Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro

Article abstract of DOI:10.1021/jf0002995

Prenylated flavonoids found in hops and beer, i.e., prenylchalcones and prenylflavanones, were examined for their ability to inhibit in vitro oxidation of human low-density lipoprotein (LDL). The oxidation of LDL was assessed by the formation of conjugate

Full text of DOI:10.1021/jf0002995

3876
J. Agric. Food Chem. 2000, 48, 3876−3884
An tioxid a n t a n d P r ooxid a n t Action s of P r en yla ted a n d
Non p r en yla ted Ch a lcon es a n d F la va n on es in Vitr o
Cristobal L. Miranda,† J an F. Stevens,‡ Vadim Ivanov,§ Mark McCall,§ Balz Frei,§,|
Max L. Deinzer,‡ and Donald R. Buhler*,†
Department of Environmental and Molecular Toxicology, Department of Chemistry, Linus
Pauling Institute, and Department of Biochemistry and Biophysics, Oregon State University,
Corvallis, Oregon 97331
Prenylated flavonoids found in hops and beer, i.e., prenylchalcones and prenylflavanones, were
examined for their ability to inhibit in vitro oxidation of human low-density lipoprotein (LDL). The
oxidation of LDL was assessed by the formation of conjugated dienes and thiobarbituric acid-reactive
substances (TBARS) and the loss of tryptophan fluorescence. At concentrations of 5 and 25 µM, all
of the prenylchalcones tested inhibited the oxidation of LDL (50 µg protein/ml) induced by 2 µM
copper sulfate. The prenylflavanones showed less antioxidant activity than the prenylchalcones,
both at 5 and 25 µM. At 25 µM, the nonprenylated chalcone, chalconaringenin (CN), and the
nonprenylated flavanone, naringenin (NG), exerted prooxidant effects on LDL oxidation, based on
TBARS formation. Xanthohumol (XN), the major prenylchalcone in hops and beer, showed high
antioxidant activity in inhibiting LDL oxidation, higher than R-tocopherol and the isoflavone
genistein but lower than the flavonol quercetin. When combined, XN and R-tocopherol completely
inhibited copper-mediated LDL oxidation. These findings suggest that prenylchalcones and
prenylflavanones found in hops and beer protect human LDL from oxidation and that prenylation
antagonizes the prooxidant effects of the chalcone, CN, and the flavanone, NG.
Keyw or d s: Antioxidants; flavonoids; prenylated chalcones; prenylated flavanones; low-density
lipoprotein
Oxidation of low-density lipoprotein (LDL) is thought
to play a central role in atherosclerosis (Steinberg et
al., 1989; Diaz et al., 1997). Oxidation of LDL can be
initiated by catalytic transition metal ions such as
copper. During the oxidation of LDL, the polyunsatu-
rated fatty acids in the lipoprotein are rapidly converted
to lipid hydroperoxides and aldehydic breakdown prod-
ucts (Esterbauer et al., 1992). LDL oxidation also results
in the oxidative modification of the apoprotein, which
plays a role in macrophage uptake and atherogenesis
(Steinberg et al., 1989; Diaz et al., 1997; Esterbauer et
al., 1992). The mechanism by which Cu2+ oxidizes LDL
in vitro involves the oxidation of LDL-associated R-to-
copherol to the R-tocopheroxyl radical, which subse-
quently initiates lipid peroxidation (Bowry and Stocker,
1993). In addition, preformed lipid hydroperoxides, if
present in LDL, may play a role via the Cu2+-mediated
formation of lipid peroxyl and alkoxyl radicals (Ester-
bauer et al., 1992; Frei and Gaziano, 1993).
scavenge singlet oxygen, superoxide radicals, peroxyl
radicals, hydroxyl radicals, and peroxynitrite (Briviba
and Sies, 1994; Bors et al., 1997). These reactive oxygen
and nitrogen species can react with critical cellular
components such as DNA, lipids, and proteins leading
to tissue injury and contributing to chronic disease. A
high intake of flavonoids has been associated with a
lower incidence of cardiovascular diseases (Hertog et al.,
1993). The low mortality rate from cardiovascular
diseases in populations consuming a high fat diet along
with frequent drinking of red wine (the “French para-
dox”) may be accounted for, in part, by the high content
of polyphenolic compounds in red wine (Renaud et al.,
1992; Constant, 1997).
Recently, several flavonoids such as chalcones and
flavanones with prenyl or geranyl side chains have been
identified in hops and beer (Stevens et al., 1997, 1998,
1999b). As phenolic compounds, these flavonoids may
be responsible for the antioxidant activity of lager beer,
which is higher than that of green tea, red wine, or
grape juice (Vinson et al., 1999). Isoxanthohumol, xan-
thohumol, and 6-prenylnaringenin were the major pre-
nylflavonoids found in beer, accompanied by minor
amounts of 8-prenylnaringenin and 6-geranylnaringenin
(Stevens et al., 1999b). To assess the antioxidant activity
of the prenylated flavonoids, we evaluated their capacity
to inhibit Cu2+-mediated oxidation of LDL by measuring
the formation of conjugated dienes and thiobarbituric
acid-reactive substances (TBARS) and the oxidation of
tryptophan residues. The antioxidant properties of the
prenylflavonoids were compared to those of quercetin
(a flavonol), genistein (an isoflavone), chalconaringenin
Numerous flavonoids have been shown to protect LDL
from Cu2+-catalyzed oxidation (Viana et al., 1996;
Teissedre et al., 1996; Cheng et al., 1998). Flavonoids,
including chalcones (with an open C-ring), are a group
of phenolic compounds with wide distribution in the
plant kingdom. The antioxidant activities of flavonoids
are related to their ability to chelate metal ions and
* Corresponding author. Tel. no.: 541-737-1784. Fax no.:
541-737-0497.
† Department of Environmental and Molecular Toxicology.
‡ Department of Chemistry.
§ Linus Pauling Institute.
| Department of Biochemistry and Biophysics.
10.1021/jf0002995 CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/21/2000

Antioxidant and Prooxidant Prenylflavonoids
J. Agric. Food Chem., Vol. 48, No. 9, 2000 3877
) 15.5 Hz), the B-ring protons (H-2′/6 and H-3′/5′, δH 7.59 and
6.85, both doublets with J ) 8.5 Hz), and the methyl protons
of the three prenyl substituents (δH 1.75, 1.70, 1.64, 1.58, 1.41,
and 1.15, all singlets).
(a nonprenylated chalcone), naringenin (a nonpreny-
lated flavanone), and R-tocopherol. The possible interac-
tion of xanthohumol, the major prenylchalcone in beer,
with R-tocopherol to inhibit Cu2+-mediated LDL oxida-
tion was also examined.
LDL Isola tion . Heparinized blood, collected from healthy,
normolipidemic volunteers, was centrifuged at 1200g for 20
min at 4 °C to obtain plasma. LDL was isolated from plasma
using the method of Chung et al. (1986) as modified by Sattler
et al. (1994). The LDL solution obtained from the single spin
procedure was adjusted to a density of 1.067 g/mL with NaBr
and centrifuged at 540 000g for 14 h at 5 °C. The second
centrifugation step was included to concentrate the LDL and
to ensure that the preparation was free from albumin. Isolated
LDL was desalted by gel filtration [PD-10 column equilibrated
with PBS (10 mM phosphate, 150 mM NaCl, pH 7.4)] and
stored at 4 °C under nitrogen until use.
Assessm en t of Cu 2+-Ca ta lyzed LDL Oxid a tion . The
individual flavonoids (5, 12.5, or 25 µM, in ethanol) or
R-tocopherol (12.5 or 25 µM, in ethanol) were added to LDL
(diluted with PBS to a final protein concentration of 50 µg/
mL) in a total volume of 200 µL/well of a 96-well plate. Ethanol
(1 µL/well) was added to LDL in control wells. The 96-well
plate was placed inside the temperature-controlled (37 °C)
chamber of a microplate reader (SpectraMax 250, Molecular
Devices, Sunnyvale, CA), and the light absorbance at 250 nm
was recorded as zero time values. Copper sulfate (2 µM) was
then added to the individual wells. Negative control wells
contained LDL without Cu2+. To determine if the flavonoids
themselves act as prooxidants, LDL was incubated with these
compounds in the absence of Cu2+. The plate was returned to
the plate reader set at 37 °C. Conjugated diene formation in
each well was measured at 30-min intervals for 5 h by
monitoring the increase in absorbance at 250 nm (Camejo et
al., 1998). Although conjugated diene formation is usually
followed in conventional spectrophotometers using the peak
maximum at 234 nm, measurement of the absorbance at 250
nm with a microtiter spectrophotometer is also an accepted
method (Camejo et al., 1998).
MATERIALS AND METHODS
Ma ter ia ls. The chalcones XN (xanthohumol), XG (xan-
thogalenol), PX (5′- prenylxanthohumol), TP (desmethylxan-
thohumol or 2′,4′,6′,4-tetrahydroxy-3′-C-prenylchalcone), TG
(2′,4′,6′,4-tetrahydroxy-3′-C-geranylchalcone), DX (dehydrocy-
cloxanthohumol), DH (dehydrocycloxanthohumol hydrate),
MXN (4′-O-methylxanthohumol), and MCN (4′,6′-dimethoxy-
2′,4-dihydroxychalcone) and the flavanone IX (isoxanthohumol)
were isolated from hops as described by Stevens et al. (1997,
2000). The prenylated flavanones 6PN (6-prenylnaringenin),
8PN (8-prenylnaringenin), DPN (6,8-diprenylnaringenin), 6GN
(6-geranylnaringenin), and 8GN (8-geranylnaringenin) were
prepared from naringenin and 2-hydroxy-2-methylbut-3-ene
or linalool (Stevens et al., 1999a,b). The remaining chalcones
CN (chalconaringenin), TX (tetrahydroxanthohumol), and DPX
(4′-O-5′-C-diprenylxanthohumol) were prepared as described
below. Naringenin (NG), genistein (GS), quercetin (QC), and
R-tocopherol (TOC) were purchased from Sigma Chemical Co.
(St. Louis, MO). The chemical structures of the individual
flavonoids are shown in Figure 1.
CN was prepared by treatment of NG with 5% NaOH in
MeOH (50 mL) under reflux conditions for 1 h. The reaction
mixture was poured into 200 mL of 2 N HCl, and the aqueous
layer was extracted with ethyl acetate (1 × 150 mL, 2 × 100
mL). The combined ethyl acetate layers were washed with
water (3 × 100 mL), dried over anhydrous Na2SO4, and
evaporated in vacuo. CN was separated from unconverted
naringenin by preparative HPLC, yielding a yellow powder
after lyophilization. UV [λmax (MeOH)]: 365 nm. APCI-MS [m/z
(rel int)]: 273 [MH]+ (100%). NMR [δH (DMSO-d6, 600 MHz)]:
12.52 (s, OH-2′/6′), 10.38 (s, OH-4′), 10.05 (s, OH-4), 7.96 (d,
J ) 15.6 Hz, H-R), 7.65 (d, J ) 15.6 Hz, H-â), 7.52 (d, J ) 8.5
Hz, H-2/6), 6.83 (d, J ) 8.5 Hz, H-3/5), 5.84 (s, H-3′/5′).
TX was obtained from XN by hydrogenation in the presence
of Pd/C. Lyophilization of the TX peak fraction yielded a white
solid. APCI-MS [m/z (rel int)]: 359 [MH]+ (100%), consistent
with C21H26O5. APCI-MS-MS [m/z (rel int)]: 341 [MH - H2O]+
(19%), 253 [C2 - A ring, C14H21O4]+ (95%), 211 [253 - C2H2O]+
(60%), 149 [C3 - B ring, C9H9O2]+ (39%), 107 [149 - C2H2O]+
(100%). NMR [δH (DMSO-d6, 600 MHz)]: 14.19 (s, OH-2′),
10.43 (s, OH-4′), 9.13 (s, OH-4), 7.01 (d, J ) 8.3 Hz, H-2/6),
6.66 d, J ) 8.3 Hz, H-3/5), 6.05 (s, H-5′), 3.78 (s, 6′-OMe), 3.16
(t, J ) 8 Hz, H-R), 2.76 (t, J ) 8 Hz, H-â), 2.43 (t, J ) 8 Hz,
H-1′′), 1.52-1.46 (m, H-3′′), 1.29-1.26 (m, H-2′′), 0.89 and 0.88
(2 × s, Me-4′′ and Me-5′′).
After 5 h of incubation, the amount of TBARS formed in
the sample was measured as described by Buege and Aust
(1978) with some modifications. The content of each well was
transferred to a 1.5-ml microcentrifuge tube containing 20 µL
of ice-cold 50% trichloroacetic acid. The tubes were vortexed,
and then 100 µL of 1% thiobarbituric acid in 0.28% NaOH was
added to each tube. The samples were acidified by the addition
of 20 µL of 1 N HCl. The tubes were heated at 90 °C for 20
min and centrifuged at 16 000g for 5 min. An aliquot (200 µL)
of the supernatant was transferred to a 96-well plate, and the
absorbance at 535 nm was measured using a microplate reader
(SpectraMax 250).
To assess the effects of the flavonoids on Cu2+-mediated
oxidation of tryptophan residues in LDL, the fluorescence of
the LDL samples was read at excitation and emission wave-
lengths of 280 and 331 nm, respectively, in a Hitachi F-2500
spectrofluorometer (Keaney and Frei, 1994).
DPX was prepared from XN and 2-hydroxy-2-methylbut-3-
ene by following a procedure described by J ain et al. (1978).
The reaction mixture, containing XN, PX, DPX, and other
prenylated xanthohumols, was submitted to preparative HPLC.
DPX was obtained as a yellow-orange solid, Mr 490, by APCI-
MS, consistent with C31H38O5. APCI-MS-MS [m/z (rel int)]:
491 [MH]+ (100%), 435 [MH - C4H8]+ (36%), 371 [A]+ (21%),
315 [371 - C4H8]+ (20%), 247 [315 - C5H8]+ (8%), 191 [247 -
C4H8]+ (24%). Because a C-prenyl function gives rise to loss of
56 mass units (C4H8) and an O-prenyl to loss of 68 mass units
(C5H8), it was determined from the fragmentation pattern that
the compound carries two C-prenyls (one C4H8 released from
MH+ and two C4H8 neutrals from the retro Diels-Alder A-ring
fragment with m/z 371) and one O-prenyl substituent. Loss of
120 mass units from the molecular ion, [MH]+, and the absence
of a fragment with [MH - 68]+, indicated that the O-prenyl
substituent was located at one of the two free A-ring hydroxyls.
Because the 1H NMR spectrum showed a hydrogen-bonded OH
signal at δH 13.89 (OH-2′) and one other OH signal (OH-4, δH
10.10), the position of the O-prenyl substituent was determined
to be at C-4′. Other resonances were assigned to the bridge
protons (H-R and H-â, δH 7.78 and 7.73, both doublets with J
Ch ela tion of Cop p er by F la von oid s. Copper chelation
by flavonoids was determined by the spectral shifts produced
when copper ions were incubated with the test compounds.
The reaction mixtures consisted of 0.5 mL of phosphate buffer
(0.2 M, pH 7.4), 0.5 mL of acetonitrile, 50 µL of flavonoid
solution (2 mM in DMSO), 50 µL of CuSO4 solution (1 mM in
water), and He-degassed water to a total volume of 10 mL.
The Cu2+ and flavonoid concentrations in these mixtures were
5 and 10 µM, respectively. The flavonoids tested were CN, XN,
XG, TP, IX, 8PN, and NG. Spectra (200-600 nm) were
recorded immediately after preparation of the mixtures and
again after 10 min, in the presence and absence of copper ions.
Sta tistica l An a lysis. Multiple mean comparisons were
performed by one-way analysis of variance (ANOVA) followed
by the least significant difference (LSD) test using the SAS
computer software. Statistical significance was accepted if the
null hypothesis was rejected at the p < 0.05 level.

3878 J. Agric. Food Chem., Vol. 48, No. 9, 2000
Miranda et al.
F igu r e 1. Chemical structures of flavonoids and R-tocopherol: CH, chalcone; FL, flavanone; XN, xanthohumol; XG, xanthogalenol;
TP, 2′,4′,6′,4-tetrahydroxy-3′-C-prenylchalcone (desmethylxanthohumol); TG, 2′,4′,6′,4-tetrahydroxy-3′-C-geranylchalcone); DX,
dehydrocycloxanthohumol; DH, dehydrocycloxanthohumol hydrate; PX, 5′-prenylxanthohumol; TX, tetrahydroxanthohumol; DPX,
4′-O-5′-C-diprenylxanthohumol; CN, chalconaringenin; IX, isoxanthohumol; 6PN, 6-prenylnaringenin; 8PN, 8-prenylnaringenin;
DPN, 6,8-diprenylnaringenin; NG, naringenin; GS, genistein; MCN, 4′,6′-dimethoxy-2′,4-dihydroxychalcone; MXN, 4′-O-
methylxanthohumol; 6GN, 6-geranylnaringenin, 8GN, 8-geranylnaringenin; QC, quercetin; TOC, R-tocopherol.

Antioxidant and Prooxidant Prenylflavonoids
J. Agric. Food Chem., Vol. 48, No. 9, 2000 3879
F igu r e 2. Conjugated diene formation in human LDL (50 µg
protein/mL) incubated with 2 µM Cu2+ in the presence or
absence of prenylated or nonprenylated chalcones at a con-
centration of 5 µM (A) or 25 µM (B). Results represent the
mean of three determinations.
F igu r e 3. Conjugated diene formation in human LDL (50 µg
protein/mL) incubated with 2 µM Cu2+ in the presence or
absence of prenylated or nonprenylated flavanones and isofla-
vone (GS) at a concentration of 5 µM (A) or 25 µM (B). Results
represent the mean of three determinations.
RESULTS
At a concentration of 25 µM, quercetin, XN (Figure
2B), and several other prenylchalcones (data not shown)
completely inhibited the Cu2+-mediated formation of
conjugated dienes during the 5-h incubation period.
MXN, which contains two methoxy groups, was almost
as inhibitory as XN, which contains only one methoxy
group (Figure 2B). However, the nonprenylated chal-
cone, CN, at 25 µM showed only a slight inhibitory
activity toward conjugated diene formation, whereas the
nonprenylated chalcone, MCN, which contains two
methoxy groups, strongly inhibited (Figure 2B).
Effects of P r en yla ted Ch a lcon es on Con ju ga ted
Dien e F or m a tion in LDL. When freshly isolated
human LDL (50 µg protein/mL) was incubated with 2
µM Cu2+ at 37 °C, the typical time course of conjugated
diene formation was observed (Esterbauer et al., 1992),
consisting of a lag phase, a propagation phase, and a
decomposition phase (Figure 2). Under these incubation
conditions, the lag time of LDL oxidation in the absence
of flavonoids was approximately 2.0 h, and the maxi-
mum absorbance was about 0.19 absorbance units,
reached after 4.5 h of incubation (Figure 2). Addition of
prenylchalcones (5 µM) increased the lag time and
decreased the rate of conjugated diene formation during
the propagation phase (Figure 2A). The three most
effective prenylchalcones were TG, XN, and TP, which
decreased conjugated diene formation by more than 70%
after 5 h of incubation compared to the vehicle control
(LDL and Cu2+). The nonprenylated chalcone, CN, had
no effect on LDL oxidation whereas QC (positive control)
completely prevented the formation of conjugated dienes
in LDL incubated for up to 5 h with Cu2+ (Figure 2A).
Furthermore, when LDL was incubated with the fla-
vonoids for 5 h in the absence of Cu2+, no increase in
conjugated diene formation was observed (data not
shown).
Effects of P r en yla ted F la va n on es on Con ju ga ted
Dien e F or m a tion in LDL. In contrast to the prenyl-
chalcones (Figure 2A), the prenylflavanones at a con-
centration of 5 µM had very little effect on Cu2+
-
mediated LDL oxidation, except for 8GN (Figure 3A).
At a concentration of 25 µM, the prenylflavanones were
more effective at inhibiting LDL oxidation (Figure 3B).
The nonprenylated flavanone, NG, at 25 µM also slightly
increased the lag time of conjugated diene formation but
was considerably less effective than the prenylfla-
vanones. Interestingly, NG and the prenylflavanones
investigated, except for 6PN, appeared to exert a slight
prooxidant effect during the initial 30 min of incubation
of LDL with Cu2+. In fact, 25 µM 8GN exerted a strong

3880 J. Agric. Food Chem., Vol. 48, No. 9, 2000
Miranda et al.
Ta ble 1. Effect of P r en yla ted a n d Non p r en yla ted
F la von oid s on Cu 2+-Dep en d en t Lip id P er oxid a tion in
Hu m a n Low -Den sity Lip op r otein (LDL)a
TBARS (A535
)
addition to LDL
5 µM flavonoid
0.030 ( 0.002
25 µM flavonoid
0.020 ( 0.001
none
Cu2+ (control)
0.081 ( 0.001a (100) 0.075 ( 0.001a (100)
Cu2+ + chalcone
CN
MCN
TP
XG
XN
PX
MXN
0.121 ( 0.003b (140) 0.112 ( 0.001b (149)
0.080 ( 0.002a (98.8) 0.044 ( 0.004c (58.7)
0.097 ( 0.003c (120) 0.030 ( 0.001d (40.0)
0.083 ( 0.003a (102) 0.022 ( 0.001e (29.3)
0.055 ( 0.002d (67.9) 0.020 ( 0.001e,f (26.6)
0.026 ( 0.001e (32.1) 0.005 ( 0.001g,h (6.7)
0.040 ( 0.001f (49.4) 0.019 ( 0.001e,f (25.3)
0.042 ( 0.001f (51.9) 0.012 ( 0.001i (16.0)
TG
Cu2+ + flavanone
NG
8PN
DPN
8GN
0.062 ( 0.002g (76.5) 0.120 ( 0.003j (160)
0.080 ( 0.001a (98.8) 0.032 ( 0.001d (42.7)
0.076 ( 0.001h (93.8) 0.056 ( 0.004k (74.7)
0.062 ( 0.002g (76.5) 0.009 ( 0.001h,i (12.0)
0.068 ( 0.001i (83.9) 0.048 ( 0.001c (64.0)
IX
Cu2+ + isoflavone
GS
0.050 ( 0.001j (61.7) 0.069 ( 0.001l (92.0)
0.019 ( 0.003k (23.4) 0.014 ( 0.001f,i (18.7)
Cu2+ + flavonol
QC
a
Results are expressed as mean values ( SE for four determi-
nations. TBARS analysis was performed on human LDL (50 µg
protein/mL) exposed to 2 µM Cu2+ and 5 or 25 µM flavonoid for
24 h at 25 °C. Values in the same column followed by the same
letter are not significantly different at p < 0.05. Values in
parentheses indicate percent of control (LDL plus Cu2+).
prooxidant effect, with no observable lag phase of diene
conjugation (Figure 3B). Unlike the prenylchalcones,
none of the prenylflavanones was able to completely
prevent the oxidation of LDL catalyzed by Cu2+ during
the 5-h incubation period.
Effects of P r en yla ted Ch a lcon es a n d F la va n on es
on TBARS F or m a tion in LDL. The effects of prenyl-
chalcones on Cu2+-mediated LDL oxidation were further
evaluated by measuring TBARS levels after incubation
for 5 h at 37 °C. At a concentration of 5 µM, the
prenylchalcones and QC were ranked as follows (using
ANOVA) in terms of their ability to inhibit TBARS
formation: QC > TG > PX, XN, TP, TX > DH, MXN,
DX, XG, DPX. These data are in full agreement with
those on conjugated diene formation (see Figure 2A).
The nonprenylated chalcone, CN, had no inhibitory
effect at 5 µM and at 25 µM significantly increased
TBARS formation by 6%. In contrast to the prenylchal-
cones, the prenylflavanones at a concentration of 5 µM
had no inhibitory effect, except for 8GN and DPN. At a
concentration of 25 µM, the prenylflavanones and GS
significantly inhibited TBARS formation and were
ranked as follows: 8GN > GS > DPN, 6GN, 8PN > IX,
6PN. These data are consistent with those on diene
conjugation (see Figure 3). The nonprenylated fla-
vanone, NG, at 25 µM, significantly increased TBARS
levels by 13%.
The effects of the flavonoids on TBARS formation
were also assessed following incubation of LDL with 2
µM Cu2+ at 25 °C for 24 h (Table 1). At 5 µM, the
prenylchalcones PX, MXN, TG, and XN and the pre-
nylflavanones 8GN, IX, and DPN significantly inhibited
LDL oxidation. XG and 8PN had no effect, whereas CN
and TP stimulated TBARS formation. At 25 µM, CN and
NG acted as strong prooxidants and significantly in-
creased TBARS levels by 49 and 60%, respectively,
while MCN acted as an antioxidant (Table 1). The
F igu r e 4. Conjugated diene formation in human LDL (50 µg
protein/mL) incubated with 2 µM Cu2+ in the presence or
absence of xanthohumol or R-tocopherol, alone or in combina-
tion, at a concentration of 12.5 µM (A) or 25 µM (B). Results
represent the mean of three determinations.
chalcones with prenyl (PX, MXN, XN, XG, and TP) or
geranyl (TG) side chains significantly inhibited Cu2+
-
induced formation of TBARS, as did the flavanones with
prenyl (8PN, IX, and DPN) or geranyl (8GN) side chains.
At 25 µM, GS was less effective than the prenylchal-
cones in inhibiting TBARS formation, whereas QC was
as inhibitory as XN, MXN, TG, and 8GN (Table 1).
Effects of r-Tocop h er ol, Alon e or in Com bin a -
tion w ith XN, on LDL Oxid a tion . R-Tocopherol (TOC)
is known to protect LDL from Cu2+-mediated oxidation,
if the Cu2+:LDL ratio is relatively high (Bowry and
Stocker, 1993; Keaney and Frei, 1994). It was of interest
to compare the relative antioxidant activity of TOC and
XN, the major chalcone in hops and in some beers. TOC
was used at concentrations of 12.5 and 25 µM, which
are within the physiological range in human plasma.
At 12.5 µM, TOC extended the lag time of conjugated
diene formation from 1.2 to 2.5 h (Figure 4A). In
contrast, 12.5 µM XN extended the lag time over 5 h.
Equimolar concentrations (12.5 µM) of TOC and XN
inhibited conjugated diene formation to a similar extent
as XN alone throughout the 5-h incubation period
(Figure 4A). At 25 µM, TOC alone or XN alone almost
completely inhibited the formation of conjugated dienes
(Figure 4B). When TOC and XN were combined to-

Antioxidant and Prooxidant Prenylflavonoids
J. Agric. Food Chem., Vol. 48, No. 9, 2000 3881
F igu r e 5. Effects of xanthohumol (XN) and R-tocopherol (TO),
alone or in combination, on Cu2+-mediated LDL oxidation as
assessed by the formation of thiobarbituric acid-reactive
substances. XN and TO were used at low (12.5 µM each, XN-L
and TO-L) or high (25 µM each, XN-H and TO-H) concen-
trations in reaction mixtures containing LDL (50 µg protein/
mL) and 2 µM Cu2+. Results represent the mean ( SE of three
determinations. Bars labeled with different letters are signifi-
cantly different from each other, p < 0.05.
F igu r e 6. Tryptophan fluorescence (excitation ) 280 nm,
emission ) 331 nm) in human LDL (50 µg protein/mL)
incubated with 2 µM Cu2+ in the presence or absence of 25
µM flavonoids.
gether, there was complete inhibition of conjugated
diene formation (Figure 4B).
The effects of XN, alone or in combination with TOC,
on TBARS formation following 5 h of incubation of LDL
with 2 µM Cu2+ are shown in Figure 5. At 12.5 µM, TOC
appeared to slightly increase TBARS levels, whereas XN
strongly inhibited. TBARS formation was significantly
lower in the presence of both TOC and XN compared to
each compound alone. Similar results were obtained at
the higher concentration (25 µM) of XN and TOC
(Figure 5).
Tr yp top h a n Oxid a tion in LDL. The reduction in
tryptophan fluorescence (excitation/emission ) 280/331
nm) was used to evaluate the protective effects of
flavonoids on Cu2+-mediated protein oxidation in LDL
during incubation at 37 °C. Copper causes rapid oxida-
tion of LDL tryptophan residues which can be monitored
fluorometrically (Giessauf et al., 1995). As shown in
Figure 6, copper (2 µM) caused an almost linear
decrease in LDL tryptophan fluorescence with increas-
ing time of incubation. The flavonoids XN, TP, TG, and
QC inhibited tryptophan oxidation, in contrast to CN,
NG, 8PN, and IX.
Ch ela tion of Cu 2+ by F la von oid s. Flavonoids dis-
play characteristic spectra that undergo changes upon
interaction with metal ions. For example, the chalcone
butein exhibits maxima at 260 and 395 nm, which shift
to 286 and 454 nm in the presence of Cu2+ ions (Cheng
et al., 1998). In the present study, however, little or no
change of the spectra was observed for XN, IX, 8PN,
MXN, PX, and DPX (data not shown). The 2′,6′-dihy-
droxychalcones CN, XG, and TP developed new maxima
around 290 nm over a 10-min period, which was
attributed to conversion of the chalcones to their iso-
meric flavanones rather than chelation of copper ions.
phase. Furthermore, the prenylchalcones inhibited the
formation of TBARS and the oxidation of tryptophan
residues in LDL, providing additional evidence for
antioxidant activity of these compounds. The prenylfla-
vanones also displayed antioxidant activity but were not
as effective as the prenylchalcones. These findings
suggest that prenylchalcones and, to a lesser extent,
prenylflavanones provide protection against oxidative
modification of LDL.
Prenylated chalcones and flavanones are found in
hops and beer. Vinson et al. (1995, 1999) compared
several beverages, including beer, for antioxidant activ-
ity toward Cu2+-induced LDL oxidation. On the basis
of the formation of TBARS, the antioxidant activity of
the beverages was ranked as follows: black tea > coffee
> prune juice ) beer > green tea > orange juice > red
wine > tangerine juice > red grape juice > white grape
juice > grapefruit juice (Vinson et al., 1999). The
antioxidant activity of the fruit juices and tea could be
explained by their high content of polyphenolic fla-
vonoids. However, the specific compounds responsible
for the antioxidant activity in beer have not been
identified. The prenylated chalcone XN and the preny-
lated flavanones IX, 6PN, 8PN, and 6GN, are polyphe-
nolic compounds recently identified in beer (Stevens et
al., 1999b). The concentrations of these prenylflavonoids
vary with the type of beer (Stevens et al., 1998). For
example, XN and IX, a flavanone isomer of XN, have
been reported to be present in beer at concentrations of
0.69 mg/L (1.95 µM) and 3.44 mg/L (9.7 µM), respec-
tively, and the total prenylflavonoid content of beer
could be as high as 4 mg/L, or approximately 11 µM
(based on the molecular weight of XN and IX of 354 Da).
The prenylflavanone constituents of beer do not origi-
nate directly from hops but from the isomerization of
the prenylchalcones in hops during the brewing process
(Stevens et al., 1999b). These prenylflavonoids and other
phenolic compounds, such as glycosides of kaempferol
and quercetin and catechins (Vinson et al., 1995; Sa¨-
DISCUSSION
The results of the present study show that prenylated
chalcones are capable of inhibiting Cu2+-mediated oxi-
dation of LDL. The prenylchalcones prolonged the lag
phase of conjugated diene formation and reduced the
rate of LDL lipid peroxidation during the propagation

3882 J. Agric. Food Chem., Vol. 48, No. 9, 2000
Miranda et al.
gesser et al., 1996), may all contribute to the antioxidant
activity of beer.
antioxidant activities of substituted naringenins and
chalconaringenins.
Studies on structure-activity relationships have shown
that the presence of hydroxyl groups and methoxy
groups in the A and B rings appear to be important in
antioxidant and free radical scavenging activities of
chalcone compounds (Anto et al., 1995). Butein, a
chalcone with a catechol in the B ring and two hydroxyl
The antioxidant activity of the prenylflavonoids may
also involve the scavenging of lipid peroxyl radicals,
resulting in the inhibition of the propagation of lipid
peroxidation in LDL. Like other flavonoids, the preny-
lated chalcones and flavanones contain phenolic hy-
droxyl groups that likely act as hydrogen donors in the
scavenging of free radicals (Neuzil et al., 1997). Evidence
for the ability of prenylchalcones to scavenge peroxyl
radicals is provided by the inhibition of lipid peroxida-
tion in rat liver microsomes induced by the radical
generator AAPH (Rodriguez et al., 1999). In addition,
prenylflavonoids may neutralize preformed lipid hydro-
peroxides; e.g., the peroxides may be reduced by hydro-
gens donated from phenolic hydroxy groups.
R-Tocopherol, the most abundant lipid-soluble anti-
oxidant in human plasma and lipoproteins, exerts
antioxidant and prooxidant activity in isolated LDL
depending on the incubation conditions (Bowry and
Stocker, 1993; Neuzil et al., 1997; Kontush et al., 1996).
Under strong oxidative conditions in the presence of
high amounts of Cu2+, TOC supplementation shows
antioxidant activity. Under conditions of mild oxidation,
TOC acts as an antioxidant in the presence of co-
antioxidants, such as ubiquinol-10 and ascorbate, but
as a prooxidant in their absence (Bowry and Stocker,
1993; Neuzil et al., 1997). Ascorbate is known to
regenerate TOC from the potentially prooxidant R-to-
copheroxyl radical. In the present study, we found
evidence for an interaction between TOC and a prenyl-
chalcone, XN, in the protection against Cu2+-mediated
LDL oxidation. When combined, TOC and XN inhibited
the formation of conjugated dienes and TBARS to a
greater extent than either TOC or XN alone. Further
studies are required to determine whether this repre-
sents a synergistic interaction or an additive effect and
whether XN can regenerate TOC from the R-toco-
pheroxyl radical.
groups in the A ring, is a potent inhibitor of Cu2+
-
mediated oxidation of LDL (Cheng et al., 1998). Another
chalcone, isoliquiritigenin, with two hydroxyl groups in
the A ring and one hydroxyl group in the B ring (phenol
B ring), also is a strong inhibitor of 2,2′-azobis(2-
amidinopropane) dihydrochloride (AAPH)-induced oxi-
dation of LDL (Vaya et al., 1997). The prenylated
chalcone isolated from licorice roots, isoprenylchalcone,
likewise inhibited LDL oxidation induced by AAPH
(Vaya et al., 1997).
In the present study, we have shown that prenyl
groups may have a marked influence on the antioxidant
activities of the different prenylflavonoids containing
phenol B rings. Specifically, prenylation may endow
hydroxylated chalcones with enhanced antioxidant ac-
tivity. This is exemplified by TP and TG that showed
very high antioxidant activity as opposed to CN, a
nonprenylated hydroxylated chalcone. CN even acted
as a prooxidant by increasing TBARS formation during
incubation of LDL with Cu2+. The addition of a methoxy
group also confers antioxidant activity to the chalcones,
since 25 µM MCN (a chalcone with two methoxy
groups), unlike 25 µM CN, effectively inhibited forma-
tion of conjugated dienes and TBARS. The prenyl and
geranyl groups also appeared to be important in the
antioxidant activity of flavanones at high concentration
(25 µM) because the nonprenylated flavanone, NG, acted
as a prooxidant, whereas 8PN, DPN, and 8GN inhibited
LDL oxidation.
The mechanisms by which prenyl and methoxy groups
enhance the antioxidant capacity of chalcones and
flavanones remain to be established. It is conceivable
that C-prenylation and O-methylation add lipophilicity
to the flavonoids, NG and CN, which could result in an
increase of the flavonoid concentration in the lipophilic
environment within the LDL particle due to “lipid-
lipid” partitioning or noncovalent interactions. Model
experiments with XN and IX have shown that the
“solubility” of both flavonoids is much higher in wort
(27 and 122 µM, respectively) than in water (3.7 and
14 µM), which indicates that a significant proportion of
these flavonoids is complexed in wort with macromol-
ecules, mainly carbohydrates and proteins (Stevens et
al., 1999a). It is in the lipophilic environment of the LDL
particles where peroxidation of unsaturated fatty acids
takes place, and hence, protection by the prenylated
flavonoids against lipid peroxidation is most effective.
Like R-tocopherol, flavonoids may behave as prooxi-
dants under certain conditions. The flavonols quercetin
and myricetin accelerate the generation of hydroxyl
radicals in reaction mixtures containing hydrogen per-
oxide and Fe3+-EDTA, as measured by deoxyribose
degradation (Laughton et al., 1989). Depending upon
the number of hydroxyl groups in their backbone
structure, flavones, isoflavones, or flavanones may exert
prooxidant activities in the presence of Cu2+ ions (Cao
et al., 1997). Catechins found in green tea, such as (-)-
epicatechin and (-)-epigallocatechin, accelerate Cu2+
-
mediated LDL oxidation during the propagation phase
(Yamanaka et al., 1997). Other flavonoids also acceler-
ate Cu2+-mediated LDL oxidation when added during
the propagation phase of lipid peroxidation (Otero et al.,
1997). Upon activation by peroxidases, the flavanone
NG and the flavone apigenin become prooxidants and
cause lipid peroxidation and extensive oxidation of
glutathione (Galati et al., 1999) and NADH (Chan et
al., 1999) at physiological pH. In the presence of a
peroxidase, flavonoid compounds with phenol B rings
may form prooxidant phenoxyl radicals, which during
redox cycling can generate superoxide radicals and
hydrogen peroxide. Naringenin also induced a concen-
tration-dependent peroxidation of membrane lipids
concurrent with DNA strand breaks in isolated rat liver
nuclei (Sahu and Gray, 1997). In the present study, NG
and CN, with phenol B rings, showed a tendency to act
Copper chelation has been proposed as a possible
mechanism for the antioxidant activities of quercetin
(Afanas’ev et al., 1989) and butein (Cheng et al., 1998).
Though representatives of different flavonoid classes,
quercetin and butein have a B ring catechol moiety in
common, which is known to be important for Cu2+
-
chelate formation (Brown et al., 1998). None of the
flavonoids investigated in this study, however, carry
3′,4′-dihydroxy substituents on the B ring, which may
explain why only small spectral shifts or no shifts were
observed in the presence of copper ions. Copper chela-
tion is therefore considered to play a minor role in the

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activity was not observed in chalcones and flavanones
with prenyl or geranyl groups, suggesting that these
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ABBREVIATIONS USED
CN, chalconaringenin; DH, dehydrocycloxanthohumol
hydrate; DPN, 6,8-diprenylnaringenin; DPX, 4′-O-5′-C-
diprenylxanthohumol; DX, dehydrocycloxanthohumol;
6GN, 6-geranylnaringenin; 8GN, 8-geranylnaringenin;
GS, genistein; IX, isoxanthohumol; MCN, 4′,6′-dimethoxy-
2′,4-dihydroxychalcone; MXN, 4′-O-methylxanthohumol;
NG, naringenin; 6PN, 6-prenylnaringenin; 8PN, 8-pre-
nylnaringenin; PX, 5′- prenylxanthohumol; QC, quer-
cetin; TG, 2′,4′,6′,4-tetrahydroxy-3′-C-geranylchalcone;
TOC, R-tocopherol; TP, 2′,4′,6′,4-tetrahydroxy-3′-C-pre-
nylchalcone (desmethylxanthohumol); TX, tetrahydrox-
anthohumol; XG, xanthogalenol; XN, xanthohumol.
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M. B.; Kromhout, D. Dietary antioxidant flavonoids and risk
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This work was supported by the Linus Pauling Institute at
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