Almond Skin Extracts and Chlorogenic Acid Delay Replicative Aging by Enhanced Oxidative Stress Response Involving SIR2 and SOD2 in Yeast

: Almond ( Prunus dulcis (Mill.) D.A.Webb) is one of the largest nut crops in the world. Recently, phenolic compounds, mostly stored in almond skin, have been associated with much of the health-promoting behavior associated with their intake. The almond skin enriched fraction obtained from cold-pressed oil residues of the endemic Moroccan Beldi ecotypes is particularly rich in chlorogenic acid. In this study, both almond skin extract (AE) and chlorogenic acid (CHL) supplements, similar to traditional positive control resveratrol, significantly increased the replicative life-span of yeast compared to the untreated group. Our results showed that AE and CHL significantly reduced the production of reactive oxygen and nitrogen species (ROS/RNS), most likely due to their ability to maintain mitochondrial function during aging, as indicated by the maintenance of normal mitochondrial membrane potential in treated groups. This may be associated with the observed activation of the anti-oxidative stress response in treated yeast, which results in activation at both gene expression and enzymatic activity levels for SOD2 and SIR2, the latter being an upstream inducer of SOD2 expression. Interestingly, the differential gene expression induction of mitochondrial SOD2 gene at the expense of the cytosolic SOD1 gene confirms the key role of mitochondrial function in this regulation. Furthermore, AE and CHL have contributed to the survival of yeast under UV-C-induced oxidative stress, by reducing the development of ROS / RNS, resulting in a significant reduction in cellular oxidative damage as evidenced by decreased membrane lipid peroxidation, protein carbonyl content and 8-oxo-guanine formation in DNA. Together, these results demonstrate the interest of AE and CHL as new regulators in the replicative life-span and control of the oxidative stress response of yeast. mechanisms of action at the molecular investigated.


Introduction
Intakes of fruit, vegetables, seeds, and nuts have been associated with lower risks of chronic and age-related degenerative diseases [1][2][3]. Diverse phytochemicals (i.e., plant non-nutrient compounds) such as carotenoids, phenolics or flavonoids have been related to these protective effects. Particularly, due to their numerous health benefits, interest in the consumption of nuts as a food rich in healthy nutrients has increased in recent decades, and today, almonds are among the most popular nut trees [3]. In addition to the beneficial action attributed to their specific lipid profile, almond skin is also a rich source of antioxidant phenolic compounds that have also attracted attention in recent years [3,4]. Interestingly, after cold-pressed oil extraction, most of the antioxidant phenolic compounds accumulated in the almond skin are retained in a skin-enriched by-product, the so-called coldpressed oil residue, which makes it an attractive raw material for the extraction and recovery of these natural antioxidant phenolics [3][4][5][6]. Morocco, the world's fourth-largest producer, has produced more than 100,000 tons of unshelled almonds in recent years. Many native almond trees (local ecotypes called Beldi) are still grown in the Eastern Morocco region [7,8]. These local almond ecotypes are known to accumulate higher levels of tocopherol and phenolic antioxidants compared to other varieties grown in the same region [6,8]. In the Eastern region of Morocco, almond trees occupy a surface of 26,000 ha producing ca 15% of the national production out of almonds. This production generates an important part of the by-products, in particular those residues of cold pressed almond oil, enriched with antioxidant phenolic compounds. Recently, an ultrasound-assisted extraction method was developed to obtain an almond extract from Beldi ecotypes rich in chlorogenic acid and other related phenolic acids [6].
Aging is a complex biological process involving multiple actors and controlled by a variety of genetic and/or environmental factors [9]. A variety of hypotheses have been suggested to explain the mechanism of aging, including the theory free radicals of aging proposed by Pr Harman in 1956 [10,11], which was certainly the most widely studied. This theory continues to be revised, and so far, it remains a sound theory for the aging process [9,10]. The theory explains that aging may be caused by the cumulative oxidative stress, leading to oxidative damage to various macromolecules (membrane lipids, proteins, DNA) within the cell, which may lead to cell death and possibly to the death of the organism [10]. This theory therefore suggests that antioxidants capable of scavenging ROS (reactive oxygen species) and/or RNS (reactive nitrogen species) are effective in delaying the aging process. Studies have shown, in good agreement, that various antioxidants of plant origin, in particular polyphenols, could have a therapeutic potential for aging and age-related diseases [12][13][14][15]. Evidence that phytochemicals such as resveratrol and quercetin have extended the lifespan of different models, acting through a well-conserved mechanism, has been identified, first in yeast and then confirmed in other models such Caenorhabditis elegans, Drosophila melanogaster and mice [12,16,17]. Yeast have emerged as an effective tool to identify anti-aging compounds [18,19]. This is not surprising given the high level of gene conservation and aging mechanism between the yeast and humans [18,20], as well as the successful identification of the candidate anti-aging test substance after their evaluation in yeast [12][13][14][15]18].
Resveratrol (RES) is the most studied anti-aging plant polyphenol, first reported to delay or mitigate aging in yeast by activation of Sir2 (silent information regulator 2) [12]. REV action on sirtuins (conserved orthologs of Sir2) has been confirmed in other models [16,17]. Sirtuins are a conserved family of nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylases, and interestingly some compelling evidence has linked their action to ROS and aging, in particular to the ROS-driven mitochondria-mediated hormetic response [21]. Other potential anti-aging plant polyphenols and their mechanisms of action at the molecular level should be investigated. Such studies may provide important information for the use and development of anti-aging plant sources and derived compounds, and may reveal mechanisms to pave the way for anti-aging drugs development. The present study demonstrated the impact of an extract from almond skin and its main component, chlorogenic acid, on the lifespan extension in yeast and described their actions on oxidative stress.

Chemicals
All solvents used in this study were of an analytical grade (Thermo Scientific, Illkirch, France). Chlorogenic acid (CHL) and resveratrol (RES) standard was purchased from LGC Standard (Molsheim, France). Other chemicals and reagents have been purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France).

Almond extract preparation
Almond oil residues were obtained from Moroccan almonds (Beldi local ecotypes) grown in Ain Sfa (34°46'42.4" N, 002°09'28.9' W), a pilot site located in Eastern Morocco. Almond trees grown under the conditions described by Melhaoui et al. [8]. The almonds were then crushed using an oil screw press (KOMET DD85G, IBG Monforts Oekotec GmbH & Co. KG, Monchengladbach, Germany) and the resulting residue was ground to approximately 100-150 μm of particulate using a blender equipped with rotating blades (Grindomix GM 200 blender, Retsch France, Eragny, France). This material was further subjected to an optimized and validated ultrasound-assisted extraction protocol [6]. Almond skin extract (AE) was obtained by ultrasound-assisted extraction completed in an ultrasonic bath (USC1200TH, Prolabo, Sion, Switzerland) consisting of a tank with an internal dimension of 300x 240x 200 mm with an electrical power of 400W corresponding to an acoustic power of 1W/cm 2 and a maximum heating power of 400W. Sample was placed in 50-mL quartz tubes equipped with a vapor condenser and was suspended in 10 ml of aqueous EtOH 53.0% (v/v) as extraction solvent using a liquid-to-solid ratio of 10:1 mL/g DW (dry weight). During extraction, optimized and validated conditions -with a US frequency of 27.0 kHz during 29.4 min and an extraction temperature of 45 ° C -were applied [6]. The similar extraction protocol was also used to quantify CHL and RES in yeast cells and the culture medium.

HPLC analysis
After extraction, each extract was centrifuged at 3,000 rpm for 15 min and the resultant supernatant was filtered with a syringe filter (0.45 µm, Millipore, Molsheim, France) prior to HPLC analysis. Separation was done by HPLC (High-Performance Liquid Chromatography) by the use of a complete Varian HPLC system consisting of: Prostar 230 pump, Metachem Degasit, Prostar 410 autosampler, Prostar 335 Photodiode Array Detector (PAD) and driven by Galaxie version 1.9.3.2 software (Varian, Les Ulis, France). An RP18 column (Purospher RP-18; 250 x 4.0 mm; internal diameter: 5 µm; Merck Chemicals, Molsheim, France) was used for separation at a temperature of 35 °C. The mobile phase consisted of a mixture of 2 solvents A (HPLC grade water with 0.2 % (v / v) acetic acid) and B (HPLC grade methanol). The non-linear gradient applied for separation was: 8% B (0 min), 12% B (11 min), 30% B (17 min), 33% B (28 min), 100% B (30-35 min), 8% B (36 min) at a flow rate of 1 ml/min. A re-equilibrating time of 10 minutes was applied between each injection. Compound detection was set at 295 and 325 nm (corresponding to the λmax of the main compounds) [6].

Yeast culture conditions and life-span assay
Yeast strain DBY746 (MATα leu2-3,112 his3Δ1 trp1-289a ura3-52 GAI+) was used. The culture was initiated from frozen stock plated onto a YPD (yeast extract peptone dextrose) medium (Sigma-Aldrich, Saint-Quentin Fallavier, France). After incubation at 30 ° C for 2-3 days, a single colony was incubated into 1 mL of SDC (synthetic complete dextrose) medium [22] and incubated overnight with shaking (220 rpm) at 30 °C. The overnight culture was then diluted into approximately 10 mL of fresh SDC medium to an absorbance value of 600 nm of 0.1 and incubated with shaking (220 rpm) at 30 °C. This time point is considered to be day 0 of chronological aging. Yeast chronological life-span (CSL) assay in liquid culture was used as described by Hu et al. [22]. Briefly, starting on day 3, aliquots of 10 μL were removed from the flask, diluted 10,000 times in sterile water, 10 μL of diluted culture were placed on YPD plates, incubated at 30 °C for 2-3 days, and the colony forming unit (CFU) numbers were determined. The microcolonies formed on the YPD plates were observed under a microscope, and the daughter cells were quantified. The CFU number at day 3 is considered to be the 100% survival. CHL (5, 10 and 25 µM final concentrations) AE (ca 1 mg/mL dry extract corresponding to 25 µM of CHL final concentration), as well as positive control RES (10 µM final concentration) were dissolved in cell culture grade dimethyl sulfoxide (DMSO; Sigma-Aldrich, Saint-Quentin Fallavier, France). Final DMSO concentration was 0.1% (v/v). Control yeast was inoculated with the same DMSO concentration.

Reactive oxygen and nitrogen species measurement
The dihydrorhodamine-123 (DHR-123) fluorescent dye (Sigma-Aldrich, Saint-Quentin Fallavier, France) was used to determine the level of reactive oxygen and nitrogen species as described by Nazir et al. [23]. Approximately 10 8 yeast cells grown in the presence of AE, CHL or RES or DMSO (control cells) were washed twice with PBS, and then resuspended in PBS containing 0.4 μM DHR-123 and incubated during 10 min in the dark at 30 °C. After washing with PBS twice, the fluorescence signal (λex = 505 nm, λem = 535 332 nm) was detected using the VersaFluor Fluorimeter (Biorad, Marnesla-Coquette, France).

Enzymatic SIRT1/SIR2 and total SOD activities determinations
For proteins extraction, approximately 10 8 yeast cells have been washed 3 times with PBS. Then 1mL of PBS was added and the mixture was subjected to 3 freeze and thaw cycles using liquid nitrogen. The cell lysate was then centrifuged at 10,000 g at 4 °C for 15 min, and the supernatant was used to prepare the sample solution by dilution with PBS. Proteins were quantified using the Qubit Protein Assay Kit following the manufacturer's instructions and using the Qubit fluorimeter (Thermo Scientific, Illkirch, France).
Total SOD activity was measured using the Superoxide Dismutase Activity kit following the manufacturer's instructions (Thermo Scientific, Illkirch, France). SIRT1/SIR2 activity was determined using the SIRT1 Assay Kit (Sigma-Aldrich, Saint-Quentin Fallavier, France) following the manufacturer's instructions and using a Versafluor fluorimeter (Biorad, Marnes-la-Coquette, France).

UV-induced oxidative stress and survival (cell viability) to oxidative stress evaluation
UV-induced oxidative stress in yeast strain DBY746 (MATα leu2-3,112 his3Δ1 trp1-289a ura3-52 GAI+) grown on YPD medium was obtained as described by Nazir et al., [23]. Yeast cells were irradiated with 106.5 J/m 2 UV-C (254 nm) under a Vilber VL-6.C filtered lamp (Thermo Fisher Scientific, Villebon-sur-Yvette, France). After overnight incubation at 30 °C yeast cells were subjected to survival assay and further analyses. Survival was estimated by counting the colony forming unit as described in paragraph 2.4.

Membrane lipid peroxidation evaluation
The thiobarbituric acid (TBA; Sigma Aldrich, Saint-Quentin Fallavier, France) method described by Garros et al. [26] was used for the measurement of membrane lipid peroxide. Briefly, about 10 8 yeast cells ground in double distilled water were centrifuged for 10 min at 10,000 xg. The supernatant (75 μL) was mixed with 25 μL of SDS (3% (w/v)), 50 μL of TBA (3% (w/v) prepared in 50 mM of NaOH) and 50 µL of HCl (23% (v/v)). Mixing was performed between each addition. The final mixture was heated for 20 min at 80 ° C, cooled on ice, and the absorbance was measured at 532 nm (A532). Non-specific absorbance measured at 600 nm (A600) was subtracted. Standard curve was prepared using 1,1,3,3, tetramethoxypropane to measure concentrations of TBARS in the samples.

Protein carbonylation level estimation
Total proteins were extracted from about 10 8 yeast cells as described in paragraph 2.8. Protein carbonyl content was determined using Protein Carbonyl ELISA kit following the manufacturer's instructions (Cell BioLabs, Paris, France).

8-oxo-guanine level estimation
DNA was extracted from about 10 8 yeast cells with Yeast DNA Extraction Reagent Kit following manufacturer's instructions (Thermo Scientific, Illkirch, France) and 8-oxo-guanine content was determined with the 8-OHdG DNA Damage ELISA kit following the manufacturer's instructions (Cell BioLabs, Paris, France).

Statistical analysis
Means and standard deviations of at least 4 separate (biologically independent) replicates were used to present the data. Significant differences between groups in all experiments were determined by ANOVA, followed by two-tailed multiple t-tests with Bonferroni correction performed with XL-STAT 2019 biostatistics software (Addinsoft, Paris, France).

Yeast lifespan extension induced by almond skin extract chlorogenic acid is accompanied by a reduction of reactive oxygen/nitrogen species
In order to evaluate the potential anti-aging effects of almond extract and chlorogenic acid, we followed the chronological aging of wild type yeast (Saccharomyces cerevisiae, strain DBY746) previously used for this purpose [27]. This wild type strain has a short mean life-span of less than 7 days when aged in the SDC medium [27], which is advantageous for studying the impact of plant extracts on the life span of the yeast. High purity commercial chlorogenic acid (CHL) standard was used instead of purifying it to avoid purity problems and possible cross-contamination. CHL was tested at three different concentrations: 5, 10 and 25 μM. The almond skin extract (AE) was prepared from cold-pressed oil residues of a local Beldi ecotype from Eastern Morocco rich in chlorogenic acid following an optimized ultrasound-assisted extraction procedure [6]. This AE was used as a raw material to evaluate the potential anti-aging action of almond skin phenols, but also to detect possible synergistic or antagonistic effects with chlorogenic acid due to the presence of other phenolics in the extract. The HPLC chromatogram and phytochemical characterization of this extract are shown in Figures S1 and S1, respectively. In addition to chlorogenic acid, this AE also contained substantial amounts of protocatechuic acid, p-hydroxybenzoic acid and p-coumaric acid. The AE was tested at a final concentration of 1 mg/mL corresponding to a concentration of approximately 25 μM of chlorogenic acid corresponding to the highest evaluated CHL concentration. Prior to their application to yeast cells to assess their impact on life-span, the absence of any significant growth and viability effects of AE and CHL at their respective tested concentrations was evaluated in order to avoid any bias resulting from possible toxic or antifungal activities (Table S2).
The results of the replicative life-span test of the DBY746 strain of yeast are shown in Figure 1.  In our hands, the mean life-span of the CTL DBY746 yeast aged in the SDC was 5.84 ± 0.16 days. As expected, there were clearly positive effects of RES on the replicative DBY746 life-span raising it to 8.49 ± 0.17 days (Figure 1a). AE (7.44 ± 0.16 days) as well as CHL at doses of 10 (7.60 ± 0.17 days) and 25 (8.15 ± 0.08 days) µM significantly extended the replicative lifespan of the yeast strain ( Figure  1a). CHL at a dose of 5 μM was not sufficient to significantly affect the lifespan of the yeast strain (Figure 1a). Given the concentration of CHL present in EA, and although the almond extract has antiaging activity, these results may suggest the presence of antagonistic compounds in this extract, which may hinder the action of CHL. Interestingly, this result is consistent with previous observations that, although some supplements may be effective, the majority of evidence has shown that either simple or complex combinations of supplements are mostly ineffective in preventing the occurrence or progression of major causes of disease [32]. Here, AE increased the lifespan of yeast by 27.5 %, while a dose-dependent effect was observed for CHL and a concentration of 25 µM was necessary to reach a life-span extension similar to that observed with the RES positive control ( Figure  1a). The difference in concentration between the RES and the CHL required to achieve the same effect may result from a slightly lower CHL efficiency. However, it may also be the consequence of a lower absorption of CHL compared to RES. The bioavailability of a natural product is of paramount importance so that it can also effectively play its role in the cell. Absorption of RES has been reported to be highly effective in various models, including animal models and humans [33]. To have an idea about the absorption of RES vs. CHL, we measured their respective concentrations in the yeast cells as well as in the culture medium 6 hours after their addition. Here, six hours after their addition, RES was absorbed more effectively by yeasts than by CHL (Table S3). This could indicate that part of the difference observed for the dose levels needed to achieve a similar increase in lifespan for these two compounds is likely to be based on this difference in absorption. Few data are available on absorption kinetics in yeast and, more specifically, on transport proteins that could be involved in RES or CHL uptake. In view of its polyhydric alcohol nature, such as arabinose, bacterial arabinose-H + transport protein araE has been proposed to be able to transport RES [34]. Expressed in yeast, this transporter enhanced the accumulation of RES, but without transporting it directly [35]. Similar polyol transporters involved in stress response have been deciphered in yeast [36] and a similar mechanism involving endogenous protein transporters could be involved in the accumulation of RES. To date, no protein transporters for CHL uptake in yeast have been described. In addition to the involvement of protein transporters, passive transport could also be considered, since passive encapsulation of RES and CHL in yeast cells acting rapidly over 4 hours of incubation has been reported [37,38]. Absorption (i.e. encapsulation efficiency in that case) varied according to the concentration in the external medium and the purity of the compound, as well as the temperature, indicating that the process could occur through passive diffusion [37,38]. In line with our data, the authors reported a passive uptake efficiency of RES higher than that of CHL [37,38].
ROS and RNS are metabolism by-products that are physiologically and continuously generated in mitochondria. Oxidative alterations to biomolecules increase with age, and are an obvious outcome of redox imbalance [21,39]. CHL is a phenolic compound that has antioxidant effects [6]. Because there was no difference in the NAD + /NAD(H) ratio estimated as described by Lin et al. [40] (data not shown), we considered that CHL and AE could extend the replicative lifespan of the yeast through antioxidative action. The production of reactive oxygen and nitrogen species (ROS and RNS) was therefore evaluated using the dihydrorhodamine 123 (DHR123) probe (Figure 1b). ROS and RNS production increased during aging when yeast was aged (on day 5 of cultivation) compared to young yeast (on day 2 of cultivation) (Figure 1b). As observed for RES, AE and CHL treatments resulted in only moderate ROS and RNS production, which at day 5 was only slightly higher than the level measured in young yeast culture. At the same time, mitochondria membrane potential (ΔΨm) evaluation, using 3-3'-dihexyloxacarbocyanineiodide (DiOC6 (3)), suggested a loss of mitochondrial function in aged yeasts, while both AE and CHL were able to maintain a functional ΔΨm value as for young yeast ( Figure S2). A similar effect has been observed for RES ( Figure S2). These results suggested that AE and CHL might act by controlling the mitochondrial-mediated aging process of ROS / RNS. This hypothesis was further tested hereafter. Apple extracts (containing CHL) have been reported to increase yeast lifespan by reducing the levels of reactive oxygen species and cell sensitivity to oxidative stress through a mechanism involving mitochondria [41,42].

Almond skin extract and chlorogenic acid activated expression of genes involved in oxidative stress resistance
Our next goal was to decipher the molecular mechanism underlying the action of AE and CHL on the life-span of the yeast. In particular, their effects on both the silent information regulator 2 (SIR2) and the superoxide dismutases (SOD1 and SOD2) steady-state mRNA levels and corresponding enzyme activity were considered. SIR2 belongs to a conserved family of Nicotinamide Adenine Dinucleotide (NAD + )-dependent protein deacetylases and convincing evidence has connected its activity with ROS and aging, in particular with ROS-driven mitochondrial-mediated response [21]. RES was one of the first recorded plant polyphenols to delay or reduce yeast aging through activation of SIR2 activity [12]. SODs encoded for the antioxidant stress genes which are involved in ROS scavenging. SOD1 is a Cu/Zn-SOD localized in the cytoplasm, while SOD2 is a mitochondrial Mn-SOD. SOD2 is an effective ROS scavenger, playing an important role in antioxidant response [43], and has been correlated with the control of life span in yeast [44].
First, the steady-state mRNA level of these genes was monitored by RT-qPCR as shown in Figure  2. Expression was normalized with TUB1 gene. Y CTL was young yeast (on day 2 of cultivation) and A CTL was aged yeast (on day 5 of cultivation). AE, almond extract (1 mg/mL). CHL: chlorogenic acid at 3 concentrations (CHL5, CHL10 and CHL25 corresponding to chlorogenic acid addition at 5, 10 and 25 µM, respectively). E-Resveratrol (RES, 10 µM) was used as control antiaging drug. Values are means ± standard deviations (SD) of 4 independent experiments. Different letters represent significant differences between the various extraction conditions (p < 0.05).
Aged yeast showed a significantly lower expression of the SIR2, SOD1 and SOD2 genes compared to young yeast, whereas, as already observed, the positive control RES could reverse that trend [28][29][30][31]. The expression of the SIR2, SOD1 and SOD2 genes was also significantly increased by AE and CHL. CHL notably induced an activation of the SIR2 gene expression similar to that of RES (Figure 2a). A very slight induction of SOD1 gene expression was observed (Figure 2b), while the expression of the SOD2 gene showed a more notable increase in presence of AE and CHL (Figure 2c). As for the expression of the SIR2 gene, a dose-dependent response was observed with CHL. Especially, CHL induced significantly more the expression of the SOD2 gene than RES.
In addition, the SIRT1/SIR2 and SOD assays were used to determine the effect on both SIRT1/SIR2 enzyme activities (Figure 3).

Figure 3.
Effects of AE and CHL on SIRT1/SIR2 (a) and total SOD (b) enzyme activities. Y CTL was young yeast (on day 2 of cultivation) and A CTL was aged yeast (on day 5 of cultivation). AE, almond extract (1 mg/mL). CHL: chlorogenic acid at 3 concentrations (CHL5, CHL10 and CHL25 corresponding to chlorogenic acid addition at 5, 10 and 25 µM, respectively). E-Resveratrol (RES, 10 µM) was used as control antiaging drug. In Y (young yeast culture), actual SIRT1/SIR2 enzyme activity was equal to 4564.4 FU/mg protein, whereas total SOD activity was 35.6 units/mg protein. The enzyme activity was expressed as a percentage of Y CTL. Values are means ± standard deviations (SD) of 4 independent experiments. Different letters represent significant differences between the various extraction conditions (p < 0.05).
The activation of SIR2 gene expression was further confirmed at enzyme level using a commercial SIRT1/SIR2 assay kit (Figure 3a). Aged yeast had a low SIRT1/SIR2 activity, while both young and RES treated yeast showed higher significantly SIRT1/SIR2 activity. In the same way, a 43.5 % increase in SIRT1/SIR2 activity was measured in the presence of AE, in line with the RT-qPCR analysis. A dose-dependent increase (up to 76.5%) in SIRT1/SIR2 activity was observed in the presence of CHL, resulting in statistically identical activation to that observed in RES.
Similarly, a comparable activation profile with total SOD activity was observed (Figure 3b), with an even more pronounced activation observed for AE and CHL (up to 81.8% increase for CHL at a dose of 25 μM). It can also be noted that the observed increase in total SOD activity was more consistent with mitochondrial SOD2 gene expression than with mitochondrial SOD1 gene expression. Again, an important role of mitochondria can be assumed in this regulation. Various plant polyphenols have been investigated in the yeast model. Phloridzin, an apple polyphenol, has previously been shown to induce the expression of the genes SIR2, SOD1 and SOD2 [45], whereas hesperidin, a flavonoid derived from the genus Citrus, and nolinospiroside F, a steroidal saponin isolated from Ophiopogon japonicus, have been reported to induce gene expression of SOD1 and SOD2 [46,47]. All of these gene expression activations resulted in an effective increase in SIRT1/SIR2 and/or total SOD enzyme activity [45][46][47]. In various models, SIRT1/SIR2 induced SOD2 expression by deacetylation resulting in both PGC-1α and FOXO activation [42,48]. The activation of SOD2 gene expression by various plant-derived natural products was associated with the extension of the life-span in yeast [45][46][47]. Here, the highest gene expression induction observed for the SOD2 gene can be associated with its mitochondrial function, our observed effect of AE and CHL maintaining the functional ΔΨm value (Figure S2), and also in line with the literature data showing its role as an effective ROS scavenger in the antioxidant response [43] and in the control of life-span in yeast [44].
In other models, CHL has been reported to activate the FOXO transcription factors, resulting in an increase in the lifespan of C. elegans (Zheng et al., 2016), which is known to be regulated by SIRT1/SIR2 activity [42,48]. In humans, CHL has been shown to reverse the aging effect on cognitive functions, in particular by improving the attention, performance and memory functions of elderly people (Kato et al., 2018). However, the molecular mechanism is still unknown, even if the antioxidant activity of CHL can be assumed.

Almond skin extract and chlorogenic acid increased yeast survival to oxidative stress induced by UV-C and reduced oxidative cell damages
Overall, the previous results suggest that AE and CHL may extend the replicative lifespan of the yeast through an antioxidant mechanism. The impact of UV-C-induced oxidative stress on DBY746 yeast was examined to test this hypothesis using our published protocol [6,23,26]. The resulting yeast survival, estimated with a quantitative colony counting assay [49], and ROS/RNS production are presented in Figure 4. significantly reduce ROS / RNS production (Figure 4b). The protection effect of CHL against oxidative stress was within the range of RES. Similar assay, but inducing oxidative stress by H2O2, was used to assess the anti-aging action of nolinospiroside F and phloridzin in yeast [45,47].
These results were consistent with the free radical theory of aging in which the accumulation of ROS and RNS as by-products of aerobic activity in living organisms shortens their lifespan [10,11]. At cellular level, this oxidative stress can lead to oxidative damages to various macromolecules within the cell, which might lead to cell death and possibly to the death of the organism (Harman, 2003) (Figure 5a). Next, to check our protective antioxidant hypothesis, we evaluate the possible protective effects of AE and CHL at different cellular levels including membrane lipids, proteins and DNA ( Figure 5).

Figure 5.
Schematic view of the different ROS / RNS-induced oxidative damages to membrane lipids, proteins and DNA (a), and effects of AE and CHL on malondialdehyde (MDA) levels measured by TBARS assay (b), protein carbonyl contents determined by ELISA assay (c) and 8-oxo-Guanine formation determined by ELISA assay (d). AE, almond extract (1 mg/mL). CHL: chlorogenic acid at 3 concentrations (CHL5, CHL10 and CHL25 corresponding to chlorogenic acid addition at 5, 10 and 25 µM, respectively). E-Resveratrol (RES, 10 µM) was used as control antiaging drug. Values are means ± standard deviations (SD) of 6 independent experiments. Different letters represent significant differences between the various extraction conditions (p < 0.05).
First, TBARS assay measuring malondialdehydes (MDA) was used (Figure 5b). MDA, the main degradation products of polyunsaturated lipids induced by ROS, is an oxidative stress biomarker in organisms and can cause damage to membranes [47]. Here, levels of MDA in yeast supplemented by AE and CHL decreased significantly, indicating that both can serve as efficient cell membrane defenses against oxidative stress. Several plant natural products and / or extracts have been reported to reduce MDA production under oxidative stress in yeast [45,47].
Protein carbonyls can form covalent adducts with cellular components that can result in structural alterations and alter their function. Although ROS and RNS are highly reactive and cause site-specific injury, protein carbonyls are more stable and may spread to distant sites, increasing oxidative damage [50]. The protection offered by AE and CHL against UV-C-induced protein carbonylation was demonstrated using the ELISA assay (Figure 5c). The content of carbonyl protein was clearly reduced by returning to levels close to the baseline control level (Figure 5c). It has been shown that the carbonyl groups may result from ROS alteration of the amino acid side chains or reactions with lipid peroxidation products that affect the protein structure [50]. This suggests that AE and CHL could reduce the carbonyl protein content both by scavenging ROS / RNS as well as by decreasing the lipid peroxidation process. In line with our observation, CHL has been proposed to reduce the protein carbonyl content in human fibroblasts and keratinocytes cells exposed to UV radiation [50].
8-Oxo-Guanine is one of the most common DNA lesions caused by ROS [51]. This oxidative modification of guanine is potentially highly mutagenic, as it may result in uneven pairing with adenine resulting in substitutions of G to T and C to A in the genome [52]. Protection from AE and CHL supplementation in yeast against UV-C-induced 8-Oxo-Guanine formation was evaluated using the ELISA assay (Figure 5c). As a result of AE and CHL, the 8-Oxo-Guanine content was significantly reduced (Figure 5c). This suggests that AE and CHL could effectively protect DNA from oxidative damage. This has never been seen in vivo in the yeast model to the best of our knowledge. Future work on determining the anti-mutagenic ability of AE and CHL will be undertaken. Overall, our results show that both AE and CHL offer a very complete protection against oxidative stress that can lead to an increase in the life span of the yeast.

Conclusions
In this study, using the yeast model, we demonstrate the interest of almond skin extract (AE) and chlorogenic acid (CHL) as new regulators that make a significant contribution to extending the replicative life-span and improving survival under oxidation conditions. This may be linked to their ability to trigger anti-oxidative reactions in yeast during aging and to respond to UV-induced oxidative stress. In both physiological conditions, the AE-and CHL-treated groups showed a reduced production of reactive oxygen and nitrogen species (ROS / RNS). Increased gene expression in SOD2 as well as of total SOD (superoxide dismutase) activity was recorded during aging in AE and CHL groups. The differential effect at the level of gene expression, with a more pronounced induction of the mitochondrial SOD2 gene at the expense of the cytosolic SOD1 gene, indicates the key role of mitochondrial function in this regulation, since normal mitochondrial membrane potential was also maintained during aging in treated groups. Similar activation was also observed for SIR2, an upstream inducer of SOD2 expression, gene expression and enzymatic activity, thus reinforcing our oxidative protection hypothesis. This role was further confirmed by the use of UV-induced oxidative stress, where AE and CHL improved yeast survival by reducing ROS / RNS production, leading to a significant reduction in cellular oxidative damage as demonstrated by decreased membrane lipid peroxidation, protein carbonyl content and 8-oxo-guanine formation in DNA. The anti-aging effects of AE and CHL should be confirmed in vivo using different models in future experiments. Their antimutagenic potential should also merit future experiments. This study demonstrates the interest of almonds as a functional food that protects cells from aging and oxidative stress.

Supplementary Materials:
The following are available online at www.mdpi.com/xxx/s1, Figure S1: (a) HPLC chromatogram (detection set at 325 nm) of the almond skin extract (Beldi genotype grown in the Ain Sfa (34°46'42.4''N, 002°09'28.9''W) pilot location in the eastern Morocco) prepared by ultrasound-assisted extraction USAE. (b) Structures and corresponding numbers on the HPLC chromatogram of the main phenolic compounds considered in this study: protocatechuic acid (1), p-hydroxybenzoic acid (2), chlorogenic acid (3) and p-coumaric acid (4). Figure S2: Mitochondria integrity estimated by mitochondrial potential (ΔΨm) variation. Table S1: Absolute quantification of chlorogenic acid (and other phenolic acids 1 ) contents in almond 2 skin extract. Table  S2: Growth index and viability of yeast cells under the different treatment conditions determined 48h after treatment. Table S3: Estimation of chlorogenic acid and E-resveratrol uptake by yeast cell determined 6h after their additions in culture medium.