Carcinogenesis vol.20 no.9 pp.1737–1745, 1999
Anti-tumor-promoting activity of a polyphenolic fraction isolated
from grape seeds in the mouse skin two-stage initiation–promotion
protocol and identification of procyanidin B5-39-gallate as the
most effective antioxidant constituent
Jifu Zhao1, Jiannong Wang2, Yingjie Chen2 and
Rajesh Agarwal1,3,4
1Center for Cancer Causation and Prevention, AMC Cancer Research
Center, Denver, CO 80214, USA, 2Shenyang Pharmaceutical University,
Shenyang 110015, People’s Republic of China and 3University of Colorado
Cancer Center, University of Colorado Health Sciences Center, Denver, CO
80262, USA
4To whom correspondence should be addressed at: AMC Cancer Research
Center, 1600 Pierce Street, Denver, CO 80214, USA
Email: [email protected]
Procyanidins present in grape seeds are known to exert
anti-inflammatory, anti-arthritic and anti-allergic activities,
prevent skin aging, scavenge oxygen free radicals and
inhibit UV radiation-induced peroxidation activity. Since
most of these events are associated with the tumor promotion stage of carcinogenesis, these studies suggest that grape
seed polyphenols and the procyanidins present therein
could be anticarcinogenic and/or anti-tumor-promoting
agents. Therefore, we assessed the anti-tumor-promoting
effect of a polyphenolic fraction isolated from grape seeds
(GSP) employing the 7,12-dimethylbenz[a]anthracene
(DMBA)-initiated and 12-O-tetradecanoylphorbol 13-acetate
(TPA)-promoted SENCAR mouse skin two-stage carcinogenesis protocol as a model system. Following tumor initiation with DMBA, topical application of GSP at doses of
0.5 and 1.5 mg/mouse/application to the dorsal initiated
mouse skin resulted in a highly significant inhibition of
TPA tumor promotion. The observed anti-tumor-promoting
effects of GSP were dose dependent and were evident in
terms of a reduction in tumor incidence (35 and 60%
inhibition), tumor multiplicity (61 and 83% inhibition) and
tumor volume (67 and 87% inhibition) at both 0.5 and
1.5 mg GSP, respectively. Based on these results, we
directed our efforts to separate and identify the individual
polyphenols present in GSP and assess their antioxidant
activity in terms of inhibition of epidermal lipid peroxidation. Employing HPLC followed by comparison with
authentic standards for retention times in HPLC profiles,
physiochemical properties and spectral analysis, nine individual polyphenols were identified as catechin, epicatechin,
procyanidins B1–B5 and C1 and procyanidin B5-39-gallate.
Five of these individual polyphenols with evident structural
differences, namely catechin, procyanidin B2, procyanidin
B5, procyanidin C1 and procyanidin B5-39-gallate, were
assessed for antioxidant activity. All of them significantly
inhibited epidermal lipid peroxidation, albeit to different
levels. A structure–activity relationship study showed that
with an increase in the degree of polymerization in
Abbreviations: C, catechin; DMBA, 7,12-dimethylbenz[a]anthracene; EC,
epicatechin; ECG, epicatechin 3-gallate; EGC, epigallocatechin; EGCG, epigallocatechin 3-gallate; GSP, polyphenolic fraction isolated from grape seeds;
MDA, malondialdehyde; MS, mass spectra; TBA, thiobarbituric acid; TPA,
12-O-tetradecanoylphorbol 13-acetate.
© Oxford University Press
polyphenol structure, the inhibitory potential towards lipid
peroxidation increased. In addition, the position of linkage
between inter-flavan units also influences lipid peroxidation
activity; procyanidin isomers with a 4–6 linkage showed
stronger inhibitory activity than isomers with a 4–8 linkage.
A sharp increase in the inhibition of epidermal lipid
peroxidation was also evident when a gallate group was
linked at the 39-hydroxy position of a procyanidin dimer.
Procyanidin B5-39-gallate showed the most potent antioxidant activity with an IC50 of 20 µM in an epidermal
lipid peroxidation assay. Taken together, for the first time
these results show that grape seed polyphenols possess high
anti-tumor-promoting activity due to the strong antioxidant
effect of procyanidins present therein. In summary, grape
seed polyphenols in general, and procyanidin B5-39-gallate
in particular, should be studied in more detail to be
developed as cancer chemopreventive and/or anticarcinogenic agents.
Introduction
Cancer is a leading cause of mortality world wide (1) and,
therefore, a major focus of research has been the chemoprevention of cancer (2,3). This approach is a means of cancer control
where the induction of this disease can be totally prevented
or the rate of development slowed or reversed partially or
substantially by the administration of one or more naturally
occurring or synthetic chemical agents (2,3). Fruits, vegetables
and common beverages, as well as several herbs and plants
with diverse pharmacological properties, have been shown to
be rich sources of chemicals with potential for chemoprevention
of various cancers (4–12 and references therein). Based on
epidemiological data followed by animal studies, or vice versa,
many new classes of chemical compounds are being evaluated
in clinical trials as chemopreventive agents against several
malignancies (2–12). At present, ~30 classes of chemicals with
cancer preventive effects have been described; most of them
have practical implications in reducing cancer incidence in
human populations (13). Among these, naturally occurring
polyphenolic antioxidants have received increasing attention
in recent years (14–16 and references therein). Employing
different long-term experimental tumorigenesis protocols,
several studies have demonstrated the cancer preventive effects
of polyphenolic antioxidants (14–18). The cancer chemopreventive effects of polyphenolic antioxidants is specifically
important since environmental pollutants, radiation, pesticides,
certain medications, contaminated water and deep fried and
spicy foods and UV radiation, as well as physical stress,
exhibit the ability to produce enormous amount of free radicals
which cause many diseases, including tumor promotion and
cancer (19–23).
1737
J.Zhao et al.
Grapes are one of the most widely consumed fruits in the
world. Grapes are rich in polyphenols and ~60–70% of grape
polyphenols exist in grape seeds. The grape seed polyphenols
are flavan-3-ol derivatives and are colorless in the pure state.
Only 4% of grape polyphenols exist in grape pulp. In grape
skin there is another type of polyphenol, called anthocyanins,
which usually have a purple color and amount to ~30% of
total polyphenols in grapes (24). Some of the individual
polyphenols in grape seeds are also present in tea, such as
catechin (C) and epicatechin (EC) (25), but most of the grape
seed polyphenols are quite different from tea polyphenols.
Besides EC and C, grape seeds are rich in dimers, trimers
and other oligomers of flavan-3-ols (26), whereas most tea
polyphenols are monomers, such as C, EC, epigallocatechin
3-gallate (EGCG), epigallocatechin (EGC) and epicatechin
3-gallate (ECG) (25). The combined name for monomers of
the derivatives of flavan-3-ol is catechins and for dimers and
other oligomers is procyanidins (or proanthocyanidins).
Recent studies have shown that procyanidins in grape
seeds possess anti-inflammatory, anti-arthritic and anti-allergic
activities and prevent heart disease and skin aging (27). In
other studies, grape seed polyphenols have been shown to
exert a much stronger oxygen free radical scavenging effect
than vitamins C and E (28,29) and to prevent UVC-induced
peroxidation (30). Oral administration of grape seed procyanidins at a dose of 2 mg/kg three times daily for 6 days inhibits
carrageenin- or dextran-induced hind paw edema, stabilizes
the capillary wall and prevents the increase in capillary
permeability caused by local cutaneous application of xylene
(31). Oral administration of grape seed polyphenols also
significantly improves visual performance in humans (32). In
addition to these studies, wine consumption has been reported
to have many beneficial health effects (33,34). Wine may also
be a source of grape seed polyphenols. For example, in red
wine production the alcohol generated from sugars in the
grapes can extract some of the grape seed polyphenols into
the wine. With regard to cancer chemopreventive effects of
grape seed polyphenols, the only study we have come across
was an abstract presented at the Annual Meeting of the
American Association for Cancer Research showing that oral
administration of 1% grape seed extract in the diet inhibits
APC mutation-associated intestinal adenoma formation in Min
mice (35). Regarding epidemiology, a case–control study
showed that increased consumption of grapes is associated
with reduced cancer risk (36).
In our continuing efforts to identify and define the cancer
chemopreventive and/or anticarcinogenic potential of newer
naturally occurring polyphenolic antioxidants (16,37–43) and
in the light of the fact that grape seed polyphenols possess
strong anti-inflammatory and antioxidant activities, studies
were performed to assess the anti-tumor-promoting effect of
a polyphenolic fraction isolated from grape seed (hereafter
referred to as GSP) on 12-O-tetradecanoylphorbol 13-acetate
(TPA) tumor promotion in the 7,12-dimethylbenz[a]anthracene
(DMBA)-initiated SENCAR mouse skin two-stage initiation–
promotion model. The selection of anti-promotion studies with
GSP was based on the reasoning that it is possibly a better
approach to prevent cancer at the promotion stage since: (i)
the promotion stage of multistage carcinogenesis is reversible
at an early stage; (ii) the initiation stage of multistage carcinogenesis is irreversible and possibly unavoidable because of
our continuous exposure to carcinogenic chemical and physical
agents (43–45). It is important to emphasize that carcinogenesis
1738
is a multistage process in experimental models and possibly
in human cancer induction, development and propagation
(44,45). Human malignancies, however, also appear to involve
a gradual accumulation of genetic changes over a period of
years. Based on the results obtained showing that GSP affords
significant protection against tumor promotion and that at
much lower doses than those employed in most of anti-tumorpromotion studies in the same model system and under identical
conditions, we proceeded to systematically separate, identify
and define the antioxidant activity of polyphenols present in
GSP. An effort was also made to determine a structure–activity
relationship between the polyphenols present in GSP and their
antioxidant potential in terms of inhibition of epidermal lipid
peroxidation.
Materials and methods
Chemicals and equipment
DMBA and thiobarbituric acid (TBA) were purchased from Sigma Chemical
Co. (St Louis, MO). TPA was from LC laboratories (Woburn, MA). All other
chemicals and reagents used in the studies were obtained in the purest form
available commercially. Proton nuclear magnetic resonance (1H-NMR) and
carbon nuclear magnetic resonance (13C-NMR) were recorded on a JEOL
GSX 400 spectrometer, with chemical shifts (δ) shown in p.p.m. with
tetramethylsilane as internal standard. Mass spectra (MS) were measured on
a JEOL JMS-SX 102 mass spectrometer. UV spectra were measured in a
Shimadzu UV-260 spectrophotometer. Infrared spectra were recorded in a
Perkin-Elmer IR-27G spectrometer. HPLC was performed using a Shimadzu
LC-10AD liquid chromatography system equipped with a UV detector
(Chelmaford, MA).
Preparation of GSP
Grapes, as large clusters with red berries, were bought from a local supermarket
in the autumn and identified as Vitis vinifera. Grape seeds were removed from
the grapes, air dried for 1 week and milled to a particle size of ,0.4 mm.
The grape seed powder (100 g) was macerated for 12 h at room temperature
three times with 800 ml of 100 mM acetate buffer, pH 4.8, in water/acetone
(30:70 v/v), each time. The three macerates were combined and concentrated
until no acetone was left using a rotary evaporator under reduced pressure
and a water bath temperature ,35°C. The concentrated solution was extracted
four times with 200 ml of ethyl acetate each time. The four ethyl acetate
extracts were combined, evaporated to remove ethyl acetate and GSP was
obtained as a lyophilized powder. The total polyphenolic content of GSP in
terms of catechins and procyanidins was determined by a vanillin–HCl
colorimetric method (46) using catechin and procyanidin B2 as authentic
standards. Briefly, 2 vol of reagent A (10% w/v vanillin in methanol) and
1 vol of reagent B (35% HCl in water) were mixed to generate reagent C. To
0.4 ml of sample solution in 50% aqueous methanol (10–75 µg/ml w/v) was
added 1.25 ml of reagent C and the tubes were left at room temperature for
15 min. The absorbance of the solutions was then read at 500 nm and matched
with a standard curve drawn using catechin and procyanidin B2 as authentic
standards. The GSP preparation thus obtained contains 95% (w/w) polyphenols
in terms of catechins and procyanidins and was highly soluble in 40% aqueous
acetone (v/v), methanol, ethanol, acetone and ethyl acetate but insoluble in
water and chloroform. This GSP preparation was used in anti-tumor-promotion
and lipid peroxidation studies and was further fractionated into individual
polyphenols for their identification and bulk preparation as detailed later for
other studies.
Animals and anti-tumor-promotion study
Four- to six-week-old female SENCAR mice were purchased from Charles
River Laboratories (Kingston, NY). The animals were housed five per cage
at 24 6 2°C and 50 6 10% relative humidity and subjected to a 12 h light/
12 h dark cycle. They were acclimatized for 1 week before use and fed a
Purina chow diet and water ad libitum. Prior to the study, the dorsal side of
the skin was shaved using electric clippers and mice with the hair cycle in
the resting phase were used in the experiment. The animals were randomly
divided into five groups of 20 animals each. Mice in groups I–III were treated
topically with a single 10 µg/mouse dose of DMBA in 0.1 ml of acetone as
tumor initiator on the dorsal shaved skin. One week later, animals in group I
were treated topically with 0.1 ml of acetone and groups II and III with 0.5
and 1.5 mg/mouse doses of GSP per application, respectively, in 0.1 ml of
acetone. Thirty minutes following these treatments, animals in groups I–III
were treated topically with a 2 µg dose of TPA in 0.1 ml acetone/mouse/
Grape seed polyphenol anti-tumor-promotion
application. The pre-treatment regimen for GSP was based on our studies
with other antioxidants showing that their topical application 30 min prior to
that of TPA produces the maximum inhibitory effect against TPA tumor
promotion in identical protocols (16,25,37,43,47). The TPA alone or two
doses of GSP plus TPA treatments were repeated twice per week up to the
termination of the experiment at 20 weeks from the start of DMBA. The
animals in group IV were treated with 0.1 ml of acetone alone and served as
a negative control for any spontaneous tumor induction. To assess whether
GSP alone produces tumor promoting effects, the animals in group V were
initiated with a 10 µg dose of DMBA and 1 week later treated with 1.5 mg
GSP/mouse/application twice a week up to the end of the experiment at 20
weeks. Animals in all the groups were watched for food and water consumption
and any apparent signs of toxicity, such as weight loss or mortality, during
the entire period of the study. Skin tumor formation was recorded weekly and
tumors .1 mm in diameter were included in the cumulative total if they
persisted for 2 weeks or more. Latent periods for the onset of tumors in
various groups were computed and the tumors were diagnosed histologically
at the termination of the experiment. At this point, the total tumor volume on
the back of each mouse was also recorded.
HPLC separation of individual polyphenols present in GSP
The GSP preparation was separated by analytical HPLC (for the identification
of individual polyphenols) and semi-preparative HPLC (for the bulk preparation of individual polyphenols for other studies), using an ESA C18 analytical
column (3 µm, 4.63150 mm) or a Waterman ODS semi-preparative column
(10 µm, 9.43500 mm), respectively. The HPLC mobile phase contained
solvent A (5% v/v acetonitrile in 100 mM acetate buffer, pH 4.8) and solvent
B (70% v/v acetonitrile in 100 mM acetate buffer, pH 4.8). The linear gradient
system employed, at room temperature, was: 0–5 min, 100% solvent A;
5–10 min, 100% solvent A to 75% solvent A and 25% solvent B; 10–35 min,
75% solvent A and 25% solvent B to 50% of solvents A and B; 35–40 min,
50% of solvents A and B to 30% solvent A and 70% solvent B; 45–50 min,
30% solvent A and 70% solvent B to 100% solvent B; 51 min, stop of run.
The solvent flow rates throughout the run were 0.6 and 1.5 ml/min in the
analytical and semi-preparative HPLC, respectively. The column eluate was
monitored at 270 nm UV absorbance in each case and individual polyphenol
peaks were collected and identified by comparing HPLC retention times with
authentic compounds, physiochemical properties and UV, i.r., MS, 1H-NMR
and 13C-NMR spectral analyses.
Epidermal lipid peroxidation assay
SENCAR mice were killed with CO2, the dorsal shaved skin excised and
epidermal microsomes were prepared as described earlier (48). The microsomes
were washed with and suspended in 0.1 M phosphate buffer, pH 7.4, containing
0.1 mM MgCl2. The generation of malondialdehyde (MDA) was employed
as a marker of lipid peroxidation and estimated by the method of Wright et al.
(49) with some modifications described earlier (50). The final concentration of
MDA was calculated using a molar extinction coefficient of 1.563105/M/cm.
For those incubations which involved GSP, vitamin C, vitamin E or individual
polyphenols present in GSP they were dissolved in acetone and added to the
incubation mixture prior to the start of incubation; the control incubations
contained the same amount of acetone. The final concentration of acetone in
the incubation mixture did not exceed 4% (v/v). Inhibition of lipid peroxidation
by the agent under test was determined by measuring the reduction in
the amount of MDA formed in these incubations compared with acetone
alone controls.
Statistical analysis
In tumorigenesis experiments, the statistical significance of difference in terms
of tumor incidence and multiplicity between the TPA and GSP 1 TPA groups
was evaluated by the χ2 and Wilcoxon rank sum tests, respectively. For tumor
volume data and lipid peroxidation studies, Student’s t-test was used to assess
the statistical significance of difference.
Results
Anti-tumor-promoting effect of GSP on TPA tumor promotion
Topical application of GSP prior to TPA application resulted
in a highly significant inhibition of TPA tumor promotion in
DMBA-initiated SENCAR mouse skin. The preventive effect
of GSP was dose dependent and was evident as a significant
reduction in tumor incidence, tumor multiplicity and tumor
volume (Figure 1A and B and Table I). In terms of any toxic
effects of topical application of GSP, as monitored by weight
gain profile, no noticeable difference was observed between
Fig. 1. Dose-dependent inhibitory effect of a polyphenolic fraction isolated
from grape seeds (GSP) on TPA tumor promotion in DMBA-initiated
SENCAR mouse skin. As detailed in Materials and methods, 1 week after
topical application of 10 µg/mouse DMBA as tumor initiator, TPA was
applied twice per week at a dose of 2 µg/mouse with or without prior
application of GSP at a dose of 0.5 or 1.5 mg/mouse. These treatments were
continued up to the end of the experiment at 20 weeks from the start of
DMBA. The percentage of mice with tumors (A), number of tumors per
mouse (B) and the body wt (g) per mouse (C) were plotted as a function of
the number of weeks on test. The percentage of mice with tumor data are
from 20 mice per group; the tumors per mouse data are means 6 SD of 20
mice per group and the body weight gain profile data are also means 6 SD
of 20 mice per group, throughout the experiment in each case.
the two doses of GSP and non-GSP-treated animals throughout
the experiment (Figure 1C). In addition, the mean water
consumption per animal per day was also comparable between
the two GSP-treated and non-GSP-treated groups of animals
(data not shown). Together, these observations suggest that
topical application of GSP, at least at the doses employed in
the present study, does not produce any apparent toxicity
during the entire period of the experiment.
In terms of its anti-tumor-promoting effects, when the data
were analyzed for tumor incidence, as shown in Figure 1A,
topical application of GSP prior to that of TPA to DMBAinitiated SENCAR mouse skin resulted in a dose-dependent
protection throughout the experiment. Compared with the nonGSP-treated positive control group of mice, the time of
appearance of the first tumor was delayed by 1 and 2 weeks
in the 0.5 and 1.5 mg GSP-treated animals, respectively. When
these data were assessed in the middle of the experiment at
10 weeks (Figure 1A), compared with 100% of mice with skin
tumors in the non-GSP-treated group, only 35 and 20% of
1739
J.Zhao et al.
Table I. Topical application of GSP prior to TPA results in a significant
protection against tumor volume (mm3) in TPA tumor promotion in DMBAinitiated SENCAR mouse skina
Treatment protocolb
Total no. of
tumors
Total tumor
volume
DMBA 1 TPA
384
20736
DMBA 1 (0.5 mg GSP 1 148 (61.4%)c 6956 (66.5%)
TPA)
DMBA 1 (1.5 mg GSP 1 66 (82.8)c
2640 (87.3%)
TPA)
Tumor volume
per tumorbearing mouse
1037 6 261
535 6 103
(48.4%)d
330 6 83
(68.2%)d
aThe data shown in each column are those obtained at the end of the tumor
experiment at 20 weeks. The values in parentheses in columns 2–4 represent
percent inhibition.
bThe doses of DMBA and TPA used were 10 and 2 µg/mouse, respectively.
The indicated doses of GSP per mouse were applied topically 30 min prior
to TPA in each case.
cHighly significant versus positive control, P , 0.001 (Wilcoxon rank sum
test).
dHighly significant versus positive control, P , 0.001 (Student’s t-test).
animals in the 0.5 and 1.5 mg GSP-treated groups, respectively,
had tumors, accounting for 65 and 80% inhibition (P , 0.001,
χ2 test) of tumor incidence, respectively. In fact, 100% tumor
incidence was observed in the positive control group as early
as 9 weeks of the protocol. At the termination of the experiment
at 20 weeks, compared with 100% of animals with skin tumors
in the positive control group, only 65 and 40% of the animals
in the 0.5 and 1.5 mg GSP-treated groups exhibited skin
papillomas, accounting for 35 and 65% inhibition (P , 0.001,
χ2 test) of tumor incidence, respectively (Figure 1A).
Similarly, when the tumor data were evaluated for tumor
multiplicity (cumulative number of tumors per group or number
of tumors per mouse), beginning with the first tumor appearance
up to termination of the experiment, both the doses of GSP
tested showed a highly significant protection against TPA
tumor promotion in SENCAR mouse skin (Figure 1B). At the
termination of the experiment, compared with 19.2 6 3.3
(mean 6 SD of 20 mice) tumors/mouse in the non-GSPtreated positive control group, only 7.4 6 2.9 and 3.3 6 3.3
(mean 6 SD of 20 mice in each case) tumors/mouse were
observed in the 0.5 and 1.5 mg GSP-treated groups, respectively, accounting for 61 and 83% protection (P , 0.001,
Wilcoxon rank sum test), respectively (Figure 1B). When the
tumor promotion data were analyzed in terms of tumor volume,
compared with the positive control group, the total tumor
volume and tumor volume per mouse were found to be
significantly lower (50–87%, P , 0.001, Student’s t-test) in
the 0.5 and 1.5 mg GSP-treated groups (Table I). However,
GSP treatment did not show a significant inhibition of tumor
growth, as is evident in a marginal reduction (12 and 26%,
P , 0.1, Student’s t-test) in tumor volume per tumor in the
0.5 and 1.5 mg GSP-treated groups of mice, respectively,
compared with the positive control group. The tumors in
each group of mice were histologically identified as benign
papillomas (data not shown). The animals initiated with DMBA
and promoted twice weekly with 1.5 mg GSP were devoid of
any skin tumors throughout the experiment (data not shown),
suggesting that GSP itself is not a tumor promoter.
Composition of individual polyphenols in GSP
Based on the results showing that GSP affords a highly
significant protection against tumor promotion, we focused
1740
our efforts on separating and identifying individual polyphenols
present in GSP. Efforts were then made to isolate bulk amounts
of the individual polyphenols from GSP by semi-preparative
HPLC and perform studies to assess their antioxidant potential
using the epidermal lipid peroxidation assay. Dry grape seeds
(100 g) were extracted as described in Materials and methods,
which resulted in 3.1 g (3.1% w/w of dry grape seeds) of total
GSP preparation. The polyphenol quantification method (46)
showed that this GSP preparation contained 95% polyphenols
(w/w). The individual polyphenols were separated by analytical
HPLC and then identified, followed by their quantification.
Photodiode array detection has shown that GSP contains C,
EC, gallic acid, EGC, dimers of C and/or EC [for example,
procyanidin B1 (EC-4β-8-C), procyanidin B2 (EC-4β-8-EC),
procyanidin B3 (C-4α-8-C), procyanidin B4 (C-4α-8-EC) and
procyanidin B5 (EC-4β-6-EC)], trimers of C and/or EC [for
example, procyanidin C1 (EC-4β-8-EC-4β-8-EC), procyanidin
T2 (EC-4β-8-EC-4β-8-C) and procyanidin T3 (EC-4β-6-EC4β-8-EC)] and polyphenolic esters of gallate (for example,
procyanidin B5-39-gallate, EGCG, ECG and procyanidin B2
39-gallate) (26). In the present study, we used UV detection
to separate and identify individual polyphenols by HPLC. As
shown in Figure 2, using a UV detector with absorbance at
270 nm, the analytical HPLC profile of GSP showed several
peaks. To identify these polyphenols, they were collected as
individual peaks and lyophilized for analysis. Based on the
retention times with authentic compounds and physiochemical
properties and spectral evidence, nine of these peaks were
identified as procyanidin B3, procyanidin B1, catechin, procyanidin B4, procyanidin B2, epicatechin, procyanidin C1, procyanidin B5-39-gallate and procyanidin B5 (Figure 2). For
example, peak 5 in the HPLC profile (Figure 2), which
was an amorphous powder, gave an orange-red color with
anisaldehyde–sulfuric acid reagent and a dark-blue color with
ferric chloride reagent; these are typical properties of procyanidins (51). In spectral analysis we observed: UV λmax nm
(logε): 212 (4.8), 230sh (4.5), 281 (3.9); i.r. νmax cm–1: 3350
(OH), 1600, 1500, 1440 (aromatic ring); positive ion FABMS m/z: 579 [M1H]1; 1H-NMR (methanol-d4) δ: 7.08 [1H,
br s], 6.88 [1H, br s], 6.73–6.77 [4H], 5.89–6.05 [3H], 5.05
[1H, br s], 4.92 [1H, br s], 4.62 [1H, br s], 4.26 [1H], 3.90
[1H], 2.91 [1H], 2.78 [1H]; 13C-NMR (methanol-d4) δ: 155.2,
157.2 [2C], 158.5, 159.1 [2C], 146.4 [2C], 146.5 [2C], 132.8,
133.4, 120.1 [2C], 116.7 [2C], 116.1 [2C], 108.1, 102.1, 101.3,
97.1, 98.0, 98.1, 80.6, 77.9, 74.3, 67.8, 37.7, 30.5. All the
spectral data are identical to those of procyanidin B2 (51)
and, therefore, we identified peak 5 in the HPLC profile as
procyanidin B2. Similar criteria were used for the identification
of other individual polyphenols, detailed elsewhere (51). The
structures of nine identified individual polyphenols present in
GSP are shown in Figure 3 and their total quantities in GSP
are listed in Table II. As shown in Table II, so far we have
identified 60.2% (w/w) of total polyphenols present in GSP
and identification of the remainder is in progress. Peaks 8 and
9 in the HPLC profile (Figure 2), procyanidin B5-39-gallate
and procyanidin B5, respectively, represent a relatively major
portion of GSP (Figure 2 and Table II). As can be seen from
the data provided in Table II, the amounts of individual
polyphenols quantified are not consistent with the peak heights
shown in the HPLC profile detected by UV detector (Figure
2). This is due to the fact that the monomers, dimers, trimers
and other oligomer forms of C and/or EC have almost the
same molar absorbances, but the molecular weight of the
Grape seed polyphenol anti-tumor-promotion
Fig. 2. Reverse phase analytical HPLC profile of GSP. The details of HPLC conditions, column and linear gradient run are described in Materials and
methods. The identification of each peak in the profile, as labeled, is based on their retention times with authentic standards and their physiochemical and
spectral analyses.
Fig. 3. Chemical structures of nine individual polyphenols identified in GSP.
trimer is three times greater than the monomer. This means
that the trimer represents three times more weight than the
monomer even though it has a comparable UV absorbance. It
is important to mention here that the contents of individual
polyphenols present in grape seeds may vary depending on
the origin, season and climate, however, in all the grape seed
extracts the concentration of procyanidins was much higher
than that of catechins and/or epicatechins.
Inhibition of epidermal lipid peroxidation by GSP and individual polyphenols isolated from GSP
It has been well documented that oxidative stress caused by
tumor promoters contributes largely to their tumor promoting
potential (52,53). The phorbol ester TPA has been studied
extensively in this regard and has been shown to cause
oxidative stress as one of its multifactorial mechanisms of
tumor promotion (52–54). If not scavenged and/or balanced
1741
J.Zhao et al.
Table II. Composition of individual polyphenols identified in GSPa
Peak no.
Compoundb
Percent of total GSP (w/w)c
1
2
3
4
5
6
7
8
9
None
Procyanidin B3
Procyanidin B1
Catechin
Procyanidin B4
Procyanidin B2
Epicatechin
Procyanidin C1
Procyanidin B5-39-gallate
Procyanidin B5
Total polyphenols identified
3.0
0.7
5.0
2.0
3.5
6.0
6.0
15
19
60.2
aGSP was subjected to reverse phase analytical HPLC as detailed in
Materials and methods.
bThe identification of each polyphenol listed here is based on their retention
time with authentic compounds in an HPLC run and their spectral analyses
as detailed in Materials and methods and Results. The quantification of each
polyphenol was done using peak area under curve analysis and comparison
with standards.
cThe calculations are based on considering total GSP as 100%.
by cellular antioxidant molecules and enzymes, oxidative stress
ultimately leads to a highly reactive oxygen species, the
hydroxyl radical, that attacks cellular targets such as DNA and
protein as well as lipid-rich membranes (55). The formation
of lipid peroxides via the lipid peroxidation process and their
inhibition in biological membranes have been explored as a
useful system to assess both oxidant and antioxidant activity
of endogenous as well as exogenous agents (55,56). In the
present study, we used the epidermal lipid peroxidation assay
to: (i) evaluate whether the observed inhibitory effects of GSP
on tumor promotion are associated with the potent antioxidant
activity of GSP; (ii) assess and identify the most potent
polyphenolic antioxidant present in GSP and derive a structure–
activity relationship in terms of antioxidant activity for different
polyphenols present in GSP; (iii) assess the magnitude of
antioxidant activity of GSP and individual polyphenols present
therein as compared with the well-known antioxidants vitamins
C and E.
As shown in Figure 4, in vitro addition of GSP to lipid
peroxidation incubation mixtures resulted in a highly significant
to complete inhibition of epidermal lipid peroxidation in a
dose-dependent manner. When these results were compared
with those obtained for the inhibition of epidermal lipid
peroxidation by vitamin E, GSP showed much stronger inhibition at all three doses tested (Figure 4). For example, compared
with 44% inhibition by vitamin E, GSP showed 65% inhibition
(P , 0.001, Student’s t-test) of lipid peroxidation in an
epidermal microsomal suspension at 80 µg/ml (Figure 4). A
higher concentration of GSP (120 µg/ml) showed complete
inhibition (P , 0.0001, Student’s t-test) of epidermal lipid
peroxidation, as compared with 70% inhibition by vitamin E
(Figure 4).
With regard to individual polyphenols isolated from GSP,
we selectively assessed the antioxidant activity of polyphenols
based on an evident structural difference. For example, catechin, procyanidin B2, procyanidin B5, procyanidin C1 and
procyanidin B5-39-gallate were evaluated for their inhibitory
activity on epidermal lipid peroxidation and compared with
vitamins C and E. All these agents showed a highly significant
(P , 0.01–0.0001, Student’s t-test) inhibition of epidermal
lipid peroxidation in a concentration-dependent manner, albeit
to different degrees (Table III). In each case, the observed
1742
Fig. 4. Inhibitory effect of GSP on epidermal lipid peroxidation and its
comparison with vitamin E. Epidermal microsomes were prepared from
SENCAR mouse epidermis and a microsomal suspension (2 mg protein) in
0.1 M phosphate buffer, pH 7.4, containing 0.1 mM MgCl2 was incubated
for 1 h at 37°C in the presence of ferric ions (1 mM FeCl3) and ADP
(5 mM) in a total volume of 1 ml. The incubation mixtures were added with
acetone alone or varying concentrations of GSP or vitamin E (40, 80 and
120 µg/ml) in acetone. The final concentration of acetone in an incubation
mixture did not exceed 4% (v/v). The reaction was terminated with 10%
trichloroacetic acid followed by 1.2 ml of 0.5% TBA. The generation of
MDA measured at 532 nm absorbance was employed as a marker of lipid
peroxidation and calculated using a molar extinction coefficient of
1.563105/M/cm as detailed in Materials and methods. The data shown as
percentage inhibition are from means (6 SD of ,10%) of three
independent assays, each done in triplicate.
Table III. Concentration-dependent inhibition of epidermal lipid
peroxidation by individual polyphenols present in GSP and vitamins C and
Ea
Compound
Vitamin C
Vitamin E
Catechin
Procyanidin
Procyanidin
Procyanidin
Procyanidin
Concentration in mM (% inhibition)b
0.3 (19)
0.2 (26)
0.3 (38)
B2
0.1 (35)
B5
0.02 (33)
C1
0.01 (35)
B5-39-gallate 0.005 (37)
0.5 (34)
0.4 (40)
0.5 (61)
0.2 (52)
0.05 (44)
0.02 (43)
0.01 (42)
0.7 (51)
0.7 (67)
0.7 (90)
0.4 (74)
0.07 (49)
0.03 (48)
0.02 (53)
0.8 (62)
1.0 (100)
1.0 (100)
0.5 (87)
0.1 (58)
0.04 (60)
0.04 (100)
aThe lipid peroxidation assay was done as detailed in Materials and
methods. In each case, the data shown is from three independent studies
with ,10% variation over the mean value; each assay was done in
triplicate.
bThe percentage inhibition shown in parentheses is based on the data
obtained for controls without any compound under test, but with vehicle.
inhibitory effect of individual polyphenols was much stronger
than those with vitamins C and E (Table III). When a
structure–activity relationship was sought between individual
polyphenols, there was an obvious proportionality between
inhibition of lipid peroxidation and the degree of polymerization (Table III). For example, catechin (monomer) , procyanidin B2 (dimer) , procyanidin C1 (trimer). In addition, the
position of the linkage between inter-flavan units also influenced lipid peroxidation activity (Table III); 4–6 linkage
isomers (e.g. procyanidin B5, structure shown in Figure 3)
showed stronger inhibitory activity than 4–8 linkage isomers
(e.g. procyanidin B2, structure shown in Figure 3). Moreover,
when a gallate group was linked to the 39-OH of a dimer (e.g.
procyanidin B5-39-gallate), the inhibitory activity against lipid
peroxidation increased sharply (Table III). It is important to
Grape seed polyphenol anti-tumor-promotion
Fig. 5. IC50 (mM) of individual polyphenols present in GSP and vitamins C and E in the epidermal lipid peroxidation assay. The data shown are those
derived from a concentration response tested with each compound and shown in Table III. Each bar represents the mean 6 SD of three independent assays in
concentration determination studies; each assay was done in triplicate (Table III).
mention here that we tested several different concentrations
of these polyphenols and vitamins C and E to obtain a
measurable inhibition of epidermal lipid peroxidation. However, only selected data showing a concentration response are
listed in Table III. Based on the data shown in Table III
deriving a concentration response, the IC50 was calculated for
individual polyphenols and compared with those for vitamins
C and E. As shown in Figure 5, the IC50 for polyphenols
present in GSP was much lower than those for vitamins C and
E and followed the order: vitamin C , vitamin E , catechin
, procyanidin B2 , procyanidin B5 , procyanidin C1 ,
procyanidin B5-39-gallate (Figure 5). Overall, procyanidin B539-gallate showed the strongest anti-lipid peroxidation activity
with an IC50 of 0.02 mM. We would like to emphasize here
that the structure–activity response shown in terms of inhibition
of lipid peroxidation represents only one of the several antioxidant parameters. More studies in other assays are warranted
based on the results of the present work.
Discussion
The central finding of the present study is that a polyphenolic
fraction isolated from grape seeds affords significant protection
against tumor promotion in the mouse skin tumorigenesis
model and that this effect of GSP may largely be due to the
exceptionally high antioxidant activity of procyanidins present
therein. A wide range of studies have shown that naturally
occurring polyphenolic antioxidants, specifically those present
in fruits and vegetables, common beverages such as green tea
and several herbs and plants with diverse pharmacological
activities, are promising classes of cancer chemopreventive
agents, possibly due to their strong anti-tumor-promoting
potential (43,47,57–59). Comparing the anti-tumor-promoting
effects of GSP observed in the present study with those
published for other agents, it is important to highlight here
that the doses of GSP used (0.5 and 1.5 mg) are much lower
than those (~3–18 mg) used in other studies, however, the
anti-tumor-promoting effects are comparable. When compared
with green tea polyphenols and the major component EGCG
present therein, GSP contains unique procyanidins which are
typical polyphenols with a larger molecular weight than tea
polyphenols. Another characteristic of GSP is that it is a
mixture of monomers, dimers, trimers and other oligomers of
catechin and/or epicatechin, whereas tea polyphenols are
only monomers of catechins and epicatechins with gallate
substitution (25). It is plausible that dimerization and trimerization of catechins and epicatechins lead to a sharp increase
in their anti-tumor-promoting potential, possibly due to a
significant increase in their antioxidant activity. This suggestion
is supported by our lipid peroxidation findings and the antitumor-promoting effects of GSP at much lower doses than
those used in the case of green tea polyphenols reported
earlier (47,57).
After establishing the cancer chemopreventive effect of GSP
in terms of inhibition of tumor promotion, we continued our
efforts to identify the individual polyphenols present therein
and assess their antioxidant activity. Using reverse phase
analytical HPLC, nine pure individual polyphenolic compounds
were separated from GSP and their structures were identified
by comparing HPLC profiles with standard compounds and
interpreting the spectroscopic evidence. Based on the standardization of analytical HPLC conditions, semi-preparative HPLC
was used as a more powerful and quick way to separate greater
quantities of individual polyphenols in the pure form for
further studies. These pure compounds are being used to
further investigate the structure–activity relationship in continuing studies on the cancer chemopreventive effects of GSP and
the polyphenols present therein. To the best of our knowledge,
ours is the first study reporting the chemopreventive effects
of GSP on tumor promotion in an experimental model of
tumorigenesis and establishing the structure–activity relationship in terms of antioxidant activity of polyphenols present
in GSP.
It is widely believed that inhibition of tumor promotion is
a better strategy in cancer chemoprevention than inhibition of
tumor initiation because initiation is a short irreversible event
whereas promotion is a long cumulative process that is
reversible during the initial stage (44,45). With the notion that
long-term targeting of the tumor promotion stage could be
a useful strategy for the prevention of cancer (11), daily
consumption of vegetables and fruits has already been associ1743
J.Zhao et al.
ated with a reduced risk for several human malignancies
(4–16). Our results provide additional support for this strategy
as well as for the already existing epidemiological and laboratory studies (35,36) reporting that grape seed polyphenols have
beneficial effects against several diseases, including cancer. In
summary, based on the results shown in the present study,
grape seed polyphenols in general, and procyanidin B5-39gallate in particular, should be studied in more detail to be
developed as cancer chemopreventive and/or anticarcinogenic agents.
Acknowledgements
This study was supported in part by USPHS grant CA64514 and AMC Cancer
Research Center Institutional Funds (to R.A.).
References
1. American Cancer Society Inc. (1998) Cancer Facts and Figures—1998.
American Cancer Society, www.cancer.org.
2. Singh,D.K. and Lippman,S.M. (1998) Cancer chemoprevention, Part 1:
retinoids and carotenoids and other classic antioxidants. Oncology, 12,
1643–1659.
3. Singh,D.K. and Lippman,S.M. (1998) Cancer chemoprevention, Part 2:
hormones, nonclassic antioxidant natural agents, NSAIDs and other agents.
Oncology, 12, 1787–1800.
4. Kelloff,G.J., Boone,C.W., Crowell,J.A., Steele,V.E., Lubet,R. and Sigman,
C.C. (1994) Chemopreventive drug development: prospects and progress.
Cancer Epidemiol. Biomarkers Prevent., 3, 85–98.
5. Kelloff,G.J., Boone,C.W., Crowell,J.A., Nayfield,S.G., Hawk,E.,
Malone,W.F., Steele,V.E., Lubet,R.A. and Sigman,C.C. (1996) Risk
biomarkers and current strategies for cancer chemoprevention. J. Cell.
Biochem., 25S, 1–14.
6. Birt,D.F., Pelling,J.C., Nair,S. and Lepley,D. (1996) Diet intervention for
modifying cancer risk. Prog. Clin. Biol. Res., 395, 223–234.
7. Morse,M.A. and Stoner,G.D. (1993) Cancer chemoprevention: principles
and prospects. Carcinogenesis, 14, 1737–1746.
8. Lipkin,M. (1997) New rodent models for studies of chemopreventive
agents. J. Cell. Biochem., 28–29 (suppl.), 144–147.
9. Conney,A.H., Lou,Y.R., Xie,J.G., Osawa,T., Newmark,H.L., Liu,Y.,
Chang,R.L. and Huang,M.T. (1997) Some perspectives on dietary inhibition
of carcinogenesis: studies with curcumin and tea. Proc. Soc. Exp. Biol.
Med., 216, 234–245.
10. Goodman,G.E. (1997) The clinical evaluation of cancer prevention agents.
Proc. Soc. Exp. Biol. Med., 216, 253–259.
11. Boone,C.W., Bacus,J.W., Bacus,J.V., Steele,V.E. and Kelloff,G.J. (1997)
Properties of intraepithelial neoplasia relevant to the development of
cancer chemopreventive agents. J. Cell. Biochem., 28–29 (suppl.), 1–20.
12. Hong,W.K. and Sporn,M.B. (1997) Recent advances in chemoprevention
of cancer. Science, 278, 1073–1077.
13. Wattenberg,L.W. (1997) An overview of chemoprevention: current status
and future prospects. Proc. Soc. Exp. Biol. Med., 216, 133–141.
14. Perchellet,J.-P. and Perchellet,E.M. (1989) Antioxidants and multistage
carcinogenesis in mouse skin. Free Radic. Biol. Med., 7, 377–408.
15. Dragsted,L.O. (1998) Natural antioxidants in chemoprevention. Arch.
Toxicol., 20 (suppl.), 209–226.
16. Mukhtar,H. and Agarwal,R. (1996) Skin cancer chemoprevention. J. Invest.
Dermatol. Symp. Proc., 1, 209–214.
17. Hocman,G. (1989) Prevention of cancer: vegetables and plants. Comp.
Biochem. Physiol., 93B, 201–212.
18. Yang,C.S., Lee,M.J., Chen,L. and Yang,G.Y. (1997) Polyphenols as
inhibitors of carcinogenesis. Environ. Health Perspect., 105 (suppl. 4),
971–976.
19. Stadtman,E.R. (1992) Protein oxidation and aging. Science, 257, 1220–
1224.
20. Agarwal,R., Katiyar,S.K., Khan,S.G. and Mukhtar,H. (1993) Protection
against ultraviolet B radiation-induced effects in the skin of SKH-1 hairless
mice by a polyphenolic fraction isolated from green tea. Photochem.
Photobiol., 58, 695–700.
21. Kehrer,J.P. (1993) Free radicals as mediators of tissue injury and disease.
Crit. Rev. Toxicol., 23, 21–48.
22. Lahiri,M., Mukhtar,H. and Agarwal,R. (1999) Reactive intermediates and
skin cancer. In Kitchin,K.T. (ed.) Carcinogenicity: Testing, Predicting and
Interpreting Chemical Effects. Marcel Dekker, New York, NY, pp. 679–714.
1744
23. Ames,B.N., Gold,L.S. and Willett,W.C. (1995) The causes and prevention
of cancer. Proc. Natl Acad. Sci. USA, 92, 5258–5265.
24. Singleton,V.L. (1992) Tannins and the qualities of wines. In Laks,P.E. and
Hemingway,R.W. (eds) Plant Polyphenols. Plenum Press, New York, NY,
pp. 859–880.
25. Agarwal,R., Katiyar,S.K., Zaidi,S.I.A. and Mukhtar,H. (1992) Inhibition
of skin tumor promoter-caused induction of epidermal ornithine
decarboxylase in SENCAR mice by polyphenolic fraction isolated from
green tea and its individual epicatechin derivatives. Cancer Res., 52,
3582–3588.
26. Bartolome,B., Hernandez,T., Bengoechea,M.L., Quesada,C., GomezCordoves,C. and Estrella,I. (1996) Determination of some structural
features of procyanidins and related compounds by photodiode-array
detection. J. Chromatogr. A, 723, 19–26.
27. Maffei Facino,R., Carini,M., Aldini,G., Berti,F., Rossoni,G.,
Bombardelli,E. and Morazzoni,P. (1996) Procyanidines from Vitis vinifera
seeds protect rabbit heart from ischemia/reperfusion injury: antioxidant
intervention and/or iron and copper sequestering ability. Planta Med., 62,
495–502.
28. Bagchi,D., Garg,A., Krohn,R.L., Bagchi,M., Tran,M.X. and Stohs,S.J.
(1997) Oxygen free radical scavenging abilities of vitamin C and E and a
grape seed proanthocyanidin extract in vitro. Res. Commun. Mol. Pathol.
Pharmacol., 95, 179–189.
29. Bagchi,D., Garg,A., Krohn,R.L., Bagchi,M., Bagchi,D.J., Balmoori,J. and
Stohs,S.J. (1998) Protective effects of grape seed proanthocyanidins
and selected antioxidants against TPA-induced hepatic and brain lipid
peroxidation and DNA fragmentation and peritoneal macrophage activation
in mice. Gen. Pharmacol., 30, 771–776.
30. Bouhamidi,R., Prevost,V. and Nouvelot,A. (1998) High protection by grape
seed proanthocyanidins (GSPC) of polyunsaturated fatty acids against
UV-C induced peroxidation. C.R. Acad. Sci. III, 321, 31–38.
31. Zafirov,D., Bredy-Dobreva,G., Litchev,V. and Papasova,M. (1990)
Antiexudative and capillaritonic effects of proanthocyanines isolated from
grape seeds (V. vinifera). Acta Physiol. Pharmacol. Bulg., 16, 50–54.
32. Corbe,C., Boissin,J.P. and Siou,A. (1988) Light vision and chorioretinal
circulation. Study of the effect of procyanidolic oligomers. J. Fr.
Ophthalmol., 11, 453–460.
33. Halpern,M.J., Dahlgren,A.L., Laakso,I., Seppanen-Laakso,T., Dahlgren,J.
and McAnulty,P.A. (1998) Red-wine polyphenols and inhibition of platelet
aggregation: possible mechanisms and potential use in health promotion
and disease prevention. J. Int. Med. Res., 26, 171–180.
34. Soleas,G.J., Diamandis,E.P. and Goldberg,D.M. (1997) Wine as a biological
fluid: history, production and role in disease prevention. J. Clin. Lab.
Anal., 11, 287–313.
35. Arii,M., Miki,M., Hosoyama,R., Ariga,H., Yamaji,N. and Kataoka,S. (1998)
Chemopreventive effect of grape seed extract on intestinal carcinogenesis in
the APCMin mouse. Proc. Am. Assoc. Cancer Res., 39, 20.
36. Zheng,T., Boyle,P., Willett,W.C., Hu,H., Dan,J., Evstifeeva,T.V., Niu,S.
and MacMahon,B. (1993) A case–control study of oral cancer in Beijing,
People’s Republic of China. Associations with nutrient intakes, foods and
food groups. Eur. J. Cancer B Oral Oncol., 29B, 45–55.
37. Agarwal,R., Katiyar,S.K., Lundgren,D.W. and Mukhtar,H. (1994)
Inhibitory effect of silymarin, an anti-hepatotoxic flavonoid, on
12-O-tetradecanoylphorbol-13-acetate-induced
epidermal
ornithine
decarboxylase activity and mRNA in SENCAR mice. Carcinogenesis, 15,
1099–1103.
38. Katiyar,S.K., Korman,N.J., Mukhtar,H. and Agarwal,R. (1997) Protective
effects of silymarin against photocarcinogenesis in mouse skin model.
J. Natl Cancer Inst., 89, 556–566.
39. Zi,X., Mukhtar,H. and Agarwal,R. (1997) Novel cancer chemopreventive
effects of a flavonoid antioxidant silymarin: inhibition of mRNA expression
of an endogenous tumor promoter TNFα. Biochem. Biophys. Res.
Commun., 239, 334–339.
40. Zi,X., Feyes,D.K. and Agarwal,R. (1998) Anti-carcinogenic effect of a
flavonoid antioxidant silymarin in human breast cancer cells MDA-MB
468: induction of G1 arrest through an increase in Cip1/p21 concomitant
with a decrease in kinase activity of CDKs and associated cyclins. Clin.
Cancer Res., 4, 1055–1064.
41. Zi,X., Grasso,A.W., Kung,H.-J. and Agarwal,R. (1998) A flavonoid
antioxidant silymarin inhibits activation of erbB1 signaling and induces
cyclin-dependent kinase inhibitors, G1 arrest and anti-carcinogenic effects
in human prostate carcinoma DU145 cells. Cancer Res., 58, 1920–1929.
42. Ahmad,N., Gali,H., Javed,S. and Agarwal,R. (1998) Skin cancer
chemopreventive effects of a flavonoid antioxidant silymarin are mediated
via impairment of receptor tyrosine kinase signaling and perturbation in
cell cycle progression. Biochem. Biophys. Res. Commun., 248, 294–301.
Grape seed polyphenol anti-tumor-promotion
43. Lahiri-Chatterjee,M., Katiyar,S.K., Mohan,R.R. and Agarwal,R. (1999) A
flavonoid antioxidant, silymarin, affords exceptionally high protection
against tumor promotion in SENCAR mouse skin tumorigenesis model.
Cancer Res., 59, 622–632.
44. Agarwal,R. and Mukhtar,H. (1991) Cutaneous chemical carcinogenesis. In
Mukhtar,H. (ed.) Pharmacology of the Skin. CRC Press, Boca Raton, FL,
pp. 371–387.
45. DiGiovanni,J. (1992) Multistage carcinogenesis in mouse skin. Pharmacol.
Ther., 54, 63–128.
46. Garcia,L.A., Velez,A.J. and de Rozo,M.P. (1985) Extraction and
quantification of polyphenols from coffee pulp. Arch. Latinoam. Nutr., 35,
491–495.
47. Katiyar,S.K., Agarwal,R., Wood,G.S. and Mukhtar,H. (1992) Inhibition of
12-O-tetradecanoylphorbol-13-acetate-caused tumor promotion in 7,12dimethylbenz[a]anthracene-initiated SENCAR mouse skin by a
polyphenolic fraction isolated from green tea. Cancer Res., 52, 6890–6897.
48. Agarwal,R.,
Wang,Z.Y.,
Bik,D.P.
and
Mukhtar,H.
(1991)
Nordihydroguaiaretic acid, an inhibitor of lipoxygenase, also inhibits
cytochrome P450-mediated monooxygenase activity in rat epidermal and
hepatic microsomes. Drug Metab. Dispos., 19, 620–624.
49. Wright,J.R., Colby,H.D. and Miles,P.R. (1981) Cytosolic factors which
affect microsomal lipid peroxidation in lung and liver. Arch. Biochem.
Biophys., 206, 296–304.
50. Katiyar,S.K., Agarwal,R. and Mukhtar,H. (1994) Inhibition of spontaneous
and photo-enhanced lipid peroxidation in mouse epidermal microsomes
by epicatechin derivatives from green tea. Cancer Lett., 79, 61–66.
51. Wang,J., Chen,Y., Hano,H. and Nomura,T. (1999) Studies on the
constituents of grape seeds. Chem. Pharm. Bull., in press.
52. Cerutti,P.A. (1994) Oxy-radicals and cancer. Lancet, 344, 862–863.
53. Kensler,T.W. and Taffe,B.G. (1989) Role of free radicals in tumor promotion
and progression. Prog. Clin. Biol. Res., 298, 233–248.
54. Perchellet,E.M. and Perchellet,J.P. (1989) Characterization of the
hydroperoxide response observed in mouse skin treated with tumor
promoters in vivo. Cancer Res., 49, 6193–6201.
55. Park,J.W. and Floyd,R.A. (1992) Lipid peroxidation products mediate the
formation of 8-hydroxydeoxyguanosine in DNA. Free Radic. Biol. Med.,
12, 245–250.
56. Girotti,A.W. (1985) Mechanisms of lipid peroxidation. J. Free Radic. Biol.
Med., 1, 87–95.
57. Huang,M.T., Ho,C.T., Wang,Z.Y. et al. (1992) Inhibitory effect of topical
application of a green tea polyphenol fraction on tumor initiation and
promotion in mouse skin. Carcinogenesis, 13, 947–954.
58. Agarwal,R., Katiyar,S.K. and Mukhtar,H. (1994) Skin cancer
chemoprevention by naturally occurring polyphenols. In Mukhtar,H. (ed.)
Skin Cancer: Mechanism and Human Relevance. CRC Press, Boca Raton,
FL, pp. 391–399.
59. Wei,H., Bowen,R., Zhang,X. and Lebwohl,M. (1998) Isoflavone genistein
inhibits the initiation and promotion of two-stage skin carcinogenesis in
mice. Carcinogenesis, 19, 1509–1514.
Received March 23, 1999; revised May 12, 1999; accepted May 13, 1999
1745