EUROPEAN JOURNAL OF DRUG METABOLISM AND PHARMACOKINETICS, 1983, Vol. 8, No 1, p.

77-84
Identification of the major urinary metabolites of (+)-catechin
and 3-0-methyl-(+)-catechin in man
M. WERMEILLE*, E. TURIN* and L.A. GRIFFITHS**
* Research and Development Department, Zyma SA, Nyon. Switzerland
** Department of Biochemistry, University of Birmingham, Birmingham, UK
Received for publication: December 2, 1981
Key words: (+)-catechin, 3-0-methyl-(+}-catechin, metabolites, flavonoid, man
SUMMARY
Following oral administration of (+}-catechin and 3-0-methyl-(+)-catechin to human volunteers the major urinary meta-
bolites were shown to be the glucuronides of 3' -O-methyl-(+)-catechin and 3,3' -O-dimethyl-(+)-catechin respectively. Isolations
from urine and from synthetic products have been carried out by semi-preparative high performance liquid chromatography;
definitive elucidations of structures have been carried out by gas chromatography-mass spectrometry and nuclear magnetic
resonance spectroscopy.
INTRODUCTION
The flavanol, (+}-catechin +}-Cyanidanol-3)
which has been reported to be effective in the
therapy of acute viral hepatitis (I) was earlier ob-
served to be in part degraded to phenolic acids and
hydroxy phenyl-y-valerolactones in the rat (2), (3)
and in man (4). Recently however evidence has been
presented that (+)-catechin is also metabolized in
the liver of the rat to 3' -O-methyl-(+)-catechin
glucuronide and that this conjugate is a major meta-
bolite in both the bile (5) and in the urine (6) of the
rat. Similarly, following the administration of 3-0-
methyl-(+)-catechin to the rat, the biliary and urinary
excretion of 3,3. (or 4' }-O-dimethyl-(+)-catechin
glucuronide was observed (7). Evidence is now pre-
sented that 3' -O-methyl-(+)-catechin and 3,3'-0-
dimethyl-( +)-catechin are present also in human
urine following the oral administration of (+)-
catechin and 3-0-methyl-(+)-catechin respectively to
human volunteers. Identification of these aglycones
Send reprint requests 10 : Dr M. Wermeille, Research
and Development Department/ Zyma SA, CH-1260 Nyon,
Switzerland.
was effected by gas chromatography - mass spectro-
metry and by nuclear magnetic resonance techniques.
EXPERIMENTAL
High performance liquid chromatography
(HPLC)
a) Isolation of 3' and 4 '-O-methyl-(+)-catechin from
the methylation products of (+)-catechin
A synthetic mixture (500mg) of O-methyl-catechin
ethers substituted on ring B and containing 3 major
products: I - 64%II - 32%and III - 4%was obtained
by a new method of regiospecific methylation of (+)-
catechin (8). Semi-preparative HPLC separations
were carried out on a Waters System (Waters
Associates Inc., Milford, MA, USA) incorporating a
u-Bondapak C18 column (particles of 10 urn, 30 em
length, 7.8 mm int. diam.), a M-6000-A chromato-
graphy pump, a U6K sample injector and a M-440
UV-detector fixed at 280 nm. The samples, con-
sisting of 500 ul of I % solution of the synthetic
mixture, were eluted with methanol-l % aq. acetic
acid (18:82 / v:v) at a flow rate or 6 ml/min. The
78 European Journal ofDrug Metabolism and Pharmacokinetics, 1983, No 1
fractions containing substances I and II were collected
separately. Twentyfive samples were injected and the
pooled fractions neutralized with NaOH IN. Me-
thanol was then evaporated and the substances I and
II in the remaining aqueous solutions were applied
to the HPLC column which was washed overnight
with water at a flow rate of 3 ml/min., this step is
necessary to eliminate residual sodium acetate. The
products I and II eluted with methanol were dried
over phosphorus pentoxide.
b) Isolation of 3.3 '-O-dimethyl-(+)-catechin from
human urine
Fractions (20 ml) of urine from 4 healthy volun-
teers each having received per os 1.000 g of 3-0-
methyl-(+}-catechin, were collected between 1-4hours
after administration. These samples were hydrolysed
with p-glucuronidase for 36 hours at 37C and
extracted 3 times with 50 ml dichloroethane. After
evaporation of the organic layer the residue was
redissolved in 4 ml of a mixture dichloromethane /
ethanol : 1/1. The final purification of a small
amount (- 10 mg) of the metabolite was carried out
under the same HPLC conditions as above. The
volume of injection was however only 50 Ill, and the
composition of the elution mixture was methanol -
I % aq. acetic acid 27.5:72.5 / v:v.
Nuclear magnetic resonance (NMR)
IH-NMR spectra were determined on a Broker
WH-360 (Bruker Inst. Co. Frankfurt, W-Germany)
apparatus equipped with Fourier transform; the
solutions were examined in DMSO-d 6 under the
following conditions: spectral width 3600 Hz; lock
2D; points 32 K/16 K; accumulation 32; reference:
TMS.
Gas chromatography - mass spectrometry
(GC-MS)
Gas chromatographic - mass spectrometric ana-
lysis was carried out on a system consisting of a HP-
5710 chromatograph (Hewlett-Packard, Palo Alto,
Calif. USA) coupled with a mass spectrometer VG
Micro-mass 305 (magnetic scan) (VG Organic Ltd.
Altrincham, Cheshire, UK). One milliliter of urine,
from volunteers having received per os one gram of
(+)-catechin or 3-0-methyl-(+)-catechin, was extracted
with 2 ml of ethyl acetate. After evaporation to
dryness the residue was redissolved in pyridine and
trimethylsilylated with N,O-bis(trimethylsilyl)-tri-
fluoracetamide, BSTFA (Fluka AG, Buchs, Swit-
zerland). One microliter of this solution was injected
onto the OV-17 3% on Gas Chrom Q 80-100 mesh
column, into diam. 2 mm, length 3 m at 250C
flushed with N2 at a flow-rate of 40 ml/min. The
connection from the column to the spectrometer
consists of a jet separator interface. The conditions
for mass spectrometry were as follows: resolution:
1000; temperature source: 210DC; electron energy 70
eV. The VG 2000 Data System was used for com-
putation of data.
RESULTS AND DISCUSSION
Isolation and identification of metabolites
In the urine of six healthy volunteers each having
received per os one gram of (+)-catechin, gas
chromatographic analysis has shown the presence of
the unchanged compound (average amount recovered:
- 0.1% of the dose administered) as well as its
glucuronide conjugate (- 11% of the dose). More-
over the presence of a metabolite as an aglycone (-
0.25% of the dose) and of the corresponding glu-
curonide (- 7% of the dose) has been detected. In
the urine of four healthy volunteers each having
received one gram of 3-0-methyl-(+)-catechin an
analogous free aglycone and glucuronide conjugate
have been detected (- 0.4% and - 30% of the dose
respectively plus - 0.15% and - 20% of the dose
for the main metabolite and its conjugate. Urines
have been collected each hour between 0-4 hours,
and each two hours between 4-12 hours. Apparent
half-life of elimination is about 2 hours for (+)-
catechin and I hour for 3-0-methyl-(+}-catechin.
In order to establish the identification of these
metabolites, two different isolation procedures have
been investigated. As shown on scheme I the structure
of the metabolite of (+)-catechin has been deter-
mined through a comparison of the GC spectra of
the metabolite in urine (after extraction and tri-
methylsilylation) with a synthetic reference com-
pound. This substance has been obtained by chemical
synthesis following a new unpublished procedure (8)
which has produced an isomeric mixture of 3' and
4' -O-methyl-(+)-catechin as well as 3' ,4'-di-Oemethyl-
(+)-catechin. Therefore a semi-preparative method
was applied in order to obtain 10mg of each of the
pure methyl ethers of (+)-catechin for NMR chara-
cterisation and for the ultimate GC-comparison with
the urine extract.
M. Wermeilleand al., Identification of the metabolites of (+) - catechin and - catechin in man
79
SCHEME 1
(+)-CATECHIN
METABOLISM CHEMICAL
IN MAN METHYLATION
1 1
The procedure followed for the identification of
the main metabolite observed in the urine of volun-
teers having received 3-0-methyl-(+)-catechin is shown
in scheme 2. A semi-preparative method has been
developed in order to isolate at least 10 mg of the
main metabolite from urine samples. The chemical
structure is then elucidated with NMR spectroscopy
and GC-MS analysis.
SCHEME 2
!
GC - COMPARISON BETWEEN
BIOLOGICAL AND SYNTHETIC SUBSTANCES
Identification by mass spectrometry
The fragmentation pattern of the trimethylsilylated
metabolite of (+)-catechin from a urine extract is
interpreted as follows. The observed molecular ion
mle = 592 corresponds to a tetra-trimethylsilylated-
monomethylated derivative of catechin.
Retro-Diels-Alder fragmentation gave an ion
mle = 310 typical for a monomethylated catechin
possessing a methyl substituent at 3' or 4'. The
fragment, mle =209 also indicated the presence of a
methyl group on the B ring. The MS technique
however does not distinguish between 3' or 4'
methyl substitution.
The observed fragmentation pattern of the tri-
methylsilylated metabolite of 3-0-methyl-(+)-catechin
is interpreted in the same way. The molecular ion
mle = 534 corresponds to a tri-trimethyl-silylated-
dimethyl-catechin. An analagous mechanism of
fragmentation as described above indicates that the
methyl groups occur at positions 3,3' or 3,4'.
1
NMR
ANALYSIS
Differenciation of
3' or 4' methylation
on Bring
ISOMERS OF
METHYLCATECHI N
GC - MS
ANALYSIS
1
3' or 4' methylation
on Bring
METABOLITE AS
GLUCURONIDE OF
FREE FORM
1
1) hydrolysis
2) trimethylsilylation
Identification of metabolite
as 3'-O-methyl-(+)-catechin
3-0-METHYL-(+)-CATECHIN
METABOLISM
IN MAN
1
METABOLITE AS
GLUCURONIDE OR
IN FREE FORM
1
1) hydrolysis
2) trimethylsilylation
CHEMICAL
METHYLATION
NOT AVAILABLE
+
1) hydrolysis
2) isolation
3) purification
(HPLC)
NMR-identification
On figure I, the IH-NMR spectrum of 3' -0-
methyl-( +)-catechin (I), is presented. On table 1 the
chemical shifts and coupling constants of this pro-
duct and of (+)-catechin, 4' -O-methyl-(+)-catechin
(II) and 3' ,4' -O-dimethyl-(+)-catechin (III) are re-
ported.
The NMR signals for the H2', H5' and H6'
protons on the B ring can be interpreted as follows:
Identification of metabolite
as 3,3'-O-dimethyl-(+)-catechin
GC - MS
ANALYSIS
3' or 4' methylation
on Bring
NMR
ANALYSIS
1
Differenclation of
3' or 4' methylation
on Bring
- H2' : doublet on account of the meta-coupling
with H6'; Je2 Hz
- H5' : doublet on account of the ortho-coupling
with H6'; Jeg Hz
- H6' : doublet of doublet on account of the
coupling with H2' and H5'.
Theoretically H2' and H5' should be doublet of
doublet on account of their para-coupling; the
coupling constant however is < I Hz and therefore
80
European Journal of Drug Metabolism and Pharmacokinetics, 1983, No 1
I
9
I
8
H'

I
7
I
6
I
5
I
4
I
3
I
,
H'
6
!

ppm
I
6.8
i
1S.8
ppm
Fig. 1 I H-NMR spectra at 360 MHz of the metabolite of (+)-catechin, 3'-O-methyl-(+)-catechin (Figure Ia) and the enlarged
section of it representing the aromatic protons at B ring (Figure Ib).
M. Wermeilleand al., Identification of the metabolites of (+) - catechin and - catechin in man
Table I : IH-NMR (360 MHz) spectral data relating to (+)-catechin and its methyl ethers
81
tS- 6
Me
.ow{$:-
6
e
'0.
,0W, ,'0'
HO I .'
3 0' 5'
WI'I.
OMe
I .
I .
I .3
I .
, OH , OH
'. OH
, OH
H Hex, Heq.
HO Hex, Heq.
HO Hex, Heq. HO Hex,Heq.
Proton Ii (ppm) Nb, MulL' J (Hz) s (ppm) Nb, Mull. J (Hz) Ii (ppm) Nb, Mull. J (Hz) Ii (ppm) Nb, Mull. J (Hz)
H. ax 2,37 1 d of d f 16,0 (H. eq)
2,37 1 d of d
{ 15,9
2,37 I d of d
{ 16,1
2,38 1 d of d
{ 16,0
7,9 (H
J
) 8,5 8,1 8,5
H. eq 2,67 I d of d { 16,0 (H. ax)
2,74 1 d of d
{ 15,9
2,66 I d of d
{ 16,1
2,74 1 d of d
{ 16,0
5,1 (H
J
) 5,6 5,3 5,4
H, 3,84 I m 3,91 I m 3,84 I m 3,92 I m
H, 4,50 I d 7,3 4,52 I d 8,0 4,53 1 d 7,4 4,57 I d 8,(
H, 5,71 I d 2,0 5,71 I d 2,3 5,71 I d 2,1 5,72 I d 2,3
H, 5,90 1 d 2,0 5,91 1 d 2,3 5,91 I d 2,1 5,92 1 d 2,3
Hz'
6,74 I d 1,5 (H
6
, ) 6,91 1 d 1,0 6,77 I d 1,9 6,96 I d 1,6
H
s'
6,70 I d 8,0 (H
6
, ) 6,75 I d 8,2 6,87 I d 8,5 6,93 I d 8,4
H
6
, 6,61 I d of d
{ 8,0 (H
s')
6,78 I d of d
C,3
6,73 1 d of d
{ 8,4
6,89 I d of d
{ 8,3
1,5 (Hz') 1,5 2,0 i.e
OH (3) 4,89 1 s broad -
4,91 1 s broad - 4,90 1 s broad - 4,93 1 d 5,5 (H
J
OH phenol 8,9-9,2 4
- - 9,1 3 bump - - 9 3 bump - 8,99 I s -
9,24 I s -
OMe (3') - -
- - 3,76 3 s - - - - -
3,75 6 s -
OMe (4') - - - - - - - - 3,75 3 s -
not perceptible on the spectra. The enlarged portion
of the spectrum corresponding to these three pro-
tons is shown on figure la. It can be seen that the
second order contribution may be important - dis-
symetry of the doublet and that the spectra present
significant differences due to the substitution of the
methyl(s) on the -OH function(s). The ortho and
para positions on an aromatic ring are highly
activated by an -OH substituent. In terms of NMR
spectroscopy, this means that the electronic density
will be reinforced at the ortho and para positions.
The -OMe substituent on an aromatic ring is a less
active function than -OH. For this reason, it can be
explained (partly) that the resonance of protons
from B ring in the case of 3' ,4 .-dimethylated
catechin is located at a lower field than the re-
sonance of the same protons in catechin.
If the substitution of -OH through -OMe has
occured only on the 3' position, the chemical shift
to a lower field is significant only for H2' (ortho)
and H6' (para). As reported on table I, this phe-
nomenon is observed for the 3' -O-methyl-(+)-catechin:
0,17 ppm for H2' and H6'; only 0,05 ppm for H5'.
The substitution on 4' position of a -OH through a -
OMe function produces a significant chemical shift
for the H5' (ortho) proton of 0,17 ppm.
In order to determine the structure of the urinary
metabolite, urine extracts are compared with the two
potential metabolites 3' or 4' -O-methyl-(+)-catechin.
The GC chromatogram exhibits differences of re-
tention time between these two trimethylsilylated
molecules; the retention time of 3' -O-methyl-( +)-
catechin was 11.5 minutes and retention time of
4' -O-methyl-(+)-catechio was 10.I minutes. Con-
sequently the metabolite was identified as 3'-0-
methyl-(+)-catechin.
The NMR spectra of the metabolite of 3-0-
methyl-( +)-catechio is shown 00 figure 2. 00 table
82
European Journal of Drug Metabolism and Pharmacokinetics, 1983, No 1
r
9
,
8
r
7
r
6
,
5
r
4
I
3
r
2
I ..
o ppm
H'
i
H' + H'

I
e.9
I
e.8
I
e.7
I
ppm
Fig. 2 : IH-NMR spectra at 360 MHz of the metabolite of 3-0-methyl-(+)-catechin, 3,3' -O-dimethyl-(+)-catechin (Figure
13) and the enlarged section of it representing the aromatic protons at B ring (Figure 2b).
M Wermeille and al., Identification of the metabolites of (+) - catechin and 3-Q-methyl-(+) - catechin in man
83
Table II : IH-NMR (360 MHz) spectral data relating to 3-0-methyl-(+)-catechin and its metabolite, 3,3' -O-dimethyl-( +)-
catechin.
. OH
OM.
H o ~ H
H O ~ : '...OH
.. 3 6' I
,OMe
4
, OMe
H Hex. Heq. HO Hex. H
eq
.
Proton I) (ppm) Nb Mult.! J (Hz) I) (ppm) Nb Mult. J (Hz)
H
4
ax 2,42 1 d of d { 16,0 (H
4eq)
2,40 1 d of d
{ 16,0 (H 4eq)
6,5 (H 3) 7,2 (H
3
)
H
4
eq 2,63 I d of d { /6,0 (H 4eq)
2,70 / d of d
{ 16,0 (H 4ax)
4,6 (H
3
) 4,9 (H 3)
H
3
3,62 1 m 3,73 I m
H
2
4,73 I d 6,6 4,75 / d 6,9
H
6
5,72 1 d 2,2 5,72 I d 2,4
H
s
5,90 I d 2,2 5,91 1 d 2,4
H
2
6,72 1 d 1,4 6,92 I s
H5'
6,69 I d 8,2
}
H
6

6,59 1 d of d
{ 8,2 (H 5')
6,73 2 2nd order
1,4(H
2,)
6,75
OMe (3) 3,17 3 s -
3,16 3 s -
OMe (3') - - - -
3,74 3 s -
{ 8,87
2 s
-
{ 9,00
2 bump
-
OH phenol. 8,98 I s
-
9,23 1 s
-
9,28 I bump
-
H2 O 3,38 3,36
DMSO-d
6
2,50 2,50
I d = doublet, m = multiplet, s = singlet
II chemical shifts and coupling constants are re-
ported. The same arguments as were developed
above are applied for the location of the methyl
substitution. The chemical shifts of protons on the B
ring between 3-0-methyl-(+)-catechin and its meta-
bolite are for H2' equal to 0,10 ppm, H5' =0,05ppm
and H6' = 0, 15 ppm. The main chemical shifts
observed for protons H2' and H6' (at least 0,15
ppm) indicates methylation at position 3'.
COMPARISON OF THE METHYLATION
PATHWAY IN MAN AND ANIMALS
The evidence presented indicates that the me-
thylation pathway for the metabolism of (+)-catechin
and 3-0-methyl-{+)-catechin observed in the rat (5),
(7) is also operative in man and it is of interest that
in both species the major product is a 3' -O-methyl
84
European Journal ofDrug Metabolism and Pharmacokinetics, 1983, No 1
ether. The current investigation has also provided
firm evidence based on the NMR technique that the
dirnethyl-t-l-j-catechin excreted in human urine fol-
lowing 3-0-methyl-(+)-catechin administration is the
3,3' -O-dimethyl ether.
Since an earlier study on the metabolism of (+)-
catechin in the rat (5) showed that the methylation
of (+)-catechin occurred in the liver, the site of
action of this liver-protective agent, this metabolic
conversion may be of considerable pharmacological
significance and it appears to be of some impor-
tance that the biological activity of the 3' -O-methyl
ethers relative to the parent flavanols should be
explored.
- In the rat, the formation of glucuronides of the
methyl ethers of both flavanols facilitated ready ex-
cretion in both bile and urine (5), (6), (7). Whether
these metabolites are similarly excreted in human
bile remains to be established.
ACKNOWLEDGMENTS
The authors wish to thank Mrs P. Gomez, E. Machoud
and G. Perissinotto for skilled technical assistance, Messrs.
A. Aellig and L. Balant for providing the biological
samples, Mr. A. Albert for the synthetic products and Mr.
D. Fraisse (Centre de Spectrometric de masse de Lyon) for
the GC-MS analysis.
REFERENCES
I. Blum A.L., Berthet P., Doelle W., Goebell H., Kortum
K., Pelloni S., Peter P., Poulsen H., Strohmeyer G.,
Tygstrup N. (1977): Treatment of acute viral hepatitis
with (+)-Cyanidanol-3. Lancet 2. 1153-1155.
2. Griffiths L.A. (1964): Studies on flavonoid metabolism:
Identification of the metabolites of (+)-catechin in rat
urine. Biochem. J. 92. 173-179.
3. Das N.P., Griffiths L.A. (1969): Metabolism of (+)-
[ 14C}catechin in the rat and guinea pig. Biochem. J.
115. 831-836.
4. Das N.P. (1971): Studies on flavonoid metabolism: Ab-
sorption and metabolism of (+)-catechin in man.
Biochem. Pharmacol. 20. 3435-3445.
5. Shaw I.C., Griffiths L.A. (1980): Identification of the
major biliary metabolite of (+)-catechin in the ral.
Xenobiotica 10. 905-911.
6. Hackett A.M., Shaw I.C., Griffiths L.A. (1982): 3'-0-
Methyl-(+)-catechin glucuronide and 3' -O-Methyl-(+)-
catechin sulphate: New urinary metabolites of (+)-
catechin in the rat. Experientia 38. 538-539.
7. Hackett A.M., Griffiths L.A. (1981): The metabolism
and excretion of 3-0-Methyl-(+)-catechin in the rat,
mouse and marmoset. Drug Metabolism and Disposition
9. 54-59.
7. Albert A., Courbat P. (1981): Personal Communication,
Zyma SA.