Antinociceptive activity of the ethanolic extract of Trixis angustifolia DC

Abstract The antinociceptive activity of the ethanolic extract of Trixis angustifolia DC. (EETx) was investigated using the acetic acid-induced writhing and the hot-plate tests in mice. In the acetic acid-induced writhing test, mice treated with EETx (50, 100 and 200 mg/kg, p.o.) exhibited reduced writhing (38%, 67%, and 74%, respectively). In the hot-plate test, the three doses administrated increased the nociceptive response time. The phytochemical analysis of EETx led to the isolation of three known compounds, hygric acid (1), 5,6-Dihydroxy-7,8,4'-trimethoxyflavone (2) and xanthomicrol (3). Compound 1 was identified for the first time in this species. These results demonstrate that T. angustifolia has potential central and peripheral antinociceptive effects and support the ethnomedicinal use of this plant. Graphical Abstract


Introduction
Trixis angustifolia DC. (Asteraceae) is an endemic plant of Mexico widely distributed in the central and the north part of the country (Villaseñor 2016). This plant shares morphological characteristics with other plants commonly known as " arnica" or " arnica del pa ıs", which are used as analgesic and anti-inflammatory remedies (Waizel-Bucay and Cruz-Ju arez 2014). Consequently, the plant has several established uses in Traditional Mexican medicine. However, only the antibacterial, hypoglycemic and hypolipidemic effects have been demonstrated (S anchez-Ch avez et al. 2019;Salazar-G omez et al. 2019aSalazar-G omez et al. , 2019b and no scientific reports demonstrating its antinociceptive activity have been published so far. A previous phytochemical study of the aerial parts of T. angustifolia resulted in the isolation and characterization of six flavones: pebrellin, salvigenin, gardenin B, xanthomicrol, cirsimaritin, and 3 0 -demethoxysudachitin (S anchez-Ch avez et al. 2019), some of which have been shown to have anti-inflammatory activity (Shin et al. 2017;Guo et al. 2018). Therefore, the present study was conducted to investigate the antinociceptive potential of the ethanolic extract of T. angustifolia (EETx) and to identify the main components present in the extract.

Results and discussion
The oral administration of EETx at 1000 and 2000 mg/kg did not cause any sings of toxicity or mortality during the 14-day acute toxicity test. In addition, no significant pathological changes in vital organs were observed by macroscopic examination. Therefore, the EETx is well-tolerated with LD 50 > 2000 mg/kg, indicating that it is reasonably safe in vivo (OECD 2001).
The antinociceptive effect of the EETx was evaluated using two classical experimental pain models: acetic acid-induced writhing and hot-plate tests. The intraperitoneal injection of acetic acid induces the release of endogenous mediators that stimulate pain nerve endings, causing abdominal constriction response in mice (Ikeda et al. 2001). Treatment with EETx at the doses of 50, 100, and 200 mg/kg reduced mean writhing by 38%, 67%, and 74%, respectively, compared with the sodium bicarbonate (vehicle) control group ( Figure S1, supplementary material). The EETx at 200 mg/kg had a significant protective effect similar to the NSAID indomethacin at 10 mg/kg, which produced 77% decreases in the number of writhing episodes. These results suggest that the ability of EETx to attenuate nociception in the abdominal constriction could derive from the suppression of the synthesis and/or release of prostaglandins by the COX pathway (Satyanarayana et al. 2004) or from inhibition of the production of inflammatory mediators (e.g., TNF-a, IL-1b, and IL-8) released into the peritoneal cavity (Ribeiro et al. 2000).
The hot plate test was used to test the central antinociceptive potential by measuring the nociceptive response time (RT) and percent maximal possible effect (%MPE). All three doses of EETx increased the RT and %MPE in the 2nd and 3rd h compared to the sodium bicarbonate (vehicle) control group (Table S1, supplementary material). The treatment with EETx at 200 mg/kg increased the RT in the 3rd h, similar to acetylsalicylic acid (100 mg/kg; used as a positive control). These results suggest that compounds present in EETx might also have centrally mediated activity.
The phytochemical analysis of EETx led to the isolation and identification of three known compounds: a proline derivative and two O-methylated flavonoids. Compound 1 was isolated as fine needles. Based on ESI (À) mass spectrometry, a molecular ion at m/z 128.0719 was identified in agreement with the molecular formula C 6 H 11 NO 2 ( Figure S2, supplementary material). The analysis of 1 H NMR spectrum revealed proline ring [d 3.81-3.75 (1 m, 1H), 3.74-3.67 and 3.14-3.05 (2 m, 2H), 2.51-2.42 and 2.15-2.06 (2 m, 2H), 2.15-2.06 and 2.01-1.92 (2 m, 2H)] and a singlet signal at d 2.91 for three hydrogens of N-methyl group ( Figure S3, supplementary material). 1 H and 13 C NMR ( Figure S4, supplementary material) assignments were carried out with the aid of the detailed 2 D analyses (gHSQC) ( Figure S5, supplementary material) and the resulting NMR evidence revealed a compound defined as hygric acid (Aurelio et al. 2003). To the best of our knowledge, the presence of 1 has never been reported before in plants from the Trixis genus. Additionally, two previously reported compounds, 5,6-Dihydroxy-7,8,4 0 -trimethoxyflavone (2) and xanthomicrol (3) were also identified in the EETx by comparisons of their NMR spectroscopy with those reported earlier (S anchez-Ch avez et al. 2019). Previous studies confirmed the association of opioid system in the antinociceptive effect of some O-methylated flavonoids (Shajib et al. 2018) and natural proline derivatives such as N-methyl-(2S,4R)-trans-4-hydroxy-l-proline have been found to possess anti-inflammatory properties (de Aquino et al. 2017). Although there is no possibility at this stage to identify the exact constituent(s) responsible for the antinociceptive activity of T. angustifolia, compounds 1, 2, and 3 presented in the EETx could contribute to the biological effect observed in the present study. Thus, further studies are necessary to validate this hypothesis and identify the mechanism of action involved in the antinociceptive activity of EETx.