Development and application of high resolution mass spectrometry analytical method to study and identify the photoinduced transformation products of environmental pollutants.

Terpenes are among the major causes of pleasant or unpleasant odors close to active or inactive landfills. We studied R-limonene and p-cymene environmental degradation products using the heterogeneous photocatalysis mediated by titanium dioxide to explore the odor pollution. The aim of the study was the development of mass spectrometry based methods both hyphenated with GC and HPLC to identify and characterize transformation products (TPs) derived from photodegradation of R-limonene and p-cymene. With the GC-MS method we identified three TPs for R-limonene and two for p-cymene comparing the obtained mass spectra with those in the NIST library. While with HPLC-MS method, thanks to the use of the high resolution of MS tool, we recognized four and five TPs for R-limonene and p-cymene respectively. No p-cymene was detected as R-limonene transformation product. The methods developed were then applied to real environmental samples coming from landfills active (Lan1) or inactive (Lan2 and Lan3) located in northern Italy. R-limonene was detected in the active landfill (Lan1 at the concentration of 2.35 µg/mL) together with one of its TPs and one TP derived from p-cymene. p-Cymene was detected in the other two inactive landfills (Lan2 and Lan3 concentrations 0.025 and 0.15 µg/mL, respectively) together with one of its TP and two TPs coming from R-limonene photodegradation. The finding of TPs together with R-limonene and p-cymene both in active and inactive landfills point out the attention on the reduction of these molecules in the environment to reduce pollution and human risks.

Synthesis and crystal structure of a new chiral α-amino-oxime nickel(II) complex.

A dinuclear nickel complex with (S)-limonene based amino-oxime ligand has been isolated and its crystal structure determined. The resolved structure of dichloridobis-{(2S,5R)-2-methyl-5-(prop-1-en-2-yl)-2-[(pyridin-2-yl)methyl-amino]-cyclo-hexan-1-one oxime}dinickel(II), [Ni2Cl2(C16H23ClN3O)2], at 100 K has monoclinic (P21) symmetry. The two NiII ions in the dinuclear complex are each coordinated in a distorted octa-hedral environment by three nitro-gen atoms, a terminal chloride and two µ bridging chlorides. Each oxime ligand is coordinated to nickel(II) by the three nitro-gen atoms, leading to two five-membered chelate rings, each displaying an envelope conformation. In the crystal, numerous inter-molecular and intra-molecular hydrogen bonds lead to the formation of a three-dimensional network structure.

Ougan juice debittering using ultrasound-aided enzymatic hydrolysis: Impacts on aroma and taste.

The optimal sonication conditions (40 kHz, 80 W/L and 60 min) during Ougan juice debittering by Aspergillus niger koji extract were established. Enzymatic hydrolysis degrees of naringin and limonin were enhanced to 89.90% and 36.16%, and enzymatic hydrolysis time was shortened by 33%. Sonication significantly enhanced activities of α-l-rhamnosidases, ß-glucosidases and limoninases from A. niger koji extract and facilitated break of CO bonds in naringin (p < 0.05). These accounted for the enhanced enzymatic hydrolysis degrees and velocities of bitter compounds. Meanwhile, sonication lowered 40%, 7% and 21%, 13%, 11%, 25% of bitter, sour tastes and green, citrus-like, floral, woody notes, but enhanced 18% and 15% of fruity and sweet notes, resulting in 38% and 33% increases in over-all taste and aroma scores. Lowered levels of bitter compounds, organic acids, green, citrus-like, floral, woody aroma compounds and enhanced levels of fruity, sweet aroma compounds caused by sonication accounted for the flavor improvements.

Urea-Based Low-Molecular-Weight Pseudopeptidic Organogelators for the Encapsulation and Slow Release of (R)-Limonene.

Low-molecular-weight compounds containing alkylurea fragments attached to the amino end of different miminalistic pseudopeptidic structures have been shown to be excellent organogelators in a variety of organic solvents and liquid organic compounds of different nature. The formation of gels in this work is defined through rheological measurements for those cases where G' > G''. Both the topology and the symmetry of the corresponding urea compounds play a role in defining their organogelator behavior. This can also be tuned by the presence of additional supramolecular guests, as is the case for suberic acid. These compounds also achieve the gelation of relevant active substances such as terpene natural oils and complex mixtures of flavors and fragrances. This provides a simple and mass-efficient supramolecular system for the quantitative encapsulation of active substances, without the need for any additional solvent or complex processes, and their consequent controlled release.

Time-integrated thermal desorption for quantitative SIFT-MS analyses of atmospheric monoterpenes.

A new time-integrated thermal desorption technique has been developed that can be used with selected ion flow tube mass spectrometry, TI-TD/SIFT-MS, for off-line quantitative analyses of VOCs accumulated onto sorbents. Using a slow desorption temperature ramp, the absolute amounts of desorbed compounds can be quantified in real time by SIFT-MS and constitutional isomers can be separated. To facilitate application of this technique to environmental atmospheric monitoring, method parameters were optimised for quantification of the three common atmospheric monoterpenes: ß-pinene, R-limonene and 3-carene. Three sorbent types, Tenax TA, Tenax GR and Porapak Q, were tested under 26 different desorption conditions determined by the "design of experiment", DOE, systematic approach. The optimal combination of type of sorbent, bed length, sampling flow rate, sample volume and the initial desorption temperature was determined from the experimental results by ANOVA. It was found that Porapak Q exhibited better efficiency of sample collection and further extraction for total monoterpene concentration measurements. On the other hand, Tenax GR or TA enabled separation of all three monoterpenes. The results of this laboratory study were tested with the sample accumulated from a branch of a Pinus nigra tree. Graphical abstract.

Review of toxicological assessment of d-limonene, a food and cosmetics additive.

R-(+)-limonene (d-limonene) is a commonly used flavor additive in food, beverages and fragrances for its pleasant lemon-like odor. Considering its increasing applications, it's necessary to understand toxicological effects and risk associated with its use. R-(+)-limonene is rapidly absorbed in experimental animals and human beings following oral administration. In humans, it gets distributed to liver, kidney, and blood resulting in the formation of metabolites like perillic acid, dihydroperillic acid, limonene-1,8-diol and limonene-1,2 diol. Important toxic effects primarily reported in rodents are severe hyaline droplet nephrotoxicity (only in male rats due to specific protein α2u-globulin; however, this effect isn't valid for humans), hepatotoxicity and neurotoxicity. R-(+)-limonene does not show genotoxic, immunotoxic and carcinogenic effects. Substantial data is available about limonene's stability after treatment with thermal and non-thermal food processing techniques; however, information about toxicity of metabolites formed and their safe scientific limits is not available. In addition, toxicity of limonene degradation products formed during storage of citrus juices isn't known. Based on all available toxicological considerations, R-(+)-limonene can be categorized as low toxic additive. More detailed studies are required to better understand interaction of limonene with modern food processing techniques as well as degradation products generated and toxicity arising from such products.

The Effect of Combining Natural Terpenes and Antituberculous Agents against Reference and Clinical Mycobacterium tuberculosis Strains.

Background: On account of emergence of multi- and extensively drug-resistant Mycobacterium tuberculosis (Mtb) strains, combinations of drugs with natural compounds were tested to search for antibiotic activity enhancers. In this work we studied terpenes (α-pinene, bisabolol, ß-elemene, (R)-limonene, (S)-limonene, myrcene, sabinene), which are the main constituents of essential oil obtained from Mutellina purpurea L., a plant with described antitubercular activity, to investigate their interactions with antibiotics against reference Mtb strains and multidrug-resistant clinical isolates. Methods: The serial dilution method was used to evaluate the minimal inhibitory concentration (MIC) of tested compounds, while the fractional inhibitory concentration index (FICI) was calculated for characterization of interactions. Moreover, IC50 values of tested compounds were determined using monkey kidney epithelial cell line (GMK). Results: The combinations of all studied terpenes with ethambutol or rifampicin resulted in a synergistic interaction. Bisabolol and (R)-limonene decreased the MIC for rifampicin at least two-fold for all tested strains, however no synergistic action was observed against virulent strains. The tested terpenes showed slight (bisabolol) or no cytotoxic effect against normal eukaryotic cells in vitro. Conclusions: The obtained enhanced activity (FICI < 0.5) of ethambutol and rifampicin against H37Ra strain under the influence of the studied terpenes may be correlated to the capability of essential oil constituents to modify bacterial resistance mechanisms in general. The observed differences in avirulent and virulent bacteria susceptibility to terpenes tested separately and in combinations with antibiotics can be correlated with the differences in the cell wall structure between H37Ra mutant and all virulent strains.

Herbicidal Activities of Some Allelochemicals and Their Synergistic Behaviors toward Amaranthus tricolor L.

Seven allelochemicals, namely R-(+)-limonene (A), vanillin (B), xanthoxyline (C), vanillic acid (D), linoleic acid (E), methyl linoleate (F), and (±)-odorine (G), were investigated for their herbicidal activities on Chinese amaranth (Amaranthus tricolor L.). At 400 µM, xanthoxyline (C) showed the greatest inhibitory activity on seed germination and seedling growth of the tested plant. Both vanillic acid (D) and (±)-odorine (G) inhibited shoot growth, however, apart from xanthoxyline (C), only vanillic acid (D) could inhibit root growth. Interestingly, R-(+)-limonene (A) lightly promoted root length. Other substances had no allelopathic effect on seed germination and seedling growth of the tested plant. To better understand and optimize the inhibitory effects of these natural herbicides, 21 samples of binary mixtures of these seven compounds were tested at 400 µM using 0.25% (v/v) Tween® 80 as a control treatment. The results showed that binary mixtures of R-(+)-limonene:xanthoxyline (A:C), vanillin:xanthoxyline (B:C), and xanthoxyline:linoleic acid (C:E) exhibited strong allelopathic activities on germination and seedling growth of the tested plant, and the level of inhibition was close to the effect of xanthoxyline (C) at 400 µM and was better than the effect of xanthoxyline (C) at 200 µM. The inhibition was hypothesized to be from a synergistic interaction of each pair of alleochemicals. Mole ratios of each pair of allelochemicals ((A:C), (B:C), and (C:E)) were then evaluated, and the best ratios of the binary mixtures A:C, B:C and C:E were found to be 2:8, 2:8, and 4:6 respectively. These binary mixtures significantly inhibited germination and shoot and root growth of Chinese amaranth at low concentrations. The results reported here highlight a synergistic behavior of some allelochemicals which could be applied in the development of potential herbicides.

Explaining the antioxidant activity of some common non-phenolic components of essential oils.

Limonene, linalool and citral are common non-phenolic terpenoid components of essential oils, with attributed controversial antioxidant properties. The kinetics of their antioxidant activity was investigated using the inhibited autoxidation of a standard model substrate. Results indicate that antioxidant behavior of limonene, linalool and citral occurs by co-oxidation with the substrate, due to very fast self-termination and cross-termination of the oxidative chain. Rate constants kp and 2kt, (M-1s-1) at 30°C were 4.5 and 3.5×106 for limonene, 2.2 and 9.0×105 for linalool and 39 and 1.0×108 for citral. Behavior is bimodal antioxidant/pro-oxidant depending on the concentration. Calculations at the M05/6-311+g(2df,2p) level indicate that citral reacts selectively at the aldehyde C-H having activation enthalpy and energy respectively lower by 1.3 and 1.8kcal/mol compared to the most activated allyl position. Their termination-enhancing antioxidant chemistry might be relevant in food preservation and could be exploited under appropriate settings.

R-Limonene metabolism in humans and metabolite kinetics after oral administration.

We studied the R-limonene (LMN) metabolism and elimination kinetics in a human in vivo study. Four volunteers were orally exposed to a single LMN dose of 100-130 µg kg-1 bw. In each case, one pre-exposure and subsequently all 24 h post-exposure urine samples were collected. From two subjects, blood samples were drawn up to 5 h after exposure. The parent compound was analysed in blood using headspace GC-MS. The metabolites cis- and trans-carveol (cCAR), perillyl alcohol (POH), perillic acid (PA), limonene-1,2-diol (LMN-1,2-OH), and limonene-8,9-diol (LMN-8,9-OH) were quantified in both blood and urine using GC-PCI-MS/MS. Moreover, GC-PCI-MS full-scan experiments were applied for identification of unknown metabolites in urine. In both matrices, metabolites reached maximum concentrations 1-2 h post-exposure followed by rapid elimination with half-lives of 0.7-2.5 h. In relation to the other metabolites, LMN-1,2-OH was eliminated slowest. Nonetheless, overall renal metabolite elimination was completed within the 24-h observation period. The metabolite amounts excreted via urine corresponded to 0.2 % (cCAR), 0.2 % (tCAR), <0.1 % (POH), 2.0 % (PA), 4.3 % (LMN-1,2-OH), and 32 % (LMN-8,9-OH) of the orally administered dose. GC-PCI-MS full-scan analyses revealed dihydroperillic acid (DHPA) as an additional LMN metabolite. DHPA was estimated to account for 5 % of the orally administered dose. The study revealed that human LMN metabolism proceeds fast and is characterised by oxidation mainly of the exo-cyclic double bond but also of the endo-cyclic double bond and of the methyl side chain. The study results may support the prediction of the metabolism of other terpenes or comparable chemical structures.

Physical stability of R-(+)-Limonene emulsions stabilized by Ulva fasciata algae polysaccharide.

The physical stability of R-(+)-Limonene emulsions stabilized by Ulva fasciata polysaccharide (UFP) was investigated in this study. Emulsion physical stability was evaluated under different polysaccharide concentrations (1%-5%, wt/wt) and pH values (3.0-11.0). The stability of R-(+)-Limonene emulsions was demonstrated by droplet size distribution, rheological properties, zeta potential and visual phase separation. R-(+)-Limonene emulsions displayed monomodal droplet size distributions, high absolute values of zeta potential and good storage stability when 3% (wt/wt) UFP was used. The rheological properties and stability of R-(+)-Limonene emulsions appeared to be dependent on polysaccharide concentration. The emulsion stability was impacted by pH. Higher zeta potential (-52.6mV) and smaller mean droplet diameter (2.45µm) were achieved in neutral liquid environment (pH 7.0). Extreme acidity caused the flocculation of emulsions, which was manifested as phase separation, while emulsions were quite stable in an alkaline environment. Through comparing the stabilities of emulsions stabilized by different emulsifiers (i.e. UFP, GA and Gelatin), the result suggested that UFP was the best emulsifying agent among them.

Fumigant and Repellent Activity of Limonene Enantiomers Against Tribolium confusum du Val.

The use of pesticides, as carried out in the last 50 years, caused several negative environmental and human health consequences, leading to the development of alternative techniques to control pests, such as the use of compounds of plant origin. In this study, we assessed the fumigant and repellent activity of both the enantiomers of limonene, a monoterpene usually found in many plant species, against Tribolium confusum du Val. We tested both molecules at different doses, air temperatures, and in absence and presence of flour. R-(+)-limonene resulted more effective than S-(-)-limonene; indeed, it was able to reach 100% of efficacy at a concentration of 85 mg/L air when tested at different temperatures without flour. Data showed a positive relationship between efficacy and temperature, and a negative effect of the presence of debris on the bioactivity of limonene. Furthermore, repellency trials reported a higher activity of R-(+)-limonene compared to the other enantiomer.

Optimisation of α-terpineol production by limonene biotransformation using Penicillium digitatum DSM 62840.

BACKGROUND: In this study, (R)-(+)-limonene biotransformation using three fungal strains was compared. Penicillium digitatum DSM 62840 was distinguished for its capacity to transform limonene into α-terpineol with high regioselectivity. Growth kinetics in submerged liquid culture and the effects of growth phase and contact time on biotransformation were studied using this strain. Substrate concentration, co-solvent selection, and cultivation conditions were subsequently optimised. RESULTS: The maximum concentration of α-terpineol (833.93 mg L(-1)) was obtained when the pre-culture medium was in medium log-phase by adding 840 mg L(-1) substrate dissolved in ethanol and cultivation was performed at 24 °C, 150 rpm, and pH 6.0 for 12 h. Addition of small amounts of (R)-(+)-limonene (84 mg L(-1)) at the start of fungal log-phase growth yielded a 1.5-fold yield of α-terpineol, indicating that the enzyme was inducible. CONCLUSION: Among these three strains tested, P. digitatum DSM 62840 was proved to be an efficient biocatalyst to transform (R)-(+)-limonene to α-terpineol. Further studies revealed that the optimal growth phase for biotransformation was in the medium log phase of this strain. The biotransformation represented a wide tolerance of temperature; α-terpineol concentration underwent no significant change at 8-32 °C. The biotransformation could also be performed using resting cells.

Self-assembled nanostructured aqueous dispersions as dermal delivery systems.

Due to their high interfacial area and capability of loading hydrophobic, hydrophilic and amphiphilic drugs, self-assembled nanoparticles are the subject of much attention in view of an application of these dispersions as carrier systems for a variety of different active ingredients. Therefore, the effect of the internal nanostructure of oil-loaded monoglyceride-based nanoparticles on the dermal delivery of diclofenac sodium was investigated. The different self-assembled phases of the nanostructured aqueous dispersions were characterized by small angle X-ray scattering (SAXS). The influence of the different phases ranging from cubic-bicontinuous, over hexagonal and cubic-micellar phases to emulsified microemulsions on the dermal delivery of the incorporated active was examined by Franz-type diffusion cell and in vitro tape stripping experiments on porcine skin. These studies revealed a dependency of the skin permeation of diclofenac sodium on the formulation's internal structure, which could be modified by varying the amount of R-(+)-limonene in the oil phase. A superiority of the emulsified microemulsion, possessing the highest amount of R-(+)-limonene, over cubic or hexagonal phases was evidenced in terms of dermal drug delivery.

Intra-molecular enantiomerism in R-(+)-Limonene as evidenced by the differential bond polarizabilities.

We propose an algorithm to obtain the differential bond polarizabilities from the Raman and Raman optical activity (ROA) spectral intensities. The signs of the differential bond polarizabilities of R-Limonene demonstrate that there is intra-molecular enantiomerism in its six membered ring structure. That is, the signs of the differential bond polarizabilities around the six membered ring are inversed under an intra-molecular mirror reflection. This is similar to what happens in the right and left handed chiral isomers under a mirror conversion.

Novel R-(+)-limonene-based thiosemicarbazones and their antitumor activity against human tumor cell lines.

In an attempt to develop potent and selective antitumor agents, a series of novel thiosemicarbazones derived from a natural monoterpene R-(+)-limonene was synthesized and their antitumor activity was evaluated. Overall, the majority of tested compounds exhibited considerable inhibitory effects on the growth of a wide range of cancer cell lines. Almost all of tested thiosemicarbazones were especially sensitive to prostate cells (PC-3). Derivatives 5, 6, 8, 9, 10, 11 and 13 presented the most potent antitumor activity against PC-3 cells. These compounds showed lower value of GI50 (0.04-0.05 µM) than the reference drug paclitaxel, besides a high selectivity for the same cell line. The 4-fluorobenzaldehyde derivative 10 was the most selective compound for prostate cells, while 2-hydroxybenzaldehyde derivative 8 was the most active compound, with potent antitumor activity against all tested cell lines.

Effect of Laurus nobilis L. Essential Oil and its Main Components on α-glucosidase and Reactive Oxygen Species Scavenging Activity.

The present study was designed to determine the effects of the essential oil of Laurus nobilis L. (Lauraceae) and its three main components on α-glucosidase and reactive oxygen species scavenging activity. The chemical composition of the essential oil from Laurus nobilis L. leaves was analyzed by GC/GC-MS and resulted in the identification of 29 compounds, representing 99.18% of the total oil. 1,8-cineole (68.82%), 1-(S)-α-pinene (6.94%), and R-(+)- limonene (3.04%) were determined to be the main components. The antioxidant features of the essential oil and its three main components were evaluated using inhibition of 2,2-diphenyl-1- picrylhydrazyl, hydroxyl, and superoxide radicals, inhibition of hydrogen peroxide and lipid peroxidation assays. The results show that the DPPH, hydroxyl, and superoxide radical as well as hydrogen peroxide scavenging activities of the essential oil are greater than the positive controls and the three main components of the oil when tested independently. The inhibition of lipid peroxidation by the oil occurred less frequently than with 1,8-cineole and R-(+)- limonene alone, but the effects were more pronounced than those seen with 1-(S)-α-pinene and the positive controls. An α-glucosidase inhibition assay was applied to evaluate the in-vitro antidiabetic activity of the essential oil. IC50-values were obtained for laurel essential oil, 1, 8-cineole, 1-(S)-α-pinene, and R-(+)-limonene: 1.748 µL/mL, 1.118 µL/mL, 1.420 µL/mL and 1.300 µL/mL, respectively. We also found that laurel essential oil and 1, 8-cineole inhibited the α-glucosidase competitively while 1-(S)-α-pinene and R-(+)-limonene were uncompetitive inhibitors.

Sensitive monitoring of monoterpene metabolites in human urine using two-step derivatisation and positive chemical ionisation-tandem mass spectrometry.

A gas chromatographic-positive chemical ionisation-tandem mass spectrometric (GC-PCI-MS/MS) method for the simultaneous determination of 10 oxidative metabolites of the monoterpenoid hydrocarbons α-pinene, (R)-limonene, and Δ(3)-carene ((+)-3-carene) in human urine was developed and tested for the monoterpene biomonitoring of the general population (n=36). The method involves enzymatic cleavage of the glucuronides followed by solid-supported liquid-liquid extraction and derivatisation using a two-step reaction with N,O-bis(trimethylsilyl)-trifluoroacetamide and N-(trimethylsilyl)imidazole. The method proved to be both sensitive and reliable with detection limits ranging from 0.1 to 0.3 µg L(-1). In contrast to the frequent and distinct quantities of (1S,2S,4R)-limonene-1,2-diol, the (1R,2R,4R)-stereoisomer could not be detected. The expected metabolite of (+)-3-carene, 3-caren-10-ol was not detected in any of the samples. All other metabolites were detected in almost all urine samples. The procedure enables for the first time the analysis of trace levels of a broad spectrum of mono- and bicyclic monoterpenoid metabolites (alcohols, diols, and carboxylic acids) in human urine. This analytical procedure is a powerful tool for population studies as well as for the discovery of human metabolism and toxicokinetics of monoterpenes.