Keywords
Olea europaea
- Oleaceae - endoplasmic reticulum - oxidative damage - neurons - lead
Introduction
Neurotoxicity indicates adverse effects that affect the nervous system and that result
from exposure to potentially toxic substances [1 ]. Environmental pollution has increased daily exposure to often harmful chemicals
such as metals, solvents, pesticides, and other contaminants [2 ]. Among these, heavy metals easily spread in the food chain, leading to alterations
of biological macromolecules such as DNA, proteins, and lipids [3 ]. The accumulation of heavy metals can cause damage to the nervous system: some metals
can cross the blood–brain barrier, reach the central nervous system, and cause neurotoxicity
or neurodegeneration. Many of these metals have been linked to devastating neurological
disorders, such as mental retardation [4 ], Alzheimerʼs disease [5 ], Parkinsonʼs disease [6 ], multiple sclerosis [7 ], and amyotrophic lateral sclerosis [8 ]. Lead (Pb) is present in trace amounts in soil, plants, and water; anthropic activity
is responsible for the increased exposure to lead. Chemically, the lead diffused in
the environment is in the oxidation state + 2 (Pb2+ ), and its accumulation occurs due to its inability to degrade and its subsequent
absorption into the soil [9 ], [10 ]. Prolonged exposure to lead can cause a variety of health disorders [11 ].
Numerous data in the literature have shown that consuming olive oil and its valuable
components could reduce neurotoxicity and degenerative conditions [12 ]. Olive oil has a predominant role in the prevention of chronic and/or degenerative
diseases, demonstrating antioxidant, anti-inflammatory, anti-obesity, anti-diabetic,
cardioprotective, antisteatotic, anticancer, antimicrobial, and neuroprotective effects
[13 ]; 98 – 99% of olive oil (saponifiable fraction) consists of monounsaturated fatty
acids (MUFA), polyunsaturated acids (PUFA), and short-chain saturated fats (SFA).
The remaining 1 – 2% (unsaponifiable fraction) includes sterols, pigments, aliphatic
alcohols, tocopherols (vitamin E), squalene, terpenoids, and phenolic compounds [14 ]. These latter are characterized by a broad spectrum of beneficial effects on human
health [15 ], and the most present in olive
oil are oleuropein, hydroxytyrosol, and oleacein. Oleuropein is an ester of elenolic
acid and belongs to the family of secoiridoids; its degradation leads to the formation
of two dialdehydic-form molecules: 3,4-DHPEA-EDA (oleacein) and 3,4-DHPEA (hydroxytyrosol)
[16 ]. Oleuropein and hydroxytyrosol have been extensively studied, and their antioxidant
effect is particularly recognized; while initially, less attention has been given
to oleacein, it is the most abundant secoiridoid in the fruit and leaves of olive
trees. To date, knowledge about oleacein has greatly increased, and there are numerous
data available in the literature on this subject. Oleacein plays an important anti-proliferative
and anti-metastatic role, as demonstrated by an interesting study conducted precisely
on the SH-SY5Y cell line [17 ]. This study showed that oleacein can exercise important anti-cancer effects, reducing
cell proliferation, by blocking
the cell cycle in the S phase and inducing apoptotic death through increased expression
of Bax and p53. In addition, oleacein exerts an important anti-inflammatory activity
[18 ], being thus a possible alternative pharmacological strategy against cancer and inflammation.
The traditional method of obtaining extra-virgin olive oil is its extraction from
olives, the fruit of the olive tree. This plant (Olea europaea L.) is an evergreen fruit tree belonging to the Oleaceae family and the genus Olea . The olive tree is a typically thermophilic and heliophilous plant, initially cultivated
almost exclusively in the Mediterranean countries with mild winters and warm summers,
although later it was successfully planted also in other countries with similar climates,
such as Australia, Argentina, California, South Africa, and New Zealand. To date,
the five countries with the largest olive-growing areas are Spain, Greece, Italy,
Tunisia, and Turkey.
The fruit is a yellow-green to black-purple drupe, formed by a “fleshy” part (pulp
that contains oil) and by the woody core containing the seed. From the pressing and
crushing of olives comes olive oil [19 ], [20 ]: the processing of olive oil generates different types of waste such as leaves and
branches from tree pruning, solid waste from the mill, wastewater from oil mills,
and olive kernels. In recent years, the trend has been to develop energy or economic
gain from these wastes [21 ]. For example, olive leaves are characterized by a very rich composition that includes
natural bioactive and phenolic compounds, which possess a health promotion potential.
The organic matter of olive leaves is variable and represents about 38% of their weight
[22 ], [23 ], [24 ], [25 ]. The amount of
polyphenols in olive leaves is higher than that of olive oil (15 – 70 mg/g fresh weight)
[26 ], [27 ], [28 ], [29 ].
In this manuscript, two extracts have been used and compared: a) the extract from
the leaves of Olea europaea L, cultivar Coratina, (OE); b) the extract obtained from OE and derived from a further
sonication process (s-OE). Most drugs used to ensure an adequate therapeutic response
against various pathological conditions are characterized by poor aqueous solubility
that reduces bioavailability. This phenomenon is also demonstrated in herbal medicines
[30 ]. The protocol developed to obtain an extract from the leaves of Olea Europaea provides for its final dissolution in a hydroalcoholic solution consisting of water
and ethanol (30 : 70) [31 ], [32 ]. Since ethanol could be toxic if taken in high concentrations, it would be desirable
to reduce the amount of alcohol and still ensure its dissolution. For this reason,
in this experimental study, we subjected the extract to a
sonication process, which improved its dissolution, allowing it to reduce the amount
of ethanol in the hydroalcoholic solution used.
Therefore, the objectives of this experimental work were as follows:
Generate an innovative extract (s-OE);
Test both extracts (OE and s-OE) on an in vitro model of neurotoxicity induced in human neurons following lead exposure;
Study the mechanisms underlying the effects generated by lead to interpret the action
of OE and s-OE extracts correctly.
Results
Hydroxytyrosol and oleuropein in OE and s-OE
As can be seen in [Fig. 1 ] and [Fig. 2 ], the concentrations of hydroxytyrosol and oleuropein are the same in both OE and
s-OE and represent, respectively, 6.38% and 42.17% w/w of the extracts.
Fig. 1 LC-HRMS full-scan chromatogram (a) and LC-HRMS PRM chromatograms of (b ) hydroxytyrosol and (c ) oleuropein in OE and s-OE dry extracts.
Fig. 2 MSMS spectra of (a ) hydroxytyrosol (153.0557 m/z) and (b ) oleuropein (539.1170 m/z) in OE and s-OE extracts.
s-OE preparation
s-OE extract was produced using sonication and the best sonication time was established
by specific viability experiments, in which OE was sonicated for a different duration
of time ([Fig. 3 a ]). Since the sonication time of 10′ did not cause changes in cell viability compared
to untreated cells, we chose this time to get s-OE. The viability of cells treated
with OE or s-OE is indicated in [Fig. 3 b ]: no significant variation was found, and we chose to use a concentration of 25 µg/mL
for both extracts as this concentration was the last to generate coincident values.
Fig. 3 Experiments of cell viability. In panel a , the OE extract was subjected to different sonication times, and its viability values
were obtained. In panel b , the effects of OE and s-OE on cell viability were compared. Three independent experiments
were carried out, and the values are expressed as the mean ± standard deviation (sd).
* denotes p < 0.05 vs. CTRL; ** denotes p < 0.01 vs. CTRL. The Student test was applied.
Total polyphenols and flavonoids
In [Table 1 ], the total content of polyphenols and flavonoids in OE and s-OE extracts are represented.
From the different gallic acid concentrations, the regression equation for the polyphenol
content gave 65.2 ± 3.93 mg and 66.4 ± 2.36 mg gallic acid equivalents (GAE)/g dry
weight for OE and s-OE, respectively. The total flavonoid content in plant extracts
was calculated as mg Rutin equivalents (RE)/g dry weight and was equal to 8.23 ± 1.31
and 7.29 ± 2.34 mg for OE and s-OE, respectively.
Table 1 Measurement of phytochemical compound concentration
Extract
Extract Polyphenols
Flavonoids
OE
65.2 ± 3.93 mg
8.23 ± 1.31 mg
s-OE
66.4 ± 2.36 mg
7.79 ± 2.34 mg
Scavenging activity
To determine the in vitro antioxidant effectiveness of OE and s-OE and evaluate which extract had more marked
antioxidant activity, three tests were used: the DPPH (1,1-diphenyl-2-picrylhydrazyl),
reducing power, and the ferrous ions chelating activity assays. The DPPH test showed
that both extracts have free radical scavenging activity, which is lower than ascorbic
acid but still high. After measuring the absorbance, the inhibitory concentration
IC50 of OE and s-OE was calculated. The IC50 of OE was found to be 0.026 ± 0.00 029 mg/mL, while the IC50 of s-OE was 0.019 ± 0.0032 mg/mL ([Fig. 4 a ]). As shown in [Fig. 4 b ], OE and s-OE have a high reducing power that is slightly lower than ascorbic acid.
Between the two extracts considered, s-OE showed a statistically higher effect than
OE. In the Fe2+ chelating activity test ([Fig. 4 c ]), OE
and s-OE showed the same trend as the other assays, and s-OE showed significantly
more activity at all test concentrations. In these experiments, ascorbic acid (500 µM,
24 h) was used as a positive control. In conclusion, tests used to measure the antioxidant
capacity of extracts suggest that s-OE works more than OE.
Fig. 4 Determination of OE and s-OE antioxidant activity. The DPPH, reducing power, and
the ferrous ions chelating activity assays are reported in panels a, b , and c , respectively. Calculation of IC50 was made using GraphPad Prism software, based on the model described by Chou et al.,
2005 [52 ]. Ascorbic acid (500 µM, 24 h) was used as positive control. Three independent experiments
were carried out. ** denotes p < 0.01 vs. the respective value on the OE curve; ***
denotes p < 0.001 vs. the respective value on the OE curve; § denotes p < 0.05 vs.
OE. A Tukey–Kramer comparison test followed variance analysis (ANOVA).
Antioxidant property on cell line
After evaluating the antioxidant activity of the extracts on the powders obtained,
we also studied this property on the chosen cell line. As can be seen in [Fig. 5 a ], pretreatment with OE and s-OE alone did not result in any accumulation of ROS,
reproducing a situation similar to that of untreated cells. In these experiments,
hydrogen peroxide was used as a positive control and cells exposed to H2 O2 (120 µM, 20′) showed an accumulation of ROS evidenced by the shift to the right of
the fluorescent peak, compared to the control cells. Lead treatment caused a lower
ROS production than H2 O2 but was statistically significant. Finally, pretreatment with extracts, followed
by lead exposition, reduced the accumulation of ROS, and s-OE worked better than OE,
with a significant reduction. In [Fig. 5 b ], the respective quantification is shown. Antioxidant properties were also assessed
by measuring the malondialdehyde levels, a toxic by-product of lipid peroxidation,
and the effects generated by the OE and s-OE extracts. [Fig. 5 c ] shows the results obtained, which have followed the same trend of ROS measurement.
Fig. 5 ROS accumulation and malondialdehyde levels. In panel a , the accumulation of ROS measured with the cytofluorometer is shown. Each box refers
to a treatment as indicated: the x-axis represents the fluorescence of the fluorochrome
Fitc connected to our fluorescent probe, while the y-axis is relative to the number
of cells that we decided to acquire (10 000). At the top of each box, there is a marker
(M3), which is arbitrarily drawn in the control and kept the same for all other samples.
The part of the peak included in M3 is indicated by a numerical percentage. In panel
b , the respective quantification, obtained from comparing the percentages, is represented.
The control percentage is arbitrarily made equal to 1 and the other values are related
to it. Panel c represents the levels of malondialdehyde generated by the different treatments. Three
independent experiments were carried out, and the values are expressed as the mean
± standard
deviation (sd). * denotes p < 0.05 vs. CTRL; ** denotes p < 0.01 vs. CTRL; § denotes
p < 0.05 vs. lead. Analysis of variance (ANOVA) was followed by a Tukey–Kramer comparison
test.
The activity of primary antioxidant enzymes
When mammalian cells are subjected to oxidative stress, some antioxidant enzymes are
expressed with the role of reducing and/or resolving the stressful condition and surviving.
Between these, catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase
(GSH-Px) are the most known. We have measured their activity under the described test
conditions. [Fig. 6 ] shows the results of the activities of CAT, SOD, and GSH-Px), in panels a, b , and c , respectively. As it is possible to observe, the activity of enzymes has been measured
at different treatment times (0, 3, 6, 9, 12, 24, and 30 hours), and in the right
part of the three panels is represented the magnification of the left panels and the
statistical diversity. The results obtained by the activity of the three enzymes showed
the same trend but with different orders of magnitude: the extracts alone did not
alter the enzymatic activity; the treatment with lead significantly
reduces it, while co-treatment improves it, compared to treatment with the lead. Again,
s-OE appeared more functional than OE.
Fig. 6 CAT, SOD, and GSH-Px activities. The activities of these enzymes are represented
in panels a, b , and c respectively. The left part of the panels shows the values of the activities, while
the right part shows the magnification of the left part and the statistical analysis.
The enzymatic activity was followed at different times (3, 6, 9, 12, 24 and 30 h).
Three independent experiments were carried out, and the values were expressed as the
mean ± standard deviation (sd). * denotes p < 0.05 vs. the time 0; ** denotes p < 0.01
vs. the time 0; § denotes p < 0.05 vs. OE + Pb2+ . Analysis of variance (ANOVA) was followed by a Tukey–Kramer comparison test.
OE and s-OE protect against lead-induced damage
In [Fig. 7 a ], a significant reduction in cell viability, following exposure to increasing concentrations
of lead, is represented. Treatment with the chosen concentration of lead (25 µM) resulted
in a reduction in viability of about 40% compared to untreated cells. Interestingly,
both extracts used have protected against lead-induced neurotoxicity, increasing cell
viability. The concentration of 25 µg/mL of extract s-OE protected about 10% more
than OE. In these experiments, treatment with beta-amyloid peptide (Aβ ), the major constituent of amyloid plaques in Alzheimerʼs disease, was used as a
positive control. Treatment with lead has also been associated with a reduction in
lipid production, as demonstrated by appropriate cytfluorimeter readings. Specifically,
lead reduced lipid levels by about 50% compared to control cells; moreover, while
OE and s-OE administered alone did not alter lipid content, assuming a behavior like
untreated
cells, co-treatment with lead restored lipid reduction and s-OE worked significantly
better than OE. This effect was shown in [Fig. 7 b ], while in [Fig. 7 c ], the respective quantification is represented.
Fig. 7 The treatment with lead has induced cellular damage: viability and lipid decrease.
Panel a shows the significant reduction in viability following exposure to increasing concentrations
of lead. The concentration chosen was 25 µM, which was able to determine a reduction
in viability of 40% compared to untreated cells. Treatment with Aβ was used as a positive control. In panel b , a cytofluorometric reading is represented that highlights the reduction in lipid
synthesis induced by lead exposure. In particular, the lipid reduction has been evidenced
by the shift on the left of the fluorescent cellular peak, compared to untreated cells.
In every single box of panel b , the x-axis represents the fluorescence of fluorochrome PE linked to our fluorescent
probe, while the y-axis is relative to the number of cells that we have decided to
acquire (20 000). At the top of each box, there is a marker (M3), which is arbitrarily
drawn in the
control and kept the same for all other samples. The part of the peak included in
M3 is indicated by a numerical percentage. In panel c , the respective quantification, obtained from comparing the percentages, is represented.
The control percentage is arbitrarily made equal to 1, and the other values are related
to it. Three independent experiments were performed, and the values were expressed
as the mean ± sd. * denotes p < 0.05 vs. the control; ** denotes p < 0.01 vs. the
control; *** denotes p < 0.001 vs. the control. ° denotes p < 0.05 vs. the corresponding
concentration of OE + Pb2+ ; °° denotes p < 0.01 vs. the corresponding concentration of OE + Pb2+ ; § denotes p < 0.05 vs. Pb2+ . Variance analysis (ANOVA) was followed by a Tukey–Kramer comparison test.
Olea europaea extracts reduce endoplasmic reticulum stress: calcium levels and GRP-78
expression
In [Fig. 8 a ], the cytosolic level of calcium was represented, and its variations are due only
to the ion coming from the endoplasmic reticulum. Treatment with thapsigargin (TAPSI),
a noncompetitive Ca2+ ATPase sarco/endoplasmic reticulum inhibitor (SERCA), increased cellular calcium
levels, forcing its release from organelle, and this effect was visible with an increase
in basal calcium levels, compared to untreated cells. In [Fig. 8 b ], it was shown that treatment with OE and s-OE alone did not affect basal calcium
and the respective curves overlapping those of untreated cells. The pretreatment with
OE or s-OE, followed by lead exposure, reduced basal calcium levels compared to levels
achieved with lead. Again, s-OE was more efficient than OE. This latter is shown in
[Fig. 8 c ]. Finally, the expression of GRP-78, the main chaperone of the endoplasmic reticulum,
was evaluated
to highlight any alterations of this organelle: as you can appreciate in [Fig. 8 d ], the treatment with lead significantly increased its expression, while pretreatment
with OE and s-OE, followed by exposure to lead, reduced it, demonstrating protection
from heavy metal. When the extracts of interest were tested alone, they were not responsible
for altering the expression of GRP-78.
Fig. 8 Measurement of cytosolic calcium. Untreated cells consist of a specific level of
calcium ions, which is expected to increase because of TAPSI exposure, as indicated
by the black arrow. Subsequently, treatment with EGTA, a chelating agent, reduces
calcium levels, as represented by the red arrow. Exposure to lead increased the basal
calcium levels compared to untreated cells. Panel a represents an experimental model that testifies to the operation of this method.
Panel b highlights the calcium levels generated by treatment with OE and s-OE alone and compares
them to those generated by TAPSI and Pb2+ . In panel c , calcium levels after pretreatment with OE/s–OE and lead exposure are shown. The
figure reports a representative experiment. Panel d shows the modulation of the expression of GRP-78 in our experimental model, normalized
for the housekeeping protein actin. The respective quantification is highlighted under
Western
blotting. Three independent experiments were performed, and the values were expressed
as the mean ± sd. ** denotes p < 0.01 vs. the control; § denotes p < 0.05 vs. lead.
A representative image is shown. Analysis of variance (ANOVA) was followed by the
Tukey–Kramer comparison test.
Lead treatment results in impaired cell cycle
Lead significantly altered the cell cycle profile, blocking cells in the Sub-G0 phase
and reducing cell viability at the expense of the G0/G1 phase. As can be seen in [Fig. 9 a ] and [b ], the Sub-G0 phase was absent in cells not treated or treated with OE and s-OE alone
(3%, 2%, and 2%, respectively). In contrast, this phase increased to 61% after treatment
with lead. The G0/G1 phase is reduced from about 62% (in control cells, in OE, and
in s-OE) to 11% (in the lead-treated sample). Pretreatment with s-OE extract, before
lead exposure, significantly restored the lead-altered cell cycle, bringing the Sub-G0
phase to 5% and the G0/G1 phase to 41%, in a condition similar to untreated or OE-
and s-OE-treated cells alone. OE extract slightly improved the cell cycle profile
(Sub-G0 = 38%; G0/G1= 30%) by only slightly overlapping the control cells. The quantification
of the phases of the cell cycle is shown in [Fig. 9 b ].
Fig. 9 Exposure to lead alters the cell cycle profile. This figure shows the cell cycle
obtained by cytofluorometric analysis. In every single box of panel a , the x-axis represents the fluorescence of the propidium iodide, while the y-axis
is relative to the number of cells that we have decided to acquire (30 000). In panel
b , the respective quantification is shown. Three independent experiments were performed,
and the values were expressed as the mean ± sd. * denotes p < 0.05 vs. Sub-G0 phase
of the control; ** denotes p < 0.01 vs. Sub-G0 phase of the control; *** denotes p < 0.001
vs. Sub-G0 phase of the control. ° denotes p < 0.05 vs. G0/G1 phase of the control;
°° denotes p < 0.01 vs. G0/G1 phase of the control; °°° denotes p < 0.001 vs. G0/G1
phase of the control. § denotes p < 0.05 vs. G2/M phase of the control. Variance analysis
(ANOVA) was followed by a Tukey–Kramer comparison test.
The deleterious effects of lead are caused by oxidative damage
The smooth endoplasmic reticulum is presumably involved in the damage induced by lead
exposure, as demonstrated by the alteration of lipid and calcium levels and the modulation
of the GRP-78′s expression. N-acetylcysteine (NAC, 2 mm for 6 hours), a known reducing
agent with strong antioxidant properties, has shown an important role in adverse events
generated by lead exposure. As shown in [Fig. 10 ], when administered alone, NAC behaved in a similar way to untreated cells, while
cell co-treatment with NAC and lead generated protection against lead-induced damage,
reporting all values like untreated cells. Protection generated by NAC has been observed
not only for lipid and calcium levels but also for GRP-78 expression, demonstrating
a direct involvement of oxidative damage formed by lead.
Fig. 10 Effects of NAC on lead-induced damage. Panel a showed the effect of NAC on the lead-induced reduction in lipid levels, while panel
b highlighted the respective quantification. Calcium ion levels after treatment with
NAC are represented in panel c . Finally, panel d reported the expression of the protein GRP-78 after co-treatment with NAC–lead. Values
of three independent experiments are indicated, and a representative image is shown.
** denotes p < 0.01 vs. CTRL; § denotes p < 0.05 vs. lead. Analysis of variance (ANOVA)
was followed by the Tukey–Kramer comparison test.
Discussion and Conclusions
Discussion and Conclusions
The first objective of this manuscript was to create an innovative extract, always
obtained from olive leaves like OE but subject to a sonication process (s-OE). It
has been reported that the use of sonication could intensify the dissolution process,
increasing the solubility and water dispersion rate of the sonicate compound [33 ]. Ultrasonic homogenization involves a wide variety of effects, and its action depends
on sonication conditions, especially sonication time and sonication power [34 ]. The increased solubility of s-OE after sonication can be explained by several factors
such as changes in particle size, morphology, and degree of crystallinity [35 ]. During the sonication process, the extract was always kept in a container containing
ice to prevent its overheating, with hypothetical breakage of its chemical components,
and because it has been shown that the combination of sonication in
an ice bath increases the efficiency of solubility [36 ]. In addition, the sonication process has also made it possible to reduce the amount
of EtOH used, passing from a hydro-alcoholic solution (EtOH: H2 O = 70 : 30) to another (EtOH: H2 O = 50 : 50). A reduced amount of EtOH makes s-OE safer and more sustainable [37 ]. As prolonged exposure to ethanol causes numerous health-damaging effects [38 ], the reduction in this alcohol in the preparation of s-OE contributes to the achievement
of environmental sustainability [39 ]. Finally, it should also be noted that the extract s-OE is more effective than OE
in the protection from neurotoxicity induced by lead exposure. Presumably, the best
dissolution of the extract has enhanced its functionality. Most of the literature
shows that lead exposure causes oxidative damage accompanied by mitochondrial
alteration, imbalance of the oxidant-antioxidant system, cytochrome c leakage, and
apoptosis [40 ], [41 ]. In our experimental model, we appreciated oxidative damage caused by exposure to
lead, as demonstrated by an accumulation of ROS, an increase in the levels of malondialdehyde,
and an alteration of the levels of activity of the main antioxidant enzymes. Moreover,
our experimental work has also shown that the neurotoxicity induced by exposure to
lead involves the dysfunction of the cellular organelle endoplasmic reticulum: an
alteration of the calcium preserved in this organelle and the reduction in lipid synthesis,
two aspects governed generally by the smooth endoplasmic reticulum [42 ]. The involvement of the endoplasmic reticulum has been also confirmed by the modulation
of the expression of GRP78, a chaperone that plays a key role in the control of cellular
stress, in the folding of
proteins, in the degradation of unfolded proteins, and in endoplasmic reticulum lumen
quality control [43 ]. A consequence of lead exposure and calcium leakage from the endoplasmic reticulum
is also an impaired distribution of cell cycle phases [44 ] with a significant increase in cell death in the Sub-G0 phase. This assumption could
be justified by a dysregulation of the cell cycle, dependent on cellular calcium [45 ]. However, the mechanism involving the endoplasmic reticulum results as a consequence
of the oxidative damage generated by lead. We hypothesize that lead exposure determines
the production and accumulation of reactive species, responsible for an alteration
of the endoplasmic reticulum, altering the physiological cross-talk between mitochondria
and endoplasmic reticulum [46 ]. This hypothesis seems to be confirmed by the results obtained with the
pretreatment with the antioxidant N-acetylcysteine, which has restored almost completely
all lead-induced dysfunctions, reverting lipid and calcium ion reduction as well as
avoiding endoplasmic reticulum involvement, as demonstrated by GRP-78 expression after
NAC-lead co-treatment. Since pretreatment with OE and s-OE can significantly reduce
the damage caused by lead (s-OE OE), they are expected to have an antioxidant effect.
The antioxidant activity of the extracts could be justified both by the presence of
total polyphenols-flavonoids and by the high concentration of oleuropein and hydroxytyrosol
present in the extracts. All these compounds are particularly known for their protective
role in oxidative damage [47 ]. The results obtained are interesting and promising, but they belong to a preliminary
study conducted in vitro (on OE and s-OE extracts and a cell line). This research has led us for the first
time to the knowledge of s-OE and the
use of both extracts in protection from harmful lead exposure. However, further research
is needed to confirm these effects in vivo under conditions that include all physiological processes. For example, it would
be desirable to conduct investigations on the bioavailability of these extracts even
after ingestion, digestion, and expulsion [48 ], [49 ]. The concentration of extracts used in this work (25 µM) was chosen because it was
safe and non-toxic in the models considered. Although in studies in vivo the concentration of extracts used will be higher than that represented in this manuscript,
this research will have been useful in highlighting the mechanisms involved in protecting
the extracts against lead. If the results obtained are confirmed also in vivo , the use of Olea europea could increase considerably, and this plant could guarantee further specific remedies
for human health. In
conclusion, we can say that:
Sonicated extract (s-OE) works better than OE. Therefore, the use of sonication is
promising and could justify further research to understand if the increased solubility
and dissolution rate can also affect the bioavailability of the extract in vivo .
Lead exposure causes damage to the mitochondria-reticulum endoplasmic unit in human
neurons, which can be attributed to oxidative damage.
Materials and Methods
Plant materials and sample preparation
The leaves of the olive tree (Olea europaea , cultivar Coratina) were collected in Roccelletta di Borgia, Calabria, Italy, latitude
20°25′47″N and longitude 34°27′36″E (February 2023, temperature 9,2 °C). The taxonomic
identification was confirmed by full professor Salvatore Ragusa, University “Magna
Graecia” of Catanzaro (author of this manuscript). The voucher specimen was deposited
in the Department of Health Sciences, University “Magna Graecia” of Catanzaro under
the following accession number Olea Europaea L., cv. Coratina: 12. Several washes with MilliQ water were carried out to remove
pollution or other contaminants from leaves. Then, 100 g of dried and milled leaves
and 800 mL of water were placed in a Pyrex round-bottomed flask equipped with a jacketed
coiled condenser in a domestic microwave oven [31 ]. After extraction, performed by irradiation at 800 W for 10 min, leaves were filtered,
and the obtained solution was dried
under pressure. To eliminate the solid residue, the extract was filtered with acetone,
and the solution was evaporated under pressure to obtain the crude extract (OE). Subsequently,
the OE was placed in dark bottles and stored at 4 °C before being used. To carry out
the experiments, the OE was dissolved in a hydroalcoholic solution composed of EtOH : water
(70 : 30).
s-OE preparation
s-OE was obtained by dissolving the OE powder in a hydroalcoholic solution in which
the ratio of EtOH to water was 50 : 50 and then subjected to a sonication treatment
(frequency of 15 kHz, power 470 W for 10 min, Bandelin electronic) under constant
stirring (150 rpm). s-OE was always kept in a container containing ice. s-OE was placed
in dark bottles and stored at 4 °C before being used for experimentation. The choice
of power to be used was justified by previous experiments in our laboratory.
UHPLC-UV-ESI-HRMS analysis
The quantification of hydroxytyrosol and oleuropein in Coratina leaves dry extract
was performed by reverse-phase ultra-high-performance liquid chromatography followed
by electrospray high-resolution mass spectrometry, with ionization in negative mode,
as reported by Frisina et al. [32 ]. Chromatography separation was performed using a Dionex Ultimate 3000 RS (Thermo
Scientific), equipped with a Hypersil Gold C18 column (100 × 2.1 mm, 1.9 µm particle
size, Thermo Scientific), which was thermostated at 24 °C. The chromatographic column
was equilibrated in 98% solvent A (ultrapure water containing 0.1% of formic acid)
and 2% solvent B (methanol). The flow rate on the column was maintained at 300 µl · min−1 and the concentration of solvent B was linearly increased from 2% to 23% in 6 min,
remaining in isocratic for 5 min, then linearly increased from 23% to 50% in 7 min
and from 50% to 98% in 5 min, remaining in isocratic for 6 min, and
finally returned to 2% in 6 min, remaining in isocratic for 3 min. The UV/VIS detector
was set at 235, 254, 280, and 330 nm. Mass detection was performed by a high-resolution
Q-Exactive orbitrap mass spectrometer (Thermo Scientific). In each scan, the negative
exact mass [M – H]− of hydroxytyrosol (153.0557 m/z) and oleuropein precursors (539.1170 m/z) were selected
in parallel reaction monitoring. Hydroxytyrosol and oleuropein were characterized
according to the corresponding HRMS spectra, accurate masses, characteristic fragmentations,
and retention times. Xcalibur software (version 4.1) was used for instrument control,
data acquisition, and data analysis.
Measurement of polyphenols content
The total polyphenols were quantified by the Folin–Ciocalteu assay using gallic acid
99% (Sigma-Aldrich) as the reference standard. The extracts OE or s-OE were prepared
by adding 20 mL of ethanol/water 50 : 50 (w/w) to 1 g of powder to obtain absorbance
values within the linearity range of the calibration curve. The mixture obtained was
stirred for 24 h at 20 °C. Then, 400 µL of extract was added to 0.8 mL of Folin–Ciocalteu
reagent diluted 10 times. After 3 minutes, 0.8 mL of sodium carbonate 7% (w/v) was
added, and the mixture was allowed to stand for 2 h with constant stirring. The absorbance
was measured at 760 nm with a Prism V-1200 spectrophotometer. The total phenolic content
was determined from the linear equation of a standard curve prepared with different
concentrations of gallic acid, and the results were expressed in mg of gallic acid
equivalents per g of dry weight.
Measurement of flavonoid content
The flavonoid content of the extracts was measured by the colorimetric technique of
aluminum chloride. Specifically, 1 mL of extract was mixed with 1 mL of 2% aluminum
chloride in methanol. After 30 min, the absorbance at 510 nm was measured, and the
rutin equivalents (mg RE/g extract) were used to represent the estimated content of
flavonoids.
Free radical scavenging activity
The free radical scavenging activity of both extracts was determined using the stable
radical 2,20 -diphenyl-1 picrylhydrazyl (DPPH) in which samples were tested at different
concentrations (0.01 – 0.32 mg/mL) [50 ]. Experimentally, an aliquot (0.5 mL) of samples was added to 3 mL of DPPH solution,
keeping the mixture in the dark for 20 min. Subsequently, the absorbance was read
by a UV-Vis spectrophotometer (Multiskan GO, Thermo Scientific) at 517 nm and at room
temperature. Ascorbic acid was used as a reference. The results are reported as an
average percentage of radical scavenging activity (%) and were expressed also as %
inhibition value and IC50 .
Reducing power assay
This method is based on the principle that substances with a reducing potential react
with potassium ferricyanide (Fe3+ ) to form potassium ferrocyanide (Fe2+ ). The reducing power of OE and s-OE extracts has been evaluated by spectrophotometric
detection of the Fe3+ -Fe2+ transformation method [51 ]. The samples were tested at different concentrations (0.01 – 0.32 mg/mL); different
amounts of samples in 1 mL solvent were mixed with 2.5 mL phosphate buffer (0.2 M,
pH 6.6) and 2.5 mL potassium ferricyanide at 1% [K3Fe(CN)6]. The mixture was incubated
at 50 °C for 20 min. The solution was quickly cooled, mixed with 2.5 mL of 10% trichloroacetic
acid, and centrifuged at 1811 rcf for 10 min. The resulting supernatant was mixed
with 2.5 mL of distilled water and 0.5 mL of 0.1% fresh ferric chloride (FeCl3 ), and the absorbance was measured at 700 nm after 10 min. Ascorbic acid was used
as a reference.
The reducing potential was expressed in ascorbic acid equivalent (ASE/mL).
Ferrous ions (Fe2+ ) chelating activity
The Fe2+ ions have pro-oxidant properties capable of determining lipid oxidation resulting
in cellular alterations, and chelating agents can reduce these damages. The chelating
activity can be studied by spectrophotometric evaluation of the inhibition of the
Fe2+ -ferrozine complex in which the agents reduce the formation of this complex: the reduction
in the color is proportional to the chelating activity produced. Experimentally different
concentrations of each sample in 1 mL solvent were mixed with 0.5 mL methanol and
0.05 mL of 2 mM FeCl2 . The mixture obtained was vigorously mixed and maintained at room temperature for
10 min. The absorbance of the solution was measured spectrophotometrically at 562 nm.
Cell cultures
A cell line of human neurons (SH-SY5Y) was acquired from the American Type Culture
Collection and kept in Eagleʼs minimum essential medium supplemented with nonessential
amino acids, 10% fetal bovine serum, penicillin (100 IU/mL), and streptomycin (100 µg/mL).
The cell line was cultivated in a 5% humidified CO2 atmosphere at 37 °C. To differentiate SH-SY5Y cells, 10 µM of all-trans-retinoic
acid was used for 5 days. When the cells reached 60% confluence, they were treated
with OE or s-OE 25 µg/mL for 24 h. In neurotoxicity-induced studies, the cells were
pretreated with OE or s-OE and then exposed to Pb2+ for an additional 24 h. Finally, in experiments conducted to study the antioxidant
effect of NAC, the cells were treated with NAC, 2 mM, for 6 hours and successively
exposed to Pb2+ for an additional 24 h.
Measurement of cell viability through MTT test
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to evaluate
cell viability in a colorimetric assay. Then, 8 × 103 cells were grown in 96-well plates, and the treatments were performed as indicated.
The medium was replaced with a phenol-free medium containing an MTT solution (0.5 mg/mL).
Finally, after 4 h incubation, 100 µL of 10% SDS was added to solubilize formazan
crystals, and the optical density was measured at wavelengths of 540 and 690 nm using
a spectrophotometric reader (X MARK Microplate Bio-Rad).
Measurement of ROS accumulation in cells
The molecule H2 DCF-DA easily enters cells and is subsequently cleaved by intracellular esterases
to form H2 DCF, which is not more able to leave cells and, if oxidized, binds to ROS, forming
the compound DCF highly fluorescent. Therefore, the quantification of the DCF probe
provides the content of ROS in cells. Experimentally, SH-SY5Y cells were seeded in
96-well microplates with a density of 6 × 104 . The following day, they were pretreated with OE and s-OE at a concentration of 25 mg/mL
for 24 hours. At the end of the treatment, the growth medium was replaced with a fresh
medium containing H2 DCF-DA (25 µM), and after 30 minutes of exposure to 37 °C, the cells were washed with
PBS, centrifuged, resuspended in PBS, and exposed or not to H2 O2 (120 µM, 20 min). Fluorescence was evaluated by cytometric analysis (FACS Accury,
Becton Dickinson).
Catalase, superoxide dismutase, and glutathione peroxidase activities
Following the treatments carried out as described, the activities of CAT, SOD, and
GSH-Px were measured in SH-SY5Y cells. For this purpose, the respective kits (UK,
Cambridge, and Abcam) were used according to the manufacturersʼ instructions. The
CAT enzyme converts hydrogen peroxide into another compound that can be measured at
570 nanometers, whose activity is inversely proportional to the measured signal. The
SOD enzyme converts superoxide radicals into hydrogen peroxide and molecular oxygen.
When measuring its activity, superoxide anions react with a specific probe to produce
a water-soluble formazanic dye. The higher the activity of SOD in the sample, the
lower the production of dye formazan. Finally, GSH-Px converts hydrogen peroxide into
water in a reaction involving glutathione and determines the consumption of NADPH.
NADPH reduction is measured at 340 nanometers and is proportional to Gpx activity.
Lipid quantification by flow cytometry
Nile red (excitation/emission ~ 552/636) is a dye that binds tightly to cellular lipids.
At the end of the appropriate treatments, the neurons were collected and adequately
incubated with 1 µg/mL of Nile red dye for 15 min in the dark and protected from light.
Subsequently, the cells were washed in PBS (pH = 7.4) and immediately read in the
flow cytometer FACS Accury (Becton Dickinson).
Cell lysis and immunoblot analysis
The cells SH-SY5Y, grown in 100 mm plates, were washed with PBS and lysed with a preheated
lysis buffer containing 50 mM TrisCl, pH 6.8, 2% SDS, and a mixture of protease inhibitors.
The protein concentration in cell lysates was determined from the DCA protein assay;
the samples were boiled and filled with SDS-polyacrylamide gel (10%). After electrophoresis,
polypeptides were transferred, and specific antibodies were used to reveal the respective
antigens. The primary antibodies were incubated overnight at 4 °C followed by a secondary
antibody conjugated with horseradish peroxidase for 1 hour at room temperature. The
blots have been developed by advanced chemiluminescence procedures. The following
primary antibodies were used: a monoclonal antibody for GRP-78 made in the rabbit
(ab21685, abcam) at 1 : 1000 dilution and a monoclonal anti-actin antibody made in
the mouse at dilution 1 : 5000. Secondary antibodies conjugated with horseradish peroxidases
made in rabbit or mouse
were used at 1 : 10 000 dilution.
Intracellular calcium measurements
The rhodamine 2 fluorescent probe is frequently used to measure the intracellular
calcium since it binds to this ion. The neurons, after being treated as indicated,
were exposed to Rhod 2 for 1 h at 25 °C and protected from light. At the end of the
exposure time, they were washed in a calcium- and magnesium-free solution to prevent
the entering of calcium from the extracellular environment and subjected to flow cytometric
analysis. The cells were also treated with a decoupling of mitochondrial oxidative
phosphorylation, carbonylcianuro-4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 µM),
and oligomycin (1 µg/mL), a mitochondrial ATPase inhibitor. Both treatments may exclude
mitochondrial calcium involvement. Thus, any movement of calcium was related to endoplasmic
reticulum calcium. A cytofluorometer reading (Accury, Becton Dickinson) provides the
basal concentration of the calcium ion in cells.
Cell cycle analysis
The cell cycle refers to the events through which a cell grows, replicates its genome,
and eventually divides into two daughter cells through the process of mitosis. Briefly,
SH-SY5Y cells were seeded in 100 mm plates. After the treatments carried out as described,
the cells were collected, washed twice with PBS, and fixed in 70% cold ethanol for
at least 2 hours at 20 °C. After fixation, the cells were washed with PBS and incubated
with 1 mL PBS containing 0.5 mg/mL RNAse A and 0.5% Triton X-100 for 30 minutes at
37 °C. Finally, the cells were stained with 50 mg/mL of propidium iodide and analyzed
by flow cytometry using a FACS Canto II system (BD Biosciences). The various phases
of the cell cycle were determined using FACS Diva software v. 6.1.3 (BD Biosciences).
Contributorsʼ Statement
J. M., S. B., and G. D. have conceptualized and designed the manuscript; J. M., F. B.,
S. R. have collected and produced data; J. M. and L. G. have interpreted data; J. M.,
and A. L. dealt with the statistical analysis; A. L., R. C., E. P., V. M., and S. R. have
participated in the original draft preparation and curated the manuscript; J. M. and
G. D. have written, revised, and supervised the manuscript.
