Optimization of a Green Extraction Technique for Kratom (Ketum) Leaf Extract via Accelerated Solvent Extraction: Phytochemical Profiles, Cytotoxicity, and Antinociceptive Activity.

Kratom (Mitragyna speciosa Korth.) has gained increasing scientific interest for its potential in pain management and addiction treatment. This study employs a green chemistry approach to optimize the extraction of kratom leaves by using Accelerated Solvent Extraction (ASE) with an ethanol-water binary solvent system. The goal was to improve the yield and potency of key bioactive compounds, especially mitragynine. Optimization was performed using One-Factor-at-a-Time (OFAT) analysis and Response Surface Methodology (RSM) employing a Box-Behnken Design (BBD). The optimal extraction conditions were determined to be an 8 min extraction time, 60 °C temperature, and 40% ethanol concentration, which resulted in mitragynine content of 4.66%, total phenolic content of 212.69 GAE mg/g, and total flavonoid content of 126.13 QE mg/g. The safety profile of the optimized ASE kratom leaf extract was evaluated using MTT cytotoxicity assay, which revealed selective cytotoxicity against HepG2 liver cancer cells (IC50 = 7.69 μg/mL), while showing no cytotoxicity toward HL-7702 normal liver cells (IC50 > 200 μg/mL). Antinociceptive activity was tested in BALB/c albino mice using the hot-plate test, where the optimized ASE kratom leaf extract demonstrated analgesic effects at dosages of 100, 200, and 500 mg/kg. Phytochemical profiling combining NMR and UPLC-ESI-QTOF-MS/MS identified several known kratom constituents, including mitragynine and its congeners as well as bioactive flavonoids such as isoquercitrin and rutin. The optimized ASE method using a green ethanol-water system produces kratom extracts with promising safety and therapeutic potential, though further work is needed to refine and scale the approach for broader phytopharmaceutical use.