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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.), inga (Inga edulis Mart.), and uchi (Sacoglottis uchi
Huber) by UHPLC-ESI-MS/MS

Flavio 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, inga, and uchi are Amazon fruits consumed specially in the North region of Brazil. Due to
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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
Flavio 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, inga, 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 ET AL.

1 | INTRODUCTION Inga, also known as inga-cipo


 , belonging to the family Fabaceae, is
naturally distributed along South America, with major concentration in
A balanced diet based on the regular consumption of fruits has been the Amazon estuary (Dias, Souza, & Rogez, 2010). This fruit has a
associated with many pharmacological benefits (Koolen, Silva, Gozzo, sweet flavor and softness of the pulp, being appreciated by the local
Souza, & Souza, 2013). Specially, prevention of cardiovascular diseases, population. Some medicinal properties are attributed to a regular intake
inflammations, and aging-related disorders can also be correlated with of inga, such as the cure of arthritis, diarrhea, and rheumatism (Duke,
intake of fruits, especially those which are rich in phenolic compounds 1975). These fruits are cylindrical in shape with 2540 cm in diameter,
and vitamins (Huang, Ou, & Prior, 2005). weighing between 100 and 800 g, including the pod and usually
Phenolic compounds are attractive due to their antioxidant activity contain between 8 and 15 seeds (Figure 1c) (Duke, 1975). The inga is
that reduces oxidative stress and prevents or delays oxidation by scav- also known as ice-cream bean (alluding to its pulps characteristics) and
enging free radicals (Koolen et al., 2013). The use of antioxidants it is used to prepare an alcoholic beverage from the aril, where indige-
derived from natural resources is gaining attention due to their health nous people from Panama and Colombia (Duke, 1970) consume the
benefits, which include prevention of cardiovascular diseases, beverage, called cachiri, at a festival of same name.
inflammations, and aging-related disorders (Aqil et al., 2012). These Chemically, those fruits are poorly studied and their properties
compounds have been observed in amazonic plants as bacuri (Platonia such as: antimicrobial, anticonvulsant, antiviral, antioxidant, antidepres-
insignis Mart.), inga (Inga edulis Mart.), and uchi (Sacoglottis uchi Huber) sant, wound healing, and cytotoxic activities have been the subject of
[sin. Endopleura uchi] (Gomes, Ghica, Rodrigues, Gil, & Oliveira-Brett,  nior et al.,
several pharmacological and toxicological studies (Costa Ju
2016; Silva, Rogez, & Larondelle, 2007). nior et al., 2010). Bacuri has demonstrated its wealth in
2013; Santos Ju
Uchi, belonging to the family Humiriaceae, is native from the polyisoprenylated benzophehones and xanthones, responsible for sev-
Amazon region and it is popularly known as uchi, uxi, uxi-amarelo, ~ a, Jancovski, & Kennelly, 2009; Diderot,
eral biological activities (Acun
pururu, uxi-liso, uxi-ordinario, or uchi-pucu (Silva, Oliveira, Tomonasa, Silvere, & Etienne, 2006; Kumar, Sharma, & Chattopadhyay, 2013), and
& Nunomura, 2009). These fruits are ellipsoid in shape with 2.57 cm also in polyphenolic molecules, compounds of great interest for having
in diameter, and normally contain one or two seeds. The skin of the antioxidant activity (Gomes et al., 2016; Magalh~aes, Lima, Marinho, &
uchi fruit is thin, tender, and brown in appearance (Figure 1a). The pulp Ferreira, 2007). Only diterpenes, xanthones, and fatty acids from the
presents a yellow-brownish color and a rough-like texture, being the seeds (Yamaguchi et al., 2014) and monoterpenes for the pulp were
sole edible part of uchi. In addition, its rich flavor is unique, not compa- found as constituents of bacuri (Borges & Rezende, 2000). Regarding
rable with any known fruit. Recent studies point to popular uses of inga, it has not been reported in the literature data about isolated or
^a, 1984; Nazir et al., 2007; Nunomura,
S. uchi as a medicinal plant (Corre identified compounds from its fruits. However, data were reported
Oliveira, Silva, & Nunomura, 2009; Silva et al., 2009) reinforcing the about isolated phenolic compounds from the inga leaves, and the eval-
idea that this fruit may be a functional food source. uation of the antioxidant capacity (Dias et al., 2010; Silva et al., 2007).
Bacuri, belonging to the family Clusiaceae, is naturally distributed Concerning to uchi, the literature has reported a high content of fatty
along the south of the Amazon forest, with major concentration in the acids in the fruits, predominantly the oleic acid (Marx, Andrade, Zoghbi,
Amazon estuary being probably domesticated by the Amazon & Maia, 2002) and carotenoids, mainly trans-b-carotene (Magalh~aes
Amerindians (Rogez et al., 2004). The local population appreciates this et al., 2007). Bergenin was also isolated from uchi pulps and barks and
fruit by the unique aroma and flavor of the edible part (pulp) it has been reported that this compound have several biological activ-
(Yamaguchi, Pereira, Lima, & Veiga-Junior, 2014) and by its the medici- ities (Magalh~aes et al., 2007; Nazir et al., 2007; Silva et al., 2009).
nior et al., 2010). The fruits are ovoid in shape
nal properties (Costa Ju Due to the lack of knowledge regarding their chemical composition
(715 cm in diameter, from 200 to 1,000 g of fresh weight), and and the potential as antioxidants source and functional food, this
normally contain three to five big seeds. The rind is thick (13 cm), pale work aimed the determination of the phenolic composition by ultra-
yellow to brownish yellow, tough and somewhat elastic, and exudes a high liquid chromatography coupled to tandem mass spectrometry
yellow latex when pressed (Figure 1b) (Rogez et al., 2004). (UHPLC-MS/MS) of the Amazon fruits bacuri, inga, and uchi. In

FIGURE 1 (a) Uchi (Sacoglottis uchi Huber), (b) bacuri (Platonia insignis Mart.), and (c) inga (Inga edulis Mart.)
DE FREITAS ET AL. | 3 of 10

addition, this study also displays the chemical fractionation of uchi pulp supernatant was collected, placed in glass centrifuge tubes and flushed
extract for the isolation of bergenin and further chemical modifications with nitrogen gas. All extractions were performed in triplicate. Addi-
for antimicrobial activity enhancement evaluation. 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 | MATERIALS AND METHODS
2.3 | Determination of TPC
2.1 | Standard chemicals
The TPC of the studied fruits pulps was determined using the Folin
Antioxidant compounds, p-coumaric acid, (1)-catechin, (2)-epicatechin, Ciocalteu reagent (Singleton & Rossl, 1965). Each of the crude extracts
citric acid, gallic acid, vanillic acid, isorhamnetin, and luteolin used as (1 mg) was dissolved in pure water (25 mL). Aliquots (250 lL) were
standards were purchased from Sigma Aldrich (St. Louis, USA). The mixed with 1 mL of the FolinCiocalteu reagent and 1 mL of a 10%
FolinCiocalteu reagent (2,4,6-tris(2-pyridyl)-s-triazine) (TPTZ) and (m/v) Na2CO3 solution. Samples were then incubated at 308C for 1.5 h
resazurin dye was obtained from Fluka-Chemie (Buchs, Switzerland). and the absorbance of each sample was measured at 765 nm. The TPC
The aluminum chloride, sodium carbonate, sodium acetate, benzyl was calculated from a calibration curve, using gallic acid as standard
bromide, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,20-Azinobis-3-ethyl- (1.257.5 lg/mL). TPC contents were expressed in mg gallic acid equiv-
benzo thiazoline-6-sulfonic acid, diammonium salt (ABTS), o-dianisidine alent (GAE)/100 g fresh pulps.
color reagent (DIAN), glucose oxidase and peroxidase enzyme solution
(PGO), acarbose, p-nitrophenyl octanoate (NPC), porcine pancreas 2.4 | Determination of total flavonoid content
lipase (Type II, from porcine pancreas, 100400 units/mg protein),
The total flavonoid content (TFC) was measured by the AlCl3 method
TrisHCl buffer (pH 8.5), and a-glucosidase from Saccharomyces cerevi-
(Lamaison & Carnet, 1990). The extracts (0.5 mg) were dissolved in
siae (Type I; 10 U/mg protein) were purchased from Sigma-Aldrich
pure water (25 mL). Aliquots (1.5 mL) of the solutions were added to
(Steinheim, Germany). Silica gel (70230 mesh) from Silicycle, Mueller
equal volumes of a solution 6% (m/V) AlCl3H2O. The mixture was vig-
Hinton agar was purchased from Difco. Monosaccharides, glucose,
orously shaken, and absorbance was read at 367.5 nm after 10 min of
fructose, saccharose, arabinose, and ramnose used to evaluate the
incubation. TFC were expressed in mg quercetin equivalent (QUERE)/
matrix effect were purchased from Merck (Darmstadt, Germany).
100 g fresh pulp.
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
2.5 | Antimicrobial assays
Milli-Q gradient system (Millipore, Milford, USA). Minimal inhibitory concentrations (MICs) were determined by the
microbroth-dilution assay, as recommended by the U.S. National
2.2 | Sample preparation Committee for Clinical Laboratory Standards (NCCLS). Assays were
performed into 96-well plates supplied with 100 lL of Mueller
Fresh inga (2.0 kg), bacuri (1.2 kg), and uchi (5.8 kg) fruits were pur-
Hinton Broth culture medium, 100 lL of each sample solution, and 5
chased at Adolpho Lisboa market in Manaus, a city located in the heart
lL of test bacterial suspensions at 1.0 3 107 UFC/mL (Koolen,
of the Amazon region of Brazil. The ripeness state of each fruit was
Soares, Silva, Almeida, & Souza, 2012). The pulp extracts were
carefully checked, where the only ones proper for consumption were
dissolved in DMSO at initial concentration of 500 lg/mL being
selected (ripe fruits). The maturity degree was determined according to
subsequently diluted until 3.75 lg/mL. The incubation was made at
size, color, and firmness. Pulps of bacuri, uchi, and inga were manually
378C during 24 hr. The microorganisms tested were: Bacillus cereus,
separated from the peels and seeds, and only the pulps were submitted
Candida albicans, Candida tropicalis, Escherichia coli, Staphylococcus
to extraction. Antioxidant compounds were extracted from the studied
aureus, and Streptococcus mutans hospital wild colonies. The bioac-
fruit pulps using an ETHOS 1 microwave extraction system (Milestone,
tivities were recorded as blue coloration in the wells after use of
Shelton, USA). After a preliminary evaluation of the total phenolic con-
resazurin dye. The bacteriostatic or bactericidal effects of the
tent (TPC) (data not shown) in different extraction conditions, an opti-
metabolites were observed by inoculation of the well materials on
mized extraction method was obtained. The potency was 600 W and
Mueller Hinton Agar plates after the tests. Positive controls tetracy-
irradiation time 60 s, temperature 458C, where an amount of 2.5 g of
cline (4 lg/mL) and ketoconazole (4 lg/mL) with the negative con-
fruit pulps was extracted with 37.5 mL of an aqueous EtOH solution
trol DMSO were used during the tests.
(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
2.6 | Antioxidant assays
recovery for bacuri (105.8 mg, 4.23%), inga (42.6 mg, 1.70%), and uchi
(129.3 mg, 5.17%). Teflon PFA (perfluoroalkoxy) vessels with 100 mL The antioxidant capacity of the extracts of bacuri, inga, and uchi were
of capacity were used. After extraction, the liquid was filtered under evaluated by DPPH and ABTS assays. The DPPH and ABTS assays
reduced pressure and allowed to cool to room temperature (ca., 258C), were performed in triplicate and in the same conditions used in a
and submitted to centrifugation at 4,000 rpm for 15 min. The 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 the product ions by MS/MS experiments. Then, analyses were con-
734 nm. For both assays, the percentage of inhibition was calculated ducted by selected reaction monitoring (SRM) in the negative ion
according to the equation: % Inhibition 5 100 2 (absorbance/average mode monitoring two transitions for each standard compound using
absorbance of control) 3 100, and expressed as trolox equivalent (TE) 20 ms of dwell time. The settings of the mass spectrometer were
per gram of fresh pulp. optimized for each SRM transition compound analysis and are
shown in a previous study (Bataglion, Silva, Eberlin, & Koolen,
2.7 | Enzymatic inhibition analysis 2015). Data were acquired and processed by Labsolution software
(v. 5.53 SP2, Shimadzu).
2.7.1 | a-Glucosidase inhibition assay
The method was previously validated according to the U.S. Food
For this procedure, a previously described methodology was employed ^ncia Nacional de
and Drug Administration (FDA) and by the Age
(Iauk et al., 2015). In brief, extracts and controls were solubilized in Vigil^ancia Sanitaria (ANVISA) guidelines over three consecutive days
DMSO and added to a maltose (40 mg/mL) in 50 mM of sodium ace- for linearity, LOD, LOQ, inter- and intraday accuracy and precision, and
tate buffer solution. The addition of a-glucosidase (100 mg/mL) solution recovery. Details about how validation method was conducted are
started the reaction. After 30 min of incubation at 378C, the reaction available in elsewhere (Bataglion et al., 2015). Stock solutions of each
was stopped by adding perchloric acid (4.2%, m/v). The generation of standard compound (1 mg/mL) were prepared and stored in methanol
glucose was quantified by the reduction of the DIAN. The supernatant at 48C. An intermediate solution containing all standard compounds
of the reaction was mixed with DIAN and PGO system-color reagent (1 mg/mL) was prepared in methanol and dilutions from this solution
and was left to incubate at 378C for 30 min. The absorbance was meas- were done at nine different levels for calibration curves and method
ured at 500 nm. Gallic acid was used as positive control. As a measure validation. A stock solution of internal standard (IS, 20 mg/mL) was pre-
of inhibitory potency, the concentration required for 50% inhibition of pared and stored in methanol and dilutions were done to reach a final
enzyme activity (IC50) was determined. concentration of 500 ng/mL in the calibration curves, which were gen-

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

Inga 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 bacuri, ing
a, and uchi are shown in Table 1.
Uchi displayed the highest amount of TPC (33.38 6 1.51 mg GAE/
MHz, CD3OD): dC 164.6 (C-2), 118.7 (C-3), 111.8 (C-4), 152.7 (C-5),
100 g fresh pulp), followed by bacuri (23.28 6 1.28 mg GAE/100 g
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 inga (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 ),
the obtained results with those previously reported for some Amazon
135.9 (C-20 ), 127.5 (C-30 ), 128.5 (C-40 ), 128.2 (C-50 ), 128.5 (C-60 ), 127.5
fruits, bacuri, uchi, and inga display lower TPC values than buriti
(C-70 ), 76.1 (C-100 ), 136.4 (C-200 ), 127.5 (C-300 ), 128.5 (C-400 ), 128.2
Mauritia flexuosa L. f. (378.07 mg GAE/100 g extract), composed mainly
(C-500 ), 128.5 (C-600 ), and 127.5 (C-700 ).
of quinic, protocatechuic, chlorogenic and p-coumaric acids (Koolen
et al., 2013), tucum~
aAstrocaryum acualeatum G. Mey. (456.8 mg
2.10 | Statistical analysis GAE/100 g fresh weight), where ascorbic acid and carotenoids are the
major constituents (Barreto, Benassi, & Mercadante, 2009) and aa-
All analyses were run in triplicate and the results were expressed as
do-AmazonasEuterpe precatoria, (26.45 mg GAE/100 g dry weight),
mean 6 SD. Differences between means were first analyzed using the
where it was observed that this fruit consists mainly of quercetin and
ANOVA test and then post hoc Tukey test (p < 0.05).
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).
3 | RESULTS AND DISCUSSION
Flavonoids, including flavones, flavanols, and condensed tannins,
are plant secondary metabolites. Consumption of vegetables and fruits
3.1 | Total phenolic and flavonoid content (TPC and
containing flavonoid has been associated to protection against heart
TFC)
disease and cancer (Juan & Chou, 2010). The results for TF, also
Phenolic compounds are considered the most antioxidant active observed at Table 1, showed that uchi extract presented the highest
metabolites from plants and they have the ability to donate electrons amount 19.44 6 0.87 mg QUERE/100 g fresh pulp), followed by bacuri
or hydrogen to form stable radical intermediates (Bors, Michel, & (15.34 6 0.91 mg QUERE/100 g fresh pulp) and ing
a (1.75 6 0.22 mg
Stettmaier, 2001). The quantification of TPC was conducted using the 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, inga, 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
inga 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, inga, and uchi
0.99 mg TE/100g) and inga (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, inga 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). Inga 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, inga 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 inga are inactive, whereas uchi pulp
DE FREITAS ET AL. | 7 of 10

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 FIGURE 3 Representative chromatogram obtained from a
overnight at the same conditions (Bajracharya, 2015). 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)
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 high concentrations. According to Bataglion et al. (2015) is the valida-
walls was observed, motivating its evaluation. After purification steps, tion of this method was satisfactory (Figure 4).
this crystalline compound was identified as being a trihydroxybenzoic After validated, the method was applied aiming the targeted
acid glycoside known as bergenin by means of nuclear magnetic reso- quantitative analysis of selected main compounds present in bacuri,
nance analysis (NMR) in comparison with published data (Nazir, Koul, ing
a, and uchi pulps. The chromatogram and concentrations of the
Qurishi, Najar, & Zargar, 2011). This substance displays several biologi- compounds investigated for the analyzed fruit extracts are shown in
 nior et al., 2013), anti-
cal activities such as antiinflammatory (Costa Ju Figure 3 and Table 3, respectively. The citric acid (2) was the domi-
~ a et al., 2009), hepatoprotective (Diderot et al., 2006),
oxidant (Acun nant organic acid for bacuri, inga, and uchi, (14.36, 10.40, and 6.58
anti-HIV (Kumar et al., 2013), and antimicrobial (Genovese, Pinto, mg/g of fresh fruit pulp, respectively). These values were much higher
Souza, & Lajolo, 2008), but it presents a poor antimicrobial activity than those found for Actinidia fruits analyzed by Wojdyo, Nowicka,
(Nazir et al., 2011). After reaction with potassium carbonate and benzyl Oszmianski, and Golis (2017) ranging from 0.057 to 0.095 mg/g of
bromide in dimethylformamide yielded the compound 5,7-dibenzylber- fresh fruit for 2. Abdel-Salam et al. (2014) suggest an antioxidant and
genin (Figure 2). antiinflammatory effect for orally given citric acid at 12 g/kg in brain
Hereby, we report the moderate antimicrobial activity of the tissue and also have demonstrated a beneficial hepatic protective
dibenzyl derivative against Staphylococcus aureus with a MIC at 125 effect at this dose range. Others studies have suggested the use of
mg/mL. Nazir et al. (2011) have synthesized compounds from bergenin this compound as an effective means of treating oxalate stones
via chemoenzymatic routes, obtaining acyl derivatives showing MIC at (Haleblian et al., 2008; Kang et al., 2007). Moreover, citric acid had
2,000 mg/mL against the same bacteria. From this, it can be concluded also been described as driver of thermogenesis, reducing obesity risk
that the derivatization step performed has increased the antimicrobial
(Ferrara, 2007). Catechins were only encountered in ing
a fruit pulp,
potential of bergenin, and consequently from the uchi pulp extract.
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/
3.5 | UHPLC-MS/MS analysis g fresh fruit for 4) was observed as a minor component, being those
An UHPLC-ESI-MS/MS method was developed, validated, and then amounts lower than for other typical Amazonian fruits, such as buriti
applied for simultaneous quantification of antioxidant compounds pres- (Bataglion, Silva, Eberlin, & Koolen, 2014). Maeda et al. (2003)
ent in fruits in a previous study (Bataglion et al., 2015). Herein, this showed that catechins can inhibit the invasion and proliferation of
method was slightly modified for quantifying antioxidant compounds in the smooth muscle cells in the arterial wall, contributing to decrease
bacuri, inga, and uchi. Once the chromatographic and mass spectrome- down the formation of the atheromatous lesion. Gallic acid (1), an
try parameters were set, a standard mixture solution of compounds antioxidant compound with many biological properties (Yamaguchi
present in the fruits was analyzed and the obtained chromatogram in et al., 2014), was exclusively detected and quantified by our method
the optimum conditions is shown in the Figure 3. in the edible pulp of uchi (0.36 mg/g of fresh fruit pulp for 1), lower
The optimized method was validated through evaluating the linear- than that found for the dry weight pulp (DWP) of cashew apple (Ana-
ity of calibration curves, recovery, and intra- and interday accuracies cardium occidentale) and camu-camu (Myrciaria dubia) (Bataglion et al.,
and precisions. For the compounds found in bacuri, inga, and uchi, 2015). Additionally, the phenolic p-coumaric acid was found to be a
LOD and LOQ values are showed at Table 3, and indicate a satisfactory minor component in bacuri (0.07 mg/g of fresh fruit pulp for 6) and
sensitivity toward each chosen standard. Accuracy and precision uchi (0.01 mg/g of fresh fruit pulp for 6). Luteolin, a common flavonoid
parameters were evaluated using quality control (QC) samples contain- widespread in fruits, was only found in uchi pulps (0.10 mg/g of fresh
ing a mixture of sugars and the target compounds at low, medium, and 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. | 9 of 10

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