Chemical Composition, Antifungal, and Cytotoxicity Activities of Inga laurina (Sw.) Willd Leaves

The species Inga laurina is native to the Brazilian Cerrado. There are no studies about the chemical composition and biological activities of extracts of this endangered species. The ethanolic extract and its successive fractions are rich in phenolic compounds and presented good antifungal activities. HPLC/MS-MS/MS and H1/C13 analysis led to the identification of seventeen compounds, most of which are gallic acid derivatives, myricetin and quercetin glycosides. The ethyl acetate fraction (EAF) contained high levels of total phenolics, expressed in milligrams of gallic acid equivalents per gram of extract (475.3 ± 1.9 mg GAE gextract−1) and flavonoids expressed in milligrams of quercetin equivalents per gram of extract (359.3 ± 10.6 mg QE gextract−1). This fraction was active against fungi of the Candida genus. The EAF showed MIC value 11.7 μg mL−1 against C. glabrata and a selectivity index of 1.6 against Vero cells. The flavonol glycoside myricetin-3-O-rhamnoside was isolated for the first time from the Inga laurina. These results make I. laurina a promising plant as a source of pharmaceutical and biological active antifungal compounds.


Introduction
Among the plants in danger of extinction from Brazilian savannah biome (Cerrado), the Inga spp. (Fabaceae) are found mainly in neotropical areas and many plants from this family have been reported to possess biological activities and several are used in folk medicine [1]. Leaves and roots of I. strigillosa are used by the indigenous tribes of the Amazon region for skin wounds, the flowers of I. cecropietorum are used for earache, and the flowers of I. rubiginosa are used against nasal congestion [2].
There are 131 species of this genus in Brazil, with 51 of them being endemic [3]. Several biological activities have been observed in Inga species, such as the antioxidant activity of I. edulis [4][5][6] and I. verna [2], the antifungal activity of I. marginata [7], the antimicrobial activity of I. fendleriana [8], the allelochemical effects in I. umbellifera [9], and the antitumoral activity in I. marginata [10].
The sp. I. laurina is found not only in the Cerrado biome but in different regions of Brazil: Amazon, Caatinga, and the Atlantic Coast. This plant is easily found in Brazil, but there are a few studies about its essential oil. The present work is based on a doctorate thesis and the aim of the present work was to identify the bioactive metabolites by Mass Spectrometry and to evaluate the antifungal activity against Candida genus and the cytotoxic activity on Vero cells ATCC CCL 81 using ethanolic extract and fractions of I. laurina leaves. Gates presented mainly phenolic compounds, as flavonoid glycosides of quercetin derivatives [11].

Extracts Preparation.
Leaves were dried at 35 ∘ C and then shredded in a knife mill. The extract was prepared by maceration at room temperature with ethanol (95%). The mass of 900.0 grams of leaves were extracted over 48 hours (3x). Filtrates were joined, concentrated under vacuum (under 40 ∘ C), and freeze-dried (Lyophilizer LS 3000, TERRONI, Brazil). A yield of 54.0 grams was obtained. The ethanolic extract (EE) was stored under refrigeration until analysis.

Liquid-Liquid Extraction.
The EE (42.0 g) was redissolved in 570.0 mL of methanol:water (9:1) and subjected to liquid-liquid extraction using solvents of increasing polarities (hexane, chloroform, ethyl acetate, and n-butanol). Three extractions were performed using 300.0 mL of solvent per extraction. Fractions were concentrated to dryness giving hexane (12.2 g), chloroform (4.1 g), ethyl acetate (3.2 g), and n-butanol (7.4 g) fractions. These fractions were freeze-dried and stored under refrigeration until analysis.

Spectrophotometric Analysis of Total
Phenolics, Proanthocyanidins, and Flavonoids Contents. Total phenolics were determined by the Folin-Ciocalteu method described by Morais et al. [29] and the results were expressed in mg of gallic acid equivalents by gram of dry extract (mg GAE g extract -1 ). Proanthocyanidin content was determined by the sulfuric vanillin method according to Morais et al. [29] and expressed as milligrams of catechin equivalents per gram of dry extract (mg CE g extract -1 ). Flavonoid content was determined as described by Woisky and Salatino [30] and expressed in mg of quercetin equivalents per gram of dry extract (mg QE g extract -1 ). The readings were taken in a Thermo Scientific Genesys-10S spectrophotometer. All analyses were performed in triplicate.  [31]. This assay way followed the methods of Nunes et al. 2016 [11]. The Amphotericin B and strains ATCC 22019 (Candida parapsilosis) and ATCC 6258 (Candida krusei) were used as quality controls.

Antifungal
2.6. Cytotoxicity Assay (CC 50 ) Using Vero Cells. The cell viability test was performed with Vero cells (ATCC CCL 81) (kidney fibroblasts, African green monkey). The cytotoxic activity was performed using the microplate dilution method [32]. Cell viability was calculated from the absorbance of each concentration tested according to the growth control. The cytotoxic concentration (CC 50 ) (concentration that presents 50% cell viability) was calculated by means of a dose-response graph with nonlinear regression [33]. Controls of growth, solvent, samples, negative control (100% lysed cells), and control of the medium were performed. The assays were performed in triplicate. The selectivity index (SI) of each sample was defined as the ratio between the logarithm of CC 50 and the MIC against each strain (SI = log[CC50]/[MIC]) [34,35].

Ethyl Acetate Fraction (EAF) Isolation and Fractionation.
Due to the fact that EAF of I. laurina showed promising results for inhibiting antifungal growth, it was selected for refractionation. Thus, a sample of EAF (0.60 g) was submitted to Column Chromatography with Sephadex LH-20 (34.

High Performance Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry (HPLC-ESI/MS ).
The assays were carried out in a Liquid Chromatography system (Agilent Infinity 1260) coupled to a High Resolution The Scientific World Journal 3  . Nitrogen (N 2 ) was used as a drying gas at a flow 8 L min −1 and as a nebulizing gas at a pressure of 58 psi. The nebulizer temperature was set at 220 ∘ C with a potential of 4.5 kV used on the capillary.

Nuclear Magnetic Resonance (NMR).
NMR spectra were carried out in the Bruker Model Ascend6 400 Avance III HD (9.2 Tesla) spectrometer. The samples were solubilized in deuterated dimethyl sulfoxide (DMSO-d 6 ) and tetramethylsilane (TMS) was used as an internal standard. Analyses were performed at 400 MHz for 1 H NMR and at 100 MHz for 13 C NMR. The following NMR analyses were performed: 1 H, 13 C, DEPT-135, COSY, and HSQC.

High Performance Liquid Chromatography (HPLC).
The EAF and subfractions from the leaves were analyzed by High Performance Liquid Chromatography coupled to a diode array detector (HPLC-DAD). A Shimadzu Chromatography system, model LC-6AD with C18 reverse phase column (Phenomenex Luna model, 4.6 mm internal diameter, 25 cm long, 5 m particles with 100Å diameter), was used. A volume of 20 L of methanol solution was injected at 3,000 g mL −1 of EAF and 1,000 g mL −1 of subfraction (F1-F8). Deionized water (phase A) and methanol (phase B) were used as mobile phases using the following program: 50% B (25 min) at a flow rate of 0.8 ml min −1 .

Statistical
Analysis. The analyses were performed in triplicate and the results were evaluated using the Analysis of Variance (ANOVA) method. The results were considered statistically different when the significance level was lower than 5% (P<0.05). The Tukey test was used to determine the significant differences between the averages. Analyses were performed using the SigmaPlot 11.0 program.

Phenolics, Proanthocyanidins, and Flavonoids Contents.
Spectrophotometric results for extracts and fractions of I. laurina leaves are shown in Table 1.
EE, EAF, and BF fractions presented the highest contents of total phenolics. The difference in these values can be explained by the polarity of the solvents used [6]. Polar solvents such as ethanol, ethyl acetate, and n-butanol are more able to extract phenolic compounds. EE and EAF have higher levels of total phenolics when compared to those of I. marginata, which has values of 31.63 mg and 8.37 of GAE g extract -1 , respectively [36]. The methanolic extract (50%) of I. edulis presented total phenolics of 496.5 mg of GAE g extract -1 , which is higher than that of EE from I. laurina [4]. Dias, Souza, and Rogez [6] reported values of 15.8 and 357.5 mg GAE g extract -1 of total phenolics for hexane and water fractions, respectively, for the acetone:water:acetic acid extract (70:28:2 v:v:v). The values of proanthocyanidins were lower when compared with total phenolics. No data were found for this technique in the literature concerning levels of proanthocyanidins for I. species.
The fractions CF and EAF presented the highest values of flavonoids among the other fractions. The good result obtained with the less polar fraction (chloroform) suggests the presence of aglycone flavonoids. Flavonols and flavones were confirmed by the test of complexation with aluminum ions (Al 3+ ). This complexation reaction allows the quantification of flavonoids by reading the absorbance of the solution. In this reaction, the aluminum chloride shifts the wavelengths of bands I and II to a higher wavelength, a bathochromic shift [37]. EE has higher level of flavonoids when compared to that presented by I. marginata, whose value is 118 mg QE gextract −1 [38].

Antifungal and Cytotoxicity Activities.
Values of MIC (minimum inhibitory concentration) for antifungal activity in g mL −1 and cytotoxic activity in CC 50 (cytotoxic concentration), in g mL −1 , for extract and fractions of leaves of I. laurina are shown in Table 2.
Fractions EE, EAF, and BF were the most active against the evaluated microorganism, showing MIC values lower than 100 g mL −1 , as shown in Table 2. In general, the BF Table 2: Results of antifungal activity (expressed as MIC in g mL −1 ), cytotoxic activity (expressed as CC 50 in g mL −1 ), and selectivity index for extract and fractions of I. laurina leaves.

MIC ( g mL -)
Microorganisms fraction showed good activity against C. albicans (MIC 11.7 g mL −1 ) and for C. glabrata (MIC 23.4 g mL −1 ); the EE and EAF fractions were also very effective against C. glabrata (MIC 11.7 g mL −1 ); and the CF fraction had no antifungal activity at the tested concentrations (MIC above 3,000 g mL −1 ). The EE of the leaves from B. laevifolia showed good results for antifungal activity, showing MIC values of 31, 63, and 63 g mL −1 for C. albicans, C. tropicalis, and C. glabrata, respectively [11].
Regarding the cytotoxic activity, the lower the CC 50 value is, the higher the cytotoxicity against Vero cells will be, because a small concentration of the sample would inhibit the growth of cells by 50%. To correlate the antifungal activity with the cytotoxic concentration, the selectivity index (SI) was calculated. The SI indicates whether the sample is more selective for antifungal activity or more toxic for Vero cells. The more positive the SI value, the greater the selectivity to inhibit fungal growth; a negative value indicates that the sample is more toxic to Vero cells than selective for the inhibition of antimicrobial growth [34]. Therefore, the fractions EE, EAF, and BF were the most selective to the tested microorganisms, as they presented positive SI values for all of the fungi of the genus Candida; the best values were 1.5, 1.6, and 1.6, respectively. Nunes et al. [11] also found positive SI values for EE and n-butanol partition from B. laevifolia leaves, for the same microorganisms tested in the Table 2. The best results were found for E, which showed a positive SI value of 1.2, 0.9, and 0.9 for C. albicans, C. tropicalis, and C. glabrata, respectively.
Other Inga species were studied regarding antifungal activity. The EE of leaves of I. vera showed low inhibition against C. albicans, with an inhibition zone of 7 to 15 mm (i.d.), whereas a good inhibition would be larger than 20 mm [39]. The ethanolic extract of I. marginata leaves showed no antifungal activity against C. albicans and C. krusei but showed activity against C. tropicalis [40]. The promising antifungal activity of I. laurina can be related to the presence of phenolic compounds. The extracts and fractions showed a high quantity of total phenolics and flavonoids and these compounds have been frequently reported as potential antifungal agents [41][42][43][44][45].

High Performance Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry (HPLC-ESI/MS 2 )
Analysis. The EAF presented the highest level of phenolic compounds and flavonoids compared to the other fractions and showed good results for antifungal activity. Therefore, this polar fraction was submitted to High Performance Liquid Chromatography coupled to Electrospray Ionization (HPLC-ESI) analysis and sequential Mass Spectrometry (MS/MS) in the negative mode. The total ion chromatogram of EAF obtained by HPLC-ESI is shown in Figure 1. Thirteen compounds could be identified in this fraction using this technique (Table 3 and Figure 2).
Column fractions (F1-F8) obtained from EAF were also analyzed by HPLC-ESI/MS 2 . Five different compounds were identified in addition to those identified in EAF (Table 3); their structures are shown in Figure 3. The compound myricetin-3-O-acetyl-rhamnoside (compound XIV) was identified in fraction F2 (m/z 505), digalloylquinic acid (compound XV, m/z 495) and myricetin-3-O-rhamnose-3 -O-rhamnoside (compound XVI, m/z 609) were identified in F5, trigalloylquinic acid (compound XVII, m/z 647) was identified in the F6, and vanillic acid (compound XVIII, m/z 167) was identified in F7 (Table 3). See Scheme S1 in the Supplementary Material for the comprehensive flowchart of purification, identification, and isolation of compounds from the EAF.
The ion m/z 609 (compound XVI) forms the fragments m/z 463, 316, and 178 (Figure 4), which correspond to compound VIII, which was confirmed to be myricetin-3-Orhamnoside ( Figure 2). The loss of 146 Da (rhamnosyl group) of ion m/z 609 forms the ion m/z 463, which loses a further 146 Da to form the ion m/z 316. This finding is in accordance with the structure proposed for the ion m/z 609, which has 2 rhamnosyl groups bonded to the myricetin aglycone. The literature reports a structure for myricetin-3 -O-rhamnose-3-O-galactoside [46]; therefore, a similar structure carrying The Scientific World Journal 5 Vanillic acid (XVIII) The Scientific World Journal   The Figures S1 to S16 show mass spectrum of phenolic compounds acquired by HPLC-ESI/MS 2 . Figures S17 to S22 show fragmentation patterns.

Structural Determination and Characterization of the Isolated Compound from Fraction F4 (Myricetin-3-O-Rhamnoside).
Fraction F4 was also analyzed by HPLC to check its purity. The HPLC chromatogram indicated the presence of only one intense peak in 8.1 min, while the UV/Vis spectrum shows two bands of absorption, characteristic for flavonoids (258 and 353 nm). See Figure S23 in the Supplementary Material to check the chromatogram and UV/Vis spectrum of F4.
The high resolution mass spectrum and NMR spectra confirmed the structure of the isolated compound in fraction 4 as myricetin-3-O-rhamnoside, a glycosylated flavonoid with crystalline aspects, with the molecular formula C 21 H 20 O 12 and MW 464.38 gmol −1 (compound VIII, Figure 2). There are no records for its melting point in the literature, but it decomposes close to 200 ∘ C and its color changes from yellow to orange and black.  (Figure 6). The spectrum in Figure 5 is in good agreement with Saldanha et al. [17], where flavonoids in Myrcia bella Cambess were identified by using the same technique as in the present work (MS/ESI-MS/MS). The fragmentation pathway of myricetin-3-O-ramnoside corresponds to that proposed by [19] when they studied the methanol extract of Pistacia lentiscus leaves.
1 H NMR spectra showed 3 different signals attributed to aromatic hydrogens, two doublets were observed at 6.20 and 6.37, both with J = 2.0 Hz, typical of meta -couplings of carbons C-6 and C-8, respectively, in ring A. There is an intense singlet in 6.89 attributed to hydrogens H-2 and H-6 of ring B, which are equivalent. The signal at 12.00 corresponds to the hydroxyl hydrogen of carbonyl C-5, which has a hydrogen bond with a carbonyl carbon C-4, corresponding to a chelated hydroxyl.
In HSQC contour map analysis, it was possible to observe a signal at 5.2 (1H, d, J = 1.2 Hz), typical of a hydrogen bound to the anomeric carbon ( 101.94, C-1 ) of rhamnose. This coupling constant value is typical of hydrogen in the equatorial position of the glycosidic ring which is coupled with the hydrogen H-2 (axial-equatorial or equatorial-equatorial). The DEPT-135 and 13 C NMR spectra, respectively, allowed confirmation of the carbon signals in the aromatic region. The correlations of the hydrogens of rhamnose were attributed through the COSY spectrum. Thus, it can be inferred that the isolated compound is myricetin-3-O-rhamnoside [4,47] (Figure 7). See Figures S24-S29 and Tables S1-S2 in the Supplementary Material to check the NMR spectra and NMR chemical shifts, respectively, of F4.
Myricetin-3-O-rhamnoside inhibited the growth of all yeasts as shown in Table 2 at a concentration of 93.8 g mL −1 . This result confirms the antifungal activity for this compound. Salazar-Aranda and coworkers [48] also observed an MIC higher than 83 g mL −1 for C. albicans and C. tropicalis, although the MIC values were higher for C. glabrata, 3.9 g mL −1 , and 7.8 g mL −1 for clinical isolates of yeast.
The isolated compound presents the same MIC of EAF for the evaluated microorganism, except for C. glabrata, which has a lower value (11.7 g mL − ). From these results, it is possible to infer that myricetin-3-O-rhamnoside can contribute markedly to the antifungal activity observed for EAF against C. albicans and C. tropicalis, since they presented the same MIC values.

Conclusions
The EE from the leaves of Inga laurina and its fractions are rich in phenolic compounds and present good antifungal activities. HPLC/MS-MS/MS and H1/C13 analysis made it possible to identify seventeen compounds, most of which are gallic acid derivatives and myricetin and quercetin glycosides. The EAF contained a high level of total phenolics (475.3 ± 1.9 mg GAE g extract -1 ) and flavonoids (359.3 ± 10.6 mg QE g extract -1 ) and was active against fungus from the Candida genus. The best MIC results found for C. albicans, C. glabrata, and C. tropicalis were 11.7, 11.7, and 46.8 g mL −1 , respectively, and the best selectivity indexes for the three microorganisms, using Vero cells, were 1.6, 1.3, and 1.0, respectively. These results make I. laurina a promising plant for advanced antifungal studies.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request. All data in our manuscript is available for readers.

Conflicts of Interest
The authors declare no conflicts of interest.

Acknowledgments
This work was supported by the Foundation for Research Support of the Minas Gerais State (FAPEMIG-Brazil; APQ-01178-11). This study was financed in part by the Coordination for the Improvement of Higher Education Personnel (CAPES), Finance Code 001. The authors also thank the Nanobiotechnology Laboratory (IBTEC-UFU) for the UHPLC-MSn assays, the Chemistry Institute of the Federal University of Uberlândia (IQUFU) for the supporting infrastructure, and Professor Dr. Glein Monteiro de Araújo (Institute of Biology-UFU) for plant identification. This manuscript is based on the thesis of the authors.

Supplementary Materials
Supplementary Scheme S1: flowchart for purification, identification, and isolation of compounds from the leaves of I.