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Biosynthesis and emission of terpenoid volatiles from Arabidopsis flowers.

Arabidopsis is believed to be mostly self-pollinated, although several lines of genetic and morphological evidence indicate that insect-mediated outcrossing occurs with at least a low frequency in wild populations. Here, we show that Arabidopsis flowers emit both monoterpenes and sesquiterpenes, potential olfactory cues for pollinating insects. Of the 32 terpene synthase genes in the Arabidopsis genome, 20 were found to be expressed in flowers, 6 of these exclusively or almost exclusively so. Two terpene synthase genes expressed exclusively in the flowers and one terpene synthase gene expressed almost exclusively in the flowers were characterized and found to encode proteins that catalyze the formation of major floral volatiles. A beta-glucuronidase fusion construct with a promoter of one of these genes demonstrated that gene expression was restricted to the sepals, stigmas, anther filaments, and receptacles, reaching a peak when the stigma was receptive to cross pollen. The observation that Arabidopsis flowers synthesize and emit volatiles raises intriguing questions about the reproductive behavior of Arabidopsis in the wild and allows detailed investigations of floral volatile biosynthesis and its regulation to be performed with this model plant system.

Alkyl and Aryl Transferases↗

Functional identification of rice syn-copalyl diphosphate synthase and its role in initiating biosynthesis of diterpenoid phytoalexin/allelopathic natural products.

Rice produces a number of phytoalexins, and at least one allelopathic agent, from syn-copalyl diphosphate (CPP), representing the only known metabolic fate for this compound. Thus, the class II terpene synthase that converts the universal diterpenoid precursor geranylgeranyl diphosphate to syn-CPP catalyzes the committed step in biosynthesis of these natural products. Here the extensive sequence information available for rice was coupled to recombinant expression and functional analysis to identify syn-copalyl diphosphate synthase (OsCPSsyn). In addition, OsCPSsyn mRNA was found to be specifically induced in leaves by conditions that stimulate phytoalexin biosynthesis. Therefore, transcription of OsCPSsyn seems to be an important regulatory point for controlling the production of these defensive compounds. Finally, alignments carried out with OsCPSsyn revealed that class II terpene synthases exhibit a sequence conservation pattern substantially different from that of the prototypical class I enzymes. One particularly notable feature is the specific conservation of the functionally cryptic 'insertional' sequence element in class II terpene synthases, indicating that this region is important for the corresponding cyclization reaction.

Alkyl and Aryl Transferases↗

Missing OH reactivity in a forest: evidence for unknown reactive biogenic VOCs.

Forest emissions of biogenic volatile organic compounds (BVOCs), such as isoprene and other terpenes, play a role in the production of tropospheric ozone and aerosols. In a northern Michigan forest, the direct measurement of total OH reactivity, which is the inverse of the OH lifetime, was significantly greater than expected. The difference between measured and expected OH reactivity, called the missing OH reactivity, increased with temperature, as did emission rates for terpenes and other BVOCs. These measurements are consistent with the hypothesis that unknown reactive BVOCs, perhaps terpenes, provide the missing OH reactivity.

Aerosols↗

Work related impairment of nasal function in Swedish woodwork teachers.

OBJECTIVE: To study the relation between exposure and nasal function in woodwork teachers. METHODS: 39 selected woodwork teachers employed full time and 32 control subjects (other school personnel) were examined at the beginning and at the end of a working week with symptom rating, nose and throat inspection, rhinomanometry, nasal mucociliary clearance test, and a smell identification test. During one working day of the same week climate, dust and terpene concentrations were measured in all 39 schools. RESULTS: The ventilation rate was highest in rooms with mechanical ventilation. Range of total dust (personally sampled) was 0.12-1.18 mg/m3, respirable dust 0.02-0.21 mg/m3, and terpenes (area sampled) 0.02-6.8 mg/m3. In contrast to the control subjects, the woodwork teachers had more nasal symptoms on the Thursday afternoon than on the Monday morning, especially those working in rooms without mechanical ventilation. Their mucociliary clearance worsened during the week (mean increase 4 min, P < 0.001). A small impairment of olfactory function was also found, but their rhinomanometric values did not change significantly. Nasal symptoms correlated weakly with the percentage of respirable dust in the total dust. Otherwise there were no significant dose-effect relations between measured dust or terpene concentrations and nasal tests. CONCLUSIONS: The woodwork teachers had mainly reversible nasal complaints, impaired nasal mucociliary clearance and olfactory function related to the work environment, with dust concentrations below the Swedish threshold limit value of 2 mg/m3.

Adult↗

Pharmaceutical quality of different Ginkgo biloba brands.

Ginkgo biloba-containing brands are one of the top sellers within the growing market for herbal remedies in many European countries as well as in the USA. In the consumers' interest, these brands should feature a certain quality and should be transparent in quality claims. In this investigation, a variety of products on the USA market was studied with respect to pharmaceutical quality, such as quantity of constituents and in-vitro dissolution. In terms of the content of active substances, flavone glycosides ranged from 24% to 36% and terpene lactones from 4% to 11%. With ginkgolic acids, there was a very large range, from < 500 ppm to about 90000 ppm. Comparing the dissolution rates of terpene lactones and flavone glycosides within the single products, most were approximately the same. Thus, terpene lactones and flavone glycosides were released from these products and dissolved at the same rate in most cases. Furthermore, most of the products investigated released more than the required 75% of the content of both components within 30 min. However, several products showed clear and relevant differences in dissolution rates to the rest (e.g. < 75% within 30 min or even less than 25% after 60 min in one case, indicating much poorer pharmaceutical quality). Beside the comparability respectively standardisation of the extracts used, the in-vitro dissolution of the relevant constituents should be similar to other drugs to guarantee comparable in-vivo performance of herbal products. An important step in standardising pharmaceutical quality is the pharmacopoeial monograph for Ginkgo biloba extract in Germany, standardising the content of pharmacologically relevant substances (flavone glycosides 22-27% and terpenlactones 5-7%, 2.8-3.4% ginkgolides A, B, C and 2.6-3.2% bilobalide thereof). Many of the investigated products, which refer to the German Commission E (of the Federal Institute for Drugs and Medicinal Devices) monograph, are not in accordance with this specification. Thus, they can not be considered to be pharmaceutically equivalent.

Chromatography, High Pressure Liquid↗

Effect of pretreatment of skin with cyclic monoterpenes on permeation of diclofenac in hairless rat.

The promoting effect of d-limonene and l-menthol on the percutaneous absorption of nonionized and ionized diclofenac (DF) was investigated employing a pretreatment method. After the pretreatment of hairless rat skin with an ethanol buffer solution containing terpenes, the permeation study was performed in vitro. The permeability coefficients of nonionized (Pn) and ionized diclofenac (Pi) were calculated under the assumption that the total flux was composed of individual fluxes of nonionized and ionized DF. In the case of pretreatment with d-limonene, both Pn and Pi values were increased dramatically. However, the promoting magnitude was not affected by an extension of the pretreatment period. In contrast, when the skin was pretreated with l-menthol, the Pn and Pi values increased gradually as the pretreatment period increased. Based on the measurement of the solubility of terpenes in the pretreatment solution, the difference in promoting efficiency could arise from the difference in thermodynamic activity of the terpenes.

Animals↗

Adjuvant influence on glyphosate efficacy in the presence of CA2+.

Calcium ion in the spray water diminishes the efficacy of glyphosate. Ammonium sulphate (AS) is long shown to be the most efficacious remedy to this antagonism. However, it is sometimes claimed that acidified lecithins or terpenic alcohols can also be used to this aim, and even ethoxylated amines (EA) provided that calcium concentrations are not higher than 5 mM. We investigated these claims by examining dose-response curves of barley plants treated with glyphosate at various calcium concentrations in the presence of these adjuvants. Calcium ion increased the slope of the curves and ED50 values, indicating a decrease in efficacy. Acidified lecithins and terpenic alcohols did not modify the response of barley to glyphosate observed in the presence of calcium ion. AS restored the ED50 and slope values observed in the absence of calcium ion. It was concluded that the inhibition of glyphosate efficacy by calcium ion was not relieved by terpenic alcohols and acidified lecithins but was completely reverted by AS. EA decreased ED50 and ED90 values of the response curves established in the presence of calcium ion without influencing the slopes (which remained high). As a consequence, at calcium concentrations up to 5 mM the response curves obtained with AS and EA intersected. It followed that in the presence of EA, ED50 values were higher than AS, whereas ED90 values were lower. By contrast, at 10 mM calcium the response curves did not intersect and consequently, in the presence of EA, ED50 and ED90 values were both higher than AS. Since ED90 relates to efficacies sought in the field, our experiments explain why at low calcium concentrations EA may appear as a better remedy than AS. Glyphosate formulations containing ammonium sulphate or a higher concentration in surfactants than usual glyphosate formulations were also studied. In the presence of calcium ion they behaved in a way which was in agreement with the above observations.

Alcohols↗

[Determination of chemical components of volatile oil from Cuminum cyminum L. by gas chromatography-mass spectrometry].

Volatile oil was extracted from Cuminum cyminum L. by using steam distillation. More than sixty peaks were separated and 49 compounds were identified by gas chromatography-mass spectrometry (GC-MS). The relative amounts of the components were determined by area normalization method. Among the 49 compounds identified, there were 16 hydrocarbons and 32 oxygenated compounds. The main compnents were cuminal and safranal (accounting for 32.26% and 24.46% respectively in the components identified). The other nine compounds with contents all over 1%, were monterpenes, sesquiterpenes, aromatic aldehydes and aromatic oxides etc. The other components with relatively small amounts were chiefly terpenes, terpenols, terpenals, terpenones, terpene esters and aromatic compounds. It is good to separate polar and apolar components in the volatile oil from Cuminum cyminum L. on the GC capillary column of moderate polarity.

Cuminum↗

Effect of nerodilol, carvone and anethole on the in vitro transdermal delivery of selegiline hydrochloride.

The aim of the study was to investigate the effect of terpene enhancers (nerodilol, carvone or anethole) on the in vitro transdermal delivery of selegiline hydrochloride with a broad objective of developing a membrane-moderated transdermal therapeutic system (TTS). The in vitro permeation studies were carried across the rat epidermis from hydroxypropyl methylcellulose (HPMC) gel drug reservoir containing selected concentrations of nerodilol, carvone or anethole and selegiline hydrochloride. The amount of selegiline hydrochloride permeated during the 24 h of the study (Q24) from HPMC gel drug reservoir without terpene enhancer was 2169 +/- 50 microg/cm2 and the corresponding flux of the drug was 92 +/- 1 microg/cm2 x h. The amount of drug permeated and its flux increased with an increase in terpne concentration in HPMC gel drug reservoir. Nerodilol provided an approximately 3.2-fold increase in the flux of selegiline hydrochloride followed by carvone with a 2.8-fold increase, and anethole with a 2.6-fold increase. It is concluded that the terpene nerodilol, carvone and anethole produced a marked penetration enhancing effect on the in vitro transdermal delivery of selegiline hydrochloride that could possibly be used in the formulation of membrane-moderated TTS.

Administration, Cutaneous↗

The mechanism of cyclic monoterpene inhibition of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase in vivo in the rat.

Seventeen hours after a single oral dose of the cyclic monoterpenes cineole or menthol, rat liver 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity was inhibited by up to 70%. The transient nature of this effect (no inhibition 41 h after dosing) was compatible with the rapid metabolism and excretion of these terpenes. Neither menthol, and its major metabolite, menthylglucuronide, nor cineole acted as direct inhibitors of HMG-CoA reductase activity in vitro, although menthol was found to bind to liver microsomes Ks approximately 0.1 mM). Unlike the short term effects of dietary cholesterol, terpene administration did not affect HMG-CoA reductase activity by modulation of the lipid microenvironment of the enzyme. Thus, following menthol or cineole treatment, we found no deviations from the normal kinetic responses to changes in temperature or in concentration of HMG-CoA. Furthermore, the inhibitory effect was still seen after solubilization of the enzyme from microsomes. The loss of HMG-CoA reductase activity was not associated with increased phosphorylation of the enzyme. Immunotitration of HMG-CoA reductase from terpene-treated rats showed that activity loss was due to less enzyme molecules (together with some possibly "cripple" enzyme), indicating that rates of enzyme synthesis or degradation had been altered. Since menthol inhibition of reductase was still observed in rats deprived of foods, we conclude that the effect is not mediated by those hormones whose concentration is changed during fasting (insulin, glucagon, and adrenaline).

Animals↗

Triterpene antioxidants from ganoderma lucidum.

Ganoderma lucidum was studied for its antioxidative activity by bioassay guided isolation in conjunction with in vitro tests. The powdered crude drug was treated with boiling water and the aqueous extract (Ex1) was further separated to obtain terpene and polysaccharide fractions. The two fractions and Ex1 were screened for their antioxidative effect against pyrogallol induced erythrocyte membrane oxidation and Fe (II)-ascorbic acid induced lipid peroxidation. All tested samples showed antioxidative activities in a dose dependent manner and the terpene fraction was found to possess the highest effect compared with the others. Chemical isolation of the terpene fraction resulted in the detection of ganoderic acids A, B, C and D, lucidenic acid B and ganodermanontriol as major ingredients.

Animals↗

Effect of lipid bilayer alteration on transdermal delivery of a high-molecular-weight and lipophilic drug: studies with paclitaxel.

Skin forms an excellent barrier against drug permeation, due to the rigid lamellar structure of the stratum corneum (SC) lipids. Poor permeability of drugs can be enhanced through alteration in partition and diffusion coefficients, or concentration gradient of drug with an appropriate choice of solvent system, along with penetration enhancers. The aim of the current investigation was to assess applicability of lipid bilayer alteration by fatty acids and terpenes toward the permeation enhancement of a high-molecular-weight, lipophilic drug, paclitaxel (PCL) through rat skin. From among the fatty acids studied using ethanol/isopropyl myristate (1:1) vehicle, no significant enhancement in flux of PCL was observed (p > 0.05). In the case of cis mono and polyunsaturated fatty acids lag time was found to be similar to control (p > 0.05). This suggests that the permeation of a high-molecular-weight, lipophilic drug may not be enhanced by the alteration of the lipid bilayer, or the main barrier to permeation could lie in lower hydrophilic layers of skin. A significant increase in lag time was observed with trans unsaturated fatty acids unlike the cis isomers, and this was explained on the basis of conformation and preferential partitioning of fatty acids into skin. From among the terpenes, flux of PCL with cineole was significantly different from other studied terpenes and controls, and after treatment with menthol and menthone permeability was found to be reduced. Menthol and menthone cause loosening of the SC lipid bilayer due to breaking of hydrogen bonding between ceramides, resulting in penetration of water into the lipids of the SC lipid bilayer that leads to creation of new aqueous channels and is responsible for increased hydrophilicity of SC. This increased hydrophilicity of the SC bilayer might have resulted in unfavorable conditions for ethanol/isopropyl myristate (1:1) along with PCL to penetrate into skin, therefore permeability was reduced. The findings of this study suggest that the permeation of a high-molecular-weight and lipophilic drug cannot be enhanced through bilayer alteration by penetration enhancers, and alteration in partitioning of drug into skin could be a feasible mode to enhance the permeation of drug.

Administration, Cutaneous↗

A novel sex-specific and inducible monoterpene synthase activity associated with a pine bark beetle, the pine engraver, Ips pini.

Ecological interactions of conifers and coniferophagous bark beetles are determined in part by terpenoids (isoprenoids), which are major defense metabolites of conifer oleoresin. Curiously, similar compounds are important aggregation pheromones of conifer-attacking bark beetles. Terpene synthases are responsible for generating the enormous variety of terpenoid carbon skeletons found in nature. These catalysts convert short-chain prenyl diphosphates to a diverse assortment of hemiterpene, monoterpene, sesquiterpene, and diterpene natural products. While terpene synthases have frequently been characterized from plant and microbial sources, they have not yet been described in animals. Here we report the discovery of a monoterpene synthase activity in an insect, the pine engraver, Ips pini (Say) (Coleoptera: Scolytidae). Cell-free assays of I. pini revealed that geranyl diphosphate (GDP) is converted to the acyclic monoterpene myrcene in whole-body extracts from males, but not females. Furthermore, the monoterpene synthase activity in males can be induced by prior treatment with juvenile hormone III (JH III) or by feeding on phloem from the host trees, Jeffrey pine (Pinus jeffreyi Grev. & Balf.) or red pine (Pinus resinosa Ait.). The sex-specificity and endocrine induction of this activity argue for its involvement in the biosynthesis of monoterpenoid pheromones mediated by enzymes from insect tissue. This discovery is the first example of a monoterpene synthase in the Metazoa and evokes exciting new questions about the origin, evolution, and occurrence of terpene synthases.

Animals↗

Screening of the antibacterial effects of a variety of essential oils on respiratory tract pathogens, using a modified dilution assay method.

The purpose of this study was to examine the antibacterial effects of a wide variety of essential oils on major respiratory tract pathogens. The antibacterial activity of 14 essential oils and their major components was evaluated by agar-plate dilution assay under sealed conditions, with agar used as a stabilizer for homogeneous dispersion. Of the selected strains of four major bacteria causing respiratory tract infection, Haemophilus influenzae was most susceptible to the essential oils, followed by Streptococcus pneumoniae and Streptococcus pyogenes. Staphylococcus aureus was less susceptible. No cross-resistance was observed between penicillin-sensitive and penicillin-resistant S. pneumoniae. Escherichia coli, used as a control bacterium, showed the lowest susceptibility. Essential oils containing aldehyde or phenol as a major component showed the highest antibacterial activity, followed by the essential oils containing terpene alcohols. Other essential oils, containing terpene ketone, or ether, had much weaker activity, and an oil containing terpene hydrocarbon was inactive. Based on these findings, thyme (wild, red, and geraniol types), cinnamon bark, lemongrass, perilla, and peppermint oils were selected for further evaluation of their effects on respiratory tract infection.

Agar↗

De novo synthesis of monoterpenes by Saccharomyces cerevisiae wine yeasts.

This paper reports the production of monoterpenes, which elicit a floral aroma in wine, by strains of the yeast Saccharomyces cerevisiae. Terpenes, which are typical components of the essential oils of flowers and fruits, are also present as free and glycosylated conjugates amongst the secondary metabolites of certain wine grape varieties of Vitis vinifera. Hence, when these compounds are present in wine they are considered to originate from grape and not fermentation. However, the biosynthesis of monoterpenes by S. cerevisiae in the absence of grape derived precursors is shown here to be of de novo origin in wine yeast strains. Higher concentration of assimilable nitrogen increased accumulation of linalool and citronellol. Microaerobic compared with anaerobic conditions favored terpene accumulation in the ferment. The amount of linalool produced by some strains of S. cerevisiae could be of sensory importance in wine production. These unexpected results are discussed in relation to the known sterol biosynthetic pathway and to an alternative pathway for terpene biosynthesis not previously described in yeast.

Acyclic Monoterpenes↗

Analysis of ginkgolides and bilobalide in food products using LC-APCI-MS.

A method was developed for the extraction and quantification of five marker compounds characteristic of Ginkgo biloba. Five ginkgo terpene trilactones: bilobalide and ginkgolides A, B, C, and J, were selected as marker compounds for this study. Initial studies produced a simple methanol extraction method for determination of gingko markers in solid dietary supplements. Five dietary supplements were analyzed and the results were later compared to the concentrations detected in the analysis of beverages. Beverage samples were prepared by extracting the ginkgo terpene trilactones using an optimized solid phase extraction (SPE) method. The extracts were analyzed using LC-atmospheric pressure chemical ionization (APCI)-MS in the negative ionization mode. The limits of detection of the extraction method ranged from 6.8 to 3.2 ng mL(-1). Using the optimized method, 14 drinks and 3 tea products were analyzed. Concentrations of total marker compounds in drinks ranged between 1685 and 21.4 ng mL(-1) with individual ginkgo terpene trilactones being detected at ppb concentrations. Analysis of brewed tea products presented much higher total marker compound concentrations ranging from 8.12 and 16.6 microg mL(-1). Analytical results reproducibility data, and recovery of the SPE method are presented.

Beverages↗

The sesquiterpene hydrocarbons of maize (Zea mays) form five groups with distinct developmental and organ-specific distributions.

The sesquiterpene hydrocarbon chemistry of maize (Zea mays) inbred line B73 was analyzed by both direct solvent extraction and headspace sampling. In seedlings, 15 olefinic compounds were identified, and 21 olefins were detected in mature plants after anthesis. Both solvent extracts and collections of headspace terpenes were found to contain the same compounds in the same relative proportions suggesting that there is no selective barrier to release from plants. Approximately 25% of the stored pool was found to be released from young seedlings per hour. The individual sesquiterpenes varied extensively in their abundance among different organs and developmental stages. Compounds could be divided into five different groups such that the members of each group always occur together in the same constant ratios to one another. Each group has a distinct distribution pattern. Group A includes the two dominant compounds, (E)-beta-farnesene and alpha-bergamotene, and appears only after herbivore damage in seedlings, but is constitutively present in the leaves and husks after anthesis. The major compounds of group B, including alpha-copaene, germacrene D and delta-cadinene, were present throughout the seedling but found only in husks of mature plants. The group C compounds, beta-bisabolene and an unknown sesquiterpene olefin, are restricted to the roots. The presence of group D and E compounds was confined to the leaves and husk of mature plants. The complex sesquiterpene mixture of group D is identical to the products formed by the previously identified terpene synthase TPS4, suggesting that each of the four other sesquiterpene hydrocarbon mixtures may also represent the products of a single terpene synthase.

Seedlings↗

Volatile organic compounds in dwelling houses and stables of dairy and cattle farms in Northern Germany.

Farmers are exposed to a complex mixture of airborne substances which can represent a health hazard. Especially animal production on a farm can be a risk factor for respiratory diseases. Most studies in this context focused on bioaerosols, compounds attached to bioaerosols or on gases such as ammonia or hydrogen sulfide. Less attention was paid to volatile organic compounds (VOCs) which may also cause respiratory diseases. This pilot study presents results of measuring VOCs in the air of the dwellings and stables, as well as in the outdoor environment, of cattle farms in Northern Germany in spring and autumn. Farmers on all selected farms complained of symptoms such as asthma, rhinitis and conjunctivitis which occurred especially during work in the stable but also in the dwellings. The mean concentration of total VOC (TVOC) for the outdoor environment, the dwellings, and the stables were 100 microg m(-3), 763 microg m(-3), 322 microg m(-3) in spring and 143 microg m(-3), 544 microg m(-3), 595 microg m(-3) in autumn, respectively. There was no significant difference in season. TVOC concentrations in dwellings on farms were elevated when compared to dwellings in other environments, probably because of an additional exposure to fuels and chemicals used in agriculture. Aliphatic and alicyclic hydrocarbons, aromatic compounds, and terpenes were main compounds of the dwellings and ketones, alcohols, and esters of the stables. Terpene concentrations in the stables were low probably because straw, which was not a strong terpene emitter, was used instead of sawdust as floor covering. Large amounts of methylethylketone were encountered in the air of one stable, probably from animal exhalation caused by increased animal activity. However, it was unlikely that this caused respiratory symptoms. Generally it was likely that the concentrations of VOCs were too low to have health effects on their own. On the other hand, the VOC concentrations were in a multifactor concentration range in which health effects could occur depending on the interaction with other exposure factors. Fungal spores and bacteria were observed in addition to VOCs at the examined locations. Therefore it is possible that the observed VOCs played a role in exacerbating respiratory symptoms in this multifactor exposure.

Aerosols↗