# Chemical Composition, Antifungal, and Cytotoxicity Activities ofInga laurina (Sw.) Willd Leaves

Authors: Carla de Moura Martins; Sérgio A. L. de Morais; Mário M. Martins; Luís C. S. Cunha; Cláudio V. da Silva; Carlos H. G. Martins; Luís F. Leandro; Alberto de Oliveira; Francisco J. T. de Aquino; Evandro A. do Nascimento; Roberto Chang
Journal: The Scientific World Journal
 (2019)

---

## Abstract

The speciesInga 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 theCandida genus. The EAF showed MIC value 11.7 μg mL−1 againstC. glabrataand a selectivity index of 1.6 against Vero cells. The flavonol glycoside myricetin-3-O-rhamnoside was isolated for the first time from theInga laurina. These results makeI. laurina a promising plant as a source of pharmaceutical and biological active antifungal compounds.


---

## Body

## 1. Introduction

Among the plants in danger of extinction from Brazilian savannah biome (Cerrado), theInga 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 ofI. strigillosa are used by the indigenous tribes of the Amazon region for skin wounds, the flowers ofI. cecropietorum are used for earache, and the flowers ofI. 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 inInga species, such as the antioxidant activity ofI. edulis[4–6] andI. verna[2], the antifungal activity ofI. marginata[7], the antimicrobial activity ofI. fendleriana[8], the allelochemical effects inI. umbellifera [9], and the antitumoral activity inI. 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 againstCandida genus and the cytotoxic activity on Vero cells ATCC CCL 81 using ethanolic extract and fractions ofI. laurina leaves.A few studies of plants from the Brazilian savannah have identified bioactive metabolites by Mass Spectrometry. The leaves of the plantBanisteriopsis laevifolia (A. Juss.) B. Gates presented mainly phenolic compounds, as flavonoid glycosides of quercetin derivatives [11].


## 2. Materials and Methods

### 2.1. Plant Material

Leaf samples ofI. laurina (Sw.) Willd, Fabaceae, were collected near the BR-050 in Uberlândia, Minas Gerais State, Brazil (18°59′13.96′′S; 48°12′42.16′′W) in the month of February (2012). The specimens were identified by Prof. Dr. Glein Monteiro de Araújo, from the Biology Institute, Federal University of Uberlândia. An exsiccate was deposited at Herbarium Uberlandense (HUFU), under number 64050.


### 2.2. 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.


### 2.3. 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, andn-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), andn-butanol (7.4 g) fractions. These fractions were freeze-dried and stored under refrigeration until analysis.


### 2.4. 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 gextract-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 gextract-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 gextract-1). The readings were taken in a Thermo Scientific Genesys-10S spectrophotometer. All analyses were performed in triplicate.


### 2.5. Antifungal Activity

#### 2.5.1. Microbial Strains and Minimum Inhibitory Concentration (MIC)

The following microorganisms obtained from the American Type Culture Collection (ATCC, Rockville MD, USA) were used:Candida albicans (ATCC 28366),Candida tropicalis (ATCC 13803), andCandida glabata (ATCC 15126). The MIC determination for antifungal assay was performed according to the CLSI (Clinical and Laboratory Standard Institute) using the broth dilution assay method [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.


### 2.6. Cytotoxicity Assay (CC50) 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 (CC50) (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 CC50 and the MIC against each strain (SI = log[CC50]/[MIC]) [34, 35].


### 2.7. Ethyl Acetate Fraction (EAF) Isolation and Fractionation

Due to the fact that EAF ofI. 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 (34.0 g, 30.0 × 3.0 cm). The column was eluted with ethyl acetate:methanol in a stepwise gradient (300.0 mL 90:10; 300.0 mL 80:20; 300.0 mL 70:30; 200.0 mL 60:40; 200.0 mL 50:50; 200.0 mL 40:60; 200.0 mL 30:70; 100.0 mL 20:80; 100.0 mL 10:90). At the end, the column was washed with 300.0 mL of MeOH. The collected fraction volume was 15.0 mL and fractions with the same profile on TLC chromatograms were joined together. The fractionation of EAF yielded 8 subfractions (F1 to F8). Fractionation was repeated with another 0.60 g of EAF to obtain a higher mass of fraction. Both fractionations yielded about 500.0 mg of pure myricetin-3-O-rhamnoside in fraction 4 after TCL monitoring.Myricetin-3-O-rhamnoside: M.p.: decomposes near 200°C. UV (MeOH): λmax 258 and 353 nm; 1H NMR (DMSO-d6, 400 MHz): δ 6.20 (1H,d,J = 2.0 Hz, H-6), 6.35 (1H,d,J = 2.0 Hz, H-8), 6.89 (2H,s, H-2′e H-6′), 5.20 (1H,d,J 1.2 Hz, H-1′′), 3.98 (1H,s, H-2′′), 3.55 (1H,dd,J 1.2 e 9.3 Hz, H-3′′), 3.16 (1H,m, H-4′′), 3.37 (1H,m, H-5′′), 0.84 (3H,d,J 6.2 Hz, H-6′′); 13C NMR (DMSO-d6, 100 MHz,): δ 156.4 (C-2), 136.5 (C-3), 177.8 (C-4), 161.3 (C-5), 98.7 (C-6), 164.2 (C-7), 93.5 (C-8), 157.5 (C-9), 104,1 (C-10), 119.6 (C-1′), 107.9 (C-2′), 145.8 (C-3′), 134.3 (C-4′), 145.8 (C-5′), 107.9 (C-6′), 101.9 (C-1′′), 70.0 (C-2′′), 70.4 (C-3′′), 71.3 (C-4′′), 70.6 (C-5′′), 17.5 (C-6′′). HPLC-ESI/MS2m/z: 463.0889 ([M – H]–, C21H19O12– calc. 463.0882).


### 2.8. High Performance Liquid Chromatography–Electrospray Ionization–Tandem Mass Spectrometry (HPLC-ESI/MSn)

The assays were carried out in a Liquid Chromatography system (Agilent Infinity 1260) coupled to a High Resolution Mass Spectrometer with Quadrupole Time of Flight (QTOF) (Agilent® model 6520 B) with an Electrospray Ionization source (ESI). The chromatographic parameters were Agilent Zorbax column, 2.1 mm internal diameter, 5 cm long, 1.8μm particles, mobile phase: water acidified with formic acid (0.1%, v v−1) (A) and methanol (B), with the following solvent gradient system: 2% B (0 min), 98% B (0-15 min); 100% B (15-17 min); 2% B (17-22 min). Nitrogen (N2) 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.


### 2.9. Nuclear Magnetic Resonance (NMR)

NMR spectra were carried out in the Bruker Model Ascend™ 400 Avance III HD (9.2 Tesla) spectrometer. The samples were solubilized in deuterated dimethyl sulfoxide (DMSO-d6) and tetramethylsilane (TMS) was used as an internal standard. Analyses were performed at 400 MHz for 1H NMR and at 100 MHz for 13C NMR. The following NMR analyses were performed: 1H, 13C, DEPT-135, COSY, and HSQC.


### 2.10. 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, 5μm particles with 100 Å diameter), was used. A volume of 20 μL of methanol solution was injected at 3,000 μg mL−1 of EAF and 1,000 μg 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.


### 2.11. 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.


## 2.1. Plant Material

Leaf samples ofI. laurina (Sw.) Willd, Fabaceae, were collected near the BR-050 in Uberlândia, Minas Gerais State, Brazil (18°59′13.96′′S; 48°12′42.16′′W) in the month of February (2012). The specimens were identified by Prof. Dr. Glein Monteiro de Araújo, from the Biology Institute, Federal University of Uberlândia. An exsiccate was deposited at Herbarium Uberlandense (HUFU), under number 64050.


## 2.2. 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.


## 2.3. 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, andn-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), andn-butanol (7.4 g) fractions. These fractions were freeze-dried and stored under refrigeration until analysis.


## 2.4. 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 gextract-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 gextract-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 gextract-1). The readings were taken in a Thermo Scientific Genesys-10S spectrophotometer. All analyses were performed in triplicate.


## 2.5. Antifungal Activity

### 2.5.1. Microbial Strains and Minimum Inhibitory Concentration (MIC)

The following microorganisms obtained from the American Type Culture Collection (ATCC, Rockville MD, USA) were used:Candida albicans (ATCC 28366),Candida tropicalis (ATCC 13803), andCandida glabata (ATCC 15126). The MIC determination for antifungal assay was performed according to the CLSI (Clinical and Laboratory Standard Institute) using the broth dilution assay method [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.


## 2.5.1. Microbial Strains and Minimum Inhibitory Concentration (MIC)

The following microorganisms obtained from the American Type Culture Collection (ATCC, Rockville MD, USA) were used:Candida albicans (ATCC 28366),Candida tropicalis (ATCC 13803), andCandida glabata (ATCC 15126). The MIC determination for antifungal assay was performed according to the CLSI (Clinical and Laboratory Standard Institute) using the broth dilution assay method [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.


## 2.6. Cytotoxicity Assay (CC50) 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 (CC50) (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 CC50 and the MIC against each strain (SI = log[CC50]/[MIC]) [34, 35].


## 2.7. Ethyl Acetate Fraction (EAF) Isolation and Fractionation

Due to the fact that EAF ofI. 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 (34.0 g, 30.0 × 3.0 cm). The column was eluted with ethyl acetate:methanol in a stepwise gradient (300.0 mL 90:10; 300.0 mL 80:20; 300.0 mL 70:30; 200.0 mL 60:40; 200.0 mL 50:50; 200.0 mL 40:60; 200.0 mL 30:70; 100.0 mL 20:80; 100.0 mL 10:90). At the end, the column was washed with 300.0 mL of MeOH. The collected fraction volume was 15.0 mL and fractions with the same profile on TLC chromatograms were joined together. The fractionation of EAF yielded 8 subfractions (F1 to F8). Fractionation was repeated with another 0.60 g of EAF to obtain a higher mass of fraction. Both fractionations yielded about 500.0 mg of pure myricetin-3-O-rhamnoside in fraction 4 after TCL monitoring.Myricetin-3-O-rhamnoside: M.p.: decomposes near 200°C. UV (MeOH): λmax 258 and 353 nm; 1H NMR (DMSO-d6, 400 MHz): δ 6.20 (1H,d,J = 2.0 Hz, H-6), 6.35 (1H,d,J = 2.0 Hz, H-8), 6.89 (2H,s, H-2′e H-6′), 5.20 (1H,d,J 1.2 Hz, H-1′′), 3.98 (1H,s, H-2′′), 3.55 (1H,dd,J 1.2 e 9.3 Hz, H-3′′), 3.16 (1H,m, H-4′′), 3.37 (1H,m, H-5′′), 0.84 (3H,d,J 6.2 Hz, H-6′′); 13C NMR (DMSO-d6, 100 MHz,): δ 156.4 (C-2), 136.5 (C-3), 177.8 (C-4), 161.3 (C-5), 98.7 (C-6), 164.2 (C-7), 93.5 (C-8), 157.5 (C-9), 104,1 (C-10), 119.6 (C-1′), 107.9 (C-2′), 145.8 (C-3′), 134.3 (C-4′), 145.8 (C-5′), 107.9 (C-6′), 101.9 (C-1′′), 70.0 (C-2′′), 70.4 (C-3′′), 71.3 (C-4′′), 70.6 (C-5′′), 17.5 (C-6′′). HPLC-ESI/MS2m/z: 463.0889 ([M – H]–, C21H19O12– calc. 463.0882).


## 2.8. High Performance Liquid Chromatography–Electrospray Ionization–Tandem Mass Spectrometry (HPLC-ESI/MSn)

The assays were carried out in a Liquid Chromatography system (Agilent Infinity 1260) coupled to a High Resolution Mass Spectrometer with Quadrupole Time of Flight (QTOF) (Agilent® model 6520 B) with an Electrospray Ionization source (ESI). The chromatographic parameters were Agilent Zorbax column, 2.1 mm internal diameter, 5 cm long, 1.8μm particles, mobile phase: water acidified with formic acid (0.1%, v v−1) (A) and methanol (B), with the following solvent gradient system: 2% B (0 min), 98% B (0-15 min); 100% B (15-17 min); 2% B (17-22 min). Nitrogen (N2) 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.


## 2.9. Nuclear Magnetic Resonance (NMR)

NMR spectra were carried out in the Bruker Model Ascend™ 400 Avance III HD (9.2 Tesla) spectrometer. The samples were solubilized in deuterated dimethyl sulfoxide (DMSO-d6) and tetramethylsilane (TMS) was used as an internal standard. Analyses were performed at 400 MHz for 1H NMR and at 100 MHz for 13C NMR. The following NMR analyses were performed: 1H, 13C, DEPT-135, COSY, and HSQC.


## 2.10. 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, 5μm particles with 100 Å diameter), was used. A volume of 20 μL of methanol solution was injected at 3,000 μg mL−1 of EAF and 1,000 μg 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.


## 2.11. 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.


## 3. Results and Discussion

### 3.1. Phenolics, Proanthocyanidins, and Flavonoids Contents

Spectrophotometric results for extracts and fractions ofI. laurina leaves are shown in Table 1.Table 1
Total phenolics, proanthocyanidins, and flavonoids for extracts and fractions ofI. laurina leaves.
Samples TP (mg GAEgextract-1) P (mg CEgextract-1) F (mg QEgextract−1) EE 127.7 ± 0.1 76.7 ± 1.3a 133.1 ± 3.5 HF 35.0 ± 0.1 20.2 ± 1.9 33.6 ± 2.1a CF 82.2 ± 1.0 43.3 ± 4.5 205.9 ± 9.2 EAF 475.3 ± 1.9 68.1 ± 4.4a 359.3 ± 10.6 BF 135.9 ± 1.5 59.7 ± 4.1 24.2 ± 1.2a EE = ethanol extract; HF = hexane fraction; CF = chloroform fraction; EAF = ethyl acetate fraction; BF =n-butanol fraction; TP = total phenolics; GAE = gallic acid equivalent; P = proanthocyanidins; CE = catechin equivalent; F = flavonoids; QE = quercetin equivalent. Results are presented as mean ± standard deviation for the triplicate assays. The analyses with the same letters did not show a significant difference between the averages by 5% Tukey test.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, andn-butanol are more able to extract phenolic compounds. EE and EAF have higher levels of total phenolics when compared to those ofI. marginata, which has values of 31.63 mg and 8.37 of GAE gextract-1, respectively [36]. The methanolic extract (50%) ofI. edulispresented total phenolics of 496.5 mg of GAE gextract-1, which is higher than that of EE fromI. laurina [4]. Dias, Souza, and Rogez [6] reported values of 15.8 and 357.5 mg GAE gextract-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 forI. 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 (Al3+). 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 byI. marginata, whose value is 118 mg QE gextract−1 [38].


### 3.2. Antifungal and Cytotoxicity Activities

Values of MIC (minimum inhibitory concentration) for antifungal activity inμg mL−1 and cytotoxic activity in CC50 (cytotoxic concentration), in μg mL−1, for extract and fractions of leaves ofI. laurina are shown in Table 2.Table 2
Results of antifungal activity (expressed as MIC inμg mL−1), cytotoxic activity (expressed as CC50 in μg mL−1), and selectivity index for extract and fractions of I. laurinaleaves.
MIC ( μ g mL −1 )  Microorganisms EE HF CF EAF BF  C. albicansATCC 28366 46.8 1,500 >3,000 93.8 11.7 C. glabrataATCC 15126 11.7 187.5 >3,000 11.7 23.4 C. tropicalisATCC 13803 93.8 3,000 >3,000 93.8 46.8  CC 50 ( μ g mL −1 )  Vero cells 352 ± 5 384 ± 9 >512 >512 >512  SI  Microorganisms EE HF CF EAF BF  C. albicansATCC 28366 0.9 - 0.6 > - 0.8 > 0.7 > 1.6 C. glabrataATCC 15126 1.5 0.3 > - 0.8 > 1.6 > 1.3 C. tropicalisATCC 13803 0.6 - 0.9 > - 0.8 > 0.7 > 1.0 EE = ethanol extract; HF = hexane fraction; CP = chloroform fraction; EAF = ethyl acetate fraction; BF =n-butanol fraction. SI: selectivity index. ATCC: American Type Culture Collection.Fractions EE, EAF, and BF were the most active against the evaluated microorganism, showing MIC values lower than 100μg mL−1, as shown in Table 2. In general, the BF fraction showed good activity againstC. albicans (MIC 11.7 μg mL−1) and forC. glabrata (MIC 23.4 μg mL−1); the EE and EAF fractions were also very effective againstC. glabrata(MIC 11.7 μg mL−1); and the CF fraction had no antifungal activity at the tested concentrations (MIC above 3,000 μg mL−1). The EE of the leaves fromB. laevifolia showed good results for antifungal activity, showing MIC values of 31, 63, and 63 μg mL−1 forC. albicans, C. tropicalis,andC. glabrata, respectively [11].Regarding the cytotoxic activity, the lower the CC50 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 genusCandida; the best values were 1.5, 1.6, and 1.6, respectively. Nunes et al. [11] also found positive SI values for EE andn-butanol partition fromB. 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 forC. albicans, C. tropicalis,andC. glabrata, respectively.OtherInga species were studied regarding antifungal activity. The EE of leaves ofI. vera showed low inhibition againstC. 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 ofI. marginata leaves showed no antifungal activity againstC. albicansandC. krusei but showed activity againstC. tropicalis [40]. The promising antifungal activity ofI. 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 [41–45].


### 3.3. High Performance Liquid Chromatography–Electrospray Ionization–Tandem Mass Spectrometry (HPLC-ESI/MS2) 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 Figure1. Thirteen compounds could be identified in this fraction using this technique (Table 3 and Figure 2).Table 3
Phenolic compounds identified in EAF and subfractions fromI. laurina by HPLC-ESI/MS2.
Fractions/Subfractions t R(min) [M – H] – Exact mass Error (ppm) Fragments  m/z Molecular formula Compound References EAF 2.9 169.0154 169.0142 -7.1 125 C 7 H 6 O 5 Gallic acid (I) [12, 13] 5.4 305.0663 305.0667 1.3 261, 219, 167/165, 125 C 1 5 H 1 4 O 7 Gallocatechin (II) or Epigallocatechin (III) [14, 15] 6.9 183.0300 183.0299 -0.5 168, 124 C 8 H 8 O 5 Methyl derivative of gallic acid (IV) [12] 9.1 197.0468 197.0455 -6.6 169, 124/123 C 9 H 1 0 O 5 Ethyl gallate (V) [14, 16] 10.1 631.0956 631.0941 -2.4 479, 316, 169 C 2 8 H 2 4 O 17 Myricetin-O-(O-galloyl)-hexoside (glycoside or galactoside) (VI) [17, 18] 10.7 479.0826 479.0831 1.0 316, 178 C 2 1 H 1 9 O 13 Myricetin-3-O-galactoside (VII) [17, 18] 11.1 463.0904 463.0888 -3.5 316, 178 C 2 1 H 1 9 O 12 Myricetin-3-O-rhamnoside (VIII) [17] 12.2 615.1007 615.0992 -2.4 463, 316/317, 178 C 2 8 H 2 4 O 16 Myricetin galloyl rhamnoside (IX) [18, 19] 12.4 317.0318 317.0303 -4.7 178, 151 C 1 5 H 1 0 O 8 Myricetin (X) [13, 14, 20] 12.7 447.0955 447.0933 -4.9 300/301 C 2 1 H 2 0 O 11 Quercetin-3-O-rhamnoside (XI) [17, 18] 13.5 599.1044 599.1042 -0.3 447, 301, 169, 151 C 2 8 H 2 4 O 15 Quercetin 3-O-alpha-(2′′-galloyl)rhamnoside (XII) [21, 22] 13.8 301.0357 301.0354 -1.0 178/179, 151 C 1 5 H 1 0 O 7 Quercetin (XIII) [14] F2 10.8 505.0999 505.0988 - 2.2 463, 316, 271, 163 C 2 3 H 2 2 O 13 Myricetin-3-O-acetyl-rhamnoside (XIV) [23, 24] F5 7.0 495.0786 495.0780 - 1.2 343, 169 C 2 1 H 2 0 O 14 Digalloylquinic acid (XV) [25–27] F5 10.3 609.1482 609.1461 - 3.4 463, 316, 178 C 2 7 H 3 0 O 16 Myricetin-3-O- rhamnoside -3′-O-rhamnoside (XVI) Structure proposed by the author F6 7.7 647.0885 647.0890 0.8 495, 343, 325, 169 C 2 8 H 2 4 O 18 Trigalloylquinic acid (XVII) [26] F7 8.3 167.0355 167.0350 - 3.0 108 C 8 H 8 O 4 Vanillic acid (XVIII) [28]Figure 1
Total ion chromatogram of EAF obtained by HPLC-ESI.Figure 2
Compounds identified in the EAF.Column fractions (F1-F8) obtained from EAF were also analyzed by HPLC-ESI/MS2. 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 (compoundXIV) was identified in fraction F2 (m/z 505), digalloylquinic acid (compoundXV,m/z 495) and myricetin-3-O-rhamnose-3′-O-rhamnoside (compoundXVI,m/z 609) were identified in F5, trigalloylquinic acid (compoundXVII,m/z 647) was identified in the F6, and vanillic acid (compoundXVIII,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.Figure 3
Compounds identified in subfractions F2-F7.The ionm/z 609 (compoundXVI) forms the fragmentsm/z 463, 316, and 178 (Figure 4), which correspond to compoundVIII, which was confirmed to be myricetin-3-O-rhamnoside (Figure 2). The loss of 146 Da (rhamnosyl group) of ionm/z 609 forms the ionm/z 463, which loses a further 146 Da to form the ionm/z 316. This finding is in accordance with the structure proposed for the ionm/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 two rhamnosyl groups in positions 3 and 3′ of the aglycone was proposed: myricetin-3-O-rhamnose-3′-O-rhamnoside. No reports on the identification of compounds II, III, VI, VII, IX, XII, XIV, XV, XVI, or XVII in theInga species have been found.Figure 4
MS/MS spectrum of ionm/z 609.The FiguresS1 to S16 show mass spectrum of phenolic compounds acquired by HPLC-ESI/MS2. Figures S17 to S22 show fragmentation patterns.


### 3.4. 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 FigureS23 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 C21H20O12 and MW 464.38 gmol−1 (compoundVIII, 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.Figure5 shows the high resolution mass spectrum of F4; it is possible to verify the molecular weight of the compound [M – H]- at ionm/z 463.0889 (acquired mass). The exact mass of compoundVIII wasm/z 463.0882 (C21H19O12)-, with an error of 1.5 ppm. The peak atm/z 927.1829 corresponds to the ioncluster formation ofVIII.Figure 5
High resolution mass spectra of fraction F4.The main fragmentation of VIII is the loss of the rhamnoside radical, yielding the ion radical atm/z 316 [M- – 147]-; with lower intensity, the aglycone can be produced (M- –146]- after the loss of an unsaturated rhamnoside of mass 146 Da (Figure 6). The spectrum in Figure 5 is in good agreement with Saldanha et al. [17], where flavonoids inMyrcia 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 ofPistacia lentiscus leaves.Figure 6
MS/MS spectrum of the ionm/z 463.1H NMR spectra showed 3 different signals attributed to aromatic hydrogens, two doublets were observed at δ 6.20 and δ 6.37, both withJ = 2.0 Hz, typical of meta -couplings of carbons C-6 and C-8, respectively, in ring A. There is an intense singlet in δ 6.89 attributed to hydrogens H-2′ and H-6′ of ring B, which are equivalent. The signal at δ 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 atδ 5.2 (1H,d,J = 1.2 Hz), typical of a hydrogen bound to the anomeric carbon (δ 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 13C 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.Figure 7
Structure of myricetin-3-O-rhamnoside compound isolated from the fraction F4.Myricetin-3-O-rhamnoside inhibited the growth of all yeasts as shown in Table 2 at a concentration of 93.8 μg mL−1. This result confirms the antifungal activity for this compound. Salazar-Aranda and coworkers [48] also observed an MIC higher than 83 μg mL−1 forC. albicans andC. tropicalis, although the MIC values were higher forC. glabrata, 3.9 μg mL−1, and 7.8 μg mL−1 for clinical isolates of yeast.The isolated compound presents the same MIC of EAF for the evaluated microorganism, except forC. glabrata, which has a lower value (11.7 μg mL-). From these results, it is possible to infer that myricetin-3-O-rhamnoside can contribute markedly to the antifungal activity observed for EAF againstC. albicans andC. tropicalis, since they presented the same MIC values.


## 3.1. Phenolics, Proanthocyanidins, and Flavonoids Contents

Spectrophotometric results for extracts and fractions ofI. laurina leaves are shown in Table 1.Table 1
Total phenolics, proanthocyanidins, and flavonoids for extracts and fractions ofI. laurina leaves.
Samples TP (mg GAEgextract-1) P (mg CEgextract-1) F (mg QEgextract−1) EE 127.7 ± 0.1 76.7 ± 1.3a 133.1 ± 3.5 HF 35.0 ± 0.1 20.2 ± 1.9 33.6 ± 2.1a CF 82.2 ± 1.0 43.3 ± 4.5 205.9 ± 9.2 EAF 475.3 ± 1.9 68.1 ± 4.4a 359.3 ± 10.6 BF 135.9 ± 1.5 59.7 ± 4.1 24.2 ± 1.2a EE = ethanol extract; HF = hexane fraction; CF = chloroform fraction; EAF = ethyl acetate fraction; BF =n-butanol fraction; TP = total phenolics; GAE = gallic acid equivalent; P = proanthocyanidins; CE = catechin equivalent; F = flavonoids; QE = quercetin equivalent. Results are presented as mean ± standard deviation for the triplicate assays. The analyses with the same letters did not show a significant difference between the averages by 5% Tukey test.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, andn-butanol are more able to extract phenolic compounds. EE and EAF have higher levels of total phenolics when compared to those ofI. marginata, which has values of 31.63 mg and 8.37 of GAE gextract-1, respectively [36]. The methanolic extract (50%) ofI. edulispresented total phenolics of 496.5 mg of GAE gextract-1, which is higher than that of EE fromI. laurina [4]. Dias, Souza, and Rogez [6] reported values of 15.8 and 357.5 mg GAE gextract-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 forI. 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 (Al3+). 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 byI. marginata, whose value is 118 mg QE gextract−1 [38].


## 3.2. Antifungal and Cytotoxicity Activities

Values of MIC (minimum inhibitory concentration) for antifungal activity inμg mL−1 and cytotoxic activity in CC50 (cytotoxic concentration), in μg mL−1, for extract and fractions of leaves ofI. laurina are shown in Table 2.Table 2
Results of antifungal activity (expressed as MIC inμg mL−1), cytotoxic activity (expressed as CC50 in μg mL−1), and selectivity index for extract and fractions of I. laurinaleaves.
MIC ( μ g mL −1 )  Microorganisms EE HF CF EAF BF  C. albicansATCC 28366 46.8 1,500 >3,000 93.8 11.7 C. glabrataATCC 15126 11.7 187.5 >3,000 11.7 23.4 C. tropicalisATCC 13803 93.8 3,000 >3,000 93.8 46.8  CC 50 ( μ g mL −1 )  Vero cells 352 ± 5 384 ± 9 >512 >512 >512  SI  Microorganisms EE HF CF EAF BF  C. albicansATCC 28366 0.9 - 0.6 > - 0.8 > 0.7 > 1.6 C. glabrataATCC 15126 1.5 0.3 > - 0.8 > 1.6 > 1.3 C. tropicalisATCC 13803 0.6 - 0.9 > - 0.8 > 0.7 > 1.0 EE = ethanol extract; HF = hexane fraction; CP = chloroform fraction; EAF = ethyl acetate fraction; BF =n-butanol fraction. SI: selectivity index. ATCC: American Type Culture Collection.Fractions EE, EAF, and BF were the most active against the evaluated microorganism, showing MIC values lower than 100μg mL−1, as shown in Table 2. In general, the BF fraction showed good activity againstC. albicans (MIC 11.7 μg mL−1) and forC. glabrata (MIC 23.4 μg mL−1); the EE and EAF fractions were also very effective againstC. glabrata(MIC 11.7 μg mL−1); and the CF fraction had no antifungal activity at the tested concentrations (MIC above 3,000 μg mL−1). The EE of the leaves fromB. laevifolia showed good results for antifungal activity, showing MIC values of 31, 63, and 63 μg mL−1 forC. albicans, C. tropicalis,andC. glabrata, respectively [11].Regarding the cytotoxic activity, the lower the CC50 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 genusCandida; the best values were 1.5, 1.6, and 1.6, respectively. Nunes et al. [11] also found positive SI values for EE andn-butanol partition fromB. 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 forC. albicans, C. tropicalis,andC. glabrata, respectively.OtherInga species were studied regarding antifungal activity. The EE of leaves ofI. vera showed low inhibition againstC. 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 ofI. marginata leaves showed no antifungal activity againstC. albicansandC. krusei but showed activity againstC. tropicalis [40]. The promising antifungal activity ofI. 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 [41–45].


## 3.3. High Performance Liquid Chromatography–Electrospray Ionization–Tandem Mass Spectrometry (HPLC-ESI/MS2) 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 Figure1. Thirteen compounds could be identified in this fraction using this technique (Table 3 and Figure 2).Table 3
Phenolic compounds identified in EAF and subfractions fromI. laurina by HPLC-ESI/MS2.
Fractions/Subfractions t R(min) [M – H] – Exact mass Error (ppm) Fragments  m/z Molecular formula Compound References EAF 2.9 169.0154 169.0142 -7.1 125 C 7 H 6 O 5 Gallic acid (I) [12, 13] 5.4 305.0663 305.0667 1.3 261, 219, 167/165, 125 C 1 5 H 1 4 O 7 Gallocatechin (II) or Epigallocatechin (III) [14, 15] 6.9 183.0300 183.0299 -0.5 168, 124 C 8 H 8 O 5 Methyl derivative of gallic acid (IV) [12] 9.1 197.0468 197.0455 -6.6 169, 124/123 C 9 H 1 0 O 5 Ethyl gallate (V) [14, 16] 10.1 631.0956 631.0941 -2.4 479, 316, 169 C 2 8 H 2 4 O 17 Myricetin-O-(O-galloyl)-hexoside (glycoside or galactoside) (VI) [17, 18] 10.7 479.0826 479.0831 1.0 316, 178 C 2 1 H 1 9 O 13 Myricetin-3-O-galactoside (VII) [17, 18] 11.1 463.0904 463.0888 -3.5 316, 178 C 2 1 H 1 9 O 12 Myricetin-3-O-rhamnoside (VIII) [17] 12.2 615.1007 615.0992 -2.4 463, 316/317, 178 C 2 8 H 2 4 O 16 Myricetin galloyl rhamnoside (IX) [18, 19] 12.4 317.0318 317.0303 -4.7 178, 151 C 1 5 H 1 0 O 8 Myricetin (X) [13, 14, 20] 12.7 447.0955 447.0933 -4.9 300/301 C 2 1 H 2 0 O 11 Quercetin-3-O-rhamnoside (XI) [17, 18] 13.5 599.1044 599.1042 -0.3 447, 301, 169, 151 C 2 8 H 2 4 O 15 Quercetin 3-O-alpha-(2′′-galloyl)rhamnoside (XII) [21, 22] 13.8 301.0357 301.0354 -1.0 178/179, 151 C 1 5 H 1 0 O 7 Quercetin (XIII) [14] F2 10.8 505.0999 505.0988 - 2.2 463, 316, 271, 163 C 2 3 H 2 2 O 13 Myricetin-3-O-acetyl-rhamnoside (XIV) [23, 24] F5 7.0 495.0786 495.0780 - 1.2 343, 169 C 2 1 H 2 0 O 14 Digalloylquinic acid (XV) [25–27] F5 10.3 609.1482 609.1461 - 3.4 463, 316, 178 C 2 7 H 3 0 O 16 Myricetin-3-O- rhamnoside -3′-O-rhamnoside (XVI) Structure proposed by the author F6 7.7 647.0885 647.0890 0.8 495, 343, 325, 169 C 2 8 H 2 4 O 18 Trigalloylquinic acid (XVII) [26] F7 8.3 167.0355 167.0350 - 3.0 108 C 8 H 8 O 4 Vanillic acid (XVIII) [28]Figure 1
Total ion chromatogram of EAF obtained by HPLC-ESI.Figure 2
Compounds identified in the EAF.Column fractions (F1-F8) obtained from EAF were also analyzed by HPLC-ESI/MS2. 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 (compoundXIV) was identified in fraction F2 (m/z 505), digalloylquinic acid (compoundXV,m/z 495) and myricetin-3-O-rhamnose-3′-O-rhamnoside (compoundXVI,m/z 609) were identified in F5, trigalloylquinic acid (compoundXVII,m/z 647) was identified in the F6, and vanillic acid (compoundXVIII,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.Figure 3
Compounds identified in subfractions F2-F7.The ionm/z 609 (compoundXVI) forms the fragmentsm/z 463, 316, and 178 (Figure 4), which correspond to compoundVIII, which was confirmed to be myricetin-3-O-rhamnoside (Figure 2). The loss of 146 Da (rhamnosyl group) of ionm/z 609 forms the ionm/z 463, which loses a further 146 Da to form the ionm/z 316. This finding is in accordance with the structure proposed for the ionm/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 two rhamnosyl groups in positions 3 and 3′ of the aglycone was proposed: myricetin-3-O-rhamnose-3′-O-rhamnoside. No reports on the identification of compounds II, III, VI, VII, IX, XII, XIV, XV, XVI, or XVII in theInga species have been found.Figure 4
MS/MS spectrum of ionm/z 609.The FiguresS1 to S16 show mass spectrum of phenolic compounds acquired by HPLC-ESI/MS2. Figures S17 to S22 show fragmentation patterns.


## 3.4. 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 FigureS23 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 C21H20O12 and MW 464.38 gmol−1 (compoundVIII, 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.Figure5 shows the high resolution mass spectrum of F4; it is possible to verify the molecular weight of the compound [M – H]- at ionm/z 463.0889 (acquired mass). The exact mass of compoundVIII wasm/z 463.0882 (C21H19O12)-, with an error of 1.5 ppm. The peak atm/z 927.1829 corresponds to the ioncluster formation ofVIII.Figure 5
High resolution mass spectra of fraction F4.The main fragmentation of VIII is the loss of the rhamnoside radical, yielding the ion radical atm/z 316 [M- – 147]-; with lower intensity, the aglycone can be produced (M- –146]- after the loss of an unsaturated rhamnoside of mass 146 Da (Figure 6). The spectrum in Figure 5 is in good agreement with Saldanha et al. [17], where flavonoids inMyrcia 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 ofPistacia lentiscus leaves.Figure 6
MS/MS spectrum of the ionm/z 463.1H NMR spectra showed 3 different signals attributed to aromatic hydrogens, two doublets were observed at δ 6.20 and δ 6.37, both withJ = 2.0 Hz, typical of meta -couplings of carbons C-6 and C-8, respectively, in ring A. There is an intense singlet in δ 6.89 attributed to hydrogens H-2′ and H-6′ of ring B, which are equivalent. The signal at δ 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 atδ 5.2 (1H,d,J = 1.2 Hz), typical of a hydrogen bound to the anomeric carbon (δ 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 13C 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.Figure 7
Structure of myricetin-3-O-rhamnoside compound isolated from the fraction F4.Myricetin-3-O-rhamnoside inhibited the growth of all yeasts as shown in Table 2 at a concentration of 93.8 μg mL−1. This result confirms the antifungal activity for this compound. Salazar-Aranda and coworkers [48] also observed an MIC higher than 83 μg mL−1 forC. albicans andC. tropicalis, although the MIC values were higher forC. glabrata, 3.9 μg mL−1, and 7.8 μg mL−1 for clinical isolates of yeast.The isolated compound presents the same MIC of EAF for the evaluated microorganism, except forC. glabrata, which has a lower value (11.7 μg mL-). From these results, it is possible to infer that myricetin-3-O-rhamnoside can contribute markedly to the antifungal activity observed for EAF againstC. albicans andC. tropicalis, since they presented the same MIC values.


## 4. Conclusions

The EE from the leaves ofInga 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 gextract-1) and flavonoids (359.3 ± 10.6 mg QE gextract-1) and was active against fungus from theCandida genus. The best MIC results found forC. albicans, C. glabrata,andC. tropicalis were 11.7, 11.7, and 46.8 μg 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 makeI. laurina a promising plant for advanced antifungal studies.



---

*Source: 9423658-2019-02-03.xml*