5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-benzopyrone

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:

1993.

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

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Apin Chemicals Ltd., Oxon, UK.

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: around 200 g

Route of administration:

oral: gavage

Vehicle:

other: arabic gum

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: suspension in arabic gum.

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

po.

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

5-hydroxyflavone (100mg/kg po)

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, blood.
- Time and frequency of sampling: urine was collected for the 24 hr after the treatment using metabolic cages; urine samples were pooled. Blood extracts were obtained from treated rats at various times after gavage. Animals were killed at the various times after treatment and whole blood collected (3-4 ml). All the biological samples were immediately frozen at -20ºC until use.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine
- Time and frequency of sampling: urine was collected for the 24 hr after the treatment using metabolic cages.
- From how many animals: number not specified, urine samples were pooled.
- Method type(s) for identification: HPLC-DAD (Perkin-Elmer, LC250, equipped with a 10 x 0.46 cm column filled with Nucleosil 5CN stationary phase, 5um; MachereyNagel, Düren, Germany), detection at 345 nm. Identification of metabolites was achieved by one and two-dimensional NMR experiments.

TREATMENT FOR CLEAVAGE OF CONJUGATES: Samples to be analyzed were incubated as such in duplicate in a 50 mM sodium phosphate buffer (pH 5.5) in the presence of 1000 units/mI of b-glucuronidase (Sigma) (final volume, 200ul) for 1 hr at 37ºC. Controls were run simultaneously under the same conditions, except for b-glucuronidase, which was replaced by the same volume of buffer. The reactions were stopped by adding 400ul of acetonitrile and centrifuged. Fifty ul of both supematants were injected and analyzed by HPLC.

Details on absorption:

No unchanged flavonoid could be detected, while under similar conditions nearly 100% of the aglycone was recovered from control blood spiked with diosmetin. Diosmetin is rapidly metabolized to several glucuronides, probably at the level of the intestinal mucosa. The 2 main metabolites appeared within few minutes of treatment, and plateaued after 2-6h. After 24h, the metabolites were no longer detectable in circulating blood. Treatment with b-glucuronidase of both blood and urine led to the disappearance of all metabolites and to the concomitant appearance of diosmetin.

Details on excretion:

In 24h urine samples, the same four metabolites found in blood could be seen, plus an extra peak, present in control urines. Treatment with b-glucuronidase of both blood and urine led to the disappearance of all metabolites and to the concomitant appearance of diosmetin.

Metabolites identified:

yes

Details on metabolites:

At least 4 metabolites with spectra fitting flavonoid structures, (not present in controls) could be isolated from the blood samples: 4 glucuronides were found, the main two metabolites being diosmetin 3'-glucuronide, and a diosmetin diglucuronide.

Conclusions:

Diosmetin is rapidly metabolized to several glucuronides, probably at the level of the intestinal mucosa, inmmediately after administration. Four metabolites (diosmetin glucuronides) appear in blood within few minutes and plateau at 2-6h, and are eliminated within 24h. The same metabolites can be found in urine after 24h.

Executive summary:

A study of the metabolic fate of diosmetin after oral administration was performed on male Sprague-Dawley rats, following basic scientific principles (no GLP). Based on the results obtained, it can be stated that diosmetin is rapidly metabolized to several glucuronides, probably at the level of the intestinal mucosa, inmmediately after administration. Four metabolites (diosmetin glucuronides) appear in blood within few minutes and plateau at 2-6h, and are eliminated within 24h. The same metabolites can be found in urine after 24h.

Reference 2

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1993.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Objective of study:

toxicokinetics

Qualifier:

no guideline available

Principles of method if other than guideline:

Model of rat liver perfusion (Garattini et al. 1973).

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by Geymonat S.p.A. (Italy).
- Purity: analytical grade

Radiolabelling:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Male Crl:CD (SD) BR rats
- Weight at study initiation: 250 ± 4 g
- Diet: 'open formula' standard diet (Altromin MT, Rieper, Italy) ad libitum
- Water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 0.5
- Humidity (%): 55
- Photoperiod (hrs dark / hrs light): 12/12 cycle

Vehicle:

water

Details on exposure:

The flavonoids, dissolved in 0.5 N NaOH, were added to the perfusion medium (0.25%v/v) at the concentration which showed a protective effect in cultured rat hepatocytes (25p)

Duration and frequency of treatment / exposure:

Single dose, 90min exposure.

Dose / conc.:

1 other: µM

Remarks:

1 - 10 µM

Dose / conc.:

10 other: µM

Remarks:

1 - 10 µM

No. of animals per sex per dose / concentration:

Not specified.

Control animals:

yes

Details on study design:

- Liver perfusion: Livers were isolated by a previously-descrlbed surgical technique (Bartosek et al. 1972) under pentobarbital sodium anesthesia (50mg/kg body weight, i.p.), and the biliary duct and portal vein were cannulated. The perfusion medium consisted of Krebs-Ringer bicarbonate buffer p H 7.4 containing 0.1% (w/v) glucose, 4%) (w/v) BSA and washed human red blood cells as the oxygen carrier at a concentration which gives a haematocrit of 13-1 5%. A constant medium/liver tissue ratio (9 ml/g) and constant flow (1 ml/g liverlmin) were used. The livers were perfused for 90min.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: liver, bile
- Time and frequency of sampling: 90-min perfusion period
- The following parameters were studied during the 90-min perfusion period: the concentrations of diosmin and diosmetin, the activity of lactate dehydrogenase (LDH) and urea levels by standard kits. Bile flow and excretion of the flavonoids were determined as previously described (Cova et al. 1992). At the end of perfusion period the liver was weighed and one lobe was used to measure ATP levels. Part of the remaining tissue was homogenized with 0.25M sucrose-phosphate buffer p H 7.4 and deproteinated with 50% (w/v) trichloroacetic acid to determine glutathione (CSH) as non-protein sulphydryl groups in the supernatant fraction. Biochemical parameters: Urea, LDH, GSH and ATP.
- Method type(s) for identification: HPLC. Diosmin and diosmetin were extracted by adding 100µL DMSO to 100µL perfusion medium, shaking for 30min and then centrifuged at 1600g for 10min, and 50uL of the resultant supernatant were injected into the HPLC apparatus. Bile (25µL) was analysed by HPLC without any treatment.
- Conditions: Jasco PU-980 HPLC apparatus in isocratic conditions. A Lichrospher 100RP 18 column (5µm; 4 x 125mm) was used with a mobile phase of methanol/acetic acid/water (40/7/53) and a flow rate of 1ml/min. The eluate was monitored for absorbance at 345nm, and the retention times were 2.6 and 11min for diosmin and diosmetin, respectively.
- Limits of detection and quantification: Recoveries of the flavonoids were 100 and 90% in perfusion and bile, respectively.

Statistics:

The Easyfit computer program (Sacchi Landriani et al. 1983) was used to evaluate individual pharmacokinetic parameters from the perfusion data. T h e medium concentration-time curves fitted a two-compartment model. The biexponential equation was chosen according to the statistical method MAICE (minimum Akaike’s Information Criterion estimation) (Yamaoka et al. 1978). Kinetic and biochemical parameters were compared using one-way analysis of variance. Statistical differences were accepted at the p < 0.05 confidence level.

Type:

excretion

Results:

The disappearance curves of diosmin and diosmetin from the perfusion medium followed a two-compartment model. Both substances rapidly disappeared from the perfusion medium and were no longer detectable after 45 min.

Details on excretion:

Liver: Diosmetin, even though it takes longer than diosmin to disappear from the perfusion medium, is actively metabolized by the liver in a short time, with many peaks appearing in bile.

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 19.65 ± 1.43 min

Remarks:

half-life of the λz phase.

Key result

Test no.:

#1

Toxicokinetic parameters:

AUC: 307 ± 15

Key result

Test no.:

#1

Toxicokinetic parameters:

other: CL = 3.63 ± 0.17 ml/min

Remarks:

hepatic clearance.

Test no.:

#1

Toxicokinetic parameters:

other: λz = 0.036 ± 0.003 min-1

Remarks:

slope of the last linear phase of the log blood concentration vs. time plot.

Metabolites identified:

yes

Details on metabolites:

When diosmetin was added to the perfusion medium it was not detectable in bile at any interval, but there were many peaks with a retention time < 11min, corresponding to more polar metabolites, which are both glucuronides and sulphates of diosmetin. Diosmetin derived from these conjugates decreased with time like the original peaks of metabolites, and amounted to approximately 31% of the initial amount in the perfusion medium.

Conclusions:

Diosmetin is actively metabolized by the liver in a short time, with many peaks appearing in bile, but not the test item itself.

Executive summary:

The metabolism of the test item was studied in vitro, on a model of rat liver perfusion, following basic scientific principles (no GLP). Under test conditions, diosmetin is actively metabolized by the liver in a short time, with many peaks appearing in bile, even though it takes longer than diosmin to disappear from the perfusion medium. The main toxicokinetic parameters in liver were t(1/2) = 19.65 ± 1.43 min and AUC = 307 ± 15 nmol/ml·min. Based on the available information, no bioaccumulation potential is expected.

Description of key information

Weight of evidence: In an in vivo study on the metabolic fate of diosmetin, it was found that it was rapidly metabolized to several glucuronides inmediately after administration, yielding four glucuronides that appeared in blood within few minutes. These reached a plateau at 2-6h, and were eliminated within 24h. The same metabolites could be found in urine after 24h. In an in vitro study, the metabolism of diosmin was studied on a model of rat liver perfusion, following basic scientific principles (no GLP). Diosmetin was found to be actively metabolized by the liver in a short time, with many peaks appearing in bile, even though it took longer than diosmin to disappear from the perfusion medium. The main toxicokinetic parameters in liver were t(1/2)= 19.65 ± 1.43 min and AUC = 307 ± 15 nmol/ml·min. Based on the available information, no bioaccumulation potential is expected.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

Weight of evidence: An in vivo study of the metabolic fate of diosmetin after oral administration was performed on male Sprague-Dawley rats by Boutin (1993), following basic scientific principles (no GLP). Diosmetin was rapidly metabolized to several glucuronides, probably at the level of the intestinal mucosa, inmmediately after administration. Four metabolites (diosmetin glucuronides) appeared in blood within few minutes and plateau at 2-6h, and were eliminated within 24h. The same metabolites could be found in urine after 24h. In an in vitro study by Perego et al (1993), the metabolism of diosmin was studied on a model of rat liver perfusion, following basic scientific principles (no GLP). Diosmetin was found to be actively metabolized by the liver in a short time, with many peaks appearing in bile, even though it took longer than diosmin to disappear from the perfusion medium. The main toxicokinetic parameters in liver were t(1/2)= 19.65 ± 1.43 min and AUC = 307 ± 15 nmol/ml·min. Based on the available information, no bioaccumulation potential is expected.