Seasonal Variation in the Chemical Composition and Antimicrobial Activity of Essential oil Obtained from Moroccan Lavender Lavandula maroccana Murb. Collected from the Wild

Abstract

Background: Lavandula maroccana Murb. is renowned for its valuable essential oil (EO), with a complex chemical composition and potent antimicrobial properties, which can be subjected to environmental influences. The objective of the present study was to assess how seasonal variations affect the yield, chemical composition and antimicrobial activity of Lavandula maroccana Murb. EO. Methods: The plant materials were collected throughout the four seasons and subjected to EO extraction using hydro-distillation method. The chemical composition was analyzed using gas chromatography/mass spectrometry (GC/MS), while the antimicrobial properties were evaluated using disc diffusion and microdilution assays against four yeasts (Candida albicans, C. glabrata, C. krusei, and C. parapsilosis), and six pathogenic bacteria (Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae). Results: The highest EO yield was recorded from the samples collected in autumn (0.19%), while the spring harvest yielded the lowest EO production (0.06%). The GC/MS analysis identified 41 different compounds, with notable variations in the major compound, carvacrol, which ranged from 29.33% to 60.60%. The summer collections contained the highest concentration of carvacrol. Antimicrobial testing, revealed that EOs obtained from all seasons displayed substantial activity against both bacteria and yeast strains. Notably, the summer-harvested EO showed the most potent antimicrobial effects, with inhibition zone and MIC values ranging from 15.32 ± 0.45 mm to 24.09 ± 0.65 mm and 0.16 mg/mL to 2.50 mg/mL, respectively. Conversely, the spring-harvested EO showed the least antimicrobial activity. Conclusion: These findings emphasize the importance of harvesting L. maroccana during the summer and/or autumn to maximize the EO yield and carvacrol content, thereby enhancing its antimicrobial effectiveness.

Keywords

seasonal variation essential oils yield phytocompounds antimicrobial activity

Introduction

The widespread use of pharmaceutical antimicrobials has led to the emergence and spread of drug-resistant microorganisms, ushering in a post-antibiotic era where common bacterial infections have become challenging to treat.¹ It has been reported that over 700,000 individuals die annually due to drug-resistant infections, with projections suggesting this number could exceed 10 million deaths by 2050.² This concerning situation has become a significant public health challenge, reason for which there is a growing need to develop and implement alternative effective strategies. As a result, an emerging approach to combat the antimicrobial resistance involves the utilization of certain natural products derived from various plant sources.^3-6 Among these plant-derived natural products, essential oils (EOs) have gained considerable attention for their potential as antimicrobial agents, offering a safe alternative to conventional antimicrobials. It has been reported that EOs exhibit a broad spectrum of antimicrobial activity against bacteria, fungi, and viruses.^7-9 Their antimicrobial effect is mainly attributed to their lipophilic characteristics and the complex mixture of bioactive compounds they contain, including terpenes, phenolics, and other volatile components. These bioactive compounds can act synergistically to disrupt microbial cell membranes, interfere with microbial enzymes, and alter cellular structures, leading to microbial death or inhibition.^8,10,11 Importantly, the diverse chemical constituents of EOs make difficult for microorganisms to develop resistance, providing a significant advantage over conventional antimicrobial agents. However, it is important to note that the chemical composition and consequently the biological activities of EOs can vary depending on several environmental factors, including seasonal climatic changes. Many previous studies have demonstrated that climatic changes among seasons can affect the quantity and quality of chemical constituents of plant EOs, and that affect greatly their biological activity.^12-14 For instance, the EOs from Coleus amboinicus Lour (synonym Plectranthus amboinicus) were gathered across various seasons, with the highest EO yield was recorded in summer, while the highest amounts of carvacrol and thymol in autumn and spring, respectively. Oxygenated monoterpenes and sesquiterpenes were more prevalent during the summer, whereas monoterpene and sesquiterpene hydrocarbons were more abundant in winter.¹⁴ The aerial parts of Mesosphaerum suaveolens (L.) Kuntze harvested during the dry season resulted in a high quantity of EO, exhibiting the highest concentration of 1,8-cineole and demonstrating an interesting larvicidal activity against Aedes aegypti larvae, compared to other harvest seasons.¹³ In another study, The EO extracted from Mentha longifolia L. during the spring season yielded the highest amount of EO, demonstrating greater antibacterial, antioxidant, and anti-cholinesterase activities, and containing different major compounds compared to EO obtained in winter.¹²

Lavandula maroccana (syn. L. abrotanoides var. attenuatta Ball.), commonly referred to as Moroccan lavender or “Kohila” by the local communities, is an endemic species with significant medicinal value. The species grows widely in the western High Atlas in arid and semi-arid bioclimates.^15,16 It prospers in rocky, dry soils and is predominantly found in mountainous regions that offer full sun exposure and excellent drainage. The aerial parts of this Moroccan lavender have traditionally been used by the Moroccan people as a dry powder or decoction to treat gastro-intestinal disorders and pulmonary infections.¹⁷ Its EO has been extensively researched and is renowned for its promising antimicrobial and antiseptic properties.^18-21 These activities have been linked to its richness in phenolic monoterpene carvacrol, alongside other compounds. However, significant variability in the content of this phenolic monoterpenoid has been shown in the Moroccan lavender EOs across different previous studies, linked probably to the time of harvesting of the plant materials. This variability has been accompanied by fluctuations in their antimicrobial property.^19,21,22 Taking into account these considerations, understanding the effect of harvesting time on the chemical composition and antimicrobial potency of L. maroccana EO becomes imperative for optimizing its application as an antimicrobial agent. Therefore, this study aimed to investigate, for the first time, the yield, chemical composition and antimicrobial activity of L. maroccana EO harvested across the four distinct seasons.

The main objective of this study is to determine the optimal season for extracting L. maroccana EO to maximize its antimicrobial effectiveness against some resistant pathogenic bacteria and yeasts. The findings have substantial practical implications for enhancing harvesting strategies. These improvements could significantly increase the therapeutic effectiveness and commercial value of this Moroccan lavender EO.

Material and Methods

Chemicals and Culture Media

Hexane of analytical grade (99.8%), dimethyl sulfoxide (DMSO) and antibiotics, used in the GC-MS analysis and antimicrobial test, were purchased from Sigma-Aldrich, Steinheim, Germany. The culture medium, Sabouraud Dextrose Agar (SDA), Sabouraud Dextrose Broth (SDB), Mueller Hinton Agar (MHA) and Mueller Hinton Broth (MHB) were obtained from Biokar Diagnostics, France.

Plant Sampling, EOs Extraction and GC/MS Analysis

Lavandula maroccana aerial parts were collected from Asni (31°05’N/08°07’W), located in the High Atlas region, at an altitude of 1051 meters above sea level, approximately 65 km from Marrakech city. Plant materials were collected monthly throughout each season: autumn (September, October, November), winter (December, January, February), spring (March, April, May), and summer (June, July, August) during the early morning hours of the year 2021/2022. The collected materials from each month were combined to represent their respective seasons. The plant materials were authenticated at the Laboratory of Microbial Biotechnologies, Agrosciences and Environment, where voucher specimen of each season was deposited under the numbers LM031, LM032, LM033 and LM034, respectively. Plant materials, collected monthly throughout the year, were air-dried and then stored in dark plastic bags in the refrigerator at 4 °C. 200 g of dry plant materials, collected over three successive months representing each season, were subjected to EO extraction through a three-hour hydro-distillation process using a Clevenger apparatus and 2 liters of distilled water. The resulting EOs were dried over anhydrous sodium sulfate, weighed to calculate the yield and stored in dark storage vials at 4 °C. For each sample, the EO extraction was performed in triplicate.

GC/MS Analysis

The EO analysis was conducted using gas chromatography-mass spectrometry (GC-MS) with a Thermo trace 1300 gas chromatograph equipped with ISQ mass selective (single quadrupole mass spectrometer detector), and a capillary column TG-5MS (30 m of length, 0.25 mm internal diameter and 0.25 mm film thickness). A volume of 2 µL of each EO sample, diluted in the hexane, was injected using a 1:50 split mode ratio. Helium was the carrier gas used at a flow rate of 1 mL/min. The injector temperature was set at 260 °C. The column temperature was programed from 100 °C to 260 °C at the rate of 4 °C/min and then held at 246 °C for 10 min. The transfer line and ion source temperatures were maintained at 230 °C. Electron ionization mass spectra obtained at 70 eV. Mass spectra were scanned in the range of 41 to 550. The compounds in the EOs were identified by matching the obtained mass spectra with data from the Wiley and NIST libraries, as well as standards of the main components, and by calculating and comparing the retention indices (RI) with those reported by the Adams terpene library.²³

Antimicrobial Activity

Microorganisms

The antimicrobial activity of the EOs from L. maroccana harvested during different seasons, was compared against four Candida strains (C. albicans CCMM-L4, C. glabrata CCMM-L7, C. krusei CCMM-L10, and C. parapsilosis CCMM-L18), as well as six pathogenic bacteria. Among the bacteria tested were three Gram-positive strains (Staphylococcus aureus CCMMB3, Micrococcus luteus ATCC 10,240, Bacillus subtilis ATCC 9524) and three Gram-negative strains (Escherichia coli ATCC 8739, Pseudomonas aeruginosa DSM 50090, and a clinical isolate of Klebsiella pneumoniae).

Antimicrobial Screening

The antimicrobial activity of the four investigated EOs was initially assessed using the EUCAST disc diffusion antimicrobial susceptibility testing method.²⁴ Microbial suspensions were uniformly spread on either MHA or SDA, depending on the targeted strains. Sterile 6 mm discs were impregnated with 10 µL of EO sample and placed on the surface of the inoculated plates. Positive controls, including ciprofloxacin and fluconazole (10 μg/disc), were also included. Plates containing Candida strains were incubated for 48 h at 28 °C, while those containing bacteria were incubated for 24 h at 37 °C. The diameter of the inhibition zones around the discs was measured in millimeters.

Broth Microdilution Assay

Following the CLSI guidelines,^25,26 the minimum inhibitory concentration (MIC) of the four investigated EOs was determined using the broth microdilution assay. Initially, two-fold serial dilutions of EOs ranging from 160 to 0.02 mg/mL were prepared in MHB or SDB using DMSO at a final concentration of 2%. Next, microwells were inoculated with 100 μL of adjusted yeast (1-2 × 10³) or bacterial suspensions (10⁸ CFU/mL), followed by the addition of 100 μL of the prepared dilutions. Microplates containing ciprofloxacin and fluconazole were used as positive controls, while DMSO served as a negative control. The MIC values were determined visually after incubating the microplates containing bacteria for 24 h at 37 °C and those containing fungi for 48 h at 28 °C. Once the MIC values were determined, 100 μL of the contents from each clear microwell were sub-cultured onto MHA or SDA plates, depending on the targeted strains. The plates were then incubated under the previously described conditions. The minimum microbicidal concentration (MMC) values were defined as the lowest concentrations of the EO samples that resulted in fungicidal (MFC) and bactericidal (MBC) effects, respectively.

Statistical Analysis

The results of EO yields, chemical compositions and antimicrobial screening were analyzed using one-way analysis of variance (ANOVA) in SPSS 12.0 software. Post-hoc comparisons of means were performed using the Tukey's test at a significance level of p < 0.05.

Results

Yield of EO

The results showed that the production of EOs varied significantly with the harvesting time (Figure 1). The highest EO production was recorded in plants collected in autumn with a yield of 0.19%. Whereas summer and spring showed the lowest EO yield with values of 0.09% and 0.06%, respectively. Plant materials collected in winter presented the intermediate EO yield value (Figure 1).

Figure 1.

Eo yields of L. maroccana harvested during different seasons. Different letters indicate significant differences at p ≤ 0.05 according to Tukey's tests.

Figure 2.

EO chromatograms of L. maroccana harvested during different seasons (a: autumn; b: winter; c: spring; d: summer).

Chemical Composition

The chemical composition of the EOs extracted from L. maroccana collected during different seasons was determined by GC-MS and the results are presented in Figure 2 and Table 1. In total, 41 different compounds were identified, accounting for 97.12% - 98.96% of the total volatile oil compositions. The oxygen-containing monoterpenes were the predominant compound class in the EOs across all seasons, accounting for between 60.65% and 75.59% of the total analyzed EO. The main constituents consistently identified in all EOs include carvacrol (29.33% - 60.60%), p-cymen-8-ol (4.20% - 5.88%), 2-methoxy-4-vinylphenol (3.45% - 8.90%), camphor (0.57% - 10.55%), linalool (2.05% - 4.73%), and carvacrol methyl ether (1.50% - 10.36%). The comparison between the harvest seasons revealed significant quantitative variations for some compounds, particularly the phenolic monoterpene carvacrol. The highest percentage of carvacrol was observed in EOs obtained from plant materials collected in summer (60.6%), followed by autumn (53.35%), winter (40.44%), and spring (29.33%). Similarly, α-farnesene was most abundant in EOs derived from plants harvested in summer (4.65%), while its percentage drops significantly to 0.18% in spring. Conversely, certain terpenoid compounds, such as borneol (5.65%), linalool (4.73%), and carvacrol methyl ether (10.36%), were found in higher concentrations in EOs derived from plant materials harvested in spring, compared to other seasons. The concentrations of camphor (5.55% - 10.55%) and 2-methoxy-4-vinylphenol (7.75% - 8.90%) were highest in the autumn and winter harvests, and were less abundant in the remaining seasons. The content of p-cymen-8-ol (4.20% - 5.88%) did not show a significant difference across all studied EOs. Overall, these findings showed that EO obtained from L. maroccana harvested in summer was mainly dominated by carvacrol (60.6%), p-cymen-8-ol (5.35%), α-farnesene (4.65%), and 2-methoxy-4-vinylphenol (3.45%). In autumn, the EO was primarily composed of carvacrol (53.35%), camphor (10.55%), 2-methoxy-4-vinylphenol (7.75%), and p-cymen-8-ol (4.20%). The plant EOs harvested in spring was characterized by the dominance of carvacrol (29.33%), carvacrol methyl ether (10.36%), borneol (5.65%), 2-methoxy-4-vinylphenol (5.49%), linalool (4.73%) and p-cymen-8-ol (4.61%). Whereas, In the winter harvest, the predominant constituents were carvacrol (40.44%), 2-methoxy-4-vinylphenol (8.90%), p-cymen-8-ol (5.88%) and camphor (5.55%).

Table 1.

Chemical Composition of L. Maroccana EOs Harvested During Different Seasons.

RT^a	LRI^b	RI^c	Compound	Relative abundance (%)
RT^a	LRI^b	RI^c	Compound	Autumn	Winter	Spring	Summer
2.12	839	839	Furfural (5)	1.05 ± 0.03^a	1.25 ± 0.03^a	1.34 ± 0.00^a	0.65 ± 0.17^a
2.18	846	852	(E)-Hex-2-enal (5)	1.55 ± 0.10^c	2.07 ± 0.05^b	3.12 ± 0.02^a	1.70 ± 0.20^bc
2.42	901	902	Heptanal (5)	0.15 ± 0.03^b	0.56 ± 0.07^a	0.28 ± 0.01^b	0.15 ± 0.03^b
2.56	-	914	Dec-3-yn-2-ol (1)	0.22 ± 0.05^b	0.67 ± 0.07^a	0.19 ± 0.01^b	0.14 ± 0.04^b
2.94	952	960	Benzaldehyde (5)	0.35 ± 0.03^b	0.70 ± 0.07^a	0.57 ± 0.02^a	0.32 ± 0.02^b
3.08	988	998	β-Myrcene (2)	0.46 ± 0.03^b	0.61 ± 0.04^b	0.56 ± 0.02^b	1.90 ± 0.07^a
3.56	1008	1003	3-Carene (2)	0.15 ± 0.03^c	0.57 ± 0.02^a	0.36 ± 0.03^b	0.12 ± 0.01^c
3.61	1026	1023	Eucalyptol (1)	0.06 ± 0.03^c	0.57 ± 0.06^b	0.81 ± 0.03^a	0.02 ± 0.00^c
3.68	1032	1030	β-Ocimene (2)	0.45 ± 0.03^bc	0.95 ± 0.03^b	0.29 ± 0.02^c	3.40 ± 0.27^a
3.80	-	1046	Benzeneacetaldehyde (5)	1.09 ± 0.07^a	1.20 ± 0.13^a	1.26 ± 0.09^a	0.12 ± 0.02^b
4.10	1080	1071	Linalool oxide (1)	0.11 ± 0.01^c	0.91 ± 0.06^a	0.97 ± 0.03^a	0.60 ± 0.07^b
4.32	1086	1081	Terpinolene (2)	0.45 ± 0.03^c	2.28 ± 0.01^a	0.27 ± 0.02^d	1.45 ± 0.04^a
4.38	1095	1096	Linalool (1)	2.25 ± 0.10 ^bc	2.05 ± 0.03 ^c	4.73 ± 0.18 ^a	2.65 ± 0.03 ^b
4.44	1100	1104	Nonanal (5)	1.04 ± 0.03^a	0.90 ± 0.06^a	1.03 ± 0.02^a	0.26 ± 0.04^b
5.24	1137	1131	cis-Verbenol (1)	0.60 ± 0.07^b	0.71 ± 0.06^b	1.29 ± 0.01^a	0.39 ± 0.06^c
5.29	1141	1139	Camphor (1)	10.55 ± 0.15 ^a	5.55 ± 0.05 ^b	4.21 ± 0.06 ^c	0.57 ± 0.05 ^d
5.41	1165	1156	Borneol (1)	0.75 ± 0.03 ^c	1.24 ± 0.03 ^b	5.65 ± 0.10 ^a	0.34 ± 0.03 ^d
5.81	1168	1170	2-Isopropenyl-5-methylhex-4-enal (1)	0.22 ± 0.05^b	0.73 ± 0.11^a	0.84 ± 0.04^a	0.36 ± 0.04^b
5.93	1179	1180	p-Cymen-8-ol (1)	4.20 ± 0.13 ^b	5.88 ± 0.05 ^a	4.61 ± 0.26 ^b	5.35 ± 0.10 ^a
6.07	1199	1195	γ-Terpineol (1)	0.55 ± 0.03^c	0.98 ± 0.01^b	1.78 ± 0.15^a	0.56 ± 0.04^c
6.50	1204	1207	D-Verbenone (1)	0.75 ± 0.03^b	0.99 ± 0.01^a	0.64 ± 0.04^b	0.45 ± 0.03^c
6.64	1235	1225	(E,E)-2,6-Dimethylocta-35,7-triene-2-ol (1)	0.25 ± 0.03^b	0.49 ± 0.07^a	0.52 ± 0.05^a	0.17 ± 0.02^b
6.70	1238	1230	3-Cyclohexene-1-propanal (5)	1.15 ± 0.03^b	1.41 ± 0.06^a	0.45 ± 0.03^c	0.30 ± 0.07^c
7.10	1241	1250	Carvacrol methyl ether (5)	1.50 ± 0.00 ^c	3.12 ± 0.08 ^b	10.36 ± 0.09 ^a	1.58 ± 0.06 ^c
7.44	1249	1259	Piperitone (1)	0.16 ± 0.04^c	0.76 ± 0.06^a	0.38 ± 0.04^b	0.65 ± 0.03^a
7.91	1269	1269	Perillaldehyde (1)	0.42 ± 0.01^b	0.83 ± 0.04^a	0.49 ± 0.04^b	0.11 ± 0.01^c
8.17	1289	1285	Thymol (1)	0.78 ± 0.01^b	0.83 ± 0.04^b	3.94 ± 0.17^a	0.34 ± 0.03^c
8.41	1298	1299	Carvacrol (1)	53.35 ± 0.32 ^b	40.44 ± 0.29 ^c	29.33 ± 0.89 ^d	60.6 ± 0.39 ^a
8.81	1309	1315	2-Methoxy-4-vinylphenol (5)	7.75 ± 0.17 ^b	8.90 ± 0.07 ^a	5.49 ± 0.33 ^c	3.45 ± 30 ^d
9.92	1346	1335	3-Allyl-2-methoxyphenol (1)	0.35 ± 0.03^a	0.27 ± 0.02^a	0.25 ± 0.03^a	0.34 ± 0.03^a
10.57	1383	1372	Damascenone (5)	0.20 ± 0.00^b	0.31 ± 0.06^ab	0.35 ± 0.03^a	0.21 ± 0.01^b
11.59	1417	1406	cis-Caryophyllene (4)	0.40 ± 0.07^b	0.48 ± 0.01^b	0.46 ± 0.03^b	1.25 ± 0.17^a
13.41	1432	1443	trans-α-Bergamotene (4)	0.25 ± 0.03^c	0.59 ± 0.02^b	1.92 ± 0.05^a	0.15 ± 0.03^c
13.56	1487	1477	trans-β-Ionone (5)	0.10 ± 0.00^b	0.50 ± 0.07^a	0.23 ± 0.05^b	0.09 ± 0.00^b
13.63	1500	1490	Bicylogermacrene (4)	0.06 ± 0.01^ab	0.33 ± 0.09^a	0.18 ± 0.01^ab	0.20 ± 0.00^ab
13.78	1505	1501	α-Farnesene (4)	1.10 ± 0.07 ^b	0.56 ± 0.90 ^c	0.18 ± 0.03 ^d	4.65 ± 0.10 ^a
14.30	1532	1528	Dihydroactinidiolide (5)	0.13 ± 0.02^b	0.51 ± 0.07^a	0.17 ± 0.02^b	0.12 ± 0.02^b
14.58	1544	1535	7-epi-cis-sesquisabinene hydrate (3)	0.30 ± 0.00^bc	0.75 ± 0.10^b	2.59 ± 0.21^a	0.19 ± 0.06^c
15.81	1577	1587	Spathulenol (3)	1.90 ± 0.07^b	2.79 ± 0.06^a	2.91 ± 0.12^a	1.02 ± 0.02^c
15.97	1582	1595	Caryophyllene oxide (3)	1.22 ± 0.08^b	1.90 ± 0.07^a	1.41 ± 0.26^ab	0.98 ± 0.05^b
22.64	1845	1851	6,10,14-Trimethylpentadecan-2-one (5)	0.55 ± 0.03^a	0.80 ± 0.00^a	0.65 ± 0.09^a	0.73 ± 0.16^a
Oxygen-containing monoterpenes (1)				75.59 ± 0.55^a	60.65 ± 0.34 ^d	73.66 ± 0.27 ^b	63.94 ± 0.20 ^c
Monoterpene hydrocarbons (2)				1.51 ± 0.12 ^c	1.49 ± 0.09 ^c	6.87 ± 0.25 ^a	4.42 ± 0.06 ^b
Oxygen-containing sesquiterpenes (3)				3.42 ± 0.15 ^c	6.92 ± 0.59 ^a	2.19 ± 0.01 ^d	5.44 ± 0.02 ^b
Sesquiterpene hydrocarbons (4)				1.82 ± 0.11 ^c	2.74 ± 0.13 ^b	6.25 ± 0.24 ^a	1.96 ± 0.18 ^c
Others (5)				16.61 ± 0.09 ^c	25.32 ± 0.76 ^a	9.71 ± 0.39 ^d	22.26 ± 0.40 ^b
Total				98.96 ± 0.29 ^a	97.12 ± 1.90 ^a	98.70 ± 1.13 ^a	98.03 ± 0.46 ^a

RT: Retention time.

LRI: Retention indices from literature.

RI: Retention indices relative to n-alkanes (C7–C30) on the TG-5MS capillary column.

Means followed by the same letter are not significantly different.

Antimicrobial Activity

The first antimicrobial screening of EOs of L. maroccana harvested during different seasons (summer, autumn, winter, and spring) was conducted by measuring the zones of microbial growth inhibition of tested strains, and the comparative results are summarized in Table 2. The results showed that L. maroccana EOs exhibited generally significant and varying degrees of antimicrobial activity against bacterial and yeast strains tested, depending on the season of harvest. The growth inhibition diameters observed for the tested strains were in the range of 8.23 ± 0.33 mm - 24.09 ± 0.65 mm. Interestingly, these values were generally similar to those obtained with standard antibiotics ciprofloxacin and fluconazole (Table 2). Comparatively, the EO from the plants collected in summer and autumn generally exhibited the highest antimicrobial activity against both bacteria and yeast strains, with inhibition zone diameters ranging from 15.32 ± 0.45 mm to 24.09 ± 0.65 mm and from 14.98 ± 0.05 mm to 23.43 ± 0.49 mm, respectively. The plant EOs harvested in spring showed the significantly smaller inhibition zone diameters (8.23 ± 0.33 mm - 19.11 ± 1.18 mm), reflecting a reduced antimicrobial effectiveness against all tested microbial strains. The EO of the plant materials collected in winter exhibited intermediate inhibition zone diameters, which were not significantly different from those of the EO of plants collected in autumn.

Table 2.

Inhibition Zone Diameters (mm) of EO of L. Maroccana Harvested During Different Seasons.

Microorganisms	Summer	Autumn	Winter	Spring	ATM
Staphylococcus aureus	20.58 ± 0.52^a	19.79 ± 0.53^ab	18.50 ± 0.33^ab	17.83 ± 0.55^b	19.43 ± 1.20^ab
Micrococcus luteus	24.09 ± 0.65^a	23.43 ± 0.49^ab	20.66 ± 0.88^bc	18.83 ± 0.77^c	20.39 ± 1.22^c
Bacillus subtilis	21.27 ± 0.48^a	20.93 ± 0.08^a	20.27 ± 0.36^ab	18.43 ± 0.29^c	19.18 ± 0.54^bc
Escherichia coli	18.66 ± 0.44^a	18.83 ± 0.55^a	17.66 ± 0.44^a	14.86 ± 0.17^b	13.22 ± 0.33^c
Klebsiella pneumoniae	19.33 ± 0.44^a	19.00 ± 0.66^a	17.66 ± 0.88^ab	16.33 ± 0.88^b	9.65 ± 0.28^c
Pseudomonas aeruginosa	15.32 ± 0.45^a	14.98 ± 0.05^a	12.53 ± 0.35^b	8.23 ± 0.33^c	9.38 ± 0.55^c
Candida albicans	21.58 ± 0.82^a	21.25 ± 0.60^a	19.33 ± 1.11^ab	18.26 ± 0.48^b	21.35 ± 0.06^a
Candida glabrata	22.44 ± 1.21^a	21.44 ± 0.36^ab	19.75 ± 1.17^abc	17.64 ± 0.23^c	19.44 ± 0.23^bc
Candida krusei	21.84 ± 1.00^ab	22.46 ± 0.36^a	20.72 ± 0.96^ab	19.11 ± 1.18^b	14.37 ± 0.06^c
Candida parapsilosis	21.80 ± 0.56^b	21.69 ± 0.67^b	19.56 ± 0.75^bc	17.96 ± 0.68^c	24.58 ± 0.52^a

ATM (Antimicrobial): The used reference antimicrobials were ciprofloxacin for bacteria and fluconazole for yeasts. Means followed by the same letter within a row are not significantly different.

The antimicrobial activity of L. maroccana EOs harvested during different seasons was also evaluated in terms of MIC and MMC, as shown in Table 3. Following the same trend as the inhibition zone diameters, the results showed that all EOs of L. maroccana obtained from different season exhibit significant antimicrobial potency against all tested bacterial and yeast strains. Remarkably, this activity was generally higher than that observed for the standard antibiotic ciprofloxacin against K. pneumoniae, lower against other bacteria strains, and similar to that of the antifungal fluconazole. However, comparing the results, it appears that the antimicrobial activity of L. maroccana EOs varied considerably depending on the harvesting season. Indeed, the EO showed the strongest antibacterial activity overall during the summer, exhibiting the lowest MIC values against all tested strains, particularly for the Gram-positive bacteria (S. aureus: 0.313 mg/mL, M. luteus: 0.313 mg/mL, and B. subtilis: 0.156 mg/mL), indicating higher potency. This activity remained relatively high for the EOs obtained from the plant materials harvested in autumn, with a slight increase in MIC and MMC values (ranging from 0.625 mg/mL to 2.5 mg/mL) compared to summer. However, the MIC and MMC values have been increased significantly in the plant EOs harvested in winter and spring (0.625 mg/mL - 20 mg/mL), indicating a reduction in the antimicrobial effectiveness (Table 3). Comparing the sensitivity of the tested microbial strains to L. maroccana EOs, it appears that Gram-positive bacteria were generally more sensitive, particularly to the EOs of plants collected in summer. The yeast strains showed intermediate sensitivity, while Gram-negative bacteria appeared less sensitive to the effect of EOs. Among Gram-negative bacteria tested, P. aeruginosa demonstrated the highest resistance, with MIC values of 2.5 mg/mL for plant EOs collected in summer and autumn, increasing to 10 mg/mL for those harvested in winter and spring.

Table 3.

MIC and MMC of EO of L. Maroccana Harvested at Different Seasons.

Microorganisms	Summer		Autumn		Winter		Spring		ATM
Microorganisms	MIC	MMC	MIC	MMC	MIC	MMC	MIC	MMC	MIC
Staphylococcus aureus	0.31	1.25	1.25	1.25	2.50	2.50	1.25	2.50	0.04
Micrococcus luteus	0.31	0.31	0.63	0.63	0.63	0.63	1.25	1.25	0.02
Bacillus subtilis	0.16	0.31	0.31	0.313	0.63	0.63	0.63	1.25	0.02
Escherichia coli	0.63	0.63	0.63	1.25	5.00	5.00	5.00	5.00	0.08
Klebsiella pneumoniae	0.31	0.63	0.31	0.63	1.25	1.25	1.25	1.25	1.28
Pseudomonas aeruginosa	2.50	5.00	2.50	2.50	10.0	20.0	10.0	20.0	0.32
Candida albicans	0.63	1.25	0.63	1.25	2.50	2.50	2.50	2.50	0.64
Candida glabrata	0.31	0.63	0.63	1.25	2.50	2.50	1.25	2.50	1.28
Candida krusei	0.31	0.63	0.63	1.25	2.50	2.50	1.25	2.50	1.28
Candida parapsilosis	0.63	1.25	0.63	1.25	2.50	2.50	2.50	2.50	0.64

MIC: Minimum inhibitory concentration in mg/mL; MMC: Minimum microbicidal concentration in mg/mL; ATM (Antimicrobial): The used reference antimicrobials were ciprofloxacin for bacteria and fluconazole for yeasts.

Discussion

Plant EOs have recently emerged as promising natural antimicrobials to combat the threat of antibiotic resistance. Previous studies have highlighted the interesting antimicrobial potential of L. maroccana EOs, emphasizing their particular chemical compositions and their potential for use as alternative natural antimicrobial or in combination therapy to restore the efficacy of some conventional antibiotics.^22,27 The present study is the first to investigate the effect of seasonal changes on the yield, chemical composition, and antimicrobial potency of L. maroccana EO against some human pathogenic bacteria and yeast strains. The EO yields of L. maroccana obtained in this study ranged from 0.06% to 0.19%, which are consistent with values previously reported in the literature.^21,27,28 The oil yield was significantly higher in plants collected in autumn (0.19%) compared to other seasons. The increased EO production in this season can be linked to the plant's enhanced maturity, heightened metabolic activity, and favorable climate conditions for the accumulation of secondary metabolites. This result aligns with reports for many medicinal plants where the highest EO yields were generally observed in samples collected during autumn and/or winter.^29-32 Regarding the GC/MS results, quantitative differences in volatile compounds of EOs extracted from plants harvested in different seasons were detected, confirming the influence of seasonal variation on the production of secondary metabolites. Numerous previous studies have consistently shown that the biochemical profiles of plant oils vary significantly depending on the time of year.^12-14 This variability is primarily due to seasonal changes in environmental factors, which all impact plant metabolism and consequently, the synthesis of secondary metabolites. The major compound identified in our EOs from all seasons was the oxygenated monoterpene carvacrol. This finding is consistent with previously published data, which also documented carvacrol as the main constituent in L. maroccana EOs.^20,21,27,28 The highest carvacrol content was observed in plant EOs harvested in summer, which exceeded 60% of the total oil content. The high content of carvacrol observed during the dry season (summer) can be attributed to increased enzymatic activity involved in monoterpenoid synthesis, which serves as the plant's adaptive response to water and environmental stresses. It is well established that water scarcity during the dry season can upregulate the enzyme terpene synthase, which is directly involved in the biosynthesis of monoterpenoids such as carvacrol. The accumulation of carvacrol under such stressful conditions can not only help in managing water loss by reducing transpiration but also play a role in defending against herbivores and pathogens, and help mitigate oxidative stress caused by reactive oxygen species produced during these conditions.³³ This finding is in line with previous reports which showed a progressive increase in the proportion of carvacrol in EOs of Thymus and Origanum spp. according to the increased dry conditions.^34,35 Other constituents, such as p-cymen-8-ol (4.20% - 5.88%), 2-methoxy-4-vinylphenol (3.45% - 8.90%), camphor (0.57% - 10.55%), linalool (2.05% - 4.73%) and carvacrol methyl ether (1.50% - 10.36%) exhibited fluctuations between harvesting seasons. This seasonal variation in the content of the main EO compounds is consistent with findings from many previously published studies on some aromatic and medicinal plants.^{13,29,31,32,36,37} It is well known that the chemical profile of plant EOs is dependent on many climatic factors, such as temperature, day length and photoperiod.^38,39

In terms of antimicrobial activity, the EOs extracted from L. maroccana collected during the four seasons exhibited interesting antimicrobial properties against all tested bacterial and yeast strains. These results are consistent with previous studies that have highlighted the antimicrobial potential of Lavandula EOs including L. maroccana.^{18,19,28,40,41} Furthermore, it appears that all EOs derived from this Moroccan lavender exhibited greater potency against Gram-positive bacteria and yeasts compared to Gram-negative strains. This difference in efficacy can be explained by the structural characteristics of Gram-negative bacteria, which possess an outer membrane composed of lipoproteins and lipopolysaccharides. This outer membrane acts as a selectively permeable barrier, regulating access to the underlying cell structures and thereby providing additional protection against antimicrobial agents.⁴² The EO from the summer harvest exhibited the strongest antimicrobial activity, followed by that from the autumn harvest, while the spring harvest showed the lowest effectiveness. The enhanced antimicrobial activity of EO from summer harvest can be attributed to the higher concentration of active compounds, primarily the phenolic monoterpene carvacrol, which is consistent with previous reports.^43-45 In fact, numerous studies have established a strong correlation between the carvacrol content in EOs and their antimicrobial efficacy.^7,28,46 The antibacterial activity of carvacrol has been linked, in part, to its hydrophobic nature, which allows it to integrate into the lipid bilayer of cell membranes, altering their permeability. This disruption in membrane integrity results in cell membrane damage, impairing the bacteria's ability to maintain essential cellular functions.^47,48 Additionally, carvacrol exhibits antifungal action by inducing fungal membrane instability, primarily through its interaction with and binding to ergosterol, an essential component of fungal cell membranes. This interaction disrupts membrane integrity and function, ultimately leading to fungal cell death.^49,50 Furthermore, carvacrol has been shown to downregulate the expression levels of genes associated with the synthesis of secreted aspartyl proteinases (SAPs) in Candida species.⁵¹ These enzymes play a crucial role in the adherence of Candida cells to the host cell surface and in causing tissue damage. The presence of other monoterpenoid components such as p-cymene-8-ol, camphor, borneol and linalool, along with sesquiterpenes, even at low concentrations, can contribute substantially to the overall antimicrobial activity. The mechanism of action generally involves multiple pathways, including disruption of microbial cell membranes, inhibition of enzyme activities, and interference with microbial metabolism.^52,53

This study provides crucial insights into the optimal season for harvesting L. maroccana, essential for maximizing the yield and quality of its EOs. The findings support sustainable harvesting practices and improve the efficacy of this Moroccan Lavender EO, making it highly suitable for application in aromatherapy, perfumery, and natural medicine. This approach offers significant ecological and commercial benefits, optimizing the resource's environmental sustainability and economic value. However, a significant limitation in this study is that the plants sampled from only one single geographical location and the seasonality was investigated over a only one year. To comprehensively understand the impact of varied environmental factors on the chemical composition and antimicrobial activity of these valuable medicinal plants, further research should involve multiple populations across different locations and extend over at least two successive years.

Conclusion

In conclusion, the present study highlighted the significant influence of seasonal variation on the yield, chemical profile, and antimicrobial activity of L. maroccana EOs against human pathogenic yeasts and bacteria. The highest EO yield was obtained from the autumn harvest (0.19%), while spring harvest presented the lowest EO production. The chemical profiles revealed some quantitative differences, notably in the major compound carvacrol, which reached its highest content of over 60% of the total oil from materials collected in summer. Regarding antimicrobial activity, the EO displayed significant efficacy against both bacterial and yeast strains. The summer-harvest EO exhibited the most potent antimicrobial effects, whereas the spring-harvested EO demonstrated the lowest activity. These findings underscore the importance of considering the harvest timing of L. maroccana to optimize its antimicrobial efficacy. The broader implications of this work suggest that selecting the appropriate harvest season could enhance the effectiveness of these EOs in combating microbial infections. Moreover, the results open avenues for further research into the mechanisms driving seasonal variations in EO composition and activity. Exploring the potential of L. maroccana EOs in combination therapies could offer new strategies for addressing antibiotic resistance, making this research highly relevant for both clinical and agricultural applications.

Footnotes

Acknowledgments

The authors thank the ‘’city of innovation’’ for providing some analysis.

Author Contributions

BS: methodology, data collection, data analysis, writing the original manuscript, revision. IA: statistical analysis, data analysis, revision. AA: methodology, data analysis, revision. All authors read and approved the final version of the manuscript.

Data Availability

All the datasets used and analyzed during the current study are presented in this manuscript.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical Approval

Ethical approval is not applicable to this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

ORCID iD

Bouchra Soulaimani

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