Tetraclinis articulata (Vahl) Mast., Mentha pulegium L., and Thymus zygis L. essential oils: Chemical composition, antioxidant and antifungal properties against postharvest fungal diseases of apple, and in vitro, in vivo, and in silico investigation

Faiza El Hajli; Said Chakir; Saoussan Annemer; Amine Assouguem; Fouzia Elaissaoui; Riaz Ullah; Essam A. Ali; Ravish Choudhary; Khalil Hammani; Rachid Lahlali; Ghizlane Echchgadda

doi:10.1515/chem-2025-0131

Artikel Open Access

Tetraclinis articulata (Vahl) Mast., Mentha pulegium L., and Thymus zygis L. essential oils: Chemical composition, antioxidant and antifungal properties against postharvest fungal diseases of apple, and in vitro, in vivo, and in silico investigation

Faiza El Hajli , Said Chakir , Saoussan Annemer , Amine Assouguem , Fouzia Elaissaoui , Riaz Ullah , Essam A. Ali , Ravish Choudhary , Khalil Hammani , Rachid Lahlali und Ghizlane Echchgadda

Veröffentlicht/Copyright: 6. März 2025

Veröffentlicht von

Veröffentlichen auch Sie bei De Gruyter Brill

Manuskript einreichen Informationen für Autor*innen Erkunden Sie dieses Fachgebiet

Aus der Zeitschrift Open Chemistry Band 23 Heft 1

Graphical abstract

Abstract

Essential oils (EOs) are bioactive chemicals derived from the extraction of aromatic and medicinal plants. They have many benefits, including antioxidant and antifungal activity. The present study aims to evaluate the phytochemical composition of Tetraclinis articulata (Vahl) Mast., Mentha pulegium L., and Thymus zygis L. with biological evaluation and its molecular docking study. The three species studied were collected from the Taza region in north-eastern Morocco. The chemical composition of the EOs was analyzed by gas chromatography coupled with mass spectrometry, and the antioxidant activity was evaluated by 2,2-diphenyl-1-picrylhydrazyl analysis. The antifungal activity of the studied EOs was tested against apple rot fungi at different concentrations in vitro and in vivo against Botrytis cinerea and Penicillium expansum. Fruit quality parameters and molecular docking of biological activities were also evaluated. The results of this study showed that the EOs of T. articulata (Vahl) Mast. were rich in monoterpenic hydrocarbons (57.71%), whereas M. pulegium L. and T. zygis L. were rich in oxygenated monoterpenes (95.54%). In this study, the EO of T. zygis L. was found to have the highest antioxidant potency (IC₅₀ = 7.60 ± 0.48 µg/mL), and the efficacy of the EO of this plant against the two pathogens tested is greater than that of the other two plants. Molecular docking results showed important activity, including ligand binding affinity to the active site of the receptor of each fungal strain, hydrogen bonds, hydrophobic bonds, and interactions for antifungal and antioxidant activities.

Keywords: molecular docking; GC-MS analysis; apple diseases; essential oils; quality parameter; antifungal and antioxidant properties

Abbreviation

T. zygis L.: Thymus zygis L.
T. articulata (Vahl) Mast.: Tetraclinis articulata (Vahl) Mast.
M. pulegium L.: Mentha pulegium L.
DPPH: 2,2-diphenyl-1-picrylhydrazyl
B. cinerea: Botrytis cinerea
P. expansum: Penicillium expansum
PDA: Potato dextrose agar
TSS: Total soluble solids
TA: Titratable acidity
MI: Maturity index
EO: essential oil
IC₅₀: value of the 50% scavenging activity of the DPPH scavenging effect

1 Introduction

Microorganisms can play a role in the composition of food and are, in many cases, essential for its ripening. On the other hand, their proportion is important as this can contribute to the deterioration of food products, with harmful effects on human health and the economy [1]. In recent years, scientists have devoted their attention to the necessity of enhancing food production to support the exponential growth of global population numbers [2]. It is widely acknowledged that fruits and vegetables are essential elements of the human diet due to their role in providing essential minerals and vitamins, as well as other vital substances [3]. They are often susceptible to microbial attack during storage, particularly from fungi and bacteria [4]. Apple is a fruit of great economic and cultural importance, cultivated in almost all temperate zones worldwide. It is possible to consume the entire fruit, except the seeds. Additionally, numerous other products are manufactured from these fruits, including juice, jam, wine, cider, tea, and dried apples [5]. In Morocco, the area dedicated to apple production is estimated to be approximately 48,671 ha [6]. It has been postulated that fungal diseases result in a loss of between 5 and 25% of global apple yield during postharvest storage and marketing. Nevertheless, during storage, apple fruit is susceptible to substantial losses, estimated to be between 20 and 60%. These losses are primarily caused by fungal infections [3]. Pathogens that emerge in the post-harvest period have the potential to degrade the quality and nutritional value of fresh products. Additionally, they can cause nutrient losses and the formation of mycotoxins, which can result in significant reductions in the market value of these products [7]. The most important fungal pathogens that manifest post-harvest on fruit are those caused by Gloeosporium, Rhizopus, Penicillium, Botrytis, Alternaria, Mucor, and Monilinia spp [8]. Among these pathogens, Botrytis cinerea and Penicillium expansum are the most damaging, causing grey rot, blue rot, and lenticella rot diseases, respectively [9]. Synthetic fungicides are frequently employed in the control of grey mold disease; however, several limitations have been placed upon their utilization due to the detrimental impact they exert upon environmental and human health, resulting from the accumulation of their residues in food [10]. Furthermore, the intensified utilization of these chemical compounds has a detrimental impact on the ecosystem. This is because the residual substances remain in the soil, are transported, and eventually enter the groundwater, which results in adverse effects on the diversity of flora and fauna [11]. As a result, research conducted by government laboratories is focused on the development of biological approaches that are both safe and environmentally friendly. These include microbial antagonists and natural compounds that can be used to combat post-harvest diseases of fruits [12].

Plants have been identified as a significant source of chemical and pharmaceutical compounds on Earth, with an exceptional ability to produce an extensive range of chemical substances [13]. Medicinal and aromatic plants (MAPs) are employed as plants, plant parts, or essential oils (EOs). They are utilized in pharmacy, cosmetology, perfumes, and the food industry, among other areas [14]. A current tendency is toward the utilization of eco-friendly and more secure substitutes for the control of plant illnesses. A strategy of biological control against fungal pathogens represents an alternative methodology that minimizes hazards to the environment and human well-being [10]. Several studies have demonstrated the efficacy of EOs derived from aromatic plants in the control of a range of plant pathogens. These oils may, therefore, represent a viable alternative to chemical pesticides for the protection of plants. In particular, several studies have indicated that EOs possess antifungal properties against fungal pathogens, including B. cinerea and P. expansum [4,15].

This study aimed to explore the potential for valorizing Moroccan MAPs, with an emphasis on identifying new natural products that could serve as bioactive ingredients. In addition, the study aimed to characterize the primary components of EOs extracted from the leaves of Tetraclinis articulata (Vahl) Mast., Mentha pulegium L., and Thymus zygis L., to assess their capacity to inhibit fungal growth. This assessment was conducted in particular against two fungal strains, namely B. cinerea and P. expansum. Indeed, evaluating molecular docking results is essential for identifying molecules that can effectively inhibit free radicals and fungal strains causing apple rot. By integrating computational docking with experimental validation, we can develop optimized solutions to combat these issues. This approach aims to prevent apple rot and enhance food safety by targeting the underlying causes of spoilage.

2 Materials and methods

2.1 Chemicals and reagents

Malic acid, ascorbic acid, NaOH, glycerol, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were obtained from Sigma-Aldrich. Potato dextrose agar (PDA) culture medium was purchased from Merck (Darmstadt, Germany). Methanol was obtained from Biosolve (Dieuze, France). Distilled water was obtained by a Milli-Q water purification system (Millipore, Bedford, MA, USA).

2.2 Plant material and extraction of EOs

The aerial parts of the plant species T. articulata (Vahl) Mast., M. pulegium L., and T. zygis L. were utilized in the present study. These were collected in April 2024 from the northeast of Morocco (Taza). Subsequently, the plants were identified by Mrs Ghizlane Echchgadda, a professor at the National School of Agronomy (ENA), Meknes (Morocco). To protect the plants from light, they were kept in darkness and dried at room temperature for 5 days. Subsequently, the plants were crushed and maintained at a temperature of +4°C until analysis. For the hydrodistillation extraction, 100 g of powder from the aerial parts of T. articulata (Vahl) Mast., M. pulegium L., and T. zygis L. were placed separately in a flask containing 1 L of distilled water. The mixture was then placed on a Clevenger apparatus for 3 h.

2.3 Gas chromatography coupled with mass spectrometry (GC-MS) analysis

The bioactive components present in the T. articulata (Vahl) Mast., M. pulegium L., and T. zygis L. plants were determined by GC-MS analysis using the protocol described by Doukkali et al. [16]. The components of these oils were then compared using standard reference databases, including NRS libraries, NIST98, and Wiley275.

2.4 Antioxidant activity: DPPH radical scavenging method

To determine the antioxidant properties of EOs from T. articulata (Vahl) Mast., M. pulegium L., and T. zygis L., the researchers employed a previously described methodology [17] using DPPH. Upon reacting with the stable radical, DPPH, the antioxidants transform it into 1,1-diphenyl-2-picryl hydrazine, which is colorized a deep violet. This results in a deterioration in color. Subsequently, a methanolic solution of DPPH (with a concentration of 0.02 mM) was added to the series of samples analyzed with an initial concentration of 1 mg/mL, which were then incubated in total darkness at room temperature for 30 min. Subsequently, the absorbance density of the samples was measured at 517 nm. These data were then used to calculate the DPPH scavenging capacity of the oils using the following formula (1):

(1) DPPH activity ( % ) = A 0 – A S A 0 × 100 .

The absorbance value of the negative control (A ₀) is used as the benchmark for the absorbance of the sample tested at 30 min (A _S). Ascorbic acid was used as the positive control, with the scavenging activity of samples being expressed in terms of the IC₅₀ – the highest value of the 50% scavenging activity of the DPPH scavenging effect.

2.5 Antifungal activity assays

2.5.1 In vitro antifungal tests

The antifungal activities of B. cinerea and P. expansum were analyzed using samples of infected apples, which were isolated and identified at the National School of Agriculture in Meknes, Morocco. The fungal strains were preserved on PDA culture medium at 4°C and 1 mL of glycerol at 20°C. An in vitro contact test was performed following the protocol outlined by Ghnaya et al. [14] to assess the antifungal activity of the oils in question. Sterile Petri dishes containing 20 mL of PDA at 50°C were prepared with the EO solutions, each with a concentration of 12.5, 25, 50, and 100 μL/mL, prepared in 1 mL of Tween 20 (0.1% v/v), added to the agar. A 5 mm mycelial disc from a 7-day-old culture was placed at the center of each PDA-containing Petri dish. The dishes were then incubated in the dark at 25°C for 7 days, during which the percentage inhibition was calculated. The control was a PDA Petri dish containing Tween 20 (0.1%) and no EO, which was incubated in the dark at the same temperature for the same duration. The percentage of inhibition was calculated using the following formula:

(2) % Inhibition = ( C − T ) / C × 100 .

The mean value for the hyphal extension of the controls (C) was calculated as the mean of three replicates. The mean value for the hyphal extension of the EO-treated plates (T) was calculated similarly.

2.5.2 In vivo antifungal tests

Three concentrations of EOs from plant species with a significant effect were studied based on the results of the in vitro tests. First, apple fruits were washed, disinfected, and cut on the equatorial side with a sterile Pasteur pipette (3 mm wide and 2 mm deep) and then treated with 20 µL of the plant EOs studied separately at concentrations of 12.5, 25, 50, and 100 µL/mL against P. expansum and B. cinerea, respectively [4]. After 2 h of incubation at room temperature, each wound was infected with 20 µL of P. expansum and B. cinerea conidial suspension calibrated at 1 × 10⁴ spores/mL [18]. Controls received the same dose of EOs and were exposed under the same conditions. All fruits were then placed in plastic bags and stored for 10 days in the growth room chamber at 20°C and 95% relative humidity (RH). For each treatment, three apples with two wounds were used. The experiment was carried out two times in duplicate. Disease severity was evaluated as follows:

(3) [ ( Mean lesion diameter of treatment / mean lesion diameter of control ) ] × 100 = disease severity ( % ) .

2.6 Quantification of quality parameters

2.6.1 Weight loss

Following the previously described approach, healthy fruits were selected, numbered, and treated. Immediately after treatment and again after 10 days, the weight of each fruit was measured. The control was an untreated apple. It was compared with artificially inoculated and treated apples. The reduction in weight of each fruit was measured according to the following formula [4,19]:

(4) [ ( Initial weight − sample weight ) / initial weight ] × 100 .

2.6.2 Total soluble solids (TSS)

A hand-held digital refractometer model PAL-1 (Atago, Tokyo Tech., Tokyo, Japan) was used to determine TSS. After 10 days of incubation, the results were expressed in % Brix by evaluating the refractive index of the fruit juice [20].

2.6.3 Fruits firmness

For all treatments, including controls artificially inoculated with the fungus but not treated, the firmness of each apple was measured in the center of the apple using a texture analyzer (PCE-FM200, Force Gauge) combined with a flat probe of 6 and 12 mm diameter [20].

2.6.4 Titratable acidity (TA)

The TA, expressed as a percentage of malic acid per liter of juice, was measured for each treatment after 10 days of incubation. This was done by titrating 10 mL of diluted apple fruit juice (50 mL SDW, 0.1 M NaOH, pH 8.3) using the standard method [8].

2.6.5 Maturity index (MI)

The MI was calculated as the ratio of TSS to TA [8,21].

2.7 Molecular docking analysis

The study aimed to identify the bioactive compounds in the EOs of the studied plants and to determine the inhibitory effects of these compounds on target proteins involved in apple disease using molecular modeling.

2.7.1 Preparation of the ligands

First, the five molecules were initially downloaded from the PubChem website in SMILES format and then converted into PDBQTs with the AutoDockTools software [22]. The 2D structure, name, and identifier of the investigated molecules are provided in Table 1.

Table 1

Names and PubChem IDs of the compounds tested

T. articulata (Vahl) Mast.	ID	M. pulegium L.	ID	T. zygis L.	ID
α-Pinene	6654	l-Menthone	26447	Thymol	6989
δ-3-Carene	26049	Pulegone	442495	Carvacrol	28093
δ-Cadinene	441005			γ-Terpinene	7461
Thymol	6989			o-Cymene	10703
Cedrol	65575
β-Caryophyllene	5281515

2.7.2 Protein target discovery

The 3D structure (Figure 1) of the protein receptors was obtained from the RCSB PDB website [23] (http://www.rcsb.org/pdb), PDB ID: code 3WH1 for B. cinerea, and latent apple tyrosinase (MdPPO1) with PDBID ID code: 6ELS, while the National Centre for Biotechnology Information [24] (https://www.ncbi.nlm.nih.gov/protein/) provided the amino acid sequence of the GMC oxidoreductase protein from P. expansum, code XP_016595360.1 [25–27].

Figure 1

The crystal structure of receptors 3WH1 XP_016595360.1 for antifungal activity (a and b) and receptor 6ELS (c) for antioxidant activity in pdb form.

2.7.3 ADME-T prediction

ADMET was used for the evaluation of the pharmacokinetic properties of the active constituents of the studied botanicals based on their molecular composition. ADMET stands for absorption, distribution, metabolism, excretion, and toxicity [28]. This analysis was performed using the pkCSM [29] web server (pkcsm), available at https://biosig.lab.uq.edu.au/pkcsm/prediction.

2.7.4 Lipinski rule

Lipinski et al. [30] identified four ADMET properties, known as “The Fivefold Rule.” The last one is authentic for the assessment of similarity to a drug and for the determination of whether or not it is orally bioavailable. The rule of five includes the following criteria: molecular weight (MW) less than 500, octanol/water partition coefficient (log P) ≤5, number of hydrogen bond donors (HBD) ≤5, number of hydrogen bond acceptors (HBA) ≤10, and number of rotating bonds (RB) ≤ 10.

2.7.5 Molecular docking

To analyze the interaction between the receptors and the ligand-binding pockets of the derivatives, a docking study was carried out with the aid of AutoDockTools, Discovery Studio, and Pymol software.

2.8 Statistical analysis

The results were expressed as the mean of the three independent replicates of the experiments. The significance of the differences between the obtained means and treatments was tested using ANOVA with Dunnett post-hoc test with GraphPad Prism version 6.07 for Windows (GraphPad Software, San Diego, CA, USA, www.graphpad.com).

3 Results and discussion

3.1 The majority component of EOs

The oil is characterized by a pale yellow color and a strong balsamic odor. The average EO yields from the leaves of T. articulata (Vahl) Mast., M. pulegium L., and T. zygis L. are 1.2, 1.04, and 3%, respectively. The compounds present in each EO from the plants were identified through mass spectrometry and linear retention indices (Figures 2 and 3). The present analysis of the EO from T. articulata (Vahl) Mast. leaves identified up to 24 compounds. The total amount was estimated at 100%. As illustrated in Table 2, the major compounds were α-pinene (53.69%), 6,9-guaiadiene (4.55%), δ-cadinene (4.28%), β-caryophyllene (3.82%), trans-cadina-1(6),4-diene (3.36), and thymol (2.97%). T. articulata (Vahl) Mast. was rich in monoterpene hydrocarbons (57.71%).

Figure 2

Chromatogram of the major components of (a) T. articulata (Vahl) Mast. and (b) M. pulegium L.

Figure 3

Chromatogram of the major components of (a) T. zygis L.

Table 2

Chemical composition of EOs obtained from T. articulata (Vahl) Mast., M. pulegium L., and T. zygis L.

	*RT	*RI	*RI lit	Concentration (% peak area)
Compounds	*RT	*RI	*RI lit	T. articulata (Vahl) Mast.	M. pulegium L.	T. zygis L.
Tricyclene	6.11	919	926	—	—	0.39
α-Thujene	7.65	922	930	—	—	0.39
α-Pinene	7.88	936	939	53.69
Myrcene	8.10	975	990	—	0.95	—
β-Myrcene	9.47	984	986			0.66
α-Phellandrene	10.21	1002	1006	—	—	0.19
δ-3-Carene	10.08	1012	1011	1.95	—	1.18
α-Terpinene	10.34	1018	1017	1.14	—	—
o-Cymene	10.54	1021	1019	—	—	6.24
p-Cymene	10.62	1026	1024	0.93	0.48	—
β-Z-ocimene	10.70	1040	1039	—	—	0.13
γ-Terpinene	11.57	1060	1059	—	0.41	10.52
Linalool	12.78	1097	1096	—	—	0.90
Menthone	14.47	1151	1162	—	4.39	—
iso-Menthone	14.74	1154	1154	—	0.82	—
Neo-Menthol	14.89	1164	1171	—	2.28	—
cis-Isopulegone	15.04	1174	1176	—	0.59	—
Terpinen-4-ol	15.22	1175	1177	—	—	0.44
α-Terpineol	15.34	1187	1188	—	1.43	0.78
cis-Carveol	16.20	1224	1227	—	2.00	—
Citronellol	16.48	1232	1234	1.13	—	—
Pulegone	16.86	1236	1237	—	24.82	—
Piperitone	17.29	1249	1251	—	6.76	—
d-Carvone	17.71	1250	1252	—	0.66	—
Thymol	18.49	1289	1290	2.97	—	16.28
Menthyl acetate	18.50	1292	1294	—	4.06	—
Carvacrol	18.66	1296	1299	—	—	60.11
iso-Methyl acetate	18.80	1307	1304	—	0.24	—
Piperitenone	19.64	1341	1343	—	47.49	—
β-Elemene	21.01	1397	1395	1.29	—	—
E-Caryophyllene	21.84	1419	1417	—	—	1.64
β-Caryophyllene	21.84	1423	1420	3.82	—	—
6,9-Guaiadiene	22.03	1445	1448	4.55	—	—
α-Humulene	22.43	1451	1454	—	0.27	—
α-Caryophyllene	22.74	1455	1458	1.47	—	—
trans-Cadina-1(6),4-diene	23.37	1473	1477	3.36	—	—
γ-Muurolene	23.64	1481	1484	1.24	—	—
β-Selinene	22.71	1485	1494	—	0.44	—
trans-Muurola-4(14),5-diene	23.73	1490	1494	0.87	—	—
α-Murolene	23.77	1492	1495	1.00	—	—
Bicyclogermacrene	23.81	1499	1505	—	0.48	—
δ-Cadinene	24.26	1517	1524	4.28	—	—
cis-Calamenene	24.34	1524	1527	0.93	—	—
trans-Cadina-1,4-diene	24.43	1530	1536	1.15	—	—
α-Calacorene	25.01	1549	1547	2.38	—	—
β-Calacorene	25.39	1570	1568	0.92	—	—
Caryophyllene oxide	26.07	1586	1583	0.88	0.44	0.24
Cedrol	26.87	1596	1598	1.87	—	—
Humulene epoxide II	26.87	1609	1608	—	0.98	—
1-Epi-cubenol	27.37	1632	1618	3.06	—	—
α-Cadinol	28.13	1662	1659	3.02	—	—
Cadalene	36.17	1674	1679	2.12	—	—
	Monoterpene hydrocarbons			57.71	1.84	19.7
	Oxygenated monoterpenes			4.1	95.54	78.51
	Sesquiterpene hydrocarbons			29.38	1.19	1.64
	Oxygenated sesquiterpenes			8.83	1.42	0.24
	Total identified (%)			100	100	100

*RI: retention indices calculated experimentally using homologous series of C8–C28 alkanes. *RI Lit: retention indices from the literature [31–34]. *RT: retention time. Compounds and their percentage value were determined from the chromatograms of three experiments obtained on an HP-5 MS column. –: Absence.

All the compounds identified in the sample were found to be derived from the EO extracted from the leaves of M. pulegium. The most significant compound identified was piperitenone, representing 47.49%. The second largest compound present was pulegone, representing 24.82% of the total. According to GC-MS analysis, this oil is rich in oxygenated monoterpenes. The percentage is 95.54%. Regarding the findings of T. zygis L. EO, 15 compounds were identified, resulting in a 100% success rate. Carvacrol was identified as the primary compound, representing 60.11% of the total, followed by thymol (16.28%), γ-terpinene (10.52%), and o-cymene (6.24%). In addition, 78.51% of this oil is represented by oxygenated monoterpenes.

The chemical composition of EOs of T. articulata (Vahl) Mast., M. pulegium L., and T. zygis L. was described in several published articles. In a Tunisian study, 43 compounds were present in the chromatographic profiles obtained from the EOs of T. articulata (Vahl) Mast., the main ones being α-pinene, bornyl acetate, β-caryophyllene, and caryophyllene oxide, and bornyl acetate was the major compound identified in the three phenological stages [10]. The main chemical identified in T. articulata (Vahl) Mast. from Essaouira province [13] was bornyl acetate (41.80%), followed by α-pinene (17.91%), camphor (15.97%), and limonene (5.51%), and this oil was rich in oxygenated monoterpenes (63.4%) and monoterpene hydrocarbons (33.98%). These results are in line with the results of the present study. In the case of M. pulegium L., the chemical composition of the studied oil of Ouazzane, Morocco, 20 compounds were identified, representing 98.91% of the total oil obtained. R-(+)-pulegone 76.35%, carvone 5.84%, dihydrocarvone 5.09%, and octanol-3 2.25% were the main compounds of the oil [35]. In fact, the predominance of pulegone was observed in the described compositions of EO of M. pulegium L. in different countries [36,37]. These results are not consistent with those of the present study, in which piperitenone was the major compound present in M. pulegium L. In India [38] and in Algeria [39], this oil is characterized by the dominance of puleggone, which is 65.9–83.1 and 43.3–87.3%, respectively, but other oils are characterized by piperitenone 83.7–97.2% in Greece [40]. Concerning T. zygis L., EOs from Ifrane to Tigrigra (Middle Atlas) are characterized by a predominance of δ-terpineol with 27.64% [41] and monoterpene fraction (94.18%) compared to sesquiterpene (5.47%). In Spain [42] and Serbia [43], the EO of this species is characterized by high concentrations of thymol, 48.59 and 35%, respectively. Furthermore, a study conducted at Khenifra [44] showed that three major compounds, thymol (40.67%), p-cymene (26.07%), and isoborneol (13.62%), account for 65.86% of T. zygis EOs, and the identified chemical compounds of this EO were classified as oxygenated monoterpenes (62.52%). According to the literature, there is variation in the chemical composition of EOs. Many factors can influence the composition of EOs, including environmental (temperature, humidity, day length, soil type, and altitude), genetic, nutrient, and water availability [44]. This variability is further dependent on the collection date and the phenological state of plants at harvest [45].

3.2 In vitro antioxidant analysis

The evaluation of the potential of a medicinal plant is typically based on assessing its antioxidant properties and related activities. The results of the antioxidant activity of the EOs of the plants under study are presented in Figures 4 and 5.

Figure 4

DPPH free radical scavenging activities of T. articulata (Vahl) Mast., M. pulegium L., and T. zygis L.

Figure 5

DPPH free radical scavenging activities of T. articulata (Vahl) Mast., M. pulegium L., T. zygis L., and ascorbic acid.

The radical scavenging activity (DPPH) of EOs was measured by spectrophotometry at 517 nm according to the reduction of the DPPH radical, characterized by a change in color from violet to yellow. From the results, it was possible to plot the inhibition percentage (%) as a function of the concentration of EOs (Figures 4 and 5). In this study, it was found that the EO of T. zygis L. exhibited the greatest antioxidant potency (IC₅₀ = 7.60 ± 0.48 µg/mL) compared to the other EOs, followed by T. articulata (Vahl) Mast. EO (IC₅₀ = 10.81 ± 0.05 µg/mL), and finally, M. pulegium L. EO, which showed DPPH reduction with an IC₅₀ of 14.80 ± 0.02 µg/mL, these EOs have moderate antioxidant potential (IC₅₀ = 1.84 ± 0.035 µg/mL for ascorbic acid). These results show that the percentage of free radical inhibition increased with increasing concentration of EO in the plants studied. Based on the IC₅₀ values, the inhibition percentage of ascorbic acid was greater than that of the three EOs for all concentrations tested (Figure 5).

The antioxidant capacity of a compound is inversely proportional to the IC₅₀ value of the compound. This indicates the amount of antioxidants required to reduce free radical concentration by 50%. This concentration is determined graphically. It is expressed in µg/mL [46]. Based on previous studies, the IC₅₀ of T. zygis L. EO measured by DPPH assay was 2.46 µg/mL [46] higher than that obtained in this study. For T. zygis L., the high percentage of inhibition is correlated with the chemical composition of the EO. In particular, for high levels of carvacrol, which is known to have a high antioxidant power, there is a correlation between the chemical composition and the observed biological activity. In addition, the superior antioxidant power of EOs with phenolic chemotypes (carvacrol, thymol) has been demonstrated in several studies [46,47]. In a further study, the EO extracted from the aerial part of T. articulata (Vahl) Mast. from Morocco was found to have a very low level of antioxidant activity [48]. A previous study in Morocco [49], with a DPPH test IC₅₀ in the order of 12.05 × 103 ± 0.24 μg/mL, which is significantly reduced compared to the standard (IC₅₀ = 4.20 ± 0.02 μg/mL), also showed that these EOs have low antioxidant activity. Several authors have investigated the antioxidant activity of T. articulata (Vahl) Mast. The majority of these studies are in agreement with our results. In the case of M. pulegium L., collected in the region of Ouazzane in Morocco, the IC₅₀ value for the antioxidant activity against the DPPH radical was found to be 7.659 mg/mL [35]. Based on the IC₅₀ values obtained in the study by Zekri et al. [36], the strongest effect of antioxidant activity was found with M. pulegium L. EO (IC₅₀ = 15.93 mg/mL). In addition, these results substantially contradict the results of the present study. However, a study conducted in Saudi Arabia showed that the antioxidant activity of M. pulegium L. EO had an IC₅₀ of 21.13 ± 0.01 μg/mL. In summary, the presence of minor or major chemical compounds, as well as the synergy between some or all of these molecules, determine the very significant antioxidant activity of EO [44]. Finally, it is suggested that the antifungal (fungistatic) and antioxidant activities of EOs may be due to the synergistic action of two or more compounds rather than a single characteristic molecule [50].

3.3 Antifungal effects of EOs on mycelial growth under in vitro conditions

The EOs of the three plant species tested were evaluated for their effect on the inhibition of mycelial growth of P. penicillium and B. cinerea. Significant antifungal activity (p < 0.05) was observed for all treatments during the 7-day incubation period (Table 3 and Figure 6). This study showed that all EOs tested had the highest antifungal activity at 100 µL/mL concentration. It was noted that the rate of mycelial growth was significantly reduced in the four concentrations studied, ranging from 31.12 ± 0.99 to 94.44 ± 0.76% (Table 3). However, the potential of the T. zygis L. and M. pulegium L. EOs for the inhibition of both pathogens tested was higher than that of the T. articulata (Vahl) Mast. EO.

Table 3

Effect on P. expansum and B. cinerea mycelial growth in vitro of different concentrations of EOs incubated at 25°C for 7 days

Inhibition of mycelial growth (%)
EO concentration (µL/mL)
Plant species	12.5		25		50		100
	P. expansum	B. cinerea	P. expansum	B. cinerea	P. expansum	B. cinerea	P. expansum	B. cinerea
T. articulata (Vahl) Mast.	33.22 ± 1.14^b	31.12 ± 0.99^a	39.34 ± 0.44^a	37.11 ± 1.76^a	64.78 ± 0.21^a	56.67 ± 1.23^a	70.89 ± 2.01^a	69.12 ± 0.13^a
M. pulegium L.	31.45 ± 0.74^a	33.34 ± 0.31^b	40 ± 0.00^b	43.34 ± 1.07^b	67.44 ± 0.11^b	66.66 ± 0.04^b	80.78 ± 0.41^b	72.22 ± 1.15^b
T. zygis L.	34.45 ± 0.41^c	44.45 ± 0.76^c	55.56 ± 0.28^c	62.22 ± 0.52^c	78.89 ± 1.07^c	83.34 ± 0.31^c	91.78 ± 0. 93^c	94.44 ± 0.76^c

Mean values followed by different letters within the column are significantly different according to Tukey’s test (p < 0.05).

Figure 6

In vitro antifungal activity of B. cinerea of EOs derived from T. articulata (Vahl) Mast. (a), and of EOs derived from T. zygis L. (b) treated with varying concentrations (12.5, 25, 50, and 100 µL/mL). (c) Comparison between the concentrations (100 µL/mL) of T. zygis L. and M. pulegium L. EOs against P. expansum after 7 days of incubation at 25°C.

The inhibitory effect of the T.zygis L. EO against both pathogens tested is greater than that of the other two plants (Table 3). In the case of B. cinerea, the significant inhibitory effect on mycelial growth was observed with the EOs of T. zygis L. (44.45 ± 0.76%) at a concentration of 12.5 µL/mL and M. pulegium L. (43.34 ± 1.07%) at a concentration of 25 µL/mL. In addition, the lowest mycelial growth inhibition was observed for T. articulata (Vahl) Mast. (31.12 ± 0.99%) and M. pulegium L. (33.34 ± 0.31%) at a concentration of 12.5 µL/mL. For P. expansum, the significant inhibitory effect on mycelial growth was observed with the EOs of T. zygis L. (55.56 ± 0.28%) and M. pulegium L. (40 ± 0.00%) at a concentration of 12.5 µL/mL. With the same pathogen, the lowest inhibition of mycelium growth was observed with M. pulegium L. (31.45 ± 0.74%) at a concentration of 12 µL/mL and T. articulata (Vahl) Mast. (39.34 ± 0.44%) at a concentration of 25 µL/mL. However, at a concentration of 50%, T. articulata (Vahl) Mast. and M. pulegium L. showed moderate antifungal activities against both strains. Conversely, at the same concentration, T. zygis L. showed a remarkable inhibitory effect on both strains: 78.89 ± 1.07% for P. expansum and 83.34 ± 0.31% for B. cinerea. Furthermore, the highest concentration of 100 µL/mL showed a strong reduction in mycelial growth with P. expansum (91.78 ± 0.93%) and B. cinerea (94.44 ± 0.76%) for T. zygis L. Additionally, EC50 values corresponding to a 50% reduction in mycelial development over 7 days were obtained using linear regression equations (Table 4). The highest EC₅₀ values were calculated for B. cinerea by T. articulata (Vahl) Mast (38.09 µL/mL) and for P. expansum by M. pulegium L. (37.68 µL/mL). In addition, the EC₅₀ values varied from plant to plant. The lowest EC₅₀ values were observed with the EOs of T. zygis L. for B. cinerea (15.04 µL/mL) and P. expansum (20.98 µL/mL). The correlation between the fungicidal and fungistatic behavior of the EO was evaluated by the detection of the re-growth of inhibited mycelia discs after their transfer to the untreated PDA medium. If there was no growth, it was called a fungicidal effect, and if the opposite is true, it was called a fungistatic effect. In the present study, according to Znini et al. [15], it is not possible to determine the MI and the fungicidal and fungistatic effect of the oils studied because the percentage of inhibition of the fungal strains does not reach 100%.

Table 4

EC₅₀ values equivalent to 50% inhibition of mycelial growth

EC₅₀ (µL/mL)
Mycelial growth	Plant species
Mycelial growth	T. articulata (Vahl) Mast.	M. pulegium L.	T. zygis L.
B. cinerea	36.58	38.09	15.04
P. expansum	37.68	34.35	20.98

To evaluate the potential of plant EOs in the formulation of novel antifungal agents that could be used to treat post-harvest infections, numerous studies have been conducted [3,4,15]. According to several studies, pesticides have several disadvantages, including persistent residues in food. These factors, combined with public awareness of the risks posed by chemicals, have made it essential to develop safe and environmentally acceptable alternatives to pesticides [3,51]. As a percentage of all plant diseases, 30% are caused by phytopathogenic fungi and can have a major impact on crops during the cultivation, post-harvest, and storage phases [52]. Indeed, against P. expansum and B. cinerea phytopathogenic fungi, the effects of a consistent number of different EOs have been investigated. A study by Rguez et al. [10] showed that at a concentration of 100 μg mL⁻¹ for the flowering stage EO and 200 μg mL⁻¹ for the vegetative and fruiting stages EO, T. articulata (Vahl) Mast. EO extracted at the three phenological stages completely inhibited in vitro mycelial growth of B. cinerea. According to another study [10], the results obtained can be explained as follows: the presence of the pure compound α-pinene was more effective in reducing germination and altering the morphology of B. cinerea conidia than the main component of T. articulata (Vahl) Mast. EO, bornyl acetate (MIC 500 μg/mL), and α-pinene at a concentration of 250 μg/mL caused the swelling of B. cinerea conidia, which is probably related to a change in membrane permeability. In line with this, Caccioni and Guizzardi [53] demonstrated that EOs with a high content of α-pinene have fungicidal properties against various pathogens. Another study in Tunisia [14] showed that the two most sensitive species, B. cinerea (71.17%) and F. avenaceum (56.89%), showed a significant reduction in mycelial growth after treatment with T. articulata (Vahl) Mast., EO; these results are in agreement with the present study. Ghnaya et al. [14] also reported the antimycotic activity of T. articulata (Vahl) Mast. EO could result from the high proportion of monoterpene and, to a lesser degree, from other minor or trace components in the oil. It is also possible that there are synergistic and antagonistic interactions between the constituents. Pardavella et al. [54] suggested that the antifungal activity of EOs might result from the formation of hydrogen bonds between the hydroxyl group of the oil phenolics and the active sites of the targeted enzymes. In the case of M. pulegium, in vitro results showed that EO inhibited the fungal growth of B. cinerea at all concentrations tested [55]. In addition, Montenegro et al. [56] showed that the EO of M. pulegium L. has moderate activity, and isopulegol is significantly the most active growth inhibitor against B. cinerea mycelial growth and had acceptable antifungal activity against P. expansum under test [57,58]. In contrast, in another study, M. pulegium and S. mutica completely inhibited the growth of all fungi tested, including B. cinerea and P. expansum [59]. This EO antifungal activity is due to the chemical composition of the oil, and M. pulegium L. was found to be very rich in pulegone, the main contributor to biological activities [35].

For T. zygis L., EO showed a high level of activity against the growth of the post-harvest phytopathogenic fungi B. cinerea and P. expansum, obtaining MGI values ranging from 90 to 100% in almost all the two fungi tested [60]. Furthermore, thyme oil, which is rich in carvacrol, is effective as an antifungal agent in the control of B. cinerea, a major cause of stem and fruit rot during pre- and post-harvest in horticultural, fruit, and vegetable crops [61]. Carvacrol, linalyl acetate, and thymol are the main constituents of this plant, and studies have shown that the EOs from the plants tested are very effective in inhibiting the growth and reducing the spore germination of the fungi tested, particularly P. expansum and B. cinerea [59]. Buonsenso et al. [50] showed that the effect of EO components on inhibiting P. expansum mycelial growth in vitro and reducing the incidence and severity of blue mold on apples can be evaluated by taking into account the abundance of volatile molecules in the cabinet atmosphere. In addition to the high activity of thymus-type oils, the presence of phenolic components such as thymol and carvacrol may explain the high antifungal activity of these EOs. It would appear possible that the phenolic components may affect cell wall enzymes such as chitin synthase/chitinase as well as fungal β-glucanases [49,62,63]. It is crucial to conduct in vitro experiments on plant extracts or EOs as a fundamental preliminary step in investigating their antifungal efficacy against post-harvest pathogens. However, it is essential to recognize that the results of such studies may guarantee that the same outcomes will be observed in vivo. To ensure the reliability of any in vitro findings, it is vital to conduct in vivo experiments to determine whether the successful results observed in vitro can be replicated in a living organism.

3.4 Effect of EOs on rot apple disease severity

The effect of biological treatments on the development of rot lesions on apple fruit caused by P. expansum and B. cinerea is shown in Figure 7.

Figure 7

Rot lesions on apple fruit caused by P. expansum (a)–(e) and B. cinerea (f)–(j) treated with EOs of T. articulata (Vahl) Mast., M. pulegium L., and T. zygis L. after 10 days of incubation at 25°C at a concentration of 1 × 10⁴ spores/mL. Fruit treated with sterile distilled water only: control (a and f), fruit treated with 12.5 µL/mL of EO (b) and (g), fruit treated with 25 µL/mL of EO (c) and (h), fruit treated with 50 µL/mL of EO (d) and (h), and fruit treated with 100 µL/mL of EO (e) and (j).

The data presented in Table 5 indicate that all treatments showed a significant concentration-dependent efficacy. The severity of rot lesions on apple fruit was reduced at concentrations of 12.5 and 25 µL/mL to 62.67 and 31.66% for P. expanqum, but for B. cinerea, the reductions of rot lesions were 59.23 and 29.03%, respectively, with EOs of T. zygis L. In addition, the same EO at 50 µL/mL showed a highly significant reduction in severity, with values of 11.33% for P. expansum and 09.19% for B. cinerea, followed by M. pulegium L. At 100 µL/mL, T. zygis L. suppressed the severity of the disease in artificially wounded and inoculated fruits, with 100% inhibition of B. cinerea. In the case of T. articulata (Vahl) Mast. and M. pulegium L., results showed that EOs extracted from the studied plants moderately reduced disease severity on apple rot at concentrations of 12.5 and 50 µL/mL compared to imazalil 1 ppm for P. expanqum and B. cinerea. At a concentration of 50 µL/mL, T. articulata (Vahl) Mast. and M. pulegium L. significantly reduced disease severity, and at 100 µL/mL, they were highly active for both pathogenic agents.

Table 5

In vivo effects of EOs on the mycelial growth of B. cinerea and P. expansum evaluated over 10 days at 25°C

Disease severity (%)
EO concentration (µL/mL)
Plant species	12.5		25		50		100
	P. expansum	B. cinerea	P. expansum	B. cinerea	P. expansum	B. cinerea	P. expansum	B. cinerea
T. articulata (Vahl) Mast.	77.05 ± 0.63^d	81.50 ± 0.32^d	47.12 ± 0.05^d	54.43 ± 0.61^d	19.65 ± 0.38^d	24.07 ± 0.28^d	09.87 ± 0.06^d	12.56 ± 0.17^d
M. pulegium L.	73.35 ± 0.83^c	70.43 ± 0.74^c	36.87 ± 0.09^c	46.87 ± 0.09^c	13.45 ± 0.60^c	21.65 ± 0.88^c	09.00 ± 0.02^c	11.35 ± 0.22^c
T. zygis L.	62.67 ± 0.12^b	59.23 ± 0.14^a	31.66 ± 0.45^b	29.03 ± 0.27^b	11.33 ± 0.55^b	09.19 ± 0.32^b	06.54 ± 0.35^b	00.00 ± 0.00^b
Imazalil 1 ppm	42.64 ± 0.02^a	37.36 ± 1.46^a	29.02 ± 0.16^a	25.53 ± 0.18^a	08.15 ± 0.24^a	06.23 ± 0.13^a	0.00 ± 0.00^a	0.00 ± 0.00^a

Mean values followed by different letters within the column are significantly different according to Tukey’s test (p < 0.05).

The effect of biological treatments on the development of rot lesions on apple fruit caused by P. expansum and B. cinerea is shown in Figure 6. The data presented in Table 5 indicate that all treatments showed significant concentration-dependent efficacy. The severity of rot lesions on apple fruit was reduced at concentrations of 12.5 and 25 µL/mL to 62.67 and 31.66% for P. expanqum, but for B. cinerea, the reductions of rot lesions were 59.23 and 29.03%, respectively, with EOs of T. zygis L. In addition, the same EO at 50 µL/mL showed a highly significant reduction in severity, with values of 11.33% for P. expansum and 09.19% for B. cinerea, followed by M. pulegium L. At 100 µL/mL, T. zygis L. suppressed the severity of the disease in artificially wounded and inoculated fruits, with 100% inhibition of B. cinerea. In the case of T. articulata (Vahl) Mast. and M. pulegium L., results showed that EOs extracted from the studied plants moderately reduced disease severity on apple rot at 12.5 and 50 µL/mL compared to imazalil 1 ppm for P. expanqum and B. cinerea. At a concentration of 50 µL/mL, T. articulata (Vahl) Mast. and M. pulegium L. significantly reduced disease severity, and at 100 µL/mL, they were highly active for both pathogenic agents. Therefore, in vitro testing of plant extracts is an established starting point for evaluating the antifungal potential of plant extracts against post-harvest pathogens, but in vivo testing is required to see if the successful results of in vitro testing can be confirmed. In agreement with the present findings, Marandi et al. [64] showed that Thymus kotschyanus oil had a greater effect in reducing disease severity (%) on pear fruits inoculated with B. cinerea and P. expansum. The high antifungal activity of T. kotschyanus oil can be related to both its phenolic components (carvacrol and thymol) and also to the synergism of its minor and major components. Several studies have shown that EOs extracted from thyme are highly effective in controlling post-harvest diseases of apple fruit. For example, Fathi et al. [65] found that T. vulgaris EO at concentrations above 400 µL/L completely inhibited the growth of P. expansum and B. cinerea, and after 15 days of storage showed that thyme EO at 10% had a statistically significant effect on P. expansum, while treatments with 1 and 10% EOs were statistically similar to the chemical control for B. cinerea [66].

In the case of mint, in vivo tests have shown the efficacy of M. pulegium L. EO in the complete suppression of gray mold on strawberries previously inoculated with B. cinerea conidia [67], and these results are in line with the results of this study. Previous work has shown the high efficacy of the EO of M. pulegium L., as it is dominated by a ketone that is more active against microbial agents than terpene oxides [68] and has been shown to have significant antifungal activity against a wide range of pathogens, including P. expansum [69]. For T. articulata (Vahl) Mast., EO extracted at the flowering stage, in vivo antifungal assays showed that it was effective in inhibiting B. cinerea infection on tomato fruit at a low concentration of 100 μg mL⁻¹. This supports its use in the control of this pathogen in post-harvest conditions [10]. Recent studies have confirmed the efficacy of EOs in controlling gray mold [10,15,67,70]. Furthermore, the research conducted by Chebli et al. [71] has indicated that major compounds have an inhibitory effect on mycelial growth. However, the biological activity observed may result from a combined or synergistic effect of minor compounds [72]. Conversely, when the EOs are in their entirety and at higher concentrations, the EO’s activity is the result of both its major compounds and the synergistic effect of minor compounds [67]. In addition to their direct antifungal activity, numerous studies have demonstrated that EOs can also enhance plant growth and stimulate the defense mechanisms of plants, which are related to the antioxidant system and phenylpropanoid pathway. This can facilitate the production of pathogen-fighting proteins, enabling plants to overcome infections [73].

3.5 Fruit quality parameters

As can be seen in Tables 6–8, EOs produced by these plants had a significant effect on apple quality parameters, depending on their concentration. Fruit firmness, weight loss, TSS, TA, and MI were assessed after 10 days of storage of apples from each treatment.

Table 6

Effect of T. articulata (Vahl) Mast. EO treatment 10 days after incubation at 25°C on the quality parameters of apples

T. articulata (Vahl) Mast. EO (µL/mL)	Weight loss (%)		Firmness (N)		TSS %		TA (g malic acid/L)		MI
T. articulata (Vahl) Mast. EO (µL/mL)	P. expanse	B. cinerea	P. expansum	B. cinerea	P. expansum	B. cinerea	P. expansum	B. cinerea	P. expansum	B. cinerea
Inoculated fruit	6.45 ± 0.61^e	6.98 ± 0.05^e	3.37 ± 0.7^d	3.52 ± 1.1^d	8.56 ± 0.20^e	8.32 ± 0.08^e	4.85 ± 0.12^d	3.98 ± 0.05^c	1.76	2.09
12.5	6.19 ± 0.12^e	6.82 ± 0.2^e	4.23 ± 1.3^c	4.19 ± 0.9^cd	8.87 ± 0.15^e	8.98 ± 0.03^e	4.18 ± 0.31^d	3.23 ± 0.45^c	2.12	2.78
25	4.26 ± 0.01^d	5.87 ± 1.5^d	5.02 ± 0.2^bc	4.98 ± 0.04^c	9.23 ± 0.64^d	9.77 ± 0.52^d	3.87 ± 0.11^cd	2.98 ± 0.39^bc	2.38	3.27
50	2.61 ± 0.88^c	3.76 ± 0.9^c	5.89 ± 0.07^b	5.45 ± 0.13^b	10.34 ± 0.06^c	11.2 ± 0.25^c	3.11 ± 0.13^c	2.34 ± 0.72^b	3.32	4.79
100	1.63 ± 0.3^b	2.54 ± 0.4^b	6.78 ± 0.06^a	6.16 ± 0.07^ab	11.66 ± 0.23^b	12.09 ± 0.35^b	2.45 ± 0.01^b	1.76 ± 0.17^a	4.75	6.86
Untreated fruit	0.08 ± 0.8^a	0.08 ± 0.1^a	6.89 ± 0.5^a	6.89 ± 0.06^a	13.7 ± 0.17^a	13.7 ± 0.05^a	0.87 ± 0.06^a	1.32 ± 0.02^a	15.74	10.37

Data represent mean ± standard deviation (SD). In each column, values having different letters are significantly different according to Tukey’s test (p < 0.05).

Table 7

Effect of M. pulegium L. EO treatment 10 days after incubation at 25°C on quality parameters of apples

M. pulegium L. EO (µL/mL)	Weight loss (%)		Firmness (N)		TSS %		TA (g malic acid/L)		MI
M. pulegium L. EO (µL/mL)	P. expansum	B. cinerea	P. expansum	B. cinerea	P. expansum	B. cinerea	P. expansum	B. cinerea	P. expansum	B. cinerea
Inoculated fruit	6.45 ± 0.61^e	6.98 ± 0.05^e	3.37 ± 0.7^d	3.52 ± 1.1^d	8.56 ± 0.20^e	8.32 ± 0.08^f	4.85 ± 0.12^de	3.98 ± 0.05^ef	1.76	2.09
12.5	6.03 ± 0.3^e	6.76 ± 0.13^e	4.29 ± 0.04^c	4.05 ± 0.87^cd	9.01 ± 0.77^d	9.16 ± 0.04^e	4.11 ± 0.21^d	3.09 ± 0.03^e	2.19	2.96
25	3.45 ± 0.03^d	4.09 ± 0.01^d	5.02 ± 0.09^b	4.89 ± 1.12^c	9.87 ± 0.65^c	10.97 ± 0.04^d	3.56 ± 0.53^c	2.75 ± 0.02^cd	2.77	3.98
50	2.41 ± 0.09^c	3.34 ± 0.02^c	5.98 ± 0.13^b	5.76 ± 0.23^b	10.76 ± 0.12^c	11.65 ± 0.07^c	2.99 ± 0.32^bc	2.13 ± 0.07^c	3.59	5.42
100	1.23 ± 0.12^b	2.68 ± 0.23^b	6.80 ± 0.54^a	6.43 ± 0.52^a	11.87 ± 0.08^b	12.27 ± 0.06^b	2.22 ± 0.08^b	1.65 ± 0.11^b	5.34	7.43
Untreated fruit	0.08 ± 0.8^a	0.08 ± 0.1^a	6.89 ± 0.5^a	6.89 ± 0.06^a	13.7 ± 0.17^a	13.7 ± 0.05^a	0.87 ± 0.06^a	1.32 ± 0.02^a	15.74	10.37

Data represent mean ± standard deviation (SD). In each column, values having different letters are significantly different according to Tukey’s test (p < 0.05).

Table 8

Effect of T. zygis L. EO treatment 10 days after incubation at 25°C on quality parameters of apples

T. zygis L. EO (µL/mL)	Weight loss (%)		Firmness (N)		TSS %		Titrable acid (g malic acid/L)		MI
T. zygis L. EO (µL/mL)	P. expansum	B. cinerea	P. expansum	B. cinerea	P. expansum	B. cinerea	P. expansum	B. cinerea	P. expansum	B. cinerea
Inoculated fruit	6.45 ± 0.61^f	6.98 ± 0.05^f	3.37 ± 0.7^e	3.52 ± 1.1^e	8.56 ± 0.20^f	8.32 ± 0.08^f	4.85 ± 0.12^ef	3.98 ± 0.05^e	1.76	2.09
12.5	5.04 ± 0.12^e	4.27 ± 0.56^e	4.66 ± 0.04^d	4.45 ± 0.32^d	9.14 ± 0.12^e	9.73 ± 0.01^d	4.02 ± 0.45^e	2.98 ± 0.09^cd	2.27	3.26
25	3.09 ± 0.03^d	2.81 ± 0.14^d	5.19 ± 0.09^c	5.12 ± 0.23^c	10.17 ± 0.07^d	11.06 ± 0.23^c	3.32 ± 0.27^d	2.45 ± 0.04^c	3.06	4.51
50	2.06 ± 0.01^c	1.64 ± 0.98^c	6.09 ± 0.04^ab	5.98 ± 0.13^bc	11.72 ± 0.21^c	12.18 ± 0.15^b	2.43 ± 0.04^c	1.98 ± 0.88^bc	4.82	6.15
100	1.04 ± 0.02^b	0.63 ± 0.23^b	6.84 ± 0.02^a	6.23 ± 0.09^b	12.24 ± 0.02^b	13.12 ± 0.09^a	1.89 ± 0.12^b	1.45 ± 0.54^ab	6.47	9.04
Untreated fruit	0.08 ± 0.8^a	0.08 ± 0.1^a	6.89 ± 0.5^a	6.89 ± 0.06^a	13.7 ± 0.17^a	13.7 ± 0.05^a	0.87 ± 0.06^a	1.32 ± 0.02^a	15.74	10.37

Data represent mean ± standard deviation (SD). In each column, values having different letters are significantly different according to Tukey’s test (p < 0.05).

Results showed a significant effect of the different treatments on weight loss (%). For both P. expansum and B. cinerea strains, all treatments reduced weight loss compared to the control. From one concentration to another, there was also a significant reduction in weight loss. However, T. zygis L. EO showed an interesting reduction of up to 0.63% at a concentration of 100 µL/mL for B. cinerea and 1.23% for P. expansum. Fruit firmness measurements showed that the EOs tested had an appreciable effect in maintaining fruit firmness for both fungal strains. The significant effects are shown in Tables 6–8. For all concentrations, the three EOs showed values between 4 and 6 N. The best preservative activity was shown by the EO of T. zygis L. for P. expansum at all concentrations, with the highest value (6.84 N) at 100 µL/mL in proximity to the untreated fruit and the lowest (4.66 N) at 12.5 µL/mL compared to the control (3.37 N). For TSS, apples inoculated with both fungi showed a slight decrease. When treated with T. articulata (Vahl) Mast., fruits inoculated with B. cinerea showed a lower TSS value of 8.87 °Brix at a concentration of 12.5 µL/mL compared to the control (13.7 °Brix) EO compared to the control (13.7 °Brix). Furthermore, the highest TSS value for B. cinerea was shown with T. zygis L. EO at a concentration of 100 µL/mL with a value of 13.12 °Brix. TA levels for all EOs tested were measured, and there was a significant effect of treatment, with TA levels increasing proportionally with increasing EO concentration. In addition, slight variations between treatments were highlighted for the evaluation of MI. In the case of fruits infected with P. expansum, the M. pulegium L. EO showed an MI value of 1.76 at a concentration of 12.5 µL/mL and an MI value of 5.34 at a concentration of 100 µL/mL, compared with a value of 15.74 MI for control fruits. On the other hand, fruits inoculated with B. cinerea had an MI value of 2.96 at a concentration of 12.5 µL/mL and an MI value of 10.37 for the control. The results of the present study agree with Cherrate et al. [4], who found that T. vulgaris effectively controls blue and gray mold in apples without adversely affecting apple quality parameters, making it an ideal candidate for future commercial use.

In addition, El Khetabi et al. [74] showed that thyme EO improved apple fruit quality, such as weight loss, TA, TSS, and MI, and also had a positive effect on maintaining fruit firmness. In line with the present study, Santoro et al. [75] showed that the application of thyme EO vapor is a promising tool for post-harvest loss reduction and quality preservation of peaches and nectarines. In addition, EO fumigation was found not to affect the overall fruit quality but was beneficial in reducing weight loss and preserving ascorbic acid and carotenoids. Indeed, a significant difference between the weight loss of strawberries treated or not with M. pulegium L. EOs against B. cinerea was shown by Aouadi et al. [67]. The reduction in weight loss during post-harvest storage of fruits is probably due to the formation of a protective layer on the surface of the fruit orifices, which prevents water loss by minimizing metabolic activity, respiration, and transpiration [76]. The present results clearly show that the use of EO affects biochemical changes in the fruit, although the mechanism of this effect is still unclear. However, to assess the cost and efficacy of these EOs on a wide range of post-harvest diseases, further studies are required.

While previous studies have explored the use of EOs for combating apple diseases, our research distinguishes itself by focusing on a largely unexplored region and the use of different aromatic and medicinal plants. Specifically, our study concentrates on the development of these plants in the Taza region, an area where, to our knowledge, no previous research has been conducted on their potential for addressing post-harvest apple diseases. In comparison, in 2013, Znini et al. [15] investigated the use of Warionia saharae EO as a solution to apple diseases. In 2021, Znini et al. [77] investigated the antifungal properties of Teucrium luteum EO using liquid- and vapor-phase methods. The present research, however, extends these studies by evaluating the biological properties of EOs from three different plants: T. zygis L., M. pulegium L., and T. articulata (Vahl) Mast. Unlike previous studies, we employ in vitro, in vivo, and in silico methods, providing a more comprehensive analysis of the bioactive molecules responsible for their antioxidant and antifungal activities against B. cinerea and P. expansum. This combined approach allows us to confirm the efficacy of these plants in a way that previous studies have not, offering new insights into potential solutions for apple diseases.

In summary, a number of studies have investigated the efficacy of EOs and plant extracts in controlling post-harvest apple diseases, particularly fungal infections that affect fruit quality and shelf-life. This research has shown that many EOs have significant antifungal properties, both in the laboratory (in vitro) and under real-life conditions (in vivo). These results are particularly interesting in the context of the search for alternative, natural solutions to control post-harvest diseases to replace or complement the chemical products often used in agriculture [3,4,74,77–79].

The diversity of biological activities observed in the oils studied is shown in Table 9. This suggests that these oils have many properties that can affect different aspects of health and biological functioning. These biological activities may include antimicrobial, anti-inflammatory, antioxidant, and analgesic effects, as well as influencing metabolic regulation, heart health, and managing oxidative stress. These properties reflect the potential of these oils to contribute to disease prevention and general well-being. However, certain oils may have stronger antimicrobial effects, while others may offer better protection against inflammation or oxidative damage that can lead to chronic disease. Understanding these different effects is crucial to determining the most effective use of each oil in different health contexts.

Table 9

Comparative studies of biological activities of T. articulata (Vahl) Mast., M. pulegium L., and T. zygis L.

EOs	Biological activities
T. articulata (Vahl) Mast.	T. articulata (Vahl) Mast. EO is rich in oxygenated and monoterpene hydrocarbons [13]. The current plants have important antioxidant and antimicrobial activities, and genotoxicity [80–82] A study by Labhar et al. shows that T. articulata (Vahl) Mast. leaf extracts are potent antioxidant and natural anti-inflammatory agents [83] In order to provide a stronger biochemical rationale for the activity of the ethyl acetate extract obtained from the bark of T. articulata (Vahl) Mast., the cytotoxic activity was first assessed by in vitro analyses, followed by a confirmatory in silico study [84]
M. pulegium L.	In the EOs of M. pulegium L., 26 compounds were identified. Pulegone was the major constituent of the EO, followed by menthone, piperitone, limonene, and cis-isopulegone. This oil is highly effective in terms of biological activities such as antioxidant, antifungal, and anti-inflammatory activities in vivo [85] Recently, EOs from M. pulegium L. have shown potent insecticidal activity, making them new candidates for pest management strategies with minimal adverse effects. M. pulegium L. showed acute contact toxicity against third-instar larvae of P. interpunctella [86] M. pulegium L. is a source of green nanoparticles used for antibacterial, antifungal, anticancer, and antioxidant applications [87]
T. zygis L.	GC/MS analysis identified 54 constituents representing 92.65% T. zygis. The major components found in the EOs of T. zygis are δ-terpineol (27.64%), followed by δ-3-carene (15.7%), thymol (14.17%), and dehydrolinalool (4.99%) [41] Thymus zygis L. exhibits antimicrobial (in vitro and in situ) and antibiofilm activities with potential antiproliferative effects [88], antifungal [89], and antioxidant [41,44], and anticancer activities [90]

3.6 In silico experiments

3.6.1 ADMET prediction

The ADMET predictions for each molecule are based on several factors, including how the molecule will be absorbed, distributed, metabolized, excreted, and toxic. The results of the ADMET predictions for each molecule are shown in Table 10. A high absorption rate of more than 90% was observed for all molecules studied. In addition, parameters such as blood–brain barrier (BBB) and central nervous system (CNS) permeability are considered to assess distribution [91]. In particular, molecules with an unbound fraction greater than −0.44 could cross the BBB because their Log BB is greater than −1 and the CNS permeability is outside the range of −2 to −3 [92]. Therefore, the carvacrol molecule will not be able to cross these barriers [93]. The enzymatic metabolism of a drug in the body reflects its chemical biotransformation [94]. The elimination of xenobiotics is mediated by CYP enzymes, which are present in all tissues of the body [95]. As a result, the compounds must act as both substrates and inhibitors of CYP. In recent years, properties such as absorption, distribution, metabolism, excretion, and toxicity (ADMET) have become a major focus in evaluating the impact or dangers of these substances on the human organism, even when they are not intended for direct human use. This assessment is due to the fact that the environment around us contains many small-molecule compounds that have an impact on our health, such as drugs, pesticides, nutritional additives, chemicals used in industry, and environmental pollutants [96]. The ADMET test, which shows that all molecules tested were non-toxic, is commonly used to assess the toxicity of compounds.

Table 10

Results of the ADMET test

Ligand	Distribution		Metabolism								Excretion	Toxicity
	Intestinal absorption in human	Blood brain barrier permeability	CNS permeability	Substrate		Inhibitor					Total clearance	AMES toxicity
	Intestinal absorption in human	Blood brain barrier permeability	CNS permeability	2D6	3A4	1AE	2C19	2C9	2D6	3A4
	Numeric (% Absorbed)	Numeric (log BB)	Numeric (log PS)	Categorical (Yes/No)							Numeric (log mL/min/kg)	AMES toxicity
α-Pinene	96.041	0.791	−2.201	NO	NO	NO	NO	NO	NO	NO	0.043	NO
3-Carene	96.438	0.759	−2.26	NO	NO	NO	NO	NO	NO	NO	0.037	NO
Thymol	90.843	0.407	−2.664	NO	NO	YES	NO	NO	NO	NO	0.211	NO
β-Caryophyllene	94.845	0.733	−2.172	NO	NO	NO	NO	NO	NO	NO	1.088	NO
δ-Cadinene	96.128	0.773	−2.945	NO	NO	NO	NO	NO	NO	NO	1.182	NO
Cedrol	93.844	0.627	−2.216	NO	YES	YES	YES	NO	NO	NO	0.837	NO
l-Menthone	97.324	0.607	−2.155	NO	NO	NO	NO	NO	NO	NO	0.244	NO
Pulegone	96.77	96.77	−2.411	NO	NO	NO	NO	NO	NO	NO	0.752	NO
Carvacrol	90.843	0.407	−1.664	NO	NO	YES	NO	NO	NO	NO	0.259	NO
γ-Terpinene	96.219	0.754	−2.049	NO	NO	NO	NO	NO	NO	NO	0.756	NO
o-Cymene	93.882	0.48	−2.397	NO	NO	YES	NO	NO	NO	NO	0.259	NO

3.6.2 Lipinski rules

The results obtained are shown in Table 11, where all the designed compounds met the established criteria according to Lipinski’s Rule of Five. This means that all parameters, such as molecular weight (MW), number of rotatable bonds (RB), number of hydrogen donors (HBD), number of hydrogen acceptors (HBA), and octanol/water partition coefficient (Log P), are within acceptable ranges, and therefore it is reasonable to conclude that these molecules, including the larger ones, have a good pharmacokinetic profile.

Table 11

Results of the Lipinski rules

Molecule	MW	HBD	HBA	Log P	RB
Thymol	150.221	1	1	2.82402	1
δ-Cadinene	204.357	0	0	4.7252	1

3.6.3 Molecular docking

A complementary in silico study was performed to better understand the interactions between the different molecules and crystal structures after an experimental study of the antifungal activity of three objective plants: the crystal structure of chitinase GH19 (3wh1) from B. cinerea and that of GMC oxidoreductase from P. expansum (XP_016595360.1). The selection of the GH19 (3wh1) receptor was made based on its crucial role in the enzymatic conversion of chitin, a key component of fungal cell walls, and its involvement in the biocontrol of B. cinerea. The present study aimed to explore how specific ligands could interact with this receptor, offering insights into possible biocontrol strategies. In addition, we selected the XP_016595360.1 receptor in P. expansum due to its essential role in the detoxification of toxic organic compounds, particularly through oxidative coupling reactions. This enzyme catalyzes the transfer of electrons from a reduced organic substrate (donor) to an acceptor, a process that is vital for the survival and adaptation of P. expansum in hostile environments. In order to conduct a molecular docking study of antioxidant activity, the enzyme latent apple tyrosinase was selected. Tyrosinases are a subclass of the polyphenol oxidase (PPO) family of enzymes, which are primarily responsible for the browning of fruits. Molecular docking results, including the binding affinity of the ligand to the receptor active site of each fungal strain, hydrogen bonds, hydrophobic bonds, and interactions, as well as for the latent apple tyrosinase (MdPPO1) 6ELS.pdb receptor for antioxidant activity, have been classified, as shown in Table 12.

Table 12

Receptor-bioactive molecule binding affinities (kcal/mol) of T. articulata (Vahl) Mast., M. pulegium L., and T. zygis L. for antifungal (receptors: XP_016595360.1 and 3wh1) and antioxidant (receptors: 6ELS.pdb) activities

Molecules	B. cinerea (3wh1)	P. expansum (XP_016595360.1)	Latent apple tyrosinase (6ELS)
Molecules	Free binding energy (kcal/mol)
α-Pinene	−3.9	−5.5	−4.8
δ-3-Carene	−4.1	−6.0	−4.7
β-Caryophyllene	−5.9	−7.0	−6.1
δ-Cadinene	−6.2	−7.4	−6.0
Cedrol	−5.7	−6.5	−5.9
l-Menthone	−4.1	−5.8	−4.8
Pulegone	−4.4	−6.1	−5.0
Thymol	−5.6	−6.3	−4.8
Carvacrol	−4.1	−6.0	−4.8
γ-Terpinene	−4.2	−6.2	−4.6
o-Cymene	−4.1	−5.8	−4.9

Bold values indicate the molecules with the highest affinity in the ligand-receptor reaction.

This study aimed to better understand the mode of action of bioactive molecules in the control of pathogenicity and oxidation by identifying the ligands that are expected to block their activities (Figures 8 and 9). Docking predicted that the molecules specifically bind to the active site of the fungal infection protein [97]. In the present study, molecular docking of eight bioactive molecules from the plant T. articulata (Vahl) Mast. showed that all ligands interacted with the active sites of the receptors and inhibited the activity of the fungi B. cinerea and P. expansum. All ligands showed significant binding energy with the active pocket of the protein (3wh1). This energy varied between −3.9 and −6.2 kcal/mol for B. cinerea and between −5.5 and −7.4 kcal/mol for P. expansum. However, the δ-cadinene molecule showed the best affinity with both strains with energy values of −7.4 and 6.2 kcal/mol for XP_016595360 1 and 3wh1 by successively forming hydrophobic bonds with amino acids (ILE 163), (TRP103), (PHE 139), and (ILE 99) of chitinase GH19 (3wh1) from the fungal strain B. cinerea, and with (LEU 600), (VAL 604), (ALA 589), (PRO 557), (ALA 322), (ILE 323), and (ARG 126) with that of GMC oxidoreductase from the P. expansum strain (XP_016595360. 1), as shown in Figures 8 and 9. In the case of the plant T. zygis L., the molecular docking study showed that the thymol molecule is strongly bound to the active site of the chitinase protein GH19 with an energy value of −5.6 kcal/mol and to the GMC oxidoreductase protein of the P. expansum strain (XP_016595360.1) with an energy value of −6.3 kcal/mol. The ligand–receptor coupling is achieved by the formation of hydrogen and hydrophobic bonds with the amino acids (TRP 103), (ILE 99), (GLN 100), and (PHE 139) for the chitinase and with (VAL 694), (ALA 589), and (HIS 556) for the GMC oxidoreductase protein (Figure 9).

Figure 8

2D interaction of bioactive molecules with high affinity (1 and 2) to B. cinerea (3wh1.pdb) and (3 and 4) P. expansum (XP_016595360.1.pdb).

Figure 9

Hydrogen bond interaction (a) and hydrophobic bond interaction (b) of δ-cadinene with the 3wh1.pdb receptor of B. cinerea (1) and XP_016595360.1.pdb receptor (2) of P. expansum observed in 3D. Furthermore, hydrogen bond interaction (a) and hydrophobic bond interaction (b) of thymol with the 3wh1.pdb receptor (3) and XP_016595360.1.pdb receptor (4).

To ascertain the potential antioxidant properties of the compounds, the present study will aim to establish a correlation between their binding affinities and their capacity to reduce oxidative stress and protect the fruit against damage. Table 12 illustrates the affinity of the molecules under investigation for the latent apple tyrosinase receptor (MdPPO1), with β-caryophyllene exhibiting the highest energy score of 6.1 kcal/mol. This affinity is a consequence of the formation of specific hydrophobic bonds between the ligand and the protein, as illustrated in Figure 10, which involve amino acids (ALA222), (TYR220), (LEU104), and (LEU111). The findings indicate that β-caryophyllene exerts a pronounced interaction with the enzyme latent apple tyrosinase (MdPPO1), which is implicated in the enzymatic browning of apples. This process is associated with the oxidation of phenolic compounds into quinones, resulting in the formation of brown pigments. In addition, the δ-cadinene molecule also exhibits a considerable binding affinity, with a score of −6.0 kcal/mol. This occurs through the formation of hydrophobic bonds with the amino acids (LYS 80), (LYS69), (PHE 79), and (ARG264), as illustrated in Figure 10. This suggests that δ-cadinene could effectively inhibit the enzyme by stabilizing its active site via hydrophobic bonds, which could help to delay browning and increase antioxidant activity by protecting phenolic compounds from oxidation.

Figure 10

2D interaction, hydrogen bond interaction, and hydrophobic bond interaction of β-caryophyllene with the latent apple tyrosinase (MdPPO1) 6ELS.pdb receptor (a). Furthermore, 2D interaction and hydrophobic-bond interaction of δ-cadinene with the latent apple tyrosinase (MdPPO1) 6ELS.pdb receptor (b).

The remaining molecules, including cedrol, pulegone, o-cymene, and α-pinene, demonstrated a moderate affinity for the latent tyrosinase receptor (MdPPO1). This study also demonstrates that δ-cadinene exhibits the highest affinity with both B. cinerea and P. expansum strains, with energy values of −7.4 and 6.2 kcal/mol for XP_016595360.1 and 3WH1, respectively. This is achieved by successively forming hydrophobic bonds with the latent tyrosinase receptor (MdPPO1). These findings provide further evidence that these molecules may play a crucial role in reducing oxidation. Consequently, the current molecule exhibits dual antioxidant and antifungal properties, effectively preventing apple rot.

The inhibition of mycelial growth and germination of spores by EOs may help limit pathogen spread by reducing spores in the storage atmosphere and on surfaces. As a result, EOs may have antifungal activity against B. cinerea and may be used as an ideal treatment in future plant disease prevention programs to eliminate fungal growth [70]. Previous studies have shown that the antifungal activity of EOs is strongly related to their chemical composition [98]. Indeed, phenolic molecules, such as thymol and carvacrol, or aldehydic molecules, such as p-anisaldehyde and ketones, are effective at inhibiting pathogen growth. EOs from thyme (Thymus vulgaris) and savory (Satureja montana), consisting mainly of thymol and carvacrol, are highly effective in controlling fungal pathogens [50]. In previous studies [61], thymol demonstrated potent antifungal activity against the target B. cinerea at minimum inhibitory concentrations of 65 mg/L and minimum fungicidal concentrations of 100 mg/L. In addition, under scanning electron microscopy, thymol significantly altered the morphology of B. cinerea hyphae by causing damage and distortion of the mycelium. The mechanism of antifungal activity involves hydrophobic compounds interacting with fungal cell membrane lipids [61]. Indeed, the principle of EOs is based on their ability to penetrate the cell membrane through the lipid bilayer, resulting in increased cell permeability, leading to cell death and inhibition of fungal sporulation and germination [99]. Another hypothesis is that fungal plasma membrane ergosterol synthesis is also involved [50]. This hypothesis is based on the loss of membrane integrity, which in turn can induce alterations in the electron transport chain, nutrient absorption, protein and nucleic acid synthesis, inhibition of enzymes essential for energy metabolism, coagulation of cell contents, and ultimately cell death [100,101].

To assess the ligand–receptor interaction of antioxidant activity, we used the PPO receptor (latent apple tyrosinase) found in apples. The enzymes known as oxidoreductases are a large group of enzymes that catalyze the transfer of electrons from one molecule to another (a reducing molecule or electron donor) (the oxidant or electron acceptor). Most of these enzymes require an electron donor, an electron acceptor, or two additional cofactors [102]. One of these enzymes is the latent tyrosinase found in apples. In addition, tyrosinases are type 3 copper enzymes of the PPO family, capable of catalyzing both the ortho-hydroxylation of monophenols and their subsequent oxidation to o-quinones, which are precursors for the biosynthesis of pigments such as melanin. The first plant protyrosinase from Malus domestica (MdPPO1) was recombinantly expressed in the latent form [103]. PPO plays a significant role in the browning and oxidation processes in plants, and its inhibition could have beneficial effects, particularly in preventing oxidative damage in biological systems or preserving food products.

Previous studies [104] confirmed these results by measuring the decrease in the intrinsic fluorescence intensity on membrane-bound polyphenol oxidases (mPPO) due to inhibitors having a single class of the inhibition site on mPPO with fluorescence spectroscopy. The active center’s important binding sites were amino acid residues Him 180, Him 201, Him 366, Cys 184, Glu 328, and Asn 333. It could hence be inferred that these were the sites at which the inhibitors were allosterically bound at the active center of mPPO through hydrogen bonding and ion contacts.

Sun et al. [105] identified the production of tyrosinase inhibitors from a range of natural materials, including phenolic and polyphenolic compounds, benzaldehyde derivatives, long-chain fatty acids, steroidal compounds, and other natural materials. These findings align with the results of the present study. The findings of the present study indicate that EOs contain active molecules that inhibit tyrosinase. Rezaeinasab et al. [106] demonstrated that EO extracted from the thymus could be a significant source of chemicals with antioxidant potential, which is characterized by the suppression of ROS-producing proteins such as lipoxygenase and xanthine oxidase. Furthermore, the most active in silico compound was found to be carvacrol in a study by Jianu et al. [107]. In conclusion, the phytochemical profile of the EOs of the three species under investigation demonstrated a range of biological properties that could be employed to protect apples against rot.

4 Conclusion

The present study investigated the antifungal activity of EOs extracted from three medicinal plants used and concluded that they have significant in vitro and in vivo effects against P. expansum and B. cinerea pathogens and can be used as natural bio-fungicides. Furthermore, the EO of T. zygis L. was found to have the best antioxidant and antifungal activities in vitro and in vivo among the three plants used without affecting apple quality parameters (weight loss, fruit firmness, TSS, and TA). The richness of this oil in bioactive molecules may explain these results. However, further phytochemical research is needed to establish the efficacy of EOs as potential botanical pesticides and as a safer, cleaner alternative to protect fruit from fungal attacks. It is also necessary to identify and explain the mode of action of the bioactive molecules present in medicinal plants that are responsible for their antimicrobial properties.

Acknowledgments

The authors wish to thanks Researchers Supporting Project Number (RSP2025R45) at King Saud University Riyadh Saudi Arabia for financial support.

Funding information: The research was financially supported by Researchers Supporting Project Number (RSP2025R45) at King Saud University.
Author contributions: S. Chakir and G. Echchgadda: supervision, validation, and correction of the manuscript. F. El Hajli: conceptualization, methodology, data curation, writing – original draft, and writing. A. Assouguem, F. Elaissaoui, S. Annemer, R. Ullah, E. A. Ali, and R. Choudhary: Software, review, and editing. K. Hammani and R. Lahlali: manuscript correction and supervision. All authors have read and agreed to the published version of the manuscript.
Conflict of interest: The authors declare no conflicts of interest.
Ethical approval: The conducted research is not related to either human or animals use.
Data availability statement: All related data are within the manuscript.

References

[1] Baghloul F, Mansori R, Djahoudi A. In vitro antifungal effect of Rosmarinus officinalis essential oil on Aspergillus Niger. Natl J Physiol Pharm Pharmacol. 2016;7(3):285. 10.5455/njppp.2017.7.7021513102016.Suche in Google Scholar

[2] Abi-Ayad FZ, Abi-Ayad M, Lazouni HA, Rebiahi SA. Evaluation of Tetraclinis articulata essential oil from Algeria flora as a potential source of antifungal activity and study of its chemical composition. J Indian Acad Wood Sci. 2013;10(1):9–15.10.1007/s13196-013-0086-7Suche in Google Scholar

[3] El Khetabi A, El Ghadraoui L, Ouaabou R, Ennahli S, Barka EA, Lahlali R. Antifungal activities of aqueous extracts of Moroccan medicinal plants against monilinia spp. Agent of Brown rot disease. J Nat Pest Res. 2023;5:100038. 10.1016/j.napere.2023.100038.Suche in Google Scholar

[4] Cherrate M, Echchgadda G, Amiri S, Ezrari S, Radouane N, Oulad El Majdoub Y, et al. Biological control of major postharvest fungal diseases of apple using two Lamiaceae extracts. Arch Phytopathol. 2022;55(20):2356–81. 10.1080/03235408.2023.2166379.Suche in Google Scholar

[5] El Ouadi Y, Bendaif H, Assaggaf H, Abdallah EM, Mekkaoui M, Mrabti HN. Efficacy of Pelargonium graveolens essential oils against some postharvest fungal diseases of apple. Adv Life Sci. 2022;9(2):195–201.Suche in Google Scholar

[6] Moinina A, Lahlali R, Boulif M. Important pests, diseases and weather conditions affecting apple production in Morocco: Current state and perspectives. Rev Maroc Des sci agron ET vétérinaires. 2019;7(1):71–87.Suche in Google Scholar

[7] De Corato U, Salimbeni R, De Pretis A, Avella N, Patruno G. Antifungal activity of crude extracts from brown and red seaweeds by a supercritical carbon dioxide technique against fruit postharvest fungal diseases. Postharvest Biol Technol. 2017;131:16–30. 10.1016/j.postharvbio.2017.04.011.Suche in Google Scholar

[8] Lyousfi N, Lahlali R, Letrib C, Belabess Z, Ouaabou R, Ennahli S. Improving the biocontrol potential of bacterial antagonists with salicylic acid against brown rot disease and impact on nectarine fruits quality. Agron. 2021;11:209. 10.3390/agronomy11020209.Suche in Google Scholar

[9] Annemer S, Ez zoubi Y, Satrani B, Stambouli H, Assouguem A, Ullah R, et al. Simultaneous hydrodistillation of Cedrus atlantica Manetti and Salvia rosmarinus Spenn: Optimization of anti-wood-decay fungal activity using mixture design methodology. ACS Omega. 2023 Aug;8(30):27030–43. [cited 2024 May 20] https://pubs.acs.org/doi/full/10.1021/acsomega.3c01970.10.1021/acsomega.3c01970Suche in Google Scholar PubMed PubMed Central

[10] Rguez S, Slimene IB, Abid G, Hammemi M, Kefi A, Elkahoui S. Tetraclinis articulata essential oil reduces Botrytis cinerea infections on tomato. Sci Hortic. 2020;266:109–291. 10.1016/j.scienta.2020.109291.Suche in Google Scholar

[11] Palou L, Ali A, Fallik E, Romanazzi G. GRAS, plant- and animal-derived compounds as alternatives to conventional fungicides for the control of postharvest diseases of fresh horticultural produce. Postharvest Biol Technol. 2016;122:41–52. 10.1016/j.postharvbio.2016.04.017.Suche in Google Scholar

[12] Annemer S, Ez-zoubi A, Ez zoubi Y, Satrani B, Stambouli H, Assouguem A, et al. Optimization and antifungal efficacy against brown rot fungi of combined Salvia rosmarinus and Cedrus atlantica essential oils encapsulated in Gum Arabic. Sci Rep. 2023 Nov;13(1):1–18. [cited 2024 May 20] https://www.nature.com/articles/s41598-023-46858-7.10.1038/s41598-023-46858-7Suche in Google Scholar PubMed PubMed Central

[13] El Hachlafi N, Fikri-Benbrahim K, Al-Mijalli SH, Elbouzidi A, Jeddi M, Abdallah EM, et al. Tetraclinis articulata (Vahl) Mast. essential oil as a promising source of bioactive compounds with antimicrobial, antioxidant, anti-inflammatory and dermatoprotective properties: In vitro and in silico evidence. Heliyon. 2024;10(1):e23084. 10.1016/j.heliyon.2023.e23084.Suche in Google Scholar PubMed PubMed Central

[14] Ghnaya AB, Amri I, Hanana M, Gargouri S, Jamouss B, Romane A, et al. Tetraclinis articulata (Vahl.) Masters essential oil from Tunisia: Chemical characterization and evaluation of herbicidal and antifungal activities. Ind Crops Prod. 2016;83:113–7. 10.1016/j.indcrop.2015.12.026.Suche in Google Scholar

[15] Znini M, Cristofari G, Majidi L, El Harrak A, Paolini J, Costa J. In vitro antifungal activity and chemical composition of Warionia saharae essential oil against 3 apple phytopathogenic fungi. Food Sci Biotechnol. 2013;22:113–9.10.1007/s10068-013-0056-2Suche in Google Scholar

[16] Doukkali L, Tahiri A, Askrane L, Tazi B, Guenoun F, Ezrari S, et al. Chemical composition and antibacterial activity of Moroccan Artemisia mesatlantica and A. absinthium essential oils against fire blight caused by Erwinia amylovora. Not Sci Biol. 2022;14(2):11173. 10.55779/nsb14211173.Suche in Google Scholar

[17] Mrabti HN, Doudach L, Kachmar MR, Khali Z, Mrabti NN, Benrahou KB, et al. Phenolic content, antibacterial, antioxidant, and toxicological investigations of Erodium guttatum (Geraniaceae) collected from the Northeast of Morocco. Turk J Bot. 2021;45(8):739–49. 10.3906/bot-2107-29.Suche in Google Scholar

[18] Palou L, Usall J, Smilanick JL, Aguilar MJ, Viñas I. Evaluation of food additives and low-toxicity compounds as alternative chemicals for the control of Penicillium digitatum and Penicillium italicum on citrus fruit. Pest Manag Sci. 2002;58(5):459–66.10.1002/ps.477Suche in Google Scholar PubMed

[19] Sangwanich S, Sangchote S, Leelasuphakul W. Biocontrol of Citrus green mould and postharvest quality parameters. Int Food Res J. 2013;20(6):3381.Suche in Google Scholar

[20] Qin X, Xiao H, Xue C, Yu Z, Yang R, Cai Z, et al. Biocontrol of gray mold in grapes with the yeast Hanseniaspora uvarum alone and in combination with salicylic acid or sodium bicarbonate. Postharvest Biol Technol. 2015;100:160–16.10.1016/j.postharvbio.2014.09.010Suche in Google Scholar

[21] Pobiega K, Przybył JL, Żubernik J, Gniewosz M. Prolonging the shelf life of cherry tomatoes by pullulan coating with ethanol extract of propolis during refrigerated storage. Food Bioprocess Technol. 2020;13(8):1447–61. 10.1007/s11947-020-02487-w.Suche in Google Scholar

[22] Trivedi HD, Patel BY, Patel PK, Sagar SR. Synthesis, molecular modeling, ADMET, and fastness studies of some quinoline encompassing pyrimidine azo dye derivatives as potent antimicrobial agents. Chem Data Collect. 2022;4:100923. 10.1016/j.cdc.2022.100923.Suche in Google Scholar

[23] RCSB PDB website: Available at http://www.rcsb.org/pdb (Date accessed: 10 September 2024).Suche in Google Scholar

[24] National Centre for Biotechnology Information: https://www.ncbi.nlm.nih.gov/protein/ (Date accessed: 10 September 2024).Suche in Google Scholar

[25] Ballester AR, Marcet-Houben M, Levin E, Sela N, Selma-Lázaro C, Carmona L, et al. Genome, transcriptome, and functional analyses of Penicillium expansum provide new insights into secondary metabolism and pathogenicity. Mol Plant Microbe Interact. 2015;28:232–48.10.1094/MPMI-09-14-0261-FISuche in Google Scholar PubMed

[26] Tannous J, Snini SP, El Khoury R, Canlet C, Pinton P, Lippi Y, et al. Patulin transformation products and last intermediates in its biosynthetic pathway, E- and Z-ascladiol, are not toxic to human cells. Arch Toxicol. 2017;91:2455–67.10.1007/s00204-016-1900-ySuche in Google Scholar PubMed

[27] Li B, Chen Y, Zong Y, Shang Y, Zhang Z, Xu X, et al. Dissection of patulin biosynthesis, spatial control and regulation mechanism in Penicillium expansum. Env Microbiol. 2019;21:1124–39. 10.1111/1462-2920.14542.Suche in Google Scholar PubMed

[28] Sing LC, Roy A, Hui LY, Mun CS, Rajak H, Karunakaran R, et al. Multi-targeted molecular docking, drug-likeness and ADMET studies of derivatives of few quinoline-and acridine-based FDA-approved drugs for anti-breast cancer activity. Struct Chem. 2022;33(3):649–69. 10.1007/s11224-022-01878-3.Suche in Google Scholar

[29] Pkcsm: https://biosig.lab.uq.edu.au/pkcsm/prediction (Date accessed: 10 September 2024)Suche in Google Scholar

[30] Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev. 1997;23(1–3):3–25.10.1016/S0169-409X(96)00423-1Suche in Google Scholar

[31] Zrira S, Benjilali B, Elamrani A. Chemical composition of the sawdust oil of Moroccan Tetraclinis articulata Vahl. J Essent Oil Res. 2005;17(1):96–7.10.1080/10412905.2005.9698842Suche in Google Scholar

[32] Ait-Ouazzou A, Lorán S, Arakrak A, Laglaoui A, Rota C, Herrera A, et al. Evaluation of the chemical composition and antimicrobial activity of Mentha pulegium, Juniperus phoenicea, and Cyperus longus essential oils from Morocco. Food Res Int. 2012;45(1):313–9.10.1016/j.foodres.2011.09.004Suche in Google Scholar

[33] Ez-Zriouli R, El Yacoubi H, Imtara H, El-Hessni A, Mesfioui A, Tarayrah M, et al. Chemical composition and antimicrobial activity of essential oils from Mentha pulegium and Rosmarinus officinalis against multidrug-resistant microbes and their acute toxicity study. Open Chem. 2022;20(1):694–702. 10.1515/chem-2022-0185.Suche in Google Scholar

[34] Gourich AA, Bencheikh N, Bouhrim M, Regragui M, Rhafouri R, Drioiche A, et al. Comparative analysis of the chemical composition and antimicrobial activity of four moroccan north middle atlas medicinal plants’ essential oils: Rosmarinus officinalis L., Mentha pulegium L., Salvia officinalis L., and Thymus zygis subsp. gracilis (Boiss.) R. Morales. Chemistry. 2022;4(4):1775–88. 10.3390/chemistry4040115.Suche in Google Scholar

[35] Allali A, Rezouki S, Fadli A, El Moussaoui A, Bourhia M, Salamatullah AM, et al. Chemical characterization and antioxidant, antimicrobial, and insecticidal properties of essential oil from Mentha pulegium L. Evid Based Complement Altern Med. 2021;1:1108133. 10.1155/2021/1108133.Suche in Google Scholar PubMed PubMed Central

[36] Zekri N, Elazzouzi H, Ailli A, Gouruch AA, Radi FZ, El Belghiti MA, et al. Physicochemical characterization and antioxidant properties of essential oils of M. pulegium (L.), M. suaveolens (Ehrh.) and M. spicata (L.) from Moroccan Middle-Atlas. Foods. 2023;12(4):760. 10.3390/foods12040760.Suche in Google Scholar PubMed PubMed Central

[37] Assaggaf H, El Hachlafi N, Elbouzidi A, Taibi M, Benkhaira N, El Kamari F, et al. Unlocking the combined action of Mentha pulegium L. Essential Oil and Thym Honey: In vitro pharmacological activities, Molecular docking, and in vivo Anti-Inflammatory effect. Heliyon. 2024;10:e31922. 10.1016/j.heliyon.2024.e31922.Suche in Google Scholar PubMed PubMed Central

[38] Agnihotri VK, Agarwal SG, Dhar PL, Thappa RK, Kapahi BK, Saxena RK, et al. Essential oil composition of Mentha pulegium growing wild in the north-western Himalayas India. Flavour Fragr J. 2005;2:607–10.10.1002/ffj.1497Suche in Google Scholar

[39] Beghidja N, Bouslimani N, Benayache F, Banayache S, Chalchat JC. Composition of the oils from Mentha pulegium Grown in different areas of the east of Algeria. Khim Prir Soedin. 2007;4:394–5.10.1007/s10600-007-0170-6Suche in Google Scholar

[40] Kokkini S, Handilou E, Karousou R, Lanaras T. Variations of pulegone content in pennyroyal (Mentha pulegium L.) plants growing wild in Greece. J Essent Oil Res. 2002;14:224–7.10.1080/10412905.2002.9699830Suche in Google Scholar

[41] Aabouch F, Satrani B, Ameggouz M, Ettaleb I, Assouguem A, Kara M, et al. Wild Thymus zygis L. ssp. gracilis and Eucalyptus camaldulensis Dehnh.: Chemical composition, antioxidant and antibacterial activities of essential oils. Open Chem. 2024;22(1):20240050. 10.1515/chem-2024-0050.Suche in Google Scholar

[42] Ballester-Costa C, Sendra E, Fernández-López J, Pérez-Álvarez JA, Viuda-Martos M. Chemical composition and in vitro antibacterial properties of essential oils of four Thymus species from organic growth. Ind Crop Prod. 2013;50:304–11.10.1016/j.indcrop.2013.07.052Suche in Google Scholar

[43] Marinkovića J, Mitić Ćulafićb D, Nikolićb B, Đukanovićb S, Markovićc T, Tasića G, et al. Antimicrobial potential of irrigants based on essential oils of Cymbopogon martinii and Thymus zygis towards in vitro multispecies biofilm cultured in ex vivo root canals. Arch Oral Biol. 2020;117:104–842. 10.1016/j.archoralbio.2020.104842.Suche in Google Scholar PubMed

[44] Chroho M, Rouphael Y, Petropoulos SA, Bouissane L. Carvacrol, and thymol content affects the antioxidant and antibacterial activity of Origanum compactum and Thymus zygis essential oils. Antibiotics. 2024;13(2):139. 10.3390/antibiotics13020139.Suche in Google Scholar PubMed PubMed Central

[45] Bouyahya A, Jamal A. Origanum compactum Benth: A review on phytochemistry and pharmacological properties. Med Aromat Plants. 2016;5:1000252.10.4172/2167-0412.1000252Suche in Google Scholar

[46] Radi FZ, Bouhrim M, Mechchate H, Al-Zahrani M, Qurtam AA, Aleissa AM, et al. Phytochemical analysis, antimicrobial and antioxidant properties of Thymus zygis L. and Thymus willdenowii Boiss. essential oils. Plants. 2021;11(1):15. 10.3390/plants11010015.Suche in Google Scholar PubMed PubMed Central

[47] Dandlen SA, Lima AS, Mendes MD, Miguel MG, Faleiro ML, Sousa MJ, et al. Antioxidant activity of six portuguese thyme species essential oils. Flavour Fragr J. 2010;25:150–5.10.1002/ffj.1972Suche in Google Scholar

[48] Rabib H, Elagdi C, Hsaine M, Fougrach H, Koussa T, Badri W. Antioxidant and antibacterial activities of the essential oil of Moroccan Tetraclinis articulata (Vahl) Masters. Biochem Res Int. 2020;1:9638548. 10.1155/2020/9638548.Suche in Google Scholar PubMed PubMed Central

[49] El Jemli M, Kamal R, Marmouzi I, Doukkali Z, Bouidida EH, Touati D, et al. Chemical composition, acute toxicity, antioxidant and anti-inflammatory activities of Moroccan Tetraclinis articulata L. J Tradit Complement Med. 2017;7(3):281–7.10.1016/j.jtcme.2016.06.006Suche in Google Scholar PubMed PubMed Central

[50] Buonsenso F, Schiavon G, Spadaro D. Efficacy and mechanisms of action of essential oils’ vapours against blue mould on apples caused by Penicillium expansum. Int J Mol Sci. 2023;24:2900. 10.3390/ijms24032900.Suche in Google Scholar PubMed PubMed Central

[51] Pateiro M, Munekata PES, Sant’Ana AS, Domínguez R, Rodríguez-Lázaro D, Lorenzo JM. Application of essential oils as antimicrobial agents against spoilage and pathogenic microorganisms in meat products. Int J Food Microbiol. 2021;337:108966. 10.1016/j.ijfoodmicro.2020.108966.Suche in Google Scholar PubMed

[52] Raveau R, Fontaine J, Lounès-Hadj SA. Essential oils as potential alternative biocontrol products against plant pathogens and weeds: A review. Foods. 2020;9(3):365. 10.3390/foods9030365.Suche in Google Scholar PubMed PubMed Central

[53] Caccioni RLD, Guizzardi M. Inhibition of germination and growth of fruit and vegetable postharvest pathogenic fungi by essential oil components. J Essent Oil Res. 2011;6(2):173–9.10.1080/10412905.1994.9698349Suche in Google Scholar

[54] Pardavella I, Daferera D, Tselios T, Skiada P, Giannakou I. The use of essential oil and hydrosol extracted from Cuminum cyminum seeds for the control of Meloidogyne incognita and Meloidogyne javanica. Plants. 2020;10(1):46. 10.3390/plants10010046.Suche in Google Scholar PubMed PubMed Central

[55] Behdani M, Pooyan M, Abbasi S. Evaluation of antifungal activity of some medicinal plants essential oils against Botrytis cinerea, causal agent of postharvest apple rot, in vitro. Intl J Agri Crop Sci. 2012;4(14):1012–6.Suche in Google Scholar

[56] Montenegro I, Said B, Godoy P, Besoain X, Parra C, Díaz K, et al. Antifungal activity of essential oil and main components from Mentha pulegium growing wild on the Chilean central coast. Agron. 2020;10(2):254. 10.3390/agronomy10020254.Suche in Google Scholar

[57] Bahri BA, Mechichi G, Rouissi W, Jilani IBH, Ghrabi-Gammar Z. Effects of cold-storage facility characteristics on the virulence and sporulation of Penicillium expansum and the efficacy of essential oils against blue mold rot of apples. Folia Hortic. 2019;31(2):301–17. 10.2478/fhort-2019-0024.Suche in Google Scholar

[58] Chaleshtori ZZ, Bonyadian M, Moshtaghi H, Ebrahimi A. Antifungal effects of essential oils of Zataria multiflora, Mentha pulegium, and Mentha piperita. J Food Qual Hazards Control. 2021;8:41–4. 10.18502/jfqhc.8.1.5462.Suche in Google Scholar

[59] Mohammadi P, Lotfi N, Naseri L, Etebarian HR. Antifungal activities of essential oils from some Iranian medicinal plants against various post-harvest molds. J Med Plants Res. 2013;7(23):1699–708.Suche in Google Scholar

[60] Santamarina MP, Ibáñez MD, Marqués M, Roselló J, Giménez S, Blázquez MA. Bioactivity of essential oils in phytopathogenic and post-harvest fungi control. Nat Prod Res. 2017;31(22):2675–9.10.1080/14786419.2017.1286479Suche in Google Scholar PubMed

[61] Zhang J, Ma S, Du S, Chen S, Sun H. Antifungal activity of thymol and carvacrol against postharvest pathogens Botrytis cinerea. J Food Sci Technol. 2019;56:2611–20. 10.1007/s13197-019-03747-0.Suche in Google Scholar PubMed PubMed Central

[62] Adams S, Kunz B, Weidenbörner M. Mycelial deformations of Cladosporium herbarum due to the application of Eugenol and Carvacrol. J Essent Oil Res. 1996;8:535–40.10.1080/10412905.1996.9700682Suche in Google Scholar

[63] Kumar A, Shukla R, Singh P, Prasad CS, Kishore R. Assessment of Thymus vulgaris L. essential oil as a safe botanical preservative against postharvest fungal infestation of food commodities. Innov Food Sci Emerg Technol. 2008;9:575–80.10.1016/j.ifset.2007.12.005Suche in Google Scholar

[64] Marandi RJ, Hassani A, Ghosta Y, Abdollahi A, Pirzad A, Sefidkon F. Control of Penicillium expansum and Botrytis cinerea on pear with Thymus kotschyanus, Ocimum basilicum, and Rosmarinus officinalis essential oils. J Med Plants Res. 2011;5(4):626–34.Suche in Google Scholar

[65] Fathi Z, Hassani A, Ghosta Y, Abdollahi A, Meshkatalsadat MH. The potential of thyme, clove, cinnamon, and ajowan essential oils in inhibiting the growth of Botrytis cinerea and Monilinia fructicola. J Essent Oil-Bear Plants. 2012;15:38–47.10.1080/0972060X.2012.10644017Suche in Google Scholar

[66] Lopez-Reyes JG, Spadaro D, Prelle A, Garibaldi A, Gullino ML. Efficacy of plant essential oils on postharvest control of rots caused by fungi on different stone fruits in vivo. J Food Prot. 2013;76:631–9.10.4315/0362-028X.JFP-12-342Suche in Google Scholar PubMed

[67] Aouadi G, Grami LK, Taibi F, Bouhlal R, Elkahoui S, Zaagueri T, et al. Assessment of the efficiency of Mentha pulegium essential oil to suppress contamination of stored fruits by Botrytis cinerea. J Plant Dis Prot. 2022;129(4):881–93. 10.1007/s41348-022-00623-6.Suche in Google Scholar

[68] Osama Saeed M, Wand Khalis A, Tais Al-Hulitan A. The effect of infestation by the confused flour beetle (Tribolium confusum Duv.) on specifications of wheat flour. J Agri Sci Tech A. 2012;2(5A):696.Suche in Google Scholar

[69] Uwineza MS, El Yousf B, Lamiri A. In vitro antifungal activity of essential oils of Mentha pulegium, Eugenia aromatica and Cerdrus atlantica on Fusarium culmorum and Bipolaris sorokiniana. Rev Maroc Prot Plantes. 2018;12:19–32.Suche in Google Scholar

[70] Soylu EM, Kurt Ş, Soylu S. In vitro and in vivo antifungal activities of the essential oils of various plants against tomato grey mould disease agent Botrytis cinerea. Int J Food Microbiol. 2010;143(3):183–9.10.1016/j.ijfoodmicro.2010.08.015Suche in Google Scholar PubMed

[71] Chebli B, Achouri M, Idrissi Hassani LM, Hmamouchi M. Chemical composition and antifungal activity of essential oils of seven Moroccan Labiatae against Botrytis cinerea Pers: Fr. J Ethnopharmacol. 2003;89:165–9.10.1016/S0378-8741(03)00275-7Suche in Google Scholar PubMed

[72] Bouzouita N, Kachouri F, Ben Halima M, Chaabouni MM. Chemical composition and antioxidant, antimicrobial and insecticidal activities of Juniperus phoenicea essential oil. J Chem Soci Tunis. 2008;10:119–25.Suche in Google Scholar

[73] Banani H, Olivieri L, Santoro K, Garibaldi A, Gullino ML, Spadaro D. Thyme and savory essential oil efficacy and induction of resistance against Botrytis cinerea through priming of defense responses in apple. Foods. 2018;7(2):11. 10.3390/foods7020011.Suche in Google Scholar PubMed PubMed Central

[74] El Khetabi A, Ezrari S, El Ghadraoui L, Tahiri A, Ait Haddou L, Belabess Z, et al. In vitro and in vivo antifungal activities of nine commercial essential oils against brown rot in apples. Hortic. 2021;7(12):545. 10.3390/horticulturae7120545.Suche in Google Scholar

[75] Santoro K, Maghenzani M, Chiabrando V, Bosio P, Gullino ML, Spadaro D, et al. Thyme and savory essential oil vapor treatments control brown rot and improve the storage quality of peaches and nectarines, but could favor gray mold. Foods. 2018;7(1):7.10.3390/foods7010007Suche in Google Scholar PubMed PubMed Central

[76] Shafee M, Taghavi TS, Babalar M. Addition of salicylic acid to nutrient solution combined with postharvest treatments (hot water, salicylic acid, and calcium dipping) improved postharvest fruit quality of strawberry. Sci Hortic. 2010;124:40–4.10.1016/j.scienta.2009.12.004Suche in Google Scholar

[77] Znini M. Chemical composition, liquid and vapor-phase antifungal activities of essential oil of Teucrium luteum subsp. flavovirens against three postharvest phytopathogenic fungi. Int J Fruit Sci. 2021;21(1):400–12.10.1080/15538362.2021.1895033Suche in Google Scholar

[78] Assouguem A, Lahlali R, Joutei AB, Kara M, Bari A, Aberkani K, et al. Assessing Panonychus ulmi (Acari: Tetranychidae) infestations and their key predators on Malus domestica borkh in varied ecological settings. Agronomy. 2024;14(3):457.10.3390/agronomy14030457Suche in Google Scholar

[79] Oucheikh L, Ou-Ani O, Moujane S, Ansari A, Oubair A, Znini M. Chemical composition, in vitro antifungal activity, DFT, molecular docking and molecular dynamics simulation studies of the essential oil from Anvillea gracinii subsp. radiata (Coss. & Durieu) Anderb. J Essent Oil Res. 2023;35(1):35–50.10.1080/10412905.2022.2109767Suche in Google Scholar

[80] Ayed A, Caputo L, De Feo V, Nazzaro F, Fratianni F, Amri I, et al. Essential oils of Tunisian Tetraclinis articulata (Vahl) Mast.: Chemical and biological insights. Chem & Biodivers. 2024;e202401618.10.1002/cbdv.202401618Suche in Google Scholar PubMed PubMed Central

[81] Benoutman A, Erbiai EH, Ettakifi H, Legssyer M, Handaq N, Makrane H, et al. Phytochemical analysis, antioxidant, antifungal activity, and genotoxicity of essential oil and solvent extracts of Tetraclinis articulata leaves from northern Morocco. Lett Appl NanoBioScience. 2024;13(1):1–19.10.33263/LIANBS131.050Suche in Google Scholar

[82] Asbabou A, Hanane T, Gourich AA, Siddique F, Drioiche A, Remok F, et al. Phytochemical profile, physicochemical, antioxidant and antimicrobial properties of Juniperus phoenicea and Tetraclinis articulata: in vitro and in silico approaches. Front Chem. 2024;12:1397961.10.3389/fchem.2024.1397961Suche in Google Scholar PubMed PubMed Central

[83] Labhar A, Ahidar N, El-Mernissi Y, Benamari O, Alla KA, Jahjah S, et al. Phytochemical, antioxidant, and anti-inflammatory properties of Tetraclinis articulata (Vahl) Masters extracts from Al Hoceima province. In E3S Web of Conferences. Vol. 527, 2024. p. 01016.10.1051/e3sconf/202452701016Suche in Google Scholar

[84] Jlizi S, Oulad El Majdoub Y, Zardi-Bergaoui A, Cacciola F, Arena K, Mondello L, et al. HPLC-PDA/ESI-MS determination of the polyphenolic profile and evaluation of the cytotoxic effect of Tetraclinis articulata bark using in vitro and in silico methods. Plant Biosyst-An Int J Dealing All Asp Plant Biol. 2023;157(5):970–7.10.1080/11263504.2023.2231930Suche in Google Scholar

[85] Messaoudi M, Rebiai A, Sawicka B, Atanassova M, Ouakouak H, Larkem I, et al. Effect of extraction methods on polyphenols, flavonoids, mineral elements, and biological activities of essential oil and extracts of Mentha pulegium L. Molecules. 2021;27(1):11.10.3390/molecules27010011Suche in Google Scholar PubMed PubMed Central

[86] Moullamri M, Rharrabe K, Annaz H, Laglaoui A, Alibrando F, Cacciola F, et al. Salvia officinalis, Lavandula angustifolia, and Mentha pulegium essential oils: insecticidal activities and feeding deterrence against Plodia interpunctella (Lepidoptera: Pyralidae). J Essent Oil Bear Plants. 2024;27(1):16–33.10.1080/0972060X.2023.2301699Suche in Google Scholar

[87] Dousari AS, Hosseininasab SS, Akbarizadeh MR, Naderifar M, Satarzadeh N. Mentha pulegium as a source of green synthesis of nanoparticles with antibacterial, antifungal, anticancer, and antioxidant applications. Sci Horticulturae. 2023;320:112215.10.1016/j.scienta.2023.112215Suche in Google Scholar

[88] Vukić MD, Čmiková N, Hsouna AB, Saad RB, Garzoli S, Schwarzová M, et al. Thymus Zygis, valuable antimicrobial (in vitro and in situ) and antibiofilm agent with potential antiproliferative effects. Plants. 2023;12(23):3920.10.3390/plants12233920Suche in Google Scholar PubMed PubMed Central

[89] Yan J, Wu H, Chen K, Feng J, Zhang Y. Antifungal activities and mode of action of Cymbopogon citratus, Thymus vulgraris, and Origanum heracleoticum essential oil vapors against Botrytis cinerea and their potential application to control postharvest strawberry gray mold. Foods. 2021;10(10):2451.10.3390/foods10102451Suche in Google Scholar PubMed PubMed Central

[90] Rautela J, Rawat S, Jakhmola V. Advanced anticancer application of Thymus Zygis essential oil (EOT). In Pharmacological aspects of essential oils. CRC Press; 2024. p. 153–61.10.1201/9781003389774-11Suche in Google Scholar

[91] Martin CR, Leith GA, Kittikhunnatham P, Park KC, Ejegbavwo OA, Mathur A, et al. Heterometallic actinide‐containing photoresponsive metal‐organic frameworks: dynamic and static tuning of electronic properties. Angew Chem Int Ed Engl. 2021;60(15):8072–80. 10.1002/anie.202016826.Suche in Google Scholar PubMed

[92] Shi Y. Support vector regression-based QSAR models for prediction of antioxidant activity of phenolic compounds. Sci Rep. 2021;11(1):8806. 10.1038/s41598-021-88341-1.Suche in Google Scholar PubMed PubMed Central

[93] Mkhayar K, Daoui O, Elkhattabi S, Chtita S, Elkhalabi R. In silico molecular investigations of derived cyclohexane-1, 3-dione compounds as potential inhibitors of protein tyrosine kinase C-met: 2D QSAR, molecular docking, and ADMET. In International Conference on Intelligent Systems and Computer Vision (ISCV), Fez, Morocco; 18-20 May 2022.10.1109/ISCV54655.2022.9806058Suche in Google Scholar

[94] Barret R. Medicinal chemistry: fundamentals. United Kingdom: Elsevier; 2018.Suche in Google Scholar

[95] Tekade M, Pingale PL, Wani SP, Rajpoot K, Sreeharsha N, Deshpande M, et al. Clinical detoxification of the body from chemical toxicants. In Essentials of pharmatoxicology in drug research. Vol. 1, Academic Press; 2023. p. 469–505.10.1016/B978-0-443-15840-7.00012-9Suche in Google Scholar

[96] Cheng F, Li W, Liu G, Tang Y. In silico ADMET prediction: recent advances, current challenges and future trends. Curr Top Med Chem. 2013;13(11):1273–89.10.2174/15680266113139990033Suche in Google Scholar PubMed

[97] Toubi Y, Abrigach F, Radi S, Souna F, Hakkou A, Alsayari A, et al. Synthesis, antimicrobial screening, homology modeling, and molecular docking studies of a new series of schiff base derivatives as prospective fungal inhibitor candidates. Molecules. 2019;24:3250. 10.3390/molecules24183250.Suche in Google Scholar PubMed PubMed Central

[98] Schiavon G, Garello M, Prencipe S, Meloni GR, Buonsenso F, Spadaro D. Essential oils reduce grey mould rot of apples and modify the fruit microbiome during postharvest storage. J Fungi. 2023;9:22. 10.3390/jof9010022.Suche in Google Scholar PubMed PubMed Central

[99] Nazzaro F, Fratianni F, Coppola R, Feo VD. Essential oils and antifungal activity. Pharmaceuticals. 2017;10:86.10.3390/ph10040086Suche in Google Scholar PubMed PubMed Central

[100] da Silva BD, Campos Bernardes P, Fontes Pinheiro P, Fantuzzi E, Roberto CD. Chemical composition, extraction sources and action mechanisms of essential oils: Natural preservative and limitations of use in meat products. Meat Sci. 2021;176:108463. 10.1016/j.meatsci.2021.108463.Suche in Google Scholar PubMed

[101] Maurya A, Prasad J, Das S, Dwivedy AK. Essential oils and their application in food safety. Front Sustain Food Syst. 2021;5:653420. 10.3389/fsufs.2021.653420.Suche in Google Scholar

[102] Athiappan M, Kumar SD, Umamaheswari S, Rajaprabu M. Rhizosphere engineering through pesticides-degrading beneficial bacteria. In Rhizosphere engineering. Academic Press; 2022. p. 239–57.10.1016/B978-0-323-89973-4.00014-4Suche in Google Scholar

[103] Kampatsikas I, Bijelic A, Pretzler M, Rompel A. In crystallo activity tests with latent apple tyrosinase and two mutants reveal the importance of the mutated sites for polyphenol oxidase activity. Acta Crystallogr Sect F: Struct Biol Commun. 2017;73(8):491–9.10.1107/S2053230X17010822Suche in Google Scholar PubMed PubMed Central

[104] Han QY, Liu F, Wen X, Ni YY. Kinetic, spectroscopic, and molecular docking studies on the inhibition of membrane-bound polyphenol oxidase from Granny Smith apples (Malus domestica Borkh.). Food Chem. 2021;338:127928.10.1016/j.foodchem.2020.127928Suche in Google Scholar PubMed

[105] Sun L, Guo Y, Zhang Y, Zhuang Y. Antioxidant and antityrosinase activities of phenolic extracts from rape bee pollen and inhibitory melanogenesis by cAMP/MITF/TYR Pathway in B16 Mouse Melanoma cells. Front Pharmacol. 2017;9(8):104–9.10.3389/fphar.2017.00104Suche in Google Scholar PubMed PubMed Central

[106] Rezaeinasab M, Benvidi A, Gharaghani S, Abbasi S, Zare HR. Electrochemical investigation of the inhibition effect of carvacrol on xanthine oxidase activity merging with theoretical studies. Process Biochem. 2019;83:86–95.10.1016/j.procbio.2019.03.014Suche in Google Scholar

[107] Jianu C, Rusu LC, Muntean I, Cocan I, Lukinich-Gruia AT, Goleț I, et al. In vitro and in Silico evaluation of the antimicrobial and antioxidant potential of Thymus pulegioides essential oil. Antioxidants. 2022;11(12):2472.10.3390/antiox11122472Suche in Google Scholar PubMed PubMed Central

Received: 2024-10-20

Revised: 2024-12-18

Accepted: 2024-12-30

Published Online: 2025-03-06

This work is licensed under the Creative Commons Attribution 4.0 International License.

Artikel in diesem Heft

https://doi.org/10.1515/chem-2025-0131

Schlagwörter für diesen Artikel

molecular docking; GC-MS analysis; apple diseases; essential oils; quality parameter; antifungal and antioxidant properties

Creative Commons

BY 4.0