**Natural Food Supplements Reduce Oxidative Stress in Primary Neurons and in the Mouse Brain, Suggesting Applications in the Prevention of Neurodegenerative Diseases**

**Miriam Bobadilla , Josune García-Sanmartín and Alfredo Martínez \***

Oncology Area, Center for Biomedical Research of La Rioja (CIBIR), 26006 Logroño, Spain; mbobadilla@riojasalud.es (M.B.); jgarcias@riojasalud.es (J.G.-S.) **\*** Correspondence: amartinezr@riojasalud.es; Tel.: +34-941-278-775

**Abstract:** Neurodegenerative diseases pose a major health problem for developed countries, and stress has been identified as one of the main risk factors in the development of these disorders. Here, we have examined the protective properties against oxidative stress of several bioactive natural food supplements. We found that MecobalActive®, Olews®, and red and white grape seed polyphenol extracts may have a neuroprotective effect in vitro, both in the SH-SY 5Y cell line and in hippocampal neuron cultures, mainly by reducing reactive oxygen species levels and decreasing caspase-3 activity. In vivo, we demonstrated that oral administration of the supplements reduces the expression of genes involved in inflammation and oxidation mechanisms, whereas it increments the expression of genes related to protection against oxidative stress. Furthermore, we found that preventive treatment with these natural extracts increases the activity of antioxidant enzymes and prevents lipid peroxidation in the brain of stressed mice. Thus, our results indicate that some natural bioactive supplements may have important protective properties against oxidative stress processes occurring in the brain.

**Keywords:** oxidative stress; ROS; neurodegenerative diseases; red grape polyphenol extract; white grape seed polyphenol extract; MecobalActive®; Olews®

## **1. Introduction**

The increasing population lifespan in developed countries is leading to a higher incidence of age-related illnesses, including neurodegenerative diseases (ND) [1]. NDs are characterized by a progressive loss of selectively vulnerable neuron populations in specific brain areas [2]. NDs encompass a heterogeneous group of chronic disorders that include, among others, Alzheimer's disease (AD) and other dementias, Huntington´s disease, Parkinson´s disease, multiple sclerosis, human prion, and motoneuron diseases [3–6]. Unfortunately, all these diseases are untreatable at the moment, and, in terms of human suffering and economic and social costs, they represent the fourth cause of global disease burden in developed countries [1].

The current literature clearly shows that oxidative stress is one of the main risk factors for AD [7]. The balance between the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), on the one hand, and of antioxidant substances on the other, is critical for a correct cell function [8]. When unbalanced, the overproduction of ROS and RNS, combined with failing antioxidant defenses, causes oxidative stress [9]. For instance, in AD, a clear diminution of antioxidant activity occurs, which leads to the accumulation of oxidative damage [10]. Additionally, decreased levels of antioxidants such as vitamin C and E and uric acid are observed in AD patients. Many studies have demonstrated that the production of excessive ROS and signs of oxidative stress were detected in the brains of these patients [11,12]. Furthermore, there is evidence that mitochondrial damage resulting in an increased production of ROS contributes to the early stages of the disease prior to the onset of clinical symptoms [9,13]. For these reasons, numerous scientific studies suggest

**Citation:** Bobadilla, M.; García-Sanmartín, J.; Martínez, A. Natural Food Supplements Reduce Oxidative Stress in Primary Neurons and in the Mouse Brain, Suggesting Applications in the Prevention of Neurodegenerative Diseases. *Antioxidants* **2021**, *10*, 46. https://doi.org/10.3390/ antiox10010046

Received: 4 December 2020 Accepted: 24 December 2020 Published: 2 January 2021

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**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/).

that diets rich in antioxidants may be helpful in preventing, postponing, or controlling the progression of AD [14,15].

To date, there is no effective treatment for these degenerative diseases. Some drugs are used for relieving the symptoms, although they usually generate many side effects and have limited efficacy [16]. Therefore, in order to develop novel preventive therapies, a large number of natural plant extracts have been tested as neuroprotective agents [17]. In nature, there are multiple compounds, including polyphenols, flavonoids, and vitamins, which are capable of counteracting the harmful effects of oxidative stress and reducing the risk of developing NDs [7,18]. Special attention has been paid to flavonoids, a type of polyphenolic compounds that are abundantly present in fruits, vegetables, red and white grapes, and green tea [1]. Flavonoids are nutrients with beneficial health effects derived from their antioxidant and anti-inflammatory properties [19,20]. There is now extensive scientific literature describing the beneficial effects of flavonoids in disease prevention [21,22].

The purpose of the present study was to investigate the protective properties against oxidative stress of several bioactive natural food supplements in vitro and in vivo. The addition of these supplements to commonly used food staples may provide a new and affordable strategy for the prevention of NDs.

#### **2. Materials and Methods**

#### *2.1. Cell Culture*

Human neuroblastoma SH-SY 5Y cell line was obtained from the American Tissue Culture Collection (ATCC, Manassas, VA, USA). Cells were grown in Dulbecco's Modified Eagle's Medium (DMEM)-F12 medium (Hyclone, Logan, UT, USA) with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA), 1% penicillin/streptomycin (Gibco), and maintained at 37 ◦C, 5% CO2. Cell culture medium was changed thrice a week.

The cell line was authenticated by STR profiling (IDEXX BioAnalytics, Kornwestheim, Germany).

#### *2.2. Primary Hippocampal Neuron Isolation and Culture*

Mouse hippocampal neurons were isolated from postnatal day 1 (P1) C57BL/6J mice, as described [23], with slight modifications. Briefly, the hippocampus was dissected in Hank's balanced salt solution (HBSS) and incubated at 37 ◦C for 15 min with trypsin/ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, St. Louis, MO, USA). After 3 washes in HBSS, tissue was triturated using a sterile 9-inch Pasteur pipette. HBSS was replaced with Neurobasal plating medium (neurobasal medium, Gibco) containing B27 supplement (1:50) (Gibco), 0.5-mM glutamine solution (Gibco), penicillin/streptomycin (Gibco), 1-mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Hyclone), and 10% heat-inactivated donor horse serum (Gibco). Neuroblasts were plated on poly-Dlysine-coated glass coverslips (p96) at a density of 3 × 10<sup>4</sup> cells/well and placed in a 37 ◦C, 5% CO<sup>2</sup> incubator overnight. Next day, in vitro neurobasal plating medium was replaced with neurobasal feeding medium (neurobasal medium containing B27 Supplement (1:50), 0.5-mM glutamine solution, penicillin/streptomycin (1:200), and 1-mM HEPES). Half of the feeding medium was replaced with fresh medium every 4 days.

#### *2.3. Natural Extracts*

Six commercial natural food supplements were used in this study. They included red grape polyphenol and white grape seed polyphenol extracts (generously provided by Alvinesa Natural Ingredients, Daimiel, Ciudad Real, Spain), extracts from the olive tree (Olews®), citicoline, MecobalActive®, and Cardiose® (all generously provided by HealthTech Bio Actives, Barcelona, Spain).

Red grape polyphenol and grape seed polyphenol extracts, from Alvinesa Natural Ingredients, are entirely constituted by phenolic compounds (premium selected blending of monomers, dimers, oligomers, and polymers) and have a unique formulation that facilitates direct absorption of the phenolic compounds by the small intestine. All these extracts are currently used as commercial supplements approved for human consumption. Some of these extracts have demonstrated their antioxidant properties in other contexts [24].

#### *2.4. Preparation of Aluminum Maltolate*

Aluminum maltolate (Al(mal)3) was prepared according to published procedures [25]. AlCl3·6H2O was dissolved in distilled water to a final concentration of 80 mM. Maltolate was dissolved in phosphate-buffered saline (PBS) to a final concentration of 240 mM. The solutions were mixed in equal volumes, and pH was adjusted to 7.4, inducing the precipitation of Al(mal)<sup>3</sup> crystals. All solutions were filtered using 0.22-µm syringe filters just before use.

#### *2.5. Cell Proliferation Assay*

Cell proliferation was analyzed using the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA), following the manufacturer's instructions. Cells were seeded in 96-well plates at a density of 3 × 10<sup>4</sup> cells per well, allowed to attach for 24 h, and exposed to different concentrations of natural bioactive extracts with or without 125-µM Al(mal)<sup>3</sup> for 72 h. The MTS reagent (3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was added for 4 h, and absorbance was examined at 490 nm using a microplate reader (POLARstar Omega, BMG Labtech, Ortenberg, Germany). The GI<sup>50</sup> (growth inhibition of 50% of cells) values of the different compounds were determined using nonlinear regression plots with Prism 8.3.0 (GraphPad Software, San Diego, CA, USA).

#### *2.6. Measurement of Intracellular ROS Levels*

The levels of ROS were determined in cell cultures by using the cellular ROS assay kit (ab113851, Abcam, Cambridge, UK), following the manufacturer's instructions. Briefly, SH-SY 5Y cells (8 × 10<sup>3</sup> cell/well) were incubated with different concentrations of natural bioactive extracts with or without 125-µM Al(mal)3 for 48 h, followed by an incubation with 25-µM 2′ -7′dichlorofluorescin diacetate (DCFH-DA) for 45 min at 37 ◦C in the dark. After two washes with PBS, DCFH-DA was detected by fluorescence spectroscopy, with excitation/emission at 485/535 nm in a microplate reader (POLARstar Omega).

#### *2.7. Caspase-3 Activation Assay*

Levels of caspase-3 were determined in cell cultures by using the caspase-3 colorimetric assay kit (K106-100; BioVision Inc., Milpitas, CA, USA), following the manufacturer's instructions and previous studies [26]. Briefly, enzyme reactions were performed in 96-well microplates, and 50 µL of cell lysate was added to each reaction mixture. Absorbance at 405 nm was measured using a plate reader (POLARstar Omega).

#### *2.8. Measurement of Nitrite and Nitrate Concentrations*

Cell media were collected and analyzed for their nitrite and nitrate contents by using the nitrite/nitrate colorimetric assay (780001, Cayman Chemicals, Ann Arbor, MI, USA), following the manufacturer's instructions. NO<sup>X</sup> (nitrite + nitrate) concentrations were determined by measuring absorbance at 540 nm using a microplate reader (POLARstar Omega). Cell media nitrate concentrations were calculated by subtracting the concentrations of cell media nitrite from the NO<sup>X</sup> concentrations.

#### *2.9. Restrain Stress and In Vivo Treatments*

Six-week-old C57BL/6J mice (Charles-River) were used for this assay. Mice were housed under standard conditions at a temperature of 22 ◦C (±1 ◦C) and a 12-h light/dark cycle with free access to food and water.

Mice were subjected to an acute model of stress by immobilization, as previously described [4,27], by placing them inside 50-mL conical tubes with no access to food or water for the indicated periods of time. Adequate ventilation was provided by several air holes (0.5 cm in diameter) drilled into the conical end of the tube and at its sides. The tubes prevented forward, backward, or rotational movements of the mice. Due to the corticosterone circadian rhythm [28], restraint stress was started at the same time of the day (9:00 a.m.) in all experiments.

In a pilot study, mice were subjected to restraint for 0, 2, 4, or 6 h, and stress markers were measured (see below). A period of 6h was chosen as the optimal time of restraint for further experiments.

Mice were randomly divided into different experimental groups (n = 8 per group) and received different doses of the natural extracts (or PBS as a control) in 200 µL by oral gavage during 5 consecutive days (Table 1). On the 6th day, mice were subjected to 6 h of restraint stress and immediately sacrificed. The whole brain was dissected out. The olfactory bulbs and the cerebellum were removed, and the remaining tissue was divided into two equal halves by a sagittal section. Each half was frozen separately in liquid N<sup>2</sup> and stored at −80 ◦C. One side was used for RNA extraction and the other one for antioxidant enzyme analysis (see below).

**Natural Extract Dose References** Red grape 100 mg/kg [29,30] 300 mg/kg White grape 100 mg/kg [29,30] 300 mg/kg MecobalActive® <sup>65</sup> <sup>µ</sup>g/kg [31] <sup>135</sup> <sup>µ</sup>g/kg Olews® 300 mg/kg [32,33] 600 mg/kg

**Table 1.** Food supplements and concentrations used for the in vivo study.

#### *2.10. Quantitative Real-Time PCR*

Total RNA was isolated from mouse brains and purified as described [34]. Briefly, total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA), with the DNase digestion step performed (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Resulting RNA (5 µg) was reverse-transcribed using the Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen), and the synthesized cDNA was amplified using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Transcripts were amplified by real-time PCR (7300 Real-Time PCR System, Applied Biosystems). At the end, a dissociation curve was implemented from 60 to 95 ◦C to validate the amplicon specificity. For each transcript, a specific calibration curve of cDNA was included to analyze the expression of NADPH oxidase 2 (NOX-2), heme oxygenase (decycling) 1 (HMOX-1), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and nuclear factor erythroid 2-related factor 2 (Nrf-2). All measurements were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene. Specific primers are shown in Table 2.

**Table 2.** Primers used in this study. Annealing temperature was 60 ◦C for all transcripts.


#### *2.11. Thiobarbituric Acid Reactive Substances (TBARS), Superoxide Dismutase (SOD), and Catalase Activity*

For the determination of oxidative stress parameters and antioxidant components in the brain, frozen tissues were homogenized in radioimmunoprecipitation assay (RIPA) buffer (Thermo Scientific, Waltham, MA, USA) supplemented with complete and phospho STOP (Roche, Basel, Switzerland) protease inhibitors. Lipid peroxidation was determined using a commercial TBARS assay kit (CA995, Canvax, Córdoba, Spain). The final malondialdehyde (MDA) products were detected by fluorescence spectroscopy, with excitation/emission at 530/590 nm in a microplate reader (POLARstar Omega). Levels of superoxide dismutase (SOD) activity were determined using an SOD assay kit (CA061, Canvax), according to the manufacturer's protocols. Absorbance at 450 nm was measured using a POLARstar Omega plate reader. Catalase activities were determined using a commercial catalase activity assay kit (CA063, Canvax) following the manufacturer's instructions. Enzyme activity was detected by fluorescence spectroscopy, with excitation/emission at 530/590 nm in a microplate reader (POLARstar Omega).

#### *2.12. Statistical Analysis*

All datasets were analyzed for normalcy and homoscedasticity. Normal data were analyzed by one-way ANOVA and Dunnett's multiple comparison post-hoc test. Data that did not follow a normal distribution were compared by the Kruskal-Wallis test, followed by the Mann Whitney post-hoc test. Analyses were performed with GraphPad Prism version 8.3.0 (GraphPad Software). A *p*-value < 0.05 was considered statistically significant.

#### **3. Results**

#### *3.1. Olews® and Red and White Grape Extracts Have Neuroprotective Effects on the SH-SY 5Y Cell Line*

To test whether the natural extracts used in this study have an antioxidant capacity, in a first approach, we tested them in vitro on the human neuroblastoma cell line SH-SY 5Y.

First, we tested the activity of the chosen supplements (Cardiose®, Olews®, citicoline, MecobalActive®, and red and white grape extracts) on the SH-SY 5Y cell line to study their potential toxicity. The cells were exposed to increasing concentrations of extracts for 72 h, and the cell number was determined by colorimetric methods.

Interestingly, two different behaviors were observed: (A) extracts that did not elicit significant changes in the number of cells, as with Cardiose® and citicoline (Figure 1A,C), and (B) extracts that induced a dose-dependent toxicity, as observed with MecobalActive®, Olews®, and red grape and white grape seed extracts. The GI<sup>50</sup> for these substances were 126, 73, 76, and 134 µg/mL, respectively (Figure 1B,D–F).

Then, we introduced a chemical inducer of cellular stress to assess the neuroprotective effects of the natural extracts. Al(mal)<sup>3</sup> is a compound that elicits neurotoxicity by inducing mitochondrial membrane potential changes, elevated reactive oxygen species, DNA damage, and apoptosis in SH-SY 5Y cells [35]. Before checking the food supplements, we established the time and concentration curves of Al(mal)<sup>3</sup> toxicity on the SH-SY 5Y cells. The concentration course studies were carried out at 24 h, 48 h, and 72 h after starting treatment with Al(mal)3. We observed that cell death was dose and time-dependent. The GI<sup>50</sup> concentrations for 24 h, 48 h, and 72 h were 482.60, 85.20, and 53.78 µM, respectively (Figure 2).

Given these results, we chose 72 h and 125-µM Al(mal)<sup>3</sup> to perform all in vitro studies involving the stressor. For this, we pretreated the SH-SY 5Y cells with the extracts for 1 h and then exposed them to Al(mal)3. After 72 h of incubation, the cell number was assessed.

In the presence of Al(mal)3, Cardiose®, citicoline, and MecobalActive® did not significantly improve cell survival (Figure 1A′ ,C′ ,D′ ). On the other hand, Olews® and red and white grape extracts presented a slight recovery of cell proliferation at the highest doses, with GI<sup>50</sup> values of 47, 930, and 1598 µg/mL, respectively (Figure 1B′ ,E′ ,F′ ).

– ′– ′ 's multiple comparison ∗ ∗∗ ∗∗∗ **Figure 1.** Neuroprotective effects of the extracts on the SH-SY 5Y cell line. Dose-response curve effects of the extracts on the SH-SY 5Y cell line. Cells were incubated with different concentrations of Cardiose ® (**A**), Olews ® (**B**), citicoline (**C**), MecobalActive ® (**D**), red grape (**E**), or white grape extracts (**F**) for 72 h in the absence (**A**–**F**) or presence (**A**′ –**F** ′ ) of 125-µM Al(mal)<sup>3</sup> . Data are normalized and expressed as a percentage of the over-basal response (mean ± SEM). Significant differences were analyzed on data from eight different experiments; one-way ANOVA and Dunnett's multiple comparison post-hoc test were used for statistical analysis. \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001 versus cells or Al(mal)<sup>3</sup> treatment. Cs: control cells, not exposed to Al(mal)<sup>3</sup> .

's multiple comparison post **Figure 2.** Dose-response curves of the stressor on the SH-SY 5Y cell line. Cells were incubated with different concentrations of Al(mal)<sup>3</sup> for 24, 48, or 72 h. Data are normalized and expressed as a percentage of the over-basal response (mean ± SEM). Significant differences were analyzed on data from eight different experiments; one-way ANOVA and Dunnett's multiple comparison post-hoc test were used for statistical analysis; 24 h, *p* < 0.00001; 48 h, *p* < 0.0001; and 72 h, *p* < 0.0001.

′ ′ ′

′ ′ ′

–

#### *3.2. Olews ®, MecobalActive ®, and Red and White Grape Extracts Have Neuroprotective Effects on Neuroblasts In Vitro*

The cytotoxic activity shown for some of the extracts on the tumor cell line led us to ask whether this was specifically an antitumor effect or was due to a broader toxicity. To answer this question, we repeated the experiments using primary cultures of mouse hippocampal neuroblasts.

As with the tumor cells, we first tested the activity of the food supplements on hippocampal neuron cultures. As with the SH-SY 5Y cells, we observed a potent and dose-independent toxicity when we added Cardiose ® and citicoline to the cell cultures (Figure 3A,C). However, the toxicity was dose-dependent after adding Olews ®, Mecobal-Active ®, and red grape and white grape seed extracts, with EC<sup>50</sup> values of 16.8, 28.5, 18.2, and 259 µg/mL, respectively (Figure 3B,D–F).

– ′– ′ 's multiple ∗ ∗∗ ∗∗∗ **Figure 3.** Neuroprotective effects of the extracts on hippocampal neuron cultures. Dose-response curves of the extracts on hippocampal neuron cultures. Cells were incubated with different concentrations of Cardiose ® (**A**), Olews ® (**B**), citicoline (**C**), MecobalActive ® (**D**), red grape (**E**), or white grape extracts (**F**) for 72 h in the absence (**A**–**F**) or presence (**A**′ –**F** ′ ) of 125-µM Al(mal)<sup>3</sup> . Data are normalized and expressed as a percentage of the over-basal response (mean ± SEM). Significant differences were analyzed on data from eight different experiments; one-way ANOVA and Dunnett's multiple comparison post-hoc test were used for statistical analysis. \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001 versus cells or Al(mal)<sup>3</sup> treatment. Cs: control cells, not exposed to Al(mal)3.

Next, to study the neuroprotective effects of the natural extracts, we pretreated hippocampal cells with the extracts, and then, we exposed them to Al(mal)3. Seventy-two h later, the cell numbers were assessed for all experimental conditions. Olews ® and red and white grape extracts presented a slight but significant recovery of the number of cells with the highest doses, with GI<sup>50</sup> values of 85, 400, and 800 µg/mL, respectively

′ ′ ′

(Figure 3B ′ ,E ′ ,F ′ ). In the case of Cardiose ®, citicoline, and MecobalActive ®, there was higher protection by the lower concentrations (7.8 to 15.6 µg/mL) (Figure 3A′ ,C ′ ,D′ ). Taken together, these results suggest that Cardiose ®, Olews ®, citicoline, MecobalActive ®, and red and white grape extracts may have certain neuroprotective roles on neuroblasts in vitro.

#### *3.3. MecobalActive ®, Olews ®, and Red and White Grape Extracts Treatment Reduces ROS Levels and Caspase-3 Activity*

Previous studies found that Al(mal)<sup>3</sup> induces neurotoxicity in SH-SY 5Y cells by disrupting the levels of ROS and by inducing apoptosis [35]. To find out the mechanisms mediating the neuroprotection role in vitro of Olews ®, MecobalActive ®, and red and white grape extracts, we studied both mechanisms in depth. For each extract, we selected a concentration closer to its GI50.

The ROS measurements indicated that there were no increases in ROS activity elicited by the supplements (Figure 4A). On the other hand, Al(mal)<sup>3</sup> produced a four-fold increase in ROS activity, as expected (Figure 4A). The ROS levels decreased very significantly when any of the supplements were added in combination with Al(mal)<sup>3</sup> (Figure 4A).

∗∗ **Figure 4.** Reactive oxygen species (ROS) levels (**A**) and caspase-3 activity (**B**) on SH-SY 5Y cells after extract treatment. Cells were treated with red grape, white grape, MecobalActive ®, or Olews ® for 48 h in the absence or presence of 125-µM Al(mal)<sup>3</sup> . ROS activity (**A**) was quantified by measuring the fluorescence at 535/590 nm. Caspase-3 activity (B) was quantified by measuring the absorbance at 405 nm. Values are presented as mean ± SEM from at least three independent experiments; Kruskal-Wallis test followed by Mann Whitney post-hoc test were used for statistical analysis. ### *p* < 0.001 versus untreated cells and \*\* *p* < 0.01 versus Al(mal)3. Abbreviations: Mal-Al: Al(mal)3.

> In a similar way, the supplements had no effect on the caspase-3 levels of the treated cells, but they greatly and significantly reduced the Al(mal)3-induced caspase-3 levels (Figure 4B). No differences were found in the nitrite or nitrate levels (data not shown), indicating that Al(mal)<sup>3</sup> does not influence the RNS.

#### *3.4. Immobilization for Six h Causes Oxidative Stress in Mouse Brains*

Based on our previous findings, we hypothesized that the oral administration of these natural supplements could prevent the appearance of oxidative stress in the brain. Before starting the formal experiments, we investigated which was the shortest period of immobilization needed to cause detectable stress in the mouse brain. For this, the animals were immobilized for zero (control), two, four, or six h, and the mRNA expression of the inflammatory markers IL-6 and TNF-alpha, as well as the oxidation marker NOX-2, were determined in the brain tissue.

We observed a statistically significant increase in the expression of IL-6 (1.7-fold) (Figure 5A), NOX-2 (two-fold) (Figure 5B), and TNF-alpha (2.2-fold) (Figure 5C) only after six h of immobilization. Shorter immobilization times did not result in the significant

#### modification of these markers (Figure 5). For this reason, we chose six h as the optimal immobilization time for further experiments.

∗ **Figure 5.** Immobilization causes oxidative stress in mouse brains. Mice were immobilized for different times: 0 (CN), 2, 4, or 6 h. The mRNA expression of IL-6 (**A**), NOX-2 (**B**), and TNF-alpha (**C**) were quantified by real-time (RT)-PCR. The mRNA expression was normalized with GAPDH. All data were related to that from the control and are expressed as a fold change. Values are presented as mean ± SEM from at least three independent experiments. Kruskal-Wallis test followed by Mann Whitney post-hoc test were used for statistical analysis. \* *p* < 0.05 versus CN. Abbreviations: CN: control.

#### *3.5. Oral Administration of Natural Extracts Provides Protection against Oxidative Stress*

– Four natural extracts were selected based on their in vitro behavior and inoculated: red grape, white grape, MecobalActive ®, and Olews ®, each of them at two different concentrations (Table 1). In agreement with our previous results (Figure 5), immobilization stress significantly increased the expression of IL-6 and TNF-alpha when compared to the control (2.5-fold and two-fold respectively) (Figure 6A,B). The administration of the extracts resulted in a statistically significant diminution of the expression of both genes in all used conditions (Figure 6A,B). For some of the extracts, specifically red grape, MecobalActive ® , and Olews ®, we found values very close to those obtained in the control animals. In addition, we also studied the expressions of NOX-2 and HMOX-1. These genes are involved in oxidation mechanisms, and they increase in the brain of mice subjected to stress [4]. The administration of natural extracts significantly decreased the immobilization-increased expression of both NOX-2 and HMOX-1 (Figure 6C,D). In the same way that occurred with inflammatory cytokines, the extracts brought the expression of both genes to levels very similar to those found in the animals without stress. Finally, we also analyzed Nrf-2 expression. This molecule is a transcription factor that regulates the expression of numerous antioxidant genes. Numerous authors have described Nrf-2 expression as a protective mechanism for oxidative stress [36–38]. As expected, immobilization stress reduced Nrf-2 expression (Figure 6E), and all extracts restored Nrf-2 expression to control or even to higher levels, indicating a potent antioxidant effect (Figure 6E).

#### *3.6. Preventive Treatment with Natural Extracts Increases Antioxidant Enzyme Activity in the Brain*

To verify the possible protective role of these extracts in oxidative stress, we studied the activity of two antioxidant enzymes, catalase and superoxide dismutase (SOD), in the mouse brains.

It has been described that stress causes a decrease in catalase activity in the mouse brain [4]. First, we confirmed that our experimental model of acute stress was able to reproduce these results. Indeed, we observed a significant reduction in catalase activity in stressed mice compared to nonstressed animals (Figure 7A). Furthermore, the administration of natural extracts led to a statistically significant increase in the levels of catalase activity after the addiction of the red grape extract, MecobalActive ®, and Olews ®. No differences were seen after the treatment with white grape extracts (Figure 7A). SOD is

one of the most important antioxidant enzymes in cells. It catalyzes the dismutation of the superoxide anion into hydrogen peroxide and molecular oxygen [39]. As with catalase activity, stress caused a significant decrease in SOD activity in the mouse brains (Figure 7B). Interestingly, the administration of natural extracts: red grape, white grape, MecobalActive ®, and Olews ® significantly increased the activity of the SOD enzyme in all used conditions (Figure 7B).

t's multiple comparison post ∗ ∗∗ **Figure 6.** Natural extracts protect against oxidative stress. Red and white grape seed extracts, MecobalActive ®, and Olews ® were administered orally during 5 consecutive days. Then, mice were immobilized for 6 h. The mRNA expressions of IL-6 (**A**), TNF-alpha (**B**), NOX-2 (**C**), HMOX1 (**D**), and Nrf2 (**E**) were quantified in mouse brains by RT-PCR. Gene expression was normalized with GAPDH. All data were normalized to levels found in nonstressed mice (normal) and are expressed as a fold change. Values are presented as mean ± SEM from eight experimental animals. One-way ANOVA and Dunnett's multiple comparison post-hoc test were used for statistical analysis. \* *p* < 0.05 and \*\* *p* < 0.01 versus normal mice, and # *p* < 0.05 and ## *p* < 0.01 versus restrained mice (stress).

t's multiple comparison post ∗ **Figure 7.** Natural extracts increase the activity of antioxidant enzymes. Mouse brains were isolated, and the catalase activity (**A**), SOD activity (**B**), and TBARS (**C**) were analyzed. The values are presented as mean ± SEM from eight experimental animals. One-way ANOVA and Dunnett's multiple comparison post-hoc test were used for statistical analysis. \* *p* < 0.05 versus normal mice, and # *p* < 0.05 and ## *p* < 0.01 versus restrained (stress) mice. Abbreviations: SOD: superoxide dismutase; TBARS: thiobarbituric acid reactive substances.

#### *3.7. Treatment with Natural Extracts Prevents the Formation of Lipid Peroxidation Products in the Brain*

Lipid peroxidation, an oxidative degradation of cellular lipids, is another important parameter to take into account when studying oxidative stress [40]. We measured the MDA levels present in the mouse brain. Acute stress more than doubled the MDA levels when compared with the nonstressed control group (Figure 7C). In addition, a treatment with any of the extracts drastically reduced MDA levels in the brain tissue, which reached levels very similar to those found in the animals without stress (Figure 7C).

#### **4. Discussion**

NDs pose a major health problem for developed countries, and this situation will progressively worsen due to a rapidly ageing population. Stress is known as the "21st century disease" and has been identified as one of the main risk factors in the development of NDs [41]. In this context, the use of natural bioactive extracts has been postulated as a possible preventive treatment of NDs due to their antioxidant power, which is able to reduce stress efficiently [42].

In this work, we found that natural bioactive supplements such as MecobalActive®, Olews®, and red and white grape seed extracts may have neuroprotective effects in vitro, both in the SH-SY 5Y cell line and in hippocampal neuron cultures, mainly by reducing ROS levels and decreasing caspase-3 activity. In vivo, we demonstrated that oral administration of the supplements for just five days reduces the expression of genes involved in inflammation and oxidation mechanisms, whereas it increments the expression of genes related to protection against oxidative stress. Furthermore, we found that a preventive treatment with these natural extracts increases the activity of antioxidant enzymes and prevents lipid peroxidation in the brains of stressed mice.

We found that Olews®, MecobalActive®, and red and white grape seed extracts show a dose-dependent toxicity in SH-SY 5Y cells. Similar results have been described in previous studies. For instance, grape seed extracts were toxic for cell line PC12 at concentrations higher than 200 µg/mL [12]. Similar extracts exhibited a dose-dependent toxicity for oral cancer cell line Ca9-22, which was very significant at doses higher than 100 µg/mL [43]. All these results have been obtained on tumor cell lines, and some authors have proposed that natural antioxidant extracts have an antitumoral capacity [44]. This is why we decided to test the extracts in a primary culture of mouse neurons. To the best of our knowledge, this is the first time that antioxidant extracts were tested in primary cultures, and we were surprised to find that this cellular toxicity also affected the nontransformed cells. Furthermore, with some extracts, the doses needed to elicit a significant antistress response were higher than the GI<sup>50</sup> value, suggesting that the same treatment was simultaneously cytotoxic and antioxidant. This can be explained if we realize that these extracts are not constituted by a pure substance, but they are a mixture of several chemicals. It is easy to envision a situation in which one or several of the components are cytotoxic, whereas others are antioxidant and, thus, cytoprotective in the presence of a stressor.

This cytotoxic behavior of the extracts seems to be at odds with the approval of these substances for human consumption and their ample use with no reported side effects. We need to understand that these extracts are approved for oral use (and not as injectable drugs), and therefore, we need to take into consideration the digestive and absorption processes. Digestion could destroy and/or modify some of the extracts´ components, whereas absorption would take only specific substances in such a way that the potentially cytotoxic molecules never reach normal neurons. The vast majority of antioxidant substances need to be fermented by the microbiota of either the small intestine or the colon to achieve optimal absorption [45]. Specifically, Cardiose® contains a flavonoid, diglycoside, that cannot be absorbed in the small intestine. It must proceed to the colon, where it is fermented prior to absorption [46]. Oleuropein, the main component of Olews®, is poorly absorbed in vitro [47], although it is fermented by intestinal bacteria, which facilitates intestinal absorption [48]. MecobalActive® needs a carrier protein that serves as a mediator for its intestinal absorption [49]. In the case of grape seed extracts, they need to be digested before reaching circulation [50]. Furthermore, simulated digestion experiments suggest that grape seed extracts are stable in acid-based environments, such as the stomach, but are processed under a simulation of duodenal conditions [51]. Therefore, we have to be cautious when interpreting in vitro results, paying more attention to in vivo studies, which should be more informative about the antioxidant neuroprotector effects of tested supplements.

Oxidative stress is recognized as a very significant contributor to the pathogenesis of many devastating NDs [52]. In particular, mitochondrial dysfunction leads to the aberrant production of ROS, which are capable of oxidizing lipids and proteins, ultimately causing cell death [53]. We used Al(mal)<sup>3</sup> to induce neurotoxicity, because it is able to induce mitochondrial membrane potential changes, elevate the ROS, and promote apoptosis in neuron cells [54]. Here, we found that Olews®, MecobalActive®, and red and white grape extracts reduce Al(mal)3–induced ROS in SH-SY 5Y cells. In addition, Al(mal)<sup>3</sup> causes caspase-3 activation, thus inducing apoptosis and, subsequently, cell death [54]. We also demonstrated that Olews®, MecobalActive®, and red and white grape extracts were able to reduce Al(mal)3-induced caspase-3 activity. In summary, our results suggest that these natural extracts may play certain antioxidant neuroprotective roles in vitro.

Excessive stress can provoke oxidative stress damage, and the brain tissue has been described as more susceptible to oxidation than other organs [55]. The use of stress models is supported by substantial evidence implicating stress as a precipitating factor for several neuropsychiatric disorders [56]. Most authors in the field use six h of immobilization for their stress-inducing experiments [4,57], but no information of what happens at shorter times is available. We ran a time course and measured the levels of inflammatory cytokines and NOX-2 in brain tissue after two, four, and six h of immobilization. The differences were statistically significant only after the longest exposure (six h), indicating that shorter times do not generate measurable changes in gene expressions in the mouse brain.

Acute restraint stress stimulates several cellular events, resulting in enhanced ROS production [58]. While intracellular ROS serve mainly for host defense against infectious agents, redox-sensitive signal transduction, and other cellular processes, the extracellular release of ROS damages surrounding tissues and triggers inflammatory processes [59] that finally enhance the lipopolysaccharide (LPS)-mediated production of proinflammatory cytokines IL-1β, IL-6, and TNF-α [60,61]. NOX2 is well-known for generating superoxide molecules under oxidative stress-mediated circumstances. Furthermore, HMOX1 acts as a heat shock protein and is induced by oxidative stress [62]. HMOX1 and NOX-2 expressions are upregulated in the stressed brain [63] and in experimental models of NDs [64]. On the contrary, nuclear factor Nrf-2 induces the expression of antioxidant genes that eventually provoke an anti-inflammatory expression profile that is crucial for the initiation of healing [65]. In accordance with this general pathway, we described that the administration of all extracts used in the study (red grape, white grape, MecobalActive®, and Olews®) prevents the expression of genes involved in inflammation and oxidation mechanisms, while increasing the expression of genes related to protection against oxidative stress, thus identifying them as efficient inhibitors of stress-related cellular damage.

Similarly, restraint stress in rodents precipitates many neurochemical, hormonal, and behavioral abnormalities that are often associated with an imbalance in the brain's intracellular redox state. Numerous studies have reported that restraint stress enhances lipid peroxidation and decreases antioxidant enzyme activities in rodents [58,66]. To prevent oxidative stress damage, most organisms are equipped with antioxidant mechanisms. SOD and catalase are the best-known antioxidant enzymes [4]. We found that a pretreatment with the extracts increased the activity of catalase and SOD when compared to stressed mice. On the other hand, lipid peroxidation is the oxidative degradation of lipids [67]. MDA is one of the final products of polyunsaturated fatty acid peroxidation in cells. An increase in free radicals causes the overproduction of MDA, which is commonly used as a marker of oxidative stress [68]. In agreement with this, we found that MDA levels significantly increased in the brains of stressed animals but were very efficiently normalized by oral administration of the supplements.

#### **5. Conclusions**

Taken together, our results suggest that some natural bioactive supplements (specifically, Olews®, MecobalActive®, and red and white grape seed extracts) may have important protective properties against oxidative stress processes occurring in the brain. Since oxidative stress has a critical role in the development of NDs, we propose the addition of these natural supplements to commonly used food staples as a possible global preventive treatment for NDs.

**Author Contributions:** Conceptualization, M.B. and. A.M.; methodology, M.B., J.G.-S.; validation, M.B., J.G.-S., and A.M.; formal analysis, M.B., J.G.-S., and A.M.; investigation, M.B., J.G.-S., and A.M.; writing—original draft preparation, M.B.; writing—review and editing, A.M.; supervision, A.M.; and funding acquisition, A.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Spanish Ministry of Science, Innovation, and Universities (RTC-2017-6424-1) and co-funded by the European Regional Development Fund (FEDER).

**Institutional Review Board Statement:** All procedures involving animals were carried out in accordance with the European Communities Council Directive (2010/63/EU) and Spanish legislation (RD53/2013) on animal experiments and with approval from the ethical committee on animal welfare of our institution (Órgano Encargado del Bienestar Animal del Centro de Investigación Biomédica de La Rioja, OEBA-CIBIR, procedure number AMR14, date of approval: 24 February 2020).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to their large volume and little interest.

**Acknowledgments:** We gratefully acknowledge Alvinesa Natural Ingredients and HealthTech Bio Actives for generously providing the food supplements free of charge.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

#### **References**


## *Review* **Olive Polyphenols: Antioxidant and Anti-Inflammatory Properties**

**Monica Bucciantini 1,\*, Manuela Leri <sup>1</sup> , Pamela Nardiello <sup>2</sup> , Fiorella Casamenti <sup>2</sup> and Massimo Stefani <sup>1</sup>**


**Abstract:** Oxidative stress and inflammation triggered by increased oxidative stress are the cause of many chronic diseases. The lack of anti-inflammatory drugs without side-effects has stimulated the search for new active substances. Plant-derived compounds provide new potential anti-inflammatory and antioxidant molecules. Natural products are structurally optimized by evolution to serve particular biological functions, including the regulation of endogenous defense mechanisms and interaction with other organisms. This property explains their relevance for infectious diseases and cancer. Recently, among the various natural substances, polyphenols from extra virgin olive oil (EVOO), an important element of the Mediterranean diet, have aroused growing interest. Extensive studies have shown the potent therapeutic effects of these bioactive molecules against a series of chronic diseases, such as cardiovascular diseases, diabetes, neurodegenerative disorders and cancer. This review begins from the chemical structure, abundance and bioavailability of the main EVOO polyphenols to highlight the effects and the possible molecular mechanism(s) of action of these compounds against inflammation and oxidation, in vitro and in vivo. In addition, the mechanisms of inhibition of molecular signaling pathways activated by oxidative stress by EVOO polyphenols are discussed, together with their possible roles in inflammation-mediated chronic disorders, also taking into account meta-analysis of population studies and clinical trials.

**Keywords:** EVOO polyphenols; oxidative stress; inflammation

#### **1. Introduction**

The increasing extension in life expectancy of humans in advanced countries matches a higher prevalence of a number of lifestyle- and age-associated pathological conditions such as cancer, systemic and neurodegenerative diseases, amyloid diseases, particularly Alzheimer's disease (AD) and Parkinson's (PD) disease, cardiovascular diseases (CVDs) and metabolic diseases including metabolic syndrome (MetS); the latter includes, in addition to type 2 diabetes mellitus (T2DM), CVDs and non-alcoholic hepatitis. These pathologies are characterized by several common features, including, among others, derangement of proteostasis and the redox equilibrium and a remarkable inflammatory response that heavily impair the biochemical and functional features of the affected tissues. Moreover, at present, these pathologies, particularly amyloid diseases, lack effective therapies; it is then evident that, in the light of the latter aspect, prevention appears as the best tool to reduce the risk of these pathological conditions. Accordingly, medical research has progressively focused on the importance of lifestyle. Physical exercise, mental activity and diet, intended as the complex of foods and nutrients taken daily by a person, are three pillars of a healthy lifestyle.

The Mediterranean diet (MD) has been the subject of a huge amount of studies on its properties to prevent different chronic-degenerative diseases from the first evidence from the early 1960s suggesting an association between the alimentation of Mediterranean people and their low cardiovascular mortality [1]. An increasing number of epidemiological

**Citation:** Bucciantini, M.; Leri, M.; Nardiello, P.; Casamenti, F.; Stefani, M. Olive Polyphenols: Antioxidant and Anti-Inflammatory Properties. *Antioxidants* **2021**, *10*, 1044. https:// doi.org/10.3390/antiox10071044

Academic Editors: Rui F. M. Silva and Lea Pogaˇcnik

Received: 29 May 2021 Accepted: 24 June 2021 Published: 29 June 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/).

and observational studies confirm that the Mediterranean diet (MD) is associated with aging well, a condition where the prevalence of diseases including MetS, CVDs, cancer and cognitive decline appears significantly reduced [2]. The MD can be considered as the heritage of a complex socio-economic development of the Mediterranean populations over past centuries, and includes practices resulting from agricultural, social, territorial and environmental factors intimately associated with the culture and lifestyle of these populations. Recently, modifications of the classical MD have been proposed by the Mediterranean Diet Foundation Expert Group [3]. The new MD pyramid, in addition to the presence of a specific content of characteristic foods (low meat/fish, high fruit, vegetables and carbohydrates, presence of red wine, use of olive oil as main lipid source, moderate caloric intake), also emphasizes the importance of other lifestyle-associated elements, such as moderation, seasonality, adequate rest, conviviality, and physical exercise. The new pyramid also reflects the changes that the MD is undergoing, at the present, within the Mediterranean societies in relation to various geographical, cultural and socio-economic contexts. The high value of the MD and its associated lifestyle was recognized in 2010 by UNESCO, who inscribed the MD in the list of the Intangible Cultural Heritage of Humanity (https://ich.unesco.org/en/RL/mediterranean-diet-00884, accessed on 2013).

An important feature of the MD is the daily consumption of a vast array of phytonutrients including vitamins and plant phenols, which provides its similarities with the Asian diet. In particular, plant polyphenols interfere with multiple signaling pathways involved in protein homeostasis, in the inflammatory response, and in the regulation of both metabolism and the antioxidant defenses [4–6], often recalling a caloric restriction (CR) regimen positively affecting, among others, whole body metabolism, mitochondrial turnover, oxidative stress and the inflammatory and neuroinflammatory response, where autophagy plays an important role [7,8]. Polyphenols can reach these effects by counteracting, at the molecular level, signaling pathways responsible for the cascade reactions involved in aging [9,10]. Overall, present data support the idea that different plant polyphenols, including those from the olive tree, are able to mimic CR effects and to modulate the expression of pro- and anti-apoptotic factors, also through epigenetic modifications [11], thus affecting the same, or very similar, cellular targets. Accordingly, plant polyphenols can be proposed as a useful tool for the prevention and/or treatment of aging-associated diseases connected with chronic inflammation or transcriptional, redox or metabolic derangement [12].

An increasing number of preclinical studies, population studies and clinical trials suggest that adherence to the MD, with particular emphasis on its content of plant polyphenols, often referred to as biophenols, reduces metabolic pathologies and aging-associated deterioration, where derangement of redox homeostasis and an excessive inflammatory response often play pivotal roles. Biophenols are found in many foods of plant origin that play pivotal roles in the MD, including red wine, extra virgin olive oil (EVOO), green tea, spices, berries and aromatic herbs. The content of polyphenols in these foods and their bioavailability are quite low; however, the daily consumption, throughout one's lifetime, of these foods ensures a reduced, yet continuous, intake of polyphenols, providing a rationale for the association between the dietary content of the latter and a significant reduction in the incidence of aging-associated pathologies reported by many population/epidemiological studies and clinical trials [13,14].

A wealth of recent studies has highlighted the fact that, in several aging-associated pathologies such as amyloid diseases, CVDs and MetS, plant polyphenols do not simply interfere with a single step of disease pathogenesis (protein/peptide aggregation, the inflammatory response, the redox/metabolic equilibrium, the proteostasis balance); rather, their positive biological and functional outcomes result from multi-target effects leading to the restoration of altered homeostatic systems in cells and tissues. In addition, the chemical similarities of these structurally distinct molecules can explain why they can induce similar effects. Among others, the importance of natural polyphenols for health has been associated with their remarkable antioxidant power elicited through the modulation of oxidative pathways. The latter can result from interference with enzymes, proteins, receptors, transcription factors and several signaling pathways [4,15]. The ability of plant polyphenols to interfere with biochemical homeostasis has also been taken into consideration [14], and epigenetic modifications of chromatin have been reported to also be involved in these effects [16,17]. Actually, recent research is providing increasing information on the biochemical, cellular and epigenetic modifications induced by several plant polyphenols and the resulting modulation of the homeostasis of key cellular processes such as metabolism, energy balance, redox equilibrium, proteostasis, cell signaling, the inflammatory response, and the control of oxidative stress and of gene expression. The knowledge stemming from these data will allow us to better understand the beneficial effects, for human wellness, of the MD, the importance of its content in plant polyphenols and the role of the latter in disease prevention and, possibly, therapy.

The rising interest in natural polyphenols has resulted in a large number of studies on their medicinal efficacy, carried out not only in cultured cells but also in model organisms and in humans. More recently, an increasing number of studies have also appeared on the biochemical and biological effects of olive polyphenols. The polyphenols elaborated by the olive tree (*Olea europaea*) are present prevalently in the leaves and drupes of the tree and are important as phytoalexins, molecules that the plant elaborates for defense against invasions by microbes and fungi and to discourage leaf-eating insects. EVOO contains over 30 phenolic compounds, including the most represented oleuropein, both in the glycated and in the aglycone (OLE) form, verbascoside, oleocanthal, hydroxytyrosol (HT), tyrosol, and others (see next section). The healthy value of EVOO and olive leaf extracts has been recognized for a long time and scientifically investigated in the last couple of centuries. More recent studies have focused on the biological properties of these molecules, including the antimicrobial, hypoglycemic, vasodilator, antihypertensive, antioxidant and anti-inflammatory ones, whose clinical importance was first reported in 1950 [18]. These properties have led to the inclusion of the alcoholic extract (80%) of olive leaves containing, in addition to minor components, OLE, HT, caffeic acid, tyrosol, apigenin and verbascoside in the European Pharmacopoeia (Ph. Eur.) [19,20].

The molecular determinants of the protection by olive polyphenols against several aging-associated and chronic degenerative conditions, including T2DM [21–23] and nonalcoholic fatty liver disease [24–28], have been extensively investigated in the last 20 years. OLE, HT and other olive polyphenols protect cells against oxidative damage resulting from redox dyshomeostasis [29,30] and an excessive inflammatory response [31], among the main determinants of age-related pathologies such as cancer, T2DM, MetS, osteoporosis and neurological diseases [27,32]. Most of these effects have been associated with the ability of polyphenols to control cell signaling and pathways, to modulate the activity of transcription factors, and to affect gene expression; these nutrigenomic properties of EVOO polyphenols have been recently reviewed [33,34]. Finally, population studies have provided evidence of a significant association between MD, EVOO consumption, and reduced risk of both CVD [35] and cognitive decline [2]. A recent review of the scientific literature focused on clinical trials and population studies has confirmed that the MD and the fortification of the foods with olive leaf extracts protect significantly against several aging-associated degenerative diseases and cancer [36–38]. Accordingly, plant polyphenols are increasingly taken into consideration, as such or their molecular scaffolds, as the starting component to develop new drugs especially designed to combat several chronic degenerative pathologies, including aging-associated neurodegeneration [36,39].

Here, the results of studies on the polyphenols produced by the olive tree and found in EVOO will be reviewed, with a special focus on the antioxidant and anti-inflammatory properties of these molecules. The effects of olive polyphenols in cell and animal models of aging-associated pathologies, including CVDs, MetS and neurodegenerative diseases, the molecular mechanisms underlying these properties, the currently available population studies and clinical trials, and the most recent advances in their possible use to combat neurodegenerative diseases will also be treated.

#### **2. Olive Polyphenols: A Group of Molecules with Shared Chemical and Biological Properties: Structure, Abundance and Bioavailability**

Biophenols are a family of over 8000 polyphenolic structures (those presently described) found in almost all plant families mainly as secondary metabolites, including several hundred isolated from edible plants [36,40]. These molecules include non-flavonoids or flavonoids; the latter are further classified as flavonols, flavononols, flavones, anthocyanins, procyanidins, phenolic acids, stilbenes and tannins on the basis of the number of hydroxyls in the molecule and the type and the position of other substituents [41]. The plant sources of plant polyphenols are, among others, bark, leaves, fruits, spices, berries, vegetables, roots, nuts and seeds, herbs, and whole grain products, from which they are transferred in processed foods of plant origin, including EVOO, red wine, green tea, coffee and turmeric. These compounds are characterized by a broad spectrum of biological activities and exert positive effects in a large number of human diseases, including cancer, CVDs, T2DM and neurodegenerative conditions, with molecular mechanisms often related to their antioxidant activity. In the case of EVOO, its healthy properties have been associated with its peculiar chemical composition [42]. EVOO contains both major components (triglycerides and other fatty acid derivatives where mainly monounsaturated fatty acids, in particular oleic acid, are present) and minor components (over 230 different chemicals including aliphatic and triterpenic alcohols, phytosterols, hydrocarbons, tocopherols, and polyphenols) [43]. In the past, the health effects of EVOO were attributed mainly to the presence of oleic acid; however, more recently, attention has been focused on phenolics, a class of bioactive compounds including phenolic acids, phenolic alcohols, flavonoids, secoiridoids and lignans [44].

In particular, olive tree polyphenols include flavonols, lignans and glycosides. Olive glycosides are iridoids, geraniol-derived monoterpenes, whose chemical structure results from a cyclopentane ring fused to a six-member heterocycle with an oxygen atom. In particular, the bicyclic H-5/H-9β, β-*cis*-fused cyclopentanepyran ring system is the most common structural feature and the basic skeletal ring of iridoids. Cleavage of the cyclopentane ring of iridoids produces seco-iridoids, while cleavage of the pyran ring produces iridoid derivatives [45]. Iridoids and secoiridoids, mainly in the glycated form, are found in many medicinal plants belonging to the subclass Asteridae that includes several plant families, particularly Oleaceae.

The polyphenols produced by the olive tree are found in the lipid fraction and in the water fraction (dispersed as minute droplets) of olive oil mainly in the glucose-free form (aglycones), resulting from deglycosylation by plant glycosidases during olive squeezing. The most abundant secoiridoid in olive oil is 3,4-dihydroxyphenylethanol-elenolic acid (3,4-DHPEA-EA), whose glucose-bound form is commonly known as oleuropein; the latter is the main cause of the bitter taste of olive leaves and drupes. Other secoiridoids include oleuropein derivatives, both in the glucose-bound form or as aglycones, such as the dialdehydic form of decarboxymethyl elenolic acid bound to either HT (3,4-dihydroxyphenylethanol-elenolic acid dialdehyde, 3,4-DHPEA-EDA, also known as oleacein) or to tyrosol (p-hydroxyphenylethanol-elenolic acid dialdehyde, p-HPEA-EDA, also known as oleocanthal, or ligstroside aglycone [46,47] (Figure 1). Oleocanthal produces the burning sensation in the back of the throat that accompanies the consumption of freshly squeezed EVOO. Olive oil also has a rich composition in simple phenols; these include tyrosol (p-hydroxyphenylethanol, p-HPEA) and hydroxytyrosol (3,4-dihydroxyphenylethanol, 3,4-DHPEA, DOPET), two phenolic alcohols mostly derived from their secoiridoid precursors. Olive polyphenols also include verbascoside, the caffeoylrhamnosylglucoside of HT, 1-acetoxypinoresinol and pinoresinol (two lignans).

**Figure 1.** Chemical structure of oleuropein (**a**), oleuropein aglycone (**b**), hydroxytyrosol (**c**), oleacein (**d**), oleocanthal (**e**).

Olive polyphenols are considered to be responsible for some of the recognized pharmacological properties of the olive tree (anti-atherogenic, antihepatotoxic, hypoglycemic, anti-inflammatory, antitumoral, antiviral, analgesic, purgative and immunomodulatory activities) [28,48,49], together with the protection against aging-associated neurodegeneration [29]. For these reasons, the EVOO quality depends not only on the content in free fatty acids resulting from triacylglycerol breakdown (acidity), but also on its content in polyphenols, the molecules responsible for its taste and for many of its healthy properties. Several factors affect the content of polyphenols in olive oil; these include olive cultivar, environmental cues (altitude, meteorological factors and irrigation), cultivation practices, and ripening stage of the fruits [50], together with extraction techniques, systems to separate oil from olive pastes. The conditions of storage (temperature, time) are also of importance, affecting the rate of oxidation/photooxidation reactions and the deposition of suspended water particles rich in polyphenols [51]. Under optimal conditions, the content of polyphenols in EVOO can exceed 60 mg/100 g.

The normal daily dietary intake of plant polyphenols is in the 0.1–1.0 g range; however, the bioavailability of these molecules, including the olive ones, in humans is poor due to reduced intestinal absorption and fast biotransformation that favors their urinary excretion. In addition, in the case of the brain, the circulating polyphenols must also cross the blood– brain barrier before reaching the parenchyma. With few exceptions, polyphenol aglycones can be partially absorbed in the small intestine by passive diffusion [52] much better than their glycated counterparts [53], although important amounts proceed to the large intestine to be eliminated [54]. A review of many studies on polyphenol bioavailability reported a 0 to 4.0 µmol/L plasma concentration of total metabolites produced from the oral administration of 50 mg aglycone equivalents of a polyphenol [55]. After intestinal absorption and passage to the lymph, most polyphenols undergo phase I and phase II metabolism, with substantial biotransformation and production of methylated, sulphated, hydroxylated, thiol-conjugated and glucuronide derivatives and degradation products [56]. These modifications alter the chemical properties of plant polyphenols, favor their excretion and, possibly, provide them new biological activities [57]. The importance of the colonic microflora for polyphenol bioavailability, due to its ability to metabolize and chemically modify polyphenols, has been reported recently [55]. Anyway, recent studies indicate that plant, including olive, polyphenols are absorbed in discrete amounts from the intestine

and rapidly distributed through the blood flow to the whole organism, including the brain, both in rats [58,59] and in humans [60,61]. Plant polyphenols do interact with, and cross, synthetic and cell membranes. The interaction of oleuropein aglycone with synthetic phospholipid membranes favored by the presence of anionic lipids has been reported in a very recent study [62]. Another study reported that several polyphenols (the olive ones were not included) protected the mitochondria against membrane permeabilization by amyloid oligomers, suggesting some interference with oligomers' interaction with the membrane [63]. Finally, oleuropein aglycone (OLE) was the main polyphenol found in breast cancer cells treated with an olive leaf extract in a recent metabolite-profiling study, suggesting its ability to cross the plasma membrane of these cells [64].

Due to the rising interest in natural phenols as possible new drugs, strategies to improve their bioavailability are under study, with encapsulation being probably the most actively investigated, in some cases with encouraging results [65,66]. Most of these molecular tools have not been tested in clinical trials, yet this strategy appears promising to improve the efficacy of natural phenols as drugs while reducing the amount of the administered dose. Actually, accurate studies on the effective dose of olive polyphenols to be administered daily to humans to obtain significant protection are still lacking; at any rate, the amount of OLE and other plant polyphenols taken daily in foods appears not adequate to ensure a dose suitable to produce short-term acute effects. However, clinical and experimental evidence indicates that a continuous consumption of moderate amounts of these molecules can be effective in the long term; this can also result in the accumulation in body tissues of these lipophilic molecules, leading to a low-intensity continuous stimulus of cell defenses against amyloid deposition, protein and metabolism dyshomeostasis, oxidative stress and other alterations underlying age-associated pathologies. These effects, although not proven experimentally, could, at least in part, explain the healthy properties of the MD. Nevertheless, the intake of moderate amounts of olive, and other plant, polyphenols provided by a typical MD supports the usefulness of the integration of polyphenol-enriched olive leaf extracts and other polyphenol-enriched nutraceuticals that can intensify, in the short term, the beneficial effects of these molecules.

#### **3. Antioxidant and Anti-Inflammatory Properties of Olive Polyphenols in Animal Models**

It is widely recognized that oxidative and nitrosative stress as well as inflammation are the major abnormalities underlying neurodegeneration and that antioxidant molecules, such as olive oil polyphenols, restore neuronal function through the amelioration of the redox status. Some beneficial effects of the MD have been associated with the consumption of EVOO polyphenols; these include antioxidant, hypoglycemic, antimicrobial, antiviral, antitumor, cardioprotective, neuroprotective, antiaging and anti-inflammatory activities [67,68]. It has been reported that EVOO polyphenols are protective against cognitive impairment associated with aging and neurodegenerative diseases due to their ability to protect DNA against oxidative stress, to inhibit mitochondrial dysfunction and to attenuate lipid peroxidation by scavenging free radicals, thus sustaining endogenous antioxidant stability [69,70]. They are also able to inhibit amyloid β (Aβ) and τ protein aggregation and toxicity, the main causes of the neurodegenerative cascade in AD [39,71,72]. EVOO polyphenols participate in the redox balance of the cell as antioxidants and as mild pro-oxidants, with ensuing upregulation of the antioxidant defenses of the cell. Accordingly, they can be considered as hormetic factors. For instance, in the presence of peroxidases, HT can undergo a redox cycling that generates superoxide [70], and tyrosol also increases *C. elegans* lifespan by activating the heat shock response [71]. It was reported that HT reduces brain mitochondrial oxidative stress and neuroinflammation in AD-prone transgenic mice by the induction of Nrf2-dependent gene expression [72]. The eight-week administration of oleuropein (60 mg/kg/day) improved mitochondrial function and reduced oxidative stress by activating the Nrf2 pathway in SHR rats [73]. Furthermore, tyrosol (240 mg/kg) was found to be protective against LPS-induced acute lung injury *through the* inhibition of NF-κB and the activation of AP-1 and of the Nrf-2 pathway [74]. EVOO polyphenols

also enhance Nrf-2 activation at the hepatic level and the ensuing release of antioxidant enzymes [75]. Nrf2 is considered the principal regulator of redox homeostasis and its activation inhibits pro-inflammatory mediators such as cytokines, COX-2 and iNOS [76]. EVOO polyphenols limit inflammation by reducing the expression/activity of the transcription factors NF-κB and AP-1 [77] thanks to their free radical scavenging and radical chain breaking capacity and to the reduced formation of ROS and RNS. Moreover, HT inhibits the development of the inflammatory cascade following LPS and carrageenan injection through downregulation of the levels of pro-inflammatory cytokines (TNF-α and IL-1β), COX2, iNOS, NO, PGE2 and NF-kB and reducing DNA damage [78–80]. It was reported that the co-injection of OLE (450 µM) with Aβ42 (50 µM) into the nucleus basalis magnocellularis (NBM) of adult rats interfered with Aβ aggregation and significantly counteracted Aβ toxicity against choline acetyltransferase-positive neurons of the NBM and reduced astrocyte and microglia activation [81]. Another study reported that OLE protects transgenic *C. elegans* strains, constitutively expressing Aβ3-42, by reducing Aβ plaque load and motor deficits [82]. Interestingly, significant anti aggregation and neuroprotective effects of a diet supplemented with OLE, HT or a mix of polyphenols from olive mill wastewater were reported in the TgCRND8 mouse model of Aβ deposition. In these transgenic mice, a significant improvement in cognitive functions and a significant reduction in Aβ plaque number, size, and compactness were found in 3- and 6-month-old mice (at the early and intermediate stage of Aβ deposition, respectively) fed for 8 weeks with the OLE-supplemented diet [83–85]. A significant improvement in synaptic function and a significant reduction in the number, size and compactness of both Aβ42 and its 3-42 pyroglutamylated derivative (pE3-Aβ) deposits occurred even when the treatment was started at 10 months, when these mice display increased brain deposits of Aβ and, in particular, of pE3-Aβ in the cortex and hippocampal areas. These data indicate that oral diet supplementation with OLE not only results in the prevention of amyloid deposition but also in the disaggregation of preformed plaques and in a reduction in pE3-Aβ generation [85]. The effect of OLE against Aβ peptide aggregation was dose-dependent and could be reproduced by diet supplementation with a mix of polyphenols from olive mill wastewater or by HT administered at the same dose as that of pure OLE [84,86]. Interestingly, the treatment with OLE (50 mg/kg of diet for 8 weeks) astonishingly activated neuronal autophagy even in TgCRND8 mice at an advanced stage of pathology. In these animals, histone 3 acetylation on lysine 9 (H3K9) and histone 4 acetylation on lysine 5 (H4K5) were increased in the cortex and the hippocampus; such an increase matched both a decrease in HDAC2 expression and a significant improvement in synaptic function [85].

It is known that abnormal acetylation takes place in memory and learning disorders such as AD, where a significant increase in HDAC2 inhibits gene expression at specific loci, such as those involving autophagy markers [87]. In addition to the induction of an intense and functional autophagic response in the cortex, other relevant biological effects of OLE were uncovered in the TgCRND8 model; these include increased microglia migration to the plaques for phagocytosis, enhanced hippocampal neurogenesis and reduced astrocyte reaction [83,88]. OLE induced autophagy through the increase in cytosolic levels of Ca2+ and the subsequent activation of the enzyme complex AMPK by Ca2+/Calmodulin Protein Kinase Kinase β (CaMKKβ) and the ensuing increase in phosphorylation of mammalian target of rapamycin (mTOR) with mTOR inhibition [89]. These data support the idea that autophagy activation by OLE and other olive polyphenols proceeds via modulation of the AMPK–mTOR axis, similarly to data reported for other plant polyphenols [90]. TgCRND8 mice fed with a diet supplemented with OLE or HT (50 mg/kg of food) exhibited increased levels of Beclin-1 and LC3 autophagic markers in the soma and dendrites of neurons of the somatosensory/parietal and entorhinal/piriform cerebral cortex, together with improved autophagosome/lysosome fusion [83,86]. Furthermore, the significant accumulation of PAR polymers and the increase in PARP1 expression found in the cortex at the early (3.5 months) and intermediate (6 months) stage of Aβ deposition in the TgCRND8 mice were rescued to control values by OLE supplementation. OLE-induced reduction in PARP1

activation was paralleled by the overexpression of SIRT1, and by a decrease in the proinflammatory NF-κB and the pro-apoptotic p53 marker [88].

The ability of EVOO polyphenols to modulate the action of NF-kB was observed both in vitro and in vivo in different tissues. In vivo, HT attenuated apoptosis in rat brain cells by modulating the levels of caspase-3 and NF-kB p65 subunit [91]; in high-fat diet (HFD)-fed C57BL/6 J male mice, daily doses of HT (5.0 mg/kg) attenuated the increment of NF-κB and SREBP 1c, and increased the activity of Nrf2 and PPAR-γ in the liver [92]. In female BALB/c mice, an EVOO-supplemented diet was protective in the management of induced systemic lupus erythematosus disease, likely through the inhibition of the MAPK, JAK/STAT, and NF-κB pathways in splenocytes [93]. One of the most studied upstream constituents of the NF-κB signaling pathway is the activation of the mitogenactivated protein kinases (MAPKs) [94]. In the TgCRND8 mice, an HT-supplemented diet modulated MAPK signaling by activating ERK and downregulating SAPK/JNK expression, a mechanism that may underlie memory improvements in these mice [86]. These data agree with other findings suggesting an involvement of ERK stimulation in memory formation and synaptic plasticity. In the C57BL/mouse model of AD, the administration of HT and its acetylated derivative significantly improved spatial memory deficits induced by the intracerebral injection of Aβ42 plus ibotenic acid. The latter affected the Bcl-2/Bad levels, activated caspase/cytochrome-dependent apoptosis, and downregulated pro-survival genes also involved in memory functions (Sirt-1, CREB, and CTREB target genes), whereas HT administration alleviated these alterations [95]. Taken together, these data suggest that OLE and/or its metabolite, HT, can be effective to combat cellular alterations underlying AD symptoms in the absence of undesirable side effects.

Finally, HT was shown to inhibit the toxicity associated with α-synuclein aggregation in PD [96]; HT and OLE improved spatial working memory and energetic metabolism in the brain of aged mice [97]; and HT decreased oxidative stress in the brain of *db/db* mice, a widely used human T2DM animal model, by improving mitochondrial function and inducing phase II antioxidative enzymes through the activation of the Nrf2–ARE pathway [98].

To date, less data have been reported for oleocanthal. Recently, in vitro and in vivo studies reported that oleocanthal enhances β-amyloid clearance as a potential neuroprotective mechanism [99,100].

#### **4. Antioxidant Properties of Olive Polyphenols: Molecular Mechanisms**

The overproduction of ROS correlates with lipid, protein or DNA damage involved in the onset of degenerative diseases; accordingly, cell defenses against a rise in ROS are fundamental [101]. Antioxidants inhibit oxidation; therefore, to react to oxidative stress, organisms maintain complex systems of antioxidants, primarily glutathione (GSH). Unfortunately, only a few drugs and biological molecules, such as vitamins, have been reported to act as antioxidants, yet with possible side effects [102,103].

Nowadays, researchers are focusing their attention on the antioxidant properties of natural compounds, without relevant side effects. In particular, the importance of the antioxidant activity of lipophilic and hydrophilic phenols in EVOO has emerged [104]. This fraction is physiologically produced by plants to react against the injuries produced by various pathogens or insects [28,105]. The antioxidant activity of the major phenolic components of EVOO, OLE and HT is related to their relative bioavailability with an appreciable level of absorption, fundamental to exert their metabolic and pharmacokinetic properties [49]. In molecular terms, OLE and HT, with their catecholic structure, behave as antioxidants in different ways: (i) by scavenging the peroxyl radicals and breaking peroxidative chain reactions, generating very stable resonance structures [106]; and (ii) by acting as metal chelators, therefore, preventing the copper sulphate-induced oxidation of low-density lipoproteins [107]. The activity of OLE and HT as metal chelators could be attributed to the ability of the hydroxyl groups to behave as electron donors and to the ensuing formation of intramolecular hydrogen bonds with free radicals [32]. However,

the scavenging activity of OLE and HT was also assessed in non-metal oxidation systems. Indeed, data obtained in vitro highlight the ability of polyphenols to reduce the inactivation of catalase (CAT) by hypochlorous acid (HOCl); this effect protects against atherosclerosis following LDL oxidation by HOCl through apoB-100 chlorination [108]. Moreover, HT has been reported to improve the redox status of the cell by increasing the levels of GSH [109].

Recently, the oxidative damage in age-related diseases turned out to be primarily caused by reduced levels of the transcriptional Nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) [110], and it was proposed as a therapeutic target for metabolic syndromes, including obesity, due to its behavior as a mediator of general adaptive responses of the cell, including proteostasis and inflammation [111,112]. However, the pivotal role of Nrf2 is involved in the regulation of protection against oxidation [113]. Following Nrf2 activation and consequently its translocation to the nucleus, Nrf2 binds to antioxidant response elements (ARE); after binding, it acts on the transcriptional expression of several antioxidant enzymes, including superoxide dismutase (SOD), c-glutamylcysteine synthetase (c-GCS), glutathione S-transferase (GST) and NADPH quinone oxidoreductase-1 (NQO1) [114].

EVOO polyphenols have been reported to interact with Nrf2 and with Nrf2-controlled enzymes. In vivo studies showed that EVOO polyphenols increased, at the mRNA level, the expression of Nrf2 and of its targets paraoxonase-2 (PON2), c-GCS, NQO1, and GST in the heart tissue of senescence-accelerated mouse-prone 8, whose diet included 10% olive oil [115]. These effects have been ascribed to HT. Indeed, a model of metabolic alterations, the high-fat diet (HFD)-fed male mice C57BL/6J, supplemented with HT (5.0 mg/kg), displayed a reduction in oxidative stress by restoring Nrf2 and the activity of the peroxisome proliferator-activated receptor-α (PPAR-α) to normal levels [116]. The same results were obtained when the same model was supplemented with the highest dose of HT (10–50 mg/kg/day), which also resulted in an increase in GST activity in the liver and in the muscle [117]. Finally, spontaneously hypertensive rats fed with OLE (60 mg/kg/day) showed increased levels of Nrf2-dependent phase II enzymes, such as NQO-1 [77]. Anyway, in spite of these and other data, the molecular mechanisms controlled by EVOO polyphenols in terms of antioxidant activity are not still clear; in fact, the reported effects were probably determined by the tissue localization of the enzyme and by the different concentrations of phenols used. Indeed, differently from previous data, in 60-day-old Wistar male rats fed with 7.5 mg/kg/day HT, oxidative stress was increased in heart tissue, probably due to the high concentration used [118]. The latter finding is not surprising; in fact, OLE and HT exert anti-proliferative and pro-apoptotic effects on tumor cells in vitro, inducing an accumulation of hydrogen peroxide mediated by the high doses [119,120].

The activity of EVOO polyphenols on Nrf2 signaling and on the levels of several antioxidant enzymes, such as γ-glutamyl-cysteinyl-ligase (γ-GCL) and SOD, was also reported in in vitro experiments with LPS-treated macrophages [121] and cancer cells [122]. Furthermore, it is widely reported that OLE and HT act on AMPK signaling, and the latter has been considered as an attractive therapeutic target for antioxidant activity. In fact, AMPK signaling plays a fundamental role in the cell defense system against ROS by direct phosphorylation of human FoxO1 (forkhead box O1) at Thr649, with the ensuing increase in FoxO1-dependent transcription of Mn-superoxide dismutase (MN-SOD) and CAT [123].

In conclusion, the data reported in the present and in the previous paragraph convincingly support the idea that EVOO polyphenols, in particular OLE and HT, exert antioxidant activity by interfering with different cellular pathways (Figure 2).

**Figure 2.** Main antioxidative effects of EVOO polyphenols.

#### **5. Anti-Inflammatory Properties of Olive Polyphenols: Molecular Mechanisms**

Inflammation is an essential defense mechanism of the organism by which the immune system recognizes and eliminates harmful agents and infected cells and promotes tissue repair to restore body homeostasis. This process is integrated into many coordinated functions and involves transiently elevated levels of cytokines able to activate both the innate and the adaptive immune systems. The inability to regulate an inflammatory response has multiple detrimental consequences for the organismal homeostasis; when the inflammatory response persists, a shift towards a long-term unresolved and uncontrolled immune response, known as chronic inflammation, involving macrophage- and lymphocyte-accumulated leukocytes does occur, and this results in local or systemic damage to the tissue or organs and in the degradation of normal physiologic function. Chronic inflammation is causally associated with disease onset or progression and increases with age. Indeed, the levels of cytokines, chemokines as well as the expression of genes involved in inflammation are higher in older people or in patients with autoimmune diseases that show a greater propensity to metabolic syndrome, cardiovascular disease and other chronic conditions such as frailty, multimorbidity and a decline in physical and cognitive function. Accordingly, interventions that target inflammatory pathways and restore a deregulated inflammatory response are promising strategies to prevent disease progression.

Convincing evidence highlights that a regular intake of food rich in polyphenols may reduce the risk for the growth of chronic diseases, including obesity, diabetes mellitus and cardiovascular diseases. This healthy effect results largely from the anti-inflammatory power of the polyphenolic compounds that is expressed by various mechanisms such as antioxidant activity (see previous paragraph) and the modulation of signaling pathways and transcriptional events (Figure 3).

**Figure 3.** EVOO polyphenols in inflammation inhibition and their healthy effects in aging.

κ β α β κ κ κ α α κ In a rheumatoid arthritis model, the EVOO phenolic extracts showed joint protective properties and reduced proinflammatory mediators by the inhibition of MAPK and NF-κB signaling in activated synovial fibroblasts [124]. In this model, a polyphenolic extract also inhibited IL-1β-induced matrix metalloproteinases, TNF-α and IL-6 production, as well as IL-1β-induced cyclo-oxygenase-2 (COX-2) and microsomal PGE synthase-1 (mPGES-1) [125]. Research on the inflammatory responses in primary human keratinocytes showed that HT and its acetate ester (HTy-Ac), a natural hydroxytyrosyl derivative found in olive oil, interfere with NF-κB signaling by reducing the degradation of IκB (Inhibitor of kB), the nuclear translocation of NF-κB, its recruitment at the promoter, and the ensuing gene transcription. In addition, in this case EVOO polyphenols efficiently attenuated the expression of pro-inflammatory mediators such as thymic stromal lymphopoietin (TSLP) and the expression of several inflammation-related genes, as well as different TSLP isoforms and IL-8, thus restraining harmful processes set off by activated keratinocytes [126]. In endothelial cells, the EVOO phenolic fraction significantly reduced VEGF-induced angiogenic responses and NADPH-oxidase activity dose-dependently, resulting in the inhibition of the expression of Nox2, Nox4, MMP-2 and MMP-9 [127]. Luteolin, one of main phenolic compounds in olive oil, was able to reduce Nox4 and p22phox expression in endothelial cells treated with TNF-α and the TNF-α-induced adhesion of monocytes to human endothelial cells, a key event in the onset of vascular inflammation. The role of luteolin as an inhibitor of this inflammatory event was mediated by suppressing the expression of adhesion molecules, such as MCP-1, ICAM-1 and VCAM-1, and NF-κB signaling. Similar results were also reported with HT, tyrosol, taxifolin and OLE, which were able to inhibit angiogenesis through their inhibition of VEGFR-2 at specific phosphorylation sites [128].

In peripheral blood mononuclear cells and in endothelial cells, HT modulated the inflammatory process through a reduction in the levels of MMP-9, prostaglandin, PGE2 and tromboxanes (TX), by inhibiting COX-2 (but not COX-1). The mechanism suggested for the action of HT, tyrosol and their secoiridoid derivatives (oleacein and oleocanthal) on the inflammatory process is similar to that reported for selective inhibitors of COX-2, such as nonsteroidal anti-inflammatory drugs (NSAIDs) [110,129,130].

Recently, a protective effect, at the intestinal level, of EVOO polyphenols, in terms of the prevention of redox unbalance and of slowdown of the onset and progression of chronic intestinal inflammation, has been described in the human colon adenocarcinoma cell line (Caco-2). In these cells, the phenolic extract allowed the reversion of the oxysterolsdriven activation of JNK and p38 and the following phosphorylation of IkB. The inhibition of the NF-kB pathway, iNOS induction and the reduction in IL-6, IL-8 and NO levels were detected after oxysterol stimulation in the presence of the phenolic extract. HT and its metabolites, hydroxytyrosol sulfate, 4-glucuronide and 3′ -glucuronide, were able to inhibit the endothelial activation and expression of VCAM-1 and ICAM-1 in the endothelial

cells of the human umbilical vein or in the intestinal Caco-2 cells stimulated by LPS or TNF-α or IL-1beta [131–135]. Further evidence has shown that olive oil polyphenols were particularly efficient against LPS-induced inflammation in human macrophages (THP-1 cells) by restoring a normal level of some inflammatory factors such as IL-6, IL-1β and MCP-1 [136].

Oleocanthal, in a dose-dependent manner, induced the inhibition of COX-1 and COX-2 more efficiently than ibuprofen [137]. Tyrosol and hydroxyl-isocroman compounds, a class of ortho-diphenols present in EVOO, displayed an inhibitory effect on NO release and on the arachidonate cascade and the eicosanoid synthesis (PGE2 and LTB4) in cultured macrophages (RAW 264.7) stimulated by phorbol-12-myristate-13-acetate esters [136]. 1-Phenyl-6,7-dihydroxy-isochroman, through the suppression of NF-κB activation and a decrease in COX-2 synthesis, efficiently inhibited the production of TXA2, PGE2, and TNF-α in LPS-primed human monocytes [138]. The data reported above suggest the use of HT or its derivatives as possible innovative drugs to be used in the control of inflammation and of the immune response.

The crucial role of the gut microbiota on the general inflammatory status and cardiovascular, metabolic, and even brain health is becoming more and more convincing via the gut–brain axis. Accumulating data support the beneficial efficacy of EVOO polyphenols on gut microbiota and intestinal immunity. EVOO polyphenols exhibit antibacterial and bacteriostatic effects against pathogenic intestinal microflora, improve the growth of beneficial bacterial strains, and indirectly increase the production of microbially produced short-chain fatty acids (SCFAs), which exhibit anti-inflammatory effects and modulate gene expression through epigenetic mechanisms [139–141]. Moreover, SCFAs are potent activators of GPR43 and play an important role in blood glucose regulation [142].

Olive oil polyphenols, such as HT and other compounds generated from certain bacterial species (e.g., Lactobacillus) favored by EVOO polyphenols, can act as ligands of the aryl hydrocarbon receptor (AhR) that represent a key element in the status of mucosal immunity and in the homeostasis of the gut barrier [143]. Furthermore, as an AhR agonist, HT was shown to favor the induction of angiogenic genes in hypoxic MCF-7 cells and to contribute to slow cancer progression and metastasis [144]. Taken together, these data suggest a significant protective effect by EVOO polyphenols at the intestinal level, supporting the link between diet and the pathogenesis and development of inflammatory bowel diseases [145]. It is worth noting that Lactobacillus and Bifidobacterium are often greatly reduced in patients with AD and in elderly people. These bacterial types, whose populations are increased following EVOO consumption, produce γ-aminobutyric acid (GABA), thus influencing the GABAergic firing pattern in the brain through enteric and vagal systems [146]. In addition, EVOO may protect cognitive performance via its antibacterial activity towards defined pathogenic species of bacteria considered as a key element for AD in the pathogen interaction hypothesis [147].

#### **6. Clinical Trials Highlighting the Antioxidant and Anti-Inflammatory Properties of Olive Polyphenols**

Many clinical trials and population studies provide important data on the consistent and efficacious protection resulting from a prolonged olive oil intake against the insurgence of aging-associated pathologies, such as neurodegeneration, cardiovascular diseases, metabolic diseases and cancer. Taking into consideration all the results from these epidemiological studies supporting a causal link between the intake of olive oil polyphenols and effective benefits, in November 2011 the European Food Safety Authority (EFSA) approved two health claims regarding the salutary role of olive oil consumption. The claims recommend the use of olive oil to substitute saturated fats to maintain regular blood cholesterol levels and to protect blood lipids from oxidation. These protective effects can be achieved by the intake of at least 20 g of EVOO or the consumption of 5.0 mg of HT or its derived compounds every day (e.g., oleuropein complex and tyrosol) (http://www.efsa.europa.eu/, accessed on 2012).

One of the most remarkable large dietary intervention randomized trials was the Prevención con Dieta Mediterránea (PREDIMED) trial, carried out in Spain. This trial involved 7447 participants at high cardiovascular risk, or with T2DM or ≥3 major risk factors, including smoking, hypertension, elevated LDL-C and low HDL cholesterol levels, overweight or obesity, or with a family history of premature coronary heart disease [148–150]. The results from this trial, at a median of 4.8 years' follow-up, showed that the group following the Mediterranean diet supplemented with EVOO or nuts showed a 30% lower risk of developing cardiovascular pathologies, such as myocardial infarction, stroke, and consequent death, with respect to the group assigned to a low-fat diet.

In a subset of the PREDIMED trial, cognitive performance was also evaluated, with the conclusion that an EVOO-enriched MD significantly improved cognition [151–153]. In another subset of the PREDIMED study, the breast cancer incidence was also investigated in the same cohort. A 68% reduction in the risk of developing cancer was observed in the EVOO group [154]. In addition, results from a subsample (*n* = 990) of the PREDIMED trial indicated that a continuous intake of VOO containing a high phenolic content, instead of other types of olive oils, was efficient in preserving LDL from oxidation and in increasing the levels of HDL-cholesterol. A controlled, double blind, cross-over, randomized, clinical trial using olive oils with different phenolic concentrations (from 0 mg/L for refined olive oil, ROO, to 629 mg/L for VOO) was conducted in 30 healthy volunteers for 3 weeks, preceded by two-week washout periods. After VOO ingestion, LDL, HT monosulfate and homovanillic acid sulfate, but not tyrosol sulfate, levels were increased, while the concentrations of biomarkers of oxidative stress, including oxidized LDL (oxLDL), conjugated dienes, and hydroxy fatty acids, decreased. ROO ingestion did not affect the levels of LDL phenols and oxidation markers [155,156].

Another randomized, controlled, parallel-arm, clinical trial was carried out to compare the effects of olive oil with high (EVOO) or low (ROO) polyphenol levels in patients undergoing coronary angiography. Forty patients with at least one classic cardiovascular risk factor were randomly divided in two groups and received 25 mL EVOO or ROO daily for 6 weeks. At the end of treatment, the group that received high-polyphenol olive oil had a significant reduction in plasma LDL-cholesterol and plasma CRP. This also resulted in an increased production of inflammatory cytokines, such as IL-10, in LPSstimulated ex vivo whole blood. Daily uptake of EVOO in subjects under pharmacological treatment could further improve LDL-cholesterol and markers of inflammation [157]. Similar beneficial effects have been demonstrated by "The Three-City Study", carried out in 2009 on 8000 elderly subjects. This first report correlated olive oil consumption with a reduced risk of visual memory decline in a population over 65 years old [35].

The positive impact of EVOO versus low-polyphenol olive oil on markers of CVD risk in a healthy Australian cohort was investigated in a double-blind randomized cross-over study (OLIVAUS). The trial examined markers of CVD risk related to cholesterol transport and metabolism, LDL oxidation, blood pressure (peripheral and central), arterial stiffness, inflammation, and cognitive performances in 50 healthy participants subjected to three weeks of daily administration of EVOO compared to a low-polyphenol olive oil [158].

A cross-over controlled trial (ISRCTN09220811), the EUROLIVE (Effect of Olive Oil Consumption on Oxidative Damage in European Populations) study, was carried out in 25 healthy European men (20–59 years). The participants consumed 25 mL raw olive oil with low or high polyphenol content daily for 3 weeks. The interventions were preceded by a two-week washout period. Then, the effects of olive oil polyphenol intake on plasma LDL concentrations and atherogenicity were evaluated, whereas the effects on lipoprotein lipase (LPL) gene expression were checked in another subset study of EUROLIVE on 18 men. The data obtained from this study showed a decrease in plasma concentrations of apo B-100 and of total and small LDL particles together with the LDL oxidation lag time and LPL gene expression [159]. Another EUROLIVE study confirmed that olive polyphenols increase human HDL functionality, favoring HDL-mediated cholesterol efflux from macrophages [160].

The association between olive oil intake and T2DM incidence in the US population resulted from a 22-year follow-up study involving 59,930 35–65-year-old women from the Nurses' Health Study and 85,157 26–45-year-old women from the NHS II, free of diabetes, CVDs and cancer at baseline. The results suggested that higher olive oil intake was correlated with a moderately reduced risk of T2DM, while the risk increased in women consuming other types of fats and salad dressings [161].

Another short-time study highlighted the effect of EVOO on post-prandial levels of glucose and LDL-cholesterol. Post-prandial glycemic and lipid profiles were investigated in 25 healthy subjects randomly assigned in a cross-over design to a Mediterranean diet supplemented with or without 10 g EVOO/day. The results showed that EVOO improved post-prandial glucose and LDL-cholesterol levels, suggesting an anti-atherosclerotic effect of the MD [162]. Furthermore, the same trial revealed that EVOO consumption resulted in high GLP-1 and gastric inhibitory peptide (GIP) levels in the circulatory system, while in another trial with type 1 diabetes (T1D) patients, an increase in gastric emptying and GPL-1 secretion was observed together with reduced glucose absorption through glucose– lipid competition that can contribute to a lower glycemic response [163]. In addition, an acute intake of EVOO resulted in a significant reduction in the levels of plasma glucose, triglyceride, apolipoprotein B-48, and dipeptidyl peptidase-4 activity and in a significant increase in the peripheral blood levels of insulin and glucagon-like peptide 1 (GLP-1), as revealed by a randomized trial of 30 participants with impaired fasting glucose levels [164].

The MICOIL pilot study was published on 10 November 2020. The trial confirmed that the long-standing benefits against cognitive impairment of polyphenol-enriched olive oil are greater than those granted by "simple" EVOO. The clinical trial divided participants with mild cognitive impairment (MCI) into three randomized groups. Genetic predisposition to AD was taken into account to obtain a homogenous baseline. Each group followed a unique diet: The first group received 50 mL/day of high-polyphenol olive oil, while following an MD. The second group received 50 mL/day of olive oil with moderate phenolic content, along with an MD. The third group only followed a normal MD. Long-term consumption of early harvest high phenolic or moderate phenolic EVOO was associated with an important amelioration of cognitive performance, as opposed to the low phenolic content MD, independent of the presence of genetic predisposition [165]. In 2010, a study on 20 patients with MetS showed that the acute intake of VOO was able to reduce the postprandial inflammatory response and the expression of several pro-inflammatory genes, mainly by decreasing the activation of NF-kB, of the activator protein-1 transcription factor complex AP-1, cytokines, mitogen-activated protein kinases (MAPKs) or arachidonic acid pathways, secondary to the reduction in LPS intestinal absorption following a high-fat meal [166,167].

To describe the exact role of olive oil in the metabolic changes reported above, a network meta-analysis of 30 human intervention studies totalizing 7688 subjects has been performed [168]. Using this approach, it was shown that the effect of olive oil on glycemia and blood lipids cannot be distinguished from the impact of MD adherence. Indeed, the administration of olive polyphenols in the dose suggested by EFSA does not modify glycemia levels, while it ameliorates insulin sensitivity [169]. These data are in accordance with the reported evidence of a direct action of polyphenols on the pancreas [170] and with the improvement of insulin secretion through the anti-inflammatory activity of oleic acid [171]. The only clear effect of the intake of a high-polyphenol olive oil was on HDLcholesterol levels, on LDL and nucleic acid oxidation and on the plasma antioxidant activity, in agreement with previous meta-analyses [172].

Finally, an MD supplemented with polyphenol-rich EVOO has probiotic effects promoting the growth of bacteria of the Lactobacillus and Bifidobacterium types. These data result from different studies where overweight/obese participants and patients with HIV or with hypercholesterolemia consumed 40–50 g/day of EVOO for 12 weeks [172–174].

#### **7. Conclusions and Future Perspectives**

Several data highlight the ability of olive oil polyphenols to counteract aging and to protect against the insurgence of aging-associated pathologies, such as neurodegeneration, cardiovascular and metabolic diseases, and cancer, in part associated with derangement of redox homeostasis and proteostasis. However, recent research supports the idea that the health-promoting properties of olive oil polyphenols go well beyond their anti-amyloid and antioxidant power reported previously, highlighting their multi-target effects.

The claimed benefits of olive oil polyphenols have been supported by positive and encouraging results from many preclinical studies both in vitro and in animal models, as well as by population surveys and clinical trials often involving large numbers of participants. However, to date, there are still some doubts to resolve and therefore definitive results are lacking, even for the bioavailability of these molecules and their effective beneficial dose. In particular, further research is needed to better describe at the molecular and genetic levels the effects of olive polyphenols in several investigated biological systems to provide solid and definitive proof of their positive effects in a number of human pathologies. It must also be considered that the health benefits in humans most likely do not depend on the consumption of a single polyphenol but are the result of a variety of synergistic mechanisms of a combination of several polyphenols or other plant components.

Each factor affecting the bioavailability, bioaccessibility and bioactivity of polyphenols should also be considered. This is crucial because the bioavailability of these molecules is influenced by many factors, including phenolic structure, food processing, the food matrix, and the organism (microbiota composition, efficiency of detoxification mechanisms); furthermore, all these factors can interact with each other, modulating polyphenol bioavailability. Moreover, the latter, and thus the efficacy of these compounds, can be improved by administration in combination with other phytochemicals or drugs or in polyphenol-loaded nanotechnology-based delivery systems.

Finally, it might be more relevant and interesting to investigate the relationship between EVOO polyphenols and the gut microbiota to obtain further dietary indications. In fact, the dietary polyphenols/gut microbiota relation is a bi-directional one. On the one hand, polyphenols can affect the composition of gut microbiota; on the other hand, the microbiota is able to metabolize these molecules into bioactive compounds. Expanding knowledge on the effects of dietary polyphenols on the intestinal microbiota and the relative mechanisms of action and ensuing consequences, in addition to pharmacokinetics and pharmacodynamics of EVOO polyphenols, will be essential to better assess the effective doses and the levels reached by these molecules in different tissues and organs following different routes of introduction.

**Author Contributions:** M.S., F.C., P.N., M.L. and M.B. wrote and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by "MONICABUCCIANTINI RICATEN21—Functional food against neurodegenerative disorder.

**Acknowledgments:** M.L. was supported by Fondazione Umberto Veronesi.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Review* **Therapeutic Potential of Polyphenols in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia**

**Valentina Novak <sup>1</sup> , Boris Rogelj 1,2 and Vera Župunski 1,\***


**\*** Correspondence: vera.zupunski@fkkt.uni-lj.si

**Abstract:** Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are severe neurodegenerative disorders that belong to a common disease spectrum. The molecular and cellular aetiology of the spectrum is a highly complex encompassing dysfunction in many processes, including mitochondrial dysfunction and oxidative stress. There is a paucity of treatment options aside from therapies with subtle effects on the post diagnostic lifespan and symptom management. This presents great interest and necessity for the discovery and development of new compounds and therapies with beneficial effects on the disease. Polyphenols are secondary metabolites found in plant-based foods and are well known for their antioxidant activity. Recent research suggests that they also have a diverse array of neuroprotective functions that could lead to better treatments for neurodegenerative diseases. We present an overview of the effects of various polyphenols in cell line and animal models of ALS/FTD. Furthermore, possible mechanisms behind actions of the most researched compounds (resveratrol, curcumin and green tea catechins) are discussed.

**Keywords:** ALS; FTD; polyphenols; neurodegeneration; resveratrol; curcumin; catechin; EGCG

#### **1. Introduction**

With the ageing population, the treatment and management of neurodegenerative diseases is a major and increasing challenge for health care systems and societies around the world [1]. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects motor neurons, resulting in deterioration of motor function, and frontotemporal dementia (FTD) is a neurodegenerative disorder characterised by changes in personality, behaviour, and language. The development of both diseases is a progressive and ultimately fatal multistep process with a complex genetic and molecular background. Despite extensive research efforts, only two treatment options with limited effects on survival and motor function are currently approved for ALS. The vast majority of compounds researched as possible ALS therapies until today were found to be ineffective in clinical trials, highlighting the need for further research [2]. Currently, only symptomatic treatments with limited effects are available for FTD [3].

Polyphenols are natural compounds whose neuroprotective effects have been demonstrated in various models of neurodegenerative diseases such as Alzheimer's and Parkinson's disease. These compounds are being explored for possible dietary intervention and supplementation as preventive measures against neurodegenerative diseases, and also as possible candidates for therapies to slow disease progression and alleviate symptoms [4]. Due to the lack of disease-changing treatments for ALS/FTD and the growing interest in natural compounds as therapeutic agents, this article reviews an intriguing topic of potential use of polyphenols in the development of treatments for ALS/FTD symptoms.

**Citation:** Novak, V.; Rogelj, B.; Župunski, V. Therapeutic Potential of Polyphenols in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. *Antioxidants* **2021**, *10*, 1328. https://doi.org/10.3390/antiox 10081328

Academic Editor: Stanley Omaye

Received: 12 July 2021 Accepted: 20 August 2021 Published: 23 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/).

#### *1.1. Amyotrophic Lateral Sclerosis and Frontotemporal Dementia*

ALS is a neurodegenerative disease characterised by progressive loss of both upper and lower motor neurons. Initial signs of the disease may include weakness of the limbs (in spinal-onset ALS) or difficulties with speech and swallowing (in bulbar-onset ALS) [5]. Disease progression eventually leads to paralysis and death from respiratory failure, on average 24 to 50 months after onset [6–10]. The worldwide incidence of ALS is 1.75 with a reported mean age at diagnosis between 51 and 69 years [11,12]. ALS cases can be divided into the familial form of the disease (fALS, 5–15% of patients), where there is a clear family history, and the predominant sporadic form (sALS) [13]. Frontotemporal dementia (FTD) is a type of dementia primarily associated with alterations in the frontal and temporal lobes. Symptoms manifest as changes in behaviour, personality, language, and motor skills [14,15]. The incidence of FTD is 1.6 and the mean age of onset is 65 years [16]. FTD can be divided into one behavioural (bvFTD) and two language variants (or primary progressive aphasias (PPA)) [14]. Mean survival time for most forms of FTD is approximately 8 years [17]. Up to 40% of FTD patients have a family history of the disease [18,19].

Clinical, genetic, pathological and biochemical data show that there is an overlap between ALS and FTD. First observations that ALS and FTD might be connected were made in the early 1990s [20,21]. Data show that about half of ALS patients have cognitive impairment and 15% meet the criteria for FTD [22,23]. Similarly, about 30% of patients with FTD develop signs of motor dysfunction and 10–15% have ALS [24,25]. The discovery of common genetic causes and biological mechanisms further confirmed that ALS and FTD are closely associated (Figure 1) [5,26].

**Figure 1.** Genes involved in pathologies along the ALS-FTD spectrum. The most common genetic causes of the disease are highlighted in bold.

ALS and FTD pathologies are multistep processes that affect many aspects of cellular activity. The most prominent pathological hallmark of both ALS and FTD are changes in protein homeostasis, including protein misfolding and aggregation, altered localisation, and defects in autophagic and proteasomal degradation. The combination of these mechanisms leads to the formation of toxic cytoplasmic inclusions in motor neurons and surrounding cells. Proteins that predominantly form these structures are two RNA-binding proteins, TAR DNA binding protein (TDP-43, protein product of *TARDBP*), and fused in sarcoma (FUS), microtubule-associated protein tau (gene *MAPT*), and superoxide dismutase 1 (SOD1) [27,28]. The correlation between pathology and genetics is complex [29–31]. Pathologically, 97% of ALS cases have pathognomonic TDP-43 aggregates, while only 1% of those are associated with mutations in TDP-43 and in the rest TDP-43 is not mutated. A total of 1% of ALS shows FUS aggregates, all of which are associated with mutations in FUS. Mutations in FUS or TDP-43 are extremely rare in FTD; however, 50% of FTD have TDP-43 aggregates and 10% of FTD have FUS aggregates. A total of 40% of FTD is tau aggregates. Impairments in protein turnover and clearance are also observed. Mutations in genes associated with different stages of autophagy are also causative for ALS/FTD, from autophagy regulating activities of C9ORF72 to impaired functions of autophagic receptors SQSTM1 and optineurin [32–37].

In healthy cells, TDP-43 and FUS are predominantly nuclear RNA/DNA-binding proteins with functions in RNA splicing, transcription, microRNA biogenesis, and mRNA transport [38–47]. Both play important parts in ribonucleoprotein coacervates that form membrane-less organelles such as stress granules in the cytoplasm and paraspeckles in the nucleus [48,49]. In ALS/FTD, FUS or TDP-43 mislocalise to the cytoplasm and form aggregates that are most likely toxic, although loss of function from the nucleus may also be the key disease-causing factor. This mislocalisation is instigated by a number of disruptions, including dysfunctions in proteostasis, nucleocytoplasmic shuttling, and the cellular stress response [50–52]. Upon stress, TDP-43, FUS, and some other ALS-associated RNA-binding proteins separate into stress granules, which may be the first step in the formation of insoluble aggregates [53,54]. Another common factor in the disruption of RNA metabolism is G4C2 repeat expansions in the C9ORF72 gene, which are the most common cause of familiar forms of ALS/FTD [55–57]. The repeats form stable nucleic acid secondary structures known as G-quadruplexes, hairpin loops, and i-motifs, that sequester RNA-binding proteins and form nuclear foci similar to paraspeckles, or can be translated into toxic dipeptide repeats via repeat-associated non-ATG translation [58–65].

Mitochondria play a central role in neurons, primarily fulfilling high needs for energy. ALS/FTD-associated changes include defects in oxidative phosphorylation and calcium homeostasis, elevated production of ROS, structural impairments, and reduced clearance of damaged mitochondria [66]. Changes in mitochondrial morphology are observed in cells overexpressing mutant SOD1, FUS, or TDP-43 [67–70]. The increased localisation of mutant SOD1 in the mitochondrial intermembrane space causes mitochondrial dysfunction and toxicity to neurons [71–73]. Overall, mitochondrial changes result in decreased electron transport chain activity and reduced ATP production [66]. Moreover, oxidative stress has been proposed to be crucial in ALS pathogenesis and has been well documented in patient samples [74–76].

#### *1.2. Currently Used Therapies for ALS/FTD*

Treatments currently in clinical trials for ALS/FTD were comprehensively reviewed by Liscic et al. [26]. Therapeutic targets include a reduction in glutamate excitotoxicity and protein aggregation, upregulation of certain heat shock proteins, and activation of troponin in skeletal muscle. Interesting novel strategies for ALS/FTD treatment may also come from stem cell therapy, non-invasive brain stimulation, and the growing knowledge of the influence of the gut microbiota on the development of neurological diseases [26]. Currently, only two drugs are approved for the treatment of ALS. Riluzole was approved for clinical use in 1995 and trials observed reduced one year mortality and slower deterioration of muscle function [26,77,78]. The mechanisms behind the beneficial effects of riluzole are not entirely clear. Different neuroprotective actions have been proposed, such as inhibition of glutamate excitotoxicity, blockade of Ca2+- or Na<sup>+</sup> -ion channels, and modulation of GABA pathways [79]. In recent years, some countries have also approved the use of edaravone (also known as MCI-186 or Radicava) for the treatment of ALS [26]. Its actions could benefit a subgroup of patients with early onset and rapidly progressive disease [80]. Edaravone is thought to act as an antioxidant and free radical scavenger, but the mechanisms are not well understood [81]. There are currently no approved direct treatments for FTD, other than symptom management [82].

#### **2. Therapeutic Potentials of Polyphenols in ALS/FTD**

Many potential therapeutic compounds have antioxidant and anti-inflammatory properties. Polyphenols (Figure 2) are a diverse group of naturally occurring compounds with a characteristic chemical structure that has one or more phenolic rings. They are found in plant foods such as fruits, vegetables, and whole grains [83,84]. In plants, polyphenols are categorised as secondary metabolites and have functions in normal growth as well as in the plant defense system [85]. They are synthesised in the shikimate and phenylpropanoid pathways [86]. Many different polyphenols have been described to have neuroprotective effects in mammalian cell and animal models of ALS/FTD [87]. In this review, the focus will be on resveratrol, epigallocatechin gallate (EGCG), and curcumin (Figure 2). We will also explore the effects of some other flavonoids and phenolic acids in the context of ALS/FTD.

**Figure 2.** Classification of polyphenols with structural formulas of epigallocatechin gallate (EGCG), resveratrol and curcumin.

#### *2.1. Resveratrol*

′ Resveratrol (3,5,4′ -trihydroxystilbene) is a polyphenol found in grapes, red wine, berries, and peanuts [88]. Both cis- and trans- isomers occur naturally, with trans-form being the focus in terms of potential neuroprotective activity [89]. Effects of resveratrol in ALS were first demonstrated in neuronal cell lines expressing the SOD1G93A mutant [90–92]. Resveratrol treatment halved the cell death observed as a consequence of SOD1-mediated toxicity [90]. Treatments of mouse motor neuron cells NSC34 expressing SOD1G93A showed a minor dose-dependent improvement in cell viability and a simultaneous reduction in the concentration of cytosolic ROS [91]. Administration of resveratrol protected rat cortical motor neurons from the toxic effects of cerebrospinal fluid (CSF) from ALS patients [93]. Further studies in mice ALS models expressing mutant SOD1G93A showed conflicting results, which are probably a consequence of different protocols on dosing and route of administration. Chronic oral administration of resveratrol at 25 mg/kg/day did not improve motor abilities and life span of ALS mice [94]. On the other hand, intraperitoneal injections of 20 mg/kg/twice a week improved survival and delayed the onset of ALS [95]. A similar positive effect on survival and motor function was observed with a higher dose (160 mg/kg/day) administered orally [96]. The neuroprotective effects of resveratrol in ALS mice have been further demonstrated in coadministration with other potential therapeutics [97,98]. Resveratrol has also been researched in models of tauopathies, a hallmark of FTD, but the overall effects on tau aggregation are inconclusive [99].

The predominant mechanism behind the neuroprotective effect of resveratrol is the activation of SIRT1, a NAD<sup>+</sup> -dependent protein deacetylase [90,92,95,96,100]. Structural studies suggested a mechanism in which resveratrol acts as an adaptor for the interaction between the peptide substrate and SIRT1 [101]. Many downstream mechanisms of SIRT1 targets have been proposed as possible mediators of the beneficial effects. SIRT1 deacetylates p53 [90,96], a known tumor suppressor protein involved in mechanisms of motor neuron cell death [102]. Resveratrol treatment upregulates factors involved in mitochondrial biogenesis, which could improve altered energy metabolism observed in ALS [92,96]. SIRT1 also targets HSF1 (heat shock factor 1) that activates several heat shock proteins. Their activity as chaperones possibly mitigates formation of toxic protein aggregates [95]. Normalisation of autophagic flux was also observed in resveratrol-treated ALS mice, but it is not clear whether SIRT1 is involved [96]. Independent of SIRT1, resveratrol can also activate AMPK (AMP-activated protein kinase) [96,103] that has downstream targets involved in neuroprotective mechanisms [104]. Moreover, a molecular mechanistic study on

SOD1G93A showed a stabilising effect of resveratrol that could impede the aggregation of mutant protein [105]. A similar inhibitory effect was observed in aggregation studies of wt SOD1 [106].

#### *2.2. Curcumin*

Curcumin (diferuloylmethane) is the predominant curcuminoid found in turmeric (*Curcuma longa*), which is widely used in traditional Indian medicine. The potential benefits of curcumin are being explored in many neurodegenerative diseases. In models of Alzheimer's and Parkinson's disease, curcumin can reduce oxidative stress, affect toxic protein aggregation, and protect against apoptosis [107,108].

Regarding ALS, curcumin was shown to impede aggregation of reduced wt SOD1 in vitro by binding its aggregation prone regions. Curcumin-bound SOD1 aggregates were smaller, unstructured, and less cytotoxic [109]. A similar effect of inhibiting beta-sheet formation and aggregation was observed with tau, a protein involved in FTD [110]. In contrast, the binding of curcumin to tau aggregates was not observed in post-mortem brain tissue sections from FTD patients [111].

Curcumin presents a challenge forin vivo use due to its poor absorption, fast metabolism, and rapid elimination. Several strategies can be utilised to overcome the low oral bioavailability of curcumin [112]. The protective effect of an analogue, dimethoxy curcumin, was demonstrated in a neuronal cell line expressing TDP-43 mutants Q331K or M337V. Dimethoxy curcumin restored mitochondrial damage by improving transmembrane potential, increasing electron transfer chain complex I activity, and upregulating UCP2 (uncoupling protein 2) [113]. The same compound also improved abnormally high excitability of cells expressing mutant TDP-43 [114]. Furthermore, an improved curcumin analogue, monocarbonyl dimethoxycurcumin C, prevented aggregation of mutant TDP-43 and reduced oxidative stress, possibly due to increased expression of heme oxygenase-1 [115].

Another approach to improve the bioavailability of curcumin is delivery using nanoparticles. The potential for ALS treatment was demonstrated with curcumin-loaded inulin-D-alfa-tocopherol succinate micelles, which were effectively delivered into mesenchymal stromal cells [116]. Furthermore, the efficiency of a turmeric supplement in nanomicelles was tested in a clinical trial involving 54 ALS patients treated primarily with riluzole. Nanocurcumin improved the survival probability of the patients, but did not significantly improve their motor function [117].

#### *2.3. Catechins*

Green tea, produced from the leaves and buds of *Camellia sinensis*, is rich in polyphenols catechins, predominantly (−)epigallocatechin gallate (EGCG), but also (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG), (−)-epicatechin (EC), and (+)-catechin [118]. In ALS models, EGCG has been shown to protect motor neuron cells from oxidative stress and mitochondrial damage [119]. Presymptomatic oral supplementation of EGCG at doses of at least 2.9 mg EGCG/kg body weight in SOD1G93A mice significantly delayed symptom onset, improved motor function, and increased lifespan [120,121].

EGCG likely acts by upregulating a prosurvival signaling pathway PI3K/Akt. Among other pathways, PI3K/Akt regulates the activity of GSK-3. Increased GSK-3 levels are associated with the formation of neurofibrillary tangles and neuronal death. In addition, GSK-3 induces apoptosis through downstream signaling, including mitochondrial damage and caspase-3 activation. It was shown that Akt phosphorylates GSK-3, resulting in less mitochondrial damage [119]. Observations in ALS mice further confirm an increase in PI3K/Akt and a decrease in death signals such as caspase-3, cytosolic cytochrome c, and cleaved PARP (poly (ADP-ribose) polymerase) [120]. EGCG also has antioxidant and anti-inflammatory effects on microglia and astrocytes [121]. In addition, it can decrease lipid peroxidation, but has no effect on iron metabolism despite its presumed chelating abilities [122]. A molecular docking study showed the potential of EGCG to reduce mutant SOD1 aggregates [123]. In vitro studies confirmed an inhibitory effect on apo-SOD1 aggre-

gation [124]. It has also been shown that the addition of EGCG induces oligomerisation of TDP-43 and inhibits its degradation into toxic aggregation-prone fragments [125]. In FTD, inhibition of tau filament formation was observed for ECG, but not for EC [126].

#### *2.4. Other Flavonoids*

In addition to green tea catechins, several other flavonoids have been tested in ALS/FTD models. Presymptomatic administration of 2 mg/kg body weight of an anthocyanin-enriched strawberry extract with callistephin (pelargonidin 3-glucoside) as the predominant component delayed ALS onset, preserved grip strength, and prolonged survival in SOD1G93A mice [127]. Oral supplementation of fisetin (3,3,4,7-tetrahydroxyflavone) improved motor functions, delayed disease onset, and increased survival in SOD1G93A mice (at a dosage of 9 mg/kg) and SOD1G85R *Drosophila melanogaster*. The predominant mechanism behind the activity of fisetin in motor neuron cell lines expressing SOD1G93A appears to be the activation of the ERK pathway involved in the regulation of cell survival. Moreover, fisetin decreased both wt and mutant SOD1 levels in cells, possibly by activating autophagy [128].

A computational study confirmed the binding of kaempferol (3,4′ ,5,7-tetrahydroxyflavone) and kaempferide to mutant SOD1G85R [129]. Both compounds were experimentally shown to have antioxidant properties and could reduce the formation of SOD1G85R aggregates in N2a mouse neuroblastoma cells. Kaempferol could act via increased phosphorylation of AMPK and downstream induction of autophagy [130]. The antioxidant effect of quercetin (3,3′ ,4′ ,5,7 pentahyroxyflavone) was first observed in lymphoblast cell lines from ALS patients [131]. In vitro tests showed that quercetin glycosides, namely quercitrin and quercetin 3-beta-d-glucoside, inhibit misfolding and aggregation of SOD1A4V mutant [132]. A similar effect on aggregation was observed with quercetin and baicalein [133]. Furthermore, preventive administration of quercetin in rats reduced oxidative stress, defective mitochondria, and brain cell death caused by aluminium exposure [134].

SOD1G93A mice treated with 5 mg/kg 7,8-dihydroxyflavone exhibited significantly improved motor performance and increased numbers of spinal motor neurons compared with untreated animals [135]. Interestingly, it was observed that treatment with 16 mg/kg genistein (4′ ,5,7-trihydroxyisoflavone) had a protective effect on disease progression in male SOD1G93A mice [136]. In contrast, in further studies, a delay in symptoms and higher survival of motor neurons was observed in both sexes, possibly due to anti-inflammatory effects and restored autophagy [137]. Twice-daily administration of 700 mg luteolin (3′ ,4′ ,5,7 tetrahydroxyflavone) in combination with palmitoylethanolamide showed some improvement of symptoms in patients with FTD [138].

#### *2.5. Phenolic Acids and Derivatives*

Phenolic acids are found in fruits, coffee, tea, and grains. Their diverse neuroprotective effects make them interesting candidates for better ALS therapies. It has been reported that protocatechuic acid administration at 100 mg/kg in SOD1G93A mice prolongs survival, improves motor function, and reduces gliosis [139]. Caffeic acid phenethyl ester (CAPE) showed a dose-dependent improvement in survival and a simultaneous reduction in cytosolic ROS in the NCS34 cell line expressing SOD1G93A. CAPE decreased the activation of the oxidative stress-associated transcription factor NF-κB and activated the antioxidant response element (ARE) [91]. Further studies in SOD1G93A mice confirmed that daily administration of 10 mg/kg CAPE after disease onset slowed symptom progression and prolonged survival. A reduction in glial activation and phospho-p38 levels was observed as a result [140]. Gallic acid and wedelolactone improved locomotor function and motor learning abilities in an aluminium or quinolinic acid-induced rat model of sALS. The effects may be due to a reduction in inflammatory cytokines, normalisation of L-glutamate levels, and decreased activation of caspase-3 [141,142]. Rosmarinic acid, the main compound in rosemary (*Rosmarinus officinalis*) extract, reduced weight loss, improved motor performance, and prolonged survival of SOD1G93A mice [143,144]. The effects of treatment with higher

doses were compared with the established ALS therapeutic agent riluzole, but were not found to be more effective [144].

## *2.6. Overview of Potential Therapeutic Effects of Polyphenols in ALS and FTD*

We have summarised the therapeutic implications of polyphenols, including their proposed mechanisms in animal and cell line models of ALS and FTD (Table 1). The predominant mechanism behind the neuroprotective role of resveratrol is the activation of SIRT1. Its downstream targets may impact processes such as neuronal survival, mitochondrial biogenesis, and prevention of protein aggregate formation, all of which contribute to the observed delay in symptoms and increased viability in ALS models [90,92,95,96]. Curcumin derivatives show neuroprotective value through several mechanisms, such as restoring mitochondrial functions, normalising cell excitability, and preventing the formation of toxic protein aggregates [113–115]. Green tea catechin EGCG has been observed to upregulate a prosurvival signaling pathway PI3K/Akt and decrease signals leading to cell death, such as activation of caspase-3, which is associated with apoptosis [119,120]. Both resulted in the delayed onset of ALS and increased survival in mice models treated with EGCG [120,121]. Fisetin acts by activating the ERK pathway, which modulates cell survival and upregulates HO-1, both of which contribute to the cellular response against oxidative stress [128]. Another mechanism exerted by some polyphenols is the downregulation of the NF-κB pathway that, overall, has an anti-inflammatory effect [91].


**Table 1.** Therapeutic implications of different polyphenols in ALS and FTD models.


**Table 1.** *Cont.*

The importance of the gut–brain axis in ALS/FTD has been recognised. On the one hand, polyphenols may serve as prebiotics and alter the gut microbiota, affecting disease pathogenesis [146], (for a detailed review, see [147]). On the other hand, certain polyphenols such as EGCG are degraded by some gut microbiota, which reduces their bioavailability [148,149]. However, some metabolites do target the brain and have beneficial effects on neurons [118,150].

#### **3. Conclusions**

Polyphenols offer new possibilities for the development of therapies for ALS/FTD. However, more research is needed in this field, including strategies for effective targeting and delivery to the site of action. When evaluating the therapeutic potential of polyphenols, we must also consider their uptake in the gut, degradation by the microbiota, and the delivery to the brain. Therefore, it is important whether polyphenols are consumed or administered intravenously and how well they can cross the blood–brain barrier [151,152]. Another hurdle for potential ALS/FTD medication is translating findings from animal models into successful clinical trials. Additional aspect of potential variability in successful treatment lies in the use of purified polyphenols or plant extracts that may act synergistically. Most of the findings reviewed here come from various successful preclinical stages and have yet to be tested in humans. Nevertheless, polyphenols have the potential to improve the treatment of ALS/FTD, either through the development of new drugs or as dietary supplements.

**Funding:** This work was funded by Slovenian Research Agency grants (P4-0127, P1-0207, J3-9263, J3-8201, J7-9399 and N3-0141) and CRP-ICGEB research grant (CRP/SVN19-03).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Differential Effects of Polyphenols on Insulin Proteolysis by the Insulin-Degrading Enzyme**

**Qiuchen Zheng † , Micheal T. Kebede † , Bethany Lee † , Claire A. Krasinski, Saadman Islam, Liliana A. Wurfl, Merc M. Kemeh, Valerie A. Ivancic, Charles E. Jakobsche, Donald E. Spratt and Noel D. Lazo \***

> Gustaf H. Carlson School of Chemistry and Biochemistry, Clark University, Worcester, MA 01610, USA; qzheng@clarku.edu (Q.Z.); mkebede@clarku.edu (M.T.K.); blee@clarku.edu (B.L.); ckrasinski@clarku.edu (C.A.K.); sislam@clarku.edu (S.I.); lwurfl@clarku.edu (L.A.W.); mkemeh@clarku.edu (M.M.K.); vivancic@clarku.edu (V.A.I.); cjakobsche@clarku.edu (C.E.J.); dspratt@clarku.edu (D.E.S.)

**\*** Correspondence: nlazo@clarku.edu

† These authors contributed equally to this work.

**Abstract:** The insulin-degrading enzyme (IDE) possesses a strong ability to degrade insulin and Aβ42 that has been linked to the neurodegeneration in Alzheimer's disease (AD). Given this, an attractive IDE-centric strategy for the development of therapeutics for AD is to boost IDE's activity for the clearance of Aβ42 without offsetting insulin proteostasis. Recently, we showed that resveratrol enhances IDE's activity toward Aβ42. In this work, we used a combination of chromatographic and spectroscopic techniques to investigate the effects of resveratrol on IDE's activity toward insulin. For comparison, we also studied epigallocatechin-3-gallate (EGCG). Our results show that the two polyphenols affect the IDE-dependent degradation of insulin in different ways: EGCG inhibits IDE while resveratrol has no effect. These findings suggest that polyphenols provide a path for developing therapeutic strategies that can selectively target IDE substrate specificity.

**Keywords:** polyphenols; resveratrol; epigallocatechin-3-gallate; insulin-degrading enzyme

#### **1. Introduction**

Insulin-degrading enzyme (IDE), aka insulysin, is a ubiquitous zinc-dependent protease belonging to the M16 family of metalloendopeptidases [1]. Members of this family form a catalytic chamber, also referred to as crypt, the volume of which in IDE (~15,700 Å<sup>3</sup> [2]) limits the length of substrates to less than 70 amino acids [3]. Biophysical studies using X-ray crystallography [2,4,5] and cryogenic electron microscopy [6] have revealed mechanisms by which IDE encloses and degrades a diverse group of metabolically important and pathologically relevant substrates including insulin, a key hormone for glucose metabolism, amyloid-β(1-42) (Aβ42), which self-assembles to form the proximate neurotoxic assemblies in Alzheimer's disease (AD) [7], and islet amyloid polypeptide (IAPP), which self-assembles to form pancreatic amyloid in type 2 diabetes (T2D) [8]. IDE consists of two bowl-shaped halves, IDE-N and IDE-C, held together by an unstructured linker (Figure 1a). The flexibility of the linker allows IDE to exist in two major conformational states during its catalytic cycle: an open conformational state, which facilitates substrate entry and anchoring to IDE's exosite; and a closed conformational state, which expedites substrate degradation. IDE bears the HXXEH catalytic motif arranged so that the two histidine residues (His108 and His112) coordinate Zn2+ and the catalytic role of Glu111 in the hydrolysis of peptide bonds is facilitated (Figure 1b).

Given IDE's ability to degrade insulin, IAPP and Aβ42, it is no surprise that there has been strong academic and pharmaceutical interest in developing small molecules that modulate IDE's activity [9–12]. Several small-molecule and peptidic IDE inhibitors have been developed and investigated for their effects on insulin levels in cells or mice. Ii1 [13]

**Citation:** Zheng, Q.; Kebede, M.T.; Lee, B.; Krasinski, C.A.; Islam, S.; Wurfl, L.A.; Kemeh, M.M.; Ivancic, V.A.; Jakobsche, C.E.; Spratt, D.E.; et al. Differential Effects of Polyphenols on Insulin Proteolysis by the Insulin-Degrading Enzyme. *Antioxidants* **2021**, *10*, 1342. https:// doi.org/10.3390/antiox10091342

Academic Editors: Rui F. M. Silva and Lea Pogaˇcnik

Received: 26 July 2021 Accepted: 23 August 2021 Published: 25 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/).

and P12-3A [14] inhibited degradation of extracellular insulin in cells and fibroblasts, respectively. BDM44768 [15], B35 [16], and 6bk [17] elevated plasma insulin levels in mice. Despite these advances, significant challenges remain, as noted in a recent review by Leissring et al. [12]. First and foremost, inhibiting IDE may lead to increased levels of IAPP and Aβ, increasing the risk for T2D and AD, respectively (Figure 2).

β **Figure 1.** X-ray structure of insulin-degrading enzyme in its closed conformational state (PDB: 4PES). (**a**) IDE is composed of an N-terminal half (IDE-N) containing domains I and II, and a C-terminal half (IDE-C) containing domains III and IV. The two halves are joined by an unstructured linker (magenta). Domain I contains the HXXEH zinc-binding and catalytic motif. Domain II contains the exosite (yellow) in an anti-parallel β-sheet conformation. (**b**) The HXXEH motif includes H108 and H112 that coordinate Zn2+ and the catalytically important glutamate (E111) residue. When E111 is mutated to a glutamine as in 4PES, the enzyme becomes inactive. β

Improved Glucose Tolerance

**Figure 2.** Beneficial and deleterious consequences of the pharmacological inhibition of IDE. A beneficial consequence of the inhibition of IDE is increased levels of insulin, leading to an improvement in glucose tolerance. Deleterious consequences include increased levels of Aβ42 and IAPP, leading to the formation of cytotoxic assemblies in the brain and pancreas, respectively.

An attractive alternative to the development of small-molecule inhibitors of IDE is a nutritional strategy that implicates polyphenols. Polyphenols are naturally occurring compounds that possess antioxidant [18–20], anti-inflammatory [19–21], and antiamyloidogenic [22] properties. As such, polyphenols have been hypothesized to prevent T2D [23] and AD [24]. Recently, we showed that IDE's activity toward Aβ42 is sustained in the presence of resveratrol (Figure 3a) [25]. The effect of resveratrol on the IDEdependent degradation of insulin, the enzyme's most physiologically important substrate, however, remains unknown. In this work, we used a combination of chromatographic and spectroscopy-based kinetic analysis to investigate the effects of resveratrol on IDE's activity toward insulin. For comparison, similar experiments were conducted in the presence of epigallocatechin-3-gallate (EGCG, Figure 3b), which is approximately two times larger than resveratrol, and is similar in size to BDM44768 [15]. Our results show that EGCG inhibits the IDE-dependent degradation of insulin, whereas resveratrol has no effect, presumably because of its smaller size. The implications of these results in the development of IDE-centric therapeutic strategies for T2D and AD are discussed.

**Figure 3.** Chemical structures of polyphenols used in this study. (**a**) Resveratrol (C14H12O<sup>3</sup> ) is found in red wine and red grapes. (**b**) Epigallocatechin-3-gallate (C22H18O11) is found in green tea.

#### **2. Materials and Methods**

#### *2.1. Insulin-Degrading Enzyme Expression and Purification*

The bacterial expression vector encoding IDE fused to GST was kindly provided by Dr. Malcolm A. Leissring of the University of California at Irvine. GST-IDE was overexpressed in *E. coli* BL21-CodonPlus RIL cells (EMD Biosciences Inc., San Diego, CA, USA), and purified as previously described [25–27]. Cleavage of the GST tag was accomplished using GST PreScission protease and further purification of IDE was accomplished by standard gel filtration [25–27]. UV absorbance at 280 nm was used to determine the concentration of IDE using an extinction coefficient of ε280nm = 113,570 M−<sup>1</sup> cm−<sup>1</sup> [28].

− −

#### *2.2. Preparation of Stock Solutions*

− −

ε <sup>−</sup> <sup>−</sup> ε <sup>−</sup> <sup>−</sup> ε Recombinant human insulin (99% pure), EGCG (>98% pure), and resveratrol (>99% pure) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of insulin and EGCG were prepared in 50 mM Tris buffer (pH 7.4). Stock solutions of resveratrol were prepared in 100% ethanol. The concentrations of the stock solutions were detected by UV absorbance at the following wavelengths: 276 nm for insulin (ε276nm = 6190 M−<sup>1</sup> cm−<sup>1</sup> ) [29]; 275 nm for EGCG (ε275nm = 11,500 M−<sup>1</sup> cm−<sup>1</sup> ) [30]; and 306 nm for resveratrol (ε306nm = 15,400 M−<sup>1</sup> cm−<sup>1</sup> ) [31]. All stock solutions were freshly prepared, and used immediately after preparation.

#### *2.3. Circular Dichroism Spectroscopy*

2.3.1. Monitoring the Loss of Insulin's Helical Circular Dichroic Signals with Digestion Time

We used circular dichroism spectroscopy to detect the changes in insulin's secondary structure as its proteolysis proceeds. All far UV circular dichroic (CD) spectra were recorded at 37 ◦C using a JASCO J-815 spectropolarimeter equipped with a Peltier temperature control unit. Each sample was prepared with a volume of 200 µL. The substrate-to-enzyme molar ratio of insulin-to-IDE was 100:1 (20 µM insulin: 0.2 µM IDE) for limited proteolysis [26], and the molar ratio of insulin-to-polyphenol was set as 1:2 (20 µM insulin: 40 µM EGCG or resveratrol), similar to the molar ratio of Aβ42-to-resveratrol we previously used [25]. We determined that by setting the insulin concentration at 20 µM, CD spectra of good signal-to-noise ratios were recorded in the far UV range (i.e., from 260 to 198 nm). All samples were incubated in quartz cuvettes with a path length of 1 mm. Each CD spectrum was recorded from 260 to 198 nm using an averaging time of 1 s and four accumulations. All samples were kept at 37 ◦C in between recording of spectra. The Savitzsky–Golay method with convolution width equal to 7 was applied to smoothen all CD spectra.

#### 2.3.2. Kinetic Parameters from Insulin's Helical Circular Dichroic Ellipticity at 222 nm

Steady-state kinetic parameters for the IDE-dependent degradation of insulin were determined using insulin's helical circular dichroic ellipticity at 222 nm ([*θ*obs(222nm)]), as described in detail elsewhere [26]. Briefly, seven substrate solutions with concentrations of 15, 20, 25, 30, 50, 80, and 110 µM were prepared in 50 mM Tris buffer (pH 7.4). EGCG or resveratrol was added at a concentration of 20 or 40 µM. Proteolysis was initiated with the addition of IDE at a concentration of 1 µM. After mixing, the solution was transferred into a 1-mm path length quartz cuvette and loaded into the sample holder of our circular dichroism spectrometer that was pre-warmed to 37 ◦C. After a brief equilibration period, [*θ*obs(222nm)] was recorded for 5 min. The real-time [*θ*obs(222nm)] data were then used to calculate the amount of digested insulin ([DI]) using the equation below:

$$[\text{DI}]\_t = [\text{I}]\_0 \times \left(1 - \frac{\left[\theta\_{\text{obs}(222\text{nm})}\right]\_t}{\left[\theta\_{\text{obs}(222\text{nm})}\right]\_0}\right) \tag{1}$$

where [DI]*<sup>t</sup>* is the amount of digested insulin at real-time *t*; [I]<sup>0</sup> is the initial amount of undigested insulin; and [*θ*obs(222nm)]*<sup>t</sup>* and [*θ*obs(222nm)]<sup>0</sup> are the observed ellipticity at realtime *t* and observed initial ellipticity at time = 0, respectively. Plots of [DI] against time were used to determine the initial rates (*V*0) of insulin proteolysis by IDE. Michaelis–Menten (*V<sup>0</sup>* plotted against [*S*], Lineweaver–Burk (1/*V<sup>0</sup>* plotted against 1/[*S*]), and Hanes–Woolf ([*S*]/*V<sup>0</sup>* plotted against [*S*]) plots were then constructed from which the kinetic constants *K*M, *V*max, *k*cat and *k*cat/*K*<sup>M</sup> were determined.

#### *2.4. Reversed Phase High Performance Liquid Chromatography*

Proteolysis samples were prepared for RP HPLC analysis by setting the substrateto-enzyme molar ratio to 100:1, and the polyphenol-to-insulin molar ratio to 2:1. These included: (1) insulin + EGCG + IDE (20 µM insulin + 40 µM EGCG + 0.2 µM IDE); and (2) insulin + RES + IDE (20 µM insulin + 40 µM RES + 0.2 µM IDE). Control samples that only contain insulin + IDE (20 µM insulin + 0.2 µM IDE) were also prepared. All reactions were started by the addition of IDE followed by incubation at 37 ◦C. Aliquots (200 µL) of each digests were removed at the desired time points and quenched by acidification to low pH with 15 µL of 5% (*v/v*) trifluoroacetic acid in water.

The IDE-dependent proteolysis of insulin was monitored using a Varian Pro Star 210 HPLC system, equipped with a ProStar 325 Variable Wavelength UV–Visible Detector. RP HPLC fractionation of insulin digests was carried out at room temperature using an analytical Agilent AdvanceBio mAb C4 column. Solvents A and B were 0.1% (*v/v*) formic

acid in H2O and 0.1% (*v/v*) formic acid in acetonitrile, respectively. Aliquots (20 µL) of solutions containing insulin digests were injected into the HPLC manually and eluted with a 20-min linear gradient of 0–100% B at a flow rate of 1 mL/min. The elution of analytes was monitored by UV absorbance at 214 and 254 nm. μ

#### **3. Results and Discussion**

Insulin's CD spectrum indicates a predominantly α-helical structure as indicated by the following features [32]: (1) a positive π → π\* band at 195 nm, polarized ⊥ to the helix axis; (2) a negative π → π\* band at 208 nm, polarized kto the helix axis; and (3) a negative n → π\* band at 222 nm, also polarized kto the helix axis. Recently, we showed that the CD spectrum of insulin is sensitive to the extent of IDE-dependent degradation [26]. In particular, we noted that as proteolysis occurs, the intensities of insulin's helical CD signals decrease with an increase in digestion time. The complete degradation of insulin by IDE is indicated by a CD spectrum with a single minimum near 198 nm, consistent with the dominant presence of unstructured or random coil peptides [26]. ‖ ‖

Figure 4 presents the CD spectra of insulin IDE digests in the absence of polyphenols and in the presence of EGCG or resveratrol. The time-dependent spectra recorded in the absence of polyphenols (Figure 4a) were similar to those we reported recently [26]. The intensities of the signals at 222 and 208 nm decreased with time, indicating the progressive loss of α-helical insulin. Similar results were obtained in the digestions that contained resveratrol (Figure 4b), which suggest that the polyphenol has no effect on the IDE-dependent degradation of insulin. In sharp contrast, insulin's CD helical signals persisted in the presence of EGCG (Figure 4c), indicating inhibition of the proteolysis of insulin by IDE. We noted, however, that the intensities of the helical signals, particularly at 222 and 198 nm, decreased, suggesting that the inhibition is partial and not complete.

μ **Figure 4.** CD spectra of IDE-dependent degradation of insulin in the absence and presence of polyphenols at 37 ◦C. (**a**) Spectra recorded for insulin digests that do not contain polyphenols (control samples) show the progressive loss on insulin's helical CD signals at 222, 208, and 198 nm, consistent with the loss of α-helical structure. (**b**) Spectra recorded for insulin digests in the presence of resveratrol were similar to the spectra recorded for the control samples, indicating that the polyphenol had no effect on IDE's activity toward insulin. (**c**) Spectra recorded for insulin digests in the presence of EGCG showed that the helical signals of insulin persist, indicating that EGCG inhibits insulin proteolysis by IDE. The insulin concentration in all samples was set at 20 µM in 50 mM Tris buffer (pH 7.4). The substrate-to-enzyme molar ratio and the polyphenol-to-insulin molar ratio were set to 100:1 and 2:1, respectively. All samples were kept at 37 ◦C in between spectral acquisition.

We also used reversed phase HPLC to monitor the proteolytic activity of IDE toward insulin over the same incubation periods used in the digestions that yielded the CD spectra shown in Figure 4. Representative chromatograms of control samples are shown in Figure 5a. As the digestion time increased, the intensity of the insulin peak decreased

and peaks at shorter retention times appeared due to the production of insulin fragments. Digestion is complete within 24 h. Figure 5b presents chromatograms of the digests in the presence of resveratrol. With the exception of the peak for resveratrol, the chromatograms were similar to those of the control samples. Insulin degradation was also complete within 24 h. Figure 5c shows the chromatograms of the digests in the presence of EGCG. In contrast to the corresponding chromatograms in Figure 5a,b, the chromatograms of the 24- and 48-h digests showed the presence of the insulin peak indicating inhibition of IDE-dependent degradation. The intensity of the insulin peak, however, decreased with time, suggesting that the inhibition of IDE was not complete. The intensity of the peak for EGCG decreased with incubation time, presumably because it epimerizes to gallocatechin-3-gallate and/or dimerizes to form digallate dimers [22,33]. Our cumulative CD (Figure 4) and RP HPLC (Figure 5) results clearly demonstrate that resveratrol has no effect on IDE's activity toward insulin. EGCG, on the other hand, partially inhibits IDE.

Next, the steady-state kinetic parameters for the IDE-dependent degradation of insulin in the absence and presence of polyphenols were determined using insulin's helical CD ellipticity at 222 nm ([*θ*obs(222nm)]). Because our analysis was limited to 5 min of digestion time, complications due to the epimerization and oxidation of EGCG were circumvented. Figures S1a and S2a present representative real-time ellipticity data obtained from proteolysis experiments in the presence of resveratrol and EGCG, respectively. As digestion proceeded, [*θ*obs(222nm)] became less negative indicating loss of helicity. We calculated the amount of digested insulin [DI]*<sup>t</sup>* using Equation (1) and these were plotted against digestion time (Figures S1b and S2b). Linear regression analysis yielded *V*0, the initial velocity (or initial rate) of insulin proteolysis. Figure 6a,b present the resulting Michaelis–Menten plots for the digestions carried out in the presence of resveratrol and EGCG, respectively, at polyphenol concentrations of 0 (control), 20, and 40 µM. Lineweaver–Burk and Hanes–Woolf plots for the digestions in the presence of resveratrol and EGCG are shown in Figures S3 and S4, respectively. The Michaelis–Menten (Figure 6a), Lineweaver–Burk (Figure S3), and Hanes– Woolf (Figure S3) plots for resveratrol are invariant to concentration, indicating that the polyphenol has no effect on IDE's activity. In sharp contrast, the corresponding plots for EGCG clearly indicate that IDE's activity decreased as the concentration of the polyphenol increased (Figure 6b and Figure S4). Table 1 summarizes the kinetic constants obtained from the Michaelis–Menten plots. Similar values were obtained from the Lineweaver–Burk (Table S1) and Hanes–Woolf (Table S2) plots. IDE's catalytic efficiency, as given by *kcat/KM*, was not affected by resveratrol. In sharp contrast, *kcat/K<sup>M</sup>* decreased by 54 and 67% relative to the control in the presence of 20 and 40 µM of EGCG, respectively. The Lineweaver–Burk plots (Figure S4a) show straight lines with different slopes and a common intercept that is not on the 1/*V*<sup>0</sup> axis, suggesting mixed inhibition by EGCG. Mixed inhibitors bind at a site separate from the enzyme's active site, but may bind to either the enzyme or enzyme-substrate complex [34]. A schematic representation of the mixed inhibition by IDE is shown in Scheme 1. θ θ

IDE + Insulin IDE • Insulin •EGCG IDE + P + + EGCG EGCG IDE • EGCG + Insulin IDE • Insulin

**Scheme 1.** Schematic representation of the mixed inhibition of IDE-dependent degradation of insulin by EGCG. EGCG binds to either IDE or IDE•insulin complex, leading to a decrease in catalytic activity.

**Figure 5.** RP HPLC analysis of IDE-dependent degradation of insulin in the absence and presence of polyphenols at ◦C. (**a**) Chromatograms of insulin digests in the absence of polyphenols (control samples) show the progressive loss of the insulin peak over the digestion period. This loss is accompanied by new peaks at shorter retention times due to the formation of insulin fragments. The chromatogram recorded for the 24-hr digest shows the absence of the insulin peak, indicating complete degradation of insulin. (**b**) Chromatograms of insulin digests in the presence of resveratrol are similar to the chromatograms in (**a**) with the exception of the peak for the polyphenol at a retention time of 7.5 min. Digestion of insulin is complete within 24 h. (**c**) Chromatograms of insulin digests in the presence of EGCG show that the digestion of insulin was not complete, even after 48 h of digestion. The intensity of the insulin peak, however, decreased with digestion time, suggesting that the inhibition by EGCG was partial and not complete. The intensity of the peak for EGCG decreased with incubation time presumably because of epimerization and/or dimerization.

**Figure 6.** Kinetics of IDE-dependent degradation of insulin in the absence and presence of polyphenols at 37 ◦C. (**a**) Michaelis– Menten plots for insulin digests in the absence and presence of resveratrol were similar to one another, indicating that the polyphenol has no effect on IDE's activity toward insulin. (**b**) Corresponding plots for the digestions in the absence and presence of EGCG clearly show that IDE's activity decreased as the concentration of EGCG increased. Plots for polyphenol concentrations of 0, 20, and 40 µM are shown in green, red, and blue, respectively. Each data point represents the mean from three trials. The solid lines are fits to the Michaelis–Menten equation.


**− − − Table 1.** Steady-state kinetic parameters for the IDE-dependent proteolysis of insulin determined from Michaelis–Menten plots.

\* Control experiments to test the effect of ethanol that was used to dissolve resveratrol.

In vivo studies on animals have identified beneficial effects of EGCG including decreased adipose mass [35], reduction in body weight [36], and improvement in glucose homeostasis [35–37]. The effects of EGCG on glucose metabolism associated with T2D have been reported to be mediated in several ways including decreased gluconeogenesis [38], increased insulin sensitivity [39], and increased glucose uptake in skeletal muscle [40], which is an important regulator of glucose homeostasis [41]. The direct molecular targets of EGCG in vivo are not known. This work shows that EGCG directly targets IDE, and in doing so, the IDE-dependent degradation of insulin is inhibited, providing molecular basis for the improvement in glucose homeostasis observed in the in vivo studies.

How might our resveratrol results be used in the development of therapeutic and/or preventive strategies for AD? Recently, we showed that resveratrol sustains IDE activity toward Aβ42 monomer [25]. This conclusion, together with this work's finding that resveratrol does not affect IDE's activity toward insulin, has an important implication. Given that insulin and Aβ42 are IDE's most physiologically important and most pathologically

significant substrates in the brain, respectively, resveratrol is an IDE substrate-selective activator. Resveratrol's selectivity toward Aβ42 and its ability to cross the blood–brain barrier [24,42,43] suggest that this polyphenol can potentially act as an ideal naturallyoccurring molecule for an IDE-centric therapeutic for AD.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/antiox10091342/s1, Figure S1: Early-stage kinetics of IDE-dependent degradation of insulin in the absence and presence of resveratrol using insulin's observed ellipticity at 222 nm [*θ*obs(222nm)], Figure S2: Early-stage kinetics of IDE-dependent degradation of insulin in the absence and presence of EGCG using observed ellipticity at 222 nm [*θ*obs(222nm)], Figure S3: Kinetics of IDE-dependent degradation of insulin in the absence and presence of resveratrol at 37 ◦C, Figure S4: Kinetics of IDE-dependent degradation of insulin in the absence and presence of EGCG at 37 ◦C, Table S1: Steady-state kinetic parameters of the IDE-dependent proteolysis of insulin determined from Lineweaver–Burk plots, Table S2: Steady-state kinetic parameters of IDE-dependent proteolysis of insulin determined from Hanes–Woolf plots.

**Author Contributions:** Conceptualization and overall direction of the project, N.D.L.; Circular dichroism and kinetic studies, Q.Z., B.L., S.I., L.A.W., C.A.K., V.A.I., M.M.K., RP HPLC, M.T.K. and C.E.J.; IDE production, B.L., V.A.I., C.A.K. and D.E.S.; Writing—original draft preparation, Q.Z., M.T.K. and N.D.L.; Writing—review and editing, all authors. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Institute on Aging through R15AG055043 to N.D.L.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article.

**Acknowledgments:** The authors thank Malcolm A. Leissring of the University of California at Irvine for providing the bacterial expression vector encoding IDE fused to GST.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


## *Article* **Central Administration of Ampelopsin A Isolated from** *Vitis vinifera* **Ameliorates Cognitive and Memory Function in a Scopolamine-Induced Dementia Model**

**Yuni Hong 1,2,†, Yun-Hyeok Choi 3,†, Young-Eun Han <sup>1</sup> , Soo-Jin Oh 1,4, Ansoo Lee 1,2, Bonggi Lee <sup>5</sup> , Rebecca Magnan <sup>6</sup> , Shi Yong Ryu <sup>7</sup> , Chun Whan Choi 3,\* and Min Soo Kim 1,2,\***


**Abstract:** Neurodegenerative diseases are characterized by the progressive degeneration of the function of the central nervous system or peripheral nervous system and the decline of cognition and memory abilities. The dysfunctions of the cognitive and memory battery are closely related to inhibitions of neurotrophic factor (BDNF) and brain-derived cAMP response element-binding protein (CREB) to associate with the cholinergic system and long-term potentiation. *Vitis vinifera*, the common grapevine, is viewed as the important dietary source of stilbenoids, particularly the widelystudied monomeric resveratrol to be used as a natural compound with wide-ranging therapeutic benefits on neurodegenerative diseases. Here we found that ampelopsin A is a major compound in *V. vinifera* and it has neuroprotective effects on experimental animals. Bath application of ampelopsin A (10 ng/µL) restores the long-term potentiation (LTP) impairment induced by scopolamine (100 µM) in hippocampal CA3-CA1 synapses. Based on these results, we administered the ampelopsin A (10 ng/µL, three times a week) into the third ventricle of the brain in C57BL/6 mice for a month. Chronic administration of ampelopsin A into the brain ameliorated cognitive memory-behaviors in mice given scopolamine (0.8 mg/kg, i.p.). Studies of mice's hippocampi showed that the response of ampelopsin A was responsible for the restoration of the cholinergic deficits and molecular signal cascades via BDNF/CREB pathways. In conclusion, the central administration of ampelopsin A contributes to increasing neurocognitive and neuroprotective effects on intrinsic neuronal excitability and behaviors, partly through elevated BDNF/CREB-related signaling.

**Keywords:** ampelopsin A; *Vitis vinifera*; memory behavioral tests; long-term potentiation; CREB/ BDNF signals

## **1. Introduction**

Alzheimer's disease (AD) is the most common neurodegenerative disease with progressive memory loss and cognitive decline in the elderly [1]. The pathological hallmarks of AD include amyloid-β protein accumulation, tau protein aggregation, excessive oxidative stress, and cholinergic dysfunction [2]. Cholinergic circuits have been implicated in cognitive functioning, especially in hippocampus-dependent memory formation, through

**Citation:** Hong, Y.; Choi, Y.-H.; Han, Y.-E.; Oh, S.-J.; Lee, A.; Lee, B.; Magnan, R.; Ryu, S.Y.; Choi, C.W.; Kim, M.S. Central Administration of Ampelopsin A Isolated from *Vitis vinifera* Ameliorates Cognitive and Memory Function in a Scopolamine-Induced Dementia Model. *Antioxidants* **2021**, *10*, 835. https://doi.org/10.3390/ antiox10060835

Academic Editors: Rui F. M. Silva and Lea Pogaˇcnik

Received: 24 April 2021 Accepted: 19 May 2021 Published: 24 May 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/).

the modulation of hippocampal synaptic plasticity and transmission [3]. Several studies revealed that deficits in cholinergic signaling, including cholinergic neurons, acetylcholine (ACh), and its receptors were observed in the brain of AD patients [4]. Thus, acetylcholinesterase (AChE) inhibitors, such as donepezil, have become major therapeutic targets for AD treatment, by increasing the availability of ACh at cholinergic synapses within a short period [5]. However, the benefits of current treatments remain controversial due to their lack of efficacy and critical side-effect for long-term use [6].

Scopolamine is a competitive antagonist of ACh at muscarinic receptors which are the main factors underlying the learning process and memory formation by regulating hippocampal synaptic plasticity [7,8]. The scopolamine-induced memory impairment has been widely used as an experimental animal model for the screening of novel therapeutics in AD [9]. Furthermore, the scopolamine appears to be associated with a significant reduction in the expression of brain-derived neurotrophic factor (BDNF) and cAMP-response element-binding protein (CREB) coupled with BDNF activation in the hippocampus [10]. CREB modulates memory formation, consolidation, and long-term memory persistence by positively controlling BDNF expression in the hippocampus [11,12]. Thus, the CREB/BDNF signaling pathways have been suggested as a potential target for the prevention of AD [13].

*V. vinifera*, the common grapevine, is viewed as the important dietary source of stilbenoids, particularly the widely-studied monomeric resveratrol [14]. Resveratrol has emerged as a natural compound with wide-ranging therapeutic benefits on cancer, cardiovascular, inflammatory, metabolic, and neurodegenerative diseases [15]. Even though resveratrol can be naturally oligomerized to achieve enhanced bioactivity with better potency and selectivity, less attention has been paid to resveratrol oligomers [16,17]. Resveratrol oligomers such as the ampelopsin A have shown promise in the treatment of AD by the interference of neurodegenerative processes, including amyloid cascade, α-synuclein cascade, oxidative damage, and cytotoxicity [18–20]. Furthermore, some stilbenoids were assessed for their anti-AChE activity and appeared to be potent AChE inhibitors from natural sources [21–23]. Among stilbenoids from extracts of *V. vinifera*, resveratrol and ampelopsin A exhibited more potent anti-amyloidogenic activity than the others [24]. Despite these findings, there is limited evidence evaluating the in vivo neuropharmacological activities of ampelopsin A. Thus, we primarily focused on the anti-amnesic potential of ampelopsin A in the scopolamine-injected mice with memory impairment. The actions of ampelopsin A were further examined at the molecular level by assessing the activity of the cholinergic system as well as the expression of CREB/BDNF signals in the hippocampus.

#### **2. Materials and Methods**

#### *2.1. General Procedures and Plant Material*

Proton nuclear magnetic resonance (1H-NMR), and carbon nuclear magnetic resonance ( <sup>13</sup>C-NMR) spectra were performed on a Bruker (Rheinstetten, Germany) AM 300 NMR spectrometer using TMS as an internal standard. Column chromatography was conducted using a Silica gel 60 (70~230 mesh, Merck KGaA, Darmstadt, Germany), ODS-A (12 nm S-7 µm, YMC GEL, Kyoto, Japan), and Preparative HPLC was performed on LC-8A (Shimadzu, Kyoto, Japan). Thin-layer chromatography analysis was performed on Silica gel 60 F<sup>254</sup> (Merck KGaA, Darmstadt, Germany) and spots were detected under a UV lamp followed by a 10% H2SO<sup>4</sup> reagent. The stem bark of *V. vinifera* was harvested in October 2020 from the vineyard, Hwaseong-si, Gyeonggido, Korea. A voucher specimen (G095) was deposited at the Bio-center, Gyeonggi Institute of Science and Technology Promotion, Suwon, South Korea.

#### *2.2. Spectroscopy of Isolated Ampelopsin A from the Stem Bark of V. vinifera*

Brown amorphous powder; <sup>1</sup>H NMR (300 MHz, acetone-*d*6) *δ*: 7.09 (2H, d, *J* = 8.8, H-2′ , 6′ ), 6.88 (2H, d, *J* = 8.3 Hz, H-2, -6), 6.75 (2H, d, *J* = 8.8 Hz, H-3′ , 5′ ), 6.64 (1H, d, *J* = 1.9 Hz, H-14), 6.62 (2H, d, *J* = 8.3 Hz, H-3, 5), 6.42 (1H, d, *J* = 2.5 Hz, H-12′ ), 6.21 (1H, br s, H-14′ ), 6.14 (1H, br d, *J* = 1.9 Hz, H-12), 5.45 (1H, d, *J* = 4.9 Hz, H-7), 5.42 (1H, br d,

*J* = 4.9 Hz, H-8), 5.42 (1H, d, *J* = 11.3 Hz, H-7′ ), 4.15 (1H, br d, *J* = 11.3 Hz, H-8′ ); <sup>13</sup>C NMR (75 MHz, acetone-*d*6) *δ*: 159.5 (C-9), 158.3 (C-11), 158.3 (C-13′ ), 157.9 (C-2′ , 6′ ), 156.7 (C-11′ ), 155.5 (C-9), 142.5 (C-9′ ), 139.8 (C-7), 132.0 (C-13), 130.3 (C-1), 129.3 (C-14), 129.3 (C-3, 5), 128.1 (C-2), 118.3 (C-8), 117.7 (C-10′ ), 115.4 (C-1′ ), 115.4 (C-4′ ), 114.9 (C-3), 109.9 (C-12), 104.9 (C-14′ ), 100.9 (C-12′ ), 96.5 (C-10), 87.8 (C-7′ ), 70.6 (C-6), 48.9 (C-8′ ), 43.3 (C-5). ESI-MS (positive ion mode): m/z 471 [M + H]<sup>+</sup> .

#### *2.3. Slice Preparation and Electrophysiology*

Young adult mice (C57BL/6J, age 5–6 weeks) were anesthetized with isoflurane. The brain was quickly removed and immersed in an ice-cold oxygenated high-magnesium artificial cerebral spinal fluid (aCSF) composed of (mM): 130 NaCl, 24 NaHCO3, 3.5 KCl, 1.25 NaH2PO4, 1 CaCl2, 3 MgCl2, and 10 glucose saturated with 95% O<sup>2</sup> and 5% CO2, at pH 7.4. The brain was attached to the stage of a vibratome (DSK Linear Slicer PRO 7, Dosaka EM, Kyoto, Japan) and 300 µm thickness of transverse slices were cut and recovered in an incubation chamber at room temperature for one hour before recording, in standard oxygenated aCSF composed of (mM): 130 NaCl, 24 NaHCO3, 3.5 KCl, 1.25 NaH2PO4, 1.5 CaCl2, 1.5 MgCl2, and 10 glucose saturated with 95% O<sup>2</sup> and 5% CO2, at pH 7.4. Slices were placed on the microscope stage and superfused with oxygenated aCSF at room temperature. Whole-cell patch recordings were obtained from CA1 pyramidal neurons in voltage-clamp configuration using a Multiclamp700b (Molecular Devices, Sunnyvale, CA, USA) and a borosilicate patch pipette of 5–7 MΩ resistance. The internal pipette solution for voltage-clamp recordings consisted of (mM): 140 Cs-MeSO4, 10 HEPES, 7 NaCl, 4 Mg-ATP, and 0.3 Na3-GTP with 1 mM QX314. All neurons included in this study have a resting membrane potential below −55 mV, had an access resistance in 10–20 MΩ, and showed only minimal variation during the recordings. Records were filtered at 2 kHz and digitized at 10 kHz using a Digidata1322A (Molecular Devices, CA, USA). The evoked excitatory postsynaptic current (eEPSCs) was recorded by applying 100 µs current injection (1–200 µA) to a bipolar stimulating electrode placed in the CA1 stratum radiatum of schaffer collateral pathway and analyzed using pCLAMP10 software (Axon Instruments, Burlingame, CA, USA). For LTP recordings, electrical stimulations were given as theta-burst stimulation, consisting of 3 trains containing 4 pulses 15 bursts (each with 4 pulses at 100 Hz) of stimuli delivered every 200 ms.

#### *2.4. Animals*

Male C57BL/6J mice (8 weeks old; 25–30 g) were purchased from Orient Bio Inc. (Seungnam, Korea). The animals were housed in a room with constant temperature (23 ± 1 ◦C) and humidity (50 ± 10%) under a 12 h light/dark cycle, and were fed with food and water ad libitum. The experimental procedure was approved by the Institutional Animal Care and Use Committee (IACUC Approval No. KIST-2020-014) and the Institutional Biosafety Committee (IBC), and was conducted in accordance with relevant guidelines and regulation of the IACUC and the IBC in the Korea Institute of Science and Technology (KIST).

#### *2.5. Surgical Procedure and Treatments*

After one week of acclimatization, mice underwent stereotaxic surgery for implantation of a cannula in the brain as previously described [25]. Using a stereotaxic frame (Kopf Instruments, Tujunga, CA, USA), a 26-gauge guide cannula (Plastics One, Roanoke, VA, USA) was inserted into the third-ventricle (3 V, coordinates: 2.0 mm posterior to the bregma, 5.3 mm below the surface of skull). Following a week recovery period, mice were randomly divided into three groups (*n* = 5 per group): control (Con, PBS as a vehicle), scopolamine + vehicle (Scop + Veh, PBS as a vehicle), and scopolamine+ampelopsin A (Scop + AmpA) pretreatment group. All groups were administrated three times a week with either 0.5 µL of phosphate-buffered saline (PBS, as a vehicle) or 0.5 µL ampelopsin A (10 ng/µL, dissolved in PBS) for one month. One month later, scopolamine + vehicle (Scop + Veh) and scopolamine + ampelopsin A (Scop + AmpA) pretreatment groups received 0.8 mg/kg scopolamine ((-)-scopolamine hydrobromide trihydrate, dissolved in 0.9% saline, i.p.) and the control group was injected with 0.9% saline (i.p.) before each behavioral test. The experimental schedule of chemical administration and behavioral tests is shown in Figure 3a.

#### *2.6. Behavioral Tests*

All behavioral tests were performed in the behavior testing room. An Anymaze video-tracking system (Stoelting) equipped with a digital camera connected to a computer was used. Following behavioral tests, open field (locomotion), novel object recognition, and passive avoidance were conducted. The study was carried out in compliance with the ARRIVE guidelines. During the behavioral tests, mice were centrally administered PBS or ampelopsin A before every behavioral test, including habituation and test session (60 min before). Subsequently, mice were treated (i.p.) with saline or scopolamine before every behavioral test (30 min before).

#### 2.6.1. Open Field Test

The open-field test (locomotion) was performed as previously described with slight modifications [26]. Specifically, the mouse was located in the center of an open field chamber (40 cm length × 40 cm width × 50 cm height) and was habituated for 20 min. Each mouse was replaced in the same chamber 24 h later. The movements of the mouse were recorded for 10 min and then analyzed via a digital camera connected to the Any-Maze animal tracking system software (Stoelting, Wood Dale, IL, USA). The total distance moved (meters) and the time (seconds) spent in the center/outer of the open field were measured.

#### 2.6.2. Novel Object Recognition Test

The novel object recognition test was performed as described in the previous study [27]. Specifically, the mouse was located in a square arena (40 cm length × 40 cm width × 50 cm height) equipped with a digital camera and was allowed to familiarize with the environment for 10 min before the test. During the first session (familiarization session), two identical objects were put against the center of the opposite wall and the mouse was allowed to explore the objects for 20 min. During the second session (test session), one of the identical objects was replaced by a novel object, and the mouse was allowed to explore the objects for 10 min. In the familiarization session, the mouse contacted with two yellow square-based pyramids(8 cm × 8 cm × 6.5 cm) while in the test session it was with a yellow cube (7 cm × 7 cm × 7 cm) and a yellow square-based pyramid. The amount of time that the mouse spent exploring each object was monitored and analyzed using an ANY-maze video-tracking system (Stoelting, USA). A discrimination index was calculated as (novel − familiar object exploration time)/(novel + familiar object exploration time).

#### 2.6.3. Passive Avoidance Test

The passive avoidance test was performed as previously described [28] using an Avoidance System (B.S Technolab INC., Seoul, Korea). The apparatus (48 cm length × 23 cm width × 28 cm height) consisted of light and dark chambers separated by a gate. On the first day, the mouse was allowed to explore both compartments freely for 10 min. On the following day (training), the mouse was placed in the light compartment and 60 s later the gate was opened. Once the mouse entered the dark compartment, the door was closed and an electrical foot shock (0.3 mA, 3 s) was delivered through the floor. After 24 h (probe trial), the mouse was placed again in the light compartment and then the gate was lifted 60 s later. The step-through latency, or time taken for the mouse to enter the dark compartment, was scored 300 s as the upper limit.

#### *2.7. ChAT Activity*

The Choline Acetyltransferase (ChAT) activity in the hippocampus was determined using ChAT Activity Assay Kit (Elabscience, Huston, TX, USA) according to the manufacturer's protocol. Absorbance at 324 nm was measured using a Tecan Infinite 200 microplate reader (Tecan, Männedorf, Switzerland). Enzyme activity was calculated using the following formula: Enzyme activity: (unit/mg protein) = [(∆A324)/ × 16.6]/(1.98 × 10−<sup>5</sup> nM−<sup>1</sup> cm−<sup>1</sup> × 24)/[protein concentration (mg/mL)]. Protein concentrations were assayed using a Quick Start Bradford Protein Assay kit (Bio-Rad, Hercules, CA, USA).

#### *2.8. Ach Level and AChE Activity*

The part of the hippocampus was homogenized on ice using RIPA buffer (Merck KGaA, Darmstadt, Germany) and the homogenates were centrifuged at 16,000× *g* for 20 min, then the supernatant was collected to analyze acetylcholine (Ach) level and acetylcholinesterase (AChE) activity using an Amplex Red Ach/AChE Assay Kit (Invitrogen, Waltham, MA, USA) in accordance with the manufacturer's protocol. Absorbance at 563 nm was measured using a Tecan Infinite 200 microplate reader (Tecan, Männedorf, Switzerland). Hippocampal Ach level and AChE activity were calculated from a standard curve.

#### *2.9. Quantitative Real-Time PCR Analysis*

The total RNA from the hippocampus tissue was extracted using Trizol reagent (Invitrogen Life Technologies, Waltham, MA, USA) and cDNA synthesized using a SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions. Complementary DNA amplification was performed using Power SYBR Green PCR Master Mix kit (Applied Biosystems, Waltham, MA, USA) and primers with the following sequences: *Bdnf* (NM\_007540), 5′ -TCATACTTCGGTTGCATGA AGG-3′ and 5′ -AGACCTCTCGAACCTGCCC-3′ ; *TrkB* (NM\_001025074), 5′ -CTGGGGCTTA TGCCTGCTG-3′ and 5′ - AGGCTCAGTACACCAAATCCTA-3′ ; *Akt1* (NM\_001165894), 5′ - ATGAACGACGTAGCCATTGTG-3′ and 5′ -TTGTAGCCAATAAAGGTGCCAT-3′ ; *Creb1* (NM\_009952), 5′ -AGCAGCTCATGCAACATCATC-3′ and 5′ -AGTCCTTACAGGAAGACTG AACT-3′ ; *iNOS* (NM\_010927), 5′ -GGCAGCCTGTGAGACCTTTG-3′ and 5′ -TGCATTG GAAGTGAAGCGTTT-3′ ; *Chrm1* (NM\_001112697), 5′ -AGTGGCATTCATCGGGATCA-3′ and 5′ -CTTGAGCTCTGTGTTGACCTTGA-3′ ; *Ache*(NM\_009599), 5′ -AGAAAATATTGCAG CCTTTG-3′ and 5′ -CTGCAGGTCTTGAAAATCTC-3′ ; *CaMK2* (NM\_177407), 5′ -GAATCTGC CGTCTCTTGAA-3′ and 5′ -TCTCTTGCCACTATGTCTTC-3′ ; *Bcl2* (NM\_177410), 5′ -AGCTGC ACCTGACGCCCTT-3′ and 5′ -GTTCAGGTACTCAGTCATCCAC-3′ ; *Bax* (NM\_007527),5′ - CGGCGAATTGGAGATGAACTG-3′ and 5′ -GCAAAGTAGAAGAGGGCAACC-3′ ; *Actb* (NM\_007393), 5′ -GGCTGTATTCCCCTCCATCG-3′ and 5′ -CCAGTTGGTAACAATGCCATG T-3′ . The StepOne Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) for quantitative PCR (qPCR) was used for quantitative real-time PCR. PCR results were normalized to those of the control genes encoding β-actin (*Actb*).

#### *2.10. Western Blot Analysis*

The part of the hippocampus was homogenized on ice using RIPA buffer (Sigma, Germany) and the homogenates were centrifuged at 16,000× *g* for 20 min, then the supernatant was collected. The protein concentration was determined as mentioned above. 30 µg of proteins were separated by 10% polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, Burlington, MA, USA). After blocking in 5% skim milk, the membrane was incubated with rabbit anti-BDNF (1:800, Abcam, Cambridge, UK), rabbit anti-phospho CREB (pCREB; 1:2000, Abcam, Cambridge, UK), mouse anti-CREB (1:1000, Invitrogen, Waltham, MA, USA), and mouse anti- β-actin (1:1000, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 ◦C. The membranes were washed and incubated for 1 h with HRP conjugated anti-rabbit (Abcam, Cambridge, UK) or anti-mouse IgG antibody (Enzo Life Sciences, Farmingdale, NY, USA). The bands were visualized using Image Quant LAS 4000 (GE Healthcare, Chicago, IL, USA) with ECL reagent (Amersham, Little Chalfont, UK), and the intensity was quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).

#### *2.11. Statistical Analysis*

Experimental values were shown as mean ± standard error of the mean (S.E.M.) and evaluated with one-way ANOVA followed by Dunnett's test. The statistical analysis was performed using the GraphPad PRISM software (GraphPad Prism Software Inc., version 8, San Diego, CA, USA). *p*-values of <0.05 were deemed significant.

#### **3. Results**

#### *3.1. Isolation and Determination of Compound from the Stem Bark of V. vinifera*

The stem bark of *V. vinifera* (3.0 kg) was extracted twice with 15 L of 70% ethanol (EtOH) by two times at room temperature (each time for 2 days). After filtration with a cotton ball, the filtrate was combined and evaporated to dryness to give 221.4 g of dark syrupy extract. The extracts were suspended in distilled water and then partitioned CH2Cl<sup>2</sup> (5.0 L × 3), EtOAc (5.0 L × 3), and n-butanol (5.0 L × 3) to give CH2Cl<sup>2</sup> (69.1 g), EtOAc (140.3 g), n-butanol (1.5 g), and water-soluble fractions (2.1 g), (Figure 1a). The EtOAc soluble fraction was subjected to silica gel (2.0 kg) column (10 × 60 cm) chromatography, eluted with MeOH in CH2Cl<sup>2</sup> in a step-gradient manner from 1% to 50% to give six fractions (F1: 11.0 g, F2: 13.3 g, F3: 9.4 g, F4: 65.3 g, F5: 13.1 g, and F6: 36.7 g). Fraction F3 (9.4 g) was separated by MPLC chromatography that used gradient mixtures as eluents (F31–F38). F34 was also purified in a similar manner with RP-18 preparative HPLC eluted with MeOH in H2O (1% to 100%) in a stepwise gradient, which finally gave 8.2 mg of compound (Purity 97% in HPLC). The molecular formula of the compound was confirmed by Mass spectrum as C28H20O7, consisting of 19 degrees of unsaturation (Figure 1c). The <sup>1</sup>H-NMR spectrum of the compound indicated five pairs of peaks. Two pairs appeared at δ 6.88/6.62 and 7.09/6.75, each peak with characteristic *ortho* and *meta* couplings; Each peak has an integral value of two. They were assigned to the protons of two *para*-disubstituted aromatic rings, A and A′ . Two other pairs resonating at δ 6.64/6.14 and 6.42/6.21 were assigned as meta protons on two tetra substituted aromatic rings, B and B′ (Figure 1b). With the above data and comparison with literature [29], the compound was identified as ampelopsin A.

#### *3.2. Bath Application of Ampelopsin A Increases the Neuronal Excitability of Hippocampal Neurons*

Long-term potentiation (LTP) is a long-lasting increase of postsynaptic responses following electrical stimulation such as theta-burst stimulation (TBS) or a brief, highfrequency stimulation (HFS), leading to an enhancement in the strength of excitatory synaptic transmission and it is considered the generally studied form for examining the synaptic mechanism of learning and memory in the brain [30,31]. We examined whether the bath application of ampelopsin A to the brain rescues the scopolamine-induced deficit in hippocampal LTP. For the measurement of hippocampal LTP, a single dose of ampelopsin A (10 ng/µL) was applied to the hippocampal slices which were perfused with artificial cerebrospinal fluid (aCSF) containing either DMSO vehicle or scopolamine (100 µM) during the baseline recording and for an additional 20 min after LTP induction. In control mice, TBS (consisting of 3 trains containing 4 pulses 15 bursts) induces a robust increase in the percentage of normalized excitatory postsynaptic current (EPSC) (Figure 2a). Scopolamine treatment markedly decreased the mean of normalized EPSC relative to the control group (Figure 2b, *p* < 0.05). In the scopolamine and ampelopsin A combined treatment group, the mean of normalized EPSC significantly increased relative to the scopolamine only treatment group (*p* < 0.05).

**Figure 1.** Isolation and structural analysis of ampelopsin A isolated from *V. vinifera*. (**a**) Extraction and purification procedures of ampelopsin A from stembark of *V. vinifera* and its molecular structure. (**b**) <sup>1</sup>H-NMR and <sup>13</sup>C-NMR (300 MHz, acetone-*d*<sup>6</sup> ) spectrums of ampelopsin A from stembark of *V. vinifera*, (**c**) Mass spectrum of ampelopsin A from stembark of *V. vinifera*.

**Figure 2.** Protective effect of ampelopsin A in hippocampal LTP. A change in the EPSC slope was monitored following LTP induction by theta-burst stimulation (TBS) at SC-CA1 synapses in the hippocampus. The magnitude of LTP was quantified as an increase in the EPSC amplitude relative to the baseline. (**a**) Averaged traces of normalized EPSC amplitude in control, scopolamine (100 µM) with DMSO vehicle, and scopolamine with ampelopsin A (10 ng/µL) group (scale bars, 30 pA or 50 pA, 30 ms). Scopolamine with DMSO or ampelopsin A was treated during the baseline recording and for an additional 20 min after LTP induction by bath application. (**b**) Bar graph of the means for the normalized EPSC amplitude recorded last 5 min, calculated from the data in Figure 2a. Values are expressed as means ± SEM (*n* = 4). \* *p* < 0.05; one-way ANOVA with Kruskal-Wallis test with Dunnett's multiple comparisons test.

#### *3.3. Administration of Ampelopsin A into the 3 V Increased Cognitive Memory Behaviors*

Based on the rescue effects of ampelopin A in EPSC measurements, we tried to figure out whether central administration of ampleopsin A restores cognition and memory function of the animals which are impaired by scopolamine injection. We designed the experimental schedules (Figure 3a) and conducted stereotaxic surgery on animals which were implanted cannulas into the third ventricle of the brain. We applied the same dose of ampleopsin A (10 ng/µL) via a cannula into the third ventricle of the experimental animals for one month while other animals were applied the PBS as a vehicle in the same way. After this pre-treatment of ampelopsin A (AmpA) over one month, we injected scopolamine (Scop, 0.8 mg/kg, i.p.) into Scop + Veh (pre-treatments of a vehicle then injected the scopolamine) and Scop + AmpA (pre-treatment of ampelopsin A then injected the scopolamine) group before each behavioral test (30 min before).

' **Figure 3.** Central administration of ampelopsin A changes the locomotion. Male C57BL/6J mice (10 weeks old) were given 0.5 µL ampelopsin A (10 ng/µL, three times a week) or the same volume of the vehicle (PBS) via the third-ventricle of the brain for one month. 30 min before the behavioral tests, scopolamine (0.8 mg/kg, i.p.) was administered to each group; Scop + Veh (pre-treatment of PBS as a vehicle, scopolamine injection on the experimental day) and Scop + AmpA (pre-treatment of ampelopsin A, scopolamine injection on the experimental day) groups. The control group (Con) was pre-treated with the vehicle into 3 V then was injected PBS (i.p.) instead of scopolamine on the experimental day. (**a**) The schematic timeline of the experiments. (**b**) Total distance and (**c**) time spent exploring the center zone were measured by an open field test (locomotion). Values are expressed as means ± SEM (*n* = 5). \*\* *p* < 0.01, \*\*\* *p* < 0.001; one-way ANOVA with Dunnett's multiple comparisons test.

#### 3.3.1. Open Field Test

An open field test performed before other behavioral analyses ensured that scopolamine worked properly by using its anxiogenic [32] and locomotor stimulant properties [33]. In the open field test, the scopolamine-injected group (Scop + Veh) showed significantly increased total distance traveled by the mice and shortened the time spent in the center zone compared with the control group (Figure 3b, F(2,12) = 12.64 and Figure 3c, F(2,12) = 14.71, *p* < 0.001). In addition, no significant differences were observed between the Scop + Veh group and Scop + AmpA group. It reveals that ampelopsin A was not associated with anxiety and hyper locomotion caused by scopolamine.

#### 3.3.2. Novel Object Recognition Test

We conducted a novel object recognition test and passive avoidance test to confirm the restoration of cognition and memory abilities by the administration of ampelopsin Ain memory-impaired models. The time spent with the novel object divided by the total time devoted to exploring both objects, expressed as the discrimination index, was shortened in the Scop + Veh groups than the control group (Figure 4a). However, the Scop + AmpA group markedly increased the discrimination index, indicating that ampelopsin A treatment ameliorated scopolamine-induced recognition memory impairment (F(2,12) = 4.811, *p* < 0.05).

**Figure 4.** Central administration of ampelopsin A improves cognitive memory behaviors. Male C57BL/6J mice (10 weeks old) were given 0.5 µL ampelopsin A (10 ng/µL, three times a week) or the same volume of the vehicle (PBS) via the thirdventricle of the brain for one month. 30 min before the behavioral tests, scopolamine (0.8 mg/kg, i.p.) was administered to each group; Scop + Veh (pre-treatment of PBS as a vehicle, scopolamine injection on the experimental day) and Scop + AmpA (pre-treatment of ampelopsin A, scopolamine injection on the experimental day) groups. The control group (Con) was pre-treated with the vehicle into 3 V then was injected PBS (i.p.) instead of scopolamine on the experimental day. In the novel object recognition test (**a**), the discrimination index showed the percent time spent with the novel object. In the passive avoidance test (**b**), mice were trained that once the mouse entered the dark compartment, the door was closed, and an electrical foot shock (0.3 mA, 3 s) was delivered through the floor (training session). After 24 h, the moving time to a darkened chamber in a shock-motivated was recorded as a latency time in the test session. Values are expressed as means ± SEM (*n* = 5). \* *p* < 0.05, \*\* *p* < 0.01; one-way ANOVA with Dunnett's multiple comparisons test. 132

#### 3.3.3. Passive Avoidance Test

In the step-through passive avoidance test, during the training trial, step-through latency was statistically the same amongst all the groups (Figure 4b, F(2,12) = 0.8304, *p* = 0.459). The Scop + Veh group showed a significant decrease in step-through latency in comparison with the control group (F(2,12) = 8.671, *p* < 0.005). A significant increase of step-through latency was presented in the Scop + AmpA group, suggesting that ampelopsin A recovered scopolamine-induced memory impairment in the experimental animals. Interestingly, the Scop + AmpA group showed similar levels with the control group. It means that scopolamine impairments did not work on memory dysfunction by chronic treatments of ampelopsin A.

#### *3.4. Administration of Ampelopsin A Ameliorates Cholinergic Dysfunction*

To elucidate the possible molecular mechanisms of ampelopsin A, the levels of acetylcholine and the activities of choline acetyltransferase and acetylcholinesterase that are involved in the acetylcholine metabolism were measured. The hippocampi of mice given scopolamine significantly decreased acetylcholine (ACh) contents and the levels of choline acetyltransferase (ChAT) were reduced by the scopolamine injection. These levels of the ACh and ChAT were recovered in the Scop+AmpA groups (Figure 5a, F(2,9) = 4.602, *p* < 0.05 and Figure 5b, F(2,9) = 1.308). In contrast, the levels of acetylcholinesterase (AChE) activities were increased in the Scop + Veh groups but significantly decreased in the Scop + AmpA groups (Figure 5c, F(2,9) = 4.602, *p* < 0.05). We also measured gene expressions of muscarinic acetylcholine receptor (*Chrm1*, F(2,9) = 2.935) and the acetylcholinesterase (*Ache*, F(2,10) = 6.650, *p* < 0.05). These genes were also changed by the central administration of ampelopsin A (Figure 5d).

#### *3.5. Administration of Amplopsin A Elevates BDNF-Related Signaling in the Hippocampus*

To further elucidate the underlying molecular mechanisms of ampelopsin A, the mRNA and protein expression of CREB/BDNF-related signaling were determined. The CREB1 (F(2,9) = 11.30, *p* < 0.001), BDNF (F(2,9) = 5.912, *p* < 0.05), CaMK2 (F(2,9) = 4.285, *p* < 0.05), Akt (F(2,9) = 6.626, *p* < 0.05), and TrkB (F(2,10) = 7.323, *p* < 0.05) mRNA levels were significantly down-regulated by the Scop + Veh group compared with the control group but were up-regulated in the Scop + AmpA group (Figure 6a). Consistently, scopolamine injection decreased protein levels of BDNF and phosphorylation of CREB in the hippocampus, and the administration of ampelopsin A effectively increased BDNF and pCREB protein levels compared with the administration of scopolamine (Figure 6c, F(2,9) = 8.8, *p* < 0.01 and Figure 5d, F(2,9) = 4.772, *p* < 0.05).

#### *3.6. Antioxidant and Anti-Apoptotic Effects on the Hippocampus by Ampelopsin A*

Ampelopsin has been known to have antioxidant and anti-apoptotic activities [34,35]. We examined whether central administration of ampelopsin A is responsible for antioxidant and anti-apoptotic effects. The Scop + Veh group significantly increased the mRNA levels of iNOS compared with the control group. This increase was attenuated when ampelopsin A was administrated (Figure 6b, F(2,12) = 7.658, *p* < 0.01). We measured pro-apoptotic and anti-apoptotic effects by the treatments of ampelopsin A. The Scop + Veh group showed a significant increase of Bax (F(2,12) = 7.658, *p* < 0.01) as a pro-apoptotic marker, then it was attenuated by the treatment of ampelopsin A. In contrast, anti-apoptotic Bcl-2 expression altered its expression (F(2,10) = 6.662, *p* < 0.05) then was restored by the treatments of ampelopsin A. The Administration of ampelopsin A recovered apoptotic gene expression in scopolamine-injected mice. These antioxidant and anti-apoptotic effects also support neuroprotective effects of ampelopsin A administration.

– β ' **Figure 5.** Inhibitory effect of ampelopsin A against scopolamine-induced cholinergic dysfunction. Male C57BL/6J mice (10 weeks old) were given 0.5 µL ampelopsin A (10 ng/µL, three times a week) or the same volume of the vehicle (PBS) via the third-ventricle of the brain for one month. Mice were sacrificed and hippocampi were isolated for measurements of cholinergic parameters and mRNA expression. 30 min before the mice sacrifice, scopolamine (0.8 mg/kg, i.p.) was administered to each group; Scop + Veh (pre-treatment of PBS as a vehicle, scopolamine injection on the experimental day) and Scop + AmpA (pre-treatment of ampelopsin A, scopolamine injection on the experimental day) groups. The control group (Con) was pre-treated with the vehicle into 3 V then was injected PBS (i.p.) instead of scopolamine on the experimental day. (**a**–**c**) Acetylcholine levels and acetylcholinesterase and choline acetyltransferase activities in the hippocampus are shown. (**d**) *Chrm1* and *Ache* mRNA levels determined by real time-PCR. Gene expression was normalized to that of β-actin. Au means the arbitrary units. Values are expressed as means ± SEM (*n* = 4). \* *p* < 0.05, \*\* *p* < 0.01; one-way ANOVA with Dunnett's multiple comparisons test.

– β β ' **Figure 6.** Increase of BDNF-related and anti-apoptotic signaling by central administration of ampelopsin A. Male C57BL/6J mice (10 weeks old) were given 0.5 µL ampelopsin A (10 ng/µL, three times a week) or the same volume of the vehicle (PBS) via the third-ventricle of the brain for one month. Mice were sacrificed and hippocampi were isolated for measurements of mRNA and protein expression. 30 min before the mice sacrifice, scopolamine (0.8 mg/kg, i.p.) was administered to each group; Scop + Veh (pre-treatment of PBS as a vehicle, scopolamine injection on the experimental day) and Scop + AmpA (pre-treatment of ampelopsin A, scopolamine injection on the experimental day) groups. The control group (Con) was pre-treated with the vehicle into 3 V then was injected PBS (i.p.) instead of scopolamine on the experimental day. (**a**) Alterations in the expression of *Bdnf*, *Creb1*, *CaMK2*, *Akt*, and *Trkb* were determined by real time-PCR (*n* = 4). (**b**) *Bcl2*, *Bax*, and *iNOS* mRNA levels determined by real time-PCR (*n* = 4–5). Gene expression was normalized to that of β-actin. Au means the arbitrary units. (**c**) Quantification of BDNF/β-actin and (**d**) phosphorylated CREB/CREB intensity (*n* = 4). Values are expressed as means ± SEM. \* *p* < 0.05, \*\* *p* < 0.01; one-way ANOVA with Dunnett's multiple comparisons test.

#### **4. Discussion**

Resveratrol (3,5,4-trihydroxystilbene) is a naturally occurring polyphenol that has attracted the attention of many chemists and pharmacologists due to its diverse biological activities such as chemopreventive, antimicrobial, antioxidant, and anti-inflammatory actions [36–39]. Grapevine is known as an important source of resveratrol and many resveratrol derivatives [40]. The previous other studies showed that extracts, resveratrols, from *V. vinifera* stembark protected the brain cell dysfunction by inhibiting the aggregation of amyloid-β and against α-synuclein cytotoxicity [18–20]. Among these extracts, a dimer of resveratrol from *V. vinifera*, ampelopsin A, exhibited more potent anti-amyloidogenic activity than the others [24]. However, it was questionable whether ampelopsin A works on cognitive

function for neuroprotective activities in the animal models. Based on the current study, it is quite clear that the brain administration (3 V) of ampleopsin A significantly improved cognitive behaviors, enhanced synaptic transmission, and the cholinergic system in scopolamine-induced memory dysfunction. The underlying mechanisms include but are not limited to the broad up-regulation of genes associated with CREB-BDNF signaling pathways.

In this study, we administrated the relatively low dose of ampelopsin A (10 ng) into the mice brain compared to other references' uses (µg or mg) [41,42]. The brain responded to this low dose of ampelopsin A to initiate neuroprotective effects in cognition and memory. In addition, we found that chronic administration of ampelopsin A efficiently improved cognition and memory functions whereas acute administration of ampelopsin A did not improve these functions in the experimental animals (data not shown). Usually, chronic treatments were considered as over 10 days to 12 weeks [41,43,44], and similar central treatments for one month showed increases in memory functions [45]. To do so, chronic (a month) and low-dose treatments of ampelopsin A contribute to a change of cognitive and memory abilities.

Cholinesterase (ChE) contributes to the short half-life of released ACh, and it terminates cholinergic neurotransmission by the hydrolysis of ACh in turn. The inhibition of ChEs expression slows down the breakdown of ACh, thereby prolonging ACh presence at synaptic cleft to stimulate their muscarinic receptors. Based on these facts, two major ChEs, AChE and butyrylcholinesterase (BuChE), have been potential targets in AD therapy [46,47]. BuChE is considered to play supportive role in the brain because AChE predominates over BuChE activity [48]. BuChE is also distributed in the hippocampus, but at lower levels than AChE which is mainly located in the synaptic cleft and synaptic membranes in normal status [49,50]. Since our study is the first study suggesting the ampelopsin A as a ChE inhibitor, we focused on the inhibitory activity of AChE as the hippocampal cholinergic mediation. As BuChE has brought much attention compensating for the action of AChE in cognitive impairment, further studies will establish the detailed influence of stilbenoids on BuChE for a beneficial feature in AD treatment [51–53].

Scopolamine, a muscarinic acetylcholine receptor antagonist, is a commonly used chemical that impairs learning and memory in animal models. Scopolamine-induced deficits in a battery of cognitive function are important for comparison of sensitivity and specificity to find therapeutic candidates for neurodegenerative diseases [54,55]. The exact mechanism of scopolamine action to ACh, ChAT, and ChE remains poorly understood. Since scopolamine has been used in the standard cognitive impairment model, there were a lot of literatures to show that the effects of scopolamine treatment can induce cognitive deficit through decreasing ACh contents and ChAT activities while increasing AChE activities in the hippocampus [10,56,57]. The stilbenoids, including ampelopsin A, have been studied as the potent AChE inhibitors for developing AD-targeting drugs [21–23]. Ampelopsin A may be considered as an AD-targeting drug by its anti-AChE activity [24]. In addition, this cholinergic system contributes to neurogenesis in the hippocampus via the CREB/BDNF signaling which is responsible for long-term memory formation [58,59]. Based on our study, administration of ampelopsin A delayed deficit of cholinergic cognitive memory and ameliorated long-term memory by restoring CREB/BDNF signaling. Therefore, ampelopsin A might be considered a strong candidate for treating AD to recover acetylcholine cascades in the hippocampus with reduced symptoms [10,60,61].

The avoidance reaction of an experimental mouse is important for the acquisition of extinction memory. In the passive avoidance test, the animal learns to avoid an unpleasant stimulus by hindering locomotion and investigation [62]. Additionally, treatments with an anti-BDNF antibody or BDNF antisense mRNA produce memory dysfunction in concurrence with a loss of LTP and ERK signaling [62–64]. To do so, the hippocampal BDNF-TrkB signaling is required for the acquisition and consolidation of conditioned fear [65,66]. In addition, hippocampus-specific deletion of BDNF lessens fear extinction, while hippocampal BDNF accelerates the acquisition of extinction memory [67]. BDNF is one of the crucial factors to form fear extinction memory [62,67,68]. Our study showed

that ampelopsin A significantly increased the avoidance reaction of experimental mice and up-regulated hippocampal BDNF/CREB cascades, including BDNF, CREB1, CaMK2, Akt, and TrkB. Although cognition and memory functional mechanisms mediated by each gene may be different, these genes are closely associated with avoiding aversive stimulus in the memory regions. We assumed that ampleopsin A may stimulate BDNF-CREB signaling in the hippocampus for increases of memory function although more detailed experiments are necessary. In addition, scopolamine induces an increase in neuro-inflammation (iNOS) and apoptosis (Bax) while it inhibits anti-apoptotic factors (Bcl-2) [69]. Our study showed that ampelopsin A has neuroprotective effects by reversing molecular and cell damages released from neuroinflammation and apoptosis.

Long-term potentiation (LTP) represents a long-lasting increase in the efficacy of excitatory synaptic transmission, and it is widely used to measure a cellular mechanism of learning and memory in the brain [30,31]. Among all neurotransmitters and trophic factors, BDNF and glutamate are mostly related to memory function [62]. BDNF directly works on depolarizing neurons by enhancing glutamatergic transmission for inducing phosphorylation of NMDA receptor through its TrkB receptors [70]. The BDNF utilizes positive regulations on LTP in memory formation at the cellular level. In addition, impairment of LTP in mutant mice lacking BDNF was rescued by recombinant BDNF application [71]. The endogenous BDNF is necessary for LTP formation which comes out from presynaptic neurons and BDNF-dependent LTP formation is responsible for protein synthesis [71,72]. The BDNF mediates the translation of protein synthesis via several intracellular signaling pathways including Akt and PI3K, kinases involved in cell growth, survival, differentiation, and intracellular trafficking. Our study showed that the chronic administration of ampelopsin A into the brain rescues the scopolamine-induced deficit in hippocampal LTP through BDNF activation. The recovered capability of LTP in the brain is important for brain protection in neurodegenerative diseases. Chronic administration of ampelopsin A might be considered a therapy for neurodegenerative disease by recovering functional LTP in the brain.

#### **5. Conclusions**

The central administration of ampelopsin A ameliorates scopolamine-induced cognitive impairment in the brain. These effects of ampelopsin A might be related to restored LTP through BDNF activation.

**Author Contributions:** Y.H. co-designed and performed mice surgery, treatments, qPCR, behavioral tests, cholinergic experiments and prepared all figures; Y.-H.C. did LC/MS and NMR, and A.L. conducted data analysis of LC/MS and NMR; Y.-E.H. and S.-J.O. performed electrophysiology and data analysis; R.M. performed preliminary experiments about AmpA's central treatments; B.L., S.Y.R., C.W.C., and M.S.K. performed all data analysis and wrote the paper; C.W.C. and M.S.K. conceived of the hypothesis and designed the project, and all authors participated in discussions. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Bio and Medical Technology Development Program of National Research Foundation (NRF) funded by the Ministry of Science and ICT (NRF-2020M3A9D8039920 and NRF-2018M3C7A1056897).

**Institutional Review Board Statement:** The experimental procedure was approved by the Institutional Animal Care and Use Committee (IACUC Approval No. KIST-2020-014) and the Institutional Biosafety Committee (IBC), and was conducted in accordance with relevant guidelines and regulation of the IACUC and the IBC in the Korea Institute of Science and Technology (KIST).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All datasets of this study are generated in the article.

**Conflicts of Interest:** The authors declare that they have no conflicts of interest with the contents of this article.

#### **References**


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