BCTC

Mentha piperita L. (Lamiaceae), commonly known as peppermint, has a long history in traditional medicine. Its diverse essential oil composition, with menthol as a key bioactive component, supports a range of medicinal applications, including pruritus relief. This suggests a potential role in modulating itch via neuropharmacological mechanisms.

This study aimed to investigate the anti-pruritic mechanisms of menthol by assessing its effects on scratching behavior and spinal microglial activation in a mouse model of pruritus.

Male ICR mice were used for the compound 48/80 and GRP_18-27-induced scratching models (n = 5-17/group, N = 179 total). C57BL/6 wild-type (n = 5/group, N = 10 total) and Trpm8^-/- mice (n = 7/group, N = 14 total) were used to confirm TRPM8 involvement. Mice received a single topical application of 8 % menthol to the forepaws for 10 s. Pruritus was induced either by subcutaneous (s.c.) injection of compound 48/80 (5 mg/kg in ICR mice or 20 mg/kg in C57BL/6 and Trpm8^-/- mice) or by GRP_18-27 (3.5 mg/kg) into the nape of the neck. Scratching behavior was recorded for 30 min (compound 48/80) or 60 min (GRP_18-27). Minocycline, N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide (BCTC), U50488 and nor-binaltorphimine (nor-BNI) were administered intraperitoneally (i.p.) to elucidate underlying mechanisms. Spinal microglial activation was assessed by Iba1 immunohistochemistry and by Iba1/phospho-p38 co-labeling with confocal microscopy. Western blotting quantified CD11b in the spinal cord.

Scratching behavior was markedly elevated in mice treated with compound 48/80. Topical application of 8 % menthol significantly reduced scratching by approximately 63.02 % compared with compound 48/80 alone (p < 0.001). This antipruritic effect was blocked by BCTC (a TRPV1/TRPM8 antagonist, p < 0.001), and menthol had no effect in Trpm8^-/- mice, strongly suggesting a TRPM8-dependent mechanism. The decrease in scratching behavior by 8 % menthol was not significantly affected by nor-BNI, a κ-opioid receptor antagonist. Minocycline (a microglial inhibitor) pretreatment also reduced scratching (by approximately 24.71 %, p < 0.05). Histological analysis revealed that compound 48/80 induced marked microglial activation (characterized by enlarged cell bodies and thickened processes) attenuated by menthol. Confocal imaging showed increased phospho-p38/Iba1 co-expression after compound 48/80 treatment, which was also reduced by topical menthol application. Western blot showed the upregulation of CD11b in compound 48/80-treated mice, and this upregulation was decreased by topical menthol application. In addition, topical 8 % menthol also significantly reduced GRP_18-27-induced scratching (by approximately 60.94 %, p < 0.05).

Topical menthol application effectively reduces pruritus in a mouse model via a TRPM8-dependent mechanism that inhibits spinal microglial activation. These findings support the traditional use of peppermint for itch relief and highlight TRPM8 and microglia as therapeutic targets for pruritus management.

Diosmin, a flavonoid glycoside with reported anti‐inflammatory effects, was evaluated for its potential analgesic activity using in vivo and in silico studies.

The antinociceptive effect of diosmin was evaluated in mice using capsaicin‐ and formalin‐induced paw‐licking tests as well as hot plate and tail flick assays. Target prediction using Similarity Ensemble Approach (SEA) and molecular docking were conducted to validate diosmin's interaction with Transient Receptor Potential Vanilloid 1 (TRPV1) and acetylcholinesterase (AChE) in silico.

Diosmin (60 and 30 mg/kg) significantly reduced pain behaviour by 90.8% and 83.2%, respectively, in the capsaicin‐induced paw‐licking test, while BCTC, a TRPV1 antagonist, significantly blocked diosmin action. In the formalin test, atropine significantly blocked the action of diosmin at the early phase. Diosmin at 60 mg/kg increased hot plate latency by 104.8%. In the tail flick test, diosmin at 60 and 30 mg/kg increased latency by 103.7% and 90.6%, respectively, indicating central analgesic effects. No significant alterations in body temperature were observed. Docking studies confirmed diosmin's favourable binding to the active site of AChE with a binding energy of −9.77 kcal/mol. Diosmin engages the TRPV1 vanilloid pocket mainly through hydrophobic interactions.

Diosmin exerts central and peripheral antinociceptive effects mediated, at least partially, through TRPV1 antagonism and cholinergic pathways.