Biological actions and molecular mechanisms of Sambucus nigra L. in neurodegeneration: A cell culture approach

molecules Article Biological Actions and Molecular Mechanisms of Sambucus nigra L. in Neurodegeneration: A Cell Culture Approach Olga Palomino 1, Ana García-Aguilar 1, Adrián González 1, Carlos Guillén2,3 , Manuel Benito 2,3 and Luis Goya 4,*   Citation: Palomino, O.; García-Aguilar, A.; González, A.; Guillén, C.; Benito, M.; Goya, L. Biological Actions and Molecular Mechanisms of Sambucus nigra L. in Neurodegeneration: A Cell Culture Approach. Molecules 2021,26, 4829. https://doi.org/10.3390/ molecules26164829 Academic Editors: Maria JoséRodríguez-Lagunas and Malen Massot-Cladera Received: 29 June 2021 Accepted: 7 August 2021 Published: 10 August 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Department of Pharmacology, Pharmacognosy and Botany, Faculty of Pharmacy, University Complutense of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain; [email protected] (O.P.); [email protected] (A.G.-A.); [email protected] (A.G.) 2 Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University Complutense of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain; [email protected] (C.G.); [email protected] (M.B.) 3Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM), Instituto de Salud Carlos III, 28029 Madrid, Spain 4Department of Metabolism and Nutrition, Institute of Science and Food Technology and Nutrition (ICTAN—CSIC), 28040 Madrid, Spain *Correspondence: [email protected]; Tel.: +34-91-549-2300 Abstract: Sambucus nigra flowers (elderflower) have been widely used in traditional medicine for the relief of early symptoms of common cold. Its chemical composition mainly consists of polyphenolic compounds such as flavonoids, hydroxycinnamic acids, and triterpenes. Although the antioxidant properties of polyphenols are well known, the aim of this study is to assess the antioxidant and protective potentials of Sambucus nigra flowers in the human neuroblastoma (SH-SY5Y) cell line using different in vitro approaches. The antioxidant capacity is first evaluated by the oxygen radical absorbance capacity (ORAC) and the free radical scavenging activity (DPPH) methods. Cell viability is assessed by the crystal violet method; furthermore, the intracellular ROS formation (DCFH-DA method) is determined, together with the effect on the cell antioxidant defenses: reduced glutathione (GSH) and antioxidant enzyme activities (GPx, GR). On the other hand, mTORC1 hyperactivation and autophagy blockage have been associated with an increase in the formation of protein aggregates, this promoting the transference and expansion of neurodegenerative diseases. Then, the ability of Sambucus nigra flowers in the regulation of mTORC1 signaling activity and the reduction in oxidative stress through the activation of autophagy/mitophagy flux is also examined. In this regard, search for different molecules with a potential inhibitory effect on mTORC1 activation could have multiple positive effects either in the molecular pathogenic events and/or in the progression of several diseases including neurodegenerative ones. Keywords: Sambucus nigra; neurodegeneration; polyphenols; human neuroblastoma; cell culture; autophagy 1. Introduction Sambucus nigra L., (elderflower) is a well-known herbaceous species of the Caprifoliaceae family spontaneously growing in Europe, West Asia, and North America. Elderflower in the form of herbal tea has been used for the relief of symptoms of common cold, and also as an hypoglycemic, purgative, diuretic and diaphoretic treatment [1–4]. Elder fruits or juices have also been used in the food area, in the processing of jams or jellies or to produce wine. In contrast, leaves and stems from the plant are considered toxic due to their content in cyanogenic glycosides (i.e., sambunigrin and prunasin) and m-hydroxysubstituted glycosides (i.e., zierin and holocalin) which, after hydrolysis, are able to release cyanide [5]. Molecules 2021,26, 4829. https://doi.org/10.3390/molecules26164829 https://www.mdpi.com/journal/molecules Molecules 2021,26, 4829 2 of 18 S. nigra flower extracts have shown to exert different biological activities. Izzo et al. [6] confirmed the antibacterial activity of elderflower extracts against both Gram-positive and Gram-negative bacteria; also, isolated compounds from elderflower have shown immunomodulatory activity through an inhibition of macrophage release of pro-inflammatory cytokines, this effect being attributed to the inhibition of activation of NF-kB and phosphatidylinositol-3-kinase (PI3K) [ 7 ]. An aqueous extract of elderflower was able to significantly increase glucose uptake, glucose oxidation, and gluconeogenesis in rat abdominal muscle. Pancreatic cells treated with the elderflower extract showed a dosedependent stimulatory effect on insulin secretion [ 8 ]. Elderflower infusion also exerted a diuretic effect in experimental animal that was even superior to the one observed with theophylline (5 mg/kg body weight) [9,10]. The biological properties of elderflower have been related to its high content in polyphenolic compounds which are known for their antioxidant potential activity in both in vitro and in vivo studies: flavonoids, including quercetin, rutin, and kaempferol among others; hydroxycinnamic acids such as chlorogenic (CGA), caffeic (CA), and protocateuchic acids. A large number of studies showed the anti-inflammatory, antiviral, antiallergic, vasoprotective and anti-carcinogenic properties exerted by this kind of natural compounds [ 5 , 11 ], including the beneficial effects of CGA as an antioxidant, hepatoprotective, and hypoglycemic agent. Regarding neurodegenerative diseases, protein aggregates and dysfunctional/damaged organelles are the most common feature that alter cell homeostasis and trigger neurodegeneration. Compelling evidence indicates that the mammalian target of rapamycin (mTOR) is a central cell growth-regulating kinase that exists in two different protein complexes, mTORC1 and mTORC2, and it is implicated in multiple essential processes including cell growth and protein synthesis in response to nutrients, growth factors, and cellular energy conditions, and the development and function of the nervous system. The induction of mTOR leads to the phosphorylation and activation of many target proteins related to translational machinery, ribosomal biogenesis, and cell growth, including the p70 ribosomal S6 kinase and eukaryotic initiation factor 4E binding protein (4EBP). Moreover, mTORC1 negatively regulates autophagy, which is a lysosomal degradation process of eukaryotic cells for clearing out a broad range of cytotoxic proteins and damaged or dysfunctional organelles, which contributes to maintain cytoplasmic quality control [ 12 ]. In this context, stimulating autophagy contributes to neuroprotection by reducing oxidative stress, representing a new therapeutic approach in the prevention and treatment of neurodegenerative diseases [ 13 ]. For instance, autophagy-inducing agents such rapamycin, a relatively selective inhibitor of mTORC1, reduce intracellular aggregates of toxic proteins in neurodegenerative diseases, such as β -amyloid levels in Alzheimer’s disease [ 14 ], α -synuclein in Parkinson’s disease [ 15 ], or huntingtin species in Huntington’s disease [ 16 ]. In addition, due to their biological properties, polyphenols including resveratrol, quercetin, and catechins activate autophagy and act as neuroprotective agents in several disease models. In contrast, mTORC1 signaling is highly linked to oxidative stress and brain disorders, including brain tumors and neurodegenerative diseases. The modulation of mTORC1 signaling by different extracts from elderflower has not been evaluated. Hyperactivation of the mTORC1 pathway is linked to a disrupted clearance of protein aggregates by autophagy during neurodegeneration, so the aqueous extract of elderflower could exert a beneficial effect by preventing neurodegeneration through a significant reduction of mTORC1 signaling on SH-SY5Y cells. Thus, in this work, a human neuroblastoma (SH-SY5Y) cell line was chosen as a cell culture model of nervous cells and treatment with a strong pro-oxidant, tert-butylhydroperoxide (t-BOOH), was used to reproduce an in vivo condition of oxidative stress in order to study the possible protective mechanisms through which Sambucus nigra flower extracts could protect the cell function. Therefore, the aim of this work was to firstly evaluate the antioxidant potential of different S. nigra flowers extracts in relation to their phenolic profile; a human neuroblas- Molecules 2021,26, 4829 3 of 18 toma cell line, SH-SY5Y was used as a cell culture model and in order to study the possible protective mechanisms exerted by a well-characterized extract of S. nigra on cell function. 2. Results 2.1. Antioxidant Activity In this study, the antioxidant activity of the three different extracts from elderflower were first evaluated by the ORAC assay, which uses Trolox as a water-soluble analogue of vitamin E and is chosen as a positive control in all the assays conducted in this work. Trolox is able to decrease ROS production, to prevent cytotoxicity in human cancer cell lines, and to rescue cells from apoptotic death [ 17 , 18 ]. Then, the oxygen scavenging activity of the samples was determined by the DPPH assay. This activity may be expressed as the inhibitory concentration 50 (IC 50 ), which expresses the antioxidant concentration required to obtain 50% radical inhibition, or as the antioxidant efficiency (AE), which is calculated as 1/IC50. Table 1shows the results for ORAC (as Trolox Equivalents—TE) and DPPH values, which are expressed as IC50 and AE for S. nigra extracts. Table 1. ORAC and DPPH values for S. nigra extracts. Values are mean ± SD, n= 3. Different letters indicate statistically significant differences (p< 0.05) among groups. Extract ORAC µmol TE/mg IC50 (mg/mL) AE Aqueous 1.38 b±0.36 8.17 a±0.30 0.122 a±0.004 Ethanolic 1.13 b±0.28 7.93 a±0.55 0.126 a±0.009 Methanolic 0.66 a±0.03 11.66 b±2.05 0.089 b±0.022 Results indicate that the aqueous and ethanolic extracts exert statistically significant higher antioxidant capacity as measured by the ORAC method when compared to the methanolic one, although their antioxidant ability is moderate. Thus, the following studies will be conducted with the aqueous and ethanolic extracts. 2.2. Chemical Profile The main polyphenols identified in S. nigra aqueous and ethanolic extracts by HPLC are listed in Table 2, together with their relative content. Table 2. HPLC profile and content of selected polyphenols from S. nigra flowers extracts. Data are mean ±SD, n= 3. Compound Retention Time (min) Content (mg/mL Extract) Aqueous Extract Ethanolic Extract 1 Myricetin 32.61 12.61 ×10−3±0.76 9.05 ×10−3±0.65 2. Quercetin 40.15 0.45 ×10−3±0.05 1.65 ×10−3±0.08 3. Caffeic acid 41.48 1.67 ×10−3±0.09 0.53 ×10−3±0.01 4. Chlorogenic acid 42.42 0.91 ×10−3±0.01 0.95 ×10−3±0.03 5. Protocateuchic acid 43.56 4.87 ×10−3±0.08 7.01 ×10−3±0.12 6. Rutin 48.50 0.70 ×10−3±0.01 1.12 ×10−3±0.04 7. Kaempferol 55.00 0.91 ×10−3±0.02 2.50 ×10−3±0.11 Chromatographic profiles of elder fruit extracts show slight differences in their composition: myricetin is the main polyphenol in the aqueous extract (44%), followed by protocateuchic acid (17%); myricetin is also the main polyphenol in the ethanolic extract (28%), but with a lower percentage and closer to the protocautechic acid content (21%) (Figure 1). These results are in agreement with those previously published [ 11 , 19 – 21 ] ensuring the integrity and quality of the tested samples. Molecules 2021,26, 4829 4 of 18 Molecules 2021, 26, x FOR PEER REVIEW 4 of 18 (Figure 1). These results are in agreement with those previously published [11,19–21] ensuring the integrity and quality of the tested samples. (a) (b) Figure 1. Representative chromatographic profile of (a) aqueous and (b) ethanolic extracts from S. nigra flowers, under the described analytical conditions measured at 330 nm. See identification of peaks in Table 2. 2.3. Cell Viability and mTORC1 Signaling Results from crystal violet assay did not show any cytotoxic effects of aqueous extract from S. nigra in SH-SY5Y cells at all the tested concentrations (1–500 µg/mL). However, S. nigra methanolic and ethanolic extracts at high concentrations significantly reduced cell viability in a dose-dependent manner. Specifically, methanolic and ethanolic extracts from S. nigra significantly decreased cell number at 500 µg/mL and from 50 to 500 µg/mL, respectively (Figure 2A). In all the experiments assessed, cell viability was not significantly affected with respect to the control condition when S. nigra extract concentration was from 1 to 25 µg/mL. Accordingly, biological actions and molecular mechanisms of extracts were analyzed using this range of concentrations. In order to gain better insight into the molecular mechanisms of S. nigra flowers in SH-SY5Y cells, the activity of mTORC1 signaling pathway was evaluated by the quantification of the phosphorylation state of threonine-389 and protein levels of its downstream effector p70S6K. The results demonstrated that the aqueous extract of elderflower significantly reduced the phosphorylation of p70 protein compared to control cells, which indicated that mTORC1 signaling is inhibited in response to all the range of concentration assessed. In contrast, methanolic and ethanolic extracts of elderflower reduced or did not affect the activity of the mTORC1 signaling pathway (Figure 2B). Moreover, since rapamycin is a potent inhibitor of mTORC1 activity and thus reduced cell proliferation [22–24], we also used this drug as a positive control to inhibit mTORC1 signaling in the presence or absence of the mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazine (CCCP) in SH-SY5Y cells (Figure 2C). Figure 1. Representative chromatographic profile of ( a ) aqueous and ( b ) ethanolic extracts from S. nigra flowers, under the described analytical conditions measured at 330 nm. See identification of peaks in Table 2. 2.3. Cell Viability and mTORC1 Signaling Results from crystal violet assay did not show any cytotoxic effects of aqueous extract from S. nigra in SH-SY5Y cells at all the tested concentrations (1–500 µ g/mL). However, S. nigra methanolic and ethanolic extracts at high concentrations significantly reduced cell viability in a dose-dependent manner. Specifically, methanolic and ethanolic extracts from S. nigra significantly decreased cell number at 500 µ g/mL and from 50 to 500 µ g/mL, respectively (Figure 2A). In all the experiments assessed, cell viability was not significantly affected with respect to the control condition when S. nigra extract concentration was from 1 to 25 µ g/mL. Accordingly, biological actions and molecular mechanisms of extracts were analyzed using this range of concentrations. In order to gain better insight into the molecular mechanisms of S. nigra flowers in SHSY5Y cells, the activity of mTORC1 signaling pathway was evaluated by the quantification of the phosphorylation state of threonine-389 and protein levels of its downstream effector p70S6K. The results demonstrated that the aqueous extract of elderflower significantly reduced the phosphorylation of p70 protein compared to control cells, which indicated that mTORC1 signaling is inhibited in response to all the range of concentration assessed. In contrast, methanolic and ethanolic extracts of elderflower reduced or did not affect the activity of the mTORC1 signaling pathway (Figure 2B). Moreover, since rapamycin is a potent inhibitor of mTORC1 activity and thus reduced cell proliferation [ 22 – 24 ], we also used this drug as a positive control to inhibit mTORC1 signaling in the presence or absence of the mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazine (CCCP) in SH-SY5Y cells (Figure 2C). Molecules 2021,26, 4829 5 of 18 Molecules 2021, 26, x FOR PEER REVIEW 5 of 18 Figure 2. Effect of aqueous, methanolic, and ethanolic extracts from S. nigra flowers on cell number (A) and mTORC1 signaling (B) in SH-SY5Y cells. (C) Western blots from SH-SY5Y cells treated with the mitochondrial uncoupler CCCP (20 µM) for 2 h in the presence or absence of 40 nM of rapamycin for 24 h with densitometric quantification of p-p70. We used rapamycin (RAPA) as a positive control to inhibit mTORC1 signaling. Values are means ± SD. n = 3. Values are expressed as a fold change (F.C.) relative to control condition. Different letters indicate statistically significant differences (p < 0.05) among groups. In panel C, p values are indicated compared to the indicated control. 2.4. ROS Production Both aqueous and ethanolic extracts were first tested in unstressed cells to check their direct antioxidant potential. Figure 3A shows that 25 and 50 µg/mL of both extracts significantly reduced the steady-state concentration of ROS, indicating that the amount of phenolic compounds in both doses was enough to decrease the basal ROS production in cultured SH-SY5Y. In this assay, cocoa flavonoid epicatechin was used as a positive control for ROS quenching in SH-SY5Y submitted to oxidative stress [25]. When neuroblastoma cells in culture were treated with 100 µM t-BOOH for 21 h or 200 µM t-BOOH for 3 h, the 2-fold increase of intracellular ROS concentration was indicative of a clear situation of oxidative stress (Figure 3B,C). Interestingly, this intense rise of ROS levels was significantly reduced when SH-SY5Y cells were pre-treated with tested concentrations of both extracts for 21 h prior to the oxidative challenge with 200 µM t-BOOH for 3 h (Figure 3B), and in both cases, chemo-protective response against ROS increase was dose-dependent. A similar dose-dependent reduction of t-BOOH-induced ROS over-production was observed when cultured neuroblastoma cells where co-treated for 24 h with 100 µM t-BOOH and either aqueous or ethanol extracts (Figure 3C). Figure 2. Effect of aqueous, methanolic, and ethanolic extracts from S. nigra flowers on cell number ( A ) and mTORC1 signaling ( B ) in SH-SY5Y cells. ( C ) Western blots from SH-SY5Y cells treated with the mitochondrial uncoupler CCCP (20 µM) for 2 h in the presence or absence of 40 nM of rapamycin for 24 h with densitometric quantification of p-p70. We used rapamycin (RAPA) as a positive control to inhibit mTORC1 signaling. Values are means ± SD. n= 3. Values are expressed as a fold change (F.C.) relative to control condition. Different letters indicate statistically significant differences (p< 0.05) among groups. In panel C, pvalues are indicated compared to the indicated control. 2.4. ROS Production Both aqueous and ethanolic extracts were first tested in unstressed cells to check their direct antioxidant potential. Figure 3A shows that 25 and 50 µ g/mL of both extracts significantly reduced the steady-state concentration of ROS, indicating that the amount of phenolic compounds in both doses was enough to decrease the basal ROS production in cultured SH-SY5Y. In this assay, cocoa flavonoid epicatechin was used as a positive control for ROS quenching in SH-SY5Y submitted to oxidative stress [ 25 ]. When neuroblastoma cells in culture were treated with 100 µ M t-BOOH for 21 h or 200 µ M t-BOOH for 3 h, the 2-fold increase of intracellular ROS concentration was indicative of a clear situation of oxidative stress (Figure 3B,C). Interestingly, this intense rise of ROS levels was significantly reduced when SH-SY5Y cells were pre-treated with tested concentrations of both extracts for 21 h prior to the oxidative challenge with 200 µ M t-BOOH for 3 h (Figure 3B), and in both cases, chemo-protective response against ROS increase was dose-dependent. A similar dose-dependent reduction of t-BOOH-induced ROS over-production was observed when cultured neuroblastoma cells where co-treated for 24 h with 100 µ M t-BOOH and either aqueous or ethanol extracts (Figure 3C). Molecules 2021,26, 4829 6 of 18 Molecules 2021, 26, x FOR PEER REVIEW 6 of 18 Figure 3. (A) Direct effect, (B) Pre-treatment, and (C) Co-treatment effects of aqueous and ethanolic extracts from S. nigra flowers on ROS generation in SH-SY5Y cells. Values are means ± SD, n = 4. Values are expressed as a percent relative to control condition. Different letters indicate statistically significant differences (p < 0.05) among groups. 0 20 40 60 80 100 120 140 C 5 ug 10 ug 25 ug 50 ug % ROS Sambucus nigra extract (µg/mL) A) EtOH extract Aqueous extract a ab b cc cc c 0 20 40 60 80 100 120 140 160 180 200 Control t-BOOH 10uM Epi 5 ug 10 ug 25 ug 50 ug % ROS Sambucus nigra extract (µg/mL) B) EtOH extract Aqueous extract a b e ddc, dcb ba b 0 50 100 150 200 250 Control t-BOOH 5 ug 10 ug 25 ug 50 ug % ROS Sambucus nigra extract (µg/mL) C) EtOH extract Aqueous extract a bb f g g c,dd c e Figure 3. ( A ) Direct effect, ( B ) Pre-treatment, and ( C ) Co-treatment effects of aqueous and ethanolic extracts from S. nigra flowers on ROS generation in SH-SY5Y cells. Values are means ± SD, n= 4. Values are expressed as a percent relative to control condition. Different letters indicate statistically significant differences (p< 0.05) among groups. Molecules 2021,26, 4829 7 of 18 2.5. GSH Concentration When SH-SY5Y cells were submitted to a situation of oxidative stress by the administration of 200 µ M t-BOOH for 3 h or 100 µ M t-BOOH for 21 h, cell concentration of GSH decreased to around 60% of basal levels (Figure 4A,B). This significant decrease of GSH was partially reversed by a pre-treatment of cells with 25 µ g/mL of aqueous extract and with all three doses of the ethanol extract (Figure 4A). Similarly, co-treatment of neuroblastoma cells with 10 or 25 µ g/mL of any of the two extracts, aqueous and ethanol, evoked a partial but significant protection against the t-BOOH-induced GSH depletion (Figure 4B). This result unequivocally indicates that the presence in the culture media of the antioxidant compounds contained in both extracts protects SH-SY5Y cells against the loss of reducing power in a situation of oxidative stress. Although Ramiro-Puig et al. [ 25 ] did not test the effect of epicatechin on GSH depletion, pre-treatment of SH-SY5Y with 10 µ M of the cocoa flavanol resulted in a complete GSH recovery after stress. Molecules 2021, 26, x FOR PEER REVIEW 7 of 18 2.5. GSH Concentration When SH-SY5Y cells were submitted to a situation of oxidative stress by the administration of 200 µM t-BOOH for 3 h or 100 µM t-BOOH for 21 h, cell concentration of GSH decreased to around 60% of basal levels (Figure 4A,B). This significant decrease of GSH was partially reversed by a pre-treatment of cells with 25 ug/mL of aqueous extract and with all three doses of the ethanol extract (Figure 4A). Similarly, co-treatment of neuroblastoma cells with 10 or 25 ug/mL of any of the two extracts, aqueous and ethanol, evoked a partial but significant protection against the t-BOOH-induced GSH depletion (Figure 4B). This result unequivocally indicates that the presence in the culture media of the antioxidant compounds contained in both extracts protects SH-SY5Y cells against the loss of reducing power in a situation of oxidative stress. Although Ramiro-Puig et al. [25] did not test the effect of epicatechin on GSH depletion, pre-treatment of SH-SY5Y with 10 µM of the cocoa flavanol resulted in a complete GSH recovery after stress. Figure 4. Protective effect of (A) Pre-treatment or (B) Co-treatment of aqueous and ethanolic extracts from S. nigra flowers on GSH levels of SH-SY5Y cells. Values are means ± SD, n = 4. Values are expressed as a percent relative to activity of control condition. Different letters indicate statistically significant differences (p < 0.05) among groups. 0 20 40 60 80 100 120 C t-BOOH 10uM Epi 5ug 10ug 25ug % GSH Sambucus nigra extract (µg/mL) EtOH extract Aqueous extract dd a aa bb cc A) a 0 20 40 60 80 100 120 C t-BOOH 5 ug 10 ug 25 ug % GSH Sambucus nigra extract (µg/mL) B) EtOH extract Aqueous extract a bbcd e f g Figure 4. Protective effect of ( A ) Pre-treatment or ( B ) Co-treatment of aqueous and ethanolic extracts from S. nigra flowers on GSH levels of SH-SY5Y cells. Values are means ± SD, n= 4. Values are expressed as a percent relative to activity of control condition. Different letters indicate statistically significant differences (p< 0.05) among groups. Molecules 2021,26, 4829 8 of 18 2.6. GPx Activity Treatment of SH-SY5Y cells with a challenge of 200 µ M t-BOOH for 3 h or 100 µ M t-BOOH for 21 h evoked a significant increase in the activity of this antioxidant defense enzyme as a rational response to the induced ROS overproduction (Figure 5A,B). Pretreatment of neuroblastoma cells with all three concentrations of any of the two extracts for 21 h before the onset of the condition of oxidative stress resulted in a partial although significant reduction of GPx activity to reach values that were between those of controls and stressed cells (Figure 5A). A very similar result was obtained when cells were cotreated for 24 h with tested concentrations of extracts and the pro-oxidant (Figure 5B). Pre-treatment with flavonoid epicatechin at 10 µ M evoked a full recovery of GPx activity after the oxidative stress in SH-SY5Y. Molecules 2021, 26, x FOR PEER REVIEW 8 of 18 2.6. GPx Activity Treatment of SH-SY5Y cells with a challenge of 200 µM t-BOOH for 3 h or 100 µM tBOOH for 21 h evoked a significant increase in the activity of this antioxidant defense enzyme as a rational response to the induced ROS overproduction (Figure 5A,B). Pretreatment of neuroblastoma cells with all three concentrations of any of the two extracts for 21 h before the onset of the condition of oxidative stress resulted in a partial although significant reduction of GPx activity to reach values that were between those of controls and stressed cells (Figure 5A). A very similar result was obtained when cells were cotreated for 24 h with tested concentrations of extracts and the pro-oxidant (Figure 5B). Pretreatment with flavonoid epicatechin at 10 µM evoked a full recovery of GPx activity after the oxidative stress in SH-SY5Y. 0 20 40 60 80 100 120 140 160 180 200 C t-BOOH 10 µM Epi 5 µg 10 µg 25 µg % GPx Sambucus nigra extract (µg/mL) a EtOH extract Aqueous extract a d bbc c c A b,c 0 20 40 60 80 100 120 140 160 180 200 C tBOOH 5 µg 10 µg 25 µg % GPx Sambucus nigra extract (µg/mL) BEtOH extract Aqueous extract a bb b,c ccc d Figure 5. Cont. Molecules 2021,26, 4829 9 of 18 Molecules 2021, 26, x FOR PEER REVIEW 9 of 18 Figure 5. Protective effect of aqueous and ethanolic extracts from S. nigra flowers on the enzymatic antioxidant defenses of SH-SY5Y cells. (A,C) Effect of co-treatment of S. nigra extracts on GPx and GR values. (B,D) Effect of pre-treatment of S. nigra extracts on GPx and GR values. Values are expressed as a percent relative to activity of control condition and are means ± SD, n = 4. Different letters indicate statistically significant differences (p < 0.05) among groups. 2.7. GR Activity Similar to what was observed in GPx assay, the t-BOOH insult provoked a 30% raise in GR activity to recover the increased oxidized glutathione produced by GPx. Contrary to what was observed with GPx, none of the concentrations of any extract was able to reduce the enhanced GR activity when cells were pre-treated before the stress (Figure 5C). However, GR was reversed to control values when neuroblastoma cells were co-treated with the three tested doses of ethanolic extract as well as with 5 and 25 µg/mL of the aqueous extract (Figure 5D). As in the case of GPx, pre-treatment of SH-SY5Y with 10 µM epicatechin reversed GR activity after the stress. 2.8. Autophagy To explore whether the autophagy process is implicated in the protection exerted by these extracts toward an oxidative insult, SH-SY5Y cells were stimulated with t-BOOH, which is an inducer of oxidative stress. Our results indicated that t-BOOH inhibited the phosphorylation of the unc51-like kinase 1 (ULK1) complex at serine 757 mediated by 0 20 40 60 80 100 120 140 160 180 C tBOOH 10 µM Epi 5 µg 10 µg 25 µg % GR Sambucus nigra extract (µg/mL) EtOH extract b aa b cc b,c c d CAqueous extract 0 20 40 60 80 100 120 140 160 180 C tBOOH 5 µg 10 µg 25 µg % GR Sambucus nigra extract (µg/mL) D EtOH extract Aqueous extract a b c cc b,c b d Figure 5. Protective effect of aqueous and ethanolic extracts from S. nigra flowers on the enzymatic antioxidant defenses of SH-SY5Y cells. ( A , C ) Effect of co-treatment of S. nigra extracts on GPx and GR values. ( B , D ) Effect of pre-treatment of S. nigra extracts on GPx and GR values. Values are expressed as a percent relative to activity of control condition and are means ± SD, n= 4. Different letters indicate statistically significant differences (p< 0.05) among groups. 2.7. GR Activity Similar to what was observed in GPx assay, the t-BOOH insult provoked a 30% raise in GR activity to recover the increased oxidized glutathione produced by GPx. Contrary to what was observed with GPx, none of the concentrations of any extract was able to reduce the enhanced GR activity when cells were pre-treated before the stress (Figure 5C). However, GR was reversed to control values when neuroblastoma cells were co-treated with the three tested doses of ethanolic extract as well as with 5 and 25 µ g/mL of the aqueous extract (Figure 5D). As in the case of GPx, pre-treatment of SH-SY5Y with 10 µ M epicatechin reversed GR activity after the stress. 2.8. Autophagy To explore whether the autophagy process is implicated in the protection exerted by these extracts toward an oxidative insult, SH-SY5Y cells were stimulated with t-BOOH, which is an inducer of oxidative stress. Our results indicated that t-BOOH inhibited the Molecules 2021,26, 4829 16 of 18 4.12. Antibodies The following antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA): anti-LC3B #4108, anti-p70S6K #9202, anti-phospho-p70S6K (Thr389), #9205, antiphospho-ULK1 (Ser 757) (#14202) and anti-ULK1 (#8054). From Sigma-Aldrich, antiβ -actin (A5316) and anti-α-tubulin (T6199) were used. 4.13. Statistics Statistical analysis of data was as follows: prior to analysis, the data were tested for homogeneity of variances by the Levene test; for multiple comparisons, one-way ANOVA was followed by a Bonferroni test when variances were homogeneous or by a Tamhane test when variances were not homogeneous. The level of significance was p< 0.05. A SPSS version 23.0 program has been used. Author Contributions: O.P. obtained the plant extracts and performed phytochemical analysis; A.G. performed preliminary studies on antioxidant activity; A.G.-A., C.G. and M.B. performed the assays related to autophagy, mTOR, and cell viability; L.G. designed the study and performed the analysis related to the antioxidant defenses. All the authors wrote and reviewed the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest. Sample Availability: Samples of the plant and standard compounds are available from the authors. References 1. European Medicines Agency Monograph. 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