Cytotoxic, Scolicidal, and Insecticidal Activities of Lavandula stoechas Essential Oil

Abstract

Essential oils (EOs) have recently attracted more interest due to their insecticidal activities, low harmfulness, and rapid degradation in the environment. Therefore, Lavandula steochas (L. steochas) essential oil was assessed for its chemical constituents, in vitro cytotoxicity, and scolicidal, acaricidal, and insecticidal activities. Using spectrometry and gas chromatography, the components of L. steochas EOs were detected. Additionally, different oil concentrations were tested for their anticancer activities when applied to human embryonic kidney cells (HEK-293 cells) and the human breast cancer cell line MCF-7. The oil’s scolicidal activity against protoscolices of hydatid cysts was evaluated at various concentrations and exposure times. The oil’s adulticidal, larvicidal, and repelling effects on R. annulatus ticks were also investigated at various concentrations, ranging from 0.625 to 10%. Likewise, the larvicidal and pupicidal activities of L. steochas against Musca domestica were estimated at different concentrations. The analyses of L. steochas oil identified camphor as the predominant compound (58.38%). L. steochas oil showed significant cytotoxicity against cancer cells. All of the tested oil concentrations demonstrated significant scolicidal activities against the protoscoleces of hydatid cysts. L. steochas EO (essential oil) showed 100% adulticidal activity against R. annulatus at a 10% concentration with an LC₅₀ of 2.34%, whereas the larvicidal activity was 86.67% and the LC₅₀ was 9.11%. On the other hand, the oil showed no repellent activity against this tick’s larva. Furthermore, L. steochas EO achieved 100% larvicidal and pupicidal effects against M. domestica at a 10% concentration with LC₅₀ values of 1.79% and 1.51%, respectively. In conclusion, the current work suggests that L. steochas EO could serve as a potential source of scolicidal, acaricidal, insecticidal, and anticancer agents.

1. Introduction

In developing countries, parasitic diseases, associated with both ectoparasites and endoparasites, represent a severe hazard to both human and animal health [1]. Cystic echinococcosis (CE) is caused by the larval stage (hydatid cyst) of Echinococcus granulosus, which can develop in the liver, heart, lungs, brain, spleen, bone, and kidneys of the host, and can be fatal [2,3]. In several endemic areas, the incidence rate of CE may vary from 1 to 200 per 100,000 persons annually [4]. Currently, a range of chemical scolicidal compounds, such as benzimidazole derivatives, are used to deactivate hydatid cyst protoscolices [5,6]. However, these chemicals cause a variety of negative side effects, including impairments in liver function, leucopenia, and abdominal pain [6,7]. A perfect scolicidal agent is one that remains stable after being diluted with cyst fluid, eliminates cyst protoscolices, is non-toxic, causes no harm to the tissue of the host, is inexpensive, and is easily accessible [3,7,8,9].

The use of scolicidal compounds is essential to the therapeutic treatment of hydatid cysts and helps prevent the spread of the protoscoleces through surgery [2]. Due to the adverse effects often associated with the use of protoscolicidal agents during hydatid cyst surgery, more emphasis is now placed on the toxicity of these agents and the search for safe alternatives [10]. Significant amounts of research have recently been directed towards examining herbal extracts as a source of novel, powerful, and non-toxic anti-scolicidal compounds [11]. Numerous plant extracts and their essential oils, including Mentha pulegium, Curcuma longa, Allium sativum, Nigella sativa, Zataria multiflora, Salvadora persica, Origanum minutiflorum, and Zingiber officinale have been shown to carry significant scolicidal effects [2,8,11,12,13]

Globally, ectoparasites pose a significant risk to both the economy and animal health [14]. Ectoparasitic infestation has been linked to a variety of health issues, including anemia, weight loss, abscesses, and tissue damage. It can also serve as a vector for several deadly diseases of great concern to livestock [14,15]. Among the varieties of ectoparasites, ticks are considered a main threat due to the severe irritation, anemia, paralysis, and toxicosis they can cause, as well as the fact that they can transmit diseases such as anaplasmosis, theileriosis, and babesiosis [16,17,18,19]. Similarly, one of the most globally prevalent arthropods with medicinal and veterinary significance is the house fly (Musca domestica L.) [20,21]. This species is known to harbor more than 100 types of microorganisms, including bacteria, viruses, parasites, worms, and protozoa, which can lead to serious and potentially fatal diseases in both humans and domestic animals [22,23].

Chemically derived drugs are the primary approach used globally to manage ectoparasites and endoparasites affecting different kinds of animals. This has resulted in several serious problems, including the development of resistance [24,25], toxic damage to non-specified organisms, and environmental pollution [26]. Consequently, new eco-friendly alternatives are being introduced into strategic parasite-monitoring programs [27]. The usage of essential oils and plant extracts as insect control agents has become the subject of intensive investigation in a number of countries because of the efficiency of their insecticidal and acaricidal effects, which have negligible environmental impacts [28,29]. These oils comprise combinations of chemical substances that are toxic to insects, and toxicity operates via a number of mechanisms including enzyme inhibition and protein denaturation [30]. It is known that several plants in the Mediterranean region possess insecticidal and acaricidal properties [31]. The genus “Lavandula” (Lamiaceae) is a wild plant found in the Mediterranean basin that comprises over 34 species and is well-known for having insecticidal effects against different species [32,33]. Along with being employed in conventional treatment, different species of genus “Lavandula” are also utilized in the pharmaceutical and cosmetic sectors [34,35]. One of the most widely studied and used lavender species in the world is Lavandula stoechas (L. stoechas) [36]. Some studies have focused on the antibacterial [37,38], antifungal [39,40], and antioxidative [40,41] characteristics of L. stoechas. Correspondingly, the objectives of the current study were to determine the following: (i) the total chemical constituents; (ii) the in vitro cytotoxic activities of L. steochas; (iii) the in vitro scolicidal activity of L. steochas against the protoscoleces of hydatid cysts; (iv) the in vitro adulticidal, larvicidal, and repellent activities of L. steochas against R. annulatus ticks; and (v) the in vitro larvicidal and pupicidal activities of L. steochas against M. domestica.

2. Materials and Methods

2.1. Plant Material

In August 2017, the aerial components of Lavandula stoechas subsp. Stoechas were gathered from the city of Elmal-Antalya, and identification was performed by the plant taxonomist Dr. Aşkn Akpulat from the Faculty of Education, Cumhuriyet University, Sivas, Turkey. A voucher specimen was deposited at the Herbarium of the Department of Biology, Cumhuriyet University (CUFH). The leaves of the gathered plants were removed from the stems and flowers, dried in the shade, and then crushed until they could pass through a 2 mm mesh.

2.2. Essential Oil Extraction

Dried and finely crushed leaves (100 g) were hydrodistilled for 3 h in a Clevenger-type distillation apparatus with 2 L of double-distilled water [41,42]. The produced EOs were filtered, dried over anhydrous sodium sulfate, and kept at 4 °C until use.

GC–MS analysis of Lavandula stoechas essential oil was performed using a Trace Ultra gas chromatograph, (GC) coupled with a DSQ II mass spectrometer (MS; Thermo Scientific). The compounds were separated on a TR-5MS (30 m × 0.25 mm × 0.25 μm) capillary column (Thermo Scientific), operating on a temperature program of 60 to 250 °C with an elevation speed of 3 °C/min and a helium flow rate of 1 mL/min. The injector and MS transfer line temperatures were set at 220 and 250 °C, respectively. The samples were prepared via the dilution of 1 mg of EO in 1 mL of acetone. In total, 1 µL of the diluted sample was injected manually in the splitless mode. The MS was operated in the EI mode at 70 eV. The ion source temperature was 240 °C, and the mass spectra were acquired in the scan mode based on a mass range of 35–400. We tentatively identified the compounds based on comparisons of their relative retention indexes and mass spectra with corresponding data found in the literature and different databases [42]. A series of n-alkanes (C8–C24) was used in the determination of the relative retention index (RRI). Relative percentages of the compounds were obtained electronically from area percentage data.

2.3. Cytotoxic Activity of L. stoechas

Cell Culture

Human embryonic kidney cells (HEK-293 cells) and the human breast cancer cell line MCF-7 were cultured in a DMEM culture medium accompanied with 10% fetal bovine serum (FBS), 0.2% sodium bicarbonate, and an antibiotic/antimycotic solution. The cells were grown in a CO₂ incubator (5% CO₂–95% atmosphere) at ±37 °C and a high humidity [43]. The trypan blue dye exclusion assay was used to determine the vitalities of all cell lines, and batches of cells with over 98% cell viability were utilized.

2.4. Cytotoxicity Assessment by MTT Assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide Tetrazolium)

The cytotoxicity assessment was performed according to the protocol set out by Siddiqui et al. [43]. In brief, the cells were plated in 96-well culture plates and adhered for 24 h in a CO₂ incubator at ±37 °C. Then, the cells were exposed to L. steochas oil in several concentrations (0.0156–1%) for 24 h. Following exposure, 10 mL of MTT (5 mg/mL of stock) was added to each well, and the plates were then incubated for a further 4 h in a CO₂ incubator. After the supernatant was discarded, 200 mL of DMSO was added to each well and thoroughly mixed. The plates were read at a wavelength of 550 nm. The cytotoxic activity of L. steochas oil against the cancer cell line was inferred from the estimated LC₅₀ value.

2.5. Scolicidal Activity

Collection of Protoscoleces from Hydatid Cysts and the Viability Test

Using sheep livers that were naturally infected with hydatid cysts, the careful excision of the hydatid was performed. The hydatid fluid samples were aspirated with protoscoleces and kept in glass cylinders for 20 min to allow the protoscoleces to settle. The supernatant fluid was then discarded and the reaming protoscoleces were washed three times in saline solution. An eosin stain at 0.1% was used to confirm the viability of the protoscoleces. Staining was performed for five minutes and the protoscoleces that did not take on the dye were considered alive, while those that were stained were considered dead [44]. Protoscoleces with a viability of 95% were selected for further study.

2.6. Determination of In Vitro Scolicidal Activity

To assess the in vitro activity, the protoscoleces treated with three concentrations of essential oil (0.025, 0.05, and 0.1%) were evaluated. A total of 2 mL of each concentration was placed into three tubes to which ~5 × 10³ protoscolices were added, which were gently mixed. Then, the three tubes of each concentration (nine in total) were incubated at ±37 °C for 1, 3, and 5 min, respectively. After incubation, the supernatant was carefully removed to avoid unsettling the remaining protoscoleces. Then, the settled protoscoleces were gently mixed with 1 mL of 0.1% eosin. Stained protoscoleces were then examined under a microscope five minutes later to determine their vitality. A group of at least 5 × 10³ protoscoleces in 2 mL of distilled water with no oil treatment was used as the control.

2.7. Acaricidal activity of L. steochas against Larvae and Adult of R. annulatus

In various villages in the Beni-Suef province of central Egypt, and further south towards Cairo (29°04′N, 31°05′E), adult engorged females of R. annulatus were collected from naturally infected cattle. The collected ticks were taken to the Parasitology Lab at the Faculty of Veterinary Medicine, Beni-Suef University. The ticks were identified according to the process of Estrada-Pena et al. [45]. A portion of the collected ticks were employed in the adult immersion test, while the remaining were incubated at 27 ± 1.5 °C and 70–80% relative humidity (RH) to produce eggs, which were then allowed to develop into larvae for use in subsequent bioassays.

2.8. Adult Immersion Test of R. annulatus

L. stoechas essential oil was evaluated for adulticidal activities against adult female R. annulatus ticks taken from naturally infected cattle. This assay was performed following the method of Drummond et al. [46]. This test was performed over five replicates (ten ticks/replicate) for each concentration. The ticks of the control group were treated with ethanol 70% and deltamethrin 5%. Briefly, the ticks were immersed in 10 mL of each concentration in a Petri dish with a diameter of 7 cm at room temperature with occasional gentle agitation. After 2 min, the solution was discarded, and the female ticks were removed and gently dried on a paper towel. For ovipositioning, the treated ticks were maintained in a BOD incubator at a temperature of 27 ± 2 °C and a relative humidity of 80 ± 10%. On day 14 post-application (PA), the eggs deposited by the treated ticks were collected and weighed. After 14 days, the number of dead adult ticks was determined, and the egg production index (EPI) was calculated for the ticks that were still alive [47,48]. Egg production index (EPI) = weight of egg mass/initial weigh of engorged female × 100.

2.9. Larvicidal Activity against R. annulatus

The larvicidal activity of L. stoechas essential oil was assessed via application against the larvae of R. annulatus ticks using the larval packet technique (LPT) with the modifications suggested by Matos et al. [49]. In brief, about one hundred ten-day-old larvae were placed on the center of 7 × 7 cm filter paper, which was impregnated with 100 μL of each concentration and then folded into a pocket shape. After 24 h, the packets were checked to assess the mortality rates—motionless larvae were considered dead. The filter paper of the control groups was impregnated with ethanol (70%) and deltamethrin 5%. This test was conducted three times for each concentration.

2.10. Repellent Activity against R. annulatus

This bioassay was based on the vertical migration behavior of tick larvae and was modeled after that elucidated by Wanzala et al. [50] with some modifications. In this assay, we used a device consisting of two aluminum rods (0.7 × 15 cm) and filter paper (7 × 7 cm) impregnated with 200 μL (covering approximately 28 cm²) of the different concentrations. The treated filter paper was clipped to one rod, while on the other rode, filter paper impregnated with 70% ethanol was clipped, and this acted as a negative control. Nearly 30 ten-day-old R. annulatus larvae were placed at the base of each rod; the rods were observed after 15 min and after 1 h, and then followed up 4 h post-application. Larvae that were found on the tops of the impregnated filter paper were not considered repelled, while those at the base of the impregnated filter paper (the uncovered part of the rod) were considered repelled. This test was performed five times for each concentration.

$$\mathrm{The} \mathrm{repellence} (\%)=\frac{\mathrm{NC} – \mathrm{NT}}{\mathrm{NC} }\times 100$$

NC = number of larvae on the negative control; NT = number of larvae on the treated paper

2.11. Insecticidal Activity against Musca domestica

Rearing of Housefly Colony

Adult house flies were collected from a farm in Beni-Suef province, Egypt. The collected house flies were taken to the Parasitology Lab at the Faculty of Veterinary Medicine, Beni-Suef University. The flies were kept in plastic jars (35 × 15 cm) at 28 ± 2 °C and 60–70% relative humidity (RH), covered with muslin cloth. A cotton swab soaked in milk (10% w/v) was introduced as food to the adult flies, and this also served as a substratum for oviposition. For hatching and larval development, the eggs were transferred to a different set of jars containing animal feed or cotton swabs soaked in milk. Similarly, pupae were collected and maintained in a separate container until they emerged as adults. Larvae and pupae were used in the bioassays, as recommended by Jesikha [51] and Abdel-Baki et al. [52].

2.12. Larvicidal Bioassay against Musca domestica

The residual film method, as set out by Busvine [53] and modified by Palacios et al. [54], was used to evaluate the larvicidal activity of L. stoechas essential oil. Briefly, 1 mL of each test solution was applied to filter paper discs placed in Petri dishes (90 mm diameter) in such a way as to generate a homogenous film. The treated Petri dishes were first air-dried for a short time to let the solvent evaporate, then the larvae (n = 10) were released, and finally the Petri dishes were kept under observation in the laboratory for 24 h. The positive control group was treated with deltamethrin at a concentration of 2 L/mL, while the negative control group was treated with acetone. Three replicates of each test were performed.

$$\mathrm{Percentage} \mathrm{mortality}=\frac{\mathrm{Number} \mathrm{of} \mathrm{dead} \mathrm{larvae}}{\mathrm{Number} \mathrm{of} \mathrm{larvae} \mathrm{introduced}}\times 100$$

2.13. Pupicidal Bioassay against Musca domestica

In this bioassay, ten 2- to 3-day-old pupae were placed in a glass Petri dish. They then received a single application of 10 µL of each test solution [55]. Acetone was given to the negative control group, while the positive control group received deltamethrin at a concentration of 2 µL/mL. The Petri dishes were put in an incubator set to 28 ± 2 °C and 75-85% relative humidity. The treated pupae were monitored for six days to evaluate the emergence of adults. Three replicates of each test were performed. The adult emergence rate was evaluated following the method of Kumar et al. [56,57] and the percentage of inhibition rate (PIR) was calculated using the following equation:

$$\mathrm{PIR}=\frac{\mathrm{Number} \mathrm{of} \mathrm{newly} \mathrm{emerged} \mathrm{insect} \mathrm{in} \mathrm{control}-\mathrm{Number} \mathrm{of} \mathrm{newly} \mathrm{emerged} \mathrm{insect} \mathrm{in} \mathrm{the} \mathrm{treated}}{\mathrm{Number} \mathrm{of} \mathrm{newly} \mathrm{emerged} \mathrm{insect} \mathrm{in} \mathrm{control}}\times 100$$

2.14. Statistics

For each treatment, three to five replicates were carried out and mean ± SE values were calculated. ANOVA was used to analyze larval mortality, followed by Duncan’s multiple range test (p < 0.05). To determine the LC50 and LC90 values, as well as their 95% confidence limits, probit analyses were used [58]. SPSS for Windows (version 22.0) was used to conduct all statistical analyses.

3. Results

3.1. Chemical Composition of the Essential Oil

Hydrodistillation yielded a pale-yellow essential oil. The yield was 1.9% (v/w). Thirty-six compounds were identified, accounting for 95.56% of the EO’s total volatile fraction. The GC–MS analyses of the L. stoechas essential oil showed that the predominant compound was camphor (58.38%), followed by fenchone (18.15%) and eucalyptol (6.93%). The fraction of oxygenated monoterpenes constituted almost 87% of the oil, while hydrocarbon monoterpenes and sesquiterpenes constituted about 4.8% and 3.4%, respectively (

Table 1. Chemical composition of the essential oil of Lavandula stoechas.

Peak No	RT (min)	R.I.ex	R.I.lt	COMPOUND	%
1	8.33	930	939	α-Pinene	0.55	MH
2	9.14	945	954	Camphene	2.04	MH
3	9.36	960	960	Thuja-2,4(10)-diene	0.68	MH
4	10.38	987	990	β-Myrcene	0.22	MH
5	11.04	998	1130	2,6-Dimethyl-1,3,5,7-octatetraene Unknown &	0.53	other
6	11.80	1021	1024	p-cymene	0.19	MH
7	11.99	1024	1029	Limonene	1.13	MH
8	12.09	1029	1031	Eucalyptol	6.93	OM
9	13.14	1057	1059	γ-Terpinene	Trace *	MH
11	13.68	1069	1072	cis-Linalool oxide (furanoid)	Trace *	OM
12	14.3	1083	1086	trans-Linalool oxide (furanoid)	Trace *	OM
13	14.69	1088	1086	Fenchone	18.15	OM
14	15.99	1122	1116 (endo) or 1121 (exo)	Fenchol **	Trace *	OM
15	16.29	1129	1126	α-Campholene aldehyde	Trace *	OM
16	17.40	1151	1146	Camphor	58.38	OM
17	18.46	1175	1169	Borneol	0.12	OM
18	18.69	1181	1177	Terpinen-4-ol	Trace *	OM
19	19.38	1195	1188	α-Terpineol	0.13	OM
21	19.53	1197	1195	Myrtenal	0.45	OM
22	20.10	1210	1205	Verbenone	0.33	OM
23		1214	1220	Fenchyl acetate **	Trace *	Mester
24	21.71	1248	1243	Carvone	0.17	OM
25	23.13	1279	1288	Bornyl acetate	1.40	Mester
26	24.81	1318	1326	Myrtenyl acetate	0.40	Mester
27	25.66	1338	1348	α-Cubebene	Trace *	HS
28	26.79	1363	1376	Isoledene?	0.23	HS
29	27.00	1368	1376	α-Copaene	0.10	HS
30	27.23	1373	1544	Isolongifolene-4,5,9,10-dehydro?? Unknown &	0.37	HS
31	31.07	1465	1476	Cadina-1(6),4-diene, trans	0.21	HS
32	31.84	1484	1496	Viridiflorene (syn. Ledene)	0.93	HS
33	32.15	1490	1500	α-Muurolene	0.41	HS
34	32.35	1496	1517	α-dehydro Himachalene?	Trace *	HS
35	32.92	1509	1523	δ-Cadinene	0.74	HS
36	33.19	1516	1529	cis-Calamenene	0.21	HS
37	33.59	1527	1534	trans-Cadina-1,4-diene	0.24	HS
38	34.04	1538	1545	α-Calacorene	0.35	HS
39	36.99	1615	1623	α-Corocalene	0.10	HS
40	39.21	1673	1676	Cadalene	0.10	HS
				Traces *	0.36
				Total (without traces = 0.36%)	95.79

RT: Retention time. R.I._ex: Experimental retention index calculated on Rt-5MS column. R.I._lt: Retention index from the literature for relative columns. * Components representing less than 0.1%. ** Correct isomer not identified. Unknown ^&: main m/z is given (% relative intensity). Comp No 5: 91 (100), 119 (58), 77 (40), 134 (32). Comp No 30: 143 (100), 157 (80), 200 (55), 128 (40), 185 (34). MH: Monoterpene hydrocarbon. OM: Oxygenated monoterpene. HS: Hydrocarbon sesquiterpene.

and Figure 1).

3.2. Cytotoxicity Assessment

Cell viability was affected by the highest concentrations of lavender, whereby the concentrations of 0.25, 0.5, and 1.0% showed toxicity against 80% of HEK-293 (Figure 2). Additionally, this compound was shown to be safe for normal cells up to a concentration of 0.0625%, at which point a strong cytotoxic effect appeared at around 0.125%. Lavender oil showed clear cytotoxic effects on the MCF-7 cell line, even at a low concentration of 0.0625%, causing cell death at a rate of 30%, while high concentrations caused significant cell death (Figure 3).

3.3. In Vitro Scolicidal Activity

Table 2. Scolicidal effect of Lavandula stoechas essential oil on the viability of E. granulosus protoscolices.

Concentrations (%)	Mortality Rates after Exposure (%) (Mean ± SE)
	1 min	3 min	5 min
0.025%	33.66 ± 1.52 c	50.4 ± 1.24 c	88.07 ± 1.33 b
0.05 %	41.07 ± 1.32 b	72.47 ± 1.76 b	98.17 ± 1.04 a
0.1 %	74.7 ± 1.99 a	95.5 ± 1.19 a	100.00 ± 0.00 a
Control	3.03 ± 0.29 d	4.1 ± 0.32 d	5.2 ± 0.23 c

Means within the same column followed by different superscripts are significantly different (Duncan’s multiple range test: p ≤ 0.05). LC = lethal concentration, CL = confidence limit, X² = chi square, df = degree of freedom.

displays the scolicidal effects of the L. stoechas essential oil at various concentrations and with different exposure times. At a concentration of 0.025%, the scolicidal effectivity values of L. stoechas oil were 33.66, 50.4, and 88.07% after 1, 3, and 5 min, respectively. The values at a concentration of 0.05% were 41.07, 72.47, and 98.17% after 1, 3, and 5 min respectively. After 3 and 5 min of exposure, an oil concentration of 0.1% caused 95.5% and 100% mortality, respectively (Figure 4). Overall, the scolicidal activity of the oil was clearly concentration- and time-dependent.

3.4. Acaricidal Activity L. stoechas EO against Adult and Larvae of R. annulatus Ticks

L. stoechas EO showed significant adulticidal activity against R. annulatus ticks, especially at concentrations of 5 and 10%, showing tick mortality rates of 86.66 and 100%, respectively, and the LC₅₀ was 2.34%. Moreover, the egg production index of the treated groups showed lower values compared to those of the control, i.e., the untreated ticks (

Table 3. Adulticidal and lethal concentrations (LC₅₀, LC₉₀) of Lavandula stoechas against R. annulatus adult ticks.

Concentrations %	Mortality % M ± SE	Egg Production Index (EPI)	LC50 (95% CL)	LC90 (95% CL)	χ2 (df = 3)	p
0.625	0.00 ± 0.00 e	35.83 ± 0.60 b	2.34 (2.13–2.57)	5.00 (4.39–5.89)	3.861	0.277
1.25	16.66 ± 3.33 d	31.80 ± 1.80 c
2.5	56.67 ± 6.67 c	26.00 ± 0.58 d
5	86.66 ± 3.33 b	24.33 ± 0.67 d
10	100.00 ± 0.00 a	0.00 ± 0.00 e
Deltamethrin 2 uL/mL	26.67 ± 3.33 c	23.33 ± 0.88 d	-	-	-	-
Ethyl alcohol 70%	0.00 ± 0.00 e	43.00 ± 1.00 a	-	-	-	-

Means within the same column followed by different superscripts are significantly different (Duncan’s multiple range test: p ≤ 0.05). LC = lethal concentration, CL = confidence limit, X² = chi square, df = degree of freedom.

).

Regarding larval toxicity, L. stoechas oil achieved a larvicidal activity of 86.7% at the highest concentration (10%), with an LC₅₀ of 9.11% (

Table 4. Larvicidal activity and lethal concentrations (LC₅₀, LC₉₀₎ of Lavandula stoechas, against larvae of R. annulatus.

Concentrations %	Mortality % M ± SE	LC50 (95% CL)	LC90 (95% CL)	χ2 (df = 3)	p
0.625	6.67 ± 1.67 f	3.82 (3.32–4.45)	15.53 (11.94–22.07)	5.43	0.143
1.25	15.00 ± 2.89 e
2.5	35.00 ± 2.88 c
5	51.66 ± 1.67 b
10	86.67 ± 1.66 a
Deltamethrin 2 uL/mL	22.66 ± 2.66 d	-	-	-	-
Ethyl alcohol 70%	5.33 ± 1.66 f	-	-	-	-

Means within the same column followed by different superscripts are significantly different (Duncan’s multiple range test: p ≤ 0.05). LC = lethal concentration, CL = confidence limit, X² = chi square, df=degree of freedom.

).

Moreover, regarding the repellence activity of L. stoechas, we found a weak repellency in the first hour, even at the highest concentration of 10%, and no repellent effect was seen in the succeeding hours (S Table 1). There were no significant differences in results between the low concentrations and the control, treated with 70% ethyl alcohol.

3.5. Insecticidal Effect of L. stoechas against Larvae and Pupae of Musca domestica

The larval toxicity of L. stoechas oil, assessed via the residual film method, increased significantly with increasing concentrations. Within 24 h after application, the lavender-treated groups showed significant mortality at 5 and 10% concentrations, with rates of 93.33 and 100%, respectively, and an LC₅₀ value of 1.79%. The treated larvae died within 24 h, with clear blackening of the cuticles. Moreover, in the deltamethrin-treated and negative control groups, no larvicidal activities were observed whatsoever (

Table 5. Larvicidal activity, and lethal concentrations (LC₅₀, LC₉₀) of Lavandula stoechas, against larvae of Musca domestics.

Concentrations %	Mortality % (Mean ± SE)	LC50 (95% CL)	LC90 (95% CL)	χ2 (df = 3)	p
0.625	6.67 ± 3.33 d	1.79 (1.62–1.98)	4.29 (3.72–5.13)	0.845	0.839
1.25	30.00 ± 5.77 c
2.5	66.66 ± 3.33 b
5	93.33 ± 3.33 a
10	100.00 ± 0.00 a
Deltamethrin 2 uL/mL	0.00 ± 0.00 d	-	-	-	-
Ethyl alcohol 70%	0.00 ± 0.00 d	-	-	-	-

Means within the same column followed by different superscripts are significantly different (Duncan’s multiple range test: p ≤ 0.05). LC = lethal concentration, CL = confidence limit, X² = chi square, df = degree of freedom.

).

Regarding pupal toxicity, L. stoechas oil achieved a percentage inhibition rate (PIR), ranging from 13.33% to 100% at various concentrations after six days of application with an LC₅₀ value of 1.51%. Moreover, a concentration of 10% L. steochas essential oil led to the complete inhibition of adult emergence, and the dead pupae displayed darker colors (

Table 6. Percentage inhibition rate (PIR) of Lavandula stoechas against housefly pupae in contact toxicity assay.

Concentrations %	PIR % (Mean ± SE)	LC50 (95% CL)	LC90 (95% CL)	χ2 (df = 3)	p
0.625	13.33 ± 3.33 d	1.51 (1.35–1.68)	3.94 (3.38–4.78)	1.667	0.644
1.25	36.66 ± 6.67 c
2.5	76.67 ± 3.33 b
5	93.33 ± 6.66 a
10	100.00 ± 0.00 a
Deltamethrin 2 uL/mL	0.00 ± 0.00 e	-	-	-	-
Ethyl alcohol 70%	0.00 ± 0.00 e	-	-	-	-

Means within the same column followed by different superscripts are significantly different (Duncan’s multiple range test: p ≤ 0.05). LC = lethal concentration, CL = confidence limit, X² = chi square, df = degree of freedom.

).

4. Discussion

Synthetic chemicals have been widely used to control parasitic infections, but their indiscriminate and excessive usage has resulted in drug resistance, as well as detrimental effects to the environment and food supply [25,26,57]. Plant essential oils (and/or active components) can be used as natural alternatives or adjuncts to current therapies used against a variety of ectoparasites and endoparasites of medical/veterinary significance [52,58,59,60,61]. The use of essential oils as therapeutic agents is more affordable, effective, and safe [14,62]. As such, the present work was designed to investigate the in vitro acaricidal, insecticidal, and scolicidal activities of L. steochas essential oil against certain cell lines, as well as its safety.

Our GC–MS analyses revealed camphor and fenchone as the main components of L. steochas essential oil, accounting for 58.38% and 18.15%, respectively, followed by 6.93% eucalyptol and 2.04% camphene. These outcomes agree with those of several previous studies, which showed that the predominant components of L. steochas oil are camphor and fenchone [63,64,65,66,67]. The timing of plant collection, the duration of hydrodistillation, the selection of plant parts to be used, and environmental factors were all found to have a significant impact on the essential oil’s yield and composition [68].

The in vitro cytotoxic activity of L. steochas EO against human embryonic kidney cells (HEK-293 cells) and human breast cancer cell line (MCF-7) MCF-7 cells was assessed in the present investigation. The results demonstrate that L. steochas oil was extremely cytotoxic to HEK-293 and MCF-7 cells, even at low concentrations. Our results corroborate those found in [69], who noted that L. stoechas flowers were cytotoxic to Allium cepa root-tip meristem cells. According to Siddiqui et al. [70], the cytotoxic effect of L. stoechas EO, perhaps resulting from apoptosis, apparently induces deformations in the nuclei and cell membrane. The ethanolic fraction of L. stoechas has an anticancer effect, which may be attributed to the presence of phytosterols [70]. Furthermore, it strongly inhibits the growth of human gastric adenocarcinoma (AGS), melanoma MV3, and breast cancer MDA-MB-231 cells, with median inhibitory concentrations (IC50) of 0.035 ± 0.018, 0.06 ± 0.022, and 0.259 ± 0.089 µL/mL, respectively [71].

We assessed the in vitro scolicidal efficacy of L. steochas EO at a variety of concentrations and exposure times on the protoscoleces of hydatidosis. The findings show that all tested concentrations of L. steochas EO had significant scolicidal activities, with 98.17% and 100% mortality achieved at doses of 0.05% and 0.1%, respectively, at 5 min post-treatment. The results of the current investigation suggest that L. steochas is a potential natural source of novel protoscolicidal agents that could be used in hydatid cyst surgery.

The current study revealed that L. steochas EO induced 100% mortality in R. annulatus adult ticks at concentration of 10%, with an LC₅₀ value of 2.34%. Regarding the larvicidal activity, 86.67% larval death was achieved at a concentration of 10% with an LC₅₀ of 9.11%. Similarly, L. stoechas EO showed significant acaricidal activities against adults and larvae of the Hyalomma suspense tick [10]. Other species of lavender have acaricidal activities. Lavandula angustifolia is effective against Rhipicephalus (Boophilus) annulatus) [11], and Lavandula luisieri has larvicidal effects against Hyalomma lusitanicum [12]. Additionally, Sertkaya et al. [13] found that L. steochas EO had an acaricidal effect against the red spider mite Tetranychus cinnabarinus. It was surprising, however, to see that L. steochas essential oil has no repellent effect against larvae of R. annulatus. Other studies did identify a repellent effect of L. steochas and other types of lavender. Lavandula angustifolia shows repellency against Hyalomma marginatum adults [72] and nymphs of Ixodes ricinus (L.) (Acari: Ixodidae) [73]. This discrepancy may be due to the tick species and tick stages used in our tests.

In terms of the insecticidal activity against M. domestica, our results indicate that L. steochas EO exhibits maximum efficacy (100%) against house fly larvae at a concentration of 10% with an LC₅₀ of 1.79% at 24 h post-application. Additionally, a 10% concentration of L. steochas EO showed the highest level of toxicity against house fly pupae, and completely inhibited adult emergence (100% PIR). Essential oils from the genus Lavandula have also shown insecticidal efficacy against several insect species. The essential oil of L. stoechas, a member of this genus, showed significant toxic effects against the adults and/or larvae of Anopheles labranchiae, Culex pipiens molestus, and Orgyia trigotephras [33,67,74]. The essential oils of L. dentate and L. angustifolia, also belonging to the same genus, exhibited significant insecticidal actions against larvae of M. domestica and Chrysoma albiceps and have thus been suggested for use as a safe and effective natural means to control these dipterans [75,76,77,78,79,80]. Bosly [79] observed morphological abnormalities in M. domestica larvae after treatment with Lavandula spp. EOs and attributed these deformities to hormonal imbalances that interrupt insect metamorphosis. Meanwhile, Khater and Khater [81] found that the oil may limit larval motility and prevent the larvae from constricting during the pupal stage, thus contributing to the observed deformities. According to Conti et al. [31] and Sajfrtova et al. [82], the concentration of volatile compounds in the oil is directly related to the toxic effects found. It is difficult to compare the efficacy of essential oils across different studies because the methods used for oil extraction vary greatly, and this impacts the subsequent essential oils’ efficacy [79].

Generally, the presence of volatile components in essential oils is largely responsible for the oils’ acaricidal, insecticidal, and cytotoxic actions [80,81,82]. For instance, the main component of L. stoechas EO, camphor, has been proven to have insecticidal effects [83,84] and creates a fragrant vapor that repels mosquitoes [66,67]. Another key ingredient in L. stoechas EO, camphene, also has insecticidal activities resulting from its ability to repel insects, including flies and moths [63,64,83,84]. All of this can thus explain L. stoechas’ effectiveness as an acaricidal, insecticidal, scolicidal, and anticancer agent.

In conclusion, L. stoechas EO has potential applicability as a potent protoscolicidal agent with high effectiveness at low doses and in shorter times. However, more research is required to fully assess the potential use of this oil in the prevention and treatment of cystic echinococcosis, including via in vivo assays and its main components. Additionally, this oil shows significant activity against M. domestica and R. annulatus ticks, which pose a substantial risk to public health; camphor is the first known substance to exhibit this activity. The mechanisms of action of these essential oils are poorly understood. One of the theories is that the monoterpenes operate on other sensitive locations, such as the neurological system, but further research is required to verify and expand on this.