| Objective:The research objects of this study were Shanxi authentic medicinal herbs Semen Zizyphi Spinosae and Radix Polygala,and they are often used together as a herbal pair.In this study,the effective components of this herbal pair and the mechanisms of the sedative and tranquilizing effect of them were studied,aiming to provide a theoretical basis for further development and utilization of the two medicinal herbs.Methods:The total flavonoids and total saponins yielded from the herbal pair were used as the index to investigate and evaluate the influence of ethanol concentration,liquid-solid ratio,extraction time and extraction temperature on the yield.According to the response surface test,the optimum extraction process of total flavonoids and total saponins was obtained.D101 macroporous adsorption resin was used to enrich total flavonoids and total saponins.The optimum enrichment process of the herb pair was obtained by investigating the sample loading,eluent concentration and eluent dosage.Contents of spinosin,jujuboside A and senegenin in freeze-dried powder of enrichment parts were determined by HPLC.Pharmacological experiments were carried out to evaluate the sedative and tranquilizing effect of the effective components of this herbal pair.Mice were divided into control group,and treatment groups of high,middle and low dose(23.0,11.5,and 5.8mg·kg-1 respectively in terms of the total flavones and total saponins contained in the herb pair effective components).Subthreshold and suprathreshold dose pentobarbital sodium experiments were conducted to study the sedative and tranquilizing effect of the effective components.The mechanisms of sedative and tranquilizing effect of the effective components of this herbal pair were studied by mice model of insomnia,which were produced by PCPA(350 mg·kg-1).Mice were divided into normal group,model group,compound Suanzaoren capsule positive group(60.0 mg·kg-1),and high,middle and low dose effective components groups(23.0,11.5,and 5.8 mg·kg-1 respectively in terms of the total flavones and total saponins contained in the effective components).The homogenate of brain tissue of mice was obtained to study the ratio of sleep related substances,5-hydroxytryptamine?5-HT?,norepinephrine?NE?,dopamine?DA?and 4-aminobutyric acid?GABA?to the total brain tissue protein,which were all determined by ELISA,for the purpose of illuminating the possible mechanisms of the effect of the herbal pair.The mechanisms of the sedative and tranquilizing effect of the effective components were also investigated by methods of serum metabolomics.Serum metabolites of mice from the normal group,model group and high,medium and low dose effective components groups were studied by gas chromatography-time-of-flight mass spectrometry?GC-TOF-MS?,and differential metabolites between groups were obtained.The differential metabolites were then used to predict the related metabolic pathways via online analysis platform?Http://www.metaboanalyst.ca/?.Results:The optimum extraction conditions for total flavonoids and total saponins were as follows:liquid-solid ratio of 10 mL·g-1,ethanol concentration of 65%,temperature of 70?,extraction time of 60 minutes.The average yield of total flavonoids and total saponins were 2.60%and 9.40%respectively.The optimum elution process was as follow.D101 macroporous resin was used for adsorption of 9.12 mg total flavonoids and 36.32 mg total saponins for every 8 mL.Gradient elution was used,followed by 8 bv elution with distilled water and 8 bv elution with 30%ethanol.The eluent was recorded as EI30.The content of total flavonoids and total saponins in EI30 were 37.5%and 24.6%respectively.The HPLC detected contents of spinoside and senegenin were 1.61%and 0.24%respectively in the EI30 freeze-dried powder,but no jujuboside A was detected.The results of pharmacological experiments showed that compared with the control group,high dose EI30 group was with increased proportion of sleeping within 30 minutes?P=0.027<0.05?.High,medium and low dose groups were all with shortened sleep latency and prolonged sleeping time?P<0.01?.The results of investigation of mechanism via PCPA induced mice model of insomnia showed that compared with the normal group,the mice model of insomnia were successfully produced.Compared with the model group,proportion of 5-HT in brain tissue of mice treated with EI30 increased significantly?P<0.01?,and proportion of NE in brain tissue of high dose EI30 group decreased significantly?P<0.01?.Also compared with the model group,proportion of GABA in total protein was significantly decreased in high dose EI30 group?P<0.05?,and proportion of DA in total protein in medium dose EI30 group was significantly lower?P<0.05?.The results from serum metabolomics were that 50 markers of PCPA induced insomnia were found.And there were 17 differential metabolites found between normal group and model group,and between model group and high dose group.The sedative and tranquilizing effect of EI30 intervention on PCPA induced mice model of insomnia was mainly working through five metabolic pathways:Valine,leucine and isoleucine biosynthesis,glycerolipid metabolism,alanine,aspartic and glutamic acid metabolism,phenylalanine metabolism,and cysteine and methionine metabolism.EI30 can regulate PCPA caused disorders in the biosynthesis of valine,leucine and isoleucin,and phenylalanine metabolism.Conclusion:The herbal pair Semen Zizyphi Spinosae and Radix Polygala has good sedative and tranquilizing effect,and the mechanism of this effect is possibly related to the regulation of the ratio of neurotransmitters 5 HT,NE,GABA,and DA in the total protein of brain,and the regulation of valine,leucine,and isoleucine biosynthesis,and phenylalanine metabolism.This study contributes to the research of the herbal pair Semen Zizyphi Spinosae and Radix Polygala and can serve as the basis for the future development and utilization of the herbal pair. |
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