foods
Article
Chemical Fingerprinting, Aorta Endothelium Relaxation Effect,
and Enzymatic Inhibition of Canelo (Drimys winteri J. R.
Forst. & G. Forst, (D.C) A. Gray, Family Winteraceae) Fruits
Ruth E. Barrientos 1,† , Javier Romero-Parra 2 , Fredi Cifuentes 3,4,† , Javier Palacios 5 ,
Néstor Jaime Romero-Jola 6 , Adrián Paredes 7,8 , Gabriel Vargas-Arana 9,10, * and Mario J. Simirgiotis 1, *
Citation: Barrientos, R.E.;
Romero-Parra, J.; Cifuentes, F.;
Palacios, J.; Romero-Jola, N.J.;
Paredes, A.; Vargas-Arana, G.;
Simirgiotis, M.J. Chemical
Fingerprinting, Aorta Endothelium
Relaxation Effect, and Enzymatic
Inhibition of Canelo (Drimys winteri J.
R. Forst. & G. Forst, (D.C) A. Gray,
Family Winteraceae) Fruits. Foods
2023, 12, 2580. https://doi.org/
10.3390/foods12132580
Academic Editor: J. Scott Smith
Received: 29 May 2023
Revised: 26 June 2023
Accepted: 29 June 2023
Published: 1 July 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Foods 2023, 12, 2580. https://doi.org/10.3390/foods12132580
1 Instituto de Farmacia, Facultad de Ciencias, Universidad Austral de Chile, Valdivia 5090000, Chile;
ruth.barrientos@alumnos.uach.cl
2 Departamento de Química Orgánica y Fisicoquímica, Facultad de Ciencias Químicas y Farmacéuticas,
Universidad de Chile, Santiago 6640022, Chile; javier.romero@ciq.uchile.cl
3 Laboratorio de Fisiología Experimental, Instituto Antofagasta, Universidad de Antofagasta,
Antofagasta 1270300, Chile; fredi.cifuentes@uantof.cl
4 Departamento Biomédico, Facultad Ciencias de la Salud, Universidad de Antofagasta,
Antofagasta 1240000, Chile
5 Laboratorio de Bioquímica Aplicada, Facultad de Ciencias de la Salud, Universidad Arturo Prat,
Iquique 1110939, Chile; clpalaci@unap.cl
6 Departamento de Sanidad Animal, Facultad de Medicina Veterinaria y Zootecnia, Universidad del Tolima,
Ibagué 730001, Colombia; njromeroj@ut.edu.co
7 Laboratorio de Química Biológica, Instituto Antofagasta, Universidad de Antofagasta,
Antofagasta 1270300, Chile; adrian.paredes@uantof.cl
8 Departamento de Química, Facultad de Ciencias Básicas, Universidad de Antofagasta,
Antofagasta 1240000, Chile
9 Laboratorio de Química de Productos Naturales, Instituto de Investigaciones de la Amazonía Peruana,
Avenue Abelardo Quiñones, Iquitos 16001, Peru
10 Facultad de Industrias Alimentarias, Universidad Nacional de la Amazonía Peruana, Iquitos 16001, Peru
* Correspondence: gvargas@iiap.gob.pe (G.V.-A.); mario.simirgiotis@uach.cl (M.J.S.);
Tel.: +56-63-63233257 (M.J.S.)
† These authors contributed equally to this work.
Abstract: Drimys winteri J.R. Forst. & G. Forst (D.C) G. Gray, var. chilensis (canelo) is an endemic tree
from Chile. Since pre-Columbian times, it has produced a fruit known as the canelo pepper, (pimienta
de canelo) or Foye pepper, which can be used as a spice. The chemical and biological analysis of
canelo fruits is reported for the first time in this study, that is, its phenolic fingerprinting by UHPLC-
PDA- Q-orbitrap MS, the antioxidant activity, the enzymatic inhibitory activity, and its relaxation
effects on rat aorta. The proximal composition and the mineral content (Ca: 1.45 ± 0.03 mg/100 g;
Mg: 7.72 ± 0.03 mg/100 g; Fe: 4.54 ± 0.21 mg/100 g; Zn: 2.99 ± 0.02 mg/100 g; Mn: 1.08 ± 0.03 mg/100 g;
Cu: 0.82 ± 0.02 mg/100 g; K: 53.03 ± 0.20 mg/100 g; Na: 0.087 ± 0.00 mg/100 g) are also reported.
The canelo fruits showed a total phenolic content of 57.33 ± 0.82 mg GAE/g dry weight. In addition,
the total flavonoid content was 38.42 ± 1.32 mg equivalent of QE/g dry weight. The antioxidant
activity was evaluated by employing DPPH and ABTS methods (IC50 of 6.65 ± 0.5 and 9.5 ± 0.05 µg/mL,
respectively), ORAC (25.33 ± 1.2 µmol Trolox/g dry plant) and FRAP (45.56 ± 1.32 µmol Trolox/g
dry plant). The enzymatic inhibition of acetylcholinesterase, butyrylcholinesterase, and tyrosinase
(IC50 : 1.94 ± 0.07, 2.73 ± 0.05, and 9.92 ± 0.05 µg extract/mL, respectively) is also reported. Canelo
extract led to an 89% relaxation of rat aorta. Our results confirm that D. winteri fruits are a rich source
of secondary metabolites and can inhibit enzymes associated with neurodegenerative diseases; the
results also suggest that canelo may induce a potentially hypotensive effect in rat aorta. The study
demonstrates the medicinal properties of canelo fruit and spice.
Keywords: endemic plants; anticholinesterase activity; hypotensive effects; neglected spice; pimiento;
Mapuche; Foye
https://www.mdpi.com/journal/foods
Foods 2023, 12, x FOR PEER REVIEW 2 of 23

Foods 2023, 12, 2580 2 of 19

1. Introduction
Several species of the genus Drimys, (Winteraceae) are widely distributed in the
1. Introduction
Americas (fromspecies
Several Mexicoofinthe thegenus
North Drimys,
to Chile (Winteraceae)
and Argentinaare in the south).
widely Drimys confer-
distributed in the
tifolia Phil. is endemic to the Juan Fernandez Islands, whileDrimys
Americas (from Mexico in the North to Chile and Argentina in the south). Drimys confertifolia angustifolia Miers is
located
Phil. isinendemic
Brasil, Drimys
to the Juan brasiliensis
Fernandez Miers is found
Islands, whileinDrimys
Brasil and México,
angustifolia Drimys
Miers andinain
is located
(Reiche),
Brasil, Drimys brasiliensis Miers is found in Brasil and México, Drimys andina (Reiche),Dri-
R. A. Rodr. and Quez., and syn. D. winteri var. andina are endemic of Chile, R. A.
mys granadensis
Rodr. and Quez., L.f. and
is found
syn. inD. México and andina
winteri var. Perú, andare two varieties
endemic of D.Drimys
of Chile, winteri granadensis
J. R Forst
etL.f.
G. Forst, D. winteri
is found in Méxicovar. winteri,
and Perú, areand
foundtwoinvarieties
Southwesternof D. Patagonia
winteri J. R(45°44′′–55°58′S).
Forst et G. Forst,
D.D.winteri
winterivar.
var.chilensis
winteri,(DC.) A. Gray
are found is widespreadPatagonia
in Southwestern across Chile and
(45◦ 44 Argentina
00 –55 ◦ 580 S). D. (30°2′′–
winteri
var. chilensis (DC.) A. Gray is widespread across Chile and Argentina (30 2 –46 2500 S).
46°25′′S). The latter is an evergreen endemic tree that grows in the Coquimbo ◦ 00
and ◦
Aysén
regions
The latterof Chile
is an and is mostendemic
evergreen abundant treeinthat
thegrows
Araucania
in theand Los Ríosand
Coquimbo regions
Aysén[1,2]. Thisof
regions
endemic tree (Figure 1), commonly named canelo in Spanish
Chile and is most abundant in the Araucania and Los Ríos regions [1,2]. This endemic treeand Foye in the Mapu-
dungun
(Figurelanguage,
1), commonly is considered
named canelosacred in and medicinal
Spanish andin the indigenous
Foye in the Mapudungun Mapuche language,culture.
Inistraditional
considered Mapuche
sacred and medicine,
medicinala leafin infusion was used
the indigenous to relieve
Mapuche birth and
culture. stomach
In traditional
pains,
Mapuchewhilemedicine,
an infusion of the
a leaf bark was
infusion wasusedusedto totreat cancer
relieve birthandandother
stomach diseases
pains, [3]. Plantan
while
species
infusionfromof the
the genus
bark wasDrimys usedhave beencancer
to treat reported andtoother
produce bioactive
diseases compounds
[3]. Plant speciessuch from
asthe
essential oils, terpenes, and flavonoids [4]. To date, 18 drimane-type
genus Drimys have been reported to produce bioactive compounds such as essential sesquiterpenoids
have
oils,been characterized
terpenes, for Drimys
and flavonoids [4]. winteri
To date,var.18 chilensis,
drimane-typeas well as various lignanes
sesquiterpenoids havesuchbeen
ascharacterized for Drimys
sesamin, (−) cubebin, and winteri var. chilensis,
eudesmin. as well
In addition, as various
according lignanes
to the such many
literature, as sesamin,
fla-
(−) cubebin,
vonoids such as and eudesmin.
apigenin, In addition,
luteolin, according
kaempferol, to the literature,
quercitrin, many flavonoids
taxifolin, quercetin, cirsimari- such
as fisetin,
tin, apigenin,
andluteolin,
astilbin kaempferol,
have been reportedquercitrin,andtaxifolin,
isolated quercetin,
but only from cirsimaritin,
bark andfisetin,leaves andof
astilbin have
this endemic tree [5].been reported and isolated but only from bark and leaves of this endemic
tree [5].

Figure 1. Canelo tree growing in Valdivia, Chile. Sealed commercial product. Zone of distribution of
Figure 1. Canelo tree growing in Valdivia, Chile. Sealed commercial product. Zone of distribution
the plant in Chile (green).
of the plant in Chile (green).
Our group has previously reported on extracts and some pure compounds from
various group
Our endemichasspecies
previously reported
in Chile that on extracts
have potentand some pure
enzymatic compounds
inhibitory from
effects or var-
other
ious endemic linked
bioactivities speciestoinameliorating
Chile that have potent enzymatic
noncommunicable inhibitory
diseases (NCDs) effects or other bio-
and neurodegenera-
activities linked
tive diseases to hypertension.
and ameliorating noncommunicable diseasesGreigia
An example is the Chilean (NCDs) and neurodegenera-
sphacelata (Ruiz and Pav.)
tive
Regel [6] fruits showed potent inhibition against AChE and BChE, sphacelata
diseases and hypertension. An example is the Chilean Greigia and some (Ruiz and
metabolites
Pav.) Regel [6] fruits showed potent inhibition against AChE and BChE,
tentatively identified by UHPLC-MS for G. sphacelata have been linked to neuroprotectiveand some
metabolites tentatively
activity. Recently, identified
the berries by UHPLC-MS
of Azara dentata Ruizfor G. Pav.
and sphacelata
[7] werehave been
found to linked to
have good
neuroprotective activity.
inhibitory potential Recently,
against the berries
AChE, BChE, of Azara dentata
and tyrosinase, while someRuiz bioactive
and Pav.compounds
[7] were
found
such to
as have good inhibitory
coumarins, potential
anthocyanins, against
phenolic AChE, BChE,
compounds, andand tyrosinase,
flavonoids, werewhile somein
detected
bioactive compounds such as coumarins, anthocyanins, phenolic compounds,
it by high-resolution UHPLC-MS. The fruits of D. winteri are commonly used to produce and
canelo pepper, which has a slight itch, is a very aromatic spice, and is rarely consumed
Foods 2023, 12, 2580 3 of 19

by humans. The research on canelo has focused on its medicinal character; however, is
it necessary to carry out the fingerprinting of chemical constituents and the evaluation
of biological activities of canelo fruits with the aim of contributing to the food industry
and to the formulation of nutraceuticals or dietary supplements. No previous research
about canelo fruit enzymatic inhibition potential or studies relating to its ability to produce
vascular relaxation effects have been reported so far.
The goals of this study are to conduct a proximal analysis and to measure the min-
eral content of D. winteri fruits; to analyze the phenolic fingerprint by UHPLC-PDA-MS;
to assess canelo fruit antioxidant activity; to evaluate its enzymatic inhibition potential
against acetylcholinesterase, butyrylcholinesterase, and tyrosinase; and to determine the
hypotensive potential of canelo fruits.

2. Materials and Methods

2.1. Chemicals
Deionized pure water (<5 µg/L TOC), used for all experiments, was produced in a
water system Arium 126 61316-RO coupled to an Arium 611 UV unit (Sartorius, Goettin-
gen, Germany). Methanol (HPLC grade) and formic acid (p.a. for mass spectrometry)
were obtained from J. T. Baker (Phillipsburg, NJ, USA. Pure gallic acid (purity > 95%),
2,20 -azinobis(3-ethylbenothiazoline-6-sulfonic acid) diammonium salt (ABTS), 6-hydroxy-
2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) (purity > 95%), iron (III) chloride hex-
ahydrate, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteu reagent 2,4,6-tri(2-pyridyl)-
s-triazine (TPTZ), aluminum chloride, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES), disodium salt, adenosine 50 -triphosphate (ATP), ethylenediaminetetraacetic acid
(EDTA), butyrylcholinesterase from equine serum, and acetylcholinesterase from Electropho-
rus electricus (electric eel) were obtained from Sigma-Aldrich® (Santiago, Chile). Sodium
nitrite, sodium carbonate, potassium persulphate, calcium chloride, and sodium hydroxide
were obtained from Merck® (Santiago, Chile). Standard grade kojic acid, galantamine, zileu-
ton, sodium acetate trihydrate, plus other HPLC reagents including acetone, chloroform,
ethyl acetate, and n-hexane were obtained from Merck® (Santiago, Chile). HPLC standards
(quercetin, chlorogenic acid gallic acid, caffeic acid, ferulic acid, vicenin 2, quercetin 3-O-
glucoside, isovitexin, orientin, kaempferol, cirsimaritin, artemisinin, vitexin, eriodictyol,
purity 95% by HPLC) were obtained from Phytolab (Vesten-Bergsgreuth, Germany), Merck®
(Santiago, Chile), Biopurify (Chengdu, China), BOCSY (Shirley, NY, USA), or Extrasynthèse
(Genay, France).

2.2. Plant Material

Fruits of Drimys winteri J.R. Forst. and G. Forst. var. chilensis (DC.) A. Gray (family
Winteraceae) were collected manually from the months of March–April of the year 2021
in Valdivia, the capital city of the Los Ríos region, near the road on the way to Oncol
Park, Chile. The mature fruits were washed, dried at room temperature to obtain pimienta
mapuche (Mapuche pepper, Figure 1), and then stored in a dark and cool place sealed
without air at an ambient temperature (25 ◦ C). A voucher specimen was placed in the
“Laboratorio de Productos Naturales of the Universidad Austral de Chile” (voucher number
DW-7-11-21). A total of 5 g of milled canelo fruits were extracted with 20 mL of ethanol:
water 1:1 v/v and the solvent evaporated in vacuo at 45 ◦ C and the remaining water
lyophilized to yield 488 mg of a brown extract. The extract was kept at −80 ◦ C in an
ultra-freezer for further dissolution and analyses.

2.3. Determination of Proximal Composition and Mineral Content

Procedures established by the AOAC were employed in all determinations [8,9].
Moisture content was acquired by drying the fresh fruit samples using an oven at a constant
weight. The crude protein content was measured using the Kjeldahl method (N × 6.25)
using a Kjelmaster k375 (Büchi Labortechnik AG, Flawil, Switzerland) device and the
fiber content by gravimetry after hydrolysis of the samples, while the total lipid content
Foods 2023, 12, 2580 4 of 19

was obtained using the Soxhlet procedure (Sigma-Aldrich, Saint Louis, MO, USA) using
petroleum ether as solvent. The ash content was measured using muffle incineration
in a furnace at 550 ± 15 ◦ C. Total carbohydrates were 100 − (g water + g protein + g
fiber + g fat + g ash). Results are expressed in g per 100 g fresh weight (g/100 g fw). For
the mineral content, the spice was dried to ash at 550 ◦ C [9]. The ash was boiled with 20%
hydrochloric acid (10 mL) and then filtered and fit to 100 mL with deionized water. Levels
of the minerals calcium (Ca), potassium (K), iron (Fe), magnesium (Mg), sodium (Na),
zinc (Zn), manganese (Mn), and copper (Cu) were determined using atomic absorption
spectroscopy (Varian AA240, Belrose, Australia), previously set with standard solutions
with known amounts of the minerals being determined using flames of air-acetylene and
nitrous oxide-acetylene, with the latter only being used for calcium analysis. Hollow
cathode monometallic lamps were utilized for each element analyzed. All analyses were
performed in triplicate.

2.4. Ultrahigh Liquid Chromatography Orbitrap MS Analysis (UHPLC OT-MS)

Analytical chromatography was run using a C-18 HPLC column (150 mm × 4.5 mm
ID Acclaim, 2.5 µm, Thermo, Bremen, Germany) at 25 ◦ C. The detection wavelengths were
285, 255, 335, and 355 nm, and PDA diode array detectors were set from 200 to 700 nm.
Mobile phases were: 2% formic aqueous solution (A) and acetonitrile 2% formic acid (B).
The gradient program was first at 5% B (zero time); up to 7% B for 5 min; then to 35%
B for 15 min; maintained 35% B for 17 min; went to 80% B for 7 min; maintained 80% B
for 10 min; and finally returned to initial conditions in 15 min for column equilibration
before the next injection. The flow rate was 0.90 mL/min., and the injection volume was
15 µL. Standards of methanol and the extract were kept at 15 ◦ C in the autosampler. The
spectrometer parameters were set as previously reported. Briefly, the flow rate of sheath
gas was 75 units; auxiliary gas unit flow rate was 25; capillary temperature was 420 ◦ C;
auxiliary gas heater temperature was 520 ◦ C; spray voltage was 2500 V (for ESI− ); and S
lens was RF level 32. Full scan data both positive and negative were acquired at a resolving
power of 70,000 FWHM at m/z 200, a scan range of m/z 70–1000, automatic gain control
(AGC) was set at 3 × 106 , and the time of injection was set to 200 ms. The system was
coupled to MS with a heated ionization HESI II probe. The nitrogen gas (purity > 99.99%)
was obtained from a Genius NM32LA (Peak Scientific, Billerica, MA, USA) machine and
used as a collision and damping gas. The mass calibration for Orbitrap was performed
twice a day to ensure the accuracy of an operating mass equal to 5 ppm. Mass calibration
was acquired in both negative and positive modes with an accuracy equal to 5 ppm, as
previously reported [6].

2.5. Antioxidant Activity and Flavonoid and Phenolics Contents

The total phenolic content (TPC) of D. winteri fruit extract was explored by the colori-
metric method with Folin–Ciocalteu reagent, and the total flavonoid content (TFC) was
acquired by the AlCl3 method [10]. The TPC results are expressed as mg gallic acid equiva-
lent (GAE)/100 g dry weight and TFC is expressed as mg quercetin equivalent of QE/100 g
dry weight. The measurements were obtained in triplicate and the results reported as the
mean ± SD.
The antioxidant capacity of D. winteri fruit extract was determined through different
methods: DPPH radical inhibition, ABTS radical inhibition, oxygen radical absorbance
capacity (ORAC), and Ferric ion reducing antioxidant power (FRAP).
The DPPH inhibition of D. winteri fruit extract was obtained according to a method
previously reported [11]. The analysis was performed in triplicate and the results reported
as IC50 (µg extract/mL) of the mean ± SD.
The ABTS inhibition produced by D. winteri fruits was measured according to the
previous method [12]. The measurements were performed in triplicate. The ABTS results
are reported as IC50 (µg extract/mL) of the mean ± SD.
Foods 2023, 12, 2580 5 of 19

The FRAP assay of D. winteri was performed as previously described [13]. The analysis
was performed in triplicate and the results are expressed as µmol Trolox/g dry plant. The
results were acquired by linear regression with Trolox (0–120 µM).
The ORAC capacity was performed according to the previous method described [14].
Values were obtained by a regression equation between the sample concentration or Trolox
and area under the fluorescein decay curve. The results are described as µmol Trolox/g
dry plant and reported as mean ± SD.

2.6. Determination of Inhibitory Enzymatic Activity

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitions were per-
formed in vitro according to the Ellman method. The enzymes were dissolved in Tris-HCl
buffer (50 mM, pH 8.0), and 5-dithiol-bis (2-nitrobenzoic acid) was prepared in buffer. The
D. winteri fruit extract was prepared at a concentration of 2 mg per milliliter in buffer.
The sample was mixed with the enzyme. To start the reaction, the respective substrate
(acetyl-thiocholine iodide or butyryl-thiocholine chloride) was added. The absorbance at
405 nm was recorded for 30 min at 37 ◦ C. Galantamine was used as the positive control.
The dopachrome method was used for assessing tyrosinase inhibition. In each well
were mixed, 20 µL of D. winteri fruit extract with 30 µL of PBS (67 mM, pH 6.8), 40 µL of
tyrosinase 100 U/mL, and 40 µL of the substrate L-DOPA 2.5 mM. The absorbance was
recorded at 492 nm. Kojic acid was the positive control substance.
All measurements of enzymatic inhibition by D. winteri fruit extract were acquired in
triplicate and the results expressed as IC50 (mean ± SD) in µg extract per mL.

2.7. Docking Simulations

The partial charges plus the geometries of all compounds shown were fully set using
the DFT method with set B3LYP-6-311G+ (d p) as the standard basis [10,15] in Gaussian 09W
software [16]. Then, deprotonations and energetic minimizations were acquired using the
LigPrep tool in Maestro Schrödinger suite v.11.8 (Schrödinger, LLC, California, USA) [17].
Torpedo Californica acetylcholinesterase structure (TcAChE; PDBID: 1DX6 code [18]), plus
human butyrylcholinesterase (hBChE; PDBID: 4BDS code [19]) and the tyrosinase obtained
from Agaricus bisporus mushroom (tyrosinase; PDBID: 2Y9X code [20]) were obtained from
the Protein Data Bank RCSB PDB [21]. Enzyme optimizations were obtained using the
Protein Preparation Wizard from Maestro software, where water molecules and ligands of
the crystallographic protein active sites were removed. In the same way, all polar hydrogen
atoms at pH 7.4 were added. Appropriate ionization states for acid and basic amino acid
residues were considered. The OPLS3e force field was employed to minimize protein energy.
The enclosing box size was set to a cube with sides of 26 Å length. The presumed catalytic
sites of each enzyme in the centroid of selected residues were chosen, considering their
accepted catalytic amino acids: Ser200 for acetylcholinesterase (TcAChE) [22,23], Ser198
for butyrylcholinesterase (hBChE) [24,25], and His263 for tyrosinase [20,26,27]. The glide-
induced fit docking protocol was employed for the final pairings [28]. Compounds were
pointed by the Glide scoring function in the extra-precision mode (Glide XP; Schrödinger,
LLC) [29] and were selected by the best scores and best RMS values (cutting criterion: less
than 1 unit) to obtain the potential intermolecular interactions between the enzymes and
compounds plus the binding mode and docking descriptors. The different complexes were
shown using a visual molecular dynamics program (VMD) and Pymol [30].

2.8. Isolation of Rat Aorta and Vascular Reactivity Assays

Animals used in this study were male Sprague Dawley rats, aged 6–8 weeks old,
n = 3, weighing 170–200 g. The experimental procedure was approved (CEIC #366/2022)
and performed in accordance with the local ethics research committee of Universidad
de Antofagasta. Animals were randomized and caged at room temperature and 45–51%
humidity. Rats were maintained with unrestricted access to water and free food. Animals
were collected for cervical dislocation. Then, the aortas were kept in a Krebs–Ringer
Foods 2023, 12, 2580 6 of 19

bicarbonate solution, and the aortic rings (2–3 mm) were dissected and placed in an organ
bath. Before the assays, the vascular endothelium integrity was assessed with 10−5 M
acetylcholine (Ach). To evaluate the relaxation effect of the ethanolic extract of D. winteri
fruits, the aortic rings were precontracted with 10−6 M phenylephrine (PE). After the
vascular plateau, increasing concentrations of the D. winteri extract were added to the organ
bath. To evaluate the relaxation role of the endothelium of D. winteri extract, the protocol
used consisted of removing the endothelium from the aortic rings, then adding increasing
concentrations of extract. To evaluate the relaxation effect produced by the fruits, in the
organ bath, aortic rings were initially contracted with 10−6 M phenylephrine (PE). Once the
vascular plateau was reached, cumulative concentrations of D. winteri extract were added
to the organ bath to record the relaxation effect. The effect of canelo fruit extract on the
contractile response to KCl (10–60 mM) and PE (10−10 –10−5 M) was also evaluated using
a different methodology. The rings were treated with a single concentration of D. winteri
fruit extract (100 µg/mL) and incubated for 20 min prior to contraction of the aortic rings
with increasing concentrations of KCl or PE.

2.9. Statistical Analysis

The data statistical analysis was carried out using 1-way or 2-way analysis of variance
(ANOVA) and the post hoc Dunnett test. In addition, the EC50 or IC50 determination was
acquired using sigmoidal nonlinear regression by the Graph Pad Prism version 5.0 software
package (GraphPad Software, Inc., La Jolla, CA, USA). Statistical significance was set at
p = 0.05.

3. Results and Discussion

3.1. Proximal Composition and Minerals of D. winteri Fruits
Table 1 shows the proximal composition and mineral content of D. winteri fruits.
It is mandatory to check the full content of metals in foods. The results of a proximal
composition showed that canelo fruits are rich in carbohydrates (58%) and have a high
protein content (13%), and the mineral content showed that the fruits are rich in magnesium
(77 mg/kg) and potassium (530 mg/kg) but lower in sodium, which make this spice
very good for the elderly and better than other fruits such as kiwis or local azaras [7].
Black pepper showed 2636.97 mg/kg Ca, 10,006.04 mg/kg K, and 1486.94 mg/kg mg;
however, it also showed 1892.12 mg/kg Na [31], while the famous spice Capsicum annuum
showed 1160.04 mg/kg Ca, 1253.65 mg/kg mg, and 16,143.58 mg/kg Na [31]. Mg and Ca
are important minerals for bone formation, supporting heart functions, relaxing muscle,
metabolizing glucose, and conducting memory [32].

Table 1. Proximal composition (%) and mineral content (mg/100 g) of D. winteri fruits.

Composition D. winteri
Moisture 15.2 ± 0.10
Ash 6.42 ± 0.26
Fatty matter 0.42 ± 0.02
Crude protein 12.74 ± 0.01
Crude fiber 7.26 ± 0.17
Carbohydrates 57.96 ± 0.01
Minerals
Ca 1.45 ± 0.03
Mg 7.72 ± 0.03
Fe 4.54 ± 0.21
Zn 2.99 ± 0.02
Mn 1.08 ± 0.03
Cu 0.82 ± 0.02
K 53.03 ± 0.20
Na 0.087 ± 0.00
Each value represents the mean ± SD of triplicates. Values are statistically different (p < 0.05).
 266.14767,
64 20.15 253–292–311 Zinniol C15H21O4− 265.14450 265.14344 4.017
158.84607
65 20.51 206–255–271 Apigenin 7-O-methyl ether C16H11O5− 283.06116 283.06010 3.735 268.03784
312.97437,
Foods 2023, 12, 2580 7 of 19
291.35034,
66 21.39 255–292 Octadecanedioic acid C18H33O4− 313.23865 313.23734 4.187
270.84320,
215.00909
3.2. HPLC-PDA-MS Identification of the Ethanolic Extract from Canelo Fruits
274.50204,
The ethanolic extract of canelo dried fruits (pepper) was analyzed using high-resolution
Hydroxyoctadecatrienoic 2 and221.15472,
67 22.29 255–292 UHPLC Q orbitrap mass analyses. C18HThe chromatogram
29O3− 293.21228 is293.21112
shown in Figure
3.953 the detailed
acid
comprehensive MS analysis is shown in Table 2 and commented upon below. 183.12144,
Figure 3
shows some structures and Figure S2 (Supplementary Materials) shows full MS 171.08131
spectra of
* representative compounds.
Identified by co-spiking experiments using authentic standard compounds.

100
95
90
85
80
75
70
65
Relative Abundance

60
55
50
45
40
35
30
25
20
15
10
5
0
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Time (min)

Figure
Figure2.2.UHPLC
UHPLCQQorbitrap
orbitraptotal
totalion
ioncurrent
currentchromatogram
chromatogramofofD.D.winteri
winteridried
driedfruit
fruit(pepper)
(pepper)
hydroethanolic extract.
hydroethanolic extract.

Table 2. HPLC-PDA-MS identification of hydroethanolic extract from canelo fruits (Drimys winteri).

Measured Theoretical
Peak Number Retention Time UV Max Tentative Identification Molecular Formula [M-H] Accuracy (ppm) Ions MSn
Mass (m/z) Mass (m/z)

1 1.38 227–272 Gluconic acid C6 H11 O7 − 195.05058 195.04993 3.351 -

2 1.43 229–274 Malic acid C4 H5 O5 − 133.01355 133.01315 3.008 -
3 1.80 227 Citric acid C6 H7 O7 − 191.01933 191.01863 3.684 -
Dihydroxybenzoic acid glucoside 153.05591, 109.2866,
4 2.75 273 C13 H15 O9 − 315.07248 315.07106 4.509
(Protocatechuic acid 4-O-glucoside) 108.60805
239.37044, 179.71397,
Hydroxybenzoic acid hexoside (Salicylic
5 2.85 273 C13 H15 O8 − 299.07718 299.07614 3.461 137.47914, 136.52159,
acid 4-O-glucoside)
119.98631
− 430.82227, 255.95665,
6 3.21 196–227 Unknown C18 H31 O13 455.17709 455.17592 2.584
215.00977, 144.00832
7 3.92 196–204–269 Unknown C15 H21 O7 − 313.12918 313.12818 3.198 -
3,4-Dihydroxybenzoic acid (protocatechuic
8 5.07 227–265 C7 H5 O4 − 153.01869 153.18242 2.987 -
acid)
9 7.15 227–257–312 3-O-Caffeoylquinic acid * C16 H17 O9 − 353.08774 353.08671 2.916 -
354.11142, 341.99585,
10 7.72 227–257–310 Hydroxycaffeoyl quinic acid C16 H19 O10 − 371.09866 371.09727 3.746 191.15846, 173.71552,
135.04477
179.78584, 160.84163,
11 7.86 227–257–312 Caffeoyl acid hexoside C15 H17 O9 − 341.08786 341.08671 3.377
135.04446
12 8.24 227–280–309 3,4-Dihydroxybenzaldehyde C7 H5 O3 − 137.02373 137.02332 2.969
13 8.47 Unknown C19 H25 O13 − 461.12979 461.12897 1.787 -
14 8.97 214–280–311 Ferulic acid * C10 H9 O4 − 193.05020 193.04954 3.450 -
191.14720, 172.27388,
15 9.06 214–280–312 3-O-p-Coumaroyl quinic acid C16 H17 O8 − 337.09283 337.09179 3.087
162.83879, 119.16192
16 9.29 - Quinic acid C7 H11 O6 − 191.05563 191.05501 3.240
191.05560, 135.04462,
17 9.30 227–257–310 5-O-Caffeoyl quinic acid C16 H17 O9 − 353.08783 353.08671 3.175
133.99362
533.58563, 515.55426,
461.33383, 353.01492,
18 9.30 227–257–311 Caffeoyl quinic acid dimer C32 H35 O18 − 707.18042 707.18234 2.721
323.06729, 242.81560,
191.05563
163.03905, 162.83839,
19 9.43 214–280–325 p-Coumaroyl acid hexoside C15 H17 O8 − 325.09293 325.09179 3.482
119.04916
475.91458, 412.85297,
20 9.63 247–315–337 200 -O-(300 0 ,400 0 -Dimethoxybenzoyl) vitexin C27 H31 O15 − 595.16620 595.16575 0.760
292.31461
21 9.66 265 Eudesmin C22 H25 O6 − 385.16354 385.16456 −2.651 -
22 9.81 247–267–324 Caffeic acid * C9 H7 O4 − 179.03442 179.03389 3.008 -
23 9.85 247–267 Ferulic acid 3-O-glucoside C16 H19 O9 − 355.10352 355.10236 3.258 193.81488, 178.68074
24 9.93 248–270-332 Isovitexin 200 -O-beta-D-glucoside C27 H29 O15 − 593.15063 593.15010 0.906 413.05600
173.67874, 163.36201,
25 10.15 214–280-323 5-O-p-Coumaroyl quinic acid C16 H17 O8 − 337.09299 337.09179 3.540
119.62347
423.92548, 301.42798,
26 10.26 280 Eriodictyol 7-O-hexoside C21 H21 O11 − 449.10904 449.10784 2.675 287.62851, 259.05487,
174.98462, 151.24983
27 10.49 250–325 3-O-Feruloylquinic acid C17 H19 O9 − 367.10355 367.10236 3.235 191.03110, 178.99828
Foods 2023, 12, 2580 8 of 19

Table 2. Cont.

Measured Theoretical
Peak Number Retention Time UV Max Tentative Identification Molecular Formula [M-H] Accuracy (ppm) Ions MSn
Mass (m/z) Mass (m/z)

315.07285, 288.19870,
28 10.50 250 Myricetin * C15 H9 O8 − 317.03009 317.02919 2.828
178.08804
29 10.70 249–283 4-Hydroxycinnamic acid C9 H7 O3 − 163.03955 163.03897 3.558 -
30 10.71 206–249–300 5-O-Feruloylquinic acid C17 H19 O9 − 367.10376 367.10236 3.817 -
31 10.89 251–304–329 Sophoraflavonoloside C27 H29 O16 − 609.14545 609.14557 0.189 255.02881, 151.06294
341.30762, 323.05405,
32 10.98 249–264–334 Isovitexin * C21 H19 O10 − 431.09727 431.09727 2.941
311.62509, 283.10669,
33 11.12 255–300–351 Isoquercitrin (Quercetin 3-O-glucoside) C21 H19 O12 − 463.08832 463.08710 2.888 300.02777, 151.00316
34 11.32 249–281–322 Kaempferol-3-O-rutinoside C27 H29 O15 − 593.15057 593.15010 0.805 287.44540
35 11.33 249–283–324 Unknown C19 H25 O10 − 413.14551 413.14422 3.109 -
341.05933, 311.35144,
36 11.36 248–267–334 Vitexin * C21 H19 O10 − 431.09836 431.09727 2.517
283.06186
327.28265, 284.03296,
37 11.45 250–285–304 Kaempferol 3-O-galactose C21 H19 O11 − 447.09341 447.09219 2.743
255.02979, 226.90129
385.14127, 300.02771,
301.05417, 302.11899,
38 11.53 255–300–351 Avicularin C20 H17 O11 − 433.07779 433.07654 2.890
271.15466, 255.48294,
227.09221, 151.03943
39 11.55 253–288–311 Taxifolin C15 H11 O7 − 303.05078 303.04993 2.811 284.30374, 125.87235
357.95270, 315.36295,
40 11.62 254–354 Isorhamnetin 3-O-glucoside C22 H21 O12 − 477.10400 477.10275 2.623
313.99323
41 11.63 - Unknown C17 H29 O10 − 393.17676 393.17552 3.139
447.09274, 301.03470,
42 11.69 256–348 Luteolin 5-O-glucoside C21 H19 O11 − 447.09351 447.09219 2.948 300.02780, 271.02429,
151.00296
284.03210, 255.0317,
43 11.87 265–365 Kaempferol 3-O-pentoside C20 H17 O10 − 417.08279 417.08162 2.808
227.07626
357.40594, 327.08704,
44 11.91 251–288–332 Isoorientin (Luteolin-6-C-glucoside) * C21 H19 O11 − 447.09335 447.09219 2.607
285.13358
45 12.05 250–281–311 Astilbin * C21 H21 O11 − 449.10934 449.10784 3.355 284.37854, 151.61490
46 12.08 251–281 Diosmetin 7-O-glucose C22 H21 O11 − 461.10898 461.10784 2.473 301.03540, 299.37292
342.00220, 261.29782,
Hexenyl-3-hydroxy-3-methyl-glutaryl
47 12.20 - C18 H29 O10 − 405.17676 405.17552 3.0464 178.37297, 160.84148,
hexoside
125.87243, 101.02345
314.09686, 299.15054,
48 12.35 250–281-311 30 -O-Methylviolanone C18 H17 O6 − 329.10651 329.10251 −12.139 285.11633, 162.83897,
161.40355
49 13.05 251–267–280 Lonchocarpenin C27 H27 O6 − 447.17923 447.18021 −2.203 215.00941
50 13.20 251–280–285 Artemisinin * C15 H21 O5 − 281.13947 281.13835 3.966 -
298.55157, 287.13138,
51 13.78 252–274–281 2-Hydroxyenterodiol C18 H21 O5 − 317.14285 317.13890 −12,455
267.64462, 258.56961
52 13.83 283 Eriodictyol * C15 H11 O6 − 287.05597 287.05501 3.326 151.46794, 134.95647
53 14.01 253–274–283 Fisetin C15 H9 O6 − 285.04028 285.03936 3.223 -
54 14.38 251–311 Quercetin * C15 H9 O7 − 301.03549 301.03428 4.029 284.31473, 151.00281
270.46667, 151.20018,
55 14.74 252–288–304 Isorhamnetin * C16 H11 O7 − 315.05096 315.04993 3.286
108.47343, 107.73669
9,12,13-Trihydroxy-10,15-octadecadienoic
56 16.40 251–277–283 C18 H31 O5 − 327.21762 327.21660 3.134 211.13336, 229.13232
acid
57 17.35 249–288 Kaempferol * C15 H9 O6 − 285.04028 285.03936 3.223 133.25186, 117.21797
251.12869, 249.92970,
58 18.10 245–285–311 Leptospermone C15 H21 O4 − 265.14441 265.14344 3.672
196.82272
228.57948, 215.00943,
59 18.28 206–251–286 Pinellic acid C18 H33 O5 − 329.23334 329.23225 3.301
171.10269
60 19.34 254–288–324 Diosmetin C16 H11 O6 − 299.05621 299.05501 4.009 284.03271
297.04083, 296.52487,
61 19.40 253–279 Cirsimaritin * C17 H13 O6 − 313.07178 313.07066 3.554
283.02441
62 19.93 252–286–311 Autumnolide C15 H19 O5 − 279.12387 279.12455 4.194 -
191.14365, 163.11220,
63 19.96 254–290–311 Unknown C14 H19 O3 − 235.13377 235.13287 3.840
144.00845, 119.04957
64 20.15 253–292–311 Zinniol C15 H21 O4 − 265.14450 265.14344 4.017 266.14767, 158.84607
65 20.51 206–255–271 Apigenin 7-O-methyl ether C16 H11 O5 − 283.06116 283.06010 3.735 268.03784
312.97437, 291.35034,
66 21.39 255–292 Octadecanedioic acid C18 H33 O4 − 313.23865 313.23734 4.187
270.84320, 215.00909
274.50204, 221.15472,
67 22.29 255–292 Hydroxyoctadecatrienoic acid C18 H29 O3 − 293.21228 293.21112 3.953
183.12144, 171.08131

* Identified by co-spiking experiments using authentic standard compounds.

3.2.1. Phenolic Acids

The presence of several coumaroyl feruloyl or caffeoyl derivatives was detected
(Table 2). Caffeoyl quinic acids and their glucosyl derivatives are considered beneficial
for human health, mainly due to their antioxidant and anti-inflammatory properties [33].
Peaks 4 and 5 were identified as dihydroxybenzoic acid glucoside (protocatechuic acid 4-O-
glucoside, C13 H15 O9 − ) and hydroxybenzoic acid glucoside (salicylic acid 4-O- glucoside,
C13 H15 O8 − ), while peak 8 was identified as 3,4-dihydroxybenzoic acid (C7 H5 O4 − ), peak
9 with a pseudomolecular ion at m/z: 353.08774 as 3-O-caffeoylquinic acid (C16 H17 O9 − ),
peak 10 as hydroxycaffeoyl quinic acid, peak 11 as caffeoyl acid hexoside (C15 H17 O9 − ),
peak 14 as ferulic acid, peak 15 as 3-O-p-coumaroyl quinic acid, peak 16 as quinic acid,
peak 17 as 5-O-caffeoyl quinic acid, peak 18 as the dimer derivative caffeoyl quinic acid
dimer, peak 19 as p-coumaroyl acid hexoside, while peaks 22, 23, 25, 27, 29, and 30 were
caffeic acid, ferulic acid 3-O-glucoside, 5-O-p-coumaroyl quinic acid, 3-O-feruloylquinic
acid, 4-hydroxycinnamic acid and 5-O-feruloylquinic acid, respectively. Finally, peak 51
was assigned as 2-hydroxyenterodiol and peak 64 as zinniol (C15 H21 O4 − ).
Foods 2023,12,
Foods2023, 12,2580
x FOR PEER REVIEW 129 of
of 19
23

Figure3.3.Structures
Figure Structuresof
ofrepresentative
representativecomponents
componentsdetected
detectedinincanelo
canelofruits.
fruits.

3.2.2.
3.2.1.C-glycosyl Flavonoids
Phenolic Acids
These classes of
The presence of flavonoids are important
several coumaroyl in or
feruloyl thecaffeoyl
diet because they showed
derivatives anti-
was detected
inflammatory,
(Table 2). Caffeoyl antidiabetic, anxiolytic,
quinic acids and theirantispasmodic, and hepatoprotective
glucosyl derivatives are consideredactivities [34].
beneficial for
Peak 32 with anion at m/z: −
human health, mainly due431.09727 was assigned
to their antioxidant and as anti-inflammatory
isovitexin (C21 H19 O 10 ) showing
properties [33].
diagnostic
Peaks 4 and C-glycosyl flavone fragments
5 were identified at m/z: 341.30762,
as dihydroxybenzoic 323.05405,(protocatechuic
acid glucoside 311.62509, 283.10669,
acid 4-
while peak 36 as its isomer vitexin, and peak 20 as 2 00 -O-(3000 ,4000 -dimethoxybenzoyl) vitexin
O-glucoside, C13H15O9 ) and hydroxybenzoic acid glucoside (salicylic acid 4-O- glucoside,
−

(C H1531OO8−15 − ). Peak 24 was tentatively identified as isovitexin 2”-O-beta-D-glucoside and9

C27
13H ), while peak 8 was identified as 3,4-dihydroxybenzoic acid (C7H5O4−), peak
peak 44 as isoorientin (luteolin-6-C-glucoside).
with a pseudomolecular ion at m/z: 353.08774 as 3-O-caffeoylquinic acid (C16H17O9 ), peak −

10 as hydroxycaffeoyl quinic acid, peak 11 as caffeoyl acid hexoside (C15H17O9−), peak 14

3.2.3. O-glycosyl Flavonoids and Aglycones
as ferulic acid, peak 15 as 3-O-p-coumaroyl quinic acid, peak 16 as quinic acid, peak 17 as
Several Oquinic
5-O-caffeoyl glycosyl
acid,flavonoids
peak 18 aswere detected,
the dimer together
derivative with some
caffeoyl quinic of acid
theirdimer,
aglycones,
peak
most of them with a myriad of biological activities [35]. Peak
19 as p-coumaroyl acid hexoside, while peaks 22, 23, 25, 27, 29, and 30 were caffeic acid,26 was identified as
the flavanone eriodictyol 7-O-hexoside (C H O − ), peak 28 as myricetin, peak 31 as
ferulic acid 3-O-glucoside, 5-O-p-coumaroyl 21 21quinic
11 acid, 3-O-feruloylquinic acid, 4-
sophoraflavonoloside
hydroxycinnamic acid(Cand 27 H29 O16 − ), peak 33 asacid,
5-O-feruloylquinic isoquercitrin
respectively.and peaks
Finally,34,peak
37, and 43
51 was
as kaempferol derivatives: kaempferol-3-O-rutinoside (C15 H2129OO −
assigned as 2-hydroxyenterodiol and peak 64 as zinniol (C 27H ). ), kaempferol 3-O-
4−15
galactopyranoside (C21 H19 O11 − ), and kaempferol 3-O-pentoside (C20 H17 O10 − ). Peaks
38–40, 42, 45, 46, 52–55,
3.2.2. C-glycosyl and 57, 6061, and 65 were assigned as: avicularin, taxifolin, isorham-
Flavonoids
netin 3-O-glucoside, luteolin 5-O-glucoside, diosmetin 7-O-beta-D-glucopyranoside, eriod-
These classes of flavonoids are important in the diet because they showed anti-
ictyol, fisetin, quercetin, isorhamnetin, kaempferol, diosmetin, cirsimaritin, and apigenin
inflammatory, antidiabetic, anxiolytic, antispasmodic, and hepatoprotective activities
7-O-methyl ether.
[34]. Peak 32 with anion at m/z: 431.09727 was assigned as isovitexin (C21H19O10−) showing
diagnostic C-glycosyl flavone fragments at m/z: 341.30762, 323.05405, 311.62509,
Foods 2023, 12, 2580 10 of 19

3.2.4. Isoflavones
Peak 48 with a parent ion at m/z: 329.10651 was identified as 30 -O-methylviolanone
(C18 H17 O6 − ).

3.2.5. Sesquiterpenes
Peak 50 with a pseudomolecular ion at m/z: 281.13947 was assigned as the antipar-
asitic compound artemisinin (C15 H21 O5 − ) and peak 62 as autumnolide (C15 H19 O5 − ).
Artemisinin is a very active compound and is key for malaria treatment. Its efficacy also
broadens to unrelated phylogenetically parasitic infections, such as schistosomiasis [36].
Some drimanes sesquiterpenes isolated from the related species Drimys brasiliensis were
devoid of antiparasitic activity [37].

3.2.6. Fatty Acids

Peak 57 with an ion at m/z: 327.21762, was identified as 9,12,13-trihydroxy-10,15-
octadecadienoic acid (C18 H31 O5 − ), peak 59 as pinellic acid (C18H33O5 −), and peaks 66 and 67
as octadecanedioic acid (C18H33O4 −) and hydroxyoctadecatrienoic acid (C18H29O3 −), respectively.

3.2.7. Other Compounds

Peaks 1–3 were tentatively identified as gluconic malic and citric acids, respectively.
Peak 12 was assigned as the aldehyde 3,4-dihydroxybenzaldehyde (C7 H5 O3 − ). Peak 58
with an ion at m/z: 265.14441 was assigned as the β-triketone leptospermone (C15 H21 O4 − ).
Peak 21 was tentatively identified as the lignin eudesmin (C22 H25 O6 − ). Peak 47 with an
ion at m/z: 405.17676 was identified as the sugar derivative hexenyl-3-hydroxy-3-methyl-
glutaryl hexoside (C18 H29 O10 − ). Peak 49 with an ion at m/z: 447.17923 was assigned as the
coumarin lonchocarpenin (C27 H27 O6 − ).

3.3. Total Phenolic and Flavonoid Contents and Antioxidant Activity

Phenolic compounds are one of the biggest groups of natural compounds and in this
work the total flavonoid-phenolic contents of D. winteri fruits were measured (Table 3). The
antioxidant activity of D. winteri fruits was determined through different methods (Table 3).
The antioxidant capacity of D. winteri fruits hydroethanolic extract was good in the different
assays evaluated, and this result is supported by the diversity in phenolic and flavonoids
detected, as shown in Table 3. Caffeoyl and feruloyl or coumaroyl quinic acid derivatives
or glycosides are the main antioxidant compounds [38], together with flavonoids present
in fruits and vegetables, and in this study several compounds detected belong to these
groups, (TPC 57.33 mg GAE/g and TFC: 38.42 mg QE/g) which support the antioxidant
activity of the fruits (Table 3). In comparison, the pericarp of black pepper (Piper nigrum)
was reported to contain total phenol-flavonoid contents of 1421.95 ± 22.35 mg GAE/100 g
and 983.82 ± 8.19 mg CE/100 g, respectively; however, for normal black and white pepper
the TFCs were 11- 654 for black and 447 mg GAE/100 g for white pepper [32].

Table 3. Total phenolic content (TPC), total flavonoid content (TFC), and antioxidant capacities of
D. winteri hydroethanolic extract.

Assay TPC a TFC b DPPH c ABTS c ORAC d FRAP d

Canelo pepper 57.33 ± 0.82 38.42 ± 1.32 6.65 ± 0.5 9.5 ± 0.05 25.33 ± 1.2 45.56 ± 1.32
Gallic acid - - 14.32 ± 0.5 1.67 ± 0.25 - -
aTPC expressed as mg gallic acid equivalent GAE/g dry weight; b TFC expressed as mg equivalent of QE/g dry
weight; c IC50 expressed as µg/mL; d expressed as µmol Trolox/g dry plant.

3.4. Enzymatic Inhibitory Activity

Neurodegenerative diseases are increasing worldwide, and the main problem is
the disability associated with the disease progression. Common treatments include the
use of enzymatic inhibitors agents in Alzheimer’s disease as well as Parkinson’s disease.
Foods 2023, 12, 2580 11 of 19

Cholinergic neurotransmission decreases with the progression of Alzheimer’s disease,

and currently, pharmacological treatment (donepezil, galantamine, and rivastigmine) is
related to the inhibition of acetylcholinesterase to decrease choline metabolism in the central
nervous system, which has the effect of increasing the presence of this neurotransmitter.
D. winteri fruit extract showed promising results related to the enzymatic inhibitory ac-
tivity of both enzymes AChE and BChE (Table 4). Moreover, the extract of D. winteri
causes a potent inhibition of tyrosinase. Previously, our group reported the inhibition of
AChE, BChE, and tyrosinase for several endemic and native species from Chile. For exam-
ple, endemic A. dentata’s berry ethanolic extract showed an IC50 of 2.87 ± 0.23 µg/mL,
6.73 ± 0.07 µg/mL, and 9.25 ± 0.15 µg/mL against AChE, BChE, and tyrosinase, re-
spectively [7], while the n-hexane extract of the endemic G. pinifolium plant showed an
IC50 of 4.58 ± 0.04 µg/mL, 23.44 ± 0.03 µg/mL, and 9.25 ± 0.15 µg/mL against AChE,
BChE, and tyrosinase, respectively. G. pinifolium ethyl acetate extract showed AChE (IC50
6.43 ± 0.03 µg/mL), BChE (IC50 33.25 ± 0.02 µg/mL), and tyrosinase (IC50
12.32 ± 0.21 µg/mL) [39]. Canelo fruits showed better activity against AChE and BChE than
the bark infusion of native Weinmannia trichosperma, with an IC50 of 3.13 ± 0.03 µg/mL
for AChE and 2.94 ± 0.08 µg/mL for BChE [40]. In addition, some constituents of canelo
detected in this study, such as several caffeoyl quinic acid derivatives, proved previously to
have anti AChE and antityrosinase activity [41], as well as some of the flavonoids detected
in our canelo fruits such as the flavanone glycoside astilbin [40].

Table 4. Enzymatic inhibitory activity (IC50 in µg/mL) of D. winteri hydroethanolic extract against
acetylcholinesterase, butyrylcholinesterase, and tyrosinase.

Assay AChE BChE Tyrosinase

Canelo 1.94 ± 0.07 2.73 ± 0.05 9.92 ± 0.05
Galantamine 0.26 ± 0.02 3.82 ± 0.02 -
Kojic acid - - 3.51 ± 0.02

3.5. Docking Simulations

Docking simulations were carried out for compounds shown in Figures 4 and 5; each
one of them turned out to be the most characteristic and abundant species according to
the UHPLC chromatogram (Figure 2) obtained from the fruit extract. Of all compounds
identified from the D. winteri fruit hydroethanolic extract, artemisinin, eriodictyol 7-O-
hexoside, 200 -O-(3000 ,4000 -Dimethoxybenzoyl) vitexin, 5-O-p-coumaroyl quinic acid, 3-O-
caffeoylquinic acid, and hydroxy-caffeoylquinic acid, as well as the known cholinesterase
and tyrosinase inhibitors, galantamine and kojic acid, respectively, were subjected to
docking assays into the acetylcholinesterase catalytic site, butyrylcholinesterase catalytic
site, and tyrosinase catalytic site. We aimed to analyze the molecular interactions between
the different enzymes and the chosen derivatives, as well as to obtain the energy docking
descriptors. The latter was performed to rationalize the inhibition activities obtained by
the extract (Table 4).

3.5.1. Acetylcholinesterase (TcAChE) Docking Results

All chosen compounds shown in Table 5 mainly perform hydrogen bond interac-
tions with the different residues of the acetylcholinesterase catalytic site. Eriodictyol
7-O-hexoside and 200 -O-(3000 ,4000 -dimethoxybenzoyl) vitexin showed the best binding energy
values of −18.206 kcal/mol and −16.406 kcal/mol, respectively. On the other hand, the
other remaining compounds (artemisinin, 5-O-p-coumaroyl quinic acid, 3-O-caffeoylquinic
acid, and hydroxy caffeoylquinic acid) showed moderate binding energies in a range
between −8.030 kcal/mol and −12.964 kcal/mol. As was mentioned above, all com-
pounds execute hydrogen bond interactions; nonetheless, eriodictyol 7-O-hexoside and
200 -O-(3000 ,4000 -dimethoxybenzoyl) vitexin perform extra π-π and T-shaped interactions,
which could explain their energy profiles compared to the other derivatives.
 caffeoylquinic acid, and hydroxy-caffeoylquinic acid, as well as the known cholinesterase
and tyrosinase inhibitors, galantamine and kojic acid, respectively, were subjected to
docking assays into the acetylcholinesterase catalytic site, butyrylcholinesterase catalytic
site, and tyrosinase catalytic site. We aimed to analyze the molecular interactions between
Foods 2023, 12, 2580
the different enzymes and the chosen derivatives, as well as to obtain the energy docking
12 of 19
descriptors. The latter was performed to rationalize the inhibition activities obtained by
the extract (Table 4).

Figure 4. Predicted binding mode and predicted intermolecular interactions between selected
Figure 4. Predicted binding mode and predicted intermolecular interactions between selected
compounds in D. winteri fruit extract and the residues of Torpedo Californica acetylcholinesterase
compounds in D. winteri fruit extract and the residues of Torpedo Californica acetylcholinesterase
(TcAChE) catalytic site. Yellow dotted lines indicate hydrogen bond interactions, cyan dotted lines
(TcAChE)
represent catalytic site. Yellow
π-π interactions, dotteddotted
magenta lines indicate hydrogen
lines represent bond interactions,
T-shaped interactions,cyan
and dotted lines
grey dotted
represent π-π interactions, magenta dotted lines represent T-shaped interactions, and grey
lines represent hydrophobic interactions. (A) Artemisinin into the catalytic site; (B) eriodictyol 7-O- dotted
Foods 2023, 12, x FOR PEER REVIEWlines represent hydrophobic interactions. (A) Artemisinin into the catalytic site; (B) eriodictyol 16 of 23
hexoside into the catalytic site; (C) 2”’-O-(3”’,4”’-dimethoxybenzoyl) vitexin into the catalytic 7-O- site;
hexoside into the catalytic
(D) 5-O-p-coumaroyl site;
quinic acid 2000the
(C)into -O-(3 000 ,4000 -dimethoxybenzoyl) vitexin into the catalytic site;
catalytic site; (E) 3-O-caffeoylquinic acid into the catalytic
site;5-O-p-coumaroyl
(D) (F) hydroxy caffeoylquinic
quinic acidacid
intointo
the the catalytic
catalytic site;site.
(E) 3-O-caffeoylquinic acid into the catalytic
site; (F) hydroxy caffeoylquinic acid into the catalytic site.

Figure 5. Predicted binding mode and predicted intermolecular interactions between selected
Figure 5. Predicted binding mode and predicted intermolecular interactions between selected com-
compounds in D. winteri fruit extract and the residues of human butyrylcholinesterase (hBChE)
pounds in D. winteri fruit extract and the residues of human butyrylcholinesterase (hBChE) catalytic
catalytic site. Yellow dotted lines indicate hydrogen bond interactions, cyan dotted lines represent
site. Yellow dottedand
π-π interactions, lines indicate
grey dottedhydrogen bond interactions,
lines represent hydrophobiccyan dotted lines
interactions. (A)represent
Artemisinin interac-
π-π into the
tions, and grey dotted lines represent hydrophobic interactions. (A) Artemisinin
catalytic site; (B) eriodictyol 7-O-hexoside into the catalytic site; (C) 2”-O-(3’’’,4’’’- into the catalytic
site; (B) eriodictyol 7-O-hexoside
dimethoxybenzoyl) into
vitexin into the the catalytic
catalytic site; (D) (C) 200 -O-(3000 ,4000quinic
site;5-O-p-coumaroyl -dimethoxybenzoyl) vitexin
acid into the catalytic
into
site; the
(E) catalytic site; (D) 5-O-p-coumaroyl
3-O-caffeoylquinic quinic acid
acid into the catalytic site;into
(F)the catalytic
hydroxy site; (E) 3-O-caffeoylquinic
caffeoylquinic acid into the
catalytic
acid into site.
the catalytic site; (F) hydroxy caffeoylquinic acid into the catalytic site.

3.5.1. Acetylcholinesterase (TcAChE) Docking Results

All chosen compounds shown in Table 5 mainly perform hydrogen bond interactions
with the different residues of the acetylcholinesterase catalytic site. Eriodictyol 7-O-
hexoside and 2”-O-(3”’,4”’-dimethoxybenzoyl) vitexin showed the best binding energy
values of −18.206 kcal/mol and −16.406 kcal/mol, respectively. On the other hand, the other
remaining compounds (artemisinin, 5-O-p-coumaroyl quinic acid, 3-O-caffeoylquinic
Foods 2023, 12, 2580 13 of 19

Table 5. Binding energies obtained from docking experiments of selected compounds from the
D. winteri fruit extract, as well as the known inhibitors galantamine and kojic acid over acetyl-
cholinesterase (TcAChE), butyrylcholinesterase (hBChE), and tyrosinase.

Binding Energy Binding Energy Binding Energy

Compound (kcal/mol) (kcal/mol) (kcal/mol)
Acetylcholinesterase Butyrylcholinesterase Tyrosinase
Artemisinin −8.030 −6.585 −4.203
Eriodictyol 7-O-hexoside −18.206 −14.486 −8.423
200 -O-(3000 ,4000 -Dimethoxybenzoyl) vitexin −16.406 −14.778 −9.218
5-O-p-Coumaroyl quinic acid −10.369 −9.509 −10.074
3-O-Caffeoylquinic acid −12.566 −10.910 −9.898
Hydroxy caffeoylquinic acid −12.964 −11.425 −10.429
Galantamine −12.989 −7.125 -
Kojic acid - - −6.050
Torpedo californica acetylcholinesterase: TcAChE; human butyrylcholinesterase: hBChE.

Eriodictyol 7-O-hexoside carries out seven hydrogen bond interactions through its
different hydroxyl groups (–OH). Three hydroxyl functions of the saccharide moiety were
responsible for five hydrogen bonding interactions, where the involved amino acids were
Tyr116, Tyr130, Glu199, and His440. This feature indicates that the polar sugar framework
plays an important role in binding energy and acetylcholinesterase inhibition. The other two
mentioned hydrogen bond interactions are performed through the two hydroxyl groups
that the catechol cores bear and the residues of Gly119 and Ser200 (Figure 4A). Similar to
the eriodictyol derivative, the 7-O-hexoside derivative 2”-O-(3”’,4”’-dimethoxybenzoyl)
vitexin also executes five hydrogen bond interactions through its hydroxyl functions and
the oxygen atom adjacent to the anomeric carbon of the saccharide motif with amino acids
Gly117, Gly118, Tyr130, and Gly441, as well as two more interactions with the –OH func-
tions of the catechol at position 3- of the chromone framework (Figure 4B). As previously
mentioned, these two compounds perform extra π-π interactions that the others derivatives
do not exhibit; in this sense, eriodictyol 7-O-hexoside performs two π-π interactions with
Phe330 and Trp233, as well as a T-shaped interaction with Phe330, while 200 -O-(3000 ,4000 -
dimethoxybenzoyl) vitexin only performs one π-π interaction with Phe330 and a T-shaped
interaction with Tyr121, explaining the possible different binding energies observed for
both compounds (Table 5).
The structural quinic acid related derivatives 5-O-p-coumaroyl quinic acid, 3-O-
caffeoylquinic acid, and hydroxy caffeoylquinic acid displayed similar binding energy
values (−10.369 kcal/mol, −12.566 kcal/mol and −12.964 kcal/mol, respectively, see
Table 5). 5-O-p-coumaroyl quinic acid had the lowest ability to fit into the acetylcholinesterase
catalytic site. This could be due to the lower number of hydrogen bond interactions in these
derivative forms compared to 3-O-caffeoylquinic acid and hydroxy caffeoylquinic, which
exhibit seven and six hydrogen bond interactions, respectively, with acetylcholinesterase
catalytic residues. The hydrogen bond interactions carried out by 5-O-p-coumaroyl quinic
acid are among its two –OH groups (one at the aromatic phenyl ring and the other at
the sugar moiety) and the amino acids Asp72 and His440, but two more interactions can
be seen between the two oxygen atoms of the ester function and the residues of Glu199
and Ty130 (Figure 4D). The quinic acid-related derivatives, 3-O-caffeoylquinic acid and
hydroxy caffeoylquinic acid, are arranged in a similar manner as the acetylcholinesterase
catalytic site, nearly overlapping. Therefore, both derivatives perform hydrogen bond
interactions with almost the same amino acids: Gln69, Tyr121, Tyr130, Glu199, and His440.
It is noteworthy that both derivatives perform hydrogen bond interactions with the residue
of Tyr121 through their carboxylate groups presented in their corresponding sugar cores
(Figure 4E,F). Finally, artemisinin, due to its hydrophobic structure lacking aromatic rings,
shows mainly these types of interactions, which are weaker than hydrogen bond interac-
tions. Thus, artemisinin, exhibiting a −8.030 kcal/mol binding energy, only shows one
Foods 2023, 12, 2580 14 of 19

hydrogen bond interaction with Tyr121 and some hydrophobic interactions with Phe330
and Phe331, among other amino acids.

3.5.2. Butyrylcholinesterase (hBChE) Docking Results

Selected compounds from the D. winteri fruit extract subjected to docking experi-
ments on the butyrylcholinesterase catalytic site showed good binding energies. Indeed,
Table 4 shows that a canelo half-maximal inhibitory concentration value (BChE IC50 ) is in
the same magnitude order as the known inhibitor galantamine (2.73 ± 0.05 µg/mL and
3.82 ± 0.02 µg/mL, respectively). As in acetylcholinesterase docking assays, hydrogen bond
interactions predominate among all compounds and the different amino acids of the cat-
alytic pocket. Once again, eriodictyol 7-O-hexoside and 200 -O-(3000 ,4000 -dimethoxybenzoyl)
vitexin exhibited the best binding energy profiles. Indeed, the structures of these two
analogues are hosts to the butyrylcholinesterase catalytic site in similar forms. 5-O-p-
coumaroyl quinic acid, 3-O-caffeoylquinic acid, and hydroxy caffeoylquinic acid showed
similar binding energies, while artemisinin displayed the lowest value (Table 4).
The component 200 -O-(3000 ,4000 -dimethoxybenzoyl) vitexin, which was the best com-
pound assayed in our docking experiments in terms of energy, performed five hydrogen
bond interactions and one π-π intermolecular bonding. The hydrogen bond interactions of
this derivative were executed through the different hydroxyl groups that the chromone,
phenyl, and saccharide moieties bear. The implicated residues are Tyr128, Glu197, Ser198,
Leu286, and Pro285. Likewise, the observed π-π interaction occurs between the catechol
ring and the indole framework of the Tpr82 amino acid (Figure 5C). Considering the
similar arrangement between 200 -O-(3000 ,4000 -dimethoxybenzoyl) vitexin and eriodictyol
7-O-hexoside in the butyrylcholinesterase catalytic pocket, it can be seen in Figure 5B,C
that both derivatives perform their different interactions with many of the same amino
acids. In this sense, eriodictyol 7-O-hexoside performs seven hydrogen bond interactions,
and six of them are carried out among the hydroxyl groups and amino acids Asp70, Thr120,
Tyr128, Glu197, Pro285, and Tyr332. The last one is performed by the oxygen atom of the
carbonyl group presented in the chromone structure with Tyr440. As with 200 -O-(3000 ,4000 -
Dimethoxybenzoyl) vitexin, the π-π interaction executed by eriodictyol 7-O-hexoside occurs
between Trp82 and the catechol core. Although eriodictyol 7-O-hexoside performs two
more hydrogen bond interactions than 200 -O-(3000 ,4000 -Dimethoxybenzoyl) vitexin, the lat-
ter exhibits best binding energy (−14.486 kcal/mol and −14.778 kcal/mol, respectively).
Nonetheless, the energy descriptors for both derivatives are in the same range and do not
represent a significant difference (Table 4).
5-O-p-Coumaroyl quinic acid showed five hydrogen bond interactions, and no π-π
interactions were observed in docking results. The hydrogen bond interactions were per-
formed by the hydroxyl groups and the carboxylate function at the sugar core. Indeed,
through the two oxygen atoms of carboxylates, three hydrogen bond interactions were
observed with residues Gly116, Ser198, and His483. The remaining interactions were per-
formed with the amino acid Pro285 and the two hydroxyls of the sugar motif (Figure 5D).
3-O-Caffeoylquinic acid and hydroxy caffeoylquinic acid, as with eriodictyol 7-O-hexoside
and 2”-O-(3”’,4”’-dimethoxybenzoyl) vitexin, showed similar binding energy values (see
Table 4). Both quinic acid-related derivatives possess similar structures and settle in a
similar manner into the butyrylcholinesterase catalytic site, and thereby shared their hydro-
gen bond interactions with many residues. 3-O-caffeoylquinic acid performed hydrogen
bond interactions with Gly117, Tyr128, Ser198, Pro285, Ser287, and His438, while hydroxy
caffeoylquinic acid performed the same type of interactions with the residues of Trp82,
Glu197, Ser198, Pro285, and His438 (Figure 5E,F).
Once again, artemisinin showed low binding energy, which could be attributed to
the scarce interactions it generates within the catalytic site. In Figure 5A, artemisinin
performs two hydrogen bond interactions between the oxygen atom of the ester group
and the residues of Gly116 and Gly117, plus hydrophobic interactions with His438, Tro82,
and Phe329.
Foods 2023, 12, 2580 15 of 19

3.5.3. Tyrosinase Docking Results

Inhibition assays of D. winteri fruit hydroethanolic extract over tyrosinase showed similar
potency to the known inhibitor kojic acid at the same magnitude (9.92 ± 0.05 µg/mL and
3.51 ± 0.02 µg/mL, respectively). In this context, binding energies of selected compounds
that underwent docking assays resulted in the same range as the energy of kojic acid
(Table 4).
Concerning the interactions (intermolecularly) of selected compounds, docking de-
scriptors suggest that the principal inhibitory activities would lie in hydroxy caffeoylquinic
acid and 5-O-p-coumaroyl quinic acid. On the other hand, artemisinin, as in acetyl-
cholinesterase and butyrylcholinesterase, showed a deficient binding energy of
Foods 2023, 12, x FOR PEER REVIEW−4.203 kcal/mol, performing mainly hydrophobic interactions such as the one shown 19 of 23
with His85 in Figure 6A.

Figure 6. Predicted mode of binding and predicted inter-molecular interactions of selected

Figure 6. Predicted mode of binding and predicted inter-molecular interactions of selected com-
compounds in D. winteri fruit extract and the residues of human Agaricus bisporus mushroom
pounds in D. winteri fruit extract and the residues of human Agaricus bisporus mushroom tyrosinase
tyrosinase catalytic site. Yellow dotted lines indicate hydrogen bond interactions, cyan dotted lines
catalytic
representsite.
π-πYellow dotted lines
interactions, indicate
magenta hydrogen
dotted lines bond interactions,
represent cyan dotted
π-π interactions, lines
red represent
dotted lines
π-π interactions,
represent π-cationmagenta dottedblue
interactions, linesdotted
represent
linesπ-π interactions,
represent red dotted
salt bridge lines represent
interactions, and greyπ-cation
dotted
interactions,
lines represent blue dotted lines
hydrophobic represent salt
interactions. bridge interactions,
(A) Artemisinin and greysite;
into the catalytic dotted lines represent
(B) eriodictyol 7-O-
hexoside intointeractions.
hydrophobic the catalytic(A) site; (C) 2”-O-(3’’’,4’’’-dimethoxybenzoyl)
Artemisinin vitexin into
into the catalytic site; (B) eriodictyol the catalytic
7-O-hexoside intosite;
the
catalytic site; (C) 200 -O-(3
(D) 5-O-p-coumaroyl 000 ,4000acid
quinic into the catalyticvitexin
-dimethoxybenzoyl) site; (E) 3-O-caffeoylquinic
into the catalytic site; acid into the catalytic
(D) 5-O-p-coumaroyl
site; (F)acid
quinic hydroxy caffeoylquinic
into the catalytic site; acid(E)
into the catalytic site. acid into the catalytic site; (F) hydroxy
3-O-caffeoylquinic
caffeoylquinic acid into the catalytic site.
Eriodictyol 7-O-hexoside executes three hydrogen bond interactions with three
hydroxyl groups7-O-hexoside
Eriodictyol of the saccharide
executesframework and amino
three hydrogen bond acids Ser282,with
interactions Gly281,
three and
hy-
Arg268. Through its aromatic rings (chromone and phenyl scaffolds),
droxyl groups of the saccharide framework and amino acids Ser282, Gly281, and Arg268. this derivative also
performsits
Through two π-π interactions
aromatic with His263
rings (chromone and Phe264,
and phenyl as well
scaffolds), as a π-cation
this derivative alsointeraction
performs
between
two the benzenewith
π-π interactions ringHis263
of theandchromone
Phe264, asand Arg268
well (Figure interaction
as a π-cation 6B). Intermolecular
between
the benzene between
interactions ring of the chromone and Arg268 (Figurevitexin
2”-O-(3’’’,4’’’-dimethoxybenzoyl) 6B). Intermolecular interactions
and the catalytic residues
between 200 -O-(3
of tyrosinase 000 ,4000 -dimethoxybenzoyl) vitexin and the catalytic residues of tyrosinase
are predominantly hydrogen bond interactions, as well as one π-π
are predominantly
interaction betweenhydrogen bond interactions,function
the 3,4-dimethoxybenzoyl as well (which
as one π-π interaction
is attached between
to one of the
the 3,4-dimethoxybenzoyl function (which is attached to one of the
oxygens of the sugar moiety) and Phe 264 residue. The amino acids involved in the oxygens of the sugar
moiety)
hydrogen andbondPhe 264 residue. The
interactions amino acids
mentioned aboveinvolved in theAsn260,
are Glu256, hydrogen bond interactions
Arg268, and Val283
mentioned
(Figure 6C).above are Glu256, Asn260, Arg268, and Val283 (Figure 6C).
The
The quinic
quinic acid-related
acid-related derivatives,
derivatives, 5-O-p-coumaroyl
5-O-p-coumaroyl quinic
quinic acid,
acid, 3-O-caffeoylquinic
3-O-caffeoylquinic
acid,
acid, and
and hydroxy
hydroxy caffeoylquinic
caffeoylquinic acid,
acid, are
are accommodated
accommodated in in the
the tyrosinase
tyrosinase catalytic
catalytic site
site
in
in a similar manner. Moreover, since tyrosinase bears two copper cations leading to aa
a similar manner. Moreover, since tyrosinase bears two copper cations leading to
binuclear copper binding site, these metal atoms could play a key role in the stabilization
of the different tested inhibitors. In the case of the quinic acid-related derivatives, since all
of them possess deprotonate carboxylate groups in their structures, this functional
chemical group interacts with copper cations in the catalytic site through salt bridge
interactions (Figure 6D–F). In addition to the salt bridge interactions of the three
 Foods 2023, 12, 2580 16 of 19

binuclear copper binding site, these metal atoms could play a key role in the stabilization
of the different tested inhibitors. In the case of the quinic acid-related derivatives, since
all of them possess deprotonate carboxylate groups in their structures, this functional
chemical group interacts with copper cations in the catalytic site through salt bridge
interactions (Figure 6D–F). In addition to the salt bridge interactions of the three derivatives
already described all of them also perform hydrogen bond interactions. 5-O-p-coumaroyl
quinic acid performs hydrogen bond interactions with His244 and Asn260. Similarly, 3-O-
caffeoylquinic acid and hydroxy caffeoylquinic acid perform hydrogen bond interactions
with Asn260. Additionally, another hydrogen bond interaction is formed with Val283
Foods 2023, 12, x FOR PEER REVIEWthrough the oxygen atom of the carbonyl group in the aliphatic chain that separates the 20 of 2
aromatic ring from the sugar scaffolds of both compounds (Figure 6E,F).

3.6. Effect of D. winteri on Vascular Relaxation

First, the ability of D. winteri fruit to induce vascular relaxation in rat aortic rings wa
First, the ability of D. winteri fruit to induce vascular relaxation in rat aortic rings was
evaluated. Experimental research revealed that D. winteri fruit would likely produce a
evaluated. Experimental research revealed that D. winteri fruit would likely produce a
hypotensiveeffect.
hypotensive effect.
TheThe relaxation
relaxation produced
produced byµg/mL
by 100 100 µg/mL was 64was
± 2%,64and
± 2%,
whenandthewhen th
concentration was increased to 1000 µg/mL, the percentage of vascular relaxation was was 115
concentration was increased to 1000 µg/mL, the percentage of vascular relaxation
± 1%.
115 ± When
1%. When the endothelium
the endotheliumwas was
denuded of rubbing,
denuded of rubbing, vascular relaxation
vascular was
relaxation negligible
was
The half-maximal
negligible. effective
The half-maximal concentration
effective (EC(EC
concentration 50) 50
for
) forcanelo fruitwas
canelo fruit wassignificantly
significantly (p <
(p < 0.001)
0.001) different
different ± 1 µg/mL
89 ±891 µg/mL in intact
in intact aortaaorta versus
versus 490 ±490 ± 1 µg/mL
1 µg/mL in denuded
in denuded endothelium
endothelium (Rubbed)
(Rubbed) (Figure 7). (Figure 7).

Figure7.7.Original
Figure Original record
recordof vascular
of vascularrelaxation
relaxation D. winteri
usingusing extract extract
D. winteri in intactinratintact
aorticrat
rings
aortic ring
(Endo) (A) or denuded endothelium (Rubbed) (B). Concentration-response
(Endo) (A) or denuded endothelium (Rubbed) (B). Concentration-response curve for D. curve for D. winteri fruit
winteri frui
ethanolic
ethanolicextract
extractin intact aorticaortic
in intact rings rings
(endo)(endo)
or denuded endothelium
or denuded (rubbed) (C).
endothelium The rat aorta
(rubbed) was rat aort
(C). The
pre-contracted with 10 −6 M PE for 10 min, then every 7 min, rising concentrations of ethanolic extract
was pre-contracted with 10−6 M PE for 10 min, then every 7 min, rising concentrations of ethanoli
(0.1 to 1000
extract µg/mL)
(0.1 to 1000 were added were
µg/mL) to the added
bath. Data
to are
thethe mean
bath. ± SEM
Data are ofthe
three to five
mean independent
± SEM of three to fiv
experiments. * p < 0.05; *** p < 0.001 vs. endo.
independent experiments. * p < 0.05; *** p < 0.001 vs. endo.
After it was established that D. winteri fruit can cause a potential hypotensor effect,
After
further it was
research wasestablished
conducted that D. winteri
to determine fruit can
whether thiscause
effect a
waspotential
linked to hypotensor
vascular effect
further research
contraction. was conducted
Phenylephrine to determine
or potassium chloride were whether this effect
used to cause aortic was linked to vascula
ring contraction.
contraction. Phenylephrine or potassium chloride were used to cause aortic ring
contraction. The maximal vascular contractive response to KCl (60 mM) was decreased by
canelo incubation: 158 ± 8% control vs. 84 ± 5% with 100 µg/mL of D. winteri ethanoli
extract; p < 0.001. The half-maximal inhibitory concentration (IC50) did not differ between
Foods 2023, 12, 2580 17 of 19

The maximal vascular contractive response to KCl (60 mM) was decreased by canelo
incubation: 158 ± 8% control vs. 84 ± 5% with 100 µg/mL of D. winteri ethanolic extract;
p < 0.001. The half-maximal inhibitory concentration (IC50 ) did not differ between the
control and experimental samples (Figure 7). The maximal vascular contractile response to
phenylephrine (10−5 M) was reduced after incubation with D. winteri: 171 ± 12% control vs.
42 ± 7% with 100 µg/mL of D. winteri ethanolic extract; p < 0.001. In a similar way, the IC50
to phenylephrine was significantly different (p < 0.05) between control and experimental
Foods 2023, 12, x FOR PEER REVIEW
samples: 176 ± 1 µg/mL control vs. 57 ± 1 µg/mL with 100 µg/mL of canelo fruit21extract of 23

(Figure 8).

Figure 8.
Figure D.winteri
8. D. winterifruit
fruit decreases
decreases the
the vascular
vascular contractile
contractile response
response in
in intact
intact aorta.
aorta. Tissue
Tissue was
was
contractedwith
contracted withaccumulative
accumulative concentrations
concentrations of of
KClKCl (10–60
(10–60 mM)mM) or phenylephrine
or phenylephrine −10 to−−5
(from(from 10
to −M]).
[log 5 [logAortic rings were
M]). Aortic ringspre-incubated with D.
were pre-incubated winteri
with extractextract
D. winteri (100 µg/mL) for 20 for
(100 µg/mL) min,
20then
min,
accumulative concentrations
then accumulative of KCl of
concentrations (A)KCl
or phenylephrine (B) were(B)
(A) or phenylephrine added.
wereData are the
added. average
Data ±
are the
SEM of four to five independent experiments. *** p < 0.001 vs. control.
average ± SEM of four to five independent experiments. ** p < 0.01, *** p < 0.001 vs. control.

4.4.Conclusions
Conclusions
Inthis
In this study, the
the metal
metalcontent
contentandandproximal
proximalcomposition
composition analysis
analysis D. D.
of of winteri fruit
winteri are
fruit
reported
are for the
reported for first time.time.
the first The chemical fingerprints
The chemical of canelo
fingerprints fruits, fruits,
of canelo determined by HPLC-
determined by
MS, are reported.
HPLC-MS, The phenolic
are reported. The fingerprinting yielded 67 compounds,
phenolic fingerprinting yielded 67 mainly
compounds,phenolic acids
mainly
and different types of flavonoids. Interestingly, the antiparasitic compound
phenolic acids and different types of flavonoids. Interestingly, the antiparasitic compound artemisinin
was also detected.
artemisinin was alsoThis fruit showed
detected. interesting
This fruit contents ofcontents
showed interesting phenolics of and flavonoids,
phenolics and
which is consistent with the number of this class of compounds
flavonoids, which is consistent with the number of this class of compounds detected detected by HPLC-MS. by
A potent antioxidant
HPLC-MS. activity wasactivity
A potent antioxidant found forwasD.found
winteriforfruits, which fruits,
D. winteri was evaluated
which was by
employingbyDPPH
evaluated and ABTS
employing DPPH methods
and ABTScombined
methods with ORAC and
combined withFRAP.
ORAC Moreover,
and FRAP. the
capacity of D. winteri fruit extract to induce vascular relaxation in rat aortic
Moreover, the capacity of D. winteri fruit extract to induce vascular relaxation in rat aortic rings was
evaluated,
rings and the results
was evaluated, and the showed
resultsthat this food
showed ingredient
that this could produce
food ingredient could aproduce
potentiala
hypotensive effect because it decreased the vascular response in intact aortic
potential hypotensive effect because it decreased the vascular response in intact aortic rings rings in an
endothelium
in an endotheliumdependent manner.
dependent The capacity
manner. of canelo
The capacity fruits to
of canelo inhibit
fruits enzymes
to inhibit related
enzymes
to non-communicable diseases, such as Parkinson’s, and Alzheimer’s disease, is reported
related to non-communicable diseases, such as Parkinson’s, and Alzheimer’s disease, is
for the first time. It was observed that D. winteri fruit extract inhibits acetylcholinesterase,
reported for the first time. It was observed that D. winteri fruit extract inhibits
butyrylcholinesterase, and tyrosinase enzymes. This study highlights the benefits of these
acetylcholinesterase, butyrylcholinesterase, and tyrosinase enzymes. This study highlights
native species used as pepper prepared with local Mapuche fruits with nutritional and
the benefits of these native species used as pepper prepared with local Mapuche fruits with
health-promoting properties and could boost their consumption.
nutritional and health-promoting properties and could boost their consumption.
Supplementary Materials: The following supporting information can be downloaded at: https://www.
Supplementary Materials: The following supporting information can be downloaded at:
mdpi.com/article/10.3390/foods12132580/s1, Figure S1 (a–h). UHPLC Q Orbitral full MS spectra
www.mdpi.com/xxx/s1, Figure S1 (a-h). UHPLC Q Orbitral full MS spectra and structures of
and structures of representative compounds, Peaks 33, 34, 36, 37, 38, 42, 49 and 50; Figure S2. ACHe
representative compounds, Peaks 33, 34, 36, 37, 38, 42, 49 and 50; Figure S2. ACHe inhibition graph
inhibition
by standardgraph by standard
Galantamine (IC50Galantamine (IC50
0.266 mg/mL); 0.266S3.
Figure mg/mL); Figurefor
Trolox curve S3.ORAC;
TroloxFigure
curve for
S4. ORAC;
Gallic
Figure S4. Gallic acid curve for total
acid curve for total phenolic content. phenolic content.

Author Contributions: Conceptualization, M.J.S., G.V.-A., and F.C., methodology, A.P., R.E.B.,
N.J.R.-J., and G.V.-A.; software, R.E.B. and J.R.-P.; validation, J.R.-P., and J.P.; formal analysis, A.P.
and F.C.; investigation, R.E.B.; resources G.V.-A.; data curation, J.R.-P. and N.J.R.-J., writing—
original draft preparation, M.J.S.; J.P., and R.E.B. and J.P.; writing—review and editing, F.C., J.P.,
Foods 2023, 12, 2580 18 of 19

Author Contributions: Conceptualization, M.J.S., G.V.-A. and F.C.; methodology, A.P., R.E.B., N.J.R.-J.
and G.V.-A.; software, R.E.B. and J.R.-P.; validation, J.R.-P., and J.P.; formal analysis, A.P. and F.C.;
investigation, R.E.B.; resources, G.V.-A.; data curation, J.R.-P. and N.J.R.-J.; writing—original draft
preparation, M.J.S., J.P., R.E.B. and J.P.; writing—review and editing, F.C., J.P. and M.J.S.; supervision,
M.J.S.; project administration, M.J.S. and G.V.-A.; funding acquisition, M.J.S. and G.V.-A. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was supported by FONDECYT 1220075 and FONDECYT 1200610 to M.J.S and
J.P., respectively; R.B. received funds from ANID PFCHA/Beca Doctorado Nacional/2019-21191978.
Data Availability Statement: Raw HPLC-MS data and other experimental data are available
upon request.
Acknowledgments: Mario J. Simirgiotis acknowledge the CYTED network “P320RT0186—Aprovechamiento
sostenible de recursos biomásicos vegetales iberoamericanos en cosmética (BIOLATES)”. Authors
acknowledge fondequip EQM170172 and EQM140002.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Rodríguez, R.; Marticorena, C.; Alarcón, D.; Baeza, C.; Cavieres, L.A.; Finot, V.; Fuentes, N.; Kiessling, A.; Mihoc, M.; Pauchard,
A.; et al. Catálogo de Las Plantas Vasculares de Chile. Gayana Bot. 2018, 75, 1–430. [CrossRef]
2. Novoa, P.; An-Der Fuhren, F.; Gonzales, C. Drimys winteri J.R. Forst. & G. Forst. var. chilensis (DC.) A. Gray; 2015. Available
online: https://clasificacionespecies.mma.gob.cl/wp-content/uploads/2019/10/Drimys_winteri_FIN_13RCE.pdf (accessed on
27 June 2023).
3. Houghton, P.; Manby, J. Medicinal Plants of the Mapuche. J. Ethnopharmacol. 1985, 13, 89–103. [CrossRef] [PubMed]
4. Muñoz-Concha, D.; Vogel, H.; Razmilic, I. Variation of Chemical Compounds in Leaves of Drimys spp. (Magnoliophyta:
Winteraceae) Populations in Chile. Rev. Chil. Hist. Nat. 2004, 77, 43–50. [CrossRef]
5. Muñoz, O.; Tapia-Merino, J.; Nevermann, W.; San-Martín, A. Phytochemistry and Biological Properties of Drimys winteri JR et G.
Forster Var Chilensis (DC) A. Bol. Latinoam. Caribe Plantas Med. Aromat. 2021, 20, 443–462. [CrossRef]
6. Barrientos, R.E.; Ahmed, S.; Cortés, C.; Fernández-Galleguillos, C.; Romero-Parra, J.; Simirgiotis, M.J.; Echeverría, J. Chemical
Fingerprinting and Biological Evaluation of the Endemic Chilean Fruit Greigia sphacelata (Ruiz and Pav.) Regel (Bromeliaceae) by
UHPLC-PDA-Orbitrap-Mass Spectrometry. Molecules 2020, 25, 3750. [CrossRef]
7. Cuesta, L.; Palacios, J.; Barrientos, R.E.; Gómez, J.; Castagnini, J.M.; Barba, F.J.; Tapia, A.; Paredes, A.; Cifuentes, F.; Simirgiotis,
M.J. UHPLC-MS Phenolic Fingerprinting, Aorta Endothelium Relaxation Effect, Antioxidant, and Enzyme Inhibition Activities of
Azara Dentata Ruiz & Pav Berries. Foods 2023, 12, 643.
8. Official Methods of Analysis of AOAC International, 18th ed.; Revision 3; AOAC International: Rockville, MD, USA, 2006.
9. Vargas-Arana, G.; Merino-Zegarra, C.; del-Castillo, Á.M.R.; Quispe, C.; Viveros-Valdez, E.; Simirgiotis, M.J. Antioxidant,
Antiproliferative and Anti-Enzymatic Capacities, Nutritional Analysis and UHPLC-PDA-MS Characterization of Ungurahui Palm
Fruits (Oenocarpus bataua Mart) from the Peruvian Amazon. Antioxidants 2022, 11, 1598. [CrossRef]
10. Petersson, G.A.; Bennett, A.; Tensfeldt, T.G.; Al-Laham, M.A.; Shirley, W.A.; Mantzaris, J. A Complete Basis Set Model Chemistry. I.
The Total Energies of Closed-Shell Atoms and Hydrides of the First-Row Elements. J. Chem. Phys. 1988, 89, 2193–2218. [CrossRef]
11. Tapia, A.; Rodriguez, J.; Theoduloz, C.; Lopez, S.; Egly, G.; Schmeda-Hirschmann, G.; Feresin, G.E.; Schmeda-Hirschmann, G.
Free Radical Scavengers and Antioxidants from Baccharis grisebachii. J. Ethnopharmacol. 2004, 95, 155–161. [CrossRef]
12. Re, R.; Pellegrinia, N.; Proteggente, A.; Pannalaa, A.; Yang, M.; Rice-Evans, C. Antioxidant Activity Applying an Improved ABTS
Radical Cation Decolorization Assay. Free Radic. Biol. Med. 1999, 26, 1231–1237. [CrossRef]
13. Benzie, I.F.F.; Strain, J.J. The Ferric Reducing Ability of Plasma (FRAP) as a Measure of “Antioxidant Power”: The FRAP Assay.
Anal. Biochem. 1996, 239, 70–76. [CrossRef] [PubMed]
14. Huang, D.; Ou, B.; Hampsch-Woodill, M.; Flanagan, J.A.; Prior, R.L. High-Throughput Assay of Oxygen Radical Absorbance
Capacity (ORAC) Using a Multichannel Liquid Handling System Coupled with a Microplate Fluorescence Reader in 96-Well
Format. J. Agric. Food Chem. 2002, 50, 4437–4444. [CrossRef] [PubMed]
15. Adamo, C.; Barone, V. Toward Reliable Density Functional Methods without Adjustable Parameters: The PBE0 Model Seeking for
Parameter-Free Double-Hybrid Functionals: The PBE0-DH Model Accurate Excitation Energies from Time-Dependent Density
Functional Theory: Assessing the PBE0. Cit. J. Chem. Phys. 1999, 110, 2889. [CrossRef]
16. Frisch, A. Gaussian 09W Reference 2009; Gaussian Inc.: Wallingford, CT, USA, 2009.
17. Release, S. Maestro, Version 11.8. Schrodinger, LLC, New York; Scientific Research Publishing: Wuhan, China, 2018.
18. Greenblatt, H.M.; Kryger, G.; Lewis, T.; Silman, I.; Sussman, J.L. Structure of Acetylcholinesterase Complexed with (-)-
Galanthamine at 2.3 Å Resolution. FEBS Lett. 1999, 463, 321–326. [CrossRef]
19. Nachon, F.; Carletti, E.; Ronco, C.; Trovaslet, M.; Nicolet, Y.; Jean, L.; Renard, P.Y. Crystal Structures of Human Cholinesterases
in Complex with Huprine W and Tacrine: Elements of Specificity for Anti-Alzheimer’s Drugs Targeting Acetyl- and Butyryl-
Cholinesterase. Biochem. J. 2013, 453, 393–399. [CrossRef]
Foods 2023, 12, 2580 19 of 19

20. Ismaya, W.T.; Rozeboom, H.J.; Weijn, A.; Mes, J.J.; Fusetti, F.; Wichers, H.J.; Dijkstra, B.W. Crystal Structure of Agaricus bisporus
Mushroom Tyrosinase: Identity of the Tetramer Subunits and Interaction with Ropolone. Biochemistry 2011, 50, 5477–5486.
[CrossRef]
21. Berman, H.M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T.N.; Weissig, H.; Shindyalov, I.N.; Bourne, P.E. The Protein Data Bank.
Nucleic Acids Res. 2000, 28, 235–242. [CrossRef]
22. Sussman, J.L.; Harel, M.; Frolow, F.; Oefner, C.; Goldman, A.; Toker, L.; Silman, I. Atomic Structure of Acetylcholinesterase from
Torpedo Californica: A Prototypic Acetylcholine-Binding Protein. Science 1991, 253, 872–879. [CrossRef]
23. Silman, I.; Harel, M.; Axelsen, P.; Raves, M.; Sussman, J. Three-Dimensional Structures of Acetylcholinesterase and of Its
Complexes with Anticholinesterase Agents. In Proceedings of the Structure, Mechanism and Inhibition of Neuroactive Enzymes;
Portland Press Limited.: London, UK, 1994; Volume 22, pp. 745–749.
24. Nicolet, Y.; Lockridge, O.; Masson, P.; Fontecilla-Camps, J.C.; Nachon, F. Crystal Structure of Human Butyrylcholinesterase and of
Its Complexes with Substrate and Products. J. Biol. Chem. 2003, 278, 41141–41147. [CrossRef]
25. Tallini, L.R.; Bastida, J.; Cortes, N.; Osorio, E.H.; Theoduloz, C.; Schmeda-Hirschmann, G. Cholinesterase Inhibition Activity,
Alkaloid Profiling and Molecular Docking of Chilean Rhodophiala (Amaryllidaceae). Molecules 2018, 23, 1532. [CrossRef]
26. Da Silva, A.P.; Silva, N.d.F.; Andrade, E.H.A.; Gratieri, T.; Setzer, W.N.; Maia, J.G.S.; da Silva, J.K.R. Tyrosinase Inhibitory Activity,
Molecular Docking Studies and Antioxidant Potential of Chemotypes of Lippia origanoides (Verbenaceae) Essential Oils. PLoS
ONE 2017, 12, e0175598. [CrossRef] [PubMed]
27. Chen, J.; Ye, Y.; Ran, M.; Li, Q.; Ruan, Z.; Jin, N. Inhibition of Tyrosinase by Mercury Chloride: Spectroscopic and Docking Studies.
Front. Pharmacol. 2020, 11, 81. [CrossRef] [PubMed]
28. Sherman, W.; Day, T.; Jacobson, M.P.; Friesner, R.A.; Farid, R. Novel Procedure for Modeling Ligand/Receptor Induced Fit Effects.
J. Med. Chem. 2006, 49, 534–553. [CrossRef] [PubMed]
29. Friesner, R.A.; Murphy, R.B.; Repasky, M.P.; Frye, L.L.; Greenwood, J.R.; Halgren, T.A.; Sanschagrin, P.C.; Mainz, D.T. Extra
Precision Glide: Docking and Scoring Incorporating a Model of Hydrophobic Enclosure for Protein-Ligand Complexes. J. Med.
Chem. 2006, 49, 6177–6196. [CrossRef]
30. DeLano, W.L. The PyMOL Molecular Graphics System. Available online: http://www.pymol.org (accessed on 27 June 2023).
31. Özcan, M.M.; Akbulut, M. Estimation of Minerals, Nitrate and Nitrite Contents of Medicinal and Aromatic Plants Used as Spices,
Condiments and Herbal Tea. Food Chem. 2008, 106, 852–858. [CrossRef]
32. Lee, J.G.; Chae, Y.; Shin, Y.; Kim, Y.J. Chemical Composition and Antioxidant Capacity of Black Pepper Pericarp. Appl. Biol. Chem.
2020, 63, 35. [CrossRef]
33. Alcázar Magaña, A.; Kamimura, N.; Soumyanath, A.; Stevens, J.F.; Maier, C.S. Caffeoylquinic Acids: Chemistry, Biosynthesis,
Occurrence, Analytical Challenges, and Bioactivity. Plant J. 2021, 107, 1299–1319. [CrossRef]
34. Courts, F.L.; Williamson, G. The Occurrence, Fate and Biological Activities of c-Glycosyl Flavonoids in the Human Diet. Crit. Rev.
Food Sci. Nutr. 2015, 55, 1352–1367. [CrossRef]
35. Roy, A.; Khan, A.; Ahmad, I.; Alghamdi, S.; Rajab, B.S.; Babalghith, A.O.; Alshahrani, M.Y.; Islam, S.; Islam, M.R. Flavonoids a
Bioactive Compound from Medicinal Plants and Its Therapeutic Applications. Biomed Res. Int. 2022, 2022, 5445291. [CrossRef]
36. Krishna, S.; Bustamante, L.; Haynes, R.K.; Staines, H.M. Artemisinins: Their Growing Importance in Medicine. Trends Pharmacol.
Sci. 2008, 29, 520–527. [CrossRef]
37. Claudino, V.D.; da Silva, K.C.; Filho, V.C.; Yunes, R.A.; Monache, F.D.; Giménez, A.; Salamanca, E.; Gutierrez-Yapu, D.; Malheiros,
A. Drimanes from Drimys brasiliensis with Leishmanicidal and Antimalarial Activity. Mem. Inst. Oswaldo Cruz 2013, 108, 140–144.
[CrossRef] [PubMed]
38. Hung, T.M.; Na, M.K.; Thuong, P.T.; Su, N.D.; Sok, D.E.; Song, K.S.; Seong, Y.H.; Bae, K.H. Antioxidant Activity of Caffeoyl Quinic
Acid Derivatives from the Roots of Dipsacus asper Wall. J. Ethnopharmacol. 2006, 108, 188–192. [CrossRef] [PubMed]
39. Barrientos, R.E.; Ibáñez, E.; Puerta, A.; Padrón, J.M.; Paredes, A.; Cifuentes, F.; Romero-Parra, J.; Palacios, J.; Bórquez, J.;
Simirgiotis, M.J. Phenolic Fingerprinting and Bioactivity Profiling of Extracts and Isolated Compounds from Gypothamnium
pinifolium Phil. Antioxidants 2022, 11, 2313. [CrossRef] [PubMed]
40. Barrientos, R.; Fernández-Galleguillos, C.; Pastene, E.; Simirgiotis, M.; Romero-Parra, J.; Ahmed, S.; Echeverría, J. Metabolomic
Analysis, Fast Isolation of Phenolic Compounds, and Evaluation of Biological Activities of the Bark From Weinmannia trichosperma
Cav. (Cunoniaceae). Front. Pharmacol. 2020, 11, 780. [CrossRef]
41. Trendafilova, A.; Ivanova, V.; Rangelov, M.; Todorova, M.; Ozek, G.; Yur, S.; Ozek, T.; Aneva, I.; Veleva, R.; Moskova-Doumanova,
V.; et al. Caffeoylquinic Acids, Cytotoxic, Antioxidant, Acetylcholinesterase and Tyrosinase Enzyme Inhibitory Activities of Six
Inula Species from Bulgaria. Chem. Biodivers. 2020, 17, e2000051. [CrossRef]

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.