Antimutagenic and Antioxidant Properties of Phenolic Fractions
from Andean Purple Corn (
Zea mays
L.)
R
OMINA
P
EDRESCHI
†
AND
L
UIS
C
ISNEROS
-Z
EVALLOS
*
Department of Horticultural Sciences, Texas A&M University, College Station, Texas 77843-2133
The antimutagenic and antioxidant properties of various phenolic fractions obtained from Andean
purple corn were examined by the Ames test and the DPPH antiradical assay. An anthocyanin-rich
water fraction (WF) and an ethyl acetate fraction (EAF) showed a dose-dependent antimutagenic
behavior against the food mutagen Trp-P-1 with IC
50
values of 321.7
(
21.36 and 95.2
(
10.95
µ
g
of chlorogenic acid equiv/plate, respectively, indicating that EAF was a more potent antimutagen.
The antioxidant activities for WF and EAF were 1.019
(
0.05 and 0.838
(
0.11
µ
g of Trolox equiv/
µ
g of phenolics, respectively. Further fractionation of WF and EAF revealed an ethyl acetate
subfraction, EA-IV, with high antimutagen potency that contained a quercetin derivative. The
mechanism of antimutagenic action of the WF is predominantly a blocking effect on the S-9 Mix
activation system of the mutagen, whereas for the EAF, it is a dual mechanism involving blocking of
the S-9 Mix and a scavenging action on Trp-P-1 electrophiles.
Purple corn extract; phenolic compounds; antimutagenic activity; antioxidant activity;
mechanism of antimutagenic action
INTRODUCTION
The generation of heterocyclic aromatic amines that are highly
mutagenic in the salmonella/reversion assay or Ames test during
cooking of proteinaceous foods is very well documented (1),
including IQ (2 amino-3-methylimidazo [4,5-f] quinoline), MeIQx
(2 amino-3,8-dimethylimidazo [4,5-f] quinoxaline), PhIP (2-
amino-1-methyl-6-phenylimidazo [4,5-b] pyridine), and Trp-
P-1 (3-amino-1,4-dimethyl-5h-pyrido[4,3-b]indole). The het-
erocyclic amine Trp-P-1 has been involved in several types of
DNA damage that lead to genetic alterations. The accumulation
of genetic alterations can lead normal cells to become cancer
cells (3). Trp-P-1 is a direct acting mutagen toward Salmonella
Typhimurium. The N-hydroxy form of Trp-P-1 is metabolically
activated to N-O-acetyl-Trp-P-1, which damages DNA by the
formation of DNA adducts that can end up in genetic mutations.
Trp-P-1 can also produce reactive oxygen species (ROS) that
can cause oxidative DNA damage (4).
Food is a complex mixture of different components, some
of which act as antimutagens (5). The ability of different
vegetable and fruit juices to act as antimutagens has been tested
(1, 5), and phenolic compounds present in such juices were
considered responsible for the antimutagenic activity (6-8). The
antimutagenic action of phenolic compounds against the food
mutagen Trp-P-1 has been reported for sweet potato (9).
Purple corn (maiz morado in Spanish) has been used by
people from the Andes to color foods and beverages for
centuries. In addition, a refreshing drink called “chicha morada”
is prepared by immersing the cobs in boiling water. The already-
known antioxidant and anticarcinogenic properties of purple
corn, in addition to its coloring attributes, make it an attractive
crop for the Nutraceutical and Functional Food Market (10-
12). More recently, purple corn extracts were tested for anti-
obesity activity and amelioration of hyperglycemia (13). In
addition, purple corn color did not show any hepatotoxicity or
nephrotoxicity in mice depleted of glutathione by pretreatment
with buthionine sulfoximine at a dose of 4500 mg/kg (14).
Previous investigations of purple corn bioactivities have
mainly been focused on its anthocyanins. A purple corn color
extract has been shown to inhibit colorectal carcinogenesis in
male F344 rats pretreated with 1,2-dimethylhydrazine and PhIP
(10). The inhibition was attributed only to anthocyanins present
in the purple corn color. However, purple corn has a significant
amount of phenolic compounds other than anthocyanins, includ-
ing mainly phenolic acids and flavonols (15). The roles of other
phenolic compounds present in the purple corn color extract
have been ignored up to now.
The objective of this study was to fractionate the different
phenolic compounds present in Andean purple corn and to
determine their specific roles as antimutagens and antioxidants.
The results from this work will provide important information
for the food industry with respect to the use of the purple corn
extracts not only as a colorant but also as a source of health-
promoting compounds.
MATERIALS AND METHODS
Sample Material, Standards, and Reagents. Purple corn extract
(PCE) was kindly provided by Fitofarma (Lima, Peru). The PCE was
* To whom correspondence should be addressed. Tel.: 979-8453244.
Fax: 979-8450627. E-mail: lcisnero@taexgw.tamu.edu.
†
Present address: Katholieke Universiteit Leuven, Belgium.
J. Agric. Food Chem. 2006, 54, 4557
−
4567
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10.1021/jf0531050 CCC: $33.50
© 2006 American Chemical Society
Published on Web 06/02/2006
obtained from purple corn grown in Arequipa, Peru. The powder extract
was prepared by extraction of ground cobs (mesh 60) in a 60% aqueous
ethanol solution at room temperature for 48 h (1 kg of cobs/7 L of
solvent). The obtained extract was filtered and spray-dried (180
°
C
inlet and 85
°
C outlet temperatures) using maltodextrins as carrier (0.5
kg of maltodextrin/100 L of extract). One kilogram of dried PCE
contained
∼40% maltodextrin.
For the spectrophotometric procedures, Folin-Ciocalteu reagent,
sodium carbonate (Na
2
CO
3
), chlorogenic acid, Trolox, and 2,2-diphenyl-
1-picrylhydrazyl (DPPH) were purchased from Sigma Chemical Co.
(St. Louis, MO). Methanol was reagent grade, and Nanopure water
was used. For the Ames test, Salmonella Typhimurium TA98 (TA98)
and the S-9 Mix activation system were purchased from Molecular
Toxicology Inc. (Boone, NC). The S-9 Mix was composed of liophilized
Aroclor 1254 induced-male Sprague Dawley rat liver fraction S9 (40
mg of protein/mL) at a concentration of 0.04 mL of fraction S9/mL of
S-9 Mix, NADPH Regenesys A (composed of 5 mM glucose-6-
phosphate, 8 mM MgCl
2
, and 33 mM KCl in 100 mM sodium
phosphate buffer, pH 7.4), and NADPH Regenesys B (composed of 4
mM NAD). MgSO
4
‚7H
2
O, citric acid‚H
2
O, K
2
HPO
4
, NaNH
4
HPO
4
‚
4H
2
O, sodium chloride, ampicillin, and glucose were purchased from
Fisher Scientific (Fair Lawn, NJ).
D
-Biotin and
L
-histidine‚HCl‚H
2
O
were obtained from Sigma Chemical Co. (St. Louis, MO). Agar was
purchased from Difco (Kansas, MO), and Oxoid no. 2 nutrient broth
was obtained from Oxoid (Ogdensburg, NY). Reagent-grade DMSO
was used. The food mutagen Trp-P-1 was purchased from Toronto
Research Chemicals Inc. (Downsview, Canada).
For the phenolic compound fractionation, Toyopearl HW-40 and
Sephadex LH-20 were purchased from Sigma Chemical Co. (St. Louis,
MO). Reagent-grade methanol and ethyl acetate were used. For the
HPLC procedure, cyanidin-3-glucoside, pelargonidin-3-glucoside, pe-
onidin-3-glucoside, delphinidin-3-glucoside, petunidin-3-glucoside, and
malvidin-3-glucoside were purchased from Polyphenols Laboratories
AS (Sandnes, Norway). Cyanidin, pelargonidin, and peonidin were
obtained from ChromaDex (Santa Clara, CA). Phenolic acids (vanillic,
p-coumaric, protocatechuic, ferulic, and benzoic acids), flavonols
(quercetin, rutin, myricetin, kaempferol), flavanone (hesperidin), and
flavones (apigenin, luteolin) were purchased from Sigma Chemical Co.
(St. Louis, MO).
Isolation of a Total Phenolic Fraction (TPF). A TPF from a PCE
was obtained by using reverse-phase C
18
cartridges (Sep-Pak cartridges)
(Waters Corp., Milford, MA). A 0.2-g sample of PCE was dissolved
in 500 mL of distilled water. The Sep-Pak cartridge was conditioned
with 50 mL of methanol followed by the addition of 50 mL of distilled
water. The sample was loaded, and the phenolic compounds already
attached in the matrix were eluted with 100 mL of methanol containing
0.1% TFA. Water-soluble impurities were eluted during loading of the
cartridge. Methanol was evaporated in a rotoevaporator (Bu¨chi,
Switzerland) under vacuum at 40
°
C and 240 mbar of pressure and
then resuspended in distilled water, frozen at -80
°
C, and freeze-dried
in a freeze-dryer from FTS Systems, Inc. (Stone Ridge, NY) at -50
°
C and 200
µmHg of pressure.
Fractionation of Phenolic Compounds. A detailed diagram of the
fractionation of the PCE phenolic compounds is presented in Figure
1. Fractionation of 0.5 g of powder PCE was accomplished following
dissolution in 100 mL of distilled water. The PCE solution was
combined with 400 mL of ethyl acetate in a separation funnel. The
mixture was agitated and left to stand until two phases were observed.
The anthocyanin-rich phase was freeze-dried (at -50
°
C and 200
µmHg
of pressure), and the ethyl acetate phase was concentrated under vacuum
(at 40
°
C and 240 mbar of pressure) and resuspended in DMSO or
methanol.
The anthocyanin-rich water fraction (WF) was further fractionated
using a 50 cm
× 2.5 cm Toyopearl HW40 gel permeation column
preequilibrated with 20% methanol containing 0.1% TFA (20 h at 2
mL/min). A total of 0.1 g of the powdered WF was dissolved in 5 mL
of 20% methanol containing 0.1% TFA. Compounds present on the
column were eluted with 300 mL of 20% methanol, 600 mL of 40%
methanol, 300 mL of 60% methanol, 300 mL of 80% methanol, and
750 mL of 100% methanol at a flow rate of 1 mL/min, and 180 fractions
(12.5 mL/each) were collected. Compound elution was monitored at
280 nm and 520 nm using a Hewlett-Packard 8452A diode-array
spectrophotometer. The subfractions obtained were concentrated to
evaporate the solvent and then resuspended in water and freeze-dried.
Following solvent removal, the ethyl acetate fraction (EAF) was
resuspended in methanol and loaded on a 50
× 2.5 cm Sephadex LH-
20 column previously equilibrated with methanol for 12-20 h at 1.0
mL/min. Individual compounds were eluted at a flow rate of 0.5 mL/
min, and 200 fractions (5 mL/each) were collected. Compound elution
was monitored in each individual fraction at several wavelengths,
namely, 280, 320, 360, and 520 nm, using a Hewlett-Packard 8452A
diode-array spectrophotometer. The subfractions obtained were con-
centrated by evaporating the solvent and were then resuspended in
DMSO. Total phenolics were quantified in each fraction by an
adaptation of the Folin-Ciocalteau method and are expressed as
chlorogenic equivalents (16). Antioxidant activity was quantified by
Figure 1.
Fractionation of phenolic compounds from a purple corn crude extract (PCE).
4558
J. Agric. Food Chem., Vol. 54, No. 13, 2006
Pedreschi and Cisneros-Zevallos
the 2,2
′
-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method
and are expressed as Trolox equivalents ( 17). The Folin-Ciocalteau
phenolic contents of PCE and the C-18-purified TPF were 21.3 ( 0.5
and 51.7 ( 0.9 g of chlorogenic acid equiv/100 g of PCE or TPF
powder, respectively, and the phenolic contents of WF and EAF
obtained from a purified TPF were 59.7 ( 0.8 and 34.0 ( 0.3 g of
chlorogenic acid equiv/100 g of WF or EAF fraction, respectively.
Toxicity and Mutagenic and Antimutagenic Activities. The Ames
test (18) was used to test the mutagenic and antimutagenic activities
of the PCE, TPF, WF, and EAF and their respective subfractions.
Salmonella Typhimurium TA98, “S-9 Mix activation system”, and the
Trp-P-1 food mutagen were included in the test. Toxicity assays under
the same conditions as used for the Ames test were performed to
determine the maximum concentrations of chemical compounds
(expressed as micrograms of chlorogenic acid equivalents per plate)
that could be added without exerting any toxic effect on the bacteria
used in the Ames test. A lyophilized disk of TA98 was added to 25
mL of nutrient broth (Oxoid no. 2) and incubated for 10-16 h at 37
°
C and under agitation at 200 rpm in a shaking incubator. Optimal
assay conditions existed when the cultures exhibited optical densities
of 1.2-1.4 at 660 nm. The procedure included incubation in a sterile
12
× 75 mm tube with 0.1 mL of DMSO, 0.1 mL of extract or chemical
substance to be tested, 0.5 mL of S-9 Mix activation system (20
µL/
plate), and 0.1 mL of TA98. The mix was shaken gently and incubated
for 20 min at 37
°
C. Then, 2 mL of top agar was added with thorough
mixing and serially diluted with peptone water to create logarithmic
differences in cell concentrations. Diluted test suspensions were plated
on nutrient agar plates using the drop surface technique and incubated
for 12-15 h at 37
°
C. Well-separated colonies on 20
µL drops were
counted having colonies between 3 and 30 using a magnifier. Results
are expressed as cfu/mL.
To test mutagenic activity, 0.1 mL of DMSO (mutagen solvent),
0.1 mL of compound to be tested, 0.5 mL of S-9 Mix activation system,
and 0.1 mL of TA98 were incubated together for 20 min at 37
°
C.
Then, 2 mL of top agar was added, and the whole solution thoroughly
mixed. The solution was poured on minimal-glucose agar plates,
allowed to harden, and incubated at 37
°
C for 48 h. Under these
conditions, the Salmonella Typhimurium TA98, which carries a
defective or mutant gene making it unable to synthesize histidine, can
regain its gene function, and revertant cells will grow in the histidine-
lacking medium. An assay blank was formed by combining 0.1 mL of
DMSO with 0.1 mL of extract solvent (water or DMSO) prior to
addition of 0.5 mL of S-9 Mix activation system and 0.1 mL TA98.
Blank incubations were used to determine the spontaneous rate of
bacterial mutation (revertants). Reversion rates in blank incubations
were used as negative controls for the assay. The number of spontaneous
revertants was expected to be between 20 and 60 colonies per plate.
The spontaneous reversion rate was subtracted from the raw treatment
values prior to statistical analysis. Mutagenic activity was tested for
the PCE, TPF, WF, EAF and their respective subfractions.
To test antimutagenic activity, 0.1 mL of Trp-P-1 (0.75
µg/mL
solution), 0.1 mL of the compound to be tested, 0.5 mL of S-9 Mix
activation system, and 0.1 mL of TA98 were placed in a sterile 12
×
75 mm tube and preincubated for 20 min at 37
°
C. Then, 2 mL of
molten top agar was added, and the solution was thoroughly mixed
and poured on minimal-glucose agar plates and incubated at 37
°
C for
48 h, after which revertants were counted. A positive control consisting
of 0.1 mL of Trp-P-1, 0.1 mL of extract solvent, 0.5 mL of S-9 Mix,
and 0.1 mL of TA98 treated as above was included in the test. A control
used 0.075
µg of Trp-P-1 per plate and normally showed 794 ( 189
revertants per plate.
Antimutagenic activity was calculated as percentage inhibition of
mutagenic activity
For PCE, TPF, WF, and EAF, antimutagenic activity was tested in
the range 0-850
µg of chlorogenic acid equiv/plate. However, for the
WF and EAF subfractions, antimutagenic activity was assayed only at
a specific concentration determined from the dose-response curves
for the WF and EAF. IC
50
values were determined from dose-response
curves and corresponded to the concentration of the tested compound
(micrograms of chlorogenic acid equivalents per plate) required for
50% inhibition of mutagenic activity.
Mechanism of Antimutagenic Action. To account for the mech-
anism of antimutagenic action, the approach described by Hour and
others (19) was utilized for the WF, EAF, and the subfraction that
showed the highest antimutagenic activity. The treatments tested were
as follows:
(I) Blank. 0.1 mL Trp-P-1 (0.75
µg/mL) was incubated with 0.5
mL of S-9 Mix, 0.1 mL of TA 98, and 0.1 mL of DMSO at 37
°
C for
30 min without the antimutagen.
(II) Mutagen and Then Antimutagen. S-9 Mix (0.5 mL) and TA98
(0.1 mL) were mixed with 0.1 mL of Trp-P-1 (0.75
µg/mL) at 37
°
C
for 15 min, and then 0.1 mL of tested compound was added and
incubated at 37
°
C for an additional 15 min.
(III) Antimutagen and Then Mutagen. S-9 Mix (0.5 mL) and TA98
(0.1 mL) were preincubated with 0.1 mL of tested compound at 37
°
C
for 15 min, and then 0.1 mL of Trp-P-1 (0.75
µg/mL) was added and
incubated for an additional 15 min at the same temperature.
(IV) All Components and Then Bacteria. Trp-P-1 (0.1 mL), the tested
compound (0.1 mL), and S-9 Mix (0.5 mL) were incubated for 15 min
at 37
°
C, and then 0.1 mL of TA98 was added and incubated at the
same temperature for an additional 15 min.
(V) All Components Simultaneously. Trp-P-1 (0.1 mL), the tested
compound (0.1 mL), TA98 (0.1 mL), and S-9 Mix (0.5 mL) were
incubated at 37
°
C for 30 min.
The WF was tested at a concentration equivalent to 200
µg of
chlorogenic acid/plate. The EAF was tested at a concentration equivalent
to 90
µg of chlorogenic acid/plate. The subfraction with the highest
antimutagenic activity was tested at a concentration equivalent to 50
µg of chlorogenic acid/plate.
HPLC Analysis. Phenolic compounds were separated using a binary
Waters 515 HPLC system, a Waters 717 plus autosampler automated
gradient controller, an SP8792 temperature controller, and a Waters
996 photodiode-array detector. For peak integration, Millenium
32
software from Waters (Milford, MA) was used. An Atlantis C
18
5-
µm,
4.6 mm
× 150 mm column and a 4.6 mm × 20 mm guard column
were used for the separation of phenolic compounds. The mobile phase
was composed of solvent A (Nanopure water adjusted to pH 2.3 with
2 N HCl) and solvent B (HPLC-grade acetonitrile). The elution was as
follows: isocratic conditions from 0 to 5 min with 85% A and 15% B;
linear gradient conditions from 5 to 30 min starting with 85% A and
ending with 0% and starting with 15% B and ending with 100%; and
then, isocratic conditions from 30 to 35 min with 0% A and 100% B.
The flow rate was 1 mL/min, and a 10-
µL sample was injected (20).
The temperature of the column was kept at 35
°
C. Phenolic compounds
were identified on the basis of retention time and UV-visible spectral
data compared to known standards as described by Pedreschi and
Cisneros-Zevallos (15). Acylated forms were deduced by performing
basic hydrolysis according to the method described by Pedreschi and
Cisneros-Zevallos (15).
Statistical Analysis. In all cases, five replicates were used to test
mutagenic and antimutagenic activities. For the other assays, six
replicates were used. Replicates were obtained from several fraction-
ations of the PCE. Results were processed by using the one-way
variance analysis (ANOVA). Differences at p < 0.05 were considered
to be significant. A Tukey for comparison of multiple means and a
χ
2
test for comparison of dose-response curves were used. SPSS software
(SPSS Inc., 2002) was used to run all specific statistical analysis.
RESULTS AND DISCUSSION
Purple Corn Extract (PCE) and the C-18 Purified
Phenolic Fraction (TPF). The purple corn extract (PCE) and
the C-18-purified phenolic fraction (TPF) showed antimutagenic
activities with similar trends of dose-response inhibitions
against the activated mutagen Trp-P-1 for a range of phenolic
concentrations of 0-850
µg/plate (Figure 2a). The curves were
Inhibition (%) )
100
×
[
1 -
(
treatment - negative control
positive control - negative control
)
]
Purple Corn Antioxidant and Antimutagenic Properties
J. Agric. Food Chem., Vol. 54, No. 13, 2006
4559
slightly different (
χ
2
test, p < 0.05), and the gap observed could
be due to a slight inhibitory effect of other compounds present
in the PCE such as maltodextrins. The antimutagenic activities
of PCE and TPF reached maxima of 96.02 ( 1.2% and 97.2 (
0.2%, respectively, for a phenolic concentration of 850
µg/plate.
No mutagenic (
∼20-35 revertants/plate) or cell toxicity effects
(
∼10
6
-10
7
cfu/mL microbial growth) were observed in the
range of concentrations tested. The antimutagenic activity of
phenolic compounds has been extensively reported (7-9, 21-
24); however, antimutagenic properties of purple corn due to
Figure 2.
Inhibition of mutagenic activity on TA98 against the food mutagen Trp-P-1 by (a) PCE and TPF and (b) WF and EAF in the range 0
−
850
µ
g
of chlorogenic acid equiv/plate. (c) IC
50
and antioxidant values for PCE, TPF, WF, and EAF.
4560
J. Agric. Food Chem., Vol. 54, No. 13, 2006
Pedreschi and Cisneros-Zevallos
phenolic compounds have not been characterized before. Ad-
ditionally, the specific antioxidant activities for PCE and TPF
were determined as 0.954 ( 0.05 and 1.107 ( 0.016
µg of
Trolox equiv/
µg of phenolics, respectively. These antioxidant
activities on a phenolic basis are similar to or higher than those
of blueberries (11). There is a need to determine whether the
antioxidant activity plays a role in the observed antimutagenic
properties.
Water and Ethyl Acetate Phenolic Fractions from PCE.
The two main fractions obtained by partition, the anthocyanin-
rich WF and the EAF, showed dose-dependent antimutagenic
activities in the range of concentrations used (Figure 2b). No
mutagenic effects were observed for either fraction (
∼20-50
revertants/plate); however, toxic effects were seen for the EAF
only at concentrations >320
µg/plate (a decrease from 10
7
to
10
5
cfu/mL). These results will restrict the use of the Ames
test to a certain concentration range for the EAF, but they also
suggest the possibility of investigating the type of phenolics
present in the EAF as natural antimicrobials. Inhibitions of
mutagenic activity against the food mutagen Trp-P-1 for WF
and EAF were 92.09 ( 1.8% and 89.5 ( 1.6% for phenolic
concentrations of 850 and 320
µg/plate, respectively.
The antioxidant activities for the WF and EAF was deter-
mined as 1.019 ( 0.05 and 0.838 ( 0.11
µg of Trolox/µg of
phenolics, respectively. A higher antioxidant activity of the WF
was expected, as this fraction is rich in anthocyanins and these
types of phenolic compounds have been reported to have higher
antioxidant activities than phenolic acids and flavonols mainly
present in EAF (25, 26).
The EAF seemed to contain the most bioactive antimutagens,
requiring the lowest phenolic concentration to obtain a 50%
inhibition in mutagenic activity (IC
50
value of 95.2 ( 10.95
µg/plate) compared to PCE, TPF, and WF (Figure 2c). However,
there was no clear relationship between antioxidant and anti-
mutagenic activities. Further fractionation of the WF and EAF
was performed to determine the specific phenolic compounds
in the PCE associated with the responses.
WF Subfractionations. Fractionation of the anthocyanin-rich
WF on a Toyopearl HW40 column yielded five subfractions
(W-I, W-II, W-III, W-IV, and W-V), which were characterized
at 280 and 520 nm (Figure 3a). The five subfractions showed
no toxicity (
∼10
6
cfu/mL microbial growth) or mutagenic effects
(
∼20-25 revertants/plate) when tested at 200 µg/plate. It was
reported previously that anthocyanins do not exert toxic effects
in the Ames test (27).
All of the water subfractions showed antimutagenic activities.
The antimutagenic activities of subfractions W-I to W-IV were
in similar ranges (
∼35-46%, not significantly different, p >
0.05) with fractions W-III and W-IV having slightly lower
values. On the other hand, subfraction W-V was significantly
lower (p < 0.05) and the least efficient antimutagen compared
to the other subfractions (
∼17%, p < 0.05) (Figure 3b). The
antimutagenic activity of anthocyanins using the Ames test has
been reported previously for different crops and mutagens (22,
Figure 3.
(a) Fractionation of the anthocyanin-rich WF on a Toyopearl HW-40 column and (b) antioxidant and percent inhibition of mutagenic activity for
the water subfractions on TA98 against the food mutagen Trp-P-1. Antimutagenic activity was tested at a concentration of 200
µ
g of chlorogenic acid
equiv/plate.
Purple Corn Antioxidant and Antimutagenic Properties
J. Agric. Food Chem., Vol. 54, No. 13, 2006
4561
24). In general, all five water subfractions showed similar
antioxidant activities, ranging from
∼0.880 to 1.050 µg of
Trolox/
µg of phenolics ( Figure 3b), indicating that there was
no clear relationship between anthocyanin antioxidant activities
and antimutagenic properties.
The HPLC-DAD characterization revealed the presence of
glucoside forms of cyanindin, peonidin, pelargonidin, and their
respective acylated counterparts (Figure 4). Two or four
anthocyanins were present in each subfraction with the exception
of WF-V, which had only one type. WF subfractions showed
mainly peonidin-3-glucoside in W-I (
∼81% relative A
520nm
),
cyanidin-3-glucoside in W-II (
∼95% relative A
520nm
), peonidin-
3-glucoside and acylated cyanidin-3-glucoside in W-III (
∼38%
and 36% relative A
520nm
, respectively), acylated cyanidin-3-
glucoside in W-IV (
∼76% relative A
520nm
), and acylated
peonidin-3-glucoside in W-V (
∼100% relative A
520nm
) (Table
1). These results suggest that the presence of acylated antho-
cyanins alone or in larger amounts in a mixture (e.g., 100%
and 83% for W-V and W-IV, respectively) reduces the anti-
mutagenic efficiency of the water subfractions in purple corn.
On the other hand, the presence of only anthocyanin glucosides
in a mixture (e.g., 100% for both W-I and W-II) can enhance
the antimutagenic potency of the water subfractions. This
enhanced potency is independent of the type of aglycons present
in the glucoside forms of the W-I and W-II mixtures.
The obtained results differ from those reported for cyanidin-
and peonidin-derived anthocyanins from purple-fleshed sweet
potato (9). In that study, acylated anthocyanins showed similar
or higher antimutagenic potencies compared to deacylated
anthocyanins, relating them to the aglycons present and the
acylating groups. For example, cyanindin-derived anthocyanins
had a stronger potency than peonidin-derived anthocyanins, and
the acylating groups, caffeic and ferulic acid, showed high
antimutagenicity effects as well. It is likely that the observed
Figure 4.
HPLC profile for the water subfractions at 520 nm. Samples were injected at a concentration of 200
µ
g of chlorogenic acid equiv/mL.
Table 1.
Relative Percentage Contributions of the Different Peaks Obtained in the Water Subfractions
subfraction
peak
no.
retention
time (min)
λ
max
(nm)
relative
A
520nm
a
(%)
compound
W-I
1
5.45
202.4, 279.1, 515.8
18.93
cyanidin-3-glucoside
3
8.033
198.9, 279.1, 515.8
81.07
peonidin-3-glucoside
W-II
1
5.45
202.4, 279.1, 515.8
95.49
cyanidin-3-glucoside
2
6.891
201.3, 275.5, 508.5
4.51
pelargonidin3-glucoside
W-III
1
5.483
202.4, 279.1, 515.8
17.49
cyanidin-3-glucoside
2
7.002
201.3, 275.5, 508.5
8.18
pelargonidin-3-glucoside
3
8.335
198.9, 279.1, 515.8
37.89
peonidin-3-glucoside
4
14.822
203.4, 279.1, 515.8
36.44
acylated cyanidin-3-glucoside
W-IV
1
5.478
202.4, 279.1, 515.8
12.49
cyanidin-3-glucoside
3
9.114
198.9, 279.1, 515.8
4.41
peonidin-3-glucoside
4
12.944
203.4, 279.1, 515.8
75.92
acylated cyanidin-3-glucoside
5
14.476
202.4, 276.7, 508.5
7.18
acylated pelargonidin-3-glucoside
W-V
6
14.929
202.0, 279.1, 515.8
100
acylated peonidin-3-glucoside
a
Percentage area is defined as the integrated area of a single peak divided by the total area of all integrated peaks.
4562
J. Agric. Food Chem., Vol. 54, No. 13, 2006
Pedreschi and Cisneros-Zevallos
differences are related to the structural forms of the anthocyanins
in sweet potato, which have more complex glycoside groups
and the presence of acylating phenolic acids, whereas purple
corn anthocyanins have simple glucose attached and malonic
acid as acylating groups (16, 28, 29).
More work is needed to understand the structure-bioactive
property relationship of anthocyanins. Some features such as
molecule polarity by the introduction of hydroxyl groups can
reduce antimutagenic activity (21) while enhancing antioxidant
activity (30, 31).
EAF Subfractionations. Fractionation of EAF on a Sephadex
LH-20 column yielded four subfractions (EA-I, EA-II, EA-III,
and EA-IV), which were characterized at 280, 320, 360, and
520 nm (Figure 5a). The four subfractions showed no toxicity
effects (
∼10
7
cfu/mL microbial growth) or mutagenic effects
(
∼20-45 revertants/plate) at a phenolic concentration of 90 µg/
plate. In general, all four EAF subfractions showed antimu-
tagenic activity against the food mutagen Trp-P-1 (Figure 4b).
The antimutagenic activities of the EAF subfractions were in
the range of
∼24-81% and presented the following descending
order: EAF-IV > EAF-III
≈ EAF-I > EAF-II. Subfraction EA-
IV was the most potent antimutagen compared to the other
subfractions (p < 0.05), followed by EAF-III, and they showed
inhibition of mutagenic activities of 81.0 ( 4.8% and 38.1 (
11.6%, respectively.
The antioxidant activities for the EAF subfractions were in
the range of
∼0.530-1.330 µg of Trolox/µg of phenolics and
decreased as follows: EAF-IV > EAF-III
≈ EAF-II > EAF-I.
Once again, subfraction EA-IV was the highest in antioxidant
activity among the different EAF subfractions (p < 0.05),
followed by EAF-III, with values of 1.330 ( 0.104 and 1.020
( 0.180 µg of Trolox/µg of phenolics, respectively.
The HPLC-DAD characterization revealed the presence of
simple phenolic acids, flavonols, a flavanone, and bound
hydroxycinnamic acid forms. These phenolic compounds were
present in mixtures of three or six phenolic compounds in each
subfraction with the exception of EAF-IV, which had only one
type of phenolic compound (Figure 6). Results revealed the
presence of mainly bound hydroxycinnamic acid forms in EA-I
(100% relative A
280nm
), phenolic acids in EA-II (
∼53% relative
A
280nm
), quercetin derivatives in EA-III (
∼80% relative A
280nm
),
and a quercetin derivative in W-IV (100% relative A
280nm
) (Table
2). These results suggest that the presence of quercetin deriva-
tives alone or in larger amounts in a mixture (e.g., 80-100%
in EA-III and EA-IV) enhances the antimutagenic efficiency
of the ethyl acetate subfractions from purple corn extracts. On
Figure 5.
(a) Fractionation of the ethyl acetate fraction EAF on a Sephadex LH-20 column and (b) antioxidant and percent inhibition of mutagenic activity
for the ethyl acetate subfractions on TA98 against the food mutagen Trp-P-1. Antimutagenic activity was tested at a concentration of 90
µ
g of chlorogenic
acid equiv/plate.
Purple Corn Antioxidant and Antimutagenic Properties
J. Agric. Food Chem., Vol. 54, No. 13, 2006
4563
the other hand, the presence of phenolic acids in a mixture (e.g.,
53-100% in EA-II and EA-I) reduces the antimutagenic potency
of the ethyl acetate subfractions. EA-IV, the most bioactive
subfraction, shows a quercetin derivative similar to a rutin-like
molecule as determined from UV-visible spectral data (Figure
6, Table 2). Rutin has been reported as an excellent antimutagen
against the mutagens IQ (2-amino-3-methyl-imidazo[4,5-f]-
quinoline) and a type of benzo[a] pyrene (21, 32). Furthermore,
quercetin has been shown to have a higher DPPH antioxidant
activity than several anthocyanidins and phenolic acids (30).
The quercetin derivative in the present study showed both high
antioxidant and antimutagenic properties.
To account for the relative contributions of the EAF and WF
in terms of bioactivity, it is important to know the relative
contribution of each fraction to the total phenolics present in
the PCE. In this study, the C-18-purified TPF represented
∼21.86% of PCE by weight after recovery from the Sep-Pak
cartridge. This TPF was partitioned in water/ethyl acetate,
yielding purified WF and EAF that represented
∼74.6% and
22.3% of TPF by weight. According to this result, PCE is an
important source of nonanthocyanin phenolic compounds that
had been ignored in previous studies in that bioactivity was
tested and attributed only to the anthocyanins (10, 13).
Mechanism of Antimutagenic Action. The TA98 cells were
subjected to five different treatments to determine the antimu-
tagenic mechanism of action of the anthocyanin-rich WF, EAF,
and EA-IV (Figure 7). Several mechanisms have been proposed
for the action of phenolic compounds as antimutagens; however,
two main mechanisms in the Ames test include the inhibition
of enzyme systems such as the cytochrome-P450-dependent
bioactivation of the various mutagens and the scavenging of
metabolically generated mutagenic electrophiles (26). In addi-
tion, a third proposed mechanism might include the blocking
of the mutagen transfer into the cytosol by phenolic binding or
insertion into the transporters of the outer membrane of the cell
(19).
The controls, which included a preincubation of bacteria
TA98 cells and the activated mutagen Trp-P-1, showed mu-
tagenic effects of
∼600 revertants/plate for all three experiments
(treatment I). The WF showed antimutagenic activity when it
was supplied after the preincubation of bacteria and the activated
Trp-P-1 (treatment II), suggesting a scavenging action of the
anthocyanins against the generated mutagenic electrophiles
(Figure 7a). The WF also showed a higher antimutagenic effect
(p < 0.05) when it was preincubated with the bacteria and the
S-9 Mix before exposure to Trp-P-1, suggesting that anthocya-
nins can interfere with the S-9 Mix enzymes responsible for
the mutagen activation (treatment III). A similar response was
obtained (p > 0.05) when the WF, S-9 Mix, and Trp-P-1 were
preincubated together before exposure to the bateria cells
(treatment IV), indicating this time that anthocyanins might
preferentially act on the enzyme systems. Treatment V, which
corresponded to a 30-min incubation of all of the components
together showed mutagenic suppression comparable to those
Figure 6.
HPLC profile for the ethyl acetate subfractions at 280 nm. Samples were injected at a concentration of 90
µ
g of chlorogenic acid equiv/mL.
Table 2.
Relative Percentage Contribution of the Different Peaks
Obtained in the ethyl acetate subfractions
subfrac-
tion
peak
no.
retention
time (min)
λ
max
(nm)
relative
A
280nm
a
(%)
compound
EA-I
1
′
20.658
198.9, 324.3
16.28
hydroxycinnamic derivative
b
2
′
21.367
200.1, 315.9
5.36
hydroxycinnamic derivative
b
3
′
22.495
198.9, 310
78.36
hydroxycinnamic derivative
b
EA-II
4
′
4.394
204.8, 259, 293.3
21.21
protocatechuic acid
5
′
13.78
202.4, 224.8, 308.8
15.79
p-coumaric acid
6
′
18.269
197.8, 288.6
14.43
hesperitin derivative
7
′
18.489
197.8, 291, 315.9
31.98
unknown
3
′
22.150
198.9, 310
11.35
hydroxycinnamic derivative
b
8
′
22.538
197.8, 326.7
5.24
hydroxycinnamic derivative
b
EA-III
9
′
5.273
202.4, 279.1, 515.8
19.68
cyanidin-3-glucoside
10
′
15.278
202.4, 254.2, 353
16.15
quercetin derivative
11
′
16.409
198.9, 253.1, 351.8
64.18
quercetin derivative
EA-IV
10
′
15.302
202.4, 254.2, 353
100
quercetin derivative
a
Percentage area is defined as the integrated area of a single peak divided by
the total area of all integrated peaks. Peak absorbances at other wavelengths
were also measured but were not presented to simplify the analysis.
b
Bound
hydroxycinnamic forms composed of ferulic and
p
-coumaric derivatives (
12
).
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J. Agric. Food Chem., Vol. 54, No. 13, 2006
Pedreschi and Cisneros-Zevallos
Figure 7.
Mechanisms of antimutagenic action for (a) WF, (b) EAF, and (c) EA-IV. The concentrations used were 200, 90, and 50
µ
g of chlorogenic
acid equiv/plate.
Purple Corn Antioxidant and Antimutagenic Properties
J. Agric. Food Chem., Vol. 54, No. 13, 2006
4565
of treatments III and IV ( p > 0.05). The latter treatment would
confirm that the predominant mechanism of antimutagenic
action by the WF is due to inhibition of the S-9 Mix enzymes
responsible for the mutagen activation of Trp-P-1. This pre-
dominant mechanism would explain the lack of correlation
between the antioxidant and antimutagenic activities observed
before for purple corn anthocyanins.
On the other hand, both the EAF and the subfraction EA-IV
showed similar trend behaviors in the inhibition of the mutagenic
activity of Trp-P-1 (Figure 7b,c). Results for treatments II, III,
and IV showed trends similar to those obtained for the WF.
However, treatment V resulted in a further reduction of
mutagenic activity (p < 0.05), indicating that the nonanthocya-
nin phenolic compounds in EAF and EA-IV were involved in
further scavenging of mutagen electrophiles due most likely to
the longer period of incubation of the different components of
the assay. These results suggest that nonanthocyanin compounds
from the EAF and EA-IV show a dual antimutagenic mechanism
of action involving both enzyme inactivation and the scavenging
of reactive electrophiles that take place in different proportions
compared to the WF. In addition, this dual mechanism would
also explain the enhanced antioxidant and antimutagenic activi-
ties observed before for the EA-IV subfraction.
The predominant mechanism of action would be dependent
not only on the phenolic structures but also on the reaction
kinetics taking place among the different components of the
Ames test. This will include the reaction kinetics of Trp-P-1
and the S-9 Mix, the antimutagen with the S-9 Mix, the
antimutagen and the generated electrophiles, and these with the
cells. Further studies are needed to determine the real impact
of reaction kinetics.
In general, this study has shown that phenolic compounds
present in Andean purple corn have antimutagenic properties.
The phenolic compounds present in the ethyl acetate fractions
were mainly composed of phenolic acids and flavonols and were
more potent antimutagens than the anthocyanins present in the
water fraction. Quercetin derivatives are responsible for this
enhanced activity. No toxic effects were seen at doses lower
than 350
µg of chlorogenic acid equiv/plate. The antimutagenic
mechanism of action of purple corn phenolic compounds
involved enzyme inactivation and scavenging of electrophiles
and depended on the phenolic fraction. This work states the
importance of considering the phenolic compounds other than
anthocyanins in biological studies when using Andean purple
corn extracts.
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