BioMed Research International

BioMed Research International / 2014 / Article

Research Article | Open Access

Volume 2014 |Article ID 549510 |

Carlota Gómez-Rincón, Elisa Langa, Paula Murillo, Marta Sofía Valero, César Berzosa, Víctor López, "Activity of Tea Tree (Melaleuca alternifolia) Essential Oil against L3 Larvae of Anisakis simplex", BioMed Research International, vol. 2014, Article ID 549510, 6 pages, 2014.

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

Academic Editor: Gilbert Lefèvre
Received27 Feb 2014
Accepted08 May 2014
Published25 May 2014


Nematicidal activity of Melaleuca alternifolia essential oil, commonly known as tea tree oil (TTO), was assayed in vitro against L3 larvae of Anisakis simplex. The results showed a mortality of 100% for concentrations between 7 and 10 μL/mL after 48 h of incubation, obtaining an LD50 value of 4.53 μL/mL after 24 hours and 4.27 μL/mL after 48 hours. Concentration-dependent inhibition of acetylcholinesterase was observed for tea tree essential oil showing inhibition values of 100% at 100 μL/mL. This fact suggests that TTO may act as an AChE inhibitor. Terpinen-4-ol was discarded as main larvicide compound as it did not show larvicidal or anticholinesterase activity. The data obtained suggest that the essential oil of Melaleuca alternifolia may have a great therapeutic potential for the treatment of human anisakiasis.

1. Introduction

In the last years, the implication of Anisakis simplex in gastric, intestinal, and allergic clinical disorders has significantly increased [1]. More than 90% of anisakiasis cases are reported in Japan [2]. However, the high human consumption together with the high Anisakis prevalence in seafood from coastal areas of Europa [3, 4] suggests that anisakiasis may be an underdiagnosed illness [13, 57].

Larval endoscopic extraction is the preferred treatment of gastric and intestinal anisakiasis but if the larva is not accessible surgical treatment should be necessary [1]. Pharmacological treatments with antibiotics, anticholinergics, and/or corticosteroids have been occasionally used but their efficacy remains unclear [6, 8, 9]. On the other hand, some studies have suggested that treatment with nematicides as albendazole may constitute an effective therapy [1013]. However, the worldwide development of resistance to chemical anthelmintic drugs [14] and a current social demand for natural therapeutic products has increased the search for new biocide molecules. Plant products are the basis of traditional medicine and the only health resource available in many communities from developing countries. Otherwise, several plant essential oils and their derived compound have shown biocidal activity against Anisakis spp. [1519].

Tea tree oil (TTO) is the essential oil obtained by hydrodistillation from Melaleuca alternifolia, a species native from New South Wales, Australia [20]. Tea tree oil is a natural product derived from the Australian native plant Melaleuca alternifolia that has shown multiple biological activities such as anti-inflammatory, antitumoral, and biocidal properties [21]. Tea tree oil is an effective bactericide [22], fungicide [23], antiviral [24], and insecticidal agent [25, 26]. Furthermore, several studies have demonstrated its effectiveness against protozoa parasites as Leishmania major or Trypanosoma brucei [27]. But there is no evidence of the effect that this substance might have on parasite nematodes.

Many nematicides act through an inhibition of parasite motility. The motility blockade causes inability to remain on the host favouring the parasite elimination. One of the mechanisms affecting this parasite function is the effect on acetylcholine and/or acetylcholinesterase activity. Levamisole acts as an agonist on nicotinic acetylcholine receptors at the nematode neuromuscular junction, leading to sustained neuromuscular depolarization and spastic paralysis [28, 29]. In this line, Mills et al. [30] propose that the pediculicide effect of TTO could be due to, at least in part, anticholinesterase activity of its two main components terpinen-4-ol and 1,8 cineole.

The aim of the present study was to analyze the activity of tea tree essential oil and its main component terpinen-4-ol against Anisakis simplex third stage larvae (L3) and to explore the inhibition of acetylcholinesterase as possible mechanism of action.

2. Materials and Methods

2.1. Essential Oil

Tea tree essential oil was supplied by Pranarôm. Although the essential oils are chemically characterized by Pranarôm International, they were analysed by GC-MS on an Agilent 6890N Network GC system coupled to a 5973 Network Mass Selective Detector, accelerating voltage −69.9 eV, recoding masses of 35.00–400.00. GC conditions are as follows: injector temperature: 150°C; temperature programme: starts from 50°C, 20°C/min to 300°C; column: HP5MS (5% phenylmethylsiloxane) capillary, 30.0 m × 250 μL × 0.25 μm nominal; carrier gas: helium at 1.0 mL/min. A NIST library was used for comparison of MS data.

2.2. In Vitro Larvicidal Activity

Anisakis simplex L3 was isolated from the intermediary host Micromesistius poutassou (blue whiting) purchased from several fish markets in Zaragoza. Worms were washed several times with sterile solution of 0.9% NaCl and identified under light microscope according to morphological features [31, 32]. Only intact Anisakis simplex s.l, L3 with length > 2.0 cm were used. Ten larvae were introduced in each well of polystyrene plates with 2 mL of sterile saline solution containing different concentration of the test solutions. The final concentrations of Melaleuca alternifolia essential oil were: 10, 7, 5, 4, 3, 2, 1, and 0.5 μg/mL. For terpinel-4-ol the final concentrations tested were 0.1, 1, and 10 μg/mL. The parasites were incubated at 37°C in 5% CO2. Each dilution, together with saline solution negative control, was tested by triplicates on three different days. Levamisole (100 μL/mL) was used as the reference antiparasitic drug. Larvae were examined at 24 h and 48 h under microscope and immobile L3 were considered dead. In order to evaluate the biocidal activity of the samples, an average mortality was determined. LC50 was calculated using nonlinear regression (GraphPad Prism 5).

2.3. Inhibition of Acetylcholinesterase

The inhibition of acetylcholinesterase (AChE) was determined by the Ellman method [33] with some modifications. The AChE activity was measured using a 96-microplate, each well containing 25 μL of 15 mM ATCI in Millipore water, 125 μL of 3 mM DTNB in buffer C (50 mM Tris-HCl, pH 8, 0.1 M NaCl, 0.02 M MgCl26H2O), 50 μL buffer B (50 mM Tris-HCl, pH 8, 0.1% bovine serum), and 25 μL of test compound. Every concentration tested of TTO (0.1, 1, 10, and 100 μL/mL), terpinen-4-ol (0.1; 1 and 10 μL/mL), and Levamisole (0.1, 1, 10, and 100 μL/mL) was diluted in DMSO and tested in triplicates. Then, 25 μL 0.22 U/mL AChE was added and the absorbance was measured eight times every 13 s at 405 nm.

2.4. Statistical Analyses

Data were subjected to analysis of variance, and mean comparison was performed by one-way ANOVA plus Scheffé multiple comparisons . The statistical analysis was performed using PASW Statistics 18 program.

3. Results

3.1. Composition of Melaleuca alternifolia Essential Oil

Eight main compounds were detected in the essential oil analyzed by GC-MS (Table 1). The compounds and their abundance are consistent with the chemical characterization provided by Pranarôm International.


Eucalyptol (1,8 cineole)3.6

3.2. In Vitro Larvicidal Activity

Tea tree essential oil showed a significant dose-dependent lethal effect on Anisakis simplex L3 (Table 2). Concentrations of 5 μL/mL and above impair the larvae survival after 24 h exposure . Otherwise, 4 μL/mL of TTO required 48 h to reduce larval vitality. The most effective concentrations were 10 μL/mL, which showed a total lethal effect at 24 h and 7 μL/mL that caused 93% and 100% mortality after 24 h and 48 h incubation. Terpinen-4-ol did not show any larvicide effect at tested concentration. Levamisole, an anthelmintic drug used as positive control, was highly effective causing a 100% larval mortality after 24 h.

Compound Concentration (μL/mL) Average mortality (%)  
( L3)
24 h 48 h


Melaleuca alternifolia EO10

Terpinen-4-ol 1000

Indicates values that differ from the control ( ).
Levamisole was used as positive control reference drug at the dose of 100 μg/mL obtaining 100% of mortality after 24 h.
n: total number of L3 used to test every concentration.

The logistic regression of data revealed a dose-response effect for the TTO that showed LD50 values of 4.53 and 4.27 μL/mL after 24 and 48 h (Figure 1).

3.3. Inhibition of Acetylcholinesterase Assay

Concentration-dependent inhibition of AChE was observed for tea tree essential oil and levamisole (Table 3). TTO showed inhibition values of 100% at 100 μL/mL and were significantly more effective than the AChE inhibitor levamisole at the same concentration . However, terpinen-4-ol, the main component of TTO, did not show inhibition of AChE at the tested concentrations.

Compound Concentration (μL/mL)% Inhibition

Melaleuca alternifolia essential oil0.1

Terpinen-4-ol 0.10

Levamisole 10

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.

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.

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.

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].

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.

5. Conclusions

According to our results Melaleuca alternifolia essential oil showed a remarkable in vitro nematicidal effect for concentrations between 7 and 10 μL/mL. Concentration-dependent inhibition of acetylcholinesterase was observed, suggesting that TTO may act as an AChE inhibitor. Terpinen-4-ol was discarded as main larvicide compound. Data obtained suggest that the essential oil of Melaleuca alternifolia may be a therapeutic tool for human anisakiasis.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.


Project is carried out by Research Group GIMACES E02, Grupo Consolidado de Investigación Aplicada, Gobierno de Aragón, supported by Gobierno de Aragón and Fondo Social Europeo “Construyendo Europa desde Aragón.”


  1. N. S. Hochberg and D. H. Hamer, “Anisakidosis: perils of the deep,” Clinical Infectious Diseases, vol. 51, no. 7, pp. 806–812, 2010. View at: Publisher Site | Google Scholar
  2. M. T. Audicana and M. W. Kennedy, “Anisakis simplex: from obscure infectious worm to inducer of immune hypersensitivity,” Clinical Microbiology Reviews, vol. 21, no. 2, pp. 360–379, 2008. View at: Publisher Site | Google Scholar
  3. E. Madrid, M. T. Galán-Puchades, and M. V. Fuentes, “Risk analysis of human anisakidosis through the consumption of the blue whiting, micromesistius poutassou, sold at Spanish supermarkets,” Foodborne Pathogens and Disease, vol. 9, no. 10, pp. 934–938, 2012. View at: Publisher Site | Google Scholar
  4. F. J. Rello, F. J. Adroher, R. Benítez, and A. Valero, “The fishing area as a possible indicator of the infection by anisakids in anchovies (Engraulis encrasicolus) from southwestern Europe,” International Journal of Food Microbiology, vol. 129, no. 3, pp. 277–281, 2009. View at: Publisher Site | Google Scholar
  5. J. Y. Chai, K. D. Murrell, and A. J. Lymbery, “Fish-borne parasitic zoonoses: status and issues,” International Journal for Parasitology, vol. 35, no. 11-12, pp. 1233–1254, 2005. View at: Publisher Site | Google Scholar
  6. L. Ramos, C. Alonso, M. Guilarte, J. Vilaseca, J. Santos, and J. R. Malagelada, “Anisakis simplex-induced small bowel obstruction after fish ingestion: preliminary evidence for response to parenteral corticosteroids,” Clinical Gastroenterology and Hepatology, vol. 3, no. 7, pp. 667–671, 2005. View at: Publisher Site | Google Scholar
  7. I. Mladineo, V. Poljak, V. Martínez-Sernández, and F. M. Ubeira, “Anti-anisakis IgE seroprevalence in the healthy Croatian Coastal Population and associated risk factors,” PLoS Neglected Tropical Diseases, vol. 8, no. 2, Article ID e2673, 2014. View at: Google Scholar
  8. T. Matsui, M. Iida, and M. Murakami, “Intestinal anisakiasis: clinical and radiologic features,” Radiology, vol. 157, no. 2, pp. 299–302, 1985. View at: Google Scholar
  9. M. A. M. Verhamme and C. H. R. Ramboer, “Anisakiasis caused by herring in vinegar: a little known medical problem,” Gut, vol. 29, no. 6, pp. 843–847, 1988. View at: Google Scholar
  10. J.-F. Magnaval, A. Berry, and M. Nadrigny, “Anaphylactic shock revealing anisakiasis,” Presse Medicale, vol. 31, no. 28, pp. 1309–1311, 2002. View at: Google Scholar
  11. D. A. J. Moore, R. W. A. Girdwood, and P. L. Chiodini, “Treatment of anisakiasis with albendazole,” The Lancet, vol. 360, no. 9326, p. 54, 2002. View at: Publisher Site | Google Scholar
  12. E. Pacios, J. Arias-Diaz, J. Zuloaga, J. Gonzalez-Armengol, P. Villarroel, and J. L. Balibrea, “Albendazole for the treatment of Anisakiasis ileus,” Clinical Infectious Diseases, vol. 41, no. 12, pp. 1825–1826, 2005. View at: Publisher Site | Google Scholar
  13. J. Arias-Diaz, J. Zuloaga, E. Vara, J. Balibrea, and J. L. Balibrea, “Efficacy of albendazole against Anisakis simplex larvae in vitro,” Digestive and Liver Disease, vol. 38, no. 1, pp. 24–26, 2006. View at: Publisher Site | Google Scholar
  14. A. K. Rana and S. Misra-Bhattacharya, “Current drug targets for helminthic diseases,” Parasitology Research, vol. 112, no. 5, pp. 1819–1831, 2013. View at: Publisher Site | Google Scholar
  15. C. Goto, S. Kasuya, K. Koga, H. Ohtoma, and N. Kagei, “Lethal efficacy of extract from zingiber-officinale (traditional chinese medicine) or 6 -shogaol and 6 -gingerol in anisakis larvae in-vitro,” Parasitology Research, vol. 76, no. 8, pp. 653–656, 1990. View at: Google Scholar
  16. I. Hierro, A. Valero, and M. C. Navarro, “In vivo larvicidal activity of monoterpenic derivatives from aromatic plants against L3 larvae of Anisakis simplex s.l,” Phytomedicine, vol. 13, no. 7, pp. 527–531, 2006. View at: Publisher Site | Google Scholar
  17. M. C. Navarro, M. A. Noguera, M. C. Romero, M. P. Montilla, J. M. González de Selgas, and A. Valero, “Anisakis simplex s.l.: Larvicidal activity of various monoterpenic derivatives of natural origin against L3 larvae in vitro and in vivo,” Experimental Parasitology, vol. 120, no. 4, pp. 295–299, 2008. View at: Publisher Site | Google Scholar
  18. R. J. Lin, C. Y. Chen, J. D. Lee, C. M. Lu, L. Y. Chung, and C. M. Yen, “Larvicidal constituents of Zingiber officinale (Ginger) against Anisakis simplex,” Planta Medica, vol. 76, no. 16, pp. 1852–1858, 2010. View at: Publisher Site | Google Scholar
  19. M. D. C. Romero, A. Valero, J. Martín-Sánchez, and M. C. Navarro-Moll, “Activity of Matricaria chamomilla essential oil against anisakiasis,” Phytomedicine, vol. 19, no. 6, pp. 520–523, 2012. View at: Publisher Site | Google Scholar
  20. G. Swords and G. L. K. Hunter, “Composition of Australian tea tree oil (Melaleuca alternifolia),” Journal of Agricultural and Food Chemistry, vol. 26, no. 3, pp. 734–737, 1978. View at: Google Scholar
  21. C. F. Carson, K. A. Hammer, and T. V. Riley, “Melaleuca alternifolia (tea tree) oil: a review of antimicrobial and other medicinal properties,” Clinical Microbiology Reviews, vol. 19, no. 1, pp. 50–62, 2006. View at: Publisher Site | Google Scholar
  22. C. F. Carson and T. V. Riley, “Antimicrobial activity of the essential oil of Melaleuca alternifolia,” Letters in Applied Microbiology, vol. 16, no. 2, pp. 49–55, 1993. View at: Google Scholar
  23. F. D. D'Auria, L. Laino, V. Strippoli et al., “In vitro activity of tea tree oil against Candida albicans mycelial conversion and other pathogenic fungi,” Journal of Chemotherapy, vol. 13, no. 4, pp. 377–383, 2001. View at: Google Scholar
  24. P. Schnitzler, K. Schön, and J. Reichling, “Antiviral activity of Australian tea tree oil and eucalyptus oil against herpes simplex virus in cell culture,” Pharmazie, vol. 56, no. 4, pp. 343–347, 2001. View at: Google Scholar
  25. J. Heukelbach, D. V. Canyon, F. A. Oliveira, R. Muller, and R. Speare, “In vitro efficacy of over-the-counter botanical pediculicides against the head louse Pediculus humanus var capitis based on a stringent standard for mortality assessment,” Medical and Veterinary Entomology, vol. 22, no. 3, pp. 264–272, 2008. View at: Publisher Site | Google Scholar
  26. E. di Campli, S. di Bartolomeo, P. Delli Pizzi et al., “Activity of tea tree oil and nerolidol alone or in combination against Pediculus capitis (head lice) and its eggs,” Parasitology Research, vol. 111, no. 5, pp. 1985–1992, 2012. View at: Publisher Site | Google Scholar
  27. J. Mikus, M. Harkenthal, D. Steverding, and J. Reichling, “In vitro effect of essential oils and isolated mono- and sesquiterpenes on Leishmania major and Trypanosoma brucei,” Planta Medica, vol. 66, no. 4, pp. 366–368, 2000. View at: Publisher Site | Google Scholar
  28. R. J. Martin and A. P. Robertson, “Mode of action of levamisole and pyrantel, anthelmintic resistance, E153 and Q57,” Parasitology, vol. 134, no. 8, pp. 1093–1104, 2007. View at: Publisher Site | Google Scholar
  29. R. J. Martin, S. Verma, M. Levandoski et al., “Drug resistance and neurotransmitter receptors of nematodes: recent studies on the mode of action of levamisole,” Parasitology, vol. 131, pp. S71–S84, 2005. View at: Publisher Site | Google Scholar
  30. C. Mills, B. J. Cleary, J. F. Gilmer, and J. J. Walsh, “Inhibition of acetylcholinesterase by Tea Tree oil,” Journal of Pharmacy and Pharmacology, vol. 56, no. 3, pp. 375–379, 2004. View at: Publisher Site | Google Scholar
  31. G. Hartwich, “Keys to genera of the Ascaridoidea,” in CIH Keys to the nemaTode Parasites of Vertebrates, R. C. Anderson, A. G. Chabaud, and S. Wilmott, Eds., vol. 2, pp. 1–15, Commonwealth Agricultural Bureaux, Slough, UK, 1974. View at: Google Scholar
  32. J. A. Petter and C. Maillard, “Larves d'Ascarides parasites de Poissons en Méditerranée occidentale,” Bulletin du Museum National d'Histoire Naturelle, vol. 10, pp. 347–369, 1988. View at: Google Scholar
  33. G. L. Ellman, K. D. Courtney, V. Andres Jr., and R. M. Featherstone, “A new and rapid colorimetric determination of acetylcholinesterase activity,” Biochemical Pharmacology, vol. 7, no. 2, p. 88, 1961. View at: Google Scholar
  34. M. Pellegrini, R. Occhini, G. Tordini, C. Vindigni, S. Russo, and G. Marzocca, “Acute abdomen due to small bowel anisakiasis,” Digestive and Liver Disease, vol. 37, no. 1, pp. 65–67, 2005. View at: Publisher Site | Google Scholar
  35. P. Nenoff, U. F. Haustein, and W. Brandt, “Antifungal activity of the essential oil of Melaleuca alternifolia (tea tree oil) against pathogenic fungi in vitro,” Skin Pharmacology, vol. 9, pp. 388–394, 1996. View at: Google Scholar
  36. M. C. Navarro-Moll, M. C. Romero, M. P. Montilla, and A. Valero, “In vitro and in vivo activity of three sesquiterpenes against L3 larvae of Anisakis type I,” Experimental Parasitology, vol. 127, no. 2, pp. 405–408, 2011. View at: Publisher Site | Google Scholar
  37. L. E. Homer, D. N. Leach, D. Lea, L. Slade Lee, R. J. Henry, and P. R. Baverstock, “Natural variation in the essential oil content of Melaleuca alternifolia Cheel (Myrtaceae),” Biochemical Systematics and Ecology, vol. 28, no. 4, pp. 367–382, 2000. View at: Publisher Site | Google Scholar
  38. C. F. Carson, B. J. Mee, and T. V. Riley, “Mechanism of action of Melaleuca alternifolia (tea tree) oil on Staphylococcus aureus determined by time-kill, lysis, leakage, and salt tolerance assays and electron microscopy,” Antimicrobial Agents and Chemotherapy, vol. 46, no. 6, pp. 1914–1920, 2002. View at: Publisher Site | Google Scholar
  39. S. Echeverrigaray, J. Zacaria, and R. Beltrão, “Nematicidal activity of monoterpenoids against the root-knot nematode meloidogyne incognita,” Phytopathology, vol. 100, no. 2, pp. 199–203, 2010. View at: Publisher Site | Google Scholar
  40. J. A. Lewis, C. H. Wu, H. Berg, and J. H. Levine, “The genetics of levamisole resistance in the nematode Caenorhabditis elegans,” Genetics, vol. 95, no. 4, pp. 905–928, 1980. View at: Google Scholar
  41. I. D. Harrow and K. A. F. Gration, “Mode of action of the anthelmintics morantel, pyrantel and levamisole on muscle-cell membrane of the nematode ascaris-suum,” Pesticide Science, vol. 16, pp. 662–672, 1985. View at: Google Scholar
  42. I. Hierro, A. Valero, P. Pérez et al., “Action of different monoterpenic compounds against Anisakis simplex s.l. L3 larvae,” Phytomedicine, vol. 11, no. 1, pp. 77–82, 2004. View at: Publisher Site | Google Scholar
  43. K. A. Hammer, C. F. Carson, T. V. Riley, and J. B. Nielsen, “A review of the toxicity of Melaleuca alternifolia (tea tree) oil,” Food and Chemical Toxicology, vol. 44, no. 5, pp. 616–625, 2006. View at: Publisher Site | Google Scholar
  44. M. Russell, “Toxicology of tea tree oil,” in Tea Tree: the Genus Malaleuca, I. Sputhwell and R. Lowe, Eds., vol. 9, pp. 191–201, Harwood Academic Publishers, Amsterdam, The Netherlands, 1999. View at: Google Scholar
  45. G. Ramage, S. Milligan, D. F. Lappin et al., “Antifungal, cytotoxic, and immunomodulatory properties of tea tree oil and its derivative components: potential role in management of oral candidosis in cancer patients,” Frontiers in Microbiology, vol. 3, article 220, 2012. View at: Publisher Site | Google Scholar

Copyright © 2014 Carlota Gómez-Rincón et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Related articles

No related content is available yet for this article.
 PDF Download Citation Citation
 Download other formatsMore
 Order printed copiesOrder

Related articles

No related content is available yet for this article.

Article of the Year Award: Outstanding research contributions of 2020, as selected by our Chief Editors. Read the winning articles.