Enzymatic catalysis in supercritical carbon dioxide: Comparison of different lipases and a novel esterase

Summary The resolution of racemic citronellol and menthol by enzymatically catalyzed transesterification in supercritical carbon dioxide (SC-CO₂) was investigated. Different lipases and an esterase in connection with various acylating reagents were employed. While the transesterification of (±)-menthol was reasonably fast and gave high enantiomeric excess, resolution of (±)-citronellol was not feasible.     

Ciliotoxicity of intranasal formulations: Menthol enantiomers

It is shown, using in vitro measurements of beat frequency of rat tracheal cilia, that (+)-(1S, 2R, 5S)-menthol and (?)?(1R, 2S, 5R)?menthol have equipotent ciliotoxicities despite the fact that inhalation of menthol vapours from the crystals of the pure enantiomers show clearly that the (?)?(1R, 2S, 5R)-menthol produces a more potent cooling sensation. Differential scanning calorimetry demonstrates the formation of a racemic compound when equimolar amounts of the two enantiomers are admixed. © 1993 Wiley-Liss, Inc.     

Menthol‐induced action potentials in Conocephalum conicum as a result of unspecific interactions between menthol and the lipid phase of the plasma membrane

Our previous study has shown that the liverwort Conocephalum conicum generates action potentials (APs) in response to both temperature drop and menthol, which are also activators of the TRPM8 (transient receptor potential melastatin 8) receptor in animals. Not only similarities but also differences between electrical reactions to menthol and cooling observed in the liverwort aroused our interest in the action of menthol at the molecular level. Patch‐clamp investigations have shown that menthol causes a reduction of current flowing through slow vacuolar (SV) channels to 29 ± 10% of the initial value (n = 9); simultaneously, it does not influence magnitudes of currents passing through a single SV channel. This may point to an unspecific interaction between menthol and the lipid phase of the membrane. An influence of menthol on lipid organization in membranes was investigated in two‐component monomolecular layers formed with menthol and dipalmitoylphosphatidylcholine (DPPC) at the argon–water interface. Analyses of the mean molecular area parameters vs the molar fraction of the menthol component have shown over‐additivity (approximately 20 Ų) in the region of high molar fractions of menthol. Infrared absorption spectroscopy studies have shown that menthol, most probably, induces breaking of a hydrogen bond network formed by ester carbonyl groups and water bridges in the lipid membrane and binds to the polar head group region of DPPC. We conclude that the disruption in the lipid phase of the membrane influences ion channels and/or pumps and subsequently causes generation of APs in excitable plants such as C. conicum.     

Impact of Menthol on Growth and Photosynthetic Function of Breviolum Minutum (Dinoflagellata,Dinophyceae, Symbiodiniaceae) and Interactions with its Aiptasia Host

Environmental change, including global warming and chemical pollution, can compromise cnidarian‐(e.g., coral‐) dinoflagellate symbioses and cause coral bleaching. Understanding the mechanisms that regulate these symbioses will inform strategies for sustaining healthy coral–reef communities. A model system for corals is the symbiosis between the sea anemone Exaiptasia pallida (common name Aiptasia) and its dinoflagellate partners (family Symbiodiniaceae). To complement existing studies of the interactions between these organisms, we examined the impact of menthol, a reagent often used to render cnidarians aposymbiotic, on the dinoflagellate Breviolum minutum, both in culture and in hospite. In both environments, the growth and photosynthesis of this alga were compromised at either 100 or 300 µM menthol. We observed reduction in PSII and PSI functions, the abundances of reaction‐center proteins, and, at 300 µM menthol, of total cellular proteins. Interestingly, for free‐living algae exposed to 100 µM menthol, an initial decline in growth, photosynthetic activities, pigmentation, and protein abundances reversed after 5–15 d, eventually approaching control levels. This behavior was observed in cells maintained in continuous light, but not in cells experiencing a light–dark regimen, suggesting that B. minutum can detoxify menthol or acclimate and repair damaged photosynthetic complexes in a light‐ and/or energy‐dependent manner. Extended exposures of cultured algae to 300 µM menthol ultimately resulted in algal death. Most symbiotic anemones were also unable to survive this menthol concentration for 30 d. Additionally, cells impaired for photosynthesis by pre‐treatment with 300 µM menthol exhibited reduced efficiency in re‐populating the anemone host.     

Phylogenetic Analysis of Selected Menthol-Producing Species Belonging to the Lamiaceae Family

Menthol is an organic compound with diverse medicinal and commercial applications, and is made either synthetically or through extraction from mint oils. The aim of the present study was to investigate menthol levels in selected menthol-producing species belonging to the Lamiaceae family, and to determine phylogenetic relationships of menthol dehydrogenase gene sequence among these species. Three genus of Lamiaceae, namely Mentha, Salvia, and Micromeria, were selected for phytochemical and phylogenetic analyses. After identification of each species based on menthol dehydrogenase gene in NCBI, BLAST software was used for the sequence alignment. MEGA4 software was used to draw phylogenetic tree for various species. Phytochemical analysis revealed that the highest and lowest amounts of both essential oil and menthol belonged to Mentha spicata and Micromeria hyssopifolia, respectively. The species Mentha spicata and Mentha piperita, which were assigned to one cluster in the dendrogram, contained the highest amounts of essential oil and menthol while Micromeria species, which was in the distinct cluster and placed in the farther evolutionary distance, contained the lowest amount of essential oil and menthol. Phylogenetic and phytochemistry analyses showed that essential oil and menthol contents of menthol-producing species are associated with menthol dehydrogenase gene sequence.     

Propagation of Biochirality: Crossovers and Nonclassical Crystallization Kinetics of Aspartic Acid in Water

All experimental procedures discussed could be treated as a screening tool for probing the existence of molecular association among the chiral molecules and the solvent system. The molecular association phases of a racemic conglomerate solution (CS) and a racemic compound solution (RCS), and the templating effect of aspartic acid solid surface were observed to minimize the chance of redissolving racemic conglomerate and racemic compound aspartic acid in water and reforming an RCS in crossovers experiments. Only 1 %wt% of l‐aspartic acid was adequate enough to induce a transformation from a racemic compound aspartic acid to a racemic conglomerate aspartic acid. This would make the propagation of biochirality more feasible and sound. However, tetrapeptide, (l‐aspartic acid)₄, failed to induce enantioseparation as templates purely by crystallization. Nonclassical crystallization theory was needed to take into account the existence of a CS. Fundamental parameters of the crystallization kinetics such as the induction time, interfacial energy, Gibbs energetic barrier, nucleation rate, and critical size of stable nuclei of: (i) racemic compound aspartic acid, (ii) racemic compound aspartic acid seeded with 1 %wt% l‐aspartic acid, (iii) racemic conglomerate aspartic acid, and (iv) l‐aspartic acid were evaluated and compared with different initial supersaturation ratios. Morphological studies of crystals grown from the crystallization kinetics were also carried out.Chirality 25:768–779, 2013. © 2013 Wiley Periodicals, Inc.     

外消旋薄荷醇对映选择性酶促酯化中酸酐与羧酸的比较研究

利用脂肪酶在有机溶剂中催化对映选择性酯化反应对外消旋薄荷醇进行了有效的光学拆分。对分别使用酸酐和相应的游离羧酸作酰基给体时的反应性能进行了比较。发现酸酐的反应性远高于对应的游离羧酸,但在酶的催化作用下酸酐易水解成为游离羧酸;在微水系统中使用过高浓度的酸酐会导致酶缺水而失活,同时会促进手性醇的非选择性酯化,从而降低产物的光学纯度。然而,在连续流加丙酸酐的半批式反应系统中,所有这些缺点均可有效地克服。与使用游离丙酸的批式反应系统相比,ｄｌ-薄荷醇的反应时间缩短了一半,酶的稳定性大幅度提高,而产物ｌ薄荷醇酯的光学纯度不相上下（＞９８％ｅ）。     

Effect of chiral enhancers on the permeability of optically active and racemic metoprolol across hairless mouse skin

The stratum corneum, the rate‐limiting barrier in transdermal drug delivery, is chiral in nature and enantiomers behave differently with respect to their transport across the skin, resulting in enantioselective permeation. The permeation characteristics of individual enantiomers of metoprolol free base (MB) were investigated using hairless mouse skin. The influence of chiral permeation enhancers, l‐menthol and (±)‐linalool, on the permeation of MB was also investigated. In the absence of enhancers, the permeation profiles of R‐ and S‐MB from donor solutions containing either RS‐MB or pure enantiomers are comparable (p < 0.05). In presence of enhancers, l‐menthol and (±)‐linalool, the flux values were increased 2.4‐ to 3.0‐fold, respectively, and the permeation profiles of R‐ and S‐MB from donor solutions containing RS‐MB are comparable (p < 0.05). However, when donor vehicle contains pure enantiomers, the permeation enhancing effect of l‐menthol on S‐MB was significantly higher (by 25%) than on R‐MB (p < 0.05). Further, in presence of l‐menthol, the flux of S‐MB from donor solution containing pure S‐MB was 35% higher than the flux of RS‐MB from racemate. No such effect was seen with (±)‐linalool. In all the investigations, no enantiomeric inversion was observed during the permeation process. The lag times were shorter in the case of l‐menthol compared with (±)‐linalool. Chirality 11:536–540, 1999. © 1999 Wiley‐Liss, Inc.     

Behavioural induction in Drosophila: timing and specificity

Experiments reported in this paper investigate the properties of a change in the responsiveness of adult Drosophila melanogaster induced by exposure to different rearing media. This effect has previously been described as habituation or associative learning. Exposure to food medium containing 0.08% menthol induced a positive response to menthol odour in a T-maze olfactometer. A brief (one hour) exposure to mentholic food just before testing was sufficient to induce a change in responsiveness. The effect did not persist through periods of more than an hour of separation from mentholic medium.Effects induced by exposure to a single compound were not specific to that compound alone. Menthol-reared flies (MRFs) differed from plain reared flies (PRFs) in their responsiveness to the odours of benzaldehyde and ethyl acetate, as well as menthol, and exposure to ethyl acetate induced a change in response to menthol odour.That there was an induced positive response to menthol in MRFs suggests that conventional habituation is insufficient to explain the induced change in responsiveness, but the generalised nature of this behavioural induction in MRFs is hard to explain in terms of associative learning. The mechanism underlying the induction remains elusive.     

Menthol tolerant clones of Mentha arvensis: approach for in vitro selection of menthol rich genotypes

In vitro raised shoots of Mentha arvensis L. were screened for menthol tolerance level by growing them in media containing 0–100 g ml^–1 menthol. A total of 2850 regenerated shoots were step wise screened for menthol tolerance at the concentrations of 50 g ml^–1 followed by 60 and 70 g ml^–1. In this screening, only 30 individual regenerated shoots were able to survive. The clones from the primary screen were inoculated into rooting medium and, after rooting, transferred to pots in the greenhouse. Ultimately, these 30 menthol tolerant clones were multiplied and grown in the field in replicated plots of 2.5×2.5 m sizes. Twigs of 30 clones from the replicated trials were rechecked for tolerant phenotypes at a concentration of 70 g ml^–1 menthol wherein, these survived even after 7 days (secondary screening). These clones were checked for oil and menthol content and were found to be better than the control plants. Out of these 30 plants, five tolerated 80 g ml^–1 menthol (tertiary level screening) and were found to contain the highest amount of menthol per g leaf biomass. Molecular analysis through RAPD showed distinct variation in the profiles of these five plants, in comparison to the control. Using this method the relationship between the primer OPT 04, menthol tolerance and high menthol content character of the genotype was established. Further, a cultivar `Saksham' was released from the selections by CIMAP for superior performance.     

Isomerization and Racemization of Menthols

The isomerization and racemization of menthol isomers have been investigated with copper chromite and Raney nickel catalyst or sodium mentholates. The equilibrium concentration has been found to correspond to 55~56% of menthol with the catalytic isomerization, whereas to 74~75% of menthol with sodium mentholates. From these data, the free energy difference between equatorial and axial group in menthols is calculated to be about 0.5~0.6 Kcal./mole for hydroxyl and 1.4~1.5 Kcal./mole for methyl at 200°C.     

Cold‐ and menthol‐evoked membrane potential changes in the moss Physcomitrella patens: influence of ion channel inhibitors and phytohormones

Microelectrode measurements carried out on leaf cells from Physcomitrella patens revealed that a sudden temperature drop and application of menthol evoked two types of different‐shaped membrane potential changes. Cold stimulation evoked spike‐type responses. Menthol depolarized the cell membrane with different rates. When it reached above 1 mV s^?1, the full response was recorded. Characteristic for the full responses was also a few‐minute plateau of the membrane potential recorded after depolarization. The influence of inhibitors of calcium channels (5 mM Gd³⁺), potassium channels (5 mM Ba²⁺), chloride channels (200 μM Zn²⁺, 50 μM niflumic acid) and proton pumps (10 μM DES), an activator of calcium release from intracellular stores (Sr²⁺), calcium chelation (by 400 μM EGTA) and phytohormones (50 μM auxin, 50 μM abscisic acid (ABA), 500 μM salicylic acid) on cold‐ and menthol‐evoked responses was tested. Both responses are different in respect to the ion mechanism: cold‐evoked depolarizations were influenced by Ba²⁺ and DES; in turn, menthol‐evoked potential changes were most effectively blocked by Zn²⁺. Moreover, the effectiveness of menthol in generation of full responses was reduced after administration of auxin or ABA, i.e. phytohormones known for their participation in responses to cold and regulation of proton pumps. The effects of DES indicated that one of the main conditions for generation of menthol‐evoked responses is inhibition of the proton pump activity. Our results indicate that perception of cold and menthol by plants proceeds in different ways due to the differences in ionic mechanism and hormone dependence of cold‐ and menthol‐evoked responses.     

Comparative Activities of the S-Enantiomer and Racemic Forms of Equol on Bone Fragility in Ovariectomized Mice

We compared the effects of the S-enantiomer and racemic forms of equol on bone using ovariectomized (OVX) mice. Femoral bone mineral density and bone strength decreased in the OVX mice, but not in OVX mice administered 0.5 mg/d S-equol. This, however, did not hold for racemic equol. Serum and urine S-equol concentrations were higher in the mice administered S-equol than in those administered racemic equol. These results suggest that the inhibitory effects of S-equol on bone fragility in OVX mice are greater than those of racemic equol.     

Positive correlation between menthol content andin vitro menthol tolerance inMentha arvensis L. cultivars

Menthol is a highly valued monoterpene produced by Japanese mint (Mentha arvensis) as a natural product with wide applications in cosmetics, confectionery, flavours, beverages and therapeutics. Selection of high menthol yielding genotypes is therefore the ultimate objective of all genetic improvement programmes inMentha arvensis. A positive correlation was observed in the present study between menthol content in oils of evaluated genotypes and the level of tolerance to externally supplied menthol of explants of these genotypes in culture medium. The easy use of this relationship as a selectable biochemical marker opens the practical applicability of largescalein vitro screening of the germplasm, clones and breeders' material for selection of elite genotypes.     

A unique interspecific hybrid spearmint clone with growth properties of Menthaarvensis L. and oil qualities of Mentha spicata L.

Crossing blocks of Mentha arvensis cv Kalka (menthol mint without carvone) and Mentha spicata cv Neera (carvone mint without menthol) in alternate rows (2:1::Kalka:Neera), and pollination of florets of Kalka with that from Neera, yielded a carvone-rich variant among the open-pollinated seedlings. The variant possesses the hybrid phenotype, including the vigorous M. arvensis growth habit and the synthesis of rich oil aroma supplemented with a menthol tinge (carvone 64%–76% against 58% for the normal carvone mint cv Neera), and thus a novel combination of the essential oil. Chromosome counts and random amplified polymorphic DNA analysis confirmed that this spearmint variant, designated as the variety Neerkalka, is a unique interspecific hybrid (2n=5x=60) of M. arvensis cv Kalka (2n=8x=96) and M. spicata cv Neera (2n=2x=24). Vegetative multiplication of the hybrid was facilitated by its underground sucker-reproducing ability which is otherwise absent in spearmints. The per cent improvement in the variant ranged from 31–97 for herbage yield and 95–317 for oil yield over the standard spearmint varieties (MSS-5, Arka and Neera), with per hectare oil yields of 125.0 kg, 139.0 kg and 65.0 kg, respectively. Received: 25 March 2000 / Accepted: 3 July 2000     

A Green and Sustainable Approach: Celebrating the 30th Anniversary of the Asymmetric l‐Menthol Process

Takasago has been devoted to producing l‐menthol since 1954, and our long history of manufacturing this important aroma chemical is reviewed here. The current asymmetric catalytic process had its 30th anniversary in 2013. Our l‐menthol process is considered carbon‐neutral, and, therefore, ‘green’ and sustainable. It uses renewable myrcene obtained from gum rosin as a starting material. In addition, the Rh‐BINAP (=2,2′‐bis(diphenylphosphino)‐1,1′‐binaphthyl) catalytic system is highly efficient. This pathway not only leads l‐menthol, but a variety of 100% biobased aroma chemical products as well. By measuring the ¹⁴C levels in a material, one can determine the percentage of carbon that is biobased. This biobased assay, described as the ratio plant‐derived C/fossil‐derived C, can clarify how renewable a product really is. This will be highlighted for several of Takasago's key aroma chemicals.     

Determination of felodipine, its enantiomers, and a pyridine metabolite in human plasma by capillary gas chromatography with mass spectrometric detection

Sensitive methods based on capillary gas chromatography (GC) with mass spectrometric (MS) detection in a selected-ion monitoring mode (SIM) for the determination of racemic felodipine, its enantiomers, and a pyridine metabolite in human plasma are described. Following liquid-liquid extraction from plasma, enantiomers of felodipine were separated on a chiral HPLC column (Chiralcel OJ) and fractions containing each isomer were collected on a continuous basis using a fraction collector. These fractions were later analyzed by GC-MS-SIM. A similar method based on GC-MS-SIM detection was developed for the determination of racemic felodipine and its pyridine metabolite with a minor modification of sample preparation. The limits of quantitation in plasma were 0.1 ng/ml for both the R(+)- and S(−)-enantiomers of felodipine and 0.5 ng/ml for both racemic felodipine and its pyridine metabolite. The stereoselective assay was used to support a clinical study with racemic felodipine, and was capable of analyzing more than 30 plasma samples per day.     

Plant protection in sustainable systems of agriculture

Abstract

A comprehensive evaluation of 13 plant essential oils namely: caraway, chenopodium, cinnamon, clove, eucalyptus, garlic, geranium, lemon, matrecary, peppermint, rose, rosemary and thyme and 14 of their monoterpenoidal constituents: borneol, camphor, carvacrol, carveol, carvone (R and S), chlorothymol, cineol, cinnamaldehyde, citronellol, eugenol, geraneol, menthol, and thymol was investigated for their antibacterial activity against the two phytopathogenic bacteria, Agrobacterium tumefaciens and Erwinia carotovora var carotovora. Trials for improving the antibacterial activity by either structure modification or mixing with the three synergists: piperonyl butoxide, Triton X-100 and Tween 20 were achieved. New N-methyl carbamate derivatives based on monoterpenoids were introduced. Cinnamon, clove, chenopodium, caraway, rosemary and thyme oils proved to possess good antibacterial activity. Thymol, chlorothymol, and carvacrol were potent against the tested bacteria and quite close to the effect of the standard. Conversion of menthol into its carbamate derivative gave good enhancement in the bactericidal activity at all concentrations compared to menthol itself. Also, the carbamate derivatives of β-citronellol and borneol improved the bactericidal activity against E. carotovora, but decreased it against A. tumefaciens. Synergistic antibacterial activity of the tested monoterpenoids was observed when combined with each of the three synergists particularly with Triton X-100.     

Effects of Menthol on Nicotine Pharmacokinetic,Pharmacology and Dependence in Mice

Although menthol, a common flavoring additive to cigarettes, has been found to impact the addictive properties of nicotine cigarettes in smokers little is known about its pharmacological and molecular actions in the brain. Studies were undertaken to examine whether the systemic administration of menthol would modulate nicotine pharmacokinetics, acute pharmacological effects (antinociception and hypothermia) and withdrawal in male ICR mice. In addition, we examined changes in the brain levels of nicotinic receptors of rodents exposed to nicotine and menthol. Administration of i.p. menthol significantly decreased nicotine’s clearance (2-fold decrease) and increased its AUC compared to i.p. vehicle treatment. In addition, menthol pretreatment prolonged the duration of nicotine-induced antinociception and hypothermia (2.5 mg/kg, s.c.) for periods up to 180 min post-nicotine administration. Repeated administration of menthol with nicotine increased the intensity of mecamylamine-precipitated withdrawal signs in mice exposed chronically to nicotine. The potentiation of withdrawal intensity by menthol was accompanied by a significant increase in nicotine plasma levels in these mice. Western blot analyses of α4 and β2 nAChR subunit expression suggests that chronic menthol impacts the levels and distribution of these nicotinic subunits in various brain regions. In particular, co-administration of menthol and nicotine appears to promote significant increase in β2 and α4 nAChR subunit expression in the hippocampus, prefrontal cortex and striatum of mice. Surprisingly, chronic injections of menthol alone to mice caused an upregulation of β2 and α4 nAChR subunit levels in these brain regions. Because the addition of menthol to tobacco products has been suggested to augment their addictive potential, the current findings reveal several new pharmacological molecular adaptations that may contribute to its unique addictive profile.