2'-(β-D-glucopyranosyloxy)-4',6'-dihydroxy-3-(4-hydroxyphenyl)propiophenone

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Objective of study:

absorption

metabolism

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Absorption and metabolism of phloretin and phloridzin (phloretin 2'-O-glucose) were investigated in rats after in situ perfusion of jejunum plus ileum (15 nmol/min) for 30 min.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

2'-(β-D-glucopyranosyloxy)-4',6'-dihydroxy-3-(4-hydroxyphenyl)propiophenone

EC Number:

200-487-1

EC Name:

2'-(β-D-glucopyranosyloxy)-4',6'-dihydroxy-3-(4-hydroxyphenyl)propiophenone

Cas Number:

60-81-1

Molecular formula:

C21H24O10

IUPAC Name:

2'-(β-D-glucopyranosyloxy)-4',6'-dihydroxy-3-(4-hydroxyphenyl)propiophenone

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sigma (L’Isle D’Abeau, Chesnes, France).
- Other identifiers: phloretin 2'-O-glucose.

Radiolabelling:

no

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Institut National de la Recherche Agronomique, de Clermont-Ferrand/Theix, 63122 Saint Genès Champanelle, France.
- Weight at study initiation: ~150 g
- Housing: 2 animals per cage
- Diet (e.g. ad libitum): standard semipurified diet (730 g/kg wheat starch, 150 g/kg casein, 60 g/kg mineral mixture, 10 g/kg vitamin mixture and 50 g/kg corn oil), access to food from 8:00 to 16:00 h.
- Water ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Photoperiod (hrs dark / hrs light): 12/12 h cycle, dark period from 8:00 to 20:00 h

Administration / exposure

Route of administration:

other: perfusion of the jenjum plus ileum.

Duration and frequency of treatment / exposure:

30min.

Doses / concentrations

Dose / conc.:

15 other: nmol/min

No. of animals per sex per dose / concentration:

6

Control animals:

no

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
Rats were anesthetized with sodium pentobarbital (40 mg/kg body) 18 h after the beginning of the meal and were kept alive during the perfusion period. After cannulation of the biliary duct, a perfusion of jejunal 1 ileal segments of intestine (from 5 cm distal from the flexura duodenojejununalis to the valvula ileocoecalis) was prepared by installing cannulas at each extremity. This segment was continuously perfused in situ at a flow rate of 1 mL/min, for 30 min, with a buffer containing: KH2PO4 (5 mmol/L), K2HPO4 (2.5 mmol/L), NaHCO3 (5 mmol/L), NaCl (50 mmol/L), KCl (50 mmol/L), CaCl2(2 mmol/L), MgCl2 (2 mmol/L), pH 6.7, glucose (8 mmol/L) and taurocholic acid (1 mmol/L), at 37°C. The intestine was washed of its content during the first 15 min. All the molecules used were stable in the buffer throughout the perfusion period. Aliquots of effluent were directly collected at the exit of the ileum in plastic tubes (1.5 mL) during the last 5 min of perfusion.
- Tissues and body fluids sampled: blood, plasma, bile.
- Time and frequency of sampling: At the end of the experiment, blood samples were withdrawn from the mesenteric vein (corresponding to the perfusate segment of the intestine) and abdominal aorta into heparinized tubes. Plasma and perfusate samples were acidified with 10 mmol/L acetic acid and stored at -20°C.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: intestinal effluent, bile
- From how many animals: 6
- Method type(s) for identification: HPLC-UV. The HPLC system used consisted of an autosampler (Kontron 360), an ultraviolet detector (set at 370nm for flavonols and at 280 nm for dihydroxychalcones) and a software system for data recording and processing. The system was fitted with a 5 μm C-18 Hypersil BDS analytical column (150 3 4.6 mm; Life Sciences International, Cergy, France). The mobile phase consisted of water/H3PO4 (99.5: 0.5; solvent A) and acetonitrile (solvent B). To visualize and separate the conjugated metabolites of flavonoids, the chromatographic conditions were as follows (flow rate: 1 mL/min): 0–2 min: solvent A 85%/solvent B 15%; 2–22 min: linear gradient from solvent A 85%/solvent B 15% to solvent A 60%/solvent B 40%; 22–24 min: solvent A 60%/solvent B 40%; 24–27 min: return to initial mobile phase conditions, then equilibration for 8 min.

TREATMENT FOR CLEAVAGE OF CONJUGATES: Plasma and perfusate samples were spiked with internal standard, acidified with acetic acid (to pH 4.9), and treated for 30 min at 37°C in the absence (unconjugated forms) or in the presence (total forms) of 5x10^6 U/L b-glucuronidase and 2.5x10^5 U/L sulfatase. The reactions were stopped by adding of 2.85 volumes of acetone and the resulting mixtures were centrifuged for 4 min at 14000xg. Supernatant (20 μL) was injected and analyzed by HPLC with UV detector.

Statistics:

Values are means ± SEM, and the differences were determined by one-way ANOVA coupled with the Student-Newman-Keuls multiple comparison test. Differences with P < 0.05 were considered significant. The statistical analysis of the glucose absorption was realized by one-way ANOVA coupled with the Tukey post-hoc test.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Phloridzin was perfused and found in the effluent as native form (2.67 ± 0.23 nmol/min) and also as aglycone (6.77 ± 0.50 nmol/min). This result suggests that hydrolysis of phloridzin to phloretin is not a limiting step for its absorption, because 80% of this glucoside was hydrolyzed. The analysis of HLPC profiles indicated that the conjugated forms of phloridzin found in the effluent corresponded to those recovered after phloretin perfusion, which strongly suggests that the hydrolysis of phloridzin preceded its metabolism by the intestinal conjugative enzymes.
Phloridzin absorption by the intestinal wall was markedly lower than that measured when the aglycone was perfused (1.11 ± 0.50 nmol/min vs. 3.60 ± 0.20 nmol/min; P < 0.05, respectively). Moreover, the flux of the secretion of conjugated metabolites was significantly reduced when phloridzin was perfused instead of phloretin.

Details on distribution in tissues:

A large proportion of the phloretin released from phloridzin hydrolysis diffused directly into the effluent, which could be related to extracellular hydrolysis.

Details on excretion:

When phloridzin was perfused, excretion of the aglycone form of phloridzin was 6.77 ± 0.50 nmol/min.

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Phloridzin was perfused and found in the effluent as native form (2.67 ± 0.23 nmol/min) and also as aglycone (6.77 ± 0.50 nmol/min); both the aglycone amount and the conjugated forms of phloridzin recovered corresponded to those recovered after phloretin perfusion. This result suggests that hydrolysis of phloridzin to phloretin is not a limiting step for its absorption, because 80% of this glucoside was hydrolyzed, and that the hydrolysis of phloridzin precedes its metabolism by the intestinal conjugative enzymes.

Applicant's summary and conclusion

Conclusions:

Phloridzin is rapidly hydrolysed to phloretin (80%, not a limiting step), and then conjugated. After perfusion, phloridzin was found in the effluent in its native form, as the aglycone and as its conjugated forms. Excretion of the aglycone form of phloridzin was 6.77 ± 0.50 nmol/min. The results show that the absorption of flavonoid glucosides was highly dependent on the mechanisms involved in their hydrolysis and on the activity of their intestinal conjugation.

Executive summary:

The study of the intestinal absorption and metabolism of phloridzin (phloretin 2'-O-glucose) was investigated in rats after in situ perfusion of jejunum plus ileum (15 nmol/min) for 30 min (in situ perfusion model), following basic scientific principles (no GLP). Based on the results, it can be stated that phloridzin is rapidly hydrolysed to phloretin (80%, not a limiting step), and is then conjugated. After perfusion, phloridzin was found in the effluent in its native form, as the aglycone and as its conjugated forms. Excretion of the aglycone form of phloridzin was 6.77 ± 0.50 nmol/min. The results show that the absorption of flavonoid glucosides was highly dependent on the mechanisms involved in their hydrolysis and on the activity of their intestinal conjugation.