UVR-induced phototoxicity mechanism of methyl N-methylanthranilate in human keratinocyte cell line.

Fragrances are used in almost every cosmetic product. International Fragrance Association (IFRA) is the regulatory body that controls the use of fragrances in cosmetic products. Methyl-N-methylanthranilate (DMA) is a naturally derived fragrance, commonly used in cosmetic products such as fine perfumes, skin care products, etc. But there is a lack of detailed study in terms of its phototoxic and photogenotoxicity mechanisms under UVA/sunlight exposure. In this study, we have shown that DMA photodegrades in 4 h under UVA (1.5 mW/cm2) and sunlight. DMA (0.0001%-0.0025%) significantly reduced the cell viability as demonstrated by NRU and MTT assays in a dose-dependent manner under UVA (5.4 J/cm2) and sunlight (1 h) exposure in the HaCaT cell line. It generated excessive intracellular ROS (superoxide anion radical via type-I photodynamic reaction), resulting in lysosomal destabilization and mitochondrial membrane depolarization. Photo-activated DMA caused DNA fragmentation as observed by olive tail moment. DNA double-strand breaks was demonstrated by phosphorylation of H2AX-histone protein and formation of photo-micronuclei in skin keratinocytes. Additionally, photo-activated DMA upregulated the oxidative stress marker gene hemeoxygenase-1 and apoptotic marker genes (cytochrome-C, caspase-3, caspase-9). Activated caspase-cascade pathway established that photo-activated DMA can potentially trigger apoptosis in the human skin cells.

Metabolomic insights into the inhibition mechanism of methyl N-methylanthranilate: A novel quorum sensing inhibitor and antibiofilm agent against Pseudomonas aeruginosa.

The quorum sensing (QS) inhibition effect of methyl N-methylanthranilate (MMA) from Pericarpium Citri Reticulatae Chachiensis against foodborne pathogen Pseudomonas aeruginosa was reported for the first time. MMA effectively attenuated QS related virulence factors production and biofilm formation, while suppressed expression of a dozen of QS related genes. Untargeted LC-MS metabolomics revealed 108 significantly altered metabolites after MMA treatment. They indicated that MMA addition reduced the efficiency of TCA cycle and antioxidant systems, disturbed amino acid and nucleotide metabolism, increased unsaturated fatty acid and decreased peptidoglycan components, which might ultimately attenuate P. aeruginosa pathogenicity and restrain biofilm formation. Physiological characterization confirmed the compromised membrane integrity and increased intracellular oxidative stress after MMA treatment. Furthermore, metabolomics data implied that MMA inhibition on QS might exert through disrupting QS autoinducer PQS biosynthesis, which was supported by molecular docking. Our data indicated that MMA could be used as a novel QS inhibitor and anti-biofilm agent to improve food safety. It also provided new insight in the possible underlying inhibition mechanism of MMA and the response of P. aeruginosa to MMA.

Fermentative N-Methylanthranilate Production by Engineered Corynebacterium glutamicum.

The N-functionalized amino acid N-methylanthranilate is an important precursor for bioactive compounds such as anticancer acridone alkaloids, the antinociceptive alkaloid O-isopropyl N-methylanthranilate, the flavor compound O-methyl-N-methylanthranilate, and as a building block for peptide-based drugs. Current chemical and biocatalytic synthetic routes to N-alkylated amino acids are often unprofitable and restricted to low yields or high costs through cofactor regeneration systems. Amino acid fermentation processes using the Gram-positive bacterium Corynebacterium glutamicum are operated industrially at the million tons per annum scale. Fermentative processes using C. glutamicum for N-alkylated amino acids based on an imine reductase have been developed, while N-alkylation of the aromatic amino acid anthranilate with S-adenosyl methionine as methyl-donor has not been described for this bacterium. After metabolic engineering for enhanced supply of anthranilate by channeling carbon flux into the shikimate pathway, preventing by-product formation and enhancing sugar uptake, heterologous expression of the gene anmt encoding anthranilate N-methyltransferase from Ruta graveolens resulted in production of N-methylanthranilate (NMA), which accumulated in the culture medium. Increased SAM regeneration by coexpression of the homologous adenosylhomocysteinase gene sahH improved N-methylanthranilate production. In a test bioreactor culture, the metabolically engineered C. glutamicum C1* strain produced NMA to a final titer of 0.5 g·L-1 with a volumetric productivity of 0.01 g·L-1·h-1 and a yield of 4.8 mg·g-1 glucose.

Distribution of methyl and isopropyl N-methylanthranilates and their metabolites in organs of rats treated with these two essential-oil constituents.

Two volatile alkaloids, methyl (MMA) and isopropyl N-methylanthranilates (IMA), identified in the essential oil of Choisya ternata Kunth (Rutaceae), have been proven to possess polypharmacological properties (antinociceptive, anti-inflammatory, gastro-, hepato-, nephroprotective activities, anxiolytic and antidepressant properties, and likewise an effect on diazepam-induced sleep). In the continuation of our investigation of their urinary-metabolite profiles, we performed GC-MS analyses of the diethyl-ether extracts of selected tissues (liver, kidneys, heart, brain, lungs, quadriceps femoris muscle, and spleen) of rats intraperitoneally treated with MMA or IMA (2 g kg-1). Organ-metabolite profiles of MMA and IMA were qualitatively mutually analogous (varying only in the alcohol moiety of the metabolites), and generally analogous to their urinary-metabolite profiles. The greatest diversity and the highest overall amount of anthranilate metabolites was found in the hepatic tissue. The principal anthranilate-related compounds in the organs of rats treated with MMA, among 12 detected, were the products of ester hydrolysis, N-methylanthranilic and anthranilic acids. In the tissues of IMA-treated rats, among 16 compounds, the most abundant ones were the unmetabolized IMA and N-methylanthranilic acid. A collection of the compositional data regarding the anthranilate-related metabolites was statistically treated by multivariate statistical analysis that provided a better insight into the possible biotransformation pathways.

Potential of natural repellents methylanthranilate and anthraquinone applied on maize seeds and seedlings against house sparrow (Passer domesticus) in captivity / Potencial de repelentes naturais, metilantranilato e antraquinona aplicados em sementes de milho e mudas contra o pardal (Passer domesticus) em cativeiro

Abstract Various bird pests caused severe economic losses to valuable crops and fruit orchards all over the world. Among the birds, house sparrow is also considered to cause heavy plunder, not only to seeds of crops but also seedlings especially in organic farming. In present study two bird repellents, methylanthranilate and anthraquinone tested against house sparrows on maize seeds and seedlings in aviary conditions. Trial group in aviary-I, the treated maize seeds and seedlings with different doses of both bird repellents, control group in aviary-II, untreated seeds and seedlings were provided for three hours in the early morning. In each aviary, two closed circuit cameras were also installed to monitor the behavioral responses against different concentrations of both chemical repellents. Statistical analysis showed that there existed highly significant (P<0.01) variations among the trial and control groups for seeds and seedlings. By comparing both repellents, significant (P<0.05) differences were detected and anthraquinone showed better efficacy when compared to methylanthranilate, but in maize seedlings both repellents equal repellent properties. Non-significant (P>0.05) differences were observed in different grading of both natural chemical repellents for maize seeds while significant (P<0.05) variations were noticed for maize seedlings when provided to sparrows. By videotaped behavior sparrows presented manifest head juddering and feather upsetting activities by consumption of treated seeds and seedlings with higher concentrations of both natural bird repellents.

Resumo Várias pragas de aves causaram graves perdas econômicas para cultivos valiosos e pomares de frutas em todo o mundo. Entre os pássaros, o pardal da casa também é considerado um grande saqueo, não só para as sementes das culturas, mas também para as mudas, especialmente na agricultura orgânica. No presente estudo, dois repelentes de aves, metilantranilato e antraquinona testados contra pardais de casa em sementes de milho e mudas em condições de aviário. O grupo de ensaio em aviary-I, as sementes de milho tratadas e as mudas com diferentes doses de repelentes de aves, grupo de controle em aviary-II, sementes não tratadas e mudas foram fornecidas por três horas no início da manhã. Em cada aviário, duas câmeras de circuito fechado também foram instaladas para monitorar as respostas comportamentais contra diferentes concentrações de ambos os repelentes químicos. A análise estatística mostrou que existiam variações altamente significativas (P<0,01) entre os grupos de teste e controle para sementes e mudas. Ao comparar os dois repelentes, detectaram-se diferenças significativas (P<0,05) e a antraquinona apresentou maior eficácia quando comparada ao metilantranilato, mas em mudas de milho, ambos os repelentes são iguais às propriedades repelentes. As diferenças não significantes (P>0,05) foram observadas em diferentes classificações de repelentes químicos naturais para sementes de milho, enquanto as variações significativas (P<0,05) foram observadas para as mudas de milho quando fornecidas aos pardais. Por um comportamento gravado em video, os pardais apresentaram manifestações de cabeça e vibrações de penas por consumo de sementes tratadas e mudas com maiores concentrações de repelentes de aves naturais.

Distinct urinary metabolite profiles of two pharmacologically active N-methylanthranilates: Three approaches to xenobiotic metabolite identification.

Two volatile alkaloids, isopropyl N-methylanthranilate (IMA) and methyl N-methylanthranilate (MMA), present in the human diet and cosmetic products, were recently demonstrated to possess important pharmacological activities. While MMA is considered to be phototoxic, there is scarce data on the toxicity of IMA. Herein, we analyzed urinary metabolites of IMA and MMA in rats (200 mg kg-1, i.p., 7 days) by combining three different approaches: 1) preparative chromatography, 2) synthesis, and 3) SPR. The preparative approach, Sephadex LH-20 chromatography of the extract of urine samples of IMA treated animals, in conjunction with NMR, enabled the identification of 16 different anthranilate derivatives, among which products of aromatic core hydroxylation (isopropyl 5-hydroxy-N-methylanthranilate, isopropyl 5-hydroxyantranilate, isopropyl 3-hydroxyantranilate) were the major ones. The first application of the synthetic/combinatorial approach led to a successful identification of MMA metabolites, where 2-(methylamino)benzamide and N-methylanthranilic acid were the principal ones, among 14 others. Generally, MMA and IMA undergo analogous biotransformation pathways; however, MMA predominantly underwent chemical conversions of the ester group, i.e. transformation into derivatives of anthranilamide and anthranilic acid, while the major metabolic pathway of IMA was hydroxylation of the aromatic core. Additionally, pathohistological examinations revealed no signs of liver toxicity, or other signs of toxicity.

Methyl N-methylanthranilate: major compound in the defensive secretion of Typhloiulus orpheus (Diplopoda, Julida).

The defensive secretion of the julid diplopod Typhloiulus orpheus contains methyl N-methylanthranilate (MNMA), an ester of N-methylanthranilic acid that comprises more than 99% of secretion of this species. MNMA is accompanied by small amounts of methyl anthranilate and two benzoquinones (2-methyl-1,4-benzoquinone and 2-ethyl-1,4-benzoquinone, respectively). MNMA is a known intermediate in the biosynthesis of both benzoquinones (as present in defensive secretions of juliformians) and glomerin-like quinazolines (chemical defense in Glomerida). The compound may have evolved independently in the pathway to glomeridan chemistry, or may even represent a pivotal branching point in the pathway to different chemical classes of diplopod defensive chemistry.

Potential of natural repellents methylanthranilate and anthraquinone applied on maize seeds and seedlings against house sparrow (Passer domesticus) in captivity

Abstract Various bird pests caused severe economic losses to valuable crops and fruit orchards all over the world. Among the birds, house sparrow is also considered to cause heavy plunder, not only to seeds of crops but also seedlings especially in organic farming. In present study two bird repellents, methylanthranilate and anthraquinone tested against house sparrows on maize seeds and seedlings in aviary conditions. Trial group in aviary-I, the treated maize seeds and seedlings with different doses of both bird repellents, control group in aviary-II, untreated seeds and seedlings were provided for three hours in the early morning. In each aviary, two closed circuit cameras were also installed to monitor the behavioral responses against different concentrations of both chemical repellents. Statistical analysis showed that there existed highly significant (P 0.01) variations among the trial and control groups for seeds and seedlings. By comparing both repellents, significant (P 0.05) differences were detected and anthraquinone showed better efficacy when compared to methylanthranilate, but in maize seedlings both repellents equal repellent properties. Non-significant (P>0.05) differences were observed in different grading of both natural chemical repellents for maize seeds while significant (P 0.05) variations were noticed for maize seedlings when provided to sparrows. By videotaped behavior sparrows presented manifest head juddering and feather upsetting activities by consumption of treated seeds and seedlings with higher concentrations of both natural bird repellents.

Resumo Várias pragas de aves causaram graves perdas econômicas para cultivos valiosos e pomares de frutas em todo o mundo. Entre os pássaros, o pardal da casa também é considerado um grande saqueo, não só para as sementes das culturas, mas também para as mudas, especialmente na agricultura orgânica. No presente estudo, dois repelentes de aves, metilantranilato e antraquinona testados contra pardais de casa em sementes de milho e mudas em condições de aviário. O grupo de ensaio em aviary-I, as sementes de milho tratadas e as mudas com diferentes doses de repelentes de aves, grupo de controle em aviary-II, sementes não tratadas e mudas foram fornecidas por três horas no início da manhã. Em cada aviário, duas câmeras de circuito fechado também foram instaladas para monitorar as respostas comportamentais contra diferentes concentrações de ambos os repelentes químicos. A análise estatística mostrou que existiam variações altamente significativas (P 0,01) entre os grupos de teste e controle para sementes e mudas. Ao comparar os dois repelentes, detectaram-se diferenças significativas (P 0,05) e a antraquinona apresentou maior eficácia quando comparada ao metilantranilato, mas em mudas de milho, ambos os repelentes são iguais às propriedades repelentes. As diferenças não significantes (P>0,05) foram observadas em diferentes classificações de repelentes químicos naturais para sementes de milho, enquanto as variações significativas (P 0,05) foram observadas para as mudas de milho quando fornecidas aos pardais. Por um comportamento gravado em video, os pardais apresentaram manifestações de cabeça e vibrações de penas por consumo de sementes tratadas e mudas com maiores concentrações de repelentes de aves naturais.

Effect of two esters of N-methylanthranilic acid from Rutaceae species on impaired kidney morphology and function in rats caused by CCl4.

AIMS: Herein we investigated the potential protective effects of methyl N-methylanthranilate (MA) and isopropyl N-methylanthranilate (IA), two naturally occurring plant constituents from Rutaceae taxa, in a rat model of acute intoxication with carbon tetrachloride (CCl4) by tracking changes in kidney tissue morphology and function. MAIN METHODS: The antioxidant capacity of IA and MA was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid radical cation (ABTS(+)) assays and superoxide-scavenging test. Wistar rats were treated daily with MA and IA for seven days in a dose of 200mg/kg. Twenty-four hours after a CCl4 injection, rats were sacrificed and blood samples were used for the evaluation of urea and creatinine. Kidney tissue specimens were stained with hematoxylin and eosin, periodic acid-Schiff and Jones stain and evaluated for morphological changes. Quantification of structural changes determined by histological analysis of kidney tissue was assessed by a morphometric analysis of glomeruli using ImageJ software. KEY FINDINGS: IA and MA applied in high doses on their own did not cause any significant damage to kidney tissue. A pretreatment with MA prior to the administration of CCl4 significantly prevented the increase of serum levels of decreased kidney function markers, while that of IA did not. Histopathological evaluation of the kidneys also revealed that MA reduced the incidence of kidney lesions. SIGNIFICANCE: Our experiments showed that methyl-, and not isopropyl-, N-methylanthranilate possesses a protective potential against CCl4-induced kidney damage in rats. The results are of interest due to the presence of natural or synthetic methyl N-methylanthranilate in the human diet and their potent analgesic properties.

Methyl and isopropyl N-methylanthranilates attenuate diclofenac- and ethanol-induced gastric lesions in rats.

AIMS: Two natural alkaloids, methyl (M) and isopropyl (I) N-methylanthranilates, with recently demonstrated significant pharmacological activities, were assayed for their possible overall effect on intact gastric mucosa and their protective properties towards the onset of gastric lesions induced by diclofenac (a non-steroidal anti-inflammatory drug, NSAID) or ethanol. MAIN METHODS: The influence of I and M on gastric mucosa integrity was assessed by oral administration in doses of 200mg/kg. The gastroprotective action of I and M in doses of 50, 100 and 200mg/kg was analyzed in the diclofenac and ethanol-induced gastric lesion models in rats. After the treatment, the stomachs of the animals were analyzed (captured by a digital camera). Ulcer scoring, morphometric and histopathological analyses of the stomachs were done. KEY FINDINGS: The oral application of these compounds on their own, even in quite high doses (200mg/kg) did not induce gastric lesions. Both alkaloids exerted a very strong antiulcer activity, even in low doses (50mg/kg), by decreasing the number of lesions caused by the application of either diclofenac or ethanol, eliminating them completely or reducing them to a form of mucosal hyperemia. SIGNIFICANCE: Their possible mechanism of action was discussed and due to their many positive properties including anxiolytic, antidepressant, antinociceptive, anti-inflammatory and gastroprotective activities, as well as a cheap and simple synthetic route for their preparation, methyl and isopropyl N-methylanthranilates, both alike, might represent a cost effective alternative sought for in the treatment of peptic ulcers and/or new safer NSAIDs for pain management.