Phytochemistry Letters 40 (2020) 109–120

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Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol

Antifungal and cytotoxicity activities and new proanthocyanidins isolated

from the barks of Inga laurina (Sw.) Willd
Carla de Moura Martins a, Sérgio A.L. de Morais b, Mário M. Martins b, Luís C.S. Cunha c,
Cláudio V. da Silva d, Thaise Lara Teixeira d, Mariana B. Santiago e, Francisco J.T. de Aquino b,
Evandro A. Nascimento b, Roberto Chang b, Carlos H.G. Martins e, Alberto de Oliveira, Ph.D. b, *
a
Chemistry Nucleus, Goiano Federal Institute-Campus Morrinhos, 75650-000, Morrinhos, GO, Brazil
b
Natural Products Research Nucleus (NuPPeN), Federal University of Uberlândia, 38400-902, Uberlândia, MG, Brazil
c
Bioprospecting Center for Natural Products (NuBiProN), Chemistry Department, Federal Institute of Triângulo Mineiro, Uberaba, Brazil
d
Institute of Biomedical Sciences-Laboratory of Trypanosomatids, Federal University of Uberlândia, 38405-320, Uberlândia, MG, Brazil
e
Institute of Biomedical Sciences, Laboratory of Antimicrobial Testing, Federal University of Uberlândia, 38405-320, Uberlândia, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: The Inga genus comprises approximately 300 species that can be found from Mexico to northern Argentina. In
Inga laurina folk medicine, Inga species are used to treat various diseases. The species Inga laurina is widely found in the
Proanthocyanidins Brazilian flora; however, there are few studies about its biological activity and chemical composition. The main
Antifungal activity
purpose of this study was to identify and isolate the chemical constituents of Inga laurina barks and to evaluate
Cytotoxicity
Total phenolics
the antifungal and cytotoxic activities. The total content of phenolics, proanthocyanidins, and flavonoids from
the barks of Inga laurina were performed by spectrophotometric methods and the ethyl acetate (EAF) and n-
butanol (BF) fractions showed the best results. Eleven compounds were identified in EAF by HPLC-ESI(− )-MS/
MS, which showed good antifungal activity with MIC values of 23.4 and 46.8 μg mL− 1, evaluated by broth
microdilution method. Five new compounds of the genus Inga were isolated for the first time. Three of these
compounds were isolated and reported on the literature for the first time: a proanthocyanidin B-type, galloca­
techin-(4α→8)-4’-O-methylgallocatechin (XI) and two proanthocyanidins A-type, gallocatechin-(2→O→7,4→8)-
4’-O-methylgallocatechin (XII) and gallocatechin-3-O-galloyl-(2→O→7,4→8)-4’-O-methylgallocatechin (XIII).
The chemical study of the plant bark showed that this species is rich in phenolic compounds and it has great
potential for the discovery of new bioactive compounds.

1. Introduction
The Fabaceae family, to which the Inga genus belongs, is the third
largest family of Angiosperms, behind Asteraceae and Orchidaceae. The
plants of this family are widely distributed in Brazil and can be found in
several states, such as Amazonas, Ceará, Goiás, Minas Gerais and Par­
aná. This family has 222 genus and 2807 species present in the country
and 15 genus and 1508 species are endemic (Pereira et al., 2019).
Among the several species found in the Cerrado, the genus Inga belongs
to this family and has several species that present biological activities.
Some species of Inga have already been studied and the chemical and
biological review show that this genus is promising in the search for new
active substances for drug manufacturing (Álvarez et al., 1998; Dias
et al., 2010; Lokvam et al., 2006; Pinto et al., 2010; Pistelli et al., 2009;
Silva et al., 2007; Souza et al., 2007; Vivot et al., 2001).
In previous studies with Inga genus, the phenolic compounds were
the main substances identified. In the leaves of I. umbellifera, flavonoids
and proanthocyanidins derived from catechin were isolated (Lokvam
et al., 2004). From I. goldmani, a trimeric proanthocyanidin and
trans-4-methoxypipecolic acid was isolated from I. paterno (Lokvam and
Kursar, 2005; Morton et al., 1991). From the leaves and barks of I. edulis
metabolites derived from catechin, myricetin, quercetin, and terpenes
derived from stigmasterol were isolated (Tchuenmogne et al., 2013).
The phenolic compounds are a large class of metabolites found in
plants and they have a broad structural diversity. More than 8000
chemical structures are known, from simple compounds like phenolic
acids to more complex compounds like tannins. They are involved in
plant defense mechanism, protecting them from UV radiation and

* Corresponding author.
E-mail address: alberto@ufu.br (A. de Oliveira).

https://doi.org/10.1016/j.phytol.2020.10.001
Received 29 May 2020; Received in revised form 18 August 2020; Accepted 2 October 2020
Available online 20 October 2020
1874-3900/© 2020 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
C. de Moura Martins et al. Phytochemistry Letters 40 (2020) 109–120

pathogens (Dai and Mumper, 2010). The barks are organs that have been
extensively studied due the medicinal properties of phenolic com­
pounds, several biological effects have already been studied as antioxi­
dant, anti-inflammatory, antibacterial and antiviral activities (Tanase
et al., 2019a, Tanase et al., 2019). Flavonoids, phenolic acids and tan­
nins are examples of substances that may retard the oxidative process of
reactive oxygen species (Laguerre et al., 2007), have antimicrobial
(Allaker and Douglas, 2009) and antifungal activities (Martins et al.,
2015; Teodoro et al., 2015). This demonstrates the great diversity of
studies that can be carried out with this class of compounds and also the
importance of conducting research to discover new compounds.
The plant Inga laurina is widely found in Brazilian flora and there are
few studies on biological activity, identification or isolated compounds
of it (Cruz et al., 2016; Furtado et al., 2014; Lokvam et al., 2007; Macedo
et al., 2011; Macedo et al., 2007; Macedo et al., 2016; Marqui, 2011;
Ramos et al., 2012). Recently, the authors reported the analysis of ex­
tracts and fractions of leaves of I. laurina. The results of the antifungal
and cytotoxic activities of the samples were promising. Seventeen
compounds were identified by (HPLC-ESI(–)-MS/MS) in the subfractions
of ethyl acetate fraction leaves and the flavonol glycoside
miricetin-3-O-ramnoside was isolated for the first time in the I. laurina
(Martins et al., 2019). The aim of this work was to evaluate the anti­
fungal and cytotoxic activities of the ethanolic extract (EE) and fractions
of I. laurina barks and identify the phenolic compounds in the samples by
HPLC-ESI(–)-MS/MS and 1H/13C NMR techniques.
2. Results and discussion
2.1. Total phenolic, proanthocyanidin and flavonoid contents
The results of total phenolic contents of extracts and fractions of
I. laurina barks are presented in Table 1.
The EE, EAF, BF and MF samples presented the highest contents of
total phenolics and proanthocyanidin compared to the flavonoid con­
tents, while the MF fraction was the best sample to extracted flavonoids.
These results can be explained by the higher polarity of the solvent
which solubilizes higher amount of polar substances. The total pheno­
lics, proanthocyanidins and flavonoids contents of I. laurina leaves have
been recently reported by Martins et al. (2019). Comparing the results of
total phenolics and proanthocyanidins of I. laurina leaves and barks, it
was observed that the bark of this plant has higher contents of phenolic
compounds and proanthocyanidins. On the other hand, the leaves have
higher contents of flavonoids.
shown in Table 2. The EE, EAF-II, BF and MF were the samples with the
best results for antifungal activity, once they inhibited fungal growth at
concentrations below 100 μg mL− 1 (Table 2). The subfraction EAF-I
showed good activity only for C. glabrata, with MIC of 23.4 μg mL− 1.
Comparing the extracts and fractions of leaves and barks, the good
antifungal activity of I. laurina may be related to the presence of
phenolic compounds in the samples (Martins et al., 2019). The extracts
and fractions of barks and leaves of the plant presented high amount of
total phenolics and proanthocyanidins and these classes of compounds
are reported in the literature as potential antifungal agents (Cos et al.,
2004; Daglia, 2012; Martins et al., 2015; Sanches et al., 2005; Tanase
et al., 2019a; Teodoro et al., 2015). The EAF-II was the most selective
sample to inhibit fungal growth because it presented the most positive
value for SI (SI > 1.0).
2.3. High performance liquid chromatography – electrospray ionization –
tandem mass spectrometry (HPLC-ESI(− )-MS/MS) in negative mode
analysis
The EAF-I and EAF-II showed better chromatographic separation by
TLC and the EAF-II presented good results for antifungal activity and the
best selectivity indexes. Therefore, these samples were chosen for
identification and isolation of bioactive substances. The total ion chro­
matograms of the fractions are shown in Figures S1 and S2 of supple­
mentary material.
Thirteen compounds were identified in the EAF-I and EAF-II from the
MS/MS spectra and by comparison with literature data (Table 3).
The identification of the compounds by HPLC-ESI(− )-MS/MS of the
EAF corroborated the results of proanthocyanidins and flavonoids con­
tents. In the bark, flavonoids derived from epi or gallocatechin
belonging to the class of flavan-3-ols were identified, thus presenting
negative tests for the determination of flavonoids content, due to the
absence of carbonyl in C4 of ring C. Compounds I, II and III were also
identified in the EAF of the leaves (Martins et al., 2019). Through
HPLC-ESI(− )-MS/MS it is not possible to distinguish, e.g., gallocatechin
from epigallocatechin; therefore, these compounds are assigned to (epi)
gallocatechin.
Table 2
Results of antifungal activity (expressed as MIC in μg mL− 1), and cytotoxic ac­
tivity (expressed as CC50, in μg mL− 1), and selectivity index for extract and
fractions of I. laurina barks.
MIC (μg mL− 1)

Microorganisms EE CF EAF-I EAF- BF MF

2.2. Antifungal and cytotoxicity activities II

C. albicans ATCC 46.8 1,000 500 23.4 46.8 11.7

The results of MIC (Minimum Inhibitory Concentration) for anti­ 28366
fungal activity in μg mL− 1, and cytotoxic activity in CC50 (cytotoxic C. glabrata ATCC 48.8 >1,000 23.4 23.4 46.8 11.7
concentration), in μg mL− 1 for extract and fractions of I. laurina barks are 15126
C. tropicalis ATCC 24.4 1000 1,000 46.8 46.8 23.4
13803
Table 1
Total phenolic, proanthocyanidin and flavonoid contents for extracts and frac­ CC50 (μg mL¡1)
tions of I. laurina barks. Vero cells 152 ± 343 ± 374 ± >512 163 ± 101 ±
Samples 1
TP (mg GAE g−extract ) 1
P (mg CE g−extract ) 1
F (mg QE g−extract ) 7 22 12 21 21

EE 384.5 ± 7.8a 449.6 ± 12.6a 5.5 ± 0.1 SI

CF 26.0 ± 4.0 27.6 ± 1.2 6.5 ± 0.2a Microorganisms EE CF EAF-I EAF- BF MF
EAF-I 291.5 ± 8.0 262.2 ± 8.2 4.2 ± 0.3b II
EAF-II 477.9 ± 9.3 467.2 ± 12.4a,b 4.4 ± 0.1b C. albicans ATCC 0.5 − 0.5 − 0.1 > 1.3 0.5 0.9
BF 400.6 ± 3.2 492.4 ± 11.9b 6.8 ± 0.5a 28366
MF 346.0 ± 1.8 a 341.8 ± 15.8 8.2 ± 0.5 C. glabrata ATCC 0.5 > − 0.5 1.2 > 1.3 0.5 0.9
EE = ethanol extract; CF = chloroform fraction; EAF = ethyl acetate fraction; BF 15126
C. tropicalis ATCC 0.8 − 0.5 − 0.4 > 1.0 0.5 0.6
= n-butanol fraction; MF = methanol fraction; TP = total phenolics; GAE = gallic
13803
acid equivalent; P = proanthocyanidins; CE = catechin equivalent; F = flavo­
noids; QE = quercetin equivalent. Results are presented as mean ± standard EE = ethanol extract; CF = chloroform fraction; EAF = ethyl acetate fraction; BF
deviation for the triplicate assays. The analysis with the same letters did not = n-butanol fraction; MF = methanol fraction. SI: selectivity index. ATCC =
show significant difference between the averages by the Tukey test at 5%. American Type Culture Collection.

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C. de Moura Martins et al. Phytochemistry Letters 40 (2020) 109–120

Table 3
Phenolic compounds identified in EAF-I and EAF-II from I. laurina barks by HPLC-ESI(− )-MS/MS.
Fractions tR [M − H]– Exact Δ Fragments Molecular Compound References
mass (ppm) m/z formula

(Abdel-Hameed et al., 2013;

2.7 169.0145 169.0142 –1.8 125 C7H6O5 gallic acid (I) Fracassetti et al., 2013; Kumar and
Kumar, 2017; Martins et al., 2019)
261, 219, 167, (Martins et al., 2019; Mena et al.,
5.2 305.0645 305.0667 7.2 C15H14O7 (epi)gallocatechin (II)
125 2012; Sun et al., 2007)
(Garcia et al., 1993; Henriques et al.,
6.4 319.0829 319.0823 –1.9 166, 137/138 C16H16O7 4’-O-methyl-(epi)gallocatechin (III) 2016; Maregesi et al., 2010;
Rodrigues, 2011)
(Dou et al., 2007; Henriques et al.,
7.7 457.0772 457.0776 0.9 305, 169 C22H18O11 (epi)gallocatechin-3-O-gallate (IV)
2016; Savić et al., 2014)
EAF-I (Martins et al., 2019; Savić et al.,
8.4 197.0457 197.0455 –1,0 169, 124 C9H10O5 ethyl gallate (V) 2014; Sun et al., 2007; Wyrepkowski
et al., 2014)
289, 245, 169,
9.1 441.0827 441.0827 0 C22H18O10 (epi)catechin-3-O-gallate (VI) (Dou et al., 2007; Savić et al., 2014)
125
319, 183, 169, 4’-O-methyl-(epi)gallocatechin-3-O-gallate
9.3 471.0922 471.0933 2.3 C23H12O11 (Henriques et al., 2016)
125 (VII)
[(epi)gallocatechin-3-O-(3’’,4’’-O-
305, 287, 197, (Chiu and Lin, 2005; Kirita et al.,
10.3 485.1083 485.1089 1.2 C24H22O11 dimethyl or (epi)gallocatechin-3-O-
161, 125 2015)
(3’’,5’’-O-dimethyl) gallate] (VIII)
13.6 163.0398 163.0401 1.8 119 C9H8O3 p-coumaric acid (IX) (Sun et al., 2007)
261, 219, 167,
1.1 305.0662 305.0667 1.6 C15H14O7 (epi)gallocatechin (II) (Mena et al., 2012; Sun et al., 2007)
125
(Abdel-Hameed et al., 2013;
2.7 169.0142 169.0142 0 125 C7H6O5 gallic acid (I) Fracassetti et al., 2013; Kumar and
Kumar, 2017; Martins et al., 2019)
483, 441, 423, proanthocyanidin B4 (X) (Dou et al., 2007; Maggi et al., 2016;
3.3 609.1242 609.1250 1.3 C30H26O14
305, 177, 125 [dimer: two units of (epi)gallocatechin] Riehle et al., 2013)
497, 455, 437, dimer: (epi)gallocatechin and 4’-O-methyl-
5.1 623.1402 623.1406 0.6 C31H30O14 (Henriques et al., 2016)
303, 125 (epi)gallocatechin (XI)
(Garcia et al., 1993; Henriques et al.,
181, 166, 137,
6.8 319.0830 319.0823 –2.2 C16H16O7 4’-O-methyl-(epi)gallocatechin (III) 2016; Maregesi et al., 2010;
125
Rodrigues, 2011)
EAF-II
(Dou et al., 2007; Henriques et al.,
7.6 457.0789 457.0776 –2.8 305, 169 C22H18O11 (epi)gallocatechin-3-O-gallate (IV)
2016)
125, 301, 437, gallocatechin-(2→O→7,4→8)-4′ -O- (Esatbeyoglu et al., 2013; Zang et al.,
8.1 621.1208 621.1250 6.8 C31H26O14
495, 603 methylgallocatechin (XII) 2013)
(Martins et al., 2019; Sun et al., 2007;
8.4 197.0463 197.0455 –4.1 169, 124 C9H10O5 ethyl gallate (V)
Wyrepkowski et al., 2014)
289, 245, 169,
9.1 441.0848 441.0827 –4.8 C22H18O10 (epi)catechin-3-O-gallate (VI) (Dou et al., 2007)
125
125, 301, 437, gallocatechin-3-O-galloyl-(2→O→7, 4→8)- (Esatbeyoglu et al., 2013; Zang et al.,
9.2 773.1270 773.1359 11.5 C38H30O18
495, 603, 621 4′ -O-methylgallocatechin (XIII) 2013)
319, 183, 169, 4’-O-methyl-(epi)gallocatechin-3-O-gallate
9.3 471.0955 471.0933 –4.7 C23H12O11 (Henriques et al., 2016)
125 (VII)

2.4. Isolation and identification of the compounds II, III, XI, XII and XIII
Compounds II, XI, XII were isolated from subfractions EAF-II-4.
Compound XIII was isolated from subfraction EAF-II-5. Compound III
was isolated from the fraction EAF-I-2 (Fig. 1).
There are no reports in the literature of these compounds in Inga
species, so this is the first time these compounds have been identified
and/or isolated in the genus Inga. Searching the compounds XI, XII and
XIII in the literature, no data on these compounds (molecular formula,
molecular weight, nomenclature, etc.) were found; therefore, these
compounds are reported herein for the first time.
Compound III was identified as 4′ -O-methylgalocatechin, molecular
mass 320.09 g mol− 1 (C16H16O7) (Fig. 1) and was characterized by NMR
and ESI(− )-MS/MS. The mass of the molecular ion [M − H]– m/z
319.0810 (acquired mass) was obtained from the high-resolution mass
spectrum. The exact mass was m/z 319.0823 (Δ –4.1). The peaks at m/z
639.1690 and m/z 959.2526 correspond to the ion cluster formation of
this compound. The high-resolution mass spectra, the major fragments
of the ion m/z 319, and the proposed mechanism fragmentation for this
compound are shown in Figures S3–S5, respectively, of the Supple­
mentary Material.
In the 1H and 13C NMR spectra the chemical shifts (δ) of the
hydrogens and carbons of this compound were attributed. The position
of the methyl group was confirmed by the HMBC contour map analysis
in which the hydrogen of the methoxy group at δ 3.65 (singlet) was
found to correlate with C-4’B at δ 135.0. The doublet at δ 4.44 (1H, J2-3
= 7.2 Hz) was assigned to H-2C and the multiplet at δ 3.78 (1 H) to H-3C.
The calculated value of the coupling constant was characteristic for 2.3-
trans configurations, which proves that this compound is a derivative of
galocatechin and not epigalocatechin (2.3-cis configurations) (Fig. 1)
(Esatbeyoglu et al., 2013; Schmidt et al., 2011). See Figures S6-S11 and
Tables S1-S2 of Supplementary Material for NMR spectra and chemical
shifts, respectively, of compound III.
From the EAF-II-4 by semi preparative HPLC, compounds with
retention time at 13 min (compound II, 5.9 mg), 14 min (compound XI,
5.2 mg) and 24 min (compound XII, 3.8 mg) were isolated (Fig. 1). In the
UV–vis spectrum of the EAF-II-4 and EAF-II-5, a band in the region at
approximately 271 nm can be observed, suggesting that the compounds
of these fractions also belong to flavonoid class such as compound III.
The chromatograms of semipreparative HPLC of EAF-II-4 and EAF-II-5
can be seen in Figures S12-S13 of the Supplementary Material.
Compound II was identified as gallocatechin with a molecular mass
of 306.07 g mol− 1 (C15H14O7). The high resolution mass spectrum of this
compound, the proposed fragmentation and the 1H and 13C NMR spectra

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C. de Moura Martins et al.
Fig. 1. Chemical structure of the compounds isolated from EAF-I-2, EAF-II-4 and EAF-II5.
are well known in the literature and are presented in the Supplemental
Material (Figures S14–S21 and Tables S3 and S4) (Mena et al., 2012; Sun
et al., 2007). From the 1H NMR spectrum the doublet at δ 4.42 (1H, J =
7.0 Hz) was assigned to H-2C and the multiplet at δ 3.78 (1 H) to H-3C.
The value of the coupling constant for H-2C and H-3C is characteristic of
hydrogens with 2,3-trans configuration, which proves that this com­
pound is galocatechin and not epigallocatechin (Esatbeyoglu et al.,
2013).
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Phytochemistry Letters 40 (2020) 109–120
Compound XI was identified as a B-type proanthocyanidin, gallo­
catechin-(4α→8)-4′ -O-methylgallocatechin, with a molecular mass of
624.15 g mol− 1 (C31H28O14). The mass of the molecular ion [M − H]–
was m/z 623.1370 (acquired mass) and the exact mass was m/z
623.1406 (Δ 5.8 ppm). The mass spectrum and sequential mass spec­
trum of the ion m/z 623 are described in the Supplementary Material
(Figures S22 and S23). The proposed ESI(–)-MS/MS fragmentation
mechanism for this compound is shown in Fig. 2. The formation of the
C. de Moura Martins et al.
Fig. 2. Proposed ESI(–)-MS/MS fragmentation mechanism of gallocatechin-(4α→8)-4′ -O-methylgallocatechin (XI).
ions m/z 455 [M – H – 168]– and m/z 167 [M – H – 456]– occurs through
the retro Diels-Alder (RDA) mechanism in the ring C of the unit I of the
proanthocyanidin. The formation of the ion m/z 437 occurs from the
elimination of a molecule of water from the ion m/z 455. From the
quinone methide mechanism (QM) of unit I occurs the formation of the
ions m/z 303 [M – H – 320]– and m/z 319 [M – H – 304]– and through the
QM mechanism and heterocyclic ring cleavage (HRC) of the ion m/z 623
occurs the formation of the ions m/z 125 [M – H – 498]– and m/z 497 [M
– H – 126]– (Demarque et al., 2016; Rodrigues et al., 2007).
No data were found in the literature of NMR analysis for this com­
pound. Therefore, NMR spectra of compound XI were compared with
(–)-galocatechin-(4α→8)-(–)-epigalocatechin and epigalocatechin-
(4β→8)-4′ -O-methylgalocatechin (Janecki and Kolodziej, 2010; Schmidt
et al., 2011). These proanthocyanidins have similar structures with
compound XI. In the 1H NMR spectrum there are five signals related to
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Phytochemistry Letters 40 (2020) 109–120
aromatic hydrogens in the upper ring; the two doubles at δ 5.65 and 5.59
(1H, J = 2.0 Hz) were assigned to H-6A and H-8A, respectively, con­
firming the coupling at meta position in ring A. The singlet at δ 6.35 (2H)
was assigned to H-2’B and H-6’B and indicates that ring B is trisubsti­
tuted. Therefore, the upper ring is an epigallocatechin or gallocatechin
unit. The singlet at δ 5.84 (1H) was assigned to H-6D and the singlet at δ
6.41 (2H) was assigned to H-2’E and H-6’E and indicates that ring B is
trisubstituted and therefore the lower ring is also an epi- or galocatechin
unit. The position of the methoxy group was confirmed by analysis of the
HMBC contour map, where the hydrogens at δ 3.67 (CH3O-4’E) correlate
with C-4’E at δ 134.9. Confirmation of the bond between C-4C and C-8D
was made by analysis of HMBC contour map, in which the correlation
was observed for H-4C with C-7D and C-8D. The orientation of the fla­
vanil unit at C-4C as α was confirmed by assigning the chemical shift of
C-2C with δC in 82.6. If the orientation was β this chemical shift would be
C. de Moura Martins et al.
around 77.0 (Esatbeyoglu et al., 2013; Fletcher et al., 1977; Schmidt
et al., 2011). The constant coupling of H-3C and H-4C with J3,4 = 8.8 Hz
also confirms an α orientation. The doublet at δ 4.06 (1H, J = 9.3 Hz)
was assigned to H-2C and the triplet at δ 4.24 (1H, J = 6.9 and 8.8 Hz), to
H-3C. The doublet at δ 4.28 (1H, J = 8.2 Hz) was assigned to H-2 F and
the multiplet at δ 3.79 (1 H), to H-3 F. The values of J2-3 with high values
for H-2 and H-3 in the upper and lower structure are characteristic of
hydrogens with 2.3-trans configuration, indicating that the compound is
a gallocatechin dimer (Haslam, 1998).
Epigallocatechin-(4β→8)-4’-O-methylgallocatechin isolated from
the ethanolic extract of Parapiptadenia rigida bark has been reported in
the literature (Schmidt et al., 2011). A proanthocyanidin with a gallo­
catechin in the upper unit, interconnected via 4Cα-8D to a methyl­
gallocatechin unit, to the best of our knowledge, has not been reported
(see Figures S24–S29 and Tables S5–S6 in the Supplementary Material
for NMR spectra and chemical shifts, respectively, of compound XI).
Compound XII was identified as gallocatechin-(2→O→7,4→8)-4’-O-
methylgallocatechin (XII) a A-type proanthocyanidin. This compound
has molecular mass 622.13 g mol− 1 (C31H26O14). The mass of the mo­
lecular ion [M − H]– was m/z 621.1208 (acquired mass) and the exact
mass was m/z 621.1250 (Δ 6.8 ppm). The mass spectrum and sequential
mass spectrum of the ion m/z 621 are described in Figures S30 and S31
of the Supplemental Material, respectively. The proposed ESI(–)-MS/MS
fragmentation mechanism for compound XII is shown in Fig. 3.
Through the heterocyclic cleavage mechanism on ring C of the
proanthocyanidin occurs the formation of ions m/z 495 [M – H – 126]–
and m/z 125 [M – H – 496]–. From the QM mechanism on ring A occurs
the formation of ions m/z 301 [M – H – 320]– and m/z 319 [M – H –
302]– (Demarque et al., 2016; Rodrigues et al., 2007) characteristic of an
Fig. 3. Proposed ESI(–)-MS/MS fragmentation mechanism of gallocatechin-(2→O→7,4→8)-4′ -O-methylgallocatechin (XII).
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Phytochemistry Letters 40 (2020) 109–120
epi or gallocatechin and an epi or methylgallocatechin.
No NMR results were found in the literature for this compound. So
the NMR signals of compound XII were compared with those of
(–)-epigallocatechin-(2β→O7,4β→8)-(+)-catechin and (–)-epi­
gallocatechin-(2β→O→7,4β→8)-(–)-gallocatechin, A-type proanthocya­
nidins that have similar structures to compound XII (Esatbeyoglu et al.,
2013; Schmidt et al., 2011; Zang et al., 2013). B-type proanthocyanidins
are interconnected via a single 4C-8D or 4C-6D bond, while A-type
proanthocyanidins are interconnected by an additional ether-type bond
between 2C-O-7D or 2C-O-5D (Esatbeyoglu et al., 2013). The doublet at
δ 3.78 (1H, J = 3.4 Hz) was assigned to H-3C and the doublet at δ 4.20
(1H, J = 3.0 Hz) to H-4C. The relatively low values of coupling constants
for H-3C to H-4C interactions in ring C are characteristic of A-type
proanthocyanidins (Esatbeyoglu et al., 2013; Foo et al., 2000; Jacques
et al., 1974). The two doublets at δ 5.88 and 5.85 (1H, J = 1.9 Hz) were
assigned to H-6A and H-8A, which are coupled at meta position in the
aromatic ring.
By NOE interactions in the NOESY contour map, the H-8A (δ 6.02)
correlates with H-2′ B and H-6′ B and these correlations confirm the meta
substitution in ring A (Fig. 4) (Esatbeyoglu et al., 2013). The singlet at δ
6.52 (H-2′ B and H-6′ B) indicates that ring B is trisubstituted. Therefore,
the upper ring is an epi or gallocatechin unit. The assignments of the
2C-O-7D and 4C-8D interflavanoid bonds were determined by NOE in­
teractions and HMBC correlations. From the NOESY contour map, the
H-6′ B interact with H-6D and these correlations confirm that the bond
should be 2C→O→7D (Fig. 4) (Esatbeyoglu et al., 2013). The NOE in­
teractions between H-2’E/H-6’E with H-6A indicate that the other
interflavanoid bond is 4C→8D (Barreiros et al., 2000; Fletcher et al.,
1977; Schmidt et al., 2011). HMBC correlations also confirmed that
C. de Moura Martins et al.
Fig. 4. Some NOE interactions that confirm the nature of the 2C→O→7D and
4C→8D bonds and the structure of compound XII.
bonding occurs between 4C→8D. Thus H-4C correlates with C-7D (δ
150.7), C-9D (δ 154.3), and C-8D (δ 105.4). In addition, H-6D has
correlated with C-5D (δ 150.5), C-7D (δ 150.7), C-8D (δ 105.4), and
C-10D (δ 101.5).
The orientation of the hydrogens H-3C and H-4C as 3.4-cis or 3.4-
trans was determined from NOE interactions. According to Cronjé et al.
(1990), A-type proanthocyanidins cannot be distinguished based on
coupling constants, as the values are very similar, but can be determined
by NOESY contour map analysis. When H-3C presents a NOE interaction
with H-6D, this correlation is characteristic of a 3.4-trans orientation,
independent of the conformation of the rigid system of bicyclic ring of
the A-type proanthocyanidin to be conformation α or β (Cronjé et al.,
1990; Esatbeyoglu et al., 2013; Schmidt et al., 2011). Therefore, if the
orientation of the hydrogens H-3C and H-4C are trans, the upper struc­
ture will be epigallocatechin (HO-3C and ring B in cis conformation);
however, if the orientation is cis the upper structure must be a galloca­
techin (HO-3C and ring B in trans conformation). In compound XII, NOE
interaction was not observed between H-3C with H-6D characteristic of
a 3.4-trans orientation; therefore, the interaction of H-3C with H-4C is
cis, and independently of the rigid ring conformation, the upper struc­
ture will be a gallocatechin. NOE interactions between H-2 F with H-3C,
H-2′ E with H-4C and H-3C with H-4C also corroborate to a 3,4-cis
orientation (Fig. 4).
The H-2F doublet (δ 4.49) with the coupling constant of 7.8 Hz in the
lower unit is characteristic of trans-diaxial hydrogens and the C-2F signal
(δ 82.1) confirms the C–O bond (Esatbeyoglu et al., 2013). The singlet
at δ 6.45 (H-2′ E and H-6′ E) indicates that ring E is trisubstituted. The
position of the methoxy group (CH3O-4’E) in the lower structure was
confirmed by the HMBC contour map analysis, in which methyl hy­
drogens (CH3, δ 3.69, s) correlate with C-4’E at δ 135.0. Equal chemical
shifts for H-2’E and H-6’E (δ 6.45, s) confirm that the methoxy group is
in the 4’E position and that the high values of the coupling constants J2-3
for H-2 F and H-3 F (J = 7.8 Hz) are characteristic of trans-diaxial hy­
drogens; therefore, we conclude that the lower unit is
115
Phytochemistry Letters 40 (2020) 109–120
4’-O-methylgallocatechin (Esatbeyoglu et al., 2013; Schmidt et al.,
2011). To the best of our knowledge this compound is being reported for
the first time. See Figures S32–S38 and Tables S7–S8 in the Supple­
mentary Material for NMR spectra and chemical shifts, respectively, of
compound XII.
Compound XIII (1.7 mg) was identified as gallocatechin-3-O-galloyl-
(2→O→7, 4→8)-4’-O-methylgallocatechin, a galloyl derivative of com­
pound XII. This compound has molecular mass of 774.14 g mol− 1
(C38H30O18). The mass of the molecular ion [M − H]– was m/z 773.1285
(acquired mass) and exact mass was m/z 773.1359 (Δ 9.6 ppm). The
mass spectrum and sequential mass spectrum of the ion m/z 773 are
described in Figures S39 and S40 in the Supplemental Material,
respectively. Proposed ESI(–)-MS/MS fragmentation mechanism is
shown in Fig. 5.
The loss of the galloyl group (m/z 152) through the QM mechanism
form the ion m/z 621 [M – H – 152]– (compound XII); then, the loss of a
molecule of water lead to the formation of the ion m/z 603. From the ion
m/z 603, the ions m/z 437 and two dimers m/z 301a and m/z 301b were
formed. These dimers correspond to an epi or gallocatechin unit and an
epi or methylgallocatechin derivative (319 – H2O), respectively, con­
firming the presence of these units in the compound.
In the 1H NMR spectra, the chemical shifts (δ) of the hydrogens and
carbons of this compound were attributed. Due to the low mass obtained
from compound XIII, the 13C NMR spectrum could not be obtained. In
this way carbon signals linked to hydrogen were assigned by HSQC.
Therefore, for elucidation, HPLC-ESI(–)-MS/MS and 1H NMR, COSY,
HSQC and NOESY analysis were performed. Table 4 shows the chemical
shifts (δ) of hydrogens and the hydrogen carbons signals assigned by
HSQC of compound XIII compared with XII.
The doublet at δ 5.15 (1H, J = 3.2 Hz) was assigned to H-3C and the
doublet at δ 4.51 (1H, J = 3.3 Hz) to H-4C; the coupling constants are
characteristic of A-type proanthocyanidins as well as compound XII
(Esatbeyoglu et al., 2013; Foo et al., 2000; Jacques et al., 1974). The
signal of a singlet (δ 6.64; 2H) in the aromatic region relative to com­
pound XII is attributed to H-2G and H-6G (equivalents) of the galloyl
group. By comparing the chemical shifts of the hydrogen, it was
concluded that the galloyl group is linked to the upper unit of the
proanthocyanidin because the H-3C signal is shifted to low field
(Table 4) (Hashimoto et al., 1989; Schmidt et al., 2011). The orientation
of H-3C and H-4C were determined as 3.4-cis from NOE interactions, as
in the case of compound XII. In this structure, H-3C also showed no
interaction with H-6D in the NOE contour map, indicating a 3.4-cis
orientation (Cronjé et al., 1990; Schmidt et al., 2011). NOE interactions
between H-2 F and H-3C, and H-2’E and H-4C also corroborate to a
3.4-cis orientation. Therefore, the upper structure is a gallocatechin. The
singlet signal at δ 3.67 (3H) indicates the presence of a methoxy group
(CH3O-4’E), suggesting that the proanthocyanidin has a unit of meth­
ylgallocatechin derivative, as in compound XII. To the best of our
knowledge, this compound is being reported for the first time. See Fig­
ures S41–S44 in the Supplementary Material for NMR spectra of com­
pound XIII.
To identify the absolute configuration of these compounds, it would
be necessary to measure Circular Dichroism (CD), which is an analytical
technique used to analyze chirality in molecules through their optical
activity (Barrett et al., 1979), but unfortunately it was not possible to
recover samples of NMR studies, making it impossible to do these
measurements.
The species I. laurina is a promising for the discovery of new com­
pounds, especially phenolic compounds, which are bioactive substances
of pharmacological interest. Future studies with the plant can be per­
formed to isolate more compounds and in greater quantities to perform
antifungal tests with isolated substances that can be researched for
medicinal purposes.
C. de Moura Martins et al. Phytochemistry Letters 40 (2020) 109–120

Fig. 5. Proposed ESI(–)-MS/MS fragmentation mechanism of gallocatechin-3-O-galloyl-(2→O→7, 4→8)-4′ -O-methylgallocatechin (XIII) m/z 773.

Table 4 Table 5
Comparative analysis of 1H NMR (100 MHz) and HSQC (carbons) data of Results of antifungal activity (expressed as MIC in μg mL− 1) of compound III
compounds XII and XIII. from I. laurina.
1 1
Position H NMR H NMR HSQC Samples Microorganisms
(400 MHz, (400 MHz,
C. albicans C. tropicalis C. glabrata
DMSO-d6) DMSO-d6)
ATCC 28366 ATCC 13803 ATCC 15126
δ1; m2; J3 δ1; m2; J3
4’-O-methylgallocatechin (III) >3,000 >3,000 >3,000
(XIII) (Compound XII)
EAF-I 500 1,000 23.4
Compound Compound XII
XIII (δ) (δ)

3C 5.15 d (3.2) 3.78 d (3.4) 70.2 66.2 combination (synergism) of the flavonoids present in the EAF-I of the
4C 4.51 d (3.3) 4.20 d (3.0) 29.3 27.7 bark is important for activity against Candida species, especially for
6A 5.98 d (1.4) 5.88 d (1.9) 94.0 94.2
8A 5.92 d (1.5) 5.85 d (1.9) 95.5 94.3
C. glabrata, which was the species most sensitive to the fraction of origin.
2G 6.64 s – 108.5 – The compound mycetin-3-O-ramnoside, isolated from the leaves of the
6G 6.64 s – 108.5 – plant, showed better results for fungal inhibition (MIC = 93.8 μg mL− 1)
2’B 6.55 s 6.52 s 105.2 106.0 compared to compound III, having a greater contribution to the anti­
6’B 6.55 s 6.52 s 105.2 106.0
fungal activity of the ethyl acetate fraction (Martins et al., 2019).
2F 4.59 d (7.0) 4.49 d (7.8) 82.0 82.1
3F 3.77 m 3.80 m 67.2 66.4
4a F 2.70 m 2.73 dd (16.2 and 28.8 (CH2) 28.6 (CH2) 3. Materials and methods
5.5)
4b F 2.33 m 2.42 dd (16.3 and 28.8 (CH2 28.6 (CH2)
3.1. Plant material
8.5)
6D 6.03 s 5.98 s 96.2 96.0
2’E 6.40 s 6.45 s 106.1 106.8 The barks of I. laurina (Sw.) Willd were collected in Uberlândia,
6’E 6.40 s 6.45 s 106.1 106.8 Minas Gerais state, Brazil (18◦ 59′ 13.96′ ’S; 48◦ 12′ 42.16′ ’W) in the
CH3 3.67 s 3.69 s 60.1 59.7 month of February. A specialist identified the specimens and an exsic­
Note: δ1: chemical shift in ppm using TMS as internal standard; m2: multiplicity cate was deposited at Herbarium Uberlandenses (HUFU), under the
(s = singlet, d = doublet, m = multiplet); J3: coupling constant expressed in Hz. number 64050. The study with this species was registered on the Na­
tional System of Genetic Resource Management and Associated Tradi­
2.5. Antifungal activity of compound III tional Knowledge for Research (SYSGEN) under the code A417AD0.

The antifungal activity assay was performed for the isolated com­ 3.2. Extracts preparation and chromatographic fractionation of bark
pound III (Table 5). When analyzed alone, the compound III showed no extract
activity because the MIC value was greater than 3,000 μg mL− 1. This
result was higher than the origin fraction (EAF-I), suggesting that the The ethanolic extracts (EE) were prepared using 1,400.0 g of dried

116
C. de Moura Martins et al.
barks by maceration at room temperature with ethanol (95 %). Eight
successive extractions were performed at 48 h intervals for each repe­
tition. At the end of the extraction process, filtrates were joined,
concentrated under vacuum (under 40 ◦ C) and freeze-dried (Lyophilizer
LS 3000, TERRONI, Brazil). A yield of 234.0 g grams was obtained of EE,
which was stored under refrigeration until analysis. The EE (58.0 g) was
used in the fractionation and was subjected to column chromatography
on silica gel 60 (300.0 g) (Scheme S1).
Increasing polarities of solvents were used in the fractionation
(hexane, chloroform, ethyl acetate, n-butanol, methanol, and water) and
seven fractions were obtained. The ethyl acetate fractions (EAF-I and
EAF-II) were most active and were then subjected to new fractionation
processes. The EAF-I has green coloration and the EAF-II, red coloration.
Fractions were concentrated and frozen-dried and stored under refrig­
eration until analysis. See Scheme S1 in the Supplementary Material for
a comprehensive flowchart of purification, quantification, identification
and isolation of compounds from the EAF-I and EAF-II fractions.
3.3. Analysis of total phenolics, proanthocyanidins and flavonoids
The quantification of total phenolic and proanthocyanidins content
was performed using spectrophotometric methods (Morais et al., 2008).
The Folin-Ciocalteu method was used in quantification of total phenolic
contents and the results were expressed in mg of gallic acid equivalents
1
by gram of dry extract (mg GAE g−extract ). The vanillin sulfuric method
was used to determine the proanthocyanidins content and the results
were expressed as milligrams of catechin equivalents per gram of dry
1
extract (mg CE g−extract). Flavonoid content was determined according to
Woisky and Salatino (1998) and the results were represented in mg of
quercetin equivalents per gram of dry extract (mg QE g-1 extratct). The
analysis was performed in triplicate and carried out in Thermo Scientific
Genesys-10S spectrophotometer.
3.4. Antifungal and cytotoxic activities
The following ATCC (American Type Culture Collection) yeast were
used: Candida albicans (ATCC 28366), C. tropicalis (ATCC 13803), and
C. glabata (ATCC 15126). The biological analysis was performed ac­
cording to the methodology describe by Nunes et al. (2016).
The MIC determination for antifungal assay was performed accord­
ing to the CLSI (Clinical and Laboratory Standard Institute) using the
broth dilution assay method (CLSI, 2008). Extract stock solutions and
partitions were prepared in 5% DMSO, and twofold serial dilutions were
prepared in RPMI in 96-well microtiter plates (Corning Incorporated,
Corning, NY, USA). The final concentrations ranged from 0.98 to 2.000 g
mL− 1. Yeasts (100 μL) were added to each well in the microtiter plates.
The growth control contained medium and inoculum. Blank controls
contained medium only. The microtiter plates were then incubated at 35
◦
C and the endpoints were read after 48 h. The Amphotericin B (final
concentration of 0.031–8 μg mL− 1) and strains ATCC 22019 (Candida
parapsilosis) and ATCC 6258 (Candida krusei) were used as quality
controls.
Cell viability test was performed with Vero cells (ATCC CCL 81)
(kidney fibroblasts, African green monkey). The samples to be tested
were dissolved in methanol and diluted in DMEM (Dulbecco’s modified
Eagle’s medium, Sigma–Aldrich) supplemented culture medium,
obtaining a stock solution at a concentration of 640 μg mL− 1. The final
methanol concentration of the stock solution did not exceed 3%.
The cytotoxic activity was performed using the microplate dilution
method. A solution containing 1 × 106 Vero cells in 10.0 mL of sup­
plemented DMEM was prepared, and 100 μL of this solution was
pipetted into each well. The plate was incubated for 6 h at 37 ◦ C, in a
humidified atmosphere at 5% CO2. Then, the culture medium was
removed, and the samples were added at concentrations 512, 256, 128,
64, 32, 16, 8, 4 μg mL− 1. The final volume of each well was 100 μL and
the amount of cells present in each well was 1 × 104 cells. The plate was
117
Phytochemistry Letters 40 (2020) 109–120
incubated for 48 h at 37 ◦ C in a humidified atmosphere with 5% CO2.
Then, 10 μL of resazurin solution (3 mM) in PBS (Phosphate Buffered
Saline) was added to each well (Rolon et al., 2006). The plates were
incubated again for 24 h under the same conditions. Absorbance read­
ings were performed at 595 nm on a microplate spectrophotometer
(Camberra Packard). The cytotoxic concentration (CC50) was deter­
mined by means of a dose-response graph with non-linear regression
(Pillay et al., 2007). The selectivity index was calculated using the
equation (SI = log[CC50]/[MIC]) (Case et al., 2006).
3.5. Ethyl acetate fraction (EAF) isolation and fractionation
The EAF-I (0.40 g) was submitted to column chromatography with
C18-type modified silica (60.0 g, 30.0 × 3.0 cm) using methanol as
mobile phase resulting in 12 sub-fractions (EAF-I-1 to EAF-I-12). The
EAF-I-2 showed only one spot on TLC and was purified using HPLC semi
preparative, producing the compound 4’-O-methylgallocatechin (III).
4’-O-methylgallocatechin (III, 45.0 mg): 1H NMR (DMSO-d6, 400
MHz): δ 6.28 (2H, s, H-2’and H-6’), 5.89 (1H, d, J 2.2 Hz, H-6), 5.70 (1H,
d, J 2.2 Hz, H-8), 4.44 (1H, d, J 7.2 Hz, H-2), 3.78 (1H, m, H-3), 3.65 (3H,
s, CH3), 2.60 (1H, dd, J 5.2 and 16.1 Hz, H-4a), 2.34 (1H, dd, J 7.8 and
16.1 Hz, H-4b); 13C NMR (DMSO-d6, 100 MHz,): δ 80.8 (C-2), 66.3 (C-3),
27.5 (C-4), 156.2 (C-5), 95.1 (C-6), 156.5 (C-7), 93.8 (C-8), 155.1 (C-9),
98.9 (C-10), 134.8 (C-1’), 106.1 (C-2’), 150.3 (C-3’), 135.0 (C-4’), 150.3
(C-5’), 106.1 (C-6’), 59.6 (CH3). HPLC-ESI(–)-MS/MS: m/z 319.0810 [M
− H]– (calc. for C16H15O–7: 319.0823).
The EAF-II was submitted to column chromatography with C18-type
modified silica (100.0 g of C18 and 1.0 g of the sample) using 70.0 mL of
MeOH:H2O solution in the same proportion. The fractionation of EAF-II
resulted 10 sub-fractions (EAF-II-1 to EAF-II-10). The fractions EAF-II-4
and EAF-II-5, which had a higher mass, were purified using HPLC
semipreparative, yielding four pure compounds:
Gallocatechin (II, 5.9 mg): 1H NMR (DMSO-d6, 400 MHz): δ 6.24
(2H, s, H-2’and H-6’), 5.88 (1H, d, J 1.6 Hz, H-6), 5.69 (1H, d, J 1.6 Hz,
H-8), 4.42 (1H, d, J 7.0 Hz, H-2), 3.78 (1H, m, H-3), 2.60 (1H, dd, J 5.1
and 16.0 Hz, H-4a), 2.34 (1H, dd, J 7.7 and 16.0 Hz, H-4b); 13C NMR
(DMSO-d6, 100 MHz,): δ 81.0 (C-2), 66.3 (C-3), 27.4 (C-4), 156.2 (C-5),
95.0 (C-6), 156.4 (C-7), 93.8 (C-8), 155.3 (C-9), 98.9 (C-10), 129.8 (C-
1’), 106.0 (C-2’), 145.6 (C-3’), 132.5 (C-4’), 145.6 (C-5’), 106.0 (C-6’).
HPLC-ESI(–)-MS/MS: m/z 305.0643 [M − H]– (calc. for C15H13O–7:
305.0667).
Gallocatechin-(4α→8)-4’-O-methylgallocatechin (XI, 5.2 mg): 1H
NMR (DMSO-d6, 400 MHz): δ 4.06 (1H, d, J 9.3 Hz, H-2C), 4.24 (1H,
broad triplet, J 6.9 and 8.8 Hz, H-3C), 4.18 (2H, dd, J 4.2 and 6.8 Hz, H-
4C), 5.65 (1H, d, J 2.0 Hz, H-6A), 5.59 (1H, d, J 2.0 Hz, H-8A), 6.35 (2H,
s, H-2’B and H-6’B), 4.28 (1H, d, J 8.2 Hz, H-2F), 3.79 (1H, m, H-3F),
2.77 (1H, dd, J 6.0 and 16.1 Hz, H-4aF) 2.37 (2H, dd, J 8.6 and 16.3 Hz,
H-4bF), 5.84 (1H, s, H-6D), 6.41 (2H, s, H-2’E and H-6’E), 3.67 (3H, s,
CH3); 13C NMR (DMSO-d6, 100 MHz,): δ 82.6 (C-2C), 70.8 (C-3C), 36.9
(C-4C), 156.0 (C-5A), 95.9 (C-6A), 157.4 (C-7A), 94.1 (C-8A), 155.5 (C-
9A), 106.2 (C-10A), 132.6 (C-1’B), 106.9 (C-2’B), 145.4 (C-3’B), 130.1
(C-4’B), 145.4 (C-5’B), 106.9 (C-6’B), 81.3 (C-2F), 66.6 (C-3F), 29.6 (C-
4aF and C-4bF), 153.3 (C-5D), 95.9 (C-6D), 153.5 (C-7D), 108.6 (C-8D),
154.6 (C-9D), 99.0 (C-10D), 135.2 (C-1’E and C-4’E), 107.0 (C-2’E and
C-6’E), 150.1 (C-3’E and C-5’E), 59.6 (CH3). HPLC-ESI(–)-MS/MS: m/z
623.1370 [M − H]– (calc. for C31H27O–14: 623.1406).
Gallocatechin-(2→O→7,4→8)-4’-O-methylgallocatechin (XII,
3.8 mg): 1H NMR (DMSO-d6, 400 MHz): δ 3.78 (1H, d, J 3.4 Hz, H-3C),
4.20 (1H, d, J 3.0 Hz, H-4C), 5.88 (1H, d, J 1.9 Hz, H-6A), 5.85 (1H, d, J
1.9 Hz, H-8A), 6.52 (2H, s, H-2’B and H-6’B), 4.49 (1H, d, J 7.8 Hz, H-2
F), 3.80 (1H, m, H-3 F), 2.73 (1H, dd, J 5.5 and 16.2 Hz, H-4aF) 2.42 (1H,
dd, J 8.5 and 16.3 Hz, H-4bF), 5.98 (1H, s, H-6D), 6.45 (2H, s, H-2’E and
H-6’E), 3.69 (3H, s, CH3); 13C NMR (DMSO-d6, 100 MHz,): δ 98.5 (C-2C),
66.2 (C-3C), 27.7 (C-4C), 155.9 (C-5A), 96.2 (C-6A), 156.5 (C-7A), 94.4
(C-8A), 152.8 (C-9A), 102.4 (C-10A), 133.1 (C-1’B), 106.0 (C-2’B),
145.1 (C-3’B), 129.8 (C-4’B), 145.1 (C-5’B), 106.0 (C-6’B), 82.1 (C-2F),
C. de Moura Martins et al.
66.4 (C-3F), 28.6 (C-4aF and C-4bF), 150.5 (C-5D), 94.6 (C-6D), 150.7
(C-7D), 105.4 (C-8D), 154.3 (C-9D), 101.5 (C-10D), 134.0 (C-1’E), 106.8
(C-2’E and C-6’E), 150.2 (C-3’E and C-5’E), 135.0 (C-4’E), 106.8 (C-
6’E), 59.7 (CH3). HPLC-ESI(–)-MS/MS: m/z 621.1208 [M − H]– (calc. for
C31H25O–14: 621.1250).
Gallocatechin-3-O-galloyl-(2→O→7,4→8)-4’-O-methyl­
gallocatechin (XIII, 1.8 mg): 1H NMR (DMSO-d6, 400 MHz): δ 5.15 (1H,
d, J 3.2 Hz, H-3C), 4.51 (1H, d, J 3.3 Hz, H-4C), 5.98 (1H, d, J 1.4 Hz, H-
6A), 5.92 (1H, d, J 1.5 Hz, H-8A), 6.64 (2H, s, H-2 G and H-6 G), 6.55
(2H, s, H-2’B and H-6’B), 4.59 (1H, d, J 7.0 Hz, H-2F), 3.77 (1H, m, H-
3F), 2.70 (1H, m, H-4aF), 2.43 (1H, m, H-4bF), 6.03 (1H, s, H-6D), 6.40
(2H, s, H-2’E and H-6’E), 3.67 (3H, s, CH3). 13C NMR (HSQC): δ 70.2 (C-
3C), 29.3 (C-4C), 94.0 (C-6A), 95.5 (C-8A), 108.5 (C-2 G and C-6G),
105.2 (C-2’B and C-6’B), 82.0 (C-2 F), 67.2 (C-3F), 28.8 (C-4aF and C-
4bF), 96.2 (C-6D), 106.1 (C-2’E and C-6’E), 60.1 (CH3). HPLC-ESI
(–)-MS/MS: m/z 773.1285 [M − H]– (calc. for C38H29O–18: 773.1359).
3.6. Performance liquid chromatography – electrospray ionization –
tandem mass spectrometry (HPLC-ESI(-)-MS/MS) operating in negative
mode
HPLC-ESI(-)-MS/MS conditions were the same described earlier as
Martins et al. (2019). The analysis was performed using a Liquid Chro­
matography (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. The mobile phase was
water/ formic acid (0.1 %, v v− 1) (A) and methanol (B), the drying gas
(N2) was used at a flow 8 L min− 1, the nebulizer temperature was set at
220 ◦ C and a potential of 4.5 kV was used on the capillary.
3.7. Nuclear magnetic resonance (NMR)
The analysis was performed in NMR Bruker spectrometer Model
Ascend™ 400 Avance III HD (9.2 T). Deuterated dimethyl sulfoxide
(DMSO-d6) was used as solvent, and tetramethylsilane (TMS) as an in­
ternal standard. Analysis were performed at 400 MHz for 1H NMR and at
100 MHz for 13C NMR. The NMR analyses were performed by 1H, 13C,
DEPT-135, COSY, NOESY, HSQC, and HMBC (Martins et al., 2019).
3.8. High performance liquid chromatography (HPLC)
The EAF-I and EAF-II fractions were analyzed by High Performance
Liquid Chromatography coupled to a Diode Array Detector (HPLC-
DAD). HPLC-DAD conditions were the same as described earlier by
Martins et al. (2019). A volume of 20 μL of methanol solution was
injected at 1000 μg mL− 1. Deionized water (phase A) and methanol
(phase B) were used as mobile phases using the following program: 10 %
B (5 min), 20 % of B (10 min); 30 % B (25 min); 70 % B (40 min) and 100
% B (55 min) at a flow rate of 8.0 mL min− 1. For the purification of EAF-I
and EAF-II was used C18 semipreparative reverse phase column (Phe­
nomenex, Synergi Hydro-RP model, 21.20 mm internal diameter, 10 cm
long, 4 μm particles with 80 Å of diameter). A volume of 100 μL of
methanol solution of the fractions was injected (90 mg mL− 1 of EAF-I-2,
146 mg mL− 1 of EAF-II-4 and 100 mg mL− 1 of EAF-II-5).
3.9. Statistical analysis
The chemical analysis was performed in triplicate and the results
were compared using the Analysis of Variance (ANOVA) method. The
results statistically different had the significance level lower than 5% (P
< 0.05), and to determine the significant differences between the av­
erages, the Tukey test was used. The SigmaPlot 11.0 program was used
for statistical analysis.
118
Phytochemistry Letters 40 (2020) 109–120
4. Conclusions
EE, EAF-II and BF fractions were the samples with the highest levels
of phenolic compounds and proanthocyanidins. These fractions had
good inhibition of fungi growth from Candida genus with MIC values of
24.4 μg mL− 1 against C. tropicalis (EE), 23.4 μg mL− 1 against C. albicans
and C. glabrata (EAF-II), and 46.8 μg mL− 1 against C. tropicalis,
C. albicans and C. glabrata (BF). These results may provide scientific
support for the medicinal use of this species. Flavonoids, type flavan-3-
ols, derived from gallocatechin, were identified in EAF-I and EAF-II
fractions, and five new compounds were isolated from genus Inga for
the first time: 4′ -O-methylgallocatechin (III), gallocatechin (II), gallo­
catechin-(4α-8)-4′ -O-methylgallocatechin (XI), gallocatechin-
(2→O→7,4→8)-4′ -O-methylgallocatechin (XII) and gallocatechin-3-O-
galloyl-(2→O→7,4 →8)-4’-O-methylgallocatechin (XIII), which were
identified by NMR and ESI(–)-MS/MS techniques. The plant I. laurina
promises to be a source of phenolic compounds with antifungal potential
of biological interest, especially as antifungals. The compounds XI (B-
type proanthocyanidin), XII and XIII (A-type proanthocyanidins) were
isolated and identified and were reported for the first time.
Author contribution statement
Carla de Moura Martins performed most of the experiments,
analyzed the data, and wrote the article. Mário M. Martins performed
the HPLS-ESI/MSn analysis and the cytotoxicity assay. Thaise Lara
Teixeira and Cláudio V. da Silva supervised the cytotoxicity assay. Luís
C. S. Cunha, Mariana B. Santiago, and Carlos. H. G. Martins performed
the antifungal experiments. Sérgio A. L. de Morais and Alberto de Oli­
veira supervised the entire project and helped analyze the data. Fran­
cisco J. T. de Aquino, Evandro A. do Nascimento and Roberto Chang
contributed to analysis tools.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgements
This study was supported by the Foundation for Research Support of
the State of Minas Gerais (FAPEMIG-Brazil; APQ-01612-18 awarded to
A.O.) and by the Coordination for the Improvement of Higher Education
Personnel - Brazil (CAPES) - Finance Code 001. The authors thank the
Nanobiotechnology Laboratory (IGEB-UFU) for the HPLC-MSn assays
and the Chemistry Institute of the Federal University of Uberlândia
(IQUFU) for the supporting infrastructure. This manuscript is based on
the thesis of the authors.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.phytol.2020.10.001.
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