Optimization of Extraction Process and Neuroprotective Activities of Flavonoids from Mentha haplocalyx Briq. with Subcritical Water

Objective: This study aims to optimize the subcritical water extraction process for flavonoids from Mentha haplocalyx Briq. (mint) and assess the mitigating effects of peppermint flavonoids on H₂O₂-induced adrenal pheochromocytoma (PC12 cell) injury in rats. Methods: The subcritical water extraction process for mint flavonoids was optimized using single-factor and Box-Behnken response surface tests. A comparison was made with reflux extraction, ultrasonic-assisted extraction, and supercritical CO₂ extraction methods. In vitro cell culture of PC12 cells was employed to establish an H₂O₂-induced PC12 cell injury model. The neuroprotective activity of mint flavonoids was analyzed using the MTT (methyl thiazolye tetrazolium, MTT) method. Results: The optimized extraction conditions were determined to be 142 ℃, 38 min, and 21:1 mL/g, resulting in a mint flavonoid yield of 14.07%±0.23%. This yield was significantly higher (P<0.001) than that of reflux extraction (334% increase), ultrasonic-assisted extraction (75% increase), and supercritical CO₂ extraction (173% increase). Mint flavonoids, within the concentration range of 1~100 μg/mL, significantly enhanced the survival rate of PC12 cells from 53.61%±0.64% to 78.49%±0.84% compared to the H₂O₂ group (P<0.001), indicating substantial alleviation of cellular oxidative injury. Conclusion: The study established an environmentally friendly, efficient, and reliable subcritical water extraction process for mint flavonoids. The extracted mint flavonoids demonstrated significant protective effects against H₂O₂-induced PC12 cell injury (P<0.01).