| Most Bupleurum species are perennial herbs, up to150cm in height with compoundumbels. The fowers are bisexual, yellowish or rarely purplish with fve stamensand thefruits occur mostly as cremocarps. Leaves are simple, long, slender and alternate withentire margin. The genus is represented by180-190species, which are widely distributedin the Northern Hemisphere and commonly used in Eurasia and North Africa for theirmedicinal properties. Dried roots of B. scorzonerifolium Willd.and Bupleurum chinenseDC were selected as the plant sources of Bupleurum in Chinese Pharmacopoeia (2010).In Asian traditional medicine, several Bupleurum species have been used either alone orin combination with other ingredients for the treatment of common cold, infammatorydisorders, hepatitis, cancer and fever. Bupleuri Radix, with a Chinese name Chaihu, isrecorded as the roots of Bupleurum chinense and B. scorzonerifolium (Apiaceae) in heChinese Pharmacopoeia, which has been widely practiced to reat infuenza, fever,infammation, malaria and menstrual disorders. Even worse, B. longiradiatum, widelydistributed in northeastern mainland China. The misuse of B. longiraiatum as Chaihu hadcaused several cases of human poisoning, and at least three people died afteradministration of this plant,which showed symptoms such as serious nausea, vomiting,twitching, opisthotonus, and so on. Modern pharmacological studies confirmed thatpolyacetylenes were the major neurotoxic compounds in the plant. And they are widelydistributed in the food plants and herbs. These compounds have potential pharmacologicalactivity, but also has the potential neurotoxicity, hence it is need the further study of itstoxicity, which is of great significance for human health and safety applications for herbs.So far, these compounds have been reported only that its neurotoxic effects, themechanism of toxicity is not studied enough, the role of the target organ and the target sitehave not been reported. In addition, other toxic effects are still poorly understood for thesecompounds. Although there are mature methods about chemical toxicity study for thesafety evaluation,these methods mainly concentrated in the acute and long-term toxicitytest. The indexes mainly focus on the morphological changes and biochemical indexes,which cannot reveal the characteristics of drug toxicity comprehensivly. therefore, we needto establish new research methods to clarify its toxic effects. In this study, we have isolatedand idetified the toxic compound Bupleurotoxin from plants of B. longiradiatum, toexplore toxic mechanism of this kind of compounds. The toxicity study was based on Metabonomics, which is aimed to identify toxicity biomarkers and to illustrate organismon the overall response of the compounds; In this study, the acute toxicity found themedian lethal dose of bupleurotoxin was3mg/kg. Metabolomics studies have identified17markers in serum, and11markers in urine, these biomarkers are ralated to tryptophanmetabolism, purine metabolism, inflammation, indicating the the toxicity of bupleurotoxinare closely related to kynurenine pathway, oxidative stress and inflammation. Furthermechanisms studies found that bupleurotoxin inhibited the GABA receptors resulting inthe generation of neuronal excitability, excessive discharge of nerve cells, a large influx ofcalcium ions, resulting in the death of nerve cells, and GABA receptors is the initial effecttargets of bupleurotoxin. Thus, this study revealed the mechanisms of toxicity ofbupleurotoxin, it is of great significance for for the study of mechanisms of toxicity ofpolyacetylenes.This thesis consists of5chapters, the chapter2-5as follows:The second chapter: Study on acute toxicity of Bupleurotoxin. In this study, thebupleurotoxin was isolated from total extract of B. longiradiatum plant by various columnchromatography, and the structure identification based on spectra data. The LD50for acutetoxicity test is used the bliss method.The third chapter: The metabonomic study of the toxicity of bupleurotoxin.In the study of metabolomics, bupleurotoxin was injected to mice by intraperitonealinjection, There were obvious poisoning symptoms of bupleurotoxin treated mice. Ametabolomic approach using reversed-phase liquid chromatography/quadrupoletime-of-flight mass spectrometry (LC-Q-TOF-MS) was developed. By combining withpartial least squares discriminant analysis (PLS-DA),17biomarkers in mice serum wereidentified. Therse identified biomarker are mainly related to tryptophan metabolism, purinemetabolism, inflammation, and oxidative injury process. It is demonstratd that kynureninepathway, oxidative stress, inflammatory reaction is closely related to pathological changesof bupleurotoxin treated mice. On the other hand, we also investigated the toxic effect ofbupleurotoxin using metabolomic method in mice urine,11biomarkers in mice serum wereidentified by combining with OPLS. The regulated biomarkers are mainly related to thefatty acid metabolism, Phenylalanine metabolism, PGE2biosynthesis and inflammationpathways. These biomarkers demonstratd that lipid peroxidation, oxidative stress,inflammatory reaction are closely related to pathological changes of bupleurotoxin treatedmice. These results provide some information for the study mechanism of bupleurotoxin. The fouth chapter: the relationship between TH1and TH2immune imbalance andneurotoxicity. In the study, we found a great relationship between TH1/TH2immuneimbalance and neurotoxicity. Some cytokines of poisoning mice serum were significantlyhigher than that in normal group, which explains the bupleurotoxin disrupted TH1/TH2immune balance, and then the inflammation cytokines may cause disorder of tryptophanmetabolism.The fifth chapter: the neurotoxicity of bupleurotoxin and related receptors. In ourstudy,we found that bupleurotoxin can inhibit activity of GABA receptor, which can causethe biological cascade reaction resulting in the excitability of nerve, calcium influx, leadingto neuronal cell death. |
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