Context:Anisakis Dujardin 1845 (Anisakidae) nematodes can cause gastrointestinal and allergic diseases when humans eat raw or undercooked seafood containing larvae. There is currently no drug available in the market against this parasitic disease, and the study of plant-derived molecules could be useful in the discovery of effective compounds. Objective: This research assesses the biocidal activity of a range of essential oils (EOs) from some Mediterranean plants against larvae found in the musculature of fresh fish. Materials and methods: EOs composition was analyzed by gas chromatography-mass spectroscopy. All the EOs were diluted at 5% v/v in olive oil to cover the fish with the solutions for 24 h. The larvae that abandoned the muscle and the larvae obtained from the artificial digestion of the fish were collected. Controls were carried out in parallel. Furthermore, Wistar rats were infected with the live larvae collected from the in vitro trials in order to find any larvae that may have penetrated the gastrointestinal wall. Results: Between 60.8% and 87.6% of parasites treated with EOs abandoned the fish muscle, and the highest in vitro mortality rate was achieved with oregano EO (53.9%). Rats previously treated with oregano, cumin and Spanish lavender EOs showed no detectable lesions in the digestive tract due to the infection with larvae. Conclusions: Oregano (Origanum vulgare L. Lamiaceae), cumin (Cuminum cyminum L. Apiaceae) and Spanish lavender (Lavender stoechas L. Lamiaceae) EOs could be used as promising ingredients in the development of products for the control of anisakiasis.
Keywords: Anisakis larvae; components of essential oils; prophylactic action; biocidal activity; Mediterranean plants
The consumption of marine fish has increased over recent decades, raising the number of cases of human anisakidosis. The main agent responsible for this parasitosis is the third larval stage (L3) of Anisakis Dujardin, 1845 (Anisakidae), nematodes which are widely found in fish and cephalopods, with high mean prevalence and intensity rates in many species of commercial interest (Chía et al. [
The host used in the different trials was blue whiting (Micromesistius poutassou) from the north of Spain, a fish with a high prevalence of parasitization by Anisakis L3 larvae, as shown by various authors (Chía et al. [
The following EOs were tested against the Anisakis L3 larvae found in the muscle of the blue whiting: Spanish lavender (Lavandula stoechas L. Lamiaceae), cumin (Cuminum cyminum L. Apiaceae), lavender (Lavandula spica L. Lamiaceae), marjoram (Origanum majorana L. Lamiaceae), oregano (Origanum vulgare L. Lamiaceae), rosemary (Rosmarinus officinalis L. Lamiaceae) and thyme (Thymus vulgaris L. Lamiaceae). These products were provided by Sensient Fragrances (Granada, Spain). The composition of the EOs was analyzed by gas chromatography–mass spectroscopy (GC–MS) on an Agilent 7890A System (Santa Clara, CA) coupled with a Waters Quattro Micro GC mass spectrometer (Cerdanyola del Vallès, Spain). The column used was a phenyl dimethylpolysiloxane capillary column ZB-5MS (30 m × 0.25 μm thickness), supplied by Phenomenex (Torrance, CA), with helium as the carrier gas (flow rate = 1 mL/min). The samples were injected using the split mode (split ratio 1:1000), with injector temperature at 200 °C. Oven temperature was programed from 50 °C (2.5 min) to 200 °C at a rate of 4 °C/min, then kept at 200 °C for 8 min. Mass spectra were acquired in electron impact mode (70 eV). Components were identified by mass spectral fragmentation, by comparing Kovats-calculated (Dabrio [
All the EOs were diluted at 5% v/v in olive oil. The presence of parasitic larvae in the muscle of the hosts was confirmed by direct observation. About 7–9 fish were used for each EO, which were gutted, washed and placed in glass containers covered with each essential oil solution. The containers were closed and kept for 24 h at 4 °C. The larvae that had abandoned the muscle were then collected, and the fish was then washed and immediately submitted to artificial digestion in a pepsin-HCl solution (pH 2–2.4) for 45 min at 36 °C. Following this, the larvae were counted, both those found loose in the container and those obtained from the digestion of the muscle. Controls were carried out in parallel, submerging the fish in olive oil (carrier), in order to verify that it has no effect on the larvae. The viability of the parasites was determined in saline solution 0.9% at 24 h under a stereoscopic microscope, applying the following criteria of mobility: dead if no movement or response to stimulus were observed, apparently alive if there was movement when stimulated with a brush and alive if there was spontaneous movement.
Forty-four female Wistar rats (weighing 140–150 g) were infected via a gastric probe with 4–8 active larvae mixed with 0.5 mL of water. The trials were conducted according to the following protocol: (a) many animals were infected with the live larvae that had abandoned the muscle during the immersion period in each of the EOs, (b) another lot consisted of animals infected with the larvae recovered from the fish after treatment and subsequent digestion. In order to establish the potential influence of the carrier oil on the viability of the larvae, a control lot was designed consisting of rats infected with the larvae obtained following the digestion of the fish submerged only in olive oil. Four hours later, the animals were sacrificed and immediately their organs were examined in detail to find any larvae that may have penetrated the wall of the gastrointestinal tract. Lastly, the digestive system was removed and opened under a stereoscopic microscope, recording the location of the larvae and the number of parasites and lesions found.
This study was conducted according to the principles specified in the Declaration of Helsinki, and under the Directive 2010/63/EU of the European Parliament and of the Council of the European Union (22 September 2010) and the Spanish Legislation (RD 53/2013).
Table 1 shows the main components of the oils tested.
Table 1. Qualitative and quantitative composition of the essential oils assayed against Anisakis larvae performed by GC-MS.
Compound (%) Spanish lavender Cumin Lavender Marjoram Oregano Rosemary Thyme α-Thujene 0.49 0.02 α-Pinene 1.06 0.28 0.53 1.51 0.30 13.47 0.05 Camphene 1.30 0.24 0.37 5.86 0.06 Sabinene 1.54 β-Pinene 7.30 0.55 2.38 2.39 β-Myrcene 0.33 0.77 0.63 1.58 0.17 α-Terpinene 0.47 0.13 12.25 0.18 0.49 3.32 2.15 17.74 Limonene 0.75 0.18 1.83 3.06 0.04 1,8-Cineole 5.17 19.58 68.05 27.98 0.14 Ocimene 0.48 Δ4-Carene 10.47 1.30 0.41 Linalilo oxide 0.28 0.37 0.23 0.10 Fenchone 48.68 0.09 Linanool 52.59 9.53 0.91 1.20 1.39 Camphor 39.98 17.33 0.73 23.32 Lavandulol 1.79 Santolina alcohol 1.25 Borneol 1.71 0.76 0.09 4.97 0.47 Terpinen-4-ol 0.39 0.35 0.84 0.46 1.11 α-Terpineol 0.82 4.83 0.24 Isobornil format 0.05 Cuminaldehyde 34.11 Linalilo anthranilate 2.29 Felledral 0.72 Bornyl formate 1.89 Δ2-Caren-10-al 20.78 1.64 0.04 Δ3-C10-al 11.80 Thymol 0.22 76.31 Carvacrol 88.39 2.41 Terpenyl acetate 1.00 Aromadendrene 1.01 2.42 3.26 0.29
Examination under the microscope revealed that the 784 Anisakis L3 larvae obtained from the muscle of M. poutassou belonged to Type I.
In the trials conducted with the parasitized fish treated with the Eos, a displacement of the larvae was generally observed from the parasitized tissues to the container in which the test was conducted. This migration was above 60% in all cases. Moreover, all the EOs tested produced a drop in the survival rate of the larvae. The highest total mortality rate was detected for oregano EO (53.9%), with the larvae showing severe oesophageal and intestinal deterioration (Figure 1). Cumin, thyme and Spanish lavender EOs proved less lethal than the oregano one (20.3, 11.4 and 3.5% mortality rates, respectively). For the lavender, marjoram and rosemary EOs the survival rate was 100%. For the larvae that remained alive, a reduction in spontaneous movement was detected, with the maximum reduction (65.8%) corresponding to larvae treated with cumin EO and the minimum (12.3%) to Spanish lavender EO; results in between these values were obtained for thyme (53.4%), lavender (31.7%) and oregano (31.4%) EOs. None of the larvae treated with marjoram and rosemary EOs required stimulation to be actively mobile. In the controls, all the larvae displayed great mobility. Table 2 displays data related to the larvae that abandoned the fish muscle or remained in it, and their survival rates and mobility following exposure to the different EOs.
Graph: Figure 1. Larva L3 of Anisakis after being treated with essential oil of oregano in vitro. It can be observed in oesophageal and intestinal damage.
Table 2. In vitro study of the effect of the EOs against the L3 of Anisakis type I.
Total L3 Container Muscle Total NS S X Total NS S X Spanish lavender 57 41 (71.9%) 32 (78.1%) 7 (17.1%) 2 (4.9%) 16 (28.1%) 16 (100%) – – Cumin 237 196 (82.7%) 28 (14.3%) 123 (62.8%) 45 (23.0%) 41 (17.3%) 5 (12.2%) 33 (80.5%) 3 (7.3%) Lavender 126 107 (84.9%) 71 (66.4%) 36 (33.7%) – 19 (15.1%) 15 (79.0%) 4 (21.1%) – Marjoram 89 78 (87.6%) 78 (100%) – – 11 (12.4%) 10 (100%) – – Oregano 102 62 (60.8%) – 9 (14.5%) 53 (85.5%) 40 (39.2%) 15 (37.5%) 23 (57.5%) 2 (5%) Rosmary 49 34 (69.4%) 33 (100%) – – 15 (30.6%) 15 (100%) – – Thyme 88 69 (78.4%) 21 (30.4%) 38 (55.1%) 10 (14.5%) 19 (21.6%) 10 (52.6%) 9 (47.4%) –
1 NS: L3 alive with no stimulus; S: L3 alive with stimulus; X = L3 dead.
This study was influenced by the in vitro results in terms of the number of larvae found alive and active following exposure of the fish to the different EOs. A total of 44 animals were infected with 218 larvae obtained in the in vitro trials, but the parasites only penetrated the digestive tract wall in five cases (11.4%). One rat had a lesion consisting of a tunnel measuring 2 × 1.5 mm with slight bleeding, located in the greater curvature of the stomach, and the larva was found partially embedded at the end of the tunnel. This lesion was caused by one of the L3 exposed to lavender EO that was collected from the container after abandoning the muscle.
The larvae treated with rosemary EO caused a similar tunnel-shaped lesion, although of greater length (4 × 1 mm), and a small ulcer (1.5 × 1 mm) surrounded by a bleeding halo; the larvae, as in the previous case, were found embedded in the greater curvature of the stomach. A third ulcer was also observed in the small intestine accompanied by signs of bleeding. The first lesion was caused by one of the L3 that had abandoned the fish muscle; the other two were caused by L3 that had remained in the muscle. Furthermore, L3s treated with rosemary EO and taken from the muscle produced two small ulcers (1 × 1 mm) in another infected rat; in one of these ulcers the parasite was found stuck in the gastric wall of the greater curvature, showing no visible signs of bleeding, and in the other it was located in a reddish area of the small intestine.
In one of the animals infected with larvae exposed to thyme EO and collected from the container, a lesion of 2 × 1 mm was found in the gastric fundus. In another rat, one larva was found in the body cavity with no visible lesions; this larva had been treated with marjoram EO and had abandoned the fish muscle during the process.
All the parasites recovered from the rats that had been taken from the fish muscle treated with EOs were found alive, except for the 22.9%, which had been in contact with cumin EO. The greatest number of larvae were recovered from the small intestine (49.1%), followed by the stomach (41.3%) and only 6.0% from the cecum. Table 3 shows the number of rodents infected and larvae inoculated, the lesions produced and their locations. With regard to the control lot, made up of animals that were infected with larvae from untreated fish muscle subjected to digestion, lesions were observed in all cases. These consisted of tunnels and ulcers similar to those described above and were found mainly in the gastric body (72.7%) and the remaining 27.3% in the small intestine (Table 3).
Table 3. Sinopsis of the in vivo trials.
Product Origin of L3 No of rodents No of L3 inoculated Lesions (number)/Location Control Muscle 6 36 11/8 Stomach and 3 small intestine Spanish lavender Container 3 16 – Muscle 2 10 – Cumin Container 7 28 – Muscle 2 5 – Lavender Container 4 25 1/Stomach Muscle 3 15 – Marjoram Container 6 30 – Muscle 2 10 1/Stomach Oregano Container – – – Muscle 3 15 – Rosmary Container 4 20 2/Stomach Muscle 3 14 2/Stomach and small intestine Thyme Container 3 17 1/Stomach Muscle 2 10 –
Human anisakidosis presents a risk to public health that must be reduced through basic prevention. Steps to control the disease include various measures and checks to detect and kill the larvae in fish, thus preventing infection of the consumer. The main recommendations are freezing the fish or exposing it to temperatures over 60 °C. However, essential oils have long been recognized as effective plant-based antimicrobials and have been used to preserve foods for centuries, thus they could be good alternatives to the abovementioned methods to protect against anisakidosis. Indeed, all seven essential oils tested in this study did somehow appear to stimulate a high percentage of the Anisakis L3 in the fish musculature to abandon it and migrate into the container (Table 2): the values were ≥70% for the lavender, Spanish lavender, marjoram, cumin, thyme and rosemary EOs, and slightly lower for the oregano one (60.8%). These data are of particular interest given that the muscle is the part of the fish that humans consume, and thus the reduced presence of parasites in this part would reduce the risk of contracting this disease. Furthermore, during this process a certain number of larvae in the fish died or lost spontaneous mobility on coming into contact with the essential oils (Table 2): the two most effective EOs in this sense were cumin and oregano, for which only 13.9% and 14.7% of the larvae, respectively, exhibited spontaneous movement. The favourable results obtained for these two EOs against Anisakis larvae are in keeping with those described by Valero et al. ([
With regard to the in vivo trials, the action of oregano, cumin and Spanish lavender EOs on the L3s in the fish muscle resulted in the larvae not causing any lesions in the animals' digestive tract. The antimicrobial activity of essential oils, which are markedly lipophilic compounds, is related to their chemical composition, in which there is a predominance of terpenic derivatives (mono- and sesquiterpenes). Although its mechanism of action has not yet been fully explained, it is likely that in most cases it involves disruptions of the membrane, increasing the permeability of the bacterial cytoplasmic membranes, and of the mitochondrial membranes in eukaryotic organisms (Cowan [
In the case of cumin EO, the activity of its main component, cuminaldehyde, against various pathogenic agents including Anisakis L3 larvae has been described, although the exact way in which it acts is still unknown (Hierro et al. [
With regard to the action of Spanish lavender EO, we should point out that although it did not show larvicidal action in the in vitro studies, in the in vivo trials no lesions were observed in the infected animals treated with this EO. The chemical composition identified in this oil was: fenchone (48.7%), camphor (40.0%) and 1,8-cineole (5.2%), so the EO analyzed could be considered a camphor-fenchone chemotype, according to Granger et al. ([
It should also be highlighted that in the case of the larvae treated with marjoram EO, although one larva was found in the body cavity of the animal, there were no visible signs of lesions in the digestive tract. This leads us to speculate that this EO, whose main component is 1,8-cineole (68.1%), may have favoured the reabsorption of the haemorrhage. This could be due to the anti-inflammatory activity of 1,8-cineole which, in agreement with the results obtained by Abu-Darwish et al. ([
The essential oils tested in this work provoked the migration of the Anisakis larvae out of the fish, therefore they might well be added to the marinated fish plates. This fact, together with the biocidal action of some of them, particularly Spanish lavender, cumin and oregano EOs, are promising active ingredients that could be used in the development of effective products for the control of anisakidosis. Further studies on safety and palatability of the marinating with essential oils should be carried out.
The results shown in this article are part of the doctoral thesis of Magdalena Gómez-Mateos Pérez.
The authors wish to thank Dr. V. Corpas-López for English revision of this manuscript.
None of the authors has any conflict of interests regarding this study.
This work was funded by the Research Groups grant from the Junta de Andalucía, Spain (BIO-243).
By Magdalena Gómez-Mateos Pérez; Concepción Navarro Moll; Gema Merino Espinosa and Adela Valero López
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