Kratom (Mitragyna speciosa) as a Phytochemical-Based Natural Product Exhibiting Opioid-like Analgesic Effects with Reduced Tolerance and Dependence Liability via TLR4-Associated Neuroimmune Modulation

Kratom (Mitragyna speciosa) is a botanical candidate for pain management with potentially reduced opioid-related risks, partly through modulation of neuroimmune pathways involving Toll-Like Receptor 4 (TLR4). This study aimed to characterize the phytochemical profile of kratom ethanol extract and evaluate its effects on TLR4 signalling, neuroinflammatory cytokines, analgesic activity, withdrawal behaviours, and organ safety in morphine-dependent mice. Metabolite profiling was conducted using UHPLC–Q-Exactive Orbitrap HRMS, followed by molecular docking of major constituents to the TLR4 complex. In vivo assessments included flow cytometry and gene expression analyses of TLR4-mediated cytokines (NF-κB, IL-1β, IL-6), behavioural assays for antinociception, endurance, and withdrawal symptoms, and histopathological and biochemical evaluation of liver, kidney, and spleen tissues. More than 100 metabolites were identified, including mitragynine and flavonoids such as rutin and isoquercetin, which showed interactions with key TLR4 residues. Selected fractions suppressed pro-inflammatory cytokine expression, increased tail-pinch latency comparable to morphine, reduced withdrawal manifestations, and demonstrated nephroprotective and immunomodulatory effects, although mild reversible hepatic alterations were observed in specific fractions. Overall, kratom ethanol extract exhibited fraction-dependent analgesic and anti-neuroinflammatory activities associated with TLR4 modulation, supporting its potential as a botanical analgesic candidate while emphasizing the importance of safety optimization and standardized fraction development.