Europ.J.clin.Pharmacol. 9, 229-234 (1975) © by Springer-Verlag 1975
Disposition of Quercetin in Man after Single Oral and Intravenous Doses* R. Gugler, M. Leschik and H. J. Dengler
Departments Germany
of Medicine,
Received: January
29,
Universities
of Giessen
1975, and in revised
and Bonn,
form: May
12,
Federal
Republic
1975, accepted: May
of
15,
1975
Summary. The p h a r m a c o k i n e t i c s of quercetin, a flavonoid, have been studied in 6 v o l u n t e e r s after single intravenous (1OO mg) and oral (4 g) doses. The data after iv a d m i n i s t r a t i o n were a n a l y z e d a c c o r d i n g to a two c o m p a r t m e n t open model w i t h half lives of 8.8 ± 1.2 min for the ~ p h a s e and 2.4 ± 0.2 h for the 8 p h a s e (predominant half life), respectively. Protein b i n d i n g was >98%. The apparent volume of d i s t r i b u t i o n was small at 0.34 ± 0.03 I/kg. Of the intravenous dose 7.4 ± 1.2% was e x c r e t e d in urine as a c o n j u g a t e d metabolite, and 0.65 ± O.1% was e x c r e t e d unchanged. After oral a d m i n i s t r a t i o n no m e a s u r a b l e plasma c o n c e n t r a t i o n s could be detected, nor was any q u e r c e t i n found in urine, either u n c h a n g e d or in a m e t a b o l i z e d form. These results exclude a b s o r p t i o n of more than I% of u n c h a n g e d drug. Recovery in faeces after the oral dose was 53 ± 5%, which suggests e x t e n s i v e d e g r a d a t i o n by m i c r o o r g a n i s m s in the gut. The data obtained show that oral a d m i n i s t r a t i o n of flavonoids may be of q u e s t i o n a b l e value. Key words: Quercetin, metabolism, man.
flavonoids,
pharmacokinetics,
Between 600 and 800 flavonoids have been isolated (I) and numerous investigations have been carried out to show their clinical effectiveness, e s p e c i a l l y in the treatment of p e r i p h e r a l v a s c u l a r diseases. A recent report of a s y m p o s i u m on flavonoids (2) stressed their various effects on blood vessels and connective tissue. E x c e l l e n t studies by Griffiths, Das and their co-workers (3-8) have d e s c r i b e d the m e t a b o l i s m of radio-labelled Catechin, apigenin, m y r i c e t i n and flavanone in the rat, guinea pig and man. A l t h o u g h Das (8) d e m o n s t r a t e d that after oral a d m i n i s t r a t i o n of catechin, as much as 7.5% appeared u n c h a n g e d in urine, the b i o a v a i l a b i l i t y of flavonoids has been q u e s t i o n e d by some investigators (9-11),in p a r t i c u l a r of quercetin, rutin and h y d r o x y - e t h y l r u t o s i d e s . All the results available now have been based on urine data, and d e t e c t a b l e con-
absorption,
disposition,
centrations of any flavonoid in human plasma have never been reported. The p r e s e n t study was d e s i g n e d to investigate the p h a r m a c o k i n e t i c s of quereetin, a key flavonoid, in man after intravenous and oral administration, using a specific and highly sensitive assay m e t h o d recently d e v e l o p e d in this laboratory (12).
MATERIALS
AND M E T H O D S
1. Experimental Design
6 volunteers (4 male, 2 female) participated in the study after giving informed consent. They were b e t w e e n 21 and 32 years of age and were c o n s i d e r e d healthy from the results of a history, physical e x a m i n a t i o n and routine laboratory tests. A. Intravenous Administration. Q u e r c e t i n
* Supported by grant Gu 86/3 from the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, Germany (FRG).
100 mg was a d m i n i s t e r e d i n t r a v e n o u s l y by an infusion pump to 6 subjects over a period of 5 minutes. Blood pressure
230
°
OH
OH
O
Fig. I. Structtlral for~lula of quercetin
and heart rate were m o n i t o r e d during the infusion p e r i o d and for I hour thereafter. Any drug r e m a i n i n g in the ampoule and the i n f u s i o n s y s t e m was w a s h e d out and the a m o u n t d e t e r m i n e d for subtraction from the total dose. Blood samples 12 ml were o b t a i n e d before and 5, 10, 20, 40, 80, 160, 320 and 540 min after the infusion. Urine was c o l l e c t e d in fractions of 0-80, 80-160, 160-320, 320540 min and 540 min to 24 h. A f t e r fasting overnight, on separate o c c a s i o n s 4 of the 6 v o l u n t e e r s r e c e i v e d single doses of q u e r c e t i n 4 g (gelatin capsules each of 500 mg quercetin), together with 250 ml of water. Blood was taken 20, 40, 80, 160, 320, 540 min and 24 h after ingestion and urine c o l l e c t e d as d e s c r i b e d for intravenous a d m i n i s t r a t i o n . In addition, faeces were c o l l e c t e d over 72 hours and, f o l l o w i n g 500 or 1OOO fold d i l u t i o n of an aliquot, assayed for q u e r c e t i n by the same p r o c e d u r e as used for p l a s m a or urine. B. Oral Administration.
2. Chemical Methods
T e t r a p h e n y l d i b o r o x i d e was a gift from Gebr. Schwabe, Karlsruhe, Germany. Q u e r c e t i n was also kindly supplied by Gebr. Schwabe as ampoules c o n t a i n i n g 1OO mg of the c o m p o u n d in pure ethanol, to be mixed w i t h 20 ml of an alkaline (pH 8.5) aqueous solution no more than I h o u r b e f o r e use. Q u e r c e t i n crystals were p u r c h a s e d from Serva, Heidelberg, Germany, and p a c k e d into g e l a t i n capsules. Both the iv and the oral p r e p a r a t i o n were tested for purity by their fluorescence spectra (12) and thin layer c h r o m a t o g r a p h y (toluene/formic acid/ formic acid ethyl ester, 50:10:40); and the content of the ampoules was checked by the m e t h o d e m p l o y e d in the study
(12). A. Quercetin. Q u e r c e t i n c o n c e n t r a t i o n s were m e a s u r e d according to a r e c e n t l y published fluorimetric method which allows d e t e r m i n a t i o n of 0.1 ~g/ml of the compound in b i o l o g i c a l fluids (12). In brief, to 2 ml of p l a s m a or urine 1.5 ml
of a I M s o d i u m citrate b u f f e r pH 2.0 were added, and the compound e x t r a c t e d into 8 ml of w a t e r - s a t u r a t e d m e t h y l ethylketone. 6 ml of the organic solvent were e v a p o r a t e d to near dryness, the residue d i s s o l v e d in 0.5 ml of acetone and r e a c t e d with 0.6 mg of t e t r a p h e n y l diboroxide. The c o m p l e x formed was adjusted to pH 3.0 w i t h 2 ml of a 0.2 M s o d i u m citrate buffer and e x t r a c t e d into 6 ml of d i i s o p r o p y l e t h e r . An aliquot of the organic layer was used for f l u o r i m e t r i c m e a s u r e m e n t at e x c i t a t i o n and emission w a v e l e n g t h s of 445 and 500 nm, respectively. I d e n t i f i c a t i o n of c o n j u g a t e d q u e r c e t i n was a c h i e v e d in the f o l l o w i n g ways: 1. A 5 ml aliquot of urine (Subject 2, 0-80 min) was adjusted to pH 5.3 w i t h an equal volume of a 0.2 M a c e t a t e buffer and incubated for 12 hours with 0.2 ml of a ~ - g l u c u r o n i d a s e - a r y l s u l p h a tase solution (Boehringer, Mannheim, Germany) at 37oc in a m e t a b o l i c shaker. A s s a y i n g the same urine before and after i n c u b a t i o n gave the amount of free and total quercetin, respectively. The amount of the conjugate was o b t a i n e d by s u b t r a c t i n g the free q u e r c e t i n from the total q u e r c e t i n formed after hydrolysis. A blank urine incubated in the same way did not reveal any f l u o r e s c e n c e interfering with quercetin. The time course of the h y d r o l y s i s showed that the r e a c t i o n was c o m p l e t e d after 10 hours. Q u e r c e t i n added to a blank urine was not d e g r a d e d after 10 hours. Acid hydrolysis in b o i l i n g water for 30 m i n u t e s gave the same results, but this m e t h o d was a b a n d o n e d because of the high blank fluorescence, w h i c h inh i b i t e d e s t i m a t i o n of low q u e r c e t i n concentrations. Urine 2 ml from Subject 2's 0-80 m i n c o l l e c t i o n period, and 2 ml of hydrolyzed urine from the same sample were each d i l u t e d with 18 ml of d i s t i l l e d w a t e r and placed on 10 x 60 mm glass columns p a c k e d with p o l y a m i d e 80-100 mesh (Serva, Heidelberg, Germany). E l u t i o n of q u e r c e t i n and its c o n j u g a t e was performed with m e t h a n o l / 1 N acetic acid (20:1), the eluate e v a p o r a t e d to near dryness, the residue applied to a silica gel thin layer plate and d e v e l o p e d in the system t o l u e n e / f o r m i c a c i d / f o r m i c acid ethyl ester (50:10:40). In a d d i t i o n to quercetin, w h i c h showed an Rf of 0.45, in the u n h y d r o l y z e d urine sample large amounts of a compound w i t h the colour c h a r a c t e r i s t i c s of q u e r c e t i n remained at the origin. When the urine had been hydroiyzed, the o r i g i n was free of this compound. Q u e r e e t i n from e i t h e r sample
B. Quercetin Conjugate.
231
was subsequently eluted from the plate with acidified methanol and analyzed fluorimetrically as d e s c r i b e d u n d e r 'Methods'. The results were identical with those obtained by the original e x t r a c t i o n m e t h o d a n d e x h i b i t e d a 10 t i m e s h i g h e r c o n c e n t r a t i o n of q u e r c e t i n in t h e h y d r o l y z e d t h a n the u n h y d r o l y z e d urine. 3. Protein B i n d i n g
Protein binding was determined by gel chromatography on S e p h a d e x G 25 a c c o r d i n g to the p r o c e d u r e o f K r i e g l s t e i n and Kuschinsky (13). A f r e s h p o o l e d plasma solution from healthy volunteers w i t h a q u e r c e t i n c o n c e n t r a t i o n of 8 ~g/ m l w a s used.
Table i. Plasma concentrations (pg/ml) of quereetin following intravenous administration of i00 mg Subject
5
iO
20
Time 40
(min) 80
1
3.3
2.5
1.8
1.2
0.94
2
3.0
1.2
0.9
0.5
3
2.1
1.5
0.8
NDa
4
5.1
3,1
2,1
160
320
540
0.64
-
0.22
0.15
0.08 -
0.54
0.36
-
1.3
0.64
0.35
0.09 -
5
5.0
1.8
1,2
0.6
0.47
0.36
0.22 0.ii
6
3.8
2.0
1,0
0.4
0.21
0.15
0.08 -
Mean 3.7
2.0
1.3
0.8
0.50
0.33
O.12 0.II
SE 0.4 0.3 0.2 a = not determined
0.2
0.iO
0.07
0.03 -
lo-
RESULTS Administration route was well
of q u e r c e t i n tolerated by
by either all subjects.
~
3
E i,
i. Plasma Levels
Individual plasma levels after intrav e n o u s i n j e c t i o n of 1OO m g o f q u e r c e t i n a r e s h o w n in T a b l e I; a n d the t y p i c a l t i m e c o u r s e o f t h e p l a s m a l e v e l in t w o subjects plotted on a semilogarithmic s c a l e is s h o w n in Fig. 2. In a l l s u b j e c t s t h e r e w a s a r a p i d d e c l i n e of t h e first slope over 20-40 min, followed by a second and less rapidly declining s l o p e , w h i c h p r o v e d to be l i n e a r at l e a s t o v e r 120 m i n o r 3 t i m e p o i n t s of measurement, u n t i l t h e l o w e r l i m i t of s e n s i t i v i t y of t h e m e t h o d w a s r e a c h e d . Every plasma level curve could be f o l l o w e d a t l e a s t f o r 160 m i n , in t h r e e v o l u n t e e r s f o r 3 2 0 a n d in o n e f o r 5 4 0 min. T h e p l a s m a c o n c e n t r a t i o n 10 m i n a f t e r i n j e c t i o n w a s 3 . 6 9 ± 0 . 4 7 pg/ m l (mean ± S . E . ) , a n d a f t e r 40 m i n 0 . 7 6 ± 0 . 2 0 pg/ml. Following oral administration of q u e r c e t i n 4 g to 4 s u b j e c t s (50 to 65 m g per kg with individual body weights betw e e n 61 a n d 8 0 kg) n o m e a s u r a b l e p l a s m a c o n c e n t r a t i o n c o u l d be f o u n d at a n y time. T h i s i m p l i e d that, if a n y q u e r c e t i n at a l l w e r e p r e s e n t in p l a s m a , the c o n c e n t r a t i o n w a s b e l o w t h e s e n s i t i v i t y of the m e t h o d , w h i c h w a s 0 . 1 ~ g / m l .
c
i 03 ¸
a--
0.03 1
J ........
°°~0 40
160
I
320 Time (min)
............
I
5&O
Fig. 2. Log quercetin plasma concentration versus time in subjects 5 (= --e) and 6 ( ~ ) following intravenous administration of iOO mg
6 o 73
5¸
$4
Disposition of Quercetin in Man after Single Oral and Intravenous Doses* R. Gugler, M. Leschik and H. J. Dengler
Departments Germany
of Medicine,
Received: January
29,
Universities
of Giessen
1975, and in revised
and Bonn,
form: May
12,
Federal
Republic
1975, accepted: May
of
15,
1975
Summary. The p h a r m a c o k i n e t i c s of quercetin, a flavonoid, have been studied in 6 v o l u n t e e r s after single intravenous (1OO mg) and oral (4 g) doses. The data after iv a d m i n i s t r a t i o n were a n a l y z e d a c c o r d i n g to a two c o m p a r t m e n t open model w i t h half lives of 8.8 ± 1.2 min for the ~ p h a s e and 2.4 ± 0.2 h for the 8 p h a s e (predominant half life), respectively. Protein b i n d i n g was >98%. The apparent volume of d i s t r i b u t i o n was small at 0.34 ± 0.03 I/kg. Of the intravenous dose 7.4 ± 1.2% was e x c r e t e d in urine as a c o n j u g a t e d metabolite, and 0.65 ± O.1% was e x c r e t e d unchanged. After oral a d m i n i s t r a t i o n no m e a s u r a b l e plasma c o n c e n t r a t i o n s could be detected, nor was any q u e r c e t i n found in urine, either u n c h a n g e d or in a m e t a b o l i z e d form. These results exclude a b s o r p t i o n of more than I% of u n c h a n g e d drug. Recovery in faeces after the oral dose was 53 ± 5%, which suggests e x t e n s i v e d e g r a d a t i o n by m i c r o o r g a n i s m s in the gut. The data obtained show that oral a d m i n i s t r a t i o n of flavonoids may be of q u e s t i o n a b l e value. Key words: Quercetin, metabolism, man.
flavonoids,
pharmacokinetics,
Between 600 and 800 flavonoids have been isolated (I) and numerous investigations have been carried out to show their clinical effectiveness, e s p e c i a l l y in the treatment of p e r i p h e r a l v a s c u l a r diseases. A recent report of a s y m p o s i u m on flavonoids (2) stressed their various effects on blood vessels and connective tissue. E x c e l l e n t studies by Griffiths, Das and their co-workers (3-8) have d e s c r i b e d the m e t a b o l i s m of radio-labelled Catechin, apigenin, m y r i c e t i n and flavanone in the rat, guinea pig and man. A l t h o u g h Das (8) d e m o n s t r a t e d that after oral a d m i n i s t r a t i o n of catechin, as much as 7.5% appeared u n c h a n g e d in urine, the b i o a v a i l a b i l i t y of flavonoids has been q u e s t i o n e d by some investigators (9-11),in p a r t i c u l a r of quercetin, rutin and h y d r o x y - e t h y l r u t o s i d e s . All the results available now have been based on urine data, and d e t e c t a b l e con-
absorption,
disposition,
centrations of any flavonoid in human plasma have never been reported. The p r e s e n t study was d e s i g n e d to investigate the p h a r m a c o k i n e t i c s of quereetin, a key flavonoid, in man after intravenous and oral administration, using a specific and highly sensitive assay m e t h o d recently d e v e l o p e d in this laboratory (12).
MATERIALS
AND M E T H O D S
1. Experimental Design
6 volunteers (4 male, 2 female) participated in the study after giving informed consent. They were b e t w e e n 21 and 32 years of age and were c o n s i d e r e d healthy from the results of a history, physical e x a m i n a t i o n and routine laboratory tests. A. Intravenous Administration. Q u e r c e t i n
* Supported by grant Gu 86/3 from the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, Germany (FRG).
100 mg was a d m i n i s t e r e d i n t r a v e n o u s l y by an infusion pump to 6 subjects over a period of 5 minutes. Blood pressure
230
°
OH
OH
O
Fig. I. Structtlral for~lula of quercetin
and heart rate were m o n i t o r e d during the infusion p e r i o d and for I hour thereafter. Any drug r e m a i n i n g in the ampoule and the i n f u s i o n s y s t e m was w a s h e d out and the a m o u n t d e t e r m i n e d for subtraction from the total dose. Blood samples 12 ml were o b t a i n e d before and 5, 10, 20, 40, 80, 160, 320 and 540 min after the infusion. Urine was c o l l e c t e d in fractions of 0-80, 80-160, 160-320, 320540 min and 540 min to 24 h. A f t e r fasting overnight, on separate o c c a s i o n s 4 of the 6 v o l u n t e e r s r e c e i v e d single doses of q u e r c e t i n 4 g (gelatin capsules each of 500 mg quercetin), together with 250 ml of water. Blood was taken 20, 40, 80, 160, 320, 540 min and 24 h after ingestion and urine c o l l e c t e d as d e s c r i b e d for intravenous a d m i n i s t r a t i o n . In addition, faeces were c o l l e c t e d over 72 hours and, f o l l o w i n g 500 or 1OOO fold d i l u t i o n of an aliquot, assayed for q u e r c e t i n by the same p r o c e d u r e as used for p l a s m a or urine. B. Oral Administration.
2. Chemical Methods
T e t r a p h e n y l d i b o r o x i d e was a gift from Gebr. Schwabe, Karlsruhe, Germany. Q u e r c e t i n was also kindly supplied by Gebr. Schwabe as ampoules c o n t a i n i n g 1OO mg of the c o m p o u n d in pure ethanol, to be mixed w i t h 20 ml of an alkaline (pH 8.5) aqueous solution no more than I h o u r b e f o r e use. Q u e r c e t i n crystals were p u r c h a s e d from Serva, Heidelberg, Germany, and p a c k e d into g e l a t i n capsules. Both the iv and the oral p r e p a r a t i o n were tested for purity by their fluorescence spectra (12) and thin layer c h r o m a t o g r a p h y (toluene/formic acid/ formic acid ethyl ester, 50:10:40); and the content of the ampoules was checked by the m e t h o d e m p l o y e d in the study
(12). A. Quercetin. Q u e r c e t i n c o n c e n t r a t i o n s were m e a s u r e d according to a r e c e n t l y published fluorimetric method which allows d e t e r m i n a t i o n of 0.1 ~g/ml of the compound in b i o l o g i c a l fluids (12). In brief, to 2 ml of p l a s m a or urine 1.5 ml
of a I M s o d i u m citrate b u f f e r pH 2.0 were added, and the compound e x t r a c t e d into 8 ml of w a t e r - s a t u r a t e d m e t h y l ethylketone. 6 ml of the organic solvent were e v a p o r a t e d to near dryness, the residue d i s s o l v e d in 0.5 ml of acetone and r e a c t e d with 0.6 mg of t e t r a p h e n y l diboroxide. The c o m p l e x formed was adjusted to pH 3.0 w i t h 2 ml of a 0.2 M s o d i u m citrate buffer and e x t r a c t e d into 6 ml of d i i s o p r o p y l e t h e r . An aliquot of the organic layer was used for f l u o r i m e t r i c m e a s u r e m e n t at e x c i t a t i o n and emission w a v e l e n g t h s of 445 and 500 nm, respectively. I d e n t i f i c a t i o n of c o n j u g a t e d q u e r c e t i n was a c h i e v e d in the f o l l o w i n g ways: 1. A 5 ml aliquot of urine (Subject 2, 0-80 min) was adjusted to pH 5.3 w i t h an equal volume of a 0.2 M a c e t a t e buffer and incubated for 12 hours with 0.2 ml of a ~ - g l u c u r o n i d a s e - a r y l s u l p h a tase solution (Boehringer, Mannheim, Germany) at 37oc in a m e t a b o l i c shaker. A s s a y i n g the same urine before and after i n c u b a t i o n gave the amount of free and total quercetin, respectively. The amount of the conjugate was o b t a i n e d by s u b t r a c t i n g the free q u e r c e t i n from the total q u e r c e t i n formed after hydrolysis. A blank urine incubated in the same way did not reveal any f l u o r e s c e n c e interfering with quercetin. The time course of the h y d r o l y s i s showed that the r e a c t i o n was c o m p l e t e d after 10 hours. Q u e r c e t i n added to a blank urine was not d e g r a d e d after 10 hours. Acid hydrolysis in b o i l i n g water for 30 m i n u t e s gave the same results, but this m e t h o d was a b a n d o n e d because of the high blank fluorescence, w h i c h inh i b i t e d e s t i m a t i o n of low q u e r c e t i n concentrations. Urine 2 ml from Subject 2's 0-80 m i n c o l l e c t i o n period, and 2 ml of hydrolyzed urine from the same sample were each d i l u t e d with 18 ml of d i s t i l l e d w a t e r and placed on 10 x 60 mm glass columns p a c k e d with p o l y a m i d e 80-100 mesh (Serva, Heidelberg, Germany). E l u t i o n of q u e r c e t i n and its c o n j u g a t e was performed with m e t h a n o l / 1 N acetic acid (20:1), the eluate e v a p o r a t e d to near dryness, the residue applied to a silica gel thin layer plate and d e v e l o p e d in the system t o l u e n e / f o r m i c a c i d / f o r m i c acid ethyl ester (50:10:40). In a d d i t i o n to quercetin, w h i c h showed an Rf of 0.45, in the u n h y d r o l y z e d urine sample large amounts of a compound w i t h the colour c h a r a c t e r i s t i c s of q u e r c e t i n remained at the origin. When the urine had been hydroiyzed, the o r i g i n was free of this compound. Q u e r e e t i n from e i t h e r sample
B. Quercetin Conjugate.
231
was subsequently eluted from the plate with acidified methanol and analyzed fluorimetrically as d e s c r i b e d u n d e r 'Methods'. The results were identical with those obtained by the original e x t r a c t i o n m e t h o d a n d e x h i b i t e d a 10 t i m e s h i g h e r c o n c e n t r a t i o n of q u e r c e t i n in t h e h y d r o l y z e d t h a n the u n h y d r o l y z e d urine. 3. Protein B i n d i n g
Protein binding was determined by gel chromatography on S e p h a d e x G 25 a c c o r d i n g to the p r o c e d u r e o f K r i e g l s t e i n and Kuschinsky (13). A f r e s h p o o l e d plasma solution from healthy volunteers w i t h a q u e r c e t i n c o n c e n t r a t i o n of 8 ~g/ m l w a s used.
Table i. Plasma concentrations (pg/ml) of quereetin following intravenous administration of i00 mg Subject
5
iO
20
Time 40
(min) 80
1
3.3
2.5
1.8
1.2
0.94
2
3.0
1.2
0.9
0.5
3
2.1
1.5
0.8
NDa
4
5.1
3,1
2,1
160
320
540
0.64
-
0.22
0.15
0.08 -
0.54
0.36
-
1.3
0.64
0.35
0.09 -
5
5.0
1.8
1,2
0.6
0.47
0.36
0.22 0.ii
6
3.8
2.0
1,0
0.4
0.21
0.15
0.08 -
Mean 3.7
2.0
1.3
0.8
0.50
0.33
O.12 0.II
SE 0.4 0.3 0.2 a = not determined
0.2
0.iO
0.07
0.03 -
lo-
RESULTS Administration route was well
of q u e r c e t i n tolerated by
by either all subjects.
~
3
E i,
i. Plasma Levels
Individual plasma levels after intrav e n o u s i n j e c t i o n of 1OO m g o f q u e r c e t i n a r e s h o w n in T a b l e I; a n d the t y p i c a l t i m e c o u r s e o f t h e p l a s m a l e v e l in t w o subjects plotted on a semilogarithmic s c a l e is s h o w n in Fig. 2. In a l l s u b j e c t s t h e r e w a s a r a p i d d e c l i n e of t h e first slope over 20-40 min, followed by a second and less rapidly declining s l o p e , w h i c h p r o v e d to be l i n e a r at l e a s t o v e r 120 m i n o r 3 t i m e p o i n t s of measurement, u n t i l t h e l o w e r l i m i t of s e n s i t i v i t y of t h e m e t h o d w a s r e a c h e d . Every plasma level curve could be f o l l o w e d a t l e a s t f o r 160 m i n , in t h r e e v o l u n t e e r s f o r 3 2 0 a n d in o n e f o r 5 4 0 min. T h e p l a s m a c o n c e n t r a t i o n 10 m i n a f t e r i n j e c t i o n w a s 3 . 6 9 ± 0 . 4 7 pg/ m l (mean ± S . E . ) , a n d a f t e r 40 m i n 0 . 7 6 ± 0 . 2 0 pg/ml. Following oral administration of q u e r c e t i n 4 g to 4 s u b j e c t s (50 to 65 m g per kg with individual body weights betw e e n 61 a n d 8 0 kg) n o m e a s u r a b l e p l a s m a c o n c e n t r a t i o n c o u l d be f o u n d at a n y time. T h i s i m p l i e d that, if a n y q u e r c e t i n at a l l w e r e p r e s e n t in p l a s m a , the c o n c e n t r a t i o n w a s b e l o w t h e s e n s i t i v i t y of the m e t h o d , w h i c h w a s 0 . 1 ~ g / m l .
c
i 03 ¸
a--
0.03 1
J ........
°°~0 40
160
I
320 Time (min)
............
I
5&O
Fig. 2. Log quercetin plasma concentration versus time in subjects 5 (= --e) and 6 ( ~ ) following intravenous administration of iOO mg
6 o 73
5¸
$4
