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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE331004 | GEO | 2026/05/16

REPOSITORIES: GEO

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Project description:Background: Of the many neurotransmitters in humans, gamma-aminobutyric acid (GABA) shows potential for improving several mental health indications such as stress and anxiety. The microbiota-gut-brain axis is an important pathway for GABAergic effects, as microbially-secreted GABA within the gut can affect host mental functionhealth outcomes. Understanding the molecular characteristics of GABA production by microbes within the gut can offer insight to novel therapies for mental health. Results: Three strains of Levilactobacillus brevis with syntenous glutamate decarboxylase (GAD) operons were evaluated for overall growth, glutamate utilization, and GABA production in typical synthetic growth media supplemented with monosodium glutamate (MSG). Levilactobacillus brevis Lbr-6108 (Lbr-6108) and Levilactobacillus brevis Lbr-35 (Lbr-35) had similar growth profiles but differed significantly in GABA secretion and acid resistance. Lbr-6108 produced GABA early, within the growth phase, and produced significantly more GABA than Lbr-35 and the type strain Levilactobacillus brevis ATCC 14689 after the stationary phase. The global gene expression during GABA production was determined by RNA sequencing at several timepoints. The GAD operon, responsible for GABA production and secretion, activated in Lbr-6108 after only six hours of fermentation and continued throughout the stationary phase. Furthermore, Lbr-6108 activated many different acid resistance mechanisms concurrently, which contribute to acid tolerance and energy production. In contrast, Lbr-35, which has a genetically similar GAD operon, including two copies of the GAD gene, showed no upregulation of the GAD operon, even when cultured with MSG. Conclusions: This study is the first to evaluate whole transcriptome changes in L. brevis during GABA production over multiple timepoints. The concurrent expression of multiple acid-resistance mechanisms reveals niche-specific metabolic functionality between common human commensals and highlights the complex regulation of GABA metabolism in this important microbial species. Furthermore, the increased and rapid GABA production of Lbr-6108 highlights the strain’s potential as a therapeutic and the overall value of screening microbes for effector molecule output.

Project description:Although at the organismal level sleep is defined as a behavioral state, at the level of the cerebral cortex sleep has a distinct local and use-dependent aspect. This observation raises the question whether sleep is a functional property of a complex brain or occurs at the level of neuronal assemblies with populations that were active more during wakefulness needing more intense sleep to recover. Here we show that primary cortical cultures have the capacity to change between sleep- and wake-like states that share key signatures with their in vivo counterparts. Cortical cultures initially exhibit random firing activity that is gradually replaced by a M-bM-^@M-^\sleep-likeM-bM-^@M-^] synchronized burst-pause firing activity as neurons mature and make connections. When stimulated with excitatory neurotransmitters, transient tonic firing is observed, followed by the reappearance of a M-bM-^@M-^\sleep-likeM-bM-^@M-^] state. Besides electrophysiological similarities also the transcriptional profile of stimulated cortical cultures greatly resembles that of the cortex of sleep deprived animals. We then used our in vitro model to map the metabolic pathways activated by the M-bM-^@M-^\wake-likeM-bM-^@M-^] state and found evidence for increased lysolipid release, strongly suggesting that sleep plays a role in neuronal membrane homeostasis. With our in vitro model the cellular and molecular consequences of sleep loss and the genetic determinants of disturbed sleep can now be investigated in a dish. Keywords: stress response For the in vivo transcriptome analyses mice were sleep deprived for 6 hours and brain harvested immediately afterwards. For the in vitro analyses, cortical cultures were stimulated with a neurotransmitter cocktail and cells harvested 3 hours afterwards. Control mice were kept undisturbed in their home cage and control cultures were sham stimulated with water. Also, another set of sleep deprived mice as well as stimulated cultures were allowed to recover before being sampled the next day at the time of day at which sleep deprivation or stimulation started the day before).

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

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