Composition of Nonextractable Polyphenols from Sweet
Cherry Pomace Determined by DART-Orbitrap-HRMS and Their In
Vitro and In Vivo Potential Antioxidant,
Antiaging, and Neuroprotective Activities
Sweet cherry pomace is an important source of phenolic compounds with beneficial health properties. As after the extraction of phenolic compounds, a phenolic fraction called nonextractable polyphenols (NEPs) remains usually retained in the extraction residue, alkaline and acid hydrolyses and enzymatic-assisted extraction (EAE) were carried out in this work to recover NEPs from the residue of conventional extraction from sweet cherry pomace. In vitro and in vivo evaluation of the antioxidant, antihypertensive, antiaging, and neuroprotective capacities employing Caenorhabditis elegans was achieved for the first time. Extractable phenolic compounds and NEPs were separated and identified by families by high-performance thin-layer chromatography (HPTLC) with UV/Vis detection. A total of 39 phenolic compounds were tentatively identified in all extracts by direct analysis in real-time high-resolution mass spectrometry (DART-Orbitrap-HRMS). EAE extracts presented the highest in vitro and in vivo antioxidant capacity as well as the highest in vivo antiaging and neuroprotective capacities. These results showed that NEPs with interesting biological properties are retained in the extraction residue, being usually underestimated and discarded.
Introduction<!>Chemicals and Samples<!>Conventional Extraction
of Extractable Polyphenols<!>Enzymatic-Assisted Extraction<!>Acid and Alkaline Hydrolyses<!>Total Phenolic and Proanthocyanidin Contents<!>High-Performance Thin-Layer
Chromatography
Separation of Extractable Polyphenols and NEPs<!>DART-Orbitrap-HRMS Analysis<!>Antioxidant
Capacity Determination<!>Antihypertensive Capacity<!>Elastase Inhibition Activity<!>Acetylcholinesterase Inhibition
Activity
Assay<!>Caenorhabditis
elegans Strains
and Maintenance<!>Antioxidant Response
in C. elegansIn Vivo Assay<!>Health
Span in C. elegansIn Vivo Assay<!>Neuroprotective
Capacity of Extracts in C. elegansIn Vivo Assay<!>Cell Culture, Treatments, and Cell Viability<!>Statistical Analysis<!>Results and Discussion<!>Determination of the Total Phenolic and Proanthocyanidin
Contents<!><!>Determination of the Total Phenolic and Proanthocyanidin
Contents<!>Chromatographic Separation by HPTLC-UV/Vis
of EPPs and NEPs Obtained from Conventional Extraction, Acid and Alkaline
Hydrolyses, and EAE Using HBN Methodology<!><!>Chromatographic Separation by HPTLC-UV/Vis
of EPPs and NEPs Obtained from Conventional Extraction, Acid and Alkaline
Hydrolyses, and EAE Using HBN Methodology<!>Tentative Identification by DART-Orbitrap-HRMS
of EPPs and NEPs Obtained by Conventional Extraction, Acid and Alkaline
Hydrolyses, and EAE Using HBN Methodology<!><!>Tentative Identification by DART-Orbitrap-HRMS
of EPPs and NEPs Obtained by Conventional Extraction, Acid and Alkaline
Hydrolyses, and EAE Using HBN Methodology<!>Antihypertensive Capacity<!><!>Antioxidant Capacity<!><!>Antioxidant Capacity<!>Antiaging Capacity<!><!>Antiaging Capacity<!><!>Antiaging Capacity<!>Neuroprotective
Capacity<!><!>Neuroprotective
Capacity<!>Cytotoxic and Proliferative Effects in HepG2,
HFF-1, SKOV3, and HT-29 Cell Lines of EPPs Obtained by Conventional
Extraction and NEPs Obtained by Alkaline Hydrolysis and EAE from Sweet
Cherry Pomace<!><!>Cytotoxic and Proliferative Effects in HepG2,
HFF-1, SKOV3, and HT-29 Cell Lines of EPPs Obtained by Conventional
Extraction and NEPs Obtained by Alkaline Hydrolysis and EAE from Sweet
Cherry Pomace<!>Conclusions<!><!>Author Contributions<!>
<p>Oxidative stress is characterized by the uncontrolled formation
of reactive oxygen species (ROS) and an imbalance in the biological
system's capacity to repair the cellular damage that increases
with aging. The increase in ROS levels induces lipid peroxidation
in cell membranes and initiates neuronal dysfunction and neuronal
death causing different diseases such as Alzheimer's or Parkinson's
diseases, among other pathological situations.1,2 Alzheimer's
disease is characterized by an accumulation of intraneuronal filaments
formed by the microtubule-associated protein tau, acetylcholine degradation,
and aggregation of amyloid-β protein (a pro-inflammatory agent)
in the brain parenchyma and cerebral blood vessels, which are associated
with the loss of neurons and their functions, a process increased
in the brain with aging.3,4 Inhibitors of the acetylcholinesterase
enzyme, such as tacrine or galantamine, are the most used medications
for the treatment of Alzheimer's disease delaying the degradation
of released acetylcholine by enhancing cholinergic neurotransmission.
Also, different researchers have demonstrated that patients receiving
anti-inflammatory therapies have decreased risk for developing Alzheimer's
disease, and antihypertensive medications are used to increase blood
flow in patients with Alzheimer's disease.5−8 However, current drugs employed
to mitigate some of the symptoms of Alzheimer's disease cause
undesirable secondary effects (nausea, diarrhea, insomnia, etc.).9 For this reason, there is growing interest in
finding alternative treatments from natural sources to prevent damage
to cells by ROS and acetylcholine degradation. In this sense, fruits
are recognized for their high concentrations of natural antioxidants
and anti-inflammatory and antihypertensive compounds, such as phenolic
compounds.10,11 These compounds have demonstrated
important antioxidant, anti-inflammatory, and antihypertensive effects,
playing a relevant role in the prevention of neurological pathologies
like Alzheimer's disease.3,9,12−16 Phenolic compounds are mainly concentrated in fruit peels, which
causes their recovery in industry processing to be low because peels
are considered waste material.17,18 In particular, the
processing of sweet cherries (Prunus avium L.) generates
a high amount of byproducts since the global production of this fruit
is about 2.2 million tons.19 Interestingly,
several researchers have demonstrated that sweet cherry pomace could
be a valuable source of bioactive compounds.20,21</p><p>Sweet cherry has been described as a source of phenolic compounds
with antioxidant capacity, which include hydroxycinnamates, anthocyanins,
catechins, and flavonols.22,23 In addition, proanthocyanidins
and flavonoid compounds found in sweet cherries have been shown to
reduce risk of Alzheimer's disease by reducing oxidant stress
and the production of β-amyloid, protecting neuronal cells.22 Regarding sweet cherry pomace, Dominguez-Rodriguez
et al. (2021) described extracts with phenolic compounds with antioxidant
and antihypertensive capacities.21 Also,
antioxidant extractable polyphenols were obtained from sweet cherry
pomace by pulsed electric fields.24 However,
the studies about the characterization and analysis of the bioactivity
of phenolic compounds from sweet cherry pomace are very limited.21</p><p>Usually, phenolic compounds are obtained
from foods by aqueous
and organic solvents.13 Nevertheless, the
analysis of phenolic compounds from different matrices omits other
phenolic compounds that are retained in the residue of the food matrix
after aqueous–organic extraction.26 This underestimated fraction corresponds to nonextractable polyphenols
(NEPs), which are low molecular weight polyphenols called hydrolyzable
polyphenols associated with macromolecules such as proteins or dietary
fiber or high molecular weight polyphenols, which are mostly nonextractable
proanthocyanidins.26 These compounds interact
with the food matrix by hydrogen and covalent bonds or hydrophobic
interactions or even extractable polyphenols could be associated with
NEPs.26</p><p>To recover NEPs from the
aqueous–organic extraction residue,
alkaline hydrolysis, acid hydrolysis, or enzymatic-assisted extraction
(EAE) methods are employed. Alkaline and acid hydrolyses are the most
used extraction methodologies to obtain NEPs.27 Nevertheless, in these extraction techniques, some phenolic compounds
are not stable to the high and low pH.27,28 For these
reasons, EAE has been reported as a sustainable treatment where the
residue of the extraction does not receive any excessive pH alteration,
being more selective and efficient to release NEPs from the food matrix
than acid and alkaline hydrolyses.28 Casein
protease, esterase, endogalacturonase, cellulase, pectinase, tannase,
and α-amylase enzymes have been employed to release NEPs from
the residue.29−31</p><p>Regarding the characterization of NEPs, preparative
high-performance
liquid chromatography in the reversed-phase mode (RP-HPLC), high-speed
counter-current chromatography (HSCCC), or normal phase HPLC (NP-HPLC)
coupled to UV/Vis detectors, electrospray ionization mass spectrometry
(ESI-MS), or matrix-assisted laser desorption ionization/time-of-flight
mass spectrometry (MALDI-TOF-MS) have been used.32−35 Nevertheless, NEPs from sweet
cherry pomace have not been characterized to date. High-performance
thin-layer chromatography (HPTLC) could be an interesting alternative
to separate NEPs from different samples in a unique analysis to be
subsequently characterized by direct analysis in real-time and acquiring
high-resolution mass spectra (DART-HRMS) with high accuracy and precision
isotopic abundance measurements with Orbitrap analyzer.36</p><p>The determination of the phenolic composition
of the extracts is
crucial to obtain a broad knowledge about what type of phenolic compounds
exert the in vitro and in vivo beneficial
properties. Even though the beneficial health properties exhibited
by NEPs from sweet cherry pomace have been reported, their toxicological
effect has not been evaluated. Thus, before extracts rich in NEPs
can be included in clinical studies or used as ingredients in any
product, their toxicity must be tested because some of their compounds
may be potentially toxic or carcinogenic.37</p><p>The traditional animal model for the in vivo study
of the bioactivity of phenolic compounds is a rodent, primarily rats
and mice. However, Caenorhabditis elegans (C. elegans) is an attractive animal model extensively used
for research involving aging and neurodegenerative diseases for which
research approval by Animal Care and Use Committees is not required.38 This nematode, length of 1 mm, has 65–80%
of genes associated with humans.39 Results
obtained with this nematode are consistent with those from other animals
such as rodents enabling subsequent preclinical and clinical assays
to be more focused.40 The antiaging capacity
of phenolic compounds of juice from sour cherries has been studied
through C. elegans, but to our knowledge, a study
about the in vivo bioactivity of NEPs obtained from
sweet cherry pomace has not been reported in the literature.41</p><p>Therefore, the main aim of this work was
to revalorize sweet cherry
pomace evaluating the efficiency of the extraction of NEPs by acid,
alkaline, and EAE methods (obtaining three extracts, one with high
bioactivity, another with high polyphenol content, and another with
high antioxidant polyphenol content) to be compared with extractable
polyphenol fraction estimating the contribution to the in
vitro and in vivo antioxidant, antihypertensive,
antiaging, and neuroprotective capacities. In vivo assays were carried out using C. elegans as an
experimental animal model. Additionally, the characterization of the
extracts was achieved by HPTLC–UV/Vis to classify the separated
phenolic compounds by families and by DART-Orbitrap-HRMS to obtain
a rapid and tentative phenolic fingerprint of the extracts.</p><!><p>Ethanol, acetone
(99.9%), formic acid (98–100%), and hydrochloric acid (37%)
of HPLC grade were supplied by Scharlab Chemie (Barcelona, Spain),
and methanol (99.99%), butanol, and sulfuric acid were supplied by
Fisher Scientific (Leicestershire, UK). Gallic acid, epicatechin,
vanillin, sodium carbonate, sodium hydroxide, sodium chloride, Folin–Ciocalteu
reagent, 4-dimethylaminocinnamaldehyde (DMAC), 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic
acid (Trolox), potassium persulfate, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic
acid) diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH•), iron(III) chloride, ethanolamine, 1,10-phenanthroline,
captopril, N-succinyl-Ala-Ala-Ala-p-nitroanilide, elastase from porcine pancreas, 5,5-dithiobis[2-nitrobenzoic
acid], acetylcholinesterase (AChE), acetylthiocholine iodide (ATCI),
galantamine, trifluoroacetic acid (TFA), angiotensin-converting enzyme
(ACE) from rabbit lung, hippuryl-histidyl-leucine (HHL), and 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic
acid (HEPES) were obtained from Sigma-Aldrich (Saint Louis, MO, USA).
Dipotassium hydrogen phosphate and sodium dihydrogen phosphate dihydrate
were supplied from Merck (Darmstadt, Germany). Ethyl acetate, toluene,
and sodium sulfate were provided by Penta (Chrudim, Czech Republic).</p><p>Ultrapure water (18.2 MÎ©/cm) was generated with a Millipore
system (Millipore, Billerica, MA, USA). Depol 740 L, Promod 439 L,
and Pectinase 62 L enzymes were kindly donated by the company Biocatalysts
Limited (Cardiff, UK).</p><p>Sweet cherries belonging to Prunus
avium L., Early
Lory variety, Rosaceae family were collected in 2019 from La Almunia
de Doña Godina (Zaragoza, Spain). To obtain the fruit pomace,
the fruits were washed, destemmed, destoned, and pressed manually.
Finally, pomace was ground in a commercial blender and stored at −20
°C until analysis.</p><!><p>Extractable polyphenols were obtained
based on the method in a
previous study performed by Dominguez-Rodriiguez et al. (2021).21 Briefly, 20 mL of methanol/water (50:50 v/v)
acidified with 2 M HCl (pH 2) was added to 15 g of cherry pomace and
incubated for 1 h at room temperature with shaking. After that, the
extract was centrifuged for 10 min at 2100g to obtain
the supernatant. The extraction residue was mixed with 20 mL of acetone/water
(70:30, v/v), shaken for 1 h at room temperature, and centrifuged
at 2100g for 10 min. Finally, both supernatants were
combined and stored at −20 °C until analysis, and the
extraction residue was stored to be used for the extraction of NEPs.
Samples were prepared in triplicate.</p><!><p>EAE
was carried out according to the optimal extraction conditions obtained
in the experimental designs performed by our research group to extract
high content of bioactive NEPs (HBN extract) from sweet cherry pomace
employing three different enzymes (Depol 740L (Depol) with β-glucanase
activity, Promod 439L (Promod) with protease and polygalacturonase
activities, and Pectinase 62L (Pectinase) with pectin lyase activity).21 In this previous work, a Box–Behnken
experimental design was used for each enzyme to determine the influence
of enzyme concentration, pH, extraction time, and temperature in the
NEP extraction from the extraction residue of sweet cherry pomace.
Extractions were achieved using phosphate buffer (100 mM) as extraction
solvent, 0.38 g of sample/mL, and enzyme concentrations of 90 Î¼L
of Depol enzyme, 140 Î¼L of Promod enzyme, and 2 Î¼L of
Pectinase per gram of sample according to the method of Dominguez-Rodriguez
et al. (2021).21</p><p>The design consisted
of 29 randomized runs for each enzyme with three levels and five central
points. The response variables were total phenolic content (Folin–Ciocalteu
method), total proanthocyanidin content (DMAC, vanillin, and butanol/HCl
assays), antioxidant capacity (DPPH, Trolox equivalent antioxidant
capacity (TEAC), and the capacity to inhibit the hydroxyl radical
assays), and antihypertensive capacity (ACE inhibition assay). The
evaluation of the adequacy of fitted models settled between parameters
to optimize and the different responses was carried out by analysis
of variance (ANOVA).</p><p>Using this experimental design, the theoretical
optimal extraction
conditions to obtain extracts with high bioactivity (HB extract) and
high content of phenolic compounds and PAs (TPA extract) from sweet
cherry pomace were also calculated by using graphical and numerical
methods based on the criteria of the desirability function and the
response surface plots. Table S1 shows
the theoretical optimal extraction conditions to obtain HBN, HB, and
TPA extracts of each enzyme obtained from the experimental designs
performed in a previous study.21 The EAEs
with Depol, Promod, and Pectinase enzymes were performed in triplicate
under the theoretical optimal extraction conditions obtained from
the experimental design to corroborate the study.</p><!><p>Acid
hydrolysis as described by Hartzfeld et al. (2002) was employed to
extract NEPs from the residue of cherry pomace with some modifications.42 Briefly, 0.38 g of extraction residue was mixed
with 1 mL of methanol/H2SO4 (90:10, vol %) by
shaking for 20 h at 85 °C in a thermoreactor (Spectroquant TR420,
Merck, Germany). Then, the extracts were submitted to centrifugation
at 3000g for 10 min, and the supernatants were collected.
Subsequently, extracts were washed twice with distilled water, and
the final volume was adjusted to 2 mL. Finally, 200 Î¼L of ethanolamine
was added with agitation, and pH was adjusted to 5.5.</p><p>On the
other hand, alkaline extraction was carried out as previously reported
by Arranz and Saura-Calixto (2010) for the extraction of NEPs.43 Extraction residue (9.38 g) was mixed with 25
mL of 2 M NaOH for 4 h at room temperature. In order to neutralize
the mixture, an appropriate amount of hydrochloric acid was added
(pH 3.0). Acid and alkaline hydrolyses were conducted in triplicate.</p><!><p>Total phenolic content was determined following the Folin–Ciocalteu
(FC) method based on the work by Kosar et al. (2005), and proanthocyanidin
content was determined according to DMAC, vanillin, and butanol/HCl
assays used by Montero et al. (2013), Gu et al. (2008), and Pérez-Jiménez
et al. (2009), respectively, employing a Cary 8454 UV–vis spectrophotometer
(Agilent Technologies, Palo Alto, CA, USA).29,44−46 The results were expressed as milligrams of epicatechin
per 100 g of sample.</p><!><p>The extracts
were preconcentrated with ethyl acetate to obtain greater band intensity
on the TLC plate and greater signal intensity in the DART-Orbitrap-HRMS
analysis according to the method of Dominguez-Rodriguez et al. (2021).47 The liquid was evaporated, and the residue was
reconstituted in 200 Î¼L of methanol to be injected into the
HPTLC system and for the analysis by DART-Orbitrap-HRMS.</p><p>Six
samples were applied in a volume of 10 Î¼L using a semiautomatic
applicator (CAMAG LINOMAT 5, Muttenz, Switzerland) with an HPTLC syringe
of 100 Î¼L (Hamilton, Bonaduz, Switzerland) employing 6 mm of
band length with a distance between tracks of 15.4 mm on normal phase
(NP) HPTLC plates (HPTLC Silica Gel 60 F254 Plates 20 cm × 10
cm). A CAMAG (Muttenz, Switzerland) instrument was used to separate
extractable polyphenols and NEPs from sweet cherry pomace extracts.</p><p>Chromatography separation was performed following the method described
by Dominguez-Rodriguez et al. (2021) where ethyl acetate–toluene–formic
acid–methanol (6:6:1.6:0.4, v/v/v/v) was employed as the mobile
phase.47 Development took 40 min, and the
plate was removed from the chamber and dried in a TLC heater at 60
°C for 15 min.</p><p>Spectral analysis was performed in a TLC
Scanner (CAMAG) from 200
to 800 nm obtaining the retention factors (Rf), peak areas in absorbance units (AU), and wavelengths at absorption
maximum in nanometers of separated substances. Then, the developed
plate was sprayed in a derivatizer (Camag, Muttenz, Switzerland) using
2 mL of 10% H2SO4 in methanol and dried using
the TLC heater at 60 °C. Before and after derivatization, digital
pictures were taken under 254 and 366 nm UV light and white light
above the plate using a TLC visualizer (CAMAG, Muttenz, Switzerland)
equipped with a 12 bi-bit charge-coupled device (CCD) digital camera.</p><!><p>Extractable
polyphenols (EPPs) obtained by conventional extraction and NEPs recovered
by alkaline hydrolysis and EAE with Promod, Depol, and Pectinase enzymes
from sweet cherry pomace were tentatively identified by DART-Orbitrap-MS.
DART ionization was performed in a DART-Standardized Voltage and Pressure
Adjustable (SVPA) device using the method described by Falk et al.
(2018).48 The DART ion source worked in
negative and positive ionization modes with helium ionizing gas at
0.55 MPa pressure, 350 °C beam temperature, and 350 V grid electrode
voltage. High-resolution mass spectral (HRMS) measurements were performed
on an Orbitrap mass spectrometer (Thermo Fischer Scientific, Bremen,
Germany) coupled to an ion source through an interface evacuated with
a diaphragm pump. The linear ion trap mass spectrometer settings were
as follows: capillary voltage 50 V; tube lens voltage 100 V; skimmer
voltage 18 V; capillary temperature 300 °C.</p><p>To perform
data acquisition and processing, the Xcalibur software (Thermo Fischer
Scientific, Germany) with DART web-based module was employed. The
acquisition rate was set to 2 spectra per second providing resolution
of 120 000 full width at half-maximum (fwhm) at m/z 200.</p><p>Liquid extracts were pipetted (10
Î¼L) onto DART-QuickStrip
plates for the analysis while residues of the extractions (solid sample)
were analyzed employing tweezers.</p><!><p>The DPPH radical scavenging capacity
was determined using the method
described by Brand-Williams et al. (1995).49 The concentration to decrease the initial DPPH concentration by
50% (EC50) was calculated by plotting the percentage of
remaining DPPH on a graph against the sample concentration using a
calibration curve of DPPH. Thereby, a greater EC50 implies
less antioxidant capacity in extracts.</p><p>Also, the TEAC assay
was applied following the method of Re et al. (1999).50 Trolox was used as the reference standard to express the
results as TEAC (Trolox equivalent antioxidant capacity) values (mmol
Trolox/g extract) employing a standard curve. The TEAC values were
obtained from four different concentrations of each extract giving
a linear response between 20% and 80% compared with the initial absorbance.
Analyses were done in triplicate for each extract.</p><p>On the other
hand, a hydroxyl radical assay based on the protocol
of Dominguez-Rodriguez et al. (2021) was employed to determine the
capacity to inhibit the formation of hydroxyl radicals.21 The results were expressed as % inhibition of
hydroxyl radical formation.</p><!><p>Angiotensin-converting
enzyme (ACE) inhibition was used to determine antihypertensive capacity
from cherry pomace following the method of Geng et al. (2010) with
some modifications.21,51 Results were expressed as a percentage
of ACE inhibition using the following equation:where Acontrol is the area under the peak of HA (hippuric acid) in the control
and Asample is the area under the peak
of HA in the sample.</p><p>Moreover, the concentration required for
the 50% inhibition of ACE activity (IC50) was calculated
for the extracts obtained under the optimal conditions by EAE and
the extracts performed by conventional extraction and acid and alkaline
hydrolysis.</p><!><p>Elastase
inhibition activity assay based on Azmi et al. (2014) with some modifications
was employed to determine the antiaging capacity of the extracts.52 Briefly, 100 Î¼L of 0.2 mM Tris-HCl buffer
(pH 8.0), 25 Î¼L of 10 mM N-succinyl-Ala-Ala-Ala-p-nitroanilide dissolved in the Tris-HCl buffer, and 50
Î¼L of extract were mixed. After incubation for 15 min at 25
°C, absorbance was measured at 410 nm. Then, 25 μL of 0.3
units/mL elastase was added and incubated for another 15 min at 25
°C and the absorbance was read at 410 nm in a Cary 8454 UV–Vis
spectrophotometer (Agilent Technologies, Palo Alto, CA, USA). Epicatechin
(0.7 mg/mL) was used as a positive control. The results were expressed
as % of elastase inhibition activity employing the following equation:where C is the absorbance
of the extract after incubation with the enzyme, D is the absorbance of the extract after incubation without enzyme, A is the absorbance of the control after incubation with
enzyme and B is the absorbance of the control after
incubation without enzyme.</p><!><p>Acetylcholinesterase (AChE) inhibition activity was
measured using Ellman's method as described by Mathew and Subramanian
(2014) with some modifications.53 In brief,
100 Î¼L of 3 mM of DTNB (5,5-dithiobis[2-nitrobenzoic acid])
dissolved in 50 mM Tris-HCl buffer (pH 8.0) containing 0.1 M NaCl
and 0.02 M MgCl2, 20 Î¼L of 0.26 U/mL AChE dissolved
in 0.1% BSA (bovine serum albumin) in buffer, 640 Î¼L of buffer,
and 20 Î¼L of the extract were mixed. After incubation for 15
min at 25 °C, absorbance was measured at 412 nm in a Cary 8454
UV–Vis spectrophotometer (Agilent Technologies), which was
treated as the control. Then, the enzymatic reaction was started by
the addition of 15 mM ATCI (acetylthiocholine iodide) dissolved in
water, and the absorbance was read at 412 nm until the reaction completed
(45 min). Galantamine (100 Î¼M) was used as the positive control.
The results were expressed as % inhibition of AChE employing the following
equation:where Abscontrol is the absorbance
containing all reagents except ATCI and Abssample is the
absorbance of the solution prepared after completing the enzymatic
reaction with ATCI.</p><!><p>C. elegans strain N2, var.
Bristol (wild-type), and the transgenic strain CL4176 (smg-1ts [pAF29(myo-3/Aβ1–42/let
UTR)+pRF4(rol-6(su10069))]) were obtained from the Caenorhabditis Genetics Center at the University of Minnesota. N2 worms were maintained
at 20 °C, while strain CL4176 was maintained at 16 °C, both
on Nematode Growth Medium (NGM) plates (agar 17.5 g/L, sodium chloride
3.0 g/L, peptone 2.5 g/L, and cholesterol 0.005 g/L) with Escherichia coli strain OP50 as the normal diet for nematodes
for all experimental assays.</p><!><p>The wild-type strain N2
of C. elegans (var. Bristol) was used as an in vivo model to evaluate the antioxidant capacity of the
extracts. The experiment was performed as described by Martorell et
al. (2011).54 To obtain age-synchronized
nematodes, eggs were isolated from gravid adults and hatched overnight
in NGM plates. NGM plates were supplemented with different sweet cherry
extracts at two different concentrations (extracts were diluted in
5% DMSO at 100 and 400 Î¼g/mL) using NGM medium as control and
vitamin C (10 Î¼g/mL) as positive control. Worms (50 worms/fed
condition) were incubated at 20 °C under these conditions. Once
the adult phase was achieved (5 days), nematodes were transferred
to a basal medium containing 2 mM H2O2 to induce
oxidative stress. After 5 h of incubation, the total number of worms
that survived the treatment was counted. A test was conducted as a
screening due to the high reproducibility of the assay (one assay
was performed for each extract).</p><!><p>Aging is characterized by
a loss of body movement. Like humans, C. elegans lose
movement with aging, and they can move only their heads. For this
reason, the mobility of nematodes was evaluated as an aging-related
parameter. An automated system based on artificial vision was used
to score the activity of worms under the different treatments during
the first 4 days of adulthood.</p><p>Age-synchronized nematodes of
wild-type strain N2 were used for the antiaging assay using 96-well
plates with solid medium (NGM). To find the optimum extract dose,
nematodes were cultured using three different amounts of extracts
(10, 20, and 30 Î¼L for all the extracts, except for the acid
extract, tested at amounts of 2.5, 5.0, and 10.0 Î¼L to avoid
a detrimental effect on C. elegans due to the pH).
A control condition without extract was included.</p><p>Mobility was
tracked during 4 days at 20 °C, and the fold
change of worm mobility (activity treatment/activity control) was
estimated each day to normalize data.</p><!><p>The evaluation
of the neuroprotective capacity of the extracts on C. elegans was carried out with the transgenic C. elegans strain
CL4176, which can produce the neurotoxic peptide amyloid β-peptide
in either neurons or body wall muscle.55 The paralysis produced by the expression of the human amyloid β-peptide
in the C. elegans strain was measured. Age-synchronized
worms were cultured in NGM as control and NGM supplemented with each
sample at three different volumes in the plate (100, 200, and 300
Î¼L excepting acid extract, which was added in a concentration
of 10, 25, and 50 Î¼L to avoid detrimental effects in the development
of nematodes) at 16 °C until L3 stage (larval stage 3 that corresponds
to 9 h after fertilization). At this time, transgene expression was
induced in nematodes by up-shifting the temperature from 16 to 25 °C.
Worms were maintained at 25 °C until 100% of worms became paralyzed.
Paralysis in induced worms was compared with noninduced worms (maintained
at 16 °C until the end of the paralysis assay).</p><p>Ginkgo biloba EGb761 (100 μg/mL) was used as positive
control. Assays were performed in duplicate.</p><!><p>All the
cells used in this study were obtained from the American
Type Culture Collection ATCC (Rockwell, MD, USA) and cultured in an
incubator at 37 °C with 5% CO2 saturation and 95%
humidity in their culture medium.</p><p>Hepatocarcinoma HepG2, primary
dermal fibroblast HFF-1, and human ovarian cancer SKOV3 cell lines
were maintained in Dulbecco's modified Eagle's medium
(DMEM); colon adenocarcinoma HT-29 cells were maintained in MacCoy's
5A medium. All cell lines were supplemented with 10% fetal bovine
serum and antibiotics.</p><p>The cell lines mentioned above were used
to determine the in vitro cytotoxic effect of conventional,
alkaline, acid,
and EAE extracts at different concentrations (0.380, 0.285, 0.095,
0.038, 0.019, and 0.0095 mg/mL extract) on the cell viability by the
MTT [3-(4,5-dimethylthiazole-2-yl)2,5-diphenyltetrazolium bromide]
assay as described by Hernández-Corroto et al. (2018) with
some modifications.56 Briefly, cells were
seeded at a density of 5000 cells/well in a 96-well plate and incubated
with 10 Î¼L of extract for 24 h. Afterward, 10 Î¼L of MTT
stock solution at 5 mg/mL in phosphate buffer was added to each well
and incubated for 6 h. Then, the culture medium was removed and formazan
crystals were dissolved with 100 Î¼L of DMSO. Finally, absorbance
was measured at 570 nm. Results were expressed as a percentage of
cell viability after 24 h concerning the control according to the
following formula:where Abssample and Abscontrol are the absorbances of remaining
formazan when cells were treated
with the extracts and extraction solvent, respectively.</p><!><p>The program
Statgraphics Centurion XVII (Statistical Graphics Corp., USA) was
used for statistical analysis. Analysis of variance (ANOVA) by Fisher's
exact test to discriminate on the least significant difference LSD
(p ≤ 0.05) was used to compare differences
in total phenolic content and total proanthocyanidin content of cherry
pomace extracts as well as in antioxidant, antihypertensive, antiaging,
and neuroprotective capacities for in vitro assays.
To compare the effect on paralysis protection of each sample versus
control-fed nematodes, one-way ANOVA and Tukey's multiple comparison
tests were applied. All statistical analyses for in vivo assays were performed in GraphPad Prism 9 statistical software package.</p><!><p>In this work, EAE
and alkaline and acid hydrolyses were employed
to release NEPs from conventional extraction residue of cherry pomace
to determine the total phenolic and proanthocyanidin contents and
evaluate the in vitro and in vivo antioxidant, antihypertensive, antiaging, and neuroprotective capacities
of these extracts.</p><p>The optimal extraction conditions to obtain
HBN, TPA, and HB extracts
by EAE (see Table S1) from sweet cherry
pomace were obtained from a Box–Behnken experimental design
previously elaborated by our research group to determine the influence
of enzyme concentration, time, temperature, and pH on the total phenolic
and proanthocyanidin contents, antioxidant capacity, and antihypertensive
capacity.21 HBN theoretical values obtained
from the experimental design were experimentally verified by Dominguez-Rodriguez
et al. (2021).21 In addition, theoretical
optimal values obtained for total phenolic content (TPC), PA contents,
and antioxidant and antihypertensive capacities from the optimal extraction
conditions to obtain TPA and HB extracts from the residue of conventional
extraction of cherry pomace were submitted for validation through
an experimental analysis in this study.</p><p>Table S2 shows the theoretical optimal
values from FC, DMAC, vanillin, and butanol/HCl assays that should
be obtained under the optimal extraction conditions for Depol, Promod,
and Pectinase enzymes to obtain TPA extracts along with a range of
values within which the experimental values must be included. Experimental
values were lower than the theoretical ones except for Promod enzyme,
for which experimental value was within the range of the predictive
model in DMAC assay (see Table S2). The
experimental analysis was carried out with sweet cherries harvested
in 2019, while theoretical values were obtained from sweet cherries
harvested in 2018. The low experimental results obtained in this analysis
compared with theoretical results may be because the cherries were
harvested in different years with different weather conditions that
may have varied the content of phenolic and proanthocyanidin compounds
as well as their biological activity.</p><p>On the other hand, experimental
values of DPPH and TEAC assays
for HB extracts were within the range of the predictive model for
each enzyme (see Table S3). However, experimental
antioxidant values from hydroxyl radical assay were lower for Promod
and Depol enzymes and higher for Pectinase enzyme than the theoretical
ones.</p><p>In general, the predictive model from experimental design
allows
obtaining a good prediction for the antioxidant capacity using DPPH
and TEAC assays to obtain HB extracts. However, this predictive model
did not allow a good prediction of the TPC and PA values for TPA extracts.</p><!><p>Table 1 shows the TPC values of the extracts obtained by conventional extraction,
alkaline and acid hydrolyses, and EAE from sweet cherry pomace. As
it can be seen, results were statistically different (p ≤ 0.05) among extraction techniques.</p><!><p>Letters
(a, b, c, d, e, f,
g, h, i, j, k, l) show the significant differences among extraction
methods of NEPs (p ≤ 0.05).</p><!><p>The richest extract in terms of
TPC was achieved by acid hydrolysis
followed by EAE with Depol enzyme from HBN and TPA extracts. The high
phenolic content in acid hydrolysis could be due to the low pH employed
in the treatment that allowed release of NEPs and other compounds
trapped in the cores or conjugated to cell walls with macromolecules.57 Additionally, other reducing compounds different
from phenolic compounds could be released from the cell wall of the
extraction residue interfering in the measurement by the FC method
and thus overestimating the TPC. By contrast, conventional extract
presented the lowest TPC content observing that a high amount of phenolic
compounds were retained in the extraction residue. Regarding EAE,
HBN extracts showed higher TPC values than TPA extracts using the
three enzymes. This result suggests that HBN extract presented other
reducing agents with antioxidant capacity different from phenolic
compounds compared with TPA extract because it was optimized to obtain
high phenolic and proanthocyanidin contents and high antioxidant capacity.</p><p>On the other hand, Table 1 shows statistical differences (p ≤
0.05) among the extraction methods employed to obtain NEPs using three
different assays to measure PAs. Depol HB extract showed the highest
PA content in DMAC assay. By contrast, Depol TPA extract showed the
lowest PA content.</p><p>Regarding vanillin assay, acid hydrolysis
was the most effective
to extract PAs, while conventional extraction showed the lowest PA
content. Acid hydrolysis also showed the highest PA content in the
butanol/HCl assay. Alkaline hydrolysis showed the lowest PA content
and did not show statistical differences with Pectinase HB, Promod
HB, Pectinase TPA, Depol HB, and Depol HBN extracts (Table 1).</p><p>In the DMAC assay,
the reagent reacts specifically with compounds
with meta-oriented di- or trihydroxy phenols, as are found in PAs,
while in the vanillin assay, the aldehyde group reacts with PAs but
also with other flavonoids.58 To determine
PA content with the vanillin assay, the absorbance is measured at
510 nm, absorbing in the same region as anthocyanins and overestimating
the PA content. For this reason, the DMAC assay is preferable to measure
PA content to the vanillin assay because it is more specific.25,58 These methods are not comparable to measure polymeric polyphenols
because they are more specific for monomeric compounds being used
as reference standards, while the butanol/HCl assay is more specific
for polymeric compounds.25,58 In this sense, Table 1 shows that acid and
EAE with Depol enzyme were the most effective treatments to obtain
monomeric NEPs from conventional extraction residue, while acid treatment
was the most efficient to obtain polymeric compounds.</p><!><p>The extracts of
EPPs obtained by conventional extraction as well as the NEP extracts
recovered by acid and alkaline hydrolyses and EAE with Depol, Promod,
and Pectinase enzymes were analyzed by HPTLC to determine their phenolic
profiles. In addition, the extracts obtained by EAE under the optimal
extraction conditions to obtain HBN extracts were chosen as the most
representative extracts to characterize their NEP profiles because
under these extraction conditions it is possible to release higher
content of bioactive phenolic compounds and PAs.</p><p>Figure 1 shows the TLC plate visualization
at 254 nm after the postchromatographic reaction through the addition
of 10% of sulfuric acid in methanol to provide fluorescence for band
visualization for the subsequent isolation and identification of compounds.
The spot colors of the separated bands on the TLC plate detected by
UV–vis light allowed us to group EPPs and NEPs by families
or classes. For instance, orange-yellow, brown-green, and purple-red
spot coloration under UV light correspond to flavonoids, flavones,
and anthocyanins, respectively.35,58 In addition, phenolic
acids, such as p-coumaric acid, chlorogenic acid,
ferulic acid, or caffeic acid, have been identified in several investigations
as blue spots.35,59</p><!><p>TLC visualization at 254 nm of separated
bands of conventional
extract and nonextractable polyphenol hydrolysates from sweet cherry
pomace after derivatization (lane 1, conventional extraction; lane
2, alkaline hydrolysis; lane 3, acid hydrolysis; lane 4, EAE with
Depol enzyme; lane 5, EAE with Promod enzyme; lane 6, EAE with Pectinase
enzyme) by HPTLC using ethyl acetate–toluene–formic
acid–methanol (6:6:1.6:0.4, v/v/v/v) as mobile phase.</p><!><p>According to reference colors, the qualitative
identification of
EPPs and NEPs from the different extracts of cherry pomace by phenolic
families was carried out. HPTLC analysis showed that different classes
of phenolic compounds were observed in the extracts collected by conventional
extraction and each hydrolysis treatment detecting 29 compounds in
total. Furthermore, as can be observed in Table S4 and Figure 1, the extracts collected by conventional extraction and alkaline
and acid hydrolyses showed a higher number of spots (5, 8, and 6 compounds,
respectively). This means that a wide range of phenolic compounds
were retained in the residue of conventional extraction. Most of the
separate compounds corresponded to phenolic acids and flavonoids or
flavones by the blue and yellow-brown color of the bands (Figure 1). In this study,
conventional extraction and alkaline hydrolysis allowed us to obtain
a higher number of phenolic acids than the rest of the treatments,
exhibiting a higher number of blue bands in the HPTLC separation process
(see Figure 1). For
instance, in the extracts recovered by conventional extraction, a
phenolic acid with a blue spot (Rf value
of 0.17) with the highest intensity (283 AU) was observed (Figure S1). This phenolic acid could correspond
to neochlorogenic or chlorogenic acid because these compounds have
been found at high amounts in sweet cherry pomace.60,61 This compound, as well as the compound detected with a Rf value of 0.70, was also detected in alkaline extract
with higher intensity than conventional extract. In addition, TLC
visualization showed that EAE extracts with the three enzymes employed
presented the same spot with a Rf value
between 0.60 and 0.70 with intense blue color that was not present
in the rest of the extracts. Promod HBN extract showed the lowest
number of spots detected (2 compounds).</p><p>Concerning yellow spots,
the alkaline extract showed different
flavones in the separation by HPTLC with brown spots with Rf values of 0.28, 0.49, and 0.60. In addition,
the alkaline extract exhibited a yellow spot with the highest intensity
(368 AU) with a Rf value of 0.24, which
could be epicatechin as this compound has been found as the flavonoid
with the highest concentration in sweet cherry pomace.60 Acid hydrolysis allowed us to obtain a wide
range of flavonoids because several brown and yellow color bands were
exhibited in the HPTLC separation. On the other hand, the acid extract
did not show phenolic acids. This may be due to the extreme pH values
used in the extraction process and the fact that phenolic acids are
unstable at these low pH values.</p><p>EAE extracts showed a lower
number of bands than conventional,
alkaline, and acid extracts. However, HPTLC visualization allowed
determination of bands of different colors in EAE extracts. In fact,
HPTLC combined with DART-HRMS presented a fast separation and tentative
identification by families and specific EPPs and NEPs from cherry
pomace.</p><!><p>Table 2 summarizes the identification
by DART-HRMS of EPPs obtained by conventional extraction from sweet
cherry pomace as well as the identification of NEPs attained from
the residue of conventional extraction by alkaline hydrolysis and
EAE with Promod, Depol, and Pectinase enzymes under the optimal extraction
conditions to produce HBN extracts. The acid extract could not be
included in the analysis because ionization problems were observed
probably due to interference from the acid solvent.</p><p>As can be seen in Table 2, a total of 39 phenolic compounds were tentatively identified
by DART-Orbitrap-HRMS in sweet cherry pomace extracts. The highest
number of NEPs detected corresponded to the alkaline extract where
a total of 27 NEPs were identified. Four phenolic compounds were found
in common in all extracts: kaempferol/luteolin (number 18), (epi)catechin
(number 21), quercetin (number 25), and taxifolin (number 26). In
particular, (epi)catechin with a molecular ion at m/z 289.0701 [M – H]− presented
the highest intensity in the extracts performed by EAE with Depol
enzyme (Figure 2A).
Several researchers observed that catechin and epicatechin are present
in sweet cherries at high concentrations. Generally, epicatechin is
more concentrated in sweet cherries than catechin.60,63,64 These compounds were detected in sweet cherry
pulp as well as in its byproducts such as stems.65 Moreover, (epi)gallocatechin (number 27) with a molecular
ion at m/z 305.0652 [M –
H]− was the most intense phenolic compound identified
in the conventional extract. Nevertheless, this compound was observed
with higher intensity in the alkaline extract than in the conventional
extract. (Epi)gallocatechin has also been identified in sweet cherry
pulp and stems.66,67</p><!><p>Mass spectrum ([M – H]−) of NEPs from
sweet cherry pomace of (A) (epi)catechin from EAE with Depol enzyme
extract, (B) gallic acid from alkaline extract, (C) vestitol from
EAE with Depol enzyme extract, and (D) procyanidin B2 from EAE with
Pectinase enzyme extract.</p><!><p>A total of 20 phenolic acids were identified in DART-Orbitrap-HRMS
analysis of sweet cherry pomace extracts, being the majority class
of phenolic compounds determined in the analysis. Among them, gallic
acid (number 4) with a molecular ion at m/z 169.0140 [M – H]− was tentatively
identified as the most intense phenolic acid observed in the extracts
(Figure 2B). In particular,
this compound was observed in alkaline and EAE with Promod enzyme
extracts at a high intensity (see Table 2). Additionally, gallic acid was detected
with the highest intensity in the extracts collected by EAE with the
Pectinase enzyme. The presence of gallic acid has been described in
sweet cherry pulp as well as in stems, although it depends on the
variety of sweet cherries studied.60,66,68</p><p>Dihydroxybenzoic acid (number 1) with a molecular
ion at m/z 153.0194 [M –
H]− was tentatively identified in all extracts.
Commonly, different
hydroxybenzoic acids, such as hydroxybenzoic acid derivative, protocatechuic
acid aglycone, 2,5-dihydroxybenzoic acid, and p-hydroxybenzoic
acid, were detected in sweet cherry pulp and stems.67,69,70</p><p>On the other hand, an isoflavone with
a molecular ion at m/z 271.0954
[M – H]− was tentatively identified as vestitol
(number 17) in EAE extracts
with Depol and Pectinase enzymes (see Table 2, Figure 2C). Isoflavones are commonly found in legumes. However,
these compounds have also been found in different fruit peels such
as Mangifera pajang Korterman peels or different
varieties of passion fruits such as Passiflora edulis, Passiflora ligularis, and Passiflora mollissima peels.71,72</p><p>Concerning procyanidins, the precursor
of procyanidin B2 (number
22, Figure 2D) with
a molecular ion at m/z 289.0701
[M – H]− with charge 2 in all extracts, excepting
EAE with Depol enzyme extract, was tentatively identified as the NEP
with the highest molecular weight. This compound has been detected
in the pulps of different varieties of sweet cherries.60,64</p><p>To our knowledge, this is the first time that NEPs from sweet
cherry
pomace were separated and identified by families by HPTLC-UV/vis and
directly identified by DART-Orbitrap-HRMS. In addition, to check the
extraction efficiency of the different treatments employed in this
work to release EPPs and NEPs from sweet cherry pomace, the residues
from conventional extraction and hydrolysis treatments were analyzed
by DART-Orbitrap-HRMS. As can be seen in Table S5, hydrolysis treatments were efficient in the release of
NEPs from the residue of conventional extraction since the signal
intensities of NEPs identified by DART-HRMS in the residues of hydrolysis
treatments were lower than in the extracts.</p><!><p>Table 3 shows the concentration
of the extracts necessary for 50% inhibition of ACE activity (expressed
as IC50) the extracts with the highest antihypertensive
capacity being the ones with the lowest IC50 values. In
general, EAE extracts showed higher antihypertensive capacity than
those from conventional extraction and acid and alkaline hydrolysis.
Depol HBN extract presented the highest antihypertensive capacity
but did not show statistical differences with Pectinase TPA extract.
By contrast, Promod HBN extract showed the lowest antihypertensive
capacity compared with the rest of EAE extracts with the three enzymes
studied coinciding with conventional extraction and alkaline hydrolysis.</p><!><p>Letters (a, b, c, d, e, f,
g, h, i, j, k) show the significant differences among extraction methods
of NEPs (p ≤ 0.05).</p><!><p>The results
obtained from DPPH, TEAC, and hydroxyl radical assays are summarized
in Table 3, showing
statistical differences (p ≤ 0.05) among extraction
methods in all assays. HB extracts showed the highest antioxidant
values compared with HBN and TPA extracts with each enzyme employed
in DPPH and TEAC assays. EAE extracts showed higher antioxidant capacity
than alkaline and acid hydrolysis highlighting Promod HB and Depol
HB extracts in DPPH assay. Also, the highest antioxidant capacity
was obtained from Promod HB extract in the TEAC assay. However, DPPH
and TEAC are spectrophotometric assays where the radicals employed
are not generated in our bodies. For this reason, the results are
limited because they do not reproduce the physiological situation.
In this sense, hydroxyl radical is a potent reactive oxygen species
in the biological system. The estimation of the inhibition of hydroxyl
radical could provide an approximation of the antioxidant effect of
the extracts in our body. Results in Table 3 show that the inhibition of hydroxyl radical
depends on the hydrolysis treatment and extraction conditions employed
to obtain NEPs (p ≤ 0.05). This method demonstrated
high antioxidant capacity in cherry pomace extracts obtained by EAE.
Pectinase HBN extract showed the highest hydroxyl radical inhibition.
By contrast, hydrolysis acid extract showed the lowest hydroxyl radical
inhibition.</p><p>To verify the in vivo biological
activity of NEPs, a C. elegans model was used for
the first time to evaluate the antioxidant capacity of the extracts
on this nematode. The antioxidant power was evaluated by inducing
oxidative stress in C. elegans with H2O2 due to the effect of this pro-oxidant on the lifespan
and mortality of the worm. This way, the antioxidant effect of EPPs
and NEPs obtained by conventional, acid, alkaline, and EAE was proportional
to the survival rate of the worms.</p><p>Figure 3A,B shows
the survival percentage of the nematode population under oxidative
stress conditions when 100 Î¼g/mL and 400 Î¼g/mL, respectively
of different sweet cherry pomace extracts were added. As it can be
seen in Figure 3A,
Depol TPA extract was the most antioxidant extract as the survival
rate of C. elegans increased by 14% compared to control
(NGM), followed by Depol HB extract (12% survival rate). By contrast,
acid hydrolysis and EAE with Promod enzyme to obtain HB extracts showed
an increase in the survival of C. elegans by 6%,
as did the extracts obtained by conventional extraction and Promod
TPA. No protective activity against stress could be determined with
the rest of the extracts. On the other hand, when a higher extract
concentration (400 Î¼g/mL) was used, Depol HBN extract presented
the highest antioxidant capacity, increasing the survival rate of C. elegans by 20% compared to the control (NGM) (Figure 3B). This increase
in the survival rate of C. elegans was comparable
to the one observed with the positive control (vitamin C, 10 Î¼g/mL).
Moreover, acid hydrolysis and Depol TPA extracts displayed high antioxidant
activity. The extracts collected by EAE with Depol and by acid hydrolysis
showed higher antioxidant activity at both concentrations, 100 and
400 μg/mL, increasing the survival rate of C. elegans by around 6–20% compared with the NGM control.</p><!><p>Effects of
cherry pomace nonextractable polyphenol hydrolysates
on the survival rate of C. elegans in response to
H2O2-induced oxidative stress expressed as the
% survival rate of C. elegans in normal medium (NGM),
medium containing 10 Î¼g/mL vitamin C, or different nonextractable
polyphenol hydrolysates from cherry pomace at (a) 100 Î¼g/mL
extract and (b) 400 Î¼g/mL extract.</p><!><p>The high antioxidant capacity of Depol HBN and TPA extracts evaluated
in C. elegans showed a correlation with the TPC measured
by FC assay, Depol HBN and TPA extracts showing the highest TPC content.
These results suggest that during EAE with Depol enzyme, phenolic
compounds different from proanthocyanidins, which are not detected
in PA assays, are released from the extraction residue and showed
an important in vivo antioxidant capacity.</p><!><p>In order to measure
the antiaging capacity of the extracts from cherry pomace, two methods
were employed. The elastase inhibition activity method was used to
evaluate the in vitro antiaging capacity of the extracts
to be compared with the results obtained by the in vivo health span method.</p><p>The elastase activity exhibited by EPPs
and NEPs is shown in Table 4. The acid extract showed the highest inhibition of elastase
activity, but the value was lower than that with a concentration of
0.7 mg/mL of epicatechin. Regarding EAE, Promod enzyme resulted in
the HBN extract with the highest inhibition of elastase activity,
higher than that from conventional extraction.</p><!><p>Letters
(a, b, c, d, e, f,
g, h, i, j) show the significant differences among extraction methods
of NEPs (p ≤ 0.05).</p><!><p>On the other hand, the in vivo evaluation
of the
antiaging capacity of NEPs was evaluated for the first time by the
mobility of C. elegans under the different extracts
as an aging-related parameter. The activity of nematodes treated with
different extracts was measured daily and compared with nematodes
in control feed conditions (NGM) during the first 4 days of adulthood
(Table S6). All extracts from sweet cherry
pomace showed a positive effect on mobility using 30 Î¼L/mL of
extract except acid and Pectinase extracts, which exhibited a higher
effect employing 10 Î¼L/mL of extract (see Table S6). Therefore, the extracts were evaluated at the optimal
concentration observed in the screening test. As can be observed in Figure 4, except for Depol
TPA extract, all extracts provided a fold change value >1, indicating
a positive effect on mobility. Moreover, among extracts tested, Promod
HBN showed the lowest effect, with fold change values near to 1 and
below the activity of nematodes treated with the conventional extract.
Interestingly, nematodes treated with Pectinase HB, Pectinase TPA,
alkaline, and Pectinase HBN extracts caused an increase in worm's
mobility (fold change values 1.8–2.6).</p><!><p>Effect of nonextractable
polyphenol hydrolysates on C.
elegans health span. Fold Change mobility values (activity
treatment/activity control) are represented for the different feed
conditions.</p><!><p>In vitro and in vivo assays showed
different results on the antiaging capacity because these assays analyzed
different antiaging parameters. The in vitro assay
was focused on the determination of the elastase inhibition capacity
to prevent the drastic decrease in skin elasticity with age, while in vivo assay determines the prevention of the reduction
in the mobility caused by the aging process. However, both assays
showed that NEPs from sweet cherry pomace provide higher antiaging
capacity than EPPs recovered by conventional extraction. Promod HBN
extract was distinguished by high antiaging capacity in vitro and in vivo.</p><!><p>The AChE
inhibitory activity of the extracts was evaluated in vitro by Ellman's method to determine the potential of the extracts
to revert the cholinergic deficit in Alzheimer's disease. As
can be seen in Table 4, the acid hydrolysis extract showed the highest AChE inhibition
with a higher neuroprotective capacity than galantamine at a concentration
of 100 Î¼M. EAE extracts showed higher AChE inhibition than the
alkaline extract, except EAE with Promod enzyme to obtain HBN extracts,
which showed inhibition of 32% ± 2%. By contrast, the conventional
extract showed the lowest acetylcholinesterase inhibition.</p><p>The
neuroprotective capacity of different NEPs extracts was evaluated
for the first time in vivo in C. elegans by a model inducing paralysis of nematodes by upshifting temperature,
which induces the expression of the human amyloid β-peptide. Figure S2 shows the percentage of CL4176 worms
not paralyzed for 24, 26, 28, 30, and 32 h at three different doses
of extracts (100, 200, and 300 Î¼L) in NGM medium compared with
a positive control with EGb 761, NGM with the induction of paralysis
without extracts, and NGM without induction. Acid extract was added
at lower doses (10, 25, and 50 Î¼L) since this extract at higher
volumes affected the egg-laying and eggs were not able to hatch (Figure S2B). Results showed that Pectinase HB,
alkaline, and Depol HB extracts exhibited the most neuroprotective
effect: the percentage of nonparalyzed worms at 28 and 30 h in groups
treated at a dose of 300 Î¼L was higher than that with the rest
of the extracts (Figures S2 and 5A). The protective effect of Pectinase HB extracts
was higher than the positive control at 30 h but without significant
differences (see Figure 5A). This extract also showed a protective effect at 32 h, but it
was lower than that with the positive control (Figure 5B). By contrast, the acid extract did not
show any protective effect.</p><!><p>Percentage of not paralyzed CL4176 scored at
(A) 30 h and (B) 32
h treated with 12 different nonextractable polyphenol hydrolysates
at a dose of 300 μL (50 μL for acid extract). ****p ≤ 0.0001, **p ≤ 0.01; ns,
not significant. Statistical comparison vs NGM condition.</p><!><p>Even though acid extract provided a positive in vitro neuroprotective effect, a detrimental effect was observed in vivo on C. elegans (and possibly in
our body) due to the low pH of the extract. Thus, NEPs released from
EAE with Pectinase to obtain HB extracts exhibited the most neuroprotective
effect both in vitro and in vivo.</p><!><p>Some natural compounds may also cause health
problems in the human body due to their proliferative and cytotoxic
effects.37 Therefore, safety is critical
in the development of novel products for the pharmaceutical, cosmetic,
or food industries.73 One of the most metabolically
competent cell lines for cytotoxic assays is the human hepatocarcinoma
HepG2 because the majority of toxicological studies indicate that
toxic effects derived from natural compounds are associated with hepatotoxicity.73,74 In this manner, the HepG2 cell line provides the closest in vitro model to the human liver in cytotoxic assays.73 Thus, it is advisable to evaluate the toxicity
of natural compounds by using different cell lines. The cytotoxicity
of conventional, alkaline, acid, and EAE extracts was evaluated. In
particular, HBN extracts attained by EAE were selected as the most
representative extracts to evaluate the cytotoxic effect of EAE extracts.
The first preliminary study allowed observing that the acid extract
presented a very high cytotoxic effect hindering the measure of its
absorbance.</p><p>Figure 6 shows the cytotoxic effect on HepG2, SKOV3, and HT-29 cancer
cell lines, as well as HFF-1 primary cell line, of different concentrations
(0.3800–0.0095 mg/mL sample) of conventional, alkaline, and
EAE extracts from sweet cherry pomace. As can be seen in Figure 6A,B,C, a cytotoxic
effect was observed with alkaline extract on HepG2, SKOV3, and HT-29
cell lines, while the highest concentration of the alkaline sample
(>0.2850 mg/mL) did not present a cytotoxic effect on the HFF-1
primary
cell line (see Figure 6D). However, this extract also exhibited a proliferative effect on
the HFF-1 cell line as concentrations of 0.3800 and 0.2850 mg/mL sample
increased the cell viability compared with control. These results
suggested that cell lines differed in their sensitivity to the same
samples, which may depend on multiple cell type-specific signaling
cascades of each cell line as well as their transcription factor activities.
The proliferative effect observed with the alkaline sample treatment
could be due to the presence of determined phytochemicals released
during alkaline hydrolysis treatment, which cause injury to the liver,
colon, ovary, and skin.</p><!><p>Cytotoxic and proliferative effects of nonextractable
polyphenol
hydrolysates obtained by conventional extraction, alkaline hydrolysis,
and EAE with Promod, Depol, and Pectinase enzymes from sweet cherry
pomace at different concentrations (0.3800–0.0095 mg/mL) on
(A) hepatocarcinoma HepG2, (B) human ovarian cancer SKOV3, (C) colon
adenocarcinoma HT-29, and (D) primary dermal fibroblast HFF-1 cell
lines.</p><!><p>On the other hand, a concentration
of 0.2850 mg/mL of all samples
did not show cytotoxic and proliferative effects on SKOV3, except
for alkaline extracts (see Figure 6B). Regarding the HT-29 cell line, the highest and
the lowest concentrations of extracts attained by EAE with Pectinase
and EAE with Promod did not show cytotoxic effects (see Figure 6C).</p><p>The low cytotoxicity
and nonproliferative effect of the extracts
achieved by EAE with Promod enzyme in all cell lines studied (HepG2,
HFF-1, SKOV3, and HT-29) suggested that this extract could be the
most suitable to be included as a bioactive ingredient in future formulations
for the elaboration of products with beneficial health properties.</p><!><p>This work presents for the first
time an in vivo evaluation of the antioxidant, antiaging,
and neuroprotective capacities
of NEP extracts from cherry pomace using C. elegans as an experimental animal model where EAE extracts presented the
highest biological activities. Depol HBN, Promod HB, Pectinase HBN,
and Depol TPA extracts were highlighted as the most bioactive extracts in vitro and in vivo. Nevertheless, EAE
with Promod enzyme extract was the only one that did not present a
cytotoxic effect on HepG2, HFF-1, SKOV3, and HT-29 cell lines. The
fast HPTLC analytical method to separate extractable polyphenols and
NEPs allowed identification of these compounds by families. Furthermore,
the rapid and tentative identification of up to 39 NEPs in sweet cherry
pomace was carried out for the first time by DART-Orbitrap-HRMS. To
our knowledge, some phenolic compounds such as vestitol, scopoletin,
or procyanidin B2 had not previously been identified in this fruit
byproduct. In vitro and in vivo experiments
as well as HPTLC and DART-Orbitrap-HRMS analysis revealed that conventional
extraction is an inefficient technique to extract phenolic compounds
since phenolic compounds with important biological properties were
retained in the extraction residue. EAE is a promising alternative
to release NEPs, providing extracts with high biological capacities.
In fact, it permitted us to obtain nontoxic extracts with high in vivo antioxidant, antiaging, and neuroprotective capacities.</p><!><p>Optimal conditions to
obtain HBN, TPA and HB extracts
by EAE, theoretical values of TPC (Folin–Ciocalteu method)
and total PA content and antioxidant and antihypertensive capacity
(ACE inhibition method) obtained under optimal EAE conditions, Rf values, peak areas, and wavelengths at absorption
maximum of separated phenolic compounds by HPTLC, exact mass data
and intensity of nonextractable polyphenols, fold change values of
the mobility of C. elegans treated with the different
extracts at three doses, effect of extracts on body paralysis delay,
and UV absorption chromatograms at 280 nm with Rf values of extracts (PDF)</p><p>jf2c03346_si_001.pdf</p><!><p>G.D.R.: investigation, formal analysis, validation,
data curation, visualization,
writing–original draft. D.R.: supervision, writing–review
& editing, project administration, funding acquisition. P.M.: investigation, formal analysis, data curation, visualization,
writing–review & editing. M.P.: conceptualization,
methodology, formal analysis, resources, supervision, writing–original
draft, writing–review & editing, project administration,
funding acquisition. M.L.M.: conceptualization, methodology,
resources, supervision, writing–original draft, writing–review
& editing, project administration, funding acquisition.</p><!><p>The authors
declare no competing financial interest.</p>