Study On Preparation Technology,Quality Control And Mechanism In Treating Insomnia Of Zhizichi Decoction Granules

Objective: A design space of the extraction process of Zhizichi Decoction was established and verified based on the concept of quality by design(Qb D).The forming process for Zhizi chi Decoction Granules were optimized,and quality control was performed.The potential mechanism of treating insomnia was explored by using network pharmacology method and molecular docking technology.The therapeutic effects and mechanism of Zhizichi Decoction Granules on insomnia model rats were investigated.Methods:1.Study on preparation process:(1)The contents of geniposide,Genipin-1-gentiobioside,daidzein,genistein and dry paste yield were used as the critical quality attributes(CQAs)of the extraction process of Zhizichi Decoction.The levels of each factor were determined by single factor experiment.The critical process parameters(CPPs)were screened by Plackett-Burman experimental design,and the extraction process was optimized by BoxBehnken experimental design.Monte Carlo method was used to calculate and verify the design space based on probability.(2)With the contents of geniposide,Genipin 1-gentiobioside,daidzein and genistein as evaluation indexes,the common concentration and drying methods were compared to determine the concentration and drying process of Zhizichi Decoction.(3)Optimizing the formulation of Zhizichi Decoction Granules by single-factor experiment.2.Quality control: According to the 2020 version of "Chinese Pharmacopoeia" four general rules0104 particles required,the traits,grain size,moisture,solubility check.The Gardenia jasminoides Ellis and Sojae Semen Praeparatum were identified in Zhizichi Decoction Granules by TLC,and the content of geniposide and daidzein were was determined by HPLC.3.Network pharmacology analysis and molecular docking: Network pharmacology and molecular docking technology was used to predict the active ingredients,potential targets and approaches of Zhizichi Decoction Granules in the treatment of insomnia.4.Preliminary molecular mechanism studies: Insomnia model rats were made by PCPA,and successful rats were randomly divided into model group,diazepam group,Zhizichi Decoction Granules low,medium and high dose group,additional normal control group,continuous administration for 7d.Open field test was used to detect the movement distance,standing times and fecal particles of rats in each group.The contents of DA,5-HT,NE,IL-6,IL-1β and TNF-α in hippocampal tissues of rats were determined by ELISA method.Results: 1.The recommended operating space of parameters were as follows: the amount of solvent was 8～11 times,the extraction time was 30～84min,and the extraction times were twice.The concentration process was decompression concentration(temperature was 80℃,vacuum degree was 0.08 ～ 0.09 MPa),and the drying process was decompression drying(temperature was 80℃,vacuum degree was 0.08～0.09MPa).The molding process of Zhizichi Decoction Granules was as follows: extract powder and lactose(mass ratio of 1:2),85% ethanol as wetting agent,dosage of 30%,granulation,drying,whole grain,that is.2.The quality inspection of Zhizichi Decoction Granules met the requirements.The TLC plots of Gardenia jasminoides Ellis and Sojae Semen Praeparatum showed clear spots and good separation.The contents of geniposide and daidzein was determined by HPLC,and the established method was simple and feasible.3.The study found that the active ingredients of Zhizichi Decoction Granules,such as geniposide,Genipin-1-gentiobioside,daidzein and genistein,can act on the key core targets of IL-1β,IL-6,ESR1,NOS3.It is involved in the regulation of Neuroactive ligand-receptor interaction,GABAergic synapse and TNF signaling Pathway,playing an important role in the treatment of insomnia.Molecular docking analysis showed that the active ingredients were closely bound to their core targets,which further confirmed that they play a role in the treatment of insomnia by regulating related targets.4.Open field test results showed that the movement distance of model group was significantly increased(P<0.05),but the number of standing times and fecal particles were not significantly increased compared with normal control group(P>0.05).Compared with model group,the moving distance,standing times of diazepam group,and high-dose of Zhizichi Decoction Granules group were significantly reduced(P<0.05).Interestingly,we found that there was no statistical difference in fecal granule number among all groups(P>0.05).Compared with normal control group,the level of NE,DA,IL-6,IL-1β and TNF-α in the hippocampus of model group were significantly increased(P<0.05),while the contents of 5-HT were significantly decreased(P<0.05).However,the level of NE,DA,IL-6 and TNF-α in hippocampus of diazepam group and high-dose of Zhizichi Decoction Granules group were significantly decreased(P<0.05),and 5-HT level was increased(P<0.05).Conclusion: The preparation process of Zhizichi Decoction Granules was reasonable and feasible,the quality control method was stable,feasible and reproducible,and the therapeutic effect on insomnia model rats might be related to the regulation of the balance of 5-HT,NE and DA contents in brain,and the down-regulation of inflammatory factors IL-6 and TNF-α.