| Insomnia is increasingly becoming one of the important physical and mental health problems mainly manifested as difficulty falling asleep,waking up easily,insufficient sleep quality or time and accompanied by symptoms such as daytime mood disorders and easy fatigue.The current clinical treatment of insomnia drugs include benzodiazepines,non-benzodiazepines,certain antidepressants and antipsychotics.Because western medicine has certain disadvantages such as affecting cognition,memory and mental activity then the Chinese herbal medicine is widely accepted for treating insomnia.TCM treatment of insomnia has the characteristics of syndrome differentiation and treatment.During the course of treatment,many traditional Chinese medicines and prescriptions for treating insomnia based on syndrome differentiation are summarized.The Golden Chamber Synopsis records the first Suanzaoren Decoction for the treatment of insomnia due to deficiency of fatigue.Jujube seed can nourish liver blood,calm the heart and the nerves has a good blood nourishing effect.It is a commonly used traditional Chinese medicine for modern clinical syndrome differentiation of blood deficiency insomnia.Modern pharmacy studies have proved that the flavonoids and saponins in jujube seed are effective ingredients for insomnia.How to improve its bioavailability and promote drug absorption is a further problem that needs to be solved for jujube seed resources.Fermentation technology,as a traditional processing method mainly uses various enzymes produced during the fermentation process to fully react with the ingredients in the medicine,which has the effect of increasing the effective ingredients,improving the curative effect of the medicine or generating new substances.Based on this idea,this study used jujuboside A and jujuboside B as reference substances mainly by establishing a blood-deficiency insomnia animal model,comparing the changes of active ingredients in the finished product before and after not fermenting and the effect on the efficacy of treating insomnia.ObjectiveTo investigate the material basis and safety of different fermented products of Poria cocos and Shenqu fermented Jujube seed,establish a blood-deficiency insomnia mouse animal model,to compare the pharmacodynamic effects of jujube seed before and after fermentation in the treatment of blood-deficiency insomnia,explore the effects of different finished products before and after fermentation on monoamine neurotransmitters in the brain.Method1 Using Poria cocos and Shenqu to prepare the fermented products of wild jujube seed,using HPLC-CAD to detect the fingerprint changes and analyzing based on jujube saponin A(juA)and jujube saponin B(juB)as the reference substance before and after fermentation.And analyzed the content of wild jujube seed.The the fermented product is tested for toxicity.2 Mice model of blood deficiency insomnia was prepared by intraperitoneal injection of cyclophosphamide(CTX)and p-chlorophenylalanine(PCPA)and the mice were coordinated by general body surface status,body weight,and sleep experiment,microscopic index white blood cell(WBC),red blood cell(RBC),hemoglobin(HGB)analysis evaluation of the preparation method of blood deficiency insomnia animal model.3 To observe the effects of jujube seed before and after fermentation on blood-deficiency insomnia mice and compare them through the sleep experiment of sodium pentobarbital in cooperation with mice,the open field experiment of behavior and the analysis of microscopic indicators WBC,RBC,and HGB.4 Using high-performance liquid phase electrochemical(HPLC-ECD)detection methods to detect the monoamine neurotransmitters norepinephrine(NE),dopamine(DA),serotonin(5HT),5-Hydroxyindole acetic acid(5-HIAA)in the hippocampus and brainstem of mice.comparing the effects of jujube seed before and after fermentation on the changes of monoamine neurotransmitters in the brain of mice.Result1 Preparation,detection and toxicity experiment of different fermentation productsUsing HPLC-CAD detection method,it was found that different fermented products of wild jujube seed all have new chromatographic peaks and the content changes before and after fermentation.After the fermentation of Poria cocos,the content of juA and juB decreased,the juA of Shenqu fermented wild jujube seed decreased slightly,JuB content increased.The toxicity test results showed that the mice in each group did not die during the experimental gavage,the administration group was in good mental activity,drinking water and eating normally,there was no significant difference in body mass change in each group(P>0.05),There was no significant difference in the organ index of mice in each group(P>0.05).2 Establishment of blood deficiency insomnia modelAfter modeling,the mental state of the mice in each group decreased and the weight of the mice in each group decreased.there was a significant difference(P<0.01 or P<0.05)and there was no significant difference in the amount of food intake and water(P>0.05),the model group was no significant difference in sleep latency in mice,but there was a tendency to prolong sleep latency(P>0.05).The sleep time of mice in each group decreased significantly and the difference was statistically significant(P<0.01)indicating that the insomnia model was established.There were significant differences in WBC,RBC,and HGB in the model group(P<0.05 or P<0.01),indicating that the content of WBC,RBC,and HGB in this model decreased.3 Compare the effects of jujube seed before and after fermentation on the effects of blood deficiency insomnia miceAfter the administration,the mental activity state of each administration group gradually improved and the lips and paw nails were light red.there was no significant difference in the sleep latency of the mice in each group(P>0.05),the sleep time of the mice in each treatment group increased with significant difference(P<0.05 or P<0.01).the Shenqu fermentation group increased significantly(P<0.05),the vertical activity of the model group mice decreased,with significant difference(P<0.05),the Poria cocos fermentation group was a significant difference in vertical activity(P<0.05).there was no significant difference in different fermentation groups(P>0.05),the horizontal activity in the model group was significantly reduced(P<0.01).there were significant differences in the diazepam group and the Shenqu fermentation group(P>0.05).the staying time in the central area of the model group decreased(P<0.05)and the diazepam group,the unfermented group,the Poria fermentation group,and the Shenqu fermentation group all had significant differences(P<0.05);different strains were fermented was a significant difference in WBC between the two groups(P>0.05)and there was no significant difference before and after fermentation,the content of RBC in the model group was reduced and there was a significant difference(P<0.05),diazepam group,unfermented group,and Poria fermentation group were significant difference in the increase in the content of mice in the Shenqu fermentation group(P<0.01).There was no significant difference before and after fermentation.compared with the model group and the unfermented group there was no significant difference in each group of HBG(P>0.05).4 Detection of monoamine neurotransmitters in the brain of mice with blood deficiency and insomniaAfter administrating,the NE content in the hippocampus of the Shenqu fermentation group increased significantly with a significant difference(P<0.05).There was a significant difference between the Poria cocos fermentation group and the Shenqu fermentation group(P<0.05).The NE content of the mouse brainstem was in the unfermented group the content is significantly reduced(P<0.05),the content of DA in the hippocampus of the model group was significantly lower(P<0.05),the DA content of the hippocampus of the model group was significantly different(P<0.05).Compared with the model group,the DA content of the diazepam group,the unfermented group and the Shenqu fermentation group all decreased and there is a significant difference(P<0.05).Compared with the unfermented group,there is no significant difference in the content of mice in each group after fermentation(P>0.05).The 5-HT in the hippocampus of the mice in each group was significantly increased and the difference was significant(P<0.05 or P<0.01).The content of mice in the Poria cocos fermentation group was significantly increased(P<0.05).Poria cocos and Shenqu fermentation the 5-HIAA in the hippocampus and brainstem of mice in the group increased significantly and the difference was significant(P<0.05).After fermentating there was no significant difference between the two groups(P>0.05).Conclusion1 After fermented with Poria and Shenqu,the fingerprints and content of wild jujube seed changed,indicating that the selection of different strains for fermentation has a certain influence on the content of active ingredients in wild jujube seed,Shenqu fermented products have good safety.2 In this study,an animal model combining disease and syndrome of blood deficiency insomnia mice was established.This model can simulate the syndrome characteristics of blood deficiency insomnia and make relevant evaluations on the establishment of the model,the judgment of the attributes of blood deficiency syndromes.The model and evaluation method of the combination of disease and syndrome have been determined,which can be used as an animal model of insomnia and blood deficiency syndrome.3 The fermented jujube seed improves sleep time and promotes the treatment of insomnia by changing cognitive behavior and increasing RBC.In the sleep experiment,Shenqu fermentation can significantly prolong sleep time,in the mouse behavioral open field experiment unfermented jujube seed and Poria cocos fermentation can increase the vertical activity of mice and the Shenqu fermentation treatment effect is better in the horizontal activity.In terms of regional residence time,each treatment group has better efficacy the results of mouse whole blood analysis show that the treatment effect on RBC before and after fermentation is better but the effect of fermentation is the same as that of unfermented.4 Fermented jujube seed can act on monoamine neurotransmitters to treat insomnia.Poria cocos fermentation can treat insomnia by increasing hippocampal NE content,unfermented jujube seed has a good therapeutic effect in the brainstem and it can treat blood-deficiency insomnia by reducing NE content,insomnia mice have increased DA content in hippocampus and brainstem and fermented both before and after have a good therapeutic effect.In the hippocampus of mice,the 5-HT content in each group was increased and the 5-HT content after fermentation by Poria cocos was higher than that in the unfermented group indicating that the fermented jujube seed had better efficacy than unfermented and Poria cocos fermentation.In the brainstem 5-HT,different fermented products are better than unfermented products but the treatment effect of each group of fermentation is the same,the content of 5-HIAA in hippocampus and brainstem before fermentation is not significant and both have good treatment after fermentation.The effect is the same between the two groups of fermented products. |
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