringin and naringenin were calculated and the results show an insignificant influence of high-fat diet and insignificant accumulation of the drugs after multiple dosing. Twelve metabolites were detected in the liver and kidney microsomes of rats and humans; naringin metabolism was a complex process simultaneously catalyzed by multiple human enzymes. All evaluated species demonstrated differences in the pharmacokinetics and metabolism of naringin and naringenin. Conclusion The results can be used to design a dosage regimen, deepen understanding of mechanisms, and accelerate new drug development. Clinical Trial Registration http://www.chinadrugtrials.org.cn/eap/main, identifiers CTR20130704 and CTR20190127.


INTRODUCTION
Pharmacokinetics is the science of the kinetic processes of drug absorption and disposition (i.e., distribution, metabolism, and excretion), which can provide a scientific basis for drug product design, dosage regimen design, understanding the body's action on the drug, and relating time course of the drug in the body to its pharmacodynamics and toxic effects.
In the last 2 decades, extensive research has been devoted to developing naringin as a first-class new drug to relieve cough and reduce sputum, and the drug has been approved for clinical trials by the China Food and Drug Administration . In pharmacological studies, naringin exhibited antitussive and protective effects against acute lung injury, pulmonary fibrosis, chronic bronchitis, and cough-variant asthma (Gao et al., 2011;Liu Y. et al., 2011;Liu Y. et al., 2012;Luo et al., 2012;Nie et al., 2012a;Nie et al., 2012b;Chen et al., 2013;Luo et al., 2013;Chen et al., 2014;Jiao et al., 2015). Furthermore, naringin toxicity was assessed in Sprague-Dawley rats (Liu M. et al., 2011;Li P. et al., 2013;Li et al., 2014). In a study assessing a single p.o. bolus naringin dose (16 g kg -1 ), no apparent toxicity was documented . Additionally, no adverse effects were observed in a toxicity study of naringin (1,250 mg kg -1 day -1 for 184 days) (Liu M. et al., 2011;Li et al., 2014).
With the exception of methods available in the literature on the analysis of rat plasma (Tsai and Tsai, 2012;Wen et al., 2012;Tong et al., 2012;Li S. Q. et al., 2013;Jin et al., 2015;Lu et al., 2015;Zeng et al., 2018;Zeng et al., 2019) and human plasma and urine (Ishii et al., 2000;Xiong et al., 2014), only a few methods are reported to analyze naringin and naringenin in other types of biological matrices in rats, dogs, and humans. The present study reports the analysis of naringin and naringenin in rat, dog, and human plasma and human urine and feces. Previously, the pharmacokinetic studies of naringin or naringenin focused on rats, and naringin was often coadministered in mixtures such as herbs, fruits, and other traditional medicines (Tsai and Tsai, 2012;Tong et al., 2012;Wen et al., 2012;Li S. Q. et al., 2013;Jin et al., 2015;Lu et al., 2015). Hence, in rats, the pharmacokinetics of naringin could be influenced by other coadministered constituents. Moreover, naringin pharmacokinetics has been minimally investigated in dogs and humans. Hence, these pharmacokinetic parameters are inaccurate and insufficient to design drug dosage regimens in clinical trials. In the current study, the pharmacokinetic parameters of naringin and naringenin were estimated accurately and sufficiently, since naringin was administered alone to rats, dogs, and humans. Furthermore, the metabolic processes of naringin were studied using human excreta. Following oral administration, naringin is metabolised to the active metabolite naringenin and their hydroxylated, hydrogenated, dehydrogenated, and acetylated metabolites by intestinal microflora . Subsequently, the metabolites are transported, mainly via the portal vein, to the liver for further biotransformation to glucuronide and sulphate conjugates, which enter systemic circulation (Tsai and Tsai, 2012). Finally, naringin, naringenin, and their conjugates are secreted into bile and excreted into the feces or reabsorbed to become systemically available (i.e., through enterohepatic circulation) (Ma et al., 2006;Liu M. et al., 2012;Zeng et al., 2017). Since the glucuronide and sulphate conjugates of flavonoids were reported as precursors of bioactive metabolites (glycoside or aglycone) (Terao et al., 2011;Matsumoto et al., 2018), hydrolysis with b-glucuronidase is often used in pharmacokinetic studies of naringin or naringenin. Although the metabolism and nonclinical excretion of naringin have previously been reported, enzyme pathways and excretion were insufficiently presented in humans, hindering elucidation of the clinical mechanisms (Liu M. et al., 2012;Zeng et al., 2017). Our study focused on the metabolism of naringin with regards to enzyme pathways and its excretion in humans, which is beneficial to comprehend the mechanisms of naringin in clinical studies.
In this study, we investigated the pharmacokinetics and metabolism of naringin and naringenin in rats, dogs, and humans, following the administration of naringin. Separate bioanalytical methods were employed for the analysis of naringin and naringenin in biological samples obtained from rats, dogs, and humans. An abundance of data was utilized to calculate the pharmacokinetic parameters of naringin and naringenin in rats, dogs, and humans, including naringin administration as a single dose (oral, p.o.; intravenous, i.v.), in multiple doses, and with a highfat diet. Moreover, the metabolism of naringin and naringenin was researched in rat and human liver and kidney microsomes, indicating small species differences in metabolites, with a complex metabolic process simultaneously catalyzed by multiple human enzymes. Species differences were observed with regards to the pharmacokinetics and metabolism of naringin. This information contributes to naringin development as a new drug and understanding the in vivo mechanisms involved.

Animals and Drug Administration
All experimental procedures and protocols were conducted in accordance with the Basel Declaration and National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978), and approved by the Animal Ethics Committee of the School of Life Sciences at Sun Yat-sen University (Guangzhou, Guangdong, China; Approval number: 090108). All nonclinical experimental designs were consistent with the Technical Guidelines for Nonclinical Pharmacokinetic Studies of Drugs published by the China Food and Drug Administration. Animal studies were reported in compliance with the Animal Research: Reporting In Vivo Experiments guidelines.
Sixty healthy adult Sprague Dawley rats (180-220 g, half male and female) were purchased from the Guangdong Medical Laboratory Animal Center (Guangzhou, Guangdong, China). Thirty Beagle dogs (10-14 kg, half male and female) were purchased from the Gaoyao Kangda Laboratory Animal Science & Technology Co., Ltd. (Gaoyao, Guangdong, China). All animals were housed under humanized conditions with free access to food and water, and acclimated to the living conditions for at least one week prior to experimentation at the Laboratory Animal Center of the School of Life Sciences, Sun Yat-sen University. Environmental conditions were maintained at 20°C -25°C, 55% ± 15% relative humidity, 12-h light/dark cycles, and 12 air changes per hour. Prior to experimentation, the animals were fasted overnight with water available ad libitum. All animals were healthy based on physical examination and laboratory analyses performed before and after the experiments. At the end of the experiments, the dogs were returned to the Laboratory Animal Center of School of Life Sciences, Sun Yatsen University.

Humans and Drug Administration
Participants were healthy males and females, 18 to 40 years old, ≥50 kg weight, 19 to 25 kg m 2 body mass index. The health condition of participants was considered good based on the screened medical history, physical examination, vital signs, electrocardiogram, and laboratory examination. A 12-lead electrocardiogram and serum troponin I had to be normal at screening within 8 days of initial dosing, and serology for hepatitis C antibodies, hepatitis B surface antigen, human immunodeficiency virus (HIV) antibodies, syphilis antibodies, HIV, and blood pregnancy tests had to be negative. Clinical laboratory test results (sodium, potassium, total protein, white blood and red blood cell counts, hematocrit, hemoglobin, platelet count, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase, blood urea nitrogen, creatinine, total bilirubin, alkaline phosphatase, creatine kinase, lactate dehydrogenase, calcium, chloride, phosphorus, albumin, globulin, cholesterol, triglycerides, glucose, uric acid, and urinalysis) had to be within normal ranges, without other clinically significant abnormalities. During the study duration, participants were required to use barrier contraception with spermicide during sexual intercourse. During the trial, female participants were in the nonmenstrual, nonpregnant, and nonlactation periods, and they were nonsmokers. Participants were not planning a pregnancy within 6 months of the clinical trials, they communicated with the investigator, understood and complied with the study requirements, and signed informed consent.
The main exclusion criteria were history of a clinically significant disease, an abnormality, or drug allergies, including a history or evidence of cardiovascular, respiratory, hepatic, renal, haematologic, gastrointestinal, endocrine, metabolic, immunologic, dermatologic, neurologic, oncologic, or psychiatric illness or significant abnormalities, or any condition, surgical intervention known to interfere with drug absorption, distribution, metabolism, or excretion. Prior to clinical trials, participants were excluded if they had acute or subacute cough, mediated by neurogenic inflammation (such as cough after cold), chronic bronchitis with chronic cough, bronchial asthma, chronic obstructive pulmonary disease, pulmonary cystic fibrosis, bronchiectasis, and other respiratory diseases. Participants were excluded if they had abnormal chest radiographs or electrocardiogram parameters as follows: PR intervals >210 ms, or QRS duration >120 ms, or QTcB >460 ms (male) or QTcB >470 ms (female). In addition, participants were excluded if they planned to have a baby within 6 months posttrials. A history of significant tea or coffee consumption (> 8 cups per day) was prohibited. Participants were excluded if they had been administered with any investigational products in clinical studies four weeks prior to this trial or any other drugs, herbal products, and dietary supplements two weeks before this trial.
Briefly, the study was a randomized, double-blinded, and placebo-controlled phase I clinical trial, including a single ascending dose and multiple-dose studies of naringin tablets administered to seven groups of healthy adult subjects (10 subjects in each group, 2 received placebo). Naringin and placebo tablets were identical in appearance and participants, care providers, and those assessing outcomes were blinded to the study drug assignment. Using the Statistical Analysis System (SAS) software (Version 9.4, SAS Institute Inc., Cary, NC, USA), subject randomization and blinding/masking were performed by statisticians from the Department of Health Statistics at the Second Military Medical University. Naringin tablets were administered p.o. in single doses of 40, 80, 160, 160 (high-fat diet), 320, and 480 mg, separately, and in multiple doses of 160 mg (once on the 1 st day, three times from 2 nd to 6 th day, and once on the 7 th day).
Clinical studies (registration numbers: CTR20130704 for single-dose studies and CTR20190127 for multiple-dose study, http://www.chinadrugtrials.org.cn/eap/main) were conducted, in accordance with the International Conference on Harmonization (ICH) E6 Consolidated Guidance for Good Clinical Practice (GCP) (1996) and the US Code of Federal Regulations (CFR) 21 parts 50 and 56. The clinical study was conducted at the Beijing Hospital; the protocols and informed consent forms were approved by the Institutional Review Board at the Beijing Hospital (Beijing, China; Approval number: 2014BJYYEC-047-14; 2018BJYYEC-123-02) and participants provided written informed consent before any study-related procedures were performed, in accordance with the Helsinki Declaration (1996). Data administration was conducted by the Department of Health Statistics at the Second Military Medical University, in accordance with ICH of Technical Requirements for Pharmaceuticals for Human Use, GCP, FDA 21 CFR Part 11, and corresponding regulations.
All blood samples were transferred into heparin-treated centrifuge tubes and centrifuged for 10 min at 4,000 rpm and 4°C. All human urine samples were mixed and transferred (4 ml) into storage tubes. All biological samples were stored at −80°C.

Bioanalytical Method Validation and Samples Determination
Separate bioanalytical methods were employed for the analysis of naringin and naringenin in rat, dog, human plasma, and human urine and faecal samples. For the rat and dog plasma samples, quercetin 3-b-D-glucoside was used as an internal standard (IS) and for the human plasma, urine, and fecal samples naringin-D4 and naringenin-D4 were used as IS's. Three concentrations served as the quality control (QC) samples in these methods: one within three folds of the lower limit of quantification (LLOQ), low QC (LQC) sample, one near the center, middle QC (MQC) sample, and one near the upper boundary of the standard curve, high QC (HQC) sample, as shown in Tables S1 and S2.
In selectivity, blank samples from the appropriate biological matrices (rat plasma, dog plasma, and human plasma, urine, and feces) were obtained from at least six sources and tested for interference. The interference in each blank sample was evaluated via comparison with the respective LLOQ response. Furthermore, the peak area of interference should be within 20% of the analyte area and 5% that of IS at corresponding retention times in LLOQ.
Accuracy and precision were measured using six determinations per concentration at four concentrations, including the LLOQ, LQC, MQC, and HQC, which were performed in at least three separate runs for at least two days. Accuracy is the percentage difference from the added concentration and referred to as %Deviation. It is calculated as ([measured concentration/added concentration] * 100) -100 with negative %Deviation representing underestimation and positive %Deviation representing overestimation of the true value. Precision is the variation coefficient of the replicates. It is calculated as (standard deviation of measured concentrations/ mean of measured concentrations) * 100. The dilution integrity experiments were conducted using samples five times the concentration of the upper limit of quantification (ULOQ), named dilution QC. The dilution QC samples were diluted to 1/10 and 1/100 dilutions with interference-free biological matrices. The accuracy and precision of the dilution QC samples were evaluated to validate the dilution integrity. The acceptance criteria were set as follows: (i) mean values for accuracy should be ±15% of the actual QC values and ±20% of the actual LLOQ values; (ii) precision should be ≤15% for QCs and ≤20% for LLOQ.
A blank sample (matrix sample processed without IS) and a zero sample (matrix sample processed with IS) were analyzed, but not included in the calibration curve. The calibration curve consisted of at least eight samples containing naringin, naringenin, and IS covering the expected range ( Table S1).
The following acceptance criteria should be met: (i) accuracy is ±15% and precision is ≤15% of the calibration curve levels and accuracy is ±20% and precision is ≤20% for the LLOQ; (ii) at least six out of eight acceptable standards in a calibration curve. The linearity of each calibration curve was determined by plotting the peak area ratio (y) of the analyte/IS vs. nominal concentration of the analyte (x). The calibration curves were constructed by weighted (1/x 2 ) least-squares linear regression.
Recovery experiments were performed by comparing the analytical results for extracted samples at three concentrations (LQC, MQC, and HQC) with unextracted standard solutions. The matrix effect experiments were performed using at least six blank matrix sources at two concentrations (LQC, HQC). The matrix effect was assessed using the ratio between the extracted blank matrix spiked with standard solution and unextracted standard solutions. The difference should be within 15% in recovery and matrix effect experiments.
The stability evaluations included the expected sample handling and storage conditions, including freeze and thaw stability (four cycles), short-term stability (room temperature for 24 h before handling; the longest batch reinjection for 72 h at 5°C, processed sample for 72 h at 5°C, and whole blood at room temperature for 1 h), long-term stability (1 and 12 months at −80°C and −20°C), and standard solution stocking stability (at least 2 weeks at −20°C). Incurred sample reanalysis was performed in separate runs on different days.
The incubated solution (500 ml) of systems I-rat, II-rat, III-rat, I-human, and II-human were extracted with ethyl acetate (500 ml) for at least 3 min by vortexing and centrifuged for 10 min at 13,000 rpm and 4°C. The extraction process described above was performed twice and all supernatants were transferred into centrifuge tubes, dried with N 2 , and dissolved with methanolwater (1:1, v/v). All samples were injected into a UPLC/Q-TOF-MS system consisting of a Waters Acquity ultra performance liquid chromatography (UPLC) coupled with a Micromass micro™ quadrupole time-of-flight mass spectrometry (Q-TOF-MS) system (Waters Corp., Milford, MA, USA), equipped with an electrospray ionization source, and controlled by the MassLynx (v. 4.1) software. Separation was performed on a Waters Acquity UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 mm) at 40°C. The mobile phase consisted of 0.1% formic acid (v/v) in water (A) and methanol (B) using a linear gradient of 95% to 60% A (0-25 min), 60% to 40% A (25-35 min), 40% to 10% A (35-40 min), 10% A (40-43 min), 10% to 95% A (43-44 min), and 95% A (44-45 min). The injection volume was 5 ml with flow rate of 0.2 ml min -1 . The Q-TOF-MS worked in a full scan mode (100-1,200 m/z), selection ion mode, and product ion mode, in negative ion mode, respectively. The conditions were as follows: capillary voltage 3,000 V, sample cone voltage 30 V, extraction cone voltage 2.0 V, source temperature 100°C, desolvation temperature 350°C, cone gas (N 2 ) flow rate of 40 L/h, desolvation gas (N 2 ) flow rate of 700 L/h, low collision energy 6.0 eV, and high collision energy 20-80 eV. Compounds were detected with accurate mass using leucine enkephalin for mass correction at 554.2615 (m/z) in negative ionization mode.
For incubation system III-human, naringin solution (25 mM, 1.5 ml), blank solution or inhibitor working solution (1.5 ml), human liver microsomes (2.25 ml), and phosphate solution (100 mM, 264.75 ml) were mixed in a tube and incubated for 5 min in a water bath shaker (37°C). Five single inhibitor working solutions were separately added. Their concentrations were as follows: a-naphthoflavone (0.04 mM) for CYP 1A2, sulfaphenazole (0.8 mM) for CYP 2C9, (+/-)-N-3benzylnirvanol (0.8 mM) for CYP 2C19, quinidine (0.08 mM) for CYP 2D6, and ketoconazole (0.04 mM) for CYP 3A4/5. All protein concentrations were 0.15 mg ml -1 in the incubation system, except 0.3 mg ml -1 for the sulfaphenazole working solution. Then, NADPH (10 mM, 30 ml) was added and the mixed solution incubated for 45 min. Finally, cold acetonitrile (600 ml, containing 100 ng ml -1 flufenamic acid) was added to every tube, mixed by vortexing, centrifuged for 30 min at 14,000 g and 4°C, and the supernatants transferred into centrifuge tubes and injected into the UHPLC/MS/MS system as described above. Incubations were run in triplicate experiments. Additionally, the naringenin solution (25 mM, 1.5 ml) was also analyzed in system III as the naringin solution (25 mM, 1.5 ml) above.

Data and Statistical Analysis
The results were calculated and analyzed with Microsoft Excel (Version 2016, Microsoft Corp., Redmond, WA, USA) and reported in three significant figures. All pharmacokinetic and excretion parameters were expressed as mean ± standard deviation (SD). All mean and SD values were reported to three significant figures. The pharmacokinetic parameters were calculated using the Phoenix WinNonlin software (Version 6.4, Pharsight Corp., Cary, NC, USA) and compared using the Student's t-test (p < 0.05) in SPSS 18.0 (SPSS Inc., Chicago, IL, USA).
All stability test results were within 15% of nominal concentrations, including freeze and thaw (four cycles), shortterm (room temperature for 24 h before handling, the longest batch reinjection for 72 h at 5°C, processed sample for 72 h at 5°C , and whole blood at room temperature for 1 h), long-term (1 and 12 months at −80°C and −20°C), and standard stock solution (at least 2 weeks at −20°C) stabilities. The recovery, interference, matrix effect, and ruggedness were evaluated to satisfy requirements of the FDA (CDER) Guidance for the Industry Bioanalytical Method Validation (Rockville, MD, USA, 2001) and Guideline on Bioanalytical Method Validation (Chinese Pharmacopoeia Commission, 2015). Incurred sample reanalyses were performed, according to pertinent guidelines; more than two-thirds of the incurred sample reanalyses results were within 20%.

Nonclinical and Clinical Pharmacokinetics and Species Differences
For the single-dose studies, noncompartmental pharmacokinetic analyses were performed to obtain the following data: the area under the curve to the last quantifiable concentration (AUC 0-t ) and to infinity (AUC 0-∞ ), the time for peak plasma level (T max ), the peak plasma level (C max ), the terminal elimination half-life (t 1/2 ), the apparent volume of distribution (Vd), the apparent clearance (CL), and the mean residence time (MRT) (Tables 1  and 2). For the multiple-dose studies, pharmacokinetic parameters included the mean plasma level at steady state, T max , t 1/2 , the percent fluctuation, and the accumulation index ( Table 3). Figure 1 shows the plasma concentration-time profiles of naringin and naringenin in rats, dogs, and humans. In addition, we compared the pharmacokinetic parameters obtained from males and females (Tables 4-6).
Species differences were presented for the pharmacokinetic parameters. In the nonclinical p.o. administration studies, the rat 1 | Pharmacokinetic parameters of naringin in single-dose studies (n=10 per groups of rats, n=6 per groups of dogs, n=8 per groups of humans, mean ± SD).

Species
Dose a (mg kg -1 or mg) The route of adminstration The unit was mg ml -1 for i.v. administration groups, and ng ml -1 for p.o. administration groups. c The unit was h mg ml -1 for i.v. administration groups, and h ng ml -1 for p.o. administration groups. d Compared to 160 mg group, the acceptable level of significance was established at P < 0.05*. The unit was mg ml -1 for rats and dogs, and ng ml -1 for humans. c The unit was h mg ml -1 for rat and dogs, and h ng ml -1 for humans. d Compared to 160 mg group, the acceptable level of significance was established at P < 0.05*. 3 | Pharmacokinetic parameters of naringin and naringenin in multiple-dose studies (p.o., three times a day, n=10 per groups of rats, n=6 per groups of dogs, n=8 per groups of humans, mean ± SD).

Species
Dose (mg kg -1 or mg a ) Analyte   Compared to males, the acceptable level of significance was established at P < 0.05*. c The unit was mg ml -1 for i.v. administration groups, and ng ml -1 for p.o. administration groups. d The unit was h mg ml -1 for i.v. administration groups, and h ng ml -1 for p.o. administration groups. e There were four females in 40 mg group (one female, p.o., placebo), four females in 80 mg group (zero female, p.o., placebo), five females in 160 mg group (one female, p.o., placebo), five females in 160 mg (high-fat diet) group (one female, p.o., placebo), three females in 320 mg group (two females, p.o., placebo), and two females in 480 mg group (two females, p.o., placebo). f The high-fat diet group.
TABLE 5 | The pharmacokinetic parameters of naringenin for different sexes in single-dose studies (half male and female for rats and dogs, a mix of males and females for humans, mean ± SD).

Information of administration a Male Female b
and dog plasma concentrations of naringin increased rapidly to peaks within about 1 h (rats, T max , 1.77 ± 2.64 h and dogs, T max , 1.11 ± 0.344 h). The t 1/2 of naringin in rats was shorter than that observed in dogs (1.41 ± 0.671 h); the plasma concentrations for rats declined below 20% of C max within 1 h (naringin, t 1/2 , < 1 h), demonstrating a multiple-peak pattern. In clinical studies, plasma naringin concentration increased to maximum at about 2 h (T max , 2.09 ± 1.15 h) and decreased to 50% of C max at about 3 h (t 1/2 , 2.69 ± 1.77 h). Subsequently, owing to naringin metabolism in the human intestine, naringenin showed a considerable lag-time in human plasma (5.74 ± 2.38 h) compared to observations in rats (0.701 ± 0.523 h) and dogs (1.83 ± 0.923 h); the naringenin concentrations increased to C max at about 3.62 ± 3.19 h later in humans, compared to those in rats (2.93 ± 2.01 h) and dogs (4.17 ± 1.68 h). Naringenin t 1/2 in humans (2.60 ± 1.89 h) differed insignificantly from values obtained in rats (2.31 ± 1.21 h) and dogs (2.03 ± 0.968 h). Although the clinical naringin administration doses concurred with animal doses, according to weight and surface, AUC and C max in humans were significantly different from those observed in animals. In nonclinical and clinical studies, all naringin AUC values fluctuated and slightly increased, whereas those for naringenin were linear in terms of doses. The absolute bioavailability of naringin was 44.1% for rats (42 mg kg -1 ) and 34.4% for dogs (12.4 mg kg -1 ) in the forms of naringin and naringenin. In food-effect trials, the pharmacokinetic parameters of naringin and naringenin, including t 1/2 , T max , C max , and AUC, were insignificantly altered by a high-fat diet. As presented in Table 3, no significant accumulation of naringin and naringenin was observed in multiple-dose studies in rats, dogs, and humans. Furthermore, there were significant differences between the pharmacokinetic parameters of males and females in rats and humans. In single-dose studies, some pharmacokinetic parameters of females were significantly higher than those of males as follows: AUC (naringin, rats, p.o., 21 mg kg -1 ; naringin, humans, 160 mg (high-fat diet); and naringenin, humans, 40 mg), t 1/2 (naringenin, rats, p.o., 42 mg kg -1 ), T max (naringenin, rats, p.o., 168 mg kg -1 ), and C max (naringenin, humans, 40 mg), while few pharmacokinetic parameters of females were significantly lower than those of males (AUC of naringin, humans, 40 mg; C max of naringenin, rats, i.v., 42 mg kg -1 ). In multiple-dose studies, significantly higher parameters of females were only observed in rats (naringin, T max , 2.70 ± 1.48 h and naringenin, accumulation index, 1.79 ± 0.457).

Metabolism in Liver and Kidney Microsomes of Rats and Humans
This study focused on the phase I and II metabolism of naringin and naringenin in rat and human liver microsomes. Additionally, the metabolites were analyzed in rat kidney microsomes. Twelve metabolites of naringin and naringenin were identified in rat and human liver and kidney microsomes ( Figures S1 and S2 and Tables 7 and S5). With the exception of the methylated metabolites of naringin and naringenin, there were similar metabolites found after incubation with rat and human liver microsomes. The metabolic pathways of naringin and naringenin obtained with human liver microsomes are presented in Figure 2. Furthermore, six metabolites, including neoeriocitrin, rhoifolin, naringenin, eriodictyol, apigenin, and 5, 7-dihydroxychromone, were simultaneously measured after the blank solution or five single inhibitor working solutions were added to the incubation system III-human. Compared to the control groups, the number of metabolites decreased significantly to different degrees due to the addition of a single enzyme inhibitor to inhibitor groups ( Figure 3). The results demonstrated that CYP2C9 was a major metabolic enzyme of naringin and responsible for the six metabolites. The enzyme contributions were shown sequentially as follows: CYP2C19, four metabolites; CYP2D6, three metabolites; CYP3A4/ 5, two metabolites; and CYP1A2, one metabolite.

Excretion in Humans
For a mass balance study, urine and feces were collected after p.o. administration of naringin in clinical studies. The cumulative excretion percentages of naringin in humans were less than 20% in the form of naringin and naringenin (Table 8). To evaluate enzyme influence on the fecal naringin content and account for the entire dose administered to subjects, six fecal samples from individuals who did not receive orally administered naringin were mixed. Next, 9-fold normal saline, instead of ninefold methanolnormal saline (2:1, v/v), was added to maintain enzyme activity in the feces. Subsequently, QC procedures were performed to test naringin and naringenin stability in the feces. These samples were maintained at 37°C for 12 h. The detected naringin and naringenin amount was less than 20% of the amount added at 37°C over night, indicating poor cumulative excretion due to significant metabolism by gut enzymes.

Bioanalytical Methods
Initially, naringin-D4 and naringenin-D4 were selected to use as IS's for the analysis of naringin and naringenin in rat and dog plasma. However, during the pre-test of the nonclinical samples, naringin-D4 and naringenin-D4 showed significant levels of naringin and naringenin impurities, which prevented these compounds to use them as IS's. Instead, the structural analog quercetin 3-ß-D-glucoside was selected as an IS for the rat and dog plasma samples. In the clinical samples, quercetin 3-ß-Dglucoside IS showed an interference with 3-ß-D-glucoside in blank human plasma, therefore, quercetin 3-ß-D-glucoside could not be used as an IS in the human plasma samples. However, purified batches of naringin-D4 and naringenin-D4 became available with no naringin and naringenin impurities, therefore, naringin-D4 and naringenin-D4 were used as IS's in the clinical samples. Based on methods available in the literature in rats (Tsai and Tsai, 2012;Tong et al., 2012;Wen et al., 2012;Li S. Q. et al., 2013;Jin et al., 2015;Lu et al., 2015;Zeng et al., 2018) and humans (Ishii et al., 2000;Xiong et al., 2014), separate bioanalytical methods were used for the analysis of naringin and naringenin in rat, dog, and human samples. Compared to other methods available in the literature on human plasma and urine analysis (Ishii et al., 2000;Xiong et al., 2014), naringin-D4 and naringenin-D4 were used as IS's in this study as they were found to have less inference with the endogenous components of the human samples. Furthermore, these bioanalytical methods proved to be more reliable and robust for the quantitation of naringin and naringenin, which are important for the successful completion of nonclinical and clinical studies.

Pharmacokinetic Characteristics and Species Differences
Tables 1 and 2 demonstrated that, compared to nonclinical species, naringin pharmacokinetic processes were prolonged in humans owing to physiologic variables such as blood rate, biochemical processes, and clearance, which are related to the weight or body surface area of animal species (including humans) (Leon and Yu, 2016). Although similar to naringin, the naringenin absorption processes were affected by the size of the experimental animal; naringenin t 1/2 values for nonclinical species were close to that of humans for unknown reasons. In addition, in these animals, the AUC for naringenin administered by p.o. administration was markedly greater than that observed for i.v. administration, demonstrating prominent biotransformation of naringin into naringenin by intestinal microflora . After single-dose administration to rats, dogs, and humans, the pharmacokinetic behavior of naringenin followed linear pharmacokinetics, whereas the relationship between dose and AUC appeared nonlinear for naringin. Naringin is likely a precursor of various metabolites, maintaining a slightly fluctuating and increasing AUC for naringin and a linearly increased AUC for naringenin. The AUC for naringenin increased more rapidly with the dose in rats and humans than in dogs where the observed increase was slower. For naringenin in rats and humans, continued dose increase might saturate the enzyme pathways for its metabolism. Thus, the increase in AUC may be higher than expected as a smaller amount of naringenin is being eliminated (i.e., more naringenin is retained). Conversely, naringenin absorption in dogs could become saturated, resulting in lower than expected changes in AUC. The absolute bioavailability of naringin was 44.1% for rats and 34.4% for dogs, indicating a substantial first-pass effect of naringin in these animals. Moreover, compared with the report in aged rats (Zeng et al., 2019), the pharmacokinetic parameters of females were similarly significantly higher than those of males in rats and humans that was likely due to gender-related differences of CYP (Scandlyn et al., 2008).

Metabolism and Species Differences
Naringin metabolites have been reported in urine, feces, and intestinal microbes of rats, dogs, and humans (Liu M. et al., 2012;Zeng et al., 2017;Chen et al., 2018), indicating species difference in metabolism. Utilising phase I biotransformation reaction pathways, naringin was metabolised to naringenin and their hydroxylated, hydrogenated, and dehydrogenated metabolites in rats and dogs, whereas small amounts of neoeriocitrin were detected in human urine and feces. The common glucuronidation and sulphate conjugation (phase II) reactions were observed in rats, dogs, and humans, while methylation reactions only occurred in rats and dogs. In addition, for some fission metabolites such as 2, 4, 6trihydroxybenzoic acid, 4-hydroxybenzoic acid, hippuric acid, and 4-hydroxyhippuric acid, only trace amounts could be detected in human urine and fecal samples.
Our study focused on naringin and naringenin metabolites in liver and kidney microsomes and their CYP enzyme pathways, which could contribute to learning more about the metabolic processes in vivo, as well as species differences. With the exception of the two methylated metabolites in rats, naringin and naringenin were metabolised to the same ten metabolites, indicating a slight metabolic difference between rat and human liver microsomes. Furthermore, a blank solution or five single inhibitor working solutions were added to the incubation system III-human to evaluate naringin and naringenin metabolic enzyme pathways. The results demonstrated that naringin metabolism was a complex process catalyzed simultaneously by multiple enzymes, and may not be influenced only by a single CYP inhibitor.

Excretion and Species Differences
In previous naringin excretion studies in rats and dogs, Liu et al. reported cumulative excretion percentage of 21% for rats and 60% for dogs in the forms of naringin, naringenin, and 4hydroxyphenylpropionic acid (Liu M. et al., 2012); Zeng et al. presented a cumulative excretion percentage of 19% in the forms of naringin and naringenin, and 98% for aged rats in the forms of naringin, naringenin, and 4-hydroxyphenylpropionic acid (Zeng et al., 2019). In our clinical studies, the cumulative excretion percentage of the dose was less than 20% in the forms of naringin and naringenin. In mass balance studies, 4-hydroxyphenylpropionic acid amount was excluded owing to the unrealistic value, above 100%, obtained in humans. The observation was attributed to 4hydroxyphenylpropionic acid metabolism from other flavonoids present in various foods such as vegetables, fruits, and beans. In studies on human intestinal microbes, Chen et al. reported the metabolism of isotope-labeled naringin, which can be used to explain the entire dose of naringin administered to subjects . Naringin and naringenin were excreted within 24 h in rats and dogs, and 48 h in humans, demonstrating a prolonged excretion process in humans. Interspecies differences can be attributed to physiological variables such as blood rate, clearance, biochemical processes, and variable intestinal motility, which are, in turn, related to the weight or body surface area of different species (Leon and Yu, 2016).
In summary, the pharmacokinetics and metabolism of naringin were studied in nonclinical and clinical trials. The pharmacokinetic parameters of naringin and its active metabolite naringenin were estimated through single-dose (p.o. and i.v.) and multiple-dose studies in rats, dogs, and humans. Twelve metabolites were identified with the incubation with rat and human liver microsomes and the metabolism was demonstrated as a complex and multipathway process in human liver microsomes. Species differences were indicated in the pharmacokinetics and metabolism of naringin. Moreover, naringin data can be employed to design clinical dosage regimens, enhance knowledge on underlying mechanisms, and promote novel drug development.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/ Supplementary Material.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Ethics committees of Beijing Hospital; Beijing Hospital. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Animal Ethics Committee of the School of Life Sciences in Sun Yat-sen University; Sun Yat-sen University.