Trichomonas gallinae (T. gallinae) is a flagellated parasite of the oropharynx and causes a potentially life-threatening disease, oral trichomonosis. The main reservoirs are Columbiformes, but many avian species, including domestic and wild birds, can be infected (1). Nitroimidazoles are the drugs of choice for treatment, but several studies reported resistant strains, both, in vivo (2–5) and in vitro (3, 4, 6–8). Since the ban of the use of nitroimidazoles for prevention in the EU and the USA, there is no alternative to prevent the disease (9).

Natural products are compounds obtained from living organisms that contain secondary metabolites that help them to survive in adverse environments or even to defend against pathogens. Among them, Essential Oils (EOs) are one of the more frequently employed for diverse uses, including antioxidant, anti-inflammatory and antimicrobial activity, which encompass virus, bacteria, fungi, and parasites (10, 11). Their composition is variable depending on the species, soil conditions, fertilizers, origin, climate, and mode of extraction, as examples, and for that reason it is difficult to assess their effects. Between 10 and 60 components are usually found in their composition, although up to 200–400 different substances are described in some of them (10, 11). However, only the major components, usually 2–4, are thought to be responsible for the attributed properties. Terpenes, especially monoterpenes and sesquiterpenes, are considered the most important components of EOs.

There is little information on the use of natural products against T. gallinae and few reports were found in the literature, including some EOs from the Asteraceae and Geraniaceae families (12, 13) but also from other medicinal plants such as Cymbopogon flexuosus (14) and Dennetia tripetala (15). Besides EOs, aqueous, alkaloid and ethanol and methanol extracts from different plants have been tested against T. gallinae, including plants from Lamiaceae and Asteraceae families, such as Lavandula angustifolia, Rosmarinus officinalis and Artemisia annua, and also extracts from other plants: Allium sativum, Harungana madagascariensis, Zingiber officinale, Myrtus commuis, Zataria multiflora, Quercus persica, Lycopus europaeus, Pulycaria disenterica, Eugenia uniflora, Murraya koenigii, Peganum harmala, Clausena lansium (15–24). However, while EOs displayed activity against T. gallinae, most of the above cited extracts were less active, and only four publications mention anti-trichomonal (AT) activity of alcoholic or alkaloid extracts comparable to that of metronidazole at a similar dose, extracts from Clausena lansium (18), Peganum harmala (19), Eugenia uniflora (20), Lavandula angustifolia, and Zingiber officinale (24).

The research of natural products against Trichomonas vaginalis (T. vaginalis) is a little wider, and EOs from several plant genera, such as Lavandula, Nectandra and Nigella were tested with satisfactory anti-trichomonal effects, and around 30 compounds from potatoes, tomatoes, and several medicinal plants have been identified having anti-protozoal activities in different bioassays [revised in Friedman et al. (25)].

EOs components from the families Lamiaceae and Asteraceae have shown antimicrobial and insect repellent properties (11), but only a scarce number of publications proved their activity against trichomonads. In this study, we have analyzed the potential use as anti-T. gallinae of 36 EOs obtained by two classical methods of EOs extraction: hydrodistillation (HD) and steam distillation (SD). We have also evaluated the main compounds from these EOs against the avian parasite Trichomonas gallinae and their cytotoxic effects on African green monkey kidney cells (Vero cells).

Several anti-trichomonas EOs, extracts and compounds from plants have been tested against trichomonads, mainly against the human pathogen T. vaginalis. Among them, the Lamiaceae family with more than 230 genera, usually aromatic, and the Asteraceae family, with worldwide distribution and more than 1,600 genera, stand out (32). Species from Lavandula and Salvia (24, 33), Artemisia (12), and Thymus (34) have been more extensively explored with these parasites. In our study, the best results were obtained from L. luisieri (both populations) while lower activity was observed with other Lavandula species or varieties, such as L. angustifolia or L. x intermedia, which displayed activity against Giardia, Hexamita or T. vaginalis in other studies (33). These slight disagreement between the studies could be due to differences in the composition, since it may vary with the diverse conditions of growing, climate or part of the plant collected, but also to the selected methodology employed in the present study.

Also, a remarkable activity was observed with EOs from Salvia (S. sclarea, S. hibrida and S. officinalis) and Thymus (T. vulgaris and T. zygis) species, which agrees with the results obtained from other authors testing AT activity (32, 35). Within the list of our best AT active EOs, Origanum and Satureja species (O. vulgare and O. majorana and S. montana) are included. There are no other anti-trichomonads assays with these two genera, but some authors demonstrated their activity against other protozoa such as Leishmania and Giardia (35, 36), as well as with some fungi and bacteria (37, 38).

Satisfactory results were also obtained in our study from R. officinalis and M. suaveolens, the former plant being mentioned in one study with moderate activity against T. gallinae (22) and presenting activity against Plasmodium falciparum in another study (35). Although the AT activity was good, they were over the cutoff point value in our study (IC₅₀ <200 μg/ml), and for that reason they were not included in later experiments.

Other Asteraceae from the genus Tanacetum showed anti-trichomonal effect in previous studies (32), but in our case, only D. graveolens displayed moderate results at intermediate doses. Due to the lower AT activity, we did not include any of the Asteraceae EOs in further analysis.

Many natural products assayed against trichomonads from the Lamiaceae are EOs, while compounds from other plants were extracted employing other methods, such as ethanolic, methanolic or alkaloid extraction, as examples. Among the extracts tested against T. gallinae trophozoites, many needed high amounts of extract to obtain a good AT activity, such as the aqueous garlic (Allium sativum) extract (16), and alcoholic extracts of Lycopus europaeus and Pulicaris dysenterica (21), Quercus persica, Artemisia annua, Myrtus commuis, Zataria multiflora, R. officinalis, Allium sativum (22) and Harungana madagascariensis (23). Others showed some kind of toxicity, such as the alkaloid extract of Murraya koenigii (17). Finally, some of the previously cited assayed extracts were not fully characterized, the major components were not described, or the potential toxic effects were not tested (22), and for these reasons and the differences in composition, we could not include them in the comparison with the result of the present study.

Regarding AT active EOs, four plants have been tested against T. gallinae so far, Dinettia tripetala (Annonaceae), Artemisia sieberi (Asteraceae), Pelargonium roseum (Geraniaceae) and Cymbopogon flexuosus (Poaceae) (12–15). Except C. flexuosus (14), that did not share components with the Lamiaceae tested, the other plants EOs shared components with the ones evaluated by us and will be discussed further.

Some of the common compounds were not highly active against trichomonads when tested alone in the experiments conducted in the present study. For example, linalool was present in the EO composition of P. roseum and D. tripetala, two plants with AT activity in vitro and in vivo (13, 15). Although linalool has been suggested as having activity against Trichomonas, under our conditions, linalool was not an active AT compound, and the activity of those EOs could be due to other main components, beta-citronellol, geraniol in P. roseum EO (13), 2-phenylnitroethane in D. tripetala EO (15), ocimene, camphor, linalyl acetate, borneol, 1-8-cineol, and α-pinene in the ethanolic extracts of L. angustifolia (24), or even to synergistic effect between them. Besides, linalool showed a positive correlation with the IC₅₀ in our study, indicating that those EOs with higher amounts of linalool had lower AT activity. Besides linalool, other compounds with scarce AT activity in our study, but present in the composition of active AT EOs or extract, are camphor and 1,8-cineol in A. sieberi EO (12) or the ethanolic extract of L. angustifolia (24). In the same way that linalool, 1,8 cineole had an inverse correlation with AT activity.

Thujones, mainly α and β thujones, are the most active anti-Trichomonas compounds in our study. These compounds are present also in the composition of A. sieberi EOs (12), which was a suitable candidate to treat T. gallinae, according to in vitro and in vivo studies. Although the authors did not report toxicity in the in vivo experiment, caution should be taken with these compounds since they are recognized as neurotoxic (31).

Camphene was one of the most active against T. gallinae when tested under our conditions. This compound was present in high amounts (higher than 5%) in the EO from Salvia hibrida, one of the most active EO tested in the present study, and the abundance of this compound was higher in the EO extracted by SD (7.6%, the most active of both EOs) than the EO extracted by HD (2.8%, the least active of both EOs). Camphene was also present as a major component of A. sieberi EO, which was highly active against T. gallinae both, in vivo and in vitro (12). Camphene was slightly cytotoxic for Vero cells, but it might be useful if adjusting the dose according to the SI, since it was the highest one, excepting the potential toxic thujones.

Two compounds are included in the composition of highly active extracts against trichomonads assayed by other authors and the present study: linalyl acetate and α-pinene. They were between the main components of the AT active ethanolic extract of L. angustifolia (24) and EOs from S. sclarea, O. virens, and T. zygis. Linalyl acetate is a suitable candidate as anti-trichomonal, since the IC₅₀ for T. gallinae trophozoites was low (32.2), and the SI high (3.1), and no toxicity was found in Vero cells. Besides, in the S. sclarea EOs, linalyl acetate appears with a concentration of more than double in the HD extract (31.20% in HD vs. 14.48% in SD), which could explain the higher activity of the EO extracted by HD vs. the EO extracted by SD. On the other hand, α-pinene was present in considerable amounts in S. hibrida EO extracted by SD (6.3%), which was more active than the S. hibrida EO extracted by HD (4.3%). The anti-trypanosomatid capacity of some EOs, including Leishmania major and Trypanosoma brucei, was also attributed to α-pinene (35, 39).

Regarding β-pinene, it has been proven as an active anti-T. vaginalis compound in another study (32). Within the major compounds of the Salvia species evaluated, β-pinene were found in higher concentration in the extracts of S. officinalis and S. hibrida obtained by SD, which may explain the higher activity, compared to HD extracts. We must keep in mind the slight toxicity of the compound, but β-pinene is the third compound with better AT activity, and for that reason we still consider that both, α and β-pinenes, are good candidates as anti-trichomonads pure compounds.

There are three compounds that appeared with high abundance in several of the most active EOs evaluated in our study: p-cymene, γ-terpinene, and thymol, which were positively correlated with the AT activity. These three compounds are more abundant in EOs from T. vulgaris and T. zygis extracted by HD, which displayed higher activity when compared with the EOs extracted by SD. These compounds are also included among the major components of both species of Origanum, O. majorana and O. vulgaris. The EO of O. majorana extracted by SD, which displayed higher AT activity, had higher abundance of thymol and p-cymene, but lower amount of γ-terpinene. We found a similar situation with EOs from O. vulgare, but, in any case, IC₅₀ from all these EOs were similar, both, for O majorana (IC₅₀ = 139.7 for EO by SD vs. IC₅₀ = 158.8 for EO by HD) and for O. virens (IC₅₀ = 175.4 for EO by SD vs. IC₅₀ = 197.9 for EO by HD). The compounds were also present at high concentration in the EOs from S. montana, although the proportions were higher in the less active EO (extracted by HD). Maybe the higher concentration of carvacrol and β-caryophyllene in the EO from S. montana extracted by SD may act synergistically with the other three compounds, as it has been observed in previous studies (40). Two of these compounds, p-cymene and γ-terpinene have also shown activity against T. vaginalis (38, 41), and their combination together with thymol in the most active EOs is frequent, possibly acting in a synergistically manner, at it has been previously proven with other protozoa (40). The activity of these compounds have been demonstrated against other protozoa as well, such as p-cymene against Plasmodium falciparum (40) and thymol against T. brucei and T. cruzi (35). There is a high agreement that these three compounds are good AT compounds considering different studies and authors.

In other studies, caryophyllene oxide and β-caryophyllene were found active against T. vaginalis (32). However, under our conditions, they displayed a slight activity against T. gallinae, and for that reason they are not included within the list of better anti-T. gallinae compounds in the present study. The EO from S. officinalis extracted SD showed better AT activity, and higher abundance of β-caryophyllene. Also, the EO from S. officinalis extracted by HD had higher abundance of 1,8-cineole, a compound that is inversely correlated with the anti-T. gallinae activity in our conditions. Maybe antagonistic interactions could be acting between β-caryophyllene and 1,8-cineole.

According to our correlation analyses, assays employing α-terpinene could also be of interest and should be further studied in future assays. Also, Germacrene D, lavandulol, trans-α-necrodyl acetate, and an unidentified compound were present at percentages higher than 5% in the most active EOs tested in the present study, L. luisieri “2” extracted by SD. Lavandulol showed a negative correlation with AT activity and therefore discarded for future studies. Trans-α-necrodyl acetate is another interesting compound to evaluate due to its highly abundance (18%) in the most active EO (L. luisieri “2” by SD). However, it was not significatively correlated with the IC₅₀ of the EOs, which could be due to absence of this compound in other plants EOs.

Comparing the chemical structures of the tested terpenes it is evident that oxidation of the double bound Δ^{4, 5} of β-caryophyllene to produce caryophyllene oxide reduced both cytotoxic and AT effects. For monoterpenes, the double methylation of C7 in compounds with a bicycle-heptane structure decreases the AT activity, whereas the acetylation of the hydroxyl group in C3 for linalool increases the AT effects. Also, the position of the hydroxyl groups in the monoterpene structure affects the AT activity as can be seen by the variations between the activity of 4-terpineol and α- terpineol or between carvacrol and thymol.

Although there were a larger number of highly active EOs extracted by SD (n = 9/13) than by HD, it is difficult to recommend one or another method of extraction, since some of the better AT compounds were enriched by SD or HD in a non-consistent manner (p-cymene, linalyl acetate and γ-terpinene), while trans-α-necrodyl acetate, and β pinene were always extracted in higher proportions in EOs obtained by SD and camphene in EOs obtained by HD. Thymol was extracted in higher amounts in EOs extracted by HD with one exception, the EO from O. majorana, which surprisingly displayed 32% in the EO extracted by SD and was absent In the EO extracted by HD. However, interaction between the richness of the compounds can occur during the extraction process, making difficult the decision of which method employ.

All the tested EOs from Lamiaceae and Asteraceae, except two, displayed good anti-trichomonal activity, showing the highest activity EOs from L. luisieri, S. officinalis, S. hibrida, S. sclarea, T. vulgaris, T. zygis, O. majorana, and S. montana.

Among the tested compounds in this study, six were selected as good candidates as AT drugs based on the absence of toxicity in cell cultures and the anti-trichomonad activity in vitro: linalyl acetate, thymol, 4-terpinenol, γ-terpinene, p-cymene, and D-fenchone. Three of them also showed a good correlation between the activity of the EOs and their abundance in the EO's composition: thymol, γ-terpinene, and p-cymene. Considering all the analysis conducted, their abundance in the selected plants EOs, the cytotoxic analysis and the AT activity, we propose these compounds as good anti-trichomonads candidates. Their activity could be increased with chemical modifications. Other compounds with high AT activity were α and β-pinenes, but their toxicity should be further studied.

However, although in most cases, the majority compounds of the EOs account for their activity, in some cases the antitrichomonal activity of the compounds is lower than that of the EOs, which could be due to their complex composition, where synergistic or antagonistic relationships occur between the components. The combination of various products could be of interest.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author/s.

Conceptualization: AG-C, RM-D, MB, and MG-M. Data curation, supervision, writing, reviewing, and editing: AG-C, MB, and MG-M. Formal analysis: ID-C, MB, AG-C, and MG-M. Funding acquisition: AG-C, RM-D, and MG-M. Investigation: ID-C, IA-C, SA, and JN-R. Methodology: ID-C, IA-C, SA, JN-R, AG-C, MB, and MG-M. Resources: AG-C, JN-R, RM-D, MB, and MG-M. MG-M. Writing—original draft: ID-C and MG-M. All authors contributed to the article and approved the submitted version.

This work has been partially financed by Grants PR108/20-08 (Santander-UCM), PID2020-114207RB-I00 (Spanish Ministry of Science and innovation), ERASMUS+ European Hub on New Challenges in the Field of Essential Oils (EOHUB) and PID2019-106222RB-C31/SRA (State Research Agency, 10.13039/501100011033).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.