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Article history: Myrciaria dubia, a plant native to the Amazon region, stands out as a fruit rich in vitamin C and other
Received 22 January 2012 metabolites with nutritional potential. We evaluated the antioxidant, genotoxic and antigenotoxic poten-
Accepted 11 April 2012 tial of M. dubia juice on blood cells of mice after acute, subacute and chronic treatments. Flavonoids and
Available online 21 April 2012
vitamin C present in the fruit of M. dubia were quantified. In vitro antioxidant activity was evaluated by
DPPH assay. Blood samples were collected for analysis after treatment, and the alkaline comet assay was
Keywords: used to analyze the genotoxic and antigenotoxic activity (ex vivo analysis using H2O2). The amount of
Myrciaria dubia
vitamin C per 100 mL of M. dubia was 52.5 mg. DPPH assay showed an antioxidant potential of the fruit.
Vitamin C
Genotoxicity
No M. dubia concentration tested exerted any genotoxic effect on mice blood cells. In the ex vivo test, the
Mice juice demonstrated antigenotoxic effect, and acute treatment produced the most significant results. After
Comet assay the treatments, there was no evidence of toxicity or death. In conclusion, our data show that M. dubia
juice has antigenotoxic and antioxidant activities, though with no genotoxicity for blood cells. Neverthe-
less, more in-depth studies should be conducted to assess the safety of this fruit for human consumption.
Ó 2012 Elsevier Ltd. All rights reserved.
0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.fct.2012.04.021
2276 F.C. da Silva et al. / Food and Chemical Toxicology 50 (2012) 2275–2281
juice in mice, using three different concentrations along with other 2.6. Determination of vitamin C
toxicological parameters.
The dosage of vitamin C (ascorbic acid) was performed in accordance with the
methodology described in Farmacopéia Brasileira (2010), using a fruit sample of
0.2 g and dissolving it in a mixture of 100 mL of water free of carbon dioxide and
2. Material and methods
25 mL of sulfuric acid 10% (w/v). Then, 3 mL of starch SI were added and immedi-
ately titrated with iodine 0.05 M (Sv). Each mL of iodine 0.05 M (sV) was equal to
2.1. Animals
8.806 mg of C6H8O6.
A total of 120 adult CF-1 mice, male and female, 8–10 weeks of age and weigh-
ing 27.02 ± 2.24 g (female) and 43.46 ± 6.70 g (male), were used. Animals were 2.7. Antioxidant assay in vitro
housed in plastic cages, with ‘‘ad libitum’’ access to water and food, under a 12-h
light/dark cycle, and at a constant temperature of 22 °C ± 1 °C. Each group was com- 2.7.1. Assay using 2,2-diphenyl-1-picrylhydrazyl (DPPH)
posed by 10 mice (five males and five females) and the protocol for these experi- The antioxidant assay of plant material was determined utilizing the DPPH as-
ments was approved by the Animal Ethics Committee of the Lutheran University say as described by Rodrigues et al. (2009). From the fruit extract dissolutions mea-
of Brazil (protocol number CEP-ULBRA 2010-007A). sured at 20,000 lg/mL, a total of 21 different dilutions were prepared in test tubes
in triplicate varying from 100 to 20,000 lg/mL. In a dark environment, 0.1 mL of
each extract dilution was transferred to test tubes with 3.9 mL of the DPPH radical
2.2. Plant material (DPPH solution of 0.05 mM) and was then homogenized in a test tube shaker. For
the calculation of DPPH we took into account the dilution of the fruit extract –
Samples of M. dubia H. B. K. (McVough) (camu–camu) (Myrtaceae) fruit were 0.1 mL of each dilution of extract to 3.9 mL of DPPH solution – and utilized the rela-
collected in wetlands, quarries and along the banks of the Jarú River in the Munic- tion Cinitial Vinitial = Cfinal Vfinal. Therefore, the extract dilution tested ranged between
ipality of Jarú, in the central region of the state of Rondônia, Cachoeira Bom Jardim. 2.5 and 275 lg/mL. Glass cuvettes were prepared with samples that were moni-
Aerial parts, flowers and buds were collected for plant identification and botanical tored until absorbance had stabilized, and readings of these solutions were subse-
information. Fruit samples were stored at 20 °C upon analyses, and were admin- quently made utilizing the BIOSPECTRO spectrophotometer, model SP 220, at
istered to mice. The three concentrations of the juice (25%, 50% and 100%) were pre- 515 gm. Using the obtained absorbance values from the different fruit extract dilu-
pared always before use so that there was no loss in metabolic properties. Juice was tions, a linear model was calculated according to the measured concentration of the
administered as 0.1 mL/10 g injections. total antioxidant activity (TAA) of the M. dubia extracts. The final result was ex-
Dried samples of the plant were identified and stored in the Herbário Central da pressed in grams of fruit (edible portions)/gram of DPPH. The data analysis was car-
Universidade Federal do Mato Grosso (UFMT) under number 29173/2010. ried out by regression (line equation). The larger the consumption of DPPH per
sample, the smaller its EC50 and the larger its antioxidant activity (Sousa et al.,
2007).
2.3. Phytochemical screening
2.8. Acute toxicity test
The plant was subjected to qualitative chemical screening for the identification
of the major classes of active chemical constituents (anthraquinones, alkaloids, car- Forty mice were divided into four groups of 10 animals each (five males and five
diac glycosides, coumarins flavonoids, tannins and saponins). The phytochemical females) for the acute treatment. They received single doses of M. dubia juice (25%,
profile of M. dubia juice was determined by these colorimetric reactions followed 50% and 100%) or water (control group) by gavage. The general behavior of the mice
by chromatographic analysis to confirm positive results (Rodrigues et al., 2009). was monitored for 1 h after dosing and periodically for 48 h (Regner et al., 2011).
(4), resulting in a single DNA damage score for each animal, and consequently each icant weight gain was reported for any of the treated groups, in
group studied. Therefore, the damage index (DI) can range from 0 (completely
relation to the control group.
undamaged, 100 cells 0) to 400 (with maximum damage, 100 4). The damage
frequency (DF) was calculated based on number of cells with tail versus those with
no tails (Rodrigues et al., 2009). 3.6. Comet assay
Fig. 2. Genotoxic/antigenotoxic effect of M. dubia juice after (A) acute, (B) sub-acute, and (C) chronic treatments as evaluated by the comet assay in male and female mice.
Damage index can range from 0 (completely undamaged, 100 cells 0) to 400 (with maximum damaged 100 4). Statistically significant difference compared to control
group ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001. (One-Way ANOVA followed by Dunnett’s test).
M. dubia belongs to the Myrtaceae family, and the fruit has great parameters of acute, subacute and chronic treatment of the M. du-
nutritional potential, easily adapted and included in human diets. bia juice (25%, 50%, and 100%) in male and female mice.
However, since it is a wild fruit, investment in research in order The genotoxic and antigenotoxic action of M. dubia juice on
to better evaluate its nutritional value in relation to its antioxidant mice blood cells was analyzed using the comet assay. No concen-
and nutritional content would be relevant, especially since it is al- tration tested demonstrated genotoxic action in blood cells (Figs.
ready being consumed in regions of the Amazon Rainforest. For 2 and 3). In the present study, the vitamin C concentration found
this reason, in this work we also investigated the toxicological in M. dubia juice was 52.5 mg/10 mL of (corresponding to 0.5 mg/
F.C. da Silva et al. / Food and Chemical Toxicology 50 (2012) 2275–2281 2279
Fig. 3. Genotoxic/antigenotoxic effect of M. dubia juice after (A) acute, (B) sub-acute, and (C) chronic treatments as evaluated by the comet assay in male and female mice.
Damage frequency was calculated based on the number of cells with tail vs those with no tail. Statistically significant difference compared to control group ⁄p < 0.05,
⁄⁄
p < 0.01, ⁄⁄⁄p < 0.001. (One-Way ANOVA followed by Dunnett’s).
mL). This amount can be considered the highest level of this com- toxicity. For instance, a protective effect of vitamin C was observed
pound in fruit. It is possible that the lack of damage to the DNA of against ethyl methane sulphonate-induced genotoxicity in fish
blood cells can be credited to interactions with other metabolic (Guha and Khuda-Bukhsh, 2002).
compounds present in M. dubia juice, promoting a reduction in The ex vivo test with M. dubia juice demonstrated an antigeno-
the pro-oxidant action of vitamin C (Aranha et al., 2010). Moreover, toxic effect after acute treatment in all of the concentrations. For
several studies showed the efficacy of vitamin C in reducing geno- the subacute and chronic treatments, only the concentrations of
2280 F.C. da Silva et al. / Food and Chemical Toxicology 50 (2012) 2275–2281
50% and 100% played a modulator role in the genotoxicity induced Conflict of Interest
by the H2O2, although juice 25% exhibited a slight antigenotoxic ef-
fect. These results suggest the protective capacity of M. dubia juice The authors declare that there are no conflicts of interest.
against genetic damage in the acute treatment or with repeated
doses over time. Other fruits rich in vitamin C, like acerola (Malpig-
hia glaba L.), showed a protective effect against DNA damage Acknowledgements
caused by H2O2 (Nunes et al., 2011), and similar tests that assesses
the effect of orange juice on blood cells of rats proved that it re- This work was supported by CNPq (Conselho Nacional de
duces the DNA damage induced by the mutagenic agents methyl Desenvolvimento Científico e Tecnológico), and FAPERGS (Fun-
methanesulfonate and cyclophosphamide (Franke et al., 2005). M. dação de Amparo à Pesquisa do Estado do Rio Grande do Sul);
dubia also demonstrated antigenotoxic activity. Brazil.
The phytochemical analysis revealed the presence of saponins,
flavonoids and tannins in M. dubia juice. These compounds present
in its fruits, together with vitamin C, probably had a fundamental References
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