**1. Introduction**

Consumption of fruits, vegetables, nuts, and seeds has been associated with lower risks of chronic and degenerative diseases [1–4]. Particularly, given their many beneficial effects on human health, in recent decades, there has been growing interest in the consumption of nuts as a nutrient-rich food [3]. Produced and consumed worldwide, almond (*Prunus dulcis* (Mill.) D.A.Webb) is one of the most popular nuts. It can be consumed in the form of whole nuts, flour, and beverages proposed in the food industry. A large part of almond health benefits has been ascribed to their lipid profile [3,4]. Almond oil is also a sought-after and attractive component for many cosmetic formulations. Over the last few decades, the part of the beneficial actions for health, but also of the growing interest for industrial applications, ascribed to almond phenolics have become increasing [3–5].

The high antioxidant capacity of almond phenolics make it an attractive alternative to synthetic antioxidants. Synthetic antioxidants were largely used to maintain the oxidative stability of emulsions and commonly used in food products and pharmaceutical and cosmetic preparations. However, synthetic antioxidants such as butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT) have adverse health effects, including carcinogenesis [6–8]. Therefore, the use of some of these synthetic antioxidants is now prohibited for food applications in Japan, Canada, and Europe, and they have been removed from what is generally recognized as a safe (GRAS) list. The replacement of these widely criticized synthetic molecules with natural molecules would meet the expectations of manufacturers and consumers. Therefore, it is now important to identify the natural antioxidants with a pronounced and safer radical scavenging capacity for consumers. Despite their distinct lipophilicity profile compared to BHA or BHT, some natural antioxidant phenolics have been shown to be as effective as these synthetic antioxidants in stabilizing nonpolar systems such as bulk oil or different emulsion types [9–11], in good agreemen<sup>t</sup> with the prediction of polar paradox theory [12]. 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 [3,5,13], making this almond cold-pressed oil residue (AOR) an attractive raw material for extraction and the valorization of these natural antioxidant phenolics. In Morocco, the fifth highest ranking producer in the world, almond is the most important nut crop both in terms of area and production value. Several local genotypes, called *Beldi*, which means "from here" as opposed to acclimatized genotypes called *Romi* (i.e., from elsewhere) [14], are of special interest [15]. The almond plantations cover a total area of 150,000 ha for an average annual production estimated at 100,000 tons of unshelled products, of which 9% of this area which provides up to 14% of the Moroccan almond production is located in Eastern Morocco (Figure 1) [15]. This production generates an important part of byproducts, in particular of cold pressed almond oil residues. To date, these by-products of almond oil have been used primarily for animal feed, as litter or for energy production. However, upgrading to higher value-added sectors would significantly increase the revenue from this byproduct valuation. With an average growth rate of 5% per year since 2010 and a large profit margin, the cosmetics market is a dynamic industry. However, this sector is very competitive, with companies facing ever more restrictive environmental regulations (such as REACH in European Union), in addition to consumer pressures that push them to innovate and gradually shift to more natural products and green production methods. The functional properties of several oleaginous species of agro-waste, including their antioxidant activity, in relation to their high concentration in phenolic compounds, have been documented [9,13,16–27]. The application of the biorefinery principle to the recovery of natural antioxidants from almond by-products as cosmetic active ingredients would therefore represent a good opportunity for the almond sector compared to their current use.

**Figure 1.** Parts of almond fruits leading to cold-pressed almond oil and its residue (AOR) used as byproduct in the present study to extract phenolic compounds.

For optimal valorization of these natural co-products, the development of effective extraction methods is necessary. In the past, there were many methods developed for the extraction of natural antioxidants from various natural matrices based on conventional methods such as maceration or Soxhlet extraction. More recently, green extraction methods including microwave-assisted extraction or ultrasound-assisted extraction (USAE) have been found to be particularly effective [11,21,28–30]. These green extraction technologies have also aroused grea<sup>t</sup> interest for industrial applications, and USAE is now considered as one of the most efficient energy saving processes in terms of duration, selectivity, and reproducibility, operating under mid-extraction conditions [28]. It is accepted that the improvement in extraction efficiency obtained using the USAE is based on both acoustic cavitation and mechanical effects [28]. Indeed, ultrasound (US) produces an acoustic cavitation effect facilitating the penetration of the extraction solvent. Therefore, easier release of the intracellular contents of the plant material is observed through greater agitation of the solvent resulting in increased surface contact between the solvent and the target compound as well as increased solubility of the target compound in the solvent of extraction [28].

Here, we report on the development and validation of a USAE method for the extraction of antioxidant phenolic acids from an enriched skin fraction made up of cold pressed AOR from *Bedli* Moroccan genotypes produced in Eastern Morocco (Figure 1).

Recently, Prgomet et al. [13] have also developed a method for comparing the polyphenol fractions from different almond byproducts including the skin using almond varieties from Portugal, but using a conventional heat reflux method. An USAE method was developed by Kahlaoui et al. [18] for the extraction of polyphenols from another almond byproduct: the hulls (the part surrounding the shell itself surrounded by the thin skin; Figure 1) from Italian and Tunisian varieties. It is thus of special interest to compare our method optimized using a different genotype, but more importantly either a green extraction method or a different (by)product. The optimal extraction conditions of this USAE using ethanol as a solvent were obtained through a multivariate technique (Behnken–Box design) coupled with response surface methodology (RSM) and then validated according to international

standards of the association of analytical communities (AOAC). This USAE was applied to investigate the influence of the genetic and environment on the phenolic contents by considering three different local *Beldi* genotypes growing at three different experimental sites. Both in vitro cell free and cellular antioxidant assays were performed to evaluate the evolution of antioxidant activity of the corresponding extracts. Finally, correlations linking phytochemical profile and antioxidant activities of the extracts are presented.

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

#### *2.1. Chemicals and Reagents*

Extraction solvents (ethanol and water) used in the present study were of analytical grade (Thermo Scientific, Illkirch, France). Reagents for antioxidant assays as well as standards (chlorogenic acid, *p*-coumaric acid, protocatechuic acid and *p*-hydroxybenzoic acid) were purchased from Merck (Saint-Quentin Fallavier, France).

#### *2.2. Plant Materials and Culture Conditions*

AOR were obtained from Moroccan almonds (local ecotypes *Beldi*) grown in 3 different pilot locations in the Eastern Morocco (Sidi Bouhria (SID; 34◦4413.6" N, 002◦2015.0" W); Ain Sfa (AIN; 34◦4642.4" N, 002◦0928.9" W); Rislane (RIS; 34◦4459.8" N, 002◦2644.7" W)) using growing conditions as previously described by Melhaoui et al. [15]. Almonds were then triturated using an oil screw press (KOMET DD85G, IBG Monforts Oekotec GmbH & Co. KG, Monchengladbach, Germany) and the residues were ground to ca. 100–150 μm particles using a blender equipped with rotating blades (Grindomix GM 200 blender, Retsch France, Eragny, France) used as raw materials for USAE optimization.

#### *2.3. Ultrasound-Assisted Extraction Method Development*

USAE was completed with an ultrasonic bath (USC1200TH, Prolabo, Sion, Switzerland) composed of a 300 × 240 × 200 mm (inner dimension) tank, with electric power of 400 W corresponding to an acoustic power of 1 <sup>W</sup>/cm<sup>2</sup> and maximal heating power of 400 W. The variable frequencies of this device can be selected thanks to a frequency controller, and it also has a temperature regulator as well as an automatic digital timer. Each sample was placed in 50 mL quartz tubes equipped with a vapor condenser, and was suspended in 10 mL extraction solvent. A liquid to solid ratio of 10:1 mL/g DW (dry weight) was used and extraction was performed at 45 ◦C.

For Extraction optimization a Box–Behnken design was used and the resulting response surface plots drawn with the help of XLSTAT2019 software (Addinsoft, Paris, France). For this purpose, three variables (aqueous Ethanol (aqEtOH) concentration (X1), US frequency (X2), and extraction duration (X3)) were studied and coded at three levels (−1, 0 and +1) as described in Table 1:


**Table 1.** Identity, code unit, coded levels, and actual experimental values of each variable used for USAE of TPC from almond oil residues.

1 % volume/volume of ethanol (analytical grade) concentration in mixture with ultrapure water (HPLC grade).

The different batches were obtained by using the DOE (design of experiment) function of XLSTAT 2019 (Addinsoft, Paris, France), which take values of selective variables at different levels

(Table 2). The experiments were carried out in triplicate. Equation of the model for the extraction of total phenolics from almond oil residues was calculated using the XLSTAT 2019 DOE analysis tool (Addinsoft, Paris, France). The corresponding response surface plots were obtained with 3D option of XLSTAT 2019 (Addinsoft, Paris, France).


**Table 2.** Results of Box–Behnken experimental design of USAE of TPC from AOR.

Experimental values are means ± RSD of 3 independent replicates.

#### *2.4. Determination of Total Phenolic Content*

After extraction, each extract was centrifuged for 15 min at 5000× *g* (Heraeus Biofuge Stratos, Thermo Scientific, Illkirch, France) and the resulting supernatant filtered using a syringe filter (0.45 μm, Merck Millipore, Molsheim, France) prior to analysis.

The total phenolic content (TPC) was determined spectrophotometrically using the Folin–Ciocalteu reagen<sup>t</sup> (Merck, Saint-Quentin Fallavier, France) and according to the protocol adapted for a microplate reader described by Abbasi et al. [31]. Briefly, 10 μL of extract were homogenized with 180 μL of a mixture composed of 4% (*w*/*v*) Na2CO3 (prepared in NaOH 0.1 M), 0.02% (*w*/*v*) potassium sodium tartrate tetrahydrate and 0.02% CuSO4. Following a 10-min of incubation at 25 ◦C, 10 μL of the Folin–Ciocalteu reagen<sup>t</sup> were added, and the homogenized mixture was incubated for 30 min at 25 ◦C. Absorbance was measured at 650 nm with a spectrophotometer (BioTek ELX800 Absorbance Microplate Reader, BioTek Instruments, Colmar, France). A standard curve (0–40 μg/mL; *R*<sup>2</sup> = 0.998) of gallic acid (Merck, Saint-Quentin Fallavier, France) was used to express the TPC in mg of gallic acid equivalents per g DW (mg GAE/g DW).

#### *2.5. Validation Parameters*

Method validation was carried out using the recommendations of the association of analytical communities (AOAC) in terms of precision, repeatability, and recovery as described in detail in Corbin et al. [21].

For HPLC, 6-point calibration lines were obtained by means of diluted solutions of each authentic commercial standard (Merck, Saint-Quentin Fallavier, France). Each sample was injected three times, and arithmetic means were calculated to generate linear regression equations plotting was done by the peak areas (y) against the injected quantities (x) of each standard. Coefficients of determination (*R*2) were used for linearity verification. The limits of detection (LOD) and of quantification (LOQ) was calculated using signal-to-noise ratios of 3:1 and 10:1, respectively.
