Activity of Tea Tree (Melaleuca alternifolia) Essential Oil against L3 Larvae of Anisakis simplex

4. Discussion

The increasing worldwide incidence of anisakiasis together with the lack of effective pharmacological treatments warrants the search for new active molecules. Although in most cases anisakiosis resolved spontaneously, the severity of potential complications such as peritonitis or intestinal wall perforations, frequently surgical treatments are required [34]. On the other hand, in the current context of economic crisis, optimization of health resources is essential. Therefore the search for less invasive and expensive sanitary interventions should be a research priority in health sciences. In that sense, the use of essential oils and natural products could provide a noninvasive, inexpensive, and effective treatment for human anisakiasis.

In recent years, the biocidal activity against Anisakis L3 of several essential oils and some of its components has been studied. Romero et al. [19] demonstrated that 125 μg/mL of Matricaria chamomilla essential oil induced 100% larval mortality after 4 h in vitro and reduced the pathogenic effects in experimentally infected rats. The results obtained in our study show that TTO was effective, obtaining significant larvicidal effect at doses over 4 μL/mL. The maximal concentration tested (10 μL/mL) was 100% lethal at 24 h. Similarly, at a concentration of 7 μL/mL, larvicidal activity was 93% at 24 h and 100% after 48 h of essential oil exposure. Observed mortality for other concentrations was low; however, mortality percentages were 52% and 83% after 24 and 48 h of exposure to 5 μL/mL of EO.

Many studies have demonstrated the potent biocide effect of TTO. Many bacteria are susceptible to concentrations of 1% or less even against antibiotic-resistant strains [21]. TTO has also a fungicidal activity at concentrations ranging from 0.12 to 2% against yeast and dermatophytes [23, 35]. Although there are no previous references regarding the TTO LD50 against nematodes, the values obtained in our study (LD5024 h = 4.53 μL/mL and LD5048 h = 4.14 μL/mL) suggest that TTO could be an effective nematicide.

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Essential oils are complex mixtures of compounds from natural origin, among which the terpenes have been largely studied and have shown useful pharmaceutical properties. Several monoterpenes as carvacrol, geraniol, or citronellol (monoterpenes) [19] and sesquiterpenes as nerolidol or farnesol [36] show a high potential against Anisakis L3. The main component of the essential oil obtained from tea tree is terpinen-4-ol [37]. Different authors suggest that this monoterpene is responsible, at least in part, for the antiprotozoal [27] and bactericidal [38] effects of the tea tree. Furthermore, recent studies suggest that terpinen-4-ol inhibits the motility of the plant parasitic nematode Meloidogyne incognita [39]. However, the larvicidal effect observed in our study cannot be associated with this compound since we did not observe any effect on Anisakis larvae. Therefore, we can deduce that the effect could be related to other compounds. It seems unlikely that alpha-terpinene and 1,8 cineole are responsible for the antiparasitic activity, at least individually, since they are inactive at high concentration against the plant nematode Meloidogyne incognita [39]. Otherwise, a possible candidate may be alpha-pinene (3% concentration in our essential oil), whose activity has been recently demonstrated in vitro and in vivo against Anisakis spp. [17]. However, due to the chemical complexity of essential oils, it should be noted that many of their properties are due to the synergistic or complementary effects of several components.

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In this work we have also evaluated the AChE inhibition as a possible mechanism of action involved in the TTO nematicidal effect. Data showed that, at the same concentrations, TTO was a more effective AChE inhibitor than levamisole. These differences could be due to the fact that both drugs block the enzyme activity at different levels. Levamisole is a potent cholinergic agonist that binds preferentially to L-subtype nicotinic acetylcholine receptors in body-wall muscle causing hypercontracted paralysis, usually followed by relaxation and death [28, 29, 40, 41]. However, our results suggest that levamisole also has a moderate direct inhibitory effect on the enzyme. In this sense, tea tree essential oil seems to have a direct effect on the enzyme activity which is completely blocked at concentrations of 100 μL/mL. These results suggest that inhibition of AChE could be a possible TTO mechanism of action against Anisakis. This hypothesis is consistent with the TTO anticholinesterase activity observed by Mills et al. [30] who proposed the competitive inhibition of AChE by 1,8 cineole and terpinen-4-ol to explain the TTO insecticidal effect. However, the IC50 values were 10.30 mM and 0.04 mM for terpinen-4-ol and 1,8 cineole, respectively, concluding that both compounds are weak inhibitors of the activity of the enzyme. In our study, even though the used concentration values were similar to those used by Mills et al. [30], terpinen-4-ol did not show anticholinesterase activity. This fact, together with the absence of larvicidal effect in vitro, leads us to discard terpinen-4-ol as main active compound against Anisakis L3. However, several studies suggest that the antimicrobial activity of TTO is attributed mainly to terpinen-4-ol [38]. For this reason, we are unable to discard that this compound has an active role in anthelmintic activity, as it may contribute indirectly or synergistically to the activity of other components of TTO.

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Due to its chemical complexity, it is difficult to establish the mechanism of action involved in the biological effects of the essential oils. Therefore, the anticholinesterase effect observed in our study might be only partially responsible for the observed anthelmintic activity. It has been demonstrated that the interaction between the components of the TTO and cell membranes induces biochemical changes causing structural and functional cell integrity loss [21]. To our knowledge, there are no data about the effect of TTO on Anisakis simplex although recent studies suggest that essential oils or their constituents could cause alterations in the cuticle, muscle cells, and digestive system of L3. These effects would unleash the parasite death [16, 42] and, moreover, reduce the larval infectivity and their pathogenic effect in vivo [19].

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Tea tree essential oil has long been used topically as an antiseptic; however, there are few data about its oral safety. Published data indicated that TTO can be toxic if ingested in higher doses [43]. In vivo rat models toxicity studies showed LD50 values of 1.9–2.6 mL/kg [44]. Recent studies confirmed these data and revealed cytotoxic effects on human oral epithelial cells at concentrations of 500 μL/mL [45]. Our in vitro experiments showed a high effectiveness of TTO against Anisakis L3 at lower concentrations suggesting that this molecule could be an effective nematicide. However, these results must be supported by in vivo studies to ensure the efficacy and safety of essential oil of tea tree in treating clinical anisakiasis.

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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4055599/

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