Deep eutectic solvents (DESs) were investigating as extracting solvent for headspace single drop microextraction (HS-SDME). The extraction efficiency of ten DESs mainly composed of tetrabutylammonium bromide (N 4444 Br) and long chain alcohols was evaluated for the extraction of terpenes from six spices (cinnamon, cumin, fennel, clove, thyme and nutmeg). The DES composed of N 4444 Br and dodecanol at a molar ratio of 1:2 showed the highest extraction efficiency and was selected to conduct the extractions of terpenes in the rest of the study. HS-SDME was optimized by design of experiments. Only two parameters from the four studied showed a significant influence on the efficiency of the method: the extraction time and the extraction temperature. The optimal extraction conditions were determined by response surface methodology. All extracts were analyzed by gas chromatography coupled to mass spectrometry (GC-MS). More than 40 terpenes were extracted and identified in nutmeg, the richest extract in terpenes in this study. Quantitative analysis based on 29 standards was conducted for each extract. Good linearity was obtained for all standards (R 2 >0.99) in the interval of 1 to 500 µg/g. Limits of quantification ranged from 0.47 µg/g (borneol) to 86.40 µg/g (α-farnesene) with more than half of the values under 2 µg/g. HS-SDME is simple, rapid and cheap compared to conventional extraction methods. The use of DESs makes this extraction method "greener" and it was shown that DESs can be suitable solvents for the extraction of bioactive compounds from plants.

Context: Menthol, the main monoterpene found in Mentha piperita L. (M. piperita) is known to modulate nociceptive threshold and is present in different curative preparations that reduce sensory hypersensitivities in pain conditions. While for pulegone, a menthol-like monoterpene, only a limited number of studies focus on its putative analgesic effects, pulegone is the most abundant monoterpene present in Calamintha nepeta (L.) Savi (C. nepeta), a plant of the Lamiaceae family used in traditional medicine to alleviate rheumatic disorders, which counts amongst chronic inflammatory diseases.Objectives: Here, we analyzed the monoterpenes composition of C. nepeta and M. piperita. We then compared the putative anti-hyperalgesic effects of the main monoterpenes found, menthol and pulegone, in acute inflammatory pain conditions.Methods:C. nepeta and M. piperita extracts were obtained through pressurized liquid extraction and analyzed by gas chromatography-mass spectrometry. The in vitro anti-inflammatory activity of menthol or pulegone was evaluated by measuring the secretion of the tumour necrosis factor alpha (TNF α) from LPS-stimulated THP-1 cells. The in vivo anti-hyperalgesic effects of menthol and pulegone were tested on a rat inflammatory pain model.Results: Pulegone and menthol are the most abundant monoterpene found in C. nepeta (49.41%) and M. piperita (42.85%) extracts, respectively. In vitro, both pulegone and menthol act as strong anti-inflammatory molecules, with EC50 values of 1.2 ± 0.2 and 1.5 ± 0.1 mM, respectively, and exert cytotoxicity with EC50 values of 6.6 ± 0.3 and 3.5 ± 0.2 mM, respectively. In vivo, 100 mg/kg pulegone exerts a transient anti-hyperalgesic effect on both mechanical (pulegone: 274.25 ± 68.89 g, n = 8; vehicle: 160.88 ± 35.17 g, n = 8, p < 0.0001), thermal heat (pulegone: 4.09 ± 0.62 s, n = 8; vehicle: 2.25 ± 0.34 s, n = 8, p < 0.0001), and cold (pulegone: 2.25 ± 1.28 score, n = 8; vehicle: 4.75 ± 1.04 score, n = 8, p = 0.0003). In a similar way, 100 mg/kg menthol exerts a transient anti-hyperalgesic effect on both mechanical (mechanical: menthol: 281.63 ± 45.52 g, n = 8; vehicle: 166.25 ± 35.4 g, n = 8, p < 0.0001) and thermal heat (menthol: 3.65 ± 0.88 s, n = 8; vehicle: 2.19 ± 0.26 s, n = 8, <0.0001).Conclusion: Here, we show that both pulegone and menthol are anti-inflammatory and anti-hyperalgesic monoterpenes. These results might open the path towards new compound mixes to alleviate the pain sensation.

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