Research On The Material Basis Of Arisaema Cum Bile’s Antipyretic Convulsant Effect

Arisaema cum Bile is one of the commonly used processed products of the poisonous Chinese medicine Arisaema cum Bile.Processed by fermentation.It has the functions of clearing away heat and resolving phlegm,relieving wind and calming convulsions."Compendium of Materia Medica" has a cloud:"If a child is frightened by the wind and phlegm,the limbs are twitching,...non-biliary stars cannot be cured." Modern clinical literature also shows that Arisaema cum Bile is often used in the treatment of febrile seizures in children.Niuhuang Baolong Pills,Qizhen Pills,Niuhuang Zhenjing Pills and other Chinese patent medicines containing Arisaema cum Bile are widely used in the treatment of pediatric diseases such as acute convulsions and high fever convulsions in children.Febrile seizures are a common disease in pediatrics.Modern pharmacological studies believe that the occurrence of the disease is closely related to the process of immune inflammation.The release of a large number of inflammatory factors causes the body to heat up,which in turn causes the excitation of neurons in the hypothalamus and hippocampus,resulting in abnormal discharge.lead to febrile seizures.Long-term and repeated febrile seizures can cause damage to hippocampal neurons and induce inflammatory responses in the brain,which further lead to seizures and neuronal cell damage.Pharmacological experiments show that Arisaema cum Bile has antiinflammatory,sedative and other pharmacological effects.The research group’s previous research found that Arisaema cum Bile has a significant anticonvulsant effect,but the material basis of Arisaema cum Bile’s anti-febrile convulsant effect is still unclear,and the quality markers related to the effect of Arisaema cum Bile are unknown.Based on the literature research and the previous research of the research group,this thesis investigates the medicinal effect of Arisaema cum Bile by constructing a mouse model of febrile convulsion.And at the cellular level,by constructing lipopolysaccharide(LPS)-induced mouse brain BV-2 microglial inflammation model to evaluate the effect site and material basis of Arisaema cum Bile water extract.To provide a scientific basis for the research on the material basis of Arisaema cum Bile’s antipyretic convulsant effect.The specific research contents are as follows:1.Effect of Arisaema cum Bile water extract on febrile convulsion model miceIn this study,a febrile convulsion model was established by subcutaneous injection of 20%dry yeast suspension and pentylenetetrazol solution as the shocking agent for the first time.The rectal temperature of mice was measured with a thermometer,and images were collected to record the convulsion latency and duration of convulsions in mice.Hematoxylin-eosin(HE)tissue staining was used to evaluate the pathological manifestations of hippocampal neuron damage.Serum PGE2 and cAMP levels were detected by ELISA.Western-blot method was used to detect the protein expression levels of COX-2,iNOS,GFAP and GABAAR in mouse brain tissue,and real-time fluorescence quantitative PCR(RT-PCR)method was used to detect the inflammation of IL-6,IL-1β and TNF-α in hippocampus tissue.Factor mRNA expression levels were used as indicators.The results showed that compared with the blank control group,the rectal temperature of the mice in the model group increased significantly,and convulsions occurred at the same time.Compared with the model group,Arisaema cum Bile water extract can effectively prolong the latency of convulsions,shorten the duration of convulsions,up-regulate the expression of type A GABA receptor(GABAAR)protein in brain tissue,reduce the rectal temperature of model mice,and reduce the levels of serum cAMP and PGE2.,indicating that Arisaema cum Bile has antipyretic and antagonizing effects of febrile convulsions;no obvious changes in the morphology of hippocampal neurons,indicating that Arisaema cum Bile can protect neurons in the hippocampus from damage;at the same time,glial fibers in astrocytes in the brain The expression of acidic protein(GFAP)is down-regulated,indicating that Arisaema cum Bile has the effect of inhibiting the activation of astrocytes,and there is a certain correlation between astrocyte activation and brain injury,indicating that the anticonvulsant effect of Arisaema cum Bile may be related to the reduction of glial cells.cell increase.Arisaema cum Bile can down-regulate the expressions of nitric oxide synthase(iNOS)and cyclooxygenase-2(COX-2)proteins,and at the same time downregulate the mRNA expression levels of IL-1β,IL-6,and TNF-α in the hippocampus,indicating that Arisaema cum Bile inhibited the occurrence of inflammatory response in the brain while exerting anticonvulsant effect.Since the occurrence of inflammatory response in the brain can increase the susceptibility to convulsions,the effect of Arisaema cum Bile on febrile seizures may be related to the inhibition of inflammatory response in the brain to a certain extent.The above studies show that Arisaema cum Bile has significant anti-febrile convulsions,protects hippocampal neurons from damage,and inhibits inflammation in the brain.2.Effect of Arisaema cum Bile water extract on LPS-induced mouse brain BV-2 microglial inflammation modelBy constructing a model of BV-2 microglial inflammation induced by LPS,the substance basis of anti-febrile convulsion effect of Arisaema cum Bile water extract was investigated.,IL6,TNF-α mRNA levels of inflammatory factors as indicators to evaluate the effect.The results showed that compared with the blank control group,the NO content in the model group and the mRNA expression levels of IL-1β,IL-6 and TNF-α were significantly increased,indicating that the modeling was successful;compared with the model group,the NO content in the Arisaema cum Bile group,The mRNA expression levels of IL-1β,IL-6 and TNF-α were significantly decreased,indicating that Arisaema cum Bile water extract significantly inhibited LPS-induced release of NO from BV-2 microglia,and decreased IL-1β,The mRNA expression levels of IL-6 and TNF-α inflammatory factors further indicate that Arisaema cum Bile has the effect of inhibiting the occurrence of inflammation in the brain.This model was used for subsequent effector screening.3.Screening of effect parts of Arisaema cum Bile water extractIn order to further study the effect site of Arisaema cum Bile water extract,BV-2 microglial inflammation model was induced by LPS,NO content in cell culture supernatant was measured by Griess method,and IL-1β and IL-6 were measured by RT-PCR method.The mRNA level of TNF-α inflammatory factor was used as the index to evaluate the effect,and the anti-febrile convulsion effect site of Arisaema cum Bile water extract was screened.The results showed that compared with the model group,the Arisaema cum Bile water extract alcohol precipitation supernatant group could significantly reduce the NO content,the mRNA expression levels of IL1β,IL-6 and TNF-α inflammatory factors,but the precipitation part had no obvious effect.The alcohol precipitation supernatant of Arisaema cum Bile water extract was further separated by systematic solvent extraction,and the effect part was screened.The results showed that compared with the model group,the ethyl acetate fraction and n-butanol fraction of Arisaema cum Bile water extract significantly decreased the NO content,the mRNA expression levels of IL-1β,IL-6,and TNF-α inflammatory factors,the petroleum ether fraction and the water fraction.No apparent effect.It indicated that ethyl acetate and n-butanol were the main effect parts of Arisaema cum Bile water extract against febrile convulsion.4.Analysis and research on the effect part components of the water extract of Arisaema cum Bile by UPLC-Q-TOF-MS/MS technologyUPLC-Q-TOF-MS/MS technology was used to analyze the ethyl acetate fraction and nbutanol fraction of the supernatant of the water extract of Arisaema cum Bile,and the unknown components were deduced according to the fragmentation law.The composition database of Arisaema cum Bile was established.Through the comparison and analysis with the database,a total of 61 components were identified from the ethyl acetate part of the water extract of Arisaema cum Bile,mainly including 18 kinds of organic acids and 17 kinds of cholic acids.A total of 72 components were identified in the butanol site,including 23 cholic acids and 11 organic acids.Chole acids are mainly composed of chenodeoxycholic acid(CDCA),hyodeoxycholic acid(HDCA)and other ingredients;organic acids are mainly composed of palmitic acid,linolenic acid,stearic acid and other ingredients.The common components of the n-butanol part and the ethyl acetate part are mainly chenodeoxycholic acid(CDCA),hyodeoxycholic acid(HDCA),hydric cholic acid(HCA),linoleic acid,stearic acid,palmitic acid,etc.34 ingredients.5.Determination of bile acids in the effect parts of Arisaema cum Bile water extract and effect verificationThe content of cholic acids in ethyl acetate and n-butanol parts was studied by HPLC-ELSD method,and the methodological inspection results showed that they all met the requirements for content determination.The results of content determination showed that the main bile acids in the ethyl acetate fraction were hyodeoxycholic acid(HDCA)and chenodeoxycholic acid(CDCA)with an average content of 17.99%and 6.68%,respectively.Bile acids could not be detected in the n-butanol part,which was different from the previous mass spectrometry analysis results.It is speculated that the content of bile acids in the n-butanol part was lower than the detection limit of liquid chromatography.In vitro LPS-induced mouse brain BV-2 microglial inflammation model was used to evaluate the difference in efficacy of the ethyl acetate site and the composition of bile acids(HDCA,CDCA).The results showed that compared with the model group,the ethyl acetate site and cholic acid composition significantly reduced NO content,IL-1β,IL-6,TNF-α mRNA expression levels of inflammatory factors,but the effect of cholic acid composition was not as good as that of ethyl acetate effect site,indicating that cholic acid The component is one of the main effect components of Arisaema cum Bile,and other components such as organic acids obtained by mass spectrometry analysis may also be the effect components of Arisaema cum Bile.From the current research,it can be seen that the types of bile acids in Arisaema cum Bile are clear,with strong specificity,and the content of the components is high.The bile acids can be used as quality markers related to the effect of Arisaema cum Bile.SummarizeIn this paper,it was found that Arisaema cum Bile water extract can reduce the rectal temperature of febrile convulsion model mice,prolong the convulsion latency,shorten the duration of convulsions,and up-regulate the expression of GAB AAR protein;at the same time,it can reduce the levels of serum cAMP and PGE2,and improve the morphological changes of hippocampal neurons.,down-regulate the protein expressions of COX-2,iNOS and GFAP in brain tissue,and reduce the mRNA expression levels of IL-1β,IL-6 and TNF-α in hippocampus tissue,indicating that Arisaema cum Bile has a good anti-febrile convulsant effect.Its mechanism It may be related to antipyretic and antagonizing brain tissue inflammation.The supernatant of the alcohol precipitation of the water extract of Arisaema cum Bile has the effect of inhibiting the inflammatory response of BV-2 microglia in the mouse brain.The ethyl acetate and n-butanol parts of the alcohol-precipitated supernatant are the antifebrile convulsions of Arisaema cum Bile effect site.UPLC-Q-TOF-MS/MS analysis showed that the effect parts mainly contain bile acids and organic acids.Among them,61 components were identified in ethyl acetate,18 organic acids and 17 cholic acids.;There are 72 components identified in the n-butanol site,23 components of bile acids and 11 components of organic acids.The HPLC analysis of the content of bile acids in the ethyl acetate part showed that the main bile acids contained in this part were hyodeoxycholic acid(HDCA)and chenodeoxycholic acid(CDCA),with an average content of 17.99%and 6.68%,respectively.These two components were not detected in the n-butanol fraction.The drug efficacy verification test shows that the bile acid composition(HDCA,CDCA)has a significant anti-inflammatory effect,and is one of the main effect components of the Arisaema cum Bile water extract against febrile convulsions.The bile acids in Arisaema cum Bile have clear categories,strong specificity and high component content,and bile acids can be used as quality markers related to the effect of Arisaema cum Bile.This study will provide the basis for the quality control and clinical application of Arisaema cum Bile.