Extract of fava d'anta, obtained from the fruits of Dimorphandra mollis (Leguminosae) by solvent extraction

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

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

Radiolabelling:

no

Species:

rat

Strain:

other: SDD Wister

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 230-300 g
- Diet: synthetic diet (44.1% wheat starch, 20% casein, 5% mineral mixture, 0.8% vitamin mixture and 0.1% choline chloride)
- Water: tap water ad libitum

Route of administration:

oral: feed

Vehicle:

other: synthetic diet

Duration and frequency of treatment / exposure:

1 week

Dose / conc.:

25 mg/kg bw (total dose)

Dose / conc.:

50 mg/kg bw (total dose)

Dose / conc.:

100 mg/kg bw (total dose)

Dose / conc.:

150 mg/kg bw (total dose)

Dose / conc.:

250 mg/kg bw (total dose)

Dose / conc.:

500 mg/kg bw (total dose)

Dose / conc.:

1 000 mg/kg bw (total dose)

Dose / conc.:

1 250 mg/kg bw (total dose)

Dose / conc.:

1 500 mg/kg bw (total dose)

No. of animals per sex per dose / concentration:

Three to five rats were used for each group.

Control animals:

no

Details on study design:

Various concentrations of test item were orally administered to rats which were given the synthetic diet over a period of 1 week. The urine was collected at 24h intervals and analyzed by TLC.
On the other hand, Human intestinal bacteria were inoculated into 500 ml GAM broth containing 50mg test item, which was then incubated anaerobically at 37ºC for 5 days. 50 ml of the cultured broth was septically taken out at 12, 18, 24, 48, 72, 96 and 120h and extracted with EtOAc. This extract was assayed by TLC and Ames test (S.typhimurium TA98 and TA100).

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled : urine
- Time and frequency of sampling: 24, 48 and 72h after administration of flavonoids.
- From how many animals: (samples pooled or not)
- Method type for identification: TLC)

TREATMENT FOR CLEAVAGE OF CONJUGATES:
Each urine was diluted 2-fold. Half of the urine was adjusted to pH2 with HCI and then extracted with ethylacetate. The other half of the urine was adjusted to 2N-HCI with 5N-HCI and the mixture was refluxed for 2hs at the boiling water bath. The resultant hydrolysate was then extracted with ethylacetate.

ANALYTICAL METHOD
- Complete description: TLC (Developing solvents, CHCI3:MeOH=3 : 1).

Metabolites identified:

yes

Details on metabolites:

- Metabolites and mutagenicity on the urine of treated rats: From the urine of rats administered more than 1250 mg test item/kg body weight of rat, free quercetin was detected but the test item was not. If administered 150-1000 mg test item/kg rat, the free quercetin was not detected but by hydrolyzing the urine with 2N-HCI, the free quercetin was detected. However, in the case of administering less than 100 mg/kg, the hydrolysate of the urine was not detected and phenolic acid-like compounds were only detected.
The urine of rats administered more than 1250 mg/kg and the hydrolysate of the urine of rats adiminstered 150-1000 mg/kg were strongly mutagenic. However, the urine and its hydrolysate of rat treated with 100 mg/kg were not mutagenic.
- Metabolites of test item by human intestinal bacteria and theiir mutagenicity: The amount of quercetin was increased gradually with a corresponding decrease in the level of test item. Quercetin was increased for 12 h and then decreased gradually with a corresponding increase in the level of unidentified compounds. In the mutagenicity test, the revertants per plate of TA 98 were increased until 12 h and decreased thereafter. The sample cultured for 48hs was not mutagenic.
-Isolation and identification of the bacteria which degrade quercetin to phenolic acids: isolated the bacterium fissuring the Bringof quercetin or rutin from human feces was isolated (Q-05 Pediococcus species). Pediococcus Q-05 transformed quercetin to 3,4-dihydroxyphenylacetic acid.

Table 1. Antibacterial activity and mutagenesis of quercetin in  Salmonella/mammalian microsome assay.

Compound	Concentration (mg/ml)	His+ revertant plate		Antibacterial Activity (inhibition 100%)
		TA98	TA100
DMSO		7	35	100
Quercetin	1	106	64	100
	0.5	295	66	98
	0.1	287	60	45
	0.05	179	56	16
	0.01	185	56	0
1-Nitropyrene		318	*

Conclusions:

The test item, at concentrations higher than 100 mg/kg rat, was metabolised to quercetin, which is mutagenic according to Ames test in S. typhimurium TA98. After 12 hours quercetin was transformed to phenolic acids, which are not mutagenic by intestinal bacteria in human intestine.

Executive summary:

A study on relating the metabolism of the test item with mutagenicity was performed in rats. Three to five rats were used for each concentration group of 25, 50, 100, 150, 250, 500, 1000, 1250 or 1500 mg/kg of rat were orally administered. Urine samples were collected at 24, 48 and 72h after administration of the test item and analyzed by TLC. Also, human intestinal bacteria were inoculated into 500 ml GAM broth containing 50mg of test item and assayed according to Ames test. It was observed that Quercetin is mutagenic for TA98. In the case of rats, at  concentrations lower than 100 mg test item/kg, no test item or quercetin is absorbed, while at concentrations higher than 100 mg/kg, the presence of Quercetin (and mutagenicity) increases up to 12 hours, when it starts to  decrease as it is converted to phenolic acids, which are not mutagenic. These results suggest that the test item is metabolised by intestinal bacteria and transformed to the metabolite having novel biologial activity, such as from mutagenic to non-mutagenic by intestinal bacteria in human intestine.

Reference 2

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:

absorption

Qualifier:

no guideline followed

Principles of method if other than guideline:

6 male Sprague-Dawley rats (3 per test group and 3 per control group). To the test groups a single dose of 40.5 µmol/L was administered using a luminal media consisted of a bicarbonate-buffered sodium chloride solution. HPLC-MS was used as the analytical equipment to identify the test item and its conjugates. The distribution of the test item and its conjugates (mainly glucuronide and sulfate) was examined in the luminal effluent the vascular side and in the intestinal tissue.

GLP compliance:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

CD rats

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Age at study initiation: 40 days old
- Weight at study initiation: 170 g
- Diet: Rats were fed a cornstarch-based rutin- and quercetin-free synthetic diet (Altromin C-1000, Altromin International, GmbH) for 7 days. ad libitum
- Water: tap water ad libitum
- Acclimation period: 7 days

Route of administration:

other: Perfusion of small intestine

Vehicle:

other: Luminal media consisting of 135 mmol/L of NaCl, 20 mmol/L of NaHCO3 at pH 7.2

Details on exposure:

The test item was administered via vascular and luminal perfusion in small intestine using a luminal media consisted of a bicarbonate-buffered sodium chloride solution spiked with the test item.

Duration and frequency of treatment / exposure:

single dose. 60 minutes

Dose / conc.:

40.5 other: µmol/L

Remarks:

± 1.8 µmol/L

No. of animals per sex per dose / concentration:

3 animals for the test group and 3 animals for the control group

Control animals:

yes, concurrent vehicle

Details on study design:

Luminal media consisted of 135 mmol/L of NaCl, 20 mmol/L of NaHCO3 at pH 7.2 , and 40.5 ± 1.8 mmol/L of test item (amount applied 1214.0 ± 55.2 nmol), and no test item in the case of controls. Oxygen uptake and acid– base homeostasis were carefully controlled. Glucose, lactate, and pyruvate were determined photometrically by using enzymatic test kits. For glucose the MPR3 glucose/GOD-Perid® test kit, for lactate the MPR3 lactate test kit, and for pyruvate the MPR1 pyruvate test kit were used.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: Vascular (50 mL) and luminal (5 mL) aliquots were obtained, and the entire isolated small intestine was harvested.

METABOLITE CHARACTERISATION STUDIES
-Vascular samples: One milliliter of each vascular sample was centrifuged at 10,000 x g for 7 min. The supernatant was separated and the pellet resuspended with 0.1 mL methanol and centrifuged again at 10,000 x g for 5 min. The combined supernatants were analyzed by HPLC. Rutin showed a recovery from vascular media of 95.7 ± 6.0% (means ± SD).
-Luminal samples: After centrifugation at 700 x g for 20 min, the supernatant was separated. The pellet was extracted with ethanol and centrifuged again at 700 x g for 20 min. The volume of the combined supernatants was defined and the solution was analyzed by HPLC. Test item recovery from luminal media was 99.0 ± 0.7% (means ± SD).
-Small intestine tissue: After lyophilization of the entire small intestine, the tissue was defatted by extracting twice with 10 mL hexane and powdered using a mortar and pestle. The pellet was extracted three times with 4 mL methanol/water (1:1) and centrifuged at 2800 x g for 10 min. The extracts were pooled and the volume adjusted to 25 mL. Test item exhibited a recovery of 94.5 ± 10.2% (means ± SD).After lyophilization of the entire small intestine, the tissue was defatted by extracting twice with 10 mL hexaneand powdered using a mortar and pestle. The pellet was
extracted three times with 4 mL methanol/water (1:1) and centrifuged at 2800 x g for 10 min. The extracts were pooled and the volume adjusted to 25 mL. Test item exhibited a recovery of 94.5 ± 10.2% (means ± SD).
- From how many animals: all of them
- Method type(s) for identification: HPLC-EDC + HPLC-MS
- Limits of detection and quantification: 30 nmol/L and 90 nmol/L, respectively.

TREATMENT FOR CLEAVAGE OF CONJUGATES:
For cleavage of glucuronides, 0.06 mL of a sodium phosphate buffer (0.1 mol/L, pH 6.8) and 0.06 mL glucuronidase solution (210 Fishman units; Escherichia coli, Sigma-Aldrich) were added to 0.5 mL sample solution. Cleavage of sulfate conjugates was performed with 0.05 mL glucuronidase–sulfatase solution (97 units; Helix pomatia, Sigma-Aldrich) and 0.4 mL sample solution in an acetate buffer (0.05 mL, 0.2 mol/L, pH 4.5). Mixtures were incubated for 45 min at 37°C. (Glucuronidase: β-D-glucuronide glucuronosohydrolase, EC 3.2.1.31; sulfatase: aryl-sulfate
sulfohydrolase, EC 3.1.6.1). The applicability of the enzymatic cleavage in cleaned-up fluorocarbon emulsion was confirmed by the conversion of 4-nitrophenol glucuronide and 4-nitrophenol sulfate with β-glucuronidase and β-glucuronidase–sulfatase, respectively. The cleavage of 4-nitrophenol glucuronide resulted in 4-nitrophenol recovery of 96.6 ± 1.8% (means ± SD, N=3), while the cleavage of 4-nitrophenol sulfate resulted in a recovery of 102.0 ± 0.5% (means ± SD, N=3).

ANALYTICAL METHOD
- Complete description: The HPLC system (Sykam) consisted of an S 1100 solvent delivery system, an S 2000 HPLC controller, an S 8110 low-pressure gradient mixer, a Marathon Basic1 autoinjector (Spark) with a 100-µL loop and a Biometra Electrochemical Detector at 600 mV (Biometra). Continuous on-line monitoring and data quantitation was performed with a Chromatopac C-R6A data processor (Shimadzu). Separation was carried out on a Hypersil ODS-2 column (125 x 4.6 mm i.d., 3 µm, Muder & Wochele), with a flow rate of 1.0 mL/min. Isocratic elution of rutin with an elution time of about 6 min was achieved with tetrahydrofuran:water:formic acid (8:82:10) as eluent; quercetin eluted at 6 min with tetrahydrofuran:water:formic acid (20:70:10). An injection volume of 10 mL (30 mL in the case of quercetin) resulted in a detection limit of 30 nmol/L and a quantitation limit of 90 nmol/L, respectively. For the identification of test item and test item conjugates, a clean-up procedure and gradient HPLC system combined with an MS detector in the electrospray ionization mode (ESI-) was used.

Statistics:

Statistical differences of fluxes were determined using ANOVA and subsequent Tukey’s range test for paired observations at a procedure-wise error rate of 5%. Viability parameters were compared using Student’s t-test for unpaired observations. P values less than 0.05 were considered to indicate significant differences.

Details on absorption:

In control perfusion experiments with test item-free basic perfusion media, no flavonoids were detectable. Stability of test item and quercetin in the luminal and vascular perfusate was confirmed for 2 hr at 37°C. The vascular appearance rate of test item increased during perfusion (significantly for the first 30 min, Fig. 1). Approximately 10% of rutin administered appeared at the vascular side, chiefly as free rutin (5.6%) but also as glucuronide (2.0%) and sulfate (2.5%) -see Table 2-. Most of the luminally administered test item left the organ preparation via luminal efflux (Table 2). The main compound
in the luminal effluent was test item, accompanied by minute amounts of glucuronide.
Minute amounts of the test item were located in the intestinal tissue in the form of unchanged rutin (0.9%) and its glucuronide (0.1%) and sulfate conjugates (0.1%) (Table 2). Traces of quercetin and quercetin conjugates at concentrations close to the detection limits were found both at the luminal and vascular sides.

Metabolites identified:

yes

Details on metabolites:

About 44% of the rutin absorbed was conjugated with glucuronic acid (20%) and sulfate (24%), as observed in the vascular side.

Table 1. Viability  parameters (means ± SD, N=3) of control perfusion and test item experiments.

	Control	Test  item
Oxygen consumption (µmol x min^-1 x g^-1)	3.8±0.3	3.8±0.3
Lactate formation (µmol x min^-1 x g^-1)	9.2±5.85	7.0±2.8
Glucose consumption (µmol x min^-1 x g^-1)	9.9±2.5	6.9±1.9
Arterial pressure (mm Hg)	64±7	62±4
Arterial pH	7.5±0.1	7.5±0.0
Venous pH	7.3±0.1	7.3±0.1

 

Table 2. Distribution of rutin and rutin conjugates in the luminal, vascular, and tissue compartments after perfusion experiments of rutin with isolated rat small intestine.

	Luminal effluent		Vascular side		Intestinal side
	nmol	%^a	nmol	%^a	nmol	%^a
Rutin	1046.0±48.6	86.2±1.9	68.9±15.6	5.6±1.0	11.3±4.8	0.9±0.4
Glucuronide	2.5±0.1	0.2±0.0	24.7±6.0	2.0±0.5	0.9±0.4	0.1±0.03
Sulfate	n.d.	n.d	29.8±7.4	2.5±0.7	1.1±0.6	0.1±0.05
Total	1048.5±48.7	86.4±1.9	123.4±10.0	10.1±0.4	13.3±5.5	1.1±0.4

Test item (1214.0 ± 55.2 nmol) was applied in three perfusion experiments of 60 min. Recoveries are given as means 6 SD. Mean recovery over three experiments was 1185.3 ± 62.7 nmol (97.6 ± 2.2%). ND, under the detection limit. 
^a Based on the dosage of 1214.0 nmol.

Conclusions:

About 10% of the administered rutin appeared at the vascular side, chiefly as free rutin (5.6%), but some rutin sulfate (2.5%) and glucuronide (2.0%) were also detected. The conjugates were preferentially absorbed to the vascular side, while only traces of the glucuronide (0.2%) were found in the luminal perfusate. Minute amounts of the rutin administered were located in the intestinal tissue (1.1%) in the form of unchanged rutin and its glucuronide and sulfate conjugates. The obtained results confirm uptake of rutin in the rat small intestine.

Executive summary:

To assess the intestinal absorption and metabolism of the test item, an isolated preparation of a vascularly and luminally perfused rat small intestine was used, characterized by fully maintained tissue viability. 6 male Sprague-Dawley rats (3 per test group and 3 per control group). To the test groups a single dose of 40.5 µmol/L was administered using a luminal media consisted of a bicarbonate-buffered sodium chloride solution. HPLC-MS was used as the analytical equipment to identify the test item and its conjugates. The distribution of the test item and its conjugates (mainly glucuronide and sulfate) was examined in the luminal effluent the vascular side and in the intestinal tissue. It was found that about 10% of the administered test item appeared at the vascular side, chiefly as free test item (5.6%), but some sulfate (2.5%) and glucuronide (2.0%) conjugates were also detected. The conjugates were preferentially absorbed to the vascular side, while only traces of the glucuronide (0.2%) were found in the luminal perfusate. Minute amounts of the test item administered were located in the intestinal tissue (1.1%) in the form of unchanged test item and its glucuronide and sulfate conjugates. The obtained results confirm uptake of rutin in the rat small intestine.

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:

absorption

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

A single dose of 300ml of tomato juice fortified with tetst item (176 µmoles) was orally administered to 11 volunteers, 6 females (1 with an ileostomy) and 5 males (4 with an ileostomy). The concentrations of the test item and its metabolites were determined in plasma at 0, 1, 2, 3, 4, 5, 6, 7, 8 and 24 h post-ingestion and urine and ileal fluid were collected prior to supplementation and over 0–2, 2–5, 5–8 and 8–24 h periods post-ingestion. All of the samples were analysed by HPLC-PDA-MS2.

GLP compliance:

no

Specific details on test material used for the study:

Aliquots of tomato juice were taken for quantitative analysis of their flavonol content. Triplicate samples of 5ml of tomato juice were extracted with 5ml of 50% methanol containing 1% formic acid and 20mM sodium diethyldithiocarbamate. (+)-Catechin was used as an internal standard. This mixture was placed on a basic orbital IKA (KS 130) shaker at 350 rpm for 30 min after which it was centrifuged at 4000g at 4ºC for 10 min. The supernatant was collected and the pellet further extracted twice with 5ml methanol. The three supernatants were combined and reduced to dryness in vacuo. The dried extract was dissolved in 125 µl methanol to which 2375 µl of 1% formic acid was added and 10 µl aliquots were analysed by HPLC–PDA–MS2. The extraction recovery for the internal standard was 94±4.5%.

Radiolabelling:

no

Species:

other: Human

Sex:

male/female

Details on test animals or test system and environmental conditions:

Six healthy human subjects, (five females, one male), and five volunteers with an ileostomy, and hence no colon (four males, one female), participated in this study. They were non-smokers and not on any medication, aged between 22 and 48 years and had a body mass index of 24.5±1. All the ileostomy volunteers had their operation at least 5 years prior to the study and had minimal resection of the small intestine.

Route of administration:

oral: feed

Details on exposure:

The volunteers orally consumed 300 ml of test item fortified tomato juice.

Duration and frequency of treatment / exposure:

single dose

Dose / conc.:

176 other: µmoles

No. of animals per sex per dose / concentration:

6 females (1 with an ileostomy) and 5 males (4 with an ileostomy)

Control animals:

no

Details on study design:

- Subjects were required to follow a diet low in flavonoids and phenolics for two days prior to the study; avoiding most fruits, vegetables and beverages such as tea, coffee, fruit juices and wine. On the morning of the study, the volunteers were asked to consume 300ml of tomato juice. The tomato juice had been fortified with rutin to bring the concentration to a standard 176 µmoles.

- Extraction of flavonoids from plasma: 250 µl plasma was added dropwise to 600 ml of acetonitrile to precipitate the proteins. The mixture was vortexed for 30 s every 2 min over a 10 in period, before centrifuging at 13,000g at 4ºC for 20 min. The supernatant was retained and the pellet re-extracted as described above but with methanol instead of acetonitrile. Experiments with quercetin-3-glucuronide and quercetin-30-sulphate showed recoveries of ca. 75% with the initial acetonitrile extraction which increased by a further 10–12% with the second methanolic extraction. The two supernatants were combined and reduced to dryness in vacuo. Extracts were then dissolved in 25 µl of methanol plus 225 µl of 1% formic acid in water and centrifuged at 16,000g at 4ºC for 2 min prior to the analysis of 250 µl aliquots of the supernatant by HPLC–PDA–MS2.(+)-Catechin was used as an internal standard with 625 ng being added to acetonitrile prior to the addition of the plasma. The extraction recovery for the internal standard from the spiked samples was 85±4.9%.

- Extraction of flavonoids from ileal fluid: Ileal fluid was collected at the specified time points
where all the contents of the pouch were emptied and stored at -20ºC before transfer to -80ºC within 24 h. Prior to extraction, the ileal fluid was defrosted and thoroughly mixed. Triplicate 0.5 g samples were extracted with 3ml of 95% methanol containing 1% formic acid and 20mM sodium diethyldithiocarabamate. 5 µg (+)-catechin was added to the extraction mixture to act as an internal standard. Samples were homogenised for 5 min (Disruptor Genie, Scientific Industries) and then centrifuged at 16,000g for 15min. The supernatant was collected and the pellet rextracted twice as described above. The three supernatants were combined and reduced to dryness in vacuo. Extracts were then made up to 1ml with 50 µl methanol and 950 µl 1% formic acid. Aliquots of the centrifuged supernatant 20–100 µl were analysed by HPLC–PDA–MS2.

- Preparation of urine: Urine samples were defrosted, thoroughly mixed, centrifuged at 16,000g at 4ºC for 2min prior to injection of 200 Urine samples were defrosted, thoroughly mixed, centrifuged at 16,000g at 4ºC for 2min prior to injection of 200 µl aliquots of the supernatant into the HPLC–PDA–MS2 for analysis of flavonoids and phenolic acids.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, metabolism)
- Tissues and body fluids sampled: blood, urine, faeces.
- Time and frequency of sampling: Blood was collected at 0, 1, 2, 3, 4, 5, 6, 7, 8 and 24 h post-ingestion of the tomato juice and urine and ileal fluid were collected prior to supplementation and over 0–2, 2–5, 5–8 and 8–24 h periods after the consumption of rutin-fortified tomato juice.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: plasma, urine, ileal fluid
- From how many animals: all of them
- Method type(s) for identification: HPLC-PDA-MS2

ANALYTICAL METHOD
- Complete description: Samples were analysed on a Surveyor HPLC system comprising of a HPLC pump, PDA detector, scanning from 250 to 700nm and an autosampler cooled to 4ºC. (Thermo Finnigan, San Jose, USA). Separation was carried out using a 250 x 4.6mm I.D. 4 µm Synergi Max-RP column for flavonoids and (Phenomenex, Macclesfield, UK) eluted with a 65min gradient of 5–43% acetonitrile in 1% formic acid at a flow rate of 1ml·min-1 and maintained at 40ºC. Similar conditions were adopted for phenolic acid detection, but being less polar, a 250 x 4.6mm I.D. 4 µm Synergi Hydro-RP column and a 60 min gradient of 2–20% acetonitrile in 0.25% acetic acid was used. After passing through the flow cell of the diode array detector, the column eluate was split and 0.3ml·min-1 was directed to a LCQ DecaXP ion trap mass spectrometer fitted with either an electrospray ionisation (ESI) or an atmospheric pressure chemical ionisation (APCI) interface (Thermo Finnigan). Analysis was carried out using full scan, data dependant MS2 scanning from m/z 100 to 1000. For the ESI interface, the capillary temperature was 250ºC, sheath gas and auxiliary gas were 40 and 5 units, respectively, and the source voltage was 5 kV for negative ionisation. For the APCI interface, the capillary temperature was 130ºC, vaporizer temperature was 350ºC, sheath gas and auxiliary gas were 80 and 60 units, respectively, and the source voltage was 6 kV for negative ionisation. Test item, quercetin-3-glucuronide and isorhamnetin-3-glucuronide were all quantified by reference to standard calibration curves at 365 nm. Other flavonols were quantified in quercetin-3-glucuronide equivalents. 3-Hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid and 4-hydroxy-3-methoxyphenylacetic acid were quantified by reference to standard calibration curves at 280 nm. Other phenolic acids were quantified in 2- hydroxyhippuric acid equivalents. Peak identification was confirmed by co-chromatography and/or MS2 fragmentation data.

Statistics:

Each sample was analysed in triplicate and data were presented as mean values ± standard error (n=3).

Details on absorption:

In the 365 nm traces obtained with plasma at 0, 2 and 3 h samples and with urine at 0, 0–2 h samples no peaks corresponding to any flavonol-based compounds were detected. However, in the 4–8 h plasma samples and the 2–5 h urine samples onwards, small quantities of quercetin-based compounds were detected. A total of nine conjugated quercetin metabolites were detected in urine and two in plasma (quercetin-3-glucuronide and isorhamnetin-3-glucuronide). The plasma and urine from volunteers with an ileostomy contained none of the quercetin metabolites that were present in samples from the subjects with an intact colon. However, ileal fluid collected over a 24 h period was found to contain 151 ± 5 µmoles of unmetabolised test item which corresponds to 86 ± 3% of the amount ingested.

Details on excretion:

There were considerable inter-individual variations in the excretion of the urinary metabolites (see Table 4). 94% of the urinary metabolites of rutin were excreted in the 2–8 h period after ingestion of the test item. Most of the test item was excreted in the ileal fluid 2–5 h post ingestion of tomato juice.

Key result

Test no.:

#1

Toxicokinetic parameters:

Cmax: 12±2 nmol/l (Quercetin-3-glucuronide) and 4.3±1.5 nmol/l (Isorhamnetin-3-glucuronide)

Key result

Test no.:

#1

Toxicokinetic parameters:

Tmax: 4.7±0.3 h (as Quercetin-3-glucuronide) and 5.4±0.2 h (as Isorhamnetin-3-glucuronide)

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 5.7±0.6 nmol/l (Quercetin-3-glucuronide) and 6.9±0.3 nmol/l (Isorhamnetin-3-glucuronide)

Metabolites identified:

yes

Details on metabolites:

A total of nine conjugated quercetin metabolites were detected in urine and two in plasma. The different classes of metabolites that were detected can be summarised as follows: Quercetin monoglucuronides, Quercetin diglucuronides, Isorhamnetin-3-glucuronide, Methylquercetin diglucuronides and Quercetin glucoside glucuronides. The typical HPLC traces identifications based on MS2 spectra are summarised in Table 1. Under strictly controlled dietary conditions, a total of six potential test item catabolites, hydroxyhippuric and hydroxyphenylacetic acids, were detected in the urine of healthy volunteers (see Table 6). These catabolites were excreted in substantial amounts post-supplementation of rutin although the hydroxyphenylacetic acids, in contrast to the hydroxyhippuric acids, accumulated only in the urine of subjects with an intact colon (see Table 7)

Table 1. HPLC–MS² identification of quercetin metabolites detected in plasma and urine from human volunteers with a colon postconsumption of 300 ml tomato juice containing 176 µmoles test item^*.

Peak	tR (min)	Compound	[M-H]^- (m/z)	MS^2 fragments ions	Location
1	21.5	Methylquercetin diglucuronide	667	491([M-H]^-GlcUA), 315([M-H]^-GlcUA-GlcUA)	Urine
2	21.9	Quercetin glucoside glucuronide	639	477([M-H]^–-Glc), 463([M-H]^–-GlcUA), 301([M-H]^–-GlcUA-Glc)	Urine
3	26.8	Quercetin glucoside glucuronide	639	477([M-H]^--Glc), 463([M-H]^--GlcUA), 301([M-H]^--GlcUA-Glc)	Urine
4	29.1	Quercetin glucoside glucuronide	639	477([M-H]^--Glc), 463([M-H]^--GlcUA), 301([M-H]^--Glc-GlcUA)	Urine
5	30.5	Quercetin-3-glucuronide	477	301([M-H]^--Glc-GlcUA)	Urine, plasma
6	31.9	Quercetin diglucuronide	653	477([M-H]^--GlcUA)301([M-H]^--GlcUA-GlcUA)	Urine
7	35.7	Isorhamnetin-3-glucuronide	491	315([M-H]^--GlcUA)	Urine, plasma
8	36.7	Quercetin-4'-glucuronide	477	301([M-H]^--GlcUA)	Urine
9	39.8	Quercetin-3'-glucuronide	477	301([M-H]^--GlcUA)	Urine

^* Peak numbers and HPLC retention times refer to HPLC trace in Figure 1. tR, retention time; [M-H]^-, negatively charged molecular ion; Glc, glucosyl unit; GlcUA, glucuronyl unit.

 

Table 2. Concentration of quercetin metabolites in the plasma of six human subjects 0–24 h after the consumption 300 ml of tomato juice containing 176 µmoles test item^*.

Metabolite (peak number)	Subject	4h	5h	6h	7h	8h
Quercetin-3-glucuronide (5)	1	22±6	14±1	14±1	4.6±0.3	n.d.
	2	24±2	10±1	10±2	2.8±0.7	n.d.
	3	11±0	13±1	7.6±0.5	2.7±0.5	n.d.
	4	5.2±1.5	4.4±0.6	2.0±0.2	n.d.	n.d.
	5	n.d.	3.9±1.0	3.7±0.9	2.9±0.7	n.d.
	6	n.d.	4.6±0.7	5.2±0.4	n.d.	n.d.
	Mean	10±4	8.2±1.8	7.1±1.8	2.2±0.7	n.d.
Isorhamnetin-3-glucuronide (7)	1	4.3±0.1	8.2±0.4	10±0	4.0±0.2	n.d.
	2	5.7±0.7	5.9±0.5	7.7±2.3	2.9±0.2	3.2±0.2
	3	1.2±0.1	3.8±0.3	3.3±0.2	2.7±0.5	2.0±0.2
	4	2.2±0.2	2.9±0.2	1.7±0.2	1.2±0.2	n.d.
	5	1.8±0.1	2.1±0.0	1.2±0.0	0.9±0.1	n.d.
	6	n.d.	n.d.	n.d.	n.d.	n.d.
	Mean	2.5±0.8	3.8±1.2	4.0±1.5	2.0±0.6	0.9±0.6

* Data for the individual subjects are expressed as nmoles/l ± standard error (n=3). Mean values are expressed as nmoles/l ± standard error (n=6). No metabolites are detected in 0, 2, 3 and 24 h plasma samples. n.d., not detected. For MS² data and identification of peaks, refer to Table 1 and Figure 1.

 

Table 3. Pharmacokinetic parameters of quercetin metabolites in the plasma of human volunteers after the consumption 300 ml of tomato juice contaning 176 µmoles test item^*.

Metabolite	Cmax	Tmax	T1/2
Quercetin-3-glucuronide	12 ± 2	4.7 ± 0.3	5.7 ± 0.6
Isorhamnetin-3-glucuronide	4.3 ± 1.5	5.4 ± 0.2	6.9 ± 0.3

*C_max, maximum concentration in plasma expressed in nmoles/l; T_max, time to reach C_max expressed in hours; T_1/2, the elimination half-life of metabolites in hours. Data presented as mean values ± standard error (n=6). n.d., not detected.

 

Table 4. Quantity of metabolites in urine of six healthy human subjects 0–24 h after consumption of tomato juice containing 176 µmoles test item^*.

Subject	Metabolites (peak number)	2-5h	5-8h	8-24h	Total	% of intake
1	Methylquercetin diglucuronide (1)	94±5	35± 4	n.d.	129±9
	Quercetin glucoside glucuronide (2)	216±30	n.d.	n.d.	216±30
	Quercetin glucoside glucuronide (3)	131±26	n.d.	n.d.	131±26
	Quercetin glucoside glucuronide (4)	112±10	9±0	n.d.	121±9
	Quercetin-3-glucuronide (5)	1122±90	70±26	n.d.	1192±104
	Quercetin diglucuronide (6)	147±27	28±4	n.d.	176±23
	Isorhamnetin-3-glucuronide (7)	547±5	140±5	n.d.	686±5
	Quercetin-4'-glucuronide (8)	1198±8	23±3	n.d.	1221±10
	Quercetin-3'-glucuronide (9)	1108±62	n.d.	n.d.	1108±62
	Total metabolites	4675±90	306±30	n.d.	4981±115	2.8%
2	Methylquercetin diglucuronide (1)	6±0	15±2	n.d.	21±2
	Quercetin glucoside glucuronide (2)	11±0	5±1	n.d.	16±1
	Quercetin glucoside glucuronide (3)	2±1	n.d.	n.d.	2±1
	Quercetin-3-glucuronide (5)	71±1	67±2	4±1	142±2
	Quercetin diglucuronide (6)	7±1	12±1	n.d.	18±2
	Isorhamnetin-3-glucuronide (7)	30 ±3	40±1	60±2	129±3
	Quercetin-4'-glucuronide (8)	87±2	35±1	n.d.	122±3
	Quercetin-3'-glucuronide (9)	92±1	46±0	19±1	157±62
	Total metabolites	305±3	219±3	82±1	608±4	0.35%
3	Quercetin-3-glucuronide (5)	81±6	43±2	51±19	175±14
	Isorhamnetin-3-glucuronide (7)	10±0	20±1	34±18	64±2
	Quercetin-4'-glucuronide (8)	18±1	18±1	n.d.	36±0
	Quercetin-3'-glucuronide (9)	10±1	17±1	10±1	37±1
	Total metabolites	118±6	97±1	95±19	310±14	0.18%
4	Quercetin-3-glucuronide (5)	16±1	7±0	n.d.	23±1
	Isorhamnetin-3-glucuronide (7)	21 ±0	16±0	82±1	120±0
	Quercetin-4'-glucuronide (8)	41± 1	n.d.	n.d.	41±1
	Quercetin-3'-glucuronide (9)	4±0	n.d.	n.d.	4±0
	Total metabolites	82±2	23±1	82±1	187±2	0.11%
5	Quercetin-3-glucuronide (5)	6±2	12±2	46±3	64±5
	Isorhamnetin-3-glucuronide (7)	21±0	17±1	27±0	65±0
	Quercetin-4'-glucuronide (8)	1±0	1±0	n.d.	2±0
	Quercetin-3'-glucuronide (9)	2±0	6±0	2±0	10±1
	Total metabolites	30±1	36±2	75±3	141±5	0.07%
6	Quercetin-3-glucuronide (5)	n.d.	3±0	24±1	27±1
	Isorhamnetin-3-glucuronide (7)	4±0	7±0	n.d.	11±0
	Quercetin-3'-glucuronide (9)	n.d.	n.d.	2±0	2±0
	Total metabolites	4±0	10±0	26±1	40±1	0.02%

* Data for the individual subjects are expressed as nmoles ± standard error (n=3). n.d., not detected. For identification of peaks refer to Table 1 and Figure 1.

 

Table 5. Amount of rutin in the ileal fluid of five ileostomy subjects collected 0–24 h after consumption of tomato juice containing 176 µmoles test item^*.

Subject	0-2h	2-5h	5-24h	Total
1	21 ± 1	132 ± 1	4.7 ± 0.0	158 ± 1
2	6 ± 0	109 ± 7	40 ± 1	155 ± 6
3	11 ± 1	118 ± 3	6 ± 1	135 ± 1
4	1 ± 0	100 ± 6	43 ± 0	149 ± 1
5	n.d.	119 ± 2	37 ± 1	156 ± 3
Mean	8 ± 5	115 ± 7	26 ± 11	151 ± 5
% of intake	5 ± 1	65 ± 4	15 ± 7	86 ± 3

* Data for the individual subjects are expressed as mmoles ± standard error (n=3). Mean values are expressed as mmoles ± standard error (n=5). No rutin was detected at 0 h time point. n.d., not detected.

 

Table 6. HPLC–MS² identification of phenolic acids detected in urine samples of volunteers with and without a colon^*.

Peak	tR (min)	Compound	[M-H]^- (m/z)	MS^2 fragment ions (m/z)
1	13.6	4-Hydroxyhippuric acid	194	100, 93
2	15.9	3,4-Dihydroxyphenylacetic acid	167	123, 108
3	26.8	3-Hydroxyphenylacetic acid	151	107, 93, 121
4	29.5	3-Methoxy-4-hydroxyphenylacetic acid	181	137
5	32.2	2-Hydroxyhippuric acid	194	150
6	38.1	3-Hydroxyhippuric acid	194	150

* Peak numbers and HPLC retention times and the peaks refer to HPLC trace in Figure 3; t_R, retention time; [M-H]^-, negatively charged molecular ion.

 

Table 7. Excretion of phenolic acids in the urine of human subjects with and without a colon 0–24 h after consumption of test itemsupplemented tomato juice^*.

Phenolic acids	Colon	0–2 h	2–5 h	5–8 h	8–24 h	Total
2-Hydroxyhippuric acid	w	1.2 ± 0.3	5.3 ± 2.1	2.5 ± 0.7	8.7 ± 2.5	18 ± 5
	w/o	1.7 ± 0.7	1.1 ± 0.6	3.4 ± 1.4	3.9 ± 2.2	10 ± 3
3-Hydroxyhippuric acid	w	0.3 ± 0.1	0.8 ± 0.2	2.0 ± 0.5	2.7 ± 1.2	5.8 ± 1.6
	w/o	4.5 ± 3.6	1.8 ± 1.1	7.6 ± 4.7	6.8 ± 2.1	21 ± 11
4-Hydroxyhippuric acid	w	2.8 ± 0.6	5.8 ± 1.6	18 ± 4	35 ± 4	62 ± 6
	w/o	2.8 ± 0.9	2.9 ± 1.0	6.0 ± 1.1	19 ± 5	31 ± 7
3-Hydroxyphenylacetic acid	w	0.2 ± 0.1	0.4 ± 0.1	1.1 ± 0.3	2.7 ± 0.9	4.4 ± 1.1
	w/o	n.d.	n.d.	n.d.	n.d.	n.d.
3,4-Dihydroxyphenylacetic acid	w	n.d.	0.9 ± 0.6	3.1 ± 0.7	12 ± 3	16 ± 3
	w/o	n.d.	n.d.	n.d.	n.d.	n.d.
3-Methoxy-4-hydroxyphenylacetic acid	w	0.9 ± 0.5	2.4 ± 0.8	6.1 ± 1.4	10 ± 4	19 ±6
	w/o	n.d.	n.d.	n.d.	n.d.	n.d.

* Mean values expressed as mmoles ± standard error (n=6). For MS² data and identification of peaks, refer to Table 6; n.d., not detected. w, with, w/o, without.

Conclusions:

A total of 15 metabolites and catabolites were detected and quantified in plasma and urine following consumption of tomato juice containing 176 µmoles of the test item, which is metabolised and made bioavailable in plasma mainly as quercetin glucuronide and isorhamnetin-3-glucuronide. An additional seven metabolites are biosynthesised in the second metabolism phase and found in urine, and all metabolites were almost completely excreted after 8 hours.

Executive summary:

A study on the absorption and metabolism of the test item in humans was performed. A single dose of 300ml of tomato juice fortified with tetst item (176 µmoles) was orally administered to 11 volunteers, 6 females (1 with an ileostomy) and 5 males (4 with an ileostomy). The concentrations of the test item and its metabolites were determined in plasma at 0, 1, 2, 3, 4, 5, 6, 7, 8 and 24 h post-ingestion and urine and ileal fluid were collected prior to supplementation and over 0–2, 2–5, 5–8 and 8–24 h periods post-ingestion. All of the samples were analysed by HPLC-PDA-MS². Metabolites appeared in plasma after 4 hours of the ingestion of the test item, mainly quercetin glucuronide and isorhamnetin-3-glucuronide, in the first phase. In a second phase, and addtional 7 metabolites are present in urine. Also, 94% of the urinary metabolites of test item were excreted in the 2–8 h period after ingestion of the test item. A total of six potential test item catabolites, hydroxyhippuric and hydroxyphenylacetic acids, were detected in the urine of healthy volunteers and excreted in substantial amounts post-supplementation of test item although the hydroxyphenylacetic acids accumulated only in the urine of subjects with an intact colon, indicating that the colon is the major site of test metabolism and absorption, principally towards catabolic pathways, with the production of conjugated quercetin metabolites being a minor route.

 

Reference 4

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:

toxicokinetics

Qualifier:

no guideline followed

Principles of method if other than guideline:

Three doses  of  16, 40 and 100 mg were administered to each volunteer (12, 7 males and 5 females), who received a capsule belonging to each dose and treatment once. The concentrations of the test item and its metabolites were determined in plasma at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 and 32hours after dosing and analysed by HPLC-EDC.

GLP compliance:

no

Species:

other: Human

Sex:

male/female

Details on test animals or test system and environmental conditions:

The study population consisted on 16 healthy, non-smoking volunteers (7 females and 9 males). Subject characteristics were as follows (mean ± SD):
- age: 22.1±3.5 years
- weight: 65.2± 9.4 kg
- body mass index: 21.7±2.1 kg/m2
The subjects were checked to be in good health and were free of medication, with the exception of four women who used oral contraceptives.

Route of administration:

oral: capsule

Details on exposure:

Two daily meals were provided to the subjects at the study site for 32 hours. The subjects mantained a low quercetin diet 5 days prior to and during both study periods, which was monitored daily. Little deviation from the low quercetin diet occurred.

Duration and frequency of treatment / exposure:

32 hours

Dose / conc.:

16 other: mg

Dose / conc.:

40 other: mg

Dose / conc.:

100 other: mg

No. of animals per sex per dose / concentration:

4 females and 4 males were given the treatments in sequence quercetin aglycone-test item, and 3 females and 5 males received the treatments in the opposite order. In both groups, 2 subjects (one male and one female) discontinued the study to unrelated causes to the study compound.

Control animals:

no

Details on study design:

This was a double blind, diet-controlled, two-period cross-over study conducted to characterise and compare the pharmacokinetics of quercetin and quercetin aglycone and test item.
The study consisted of two study periods, two treatments and three different doses within both treatments. The sequence of treatments was randomised, but within a treatment the study drug was given in ascending dosages (see figure attached).
Within the treatments the wash-out interval between doses 1 and 2 was 2 days, and between doses 2 and 3 it was 3 days. Between periods the wash-out interval was 9 days.
Each volunteer received a capsule belonging to each dose and treatment once. The test drug was taken in the morning after an overnight fast with a glass of water on an empty stomach. A glass of water (200 mL) was given after 2h after taking the study drug. Lunch was served 4 h after drug administration, dinner 8 h and snacks 6h and 10 h after drug administration.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption)
- Tissues and body fluids sampled: blood and plasma.
- Time and frequency of sampling: Blood samples were collected into vacuum tubes containing ethylene diamine tetraacetic acid (EDTA) at 15 mins, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h and 32 h post-dose. A baseline blood sample was taken 20-30 min before administration of the study drug.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: plasma
- From how many animals: all of them
- Method type(s) for identification: HPLC-ECD
- Other (validation of method): Quantitation of the quercetin peak was based on the standard additions method using plasma standards containing 0, 10, 30, 90 of 150 µg/l of added quercetin. The reproducibility of the method was followed with analysis of a pooled plasma sample in triplicate in each run. Day to day variation of the reference was good (8.1%). Day to day variations of the plasma standards 10, 30, 90 and 150 µg/l were also followed and were 19.8, 15.6, 11.2 and 3.0%.

TREATMENT FOR CLEAVAGE OF CONJUGATES:
Quercetin conjugates were hydrolysed by incubating 1 ml EDTA plasma with 110 µl 0.78M sodium acetate buffer (pH 4.8), 100 µl 0.1 M ascorbic acid and 40 µl of a crude preparation from Helix pomatia containing 4000 U β-glucuronidase and 200 U sulfatase activity, for 7 h and 37ºC. After incubation, quercetin was released from protein using solid-phase extraction.

ANALYTICAL METHOD
- Determination of total quercitin in plasma: The sample was diluted with 2 ml phosphate buffer (70 mM, pH 2.4) and added to a Bond Elut C18 solid-phase extraction column, preconditioned with 6 ml methanol and 6 ml phosphate buffer. The column was washed with 9 ml phosphate buffer and 0.5 ml water. Quercetin was eluted into a conical glass tube with 2 ml methanol and dried. For removal of additional interferences,1 ml toluene-dichloromethane (80:20, v/v) and 200 µl 5.3 M acetic acid-32 mM oxalic acid (80:20, v/v, pH 2.4) were added. The tubes were vortexed for 1 min and centrifuged for 15 min at 1000g. The lower phase was used for HPLC analysis. It was performed with a HP 1090 liquid chromatograph (Hewlett-Packard, Palo Alto, calif.) a Coulochem 5100A electrochemical detector with a model 5011 analytical cell (ESA Inc., Chelmsford, Mass.) and a Inertsil ODS-3 analytical HPLC column (250 x4.0 mm I.D., 5 µm, GL Sciences, Tokyo, Japan). The mobile phase consisted of 59% methanol in phophate buffer (70 mM, pH 2.4). The flow rate of the eluent was 1 ml/min and the injection volume was 30 µl. The detector was set to 100 mV.
- Determination of plasma conjugated test item conjugations: Plasma unconjugated test item concentrations were analysed in samples obtained after ingestion of 100 mg test item. In this method the hydrolysing and the liquid-liquid extraction steps used in the analysis of total quercetin were omitted. To 0.5 ml plasma, 55 µl sodium acetate buffer (0.78 M, pH 4.8), 50 µl 0.1 M ascorbic acid and 2 ml phosphate buffer were added. Test itemwas extracted using Bond Elut C18 extraction columns and the dried eluate was dissolved into 100 µl methanol and 100 µl 5.3 M acetic acid-32 mM oxalic acid (80:20, v/v, pH 2.4). Of this, 30 µl was injected into the HPLC. The mobile phase was 45% methanol in phosphate buffer (70 mM, pH 2.4) and the flow rate was 1 ml. The test item standard eluted at 9.7 min. The electrochemical detector was set at +150mV. Quantitaion was based on the standard additions method with plasma standards containing 0, 40, 120 and 360 µg/l added test item. Within-day precision for added test item in plasma for test item concentrations 40, 120, 360 µg/l (n=3 for each concentration) was 7.8, 5.1 and 9.7% respectively. Recoveries of test item at the same concentrations were 106, 98 and 103%, respectively. the detection limit for added test item was 5 µg/l plasma.

Statistics:

The pharmacokinetic parameters were calculated using a validated commercial software package SPIHAR/PC, version 4.0, obtained from SIMED, Créteil, France. The main approach was the intent-to-treat analysis in which all randomised subjects were included in the analyses. A P value less than 0.05 was considered statistically significant, except when testing the carry-over effect when a P value of less than 0.10 was considered as statistically significant.
Baseline comparisons between sequences were made using a t-test for two independent samples. Pharmacokinetic parameters (except tmax) were analysed using variance analysis for cross-over desing where there were also repetitions within the period. The model comprised the following factors: treatment, sequence, dose and period and some of their interactions. Tmax was analysed using the Wilcoxon rank sum test. Single dose comprarisons between treatments were carried out with linear contrasts or pairwise comparisons. In addition, linear trend was fitted to AUC and Cmax in order to analyse dose-response. For the calculation of AUC(0-24), AUC(0-32) and Cmax, the data was ln-transformed. Comparison between genders of the AUC(0-24) means was performed for each dose and treatment separately using the extended Mantel-Haenszels test. Correlation between the AUC(0-24) values after the quercetin aglycone and test item treatments, was evaluated by plotting AUC(0-24) for quercetin aglycone as a function of AUC(0-24) for test item, and calculating the correlation coefficient. This was done separately for each dose.

Details on absorption:

The geometric mean AUC(0-24) of plasma total from the test item increased from 381 µg h/l (16 mg) to 1138 µg h/l (100mg). Those values were slightly superior for the geometric mean AUC(0-32). After ingestion of the test item there was great inter-individual variation in the AUC and Cmax values. It was also observed that the tmax is quite high for the test item, indicating that its absorption is very slow. Additionally, quercetin was absorbed more efficiently from the test item by females than by males and the inter-individual differences were statistically significant at all doses (P=0.021, P=0.003, P=0.008).

Key result

Test no.:

#1

Toxicokinetic parameters:

AUC: AUC(0-24) 381, 636 and 1017 µg h/l at doses 16 mg, 40 mg and 100 mg, respectively

Remarks:

95%CI= 305-478, 530-763 and 702-1473 µg h/l at doses 16 mg, 40 mg and 100 mg, respectively

Key result

Test no.:

#1

Toxicokinetic parameters:

AUC: AUC(0-32) 479, 793 and 1202 µg h/l at doses 16 mg, 40 mg and 100 mg, respectively

Remarks:

95% CI= 383-600, 672-936 and 850-1700 µg h/l at doses 16 mg, 40 mg and 100 mg, respectively

Key result

Test no.:

#1

Toxicokinetic parameters:

Cmax: 23.5, 47.6 and 89.9 µg/l at doses 16 mg, 40 mg and 100 mg, respectively

Remarks:

95% CI= 17.6-31.5, 34.4-65.8, 53.2-152.1 µg/l at doses 16 mg, 40 mg and 100 mg, respectively

Key result

Test no.:

#1

Toxicokinetic parameters:

Tmax: Mean±SD: 6.5±1.8, 7.4±2.2 and 7.5±2.2 hours at doses 16 mg, 40 mg and 100 mg, respectively

Metabolites identified:

yes

Details on metabolites:

Test item is mainly present as quercetin glururonides and/or sulfates and small amounts of free quercetin aglycone.

Table 1. Pharmacokinetic parameters of quercetin from three oral doses of the test item. AUC(_0-24): area under the plasma concentration-time curve from 0-24 h (µg hr/l, geometric means); AUC(_0-32): area under the plasma concentration-time curve from 0-32 h (µg hr/l, geometric means); C_max: maximum plasma concentration (µg/l); t_max:time to reach C_max; 95% CI:95% confidence interval.

Treatment	16 mg	40mg	100mg
AUC(0-24)	381	636	1017
95% CI	305-478	530-763	702-1473
AUC(0-32)	479	793	1202
95% CI	383-600	672-936	850-1700
Cmax	23.5	47.6	89.9
95% CI	17.6-31.5	34.4-65.8	53.2-152.1
tmax (mean±SD)	6.5±1.8	7.4±2.2	7.5±2.2

Conclusions:

The test item was slowly absorbed and it mainly metabolises to quercetin glururonides and/or sulfates. Quercetin was absorbed more efficiently from the test item by females than by males and the inter-individual differences were statistically significant

Executive summary:

A study on the absorption of the test item in humans was performed. Three doses  of  16, 40 and 100 mg were administered to each volunteer (12, 7 males and 5 females), who received a capsule belonging to each dose and treatment once. The concentrations of the test item and its metabolites were determined in plasma at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 and 32 hours after dosing and analysed by HPLC-EDC. The test item was slowly absorbed and it mainly metabolises to quercetin glururonides and/or sulfates. Quercetin was absorbed more efficiently from the test item by females than by males and the inter-individual differences were statistically significant. Based on these results, the test item by itself shows no bioaccumulation potential.

Reference 5

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:

absorption

excretion

Principles of method if other than guideline:

A single dose of 50 µmol/kg was orally administered (by gavage) to 4 male Sprague Dawley rats, and the concentrations of the test item and its metabolites were determined in plasma at 0 (predose), 0.5, 1, 2, 4, 8, 10, 12, 16 and 24 hours after dosing and analysed by HPLC-UV.

GLP compliance:

no

Radiolabelling:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Inc., Atsugi, Japan
- Age at study initiation: 6 weeks old
- Weight at study initiation: 145-176 g
- Housing: air-conditioned room
- Diet: AIN-76A diet (Oriental Yeast Co., Tokyo, Japan) ad libitum
- Water: tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3 °C
- Photoperiod (hrs dark / hrs light): 12 h dark/12 light
- Fasting period: overnight

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Duration and frequency of treatment / exposure:

24h

Dose / conc.:

50 other: µmol/kg

No. of animals per sex per dose / concentration:

4

Control animals:

no

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption and excretion)
- Tissues and body fluids sampled: urine, plasma and blood
- Time and frequency of sampling: Blood collection from one rat was carried out twice. Plasma was obtained by centrifugation. Urine samples were collected for 24 h using metabolic cages, and each excretion volume was measured.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: plasma and urine
- From how many animals: all of them
- Method type(s) for identification: HPLC-UV

TREATMENT FOR CLEAVAGE OF CONJUGATES (if applicable):
Plasma (0.5 mL) and urine were acidified with the same volume of 1 M acetate buffer (pH 4.5) and preincubated for 2 min at 37 °C. Solutions were treated with 5.4 x 102 units/mL beta-glucuronidase and 0.2 x 102 units/mL sulfatase for 20 min at 37 °C, and then 0.5 mL of 0.01 M oxalic acid was added. The mixtures were centrifuged for 5 min at 8000 rpm. Supernatants were applied to a Sep-Pak C18 cartridge. After the cartridge had been washed with 0.01 M oxalic acid and distilled water, the methanol eluate was obtained. The eluate was evaporated to dryness, and the residue was dissolved in 100 µL of methanol.

ANALYTICAL METHOD
- Complete description: HPLC analysis was performed according to the method described previously with some modification. After centrifugation for 2 min at 0 °C at 15000 rpm, the supernatants were analyzed chromatographically by a JASCO HPLC system (PU-1580, CO-1565 and As-1559, Tokyo, Japan) using a 250 x 4.6 mm i.d. Capcell Pak C18-UG120 column (Shiseido, Tokyo, Japan) and UV detection at 372 nm (multiwavelength detector MD-1510, JASCO, Tokyo, Japan). The mobile phase contained the following: solvent A, 10% methanol with 1% acetic acid; solvent B, 70% methanol with 1% acetic acid. Gradient conditions were as follows: A/B ) 100-70/30 for 0-15 min; 70/30-65/35 for 15-20 min; 65/35-50/50 for 20-30 min; 50/50-0, for 30-45 min; 0 for 45-55 min; 0-100 for 55-55.1 min; 100 for 55.1-65 min. The column temperature was maintained at 35 °C, and the flow rate was 1 mL/min. The retention time of tamarixetin was almost the same as that of isorhamnetin under these HPLC conditions. Therefore, quercetin and tamarixetin (as a methylated quercetin) were quantified by measuring the peak areas based on calibration plots of the peak area of standard quercetin and tamarixetin at various concentrations. The recovery with this method using a standard was >95%.

Type:

absorption

Results:

The test item was absorbed slowly and reached the highest plasma levels of 0.85 ± 0.14 nmol/mL at 8 h after dosing.

Type:

excretion

Results:

After 12 hours, the excretion of the test item is complete.

Details on absorption:

The concentrations of quercetin in rat plasma with time after administration of the test item in 0.5% CMC-Na by gastric intubation is shown in Figure 3A. All plasma samples were treated with beta-glucuronidase/sulfatase. The test item was absorbed slowly and reached the highest plasma levels of 0.85 ± 0.14nmol/mL at 8 h after dosing. The plasma level of methylated quercetin (as tamarixetin) showed a change similar to that of quercetin.

Details on excretion:

The mean 24 h urinary excretions of the test item (means ±SE, n=4) was 32.4±14.1 and 50.7 ± 20.2 nmol as quercetin and as tamarixetin, respectively. The area under the concentration time curve (AUC) for the test item is 4.13 nmol/h as quercetin and 1.91 nmol/h as methylated quercetin. The ratio of excretion to ingestion of the test item was quite low (0.38 ± 0.16%).

Key result

Test no.:

#1

Toxicokinetic parameters:

AUC: 4.13 nmol/h in plasma (0-24) as quercetin and 1.91 nmol/h in plasma (0-24) as methylated quercetin

Metabolites identified:

yes

Details on metabolites:

The metabolites found were quercetin and methylated quercetin and were determined in plasma at 0 (predose), 0.5, 1, 2, 4, 8, 10, 12, 16 and 24 hours (see Table 1 and Figure attached)

Table 1. Urinary Excretion and Total Area under the Plasma Concentration−Time Curve of Quercetin and Methylated Quercetin in Rat Administered Quercetin and Its Glycosides

supplement	metabolite	urinary excretion (nmol/24 h urine)	excretion/ingestion (%)	AUC 0 f 24 h (nmol/h)
quercetin	quercetin	32.4 ± 14.1	0.38 ± 0.16	4.13
	methylated quercetin	50.7 ± 20.2	0.6 ± 0.23	1.91
test item	quercetin	59.1 ± 10.3	0.75 ± 0.12	9.97
	methylated quercetin	173.4 ± 40.2	2.2 ± 0.50	9.75
αG-rutin	quercetin	95.3 ± 41.3	1.16 ± 0.52	20.25
	methylated quercetin	248.3 ± 100.8	3.03 ± 1.28	73.3
αG-rutin and test item	quercetin	92.8 ± 28.8	1.2 ± 0.40	25.44
	methylated quercetin	232.2 ± 69.2	2.99 ± 0.97	71.62

Conclusions:

The test item was absorbed slowly and excreted quickly (12h), probably due to its lack of solubility in water.

Executive summary:

A study on the absorption and excretion of the test item in rats was performed. A single dose of 50 µmol/kg was orally administered (by gavage) to 4 male Sprague Dawley rats, and the concentrations of the test item and its metabolites were determined in plasma at 0 (predose), 0.5, 1, 2, 4, 8, 10, 12, 16 and 24 hours after dosing and analysed by HPLC-UV.
The test item was absorbed slowly and reached the highest plasma levels of 0.85 ± 0.14 nmol/mL at 8 h after dosing. The test item was totally excreted after 12 hours. The AUC values for the test item were 4.12 nmol/h in plasma (0-24) as quercetin and 1.91 nmol/h in plasma (0-24) as methylated quercetin, .Based on these results, the test item by itself shows no bioaccumulation potential.

Reference 6

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:

absorption

excretion

Principles of method if other than guideline:

A single dose of 50 µmol/kg was orally administered (by gavage) to 4 male Sprague Dawley rats, and the concentrations of the test item and its metabolites were determined in plasma at 0 (predose), 0.5, 1, 2, 4, 8, 10, 12, 16 and 24 hours after dosing and analysed by HPLC-UV.

GLP compliance:

no

Radiolabelling:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Inc., Atsugi, Japan
- Age at study initiation: 6 weeks old
- Weight at study initiation: 145-176 g
- Housing: air-conditioned room
- Diet: AIN-76A diet (Oriental Yeast Co., Tokyo, Japan) ad libitum
- Water: tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3 °C
- Photoperiod (hrs dark / hrs light): 12 h dark/12 light
- Fasting period: overnight

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Duration and frequency of treatment / exposure:

24h

Dose / conc.:

50 other: µmol/kg

No. of animals per sex per dose / concentration:

4

Control animals:

no

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption and excretion)
- Tissues and body fluids sampled: urine, plasma and blood
- Time and frequency of sampling: Blood collection from one rat was carried out twice. Plasma was obtained by centrifugation. Urine samples were collected for 24 h using metabolic cages, and each excretion volume was measured.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: plasma and urine
- From how many animals: all of them
- Method type(s) for identification: HPLC-UV

TREATMENT FOR CLEAVAGE OF CONJUGATES (if applicable):
Plasma (0.5 mL) and urine were acidified with the same volume of 1 M acetate buffer (pH 4.5) and preincubated for 2 min at 37 °C. Solutions were treated with 5.4 x 102 units/mL beta-glucuronidase and 0.2 x 102 units/mL sulfatase for 20 min at 37 °C, and then 0.5 mL of 0.01 M oxalic acid was added. The mixtures were centrifuged for 5 min at 8000 rpm. Supernatants were applied to a Sep-Pak C18 cartridge. After the cartridge had been washed with 0.01 M oxalic acid and distilled water, the methanol eluate was obtained. The eluate was evaporated to dryness, and the residue was dissolved in 100 µL of methanol.

ANALYTICAL METHOD
- Complete description: HPLC analysis was performed according to the method described previously with some modification. After centrifugation for 2 min at 0 °C at 15000 rpm, the supernatants were analyzed chromatographically by a JASCO HPLC system (PU-1580, CO-1565 and As-1559, Tokyo, Japan) using a 250 x 4.6 mm i.d. Capcell Pak C18-UG120 column (Shiseido, Tokyo, Japan) and UV detection at 372 nm (multiwavelength detector MD-1510, JASCO, Tokyo, Japan). The mobile phase contained the following: solvent A, 10% methanol with 1% acetic acid; solvent B, 70% methanol with 1% acetic acid. Gradient conditions were as follows: A/B ) 100-70/30 for 0-15 min; 70/30-65/35 for 15-20 min; 65/35-50/50 for 20-30 min; 50/50-0, for 30-45 min; 0 for 45-55 min; 0-100 for 55-55.1 min; 100 for 55.1-65 min. The column temperature was maintained at 35 °C, and the flow rate was 1 mL/min. The retention time of tamarixetin was almost the same as that of isorhamnetin under these HPLC conditions. Therefore, quercetin and tamarixetin (as a methylated quercetin) were quantified by measuring the peak areas based on calibration plots of the peak area of standard quercetin and tamarixetin at various concentrations. The recovery with this method using a standard was >95%.

Type:

absorption

Results:

The test item was absorbed slowly and reached the highest plasma levels of 1.30 ± 0.33 nmol/mL and when mixed with αG-Rutin, it was absorbed much faster, 2.07 ± 0.78 nmol/mL, both at 8 h after dosing.

Type:

excretion

Results:

After 24 hours, there is no more presence of the test item, but when is combined with αG-Rutin, the excretion is not complete.

Details on absorption:

The concentrations of quercetin in rat plasma with time after administration of the test item in 0.5% CMC-Na by gastric intubation is shown in Figure 3A. All plasma samples were treated with beta-glucuronidase/sulfatase. The test item was absorbed slowly and reached the highest plasma levels of 1.30 ± 0.33 nmol/mL at 8 h after dosing. The mixture of the test item and αG-rutin showed three peaks of 0.87 ± 0.14, 2.07 ± 0.78, and 2.96 ± 1.80 nmol/mL at 30 min, 8 h, and 12 h after dosing, respectively. The concentration of quercetin in rat plasma was lower in rats administered the test item than in those administered the mixture of the test item and αG-rutin. The plasma level of methylated quercetin (as tamarixetin) showed a change similar to that of quercetin in both groups (test item and mixture test item+αG-rutin).

Details on excretion:

The mean 24 h urinary excretions of the test item and the mixture of test item and αG-rutin
(means ±SE, n=4) were 59.1±10.3 and 92.8 ± 28.8 nmol as quercetin, respectively, and 173.4 ± 40.2, and 232.2 ± 69.2 nmol as tamarixetin, respectively. The area under the concentration time curve (AUC) for the mixture of test item and αG-Rutin is higher than for the test item alone. The ratio of excretion to ingestion αG-rutin is absorbed quicker than the test item. The supply of the test item and αG-rutin in combination resulted in the maintenance of a high plasma concentration of quercetin metabolites.

Key result

Test no.:

#1

Toxicokinetic parameters:

AUC: 9.97 nmol/h in plasma (0-24) as quercetin and 9.75 nmol/h in plasma (0-24) as methylated quercetin

Metabolites identified:

yes

Details on metabolites:

The metabolites found were quercetin and methylated quercetin and were determined in plasma at 0 (predose), 0.5, 1, 2, 4, 8, 10, 12, 16 and 24 hours (see Table 1 and Figure attached)

Table 1. Urinary Excretion and Total Area under the Plasma Concentration−Time Curve of Quercetin and Methylated Quercetin in Rat Administered Quercetin and Its Glycosides

supplement	metabolite	urinary excretion (nmol/24 h urine)	excretion/ingestion (%)	AUC 0 f 24 h (nmol/h)
quercetin	quercetin	32.4 ± 14.1	0.38 ± 0.16	4.13
	methylated quercetin	50.7 ± 20.2	0.6 ± 0.23	1.91
test item	quercetin	59.1 ± 10.3	0.75 ± 0.12	9.97
	methylated quercetin	173.4 ± 40.2	2.2 ± 0.50	9.75
αG-rutin	quercetin	95.3 ± 41.3	1.16 ± 0.52	20.25
	methylated quercetin	248.3 ± 100.8	3.03 ± 1.28	73.3
αG-rutin and test item	quercetin	92.8 ± 28.8	1.2 ± 0.40	25.44
	methylated quercetin	232.2 ± 69.2	2.99 ± 0.97	71.62

Conclusions:

The test item was absorbed slowly and excreted quickly (24h) but when the absorption efficiency increases when mixed with αG- Rutin, due to its higher water solubility.

Executive summary:

A study on the absorption and excretion of the test item in rats was performed. A single dose of 50 µmol/kg was orally administered (by gavage) to 4 male Sprague Dawley rats, and the concentrations of the test item and its metabolites were determined in plasma at 0 (predose), 0.5, 1, 2, 4, 8, 10, 12, 16 and 24 hours after dosing and analysed by HPLC-UV.
The test item was absorbed slowly and reached the highest plasma levels of 1.30 ± 0.33 nmol/mL at 8 h after dosing and when mixed with water-soluble αG-Rutin, it was absorbed much faster, reaching a peak at 2.96 ± 1.80 nmol/mL, at 12 h after dosing. The test item was totally excreted after 24 hours, but in mixture with αG-Rutin, it was still present after that time. The AUC values for the test item were 9.97 nmol/h in plasma (0-24) as quercetin and 9.75 nmol/h in plasma (0-24) as methylated quercetin, but those values were much higher when mixed with αG-Rutin (25.44 and 71.62 nmol/h in plasma).Based on these results, the test item by itself shows no bioaccumulation potential, but it does when mixed with the water-soluble αG-Rutin.

Description of key information

Weight of evidence approach.

In a study on the absorption and excretion of rutin and quercetin in rats, rutin was absorbed slowly and excreted quickly (24h) but when the absorption efficiency increases when mixed with αG- Rutin, due to its higher water solubility. Quercetin was absorbed slowly and excreted quickly (12h), probably due to its lack of solubility in water.

 

In a study on the absorption of rutin in humans, the test item was slowly absorbed, and it mainly metabolises to quercetin glururonides and/or sulfates. Quercetin was absorbed more efficiently from the test item by females than by males and the inter-individual differences were statistically significant.

 

In a study on the absorption and metabolism of rutin in humans, a total of 15 metabolites and catabolites were detected and quantified in plasma and urine following consumption of tomato juice containing 176 µmoles of rutin, which is metabolised and made bioavailable in plasma mainly as quercetin glucuronide and isorhamnetin-3-glucuronide. An additional seven metabolites are biosynthesised in the second metabolism phase and found in urine. All metabolites were almost completely excreted after 8 hours.

 

In another absorption study, uptake of rutin in the rat small intestine was confirmed. About 10% of the administered rutin appeared at the vascular side, chiefly as free rutin (5.6%), but some rutin sulfate (2.5%) and glucuronide (2.0%) were also detected. The conjugates were preferentially absorbed to the vascular side, while only traces of the glucuronide (0.2%) were found in the luminal perfusate. Minute amounts of the rutin administered were located in the intestinal tissue (1.1%) in the form of unchanged rutin and its glucuronide and sulfate conjugates.

 

In a study on the metabolism of rutin, the test item, at concentrations higher than 100 mg/kg rat, was metabolised to quercetin, which is mutagenic according to Ames test in S. typhimurium TA98. After 12 hours, quercetin was transformed to phenolic acids, which are not mutagenic by intestinal bacteria in human intestine.

 

Rutoside, isoquercetin and kaempferol-3-O-rutinoside, three of the main components of the substance (91% of total content) are flavonol glycosides. Quercetin, the fourth main component, is a flavonol and it is the aglycone form of the two main components, rutoside and isoquercetin (90% of total content).

Glycosides, when ingested, are hydrolysed by the intestinal microflora, yielding the corresponding aglycones. Thus, absorption of these substances in significant amount will only occur via their hydrolysed derivatives (i.e., quercetin for the two main components).

Aglycones are absorbed mainly in the bacterially colonized segments of the gastrointestinal tract and are partly conjugated with glucuronic acid and/or sulphate and partly further metabolized by bacterial ring cleavage (of the flavonoids C-ring). Then, the glucuronates, sulphates and bacterial degradation products (phenols and phenolic compounds) are all excreted via the bile and urine (EFSA Journal 2010; 8(9):1065).

As the metabolic pathway is very similar, all these substances are expected to have similar toxicological properties.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential