Bio-molluscicidal potential and biochemical mechanisms of clove oil and its main component eugenol against the land snail, Theba pisana.

The land snail, Theba pisana is a serious pest that adversely affects various crops in sustainable agriculture. Essential oils and their constituents represent an environmentally sound alternative to synthetic pesticides. Our study aimed to investigate the lethal and sub-lethal toxicity of clove oil and its main component eugenol to understand the mechanisms underlying its toxic action against T. pisana. The GC-MS profile of the clove oil composition was characterized. In the laboratory experiment, LD50 of clove oil and eugenol via the contact testing were determined after 48 and 72 h. Moreover, sub-lethal effects of clove oil or eugenol on the survivors following the exposure of snails to the 25 and 50% of the LD50/48 and 72 h were evaluated through using snail tissues for biochemical measurments. The GC-MS analysis showed that eugenol (64.87%) was the major constituent present in the oil. The results also showed that LD50 values at 48 and 72 h were 2006.5 and 1493.5 µg/g b.w for oil and 239.6 and 195.3 µg/g b.w for eugenol, respectively. Compared to control, the sub-lethal effects of clove oil or eugenol at 48 and 72 h showed a significant increase in reduced glutathione (GSH) levels. Catalase (CAT) and glutathione-S-transferase (GST) activities significantly elevated in oil- or eugenol-treated snails, except at low dose after 48 h. After two exposure times, snails exposed to oil or eugenol at both sub-lethal effects had considerably higher γ-glutamyltransferase (γ-GT) and aspartate aminotransferase (AST) activities. Moreover, markedly augmentation in alkaline phosphatase (ALP) and alanine aminotransferase (ALT) activities at all exposure times, with the exception of snails treated with low dose of eugenol after 48 h was observed. Both clove oil and eugenol at the tested doses caused a significant inhibition in acetylcholinesterase (AChE) activity at two exposure times. Our findings highlight the potential of clove oil and eugenol, as an efficient natural molluscicide alternative to its synthetic counterparts for snail control.

Terpenoid constituents of cinnamon and clove essential oils cause toxic effects and behavior repellency response on granary weevil, Sitophilus granarius.

This study evaluated toxic effects, repellency and respiration rate caused by terpenoid constituents of cinnamon and clove essential oils and against Sitophilus granarius L. (Coleoptera: Curculionidae). The lethal concentrations (LC50 and LC90), repellent effect, and behavior repellency response on adults of S. granarius after exposure to six concentrations of each essential oil and terpenoids were evaluated. The chemical composition of the cinnamon oil was also determined and primary compounds were eugenol (10.5%), trans-3-caren-2-ol (10.2%), benzyl benzoate (9.99%), caryophyllene (9.34%), eugenyl acetate (7.71%), α-phellandrene (7.41%), and α-pinene (7.14%). In clove essential oil, the primary compounds were eugenol (27.1%), caryophyllene (24.5%), caryophyllene oxide (18.3%), 2-propenoic acid (12.2%), α-humulene (10.8%), γ-cadinene (5.01%), and humulene oxide (4.84%). Cinnamon and clove essential oil was toxic to S. granarius. In toxic terpenoids compounds, eugenol has stronger contact toxicity in S. granarius than caryophyllene oxide, followed by α-pinene, α-humulene, and α-phellandrene. Insects reduced their respiratory rates after being exposed to essential oil terpenoids and avoided or reduced their mobility on terpenoid-treated surfaces. Cinnamon and clove essential oil, and their terpenoid constituents were toxic and repellent to adult S. granarius and, therefore, have the potential to prevent or retard the development of insecticide resistance.

Molecular and structural changes induced by essential oils treatments in Vicia faba roots detected by genotoxicity testing.

Over the last few years, there has been an increased interest in exploiting allelopathy in organic agriculture. The aim of this investigation was to examine the effects of essential oil mixtures in order to establish their allelopathic use in agriculture. Two mixtures of essential oils consisting respectively of tea tree oil (TTO) and clove plus rosemary (C + R) oils were tested. Phytotoxicity and genotoxicity tests on the root meristems of Vicia faba minor were performed. A phytotoxic influence was particularly relevant for C + R mixture, while genotoxicity tests revealed significant results with both C + R oil mixture and TTO. Phenotypic analysis on Vicia faba minor primary roots following C + R oil mixture treatment resulted in callose production, an early symptom attributed to lipid peroxidation. The approach described in this study, based on genotoxicity bioassays, might identify specific DNA damage induced by essential oil treatments. These tests may represent a powerful method to evaluate potential adverse effects of different mixtures of essential oils that might be useful in alternative agriculture. Future studies are focusing on the positive synergism of more complex mixtures of essential oils in order to reduce concentrations of potentially toxic components while at the same time maintaining efficacy in antimicrobial and antifungal management.

Toxicological effects of pet food ingredients on canine bone marrow-derived mesenchymal stem cells and enterocyte-like cells.

We developed an in vitro method to assess pet food ingredients safety. Canine bone marrow-derived mesenchymal stem cells (BMSC) were differentiated into enterocyte-like cells (ELC) to assess toxicity in cells representing similar patterns of exposure in vivo. The toxicological profile of clove leave oil, eugenol, guanosine monophosphate (GMP), GMP + inosine monophosphate, sorbose, ginger root extract, cinnamon bark oil, cinnamaldehyde, thyme oil, thymol and citric acid was assessed in BMSC and ELC. The LC50 for GMP + inosine monophosphate was 59.42 ± 0.90 and 56.7 ± 3.5 mg ml(-1) for BMSC and ELC; 56.84 ± 0.95 and 53.66 ± 1.36 mg ml(-1) for GMP; 0.02 ± 0.001 and 1.25 ± 0.47 mg ml(-1) for citric acid; 0.077 ± 0.002 and 0.037 ± 0.01 mg ml(-1) for cinnamaldehyde; 0.002 ± 0.0001 and 0.002 ± 0.0008 mg ml(-1) for thymol; 0.080 ± 0.003 and 0.059 ± 0.001 mg ml(-1) for thyme oil; 0.111 ± 0.002 and 0.054 ± 0.01 mg ml(-1) for cinnamon bark oil; 0.119 ± 0.0004 and 0.099 ± 0.011 mg ml(-1) for clove leave oil; 0.04 ± 0.001 and 0.028 ± 0.002 mg ml(-1) for eugenol; 2.80 ± 0.11 and 1.75 ± 0.51 mg ml(-1) for ginger root extract; > 200 and 116.78 ± 7.35 mg ml(-1) for sorbose. Lemon grass oil was evaluated at 0.003-0.9 in BMSC and .03-0.9 mg ml(-1) in ELC and its mechanistic effect was investigated. The gene toxicology studies showed regulation of 61% genes in CYP450 pathway, 37% in cholestasis and 33% in immunotoxicity pathways for BMSC. For ELC, 80% for heat shock response, 69% for beta-oxidation and 65% for mitochondrial energy metabolism. In conclusion, these studies provide a baseline against which differential toxicity of dietary feed ingredients can be assessed in vitro for direct effects on canine cells and demonstrate differential toxicity in differentiated cells that represent gastrointestinal epithelial cells.

Sublethal Exposure to Clove and Cinnamon Essential Oils Induces Hormetic-Like Responses and Disturbs Behavioral and Respiratory Responses in Sitophilus zeamais (Coleoptera: Curculionidae).

Essential oils have been suggested as suitable alternatives for controlling insect pests. However, the potential adaptive responses elicited in insects for mitigating the actions of these compounds have not received adequate attention. Furthermore, as is widely reported with traditional insecticides, sublethal exposure to essential oils might induce stimulatory responses or contribute to the development of resistance strategies that can compromise the management of insect pests. The current study evaluated the locomotory and respiratory responses as well as the number of larvae per grain produced by the maize weevil, Sitophilus zeamais Motschulsky, after being sublethally exposed to the essential oils of clove, Syzygium aromaticum L., and cinnamon, Cinnamomum zeylanicum L. The essential oils showed similar insecticidal toxicity (exposure route: contact with dried residues; Clove LC95 = 3.96 [2.78-6.75] µl/cm(2); Cinnamon LC95 = 3.47 [2.75-4.73] µl/cm(2)). A stimulatory effect on the median survival time (TL50) was observed when insects were exposed to low concentrations of each oil. Moreover, a higher number of larvae per grain was produced under sublethal exposure to clove essential oil. S. zeamais avoided the treated areas (in free-choice experiments) and altered their mobility when sublethally exposed to both essential oils. The respiratory rates of S. zeamais (i.e., CO2 production) were significantly reduced under low concentrations of the essential oils. We recommend the consideration of the potential sublethal effects elicited by botanical pesticides during the development of integrated pest management programs aiming to control S. zeamais.

Toxicological assessment of kretek cigarettes Part 5: mechanistic investigations, inhalation toxicity.

The biological effects of mainstream smoke (MS) from Indonesian-blended cigarettes with and without added cloves, cloves extracted with hot ethanol, and extracted cloves replenished with eugenol or clove oil were assessed in a 90-day inhalation study in rats. A separate 35-day inhalation study in rats was performed with MS from American-blended cigarettes with 0%, 2.5%, 5% or 10% added eugenol. Effects commonly seen in inhalation studies with MS were observed. These included histopathological changes indicative of irritation in the entire respiratory tract and inflammatory responses in the lung. Adding cloves to American- or Indonesian-blended cigarettes reduced the inflammatory response in the lung but with no difference between the two blend types. When the clove oil was extracted (∼ 75% reduction of eugenol achieved) from cloves, the inflammatory response in the lung was still reduced similarly to whole cloves but the severity of histopathological changes in the upper respiratory tract was less reduced. Add back of clove oil or pure eugenol reduced this response to a level similar to what was seen with whole cloves. When eugenol was added to American-blended cigarettes, similar findings of reduced lung inflammation and severity of histopathological changes in respiratory the tract was confirmed. These studies demonstrate a clear effect of cloves, and in particular eugenol, in explaining these findings.

Toxicological assessment of kretek cigarettes Part 4: mechanistic investigations, smoke chemistry and in vitro toxicity.

The smoke chemistry and in vitro toxicity of mainstream smoke (MS) was investigated in American-blended cigarettes with or without the addition of 2.5%, 5% or 10% eugenol to the tobacco and in Indonesian-blended cigarettes with and without the addition of cloves, cloves extracted with hot ethanol, and extracted cloves replenished with eugenol or clove oil. The addition of eugenol reduced the concentration of nearly all toxicants measured in MS as well as the in vitro cytotoxicity of the gas/vapor phase. Reductions were also seen in bacterial mutagenicity of the total particulate matter (TPM) assessed by the Ames Assay. The addition of extracted cloves led to increases and decreases of toxicant concentrations in MS. Replenishment with eugenol or clove oil decreased the toxicant concentrations; with most smoke constituent concentrations reduced below the concentration found in tobacco-only cigarettes. Cytotoxicity of the TPM was not affected by the clove preparations. However, GVP cytotoxicity was reduced (untreated cloves showing the highest reductions). Mutagenicity of TPM was decreased by the clove preparations. Mechanisms for the reductions, (up to 40%), are most likely due to dilution effects by eugenol, changed burning characteristics of the tobacco, and free radical scavenging by eugenol.

Modulation efficiency of clove oil nano-emulsion against genotoxic, oxidative stress, and histological injuries induced via titanium dioxide nanoparticles in mice.

Titanium dioxide nanoparticles (TiO2-NPs) have found wide applications in medical and industrial fields. However, the toxic effect of various tissues is still under study. In this study, we evaluated the toxic effect of TiO2-NP on stomach, liver, and kidney tissues and the amelioration effect of clove oil nanoemulsion (CLV-NE) against DNA damage, oxidative stress, pathological changes, and the apoptotic effect of TiO2-NPs. Four groups of male mice were subjected to oral treatment for five consecutive days including, the control group, the group treated with TiO2-NPs (50 mg/kg), the group treated with (CLV-NE) (5% of the MTD), and the group treated with TiO2-NPs plus CLV-NE. The results revealed that the treatment with TiO2-NPs significantly caused DNA damage in the liver, stomach, and kidney tissues due to increased ROS as indicated by the reduction of the antioxidant activity of SOD and Gpx and increased MDA level. Further, abnormal histological signs and apoptotic effect confirmed by the significant elevation of p53 expression were reported after TiO2-NPs administration. The present data reported a significant improvement in the previous parameters after treatment with CLV-NE. These results showed the collaborative effect of the oils and the extra role of nanoemulsion in enhancing antioxidant effectiveness that enhances its disperse-ability and further promotes its controlled release. One could conclude that CLV-NE is safe and can be used as a powerful antioxidative agent to assess the toxic effects of the acute use of TiO2-NPs.

Comparison of the effects of four anaesthetics on haematological and blood biochemical profiles in pikeperch (Sander lucioperca L.).

OBJECTIVES: The objectives of the study were to compare the effects of Propiscin, 2-phenoxyethanol, clove oil and tricaine methane sulphonate (MS 222), anaesthetics frequently used in aquaculture. DESIGN: The haematological and biochemical blood profiles of pikeperch (Sander lucioperca L.) anesthetized with Propiscin (1.5 ml L-1), 2-phenoxyethanol (0.3 ml L-1), clove oil (33 mg L-1), MS 222 (150 mg L-1) and non-anesthetized control group were tested. Each tested group was divided into two subgroups, the first subgroup was sampled in anaesthesia 10 min after application of the anaesthetic and the second one live on 24h. RESULTS: The erythrocyte count and haematocrit was significantly decreased in 2-phenoxyethanol (24 h) compared with control group (CG). The mean corpuscular haemoglobin concentration was significantly increased in 2-phenoxyethanol (10 min), Propiscin (10 min and 24 h) compared to CG. The 2-phenoxyethanol (10 min and 24 h), MS 222 (24 h), clove oil (24 h), and Propiscin (10 min and 24 h) showed significantly lower leukocyte count compared with CG. The level of glucose was significantly (p<0.05) elevated with MS 222 (10 min) and clove oil (10 min) compared with CG. The 2-phenoxyethanol (10 min and 24 h), MS 222 (24 h), clove oil (24 h), and Propiscin (24 h) showed significantly lower (p<0.01) ammonia levels compared with CG. The triacylglycerols was significantly decreased (p<0.01) with Propiscin (10 min and 24 h), MS 222 (24 h), clove oil (24 h) and with 2-phenoxyethanol (24 h) compared with CG. After 24 hours MS 222 (24 h) and Propiscin (24 h) anaesthesia, fish showed significantly lower (p<0.01) concentration of inorganic phosphate compared with CG. CONCLUSIONS: On the basis of this experiment, it appears that clove oil was associated with the lowest effects in pikeperch and therefore would be recommended as an alternative to MS 222, while Propiscin and 2-phenoxyethanol are not suitable for manipulation with pikeperch in aquaculture.

Comparison of acute toxicity of 2-phenoxyethanol and clove oil to juvenile and embryonic stages of Danio rerio.

OBJECTIVES: Anaesthetics are used in aquaculture to prevent stress and mechanical damage to fish during handling or the treatment of fish in breeding, blood sampling and other veterinary interventions. Clove oil and 2-phenoxyethanol are used in the Czech Republic in a water bath for the short-term immobilization of the fish. DESIGN: Acute toxicity tests were performed on aquarium fish Danio rerio, which is considered to be one of the model organisms most commonly used in toxicity testing. The semi-static method according to OECD No. 203 (Fish acute toxicity test) was used for testing juvenile fish. Embryo toxicity tests were performed in zebrafish embryos (D. rerio) in compliance with the OECD No. 212 methodology (Fish, short-term toxicity test on embryo and sac-fry stages). The results obtained (the number of dead individuals at particular test concentrations) were subjected to a probit analysis using the EKO-TOX 5.2 programme in order to determine LC50 clove oil and 2-phenoxyethanol values. The statistical significance of the difference between LC50 values in juvenile and embryonic stages of D. rerio was tested using the Mann-Whitney non-parametric test implemented in the Unistat 5.1 programme. RESULTS: The LC50 clove oil mean value was 18.8 +/- 5.52 mg.L-1 in juvenile D. rerio, and 15.64 +/- 3.30 mg.L-1 in embryonic stages of D. rerio. The LC50 2-phenoxyethanol mean value was 338.22 +/- 15.22 mg.L-1 in juvenile D. rerio, whereas in embryonic stages of D. rerio it was 486.35 +/- 25.53 mg.L-1. CONCLUSIONS: The study proved statistically significantly higher (p<0.01) sensitivity in juvenile fish to 2-phenoxyethanol compared to the embryonic stages. Acute toxicity values of clove oil for juvenile and embryonic stages were comparable.

Toxicity to, oviposition and population growth impairments of Callosobruchus maculatus exposed to clove and cinnamon essential oils.

The use of plant essential oils has been shown to efficiently control insect pests of stored beans, significantly reducing the threats associated with synthetic insecticides. Here, we evaluated the potential of applications of essential oils of clove, Syzygium aromaticum L., and cinnamon, Cinnamomum zeylanicum L., to control Callosobruchus maculatus, considered as one of the most cosmopolitan pests of stored beans. Using four combinations of couples (i.e., unexposed couples, exposed females, exposed males, and exposed couples), we also evaluated how sublethal exposure to these essential oils impacted C. maculatus oviposition. Bioassays results revealed that both essential oils exhibited insecticidal activities similar to the synthetic pyrethroid insecticide deltamethrin. Furthermore, oil dosage increments proportionately decreased the growth rate and reduced the losses in bean weight caused by cowpea weevils, and offspring emergence was almost abolished when parents were exposed to the LD20 of each essential oil. Finally, significant oviposition impairments were perceived only in couples where females were exposed (i.e., females exposed and exposed couples) to the LD20 of cinnamon and clove essential oils. Thus, by exhibiting similar insecticidal activities as synthetic insecticides and by significantly affecting the oviposition of sublethally exposed C. maculatus females, the cinnamon and clove essential oils represent valuable tools with potential of integration into the management of C. maculatus infestations.

Toxicity of clove essential oil and its ester eugenyl acetate against Artemia salina.

The production of compounds via enzymatic esterification has great scientific and technological interest due to the several inconveniences related to acid catalysis, mainly by these systems do not fit to the concept of "green chemistry". Besides, natural products as clove oil present compounds with excellent biological potential. Bioactives compounds are often toxic at high doses. The evaluation of lethality in a less complex animal organism can be used to a monitoring simple and rapid, helping the identification of compounds with potential insecticide activity against larvae of insect vector of diseases. In this sense, the toxicity against Artemia salina of clove essential oil and its derivative eugenyl acetate obtained by enzymatic esterification using Novozym 435 as biocatalyst was evaluated. The conversion of eugenyl acetate synthesis was 95.6%. The results about the evaluation of toxicity against the microcrustacean Artemia salina demonstrated that both oil (LC50= 0.5993 µg.mL-1) and ester (LC50= 0.1178 µg.mL-1) presented high toxic potential, being the eugenyl acetate almost 5 times more toxic than clove essential oil. The results reported here shows the potential of employing clove oil and eugenyl acetate in insecticide formulations.

Cytotoxicity of clove (Syzygium aromaticum) oil and its major components to human skin cells.

The essential oil extracted from clove (Syzygium aromaticum) is used as a topical application to relieve pain and promote healing in herbal medicine and also finds use in the fragrance and flavouring industries. Clove oil has two major components, eugenol and beta-caryophyllene, which constitute 78% and 13% of the oil, respectively. Clove oil and these components are generally recognized as 'safe', but the in-vitro study here demonstrates cytotoxic properties of both the oil and eugenol, towards human fibroblasts and endothelial cells. Clove oil was found to be highly cytotoxic at concentrations as low as 0.03% (v/v) with up to 73% of this effect attributable to eugenol. beta-caryophyllene did not exhibit any cytotoxic activity, indicating that other cytotoxic components may also exist within the parent oil.

Toxicity of clove essential oil and its ester eugenyl acetate against Artemia salina / Toxicidade do óleo essencial de cravo e seu éster acetato de eugenila contra Artemia salina

Abstract The production of compounds via enzymatic esterification has great scientific and technological interest due to the several inconveniences related to acid catalysis, mainly by these systems do not fit to the concept of “green chemistry”. Besides, natural products as clove oil present compounds with excellent biological potential. Bioactives compounds are often toxic at high doses. The evaluation of lethality in a less complex animal organism can be used to a monitoring simple and rapid, helping the identification of compounds with potential insecticide activity against larvae of insect vector of diseases. In this sense, the toxicity against Artemia salina of clove essential oil and its derivative eugenyl acetate obtained by enzymatic esterification using Novozym 435 as biocatalyst was evaluated. The conversion of eugenyl acetate synthesis was 95.6%. The results about the evaluation of toxicity against the microcrustacean Artemia salina demonstrated that both oil (LC50= 0.5993 µg.mL–1) and ester (LC50= 0.1178 µg.mL–1) presented high toxic potential, being the eugenyl acetate almost 5 times more toxic than clove essential oil. The results reported here shows the potential of employing clove oil and eugenyl acetate in insecticide formulations.

Resumo A produção de compostos via esterificação enzimática possui grande interesse científico e tecnológico devido às inúmeras inconveniências relacionadas com a catálise ácida, principalmente por estes sitemas não se adequarem ao atual termo “tecnologias limpas”. Além disso, produtos naturais como o óleo de cravo, apresentam compostos com excelentes potenciais biológicos. Compostos bioativos são quase sempre tóxicos em altas doses. A avaliação da letalidade em um organismo animal menos complexo pode ser usada para um monitoramento simples e rápido, servindo também para a identificação de compostos com potencial atividade inseticida contra larvas de insetos vetores de doenças. Neste sentido, foi determinada a toxicidade frente a Artemia salina do óleo essencial de cravo e do seu derivado acetato de eugenila obtido por esterificação enzimática com lipase Novozym 435. A conversão da reação de síntese de acetato de eugenila foi de 95,6%. Os resultados referentes à avaliação da toxicidade frente ao microcrustáceo Artemia salina demonstraram que tanto o óleo (LC50= 0,5993 µg.mL–1) quanto o éster (LC50= 0,1178 µg.mL–1) apresentam elevado potencial toxicológico, sendo que o éster apresenta aproximadamente 5 vezes mais toxicidade em relação ao óleo. Estes resultados demonstram o potencial emprego do óleo de cravo e de acetato de eugenila em formulações de inseticidas.

Daily rhythms of toxicity and effectiveness of anesthetics (MS222 and eugenol) in zebrafish (Danio rerio).

Although the chronotoxicity of xenobiotics is relatively well known in mammals, the existence of daily rhythms of drug toxicity and effectiveness in fish has been neglected to date. The aim of this research was to investigate the influence of the time (middle of the light phase [ML] versus middle of the dark phase [MD]) of exposure to two anesthetic substances (MS-222 or clove oil) commonly used with fish on the median lethal concentration (LC(50)) and swimming activity of zebrafish (Danio rerio). To this end, adult zebrafish were kept under a 12 h:12 h light-dark (LD) cycle and exposed to different concentrations of the anesthetics for 15 min at ML or MD. LC(50) calculations were performed using the Spearman-Karber program, whereas swimming activity was video-recorded and analyzed with specialized software. Zebrafish exhibited a mostly diurnal activity pattern (77.9% of activity occurring during daytime). The acute toxicity and mortality caused by MS-222 and eugenol varied with the time of exposure. For MS-222, the LC(50) was 170.6 ± 7.4 mg/L in fish exposed at ML and 215.6 ± 3.9 mg/L at MD, whereas for eugenol the LC(50) was 70.3 ± 3.1 mg/L at ML and 104.9 ± 5.4 mg/L at MD. Exposure to sublethal concentrations of MS-222 and eugenol altered the swimming patterns of zebrafish in a different manner depending on the time of exposure. Thus, the time required for decreasing swimming activity during exposure to anesthetics was shorter at ML than at MD, whereas the recovery period was longer during the day. In conclusion, these results revealed that the toxicity and effectiveness of both anesthetic substances is highest during daytime, the active phase of fish, thus suggesting a link between the daily rhythms of behavior and toxicity.