Major Individual Constituents Research Articles

Adjuvants in fungicide formulations can be skin sensitizers and cause different types of cell stress responses

New approaches based on -omics technologies can identify biomarkers and processes regulated in response to xenobiotics, and thus support toxicological risk assessments. This is vital to meet the challenges associated with “cocktail effects”, i.e. combination effects of chemicals present simultaneously in a product, our environment, and/or our body. For plant protection products (PPPs), investigations largely focus on active ingredients such as herbicides and fungicides. In this study, we have analyzed agricultural chemicals, two surfactants (poly(oxy-1,2-ethanediyl), alpha-sulfo-omega-[2,4,6-tris(1-phenylethyl)phenoxy]-, ammonium salt, POL; N,N-dimethylcapramide, NND), and one preservative, 1,2-benzisothiazol-3(2 H)-one (BEN) used as adjuvants in PPPs, and further three fungicide PPPs, Proline EC 250, Shirlan, Folicur Xpert, containing the adjuvants, and other major individual constituents (fluazinam (FLU), prothioconazole (PRO), tebuconazole (TEB)) as well as defined mixtures (“mixes”) thereof using several in vitro approaches. All investigated single agricultural chemicals were predicted as skin sensitizers using an in vitro transcriptomic assay based on a dendritic cell model. For selected chemicals and mixes, also skin sensitization potency was predicted. The preservative BEN induced significant changes in cytokine secretion and dendritic cell activation marker CD86 expression. The surfactant NND changed cytokine secretion only and the POL only affected CD86 expression. Proteomic analyses revealed unique response profiles for all adjuvants, an oxidative stress pattern response in BEN-treated cells, and differentially abundant proteins associated with cholesterol homeostasis in response to POL. In summary, we find responses to agricultural chemicals and products consistent with the dendritic cell model reacting to chemical exposure with oxidative stress, ER stress, effects on autophagy, and metabolic changes especially related to cholesterol homeostasis. After exposure to certain mixes, novel proteins or transcripts were differentially expressed and these were not detected for any single constituents, supporting the occurrence of cocktail effects. This indicates that all chemicals in a PPP can contribute to the toxicity profile of a PPP, including their skin sensitizing/immunotoxic properties.

Chemical constituents and antioxidant activity of the essential oil from leaves of Annona vepretorum Mart. (Annonaceae).

Background:Annona vepretorum (AV) is a native tree from Caatinga biome (semiarid region of Brazil) popularly known as “araticum” and “pinha da Caatinga.”Objective:This study was carried out to evaluate the chemical constituents and antioxidant activity (AA) of the essential oil from the leaves from AV (EO-Av) collected in Petrolina, Pernambuco, Brazil.Materials and Methods:Fresh leaves of AV were cut into pieces, and subjected to distillation for 2 h in a clevenger-type apparatus. Gas chromatograph (GC) analyses were performed using a mass spectrometry/flame ionization detector. The identification of the constituents was assigned on the basis of comparison of their relative retention indices. The antioxidant ability of the EO was investigated through two in vitro models such as radical scavenging activity using 2,2-diphenyl-1-picrylhydrazyl method and β-carotene-linoleate-model system. The positive controls (ascorbic acid, butylated hydroxyanisole and butylated hydroxytoluene) were those using the standard solutions. Assays were carried out in triplicate.Results:The oil showed a total of 21 components, and 17 were identified, representing 93.9% of the crude EO. Spathulenol (43.7%), limonene (20.5%), caryophyllene oxide (8.1%) and α-pinene (5.5%) were found to be the major individual constituents. Spathulenol and caryophyllene oxide could be considered chemotaxonomic markers of these genera. The EO demonstrated weak AA.

Chemical Composition of Essential Oil from the Leaves of Jatropha mutabilis (Pohl) Baill (Euphorbiaceae)

:Jatropha is an important genus belonging to the Euphorbiaceae family. The essential oil from the leaves of Jatropha mutabilis, which were collected in the semiarid of Pernambuco (Northeast Brazil), was obtained by hydrodistillation in a Clevenger-type apparatus and analyzed by Gas Chromatography-Mass Spectrometry (GC-MS). Twenty components comprising 100 % of the leaf essential oil were identified. The major individual constituents were phytol (41.38 %), (+)-spathulenol (12.81 %), epi-α-cadinol (9.27 %), (-)- caryophyllene oxide (4.79 %), germacrene D (4.46 %), α-cadinol (3.86 %) and elemol (3.64 %). This is the first report on the chemical composition of the essential oil of this species.

Biting deterrence, repellency, and larvicidal activity of Ruta chalepensis (Sapindales: Rutaceae) essential oil and its major individual constituents against mosquitoes.

The essential oil from aerial parts of Ruta chalepensis L. (Sapindales: Rutaceae) was obtained by hydrodistillation, and its chemical profile was identified using gas chromatography and gas chromatography-mass spectrometry. Compounds, 2-undecanone (43.2%), 2-nonanone (27.9%), and 2-nonyl acetate (10.6%) were the major constituents of the oil. Biting deterrent activity of R. chalepensis essential oil at 10 and 50 microg/cm2, 2-undecanone at 8.5 microg/cm2, 2-nonanone at 9 microg/cm2, and 2-nonyl acetate at 9.3 microg/cm2 was similar to DEET (N, N-diethyl-meta-toluamide) at 4.8 microg/cm2, against Aedes aegypti L. Biting deterrent activity of R. chalepensis oil at 50 microg/cm2 against Anopheles quadrimaculatus Say was statistically similar to DEET at 4.8 microg/cm2, whereas the activity was lower in the other compounds tested. In cloth patch assay, R. chalepensis essential oil was effective at 187 microg/cm2, whereas 2-undecanone was effective at 108.9 microg/cm2 against Ae. aegypti. In larval bioassays, 2-undecanone showed similar toxicity whereas toxicity of R. chalepensis essential oil and 2-nonanone was higher at 24-h posttreatment at the LD50 in An. quadrimaculatus than Ae. aegypti. This study revealed that R. chalepensis essential oil and its major compounds were active biting deterrents against Ae. aegypti at higher application rates whereas only the essential oil showed activity similar to DEET against An. quadrimaculatus. 2-undecanone was the most active compound in in vivo repellency bioassay against Ae. aegypti. Chemical composition of R. chalepensis essential oil varies because of plant production and harvest practices, and the activity level of the essential oil may depend on the source of the sample.

Pharmacokinetics of linalool and linalyl acetate in rats after repeated oral administration of silexan, an essential oil from Lavandula angustifolia flowers

Silexan is the active ingredient in Lasea® – an approved drug for the treatment of restlessness and mild anxiety in Germany. The naturally occurring enantiomers R-(-)linalool (L) and R-(-)-linalyl acetate (LA) are the main constituents of silexan representing 70 – 80% of the total oil. In previous studies we have reported on the pharmacokinetics of L and LA in rats after single oral administration of silexan or the two major individual constituents (Noldner et al., 2011). We now investigated the pharmacokinetics of L and LA after repeated application of silexan (100 mg/kg/day p.o.) over a period of 14 consecutive days. The plasma and organ concentrations of L and LA were measured by headspace GC-MS for 48h after the last administration. The results show that both single and multiple doses of silexan result in similar concentrations of L in plasma and all analyzed organs. However, the concentrations and distribution of LA were significantly different. After single administration LA was only measured in the brain while it was detected in all tissues except the plasma after repeated administration although at low concentrations. The lower LA levels are obviously due to a very quick metabolization of LA into L. In conclusion, the results demonstrate that oral administration of silexan rapidly results in high plasma and tissue levels of L without a clear tendency for accumulation after repeated application.

Repellent and Larvicidal Activity of Ruta graveolens Essential Oil and its Major Individual Constituents Against Aedes aegypti

Repellent and Larvicidal Activity of Ruta graveolens Essential Oil and its Major Individual Constituents Against Aedes aegypti

Chemical Composition of the Essential Oil of Zanthoxylum avicennae (Lam.) DC Leaves (Rutaceae) from Vietnam

The volatile components of the essential leaf oil of Zanthoxylum avicennae (Lam.) DC (Rutaceae) from Nghean province (Vietnam) were analyzed by means of capillary GC and GC-MS as well as in some cases by background subtraction of co-eluting compounds. Out of more than fifty identified compounds, the sesquiterpene hydrocarbons β-caryophyllene (17.01 %) and α-humulene (10.38 %) as well as the monoterpene hydrocarbons α-pinene (10.07 %) and β-phellandrene, were determined to be the major individual constituents of the essential oil. Further mono- and sesquiterpene hydrocarbons, such as α-terpinene, (E)-β-ocimene, λ-terpinene, germacrene D and β-selinene, were found in concentrations higher than 3.0 %.

Composition and Antimicrobial Activity of the Leaf Essential Oils of Duguetia gardneriana Mart. And Duguetia moricandiana Mart. (Annonaceae)

As part of their continuing studies on the constituents of the genus Duguetia, these authors have studied the composition and antimicrobial activity of the essential oils obtained from the leaves of two species, and the results are reported here. A hydrodistilled oils obtained from the leaves of Duguetia gardneriana and Duguetia moricandiana were analyzed by GC/MS. The oils showed a total of 33 components. Germacrene D (28.1%), viridiflorene (24.0%), β-pinene (12.6%), α-pinene (9.1%) and β-caryophyllene (5.6%) were found to be the major individual constituents of D. gardneriana oil. The leaf oil of D. moricandiana was dominated by germacrene D (44.3%), α-pinene (13.0%), viridiflorene (9.3%), β-pinene (9.2%) and β-caryophyllene (6.8%). The oils were tested for their antimicrobial activity against 11 pathogenic microorganisms using standard gel diffusion method. The essential oil of D. gardneriana showed activity against Staphylococcus aureus and Candida guilliermondii, while the essential oil of D. moricandiana was more active against Staphylococcus aureus and Candida albicans.

Qualitative and Quantitative Analysis of the Main Constituents of Radix Ilicis Pubescentis by LC-Coupled with DAD and ESIMS Detection

A sensitive and selective high performance liquid chromatographic method coupled with DAD detection is presented for quality control of Radix Ilicis Pubescentis. By means of this analytical procedure the major individual constituents (ilexoside O, ilexgenin A, ilexsaponin A1, ilexsaponin B1, liriodendin and acanthoside B) could be quantified simultaneously. LC-ESI-MS was applied for identification of the six compounds in the plant by comparing their m/z value and retention times with those of selected standards. For quantitative analysis, the extraction procedure and the extraction solvent were optimized in order to ensure the exhaustive extraction of the plant material. The HPLC conditions were evaluated and optimized for the exact quantification of all six individual compounds. Chromatographic separation was carried out on a C18 column using gradient elution with acetonitrile and 0.1% phosphoric acid as the mobile phase. Detection was carried out using a photodiode array detector. The calibration curves for determination of the six constituents showed good linearity over the investigated ranges (r2>0.999). Measurement of intra-day and inter-day variability (expressed as RSD value) was conducted to assess precisions of the method, and RSD (%) of intra- and inter-day variation were between 1.56-3.36% and 1.61-3.58%, respectively. The recoveries of the six compounds were between 96.4-102.2%, with RSD (%) values ranging from 1.7-3.8%. These validation results demonstrated the suitability of the method for the precise and accurate determination of the main constituents in Radix Ilicis Pubescentis. The method was successfully applied for quality evaluation of 12 batches of Radix Ilicis Pubescentis obtained from different regions of southern China. The contents of the six major constituents varied significantly due to their different origins, which can be used as an aid to assessing the quality of Radix Ilicis Pubescentis.

Optimization and Validation of an HPLC–Method for Quality Control of Pueraria Lobata Root

Pueraria lobata Willd. (Fabaceae) is a widely used medicinal plant, known as “Kudzu” and “Ge” in Japanese and Chinese traditional medicine, respectively. P. lobata is a rich source of isoflavones with phytoestrogenic properties, and its commercial use is widespread. In this study the optimization and validation of an HPLC-method for quality control of Pueraria root material is presented. By means of this analytical method the major individual constituents, i.e. the isoflavone 8- C-glycosides hydroxypuerarin, puerarin, methoxypuerarin and xylosylpuerarin, the 7- O-glycosides daidzin and genistin, and the aglycones daidzein and genistein could be quantified. The extraction procedure and the extraction solvent composition were optimized in order to ensure the exhaustive extraction of the plant material. The HPLC - conditions were evaluated and optimized for the exact quantification of all individual compounds. The HPLC analysis was carried out on a Agilent XDB RP C18 (150 × 4.6) column eluted with a binary system consisting of water (+ 0.01% formic acid) and methanol (+ 0.01% formic acid) using a linear gradient; detection was at 262 nm. Daidzin, daidzein, genistin and genistein were used as external standards. Due to the great difference in content between the C-glycosides on the one hand, and daidzin, genistin and the aglycones on the other, two separate HPLC runs were necessary for a complete analysis. The final method was fully validated according to the ICH guidelines in terms of linearity, precision and accuracy. Linear relationships for the responses of all four standards were proven. The method was shown to be repeatable for the individual compounds, i.e. RSD% betweendays values were within 2 to 7.5%. The accuracy of the method was demonstrated to be equal to 100% by recovery experiments. These validation results demonstrated the suitability of the method for the precise and accurate determination of Pueraria root isoflavones.

The Composition and Antimicrobial Activity of Stem Bark Essential Oil of Goniothalamus cardiopetalus (Bl.) Hook.f. et Thoms.

A hydrodistilled oil obtained from the stem bark of Goniothalamus cardiopetalus (Annonaceae) was analyzed by GC/MS. The oil showed a total of 60 components of which 40 compounds representing 67.1% of the oil were identified. Linalool (11.7%), α-pinene (7.0%), trans-pinocarveol (5.2%), caryophyllene oxide (5.0%) -terpineol (4.9%), guaiol (4.4%) camphor (3.9%) and bornyl acetate (3.9%) were found to be the major individual constituents of the oil. The antimicrobial activity of the oil was tested against a panel of 17 bacterial and six fungal strains by the disc diffusion method. The oil inhibited the growth of all test organisms at varying levels and the minimal inhibitory concentrations (MIC) were also determined.

Antifungal activity of peppermint and sweet basil essential oils and their major aroma constituents on some plant pathogenic fungi from the vapor phase.

The vapors of peppermint oil and two of its major constituents (menthol and menthone), and sweet basil oil and two of its major constituents (linalool and eugenol), were tested against Sclerotinia sclerotiorum (Lib.), Rhizopus stolonifer (Ehrenb. exFr.) Vuill and Mucor sp. (Fisher) in a closed system. These fungi cause deterioration and heavy decay of peach fruit during marketing, shipping and storage. The essential oils, their major individual aroma constituents and blends of the major individual constituents at different ratios inhibited the growth of the fungi in a dose-dependent manner. Menthol was found to be the individual aroma constituent responsible for the antifungal properties of peppermint essential oil, while menthone alone did not show any effect at all doses. In the case of basil oil, linalool alone showed a moderate antifungal activity while eugenol showed no activity at all. Mixing the two components in a ratio similar to their concentrations in the original oil was found to enhance the antifungal properties of basil oil indicating a synergistic effect.

The Root Bark Essential Oil ofUvaria narumWall.

ABSTRACT The constituents of the root bark essential oil of Uvaria narum Wall. (Annonaceae) from Kerala, India were analyzed by GC/MS. About 52 components were detected, out of which 22 were identified. Bornyl acetate (15.2%) and patchoulenone (8.1%), a tricyclic sesquiterpene ketone, were found to be the major individual constituents of the oil.

Changes in cuticular transpiration rate and cuticular lipids of oat (Avena sativa) seedlings induced by water stress

Two cultivars of oat (Avena sativa L. cvs Pendek and Stormogul II) were exposed to short periods of water‐deficit stress on five consecutive days. The plants responded to the stress by decreasing their cuticular transpiration rate. After two stress periods the cuticular transpiration rate was reduced by 30% for Pendek and by 47% for Stormogul II, and after another three stress periods by 30% and 20%, respectively. These reductions were correlated neither to changes in the total amount of what is generally called epicuticular lipids, nor to changes in any of the major individual constituents of the epicuticular lipids (alkanes, free and esterified fatty acids or free primary alcohols). After removal of the epicuticular lipids the long chain free primary alcohols of the leaves were extracted and determined. The amount of these presumably intracuticular alcohols increased after stress and changed to shorter chain length. From these results it is concluded that the intra‐ as well as the epicuticular lipids must be taken into consideration when discussing leaf surface lipids as protecting agents against water loss.