7-(2-O-(6-deoxy-α-L-mannopyranosyl)-β-D-glucopyranosyloxy)-2,3-dihydro-4',5,7-trihydroxyflavone

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

absorption

Qualifier:

no guideline followed

Principles of method if other than guideline:

A single dose of 746.7 mg/kg test item was orally administered (by gavage) to 6 male Sprague Dawley rats, and the concentrations of the test item and its metabolites were determined in plasma at 0 (predose), 5, 15, 30, 45, 60, 120, 240, 360, 480, 720 and 1440 min after dosing by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

GLP compliance:

no

Specific details on test material used for the study:

- Naringin (4',5',7-trihydroxy flavanone-7-rhamnonglucoside)
- Source: Sigma (St. Louis, MO)
- Purity: > 95%

Radiolabelling:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: College of Military Medicine Animal Research Center (Guangzhou, China).
- Weight at study initiation: 250–300 g
- Housing:
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
- Humidity (%):
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

50% PEG400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: the test item was dissolved into 127.9 mg/ml with 50% PEG400.

Duration and frequency of treatment / exposure:

single dose.

Dose / conc.:

746.7 mg/kg bw/day (nominal)

No. of animals per sex per dose / concentration:

6 male rats.

Control animals:

yes

Positive control reference chemical:

Not applicable.

Details on study design:

SAMPLE PREPARATION
- A 0.1 ml aliquot sample plasma was transferred to a clean glass tube. Ten microliters of methanol with 1000 ng/ml internal standard was added and vortexed. Then 400 μl ethyl acetate (LC grade) was added to the tube and vortexed again for 2 min. After the sample was centrifuged at 6000×g for 10 min, the upper organic layer was transferred into a 5 ml
glass tube. 200 μl ethyl acetate was added to the deposition again and then the deposition was extracted as those mentioned above again. The organic layer was mixed together and
dried with nitrogen at room temperature. The dried residue was dissolved in 100 μl of the mobile phase. A 20 μl aliquot of the sample was injected into the LC/MS/MS.

ANALYTICAL METHOD
- Separation method: HPLC system (SHIMADZU HPLC system, SHIMADZU, Columbia, MD), consisting of an SCL-10A system controller, an LC-10AD HPLC pump, a DGU 12A-degasser, and a manual injector.
- Conditions: The HPLC column (100 mm × 2.0 mm) was a 5 μm BetaBasic-C18 ODS column (KEYSTONE Bellefonte, PA). Column temperature was maintained at room temperature.
The mobile phase consisted of methanol (70%) and water (30%), which was filtered through a 0.2 μm nylon filter before use. The flow rate was 0.4 ml/min and the total run time was
3.0 min for each injection.
- Detection method: MS (Applied Biosystems MDS Sciex API3000 Triple Quadrapole mass spectrometer (MS/MS) equipped with a heated nebulizer interface (500ºC)). The mass spectrometer chose the positive ionization mode as the detection mode with the collision gas off. At the same time the mass spectrometer temperature was set at 370ºC. Under these conditions, the analytes yielded protonated molecules at m/z 581.3 for naringin, m/z 273.4 for naringenin and m/z 611.5 for hesperidin.
- Internal or external calibration: internal (standard: hesperidin, source: National Institute for the control of pharmaceutical and biological products, HPLC grade).
- Limits of detection and quantification: LOQ = 0.5 ng/ml.
- Extraction recovery (indicate if results are corrected or not for recoveries): The mean recovery values for the entire procedure were found to be 90.58, 91.64, 94.15% for naringin.
- Validation: The assay was validated in rat plasma. The retention time was 0.5, 0.5, and 1.07 min for naringin, hesperidin and naringenin, respectively. The absence of a signal at the
retention time of naringin, naringenin and hesperidin in the blank plasma established the specificity of the assay. For naringin, the accuracy values of the assay varied from 93.02 to 96.37%, the within-day precision values ranged from 3.9 to 5.3%, the between-day precision values ranged from 1.7 to 4.9%; the assay was linear from 5 to 1000 ng/ml, using 0.1 ml rat plasma, with a regression coefficient (r2) >0.9996.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: plasma.
- Time and frequency of sampling: Approximately 0.5 ml blood was withdrawn from each rat according to a schedule of 0 (predose), 5, 15, 30, 45, 60, 120, 240, 360, 480, 720 and 1440 min after dosing.

Statistics:

Data was collected and processed using Sciex Analyst 1.1. Plasma concentration–time profiles were analyzed by WinNonlin computer software, Version 4.0 (Pharsight Corporation, Mountain View, CA), using a noncompartmental method.

Preliminary studies:

Naringenin’s conjugates in the homogenates of different tissues were confirmed to be completely hydrolyzed within 2 h in the preliminary test. Meanwhile, no obvious loss of naringin was observed during this process.

Type:

absorption

Results:

the test item was quickly absorbed into the body (Tmax 45min), and declined rapidly within 2h.

Type:

excretion

Results:

After 24h, the test item was no longer detectable.

Details on absorption:

The test item was quickly absorbed into the body and could be detected 5 min after oral administration. The concentration of naringin in plasma reached nearly 2300 ng/ml in 45 min (Tmax) and declined rapidly, within 2 h. The Cmax was 3782.50 ± 986.82 ng/ml, and the AUC(0–24) was 6026.32 ± 1562.63 ng/ml h.

Details on excretion:

The test item could not be detected after 24h.

Key result

Test no.:

#1

Toxicokinetic parameters:

Cmax: 3782.50 ± 986.82 ng/ml

Remarks:

in plasma.

Key result

Test no.:

#1

Toxicokinetic parameters:

Tmax: 0.75 ± 0.25 h

Remarks:

in plasma.

Key result

Test no.:

#1

Toxicokinetic parameters:

AUC: 6026.32 ± 1562.63 ng/ml h

Remarks:

in plasma (0-24).

Metabolites identified:

yes

Details on metabolites:

The concentration of naringenin and naringenin glucuronide in plasma increased more slowly after oral administration of naringin. The Tmax of naringenin and naringenin glucuronide occurred at 9 and 7.5 h. For naringenin, the Cmax was 227.05±88.41 ng/ml, and the AUC0–24 was 1252.24±461.66 ng/ml h, whereas for naringenin glucuronide, the Cmax was 43575.00±8409.00 ng/ml, and the AUC(0–24) was 238269.00±53253.10 ng/ml h. The AUC0–24 of naringenin glucuronide was higher than naringin and naringenin, indicating that naringenin glucuronide is the main existent form in rat plasma after oral administration.

Table 1. Relevant pharmacokinetic parameters.

Parameter	Average ± S.D. (n = 6)
Naringin
Tmax (h)	0.75±0.25
Cmax (ng/ml)	3782.50±986.82
AUC [0-24] (ng/ml h)	6026.32±1562.63
MRT (h)	6.47±1.18
CL (ml/h)	0.028±0.008
HL-λz (h)	5.34±1.07
Naringenin
Tmax (h)	9.00±1.50
Cmax (ng/ml)	227.05±88.41
AUC [0-24] (ng/ml h)	1252.24±461.66
MRT (h)	8.16±1.59
CL (ml/h)	0.30±0.070
HL-λz (h)	2.98±0.65
Naringenin glucuronide
Tmax (h)	7.50±1.00
Cmax (ng/ml)	43575.00±8409.00
AUC [0-24] (ng/ml h)	238269.00±53253.10
MRT (h)	6.37±0.60
CL (ml/h)	0.0004±0.00019
HL-λz (h)	1.84±0.16

Conclusions:

The test item was quickly absorbed into the body, and could be detected 5 min after oral administration. The concentration of naringin in plasma reached nearly 2300 ng/ml in 45 min (Tmax) and declined rapidly, within 2 h. Based on these results, the test item shows no bioaccumulation potential.

Executive summary:

A study on the absorption of the test item in rats was performed. A single dose of 746.7 mg/kg test item was orally administered (by gavage) to 6 male Sprague Dawley rats, and the concentrations of the test item and its metabolites were determined in plasma at 0 (predose), 5, 15, 30, 45, 60, 120, 240, 360, 480, 720 and 1440 min after dosing by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The test item was quickly absorbed into the body and could be detected 5 min after oral administration. The Tmax was 45 min. The plasma concentration also declined quickly, within 2 h. Its metabolites, naringenin and naringenin glucuronide, were produced slowly after oral administration and Tmax for them were 9 and 7.5 h, respectively. After 24 h, no test item could be detected. Based on these results, the test item shows no bioaccumulation potential.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to other study

Objective of study:

distribution

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test item was administered orally to 48 rats (groups of 3 males and 3 females for each sampling time) at a dose of 42 mg/kg and the concentrations of naringin and its metabolites in tissue compartments were determined after 0 (pre-dose), 0.08, 0.5, 1, 2, 4, 8 and 12 h post-dose by liquid chromatography-tandem mass spectrometry (LC-MS/MS), in order to evaluate the distribution of the test item in various tissues.

GLP compliance:

no

Specific details on test material used for the study:

Naringin, for oral administration, was purified from the aqueous extract of Huajuhong in our laboratory, with a purity of 98.8 % determined by HPLC method with external standard. Naringin standard was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China).

Radiolabelling:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Male and female Sprague-Dawley rats, certified specific pathogen-free, were purchased from the Guangdong Medical Laboratory Animal Center (Guangzhou, China).
- Weight at study initiation: 200 ± 20 g
- Housing: housed in suspended plastic cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20–25 ºC
- Humidity (%): 55 ± 15 %
- Air changes (per hr): 12 air change cycles/h
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle

Route of administration:

oral: gavage

Vehicle:

not specified

Details on exposure:

After overnight fasting but with access to water, rats were orally administered by gavage with naringin at the dose of 42 mg/kg.

Duration and frequency of treatment / exposure:

single dose.

Dose / conc.:

42 mg/kg bw/day (nominal)

No. of animals per sex per dose / concentration:

3 males and 3 females per group.

Control animals:

yes

Positive control reference chemical:

Not applicable.

Details on study design:

SAMPLE PREPARATION
- An aliquot of 100μL tissue homogenate was transferred to a fresh 1.5 mL tube, incubated with 10μL β-glucuronidase/sulfatase (10 Units/μL) at 37 °C for 2 h. After additions of 20μL mobile phase, 6μL formic acid (5 %, v/v) and IS (3.9 ng) to the tube, 1 mL ethyl acetate was added 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 and evaporated at 40 °C. The residue was then reconstituted in 100μL mobile phase with vortex-mixing for 3 min, and centrifuged at 13 000 g for 10 min. Finally, the supernatant was injected into the LC-MS/MS system. The procedure of preparing samples to assay free naringenin was the same as above without the enzymolysis step.

ANALYTICAL METHOD
- Separation method: LC-MS/MS system: Agilent 1200 SL HPLC system with a G1322A vacuum degasser, a G1312B binary pump, a G1329B autosampler, and a G1316A column thermostat and an Agilent 6 410 triple quadrupole mass spectrometer (Agilent Technol., Santa Clara, CA, USA) equipped with an electrospray ionization interface.
- Conditions: The high resolution and high-throughput column used was Agilent Zorbax Eclipse Plus C18 (2.1 × 100 mm, 1.8μm). 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 40 ºC. The injection volume was 10μL and the chromatographic run-time was 5 min for each injection.
- Detection method: MS detection was performed in the negative-ion mode using multiple reaction monitoring (MRM) of m/z 579.2→271.0 for naringin, m/z 271.0→151.0 for naringenin, and m/z 463.0→299.8 for isoquercitrin as internal standard (IS). The capillary voltage was set at −4 000 V and the nebulizer gas (nitrogen) pressure was set at 30psi. The drying gas (nitrogen) was heated to 350 °C and delivered at a flow rate of 9 L/min. The fragmentor voltages for naringin, naringenin and IS were 200 V, 90 V and 130 V, and the collision energies were 35 V, 20 V and 25 V, respectively.
- Internal or external calibration: internal
- Limits of detection and quantification: LOQ = 10ng/g.
- Extraction recovery (indicate if results are corrected or not for recoveries): The extraction recoveries of naringin and naringenin were all above 71.45 %, and the matrix effects of naringin and naringenin ranged from 62.45 to 106.85 %. Results not corrected.
- The LC-MS/MS system was controlled by the MassHunter workstation software (Agilent Technol., Santa Clara, CA, USA).
- Method validation: In order to confirm the specificity of the method, endogenous matrix in different blank tissues samples from 6 individual rats were evaluated. The blank tissue homogenates from 6 individuals were mixed for the following sample preparation in assay validation. The intra- and inter-day accuracies of the method for both naringin and naringenin ranged from 91.26 to 109.72 % and from 91.98 to 106.94 %, respectively. The intra- and inter-day precisions were less than 13.72 %.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: heart, liver, kidney, spleen, lung, stomach, small intestine, trachea, muscle, fat, ovary/testis, and brain.
- Time and frequency of sampling: Animals were sacrificed at 0 (pre-dose), 0.08, 0.5, 1, 2, 4, 8 and 12 h post-dose.
- Other: After tissue samples were dissected, they were washed with physiologic saline to remove blood or content, then blotted on filter paper and weighted, followed by addition of physiologic saline at the ratio of 1:5 (w/v) and homogenization. All samples were kept at −70ºC until processing and analysis.
TREATMENT FOR CLEAVAGE OF CONJUGATES: tissue homogenate samples were incubated with 10μL β-glucuronidase/sulfatase (10 Units/μL) at 37 °C for 2 h.

Statistics:

The maximum concentrations (Cmax) and the times of maximum concentrations (Tmax) were obtained from the observed data. The areas under curve (AUC) values were calculated by use of the trapezoidal rule.

Preliminary studies:

Naringenin’s conjugates in the homogenates of different tissues were confirmed to be completely hydrolyzed within 2 h in the preliminary test. Meanwhile, no obvious loss of naringin was observed during this process.

Type:

distribution

Results:

Naringin is rapidly and widely distributed to most tissues except the brain.

Type:

excretion

Results:

After 12h, naringin was no longer detectable in all tissues examined.

Details on distribution in tissues:

After an oral administration of naringin to rats, both naringin and total naringenin were widely distributed to all tissues tested except brain. The naringin concentrations were high in stomach, small intestine, liver and trachea. High levels of total naringenin were found in liver, stomach, small intestine, kidney, lung and trachea. No significant amounts of either were found in the brain.

Details on excretion:

Naringin was undetectable (or almost) at 12h in all tissues examined, which suggested that there were no long-term accumulation in the tissues.

Test no.:

#1

Toxicokinetic parameters:

Cmax: 60941.27 ng/g

Remarks:

in stomach.

Test no.:

#1

Toxicokinetic parameters:

Tmax: 0.08 h

Remarks:

in stomach.

Test no.:

#1

Toxicokinetic parameters:

AUC: 1969.08 ng·h/g

Remarks:

in stomach.

Test no.:

#2

Toxicokinetic parameters:

other: see 'Any other information on results incl. tables'

Metabolites identified:

no

Details on metabolites:

After an oral administration of naringin, as a pure compound or as mixture, the forms detected in the circulation included native naringin as well as several major metabolites: aglycone naringenin, glucuronide and sulfate of naringenin.

The results showed the AUC decreased in tissues in the following order: for naringin, stomach, small intestine, liver, trachea, muscle, kidney, lung, fat, heart, spleen, ovary, testis, brain; and for total naringenin, liver, stomach, small intestine, kidney, trachea, lung, testis, heart, ovary, fat, spleen, muscle, brain. The Cmax of total naringenin for all tissues analyzed were obtained approximately at 2–4 h, and the Tmax of naringin varied in different tissues.

Table 1. AUC, Cmax and Tmax for naringin and total naringenin in tissues after a single oral administration of 42 mg/kg naringin to rats.

	Naringin			Total naringenin
Tissues	AUC (ng·h/g)	Cmax (ng/g)	Tmax (h)	AUC (ng·h/g)	Cmax (ng/g)	Tmax (h)
Stomach	67843.57	60941.27 ± 19260.36	0.08	1969.08	267.72 ± 92.24	1
Small intestine	17592.49	10839.06 ± 7100.04	0.08	1045.35	256.02 ± 279.16	2
Liver	634.17	231.45 ± 214.73	0.08	2212.21	507.57 ± 206.16	2
Kidney	476.15	35.15 ± 79.12	0.5	328.59	109.54 ± 193.38	2
Trachea	163.47	111.98 ± 130.01	0.08	308.13	50.96 ± 36.30	4
Lung	129.76	34.48 ± 43.47	1	251.34	54.76 ± 48.72	2
Heart	90.74	12.49 ± 25.18	2	83.30	32.49 ± 25.96	2
Fat	87.80	10.70 ± 37.05	8	56.96	8.60 ± 20.65^a	2
Muscle	57.54	32.23 ± 56.20	0.5	36.76	7.17 ± 14.68^a	2
Testis	56.82	12.70 ± 33.59	4	106.10	23.46 ± 17.20	4
Ovary	47.32	14.16 ± 13.21	2	59.59	8.80 ± 7.23^a	4
Spleen	39.20	9.48 ± 22.58^a	8	44.27	11.00 ± 13.32	2
Brain	10.38	3.46 ± 5.16^a	4	6.66	2.22 ± 5.44^a	4

Conclusions:

The test item and its metabolites were rapidly and widely distributed to all the tissues except the brain, but no accumulation was observed in the tissues examined (the test item was undetectable after 12h). Based on these results, the test item shows no bioaccumulation potential.

Executive summary:

The study of tissue distribution of the test item and its metabolites was performed in rats to examine whether they undergo selective uptake by specific organs. The test item was administered orally to 48 rats (groups of 3 males and 3 females for each sampling time) at a dose of 42 mg/kg and the concentrations of naringin and its metabolites in tissue compartments were determined after 0 (pre-dose), 0.08, 0.5, 1, 2, 4, 8 and 12 h post-dose by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The test item and its metabolites were rapidly and widely distributed to all the tissues except the brain, but no accumulation was observed in the tissues examined (the test item was undetectable after 12h). Based on these results, the test item shows no bioaccumulation potential.

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

The fate of orally administered Naringin and Naringenin was studied in rats, by studying the plasma concentrations and urinary and biliary excretion of the metabolites, and performing structural investigations of the metabolites excreted in bile and urine.

GLP compliance:

no

Specific details on test material used for the study:

Source: purchased from Sigma ChemicalCo.

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

- Weight: 240-280g

Route of administration:

oral: gavage

Vehicle:

not specified

Details on exposure:

The rats had been deprived of food for 16h before experiments.

Duration and frequency of treatment / exposure:

Single dose administration.

Dose / conc.:

130 mg/kg bw/day (nominal)

No. of animals per sex per dose / concentration:

6 for plasma studies, 6 for urinary excretion studies, 3 for biliary excretion studies.

Control animals:

no

Positive control reference chemical:

not applicable.

Details on study design:

SAMPLE PREPARATION
- Plasma samples were analyzed as follows: 0.05 ml of plasma was mixed with 0.1 ml ef 0.5M NaH2PO4 and subsequently with 2 ml of MeOH. After centrifugation, the supernatant was evaporated to dryness under a stream of nitrogen. The residue was dissolved in 0.15 ml of 0.2M NaH2PO4 and mixed with 0.1ml of IS solution (2.5 μg of hesperidin in 1 ml of MeOH). An aliquot (10 μl) of the solution was injected into the HPLC apparatus.
- The metabolites in urine and bile were determined by the following method: Each sample taken from an animal was made up to 10 ml with MeOH. A 0.1 ml of the MeOH solution was mixed with 0.1 ml of IS solution. An aliquot (10 μl) of the solution was subjected to HPLC apparatus.

ANALYTICAL METHOD
- Separation method: HPLC.
- Conditions: Nucleosil 5C-18 column held at 40ºC, the mobile phase was 0.3M phosphate buffer (pH 2.1)-MeOH (4:1.9) and the flow rate was 0.9 ml/min.
- Detection method: UV, 330 nm.
- Internal or external calibration: internal (hesperidin)
- Limits of detection and quantification: LOQ = 3-60 µg/ml for each metabolite.
- Extraction recovery (indicate if results are corrected or not for recoveries): not lower than 85%.
- Method validation: CVs were within 2-8%.

Details on dosing and sampling:

PHARMACOKINETIC STUDY
- Tissues and body fluids sampled: plasma, urine, bile.
- Time and frequency of sampling: blood samples were withdrawn from cervical vein at regular intervals; plasma was separated by centrifugation for 5 min. Urine and bile samples were taken at regular intervals for 24-48h, each sample being collected in 5 ml of MeOH (0.2M) NaH2P04 (10:1).

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, plasma, bile.
- Time and frequency of sampling: blood samples were withdrawn from cervical vein at regular intervals; plasma was separated by centrifugation for 5 min. Urine and bile samples were taken at regular intervals for 24-48h, each sample being collected in 5 ml of MeOH (0.2M) NaH2P04 (10:1).
- Method type(s) for identification: preparative HPLC on ODS column (YMCRI-355), using hesperidin as an internal standard and H2O-CH3CN as an eluent; UV spectral shifts with shift reagents, IR spectral data, mass spectrometry, 1H-NMR and 13C-NMR.
- Limits of detection and quantification: the calibration curves were linear and the recovery was not lower than 85% in the range of 3-60 µg/ml for each metabolite. As for the reproducibilities of the analysis, the CVs were within 2-8% in the range of 3-60 µg/ml.

Type:

metabolism

Results:

Naringin is absorbed as naringenin after hydrolysis to naringenin, and its main metabolite is naringenin 7-glucuronide.

Details on absorption:

Naringin is absorbed as naringenin after hydrolysis.
The maximum concentration of metabolites in plasma was achieved around 4h after administration of naringenin, then the concentrations declined until 10h after administration, when the second peaks began to appear, indicating the existence of enterohepatic recirculation of metabolites.
Metabolites with larger molecular weights preferentially transferred into bile, whereas smaller ones transferred into blood.

Details on excretion:

The total cumulative mean % of dose for all metabolites in urine was of 4% after 48h, and in bile 20% after 36h.

Metabolites identified:

yes

Details on metabolites:

- Biliary metabolites: naringenin 7-glucuronide, naringenin 7-sulfate 4'-glucuronide and naringenin 7-glucuronide 4'-sulfate.
- Urinary metabolites: naringenin 4'-glucuronide, naringenin 7,4'-disulfate,and naringenin 7-glucuronide.
- Plasma: naringenin 4'-glucuronide, naringenin 7,4'-disulfate,and naringenin 7-glucuronide.

Both naringin and naringenin gave the same metabolites when administered orally to rats. For naringin, the appearance of metabolites was considerably slower than for naringenin (first peak at 1h and second around 10h); lower concentrations of the metabolites with greater variations were observed. This indicates that naringin is absorbed as naringenin after hydrolysis.

Conclusions:

Naringin is absorbed as naringenin after hydrolysis, and its main metabolite is naringenin 7-glucuronide. Other metabolites formed are naringenin 7-sulfate 4'-glucuronide and naringenin 7-glucuronide 4'-sulfate in bile; naringenin 4'-glucuronide and naringenin 7,4'-disulfate in urine; and naringenin 4'-glucuronide and naringenin 7,4'-disulfate in plasma.

Executive summary:

The fate of the metabolites of the test item after oral administration to rats was studied following basic scientific principles. A single dose of 130 mg/kg was administered to male Wistar rats, and the effects on plasma (6 rats), bile (3 rats) and urine (6 rats) were studied. Based on the results, it can be stated that naringin is absorbed as naringenin after undergoing hydrolysis. The main metabolite found in all samples, was naringenin 7-glucuronide. The other metabolites found were naringenin 7-sulfate 4'-glucuronide and naringenin 7-glucuronide 4'-sulfate in bile; naringenin 4'-glucuronide and naringenin 7,4'-disulfate in urine; and naringenin 4'-glucuronide and naringenin 7,4'-disulfate in plasma. Concentration-time profiles showed signs of enterohepatic recirculation of metabolites.

Description of key information

Weight of evidence:

Adsorption: A study on the absorption of the test item in rats was performed by Fang et al. (2006). The test item was quickly absorbed into the body and the plasma concentration also declined quickly, not being detectable after 24h.

Distribution: A study on the tissue distribution of naringin by Zou et al. (2012) reports the test item and its metabolites being rapidly and widely distributed to all tissues except the brain, but no accumulation in tissues.

Metabolism: A study on the metabolites of naringin after oral administration in rats by Abe et al. (1993) reports naringin and naringenin yielding the same metabolites, indicating that naringin is mainly absorbed as naringenin after hydrolysis. The main metabolites found in rats were naringenin 7-glucuronide, naringenin 7 -sulfate 4'-glucuronide and naringenin 7 -glucuronide-4'-sulfate in bile; naringenin 4'-glucuronide and naringenin 7,4'-disulfate in urine; and naringenin 4'-glucuronide and naringenin 7,4'-disulfate in plasma.

Excretion: In the available studies in rats, naringin could not be detected after 24h. In the available study in humans, the amount excreted after 24h corresponds to 5 -57% of the administered dose (including metabolites).

Based on the available information, it can be concluded that there is no bioaccumulation potential for naringin.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

Weight of evidence:

- A study on the absorption of the test item in rats was performed by Fang et al. (2006) reports that test item was quickly absorbed into the body and the plasma concentration also declines quickly, not being detectable after 24h.

- A study on the tissue distribution of naringin in rats by Zou et al. (2012) reports naringin and naringenin being rapidly and widely distributed to all tissues except the brain, but no accumulation in tissues. Based on the available information, it can be concluded that there is no bioaccumulation potential for naringin.

- A study on the metabolites of naringin after oral administration in rats by Abe et al. (1993) reports naringin and naringenin yielding the same metabolites, indicating that naringin is mainly absorbed as naringenin after hydrolysis. The main metabolites found were naringenin 7-glucuronide, naringenin 7 -sulfate 4'-glucuronide and naringenin 7 -glucuronide-4'-sulfate in bile; naringenin 4'-glucuronide and naringenin 7,4'-disulfate in urine; and naringenin 4'-glucuronide and naringenin 7,4'-disulfate in plasma. Concentration-time profiles showed signs of enterohepatic recirculation of metabolites.

- A study on the metabolism of naringenin from grapefruit juice in humans by Fuhr et al. (1995) describes naringin being partly metabolised to naringenin after oral intake (creating a lag in excretion), and naringenin being rapidly and almost completely glucuronidated. The amount excreted after 24h corresponds to 5 -57% of the administered dose.