Food and Chemical Toxicology 120 (2018) 479–490

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Food and Chemical Toxicology

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In vitro antioxidant and antihypertensive compounds from camu-camu T

(Myrciaria dubia McVaugh, Myrtaceae) seed coat: A multivariate structure-
activity study
Marina Fidelisa, Jânio Sousa Santosa, Graziela Bragueto Eschera, Mariana Vieira do Carmob,
Luciana Azevedob, Marcia Cristina da Silvac, Predrag Putnikd, Daniel Granatoa,∗
a
Graduate Program in Food Science and Technology, State University of Ponta Grossa, Avenida Carlos Cavalcanti, 4748, 84030-900, Ponta Grossa, Brazil
b
Faculty of Nutrition, Federal University of Alfenas, Rua Gabriel Monteiro da Silva, 714, 37130-000, Alfenas, Brazil
c
Department of Food, Federal Institute of Education, Science and Technology from Rio de Janeiro (IFRJ), 20270-021, Rio de Janeiro, Brazil
d
Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10000, Zagreb, Croatia

A R T I C LE I N FO A B S T R A C T

Keywords: Camu-camu (Myrciaria dubia) pulp, seeds, and skin are widely known because of their nutritional properties.
Inhibition of lipid oxidation However, the seed coat has never been studied as a source of bioactive compounds. Herein, we characterized the
DPPH• phenolic composition, the antioxidant activity, and inhibition of angiotensin-converting enzyme (ACE) of three
Phenolic acids different extracts (water, propanone, and ethanol) from this residue and assessed the structure-activity using
Flavonoids
bivariate and multivariate statistical approaches. Phenolic acids and flavonoids were quantified by high-per-
Food waste
formance liquid chromatography while the ferric reducing antioxidant power (FRAP), inhibition of lipid per-
HPLC-DAD
oxidation using egg yolk and Wistar rat brain, scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical,
Folin-Ciocalteu reducing capacity (FCRC), and the inhibition of angiotensin-converting enzyme (ACE) by the
extracts were also analyzed. t-Resveratrol was found in camu-camu seed coat for the first time. The aqueous
extract had the highest total phenolic content, FRAP, DPPH•, FCRC, and inhibition of lipid oxidation using both
chemical and biological assays, while the propanone extract showed the opposite behavior but it presented
higher in vitro antihypertensive activity. The ethanolic extract exhibited intermediate values for the responses.
The association between chemical composition and the functional properties of the camu-camu seed coat ex-
tracts were revealed using correlation analysis and principal component analysis.

1. Introduction minimizes its consumption in natura (Myoda et al., 2010). As it is not

Tropical fruits are attractive to the food industry mainly because of
their unique appearance, flavor, and nutritional value (Kaneshima
et al., 2013). Among the fruits classified as berries, camu-camu has
received attention due to its high content of bioactive compounds, such
as phenolic compounds (flavonoids and ellagitannins), ascorbic acid,
and β-carotene (Inoue et al., 2008; Gonçalves et al. al., 2010; Akter
et al., 2011).
Camu-camu (Myrciaria dubia [H.B.K] McVaugh), also known as
“caçari” or “araçá d'água”, was discovered in 1958. It belongs to the
Myrtaceae family and is spontaneously present on riverbanks and lakes
of the Amazon basin, between Peru and Brazil (Zapata and Dufour,
1993; Silva and Andrade, 1997). Due to its high content of ascorbic acid
and phenolic compounds, camu-camu reveals a bitter taste, which
widespread in the entire Brazilian territory, the commercialization is
achieved on a small scale, basically in the form of frozen pulp. In
Europe, Japan, Canada, and in the United States, there is great interest
in the fruit, which is imported and its pulp transformed into sparkling
drinks, vinegar, ice cream, and sweets (Yuyama, 2011).
The seeds and peels of camu-camu represent about 40% of the fruit
(Rodrigues et al., 2001). However, these by-products are generally
discarded without taking advantage of their chemical constituents. In
this aspect, these residues may present higher antioxidant potential
when compared to pulp because most bioactive compounds are re-
tained on the fruit parts, which are treated as by-products by the food
industry (Guo et al., 2003; Myoda et al., 2010; Azevêdo et al., 2014).
Therefore, the processing of these by-products has the potential to be-
come a segment of the agribusiness contributing to a better utilization,

∗
Corresponding author.
E-mail address: dgranato@uepg.br (D. Granato).

https://doi.org/10.1016/j.fct.2018.07.043
Received 20 May 2018; Received in revised form 16 July 2018; Accepted 24 July 2018
Available online 25 July 2018
0278-6915/ © 2018 Elsevier Ltd. All rights reserved.
M. Fidelis et al.
Fig. 1. Camu-camu fruit, its seeds, seed coat, and the extracts analyzed: (CH3)2CO (propanone), H2O (water), and EtOH (ethyl alcohol).
both in the food industry and cosmetics, thus allowing its economic
valuation (Azevêdo et al., 2014; Nile et al., 2018).
Currently, the extraction of bioactive compounds from by-products
(i.e., skin and seeds) of fruits and vegetables has been increasingly
studied in order to avoid significant losses and wastes, besides re-
presenting potential benefits for applications in the industry (Sagar
et al., 2018; Lu et al., 2018; Fan et al., 2018). Studies have reported that
camu-camu seeds are composed of different classes of phenolic com-
pounds, which are widely known for their functional and biological
effects. Among these substances, phenolic acids (ellagic and syringic
acids), and flavonoids, such as quercetin, myricetin, rutin, and catechin
stand out (Reynertson et al., 2008; Akter et al., 2011; Azevêdo et al.,
2014).
Phenolic compounds from camu-camu have attracted commercial
and scientific interest mainly because of their potential to be used as
nutraceuticals (isolated compounds) and functional food (camu-camu
extracts), acting to reduce the risk of non-communicable diseases
(Azevêdo et al., 2015; Grigio et al., 2017; Donado-Pestana et al., 2018).
Myoda et al. (2010) found that the total phenolic content of camu-camu
seed aqueous and propanone extracts was three times higher than that
of acelora, demonstrating the functional potential of camu-camu seeds
as sources of bioactive compounds. Therefore, the high levels of phe-
nolic compounds combined with the antioxidant capacity identified
show that the camu-camu residue may represent a new source of
functional compounds for the improvement of human nutrition (Souza
et al., 2017).
In order to avoid food waste, in addition to the fact that the
Brazilian fruit exploitation may be of industrial interest, the objectives
of this work were to evaluate different extracts (water, propanone, and
ethyl alcohol) of camu-camu seed coat with respect to their phenolic
composition, antioxidant activity, and inhibition of angiotensin-con-
verting enzyme (ACE) and to assess the structure-activity using bi-
variate and multivariate statistical methods.
2. Materials and methods
2.1. Chemicals
Gallic, chlorogenic, syringic, p-coumaric, ferulic, rosmarinic, and
ellagic acids, quercetin-3-rutinoside (rutin), quercetin, (+)-catechin,
(−)-epicatechin, Folin-Ciocalteu's phenol reagent 2 N, 2,2-diphenyl-1-
picrylhydrazyl radical (DPPH•), pyrocatechol violet (3,3′,4-trihydrox-
yfuchsone- 2″-sulfonic acid), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ),
ferric chloride hexahydrate (FeCl3·6H2O), copper sulfate pentahydrate
(CuSO4·5H2O), vanillin, angiotensin I-converting enzyme from rabbit
lung (EC 3.4.15.1), hippuryl-L-histidyl-L-leucine substrate, and
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Food and Chemical Toxicology 120 (2018) 479–490
acetonitrile were obtained from Sigma-Aldrich (São Paulo, Brazil).
trans-Resveratrol was obtained from Extrasynthese (France). 2,4-
Dihydroxybenzoic and 2,5-dihydroxybenzoic acids were purchased
from Carl Roth (Karlsruhe, Germany). Ascorbic acid and propanone
were obtained from Biotec (Paraná, Brazil). Sulfuric acid and sodium
carbonate were obtained from Vetec (Rio de Janeiro, Brazil).
Anhydrous sodium acetate and methyl alcohol were obtained from
Anidrol (São Paulo, Brazil) while potassium hexacyanoferrate (III) was
obtained from Merck (Darmstadt, Germany). Ethyl alcohol was pur-
chased from Neon (São Paulo, Brazil). Formic acid was acquired from
Reagen (Rio de Janeiro, Brazil), and Milli-Q (São Paulo, Brazil) ultra-
pure water was used in the experiments.
2.2. Camu-camu seeds and extraction
Camu-camu fruit was cultivated in Iguape in the state of São Paulo/
Brazil, geographical coordinates 24° 41′51” south, 47° 34′16” west at
6 m altitude, and harvested in March 2017. The fruits were sanitized
(NaClO at 200 mg/L for 15 min) and the seeds removed manually. The
seeds were dried in an oven with air circulation at 35 °C for 31 h
(∼12% moisture) and then the seed coat was removed and ground in a
universe hard metal cutter mill (Ika Werke, Model M 20, USA). The
camu-camu seed coat flour was standardized with 42 Tyler mesh sieve
and stored in a glass vessel at 8 °C. The extractions were performed
using the ratio 1:20 (sample:solvent, m/v), i.e., 10 g of flour obtained
from the camu-camu seed coats were mixed with 200 mL of solvent
mixture. Overall, samples were extracted with continuous magnetic
agitation under temperature control (45 °C) in three different solvents;
ultrapure water, ethyl alcohol or propanone (Fig. 1) for 45 min and the
extracts were finally vacuum-filtered using a Büchner's funnel and im-
mediately analyzed for their phenolic composition and functional
properties.
2.3. Phenolic composition
The total phenolic content (TPC) of extracts was determined using a
colorimetric method that uses K3 [Fe(CN)6] and FeCl3·6H2O at
0.5 mmol/L (Margraf et al., 2015). Results were expressed as mg of
gallic acid equivalent per 100 g of seed coat (mg GAE/100 g). The
condensed tannins content (CT) was quantified using the vanillin-
H2SO4 method (Horszwald and Andlauer, 2011) and data were ex-
pressed as mg of (+)-catechin equivalent per 100 g of seed coat (mg
CE/100 g). The non-tannin phenolic content was estimated by sub-
tracting the CT from the TPC and results were expressed as mg/100 g.
The quantification of individual phenolic compounds was per-
formed using a high-performance liquid chromatograph (HPLC)
M. Fidelis et al. Food and Chemical Toxicology 120 (2018) 479–490

Table 1 was quantified in accordance with the methodology proposed by

Elution gradient conditions for identification and quantification of phenolic Singleton et al. (1999), with modifications to a 96-well microplate, and
compounds in camu-camu seed coat extracts by liquid chromatography. results were expressed as mg gallic acid equivalent per 100 g of seed
Phenolic acids Flavonoids and t-resveratrol coat (mg GAE/100 g). Cu2+ chelating ability was assessed using the
spectrophotometric method that employs pyrocatechol violet as the
Time (min) Phase A Phase B Time Phase A Phase B chromogen agent according to the experimental conditions proposed by
(%) (%) (min) (%) (%)
Santos et al. (2017) and results were expressed as the percentage of
0 100 0 0 100 0 formation of Cu2+-pyrocatechol violet complex.
10 97 3 5 88 12 Fe2+-induced lipid peroxidation of phospholipids and triacylgly-
15 90 10 11 45 55 cerols contained in fowl egg yolk was determined in a buffered system
17 88 12 19 35 65
(pH 7.4) at 37 °C according to the procedures described elsewhere
23 45 55 30 100 0
30 35 65 37 100 0 (Margraf et al., 2016). Results were expressed as % inhibition of lipid
35 100 0 peroxidation. For the biological antioxidant activity evaluation, male
47 100 0 Wistar rats (4 weeks old, ∼400 g) were used. Animals were fed on a
standard diet (AIN 93) and maintained on a 12 h light/dark cycle and
allowed free access to diet and water. The animals were anesthetized
Shimadzu LC-20T, equipped with DAD (diode detector array) and with ketamine and xylazine (100 mg/kg and 10 mg/kg, respectively),
fluorescence detectors, degasser system, auto sampler, and oven and euthanized by cervical dislocation. Rat brains were isolated, wa-
column. Chromatographic separation was conducted on a reverse phase shed with a NaCl (0.9 g/100 g), and homogenized with six volumes
column (C18, 150 mm × 4.6 mm, particle size 3.5 μm). For the chro- phosphate buffered solution (0.10 mol/L, pH 7.4). The homogenates
matographic separation, the gradient elution used for the quantification were cleared by centrifugation (10,000 g for 10 min state the tem-
of phenolic acids, t-resveratrol, and flavonoids are presented in Table 1. perature) and the clear supernatant was collected for analysis. Lipid
The column temperature was maintained at 40 °C for both methods, the peroxidation of brain/egg yolk homogenates (500 μL) was induced
sample injection volume was 10 μL, with the flow rate of 0.5 mL/min. using a 4 mmol/L FeSO4 solution (50 μL) at 37 °C for 45 min in a water
The mobile phase consisted of water acidified with 0.2% (v/v) formic bath. The reaction was terminated by addition of 20% v/v acetic acid
acid (mobile phase A) and acetonitrile (mobile phase B). The wave- (500 μL, pH 3.5) and the formation of thiobarbituric acid reactive
lengths used were 210, 270, 320, and 360 nm for the DAD detector and substances (TBARS) was initiated with addition of 1 mL 2-thiobarbituric
λemission = 320 nm and λextinction = 210 nm for the fluorescence de- acid (46 mmol/L). Reaction was performed at 95 ± 5 °C for 30 min in
tector. a glycerin bath and TBARS were determined by UV-VIS spectro-
The extracts were filtered through a 0.45 μm nylon membrane and photometry at 532 nm against a blank (ultrapure water). Lipid perox-
injected in triplicate for each method. For quantification of the phenolic idation was expressed as % inhibition. The biological protocol was
compounds in the samples, analytical curves were prepared in tripli- approved by the Ethics Committee for Animal Use from the State Uni-
cate, with seven evenly spaced concentrations (0.5–100 mg/L) with all versity of Ponta Grossa (Protocol 047/2017).
regression lines presenting R2 ≥ 0.997 and residuals that followed the
normal distribution (data not shown). The results of the individual 2.5. In vitro antihypertensive activity
phenolic composition were expressed as mg/100 g seed coat.
Although the use of HPLC-DAD detected t-resveratrol in the extracts The angiotensin I-converting enzyme inhibitory (ACEI) activity of
(using a standard solution of this compound), the t-resveratrol identi- the extracts was determined following a spectrophotometric assay
fication was confirmed using an Aquity-UPLC™ system (Waters, MA, (Balthazar et al., 2018). Each camu-camu seed coat extract (1 mL) was
USA) coupled with electrospray ionization mass spectrometry [ESI-MS/ diluted with 2 mL ultrapure water and homogenized. Then, 50 μL of
MS] Xevo® (Waters, MA, USA). Analysis of t-resveratrol was performed each extract were added to 200 μL of 2.5 mmol/L hippuryl-L-histidyl-L-
by reversed phase using BEH C18 column (50 × 2.1 mm i. d. and 1.7 μm leucine substrate, which contained 50 mmol/L Tris buffer and
of particle size) with a temperature of 20 °C, 10 μL of injection volume 300 mmol/L NaCl (pH 8.3). This mixture was incubated at 37 °C/3 min.
and flow rate of 0.5 mL/min. The mobile phase consisted of ultrapure A 20 μL aliquot of ACE (0.1 U/mL) was added to the mixture, which
water (solvent A) and methyl alcohol (solvent B). The elution gradient was vortexed, incubated at 37 °C/30 min, and shaken at 200 rpm. The
used was 3–100% (solvent B) in 5 min. The propanone extract (highest amount of hippuric acid produced in the reaction was used to measure
t-resveratrol content) and the pure standard were prepared in MeOH, ACE-inhibitory activity and monitored at 228 nm. Finally, the reaction
with 10 μL being injected. For the identification of t-resveratrol, MS/MS was stopped by adding 250 μL of 1 mol/L HCl and samples were filtered
detection was operated in the negative mode. Equipment was config- using a 0.22 μm nylon filter. The extent of inhibition was calculated
ured with energies of 59 V (cone) and 2.79 kV (capillary); nebulizer according to Equation (1).
nitrogen gas, 40 psi at 350 °C. The ions fragmentation occurred in the
second quadrupole with argon atmosphere, and the fragments were ACEI (%) = [(B−A)/(B−C)]×100 (1)
monitored in the third quadrupole in range of m/z 100 to 220 and data Whereby A represents the absorbance in the presence of angiotensin
were compared to a previous work (Wang et al., 2005). I-converting enzyme and the ACEI component, B represents the absor-
bance without the ACEI component, and C represents the absorbance
2.4. Antioxidant activity: chemical and biological assays without ACE.

In the current research, the camu-camu seed coat extracts were
assessed in relation to the antioxidant activity using different assays:
the ferric reducing antioxidant power (FRAP) of extracts was measured
using the method proposed by Benzie and Strain (1996) and results
were expressed as mg ascorbic acid equivalent per 100 g of seed coat
(mg AAE/100 g). The DPPH• free radical scavenging activity was ana-
lyzed using the method described by Brand-Williams et al. (1995) and
results were expressed as mg ascorbic acid equivalent per 100 g of seed
coat (mg AAE/100 g). The Folin-Ciocalteu reducing capacity (FCRC)
2.6. Statistical analysis
All measurements were conducted in triplicate and results were
expressed by the means followed by the standard deviation. One-way
ANOVA followed by the Fisher least significance difference test after
assessing the homoscedasticity (Brown-Forsythe test) was used to de-
termine statistical significant differences between the extracts
(p < 0.05). When applicable, unpaired Student-t test was used to
compare the phenolic composition of the extracts. Correlation

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coefficients (r-value) were calculated to assess which phenolic com-

pound/phenolic class exerted the antioxidant/antihypertensive activity
of the camu-camu seed coat extracts. Probability values below 5% were
regarded as significant (Nunes et al., 2015). Principal component ana-
lysis was applied using the replicates of all analyzes aiming to project
the camu-camu seed extracts and facilitate the interpretation of the
data. For this purpose, the means were autoscaled to the unit variance
using the z-score according to Equation (2) and the correlation matrix
(3 camu-camu seed coat extracts, totaling 9 replicates x 26 responses)
was used for the calculation (Granato et al., 2018).

Xij − Xj
z ij =
sj (2)

Whereby Z is the standardized value for each value of the response,

Xij represents the original value for the object (i) of measured attribute
(j), Xj is the mean value of the variable j, and sj is the standard deviation
for the response variable.
Factor loadings higher than 0.60 were used to project the extracts in
the factor plane (PC1 x PC2). The software Statistica 13.3 (Statsoft,
USA) was used in the statistical analysis.

3. Results and discussion

3.1. Total phenolic content

The results of total phenolic content, condensed tannins, and non-

tannin phenolic content of camu-camu seed coat extracts are shown in
Fig. 2. Significant differences were observed for all responses
(p < 0.001), showing that the solvent system highly affects the ex-
traction of phenolic compounds from camu-camu seed coat. It is pos-
sible to observe that the aqueous extract had the highest total phenolic
content (Fig. 2A). The ability of water to extract a greater content of
phenolic compounds may be related to the affinity between the phe-
nolic compounds present in the camu-camu seed coat and the polarity
of the extracting medium. Fujita et al. (2015) evaluated the bioactive
compounds of camu-camu pulp (Myrciaria dubia Mc Vaugh) and de-
tected ellagitannins, ellagic acid, quercetin glycosides, syringic acid,
and myricetin. Such compounds have hydroxyl groups in their chemical
structure that confer reducing capacity of the molecules. Such char-
acteristics make the total phenolic composition readily extracted using
water as the solvent. Our results obtained with the three solvents are
much higher than those reported for other agroindustrial residues, such
as Amygdalus pedunculata Pall seed coat - 781 mg GAE/100 g (Lu et al.,
2018) and lower than coffee cherry husk – 22 g GAE/100 g, peanut husk
14 g GAE/100 g, and sugarcane bagasse – 16 g GAE/100 g dry basis
(Vijayalaxmi et al., 2015).
The condensed tannins content presented the opposite behavior to
that observed in relation to the total phenolic content. As can be ob-
served in Fig. 2B, the aqueous extract showed the lowest content of
condensed tannins, whereas the ethanolic extract had the highest
concentration of the same class of compounds. The aqueous extract
presented the highest content of non-tannin phenolics (Fig. 2C), in- Fig. 2. Quantification of phenolic compounds classes of different extracts
dicating that these compounds have greater affinity with water. The (EtOH: ethyl alcohol, H2O: water, (CH3)2CO: propanone) from camu-camu seed
presence of hydrophilic phenolic compounds has already been reported coat: (A) The total phenolic content; (B) Condensed tannins content; (C) Non-
tannins phenolic content. Different letters represent statistically significant
in camu-camu pulp by Fujita et al. (2015).
differences (p < 0.05) by the Fisher LSD test.

3.2. Individual phenolic composition

soluble in aqueous medium, which justifies the highest levels in the
aqueous extract compared with the ethanolic and propanone extracts.
The contents of individual phenolic compounds quantified by HPLC-
In addition to the phenolic acids, the aqueous and ethanolic extracts
DAD are shown in Table 2. There are significant differences (p < 0.05)
had a higher content of (−)-epicatechin compared to the propanone
between the three extracts (aqueous, ethanolic, and propanone), except
extract. The presence of phenolic acids, such as gallic acid, as well as
for 2-hydroxycinnamic acid (p = 0.2415). The aqueous extract pre-
some glycosylated flavonoids has been previously reported in aqueous
sented the highest levels (p < 0.001) of gallic and chlorogenic acid.
extract of camu-camu pulp (Fujita et al., 2015). The ethanolic extract,
Accordingly, 2,4-dihydroxybenzoic and 2,5-dihydroxybenzoic acids
in turn, had the highest (p < 0.05) content of caffeic acid. Although
were only found in the aqueous extract. These compounds are highly

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Table 2
Individual composition of the phenolic acids, flavonoids, and t-resveratrol found in the camu-camu seed coat extracts.
Phenolic compounds H2O (mg/100 g) EtOH (mg/100 g) (CH3)2CO (mg/100 g) p-Value1

Gallic acid 12.57 ± 1.61a 6.86 ± 0.15b ND < 0.0001

2,5-Dihydroxybenzoic acid 15.05 ± 3.89 ND ND NA
2,4-Dihydroxybenzoic acid 2.16 ± 0.65 ND ND NA
Chlorogenic acid 46.52 ± 2.92a 5.23 ± 0.27b 5.77 ± 0.65b < 0.0001
Caffeic acid ND 2.05 ± 0.02a 2.09 ± 0.22b < 0.0001
Syringic acid ND 9.11 ± 0.28b 4.31 ± 1.01a < 0.0001
p-Coumaric acid 3.42 ± 1.06c 48.18 ± 0.44b 49.62 ± 0.05a < 0.0001
Ferulic acid 6.83 ± 0.89c 41.57 ± 1.57b 59.28 ± 0.50a < 0.0001
Rosmarinic acid 10.95 ± 0.05c 11.44 ± 0.26b 13.19 ± 0.16a < 0.0001
Ellagic acid ND 28.33 ± 0.60b 29.13 ± 0.20a < 0.0001
trans-Resveratrol 2.15 ± 0.04b 1.73 ± 0.03c 3.22 ± 0.08a < 0.0001
Rutin 3.46 ± 0.10b 13.97 ± 0.68a 12.94 ± 2.95a 0.0006
Quercetin 0.70 ± 0.01b 0.85 ± 0.03b 1.10 ± 0.01a 0.0003
(+)-Catechin 2.38 ± 0.02b 2.26 ± 0.04b 2.49 ± 0.02a 0.0033
(−)-Epicatechin 2.08 ± 0.04a 2.08 ± 0.01a 1.54 ± 0.01b < 0.0001
Total quantified (mg/100 g) 108.58 174.00 185.10 –

Note: NA = not applicable; ND = not detected; 1Probability values based on one-way ANOVA (3 extracts) or unpaired Student-t test (2 extracts). Different letters in
the same row represent significant statistical difference (p < 0.05).

caffeic acid has two hydroxyl groups in its structure, it has a higher
molecular weight compared to hydroxybenzoic acids (C3-C1), ex-
plaining the extraction of 2,5 and 2,4-dihydroxybenzoic acids by water
and not by ethyl alcohol or propanone. Regarding the quercetin-3-ru-
tinoside content, the ethyl alcohol and propanone extracted a higher
content than water to the aqueous.
Following the affinity profile by polarity, the most apolar com-
pounds were extracted by propanone. Accordingly, propanone ex-
tracted the highest levels of p-coumaric acid (49.62 mg/100 g), ferulic
acid (59.28 mg/100 g), and ellagic acid (29.13 mg/100 g), totaling the
highest content of phenolic compounds compared to the other solvents.
However, the literature lacks information regarding the individual
phenolic composition of camu-camu (Myrciaria dubia McVaugh,
Myrtaceae) seed coat extracts. Thus, this is the first report on the
quantification of phenolic compounds in camu-camu seed coats ex-
tracted by different solvents. For comparison purposes, Lu et al. (2018)
identified more that than 30 phenolic compounds in Amygdalus ped-
unculata Pall seed coat and observed a content of 203 mg/100 g, which
is comparable to the data obtained in the present study.
trans-Resveratrol was identified and quantified for the first time in
camu-camu seed coats, and its presence was confirmed in the propa-
none extract by UPLC-ESI-MS/MS (Fig. 3). Fig. 3A and D demonstrate
an ionized molecular structure of identified t-resveratrol as standard
(m/z 227.30) and propanone extract (m/z 228.19), respectively. Fig. 3B
and C demonstrate the possible fragmentations of the molecular
structure (m/z 117.66, m/z 142.87, m/z 155.66, m/z 111.16) compared
to structure of intact t-resveratrol (molecular weight = 228.23 g/mol),
respectively. These spectral data corroborate the results obtained by
Wang et al. (2005) on the fragmentation of the t-resveratrol molecular
structure (m/z 117, m/z 119 and m/z 143).
3.3. Antioxidant activity
The antioxidant activity of the different camu-camu seed coat ex-
tracts (Fig. 4) presented statistically significant differences
(p < 0.001). The aqueous extract had the highest antioxidant activity
by FRAP (7425 ± 247 mg AAE/100 g), DPPH• (2838 ± 176 mg AAE/
100 g), and the Folin-Ciocalteau reducing capacity (8522 ± 318 mg
GAE/100 g) in relation to the other extract (Fig. 4A, B and 4C). The
high antioxidant activity of the aqueous extract of camu-camu seed coat
may be related to higher (p < 0.05) total phenolic content
(3923 ± 29 mg GAE/100 g) compared to the other extracts. Among
the phenolic acids identified in the aqueous extract, chlorogenic acid
presented the highest content (46.52 ± 2.92 mg/100 g, Table 2).
Studies have shown that different foods and beverages (coffee and tea)
contain chlorogenic acid and presents functional properties, such as
antioxidant, anti-inflammatory, antihypertensive, antibacterial, cardi-
oprotective, and neuroprotective activities (Naveed et al., 2018).
Grigio et al. (2017) also observed antioxidant activity values in the
aqueous extract of the camu-camu seed - 840 mg ascorbic acid
equivalent/100 g and 245 mg ascorbic acid equivalent/100 g in FRAP
and DPPH• assays, respectively. In a study on bioactive constituents and
antioxidant activity of berries aqueous methanolic extracts from Ro-
manian Goji berries cultivars, Mocan et al. (2018) also found significant
antioxidant activity measured by the DPPH• (935 mg Trolox equivalent/
100 g extract) and FRAP assays (1652 mg Trolox equivalent/100 g ex-
tract).
The highest inhibition of the lipid peroxidation (both in egg yolk
and Wistar rat brain) was observed for the aqueous extract followed by
the ethanolic and propanone extracts (Fig. 4D and E). Both methods
used to assess the extent of lipid oxidation (TBARS) were closely cor-
related (p < 0.05) with total phenolic content, non-tannin phenolic
content, gallic acid, 2,5-dihydroxybenzoic acid, 2,4-dihydroxybenzoic
acid, chlorogenic acid, and (−)-epicatechin (Fig. 5A and B). Our results
support the findings obtained by Psotová et al. (2003) who studied the
antioxidant activity of different phenolic compounds, including 3,4-
dihydroxybenzoic acid and chlorogenic acid in rat liver homogenate.
Lipid peroxidation causes oxidative damage in the cell membrane in-
duced by reactive oxygen species (Kancheva and Kasaikina, 2012).
Pereira et al. (2014) found a reduction of lipid peroxidation in the liver
and serum of rats after consumption of tropical fruit juice containing
camu-camu. Gonçalves et al. (2014) found that the consumption of
frozen pulp extracts of camu-camu significantly increased plasma an-
tioxidant activity in diabetic rats.
The Cu2+ chelating ability of the different camu-camu seed coat
extracts ranged from 77 to 94% (Fig. 4F). These results indicate a low
ability of the phenolic compounds present in camu-camu seed coat
extracts to bind Cu2+, since there was a high percentage of formation of
the pyrocatechol violet -Cu2+ complex. Total condensed tannins and
syringic acid were associated (p < 0.05) with the Cu2+ chelating
ability (Fig. 5C). Phenolic compounds with a single OH group on the
aromatic ring and without a O-CH3 group on the benzene ring usually
do not present metal chelating properties. Accordingly, the presence of
o-dihydroxyphenil or galloyl group is necessary for the complexation of
bivalent metal ions. The presence of 4-keto group or an ortho-dihydroxy
group in ring A of flavonoids, and methoxyl/hydroxyl groups in the
ortho position of phenolic acids increases Cu2+ chelation ability,
whereas the glycosylation of OH group of phenols prevents metal ions
from binding (Riha et al., 2014; Zhou et al., 2006). Our results are in
accordance with those obtained by Karamać (2009) who found a strong

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Fig. 3. Mass spectrum of t-resveratrol pure standard (A) with its respective fragment ions (B; C) in the negative ionization mode and the identification of t-resveratrol
in the propanoic extract of camu-camu seed coats (D).
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Fig. 3. (continued)

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Fig. 4. Antioxidant activity of different extracts (EtOH: ethyl alcohol, H2O: water, (CH3)2CO: propanone) from camu-camu seed coat: FRAP (A); DPPH• (B); Folin-
Ciocalteu reducing capacity (C); inhibition of lipid peroxidation (D); inhibition of Wistar rat brain oxidation (E); and Cu2+ chelating ability (F). Different letters
comparing the camu-camu seed coat extracts represent statistically significant differences (p < 0.05).

association between tannin content in selected edible nuts and the
chelation of Cu2+, Fe2+, and Zn2+. On the contrary, our data are not
in-line with those obtained by Andjelković et al. (2006) who did not
find any significant correlation between syringic acid with Fe2+ che-
lating ability and with Santos et al. (2017) who found a significant
correlation between TPC and Cu2+ chelating ability of coffee bev-
erages.
The correlation analysis shows that gallic acid, 2,5-dihydrox-
ybenzoic acid, 2,4-dihydroxybenzoic acid, chlorogenic acid, and
(−)-epicatechin were positively and significantly correlated
(p < 0.05) with FRAP, DPPH•, and FCRC (Fig. 5D and F), corroborating
the data obtained by Xiang et al. (2014), Granato et al. (2015), and
Todorovic et al. (2017) in various food matrices (red wines, juices, and
cocoa) and food by-products (i.e., seed coat) (Fan et al., 2018; Figueroa
et al., 2018; Attree et al., 2015). The chemical structure of phenolic
compounds directly influences the antioxidant activity because of the
presence of hydroxyl groups in their structures, which are capable of
donating electrons to free radicals. This feature enables the scavenging
of free radicals and reduction of transition metals (Prior et al., 2005).
The same authors point out that, in addition to the ability to donate
electrons, phenolic compounds are also related to the ability to donate
hydrogen atoms, which can be observed through significant (p < 0.05)
and positive correlation with inhibition of lipid peroxidation assays.
t-Resveratrol content was only significantly associated with Cu2+
chelating ability, corroborating the results obtained by Xiang et al.
(2014). These authors analyzed the antioxidant activity and neuro-
protective effects in vitro on SH-SY5Y cell line of red wines added with
10-fold the normal concentration of t-resveratrol and found that the
contribution of resveratrol to antioxidant activity is negligible. On the
other hand, Macedo et al. (2013) studied the effects of red wines with
different in vitro and in vivo antioxidant activity on Wistar rats fed a
lipid-rich diet and found that a multivariate direct association between
t-resveratrol content and the oxygen radical absorbance capacity
(ORAC) in rat plasma and an inverse association with malondialdehyde
in rat liver homogenate. Therefore, it is clear that the in vitro and in vivo
functional properties of this compound depends on the protocol used to
assess the antioxidant activity and/or oxidative stress (Navarro et al.,
2018).
Another important result obtained here is the significant (p < 0.05)
correlation between the inhibition of egg yolk lipid peroxidation with
FRAP (r = 0.9261), DPPH• (r = 0.9667), and FCRC (r = 0.9727).
Similarly, significant (p < 0.05) correlation between the inhibition of
rat brain lipid oxidation was obtained with FRAP (r = 0.9119), DPPH•
(r = 0.9624), and FCRC (r = 0.9429). In addition, both antioxidant
activity methods based on the measurement of TBARS in triacylgly-
cerol-rich matrices (egg yolk and rat brain homogenate) were also

486
M. Fidelis et al.
Fig. 5. Correlation between phenolic composition and inhibition of lipid peroxidation (A), inhibition of Wistar brain oxidation (B), Cu+2 chelating ability (C), FRAP
(D), free radical scavenging activity toward DPPH (E), Folin-Ciocalteu reducing capacity (F), and in vitro antihypertensive activity (G).
significantly correlated (r = 0.9366, p < 0.001). These data are in-line
with those reported by Castro et al. (2006) who assessed the anti-
oxidant activity of α-tocopherol and Trolox using the TBARS (using rat
brain homogenate as substrate) and DPPH• assays (r = −0.99; data
expressed as IC50). Our results indicate that, for camu-camu seed coat
extracts, the use of rat brain as substrate to assess the antioxidant ac-
tivity of extracts should be pondered as chemical antioxidant methods
were well associated with the ex vivo antioxidant activity measurement.
3.4. Antihypertensive activity
Camu-camu seed coat extracts exhibited an inhibition of 28–40% of
ACE I activity (Fig. 6). In this study, the in vitro inhibition of ACE I
activity showed a significant (p < 0.05) and positive correlation with
the content of p-coumaric, rosmarinic, ferulic, caffeic, ellagic acids,
quercetin-3-rutinoside, and quercetin (Fig. 5G). These results corrobo-
rate the data obtained by Kwon et al. (2006) who investigated the
ability of different herbal extracts and standard phenolics on inhibiting
the activity of ACE I. Ellagic acid was considered one of the main re-
sponsible compounds to inhibit the ACE I activity of spray-dried camu-
camu pulp (Fujita et al., 2015).
The phenolic compounds have shown beneficial effects not only in
reducing the risk of developing hypertension, but also in normalizing
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Food and Chemical Toxicology 120 (2018) 479–490
blood pressure (Panche et al., 2016; Manach et al., 2005). Studies have
shown that phenolic compounds has several therapeutic properties in-
cluding antihypertensive (Mancuso and Santangelo, 2014). Docking
molecular experiments and the structure-activity relationship show that
phenolic acids and flavonoids inhibit ACE I. This inhibition mainly has
the interaction with the zinc ion present in the active site of the enzyme
(Al Shukor et al., 2013). According to Guerrero et al. (2012) and Larson
et al. (2010), flavonoids may be a potential source of antihypertensive
activity because of the hydroxyl and/or methoxyl group positions allied
to the double bond in the rings are responsible for increasing the ac-
tivity of ACE I.
3.5. Correlation analysis by principal component analysis (PCA)
Among the most well-known multivariate statistical tools, principal
component analysis (PCA) stands out because it differentiates the
samples by means of a two-dimensional projection (Granato et al.,
2018). Fig. 7 shows that the first principal component (PC1) explained
73% of the data variability and the second PC was able to explain other
22%, retaining roughly 95% of all variability in the experimental data.
It was observed that the aqueous extract had the highest antioxidant
capacity (DPPH•, FRAP, and FCRC), inhibition of lipid peroxidation,
total phenolics, non-tannin phenolics, (−)-epicatechin, chlorogenic
M. Fidelis et al.
Fig. 5. (continued)
Fig. 6. In vitro antihypertensive activity of different extracts (ethyl alcohol,
water, propanone) from camu-camu seed coat. Different letters represent sta-
tistically significant differences (p < 0.05) by the Fisher LSD test.
acid, 2,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, and gallic
acid. The propanone extract presented higher antihypertensive activity,
Cu2+ chelating ability, and higher levels of quercetin, quercetin-3-ru-
tinoside (rutin), t-resveratrol, ellagic, caffeic, rosmarinic, ferulic, and p-
coumaric acids. The ethanolic extract showed higher content only of
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Food and Chemical Toxicology 120 (2018) 479–490
condensed tannins, syringic acid, and (−)-epicatechin. Overall, by
means of the multivariate projections on the factor-plane, the results
clearly showed that ethyl alcohol extracted intermediate levels of most
bioactive compounds from camu-camu seed coat.
4. Conclusions
We reported, for the first time in the literature, the phenolic com-
position (with emphasis on t-resveratrol), the antioxidant activity
measured by different chemical and biological assays, and the in vitro
antihypertensive activity of different camu-camu seed coat extracts.
The aqueous extract had the highest total phenolic content, and anti-
oxidant activity, while the extract obtained with propanone showed the
opposite behavior but it presented higher in vitro antihypertensive ac-
tivity. The ethanolic extract exhibited intermediate values for the re-
sponses. In addition, the results obtained in this study revealed the
structure-activity relationship between phenolic compounds and ACE I
inhibitory activity. Overall, camu-camu seed coat is an industrial waste
that can be exploited for its bioactive compounds, suggesting promising
applications in food technology. As a final comment, in vivo studies
should be performed to corroborate the functional properties of camu-
camu seed coat extracts.
M. Fidelis et al.
Fig. 7. Principal component analysis based on the three replicates of each camu-camu seed coat extract (water, ethyl alcohol, and propanone) for the chemical
composition, antioxidant, and antihypertensive activities.
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgments
Authors thank PROAP/CAPES for funding the work, Fundação
Araucária and CAPES for the graduate scholarships, C-LABMU-UEPG
and C-LABMU-SEBISA-UEPG (Complexo de Laboratórios Multiusuários
da UEPG) for the unique infrastructure used to perform the experi-
mental work. D. Granato acknowledges CNPq for a productivity grant
(process 303188/2016-2). The help from Prof. Dr. Adriano G. Cruz in
the analysis of ECA and Pablo I. Monteiro in the HPLC analysis is also
acknowledged. P. Putnik thanks the Croatian Science Foundation - High
voltage discharges for green solvent extraction of bioactive compounds
from Mediterranean herbs (IP-2016-06-1913).
Transparency document
Transparency document related to this article can be found online at
https://doi.org/10.1016/j.fct.2018.07.043.
Appendix A. Supplementary data
Supplementary data related to this article can be found at https://
doi.org/10.1016/j.fct.2018.07.043.
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