Screening Of Active Ingredients Of Ginseng To Maintain Sleep Rhythm Homeostasis And Study On Its Mechanism Of Action

Objective: Using the Drosophila melanogaster model,combined with behavioral analysis and other methods to screen the active components of ginseng to maintain sleep rhythm homeostasis,and to clarify its mechanism of action.Methods: The water-extracted and alcohol-extracted components(SPJ)of ginseng were obtained by water extraction and non-aqueous solvent extraction,respectively,and the effective active components were determined by using the Drosophila melanogaster model combined with the DAM2 behavioral analysis system.Climbing experiments were used to analyze the effects of the said components on the motor function of Drosophila.The safety of the active components was evaluated by preference,aversion and withdrawal experiments.The effects of active ingredients on the expression of normal and sleep-deprived Drosophila brain tissue proteome were analyzed by proteomic method,combined with biological information to study the function of differentially expressed proteins,and initially explore their molecular mechanisms.The composition of ginsenosides in the active components was analyzed by HPLC,and the active ginsenoside monomers were screened through the detection and comparison of sleep conditions.RT-q PCR was used to analyze the effect of saponin monomer on the m RNA levels of key antioxidant enzymes in Drosophila brain tissue,and the content of antioxidant GSH and the activity of antioxidant enzyme SOD were analyzed by kit method;Using tert-butyl hydroperoxide(t-BHP)to establish a Drosophila model of oxidative damage-induced sleep rhythm disorders;RT-q PCR to analyze the m RNA levels of key indicators of endoplasmic reticulum stress and mitochondrial oxidative stress;using mutant Drosophila combined with Molecular docking technology was used to determine the potential targets of active ginsenoside monomers;Western blot was used to analyze the effect of active saponin monomers on the acetylation level and Sir2 expression in Drosophila brain tissue,and combined with inhibitors to explore the efficacy and mechanism signals of active ginsenoside monomers pathway;targeted metabolomics to analyze the changes of active ginsenoside monomers on neurotransmitters in Drosophila brain tissue;evaluate the safety of active ginsenoside monomers through preference,aversion and withdrawal experiments.Results: The alcohol-extracted component SPJ of ginseng can significantly increase the total sleep duration of normal and sleep deprived Drosophila models,alleviate sleep fragmentation and restore sleep amplitude,and improve the climbing ability of Drosophila without addiction and dependence.Proteomic results found that sleep deprivation mainly reduced the expression of redox balance-related proteins in Drosophila brain tissue,while SPJ could restore their expression levels to normal.The contents of ginsenosides Rc and s-Rg3 in SPJ were higher.Through comparative studies,we found that ginsenoside Rc has the strongest effect on maintaining sleep rhythm homeostasis and has antioxidant potential;ginsenoside Rc can alleviate the imbalance of sleep rhythm homeostasis caused by oxidative damage,and it was proved by gene mutation and molecular docking technology that Pink1 is a potential efficacy target of Rc.At the same time,ginsenoside Rc can inhibit the acetylation of brain tissue proteins caused by oxidative damage,and the effect depends on the expression of sir2 deacetylase.Ginsenoside Rc can promote oxidative damage in Drosophila brain tissue Biosynthetic pathways of dopamine;ginsenoside Rc did not alter preference and aversion in Drosophila,and had no withdrawal effect.Conclusion: The alcohol-extracted component SPJ of ginseng is an active ingredient for maintaining the steady state of sleep rhythm,and ginsenoside Rc is the main active monomer of the alcohol-extracted component.Ginsenoside Rc activates the expression of Pink1 and its downstream deacetylase Sir2,reduces the acetylation modification of the brain proteome,improves the antioxidant system,and alleviates the imbalance of sleep rhythm homeostasis caused by oxidative damage.In addition,ginsenoside Rc can promote the dopamine biosynthesis pathway in brain tissue and has neuroprotective potential,and it is preliminarily determined that ginsenoside Rc is not dependent and addictive.