Fruiting increases total content of flavonoids and antiproliferative effects of Cereus jamacaru D.C. cladodes in sarcoma 180 cells in vitro

Jean Carlos Vencioneck Dutra, Judá Ben-Hur de Oliveira, Vanessa Silva dos Santos, Paula Roberta Costalonga Pereira, Jean Moisés Ferreira, Maria do Carmo Pimentel Batitucci

1Laboratório de Genética Vegetal e Toxicológica, Departamento de Ciências Biológicas, Universidade Federal do Espírito Santo, 29075-910, Vitória,Espírito Santo, Brazil

2Laboratório de Biologia Molecular e Express?o Gênica, Departamento de Ciências Biológicas, Universidade Federal de Alagoas, 57309-005,Arapiraca, Alagoas, Brazil

Keywords:Mandacaru Flavonoid content DPPH ABTS Fe2+ ion chelating activity MTT assay Sarcoma 180 cells

A BSTRACT Objective: To evaluate the influence of fruiting phenological stage on total flavonoid content,antioxidant activity, and antiproliferative effects of Cereus jamacaru (C. jamacaru) (mandacaru)cladodes and fruit. Methods: Fruit and cladodes at vegetative and fruiting stage of C.jamacaru were collected. The fruit was dissected and bark, pulp, and seeds were separated.Vegetative and fruiting cladodes, together with bark, pulp, and seeds were used to obtain five hydroalcoholic extracts. The extracts were investigated for total flavonoid content, using AlCl3 colorimetric method, antioxidant activity by 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical scavenging capacity and Fe2+ ion chelating activity, and in vitro antiproliferative effects (sarcoma 180 cells) by 3-(4,5- dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Results: The extract of C. jamacaru cladodes at the fruiting stage showed higher flavonoid content compared to the other extracts.Seed extracts showed the highest antioxidant activity in 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) assays, and the extract of cladodes at vegetative stage showed better antioxidant activity in Fe2+ ion chelating activity. The extract of fruiting cladodes promoted higher antiproliferative effects compared to the other extracts.Conclusions: These findings suggest that fruiting increases the content of flavonoids and antiproliferative effects of C. jamacaru cladodes. Data reinforce the potential use of C. jamacaru cladodes and fruits as natural antioxidants and potent anticancer agent.

1. Introduction

Cereus jamacaru (C. jamacaru) D.C. (mandacaru) is a Cactaceae native from Brazil, found mainly on arid areas and is used as food and, in traditional medicine, to treat urinary infection,kidney inflammation, and rheumatism[1]. The antioxidant and antiproliferative actions of C. jamacaru have already been reported in the literature, which motivated the conduction of more researches on the effective use of this plant as a natural source of antioxidants and for the development of new drugs[2-4].

Optimization of the uses of natural products is not restricted to the identification of the vegetal actives and their biological effects.Many studies have aimed to evaluate the influence of genetic and environmental factors, crop management, and phenology on the production of phytochemicals[5-8]. The influence of phenology on the frugivory, dispersion, and germination of seeds of C. jamacaru has been reported[9]. However, there are no studies demonstrating the influence of phenology on the biological activities of this cactus,such as the antioxidant and antiproliferative activities.

In recent years, there has been a growing use of in vitro techniques to evaluate the antioxidant and antiproliferative potential of natural products. These techniques have been used for multiple and complementary measures of antioxidant and cytotoxicity activities[5,6,10,11]. In this context, antioxidant assays, such as 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and Fe2+ion chelating activity,and in vitro antiproliferative assays using cell lines, such as sarcoma 180 (murine cancer), have been efficiently used as screening tools for natural products of plant origin with potential use for health promotion and development of new drugs[5-8].

Therefore, the aim of this study was to evaluate the influence of fruiting on the total flavonoids content of extracts of C. jamacaru cladodes at the vegetative stage and fruiting, as well as their fruits,and to relate these results to the in vitro antioxidant activities and antiproliferative effects.

2. Materials and methods

2.1. Chemicals

The chemicals used in this study included Ficoll?Paque Plus(Sigma-Aldrich); RPMI 1640 culture medium (Cultilab); fetal calf serum (Gibco); Cisplatin (Fauldcispla?, Libbs); DPPH (Sigma-Aldrich); ABTS (Sigma-Aldrich); potassium persulfate (Sigma-Aldrich); 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid(Sigma-Aldrich); 3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-4,4’-disulfonic acid Na-salt (Sigma-Aldrich); iron chlorideⅡ(Sigma-Aldrich); 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H- tetrazolium bromide (MTT) (Sigma-Aldrich).

2.2. Plant material

The fruit and cladodes of C. jamacaru in the vegetative stage(without flowers or fruits) and fruiting stage (with fruit) were collected in the state of Alagoas, northeastern Brazil (9° 40’ 44.7”S, 36° 41’ 21.9” W), in July 2018. The thorns of cladodes were removed and the cladodes were later sliced with a blade. The fruit was collected together with cladodes in the fruiting stage, dissected and the bark, pulp, and seeds were obtained. The plant material was oven-dried at 50 ℃ for 24 h and vegetative and fruiting cladodes,bark, pulp, and seeds were used to obtain five hydroalcoholic extracts.

2.3. Hydroalcoholic extract

Dried plant material was powdered, macerated in EtOH/H2O(70:30 v/v) solution (dry plant: EtOH/H2O - 1:5 w/v) at room temperature (25-30 ℃), protected from the light for 3 d, filtered and stored. Vegetal material was submitted to the same conditions of maceration (plus three days of maceration) for exhaustion of the compound extraction, totaling six days of maceration. The solvent was evaporated under reduced pressure at 60 ℃ to obtain the crude extracts of C. jamacaru. The extracts were stored at 6-10 ℃ and protected from the light until its utilization. The yield of the extracts were calculated by the formula[12]:

Total extract yield (%) = FM/IM × 100

Where “FM” = final mass of dry extract (g); “IM” = initial mass of dry plant (g).

2.4. Total flavonoid content

The colorimetric method described by Jia et al.[13] was used to determine the total flavonoid content of C. jamacaru extracts. The experiment was performed in triplicate and the absorbance was detected at 430 nm, using an ELISA reader. Rutin was used as a standard for flavonoids and for this purpose a series of methanolic dilutions of rutin(500.0, 250.0, 125.0, 62.5, 31.3, 15.6, and 7.8 μg/mL) were prepared and assayed. The total flavonoid content in the extracts was expressed as rutin equivalent (μg/mL).

2.5. Antioxidant activity

2.5.1. DPPH

DPPH radical reduction method was used to evaluate the antioxidant activity of C. jamacaru extracts and ascorbic acid standard[14]. The absorbance was taken at 517 nm by ELISA reader.The test was performed in triplicate and the percentage of reduction of DPPH·radical was calculated by the equation:

% inhibition = [(AbsControl- AbsSample) / AbsControl] × 100

Where “% inhibition” is the percentage of inhibition capacity of DPPH·; “AbsControl” is the absorbance of DPPH·reaction of the control; and “AbsSample” is the absorbance of the sample.

2.5.2. ABTS

ABTS capturing method was performed to evaluate the total antioxidant activity of C. jamacaru extracts and ascorbic acid standard[15]. The absorbance was taken at 734 nm by ELISA reader.The experiment was performed in triplicate and the percentage of scavenging inhibition capacity of ABTS+·of the C. jamacaru extract was calculated by the following equation:

% inhibition = [(AbsControl- AbsSample/ AbsControl)] × 100

Where “% inhibition” is the percentage of scavenging inhibition capacity of ABTS+·; “AbsControl” is the absorbance of ABTS+·reaction of the control; and “AbsSample” is the absorbance of the sample.

2.5.3. Fe2+ chelation ions

Chelating activity of ferrous ions (Fe2+) was assayed by inhibition of ferrous-ferrozine complex formation after treatment with C.jamacaru extracts and EDTA standard[16]. The absorbance was taken at 562 nm by ELISA reader. The experiment was performed in triplicate and the percentage of ferrous ion chelating effect was calculated using the following equation:

% chelating effect = [(AbsControl- AbsSample)/AbsControl] × 100

Where “% chelating effect” is the percentage of ferrous ions (Fe2+)chelating effect; “AbsControl” is the absorbance of Fe2+reaction of the control; and “AbsSample” is the absorbance of the sample.

2.6. Cytotoxicity in vitro

2.6.1. Sarcoma 180 cells

Sarcoma 180 cells (murine sarcoma) were acquired from Banco de Células do Rio de Janeiro. In 96-well plates, sarcoma 180 cells were plated with 2×105cells per well. The cells that received C. jamacaru extracts were diluted with water at 10.0, 50.0, 100.0, 200.0, 300.0, 400.0,and 500.0 μg/mL and untreated cells (0.0 μg/mL of extract) were used as a control. Sarcoma 180 cells were cultured for 24 h, 48 h, or 72 h to evaluate the anticancer effect of C. jamacaru extracts. All protocols were approved by the Research Ethical Committee of Universidade Federal do Espírito Santo (certificate 89/2015).

2.6.2. Cell culturing methods

Culture medium RPMI 1640, supplemented with gentamicin (50.0 mg/L), amphotericin B (2.0 mg/L), and 10% of fetal calf serum at 37 ℃, 5%of CO2saturation and humid atmosphere was used to grow the sarcoma 180 cells. The cells were cultured under these conditions 24 h before starting the treatments. At the end of the treatment, the cell viability was evaluated.

2.6.3. MTT assay

MTT assay was used to determine cell viability of sarcoma 180. At the end of the treatment, the plates were centrifuged at 860 rcf for 10 min, the supernatant was discarded and 20 μL of MTT at 5 mg/mL were added to each well. 3 h later, 100 μL of DMSO were added in each well and the absorbance was detected at 590 nm by ELISA reader.The results were expressed as a relative percentage of cell viability in comparison to the untreated cells. The experiment was performed in triplicate.

2.7. Statistical analysis

Normality of the data was evaluated a priori and results were expressed as mean ± standard deviation. To quantify the total content of flavonoid in C. jamacaru extracts, a linear curve relating rutin concentrations and absorbances was performed and the equation and R2were obtained. The total flavonoid content in C. jamacaru extracts was compared by multiple t test (P＜0.05). The antioxidant capacity of the extracts at different concentrations was compared by ANOVA post hoc Tukey test (P＜0.05). To evaluate the anticancer activity of C. jamacaru extracts in vitro, the cell viability of sarcoma 180 treated with extracts at various concentrations was compared to untreated cells (0.0 μg/mL of extract) by ANOVA post hoc Dunnett’s test (P＜0.05). Principal component analysis (PCA) and Pearson correlation were performed to visualize relationships between the total flavonoid content, antioxidant activities, and anticancer effects of C. jamacaru extracts. In the PCA analysis and Pearson correlation, the values of antioxidant activities and anticancer effects at the concentration of 500.0 μg/mL were used as this concentration presented the best results for the biological activities tested.

3. Results

3.1. Flavonoid content

Table 1 summarizes the total yield and total flavonoid content of C.jamacaru extracts. The total flavonoid content was calculated using the equation y = 0.003 345x + 0.121 2 (R2= 0.994 8; P＜0.000 1). The highest total yield was observed in the pulp extract (70.34%) and C.jamacaru cladode extract at the fruiting stage presented the highest total content of flavonoids [(201.45 ± 16.54) μg/mL] among all extracts, approximately 10 times higher than the C. jamacaru cladode extract at the vegetative stage [(20.85 ± 3.78) μg/mL].

Table 1 Total extract yield and total flavonoid content of C. jamacaru extracts.

3.2. Evaluation of antioxidant activity

The results of the antioxidant evaluation of extracts are presented in Table 2. In the DPPH test, C. jamacaru seeds extract presented better antioxidant activity when compared to the other extracts, which was similar to the results of the ascorbic acid as a standard in some tested concentrations. In comparison to the other extracts, the C. jamacaru fruit pulp extract showed lower antioxidant activity in DPPH assay and no antioxidant activity was detected at concentrations of 62.5 and 125.0 μg/mL.

For the ABTS assay, C. jamacaru cladode extract at the fruiting stage was the one with the better antioxidant activity when compared to the other extracts. The extract of seeds showed antioxidant activity similar to the standard ascorbic acid at the concentration of 500.0 μg/mL. Moreover, similar to the results observed in the DPPH assay,the C. jamacaru fruit pulp extract presented the lowest antioxidant power in the ABTS assay and no antioxidant activity was detected at concentrations of 62.5 and 125.0 μg/mL.

At the evaluation of the chelating activity of Fe2+ions, the C.jamacaru cladode extract at the vegetative stage presented the highest antioxidant power in comparison to the other extracts. At concentrations of 62.5 and 125.0 μg/mL, no antioxidant activity was detected in the C. jamacaru fruit bark extract. However, at all tested concentrations of C. jamacaru seeds extract, no antioxidant activity was detected. The values of DPPH, ABTS, and Fe2+chelation, for fruiting cladodes, are higher for 250.00 μg/mL compared to 500.00 μg/mL concentration (Table 2).

3.3. Anticancer activity in vitro

Figure 1 shows the cytotoxicity of C. jamacaru extracts in sarcoma cells 180 in vitro. Compared to the group of untreated cells (0.0 μg/mL of extract), it was observed that all C. jamacaru extracts significantly reduced the viability of tumor cells after 24 h (Figure 1A), 48 h (Figure 1B), and 72 h of treatment in all concentrations(Figure 1C). At the end of 72 hours of treatment (Figure 1C), it was found that the C. jamacaru cladode extract at the fruiting stage was able to promote a more pronounced reduction in the viabilityof the tumor cells at the concentrations of 200.0, 300.0, 400.0, and 500.0 μg/mL. Likewise, the seed extract was able to promote a more pronounced reduction in the viability of tumor cells at concentrations of 300.0, 400.0, and 500.0 μg/mL.

Table 2 Antioxidant activity of C. jamacaru extracts and standards by DPPH, ABTS, and Fe2+ chelation ions assays (mean±SD).

Figure 1. Cytotoxicity of C. jamacaru extracts in sarcoma 180 cells in vitro by MTT assay.

3.4. Explorative analyses： Correlations between total flavonoid content and antioxidant and anticancer assays

PCA and Pearson correlation coefficient were used to correlate the results obtained in different assays. In PCA, it was accounted 52.23% and 25.92% in the first and second principal components,respectively, and total variance of 78.16% (Figure 2). PC1 was dominated by the following variables: total flavonoid content,DPPH, ABTS, and MTT assay after 48 h and 72 h of treatment.PC2 was dominated by the variables: total flavonoid content,Fe2+chelating activity, and MTT assay after 24 h of treatment.These results suggested that flavonoid content, DPPH, and ABTS antioxidant assays were correlated with anticancer effects after 48 h and 72 h of treatment, while Fe2+chelating activity was correlated with anticancer effect after 24 h of treatment. C. jamacaru cladode extract at fruiting stage presented the highest total flavonoid content level in comparison to the other extracts and it seemed to be the main factor related to its antioxidant activity (DPPH and ABTS assays)and anticancer effects. DPPH and ABTS assays were the main factor related to the anticancer activity exhibited for seeds extract. Added to this, Fe2+chelating activity seemed to be the main factor related to the anticancer effects exhibited for C. jamacaru cladode extract at vegetative stage and fruit pulp extract.

Table 3 presents the results of Pearson correlation analysis. Values followed by negative sign indicate an inversely proportional relation between the factors. Thus, considering that MTT assay was used to verify the viability of tumor cells after treatment with C. jamacaru extracts, negative correlations between the MTT assay and the total flavonoid content or antioxidant activities suggest increasing anticancer effect. A moderate negative correlation between anticancer effect after 24 h of treatment (MTT 24 h) and total flavonoid content level was demonstrated. Moreover, a moderate negative correlation between anticancer effect after 48 h of treatment(MTT 48 h) and total flavonoid content, DPPH, and ABTS activities was observed. The anticancer effect after 72 h of treatment (MTT 72 h) showed a moderate negative correlation with the DPPH activity and moderate positive correlation with Fe2+chelating activity.

Table 3 Pearson correlation analysis between total flavonoid content, antioxidant activities (DPPH, ABTS, and Fe2+ chelation), and anticancer effects (MTT assay) of C.jamacaru extracts.

To evaluate anticancer effects, MTT assay was performed after 24 h of treatment (MTT 24 h), 48 h of treatment (MTT 48 h), and 72 h of treatment (MTT 72 h).Values of antioxidant activities and anticancer effects at the concentration of 500.0 μg/mL were used to perform Pearson correlation.

Figure 2. PCA (scores and loading plots, biplot) based on total flavonoid content analyzed in C. jamacaru extracts (vegetative and fruiting cladodes,bark, pulp, and seed fruit extracts) and their antioxidant activities (DPPH,ABTS, and Fe2+ chelating activity) and anticancer effects (MTT assay).

4. Discussion

Phytochemicals production and accumulation in plants may differ due to genetic, environmental, and phenological factors[5,17]. The phytochemicals can accumulate in different parts of the plants[18],while the variety, processing, cooking, and growing conditions may determine the level of phytochemicals accumulated in plant tissues[19].

Studies on the phenological aspects of cacti are scarce and usually address factors related to ecology or increase in fruit and plant biomass production, as the studies of Gomes et al.[9] and Arba et al.[20]. Gomes et al.[9] performed a study relating phenology,frugivory, dispersion and germination of C. jamacaru seeds, and Arba et al.[20] investigated the effect of irrigation on plant biomass at different phenological stages in cactus of the genus Opuntia produced for commercial purposes.

C. jamacaru is a succulent plant adapted to arid regions and the fruiting stage seems to play an important modulating role in the production of flavonoids. The total flavonoid content in cladodes extract of C. jamacaru at vegetative stage (20.85 μg/mL) was higher than that observed by de Sousa Araújo et al.[21] in methanolic extract(0.59 μg/mL) and Dutra et al.[4] in hydroalcoholic extract (0.51 μg/mL).For the content of flavonoids in the bark and pulp, the values found in our study differ from those observed by Lima[22], evaluating the total yellow flavonoid in C. jamacaru fresh fruits, 3.20 and 2.35 mg/100 g in bark and pulp, respectively.

The plant material used in our study was collected in July 2018,after the fructification peak of June 2018[9]. Considering that plants grown in natural environments may show heterogenous flowering and fruiting, it was possible to find and collect in the same population C. jamacaru plants in vegetative and fruiting stage.

Compared to previous studies conducted with C. jamacaru cladodes at the vegetative stage[4,21], our study found that there is an increase in the total production of flavonoids during the fruiting stage. Our study also showed that the extract of C. jamacaru cladodes in the vegetative stage had relatively high total flavonoid content (20.85 μg/mL) compared to what Dutra et al. (0.51 μg/mL) described using C. jamacaru cladodes collected at the same region and phenological stage in September 2016[4]. Fluctuations in flavonoid content may occur due to environmental changes, as suggested by Pretti et al.[5]. However, we believe that fruiting increases plant flavonoid production and that the relatively high level of flavonoids in cladodes at the vegetative stage may have occurred due to post-fructification stage.

Several methods can be employed to assess the antioxidant capacity of vegetable extracts in vitro[23,24]. The use of a single method of antioxidant evaluation provides basic information about antioxidant properties of a sample. Therefore, the use of different methods of antioxidant evaluation is recommended, since it allows a better understanding of antioxidant properties and mechanism of action of a sample[23,25].

DPPH, ABTS, and Fe2+chelation ions assays were used to evaluate the antioxidant activity of C. jamacaru extracts in this work. A low antioxidant activity in pulp and bark extracts was observed, and in some concentrations the antioxidant activity was not detected (62.5 and 125.0 μg/mL) for DPPH and ABTS assays using pulp extract and for Fe2+chelation assay using bark extract. The studies with fruits showed that plant phenology and physiological responses to biotic and abiotic factors were able to modify the properties of potential antioxidant, such as under climate scenarios[7,26,27]. C.jamacaru is a cactus adapted to semi-arid conditions, surviving in high temperatures[28]. However, the high temperatures in which this cactus develops may degrade the phytotochemicals found in the fruits and reduce their antioxidant capacity[26], as observed in our study.

Bark extract presented higher flavonoid content and better antioxidant activity in DPPH and ABTS than pulp extract, similar to that observed by Lima[22], evaluating the flavonoid content and ABTS antioxidant activity of fresh fruits of C. jamacaru collected in the same biome.The flavonoids are phenolic compounds reported as the main factor responsible for the chelating activity[29]. The extract of C. jamacaru cladodes at the fruiting stage presented higher flavonoid content, but it did not show the best results in chelating activity. Therefore, no direct relationship was observed between total flavonoid content and chelating activity, suggesting that, as observed by dos Santos et al.[6],the antioxidant ability of plant extracts may be related to the chemical structure of flavonoids.

For the conditions tested in our study, all extracts of C. jamacaru were able to promote in vitro antiproliferative effects, demonstrating that the cladodes and the fruit of this cactus present potential use for the prevention/treatment of cancer. In vivo antiproliferative effects of cladodes extract of C. jamacaru at vegetative stage were reported in the literature[2-4]. The aqueous extract of the cladodes induces antiproliferative effects and increases cellular aberrations in Allium cepa test system cells[3] and hydroethanolic extract from the cladodes promotes antiproliferative effect on sarcoma 180 tumor cells in rodents[2,4].

Phenological stage, as well as the variation of climate, soil, and time conditions, seem to exert an important influence on the secondary metabolism, modifying the quality and properties of plant compounds,inducing changes in chemical composition[30], and interfering with the production of phenolic compounds, such as flavonoids[5,6].

Secondary metabolism products provide plant protection against herbivors, pathogen attack, protection of abiotic stresses, and can act as modulators of gene expression and signal transduction, influencing the content/availability of phytochemicals[31,32]. In addition, flowering and fruiting are also able to modulate the secondary metabolism of plants and alter production of these compounds and the biological activities related to them[6,18].

Among more than 200 000 different types of compounds produced by plants[33], there are many colored substances, pigments, such as flavonoids found in different plant organs. These secondary metabolites belong to the class of phenylpropanoids and present a wide color range,from pale-yellow to blue. The color development in fruits is considered an important evolutionary trait and is a factor that interferes in fruit quality and market value[34].

The fruits of C. jamacaru present reddish-brown barks when ripe. The pigmentation of the bark is related to compounds such as anthocyanins,a class of flavonoids responsible for the colors ranging from orange to blue found in many flowers, leaves, fruits, seeds, and other matrices[35].Anthocyanins are used as food dyes mainly in the beverage industry and in recent years the consumption of these substances has increased due to the public concern about the possible adverse effects of synthetic food dyes[36].

Following the comparison of C. jamacaru extracts at different developing stages, we suggest that the differences between the flavonoid contents and biological activities observed are related to the modulations of the primary and secondary metabolic pathways in the plants. In addition, the diversity of phenolic compounds in plants is a result of derivations in the shikimic acid pathway, a process mediated by the enzyme L-phenylalanine ammonia-lyase (PAL), an enzyme that acts on a branch point between the primary and secondary metabolism, shikimic acid and phenylpropanoid production pathways,respectively[37].

PAL acts on a limited set of molecular structures, producing several phenolic compounds, such as flavonoids, the most abundant class of plant polyphenols synthesized by the combination of the shikimic acid and acyl polylate pathways[37]. The enzymatic action of PAL madiates the synthesis of L-phenylalanine, a compound used in the production of trans-cinnamic acid, a molecule from which most of the phenolic compounds derive[38,39]. Thus, modulations in the pathway of secondary plant metabolism may induce both the production of phytochemicals and the increase in the content and/or accumulation of these substances.

Our findings indicate that during the fruiting stage there is modulation of the secondary metabolic pathway of C. jamacaru, leading to an increase in total flavonoid content. The extracts of the cladodes and the fruit of C. jamacaru presented antioxidant activity and antiproliferative effects, however no direct relationship between the flavonoid content and the biological activities was observed. This study also suggests the potential use of C. jamacaru cladodes and fruits as a source of natural antioxidants and as potent anticancer agents. These data reinforce the effectiveness of the biological activities of natural products, such as those observed in our study, favor the extensive use of C. jamacaru in cooking, industry, drug development and also assist in understanding the role of secondary metabolite pathway in metabolites synthesis.

Conflict of interest statement

The authors declare no conflict of interest.

Acknowledgements

We would like to express our gratitude to Universidade Federal do Espírito Santo.

Funding

This work was supported by grants from FAPES (Funda??o de Amparo à Pesquisa e Inova??o do Espírito Santo) - term of grant 225/2015.