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Neuroprotective and Antioxidant Enhancing Properties of Selective Equisetum Extracts

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The sterile stems belonging to the Equisetum species are often used in traditional medicine of various nations, including Romanians. They are highly efficient in treating urinary tract infections, cardiovascular diseases, respiratory tract infections, and medical skin conditions due to their content of polyphenolic derivatives that have been isolated. In this regard, this study aimed to provide the chemical composition of the extracts obtained from the Equisetum species (E. pratense, E. sylvaticum, E. telmateia) and to investigate the biological action in vitro and in vivo. For the chemical characterization of the analyzed Equisetum species extracts, studies were performed by using ultra-high-performance liquid chromatography (UHPLC-DAD). In vitro evaluation of the antioxidant activity of the plant extracts obtained from these species of Equisetum genus was determined. The neuroprotective activity of these three ethanolic extracts from the Equisetum species using zebrafish tests was determined in vivo. All obtained results were statistically significant. The results indicate that E. sylvaticum extract has a significant antioxidant activity; whereas, E. pratense extract had anxiolytic and antidepressant effects significantly higher than the other two extracts used. All these determinations indicate promising results for the antioxidant in vitro tests and neuroprotective activity of in vivo tests, particularly mediated by their active principles.
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molecules
Article
Neuroprotective and Antioxidant Enhancing Properties of
Selective Equisetum Extracts
Denisa Batir-Marin 1, Monica Boev 1, Oana Cioanca 2,*,† , Cornelia Mircea 2 ,, Ana Flavia Burlec 2 ,,
Galba Jean Beppe 3, , Adrian Spac 2, , Andreia Corciova 2, Lucian Hritcu 4,† and Monica Hancianu 2


Citation: Batir-Marin, D.; Boev, M.;
Cioanca, O.; Mircea, C.; Burlec, A.F.;
Beppe, G.J.; Spac, A.; Corciova, A.;
Hritcu, L.; Hancianu, M.
Neuroprotective and Antioxidant
Enhancing Properties of Selective
Equisetum Extracts. Molecules 2021,26,
2565. https://doi.org/10.3390/
molecules26092565
Academic Editor: Simona Rapposelli
Received: 28 February 2021
Accepted: 22 April 2021
Published: 28 April 2021
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Attribution (CC BY) license (https://
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4.0/).
1Department of Pharmaceutical Sciences, Faculty of Medicine and Pharmacy, Dunarea de Jos University,
800010 Galati, Romania; denisa.batir@ugal.ro (D.B.-M.); monica.boev@ugal.ro (M.B.)
2Faculty of Pharmacy, Grigore T. Popa University of Medicine and Pharmacy Iasi, 700115 Iasi, Romania;
cornelia.mircea@umfiasi.ro (C.M.); ana-flavia.l.burlec@umfiasi.ro (A.F.B.); adrian.spac@umfiasi.ro (A.S.);
maria.corciova@umfiasi.ro (A.C.); monica.hancianu@umfiasi.ro (M.H.)
3
Department of Biological Sciences, Faculty of Science, University of Maroua, Maroua P.O. Box 814, Cameroon;
jean.beppe-galba@fs.univ-maroua.cm
4Department of Biology, Faculty of Biology, Alexandru Ioan Cuza University of Iasi, 700506 Iasi, Romania;
hritcu@uaic.ro
*Correspondence: oana.cioanca@umfiasi.ro; Tel.: +40-232-301-815
These authors equally contributed to this work.
Abstract:
The sterile stems belonging to the Equisetum species are often used in traditional medicine
of various nations, including Romanians. They are highly efficient in treating urinary tract infec-
tions, cardiovascular diseases, respiratory tract infections, and medical skin conditions due to their
content of polyphenolic derivatives that have been isolated. In this regard, this study aimed to
provide the chemical composition of the extracts obtained from the Equisetum species (E. pratense,
E. sylvaticum
,E. telmateia) and to investigate the biological action
in vitro
and
in vivo
. For the chem-
ical characterization of the analyzed Equisetum species extracts, studies were performed by using
ultra-high-performance liquid chromatography (UHPLC-DAD).
In vitro
evaluation of the antioxi-
dant activity of the plant extracts obtained from these species of Equisetum genus was determined.
The neuroprotective activity of these three ethanolic extracts from the Equisetum species using ze-
brafish tests was determined
in vivo
. All obtained results were statistically significant. The results
indicate that E. sylvaticum extract has a significant antioxidant activity; whereas, E. pratense extract
had anxiolytic and antidepressant effects significantly higher than the other two extracts used. All
these determinations indicate promising results for the antioxidant
in vitro
tests and neuroprotective
activity of in vivo tests, particularly mediated by their active principles.
Keywords:
Equisetum pratense;Equisetum telmateia;Equisetum sylvaticum; UHPLC; antioxidant activity;
neuroprotective; polyphenolic derivatives
1. Introduction
The Horsetails (Equisetaceae) are one of the oldest groups of vascular plants. Despite
the small size of the family and a limited number of morphological characters, most species
of horsetails are characterized by very high morphological variability [
1
]. Equisetum L.
genus is comprised of approximately 15 extant species with a nearly worldwide distribu-
tion [
2
,
3
]. Research in the past decades has shown an increased interest in phytochemical
products and plant extracts, due to frequent use in the prevention and treatment of some
diseases. Several studies have demonstrated that the antioxidants found in plants are of
major interest to medicine since they protect the organism against oxidative stress, gen-
erated in the context of some diseases: atherosclerosis, ischemic cardiac disease, cancer,
Alzheimer’s disease, Parkinson’s disease, aging, and even in infectious diseases [
4
6
]. Nev-
ertheless, various tests are still underway for the majority of the positive results obtained
Molecules 2021,26, 2565. https://doi.org/10.3390/molecules26092565 https://www.mdpi.com/journal/molecules
Molecules 2021,26, 2565 2 of 18
in vitro
and/or
in vivo
, and only a few are transferable to clinical uses. Furthermore, other
lab data confirmed the traditional uses of medicinal and aromatic plants.
Equisetum species are perennial ferns from the Equisetaceae family. They have a
fertile stem bearing yellowish spores, produced in early spring. The green, photosynthetic,
strongly branched, sterile stems are produced in late spring and persist until late autumn.
The sterile strain is the medicinal product of the Equisetum arvense L. plant (Equiseti herba)
mentioned in the European Pharmacopoeia. Its well-established use is as a diuretic and
is solely based on long term usage in traditional medicine. However, the present study is
based on the phytochemical analysis of the chemical composition, but also on the
in vitro
and
in vivo
biological action of three different species of Equisetum: E. pratense Ehrh.,
E. sylvaticum L. and E. telmateia Ehrh. which are found in the wild flora of Romania.
The most widely known phytochemical compounds of the Equisetum genus are
flavonoids, phenolic acids, alkaloids, phytosterols, tannins, and triterpenoids [
7
]. However,
such compounds are common to the majority of the superior plants. Experimental data
published so far and in this paper show that polyphenolic derivatives that have been
isolated from Equisetum species can provide indirect protection by activating antioxidant
transcription in the promoter regions of genes that induce oxidative stress [
5
,
8
]. Besides,
polyphenols can modulate both the cellular signal involved in cell proliferation and cell
development itself by inhibiting inflammation and initiating the process of apoptosis of
damaged cells [9,10].
Oxygen is the essential element for maintaining cellular activities, and also for the
body in general [
11
]. Penetrated in the form of gas, through the respiratory tract, molecular
oxygen is mainly transformed into water in the biochemical processes in the respiratory
chain and thus allows the production of the energy required for cellular processes, in the
form of ATP [
11
]. Under physiological conditions, some of this oxygen is transformed into
reactive oxygen species (ROS), for which neutralization systems in the form of antioxidant
substances or enzymes are developed at the cellular level. Long-term or repeated expo-
sure of the body to oxidative stress causes the development of pathological phenomena,
including cancer [
12
14
]. In these situations, it is necessary to supplement the antioxidant
defense capacity at the cellular level by administering antioxidant vitamins (vitamins C
and E) or plant extracts that contain antioxidants [
15
,
16
]. Numerous tests to evaluate
the antioxidant action
in vitro
have been developed over time, as a preliminary step to
testing the antioxidant effects
in vivo
[
17
]. From these tests, those selected were based
on antioxidant mechanisms that can also occur in the biological environment, such as
chelation of ferrous ion, lipoxygenase inhibition, the scavenger capacity of the hydroxyl
radical, and the superoxide radical anion [18].
Neurodegenerative diseases have become increasingly intensively studied, and their
early detection is the point of interest in understanding their etiology. Most inducing
processes of neurodegeneration cause the initiation of a chemical cascade that results in
apoptosis or necrosis of neurons. This manifests itself in the form of loss of certain neuro-
logical and cognitive functions [
19
,
20
]. Most often, these degenerative manifestations are
accompanied by regenerative or neuroprotective compensatory mechanisms such as the
presence of antioxidant enzymes, neurotrophic growth factors, or peptides with a regu-
latory role [
19
]. By discovering the most common processes of neurodegeneration—and
also neuroprotection—intervention strategies can be drawn in the progress of various
neurodegenerative diseases [
21
]. Neuroprotection is a sum of strategies to protect the
nervous system against neuronal damage or degeneration caused by events such as neu-
rodegenerative diseases, cerebral ischemia, or various traumas that may occur. The goal of
neuroprotection is to limit the spread of neural apoptosis and minimize neural dysfunction
through mechanisms to maintain the integrity of cellular interactions [1921].
The present study aims to highlight the chemical composition of the extracts obtained
from the Equisetum species (E. pratense,E. sylvaticum,E. telmateia) and to investigate the
biological action in vitro and in vivo.
Molecules 2021,26, 2565 3 of 18
2. Results and Discussion
2.1. Obtaining Extracts
Sterile stems of E. pratense Ehrh., E. telmateia Ehrh. and E. sylvaticum L. were used.
Two types of total extracts were obtained using different solvents: 70% methanolic and 70%
ethanolic. To obtain extracts rich in polyphenolic compounds, the extraction with these
solvents was performed, taking into account the polar character of these compounds and
the high extractability in such solvents.
2.2. Semi-Quantitative and Qualitative Determination of the Polyphenolic Compounds
(UHPLC-DAD)
The presence of the following polyphenolic derivatives in the two types of extracts
(methanolic and ethanolic) obtained from the three investigated Equisetum samples was
confirmed by the liquid chromatography technique: chlorogenic acid, caffeic acid, ferulic
acid, as well as glycosylated derivatives of quercetin and luteolin. Such active principles
have also been identified by other authors in extracts obtained by various methods from
E. arvense [
13
,
17
,
22
,
23
]. According to the spectral and quantitative analysis, the flavonoid
components quantified in the samples are presented briefly in Table 1.
Table 1. Flavonoids identified and quantified in the investigated samples.
Compound Expressed in
mg/g Plant Product *
Sample (70% Methanolic Extract) Sample (70% Ethanolic Extract)
E.
sylvaticum E. pratense E.
telmateia
E.
sylvaticum E. pratense E.
telmateia
epicatechin 0.9010 ±0.021 0.5925 ±0.001 1.1967 ±0.001 0.1586 ±0.002 0.0263 ±0.003 0.7079 ±0.011
quercetin-3-glucoside 1.6995 ±0.001 8.2442 ±0.001 23.4765 ±0.033 41.9429 ±0.022 3.8440 ±0.001 13.6175 ±0.022
luteolin-7-glucoside 11.0456 ±0.011 1.8562 ±0.001 5.6447 ±0.021 3.7656 ±0.019 0.0378 ±0.011 1.2254 ±0.003
apigenin-7-glucoside 2.3249 ±0.013 2.2947 ±0.021 27.7463 ±0.009 7.8806 ±0.003 1.4626 ±0.004 21.0042 ±0.021
luteolin 0.6150 ±0.011 0.0344 ±0.003 0.4214 ±0.009 0.4069 ±0.011 0.0076 ±0.021 0.1455 ±0.011
quercetin 0.0658 ±0.001 0.0059 ±0.002 0.4013 ±0.022 0.0255 ±0.009 0.0071 ±0.001 0.1369 ±0.005
apigenin 0.0716 ±0.002 0.6260 ±0.009 0.9571 ±0.009 0.0591 ±0.001 0.1359 ±0.002 0.3758 ±0.001
kaempferol 1.4997 ±0.021 0.9781 ±0.009 1.4417 ±0.009 0.0216 ±0.009 0.2022 ±0.001 0.7609 ±0.004
* the results represent the average value of triplicate quantification; limit of detection (LOD): 280 ng/mL; limit of quantification (LOQ):
145 ng/mL.
Quantitatively, for the methanolic extracts, E. telmateia and E. sylvaticum are richer in
the flavonoid fraction than E. pratense, being evident that the glycosylated derivatives of
quercetin, luteolin, and apigenin are predominant in all three samples. It is noted the low
solubility of the flavonoid aglycones in the hydroalcoholic solution, but similar to the stud-
ies of other researchers, the extraction yield also varies depending on the chemical structure
of each component. Quercetin glycosides are predominant in the E. sylvaticum sample, and
in the E. telmateia sample, quercetin and apigenin glycosides represent the majority. The
noted differences between the methanolic and ethanolic extracts for the same species are
related to each compound affinity towards the solvent. Generally, higher extraction yields
are obtained for phenolic acids and glycosylated flavonoids in hydroalcoholic solutions
rather than organic solvents, which is observed in our results. Although the solvent is
one of the main factors influencing the extraction of certain compounds, the temperature
and the use of shaking or stirring can also impact the extractability. Interestingly, ultra-
sounds favored the extraction of quercetin-3-glucoside in E. sylvaticum ethanolic extract,
whereas reflux extraction in methanol 70% increased the extractability of the majority of
the flavonoid glycosides for all species. This can be explained to some extent taking into
account that ultrasounds break molecular structures, leading probably to artefacts or more
lipophilic structures which were not extracted nor identified in the investigated samples.
The concentrations of the four identified polyphenolic acids are shown in Table 2.
Molecules 2021,26, 2565 4 of 18
Table 2. Polyphenolcarboxylic acids identified in the methanolic and ethanolic extracts of Equisetum species.
Compound
Expressed in mg/g
Plant Product *
Sample (70% Methanolic Extract) Sample (70% Ethanolic Extract)
E.
sylvaticum E. pratense E.
telmateia
E.
sylvaticum E. pratense E.
telmateia
neochlorogenic acid 0.2639 ±0.001 0.0730 ±0.001 0.6381 ±0.005 0.4838 ±0.002 0.1879 ±0.002 0.3059 ±0.002
chlorogenic acid 4.3322 ±0.001 0.7491 ±0.002 10.1162 ±0.002 3.6744 ±0.001 0.7362 ±0.003 14.3814 ±0.004
caffeic acid 0.1468 ±0.002 0.2363 ±0.002 0.8590 ±0.003 0.6624 ±0.002 0.1922 ±0.001 0.3244 ±0.002
ferulic acid 0.6414 ±0.002 0.2295 ±0.005 1.0245 ±0.001 5.7962 ±0.004 0.5559 ±0.001 1.0439 ±0.003
* the results represent the average value of average value of triplicate quantification.
For the methanolic extracts, the polyphenolic acid with the highest content is chloro-
genic acid, regardless of the investigated sample, but the maximum content is found in
E. telmateia
(approx. 10 mg/g), while the proportion decreases in half in E. sylvaticum and
less than 7% in E. pratense. In general, the amount of polyphenol carboxylic acids is higher
in hydroalcoholic extracts than in methanolic extracts, a fact proved by the high content of
ferulic acid (E. sylvaticum) and chlorogenic acid (E. telmateia). Such differences could also
be related to the growing environment and the biosynthetic capacity of each species. Our
specimens were collected from three different regions in which the species grow usually in
larger amounts. E. sylvaticum was collected from the highest altitude (915 m) within the
spruce forest, E. pratense from 681 m, placed along the riverbank’s bushes and E. telmateia
from the edges of the forests at a lower altitude (259 m). More research is still undergoing
in this aspect.
2.3. In Vitro Evaluation of the Antioxidant Activity of the Plant Extracts Obtained from the Three
Species of the Equisetum Genus
2.3.1. The Chelating Capacity of the Ferrous Ion Determination
The ferrous ion is present in serum and intracellular fluid in extremely small amounts,
but when its concentration increases and its protein binding capacity is low, it may be
involved in pathological phenomena [
22
]. At the cellular level, it participates in the Fenton
and Haber-Weiss reactions of hydroxyl radical generation, one of the most aggressive
free radicals involved in the occurrence of oxidative stress [
23
]. The chelating capacity of
ferrous ions is an important indicator for the evaluation of the antioxidant activity. The
results obtained in this test are shown in Figure 1.
Molecules 2021, 26, x FOR PEER REVIEW 4 of 19
The concentrations of the four identified polyphenolic acids are shown in Table 2.
Table 2. Polyphenolcarboxylic acids identified in the methanolic and ethanolic extracts of Equisetum species.
Compound
Expressed in mg/g Plant
Product *
Sample (70% Methanolic Extract) Sample (70% Ethanolic Extract)
E.
sylvaticum E. pratense E.
telmateia
E.
sylvaticum E. pratense E.
telmateia
neochlorogenic acid 0.2639 ± 0.001 0.0730 ± 0.001 0.6381 ± 0.005 0.4838 ± 0.002 0.1879 ± 0.002 0.3059 ± 0.002
chlorogenic acid 4.3322 ± 0.001 0.7491 ± 0.002 10.1162 ± 0.002 3.6744 ± 0.001 0.7362 ± 0.003 14.3814 ± 0.004
caffeic acid 0.1468 ± 0.002 0.2363 ± 0.002 0.8590 ± 0.003 0.6624 ± 0.002 0.1922 ± 0.001 0.3244 ± 0.002
ferulic acid 0.6414 ± 0.002 0.2295 ± 0.005 1.0245 ± 0.001 5.7962 ± 0.004 0.5559 ± 0.001 1.0439 ± 0.003
* the results represent the average value of average value of triplicate quantification.
For the methanolic extracts, the polyphenolic acid with the highest content is chloro-
genic acid, regardless of the investigated sample, but the maximum content is found in E.
telmateia (approx. 10 mg/g), while the proportion decreases in half in E. sylvaticum and less
than 7% in E. pratense. In general, the amount of polyphenol carboxylic acids is higher in
hydroalcoholic extracts than in methanolic extracts, a fact proved by the high content of
ferulic acid (E. sylvaticum) and chlorogenic acid (E. telmateia). Such differences could also
be related to the growing environment and the biosynthetic capacity of each species. Our
specimens were collected from three different regions in which the species grow usually
in larger amounts. E. sylvaticum was collected from the highest altitude (915 m) within the
spruce forest, E. pratense from 681 m, placed along the riverbank’s bushes and E. telmateia
from the edges of the forests at a lower altitude (259 m). More research is still undergoing
in this aspect.
2.3. In Vitro Evaluation of the Antioxidant Activity of the Plant Extracts Obtained from the
Three Species of the Equisetum Genus
2.3.1. The Chelating Capacity of the Ferrous Ion Determination
The ferrous ion is present in serum and intracellular fluid in extremely small
amounts, but when its concentration increases and its protein binding capacity is low, it
may be involved in pathological phenomena [22]. At the cellular level, it participates in
the Fenton and Haber-Weiss reactions of hydroxyl radical generation, one of the most
aggressive free radicals involved in the occurrence of oxidative stress [23]. The chelating
capacity of ferrous ions is an important indicator for the evaluation of the antioxidant
activity. The results obtained in this test are shown in Figure 1.
Figure 1. Graphical representation of the mean values (n = 5) of the iron-chelating capacity (%) depending on the concen-
tration of the methanolic (A) and ethanolic (B) extracts used. Legend: ESm—E. sylvaticum methanolic, EPm—E. pratense
methanolic, ETm—E. telmateia methanolic; ESe—E. sylvaticum ethanolic, EPe—E. pratense ethanolic, ETe—E. telmateia eth-
anolic.
Figure 1.
Graphical representation of the mean values (n= 5) of the iron-chelating capacity (%) depending on the
concentration of the methanolic (
A
) and ethanolic (
B
) extracts used. Legend: ESm—E. sylvaticum methanolic, EPm—E.
pratense methanolic, ETm—E. telmateia methanolic; ESe—E. sylvaticum ethanolic, EPe—E. pratense ethanolic, ETe—E. telmateia
ethanolic.
At concentrations above 3 mg/mL, the methanolic extract 70% of E. sylvaticum is
more active compared to the gallic acid, this time the phyto-complex present in the extract
Molecules 2021,26, 2565 5 of 18
achieving an additional synergy compared to a single compound. The analysis of ethanolic
extracts 70% indicates again the superiority of the extract obtained from E. sylvaticum, but,
unlike the methanol one, it is not the richest in polyphenols of the three extracts analyzed.
This time the difference in efficacy between the extracts of E. telmateia and E. pratense
increases in favor of the first extract.
2.3.2. Determination of the Lipoxygenase Inhibition Capacity
Lipoxygenase (EC.1.13.11.33) is an enzyme of the oxidoreductase class that catalyzes
the oxidation of unsaturated fatty acids with the formation of peroxides [
24
]. They are
involved in oxidative phenomena at the cellular level increasing the oxidation rate of
lipids with the appearance of pathological phenomena. The enzyme is also involved in the
synthesis of inflammation mediators [
25
]. The results obtained for the evaluation of the
lipoxygenase inhibition capacity are shown in Figure 2.
Molecules 2021, 26, x FOR PEER REVIEW 5 of 19
At concentrations above 3 mg/mL, the methanolic extract 70% of E. sylvaticum is more
active compared to the gallic acid, this time the phyto-complex present in the extract
achieving an additional synergy compared to a single compound. The analysis of etha-
nolic extracts 70% indicates again the superiority of the extract obtained from E. sylvati-
cum, but, unlike the methanol one, it is not the richest in polyphenols of the three extracts
analyzed. This time the difference in efficacy between the extracts of E. telmateia and E.
pratense increases in favor of the first extract.
2.3.2. Determination of the Lipoxygenase Inhibition Capacity
Lipoxygenase (EC.1.13.11.33) is an enzyme of the oxidoreductase class that catalyzes
the oxidation of unsaturated fatty acids with the formation of peroxides [24]. They are
involved in oxidative phenomena at the cellular level increasing the oxidation rate of li-
pids with the appearance of pathological phenomena. The enzyme is also involved in the
synthesis of inflammation mediators [25]. The results obtained for the evaluation of the
lipoxygenase inhibition capacity are shown in Figure 2.
Figure 2. Graphical representation of the mean values (n = 5) of the lipoxygenase inhibition capacity (%) depending on the concen-
tration of the methanolic (A) and ethanolic extracts (B) used. Legend: ESm—E. sylvaticum methanolic, EPm—E. pratense methanolic,
ETm—E. telmateia methanolic; ESe—E. sylvaticum ethanolic, EPe—E. pratense ethanolic, ETe—E. telmateia ethanolic.
EPe, the ethanolic extract from
E. pratense
, has a better inhibitory capacity against
lipoxygenase than its own methanolic sample (EPm). Generally, comparing the values
obtained for this test for both types of extracts, we observe that ethanolic extracts are more
active than the methanolic, but lower than the chosen standard. However, at 10 mg/mL
ESe proves a similar potential with gallic acid. For both methanol and ethanolic extracts,
the best antioxidant activity determined for this test was obtained for the E. sylvaticum
species, the values recorded especially for the ethanol extract were slightly higher, but
comparable to those of gallic acid, used as a reference. All extracts obtained from the three
species of the genus Equisetum showed lower efficiency than gallic acid.
2.3.3. Determination of the Scavenger Action of the Hydroxyl Radical
The hydroxyl radical causes the initiation of oxidation reactions of unsaturated fatty
acids that are present in the structure of membrane phospholipids, and their oxidation
under conditions of oxidative stress will affect the stability of the cell membrane with the
risk of uncontrolled loss of cell contents or reduced cell viability. Last but not least, this
hydroxyl radical induces the oxidation of proteins with the modification of their spatial
structure and affects their biological functions. In vitro and in vivo studies have shown
the negative effect of plasma hydroxyl radical on the stability of fibrinogen which will be
more easily converted to fibrin, which ultimately leads to increased blood coagulation
[10,15]. The results obtained when evaluating the scavenger capacity of the hydroxyl rad-
ical are presented in Figure 3.
Figure 2.
Graphical representation of the mean values (n= 5) of the lipoxygenase inhibition capacity (%) depending
on the concentration of the methanolic (
A
) and ethanolic extracts (
B
) used. Legend: ESm—E. sylvaticum methanolic,
EPm—
E. pratense
methanolic, ETm—E. telmateia methanolic; ESe—E. sylvaticum ethanolic, EPe—E. pratense ethanolic,
ETe—E. telmateia ethanolic.
EPe, the ethanolic extract from E. pratense, has a better inhibitory capacity against
lipoxygenase than its own methanolic sample (EPm). Generally, comparing the values
obtained for this test for both types of extracts, we observe that ethanolic extracts are more
active than the methanolic, but lower than the chosen standard. However, at 10 mg/mL
ESe proves a similar potential with gallic acid. For both methanol and ethanolic extracts,
the best antioxidant activity determined for this test was obtained for the E. sylvaticum
species, the values recorded especially for the ethanol extract were slightly higher, but
comparable to those of gallic acid, used as a reference. All extracts obtained from the three
species of the genus Equisetum showed lower efficiency than gallic acid.
2.3.3. Determination of the Scavenger Action of the Hydroxyl Radical
The hydroxyl radical causes the initiation of oxidation reactions of unsaturated fatty
acids that are present in the structure of membrane phospholipids, and their oxidation
under conditions of oxidative stress will affect the stability of the cell membrane with the
risk of uncontrolled loss of cell contents or reduced cell viability. Last but not least, this
hydroxyl radical induces the oxidation of proteins with the modification of their spatial
structure and affects their biological functions.
In vitro
and
in vivo
studies have shown the
negative effect of plasma hydroxyl radical on the stability of fibrinogen which will be more
easily converted to fibrin, which ultimately leads to increased blood coagulation [
10
,
15
].
The results obtained when evaluating the scavenger capacity of the hydroxyl radical are
presented in Figure 3.
Molecules 2021,26, 2565 6 of 18
Molecules 2021, 26, x FOR PEER REVIEW 6 of 19
Figure 3. Graphical representation of the mean values (n = 5) of the scavenger capacity of the hydroxyl radical (%) depend-
ing on the concentration of the methanolic (A) and ethanolic extracts (B) used. Legend: ESm—E. sylvaticum methanolic,
EPm—E. pratense methanolic, ETm—E. telmateia methanolic; ESe—E. sylvaticum ethanolic, EPe—E. pratense ethanolic,
ETe—E. telmateia ethanolic.
Methanolic extracts have a higher scavenger capacity of the hydroxyl radical com-
pared to ethanolic ones, but without exceeding the efficiency of gallic acid used as a ref-
erence substance. The lowest scavenger activity of the hydroxyl radical was recorded for
hydro-alcoholic extracts of E. pratense. The methanolic extract of the species E. sylvaticum
showed the most important action, being slightly higher than that of the reference sub-
stance, gallic acid. Neutralization of the hydroxyl radical depends very much on the pres-
ence in the structure of the compound or scavenger compounds of groups capable of
yielding hydrogen atoms, such as OH groups in the structure of polyphenols. In ethanolic
extracts, 70%, glycosylated forms of polyphenols predominate, compared to methanolic
extracts in which aglycones containing several free OH groups and capable of reducing
action predominates.
2.3.4. Determination of the Scavenger Capacity of the Superoxide Anion
The superoxide radical anion is formed by the reduction of molecular oxygen that
accepts a single electron, most often in the side reactions of the mitochondrial respiratory
chain. Chemically, it has a medium oxidizing character but can generate hydroxyl radical
and singlet oxygen which are very strong oxidants [26,27]. The results obtained when
evaluating the scavenger capacity of the superoxide radical anion are presented in Figure
4.
Figure 4. Graphical representation of the mean values (n = 5) of the scavenger capacity of the superoxide radical anion (%)
depending on the concentration of the methanolic (A) and ethanolic (B) extracts. Legend: ESm—E. sylvaticum methanolic,
EPm—E. pratense methanolic, ETm—E. telmateia methanolic; ESe—E. sylvaticum ethanolic, EPe—E. pratense ethanolic,
ETe—E. telmateia ethanolic.
Figure 3.
Graphical representation of the mean values (n= 5) of the scavenger capacity of the hydroxyl radical (%) depending
on the concentration of the methanolic (
A
) and ethanolic extracts (
B
) used. Legend: ESm—E. sylvaticum methanolic, EPm—E.
pratense methanolic, ETm—E. telmateia methanolic; ESe—E. sylvaticum ethanolic, EPe—E. pratense ethanolic, ETe—E. telmateia
ethanolic.
Methanolic extracts have a higher scavenger capacity of the hydroxyl radical compared
to ethanolic ones, but without exceeding the efficiency of gallic acid used as a reference
substance. The lowest scavenger activity of the hydroxyl radical was recorded for hydro-
alcoholic extracts of E. pratense. The methanolic extract of the species E. sylvaticum showed
the most important action, being slightly higher than that of the reference substance,
gallic acid. Neutralization of the hydroxyl radical depends very much on the presence
in the structure of the compound or scavenger compounds of groups capable of yielding
hydrogen atoms, such as OH groups in the structure of polyphenols. In ethanolic extracts,
70%, glycosylated forms of polyphenols predominate, compared to methanolic extracts
in which aglycones containing several free OH groups and capable of reducing action
predominates.
2.3.4. Determination of the Scavenger Capacity of the Superoxide Anion
The superoxide radical anion is formed by the reduction of molecular oxygen that
accepts a single electron, most often in the side reactions of the mitochondrial respiratory
chain. Chemically, it has a medium oxidizing character but can generate hydroxyl radical
and singlet oxygen which are very strong oxidants [
26
,
27
]. The results obtained when
evaluating the scavenger capacity of the superoxide radical anion are presented in Figure 4.
Molecules 2021, 26, x FOR PEER REVIEW 6 of 19
Figure 3. Graphical representation of the mean values (n = 5) of the scavenger capacity of the hydroxyl radical (%) depend-
ing on the concentration of the methanolic (A) and ethanolic extracts (B) used. Legend: ESm—E. sylvaticum methanolic,
EPm—E. pratense methanolic, ETm—E. telmateia methanolic; ESe—E. sylvaticum ethanolic, EPe—E. pratense ethanolic,
ETe—E. telmateia ethanolic.
Methanolic extracts have a higher scavenger capacity of the hydroxyl radical com-
pared to ethanolic ones, but without exceeding the efficiency of gallic acid used as a ref-
erence substance. The lowest scavenger activity of the hydroxyl radical was recorded for
hydro-alcoholic extracts of E. pratense. The methanolic extract of the species E. sylvaticum
showed the most important action, being slightly higher than that of the reference sub-
stance, gallic acid. Neutralization of the hydroxyl radical depends very much on the pres-
ence in the structure of the compound or scavenger compounds of groups capable of
yielding hydrogen atoms, such as OH groups in the structure of polyphenols. In ethanolic
extracts, 70%, glycosylated forms of polyphenols predominate, compared to methanolic
extracts in which aglycones containing several free OH groups and capable of reducing
action predominates.
2.3.4. Determination of the Scavenger Capacity of the Superoxide Anion
The superoxide radical anion is formed by the reduction of molecular oxygen that
accepts a single electron, most often in the side reactions of the mitochondrial respiratory
chain. Chemically, it has a medium oxidizing character but can generate hydroxyl radical
and singlet oxygen which are very strong oxidants [26,27]. The results obtained when
evaluating the scavenger capacity of the superoxide radical anion are presented in Figure
4.
Figure 4. Graphical representation of the mean values (n = 5) of the scavenger capacity of the superoxide radical anion (%)
depending on the concentration of the methanolic (A) and ethanolic (B) extracts. Legend: ESm—E. sylvaticum methanolic,
EPm—E. pratense methanolic, ETm—E. telmateia methanolic; ESe—E. sylvaticum ethanolic, EPe—E. pratense ethanolic,
ETe—E. telmateia ethanolic.
Figure 4.
Graphical representation of the mean values (n= 5) of the scavenger capacity of the superoxide radical anion (%)
depending on the concentration of the methanolic (
A
) and ethanolic (
B
) extracts. Legend: ESm—E. sylvaticum methanolic,
EPm—E. pratense methanolic, ETm—E. telmateia methanolic; ESe—E. sylvaticum ethanolic, EPe—E. pratense ethanolic,
ETe—E. telmateia ethanolic.
Molecules 2021,26, 2565 7 of 18
It is noted that the antioxidant efficacy for this test is closer in value to the methanolic
extracts of E. sylvaticum and E. telmateia compared to the reference substance, gallic acid.
As with all other determinations of antioxidant action, E. pratense showed the lowest action
for both methanolic and ethanolic extract. If the presence of hydrogen donor groups in the
scavenger molecule is necessary to neutralize the hydroxyl radical, then both hydrogen
donor groups and functional groups capable of neutralizing the anion charge are needed
to neutralize the superoxide radical anion.
2.4. In Vivo Evaluation of the Neuroprotective Activity of the Plant Extracts Obtained from the
Three Species of Equisetum
Taking into account all results, the common use of the medicinal species as tinctures,
the potential use without major risks (usually given by residual solvents, such as methanol),
the environment protection and, above all, the solubility of the extracts in water, we decided
to evaluate the neuroprotective action only for the ethanolic extracts of Equisetum species
(E. pratense Ehrh., E. sylvaticum L., and E. telmateia Ehrh.) by administering the samples
to zebrafish. Moreover, in the preliminary studies we observed no toxicity or significant
behavior changes in two control groups placed in a fish tank, dosed as high as 5 mg/L.
The ethanolic extracts were completely soluble in the water without any residues, which is
essential in testing in a water tank. In this regard, using specific behavioral tests, the effects
on spatial memory (Y-maze test) and anxiety-like behavior (novel tank diving test—NTT)
were followed.
2.4.1. Novel Tank Diving Test (NTT)
The NTT test is used to assess the anxiety-like response. The position of the animal
in the tank (at the top or bottom of the tank) is considered to be an index of anxiety [
28
].
For each type of the Equisetum ethanolic extract (EPe, ESe, ETe), individual tests were
performed at both 0.5 mg/L and 1 mg/L dose.
In the NTT test, one-way ANOVA demonstrated a significant effect of the treatment
on the number of entries in the top zone of the tank in different groups (F(4,45) = 25.22,
p< 0.0001)
(Figure 5A), and the time spent in the top zone of the tank (F(4,45) = 12.16,
p< 0.0001
) (Figure 5B). Zebrafish exposed to scopolamine (Sco) exhibited a significant
decrease in the time spent in the top zone of the tank as compared to the control group
(Figure 5B, p< 0.001), suggesting high levels of anxiety. Moreover, treatment with EPe
prevented the amnesic effect of Sco, as evidenced by increases in the number of entries in
the top zone of the tank (Figure 5A, p< 0.001 for 0.5 mg/L and p< 0.0001 for 1 mg/L) as
compared to Sco-alone treated group, whereas a significant increase in the time spent in
the top zone of the tank was noticed for the 1 mg/L (Figure 5B, p< 0.001). Imipramine
(IMP, 20 mg/L), a tricyclic antidepressant, was used as a reference drug in the NTT test.
In the NTT test, one-way ANOVA demonstrated a significant effect of the treatment
on the number of entries in the top zone of the tank in different groups (F(4,45) = 16.26,
p< 0.0001) (Figure 6A), and the time spent in the top zone of the tank (F(4,45) = 15.86,
p< 0.0001) (Figure 6B). Administration of Sco induced a significant decrease in the time
spent in the top zone of the tank as compared to the control group (Figure 6B, p< 0.001),
suggesting high levels of anxiety. Moreover, treatment with ESe prevented the amnesic
effect of Sco, as evidenced by increases in the number of entries in the top zone of the tank
(Figure 6A, p< 0.0001 for 1 mg/L) as compared to the Sco-alone treated group, whereas a
significant increase in the time spent in the top zone of the tank was noticed for the 1 mg/L
(Figure 6B, p< 0.01).
Molecules 2021,26, 2565 8 of 18
Molecules 2021, 26, x FOR PEER REVIEW 8 of 19
Figure 5. The Equisetum pratense ethanolic extract (EPe, 0.5 and 1 mg/L) reduced the anxiety-like response in the scopola-
mine (Sco, 100 μM)-induced zebrafish in the novel tank diving (NTT) test. (A) represent the number of entries in the top
zone of the tank in different groups; (B) represent the time spent in the top zone of the tank (s) in different groups. For
Tukey’s posthoc analysis: Sco vs. EPe (0.5 mg/L): ## p < 0.001; Sco vs. EPe (1 mg/L): ### p < 0.0001 (A); Sco vs. EPe (1 mg/L):
## p < 0.001 (B).
In the NTT test, one-way ANOVA demonstrated a significant effect of the treatment
on the number of entries in the top zone of the tank in different groups (F(4,45) = 16.26, p
< 0.0001) (Figure 6A), and the time spent in the top zone of the tank (F(4,45) = 15.86, p <
0.0001) (Figure 6B). Administration of Sco induced a significant decrease in the time spent
in the top zone of the tank as compared to the control group (Figure 6B, p < 0.001), sug-
gesting high levels of anxiety. Moreover, treatment with ESe prevented the amnesic effect
of Sco, as evidenced by increases in the number of entries in the top zone of the tank (Fig-
ure 6A, p < 0.0001 for 1 mg/L) as compared to the Sco-alone treated group, whereas a
significant increase in the time spent in the top zone of the tank was noticed for the 1 mg/L
(Figure 6B, p < 0.01).
Figure 6. The Equisetum sylvaticum ethanolic extract (ESe, 0.5 and 1 mg/L) reduced the anxiety-like response in the scopol-
amine (Sco, 100 μM)-induced zebrafish in the novel tank diving (NTT) test. (A) represent the number of entries in the top
zone of the tank in different groups; (B) represent the time spent in the top zone of the tank (s) in different groups. For
Tukey’s posthoc analysis: Sco vs. ESe (1 mg/L): ### p < 0.0001 (A); Sco vs. ESe (1 mg/L): # p < 0.01 (B).
Figure 5.
The Equisetum pratense ethanolic extract (EPe, 0.5 and 1 mg/L) reduced the anxiety-like response in the scopolamine
(Sco, 100
µ
M)-induced zebrafish in the novel tank diving (NTT) test. (
A
) represent the number of entries in the top zone of
the tank in different groups; (
B
) represent the time spent in the top zone of the tank (s) in different groups. For Tukey’s
posthoc analysis: Sco vs. EPe (0.5 mg/L): ## p< 0.001; Sco vs. EPe (1 mg/L): ### p< 0.0001 (
A
); Sco vs. EPe (1 mg/L):
## p< 0.001 (B).
Figure 6.
The Equisetum sylvaticum ethanolic extract (ESe, 0.5 and 1 mg/L) reduced the anxiety-like response in the
scopolamine (Sco, 100
µ
M)-induced zebrafish in the novel tank diving (NTT) test. (
A
) represent the number of entries in the
top zone of the tank in different groups; (
B
) represent the time spent in the top zone of the tank (s) in different groups. For
Tukey’s posthoc analysis: Sco vs. ESe (1 mg/L): ### p< 0.0001 (A); Sco vs. ESe (1 mg/L): # p< 0.01 (B).
In the NTT test, one-way ANOVA demonstrated a significant effect of the treatment
on the number of entries in the top zone of the tank in different groups (F(4,45) = 18.21,
p< 0.0001
) (Figure 7A), and the time spent in the top zone of the tank (F(4,45) = 8.35,
p< 0.0001
) (Figure 7B). Exposure to Sco resulted in a significant decrease in the time
spent in the top zone of the tank as compared to the control group (Figure 7B, p< 0.001),
suggesting high levels of anxiety. Moreover, treatment with ETe prevented the amnesic
effect of Sco, as evidenced by increases in the number of entries in the top zone of the
tank (Figure 7A, p< 0.0001 for 0.5 mg/L and p< 0.0001 for 1 mg/L) as compared to the
Sco-alone treated group, whereas a significant increase in the time spent in the top zone of
the tank was noticed (Figure 7B, p< 0.001 for 0.5 mg/L and p< 0.01 for 1 mg/L).
Molecules 2021,26, 2565 9 of 18
Figure 7.
The Equisetum telmateia ethanolic extract (ETe, 0.5 and 1 mg/L) reduced the anxiety-like response in the scopolamine
(Sco, 100
µ
M)-induced zebrafish in the novel tank diving (NTT) test. (
A
) represent the number of entries in the top zone of
the tank in different groups; (
B
) represent the time spent in the top zone of the tank (s) in different groups. For Tukey’s
posthoc analysis: Sco vs. ETe (0.5 mg/L): ### p< 0.0001; Sco vs. ETe (1 mg/L): ### p< 0.0001 (
A
); Sco vs. ETe (0.5 mg/L):
## p< 0.001; Sco vs. ETe (1 mg/L): # p< 0.01 (B).
Our results are in line with other data reported by different authors about the anx-
iolytic effects of Equisetum extracts. Vieira et al. [
29
] demonstrated that the extract of
E. arvense
disrupted the behavioral states (fear- and anxiety-like disorders) noticed in
zebrafish.
Singh et al. [30]
reported that ethanolic extract of E. arvense exhibited anxi-
olytic effects in mice, due to the flavonoid content. Sarris et al. [
31
] nicely described
in a review of preclinical studies the anxiolytic profile of the E. arvense, describing a
mechanism that primarily involved gamma-aminobutyric acid (GABA), either via direct
receptor binding or ionic channel or cell membrane modulation. Additionally, it has been
stated that (-)-epicatechin mitigated hippocampus oxidative stress, anxiety-like behavior,
and systemic inflammation in aged mice [
32
]. Park et al. [
33
] provided evidence that
(-)-epigallocatechin-3-O-gallate reversed caffeine-induced anxiogenic-like effects in an
animal model.
Vignes et al. [34]
also reported that (-)-epigallocatechin-gallate induced
anxiolytic activity in mice which could result from an interaction with GABA(A) receptors.
Gadotti et al. [35]
demonstrated the anxiolytic effects of the flavonoid luteolin in a mouse
model of acute colitis. Luteolin compound exerted a significant antidepressant effect at a
low dose and could be considered as a novel therapeutic strategy in depression as reported
by Crupi et al. [
36
]. Furthermore, luteolin has antidepressant-like effects, partly due to the
suppression of endoplasmic reticulum stress [
37
]. Zhang et al. [
38
] reported that quercetin
exerted the beneficial or detrimental effects on the shoaling and anxiety behaviors in ze-
brafish depending on the treatment concentrations, and the underlying mechanisms are
potentially associated with neuroinflammation and neuron apoptosis. Quercetin inhibited
anxiety-like symptoms and neuroinflammation induced by lipopolysaccharide in rats
as stated by Lee et al. [
39
]. Quercetin mitigated anxiety-like behavior and normalized
hypothalamus-pituitary-adrenal axis function in a mouse model of mild traumatic brain
injury as reported by
Kosari-Nasab et al. [40]
. Furthermore, quercetin protected against
stress-induced anxiety- and depression-like behavior and improved memory in male mice
as shown by Samad et al. [
41
].
Salgueiro et al. [42]
suggested that apigenin possessed
anxioselective effects, acting on central benzodiazepine receptors as an anxiolytic agent.
Ahmad et al. [
43
] reported the ability of kaempferol to inhibit the anxiety state in rats.
Furthermore, it was demonstrated that chlorogenic acid had anxiolytic effect coupled with
antioxidant activity. Caffeic acid also produced antidepressive- and/or anxiolytic-like ef-
fects through indirect modulation of the alpha 1A-adrenoceptor system in mice as reported
by Takeda et al. [
44
]. Finally, ferulic acid through mitigation of NMDA receptor pathway
Molecules 2021,26, 2565 10 of 18
exerted an anxiolytic-like effect in mouse model of maternal separation stress [
45
]. Upon
these reports, our Equisetum extracts also exhibited an anxiolytic profile in the Sco zebrafish
model that could be attributed to their flavonoid and polyphenolcarboxylic acids content.
2.4.2. Y-Maze Test
The test is based on the tendency of zebrafish to explore a new environment and assess
whether the exploratory behavior is modulated by past experiences [46]. For each type of
the Equisetum ethanolic extract (EPe, ESe, ETe), individual tests were performed at both
0.5 mg/L and 1 mg/L dose.
In the Y-maze test, the results of the one-way ANOVA revealed a significant effect
of the treatment on the spontaneous alternation percentage (F(4,45) = 32.40, p< 0.0001)
(Figure 8A)
and time in the novel arm (% of the total time) F(4,45) = 45.50, p< 0.0001)
(Figure 8B). Administration of Sco altered the spatial memory formation, as evidenced
by a decrease in the percentage of the spontaneous alternation (p< 0.0001) (Figure 8A),
whereas the response to novelty evaluated through the time in the novel arm, was im-
paired
(p< 0.0001)
(Figure 8B). By contrast, treatment with EPe, acted against Sco-induced
amnesia as noticed by increasing the spatial memory formation (significant increase of the
spontaneous alternation percentage, p< 0.0001 for 0.5 mg/L and p< 0.0001 for 1 mg/L)
(Figure 8A) and the time in the novel arm (p< 0.0001 for 0.5 mg/L and p< 0.0001 for
1 mg/L) (Figure 8B) as compared to Sco-alone treated groups. Donepezil (DP, 10 mg/L), a
cholinesterase inhibitor, was used as a reference drug.
Molecules 2021, 26, x FOR PEER REVIEW 10 of 19
et al. [39]. Quercetin mitigated anxiety-like behavior and normalized hypothalamus-pitu-
itary-adrenal axis function in a mouse model of mild traumatic brain injury as reported
by Kosari-Nasab et al. [40]. Furthermore, quercetin protected against stress-induced anx-
iety- and depression-like behavior and improved memory in male mice as shown by Sa-
mad et al. [41]. Salgueiro et al. [42] suggested that apigenin possessed anxioselective ef-
fects, acting on central benzodiazepine receptors as an anxiolytic agent. Ahmad et al. [43]
reported the ability of kaempferol to inhibit the anxiety state in rats. Furthermore, it was
demonstrated that chlorogenic acid had anxiolytic effect coupled with antioxidant activ-
ity. Caffeic acid also produced antidepressive- and/or anxiolytic-like effects through indi-
rect modulation of the alpha 1A-adrenoceptor system in mice as reported by Takeda et al.
[44]. Finally, ferulic acid through mitigation of NMDA receptor pathway exerted an anxi-
olytic-like effect in mouse model of maternal separation stress [45]. Upon these reports,
our Equisetum extracts also exhibited an anxiolytic profile in the Sco zebrafish model that
could be attributed to their flavonoid and polyphenolcarboxylic acids content.
2.4.2. Y-Maze Test
The test is based on the tendency of zebrafish to explore a new environment and
assess whether the exploratory behavior is modulated by past experiences [46]. For each
type of the Equisetum ethanolic extract (EPe, ESe, ETe), individual tests were performed at
both 0.5 mg/L and 1 mg/L dose.
In the Y-maze test, the results of the one-way ANOVA revealed a significant effect of
the treatment on the spontaneous alternation percentage (F(4,45) = 32.40, p < 0.0001) (Fig-
ure 8A) and time in the novel arm (% of the total time) F(4,45) = 45.50, p < 0.0001)(Figure
8B). Administration of Sco altered the spatial memory formation, as evidenced by a de-
crease in the percentage of the spontaneous alternation (p < 0.0001) (Figure 8A), whereas
the response to novelty evaluated through the time in the novel arm, was impaired (p <
0.0001) (Figure 8B). By contrast, treatment with EPe, acted against Sco-induced amnesia
as noticed by increasing the spatial memory formation (significant increase of the sponta-
neous alternation percentage, p < 0.0001 for 0.5 mg/L and p < 0.0001 for 1 mg/L) (Figure
8A) and the time in the novel arm (p < 0.0001 for 0.5 mg/L and p < 0.0001 for 1 mg/L)
(Figure 8B) as compared to Sco-alone treated groups. Donepezil (DP, 10 mg/L), a cholin-
esterase inhibitor, was used as a reference drug.
Figure 8. Behavioral response to the Equisetum pratense ethanolic extract (EPe, 0.5 and 1 mg/L) treatment in the scopolamine
(Sco, 100 μM)-induced zebrafish in Y-maze test. (A) represent the spontaneous alternation percentage in different groups;
(B) represent the time in the novel arm (% of the total time) in different groups. For Tukey’s posthoc analysis: Sco vs. EPe
(0.5 mg/L): ### p < 0.0001; Sco vs. EPe (1 mg/L): ### p < 0.0001 (A); Sco vs. EPe (0.5 mg/L): ### p < 0.0001; Sco vs. EPe (1
mg/L): ### p < 0.0001 (B).
Figure 8.
Behavioral response to the Equisetum pratense ethanolic extract (EPe, 0.5 and 1 mg/L) treatment in the scopolamine
(Sco, 100
µ
M)-induced zebrafish in Y-maze test. (
A
) represent the spontaneous alternation percentage in different groups;
(
B
) represent the time in the novel arm (% of the total time) in different groups. For Tukey’s posthoc analysis: Sco vs. EPe
(0.5 mg/L): ### p< 0.0001; Sco vs. EPe (1 mg/L): ### p< 0.0001 (
A
); Sco vs. EPe (0.5 mg/L): ### p< 0.0001; Sco vs. EPe
(1 mg/L): ### p< 0.0001 (B).
In the Y-maze test, the results of the one-way ANOVA revealed a significant effect
of the treatment on the spontaneous alternation percentage (F(4,45) = 97.53, p< 0.0001)
(Figure 9A)
and time in the novel arm (% of the total time) F(4,45) = 38.77, p< 0.0001)
(Figure 9B). Administration of Sco altered the spatial memory formation, as evidenced
by a decrease in the percentage of the spontaneous alternation (p< 0.01) (Figure 9A),
whereas the response to novelty evaluated through the time in the novel arm, was impaired
(p< 0.0001) (Figure 9B). By contrast, treatment with ESe, attenuated the Sco-induced
amnesia as evidenced by increasing the spatial memory formation (significant increase of
the spontaneous alternation percentage, p< 0.0001 for 0.5 mg/L and p< 0.0001 for 1 mg/L)
(Figure 9A) and the time in the novel arm (p< 0.0001 for 0.5 mg/L and p< 0.01 for 1 mg/L)
(Figure 9B) as compared to Sco-alone treated groups.
Molecules 2021,26, 2565 11 of 18
Molecules 2021, 26, x FOR PEER REVIEW 11 of 19
In the Y-maze test, the results of the one-way ANOVA revealed a significant effect of
the treatment on the spontaneous alternation percentage (F(4,45) = 97.53, p < 0.0001) (Fig-
ure 9A) and time in the novel arm (% of the total time) F(4,45) = 38.77, p < 0.0001)(Figure
9B). Administration of Sco altered the spatial memory formation, as evidenced by a de-
crease in the percentage of the spontaneous alternation (p < 0.01) (Figure 9A), whereas the
response to novelty evaluated through the time in the novel arm, was impaired (p < 0.0001)
(Figure 9B). By contrast, treatment with ESe, attenuated the Sco-induced amnesia as evi-
denced by increasing the spatial memory formation (significant increase of the spontane-
ous alternation percentage, p < 0.0001 for 0.5 mg/L and p < 0.0001 for 1 mg/L) (Figure 9A)
and the time in the novel arm (p < 0.0001 for 0.5 mg/L and p < 0.01 for 1 mg/L) (Figure 9B)
as compared to Sco-alone treated groups.
Figure 9. Behavioral response to the Equisetum sylvaticum ethanolic extract (ESe, 0.5 and 1 mg/L) treatment in the scopola-
mine (Sco, 100 μM)-induced zebrafish in Y-maze test. (A) represent the spontaneous alternation percentage in different
groups; (B) represent the time in the novel arm (% of the total time) in different groups. For Tukey’s posthoc analysis: Sco
vs. ESe (0.5 mg/L): ### p < 0.0001; Sco vs. ESe (1 mg/L): ### p < 0.0001 (A); Sco vs. ESe (0.5 mg/L): ### p < 0.0001; Sco vs. ESe
(1 mg/L): # p < 0.01 (B).
In the Y-maze test, the results of the one-way ANOVA revealed a significant effect of
the treatment on the spontaneous alternation percentage (F(4,45) = 151.50, p < 0.0001) (Fig-
ure 10A) and time in the novel arm (% of the total time) F(4,45) = 27.01, p < 0.0001)(Figure
10B). Administration of Sco altered the spatial memory formation, as evidenced by a de-
crease in the percentage of the spontaneous alternation (p < 0.0001) (Figure 10A), whereas
the response to novelty evaluated through the time in the novel arm, was impaired (p <
0.0001) (Figure 10B). By contrast, treatment with ETe attenuated the Sco-induced amnesia
as evidenced by increasing the spatial memory formation (significant increase of the spon-
taneous alternation percentage, p < 0.0001 for 0.5 mg/L and p < 0.0001 for 1 mg/L) (Figure
10A) and the time in the novel arm (p < 0.001 for 0.5 mg/L and p < 0.0001 for 1 mg/L)
(Figure 10B) as compared to Sco-alone treated groups.
Figure 9.
Behavioral response to the Equisetum sylvaticum ethanolic extract (ESe, 0.5 and 1 mg/L) treatment in the
scopolamine (Sco, 100
µ
M)-induced zebrafish in Y-maze test. (
A
) represent the spontaneous alternation percentage in
different groups; (
B
) represent the time in the novel arm (% of the total time) in different groups. For Tukey’s posthoc
analysis: Sco vs. ESe (0.5 mg/L): ### p< 0.0001; Sco vs. ESe (1 mg/L): ### p< 0.0001 (
A
); Sco vs. ESe (0.5 mg/L):
### p< 0.0001; Sco vs. ESe (1 mg/L): # p< 0.01 (B).
In the Y-maze test, the results of the one-way ANOVA revealed a significant effect
of the treatment on the spontaneous alternation percentage (F(4,45) = 151.50, p< 0.0001)
(Figure 10A) and time in the novel arm (% of the total time) F(4,45) = 27.01, p< 0.0001)
(Figure 10B). Administration of Sco altered the spatial memory formation, as evidenced
by a decrease in the percentage of the spontaneous alternation (p< 0.0001) (Figure 10A),
whereas the response to novelty evaluated through the time in the novel arm, was impaired
(p< 0.0001)
(Figure 10B). By contrast, treatment with ETe attenuated the Sco-induced
amnesia as evidenced by increasing the spatial memory formation (significant increase of
the spontaneous alternation percentage, p< 0.0001 for 0.5 mg/L and p< 0.0001 for 1 mg/L)
(Figure 10A) and the time in the novel arm (p< 0.001 for 0.5 mg/L and p< 0.0001 for
1 mg/L) (Figure 10B) as compared to Sco-alone treated groups.
Molecules 2021, 26, x FOR PEER REVIEW 12 of 19
Figure 10. Behavioral response to the Equisetum telmateia ethanolic extract (ETe, 0.5 and 1 mg/L) treatment in the scopola-
mine (Sco, 100 μM)-induced zebrafish in Y-maze test. (A) represent the spontaneous alternation percentage in different
groups; (B) represent the time in the novel arm (% of the total time) in different groups. For Tukey’s posthoc analysis: Sco
vs. ETe (0.5 mg/L): ### p < 0.0001; Sco vs. ETe (1 mg/L): ### p < 0.0001 (A); Sco vs. ETe (0.5 mg/L): ### p < 0.0001; Sco vs.
ETe (1 mg/L): ### p < 0.0001 (B).
Supporting data from the literature indicated cognitive enhancement effects of Equi-
setum extracts. Dos Santos Jr et al. [47] demonstrated that cognitive enhancement in aged
rats after chronic administration of E. arvense L. may be attributed, at least in part, to its
antioxidant action. Itoh et al. [48] demonstrated that epicatechin increased the persistence
of long-term memory formed by conditioned taste aversion in Lymnaea stagnalis. (-)-Epi-
catechin mitigated high fat diet-induced neuroinflammation and altered behavior in mice
as reported by Kang et al. [49]. Diaz et al. [50] demonstrated that epicatechin reduced
spatial memory deficit caused by amyloid-β2535 toxicity modifying the heat shock pro-
teins in the ca1 region in the hippocampus of rats. Tan et al. [51] reported that luteolin
treatment effectively alleviated brain edema and ameliorated neurobehavioral dysfunc-
tion and memory loss in vivo. Richetti et al. [52] reported that quercetin and rutin pre-
vented scopolamine-induced memory impairment in zebrafish. Sang et al. [53] reported
that novel apigenin-rivastigmine hybrids could improve scopolamine-induced memory
impairment in zebrafish. Hussein et al. [54] evidenced the neuroprotective role of
kaempferol against chlorpyrifos-induced oxidative stress and memory deficits in rats via
GSK3β-Nrf2 signaling pathway. Furthermore, chlorogenic acid improved the spatial
memory of rats and prevented the CA1 pyramidal cell death after bilateral common ca-
rotid occlusion by increasing Bcl2, SOD2, and CD31 expressions and decreasing ET-1 ex-
pression [55]. Deshmukh et al. [56] reported that caffeic acid attenuated oxidative stress,
learning and memory deficit in intra-cerebroventricular streptozotocin induced experi-
mental dementia in rats. Finally, ferulic acid exerted Nrf2-dependent protection against
prenatal lead exposure-induced cognitive impairment in offspring mice as reported by Yu
et al. [57]. Our Equisetum extracts also exhibited a cognitive-enhancing profile in the Sco
zebrafish model that could be attributed to their flavonoid and polyphenolcarboxylic ac-
ids content.
When correlating the chemical composition with the biological tests performed in
vitro (antioxidant action, enzymatic inhibitors, radical scavenger), there is a direct reci-
procity relationship between the increased concentration of flavonoids, the doses admin-
istered, and the anxiolytic and antidepressant intensity in the fish. Moreover, considering
the high level of silicon (water-soluble silicon derivatives) of the species E. telmateia, it was
observed that the efficacy of the extracts was below the level of the other two samples.
When evaluating the effect on short-term memory, significant differences were observed
Figure 10.
Behavioral response to the Equisetum telmateia ethanolic extract (ETe, 0.5 and 1 mg/L) treatment in the scopolamine
(Sco, 100
µ
M)-induced zebrafish in Y-maze test. (
A
) represent the spontaneous alternation percentage in different groups;
(
B
) represent the time in the novel arm (% of the total time) in different groups. For Tukey’s posthoc analysis: Sco vs. ETe
(0.5 mg/L): ### p< 0.0001; Sco vs. ETe (1 mg/L): ### p< 0.0001 (
A
); Sco vs. ETe (0.5 mg/L): ### p< 0.0001; Sco vs. ETe
(1 mg/L): ### p< 0.0001 (B).
Molecules 2021,26, 2565 12 of 18
Supporting data from the literature indicated cognitive enhancement effects of Eq-
uisetum extracts. Dos Santos Jr et al. [
47
] demonstrated that cognitive enhancement in
aged rats after chronic administration of E. arvense L. may be attributed, at least in part,
to its antioxidant action. Itoh et al. [
48
] demonstrated that epicatechin increased the per-
sistence of long-term memory formed by conditioned taste aversion in Lymnaea stagnalis.
(-)-Epicatechin mitigated high fat diet-induced neuroinflammation and altered behavior
in mice as reported by Kang et al. [
49
]. Diaz et al. [
50
] demonstrated that epicatechin
reduced spatial memory deficit caused by amyloid-
β
25-35 toxicity modifying the heat
shock proteins in the ca1 region in the hippocampus of rats. Tan et al. [
51
] reported that
luteolin treatment effectively alleviated brain edema and ameliorated neurobehavioral
dysfunction and memory loss
in vivo
. Richetti et al. [
52
] reported that quercetin and rutin
prevented scopolamine-induced memory impairment in zebrafish. Sang et al. [
53
] reported
that novel apigenin-rivastigmine hybrids could improve scopolamine-induced memory im-
pairment in zebrafish. Hussein et al. [
54
] evidenced the neuroprotective role of kaempferol
against chlorpyrifos-induced oxidative stress and memory deficits in rats via GSK3
β
-Nrf2
signaling pathway. Furthermore, chlorogenic acid improved the spatial memory of rats
and prevented the CA1 pyramidal cell death after bilateral common carotid occlusion
by increasing Bcl2, SOD2, and CD31 expressions and decreasing ET-1 expression [
55
].
Deshmukh et al. [
56
] reported that caffeic acid attenuated oxidative stress, learning and
memory deficit in intra-cerebroventricular streptozotocin induced experimental demen-
tia in rats. Finally, ferulic acid exerted Nrf2-dependent protection against prenatal lead
exposure-induced cognitive impairment in offspring mice as reported by Yu et al. [
57
]. Our
Equisetum extracts also exhibited a cognitive-enhancing profile in the Sco zebrafish model
that could be attributed to their flavonoid and polyphenolcarboxylic acids content.
When correlating the chemical composition with the biological tests performed
in vitro
(antioxidant action, enzymatic inhibitors, radical scavenger), there is a direct reciprocity
relationship between the increased concentration of flavonoids, the doses administered,
and the anxiolytic and antidepressant intensity in the fish. Moreover, considering the
high level of silicon (water-soluble silicon derivatives) of the species E. telmateia, it was
observed that the efficacy of the extracts was below the level of the other two samples.
When evaluating the effect on short-term memory, significant differences were observed
that reconfirm the great effectiveness of the E. pratense and E. telmateia extracts compared
to E. sylvaticum.
3. Materials and Methods
3.1. Plant Materials
Larger quantities (2 kg) of E. pratense and E. sylvaticum were harvested from the
North-Eastern region of Romania in June–July 2017. E. telmateia (2 kg) was collected in July
2018 also from the North-Eastern region of Romania. The botanical identity was checked
and attested in the plant biology laboratory by Acad. Prof. Toma Constantin PhD from
the Biology Faculty, “Al. I. Cuza” University Iasi Romania. After identification, the raw
material was dried to a constant mass for 21 days in a single layer in a dark room at a
controlled temperature of 23
C. To obtain the extracts, the aerial parts of the Equisetum
species were used. Methanolic extracts were obtained from 10 g of homogenized 2 kg of
dry plant material with methanol 70% using the water bath and the refrigerant for 60 min.
The obtained methanol extracts were evaporated using the rotary evaporator Buchi R10
(Flavil, Switzerland) [
58
,
59
]. Ethanolic extracts were obtained from 10 g of homogenized
2 kg of dry plant material with ethanol 70% using an ultrasonic bath (Bandelin Sonorex,
Type RK 31, Berlin, Germany) at 30
C for 30 min. The mixture was filtered and then the
ethanolic extracts were evaporated. All dry extracts were brought to a constant weight
so that there was no residual water. The dried extracts were then brought into a vial and
maintained for analysis in a dry, cool, dark place [
5
,
60
]. The extracts were homogenous,
fine glassy fragments were present for the ethanolic samples. The homogeneity of the
analyzed extracts was ensured by fine powdering each sample in a glass mortar.
Molecules 2021,26, 2565 13 of 18
3.2. UHPLC Analysis of Phenolic Acids and Flavonoids
The ultra-high-performance liquid chromatography (UHPLC) analysis of the inves-
tigated extracts aimed to highlight the most important active principles in the class of
polyphenols. Thus, the analysis used the system Thermo Fischer UltiMate 3000 (Pro Analy-
sis Systems, Bucharest, Romania) coupled with a quaternary pump (LPG-3400 SD) with
degasser built-in four-channel, autosampler (allows analysis of consecutive 120 samples)
and the detector type MULTIDIODE (DAD UltiMate 3000RS). This equipment is adapted
to reach a pressure of 62 MPa/9000 psi/620 bar, which allows a fast running of the analysis
method in a relatively short time (max. 25 min). Luna PFP (150
×
4, 6
×
4) (Phenomenex,
CA, USA) was the column attached to this system. For the detection, the corresponding
data were recorded at 5 different wavelengths, using the two lamps, and the detection range
was between 190 nm and 800 nm (UV-VIS) to highlight the different types of polyphenols
present in extracts included in the study [
17
,
61
]. The used standards (purity
97% HPLC
grade, Sigma-Aldrich, Munich, Germany) were: chlorogenic acid, caffeic acid, ferulic acid,
rosmarinic acid, p-coumaric acid, epicatechin, quercetin-3-glucoside, apigenin-7-glucoside,
apigenin, quercetin, luteolin, luteolin-7-glucoside, kaempferol, and rutoside (5–10
µ
g/mL
in methanol). The solvent system consisted of a mixture of acetonitrile with 0.1% phos-
phoric acid and 0.1% phosphoric acid. Samples were injected in amounts of 5
µ
L along
with the standards. The data were integrated with the Thermo Scientific
Dionex
Chromeleon
v. 7.2.12 program, with dynamic algorithms for identifying the tracked
peaks, and then the results were compared with the databases in the specialized literature.
The identity of each compound was confirmed both using the reference spectra and the
retention time. Moreover, calibration curves (r
2
= 0.9989) were obtained for chlorogenic
acid, luteolin-7-glucoside, and quercetin-3-glucoside. Triplicate analysis was performed for
each sample. The limit of detection (LOD) was calculated at 280 ng/mL and the limit of
quantification (LOQ) was 145 ng/mL.
3.3. Antioxidant Capacity Assay
The following tests were used to study antioxidant activity: chelation of ferrous ion,
lipoxygenase inhibition, the scavenger capacity of the hydroxyl radical, and the superoxide
radical anion. The chelating capacity of the ferrous ion was determined using ferrozine
which forms a pink complex with maximum absorbance at 562 nm [
62
]. Determination
of the lipoxygenase inhibition capacity could be measured because the active compounds
present in the extracts block 15-lipoxygenase by blocking linoleic acid oxidation and
reducing absorbance at 234 nm [
63
]. Determination of the scavenger action of the hydroxyl
radical is based on the method that hydroxyl radical, formed in the reaction between ferrous
ion and hydrogen peroxide, will hydroxylate salicylic acid to form a pink-violet compound
with maximum absorbance at 562 nm [
64
]. The scavenger capacity of the superoxide anion
was determined because the superoxide radical generated by the reduced nicotinamide
adenine nucleotide-phenazine methosulfate system reduces nitro blue tetrazole to a violet-
blue formazan with a maximum absorbance at 560 nm [
65
]. All quantifications represent
the mean values
±
SD (standard deviation) for 5 consecutive determinations, calculated in
Excel, p= 0.0016, which indicates a statistic significance.
3.4. Animals and Group Division
100 adult male and female, wild-type short-fin zebrafish (Danio rerio), 3–5 months
old were obtained from Pet Product S.R.L., Bucharest, Romania. Zebrafish, approximately
1:1 female to male were maintained in three tanks of 70 L each, an automatic filtration
system, and controlled aeration. Fish were fed two times daily with NovoMalawi (JBL,
Neuhofen, Germany). The animal room was illuminated on a 14/10 h light/dark cycle.
Room temperature was maintained at 25
C
±
1
C. The water temperature was kept at
27
±
0.5
C, pH 7
±
0.15, dissolved oxygen 6
±
0.1 mg/L, total ammonia < 0.01 mg/L,
total hardness 6 mg/L, and alkalinity of 22 mg/L CaCO
3
. The 10 groups of 10 fish were as
follows: control, ethanolic extracts from E. pratense Ehrh., E. sylvaticum L., and E. telmateia
Molecules 2021,26, 2565 14 of 18
Ehrh. (0.5 mg/L and 1 mg/L, respectively), scopolamine (Sco, 100
µ
M), imipramine (IMP,
20 mg/L), and donepezil (DP, 10 mg/L). Before behavioral tests, fish (except those from the
control group) were individually immersed in Sco (100 µM) solution for 30 min to induce
amnesia, whereas exposure to Equisetum ethanolic extracts (0.5 mg/L and 1 mg/L) was
done 1 h before the behavioral tests.
The animals were observed in quarantine at least one week before use in experimental
studies. This study was carried out in strict accordance with the recommendations of the
Directive 2010/63/EU of the European Parliament and of the Council of 22 September
2010 on the protection of animals for scientific purposes. The protocol was approved by
the Faculty of Biology Ethics Committee for animal experimentation (Protocol number
02/30.06.2020). All efforts were made to minimize animal pain and suffering.
3.5. Behavioral Assays
Animal behavior was recorded using a Logitech C922 Pro HD Stream webcam (Log-
itech, Lausanne, Switzerland) and the videos were analyzed using the ANY-maze
®
behav-
ioral tracking software (Stoelting Co., Wood Dale, IL, USA).
3.5.1. Novel Tank Diving Test (NTT)
In NTT, the zebrafish exhibits robust behavioral responses to novelty-provoked anxiety.
The NTT protocol applied in this study was described before by Cachat et al. [
66
] and
Rosemberg et al. [
67
]. The testing apparatus consisted of a trapezoidal glass tank filled
with 1.5 L of home tank water and had the following dimensions: 23.9 cm along the bottom
×
28.9 cm at the top
×
15.1 cm high with 15.9 cm along the diagonal side, 7.4 cm wide at
the top, and 6.1 cm wide at the bottom. The fish were individually placed in the testing
tank and their behavior was recorded for 6 min with a webcam placed at 40 cm in the front
of the tank. The tank was virtually divided into the top zone and bottom zone, respectively.
The following parameters were analyzed: the number of entries in the top zone of the tank
and the time spent in the top zone of the tank.
3.5.2. Y-Maze Test
The zebrafish memory and response to novelty were investigated using a protocol of
the Y-maze task that was formerly described by Cognato et al. [
68
] and Zanandrea et al. [
69
].
The fish were tested in a Y-shaped glass tank, having three arms in size of 25
×
8
×
15 cm
(L
×
l
×
h) and filled with 3 L of home tank water. Different recognizable geometric shapes,
such as triangles, circles, and squares, were placed on the outer walls of each arm. The
arms of the maze were set randomly as follows: (i) the start arm (A) from which the fish
begins the test, (ii) the other arm (B) that is permanently open, and (iii) the novel arm (C)
which is blocked during the training period and open in the testing phase. The center of
the Y-maze was not taken into account for the analysis. This task was performed in two
stages separated by 1 h between them in order to assess the response to novelty and the
spatial recognition memory. During the first stage (training session), the fish was allowed
to explore the start and the other arm for 5 min, while the novel arm was kept closed. In
the second stage (testing session) the fish was placed in the start arm and was allowed to
explore the entire maze for 5 min. The time spent in the novel arm (% of total time), and
the spontaneous alternation percentage [
70
] were the behavioral endpoints examined in
this task.
3.6. Statistical Analysis
The results were presented as mean
±
standard error of the mean (E.S.M.). Behavioral
data were analyzed by one-way ANOVA followed by Tukey’s posthoc multiple comparison
test, considering treatment as a factor. All analyses were performed by GraphPad software
(GraphPad Prism 7.0, La Jolla, CA, USA) and the significance was set at p< 0.05.
Molecules 2021,26, 2565 15 of 18
4. Conclusions
The UHPLC analysis of methanolic and ethanolic extracts revealed the presence of
the following components: chlorogenic acid, caffeic acid, ferulic acid, neochlorogenic
acid, epicatechin, quercetin, quercetin-3-glucoside, luteolin, luteolin-7-glucoside, apigenin-
glucoside, and kaempferol. Of all the polyphenolic acids, chlorogenic acid was found in the
highest amount in E. telmateia species (10.12 mg/g in methanolic extract and 14.38 mg/g
in ethanolic extract). Regarding flavonoid derivatives, luteolin-glucoside (11.05 mg/g)
stands out in the methanolic extract of E. sylvaticum species, while for the ethanolic one, the
glycosides of quercetin (41.94 mg/g) stand out. For the species E. telmateia, in both extracts,
the majority were the glycosides of quercetin and apigenin.
The results obtained in the evaluation of the antioxidant action for the two types of
extracts indicate a variable capacity to reduce the oxidative processes. The most effec-
tive were the extracts from E. sylvaticum, and the weakest were those from E. pratense.
Thus, it can be highlighted that the flavonoid fraction in these plant products, through
glycosylated derivatives, influences the free radical scavenger potential to a greater extent
than the polyphenolic acid fraction. The sample, both methanolic and ethanolic of E.
sylvaticum species, is the richest in flavonoid components, which is why it showed the most
intense effects on ferrous ions, hydroxyl radical, and superoxide anion as well as in the
inhibition of lipoxygenase. This indicates an increased availability of flavonoids (through
the benzopyran-like chemical structure) to bind reactive oxygen species. Correlating the
data obtained from the evaluation of the methanolic extract with those obtained from the
analysis of the content of polyphenols and flavonoids, the E. sylvaticum extract containing
the highest number of polyphenols is about three times more active compared to the other
two extracts. This difference in activity could be explained by the type of polyphenols
present in the samples analyzed, as well as by the large difference in flavonoid content.
The
in vivo
evaluation of neuroprotective activity was performed by following up
the effects produced by the ethanolic extracts of the three species of Equisetum above
zebrafish in terms of anxiety-like behavior and short-term memory. Novel tank diving test
(NTT) analyzed the number of entries to the top zone of the tank and the time spent in
the top zone of the tank. The 1 mg/L concentration in E. pratense and E. sylvaticum had
anxiolytic and antidepressant effects higher than the 0.5 mg/L concentration. Regarding
the ethanolic extract of E. telmateia, the time spent in the top zone of the tank is higher for
the concentration of 0.5 mg/L compared to 1 mg/L. Compared to the reference substance,
IMP, the number of entries in the top zone of the tank by zebrafish was higher at the
concentration of 1 mg/L, while at the concentration of 0.5 mg/L, the number of entries was
lower, but comparable with these. The Y-maze test analyzed the percentage of spontaneous
alternation and the time spent in the novel arm (% of the total time). The differences
between the ethanolic extract concentrations of 0.5 mg/L and 1 mg/L were not significant
for the analyzed species. Compared to the reference substance, DP, both the concentrations
of 0.5 mg/L and those of 1 mg/L of the three species determined an even twice greater
stimulation of short-term memory by increasing the percentage of spontaneous alteration
and the response to novelty.
Author Contributions:
Conceptualization, D.B.-M., O.C., C.M., M.H., and L.H.; methodology, D.B.-
M., M.B., O.C., C.M., A.F.B., G.J.B., A.S., and A.C.; software, O.C., and A.S.; formal analysis, D.B.-M.,
M.B., O.C., C.M., A.F.B., G.J.B., A.S., and A.C.; investigation, D.B.-M., M.B., O.C., C.M., A.F.B., G.J.B.,
A.S., and A.C.; resources, L.H. and M.H.; writing—original draft preparation, L.H., O.C., and M.H.;
writing—review and editing, L.H., O.C., and M.H.; supervision, L.H., and M.H.; funding acquisition,
L.H., and M.H. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement:
This study was carried out in strict accordance with the
recommendations of the Directive 2010/63/EU of the European Parliament and of the Council of 22
September 2010 on the protection of animals in scientific purposes. The protocol was approved by the
Faculty of Biology Ethics Committee for animal experimentation (Protocol number 02/30.06.2020).
Molecules 2021,26, 2565 16 of 18
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data presented in this study are available on request from the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability:
Samples are available at the Pharmacognosy Department, Facultu of Pharmacy,
“Grigore T. Popa” University of Medicine and Pharmacy, Iasi, Romania.
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... This could be explained through the involvement of only some of the polyphenols found in the extract in the formation of AgNPs. Correspondingly, in the case of extracts, the best antioxidant activity was also observed for E. sylvaticum [53]. As shown in Figure 6, all AgNPs obtained from the Equisetum species showed a much more intense chelating capacity of the ferrous ion than gallic acid, which was used as standard. ...
... 387 Among the obtained nanoparticles, the lowest efficiency regarding LOX inhibition can be 388 seen for Et-AgNPs. The constituents of E. sylvaticum seem to possess the most important 389 capacity of inhibiting LOX, taking into account results from previous studies [53] and the 390 fact that only Es-AgNPs showed a higher efficiency compared to gallic acid. The hydroxyl radical is synthesized in vivo or in vitro through Fenton and Haber-393 Weiss reactions, its synthesis being more intense under hypoxic conditions. ...
... Among the obtained nanoparticles, the lowest efficiency regarding LOX inhibition can be seen for Et-AgNPs. The constituents of E. sylvaticum seem to possess the most important capacity of inhibiting LOX, taking into account results from previous studies [53] and the fact that only Es-AgNPs showed a higher efficiency compared to gallic acid. ...
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... ES (10 mg Á mL À1 ) has resulted in lipoxygenase inhibition capacity (methanolic and ethanolic extracts) and scavenger action of the hydroxyl radical of 100%. ET and ES resulted in scavenger capacity of the superoxide anion of 80% (10 mg Á mL À1 of plants in methanol) [40]. ...
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Evidence has implicated oxidative stress (OS) and inflammation as drivers of neurodegenerative pathologies. We previously reported on the beneficial effects of (−)-epicatechin (Epi) treatment on aging-induced OS and its capacity to restore modulators of mitochondrial biogenesis in the prefrontal cortex of 26-month-old male mice. In the present study using the same mouse model of aging, we examined the capacity of Epi to mitigate hippocampus OS, inflammation, hyperphosphorylation of tau protein, soluble β-amyloid protein levels, cell survival, memory, anxiety-like behavior levels and systemic inflammation. Mice were subjected to 4 weeks of Epi treatment (1 mg kg⁻¹ day⁻¹) and samples of the hippocampus were obtained. Assessments of the OS markers, protein carbonyls, and malondialdehyde levels demonstrated their significant increase (∼3 fold) with aging that were partially suppressed by Epi. The protein levels of the glial fibrillary acidic protein, inflammatory factor 1 (Iba1), pro-inflammatory cytokines, interleukins (IL-1β, IL-3, 5, 6 and 15), cyclooxygenase 2, tumor necrosis factor α, nuclear factor-activated B cells and interferon γ increase with aging and were also significantly decreased with Epi treatment. However, anti-inflammatory cytokines, IL-1ra, IL-10 and 11 decrease with aging and were restored with Epi. Epi also reversed the aging effects on the hyperphosphorylation of tau, increased soluble β-amyloid levels (∼2 fold), cellular death (as per caspase 3 and 9 activity), and reduced nerve growth factor and triggering receptor expressed on myeloid cells 2 levels. Measures of anxiety like-behavior and memory demonstrated improvements with Epi treatment. Indicators of systemic inflammation increase with aging and Epi was capable of decreasing blood inflammatory markers. Altogether, the results show a significant capacity of Epi to mitigate hippocampus OS and inflammation leading to improved brain function.
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Quercetin, a potential fish food supplement, has been reported to process many beneficial properties. However, some negative effects of quercetin have been observed, which pointed out necessity for additional studies to evaluate its safety. Therefore, the present study investigated effects of quercetin (0.01, 0.1, 1, 10, 100 and 1000 μg/L) on shoaling and anxiety behaviors through novel tank tests in zebrafish (Danio rerio). Furthermore, oxidative stress, neuroinflammation and apoptosis in the brains were examined to learn more about mechanisms of action related to quercetin. The results showed that quercetin at the lower concentrations exerted beneficial effects on shoaling and anxiety behaviors. On the contrary, when quercetin was up to 1000 μg/L, it exerted detrimental effects shown as decreases of movement and increases of anxiety behaviors. Generally, U-shaped responses of antioxidant enzyme activities (superoxide dismutase and catalase), and inversed U-shaped responses of inflammatory mediators (cyclooxygenase-2) and cytokines (interleukin-1β, interleukin-6, interleukin-10, and tumor necrosis factor α) to quercetin treatment were found in the brains. In addition, quercetin at the lower concentrations attenuated cell apoptosis, while even more apoptosis was found at the 1000 μg/L quercetin group. In conclusion, quercetin could exert beneficial or detrimental effects on the shoaling and anxiety behaviors depending on the treatment concentrations, and the underlying mechanisms are potentially associated with neuroinflammation and neuron apoptosis.
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Obesity is characterized by a condition of low-level chronic inflammation that can lead to altered cognition and behavior. The flavanol (-)-epicatechin (EC) has been shown to have anti-inflammatory actions in mouse models of diet-induced obesity. This study investigated the capacity of dietary EC to mitigate hippocampal inflammation and impaired memory in high fat diet (HFD)-fed mice. Healthy 6 weeks old male C57BL/6J mice (10 mice/group) were fed for 13 weeks either: a control diet (10% total calories from fat), a high fat diet (60% total calories from fat), or the control and high fat diets supplemented with 20 mg EC/kg body weight. Short-term object recognition memory was evaluated by the novel object recognition (NOR) task and spatial memory by the object location memory (OLM) task and the Morris water maze (MWM). After 13 weeks on the dietary treatments, HFD-fed mice developed obesity, which was not affected by EC supplementation. HFD consumption caused endotoxemia, and increases in parameters of hippocampal inflammation, i.e. mRNA levels of TLR4, Iba-1, and NOX4. All these changes were mitigated by EC supplementation. EC supplementation also significantly improved recognition memory in HFD-fed mice while neither HFD consumption nor EC supplementation affected mouse spatial memory. Overall, EC supplementation prevented short-term recognition memory impairment in HFD-induced obese mice, which could be in part due to the capacity of EC to mitigate metabolic endotoxemia and associated hippocampal inflammation and oxidative stress.