Besides their use in the treatment of diseases, several other uses are attributed to plants and their derivatives, which has been arousing interest mainly in the food and cosmetics industries. An element originating from the secondary plant metabolism that is being widely used is the essential or volatile oil, and some of its medicinal uses can be cited as: aromatherapy, antitumor, antidepressant, antimicrobial, insecticide, acaricide (Raut & Karuppayil, 2014; Teixeira et al., 2013); as herbicides, in the alternative control of phytopathogens, as allelopathic (Sharifi-Rad et al., 2017) and postharvest disease control (Combrinck et al., 2011; Lopes et al., 2023).

A species widely used both in cooking and in folk medicine that deserves attention is Thymus vulgaris L. (Lamiaceae), popularly known as thyme. Usually, thyme is used for the treatment of respiratory diseases (cough, bronchitis and asthma) in the form of tea, ointment, syrup or vapor inhalation (Javed et al., 2013), and it also has antiseptic, antimicrobial, carminative and disinfectant actions (Hosseinzadeh et al., 2015). Thyme essential oil is widely used by the cosmetic and also pharmaceutical industry, mainly for its antioxidant properties (Asbaghian et al., 2011).

The cultivation of medicinal plants for various purposes, including the production of herbal medicines, is a practice that should be increasingly improved to solve possible quality problems that may occur with plants from extractivism, for example. In this perspective, the production of bioactive plants with control of abiotic factors, is important for the generation of standardized vegetable by-products, with quality control, aiming at the constancy of chemical components, which meet the most varied needs of both producers and consumers (Morais, 2009). In this scenario, protected cultivation systems and soilless cultivation allow for adequate production planning, which consequently leads to higher productivity and improved final product quality (Andriolo, 2017).

In addition, to guarantee the population’s safe use of certain medicinal plants, preliminary tests using bio indicators should be performed, especially regarding toxicity. Among the toxicogenetic biotests, the Allium cepa (onion) test is widely used to identify possible harmful effects of numerous substances on onion root meristematic cells. In the natural environment, the first part of the plant to be exposed to toxic agents dispersed in soil and water is the root, so its analysis represents a rapid method for monitoring toxicity (Vieira & Silveira, 2018).

In this sense, this biotest is considered ideal for a preliminary verification of genotoxic and antiproliferative effects, because it presents high sensitivity and correlation with tests using animal cells (Carmo et al., 2020; Tedesco et al., 2017). In addition to its long history of use in cytotoxicological tests, other advantages of using A. cepa include its large chromosomes that are easily observable under a microscope; the ability to reveal effects (positive or negative) even at relatively low levels of interaction between the tested substance and the genetic material; and, as an in vivo test, the results obtained can be applied to the evaluation of genotoxicity in plants as well as in other eukaryotes, including man (Bonciu et al., 2018).

In view of the above, the objective of the present article was to determine the chemical composition of the essential oil of T. vulgaris grown in two seasons (winter and summer), and evaluate the potential genotoxic and antiproliferative effects by A. cepa test.

The experiment was installed in an adapted greenhouse with fertigation system, located in the Department of Plant Science, Federal University of Santa Maria (UFSM). Santa Maria is a city with Cfa climate (humid subtropical, with hot summer, and sometimes drought), according to Köeppen (Alvares et al., 2013), located in the center of the state of Rio Grande do Sul, 95 meters above sea level, having as characteristic the clear division between seasons.

One hundred Thymus vulgaris L. seedlings, produced by cuttings, were grown in pots filled with commercial Maxiplant® substrate, in two seasons, winter ٢٠١٨ (june-august) and summer 2018/2019 (december-january). The pots were on benches, inside a low-density polyethylene greenhouse with a thickness of 100 μm. Nutrient solution was supplied to the plants through a drip tape connected to a submerged pump inside a reservoir. A timer was used to schedule the supply time of the nutrient solution (three times a day). The drained solution was collected and then returned to the original reservoir in a closed system.

After 60 days in the greenhouse during the winter, the plants were collected. In order to measure the effects of seasonality, the material was collected on the day that marked the mid-season (August 06, 2018). The heat sum for the 60 days that the plants remained in the greenhouse during the winter was calculated. For the summer experiment, the sum temperature of the winter was used to define the day of collection of the plants, which was when the sum temperature of the summer reached a value close to that of winter (January 9, 2019).

2.2 Essential oil extraction and dilution

The essential oil of thyme was extracted in the Laboratory of Plant Cytogenetics and Genotoxicity (LABCITOGEN) of the Department of Biology. Immediately after collection, the fresh mass (FM) was determined by weighing 20 whole plants on analytical scales. Next, 50 grams (g) samples of fresh plants were weighed and frozen for later extraction of essential oil.

The hydrodistillation method was performed using a Clevenger apparatus for two hours, with 50 g of fresh-frozen thyme. After extraction, the EO was collected in the form of supernatant, deposited in an amber flask, weighed on an analytical balance, identified and stored at 4°C ± 2°C until its use. Dilution of the EO was performed with absolute ethanol, obtaining concentrations of 0.10% and 0.25%.

Oil samples were analyzed by gas chromatography to determine the phytochemical composition.

2.3 Allium cepa test

Cytogenotoxicity analysis was performed using the A. cepa test, which consists of examining meristematic cells from onion roots to determine mitotic indexes and to look for chromosomal changes.

For the test, seven groups of four onion bulbs (repetitions) were placed in distilled water until they emitted roots. After root emergence, each group of four onion bulbs was transferred to one of the following treatments: controls (negative control = distilled water; negative control relative to oil dilution = ethanol; and positive control = glyphosate) and diluted thyme EOs, where they remained for 24 hours. The detailed description of the treatments and controls used in this experiment is shown in Table 1.

Table 1 – Treatments used in the evaluation of the cytotoxicity and genotoxicity of the essential oil of Thymus vulgaris, by the Allium cepa test

Source: Organized by the authors

After the treatment period, all roots were collected, fixed in ethanol: acetic acid (3:1 ratio) for 24 hours, and stored in 70% ethanol under refrigeration. The slides for the analysis of meristematic cells of A. cepa roots were prepared according to the crushing method (Guerra & De Souza, 2002), with minor modifications (Pereira et al., 2022). For each treatment, two slides were prepared (repetitions). Under a Leica optical microscope, the slides were examined by the scanning method at 40X magnification. In each slide, 500 cells were analyzed, totaling 1000 cells per bulb and 4000 cells per treatment.

The meristematic cells were classified according to the stage of cell division in which they were found (interphase, prophase, metaphase, anaphase and telophase), for subsequent calculation of the mitotic index (MI). The MI was calculated by the ratio of the number of dividing cells by the total number of cells analyzed and multiplied by 100. Any chromosomal irregularities were also counted and photographed. The genotoxic index (GI) was calculated by the ratio of the number of cells with alterations by the total number of cells analyzed and multiplied by 100.

2.4 Chemical analysis of the essential oils

The chromatographic analysis was performed in the Laboratory of Plant Extractives (LABEVE) of the Department of Forest Sciences at UFSM. For the analysis, an aliquot of 2 microliter (μL) of essential oil was diluted in 1 mL of hexane (HPLC grade) in duplicate.

The chemical composition of the EO was determined using an Agilent 7890A gas chromatograph coupled to a 5075C mass spectrometer (GC-MS) with a non-polar DB5-MS capillary column (Hewlett Packard, 5% phenylmethyl siloxane, 30 m × 0.25 mm i.d. × 0.25 µm film thickness), electron ionization mode at 70 eV. Helium was the carrier gas (1.0 mL·min-1), injector and detector temperatures of 150°C and 280°C were used, respectively. The samples were injected in split inlet mode (ratio 1:100). The oven was heated at 40 °C for 4 min, and then the temperature was increased to 320 °C at a rate of 4 °C·min-1.

The identification of compounds was based on comparison of retention indices, calculated by linear interpolation, to the retention times of an n-alkane series, and a comparison of their mass spectra with authentic samples and with literature data (Adams, 2009; NIST, 2009). The relative amounts of the components were calculated based on GC peak areas, using gas chromatography with a flame ionization detector (GC-FID). The analysis parameters were the same as those presented above, except for the split ratio (1:50) and the injector and detector temperatures (300ºC).

2.5 Statistical analysis

The corresponding data from the A. cepa test were compared by the Scott-Knott test (p < 0.05), using the statistical software SISVAR 5.6 (Ferreira, 2014). The CG data were subjected to cluster analysis in the GENES software (Cruz, 2013), using the Mahalanobis distance as a dissimilarity measure, and the clustering was performed using the Tocher method.

The temperature sum corresponding to the 60 days of the winter experiment (June-August) was calculated as TS = 827.15, with an average temperature during this period of 13.3°C. In the summer experiment (December - January), the plants were collected when the temperature sum reached a value close to that of winter (TS = 836.85), completing 33 days in the greenhouse, and on these days the average temperature was 25.4 ºC. The average fresh mass per plant was 17.57 g in winter and 25 g in summer. The average essential oil yield per thyme plant was 3.70% g.MF-1 in winter (percentage in grams by weight of fresh mass), and 6.37% g.MF-1 in summer (Hister et al., 2025). Taking into account a spacing of 30 cm between plants and 45 cm between rows, in 1 hectare it would be possible to plant approximately 74 thousand thyme plants per hectare, each hectare could yield on average 372.59 kg of essential oil.

The analysis of meristematic cells of A. cepa root tips subjected to thyme essential oil (EO) treatments are listed in Table 2.

It is observed that only the treatment with 0.25% diluted EO from winter-grown plants (OE025W) showed antiproliferative cytotoxic activity, differing statistically from the negative controls (NC and NCE). Although the other treatments reduced the number of cells in cell division, this reduction in proliferation did not differ significantly from the negative controls. In this sense, only the treatment with the highest concentration of essential oil had cytotoxic effect on A. cepa root cells. Aazza et al. (2014) attribute the antiproliferative activity to the monoterpenes, thymol and carvacrol, which are predominantly present in thyme EO. Other medicinal species have also demonstrated antiproliferative activity of their essential oils, such as Vitex negundo L. (Issa et al., 2020), Rosmarinus officinalis L. (Cosme et al., 2022), Origanum vulgare L. (Husić et al., 2023) and Alpinia zerumbet (Pers.) B.L. Burtt & R.M. Sm. (Sousa et al., 2024).

Table 2 – Number of Allium cepa meristematic cells analyzed in the different treatments with Thymus vulgaris essential oil and their respective mitotic index (MI) and genotoxic index (GI), including cells in interphase, cells in division and cells with irregularities

NC = negative control; NCE = negative control in absolute ethanol; PC = positive control; EO010W = essential oil (0.10%) - extracted from fresh-frozen thyme plants cultivated in winter; EO010S = essential oil (0.10%) - extracted from fresh-frozen thyme plants cultivated in summer; EO025W = essential oil (0.25%) - extracted from fresh-frozen thyme plants cultivated in winter; EO025S = essential oil (0.25%) - extracted from fresh-frozen thyme plants cultivated in summer. *Values followed by different letters differ statistically by the Scott-Knott test (p < 0.05). Source: Organized by the authors

Regarding genotoxicity, all treatments with EO, as well as with NCE and PC controls, did not differ; however, all showed statistically significant differences when compared with the negative control in distilled water (NC). In other words, the EO treatments—regardless of concentration or climatic season tested—not only differed from the NC in water but also did not differ from the NC in ethanol (used as the oil diluent), nor from the positive control with glyphosate, a substance known to cause mutations. In this sense, although the chromosomal alterations found were low in number (an average of 1% of the cells analyzed), it is possible to affirm that thyme EO is genotoxic. Figure 1 illustrates some chromosomal alterations found in the slide analysis.

Figure 1 – Chromosomal alterations visualized in meristematic cells of onion (Allium cepa) roots treated with thyme essential oil (Thymus vulgaris L.)

Source: Authors (2019)

Caption: A) Micronucleus; B) Disorganized metaphase; C) Metaphase with missing chromosome; D) Anaphase bridge; E) Anaphase with missing chromosome and micronucleus; F) Telophase bridge. Scale: 10 µm

Regarding the quality of the essential oil from thyme plants, a chemical analysis was performed through gas chromatography (GC). The chromatographic profile resulting from the GC performed on the winter-grown and summer-grown thyme EO can be seen in Table 3.

Table 3 – Chemical composition obtained by gas chromatography of the essential oil of Thymus vulgaris plants grown in greenhouse during the winter and the summer

KIC: calculated kovats index; KIL: literature kovats index (NIST, 2009). *Clustering by Tocher method. Source: Organized by the authors

The GC analysis was able to detect 94.10% on the winter-grown thyme EO, identifying 22 chemical substances present in the oil, with thymol being the most abundant, with 52.41%. The other major compounds were γ-terpinene (17.57%), β-cymene (9.26%), β-caryophyllene (2.93%), and carvacrol (2.57%). In the summer-grown thyme EO, it was possible to identify 25 chemical compounds that represent 95.99% of the elements, with thymol again being the predominant substance, with 45.94%. The other major components differed only by the presence of α-terpinene instead of β-caryophyllene among the five most abundant elements, namely γ-terpinene (23.04%), β-cymene (8.35%), α-terpinene (2.86%), and carvacrol (2.65%). Thyme essential oil with a high thymol content is considered to be of excellent quality. According to the ISO 19817 (2017) standard, the thymol content in thyme essential oil should be between 35 and 55%. Therefore, thyme essential oil extracted under the conditions presented above provides excellent quality oil.

Corroborating the results found here, previous studies have already demonstrated the predominance of thymol in thyme essential oil (Dauqan & Abdullah, 2017; Sharangi & Guha, 2013), followed by carvacrol, p-cymene, γ-terpinene (Agili, 2014; Borugă et al., 2014; Gedikoğlu et al., 2019). This variation in the other majoritarian compounds may be linked to various environmental, nutritional, and climatic factors to which the thyme plants were subjected.

Although thymol was the majority component of thyme EO in both seasons tested, in winter it was present in even greater amounts, accounting for more than half of the chemical composition of the oil. This may be related to the cytotoxicity found only in EO from winter-grown plants. Commonly, the cytotoxic activity of some EOs is mainly due to the presence of substances such as alcohols, aldehydes and phenols (Bakkali et al., 2008), these which are part of the terpenes, being thymol a phenol of the monoterpene group.

Marco et al. (2012) tested the EO of Lippia sidoides Cham. (pepper rosemary), whose main component is thymol, on the germination of Lactuca sativa seeds and observed that there was an inhibitory cytotoxic effect. Experiments by Gedikoğlu et al. (2019) showed antimicrobial activity of thyme essential oil. Thymol can cause cell apoptosis and telomere shortening in Candida albicans (Kumar et al., 2025). Isolated thymol has also been shown to be active against Salmonella and Staphylococcus bacteria (Dauqan & Abdullah, 2017). Essential oils of oregano and rosemary pepper, with carvacrol and thymol being the major constituents of these oils, respectively, showed greater antifungal activity against four fungi that cause post-harvest diseases in mangoes (Vilela et al., 2024).

The cytotoxicity of EO in mammalian cells is caused by the induction of apoptosis and necrosis (Bakkali et al., 2008). In this regard, studies by Sertel et at. (2011) have already shown that thyme EO inhibited the growth of human oral cavity squamous cell carcinoma and Kubatka et al. (2019), in an in vitro study, revealed the antiproliferative and pro-apoptotic effects of thyme EO on mammary carcinoma cells.

Characteristics such as cytotoxicity are of great interest as it sanctions the use of EOs and other phytoproducts in a variety of practices, such as in preserving food and other agricultural products, and especially in fighting pathogens. It has been shown that EOs and some of their isolated constituents are really effective in protecting against a wide range of organisms, and can be used as antiseptics and antimicrobials for personal use and for insecticidal use in the preservation of crops or food stocks (Bakkali et al., 2008).

In the experiments presented here, up to the 0.25% concentration, thyme EO induced chromosomal alterations, although in a small amount, and it is not possible to exempt thyme EO, and possibly thymol, from potential genotoxic activity, since thymol and carvacrol alone have already demonstrated genotoxic effect in previous studies (Alves et al., 2018; Pinheiro et al., 2015). Also, the low GI may also be due to the low concentration of the EO used in the treatments.

EO extracted from fresh winter-grown thyme shows antiproliferative cytotoxic potential only at the 0.25% concentration. In addition, EO shows, albeit low, genotoxicity at the concentrations tested. Thymol is the major substance in the EO of both winter and summer thyme plants, exceeding 50% of the oil composition in winter.

These results may serve as an indicator for future studies with thyme, in the search for practical benefits that take into account its wide range of biological activity, including the ability to cause cytotoxic effects, such as inhibition of cell division and consequent cell death, along with the reduced ability to induce chromosomal alterations.