Biological Properties of Myrtus communis Essential Oil – Antioxidant,Antimicrobial and Cytotoxic Potential

Abstract

Objectives

Myrtus communis L. (Myrtaceae) is a Mediterranean aromatic plant with numerous beneficial properties. This study aimed to evaluate and investigate its antioxidant and antibacterial activities, as well as its effects on primary immune cells, astrocytes, and C6 glioma cells.

Methods

After GC-MS chemical quantification of leaf essential oil composition, HPTLC-DPPH, HPTLC-direct bioautography, and three assays that estimate metabolic activity and cell membrane function were employed to estimate its pharmacological activities.

Results

Oil analysis revealed 26 compounds, with α-pinene (41.61%) and 1,8-cineole (38.79%) being dominant constituents. Strong antioxidant activity can be primarily attributed to 1,8-cineole (80.5 ± 4.8 µg gallic acid equivalents/µL). Antibacterial evaluation showed higher susceptibility of Staphylococcus aureus (529.4 ± 41.3 µg streptomycin equivalents/µL) than Klebsiella pneumoniae (279.8 ± 13.1 µg/µL). M. communis essential oil exhibited pronounced cytotoxicity toward immune cells (peritoneal macrophages, thymocytes, and splenocytes) and primary astrocytes at the highest tested concentration (100 μg/ml), while it had no impact on C6 glioma cells. At lower concentrations, the oil exerted minimal or no cytotoxic effects, indicating a concentration-dependent and cell-type-specific response.

Conclusion

These data provide comprehensive evidence linking the chemical composition of the essential oil to its bioactivity and potential pharmacological applications, highlighting the considerable antioxidant and antibacterial activities, while emphasising the need for careful application due to its toxic potential.

Introduction

Myrtus communis L. (synonyms: Myrtus augustinii Sennen & Malag., Myrtus communis var. communis L., Myrtus communis subsp. mucronata Pers., Myrtus communis var. italica ((Mill.) Rouy & E.G.Camus, and Myrtus communis var. lusitanica Rouy) known as common myrtle or true myrtle, belongs to the Myrtaceae family. ¹ It is an evergreen, aromatic shrub to medium-sized tree native to the Mediterranean Basin and Western Asia, and introduced into Southern Africa (Cape region), Northern America (Texas, Louisiana, and California, USA), and Southern America (eg, the Caribbean, Cuba, and Puerto Rico). Common myrtle has traditionally been used for the treatment of inflammation and wounds, as well as various digestive complaints, and for genitourinary, pulmonary, and skin diseases.^2–5 It acts as a natural protective agent against different toxins to treat and prevent various toxicological disorders. A recent review ⁶ highlighted that its most significant protective properties include prominent antioxidant and hepatoprotective potential, as well as a promising ability to inhibit the growth of tumour cells.

The leaves and edible fruits contain a fragrant essential oil that is rich in bioactive compounds with diverse therapeutic potential. ² Considerable antimicrobial and antioxidant properties and moderate anticancer activity of its essential oil are, at least partly, responsible for most of the biological activities of common myrtle. ⁷ Although the myrtle essential oil is a complex mixture of compounds that varies depending on the geographical origin, time of harvest, and distillation parameters, it typically contains the following key constituents: α-pinene, 1,8-cineole (eucalyptol), myrtenyl acetate, limonene, and linalool.^2,7,8 The oil inhibits bacterial growth and biofilm formation of both Gram-positive and Gram-negative bacteria, and affects the survival and replication of fungi and viruses.^2,8 Both Klebsiella pneumoniae, an opportunistic gram-negative pathogen, and Staphylococcus aureus, the most common gram-positive bacterial pathogen in humans, are capable of forming biofilms, which contribute to antibiotic resistance and persistence of infection. ⁹ Klebsiella pneumoniae is a leading cause of healthcare-associated infections, including pneumonia, bloodstream infections, and urinary tract infections, with carbapenem-resistant strains posing a major global health threat due to limited therapeutic options. ¹⁰ Staphylococcus aureus, particularly methicillin-resistant S. aureus (MRSA), represents a serious challenge in both hospital and community settings, contributing to increased morbidity, mortality, and healthcare costs worldwide. ¹¹ There is an ever-growing pull of bacterial strains that develop resistance to the standard therapy and there is a constant need for the discovery of new drugs to combat such pathogens.

The current study aims to evaluate the effects of common myrtle leaf essential oil antimicrobial (towards K. pneumoniae and S. aureus) and antioxidant activity, alongside its impact on peritoneal macrophages, spleen and thymus lymphocytes, normal and C6 astrocytes viability, and thus to connect its potential beneficial properties and toxic effects.

Materials and Methods

Drugs and Chemicals

Sodium dihydrogen phosphate, sodium hydroxide, and sodium chloride were provided by Merck (Darmstadt, Germany). Toluene and ethyl acetate were purchased from Zorka Pharma (Šabac, Serbia). Methanol, sulfuric acid, and acetic acid were provided by Centrohem (Stara Pazova, Serbia). Dulbecco's Modified Eagle Medium (DMEM), sodium pyruvate, trypsin, EDTA, Roswell Park Memorial Institute (RPMI) 1640, Poly-L-lisine (PLL), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Crystal violet, NaCl, KCl, Na₂HPO₄, KH₂PO₄, NaHCO₃, p-Anisaldehyde, streptomycin, gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals and dimethyl sulfoxide (DMSO) were obtained from Sigma Aldrich Chemie GmbH (Steinheim, Germany). Tripton LP0042 and yeast extract LP0021 were obtained from Oxoid LTD (Basingstoke, UK), and nutrient agar was from Lab M (Bury, UK). Fetal bovine serum (FBS) and Penicillin and streptomycin (100x solution) were obtained from Gibco (Thermo Fisher Scientific Inc., USA).

Essential oil Analysis

Commercial M. communis leaf essential oil, obtained through hydrodistillation, was purchased from Health and Sleep R&D (Turkey).

The analysis was performed by a Thermo Scientific Trace 1300 GC gas chromatograph in conjunction with Thermo Scientific ISQ7000 single quadrupole mass spectrometer detector (GC-MSD). ¹² TRACE TR-5MS capillary GC column (5% phenyl 95% dimethyl polysiloxane, 30 m × 0.25 mm, 0.25 μm film thickness) (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used as the analytical column for chromatographic separation. The oil sample was diluted with acetone at a 1:500 ratio in a 2 mL vial and injected into the GC-MSD system. The run duration was 65 min. The injections were performed as follows: inlet temperature, 250 °C; injection volume, 2 μL; split ratio, 1:20. Helium was used as the carrier gas with a flow rate of 1.5 mL/min. The oven temperature was initially maintained at 30 °C for 5 min, then increased by 5 °C/min to 280 °C and held at this level for 10 min. The detector temperature was 230 °C. Chromatographic evaluations were performed using Xcalibur software. The compounds were identified by comparison with spectra reported in the Wiley 1n.l and NIST databases (National Institute of Standards and Technology) ¹³ and based on the retention indexes of the homologous series of n-alkanes (C8–C30). For each identified compound, basic, molecular, and qualifying ions were selected as part of the identification process. Results are expressed based on the pick area and comparisons of the obtained chromatograms.

HPTLC Analysis

An aliquot of 1 μL of M. communis essential oil was applied as an 8 mm band onto a 5 × 10 cm HPTLC glass plate precoated with silica gel 60 F254 (Merck, Darmstadt, Germany), using a Linomat 5 sample applicator (CAMAG, Muttenz, Switzerland). The application zone was positioned 8 mm from the lower edge of the plate, with lateral margins of at least 20 mm on both sides. Plate development was performed in a saturated 10 × 10 cm Twin Trough Chamber (CAMAG) to a migration distance of 70 mm, using a mobile phase of toluene: ethyl acetate (85:15, V/V). After development, the plate was air-dried under a stream of cold air. Visualisation of the separated constituents was achieved through chemical derivatisation. For this purpose, the chromatogram was immersed in a methanolic solution of p-anisaldehyde–sulfuric acid (ASA) reagent using a CAMAG Chromatogram Immersion Device (vertical dipping speed, 1 cm/s; immersion time, 2 s). The derivatising solution consisted of p-anisaldehyde, methanol, glacial acetic acid, and concentrated sulfuric acid in a volumetric ratio of 0.5:70:8:4. The plates were subsequently heated at 100 °C for 5–10 min to promote colour development of the bands. Photographic documentation (JPEG format), used for the analysis of the developed HPTLC plates, was conducted using a 48-megapixel camera.

HPTLC-DPPH Assay

To assess the antioxidant potential of M. communis essential oil, the chromatographic procedure was repeated under identical conditions. Following the method previously reported by Jović and coworkers, ¹⁴ the developed HPTLC plate was immersed in a 0.1% (w/V) methanolic solution of DPPH. The plate was then incubated for 30 min at room temperature in the dark. Visualisation of antioxidant activity was performed under white light, and gallic acid was used as the reference standard for comparison of radical scavenging capacity.

Antibacterial Activity

Bacteria Cultivation and Exposure to Essential Oil

The antibacterial activity of the tested essential oil was investigated using HPTLC-direct bioautography assays against K. pneumoniae ATCC 35218 and S. aureus ATCC 6538, following the procedure outlined in a previous study. ¹⁵ Bacterial strains incubated in Luria Bertani (LB) broth and incubated for were used for bioautography until the cultures reached the exponential phase (monitored by measuring the optical density (OD) at 600 nm). The developed HPTLC plates were briefly immersed (3-4 s) in the actively growing bacterial suspensions (OD = 0.6 for S. aureus and OD = 0.4 for K. pneumoniae) and subsequently incubated in a humid chamber at 37 °C for 1.5 h under aerobic conditions to allow bacterial growth on the plate surface. Antibacterial zones were detected using a 0.1% (w/V) solution of MTT in 0.1 M phosphate buffer (pH 7.2). After an additional hour of incubation, areas of bacterial growth inhibition were visualised as white zones against a bluish background. Streptomycin served as the positive control in assessing antibacterial efficacy.

Image Processing and Data Acquisition

Digital images of the chromatograms, showing the separation profile of essential oil components after ASA derivatisation, as well as those visualising antibacterial and DPPH activities, including reference standards, were analysed using the ImageJ software package (https://imagej.net/downloads). The image processing workflow followed the approach detailed in a previous study. ¹⁴ All chromatogram images were first resized to identical dimensions and converted to 8-bit grayscale format, followed by background subtraction. Using the rectangular selection tool, the chromatographic tracks were delineated, and intensity profiles were generated, producing plots of pixel intensity as a function of migration distance. The x-axis corresponded to the migration path along the chromatographic lane, while the y-axis represented pixel intensity. Peak areas of separated compounds and bioactive zones were measured with the Wand tool. To quantify the total terpenoid content (expressed as ursolic acid equivalents) and overall bioactivity (in terms of streptomycin and gallic acid equivalents), the cumulative peak area across the entire chromatographic lane was calculated. These cumulative values were then compared against calibration curves obtained from corresponding standards to determine equivalent concentrations or activities.

Method Validation

The quantification methods for ursolic acid, gallic acid, and streptomycin were validated in accordance with the guidelines provided by the International Conference on Harmonization (ICH). As outlined in a previously published procedure, ¹⁶ calibration curves were constructed by plotting the chromatographic peak areas (expressed in pixels) against known concentrations of standards applied per band (μg/band). The linearity of the response was assessed using least squares regression analysis. The sensitivity of the method was determined by calculating the limits of detection (LOD) and quantification (LOQ), based on the standard deviation of the analytical response and the slope of the respective calibration curve. All measurements were performed in triplicate.

Cytotoxic Activity Determination

Primary Cell Culture

One to 2-day-old male rat pups of the Wistar rat strain from the local colony at the Faculty of Biology of the University of Belgrade were used for primary cortical astrocyte culture preparation. Two-month-old male Wistar rats from the Vivarium of the Faculty of Medicine, University of Niš, were used for the isolation of peritoneal macrophages, thymus, and spleen lymphocytes. The animal procedures were performed in compliance with the European Communities Council Directive (2010/63/EU) and the Serbian Laboratory Animal Science Association for the protection of animals used for experimental and scientific purposes. The reporting of this study conforms to ARRIVE 2.0 guidelines regarding animal breding, selection and randomisation. ¹⁷ The animal procedures were approved by the Veterinary Administration of the Ministry of Agriculture, Forestry, and Water Management of the Republic of Serbia (authorisation reference numbers 323-07-02890/2023-05 and 323-07-08984/2022-05). Experimental procedures were conducted in accordance with the Ethical approvals for research projects from January until May 2024 in laboratories at the Faculty of Medicine, University of Nis, and from January until May 2024 in laboratories at the Faculty of Biology, University of Belgrade.

Cell culture of primary astrocytes was prepared from cerebral cortices that were mechanically dissociated by gentle pipetting under sterile conditions in cold Dulbecco's modified Eagle's medium (DMEM), supplemented with 100 IU/ml penicillin and 0.1 mg/ml streptomycin. After centrifuge/washing steps at 500 × g for 4 min, the cell suspension was seeded in Culture medium - DMEM supplemented with 10% heat-inactivated FBS, 25 mmol/l glucose, 2 mmol/l L-glutamine, 1 mmol/l sodium pyruvate, 100 IU/ml penicillin and 100 μg/ml streptomycin and grown at 37 °C in a humified incubator with 5% CO₂. Cell splitting was performed using a trypsin working solution (0.25% trypsin and 0.02% EDTA), and the cells were replated on new dishes at a density of 2 × 10⁴ cells/cm². Each cell culture was prepared from a single animal cortex; a total of 6 animals were used for this study. More than 98% of cells in the cultures were GFAP-expressing cells. ¹⁸ For further analysis, primary astrocytes were seeded on 96-well plates at a density of 20000 cells/well, until 80% of confluence, when they were ready for the treatment.

Macrophages were isolated from the rat's peritoneal cavity by washing with PBS, and the collected suspensions were further centrifuged (1500 rpm for 10 min at 4 °C). The obtained cell pellets were resuspended in RPMI supplemented with FBS and antibiotic-antimycotic. The cell density was adjusted to 2.5 × 10⁶ cells/ml, and the cells were further incubated for 1.5 h in a humidified incubator with 5% CO₂. After a certain amount of time had elapsed, non-adherent cells were removed, and fresh RPMI (100 μl) was added and used in further experiments. For spleen and thymus tissue lymphocytes, the corresponding tissues were mechanically dissociated through a strainer. The obtained suspensions were then centrifuged, and the pellets were used to prepare a cell suspension in RPMI (2.5 × 10⁶ cells/ml), which was used in the experiments.

C6 Cell Culture

The rat C6 glioma cell line (ATTC, CCL-107 ™) was defrosted and cultivated under the same conditions as previously described primary astrocytes. For this study, C6 cell line passages 3–6 were used. Cells were split once, before being seeded for further experiments, at a density of 6000 cells/well in a 96-well plate. Cells were treated at 70–80% confluence.

Cell Culture Treatment

Cells were seeded on 96-well plates. Cultures were incubated with M. communis essential oil at the indicated final concentration for 24 h. The essential oil was dissolved in DMSO to prepare a stock solution, followed by dissolution in DMEM/RPMI to obtain the indicated final concentrations of M. communis essential oil. In final treatment solutions, the highest DMSO content was less than 0.05%, the concentration that did not affect cell viability (data not shown). The concentration span of the essential oil was 0.01 to 100 μg/mL. At 24 h after treatment, cell culture media were collected, and cells on 96-well plates were subjected to cytotoxicity assays.

Cell Viability Determination

The effects of M. communis essential oil on cell viability were estimated based on metabolic activity, MTT assay, cell membrane function (crystal violet assay, CV), and lactate dehydrogenase (LDH) activity. ¹⁹ Briefly, cell metabolic activity was estimated based on the formazan crystals generated from MTT, which were dissolved in DMSO, and the optical density was measured at 570 nm using a plate spectrophotometer. For the CV assay, cells were fixed with 4% PFA, washed in PBS, and incubated with 0.5% crystal violet. The stain was dissolved in acetic acid, and the absorbance was measured at 595 nm. The activity of the LDH in the removed cell medium was determined based on the reduction of NAD⁺ to NADH. The reaction intensity was monitored at 340 nm over 5 min. The results of all three assays are presented as % of viable cells.

Statistical Analysis

The results of this study were expressed as mean values ± SD and compared using one-way analyses of variance (ANOVA). Further, Tukey's post hoc test for multiple comparisons was used. Probability values less than 0.05 (p < 0.05) were taken as statistically significant.

Results

Chemical Composition of M. communis Essential Oil

The compounds identified in the essential oil sample by GC-MS analysis are listed in Table 1 in order of their elution times from the column. A total of 26 compounds were detected, accounting for 98.68% of the entire essential oil composition. The most abundant identified constituents were α-pinene (41.61%) and 1,8-cineole (38.79%). Additionally, α-linalool, α-terpineol, α-terpinyl acetate, and linalyl acetate were present at 5.55%, 2.82%, 1.99%, and 1.57%, respectively. Other constituents present in amounts close to 1% included geranyl acetate (1.21%) and trans-caryophyllene (1.03%).

Table 1.

Relative Percentage of the Compounds Detected in M. communis Essential Oil by GC-MS.

Peak	Retention Time (min)	KI	ExpKI	Compound Name	% (based on Peak Area)
1	6.5	901	902	Isobutylisobutyrate	0.33
2	6.61	931	924	α-Pinene	41.61
3	8.2	970	966	β-Pinene	0.26
4	8.40	992	998	α-Myrcene	0.12
5	9.72	1009	1011	3-carene	0.14
6	10.34	1024	1022	p-Cymene	0.65
7	11.22	1030	1026	1,8-Cineole (Eucalyptol)	38.79
8	12.48	1050	1049	α-Ocimene	0.11
9	13.96	1071	1067	γ-Terpinene	0.04
10	14.92	1085	1081	α-Terpinolen	0.13
11	15.69	1099	1097	α-Linalool	5.55
12	17.64	1146	1142	trans-Pinocarveol	0.15
13	18.02	1146	1142	4-Terpineol	0.18
14	20.94	1172	1172	α-Terpineol	2.82
15	21.27	1194	1189	Myrtenol	0.21
16	22.98	1200	1199	p-Allylanisol	0.10
17	25.60	1240	1239	Linalyl acetate	1.57
18	26.04	1278	1274	Carvacrol	0.06
19	28.04	1295	1297	trans-Pinocarveyl acetate	0.42
20	29.50	1333	1327	α-Terpinyl acetate	1.99
21	29.74	1377	1376	Neryl acetate	0.12
22	31.02	1382	1380	Geranyl acetate	1.21
23	33.58	1405	1401	iso-Cariophyllene	1.03
24	34.04	1410	1415	Methyl eugenol	0.19
25	34.59	1451	1452	α-Humulene	0.55
26	39.94	1570	1567	Caryophyllene oxide	0.35
Non-identified compounds					1.32

*KI - Kovats Index Literature, *ExpKI - experimental Kovats Index.

HPTLC Fingerprinting and Chemical Profile of M. communis Essential Oil

The characterisation and identification of secondary metabolites present in the essential oil of M. communis were performed using HPTLC. The resulting fingerprint profile, following derivatisation with ASA reagent, is presented in Figure 1a. The applied chromatographic system, which employed silica gel as the stationary phase and a toluene: ethyl acetate mixture (85:15, V/V) as the mobile phase, enabled the optimal separation of metabolites, visualised as distinct brown, violet, and pink-coloured zones. Among these, the zones with hR_F values of 33 and 50 were the most prominent based on colour intensity. Summed peak areas and the calibration curve were used to express the total terpenoid content as ursolic acid equivalents, which is presented along with the validation parameters of the ursolic acid calibration curve in Table 2.

Figure 1.

HPTLC fingerprint profiles of M. communis essential oil visualised under white light after derivatisation with: a) ASA reagent; b) DPPH solution; c) S. aureus; d) K. pneumoniae. Mobile phase, toluene: ethyl acetate (85:15, V/V).

Table 2.

Total Terpenoid Content, Antioxidant Capacity, and Antibacterial Activity of M. communis Essential Oil Expressed as Standard Equivalents (µg/µL), with Corresponding Regression Data for Each HPTLC Method.

Method	M. communis Activity (μg of Standard Equivalent/μL of Oil)	Standard	Regression Equation	R2	Linear Range (μg)	LOD (μg)	LOQ (μg)
TTC	352.3 ± 21.5	Ursolic acid	y = 1008219x + 2103241	0.992	1–7	0.7	2.1
DPPH	80.5 ± 4.8	Gallic acid	y = 2121957x + 9855851	0.995	1–8	0.6	1.9
S. aureus	529.4 ± 41.3	Streptomycin	y = 522572x + 979338	0.995	10–45	3.4	10.4
K. pneumoniae	279.8 ± 13.1		y = 416142x + 3009649	0.998	10–40	1.8	5.3

Antioxidant Activity of M. communis Essential Oil

The antioxidant activity of the essential oil was evaluated using HPTLC combined with the DPPH assay. The resulting chromatogram is shown in Figure 1b, where the separated components of the oil appear as yellow zones against a reddish background. One intensely yellow-stained zone with an hR_F value of 50 corresponds to the oil component with the highest antioxidant potential. Additionally, several other zones exhibiting weaker radical scavenging activity were also identified. Following the same approach used for quantifying the total terpenoid content, the antioxidant potential of the essential oil was quantified and expressed in terms of gallic acid equivalents (Table 2).

Antibacterial Activity of M. communis Essential Oil

The antibacterial activity of the M. communis essential oil against S. aureus and K. pneumoniae was assessed by coupling HPTLC with the MTT assay (Figure 1c and 1d). HPTLC plates containing the separated components of the essential oil were exposed to bacterial suspensions and incubated, allowing bacterial growth to occur directly on the plates. Zones corresponding to antibacterial compounds in the oil appeared as white areas against a bluish background. The bioautograms obtained for both tested bacterial strains revealed prominent antibacterial activity associated with zones at hR_F values of 5, 20, 50, and 95. However, the bioautogram corresponding to S. aureus exhibited significantly more intense inhibition zones, indicating a higher susceptibility of S. aureus compared to K. pneumoniae. Additionally, a zone at hR_F 80 showed pronounced activity against S. aureus, while no activity was observed against K. pneumoniae. Following the same approach as applied for the quantification of total terpenoid content and antioxidant activity, the antibacterial activity was quantified and expressed as streptomycin equivalents, a reference standard to which both bacterial strains are susceptible (Table 2).

Both peritoneal macrophages and splenocytes showed a marked decrease in cell viability when exposed to the highest tested concentration of the oil (100 µg/ml), with cell viability decreased in the MTT assay to ∼43% and significant decreases in the CV assay (∼20%) and high LDH leakage (∼21%) (Table 3). At intermediate concentrations (10-1 µg/ml), viability, as estimated through the MTT assay, was not significantly altered and remained close to the control. Interestingly, in both assays estimating cell membrane function, the intermediate concentration also pointed to decreased cell viability. Thymocytes displayed moderate sensitivity to the exposed essential oil, with MTT viability reduced to ∼54% at 100 µg/ml and consistently decreased CV and LDH values (75-88%). This effect was visible even at lower doses, indicating these cells are relatively more sensitive to the oil compared to others (Table 3). Primary astrocytes exhibited a sharp decrease in viability only at the highest dose (100 µg/ml), with MTT viability reduced to ∼9.6% and in CV (∼24%) and LDH (∼19%) (Table 3). However, at lower concentrations (10 µg/ml and lower), astrocytes maintained viability near control values. Application of increasing concentrations of the examined essential oil did not produce any significant changes in C6 astrocytes’ ability to metabolise MTT, nor did it affect cell membrane permeability measured through CV absorption and LDH leakage (Table 3).

Table 3.

Effect of M. communis Essential Oil in Different Concentrations on Cell Viability (Given in Percents, %) Estimated Using various Methods.

Cell Type	Method for Viability	M. communis Essential Oil (μg/ml)					Control
		100	10	1	0.1	0.01	-
Peritoneal macrophages	MTT	43.1 ± 0.7*	108.8 ± 6.4	113.8 ± 5.7	107.4 ± 6.6	103 ± 1.7	100 ± 5.6
	CV	19.9 ± 7.9*	47.2 ± 1.4*	51.8 ± 3.5*	71.3 ± 12.2*	86.9 ± 5.9**	100 ± 8.9
	LDH	21.4 ± 8.2*	37.5 ± 5.7*	58.2 ± 9.1*	77.5 ± 15.8**	90.1 ± 10.4**	100 ± 9.2
Thymocytes	MTT	53.7 ± 1.5*	96.4 ± 2.6	91.6 ± 9.4	96.9 ± 4.50	96.3 ± 10.9	100 ± 3.6
	CV	81.6 ± 12.3**	87.6 ± 5.5**	88.1 ± 0.9**	89.5 ± 5.9**	96.6 ± 3.9	100 ± 3.7
	LDH	75.4 ± 9.5**	78.1 ± 7.4**	81.8 ± 4.5**	80.5 ± 7.4**	93.8 ± 8.5	100 ± 5.4
Splenocytes	MTT	43.6 ± 5.7	118.2 ± 16.1	113.8 ± 15.3	107.4 ± 11.7	103.3 ± 8.4	100 ± 5.1
	CV	19.9 ± 7.9*	47.2 ± 1.2*	51.9 ± 3.5*	63.2 ± 13.5*	75.8 ± 11.4**	100 ± 8.9
	LDH	21.5 ± 10.2*	35.7 ± 8.4*	42.2 ± 13.5*	65 ± 12.2*	71.7 ± 10.3**	100 ± 10.4
Astrocytes	MTT	9.6 ± 3.9*	95.4 ± 5.1	96.6 ± 5.4	99.1 ± 3.7	99.7 ± 3.1	100 ± 2.2
	CV	24.3 ± 1.7*	111.3 ± 17.1	127.1 ± 6.9**	128.1 ± 14.5**	128.1 ± 15.2**	100 ± 8.8
	LDH	19.1 ± 4.5*	79.2 ± 13.5**	80.5 ± 25.2**	84.9 ± 19.9**	88.4 ± 17.7**	100 ± 12.1
C6	MTT	93.1 ± 2.6	104.1 ± 6.7	101.0 ± 4.2	93.7 ± 1.3	102.8 ± 2.1	100 ± 3.2
	CV	96.6 ± 1.8	102.4 ± 1.1	100.8 ± 0.3	100.7 ± 0.4	101.2 ± 0.4	100 ± 0.5
	LDH	95.1 ± 1.2	100 ± 10.1	100 ± 14.2	100 ± 8.4	100 ± 5.9	100 ± 7.5

Values are given as mean percent (%) of live cells ± SD (n = 6) and were compared using One-way ANOVA followed by Tukey's post hoc test for group comparison: ***p < 0.05, **p < 0.01, *p < 0.001 versus control

Discussion

To date, numerous studies have confirmed the wide range of therapeutic properties of myrtle essential oil, including antibacterial, antifungal, antioxidant, cytotoxic, etc These therapeutic properties can be directly attributed to the unique chemical constituents of the oil, especially terpenoids. ²⁰ These data support the findings of the present study, particularly in terms of the high content of these terpenoids and the observed antibacterial and antioxidant potential of the analysed essential oil. The composition profile of the analysed essential oil is consistent with some previous reports that identified α-pinene and 1,8-cineole as the major constituents of the essential oil isolated from leaves, stems, and flowers.^21,22 On the other hand, other studies have reported variations in the relative abundance of oil constituents. For instance, essential oil from Iranian myrtle showed a higher content of 1,8-cineole compared to α-pinene, ²³ while the Turkish sample was dominated by linalool and linalyl acetate. ²² Such variations in essential oil composition of the same plant species can be attributed to several factors, including environmental conditions, harvesting season, vegetative stage of the plant, and the specific extraction method used. ²⁴ During the flowering phase, the leaf essential oil of M. communis was predominantly composed of α-pinene (3.8-23.0%), 1,8-cineole (9.9-20.3%), limonene (5.5-17.8%), linalool (12.3-17.6%), and α-terpinyl acetate (1.8-7.0%). ²⁵ At the fruit ripening stage, the chemical profile remained similar, with higher proportions of 1,8-cineole (24.0-36.1%) and α-pinene (22.1-22.5%), along with linalool, limonene, linalyl acetate, α-terpinyl acetate, and geranyl acetate in smaller amounts. ² In addition, these compositional differences may, to some extent, influence the biological activity of the oils. This was demonstrated in the study where the oil from plants originating from Turkey exhibited slightly higher antibacterial activity than that of the oil obtained from plants growing in Italy. ²²

In order to evaluate the antibacterial activity and antioxidant potential of the essential oil, the HPTLC method was applied in combination with bioautography. This approach offers several advantages over conventional microbiological methods, such as agar diffusion and dilution techniques, as well as over the spectrophotometric radical scavenging assay. One of the main advantages is the ability to obtain direct information on the activity of individual components that have been separated on a thin-layer plate, resulting in improved time efficiency, simplicity, and cost-effectiveness of the analysis. ²⁶ Furthermore, after on-surface detection of bioactive compounds, HPTLC can be successfully coupled with additional structure elucidation techniques, such as absorption or fluorescence spectroscopy, attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, mass spectrometry (MS), and nuclear magnetic resonance (NMR), for comprehensive structural characterisation. ²⁷

In this study, the essential oil was also analysed using HPTLC with silica gel as the stationary phase and a toluene: ethyl acetate (85:15, V/V) mobile phase, which was found to be suitable for optimal separation of the oil components. Visualisation of the separated zones was performed by observing their colouration under white light after derivatisation with ASA reagent. ASA is a widely used reagent for visualising natural products, producing spots of varying colour depending on the compound class and concentration. ²⁸ Considering the fingerprint profile and the specific colouration of separated zones, literature data may provide indicative trends regarding the chromatographic behaviour of natural product classes, but in the absence of reference standards, unequivocal compound identification is not possible. Given that no authentic reference standards were applied for HPTLC analysis in this study and that a different mobile phase was used compared to previous studies, the assignment of individual zones to specific compounds remains tentative and is discussed only at the level of compound classes. Since 1,8-cineole and α-pinene were identified as the major constituents of the analyzed oil according to GC-MSD results, these compounds would, in principle, be expected to contribute to the more intense coloured zones observed after ASA derivatization. Literature reports indicate that oxygenated and hydrocarbon monoterpenes, such 1,8-cineole and α-pinene, typically produce a burgundy to brown zones upon ASA derivatization and visualisation under white light.^29,30 However, the obtained fingerprint chromatogram showed several brownish zones at hR_F values of 35, 50, 60, and 80. Therefore, it is not possible to reliably associate a specific zone with a particular compound identified by GC analysis. However, previous studies employing normal-phase systems have shown that oxygenated monoterpenes generally migrate at lower hR_F values than hydrocarbon monoterpenes, which is consistent with the polarity-driven separation observed in this study. ²⁹ In the same context, terpene alcohols such as α-terpineol, linalool, and geraniol, which are more polar, are expected to appear at lower hR_F values as reddish to brown zones. ³⁰ Conversely, their corresponding acetate esters, due to reduced polarity, are likely to elute at intermediate hR_F values.

In accordance with the obtained results, the only intense zone observed at hR_F 50 on the chromatogram following the DPPH assay can be attributed to one or more major constituents of the oil or their combined effect. Literature data indicate that several major monoterpenes commonly present in essential oil, including oxygenated monoterpenes, especially 1,8-cineole, may contribute to DPPH and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity.^31,32 It has also been confirmed that α-pinene exhibits pronounced DPPH radical scavenging activity. ³² Additionally, significant antioxidant activity has been reported for trans-caryophyllene. However, due to its low abundance in the analyzed oil, its contribution to the total antioxidant potential can be considered negligible. Moreover, the presence of a phenolic fraction, such as thymol or carvacrol, compounds well known for their strong DPPH radical scavenging capacity, further enhances the overall antioxidant effect of essential oils. ³³ Overall, the essential oil exhibited considerable antioxidant potential, quantified as 80.5 ± 4.8 µg gallic acid equivalents per µL of oil.

The MTT bioassay applied in this study relies on the activity of bacterial oxidoreductases, which reduce the yellow tetrazolium salt to blue-violet formazan, thereby indicating the metabolic activity of viable cells. S. aureus and K. pneumoniae were selected as test organisms due to the increasing difficulty of treating infections caused by these pathogens, stemming from their escalating resistance to conventional antimicrobial agents. S. aureus, a Gram-positive pathogen, is associated with a broad range of diseases, from superficial skin and soft tissue infections to severe conditions such as pneumonia, bacteremia, and osteomyelitis, with methicillin-resistant S. aureus (MRSA) representing a major clinical concern. ³⁴ In contrast, K. pneumoniae, a Gram-negative bacterium, is responsible for serious infections including pneumonia, urinary tract infections, bacteremia, meningitis, and hepatic abscesses. ³⁵ Both species are widely distributed in the environment, with particularly high pathogenic potential in vulnerable populations such as neonates, the elderly, and immunocompromised individuals. ³⁶

Quantification of antibacterial activity, expressed as μg streptomycin equivalents per μL of oil, revealed that S. aureus (529.4 ± 41.3 μg/μL) was nearly twice as susceptible as K. pneumoniae (279.8 ± 13.1 μg/μL). This finding is consistent with the known structural differences between Gram-positive and Gram-negative bacteria. The outer membrane of Gram-negative species, enriched in lipopolysaccharides, forms a hydrophilic permeability barrier that limits the diffusion of hydrophobic molecules, thereby reducing antibacterial efficacy. ³⁷ Similar trends of activity were observed for myrtle leaf essential oil using the disc diffusion method, ³⁸ where the oil exerted greater activity towards S. aureus than to K. pneumoniae. The discrepancies in the activity of M. communis essential oil were found for other bacterial strains, such as Escherichia coli, K. pneumoniae, Enterobacter cloacae, Proteus rettgeri, P. aeruginosa, as well.^39–41

The ability of the EO to inhibit the growth of the two studied microorganisms (Table 2) showed to be in the range of that of the standard antibiotic streptomicin. In the case of S. aureus the MIC value for streptomycin has been long ago found to vary and can range from as little as 100 ug/ml to more than 5000 ug/ml. ⁴² The activity of the essential oil in this case can be considered moderate, but still comparable to the standard antibiotic. In the case of K. pneumoniae the resistance profile also varies and has a broad range ⁴³ meaning that a careful selection of antibiotic for the treatment is required. From the perspective of the essential oil activity it can be noted that its potential is close to the one needed for streptomycin to act in average resistant bacteria. Finding the alternative agents with antibacterial properties in nature, such as essential oils, is of great importance not only for treating the bacteria infecting humans, but also for treating bacteria that can cause food spoilage. ⁴⁴

Although myrtle essential oil was found to generally exhibit lower antibacterial activity against Gram-positive bacteria than against Gram-negative bacteria, its main bactericidal components, 1,8-cineole, β-linalool, eugenol, and α-terpineol, are well known for their antibacterial activity.^24,45 These data are consistent with the bioautography results of the present study, where the spots appearing at lower hR_F values, corresponding to these more polar compounds, displayed pronounced antibacterial activity against both tested strains. In the case of K. pneumoniae, the absence of a zone with hR_F at 80, which corresponds to the nonpolar fraction of the essential oil, potentially correlates with its greater resistance to hydrophobic compounds compared to S. aureus.

Immune system cells located at different sites of the body (peritoneal cavity, spleen, etc) serve as scavengers for foreign bodies, eg, bacteria. ⁴⁶ Myrtus communis essential oil demonstrated pronounced cytotoxicity in primary immune cells (peritoneal macrophages, thymocytes, and splenocytes), with astrocytes also showing significant vulnerability at the highest dose, indicating their heightened sensitivity to the treatment. In contrast, C6 glioma cells maintained stable metabolic activity and membrane integrity across all concentrations, highlighting their relative resistance and suggesting that the cytotoxic response of the oil is both dose-dependent and cell-type-specific. C6 glioma cells exhibit greater resistance when exposed to the highest concentration of M. communis essential oil, which induced cytotoxicity in primary astrocytes, probably due to their enhanced metabolic flexibility and elevated antioxidant defence mechanisms. ⁴⁷ Unlike primary astrocytes, C6 cells demonstrate increased glucose uptake and a highly glycolytic phenotype, which supports rapid proliferation and improved stress adaptation. Additionally, C6 cells, a type of glioblastoma, are more resistant to redox anticancer agents and are known to upregulate antioxidant pathways, such as Nrf2, allowing them to neutralise oxidative components more effectively than primary astrocytes. ⁴⁸ Their tumour-derived origin also contributes to altered apoptotic thresholds and detoxification capabilities, making them less sensitive to compounds that are otherwise cytotoxic to normal glial cells.

Strengths and Limitations of the Study

One of the main limitations of this study is that a single method, even a very precise and current one, such as HPTLC-DPPH assay, can not provide full insight into the sample's antioxidant capacity. On the other hand, this part of the study was included for additional clarification of showed effects of M. communis essential oil. In addition, since the complete chemical fingerprint of this sample has been obtained, strong antioxidant activity was expected based on the compounds present in the tested sample. Also, HPTLC-direct bioautography assays against K. pneumoniae and S. aureus as an antimicrobial assessment were done on strains that are relevant for numerous pathological conditions. One of the major strengths of the study lies in the complementary investigation of cytotoxicity on peritoneal macrophages, thymocytes, and splenocytes as immune cells alongside astrocytes and C6 glioma cells. In our future studies, we plan to investigate the connection between the obtained results and the traditional application of M. communis finding the active constituents that can possibly be used independently from the oil.

In this study, the cytotoxic effects of M. communis essential oil were, for the first time, investigated in well-defined primary immune cells and different glial cell types, representing a significant novelty compared with previous studies in which cytotoxicity was mainly assessed using other cellular models or tumour cell lines. Although the antioxidant and antimicrobial properties of this oil have been previously reported, they have not been examined in this manner, using a combined HPTLC bioautography approach with quantitative evaluation of the biological activity of individual fractions, allowing a more precise correlation between chemical composition and biological effects. Given the pronounced variability in essential oil composition depending on geographical origin, plant phenological stage, environmental conditions, and extraction method, comprehensive chemical characterisation (GC-MS and HPTLC) was included as an essential initial step of the study, in accordance with good research practice and fundamental principles of working with plant material, to ensure that the obtained biological results are relevant, comparable, and reliably interpreted.

Conclusions

The present study demonstrates that Myrtus communis leaf essential oil, rich in α-pinene and 1,8-cineole, possesses significant antioxidant and antibacterial activity, particularly against Staphylococcus aureus. Cytotoxicity assays indicated that the oil exhibits concentration-dependent effects on primary immune cells (macrophages, splenocytes and thymocytes) and astrocytes, while C6 glioma cells remain largely resistant, highlighting cell-type-specific sensitivity. These findings support the therapeutic potential of M. communis essential oil as a natural antioxidant and antimicrobial agent, while also emphasising the importance of careful application due to its cytotoxic effects on normal cells.