Garlic oil as a fight against histological and oxidative stress abnormalities in Wistar rats after oral inoculation of Anisakis spp. Type II (L3) (Nematoda) / [O uso do óleo de alho na luta contra anormalidades histológicas e de estresse oxidativo em ratos Winstar após inoculação oral de Anisakis spp. Tipo II (L3) (Nematoda)]

The consumption of inadequately thermally treated fish is a public health risk due to the possible propagation of Anisakis larvae and their antigenic proteins, the causative agent of the zoonotic disease anisakidosis. The present study demonstrated the physiological and histopathological changes that accompanied an oral inoculation of crude extracts from fresh and thermally treated Anisakis Type II (L3) in Wistar albino rats. Nematode worms were isolated from the marine fish Dicentrarchus labrax. They were examined and taxonomically identified using light and scanning electron microscopy. The study was performed in 6 rat groups: a control group (I), a garlic oil (GO) inoculated group (II), a fresh L3 inoculated group (III), a thermally treated L3 inoculated group (IV), a fresh L3 + GO inoculated group (V), and a thermally treated L3 + GO inoculated group (VI). It was observed that rats inoculated with fresh and thermally treated L3 crude extracts showed abnormal oxidative stress markers associated with the destruction of normal architecture of spleen and thymus. GO produced a protective effect in rat groups inoculated with L3 extracts + GO administration via the amelioration of oxidative stress markers, which was confirmed by the marked normal structure of the organs' histology. Cooking of L3 infected fish induced severe physiological and histopathological alterations compared to uncooked infected fish. The administration of garlic before and after fish eating is recommended to avoid the dangerous effect of anisakids, even if they are cooked.(AU)

O consumo de peixes tratados termicamente de forma inadequada é um risco à saúde pública devido à possível propagação das larvas de Anisakis e suas proteínas antigênicas, o agente causador da doença zoonótica anisakidose. O presente estudo demonstrou as alterações fisiológicas e histopatológicas que acompanharam a inoculação oral de extratos brutos de Anisakis Tipo II (L3) frescos e termicamente tratados em ratos Wistar albinos. Vermes nematoides foram isolados do peixe marinho Dicentrarchus labrax e foram examinados e identificados taxonomicamente usando microscopia óptica e eletrônica de varredura. O estudo foi realizado em 6 grupos de ratos: grupo controle (I), grupo inoculado com óleo de alho (GO) (II), grupo inoculado com L3 fresco (III), grupo inoculado com L3 tratado termicamente (IV), grupo inoculado com L3 + GO fresco (V), e grupo inoculado com L3 + GO tratado termicamente (VI). Observou-se que ratos inoculados com extrato bruto L3 fresco e tratado termicamente mostraram marcadores de estresse oxidativo anormais associados à destruição da estrutura normal do baço e do timo. GO produziu um efeito protetor em grupos de ratos inoculados com extrato L3 + administração de GO através da melhoria dos marcadores de estresse oxidativo, que foi confirmada pela marcante estrutura normal da histologia dos órgãos. O cozimento de peixes infectados com L3 induziu alterações fisiológicas e histopatológicas graves quando comparado com peixes infectados não cozidos. Recomenda-se a administração de alho antes e depois da ingestão do peixe para evitar o efeito perigoso dos anisakídeos, mesmo se cozidos.(AU)

The oil of garlic, Allium sativum L. (Amaryllidaceae), as a potential protectant against Anisakis spp. Type II (L3) (Nematoda) infection in Wistar rats / O óleo de alho, Allium sativum L. (Amaryllidaceae), como potencial protetor contra Anisakis spp. Infecção tipo II (L3) (Nematoda) em ratos Wistar

Abstract The consumption of inadequately thermally treated fish is a public health risk due to the possible propagation of Anisakis larvae. The present study demonstrated the physiological and histopathological changes that accompanied an oral inoculation of crude extracts from fresh and thermally treated Anisakis Type II (L3) in rats. Worms were isolated from a marine fish and examined and identified using light and scanning electron microscopy. The study was performed in 6 rat groups: control (I), garlic oil (GO) inoculated (II), fresh L3 inoculated (III), thermally treated L3 inoculated (IV), fresh L3 + GO inoculated (V), and a thermally treated L3 + GO inoculated (VI) groups. Rats inoculated with fresh and thermally treated L3 showed abnormal liver and kidney functions associated with the destruction of normal architecture. GO produced a protective effect in rat groups inoculated with L3 extracts + GO via the amelioration of liver and kidney functions, which was confirmed by the marked normal structure on histology. Cooking of L3-infected fish induced severe alterations compared to uncooked fish. The administration of garlic before and after fish eating is recommended to avoid the dangerous effect of anisakids, even if they are cooked.

Resumo O consumo de peixe inadequadamente tratado termicamente representa um risco para a saúde pública, com a possibilidade da propagação de larvas de Anisakis. O presente estudo demonstrou as alterações fisiológicas e histopatológicas acompanhadas de inoculação oral de extractos brutos de Anisakis tipo II (L3) frescos e termicamente tratados em ratos. Os vermes foram isolados de um peixe marinho, examinados e identificados por microscopia de luz e eletrônica de varredura. O estudo foi conduzido em 6 grupos de ratos: controle (I), óleo de alho (GO) inoculado (II), L3 fresco inoculado (III), L3 tratado termicamente inoculado (IV), L3 fresco + GO inoculado (V), e um grupo L3 + GO tratado termicamente inoculado (VI). Observou-se que ratos inoculados com L3 fresco e tratados termicamente mostraram funções hepáticas e renais anormais, associadas à destruição da sua arquitetura normal. GO produziu um efeito protector em grupos de ratos inoculados com extractos L3 + GO através da melhoria das funções do fígado e dos rins, o que foi confirmado pela estrutura normal marcada da sua histologia. A cozedura de peixes infectados com L3 induziu alterações mais graves do que os peixes não cozidos. Recomenda-se a administração de alho antes e depois do consumo de peixe, para evitar o efeito perigoso dos anisakids, mesmo que sejam cozidos.

Efficacy of Origanum syriacum Essential Oil against the Mosquito Vector Culex quinquefasciatus and the Gastrointestinal Parasite Anisakis simplex, with Insights on Acetylcholinesterase Inhibition.

Developing effective and eco-friendly antiparasitic drugs and insecticides is an issue of high importance nowadays. In this study, we evaluated the anthelminthic and insecticidal potential of the leaf essential oil obtained from Origanum syriacum against the L3 larvae of the parasitic nematode Anisakis simplex and larvae and adults of the mosquito Culex quinquefasciatus. Tests on A. simplex were performed by standard larvicidal and penetration assays, while mosquito toxicity was assessed relying on larvicidal, tarsal contact, and fumigation tests. To shed light on the possible mode of action, we analyzed the oil impact as acetylcholinesterase (AChE) inhibitor. This oil was particularly active on L3 larvae of A. simplex, showing a LC50 of 0.087 and 0.067 mg mL-1 after 24 and 48 h treatment, respectively. O. syriacum essential oil was highly effective on both larvae and adults of C. quinquefasciatus, showing LC50 values of 32.4 mg L-1 and 28.1 µg cm-2, respectively. Its main constituent, carvacrol, achieved larvicidal LC50(90) of 29.5 and 39.2 mg L-1, while contact toxicity assays on adults had an LC50(90) of 25.5 and 35.8 µg cm-2, respectively. In fumigation assays, the LC50 was 12.1 µL L-1 after 1 h and decreased to 1.3 µL L-1 in 24 h of exposure. Similarly, the fumigation LC50 of carvacrol was 8.2 µL L-1 after 1 h of exposure, strongly decreasing to 0.8 µL L-1 after 24 h of exposure. These results support the folk usage of Lebanese oregano as an antiparasitic agent, providing new insights about its utilization for developing new effective and eco-friendly nematocidal and insecticidal products.

Effectiveness of five flavored Tunisian olive oils on Anisakis larvae type 1: application of cinnamon and rosemary oil in industrial anchovy marinating process.

BACKGROUND: Anisakidosis is caused by the ingestion of raw or undercooked fish or cephalopods containing viable Anisakis larvae. Several natural extracts, oils, essential oils, and their compounds have been tested against Anisakis. In this study the effectiveness of Tunisian olive oil with different spices or plants (cardamom, cinnamon, ginger, laurel, and rosemary) was tested against Anisakis larvae type 1. RESULTS: For the in vitro test, larvae were submerged separately in the oils mentioned above and observed to check viability. Cinnamon oil was the most effective against parasites with lethal time (LT) scores being LT50 = 1.5 days and LT100 = 3 days, followed by rosemary. Laurel, cardamom, and ginger oils were less effective. For the ex vivo experiment, cinnamon, and rosemary oils were tested in anchovy fillets, previously artificially parasitized. Cinnamon was the most effective against parasites (dead after 4 days) as compared to rosemary (7 days). CONCLUSION: The use of cinnamon and rosemary-flavored olive oil in the industrial marinating process can be considered as an efficient alternative to the freezing process required by European Regulation EC No 853/2004 to devitalize Anisakis. © 2019 Society of Chemical Industry.

First reporting of Anisakis sp. in the Armed Snook fish (Centropomus armatus) caught and commercialized in Buenaventura, Colombia / Primer reporte de Anisakis sp. en el pez Róbalo (Centropomus armatus) capturado y comercializado en Buenaventura, Colombia

Objective: Nematodes of the Anisakidae family are parasites found in aquatic organisms. The lack of studies on anisakidosis and Anisakis in Colombia has meant this type of parasitosis is not widely known by health personnel and underreporting of the disease is highly likely. The objective of this study was to identify anisakid nematodes in the armed snook fish (Centropomus armatus) obtained by artisanal fishing and sold commercially in the coastal port city of Buenaventura. Material and methods: Morphological identification of these worms was performed using taxonomic keys and supplemented with microscopic study using the histochemical Hematoxylin-Eosin technique. Results: Nematodes of the genus Anisakis were found in 42% and the mean abundance was 2.8 in the C. armatus. Conclusions: The findings confirm the presence of Anisakis sp. in fish for human consumption in Buenaventura, the main fishing port in the Colombian Pacific region. This finding in itself warrants further investigation into the possibility of an emerging disease in Colombia.

Objetivos: Los nematodos de la familia Anisakidae son parásitos de organismos acuáticos. La falta de estudios en anisakidosis y anisakidos en Colombia ha resultado en el desconocimiento de esta infección por parte del personal de salud y un posible subregistro de la enfermedad. El objetivo de este estudio fue identificar nematodos anisakidos en el pez róbalo (Centropomus armatus), obtenido por pesca artesanal y comercializado en la localidad de Buenaventura. Materiales y métodos: La identificación morfológica se realizó usando claves taxonómicas para estos gusanos complementada con estudio microscópico mediante técnica histoquímica de Hematoxilina-Eosina. Resultados: Se encontraron nemátodos del género Anisakis en 42% y una abundancia promedio de 2.8 en el hospedero C. armatus. Conclusiones: Los hallazgos confirman la presencia de Anisakis sp. en peces de consumo humano de Buenaventura, principal puerto pesquero en la región Pacífica colombiana, lo cual abre el camino en la investigación de una posible enfermedad emergente en este país.

Molecular Epidemiology of Anisakis and Anisakiasis: An Ecological and Evolutionary Road Map.

This review addresses the biodiversity, biology, distribution, ecology, epidemiology, and consumer health significance of the so far known species of Anisakis, both in their natural hosts and in human accidental host populations, worldwide. These key aspects of the Anisakis species' biology are highlighted, since we consider them as main driving forces behind which most of the research in this field has been carried out over the past decade. From a public health perspective, the human disease caused by Anisakis species (anisakiasis) appears to be considerably underreported and underestimated in many countries or regions around the globe. Indeed, when considering the importance of marine fish species as part of the everyday diet in many coastal communities around the globe, there still exist significant knowledge gaps as to local epidemiological and ecological drivers of the transmission of Anisakis spp. to humans. We further identify some key knowledge gaps related to Anisakis species epidemiology in both natural and accidental hosts, to be filled in light of new 'omic' technologies yet to be fully developed. Moreover, we suggest that future Anisakis research takes a 'holistic' approach by integrating genetic, ecological, immunobiological, and environmental factors, thus allowing proper assessment of the epidemiology of Anisakis spp. in their natural hosts, in human populations, and in the marine ecosystem, in both space and time.

Green drugs in the fight against Anisakis simplex-larvicidal activity and acetylcholinesterase inhibition of Origanum compactum essential oil.

Anisakiasis is a fish-borne parasitic disease caused by the consumption of raw or undercooked fish, as well as cephalopods, contaminated by third instar larvae (L3) of species belonging to the genus Anisakis (Anisakidae). Origanum compactum is a small herbaceous aromatic plant endemic to Spain and Morocco. In Morocco, the plant is used under infusion to treat heart diseases and intestinal pains or as preservative for foodstuffs. This is the first time that the O. compactum essential oil is tested against the parasitic nematode Anisakis simplex. The phytochemical analysis by GC-MS revealed carvacrol (50.3%) and thymol (14.8%) as the major oil constituents. The essential oil and its major constituents carvacrol and thymol were tested against A. simplex L3 larvae isolated from blue whiting fish (Micromesistius poutassou). A. simplex mortality (%) after 24 and 48 h of treatment at 1 µl/ml was 100%, with a low LD50 compared with other essential oils and extracts, and the penetration in the agar assay was also reduced, if compared with control wells. The oil, as well as its major constituents, demonstrated a dose-dependent larvicidal activity. Inhibition of the enzyme acetylcholinesterase through a colorimetric assay in 96-well plates was used to elucidate the pharmacological mechanism as this enzyme plays a key role in nematodes neuromuscular function. Interestingly, O. compactum essential oil, carvacrol and thymol inhibited the enzyme, confirming that this could be one of the mechanisms involved in the anthelmintic activity. To the best of our knowledge, this is the first time that O. compactum essential oil is reported as a larvicidal agent against A. simplex L3 larvae.

Use of Tunisian flavored olive oil as anisakicidal agent in industrial anchovy marinating process.

BACKGROUND: Natural compounds are more frequently used against Anisakis, responsible for the important fish-borne disease anisakidosis. The aim of the study was to evaluate the effectiveness of enriched Tunisian olive oil with different spices (cumin, turmeric, clove, thyme, and lemon) against Anisakis larvae type 1. RESULTS: In vitro experiment: larvae were submerged separately in the aforementioned oils and then examined to check viability. For each oil, LT50 and LT100 were calculated. Turmeric and cumin oils are the most effective against the parasites; followed by lemon, thyme and clove oils. For the in vivo experiment, turmeric and cumin oils were tested in anchovy fillets previously artificially parasitized with L3 larvae. Cumin was the most effective against parasites (dead after 5 days) compared with turmeric (8 days). For the two oils, the resulting odor was pleasant, as was the taste, while changes in color were much more evident in turmeric fillets. CONCLUSION: All the flavored oils demonstrated a good nematodical action against Anisakis. Cumin oil was the most effective against encysted larvae. Turmeric oil showed the best activity in the in vitro experiment. The use of flavored oils in the marinating process could represent an efficient strategy to devitalize Anisakis. © 2017 Society of Chemical Industry.

Introducing a Novel Media to Improve the Recovery of Culturable Bacteria from the Fish Parasite Anisakis spp. larvae (Nematoda: Anisakidae).

This paper describes a cultivation method to increase the recovery of bacteria from the marine muscle-invading parasitic nematode larvae of Anisakis spp. These larvae hold a high and complex population of accumulated bacteria, originating from seawater, crustaceans, fish, and marine mammals, all involved in the lifecycle of Anisakis. Two in-house agars based on fish juice prepared by either mechanical or enzymatic degradation of the fish tissue, were made. The Anisakis larvae were homogenised prior to cultivation on the in-house fish juice agars and the bacterial numbers and diversity were compared to those obtained applying the commercially available Marine Agar and Iron Agar Lyngby. Bacterial colonies of unique appearance were subcultured and identified by 16S rRNA gene sequencing. Totally three of twenty identified taxa were found on the in-house fish juice agars only. Fish juice agar prepared enzymatically would be the best supplementary agar, as this agar gave significantly higher heterotrophic plate counts, compared to mechanical preparation. The enzymatically prepared fish juice gave more suitable agar quality, was more resource efficient, and had apparently increased nutrient density and availability.

Evaluation of different Mediterranean essential oils as prophylactic agents in anisakidosis.

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.

Antihelmintic effects of nutmeg (Myristica fragans) on Anisakis simplex L3 larvae obtained from Micromesistius potassou.

Anisakis simplex is a foodborne pathogen that can produce human infections and allergic reactions due to the high consumption of raw fish. The seeds of Myristica fragans (Myristicaceae), popularly known as nutmeg, are worldwide used as a culinary spice due to its flavour and properties in food preservation. A nutmeg extract was prepared, analyzed, screened for cytotoxicity and tested against Anisakis simplex L3 larvae. In order to detect the biologically active constituents of the extract, myristicin was tested on the larvae. An acetylcholinesterase inhibition bioassay was also carried out to investigate the antihelmintic mechanism of action. Our results demonstrate that nutmeg exerts antihelmintic effects on Anisakis simplex, being myristicin one of the active compounds. The extract induced a high rate of dead anisakis at concentrations between 0.5 and 0.7 mg/ml without being considered cytotoxic; however, an inhibition of acetylcholinesterase was discarded as the molecular mechanism involved in the activity.

Peppermint (Mentha piperita) and albendazole against anisakiasis in an animal model.

OBJECTIVES: Therapy against anisakiasis requires invasive techniques to extract L3 , and an effective drug against this nematode is needed. The aim of this study was to determine the efficacy of peppermint essential oil (EO) and its main components against the parasite in comparison to albendazole, a drug currently prescribed to treat anisakiasis. METHODS: We conducted in vitro experiments and studied an experimental model simulating the human infection in Wistar rats. We used polymerase chain reaction restriction fragment length polymorphism to identify A. simplex s.s. and A. pegreffii and determine any differences in their pathogenicity and susceptibility to the treatments. RESULTS: The in vitro and in vivo experiments both showed that the larvicidal activity of peppermint EO, menthol, menthone and menthyl acetate is higher than that of albendazole. Large stomach lesions were observed in 46.7% of the albendazole-treated rats, whereas no gastrointestinal lesions were detected in those treated with peppermint EO, menthol, menthyl acetate or menthone. CONCLUSIONS: In this animal model, treatment with peppermint EO or its main components was more effective than was treatment with albendazole. Lesions were more frequently produced by A. simplex s.s. larvae than by A. pegreffii larvae.

Activity of Thymus vulgaris essential oil against Anisakis larvae.

Anisakiasis is an important food-borne disease especially in countries with high fish consumption. The increase of cases of human disease and the virtual absence of effective treatments have prompted the research on new active compounds against Anisakis larvae. As well known, the disease is related to the consumption of raw or almost raw seafood products, but also marinated and/or salted fishery products, if the processing is insufficient to destroy nematode larvae can represent a risks for the consumers. In the light of the biocidal efficacy against different pathogens demonstrated for various essential oils, the aim of this work is to evaluate the effect of Thymus vulgaris essential oil (TEO) against anisakidae larvae. The TEO at 10% and 5% concentration in oil sunflower seeds, caused in vitro the death of all larvae within 14 h, with cuticle and intestinal wall damages. The results obtained showing a significant activity against Anisakis larvae, suggest further investigation on TEO as a larvicidal agent and on its potential use in the industrial marinating process.

Anthelmintic activities of aporphine from Nelumbo nucifera Gaertn. cv. Rosa-plena against Hymenolepis nana.

Nelumbo nucifera Gaertn. cv. Rosa-plena (Nelumbonaceae), commonly known as lotus, is a perennial aquatic plant grown and consumed throughout Asia. All parts of N. nucifera have been used for various medicinal purposes in oriental medicine. From the leaves of Nelumbo nucifera Gaertn. cv. Rosa-plena (an aquatic plant), liriodenine (1), lysicamine (2), (-)-anonaine (3), (-)-asimilobine (4), (-)-caaverine (5), (-)-N-methylasimilobine (6), (-)-nuciferine (7), (-)-nornuciferine (8), (-)-roemerine (9), 7-hydroxydehydronuciferine (10) and cepharadione B (11) were isolated and identification and anthelmintic activities of aporphine was evaluated against Anisakis simplex and Hymenolepis nana. This study found that the above constituents killed H. nana or reduced their spontaneous movements (oscillation/peristalsis). However, the above constituents at various concentrations demonstrated no larvicidal effect or ability to halt spontaneous parasite movement for 72 h against A. simplex, respectively. In addition, according to an assay of cestocidal activity against H. nana and nematocidal activity against A. simplex, we found that the above compounds showed greater lethal efficacy on H. nana than against A. simplex. Further investigation showed that these above constituents have effects against peroxyl radicals under cestocidal effect. Together, these findings suggest that these constituents of Nelumbo nucifera Gaertn. cv. Rosa-plena might be used as anthelmintic agents against H. nana.

Cross-reactivity between Anisakis spp. and wasp venom allergens.

BACKGROUND: Anisakiasis is caused by the consumption of raw or undercooked fish or cephalopods parasitized by live L3 larvae of nematode Anisakis spp. Larvae anchor to stomach mucosa releasing excretion/secretion products which contain the main allergens. It has been described that nematode larvae release venom allergen-like proteins among their excretion/secretion products. We investigated potential cross-reactivity between Anisakis and wasp venom allergens. METHODS: Two groups of 25 patients each were studied: wasp venom- and Anisakis-allergic patients. Sera from patients were tested by ImmunoCAP, dot-blotting with recombinant Anisakis allergens and ADVIA-Centaur system with Hymenoptera allergens. Cross-reactivity was assessed by IgE immunoblotting inhibition assays. Role of cross-reactive carbohydrate determinants (CCDs) was studied by inhibition with bromelain and periodate treatment. RESULTS: A total of 40% of wasp venom-allergic patients had specific IgE to Anisakis simplex and 20% detected at least one of the Anisakis recombinant allergens tested. Likewise, 44% of Anisakis-allergic patients had specific IgE to Vespula spp. venom and 16% detected at least one of the Hymenoptera allergens tested. Wasp venom-allergic patients detected CCDs in Anisakis extract and peptide epitopes on Anisakis allergens rAni s 1 and rAni s 9, whereas Anisakis-allergic patients only detected CCDs on nVes v 1 allergen from Vespula spp. venom. The only Anisakis allergen inhibited by Vespula venom was rAni s 9. CONCLUSIONS: This is the first time that cross-sensitization between wasp venom and Anisakis is described. CCDs are involved in both cases; however, peptide epitopes are only recognized by wasp venom-allergic patients.

Antioxidant, anti-adipocyte differentiation, antitumor activity and anthelmintic activities against Anisakis simplex and Hymenolepis nana of yakuchinone A from Alpinia oxyphylla.

BACKGROUND: Alpinia oxyphylla is a common remedy in traditional Chinese medicine. Yakuchinone A is a major constituent of A. oxyphylla and exhibits anti-inflammatory, antitumor, antibacterial, and gastric protective activities. METHODS: Antioxidant and antitumor characteristics of yakuchinone A in skin cancer cells as well as novel mechanisms for the inhibition of adipocyte differentiation, cestocidal activities against Hymenolepis nana adults, and nematocidal activities against Anisakis simplex larvae are investigated. RESULTS: Yakuchinone A presents the ability of the removal of DPPH·and ABTS+ free radicals and inhibition of lipid peroxidation. Yakuchinone A suppresses intracellular lipid accumulation during adipocyte differentiation in 3 T3-L1 cells and the expressions of leptin and peroxisome proliferator-activated receptor γ (PPARγ). Yakuchinone A induces apoptosis and inhibits cell proliferation in skin cancer cells. The inhibition of cell growth by yakuchinone A is more significant for non-melanoma skin cancer (NMSC) cells than for melanoma (A375 and B16) and noncancerous (HaCaT and BNLCL2) cells. Treatment BCC cells with yakuchinone A shows down-regulation of Bcl-2, up-regulation of Bax, and an increase in cleavage poly (ADP-ribose) polymerase (PARP). This suggests that yakuchinone A induces BCC cells apoptosis through the Bcl-2-mediated signaling pathway. The anthelmintic activities of yakuchinone A for A. simplex are better than for H. nana. CONCLUSIONS: In this work, yakuchinone A exhibits antioxidative properties, anti-adipocyte differentiation, antitumor activity, and anthelmintic activities against A. simplex and H. nana.

Activity of Matricaria chamomilla essential oil against anisakiasis.

The increase in diagnosed cases of anisakiasis and the virtual absence of effective treatments have prompted the search for new active compounds against Anisakis L(3) larvae. The biocidal efficacy against different pathogens shown by various essential oils (EO) led us to study the Matricaria chamomilla EO and two of its main components (chamazulene and α-bisabolol) against the L(3) larvae of Anisakis type I. The activity of M. chamomilla EO, chamazulene and α-bisabolol was established by in vitro and in vivo experiments. The EO (125µg/ml) caused the death of all nematodes, which showed cuticle changes and intestinal wall rupture. In the in vivo assays, only 2.2%±1.8 of infected rats treated with M. chamomilla EO showed gastric wall lesions in comparison to 93.3%±3.9 of control. Chamazulene was ineffective, while α-bisabolol showed a high activity to that of the EO in vitro tests but proved less active in vivo. These findings suggest that the larvicidal activity may result from the synergistic action of different compounds of M. chamomilla EO. Neither of the tested products induces irritative damage in the intestinal tissues. In conclusion, M. chamomilla EO is a good candidate for further investigation as a biocidal agent against Anisakis type I.

[Cloning and expression of L-like cysteine protease of Anisakis simplex].

OBJECTIVE: To clone and express the full lenth of L-like cysteine protease gene of Anisakis simplex (AsCP). METHODS: According to L-like cysteine protease encoding gene of A. simplex from GenBank EST database, specific primers were designed to amplify 3'-end of AsCP gene using rapid-amplification of cDNA ends (RACE), and the full lenth of the L-like cysteine protease gene was obtained. Specific primers were designed according to the full length of the gene. Using total RNA of A. simplex third-stage larvae, coding sequence of the AsCP gene was amplified by RT-PCR. The PCR product was digested by EcoR I and Sal I, and cloned into pET32a(+) vector. The recombinant plasmid was checked by double enzyme digestion and sequencing, and the positive recombinant plasmid was transformed into Escherichia coli BL21 (DE3). Expression of the protein induced by IPTG of gradient concentration (0.2, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mmol/L) and by the same concentration (1 mmol/L) of IPTG at different time(0, 1, 1.5, 2, 2.5, 3, 3.5, and 4 h) was conducted. The expression situation of recombinant protein was analyzed by SDS-PAGE. RESULTS: A 1211 bp of 3'-end of AsCP gene was amplified by 3'RACE, full length of the gene was 1462 bp and coding 411 amino acids. It showed 36.4% identity with the L-cysteine protease of Caenorhabditis elegans. Double enzyme digestion of the constructed recombinant plasmid pET32a(+)-AsCP showed that there was about 1150 bp fragment, the constructed recombinant plasmid was then identified by sequencing. SDS-PAGE showed that the recombinant protein (Mr 60,000) was identical with the target. IPTG showed little effect on the protein expression, and the production of protein was up to maximum after 2 hours induction. CONCLUSION: The AsCP gene has been cloned and expressed.

Larvicidal constituents of Zingiber officinale (ginger) against Anisakis simplex.

In this study, we investigated the anthelmintic activity of [10]-shogaol, [6]-shogaol, [10]-gingerol and [6]-gingerol, compounds isolated from the roots of Zingiber officinale L., Zingiberaceae (ginger), against Anisakis simplex. The above compounds kill or reduce spontaneous movement in A. simplex larvae. The maximum lethal efficacy of [10]-shogaol and [10]-gingerol was approximately 80% and 100%, respectively. We further examined the time course of compound-induced loss of mobility in A. simplex. The results showed that various concentrations of [10]-shogaol, [6]-shogaol, [10]-gingerol and [6]-gingerol have maximum effects on loss of spontaneous movement from 24 to 72 h. In addition, the time course of mortality and the percentage of loss of spontaneous movements were ascertained to determine the minimum effective doses of [10]-gingerol and [10]-shogaol. [10]-Gingerol exhibited a larger maximum larvicidal effect and greater loss of spontaneous movement than [10]-shogaol and albendazole. In addition, these constituents of Zingiber officinale showed effects against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and peroxyl radicals. These constituents of Zingiber officinale are responsible for its larvicidal activity against A. simplex.

In vivo larvicidal activity of monoterpenic derivatives from aromatic plants against L3 larvae of Anisakis simplex s.l.

In view of the lack of an effective pharmacological treatment against human anisakiosis, a disease produced by L(3) larvae of the genus Anisakis present in raw fish, we studied the in vivo larvicidal effect of certain monoterpenic derivatives against L(3) of A. simplex s.l. The aldehydic monoterpene citral and the alcoholic citronellol, when they are administered together to the larvae of the nematode at the concentration of 46.90 mg/0.5 ml in olive oil, achieve 85.90% and 67.53% dead L(3), respectively, and also stop rats suffering gastrointestinal hemorrhages produced by the larvae.