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BY 4.0 license Open Access Published by De Gruyter Open Access October 10, 2023

Prefatory in silico studies and in vitro insecticidal effect of Nigella sativa (L.) essential oil and its active compound (carvacrol) against the Callosobruchus maculatus adults (Fab), a major pest of chickpea

  • Otmane Zouirech EMAIL logo , Abdelfattah El Moussaoui , Hamza Saghrouchni , Abdel-Rhman Z. Gaafar , Hiba-Allah Nafidi , Mohammed Bourhia EMAIL logo , Farid Khallouki , Badiaa Lyoussi and Elhoussine Derwich
From the journal Open Chemistry

Abstract

To help discover a reasonable and eco-friendly insecticide, we undertook a study on the insecticidal potential of carvacrol and essential oils extracted by hydrodistillation using a Clevenger apparatus from the plant species Nigella sativa seeds essential oils of Nigella sativa (EONS) on Callosobruchus maculatus adults. Several tests including contact toxicity, repellent effect, topical contact test, and inhalation effect were conducted. Adults of C. maculatus have been exposed to the toxic effects of different concentrations of these essential oils as well as with carvacrol. The results obtained showed that both EONS and carvacrol exhibited a moderate repellent effect (class II) on C. maculatus adults. EONS showed the highest toxicity by inhalation test, with an LD50 of 13.386 and an LD95 of 33.186 μL/cm2, compared to carvacrol (LD50 = 21.509 and LD95 = 38.877 μL/cm2). The EONS by contact test exhibited more toxic effects, with an LD50 of 23.350 µL/100 g and an LD95 of 45.315 µL/100 g, compared to carvacrol (LD50 = 27.853 µL/100 g and LD95 = 45.184 µL/100 g). For the topical contact test results, carvacrol was more toxic, with an LD50 of 3.915 and an LD95 of 7.696 µL/mL, compared to EONS (LD50 = 14.509 and LD95 = 25.516 µL/mL). The high toxicity of EONS can be explained by the presence of 25.8% of o-cymene, 8.53% of cyclofenchene, and 7.71% of beta-pinene, as well as 4.6% of carvacrol, in its chemical composition. Unmitigatedly, these data suggest that the essential oils of N. sativa may present a raw material for the development of new bio-insecticidal products against C. maculatus, one of the main pests of stored foodstuffs.

1 Introduction

Insect pests of stored legumes damage our economy by infesting stored agricultural products [1,2]. According to food and agriculture organization and national agriculture strategy, loss rates of stored legume seeds are between 5 and 10% in developing countries, due to rodents and commodity insects alone [3]. C. maculatus is a major pest of chickpea grain [4]. To address this problem, for protection against insect pests in the storage of stored foodstuffs and other agricultural products, various synthetic insecticides have been used to preserve stored foodstuffs, which can be the most effective and cheapest way to control insects. However, the excessive use of chemical insecticides has negative effects; indeed, pests and insects are going to develop resistance [5,6,7]. Furthermore, the effectiveness of insecticides used against storage pests varies greatly after treatment [8], which results in the excessive use of these synthetic compounds [8]. Insecticides are also very dangerous for the environment and consumers because of their residual properties [912].

Insecticide residues can have long-term impacts on terrestrial and aquatic ecosystems, as well as on human health through the food chain, so new alternatives must be ecologically sound, cost-effective, and protect human and environmental health without residual activity. Therefore, there is a need to develop new botanical pesticides targeting a wide range of pests to help challenge environmental issues and consumer safety.

In this regard, botanical pesticides are being tested for their toxic properties against various pests of stored cereals [13,14,15], especially in the form of essential oils [16,17], which are an alternative to chemicals with patterns of insecticidal properties in response to the threat of insects, the main stock pests [20,21].

As a matter of fact, considerable research data has been conducted on the potential use of plant-derived products as a source of natural substances with great potential for application in the control of insects and other plant and animal pests, as exemplified in [22,23]. Some botanical extracts and essential oils have been reported to have toxic effects against this insect pest [17,18,19]. In addition, the toxic effects of many components of the essential oils of these aromatic plants are also used in the control of many stock pests [20].

Carvacrol is a monoterpenoid phenol occurring in the essential oils of several aromatic and medicinal plants, such as Origanum compactum, Corydothymus capitatus, Satureja montana, and Thymus vulgaris, as well as in Nigella sativa [21,22]. Due to its insecticidal activities, in addition to having a long clinical tradition of using essential oils containing it, carvacrol is widely used to target many food storage pests [23]. As a natural compound, carvacrol is degraded in nature and is safe for non-target organisms [24]. These advantages indicate that carvacrol is a viable alternative to chemical agents.

Nigella sativa is a medicinal plant native to the Mediterranean region and often nicknamed black cumin. This species belongs to the Ranunculaceae family, cultivated mainly for its miraculous seeds. Used in cooking for their spicy aromas, black cumin seeds are recognized in phytotherapy for their digestive, carminative, anthelmintic, antioxidant, anti-inflammatory, antihistaminic, and diuretic properties among many more medicinal effects [25,26,27,28,29]. Nonetheless and to the best of our knowledge, a relatively in-depth study on the insecticidal effects of N. sativa essential oils against Callosobruchus maculatus adults is still absent.

The aim of this study is to identify the chemical composition of the essential oil of N. sativa seeds and screened repellent, toxic, and developmental inhibitory effects of essential oils (EO) of N. sativa seeds as well as on of its main chemical constituents carvacrol as a constituent of interest for this study due to its potential beneficial capacities in essential oils of Nigella sativa (EONS) against the chickpea pest (C. maculatus).

2 Materials and methods

The samples of N. sativa (L.) seeds used in this study come from the region of Souk El Arbaa (Morocco) 34°39′57″ North, 5°58′54″ West. It should be noted that the seeds analyzed in this work were harvested at the end of August 2021, when the fruits were well dried.

2.1 Sample extraction

Essential oils were extracted from seeds by steam distillation in a Clevenger-type apparatus. Three distillations were carried out for each dried species (200 g), for an average duration of 3 h. The recovered essential oil was stored at 4°C in the dark.

The essential oil yield (expressed as a percentage) is calculated as the ratio of the weight of the extracted oil to the weight of the plant material used.

2.2 Identifying volatile compounds by gas chromatography–mass spectrophotometry (GC–MS)

To help determine the different components and their percentages, the essential oils were analyzed by GC–MS. Using a Shimadzu GC-2010 model GC–MS, ENOS was injected. Helium served as the carrier gas in an HP-Innowax Agilent column (30 m 0.25 mm i.d., 0.25 µm thickness). The temperature of the gas chromatography (GC) oven was maintained at 40°C, designed to rise to 260°C at a rate of 5 °C/min and then maintained at 260°C for 40 min. The temperature of the injector was 250°C. At 70 eV, the mass spectrophotometry (MS) was recorded. The mass range was m/z between 30 and 400. The phytochemical identification of terpenic compounds was performed by comparing their mass spectra with those of the Adams reference [30].

2.3 Insecticidal activity

2.3.1 Breeding the insect

Insects of C. maculatus F. (Coleoptera: Bruchidae) were maintained in the laboratory without exposure to insecticides. These insects of all sexes were reared on chickpea seeds in glass jars. Mass rearing was performed at a temperature of 25 ± 1°C, a relative humidity of 70–85%, and a photoperiod of 16:8 h (light: dark). Only adults were used for contact and fumigation bioassays. All tests were carried out under conditions identical to those of the farm.

2.4 Method for assessing insecticidal activity

2.4.1 Repellency test on adults of C. maculatus

This test consists of studying the repellent effect of EONS and carvacrol on adults of C. maculatus. Four doses were prepared by diluting each time in 1 mL of acetone the respective volumes of 0, 2.5, 10, and 20 µL/mL. Disks of Whatman No. 1 paper with a surface area of 31.80 cm² were placed in a Petri dish, and each of the prepared solutions was added to the disks, and the second one received only acetone, after evaporation of the solvent, we put together the two treated and untreated parts by an adhesive tape and we placed them in a Petri dish. The different volumes of essential oil and carvacrol 2.5, 5, 10, and 20 μL per unit area of half filter paper (i.e., 31.80 cm²) correspond to the doses 0.078, 0.157, 0.314, and 0.628 µL/cm², respectively. For the control, the washer was treated with acetone only. The average percentage repellency (PR%, equation (1)) is calculated and assigned to one of the different repellent classes, according to the classification of McDonald et al. [31].

(1) PR ( % ) = N c N t N c + N t × 100 ,

where PR% is the average percentage repellency, N c is the number of insects present on the half-disc treated with acetone only, and N t is the number of insects present on the half-disc treated with the oil solution and carvacrol.

2.4.2 Inhalation toxicity test of EONS and carvacrol on seeds against adults of C. maculatus

This test consisted of studying the effect of N. sativa essential oils and carvacrol on the mortality rate of C. maculatus adults by inhalation using the method described by Iturralde-garcía et al. and Ferreira et al. [4,32].

(2) M c ( % ) = M o M t 100 M t × 100 ,

where M c (%) is the corrected mortality percentage, M o (%) is the percentage of deaths in the treated population, and M t (%) is the percentage of deaths in control population.

2.4.3 Contact toxicity test of EONS and carvacrol on seeds against adults of C. maculatus

In jars identical to those described above, 50 g of chickpea seeds were placed on which were sprayed four doses of essential oils corresponding to the respective concentrations of 2.5, 5, 10, and 20 µL/100 g. The whole content was mixed vigorously, the jars were left open at room temperature until complete evaporation of the solvent, and then 10 adult insects of C. maculatus were introduced. The jars were closed with perforated lids and incubated in a chamber at 30°C with a relative humidity of 70–75%. Mortality was recorded after 24, 48, 72, 96, and 120 h. After this time, dead insects (i.e., insects were considered dead if no movement of legs and antennae was recorded) and survivors were separated and counted. The results given represent the average of three replicates. Mortalities in the treated boxes (Mo) were expressed according to Abbott’s formula (3) [4,32]:

(3) M c ( % ) = M o M t 100 M t × 100 ,

where M c (%) is the corrected mortality percentage, M o (%) is the percentage of deaths in the treated population, and M t (%) is the percentage of deaths in control population.

2.4.4 Topical contact toxicity test of EONS and carvacrol against C. maculatus adults

This test consisted of applying different concentrations (0, 2.5, 5, 10, and 20 µL/L) to the prothorax of C. maculatus adults [33] at a volume of 0.2 µL. Treated insects were placed in Petri dishes at a rate of 10 insects per dish and kept in the dark at room temperature. For the control, the washer was treated with acetone only. Three replicates were made for each dose, and dead insects were counted (and kept in the dishes) for 24 h. Mortalities in the treated boxes were expressed according to Abbott’s formula (2) [4,32]:

(4) M c ( % ) = M O M t 100 M t × 100 ,

where M c (%) is the corrected mortality percentage, M o (%) is the percentage of deaths in the treated population, and M t (%) is the percentage of deaths in control population.

2.4.5 Assessment of insect mortality

An insect was considered dead when an insect was found in dorsal or ventral recumbency with a stiff body. The insect showed no reaction, especially in the legs and antennae, after stimulation with a pair of spit wands.

2.5 Molecular docking

Molecular docking aims to perform a predictive ligand–receptor complex structure using computation methods [34]. This method of drug design is fast, reliable, and cost-effective [35,36]. Molecular docking has been applied on the acetylcholinesterase protein from T. californica (TcAChE), obtained from the Protein Data Bank (protein PDB: 1EVE) [37]. The water molecules, co-factors, and the complexed ligand in the target structure were deleted, and polar hydrogen atoms and Kollman charges were added, then rotatable bonds and torsion tree roots were determined. The ligand and target structures were prepared using AutoDock tools (version 1.5.6) and visualised using PyMOL Molecular Graphics System, Version 2.0 (Schrödinger, LLC) [6] and Discover Studio Visualizer 4.0 (DSV 4.0) [7]. This software is also used to determine the active site residues that have been taken from the co-crystal binding site, therefore determining the cubic grid box with X = 20, Y = 20, and Z = 20 and x_center = 5.309, y_center = 65.929, and z_center = 65.300 at a spacing of 0.375 Å, energy range of 4, and exhaustiveness of 8. The docking analyses were carried out using the AutoDockVina program [34].

2.5.1 Absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties

The toxicity and physical characteristics of studied compounds were predicted using ADMETsar server version 2 based on the relationship between the quantitative structure and activity of the molecular structure of the inhibitor.

2.6 Analytical statistics

Statistical analysis was performed using GraphPad Prism (version 8.0.1). All experiments were done in triplicates and standard deviations were reported. The variables’ normality was verified using Shapiro–Wilk tests, and the homogeneity of variances was investigated using Levene’s test. The discrepancies between the means were investigated using Tukey’s multiple comparison test and one-way analysis of variance. Differences were considered statistically significant at a probability level 0.05.

3 Results

3.1 Chemical composition of N. sativa essential oil constituents

By hydrodistillation, a yield of 0.8 ± 0.02% was obtained from the seeds of the Moroccan variety of N. sativa species. The obtained essential oil has a transparent yellow color and an aromatic smell.

The analysis of the essential oil sample obtained by GC–MS allowed us to identify 28 compounds that represent 100% of the global chemical composition (Table 1). This essential oil is mainly composed of p-Cymene (25.8%), Cyclofenchene (8. 53%), beta-Pinene (7.71%), dl-Limonene (5.86%), Nerolidol (5.78%), Isolongifolene (5.18%) l-terpine-4-ol (5.6%), alpha-Terpinene (4.67%), Carvacrol (4.6%), and Sabinene (3.78%) as major metabolites (Figure 1).

Table 1

Chemical constituents of EONS

Rt Compound name Area (%)
15.955 Cuminaldehyde 1.05
18.455 Nerolidol 5.78
19.930 l-4-Terpineol 5.6
20.530 p-Menth-2-en-1-ol 0.32
22.505 endo-Borneol 0.43
19.180 Isoeugenol 0.93
33.680 Carvacrol 4.6
17.355 Menthalactone 2.89
20.530 Carvotanacetone 0.38
18.080 Terpinyl butyrate 0.13
19.680 Endobornyl acetate 1.47
21.955 Citronellyl tiglate (E) 0.45
24.905 Linalyl benzoate 0.18
4.695 Cyclofenchene 8.53
5.035 l-Phellandrene 5.74
6.405 beta-Pinene 7.71
6.850 Sabinene 3.78
7.200 beta-Phellandrene 0.93
9.050 (+)-Limonen 1.36
9.220 dl-Limonene 5.86
9.640 beta-Phellandrene 0.43
10.410 gamma-Terpinene 2.03
12.469 O-Cymene 25.8
17.655 Bicyclogermacrene 0.49
19.180 Isolongifolene 5.18
16.455 alpha-Longipinene 2.46
8.690 alpha-Terpinene 4.67
18.130 (1-Methylethenyl)benzene 0.82
Total 100%
Figure 1 
                  GC–MS chromatogram of the N. sativa essential oil (EONS).
Figure 1

GC–MS chromatogram of the N. sativa essential oil (EONS).

3.2 Repellent activity

Table 2 shows the analysis of the repellent effect exerted by EONS and carvacrol on C. maculatus adults after 2 h exposure. The results clearly demonstrated the ability of the essential oil and carvacrol to act as repellents against C. maculatus adults.

Table 2

Repulsion (%) on EONS and carvacrol filter paper toward adults of C. maculatus

Concentration (µL/cm²) Repulsion rate (%) EONS carvacrol
0.078 13.33 ± 5.77 10 ± 0.00
0.157 20 ± 0.00 16.66 ± 5.77
0.314 33.33 ± 11.54 40 ± 10.00
0.628 50.00 ± 10.00 53.33 ± 5.77
Average repulsion rates 29.16 29.99
Repellent class II II

Furthermore, the repellent effects of EONS and carvacrol were influenced by the doses administered. In other words, the greater the quantity of substance applied, the more pronounced the repellent effect observed.

In the specific case of EONS, the results indicated moderate repellency rates, which vary according to the dose administered. The optimum dose appears to be 0.628 μL/cm2, resulting in a maximum repellency of 50.00 ± 10.00% after a 60 min exposure period.

Similarly, carvacrol also exhibited a dose-dependent repellent response. Observations revealed that at a dose of 0.628 µL/cm2, the magnitude of repellency was found to be 53.33 ± 5.77%.

The results presented in Table 2 revealed significant information concerning the repellent properties of the EONS and carvacrol studied. Using the classification proposed by McDonald et al. [31], these two substances were qualified as moderately repellent, with a repellency rate of 29.16 and 29.99%, respectively, for EONS and carvacrol. Such classification is based on an assessment of the repellent efficacy of the substances tested, considering the percentage of repellence provoked in the target organism. In this context, PR values of 29.16% for EONS and 29.99% for carvacrol indicate, although to a moderate extent, the importance of EONS and carvacrol in the control and repelling of C. maculatus adults. It is worth noting that the classification of moderately repellent suggests that EONS and carvacrol have the ability to induce a repellent response in the insects studied, but that this response is not extremely strong. The results, therefore, underline the repellent effect of these substances, which entails their potential implications for the control of C. maculatus populations in agricultural or storage contexts.

3.3 Inhalation toxicity

The aim of this assay was to evaluate the effect of EONS and carvacrol through fumigant/respiratory effect on C. maculatus adults. Figure 2 shows the cumulative and adjusted mortality rates (%) of C. maculatus adults as a function of exposure times and concentrations of EONS and carvacrol. These mortality rates increased in parallel with increasing concentrations of EONS and carvacrol. Notably, the highest concentration (20 µL/mL) resulted in 100% total mortality after 48 h of exposure. In contrast, carvacrol required 120 h of exposure to achieve 100% complete mortality.

Figure 2 
                  Evolution of the percentage of mortality of inhalation in C. maculatus adults subjected to different concentrations of EONS and carvacrol for 120 h.
Figure 2

Evolution of the percentage of mortality of inhalation in C. maculatus adults subjected to different concentrations of EONS and carvacrol for 120 h.

After the first 24 h of exposure, it becomes clear that essential oil and carvacrol have LD50 values of around 13.386 and 21.509 µL/mL, respectively. These data underline the potent inhalation toxicity of the EONS and carvacrol, specifically toward these bruchids, as summarized in Table 3.

Table 3

LD50 and LD95 values calculated based on the mortality of C. maculatus adults by the different tests after 24 h of exposure to EONS and carvacrol

Bioassays LD50 LD95 X 2
EONS Inhalation test 13.386 33.168 4.604
Contact test 23.730 45.315 1.073
Topical contact test 14.350 25.516 2.528
Carvacrol Inhalation test 21.509 38.877 1.289
Contact test 27.853 45.184 1.020
Topical contact test 3.915 7.696 1.682

X 2: Indicate confidence intervals are too wide, they do not lend themselves to calculation.

These results jointly highlight the marked efficacy of EONS and carvacrol as toxic agents against C. maculatus adults. The experiment demonstrates the lethal impact of both substances on bruchids, underlining their potential for use in pest management strategies, more particularly to protect stored chickpea seeds and stem infestations.

3.4 Contact toxicity

The toxic effect was assessed by directly applying different doses of EONS and carvacrol to chickpea seeds. Figure 3 illustrates how the cumulative and adjusted mortality percentages (%) of C. maculatus adults change as a function of the essential oil concentrations used. This figure highlights how mortality percentages vary according to the doses of essential oil applied. These results underline the potentially lethal impact of EONS on C. maculatus adults and provide evidence on how mortality evolves as a function of the concentration of essential oil used. The highest dose of EONS (20 µL/100 g) caused 100% mortality of C. maculatus adults after 72 h of exposure. However, the highest dose of carvacrol (20 µL/100 g) caused 100% mortality of C. maculatus adults after 120 h of exposure.

Figure 3 
                  Percentage mortality of adults of C. maculatus in the presence of different concentrations of EONS and carvacrol for 120 h.
Figure 3

Percentage mortality of adults of C. maculatus in the presence of different concentrations of EONS and carvacrol for 120 h.

The different values reflect the different routes of exposure. The LD50 and LD95 are different (Table 3). The low LD50 value (23.730 μL/100 g) calculated in 24 h of exposure attests to the toxicity efficiency of this essential oil (EONS) against these imagoes. The above data showed that carvacrol is highly toxic against these imagoes with an LD50 value of about 27.853 μL/100 g.

3.5 Topical contact toxicity

To assess the topical contact toxicity of EONS and carvacrol, different doses were assessed. Figure 4 shows that the cumulative and corrected mortality (%) of C. maculatus adults varied with the concentrations used. The highest dose of carvacrol (20 µL/L) caused 100% mortality of bruchids after 24 h of exposure. However, the highest dose of EONS (20 µL/L) resulted in 76.667 ± 5.664% mortality of the bruchids after 24 h of exposure.

Figure 4 
                  Evolution of mortality percentages of C. maculatus adults by tropical contact on filter paper submitted to different concentrations of EONS during 24 h.
Figure 4

Evolution of mortality percentages of C. maculatus adults by tropical contact on filter paper submitted to different concentrations of EONS during 24 h.

According to the results shown in Table 3, the low LD50 value (3.915 μL/L) calculated in 24 h of exposure by topical contact attests to the toxicity efficacy of carvacrol toward these C. maculatus adults. However, the results demonstrated a low LD50 value of 14.350 μL/L recorded for EONS.

Table 3 summarizes the LD50 and LD95 values calculated based on the mortality of C. maculatus adults by the inhalation test, contact test, and topical contact after 24 h of exposure to EONS and carvacrol. The different values reflect the different routes of exposure. The LD50 and LD95 are different. The above data show that carvacrol is much more toxic by topical contact.

3.6 Molecular docking

Table 4 shows the docking scores of the bioactive compounds of N. sativa against TcAChE. The docking pose is between carvacrol and TcAChE receptor with a binding energy of −7.5 kcal/mol and with one conventional hydrogen bond.

Table 4

Binding energies of studied molecules

Ligand Binding score (kcal/mol) Number of conventional hydrogen bond
Carvacrol −7.5 1

The analysis of the molecular docking showed that, almost all the bioactive compounds have hydrophobic interactions as well as van der Waals-type interactions. Carvacrol forms one conventional hydrogen bond with ASP72 and exhibits pi–pi T-shaped and pi–pi stacked interactions with TRP84 and PHE330, as well as pi–sigma interaction with TYR334 and TRP84 in addition to Pi–alkyl interaction with PHE331 residue (Figures 5 and 6).

Figure 5 
                  The molecular docking results of carvacrol with 1EVE protein, surfaces around ligand and 2D forms.
Figure 5

The molecular docking results of carvacrol with 1EVE protein, surfaces around ligand and 2D forms.

Figure 6 
                  3D visualization of carvacrol (red color) docked with 1EVE protein (light blue color) using PyMOL.
Figure 6

3D visualization of carvacrol (red color) docked with 1EVE protein (light blue color) using PyMOL.

The ADMET properties were used to highlight the physical characteristics of the carvacrol products, as summarized in Table 5. The ability to cause cancer (carcinogenicity) or genetic mutation (mutagenicity) was chosen to represent the toxicity of the molecule studied. The carcinogenicity and mutagenicity were found to be between 0.5142 and 0.86. Generally, predicted levels between 0.00 and 0.50 represent low toxicity probabilities, while levels greater than 0.50 indicate high toxicity probabilities [38]. The LD50 value of a toxic substance should be greater than 300 mg/kg. Acute toxicity determined a lethal dose for our molecules of the order of 3521.50 mg/kg (Table 5) [39]. The biodegradability values show that the studied molecules are biodegradable and environmental friendly.

Table 5

ADMET properties of studied molecules

Physical properties Carvacrol
Molecular weight (g/mol) 150.22
LogP 2.82
H-bond acceptor 1
H-bond donor 1
Number of atoms 11
Number of heavy atoms 6
Number of rotatable bonds 1
Acute oral toxicity (mg/kg) 3521.50
Carcinogenicity probability 0.5286
Mutagenicity probability 0.8600
Biodegradability probability 0.6000
Water solubility (mg/mL) 7.40 × 10−2
Solubility class Soluble

4 Discussion

EONS are composed by a important proportion of monoterpenes (75.79%) compared to our previous study in 2022 [22], which was amounted to 85.51% depending on the extraction period. The main constituents include p-Cymene (25.8%), Cyclofenchene (8.53%), beta-Pinene (7.71%), and dl-Limonene (5.86%). Alcohols represent 17.66% of total EONS, with Nerolidol (5.78%), l-4-terpineol (5.6%), and carvacrol (4.6%) being the major ones. The disparate results compared to our previous investigation probably depend on different factors, namely, seed maturity, growing conditions, amount of sunlight exposure, period of collection, pedoclimatic environment, ecological changes, analysis conditions, or also shelf life, among many other factors that influence the qualitative and quantitative aspects of essential oils [22]. It is important to note that drying also had a clear influence on the chemical composition of these essential oils [40]. In our study, thymoquinone, for example, one of the main components of N. sativa seeds was not detected.

According to the results obtained, we noted that the EO extracted from the aromatic and medicinal plant N. sativa (Ranunculaceae) as well as one of its main constituents, carvacrol, exerted toxic effects in a concentration-dependent manner on the adults of C. maculatus.

A large body of literature has reported evidence of the repellant activities of plant extracts or essential oils, as exemplified in [41]. Essential oils extracted from Artemisia aragonensis Lam. and Artemisia negrei L. (Asteraceae) demonstrated a significant repellent effect on the insect C. maculatus [42]. The results obtained clearly indicated that these essential oils have the capacity to repel C. maculatus adults to a marked degree. The percentages of repellence observed showed that essential oils extracted from Cinnamomum verum L. exerted a significant impact on the behavior of C. maculatus, prompting them to avoid or leave areas treated with these oils. This repellent response can also be attributed to specific compounds present in the essential oils, such as cinnamaldehyde dimethyl acetal (64.50%), which were identified as factors contributing to the repellent effect [43]. Furthermore, results suggested that essential oils extracted from Origanum compactum Benth may have promising potential as natural repellents in biological control or pest management strategies, notably for the control of C. maculatus in agricultural or food storage environments [14]. Similarly, Aggarwal et al. evaluated the repellent activity of L-menthol and its acyl derivatives against C. maculatus, Tribolium castaneum, Sitophilus oryzae, and Rhizopertha dominica, which gave repellency rates of 100, 82, 78, and 72% respectively, at a concentration of 20 µg/mL [44]. Thus, the results of previous work showed a significant repellent effect of essential oils extracted from Syzygium aromaticum on the insect C. maculatus. Used at doses of 4, 8, 16, and 32 µL diluted in 0.5 mL of acetone, these oils gave repulsion rates of 93.33 ± 11.55% after 1 h of exposure [45]. The repellent effects of these essential oils may depend on the chemical composition and sensitivity level of the insect [46]. In this context, several studies have explored the relationships between the chemical composition and the repellent activity of essential oils [47,48]. In most cases, the essential oil’s repellency activity is due to the variability of its chemical components [41,49,50,51].

EONS showed a repellent effect on C. maculatus adults and showed a repellent effect on C. maculatus. This could be explained either by the existence of repellent compounds in this essential oil or by the absence of attractive compounds, as well as by the sensitivity of bruchid [43]. Indeed, several monoterpenes have been described to have notable repellent activities against stored-food insects [50]. The repellent activity of the EONS tested is therefore explained by their richness in monoterpene and sesquiterpene compounds. From an explanatory point of view, aromatic plant compounds that act as insect repellents act as semi-chemicals that modify insect behavior through the olfactory senses of the antennae [51]. Insect repellents do this by providing a vapor barrier and deterring the arthropod from coming into contact with the surface or stimulus [52].

In regard to the repellent effect of carvacrol, a positive repulsion rate was noted. This molecule showed a repellent effect on the adults of C. maculatus. This could be explained by the sensitivity of the C. maculatus bruchid to carvacrol [15].

When studying the toxicity of EONS and carvacrol against C. maculatus adults, dose-response curves were established, exposed by contact, and by fumigation. The analysis of the results showed that both samples studied (EONS and carvacrol) are more toxic by inhalation than by contact. This can be explained by the fact that the contact test does not ensure homogenization of the number of EONS or carvacrol received by the insect, which is in accordance with Moritz’s earlier observations. There are always insects that may have escaped treatment from mass exposure [52].

Considering the LD50 and LD95, it can be seen that EONS is still more toxic than carvacrol, which was less toxic by inhalation than by contact. Because of their high volatility, they have fumigant activity that could be important for controlling insects in stored commodities [53]. The LD50 and LD95 obtained indicate that C. maculatus adults are more sensitive to EONS than to carvacrol. However, it would be difficult to assume that the insecticidal activity of this oil is limited to only some of its major constituents; it could also be due to some minority constituents or to a synergistic effect of several constituents [54]. It should also be noted that the LD50 and LD95 values were different for EONS than for carvacrol, depending on the route of exposure (inhalation, contact, and topical contact).

The dose effect of EONS and carvacrol on the mortality rate of adults of C. maculatus 24 h after ingestion exposed by contact (topical contact) showed that carvacrol was more toxic by topical contact than EONS. Our study showed that these two samples tested, namely EONS and carvacrol, are more toxic by topical contact applied directly to the thorax of the adults studied than when mixed with chickpea seeds. This would explain why the toxicity of EONS and carvacrol is greater in individual ingestion than in collective ingestion [55].

Carvacrol (monoterpenoid phenol) is an acetylcholinesterase inhibitor [56]. It has been shown to be toxic to pests including Anaphothrips obscurus [57], Mahanarva spectabilis [58], Sitophilus granaries, and Tribolium confusum [36]. Similarly, the present study also showed that EONS, together with carvacrol, affects the survival of C. maculatus adults. The observed reduction could probably be due to a direct interaction between EO and AChE or indirectly through an action on neural cells. This may be due to the fact that the chemical structure of carvacrol comprises a benzene ring with methyl and hydroxyl groups [45]. The methyl group increases the fat solubility of carvacrol, which is toxic to Lymantria larvae or Aedes aegypti larvae [59]. In addition, carvacrol has been shown to have a significant effect on acetylcholinesterase activity observed in Lymantria dispar [60].

From the two samples tested and considering these facts, it could be stated that essential oils extracted from N. sativa (L.) and carvacrol have a definite action in the control of C. maculatus. This work could therefore be continued with a view to the practical use of these essential oils and compounds in the protection of chickpea stocks.

5 Conclusion

This study was undertaken on the use of compounds derived from the essential oil of N. sativa as a potential bioinsecticide and as a potential alternative to synthetic agents currently used against the stored-food insect pest C. maculatus. However, the comparison of the repellent percentages of the tested essential oils and carvacrol revealed, despite moderate repellent effects, a significant fumigant effect of the adulticides after 5 days of exposure. Because of all the obtained results, we can suggest that EO from N. sativa can be recommended as a fumigant and as an insect repellent against C. maculatus. This approach will present several advantages for the health of the living being and for its environment as compared to the synthetic chemicals that negatively contaminate the biosphere.

Acknowledgments

The authors extend their appreciation to the Researchers Supporting Project number RSPD2023R686, King Saud University, Riyadh, Saudi Arabia.

  1. Funding information: This work is supported by the Researchers Supporting Project number RSPD2023R686, King Saud University, Riyadh, Saudi Arabia.

  2. Author contributions: The authors confirm equal responsibility for the following: study conception and design, data collection, analysis and interpretation of results, and manuscript preparation.

  3. Conflict of interest: The authors declare that there is no conflict of interest.

  4. Ethical approval: The conducted research is not related to either human or animals use.

  5. Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Received: 2023-07-18
Revised: 2023-08-16
Accepted: 2023-09-15
Published Online: 2023-10-10

© 2023 the author(s), published by De Gruyter

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

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