Optimization of complex enzymatic extraction, structural characterization, and sleep-improving effects of Lycium...

Lycium ruthenicum Murr. is an edible and medicinal plant, abundant in bioactive polysaccharides with health-promoting properties. The polysaccharides of Lycium ruthenicum Murr. (LRMP) have demonstrated a range of beneficial activities, including antioxidant, immunomodulatory, neuroprotective, and anti-aging, etc. Box-Behnken response surface methodology (RSM) was employed to optimize the complex enzymatic extraction process of LRMP and to investigate its sleep-improving effects in zebrafish. The polysaccharide structure was characterized using a series of chemical and spectroscopic analyses. A caffeine-induced zebrafish insomnia model was established and treated with LRMP. To this end, a comprehensive suite of analyses was conducted, encompassing behavioral profiling, histological examination of neurons, and quantification of molecular markers related to neurotransmission, inflammation, and oxidative stress. The optimal extraction parameters were: complex enzyme ratio (pectinase 9000 U/g, cellulase 800 U/g, papain 3200 U/g), enzymatic hydrolysis pH 5.00, temperature 40.0 °C, time 2.5 h, and solid-to-liquid ratio 1:50 (g/mL), yielding a polysaccharide extraction rate of 52.26 ± 0.10 %. LRMP was identified as an acidic heteropolysaccharide with a molecular weight range of 1.1-2690.397 kDa. In a zebrafish insomnia model, LRMP administration dose-dependently improved sleep quality by up to 82.69 % and enhanced sedative effects by 1.27-fold, obviously prolonging rest time and reducing locomotion. Mechanistically, LRMP mitigated neuroinflammation and oxidative stress by suppressing pro-inflammatory cytokines (IL-6, IL - 1β, TNF-α) and MDA while elevating IL-10 and antioxidant enzymes (SOD, GSH-Px, CAT). It also restored neurotransmitter balance by promoting 5-HT and GABA and inhibiting Glu, DA, and NE. Correspondingly, LRMP upregulated key proteins (5-HT1AR, GABAAα1, TPH2, GAD67) and activated the Nrf2 pathway, while downregulating excitatory neurotransmission (DRD1, Glu A1, TH) and inflammatory markers. Furthermore, LRMP exerted neuroprotective effects, preserving neuronal structure and increasing Nissl body counts. This study provides a theoretical foundation for the potential application of LRMP in functional foods and pharmaceuticals and offers support for the development of novel natural sleep-improving agents.