The aerial parts of Haplophyllum tuberculatum were systematically collected in February 2021 from a location near the city of Akka in southeastern Morocco. The geographical coordinates of the collection site were recorded as follows: Latitude: 29.39876053°N (29N 3252388.525 m N) and Longitude: 8.26860784°W (29N 570963.419 m E). The elevation of the collection site was measured at approximately 570,000 m. The taxonomic identification of the H. tuberculatum plant species was performed by a botanist from Sidi Mohamed Ben Abdellah University, Fez, Morocco. A reference sample of the collected plant material, labeled HT0019220211, was deposited in the herbarium of the Laboratory of Natural Substances, Pharmacology, Environment, Modeling, Health, and Quality of Life, Faculty of Sciences in Fez.

The essential oil of Haplophyllum tuberculatum was extracted using the hydrodistillation method with the Clevenger apparatus. 200 g of pre-dried aerial parts were placed in a 2 L flask, and 750 mL of water was added. Subsequently, the resulting mixture was left to boil for 3 h. The result HTEO obtained was stored at −4°C until utilization.

The mechanism of action of phytochemicals identified in HTEO was theoretically investigated by use of in silico approach. To this end, the interaction of chemicals with the FAD glucose dehydrogenase and beta-1,4-endoglucanase from A. flavus and A. niger respectively was evaluated (Eno et al., 2022; Rased et al., 2022). While the NAD(P)H Oxidase was used to investigate the mechanism of action of the concerned chemicals as antioxidant agents.

The structures of the chemical compounds identified from HTEO were retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) in structure data format (SDF) (Kim et al., 2023). Subsequently, the structures were imported to the workspace of the Schrödinger Maestro 11.5 version before being prepared using the LigPrep tool with the OPLS3 force field. Preparatory steps taken include the generation of a maximum of 32 stereoisomers and the selection of ionization states at pH 7.0 ± 2.0.

Similarly, the crystal structures of the proteins FAD glucose dehydrogenase, a beta-1,4-endoglucanase, NAD(P)H Oxidase, were retrieved in protein databank (PDB) format from the PDB (https://www.rcsb.org/) (Berman, 2000) using the PDB IDs: 4YNT, 5I77, 2CDU.

The protein preparation wizard was utilized to prepare the protein as follows: the refinement of the structure, assignment of charges and bond orders, addition of hydrogens to heavy atoms, conversion of selenomethionines to methionines, and deletion of all water molecules. Noteworthy, the OPLS3 forcefield was employed to perform the minimization of the structures using the OPLS3e force field with the root mean square deviation (RMSD) value of heavy atoms set to 3.0. Ultimately, the receptor grid generation tool was employed to delineate the binding pocket of the protein with a volumetric spacing of 20 × 20 × 20.

The insecticidal activity of H. tuberculatum EO was assessed against C. maculatus. The insects utilized were obtained from a sample of Cicer arietinum (chickpea) from a stock in the region of Fez-Meknes. To maintain a sufficient number of insects for the experiments, mass rearing was conducted in glass jars. The glass jars were placed in a controlled environment with specific conditions including the photoperiod was set to 10 h of darkness and 14 h of light, mimicking natural light cycles, the relative humidity was maintained at a saturated level of 65% ± 5%, and the temperature was kept at a constant 25°C.

The antioxidant power of EOHT was evaluated using three tests, namely, the free radical scavenging DPPH assay, according to the protocol described in the study of Mssillou et al. (2021). The ferric reducing antioxidant power (FRAP) assay, according to the protocol described in the study of El Abdali et al. (2023); the measurement of total antioxidant capacity (TAC) according to the protocol reported in the study of Allali et al. (2021).

The mortality rate of C. maculatus insect was calculated using the Abbott formula (Balouiri et al., 2015). The analysis of variance (ANOVA) of repeated measures regarding the percentage of mortality by toxicity over time was calculated for 24, 48, 72, and 96 h. The determination of LC₅₀ concentrations was done using the probit method (Hewlett, 1972) by the “IBM SPSS Program Version 21” software.

The yield of HTEO extracted by hydrodistillation was found to be 0.27%. However, it is worth noting that the yield of EO from H. tuberculatum can vary significantly and is influenced by various factors including maturity stage, genetic factors, environmental conditions, and geographic variations between habitats. This has also been observed as a phenomenon common with most aromatic and medicinal plant species. Generally, previous studies in which HTEO was extracted reported a yield that varied between 0.101% and 0.65% (Al-Burtamani et al., 2005; El-naggar et al., 2014; Bergheul et al., 2017; Sriti et al., 2017; Agour et al., 2020).

Following the extraction of HTEO, GC-MS was employed to identify the phytochemical constituent, and the chromatogram whose peaks represent the compounds present is depicted in Figure 1. The results of the analysis revealed 34 compounds which are presented in Table 1. Analysis of the results revealed sesquiterpenes to be the most abundant class of compounds, accounting for 55.70% of the total composition. Monoterpenes were found to be the second most abundant at 25.83%, while diterpenes were present in very low quantities. The majority compound of the studied essential oil is β-Caryophyllene, a sesquiterpene molecule with a percentage of (36.94%), followed by monoterpenes, α-Phellandrene (14.72%), Germacrene D (8.72%) and Eugenol (2.49%).

However, the phytochemical profile of HTEO is characterized mainly by the presence of oxygenated monoterpenes, monoterpene hydrocarbons, and non-terpene hydrocarbons, as well as oxygenated sesquiterpenes and sesquiterpene hydrocarbons (Mohammadhosseini et al., 2021). The main compounds, of the essential oils of H. tuberculatum, reported in previous studies were found to be different to those obtained in this study. According to (Hamdi et al., 2018), chromatographic analysis of the essential oil of H. tuberculatum revealed the presence of oxygenated monoterpenes (46.7%), monoterpene hydrocarbons (20.7%), and sesquiterpene hydrocarbons (0.5%). This variation could be attributed to the effect of geographical and environmental factors, as they not only influence the yield of EO but also the composition of the extracted oils. Table 2 presents some examples of the major compounds in HTEO reported in the literature.

The results of the evaluation of the antifungal activity of HTEO are presented in Table 3. HTEO exhibited inhibitory activity against all three fungal strains tested. Among the strains, A. flavus showed the highest sensitivity to HTEO, with an inhibition rate of 76.50% ± 0.60%, while A. niger exhibited moderate sensitivity with an inhibition rate of 61.22% ± 2.11%. F. oxysporum was found to be less sensitive to HTEO, with an inhibition percentage of 28.51% ± 0.69% (Figure 2). The MIC values, which indicate the minimum concentration required to inhibit the growth of the fungal strains, ranged from 20.53 ± 5.08 to 76.26 ± 5.08 mg/mL. The results of this study show that HTEO has limited antifungal efficacy against pathogenic molds.

Previous studies have also explored the antimicrobial properties of H. tuberculatum extracts. Exemplifying this is the results of the study by Abdelhhalek et al. (2020), in which they reported that the ethanolic extract of H. tuberculatum demonstrated inhibition rates of 82.96% and 93.70% against F. culmorum and Rhizoctonia solani, respectively. Additionally, two alkaloids, skimmianine and vulcanine, isolated from the aerial parts of H. tuberculatum exhibited antimicrobial effects against phytopathogenic bacteria and fungi. Skimmianine showed potent inhibitory activity against Rhizobium radiobacter, Ralstonia solanacerum, and Pectobacterium carotovorum ss. carotovorum, with a MIC of 62.5 mg/mL. In the antifungal assay, both skimmianine and vulcanine demonstrated inhibitory effects against Verticillium dahliae, F. oxysporum, and Alternaria solani (Abdelgaleil et al., 2020).

The antifungal effect of HTEO on C. albicans varies depending on the phytochemical composition of the extract and the specific strain of the fungal used. Hamdi et al. (2017a), reported that HTEO, had a MIC of 0.31 mg/mL against C. albicans ATCC 90028. In another study, the pure oil partially inhibited the growth of C. albicans ATCC 10231 with a diameter of 17.6 ± 0.3 mm (Al-Burtamani et al., 2005). Conversely, Sabry and El Sayed (2016) reported that essential oils obtained from the aerial parts of H. tuberculatum were inactive against C. albicans strain CBDN 05036. However, HTEO was also reported to exert antimycelial activity in addition to its antifungal activity. Furthermore, Al-Burtamani et al. (2005) also reported that HTEO inhibited the growth of F. oxysporum and A. flavus with MIC values lower than 1 mg/mL.

Following the docking of the phytochemicals identified in HTEO against the previously mentioned targets, their binding affinities for the proteins as revealed by their docking score presented in Table 4, were assessed. For antifungal activity, γ-Cadinene and p-Cymen-7-ol with docking scores of −6.322 and −6.302 kcal/mol, respectively, were found to possess the highest affinities for A. flavus FAD glucose dehydrogenase while Carvacrol and Viridiflorol also had the highest affinities for β-1,4-endoglucanase from A. niger with docking scores of −5.647 kcal/mol and −5.453 kcal/mol. In antioxidant activity, α-Terpinyl isobutyrate showed high affinity for the active site of NADPH oxidase with a glide score of −6.188 kcal/mol.

The Figure 3 and Figure 4 represent the 2D viewer and the 3D viewer of ligands interactions with the active sites, respectively. Analysis of the resulting complexes to delineate the interactions between them revealed that Carvacrol interacted with the active site amino acid residues of A. niger β-1,4-endoglucanase via two hydrogen bonds with residues including TYR 309 and GLY 234. In the antioxidant activity, α-Terpinyl isobutyrate established one hydrogen bonds in the active site of NADPH oxidase with residues ALA 300.

Few studies report on the in silico activities of the major compounds in the essential oil studied. However, studies related to the activities described in the present work have been identified. A previous study reported that trans-caryophyllene and eugenol had a more stable binding strength in the acetylcholinesterase enzyme of Tribolium castaneum, and there was synergy between eugenol and trans-caryophyllene when the two compounds interacted with acetylcholinesterase (Ikawati et al., 2022). On the other hand, the in silico study of the anti-gout and anti-diabetic activities of the essential oil from Piper lolot reveals that the major compound, β-caryophyllene (20.6%), exhibits inhibitory effects against the enzymes α-glucosidase, α-amylase and xanthine oxidase, with docking scores of −7.4, −6.2, and −5.8 kcal/mol, respectively (Nguyen et al., 2023).

The in silico study of the antioxidant activity of compounds in Juniperus thurifera bark essential oil shows that α-cadinol and muurolol have inhibitory activity on the active site of NADPH oxidase with a glide score of −6.041 and −5.956 kcal/mol, respectively (Lafraxo et al., 2022). Similarly, β-terpineol, carvacrol, and thymol, contained in Lavandula dentata essential oil were found to be active against NADPH oxidase, with a displacement score of −4.728, −6.17, and −6.483 kcal/mol, respectively (El Abdali et al., 2023).

The insecticidal activity of HTEO was assessed via inhalation and contact against adults of C. maculatus and the results of the study are presented in Figures 5, 6. The inhalation test showed that after 24 h, only the doses of 10 and 20 μL/100 g resulted in low percentages of mortality in C. maculatus adults, with 3.33% and 13.33% respectively. After 96 h, the 1 μL/100 g dose still had no effect, while the doses of 10 and 20 μL/100 g resulted in mortality percentages above 50%. The contact mortality test showed similar results, with the concentration of 5 μL/100 g causing 3.33% mortality after 24 h and 13.33% after 96 h. The highest dose (20 μL/100 g) resulted in 10% mortality after 24 h and 63.33% after 96 h. Overall, the contact toxicity of HTEO was higher against C. maculatus through the contact test compared to the inhalation test.

The LC₅₀ and LC₉₅ values obtained from the contact test were lower than those obtained from the inhalation test. After 24 h, the contact test yielded LC₅₀ values of 30.66 and 40.28 μL/100g, while the inhalation test resulted in LC₅₀ values of 14.59 and 14.68 μL/100 g. After 96 h, the LC₅₀ values were 27.95 and 28.40 μL/100 g for the contact and inhalation tests, respectively.

The LC₅₀ and LC₉₅ values obtained from the contact test were lower than those obtained from the inhalation test (Figure 7). Notably, after 24 h, the contact test yielded LC₅₀ values of 30.66 and 40.28 μL/100g, while the inhalation test resulted in LC₅₀ values of 14.59 and 14.68 μL/100 g. After 96 h, the LC₅₀ values were 27.95 and 28.40 μL/100 g for the contact and inhalation tests, respectively.

In the control group, C. maculatus bruchid females laid 207.33 eggs. Interestingly, the use of 20 μL/100 g of HTEO significantly reduced the emergence of bruchids by 91.34%. As depicted in Figure 8, HTEO significantly reduced the fecundity of C. maculatus. Specifically, the concentration of 5 μL/100 g resulted in a remarkable reduction in oviposition rate (48.85%) and emergence inhibition (45.15%) compared to the negative control. The concentration of 20 μL/100 g induced a significant reduction in oviposition rate (85.09%) and emergence inhibition (84.59%). 207.33 was the number of eggs laid by C. maculatus bruchid females, and the use of 20 μL/100g, significantly reduced the emergence with a rate of 91.34%. The results presented in Figure 8 show that HTEO significantly reduced fecundity in the insect pest C. maculatus. The concentration 5 μL/100g, resulted in a very remarkable rate of oviposition reduction (48.85%), and emergence inhibition (45.15%) compared to the negative control. on the other hand, the concentration of 20 μL/100 g induces a very important rate of oviposition reduction and emergence inhibition, which are respectively 85.09% and 84.59%.

The repellent effect of HTEO against the insect C. maculatus was assessed using the preferential surface method on filter paper, and the results obtained are depicted in Figure 9. It was observed that the repellent activity was dose-dependent. At the lowest concentration tested (0.016 μL/cm²), a repulsion rate of 60% was observed after 30 min, which further increased to 80% after 120 min of application. The maximum repellent effect against C. maculatus was achieved after 60 min by using a concentration of 0.315 μL/cm². These findings highlight the potent repellent properties of HTEO against C. maculatus, hence, rendering it worthy of further exploration.

To the best of our knowledge, this study is the first to investigate the insecticidal activity of HTEO against C. maculatus. To this end, the determination of toxicity by contact and inhalation test shows significant results with LC₅₀ values of 14.59 and 14.68 µL/100 g, respectively. Previous studies have been conducted on the insecticidal potentials of HTEO against other insect pests, as well as the insecticidal activities of extracts of plants belonging to the same genus or family as H. tuberculatum (Rutaceae) against C. maculatus using different protocols including fumigation, repellent and contact (Mssillou et al., 2022b).

Noteworthy, studies have shown that the insecticidal efficacy of EO extracted from various plant species varies against C. maculatus. In a study, the EO of a total of 121 plant species from 26 families were reported to possess insecticidal effects against C. maculatus (Mssillou et al., 2022b). Notably, in another study by Allali et al., the EO of Syzygium aromaticum, primarily composed of 80.26% eugenol, exhibited insecticidal activity against C. maculatus in inhalation tests, causing mortality rates of 6.67% at 24 h, 36.67% at 48 h, and 100% at 72 h. This EO also reduced oviposition and completely suppressed adult emergence (Aimad et al., 2021). Similarly, the EO of Dittrichia viscosa L., which contained bornyl acetate as the most abundant compound with 41%, resulted in 60% mortality in the contact test and 97.5% mortality in the inhalation test after 96 h of exposure to a 1 µL dose (Mssillou et al., 2022a). Also, the application of 20 μL of the EO of Dittrichia viscosa L. on C. maculatus, causes an approximately 91% reduction in the number of eggs laid by this insect. In another study, the insecticidal activity of the EO of Atalantia monophylla, containing eugenol (19.76%) and sabinene (19.57%), against C. maculatus using the fumigation test was found to result in 70.22% mortality, 85.24% repellency test, and also exhibited ovicidal activity (Nattudurai et al., 2017). Additionally, essential oils extracted from three Citrus species demonstrated insecticidal effects against C. maculatus, with LC₅₀ values of 6.33, 7.21, and 8.70 μL/L of air for C. aurantium, C. limon, and C. reticulate, respectively (Saeidi et al., 2011). It is also worth noting that HTEO has also been reported to possess insecticidal activities against other insect species infesting plant products. Exemplifying this is the contact toxicity showed by HTEO against the adults of three insect pests of cereals, namely, Sitophilus oryzae, Tribolium castaneum and Trogoderma granarium with LC₅₀ values of 0.11, 0.048, and 0.13 mg/cm², respectively (Saad et al., 2022). Additionally, HTEO extracted from aerial parts showed strong activity against Aedes aegypti larvae, specifically, the EO used had the potential to kill Aedes aegypti larvae at 125 ppm (Al-Rehaily et al., 2014).

Other extracts obtained from H. tuberculatum have also demonstrated insecticidal activities. The aqueous extract of H. tuberculatum, at concentrations of 5% and 10%, reduced egg hatching and juvenile mobility in Meloidogyne javanica, and showed a toxic effect on eggs and juveniles. At these same concentrations, the immobility of juveniles reached 100% after 48 h of exposure (Kallel et al., 2009). The methanolic extract of H. tuberculatum has also been reported to negatively impact the essential reproduction parameters of female Locusta migratoria. In this regard, the oral administration of this extract to newly emerged females (1.5 g/female), resulted in significant negative effects in terms of delay of the first oviposition, as well as reduction of fecundity and fertility (Acheuk et al., 2012).

Although the mode-of-action (MOA) of the observed HTEO-mediated antifungal and insecticidal activities remains to be elucidated and is still being explored, it is commonly believed that the complex mixture of compounds present in the EO might act synergistically, instigating several biological effects simultaneously. In this context, numerous studies have reported that the phytochemicals present in EOs can disrupt the cytoplasmic membrane and influence a range of other intracellular signaling and biological process, particularly energy generation (Swamy et al., 2016). Reduced membrane potentials, proton pump disruption, and ATP depletion have been reported as contributing factors to the reported antifungal activity (Turina et al., 2006; Chebbac et al., 2022).

Some reports have elaborated on the MOA of individual constituents present in EOs against insect pests. For example, a recent study revealed that the EO derived from Mentha arvensis exhibits a systemic MOA against Sitophilus granarius, and this was ascribed to the ability of the EO to perturb the insect’s neurological and muscular systems, intracellular aerobic respiratory processes, and cuticle, which serves as the primary protective barrier of insects (Renoz et al., 2022). Menthol and eugenol, both abundant in HTEO, have been shown to act on the octopaminergic system, triggering octopamine receptors and the phosphorylation pathway for protein kinase A in insect neurons (Hong et al., 2018; Jankowska et al., 2019). In Aphis gossypii, β-caryophyllene, another constituent enriched in HTEO, has been demonstrated to inhibit several enzymatic targets, including acetylcholine esterase, polyphenol oxidase, and carboxylesterase (Liu et al., 2010).

The results of the antioxidant activity of HTEO obtained using three tests (DPPH, FRAP and TAC) are presented in Table 5. As evident from the table, HTEO demonstrates a significant antioxidant activity with a value of 758.34 ± 3.87 mg AAE/g of EO. This potent activity can be attributed to the oil’s high content of various terpene compounds. While the EC₅₀ and IC₅₀ values of EOHT were only slightly higher compared to those of the standard compounds, they still indicate strong antioxidant activity. A comparison of the antioxidant capacity to that of the aqueous and ethanolic extracts of other samples collected from the same region revealed a higher total antioxidant capacity. Also, the aqueous extract demonstrates lower free radical scavenging activity (IC₅₀ = 0.37104 mg/mL) compared to the EO. However, both extracts show slightly higher iron-reducing activity (EC₅₀ values of 0.15554 and 0.16944 mg/mL for the aqueous and ethanolic extracts, respectively) (Agour et al., 2022).

Similar findings have been reported in other studies investigating the antioxidant activity of EO extracted from the leaves and stems of H. tuberculatum. It showed moderate antiradical activity against DPPH (IC₅₀ = 0.14 mg/mL) (Hamdi et al., 2017a). The results are consistent with a previous study by Debouba et al. (2014) (Debouba et al., 2014), which reported moderate antioxidant activity of H. tuberculatum oils against free radicals and low iron-reducing power. In an in vivo test on diabetic rats, the EO obtained from the flowers and aerial parts of H. tuberculatum significantly restored the reduced level of glutathione, indicating their antioxidant potential (Sabry and El Sayed, 2016). The antioxidant activities observed in the essential oils can be attributed to their chemical composition, particularly the high content of monoterpene compounds (25.83%). The antioxidant activities of essential oils are often associated with concepts such as additivity, antagonism, and synergy, as the presence of multiple compounds can contribute to the overall antioxidant capacity (Harkat-Madouri et al., 2015). Therefore, the high antioxidant activity of HTEO can be attributed to its chemical composition, particularly the abundance of monoterpene compounds.