G. BENKOVIĆ et al.: Metabolism of flavonoid aglycons
121
Therefore, the obtained results can be taken as a rough
estimate of the kinetics for this reaction.
only a few recombinant cytochromes P450 (CYP1A2,
CYPC19, CYP2D6 and CYP3A4) catalyze the observed
metabolic reactions of the analyzed flavonoids.
Sakuranetin undergoes sequential biotransfor-
mation, first demethylation to naringenin, then aromatic
hydroxylation to eriodictyol. At the same time, minor
product of direct aromatic hydroxylation of sakuranetin is
formed i.e. 5,3',4'-trihydroxy-7-methoxyflavanone. Both
metabolic pathways are observed in incubations with
human liver microsomes and sequential biotransfor-
mation is 10 times more catalytic effective ((0.020 ±
0.005) × 106 M–1 min–1) when compared to hydroxylation
at the positon 3' ((0.0022 ± 0.0002) × 106 M–1 min–1).
Interestingly, only minor reaction was directly linked with
cytochromes P450 used in this study i.e. CYP3A4 and
CYP1A2 for which catalytic effectiveness constants were
(0.06 ± 0.03) × 106 M–1 min–1 and (0.7 ± 0.4) × 106 M–1 min–1,
respectively.
Because of the interferences of metabolites on the
UV detector in enzyme kinetics incubations with human
liver microsomes, it was not possible to follow the main
metabolic pathway of tangeretin in which tangeretin is
demethylated to 4'-hydroxy-5,6,7,8-tetramethoxyflavone
and then hydroxylated to 3',4'-dihydroxy-5,6,7,8-tetra-
methoxyflavone. However, these interferences were not
present in incubations with recombinant enzymes. The
most important enzyme for this metabolic pathway was
CYP1A2 with catalytic effectiveness of (1.0 ± 0.2) × 106 M–1
min–1. Kinetic parameters were determined on HLM for
the reaction of single demethylation of tangeretin on ring
A for the metabolite at tR = 10.85 min. The catalytic
effectiveness constant for this reaction was (0.023 ±
0.008) × 106 M–1 min–1. This reaction was mediated by
CYP3A4 (kcat/Km = (0.21 ± 0.03) × 106 M–1 min–1).
Demethylation of tangeretin at the position 4' is catalyzed
by CYP3A4 (kcat/Km = (0.11 ± 0.02) × 106 M–1 min–1), and to
a lesser extent with CYP2D6 (kcat/Km = (0.0058 ± 0.0001) × 106
M–1 min–1).
The enzyme that is most efficient is CYP1A2. With
the exception of metabolic conversion of naringenin to
eriodictyol, CYP1A2 catalytic effectiveness, kcat/Km, was in
the range from 0.5 to 2.9 × 106 M–1 min–1, indicating the
high catalytic specificity or efficacy of this cytochrome
P450 in flavonoid metabolism. This result is in agreement
with published data on the metabolism of flavonoids
mediated by cytochromes P450, in which most authors of
the studies concluded that CYP1A2 plays a major role in
the oxidative metabolism of flavonoids in in vitro
Given the low concentrations generally achieved by
oral intake of flavonoids,
a very important kinetic
parameter to consider is the Km value. This is especially
important in sense that flavonoids are known to have very
low solubility in water and, most importantly, poor
bioavailability after oral administration. Consequently, the
usual concentrations achieved in the blood after the
flavonoids consumption from different sources are nano-
molar to micromolar, with large interindividual differences,
and most often do not exceed 10 μM values.[36] Flavonoids
for which the low Km value has been determined have a
higher chance of being metabolized in vivo after their oral
administration compared to those having higher catalytic
conversion rate (kcat) and high Km. In that sense, flavonoids
that have higher potential of competitive interactions with
other xenobiotics are: apigenin, acacetin, kaempferol, 3,7-
dihydroxyflavone and galangin; all having Km values bellow
10 μM (Table 2).
Out of ten analyzed flavonoids, seven of them are
susceptible to the metabolism mediated by CYP1A2 (Table
2), which further confirms the hypothesis that this
cytochrome P450 is the most important for the metabolism
of flavonoids in humans. However, for a better estimate of
the possible clinical significance of these metabolic
reactions, it is not enough to consider only the Km para-
meter. This parameter needs to be put in relation to the
rate constant (kcat) to provide kcat/Km ratio known as
catalytic effectiveness. E.g. kaempferol, galagnin, and
naringenin, have low Km values, but given the relatively low
rate of catalyst constant, it is expected that the extent of
these reactions will not be so significant.
DISCUSSION
In this study we have characterized kinetic parameters of
O-demethylations and aromatic hydroxylations to which
flavonoid aglycons are susceptible. The range of values for
rate constant kcat was 0.021 to 14.2 min–1. The Michaelis-
Menten constants, Km, was obtained in the range of 0.7 to
492 μM, and calculated catalytic effectiveness, kcat/Km, was
in the range from 0.0015 to 2.9 × 106 M–1 min–1. The range
of results obtained for all kinetic parameters is very wide,
and within a three-orders of magnitude. If we have in mind
the structural homogeneity of the analyzed compounds,
these results indicate a relatively high selectivity of human
liver cytochrome P450 to certain substrates from this group
of compounds. This conclusion is supported by the fact that
The range of the kcat/Km values of the studied
reactions catalyzed by human liver microsomes was
(0.0015 – 2.9) × 106 M–1 min–1. It has been previously
pointed out that the values of kcat/Km ratio catalyzed by
CYP1A2 enzymes ranged from 0.003 to 2.9 × 106 M–1 min–1.
Therefore, it is not surprising that reactions with the
highest catalytic efficiency are catalyzed by CYP1A2.
Tangeretin demethylation reactions catalyzed by CYP3A4
DOI: 10.5562/cca3528
Croat. Chem. Acta 2019, 92(1) 115–123