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Caucasian Blueberry: Comparative Study of Phenolic Compounds and


Neuroprotective and Antioxidant Potential of Vaccinium myrtillus and
Vaccinium arctostaphylos Leaves

Article in Life · December 2022


DOI: 10.3390/life12122079

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life

Communication
Caucasian Blueberry: Comparative Study of Phenolic
Compounds and Neuroprotective and Antioxidant Potential of
Vaccinium myrtillus and Vaccinium arctostaphylos Leaves
Arnold A. Shamilov 1 , Daniil N. Olennikov 2, * , Dmitryi I. Pozdnyakov 3 , Valentina N. Bubenchikova 4 ,
Ekaterina R. Garsiya 1 and Mikhail V. Larskii 5

1 Department of Pharmacognosy, Botany and Technology of Phytopreparations, Pyatigorsk


Medical-Pharmaceutical Institute, Branch of Volgograd State Medical University, Ministry of Health of
Russian Federation, 11 Kalinina Avenue, 357500 Pyatigorsk, Russia
2 Laboratory of Biomedical Research, Institute of General and Experimental Biology, Siberian Division,
Russian Academy of Science, 6 Sakhyanovoy Street, 670047 Ulan-Ude, Russia
3 Department of Pharmacology and Clinical Pharmacology, Pyatigorsk Medical-Pharmaceutical Institute,
Branch of Volgograd State Medical University, Ministry of Health of Russian Federation, 11 Kalinina Avenue,
357500 Pyatigorsk, Russia
4 Department of Pharmacognosy and Botany, Kursk State Medical University, Ministry of Health of Russian
Federation, 3 Karl Marks Street, 305000 Kursk, Russia
5 Department of Pharmaceutical Chemistry, Pyatigorsk Medical-Pharmaceutical Institute, Branch of Volgograd
State Medical University, Ministry of Health of Russian Federation, 11 Kalinina Avenue,
357500 Pyatigorsk, Russia
* Correspondence: olennikovdn@mail.ru; Tel.: +7-902-160-06-27

Citation: Shamilov, A.A.; Olennikov, Abstract: (1) Background: Two Caucasian blueberries Vaccinium myrtillus L. and Vaccinium arc-
D.N.; Pozdnyakov, D.I.; tostaphylos L. are famous berry bushes growing in the Caucasus region and used to treat neurological
Bubenchikova, V.N.; Garsiya, E.R.; diseases, but the chemistry and bioactivity of leaf extracts are still poorly studied. (2) Methods:
Larskii, M.V. Caucasian Blueberry: Phenolic compounds of V. myrtillus and V. arctostaphylos leaf extracts were profiled and quantified
Comparative Study of Phenolic by HPLC–PDA–ESI–tQ–MS. The neurotropic potential of Vaccinium extracts was studied using the
Compounds and Neuroprotective
model of middle cerebral artery permanent occlusion to determine cerebral blood flow, the area of
and Antioxidant Potential of
the brain tissue necrosis, and antioxidant enzyme activity (including superoxide dismutase, succinate
Vaccinium myrtillus and Vaccinium
dehydrogenase, and cytochrome C oxidase), as well as the concentration of TBARS. (3) Results:
arctostaphylos Leaves. Life 2022, 12,
2079. https://doi.org/10.3390/
Hydroxycinnamates and flavonoids were identified in the leaves of V. myrtillus and V. arctostaphylos,
life12122079 and the dominant metabolite of both extracts was 5-O-caffeoylquinic acid in the amount of 105–
226 mg/g. The studied extracts enhanced the cerebral hemodynamics and decreased the frequency
Academic Editor: Jianfeng Xu
of necrotic and lipooxidative processes in the brain tissue, accompanied by an increase in the activ-
Received: 12 November 2022 ity of antioxidant enzymes. The positive effect of V. arctostaphylos was stronger and exceeded the
Accepted: 8 December 2022 effectiveness of Ginkgo biloba standardized extract. (4) Conclusion: The leaf extracts of Caucasian
Published: 11 December 2022 blueberries V. myrtillus and V. arctostaphylos as a new source of hydroxycinnamates demonstrated
Publisher’s Note: MDPI stays neutral
a protective effect of the brain ischemia pathology and can be used as therapeutic agents to treat
with regard to jurisdictional claims in neurological diseases.
published maps and institutional affil-
iations. Keywords: blueberry; Vaccinium myrtillus; Vaccinium arctostaphylos; phenolic compounds; antioxidant
activity; neuroprotective activity

Copyright: © 2022 by the authors.


Licensee MDPI, Basel, Switzerland. 1. Introduction
This article is an open access article
Currently, the Vaccinium L. genus includes over 170 species [1], and there are 14 native
distributed under the terms and
species in the Russian Federation, including European blueberry (V. myrtillus L.), which
conditions of the Creative Commons
Attribution (CC BY) license (https://
grows throughout Europe outside of Central Asia, and Caucasian blueberry (V. arctostaphy-
creativecommons.org/licenses/by/
los L.), which is an endemic plant of Transcaucasia and North Caucasus [2,3]. The fruits
4.0/).

Life 2022, 12, 2079. https://doi.org/10.3390/life12122079 https://www.mdpi.com/journal/life


Life 2022, 12, x FOR PEER REVIEW 2 of 16
Life 2022, 12, 2079 2 of 15

arctostaphylos L.), which is an endemic plant of Transcaucasia and North Caucasus [2,3].
of V.fruits
The myrtillus
of V.are included
myrtillus areinincluded
pharmacopoeias of differentof
in pharmacopoeias countries
differentascountries
an astringent,
as an hy-
as-
polipidemic, and vision-improving remedy [4]. It is also known that an aqueous
tringent, hypolipidemic, and vision-improving remedy [4]. It is also known that an aque- decoction
of the
ous fruit hasofantiprotozoal,
decoction antitumor, and
the fruit has antiprotozoal, cytotoxicand
antitumor, activity [5–7].activity
cytotoxic Literature data
[5–7]. in-
Liter-
dicate that V. myrtillus is a rich source of phenolic acids [8,9], flavonoids, and
ature data indicate that V. myrtillus is a rich source of phenolic acids [8,9], flavonoids, and phenolic
glycosides,
phenolic as well as as
glycosides, triterpenoids, carotenoids,
well as triterpenoids, organic acids,
carotenoids, carbohydrates,
organic and higher
acids, carbohydrates,
fatty acids [10,11].
and higher fatty acids [10,11].
In contrast
In contrast to
to the
the European blueberry, V.
European blueberry, V. arctostaphylos
arctostaphylos is is not
not aa pharmacopoeial
pharmacopoeial plant
plant
due to insufficient knowledge of its chemical composition and pharmacological
due to insufficient knowledge of its chemical composition and pharmacological activity. activity.
Botanically, the Caucasian blueberry is a shrub or small tree up to 2–3 m high with rounded
Botanically, the Caucasian blueberry is a shrub or small tree up to 2–3 m high with
branches and large oblong leaves. Flowers in the raceme and the corolla are large whitish
rounded branches and large oblong leaves. Flowers in the raceme and the corolla are large
red. Fruits are large, globular, and black with a smooth surface. The leaves of V. arctostaphy-
whitish red. Fruits are large, globular, and black with a smooth surface. The leaves of V.
los are up to 10 cm length compared to the leaves of V. myrtillus, which are up to 3 cm
arctostaphylos are up to 10 cm length compared to the leaves of V. myrtillus, which are up
(Figure 1). To date, the chemical composition of fruits was only slightly studied [12,13],
to 3 cm (Figure 1). To date, the chemical composition of fruits was only slightly studied
and there is a need for new studies on this promising source of bioactive metabolites.
[12,13], and there is a need for new studies on this promising source of bioactive metabo-
Additionally, some phenolics have been identified in the leaves [14], and hypoglycemic
lites. Additionally, some phenolics have been identified in the leaves [14], and hypogly-
and hypotensive activities have been demonstrated for the leaf extracts [15,16].
cemic and hypotensive activities have been demonstrated for the leaf extracts [15,16].

(a) (b) (c) (d)


Figure
Figure 1.1. Vaccinium
Vaccinium spp.
spp. in
in their
their natural
natural habitat:
habitat: Vaccinium
Vaccinium arctostaphylos
arctostaphylos L.
L. (Caucasian
(Caucasian blueberry,
blueberry,
bank of Belaya river, Khamyshki village, Maykop District, Republic of Adygea) (a); Vaccinium myr-
bank of Belaya river, Khamyshki village, Maykop District, Republic of Adygea) (a); Vaccinium myrtillus
tillus L. (bank of the Cherek River, Kabardino–Balkarian Republic) (b). Dried leaves of V. arctostaph-
L. (bank of the Cherek River, Kabardino–Balkarian Republic) (b). Dried leaves of V. arctostaphylos (c)
ylos (c) and V. myrtillus (d).
and V. myrtillus (d).
Phenolics
Phenolics havehave aa significant
significant effect on the
effect on the human
human organism
organism and and are
are primarily
primarily used
used asas
effective remedies for preventing a wide range of diseases from atherosclerosis
effective remedies for preventing a wide range of diseases from atherosclerosis to ischemic to is-
chemic stroke. Phenolic
stroke. Phenolic compoundscompounds
and totaland total phenolics-based
phenolics-based phytocompositions
phytocompositions are in-
are increasingly
creasingly used in modern neurology practice, for example, for the
used in modern neurology practice, for example, for the treatment and prevention of treatment and preven-
tion of cerebral
cerebral ischemia ischemia and neurodegenerative
and neurodegenerative diseases diseases
[17,18].[17,18].
Previously,Previously, it has
it has been been
shown
shown that the addition of polyphenols to the diet contributes to
that the addition of polyphenols to the diet contributes to the development of a numberthe development ofofa
number of therapeutic benefits in patients with Parkinson’s disease, apparently
therapeutic benefits in patients with Parkinson’s disease, apparently by reducing oxidative by reduc-
ing oxidative
damage of thedamage of nigra
substantia the substantia
neurons nigra neurons
[19]. In [19].
addition, a In addition,
number a number
of studies show of that
studies
the
show that the administration of polyphenolic substances significantly
administration of polyphenolic substances significantly reduces damage to neurons during reduces damage to
neurons
ischemiaduring ischemia [20].
[20]. Moreover, Moreover,substances
polyphenolic polyphenolic aresubstances are oftenasconsidered
often considered compounds as
compounds
that eliminate that
theeliminate the hyperproduction
hyperproduction of ROS and
of ROS and oxidative oxidative
stress [21,22],stress [21,22],
and their and
neuro-
their neuroprotective
protective effects haveeffects have beenconfirmed
been clinically clinically confirmed [21]. The
[21]. The results of aresults of a prospec-
prospective cohort
tive cohort study by Gao et al. demonstrated a significant reduction
study by Gao et al. demonstrated a significant reduction in the risk of stroke when in the risk of stroke
large
when
amountslarge
of amounts of dietary polyphenols
dietary polyphenols are consumed, are particularly
consumed, particularly
citrus fruits citrus fruits [23].
and grapes and
grapes
Of note,[23].
some Ofgender
note, some genderwere
differences differences
identifiedwere identified
in this in this
work: the work: the pharmaco-
pharmacological efficacy
logical efficacy of
of polyphenols was polyphenols was higher
higher in women than in women
men [23]. than in men [23].
Life 2022, 12, 2079 3 of 15

Disorders of cerebral circulation represent a considerable medical and social problem.


According to the World Health Organization, stroke ranks second among the causes of mor-
tality and first among the causes of primary disability [24]. At the same time, approximately
80% of all stroke cases are due to ischemic brain damage [25]. One of the therapeutic options
for the adjuvant treatment of cerebral ischemia is neuroprotection, in which a certain share
is provided by herbal medicines. For example, some traditional Chinese medicine products
have a pronounced neuroprotective effect, and the polyvalence of their action is important,
affecting many pathogenetic mechanisms of cellular damage in ischemia: from oxidative
stress to metabolic homeostasis and excitotoxicity [26].
The genus Vaccinium L., along with classical plant sources of antioxidants, can serve as
a basis for the search for new biologically active compounds with neuroprotective activity;
for example, the neuroprotective properties of anthocyanins of blueberries have been
established [27]. In leaves, the main phenolic groups are proantocyanidines, flavonols, and
hydroxycinnamic acids with potential activity in therapy of Alzheimer’s and Parkinson’s
disease [28].
As part of the ongoing study of Vaccinium species [29,30], in this study, the phenolic
compounds from V. myrtillus and V. arctostaphylos leaves were profiled and quantified by
HPLC–PDA–ESI–tQ–MS assay, and the antioxidant and neuroprotection potential of total
blueberry leave extracts was estimated in brain ischemia in vivo experiments.

2. Materials and Methods


2.1. Plant Material and Chemicals
Samples of Vaccinium myrtillus L. leaves were collected on the bank of the Cherek
River, Kabardino–Balkarian Republic (22 September 2019, 43◦ 570 63.2400 N, 42◦ 590 23.8100 E,
1905 m a.s.l.; Russia; flowering stage; 5 locations, 10 samples; voucher No PALE 005073-01–
PALE 005073-10). The species were authenticated by Mikhail Goncharov, Ph.D. Biology
(Saint Petersburg State Chemical and Pharmaceutical University, Saint Petersburg, Russia).
Samples of the V. arctostaphylos L. leaves were collected from an alpine meadow in the
Republic of Adygea, Khamyshki village, Maykop District (26 September 2020, 44◦ 020 34100 N,
40◦ 100 03500 E, 698 m a.s.l.; Russia; flowering stage; 5 locations, 10 samples; voucher No
PALE 005082-01–PALE 005082). The species were authenticated by Dmitryi Shilnikov,
Ph.D. Biology (Ecological and Botanical Station “Pyatigorsk”, Botanical Institute, Russian
Academy of Sciences, Pyatigorsk, Russia). Plant material was dried at 40 ◦ C (ventilated heat
oven; 8–10 days) and stored at 3–4 ◦ C before analysis. Selected chemicals were purchased
from Sigma–Aldrich (St. Louis, MO, USA): acetonitrile for HPLC (Cat. No. 34851, ≥99.9%);
formic acid (Cat. No. F0507, ≥95%); 4-O-caffeoylquinic acid (CAS 905-99-7, Sigma-Aldrich
No. 65969, ≥98%); 5-O-caffeoylquinic acid (CAS 906-33-2, Sigma-Aldrich No. 94419, ≥98%);
caffeic acid (CAS 331-39-5, Sigma-Aldrich No. C0625, ≥98%); 4,5-di-O-caffeoylquinic acid
(CAS 14535-61-3, Sigma-Aldrich No. PHL80427, ≥95%); rutin (CAS 207671-50-9, Sigma-
Aldrich No. R5143, ≥94%); hyperoside (CAS 482-36-0, Sigma-Aldrich No. 83388, ≥97%);
isoquercitrin (CAS 482-35-9, Sigma-Aldrich No. 16,654, ≥98%); guaiaverin (CAS 22255-13-6,
Sigma-Aldrich No. 94821, ≥95%); avicularin (CAS 572-30-5, Sigma-Aldrich No. PHL80361,
≥95%); quercitrin (CAS 849061-97-8, Sigma-Aldrich No. 337951, ≥95%); quercetin-3-O-(600 -
O-acetyl)-glucoside (CAS 54542-51-7, No.1099, ≥85%, Extrasynthese, Lyon, France).

2.2. Plant Extract Preparation


Plant extract for HPLC analysis was prepared using 200 mg sample of milled material
(particle size 0.125 µm) treated by 2 mL of 70% ethanol and sonication (ultrasonic bath,
30 min, 50 ◦ C, ultrasound power 100 W, frequency 35 kHz; ×3). The ethanolic extract was
centrifuged at 6000 × g (10 min), filtered through 0.22-µm syringe filters into a measuring
flask (10 mL) and the final volume was filled up to 10 mL. The final extract was stored at
2 ◦ C before analysis. Plant extract for the pharmacological study was prepared using the
same procedure from 150 g-portion of material and drying in a vacuum oven. The total
Life 2022, 12, 2079 4 of 15

yields of V. myrtillus and V. arctostaphylos extracts were 49.3% and 40.1% of plant material
weight, respectively.

2.3. High-Performance Liquid Chromatography with Photodiode Array Detection and Electrospray
Ionization Triple Quadrupole Mass Spectrometric Detection (HPLC–PDA–ESI–QQQ–MS)
High-performance liquid chromatography with photodiode array detection and elec-
trospray ionization triple quadrupole mass spectrometric detection (HPLC–PDA–ESI–QQQ–
MS) with LC-20 Prominence liquid chromatograph coupled with an SPD-M30A photodiode
array detector, LCMS 8050 triple-quadrupole mass spectrometer and GLC Mastro C18 col-
umn (2.1 × 150 mm, 3 µm; all Shimadzu, Columbia, MD, USA) were used for the profiling
and quantification of the plant extracts, as described previously [31]. The similarity of reten-
tion time, ultraviolet and mass spectral data was used for the identification of metabolites
after comparison with the reference standards and literature data. The metrics of calibration
curves as correlation coefficient (r2 ), standard deviation (SYX ), limits of detection (LOD),
limits of quantification (LOQ), and linear range were found for calibration solution (1, 10,
25, 50, 100 µg/mL) of 11 reference standards (4-O-caffeoylquinic acid, 5-O-caffeoylquinic
acid, caffeic acid, 4,5-di-O-caffeoylquinic acid, rutin, hyperoside, isoquercitrin, guaiaverin,
avicularin, quercitrin, quercetin-3-O-(600 -O-acetyl)-glucoside) using the known method [32]
(Table 1).

Table 1. Regression equations, correlation coefficients (r2 ), standard deviation (SYX ), limits of detec-
tion (LOD), limits of quantification (LOQ), and linear ranges for 11 reference standards.

Regression Equation a LOD/LOQ Linear Range


Compound r2 SYX
a b × 106 (µg/mL) (µg/mL)

4-O-Caffeoylquinic acid 0.162 −0.011 0.9973 1.83 × 10−2 0.37/1.12 2–100


5-O-Caffeoylquinic acid 0.150 −0.010 0.9982 1.67 × 10−2 0.36/1.11 2–100
Caffeic acid 0.189 −0.017 0.9988 1.26 × 10−2 0.22/0.67 1–100
Quercetin 3-O-rutinoside (rutin) 0.085 −0.062 0.9873 3.89 × 10−2 1.51/4.57 5–100
Quercetin 3-O-galactoside (hyperoside) 0.090 −0.052 0.9891 3.52 × 10−2 1.29/3.91 4–100
Quercetin 3-O-glucoside (isoquercitrin) 0.098 −0.053 0.9889 3.37 × 10−2 1.14/3.43 4–100
Quercetin 3-O-arabinopyranoside (guaiaverin) 0.121 −0.031 0.9953 2.59 × 10−2 0.70/2.14 3–100
Quercetin 3-O-arabinofuranoside (avicularin) 0.114 −0.026 0.9927 2.80 × 10−2 0.81/2.46 3–100
Quercetin 3-O-rhamnoside (quercitrin) 0.109 −0.043 0.9912 3.16 × 10−2 0.96/2.90 3–100
Quercetin 3-O-(600 -acetyl)-glucoside 0.095 −0.050 0.9880 3.28 × 10−2 1.14/3.45 4–100
4,5-Di-O-caffeoylquinic acid 0.163 −0.016 0.9975 1.93 × 10−2 0.39/1.18 2–100
a Regression equation: y = a·x + b.

2.4. Neuroprotective Activity


2.4.1. Animals
The study was used 100 male Wistar rats, 180–200 g weight. The animals were
purchased from the laboratory animal nursery “Rappolovo” (Russia, Leningrad region)
and during the experiment were kept in polypropylene boxes by 5 rats in controlled
conditions of detention: air temperature 20 ± 20 ◦ C, humidity—55–65%, daily cycle 12 h
a day/12 h a night. The treatment of laboratory animals and their maintenance met the
requirements of Directive 2010/63/EU of the European Parliament and of the Council of
the European Union of 22 September 2010 for the protection of animals used for scientific
purposes. The local Ethics committee (Protocol of meeting No. 10 of 20 August 2021)
approved the research concept.

2.4.2. Brain Ischemia Model


Permanent focal cerebral ischemia was reproduced by right sided thermocoagulation
of the middle cerebral artery (MCA). In this research, the concept irreversible technique was
used. Rats were anesthetized by chloral hydrate (350 mg/kg, intraperitoneal). After, the
area from the right side of the eye was depilated, skin was cut, and soft tissues were pushed
apart, exposing the process of the zygomatic bone, which was deleted. Next, a trepanation
Life 2022, 12, 2079 5 of 15

hole was made with a drill and the MCA was burned with a thermocoagulator under the
place of its intersection with the olfactory tract. If possible, the anatomic structure of soft
tissues was restored. The suture was processed with a 10% povidone-iodine solution [33].

2.4.3. Study Design


Experimental animals were divided into 5 equal groups of 20 individuals (the deviation
of the body weight of rats in one experimental group did not exceed 10%). The first group
was sham operated (SO), to whose animals all sequential surgical manipulations were
applied with the exception of arterial coagulation; negative control (NC)—animals with
focal cerebral ischemia, but deprived of pharmacological support (this group received
purified water throughout the study); EGB761 is a group of rats that were treated with a
reference—standardized extract of Ginkgo biloba (EGB761; obtained from Hunan Warrant
Pharmaceuticals, Changsha, China) at a dose of 35 mg/kg [34]; VM is a group of animals
that received extract from V. myrtillus; VA is a group of animals that received extract
from V. arctostaphylos. The administered dose for the studied extracts was selected on
the basis of previous studies and amounted to 50 mg/kg. The referent and the studied
extracts were administered for 3 days after ischemia, orally, once a day. On the 4th day of
the experiment, a change in the level of local cerebral blood flow was evaluated in rats,
after which the animals were decapitated and the brain was extracted, in the tissue of
which the change in the necrosis zone was evaluated (in 10 rats from the group). The
activity of superoxide dismutase, cytochrome-c oxidase and succinate dehydrogenase was
determined in the remaining 10 individuals from the group. Changes in the concentration
of 2-thiobarbituric acid active substances (TBARS) in brain tissue were also evaluated. All
tests were performed once for each animal. A total of 20 tests were performed for one
experimental group.

2.4.4. Cerebral Blood Flow Evaluation


The average systolic velocity of cerebral blood flow was recorded in the of the MCA
area, for which a trepanation hole was made in this animal’s brain region. Rats were
anesthetized by chloral hydrate. The average systolic velocity was recorded using an
ultrasound Doppler device, a sensor USOP-010-01 with a work frequency of 25 MHz
and a MM-D-K-Minimax Doppler v.1.7. (Saint Petersburg, Russia) software for Windows,
using mathematical processing of the fast Fourier transform signal to transform the color
spectrum of the blood flow distribution [35].

2.4.5. Biomaterial Sampling and Preparation


Animal brains were used as biomaterial for analysis. The brain was homogenized in
a mechanical homogenizer Potter type in a cold system of buffer solutions that consist of
1 mM EGTA, 215 mM mannitol, 75 mM sucrose, 0.1% bovine serum albumin solution with
a 20 mM HEPES. The pH of buffer solution was 7.2. To obtain a primary homogenate, the
ratio of tissue mass/volume of buffer solution 1:7 was used. Primary brain homogenate
was separated into two parts. The first of them was used to determine the concentration of
TBARS. The second part of the homogenate was used to determine superoxide dismutase,
cytochrome c oxidase and succinate dehydrogenase activity. For that, homogenate was
centrifuged 2 min at 1100× g acceleration. The supernatant was moved to Eppendorf-
type test tubes and a percoll solution 10% concentration was layered, after which mixture
was re-centrifuged at 18,000× g for 10 min. The secondary supernatant was deleted, the
precipitate was resuspended in a buffer solution and re-centrifuged at 10,000× g for 5 min

2.4.6. Evaluation of Necrosis Zone


The size of the necrosis area of the brain was determined by the triphenyl tetrazolium
method (n = 10 from each group). The brain was extracted from skull, the cerebellum was
cut off. After, the hemispheres were separated. Both hemispheres were weighed. Next, the
hemispheres were separately homogenized in PBS and placed in test-tubes with 10 mL of
Life 2022, 12, 2079 6 of 15

1% triphenyltetrazolium chloride solution in a PBS with pH 7.4. The test-tubes were placed
in a water bath for 20 min at a temperature of 37 ◦ C. Next, the probes were centrifuged in
the mode of 3000 RCF/10 min and the resulting supernatant was removed. A total of 3 mL
of phosphate buffer and 3 mL of cooled chloroform were added to the precipitate. Shaken
for 2 min. Chloroform extract of formazan was obtained for 15 min at 4 ◦ C, shaking the
mixture every 5 min for 30 s. The optical density (492 nm) against pure chloroform was
centrifuged and measured. The necrosis zone area was expressed as a percentage of the
total mass of the hemispheres and was calculated by formula:

ε 1 M1 + ε 2 M2
x = 100 − 100%,
ε 1 ( M1 + M2 )

where x is the size of the necrosis zone as a percentage of the total mass of the brain; ε1 is
the optical density of the sample with an intact hemisphere; ε2 is the optical density of the
sample with a damaged hemisphere; M1 is the mass of the intact hemisphere; M2 is the
mass of the damaged hemisphere.

2.5. Antioxidant Potential


2.5.1. Thiobarbituric Acid Reactive Substances (TBARS) Evaluation
The concentration of TBARS was evaluated in the brain homogenate by spectrophoto-
metric method. The principle of the method is based on the spectrophotometric (at 532 nm)
determination of colored products of the condensation reaction between unsaturated alde-
hydes and 2-thiobarbituric acid. In this case, the absorbance of the solution is correlated
to the concentration of malondialdehyde. The amount of TBARS was calculated by the
malondialdehyde molar extinction coefficient (1.56 × 105 l × mol−1 × cm−1 ) value. The
obtained results were expressed in nmol/mg protein. Protein content was determined
by the Bradford method. Absorbance was recorded on Infinite F50 reader (Tecan Austria
GmbH, Grödig, Austria) [36].

2.5.2. Superoxide Dismutase (SOD) Activity


The activity of SOD was estimated by the xanthine-xanthine oxidase method. Principal
of a method is based on the determination of products of reaction of the dismutation of
the superoxide radical that a was formed during the complex oxidation and reduction of
xanthine and 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride, respectively.
The reaction mixture consisted of 0.05 mM of xanthine; 0.025 mM of 2-(4-iodophenyl)-3-(4-
nitrophenol)-5-phenyltetrazolium chloride; 0.94 mM of EDTA, 80 U/L of xanthine oxidase,
40 mM of CAPS (N-cyclohexyl-3-aminopropanesulfonic acid) buffer. The optical density
of the mixture was estimated at 505 nm. SOD activity was expressed in units/protein mg.
Protein content was determined by the Bradford method. Absorbance was recorded on
Infinite F50 reader (Tecan Austria GmbH, Grödig, Austria) [37].

2.5.3. Succinate Dehydrogenase (SDH) Activity


The SDH activity was evaluated by spectrophotometric method which based on the
reaction of the reduction of dichlorophenolindophenol that was catalyzed by succinate. To
inhibit a mitochondrial complex I activity rotenone was added to the analyzed mixture.
Absorbance of the probes was estimated at 600 nm [23]. During the analysis, standard
kits from Abcam (ab110217/MS643) were used. Absorbance was recorded on Infinite F50
reader (Tecan Austria GmbH, Grödig, Austria) [38].

2.5.4. Cytochrome-C-Oxidase (COX) Activity


The COX activity was evaluated in the reaction of cytochrome C (II) oxidation when
KCN was added in an incubation mixture. Absorbance was estimated at 500 nm (24).
Standard kits from Abcam (ab110217/MS643) were used in the analysis. Absorbance was
recorded on Infinite F50 reader (Tecan Austria GmbH, Grödig, Austria) [39].
KCN was added in an incubation mixture. Absorbance was estimated at 500 nm (24).
Standard kits from Abcam (ab110217/MS643) were used in the analysis. Absorbance was
recorded on Infinite F50 reader (Tecan Austria GmbH, Grödig, Austria) [39].

2.6. Statistical Analysis


Life 2022, 12, 2079 7 of 15
Statistical analysis of chemical data was performed by one-way analysis of variance,
and the significance of the mean difference was determined by Duncan’s multiple range
test.
2.6.Differences at p < 0.05 were considered statistically significant. The results are pre-
Statistical Analysis
sented Statistical
as mean analysis
values of± standard deviations
chemical data (S.D.). by
was performed The linear analysis
one-way regression analysis and
of variance,
generation
and the significance of the mean difference was determined by Duncan’s multiple range(Alentum
of calibration graphs were conducted using Advanced Grapher 2.2 test.
Software Inc.,atRamat-Gan,
Differences p < 0.05 were Israel). Thestatistically
considered statistical significant.
analysis ofThe theresults
resultsareofpresented
neuroprotective
as
mean values
activity ± standard
evaluation deviationsby
was processed (S.D.).
using The
thelinear regression6.0
STATISTICA analysis and generation
software (Dell Technolo-
of Inc.,
gies calibration
Roundgraphs
Rock,were conducted
TX, USA). The using Advanced
obtained Grapher
data were 2.2 (Alentum
expressed as M ±Software
SEM (mean ±
Inc., Ramat-Gan, Israel). The statistical analysis of the results
standard error of the mean). The normality was assessed by the Shapiro–Wilkof neuroprotective activity
test, the
evaluation was processed by using the STATISTICA 6.0 software (Dell Technologies Inc.,
uniformity of variance was estimated by the Leven’s test. The ANOVA with post hoc
Round Rock, TX, USA). The obtained data were expressed as M ± SEM (mean ± standard
Newman-Keusle
error of the mean). test was
The used inwas
normality post-processing
assessed by theanalysis.
Shapiro–WilkA critical level
test, the of significance
uniformity of
was p < 0.05.
variance was estimated by the Leven’s test. The ANOVA with post hoc Newman-Keusle
test was used in post-processing analysis. A critical level of significance was p < 0.05.
3. Results
3. Results
3.1. Phenolic Compounds of V. myrtillus and V. arctostaphylos Leaves
3.1. Phenolic Compounds of V. myrtillus and V. arctostaphylos Leaves
Nine phenolic compounds, including the four phenolic acids of 4-О-caffeoylquinic,
Nine phenolic compounds, including the four phenolic acids of 4-O-caffeoylquinic,
5-О-caffeoylquinic,
5-O-caffeoylquinic, caffeic, and 4,5-di-O-caffeoylquinic
caffeic, and 4,5-di-О-caffeoylquinic acids
acids as well
as well as flavonoid
as five five flavonoid
quercetin
quercetinglycosides
glycosides (quercetin-3-О-galactoside, quercetin-3-О-glucoside,
(quercetin-3-O-galactoside, quercetin-3-O-glucoside, quercetin-3-О-
quercetin-3-O-
arabinopyranoside,
arabinopyranoside, quercetin-3-О-arabinofuranoside, quercetin-3-О-rhamnoside),
quercetin-3-O-arabinofuranoside, quercetin-3-O-rhamnoside), were were
identified in V. myrtillus leaves (Figure 2, Table 2).
identified in V. myrtillus leaves (Figure 2, Table 2).

Figure 2. Cont.
Life 2022, 12, 2079 8 of 15
Life 2022, 12, x FOR PEER REVIEW 8 of 16

(c) (d) (e)

(f) (g) (h)

(i) (j) (k)

(l) (m)
Figure 2. High-performance liquid chromatography with photodiode array detection (HPLC-PDA)
Figure 2. High-performance liquid chromatography with photodiode array detection (HPLC-PDA)
chromatograms (330 nm) of leaf extracts of V. myrtillus (a) and V. arctostaphylos (b) and mass spectra
chromatograms
of compounds (330
1 (c),nm)
2 (d),of3 leaf (f), 5 (g),of6V.
(e), 4extracts myrtillus
(h), (k), V.
(a)9and
7 (i), 8 (j), 10 arctostaphylos
(l), and 11 (m).(b) and mass are
Compounds spectra
of numbered
compoundsas 1 (c), 2in(d),
listed Table3 (e), 4 (f),square
2. Blue 5 (g), indicates
6 (h), 7 (i), 8 (j), 9of(k),
location 10 (l),
[M-H] and 11 (m). Compounds are
− ion.

numbered as listed in Table 2. Blue square indicates location of [M − H]− ion.


Table 2. Retention times (t), ultraviolet (UV), and mass spectral (ESI-MS, MS/MS) data of com-
pounds 1–11 were found for leaf extracts of V. myrtillus and V. arctostaphylos.
Table 2. Retention times (t), ultraviolet (UV), and mass spectral (ESI-MS, MS/MS) data of com-
pounds 1–11 were found for leaf extracts of V. myrtillusESI-MS,
and V. arctostaphylos.
[M-
No t, min Compound * UV, λmax, nm MS/MS, m/z
H]−, m/z
No 1 3.49
t, min 4-О-Caffeoylquinic
Compound * acid UV, 322
λmax , nm 353 ESI-MS, [353]: 191, 179, 173, 135
MS/MS, m/z
2 5.51 5-О-Caffeoylquinic acid 322 [M − H]− , m/z[353]: 191, 179, 165
353
1 3 6.20
3.49 Caffeic acid acid
4-O-Caffeoylquinic 320
322 179 353 [179]: 135 179, 173, 135
[353]: 191,
2 4 5.51
10.26 Quercetin5-O-Caffeoylquinic
3-О-galactosideacid
(hyperoside) 322 355
255, 267, 463 353 [353]:
[463]: 301191, 179, 165
3 5 6.20
10.83 Caffeic acid
Quercetin 3-О-glucoside (isoquercitrin) 320
255, 267, 356 463 179 [463]: 301 135
[179]:
4 10.26 Quercetin 3-O-galactoside (hyperoside) 255, 267, 355 463 [463]: 301
6 12.72 Quercetin 3-О-arabinopyranoside (guaiaverin) 255, 267, 354 433 [433]: 301
5 10.83 Quercetin 3-O-glucoside (isoquercitrin) 255, 267, 356 463 [463]: 301
6 7 13.38
12.72 Quercetin3-O-arabinopyranoside
Quercetin 3-О-arabinofuranoside (avicularin)
(guaiaverin) 255,
255,267,
267,354
354 433 433 [433]: 301
[433]: 301
7 8 13.91
13.38 Quercetin
Quercetin 3-О-rhamnoside (quercitrin)
3-O-arabinofuranoside (avicularin) 255,
255,267,
267,352
354 447 433 [447]: 301
[433]: 301
8 9 13.91
14.58 Quercetin 3-O-rhamnoside (quercitrin)
3,5-Di-О-caffeoylquinic acid 255,322
267, 352 515 447 [515]: 353, 191, [447]:
179, 301
173
9 10 14.58
9.80 3,5-Di-O-caffeoylquinic
Quercetin 3-О-rutinosideacid
(rutin) 322 355
255, 267, 609 515 [515]:
[609]: 353,
463, 191, 179, 173
301
10 9.80 Quercetin 3-O-rutinoside (rutin) 255, 267, 355 609 [609]: 463, 301
11 15.14 Quercetin 3-O-(6′′-acetyl)-glucoside 256, 268, 351 505 [505]: 463, 301
11 15.14 Quercetin 3-O-(600 -acetyl)-glucoside 256, 268, 351 505 [505]: 463, 301
* Identification was performed by comparison of obtained retention times, UV, MS, and MS/MS
* Identification
with referencewas performed by comparison of obtained retention times, UV, MS, and MS/MS with refer-
standards.
ence standards.

Ten compounds of phenolic origin were found in V. arctostaphylos leave extracts,


Ten compounds
among them three of phenolic
phenolic origin
acids were found in V. arctostaphylos
(4-О-caffeoylquinic, leave extracts,
5-О-caffeoylquinic, among
and caffeic
them three phenolic acids (4-O-caffeoylquinic, 5-O-caffeoylquinic, and caffeic acids) and six
quercetin glycosides [quercetin-3-O-rutinoside, quercetin-3-O-galactoside, quercetin-3-O-
glucoside, quercetin-3-O-arabinopyranoside, quercetin-3-O-rhamnoside and quercetin-3-O-
(600 -acetyl)-glucoside] (Figure 3).
Life 2022, 12, x FOR PEER REVIEW 9 of 16

Life 2022, 12, 2079


acids) and six quercetin glycosides [quercetin-3-О-rutinoside, quercetin-3-О-galactoside,
9 of 15
quercetin-3-О-glucoside, quercetin-3-О-arabinopyranoside, quercetin-3-О-rhamnoside
and quercetin-3-O-(6′′-acetyl)-glucoside] (Figure 3).

OH COOH OH
O OH
OH O
OH OH HO O
O
HOOC OH O OH
OH OH O OH
OH HOOC OH OH O
OH HO OH
OH OH O
1 2 3 4
OH
OH OH OH
OH
OH OH OH
HO O
HO O HO O HO O
OH
OH O O
O OH OH O
O OH O O O OH
OH O HO OH
OH O
OH
OH O OHOH OH O
OH
OH
5 6 7 8
OH OH OH
OH
OH OH
O
OH OH O
OH HO O O HO O
O
OH O OH O CH3
HOOC O O O
OH O OH O OH
OH HO OH HO OH
O OH O OH O
9 10 11
Figure 3. Compounds 1–11 found in leaf extracts of V. myrtillus and V. arctostaphylos.
Figure 3. Compounds 1–11 found in leaf extracts of V. myrtillus and V. arctostaphylos.

Caffeoylquinicacids
Caffeoylquinic acidsandandcaffeic
caffeicacid,
acid,the
theknown
knownmetabolites
metabolitesofofV.V.myrtillus
myrtillusleaves
leaves[40],
[40], were also found in V. arctostaphylos leaves of Georgian origin [41].
were also found in V. arctostaphylos leaves of Georgian origin [41]. In a previous study, In a previous
study,
the basicthe basic quercetin
quercetin glycosides
glycosides in V. myrtillus
in V. myrtillus leaves leaves from Finland
from Finland were rutin,
were rutin, hy-
hyperoside,
peroside, guaiaverin,
guaiaverin, avicularin, avicularin, and [42],
and quercitrin quercitrin
and the[42], and
only the only
known known in
flavonoid flavonoid in V.
V. arctostaphylos
arctostaphylos leaves is quercetin [43]. Quercetin
00 3-O-(6′′-acetyl)-glucoside
leaves is quercetin [43]. Quercetin 3-O-(6 -acetyl)-glucoside was found in V. myrtillus was found in V.
myrtillus leaves for the first time as well as quercetin glycosides, which were newly dis-
leaves for the first time as well as quercetin glycosides, which were newly discovered in
covered in V. arctostaphylos leaves.
V. arctostaphylos leaves.
Results of the quantification of compounds 1–11 in V. myrtillus and V. arctostaphylos
Results of the quantification of compounds 1–11 in V. myrtillus and V. arctostaphylos leaf
leaf extracts demonstrated high contents of 5-О-caffeoylquinic acid (226.85 mg/g in V. myr-
extracts demonstrated high contents of 5-O-caffeoylquinic acid (226.85 mg/g in V. myrtillus,
tillus, 105.32 mg/g in V. arctostaphylos), quercetin 3-О-glucoside (12.02 mg/g in V. myrtillus),
105.32 mg/g in V. arctostaphylos), quercetin 3-O-glucoside (12.02 mg/g in V. myrtillus), and
and quercetin 3-О-galactoside (12.02 mg/g in V. arctostaphylos) (Table 3). Earlier, chloro-
quercetin 3-O-galactoside (12.02 mg/g in V. arctostaphylos) (Table 3). Earlier, chlorogenic
genic acid was detected as the main phenolic compound in the V. myrtillus leaves (approx-
acid was detected as the main phenolic compound in the V. myrtillus leaves (approximately
imately 52–84% of the total amount of phenols) [44,45], and quercetin and kaempferol
52–84% of the total amount of phenols) [44,45], and quercetin and kaempferol derivatives
derivatives were the predominant phenolic groups (39.2%) [46]. The water–alcoholic ex-
were the predominant
tract of V. arctostaphylosphenolic groups
leaves from (39.2%) [46].
Iran contained The water–alcoholic
chlorogenic V. arc-
extractatof97.2
acid and flavonoids
tostaphylos leaves from Iran contained
mg/g and 2.2–22.4 mg/g, respectively [15,47]. chlorogenic acid and flavonoids at 97.2 mg/g and
2.2–22.4 mg/g, respectively [15,47].
Table 3. Contents of compounds 1–11 in leaf extracts of V. myrtillus and V. arctostaphylos in mg/g of
Table
dry weight ± S.D.of compounds 1–11 in leaf extracts of V. myrtillus and V. arctostaphylos in mg/g of
3. Contents
dry weight ± S.D.
Compound V. myrtillus V. arctostaphylos
4-О-Caffeoylquinic
Compound acid <0.01
V. myrtillus 8.01 ± 0.14
V. arctostaphylos
5-О-Caffeoylquinic acid 226.85 ± 5.21 105.32 ± 2.41
4-O-Caffeoylquinic acid <0.01 8.01 ± 0.14
Caffeic acid <0.01 4.29 ± 0.08
5-O-Caffeoylquinic acid 226.85 ± 5.21 105.32 ± 2.41
Quercetin 3-О-rutinoside (rutin) <0.01 1.46 ± 0.03
Caffeic acid <0.01 4.29 ± 0.08
Quercetin 3-О-galactoside (hyperoside) 4.69 ± 0.09 3.99 ± 0.06
Quercetin 3-O-rutinoside (rutin) <0.01 1.46 ± 0.03
Quercetin 3-О-glucoside (isoquercitrin) 12.02 ± 0.24 2.38 ± 0.05
Quercetin 3-O-galactoside (hyperoside) 4.69 ± 0.09 3.99 ± 0.06
Quercetin 3-О-arabinopyranoside
Quercetin (guaiaverin)
3-O-glucoside (isoquercitrin) 1.34 ±±0.02
12.02 0.24 1.29
2.38±±0.02
0.05
Quercetin 3-О-arabinofuranoside (avicularin)
Quercetin 3-O-arabinopyranoside (guaiaverin) <0.01
1.34 ± 0.02 <0.01
1.29 ± 0.02
Quercetin
Quercetin 3-О-rhamnoside (quercitrin)
3-O-arabinofuranoside (avicularin) 2.77 ± 0.05
<0.01 1.23<0.01
± 0.02
Quercetin
Quercetin 3-O-(6′′-acetyl)-glucoside
3-O-rhamnoside (quercitrin) 2.77<0.01
± 0.05 0.70
1.23± ±
0.01
0.02
Quercetin 3-O-(600 -acetyl)-glucoside <0.01 0.70 ± 0.01
4,5-Di-O-caffeoylquinic acid <0.01 <0.01

3.2. Neuroprotective Activity of V. myrtillus and V. arctostaphylos Leaf Extracts


The study of neuroprotective activity of extracts from V. myrtillus and V. arctostaphylos
showed that their administration to animals in the post-ischemic period contributed to an
Life 2022, 12, x FOR PEER REVIEW 10 of 16

4,5-Di-О-caffeoylquinic acid <0.01 <0.01

Life 2022, 12, 2079 3.2. Neuroprotective Activity of V. myrtillus and V. arctostaphylos Leaf Extracts 10 of 15

The study of neuroprotective activity of extracts from V. myrtillus and V. arctostaphy-


los showed that their administration to animals in the post-ischemic period contributed to
anincrease
increaseinin the
the level of
of cerebral
cerebralbloodbloodflow
flow(Figure
(Figure4)4) compared
compared to to that
that of untreated
of untreated ratsrats by
by99.3%
99.3%(p (p<< 0.05)
0.05) and 115.0%
115.0% (p (p <<0.05),
0.05),respectively,
respectively,while
whilethe use
the useofof
thethe
EGB761
EGB761 refer-
reference
ence increased
increased this
this indicatorby
indicator by79.3%
79.3%(p (p<< 0.05). Of
Ofnote,
note,ininanimals
animals that
thatreceived extrac-
received extraction
tion from
from V. V. arctostaphylos,the
arctostaphylos, thelevel
levelofofcerebral
cerebralblood
blood flow
flow was higher
higher inin the
therats
ratsinjected
injected with
with
thethe reference
reference byby 19.9%
19.9% (p (p
< <0.05).
0.05).

4
CBF (sm/sec)

0
SO NC EGB761 VM VA

Figure
Figure 4. The
4. The effect
effect of V. V. myrtillus
ofmyrtillus and and V. arctostaphylos
V. arctostaphylos leaf extracts
leaf extracts on theon the cerebral
cerebral hemodynamics
hemodynamics
in in
rats with
rats withcerebral ischemia.
cerebral ischemia.Experimental
Experimentalgroups: SO—sham-operated
groups: SO—sham-operated animals; NC—negative
animals; NC—negative
control
controlanimals; EGB761—EGB761
animals; EGB761—EGB761 reference remedy
reference group;group;
remedy VM—V.VM—V. myrtillusmyrtillus
group; VA—V.
group;arcto-
VA—V. arc-
staphylos group.
tostaphylos Hashtag
group. (#) indicates
Hashtag significant
(#) indicates difference
significant (p < 0.05)(pvs.
difference the SO
< 0.05) vs.group; asterisk
the SO group;(*)asterisk
indicates significant difference (p < 0.05) vs. the NC group; delta (Δ) indicates significant difference
(*) indicates significant difference (p < 0.05) vs. the NC group; delta (∆) indicates significant difference
(p < 0.05) vs. the EGB761 group.
(p < 0.05) vs. the EGB761 group.
Additionally, the use of the studied extracts led to a decrease in the brain necrosis
Additionally, the use of the studied extracts led to a decrease in the brain necrosis
zone (Figure 5), while against the background of the administration of the extract from V.
zone (Figure 5), while against the background of the administration of the extract from
myrtillus, this indicator decreased by 28.2% (p < 0.05), and for V. arctostaphylos, it decreased
V. myrtillus, this indicator decreased by 28.2% (p < 0.05), and for V. arctostaphylos, it decreased
by 42.2% (p < 0.05). At the same time, the use of the reference contributed to a decrease in
thebybrain
42.2% (p < 0.05).
necrosis zone At the same
by 34.3% (p < time,
0.05), the usewas
which of the reference
higher contributed
than the to a for
same indicator decrease
in the brain necrosis zone by 34.3% (p < 0.05), which was higher than the
the group of animals that were injected with extraction from V. arctostaphylos by 12.1% same indicator
(p
Life 2022, 12, x FOR PEER REVIEW 11 of 16
for the group of animals that were injected with extraction from V. arctostaphylos
< 0.05). by 12.1%
(p < 0.05).

40

35

30
Necrosis zone (%)

25

20

15

10

0
SO NC EGB761 VM VA

Figure
Figure 5. The
5. The effect
effect of V. V. myrtillus
ofmyrtillus and and V. arctostaphylos
V. arctostaphylos leaf extracts
leaf extracts on theon the necrosis
necrosis zone inzone
rats in rats with
with
cerebral
cerebralischemia.
ischemia. Experimental
Experimental groups:
groups:SO—sham-operated
SO—sham-operated animals; NC—negative
animals; NC—negative control ani-
control animals;
mals; EGB761—EGB761
EGB761—EGB761 reference
reference remedy
remedy group;VM—V.
group; VM—V.myrtillus
myrtillusgroup;
group;VA—V.
VA—V.arctostaphylos
arctostaphylos group.
group. Asterisk
Asterisk (*) indicates
(*) indicates significant
significant difference
difference (p < 0.05)
(p < 0.05) vs.NC
vs. the the group;
NC group;
deltadelta (Δ) indicates
(∆) indicates significant
significant difference (p < 0.05) vs. the EGB761 group.
difference (p < 0.05) vs. the EGB761 group.
3.3. Antioixdant Potential of V. myrtillus and V. arctostaphylos Leaf Extracts
The study of changes in the antioxidant balance of brain tissue with the administra-
tion of the studied extracts showed that the use of extracts from V. myrtillus and V. arcto-
staphylos contributed to an increase in the activity of SOD by 4.1% (p < 0.05) and 76.4% (p
Life 2022, 12, 2079 11 of 15

3.3. Antioixdant Potential of V. myrtillus and V. arctostaphylos Leaf Extracts


The study of changes in the antioxidant balance of brain tissue with the administration
of the studied extracts showed that the use of extracts from V. myrtillus and V. arctostaphylos
contributed to an increase in the activity of SOD by 4.1% (p < 0.05) and 76.4% (p < 0.05),
respectively, and reduced the content of TBARS by 55.1% (p < 0.05) and 64.3% (p < 0.05),
respectively (Table 4).

Table 4. Effect of V. myrtillus and V. arctostaphylos leaf extracts on activity of mitochondrial enzymes
and TBARS in rats with cerebral ischemia.

Experimental Group SOD, U/Protein mg COX, U/Protein mg SDH, U/Protein mg TBARS, µM/Protein mg
Sham-operated animals 300.5 ± 7.1(29) 4.3 ± 0.0(47) 2.7 ± 0.062 2.4 ± 0.124
Negative control group 125.6 ± 9.5(41) # 2.1 ± 0.0(14) # 1.1 ± 0.097 # 9.8 ± 0.663 #
EGB761 reference group 190.5 ± 11.2(84) * 2.5 ± 0.1(17) * 1.6 ± 0.018 * 5.2 ± 0.541 *
V. myrtillus group 181.0 ± 10.9(37) * 2.6 ± 0.0(37) * 1.5 ± 0.09 * 4.4 ± 0.364 *
V. arctostaphylos group 221.5 ± 10.2(32) * 3.0 ± 0.0(58) * ∆α 2.1 ± 0.071 * ∆α 3.5 ± 0.193 *
Hashtag (#) indicates significant difference (p < 0.05) vs. sham-operated animals’ group; asterisk (*) indicates
significant difference (p < 0.05) vs. negative control group; delta (∆) indicates significant difference (p < 0.05) vs.
the EGB761 reference group; alpha (α) indicates significant difference (p < 0.05) vs. the V. myrtillus group.

At the same time, the use of EGB761 contributed to an increase in SOD activity by 51.7%
(p < 0.05) and a decrease in TBARS by 46.9% (p < 0.05), which was statistically significantly
lower than similar indicators of animals receiving extraction from V. arctostaphylos. Analysis
of changes in the activity of mitochondrial enzymes showed that when extracts from
V. myrtillus L. and V. arctostaphylos were used, as well as the reference, the activity of COX
increased (relative to the NC group of rats) by 23.8% (p < 0.05), 42.9% (p < 0.05), and 19.0%
(p < 0.05), respectively, whereas SDH activity increased by 36.4% (p < 0.05), 45.5% (p < 0.05),
and 90.9% (p < 0.05). Of note, in rats injected with the extract from V. arctostaphylos, the
activities of both COX and SDH were higher than in animals treated with the reference and
extract from V. myrtillus (p < 0.05).

4. Discussion
A detailed study of plant metabolites opens possibilities for selection of precise
and accurate methods to include in pharmacopeial practice. The results obtained in
this study show that the major and marker compound for both Vaccinium species was
5-O-caffeoylquinic acid (chlorogenic acid). In predicting the pharmacological activity,
phenolic acids are mostly represented by chlorogenic acid isomers (4-O-caffeoylquinic
and 5-O-caffeoylquinic acids). Moreover, the main aglycone of flavonoids is quercetin,
and its glycosides are presented by monosides (quercetin-3-O-galactoside, quercetin-3-O-
glucoside, quercetin-3-O-arabinopyranoside, quercetin-3-O-arabinofuranoside, quercetin-3-
O-rhamnoside, and quercetin-3-O-(600 -acetyl)-glucoside) and by only one bioside (quercetin-
3-O-rutinoside). Chlorogenic acids and quercetin glycosides are known neuroprotective
agents. Thus, chlorogenic acid shows a protective effect on the nitrosative stress induced
by sodium nitroprusside [48]. In vivo, chlorogenic acid decreased the infarct area in the
hypoxic–ischemic brains of rats [49]. Additionally, chlorogenic acid may be a potential agent
to treat Parkinson’s disease in the apoptosis-mediated neuronal senescence of dopaminergic
neurons [50]. The neuroprotective effects of quercetin may be used in the treatment of
Alzheimer’s disease via its antioxidant properties and providing an inhibitory effect of fibril
formation of amyloid-β aggregates [51]. These effects are useful in the therapy of pediatric
neurological diseases such as central nervous system tumors, autism, and hyperactivity
disorder [52]. Quercetin and rutin show neuroprotective effects in the cerebral ischemia
model in rats by its 4-oxo group and 2,3-double bond in the C ring [53]. Thus, the potential
neuroprotective effects of Vaccinium extracts may be related to the significant amount of
these phenolics in the Vaccinium leaves.
Life 2022, 12, 2079 12 of 15

Increasingly, natural remedies are being used as adjuvant neuroprotective therapy for
cerebrovascular disorders [54]. The neuroprotective effects of traditional Chinese medicine
(for example, ginseng preparations, as well as Scutellaria baicalensis, Withania somnifera,
Phyllanthus emblica, and Viscum album [55]) are widely known. It has been established
that the use of phytomedicines has a pleiotropic effect on brain tissue, while eliminating
almost all main pathogenetic pathways of the “ischemic cascade”: neuroinflammation,
apoptosis, energy deficiency, glutamate-calcium excitotoxicity, hyperlactatemia, and acido-
sis [55]. However, as a rule, remedies based on phytocompositions are known as effective
antioxidants [55,56].
The concept of phytotherapeutic intervention in ischemic stroke is currently being
actively studied; however, there is no doubt that this approach is only auxiliary and
cannot fully replace the “gold standard” of treatment of ischemic brain damage—effective
thrombolysis [57]. The rapidly developing direction of phytotherapeutic neuroprotection
makes it necessary to search for new drugs that meet the basic postulates of this area of
medicine and pharmacology [58]. In this work, possible neuroprotective properties of
extracts obtained from two species of representatives of the heather family, V. myrtillus
and V. arctostaphylos, were studied. Blueberry-based products are primarily known as
effective biologically active additives with pronounced antioxidant activity and tropicity
in relation to blood vessels and the cardiovascular system as a whole, which has made
it possible to use these products in the correction of vascular complications of diabetes
mellitus, atherosclerosis, and glaucoma [58]. At the same time, a similar therapeutic profile
of extracts from blueberries suggests the presence of neuroprotective activity. During
the study, it was found that the administration of extracts obtained from V. myrtillus and
V. arctostaphylos to animals with focal ischemia contributed to the improvement of cerebral
hemodynamics in the post-ischemic period, which may be associated with the restoration of
the function of collateral blood supply to the ischemic zone. A decrease in the necrotic focus
was also shown in rats receiving the analyzed extracts compared with untreated animals.
Because one of the significant mechanisms of ischemic brain damage is the development
of oxidative stress, we studied the effect of extracts from blueberries on changes in the
parameters characterizing the development of this pathological process—the activity of
superoxide dismutase and the final concentration of peroxidation products represented by
TBARS [36]. As a result, the introduction of extracts from V. myrtillus and V. arctostaphylos
increased the activity of superoxide dismutase and reduced the intensity of free radical lipid
oxidation reactions. An important aspect of brain ischemia is a violation of the energy state
of cells with a sharp drop in ATP synthesis and the induction of irreversible processes in the
form of necroptosis or apoptosis [59]. In this regard, the effect of extracts from V. myrtillus
and V. arctostaphylos on the activity of two integral enzymatic indicators of the metabolic
status of the cell, cytochrome c oxidase and succinate dehydrogenase, was evaluated.
The administration of the analyzed extracts led to an increase in the activity of these
enzymes, and the effect of the administration of the extract obtained from V. arctostaphylos
exceeded that of the reference, the classical neuroprotective herbal remedy Ginkgo biloba
extract (EGB761). Thus, based on the overall results of the set of pharmacological tests of
extracts from V. myrtillus and V. arctostaphylos, it can be assumed that these objects have
neuroprotective activity, which can be realized through antioxidant and metabolic action.
At the same time, a more promising therapeutic agent is the extraction from V. arctostaphylos,
the use of which led to a statistically significantly better pharmacological response than the
introduction of the reference and extract obtained from V. myrtillus.

Author Contributions: Conceptualization, A.A.S. and D.N.O.; methodology, A.A.S.; software, A.A.S.;
validation, D.N.O., D.I.P. and E.R.G.; formal analysis, V.N.B., E.R.G. and M.V.L.; investigation, A.A.S.,
D.N.O., D.I.P., E.R.G., M.V.L.; resources, D.N.O., E.R.G. and M.V.L.; data curation, D.I.P., V.N.B. and
M.V.L.; writing—original draft preparation, A.A.S., E.R.G.; writing—review and editing, D.N.O.;
visualization, A.A.S., E.R.G.; supervision, D.N.O.; project administration, A.A.S.; funding acquisition,
D.N.O. All authors have read and agreed to the published version of the manuscript.
Life 2022, 12, 2079 13 of 15

Funding: This research was funded by the Ministry of Education and Science of Russia, grant
numbers FSRG-2020-0019, 121030100227-7.
Institutional Review Board Statement: The study was conducted according to the guidelines of
the Declaration of Helsinki, and approved by the Russian Health Ministry (protocol code 708H,
23 August 2010) and the Ethics Committee of Institute of General and Experimental Biology (protocol
code 10, 20 August 2021).
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained within the article.
Acknowledgments: The authors acknowledge the Buryat Research Resource Center for the technical
support in chromatographic and mass-spectrometric research.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or
in the decision to publish the results.

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