Phytochemical Profile and Cosmeceutical Potential of Leaf Extracts of Two Species of the Anacardiaceae Family from the Mediterranean Scrubland: Pistacia lentiscus L. and Pistacia atlantica Desf.

Abstract

Skin aging involves oxidative stress, inflammation, and hyperpigmentation, prompting growing interest in plant-based treatments. Pistacia lentiscus L. and Pistacia atlantica Desf. (Anacardiaceae), North African pharmacopoeia species with recognized phytotherapeutic and cosmeceutical properties, were analyzed to elucidate these biological activities through their phytochemical composition and in vitro antioxidant, anti-inflammatory, and anti-hyperpigmentation potentials. Leaves were successively extracted with hexane, ethyl acetate, and methanol. The methanolic leaf extract of P. atlantica exhibited the highest total phenolic content (430.28 ± 0.01 mg GAE/g), while P. lentiscus showed the highest flavonoid content (230.00 ± 0.01 mg QE/g). LC–MS/MS analysis identified thirteen major phenolic compounds, including methyl gallate and myricitrin. Biological assays revealed that P. lentiscus exhibited the strongest antioxidant activity (IC₅₀ = 5.19 ± 0.01 µg/mL) and the highest ferric-reducing power, whereas P. atlantica showed strong inhibition of protein denaturation (139.10 ± 0.55 µg/mL). Both extracts displayed potent anti-lipoxygenase activity (IC₅₀ = 22.53 ± 0.05 and 22.67 ± 0.04 µg/mL, respectively), as well as anti-tyrosinase effects (IC₅₀ = 39.80 ± 0.08 and 38.25 ± 0.02 µg/mL, respectively). Altogether, these findings underscore the cosmetic potential of these Pistacia species and support their use as valuable raw materials for the development of dermatological treatments.

1. Introduction

Skin aging and pigmentary disorders represent major challenges in dermato-cosmetics today. These phenomena are multifactorial, involving oxidative stress, inflammation, hydration loss, and the appearance of pigmentary spots [1]. Faced with these issues, in many cultures, especially in North Africa, skin tone uniformity, particularly on the face, neck, and arms, constitutes an essential aesthetic criterion [2]. This aspiration for clearer and more homogeneous skin, often reinforced by social pressures and historical stigmas, has naturally led to an increasing demand for hyperpigmentation treatments [3,4,5]. However, this demand raises significant concerns regarding the safety of products used.

Many products marketed for skin lightening indeed contain potentially dangerous substances, such as steroids, mercury, or hydroquinone, responsible for serious side effects, including skin disorders like exogenous ochronosis or damage related to prolonged exposure to heavy metals [6]. This safety issue therefore makes the search for natural active ingredients capable of protecting and regenerating the skin, while being safer, essential. Within this context, the development of innovative dermocosmetic solutions based on plant-derived compounds has become a major research priority.

Plant extracts are increasingly recognized as valuable sources of multifunctional bioactive molecules suitable for dermocosmetic applications. Previous research has established that numerous natural phytochemicals exhibit strong antioxidant performance [7] as well as marked anti-inflammatory properties by suppressing nitric oxide production, down-regulating cyclo-oxygenase-2 expression, and limiting the release of pro-inflammatory cytokines [8]. As a recent example, benzylic alcohol derivatives isolated from Moringa oleifera seeds were shown to reduce lipopolysaccharide-stimulated nitric oxide generation through selective interaction with matrix metalloproteinase-9, thereby highlighting the therapeutic relevance of plant-derived compounds as effective anti-inflammatory agents [9]. Collectively, these bioactivities underline the crucial role of botanical ingredients in mitigating oxidative stress and chronic inflammation, two major biological drivers involved in skin aging and pigmentation disorders. Thus, plants represent promising candidates for the development of safe skin-lightening and anti-aging formulations.

Among natural candidates, Mediterranean plants of the Anacardiaceae family, particularly the genus Pistacia, are gaining increasing scientific interest due to their traditional dermatological uses in Africa and around the Mediterranean Basin [10,11,12]. P. lentiscus L. and P. atlantica Desf. are among the best-known Pistacia species, widely distributed throughout the Mediterranean and the Middle East [10]. They have been traditionally applied to treat various skin conditions, including eczema, wounds, and burns [11,13]. P. lentiscus mastic has also been used as a spice and cosmetic ingredient [14], whereas P. atlantica is recognized for its properties promoting wound healing and treating scabies, chapped lips, and hair loss [15]. Moreover, its resin and oil have demonstrated beneficial effects on wound contraction and cutaneous lesion maturation [16].

Extensive research has documented the multiple pharmacological activities of P. lentiscus and P. atlantica, revealing antibacterial [17,18], antioxidant [19,20], antidiabetic [21,22], anti-inflammatory [23,24], antitumoral [25,26], antihyperlipidemic [27], anti-acetylcholinesterase [28,29] and hepatoprotective [30,31]. Of particular relevance to cosmeceutical applications, wound-healing effects have also been demonstrated, confirming their potential for skin protection and regeneration [32,33]. However, despite this well-established diversity of biological activities, no comparative study has yet combined phytochemical profiling (using LC-MS/MS and GC-FID) with in vitro evaluation of antioxidant, anti-inflammatory, and anti-tyrosinase activities of P. lentiscus and P. atlantica leaves. This lack of integrated evidence, especially regarding their ability to modulate melanin synthesis through tyrosinase inhibition, a key mechanism in hyperpigmentation control, represents a significant knowledge gap in their cosmeceutical relevance.

The unique chemical composition of these species explains this richness of biological activities. Phytochemical studies have shown that P. lentiscus and P. atlantica are particularly rich in diverse bioactive compounds including triterpenoids, flavonoids, phenolic compounds, tannins, fatty acids, and phytosterols, which may account for their potential therapeutic properties [34,35]. Interestingly, these compounds and their concentrations can vary depending to the plant’s growth conditions and the extraction methods applied for their isolation [36]. Therefore, a detailed phytochemical investigation is crucial prior to evaluating their biological properties.

On this basis, the main objective of the present study aimed to establish a correlation between the traditional uses of P. lentiscus and P. atlantica leaf extracts and their in vivo effects, particularly in a cosmeceutical context. The phytochemical profiles of both species were first characterized through the quantification of total phenolic constituents using the Folin–Ciocalteu method and the measurement of total flavonoid contents using the aluminum chloride colorimetric method, followed by detailed analysis using Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS) to identify the main phenolic compounds. In parallel, Gas Chromatography coupled with a Flame Ionization Detector (GC-FID) was used to highlight compounds with potential skin-hydrating effects. The biological activities were evaluated through several complementary approaches: antioxidant activity was assessed using DPPH free radical scavenging and Ferric Reducing Antioxidant Power (FRAP) assays; anti-inflammatory activity was determined by measuring lipoxygenase (LOX) enzyme inhibition and protein denaturation tests; and finally, anti-hyperpigmentation potential was explored through tyrosinase inhibition evaluation. We hypothesized that differences in the phytochemical composition of P. lentiscus and P. atlantica would translate into distinct biological profiles relevant to skin protection and pigmentation control.

2. Materials and Methods

2.1. Chemicals and Reagents

All chemicals and reagents utilized in the present study were of analytical grade. Detailed specifications are provided in Table S1 (Supplementary Materials).

2.2. Sample Collection

The leaves of P. lentiscus and P. atlantica were collected in 2022 during the productive stage in Algeria, in Bouira (6°22′00″ N, 3°53′00″ E) and Batna (35°33′00″ N, 6°10′00″ E), respectively. The botanical identification was conducted by Mohamed DRIDI, a senior agricultural engineer affiliated with the Laboratory of Environment Biomonitoring (LR01/ES14), Group “Ecological Risk of Pharmaceuticals & Pesticides”, Zarzouna 7021, Tunisia. Herbarium vouchers of leaf samples from both species were deposited in the same institution under the codes P.L/LBE 22-004 and P.A/LBE 22-005. The leaves were washed and dried in the shade at room temperature (25 °C) to avoid light oxidation, weighed, and then ground using a RETSCH GM300 grinder (3000 W, 50/60 Hz). The resulting powders were stored in glass containers at 4 °C for later experimental use.

2.3. Extraction and Yield Assessment

The extracts of each plant species were obtained through maceration at room temperature (25 °C) with solvents of increasing polarities, following the method outlined by Ouahabi et al. [37]. These solvents were selected for their complementary polarities (hexane: non-polar, ethyl acetate: medium polarity, methanol: polar), allowing comprehensive and selective extraction of lipophilic to hydrophilic phytochemicals. Ten grams of powdered dried leaves was mixed with 200 mL of n-hexane, ethyl acetate, or methanol, and then subjected to magnetic stirring at room temperature for 2 h with n-hexane, and for 24 h with ethyl acetate and methanol. The extracts were then filtered under vacuum using a Büchner filter funnel with porosity 4. The filtrate was evaporated to dryness under vacuum using a rotary evaporator (Laborota 4000, Heidolph Instruments, Schwabach, Germany). The dried extracts obtained were weighed (expressed in grams, g), and all assays were performed in triplicate (n = 3). The extraction yield was subsequently determined using the following equation:

Yield (%) = (mass of dried extract/mass of dried sample) × 100

(1)

2.4. Phytochemicals

2.4.1. Quantification of Total Phenolic Constituents

The total polyphenol content in P. lentiscus and P. atlantica extracts was measured using the Folin–Ciocalteu method which quantifies the total concentration of hydroxyl groups in the sample. The protocol follows the method described by Nea et al. [38] and Singleton and Rossi [39] with slight modifications. Briefly, 200 μL of extract solution (1 mg/mL) diluted in ethyl acetate and methanol were mixed with 1 mL of Folin–Ciocalteu reagent (diluted 10-fold in deionized water), Afterwards, 800 μL of a 7.5% sodium carbonate solution was added to the reaction mixture. The samples were left to react for 1 h at ambient temperature in the dark. The absorbance of each mixture was then recorded at 765 nm using a UV–Visible spectrophotometer (Ultrospec 7000, GE Healthcare, Chalfont St Giles, UK), with the solubilization solvent serving as the blank and distilled water as the control. A calibration curve was generated using gallic acid standards at concentrations ranging from 0 to 1000 μg/mL under identical conditions. All experiments were performed in triplicate and the total phenolic content was expressed as mg of gallic acid equivalents per gram of the extract (mg GAE/g) [40].

2.4.2. Measurement of Total Flavonoid Contents

Flavonoids were quantified using a modified method described by Nair et al. [41] and Kim et al. [42]. A volume of 400 μL of methanol or ethanol acetate extract, standard solution (quercetin), solubilization solvent (blank), or distilled water (control) was mixed with 120 μL of 5% NaNO₂. After a 5 min incubation, 120 μL of 10% aluminum chloride (AlCl₃) was added, and the mixture was vigorously stirred. After 6 min, 800 μL of 1 M NaOH was added. The absorbance was immediately measured at 510 nm against the control using a UV-Vis spectrophotometer (Ultrospec 7000, GE Healthcare, Chalfont St Giles, UK).

The calibration curve was established using quercetin standards at concentrations ranging from 0 to 0.1 mg/mL under identical conditions. All measurements were performed in triplicate. The total flavonoid content was expressed as mg of quercetin equivalents per gram of the extract (mg QE/g) [40].

2.4.3. Fatty Acid GC-FID Analysis

The fatty acid methyl ester (FAME) profiles were determined by GC-FID following the method of Bourgou et al. [43] and Saha Tchinda et al. [44], with minor modifications. For analysis, 10 mg of P. lentiscus and P. atlantica extracts were incubated at 70 °C for 90 min in a mixture of n-hexane (0.2 mL) and boron trifluoride (BF₃) reagent (0.5 mL) to induce transesterification. After the reaction, a saturated NaCl solution (0.5 mL, 40%) and sulfuric acid (0.2 mL, 10% v/v) were added, followed by methyl ester extraction with 8 mL of n-hexane under agitation. Analysis was performed using an Agilent HP 6890 GC system equipped with an RT-2560 capillary column (30 m × 0.25 mm × 0.20 µm) and nitrogen carrier gas (1.6 mL/min). The temperature program included an initial hold at 170 °C (2 min), a 3 °C/min ramp to 240 °C, and a 15 min hold. Injector and detector temperatures were set and maintained at 225 °C. Methyl esters were identified by comparing their retention times to those of a certified FAME reference standard. Relative FAME percentages were calculated from peak areas and expressed as % of total fatty acids.

2.4.4. LC-MS/MS Analysis

The identification and semi-quantification of molecules were carried out by LC-MS/MS in the negative ionization mode, using an approach combining LC-QTOF for qualitative structural identification and LC-TQ for quantitative purposes using the multiple reaction monitoring (MRM) mode. Methanolic extract samples were prepared at a concentration of 10 mg/mL. As relative quantification was performed with a standard addition method, each sample was separated in two aliquots of 1 mL and an internal standard solution of flavone (0.5 mg/mL) was added to one aliquot, followed by thorough vortex mixing. The mixture was then filtered through a 0.22 µm PTFE membrane before injection.

For structural identification, exact mass data were recorded using an Agilent 1290 Infinity II LC system coupled to an Agilent 6530 Accurate-Mass Q-TOF LC/MS hybrid quadrupole time-of-flight mass spectrometer (Agilent Technologies, Santa Clara, CA, USA), equipped with an ESI source, operating in negative ionization mode. Ten µL of sample was injected onto an Acquity^® BEH C18 column (2.1 × 100 mm; 1.8 µm particle size, Waters Co., Milford, MA, USA) that was used for compound separation. A 16 min LC method was developed according to the method of Wojdylo et al. [45], with modifications. Its flow rate was maintained constant at 0.450 mL/min, with solvents A and B consisting of milliQ water and MS-grade acetonitrile, respectively, both supplemented with 0.1% formic acid. The elution gradient started at 1% solvent B and was held for 1 min. Then, a linear gradient ramp was applied to reach 100% solvent B in 11 min. This was followed by a 1.5 min isocratic wash at 100% B, then a return to 1% B for 2.5 min to re-equilibrate the column. The column and autosampler temperatures were set to 30 °C and 15 °C, respectively. The source parameters were set as follows: gas temperature 300 °C, drying gas flow 8 L/min, nebulizer pressure 35 psi, sheath gas temperature 350 °C, sheath gas flow 11 L/min, capillary voltage 3500 V and nozzle voltage 1000 V. Data were first acquired in full scan mode MS1, with a fragmentor voltage of 175 V and an m/z range from 100 to 1000. Putative polyphenols were identified based on mass errors lower than 16 ppm. Targeted MS2 was then used to break down these precursor ions into their fragments. Collision energy, isolation width, and fragment mass range were set at 35 eV, 1.3 m/z, and 100–650 m/z, respectively. MS/MS fragmentation profiles were thereby obtained for each selected precursor ion, and polyphenol identification was validated after comparison with the MassBank, PubChem databases and scientific literature.

For semi-quantification purposes, MRM transitions were recorded using an Agilent 1290 Infinity II LC system (Agilent Technologies, Santa Clara, CA, USA) coupled to an Agilent 6475 Triple Quadrupole LC-MS mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). The LC method used was the same as for LC-QTOF, to achieve similar retention times. The TQ ion source parameters were set as follows: drying gas temperature 300 °C, drying gas flow 13 L/min, nebulizer pressure 35 psi, sheath gas temperature 250 °C, sheath gas flow 11 L/min, capillary voltage 4000 V.

MRM transitions were recorded in the negative ionization mode, except for flavone (internal standard), with a dwell time of 5 ms while fragmentor voltage and collision energy were set at 150 V and 30 V, respectively. Recorded MRM transitions are presented in Table 1. Relative quantification was performed using the following formula:

Polyphenol content (A.U./mg DW) = [MRM peak area/(spiked MRM flavone peak area − non-spiked MRM flavone peak area)]/sample dry weight

(2)

With:

A.U. = Arbitrary Units

DW = Dry Weight

MRM = Multiple Reaction Monitoring

All data acquisition and processing were carried out using MassHunter^® Workstation software (Version 10.0, Agilent Technologies, Santa Clara, CA, USA).

2.5. Biological Activities

2.5.1. Antioxidant Assays

The antioxidant power of methanolic extracts from P. lentiscus and P. atlantica was evaluated through DPPH radical scavenging and the ferric reducing antioxidant power (FRAP) assays. These two methods assess the extracts’ capacity to scavenge free radicals and to reduce ferric ion (Fe³⁺) to ferrous ion (Fe²⁺) [43], in comparison, respectively, with the reference standards: ascorbic acid and Trolox.

• DPPH assay (2,2-diphenyl-1-picrylhydrazyl radical scavenging test)

The ability of the methanolic extracts of P. lentiscus, P. atlantica, and Trolox to reduce acellular free radicals was assessed by monitoring the discoloration of the DPPH solution in methanol, originally purple, following the method of Brand-Williams et al. [46], with slight modifications.

The samples were analyzed at concentrations of 5, 15, 45, and 135 µg/mL. A volume of 800 µL of each sample diluted in methanol or the standard Trolox at different concentrations was added to 2 mL of DPPH solution (0.001%) in methanol, then manually shaken. After a 30 min incubation in the dark at ambient temperature, the absorbance of each preparation was recorded using a UV/visible double-beam spectrophotometer (Ultrospec 7000, GE Healthcare, UK) at a wavelength of 517 nm. The same procedure was followed for Trolox (Cayman Chemical, Ann Arbor, MI, USA) used as the positive control, while methanol with DPPH (Alfa Aesar, Karlsruhe, Germany) was used as the blank. All experiments were performed in triplicate. The percentage of inhibition was calculated using the following formula:

Inhibition% = [(Abs _Blank – Abs _Sample)/Abs _Blank] × 100

(3)

where “Abs _Blank” refers to the absorbance of the blank and “Abs _Sample” corresponds to the absorbance of the sample (or positive control).

• Ferric reducing antioxidant power (FRAP) assay

The ferric-reducing antioxidant power of the extracts was evaluated according to the method described by Tanoh et al. [47], with some adjustments. Four dilutions of the extracts and ascorbic acid (standard, Sigma Aldrich, Taufkirchen, Germany) were prepared in methanol (12.5 to 100 µg/mL). A volume of 1000 µL of each dilution was mixed with an equal volume of phosphate buffer (200 mM, pH 6.6) and potassium ferricyanide solution (1%). The mixture was incubated at 50 °C for 20 min, then 1000 µL of trichloroacetic acid (10% v/v) was added. After centrifugation (3000 g, 10 min) at ambient temperature, the supernatant was collected and mixed with 1 mL of distilled water and 150 µL of FeCl₃ solution (0.1%).

The absorbance was measured at 700 nm using a double-beam UV/visible spectrophotometer (Ultrospec 7000, GE Healthcare, UK). This measurement relies on the increase in absorbance of the reaction mixture, reflecting the reduction of Fe³⁺ ions to Fe²⁺. Higher absorbance (compared to the blank containing phosphate buffer, potassium ferricyanide solution, trichloroacetic acid, distilled water, and FeCl₃, but without the sample or ascorbic acid) indicates greater reducing power. All samples were performed in triplicate.

2.5.2. Assessment of the Anti-Inflammatory Activities

• Bovine Serum Albumin Denaturation Assay

The anti-inflammatory activity of the methanolic extracts of P. lentiscus and P. atlantica leaves was assessed using the protein denaturation assay, as described by Kar et al. and Nea et al. [48,49], with minor modifications. Briefly, the reaction mixtures were prepared by combining 1000 µL of the methanolic extract samples with diclofenac (≥98.5 purity, Sigma-Aldrich, Buchs, Switzerland) as the reference standard, at concentrations of 400, 300, 200, and 100 μg/mL. To this, 500 µL of a 2% BSA solution in water, and 2500 µL of phosphate-buffered saline (PBS), adjusted to pH 6.3 with hydrochloric acid (HCl), were added. The tubes were incubated at room temperature for 20 min, then heated to 70 °C for 5 min, and cooled down for 10 min. The extent of BSA denaturation was assessed by measuring absorbance at 660 nm using an UV-Vis spectrophotometer (Ultrospec 7000, GE Healthcare, UK).

The assay was conducted in triplicate, and the percentage of albumin denaturation inhibition was expressed as the mean ± standard deviation (SD) relative to control, according to the following formula:

BSA denaturation% = [(Abs _Blank − Abs _sample)/Abs _blank] × 100

(4)

where “Abs _blank” presents the absorbance (Abs) of the reaction mixture without the methanolic extract, “Abs _sample” corresponds to the absorbance of the reaction mixture containing the methanolic extract, adjusted by subtracting the absorbance of the diluted methanolic extract.

• Lipoxygenase inhibition assay

The anti-inflammatory activities of methanolic extracts were evaluated according to the method of Nikhila and Sangeetha [50], with slight modifications. To summarize, the reaction mixture, containing methanolic extracts at different concentrations (100, 75, 50, and 25 μg/mL in methanol), was prepared in triplicate, along with 10 mg of lipoxygenase (≥50,000 U/mg) in 100 mL of distilled water to obtain a solution of 5000 U/mL (Sigma, Taufkirchen, Germany) and 35 μL (0.1 mg/mL) of a 0.2 M borate buffer solution (pH = 9.0). The prepared samples were incubated at 25 °C for 15 min. The reaction was initiated by adding 35 μL of a substrate solution (250 μM linoleic acid, ≥98.5 purity, Sigma-Aldrich, St. Louis, MO, USA), and then absorbance was measured at 234 nm using a UV spectrophotometer (Ultrospec 7000, GE Healthcare, UK). Quercetin (Sigma-Aldrich, Taufkirchen, Germany) was used as the positive control and prepared at the same concentration as our methanolic extract. The percentage of lipoxygenase activity inhibition was calculated as follows:

Lipoxygenase inhibition% = [(Abs _blank − Abs _sample)/(Abs _blank)] × 100

(5)

“Abs _blank” denotes the absorbance of the reaction mixture without the methanolic extract, while “Abs _sample” represents the absorbance of the mixture containing the extract, corrected by subtracting the intrinsic absorbance of the diluted methanolic extract.

2.5.3. Anti-Tyrosinase Activity Evaluation

The anti-tyrosinase activity of methanolic extracts of P. lentiscus and P. atlantica obtained by maceration was evaluated following the protocols described by Rangkadilok et al. [51] and Genva et al. [52].

Tyrosinase inhibition was assessed by incubating 100 µL of each sample, the control (1 mM kojic acid solution), or the blank (50 mM phosphate buffer, pH 6.5) with 50 µL of the tyrosinase solution (625 U/mL) and 250 µL of oxygenated buffer. The mixture was incubated at room temperature for 15 min. After incubation, the reaction mixture was transferred to a quartz cuvette, and 400 µL of substrate solution (1 mM L-DOPA) was added. Absorbance was measured at 475 nm using a spectrophotometer (Ultrospec 7000, GE Healthcare, Cambridge, UK).

Quercetin (Sigma, Germany) was used as a positive control. Each sample was tested in triplicate. The percentage of tyrosinase inhibition was calculated using the following formula:

Tyrosinase inhibition % = [(Abs _blank − Abs _sample)/Abs _blank] × 100

(6)

where “Abs _blank” corresponds to the absorbance of the blank (50 mM phosphate buffer, pH 6.5) representing 0% inhibition, and “Abs _sample” presents the absorbance of the assay reaction mixture containing the extract, kojic acid, or quercetin. The IC₅₀ values of the extracts, kojic acid, and quercetin were then calculated.

2.5.4. Data Analysis

Quantitative data were presented as mean ± SD and mean ± SEM. The Student’s t-test for unpaired series was used to compare the means of P. lentiscus and P. atlantica. Dunnett’s post hoc test was used to evaluate the differences between the pairs of means in the groups. One way and two-way ANOVA followed by the Tukey post hoc test were applied for the multiple comparison using the GraphPad Prism 8 program (San Diego, CA, USA). Additionally, the nonlinear regression feature was used to calculate the IC₅₀ values. The threshold for statistical significance was set at a p-value of 0.05.

3. Results and Discussion

3.1. Chemical Composition

3.1.1. Extraction Yields, Phenolic and Flavonoid Contents

The extraction of phenolic compounds was carried out using solvents of increasing polarities: methanol, ethyl acetate, and hexane in order to compare the extraction yields of P. lentiscus and P. atlantica leaves, along with their phenolic and flavonoid contents.

Extraction yields varied significantly depending on the solvent used (p < 0.001) (Figure 1). Methanolic extracts exhibited the highest yields, reaching 42.10 ± 1.0% for P. atlantica and 35.20 ± 1.1% for P. lentiscus, with a significant difference in favor of P. atlantica (p < 0.001). Extracts obtained with ethyl acetate showed intermediate yields of 11.10 ± 0.5% for P. atlantica and 13.90 ± 0.5% for P. lentiscus, with the latter species presenting a significantly higher yield (p < 0.001). Hexane, an apolar solvent, generated the lowest yields, with 11.20 ± 0.25% for P. atlantica and 3.33 ± 0.2% for P. lentiscus, with the yield being significantly higher for P. atlantica (p < 0.001).

The methanol yield of P. atlantica was considerably higher than that reported by Belyagoubi et al. (36.40%) [53], while for P. lentiscus, it was markedly higher than that obtained with ethanol by Remila et al. (6.09%) [25]. These observations indicate that the use of polar solvents, such as methanol, significantly increases yields and highlights that P. atlantica provides a higher yield than P. lentiscus. These results are coherent with previously reported studies and highlight the importance of solvent polarity in maximizing extraction [53,54]. Based on these extraction yields, the two most efficient and most polar solvents were selected for total polyphenol and flavonoid quantification.

Consistent with the yield obtained, methanol allowed the extraction of the highest concentrations of polyphenols and flavonoids in both species compared to extraction with ethyl acetate (p < 0.001) (Figure 2). The polyphenol content of the methanolic extracts reached 343.06 ± 0.02 mg GAE/g for P. lentiscus and 430.28 ± 0.01 mg GAE/g for P. atlantica, whereas the ethyl acetate extracts contained only 88.6 ± 0.03 mg GAE/g and 90.56 ± 0.56 mg GAE/g, respectively (Figure 2a). The inter-solvent comparison showed that methanol extracted significantly higher amounts of polyphenols than ethyl acetate in both species (p < 0.001). Furthermore, inter-species analysis revealed that both methanolic and ethyl acetate extracts of P. atlantica exhibited significantly higher polyphenol contents than those of P. lentiscus (p < 0.001). Concerning flavonoids, the methanolic extracts contained 230.00 ± 0.01 mg QE/g for P. lentiscus and 183.00 ± 0.01 mg QE/g for P. atlantica, while the ethyl acetate extracts showed markedly lower levels of 86.39 ± 0.03 mg QE/g and 74.3 ± 0.04 mg QE/g, respectively (Figure 2b). The inter-solvent comparison once again demonstrated the superior extraction capacity of methanol over ethyl acetate for flavonoids (p < 0.001). Conversely, the inter-species comparison indicated that P. lentiscus exhibited significantly higher flavonoid contents than P. atlantica in both solvent extracts (p < 0.001).

The polyphenol content of P. lentiscus foliar extract showed moderate variation compared to existing literature, being slightly higher to the findings of Amel et al. [55] (216.29 ± 20.62 mg GAE/g) but approximately 2.3-fold lower than the maximum value of 524.87 ± 1.17 mg GAE/g reported in Morocco [56].

Regarding flavonoids, the content measured in this study was significantly higher than some earlier findings, such as the 19.162 ± 0.436 mg QE/g reported by the same team [55], but remains consistent with the values obtained by Remila et al. [25] and Seddoqi et al. [56], which were around 139.38 ± 3.11 and 147.91 ± 0.04 mg QE/g, respectively.

In P. atlantica foliar extract, the flavonoid content was up to four times higher than the 51.77 ± 4.34 mg QE/g reported by Peksel et al. [57] and the range of 100.10 to 110.62 mg QE/g reported by Amri et al. [55]. In contrast, the polyphenol content was lower than the 245.60 ± 6.02 mg GAE/g observed in Turkish extracts obtained by methanol extraction using a Soxhlet apparatus [57].

These differences maybe originate from variations in extraction methods, climatic conditions, and the geographical origin of the plants [58,59]. It is widely recognized that phenolic compounds are key components of plant metabolism and tend to accumulate under stress conditions, such as drought, extreme temperatures, and pollution [60]. Moreover, the solubility of phenolic compounds depends on several factors such as their degree of polymerization, the plant part used, and soil characteristics [61,62].

Our results highlight the richness of the methanolic extract of P. atlantica and P. lentiscus in polyphenols and flavonoids. To further characterize this promising composition, the methanolic extract was analyzed by LC-MS/MS and GC-FID. Analyses by LC-MS/MS indeed enables precise identification of individual polyphenols to determine the extract’s composition and investigate the potential origin of its biological activities, while GC-FID provides information on the fatty acid profile, which may indicate potential moisturizing properties. This combined analytical approach enhances understanding of the extract’s functional properties and guides its targeted use for dermo-cosmetic applications.

3.1.2. Fatty Acid Composition

GC-FID analysis was performed to identify and compare the fatty acids present in leaf extracts of P. lentiscus and P. atlantica, which may influence the dermo-cosmetic properties of our extracts, particularly their moisturizing. As illustrated in Figure 3 and

Table A1. Assessment of the fatty acid composition of P. lentiscus and P. atlantica methanolic extracts using Gas Chromatography coupled to Flame Ionization Detector.

	Fatty Acid	Rt (min)	P. lentiscus (%)	P. atlantica (%)	p-Value
1	Caproic acid (C6:0)	3.59	0.13 ± 0.02	0.10 ± 0.06	0.54
2	Undecanoic acid (C11:0)	5.50	0.19 ± 0.07	0.15 ± 0.01	0.47
3	Lauric acid (C12:0)	7.81	0.38 ± 0.11	0.55 ± 0.31	0.42
4	Tridecanoic acid (C13:0)	8.96	0.40 ± 0.01	1.07 ± 0.13	<0.001
5	Myristic acid (C14:0)	10.31	3.63 ± 0.07	1.52 ± 0.09	<0.001
6	Pentadecanoic acid (C15:0)	11.74	nd	0.54 ± 0.23	-
7	Palmitic acid (C16:0)	13.34	18.35 ± 0.06	6.51 ± 0.02	<0.001
8	Palmitoleic acid (C16:1)	13.72	4.42 ± 0.09	1.02 ±0.05	<0.001
9	Margaric acid (C17:0)	14.88	nd	nd	-
10	Stearic acid (C18:0)	16.49	1.36 ± 0.04	0.91 ± 0.01	<0.001
11	Oleic acid (C18:1)	16.50	1.36 ± 0.03	nd	-
12	Elaidic acid (C18:1: ω9)	16.83	5.30 ± 0.11	0.72 ± 0.17	<0.001
13	Vaccenic acid (C18:1: ω7)	16.92	0.65 ± 0.01	nd	-
14	Linoleic acid (C18:2)	17.56	23.96 ± 0.04	4.86 ± 0.06	<0.001
15	γ-linolenic acid (C18:3 γ)	18.60	31.54 ± 0.01	8.60 ± 0.09	<0.001
16	Arachidic acid (C20:0)	19.63	0.61 ± 0.16	0.48 ± 0.01	0.21
17	Cis-11,14-eicosadienoic acid (C20:2)	20.76	1.24 ± 0.02	8.31 ± 0.11	<0.001
18	Cis-5,8,11,14,17-eicosapentaenoic acid (C20-5n3)	22.22	nd	3.12 ± 0.12	-
19	Behenic acid (C22:0)	22.64	1.18 ± 0.05	0.45 ± 0.02	<0.001
20	Cis-13,16-docosadienoic acid (C22:2)	23.73	2.24 ± 0.02	54.13 ± 0.09	<0.001
21	Lignoceric acid (C24:0)	25.75	2.17 ± 0.02	0.63 ± 0.13	<0.001
22	Nervonic acid (C24:1n9)	26.10	nd	4.90 ± 0.07	-
	Saturated fatty acids (SFA) [a]		29.29	14.34
	Unsaturated fatty acids (UFA) [b]		70.71	85.66
	Unsaturation ratio = UFA/SFA [c]		2.41	5.97

Rt: retention time. nd: not detected. [a]: saturated fatty acids (SFA). [b]: unsaturated fatty acids (UFA). [c]: unsaturation ratio = UFA/SFA. Data were determined as a mean ± SEM retention time of each phenolic composition in three independent experiments. SEM: standard error of the mean.

, both extracts contain much more of unsaturated fatty acids (85.66% in P. atlantica and 70.71% in P. lentiscus) than saturated fatty acids. The P. lentiscus extract was primarily composed of palmitic acid (C16:0, 18.35%), γ-linolenic acid (C18:3γ, 31.54%), and linoleic acid (C18:2, 23.96%). In contrast, P. atlantica was characterized by the predominance of cis-13,16-docosadienoic acid (C22:2), which accounted for 54.13% of its composition.

Previous studies have indicated that climatic variations can influence fatty acid desaturation, thereby altering the balance between saturated and polyunsaturated fatty acids [63]. This may explain the differences observed between the two species in our study. The higher levels of γ-linolenic acid and linoleic acid in P. lentiscus suggest a greater desaturation activity of C18 fatty acids, while the strong predominance of cis-13,16-docosadienoic acid in P. atlantica reflects a distinct elongation pathway leading to the synthesis of very-long-chain polyunsaturated fatty acids. In addition to environmental influences, species-specific genetic factors also contribute to determining lipid metabolism and composition [64]. Therefore, the higher proportion of polyunsaturated fatty acids in P. atlantica (85.66%) compared with P. lentiscus (70.71%) is likely the result of combined effects of genetic regulation and ecological adaptation.

The leaves of P. lentiscus and P. atlantica remain widely unexplored compared to other parts of the plant, such as P. lentiscus seed oil, which is predominantly composed of oleic acid [65,66,67,68]. Hexane extracts from P. lentiscus leaves reveal a predominance of palmitic and oleic acids [69]. Yet the fatty acid composition of P. atlantica leaves had not yet been evaluated, thus justifying its investigation in the present study and the evaluation of its potential value as a source of fatty acids.

These lipidic compounds, particularly unsaturated fatty acids, have moisturizing and emollient properties, promoting skin suppleness, maintaining the skin barrier, and stabilizing cell membranes [70,71,72]. Topical application of linoleic and linolenic acids has been shown to restore the skin barrier and improve the overall appearance of the skin [71,73], suggesting that extracts of P. lentiscus and P. atlantica are promising dermo-cosmetic ingredients for enhancing hydration, softness, and barrier function in innovative formulations.

3.1.3. LC-MS/MS Screening and Identification of Polyphenolic Compounds

A total of thirteen phenolic compounds, mainly flavonoids, have been identified by LC-QTOF based on their characteristic fragmentation profiles (Figure 4, Table 1). Seven compounds were detected in P. lentiscus: isoquercitrin, myricitrin, methyl gallate, myricetin-3-O-hexoside, gallocatechin, epigallocatechin gallate, and pistafolin A. In P. atlantica, ten compounds were identified: isoquercitrin, chlorogenic acid, myricitrin, methyl gallate, myricetin-3-O-hexoside, rutin, luteolin, quercetin-3-O-rhamnoside, kaempferol-3-O-galactoside (Trifolin), and quercetin-3-O-arabinoside. Among these compounds, four are common to both species and their relative abundances, show significant variations between the two species.

Table 1. Phytochemical characterization of methanolic extracts of P. lentiscus and of P. atlantica by LC-MS/MS (negative mode).

N°	Compound	Formula	MRM (m/z)	MM	Predicted M-H	M-H Obs	Mass Error (ppm)	RT	Observed Fragments	Samples	References
1	Isoquercetin	C21H20O12	463 → 301	464.0954	463.0876	463.0840	7.77	3.9	300.0290, 271.0265, 301.0370, 255.0320	PL, PA	[74], PubChem
2	Chlorogenic acid	C16H18O9	353 → 191	354.0950	353.0872	353.0846	7.49	3.1	191.0561	PA	[75], PubChem
3	Myricitrin	C21H20O12	463 → 317	464.0954	463.0876	463.0840	7.77	3.9	301.0357, 317.0309, 300.0280, 271.0262	PL, PA	[76], MassBank
4	Methyl gallate	C8H8O5	183 → 124	184.0371	183.0293	183.0282	6.31	3.1	124.0163, 123.0089, 106.0057	PL, PA	[77]
5	Myricetin 3-O-hexoside	C21H20O13	479 → 317	480.0903	479.0825	479.0784	8.59	3.6	316.0223, 271.0262, 287.0190, 317.0298, 179.0006, 151.0004	PL, PA	[78]
6	Gallocatechin	C15H14O7	305 → 125	306.0739	305.0661	305.0661	7.72	2.6	125.0243, 137.0246, 109.0288, 139.0371, 167.0330	PL	PubChem, MassBank
7	Epigallocatechin gallate	C22H18O11	457 → 169	458.0849	457.0771	457.0737	7.36	3.4	125.0248, 137.0247, 179.0355, 219.0669, 305.0671	PL	[79], MassBank
8	Pistafolin A	C28H24O18	647 → 495	648.0962	647.0884	647.0828	8.72	3.5	343.0688, 169.0149, 495.0788	PL	[80,81]
9	Rutin	C27H30O16	609 → 300	610.1533	609.1455	609.1551	−15.73	3.8	300.0275, 301.0380, 272.0316, 178.3373, 151.0027	PA	[82]
10	Luteolin	C15H10O6	285 → 133	286.0477	285.0399	285.0399	−0.06	4.5	133.0399, 151.006, 270.2072, 107.0168, 175.0380, 121.0269	PA	[83]
11	Quercetin 3-O-rhamnoside	C21H20O11	447 → 301	448.1005	447.0927	447.0959	−7.15	4.2	300.0285, 301.0346, 271.0266, 255.0302, 178.9992	PA	[84]
12	Kaempferol-3-O-galactoside (Trifolin)	C21H20O11	447 → 284	448.1005	447.0927	447.0959	−7.15	4.2	325.0602, 285.0419, 284.0280, 255.0302	PA	[85], PubChem
13	Quercetin 3-O-arabinoside	C20H18O11	433 → 301	434.0849	433.0771	433.0772	−0.27	4.1	300.0268, 271.0256, 301.0344, 255.0271	PA	[84]

MRM: Multiple reaction monitoring, MM: Monoisotopic mass, Predicted M-H: Predicted [M-H]⁻ ion, M-H obs: Observed [M-H]⁻ ion, Mass error (ppm): Mass error (parts per million), RT: Retention time, PL: P. lentiscus L., PA: P. atlantica Desf.

Table 1. Phytochemical characterization of methanolic extracts of P. lentiscus and of P. atlantica by LC-MS/MS (negative mode).

N°	Compound	Formula	MRM (m/z)	MM	Predicted M-H	M-H Obs	Mass Error (ppm)	RT	Observed Fragments	Samples	References
1	Isoquercetin	C21H20O12	463 → 301	464.0954	463.0876	463.0840	7.77	3.9	300.0290, 271.0265, 301.0370, 255.0320	PL, PA	[74], PubChem
2	Chlorogenic acid	C16H18O9	353 → 191	354.0950	353.0872	353.0846	7.49	3.1	191.0561	PA	[75], PubChem
3	Myricitrin	C21H20O12	463 → 317	464.0954	463.0876	463.0840	7.77	3.9	301.0357, 317.0309, 300.0280, 271.0262	PL, PA	[76], MassBank
4	Methyl gallate	C8H8O5	183 → 124	184.0371	183.0293	183.0282	6.31	3.1	124.0163, 123.0089, 106.0057	PL, PA	[77]
5	Myricetin 3-O-hexoside	C21H20O13	479 → 317	480.0903	479.0825	479.0784	8.59	3.6	316.0223, 271.0262, 287.0190, 317.0298, 179.0006, 151.0004	PL, PA	[78]
6	Gallocatechin	C15H14O7	305 → 125	306.0739	305.0661	305.0661	7.72	2.6	125.0243, 137.0246, 109.0288, 139.0371, 167.0330	PL	PubChem, MassBank
7	Epigallocatechin gallate	C22H18O11	457 → 169	458.0849	457.0771	457.0737	7.36	3.4	125.0248, 137.0247, 179.0355, 219.0669, 305.0671	PL	[79], MassBank
8	Pistafolin A	C28H24O18	647 → 495	648.0962	647.0884	647.0828	8.72	3.5	343.0688, 169.0149, 495.0788	PL	[80,81]
9	Rutin	C27H30O16	609 → 300	610.1533	609.1455	609.1551	−15.73	3.8	300.0275, 301.0380, 272.0316, 178.3373, 151.0027	PA	[82]
10	Luteolin	C15H10O6	285 → 133	286.0477	285.0399	285.0399	−0.06	4.5	133.0399, 151.006, 270.2072, 107.0168, 175.0380, 121.0269	PA	[83]
11	Quercetin 3-O-rhamnoside	C21H20O11	447 → 301	448.1005	447.0927	447.0959	−7.15	4.2	300.0285, 301.0346, 271.0266, 255.0302, 178.9992	PA	[84]
12	Kaempferol-3-O-galactoside (Trifolin)	C21H20O11	447 → 284	448.1005	447.0927	447.0959	−7.15	4.2	325.0602, 285.0419, 284.0280, 255.0302	PA	[85], PubChem
13	Quercetin 3-O-arabinoside	C20H18O11	433 → 301	434.0849	433.0771	433.0772	−0.27	4.1	300.0268, 271.0256, 301.0344, 255.0271	PA	[84]

MRM: Multiple reaction monitoring, MM: Monoisotopic mass, Predicted M-H: Predicted [M-H]⁻ ion, M-H obs: Observed [M-H]⁻ ion, Mass error (ppm): Mass error (parts per million), RT: Retention time, PL: P. lentiscus L., PA: P. atlantica Desf.

Our results show that these Mediterranean species possess a high phenolic content, characterized by a flavonoid-dominated composition. The chemical composition of both extracts was dominated by methyl gallate (3.72 ± 0.07 and 1.97 ± 0.12 for P. lentiscus and P. atlantica, respectively), a compound rarely reported as a major constituent in previous studies on the genus Pistacia. This composition is followed by myricitrin (1.49 ± 0.02 and 0.15 ± 0.01), myricetin-3-O-hexoside (0.12 ± 0.02 and 0.04 ± 0.02), and isoquercitrin (0.10 ± 0.02 and 0.10 ± 0.01) in P. lentiscus and P. atlantica, respectively. The high content of hydrophilic phenolic compounds detected in this extract is mainly linked to the extraction solvent. Methanol, owing to its strong polarity, preferentially dissolves polyphenols that possess multiple hydroxyl groups and therefore exhibit a polar character [86]. This solvent-driven selectivity accounts for the predominance of methyl gallate and myricetin derivatives, both of which are highly polar phenolics, in the LC-MS/MS profile of the methanolic leaf extract.

This composition partially contrasts with that reported in the literature. Numerous previous studies on the phenolic compounds of P. lentiscus and P. atlantica have revealed significant variability in their composition, influenced by the extraction and analytical methods employed, as well as by geographical factors such as climate, soil characteristics, altitude, and sun exposure.

For instance, Seddoqi et al. [56] reported, for P. lentiscus leaves collected in Morocco and analyzed by LC-MS/MS in positive mode, that epigallocatechin was the most abundant compound, whereas Rodriguez et al. [80] identified, for leaves of the same species collected in Algeria and analyzed by HPLC-ESI-QTOF in negative mode, catechin, β-glucogallin, and quercitrin gallate as the main constituents. Moreover, in another study, methyl gallate, gallic acid, rutin, and quercetin-3-O-glucoside were identified by RP-HPLC as the major constituents in the methanolic leaf extract of P. atlantica from Turkey obtained by maceration [87]. These differences highlight the uniqueness of our finding regarding methyl gallate as the major component.

Beyond methodological aspects, geo-environmental conditions may also shape phytochemical variability between samples. Phenolic biosynthesis is known to increase in response to abiotic stresses such as water deficit, intense sunlight, salinity, and thermal fluctuations, as well as seasonal variations, in addition to biotic pressures [88,89,90]. These adaptive defense mechanisms can therefore contribute to interspecific differences in phenolic profiles, reflecting the influence of the ecological conditions prevailing at the sampling sites.

The abundant presence of methyl gallate and myricetin derivatives is of particular interest given their multiple biological effects. Flavonoids are well known for their antimicrobial, anti-inflammatory, and strong antioxidant properties [91,92]. Methyl gallate, in particular, exerts various promising anti-inflammatory and dermatological effects, such as the inhibition of Cyclooxygenase-2 (COX-2) and leukotriene C4 (LTC4) production [42], an anti-aging mechanism via the inhibition of protein glycation [93], and a depigmenting effect through modulation of the Glycogen Synthase Kinase 3 beta (GSK3β/β) catenin pathway [94]. Similarly, myricetin (the aglycone of myricitrin and myricetin-3-O-hexoside) exhibits cutaneous anti-inflammatory properties and remarkable antioxidant activity, attributed to the presence of three hydroxyl groups on its B ring [95]. The richness of these extracts in such compounds therefore offers up promising application prospects in the field of skin care. Accordingly, we investigated the biological activities of these extracts.

Taken together, our findings show that the methanolic extract of P. atlantica exhibited the highest total polyphenol content, whereas P. lentiscus contained the highest flavonoid levels. Both extracts were dominated by methyl gallate and myricitrin, and displayed a high proportion of unsaturated fatty acids.

3.2. Biological Activities

Based on the chemical composition of P. lentiscus and P. atlantica leaf extracts and their traditional use in folk medicine for skin care, biological assays were conducted to evaluate their potential for dermo-cosmetic applications. Antioxidant, anti-inflammatory, and anti-tyrosinase activities were assessed to determine the suitability of these extracts as multifunctional active ingredients in innovative cosmetic formulations targeting skin protection, soothing, and brightening effects

3.2.1. Antioxidant Activities

The results of the DPPH and FRAP tests confirmed the strong antioxidant activity of the extracts from both plant species. The results of the DPPH assay (Figure 5a,

Table 2. Antioxidant, anti-inflammatory and anti-tyrosinase activities of methanolic extracts of P. lentiscus and P. atlantica.

Samples and Standards	Biological Activities IC50 (µg/mL) [a]
	DPPH	BSA Denaturation	LOX Denaturation	Anti-Tyrosinase
P. lentiscus	5.19 ± 0.01 ***	143.00 ± 0.70 ***	22.53 ± 0.05 ***	39.80 ± 0.08 ***
P. atlantica	5.61 ± 0.04 *###	139.10 ± 0.55 ***##	22.67 ± 0.04 ***ns	38.25 ± 0.02 ***###
Ascorbic acid	5.84 ± 0.05	-	-	-
Diclofenac	-	60.88 ± 0.03	-	-
Quercetin	-	-	17.91 ± 0.03
Kojic acid	-	-	-	34.28 ± 0.07

^[a] IC₅₀ value defined as half-maximal inhibitory concentration and expressed as mean ± SD (n = 3). DPPH: 2,2-diphenyl-1-picrylhydrazyl, LOX: lipoxygenase, BSA: bovine serum albumin and anti-tyrosinase. * denotes p-value < 0.05, *** p-value < 0.001, extracts vs. to standard; ^## denotes p-value < 0.01, P. lentiscus vs. P. atlantica; ^### denotes p-value < 0.001, P. lentiscus vs. P. atlantica; ^ns: no significant, comparison between P. lentiscus and P. atlantica.

) revealed the dose-dependent antioxidant activity of both extracts, with a slightly higher efficacy (p < 0.001) for P. lentiscus (IC₅₀ = 5.19 ± 0.01 µg/mL, ~11% more active) compared to P. atlantica (IC₅₀ = 5.61 ± 0.04 µg/mL, ~4% more active) relative to ascorbic acid. Both extracts demonstrated significant antioxidant activity when compared to ascorbic acid (IC₅₀ = 5.84 ± 0.05 µg/mL), with highly significant differences for P. lentiscus (p < 0.001) and significant to highly significant differences for P. atlantica (p < 0.05 to p < 0.001).

To further evaluate their antioxidant properties, the FRAP assay was performed to assess the reducing power of the extracts. The results (Figure 5b, Table 2) revealed a concentration-dependent increase in reducing activity for both extracts. P. lentiscus exhibited significantly higher FRAP values (p < 0.001) than P. atlantica at the same concentrations. At 100 µg/mL, P. lentiscus showed a reducing capacity close to that of Trolox, highlighting its strong antioxidant potential.

The strong antioxidant activity of both extracts, linked to their richness in phenolic compounds [61,96], was confirmed by our results using the DPPH assay. Therefore, the redox potential of phenolic compounds plays a crucial role in assessing the antioxidant potential of the extracts [97].

The observed variations can be largely attributed to the flavonoid richness of both extracts, known for their redox properties and their ability to donate electrons or hydrogen atoms, which is strongly associated with improved radical-scavenging and reducing capacities [98]. The higher content of these compounds in the P. lentiscus extract explains its greater DPPH·radical-scavenging activity [99]. These differences may also be reinforced by the specific predominance in P. lentiscus of other major antioxidants such as methyl gallate and myricitrin, two compounds well known for their strong antioxidant effects [95,100,101], due to their ability to neutralize the DPPH· radical and chelate iron ions [102,103,104] whose concentrations are higher in this extract.

Our findings are consistent with a previous study reporting that the methanolic extract of P. lentiscus displayed strong antioxidant activity, with an IC₅₀ value of 8 µg/mL, which is fairly close to our results (5.19 μg/mL). However, our results and those of Amessis-Ouchemoukh et al. [105] show significantly higher activity than those reported by Bourroubey et al. [106] and Hemma et al. [107], who obtained IC₅₀ of 0.06 mg/mL and 0.4 mg/mL, respectively. Furthermore, a study conducted in Morocco in 2022 [108] revealed lower activity (IC₅₀ of 27.22 µg/mL) compared to our findings. Similarly, in Algeria, Benabdellah et al. [109] reported that the 80% methanolic extract of P. atlantica showed high antioxidant capacity with an IC₅₀ of 5.61 µg/mL. These differences can be attributed to various exogenous factors, such as geographical origin, climatic conditions, harvesting period, extraction methods, and phytochemical composition of the extracts [110].

An important reducing capacity of Fe³⁺ to Fe²⁺ was also observed in both extracts. These results are consistent with previous studies carried out in Morocco and Greece, where leaf extracts from these species demonstrated, using the same method, a significantly higher iron-reducing capacity than reference standards [108,111]. This reducing power is due to their richness in polar compounds acting as hydrogen donors or electron transfer agents [112]. Moreover, the solubility of phenolic compounds, influenced by their degree of polymerization, the part of the plant used, and climatic conditions, also plays a role in this activity [61,62]. Since the two assays rely on different chemical principles, the variations observed in antioxidant activities can be explained by the nature of the reagents used: the DPPH method primarily assesses free radical scavenging ability, whereas the FRAP method evaluates reducing power [113].

The results of both antioxidant assays indicate that the leaf extracts of P. lentiscus and P. atlantica exhibit significant antioxidant activity, highlighting their promising potential as natural ingredients in anti-aging cosmetic formulations and as formulation-stabilizing agents.

3.2.2. Anti-Inflammatory Activities

Given the traditional use of Pistacia spp. to treat inflammatory skin conditions [114], the anti-inflammatory potential of P. lentiscus and P. atlantica was assessed through their ability to inhibit bovine serum albumin (BSA) denaturation and the activity of the lipoxygenase (LOX) enzyme activity, two key mechanisms involved in inflammatory processes. Both extracts demonstrated high to moderate anti-inflammatory activity, with similar overall efficacy; P. atlantica stood out for slightly greater inhibition in the BSA denaturation test, while P. lentiscus was more effective in inhibiting LOX (Figure 6). The results indicate that the protective effect against BSA denaturation was dose-dependent, with IC₅₀ values of 143.0 ± 0.70 µg/mL for P. lentiscus and 139.1 ± 0.55 µg/mL for P. atlantica. For comparison, diclofenac, used as a positive control, exhibited a stronger effect with an IC₅₀ of 60.88 ± 0.03 µg/mL. At higher concentrations (300 µg/mL), P. atlantica shows a slightly stronger inhibition than P. lentiscus (p < 0.01) (Figure 6a, Table 2). The anti-LOX activity of both extracts was strong, with IC₅₀ values of 22.53 ± 0.05 µg/mL for P. lentiscus and 22.67 ± 0.04 µg/mL for P. atlantica, with IC₅₀ values slightly higher than that of quercetin used as a standard (17.91 ± 0.03 µg/mL, ~26% less potent). The lipoxygenase inhibition curve (Figure 6b, Table 2) shows a concentration-dependent increase for both species, with values close to each other. Both extracts reached inhibition levels comparable to quercetin at 100 µg/mL. Statistical analysis revealed highly significant differences (p < 0.001) between the extracts and the positive control.

The results regarding the capacity to inhibit BSA denaturation surpass those reported by Bourroubey et al. [115]. Indeed, our extract achieved 75% inhibition at a concentration of only 400 µg/mL, while their study reported a maximum inhibition of 66.53% at a fivefold higher concentration (2000 µg/mL). Extending the comparison to other studies conducted on the same species but using different extraction solvents, Bakka et al. [116] reported that an ethyl acetate extract of P. atlantica exhibited a maximum inhibition of 97% at 10 mg/mL. In comparison, sodium diclofenac, used as the reference drug, achieved only 55.56% inhibition at the same concentration (10 mg/mL). Similarly, the ethanolic extract of P. lentiscus from southern Algeria inhibited protein denaturation by 92.65 ± 0.67% at 1000 µg/mL [117].

This enhanced potency could be attributed to the distinct climatic conditions of our collection sites, which may influence the composition and concentration of bioactive compounds. The observed anti-inflammatory effect appears to be related to the ability of the extracts to prevent the formation of autoantigens through the inhibition of protein denaturation [115]. This mechanism is thought to be attributable to the presence of bioactive compounds, especially flavonoids and tannins, which exert their effects not only through antioxidant activity but also by inhibiting pro-inflammatory enzymes, but may also modulate the expression of genes involved in the inflammatory response, representing an interesting avenue for future studies [118,119]. The inhibition of protein denaturation may also reflect a potential membrane-stabilizing effect, attenuating the amplification of the inflammatory process [120]. A similar mechanism is observed with certain nonsteroidal anti-inflammatory drugs, such as diclofenac, which bind to specific sites to prevent protein denaturation and the subsequent activation of the inflammatory cascade [121]. It should be noted that this inhibitory effect may decline at higher concentrations, likely due to the saturation of active sites or antagonistic interactions among certain polyphenols [56].

In parallel, the strong anti-inflammatory activity observed in both species may be closely linked to their phenolic profile, particularly the abundance of myricetin (the aglycone of myricitrin and myricetin-3-O-hexoside) and methyl gallate, known to act through the prevention of protein denaturation and the inhibition of LOX activity [95,122]. The anti-lipoxygenase potential of the extracts could also be attributed to other phenolic constituents, including isoquercitrin, gallic acid derivatives, and precursors of condensed tannins [114,123]. Isoquercitrin, a quercetin glycoside identified in the analyzed samples, is well known for its inhibitory affinity toward lipoxygenase [123,124]. The inhibition of this enzyme leads to a reduction in leukotriene synthesis, key mediators of inflammation, thereby limiting cell recruitment and the intensification of the inflammatory response [125]. Additionally, the antioxidant properties of the extracts, allowing the neutralization of reactive oxygen species (ROS) generated during inflammation, further support and strengthen their overall anti-inflammatory effect [106,123].

These results pave the way for improved valorization of these two extracts as natural sources of bioactive compounds, with potential applications in dermatological or cosmetic formulations aimed at reducing skin inflammation and its associated effects.

3.2.3. Anti-Tyrosinase Activity

The last aim of this study was to investigate the phenomenon of cutaneous hyperpigmentation, which manifests as the appearance of dark spots, mainly due to prolonged exposure to ultraviolet radiation or skin aging [32]. This pigmentary imbalance results from the excessive production of melanin, a pigment synthesized from L-tyrosine via a series of enzymatic reactions that are primarily regulated by three enzymes: tyrosinase, along with two associated proteins, Tyrosinase-Related Protein 1 (TRP-1) and Tyrosinase-Related Protein 2 (TRP-2) [126]. Since P. atlantica and P. lentiscus are traditionally employed in folk medicine for skin disorders, we aimed to evaluate their potential in inhibiting tyrosinase [13,14].

Methanolic extracts of the leaves of these two plant species showed dose-dependent inhibitory profiles against tyrosinase activity (Figure 7). Comparative analysis of IC₅₀ values indicated that P. atlantica exhibited slightly higher inhibitory potency (38.25 ± 0.02 µg/mL) than P. lentiscus (39.80 ± 0.08 µg/mL), although this difference was not statistically significant (Table 2). Remarkably, both extracts demonstrated comparable activity to kojic acid, the reference compound (IC₅₀ = 34.28 ± 0.07 µg/mL), with P. atlantica being only 11.58% less potent and P. lentiscus only 16.10% less potent than the reference standard (p < 0.001).

The dose–response curves revealed a notable convergence in maximal inhibitory efficacy between the tested extracts and the standard. At saturating concentrations (≈200 µg/mL), tyrosinase inhibition reached approximately 90% for all samples, suggesting a similar inhibition mechanism and a comparable efficacy plateau. These findings indicate that despite differences in potency (IC₅₀), Pistacia extracts retain a maximal inhibitory potential equivalent to that of kojic acid, a well-established pharmacological reference for this enzymatic activity.

To our knowledge, no previous studies have reported the anti-tyrosinase activity of methanolic extracts of P. lentiscus and P. atlantica. However, several studies have described this activity using other types of extracts. The 80% hydro-methanolic extract of P. atlantica leaves obtained by maceration exhibited an IC₅₀ of 153.07 ± 2.63 µg/mL [109]. Higher levels of activity were observed in other plant organs: the hydro-methanolic extract of leaf buds showed an EC₅₀ of 0.098 ± 0.00 mg/mL [19], while that of unripe fruits presented an EC₅₀ of 0.70 mg/mL [36].

Extracts obtained with different solvents have also demonstrated tyrosinase inhibitory activity, showing variability in their inhibitory potential. The ethyl acetate extract of P. lentiscus obtained by SPE exhibited an IC₅₀ of 123 µg/mL [32], while a hydro-methanolic extract achieved 78% inhibition with an IC₅₀ of 40 µg/mL, values comparable to those observed in the present study. For P. atlantica, Ghafari et al. [127] reported 59.07% inhibition, whereas Eghbali et al. [128] observed a reduction in cellular tyrosinase activity and melanin content, suggesting a potential intracellular effect.

The observed activity may be attributed to the presence of phenolic compounds and flavonoids that can interact with the active site of tyrosinase [129]. As reported by Lee et al. [130], polyphenols and flavonoids exhibit a strong inhibitory potential against tyrosinase and its related proteins (TRP-1 and TRP-2), and certain toxic and mutagenic compounds [131]. To date, polyphenols also represent the largest group of tyrosinase inhibitors [132]. It can also be assumed that the tannin content in P. lentiscus and P. atlantica may precipitate the tyrosinase enzyme, thereby inhibiting its enzymatic activity in the reaction medium. These constituents are readily soluble in methanol and have shown strong tyrosinase inhibitory activity in different plants [133,134]. Kishore et al. [135] also reported that flavonol glycosides have significant anti-tyrosinase activity.

Beyond the properties already highlighted in our study, the leaf extracts of P. lentiscus and P. atlantica also demonstrate a notable ability to modulate the mechanisms involved in skin pigmentation. This natural activity makes them promising candidates for the development of innovative, safe, and effective products aimed at evening out skin tone and preventing pigmentation disorders. with a lower risk of adverse effects compared to conventional agents such as kojic acid.

In summary, the methanolic leaf extracts of P. lentiscus exhibited stronger radical scavenging capacity, reducing power, and anti-inflammatory activity through albumin denaturation inhibition. In contrast, P. atlantica showed slightly higher anti-tyrosinase activity. Moreover, both extracts demonstrated strong LOX inhibition, further supporting their potential as natural bioactive agents for managing skin inflammation and hyperpigmentation.

4. Conclusions

This study demonstrates for the first time the complementary bioactivities of P. lentiscus and P. atlantica leaf extracts, providing a solid scientific rationale for their incorporation into cosmeceutical formulations targeting oxidative stress, inflammation, and hyperpigmentation. The LC-MS/MS profiling highlighted phytochemical richness dominated by methyl gallate and myricetin derivatives, which supports the observed antioxidant, anti-inflammatory and anti-tyrosinase effects.

P. lentiscus exhibited a stronger capacity to counteract oxidative stress and inhibit melanogenesis, while P. atlantica showed superior anti-inflammatory potential, suggesting distinct and complementary dermoprotective applications. These findings validate their traditional use and position both species as promising natural alternatives to synthetic skin-care agents, in line with the current interest in plant-derived cosmetic agents.

Future investigations should focus on comparative evaluation of methanolic versus ethyl acetate extracts to assess polarity-dependent cosmeceutical efficacy, cytotoxicity assessments to confirm safety profiles, and in silico evaluations to predict molecular interactions and biological targets. These approaches would contribute to the scientific understanding and potential utilization of these Mediterranean species by supporting the development of value-added formulations, while encouraging responsible management practices to preserve natural populations.