Optimization of the Preparation Process of Isoliensinine and the Mechanism of Its Sleep-Improving Effect in Mice

Isoliensinine is a bisbenzylisoquinoline alkaloid extracted from the dried plumules and radicles of the mature seeds of Nelumbo nucifera Gaertn. (Nymphaeaceae). As one of the primary active ingredients in the lotus plumule, it is traditionally recognized in Chinese medicine for its effects of "clearing the heart and calming the nerves." However, total synthesis of isoliensinine remains unachievable, and current extraction and isolation methods suffer from high costs and low efficiency.In this study, a rapid, efficient, and low-cost isolation and purification method was first employed. The process was optimized using Response Surface Methodology (RSM). By utilizing Medium-Pressure Preparative Liquid Chromatography (MPLC) and adjusting parameters including mobile phase ratios, eluent flow rates, sample loading capacity, and mobile phase polarity, we successfully obtained high-purity isoliensinine in large quantities while significantly improving isolation efficiency.Subsequently, an insomnia mouse model was established using p-chlorophenylalanine (PCPA). Treatment with the isolated isoliensinine confirmed its sleep-promoting effects. Further investigation into the levels of $\gamma$-aminobutyric acid (GABA) and glutamate (Glu) in the mouse hypothalamus, along with the validation of the GABAergic signaling pathway, revealed the underlying mechanism: isoliensinine promotes sleep primarily by enhancing the activity of glutamate decarboxylase (GAD). This facilitates the conversion of the excitatory neurotransmitter Glu into the inhibitory neurotransmitter GABA. The increased GABA is then transported to the synaptic cleft via vesicular GABA transporters (VGAT) to act on GABA receptors, thereby influencing downstream pathways to exert sedative effects.This research establishes a new optimized process for the isolation of isoliensinine via RSM, reducing production costs while improving efficiency. Furthermore, it validates the functional role of isoliensinine in promoting sleep by modulating GAD activity.