In Vitro Evaluation of Antiprotozoal Properties, Cytotoxicity Effect and Anticancer Activity of New Essential-Oil Based Phytoncide Mixtures

Protozoa, in both humans and animals, are one of the leading causes of disease. International programmes introduced in many countries have helped reduce the incidence of disease. However, it has recently become increasingly difficult to achieve the goals set for the coming years. One of the main reasons for this, as with other pathogenic organisms, such as bacteria and fungi, is the increasing resistance to current methods of treating and preventing infection. Therefore, new therapies with high efficacy are needed. In the present study, the novel mixtures of essential oils (EOs), clove, garlic, Ceylon cinnamon, and rosemary with organic acids (acetic, propionic, lactic) and metal ions (Cu, Mn, Zn) were tested against five selected model protozoa (Euglena gracilis, Gregarina blattarum, Amoeba proteus, Paramecium caudatum, Pentatrichomonas hominis). The cytotoxicity and potential anticancer activity of the obtained combinations were tested on the human fibroblasts (NHDF) and human cancer cell lines (A549, MCF7, LoVo, HT29). All of the mixtures showed very good antiprotozoal properties. The most efficient were the combination of clove and rosemary essential oils, mixtures of acids, and Mn ions. The LD50 values were in the range of 0.001–0.006% and the LD100 values were 0.002–0.008%. All of the tested mixtures did not show cytotoxicity against normal cells, but did show growth inhibition against cancer cell lines. The most cytotoxic against cancer cells were combinations with cinnamon essential oil. Nevertheless, the proposed combinations containing essential oils, organic acids, and metal ions have high antiprotozoal activity, with low toxicity to healthy human cells.


Introduction
Protozoa-caused diseases are a significant problem that affect millions of people around the world, directly and indirectly. The most spread and deadliest are malaria, babesiosis, human African trypanosomiasis Chagas disease and toxoplasmosis. In 2021, malaria caused 619,000 deaths globally, 95% of which were in 29 countries [1]. COVID-19, the pandemic spread all over the world, caused a long period of isolation in many countries, and between December 2019 and January 2023, caused more than 6.7 million deaths globally [2]. Diseases caused by protozoa are also a challenge in animal husbandry, such as broiler chickens, laying hens or swine, at every stage of life. Each year, there are epithelial fibroblasts and several human cancer cell lines as a widely used model.

Activity against Selected Protozoa
The LD 50 and LD 100 values of 48 different combinations were determined against the selected protozoa. Each composition is shown in the Table 1. Two common antibiotics, chloramphenicol and metronidazole, were used as reference substances. The essential oils used in the study, without organic acid or mineral salts, showed very promising results in some of the analyses, comparable to the effectiveness of antibiotics. The most promising and strongest antiprotozoal activity was obtained for clove oil. However, the other essential oils also showed potential for further research.  The mixtures containing an essential oil, a single acid and a metal ion were characterized by significantly better antiprotozoal efficacy than essential oils alone. As can be observed, the effective antiprotozoal concentration was much lower than LD 50 and LD 100 for the pure essential oils. What is more important, they were lower than LD 50 and LD 100 values obtained for the antibiotics used as a reference substances. This fact can be observed regardless of the essential oil. In compositions containing essential oil, metal ions and a single organic acid, as in the case of a single essential oil, the strongest antiprotozoal properties were shown by all of the combinations containing clove essential oil. For LD 50 , it was 0.01-0.02%, while LD 100 was 0.015-0.03%. However, for the other essential oils and their combinations with acids, the LD 50 and LD 100 values were 0.01-0.05% and 0.02-0.07%, respectively. Combinations containing garlic, cinnamon and rosemary oils, in contrast to the clove essential oil mixtures, showed relatively weak antiprotozoal activity against Pentatrichomonas hominis. The best antiprotozoal properties against all of the tested organisms were observed for combinations with lactic acid and Mn ions, for all of the essential oils used.
However, regardless of the essential oil used, the best results were obtained for the composition containing a mixture of organic acids. The combinations that presented the most powerful antiprotozoal properties were those that included clove essential oil. The LD 50 and LD 100 values for the SMMn and SMZn were in the range of 0.001-0.003% and 0.002-0.005%, respectively. The SMCu mixture shows slightly weaker properties, with LD 50 : 0.001-0.004% and LD 100 : 0.003-0.006%. Very good antiprotozoal activity was shown in all three combinations with rosemary essential oil (RMCu, RMMn and RMZn). The LD 50 values were very promising (0.001-0.003%); however, further tests showed that to reach LD 100 values, much more concentrated solutions were needed (0.004-0.009%). The remaining essential oils obtained results that were not as promising as the previous two; nevertheless, they also showed very good antiprotozoal activity, higher than that of the antibiotics currently used. Two combinations containing manganese ions (GMMn and CMMn) showed the lowest LD 50 and LD 100 values for garlic and cinnamon essential oils. All of the results are presented in Tables 2-9.

Compositions Analysis (GC-MS)
Tables 10 and 11 show that the compositions contained typical compounds for clove and garlic essential oils compared to the retention indices from the Adams and NIST databases. Table 12 presents the results of NMR ( 1 H and 13 C) analyses. Composition of Ceylon cinnamon and rosemary essential oils are presented in Tables 13 and 14. 2.2.1. Clove Essential Oil Two of the essential oils used, clove and cinnamon, were characterized by a relatively high dominance of one main compound in the composition. In the case of the clove bud essential oil, eugenol accounted for more than 70% of the content. This composition was complemented by caryophyllene (14%) eugenyl acetate (9.1%) and humulene (3.4%).

Garlic Essential Oil
The garlic essential oil did not contain a single main compound and its composition of individual molecules are relatively evenly distributed among several of them. It contained the vast majority of allicin derivatives. These were, respectively, diallyl disulphide (30.7%), diallyl trisulfide (25%) and diallyl tetrasulfide (14.5%).
Although there are many publications that describe the composition of essential oils extracted from garlic by gas chromatography, there are many doubts about the actual accuracy of such a measurement [33]. The thermal degradation of diallyl disulphides at about 150 • C (CG condition) to mono-, tri-, and terta-disulphides, as well as their rearrangement to heterocyclic thiopyranes, trithiolane or tetrathianes, has been demonstrated [34,35]. We decided to carry out a comparative analysis of garlic EO, using the mildest method, which was nuclear magnetic resonance spectroscopy (NMR). This technique does not require exposure to high temperatures and allows for the unambiguous determination of the presence of mono-and ooligothio-derivatives of diallyl compounds. The methylene group of the allyl fragment was very well separated on the 1 H NMR spectrum in the 3.1 to 3.65 ppm region. The 13 C, as well as the correlative spectra, confirmed the unequivocal identification of the compounds. We obtained different values from those presented in Table 12 for the proportion of diallyl derivatives. As a predominate, we found diallyl disulphide, which was presented in nearly 50% of the EO. Tri-and hexa-allyl disulfides were the next compounds present in the mix, which were higher than 10%. Contrary to the GC-MS profile, diallyl tetrasulfide was detected bellow the quantification limit. The share of the latter compound in the chromatographic analysis was as high as 14%. As there are no reports describing the chemical shifts of diallyl heptasulphide, we assumed the presence of this compound in the EO. Additionally, there are no EI-MS spectra or RI values in the NIST databases. NMR spectra's are presented at Figures 1 and 2.

Rosemary Essential Oil
The rosemary essential oil was characterized by the presence of four main compounds, accounting for more than 70% of its composition. Unfortunately, the relative proximity of the peaks resulting from the KI values (1032 and 1035) for limonene and eucalyptol, respectively, prevented their effective separation and verification of their intrinsic validity. However, collectively, they account for 47.9% of the composition of the essential oil of rosemary. Other constituents included in the composition of this essential oil were α-pinene (13.1%) and camphor (11.75%).

Evaluation of Biological Activity In Vitro
The assessment of the impact on cell cultures was performed using the sulforhodamine B (SRB) assay. This test is a good tool for preliminary studies. From the results of the test, both the cytotoxic and cytostatic effects of the tested compounds can be determined. The test measures both the effects on the basis of the amount of protein, and the result is not influenced by the inhibition or generation of free radicals by the compounds. The compounds tested are oils. Cancer cells, including those used in the tests, have a simplified morphological structure and physiology adapted to rapid cell division. The test compounds, oils, are, due to their nature, easily penetrated into the cells. Tumors cells, by virtue of their physiology, are unable to metabolize such compounds. This usually results in the inhibition of cell growth and, to a lesser extent, apoptosis or cytotoxicity.
As a result of the study, no cytotoxicity of the tested compounds was found in the tested concentration range. A decrease in the amount of protein was observed in the test cultures below the amount at the beginning of the test. Such effects are seen in the case of cytostics or other compounds with strong toxicity. Example photographs of cell cultures are included in the supplementary materials ( Figure S2).  show the IC 50 values for the four oils tested, together with their modifications, for normal fibroblast (NHDF) cultures and the selected tumor lines. The results obtained indicate a cytostatic effect of the tested oils. The tests showed a higher sensitivity of the tumor cells compared to the fibroblast cultures. The greatest differences in activity, seen in Table 15, are for cinnamon oil. The weakest activity was found for rosemary oil. In the case of this oil, the use of additives achieved the inhibition of cancer cell growth ( Table 18). The garlic (Table 16) and clove (Table 17) oils also showed strong effects on cancer cells and weaker effects on normal cells.

Discussion
The presented tests, with combinations containing an essential oil (clove, Ceylon cinnamon, garlic or rosemary), organic acid or its mixture (acetic, propionic and lactic acid) and metal ions (Cu, Mn or Zn), revealed antiprotozoal properties. The idea of preparing a complexed mixture was based on our previous research and test. Many scientific reports demonstrate the antiprotozoal activity of essential oils or even mixtures containing two constituents, but there is a lack of data regarding compositions containing essential oils, organic acid and metal ions. [36][37][38][39].
The combinations with clove essential oil had the best antiprotozoal properties of all the mixtures used. The clove essential oil also has very good antiprotozoal properties [40]. The obtained LD 50 and LD 100 values were between 0.1-0.3%. Santoro et al., observed significantly better properties against the various stages of the development of Trypanosoma cruzi than with eugenol alone [39]. A similar situation occurred in our study, where more unexpected values were reached for the combinations with organic acids and metal ions. The LD 50 and LD 100 were 0.001-0.04% and 0.001-0.005%, respectively. This suggests a synergistic effect between the components used. The GC-MS analysis proved that the clove essential oil used was derived from buds. This is evidenced by the content of eugenol (71.4%), trans-caryophyllene (14%) and, most importantly, the presence of eugenyl acetate (9.1%) [41][42][43].
Other very promising results were obtained for the Ceylon cinnamon essential oil. The pure essential oil was very effective. However, further tests, with combinations containing acids and metal ions, were not as exceptional as had been expected. The GC-MS analyses showed the content of the main compound, trans-cinnamaldehyde, was 54.7%. This is a relatively low content of the head compound of cinnamon essential oil, which is in the range 65-75% [44][45][46]. In the case of the tested sample, the results were comparable with those presented by Martiniaková et al. The trans-cinnamaldehyde content was similar, but the other compounds were present in comparable amounts [47].
The mixtures containing garlic essential oils showed good results. The best antiprotozoal results for the combinations with this oil were obtained against Amoeba proteus. The same results and conclusions were suggested by Behnia et al. [48]; in their studies against Entamoeba histolica, they demonstrated the antiamoeba properties of different extracts and garlic essential oil. The activity was time-dependent and the exposure for the solutions was 24 h and 48 h. These findings correspond to the results presented in our studies. Further tests showed that combinations with garlic essential oil had good antiprotozoal properties, but the lowest from the analyzed samples.
At the initial stage if the study, the rosemary essential oil did not show the best antiprotozoal properties of the tested essential oils. The values of LD 50 and LD 100 were at the level of 0.1-0.3% and 0.4-0.6%, respectively. The situation was similar for its combination with single acids. However, the combination with a mixture of acids proved to be very effective against all of the protozoa tested, reaching LD 50 : 0.001-0.007% and LD 100 : 0.003-0.009% values.
At present, many literature reports can be found on combining various types of compounds and essential oils with metal ions. In these studies, a definite enhancement of their effect can be observed [32,49]. The following results confirm that the use of the addition of metal ions and organic acids increases the antiprotozoal properties of the essential oils.
One of the most serious problems arising from the use of new substances, whether in pharmaceuticals, medicine or human and animal nutrition, is the lack of data on the toxicology of new compounds, their composition or metabolites. New formulations with very promising results have often not been used in a wider context due to side effects and toxicity. The situation is similar in the case of essential oils, where sufficiently high concentrations can cause adverse reactions or toxic effects. For example, clove essential oil may act as an irritant. On the other hand, some essential oils and their main constituents, such as Rosmarinus officinalis (camphor), Eucalyptus globulus (1,8-cineole) and Mentha species (menthone and menthol), and, in particular, Mentha pulegium (pulegone), may cause hepatic damage, convulsions and hallucinations. They are also forbidden in pregnancy due to their abortifacient effect [50,51]. In our study, the toxic effect of the prepared mixtures was tested on several cell lines and cancer cell lines, for example, NHDF (normal human dermal fibroblasts), A549 (lung cancer) or MCF7 (human breast cancer). All of the essential oils used in this study have been reported as potential anticancer agents [32,[52][53][54][55].
The results indicate the low toxicity of the tested oils towards normal cells and the inhibition of cancer cell growth. There were no results indicating strong cytotoxicity of the tested compounds, the main effect being the inhibition of cell growth. This confirms the safety of the tested oils. The results obtained may be indicative of their chemopreventive properties. Due to their volatile properties, the tested oils can easily work in the respiratory tract or digestive tract when applied indoors, in the same way as with pollutants reaching our body. The results obtained show a selective action of the oils against cancer cells, with the best effect seen for cinnamon oil (4-12 × stronger effect). Modification, through the addition of metals to the oils, also resulted in an improved anticancer effect. The addition of Zn significantly improved the activity of cinnamon oil (1.7-2.2 × potent, depending on the line tested) and is also important for its protective effect against upper respiratory tract infections.
All of the obtained results are very promising in terms of either their antiprotozoal activity or low cytotoxicity and their possession of good anticancer properties. Low cytotoxicity is the first step to determine the safety of the combinations for human and animals. Unfortunately, heavy metals can accumulate in tissues and organs and can cause several disorders. Moreover, some essential oils can cause allergic reactions and organic acids can cause skin and mucosal irritation. The wider use of the mixtures, for diseases caused by protozoa or cancer treatments, requires more tests and further investigation, e.g., in vivo trials. In vivo trials performed on animals, which would include intentional infection, require special ethic approval of the Local Ethics Committee.

Maintenance of Parasite Cultures and Evaluation of Antiprotozoal Activity
For the further tests, five different protozoa were chosen. They belong to the same taxonomic groups as the most widespread pathogenic protozoa. Three of them, Amoeba proteus, Paramecium caudatum and Euglena gracilis, represent aquatic protozoa, while Gregarina blattarum, Pentatrichomonas hominis live in the digestive track of cockroaches and humans, respectively.
All of the analyzed protozoa were cultivated under different conditions. Hay infusion was the medium for the cultivation of Paramecium [58][59][60]. For Euglena, the solution was used according to Wu et al. [61]. Pentatrichomonas hominis was kept in solution according to Chomicz et al. after it was isolated from stool samples [62]. Tetrahymena and Chilomonas were ciliates, used as an example feed for Amoeba proteus, and were cultivated in Prescott medium [63,64]. Only one protozoa was not cultivated and was isolated from the cockroaches. Gregarines, after isolation, proposed by J. Moraczewski [65], were placed in Ringer solution on a watch glass.
To determine the LD 50 and LD 100 values, different combinations and their concentrations were tested. Each time, four-fold replicates and blank were used. The Reed-Muench method was used for determining the LD 50 and LD 100 values. The protozoa were treated with different concentrations of the samples and observed on a watch glass, for 3 to 5 min.
The resulting phytoncide-metal mixture and phytoncide alone were dissolved in an aqueous solution of polysorbate 80 (0.05%) and applied to the watch glass. No biocidal activity of polysorbate 80 was observed at these concentrations. Chloramphenicol and metronidazole were used as standard substances for the control of protozoa. Antibiotics were used at a concentration of 5 mg/mL and diluted from stock solutions to achieve LD 50 and LD 100 .

Essential Oils
The essential oils were purchased from three companies. The cinnamon essential oil was provided by Food Base Kft. (Gödöllő, Hungary); the garlic essential oil from Synthite Industries Pvt., Ltd. (Kolenchery, Kerala, India); the rosemary and clove essential oil from De Monchy Aromatics Ltd. (Poole, Dorset, UK).

Chemicals and Reagents
The organic acids (acetic acid 99%, propionic acid 99.5% and lactic acid 85%) and other chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and comply with FCC and FG standards. The standards for chromatographical analyses were bought in Sigma-Aldrich (St. Louis, MO, USA), UQF (Wrocław, Poland) and Metasci (Toronto, ON, Canada), as well as our own collection of chemicals in the Department of Food Chemistry and Biocatalysis. The purity and percentage composition, according to the supplier's specification, was minimum ≥95%.

Phytoncides Mixture Preparation
Organic acids and essential oils were mixed in the same amount (100 mL). The mixture of organic acids was prepared in ratio 1:1:1. The composition was mixed, and mineral salts were added in the amount of 5 g. The salts were: manganese (II) chloride (2.18 g of ions Mn 2+ ), copper (II) carbonate hydroxide (2.87 g of ions Cu 2+ ) and zinc carbonate (2.61 g of ions Zn 2+ ). The composition was heated and then left to cool overnight. After that time, the solution of one, two or three phases was filtered through paper filter. The composition was diluted to prepare a solution in the range of 0.001% to 1.5%.

GC-MS Analysis
The profile of the essential oils investigated was evaluated using the GC-MS technique, according to the protocol [66]. The identification of all of the volatile components was based on a comparison of the mass spectra with the mass spectra of the compound obtained experimentally, available in the NIST20 database. Additionally, the retention indices (RI), obtained experimentally, were calculated using macro [67] and were compared with the RI available in the NIST20 database and the data from the literature [68]. GCMS Post-run analysis software version 4.45 (Shimadzu Company, Kyoto, Japan) and ACD/Spectrus Processor (Advanced Chemistry Development, Inc., Toronto, ON, Canada) were used to process the data. The quantification of the identified constituents was performed by calculation based on the amount of added internal standard and expressed as a percentage of the integrated peaks' area. Analysis was performed using the Shimadzu 2020 apparatus (Varian, Walnut Creek, CA, USA) equipped with a Zebron ZB-5 MSI column (30 m × 0.25 mm × 0.25 µm) column (Phenomenex, Torrance, CA, USA). The temperature of the GC oven was programmed from 50 • C to 250 • C at a rate of 3.0 • C and kept for 3 min. Scanning was performed from 35 to 550 m/z in electronic impact (EI) at 70 eV and ion source temperature 250 • C. Samples were injected at split ratio 1:10 and gas helium was used as the carrier gas at a flow rate of 1.0 mL/min. Garlic and rosemary Eos were analyzed on Varian CP-3800/Saturn 2000 apparatus (Varian, Walnut Creek, CA, USA) and compounds were separated by on Zebron ZB-5MSi (30 m × 0.25 mm × 0.25 µm) column. GC temperature program: initially 50 • C, then to 180 • C at 4.0 • C/min ratio, and finally to 250 • C at 10 • C/min ratio. As a carrier gas helium with linear velocity 35.0 cm/s; split ratio 1:10 was used. MS operational conditions: ion source temperature 250 • C; electron impact (EI) ionization at 70 eV; scanning range between 35 and 300 m/z.

NMR Measurement
The 1 H NMR and 13 C spectra of EOs were recorded in a CDCl 3 solution on a Bruker Avance™ 600 MHz spectrometer (Bruker, Billerica, MA, USA). Two different measurements, that is 25 µL (for 1 H measurement) or 250 µL for 13 C and correlative spectra), of essential oil were dissolved in 600 µL of CDCl 3 to record the spectra. The data were processed on the ACD Spectrus Processor 2021.2.1, Advanced Chemistry Development, Inc. Toronto, ON, Canada.

Cell Culture
Normal cell lines-dermal fibroblasts (NHDF) purchased from LONZA (Verviers, Belgium) and cancer cell lines A549 (lung cancer), MCF7 (breast cancer) and LOVO and HT29 (colorectal adenocarcinomas) were used in the study. Tumour lines were purchased from the European Collection of Authenticated Cell Cultures (ECACC). The cells were cultured under standard 37 • C and 5% CO 2 conditions. The cell lines were thawed for a minimum of 2 weeks prior to the start of the study and passaged after reaching full confluence. The cells were cultured in medium supplemented with 10% FBS and appropriate NHDF, HT29-DMEM, A549 and MCF7-EMEM, LOVO-DMEM/F12 media. The media were supplemented with antibiotics and L-glutamine. All of the reagents were purchased from Biological Industries-now part of Sartorius (Kibbutz Beit Haemek, Israel). Materials for culture bottles, plates, tubes were purchased from SPL Life Sciences (Pocheon, Korea).

Evaluation of Biological Activity on Cell Cultures
The activity assessment was performed according to the National Cancer Institute guidelines for screening human tumour lines and based on the basis of our own studies [69]. Tumour cell lines and normal human cells cultured in a suitable medium containing 5% fetal serum were used for the study. To prepare the cells for the experiment, the medium was harvested and inactivated with trypsin. Trypsin has the effect of detaching cells from the medium. A portion of the cells were then collected into tubes and trypsin inactivated with the medium. The quality and viability of the cells used in the study was measured using a NucleoCounter ® NC-200 reader (Chemometec, Denmark).vCell viability is measured using dedicated cassettes containing acridine orange (AO) and 4 ,6-diamidino-2-phenylindole (DAPI).
The cells were counted in line NC200 (Chemometek, Allerod, Denmark) and the final cell count after inoculation in a 96-well plate was approximately 1 million cells (1 × 10 4 cells per well). After inoculation, the plates were incubated at 37 • C, 5% CO 2 and 100% relative humidity for 24 h before phytoncides were added. After 24 h of incubation, one plate from each cell line was fixed with 50% (w/v) TCA to represent cell population measurements for each tumour line at the time of oil addition (T0). The oils were prepared at a concentration of 10 mg/mL and the volume added to each microtiter well was 1 mg/mL, 0.5 mg/mL and 0.1 mg/mL, respectively. The plates were then incubated for an additional 48 h at 37 • C, 5% CO 2 , 95% air and 100% relative humidity. After this time, the contents of the wells were fixed by adding 30 µL of 50% (w/v) TCA and incubated for 60 min at 4 • C. The supernatant was discarded and the plates were washed five times with tap water and air-dried. To each well, 100 µL of a 0.4% (w/v) solution of sulforhodamine B (SRB) in 1% acetic acid was added and plates were incubated for 10 min at room temperature. After staining, the unbound dye was removed by washing five times with 1% acetic acid and the plates were air-dried. Doxorubicin was used as the positive control, in final concentration 10 µM. The bound dye was dissolved in 10 mM Trisma Base and the absorbance was read on a MultiscanGo reader (Thermo Scientific, Waltham, MA, USA) at 515 nm.
The absorption results obtained were compared with those of the control and the T0 control. In the case of absorbance values below the T0 control, such a result provided information on the cytotoxic properties of the tested compounds. If the results were between T0 and the control value, the result indicated an inhibitory effect on cell growth.

Conclusions
The proposed new mixtures containing essential oils, organic acids and metal ions have not yet been obtained and described in the scientific literature. Those combinations containing three different active substances showed very high antiprotozoal efficacy at very low concentrations. The most effective combinations turned out to be solutions containing, in addition to essential oil and metal ions, a mixture of organic acids. The most effective combinations against the analyzed protozoa were those containing clove essential oil. Very similar results were also obtained for rosemary essential oil. A slightly lower effectiveness was characterized by mixtures with garlic and ceylon cinnamon essential oils. However, it should be noted that even the least effective combinations, in most cases, were significantly more effective than the reference substances, chloramphenicol and metronidazole. Moreover, the analyses of the cytotoxic effect against human cancer cell lines showed a very promising effect. The tests performed on normal human cell lines showed low toxic effects. However, on the other hand, the tested compositions inhibited the growth of cancer cells. The highest anticancer, or more precisely, chemopreventive properties, were obtained for the cinnamon essential oil mixtures and combinations containing zinc ions.
The collected data allow us to conclude that the discovered combinations, under in vitro conditions, have very good antiprotozoal properties and, importantly, they have low toxicity against healthy human cell lines and inhibit the growth of cancer cell lines. Further studies should be conducted to determine their potential side effects, metabolism and accumulation in tissues under in vivo conditions. Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules28031395/s1, links for the NMR spectra and GC-MS chromatograms Figure S1: MS spectrum of an unknown compound (nr 8) in clove EO; Figure S2. Evaluation of cytotoxicity of test oils (1 mg/mL) to normal human fibroblasts after 48 h of incubation.

Data Availability Statement:
The data presented in this study are available in the tables and supplementary materials.

Conflicts of Interest:
The authors declare no conflict of interest.