Toxicity, feeding deterrence, and effect of activity of 1,8-cineole from Artemisia annua on progeny production of Tribolium castanaeum (Coleoptera: Tenebrionidae)

1,8-Cineole isolated from Artemisia annua was tested against Tribolium castaneum (Herbst) for contact toxicity, fumigant toxicity, and antifeedant activity. The adults of T. castaneum were more susceptible than larvae to both contact and fumigant toxicity of 1,8-cineole, and LD50 and LC50 values of 108.4 microg/mg body weight of adult insect and 1.52 mg/liter air were found, respectively. Furthermore, 14-d-old T. castaneum larvae were more tolerant than 16- and 18-d-old larvae and adults to the contact toxicity of 1,8-cineole, but the 16- and 18-d-old larvae have similar susceptibility. In contrast, all the larvae (14-18 d old) of T. castaneum were much more tolerant than the adults to the fumigant action, but larvae of different ages had similar susceptibility. The compound 1,8-ciineole applied to filter paper at a concentration of 3.22-16.10 mg/cm2 significantly (P < 0.05) reduced the hatching of T. castaneum eggs and the subsequent survival rate of the larvae. Adult emergence was also reduced by 1,8-cineole. Feeding deterrence of 81.9% was achieved in T. castateum adults by using a concentration of 121.9 mg/g food, whereas larvae showed 68.8% at the same concentration.     

Cardiovascular effects of 1,8-cineole,a terpenoid oxide present in many plant essential oils,in normotensive rats

The cardiovascular effects of i.v. treatment with 1,8-cineole, a monoterpenic oxide present in many plant essential oils, were investigated in normotensive rats. This study examined (i) whether the autonomic nervous system is involved in the mediation of 1,8-cineole-induced changes in mean aortic pressure (MAP) and heart rate (HR) and (ii) whether the hypotensive effects of 1,8-cineole could result from its vasodilatory effects directly upon vascular smooth muscle. In both pentobarbital-anesthetized and conscious, freely moving rats, bolus injections of 1,8-cineole (0.3-10 mg/kg, i.v.) elicited similar and dose-dependent decreases in MAP. Concomitantly, 1,8-cineole significantly decreased HR only at the highest dose (10 mg/kg). Pretreatment of anesthetized rats with bilateral vagotomy significantly reduced the bradycardic responses to 1,8-cineole (10 mg/kg) without affecting hypotension. In conscious rats, i.v. pretreatment with methylatropine (1 mg/kg), atenolol (1.5 mg/kg), or hexamethonium (30 mg/kg) had no significant effects on the 1,8-cineole-induced hypotension, while bradycardic responses to 1,8-cineole (10 mg/kg) were significantly reduced by methylatropine. In rat isolated thoracic aorta preparations, 1,8-cineole (0.006-2.6 mM) induced a concentration-dependent reduction of the contraction induced by potassium (60 mM). This is the first physiological evidence that i.v. treatment with 1,8-cineole in either anesthetized or conscious rats elicits hypotension; this effect seems related to an active vascular relaxation rather than withdrawal of sympathetic tone.     

Application of solid-phase microextraction to the quantitative analysis of 1,8-cineole in blood and expired air in a Eucalyptus herbivore,the brushtail possum (Trichosurus vulpecula)

We have developed two solid-phase microextraction (SPME) methods, coupled with gas chromatography, for quantitatively analysing the major Eucalyptus leaf terpene, 1,8-cineole, in both expired air and blood from the common brushtail possum (Trichosurus vulpecula). In-line SPME sampling (5 min at 20 degrees C room temperature) of excurrent air from an expiratory chamber containing a possum dosed orally with 1,8-cineole (50 mg/kg) allowed real-time semi-quantitative measurements reflecting 1,8-cineole blood concentrations. Headspace SPME using 50 microl whole blood collected from possums dosed orally with 1,8-cineole (30 mg/kg) resulted in excellent sensitivity (quantitation limit 1 ng/ml) and reproducibility. Blood concentrations ranged between 1 and 1380 ng/ml. Calibration curves were prepared for two concentration ranges (0.05-10 and 10-400 ng/50 microl) for the analysis of blood concentrations. Both calibration curves were linear (r(2)=0.999 and 0.994, respectively) and the equations for the two concentration ranges were consistent.     

Identification of pheromone candidates for the eucalyptus weevil,Gonipterus platensis (Coleoptera,Curculionidae)

The eucalyptus weevil, Gonipterus platensis (Coleoptera, Curculionidae), is a major pest of eucalyptus plantations worldwide. To date, no pheromones have been identified for this species, despite their valuable potential as tools in monitoring or control strategies. Here we report the detection and identification of pheromones candidates of G. platensis. The weevil's volatile compounds were collected by solid phase micro extraction (SPME) and monolithic material sorption extraction (MMSE). Using Gas Chromatography coupled to Mass Spectrometry (GC/MS) analysis, eleven insect specific compounds were detected and identified: verbenene, cis-verbenol, trans-verbenol, verbenone, 2-oxo-1,8-cineole, 9-hydroxy-1,8-cineole, 2-α-hydroxy-1,8-cineole, 3-oxo-1,8-cineole, 2-β-hydroxy-1,8-cineole, 3-α-hydroxy-1,8-cineole and 7-hydroxy-1,8-cineole. Three of these compounds, verbenene, cis-verbenol and trans-verbenol, were shown to be male-specific. Antennal sensitivity towards ten compounds emitted by G. platensis was detected using Gas Chromatography–Mass Spectrometry/Electroantennographic Detection (GC-MS/EAD). Extracts from virgin males proved to be attractive to virgin females in olfactometer bioassays. Further behavioural bioassays showed that both virgin females and virgin males were attracted to the male-specific compound cis-verbenol and that virgin females were attracted to trans-verbenol. Verbenone was attractive to mated females. Regarding 2-α-hydroxy-1.8-cineole and 2-oxo-1,8-cineole, which are produced by both sexes, the alcohol was attractive to virgin males and both the alcohol and the ketone were repellant to mated females. This is, to our knowledge, the first identification of pheromones candidates in Gonipterus spp. and also the first evidence of cineole metabolites acting as semiochemicals.     

Metabolites of dietary 1,8-cineole in the male koala (Phascolarctos cinereus)

The in vivo metabolic fate of 1,8-cineole was investigated in six male koalas. Koalas were fed ad lib a diet of Eucalyptus cephalocarpa leaf with a 1,8-cineole concentration of 2.53+/-0.70% dry mass of leaf, corresponding to a 1,8-cineole intake of 2.4+/-1.1 mmol/kg (3.1+/-1.3 g). Urine and faeces were collected for 24 h and metabolites identified by GC-MS and LC-MS. Metabolites were quantified before and after hydrolysis with beta-glucuronidase to give free and total levels, respectively. Fractional recovery of ingested 1,8-cineole was 1.3+/-0.4 and 1.4+/-0.4 (mean+/-S.D.) for free and total measurements, respectively. Seven metabolites were identified and quantified: 9- and 7-hydroxycineole, 9- and 7-cineolic acid, 7-hydroxy-9-cineolic acid, 9-hydroxy-7-cineolic acid and 7,9-dicineolic acid. The hydroxycineolic acids dominated the metabolite profile (85%). 7,9-Dicineolic acid, a novel metabolite of 1,8-cineole, accounted for almost 10% of the recovered dose making it the second most abundant metabolite after 7-hydroxy-9-cineolic acid (77%). Together, the less oxidised metabolites, the hydroxycineoles and cineolic acid, accounted for only 5% of the cineole consumed. Significant conjugation only occurred with four minor, less oxidised, alcohol and carboxylic acid metabolites. We have shown that the koala detoxifies and eliminates 1,8-cineole primarily by extensive oxidation without utilising conjugation pathways.     

Inhibition of soybean lipoxygenase-1 by 10-butyryl substituted 1,8-dihydroxy-9-anthrone (butantrone)

10-Butyryl substituted 1,8-dihydroxyanthrone (butantrone) inhibited soybean lipoxygenase-1 irreversibly and more efficiently than its parent compound 1,8-dihydroxyanthrone (dithranol, anthralin) (IC50 values 0.090 mM and 1.1 mM, respectively). Intact butantrone rather than its hydrolysis product was the primary effector and the 10-butyryl moiety its site specific probe, probably directing the inhibitor to the proximity of the binding site of the lipid substrate/product.     

Inhibition of cholinesterase by essential oil from food plant

Inhibition of cholinesterase has attracted much attention recently because of its potential for the treatment of Alzheimer's disease. In this work, the anticholinesterase activities of plant oils were investigated using Ellman's colorimetric method. The results indicate that essential oils obtained from Melissa officinalis leaf and Citrus aurantifolia leaf showed high acetylcholinesterase and butyrylcholinesterase co-inhibitory activities. C. aurantifolia leaf oil revealed in this study has an IC(50) value on acetylcholinesterase and butyrylcholinesterase of 139 ± 35 and 42 ± 5 μg/ml, respectively. GC/MS analysis revealed that the major constituents of C. aurantifolia leaf oil are monoterpenoids including limonene, l-camphor, citronellol, o-cymene and 1,8-cineole.     

Essential Oils of Three Taxa of the Nepeta argolica Aggregate from Greece

The essential-oil composition of three not previously studied taxa belonging to the Nepeta argolica aggregate was examined. The oils of N. argolica ssp. malacotrichos and N. argolica ssp. vourinensis (both Greek endemics) are rich in 1,8-cineole (30.9-55.6% of the total oil composition), while those of the Balkan endemic N. spruneri are rich in either sesquiterpenes (mainly caryophyllene oxide, 14.6-19.8%) or in 4aα,7α,7aβ- and 4aα,7α,7aα-nepetalactones (total content 17.5-40.5%), accompanied by caryophyllene oxide (11.5-11.7%) and 1,8-cineole (up to 16.5%). The comparison of our results with published information concerning the related taxa of the N. argolica aggregate showed that while oils with as main components nepetalactones or 1,8-cineole are common, sesquiterpene-rich oils are reported for the first time within the aggregate.     

1,8-cineole and castor oil in sodium lauryl ether sulphate disrupt reproduction and ovarian tissue of Rhipicephalus (Boophilus) microplus

Essential and fixed oils have been researched as alternatives to chemical acaricides. The activity of volatile compounds from essential oils (1,8-cineole, citral and eugenol) at 1.0% (w/v) and fixed oil (castor oil) at 0.3% (w/v) dissolved in 2.0% (v/v) dimethyl sulfoxide (DMSO) + 0.2% (w/v) Tween 80^® was assessed against Rhipicephalus microplus using immersion tests. 1,8-cineole (29.0%) and castor oil (30.2%) had the highest reproductive inhibition rate. A second experiment was performed to verify the effect of the 1,8-cineole (10.0% w/v) and, or castor oil (0.3% w/v) on tick reproduction using different solubilizing agents. The highest reproductive inhibition was observed for the combination of 1,8-cineole/castor oil (94.1%) and 1,8-cineole in 2.0% (w/v) sodium lauryl ether sulphate (SLES) (92.8%). A third experiment showed morphological changes in R. microplus oocytes at different stages of development, as well as in pedicel cells. The most intense effects were observed when ticks were immersed in the formulation containing 1,8-cineole (10.0% w/v) and castor oil (0.3% w/v) dissolved in 2% (w/v) SLES. These findings highlight the potential of this formulation as an alternative for managing cattle ticks as their cytotoxic effects can reduce R. microplus reproductive success.     

IBV核蛋白的表达纯化及在监测中应用

鸡传染性支气管炎病毒（Infectious bronchitis virus,IBV）是禽类一种变异性很强的冠状病毒,为获得高度纯化的IBV核蛋白,建立监测IBV抗体的方法,通过RT-PCR从IBV中扩增IBV-N基因,定向连接到表达载体pGEX-KG中,转化E. coli BL21（DE3）菌株,诱导获得表达产物,SDS-PAGE分析产物大小、Western blot分析其免疫学活性,通过亲和层析法获得高度纯化的表达蛋白,建立一种检测IBV抗体的方法,并应用于临床监测。结果显示,IBV-N基因全长1230bp,GST融合蛋白表达产物大小约为80kDa,表达量约占菌体总蛋白的25%,具有良好的免疫学活性;通过GST亲和层析柱成功获得纯化蛋白,以该蛋白包被酶标板,成功地建立了一种检测血清中IBV抗体的间接ELISA方法。研究表明重组N蛋白作为IBV诊断抗原,具有制备简便、特异性强、敏感性高、重复性好的特点,可用于该病的临床监测,为该病疫情监测、发病机制研究奠定了基础。     

Metabolism of 1,8-cineole by human cytochrome P450 enzymes: identification of a new hydroxylated metabolite

Human metabolism of the monoterpene cyclic ether 1,8-cineole was investigated in vitro and in vivo. In vitro, the biotransformation of 1,8-cineole was investigated by human liver microsomes and by recombinant cytochrome P450 enzymes coexpressed with human CYP-reductase in Escherichia coli cells. Besides the already described metabolite 2alpha-hydroxy-1,8-cineole we found another metabolite produced at high rates. The structure was identified by a comparison of its mass spectrum and retention time with the reference compounds as 3alpha-hydroxy-1,8-cineole. There was a clear correlation between the concentration of the metabolites, incubation time and enzyme content, respectively. CYP3A4/5 antibody significantly inhibited the 2alpha- and 3alpha-hydroxylation catalyzed by pooled human liver microsomes. Further kinetic analysis revealed that the Michaelis-Menten K(m) and V(max) for oxidation of 1,8-cineole in position three were 19 microM and 64.5 nmol/min/nmol P450 for cytochrome P450 3A4, and 141 microM and 10.9 nmol/min/nmol P450 for cytochrome P450 3A5, respectively. To our knowledge, this is the first time that 3alpha-hydroxy-1,8-cineole is described as a human metabolite of 1,8-cineole. We confirmed these in vitro results by the investigation of human urine after the oral administration of cold medication containing 1,8-cineole. In human urine we found by GC-MS analysis the described metabolites, 2alpha-hydroxy-1,8-cineole and 3alpha-hydroxy-1,8-cineole.     

鸡传染性支气管炎病毒的RNA干扰

为探讨短的双链RNA(siRNA)对鸡传染性支气管炎病毒(IBV)增殖的干扰作用,利用软件设计siRNA1280个,75%位于Pol基因内。通过同源比较和保守性分析,筛选到针对Pol、M、N基因的12个siRNA(每个基因3～4个)作为后选目的片段,分别在Vero细胞、9日龄SPF鸡胚上进行基因干扰试验。结果,来自Pol、N靶序列的2个siRNA在Vero细胞上及鸡胚上均对IBV增殖产生明显的干扰作用,并与siRNA剂量有一定相关性,依赖于与mRNA互补的负链siRNA存在。本研究首次证实IBV增殖过程中存在siRNA干扰现象,为利用RNA干扰(RNAi)技术控制IBV提供了新手段。     

Synthesis of ‘cineole cassette’ monoterpenes in Nicotiana section Alatae: gene isolation, expression, functional characterization and phylogenetic analysis

The scent bouquets of flowers of Nicotiana species, particularly those of section Alatae, are rich in monoterpenes, including 1,8-cineole, limonene, β-myrcene, α- and β-pinene, sabinene, and α-terpineol. New terpene synthase genes were isolated from flowers of Nicotiana bonariensis, N. forgetiana, N. longiflora, and N. mutabilis. The recombinant enzymes synthesize simultaneously the characteristic 'cineole cassette' monoterpenes with 1,8-cineole as the dominant volatile product. Interestingly, amino acid sequence comparison and phylogenetic tree construction clustered the newly isolated cineole synthases (CIN) of section Alatae together with the catalytically similar CIN of N. suaveolens of section Suaveolentes, thus suggesting a common ancestor. These CIN genes of N. bonariensis, N. forgetiana, N. longiflora, and N. mutabilis are distinct from the terpineol synthases (TERs) of the taxonomically related N. alata and N. langsdorfii (both Alatae), thus indicating gene diversification of monoterpene synthases in section Alatae. Furthermore, the presence of CINs in species of the American section Alatae supports the hypothesis that one parent of the Australian section Suaveolentes was a member of the present section Alatae. Amino acid sequences of the Nicotiana CINs and TERs were compared to identify relevant amino acids of the cyclization reaction from α-terpineol to 1,8-cineole.     

Natural variation in the essential oil content of Melaleuca alternifolia Cheel (Myrtaceae)

The composition and yield of oil in 615 trees representing the natural populations of Melaleuca alternifolia, or tea tree, was investigated. A sixth distinct oil chemotype was identified. Of the six chemotypes, one chemotype is dominated by terpinen-4-ol, one by 1,8-cineole, one by terpinolene and the remaining three chemotypes are all dominated by 1,8-cineole and differ in either terpinen-4-ol or terpinolene content. Whilst most chemotypes are present throughout the distribution range, a definite correspondence of oil types with geographic location was found. Terpinen-4-ol types predominate in and around the Bungawalbin basin in the Casino area of northern New South Wales (NSW), high 1,8-cineole types predominate toward the southern end of the distribution around Grafton and terpinolene types predominate in southern Queensland. Preliminary formulae have been developed to allow comparisons of oil data obtained by steam distillation with a static headspace gas chromatography method.     

乙型肝炎病毒X蛋白结合蛋白（HBXIP）对细胞周期的影响

与乙肝病毒X蛋白（HBX）的C端结合,它具有抑制HBX活性的作用. 为进一步阐明HBXIP对细胞增殖的作用及其分子机制,构建了HBXIP的真核表达载体,并将其稳定转染至正常人肝细胞系L-O2细胞中,建立了稳定表达HBXIP蛋白的肝细胞系,命名为L-O2-hbxip. 然后,应用MTT、BrdU标记实验和流式细胞术等方法,发现HBXIP过表达后,L-O2细胞的生长速度明显加快,可促进细胞由G₁期进入到S期,表明HBXIP具有促进L-O2-hbxip细胞增殖的作用.应用免疫印迹对有关细胞周期相关蛋白进行了检测. 结果显示,HBXIP过表达时可上调细胞周期蛋白D₁、细胞周期蛋白E的表达,并下调p21和p27的表达,从而调节细胞周期,产生对细胞增殖的影响.     

Variation of Salvia fruticosa Essential Oils on the Island of Crete (Greece)

The essential oil content and composition of Salvia fruticosa (Greek sage) plants growing wild in 20 localities scattered on the island of Crete are studied. The results of our analyses have shown a noticeable variation in the essential oil content (ranging from 1.1 up to 5.1 %) and the amount of the four main oil components: 1,8-cineole (22.7 ? 64.2% of total oil), α-thujone (1.0 ? 19.2%) β-thujone (0.9 ? 25.6%) and camphor (0.8 ? 30.3%). Discriminant analysis revealed that the variation pattern of the essential oil content and the amount of the four main oil components is geographically related, following a W → E direction. Plants grown in Western Crete show a lower essential oil content and their oils are characterised by the predominance of 1,8-cineole. On the other hand, those collected from Eastern Crete exhibit higher values in essential oil content and their oils, besides 1,8-cineole, are rich in α- and β-thujone or camphor. Our findings are further discussed in relation to literature data.     

Gastroprotective Mechanisms of the Monoterpene 1,8-Cineole (Eucalyptol)

Recently, our research group identified and reported 1,8-cineole (CIN), a monoterpene that naturally occur in many aromatic plants, as one of the major constituent of the essential oil from leaves of Hyptis martiusii (EOHM), as well as characterized the gastroprotective action of this oil. The aim of this study was to investigate the mechanisms of action involved in the antiulcer and healing activity of CIN, in order to confirm its correlation with the gastroprotective effect of EOHM. Wistar rats were exposed to different protocols (acute ulceration, gastrointestinal motility and antisecretory activity). In addition, were determinated the involvement of nitric oxide and sulphydryl groups; the levels of gastric mucus, lipid peroxidation, sulphydryl groups and myeloperoxidase activity. The healing ability was evaluated by acetic acid-induced chronic ulcer and histological and immunohistochemical analysis (PCNA, Ki-67 and BrdU). The treatment with CIN inhibited ethanol-, ethanol/HCl- and indomethacin-induced gastric lesions. The highest doses of CIN inhibited gastric emptying, but did not affect intestinal transit. CIN (100 mg/kg) reduced the volume of basal but not stimulated acid secretion. CIN increased levels of mucus (89.3%), prevented depletion of –SH groups (62.6%) and reduced the level of lipid peroxidation (55.3%) and myeloperoxidase activity (59.4%) in the gastric mucosa. In chronic ulcer model, CIN reduced in 43.1% the gastric area lesion, promoted significant regeneration and restoration of the levels of mucus in glandular cells as confirmed by histological analysis; and promoted increase in cell proliferation as evidenced by reactivity for PCNA, Ki-67 and BrdU. This findings demonstrate the role of 1,8-cineole as an important ulcer healing agent and indicate the involvement of antioxidant and cytoprotective mechanisms in the gastroprotective effect of compound. This study also provides evidence that 1,8-cineole is related to the gastroprotective effect of the essential oil of Hyptis martiusii.     

Interaction of the coronavirus nucleoprotein with nucleolar antigens and the host cell

Coronavirus nucleoproteins (N proteins) localize to the cytoplasm and the nucleolus, a subnuclear structure, in both virus-infected primary cells and in cells transfected with plasmids that express N protein. The nucleolus is the site of ribosome biogenesis and sequesters cell cycle regulatory complexes. Two of the major components of the nucleolus are fibrillarin and nucleolin. These proteins are involved in nucleolar assembly and ribosome biogenesis and act as chaperones for the import of proteins into the nucleolus. We have found that fibrillarin is reorganized in primary cells infected with the avian coronavirus infectious bronchitis virus (IBV) and in continuous cell lines that express either IBV or mouse hepatitis virus N protein. Both N protein and a fibrillarin-green fluorescent protein fusion protein colocalized to the perinuclear region and the nucleolus. Pull-down assays demonstrated that IBV N protein interacted with nucleolin and therefore provided a possible explanation as to how coronavirus N proteins localize to the nucleolus. Nucleoli, and proteins that localize to the nucleolus, have been implicated in cell growth-cell cycle regulation. Comparison of cells expressing IBV N protein with controls indicated that cells expressing N protein had delayed cellular growth. This result could not to be attributed to apoptosis. Morphological analysis of these cells indicated that cytokinesis was disrupted, an observation subsequently found in primary cells infected with IBV. Coronaviruses might therefore delay the cell cycle in interphase, where maximum translation of viral mRNAs can occur.     

传染性支气管炎病毒核衣壳蛋白基因克隆及在E.coli中的表达

核衣壳蛋白基因 (N基因 )是传染性支气管炎病毒的重要结构基因 .根据已报道的序列设计引物 ,利用RT PCR技术从病毒RNA中扩增和克隆到了N基因的cDNA ,并测定了核苷酸序列 .克隆的N基因片段ORF全长 12 30bp ,编码 4 0 9个氨基酸 .将该片段序列与其他IBV病毒株比较 ,核苷酸的同一性为 87 0 %～ 98 6 %,氨基酸的同一性为 91 0 %～ 98 1%.将该cDNA亚克隆到pBV2 2 0表达载体 ,转化大肠杆菌DH5α菌株 ,Western印迹检测 ,获得了分子量约 4 5kD表达蛋白