| ObjectiveSuanzaoren Decoction was originated from Synopsis of the Golden Chamber written by Zhang Zhongjing in Han Dynasty.It is mainly used to treat asthenia,fatigue and restlessness.Jujube kernel,Poria cocos and liquorice are the medicinal materials that can be used as food,while Chuanxiong and Anemarrhena are the medicinal materials for health care.This project is devoted to develop Suanzaoren Decoction into Ningxinanshen oral liquid,which is a traditional Chinese medicine health product.The research is carried out from the aspects of its preparation process,the formulation of quality standards,the evaluation of health care function and safety,etc.the combination of classical Chinese medicine prescription and modern preparation technology will further explore the value of traditional Chinese medicine and promote the development of health industry of traditional Chinese medicine health products.The content of Suanzaoren Decoction,the improvement of sleep and the content of neurotransmitter in mice were used as the index to screen the prescription of Ningxinanshen oral liquid.Then the extraction process of Suanzaoren Decoction was optimized by response surface design experiment.On the basis of decoction,the additional agents such as clarifier,flavor modifier and preservative were selected to determine the preparation process of Ningxinanshen oral liquid.The healthy function of Suanzaoren Decoction and Ningxinanshen oral liquid in improving sleep was evaluated by normal mice model,and the change of neurotransmitter content in mice brain was detected;the safety of Ningxinanshen oral liquid was evaluated by acute toxicity experiment and maximum tolerance experiment.The TLC identification of each flavor in Ningxinanshen Oral Liquid was carried out and its HPLC content determination method was investigated to establish a preliminary quality standard.And the use of network pharmacology technology to predict the mechanism of Ningxinanshen oral liquid to improve sleep.Methods1.The mice were given two kinds of Suanzaoren Decoction with different compatibility in low,middle and high dose groups for 30 days respectively.The improvement of sleep function of the two kinds of Suanzaoren Decoction was evaluated and compared.The contents of five neurotransmitters,such as NE,5-HT,DA,GABA and Ach,were detected in the brain of the mice.The organ index of the important organs of the mice was calculated.The contents of Suanzaoren saponin A,spironolactone and Zhizhi in the two kinds of Suanzaoren Decoction were analyzed The content of saponin B ?,mangiferin,ferulic acid,liquiritigenin and ammonium glycyrrhizinate were determined,and the best prescription of Ningxinanshen oral liquid was selected.Taking the total content of Jujuboside A,spironolactone,anemarrhenoside B?,mangiferin,ferulic acid,glycyrrhizin and Ammonium Glycyrrhizinate in the water extract of Suanzaoren Decoction as the index,the extraction process of Suanzaoren Decoction was optimized by the corresponding curved surface design experiment.According to the results of single factor investigation,three factors were selected as the object of investigation: the amount of water added,the time of extraction and the times of extraction.Design-Expert 8.0.6 software optimizes the experimental data,obtains the optimal extraction process,and through the process verification,guarantees the extraction process to be stable and feasible.Taking the total content of seven control components and the clarity of the preparation as the main indicators,the types and dosage of clarifier,flavor straightener and preservative were investigated and optimized respectively,among which the clarity was checked according to the method of Pharmacopoeia clarity check(general rule 0902)and the first method of visual inspection.To determine the preparation technology of Ningxinanshen oral liquid.2.The evaluation of health function is to divide the normal mice into 7 groups.Suanzaoren Decoction and Ningxinanshen oral liquid are concentrated to low,medium and high concentrations respectively.The mice are administrated for 30 days to observe their effects on the sleep time,sleep rate and sleep latency induced by barbiturates,so as to evaluate the health function of improving sleep.Five neurotransmitters,such as NE,5-HT,DA,GABA and Ach,were detected by kit.3.After one week's feeding,the animals were randomly divided into four groups,10 in each group.The distilled water and different concentrations of Ningxinanshen oral liquid were given to mice by gavage with 0.2ml/10 g respectively to observe the response of mice in each group after administration.In order to understand the reversibility of the toxic reaction and the possible delayed toxicity,14 days were observed continuously.The maximum tolerable concentration and volume of Ningxinanshen oral liquid were given to the animals by gavage for 3 times in a single day for 14 consecutive days.The reaction of the animals was recorded in detail,the maximum tolerable amount of Ningxinanshen oral liquid was calculated,and the safety of Ningxinanshen oral liquid was evaluated.4.Seven To control the quality of Ningxinanshen oral liquid and establish the sensory index of Ningxinanshen oral liquid;to identify the medicinal materials in Ningxinanshen oral liquid by TLC;to establish the pH,microbial limit,pollutant limit and other standards of Ningxinanshen oral liquid with reference to the national standards;to establish the spironolactone and jujube in Ningxinanshen oral liquid by HPLC Methods for the determination of ginsenoside a,saponin B?,mangiferin,ferulic acid,glycyrrhizin and ammonium glycyrrhizinate.5.Use TCMsp database of TCM System Pharmacology analysis platform to find the chemical components and target points related to 5 Chinese herbs in Ningxinanshen oral liquid,and select BBB ? 0.3,obtain the target related to insomnia through durgbank database,import the target for insomnia treatment into string,and use string to build disease network.The related potential targets were enriched by using David database to analyze the enrichment of GO and KEGG metabolic pathway.Results1.The best compatibility of Ningxinanshen oral liquid is jujube seed 15 g,Poria cocos 6g,Anemarrhena 6g,Chuanxiong 6g,liquorice 3g.The best extraction process of Ningxinanshen oral liquid is to add 9 times of water,soak for 30 minutes,reflux extraction for 3 times,each time for 1.5 hours;the most suitable clarifier is HACC clarifier,when the ratio of medicine to liquid is 1:2,the best clarifier is 2% a and 6% B;0.05% sucralose is selected as the flavoring agent;0.02% ethyl nipagine is selected as the anti-corrosion agent.2.Suanzaoren Decoction and Ningxinanshen oral liquid can prolong the sleep time of mice induced by pentobarbital sodium,increase the incidence of sleep induced by pentobarbital sodium and shorten the sleep latency induced by barbital sodium.The contents of NE,5-HT,DA and GABA in the brain of normal mice were significantly increased and the content of Ach was significantly reduced by gavage of Suanzaoren Decoction and Ningxinanshen oral liquid.However,with the increase of dosage,the effect of drugs on the content of neurotransmitters was weakened.3.When the dosage of Ningxinanshen oral liquid was 60000mg/g,no animal died;after calculation,the maximum tolerance of mice to Ningxinanshen oral liquid was 250 times of the dosage of human day,indicating that Ningxinanshen oral liquid was safe.4.The TLC spots of five kinds of medicinal materials are clear,the resolution is good,there is no negative interference,and the specificity is good.The logistic regression analysis of Jujuboside A Y=1.5019X+10.2133,the logistic regression analysis of spironolactone Y=12963136.8807X-56925.7677,the logistic regression analysis of anemarrhenoside B? Y=1.5417X+5.4534,the logistic regression analysis of mangiferin Y=2×107X-130897,the logistic regression analysis of ferulic acid Y=1×107X-7653.8,the logistic regression analysis of glycyrrhizin Y=19822010.608X-100587.7811,the logistic regression analysis of Ammonium Glycyrrhizinate Y=2693854.254X-53841.2587,all correlation coefficient are higher than 0.9990.The accuracy of the method is good and it can be used for the quality control of Suanzaoren Decoction.5.In Ningxinanshen oral liquid,58 potential active ingredients and 211 potential targets related to insomnia were screened out;6 metabolic pathways were obtained by enrichment of KEGG,mainly including Neuroactive ligand-receptor interaction ?Calcium signaling pathway?CGMP-PKG signaling pathway,etc.ConclusionIn this project,the best compatibility of Ningxinanshen oral liquid was selected,and the best extraction process was determined by response surface design.The clarifier for Ningxinanshen oral liquid was HACC,the flavoring agent was sucralose and the preservative was Nipagin ethyl ester,and the best dosage was determined.Finally,Ningxinanshen oral liquid was determined The preparation process of oral liquid.Through the synergistic effect of barbital sodium and the determination of neurotransmitter content in the brain,the health care function of Suanzaoren Decoction and Ningxinanshen oral liquid was evaluated,and the consistency of the efficacy of Suanzaoren Decoction and Ningxinanshen oral liquid was confirmed,indicating that Ningxinanshen oral liquid has the effect of improving sleep.The results of acute toxicity test and maximum tolerance test showed that Ningxinanshen oral liquid was safe and nontoxic as a health product.The quality control method of Ningxinanshen oral liquid was established through the study of TLC identification and content determination.The results of network pharmacology confirmed the characteristics of Ningxinanshen oral liquid in improving sleep,such as multi-component,multi-target and multi-channel,and predicted that its possible mechanism might be related to the regulation of neurotransmitter receptor expression and the regulation of neurotransmitter content. |
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