Composition, Anticholinesterase and Antipedicular Activities of Satureja capitata L. Volatile Oil

2.1 Chemicals and reagents

Acetylthiocholine iodide and butyrylthiocholine iodide were purchased from Sigma Aldrich (Germany), while Ellman’s reagent was obtained from Sigma Aldrich (UK). Galantamine hydrobromide was obtained from Sigma Aldrich (USA) and benzyl benzoate was purchased from Sigma Aldrich (France).

2.2 Equipment

GC-MS (Shimadzu QP-5000 GC-MS, Japan), spectrophotometer UV-visible (Jenway 7315,UK), binocular stereo dissecting microscope (AmScope, SKU: SE306-A-E, USA), filter paper (Whatman no.1, USA), microwave extractor (CW-2000, China), balance (Radw, Poland) and grinder (Moulinex-Uno., China) were the instruments used in the current investigation.

2.3 Collection and preparing of plant materials

The leaves of S. capitata were collected during the flowering season in May 2017, from the Bethlehem region of Palestine. The taxonomical classification was conducted in the Pharmacognosy Laboratory at An-Najah National University and kept under the voucher specimen code: Pharm-PCT-690.

The collected leaves were separated carefully, washed with distilled water and dried for three weeks in the shade at room temperature. The obtained dried leaves then were powdered using a grinder and stored in a special jar for further use [17].

2.4 Isolation of S. capitata VO

The VO of S. capitata was isolated using the Microwave method as described by Jaradat et al. (2019) with minor modifications. Within the isolation process, the powder suspension was exposed to micro- and ultrasonic- waves to improve the extraction process. A one liter round-bottom flask containing 100 g of the dried leaf powder was placed in the Microwave extractor aparatus. In this flask, the powder was suspended in 500 ml distilled water. During the isolation process, the power of the microwave extractor apparatus was adjusted to 1000 W. The isolation process was conducted for 15 min at 100°C and was repeated three times for the same plant sample. The obtained VO was collected into a clean beaker, chemically dried using CaCl₂ and finally stored at 2-8°C. The obtained S. capitata VO yield was 1.06% v/w from the plant sample [18].

2.5 GC-MS study

GC-MS chromatograms were recorded using Shimadzu QP-5000 GC-MS (Japan). The GC was equipped with a Rtx-5 ms column (30 m long, 0.25 μm thickness and 0.250 mm inner diameter). Helium was used as a carrier gas at a flow rate of 1 ml/min. The injector temperature was 220°C. The oven temperature was programmed from 50°C (1 min hold) at 5°C/min to 130°C, then at 10^°C/min to 250°C and kept isothermally for 15 min. Transfer line temperature was 290°C. For GC-MS detection, an electron ionization system, with detector volts of 1.7 KV was used. A scan rate of 0.5 s, and scan speed of 1000 amu/sec was applied, covering a mass range from 38-450 M/Z. The chemical ingredients of the VO were identified by comparing their mass spectrometry (MS) with the reference spectra in the mass spectrometry data center of the National Institute of Standards and Technology (NIST), more than 15% of the identified compounds and by comparing their retention indices and Kovats indices in the literature [19].

2.6 Anticholinesterase assay

The inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) by the VO of S. capitata was examined using a spectrophotometric assay and Ellman’s method [20]. Briefly, 5 μl of 0.03 U/ml acetylcholinesterase was added to 0.01 U/ml butyrylcholinesterase, 5 μl of Ellman’s reagent (5,5’-dithio-bis-[2-nitrobenzoic acid]) (DTNB) and 205 μl of the VO at different concentrations (62.5, 125, 250, 500, 1000 μg/ml). The mixtures were incubated in a water bath at 30°C for 15 min. After that, 5 μl of butyrylthiocholine iodide and acetylthiocholine iodide substrates were added to these mixtures to enhance the reaction. The formation of yellow color indicated the presence of the anion of 5-thio-2-nitrobenzoate, which is a reaction output of DTNB and thiocholines, and the absorbance at 412 nm was determined using a UV-visible spectrophotometer. The assay also was carried out without the presence of the VO and the enzymes to test the non-enzymatic hydrolysis of the substrates. The mixture containing all ingredients except the VO was used as the control. In addition, galantamine, which is a potent acetylcholinesterase and butyrylcholinesterase inhibitor, was used as a reference compound to compare its activity with the VO of S. capitata.

The percentages of enzyme activity and the percentages of enzyme inhibition were calculated using the following formulas:

Where:

ΔAbs is the change in absorbance

V is the rate of the reaction in the presence of the inhibitor.

Vmax is the reaction rate without the inhibitor.

2.7 Pediculus humanus capitis collection

By using a fine-tooth anti-lice metallic comb, the adult, nymphs, and nits of Pediculus humanus capitis were collected from the scalps of 6-12 year-old children from Jenin city (Palestine) and placed in plastic tubes with covers. Children that used anti-lice agents within one month were excluded. Prior approval of the parents was obtained. The study protocol and the informed consent forms were approved by the institutional review board archived number: 1992019.

2.8 Pediculicidal activity

The VO of S. capitata was tested for anti-lice activity using the diffusion method of filter papers in Petri-dishes [21]. The VO of S. capitata was diluted to 5%, 10%, and 20% concentrations using distilled water. The adults and nymphs of P. humanus capitis were identified and separated using a binocular stereo dissecting microscope.

The adult and nymph lice, in a ratio of four to two, were separated into 21 groups with 10 lice in each group and were placed on Whatman filter papers No. 1 at the bottom of 21 Petri-dishes (100 mm x 15 mm). 0.5 ml of each of the VO concentrations was added on the filter paper using pipetes to form a thin layer on an area of 4.5 cm². A group treated with 0.5 ml distilled water was used as the control. The VO group contained 0.5 ml of S. capitata VO diluted with distilled water to three different concentrations 5%, 10%, and 20%. In addition, three groups were treated with 0.5 ml of 5%, 10%, and 20% benzyl benzoate.

All Petri-dishes were placed in a dark chamber for 1 h at 25±0.5℃ and 72±2% relative humidity, then 0.5 ml of distilled water was added and the dishes were placed in the chamber under the same conditions mentioned above for 18 h. After that, the 21 dishes were observed for any possible movement of lice using a dissecting microscope; in the absence of movement, the lice were considered dead. The study was performed in triplicate [22, 23].

2.9 Evaluation of ovicidal activity

The ovicidal activity of the S. capitata VO was evaluated by placing 35 mature viable lice nits on Whatman filter papers No. 1 with a 6 cm of diameter; these were placed in seven Petri-dishes (five nits in each dish) for all groups mentioned above. Then, 0.5 ml of each of the control and treatment solutions were added. All dishes were incubated in a dark chamber at 25±0.5℃ for 14 days, with 0.1 ml of distilled water added every 48 h to maintain moisture in the chamber. Hatching of nymphs from nits was observed by microscopy, and the percentages of hatching at day 6 and day 14 were recorded. Each ovicidal test was carried out in triplicate [22, 23].

2.10 Statistical analyses

Determination of anticholinesterase, pediculicidal and ovicidal activities of S. capitata VO was carried out in triplicate for each test. The obtained results were presented as means ± standard deviation (±SD) and were compared using unpaired t-tests. The statistical significance was considered when the p value was <0.05.

3.1 GC-MS analysis of S. capitata

Sixteen components were identified using GC-MS and the major identified VOs were carvacrol, γ-terpinene, p-cymene, and thymol, with the following percentages respectively; 24.35%, 23.34%, 18.14%, and 16.1%. The hydrocarbon and alcohol groups were the only identified phytochemical classes of the VOs, as shown in Table 1.

3.2 Anticholinesterase activity

From the standard calibration curves in Figures 1 and 2, was obtained the required equations to estimate the cholinesterase inhibition activity of S. capitata VO for AChE and BChE. The results revealed that that the VO of S. capitata has AChE and BChE inhibitory activity with IC₅₀ values of 28.24±0.97 and 92.31±1.22 μg/ml, respectively compared with the positive control galantamine which has IC₅₀ values of 5.21±0.07 and 10.33±0.37 μg/ml, respectively as shown in Table 2.

Figure 1

Correlation and regression of percentage acetylcholinesterase inhibition of Satureja capitata volatile oil and galantamine.

Figure 2

Correlation and regression of percentage butyrylcholinesterase inhibition of Satureja capitata volatile oil and galantamine.

3.3 Anti-louse effects

The obtained anti-louse results conducted on 210 head lice, and the ovicidal activity in 105 nymphs treated with three different concentrations of the VO, benzyl benzoate or the control which was distilled water, are shown in Table 3.

The eradication rate of P. humanus capitis adults and nymphs by 20% S. capiata VO was 100%; the same result was achieved with the same concentration of benzyl benzoate.

In addition, the ovicidal effect of the VO was measured by the hatching rates of P. humanus capitis nymphs, which were zero after 6 and 14 days of treatment with S. capitata VO at the 20% concentration. This result was similar to that obtained with benzyl benzoate as a reference compound.

3.4 Discussion

The therapeutic potential of traditional medicinal plants has been demonstrated throughout human history. Currently, plants still constitute an important source for the isolation of pharmacologically active compounds, which are utilized widely for pharmaceutical and medical applications [24, 25, 26]. Chemically, VOs are comprised of hydrocarbon compounds with oxygenated, hydrogenated and dehydrogenated derivatives. They have specific aromatic odors and evaporate at room temperatures. VOs are frequently used for therapeutic purposes due to their antibacterial, antifungal, anthelmintic, analgesic, antipruritic and anticancer effects [27]. These natural compounds are isolated from various plant parts and tissues and they belong to several groups of phytochemical constituents such as aldehydes, esters, ethers, hydrocarbons, phenols, ketones, and oxides [28].

As revealed by the GC-MS results, 16 phytochemical compounds were identified in S. capitata VO and the major constituents were carvacrol, γ-terpinene, p-cymene, and thymol. Carvacrol accounted for the highest percentage, which is considered a chemotype molecule for this plant species [29, 30, 31, 32].

Galantamine (Galanthamine) is a potent cholinesterase enzyme inhibitor that has been used in many countries for the treatment of the symptomatic senile dementia of the Alzheimer’s type and also has been utilized after general anesthesia to reverse the neuromuscular paralysis induced by tubocurarine-like muscle relaxants. More recently, it has been shown to attenuate drug- and lesion-induced cognitive deficits in animals [33]. Galantamine directly inhibits acetylcholinesterase activity, with weaker activity on butyrylcholinesterase [34].

In this study, in-vitro anticholinesterase activity was examined using spectrophotometric Ellman’s assay and the results showed that the VO of S. capitata has potential acetylcholinesterase inhibition effect. Whereas, the butyrylcholinesterase inhibitory effect was weak comparing with the positive control galantamine.

The inadequate and repetitive usage of anti-louse pesticides such as malathion, DDT (dichlorodiphenyltrichloroethane), carbaryl, d-phenothrin, and permethrin, which are commonly used as topical chemical drugs for the treatment of head lice infestation, has resulted in the emergence of resistance of head lice to one or more of these drugs [35]. In addition, many of these compounds such as DDT have been banned due to their long-term toxic effects [36]. Consequently, there is a need to find alternatives to these chemical drugs. Natural VOs are promising candidates because they are ecosystem friendly, easily hydrolyzed and effective against a wide range of insects [37, 38, 39]. In addition, several previous studies have demonstrated the anti-louse effects of these compounds [40, 41, 42]. However, neurotoxicity is the main sign observed in insects treated by VOs, similar effects to those seen following treatment with the chemical insecticidal agents such as carbamates and organophosphates.

In the current study, the VO of S. capitata was tested against the adults, nymphs, and nits of P. humanus capitis. The results revealed that the VO at 20% was effective against the adults, nymphs, and nits of head lice, to the same level as benzyl benzoate with a mortality rate of 100%. The VO also completely inhibited emergence and hatching of nymph by 100% following 6 and 14 days of exposure. Similar results were obtained with 20% concentration of benzoyl benzoate. The obtained results of pediculicidal and ovicidal tests confirmed that the VO of S. capitata has potential activity against lice infestation. Further in-vivo studies are required to confirm these findings, and to find the suitable pharmaceutical formulation to manufacture new drugs from the VO of S. capitata plant.

3.5 Conclusion

The VO of S. capitata can be used as a promising natural alternative to synthetic anticholinesterase drugs, which can be employed in drug development and may lead to new candidates in the field of the treatment of neurodegenerative diseases. In addition, the achieved results confirmed that this VO has potential activity against lice infestations. However, additional clinical studies are required to support these results and to find suitable pharmaceutical dosage and formulations.