Anti-Candida and Antibiofilm Activity of Selected Lamiaceae Essential Oils

Background : Candidiasis is a common oral and vaginal infection. Some papers have presented that the essential oils of Lamiaceae plants can have antifungal activity. This study aimed to investigate the activity of 7 essential oils of the Lamiaceae family with known phytochemical compositions against Candida fungi. Methods : Forty-four strains belonging to six species were tested: C. albicans , C. glabrata , C. guilliermondii , C. krusei , C. parapsilosis

Antifungal drugs are used to treat candidiasis and other yeast-like infections.However, the search for new medicines is constantly ongoing.Many natural substances, including essential oils, have antifungal properties and can be used to treat fungal diseases.Essential oils are found in many plants, including those belonging to the following families: Acoraceae, Apiaceae, Asteraceae, Cupressaceae, Geraniaceae, Illiciaceae, Lamiaceae, Lauraceae, Myristicaceae, Myrtaceae, Oleaceae, Pinaceae, Poaceae, Rosaceae, Santalaceae, and Zingiberaceae [8].Essential oils from the Lamiaceae family are particularly important in traditional medicine, pharmacology, as well as the food industry.Species in this family are rich in terpenes possessing Today we know that essential oils have been used for millennia.Archaeological evidence suggests their use as early as the Neolithic Age (before 4000 B.C.).Information about them is also contained in ancient accounts from Mesopotamia and Egypt [10].Essential oils are used in aromatherapy, bathing, massages, wound healing, as well as in the treatment of headache, muscular pain, respiratory problems, skin changes, and joint inflammation [10,11].Essential oils are likewise used in toothpastes and mouth rinses to protect against dental caries, periodontal diseases, and candidiasis [12][13][14].Furthermore, essential oils are used in the treatment of vaginitis [15,16].They have a broad range of therapeutically beneficial effects, including antioxidant and anti-inflammatory properties, as well as antibacterial, antifungal, and antibiofilm activity [17][18][19].
This study aimed to investigate the antifungal and antibiofilm activity of seven essential oils of the Lamiaceae family.In total, 44 Candida strains, mainly clinical isolates belonging to six species, were tested in vitro.Additionally, in silico toxicity prediction was performed for the main essential oils used in the investigation.

Essential Oils
Essential oils were purchased in Etja (Elbląg, Poland).All oils were stored at 4 °C, with an expiration date of 2023 or 2024.This study made use of seven essential oils from the Lamiaceae family, namely from Lavandula stoechas L., Melissa officinalis L., Mentha × piperita L., Origanum vulgare L., Rosmarinus officinalis L., Salvia officinalis L. and Thymus vulgaris L. Table 1 shows the phytochemical composition of the oils according to the data obtained from the manufacturer.

Fungal Strains
During the in vitro tests, strains from the Chair and Department of Medical Microbiology collection at the Poznań University of Medical Sciences were used.The tests were performed on C. albicans (16 strains), C. glabrata (8 strains), C. krusei (8 strains), C. parapsilosis (4 strains), C. tropicalis (4 strains), and C. guilliermondii (2 strains).All clinical strains were obtained from patients' mucous membranes and were identified using the Integral System Yeasts Plus (Graso Biotech, Starogard Gdański, Poland) biochemical test.The experiments also included the following reference strains: C. albicans ATCC 14053 and C. glabrata ATCC 66032.All species were grown at 35 °C for 24 h in Sabouraud dextrose agar (Graso Biotech).

Antimicrobial Activity (MIC)
The minimal inhibitory concentrations (MICs) of selected essential oils were determined by the micro-dilution method using 96-well plates (NUNC, USA and Nest Scientific Biotechnology, Jiangsu, China).The studies were conducted according to the methodology described in our previous publications [20,21].Briefly, 90 µL of tryptic soy broth (Graso Biotech) and 10 µL of fungi suspension were placed into each well to a final inoculum concentration of 10 6 CFU/mL.Suspension was performed using McFarland standards and microscopy [22].Before studies, essential oils were emulsified in tryptic soy broth (1:1) using Omni TH homogenizer (Omni, Kennesaw, GA, USA).
Serial dilutions of each essential oil were performed to obtain concentrations ranging from 200 to 0.1 mg/mL.The plates were incubated at 35 °C for 24 h.The MIC value was the lowest essential oil concentration that inhibited any visible fungal growth.Optical density (OD) was measured at 620 nm using a microtiter plate reader (Eppendorf, Warszawa, Poland).Additionally, 10 µL of a 1% aque-ous solution of XTT (Sigma Aldrich, Poznań, Poland) was added to each well.Microorganisms convert tetrazolium compounds to a colored water-soluble formazan product [20,21,23,24].All the experiments were performed in triplicate.

Biofilm Inhibition
The inhibition of biofilm formation by C. albicans (ATCC 14053), the C. albicans, C. glabrata and C. krusei clinical strains was evaluated by means of a crystal violet assay.The influence of essential oils at the concentrations of MIC was determined.The negative control was a sterile culture medium.At first, suspension of each yeast was performed at a concentration of 10 6 CFU/mL [25].The biofilm was formed in 96-well plates with tryptic soy broth, after the addition of the appropriate essential oil to its previously determined MIC and 10 µL of fungal suspension.The total volume in wells was 100 µL.Next, plates were incubated for 48 hours at 37 °C.After incubation, the wells were washed with 200 µL of PBS three times, and plates were dried by inverting them on absorbent paper for 15 min.Each well was fixed with 200 µL of methanol for 15 min and dried after removing it.Afterward, the wells were stained with 200 µL of 1% crystal violet solution for 20 minutes.Wells were washed thrice with PBS, dried, and 200 µL of 96% ethanol was added to dissolve the crystal violet [26].To quantify the biofilm, the optical density (OD) was measured at 620 nm, using an Elisa Reader 250 (bioMerieux, Marcy-l'Étoile, France).The percentage of biofilm biomass growth was determined using the following formula: % Biofilm growth = 100 × (Sample OD620 -Control OD620 ) / (Control OD620 )

In Silico Toxicity Prediction
The toxicity of the main compounds found in the studied Lamiaceae essential oils presented in Table 1 was determined using in silico methods.Specifically, the ProTox-II (https://tox-new.charite.de/protox_II)[27] and pkCSM (http://biosig.unimelb.edu.au/pkcsm/)[28] software were used.Studies were performing according to software manuals.

Statistics
The mean, SD and median of MIC values of essential oils against Candida strains were calculated.The Kruskal-Wallis and post-hoc tests were applied to determine the statistical significance of differences in the MICs of fungi.The results were considered significant at the level of p < 0.05.Data were tested using InStat3 software (GraphPad Software, Boston, MA, USA).

Antimicrobial Activity (MIC)
Essential oils inhibited the growth of the tested strains of Candida at concentrations of 0.1-100 mg/mL (Fig. 1).The essential oils obtained from lemon balm and oregano showed the best anti-Candida activity.The MIC value for both oils was below 3.125 mg/mL.Lavender, mint, rosemary, and thyme essential oils were also very active in the range of 0.39 to 6.25 or 12.5 mg/mL.The essential oil obtained from sage exhibited statistically the weakest activity with MIC values ranging from 3.125 to 100 mg/mL for single C. krusei and C. parapsilosis strains.These results suggest that strains of C. krusei are the least sensitive to Lamiaceae essential oils.The activity of each essential oil against various Candida strains is outlined in Table 2 and Supplementary Tables 1-7.Statistical analysis of the mean MICs values (mg/mL) for selected Lamiaceae essential oils obtained for all Candida strains is presented in Table 3.

Biofilm Inhibition
The antibiofilm study using Lamiaceae essential oils yielded various results.At the MIC values, oregano and thyme essential oils showed the strongest effect, inhibiting biofilm growth by about 90%.Lavender, mint, and rosemary oils at the MIC concentration inhibited biofilm growth by about 75-85%.The weakest antibiofilm activity was observed with lemon balm and sage oils, which destroy only about 60-70% of biofilm.Interestingly, lemon balm essential oil exhibited the strongest anti-Candida activity in the planktonic form, while it exerted a much weaker inhibitory effect in the antibiofilm study.The antibiofilm activity of the studied Lamiaceae essential oils at the MICs are presented in Fig. 2.

In Silico Toxicity Prediction
In silico toxicity studies revealed that most of the main compounds found in the Lamiaceae essential oils used in this investigation did not exhibit carcinogenicity, mutagenicity, or cytotoxicity.Only p-cymene has carcinogenic activity, while menthone exhibited mutagenic activity.16 out of 21 compounds were found to have the potential to lead to skin sensitization (Table 4, Ref. [27,28]).This means that Lamiaceae essential oils can irritate the skin and should only be applied for short periods of time.With the exception of p-cymene, all of the tested compounds have high lethal dose 50 (LD50) values, which means that they are relatively safe for use in the oral cavity, even if ingested.

Discussion
Volatile oils obtained from Lamiaceae plants contain various groups of chemical compounds such as monoterpenes, sesquiterpenes as well as phenolics.Depending on Table 4. Toxicity of the main Lamiaceae essential oil compounds determined using the in silico ProTox-II and pkCSM online the major chemical compounds, several chemotypes have been described [29].It has been well established, that the chemical composition of essential oils is influenced by environmental factors, geographical regions of plant growth, harvesting time, the stage of plant development [30].Popular extraction methods used to isolate essential oils, include conventional hydrodistillation (HD) [31][32][33], and vacuum distillation [34].These methods of distillation are described in the European Pharmacopoeia.Other methods of distillation are likewise used and include enzyme-assisted hydrodistillation [35], and water microwave assisted hydrodistillation (MAHD) techniques [36,37].However, no significant differences were found in composition of Lamiaceae essential oils after the comparison of MAHD and HD [36].Another innovative method is supercritical fluid extraction (SFE) of volatile oils, which allows for higher essential oil yield and the isolation of more chemical compounds when compared to the more traditional hydrodistillation process [38].In contrast, Rodriguez-Solana et al. [39] found that the highest extraction yields were obtained using the Soxlet and accelerated solvent (ASE) techniques (but not SFE) in case of Mentha piperita and Rosmarinus officinalis.To identify and quantity of chemical constituents of a given essential oil, the method of choice is gas chromatography with mass spectrometry (GC-MS) [33] and GC with flame ionization detector (GC-FID) [33,39].
Data found in the literature indicate considerable variability regarding the antifungal activity of essential oils.These differences are likely related to the different bio-chemical composition of essential oils, which depend on the place of harvest, soil and light conditions, and the harvest date [40].Another critical factor is the method of obtaining essential oils, which can lead to significantly different concentrations of active compounds [41].
In this study, the active concentrations of essential oils ranged between 0.1 to 100 mg/mL.Essential oil from lavender acted against Candida strains at concentrations between 0.39-6.25 mg/mL.These values differ markedly from those available in the published literature available to date.In the study by Khoury et al. [42], the MIC of Lavandula stoechas oil against C. albicans was found to be 0.5 mg/mL.This value was similar to that obtained during this investigation.However, Zuzarte et al. [43] reported that the MICs of Lavandula stoechas oil against C. albicans, C. guillermondii, C. krusei, C. parapsilosis, and C. tropicalis were very low, ranging between 0.64-2.5 µg/mL.
Based upon the results obtained in this investigation, the essential oils from melissa and oregano have the best antifungal activity (MICs from 0.2 up to 3.125 mg/mL).Other Polish studies have shown the sensitivity of yeast-like fungi to melissa oil to be in the concentration range of 0.25-2.0mg/mL.Most strains of C. albicans, C. glabrata, and C. humicola were inhibited at concentrations of 0.25-0.5 mg/mL.In contrast, MIC values for C. kefyr, C. krusei, C. lusitaniae and C. tropicalis strains were in the range of 0.5-2.0mg/mL [44].Similarly, a study from the USA found that melissa oil exhibited a MIC of 0.3 mg/mL against C. albicans [45].In research from Italy, MICs against C. krusei, C. parapsilosis, biofilm inhibition was 10 µg/mL, and Agarval et al. [58] demonstrated this effect at a MIC of 800 µg/mL.These differences may be related among others to with different composition of essential oils or other research methodology [9].It is surprising, however, that also in the case of individual chemical compounds these differences can be very large.A study of thymol on biofilm formation by Jafri and Ahmad [59] indicated that it was active at a concentration of 3.12 µg/mL.However, in the studies of Braga et al. [60] the required concentration was as high as 125 µg/mL.
A significant advantage of this investigation is the inclusion of a large number of both essential oils and Candida strains.Very often, only single reference strains are tested by other authors [42,45,50,51,54,55].Additionally, many publications do not include clinical strains [42,45,47,[49][50][51]54,55].Unfortunately, as can be seen in the tables presented above there are often notable differences in the sensitivity of fungi from the same species to the same essential oil.An excellent example of this variability is the activity of Thymus vulgaris essential oil against C. albicans, for which the MIC values range from 0.39 up to 12.5 mg/mL.Such differences cannot be readily appreciated when testing only one strain per species.
The use of several species allows for the demonstration of interspecies differences in the sensitivity to plant compounds within a given genus.One such notable example from this study is the low sensitivity of C. krusei to essential oils compared to other species of the Candida genus.Such differences cannot be elucidated by studies in which only one species of a given genus was tested [42,45,47,48,50,51,54].Furthermore, in the case of natural substances, phytochemical analysis is important, allowing for the comparison of substances originating, for example, from different regions of the world or isolated using different methods.Unfortunately, in some publications, phytochemical data are not included or data from the general literature is provided, this does not accurately reflect the actual composition of the substances used in the studies [44,52,54].
The large number of clinical Candida isolates used in this investigation lends to its broad applicability.Most of the available literature focuses on single reference strains with clinical isolates being excluded entirely [42,45,47,[49][50][51]54,55].To the best of our knowledge, this is the first study to investigate the in silico toxicity prediction of the main compounds of Lamiaceae essential oils.The present investigation also demonstrates via in silico methods that the majority of the compounds in the Lamiaceae essential oils used in this study do not exhibit toxicity.Unfortunately, there are few publications assessing the toxicity of the essential oils or their constituent compounds used in the present study.In the available studies on mice and rats, essential oils and extracts from Lamiaceae are non-toxic or slightly toxic [61][62][63].These studies lend support to the in silico findings reported here.

Conclusions
Essential oils from lemon balm and oregano exerted the most potent anti-Candida activity, with MIC values below 3.125 mg/mL.Lavender, mint, rosemary, and thyme essential oils also inhibit growth in the range of 0.39 to 6.25 or 12.5 mg/mL.The weakest activity was observed with sage essential oil, for which the MIC values ranged from 3.125 to 100 mg/mL for single C. krusei and C. parapsilosis strains.All tested essential oils can also inhibit Candida albicans biofilm formation.The majority of main compounds found in the Lamiaceae essential oils used in this study did not exhibit toxicity in silico, but probably can sensitize the skin.

Fig. 1 .
Fig. 1.Representative MIC determination setup for 6 tested Candida species on 96-well plates.Pink color indicates fungal growth.