Plant Foods Hum Nutr (2011) 66:129–135

DOI 10.1007/s11130-011-0223-7

ORIGINAL PAPER

Antigenotoxicity and Antioxidant Activity of Acerola Fruit

(Malpighia glabra L.) at Two Stages of Ripeness
Roberta da Silva Nunes & Vivian Francília Silva Kahl & Merielen da Silva Sarmento &
Marc François Richter & Letícia Veras Costa-Lotufo &
Felipe Augusto Rocha Rodrigues & Juan Andres Abin-Carriquiry &
Marcela María Martinez & Scharline Ferronatto &
Alexandre de Barros Falcão Ferraz & Juliana da Silva

Published online: 19 April 2011

# Springer Science+Business Media, LLC 2011

Abstract Genotoxic and antigenotoxic effects of acerola that unripe samples inhibit the free radical DPPH more
fruit at two stages of ripeness were investigated using mice significantly than the ripe ones. The results about determi-
blood cells. The results show that no ripeness stage of nation of compounds made using HPLC showed that unripe
acerola extracts presented any genotoxic potential to acerola presents higher levels of vitamin C as compared to
damage DNA (Comet assay) or cytotoxicity (MTT assay). ripe acerola. Thus, vitamin C and the complex mixture of
When antigenotoxic activity was analyzed, unripe fruit nutrients of Malpighia glabra L., and especially its ripeness
presented higher DNA protection than ripe fruit (red color) stages, influenced the interaction of the fruit extract with the
extract. The antioxidant capacity of substances also showed DNA. Acerola is usually consumed when ripe (red fruit),
although it is the green fruit (unripe) that has higher potential as
R. da Silva Nunes : V. F. S. Kahl : M. da Silva Sarmento : beneficial to DNA, protecting it against oxidative stress.
J. da Silva (*)
Laboratório de Genética Toxicológica—Programa de Pós-
Keywords Malpighia glabra L. . Acerola . Genotoxicity .
Graduação em Genética e Toxicologia Aplicada,
Universidade Luterana do Brasil, Antigenotoxicity . Vitamin C
92425–900 Canoas, Rio Grande do Sul, Brazil
e-mail: juliana.silva@ulbra.br Abbreviations
AEAC Ascorbic acid equivalent
R. da Silva Nunes
Centro Universitário Luterano de Ji-Paraná, antioxidant capacity
Universidade Luterana do Brasil, AOA Antioxidant activity
76.907–438 Ji-Paraná, RO, Brazil DF Damage frequency
DI Damage index
M. F. Richter
Universidade Estadual do Rio Grande do Sul, UERGS, DNA Deoxyribonucleic acid
90.010–191 Porto Alegre, RS, Brazil DMSO Dimethyl sulfoxide
DOX Doxorubicin
L. V. Costa-Lotufo : F. A. R. Rodrigues
DPPH 2,2-diphenyl-1-picrylhydrazyl radical
Laboratório de Oncologia Experimental,
Universidade Federal do Ceará, UFC, EDTA Ethylenediaminetetraacetic acid
60341–970 Fortaleza, CE, Brazil HCl Hydrochloric acid
HCT-8 Tumor line of human colon
J. A. Abin-Carriquiry : M. M. Martinez
H2O2 Hydrogen peroxide
Department of Neurochemistry,
Instituto de Investigaciones Biológicas Clemente Estable, HPLC High performance liquid
11600 Montevideo, Uruguay chromatography
H3PO4 Phosphoric acid
S. Ferronatto : A. de Barros Falcão Ferraz
NaCl Sodium chloride
Laboratório de Farmacognosia e Fitoquímica,
Universidade Luterana do Brasil, NaOH Sodium hydroxide
92425–900 Canoas, RS, Brazil MDAMDB-435 Tumor line of human breast
130
MeOH Methanol
MTT 3-(4,5-dimethyl-2-thiazole)-2,5-
diphenyl-2-H-tetrazolium bromide salt
PBS Phosphate buffered saline
RDA Recommended Dietary Allowance
SF-295 Tumor line of human nervous system
Tris Tris(hydroxymethyl)aminomethane
Introduction
The effects of the plant constituents on organisms are
almost always observed based on the popular knowledge of
traditional and repetitive use. It is known that vegetables
and fruits have different phytochemical constituents respon-
sible for therapeutic properties like antioxidant, antimutagenic
and anticarcinogenic properties [1–3].
According to Machado [4], there is a worldwide trend
towards increased consumption of tropical fruit juice.
Malpighia glabra L., popularly called “acerola” in Brazil
or “Barbados cherry” and “West Indian cherry” elsewhere,
is a native species to tropical America. It is very popularly
used due to its astringent, antifungal and antianemic
properties, as well as its nutritional capacity and vitamin
content. Chemical composition of acerola, including the
distribution of components, varies between cultivars with
environmental conditions and also with the stage of fruit
ripeness [5, 6]. The fruit ripens quickly after being picked
(within 2 to 3 days), at room temperature [5].
Acerola, due to its undeniable potential as a natural source
of different vitamins and the high potential for industrial use,
has attracted the interest of fruit growers and acquired
economic importance in several regions of Brazil [3].
The nutritional composition of fresh acerola fruits and
raw acerola juice illustrates that these products are
excellent sources of vitamin C, flavonoids, carotenoids,
precursors of vitamin A, lycopene, and contain consid-
erable amounts of thiamin, riboflavin, niacin, pantothenic
acid, calcium, iron and magnesium [7]. A portion of
100 g of fresh fruit provides 2.033% of the Recommended
Dietary Allowance (RDA) of vitamin C and 28.76% of the
vitamin A needed by an adult [7, 8].
In recent years, many studies have been conducted on
the role of reactive oxygen species in the etiology of
various diseases [9]. Vitamin C and other constituents of
acerola, like flavonoids, act as antioxidants in the human
body [10]. It has been reported that the amounts of both
vitamin C and flavonoids in acerola decrease with fruit
maturation [11]. In this scenario, the antioxidant properties
of flavonoids have drawn attention to preventive nutrition,
because food constituents protect against oxidative damage
and may also contribute to the prevention of major
Plant Foods Hum Nutr (2011) 66:129–135
pathologies such as cardiovascular disease, aging and
cancer [12]. The high ascorbic acid content and the
presence of anthocyanins highlight the importance of
acerola due to the functional ability that these compounds
exhibit to capture free radicals in the human body. A food
can be considered functional if it is demonstrated that it can
affect beneficially one or more target functions in the body,
producing the adequate nutritional effects in a way that is
both relevant to well-being and health and to reduce the risk
of a disease [13].
Due to the increasing commercialization and the grow-
ing consumption of tropical fruits, both in Brazilian and
international markets, and because tropical fruits are a
source of antioxidants, the objective of this study was to
determine the antigenotoxic potential of acerola at two
ripeness stages ex vivo using the Comet assay in mice cells.
The cytotoxic activity of the fruit and some chemical
components were also evaluated.
Materials and Methods
Origin and Samples Preparation
Ripe and unripe Malpighia glabra L. fruits were collected
in May 2008, in the district of Ubajara, CE, Brazil. The
fruits were peeled and seeds removed. The pulp extract
from ripe to unripe fruits were lyophilized. The dilutions
were made with water.
Animals and Cell Treatment for Comet Assay
Male mice, Mus musculus CF-1, weighing 30–40 g, were
supplied by the central animal house of the Lutheran
University of Brazil, ULBRA. The animals were housed in
propylene cages under 12 h light/dark cycles at constant
temperature of 23±2 °C and relative humidity of about
60%, with tap water and food ad libitum. All experiments
were approved by CONEP—Brazil (National Commission
of Ethics in Research).
The acerola cultivar used in this study was AC69 at two
stages of ripeness, ripe and unripe. The fruits were collected
randomly across the whole plant, and were sorted to
ripeness using a skin color gradient, as follows: (1) green
fruits (unripe), fruits showing a green hue on more than
75%; and (2) red fruits (ripe), those showing a red or
burgundy hue on 100% of the shell. After collection, fruits
were kept frozen and protected from light in order to
preserve their chemical and physical characteristics upon
extract preparation [14].
The pulp extract of frozen acerola was lyophilized and then
stored in a desiccator. Next, lyophilized extracts were diluted
in RPMI. The maximum concentration of fruit extracts used
Plant Foods Hum Nutr (2011) 66:129–135
for cell exposure in vitro was 2 mg/ml, which was
determined in a preliminary assessment using 1–5 mg/ml.
The dose selection was performed according to OECD
guideline 473 [15]. The dose of 2 mg/ml demonstrated
sufficient number of viable cells for both stages of ripeness.
This dose was used to prepare dilutions, at three concen-
trations (100%, 50% and 25%): C1 (2 mg/ml), C1/2 (1 mg/
ml) and C1/4 (0.5 mg/ml). The cells were treated with these
doses during 2 h. C1/2 from unripe fruit was also used for
cell exposure for 4 h, because it presented better results in the
experiments (see results), in order to evaluate if different
exposure times would play a role in the results.
Animals were sacrificed by decapitation. Four aliquots
of 100 μl of whole blood were collected from each animal
(n=4), each exposed to one of the three concentrations
used, for 2 h, at 37 °C, in stove (exposure groups of
replicated test), protected from light. One blood sample of
each animal was used as negative control (without H2O2),
with no exposure to acerola extracts [16]. Four replicated
slides were prepared for Comet assay from each exposure
group. Two slides were immersed directly in a lysis
solution and two slides exposed to H2O2 (before lysis
solution). DNA damage was induced ex vivo by exposing
the blood cells on slides to H2O2 0.25 mM. In order to
establish the protective ability of fruit extracts against
the oxidative stress induced by hydrogen peroxide, the
Comet assay was performed with slides from all the
groups. The slides were exposed to hydrogen peroxide
for 5 min (ex vivo) [16, 17]. Antigenotoxic potential was
expressed as described in Kapiszewska et al. [18], as
percentage inhibition of damage index (DI) according to
the expression: (I%): percentage of inhibition of DI ¼
½H2 O2 DI  DI of the extract with H2 O2 =½H2 O2 DI  DI
negative control  100.
Comet Assay
The alkaline Comet assay was performed according to
guidelines proposed by Tice et al. [19] with the slight
modification developed by Silva et al. [20]. Briefly, 5 μl
of blood sample were added to 95 μl of 0.75% (w/v) low-
melting point agarose, and part of the mixture was spread
on a microscope slide pre-coated with 1.5% (w/v) normal-
melting point agarose and covered with a coverslip. After
agarose solidified, coverslips were removed, and slides
were immersed in a lysis solution for at least 2 h. Slides
were subsequently incubated in freshly prepared alkaline
buffer (pH> 13) for 20 min. Electrophoresis (0.8 V/cm)
was performed in the same buffer. Every step was carried
out under indirect red light. After electrophoresis, slides
were neutralized in Tris 400 mM (pH 7.5). Slides were
then fixed, and stained with silver nitrate [21]. Cells were
scored visually according to tail size and % of DNA in the
131
tail into five classes: (1) class 0: undamaged, with no tail;
(2) class 1: with tail shorter than the diameter of the head
(nucleus); (3) class 2: with tail length between one and
two times the diameter of the head; (4) class 3: with tail
longer than two times the diameter of the head, and (5)
class 4: comets with no heads. Two parameters, damage
index (DI) and damage frequency (DF), were used in our
analyses to evaluate DNA damage. DI was calculated for
each sample and ranged from 0 (completely undamaged:
100 cells X 0) to 400 (with maximum damage: 100 cells
X 4). DF (%) corresponds to the percentage of cells with
tail (classes 1–4) in each sample. All slides were scored
blindly.
HPLC Analyses
Three milligrams of lyophilized sample was weighed and
1 ml of H20 was added and submitted to vortex agitation.
After, the sample was centrifuged for 5 min at 4 °C and
1,000 rpm. A 20 μl aliquot of the supernatant was injected
into the HPLC system. Standard solutions of quercetin
were prepared from stock solution (10 mM in MeOH). It
was diluted 1/20 with MeOH (50 μl of standard and
950 μl of MeOH), and this solution was then diluted 1/20
again, using mobile phase. The standard solution of rutin
was made by dissolving 3 mg of rutin in 1 ml of MeOH.
A 50 μl aliquot of this solution was diluted in a final
volume of 1,000 μl with MeOH. Then, a 50 μl aliquot
from the first solution was again diluted in a final
volume of 1,000 μl with mobile phase. The standard
solution of ascorbic acid (vitamin C) was prepared with
5 mg of ascorbic acid in 1 ml of H2O. It was diluted 1/20
with H2O (50 μl of standard and 950 μl of MeOH) and
then it was again diluted to 1/20, with the mobile phase
(50 μl of sample and 950 μl of the mobile phase).
Standard samples used were ascorbic acid, rutin, and
quercetin (Sigma).
Quantitative analysis of constituents was conducted by
reverse-phase HPLC using a C18 column (150 mm×
34.6 mm; phenomenex, USA) with 5 μm particle size. A
binary HPLC pump (Waters 1525) coupled with a 717
plus autosampler Waters and a photodiode array detector
Waters, and chromatography data software (Empower 2)
was utilized. The temperature of the column was set at
30 °C. The mobile phase used was: a) methanol
(MeOH); and b) 0.5 % phosphoric acid (H3PO4), pH 2
and 5% MeOH at total flow of 0.7 ml/min. The gradient
system consisted of (min/%B): 0/80, 40/0, 41/80, 47/80.
Run time was 47 min, with 1-min intervals between each
race. The eluent was monitored at 210–600 nm and
quantitative determination of flavonoids was performed
at 375 nm and ascorbic acid at 264 nm. The results were
expressed in mg/100 g of acerola.
132 Plant Foods Hum Nutr (2011) 66:129–135

Determination of Total Flavonoid Content thiazol)-2,5-diphenyl-2-H-tetrazolium bromide (MTT;

For the determination of flavonoid content, 50 mg of
lyophilized extract of each sample was dissolved in water
and diluted with ethanol in 250 ml volumetric flask. To
2.0 ml of the stock solution was added 1 ml of aluminum
chloride solution 2.5% in 25 ml volumetric flask. The
final volume was adjusted with ethanol. After 30 min,
readings at 425 nm were performed in a Shimadzu
spectrophotometer, for each solution. The total flavonoid
content was calculated using the expression: A  1:25=m½A ¼
absorbance measured; m ¼ mass of drugðgÞ [22].
DPPH Free Radical Scavenging Assay
Scavenging of DPPH free radical was measured using a
modified Yamaguchi et al. [23] method in which the
different methanolic plant extracts were added to Tris-HCl
buffer, pH 7.0, containing 250 mM DPPH dissolved in
methanol. At least six different dilutions of each extract
were tested, and allowed to stand 20 min in the dark, before
absorbance was measured at 517 nm using a Shimadzu
spectrophotometer model UV-1602PC (Kyoto, Japan). The
experiment was conducted in triplicate. Antioxidant activity
(AOA) was expressed as IC50 (inhibitory concentration in
μg/ml of samples or positive controls necessary to reduce
the absorbance of DPPH by 50% compared to the negative
control). The lower the IC50, the higher is the AOA. Results
were also expressed as AEAC (ascorbic acid equivalent
antioxidant capacity) in grams and calculated as follows:
AEAC ðμg AA=gÞ ¼ IC50ðAAÞ =IC50ðsampleÞ  1 g.
MTT Assay
Analysis of cytotoxicity by MTT method was performed
according to Skehan et al. [24]. The colorimetric analysis
is based on the conversion of the salt 3-(4,5-dimethyl-2-
Sigma M2128; 98% purity) into blue formazan, from
mitochondrial enzyme present only in metabolically active
cells. The method allows defining cytotoxicity, but not the
mechanism of action. Tumor lines used were MDAMDB-
435 (human melanoma), HCT-8 (human colon) and SF-
295 (human nervous system), which were grown in RPMI
1640. The chemotherapy doxorubicin (DOX) was used as
positive control. Acerola fruits extracts were diluted in
sterile PBS and tested as a 50 μg/ml solution. Cells were
placed in a 96-well plate at a concentration of 0.1×106
cells/ml for line SF-295 and MDAMDB-435 and 0.3×106
for line HCT-8. The plates were incubated for 72 h in a
stove at 5% of CO 2 and 37 °C. Next, they were
centrifuged and the supernatant removed. Then 150 μl of
MTT solution was added and plates were incubated for
3 h. Absorbance was read after dissolution of precipitate
with 150 μl of pure DMSO in a spectrophotometer plate at
595 nm.
Results
The Comet assay revealed that no acerola concentration
showed potential to induce DNA damage, both as DI and
DF, independently of ripeness (Table 1). When antigeno-
toxicity activity was analyzed, only unripe fruit showed
significant results in relation to the control. Concentrations
C1/2 (P<0.001) and C1/4 (P<0.01) protected DNA in
relation to H2O2 as measured by DI and DF (Table 1).
Figure 1a shows that, for the 4-h treatment, the dose C1/2
caused a significant reduction in DNA damage, in
comparison to the 2-h treatment. When antigenotoxic
potential is analyzed according to Kapiszewska et al. [18],
a DNA damage modulation of 14% was observed after the
2-h treatment, while after the 4-h treatment the modulation
was 71%.

Table 1 Comet assay in periph-

eral blood leukocytes of mice Groups Comet assay parameters
blood cells (ex vivo) treated with
acerola for 2 h with and without Without H2O2 With H2O2
hydrogen peroxide (0.25 mM).
Values are expressed as Damage index Damage frequency Damage index Damage frequency
mean±standard deviation
Control 13.50±9.87 9.06±6.25 111.40±17.78a 44.75±2.96a
Unripe acerola
**Significant difference related
to the control groups for damage C1/4 (0.5 mg/ml) 5.38±2.83 4.38±1.77 73.50±15.65*** 33.50±5.81**
index and damage frequency at C1/2 (1 mg/ml) 7.88±3.44 4.38±2.56 79.63±37.87** 31.00±7.69***
P<0.01; *** P<0.001 (one-way C1 (2 mg/ml) 8.38±8.14 5.88±6.38 102.10±14.53 38.38±5.78
ANOVA followed by Tukey Ripe acerola
Test). a Significant difference
related to the control without C1/4 (0.5 mg/ml) 14.88±2.75 11.13±3.36 112.90±7.47 48.00±1.20
hydrogen peroxide at C1/2 (1 mg/ml) 14.13±9.05 10.75±6.16 101.90±26.11 42.63±9.58
P<0.001 (One Way ANOVA C1 (2 mg/ml) 18.25±6.67 14.25±4.06 111.00±6.41 46.50±2.00
followed by Tukey Test)
Plant Foods Hum Nutr (2011) 66:129–135 133

(a) 150 Table 2 Test HPLC in unripe (green) and ripe (red) acerola diluted in
aqueous fruit extract
Damage Index (0-400)

* Sample Ascorbic acida Rutina Quercetina

100
Controlb 250.0 150.0 150.0
Unripe acerola 8,104.0 150.1 57.1
50 ***, a Ripe acerola 4,447.6 150.2 57.1
a
Results in mg/100 g of sample; b Standard sample (ascorbic acid, rutin,
and quercitin)
0
Control (H2O2) C1/2 2h C1/2 4h

The free radical scavenging of DPPH free radical effect

(b) 60 of the two samples was tested using the DPPH free radical
scavenging assay. Ascorbic acid was used as positive
Frequency Damage (%)

40 ***
20
0 presented promising antioxidant activity. It was also
Control (H2O2) C1/2 2h
Fig. 1 Comet assay in peripheral blood leukocytes of mice blood
cells (ex vivo) without treatment with acerola (control) and treated
with unripe acerola sample (C1/2: 1 mg/ml) for 2 h and 4 h, all
exposed to hydrogen peroxide (0.25 mM). (a) Damage index; and (b)
Damage frequency. Values are expressed as mean±standard deviation.
*Significant difference related to the control groups for damage
index and damage frequency at P< 0.05; ** P<0.01; *** P<0.001.
a
Significant difference related to the C1/2 2 h at P<0.01 (one-way
ANOVA followed by Tukey Test)
control, in the same assay. The IC50 values for the extracts
** are shown in Table 3. The results of the free radical
scavenging effect of ascorbic acid (IC50 = 4.03 μg/ml)
validate the assay. Although the free radical scavenging
capacities of the ripe acerola sample were lower (higher
concentration necessary to reduce the absorbance of DPPH
by 50%) than the effects of vitamin C, unripe acerola
C1/2 4h
observed that the amount of extract needed to capture the
free radical DPPH is lower in the unripe fruit (IC50 =
4.33 μg/ml) than in ripe (IC50 = 8.67 μg/ml) acerola fruit,
and that the antioxidant capacity of IC50 of unripe fruit was
twice as high as that of ripe fruit (Table 3).
The cytotoxicity (MTT) of all samples is presented in
Table 4, with their respective percentages of inhibition. No
sample of acerola showed inhibition of tumor growth. In
order to be considered a potential antitumor substance, the
inhibition has to be ≥75% in at least two tumor lines tested.

HPLC revealed that unripe acerola presented higher

level of vitamin C than ripe fruit. Considering rutin and
quercetin, no differences were observed between ripe and
unripe fruit (Table 2).
Quantitative analysis of total flavonoids shows that ripe
acerola extract presented higher flavonoid content (15.3±
0.35%) than unripe acerola extract (8.7±0.21%).
Discussion
Nowadays acerola is considered as a functional fruit and
therefore is consumed to prevent diseases or as adjuvant in
treatment strategies. The characteristics of acerola are
complex, promoting the interaction with biological organ-

Table 3 IC50 values in the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay and AEAC (antioxidant capacity equivalent to ascorbic acid) of unripe
and ripe acerola fruits (lyophilized samples diluted in water) and ascorbic acid

Sample Inhibition per concentration IC50 (μg/ml)a AEAC (μg/g)

1 μg/ml 10 μg/ml 100 μg/ml 1,000 μg/ml

Ascorbic acid 9.70 98.11 99.40 99.62 4.03±0.16 1.0000

Unripe acerola 9.75 97.94 99.21 99.73 4.33±1.67 0.9307
Ripe acerola 9.58 62.81 95.30 98.36 8.69±1.95 0.4638
a
Mean±SD from IC50 (μg/ml). Results were based on the values measured at 20 min. Ascorbic acid was used as positive control
134
Table 4 Percentage of inhibition of tumor growth of the samples on
three tumoral lines tested at single dose of 50 μg/ml. Values are
expressed as mean±standard deviation
Samples Degree of inhibition of tumor growth (DI%)
MDAMB-495a SF-295b HCT-8c
Doxorubicin 99.43±0.34 96.14±0.48 87.62±1.51
Unripe acerola 26.22±4.58 0.00±0.00 11.10±0.87
Ripe acerola 29.50±0.55 0.00±0.00 3.94±3.05
a
Human melanoma cell lines; b Human nervous system cell lines; c Human
colon cell lines
isms in many ways. Several factors are responsible for the
chemical composition of acerola, such as species, environ-
ment and also ripeness stage [5]. Samples used in this study
were of the same species, Malpighia glabra L., but from
different ripeness stages (ripe to unripe).
The results obtained in the present study show that
neither ripeness stages of acerola presented genotoxic
potential to DNA of mice cells exposed to acerola extracts
ex vivo (Table 1). No data on assessments of the
genotoxicity or damage effect of different species of acerola
was found in the literature.
When antigenotoxic activity of acerola extracts was
analyzed, unripe fruit induced higher DNA protection
than ripe fruit extract. Unripe samples (at concentrations
0.5 and 1 mg/ml) showed a protective effect to DNA
against damages caused by H2O2, though the same was
not observed for ripe samples (Table 1). One of the
reasons for the results observed was that unripe acerola
presents higher levels of vitamin C than the ripe fruit
(Table 2), which was determined by HPLC. In the U.S.,
the RDA for vitamin C was revised in the year 2000
upward from the previous recommendation of 60 mg daily
for men to women (75 mg and 90 mg, respectively) [25].
The results obtained in the preset study show that the
samples analyzed contain more vitamin C in 100 g than
the recommended value, where vitamin C content in
unripe acerola was ~98 times and in ripe samples was
~54 times higher than the values recommended by RDA.
Complementing this result, the DPPH test, which tests
antioxidant capacity of substances, revealed that unripe
samples inhibit free radical DPPH more significantly than
the ripe fruit samples, and that antioxidant activity of
unripe fruit is very similar to that of the vitamin C
(Table 3). As vitamin C is a known antioxidant agent [26],
this may have influenced unripe acerola extract in
decrease the oxidant action of hydrogen peroxide in the
mice cells, protecting DNA. Plants in general, particularly
fruits, have several compounds with antioxidant proper-
ties, which include ascorbic acid, carotenoids and poly-
Plant Foods Hum Nutr (2011) 66:129–135
phenols [27–29]. The vitamin C content in most fruits
tends to decrease during ripening [30]. Butt [31] attributes
this decrease to the action of an enzyme called ascorbic
acid oxidase (ascorbate oxidase), isolated from acerola by
Asenjo [32], who found that the enzyme activity in ripe
fruit is higher than in unripe, which may explain the losses
encountered in the course of maturation.
When the period of pretreatment of blood samples from
mice with acerola extracts was increased, it was observed
that the 4-h treatment modulated the damage five times
more expressively than the 2-h treatment with unripe
acerola extract.
According to Vendramini & Trugo [5], acerola
presents more vitamin C when unripe. In this study, it
was proved that unripe fruit actually presents higher
levels of ascorbic acid than ripe fruit, while rutin and
quercetin were detected at similar levels, independently of
ripeness. The presence of ascorbic acid and flavonoids,
especially flavonols group as rutin and quercetin, place
acerola as an indisputable source of important compounds
for human diet [7]. A sufficient number of flavonoids
have exhibited antineoplastic activity. Several recent
reviews have highlighted this activity (for a review see
Tapas et al. [12]). However, HPLC demonstrated a great
amount of rutin and quercetin, although the test of
cytotoxicity (MTT) did not demonstrate any inhibitory
potential of cell growth from acerola extracts, either ripe
or unripe (Table 4).
Thus, vitamin C and the complex mixture of nutrients of
Malpighia glabra L., and especially its ripeness stages,
influenced the interaction of the fruit extract with DNA.
Although many other factors are involved in the differences
observed, considerable evidence demonstrated that green
fruit has higher beneficial potential to DNA, protecting it
against oxidative stress. Considering these data and the fact
that acerola is usually consumed when ripe, we can suggest
that the use of acerola, especially in its initial stage of
ripeness, protects and helps repair damaged DNA. As
regards antioxidant action, unripe acerola can be pointed as
a good source of natural antioxidants that may be more
effective and cost effective than the use of dietary supple-
ments in protecting the body against oxidative damage.
Acerola, among other fruits, is a food suggested as part of a
proper diet for healthy living. However, it is believed that
quality of life cannot be achieved only by consuming a
single food.
Acknowledgements The authors also thank Nutrilite Farm (Ceará,
Brazil) for help and assistance. This work was supported by grants
from the Brazilian Agencies Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do
Rio Grande do Sul (FAPERGS) and Lutheran University of Brazil
(ULBRA).
Plant Foods Hum Nutr (2011) 66:129–135
References
1. Kusamran WR, Tepswan A, Kupradinun P (1998) Antimutagenic
and anticarcinogenic potentials of some Thai vegetables. Mutat
Res 402:247–258
2. Nakamura YK, Suganuma E, Kuijama N, Sato K, Ohtsuki K (1998)
Comparative bio-antimutagenicity of common vegetables and tradi-
tional vegetables in Kyoto. Biosci Biotechnol Biochem 62:1161–1165
3. Nogueira MEI, Passoni MH, Biso FI, Longo MC, Cardoso CRP,
dos Santos LC, Varanda EA (2006) Investigation of genotoxic and
antigenotoxic activities of Melampodium divaricatum in Salmonella
typhimurium. Toxicol In Vitro 20:361–366
4. Machado UD (1992) Nordeste-EMBRAPA: Relatório: Avaliação
e proposições. Sindicato Nacional dos Trabalhadores de Pesquisa
e Desenvolvimento Agropecuário, Brasília
5. Vendramini AL, Trugo LC (2000) Chemical composition of
acerola fruit (Malpighia glabra L.) at three stages of maturity.
Food Chem 71:195–198
6. Kawaguchi M, Tanabe H, Nagamine K (2007) Isolation and
characterization of a novel flavonoid possessing a 4,2″-glycosidic
linkage from green mature acerola (Malpighia emarginata DC.)
fruit. Biosci Biotechnol Biochem 71:1130–1135
7. USDA (National Nutrient Database for Standard) (2010) http://
search.nal.usda.gov/nalsearch/> (accessed 01.10.10)
8. Freitas CAS, Maia GA, Costa JMC, Figueiredo RW, Souza PHM
(2006) Acerola: produção, composição, aspectos nutricionais e
produtos. R Bras Agrociência 12:395–400, in portuguese
9. Halliwell B (1987) Oxidants and human disease: Some new
concepts. FASEB J 1:358–364
10. Sizer FS, Whitney EN (2003) Nutrition: concepts and controver-
sies, 9th edn. Thompson Wadsworth, Belmont
11. Hanamura T, Uchida E, Aoki H (2008) Changes of the composition
in acerola (Malpighia emarginata DC.) fruit in relation to cultivar,
growing region and maturity. J Sci Food Agric 88:1813–1820
12. Tapas AR, Sakarkar DM, Kakde RB (2008) Flavonoids as
nutraceuticals: A review. Trop J Pharm Res 7:1089–1099
13. Roberfroid M (2002) Functional food concept and its application
to prebiotics. Dig Liver Dis 34:105–110
14. Campelo E, Martins M, Carvalho I, Pedrosa E (1998) Teores de
vitamina C em polpas de acerola (Malpighia glabra L.)
congeladas. B CEPPA 16:107–113, in portuguese
15. OECD (1997) OECD guidelines for the testing of chemicals: in
vitro mammalian chromosome aberration test, revised and new
guidelines, adopted 1997. Organization for Economic Cooperation
and Development, Paris
16. Lovell DP, Omori T (2008) Statistical issues in the use of the
comet assay. Mutagenesis 23:171–182
17. Szeto YT, Collins AR, Benzie IFF (2002) Effects of dietary
antioxidants on DNA damage on lysed cells using a modified
comet assay procedure. Mutat Res 500:31–38
135
18. Kapiszewska M, Soltys E, Visioli F, Cierniak A, Zajac G (2005)
The protective ability of the Mediterranean plant extract against
the oxidative DNA damage. The role of the radical oxygen species
and the polyphenol content. J Physiol Pharmacol 56:183–197
19. Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A,
Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki YF (2000)
Single cell gel/comet assay: Guidelines for in vitro and in vivo
genetic toxicology testing. Environ Mol Mutagen 35:206–221
20. Silva J, Freitas TRO, Marinho JR, Speit G, Erdtmann B (2000)
An alkaline single-cell gel electrophoresis (comet assay) assay for
environmental biomonitoring with native rodents. Genet Mol Biol
23:241–245
21. Nadin SB, Vargas-Roig LM, Ciocca DR (2001) A silver staining
method for single-cell gel assay. J Histochem Cytochem 49:1183–
1186
22. British Pharmacopoeia (2007) Version 11.0-Cd-Rom. Her Majesty’s
Stationary Office, London
23. Yamaguchi T, Takamura H, Matoba T, Terao J (1998) HPLC
method for evaluation of the free radical-scavenging activity of
foods by using 1,1-diphenyl-2-picrylhidrazyl. Biosci Biotechnol
Biochem 62:1201–1204
24. Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica
D, Warren JT, Bokesch H, Kenney S, Boyd MR (1990) New
colorimetric cytotoxicity assay for anticancer-drug screening. J
Natl Cancer Inst 82:1107–1112
25. Higdon J (2009) Vitamin C Linnus Pauling Institute Micronut Res Opt
Health. <http://lpi.oregonstate.edu/infocenter/vitamins/vitaminC/>
(accessed 01.10.10)
26. Franke SIR, Prá D, Silva J, Erdtmann B, Henriques JAP (2005)
Possible repair action of vitamin C on DNA damage induced by
methyl methanesulfonate, cyclophosphamide, FeSO4 and CuSO4
in mouse blood cells in vivo. Mutat Res 583:75–84
27. Leong LP, Shui G (2002) An investigation of antioxidant capacity
of fruits in Singapore markets. Food Chem 76:69–75
28. Paredes-López O, Cervantes-Ceja M, Vigna-Pérez M, Hernández-
Pérez T (2010) Berries: Improving human health and healthy
aging, and promoting quality life—A review. Plant Foods Hum
Nutr 65:299–308
29. Seeram NP, Aviram M, Zhang Y, Henning SM, Feng L, Dreher M,
Heber D (2008) Comparison of antioxidant potency of commonly
consumed polyphenol-rich beverages in the United States. J Agric
Food Chem 56:1415–1422
30. Jawaheer B, Goburdhum D, Rugoo A (2003) Effect of processing
and storage of guava into jam and juice on ascorbic acid content.
Plant Foods Hum Nutr 51:1–12
31. Butt VS (1980) Direct oxidases and related enzymes. In: Stumpf
PK, Conn EE (eds), The Biochemistry of Plants: A Comprehen-
sive Treatise. Academic, New York, pp 81–123, 2
32. Asenjo CF, Penaloza A, Medina P (1960) Characterization of
ascorbase present in the fruit of the Malpighia punicifolia L.
FASEB J 19:1–1