The search for new anthelmintics requires the use of appropriate methods to test their efficacy. From this point of view, the EHT, which is used to study in vitro the ovicidal activity of drugs, offers comparable, reliable and precise results and has an advantage over other tests due to its higher sensitivity. It is suitable for an initial assessment of anthelmintic potential and forms a basis for other tests. For this reason, it is the most commonly used in vitro test for the evaluation of the anthelmintic activity of drugs, i.e. benzimidazoles, and for the detection of resistance (Babják et al., 2021). In the EHT performed in the present study, the EO of S. montana showed moderate ovicidal activity varying from 17.3–83.0%, with an IC50 value of 0.59 mg/ml. Although the maximum inhibitory effect (100%) on hatching of GINs eggs was observed in our previous study (Štrbac et al., 2022a), the IC50 value obtained in the present study is still high compared to oils from other studies (André et al., 2018; Štrbac et al., 2022b), indicating its anthelmintic potential. Indeed, this parameter is considered suitable for comparing drug-inhibitory effect of different active substances (Berrouet et al., 2020).
The results obtained for two samples of S. montana EO in present and the previous study (Štrbac et al., 2022a) indicate a high variability in the efficacy of EOs, since both samples were obtained from the same producer and had the same chemical composition. However, in addition to the known factors that can influence the presence and abundance of the compounds (geographical origin of the plant, light, rainfall, soil conditions, age and part of the plant, genetic characteristics, presence of certain organisms and microorganisms, etc.) and thus their effect, many other factors related in post-extraction period may also be involved. That esspecially reffers to the way the EOs are stored and the length of time before they are used, as they are sensitive to light, temperature and oxygen (Fokou et al., 2020; Cimino et al., 2021). The experimental parameters may also influence the results, as the method used to obtain the different concentrations was different, with the dilution method being used in the present study and highly sensitive micropipettes being used in the last one.
On the other hand, in vivo FECRT is considered the gold standard for the evaluation of drug efficacy and the detection of AR and is intended for all anthelmintic classes (Babják et al., 2021). It is the most commonly used method for evaluating the anthelmintic efficacy of drugs in practice (Denwood et al., 2023). Examination of the effect of S. montana EO in vivo was performed for the first time to our knowledge, and it also showed moderate activity with a total reduction of 33.0% on D14 after treatment. The overall efficacy of the entire formulation (EO + sunflower oil for dilution) was 40.0%. While in Farm 1 the applied oil was less effective, in Farm 2 it significantly reduced the EPG on D14 after treatment and reached an efficacy of 68.3%. A similar trend was also observed in our previous studies (Štrbac et al., 2023a; Štrbac et al., 2023b) conducted with oregano and mint EOs, where these oils were also much more effective in farm 2. This can be explained by the differences in animal husbandry (free range in comparison with the boxes), which affect the manipulation of the animals during treatment, but may also have an influence on the gastrointestinal tract of animals in general due to differences in their feeding. However, the average individual reduction was higher than that at the group level, reaching 50% in total on D14, suggesting that EOs had an effect in most of the tested animals. Preliminary toxicity studies indicate the safety of the use of winter savory in practice.
The lower efficacy in vivo of EOs in general, including S. montana, can be attributed to their unstable nature as their ingredients are prone to degradation and evaporation, which causes limited bioavailability (Cimino et al., 2021; Nehme et al., 2021). On the other hand, complexity of gastrointestinal tract of ruminants that hinder per oral applications is also involved (Hoste et al., 2008). Both of this can be improved by the use of encapsulation techniques that can protect the main active ingredients that are sensitive to various factors (oxygen, light and moisture), and prevent interaction with other compounds. In this way, the stability and bioavailability of EOs can be further increased, while reducing toxicity, volatility, odour and taste. Also important, encapsulation enables a controlled release of the active ingredients of EOs along the gastrointestinal tract (Cimino et al., 2021; Sousa et al., 2022; Yammine et al., 2023). As the dose tested did not cause any adverse effects in the animals, increasing the dose or multiple applications during several consecutive days could also be an option to improve efficacy under field conditions. Alternatively, other forms of EO application such as drenching (Ahmed et al., 2014) or the use of lick blocks with herbal compounds (Junkuszew et al., 2015) should also be considered.
According to the results of GC-MS analysis, the most important compounds responsible for the anthelmintic properties of winter savory are p-cymene, carvacrol and γ-terpinene. In a study by André et al. (André et al., 2016), the isolated carvacrol showed high anthelmintic potential in various in vitro tests, including the larval development test (LDT) and the adult worm motility test (AWMT) in addition to the EHT. This indicates that this compound is effective against different parasite stages (eggs, larvae and adult worms). Moreover, in the same study, its acetylated derivative reduced the group EPG by 65.9% at D16 after treatment in the FECRT. Its strong anthelmintic activity was also confirmed in a study by Katiki et al. (2017), in which it showed one of the highest ovicidal effects against H. contortus eggs compared to other compounds. P-cymene and γ-terpinene were not individually tested for their anthelmintic activity against GINs, but were present in some EOs with this property: p-cymene at 22.56% in Alpinia zerumbet (Macedo et al., 2013) and at 23.76% in Thymus vulgaris (Ferreira et al., 2016) as well as γ-terpinene at 20.15% in Melaleuca alternifolia (Grando et al., 2015) and at 11.42% in Citrus aurantifolia (Ferreira et al., 2018).
Not only the presence but also the percentage of the compounds in the EOs is important for the pharmacological effects. Thus, these three compounds were also the main constituents of the previously tested oregano EO (Štrbac et al., 2023a), but in a different ratio: carvacrol 76.21%, p-cymene 12.57% and γ-terpinene 2.63%. However, oregano showed a superior effect with an average group EPG reduction of 43.21% and 60.13% at D7 and D14 after treatment, respectively. This could indicate a superior activity of carvacrol compared to p-cymene, although this statement needs to be confirmed by individual testing and comparison of these compounds. Indeed, individual compounds can differ considerably in their anthelmintic activity (Katiki et al., 2017). Moreover, the dominance of one compound and its appropriate abundance (at least > 50%) with the simultaneous presence of other compounds but in lower percentages, may lead to high activity, as shown by the results of a previously conducted study with eleven EO tested in vitro (Štrbac et al., 2022a). These considerations could be useful for the future development of herbal anthelmintics.
Plant-based formulations have many advantages that can be exploited for their use in animals for various indications, including the control of GINs (Bava et al., 2021; Castagna et al., 2021; Castagna et al., 2022b). The compounds that compose them have a wide variety of pharmacological effects, belong to different chemical groups with possibly different mechanisms of action, and have therefore already shown the effect against various parasite stages of GINs (André et al., 2018; Štrbac et al., 2022b). This may also contribute to their less susceptibility to resistance development in comparison with synthetic drugs (Borges and Borges, 2016; Ferreira et al., 2018). Botanical drugs are also considered safer than synthetic, whereby environmental aspect and public health also favouring natural drugs due to their biodegradability, to challenge the residue problem (Veerakumari, 2015; Castagna et al., 2022a; Romero et al., 2022). Finally, the price of plant drugs is also considered reasonable and even cheaper than that of commercial drugs, so the procurement of these medicines should not be a problem, especially in countries with developed biodiversity (Ferreira et al., 2018), as in the case of S. montana in Serbia (Šubarević et al., 2015).
Novel anthelmintics can be used in different ways to control sheep GINs. They can be used independently, but only if they achieve sufficient efficacy under field conditions. Although a threshold of 95% is used to assess efficacy and the development of potential resistance to commercial anthelmintics in ruminants (Kaplan, 2020), some authors recommend a minimum efficacy of 90% in FECRT for new anthelmintic agents (Geurden et al., 2022). Thus, S. montana as well as EOs from other studies, are not yet suitable for independent use in the doses, formulations and route of administration examined. However, other alternative methods such as genetic selection of animals that are naturally resistant to nematodes (Tsukahara et al., 2021), pasture management and nutritional manipulations (Yoshihara et al., 2023), biological control methods (direct – use of nematophagous fungi, bacteria or even other nematodes or indirect – use of deep buntle or earthworms) (Szewc et al., 2021) and development of vaccines (Liu et al., 2023) also have some limitations despite their numerous advantages. Finally, the exclusive use of commercial drugs is also no longer suitable due to the development and spread of resistance, as well as residue issues that concerns public health and environment (Castagna et al., 2022a; Maestrini et al., 2022; de Agüero et al., 2023), as already mentioned.
Therefore, there is a broad consensus that integrated parasite management, which refers to the use of several appropriate methods of parasite control, represent the most appropriate solution for the future management of GINs in sheep (Maqbool et al., 2017; Tariq et al., 2017; Mondragón-Ancelmo et al., 2019). In this system, various plant-based anthelmintics, including EOs, may represent a valuable source in combination with other mentioned alternatives. They also can be combined with commercial drugs whose use should be based on target treatments or target selective treatments (Maurizio et al., 2023). Thus, the combination of the ethanol extract of Ananas comosus, drenched weekly for 6 weeks using a stomach tube (100 mg/kg), with 1 g of the fungal product Clonostachys rosea given daily, effectively reduced faecal egg and larva counts, larval development and the number of infectious larvae (L3) in the pasture (Ahmed et al., 2014). Carvacrol has also been shown to enhance the effect of drugs agonists of nAChR (imidazothiazoles) or agonists of GABA receptors (avermectins and piperazine), and can therefore be considered for use with these drugs to achieve a synergistic effect (Trailović et al., 2015; Marjanović et al., 2020). In contrast, the results of a study by Mesquita-Sousa et al. (2023) showed that the combination of Citrus aurantinum var. Dulcis EO and albendazole has antagonistic effect against benzimidazole resistant Haemonchus contortus, which is also possible and should be considered.