(**C**)

**Figure 1.** Effect of methanol extracts of *Moringa oleifera* leaf powder on cell viability and cell morphology. (**A**) Effect of moringa on SH-SY5Y cell viability. Cells were treated with moringa from 5 to 500 μg/mL for 24 h. (**B**) Protective effect of moringa in a hydrogen peroxide-induced oxidative stress model. Cells were pretreated with moringa from 5 to 100 μg/mL for 24 h before 100 μM H2O2 for 1 h. (**C**) Effect of moringa on SH-SY5Y cell morphology. \* *p* < 0.05 versus no extract, # *p* < 0.05 versus H2O2.

#### *3.4. Effect on Intracellular ROS Production*

The most cytoprotective and non-toxic concentrations (5, 10 and 25 μg/mL) of *Moringa oleifera* methanol extracts were selected for subsequent experiments. Hydrogen peroxide can be converted into hydroxyl radical (HO•) via Fenton reaction [42]. The intracellular production of ROS was measured using the DCFHA-DA assay. As shown in Figure 2, hydrogen peroxide significantly increased by 56% compared to no extract cells. However, pretreatments with the three assayed concentrations of *Moringa oleifera* significantly reduced hydrogen peroxide-induced ROS overproduction. Similar results were observed for moringa leaf flavonoids in bovine mammary epithelial cells against hydrogen peroxide and the compound 1-O-(4-hydroxymethylphenyl)-α-L-rhamnopyranoside from *Moringa oleifera* seeds against carbon against CCl4 [43,44].

**Figure 2.** Effect of methanol extracts of *Moringa oleifera* leaf powder on intracellular ROS overproduction. Cells were pretreated with moringa (5, 10 and 25 μg/mL) for 24 h before 100 μM H2O2 for 1 h. \* *p* < 0.05 versus no extract, # *p* < 0.05 versus H2O2.

#### *3.5. Effect on Oxidative Stress Markers*

An overproduction of ROS to the detriment of endogenous antioxidants leads to oxidative damage to biomolecules. Lipid peroxidation and GSH content are clinically significant biomarkers of oxidative stress in humans [45]. The double bonds of polyunsaturated fatty acids are highly vulnerable to ROS attack and consequently to lipid peroxidation. Malondialdehyde (MDA) is one the most investigated end product of lipid oxidation [42]. As shown in Figure 3A, hydrogen peroxide significantly increased the peroxidation of lipids by 229%. However, pretreatments with methanol extract of moringa leaf powder at 25 μg/mL significantly reduced TBARS levels (160% compared to 229% of hydrogen peroxide). Therefore, moringa leaf extract has a potent preventive effect against the peroxidation of lipids. As shown above, the methanol extract of moringa has scavenging capacity, so the effectonlipidperoxidationcanbeattributedtoadirectactiononfreeradicalsprocess[33].

**Figure 3.** Effect of methanol extracts of *Moringa oleifera* leaf powder on (**A**) lipid peroxidation and (**B**) Index Redox (GSH/GSSG). Cells were pretreated with moringa (5, 10 and 25 μg/mL) for 24 h before 100 μM H2O2 for 1 h. \* *p* < 0.05 versus no extract, # *p* < 0.05 versus H2O2.

Reduced glutathione is the major antioxidant compound in mammalian cells. The index redox (GSH/GSSG) was significantly decreased in hydrogen peroxide (IR = 0.41) compared to no extract cells (IR = 0.74). On the other hand, methanol extract of moringa at 25 μg/mL significantly increased IR to 0.578 compared to hydrogen peroxide-treated cells.

#### *3.6. Effect on Antioxidant Enzymes Activities*

The antioxidant enzymatic defense system plays a key role against free radicals. The enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) constitute the first defense system against oxidative stress. Superoxide dismutase (SOD) converts the superoxide anion radical into hydrogen peroxide. Catalase (CAT) and glutathione peroxidase (GPx) catalyze the decomposition of hydrogen peroxide to water (GPx) and to water and oxygen (CAT) [46]. In Figure 4, our study demonstrated that there is a significant decrease in the activity of these antioxidant enzymes by 53% for CAT, 51% for SOD and 65% for GPx compared to no extract cells (100%). However, pretreatments with moringa extracts reversed this impairment in the defensive system. For catalase, there was a significant increase at 10 and 25 μg/mL (82.9% and 81.2%). For SOD and GPx, the three assayed concentrations markedly improved the activity of these enzymes. The ability

of moringa to increase antioxidant enzymatic activity suggests that their polyphenols have a direct antioxidant effect [33].

**Figure 4.** Effect of methanol extracts of *Moringa oleifera* leaf powder on antioxidant enzyme activity. Cells were pretreated with moringa (5, 10 and 25 μg/mL) for 24 h before 100 μM H2O2 for 1 h. \* *p* < 0.05 versus extract, # *p* < 0.05 versus H2O2.

#### *3.7. Effect on Mitochondrial Regulation*

Mitochondria are double membrane-bound cell organelles presented in almost all cell eukaryotes. Mitochondria are involved in energy production [47]. The increased ROS generation can be associated with mitochondrial dysfunction. Mitochondria is a key target for the prevention and treatment of those oxidative stress-related neurodegenerative diseases [48]. As shown in Figure 5A, hydrogen peroxide reduced significantly mitochondrial membrane potential (MMP) by 55% compared to no extract cells (100%). However, pretreatments with 10 μg/mL and 25 μg/mL significantly increased MMP to 76% and 88%, respectively. Regarding calcium cytosolic, there was a significant increase (357 nM) after hydrogen peroxide-treated cells compared to no extract cells (88 nM). However, the pretreatment with 25 μg/mL almost reduced cytosolic calcium levels to no extract levels (95 nM) (Figure 5B). Furthermore, hydrogen peroxide significantly increased mitochondrial calcium level (1.18 relative to no extract). This increase in mitochondrial calcium level was markedly reduced after pretreatments with 10 μg/mL and 25 μg/mL of moringa extracts (1.01 and 1.03 relative to no extract, respectively) (Figure 5C). Hydrogen peroxide interacts with mitochondrial structure and function, leading to an impaired calcium level and a mitochondrial potential deficiency [49]. Altered calcium levels can affect mitochondrial function and integrity (i.e., depletion of mitochondrial DNA, release of cytochrome C,

ultrastructural lesions, morphological changes) as well as to promote the production of more ROS (i.e., peroxidation of cardiolipin), contributing to the pathogenesis of many common neurodegenerative diseases [50]. The methanol extract of moringa leaves exerts a mitochondrial protective role in neurons, showing for the first time that the mitochondria is the target of action for this medicinal plant.

**Figure 5.** Effect of methanol extracts of *Moringa oleifera* leaf powder on (**A**) mitochondrial membrane potential (**B**) levels of cytosolic calcium and (**C**) levels of mitochondrial calcium. Cells were pretreated with moringa (5, 10 and 25 μg/mL) for 24 h before 100 μM H2O2 for 1 h. \* *p* < 0.05 versus no extract, # *p* < 0.05 versus H2O2.

The major polyphenols identified in methanolic extract of leaves of *Moringa oleifera* include quercetin, kaempferol, apigenin and gallic acid [8]. Previous studies have demonstrated that, after intestinal absorption, polyphenols can reach bloodstream concentration to exert their action [51]. Particularly, these polyphenols presented in moringa have been detected and quantified in blood (quercetin at a concentration of 0.0723 ± 0.0810 μM; kaempferol at a concentration of 0.0579 ± 0.0609 μM; apigenin at a concentration of

0.0106 ± 0.0123 μM and gallic acid at a concentration of 0.007 ± 0.003 μM) [52–54]. Additionally, Nair et al. (2020) have recently demonstrated that compounds presented in moringa extracts are able to pass across Blood-brain-barrier (BBB) and to reach its pharmacological target [55]. Regarding the safe toxicological profile of moringa, doses of up to 2000 mg/kg of methanol and aqueous leaf extract were safe in animal studies [56,57].

Conversely, gallic acid is one of the most abundant phenolic compounds identified in the leaf extract [58]. Previous studies demonstrated that pretreatments with gallic acid protected SH-SY5Y cells against 6-Hydroxydopamine (6-OHDA) induced damage by attenuating mitochondrial dysfunction, reducing intracellular ROS level, and inhibiting apoptotic cell death [59]. Moreover, pretreatments with gallic acid also exerted a potent protection in a hydrogen peroxide-induced oxidative stress model in SH-SY5Y cells by decreasing intracellular ROS production, increasing REDOX activity, and inhibiting caspase-3 activation [60]. Apart from its neuroprotective effect in oxidative stress models in SH-SY5Y cells, gallic acid and its derivatives have shown to inhibit the aggregation of several amyloid proteins associated with the pathological process of many neurodegenerative diseases. Hence, the oral administration of gallic acid improved memory in an APP/PS1 transgenic mouse model by reducing Aβ1-42 aggregation [61]. Moreover, gallic acid also inhibited the insulin amyloid fibril formation in an in-vitro model [62]. Studies on the relationship between structure and inhibitors of amyloid formation have revealed that gallic acid owes its activity to both aromatic and hydroxyl groups and the presence of galloyl moiety [62,63]. In addition to gallic acid, epigallocatechin-3-gallate has shown to reduce Abeta generation in murine neuron-like cells (N2a) transfected with the human "Swedish" mutant amyloid precursor protein (APP) and in primary neurons derived from Swedish mutant APP-overexpressing mice (Tg APPsw line 2576) as well as to reduce the intensity of pancreatic amyloid fibrils in human islet amyloid polypeptide (hIAPP) in trasgenic mice [64,65]. Epigallocatechin-3-gallate exerts its action by binding transthyretin (TTR) and maintaining it in a non-aggregated soluble form [65–68]. Therefore, in our study, the neuroprotective effect of the methanol extract of moringa may be partly due to gallic acid and its derivatives.

## **4. Conclusions**

In conclusion, the current work provides preliminary evidence of the antioxidantmediated neuroprotective effects of the methanol extract of *Moringa oleifera* leaf powder. Moringa has shown to exert a protective effect against hydrogen peroxide in neuroblastoma cells by reducing ROS overproduction and lipid peroxidation levels, by increasing GSH content and antioxidant enzymes activity and by avoiding mitochondrial dysfunction. The most active concentration was 25 μg/mL. This work supports and deepens the therapeutic properties of a nutritionally rich plant. Particularly, this research delves into the molecular mechanisms by which *Moringa oleifera* methanol extract exerts its neuroprotective effect. These promising results create the need for in-vivo studies and even clinical trials to validate the physiological relevance of our in-vitro data.

**Author Contributions:** Conceptualization, E.G.-B. and M.P.G.-S.; methodology, E.G.-B., I.U.-V. and M.S.; formal analysis, E.G.-B., I.U.-V. and M.S.; investigation, E.G.-B., I.U.-V. and M.S.; writing—original draft preparation, E.G.-B.; writing—review and editing, E.G.-B., I.U.-V. and M.P.G.-S. 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.
