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In our previous study based on a whole-blood model of sepsis infected with trans-anethole (TA)-treated Staphylococcus aureus, we have found that innate immune response was more effective in comparison to non-treated cells. Due to the previous observation, in the current preliminary study, a primary adaptive immune response was analysed. This study was conducted to evaluate the expression of selected cytokine (IL1B, IL2, IL6, IL10, TNF, TGFB1, IFNG) and Toll-like receptor (TLR2) genes in lymphocytes isolated from whole human blood infected with S. aureus Newman strain treated with TA. The lymphocytes were isolated by density gradient centrifugation from blood samples infected with S. aureus, as well as from non-infected samples. Gene expression was measured using quantitative real-time PCR. The lymphocytes isolated from the blood infected with TA-treated staphylococcal cells demonstrated significantly greater IL10, IL1B, IL6, TNF and TLR2 expression. Hence, it is possible that the previously observed changes in the surface structure of TA-treated S. aureus Newman strain may significantly increase the relative expression of IL10, IL1B, IL6, TNF and TLR2 genes in lymphocytes; however, further studies are needed., (© 2021 The Society for Applied Microbiology.)

Aims: Antibacterial activities of phenylpropenes (PPs) (eugenol, isoeugenol, estragole and trans-anethole) and hydroxycinnamic acids (HCAs) (p-coumaric, caffeic and ferulic acids) were assessed against Escherichia coli and Staphylococcus epidermidis. Effect of cyclodextrin and liposome encapsulation on the PPs activity was also evaluated., Methods and Results: All PPs inhibited the bacterial growth in the hundred micromolar range, while HCAs did not, as determined by broth macrodilution. Anethole and estragole showed a higher efficiency than eugenol and isoeugenol, and E. coli was more susceptible than S. epidermidis. Hydroxypropyl-β-cyclodextrin/PP complexes and anethole-loaded Lipoid S100-liposomes were prepared by freeze-drying and ethanol injection respectively. Both formulations were substantially less active than free PPs. For instance, E. coli growth inhibition was about 14% for all HP-β-CD/PP complexes evaluated at MIC 50 values of free PPs (P < 0·05), and about 12% for liposomal anethole evaluated at minimal bactericidal concentration value of free anethole (P < 0·05)., Conclusions: Hydrophobicity appears to be crucial for PPs antibacterial activity. Encapsulation in cyclodextrin and liposome seems to retain the PPs preventing their interaction with bacteria., Significance and Impact of the Study: This study highlights the structural features of simple phenylpropanoids related to their antibacterial activity. The limitations of conventional encapsulation systems on the activity of PPs should be considered in future applications., (© 2019 The Society for Applied Microbiology.)

One strategy for overcoming infectious diseases caused by drug-resistant fungi involves combining drugs rendered inactive by resistance with agents targeting the drug resistance mechanism. The antifungal activity of n-dodecanol disappears as incubation time passes. In Saccharomyces cerevisiae, anethole, a principal component of anise oil, prolongs the transient antifungal effect of dodecanol by downregulating genes of multidrug efflux pumps, mainly PDR5. However, the detailed mechanisms of dodecanol's antifungal action and the anethole-induced prolonged antifungal action of dodecanol are unknown. Screening of S. cerevisiae strains lacking genes related to Ca2+ homeostasis and signaling identified a pmr1Δ strain lacking Golgi Ca2+-ATPase as more sensitive to dodecanol than the parental strain. Dodecanol and the dodecanol + anethole combination significantly increased intracellular Ca2+ levels in both strains, but the mutant failed to clear intracellular Ca2+ accumulation. Further, dodecanol and the drug combination reduced PMR1 expression and did not lead to specific localization of Pmr1p in the parental strain after 4-h treatment. By contrast with the parental strain, dodecanol did not stimulate PDR5 expression in pmr1Δ. Based on these observations, we propose that the antifungal activity of dodecanol is related to intracellular Ca2+ accumulation, possibly dependent on PMR1 function, with anethole enabling Ca2+ accumulation by restricting dodecanol efflux., (© FEMS 2020.)

High doses of nutmeg (seeds from Myristica fragrans Houtt.) can be abused as a psychoactive drug due to phenylpropene ingredients. During controlled abstinence, e.g., in forensic psychiatric clinics, nutmeg abuse has to be distinguished from an ingestion of other spices having phenylpropene ingredients (e.g., black pepper or garden lovage) or unintentional low-dose nutmeg intake. The aim of this study was to develop an evaluation model for the estimation of time point and amount of nutmeg abuse and differentiation from ingestion of other spices or low doses of nutmeg based on the gas chromatographic-mass spectrometric (GC-MS) analysis of urine samples. A total of 3 volunteers ingested 1.5 g of freshly ground nutmeg. No symptoms were reported. Urine samples were collected for up to 3 days. In addition, 18 blank samples from volunteers with regular diet and 2 authentic samples from forensic psychiatry patients with supposed nutmeg abuse were analyzed. All samples were analyzed by GC-MS in full scan mode. Metabolites of the nutmeg ingredients safrole, myristicin and elemicin were identified via a library search. For semi-quantitative estimations, the area ratios of the analytes to the internal standard (MDMA-d5) were normalized to the creatinine concentration. Up to 8 different metabolites were detected for at least 18 hours after intake of 1.5 g of nutmeg. In the two authentic samples, the normalized area ratios of those metabolites were 0.5-14 times the maximum reached in the intake study. Two additional metabolites could be detected in authentic samples. Probably due to ingestion of other spices, 5 of the 8 metabolites after intake of 1.5 g of nutmeg were detected in blank urine samples as well. The intake of high doses of nutmeg can be differentiated from the ingestion of other spices or low doses of nutmeg via standard GC-MS analysis of urine and application of the proposed evaluation model., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

We used quantitative nuclear magnetic resonance analyses to measure the contents of major constituents of Acorus rhizome materials used as herbal drugs. The inhibitory effects of crude n-hexane extracts and their individual constituents on in vitro acetylcholine esterase activity were evaluated. The crude extracts had unexpectedly weak inhibitory effects (46-64% inhibition at 1.0 mg/mL), despite the high content (46-64%) of β-asarone, which independently had a potent effect (IC 50 2.9 µM [0.61 µg/mL]). Further investigation revealed participation of eudesmin A, a lignan constituent, in the suppression of the inhibitory effect of β-asarone.

Aims: Quorum sensing (QS) is a cell-cell communication system used by a broad spectrum of pathogenic bacteria to control the expression of their virulence genes. The interruption of QS systems of pathogenic bacteria has been considered as a novel way to fight bacterial diseases. In this study, trans-anethole, the main component of anise (Pimpinella anisum) oil was examined for its QS inhibitor (QSI) potential in an attempt to identify novel QSI compound effective against opportunistic pathogen Pseudomonas aeruginosa., Methods and Results: The preliminary screening of QSI capacity of trans-anethole was determined using a quorum-sensing inhibitor screen (QSIS) assay. The QSIS assay indicated that trans-anethole has QSI properties. QSI capacity of trans-anethole was further confirmed by lasB-gfp fussion assay and virulence factor assays. A sub-MIC of trans-anethole reduced the expression of lasB by 57%, elastase production by 59%, protease production by 56%, pyocyanin production by 95% and swarming motility by 68% without inhibiting growth of Pseudomonas aeruginosa PA01. Molecular docking and protein-ligand interaction studies were performed to understand the molecular mechanism underlying inhibitory activity of trans-anethole. The results of these analysis suggested that trans-anethole fits within the binding site of the LasR protein of P. aeruginosa., Conclusion: Trans-anethole has the potential to inhibit QS-regulated virulence factors in P. aeruginosa by binding to LasR protein, similar to its natural ligand N-(3-oxododecanoyl)-l-homoserine lactone., Significance and Impact of the Study: In this study, for the first time, it was demonstrated that trans-anethole has the potential to disrupt bacterial communication and can be developed as a novel QSI to combat with P. aeruginosa and other clinically significant pathogens., (© 2018 The Society for Applied Microbiology.)

α-, β- and γ-asarone are naturally occurring phenylpropenes that occur in different plant families, mainly in Aristolochiaceae, Acoraceae and Lauraceae. Plants containing asarones are used as flavouring ingredients in alcoholic beverages (bitters), traditional phytomedicines and the rhizome of e.g. Acorus calamus is used to prepare tea. Although α- and β-asarone show a potential in the treatment of several diseases, previous studies have shown carcinogenicity in rodents (duodenum, liver). However, the mechanism of action remained unclear. Studies on the mutagenicity of propenylic α- and β-asarone are inconsistent and data on carcinogenicity and genotoxicity of allylic γ-asarone are lacking completely. Thus, the present study determined the mutagenicity of the three asarone isomers using the Ames fluctuation assay with and without exogenous metabolic activation (S9 mix) in the standard Salmonella typhimurium strains TA98 and TA100. A concentration dependent increase in mutagenicity could be verified for α- and β-asarone in strain TA100 in the presence of rat liver homogenate. The side-chain epoxides of α- and β-asarone, major metabolites formed in liver microsomes, caused mutations in TA100, supporting the hypothesis that epoxidation of the side chain plays a key role in mutagenicity of the propenylic alkenylbenzenes. The allylic γ-asarone, not undergoing detectable side-chain epoxidation in liver microsomes, was supposed to be activated via side-chain hydroxylation and further sulphonation, a typical pathway for other allylic alkenylbenzenes like estragole or methyleugenol. However, neither y-asarone nor 1'-OH-γ-asarone showed any mutagenic effect even in the human SULT-expressing Salmonella strains (TA100-hSULT1A1 and TA100-hSULT1C2), while 1'-OH-methyleugenol used as a positive control was mutagenic under these conditions. These results indicate that the propenylic asarones are genotoxic via metabolic formation of side-chain epoxides while the side-chain hydroxylation/sulphonation pathway is either not operative or does not lead to mutagenicity with the allylic γ-asarone., (© The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Lycoriella ingenua (Dufour) (Diptera: Sciaridae) and Coboldia fuscipes (Meigen) (Diptera: Scatopsidae) are two of the most economically important insect pests of cultivated mushrooms. The toxicities to the fly larvae of the three phenylpropanoids (methyleugenol, myristicin, and safrole) from aerial parts of Asarum sieboldii Miquel (Aristolochiaceae) were compared with those of the currently available carbamate insecticide benfuracarb. In a contact+fumigant mortality bioassay with L. ingenua and C. fuscipes larvae, methyleugenol (1.46 and 2.33 µg/cm2) was the most toxic compound, followed by safrole (2.03 and 2.59 µg/cm2) and myristicin (3.59 and 4.96 µg/cm2), based on 24-h LC50 values. The phenylpropanoids were less toxic than benfuracarb (LC50, 0.75 and 0.55 µg/cm2). In vapor-phase mortality tests with the larvae, the phenylpropanoids were consistently more toxic in closed versus open containers, indicating that the effect of the compounds was largely a result of vapor action. Global efforts to reduce the level of highly toxic synthetic insecticides in the agricultural environment justify further studies on A. sieboldii plant-derived products as potential fumigants for the control of mushroom fly populations in mushroom houses and mushroom compost., (© The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

An assessment was made of the toxicity of 12 insecticides and three essential oils as well as Bacillus thuringiensis subsp. israelensis (Bti) alone or in combination with the oil major constituents (E)-anethole (AN), (E) -cinnamaldehyde (CA), and eugenol (EU; 1:1 ratio) to third instars of bamboo forest-collected Aedes albopictus (Skuse) and rice paddy field-collected Anopheles sinensis Wiedemann. An. sinensis larvae were resistant to various groups of the tested insecticides. Based on 24-h LC50 values, binary mixtures of Bti and CA, AN, or EU were significantly more toxic against Ae. albopictus larvae (0.0084, 0.0134, and 0.0237 mg/liter) and An. sinensis larvae (0.0159, 0.0388, and 0.0541 mg/liter) than either Bti (1.7884 and 2.1681 mg/liter) or CA (11.46 and 18.56 mg/liter), AN (16.66 and 25.11 mg/liter), or EU (24.60 and 31.09 mg/liter) alone. As judged by cotoxicity coefficient (CC) and synergistic factor (SF), the three binary mixtures operated in a synergy pattern (CC, 140.7-368.3 and SF, 0.0007-0.0010 for Ae. albopictus; CC, 75.1-245.3 and SF, 0.0008-0.0017 for An. sinensis). Global efforts to reduce the level of highly toxic synthetic insecticides in the aquatic environment justify further studies on the binary mixtures of Bti and essential oil constituents described, in particular CA, as potential larvicides for the control of malaria vector mosquito populations.

Leptotrombidium pallidum (Nagoya, Miyagawa, Mitamura & Tamiya) is a primary vector of Orientia tsutsugamushi (Hyashi), the causative agent of scrub typhus. An assessment is made of the repellency to L. pallidum larvae (chiggers) of cassia bark, eucalyptus, and star anise oils and major constituents (E)-cinnamaldehyde, 1,8-cineole, and (E)-anethole of the corresponding oils. Results were compared with those of conventional repellents DEET (N,N-diethyl-3-methylbenzamide), IR3535 [(ethyl 3-[acetyl(butyl)amino]propanoate)], and permethrin. Based on the median repellent concentration (RC50) values, (E)-cinnamaldehyde, (E)-anethole, cassia bark oil, and star anise oil (RC50, 0.95-1.52 mg/cm2) exhibited significantly more potent repellency than DEET (3.85 mg/cm2). (E)-cinnamaldehyde, (E)-anethole, cassiabark oil, 1,8-cineole, and star anise oil were approximately 43, 16, 11, 8, and 4 times more effective than IR3535 (CC5, 6.51%) as judged by the median climbing distance-disturbing concentration (CC50) values. The median residual duration time of repellency (RT50) was significantly more pronounced in DEET (RT50, 323 min) than in all essential oils and constituents (108-167 min). In the light of global efforts to reduce the level of highly toxic synthetic repellents, the three essential oils and their major constituents described merit further study as potential biorepellents for the control of L. pallidum populations.

Estragole is a naturally occurring food-borne genotoxic compound found in a variety of food sources, including spices and herbs. This results in human exposure to estragole via the regular diet. The objective of this study was to quantify the dose-dependent estragole-DNA adduct formation in rat liver and the urinary excretion of 1'-hydroxyestragole glucuronide in order to validate our recently developed physiologically based biodynamic (PBBD) model. Groups of male outbred Sprague Dawley rats (n = 10, per group) were administered estragole once by oral gavage at dose levels of 0 (vehicle control), 5, 30, 75, 150, and 300mg estragole/kg bw and sacrificed after 48h. Liver, kidney and lungs were analysed for DNA adducts by LC-MS/MS. Results obtained revealed a dose-dependent increase in DNA adduct formation in the liver. In lungs and kidneys DNA adducts were detected at lower levels than in the liver confirming the occurrence of DNA adducts preferably in the target organ, the liver. The results obtained showed that the PBBD model predictions for both urinary excretion of 1'-hydroxyestragole glucuronide and the guanosine adduct formation in the liver were comparable within less than an order of magnitude to the values actually observed in vivo. The PBBD model was refined using liver zonation to investigate whether its predictive potential could be further improved. The results obtained provide the first data set available on estragole-DNA adduct formation in rats and confirm their occurrence in metabolically active tissues, i.e. liver, lung and kidney, while the significantly higher levels found in liver are in accordance with the liver as the target organ for carcinogenicity. This opens the way towards future modelling of dose-dependent estragole liver DNA adduct formation in human.

The alkenylbenzene estragole is a constituent of several herbs and spices. It induces hepatomas in rodents at high doses following bioactivation by cytochrome P450s and sulfotransferases (SULTs) giving rise to the ultimate carcinogenic metabolite 1'-sulfooxyestragole which forms DNA adducts. Methanolic extracts from different alkenylbenzene-containing herbs and spices were able to inhibit SULT activity. Flavonoids including quercetin, kaempferol, myricetin, apigenin, and nevadensin were the major constituents responsible for this inhibition with Ki values in the nano to micromolar range. In human HepG2 cells exposed to the proximate carcinogen 1'-hydroxyestragole, the various flavonoids were able to inhibit estragole DNA adduct formation and shift metabolism in favor of glucuronidation which is a detoxification pathway for 1'-hydroxyestragole. In a next step, the kinetics for SULT inhibition were incorporated in physiologically based biokinetic (PBBK) models for estragole in rat and human to predict the effect of co-exposure to estragole and (mixtures of) the different flavonoids on the bioactivation in vivo. The PBBK-model-based predictions indicate that the reduction of estragole bioactivation in rat and human by co-administration of the flavonoids is dependent on whether the intracellular liver concentrations of the flavonoids can reach their Ki values. It is expected that this is most easily achieved for nevadensin which has a Ki value in the nanomolar range and is, due to its methyl ation, more metabolically stable than the other flavonoids.

A simple, accurate and sensitive ionic liquid-based headspace single-drop microextraction procedure followed by high-performance liquid chromatography was developed and validated for the determination of camphor and trans-anethole in compound liquorice tablets. The volume of the ionic liquid microdrop was increased to 12 µL by modifying the device of the suspended drop. The stability of the microdrop and the sensitivity of the method were improved. Under the optimum experimental conditions, the calculated calibration curves gave acceptable linearity for camphor and trans-anethole with correlation coefficients of 0.9990 and 0.9998, respectively. The repeatability of the proposed method, expressed as relative standard deviation, was below 4.5% (n = 5). The limits of detection for the two target analytes were found to be 9.77 and 1.95 × 10(-2) μg/mL, respectively. In this study, the separation, purification and enrichment were achieved in one step in an airtight system, which reduced the interferences caused by other complicated constituents, increased the signal-to-noise of the method and ensured the accuracy of the results because there was no loss of volatile components. It is expected to be widely applied for sample pretreatment of volatile components with high boiling points in samples with complicated matrices such as the extractions of plants or Chinese traditional drugs.

The navel orangeworm, Amyelois transitella (Walker) (Lepidoptera: Pyralidae), is the most destructive lepidopteran pest of almonds [Prunus dulcis (Mill.) D.A.Webb] and pistachios (Pistacia vera L.) in California and is a serious problem in figs (Ficus carica L.) and walnuts (Juglans spp.). In addition to direct damage, larval feeding leaves nuts vulnerable to infection by Aspergillus spp., fungi that produce toxic aflatoxins. A potentially safe and sustainable approach for managing navel orangeworm in orchards may be to use natural essential oil synergists to interfere with this insect's ability to detoxify insecticides and phytochemicals. We tested the effects of a naturally occurring plant-derived chemical, myristicin, and a synthetic inhibitor of cytochrome P450 monooxygenases (P450s), piperonyl butoxide, on the toxicity of three insecticides (alpha-cypermethrin, tau-fluvalinate, and methoxyfenozide [Intrepid]) and a phytochemical (xanthotoxin) to A. transitella. Piperonyl butoxide significantly synergized alpha-cypermethrin and tau-fluvalinate, whereas myristicin synergized only alpha-cypermethrin. Piperonyl butoxide synergized the toxicity of xanthotoxin as early as 72 h after exposure, whereas myristicin synergized xanthotoxin after 120 h. In view of these findings and the limited availability of environmentally safe synthetic insecticides for sustainable management, particularly in organic orchards, myristicin is a potential field treatment in combination with insecticides to reduce both navel orangeworm survival and aflatoxin contamination of nuts. In addition, this study demonstrates that in A. transitella the insect growth regulator methoxyfenozide is not detoxified by P450s.

The effect of α-asarone on impairment of cognitive performance caused by amnesic drug scopolamine was investigated. Treatment with α-asarone attenuated scopolamine-induced cognitive deficits as evaluated by passive avoidance and Y-maze test. Administration of α-asarone for 15 d improved memory and cognitive function as indicated by an increase in transfer latency time and spontaneous alternation in passive avoidance and the Y-maze test respectively. To understand the action of α-asarone, the levels of acetylcholinesterase (AChE), malondialdehyde (MDA), and superoxide dismutase (SOD) in the hippocampus (Hippo) and cerebral cortex (CC) of scopolamine-induced amnesic mice were evaluated. The mice treated with Scopolamine showed increased activity of AChE, MDA and SOD levels in both the Hippo and the CC area. Treatment with α-asarone attenuated the increased activity of AChE and normalized the MDA and SOD levels in the Hippo and the CC area in the scopolamine treated amnesic mice. These results suggest that α-asarone has a beneficial effect in cognitive impairment induced by dysfunction of cholinergic system in brain through inhibition of AChE activity and by influencing the antioxidant defense mechanism.

The increasing incidence of hospital-acquired infections caused by drug-resistant pathogens, host toxicity, the poor efficacy of drugs and high treatment costs has drawn attention to the potential of natural products as antifungals in mucocutaneous infections and combinational therapies. Moreover, cellular and subcellular targets for these compounds may provide better options for the development of novel antifungal therapies. Eugenol, methyl eugenol and estragole are phenylpropanoids found in essential oil. They are known to possess pharmacological properties including antimicrobial activity. Induction of oxidative stress characterized by elevated levels of free radicals and an impaired antioxidant defence system is implicated as a possible mechanism of cell death. An insight into the mechanism of action was gained by propidium iodide cell sorting and oxidative stress response to test compounds in Candida albicans. The extent of lipid peroxidation (LPO) of cytoplasmic membranes was estimated to confirm a state of oxidative stress. Activity levels of primary defence enzymes and glutathione were thus further determined. Whereas these compounds cause fungal cell death by disrupting membrane integrity at minimum inhibitory concentrations (MIC), sub-MIC doses of these compounds significantly impair the defence system in C. albicans. The study has implications for understanding microbial cell death caused by essential oil components eliciting oxidative stress in Candida. The formation of membrane lesions by these phenylpropanoids thus appears to be the result of free radical cascade-mediated LPO., (© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The present study investigates interindividual variation in liver levels of the proximate carcinogenic metabolite of estragole, 1'-hydroxyestragole, due to variation in two key metabolic reactions involved in the formation and detoxification of this metabolite, namely 1'-hydroxylation of estragole and oxidation of 1'-hydroxyestragole. Formation of 1'-hydroxyestragole is predominantly catalyzed by P450 1A2, 2A6, and 2E1, and results of the present study support that oxidation of 1'-hydroxyestragole is catalyzed by 17beta-hydroxysteroid dehydrogenase type 2 (17beta-HSD2). In a first approach, the study defines physiologically based biokinetic (PBBK) models for 14 individual human subjects, revealing a 1.8-fold interindividual variation in the area under the liver concentration-time curve (AUC) for 1'-hydroxyestragole within this group of human subjects. Variation in oxidation of 1'-hydroxyestragole by 17beta-HSD2 was shown to result in larger effects than those caused by variation in P450 enzyme activity. In a second approach, a Monte Carlo simulation was performed to evaluate the extent of variation in liver levels of 1'-hydroxyestragole that could occur in the population as a whole. This analysis could be used to derive a chemical-specific adjustment factor (CSAF), which is defined as the 99th percentile divided by the 50th percentile of the predicted distribution of the AUC of 1'-hydroxyestragole in the liver. The CSAF was estimated to range between 1.6 and 4.0, depending on the level of variation that was taken into account for oxidation of 1'-hydroxyestragole. Comparison of the CSAF to the default uncertainty factor of 3.16 for human variability in biokinetics reveals that the default uncertainty factor adequately protects 99% of the population.

Preparations of the plant Acorus calamus (calamus or sweet flag) (A. calamus) are available via internet trade and marketed as being hallucinogenic. In 2003-2006, the Swedish Poisons Information Centre received inquiries about 30 clinical cases of intentional intoxication with A. calamus products. The present investigation aimed to identify alpha- and beta-asarone, considered active components of A. calamus, and metabolites thereof in urine samples collected in seven of these cases. To further aid the identification of asarone biotransformation products, a calamus oil preparation was incubated with the fungus Cunninghamella elegans, which is used as a microbial model of mammalian drug metabolism. Using gas chromatography-mass spectrometry (GC-MS) analysis in selected ion monitoring mode, alpha-asarone was detected in five urine samples at concentrations ranging between approximately 11 and 1150 microg/L and beta-asarone in four of those at approximately 22-220 microg/L. A previously identified asarone metabolite, trans-2,4,5-trimethoxycinnamic acid (trans-TMC), was detected in the fungus broth by liquid chromatography-tandem mass spectrometry whereas cis-TMC was tentatively identified in the human urine samples. Using GC-MS, a hydroxylated asarone metabolite was identified both in fungus broth and urine samples. However, this study demonstrated no evidence for the presence of 2,4,5-trimethoxyamphetamine, claimed as a hallucinogenic component of A. calamus. The main clinical symptom reported by the patients was prolonged vomiting that sometimes lasted more than 15 h.

The extent of bioactivation of the herbal constituent estragole to its ultimate carcinogenic metabolite 1'-sulfooxyestragole depends on the relative levels of bioactivation and detoxification pathways. The present study investigated the kinetics of the metabolic reactions of both estragole and its proximate carcinogenic metabolite 1'-hydroxyestragole in humans in incubations with relevant tissue fractions. Based on the kinetic data obtained a physiologically based biokinetic (PBBK) model for estragole in human was defined to predict the relative extent of bioactivation and detoxification at different dose levels of estragole. The outcomes of the model were subsequently compared with those previously predicted by a PBBK model for estragole in male rat to evaluate the occurrence of species differences in metabolic activation. The results obtained reveal that formation of 1'-oxoestragole, which represents a minor metabolic route for 1'-hydroxyestragole in rat, is the main detoxification pathway of 1'-hydroxyestragole in humans. Due to a high level of this 1'-hydroxyestragole oxidation pathway in human liver, the predicted species differences in formation of 1'-sulfooxyestragole remain relatively low, with the predicted formation of 1'-sulfooxyestragole being twofold higher in human compared with male rat, even though the formation of its precursor 1'-hydroxyestragole was predicted to be fourfold higher in human. Overall, it is concluded that in spite of significant differences in the relative extent of different metabolic pathways between human and male rat there is a minor influence of species differences on the ultimate overall bioactivation of estragole to 1'-sulfooxyestragole.

Basil oil and its three major active constituents (trans-anethole, estragole, and linalool) obtained from basil (Oscimum basilicum L.) were tested on three tephritid fruit fly species [Ceratitis capitata (Wiedemann), Bactrocera dorsalis (Hendel), and Bactrocera cucurbitae (Coquillett)] for insecticidal activity. All test chemicals acted fast and showed a steep dose-response relationship. The lethal times for 90% mortality/knockdown (LT90) of the three fly species to 10% of the test chemicals were between 8 and 38 min. The toxic action of basil oil in C. capitata occurred significantly faster than in B. cucurbitae but slightly faster than in B. dorsalis. Estragole acted faster in B. dorsalis than in C. capitata and B. cucurbitae. Linalool action was faster in B. dorsalis and C. capitata than in B. cucurbitae. trans-Anethole action was similar to all three species. Methyl eugenol acted faster in C. capitata and B. cucurbitae than in B. dorsalis. When linalool was mixed with cuelure (attractant to B. cucurbitae male), its potency to the three fly species decreased as the concentration of cuelure increased. This was due to linalool hydrolysis catalyzed by acetic acid from cuelure degradation, which was confirmed by chemical analysis. When methyl eugenol (B. dorsalis male attractant) was mixed with basil oil, trans-anethole, estragole, or linalool, it did not affect the toxicity of basil oil and linalool to B. dorsalis, but it did significantly decrease the toxicity of trans-anethole and estragole. Structural similarity between methyl eugenol and trans-anethole and estragole suggests that methyl eugenol might act at a site similar to that of trans-anethole and estragole and serve as an antagonist if an action site exists. Methyl eugenol also may play a physiological role on the toxicity reduction.

Mountain pine beetle (Dendroctonus ponderosae) is the most important insect pest in southern Rocky Mountain ponderosa pine (Pinus ponderosa) forests. Tree mortality is hastened by the various fungal pathogens that are symbiotic with the beetles. The phenylpropanoid 4-allylanisole is an antifungal and semiochemical for some pine beetle species. We analyzed 4-allylanisole and monoterpene profiles in the xylem oleoresin from a total of 107 trees at six sites from two chemotypes of ponderosa pine found in Colorado and New Mexico using gas chromatography-mass spectroscopy (GC-MS). Although monoterpene profiles were essentially the same in attacked and nonattacked trees, significantly lower levels of 4-allylanisole were found in attacked trees compared with trees that showed no evidence of attack for both chemotypes.

The purpose of this study, which comprised 2 experiments, was to investigate cognitive effects on odor perception. An odor was presented using an olfactometer. In Experiment 1 ("continuous" presentation), anethole, an odor unfamiliar to most Japanese individuals, was presented continuously for 1 session (20 min), whereas in Experiment 2 ("intermittent" presentation), odor stimuli were presented 60 times for a short duration (0.2 s) over 4 sessions (24 min, including 9 min of intersession intervals), in which odor duration, temperature, and humidity were strictly controlled and the odor in the nostril was removed immediately after presentation. In each session, participants were asked to continuously evaluate odor intensity. In both Experiments 1 and 2, the participants were informed that the odor was either healthy (healthy-description group) or hazardous (hazardous-description group) prior to the session. The results show that in Experiment 2 (intermittent presentation), the hazardous-description group perceived the odor as more intense than did the healthy-description group, especially during the last 2 sessions. In Experiment 1 (continuous presentation), however, no significant difference in perceived intensity was present between the 2 groups. This study demonstrates the effect of cognitive state on perceived intensity by developing an experimental setting wherein the peripheral adaptation process was reduced and central olfactory processes were emphasized.

The phenoxyacetic acid derivatives 1-6 [2-methoxy-4-(2-propenyl)phenoxyacetic acid (1); 2-methoxy-5-nitro-4-(2-propenyl)phenoxyacetic acid (2); methyl 2-methoxy-4-(2-propenyl)phenoxyacetate (3); ethyl 2-methoxy-4-(2-propenyl)phenoxyacetate (4); methyl 2-methoxy-5-nitro-4-(2-propenyl)phenoxyacetate (5); ethyl 2-methoxy-5-nitro-4-(2-propenyl)phenoxyacetate (6)] related to alpha-asarone have been reported previously as hypolipidaemic agents in diet-induced hyperlipidaemic mice. We have aimed to expand the pharmacological profile of these derivatives by investigating their hypolipidaemic activity in rats and mice under different experimental conditions. The antiplatelet activity was tested also in-vitro from blood derived from consenting healthy volunteers. In normolipidaemic rats, compounds 2, 3 and 5 at oral doses of 40 and 80 mg kg(-1) significantly decreased total cholesterol and LDL-cholesterol levels. Moreover, analogues 3 and 5 administered to hypercholesterolaemic rats at the same doses for seven days also produced a reduction in the content of these same lipoproteins. In neither case were the high-density lipoprotein cholesterol and triglyceride concentrations affected. However, practically all tested compounds were found to be hypocholesterolaemic agents, and were shown to effectively lower low-density lipoprotein cholesterol and triglyceride levels in Triton-induced hyperlipidaemic mice at oral doses of 50 and 100 mg kg(-1). In all tests, all animals appeared to be healthy throughout the experimental period in their therapeutic ranges. Triton-induced hypercholesterolaemic mice appeared to be a desirable model for this class of hypolipidaemic drugs. On the other hand, compounds 1, 2, 4 and 5 significantly inhibited ADP-induced aggregation in-vitro. These findings indicated that all of these compounds appeared to be promising for the treatment of human hyperlipidaemia and thrombotic diseases.

A quantitative high-performance thin-layer chromatographic method for determination of beta-asarone in Calamus rhizome was developed and validated. The method is suitable for proper identification of Acorus calamus. Through the use of caffeine-modified silica gel as the stationary phase and toluene-ethyl acetate (93 + 7, v/v) as the mobile phase, beta-asarone is baseline separated from its isomer alpha-asarone. Scanning densitometry with absorption measurement at 313 nm allows specific, accurate, and precise quantification of beta-asarone. The working range of 40 to 200 ng absolute of the target substance is sufficient to establish whether a given sample passes the limit test of 0.5% maximum as required by the Swiss Pharmacopoeia.

Aims: To evaluate the fungitoxic activity of the essential oil of Agastache rugosa alone and to determine its combination effect with ketoconazole against Blastoschizomyces capitatus., Methods and Results: The antifungal activities of the essential oil of A. rugosa and its main constituent estragole were investigated using the broth microdilution, disk diffusion methods and checkerboard microtitre assay. Both estragole and the essential oil exhibited strong activities against the tested fungi and showed synergism with ketoconazole against B. capitatus., Conclusions: Both estragole and the essential oil of A. rugosa have significant growth-inhibiting activities against B. capitatus showing strong synergistic effect with ketoconazole., Significance and Impact of the Study: The essential oil of A. rugosa, combined with ketoconazole, may be particularly useful against B. capitatus, a rare pathogenic fungus documented to cause severe and fatal mycoses in immunocompromised patients.

Some basil varieties are able to convert the phenylpropenes chavicol and eugenol to methylchavicol and methyleugenol, respectively. Chavicol O-methyltransferase (CVOMT) and eugenol O-methyltransferase (EOMT) cDNAs were isolated from the sweet basil variety EMX-1 using a biochemical genomics approach. These cDNAs encode proteins that are 90% identical to each other and very similar to several isoflavone O-methyltransferases such as IOMT, which catalyzes the 4'-O-methylation of 2,7,4'-trihydroxyisoflavanone. On the other hand, CVOMT1 and EOMT1 are related only distantly to (iso)eugenol OMT from Clarkia breweri, indicating that the eugenol O-methylating enzymes in basil and C. breweri evolved independently. Transcripts for CVOMT1 and EOMT1 were highly expressed in the peltate glandular trichomes on the surface of the young basil leaves. The CVOMT1 and EOMT1 cDNAs were expressed in Escherichia coli, and active proteins were produced. CVOMT1 catalyzed the O-methylation of chavicol, and EOMT1 also catalyzed the O-methylation of chavicol with equal efficiency to that of CVOMT1, but it was much more efficient in O-methylating eugenol. Molecular modeling, based on the crystal structure of IOMT, suggested that a single amino acid difference was responsible for the difference in substrate discrimination between CVOMT1 and EOMT1. This prediction was confirmed by site-directed mutagenesis, in which the appropriate mutants of CVOMT1 (F260S) and EOMT1 (S261F) were produced that exhibited the opposite substrate preference relative to the respective native enzyme.

A highly potent attachment-inhibitor, polygodial, was isolated from a hexane extract of the leaves of Tasmannia lanceolata. The attachment-inhibiting activity of polygodial against the blue mussel was increased 4-fold when used in combination with sorbic acid, anethole, and indole.

A series of alpha-asarone analogues related to clofibrate, containing an acetic acid group at C-2 of the aromatic ring, has been prepared as the acids or as the ethyl and methyl esters. The corresponding alcohols were also synthesized by reduction of the ethyl esters. The compounds were examined in hyperlipidaemic male mice to evaluate their ability to modify serum lipoprotein cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglycerides after oral administration of 40 and 80 mg kg(-1) for 6 days. Except for methyl 2-methoxy-5-nitro-4-(2-propenyl)phenoxyacetate at either dose, these clofibrate-related phenoxyacetic acid derivatives were found to have significant hypocholesterolaemic activity. Levels of low-density lipoprotein cholesterol and triglycerides were significantly reduced and those of high-density lipoprotein cholesterol were elevated. 2-Methoxy-5-nitro-4-(2-propenyl)phenoxyacetic acid was active at both doses in all the tests. Clofibrate (150 mg kg(-1)) was more potent at reducing low-density lipoprotein cholesterol. No activity was detected for the alcohol derivatives. These preliminary results suggest that this class of compound might have more promise as potential hypolipidaemic agents than other alpha-asarone derivatives. Further investigation and characterization should be performed to determine the mode of action of these agents on lipid metabolism.

Essential oils extracted by hydrodistillation from five different varieties of Ocimum basilicum L. plants (Anise, Bush, Cinnamon, Dark Opal and a commercial sample of dried basil) were examined for antimicrobial activity against a wide range of foodborne Gram-positive and -negative bacteria, yeasts and moulds by an agar well diffusion method. All five essential oils of basil showed antimicrobial activity against most of the organisms tested with the exception of Flavimonas oryzihabitans and Pseudomonas species. The inhibitory effect of Anise oil, in comparison with mixtures of the predominant components of pure linalool and methyl chavicol, against the acid-tolerant organisms, Lactobacillus curvatus and Saccharomyces cerevisiae, was examined in broth by an indirect impedance method. Synergistic effects between Anise oil, low pH (pH 4.2) and salt (5% NaCl) were determined. The antimicrobial effect of Anise oil was also assessed in a tomato juice medium by direct viable count, showing that the growth of Lact. curvatus and S. cerevisiae was completely inhibited by 0.1% and 1% Anise oil, respectively. The results of the current study indicate the need for further investigations to understand the antimicrobial effects of basil oils in the presence of other food ingredients and preservation parameters.

Basil essential oils, including basil sweet linalool (BSL) and basil methyl chavicol (BMC), were screened for antimicrobial activity against a range of Gram-positive and Gram-negative bacteria, yeasts and moulds using an agar well diffusion method. Both essential oils showed antimicrobial activity against most of the micro-organisms examined except Clostridium sporogenes, Flavimonas oryzihabitans, and three species of Pseudomonas. The minimum inhibitory concentration (MIC) of BMC against Aeromonas hydrophila and Pseudomonas fluorescens in TSYE broth (as determined using an indirect impedance method) was 0.125 and 2% (v/v), respectively; the former was not greatly affected by the increase of challenge inoculum from 10(3) to 10(6) cfu ml-1. Results with resting cells demonstrated that BMC was bactericidal to both Aer. hydrophila and Ps. fluorescens. The growth of Aer. hydrophila in filter-sterilized lettuce extract was completely inhibited by 0.1% (v/v) BMC whereas that of Ps. fluorescens was not significantly affected by 1% (v/v) BMC. In addition, the effectiveness of washing fresh lettuce with 0.1 or 1% (v/v) BMC on survival of natural microbial flora was comparable with that effected by 125 ppm chlorine.

A simple and sensitive high-performance liquid chromatography (HPLC) method for the determination of asarone in rabbit plasma has been developed. Up to 0.1 mL of plasma containing asarone was deproteinated by acetonitrile, which contained an internal standard (indomethacin). The supernatant was injected into a Nucleosil 7C18 column using acetonitrile-water-triethylamine (55:45:0.1 v/v, pH 5.4-5.5, adjusted with orthophosphoric acid) as the mobile phase and UV detection at 257 nm, followed by UV spectrum identification (between 200 and 380 nm) with a photodiode array detector. The method is rapid, easily reproduced, selective and sensitive. It was applied to pharmacokinetic studies of asarone in rabbit, after 5, 10, or 20 mg kg-1 intravenous administration. Rapid distribution followed by a slower elimination phase was observed from the plasma concentration-time curve. The plasma disposition at each dose fitted well to a two-compartment open model and the terminal disposition became much slower as the dose was increased, suggesting a nonlinear dose-dependent plasma asarone disposition.

Approximately 20% of a dose of estragole, a naturally occurring flavoring agent, was excreted in the urine of outbred male CD -1 mice as a conjuage (presumably the glucuronide) of 1'-hydroxyestragole, Estragole and its 1'-hydroxy metabolite caused significant increases in the incidences of hepatocellular carcinomas in male CD-1 mice that received the compounds by sc injection at 1-22 days of age. Estragole induced hepatocellular carcinomas by 15 months in 23 and 39% of the mice that received total doses of 4.4 and 5.2 mumoles, respectively, and lived to an age of 12 months or more. Of the 12-month survivors given a total dose of 4.4 mumoles of 1'-hydroxyestragole, 70% developed hepatocellular carcinomas; the incidence in mice that received only the vehicle (trioctanoin) was 12%. Multiple tumors ocurred in 5, 28, 64, and 0%, respectively, of the mice in each of these 4 groups. Of the mice given a total dose of 4.4 mumoles of 1'-hydroxysafrole, 59developed hepatocellular carcinomas; 39% of the mice bore multiple liver tumors. As previsously demonstrated for 1'-acetoxysafrole, 1'-acetoxyestragole and 1'-acetoxy-1-allyl-4-methoxynaphthalene reacted nonenzymatically with guanosine and inosine to form adducts. These electrophilic esters were strongly mutagenic for the Salmonella typhimurium missense mutant TA100. 1'-Acetoxyallybenzene had little or no activity in either of these tests. Attempts to demonstrate liver-mediated mutagenicty for 1'-hydroxysafrole and 1'-hydroxyestragole in the bacterial test system were unsuccessful.