Anti-inflammatory Activity, Toxicity Assessment and GC-MS Chemical Profiling of Cha-Nu-Ruk Traditional Thai Herbal Poultice for Knee Pain Management

Introduction: Thai Traditional herbal medicine employs multi-herb formulations for managing inflammatory conditions. This study investigates the anti-inflammatory properties, cytotoxicity, and chemical composition of Cha-Nu-Ruk, an eight-herb traditional poultice formulation for knee pain. Methods: Eight medicinal plants (Tiliacora triandra, Thunbergia laurifolia, Azadirachta indica, Clinacanthus nutans, Pandanus amaryllifolius, Justicia gendarussa, Sida acuta, and Zingiber cassumunar) were extracted with 70% ethanol. Anti-inflammatory activity was evaluated through nitric oxide inhibition assay using Jurkat cells. Cytotoxicity was assessed via MTT assay. Chemical profiling employed GC-MS analysis. Results: Individual plant extracts demonstrated variable anti-inflammatory activity (NO inhibition: 28.52-73.80%) with Clinacanthus nutans showing highest potency. The combined eight-herb formulation yielded 11.82±0.41% extraction efficiency with 26.2% NO inhibition. GC-MS analysis identified 21 compounds, with DMPBD (19.13% relative abundance) and β-sitosterol (1.13%). Cytotoxicity evaluation revealed acceptable safety profiles (>80% cell viability) for most extracts. Conclusions: While individual herbs showed promising anti-inflammatory potential, the traditional eight-herb combination did not demonstrate synergistic enhancement. Chemical standardization revealed significant batch-to-batch variability, highlighting the need for optimized formulation ratios and quality control protocols.