Minor furanocoumarins and coumarins in grapefruit peel oil as inhibitors of human cytochrome P450 3A4

Article abstract of DOI:10.1021/np900266m

A new cyclic acetal (1) of marmin (6′,7′-dihydroxy-7- geranyloxycoumarin), two new cyclic acetals (5, 6) of 6′,7′- dihydroxybergamottin, and the known compounds marmin (2), 7-geranyloxycoumarin (3), bergamottin (4), and 6′,7′-dihydroxybergamottin (7) were

Full text of DOI:10.1021/np900266m

1702
J. Nat. Prod. 2009, 72, 1702–1704
Minor Furanocoumarins and Coumarins in Grapefruit Peel Oil as Inhibitors of Human
Cytochrome P450 3A4
Tha¨ıs B. Ce´sar,† John A. Manthey,*,‡ and Kyung Myung‡
School of Pharmaceutical Sciences, UniVersity of Sa˜o Paulo State, Araraquara, Brazil, and the U.S. Citrus and Subtropical Products
Laboratory, USDA-Agricultural Research SerVice-South Atlantic Area, Winter HaVen, Florida 33881
ReceiVed April 30, 2009
A new cyclic acetal (1) of marmin (6′,7′-dihydroxy-7-geranyloxycoumarin), two new cyclic acetals (5, 6) of 6′,7′-
dihydroxybergamottin, and the known compounds marmin (2), 7-geranyloxycoumarin (3), bergamottin (4), and 6′,7′-
dihydroxybergamottin (7) were isolated from grapefruit peel oil. All compounds were tested for inhibitory activity
against intestinal cytochrome P450 3A4, an enzyme involved in the “grapefruit/drug” interactions in humans. Coumarins
(1-3) exhibited negligible inhibitory activity, while the furanocoumarins (4-7) showed potent in vitro inhibitory activity
with IC50 values of 2.42, 0.13, 0.27, and 1.58 µM, respectively.
Grapefruit (Citrus paradisi MacFad.) (Rutaceae) contains many
diverse bergamottin (5-geranyloxypsoralen) derivatives including
trace-occurring mixed-species dimers.1-4 Many of these compounds
have been shown to influence the oral bioavailability of prescription
drugs whose primary metabolic pathways involve the intestinal
cytochrome P450 3A4 (CYP3A4).2,3,5,6 Although most of the main
coumarins and furanocoumarins in grapefruit have been reported,7-9
many minor constituents remain uncharacterized. This is particularly
evident in chromatographic analyses of the nonvolatile constituents
of grapefruit peel oil.10,11 This paper reports our investigation of
minor-occurring furanocoumarins in nonvolatile residues of high-
vacuum distilled grapefruit peel oil and provides information on
three new acetals, including 6′,7′-marmin decanal acetal (1), 6′,7′-
dihydroxybergamottin octanal acetal (5), and 6′,7′-dihydroxyber-
gamottin decanal acetal (6), along with the isolation of the known
marmin (2), 7-geranyloxycoumarin (3), bergamottin (4), and 6′,7′-
dihydroxybergamottin (7).7-10 Neral and geranial acetals of oxy-
peucedanin, 5-(2′,3′-dihydroxyisopentyloxy)psoralen, represent simi-
lar structures reported in lime oil.11
The isolation of (furano)coumarin octanal and decanal acetals
1, 5, and 6 and the known compounds 2-4 and 7 was achieved by
sequential normal- and reversed-phase column chromatography and
semipreparative HPLC of acetone extracts of grapefruit oil non-
volatile residues. 1H and 13C NMR spectra were obtained for
compounds 1-7 in DMSO-d6. NMR data of the known compounds
2-4 and 7 were in agreement with literature values.7-13 Assign-
ments of NMR data for 3, 5, and 6 were based on published
assignments for related compounds.11,13-17
The UV spectrum of 1 closely matched the spectrum of
7-geranyloxycoumarin (3), a major coumarin in grapefruit peel oil.9
The ESIMS spectrum of 1 exhibited an [M + H]+ ion at m/z 471,
major fragment ions at m/z 315, 297, and 163, and neutral losses
of 156, 174, and 308 amu. The major fragment ions at 297 and
163 of 1 are similarly observed in the ESIMS spectrum of 3 (data
1
not shown). HRMS and the 13C and H NMR data of 1 (Tables 1
and 2, respectively) are consistent with a molecular formula of
resonance at 1.23 ppm with a large integration value and the absence
C29H42O5. The intense IR absorption band at 1733 cm-1 and the
of other terminal methyl group resonances other than those of C-10′′
of the alkane substituent and of the geranyl C-3′ and C-7′ methyl
groups. The assignment of an acetal group to C-1′′ of 1 is consistent
with the observation of a 1H NMR resonance at δ 4.77 (H-1′′, t, J
) 4.72 Hz) and the 13C NMR resonance at δ 101.2.11 The 1H NMR
spectrum revealed three signals associated with protons (H-1′ δ
4.67; H-6′ δ 3.44; H-1′′ δ 4.77) covalently bonded to three alkoxy
carbons (C-1′, C-6′, and C-1′′), whereas the 13C NMR data provided
evidence of four alkoxy carbons (C-1′ δ 65.1, C-6′ δ 83.6, C-7′ δ
78.7, and C-1′′ δ 101.2). These data supported a structure lacking
13C NMR resonance at 160.22 ppm are attributed to the presence
of a carbonyl functional group. The presence of a linear alkane
chain is suggested by the intense methylene IR vibration at 2924
cm-1 and the near absence of a methyl stretch at ∼2955 cm-1
.
1
This is in agreement with the broad H NMR methylene proton
* To whom correspondence should be addressed. Phone: 863-293-4133.
Fax: 863-299-8678. E-mail: John.Manthey@ars.usda.gov.
† University of Sa˜o Paulo State.
‡ U.S. Citrus and Subtropical Products Laboratory.
10.1021/np900266m CCC: $40.75 This article not subject to U.S. Copyright. Published 2009 by the Am. Chem. Soc. and the Am. Soc. of Pharmacogn.
Published on Web 08/18/2009

Notes
Journal of Natural Products, 2009, Vol. 72, No. 9 1703
Table 1. 13C NMR Data (δ) for 1, 5, and 6a
Table 2. 1H NMR Data for 1, 5, and 6a
δH (J in Hz)
position
1
5
6
1
2
3
4
4a
5
6
7
position
1
5
6
160.2
160.0
160.0
1
2
3
4
5
6
7
8
112.9
144.3
112.3
129.4
112.2
161.6
101.4
155.3
114.0
139.5
106.9
148.4
112.3
157.4
93.6
152.0
105.4
146.0
69.1
119.3
141.9
36.1
27.2
83.4
78.6
16.3
22.0/23.2
101.1
34.4
25.1
28.9
28.6
31.2
114.1
139.6
107.0
148.2
112.4
157.4
93.7
152.0
105.4
146.1
69.1
119.4
142.0
36.0
27.2
83.4
78.7
16.3
22.1/23.3
101.1
34.4
25.2
28.9
28.9
28.9
6.27, d (9.2)
7.98, d (9.2)
7.61, d (8.6)
6.93, dd (8.8, 2.3)
6.31, d (9.8)
8.17, d (10.0)
6.32, d (9.6)
8.19, d (9.7)
8
8a
6.98, d (2.4)
7.35, s
7.38, s
1′′′
7.33, d (2.2)
8.04, d (2.1)
5.02, dd (1.3, 6.7)
5.46, dd (6.2, 6.8)
7.34, d (2.2)
8.05, d (2.3)
5.02, dd (2.6, 6.8)
5.53, dd (6.1, 6.8)
2′′′
1′′′
2′′′
1′
1′
4.67, d (6.4)
5.46, dd (5.7, 6.6)
2′
65.1
119.1
140.9
36.0
27.2
83.6
78.7
16.4
23.5/23.3
101.2
34.3
3′
2′
4′
1.50, m
2.13, m
1.50, m
2.13, m
1.48, m
2.12, m
3.32, m
3′
5′
4′
6′
3.44, dd (9.7, 3.4)
3.35, dd (9.1, 3.9)
5′
7′
6′
3′-CH3
7′-(CH3)2
1′′
1.74, s
1.15, s
1.67, s
1.66, s
7′
1.00, s; 1.1, s
4.73, t (4.7)
1.22, m
0.99, s; 1.12, s
4.73, t (4.7)
3′-CH3
7′-(CH3)2
1′′
2′′
3′′
4′′
5′′
6′′
7′′
8′′
9′′
10′′
4.77, t (4.7)
2′′-7′′
2′′-9′′
CH3-7′′
CH3-9′′
1.23, m
1.22, m
0.84, s
0.84, s
25.2
0.85, s
28.9/28.6
28.9/28.6
28.9/28.6
28.9/28.6
31.2
a Spectra were recorded in DMSO-d6.
gamottin (7) and subsequently bergaptol. Compound 5 was thus
identified as 6′,7′-dihydroxybergamottin octanal acetal.
23.5
13.8
28.6
31.2
23.5
13.9
22.1
13.9
The UV and ESIMS spectra of 6 also suggest a furanocoumarin
structure with an [M + H]+ ion at m/z 511, with neutral losses of
156, 174, and 308 amu, along with fragment ions at m/z 355, 337,
a Spectra were recorded in DMSO-d6.
and 203. The HRMS and the H and 13C NMR data suggest a
1
protons bonded to C-7′ and the involvement of C-6′ and C-7′ in
the acetal portion of 1. The neutral loss of 156 amu in the ESIMS
spectrum is consistent with the loss of 1-decanal (C10H20O), and
the neutral loss of 174 is consistent with the loss of 1-decandiol
(C10H22O2). Mild acid hydrolysis of 1 produced 7-geranyloxycou-
marin and subsequently 7-hydroxycoumarin (umbelliferone) (data
not shown). Facile hydrolysis under mild acidic conditions supports
the acetal structure.
To further verify this structure, 1 was synthesized from 2 and
decanal using reaction conditions for the synthesis of acetals, similar
to that reported by Feger et al.11 The MS and the 1H and 13C NMR
data of the synthesized product and the originally isolated 1 were
identical. Marmin (2) was previously shown to possess an R-
configuration,18 and thus, 1 was identified as (R)-6′,7′-marmin
decanal acetal.
molecular formula of C31H42O6. Similar to the data for 5, a
comparison of the data of 6 with compounds with related chemical
structures indicates a bergaptol (5-hydroxypsoralen) moiety with a
substituted geranyl side chain. The IR spectrum as well as the 13
C
and 1H NMR spectra (Tables 1 and 2, respectively) of 6 are nearly
identical to those of 5, with the exception of two additional
methylene groups in the linear C10 alkane substituent of 6. The
neutral losses of 156 and 174 amu in the ESIMS spectrum of 6 are
consistent with the loss of 1-decanal (C10H20O) and 1-decandiol
(C10H22O2), respectively. These neutral losses were similarly
observed for 1. Mild acid treatment of 6 produced 6′,7′-dihydroxy-
bergamottin (7) and subsequently bergaptol. The facile hydrolysis
under mild acidic conditions further supports an acetal structure.
These data allowed us to propose the structure of 6′,7′-dihydroxy-
bergamottin decanal acetal.
In contrast to 1, compound 5 exhibited a UV spectrum closely
similar to the furanocoumarins bergamottin (4) and 6′,7′-dihy-
droxybergamottin (7), also isolated from the grapefruit peel oil
nonvolatile residues.7,12,13 The ESIMS spectrum of 5 exhibited an
[M + H]+ ion at m/z 483, major fragment ions at m/z 337 and 203,
and neutral losses of 146 and 280 amu. The major fragment ions
at 337 and 203 are similarly observed in the ESIMS of 4 and 7.
The HRMS and the 13C and 1H NMR data (Tables 1 and 2,
respectively) are consistent with a molecular formula of C29H38O6.
Similar to the data of 1, the IR absorption band at 1732 cm-1 and
the 13C NMR resonance at δ160.0 are attributed to a carbonyl
functional group in 5. An alkane chain in 5 is suggested by the
Compounds 5 and 6 were synthesized from 7 by combining with
octanal and decanal, respectively, using reaction conditions for the
synthesis of acetals.11 These syntheses produced single compounds
that exhibited identical ESIMS and 1H and 13C NMR spectra with
the originally isolated compounds. These results support the octanal
and decanal acetal structures proposed for 5 and 6, respectively.
The absolute configuration of 7 was previously determined as R,20
and thus the absolute configurations of 5 and 6 were similarly
assigned.
The IC50 values for the inhibition of in vitro CYP3A4 by 1-7
(Table 3) show that compounds 5 and 6 are far more potent
CYP3A4 inhibitors (IC50 values of 0.13 and 0.27 µM, respectively)
than the positive controls 4 and 7 (IC50 values of 2.42 and 1.58
µM, respectively). This may be due to the significantly higher
lipophilicity of 5 and 6 due to the additional octanal and decanal
chains. The coumarins, 1-3, are inactive as CYP3A4 inhibitors.
The ease of synthesis of 5 and 6 makes these compounds attractive
model compounds to further investigate the irreversible CYP3A4
inhibition by grapefruit juice furanocoumarins.
1
intense methylene IR vibration at 2922 cm-1 and by the H NMR
data (Table 2) in a manner similar to 1. The proton resonance of
H-4 (δ 8.17) confirms the 5-O substitution.19 Also similar to 1, the
13C and 1H NMR data are consistent with the absence of H-7′ and
with the involvement of C-6′ and C-7′ in the acetal portion of 5.
The neutral loss of 146 amu in the ESIMS spectrum is consistent
with the loss of 1-octandiol (C8H18O2), leaving the fragment ion at
m/z 337 for which we propose the structure of 6′,7′-dehydrober-
gamottin. Mild acid hydrolysis of 5 produced 6′,7′-dihydroxyber-

1704 Journal of Natural Products, 2009, Vol. 72, No. 9
Notes
The chromatograms were recorded at 310 nm and analyzed using
MassLynx software ver. 3.5 (Micromass, Division of Waters Corp.,
Beverly, MA). ESIMS analyses were performed as described previ-
ously.4
Table 3. IC50 (µM) of Cytochrome P450 3A4 Inhibition by
Compounds 1-7a
compound
IC50 values
6′,7′-marmin decanal acetal (1)
marmin (2)
7-geranyloxycoumarin (3)
NDb
ND
ND
Synthesis of 6′,7′-Marmin Decanal Acetal (1), 6′,7′-Dihydroxy-
bergamottin Octanal Acetal (5), and 6′,7′-Dihydroxybergamottin
Decanal Acetal (6). Marmin (15.0 mg), dissolved in 2 mL of acetone,
was combined with 15 µL of decanal and shaken for 15 min at RT.
The solution was injected onto a semipreparative Atlantis C18 column
equilibrated with H2O/MeCN (85:15 (v/v)). A linear gradient to 100%
MeCN was run over 60 min at 5 mL/min. The reaction resulted in 1 as
the sole product, which was collected, analyzed by HPLC-ESIMS, and
submitted to 1H and 13C NMR analyses and melting point determination.
The same methods were used for the syntheses of 5 and 6, using 6′,7′-
dihydroxybergamottin, and octanal and decanal, respectively.
6′,7′- Marmin Decanal Acetal (1): white, amorphous powder; UV
(MeOH) λmax nm (log ε) 324 (4.18); 253 (1.05) nm; IR ν(KBr) cm-1
3073, 2924, 2854, 2733, 1613, 1556, 1506, 1464, 1120, 999, 833;
HRESIMS m/z 493.2914 [M + Na]+ (calcd for C29H42O5Na, 493.2930);
bergamottin (4)
2.42 ( 0.47
0.13 ( 0.06
0.27 ( 0.08
1.58 ( 0.99
6′,7′-dihydroxybergamottin octanal acetal (5)
6′,7′-dihydroxybergamottin decanal acetal (6)
6′,7′-dihydroxybergamottin (7)
a Data represent means ( standard deviations of triplicates. b ND (not
detected) indicates a limited inhibition of CYP3A4 by a compound.
Experimental Section
General Experimental Procedures. Melting points were determined
with a Thomas-Hoover Capillary melting point apparatus. UV spectra
were recorded on a UV-2401 PC Shimadzu UV-visible spectropho-
tometer. FTIR spectra were recorded with a Perkin-Elmer Spectrum
One with compounds applied to KBr IR cards (International Crystals
Lab, Garfield, NJ). Compounds were dissolved in DMSO-d6, and the
1H and 13C NMR spectra were obtained with a 400 MHz Varian INOVA
magnet system with TMS as internal standard. ESIMS were measured
with a Waters Micromass ZQ single quadrupole LC mass spectrometer.
HRESIMS were measured with a Micromass, Inc. Autospec mass
spectrometer (University of Iowa, Iowa City, Iowa). Materials for
column chromatography were silica gel (70-230 mesh; Sigma, St.
Louis, MO) and Redisep normal- and reversed-phase C18 columns
(Teledyne Isco, Inc., Lincoln, NE), attached to either a Horizon Flash
Chromatography System (Biotage, Uppsala, Sweden) or an ISCO
Combiflash 100 Column Chromatography System with a type 11 UV
detector equipped with 340/365 filter. Preparative TLC was run with
tapered, glass precoated silica gel plates GF254 (Analtech, Newark,
NJ).
1
13C NMR see Table 1, and H NMR see Table 2.
6′,7′-Dihydroxybergamottin Octanal Acetal (5): white, amorphous
powder; UV (MeOH) λmax nm (log ε) 310 (4.05), 268 (sh), 260 (sh),
251 (4.17) nm; IR ν(KBr) cm-1 3159, 2967, 2922, 2853, 1732, 1625,
1578, 1455, 1127, 1027; HRESIMS m/z 505.2577 [M + Na]+ (calcd
1
for C29H38O6Na, 505.2566); 13C NMR see Table 1, and H NMR see
Table 2.
6′,7′-Dihydroxybergamottin Decanal Acetal (6): white, amorphous
powder; UV (MeOH) λmax nm (log ε) 314 (4.15), 267 (sh), 260 (sh),
251 (4.14); IR ν(KBr) cm-1 3160, 2967, 2924, 2854, 1734, 1612, 1579,
1456, 1127, 1074; HRESIMS m/z 533.2886 [M + Na]+ (calcd for
C31H42O6Na, 533.2879); 13C NMR see Table 1, and 1H NMR see
Table 2.
Acknowledgment. Mention of a trademark or proprietary product
is for identification only and does not imply a guarantee or warranty
of the product by the U.S. Department of Agriculture.
Grapefruit Oil Residue. The grapefruit nonvolatile residue from
vacuum-distilled oils from mixed grapefruit varieties was obtained from
an industrial source.
Assay for Inhibition of Cytochrome P450 3A4. In vitro CYP3A4
inhibition assays of the (furano)coumarins were conducted using a Vivid
CYP450 screening kit (Invitrogen, Madison, WI) according to the
manufacturer’s protocol. Test compounds were added to 100 µL of
total reaction volume at different final concentrations, which ranged
from 0.24 to 500 ng/100 µL. Two compounds known as CYP3A4
inhibitors, 4 and 7, were used as controls. All assays were performed
using a Synergy HT multidetection microplate reader (BioTek, Wi-
nooski, VT) with three replicates. The IC50 (50% inhibitory concentra-
tion) value of each compound was determined on the basis of
fluorescence measurement (emission at 485 nm and excitation at 530
nm) at 30 min after initiation of reaction. In our experiments,
fluorescence levels were monitored for 30 min using kinetic assay mode
with 3 min intervals. The levels measured at 20 min after initiation of
the reaction remained constant (data not shown), indicating a termination
of the enzymatic reactions.
References and Notes
(1) Fukuda, K.; Ohta, T.; Oshima, Y.; Ohashi, N.; Yoshikawa, M.;
Yamazoe, Y. Pharmacogenet. Genom. 1997, 7, 391–396.
(2) Guo, L. Q.; Fukuda, K.; Ohta, T.; Yamazoe, Y. Drug Metab. Dispos.
2000, 28, 766–771.
(3) Tassaneeyakul, W.; Guo, L. Q.; Fukada, K.; Ohta, T.; Yamazoe, Y.
Arch. Biochem. Biophys. 2000, 378, 356–363.
(4) Manthey, J. A.; Myung, K.; Mertens-Talcott, S.; Derendorf, H.;
Butterweck, V.; Widmer, W. W. Proc. Fla. State Hort. Soc. 2006,
119, 361–366.
(5) Wangensteen, H.; Molden, E.; Christensen, H.; Malterud, K. E. Eur.
J. Clin. Pharmacol. 2003, 58, 663–668.
(6) Paine, M. F.; Criss, A. B.; Watkins, P. B. Drug Metab. Dispos. 2004,
32, 1146–1153.
(7) Fisher, J. F.; Nordby, H. E. J. Food Sci. 1965, 30, 869–873.
(8) Fisher, J. F.; Nordby, H. E.; Waiss, A. C., Jr.; Stanley, W. L.
Tetrahedron 1967, 23, 2523–2528.
Extraction and Isolation. The nonvolatile residue obtained from
grapefruit peel oil was dissolved in acetone (100 g/L), filtered through
100SH/BX filter papers (Fisher Scientific), combined with 400 g of
silica gel, and vacuum evaporated until the furanocoumarin-loaded paste
was a free-flowing powder. The furanocoumarin-loaded silica gel was
packed in a column and washed with hexanes and EtOAc solvent
mixtures [1 L, 1:0; 2:1; 1:1; 1:2; and 0:1 (v/v)]. The resulting fractions
were separately mixed with 100 g of silica gel and dried under vacuum
to obtain powdered samples. The sample-loaded silica gel was subjected
to column chromatography using 40 and 120 g Redisep silica gel
columns and eluted successively with hexanes/EtOAc gradient mixtures
of increasing polarity. Column fractions (30 mL) were collected and
analyzed by HPLC.
Further isolations were performed by preparative TLC using solvent
systems previously described.9 Some compounds were further purified
using an Atlantis (19 × 100 mm) 5 µm semipreparative column (Waters,
Milford, MA) attached to a Waters 600 HPLC controller and 996 PDA.
Fuanocoumarin separations were performed using an 85 min linear
gradient of H2O/MeCN (40:60 to 15:85) at a flow rate of 5 mL/min.
(9) Tatum, J. H.; Berry, R. E. Phytochemistry 1979, 18, 500–502.
(10) Abulrob, A.-N.; Suller, M. T. E.; Gumbleton, M.; Simons, C.; Russell,
A. D. Phytochemistry 2004, 65, 3021–3027.
(11) Feger, W.; Brandauer, H.; Gabris, P.; Ziegler, H. J. Agric. Food Chem.
2006, 54, 2242–2252.
(12) Dreyer, D. L.; Huey, P. F. Phytochemistry 1973, 12, 3011–3013.
(13) Angioni, A.; Cabras, P.; D’Hallewin, G.; Pirisi, F. M.; Reniero, F.;
Schirra, M. Phytochemistry 1998, 47, 1521–1525.
(14) Lee, K. H.; Soine, T. O. J. Pharm. Sci. 1969, 58, 681–683.
(15) Stevenson, P. C.; Simmonds, M. S. J.; Yule, M. A.; Veitch, N. C.;
Kite, G. C.; Irwin, D.; Legg, M. Phytochemistry 2003, 63, 41–46.
(16) Adebajo, A.; Reisch, J. Fitoterapia 2000, 71, 334–337.
(17) Razdan, T. K.; Qadri, B.; Karkar, S.; Waight, E. S. Phytochemistry
1987, 26, 2063–2069.
(18) Edegger, K.; Mayer, S. F.; Steinreiber, A.; Faber, K. Tetrahedron 2004,
60, 583–588.
(19) Gonza´lez, A. G.; Breton, J. L.; Lopez Dorta, H.; Martinez Iniguez,
M. A.; Rodriguez Luis, F. An. Quim. 1973, 69, 1013–1029.
(20) Hosoi, S.; Kamiya, M.; Ohta, T. Org. Lett. 2001, 3, 3659–3662.
NP900266M