Received: 10 July 2017 | Revised: 10 September 2017 | Accepted: 26 September 2017

DOI: 10.1111/jfbc.12455

FULL ARTICLE

Biological evaluation and quantitative analysis of antioxidant

compounds in pulps of the Amazonian fruits bacuri (Platonia
insignis Mart.), ing
a (Inga edulis Mart.), and uchi (Sacoglottis uchi
Huber) by UHPLC-ESI-MS/MS

Fl
avio A. de Freitas1,2 |
jo2 | Elzalina R. Soares2 |

Rafael C. Arau
Rita C. S. Nunomura2 | Felipe M. A. da Silva1,2 | Sarah R. S. da Silva3 |
Antonia Q. L. de Souza3 | Afonso D. L. de Souza2 | Francisco Franco-Montalb

an4 |
Leonard D. R. Acho5 | Emerson S. Lima5 | Giovana A. Bataglion2 |
Hector H. F. Koolen1

1
Metabolomics and Mass Spectrometry
Research Group, Amazonas State University,
Abstract
UEA 69050-010, Manaus, Amazonas, Brazil Bacuri, ing
a, and uchi are Amazon fruits consumed specially in the North region of Brazil. Due to
2
Department of Chemistry, Federal their large consumption and the lack of knowledge regarding its chemical composition, these fruits
University of Amazonas, UFAM 69077-000, were studied in relation to their Antioxidant chemical constitution. The total phenolic content
Manaus, Amazonas, Brazil
ranged from 3.86 6 0.47 to 33.38 6 1.51 mg GAE/100 g, and the total flavonoid content ranged
3
Faculty of Agricultural Sciences, Federal
from 1.75 6 0.22 to 19.44 6 0.87 mg QUERE/100 g, where the contents showed a significant cor-
University of Amazonas, UFAM 69077-000,
Manaus, Amazonas, Brazil relation with DPPH and ABTS antioxidant assays. Thus, UHPLC-MS/MS was applied do quantify
4
Department of Medicinal & Organic selected compounds, been citric acid the most abundant for all fruits. Furthermore, samples were
Chemistry, Faculty of Pharmacy, University screened for their a-glycosidase and lipase inhibitory effects, in addition to their antimicrobial
of Granada, UGR 18071, Granada, Spain potentials. Bacuri showed the highest antioxidant and a-glycosidase inhibitory capacity (IC50 15.20
5
Faculty of Pharmacy, Federal University of lg/mL), whereas uchi and its main metabolite bergenin displayed moderate antimicrobial activities.
Amazonas, UFAM 69077-000, Manaus,
The results shed light into the potentials of Amazonian fruit sources.
Amazonas, Brazil
Practical applications
Correspondence
Fl
avio Augusto de Freitas, Department of
Plant phenolics are essential components of functional foods, due to their antioxidant and enzyme
Chemistry, Federal University of Amazonas, inhibition activities, which are directly linked to several diseases prevention. This is the first study
UFAM 69077-000, Manaus, AM, Brazil. about the quantification of antioxidant compounds in the Amazonian fruits: bacuri, ing
a, and uchi.
Email: freitas.flavio@yahoo.com.br
Although they are quite consumed in the North region of Brazil, there are no bio-products made
from them. This study aimed to elucidate the knowledge about the chemical composition and
potentialities within these fruits with the practical purpose of highlighting them to future commer-
cial applications. In addition, once we indicate their capabilities, we contribute with local
populations in respect to the production of such fruits, which still is a family activity.

KEYWORDS
antioxidant activity, bacuri, ing

a, antioxidant compounds, uchi

J Food Biochem. 2017;e12455. wileyonlinelibrary.com/journal/jfbc V

C 2017 Wiley Periodicals, Inc. | 1 of 10
https://doi.org/10.1111/jfbc.12455
2 of 10 | DE FREITAS
1 | INTRODUCTION
A balanced diet based on the regular consumption of fruits has been
associated with many pharmacological benefits (Koolen, Silva, Gozzo,
Souza, & Souza, 2013). Specially, prevention of cardiovascular diseases,
inflammations, and aging-related disorders can also be correlated with
intake of fruits, especially those which are rich in phenolic compounds
and vitamins (Huang, Ou, & Prior, 2005).
Phenolic compounds are attractive due to their antioxidant activity
that reduces oxidative stress and prevents or delays oxidation by scav-
enging free radicals (Koolen et al., 2013). The use of antioxidants
derived from natural resources is gaining attention due to their health
benefits, which include prevention of cardiovascular diseases,
inflammations, and aging-related disorders (Aqil et al., 2012). These
compounds have been observed in amazonic plants as bacuri (Platonia
insignis Mart.), ing
a (Inga edulis Mart.), and uchi (Sacoglottis uchi Huber)
[sin. Endopleura uchi] (Gomes, Ghica, Rodrigues, Gil, & Oliveira-Brett,
2016; Silva, Rogez, & Larondelle, 2007).
Uchi, belonging to the family Humiriaceae, is native from the
Amazon region and it is popularly known as uchi, uxi, uxi-amarelo,
pururu, uxi-liso, uxi-ordin
ario, or uchi-pucu (Silva, Oliveira, Tomonasa,
& Nunomura, 2009). These fruits are ellipsoid in shape with 2.57 cm
in diameter, and normally contain one or two seeds. The skin of the
uchi fruit is thin, tender, and brown in appearance (Figure 1a). The pulp
presents a yellow-brownish color and a rough-like texture, being the
sole edible part of uchi. In addition, its rich flavor is unique, not compa-
rable with any known fruit. Recent studies point to popular uses of
^a, 1984; Nazir et al., 2007; Nunomura,
S. uchi as a medicinal plant (Corre
Oliveira, Silva, & Nunomura, 2009; Silva et al., 2009) reinforcing the
idea that this fruit may be a functional food source.
Bacuri, belonging to the family Clusiaceae, is naturally distributed
along the south of the Amazon forest, with major concentration in the
Amazon estuary being probably domesticated by the Amazon
Amerindians (Rogez et al., 2004). The local population appreciates this
fruit by the unique aroma and flavor of the edible part (pulp)
(Yamaguchi, Pereira, Lima, & Veiga-Junior, 2014) and by its the medici-

nior et al., 2010). The fruits are ovoid in shape
nal properties (Costa Ju
(715 cm in diameter, from 200 to 1,000 g of fresh weight), and
normally contain three to five big seeds. The rind is thick (13 cm), pale
yellow to brownish yellow, tough and somewhat elastic, and exudes a
yellow latex when pressed (Figure 1b) (Rogez et al., 2004).
FIGURE 1 (a) Uchi (Sacoglottis uchi Huber), (b) bacuri (Platonia insignis Mart.), and (c) ing
a (Inga edulis Mart.)
ET AL.
Ing
a, also known as ing
a-cipo

, belonging to the family Fabaceae, is
naturally distributed along South America, with major concentration in
the Amazon estuary (Dias, Souza, & Rogez, 2010). This fruit has a
sweet flavor and softness of the pulp, being appreciated by the local
population. Some medicinal properties are attributed to a regular intake
of ing
a, such as the cure of arthritis, diarrhea, and rheumatism (Duke,
1975). These fruits are cylindrical in shape with 2540 cm in diameter,
weighing between 100 and 800 g, including the pod and usually
contain between 8 and 15 seeds (Figure 1c) (Duke, 1975). The ing
a is
also known as ice-cream bean (alluding to its pulps characteristics) and
it is used to prepare an alcoholic beverage from the aril, where indige-
nous people from Panam
a and Colombia (Duke, 1970) consume the
beverage, called cachiri, at a festival of same name.
Chemically, those fruits are poorly studied and their properties
such as: antimicrobial, anticonvulsant, antiviral, antioxidant, antidepres-
sant, wound healing, and cytotoxic activities have been the subject of

nior et al.,
several pharmacological and toxicological studies (Costa Ju

nior et al., 2010). Bacuri has demonstrated its wealth in
2013; Santos Ju
polyisoprenylated benzophehones and xanthones, responsible for sev-
~ a, Jancovski, & Kennelly, 2009; Diderot,
eral biological activities (Acun
Silvere, & Etienne, 2006; Kumar, Sharma, & Chattopadhyay, 2013), and
also in polyphenolic molecules, compounds of great interest for having
antioxidant activity (Gomes et al., 2016; Magalh~aes, Lima, Marinho, &
Ferreira, 2007). Only diterpenes, xanthones, and fatty acids from the
seeds (Yamaguchi et al., 2014) and monoterpenes for the pulp were
found as constituents of bacuri (Borges & Rezende, 2000). Regarding
ing
a, it has not been reported in the literature data about isolated or
identified compounds from its fruits. However, data were reported
about isolated phenolic compounds from the ing
a leaves, and the eval-
uation of the antioxidant capacity (Dias et al., 2010; Silva et al., 2007).
Concerning to uchi, the literature has reported a high content of fatty
acids in the fruits, predominantly the oleic acid (Marx, Andrade, Zoghbi,
& Maia, 2002) and carotenoids, mainly trans-b-carotene (Magalh~aes
et al., 2007). Bergenin was also isolated from uchi pulps and barks and
it has been reported that this compound have several biological activ-
ities (Magalh~aes et al., 2007; Nazir et al., 2007; Silva et al., 2009).
Due to the lack of knowledge regarding their chemical composition
and the potential as antioxidants source and functional food, this
work aimed the determination of the phenolic composition by ultra-
high liquid chromatography coupled to tandem mass spectrometry
(UHPLC-MS/MS) of the Amazon fruits bacuri, ing
a, and uchi. In
DE FREITAS ET AL.
addition, this study also displays the chemical fractionation of uchi pulp
extract for the isolation of bergenin and further chemical modifications
for antimicrobial activity enhancement evaluation.
2 | MATERIALS AND METHODS
2.1 | Standard chemicals
Antioxidant compounds, p-coumaric acid, (1)-catechin, (2)-epicatechin,
citric acid, gallic acid, vanillic acid, isorhamnetin, and luteolin used as
standards were purchased from Sigma Aldrich (St. Louis, USA). The
FolinCiocalteu reagent (2,4,6-tris(2-pyridyl)-s-triazine) (TPTZ) and
resazurin dye was obtained from Fluka-Chemie (Buchs, Switzerland).
The aluminum chloride, sodium carbonate, sodium acetate, benzyl
bromide, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,20-Azinobis-3-ethyl-
benzo thiazoline-6-sulfonic acid, diammonium salt (ABTS), o-dianisidine
color reagent (DIAN), glucose oxidase and peroxidase enzyme solution
(PGO), acarbose, p-nitrophenyl octanoate (NPC), porcine pancreas
lipase (Type II, from porcine pancreas, 100400 units/mg protein),
TrisHCl buffer (pH 8.5), and a-glucosidase from Saccharomyces cerevi-
siae (Type I; 10 U/mg protein) were purchased from Sigma-Aldrich
(Steinheim, Germany). Silica gel (70230 mesh) from Silicycle, Mueller
Hinton agar was purchased from Difco. Monosaccharides, glucose,
fructose, saccharose, arabinose, and ramnose used to evaluate the
matrix effect were purchased from Merck (Darmstadt, Germany).
HPLC grade dichloromethane, dimethylformamide, dimethylsulfoxide
(DMSO), ethanol, ethyl acetate, and methanol were from J. T. Baker
(Mexico City, Mexico). Ultrapure water (18.2 MX cm) was purified by a
Milli-Q gradient system (Millipore, Milford, USA).
2.2 | Sample preparation
Fresh ing
a (2.0 kg), bacuri (1.2 kg), and uchi (5.8 kg) fruits were pur-
chased at Adolpho Lisboa market in Manaus, a city located in the heart
of the Amazon region of Brazil. The ripeness state of each fruit was
carefully checked, where the only ones proper for consumption were
selected (ripe fruits). The maturity degree was determined according to
size, color, and firmness. Pulps of bacuri, uchi, and ing
a were manually
separated from the peels and seeds, and only the pulps were submitted
to extraction. Antioxidant compounds were extracted from the studied
fruit pulps using an ETHOS 1 microwave extraction system (Milestone,
Shelton, USA). After a preliminary evaluation of the total phenolic con-
tent (TPC) (data not shown) in different extraction conditions, an opti-
mized extraction method was obtained. The potency was 600 W and
irradiation time 60 s, temperature 458C, where an amount of 2.5 g of
fruit pulps was extracted with 37.5 mL of an aqueous EtOH solution
(80%, 15 mL/g of pulp). It was found that 15 mL/g (total volume of
37.5 mL solvent for 2.5 g of each pulp) provided the maximum extract
recovery for bacuri (105.8 mg, 4.23%), ing
a (42.6 mg, 1.70%), and uchi
(129.3 mg, 5.17%). Teflon PFA (perfluoroalkoxy) vessels with 100 mL
of capacity were used. After extraction, the liquid was filtered under
reduced pressure and allowed to cool to room temperature (ca., 258C),
and submitted to centrifugation at 4,000 rpm for 15 min. The
| 3 of 10
supernatant was collected, placed in glass centrifuge tubes and flushed
with nitrogen gas. All extractions were performed in triplicate. Addi-
tionally, a portion of uchi fruit pulps (7.12 g) was macerated at room
temperature (ca., 258C) with EtOH (100 mL/g of pulp) in triplicate.
2.3 | Determination of TPC
The TPC of the studied fruits pulps was determined using the Folin
Ciocalteu reagent (Singleton & Rossl, 1965). Each of the crude extracts
(1 mg) was dissolved in pure water (25 mL). Aliquots (250 lL) were
mixed with 1 mL of the FolinCiocalteu reagent and 1 mL of a 10%
(m/v) Na2CO3 solution. Samples were then incubated at 308C for 1.5 h
and the absorbance of each sample was measured at 765 nm. The TPC
was calculated from a calibration curve, using gallic acid as standard
(1.257.5 lg/mL). TPC contents were expressed in mg gallic acid equiv-
alent (GAE)/100 g fresh pulps.
2.4 | Determination of total flavonoid content
The total flavonoid content (TFC) was measured by the AlCl3 method
(Lamaison & Carnet, 1990). The extracts (0.5 mg) were dissolved in
pure water (25 mL). Aliquots (1.5 mL) of the solutions were added to
equal volumes of a solution 6% (m/V) AlCl3
H2O. The mixture was vig-
orously shaken, and absorbance was read at 367.5 nm after 10 min of
incubation. TFC were expressed in mg quercetin equivalent (QUERE)/
100 g fresh pulp.
2.5 | Antimicrobial assays
Minimal inhibitory concentrations (MICs) were determined by the
microbroth-dilution assay, as recommended by the U.S. National
Committee for Clinical Laboratory Standards (NCCLS). Assays were
performed into 96-well plates supplied with 100 lL of Mueller
Hinton Broth culture medium, 100 lL of each sample solution, and 5
lL of test bacterial suspensions at 1.0 3 107 UFC/mL (Koolen,
Soares, Silva, Almeida, & Souza, 2012). The pulp extracts were
dissolved in DMSO at initial concentration of 500 lg/mL being
subsequently diluted until 3.75 lg/mL. The incubation was made at
378C during 24 hr. The microorganisms tested were: Bacillus cereus,
Candida albicans, Candida tropicalis, Escherichia coli, Staphylococcus
aureus, and Streptococcus mutans hospital wild colonies. The bioac-
tivities were recorded as blue coloration in the wells after use of
resazurin dye. The bacteriostatic or bactericidal effects of the
metabolites were observed by inoculation of the well materials on
Mueller Hinton Agar plates after the tests. Positive controls tetracy-
cline (4 lg/mL) and ketoconazole (4 lg/mL) with the negative con-
trol DMSO were used during the tests.
2.6 | Antioxidant assays
The antioxidant capacity of the extracts of bacuri, ing
a, and uchi were
evaluated by DPPH and ABTS assays. The DPPH and ABTS assays
were performed in triplicate and in the same conditions used in a
previous study (Souza et al., 2016). The consumption of DPPH was
4 of 10 | DE FREITAS ET AL.

monitored by measuring the absorbance at 492 nm and for ABTS at
734 nm. For both assays, the percentage of inhibition was calculated
according to the equation: % Inhibition 5 100 2 (absorbance/average
absorbance of control) 3 100, and expressed as trolox equivalent (TE)
per gram of fresh pulp.
2.7 | Enzymatic inhibition analysis
2.7.1 | a-Glucosidase inhibition assay
For this procedure, a previously described methodology was employed
(Iauk et al., 2015). In brief, extracts and controls were solubilized in
DMSO and added to a maltose (40 mg/mL) in 50 mM of sodium ace-
tate buffer solution. The addition of a-glucosidase (100 mg/mL) solution
started the reaction. After 30 min of incubation at 378C, the reaction
was stopped by adding perchloric acid (4.2%, m/v). The generation of
glucose was quantified by the reduction of the DIAN. The supernatant
of the reaction was mixed with DIAN and PGO system-color reagent
and was left to incubate at 378C for 30 min. The absorbance was meas-
ured at 500 nm. Gallic acid was used as positive control. As a measure
of inhibitory potency, the concentration required for 50% inhibition of
enzyme activity (IC50) was determined.
the product ions by MS/MS experiments. Then, analyses were con-
ducted by selected reaction monitoring (SRM) in the negative ion
mode monitoring two transitions for each standard compound using
20 ms of dwell time. The settings of the mass spectrometer were
optimized for each SRM transition compound analysis and are
shown in a previous study (Bataglion, Silva, Eberlin, & Koolen,
2015). Data were acquired and processed by Labsolution software
(v. 5.53 SP2, Shimadzu).
The method was previously validated according to the U.S. Food
^ncia Nacional de
and Drug Administration (FDA) and by the Age
Vigil^ancia Sanit
aria (ANVISA) guidelines over three consecutive days
for linearity, LOD, LOQ, inter- and intraday accuracy and precision, and
recovery. Details about how validation method was conducted are
available in elsewhere (Bataglion et al., 2015). Stock solutions of each
standard compound (1 mg/mL) were prepared and stored in methanol
at 48C. An intermediate solution containing all standard compounds
(1 mg/mL) was prepared in methanol and dilutions from this solution
were done at nine different levels for calibration curves and method
validation. A stock solution of internal standard (IS, 20 mg/mL) was pre-
pared and stored in methanol and dilutions were done to reach a final
concentration of 500 ng/mL in the calibration curves, which were gen-

2.7.2 | Lipase inhibition assay
The inhibition activity of the pulp extracts toward lipase was evaluated
as previously described by Marrelli et al. (2012), using NPC as a
substrate that, in the presence of lipase, liberates p-nitrophenol and
octanoic acid. Briefly, Type II crude porcine pancreatic lipase was used
at a concentration of 5 mg/mL. NPC was prepared in DMSO to achieve
a concentration of 5 mM. Phenolic extracts (100 mL) were mixed with
4 mL of TrisHCl buffer (pH 8.5) and the enzyme solution (100 mL).
After incubation at 378C for 25 min, NPC (100 mL) was added and incu-
bated again at 378C for 25 min. The absorbance was read at 412 nm.
2.8 | UHPLC-ESI-MS/MS analysis
The quantification of antioxidant compounds in pulp extracts was per-
formed with a LC-MS/MS 8040 (Shimadzu, Kyoto, Japan) consisted of
a triple quadrupole mass spectrometer equipped with an electrospray
ionization (ESI) source. The chromatographic separation was performed
on a Shim-pack XR-ODS III 2.2 mm, 2.0 mm i.d., 150 mm column
(Shimadzu, Kyoto, Japan) using a binary mobile phase. Solvent A was
pure water and Solvent B was methanol HPLC grade. The gradient elu-
tion at 358C was as follows: 01 min, 5% B; 14 min, 560% B; 47
min, 6070% B; 710 min, 70100% B; 1010.50 min, 100% B;
10.5011 min, 1005% B; 1115 min, 5% B, at a flow rate of 0.4 mL/
min. The autosampler temperature was maintained at 108C and the
injection volume was 10 mL.
The ionization source parameters were as following: capillary
voltage, 3.5 kV; heat block temperature, 3008C; desolvation line
temperature, 2508C; drying gas flow (N2), 20 L/min; nebulizing gas
flow (N2), 3 L/min; collision induced dissociation gas pressure (Ar),
224 kPa. For each standard, the precursor ion [M-H] 2
was deter-
mined in full scan experiments over a m/z range of 100500, and
erated in the concentrations of 20, 50, 75, 100, 200, 400, 600, 800,
and 1,000 ng/mL of standard compounds. Based on structural similar-
ities, vanillic acid and isorhamnetin were used as IS for quantification of
phenolic acids and the flavonoid, respectively. It was previously
certified that these compounds were not present in the studied fruit
samples. For the quantitative analysis, 1 mg of each dried pulp extract
was dissolved in 1 mL of methanol 80% and then filtered through a
polyvinylidene difluoride (PVDF) membrane filter of 0.45 lm pore
before injections.
2.9 | Column chromatography of uchi extract and
chemical modifications
The crude ethanolic extract (3.7 g, 51.9%) was suspended in EtOH/
water (1:4), partitioned with hexane and CH2Cl2. The organic phase
was evaporated yielding 650 mg of crude fraction. Thus, this was sub-
jected to a silica gel (70230 mesh) column chromatography (CC). The
fractionation was performed using a gradient elution from 100:0 to
0:100, CH2Cl2-EtOAc. Fractions 3944 (EtOAc 100%) showed to be
formed by crystals. After thin layer chromatography analysis, these
fractions were pooled according to their Rf values and subjected to re-
crystallization with a mixture of EtOAc-MeOH (9:1) yielding bergenin
(9 102.8 mg) (Nunomura et al., 2009).
The isolated compound (100 mg, 0.304 mmol) was solubilized in
anhydrous dimethylformamide and reacted with K2CO3 (1.52 mmol,
5 eq.) for 15 min, under agitation and at room temperature. Further,
181.2 mL of benzyl bromide (1.52 mmol, 5 eq.) were added, and
reacted overnight at the same conditions (Duke, 1970). After the
reaction period, the products were purified over CC using an iso-
cratic elution with CH2Cl2-EtOAc (1:1) to give 9a (62.1 mg, 62%
yield).
DE FREITAS ET AL. | 5 of 10

T AB LE 1 TP and TF contents, antioxidant, enzyme inhibition, and antimicrobial activities recorded for the evaluated fruit samples, com-
pounds, and controls

Antioxidant activityc Enzyme inhibitiond Antimicrobial activitye

Lipase a-glucosidase
p < .04 p < .02
TPCa TFCb DPPH ABTS
Sample p < .04 p < .02 p < .03 p < .01 % inhibition C. albicans S. aureus

Bacuri 23.28 6 1.28 i

15.34 6 0.91 29.0 6 0.99 49.8 6 2.15 2.47 6 0.42 98.65 6 2.15
IC50e 5 15.20 6 0.96

Ing
a 3.86 6 0.47 1.75 6 0.22 1.91 6 0.21 12.2 6 0.93 5.48 6 0.63 6.36 6 0.31

Uchi 33.38 6 1.51 19.44 6 0.87 34.2 6 1.19 51.6 6 1.83 2.28 6 0.52 21.88 6 2.16 250

Bergenin 250

5,7-Dibenzyloxy-bergenin 125

Oliostate f
93.24 6 1.14

Quercetine f
99.27 6 2.80 10.65 6 1.06

Ketoconazole g
3.50

Tetracycline h
1.25
a
Total phenolic content expressed in mg GAE/100g of fresh pulp.
b
Total Flavonoids content expressed in mg QUERE/100 g of fresh pulp.
c
Values expressed in mg TE/100 g of fresh pulp.
d
Values expressed in mg TE/100 g of fresh pulp.
e
MIC values expressed in mg/mL.
f
Enzyme inhibition positive control.
g
Antifungal positive control.
h
Antibacterial positive control.
i
All values were expressed as mean 6 SD.

2.9.1 | 5,7-Dibenzyloxybergenin (9a) FolinCiocalteus method and results for the studied Amazon fruits,

White amorphous powder; ESIMS: m/z 327 [M-H] ; 2 13

C NMR (125
MHz, CD3OD): dC 164.6 (C-2), 118.7 (C-3), 111.8 (C-4), 152.7 (C-5),
149.1 (C-6), 149.7 (C-7), 127.6 (C-8), 74.3 (C-9), 80.8 (C-11), 69.7 (C-
0 fresh pulp) and ing
a (3.86 6 0.47 mg GAE/100 g fresh pulp). Comparing
12), 72.0 (C-13), 80.2 (C-14), 60.9 (CH3O-15), 61.0 (C-16), 69.9 (C-1 ),
135.9 (C-20 ), 127.5 (C-30 ), 128.5 (C-40 ), 128.2 (C-50 ), 128.5 (C-60 ), 127.5
(C-70 ), 76.1 (C-100 ), 136.4 (C-200 ), 127.5 (C-300 ), 128.5 (C-400 ), 128.2
(C-500 ), 128.5 (C-600 ), and 127.5 (C-700 ).
2.10 | Statistical analysis
All analyses were run in triplicate and the results were expressed as
mean 6 SD. Differences between means were first analyzed using the
ANOVA test and then post hoc Tukey test (p < 0.05).
3 | RESULTS AND DISCUSSION
3.1 | Total phenolic and flavonoid content (TPC and
TFC)
Phenolic compounds are considered the most antioxidant active
metabolites from plants and they have the ability to donate electrons
or hydrogen to form stable radical intermediates (Bors, Michel, &
Stettmaier, 2001). The quantification of TPC was conducted using the
bacuri, ing

a, and uchi are shown in Table 1.
Uchi displayed the highest amount of TPC (33.38 6 1.51 mg GAE/
100 g fresh pulp), followed by bacuri (23.28 6 1.28 mg GAE/100 g
the obtained results with those previously reported for some Amazon
fruits, bacuri, uchi, and ing
a display lower TPC values than buriti
Mauritia flexuosa L. f. (378.07 mg GAE/100 g extract), composed mainly
of quinic, protocatechuic, chlorogenic and p-coumaric acids (Koolen
et al., 2013), tucum~
aAstrocaryum acualeatum G. Mey. (456.8 mg
GAE/100 g fresh weight), where ascorbic acid and carotenoids are the
major constituents (Barreto, Benassi, & Mercadante, 2009) and aa-
do-AmazonasEuterpe precatoria, (26.45 mg GAE/100 g dry weight),
where it was observed that this fruit consists mainly of quercetin and
vanillic acid (Bataglion et al., 2015). However, uchi and bacuri pre-
sented values similar to that presented by watermelon (26 mg GAE/
100 g fresh pulp) (Reddy, Sreeramulu, & Raghunath, 2010).
Flavonoids, including flavones, flavanols, and condensed tannins,
are plant secondary metabolites. Consumption of vegetables and fruits
containing flavonoid has been associated to protection against heart
disease and cancer (Juan & Chou, 2010). The results for TF, also
observed at Table 1, showed that uchi extract presented the highest
amount 19.44 6 0.87 mg QUERE/100 g fresh pulp), followed by bacuri
(15.34 6 0.91 mg QUERE/100 g fresh pulp) and ing

a (1.75 6 0.22 mg
QUERE/100 g fresh pulp). Koolen et al. (2013) found in buriti
6 of 10 | DE FREITAS ET AL.

(M. flexuosa L. f.) fruits TFC ranging from 246.84 6 1.11 to 567.16 6 T AB LE 2 Correlation between total phenolic content (TPC), total
1.15 mg QUERE/100 g dried weight, where the mainly flavonoids flavonoid content (TFC), and antioxidant activity of bacuri, ing
a, and
uchi
were (1) catechin, (2) epicatechin, and luteolin. Apigenin, myricetin,
luteolin, kaempferol, and quercetin were also observed in lower Correlation R2
concentrations. TPC versus DPPH 0.98

TPC versus ABTS 0.95

3.2 | Antioxidant analysis
TFC versus DPPH 0.99
The multifunctional characteristics of phenolic compounds delivered by
TFC versus ABTS 0.97
Amazon fruits are well known due to the different geographical loca-
ABTS versus DPPH 0.99
tion, system compositions, oxidative conditions, maturity, and antioxi-
dant mechanisms (Prior, Wu, & Schaich, 2005). Thus, the antioxidant
effectiveness of a pulp extract is best measured by results of generally
bowel pain and flatulence (Fujisawa, Ikegami, Inoue, Kawabata, & Ogi-
accepted tests (e.g., spectrophotometric measurements). Based on
hara, 2005). One way to circumvent these problems would be a healthier
these conditions and different available tests, DPPH and ABTS assays
diet with vegetables, and especially fruits, being a more acceptable
were chosen to evaluate the antioxidant capacity of uchi, bacuri, and
source of glycosidase inhibitors, due to the relative safety, including low
ing
a using trolox as positive control (IC50 4.4 lg/mL). In DPPH assay,
incidences of gastrointestinal collateral effects (Benalla, Bellahcen, &
uchi pulp displayed the highest antioxidant capacity between the ana-
Bnouham, 2010). Thus, some fruits, depending on their compositions,
lyzed fruits (34.2 6 1.19 mg TE/100g), followed by bacuri (29.0 6
act as a-glucosidase inhibitors. Extracts of the fruits bacuri, ing
a, and uchi
0.99 mg TE/100g) and ing
a (1.91 6 0.21 mg TE/100g). These results
were tested in vitro for their enzyme inhibition capacities. Bacuri dis-
indicate that uchi and bacuri fruits have an antioxidant activity similar
played the highest inhibition percentage for this enzyme (98.65 6
to that presented by watermelon (32 mg TE/100 g) (Reddy et al.,
2.15%). Conversely, ing
a and uchi showed low inhibition percentage
2010). However, the values of antioxidant activity obtained in this
(6.36 6 0.31% and 21.88 6 2.16%, respectively). As bacuri was the only
work by DPPH assay were much lower than those presented by Reddy
inhibitor fruit, its IC50 was determined (15.20 6 0.96 lg/mL). In addition,
et al. (2010) for orange (167 mg TE/100 g), mango (211 mg TE/100 g),
that value is similar to the IC50 value of quercetin, 10.65 6 1.06 lg
apple (330 mg TE/100 g), and guava (891 mg TE/100 g). These results
standard/mL. Comparing this result with the inhibition of a-glucosidase
can also be associated with the ABTS assay, where uchi and bacuri
presented by muscadine (Vitis rotundifolia) 1,920 lg/mL (You, Chen,
pulps presented close-related antioxidant capacities (51.6 6 1.83 and
Wang, Jiang, & Lin, 2012) and by 10 different cultivars of fig fruits (black,
49.8 6 2.15 mg TE/100 g, respectively). Ing
a gave a lower value
red, yellow, and green) and two varietal types (fig and brevas) that
(12.2 6 0.93 mg TE/100 g extract). In the ABTS assay, the antioxidant
showed a range of IC 50 from 18.3 to 22.1 mg/mL (Wojdyo, Nowicka,
capacity of uchi and bacuri showed almost twice the value of the anti-
Carbonell-Barrachina, & Hern

andez, 2016), bacuri showed a higher inhi-
oxidant activity presented by Reddy et al. (2010) to pineapple (22 mg
bition potential than muscadine and fig fruits, showing that the fresh
TE/100 g), watermelon (23 mg TE/100 g), orange (22 mg TE/100 g),
consumption of this fruit may be beneficial.
sweetlime (26 mg TE/100 g), and banana (30 mg TE/100 g). However,
the antioxidant activity of uchi and bacuri fresh pulps was lower than 3.3.2 | Lipase inhibition assay
those presented by grapes (green) (85 mg TE/100 g), pomegranate The pancreas releases a pancreatic lipase, which hydrolyze triglycerides,
(135 mg TE/100 g), and guava (496 mg TE/100 g) (Reddy et al., 2010). turning them into glycerol and fatty acids (Birari & Bhutani, 2007). This
Based on the performed antioxidant assays, comparing with other enzyme plays an important role in the absorption of triglycerides,
fruits, bacuri and uchi presented an antioxidant capacity similar or because dietary fat is not directly absorbed (Lowe, 1994). Lipase inhibi-
higher than several fruits. It is also possible to observe a significant cor- tors have demonstrated a variety of pharmacological effects such as
relation between the TPC and TFC contents and the antioxidant anti-obesity, reduction of serum cholesterol, and diabetes and hyper-
capacity from both the DPPH (r2 5 0.98 and 0.99, respectively) and tension prevention capacities. Therefore, the pancreatic lipase is con-
ABTS (r2 5 0.95 and 0.97, respectively) assays (Table 2). sidered an important target to evaluate the ability of fruit pulps in the
inhibition, and consequently its prevention capacity (Garza, Milagro,
3.3 | Enzymatic inhibition analysis
n, & Martnez, 2011). Unfortunately, none of the
Boque, Campio
assayed fruits displayed enough inhibition capacity to be further
3.3.1 | a-Glucosidase inhibition assay
studied. Among them, ing
a had the best result, being able to inhibit
Glycemic control is an effective and long-term therapy for Type 2 diabe-
5.48 6 1.13% the activity of pancreatic lipase (Table 1).
tes, since controls glucose absorption. Glucosidase inhibitors are com-
monly prescribed to reduce postprandial hyperglycemia induced by
3.4 | Antimicrobial assays
starch digestion in the small intestine (Bolen et al., 2007). However, it
has been reported many times that synthetic glucosidase inhibitors (acar- The preliminary evaluation of the antimicrobial activity of the pulp
bose and miglitol) cause diarrhea and other intestinal disorders, such as extracts showed that bacuri and ing
a are inactive, whereas uchi pulp
DE FREITAS ET AL.
FIGURE 2 Derivatization of bergenin: In brief, an amount of
100 mg of 9 (0.304 mmol) was solubilized in anhydrous
dimethylformamide and reacted with K2CO3 (1.52 mmol, 5 eq.) for
15 min, under agitation, and at room temperature. Further, 181.2
mL of benzyl bromide (1.52 mmol, 5 eq.) were added, and reacted
overnight at the same conditions (Bajracharya, 2015).
extract displayed MIC value at 250 mg/mL against S. aureus. During the
extraction step for uchi, a formation of a crystal-like solid in the flask
walls was observed, motivating its evaluation. After purification steps,
this crystalline compound was identified as being a trihydroxybenzoic
acid glycoside known as bergenin by means of nuclear magnetic reso-
nance analysis (NMR) in comparison with published data (Nazir, Koul,
Qurishi, Najar, & Zargar, 2011). This substance displays several biologi-

nior et al., 2013), anti-
cal activities such as antiinflammatory (Costa Ju
~ a et al., 2009), hepatoprotective (Diderot et al., 2006),
oxidant (Acun
anti-HIV (Kumar et al., 2013), and antimicrobial (Genovese, Pinto,
Souza, & Lajolo, 2008), but it presents a poor antimicrobial activity
(Nazir et al., 2011). After reaction with potassium carbonate and benzyl
bromide in dimethylformamide yielded the compound 5,7-dibenzylber-
genin (Figure 2).
Hereby, we report the moderate antimicrobial activity of the
dibenzyl derivative against Staphylococcus aureus with a MIC at 125
mg/mL. Nazir et al. (2011) have synthesized compounds from bergenin
via chemoenzymatic routes, obtaining acyl derivatives showing MIC at
2,000 mg/mL against the same bacteria. From this, it can be concluded
that the derivatization step performed has increased the antimicrobial
potential of bergenin, and consequently from the uchi pulp extract.
3.5 | UHPLC-MS/MS analysis
An UHPLC-ESI-MS/MS method was developed, validated, and then
applied for simultaneous quantification of antioxidant compounds pres-
ent in fruits in a previous study (Bataglion et al., 2015). Herein, this
method was slightly modified for quantifying antioxidant compounds in
bacuri, ing
a, and uchi. Once the chromatographic and mass spectrome-
try parameters were set, a standard mixture solution of compounds
present in the fruits was analyzed and the obtained chromatogram in
the optimum conditions is shown in the Figure 3.
The optimized method was validated through evaluating the linear-
ity of calibration curves, recovery, and intra- and interday accuracies
and precisions. For the compounds found in bacuri, ing
a, and uchi,
LOD and LOQ values are showed at Table 3, and indicate a satisfactory
sensitivity toward each chosen standard. Accuracy and precision
parameters were evaluated using quality control (QC) samples contain-
ing a mixture of sugars and the target compounds at low, medium, and
| 7 of 10
FIGURE 3 Representative chromatogram obtained from a
standard mixture of the compounds by SRM mode; Gallic (1) and
citric (2) acids, (1)-catechin (3), (2)-epicatechin (4), vanillic acid
(5) (IS), p-coumaric acid (6), luteolin (7), and isorhamnetin (8) (IS)
high concentrations. According to Bataglion et al. (2015) is the valida-
tion of this method was satisfactory (Figure 4).
After validated, the method was applied aiming the targeted
quantitative analysis of selected main compounds present in bacuri,
ing

a, and uchi pulps. The chromatogram and concentrations of the
compounds investigated for the analyzed fruit extracts are shown in
Figure 3 and Table 3, respectively. The citric acid (2) was the domi-
nant organic acid for bacuri, ing
a, and uchi, (14.36, 10.40, and 6.58
mg/g of fresh fruit pulp, respectively). These values were much higher
than those found for Actinidia fruits analyzed by Wojdyo, Nowicka,
Oszmianski, and Golis (2017) ranging from 0.057 to 0.095 mg/g of
fresh fruit for 2. Abdel-Salam et al. (2014) suggest an antioxidant and
antiinflammatory effect for orally given citric acid at 12 g/kg in brain
tissue and also have demonstrated a beneficial hepatic protective
effect at this dose range. Others studies have suggested the use of
this compound as an effective means of treating oxalate stones
(Haleblian et al., 2008; Kang et al., 2007). Moreover, citric acid had
also been described as driver of thermogenesis, reducing obesity risk
(Ferrara, 2007). Catechins were only encountered in ing

a fruit pulp,
where (1)-Catechin (5.05 mg/g of fresh fruit pulp for 3) was observed
as the most abundant diasteromer, whereas (2)-epicatechin (0.06 mg/
g fresh fruit for 4) was observed as a minor component, being those
amounts lower than for other typical Amazonian fruits, such as buriti
(Bataglion, Silva, Eberlin, & Koolen, 2014). Maeda et al. (2003)
showed that catechins can inhibit the invasion and proliferation of
the smooth muscle cells in the arterial wall, contributing to decrease
down the formation of the atheromatous lesion. Gallic acid (1), an
antioxidant compound with many biological properties (Yamaguchi
et al., 2014), was exclusively detected and quantified by our method
in the edible pulp of uchi (0.36 mg/g of fresh fruit pulp for 1), lower
than that found for the dry weight pulp (DWP) of cashew apple (Ana-
cardium occidentale) and camu-camu (Myrciaria dubia) (Bataglion et al.,
2015). Additionally, the phenolic p-coumaric acid was found to be a
minor component in bacuri (0.07 mg/g of fresh fruit pulp for 6) and
uchi (0.01 mg/g of fresh fruit pulp for 6). Luteolin, a common flavonoid
widespread in fruits, was only found in uchi pulps (0.10 mg/g of fresh
fruit pulp for 7).
8 of 10 | DE FREITAS ET AL.

FIGURE 4 Intra- and interday precisions and accuracies for quality control (QC) samples at three different concentration levels (LQC: 100
ng/mL; MQC: 400 ng/mL; HQC: 800 ng/mL) for the quantified compounds

T AB LE 3 Quantified compounds and validation parameters (retention time [R.T.], linearity [R2], slope, intercept, limit of detection [LOD], and
limit of quantification [LOQ] for the analyzed compounds)

Compound [M-H]2/transition Bacuria Ing

a uchi R.T.b R2 Slope Intercept LODc LOQd

Gallic acid m/z 169/125, 79 e 0.36 3.46 0.998 0.0207 20.3714 0.043 0.144

Citric acid m/z 191/111, 172 14.36 10.40 6.58 3.26 0.998 0.0091 20.0910 0.032 0.109

(1)-Catechin m/z 289/153, 121 5.05 4.33 0.997 0.0004 20.0022 0.052 0.175

(2)-Epicatechin m/z 289/153, 121 0.06 4.67 0.998 0.0003 20.0004 0.040 0.133
f
Vanillic acid m/z 167/152, 108 IS IS IS 4.79 0.998 0.0024 20.0104 0.037 0.125

Luteolin m/z 285/151, 133 0.10 6.39 0.998 0.0049 0.0574 0.042 0.142

p-Coumaric acid m/z 163/119, 93 0.07 0.01 5.13 0.998 0.1474 21.0991 0.038 0.128

Isorhamnetin m/z 315/151, 107 IS IS IS 6.97 0.998 0.0185 0.0541 0.032 0.108
a
Values expressed in mg/g of fresh pulp.
b
Retention time.
c
Limit of detection.
d
Limit of quantitation.
e
Not present and/or below obtained LOD.
f
Internal standard.
DE FREITAS ET AL.
4 | CONCLUSIONS
Antioxidant activity evaluation revealed the antioxidant properties of
uchi, bacuri, and ing
a, but with moderate activities when compared
with other fruits. Bacuri presented the highest rate of a-glycosidase
inhibition, indicating a potential source of glucose-lowering com-
pounds. Uchi displayed weak antioxidant and antimicrobial potentials.
Thus, its main constituent was isolated and chemically-modified,
displaying an increase of 50% in the activity against S. aureus. Despite
the high consumption of ing
a in the North region, this fruit did not dis-
play the same potentials. The application of the proposed UHPLC-MS/
MS yielded the determination of six compounds in the studied fruits.
The results of this work will contribute to guide commercial applica-
tions of these fruits, and to evaluate its economic potential as natural
food sources from the Amazon region of Brazil.
AC KNOW LE DGME NT S
The authors would like to thank to Funda~ao de Amparo a Pesquisa
do Estado do Amazonas (FAPEAM) and Coordenadoria de Aperfei-
oamento de Pessoal de Nvel Superior (CAPES).
OR CID
Fl
avio A. de Freitas http://orcid.org/0000-0001-7940-4910
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SUP POR TI NG INFOR MATION
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jo RC, Soares ER,
How to cite this article: de Freitas FA, Arau
et al. Biological evaluation and quantitative analysis of antioxi-
dant compounds in pulps of the Amazonian fruits bacuri (Plato-
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a (Inga edulis Mart.), and uchi (Sacoglottis
uchi Huber) by UHPLC-ESI-MS/MS. J Food Biochem. 2017;
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