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Antiproliferative, antimutagenic and antioxidant activities of a Brazilian tropical fruit juice
LWT - Food Science and Technology xxx (2014) 1e6
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Antiproliferative, antimutagenic and antioxidant activities of a Brazilian tropical fruit juice Luciano Bruno de Carvalho-Silva a, Ana Paula Dionísio b, *, Ana Carolina da Silva Pereira c, Nedio Jair Wurlitzer b, Edy Sousa de Brito b, Giovana Anceski Bataglion c, Isabella Montenegro Brasil d, Marcos Nogueira Eberlin c, Rui Hai Liu e a
School of Nutrition, Federal University of Alfenas, Minas Gerais, Brazil Embrapa Tropical Agroindustry, Fortaleza, Ceará, Brazil Thomson Mass Spectrometry Laboratory, UNICAMP, Campinas, SP, Brazil d Department of Food Technology, Federal University of Ceará, Fortaleza, Ceará, Brazil e Department of Food Science and Institute of Comparative and Environmental Toxicology, Stocking Hall, Cornell University, Ithaca, NY, USA b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 February 2014 Received in revised form 1 April 2014 Accepted 2 April 2014 Available online xxx
This paper reports the antioxidant activity of a tropical fruit juice and its antiproliferative and antimutagenic effects. The antioxidant activity was determined by several different methods, such as ORAC, ABTS, PSC (peroxyl radical scavenging capacity) and CAA (cellular antioxidant activity). The total of phenolics and flavonoids were determined, and the phenolic compounds were identified by LC-DAD-ESIMS. The juice showed high total phenolic and flavonoid contents (838.44 30.27 g GAE/100 g and 219.45 12.27 mg CE/100 g DW, respectively). According to the PSC, CAA, ABTS and ORAC assays, the tropical fruit juice showed a high antioxidant value of 308.39 3.10 mM AEE/100 g, 26.76 4.47 mM QE/ 100 g, 167.17 4.10 mM TE/g DW and 235.90 11.90 mM TE/g DW, respectively. The proliferation of HepG2 was significantly inhibited, in a dose-dependent manner, by exposure to the tropical fruit juice. Antimutagenic activity was investigated by micronucleous test in mice, and all doses evaluated (30, 100 and 300 mg/kg b.w.) showed beneficial effects. As a consequence, these results encourage further studies on the pharmacological and functional properties of this tropical fruit juice, in order to evaluate its use as a functional food, due to its beneficial health properties. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Phytochemicals Cancer Antimutagenic activities
1. Introduction Brazil is a country that stands out because of the climatic conditions and large diversity of underexploited native and exotic fruit species. An example of this is the camuecamu (Myrciaria dubia Mc. Vaugh), a common fruit that grows near the Amazonian rivers and lakes. The camuecamu is growing in both Brazil and Peru and represents one of the richest sources of vitamin C ever studied (Genovese, Pinto, Gonçalves, & Lajolo, 2008; Rufino et al., 2010). Acai (Euterpe oleracea Mart.) is also obtained from the Amazon River basin, and is manually harvested from wild acai palms (E. oleracea) that are native to that area. This fruit has received much
* Corresponding author. Embrapa Tropical Agroindustry, Dra Sara Mesquita Street, 2270, 60511-110 Fortaleza, CE, Brazil. Tel.: þ55 85 33917327; fax: þ55 85 33917221. E-mail addresses: [email protected], [email protected] (L.B. de Carvalho-Silva).
attention in recent years due to its high antioxidant activity and its role as a “functional food”. It represents good perspectives for industrial purposes and is recognized worldwide, as shown by its increased amount of exportation (Genovese et al., 2008). Acerola (Malpighia emarginata) and the cashew-apple (Anacardium occidentale L.) also represent a good source of bioactive compounds, and offer potential uses in different juices and beverages (Rufino et al., 2010; Schauss et al., 2006). Although the acerola fruit is native to the Antilles, today Brazil is the largest producer, exporter and consumer (Rufino et al., 2010). The yellow mombin fruit (Spondias mombim L.) is greatly appreciated by Brazilian consumers due to its exotic flavor, in addition to its higher levels of phenolic and antioxidant compounds than in the majority of other fruits consumed (Tiburski, Rosenthal, Deliza, Godoy, & Pacheco, 2011). It is a well known fact that fruits contain a wide variety of antioxidant compounds, such as polyphenols that may help to protect cellular systems from oxidative damage, therefore reducing
http://dx.doi.org/10.1016/j.lwt.2014.04.002 0023-6438/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Carvalho-Silva, L. B., et al., Antiproliferative, antimutagenic and antioxidant activities of a Brazilian tropical fruit juice, LWT - Food Science and Technology (2014), http://dx.doi.org/10.1016/j.lwt.2014.04.002
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oxidative stress and modulating signal transduction pathways involved in cell proliferation and survival (Wolfe & Liu, 2008). However, different fruits have different bioactive compounds with varied antioxidant activity. In this sense, when fruits are consumed together, the total antioxidant activity may be modified via synergistic, additive, or antagonistic interaction among these components, which may change their physiological impacts (Wang, Meckling, Marcone, Kakuda, & Tsao, 2011). For this reason, the purpose of this work is to investigate the antioxidant, antiproliferative and antimutagenic potential of a Brazilian tropical fruit juice, using ‘in vitro’ and ‘in vivo’ assays. 2. Materials and methods 2.1. Tropical fruit juice An optimized formulation of a mix of tropical fruit juice was evaluated in this work. The formulation was optimized in terms of bioactive compounds by using a fractional factorial design (2k1, k ¼ 6), coupled with the response surface methodology (2k, k ¼ 5), totaling 36 assays in the first step, and 46 in the second one (data not shown). The results indicate an optimized formulation composed of 10% acerola (M. punicifolia), 5% acai (E. oleracea), 5% yellow mombin (Spondias lutea L.), 5% cashew apple (A. occidentale L.), 5% camuecamu (M. dubia), 20% pineapple (Ananas comosus L.), and 50% water. The fruit juice was adjusted with sucrose to 12 soluble solids. The tropical fruit juice was prepared, lyophilized, and stored at 20 C prior to use. 2.2. Determination of total antioxidant activity by ABTS method The antioxidant activity of the aqueous fruit juice extract was determined by the ABTS$þ assay, based on a method developed by Miller et al., (1993), with modifications proposed by Rufino et al. (2010). All values were expressed as the mean (mM of Trolox equivalents per g of sample dry weight) standard deviation (SD) of three replications. 2.3. Determination of total antioxidant activity by ORAC (oxygen radical absorbance capacity) assay The ORAC assay measures the antioxidant scavenging function against the peroxyl radical, induced by AAPH at 37 C, with fluorescein used as a fluorescent probe (Ou, Hampsch-Woodill, & Prior, 2001). The Multi-Detection microplate reader (Synergy HT, Bio-Tek Instruments Inc., Winooski, VT) was programmed to record the fluorescence of the diluted samples (25 mL) every minute after the incubation of the samples with 150 mL 40 nM fluorescein in 75 mM phosphate buffer, pH 7.4, and with the addition of 25 mL AAPH (153 mM in 75 mM phosphate buffer, pH 7.4) for 60 min. The values were expressed as mM Trolox equivalents per g of dry weight (DW) of the samples. 2.4. Measurement of antioxidant activity using peroxyl radical scavenging capacity (PSC) assay The PSC assay was as described previously by Adom and Liu (2005). The reaction was carried out at 37 C, and the fluorescence was monitored at 485 nm excitation and 538 nm emission, with the fluorescence spectrophotometer. Data was acquired with Ascent software, version 2.6 (Thermo Labsystems). The area under the average fluorescence-reaction time kinetic curve (AUC) for both the control and the samples (up to 36 min) were integrated and used as the basis for calculating the antioxidant activity. Results
were expressed as mM of vitamin C equivalents per 100 g of sample extract standard deviation (SD) of triplicate analyses. 2.5. Determination of total phenolic content The total phenolic content was determined using the FolinCiocalteu colorimetric method (Singleton, Orthofer, & LamuelaRaventos, 1999), with modifications (Yang, Meyers, Van der Heide, & Liu, 2004). Briefly, all extracts were diluted 1:10 with distilled water to obtain readings within the standard curve ranges of 0.0e600.0 mg of gallic acid mL1. The absorbance was measured at 760 nm after 90 min at room temperature by a MRX II Dynex plate reader (Dynex Technologies Inc., Chanilly, VA). All values were expressed as the mean (mg of gallic acid equivalents per 100 g of sample dry weight) SD of three replications. 2.6. Determination of total flavonoid content The total flavonoid content of the tropical fruit juice extract was determined using a modified colorimetric method (Jia, Tang, & Wu, 1999; Yang et al., 2004). Briefly, 0.25 mL of 1:10 diluted tropical fruit juice extracts were mixed with 1.25 mL of distilled water and subsequently with 75 ml of 5% sodium nitrite solution and was allowed to react for 5 min. The absorbance of the mixture was immediately measured at 510 nm against a prepared blank using an MRX II DYNEX spectrophotometer. The flavonoid content was determined by a catechin standard curve and expressed as the mean (mg of catechin equivalents e CE e per 100 g of sample dry weight) SD of triplicate extracts. 2.7. Identification of bioactive compounds by LC-DAD-ESIeMS 2.7.1. Phenolic compounds The LC-DAD-ESI/MS instrument consisted of an Varian 250 HPLC (Varian, CA) coupled with a diode array detector (DAD) and a 500MS IT mass spectrometer (Varian). A Symmetry column (C18, 3 mm, 250 2 mm, Waters) was used and the oven temperature was set at 30 C. The mobile phase consisted of a combination of A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile), a flow rate of 0.4 mL min1 was used with a linear gradient from 10% to 26% B (v/v) in 40 min, then to 65% B at 70 min, and finally to 100% B to 71 min and was maintained up to 75 min. The DAD was set at 270 and 512 nm for real-time read-out and UV/VIS spectra, from 190 to 650 nm, were continuously collected for phenolic identification. Mass spectra were simultaneously acquired using electrospray ionization in the positive and negative ionization (PI and NI) modes at low and high fragmentation voltages (80 V) for the mass range of 100e1000 amu. A drying pressure of 35 psi and gas temperature of 370 C, a nebulizer pressure of 40 psi, and capillary voltages of 3.5 kV for PI and NI were used. The LC system was directly coupled to the MSD with a 50% splitting. 2.7.2. Organic acids and sugars ESI-MS analysis was performed using a system of liquid chromatography coupled to a triple quadrupole LCMS 8040 (Shimadzu), with of an electrospray ionization source. The fruit extract samples (50 mg) were diluted in 1 mL of methanol and the mass spectra was acquired in the positive and negative ion modes by injecting 10 ml of the solutions without chromatographic separation. Ionization source conditions were: capillary voltage of 4.5 kV; nebulizing gas flow of 2 L/min, drying gas flow of 15 L min1; DL temperature of 250 C and heat block temperature of 300 C. The full scan spectra was acquired in the m/z range of 100 to 1000 amu. For the MS/MS analysis, a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer that was coupled to an ion trap LTQ (Thermo Finnigan)
Please cite this article in press as: Carvalho-Silva, L. B., et al., Antiproliferative, antimutagenic and antioxidant activities of a Brazilian tropical fruit juice, LWT - Food Science and Technology (2014), http://dx.doi.org/10.1016/j.lwt.2014.04.002
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was used. The ions of interest were selected and the collision energy was adjusted from 10 to 50 eV, depending on the fragmentation of the ion of interest. 2.8. In vitro studies 2.8.1. Cellular antioxidant activity (CAA) HepG2 cells were seeded at a density of 6 104/well on a 96well microplate in 100 mL of growth medium/well. Twenty-four hours after seeding, the growth medium was removed, and was applied PBS (Phosphate buffered saline) wash and no PBS wash protocol (Wolfe et al., 2008). Wells were treated in triplicate for 1 h with 100 mL of treatment medium containing tested fruit juice extract plus 25 mM DCFH-DA (Dichloro -fluorescein diacetate). When a PBS wash was utilized, wells were washed with 100 mL of PBS. Then 600 mM AAPH was applied to the cells in 100 mL of HBSS, and the 96-well microplate was placed into a Fluoroskan Ascent FL plate reader (ThermoLabsystems, Franklin, MA) at 37 C. Emission at 538 nm was measured after excitation at 485 nm every 5 min for 1 h (Wolfe et al., 2008). EC50 values were converted to CAA values, which are expressed as micromoles of quercetin equivalents (QE) per 100 g of DW, using the mean EC50 value for quercetin from at least three separate experiments. 2.8.2. Cytotoxicity and inhibition of proliferation assays Cytotoxicity toward HepG2 cells was measured using the method developed by Liu and Sun (2003). HepG2 cells in growth media were placed in each well of a 96-well flat-bottom plate at a density of 4.0 104 cells/well. After 24 h of incubation at 37 C with 5% CO2, the growth medium was removed, each well was washed with 100 mL of PBS and replaced by media containing different concentrations of fruit juice extract. After another 24 h of incubation, cytotoxicity was determined by the methylene blue assay (Felice, Sun, & Liu, 2009). Cytotoxicity was determined by a 10% reduction of absorbance at the 570 nm reading of each concentration compared to the control. A minimum of three replications for each sample was used to determine the cytotoxicity. Antiproliferative activity against human HepG2 liver cancer cells was determined using the method reported previously (Dewanto, Wu, & Liu, 2002). Briefly, HepG2 cells in growth media were placed in each well of a 96-well flat-bottom plate at a density of 2.5 104 cells/well. After 4 h of incubation at 37 C with 5% CO2, the growth medium was replaced by media containing different concentrations of fruit juice extract (0e25 mg/mL). After 72 h of incubation, cell proliferation was determined by the methylene blue assay (Felice et al., 2009). Cell proliferation (percent) was determined from the absorbance at the 570 nm for each concentration compared to the control. A minimum of three replications for each sample was used to determine the antiproliferative activity.
intraperitoneal doses of the dietary crude extract (DCE): 100, 300 and 1000 mg/kg, and were observed and scored daily for behavior and clinical conditions according to Ullman-Cullere and Foltz (1999) during the sequence of 15 days. The (DL50) was calculated using a linear regression. 2.9.2. Micronucleous (MN) test The micronucleous (MN) test was performed according to the guidelines of MacGregor et al. (1987) to investigate the protective effect of tropical juice extract against the clastogenicity induced by cyclophosphamide (CP). Tropical juice was administered at a dose of 100 mL by orogastric gavage for 15 consecutive days, at concentrations of 30, 100 and 300 mg/kg of body weight, that were selected based on an acute toxicity study in mice, previously described, which was higher than 1000 mg/kg. The positive groups (G1, G3, G4 and G5) received cyclophosphamide (CP) at a dose of 50 mg/kg of body weight, by an intraperitoneal injection on the 14th day, and the negative groups (G2, G6, G7 and G8) received an intraperitoneal injection with 0.9% NaCl to investigate a possible effect on spontaneous micronucleous (MN) frequencies. Experiments were performed according to distribution groups: G1 ¼ NaCl 0.9% þ CP (positive control); G2 ¼ NaCl 0.9% þ NaCl 0.9% (negative control); G3 ¼ 30 mg/kg tropical juice þ CP; G4 ¼ 100 mg/kg tropical juice þ CP; G5 ¼ 300 mg/kg tropical juice CP; G6 ¼ 30 mg/kg tropical juice þ NaCl 0.9%; G7 ¼ 100 mg/kg tropical juice þ NaCl 0.9%; G8 ¼ 300 mg/kg þ NaCl 0.9%. All animals were sacrificed on the 15th day, both femurs were removed and cleaned, and the epiphyses were cut to expose the medullary canal. The bone marrow was flushed with fetal bovine serum with the help of a syringe and cell suspension was prepared on a clean glass slide and stained with Leishman’s stain. All the slides were analyzed in a blind test using a light microscope at 1000 magnification. The frequency of micronucleated polychromated erythrocytes (MNPCEs) in each mouse was used as the experimental unit. The number of micronucleated normochromatic erythrocytes (MNNCEs) was registered at a total of 1000 normochromatic erythrocytes (NCEs) per animal. Toxicity to bone marrow was estimated by the relationship between the frequency of PCEs and NCEs. The ratio of PCEs to NCEs was determined in the first 1000 erythrocytes scored per coded slide by two independent microscopists. 2.10. Statistical analysis Statistical analysis was conducted using SigmaPlot Version 2000 (Aspire Software International, Ashburn, VA) and SPSS for Windows version 15.0 (Norussis, 2006) software. Results were subjected to ANOVA and the differences between the means were located using Tukey’s multiple comparison test. Significance was determined at P < 0.05. All data was reported as the mean SD in triplicate.
2.9. In vivo studies
3. Results and discussion
Animals: Newly weaned male Swiss albino mice were obtained and maintained under controlled temperature (22e24 C), light (12 h light/12 h dark), humidity (45e65%), and with food and water ad libitum. All mice used for experimental research had a body weight between 25 and 35 g. The UNIFAL-MG Animal Bioethical Committee approved this study under protocol number #239/2011 in accordance with the Brazilian Society of Science in Laboratory Animals.
3.1. Antioxidant activity, total phenolic and flavonoids determination of the tropical fruit juice
2.9.1. Antimutagenic assay The acute toxicity (DL50) study was done to determine which concentrations could cause the potential therapeutic activity, but not kill the animals. Three groups of five animals were treated with
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Fruits are rich in antioxidants that help in lowering the incidence of degenerative diseases such as cancer (Lim, Lim, & Tee, 2007), and can prevent or delay oxidative damage of lipids, proteins and nucleic acids through reactive oxygen species. In this study, the total antioxidant capacity of the tropical fruit juice, measured by ABTS and ORAC assays, was 167.17 4.10 and 235.90 11.90 mM Trolox/g of DW, respectively. Also, the results of antioxidant activity using the peroxyl radical scavenging capacity (PSC) assay, expressed as micromoles (mM) of vitamin C equivalents per 100 g of DW, was 308.39 3.10, with values of EC50 (mg/
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mL) ¼ 4.41 0.01. The antioxidant properties of a number of tropical fruits have been investigated individually (Rufino et al., 2010). However, bioactive compound interactions (additive, synergistic or antagonistic), have not been explored well in situations when some fruits are combined (e.g. tropical fruit juice). Furthermore, the fruit used in this work are, in some part, native and underexplored. As far as the authors know, this is the first report involving some ‘in vitro’ and ‘in vivo’ properties of a product obtained by a combination of tropical fruits, such as camuecamu, acerola and acai, for example. As described by Wang, Cao, and Prior (1996), the majority of the antioxidant activity of fruits and vegetables is due to carotenoids, ascorbic acid and polyphenols. Thus, phenolic compounds are considered to be the main contributors to the antioxidant activity of fruit juices (Frontela-Saseta et al., 2011). For this reason, we measured the phenolic content of the tropical fruit juice, with values of 838.44 30.27 mg GAE/100 g DW and 219.45 12.27 mg CE/100 g, for total phenolic and flavonoids compounds, respectively. Results were compared with those obtained by Sun, Chu, Wu, and Liu (2002), who evaluated the antioxidant and antiproliferative activities of fruits. The cranberry showed a high phenolic content (527.2 21.5 mg GAE/100 g), while fruits such as the apple (296.3 6.4 mg GAE/100 g), the red grape (201.0 2.9 mg GAE/100 g) and the strawberry (160.0 1.2 mg GAE/100 g) had medium total phenolic content. Furthermore, the tropical fruit juice analyzed in our study showed a total phenolic content higher than in some fruits commonly consumed in the United States, such as: the pineapple (94.3 1.5 mg GAE/100 g), the banana (90.4 3.2 mg GAE/100 g), peach (84.6 0.7 mg GAE/100 g), the lemon (81.9 3.5 mg GAE/100 g), the orange (81.2 1.1 mg GAE/100 g), the pear (70.6 1.6 mg GAE/100 g), and the grapefruit (49.6 2.6 mg GAE/100 g) (Sun et al., 2002). Despite the wide use of these chemical antioxidant activity assays, their ability to predict ‘in vivo’ activity is questioned for a number of reasons. Some were performed at a non-physiological pH and temperature, and none of them take into account the bioavailability, uptake, and metabolism of the antioxidant compounds. An alternative is given by ‘in vivo’ tests with animal models and human studies, which are of extreme importance because they detect the effect that an antioxidant can have on a whole organism. However, these are expensive and time-consuming and not suitable for initial antioxidant screening of foods. For this reason, the cellular antioxidant activity (CAA) was recently developed, which considers cellular uptake, distribution, and efficiency of protection against peroxyl radicals under physiological conditions (Wolfe & Liu, 2007). The cellular antioxidant activity (CAA) of the tropical fruit juice was measured using two protocols (PBS wash and no PBS wash protocol), as previously described (Wolfe & Liu, 2007), and the medium values of CAA and EC50 (cytotoxicity doses), are listed in Table 1. The results obtained for CAA and EC50 were compared with the literature. Wolfe et al. (2008) determined the cellular antioxidant activity of 25 fruits commonly consumed in the United States, and the values ranged from 3.15 0.21 to 292 11 mM of QE/100 g for CAA, and 235 16 to 2.53 0.10 mg/mL for EC50, where the wild blueberry had the highest CAA value. Compared with different fruits and vegetables in recent literature (Wolfe et al., 2008; Wolfe & Liu, 2007), our results showed lower EC50 values (Table 1) when compared with the wild blueberry and the pomegranate, although the CAA values are lower, compared to apples, cherries and plums.
Table 1 Cellular Antioxidant Activities of the tropical fruit juice extract expressed as EC50 and CAA values (mean SD, n ¼ 3). Species
Quercetin Tropical fruit juice
EC50 mmol/mla
CAA mmol QE/100 g
No PBS wash
PBS wash
No PBS wash
PBS wash
7.01 0.55 1.43 0.40
7.21 0.89 1.57 0.35
e 26.76 4.47
e 19.30 4.07
a EC50 values obtained from the no PBS wash and PBS wash protocols are not significantly different (P > 0.05).
protective effect of vegetables and fruits is attributed to the ability of the antioxidants to scavenge free radicals, preventing DNA damage and subsequent mutation (Liu et al., 2002). For this reason, the tropical fruit juice was evaluated for antiproliferative activity against HepG2 human liver cancer cells (see Fig. 1). The results indicate that the juice presented antiproliferative activity against HepG2 cell growth in a dose-dependent manner. There wasn’t significant cytotoxicity at concentrations up to 150 mg/mL, and this suggested that the antiproliferative activity was not caused by the cytotoxicity. The phenolic profile of the tropical fruit juice is shown in Fig. 2. The main compounds identified were the anthocyanins viewed in the chromatogram at 512 nm. The compounds cyanidin-3-Oglucoside, cyanidin-3-O-rutinoside, cyanidin-3-O-rhamnoside and pelargonidin-3-O-rhamnoside were identified based on their m/z comparison to values previously reported for acai and acerola (Brito et al., 2007; Schauss et al., 2006). Also, the mass spectra of the fruit juice extract shows the presence of ions of m/z 133, 175, 189 and 191, which correspond to malic, ascorbic, oxalosuccinic and citric acids, respectively. In the positive ion mode, the amino acids aamino-n-butyric acid, valine, glutamine and arginine were detected as protonated molecules as well as saccharides were detected as sodium and potassium adducts (Table 2). The ascorbic acid and the anthocyanins present in the juice contribute to the high levels of total antioxidant activity, obtained by the ORAC, ABTS, PSC and CAA methods, and consequently, may be involved in a antioxidant system to decrease the oxidative stress and the risk of cancer. 3.3. Antimutagenic test The fact that the tropical fruit juice evaluated in our work presents high antioxidant activity by all methods tested, emphasizes the importance of demonstrating their ‘in vivo’ antimutagenic
3.2. Antiproliferative activity against HepG2 and the identification of bioactive compounds by LC-DAD-ESI/MS of tropical fruit juice Endogenous oxidative DNA damage has been considered to be a significant factor in the initiation of human cancer. The cancer-
Fig. 1. Inhibition of proliferation of HepG2 human liver cancer cells by the tropical fruit juice (mean SD, n ¼ 3).
Please cite this article in press as: Carvalho-Silva, L. B., et al., Antiproliferative, antimutagenic and antioxidant activities of a Brazilian tropical fruit juice, LWT - Food Science and Technology (2014), http://dx.doi.org/10.1016/j.lwt.2014.04.002
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Fig. 2. HPLC chromatogram (520 nm) of tropical fruit juice. The peak assignments are: (1) cyanidin-3-O-glucoside, (2) cyanidin-3-O-rutinoside, (3) cyanidin-3-O-rhamnoside and (4) pelargonidin-3-O-rhamnoside.
Table 2 Identification of main compounds by ESIeMS analysis. m/z
Compound
Fragments
Positive ion mode 595 Cyanidin-3-O-rutinoside 449 Cyanidin-3-O-glucoside 433 Cyanidin-3-O-rhamnoside 417 Pelargonidin-3-O-rhamnoside 104 a-Amino-n-butyric acid 118 Valine 147 Glutamine 156 Histidine 175 Arginine 203 Monosaccharide þ Naþ 219 Monosaccharide þ Kþ 365 Disaccharide þ Naþ 381 Disaccharide þ Kþ 527 Trisaccharide þ Naþ 543 Trisaccharide þ Kþ 689 Tetrasaccharide þ Naþ 705 Tetrasaccharide þ Kþ Negative ion mode 133 Malic acid 175 Ascorbic acid 189 Oxalosuccinic acid 191 Citric acid 309 Dimer of malic and ascorbic acids
449, 287 287 287 271 58, 45, 43, 41 72 130, 84, 56 110, 93, 83 70, 60 185 201 203, 185 219, 201 365, 203, 185 381, 219, 201 527, 365, 203, 185 543, 381, 219, 201 115, 115, 127, 173, 175,
71 87, 71, 59 83, 73 111 133
activity through a micronucleous test in mice. These results showed no significant variations of body weight and food intake among the experimental groups (P > 0.05), during the study period, and indicated that the administration of tropical fruit juice, in various concentrations, showed no interference with the animal growth. The frequency of micronucleous (MN) after the administration of tropical fruit juice in polychromatic erythrocytes (MNPCEs) of bone marrow in mice is presented in Table 3. The ratio of PCE : NCE from tropical fruit juice þ0.9% NaCl groups (G2, G4 and G6) was not significantly different from a negative control group (0.9% NaCl) (P > 0.05), indicating that tropical fruit juice did not present cytotoxic properties in mice bone marrow cells at the tested doses (Table 3). The CP positive control proved to be efficient in the induction of chromosomal damage in immature erythrocytes (PCEs), because the frequency of MNPCEs present was statistically superior to the negative control (0.9% NaCl). The tropical fruit juice showed antimutagenic activity in all concentrations when compared with the positive control (P < 0.05), where the animals treated with the 30 mg/kg b.w. and 100 mg/kg b.w. of tropical fruit juice presented higher reduction followed by the 300 mg/kg b.w. (Table 3). In conclusion, it can be observed that the tropical fruit juice evaluated in this work showed high antiproliferative and antimutagenic activities, possibly attributed to its high content of bioactive compounds. As a consequence, these results encourage further studies on the pharmacological and functional properties of this tropical fruit juice, in order to evaluate its possibility as a functional food due to its beneficial health properties. Acknowledgments
Table 3 Frequency of micronucleated polychromated erythrocytes (MNPCEs) of bone marrow cells of Swiss mice in experimental groups treated with tropical fruit juice. Group Treatment
G1 G2 G3 G4 G5 G6 G7 G8
NaCl þ CP NaCl þ NaCl Tropical juice Tropical juice Tropical juice Tropical juice Tropical juice Tropical juice
Number of MNPCEs % Reduction* analyzed No. % PCEs 12,000 12,000 30 mg/kg bw þ CP 12,000 30 mg/kg bw þ NaCl 12,000 100 mg/kg bw þ CP 12,000 100 mg/kg bw þ NaCl 12,000 300 mg/kg bw þ CP 12,000 300 mg/kg bw þ NaCl 12,000
98 16 25 12 35 17 64 15
0.82 0.13 0.21 84.88a 0.10 0.29 77.78a 0.14 0.53 40.96b 0.12
CP e Cyclophosphamide. *Equal letter in same column are statistically equals if P < 0.05 by Tukey test.
The authors would like to thank to Brazilian Agricultural Research Corporation (EMBRAPA), grant number 0209010250000, the National Council for Scientific and Technological Development (CNPq), grant number 477799/2011-6, and the Coordination for the Improvement of Higher Level -or Education- Personnel (CAPES) for the financial support. References Adom, K. K., & Liu, R. H. (2005). Rapid peroxyl radical scavenging capacity (PSC) assay for assessing both hydrophilic and lipophilic antioxidants. Journal of Agricultural and Food Chemistry, 53, 6572e6580. Brito, E. S., Araujo, M. C. P., Alves, R. A., Carkeet, C., Clevidence, B. A., & Novotny, J. A. (2007). Anthocyanins present in selected tropical fruits: acerola, jambolão, jussara and guajiru. Journal of Agricultural and Food Chemistry, 55, 9389e9394.
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Antiproliferative, antimutagenic and antioxidant activities of a Brazilian tropical fruit juice Luciano Bruno de Carvalho-Silva a, Ana Paula Dionísio b, *, Ana Carolina da Silva Pereira c, Nedio Jair Wurlitzer b, Edy Sousa de Brito b, Giovana Anceski Bataglion c, Isabella Montenegro Brasil d, Marcos Nogueira Eberlin c, Rui Hai Liu e a
School of Nutrition, Federal University of Alfenas, Minas Gerais, Brazil Embrapa Tropical Agroindustry, Fortaleza, Ceará, Brazil Thomson Mass Spectrometry Laboratory, UNICAMP, Campinas, SP, Brazil d Department of Food Technology, Federal University of Ceará, Fortaleza, Ceará, Brazil e Department of Food Science and Institute of Comparative and Environmental Toxicology, Stocking Hall, Cornell University, Ithaca, NY, USA b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 February 2014 Received in revised form 1 April 2014 Accepted 2 April 2014 Available online xxx
This paper reports the antioxidant activity of a tropical fruit juice and its antiproliferative and antimutagenic effects. The antioxidant activity was determined by several different methods, such as ORAC, ABTS, PSC (peroxyl radical scavenging capacity) and CAA (cellular antioxidant activity). The total of phenolics and flavonoids were determined, and the phenolic compounds were identified by LC-DAD-ESIMS. The juice showed high total phenolic and flavonoid contents (838.44 30.27 g GAE/100 g and 219.45 12.27 mg CE/100 g DW, respectively). According to the PSC, CAA, ABTS and ORAC assays, the tropical fruit juice showed a high antioxidant value of 308.39 3.10 mM AEE/100 g, 26.76 4.47 mM QE/ 100 g, 167.17 4.10 mM TE/g DW and 235.90 11.90 mM TE/g DW, respectively. The proliferation of HepG2 was significantly inhibited, in a dose-dependent manner, by exposure to the tropical fruit juice. Antimutagenic activity was investigated by micronucleous test in mice, and all doses evaluated (30, 100 and 300 mg/kg b.w.) showed beneficial effects. As a consequence, these results encourage further studies on the pharmacological and functional properties of this tropical fruit juice, in order to evaluate its use as a functional food, due to its beneficial health properties. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Phytochemicals Cancer Antimutagenic activities
1. Introduction Brazil is a country that stands out because of the climatic conditions and large diversity of underexploited native and exotic fruit species. An example of this is the camuecamu (Myrciaria dubia Mc. Vaugh), a common fruit that grows near the Amazonian rivers and lakes. The camuecamu is growing in both Brazil and Peru and represents one of the richest sources of vitamin C ever studied (Genovese, Pinto, Gonçalves, & Lajolo, 2008; Rufino et al., 2010). Acai (Euterpe oleracea Mart.) is also obtained from the Amazon River basin, and is manually harvested from wild acai palms (E. oleracea) that are native to that area. This fruit has received much
* Corresponding author. Embrapa Tropical Agroindustry, Dra Sara Mesquita Street, 2270, 60511-110 Fortaleza, CE, Brazil. Tel.: þ55 85 33917327; fax: þ55 85 33917221. E-mail addresses: [email protected], [email protected] (L.B. de Carvalho-Silva).
attention in recent years due to its high antioxidant activity and its role as a “functional food”. It represents good perspectives for industrial purposes and is recognized worldwide, as shown by its increased amount of exportation (Genovese et al., 2008). Acerola (Malpighia emarginata) and the cashew-apple (Anacardium occidentale L.) also represent a good source of bioactive compounds, and offer potential uses in different juices and beverages (Rufino et al., 2010; Schauss et al., 2006). Although the acerola fruit is native to the Antilles, today Brazil is the largest producer, exporter and consumer (Rufino et al., 2010). The yellow mombin fruit (Spondias mombim L.) is greatly appreciated by Brazilian consumers due to its exotic flavor, in addition to its higher levels of phenolic and antioxidant compounds than in the majority of other fruits consumed (Tiburski, Rosenthal, Deliza, Godoy, & Pacheco, 2011). It is a well known fact that fruits contain a wide variety of antioxidant compounds, such as polyphenols that may help to protect cellular systems from oxidative damage, therefore reducing
http://dx.doi.org/10.1016/j.lwt.2014.04.002 0023-6438/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Carvalho-Silva, L. B., et al., Antiproliferative, antimutagenic and antioxidant activities of a Brazilian tropical fruit juice, LWT - Food Science and Technology (2014), http://dx.doi.org/10.1016/j.lwt.2014.04.002
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oxidative stress and modulating signal transduction pathways involved in cell proliferation and survival (Wolfe & Liu, 2008). However, different fruits have different bioactive compounds with varied antioxidant activity. In this sense, when fruits are consumed together, the total antioxidant activity may be modified via synergistic, additive, or antagonistic interaction among these components, which may change their physiological impacts (Wang, Meckling, Marcone, Kakuda, & Tsao, 2011). For this reason, the purpose of this work is to investigate the antioxidant, antiproliferative and antimutagenic potential of a Brazilian tropical fruit juice, using ‘in vitro’ and ‘in vivo’ assays. 2. Materials and methods 2.1. Tropical fruit juice An optimized formulation of a mix of tropical fruit juice was evaluated in this work. The formulation was optimized in terms of bioactive compounds by using a fractional factorial design (2k1, k ¼ 6), coupled with the response surface methodology (2k, k ¼ 5), totaling 36 assays in the first step, and 46 in the second one (data not shown). The results indicate an optimized formulation composed of 10% acerola (M. punicifolia), 5% acai (E. oleracea), 5% yellow mombin (Spondias lutea L.), 5% cashew apple (A. occidentale L.), 5% camuecamu (M. dubia), 20% pineapple (Ananas comosus L.), and 50% water. The fruit juice was adjusted with sucrose to 12 soluble solids. The tropical fruit juice was prepared, lyophilized, and stored at 20 C prior to use. 2.2. Determination of total antioxidant activity by ABTS method The antioxidant activity of the aqueous fruit juice extract was determined by the ABTS$þ assay, based on a method developed by Miller et al., (1993), with modifications proposed by Rufino et al. (2010). All values were expressed as the mean (mM of Trolox equivalents per g of sample dry weight) standard deviation (SD) of three replications. 2.3. Determination of total antioxidant activity by ORAC (oxygen radical absorbance capacity) assay The ORAC assay measures the antioxidant scavenging function against the peroxyl radical, induced by AAPH at 37 C, with fluorescein used as a fluorescent probe (Ou, Hampsch-Woodill, & Prior, 2001). The Multi-Detection microplate reader (Synergy HT, Bio-Tek Instruments Inc., Winooski, VT) was programmed to record the fluorescence of the diluted samples (25 mL) every minute after the incubation of the samples with 150 mL 40 nM fluorescein in 75 mM phosphate buffer, pH 7.4, and with the addition of 25 mL AAPH (153 mM in 75 mM phosphate buffer, pH 7.4) for 60 min. The values were expressed as mM Trolox equivalents per g of dry weight (DW) of the samples. 2.4. Measurement of antioxidant activity using peroxyl radical scavenging capacity (PSC) assay The PSC assay was as described previously by Adom and Liu (2005). The reaction was carried out at 37 C, and the fluorescence was monitored at 485 nm excitation and 538 nm emission, with the fluorescence spectrophotometer. Data was acquired with Ascent software, version 2.6 (Thermo Labsystems). The area under the average fluorescence-reaction time kinetic curve (AUC) for both the control and the samples (up to 36 min) were integrated and used as the basis for calculating the antioxidant activity. Results
were expressed as mM of vitamin C equivalents per 100 g of sample extract standard deviation (SD) of triplicate analyses. 2.5. Determination of total phenolic content The total phenolic content was determined using the FolinCiocalteu colorimetric method (Singleton, Orthofer, & LamuelaRaventos, 1999), with modifications (Yang, Meyers, Van der Heide, & Liu, 2004). Briefly, all extracts were diluted 1:10 with distilled water to obtain readings within the standard curve ranges of 0.0e600.0 mg of gallic acid mL1. The absorbance was measured at 760 nm after 90 min at room temperature by a MRX II Dynex plate reader (Dynex Technologies Inc., Chanilly, VA). All values were expressed as the mean (mg of gallic acid equivalents per 100 g of sample dry weight) SD of three replications. 2.6. Determination of total flavonoid content The total flavonoid content of the tropical fruit juice extract was determined using a modified colorimetric method (Jia, Tang, & Wu, 1999; Yang et al., 2004). Briefly, 0.25 mL of 1:10 diluted tropical fruit juice extracts were mixed with 1.25 mL of distilled water and subsequently with 75 ml of 5% sodium nitrite solution and was allowed to react for 5 min. The absorbance of the mixture was immediately measured at 510 nm against a prepared blank using an MRX II DYNEX spectrophotometer. The flavonoid content was determined by a catechin standard curve and expressed as the mean (mg of catechin equivalents e CE e per 100 g of sample dry weight) SD of triplicate extracts. 2.7. Identification of bioactive compounds by LC-DAD-ESIeMS 2.7.1. Phenolic compounds The LC-DAD-ESI/MS instrument consisted of an Varian 250 HPLC (Varian, CA) coupled with a diode array detector (DAD) and a 500MS IT mass spectrometer (Varian). A Symmetry column (C18, 3 mm, 250 2 mm, Waters) was used and the oven temperature was set at 30 C. The mobile phase consisted of a combination of A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile), a flow rate of 0.4 mL min1 was used with a linear gradient from 10% to 26% B (v/v) in 40 min, then to 65% B at 70 min, and finally to 100% B to 71 min and was maintained up to 75 min. The DAD was set at 270 and 512 nm for real-time read-out and UV/VIS spectra, from 190 to 650 nm, were continuously collected for phenolic identification. Mass spectra were simultaneously acquired using electrospray ionization in the positive and negative ionization (PI and NI) modes at low and high fragmentation voltages (80 V) for the mass range of 100e1000 amu. A drying pressure of 35 psi and gas temperature of 370 C, a nebulizer pressure of 40 psi, and capillary voltages of 3.5 kV for PI and NI were used. The LC system was directly coupled to the MSD with a 50% splitting. 2.7.2. Organic acids and sugars ESI-MS analysis was performed using a system of liquid chromatography coupled to a triple quadrupole LCMS 8040 (Shimadzu), with of an electrospray ionization source. The fruit extract samples (50 mg) were diluted in 1 mL of methanol and the mass spectra was acquired in the positive and negative ion modes by injecting 10 ml of the solutions without chromatographic separation. Ionization source conditions were: capillary voltage of 4.5 kV; nebulizing gas flow of 2 L/min, drying gas flow of 15 L min1; DL temperature of 250 C and heat block temperature of 300 C. The full scan spectra was acquired in the m/z range of 100 to 1000 amu. For the MS/MS analysis, a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer that was coupled to an ion trap LTQ (Thermo Finnigan)
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was used. The ions of interest were selected and the collision energy was adjusted from 10 to 50 eV, depending on the fragmentation of the ion of interest. 2.8. In vitro studies 2.8.1. Cellular antioxidant activity (CAA) HepG2 cells were seeded at a density of 6 104/well on a 96well microplate in 100 mL of growth medium/well. Twenty-four hours after seeding, the growth medium was removed, and was applied PBS (Phosphate buffered saline) wash and no PBS wash protocol (Wolfe et al., 2008). Wells were treated in triplicate for 1 h with 100 mL of treatment medium containing tested fruit juice extract plus 25 mM DCFH-DA (Dichloro -fluorescein diacetate). When a PBS wash was utilized, wells were washed with 100 mL of PBS. Then 600 mM AAPH was applied to the cells in 100 mL of HBSS, and the 96-well microplate was placed into a Fluoroskan Ascent FL plate reader (ThermoLabsystems, Franklin, MA) at 37 C. Emission at 538 nm was measured after excitation at 485 nm every 5 min for 1 h (Wolfe et al., 2008). EC50 values were converted to CAA values, which are expressed as micromoles of quercetin equivalents (QE) per 100 g of DW, using the mean EC50 value for quercetin from at least three separate experiments. 2.8.2. Cytotoxicity and inhibition of proliferation assays Cytotoxicity toward HepG2 cells was measured using the method developed by Liu and Sun (2003). HepG2 cells in growth media were placed in each well of a 96-well flat-bottom plate at a density of 4.0 104 cells/well. After 24 h of incubation at 37 C with 5% CO2, the growth medium was removed, each well was washed with 100 mL of PBS and replaced by media containing different concentrations of fruit juice extract. After another 24 h of incubation, cytotoxicity was determined by the methylene blue assay (Felice, Sun, & Liu, 2009). Cytotoxicity was determined by a 10% reduction of absorbance at the 570 nm reading of each concentration compared to the control. A minimum of three replications for each sample was used to determine the cytotoxicity. Antiproliferative activity against human HepG2 liver cancer cells was determined using the method reported previously (Dewanto, Wu, & Liu, 2002). Briefly, HepG2 cells in growth media were placed in each well of a 96-well flat-bottom plate at a density of 2.5 104 cells/well. After 4 h of incubation at 37 C with 5% CO2, the growth medium was replaced by media containing different concentrations of fruit juice extract (0e25 mg/mL). After 72 h of incubation, cell proliferation was determined by the methylene blue assay (Felice et al., 2009). Cell proliferation (percent) was determined from the absorbance at the 570 nm for each concentration compared to the control. A minimum of three replications for each sample was used to determine the antiproliferative activity.
intraperitoneal doses of the dietary crude extract (DCE): 100, 300 and 1000 mg/kg, and were observed and scored daily for behavior and clinical conditions according to Ullman-Cullere and Foltz (1999) during the sequence of 15 days. The (DL50) was calculated using a linear regression. 2.9.2. Micronucleous (MN) test The micronucleous (MN) test was performed according to the guidelines of MacGregor et al. (1987) to investigate the protective effect of tropical juice extract against the clastogenicity induced by cyclophosphamide (CP). Tropical juice was administered at a dose of 100 mL by orogastric gavage for 15 consecutive days, at concentrations of 30, 100 and 300 mg/kg of body weight, that were selected based on an acute toxicity study in mice, previously described, which was higher than 1000 mg/kg. The positive groups (G1, G3, G4 and G5) received cyclophosphamide (CP) at a dose of 50 mg/kg of body weight, by an intraperitoneal injection on the 14th day, and the negative groups (G2, G6, G7 and G8) received an intraperitoneal injection with 0.9% NaCl to investigate a possible effect on spontaneous micronucleous (MN) frequencies. Experiments were performed according to distribution groups: G1 ¼ NaCl 0.9% þ CP (positive control); G2 ¼ NaCl 0.9% þ NaCl 0.9% (negative control); G3 ¼ 30 mg/kg tropical juice þ CP; G4 ¼ 100 mg/kg tropical juice þ CP; G5 ¼ 300 mg/kg tropical juice CP; G6 ¼ 30 mg/kg tropical juice þ NaCl 0.9%; G7 ¼ 100 mg/kg tropical juice þ NaCl 0.9%; G8 ¼ 300 mg/kg þ NaCl 0.9%. All animals were sacrificed on the 15th day, both femurs were removed and cleaned, and the epiphyses were cut to expose the medullary canal. The bone marrow was flushed with fetal bovine serum with the help of a syringe and cell suspension was prepared on a clean glass slide and stained with Leishman’s stain. All the slides were analyzed in a blind test using a light microscope at 1000 magnification. The frequency of micronucleated polychromated erythrocytes (MNPCEs) in each mouse was used as the experimental unit. The number of micronucleated normochromatic erythrocytes (MNNCEs) was registered at a total of 1000 normochromatic erythrocytes (NCEs) per animal. Toxicity to bone marrow was estimated by the relationship between the frequency of PCEs and NCEs. The ratio of PCEs to NCEs was determined in the first 1000 erythrocytes scored per coded slide by two independent microscopists. 2.10. Statistical analysis Statistical analysis was conducted using SigmaPlot Version 2000 (Aspire Software International, Ashburn, VA) and SPSS for Windows version 15.0 (Norussis, 2006) software. Results were subjected to ANOVA and the differences between the means were located using Tukey’s multiple comparison test. Significance was determined at P < 0.05. All data was reported as the mean SD in triplicate.
2.9. In vivo studies
3. Results and discussion
Animals: Newly weaned male Swiss albino mice were obtained and maintained under controlled temperature (22e24 C), light (12 h light/12 h dark), humidity (45e65%), and with food and water ad libitum. All mice used for experimental research had a body weight between 25 and 35 g. The UNIFAL-MG Animal Bioethical Committee approved this study under protocol number #239/2011 in accordance with the Brazilian Society of Science in Laboratory Animals.
3.1. Antioxidant activity, total phenolic and flavonoids determination of the tropical fruit juice
2.9.1. Antimutagenic assay The acute toxicity (DL50) study was done to determine which concentrations could cause the potential therapeutic activity, but not kill the animals. Three groups of five animals were treated with
3
Fruits are rich in antioxidants that help in lowering the incidence of degenerative diseases such as cancer (Lim, Lim, & Tee, 2007), and can prevent or delay oxidative damage of lipids, proteins and nucleic acids through reactive oxygen species. In this study, the total antioxidant capacity of the tropical fruit juice, measured by ABTS and ORAC assays, was 167.17 4.10 and 235.90 11.90 mM Trolox/g of DW, respectively. Also, the results of antioxidant activity using the peroxyl radical scavenging capacity (PSC) assay, expressed as micromoles (mM) of vitamin C equivalents per 100 g of DW, was 308.39 3.10, with values of EC50 (mg/
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mL) ¼ 4.41 0.01. The antioxidant properties of a number of tropical fruits have been investigated individually (Rufino et al., 2010). However, bioactive compound interactions (additive, synergistic or antagonistic), have not been explored well in situations when some fruits are combined (e.g. tropical fruit juice). Furthermore, the fruit used in this work are, in some part, native and underexplored. As far as the authors know, this is the first report involving some ‘in vitro’ and ‘in vivo’ properties of a product obtained by a combination of tropical fruits, such as camuecamu, acerola and acai, for example. As described by Wang, Cao, and Prior (1996), the majority of the antioxidant activity of fruits and vegetables is due to carotenoids, ascorbic acid and polyphenols. Thus, phenolic compounds are considered to be the main contributors to the antioxidant activity of fruit juices (Frontela-Saseta et al., 2011). For this reason, we measured the phenolic content of the tropical fruit juice, with values of 838.44 30.27 mg GAE/100 g DW and 219.45 12.27 mg CE/100 g, for total phenolic and flavonoids compounds, respectively. Results were compared with those obtained by Sun, Chu, Wu, and Liu (2002), who evaluated the antioxidant and antiproliferative activities of fruits. The cranberry showed a high phenolic content (527.2 21.5 mg GAE/100 g), while fruits such as the apple (296.3 6.4 mg GAE/100 g), the red grape (201.0 2.9 mg GAE/100 g) and the strawberry (160.0 1.2 mg GAE/100 g) had medium total phenolic content. Furthermore, the tropical fruit juice analyzed in our study showed a total phenolic content higher than in some fruits commonly consumed in the United States, such as: the pineapple (94.3 1.5 mg GAE/100 g), the banana (90.4 3.2 mg GAE/100 g), peach (84.6 0.7 mg GAE/100 g), the lemon (81.9 3.5 mg GAE/100 g), the orange (81.2 1.1 mg GAE/100 g), the pear (70.6 1.6 mg GAE/100 g), and the grapefruit (49.6 2.6 mg GAE/100 g) (Sun et al., 2002). Despite the wide use of these chemical antioxidant activity assays, their ability to predict ‘in vivo’ activity is questioned for a number of reasons. Some were performed at a non-physiological pH and temperature, and none of them take into account the bioavailability, uptake, and metabolism of the antioxidant compounds. An alternative is given by ‘in vivo’ tests with animal models and human studies, which are of extreme importance because they detect the effect that an antioxidant can have on a whole organism. However, these are expensive and time-consuming and not suitable for initial antioxidant screening of foods. For this reason, the cellular antioxidant activity (CAA) was recently developed, which considers cellular uptake, distribution, and efficiency of protection against peroxyl radicals under physiological conditions (Wolfe & Liu, 2007). The cellular antioxidant activity (CAA) of the tropical fruit juice was measured using two protocols (PBS wash and no PBS wash protocol), as previously described (Wolfe & Liu, 2007), and the medium values of CAA and EC50 (cytotoxicity doses), are listed in Table 1. The results obtained for CAA and EC50 were compared with the literature. Wolfe et al. (2008) determined the cellular antioxidant activity of 25 fruits commonly consumed in the United States, and the values ranged from 3.15 0.21 to 292 11 mM of QE/100 g for CAA, and 235 16 to 2.53 0.10 mg/mL for EC50, where the wild blueberry had the highest CAA value. Compared with different fruits and vegetables in recent literature (Wolfe et al., 2008; Wolfe & Liu, 2007), our results showed lower EC50 values (Table 1) when compared with the wild blueberry and the pomegranate, although the CAA values are lower, compared to apples, cherries and plums.
Table 1 Cellular Antioxidant Activities of the tropical fruit juice extract expressed as EC50 and CAA values (mean SD, n ¼ 3). Species
Quercetin Tropical fruit juice
EC50 mmol/mla
CAA mmol QE/100 g
No PBS wash
PBS wash
No PBS wash
PBS wash
7.01 0.55 1.43 0.40
7.21 0.89 1.57 0.35
e 26.76 4.47
e 19.30 4.07
a EC50 values obtained from the no PBS wash and PBS wash protocols are not significantly different (P > 0.05).
protective effect of vegetables and fruits is attributed to the ability of the antioxidants to scavenge free radicals, preventing DNA damage and subsequent mutation (Liu et al., 2002). For this reason, the tropical fruit juice was evaluated for antiproliferative activity against HepG2 human liver cancer cells (see Fig. 1). The results indicate that the juice presented antiproliferative activity against HepG2 cell growth in a dose-dependent manner. There wasn’t significant cytotoxicity at concentrations up to 150 mg/mL, and this suggested that the antiproliferative activity was not caused by the cytotoxicity. The phenolic profile of the tropical fruit juice is shown in Fig. 2. The main compounds identified were the anthocyanins viewed in the chromatogram at 512 nm. The compounds cyanidin-3-Oglucoside, cyanidin-3-O-rutinoside, cyanidin-3-O-rhamnoside and pelargonidin-3-O-rhamnoside were identified based on their m/z comparison to values previously reported for acai and acerola (Brito et al., 2007; Schauss et al., 2006). Also, the mass spectra of the fruit juice extract shows the presence of ions of m/z 133, 175, 189 and 191, which correspond to malic, ascorbic, oxalosuccinic and citric acids, respectively. In the positive ion mode, the amino acids aamino-n-butyric acid, valine, glutamine and arginine were detected as protonated molecules as well as saccharides were detected as sodium and potassium adducts (Table 2). The ascorbic acid and the anthocyanins present in the juice contribute to the high levels of total antioxidant activity, obtained by the ORAC, ABTS, PSC and CAA methods, and consequently, may be involved in a antioxidant system to decrease the oxidative stress and the risk of cancer. 3.3. Antimutagenic test The fact that the tropical fruit juice evaluated in our work presents high antioxidant activity by all methods tested, emphasizes the importance of demonstrating their ‘in vivo’ antimutagenic
3.2. Antiproliferative activity against HepG2 and the identification of bioactive compounds by LC-DAD-ESI/MS of tropical fruit juice Endogenous oxidative DNA damage has been considered to be a significant factor in the initiation of human cancer. The cancer-
Fig. 1. Inhibition of proliferation of HepG2 human liver cancer cells by the tropical fruit juice (mean SD, n ¼ 3).
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Fig. 2. HPLC chromatogram (520 nm) of tropical fruit juice. The peak assignments are: (1) cyanidin-3-O-glucoside, (2) cyanidin-3-O-rutinoside, (3) cyanidin-3-O-rhamnoside and (4) pelargonidin-3-O-rhamnoside.
Table 2 Identification of main compounds by ESIeMS analysis. m/z
Compound
Fragments
Positive ion mode 595 Cyanidin-3-O-rutinoside 449 Cyanidin-3-O-glucoside 433 Cyanidin-3-O-rhamnoside 417 Pelargonidin-3-O-rhamnoside 104 a-Amino-n-butyric acid 118 Valine 147 Glutamine 156 Histidine 175 Arginine 203 Monosaccharide þ Naþ 219 Monosaccharide þ Kþ 365 Disaccharide þ Naþ 381 Disaccharide þ Kþ 527 Trisaccharide þ Naþ 543 Trisaccharide þ Kþ 689 Tetrasaccharide þ Naþ 705 Tetrasaccharide þ Kþ Negative ion mode 133 Malic acid 175 Ascorbic acid 189 Oxalosuccinic acid 191 Citric acid 309 Dimer of malic and ascorbic acids
449, 287 287 287 271 58, 45, 43, 41 72 130, 84, 56 110, 93, 83 70, 60 185 201 203, 185 219, 201 365, 203, 185 381, 219, 201 527, 365, 203, 185 543, 381, 219, 201 115, 115, 127, 173, 175,
71 87, 71, 59 83, 73 111 133
activity through a micronucleous test in mice. These results showed no significant variations of body weight and food intake among the experimental groups (P > 0.05), during the study period, and indicated that the administration of tropical fruit juice, in various concentrations, showed no interference with the animal growth. The frequency of micronucleous (MN) after the administration of tropical fruit juice in polychromatic erythrocytes (MNPCEs) of bone marrow in mice is presented in Table 3. The ratio of PCE : NCE from tropical fruit juice þ0.9% NaCl groups (G2, G4 and G6) was not significantly different from a negative control group (0.9% NaCl) (P > 0.05), indicating that tropical fruit juice did not present cytotoxic properties in mice bone marrow cells at the tested doses (Table 3). The CP positive control proved to be efficient in the induction of chromosomal damage in immature erythrocytes (PCEs), because the frequency of MNPCEs present was statistically superior to the negative control (0.9% NaCl). The tropical fruit juice showed antimutagenic activity in all concentrations when compared with the positive control (P < 0.05), where the animals treated with the 30 mg/kg b.w. and 100 mg/kg b.w. of tropical fruit juice presented higher reduction followed by the 300 mg/kg b.w. (Table 3). In conclusion, it can be observed that the tropical fruit juice evaluated in this work showed high antiproliferative and antimutagenic activities, possibly attributed to its high content of bioactive compounds. As a consequence, these results encourage further studies on the pharmacological and functional properties of this tropical fruit juice, in order to evaluate its possibility as a functional food due to its beneficial health properties. Acknowledgments
Table 3 Frequency of micronucleated polychromated erythrocytes (MNPCEs) of bone marrow cells of Swiss mice in experimental groups treated with tropical fruit juice. Group Treatment
G1 G2 G3 G4 G5 G6 G7 G8
NaCl þ CP NaCl þ NaCl Tropical juice Tropical juice Tropical juice Tropical juice Tropical juice Tropical juice
Number of MNPCEs % Reduction* analyzed No. % PCEs 12,000 12,000 30 mg/kg bw þ CP 12,000 30 mg/kg bw þ NaCl 12,000 100 mg/kg bw þ CP 12,000 100 mg/kg bw þ NaCl 12,000 300 mg/kg bw þ CP 12,000 300 mg/kg bw þ NaCl 12,000
98 16 25 12 35 17 64 15
0.82 0.13 0.21 84.88a 0.10 0.29 77.78a 0.14 0.53 40.96b 0.12
CP e Cyclophosphamide. *Equal letter in same column are statistically equals if P < 0.05 by Tukey test.
The authors would like to thank to Brazilian Agricultural Research Corporation (EMBRAPA), grant number 0209010250000, the National Council for Scientific and Technological Development (CNPq), grant number 477799/2011-6, and the Coordination for the Improvement of Higher Level -or Education- Personnel (CAPES) for the financial support. References Adom, K. K., & Liu, R. H. (2005). Rapid peroxyl radical scavenging capacity (PSC) assay for assessing both hydrophilic and lipophilic antioxidants. Journal of Agricultural and Food Chemistry, 53, 6572e6580. Brito, E. S., Araujo, M. C. P., Alves, R. A., Carkeet, C., Clevidence, B. A., & Novotny, J. A. (2007). Anthocyanins present in selected tropical fruits: acerola, jambolão, jussara and guajiru. Journal of Agricultural and Food Chemistry, 55, 9389e9394.
Please cite this article in press as: Carvalho-Silva, L. B., et al., Antiproliferative, antimutagenic and antioxidant activities of a Brazilian tropical fruit juice, LWT - Food Science and Technology (2014), http://dx.doi.org/10.1016/j.lwt.2014.04.002
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Please cite this article in press as: Carvalho-Silva, L. B., et al., Antiproliferative, antimutagenic and antioxidant activities of a Brazilian tropical fruit juice, LWT - Food Science and Technology (2014), http://dx.doi.org/10.1016/j.lwt.2014.04.002