Ana B. Miltojević

Effects of Methyl and Isopropyl N-methylanthranilates from Choisya ternata Kunth (Rutaceae) on Experimental Anxiety and Depression in Mice

Choisya ternata Kunth (Rutaceae) is a plant species used in Mexican folk medicine for its antispasmodic and simulative properties. Recently, we identified a new alkaloid, isopropyl N‐methylanthranilate, and a related one, methyl N‐methylanthranilate, from the essential oil of this species and have proven them to possess antinociceptive activity even at 0.3 mg/kg. In the present study, anxiolytic and antidepressant effects of the two compounds have been studied in open field, horizontal wire, light/dark, forced swimming and tail suspension tests, as well as the effect on the onset and duration of diazepam‐induced sleep in BALB/c mice. The volatile alkaloids (50–200 mg/kg, administered intraperitoneally), without having a muscle relaxant effect, caused a significant increase in the time the animals spent in an unsecured and putatively dangerous area when compared with the control group but had no effect on the number of crossings between the light/dark compartments. In addition to this anxiolytic activity, a significantly antidepressant‐like effect was apparent at all tested doses, which was not due to an increase in locomotive activity. The anthranilates administered on their own did not induce sleep in mice but significantly prolonged the diazepam‐induced sleep, in a dose‐dependent way, suggesting an interaction with the gamma‐aminobutyric acid receptor complex. Copyright

New volatile sulfur-containing compounds from wild garlic (Allium ursinum L., Liliaceae)

In many countries, the leaves of Allium ursinum L. (Liliaceae) are a popular substitute for garlic and, for centuries, the herb has been taken internally to treat an array of medical conditions. Herein, we report the chemical composition of 12 different A. ursinum essential-oil samples (five populations; fresh/air-/oven-dried plant material; leaves/inflorescences). GC-MS/GC-FID analyses, quantitative structure-property relationship modeling (simple 0D/1D-descriptors) of retention indices and the synthesis of selected compounds, enabled the identification of >200 different constituents, mainly organo(poly)sulfides. Some of these were new compounds (allyl (methylthio)methyl, (methylthio)methyl (Z)/(E)-1-propenyl and allyl 1-(methylthio)propyl disulfides) or were previously not detected in samples of natural origin (heptyl methyl, methyl octyl, allyl hexyl (1), allyl octyl (2) and propyl (propylthio)methyl sulfides). A multivariate statistical analysis revealed the onset of significant changes in the plant material volatile profile during the drying process.

Antinociceptive esters of N-methylanthranilic acid: Mechanism of action in heat-mediated pain.

Recently, we identified a new natural antinociceptive alkaloid ternanthranin, isopropyl N-methylanthranilate (ISOAN), from the plant species Choisya ternata Kunth (Rutaceae). In this work we concentrated on the elucidation of its mechanism of action in comparison with two other esters of this acid (methyl (MAN) and propyl (PAN)). Mice orally pre-treated with ISOAN, MAN or PAN (at 0.3, 1 and 3mg/kg) were less sensitive to chemical or thermal stimuli in different nociception models (formalin-, capsaicin- and glutamate-induced licking response, tail flick and hot plate). All compounds (1 and 3mg/kg) showed significant activity in the peripheral nociception models, as well as a dose-dependent spinal antinociceptive effect in the tail flick model. We observed that glibenclamide was able to reverse the antinociceptive effect of ISOAN in the hot plate model suggesting the involvement of K(+)ATP channels. The antinociceptive effect of MAN and PAN may be related to adrenergic, nitrergic and serotoninergic pathways. In addition, the antinociception of PAN was reverted by naloxone implying that the opioid pathway participates in its activity. The cholinergic and cannabinoid systems were found not be involved in the onset of the antinociceptive effects of any of the esters. In conclusion, isopropyl, methyl and propyl N-methylanthranilates produced significant peripheral and central antinociception at doses lower than that of morphine, the classical opioid analgesic drug, without causing toxicity.

Anti-Inflammatory Activity of Choisya ternata Kunth Essential Oil, Ternanthranin, and Its Two Synthetic Analogs (Methyl and Propyl N-Methylanthranilates)

Choisya ternata Kunth (Rutaceae) is native to North America where it is popularly known as “Mexican orange”. In this study, the anti-inflammatory effects of the essential oil (EO) obtained from the leaves of C. ternata, one of its minor components (ternanthranin—ISOAN) and its two synthetic analogues (methyl and propyl N-methylanthranilate – MAN and PAN) were evaluated. Mice pretreated with the EO (EO) obtained from C. ternata leaves (3–100 mg/kg, p.o.), ISOAN, MAN or PAN (1–30 mg/kg, p.o.) and the reference drugs, morphine (1 mg/kg, p.o.) and acetylsalicylic acid (ASA, 100 mg/kg, p.o.), were evaluated in inflammation models such as formalin and subcutaneous air pouch models, with measurement of cell migration, exudate volume, protein extravasation, nitric oxide and pro-inflammatory cytokines. The EO from C. ternata significantly inhibited the time that the animals spent licking the formalin-injected paw in the second phase of the model at their higher doses (30 and 100 mg/kg, respectively). An inhibition of the inflammatory reaction induced after subcutaneous carrageenan injection into air pouch was also observed. In this model, the EO significantly reduced cell migration, exudate volume, protein extravased, and the increase in levels of inflammatory mediators (nitric oxide, TNF-α and IL-1β). ISOAN, MAN and PAN behaved in the same fashion at much smaller doses. Also, these molecules were able to show significant effects in the reduction of paw edema (at all tested doses) when the phlogistic agent was carrageenan, bradykinin, 5-HT, PGE2, C48/80 or 12-O-tetradecanoylphorbol-acetate (TPA). None of the tested doses had any effect in reducing histamine-induced edema. Our results indicate that the EO from C. ternata and anthranilate derivatives demonstrates an anti-inflammatory effect.

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.

α‐Linalool – a marker compound of forged/synthetic sweet basil (Ocimum basilicum L.) essential oils

BACKGROUND Ocimum basilicum L. (sweet basil) is known to occur as several chemotypes or cultivars that differ in their essential oil composition. The surprising discovery of 3,7-dimethylocta-1,7-dien-3-ol, the rare α isomer of the well-known monoterpene alcohol β-linalool (3,7-dimethylocta-1,6-dien-3-ol), in samples of Serbian basil oil provoked an investigation of the origin of α-linalool in these samples. Three scenarios were considered, namely (a) the existence of a new natural chemotype, (b) an artefactual formation during the isolation procedure and (c) the case of a synthetic/forged oil. RESULTS Noteworthy amounts (15.1-16.9%) of pure α-linalool were isolated from a commercial sample of basil oil, and detailed spectral analyses (MS, IR, (1) H and (13) C NMR) unequivocally confirmed its identity. The analysis by GC and GC/MS of an additional 20 samples of different O. basilicum oils commercially available on the Serbian market or isolated from plant material cultivated in Serbia resulted in the identification of 149 compounds. The obtained compositional data were compared using multivariate statistical analysis to reveal the possible existence of a new basil chemotype. CONCLUSION The results of the chemical and statistical analyses give more pro arguments for the synthetic/forged oil hypothesis and suggest that α-linalool could be used as a marker compound of such O. basilicum oils.

Structural elucidation of thermolysis products of methyl N-methyl-N-nitrosoanthranilate

Although it is common knowledge that N-nitroso compounds are thermally (and otherwise chemically) labile, little or nothing is known about the specific reactions that occur during thermal treatment of a compound possessing this functionality. Methyl N-methyl-N-nitrosoanthranilate was found to undergo complete thermal degradation under gas chromatographic (GC–MS) conditions yielding a major unidentified coupling product and methyl N-methylanthranilate. In an attempt to elucidate the structure of the formed degradation product, a preparative scale thermolysis in an evacuated vessel at 220 °C of the nitroso compound was carried out. A chromatographic separation of the thermolysate, followed by GC–MS and NMR (and other spectral techniques) analyses enabled the identification of in total 46 different products. Among them a novel coupling product, methyl 5-methyl-6-oxo-(5H)-phenanthridine-4-carboxylate, was identified and fully spectrally characterized. Interestingly, the initially detected coupling product that formed under GC conditions, tentatively identified as dimethyl 2,2′-(1,2-dimethylhydrazine-1,2-diyl)dibenzoate, was not detected in the thermolysate. A careful consideration of the structures of the identified thermolysate constituents led us to propose major thermolysis pathways of methyl N-methyl-N-nitrosoanthranilate both in condensed and vapor phases. Generally, the identified products could be classified as those arising from fission of the N–NO bond or rather unexpectedly, the Ar–NNO bond, i.e. products related to anthranilic and benzoic acids, respectively. The latter represents a novel chemical transformation up to now unreported and unutilized. The structural diversity of the identified products and the noted marked differences between vapor and condensed phases point to the possible synthetic utility of thermolysis of carefully designed N-nitroso compounds.