Inhibition of human recombinant cytochrome P450s by curcumin and curcumin decomposition products

Article abstract of DOI:10.1016/j.tox.2007.03.007

Curcumin (diferuloylmethane) is a major yellow pigment and dietary component derived from Curcuma longa. It has potent anti-inflammatory, anticarcinogenic, antioxidant and chemoprotective activities among others. We studied the interactions of curcumin, a mixture of its decomposition products, and four of its individually identified decomposition products (vanillin, vanillic acid, ferulic aldehyde and ferulic acid) on five major human drug-metabolizing cytochrome P450s (CYPs). Curcumin inhibited CYP1A2 (IC₅₀, 40.0 μM), CYP3A4 (IC₅₀, 16.3 μM), CYP2D6 (IC₅₀, 50.3 μM), CYP2C9 (IC₅₀, 4.3 μM) and CYP2B6 (IC₅₀, 24.5 μM). Curcumin showed a competitive type of inhibition towards CYP1A2, CYP3A4 and CYP2B6, whereas a non-competitive type of inhibition was observed with respect to CYP2D6 and CYP2C9. The inhibitory activity towards CYP3A4, shown by curcumin may have implications for drug-drug interactions in the intestines, in case of high exposure of the intestines to curcumin upon oral administration. In spite of the significant inhibitory activities shown towards the major CYPs in vitro, it remains to be established, whether curcumin will cause significant drug-drug interactions in the liver, given the reported low systemic exposure of the liver to curcumin. The decomposition products of curcumin showed no significant inhibitory activities towards the CYPs investigated, and therefore, are not likely to cause drug-drug interactions at the level of CYPs.

Full text of DOI:10.1016/j.tox.2007.03.007

Toxicology 235 (2007) 83–91
Inhibition of human recombinant cytochrome P450s by
curcumin and curcumin decomposition products
Regina Appiah-Opong, Jan N.M. Commandeur∗,
Barbara van Vugt-Lussenburg, Nico P.E. Vermeulen
Division of Molecular Toxicology, Leiden/Amsterdam Center for Drug Research (LACDR), Department of Pharmacochemistry,
Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
Received 25 January 2007; received in revised form 7 March 2007; accepted 8 March 2007
Available online 15 March 2007
Abstract
Curcumin (diferuloylmethane) is a major yellow pigment and dietary component derived from Curcuma longa. It has potent anti-
inflammatory, anticarcinogenic, antioxidant and chemoprotective activities among others. We studied the interactions of curcumin,
a mixture of its decomposition products, and four of its individually identified decomposition products (vanillin, vanillic acid,
ferulic aldehyde and ferulic acid) on five major human drug-metabolizing cytochrome P450s (CYPs). Curcumin inhibited CYP1A2
(IC50, 40.0 ␮M), CYP3A4 (IC50, 16.3 ␮M), CYP2D6 (IC50, 50.3 ␮M), CYP2C9 (IC50, 4.3 ␮M) and CYP2B6 (IC50, 24.5 ␮M).
Curcumin showed a competitive type of inhibition towards CYP1A2, CYP3A4 and CYP2B6, whereas a non-competitive type of
inhibition was observed with respect to CYP2D6 and CYP2C9. The inhibitory activity towards CYP3A4, shown by curcumin may
have implications for drug–drug interactions in the intestines, in case of high exposure of the intestines to curcumin upon oral
administration. In spite of the significant inhibitory activities shown towards the major CYPs in vitro, it remains to be established,
whether curcumin will cause significant drug–drug interactions in the liver, given the reported low systemic exposure of the liver to
curcumin. The decomposition products of curcumin showed no significant inhibitory activities towards the CYPs investigated, and
therefore, are not likely to cause drug–drug interactions at the level of CYPs.
© 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cytochrome P450; Curcumin; Drug–food interactions; Enzyme inhibition
1. Introduction
Multiple drug therapy is a common therapeutic prac-
tice especially in patients with multiple complications
(Nadler et al., 2003; Hemaiswarya and Doble, 2006). If
two or more drugs with affinity for the same cytochrome
P450 (CYP) enzyme are co-administered, their biotrans-
formation may be compromised, leading to undesirable
accumulation of the drugs with toxic side effects as
possible consequence. Drug–drug interactions involv-
ing CYPs have been identified as an important cause
of adverse drug reactions and therapeutic failure (Honig
Abbreviations: CYP, cytochrome P450; HPLC, high performance
liquid chromatography; BROD, benzyloxyresorufin O-debenzylase;
MROD, methoxyresorufin O-demethylase; DBF, dibenzylfluorescein;
BQ, 7-benzyloxyquinoline; BFC, 7-benzyloxy-4-trifluoromethyl-
couma-rin; GSH, reduced glutathione; NAC, N-acetyl l-cysteine
Corresponding author. Tel.: +31 205987590; fax: +31 205987610.
E-mail address: jnm.commandeur@few.vu.nl
(J.N.M. Commandeur).
∗
0300-483X/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.tox.2007.03.007

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R. Appiah-Opong et al. / Toxicology 235 (2007) 83–91
et al., 1993; Pea and Furlanut, 2001). Drug–drug interac-
tions may be due to enzyme induction or inhibition, the
latter being more common (Hemaiswarya and Doble,
2006; Zafar and Sharif, 2003). Next to drugs, several
natural compounds have also been shown to cause sig-
nificant interactions at the level of drug-metabolizing
enzymes (Ioannides, 2002; Obach, 2000).
peutic drugs on the market include CYP1A2, CYP2D6,
CYP2B6, CYP2E1, CYP2C9 and CYP3A4 (Shimada
et al., 1994). Because curcumin has been shown to
be chemically unstable under neutral and alkaline con-
ditions (Wang et al., 1997), the inhibitory properties
of these decomposition products were also studied
as mixture and individually, if available. Degradation
products which have been identified include trans-6-(4ꢀ-
hydroxy-3ꢀ-methoxyphenyl)-2,4-dioxo-5-hexenal, and
minor products being vanillin, vanillic acid, ferulic alde-
hyde and ferulic acid (Fig. 1) (Wang et al., 1997). Apart
from reversible inhibition, mechanism-based inhibition
was also taken into consideration.
Curcumin, a polyphenolic component of turmeric
(Curcuma longa), is a yellow pigment widely used for
coloring of foods. It has been shown previously to exhibit
anticancer, antioxidant, anti-inflammatory, antiparasitic
and anti-HIV properties (Leu and Maa, 2002; Cole et al.,
2004; Vajragupta et al., 2005; Reddy et al., 2005). Cur-
cumin has also been shown to have chemoprotective,
chemopreventive and immuno-modulating properties
(Cole et al., 2004; Donatus et al., 1990; Cheng et al.,
2001). Curcumin can be considered as a safe compound,
because oral doses as high as 8 g/day administered to
humans did not result in overt side effects (Cheng et al.,
2001). Clinical trials for the use of curcumin as an anti-
cancer agent are currently ongoing (Sharma et al., 2004).
Because relatively high doses of curcumin are eval-
uated in human studies, it might be anticipated that
curcumin might cause drug–drug interactions at the level
ofintestinaland/orliverdrugmetabolism. Severalinvivo
and in vitro animal studies have shown that curcumin
can significantly modulate the activity of several drug-
metabolizing enzymes by down-regulation, induction or
by direct inhibition. Oetari et al. (1996) and Thapliyal
and Maru (2001) reported potent inhibition of rat liver
microsomal CYP1A1, CYP1A2 and CYP2B1 enzymes
by curcumin. In in vivo studies repetitive administra-
tion of curcumin to rats resulted in down-regulation of
intestinal CYP3A-enzymes, whereas hepatic and renal
CYP3A-levels were significantly induced (Zhang et al.,
2006). Also, down-regulation of esophagal CYP2B1 and
CYP2E1 was reported after intragastric treatment of rats,
whichmightpartiallyexplainthechemopreventiveactiv-
ity of curcumin against carcinogenic N-nitrosamines
(Mori et al., 2006).
2. Materials and methods
2.1. Materials
Methoxyresorufin and benzyloxyresorufin were synthe-
sized by the method of Burke et al. (1985) and the purity
was determined by high performance liquid chromatogra-
1
phy (HPLC), mass spectrometry and H NMR. The plasmid,
pSP19T7LT 2D6 containing human CYP2D6 bicistronically
co-expressed with human cytochrome P450 NADPH reductase
was kindly provided by Prof. M. Ingelman-Sundberg (Stock-
holm, Sweden). The plasmids, BMX100/h1A2 and pCWh3A4
with human cytochrome P450 NADPH reductase were kindly
donated by Dr. M. Kranendonk (Lisbon, Portugal). Expression
plasmids, pCWh2B6hNPR and pCWh2C9hNPR with human
cytochrome P450 NADPH reductase were kindly provided by
Prof. F.P. Guengerich (Nashville, TX, USA). All other chem-
icals were of analytical grade and obtained from standard
suppliers.
2.2. CYP expression and membrane isolation
The plasmids containing cDNA of five human CYPs were
transformed into Escherichia coli strain JM109. Expression
of the CYPs was carried out in 3-L flasks containing 300 mL
terrific broth (TB) with 1 mM ␦-aminolevulinic acid, 0.5 mM
thiamine, 400 ␮L/L trace elements, 100 ␮g/mL ampicillin,
1 mM isopropyl-␤-d-thiogalactopyranoside (IPTG), 0.5 mM
FeCl3 (for CYP2D6 and CYP3A4 only) and 30 ␮g/mL
kanamycin (for CYP3A4 only). The culture media were inoc-
ulated with 3 mL overnight cultures of bacteria containing
plasmids for the various CYPs. The cell cultures were incu-
bated for about 40 h at 28 ◦C and 125 rpm and CYP contents
were determined using the carbon monoxide (CO) difference
spectra as described by Omura and Sato (1964). Cells were
pelleted by centrifugation (4000 × g, 4 ◦C, 15 min) and resus-
pended in 30 mL Tris–sucrose–EDTA (TSE) buffer (50 mM
Tris–acetate buffer, pH 7.6, 250 mM sucrose, 0.25 mM EDTA).
Cells were treated with 0.5 mg/mL lysozyme prior to disrup-
tion by French press (1000 psi, 3 repeats). The membranes
containing the human CYPs were isolated by ultracentrifu-
As yet the effects of curcumin on the major human
drug-metabolizing CYPs have not been studied. Due
to the large species differences in the properties of
metabolic enzymes and metabolic profiles of drugs,
the animal studies described above are poorly pre-
dictive for the human situation (Eagling et al., 1998;
Guengerich, 1997). The present in vitro investigation
therefore was designed to assess the potential of cur-
cumin to cause drug–drug interactions via inhibition
of the five most important human drug-metabolizing
CYPs. Major human CYP isoforms responsible for the
metabolism and disposition of about 90% of the thera-

R. Appiah-Opong et al. / Toxicology 235 (2007) 83–91
85
Fig. 1. Chemical structures of curcumin and its decomposition products at pH 7.4.
gation in a Beckmann 50.2Ti rotor (60 min, 40,000 rpm, 4 ◦C),
gradient eluting from 15 to 95% of solvent B in 60 min. The
carrier flow rate was 0.4 mL/min and chromatographic peaks
of decomposition products were monitored by UV detection
(λ = 280 nm). Under these chromatographic conditions cur-
cumin and commercially available reference compounds of
possible degradation products eluted at: 28.5 min (curcumin),
14.3 min (vanillic acid), 15.2 min (vanillin), 17.9 min (ferulic
acid) and 18.5 min (ferulic aldehyde).
resuspended in TSE buffer and stored at −80 ◦C until use.
2.3. Decomposition of curcumin
Curcumin decomposition was performed according to the
method of Wang et al. (1997) in phosphate buffer of pH 7.4.
Briefly, aliquots of 20 ␮L of 5 mM curcumin (dissolved in
methanol) were added to 980 ␮L of 0.1 M potassium phosphate
buffer pH 7.4. After 1 h incubation at 37 ◦C samples were ana-
lyzed by HPLC (Jasco Separations FP 1575). Vanillin, vanillic
acid, ferulic aldehyde and ferulic acid were used as standards.
Gradient reversed-phase HPLC separations were performed
using a C18 column (150 mm × 3.2 mm, 5 ␮m particle size,
Phenomenex) and a mobile phase consisting of 0.1% acetic
acid (solvent A) and 98% methanol with 0.1% acetic acid (sol-
ventB). HPLC-gradientseparationwascarriedoutwithalinear
2.4. CYP inhibition assays
2.4.1. 7-Methoxyresorufin, 7-benzyloxyresorufin, diben-
zylfluorescein, 7-benzyloxy-4-trifluo-romethylcoumarin
and 7-benzyloxyquinoline O-dealkylation
Inhibition of the activities of the human CYP isoforms 1A2,
3A4 and 2B6, by curcumin and its decomposition products was
determined using microplate fluorimetric assays (Burke et al.,
Table 1
Experimental conditions for fluorescence CYP assays
CYP
Enzyme concentration
(nm)
Incubation
time (min)
Substrate
MRes
Substrate
concentration (␮M)
Excitation
wavelength (nm)
Emission
wavelength (nm)
1A2
3A4
13.2
10
5.0
530
586
14.3
2.8
14.3
14.3
30
10
20
30
BRes
DBF
BFC
BQ
5.0
0.5
80.0
40.0
530
485
410
410
586
535
538
538
2B6
15.3
30
BRes
20.0
530
586
MRes, methoxyresorufin; BRes, benzyloxyresorufin; DBF, dibenzylfluorescein; BQ, 7-benzyloxyquinoline; BFC, 7-benzyloxy-4-trifluoromethyl-
coumarin.

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R. Appiah-Opong et al. / Toxicology 235 (2007) 83–91
1985; Stresser et al., 2000). Incubation conditions (e.g. enzyme
concentration, substrates, incubationtime)andwavelengthsfor
detection for each assay are shown in Table 1. Under these
conditions, the product formation was linear over the periods
indicated [data not shown]. The inhibitory activity of curcumin
towards CYP3A4 was tested using four different substrates of
CYP3A4, namely DBF, BFC and BQ, because inhibition of
CYP3A4 activity is known to be substrate-dependent (Stresser
et al., 2000).
In general, the microsomal incubations were carried out in a
total volume of 200 ␮L and in the presence of 100 ␮M NADPH
(freshly prepared) in a black coaster 96-well plate. Microsomes
were preincubated for 5 min at 37 ◦C with 0.1 M potassium
phosphate buffer (pH 7.4), substrates and inhibitors (curcumin
and its decomposition products) with DMSO at a final con-
centration of 0.5% (v/v) or less in all the CYP assays. At
theseDMSOconcentrations, theactivitiesofthestudiedhuman
CYPs are only affected to a small extent (Busby et al., 1998).
Uninhibited incubations, set at 100%, always contained the
same concentration of DMSO as the inhibited incubations. Sta-
bilityofcurcumininphosphatebufferpH7.4, hasbeenreported
to be strongly improved by addition of rat liver microsomes
or cytosol, glutathione (GSH), N-acetyl l-cysteine (NAC) or
ascorbic acid (Oetari et al., 1996). Therefore, enzymes were
always added before the incorporation of curcumin in all inhi-
bition assays performed.
Initially, the inhibitory effect of curcumin and its decom-
position products on the CYP isoforms was studied at a
concentration of 300 ␮M. To study the effect of the total
mixture of curcumin decomposition products, curcumin was
first added to phosphate buffer pH 7.4 to a final concentra-
tion of 300 ␮M; under these conditions curcumin is known to
rapidly decompose (Wang et al., 1997). After 1 h, enzyme, sub-
strate and NADPH were added to determine enzyme activity
in presence of decomposition products derived from 300 ␮M
curcumin.
For IC50 determinations, concentration ranges for curcumin
used were from 0.9 to 100 ␮M and for the decomposition
products, from 2.5 to 1000 ␮M. Incubations were commenced
by the addition of 100 ␮M NADPH, maintained at 37 ◦C for
the periods defined. Reactions were terminated with 75 ␮L of
80% acetonitrile and 20% 0.5 M Tris solution or 2N NaOH
in the case of dibenzylfluorescein (DBF). Product formation
was linear for all incubation times. Concentrations of the
probe substrates in all reaction mixtures were chosen near the
Michaelis–Menten’s (Km) value for each of the CYPs tested.
The Km valuesobtained usingthealkoxyresorufinsand all other
substrates were within the range of reported literature values
(Staskal et al., 2005). All measurements were performed in
triplicate. Metabolite formation was measured spectrophoto-
metrically on a Victor2 1420 multilabel counter.
diclofenac and inhibitor. Screening for inhibitory effects of
300 ␮M concentrations of curcumin, its decomposition mix-
ture and the individual decomposition products on the CYP2C9
was done. For IC50 determinations, curcumin concentrations
used were of the range 0.4–100 and 3.9–2000 ␮M for the indi-
vidual decomposition products, vanillin, vanillic acid, ferulic
aldehyde and ferulic acid. After preincubation for 5 min at
37 ◦C, reactions were initiated by adding NADPH and termi-
nated after 10 min with the addition of 200 ␮L methanol. The
reaction mixtures were centrifuged at 14,000 rpm for 3 min.
Product formed was measured using an isocratic HPLC method
(Walsky and Obach, 2004). A C18 column (150 mm × 3.2 mm,
5 ␮m particle size, Phenomenex) was used and the carrier
flow rate was 0.6 mL/min. The mobile phase consisted of
60% (v/v) 20 mM potassium phosphate buffer (pH 7.4), 22.5%
(v/v) methanol and 17.5% (v/v) acetonitrile. Peaks were
monitored at the wavelength of 280 nm. Retention times for
4-hydroxydiclofenac and diclofenac were 5.0 and 24.1 min,
respectively.
2.4.3. Dextromethorphan O-demethylation
Inhibition of CYP2D6 activity by curcumin and its decom-
position products was evaluated by the method described
by Ko et al. (2000). Inhibitory effects of 300 ␮M con-
centrations of curcumin, its decomposition mixture and the
individual decomposition products on the CYP2D6 were first
assessed. The reaction mixture had a total volume of 500 ␮L
and consisted of 18.2 nM enzyme, 4.5 ␮M dextromethor-
phan, 90.9 ␮M NADPH, 0.1 M potassium phosphate buffer
and inhibitor. For IC50 determination, curcumin concentration
range used was 0.4–181.8 ␮M and the decomposition prod-
ucts 3.6–1818.2 ␮M. Reactions were initiated by the addition
of NADPH and allowed to proceed for 45 min before ter-
mination with the addition of 60 mM zinc sulphate solution.
ProductformedwasmeasuredusinganisocraticHPLCfluores-
cence detection method and a C18 column (100 mm × 3 mm,
5 ␮m particle size, Chromspher). The mobile phase con-
sisted of 24% (v/v) acetonitrile and 0.1% (v/v) triethylamine
adjusted to pH 3 with perchloric acid. The carrier flow rate
was 0.6 mL/min. Peaks were monitored at 280 nm (excitation)
and 310 nm (emission). The retention times of dextrorphan and
dextromethorphan were 3.4 and 24.5 min, respectively.
2.5. Type of inhibition
To determine the types of inhibition occurring in the
reactions involving CYP1A2 and CYP3A4, the substrate
concentrations used were ranging from 0.65 to 10 ␮M for
both methoxyresorufin and benzyloxyresorufin. In the case
of the other CYPs, substrate concentration ranges were from
3.1 to 50 ␮M benzyloxyresorufin (CYP2B6), 0.3 to 20 ␮M
diclofenac (CYP2C9) and 1.4 to 22.7 ␮M dextromethorphan
(CYP2D6). Fivedifferentsubstrateconcentrationswereusedin
each assay. The concentration of curcumin used for the assays
areindicatedinTable3. Reactionswerecarriedoutasdescribed
above for all CYPs.
2.4.2. Diclofenac hydroxylation
For the CYP2C9 inhibition assay, reaction mixtures in
500 ␮L total volume consisted of 49 nM enzyme, 100 ␮M
NADPH, 0.1 M potassium phosphate buffer (pH 7.4), 6 ␮M

R. Appiah-Opong et al. / Toxicology 235 (2007) 83–91
87
2.6. Mechanism-based inhibition
The potential of curcumin for mechanism-based inhibition
of CYP1A2, CYP3A4 and CYP2B6 was evaluated according
to the method of Heydari et al. (2004) with slight modifica-
tions. Briefly preincubation mixtures of total volumes 600 ␮L
contained 13–16 nM CYP enzymes, 100 ␮M NADPH and cur-
cumin solution (0, 10 and 50 ␮M). The preincubations were
performed for 20 min at 37 ◦C, and at 5 min intervals 100 ␮L
aliquots were taken to determine the remaining CYP activ-
ity. The aliquots of preincubation mixtures were added to
tubes containing 400 ␮L of the respective substrates (5 ␮M
methoxyresorufin for CYP1A2, 5 ␮M benzyloxyresorufin for
CYP3A4 and 20 ␮M benzyloxyresorufin for CYP2B6) and
NADPH (100 ␮M), and incubated as described above. After
stopping the reactions the remaining activities were determined
using a fluorescence spectrophotometer (Perkin-Elmer, Model
3000). Duplicate experiments were performed.
For CYP2D6 and CYP2C9, preincubation tubes contained
40–49 nM CYP enzyme, 100 ␮M NADPH, 50 and 20 ␮M cur-
cumin solution. Preincubations were performed for 20 min
at 37 ◦C and at time intervals of 5 min the remaining activ-
ities of the enzymes were re-assessed. Aliquots (100 ␮L) of
the 600 ␮L preincubation mixtures were transferred into tubes
containing 400 ␮L dextromethorphan (4.5 ␮M) or diclofenac
(6.0 ␮M) and NADPH (100 ␮M) and incubated at 37 ◦C for
30 or 10 min to determine remaining activity of CYP2D6 or
CYP2C9, respectively.
Fig. 2. HPLC chromatogram of the decomposition mixture of cur-
cumin after incubation for 1 h at pH 7.4: identified products are
indicated by arrows—vanillin (Van), vanillic acid (Vac), ferulic
aldehyde (Fal), ferulic acid (Fac). The major peak most likely
corresponds to trans-6-(4ꢀ-hydroxy-3-methoxyphenyl)-2,4-dioxo-5-
hexenal (Wang et al., 1997).
position products of curcumin towards human CYPs,
decomposition experiments were also performed in the
present study. Curcumin was treated with 0.1 M phos-
phate buffer of pH 7.4 at 37 ◦C for 1 h, according to the
procedure described by Wang et al. (1997). Fig. 2 shows
the HPLC chromatogram of the resulting mixture of
decomposition products of curcumin. Chromatographic
peaks observed included a major peak and seven minor
peaks, four of which were identified as vanillin, vanil-
lic acid, ferulic aldehyde and ferulic acid, by co-eluting
with commercially available reference compounds [data
not shown]. This major decomposition product with
retention time of 16.9 min most likely represents trans-6-
(4ꢀ-hydroxy-3ꢀ-methoxyphenyl)-4-dioxo-5-hexenal, as
the same procedure of decomposition was used as that
described by Wang et al. (1997).
2.7. Data analysis
Percent inhibition of CYP activity by curcumin, its decom-
position products were calculated from the ratio of the activity
oftreatedtocontrolsamples. Statisticalanalysiswasperformed
using the Student’s t-test. The enzyme kinetic parameters
(Km and Vmax) for metabolism of the various substrates and
IC50 values were analyzed using GraphPad Prism 4.0 version
(GraphPad Prism Software Inc., San Diego, CA). The inhibitor
constant (Ki) values for competitive inhibition were calculated
according to the following equation: for competitive inhibition,
Ki = Km(inhibited)[I]/(Km(uninhibited) − Km(inhibited)), where substrate
concentration is equal to the Km, and for non-competitive
inhibition, Ki = Vmax(inhibited)[I]/(Vmax(uninhibited) − Vmax(inhibited)),
where Km, Vmax, S and [I] are Michaelis constant, maximal
enzyme activity, substrate concentration and inhibitor concen-
tration, respectively.
3.2. CYP inhibition and types of inhibition
The inhibitory effects of 300 ␮M concentrations of
curcumin, four of the individual decomposition products
and a complete mixture of decomposition products, on
the activities of CYP1A2, CYP3A4, CYP2D6, CYP2C9
and CYP2B6 are shown in Fig. 3. At this concentra-
tion, curcumin appeared to inhibit almost completely
the activities of CYP3A4, CYP2C9, and CYP1A2,
and 72 and 69.1% of the activities of CYP2D6 and
CYP2B6, respectively. The complete decomposition
mixture and the four decomposition products, vanillin,
vanillic acid, ferulic aldehyde and ferulic acid, showed
milder inhibitory effects on the above CYPs. Percent-
ages of inhibition of CYP activities in the range 1–50%
wereobservedinassayswiththedecompositionproducts
3. Results
3.1. Decomposition of curcumin
Degradation of curcumin under various pH condi-
tions, and the stability of curcumin in physiological
matrices have been previously reported (Wang et al.,
1997). To identify the inhibitory potentials of the decom-

88
R. Appiah-Opong et al. / Toxicology 235 (2007) 83–91
Fig. 3. Inhibition of CYP3A4 (A), CYP1A2 (B), CYP2B6 (C), CYP2C9 (D) and CYP2D6 (E) activities by curcumin, four of its decomposition
products (each at a concentration of 300 ␮M) and a mixture of decomposition products derived from 300 ␮M curcumin after incubation for 1 h at
37 ◦C (pH 7.4). General assay conditions are described under Section 2. The charts represent mean and standard deviations of triplicate experiments.
The symbol ‘#’ indicates statistically significant difference (P < 0.05) from uninhibited reactions, as determined by Student’s t-test. Cur, curcumin;
Van, vanillin; Vac, vanillic acid; Fal, ferulic aldehyde; Fac, ferullic acid.
(Fig. 3). The decomposition mixture caused 57.4 and
74.8% inhibition of CYP3A4 and CYP2C9, respectively.
For the decomposition products showing more than
50% inhibition of enzyme activity at 300 ␮M, the IC50
values were determined (Table 2). The four decompo-
sition products of curcumin that exhibited inhibition of
the CYP isoforms, appeared to be rather weak inhibitors
as shown in Table 2. For curcumin, the inhibition
of the CYPs in decreasing order of potency was
CYP2C9 > CYP3A4 > CYP2B6 > CYP1A2 > CYP2D6.
Results obtained on inhibition of CYP3A4 by curcumin,
using the substrates 7-benzyloxyquinoline (BQ) and
7-benzyloxy-4-trifluoromethylcoumarin (BFC) could
not be analyzed due to interference by curcumin at the
wavelength for detection of the metabolites.
Enzyme kinetic parameters for all CYPs and the
corresponding types of inhibition with curcumin are
shown in Table 3. In the MROD (methoxyresorufin
O-deethylase, with CYP1A2) and BROD (benzyloxyre-
sorufin O-debenzylase, with CYP3A4, CYP2B6) assays,
the presence of curcumin resulted in an increase of
the respective Km values, while the Vmax values did
not change significantly when compared to the con-
trol experiment, thus indicating competitive inhibition
according to Michaelis–Menten’s kinetics. However,
with respect to CYP2D6 and CYP2C9 curcumin caused
significant decreases in Vmax values with no sig-
nificant changes in the Km values, thus indicating
non-competitive inhibition.
Table 2
Concentrations of curcumin and four of its decomposition products
required to reduce the activities of five different human CYPs by 50%
(IC50 value, ␮M)
Enzyme
Curcumin
Ferulic aldehyde
227.5 23.4
CYP1A2
CYP3A4
40.0 12.7
3.3. Mechanism-based inhibition
16.3 1.7a
13.9 3.4b
nd
nd
The effects of preincubation of curcumin with
NADPH-fortified CYP isozymes on inhibition potency
was also evaluated with all five human CYPs. Preincuba-
tion of curcumin for 20 min with NADPH-supplemented
CYP did not increase inhibition of CYP activities by
curcumin in any of the CYP isoforms (data not shown).
Therefore, mechanism-based inhibition does not seem
to occur with any of the CYPs studied.
CYP2D6
CYP2C9
CYP2B6
50.3 2.0
4.3 0.8
24.5 0.8
nd
259.3 22.8
nd
All values are the means standard deviation (S.D.) of at least two
experiments as described in Section 2. nd: not determined due to low
percent inhibition observed (<50%).
a
Substrates used, benzyloxyresorufin.
Substrates used, DBF.
b

R. Appiah-Opong et al. / Toxicology 235 (2007) 83–91
89
Table 3
Enzyme kinetic parameters of the effects of curcumin on human CYP activities
Enzyme
Curcumin (␮M)
Vmax (nM/min/nM)
Km (␮M)
Ki (␮M)
Type of inhibition
Competitive
CYP1A2
43.3
10.9
7.4
3.5
51.0
3.9
11.5
0.8
33.2
14.0
0.0
25.0
0.0
2.5
0.0
45.5
0.0
6.0
0.0
50.0
2.43 0.51
2.27 0.04
0.21 0.02
0.16 0.03
1.68 0.01
0.89 0.04
7.83 0.05
5.14 0.07
0.13 0.04
0.11 0.01
5.0 1.6
7.8 0.8
2.8 0.2
3.8 0.1
3.2 0.1
3.3 0.1
6.1 1.2
6.4 0.2
34.0 10.4
69.0 12.8
CYP3A4a
CYP2D6
CYP2C9
CYP2B6
Competitive
Non-competitive
Non-competitive
Competitive
Values are means S.D. of at least two experiments as described in Section 2. Curcumin concentrations used in the experiments are indicated in
the table.
a
The substrate used in this CYP3A4 inhibition assay was benzyloxyresorufin.
4. Discussion
may have contributed to the two different types of inhi-
bition observed. The insignificant inhibitory activities
of the decomposition products towards the tested CYPs,
clearly indicate that the decomposition products of cur-
cumin are not likely to cause drug–drug interaction at
the level of major drug-metabolizing CYPs. Moreover,
decomposition of curcumin is not likely to occur sig-
nificantly at the low pH in the gut, in addition to the
presence of the stabilizing factors such as enzymes and
GSH (Oetari et al., 1996).
The purpose of this study was to evaluate the
inhibitory potential of curcumin and its decomposition
products on the five important human drug-metabolizing
CYPs, namely CYP1A2, CYP3A4, CYP2B6, CYP2C9
and CYP2D6. Earlier reports on the inhibition of rat liver
microsomal CYPs by curcumin showed that curcumin
is a strong inhibitor of CYP1A and CYP2B (Oetari et
al., 1996; Thapliyal and Maru, 2001). In the present
study, curcumin and its decomposition products were
first screened at a high concentration of 300 ␮M, for their
inhibitory potential towards the five CYPs used. At the
high concentration, compounds exhibiting less than 50%
inhibitory activities were excluded from further tests
withtheparticularCYPs. Curcuminwasfoundtopossess
higher inhibitory potentials against human CYP1A2,
CYP2B6, CYP3A4, CYP2D6 and CYP2C9 than the
decomposition products. However, in contrast to CYP
inhibition data on rat mentioned above, curcumin is a less
potent inhibitor of human CYP1A2 and CYP2B6. These
results support findings that animal data may be poorly
predictive of the human situation (Eagling et al., 1998).
A recent study on inhibitory activities of Indonesian
medicinal plants, showed plant extracts of three curcuma
species possessing 65–73% inhibitory activities towards
CYP3A4and 30–54% inhibition towardsCYP2D6 (Usia
et al., 2006). Although these activities are due to the
whole extracts with components including curcumin
they lend support to our findings that curcumin signif-
icantly inhibits CYP3A4 but less significantly inhibits
CYP2D6. Curcumin inhibited CYP1A2, CYP2B6 and
CYP3A4 competitively, while non-competitive inhibi-
tion was observed with CYP2C9 and CYP2D6. The
structural difference in active sites of the enzymes used
The inhibitory potential of curcumin towards
CYP3A4 (IC50 = 16.3 ␮M and Ki = 7.4 ␮M) could have
implications for drug–drug interactions in the intestines
because of the direct exposure of the intestines to cur-
cumin upon oral administration and the high levels of
CYP3A4 in the intestinal epithelial cells. Inhibition of
CYP3A4 in the intestines upon co-administration of
drugs could result in a significantly increased bioavail-
ability of drugs, and consequently increased plasma
concentrations of drugs, with the potential result of
adverse drug reactions. Inhibition of CYP3A4 by
co-administered drugs has been shown to result in
adverse clinical drug–drug interactions, including fatal-
ities (Honig et al., 1993). For example, concomitant
intake of grapefruit juice with drugs has also been shown
to increase plasma concentrations of many drugs in
humans (Fuhr, 1998). This effect appears to be mediated
mainly by the inhibition of CYP3A4 in the intestinal
wall. It is worth noting that inhibitors of CYP3A4 are
often also known to inhibit P-glycoprotein (P-gp) func-
tion (Wacher et al., 1998), both phenomena have been
suggested to synergistically influence bioavailability of
orally administered agents. Inhibition of P-gp by cur-
cumin at an effective concentration of 15 ␮M has also
been documented (Chearwae et al., 2004). It is antici-

90
R. Appiah-Opong et al. / Toxicology 235 (2007) 83–91
pated that inhibition of CYP3A4 and P-gp by curcumin
may be advantageous in mitigating first pass elimination
of orally administered drugs (Kuppens et al., 2005).
It is still unknown whether inhibition of the activ-
ities of the presently tested human CYPs in the liver
may cause significant systemic drug–drug interactions.
As yet there are no reports on interaction between cur-
cumin and drugs at the level of hepatic CYPs in humans.
Currently available human pharmacokinetic data show
an extremely low exposure of the liver to curcumin
even at very high doses. Plasma concentrations of cur-
cumin and its metabolites in humans were found to be
in nanomolar ranges. High concentrations of curcumin
were found in the faeces (Sharma et al., 2004). This
implies that the inhibitory effect of curcumin on activ-
ities of the CYPs in the liver may be insignificant. The
inhibition parameters determined in the present study,
including IC50 and Ki values, which are important to esti-
mate the CYP-inhibitory potential of curcumin (Bapiro
et al., 2001) are relatively high (in micromolar ranges)
compared to the anticipated amounts of curcumin in the
liver. However, in recent rodent studies it was demon-
strated that repeated oral administration of curcumin
resulted in a two-fold up-regulation of both CYP3A
and P-gp in the liver, whereas a down-regulation of
these proteins was observed in the intestines (Zhang et
al., 2006). The significant increase in the area-under-
curve and decreases of oral clearances of midazolam
and celiprolol, suggest that the effects on the intestinal
activities was more significant than those on the hepatic
activities.
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