Research Of Quality Evaluation Method And Effect On Liver Function Of Bupleurum Chinense DC. Based On Metabolomics

Topic Basis:Radix Bupleuri（RB,Chinese name is Chaihu）,a commonly used herbal medicine,with the efficacy of relieving fever,smoothing liver,and lifting Yang-Qi.In the Chinese Pharmacopoeia,Chaihu is recorded as the dried roots of Bupleurum chinense DC.and Bupleurum scorzonerifolium Willd.（Umbelliferae）.Although the pharmacopoeia clearly states that medicinal sites are roots,with the rising price of RB,there are different degrees of incorporation of non-medicinal parts（aerial parts）in the medicinal materials of RB on the market.Does the incorporation of medicinal parts affect the quality of RB?What is the difference in the chemical composition between the medicinal and non-medical parts of RB?Vinegar baked Radix Bupleuri（VBRB）also records in the Chinese Pharmacopoeia.What is the difference in chemical and pharmacological effects between raw and Vinegar baked of RB?The previous study of our research group found that the chemical profile of Radix Bupleuri has changed obviously,but there is no specific quality control indicator of VBRB.The traditional efficacy of RB is a functional effect on the liver.Modern pharmacological studies have shown that RB has protective effect on the liver,but it can also lead liver injury when it was used improperly.How does the dosage or compatibility of RB have protective or damaging effects on the liver?In order to provide basis for quality evaluation and rational clinical use of RB,the chemical differences between roots and aerial parts of RB,and between raw and vinegar baked RB were compared using metabolomics.The effects of different combinations in routine treatment doses of RB were also investigated.Objective:（1）To comprehensive characterization of the chemical components and to find differential metabolites and sensitive chemical markers between roots and aerial parts of RB,raw and vinegar baked RB.The purpose is to find differential metabolites as chemical markers that control the incorporation of residual stems and control the quality of vinegar products,and to provide experimental data for the material basis for differences in efficacy between raw and vinegar baked RB.（2）To define the degree of difference in liver protection between roots and aerial parts of RB,raw and vinegar baked RB.（3）To explore the effect on the liver of RB and Xiaoyao Powder and Xiaochaihu Decoction.To clarify the effects of dose,compatibility environment and different application conditions of RB on the liver.（4）To optimize modeling dose and duration of CCl₄-induced chronic hepatic injury models.To provide an experimental basis for the application of this model.Method:（1）Phytochemical analysis was performed using the ¹H NMR and UHPLC/ESI Q-Orbitrap HRMS metabolomics techniques to determine the components of roots and aerial parts of RB,and vinegar baked RB to identification of compounds.Combining multivariate statistical methods,the differences between roots and aerial parts of RB,and raw and vinegar baked RB were analyzed to find differential metabolites.The qualitative identification and relative quantification of differential metabolites were carried out.Compounds with large differences in content were selected as quality markers for distinguishing roots and aerial parts of RB.（2）The difference of hepatoprotective efficacy between roots and aerial parts of RB,and raw and vinegar baked RB were evaluated using the CCl₄-induced mouse acute liver injury model by observing serum transaminases and liver histopathological sections,combined with liver tissue ¹H NMR metabolomics studies.The degree of difference between different RB was quantitative description from the quantity and extent of metabolite regulation.（3）The plasma transaminases of patients with depression before and after treatment with Xiaoyao Powder were determined.The degree of liver damage caused by depression was investigated.And the effect of RB on the liver of patients was studied under Xiaoyao San compatibility environment and therapeutic dose.（4）¹H NMR metabonomics approach was used to reveal the chemical difference of urine between patients with Xiao-Chaihu-Tang syndrome and healthy participants.The partial least squares method was used to establish a model to distinguish the patients with Xiao-Chaihu-Tang syndrome from the healthy controls.The different endogenous metabolites were identified.The relationship between the Xiao-Chaihu-Tang syndrome and liver diseases was investigated based on the analysis of disordered metabolic pathways,as well as the effect of Xiaochaihu Decoction on the liver during the treatment.（5）Five different doses of CCl₄ were administered to rats to induce a chronic liver injury model.The general model of animal,body weight,organ appearance,liver histopathological observation,serum biochemical test,and organ coefficient were used as indicators to conduct a standardized study of the models.The model was dynamically tracked and evaluated using urine ¹H NMR metabolomic analysis.Results:（1）The chemical components of roots and aerial parts of RB,and raw and vinegar baked RB comprehensively analyzed using ¹H NMR and UHPLC/ESI Q-Orbitrap HRMS plant metabolomics techniques.Thirty-seven compounds recognized by ¹H NMR spectra are mainly primary metabolites such as sugars and amino acids,and secondary metabolites with high content such as saikosaponin a and saikosaponin d.Thirty-two compounds identified by UHPLC/ESI Q-Orbitrap HRMS are all secondary metabolites in the plant.The ¹H NMR and UPLC/ESI Q-Orbitrap HRMS metabolomic analysis results showed that roots and aerial parts of RB could be clearly separated in the PCA score plot,indicating that the chemical composition of roots and aerial parts of RB were significantly different.Differential metabolites mainly belong to flavonoids,phenylpropanoids and saikosaponins.The largest difference in content is rutin with a difference of up to 50 times.According to the regulations in the Chinese Pharmacopoeia（2015 Edition）,Part IV0212 General Requirements for the Examination of Medicinal Materials and Pieces,the stems of RB should be considered as the impurity and mixed into the roots of RB with 3%（by weight）.The samples can be clearly separated in the PCA scatter plot,indicating that the chemical composition has changed obviously.The ¹H NMR and UPLC/ESI Q-Orbitrap HRMS techniques were used to analyze raw and vinegar baked RB,and the two samples were clearly separated in the PCA analysis scatter plot.Twelve compounds recognized by ¹H NMR spectra are mainly primary metabolites except saikosaponin a.Ten compounds identified by UHPLC/ESI Q-Orbitrap HRMS are all secondary metabolites in the plant,including saikosaponins and acetyl saponins.（2）The results of serum ALT and AST showed that they were significantly increased in the CCl₄ group,indicating successful replication of the model.Both root of RB and VBRB could significantly decreas serum transaminase（p<0.05）,which had obvious hepatoprotective effect.Although the aerial parts of RB had a tendency to reduce ALT and AST,there was no significant difference compared with the CCl₄ group,indicating that the aerial parts of RB had no hepatoprotective effects.The results of liver pathological section observation showed that liver tissue pathological damage caused by CCl₄ could restored by both root of RB and VBRB.The pathological of hepatic tissue in the group of aerial parts of RB had no obvious difference with the CCl₄ group.Fourteen different metabolites were found,involving energy metabolism,lipid metabolism,fatty acid oxidation,oxidative stress,nucleoside metabolism and amino acid metabolism.RB,aerial parts of RB,and VBRB showed effect as revealed by the amount and regulatory degree of the perturbed metabolites.The metabolism pathways analysis showed that the liver protective effect was related with energy metabolism（including the tricarboxylic acid cycle and glycolysis）,lipid metabolism,amino acid metabolism and nucleic acid metabolism,and glutathione metabolism.The results of determination of plasma ALT and AST in patients with depression showed that the vast majority were within the normal range,and transaminase values in individual patients with transiently elevated levels had no clinical diagnostic significance.Depressive patients have not yet achieved the pathological features of liver injury,but 26patients felt"sore rib discomfort"symptoms before the treatment.Xiaoyao Powder treatment for 8 weeks had no effect on transaminase,"sore-rib pain"symptoms of patients disappeared after treatment.（4）Clinical observations showed that the symptoms of"right upper abdominal pain","rib pain","right flank tingling pain"and other symptoms related to liver were improved or cured after treatment of Xiaochaihu Decoction.It has a protective effect on the liver of patients with Xiao-Chaihu-Tang syndrome.The partial least squares method was used to establish a model to distinguish the patients with Xiao-Chaihu-Tang syndrome from the healthy controls.（5）The results of organ appearance and organ coefficient showed that 0.02 mL CCl₄/100 g modelling dose was not likely to affect other systems.Serum ALT and AST measurements increased from 2nd week to 4th weeks,and decreased from 6th week.Urine metabolomics experimental results showed that CCl₄ caused significant changes in metabolic profiles in rats;and with the prolongation of modeling time,the changes became more and more significant.Conclusion:（1）The incorporation of 3%of the aerial parts in root of RB will impact on the quality of the medicinal materials significantly,indicating that the requirement of Pharmacopoeia that no more than 3%of the incorporation of crumb impurities into pieces is reasonable.Rutin can also be used as a quality marker to control the incorporation.The aerial parts of RB contains a large amount of flavonoids and phenylpropanoid compounds,and should be further studied to develop its medicinal value.（2）The protective effect of VBRB on acute liver injury of CCl₄ is stronger than that of raw RB,suggesting that VBRB should be the first choice for the treatment of liver diseases.（3）Although not reflected in the transaminase results,the depressive state may had affected the liver function of the patient,but substantial damage has not yet occurred.Xiaoyao Powder,which mainly involves soothing the liver and invigorating the spleen,can protect the liver function of the patient at doses of 30 g/day to 45 g/day,and it does not cause damage to the liver.（4）Xiaochaihu Decoctionas had a protective effect on the liver of patients on the basis of correct syndrome differentiation,with a dose of 24 g/d of RB.（5）The appropriate dosage of chronic liver injury induced by CCl₄ in rats was 0.02 mL CCl4/100 g,and the appropriate time was 4 to 6 weeks.Under this condition,rats showed obvious symptoms of chronic injury.Too much dose and too much time will not only seriously damage the liver,but also involve other organ systems and cause irreversible damage to the animal.