Evaluation of Biological Activity of Essential Oil from Teucrium polium L.

Algeria boasts a rich diversity of medicinal plants thriving in the fertile Sahel regions as well as the arid Highlands and desert zones. The focus of this study is the tomentose germander (Teucrium polium L.), a species belonging to the Lamiaceae family, collected from the Oued Melegue Daïra Ouenza region of Tebessa, in eastern Algeria.

Extraction of essential oils (E.O)
 
Hydro distillation is a method that facilitates the extraction of essential oils (EO) from fresh or dried plant material. The process involves placing 100 g of the dried plant mass into a large glass flask and adding an adequate amount of distilled water, leaving sufficient headspace to prevent overflow during boiling. The flask is then heated using a heating mantle, bringing the mixture to a boil.

As essential oils have a lower density compared to water, they float on the surface of the aqueous mixture. The essential oil layer is carefully collected and stored in well-sealed, opaque bottles at low temperature (4-5°C) to ensure preservation.
 
Microbial activity
 
Determination of antibacterial activity by the disc method
 
The sensitivity of bacterial strains to the essential oil (EO) was evaluated using the disk diffusion method. Bacterial suspensions were prepared in physiological saline from young colonies (18-24 hours old) by adjusting the turbidity to 0.5 McFarland standard. These suspended bacteria were then swabbed onto the dry surface of Mueller-Hinton agar plates using sterile swabs. Sterile Whatman No. 3 paper discs (6 mm in diameter) were placed on the inoculated agar and impregnated with 10 μL of the essential oil (Esmaeil, 2010; Darshana, 2016).

The plates were incubated at 37°C for 24 hours. The bacterial strain was considered non-sensitive to the natural substance if the inhibition zone diameter was less than 8 mm, moderately sensitive between 8 and 14 mm, sensitive between 14 and 20 mm and highly sensitive if the diameter exceeded 20 mm (Ameur et al., 2009; Dalila, 2019).
 
MICs determination of essential oils
 
A dilution series ranging from 2.5 to 20 μL/mL was prepared by dissolving the essential oil in dimethyl sulfoxide (DMSO). For each dilution, 2 mL was mixed with 18 mL of Mueller-Hinton agar cooled to 45°C. The mixtures were then poured into 90mm diameter Petri dishes and allowed to solidify at room temperature for 24 hours (Kempf, 2011).

Bacterial suspensions were prepared in physiological saline from young 18-24-hour colonies, with the turbidity adjusted to 0.5 McFarland standard. A 2-μL aliquot of each suspension was spot-inoculated onto the surface of the essential oil-incorporated agar plates and allowed to dry. The inoculated plates were subsequently incubated at 37°C for 24 hours (Benslimani, 2011). The minimum concentration preventing visible bacterial growth was recorded as the MIC.
 
In vitro anti oxydant activity
 
Capacity of trapping of the free radical “2,2-diphenyl-1- picrylhydrazyl” (DPPH)
 
The free radical scavenging capacity of the essential oil was evaluated against 2,2-diphenyl-1-picrylhydrazyl (DPPH) according to the method described by (Boulila, 2015).1 mL of the essential oil at various concentrations was mixed with 2 mL of a methanolic DPPH solution (0.04 g/L). After incubating in the dark for 60 minutes, the absorbance was measured at 517 nm using a JENWAY 6300 spectrophotometer, with methanol used as the blank.

The percentages of inhibition of the DPPH were calculated according to the formula:
 

 Aeq represents the absorbance of the methanolic DPPH solution after the addition of the antioxidant sample, once equilibrium is reached. A0 denotes the absorbance of the DPPH solution with only methanol added in the same proportions. A curve plotting the percentage of DPPH inhibition against the antioxidant concentration in micrograms per milliliter (μg/mL) enabled the determination of the median inhibitory concentration (IC50), defined as the antioxidant concentration required to reduce the initial DPPH concentration by 50%. Ascorbic acid was used as a positive control for comparison (Dridi, 2016).
 
Reduction of ferric ion
 
The reducing power was evaluated following the method described by (Oyaizu, 1986). 1 mL of essential oil concentrations (0.1, 0.2, 0.3, 0.4 and 0.5 mg/mL in methanol) was mixed with 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide solution. The mixtures were incubated at 50°C for 30 minutes. Subsequently, 2.5 mL of 10% trichloroacetic acid was added to the solutions, which were then centrifuged at 3000 rpm for 10 minutes.


After centrifugation, 2.5 mL of the supernatant from each concentration was combined with 2.5 mL of distilled water and 0.5 mL of 0.1% ferric chloride solution. The absorbance was measured at 700 nm using a JENWAY 6300 spectrophotometer. Ascorbic acid was used as a positive control, following the same experimental conditions.

The result is expressed as the effective concentration EC50, defined as the concentration of essential oil in micrograms per milliliter of the mixture (EC50 in μg/mL) required to achieve a given reducing capacity.

 

According to both the theoretical and practical results obtained in our study, the antioxidant substance demonstrated a variable antiradical activity, but it was comparatively weaker than ascorbic acid.

 

 

The authors would like to thank the entire team of the analytical chemistry laboratory at the Department of Pharmacy, Badji Mokhtar University, Annaba.

Informed consent
 
All participants provided written informed consent after being fully informed about the study.

The authors declare that there is no conflict of interest. The authors alone are responsible for the accuracy and integrity of the paper’s content.