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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
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
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].
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.
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].
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.
Figure 2. Cont.
Life 2022, 12, 2079 8 of 15
Life 2022, 12, x FOR PEER REVIEW 8 of 16
(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.
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
Life 2022, 12, 2079 3.2. Neuroprotective Activity of V. myrtillus and V. arctostaphylos Leaf Extracts 10 of 15
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
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.
References
1. WHO: The World Flora Online. Available online: http://www.worldfloraonline.org/ (accessed on 12 November 2022).
2. Takhtadjan, A. Flowering Plants, 2nd ed.; Springer Science & Business Media: Berlin/Heidelberg, Germany, 2009; pp. 188–193,
ISBN 978-1-4020-9608-2.
3. Czerepanov, S.K. Vascular Plants of Russia and Neighboring Countries; Word and Family-95: Saint-Petersburg, Russia, 1995; p. 424.
4. Kasotea, D.M.; Duncanb, G.J.; Neacsua, M.; Russella, W.R. Rapid method for quantification of anthocyanidins and anthocyanins
in human biological samples. Food Chem. 2019, 290, 56–63. [CrossRef]
5. Chan, S.W.; Tomlinson, B. Effects of bilberry supplementation on metabolic and cardiovascular disease risk. Molecules 2020,
25, 1653. [CrossRef]
6. Satoh, Y.; Ishihara, K. Investigation of the antimicrobial activity of bilberry (Vaccinium myrtillus L.) extract against periodontopathic
bacteria. J. Oral Biosci. 2020, 62, 169–174. [CrossRef]
7. Sezer, E.D.; Oktay, L.M.; Karadadaş, E.; Memmedov, H.; Gunel, N.S.; Sözmen, E. Assessing anticancer potential of blueberry
flavonoids, quercetin, kaempferol, and gentisic acid, through oxidative stress and apoptosis parameters on HCT-116 cells. J. Med.
Food. 2019, 22, 1118–1126. [CrossRef]
8. Vaneková, Z.; Vanek, M.; Škvarenina, J.; Nagy, M. The influence of local habitat and microclimate on the levels of secondary
metabolites in slovak bilberry (Vaccinium myrtillus L.) Fruits. Plants 2020, 9, 436. [CrossRef]
9. Zheng, W.; Wang, S.Y. Oxygen radical absorbing capacity of phenolics in blueberries, cranberries, chokeberries, and lingonberries.
J. Agric. Food Chem. 2003, 51, 502–509. [CrossRef]
10. Viljanen, K.; Kylli, P.; Kivikari, R.; Heinonen, M. Inhibition of protein and lipid oxidation in liposomes by berry phenolics. J. Agric.
Food Chem. 2004, 52, 7419–7424. [CrossRef]
11. Puupponen-Pimiä, R.; Nohynek, L.; Hartmann-Schmidlin, S.; Kähkönen, M.; Heinonen, M.; Määttä-Riihinen, K.; Oksman-
Caldentey, K.M. Berry phenolics selectively inhibit the growth of intestinal pathogens. J. Appl. Microbiol. 2005, 98, 991–1000.
[CrossRef]
12. Ayaz, F.A.; Ayaz, S.H.; Gruz, J.; Novak, O.; Strnad, M. Separation, characterization, and quantitation of phenolic acids in a
little-known blueberry (Vaccinium arctostaphylos L.) fruit by HPLC-MS. J. Agric. Food Chem. 2005, 53, 8116–8122. [CrossRef]
13. Lätti, A.K.; Kainulainen, P.S.; Hayirlioglu-Ayaz, S.; Ayaz, F.A.; Riihinen, K.R. Characterization of anthocyanins in Caucasian
blueberries (Vaccinium arctostaphylos L.) native to Turkey. J. Agric. Food Chem. 2009, 57, 5244–5249. [CrossRef]
14. Chkhikvishvili, I.D.; Kharebava, G.I. Chicoric and chlorogenic acids in plant species from Georgia. Appl. Biochem. Microbiol. 2001,
37, 188–191. [CrossRef]
15. Mohtashami, R.; Huseini, H.F.; Nabati, F.; Hajiaghaee, R.; Kianbakht, S. Effects of standardized hydro-alcoholic extract of
Vaccinium arctostaphylos leaf on hypertension and biochemical parameters in hypertensive hyperlipidemic type 2 diabetic patients:
A randomized, double-blind and placebo-controlled clinical trial. Avicenna J. Phytomed. 2019, 9, 44–53.
16. Nickavar, B.; Amin, G. Enzyme assay guided isolation of an α-amylase inhibitor flavonoid from Vaccinium arctostaphylos leaves.
Iran J. Pharm. Res. 2011, 10, 849–853.
17. Yang, L.; Wang, Z.M.; Wang, Y.; Li, R.S.; Wang, F.; Wang, K. Phenolic constituents with neuroprotective activities from Hypericum
wightianum. Phytochemistry 2019, 165, 112049. [CrossRef]
18. Xie, Y.; Yang, W.; Tang, F.; Chen, X.; Ren, L. Antibacterial activities of flavonoids: Structure-activity relationship and mechanism.
Curr. Med. Chem. 2015, 22, 132–149. [CrossRef]
19. Aryal, S.; Skinner, T.; Bridges, B.; Weber, J.T. The pathology of Parkinson’s disease and potential benefit of dietary polyphenols.
Molecules 2020, 25, 4382. [CrossRef]
Life 2022, 12, 2079 14 of 15
20. Kempuraj, D.; Thangavel, R.; Kempuraj, D.D.; Ahmed, M.E.; Selvakumar, G.P.; Raikwar, S.P.; Zaheer, S.A.; Iyer, S.S.; Govindarajan,
R.; Chandrasekaran, P.N.; et al. Neuroprotective effects of flavone luteolin in neuroinflammation and neurotrauma. Biofactors
2021, 47, 190–197. [CrossRef]
21. Zhou, Y.; Zhang, S.; Fan, X. Role of polyphenols as antioxidant supplementation in ischemic stroke. Oxid. Med. Cell Longev. 2021,
2021, 5471347. [CrossRef]
22. Xie, Y.; Wang, H.; He, Z. Recent advances in polyphenols improving vascular endothelial dysfunction induced by endogenous
toxicity. J. Appl. Toxicol. 2021, 41, 701–712. [CrossRef]
23. Gao, Q.; Dong, J.Y.; Cui, R.; Muraki, I.; Yamagishi, K.; Sawada, N.; Iso, H.; Tsugane, S.; Japan Public Health Center-based
Prospective study group. consumption of flavonoid-rich fruits, flavonoids from fruits and stroke risk: A prospective cohort study.
Br. J. Nutr. 2021, 126, 1717–1724. [CrossRef]
24. Kalt, W.; Cassidy, A.; Howard, L.R.; Krikorian, R.; Stull, A.J.; Tremblay, F.; Zamora-Ros, R. Recent research on the health benefits
of blueberries and their anthocyanins. Adv. Nutr. 2020, 11, 224–236. [CrossRef]
25. Rabinstein, A.A. Update on treatment of acute ischemic stroke. Continuum 2020, 26, 268–286. [CrossRef]
26. Ting, H.C.; Chang, C.Y.; Lu, K.Y.; Chuang, H.M.; Tsai, S.F.; Huang, M.H.; Liu, C.A.; Lin, S.Z.; Harn, H.J. Targeting cellular stress
mechanisms and metabolic homeostasis by chinese herbal drugs for neuroprotection. Molecules 2018, 23, 259. [CrossRef]
27. Riihinen, K.; Jaakola, L.; Kärenlampi, S.; Hohtola, A. Organ-specific distribution of phenolic compounds in bilberry (Vaccinium
myrtillus) and ‘northblue’ blueberry (Vaccinium corymbosum × V. angustifolium). Food Chem. 2008, 110, 156–160. [CrossRef]
28. Rahman, M.M.; Ichiyanagi, T.; Komiyama, T.; Sato, S.; Konishi, T. Effects of anthocyanins on psychological stress-induced
oxidative stress and neurotransmitter status. J. Agric. Food Chem. 2008, 56, 7545–7550. [CrossRef]
29. Olennikov, D.N.; Shamilov, A.A. New compounds from Vaccinium vitis-idaea. Chem. Nat. Compd. 2022, 58, 240–244. [CrossRef]
30. Olennikov, D.N.; Shamilov, A.A. Catechin-O-rhamnosides from Vaccinium vitis-idaea stems. Chem. Nat. Compd. 2022, 58, 269–273.
[CrossRef]
31. Shamilov, A.A.; Olennikov, D.N.; Pozdnyakov, D.I.; Bubenchikova, V.N.; Garsiya, E.R. Investigation of phenolic compounds at
the leaves and shoots Arctostaphylos spp. and their antioxidant and antityrosinase activities. Nat. Prod. Res. 2022, 36, 6312–6317.
[CrossRef]
32. Kashchenko, N.I.; Jafarova, G.S.; Isaev, J.I.; Olennikov, D.N.; Chirikova, N.K. Caucasian dragonheads: Phenolic compounds,
polysaccharides, and bioactivity of Dracocephalum austriacum and Dracocephalum botryoides. Plants 2022, 11, 2126. [CrossRef]
33. Pozdnyakov, D.I. 4-Hydroxy-3,5-di-tret-butyl cinnamic acid restores the activity of the hippocampal mitochondria in rats under
permanent focal cerebral ischemia. Iran. J. Basic. Med. Sci. 2021, 24, 1590–1601. [CrossRef]
34. Wang, N.; Chen, X.; Geng, D.; Huang, H.; Zhou, H. Ginkgo biloba leaf extract improves the cognitive abilities of rats with
D-galactose induced dementia. J. Biomed. Res. 2013, 27, 29–36. [CrossRef] [PubMed]
35. Voronkov, A.V.; Dyakova, I.N.; Pozdnyakov, D.I. The influence of natural compounds of polyphenolic structure on the vasodilating
function of the vascular endothelium of the rat brain in conditions of its focal ischemia. Eksp. Klin. Farmakol. 2016, 79, 7–9.
[PubMed]
36. Janero, D.R. Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative
tissue injury. Free Rad. Biol. Med. 1990, 9, 515–540. [CrossRef] [PubMed]
37. Woolliams, J.A.; Wiener, G.; Anderson, P.H.; McMurray, C.H. Variation in the activities of glutathione peroxidase and superoxide
dismutase and in the concentration of copper in the blood in various breed crosses of sheep. Res. Vet. Sci. 1983, 34, 253–256.
[CrossRef] [PubMed]
38. Wang, H.; Huwaimel, B.; Verma, K.; Miller, J.; Germain, T.M.; Kinarivala, N.; Pappas, D.; Brookes, P.S.; Trippier, P.C. Synthesis
and antineoplastic evaluation of mitochondrial complex II (succinate dehydrogenase) inhibitors derived from atpenin A5.
ChemMedChem 2017, 12, 1033–1044. [CrossRef]
39. Li, Y.; D’Aurelio, M.; Deng, J.H.; Park, J.S.; Manfredi, G.; Hu, P.; Lu, J.; Bai, Y. An assembled complex IV maintains the stability
and activity of complex I in mammalian mitochondria. J. Biol. Chem. 2007, 282, 17557–17562. [CrossRef]
40. S, tefănescu, B.E.; Szabo, K.; Mocan, A.; Crişan, G. Phenolic compounds from five Ericaceae species leaves and their related
bioavailability and health benefits. Molecules 2019, 24, 2046. [CrossRef]
41. Mzhavanadze, V.V.; Targamadze, I.L.; Dranik, L.I. Polyphenols of the leaves of Vaccinium arctostaphylos. Chem. Nat. Compd. 1971,
7, 536–537. [CrossRef]
42. Liu, P.; Lindstedt, A.; Markkinen, N.; Sinkkonen, J.; Suomela, J.-P.; Yang, B. Characterization of metabolite profiles of leaves of
bilberry (Vaccinium myrtillus L.) and lingonberry (Vaccinium vitis-idaea L.). J. Agric. Food Chem. 2014, 62, 12015–12026. [CrossRef]
43. Mzhavanadze, V.V.; Targamadze, I.L.; Dranik, L.I. Phenolic compounds of the leaves of Vaccinium arctostaphylos. Chem. Nat.
Compd. 1972, 8, 125–126. [CrossRef]
44. Tadić, V.M.; Nešić, I.; Martinović, M.; Rój, E.; Brašanac-Vukanović, S.; Maksimović, S.; Žugić, A. Old plant, new possibilities: Wild
bilberry (Vaccinium myrtillus L., Ericaceae) in topical skin preparation. Antioxidants 2021, 10, 465. [CrossRef] [PubMed]
45. Ieri, F.; Martini, S.; Innocwnti, M.; Mulinacci, N. Phenolic distribution in liquid preparations of Vaccinium myrtillus L. and
Vaccinium vitis idaea L. Phytochem. Anal. 2013, 24, 467–475. [CrossRef] [PubMed]
46. Hasaloo, T.; Sepehrifar, R.; Hajimehdipoor, H. Levels of phenolic compounds and their effects on antioxidant capacity of wild
Vaccinium arctostaphylos L. (Qare-Qat) collected from different regions of Iran. Turk. J. Biol. 2011, 35, 13. [CrossRef]
Life 2022, 12, 2079 15 of 15
47. Oszmianski, J.; Wojdyło, A.; Gorzelany, J.; Kapusta, I. Identification and characterization of low molecular weight polyphenols in
berry leaf extracts by HPLC-DAD and LC-ESI/MS). J. Agric. Food Chem. 2011, 59, 12830–12835. [CrossRef]
48. Taram, F.; Winter, A.N.; Linseman, D.A. Neuroprotection comparison of chlorogenic acid and its metabolites against mechanisti-
cally distinct cell death-inducing agents in cultured cerebellar granule neurons. Brain Res. 2016, 1648, 69–80. [CrossRef]
49. Zheng, Y.; Li, L.; Chen, B.; Fang, Y.; Lin, W.; Zhang, T.; Feng, X.; Tao, X.; Wu, Y.; Fu, X.; et al. Chlorogenic acid exerts neuroprotective
effect against hypoxia-ischemia brain injury in neonatal rats by activating Sirt1 to regulate the Nrf2-NF-κB signaling pathway.
Cell Commun. Signal. 2022, 20, 84. [CrossRef]
50. Singh, S.S.; Rai, S.N.; Birla, H.; Zahra, W.; Rathore, A.S.; Dilnashin, H.; Singh, R.; Singh, S.P. Neuroprotective Effect of chlorogenic
acid on mitochondrial dysfunction-mediated apoptotic death of DA neurons in a Parkinsonian mouse model. Oxid. Med. Cell
Longev. 2020, 2020, 6571484. [CrossRef]
51. Khan, H.; Ullah, H.; Aschner, M.; Cheang, W.S.; Akkol, E.K. Neuroprotective effects of quercetin in Alzheimer’s disease.
Biomolecules 2019, 10, 59. [CrossRef]
52. Alvarez-Arellano, L.; Salazar-García, M.; Corona, J.C. Neuroprotective effects of quercetin in pediatric neurological diseases.
Molecules 2020, 25, 5597. [CrossRef]
53. Pu, F.; Mishima, K.; Irie, K.; Motohashi, K.; Tanaka, Y.; Orito, K.; Egawa, T.; Kitamura, Y.; Egashira, N.; Iwasaki, K.; et al.
Neuroprotective effects of quercetin and rutin on spatial memory impairment in an 8-arm radial maze task and neuronal death
induced by repeated cerebral ischemia in rats. J. Pharmacol. Sci. 2007, 104, 329–334. [CrossRef]
54. Gaire, B.P. Herbal medicine in ischemic stroke: Challenges and prospective. Chin. J. Integr. Med. 2018, 24, 243–246. [CrossRef]
[PubMed]
55. Mishra, A.; Mishra, P.S.; Bandopadhyay, R.; Khurana, N.; Angelopoulou, E.; Paudel, Y.N.; Piperi, C. Neuroprotective potential of
chrysin: Mechanistic insights and therapeutic potential for neurological disorders. Molecules 2021, 26, 6456. [CrossRef] [PubMed]
56. Wang, J.; Zhang, J.; Li, S.; Huang, C.; Xie, Y.; Cao, Y. Anthocyanins decrease the internalization of TiO2 nanoparticles into 3D
Caco-2 spheroids. Food Chem. 2020, 331, 127360. [CrossRef] [PubMed]
57. Wu, S.; Wu, B.; Liu, M.; Chen, Z.; Wang, W.; Anderson, C.S.; Sandercock, P.; Wang, Y.; Huang, Y.; Cui, L.; et al. China stroke study
collaboration. Stroke in China: Advances and challenges in epidemiology, prevention, and management. Lancet Neurol. 2019, 18,
394–405. [CrossRef]
58. Silva, S.; Costa, E.M.; Veiga, M.; Morais, R.M.; Calhau, C.; Pintado, M. Health promoting properties of blueberries: A review. Crit.
Rev. Food Sci. Nutr. 2020, 60, 181–200. [CrossRef]
59. He, Z.; Ning, N.; Zhou, Q.; Khoshnam, S.E.; Farzaneh, M. Mitochondria as a therapeutic target for ischemic stroke. Free Radic.
Biol. Med. 2020, 146, 45–58. [CrossRef]