nutrients

Article
Antioxidant Status, Antidiabetic Properties and
Effects on Caco-2 Cells of Colored and Non-Colored
Enriched Extracts of Sweet Cherry Fruits
Ana C. Gonçalves 1 , Márcio Rodrigues 1,2 , Adriana O. Santos 1 , Gilberto Alves 1
and Luís R. Silva 1, *
1 CICS-UBI—Health Sciences Research Centre, University of Beira Interior, 6201-506 Covilhã, Portugal;
anacarolinagoncalves@sapo.pt (A.C.G.); marciorodrigues@fcsaude.ubi.pt (M.R.); aos@ubi.pt (A.O.S.);
gilberto@fcsaude.ubi.pt (G.A.)
2 UDI-IPG, Research Unit for Inland Development, Polytechnic Institute of Guarda, 6300-749 Guarda, Portugal
* Correspondence: luisfarmacognosia@gmail.com; Tel.: +351-275-329-077




Received: 20 September 2018; Accepted: 30 October 2018; Published: 5 November 2018


Abstract: This study aimed to compare three different extracts of Saco sweet cherry, namely the
non-colored fraction, colored fraction, and total extract concerning phenolic composition, antioxidant
and antidiabetic potential, and erythrocytes’ protection and effects on Caco-2 cells. Twenty-two
phenolic compounds were identified using high-performance liquid chromatography with diode-array
detection. Hydroxycinnamic acids were the most predominant in both the non-colored fraction
and total extract, while cyanidin-3-O-rutinoside was the main anthocyanin found in the colored
fraction. The total extract was the most effective against 1,1-diphenyl-2-picrylhydrazyl, nitric oxide,
and superoxide radicals, and in the inhibition of α-glucosidase enzyme. The colored fraction revealed
the best activity against hemoglobin oxidation and hemolysis. Regarding to Caco-2 cells, the colored
extract exhibited the highest cytotoxic effects, while the total extract was the most efficient in
protecting these cells against oxidative damage induced by tert-butyl hydroperoxide.

Keywords: sweet cherry; anthocyanins; non-colored phenolics; antioxidant activity; erythrocytes

protection; Caco-2 cells

1. Introduction
Phenolic compounds are widely distributed in nature and present strong antioxidant properties [1].
It is believed that their presence in the daily diet exerts a beneficial effect on human health,
being associated with the decrease of oxidative stress-related disorders’ occurrence [1,2]. Furthermore,
phenolic compounds reduce the rate of oxidative processes by acting as reducing agents, hydrogen
donors, singlet oxygen quenchers, and metal chelators, inhibiting the propagation of oxidizing chain
reactions caused by free radicals and protecting the human body against oxidative damage [3,4].
Recently, special attention has been paid to the use of plants and fruit extracts in the cosmetic,
food, and pharmaceutical industries due to their richness in phenolic compounds [5,6]. The extractions
to obtain fractions rich in bioactive substances are preferentially carried out using water–alcohol
mixtures, often including ethanol or methanol as extraction solvents, given their affinity with both
lipophilic and hydrophilic bioactive molecules. Ethanol is the most commonly used solvent because it
is economical, reusable, and unlike methanol, is non-toxic [5].
Extracts of sweet cherry (Prunus avium Linnaeus (L.)) have been subjected to several studies
due to their properties as health promoters [7–9]. Therefore, sweet cherry berries may have a great
potential in the management of obesity, diabetes, and related comorbidities [10]. Although these fruits

Nutrients 2018, 10, 1688; doi:10.3390/nu10111688 www.mdpi.com/journal/nutrients

Nutrients 2018, 10, 1688 2 of 20

are preferably consumed fresh, they can also be commercialized in processed products such as frozen,
canned, wine or concentrate juices, jellies, jams, and dried forms [2]. They are rich in several non-colored
(chlorogenic acids, flavan-3-ols, and flavonols) and colored (mainly cyanidin-3-O-rutinoside) phenolic
compounds, whose levels are regulated by genotype, fruit maturity, climatic conditions, and storage
conditions [6,7].
The largest European cherry-producing countries are Poland, Spain, Italy, Greece, Hungary,
and Germany [11]. In Portugal, around 15 thousand tons of cherries are produced per year. Most of
them are collected from the Fundão region, with Saco being one of the most important and oldest
cultivars [7]. Previous works with Saco proved their large array of health benefits, namely antioxidant
effects [6,7,9], anticancer activity against human cancer cells from the colon (HT-29 and HCT-15)
and stomach (MKN45) [6,9], antidiabetic properties, and capacity to confer protection to human
erythrocytes against hemoglobin oxidation and hemolysis [7].
Considering the above-mentioned reasons, the aim of this work was to improve the knowledge
on the phenolic profile of the total extract, and colored and non-colored fractions of Saco sweet
cherry from the Fundão region (Portugal). Furthermore, and knowing that colored and non-colored
compounds can interact with each other in synergistic, additive, and antagonistic ways, we also
evaluated their antioxidant potential against 1,1-diphenyl-2-picrylhydrazyl (DPPH), nitric oxide
(• NO), and superoxide (O2 •− ) radicals. The capacity of the extracts to inhibit α-glucosidase enzyme
was also determined, as well as the protection afforded against induced oxidative damage in
human erythrocytes. Additionally, the cytotoxic properties of the extracts were assessed for the
first time against human colon carcinoma cells (Caco-2) under quiescent conditions concerning their
antiproliferative activity and toxicology responses when subjected to oxidative stress induced by
tert-butyl hydroperoxide (t-BHP).

2. Materials and Methods

2.1. Chemicals and Reagents

All chemicals used were of analytical grade. Cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside,
pelargonidin-3-O-rutinoside, and peonidin-3-O-rutinoside were from Extrasynthese (Genay, France).
The other phenolics and DPPH, β-nicotinamide adenine dinucleotide (NADH), phenazine methosulfate
(PMS), nitrotetrazolium blue chloride (NBT), α-glucosidase from Saccharomyces cerevisiae (type I,
lyophilized powder), trypan blue, 2,20 -azobis (2-ethylpropionamidine) dihydrochloride (AAPH),
tert-butyl hydroperoxide (t-BHP), high-glucose Dulbecco´s Modified Eagle Medium (DMEM),
fetal bovine serum (FBS), antibiotics (10,000 U/mL penicillin, 10,000 mg/mL streptomycin),
trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA) solution, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), pyruvate, propidium iodide (PI),
bovine serum albumin (BSA), and Ribonuclease A (RNase) were purchased from Sigma-Aldrich
(St. Louis, MO, USA). N-(1-naphthyl)ethylenediamine dihydrochloride, sulfanilamide, 4-nitrophenyl-
alpha-D-glucopyranoside (pNPG), and sodium nitroprusside dihydrate (SNP) were obtained from
Alfa Aesar (Karlsruhe, Germany). Methanol and acetonitrile were acquired from Fisher Chemical
(Leicestershire, Glenfield, United Kingdom). Water was deionized using a Milli-Q water purification
system (Millipore Ibérica, S.A.U., Madrid, Spain). Caco-2 cells were from the American Type Culture
Collection (ATCC; Manassas, VA, USA).

2.2. Cherry Samples

One kg of Saco sweet cherry cultivar was collected by hand from an orchard located in Fundão
region (Portugal) at the same stage of ripeness and age (three years old), during June of 2016.
The cherries were transported to the laboratory of Health Sciences Research Centre (CICS) of the
University of Beira Interior (Covilhã, Portugal). The pits were removed, and the pulp was immediately
frozen with liquid nitrogen and maintained at −20 ◦ C. Then, the pulp was lyophilized, powdered
Nutrients 2018, 10, 1688 3 of 20

(mean particle size lower than 910 µm), and separated into three aliquots and used for the preparation
of the extracts.

2.3. Phenolic Compounds

2.3.1. Extraction
Phenolic compounds of Saco samples were extracted according to Gonçalves et al. [7]. Briefly, 1 g
of dried Saco was stirred at 300 rpm with ethanol (70:30) for 2 h, and then centrifugated at 4000 rpm for
10 min. After that, the supernatant was evaporated under reduced pressure at 30 ◦ C and the resulting
extract was dissolved with 50 mL of deionized water and placed into a C18 solid-phase extraction
(SPE) column (70 mL/10,000 mg; Macherey–Nagel, Düren, Germany) previously conditioned with
ethyl acetate, ethanol, and 0.01 mol/L HCl. After passing the sample, the column was washed with
3 mL of 0.01 mol/L HCl. Afterward the fraction I (non-colored phenolics) was eluted with 20 mL
of ethyl acetate and placed in an erlenmeyer, while the fraction II (anthocyanins) was eluted with
40 mL of ethanol containing 0.1% HCl and placed in another erlenmeyer. To obtain the fraction III
(total extract), another C18 solid-phase extraction was performed, with this one being eluted with
40 mL of ethanol containing 0.1% HCl. Finally, each fraction was evaporated to complete dryness
and frozen at −20 ◦ C until analysis. The extraction yields of non-colored fraction, colored fraction,
and total extract were 8.5 ± 0.02%, 3.6 ± 0.005%, and 13.0 ± 0.01%, respectively.

2.3.2. HPLC-DAD Analysis

Twenty microliters of each sample were analyzed on a LC model Agilent 1260 system (Agilent,
Santa Clara, CA, USA) using a Nucleosil® 100-5 C18 column (25.0 cm × 0.46 cm; 5 µm particle size
waters; Macherey-Nagel, Düren, Germany), based on a previously published method [7]. Detection
was achieved with an Agilent 1260 Infinity DAD using the ChemStation software supplied by Agilent
Technologies (Waldbronn, Germany). The non-colored fraction was re-dissolved with 4 mL of methanol,
while the colored fraction was dissolved with 20 mL of acidified water with pH 3.0. The total extract was
again eluted using an SPE column to separate the non-colored and colored phenolics (anthocyanins)
for identification and quantification. After dissolution, each fraction was filtered using a 0.45 µm
polytetrafluoroethylene membrane (Millipore, Bedford, MA, USA) prior analysis.
The detection and quantification of anthocyanins and non-colored phenolic compounds were
performed according to Gonçalves et al. [7]. The compounds of each extract were identified by
comparing their retention times and ultraviolet–visible spectra in the 200–600 nm range with the
library of spectra previously compiled by the authors. Anthocyanins were detected at 500 nm.
Flavan-3-ols and hydroxybenzoic acids were detected at 280 nm, hydroxycinnamic acids at 320 nm,
and flavonols at 350 nm. The unknown compounds 1–3 were identified as cyanidin-3-O-rutinoside.
The hydroxybenzoic acid derivative 1 was identified as p-hydroxybenzoic acid. The 3-O-caffeoylquinic
acid and hydroxycinnamic acid derivatives 1 and 2 were identified as 5-O-caffeoylquinic acid.
p-Coumaric acid derivatives 1–5 were identified as p-coumaric acid. The total phenolics and total
anthocyanins (∑) were the result of the sum of each determined compound belonged to non-colored
phenolics or anthocyanins, respectively.

2.4. Biological Acitivity Evaluation

The evaluation of the biological potential of the non-colored fraction, colored fraction,
and total extract was performed spectrophotometrically by in vitro microassays using 96-well plates.
The absorbances were measured in a microplate reader Bio-Rad Xmark spectrophotometer (Bio-Rad
Laboratories, Hercules, CA, USA).
Nutrients 2018, 10, 1688 4 of 20

2.4.1. Antioxidant Capacity

DPPH
The capacity of non-colored, colored fractions, and total extract to act as free radical scavengers
against DPPH• was evaluated as previously reported [7].

Nitric Oxide
The activity of the dried extracts against • NO was determined as previously described by
Gonçalves et al. [7].

Superoxide Radical
The effect of cherry extracts on the O2 •− -induced reduction of NBT was monitored at 562 nm.
O2 •−was generated by the PMS-NADH-O2 system, as previously reported [12].

2.4.2. α-Glucosidase Inhibitory Activity

The α-glucosidase inhibitory activity was determined at 405 nm, based on Ellman’s method,
as previously described by Silva and Teixeira [12].

2.4.3. Protective Effect in Human Erythrocytes against Oxidative Damage

Venous human blood was collected from randomized patients of Cova da Beira Hospital Centre
(Covilhã, Portugal) by antecubital venipuncture into K3 EDTA vacuum tubes. Erythrocytes were
isolated based on the procedure previously described [13].

Inhibition of Hemoglobin Oxidation

The inhibition of hemoglobin (Hb) oxidation was evaluated by monitoring the effects of the three
Saco extracts on the formation of methemoglobin after the reaction of oxyhemoglobin with peroxy
radicals (ROO• ) generated by AAPH [7,13,14]. The absorbance was read at 630 nm. Quercetin was
used as a positive control. Three experiments were performed in triplicate for each extract.

Inhibition of Hemolysis
Peroxy radicals (ROO• s) were generated using AAPH and the prevention of ROO• -induced
hemolysis of human erythrocytes was evaluated by monitoring the release of Hb after the membrane
disruption caused by the hemolytic process according to the procedure described by Chisté et al. [13]
and Gonçalves et al. [7]. The absorbance was obtained at 540 nm. Quercetin was used as a positive
control. Three experiments were performed in triplicate for each extract.

2.5. Cell Culture Conditions and Treatments

Caco-2 cells were maintained in DMEM supplemented with 10% FBS and 2% of Pen/Strep.
Cells were grown in 75 cm2 culture flasks at 37 ◦ C in a humidified air incubator with 5% CO2 . Once the
cells reached 90–95% of confluence, they were washed twice with 10 mL of phosphate-buffered saline
(PBS) and detached by gentle trypsinization (5 mL of trypsin-EDTA), and before the experiments, viable
cells were counted and suitably diluted in the adequate complete culture medium (25,000 cells/mL).
These cell culture conditions and procedures were common through all assays. For the several assays,
cells were used between passages 44 and 60. After the trypsinization and count of the cells, 200 µL of
the prepared cellular suspension (25,000 cells/mL) was seeded in 96-well plates and incubated for one
day before carrying out the viability assays. Five concentrations in the range of 50–800 µg/mL of each
extract were dissolved in medium containing 0.5% (v/v) DMSO. The final concentration of DMSO did
not affect cellular viability (data not shown).
Nutrients 2018, 10, 1688 5 of 20

2.5.1. Cytoprotection Assay

Preliminary assays were done to determine the appropriate concentration and exposure time to
t-BHP in order to evaluate the activity of the cherry extracts (data not shown). The t-BHP dilutions
were prepared with concentrations that ranged from 0.25 to 4 mM and cells were exposed for 2,
4, and 6 h. For the analysis of the obtained results, and to achieve a suitable viability decrease,
the selected exposure conditions were a time of 6 h with t-BHP at 1 mM. Cells were seeded under the
same conditions previously described, with and without t-BHP co-incubation. After cell incubation for
24 h, the medium was removed, and cells were treated with the extracts for 24 h, then the t-BHP was
added to each well plate for 6 h. The difference of the assay without incubation was that after 24 h of
incubation with extracts, the medium was completely removed, and 1 mM of t-BHP was added to each
well for 6 h. Finally, the MTT and lactate dehydrogenase (LDH) assays and the cell cycle distribution
were carried out to evaluate the effect of the extracts against the induced toxicity.

2.5.2. MTT Cell Proliferation Assay

To determine the effect of extracts on Caco-2 cells, viability was assessed using an MTT assay after
24 h of exposure of each extract at different concentrations. Then, the medium was removed, and each
well was washed with 200 µL of PBS. The metabolic activity of cells was evaluated via their capacity
to reduce the yellow MTT (0.5 mg/mL in the appropriate serum-free medium) to a blue formazan
product using 4 h of incubation at 37 ◦ C. Then, the medium containing MTT was removed and the
formazan crystals were dissolved in DMSO. The absorbance was read at 570 nm using a microplate
reader Bio-Rad Xmark spectrophotometer. Cell proliferation values were expressed as percentages
from the relative absorbance measured in the treated wells versus control wells [12,15]. A total of six
independent experiments per extract were performed. Untreated cells were used as control.

2.5.3. LDH Assay

The release of the stable cytosolic enzyme LDH into the media was spectrophotometrically
determined at 340 nm, based on the conversion of pyruvate to lactate by LDH, using NADH as
a cofactor [12]. The reaction mixture was composed of the extracts NADH and pyruvate. All solutions
were prepared in PBS (pH 7.4). The medium of each well plate treated with the extracts was collected
after 24 h of cellular exposure and the LDH was evaluated. A total of six independent experiments per
extract were performed. Untreated cells were used as a control.

2.5.4. Cell Cycle Distribution Analysis

The analysis of the cell cycle distribution of cells was determined through PI staining of fixed
and permeabilized cells. Briefly, 2.4 mL of Caco-2 cells were seeded in 12 well-plates (cell density
of 25,000 cells/mL) in complete culture medium. After 24 h, they were treated with 200, 400,
and 800 µg/mL of each extract. Untreated cells were used as a negative control. At the end of 24 h of
incubation, each well was washed with PBS and the cells were harvested using a trypsin treatment.
The resulting cell suspension was kept on ice, pelleted via centrifugation, and resuspended in 450 µL
of a cold solution of 0.5% BSA in PBS. The resulting cell suspension was kept on ice and then fixed
by gently adding ice-cold 70% ethanol (−20 ◦ C) with simultaneous vortexing. After 1 day at −20 ◦ C,
fixed cells were washed twice with PBS and resuspended in a solution of PI prepared in PBS/BSA
0.5% (50 µg/mL), passed through cell strainer filters (40 µm nylon, Falcon® , Life Sciences, Wiesbaden,
Germany) and sequentially incubated with RNase at a final concentration of 7.1 µg/mL (stock solution
in 50% glycerol, 10 mM Tris-HCl, pH 8) for 15 min in the dark. Stained cells were then acquired at a
BD Biosciences FACSCalibur flow cytometer (San Jose, CA, USA), using the 488 nm laser. Data were
analyzed using CellQuest™ Pro Software, version 5.1.1[M1] (BD Biosciences, San Jose, CA, USA).
First, singlets were selected by creating a region on the Fluorescence channel 3 (FL3)-Width/FL3-Area
Nutrients 2018, 10, 1688 6 of 20

contour plot. FL3 signal corresponds to a wavelength > 670 nm. Then, gated singlet events were plotted
on the Forward Scatter (FSC)-Height/FL3-Area contour plots for correlation of size with PI-staining.

2.6. Statistical Analysis of Results

All data were recorded as mean ± standard deviation of triplicate determinations. The HPLC-DAD
statistical phenolic comparison was made using the two-way ANOVA and the Bonferroni test. About to
the biological potential, the statistical comparison was assured by the one-way ANOVA, and the means
were classified by Tukey’s test at a 95% level of significance. To determine the correlation between the
antioxidant activity methods and the contribution of the total phenols, Pearson’s correlation coefficients
were calculated. Relative to the cellular-based assays, data from different groups were compared by
two-way ANOVA followed by Dunnett’s test (LDH and MTT assays) as a post-hoc test. Values of
p < 0.05 were considered statistically significant. All analyses were performed using Graph Pad Prism
Version 6.01 (San Diego, CA, USA).

3. Results and Discussion

3.1. Phenolic Profile

The chromatographic analysis allowed the identification and quantification of a total of 22 phenolic
compounds, including 2 hydroxybenzoic acid derivatives, 14 hydroxycinnamic acids, 1 flavonol, and 5
anthocyanins (Tables 1 and 2). All of these compounds were previously described in Saco sweet cherry
from Fundão (Portugal) [6,7]. As expected, the non-colored phenolics were only found in the total
extract and non-colored fraction. Nevertheless, both extracts showed different quantitative composition
(Table 1). The total contents of the non-colored phenolics in the total extract and non-colored fraction
were 11,069.7 and 15,220.9 µg/g of dried extract, respectively (Table 1).

Table 1. Non-colored phenolic contents of Saco sweet cherry extracts (µg/g of dried extract).

Non-Colored Phenolics Total Extract Non-Colored Fraction

Hydroxybenzoic acid derivative 1 1337.85 ± 68.16 1839.54 ± 5.09 a
Hydroxycinnamic acid derivative 1 494.32 ± 51.66 679.69 ± 71.03
Hydroxycinnamic acid derivative 2 143.45 ± 21.30 197.24 ± 29.28
Hydroxybenzoic acid derivative 2 25.08 ± 0.92 34.48 ± 1.27
3-O-Caffeoylquinic acid 1482.97 ± 54.15 2039.09 ± 74.45 a
ρ-Coumaric acid derivative 1 50.02 ± 0.55 68.78 ± 0.76
ρ-Coumaroylquinic acid nq nq
Hydroxycinnamic acid derivative 3 372.26 ± 35.99 511.86 ± 49.48
5-O-Caffeoylquinic acid 734.38 ± 44.86 1009.77 ± 61.68 a
Hydroxycinnamic acid derivative 4 2835.87 ± 143.08 3899.33 ± 196.73 a
Caffeic acid 1263.49 ± 98.92 1737.30 ± 136.01 a
p-Coumaric acid derivative 2 528.74 ± 19.83 727.02 ± 27.26
Hydroxycinnamic acid derivative 5 704.96 ± 97.52 969.31 ± 134.08 a
Hydroxycinnamic acid derivative 6 196.75 ± 16.19 270.53 ± 22.26
p-Coumaric acid 21.07 ± 1.64 28.96 ± 2.26
Hydroxycinnamic acid derivative 7 666.97 ± 67.02 917.089 ± 92.15 a
Hydroxycinnamic acid derivative 8 175.97 ± 16.59 241.95 ± 22.80
Quercetin-3-O-glucoside nq nq
Kaempferol-3-O-rutinoside nq nq
Quercetin 35.58 ± 3.73 48.93 ± 5.13
Σ 11,069.73 15,220.88
Values are expressed as mean ± standard deviation of three assays. ∑, sum of the determined non-colored phenolics;
nq, not quantified. a Significant result (p < 0.05) is indicated as vs. total extract.

With respect to phenolic acids and derivatives, they corresponded to 69.8% and 99.7% of the
total phenolic compounds determined in the total extract and non-colored fraction, respectively
(Table 1). The main compounds were hydroxycinnamic acid derivative 4 and 3-O-caffeoylquinic acid,
Nutrients 2018, 10, 1688 7 of 20

representing 17.9% and 9.4% in the total extract, and 25.6% and 13.4% in the non-colored fraction,
respectively (Table 1). These results agreed with previous works, where the authors reported that the
major compounds found in sweet cherries are hydroxycinnamic acids, mainly the 3-O-caffeoylquinic
acid being the one that was also the most abundant compound found in Saco [6,7,9].
Additionally, one flavonol was detected and identified as quercetin, corresponding to 0.23% and
0.32% of total phenolic content in total extract and non-colored fraction, respectively. No flavan-3-ols
(e.g., catechins) were detected. This fact was not surprising given that Gonçalves et al. [16] have
already observed that catechin levels decreased in Saco cultivar during storage at room temperature
(15 ± 5 ◦ C) for 6 days.
With respect to anthocyanins, there were identified and quantified five compounds (Table 2).
All of these colored compounds were previously reported in Saco sweet cherry [6,7].

Table 2. Anthocyanin contents of Saco sweet cherry extracts (µg/g of dried extract).

Anthocyanins Spectra of Absorption (nm) Total Extract Coloured Fraction

Unknown 1 500 2.99 ± 0.24 nd
Cyanidin-3-O-glucoside 500 193.48 ± 0.54 3427.93 ± 4.39 a
Cyanidin-3-O-rutinoside 500 3865.64 ± 2.95 15656.18 ± 25.71 a
Unknown 2 500 341.16 ± 2.82 nq
Pelargonidin-3-O-rutinoside 500 337.464 ± 20.19 130.39 ± 1.22 a
Peonidin-3-O-rutinoside 500 nq nd
Σ 4740.73 19,214.50
Values are expressed as mean ± standard deviation of three assays. ∑, sum of the determined anthocyanins; nd; not
detectable; nq, not quantified. a Significant result (p < 0.05) is indicated as vs. total extract.

The total amount of anthocyanins found in the total extract and colored fraction were 4740.7 and
19,214.5 µg/g of dried extract, respectively (Table 2). Despite the different amounts of each anthocyanin
observed, both extracts revealed a similar profile, being the unique difference related to the presence
of two unknown anthocyanins detected only in the total extract (Table 2). Cyanidin-3-O-rutinoside
was the main one quantified in both extracts, representing 24.5% and 81.5% of the total compounds for
the total extract and colored fraction, respectively, followed by cyanidin-3-O-glucoside. These data are
in agreement with other previously reported works [6,7,10].
Comparatively with other cherries, tart cherries are richer in cyanidin-3-O-glucosylrutinoside
(357.7 µg/g of dried extract), followed by cyanidin-3-O-rutinoside (226.1 µg/g of dried extract),
and quercetin than sweet cherries (292.6 µg/g of dried extract) [17].

3.2. Antioxidant Capacity

Reactive species, including • NO, O2 •− , and ROO• , are products of normal cellular metabolism,
playing crucial roles in signal transduction pathways, growth regulation, gene expression, and immune
responses [4]. Normally, their production in the human body is balanced by antioxidants; however,
when they are overproduced, they cause damage in DNA, lipids, and proteins, potentiating many human
diseases, including cancer, diabetes, necrosis, neurological disorders, and cardiovascular illnesses [1].
1,1-Diphenyl-2-picrylhydrazyl (DPPH) assay is widely used to determine the antioxidant activity
of single compounds and plant extracts. All extracts studied in this work revealed to be able to scavenge
DPPH radical in a concentration-dependent manner. The total extract was the most active (IC50 =
21.88 ± 0.32 µg/mL), followed by the colored and non-colored fractions (IC50 = 31.39 ± 0.60 and
210.86 ± 0.86 µg/mL, respectively). However, all extracts were less active than ascorbic acid control
(IC50 = 4.57 ± 0.16 µg/mL) (Figure 1A and Table 3). Since phenolics are good hydrogen and electron
donors and interact with each other rising the antioxidant ability, it was expected that the total extract,
which has both colored and non-colored compounds, would be the most active [18].
Nutrients 2018, 10, 1688 8 of 20

Table 3. IC25 and IC50 (µg/mL) values found in the antioxidant activity, α-glucosidase, hemoglobin
oxidation, and hemolysis assays of Saco sweet cherry extracts.
Nutrients 2018, 10, x 8 of 20
Hemoglobin
Extract DPPH• • NO O2 •− α-Glucosidase Hemolysis
Oxidation
Values
Total extractare expressed as mean
21.88 ± 0.32 ± ±standard
33.72 0.89 deviation
41.68 ± 0.72 of three assays
53.15 ± 1.32 concerning the antioxidant
34.29 ± 0.88 28.71 ± 0.73
Colored fraction 31.39 ± 0.60 a 47.44 ± 0.67 a 16.58 ± 0.27 (IC25 ) 142.02 ± 1.17 a 33.86 ± 0.70 9.44 ± 0.48
capacity
Non-colored against
fraction 1,1-diphenyl-2-picrylhydrazyl,
210.86 ± 0.86 a,b 167.96 ± 0.92 a,b
nitric oxide456.19
69.40 ± 1.22 a,b
and±superoxide
3.74 (IC25 ) radicals (DPPH•, •NO
155.13 ± 1.45 a,b 48.31 ± 1.07 a,b
and Oare
Values 2 , respectively), α-glucosidase inhibitory activity, hemoglobin oxidation and hemolysis. IC25:
•−
expressed as mean ± standard deviation of three assays concerning the antioxidant capacity against
25% inhibitory concentration.
1,1-diphenyl-2-picrylhydrazyl, a Significant
nitric oxide andresult (p < 0.05)
superoxide radicals (DPPH• ,as
is indicated • NO
vs. total
and O •− , respectively),
extract.
2
b p < 0.05 is

α-glucosidase inhibitory activity,

indicated as vs. colored fraction. hemoglobin oxidation and hemolysis. IC25 : 25% inhibitory concentration.
a Significant result (p < 0.05) is indicated as vs. total extract. b p < 0.05 is indicated as vs. colored fraction.

A B

C D

Figure 1. Antioxidant activity against (A) 1,1-diphenyl-2-picrylhydrazyl radical (DPPH• ), (B) nitric
Figure
oxide 1. Antioxidant
radical (• NO) andactivity against (A)
(C) superoxide 1,1-diphenyl-2-picrylhydrazyl
radical radical
(O2 •− ), and (D) α-glucosidase (DPPH•activity
inhibition ), (B) nitric
of
oxide
Saco radical
sweet ( NO)
•
cherry and (C) superoxide radical (O2 ), and (D) α-glucosidase inhibition activity of Saco
extracts.
•−

sweet cherry extracts.

The result obtained from the total extract is in accordance with our previous work relative to
the SacoThesweet
resultcherry
obtained DPPH from the total extract
scavenging abilityis(IC
in 50
accordance
= 16.24 ±with 0.46 our previous
µg/mL) work relativeother
[7]. Considering to the
Saco sweet
red-fruit cherrythe
extracts, DPPH
threescavenging
extracts showedability more
(IC50 =potential
16.24 ± 0.46 thanµanthocyanin-enriched
g/mL) [7]. Consideringfraction other red-of
fruit extracts,
Romina strawberrythe three
fruitsextracts
(IC50 showed
= 1590 ±more 3.54 potential
µg/mL), than while anthocyanin-enriched
the colored fraction of fraction of Romina
Saco proved to
strawberry fruits (IC = 1590 ± 3.54 µ g/mL), while the colored fraction
have similar activity to whole methanolic extract of strawberries (IC50 = 30.29 ± 0.18 µg/mL) [19].
50 of Saco proved to have similar
Inactivity
addition,to whole
our totalmethanolic
and colored extract of strawberries
extracts revealed to (ICbe50 = 30.29active
more ± 0.18than
µ g/mL) [19].petroleum
crude, In addition, our
ether,
total and colored extracts revealed to be more active than crude, petroleum
chloroform, ethyl acetate, and butanol fractions of myrtle fruits (Myrtus communis) (IC50 = 130.44 ± 12.1, ether, chloroform, ethyl
acetate,
108.3 and111.14
± 10.2, butanol fractions
± 11.1, 85.6 ± of 5.7,
myrtleandfruits
84.42 (Myrtus
± 1.8 µg/mL, communis) (IC50 = 130.44
respectively) [20]. ± 12.1, 108.3 ± 10.2,
111.14
The±antioxidant
11.1, 85.6 ± 5.7, and 84.42
capacity of the±extracts
1.8 µ g/mL,
alsorespectively)
was evaluated against • NO and O2 •− . Both radicals
[20].
The antioxidant
are present in the human capacity of theNO
body. While extracts also was
is normally evaluated
produced against
in the •NO and O2•−. Both radicals
organism as a second messenger
are as
and present
a part of in immune
the human body. its
response, While NO with
reaction is normally •−
O2 leads produced in the organism
to the production of more astoxic
a second
free
messenger
radical species,andsuchas a aspart of immune and
peroxynitrite ROO•its
response, reaction with
, increasing O2•− leads
deleterious to the
effects in production of more
cells [1]. Presently,
toxicisfree
there much radical species,
evidence aboutsuchtheas peroxynitrite
beneficial effectsand ROO•, increasing
of phenolic-rich on • NO and
deleterious
extracts O2 •−
effects in species’
cells [1].
Presently, there
elimination [7,12].is much evidence about the beneficial effects of phenolic-rich extracts on NO and •

O2•−Inspecies’
this study,elimination extracts displayed a great capacity to capture • NO in a concentration-
all Saco[7,12].
In this
dependent study,
manner all Saco
(Figure 1B andextracts
Table displayed
3). The totalaextract
great wascapacity
the most to active
capture (IC•50NO in a±concentration-
= 33.72 0.89 µg/mL),
dependent
followed by manner
the colored(Figure
and1B and Table 3).
non-colored The total
fractions (ICextract
50 = was
47.44 ± the most
0.67 and active (IC
167.96 ± = 33.72
50 0.92 ± 0.89
µg/mL,
µ g/mL), followed by the colored and non-colored fractions (IC =
respectively) (Figure 1B and Table 3). All extracts showed a better ability to scavenge NO than ascorbic
50 47.44 ± 0.67• and 167.96 ± 0.92
µ g/mL,
acid respectively)
control (IC50 = 179.69 (Figure 1B and
± 2.06 Table The
µg/mL). 3). All
totalextracts
extractshowed
was thea mostbettereffective,
ability togiven
scavenge •
the factNO
than
that theascorbic
naturalacid control (IC
combination = 179.69
of50both ± 2.06 µ g/mL).
non-colored phenolics Theand total extract was the
anthocyanins most effective,
increases the capture given
of
the fact that the natural combination of both non-colored phenolics and anthocyanins increases the
capture of radical species though electron delocalization after hydrogen donation by the hydroxyl
(OH) groups, diminishing the concentration of these undesirable species [4].
Indeed, the capacity of sweet cherries to scavenge •NO was previously reported by Jacob et al.
[21]. The authors verified a decrease in NO levels in ten healthy women 3 h after the consumption of
280 g of Bing sweet cherries. Relatively to other red-fruits, our extracts showed better activity than
Nutrients 2018, 10, 1688 9 of 20

radical species though electron delocalization after hydrogen donation by the hydroxyl (OH) groups,
diminishing the concentration of these undesirable species [4].
Indeed, the capacity of sweet cherries to scavenge • NO was previously reported by Jacob et al. [21].
The authors verified a decrease in NO levels in ten healthy women 3 h after the consumption of 280 g
of Bing sweet cherries. Relatively to other red-fruits, our extracts showed better activity than ethanolic
(IC50 = 1600 ± 0.03 µg/mL) and aqueous extracts (IC50 = 1500 ± 0.02 µg/mL) of blueberry fruits
(Vaccinium corymbosum L.) [1]. In addition, both the total extract and colored fraction proved to be more
active than ethanolic strawberry extracts (IC50 = 118 ± 45.2 µg/mL) [22]. In comparison with ethanolic
grape extracts, grapes displayed a better ability at neutralizing • NO (IC50 = 5.1 ± 0.2 µg/mL) [22].
With respect to the ability of Saco extracts to scavenge O2 •− , all of them showed activity in
a concentration-dependent effect. The results obtained from total and non-colored extracts were IC50
= 41.68 ± 0.72 and 69.40 ± 1.22 µg/mL, respectively (Figure 1C and Table 3). The colored fraction
revealed the weaker activity (IC25 = 16.58 ± 0.27 µg/mL). Even so, all the three extracts demonstrated
to be less active than ascorbic acid control (IC50 = 28.87 ± 0.38 µg/mL). The total extract was the
most active. The presence of both colored and non-colored phenolics increases and diversifies their
interactions, and intensifies the antioxidant activity [1].
The scavenging ability of sweet cherry fruits against O2 •− was already demonstrated by
Prior et al. [23]. With respect to other red fruits, our total extract exhibited similar activity to the
vitamin C rich fraction of bilberry fruits (Vaccinium myrtillus L.) (IC50 = 49 µg/mL), but less efficiency
than their flavonoid (IC50 = 21 µg/mL) and phenolic acid (IC50 = 15 µg/mL) rich fractions [24].
Moreover, our total extract and non-colored fraction proved to have higher ability to scavenge these
species when compared with ethanolic extracts of strawberry fruits (IC50 = 72.5 µg/mL), but less
activity than those of acetone (IC50 = 9.7 µg/mL). Furthermore, our total extract showed similar activity
to strawberry ethyl acetate extracts (IC50 = 40.1 µg/mL) [25].
Our work is another report that supports the strong connection between the antioxidant capacity
and the phenolic content. It is well-known that phenolic compounds can easily capture free radical
species and chelate metal ions due to their acid moiety and OH groups [18]. The phenolic OH groups on
the B-ring easily donate hydrogen atoms or an electron to radicals, stabilizing them [1]. Additionally,
the OH groups present in the unsaturated C-ring and the double bond on this ring also enhance
the antioxidant capacity [4]. Following this same order of context, it is important to emphasize the
presence of cyanidin-3-O-rutinoside in both the total extract and colored fraction. This anthocyanin
presents several OH groups in its structure, being one of the phenolics more responsible for the
biological activity of the fruits [26]. Furthermore, both colored and non-colored phenolics interact
with each other, through additive and synergistic combinations, conferring to sweet cherries great
antioxidant effects [1,18].
In fact, by Pearson’s test, positive correlations were found between the antioxidant tests and
the total amount of phenolic compounds. High correlations (r > 0.9013) were obtained between
the majority of the non-colored phenolics, anthocyanins, and antioxidant activities measured by
DPPH• , • NO and O2 •− assays. Even so, we found a negative correlation between the O2 •− scavenging
test, cyanidin-3-O-glucoside (r = −0.9818), and cyanidin-3-O-rutinoside (r = −0.9818), and between
DPPH• and • NO antioxidant assays, and pelargonidin-3-O-rutinoside (r = −0.9241 and −0.9660,
respectively). Finally, it is important to remember the possible presence of other non-determined
reducing compounds, such as organic acids, carotenoids, and volatile compounds, which may also
contribute to increasing the antioxidant potential, and that were not determined in this work.

3.3. α-Glucosidase Inhibitory Activity

Diabetes mellitus is an epidemic metabolic disease, characterized by chronic hyperglycemia and
glucose intolerance caused by defects in insulin hormone that affects millions of people worldwide [2].
One therapeutic approach is to restore blood glucose levels as close to normal as possible via
the inhibition of carbohydrate hydrolyzing-enzymes, such as α-glucosidase [27]. Over the years,
Nutrients 2018, 10, 1688 10 of 20

many phenolic compounds present in plants have shown to be capable of inhibiting the action of
α-glucosidase, thereby delaying glucose uptake [7,27].
In our study, all Saco extracts tested proved to have the capacity to inhibit the α-glucosidase
enzyme as a concentration-dependent effect. The total extract showed the most effectiveness
(IC50 = 53.15 ± 1.32 µg/mL), followed by the colored (IC50 = 142.02 ± 1.17 µg/mL) and non-colored
fractions (IC25 value of 456.19 ± 3.74 µg/mL) (Figure 1D and Table 3). The obtained IC50 values were
much lower than the acarbose control (IC50 = 389.89 ± 4.01 µg/mL), one of the therapeutic drugs most
recommended to treat type 2 diabetes but whose use is limited since it causes gastrointestinal problems
such as diarrhea, flatulence, and intestinal pain [7]. Both colored and non-colored phenolics interact
among all and with the substrate of the enzyme and create bonds with this one, thus interfering with
its action and consequently preventing the carbohydrate digestion, as well as protecting pancreatic
β-cells from oxidative stress levels, contributing to their normal functioning [28].
The antidiabetic properties of sweet cherries are already known [7,29,30]. Lachin [30] reported
that diabetic rats fed with 200 mg of cherry extract per kg of body weight for 30 days showed reduced
blood glucose and urinary microalbumin levels, proving that the ingestion of these fruits can protect
pancreatic β-cells and retard glucose absorption. Besides, Cao et al. [29] revealed that fractionated
extracts of Black Pearl sweet cherry cultivar rich in anthocyanins, hydroxycinnamic acids, and flavonols
also demonstrated antidiabetic properties by promoting cellular glucose consumption in HepG2 cells.
Comparing to the antidiabetic potential of other fruit extracts, our total extract and colored
fraction exhibited a better capacity to inhibit α-glucosidase enzyme than dried crude acetone extracts
of Ficus lutea and Ficus sycomorus (IC50 = 290 ± 111 and 217 ± 69 µg/mL, respectively) [31].
The positive results obtained with Saco extracts to inhibit α-glucosidase action are largely due
to their phenolic constituents. In these ones, the unsaturated C-ring, the linkage of the B-ring at
the position 3, the OH substitution on the B-ring, and the presence of either 3-OH and 4-CO groups
enhance their ability to inhibit α-glucosidase [3].
In addition, they interact not only synergistically and additively between them and with other
compounds, but also in both competitive and non-competitive ways with the substrate of the enzyme,
increasing the antidiabetic potential of this fruit [3,7]. Also, phenolics can bond with digestive enzymes
through hydrophobic association, inhibiting the action of them [32]. For these reasons, both colored
and non-colored phenolic compounds can promote the insulin production and the proliferation of
pancreatic β-cells and can interfere with glucose homeostasis by modifying independent mechanisms
and increasing insulin receptor-dependents (such as peroxisome proliferator-activated receptor gamma,
PPARγ), thus exerting antidiabetic actions [33].
In our study, it is possible to state that non-colored compounds have practically no significant and
notorious effects on the inhibition of α-glucosidase, whilst anthocyanins showed great effectiveness.
These results were expected, since previous studies reported that the colored compounds are the main
compounds responsible for the antidiabetic capacities revealed by cherries, mainly due to their high
antioxidant abilities (that are enhanced by the presence of the 3-OH group and the OH substitution on
the B-ring in anthocyanins) that allow the protection of insulin mechanisms against oxidative damage
and offer capacity to inhibit intestinal enzymes [3,28,34]. To reinforce this fact, the Pearson’s test was
performed and there was a high correlation between the α-glucosidase inhibitory assay and the total
amount of anthocyanins (r = 0.9929).
Additionally, the presence of rutinose in the 3-O-position of cyanidin, which already has many OH
groups, reinforces this health-promoting property [28]. Indeed, the main colored compound present in
sweet cherries, that is cyanidin-3-O-rutinoside, already has been demonstrated to have a significant
inhibitory effect in α-glucosidase activity in a concentration-dependent manner by competing with
glucose for the binding site on sodium-dependent glucose transporter (SGLT) 1, and consequently
delaying the absorption of glucose [3,35]. More recently, Adisakwattana et al. [35] reported that
cyanidin and its glycosides also create covalent and/or non-covalent interactions between their OH
groups and the polar groups (amide, guanidine, peptide, amino, and carboxyl groups) of amino
Nutrients 2018, 10, 1688 11 of 20

acids in the active site of carbohydrate digestive enzymes, stopping their action. The same work also
revealed that a single oral administration of cyanidin-3-O-rutinoside (100 and 300 mg/kg) significantly
decreases plasma glucose levels in rats at 30, 60, and 90 min after maltose and sucrose administration.
Furthermore, cyanidin-3-O-glucoside also promotes the release of insulin by pancreatic β-cells and
glucose uptake by enhancing glucose transporter GLUT 4 [33].
Non-colored compounds can also interfere with the activity of digestive enzymes and prevent
insulin resistance events [34]. Particularly chlorogenic acids already showed to be able to increase
GLUT 4 expression via the phosphatidylinositol 3-kinase (PI3K)-independent pathway, as well as to
suppress hepatic gluconeogenesis through the inhibition of glucose 6-phosphatase activity, and to
inhibit Na+ -dependent glucose transporters SGLT 1 and SGLT 2 [34]. Caffeic acid also showed ability
to enhance glucose-stimulated insulin secretion in mice pancreatic islets at concentrations from 10–10
to 10−6 M and the expression of key insulin regulatory genes INS1, INS2, PDX1, INSR, IRS1, MAFB,
and GLUT2 [36].

3.4. Protective Effects of Saco Extracts against ROO• in Human Blood Samples
Erythrocytes are highly susceptible to being attacked by reactive species due to their content in
unsaturated fatty acids, oxygen, hemoglobin, and in transition metals such as copper and iron [37].
In addition, they are more frequently exposed to oxygen than other body tissues and so they are more
vulnerable to oxidative damage, causing the oxidation of lipids and proteins in the cell membrane,
and thus inducing hemolysis [38]. Previous studies already have shown that phenolics present in
plants offer therapeutic benefits against oxidative damage in erythrocytes [7,38,39].
Figure 2A shows the protective effects of Saco extracts to avoid hemoglobin oxidation in a
concentration-dependent manner. To the best of our knowledge, this is the first study about the
capacity of colored and non-colored rich fractions of sweet cherries to prevent hemoglobin oxidation.
The colored fraction and the total extract were the most active and displayed similar activity (IC50 =
33.86 ± 0.70 and 34.29 ± 0.88 µg/mL, respectively) (Figure 2A and Table 3); however, they were seven
times less active than quercetin control (IC50 = 4.38 ± 0.42 µg/mL) analyzed in the same conditions.
The non-colored fraction showed an IC50 value of 155.13 ± 1.45 µg/mL (Figure 2A and Table 3).
As far as we know, few studies exist on the ability of fruit extracts to prevent the oxidation of
hemoglobin. Previously, we have reported that the total extract of Saco possesses the capacity to
prevent hemoglobin oxidation [7]. Hydrophilic extracts of murici fruits (Byrsonima crassifolia) also
showed this property, revealing an IC50 of 271 ± 44 µg/mL [14], being less active than Saco extracts.
The capacity of phenolic compounds to inhibit oxidative damage is closely associated with the
strong antioxidant capacity exhibited by themselves [18]. Both colored and non-colored phenolics can
penetrate into the erythrocytes’ membrane due to their lipophilic character, causing a reduction in its
fluidity and stability, and thus preventing the propagation of these harmful radicals [37]. The biological
activity of the total extract and colored fraction is enhanced by the presence of catechol rings and OH
groups, principally in cyanidin-3-O-rutinoside, increasing the potential of both extracts [40], as well
as the ability to inhibit the oxidation of hemoglobin by oxidizing the heme iron of erythrocytes [41].
In this way, they prevent enzymatic reactions and consequently oxidative events.
Another experiment was conducted in order to evaluate the capacity of Saco extracts to prevent
hemolysis. The addition to 2,20 -azobis (2-ethylpropionamidine) dihydrochloride (AAPH) generated
ROO• that is capable of attacking the membrane of erythrocytes from the outside, promoting
hemolysis [37]. As far as we know, this is the first report concerning lysis prevention by both colored
and non-colored fractions of sweet cherry fruits. The colored fraction (IC50 = 9.44 ± 0.48 µg/mL)
showed the best anti-hemolytic protection, followed by the total extract (IC50 = 28.71 ± 0.73 µg/mL)
and non-colored fraction (IC50 = 48.31 ± 1.07 µg/mL) (Figure 2B and Table 3). All extracts exhibited
less activity than quercetin control (IC50 = 1.58 ± 0.12 µg/mL) (Figure 2B and Table 3).
 hemolysis [37]. As far as we know, this is the first report concerning lysis prevention by both colored
and non-colored fractions of sweet cherry fruits. The colored fraction (IC50 = 9.44 ± 0.48 µ g/mL)
showed the best anti-hemolytic protection, followed by the total extract (IC50 = 28.71 ± 0.73 µ g/mL)
and non-colored fraction (IC50 = 48.31 ± 1.07 µ g/mL) (Figure 2B and Table 3). All extracts exhibited
Nutrients 2018, 10,
less activity 1688quercetin control (IC50 = 1.58 ± 0.12 µ g/mL) (Figure 2B and Table 3).
than 12 of 20

A B

Figure 2. Inhibition of (A) hemoglobin oxidation and (B) hemolysis by Saco sweet cherry extracts.

Saco total extract already proved to be able to prevent hemolysis [7]. Considering other fruit
extracts, Saco extracts exhibited more potential to inhibit hemolysis than aqueous extracts of strawberry
fruits (Arbutus unedo L.) (IC50 = 430.00 µg/mL) [26]. Furthermore, the colored fraction displayed
similar activity to methanolic extracts of grape fruits (Ruby Cabernet) (IC50 = 11.62 µg/mL) [39].
These results reinforce the fact that phenolic compounds, principally anthocyanins, can scavenge
free radicals before they attack the membrane of erythrocytes, diminishing their concentration,
and thereby preventing hemolysis [37]. These results were supported by another work concerning the
anti-hemolytic effects of phenolics from honey, where it was also verified that they can capture free
radicals, preventing lysis’ incident [40].

3.5. Effect of Sweet Cherry Extracts in Caco-2 Cell Viability

As referred above, it was already reported that sweet cherries have antidiabetic properties [7,29,30].
Hereupon, human colorectal adenocarcinoma Caco-2 cells were selected for two reasons: (i) they are a
model of the intestinal epithelium, since, after differentiation, they form monolayers, that reproduce
several characteristics of intestinal epithelial cells (e.g., the formation of microvillus at the apical
cell surface, the tight junctions between cells, and the expression of brush-border proteins including
digestive enzymes, transporters, and receptors; and (ii) it is important to note that after consumption
of cherries, their compounds directly contact with intestinal epithelium [15].
In a first step, and given that phenolic compounds can exert a dose-dependent cytotoxicity,
a preliminary experiment was conducted to determine the range of concentrations for which the
exposure to each extract does not affect the cellular viability. As can be seen in Figure 3, Caco-2 cells
proved to be more sensitive to the colored fraction when compared to the total extract and non-colored
fraction, exhibiting decreases concerning their cellular viability, that ranged from 93.11% (200 µg/mL)
to 73.66% (400 µg/mL) to 37.90% (800 µg/mL), and had an IC50 value of 667.84 ± 2.46 µg/mL.
Additionally, and as expected, the more significant LDH response was also obtained using the colored
fraction, mainly for the higher concentrations, such as 200, 400, and 800 µg/mL (with values of 116.5%,
126.3%, and 151.1%, respectively) (Figure 3).
colored fraction, exhibiting decreases concerning their cellular viability, that ranged from 93.11% (200
μg/mL) to 73.66% (400 μg/mL) to 37.90% (800 μg/mL), and had an IC50 value of 667.84 ± 2.46 μg/mL.
Additionally, and as expected, the more significant LDH response was also obtained using the
colored fraction, mainly for the higher concentrations, such as 200, 400, and 800 μg/mL (with values
of 116.5%,
Nutrients 126.3%,
2018, 10, 1688 and 151.1%, respectively) (Figure 3). 13 of 20

Figure 3. Effect of Saco extracts on Caco-2 cell lines viability after 24 h of exposure, assessed by
Figure 3. Effect of Saco extracts on Caco-2 cell lines viability after 24 h of exposure, assessed by 3-(4,5-
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction and lactate dehydrogenase
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
(LDH) leakage assays. Values show mean ± SEM bromide
of six(MTT) reduction
independent and performed
assays lactate dehydrogenase
in triplicate
(LDH) leakage assays. Values
# show
(* p < 0.05, ** p < 0.01 and p < 0.0001).mean ± SEM of six independent assays performed in triplicate (*
p < 0.05, ** p < 0.01 and # p < 0.0001).
The obtained data suggest that the loss of mitochondrial activity happened prior to the
The obtained
membrane’s damage data suggest
given that that the loss
the results of of mitochondrial
MTT were more activity
expressivehappened
than thosepriorof toLDH,
the
membrane’s damage given that the results of MTT were more expressive than those
thus discarding a necrotic process in the lowest concentrations (≤200 µg/mL) and its occurrence of LDH, thus
discarding
in a necrotic
the highest process(400
concentrations in the
andlowest concentrations
800 µg/mL), which was(≤200 μg/mL) and
accompanied byits
an occurrence
elevation ofinLDH the
highest
in concentrations
the culture medium (400 and 800
[42–44]. μg/mL),
Indeed, whichcan
phenolics wasinterfere
accompanied by an elevation
in different of LDH in the
steps of carcinogenesis
culture medium [42–44]. Indeed, phenolics can interfere in different steps of carcinogenesis
(promotion, initiation, and progression) acting as a blocking agent or as a cancer suppressor, inhibiting
(promotion,
the metabolicinitiation, andpre-carcinogens,
action of the progression) acting as a blockingblocking
and consequently, agent ortheastumor
a cancer suppressor,
initiation [44,45].
inhibiting the metabolic action of the pre-carcinogens, and consequently, blocking
In our work, higher concentrations of phenolics (>200 µg/mL) may induce DNA damage and necrosis the tumor
in cancer cells [43,44].
The remarkable effect of the colored extract was not surprising, taking into account that
the anthocyanins promoted cell death in cancer cells, triggering apoptosis through two major
cell-intrinsic pathways, either by binding to the death receptors on the cell surface or by promoting the
mitochondrial release of cytochrome C, inciting the depolarization of the mitochondrial membrane and
the degradation of the poly[adenosine diphosphate-ribose] polymerase 1 [46]. This fact was supported
by the positive correlation obtained by the total anthocyanin content and the inhibitory proliferation
activity (r = 0.6674). These effects were also enhanced by their three OH groups on the B-ring, which are
the main responsible for their biological activity [18]. Additionally, non-colored phenolics also exhibit
antiproliferative effects. Particularly, Yen et al. [47] verified that many phenolic compounds, including
quercetin derivatives, caffeic, chlorogenic, and p-coumaric acids, showed cytotoxicity and strong
inhibitory effects on human cancer cell growth, once again due to their antioxidant capacities.
In order to further explore the cellular mechanism of growth inhibition by cherry extracts,
a morphological assessment, and an attempt of evaluation of cell cycle were performed. Apoptosis
and necrosis represent two fundamental types of cell death. On the one hand, apoptosis causes
several morphological and biochemical changes in cells that may take hours or days. From the
morphological point of view, it is usually associated with cell shrinkage and bleb formation [47].
On the other hand, necrosis occurs suddenly, and it is characterized by mitochondrial and cellular
swelling followed by plasma membrane disruption [43]. This study was performed upon incubation
with the cherry extracts using the higher concentrations (200, 400, and 800 µg/mL), which were those
that showed promising antiproliferative activity. The control cells positively marked with PI and gave
a characteristic histogram on linear PI-fluorescence channel, but unfortunately, it was not possible
to assess the cell cycle on treated cells because the number of events was not sufficient (data not
shown) due to extensive cell death, as was apparent with observation under the optical microscope
(Figures 4 and 5). While the acquisition of controls was stopped when about 10,000 singlet events
were acquired, treated cells were acquired until no more cell suspension was left, and originated a
much lower event count. To visualize simultaneously morphological information (size) and PI-staining
intensity of events, data were plotted on Forward Scatter (FSC)-Height vs. Fluorescence channel 3
(FL3)-Area contour plots (Figure 4). PI-stained and normal sized events decreased as the concentration
increased (upright (UR) part of the plots, Figure 4B,E,H for the total extract, Figure 4C,F,I for the colored
Nutrients 2018, 10, 1688 14 of 20

fraction, and 4D,G,J for the non-colored fraction, respectively). Furthermore, in the highest tested
concentrations of the total extract and colored fraction (400 and 800 µg/mL), there was a significant
increase in the amount of debris with a size bigger than usual (upper left quadrant), which may be
evidence of necrotic processes (Figure 4E,H for the total extract, and Figure 4F,I for the colored fraction,
respectively). Beyond that, and as expected the colored fraction was the most cytotoxic, showing the
lowest event number (Figure 4I). This fact agrees with the previous result obtained through MTT and
LDH assays
Nutrients concerning
2018, 10, x the colored fraction (Figure 3). 15 of 21

Figure 4. Correlation of size with Propidium Iodide (PI)-staining contour plots obtained by flow
Figure 4. Correlation
cytometric of size
assay in Caco-2 cellswith Propidium
treated Iodide
with cherry (PI)-staining
extracts contour plots
for 24 h. Untreated obtained
cells control (A)by
andflow
cell
cytometric
treated with assay in Caco-2
200 (B–D), 400cells treated
(E–G), with
and 800 cherry
(H–J) extracts
µg/mL for 24
of each h. Untreated
extract are shown.cellsThe
control (A)were
extracts and
cell
Sacotreated with (B,E,H),
total extract 200 (B–D),
the400 (E–G),
colored and (C,F,I)
extract 800 (H–J)
andμg/mL of each extract
the non-colored extractare shown.
(D,G,J). TheThe extracts
percentage
were Saco total extract (B,E,H), the colored extract (C,F,I) and the non-colored extract
of events in each quadrant is shown for a representative sample. PI-stained and normal sized events (D,G,J). The
percentage
correspondoftoevents in each
the upper quadrant
right quadrant is (potential
shown forlivea representative sample.
cells). PI-stained PI-stained
debris (potentialand normal
apoptotic
sized
cells) events correspond
correspond to the to the upper
lower right quadrant
right quadrant. Left (potential live cells).resulting
upper (potentially PI-stained debris
from (potential
necrosis) and
apoptotic cells) correspond
lower left quadrants to the lower
are PI-negative eventsright quadrant.
(potential Left upper
cytoplasmic debris).(potentially resulting
FSC, forward scatter;from
FL3,
necrosis) and channel
fluorescence lower left
3. quadrants are PI-negative events (potential cytoplasmic debris). FSC, forward
scatter; FL3, fluorescence channel 3.

At the morphological observation of cultures under the microscope, a high amount of debris
was observed (Figure 5) that increased with the increase of concentration (Figure 5H–J). These data
were to be expected because it was already mentioned that high concentrations of phenolics can
induce acute toxicity effects on cancer cells. Therefore, this work provides more support of the toxicity
 Nutrients 2018, 10, 1688 15 of 20

At the morphological observation of cultures under the microscope, a high amount of debris was
observed (Figure 5) that increased with the increase of concentration (Figure 5H–J). These data were
to be expected because it was already mentioned that high concentrations of phenolics can induce
acute toxicity effects on cancer cells. Therefore, this work provides more support of the toxicity and
pro-oxidant effects of higher levels of phenolic compounds, which are capable of affecting cell functions,
such as growth and differentiation, causing probably both apoptosis and necrotic events. Specifically,
it was already documented that quercetin may induce an initial shock to the cells, resulting in necrosis,
followed
Nutrients by10,axreorganization of the remaining viable cells that will undergo apoptosis after prolonged
2018, 16 of 21
treatment [43]. Furthermore, Wang et al. [44], concerning the inhibitory effect of blueberry anthocyanin
effect of blueberry
extracts on melanoma anthocyanin extracts
cells, verified on melanoma
that total cells,increased
apoptotic cells verified gradually
that total atapoptotic cells
concentrations
increased
of 0–800 gradually
µg/mL inata concentrations
dose-dependent ofmanner,
0–800 μg/mL in a dose-dependent
with necrosis manner,
occurring from with necrosisof
the concentration
occurring
300 µg/mL. from the concentration of 300 μg/mL.

Figure 5. Morphological assessment of Caco-2 cells (control vs. treatment after 24 h of incubation).
(A) corresponds
Figure to the assessment
5. Morphological control, (B,E,H) correspond
of Caco-2 to Saco
cells (control vs.total extract,
treatment while
after 24 h(C,F,I) to the colored
of incubation). (A)
fraction and (D,G,J) to the non-colored one, at concentrations of 200, 400 and 800 µg/mL,
corresponds to the control, (B,E,H) correspond to Saco total extract, while (C,F,I) to the colored respectively.
As expected
fraction and considering
and (D,G,J) the data
to the non-colored one,ofatFigure 4, it wasofobserved
concentrations an 800
200, 400 and increase
μg/mL, in respectively.
debris as the
Asconcentration
expected and of each fraction the
considering increased.
data of Figure 4, it was observed an increase in debris as the
concentration of each fraction increased.
3.6. Cytoprotection Assay
3.6. Cytoprotection
The second stepAssay
was to evaluate the potential of each extract to protect the cells against the toxicity
caused by t-BHP. t-BHP is an organic peroxide metabolized by cytochrome P450 and is widely used,
The second step was to evaluate the potential of each extract to protect the cells against the
being a better alternative than hydrogen peroxide in oxidation-induced stress studies since it creates
toxicity caused by t-BHP. t-BHP is an organic peroxide metabolized by cytochrome P450 and is
widely used, being a better alternative than hydrogen peroxide in oxidation-induced stress studies
since it creates more stable radical species, particularly toxic peroxyl and alkoxyl radicals that affect
cell integrity and form covalent bonds with cellular molecules, promoting the death of cells [48].
Cells were treated with different concentrations of each extract for 24 h prior to t-BHP exposure
(1 mM, 6 h). Cellular viability was again determined by MTT and LDH leakage assays. All the extracts
exerted a dose-dependent protective effect in the MTT reduction and LDH leakage assays (Figure 6).
Nutrients 2018, 10, 1688 16 of 20

more stable radical species, particularly toxic peroxyl and alkoxyl radicals that affect cell integrity and
form covalent bonds with cellular molecules, promoting the death of cells [48].
Cells were treated with different concentrations of each extract for 24 h prior to t-BHP exposure
(1 mM, 6 h). Cellular viability was again determined by MTT and LDH leakage assays. All the extracts
exerted a dose-dependent protective effect in the MTT reduction and LDH leakage assays (Figure 6).
Pre-treatment of Caco-2 cells with each extract was seen to significantly prevent reductions in cell
viability. The total protection, when compared to stressed control cells, was almost achieved at the
concentrations of 50 and 200 µg/mL with and without co-incubation with t-BHP for the total extract
and non-colored fraction. While for the colored fraction, this one was achieved at concentrations of 50
Nutrients 2018, 10, x 17 of 21
and 100 µg/mL with and without co-incubation, respectively (Figure 6). Relative to the assay with
co-incubation,
extract, 4.98% and there werefor
14.46% observed increments
the non-colored of viability
fraction, between
and between 2.89%
1.73% and
and 25.45%
17.22% forfor
thethe total
colored
extract, 4.98% and 14.46% for the non-colored fraction, and between 1.73% and 17.22%
fraction (Figure 6). Concerning the obtained data without co-incubation, lower values of protection for the colored
fraction
were (Figureranging
observed, 6). Concerning the to
from 25.00 obtained
33.58% data without
for the co-incubation,
total extract, lower values
18.15 to 28.96% for theof protection
non-colored
were observed,
fraction, ranging
and 16.90% to from
27.02%25.00
for to 33.58%
the forfraction
colored the total(Figure
extract,6).
18.15
Thisto outcome
28.96% for the non-colored
indicates that the
presence of phenolics in the medium, even outside the cells, also offers protection against that
fraction, and 16.90% to 27.02% for the colored fraction (Figure 6). This outcome indicates the
cellular
presence
damage [44].of phenolics in the medium, even outside the cells, also offers protection against cellular
damage [44].

Figure 6. Caco-2 cellular viability assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

Figure 6. Caco-2 cellular viability assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) reduction and lactate dehydrogenase (LDH) leakage assays, after exposure to Saco
bromide (MTT) reduction and lactate dehydrogenase (LDH) leakage assays, after exposure to Saco
extracts, with and without tert-butyl hydroperoxide (t-BHP)-induced toxicity. Cells were pre-treated
extracts, with and without tert-butyl hydroperoxide (t-BHP)-induced toxicity. Cells were pre-treated
with each extract for 24 h. Insulted cells were further exposed to t-BHP (1 mM) for 6 h. Values show
with each extract for 24 h. Insulted cells were further exposed to t-BHP (1 mM) for 6 h. Values show
mean ± SEM of six independent assays performed in triplicate (* p < 0.05, ** p < 0.01 and # p < 0.0001
mean ± SEM of six independent assays performed in triplicate (* p < 0.05, ** p < 0.01 and # p < 0.0001
compared to the respective controls).
compared to the respective controls).
In both assays, the highest protection was verified with the total extract at the concentration of
In both assays, the highest protection was verified with the total extract at the concentration of
800 µg/mL, which is another support about the simultaneous interaction and synergy between colored
800 μg/mL, which is another support about the simultaneous interaction and synergy between
and non-colored phenolic compounds [44].
colored and non-colored phenolic compounds [44].
Once again and given the oxidative-stress induced by t-BHP, it was verified that there was
Once again and given the oxidative-stress induced by t-BHP, it was verified that there was an
an increase in LDH leakage, most notorious in the assay without co-incubation, revealing that the
increase in LDH leakage, most notorious in the assay without co-incubation, revealing that the
permanence of phenolics in the medium increases the protective effects on cells. Our results are in
agreement with the work of Bedoya-Ramírez et al. [49], who reported that 500 μg/mL of Colombian
coffee can increase cell viability between 34% and 45% evaluated by MTT assay.
The phenolic composition of each extract is described in Tables 1 and 2. In fact, phenolics are
greatly responsible for the observed antioxidant protective effects. As is already known,
Nutrients 2018, 10, 1688 17 of 20

permanence of phenolics in the medium increases the protective effects on cells. Our results are in
agreement with the work of Bedoya-Ramírez et al. [49], who reported that 500 µg/mL of Colombian
coffee can increase cell viability between 34% and 45% evaluated by MTT assay.
The phenolic composition of each extract is described in Tables 1 and 2. In fact, phenolics are
greatly responsible for the observed antioxidant protective effects. As is already known, mitochondria
are very susceptible to oxidative stress induced by oxygen species generated continuously in these
organelles in the course of oxidative stress [48]. Phenolics, essentially due to their hydroxyl and
carboxyl groups, exhibit promising and strong antioxidant activities being capable of capturing these
radicals and chelating metals before they attack the mitochondrial membrane [42]. Particularly, 50 µM
of quercetin derivatives already showed similar protective effects at preventing Caco-2 damage induced
by t-BHP [50].

4. Conclusions
Considering the current interest in cherries, the present study describes the phenolic constitution
and health-promoting properties of three extracts from Saco sweet cherry. A total of 22 phenolic
compounds, including 17 non-colored phenolics and 5 anthocyanins, were identified, hydroxycinnamic
acids and cyanidin-3-O-rutinoside being the main ones. In relation to biological potential, in a general
way, the total extract proved to be the most active given the interactions established between both
colored and non-colored phenolic compounds. Even so, all extracts revealed facility to scavenge free
radical species and to inhibit the α-glucosidase intestinal enzyme. Concerning human erythrocytes
protection, the colored fraction was the most effective, mainly due to the presence of many OH
groups in anthocyanins, which increases the capacity of scavenging ROO• species. On the other hand,
and relative to the Caco-2 cell study, the highest tested concentrations of each extract showed the ability
to inhibit the proliferation of Caco-2 cells mainly due to their antioxidant properties. Additionally, both
fractions and the total extract exerted a cytoprotective effect against oxidative stress induced by t-BHP
in Caco-2, improving cell viability. Once again, this protection was closely linked to the capacity of
phenolics to pass through the cellular membrane and act as direct antioxidants, capturing free radicals
and chelating metals. Our results suggest that phenolic compounds are a major factor correlated with
health benefits. However, our toxicity results justify concerns regarding the consumption of high doses
that become unsafe and harmful to human health. Therefore, further animal and human trials are
needed to unravel ensure safe dosage of sweet cherry fractions and to incite their use to enrich food
and nutraceutical or pharmaceutical products.

Author Contributions: A.C.G. performed the research and wrote the paper. M.R. and A.O.S. contributed with
new reagents and analytical tools. G.A. and L.R.S. designed the research, analyzed the data, and edited the paper.
All authors approved the manuscript in its current form.
Funding: This work is supported by FEDER funds through the POCI-COMPETE 2020-Operational Programme
Competitiveness and Internationalisation in Axis I—Strengthening research, technological development and
innovation (Project POCI-01-0145-FEDER-007491), Operational Program of the Center (Project CENTRO-
01-0247-FEDER-017547), and National Funds by FCT—Foundation for Science and Technology (Project UID/
Multi/00709/2013). Luís R. Silva was supported by a postdoc incentive grant under the Protocol signed between
the University of Beira Interior (UBI) and Banco Santander/Totta. Ana Carolina was supported by the R&D
Business Project (Project CENTRO-01-0247-FEDER-017547) co-financed by the European Regional Development
Fund (ERDF) through the Regional Operational Program of the Center (Portugal 2020).
Conflicts of Interest: The authors have declared no conflict of interest.

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