Chemical, Mineralogical, and Biological Properties of Pistacia atlantica subsp. atlantica Essential Oils from the Middle Atlas of Morocco

Abstract

This study focused on Atlas pistachio (Pistacia atlantica subsp. atlantica), an endangered species from the Moroccan Middle Atlas, analyzing its leaves to assess their antioxidant and antimicrobial activity. Essential oils (EOPA) were extracted by distillation using a Clevenger apparatus, and their phytochemical compounds were identified by gas chromatography–mass spectrometry (GC/MS). Antioxidant activity tests were carried out using the DPPH and FRAP methods. In addition, antimicrobial activity was tested against Candida albicans to determine its antifungal effect, and against two Gram-positive strains (Staphylococcus aureus and Bacillus subtilis) and three Gram-negative strains (Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae) to determine the antibacterial effect. The results show that the essential oils contained between 23 and 49 compounds, depending on the extraction area, with (-)-germacrene D as the main compound. Antioxidant activity varied by study area, with IC₅₀ ranging from 0.414 mg/mL (Amghas) to 1.520 mg/mL (Ait Naamane), and EC₅₀ from 2.132 mg/mL to 5.4 mg/mL. In terms of antimicrobial activity, Afourgah essential oils showed the best results, with significant inhibition diameters against bacteria and low MIC. In particular, Amghas essential oils inhibited Staphylococcus aureus well, while Ait Naamane essential oils were less effective. This variability in phytochemical composition, as well as antioxidant and antimicrobial activities, may be attributed to climatic differences specific to the distribution zones of the Atlas pistachio tree. This study contributes to a better understanding of the botanical and chemical characterization of the Pistacia genus, and highlights its potential as a source of bioactive agents.

1. Introduction

The genus Pistacia, part of the botanical family Anacardiaceae comprising over 70 genera and more than 600 species, is mainly found in Mediterranean regions in the southern United States, Mexico, and in the Middle East [1]. Pistacia atlantica subsp. atlantica is commonly known as “Lbtom” in Darija and “Ijj”, “Tijout”, or “Tijft” in the Berber dialects of the Middle Atlas in Morocco. This hardy, woody tree is native to north Africa and develops an impressive silhouette when mature [2]. P. atlantica Desf. has been used for various purposes over time, including culinary, medicinal, fodder, and wood-related applications [3]. This taxon, comprising both deciduous and evergreen shrubs and trees, is well known for its resin production and remarkable adaptability to extremely arid environments, typically growing to heights of 8 to 10 m [4]. Species such as Pistacia terebinthus, P. atlantica Desf., P. lentiscus L., P. vera L. and P. khinjuk Stocks are found from the Mediterranean basin to central Asia. In Morocco, three species occur naturally—P. atlantica subsp. atlantica, P. terebinthus L. and P. lentiscus L. Of these species, P. vera is the only one cultivated commercially, while the others are mainly used as rootstocks [5]. Furthermore, this genus, rich in phenolic compounds, terpenoids, monoterpenes, flavonoids, alkaloids, saponins, fatty acids, sterols and fibers, is attracting growing interest in the pharmaceutical field. Research shows that these species are traditionally used to treat conditions such as asthma, rheumatism, hypertension, diabetes, diarrhea and hemorrhoids. They also possess antioxidant, antimicrobial, antiviral, anticholinesterase, anti-inflammatory, antinociceptive, antidiabetic, antitumor, antihyperlipidemic, antiatherosclerotic and hepatoprotective properties. Different parts of Pistacia species have been studied for their various biological activities; food uses are also reported and Pistacia essential oils are used in the cosmetics, pharmaceutical and food industries [6,7,8,9,10].

This study aims to assess the therapeutic potential of Atlas pistachio (Pistacia atlantica subsp. atlantica: PA) by analyzing its medicinal properties in three distinct bioclimatic zones of the Middle Atlas in Morocco. This research aims to examine the intraspecific variability of this species, as well as its antioxidant and antimicrobial efficacy, by identifying the active compounds present in the plant’s essential oils. It also highlights the variations in therapeutic potential depending on the ecological conditions of different zones. The study could thus contribute to enhancing the value of this species while supporting its conservation, given its “near-threatened” status on the red list.

2. Materials and Methods

2.1. Plant Materials

The atlas pistachio leaves used in this study were collected in three regions of the Middle Atlas of Morocco, more precisely, in the areas of Aït Naamane (33°41′56.1″ N, 5°21′23.2″ W), Lac Afourgah (33°36′45.9″ N, 4°53′02.6″ W) and Amghas (33°68′63.9″ N, 5°37′01.1″ W), in June 2023 (Figure 1).

2.2. Mineralogical Analysis

Mineralogical analysis was carried out on the residue of a product calcined at 500 °C for 5 h. Samples weighing 2 g were taken from various parts of the plant, including empty flower heads, seeds, leaves and roots. The quantification of trace elements such as Ca, K, Mg, Na, P, Cl, Cu, Fe, Zn and Se was carried out using the aqua regia method, following the procedure described by [11] with some modifications. This method involves dissolving 0.15 g of mineral material in a mixture of hydrochloric acid and nitric acid. The mixture is then heated on a hot plate and evaporated to dryness. After this, hydrochloric acid (2 M) is added until complete dissolution, and analysis is carried out by inductively coupled plasma atomic emission spectrometry (ICP-AES) coupled with argon plasma.

2.3. Phytochemical Analysis

2.3.1. Preparation of Extracts

The harvested leaves were air-dried for two weeks, crushed and then stored in the laboratory. Extraction was carried out with 70% ethanol, chosen for its efficiency, speed, ease of evaporation and low toxicity as a green solvent.

2.3.2. Phytochemical Screening

To diagnose the chemical differences between the leaves of PA in different areas, we first carried out a phytochemical screening for the following:

• Tannins—The presence of tannins can be detected by mixing 1 mL of the extract with 1 mL of 1% FeCl₃ solution, resulting in a greenish or blackish coloration [12];
• Catechins—Boiling a mixture of 5 mL infusion and 1 mL concentrated hydrochloric acid for 15 min produces a red precipitate that dissolves in amyl alcohol, indicating the presence of catechins [12];
• Gallotannins—After heating a mixture of 30 mL infusion solution and 15 mL Stianini reagent in a water bath for 15 min, the precipitate is filtered off and the filtrate is saturated with 5 g sodium acetate powder. We then add 1 mL of 1× Cl₃ solution dropwise. The formation of a precipitate indicates the presence of gallotannins [12];
• Flavonoids—Add 1 mL infusion to 1 mL hydrochloric alcohol, then add magnesium chips and 1 mL isoamyl alcohol. A pink-orange (flavones), purple-pink (flavonone) or reddish (flavanol, flavanol) color appears in the isoamyl alcohol layer of the supernatant, indicating the presence of free flavonoids (genin) [12];
• Saponins—The presence of saponins is determined by vigorously shaking a mixture of 0.2 mL extract and 5 mL distilled water for 5 min. The persistence of foam after shaking indicates the presence of saponins [13];
• Sterols (Shalkowski test)—To identify sterols, 1 mL H₂SO₄ is added to the extract. The formation of a reddish-brown ring at the interface between the two liquids confirms the presence of sterols [14];
• Alkaloids—Two manipulations were carried out to identify [13] alkaloids.
• Manipulation 1: For the Dragendorff test, a mixture of 1 mL extract and 1 mL Dragendorff reagent was prepared. The formation of an orange cloud indicates the presence of alkaloids.
• Manipulation 2: In the Meyer test, a mixture of 1 mL extract and 1 mL Meyer’s reagent was prepared. The presence of alkaloids is indicated by a yellow color;
• Cardiac glycosides—The presence of cardiac glycosides was determined by mixing 2 mL of chloroform with 1 mL of the extract. The addition of H₂SO₄ resulted in the formation of a reddish-brown color, indicating the presence of cardiac glycosides [15];
• Oses and holosides—To detect the presence of oses and holosides, 1 mL of the extract was added to 2–3 drops of concentrated sulfuric acid. After 5 min, 3–4 drops of saturated thymol alcohol were added, producing a red coloration [16];
• Mucilage—Mucilage was detected by combining 1 mL of the extract with 5 mL of pure alcohol. The appearance of granular sediment confirmed the presence of mucilage [16].

2.3.3. Gas Chromatography Coupled with Mass Spectrometry (GC/MS)

Here, 200 g of leaf powder were air-dried for 3 h, then hydro-distilled with a Clevenger apparatus to obtain the essential oils EOPAA1 (essential oil of Pistacia atlantica in area 1), EOPAA2 (essential oil of Pistacia atlantica in area 2) and EOPAA3 (essential oil of Pistacia atlantica in area 3). This essential oil was dried over anhydrous sodium sulfate and stored at 4 °C in a dark environment. For analysis by gas chromatography–mass spectrometry, the essential oil was diluted with hexane.

The essential oil yield was calculated using the following formula:

$$R={\displaystyle \frac{M\prime }{M}}\times 100$$

(1)

where R is the essential oil yield in %, M′ represents the mass of the recovered essential oil in grams, and M is the mass of the plant material in grams.

The phytochemical characterization of the essential oil was performed by GC-MS using a nonpolar silica column. To achieve this, the operating conditions for the analysis were as follows: the initial temperature was set to 40 °C for 2 min with a ramp rate of 2 °C/min, while the final and injector temperatures were set to 260 °C for 10 min and 250 °C, respectively. In this analysis, helium gas was used as the carrier gas (1 mL/min) with a “split” mode injection. The ionization energy and ion source temperature were set to 70 eV and 200 °C, respectively, and the scan mass range was m/z 40–650. The oil was diluted in hexane solvent (10:100) before being injected with 1 µL. The chemical identification was performed using retention indices (RI) along with a comparison to the ADAMS database [17].

2.3.4. Study of Antioxidant Activity

• DPPH free radical scavenging test

To measure the antioxidant activity of the essential oils EOPAA1 (essential oil of Pistacia atlantica in area 1), EOPAA2 (essential oil of Pistacia atlantica in area 2) and EOPAA3 (essential oil of Pistacia atlantica in area 3) via the DPPH assay, we prepared ten tubes for each EO, each containing 750 µL of DPPH (4 mg DPPH in 100 mL methanol). We added 250 µL of each EO dilution, incubated the tubes in the dark for 30 min, and then measured optical density at 517 nm. The results are expressed as percentage inhibition versus a positive control (quercetin), calculated using the formula % inhibition = (A₀ − AE₀/A₀) × 100, where A₀ is the optical density of the negative control and AE₀ that of the EO [18].

• Ferric antioxidant reducing power test (FRAP)

The reducing power test was carried out according to the method described by [19], with some minor modifications; we added 750 µL of phosphate buffer and 750 µL of potassium ferricyanide to 150 µL of samples prepared in methanol. After incubation at 50 °C for 20 min, we acidified the mixture with 750 μL of trichloro acetic acid (TCA) and centrifuged it. The upper solution was mixed with distilled water and FeCl₃, and absorbance was measured at 700 nm.

• Docking studies

To explore the mechanisms of the inhibition of antioxidant activity in Pistacia atlantica subsp. atlantica leaves, a molecular docking technique was performed between the NADPH oxidase protein encoded in the protein data bank (PDB) by 2CDU.pdb and germacrene D as well as α pinene, the main compounds extracted from the essential oils of the leaves studied. Initially, the target protein was prepared by adding Gasteiger fillers and removing water molecules and any co-crystallized ligands bound to the receptor protein [20,21,22]. Next, Autodock 4.2 software was used to dock the protein prepared in PDBQT format with the main compounds extracted from Pistacia atlantica leaves. Finally, the interactions produced were visualized using Discovery Studio 2021 software in two and three dimensions [23,24,25].

2.4. Assessment of the Essential Oil Antimicrobial Properties

The antimicrobial activity of the essential oil was evaluated on fungal and bacterial strains (

Table 1. Microbial strains used for antibacterial and antifungal activity.

Staphylococcus aureus	ATCC 6633
Escherichia coli	K12
Bacillus subtilis	DSM 6333
Klebsiella pneumoniae	CIP A22
Pseudomonas aeruginosa	CIP 82.114
Candida albicans	ATCC 10231

) supplied by the Laboratory of Biotechnology in the Faculty of Sciences Fez, Morocco [26]. Two complementary methods were employed.

2.4.1. Disc Broad Casting Method

This method was used to assess the zone of inhibition of microbial growth on solid media. Petri dishes containing 20 mL of agar medium (Muller–Hinton for bacteria or malt extract for fungi) were prepared. A standardized microbial suspension (10⁶–10⁸ CFU/mL) was spread evenly over the surface of the medium. Here, 5 mm discs impregnated with 5 µL of essential oil were placed on the medium. After incubation for 48 to 72 h at 37 °C, the diameters of the inhibition zones were measured [27,28].

2.4.2. Liquid Microdilution Method

The liquid microdilution method was used to determine the minimum inhibitory concentration (MIC) of the essential oil. A 96 well microplate was used, with an appropriate medium (Muller–Hinton for bacteria or malt extract for fungi) and the essential oil diluted in DMSO (5%) added to the first column. Successive dilutions were made to establish a range of concentrations, followed by the addition of a standardized microbial suspension. After incubation for 24 h at 37 °C, the MIC was identified by adding 20 µL of 0.1% resazurin; a color change indicated microbial growth, while no change signaled complete inhibition [27].

3. Results and Discussion

3.1. Mineralogical Analysis

Table 2. Mineral content (mg/kg) of leaves from the three study areas (LPA1—leaves from Amghas, LPA2—leaves from Afourgah and LPA3—leaves from Ait Naamane).

	Ca	Cu	Fe	K	Mg	Mn	Na	P	Pb	Se	Zn
LPA1	2005.56	0.42	39.02	1164.96	553.37	2.85	55.13	144.76	0.17	<0.02	1.68
LPA2	>2250.00	0.53	34.80	1229.51	559.60	3.64	160.23	102.69	<0.02	<0.02	1.78
LPA3	>2250.00	0.49	31.67	1001.01	356.72	1.90	147.22	96.98	0.22	<0.02	1.40

highlights significant differences in the mineralogical compositions of PA leaves from three bioclimatic zones. Concentrations of mineral elements vary according to climatic stages in each zone, reflecting adaptive responses to specific environmental conditions. Potassium (K) displays remarkably high levels in all zones, exceeding the measurement limit (>2250.00 mg/kg) for leaves of LPA2 and LPA3, underlining its essential role in adaptation to stressful environments. Calcium (Ca) is also dominant, with a particularly high concentration in LPA2 (>2250.00 mg/kg), which could reinforce cell structure under dry conditions. Iron (Fe) shows notable variability, ranging from 1164.96 mg/kg (LPA1) to 1001.01 mg/kg (LPA3), suggesting sensitivity to local variations in soil availability. Minerals play a crucial role in metabolic processes, being essential for cell growth and differentiation [29]. A study of Pistacia atlantica subsp. mutica and cabulica oils in Iran revealed high concentrations of iron (Fe), reaching 382 mg/kg for cabulica and 69 mg/kg for mutica, as well as the presence of minerals such as lead and copper. PA leaves show significant levels of potassium, sodium, calcium and lithium, with traces of barium detected [30]. These observations highlight the importance of minerals in the composition of PA oils and leaves, suggesting potential implications for nutrition and health. Concerning macro and micromineral analysis, high concentrations were observed for most minerals (such as Ca, K, Mg, Na, Fe and P) in AP leaves. Divergent results from [25,26] show different percentages compared to our values. Ref. [31] notes that the order of mineral content in two true pistachio cultivars was as follows: K⁺ > Fe⁺² > Ca⁺² > Na⁺ > Zn⁺² > Mn⁺² > Cr⁺³. It should be noted that mineral concentrations in nuts are influenced by soil properties, water quality and geographical conditions.

3.2. Phytochemical Screening

The phytochemical screening of PA leaf extracts reveals the presence of various bioactive secondary metabolites, such as flavonoids, sterols, terpenes, gall tannins, saponosides and cardiac glycosides (

Table 3. Results of phytochemical screening of hydroethanolic extracts from the leaves.

Secondary Metabolites		Extract of PA
Tannins		+
Tannins	T. Catechicals	−
	T. Gallics	+
Flavonoids		+++
Saponosides		++
Sterols and terpenes		+++
Alkaloids		−
Cardiac glycosides		++
Oses and holosides		++
Mucilage		++

Very positive reaction (+++); positive reaction (++) ; moderately positive reaction (+) ; negative reaction (−).

). These compounds are known for their antioxidant, antimicrobial and cardioprotective properties [32,33].

3.3. Phytochemical Analysis by GC-MS

The method of extraction of the essential oils studied revealed yields of 0.3%, 0.25%, and 0.10% for samples from Amghas, Afourgah, and Ait Naamane, respectively. GC-MS analysis of the three essential oils (EOPAA1, EOPAA2, and EOPAA3) revealed a chemical composition dominated by hydrocarbon sesquiterpenes (ST.H) and hydrocarbon monoterpenes (MO.H), with proportions varying among samples. Totals of 34, 33, and 36 compounds were identified in EOPAA1, EOPAA2, and EOPAA3, respectively, representing total percentages of 99.9%, 99.9%, and 99.1% (

Table 4. Chemical composition of EOPA.

R.T (min)	Compounds	R.I		Chemical Class	Area (%)
		Cal	Lit		EOPAA1	EOPAA2	EOPAA3
7.59	Tricyclene	924	926	MO.H	-	-	0.4
7.67	α-Thujene	930	930	MO.H	-	0.4	-
7.90	α-Pinene	932	933	MO.H	-	6.5	9.9
8.37	Camphene	945	949	MO.H	-	1.6	1.9
9.03	Sabinene	971	975	MO.H	-	6.4	0.5
9.18	β-Pinene	977	980	MO.H	-	1.3	3.6
9.48	Myrcene	987	990	MO.H	-	1.1	1.5
10.00	α-Phellandrene	1000	1002	MO.H	-	-	0.4
10.32	α-Terpinene	1015	1018	MO.H	-	0.8	-
10.55	o-Cymene	1024	1026	MO.H	-	4.9	3.4
10.70	D-Limonene	1028	1029	MO.H	-	1.6	8.7
10.79	Eucalyptol	1030	1031	MO.O	-	6.9	-
11.57	γ-Terpinene	1055	1059	MO.H	-	1.9	-
12.39	Terpinolene	1085	1088	MO.H	-	0.8	0.8
14.05	Pinocarveol	1135	1139	MO.O	-	-	1.0
14.22	Norbornen-2-ol acetate	1085	1090	O	-	1.1	-
14.96	Borneol	1165	1169	MO.O	-	1.2	-
15.21	p-Menth-2-en-1-ol	1120	1121	MO.O	-	-	1.3
15.28	Terpinen-4-ol	1175	1177	MO.O	7.9	-	-
15.36	p-Cymen-7-ol	1287	1290	MO.O	-	-	0.7
15.60	endo-Fenchol	1285	1288	MO.O	-	-	2.5
15.63	Fenchone	1084	1086	MO.O	-	2.2	-
15.69	Terpineol	1196	1199	MO.O	0.9	8.1	-
18.15	Bornyl acetate	1285	1288	O	-	1.4	1.4
19.09	1.4-cineole	1011	1014	MO.O	-	-	0.7
19.13	Anethole	1250	1252	MO.O	-	0.9	-
19.58	Elemene	1333	1335	ST.H	1.7	1.4	-
19.81	α-Terpinyl acetate	1313	1317	O	-	0.4	-
19.89	α-Cubebene	1345	1348	ST.H	-	-	0.4
20.51	Ylangene	1420	1420	ST.H	-	-	0.4
20.67	α-Copaene	1370	1374	ST.H	-	-	1.0
20.89	β-Bourbonene	1385	1388	ST.H	-	-	0.4
21.00	β-Elemene	1588	1589	ST.H	-	0.5	-
21.10	α-Gurjunene	1407	1409	ST.H	-	-	1.2
21.92	Caryophyllene	1462	1464	ST.H	7.8	1.6	3.8
22.12	Germacrene B	1560	1561	ST.H	0.9	1.4	0.8
22.17	β-Copaene	1430	1432	ST.H	1.5	0.7	0.6
22.20	Phenol acetate	1440	1442	MO.O	-	0.4	-
22.23	β-Ionone	1432	1436	O	-	-	0.5
22.31	Aromandendrene	1440	1441	ST.H	-	0.6	0.7
22.62	Cadalene	1674	1676	ST.H	-	0.7	-
22.71	Maaliol	1566	1567	ST.O	1.0	-	-
22.73	α-Humulene	1450	1454	ST.H	-	-	2.1
22.85	Indole	1290	1291	O	-	0.6	-
22.96	Isolongifolene	1388	1390	ST.H	1.0	-	-
23.29	γ-Cadinene	1510	1513	ST.H	2.3	0.6	2.8
23.30	α-Amorphene	1484	1484	ST.H	-	-	0.7
23.46	Germacrene D	1486	1485	ST.H	26.5	10.2	5.0
23.63	γ-Muurolene	1477	1479	ST.H	-	-	2.2
23.72	Viridiflorine	1495	1496	ST.H	2.2	1.2	-
23.83	Bicyclogermacrene	1730	1734	ST.H	6.6	3.5	-
23.86	Verbenone	1206	1205	MO.O	-	-	1.1
24.24	α-Cadinene	1537	1538	ST.H	1.3	0.9	3.4
24.26	Cadinene ether	1550	1553	ST.O	-	-	6.1
24.33	cis-Calamenene	1710	1713	ST.O	-	-	1.3
24.36	δ-Cadinene	1801	1803	ST.H	5.3	2.0	-
24.85	Cadalene	1675	1676	ST.H	-	-	0.8
25.01	Elemol	1546	1549	ST.O	-	0.4	0.5
25.21	Germacra-4(15),5,10(14)-trien-1α-ol	1684	1686	ST.O	-	-	4.7
25.75	Hexenyl benzoate	1563	1566	O	1.7	-	-
26.01	Spathulenol	1575	1578	ST.O	15.6	5.5	3.8
26.06	Alloaromadendrene oxide	1640	1641	ST.H	-	0.9	0.6
26.29	Globulol	1588	1590	ST.O	2.1	1.6	0.7
26.55	Viridiflorol	1590	1592	ST.O	1.4	1.7	-
27.13	Caryophyllene oxide	1580	1583	ST.O	1.4	-	-
27.38	Junenol	1617	1619	ST.O	1.1	-	-
27.50	γ-Himachalene	1480	1482	ST.H	-	1.0	-
27.93	α-Muurolol	1642	1646	ST.O	7.9	4.2	5.9
28.00	α-Cadinol	1650	1654	ST.O	0.8	-	0.8
28.23	α-muurolene	1766	1768	ST.H	-	-	1.0
29.21	Eudesma-4,11-dien-2-ol	1807	1808	ST.O	1.0	-	1.2
32.44	Catechol	1255	1258	O	-	0.6	1.0
32.75	Isophytol	1944	1947	DT.O	-	-	2.1
33.28	Dehydro aromadendrane	1460	1462	ST.H	-	0.8	1.4
38.32	Phytol	1940	1943	DT.O	-	0.5	1.4
		Chemical class
		Monoterpene hydrocarbon (MO.H)			0	34.2	31.1
		Monoterpene oxygenated (MO.O)			8.8	19.7	7.3
		Sesquiterpene hydrocarbon (ST.H)			57.1	28	29.3
		Sesquiterpene oxygenated (ST.O)			32.3	13.4	25
		Diterpene hydrocarbon (DT.H)			0	0	0
		Diterpene oxygenated (DT.O)			0	0.5	3.5
		Others (O)			1.7	4.1	2.9
		Total (%)			99.9	99.9	99.1

R.T: retention time. R.I: retention index. Cal: calculated. Lit: literature. MO.H: monoterpene hydrocarbon. MO.O: monoterpene oxygenated. ST.H: sesquiterpene hydrocarbon. ST.O: sesquiterpene oxygenated. DT.H: diterpene hydrocarbon. DT.O: diterpene oxygenated. O: others.

and Figure 2). EOPAA1 is characterized by a high content of hydrocarbon sesquiterpenes (57.1%) and oxygenated sesquiterpenes (32.3%). The main compounds identified are germacrene D (26.5%), spathulenol (15.6%), and caryophyllene (7.8%) (Figure 3). EOPAA2 has a higher content of hydrocarbon monoterpenes (34.2%) and oxygenated monoterpenes (19.7%), with a dominance of α-pinene (6.5%), D-limonene (8.7%), and germacrene D (10.2%). EOPAA3 exhibits an intermediate profile, characterized by a notable proportion of oxygenated sesquiterpenes (25%) and a more pronounced presence of oxygenated diterpenes (3.5%). This sample is dominated by α-pinene (9.9%), D-limonene (8.7%), and cadinene ether (6.1%).

Among the most important compounds are terpenes, flavonoids, tannins, steroids, fatty acids, and essential oils, as well as other substances found in the fruits, leaves, bark of the trunk, bark of the roots, and resin of these subspecies. Some of these compounds have demonstrated various biological activities, both in vivo and in vitro, and some are the subject of patents. To date, over 150 compounds have been isolated and characterized from the subspecies of P. atlantica [34].

Our results are in line with some previous studies while diverging from other research, for example, the Moroccan study by [35], which revealed that the chemical composition of essential oils was determined by tree diameter, which is linked to tree age. Thus, terpinen-4-ol (19.00–22.33%) was more prevalent in trees with a diameter of 23.73 cm or more, while α-pinene (18.49–37.51%) predominated in trees with a diameter of 22.45 cm or less. In Iran, PA essential oil was mainly composed of α-pinene, camphene, β-pinene, D-limonene, cyclohexene and carene. As for the essential oil of P. khinjuk, it mainly contained α-pinene, β-pinene, trans-verbnol, bicycle (3.1.1.) heptane, verbene, camphene, D-limonene and α-campholenal [36].

Other studies conducted by [37] examined the intraspecific diversities of three populations of P. atlantica distributed along an aridity gradient. They found that the terpene concentration increased with aridity, with the most arid site showing higher concentrations of monoterpenes (136 μg/g dry weight) and sesquiterpenes (290 μg/g dry weight). Spathulenol and α-pinene were proposed as chemical markers of aridity.

3.4. Antioxidant Activity of EOPA in the Three Zones

Three essential oils were analyzed for their antioxidant activity using two methods, namely, DPPH and FRAP. Ascorbic acid and BHT were used as references (

Table 5. Antioxidant activity of a few synthetic antioxidants as measured by the DPPH test and FRAP assay.

Artificial Antioxidant	IC50 (mg/mL)	EC50 (mg/mL)
BHT	0.009 ± 0.0001	1.165 ± 0.114
Ascorbicacid	0.001 ± 0.001	0.003 ± 0.001

).

The results show that the antioxidant activity of Pistacia atlantica essential oils (EOPA) varies according to the bioclimatic zone. In zone 1 (semi-arid), EOPA has a mean IC₅₀ of 0.414 ± 0.010 mg/mL, while in zone 2 (subhumid to temperate winter), mean IC₅₀ values are 0.931 ± 0.008 mg/mL. In zone 3 (subhumid to cold winter), EOPA achieves a mean IC₅₀ of 1.520 ± 0.011 mg/mL. These data show gradual variations in IC₅₀ across the three bioclimatic zones (

Table 6. Antioxidant activity of essential oil extracted from PA of different areas using the DPPH protocol.

	IC50 (mg/mL)
Sample	Max	Min	Mean ± SD
EOPAA1	0.431	0.394	0.414 ± 0.010
EOPAA2	0.945	0.915	0.931 ± 0.008
EOPAA3	1.532	1.498	1.520 ± 0.011

and

Table 7. Antioxidant activity of essential oil extracted from PA of different areas using the FRAP protocol.

	EC50 (mg/mL)
Area	Max	Min	Mean ± SD
EOPAA1	2.183	2.09	2.132 ± 0.034
EOPAA2	5.18	4.6	4.895 ± 0.195
EOPAA3	5.81	4.98	5.4 ± 0.277

).

Essential oils of Pistacia atlantica (EOPAA) from three climatic zones were evaluated for antioxidant activity using the ferric reducing antioxidant power (FRAP) method. In the semi-arid zone (area 1), EOPAA1 displays the best antioxidant activity with a low EC₅₀ (~1 mg/mL). In the subhumid to temperate winter zone (area 2), activity decreases, with an EC₅₀ of 4 mg/mL for EOPAA2, while in the subhumid to cold winter zone (area 3), a similar trend is observed for EOPAA3 (~5 mg/mL). So, overall, essential oils from the leaves of the Atlas pistachio tree show a better antioxidant capacity, particularly in semi-arid environments.

Indeed, PA leaves and fruits showed comparable or significantly higher antioxidant activity than standard antioxidant compounds in various in vitro tests [38,39,40].

In contrast, essential oil from Pistacia atlantica leaves showed relatively low antioxidant activity in the DPPH assay, compared with synthetic antioxidants [41]. It has also been suggested that the roasting process may increase the antioxidant activity of fruits [42]. Little research has been devoted to assessing the antioxidant properties of essential oils from Pistacia species [9,43,44]. In general, DPPH tests have shown that these essential oils possess low free radical scavenging capacity in organic solutions. Similar observations were reported by [41].

In their work, Gourine et al. [40] examined the seasonal variations in the chemical composition and antioxidant activity of essential oil from Pistacia atlantica Desf. leaves over a period of June to October. They found that the IC₅₀ values ranged from 8.5 to 27.9 mg/mL, with antioxidant activity generally higher for essential oils from male plants. Several observations have made it possible to distinguish essential oils from the different locations studied. This differentiation is based on the percentage distribution of the main compounds present in these essential oils. It was observed that, within the same locality, some samples were particularly rich in a specific compound, while others had low or even very low levels of the same compound. For example, some samples were high in terpinen-4-ol, with values ranging from 10.83 to 15.58%, while others were very low, with levels ranging from traces to 2.13%. This observation reinforces the hypothesis of the existence of chemotypes in essential oils, which would explain the variability of results obtained for the same Pistacia atlantica essential oil [45]. The results of our study are broadly in line with those reported by [46], who identified samples rich in α-pinene, (-)-germacrene D and β- pinene. However, our work has highlighted other rarely reported major compounds, such as caryophyllene, spathulenol, eucalyptol and D-limonene. As previously mentioned, essential oils from different localities in the Moroccan Middle Atlas strongly suggest the presence of distinct chemotypes.

3.5. Docking Studies

The results of the molecular docking study (Figure 4) show that the compound (-)-germacrene D extracted from Pistacia atlantica leaves in zones 1 and 2 was complexed with the protein NADPH oxidase with a binding energy of −8.11 kcal/mol, revealing various intermolecular interactions, including four alkyl bonds detected towards the amino acid residues His10, Ala300, Ala303 and Ala11. Similar was seen for α-pinene. The main compound in zone 3 was docked with the same target protein, exhibiting a binding energy of −6.26 kcal/mol, displaying almost similar chemical interactions, such as alkyl bonds created with amino acid residues His10, Ala300, Ala11 and Ala303, as well as an additional Pi–alkyl bond produced towards the active site Ala11, as shown in Figure 4. The results obtained are in good agreement with those from our previous studies, where the two main compounds were also docked to the active sites of the responsible protein in complex with its co-crystallized ligands, such as those detected on amino acid residues Ala11 and Ala300 [25].

3.6. Assessment of the Oil’s Antimicrobial Properties

3.6.1. Agar Diffusion Method

The zones of inhibition of the various strains with the different essential oils are shown in Figure 5. The results of the antimicrobial activity of the three essential oils (EOAA1, EOAA2, EOAA3) reveal notable variations according to the bacterial and fungal strains tested. EOAA2 showed the best overall activity, notably against Bacillus subtilis, Pseudomonas aeruginosa and Candida albicans, with zones of inhibition of 16.2 mm, 15.3 mm and 10.9 mm, respectively. EOAA1 showed strong activity against Staphylococcus aureus (19.37 mm) and moderate inhibition against Escherichia coli (4.467 mm) and Klebsiella pneumoniae (7.1 mm). In contrast, EOAA3 showed weaker zones of inhibition in most tests, with the greatest activity against Escherichia coli (7.3 mm), and weaker inhibition against Staphylococcus aureus (6.033 mm) and Pseudomonas aeruginosa (5.967 mm). EOAA3 oils showed no inhibitory activity against the clinical strain Candida albicans (Figure 6). This could be explained by the absence of substances with antifungal properties [47].

The antimicrobial activity of essential oils is attributed to the various agents present, such as flavonoids, terpenes and phenols [48]. The antibacterial power of the essential oils tested is linked to the origin of the sample. The phenomenon of aridity, which varies from one area to another, could significantly influence the efficacy of these essential oils in certain cases. In addition, the activity of these oils depends on their concentration and the nature of the strains tested [49].

3.6.2. Disc Distribution Method

Figure 7 shows the minimum inhibitory concentration (MIC) results for the three Pistacia atlantica oils, revealing notable variations in their antimicrobial efficacy depending on the strains tested. The oils showed moderate inhibition against Escherichia coli (MICs of 1.875 ± 0.8839 and 1.063 ± 0.6187) and Bacillus subtilis (MICs of 1.063 ± 0.6187 to 3.75 ± 1.768). More marked activity could be observed against Staphylococcus aureus, with a low MIC of 0.4688 ± 0.221, while against Klebsiella pneumoniae and Pseudomonas aeruginosa, the MIC varied between 1.063 ± 0.6187 and 5.00 ± 0.00, indicating moderate efficacy.

Finally, the oils showed little antifungal activity against Candida albicans, with an MIC of 0.4688 ± 0.221 for some oils, and no inhibition for others. Variations in the results may be linked to climatic differences in the zones of origin of the samples. Arid or dry zones favor the production of bioactive compounds in essential oils to protect the plant, which explains their more pronounced antimicrobial activity, particularly against Staphylococcus aureus. On the other hand, less climatically stressed areas show higher MICs against strains such as Klebsiella pneumoniae and Pseudomonas aeruginosa. These results illustrate the impacts of local climate on the chemical composition of essential oils and their antimicrobial efficacy.

Compared with essential oils reported in the literature, such as P. atlantica subsp. mutica and P. khinjuk, whose MICs range from 6 to 12.5 μg/mL and 16 to 150 μg/mL, respectively, for different strains, our essential oil showed a much stronger antimicrobial activity, with significantly lower minimum inhibitory concentrations, underlining its exceptional potential. Kamrani et al. [50] studied the antibacterial effects of Iranian P. atlantica subsp. kurdica against plaque-causing microorganisms. The fruit shell extract, prepared with 70% ethanol, showed inhibitory activity against S. mutans, S. salivarius, S. sobrinus, S. sanguis and Actinomycesviscosus. MIC values for the extracts ranged from 0.71 to 0.86 mg/mL. On the other hand, the antimicrobial activity of methanolic and ethyl acetate extracts of P. atlantica Desf. leaves grown in Libya was evaluated against S. aureus, B. subtilis, E. coli, P. aeruginosa and Candida albicans [51]. The essential oils of Pistacia atlantica Desf. resin demonstrated significant in vitro antimicrobial activity against seven bacterial strains and three Candida species. They were particularly effective against Staphylococcus aureus and Enterococcus faecalis, with minimum inhibitory concentrations (MIC) below 10 µg/mL, while Candida albicans showed resistance. The evaluation was performed using the agar diffusion method at concentrations around 105 µg/mL [49].

The activity and stability of essential oils vary depending on their physical state. Certain components of essential oils are unstable in the vapor state but remain stable in aqueous environments [52]. Furthermore, research has highlighted the phenolic properties of oleoresin, suggesting that phenolic compounds interfere with the enzymatic mechanisms essential for energy production in bacteria and yeasts. These compounds can also eliminate germs by altering their molecular structures [53].

4. Conclusions

This study of the Atlas pistachio tree in three areas of the Middle Atlas has highlighted significant variability in its phytochemical and mineralogical composition, as well as its antioxidant and antimicrobial activities. The mineralogical analysis revealed variations in the mineral concentrations of the leaves, with a marked predominance of potassium across the three bioclimatic zones, reflecting the ecological specificities of each region. Phytochemical evaluation confirmed the presence of key bioactive compounds, particularly hydrocarbon monoterpenes, known for their diverse biological effects. Furthermore, the antioxidant activity tests demonstrated the notable efficacy of the essential oils, underscoring their potential as natural agents against oxidative stress. In terms of antimicrobial activity, the results showed significant efficacy against several bacterial and fungal strains. Notably, the essential oils extracted from the leaves of Afourgagh (EOAA2) exhibited the strongest activity, particularly against Bacillus subtilis, Pseudomonas aeruginosa, and Candida albicans.

These findings confirm the importance of the Atlas pistachio tree as a valuable resource for pharmaceutical applications. They also highlight the need to valorize this species while shedding light on the interspecific variability between the humid and semi-arid environments of Morocco.