Chemical analysis and evaluation of antioxidant and antimicrobial activities of fruit fractions of Mauritia flexuosa L. f. (Arecaceae)

Plant material

The fruits of M. flexuosa L. f. were collected in the Environmental Protection Area—APA -Chapada do Araripe (7°15′33.37″S 39°28′6.95″O) in October 2016, in the municipality of Crato, Ceará, Brazil. A voucher specimen was deposited in the “Herbário Caririense Dárdano de Andrade-Lima—HCDAL” at the Regional University of Cariri—URCA under register number 12.620. Authorization to collect botanical material was provided by the Authorization and Biodiversity Information System (SISBIO) of the Chico Mendes Institute for Biodiversity Conservation (ICMBio), registered under No. 61003-1.

Drugs, reagents and equipment

Only analytical grade chemicals were used in this study. Acetonitrile, ethyl alcohol, formic acid, catechin, Folin-Ciocalteu reagent and gallic acid were purchased from Merck (Darmstadt, Germany). Apigenin, rutin, orientin, luteolin, kaempferol, quercetin, 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS), Trolox, 2, 4, 6-Tri(2-pyridyl)-s-triazine (TPTZ), phenanthroline, amikacin, gentamicin, benzylpenicillin, cefotaxime, fluconazole and ketoconazole were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The antibiotics were dissolved in sterile water. High performance liquid chromatography (HPLC-DAD) was performed with a Shimadzu Prominence Auto Sampler (SIL-20A) HPLC system (Shimadzu, Kyoto, Japan), equipped with Shimadzu LC-20AT with reciprocating pumps connected to a DGU 20A5 degasser with a CBM 20A integrator, SPD-M20A diode array detector and LC solution 1.22 SP1 software.

Preparation of the fractions

The chloroform (FCB), ethyl acetate (FAB) and ethanolic (FEB) fractions were obtained in a Soxhlet apparatus. About 800 g of the fruit pulp were subjected to extractions of 6 to 8 h in each solvent, with solvent exchange in the order of polarity and after complete evaporation. Then, the solutions were concentrated on rotary evaporator (Model 801, Fisatom, Brazil) at 50 °C under reduced pressure, yielding 1.34%, 0.87% and 2.9% (w/w) of FCB, FAB and FEB, respectively.

Chemical analysis

Evaluation of the antioxidant activity

Antimicrobial assays

Evaluation of the modulating effect of MFFs in association with antimicrobial drugs

The modulating effect of the MFFs was analyzed by combining them with aminoglycosides (amikacin and gentamicin), beta-lactams (benzylpenicillin and cefotaxime) and azoles (fluconazole and ketoconazole) according to the methodology proposed by Coutinho et al. (2008). The bacterial strains Bacillus cereus INCQS 00303 and Salmonella choleraesuis INCQS 00038, and the fungal strains Candida albicans INCQS 40006, Candida krusei INCQS 40095 and Candida tropicalis INCQS 40042 were the microorganisms used. The MFFs were used at sub-inhibitory concentrations (MIC/8) obtained in specific culture medium (10%). The solutions were distributed in microdilution plates followed by addition of different concentrations of the antimicrobials obtained by serial dilution from an initial solution of 1,024 µg/mL. In the control, antibiotics were not added to the strains. The plates were incubated at 35 ± 2 °C for 24 h and read by colorimetry by the addition of 25 µL of resazurin solution (0.01%).

Statistical analysis

Differences between groups of HPLC and antioxidant assays results were assessed by an analysis of variance model and Tukey’s test. The microbiological results were expressed as geometric mean and analysis of results was applied to two-way ANOVA followed by Bonferroni posttests using GraphPad Prism 6.0 software. Results with P < 0.05 were considered as statistically significant.

Chemical analysis

Quantification of phenolic compounds and flavonoids by HPLC-DAD

The chromatographic profiles of the fractions from HPLC analyzes are shown in Fig. 1. Samples contain other compounds in smaller amounts, besides catechin (retention time -tR = 14.27 min, peak 1), caffeic acid (tR = 24.93 min, peak 2), rutin (tR = 41.07 min, peak 4), quercetin (tR = 52.09 min, peak 5), apigenin (tR = 56.38 min, peak 6), luteolin (tR = 64.11 min, peak 7) and kaempferol (tR = 67.02 min, peak 8). The HPLC analysis demonstrated variability between the total and individual values of each compound (Fig. 2), according to each fraction.

The results showed that the main phenolic compounds identified in the samples were orientin (5.29 ± 0.01–0.13 ± 0.02 mg/g), caffeic acid (3.96 ± 0.03–0.16 ± 0.01 mg/g) and quercetin (3.87 ± 0.05–1.84 ± 0.05 mg/g). In the ethyl acetate fraction, it was possible to quantify all the standards used in the analysis, whereas in the ethanolic fraction, apigenin and kaempferol were not identified, and in the chloroform fraction only catechin, caffeic acid and orientin were identified (Table 3).

Table 3:

Components of fruit pulp fractions of Mauritia flexuosa.

These results are expressed as mean ± SD (n = 3). The averages followed by different letters differ by Tukey’s test with p < 0.05, for the compounds of the same fraction.

Compounds	FCB (mg/g)	FAB (mg/g)	FEB (mg/g)	LOQ (µg/mL)	LOD (µg/mL)
Catechin	2.27 ± 0.04a	0.35 ± 0.01c	1.43 ± 0.02b	0.015	0.042
Caffeic acid	0.16 ± 0.01b	3.72 ± 0.04a	3.96 ± 0.03a	0.008	0.023
Rutin	–	1.46 ± 0.03a	1.71 ± 0.01a	0.023	0.034
Orientin	0.13 ± 0.02c	5.29 ± 0.01a	1.85 ± 0.01b	0.032	0.051
Quercetin	–	1.84 ± 0.05b	3.87 ± 0.05a	0.025	0.075
Apigenin	–	0.15 ± 0.02a	–	0.011	0.049
Luteolin	–	0.42 ± 0.02b	1.47 ± 0.04a	0.019	0.022
Kaempferol	–	3.69 ± 0.01a	–	0.003	0.029

DOI: 10.7717/peerj.5991/table-3

As can be seen, the content of caffeic acid was similar in the ethyl acetate and ethanolic fractions. However, the chloroform fraction had a more than 20-fold lower content of the compound. Significant amounts of orientin were found in the ethyl acetate fraction, but in the other fractions, the content of this compound was significantly lower. For quercetin, significantly different values were found between the ethanolic and ethyl acetate fractions (3.87 ± 0.05 mg/g and 1.84 ± 0.05 mg/g, respectively). However, this compound was not quantified in the chloroform fraction and kaempferol was identified only in the ethyl acetate fraction.

Antioxidant assays

Fe²⁺ chelating activity

The fractions studied in this work presented elevated chelating activity, whose response was concentration dependent (14–700 µg/mL) and presented significant differences when considering the same concentrations of the different samples (Fig. 3). However, considering the maximum chelation percentage, there were no significant variations, with a maximum percentage of 78.2% for FCB, 72.9% for FAB and 80.9% for FEB.

Table 4:

Antioxidant activity values of fruit pulp fractions of M. flexuosa, according to the ABTS free radical capture.

These results are expressed as mean ± SD (n = 3). The averages followed by different letters differ by Tukey’s test with a p < 0.05.

Samples	AA (μM trolox/g)
FCB	83.17 ± 3.23a
FAB	23.39 ± 0.10b
FEB	43.65 ± 0.12c

DOI: 10.7717/peerj.5991/table-4

Antimicrobial assays

Modulating activity by direct contact

The analysis of modulating activity of the fractions of M. flexuosa by direct contact with antibiotics against bacterial strains showed that the fractions caused both synergistic and antagonistic effects, as can be observed in Figs. 4 and 5. The chloroform fraction exerted a synergistic effect in combination with gentamicin against B. cereus and S. choleraesuis, reducing the MIC of this antibiotic from 512 to 256 µg/mL. A similar effect was obtained with the association of this fraction with cefotaxime against B. cereus, in which the MIC of the antibiotic was reduced from 1,024 to 256 µg/mL. On the other hand, the fraction exerted an antagonistic effect when combined with benzylpenicillin against B. cereus, increasing the MIC of the penicillin from 21.3 µg/mL to 170.6 µg/mL.

In the tests with S. choleraesuis, the FCB reduced the MIC of Amikacin from 512 µg/mL to 170.6 µg/mL, whereas the FEB decreased it from 512 µg/mL to 341.3 µg/mL. In association with cefotaxime, against the same microorganism, all the fractions exerted the same synergistic effect, decreasing the MIC of the antibiotic from 1,024 µg/mL to 512 µg/mL. Finally, when associated with benzylpenicillin against this microorganism, only FAB and FEB potentiated the action of the antibiotic, reducing its MIC from 1,024 µg/mL to 426.6 and 512 µg/mL, respectively.

In the analysis of the antifungal activity modulation, the fractions did not present significant effects, when associated to the drugs against C. albicans and C. krusei. The only significant modulation was obtained from the association of the ethyl acetate fraction with on ketoconazole against C. tropicalis. This association presented an antagonistic effect, because the MIC of the drug was increased from 213.3 to 512 µg/mL, as shown in Fig. 6. These data indicate that, at the experimental conditions of the present work, the fruits of buriti might not be useful in the modulation of resistant fungal infections.

Chemical analysis

The determination of the contents of phenolic compounds and flavonoids is relevant because the antioxidant activity of several plant extracts has been attributed to the presence of these compounds, especially flavonoids (Sousa et al., 2015). In addition, the structures of these compounds facilitate their action against several microorganisms as they form complexes with extracellular and soluble proteins, causing rupture of microbial membranes as well as enzymatic inhibition (Cowan, 1999; Cushnie & Lamb, 2005).

Flavonoids can be found in fruits, vegetables and beverages such as teas and wines (Nijveldt et al., 2001). These compounds present numerous biological activities against several diseases: inflammation, thrombosis, osteoporosis, diabetes, viral infections, liver diseases and ulcers, in addition to presenting vasodilatory effects (Tapas, Sakarkar & Kakde, 2008; Agrawal, 2011).

Structurally, polyphenols are defined as compounds having benzene rings with one or more hydroxyl substituents (Angelo & Jorge, 2007). Based on the chemical structures of the phenolic patterns used in this study (Fig. 2), it is justified that these substances are directly associated with activities such as complexation of metal ions, proteins and polysaccharides, as well as the sequestration of free radicals (Pereira & Cardoso, 2012).

These results of quantification of phenolic compounds and flavonoids by HPLC-DAD show that there are differences in the concentrations of the compounds in each fraction. In the present study, we suggest that differences in the phenolic content of the samples may be corelated with the polarity of the solvent, which influences extraction of a specific group of compounds (Najjaa et al., 2011; Milanez et al., 2018).

Orientin is a C-glycoside flavonoid that is obtained by the glycosylation of luteolin (Luteolin-8-C-glycoside). Previous studies reported the presence of this compounds in other species of the family Arecaceae (Hassanein et al., 2015; Heinrich, Dhanji & Casselman, 2011; Finco et al., 2012; Yao et al., 2012). This flavonoid is recognized for exhibiting biological activities such as antioxidant, anti-adipogenic, antithrombotic, anti-inflammatory, anti-platelet and protective against radiation (Devi et al., 2000; Kim et al., 2010; Praveena et al., 2014; Yoo et al., 2014; Lee & Bae, 2015).

Caffeic acid, also referred to as 3,4-dihydroxycinnamic acid, is among the metabolites of hydroxycinnamate and phenylpropanoids the most distributed in plant tissues (Fu et al., 2010). It is also found in other species of the Arecaceae family, where they are responsible for antimicrobial, antioxidant, anti-inflammatory and anti-angiogenic bioactivities (Chakraborty & Mitra, 2008; Beskow et al., 2014; Marques et al., 2016; Taleb et al., 2016).

Antioxidant assays

According to our data, the fractions of buriti showed higher antioxidant capacity than other traditionally important fruits of the Arecaceae family, such as: açaí (Euterpe oleracea Mart.), Carnauba (Copernicia prunifera (Mill.) HE Moore) and juçara (Euterpe edulis Mart.), since literature data report that these plants presented levels of 15.1  ± 4.1 µM Trolox/g, 10.7 ± 0.2 µM Trolox/g and 78.3 ± 13.3 µM Trolox/g, respectively (Rufino et al., 2010).

A study by Almeida et al. (2011) reported the antioxidant activity of 11 fresh fruits from the Brazilian Northeast by the ABTS capture method, obtaining values of 15.73 ± 0.01 to 0.63 ± 0.01 µM of Trolox/g. The values obtained in the present study for buriti fractions were higher than the contents reported for those 11 fruits, which are considered good antioxidants.

Excessive iron accumulation can induce significant toxicity both by generation of reactive oxygen species and because of the lack iron excretion regulation in humans (Nurchi et al., 2016). Previous studies have shown the correlation between phenolic compounds and antioxidant activity, in addition to associating the chelating activity of natural products with their composition in the samples (Loizzo et al., 2012; Koolen et al., 2013; Cândido, Silva & Agostini-Costa, 2015; Tauchen et al., 2016). In fact, the structure of phenolic compounds, such as phenolic acids and flavonoids, is decisive for radical elimination and metal chelating activities. In addition, activity of these compounds depends on the number and position of hydroxyl groups in relation to the carboxyl functional group (Tapas, Sakarkar & Kakde, 2008; Garcia-Salas et al., 2010).

According to Loganayaki & Manian (2010), primary antioxidants prevent the onset and spread of oxidative chain reactions by neutralizing free radicals, and secondary antioxidants act to suppress oxidative damage by inhibiting radical formation. Thus, the fruit pulp fractions of M. flexuosa can act both as primary and secondary antioxidants because they exhibit antioxidant activity both by inhibiting free radicals and chelating iron ions.

Antimicrobial assays

In the antibacterial assays, the fractions that had presented MICs ≥1,024 µg/mL against strains tested indicate low to moderate activities (Holetz et al., 2002). In fact, MICs ≥1,024 µg/mL present no clinically significant effects against a given bacteria, because concentrations above 1,000 µg/mL require a high dose of the natural product to achieve this concentration at plasma levels (Houghton et al., 2007). Regarding the antifungal activity, all strains presented MICs of 512 µg/mL, indicating that they present moderate antifungal activities (Matias et al., 2015).

Phenolic compounds are also recognized for their antimicrobial activities because they can interact with membrane proteins through hydrogen bonds involving the hydroxyl groups. This interaction can cause changes in membrane permeability that can lead to cell destruction (Tapas, Sakarkar & Kakde, 2008; Oussaid et al., 2017).

This study shows that the MFFs were more effectiveness against Gram-positive bacteria, corroborating with earlier reports that indicates that these microorganisms are more susceptible to the effects of plant extracts, because Gram-negative bacteria have a membrane that surrounds the cellular wall, which limits the diffusion and consequent effect of the compounds present in the extracts (Murari et al., 2008; Tian et al., 2009).

From these results, it is possible to correlate the modulating action of the fractions with their phenolic content, which is supported by studies that reported the action of these compounds as antibiotic modifiers. Lima et al. (2016) reported the drug modifying activity of phenolic acids, including caffeic acid, which potentiated the activity of antimicrobial agents against bacterial and fungal strains. Diniz-Silva et al. (2017) showed the potentiating activity of fruit flavonoids (in both aglycone and glycosylated forms) in association with norfloxacin against Staphylococcus aureus. According to the study, 3-O-glycoside flavonoids, such as hesperetin and quercetin, modulated the antibiotic resistance in this strain.

Aminoglycosides, in general, exhibit characteristic cellular toxicity as a function of their absorption into the intracellular environment, including: nephrotoxicity, ototoxicity and neuromuscular blockade (Oliveira, Cipullo & Burdmann, 2006). The combination of this class of antibiotics and natural products may be an alternative to reduce their side effects, because a significant reduction of the MIC also reduces the therapeutic dose (Figueredo et al., 2013).

Currently, the strategy used to overcome the bacterial resistance mechanism against beta-lactam antibiotics, is to associate this drugs with beta-lactamase inhibitors (Veras et al., 2017). The probability of resistance development during the treatment with antibiotics is significantly high, justifying the search for strategies to overcome these mechanisms. In this context, the development of efflux pump inhibitors that can be used as therapeutic adjuvant has been considered an attractive option (Tang, Apisarnthanarak & Hsu, 2014). Thus, due to the great pharmacological potential, natural products are important sources of compounds that can be useful in the management of infections caused by resistant microorganisms (Gibbons, 2004).

The presence of chitin contributes to the mechanical resistance of fungal cell walls, that are target of azoles, which act by interfering with ergosterol synthesis in the cell membrane of these microorganisms (Merzendorfer, 2006; Pfaller, 2012). Resistance to azoles in Candida species can occur by several mechanisms, such as presence of efflux pumps, point mutations that can alter the synthesis and cause overexpression of ergosterol and mutations that impair the membrane rupture, such as by-pass pathways (Pfaller, 2012). Therefore, these mechanisms might be associated with the results obtained by the present study.

Despite advances in prevention, diagnosis and therapy, invasive fungal infections still cause significant mortality and morbidity in immunocompromised patients and thus, antifungal resistance remains a worrying condition (Kanafani & Perfect, 2008). Currently, the therapy of these infections is limited to three main antifungal classes (azoles, echinocandins and polyenes), which present significant toxic effects, highlighting the importance of the search for new antifungal agents (Morais-Braga et al., 2016).