- Email: [email protected]
Toxicokinetics of naringin, a putative antitussive, after 184-day repeated oral administration in rats
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 1 ( 2 0 1 1 ) 485–489
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/etap
Short communication
Toxicokinetics of naringin, a putative antitussive, after 184-day repeated oral administration in rats Menghua Liu a,1 , Cuiping Yang a,1 , Wei Zou a , Xiaoling Guan b , Wenyan Zheng a , Li Lai b , Siqi Fang a , Shuxing Cai a , Weiwei Su a,∗ a
Guangzhou Quality R&D Center of Traditional Chinese Medicine, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China b National Chengdu Center for Safety Evaluation of Drugs, Chengdu 610041, China
a r t i c l e
i n f o
a b s t r a c t
Article history:
The toxicokinetic characteristics of naringin were investigated in rats that had been orally
Received 19 August 2010
administered naringin extract, a candidate for oral treatment of cough, prepared from Citrus
Received in revised form
grandis “Tomentosa”, at 50, 250, or 1250 mg/kg/day in a repeated-dose study for 1, 32, 93, or
31 December 2010
184 days. Increased values of the mean systemic exposure were approximately proportional
Accepted 24 January 2011
to increases in dose levels during all collection intervals; no saturation was observed. No
Available online 4 February 2011
significant differences in mean systemic exposure were observed between male and female rats. Results provide a reference for interpretation of toxicology findings and relevance to
Keywords:
clinical safety issues.
Toxicokinetics
© 2011 Elsevier B.V. All rights reserved.
Naringin Naringenin Rats
1.
Introduction
Naringin (4 ,5,7-trihydroxyflavanone-7-rhamnoglucoside) belongs to a family of C6 –C3 –C6 polyphenol compounds which are widely distributed in foods of plant origin. As a secondary metabolite, naringin is especially abundant in citrus plants. Up to now, extensive studies have focused on its anti-inflammatory (Lambev et al., 1980), anti-ulcer (Martin et al., 1994), antioxidation (Chen et al., 1990), anti-atherogenic (Lee et al., 2001) and cancer preventative (Schindler and Mentlein, 2006) activities. Intensive studies on naringin’s pharmacokinetics have been carried out in rats (Fang et al.,
∗
Corresponding author. Tel.: +86 20 84110808; fax: +86 20 84112398. E-mail address: [email protected] (W. Su). 1 These authors contributed equally to this work. 1382-6689/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2011.01.006
2006; Felgines et al., 2000; Li et al., 2004, 2010; Wang et al., 2006), rabbits (Hsiu et al., 2002), dogs (Mata-Bilbao et al., 2007), and even humans (Ishii et al., 1996, 1997). Oral administration of naringin is reportedly absorbed in blood both as parent and as aglycone naringenin in rats after hydrolysis of -glycoside conjugates by intestinal microflora (Felgines et al., 2000; Li et al., 2004). Naringenin in blood has been found predominately in glucuronide and sulfate forms, but rarely in its free form (Fang et al., 2006; Felgines et al., 2000; Li et al., 2004; Wang et al., 2006). Naringin extract was developed as a natural oral remedy for relief of cough and phlegm symptoms at our lab for several years. It was extracted from Citrus grandis “Tomentosa”
486
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 1 ( 2 0 1 1 ) 485–489
(Huajuhong), and has a naringin content of greater than 98%. Thorough studies were conducted on Citrus grandis’s differentiation as a medicinal substance at the molecular level (Su et al., 2010), and on naringin extract’s preparation process (Su et al., 2009), qualification and impurities (unpublished results), pharmacodynamics (Gao et al., 2010) and pharmacokinetics (Fang et al., 2006). However, toxicokinetic data for naringin are limited. The purpose of this study is to determine potential toxicokinetics of naringin extract in rats.
2.
Materials and methods
2.1.
Chemicals
Naringenin, isoquercitrin (internal standard [IS]), glucuronidase (Type H-1, 100KU) and formic acid (LC grade, 50% in H2 O) were purchased from Sigma (St. Louis, MO, USA). Naringin standard was obtained from the National Institute for the Control of Pharmaceutical and Biological Products. Methanol and ethyl acetate (both LC grade) were purchased from SK chemicals (Ulsan, Korea). Milli-Q plus water (Millipore, Bedford, MA, USA) was used throughout the study.
2.2.
Test substance
Naringin extract was purified from the aqueous extract of Huajuhong at our center; it was 98.8% pure, as determined by HPLC method with external standard.
2.3.
Animals and housing conditions
Male and female Sprague–Dawley (SD) rats (4–6 weeks old), certified specific pathogen-free, were purchased from Slack Shanghai Laboratory Animal Co., Ltd. (Shanghai, China). Animals were individually housed in suspended plastic cages and acclimated for approximately 1 week prior to the initiation of dosing with feed and water available ad libitum. Environmental conditions were maintained at 20–25 ◦ C, 55 ± 15% relative humidity, at 12-h light/dark cycles and at 10–12 air change cycles/h.
2.4.
Doses
The pharmacodynamic studies in mice showed that the lowest effective dose (LED) for naringin extract for the treatment of cough was 15.0 mg/kg. The results of acute toxicity testing showed that the oral maximum tolerated dose (MTD) was 20.4 g/kg for mice. According to these studies, a dose of 1250 mg/kg/day of naringin extract (about 300× the clinical dose of naringin extract) was selected as the high dose in this study. Doses of 250 and 50 mg/kg/day were taken as intermediate and low doses, respectively. The volume of oral administration to rats was 10 ml/kg.
2.5.
Experimental design
This study was performed at the National Chengdu Center for Safety Evaluation of Drugs (Chengdu, China). All animals used in this study received care in compliance with the guide for the Care and Use of Laboratory Animals of China. All operations were carried out under the Good Laboratory Practice
(GLP) Regulations of the State Food and Drug Administration of China. Naringin extract suspension for oral administration was prepared freshly with sterile water for injection. Following randomization into study groups, rats (10/group/sex) were administrated naringin extract by oral gavages at 50, 250, or 1250 mg/kg/day for 1, 32, 93, or 184 days with a frequency of 6 days/week. Blood samples were collected using sparse sampling after dosing at 0 (predose), 1, 2, 4, 8 and 24 h from 5 rats, and 0.5, 1.5, 3, 6 and 12 h from other 5 rats. Plasma was harvested by centrifugation at 3000 × g for 10 min and then kept frozen at approximately −70 ◦ C until analysis.
2.6.
Bioanalytical methods
Concentrations of naringin and naringenin in standards and samples were determined simultaneously by an LC–MS/MS system, which consisted of an Agilent 1200 HPLC series system and an Agilent 6410 triple quadrupole mass spectrometer (Agilent Technol., Santa Clara, CA, USA) equipped with an electrospray ionization source. The rapid resolution highthroughput column used was Agilent Zorbax eclipse plus C18 (2.1 mm × 100 mm, 1.8 m). The mobile phase was composed of methanol (52%) and 0.25% (v/v) formic acid (48%). The flow rate was 0.2 ml/min, and column temperature was maintained at 40 ◦ C. The injection volume was 10 l. Detection was performed in the negative-ion mode using multiple reaction monitoring (MRM) of the m/z 579.2/271.0 for naringin, m/z 271.0/151.0 for naringenin, and m/z 463.0/299.8 for IS. The calibration ranges were 9.78–1955.20 ng/ml for naringin and 4.07–2036.00 ng/ml for naringenin. Combined quality control standards were prepared at 19.55, 195.52, 1955.20 ng/ml for naringin and 10.18, 101.80, 1018.00 ng/ml for naringenin and analyzed with the plasma samples. The linearity of the calibration curve was obtained by plotting the peak-area ratios of naringin/naringenin to IS versus the concentrations of naringin/naringenin with 1/x2 weighted least-squares linear regression analysis. Analytical data were accepted from only the analyses during which results of the quality control standards passed acceptance criteria. The preparation method for plasma sample was modified from our former report (Fang et al., 2006). An aliquot of 50 l rat plasma was transferred to a fresh 1.5 ml tube, incubated with 10 l enzyme (-glucuronidase/sulfatase, 10 Unit/l) at 37 ◦ C for 2 h, then 6 l of 5% formic acid (v/v) and IS (3.9 ng) were added and mixed. 800 l ethyl acetate was added to the tube and vortexed for 3 min. After the sample was centrifuged at 10,000 × g for 10 min, the upper organic layer was transferred into a fresh tube. Another 400 l ethyl acetate was added to the residue and the extraction steps were repeated. The organic layer was mixed together and evaporated at 40 ◦ C; the residue was then dissolved in 100 l mobile phase with vortex-mixing for 3 min, and centrifuged at 13,000 × g for 10 min. Finally, 10 l of the supernatant was injected into the LC–MS/MS system.
2.7.
Data analysis
Concentrations of naringin and naringenin were analyzed with Agilent MassHunter Quantitative analysis software. Values of total naringin were calculated by naringin concen-
487
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 1 ( 2 0 1 1 ) 485–489
A
2500.00 day 1
2000.00
C max (nmol/mL)
tration plus that converted from naringenin according to the molar ratio. The maximum plasma concentration (Cmax ) and the time of maximum plasma concentration (Tmax ) were obtained from the observed data. The area under the plasma concentration–time curve from 0 h to 24 h (AUC0–24 h ) was calculated using the linear trapezoidal method. Comparisons between males and females in each group were analyzed using the Student’s t-test.
day 93
1500.00
Results
day 184
1000.00 500.00 0.00
3.
day 32
B
0
5
10
15
20
25
day 1
16000.00
C max (nmol/mL)
25
20000.00 18000.00
day 32
14000.00 12000.00
day 93
10000.00
day 184
8000.00 6000.00 4000.00 2000.00 0.00
0
5
10
15
Time(h)
C C max (nmol/mL)
None of the animals died during this experiment. Male and female rats presented normal behavior and food consumption at all times. Considering that after oral administration of the agent, naringin-related material found in rat plasma included parent, naringenin and glucuronide/sulfates (Hsiu et al., 2002; Wang et al., 2006); free and conjugate forms of naringenin were taken into account as total naringin to evaluate systemic oral exposure of naringin extract in rats. There were no significant differences in toxicokinetic parameters of total naringin between males and females from each oral administration phase at three different doses (P < 0.05). Therefore, we combined the toxicokinetic parameters from male and female rats together. The time-course changes of concentration and toxicokinetic parameters of total naringin following oral administration in rats are shown in Fig. 1 and Table 1, respectively. Considerable inter-individual differences were found in the plasma concentrations. On the 1st, 32nd, 93rd and 184th day after oral administration of naringin extract, maximal plasma levels of total naringin occurred at 3–4 h after dosing at 50 mg/kg/day, 6–8 h at 250 mg/kg/day, and 6–10 h at 1250 mg/kg/day, i.e., Tmax at the high dose is almost three times longer than that at low dosage. The mean systemic exposure in terms of Cmax and AUC0–24 h to naringin extract following oral administration in rats was found to be approximately proportional to the increase of dose from 50 to 1250 mg/kg/day on days 1, 32, 93 and 184 (R > 0.99). As shown in Fig. 1, there were marked differences in systemic exposure on days 32, 93 and 184 when compared with AUC0–24 h values obtained on day 1 at 250 mg/kg/day dose and 1250 mg/kg/day dose, resulting in 1.4-, 4.1-, and 2.2fold changes at the intermediate dose and 3.6-, 5.5-, 7.9-fold changes at the high dose. However, at dose 50 mg/kg/day, significant differences were found only on day 184 with about 2-fold change compared with that of day 1, and mean systemic exposure was at the same level on days 1, 32 and 93. Decreased exposure was observed over time in terms of Cmax and AUC0–24 h values at the low and intermediate dose levels. At a dosage of 50 mg/kg/day, mean AUC0–24 h values decreased from 4260.13 nmol h/l on day 1 to 3584.02 nmol h/l on day 32; at 250 mg/kg/day, mean AUC0–24 h values decreased from 113713.57 nmol h/l on day 93 to 60235.26 nmol h/l on day 184.
20
Time(h)
120000.00
day 1 day 32
100000.00
day 93
80000.00
day 184
60000.00 40000.00 20000.00 0.00
0
5
10
15
20
25
Time(h) Fig. 1 – Mean plasma concentration–time profiles of total naringin at dose of 50 (A), 250 (B), 1250 (C) mg/kg/day naringin extract during the repeated-dose study in SD rats.
4.
Discussion
Our study on the toxicokinetics of naringin extract in rats provides some insight into its concentration in different biological samples over time. Toxicokinetic characteristics and patterns must be considered in evaluating safety of naringin extract. The Tmax of total naringin at intermediate and high doses was significantly greater than that at the low dose, which was also reported by Fang et al. (2006) and Felgines et al. (2000). As orally administered naringin is absorbed mainly as aglycone after hydrolysis of -glycoside conjugates by intestinal microflora (Felgines et al., 2000; Li et al., 2004; Serra et al., 2008), more time might have been needed by intestinal bacteria to biotransform this compound, and thus would also be needed to reach maximal plasma concentration (Wang et al., 2006).
488
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 1 ( 2 0 1 1 ) 485–489
Table 1 – Toxicokinetic parameters of total naringin for naringin extract administration to rats (n = 10). TK parameters
Tmax (h)
Cmax (nmol/l)
AUC0–24 h (nmol h/l)
50 mg/kg/day Day 1 Day 32 Day 93 Day 184
3 4 4 4
1481.79 1085.30 1794.53 2368.43
4260.13 3584.02 5270.22 8900.64
250 mg/kg/day Day 1 Day 32 Day 93 Day 184
6 6 8 6
5706.51 4679.79 18982.99 12596.99
27475.46 38918.88 113713.57 60235.26
1250 mg/kg/day Day 1 Day 32 Day 93 Day 184
6 10 10 10
13464.17 47226.28 73387.24 98383.16
102626.61 371278.08 568075.10 815385.74
The increases of Cmax and AUC0–24 h in rats orally administered naringin extract were approximately proportional to the increase in dose during all collection intervals. No saturation was observed. At intermediate and high doses, exposures increased with dosing time, indicating that naringin accumulated during repeated-dose phases. These suggest significant induction occurs in rats during absorption at these two dose levels. Such finding would be a consideration in safety evaluations of naringin extract between different species. At 50 and 250 mg/kg/day dose levels, low exposures were observed on days 32 and 184 compared with days 1 and 93, respectively. These findings were also seen in a toxicokinetic study of thiazolidinedione in dogs (Sun et al., 2009). Total naringin concentration tested at 24 h after administration decreased significantly, indicating that naringin might not accumulate much for several days in a clinical study. In summary, the increases in Cmax and AUC0–24 h values of total naringin were approximately proportional to the increase in dose levels during all collection intervals; no saturation was observed. Naringin was accumulated at intermediate and high doses. No remarkable sex-related differences in mean systemic exposure to naringin extract were observed in rats. These results serve to better interpret toxicology findings, and to provide a reference for further study of naringin extract in humans.
Conflict of interest statement None declared.
Acknowledgements This work was funded by the National Natural Science Foundation of China (No. 30873422), the National Key Technology R&D Program (No. 2006BAI06A02-2) from the Ministry of Science and Technology and the National Major Scientific and Technical Special Project (No. 2011ZX09102-011-03) from the Ministry of Health of the People’s Republic of China.
references
Chen, Y.T., Zheng, R.L., Jia, Z.J., Ju, Y., 1990. Flavonoids as superoxide scavengers and antioxidants. Free Radic. Biol. Med. 9, 19–21. Fang, T.Z., Wang, Y.G., Ma, Y., Su, W.W., Bai, Y., Zhao, P.Y., 2006. A rapid LC/MS/MS quantitation assay for naringin and its two metabolites in rats plasma. J. Pharm. Biomed. Anal. 40, 454–459. Felgines, C., Texier, O., Morand, C., Manach, C., Scalbert, A., Régerat, F., Rémésy, C., 2000. Bioavailability of the flavanone naringenin and its glycosides in rats. Am. J. Physiol. Gastrointest. Liver Physiol. 279, 1148–1154. Gao, S., Li, P.B., Yang, H.L., Fang, S.Q., Su, W.W., 2010. Antitussive effect of naringin on experimentally induced cough in guinea pigs. Planta Med. doi:10.1055/s-0030-1250117. Hsiu, S.L., Huang, T.Y., Hou, Y.C., Chin, D.H., Lee Chao, P.D., 2002. Comparison of metabolic pharmacokinetics of naringin and naringenin in rabbits. Life Sci. 70, 1481–1489. Ishii, K., Furuta, T., Kasuya, Y., 1996. Determination of naringin and naringenin in human plasma by high-performance liquid chromatography. J. Chromatogr. B 683, 225–229. Ishii, K., Furuta, T., Kasuya, Y., 1997. Determination of naringin and naringenin in human urine by high-performance liquid chromatography utilizing solid-phase extraction. J. Chromatogr. B 704, 299–305. Lambev, I., Krushkov, I., Zheliazkov, D., Nikolov, N., 1980. Antiexudative effect of naringin in experimental pulmonary edema and peritonitis. Eksp. Med. Morfol. 19, 207–212. Lee, C.H., Jeong, T.S., Choi, Y.K., Hyun, B.H., Oh, G.T., Kim, E.H., Kim, J.R., Han, J.I., Bok, S.H., 2001. Anti-atherogenic effect of citrus flavonoids, naringin and naringenin, associated with hepatic ACAT and aortic VCAM-1 and MCP-1 in high cholesterol-fed rabbits. Biochem. Biophys. Res. Commun. 284, 681–688. Li, X.H., Xiong, Z.L., Lu, S., Zhang, Y., Li, F.M., 2010. Pharmacokinetics of naringin and its metabolite naringenin in rats after oral administration of Rhizoma drynariae extract assayed by UPLC–MS/MS. Chinese J. Nat. Med. 8 (1), 40–46. Li, X.L., Xiao, H.B., Liang, X.M., Shi, D.Z., Liu, J.G., 2004. LC–MS/MS determination of naringin, hesperidin and neohesperidin in rat serum after orally administrating the decoction of Bulpleurum falcatum L. and Fractus aurantii. J. Pharm. Biomed. Anal. 34, 159–166.
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Martin, M.J., Marhuenda, F., Perez-Guerrero, C., Franco, J.M., 1994. Antiulcer effect of naringin on gastric lesions induced by ethanol in rats. Pharmacology 49, 144–150. Mata-Bilbao, M., de, L., Andrés-Lacueva, C., Roura, E., Jáuregui, O., Escribano, E., Torre, C., Lamuela-Raventós, R.M., 2007. Absorption and pharmacokinetics of grapefruit flavanones in beagles. Brit. J. Nutr. 98, 86–92. Schindler, R., Mentlein, R., 2006. Flavonoids and Vitamin E reduce the release of the angiogenic peptide vascular endothelial growth factor from human tumor cells. J. Nutr. 136, 1477–1482. Serra, H., Mendes, T., Bronze, M.R., Luísa Simplíci, Ana., 2008. Prediction of intestinal absorption and metabolism of pharmacologically active flavones and flavanones. Bioorgan. Med. Chem. 16, 4009–4018. Su, C., Wong, K.L., But, P.-H.P., Su, W.W., Shaw, P.C., 2010. Molecular authentication of the Chinese Herb Huajuhong and
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related medicinal material by DNA sequencing and ISSR markers. J. Food Drug Anal. 18, 161–170. Su, W.W., Wang, Y.G., Fang, T.Z., Peng, W., Wu, Z., 2009. Uses of naringenin, naringin and salts thereof as expectorants in the treatment of cough, and compositions thereof. European Patent 1591123. Sun, N., Lu, G.C., Lin, M., Fan, G.R., Wu, Y.T., 2009. Subchronic toxicity and toxicokinetics of MCC-555, a novel thiazolidinedione, after 270-day repeated oral administration in dogs. Environ. Toxicol. Pharmacol. 27, 237–246. Wang, M.J., Lee Chao, P.D., Hou, Y.C., Hsiu, S.L., Wen, K.C., Tsai, S.Y., 2006. Pharmacokinetics and conjugation metabolism of naringin and naringenin in rats after single dose and multiple dose administration. J. Food Drug Anal. 14, 247–253.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/etap
Short communication
Toxicokinetics of naringin, a putative antitussive, after 184-day repeated oral administration in rats Menghua Liu a,1 , Cuiping Yang a,1 , Wei Zou a , Xiaoling Guan b , Wenyan Zheng a , Li Lai b , Siqi Fang a , Shuxing Cai a , Weiwei Su a,∗ a
Guangzhou Quality R&D Center of Traditional Chinese Medicine, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China b National Chengdu Center for Safety Evaluation of Drugs, Chengdu 610041, China
a r t i c l e
i n f o
a b s t r a c t
Article history:
The toxicokinetic characteristics of naringin were investigated in rats that had been orally
Received 19 August 2010
administered naringin extract, a candidate for oral treatment of cough, prepared from Citrus
Received in revised form
grandis “Tomentosa”, at 50, 250, or 1250 mg/kg/day in a repeated-dose study for 1, 32, 93, or
31 December 2010
184 days. Increased values of the mean systemic exposure were approximately proportional
Accepted 24 January 2011
to increases in dose levels during all collection intervals; no saturation was observed. No
Available online 4 February 2011
significant differences in mean systemic exposure were observed between male and female rats. Results provide a reference for interpretation of toxicology findings and relevance to
Keywords:
clinical safety issues.
Toxicokinetics
© 2011 Elsevier B.V. All rights reserved.
Naringin Naringenin Rats
1.
Introduction
Naringin (4 ,5,7-trihydroxyflavanone-7-rhamnoglucoside) belongs to a family of C6 –C3 –C6 polyphenol compounds which are widely distributed in foods of plant origin. As a secondary metabolite, naringin is especially abundant in citrus plants. Up to now, extensive studies have focused on its anti-inflammatory (Lambev et al., 1980), anti-ulcer (Martin et al., 1994), antioxidation (Chen et al., 1990), anti-atherogenic (Lee et al., 2001) and cancer preventative (Schindler and Mentlein, 2006) activities. Intensive studies on naringin’s pharmacokinetics have been carried out in rats (Fang et al.,
∗
Corresponding author. Tel.: +86 20 84110808; fax: +86 20 84112398. E-mail address: [email protected] (W. Su). 1 These authors contributed equally to this work. 1382-6689/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2011.01.006
2006; Felgines et al., 2000; Li et al., 2004, 2010; Wang et al., 2006), rabbits (Hsiu et al., 2002), dogs (Mata-Bilbao et al., 2007), and even humans (Ishii et al., 1996, 1997). Oral administration of naringin is reportedly absorbed in blood both as parent and as aglycone naringenin in rats after hydrolysis of -glycoside conjugates by intestinal microflora (Felgines et al., 2000; Li et al., 2004). Naringenin in blood has been found predominately in glucuronide and sulfate forms, but rarely in its free form (Fang et al., 2006; Felgines et al., 2000; Li et al., 2004; Wang et al., 2006). Naringin extract was developed as a natural oral remedy for relief of cough and phlegm symptoms at our lab for several years. It was extracted from Citrus grandis “Tomentosa”
486
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 1 ( 2 0 1 1 ) 485–489
(Huajuhong), and has a naringin content of greater than 98%. Thorough studies were conducted on Citrus grandis’s differentiation as a medicinal substance at the molecular level (Su et al., 2010), and on naringin extract’s preparation process (Su et al., 2009), qualification and impurities (unpublished results), pharmacodynamics (Gao et al., 2010) and pharmacokinetics (Fang et al., 2006). However, toxicokinetic data for naringin are limited. The purpose of this study is to determine potential toxicokinetics of naringin extract in rats.
2.
Materials and methods
2.1.
Chemicals
Naringenin, isoquercitrin (internal standard [IS]), glucuronidase (Type H-1, 100KU) and formic acid (LC grade, 50% in H2 O) were purchased from Sigma (St. Louis, MO, USA). Naringin standard was obtained from the National Institute for the Control of Pharmaceutical and Biological Products. Methanol and ethyl acetate (both LC grade) were purchased from SK chemicals (Ulsan, Korea). Milli-Q plus water (Millipore, Bedford, MA, USA) was used throughout the study.
2.2.
Test substance
Naringin extract was purified from the aqueous extract of Huajuhong at our center; it was 98.8% pure, as determined by HPLC method with external standard.
2.3.
Animals and housing conditions
Male and female Sprague–Dawley (SD) rats (4–6 weeks old), certified specific pathogen-free, were purchased from Slack Shanghai Laboratory Animal Co., Ltd. (Shanghai, China). Animals were individually housed in suspended plastic cages and acclimated for approximately 1 week prior to the initiation of dosing with feed and water available ad libitum. Environmental conditions were maintained at 20–25 ◦ C, 55 ± 15% relative humidity, at 12-h light/dark cycles and at 10–12 air change cycles/h.
2.4.
Doses
The pharmacodynamic studies in mice showed that the lowest effective dose (LED) for naringin extract for the treatment of cough was 15.0 mg/kg. The results of acute toxicity testing showed that the oral maximum tolerated dose (MTD) was 20.4 g/kg for mice. According to these studies, a dose of 1250 mg/kg/day of naringin extract (about 300× the clinical dose of naringin extract) was selected as the high dose in this study. Doses of 250 and 50 mg/kg/day were taken as intermediate and low doses, respectively. The volume of oral administration to rats was 10 ml/kg.
2.5.
Experimental design
This study was performed at the National Chengdu Center for Safety Evaluation of Drugs (Chengdu, China). All animals used in this study received care in compliance with the guide for the Care and Use of Laboratory Animals of China. All operations were carried out under the Good Laboratory Practice
(GLP) Regulations of the State Food and Drug Administration of China. Naringin extract suspension for oral administration was prepared freshly with sterile water for injection. Following randomization into study groups, rats (10/group/sex) were administrated naringin extract by oral gavages at 50, 250, or 1250 mg/kg/day for 1, 32, 93, or 184 days with a frequency of 6 days/week. Blood samples were collected using sparse sampling after dosing at 0 (predose), 1, 2, 4, 8 and 24 h from 5 rats, and 0.5, 1.5, 3, 6 and 12 h from other 5 rats. Plasma was harvested by centrifugation at 3000 × g for 10 min and then kept frozen at approximately −70 ◦ C until analysis.
2.6.
Bioanalytical methods
Concentrations of naringin and naringenin in standards and samples were determined simultaneously by an LC–MS/MS system, which consisted of an Agilent 1200 HPLC series system and an Agilent 6410 triple quadrupole mass spectrometer (Agilent Technol., Santa Clara, CA, USA) equipped with an electrospray ionization source. The rapid resolution highthroughput column used was Agilent Zorbax eclipse plus C18 (2.1 mm × 100 mm, 1.8 m). The mobile phase was composed of methanol (52%) and 0.25% (v/v) formic acid (48%). The flow rate was 0.2 ml/min, and column temperature was maintained at 40 ◦ C. The injection volume was 10 l. Detection was performed in the negative-ion mode using multiple reaction monitoring (MRM) of the m/z 579.2/271.0 for naringin, m/z 271.0/151.0 for naringenin, and m/z 463.0/299.8 for IS. The calibration ranges were 9.78–1955.20 ng/ml for naringin and 4.07–2036.00 ng/ml for naringenin. Combined quality control standards were prepared at 19.55, 195.52, 1955.20 ng/ml for naringin and 10.18, 101.80, 1018.00 ng/ml for naringenin and analyzed with the plasma samples. The linearity of the calibration curve was obtained by plotting the peak-area ratios of naringin/naringenin to IS versus the concentrations of naringin/naringenin with 1/x2 weighted least-squares linear regression analysis. Analytical data were accepted from only the analyses during which results of the quality control standards passed acceptance criteria. The preparation method for plasma sample was modified from our former report (Fang et al., 2006). An aliquot of 50 l rat plasma was transferred to a fresh 1.5 ml tube, incubated with 10 l enzyme (-glucuronidase/sulfatase, 10 Unit/l) at 37 ◦ C for 2 h, then 6 l of 5% formic acid (v/v) and IS (3.9 ng) were added and mixed. 800 l ethyl acetate was added to the tube and vortexed for 3 min. After the sample was centrifuged at 10,000 × g for 10 min, the upper organic layer was transferred into a fresh tube. Another 400 l ethyl acetate was added to the residue and the extraction steps were repeated. The organic layer was mixed together and evaporated at 40 ◦ C; the residue was then dissolved in 100 l mobile phase with vortex-mixing for 3 min, and centrifuged at 13,000 × g for 10 min. Finally, 10 l of the supernatant was injected into the LC–MS/MS system.
2.7.
Data analysis
Concentrations of naringin and naringenin were analyzed with Agilent MassHunter Quantitative analysis software. Values of total naringin were calculated by naringin concen-
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A
2500.00 day 1
2000.00
C max (nmol/mL)
tration plus that converted from naringenin according to the molar ratio. The maximum plasma concentration (Cmax ) and the time of maximum plasma concentration (Tmax ) were obtained from the observed data. The area under the plasma concentration–time curve from 0 h to 24 h (AUC0–24 h ) was calculated using the linear trapezoidal method. Comparisons between males and females in each group were analyzed using the Student’s t-test.
day 93
1500.00
Results
day 184
1000.00 500.00 0.00
3.
day 32
B
0
5
10
15
20
25
day 1
16000.00
C max (nmol/mL)
25
20000.00 18000.00
day 32
14000.00 12000.00
day 93
10000.00
day 184
8000.00 6000.00 4000.00 2000.00 0.00
0
5
10
15
Time(h)
C C max (nmol/mL)
None of the animals died during this experiment. Male and female rats presented normal behavior and food consumption at all times. Considering that after oral administration of the agent, naringin-related material found in rat plasma included parent, naringenin and glucuronide/sulfates (Hsiu et al., 2002; Wang et al., 2006); free and conjugate forms of naringenin were taken into account as total naringin to evaluate systemic oral exposure of naringin extract in rats. There were no significant differences in toxicokinetic parameters of total naringin between males and females from each oral administration phase at three different doses (P < 0.05). Therefore, we combined the toxicokinetic parameters from male and female rats together. The time-course changes of concentration and toxicokinetic parameters of total naringin following oral administration in rats are shown in Fig. 1 and Table 1, respectively. Considerable inter-individual differences were found in the plasma concentrations. On the 1st, 32nd, 93rd and 184th day after oral administration of naringin extract, maximal plasma levels of total naringin occurred at 3–4 h after dosing at 50 mg/kg/day, 6–8 h at 250 mg/kg/day, and 6–10 h at 1250 mg/kg/day, i.e., Tmax at the high dose is almost three times longer than that at low dosage. The mean systemic exposure in terms of Cmax and AUC0–24 h to naringin extract following oral administration in rats was found to be approximately proportional to the increase of dose from 50 to 1250 mg/kg/day on days 1, 32, 93 and 184 (R > 0.99). As shown in Fig. 1, there were marked differences in systemic exposure on days 32, 93 and 184 when compared with AUC0–24 h values obtained on day 1 at 250 mg/kg/day dose and 1250 mg/kg/day dose, resulting in 1.4-, 4.1-, and 2.2fold changes at the intermediate dose and 3.6-, 5.5-, 7.9-fold changes at the high dose. However, at dose 50 mg/kg/day, significant differences were found only on day 184 with about 2-fold change compared with that of day 1, and mean systemic exposure was at the same level on days 1, 32 and 93. Decreased exposure was observed over time in terms of Cmax and AUC0–24 h values at the low and intermediate dose levels. At a dosage of 50 mg/kg/day, mean AUC0–24 h values decreased from 4260.13 nmol h/l on day 1 to 3584.02 nmol h/l on day 32; at 250 mg/kg/day, mean AUC0–24 h values decreased from 113713.57 nmol h/l on day 93 to 60235.26 nmol h/l on day 184.
20
Time(h)
120000.00
day 1 day 32
100000.00
day 93
80000.00
day 184
60000.00 40000.00 20000.00 0.00
0
5
10
15
20
25
Time(h) Fig. 1 – Mean plasma concentration–time profiles of total naringin at dose of 50 (A), 250 (B), 1250 (C) mg/kg/day naringin extract during the repeated-dose study in SD rats.
4.
Discussion
Our study on the toxicokinetics of naringin extract in rats provides some insight into its concentration in different biological samples over time. Toxicokinetic characteristics and patterns must be considered in evaluating safety of naringin extract. The Tmax of total naringin at intermediate and high doses was significantly greater than that at the low dose, which was also reported by Fang et al. (2006) and Felgines et al. (2000). As orally administered naringin is absorbed mainly as aglycone after hydrolysis of -glycoside conjugates by intestinal microflora (Felgines et al., 2000; Li et al., 2004; Serra et al., 2008), more time might have been needed by intestinal bacteria to biotransform this compound, and thus would also be needed to reach maximal plasma concentration (Wang et al., 2006).
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Table 1 – Toxicokinetic parameters of total naringin for naringin extract administration to rats (n = 10). TK parameters
Tmax (h)
Cmax (nmol/l)
AUC0–24 h (nmol h/l)
50 mg/kg/day Day 1 Day 32 Day 93 Day 184
3 4 4 4
1481.79 1085.30 1794.53 2368.43
4260.13 3584.02 5270.22 8900.64
250 mg/kg/day Day 1 Day 32 Day 93 Day 184
6 6 8 6
5706.51 4679.79 18982.99 12596.99
27475.46 38918.88 113713.57 60235.26
1250 mg/kg/day Day 1 Day 32 Day 93 Day 184
6 10 10 10
13464.17 47226.28 73387.24 98383.16
102626.61 371278.08 568075.10 815385.74
The increases of Cmax and AUC0–24 h in rats orally administered naringin extract were approximately proportional to the increase in dose during all collection intervals. No saturation was observed. At intermediate and high doses, exposures increased with dosing time, indicating that naringin accumulated during repeated-dose phases. These suggest significant induction occurs in rats during absorption at these two dose levels. Such finding would be a consideration in safety evaluations of naringin extract between different species. At 50 and 250 mg/kg/day dose levels, low exposures were observed on days 32 and 184 compared with days 1 and 93, respectively. These findings were also seen in a toxicokinetic study of thiazolidinedione in dogs (Sun et al., 2009). Total naringin concentration tested at 24 h after administration decreased significantly, indicating that naringin might not accumulate much for several days in a clinical study. In summary, the increases in Cmax and AUC0–24 h values of total naringin were approximately proportional to the increase in dose levels during all collection intervals; no saturation was observed. Naringin was accumulated at intermediate and high doses. No remarkable sex-related differences in mean systemic exposure to naringin extract were observed in rats. These results serve to better interpret toxicology findings, and to provide a reference for further study of naringin extract in humans.
Conflict of interest statement None declared.
Acknowledgements This work was funded by the National Natural Science Foundation of China (No. 30873422), the National Key Technology R&D Program (No. 2006BAI06A02-2) from the Ministry of Science and Technology and the National Major Scientific and Technical Special Project (No. 2011ZX09102-011-03) from the Ministry of Health of the People’s Republic of China.
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