Studies On The Degradation And Dissociation Of Several Salvianolic Acids

Salvia miltiorrhiza is an important traditional Chinese medicine with blood-activating and stasis-dissolving effect. Phenolic acids in Salvia miltiorrhiza, such as Salvianolic acid B, lithospermic acid, Salvianolic acid A, Danshensu, and Salvianolic acid C, possess anti-oxidant, anti-hypertensive, anti-fibrotic and neuroprotective activities. The degradation and dissociation of salvianolic acids widely exist during the manufacturing of Salvia miltiorrhiza preparations. Salvianolic acids with different activities can generate from degradation reactions, which contribute to possess complicated pharmacological effects on multiple targets. However, these phenomena also affect the batch-to-batch consistency and stability of Salvia miltiorrhiza preparations. In this thesis, the degradation and dissociation characteristics of several salvianolic acids were investigated. The main contents and achievements are summarized as follows:1. The kinetics model of Salvianolic acid B degradation was developed. Higher pH value, higher extraction temperature, and longer extraction time led to more degradation of Salvianolic acid B. A kinetics model was developed considering the degradation of Salvianolic acid E, lithospermic acid, Salvianolic acid B, and reverse reactions of Salvianolic acid B degradation. Degradation kinetic constants were calibrated by fitting the experimental data to proposed ordinary differential equations. Small absolute average deviation values also indicated a close agreement between experimental results and calculation results. Verification experiments were carried out. The degradation kinetics rate constants of salvianolic acids in a Salvia miltiorrhiza extract were predicted using this model. The predicted values agreed well with the experimental results, which means that kinetics model was predictive.2. The kinetics model of Salvianolic acid B degradation was applied in the optimization of Salvia miltiorrhiza extraction process. Using the kinetics model, critical process parameters (CPPs) of Salvia miltiorrhiza extraction process were found to be extraction temperature, extraction time, and pH value using sensitivity analysis. A design space with a minimum probability of 90% to attain extraction process evaluation indices was calculated with the disturbances of CPPs simulated using a Monte Carlo method. The design space was verified successfully, which indicated that the developed kinetics model is predictive and could be applied in the parameter optimization of Salvia miltiorrhiza extraction process.3. A monitoring method of lithospermic acid degradation process using online UV spectroscopy was developed. According to identified degradation products, the degradation pathways of lithospermic acid were deduced, which were isomerization, hydrolysis, decarboxylation, and oxidation. The degradation of lithospermic acid can be described using a first order irreversible kinetic equation. The degradation rate of lithospermic acid increased at higher pH values and decreased when contacting with less oxygen. After decompositing the spectra collected during degradation process using MCR,3 components were obtained. They were considered to be lithospermic acid, lithospermic acid isomer and other degradation products, respectively. To obtain more components in MCR, UV spectra collected in different experiments were suggested to integrate. A quantitative model between the UV spectra and lithospermic acid concentrations was developed using PLS. The prediction performance of this model was satisfactory.4. The dissociation constants for salvianolic acidts were determined using a liquid-liquid equilibrium method. A new method to determine the pKa values of phenolic acids, such as salvianolic acids in Salvia miltiorrhiza extracts, was developed. The pKa values of phenolic acids can be fitted according to the quantitative model between aqueous phase pH values and apparent distribution coefficient values. The pKa values of several phenolic acids in a mixture with most components unknown can be determined simultaneously using this method. Distribution coefficient values of phenolic acid molecules and ions can also be obtained. Distribution coefficients and pKa values are useful data in solvent selection and parameter optimization of purification processes for phenolic acids. These data can also be used in phenolic acids related preparation processes. The pKa values of Salvianolic acid B, lithospermic acid, rosmarinic acid, Danshensu, Salvianolic acid A, and caffeic acid in Salvia miltiorrhiza extracts were obtained. The pKa values of lithospermic acid and Salvianolic acid A were reported for the first time.