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Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2005.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reference:
Composition 0
Objective of study:
metabolism
Qualifier:
no guideline available
Principles of method if other than guideline:
The aim of the study is to determine the capability of human gut bacteria to transform neohesperidin dihydrochalcone. Pure bacterial cultures or human fecal slurries were grown under strictly anoxic conditions at 37 ºC for 4-22h, and then exposed to the test item and the supernatant analysed.
GLP compliance:
no
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: purchased from Sigma (Deisenhofen, Germany).
- Identification parameters: Rt: 30.8 min, UV λmax 228 and 288 nm.
Radiolabelling:
no
Species:
other: bacteria
Strain:
other: human fecal slurries and human intestinal bacteria, E. ramulus and C. orbiscindens.
Details on test animals and environmental conditions:
The bacterial cultures (10 mL) were grown under strictly anoxic conditions at 37 ºC in 16 mL tubes using a complex medium (ST medium). In addition, a defined bicarbonate-buffered medium (medium B) supplemented with 10 or 20 mM glucose or without glucose addition was used for fermentation experiments. Both media were inoculated (5%) with fecal slurry, which was prepared by suspending 1 g of freshly collected feces anoxically in a final volume of 10 mL of ST medium. Pure cultures of Eubacterium ramulus strain wK1 (DSM 16296) and Clostridium orbiscindens strain I1 were grown in ST medium at 37 °C.
Route of administration:
other: in culture medium.
Vehicle:
DMSO
Details on exposure:
Fermentation experiments were performed by adding 100 µL of a 50mM stock solution of neohesperidin dihydrochalcone dissolved in dimethyl sulfoxide (DMSO) with a syringe to 10 mL of medium in 16mL tubes. The media were inoculated with 1-5% of a 10% fecal suspension or pure bacterial culture and incubated at 37 °C in a tube rotator. In some of the experiments, a second dose of neohesperidin dihydrochalcone was added after 43-48 h of incubation. Fermentation experiments with the intermediates of neohesperidin dihydrochalcone degradation were performed on a minor scale by addition of 20 µL of the respective stock solution dissolved in DMSO with a syringe to 2 mL of ST medium in 5 mL tubes. The medium was inoculated with 5% of an exponentially growing E. ramulus culture or 10% of an exponentially growing C. orbiscindens culture and incubated at 37 °C. In some of the experiments, the bacteria were grown for 4-22 h before addition of the flavonoid substrate. At the times indicated, aliquots of 200 µL were taken with a syringe and centrifuged for 5 min at 12000g, and the supernatant (30 µL) was analyzed without further processing by HPLC. To prove the solubility of neohesperidin dihydrochalcone and its bacterial metabolites in aqueous medium, complete aliquots and pellets resulting from centrifugation of selected samples were analyzed after lyophilization and extraction with DMSO.
Duration and frequency of treatment / exposure:
24-144h.
Dose / conc.:
100 other: µL of a 50mM stock solution of neohesperidin dihydrochalcone.
Control animals:
not specified
Details on study design:
Neohesperidin dihydrochalcone and aromatic metabolites were analyzed by reversed-phase HPLC. The HPLC system (Gynkotek, Munich, Germany) was equipped with a high-precision pump (M480G), a degasser (GT-103), an autosampler (GINA 160), a column oven (STH 585), a diode array detector (UVD 320), and a 250 × 4 mm i.d., 5 µm, LiChrospher 100 RP-18 column (Merck, Darmstadt, Germany). The column temperature was maintained at 37ºC. Aqueous 0.1% trifluoroacetic acid (TFA; solvent system A) and methanol (solvent system B) served as the mobile phase. The HPLC was run in gradient mode (solvent B from 5% to 30% in 20 min, from 30% to 50% in 5 min, from 50% to 80% in 10 min, and from 80% to 100% in 4 min) at a flow rate of 1 mL/min and detection at 280 nm. In addition, UV spectra were recorded in the range of 200-355 nm. External standard substances were used for calibration.
Selected incubation supernatants from degradation experiments were used for compound identification by electrospray ionization mass spectrometry (ESI-MS). For analysis, a triple-quadrupole mass spectrometer fitted with a Z-spray API electrospray source (Quattro II, Micromass, U.K.) was used. The model 2960 HPLC system (Waters, Milford, MA) was equipped with a 250 × 4 mm i.d., 5 µm, LiChrospher 100 RP-18 column (Merck) and a diode array detector (PDA 996). The mobile phase was a mixture of aqueous 1.6% formic acid (FA; solvent system A) and methanol (solvent system B). The gradient mode described above was used at a flow rate of 0.7 mL/min. The column temperature was maintained at 35 °C. The flow was split
6:1 prior to introduction into the mass spectrometer. MS analyses were carried out in positive ionization mode. The temperature of the ion source was maintained at 100 °C. The cone and capillary voltages used were 20 V and 3.0 kV, respectively. The desolvation temperature was 350 °C, and the desolvation gas (N2) was held at 400 L/h. In parallel with NMR analyses, the 3-(3-hydroxy-4-methoxyphenyl)-propionic acid preparation was subjected to MS with electron impact ionization (EI-MS; 70 eV) using an MS-50 spectrometer (AEI, Manchester, U.K.). EI-MS of 3-(3-hydroxy-4-methoxyphenyl)propionic acid: (m/z, rel intens) 196 (M+, 32), 150 (11), 137 (62), 91 (38), 78 (100), 63 (82).
The structure of 3-(3-hydroxy-4-methoxyphenyl)-propionic acid, formed during neohesperidin dihydrochalcone degradation by fecal samples, was elucidated by 13C and 1H NMR analysis.
Details on dosing and sampling:
METABOLITE CHARACTERISATION STUDIES
- Method type(s) for identification: The bacterial neohesperidin dihydrochalcone conversion was followed by HPLC/UV analysis of samples taken in the course of fermentation. The samples were centrifuged, and the supernatants were examined. Identification of the metabolites formed was accomplished with HPLC/DAD analysis and reference to standard substances. MS and NMR analyses were used to confirm the results.
Metabolites identified:
yes
Details on metabolites:
Fecal slurries from four different human donors converted 0.4-0.5 mM neohesperidin dihydrochalcone to equimolar amounts of 3-(3 -hydroxy-4-methoxyphenyl)propionic acid (Rt: 26.8 min, UV λmax: 225 and 285 nm). Two transient intermediates were identified as hesperetin dihydrochalcone 4′-β-D-glucoside and hesperetin dihydrochalcone. These metabolites suggest that neohesperidin dihydrochalcone is first deglycosylated to hesperetin dihydrochalcone 4′-β-D-glucoside and subsequently to the aglycon hesperetin dihydrochalcone. The latter is hydrolyzed to the corresponding 3-(3-hydroxy-4-methoxyphenyl)propionic acid and probably phloroglucinol.
Bioaccessibility testing results:
Bioavailability of neohesperidin dihydrochalcone and similar compounds depends on the activity of gut bacteria, since only bacteria are able to attack flavonoid rhamnoglucosides.

Neohesperidin dihydrochalcone could not be metabolized as the sole source of carbon, but experimented a rapid conversion within 22 h in the presence of glucose. Eubacterium ramulus and Clostridium orbiscindens were not capable of converting neohesperidin dihydrochalcone, and no other single bacterium isolated from the human gut has been identified which is able to degrade flavonoid rhamnoglucosides to the respective phenolic acids. Therefore, transformation of flavonoid glycosides must be brought about by cooperative action of different bacterial species present in the complex intestinal microbiota.

Conclusions:
Neohesperidin dihydrochalcone can be degraded by human intestinal microbiota in the presence of other carbon sources. The metabolites of such degradation were identified as hesperetin dihydrochalcone and hesperetin dihydrochalcone 4′-β-D-glucoside, which are then hydrolised to 3-(3 -hydroxy-4-methoxyphenyl)propionic acid and probably phloroglucinol.
Executive summary:

The degradation of neohesperidin dihydrochalcone by human intestinal microbiota and human fecal slurries was studied in vitro, following basic scientific principles. Under test conditions, neohesperidin dihydrochalcone can be degraded by human intestinal microbiota in the presence of other carbon sources. The metabolites of such degradation were identified as hesperetin dihydrochalcone and hesperetin dihydrochalcone 4'-ß-D-glucoside, which are then hydrolised to 3-(3 -hydroxy-4-methoxyphenyl)propionic acid and probably phloroglucinol. This transformation is thought to be brought about by cooperative action of different bacterial species present in the complex intestinal microbiota.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reference:
Composition 0
Objective of study:
toxicokinetics
Qualifier:
no guideline followed
Principles of method if other than guideline:
Study performed on male Sprague-Dawley rats by either oral (n = 6) or intravenous (n = 6) administration of the test substance and monitorization of plasma and tissue concentrations by an LC-ESI-MS system to determine main toxicokinetics parameters.
GLP compliance:
no
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Chengdu Mansite Pharmaceutical Co. Ltd. (Chengdu, China)
- Purity > 98.0%
Radiolabelling:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Laboratory Animal Center of Wenzhou Medical College (Wenzhou, China)
- Weight at study initiation: 200–220 g
- Housing: housed at Wenzhou Medical College Laboratory Animal Research Center, under controlled conditions.
- Diet: prohibited for 12 h before the experiment.
- Water: ad libitum.
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 ± 1
- Humidity (%): 55 ± 10
- Photoperiod (hrs dark / hrs light): natural light–dark cycle
Route of administration:
oral: unspecified
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: After fasting for 12 h, six rats were given a dose of 2.0 mg/kg NHDC via the sublingual vein, and the other six rats were administered with the dose of 20 mg/kg NHDC orally.
Duration and frequency of treatment / exposure:
Single dose administration, observation period 24h.
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
oral administration.
Dose / conc.:
2 mg/kg bw/day (nominal)
Remarks:
administered via the sublingual vein.
No. of animals per sex per dose:
6 males per dose.
Control animals:
no
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: blood; heart, liver, brain, lung, kidney, stomach and spleen.
- Time and frequency of sampling: Blood samples (0.3 mL) were collected from the tail vein into heparinized 1.5 mL polythene tubes at 0, 0.083, 0.167, 0.333, 0.5, 0.833, 1.333, 2.0, 2.667 and 3.333 h after intravenous administration and at 0, 0.083, 0.167, 0.333, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 10, 12, 24 h after oral administration. The samples were immediately centrifuged at 3000 g for 5 min. The plasma obtained (100 mL) was stored at 20 C until analysis.
- Three groups of 2 rats, which had been administered the test item intravenously, were euthanized by decapitation at 5 min, 0.5 and 1 h after dosing. Tissues, including the heart, liver, brain, lung, kidney, stomach and spleen were dissected and washed with cold saline. The tissues were then weighed and homogenized in cold saline solution (500 mg/mL). The obtained tissue homogenates were immediately stored at 20ºC until analysis.
- Before analysis, the plasma sample was thawed to room temperature. In a 1.5 mL centrifuge tube, an aliquot of 10 mL of the IS working solution (8.0 mg/mL) was added to 100 mL of collected plasma sample followed by the addition of 200 mL acetonitrile. The tubes were mixed by vortex for 0.5 min. After centrifugation at 14 900g for 10 min, the supernatant (10 µL) was injected into the LC-ESI-MS system for analysis.

- Analytical methods: A sensitive, simple and specific LC-ESI-MS method for the determination of NHDC in rat biological samples was developed and validated over the concentration range of 10–3000 ng/mL NHDC. Lower limit of quantification (LLOQ) for NHDC was 10 ng/mL. Mean recovery of NHDC from plasma and tissues was better than 80.3%. Coefficient of variation of intra-day and inter-day precision were both less than 15%. Method description: Biological samples were processed with one-step protein precipitation. Rutin was chosen as the internal standard (IS). Chromatographical separation was achieved on an SB-C18 (2.1 mm 150 mm, 5 mm) column with acetonitrile–0.1% formic acid in water as the mobile phase with gradient elution. Electrospray ionization (ESI) source was applied and operated in negative ion mode; selected ion monitoring mode was used for quantification using target fragment ions m/z 611.4 for NHDC and m/z 609.1 for IS.
Statistics:
Pharmacokinetic analyses and plasma concentration versus time data were analyzed by DAS software (Version 2.0, Drug Clinical Research Center of Shanghai University of T.C.M and Shanghai BioGuider Medicinal Technology Co., Ltd., China). Pharmacokinetic parameters were calculated by using the non-compartmental model.
Type:
absorption
Results:
Fast, in 0.2h plasma concentrations start to increase.
Type:
distribution
Results:
Fast and wide, Cmax 800-1100ng/mL, can cross blood-brain barrier.
Type:
excretion
Results:
Short residence time. Elimination after 24h, clearance rate 7.4 L/h/kg.
Details on absorption:
For oral administration, the plasma NHDC concentrations increased very quickly within 0.2 h, which suggests fast absorption.
Details on distribution in tissues:
The maximum plasma concentration (Cmax) of NHDC ranged from 801 to 1100 ng/mL, while the half-life (t1/2) was 1.0 ± 0.2 h. Results indicated that NHDC underwent a rapid and wide distribution into tissues within the time period examined. It was merely 5 min after i.v. administration that NHDC had already reached its Cmax in most of tissues, including the brain, showing that NHDC could effectively cross the blood–brain barrier.
Details on excretion:
Residence time of NHDC in vivo was very short, both for oral and i.v. administration. Plasma concentrations gradually decreased until 24 h. The clearance rate was 7.4 ± 2.5 L/h/kg.
Key result
Test no.:
#1
Toxicokinetic parameters:
half-life 1st: 1.0 ± 0.2 h
Key result
Test no.:
#1
Toxicokinetic parameters:
Cmax: 980.3±255.2
Key result
Test no.:
#1
Toxicokinetic parameters:
Tmax: 0.167
Key result
Test no.:
#1
Toxicokinetic parameters:
AUC: 2558.7±697.1
Metabolites identified:
not specified
Bioaccessibility testing results:
The absolute bioavailability of the test item was observed to be 21.8%.

Table 1. Single-compartmental pharmacokinetic parameters following oral dose (20 mg/kg) and intravenous administration (2 mg/kg; n=6). 

Phramacokinetic parameters

Unit

Oral Values

(mean ± SD)

I.V. Values

(mean ± SD)

Half-life (t1/2)

H

1.0 ± 0.2

0.46 ± 0.10

Peak concentration (Cmax)

µg/L·h

 

980.3 ± 255.2

 

2125.9 ± 596.3

Time to peak concentration (Tmax)

h

 

0.167 ± 0

 

0.083 ± 0

Area under concentration–time curve (AUC0-t)

µg/L·h

 

2558.7 ± 697.1

 

1204.5 ± 384.7

AUC0-

µg/L·h

 

2750.6 ± 786.2

 

1215.2 ± 389.9

Apparent volume of distribution (V)

L/kg

 

72.9 ± 18.5

 

1.2 ± 0.49

Clearance (CL)

 

L/h/kg

7.4 ± 2.5

 

1.8 ± 0.6

Mean residence time (MRT0-t)

h

 

5.1 ± 0.9

 

0.57 ± 0.07

MRT0-

h

7.2 ± 1.8

0.60 ± 0.08

Bioavailability (F)

%

21.8

 

F = [(AUCp.o)·(Dosei.v)]/[(AUCi.v)·(Dosep.o)]·100%.

Conclusions:
The test item undergoes rapid absorption (bioavailability 21.8%), rapid and wide distribution but short residence time, and rapid elimination (around 24h) after oral administration. Based on the available data, the test item shows no potential for bioaccumulation.
Executive summary:

A toxicokinetic study was performed on male Sprague-Dawley rats by either oral (n = 6, 20 mg/kg bw) or intravenous (n = 6, 2 mg/kg bw) administration of the test substance and monitorization of plasma and tissue concentrations by an LC-ESI-MS system. Under test conditions, the test item undergoes rapid absorption (bioavailability 21.8%), rapid and wide distribution but short residence time, and rapid elimination (around 24h) after oral administration, showing no signs of potential bioaccumulation.

Description of key information

Key study (study well documented, meets generally accepted scientific principles, acceptable for assessment): A pharmacokinetic study of the test item in the plasma of rats indicate that undergoes rapid absorption (bioavailability 21.8%), rapid and wide distribution but short residence time, and rapid elimination (around 24h) after oral administration, showing no signs of potential bioaccumulation. Other studies show that hydrolysis of the glycoside moiety upon ingestion is mainly due to intestinal bacteria.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

- A toxicokinetics study was performed on male Sprague-Dawley rats by either oral (n = 6, 20 mg/kg bw) or intravenous (n = 6, 2 mg/kg bw) administration of the test substance and monitorization of plasma and tissue concentrations by an LC-ESI-MS system. Under test conditions, the test item undergoes rapid absorption (bioavailability 21.8%), rapid and wide distribution but short residence time, and rapid elimination (around 24h) after oral administration, showing no signs of potential bioaccumulation.

- After oral ingestion of flavonoids, the glycoside moiety is hydrolysed and absorbed mainly as the corresponding aglycone, which may undergo conjugation with glucuronide and/or sulphate or be further degraded by intestinal bacteria, being those metabolites excreted via urine, bile or feces (EFSA Journal 2010; 8 (9): 1065).

- Another study on the degradation of neohesperidin dihydrochalcone by intestinal bacteria, showed that the test substance could not be metabolized by bacteria as the sole source of carbon, but experimented a rapid conversion within 22 h in the presence of glucose. Eubacterium ramulus and Clostridium orbiscindens were not capable of converting neohesperidin dihydrochalcone, and no other single bacterium isolated from the human gut has been identified which is able to degrade flavonoid rhamnoglucosides to the respective phenolic acids. Therefore, transformation of flavonoid glycosides must be brought about by cooperative action of different bacterial species present in the complex intestinal microbiota.