Chemical And Pharmacological Comparison Of Raw And Vinegar-baked Radix Bupleuri

Ethnopharmacology:Radix Bupleuri (RB, Chinese name is Chaihu), an herbal medicine in TCM, has been popularly used in herbal medicines in China for several thousand years. Processing of herbal drugs has been a part of the heritage of Chinese medicine for thousands of years, it plays an important role in disease prevention and control for the Chinese people, and ensures the safe and effective clinical treatment of TCM. The processing of RB with vinegar to prepare vinegar-baked Radix Bupleuri (VBRB) has a long history in the clinic of TCM. The pharmacological effects and components in the drug will change a little bit due to the vinegar-baking procedure. Previous reports showed that the contents of saikosaponin b1 (ssbl) and saikosaponin b2 (ssb2) were increased, while the saikosaponin a (ssa), saikosaponin c (ssc), and saikosaponin d (ssd) were decreased after the vinegar-baking process. And after being processed with vinegar, both the yields and compostions of volatile oil in Radix Bupleuri could be changed. It was demonstrated that VBRB had much stronger effect on soothing liver, bile secretion, and acesodyne and than that of the RB. The detoxification of VBRB was also evaluated. However, the chemical compositions of raw and processing RBs were not analysed comprehensively and systematicly, The pharmacological effects of VBRB has been evaluated to some degree by observations of histopathology examination and clinical biochemistry assessments, the mechanistic details of VBRB remain uncertain and have not been fully explained from a holistic perspective. In addition, according to Chinese Pharmacopoeia, RB should be baked with rice vinegar to give VBRB. However, there are many kinds of vinegars present in the Chinese market, and if they were used in the vinegar-baking process of herbal drugs, and the influence of different vinegars on the volatile compounds in VBRB remains unknown.OBJECTIVE:To comparison of raw and vinegar-baked RBs using many technologies. Then to compare their pharmacological effects on the liver and the toxicity effects, and find the potential biomarkers. To explore the mechanisms of therapeutic effects and toxicity in metabolite levels initially. Further more, two different VBRBs were also compared in chemical and pharmacological effects to elucidate the vinegar type on vinegar-baking process of RB. This apply theoretical basis to ensure the therapeutic effect and safety of RB and VBRB in clinic.METHODS:In this study, two different vinegars (rice vinegar and Shanxi vinegar) were used to prepare VBRB. Then the chemical difference between raw and two processed RBs were compared by based 1H-NMR fingerprinting approach. Then to investigate and compare the chemical differences of the volatile oils between crude and vinegar-processed RB samples by GC-MS coupled with multivariate data analysis. HPLC was applied to quantative analysis of saikosaponins in RB and VBRBs. In addition, their liver protective effects were also investigated using CCl4 induced liver injury animal models. Then focuses on the toxic evaluation of them in a rat model, metabonomics were used to evaluate the mechanisms of therapeutic effects and toxicity. The Two different VBRBs were also compared to elucidate the vinegar type on vinegar-baking process of RB.RESULTS:1. In the study of chemical composition, the two vinegar were compared firstly. The pH of them was different, and 1H-NMR provided the overall outlook of vinegae, which can be seen the differences of them intuitively.(1) The chemical composition between raw and two processed RBs were systematically compared by 1H-NMR spectroscopy coupled with multivariate analysis. A total of 36 metabolites were identified in the NMR based metabolic fingerprint of RBs, and clear differences could be seen between the raw and two processed RBs. The vinegar-baking process induced chemical changes included elevated contents of ssb1, ssb2, acetic acid, malic acid, as well as the decreased contents of ssa, ssd, sucrose, succinic acid, etc. And clear differences could be seen between the two processed RBs, the variations between two different VBRBs were observed in the levels of sucrose, glycine, pyruvic acid,et al.(2) GC-MS coupled with multivariate data analysis was applied to compare the volatile components between crude and two vinegar processed RBs. After vinegar-baking, not only the oil yields were decreased significantly, the chemical compositions were also changed, including the disappearance (such as β-pinene, n-nonaldehyde, and verbenol) or appearance (such as 2-nonyl acetylene, a-cubebene, and nerolidol), as well as the content increase or decrease of some volatile compounds (such as dodecanal, benzaldehyde,and guaiacol). The oil yields of two different VBRBs showed no significant difference, but differed markedly in their chemical compositions (such as Pentanal, Benzaldehyde, Stearic acid).(3) HPLC was used to quantify four saikosaponins (ssa, ssd, ssb1, ssb2) in the crude and vinegar-baked RBs, and the results were shown that the contents of ssb2 and ssb1 increased dramatically, while the ssd and ssa decreased after vinegar-baking process, this consistent with literature reports. Two different VBRBs also showed differences in the saikosaponins contents. 2. In the study of pharmacological effects, The liver protective effects against CCl4 induced mice liver injury between the raw and two processed RBs were also investigated.1H-NMR metabolomics was applied to liver analysis,9 biomarkers were identified, including alanine, phenylalanine, and taurine etc, and the corresponding metabolic pathways associated with these changes were the amino acid metabolism and taurine metabolism. Both raw and processed RBs exerted liver protective effects against CCl4 induced liver injury, which were manifested by the improvement of histopathological change, the decrease of ALT and AST, and the return of some different metabolites to normal level. These results indicated that the two vinegar-baked RBs showed better recovery trend than that of raw RB, and the mice in S-VBRB showed a better trend of recovery than R-VBRB group.3. In the study of toxicity, the traditional toxicity evaluations showed that the raw and procesed RB treatments elicited hepatotoxicity, as characterized by the elevated serum ALT, AST and ALP activity, and liver histopathologic alterations. In order to exploit the toxic mechanism of RB,1H-NMR based metabonomic technique was used to study the alterations of metabolic profiles. Some different metabolites were identified in serum, liver extract, and kidney extract. Methionine, glutamine, and glutamate could consider to be a risk factor for liver disease, and taurine, ornithine, and s-inositol, may considered as sensitive biomarkers to predict renal disease. The corresponding metabolic pathways associated with these changes were the amino acid metabolism, energy metabolism, taurine metabolism, and lipid metabolism. The raw and procesed RBs had slight toxicity effects on liver and kidney, while there were no significant differences between the three treated rats. Then based on the biochemical indicators and metabolomics, the two vinegar-baked RBs showed low toxicity than that of raw RB, and the rats in S-VBRB showed low toxicity than R-VBRB group.Conclusion:1. The raw and vinegar-baked RB differed not only in the chemical compositions but also the pharmacological and detoxification effects. The results indicated that the chemical compositions were also changed after processing. And VBRB showed better liver protection effect and low toxicity. Two different VBRBs also showed differences in the chemical compositions and biological effects, the S-VBRB had better pharmacological effect than R-VBRB.The results obtained in this study implied that the type of vinegar is important for vinegar baking. Thus, in order to ensure the therapeutic effect and safety of VBRB in clinic, the role of vinegar in the processing as well as the effect of different vinegars on the processing should be further investigated.2. In this study, NMR metabolomics were applied to compare the chemical components, pharmacological and toxicity effects between raw and vinegar-baked RBs, which reflect NMR has distinct advantages of globality and high sensitivity.