Minty aroma compounds in red wine: Development of a novel automated HS-SPME-arrow and gas chromatography-tandem mass spectrometry quantification method.

A novel automated method was developed for the quantitative determination of nine terpenoids that could contribute to the minty notes of red wine bouquet. The method couples headspace SPME-Arrow extraction with GC-MS/MS analysis. PDMS/DVB fiber was chosen for the extraction and an ionization energy of 30 eV permitted to optimize the analyte detection. The optimal sample preparation consists of a two-fold dilution of the wine sample with addition of 4 g of sodium chloride while the most suitable extraction conditions take place at 50 °C for 1 h. The method shows good linearity, intraday variations between 2 and 25%, interday variations between 7 and 23% and recoveries between 80 and 119%. The method exhibits the required low detection (between 3 and 60 ng/L) and quantification (between 6 ng/L and 200 ng/L) limits. These limits have permitted the quantification of the pool of minty terpenoids in fourteen red Bordeaux wines.

Enzyme-assisted Extraction of Bioactive Phytochemicals from Japanese Peppermint (Mentha arvensis L. cv. 'Hokuto').

In this study, we provide a method for obtaining essential oil from Mentha arvensis L. in large quantities. Three types of polysaccharide-degrading enzymes were investigated, namely cellulase A "Amano" 3, cellulase T "Amano" 4, and hemicellulase "Amano" 90. The optimum extraction conditions were the combined use of 2 wt% cellulase T and 2 wt% hemicellulase 90, and 3 h of incubation. Enzymeassisted extraction increased the amount of the essential oil from 2.2 mL to 3.0 mL, compared with the amount extracted without an enzyme.

The Influence of Essential Oil Compounds on Antibacterial Activity of Mupirocin-Susceptible and Induced Low-Level Mupirocin-Resistant MRSA Strains.

Because of the bacterial drug resistance development, it is reasonable to investigate chemical compounds capable of preventing the spread of resistance to mupirocin (MUP), commonly used in staphylococcal eradication. The objective of the study was to verify the influence of essential oil compounds (EOCs) on the antibacterial activity of MUP against mupirocin-susceptible (MupS) and induced low-level mupirocin-resistant (MupRL) methicillin-resistant Staphylococcus aureus (MRSA) strains. The following parameters were examined: MRSAMupS and MRSAMupRL susceptibility to EOCs (1,8-cineole, eugenol, carvacrol, linalool, (-)-menthone, linalyl acetate, and trans-anethole), the bacterial cell size distribution, and chemical composition by the use of Fourier Transform Infrared Spectroscopy (FTIR) and Raman spectroscopies. The MRSAMupS and MRSAMupRL strains were susceptible to all tested EOCs. 1,8-cineole and (-)-menthone showed synergistic activity against MRSAMupS in combination with mupirocin, whereas 1,8-cineole exhibited synergistic activity against MRSAMupRL as well. In-depth analysis showed that both MRSAMupS and MRSAMupRL displayed similar distributions of the bacterial cell size. The FTIR and Raman spectra of the MRSAMupS and MRSAMupRL strains showed differences in some regions. New bands in the MRSAMupRL Raman spectrum were observed. It was concluded that the use of 1,8-cineole in combination with mupirocin can increase the mupirocin activity against the MRSAMupS and MRSAMupRL strains.

Antimicrobial activity of five essential oils from lamiaceae against multidrug-resistant Staphylococcus aureus.

Analysis of Lamiaceae essential oils (EOs) by GC-FID-MS revealed the presence as the major constituents of linalool (16.8%), linalyl acetate (15.7%) in Lavandula angustifolia, menthol (29.0%), menthone (22.7%), menthyl acetate (19.2%) in Mentha x piperita, terpinen-4-ol (27.1%), (E)-sabinene hydrate (12.1%), γ-terpinene (10.0%) in Origanum majorana, α-thujone (19.5%), camphor (19.0%), viridiflorol (13.5%) in Salvia officinalis, thymol (61.9%), p-cymene (10.0%), γ-terpinene (10.0%) in Thymus vulgaris. Based on the MIC and MBC values (0.09-0.78 mg/mL) and ratio MBC/MIC showed that EO from T. vulgaris (TO) had the strong inhibitory and bactericidal effect against multidrug-resistant Staphylococcus aureus. The bacterial cells were total killed by TO at 2MIC concentration after 6 h. The higher concentrations of other EOs were needed to achieve bactericidal effects. The strong bactericidal effect of TO against these bacteria indicates the possibility of topical use of TO but it requires research under clinical conditions.

Utilization of the Japanese Peppermint Herbal Water Byproduct of Steam Distillation as an Antimicrobial Agent.

The present study provides valuable data that the herbal water byproduct of Japanese peppermint, produced during the steam distillation extraction of an essential oil, can be utilized as an antibacterial agent. The major ingredient in the herbal water from Japanese peppermint 'Hokuto' was menthol, with a concentration close to its water solubility. The herbal water produced showed excellent antibacterial efficacy against typical gram-negative and gram-positive bacterial strains of Escherichia coli and Staphylococcus aureus, respectively, and the antibacterial efficacy was maintained even when the herbal water was diluted up to an appropriate concentration of 50%. The efficacy of the herbal water against E. coli was higher than that against S. aureus, which is likely because of the difference in the efficacy of menthol against these two different bacterial strains. The excellent antibacterial efficacy of the herbal water is mainly attributed to the function of menthol, while other trace ingredients also contributed to the antibacterial efficacy. The Japanese peppermint herbal water byproduct, generally treated as industrial waste and disposed, can be easily commercialized as an antibacterial agent if efforts are made to maintain a constant menthol concentration throughout the steam distillation essential oil extraction process.

Phenolic and Volatile Composition of a Dry Spearmint (Mentha spicata L.) Extract.

The present paper reports a complete mass spectrometric characterization of both the phenolic and volatile fractions of a dried spearmint extract. Phenolic compounds were analysed by ultra-high performance liquid chromatography-electrospray ionization-mass spectrometry (UHPLC-ESI-MS(n)) and a total of 66 compounds were tentatively identified, being the widest phenolic characterisation of spearmint to date. The analysis suggests that the extract is composed of rosmarinic acid and its derivatives (230.5 ± 13.5 mg/g) with smaller amounts of salvianolic acids, caffeoylquinic acids, hydroxybenzoic acids, hydroxycinnamic acids, flavones, and flavanones. Head space solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) technique, that was applied to characterize the volatile fraction of spearmint, identified molecules belonging to different chemical classes, such as p-cymene, isopiperitone, and piperitone, dihydroedulan II, menthone, p-cymen-8-ol, and ß-linalool. This comprehensive phytochemical analysis can be useful to test the authenticity of this product rich in rosmarinic acid and other phenolics, and when assessing its biological properties. It may also be applied to other plant-derived food extracts and beverages containing a broad range of phytochemical compounds.

Glandular trichomes and essential oil characteristics of in vitro propagated Micromeria pulegium (Rochel) Benth. (Lamiaceae).

MAIN CONCLUSION: In vitro conditions and benzyladenine influenced both content and composition of micropropagated Micromeria pulegium essential oils, with pulegone and menthone being the main essential oil components. The content and chemical composition of Micromeria pulegium (Rochel) Benth. essential oils were studied in native plant material at vegetative stage and in micropropagated plants, obtained from nodal segments cultured on solid MS medium supplemented with N(6)-benzyladenine (BA) or kinetin at different concentrations, alone or in combination with indole-3-acetic acid. Shoot proliferation was achieved in all treatments, but the highest biomass production was obtained after treatment with 10 µM BA. Phytochemical analysis identified up to 21 compounds in the essential oils of wild-growing and in vitro cultivated plants, both showing very high percentages of total monoterpenoids dominated by oxygenated monoterpenes of the menthane type. Pulegone and menthone were the main essential oil components detected in both wild-growing plants (60.07 and 26.85 %, respectively) and micropropagated plants grown on either plant growth regulator-free medium (44.57 and 29.14 %, respectively) or BA-supplemented medium (50.77 and 14.45 %, respectively). The percentage of total sesquiterpenoids increased in vitro, particularly owing to sesquiterpene hydrocarbons that were not found in wild-growing plants. Differences in both content and the composition of the essential oils obtained from different samples indicated that in vitro culture conditions and plant growth regulators significantly influence the essential oils properties. In addition, the morphology and structure of M. pulegium glandular trichomes in relation to the secretory process were characterized for the first time using SEM and light microscopy, and their secretion was histochemically analyzed.

Two new menthane monoterpenes from Illicium wardii.

Two new menthane monoterpenes, wardicines A (1) and B (2), were isolated from the fruits of Illicium wardii A.C. Smith. Their structures were determined on the basis of spectroscopic data analysis. Cytotoxicities of compounds 1 and 2 against three human tumor cell lines A549, HCT-116, and CCRF-CEM were also evaluated, but no significant activities were observed for the two compounds.

Larvicidal activity of the essential oil of Youngia japonica aerial parts and its constituents against Aedes albopictus.

The aim of this study was to evaluate the larvicidal activity of the essential oil of Youngia japonica aerial parts against the larvae of Aedes albopictus and to isolate any active compounds from the oil. Gas chromatography-mass spectrometry (GC-MS) analyses revealed the presence of 31 compounds, with menthol (23.53%), α-asarone (21.54%), 1,8-cineole (5.36%), and caryophyllene (4.45%) as the major constituents. Bioactivity-directed chromatographic separation of the oil led to the isolation of menthol and α-asarone as active compounds. The essential oil of Y. japonica exhibited larvicidal activity against the fourth instar larvae of A. albopictus with an LC50 value of 32.45 µg/mL. α-Asarone and menthol possessed larvicidal activity against the fourth instar larvae of A. albopictus with LC50 values of 24.56 µg/mL and 77.97 µg/mL, respectively. The results indicate that the essential oil of Y. japonica aerial parts and the two constituents can be potential sources of natural larvicides.

Solubilization of menthol by platycodin D in aqueous solution: an integrated study of classical experiments and dissipative particle dynamics simulation.

Menthol (M) and platycodin D (PD) are the main active ingredients in Mentha haplocalyx and Platycodon grandiflorum A. DC., respectively. They are commonly used in combination in traditional Chinese medicine. In this study, laboratory experiments and computer simulations were used to investigate the solubilization of M by PD, which was believed to be one of the main causes of the synergistic effect of M. haplocalyx and P. grandiflorum A. DC. Results showed that both the method by which M was added and the concentration of PD had significant effects on the solubilization efficiency of M, and these influences were closely associated with each other. Temperature, an important environmental condition, was also found to have a significant effect on the solubilization effect of PD. These findings not only clarify the molecular basis of the solubilization effect, including amount solubilized at the macroscale and the structures of the micelles, and the drug loading mechanisms and processing at the mesoscale. This work may provide some guidance for the further development of saponins and fundamental research in the drug delivery system.

Identification of insecticidal constituents of the essential oils of Dahlia pinnata Cav. against Sitophilus zeamais and Sitophilus oryzae.

The aim of this research was to determine the chemical composition of the essential oils of Dahlia pinnata, their insecticidal activity against Sitophilus zeamais and Sitophilusoryzae and to isolate insecticidal constituents. Based on bioactivity-guided fractionation, active constituents were isolated and identified as D-limonene, 4-terpineol and α-terpineol. Essential oils and active compounds tested exhibited contact toxicity, with LD50 values ranging from 132.48 to 828.79 µg/cm(2) against S. zeamais and S. oryzae. Essential oils possessed fumigant toxicity against S. zeamais and S. oryzae with LC50 from 14.10 to 73.46 mg/L. d-Limonene (LC50 = 4.55 and 7.92 mg/L) showed stronger fumigant toxicity against target insects. 4-Terpineol (88 ± 8%) and d-limonene (87 ± 5%) showed the strongest repellency against S. zeamais and S. oryzae, respectively. The results indicate that essential oils and insecticidal constituents have potential for development into natural fumigants, insecticides or repellents for control of the stored-product insect pests.

New withanolide, acyl and menthyl glucosides from fruits of Withania coagulans Dunal.

The fruits of Withania coagulans Dunal (family: Solanaceae) are sweet, sedative, emetic, alterative and diuretic; used to treat asthma, biliousness, strangury, wounds, dyspepsia, flatulent colic, liver complaints and intestinal infections. Phytochemical investigation of the fruits yielded a withanolide tetraglucoside identified as (20S, 22R)-5alpha, 20alpha-diacetoxy-6beta-hydroxy- 1-oxowitha-2,24-dienolide-6-beta-D-glucopyranosyl-(6' --> 1")-beta-D-glucopyranosyl-(6" --> 1''')-beta-D-glucopyranosyl-(6''' --> 1''''-beta-D-glucopyranoside), a capryloyl hexaglucoside formulated as n-octanoyl-beta-D-glucopyranosyl-(6a --> 1b)-beta-D-glucopyranosyl-(6b --> c)-beta-D-glucopyranosyl-(6c --> 1d)-beta-D-glucopyranosyl-(6d --> 1e)-glucopyranosyl-(6e --> 1f)-glucopyranoside and a menthyl tetraglucoside characterized as menthol-O-alpha-L-glucopyranosyl-(2a --> 1b)-O-alpha-L-glucopyranosyl-(2b --> 1c)-O-alpha-L-glucopyranosyl-(2c --> 1d)-O-alpha-L-glucopyranoside along with three fatty acid esters, n-nonacosanyl linolenate, n-octacosanyl linolenate and n-heptacosanyl linolenate. The structures of the isolated phytoconstituents have been established on the basis of spectral data analysis and chemical means.

Menthol: a simple monoterpene with remarkable biological properties.

Menthol is a cyclic monoterpene alcohol which possesses well-known cooling characteristics and a residual minty smell of the oil remnants from which it was obtained. Because of these attributes it is one of the most important flavouring additives besides vanilla and citrus. Due to this reason it is used in a variety of consumer products ranging from confections such as chocolate and chewing gum to oral-care products such as toothpaste as well as in over-the-counter medicinal products for its cooling and biological effects. Its cooling effects are not exclusive to medicinal use. Approximately one quarter of the cigarettes on the market contain menthol and small amounts of menthol are even included in non-mentholated cigarettes. Natural menthol is isolated exclusively from Mentha canadensis, but can also be synthesised on industrial scale through various processes. Although menthol exists in eight stereoisomeric forms, (-)-menthol from the natural source and synthesised menthol with the same structure is the most preferred isomer. The demand for menthol is high and it was previously estimated that the worldwide use of menthol was 30-32,000 metric tonnes per annum. Menthol is not a predominant compound of the essential oils as it can only be found as a constituent of a limited number of aromatic plants. These plants are known to exhibit biological activity in vitro and in vivo such as antibacterial, antifungal, antipruritic, anticancer and analgesic effects, and are also an effective fumigant. In addition, menthol is one of the most effective terpenes used to enhance the dermal penetration of pharmaceuticals. This review summarises the chemical and biological properties of menthol and highlights its cooling effects and toxicity.

Chemical composition and antibacterial activity of the essential oil from Mentha requienii Bentham.

The chemical composition of essential oil obtained by hydrodistillation of the fresh aerial parts of Mentha requienii Bentham (Lamiaceae) collected on the Gennargentu Mountains (Sardinia, Italy) has been investigated by gas chromatography and gas chromatography-mass spectrometry. The main constituents that resulted were pulegone (78%), menthone (0.5%), isomenthone (18%), isopulegone (1.3%) and limonene (1.76%). In vitro antifungal activity is evaluated in order to identify new means that could be helpful in the prevention of contamination in indoor environments.

Simple preparative gas chromatographic method for isolation of menthol and menthone from peppermint oil, with quantitative GC-MS and (1) H NMR assay.

The quantitative performance of a simple home-built preparative gas chromatography (prep-GC) arrangement was tested, incorporating a micro-fluidic Deans switch, with collection of the target compound in a deactivated uncoated capillary tube. Repeat injections of a standard solution and peppermint sample were made into the prep-GC instrument. Individual compounds were eluted from the trapping capillary, and made up to constant volume. Chloronaphthalene internal standard was added in some cases. Recovered samples were quantitatively assayed by using GC-MS. Calibration linearity of GC-MS for menthol standard area response against number of injections (2-20 repeat injections) was excellent, giving R(2) of 0.996. For peppermint, menthol correlation over 2-20 repeated injections was 0.998 for menthol area ratio (versus IS) data. Menthone calibration for peppermint gave an R(2) of 0.972. (1) H NMR spectroscopy was conducted on both menthol and menthone. Good correspondence with reference spectra was obtained. About 80 µg of isolated menthol and menthone solute was collected over a sequence of 80 repeat injections from the peppermint sample, as assayed by 600 MHz (1) H NMR analysis (∼100% recovery for menthol from peppermint). A procedure is proposed for prediction of number of injections required to acquire sufficient material for NMR detection.

Classification of Greek Mentha pulegium L. (Pennyroyal) samples, according to geographical location by Fourier transform infrared spectroscopy.

INTRODUCTION: Mentha pulegium L. (pennyroyal) is one of the four most commercially important Mentha species, even it is not a cultivated plant. It can be abundantly located in the Iberian Peninsula and North African countries. In Greece it grows in the wild and it is scattered all over the country. Pennyroyal is best known for its essential oil, with Spain and Morocco being the largest producers in the world. Mid-infrared spectroscopy has been applied to determine the origin of various samples. OBJECTIVES: In this work Fourier transform infrared spectroscopy (FT-IR) combined with canonical discriminant analysis has been applied to distinguish 70 Greek pennyroyal samples according to their collection areas. MATERIAL AND METHODS: Pennyroyal nonpolar organic extracts were prepared using ultrasound-assisted solvent extraction. The spectra of the extracts were recorded in the range of 4000-400 cm(-1) and the best discrimination was achieved in the spectral region 1720-1650 cm(-1) . RESULTS: Spectral features for the discrimination of pennyroyal samples among the different collection areas occur primarily in the carbonyl region and are correlated with the main volatile constituents of the extracts (menthone, isomenthone, pulegone, piperitone). All areas were easily differentiated by canonical discriminant analysis. The percentages of correct classification and validation were 94.3 and 90.0%, respectively. CONCLUSION: The combination of FT-IR spectroscopy and multivariate analysis provides a rapid and ambient method to discriminate pennyroyal samples in terms of geographical origin.

Molecular response of Musca domestica L. to Mintostachys verticillata essential oil, (4R)+-pulegone and menthone.

Intense applications of synthetic insecticides for the control of adult Musca domestica have led to the insects developing resistance to most of them. In consequence, there is interest in new active ingredients as alternatives to conventional insecticides. Essential oils (EO) are potential tools for controlling M. domestica because of their effectiveness and their minimal environmental effects. In a fumigant assay, M. domestica adults treated with Minthostachys verticillata EO [LC(50)=0.5 mg/dm(3); majority components by SPME-GC: (4R)(+)-pulegone (67.5%), menthone (22.3%) and (4R)(+)-limonene (3.8%)], died within 15 min or less. The terpenes absorbed by the flies and their metabolites, analyzed using SPME fiber, were (4R)(+)-limonene (LC(50)=6.2 mg/dm(3)), menthone (LC(50)=1.9 mg/dm(3)), (4R)(+)-pulegone (LC(50)=1.7 mg/dm(3)) and a new component, menthofuran (LC(50)=0.3 mg/dm(3)), in a relative proportion of 12.4, 6.5, 35.9 and 44.2% respectively. Menthofuran was formed by oxidation of either (4R)(+)-pulegone or menthone mediated by cytochrome P450, as demonstrated by a fumigation assay on flies previously treated with piperonyl butoxide, a P450 inhibitor, which showed a decrease in toxicity of the EO, (4R)(+)-pulegone and of menthone, supporting the participation of the P450 oxidizing system in the formation of menthofuran. The enzymatic reaction of isolated fly microsomes with the EO or the (4R)(+)-pulegone produced menthofuran in both cases. Contrary to expectations, the insect detoxification system contributed to enhance the toxicity of the M. verticillata EO. Consequently, resistant strains overexpressing P450 genes will be more susceptible to either M. verticillata EO or (4R)(+)-pulegone and menthone.

Monoterpenes isolated from Minthostachys verticillata (Griseb.) Epling essential oil modulates immediate-type hypersensitivity responses in vitro and in vivo.

In a previous work, we have demonstrated that Minthostachys verticillata essential oil has immunomodulatory effects in vitro on cells from allergic patients. Here we characterized main components of M. verticillata essential oil and also tested if these compounds modulate In vitro and in vivo the immediate-type allergic reaction. Gas chromatography was used to identify main components of the essential oil. Pulegone (63.4 %), menthone (15.9 %), and limonene (2.1 %) were found as main classes. IL-13 levels were evaluated from lymphocytes cultures stimulated with allergen alone or combined with monoterpenes. All compounds stimulated cell proliferation but, interestingly, promoted a reduction of IL-13 values, limonene and the mixture of the three compounds being the most active. ß-Hexosaminidase release was determined from basophils to which essential oil or monoterpenes were added. We observed that, whichever combination of monoterpenes was used, ß-hexosaminidase release was diminished in all cases. Obtained values were even lower than those of antiallergic drug desloratadine. Essential oil and limonene inhibited mast cell activation and degranulation in the skin when testing passive cutaneous anaphylaxis, limonene being the most active. In conclusion, limonene was the compound that showed the most potent immunomodulatory activity. This fact suggests that it constitutes a promising natural alternative for a novel treatment of allergic diseases.

Toxicity of Schizonpeta multifida essential oil and its constituent compounds towards two grain storage insects.

BACKGROUND: The essential oil derived from the flowering aerial parts of Schizonpeta multifida was investigated for insecticidal activity against two grain storage insects, maize weevil (Sitophilus zeamais) and red flour beetle (Tribolium castaneum). RESULTS: The main constituents of the essential oil were menthone (40.34%) and pulegone (26.87%), followed by D-limonene (5.81%) and isomenthone (5.14%). Bioactivity-directed chromatographic separation on repeated silica gel columns led to the isolation of menthone and pulegone. Pulegone showed pronounced contact toxicity against S. zeamais and T. castaneum adults (7 day LD(50)= 4.46 and 1.55 µg per adult respectively) and was more toxic than menthone (7 day LD(50)=33.47 and 2.67 µg per adult respectively), while the crude essential oil had 7 day LD(50) values of 30.17 and 2.75 µg per adult respectively. Pulegone also displayed strong fumigant toxicity against S. zeamais and T. castaneum adults (7 day LC(50)=3.47 and 11.56 mg cm(-3) respectively) and was again more toxic than menthone (7 day L(50) =10.32 and 31.25 mg cm(-3) respectively), while the crude essential oil had 7 day LC(50) values of 8.33 and 26.41 mg cm(-3) respectively. CONCLUSION: The essential oil of S. multifida and its two main components may have potential to be developed as new natural fumigants/insecticides for the control of stored product insects.

Productivity and quality of volatile oil extracted from Mentha spicata and M. arvensis var. piperascens grown by a hydroponic system using the deep flow technique.

The purpose of this study was to determine the differences between spearmint (Mentha spicata L.) and Japanese mint (M. arvensis L. var. piperascens Malinv.) cultivated in either soil or nutrient solution using the deep flow technique (DFT). The differences were measured in terms of harvest period (full bloom period) and quantity and chemical components of volatile oils. The spearmint and Japanese mint were cultivated in four different nutrient formulas: plant standard nutrient, plant standard nutrient with an amino acid mixture, plant standard nutrient with a sulphur compound, and a combination of plant standard nutrient with an amino acid mixture and a sulphur compound. We observed that cultivation of spearmint and Japanese mint in nutrient solution using DFT is an effective method to provide high production of volatile oil, since it results in an earlier harvest period and higher quantity of volatile oil. We determined that for spearmint an amino acid mixture is an appropriate nutrient supplement to enhance production of volatile oil with optimum carvone content. Finally, we observed high menthol content in Japanese mint grown in all four nutrient formulas; however, supplementation with a combination of sulphur fertilisation and amino acid mixture yields the highest quantity of volatile oil.