Tissue distribution of naringenin conjugated metabolites following repeated dosing of naringin to rats

Background: Naringin is a major antioxidant in Citrus fruits and herbs. To clarify molecular forms distributed to various tissues, we investigated tissue distribution of naringin and relevant metabolites in rats after repeated dosing. Methods: Male Sprague-Dawley rats were orally administered naringin (210 mg/kg) twice daily for eight days. At 6 h post the 17th dose, various tissues including liver, kidney, heart, spleen and brain were collected and analyzed by HPLC method before and after hydrolysis with β-glucuronidase and sulfatase, individually. Results: The free forms of naringin and naringenin were not detected in all the tissues assayed. Liver contained the highest concentration of naringenin sulfates, followed by spleen, heart, brain and kidney. Naringenin glucuronides were present in liver and kidney, but not in spleen, brain and heart. Conclusion: The bioavailability of naringenin glucuronides and sulfates supported its application for personalized medicine.


Introduction
Flavonoids, major class of antioxidants, prove interesting to pharmacologists due to various beneficial bioactivities [1,2].
Naringin-containing nutraceuticals are increasingly used as dietary supplements, yet based on our prior studies reporting pharmacokinetics o f n aringin i n rabbits a nd r ats , n aringenin sulfates and glucuronides appeared predominately in the blood-stream, with no trace of naringin or naringenin detected [15,16].
Whether bioactivities of naringin and naringenin cited by earlier in vitro study can be extrapolated to in vivo effects remained unanswered [17]. Regarding major forms in various organs, previous studies report tissue distribution of naringin or naringenin in rats [18][19][20], yet these followed single dose via intravenous or oral route, findings less than consistent. We analyzed distribution of naringin and relevant metabolites in rat tissue after repeated dosing.

2.
Materials and methods

Drug administration and collection of blood and organs
Six male Sprague-Dawley rats weighing 300-350 g were purchased from the National Science Council, Taipei, and maintained in the China Medical University Animal Center.
Naringin was dispersed in warm water for oral administration (210 mg/kg twice daily) for 17 doses via gastric gavage. Finally, rats were fasted overnight before 17 th dose, blood and various organs were collected at 6 h after dosing, based on peak time of naringenin conjugates observed by our previous pharmacoki-netic study [16]. Immediately after blood collection, systemic perfusion was conducted by pumping normal saline to wash out blood. The organs including liver, kidney, spleen, heart and brain were dissected, blotted dry, accurately weighed, and frozen at -80℃. Animal study adhered to Guidebook for the Care and

Use of Laboratory Animals (2002) published by The Chinese
Society for the Laboratory Animal Science, Taiwan.

Preparation and quantitation of tissue samples
All tissue samples were lyophilized, then chopped into small pieces and milled with normal saline (300 µL/g tissue) by Potter-Elvehjem tissue grinder (Kontes Glass Co.; Vineland, NJ).
Homogenates (0.5 mL) were deproteinized with 3-fold methanol. To quantify naringenin glucuronides, 100 µL of buffer containing tissue extract was mixed with 100 µL of β-glucuronidase (1000 units/mL in pH 5 buffer), 100 µL of ascorbic acid (150 mg/mL) and incubated at 37℃ for 2 h. To quantify naringenin sulfates/glucuronides, 100 µL of the buffer containing tissue extract were mixed with 100 µL of sulfatase (containing 1000 units/mL of sulfatase and 24,940 units/mL of glucuronidase in pH 5 buffer), 100 µL of ascorbic acid (150 mg/mL), and incubated at 37℃ for 1 h. After hydrolysis, procedure was the same as described above for free form naringenin. For calibrator preparation, 100 µL of tissue standards with various concentrations of naringenin were spiked with 100 µL of pH 5 acetate buffer, 100 µL of ascorbic acid (150 mg/mL), then added to 100 µL of 0.1 N HCl. Later procedure followed that described above. Calibration graph was plotted by linear regression of peak area ratios (naringenin to internal standard) against concentrations of naringenin.

Data analysis
Concentrations of naringenin glucuronides and naringenin sulfates in each tissue were expressed in μmol per gram of tissues (μmol/g). Ratios (mL/g) of concentrations of naringin glucuronides and naringin sulfates in various organs (μmol/g) to those in serum (μmol/mL) were calculated, although units differed.

Results
Quantitation method of naringenin in each tissue was established and optimized. Good linearity was obtained at concentration 0.4-50.0 µg/mL of naringenin in each tissue.

Validation of assay indicated all coefficients of variation (CVs)
and relative errors of intra-run and inter-run analysis below 2.2% and 18.1%, respectively. The LLOQ and LOD of naringenin were 0.40 and 0.02 µg/mL, respectively, in all tissues.
Results indicated neither naringin nor naringenin in liver, spleen, heart, brain and kidney, molecular forms in tissues as naringenin sulfates and naringenin glucuronides. Table 1 plots concentrations of naringenin sulfate and naringenin glucuronide in serum and various organs 6 h post 17 th dose. Results indicate the liver with highest ratio of naringenin sulfates, followed by spleen, heart brain, and kidney. Naringenin glucuronides were present in liver and kidney, not in spleen, brain, or heart.

Table 1 -Concentrations (nmol/mL for serum and nmol/g for tissues) of naringenin glucuronides (G) and naringenin sulfates (S) in serum and various organs at 6 h following 17 th dose of naringin (210 mg/kg) in six rats.
Metabolites serum liver spleen heart brain kidney sulfates as major forms, whereas naringin and naringenin were not present, which was consistent with previous studies [15,16]. spleen, heart and brain had higher concentrations of naringin sulfates than serum by 386, 100, and 57 %, respectively. In regard to distribution of naringenin glucuronides, conversely, liver and kidney contained lower concentrations than serum by 30 and 76%, respectively.

Discussion
Quantitation Quantitation of serum and collected tissues showed that after repeated dosing, free forms of naringin and naringenin were not detected in both bloodstream and all assayed organs; major molecular forms were naringenin sulfates and naringenin glucuronides. In serum, naringenin glucuronides were predominant; in liver, spleen, heart and brain, narigenin sulfate was the major form. Findings imply naringenin glucuronides deglucuronidated and then sulfated in these organs.
Among assayed organs, the liver contained higher naringenin sulfate and naringenin glucuronide concentrations than other organs, which concurred with previous studies [19] and indicated these naringenin conjugates have higher protein binding with liver proteins. In the liver, concentration of naringenin sulfates was higher than that of naringenin glucuronides by 240%. In spleen, heart, and brain, naringenin glucuronides were not detected, meaning naringenin released through hydrolysis with sulfatase in these organs was solely from naringenin sulfates. It can thus be assumed that when naringenin glucuronides entered liver, spleen, heart, or brain from circulation, they were hydrolyzed by glucuronidase, then sulfated by sulfotransferase in these organs [21,22]. In sum, liver, spleen, heart and brain contained narigenin sulfates as principal metabolites of naringin. Therefore, bioactivities of naringenin sulfates in liver, spleen, heart and brain warrant more investigations. In the kidney, naringenin glucuronides and naringenin sulfates manifested far lower concentrations than those in serum, indicating that only a little fraction of glucuronides or sulfates had entered the kidney. This finding was not consistent with previous study reporting moderate concentrations of naringenin glucuronides detected in kidney, liver and brain at 2 h post oral dose of naringenin in rats [18]; future studies must clarify.
Our study revealing absence of naringenin in all assayed tissues was not in good agreement with prior research detecting naringenin in tissues after single dose of naringin [19]. This discrepancy might arise from variant dosage or detection method: i.e., 17 doses of naringin might modulate expression of UDP-glucuronosyltransferase or sulfotransferase and result in more extensive metabolism [23]. Repeated dosing of naringin to rats yielded wide distribution of naringenin sulfates to various organs, while naringin and naringenin reached no organs. We identified chemical nature and concentration of naringenin conjugates in tissue; these may disclose pharmacological roles of putative active metabolites after chronic dosing of naringin.