Mentha villosa Hubs., M. x piperita and their bioactives against gastrointestinal nematodes of ruminants and the potential as drug enhancers.

Parasitic diseases caused by gastrointestinal nematodes (GIN) are responsible for a major impact on ruminant welfare. Although the available anthelmintics have a safe margin of toxicity to the animals, their indiscriminate use has increased the selection of resistant parasite populations. In this scenario, essential oils (EO) stand out as a promising ecofriendly therapeutic alternative against GIN. The objective of this work was to determine the effect of the EO of Mentha villosa Hubs (MVEO) collected in 2017 and 2018, M. x piperita (MPEO) and their main components, carvone and limonene, against the third stage larvae (L3) of Haemonchus spp. and Trichostrongylus spp. The solutions, including in nanoemulsion preparations, were tested in a range of concentrations using the larval migration inhibition test (LMIT). The EO and carvone were also tested in combination with nitroxynil (NTX) to determine their effect as drug enhancers (additive or synergy). MVEO/2017, MVEO/2018, MPEO and carvone showed 70.6 (73.4 mg/mL), 86.3 (74.9 mL/mL), 95.5 (143.6 mg/mL), and 88.2 % (38.3 mg/mL) efficacy against L3, respectively. Carvone alone had approximately a 3-fold higher efficacy when compared to its concentration in each EO: 68.8 % in MVEO/2017 and 83.9 % in MVEO/2018. Limonene did not show any significant effect on inhibiting L3 migration. The combination of MPEO and NTX, and carvone and NTX showed a statistically significantly (P <  0.05) synergic and additive effect, respectively, when compared to the isolated treatment. The nanoemulsion of MVEO/2017 at 0.367 mg/mL, inhibited L3 migration by 83.1 %, demonstrating to be highly effective (concentration ratio of 1:0.004), when compared to the MVEO/2017 (70.6 % at 73.4 mg/mL) extraction. The in vitro data from the combination of MPEO or carvone plus NTX suggest that these products can be considered for in vivo experiments against the most important GIN of ruminants as drug enhancers, possibly reducing the final concentration of NTX`. The efficacy of carvone was higher (EC50 = 1.96 mg/mL) than its expected efficacy, based on its concentrations on both EO. Therefore, this component does not need the entire EO composition to exert its L3 motility action. The remarkable efficacy demonstrated by the MVEO/2017/nanoemulsion (EC50 = 0.10 mg/mL), supports its potential to be a candidate to the next-generation therapy to alleviate clinical parasite infections and combat GIN resistant populations.

Protective Effect of D-Carvone against Dextran Sulfate Sodium Induced Ulcerative Colitis in Balb/c Mice and LPS Induced RAW Cells via the Inhibition of COX-2 and TNF-α.

Ulcerative colitis (UC) is an intractable ailment, in which may chronic inflammations/ulcerations may develop in the mucosal lining of the colon with multiple recurrences. Various drugs such as steroids, immunosuppressants, and antibiotics are extensively used to treat UC. The patients suffer from adverse effects of these advanced drugs. So, they need a harmless therapeutic agent from natural sources. The therapeutic D-carvone has an anti-inflammatory action against the investigational colon cancer models. Therefore, we analyzed the effect of D-carvone on dextran sulfate sodium (DSS) provoked colitis model in mice as follows: Group I: noncolitis healthy control mice; Group II: ulcerative colitis mice models; Group III: D-carvone (40 mg/kg) + ulcerative colitis models; Group IV: sulfasalazine (50 mg/kg) + ulcerative colitis models. On the 8th day, the experimental study was terminated and serum samples and colon tissues were processed for further analysis. The effect of D-carvone at different concentration was studied on the LPS challenged RAW 264.7 cell lines. The D-carvone (40 mg/kg) treatment maintained the colon length and decreased disease activity index (DAI) score in UC animals. The increased antioxidant enzymes status and decreased oxidative stress and pro-inflammatory markers were noted in the D-carvone (40 mg/ kg) + UC mice. Histopathological study of colon tissue of D-carvone (40 mg/kg) treated UC mice displayed less mucosal damage and improved crypt integrity and goblet cells compared with DSS only provoked mice. The im-munohistochemical expression of iNOS and COX-2 was drastically diminished in the D-carvone treated UC mice. D-carvone (40 mg/kg) treatment appreciably diminished the LPS provoked NO production and pro-inflammatory modulators in the RAW 264.7 macrophage cell lines. These findings proved that D-carvone has a potential therapeutic effect to prevent LPS induced inflammation in in vitro cells and chemically induced ulcerative colitis in vivo models.

Inhalation Administration of the Bicyclic Ethers 1,8- and 1,4-cineole Prevent Anxiety and Depressive-Like Behaviours in Mice.

The anxiolytic and antidepressant-like activities of the naturally occurring monoterpene 1,8-cineole and its structural isomer 1,4-cineole were evaluated in mice via inhalation administration at doses ranging from 4 × 10-6 to 4 × 10-1 mg per 400 µL of triethyl citrate. Mice were tested for anxiety-like behaviours by using the light-dark box test (LDB) and marble-burying test (MBT) and for depression-like symptoms by using the forced swimming test (FST) and tail suspension test (TST). Diazepam and fluoxetine were used as standard drugs for anxiolytic and antidepressant tests, respectively. The results showed that 1,8-cineole at 4 × 10-4 mg, and 1,4-cineole at 4 × 10-4 and 4 × 10-3 mg significantly increased the amount of time spent in the light box and the number of entries in the light box in the LDB as well as reduced the number of marbles buried in the MBT relative to those in the control, suggesting an anxiolytic effect. Similarly, 1,8-cineole at 4 × 10-4 and 4 × 10-2 mg and 1,4-cineole at doses of 4 × 10-4 to 4 × 10-2 mg significantly reduced immobility times in the FST and TST relative to those of the control, suggesting an antidepressant activity. The role of the GABAA/benzodiazepine receptor system in the anxiolytic effects of 1,8- and 1,4-cineole was investigated through co-administration of flumazenil, a GABAergic system antagonist. Flumazenil reversed the effects of diazepam and 1,8-cineole, suggesting that 1,8-cineole affects the GABAA/benzodiazepine receptors. Collectively, the results suggest that inhaled 1,8- and 1,4-cineole prevented anxiety and depressive-like symptoms in classic mice models.

Trachyspermum ammi (L.) Sprague, superb essential oil and its major components on peptic ulcers: in vivo combined in silico studies.

BACKGROUND: Trachyspermum ammi (L.) Sprague is used for treating gastrointestinal disorders. Several studies indicated gastric antiulcer activity of T. ammi extract, yet the effect of its essential oil has not been studied on. OBJECTIVES: The present study evaluates chemical composition of T. ammi essential oil and anti-peptic ulcer effect of the essential oil as well as its three major components in ethanol induced-gastric ulcers in rats. METHODS: Primarily chemical composition of the essential oil was analyzed by gas chromatography-mass spectrometry (GC/MS). Rats received the essential oil (500, 250, 125, 62.5, 31.25 mg/kg), thymol (30, 100 mg/kg), para-cymene (100, 150 mg/kg) and gamma-terpinene (100, 150 mg/kg) using gavage tube along with ethanol 80%. Finally, dissected stomachs were assessed both macroscopically and microscopically to evaluate anti-ulcerative effect of the essential oil and the pure compounds. Moreover, molecular docking was utilized to explore the interactive behavior of the main components with active site residues of H+/K+ ATPase. RESULTS: Analysis of the essential oil indicated that para-cymene (37.18%), gamma-terpinene (35.36%) and thymol (20.51%) are the main components. Administration of different doses of the essential oil noticeably diminished the number of peptic ulcers in a dose-dependent manner. Among the main components, thymol was more potent than para-cymene and gamma-terpinene. Administration of the essential oil (500 mg/kg) and thymol (100 mg/kg) observed maximum inhibition percentage (98.58% and 79.37%, respectively). Molecular docking study provides the evidence of thymol ability to inhibit H+/K+ ATPase. CONCLUSIONS: The findings revealed that T. ammi essential oil can be applied to treat gastric ulcer as a natural agent. Graphical abstract.

Understanding the skin sensitization capacity of ascaridole: a combined study of chemical reactivity and activation of the innate immune system (dendritic cells) in the epidermal environment.

To improve the prediction of the possible allergenicity of chemicals in contact with the skin, investigations of upstream events are required to better understand the molecular mechanisms involved in the initiation of allergic reactions. Ascaridole, one of the compounds responsible for skin sensitization to aged tea tree oil, degrades into intermediates that evolve via different mechanisms involving radical species. We aimed at broadening the knowledge about the contribution of radical intermediates derived from ascaridole to the skin sensitization process by assessing the reactivity profile towards amino acids, identifying whether free radicals are formed in a reconstructed human epidermis (RHE) model and their biological properties to activate the immune system, namely dendritic cells in their natural context of human HaCaT keratinocytes and RHE. Electron paramagnetic resonance combined to spin-trapping in EpiSkin™ RHE confirmed the formation of C-radicals in the epidermal tissue from 10 mM ascaridole concentration, while reactivity studies toward amino acids showed electrophilic intermediates issued from radical rearrangements of ascaridole as the main reactive species. Activation of THP-1 cells, as surrogate for dendritic cells, that were cocultured with HaCaT was significantly upregulated after treatment with low micromolar concentrations based on cell surface expression of the co-stimulatory molecule CD86 and the adhesion molecule CD54. Placing THP-1 cells underneath the RHE allowed us to monitor which of the concentrations that produce radical(s) and/or protein antigens in the epidermal skin environment promote the activation of dendritic cells. We detected no significant upregulation of CD86/CD54 after topical RHE application of concentrations up to 30 mM ascaridole (t = 24 h) but clear upregulation after 60 mM.