Study On Degradation Of Salvianolic Acids

Relatively few researches are available on pharmaceutical process analysis of TCM.which seriously restricted the quality upgrade of final product.Safety and efficacy problem of TCM is often related to poor quality batch consistency that can be caused by the degradation of active ingredient in the drug product.Salvia miltiorrhiza Bunge(Danshen in Chinese)is common herbal medicine for many TCMs in the treatment of cardiovascular and cerebrovascular diseases.Among Salvianolic acids,known as active ingredients of Danshen,Salvianolic acid B(Sal B)is the most abundant of them.During manufacturing process and storage.Sal B undergoes degradation and transformation and its degradation prodouct.Lithospermic acid(LA),experiences further degradation.Therefore,Sal B and LA are taken as examples to explore the novel techniques and methods in pharmaceutical process analysis,to study the degradation kinetics and mechanism of active ingredients during manufacturing process and to find the key process parameters.These information is of great significance for upgrading the quality control of TCM.The major contents and achievements of this thesis are summarized as follows:1.A novel quantitative 1H NMR(Q-NMR)method was proposed for monitoring the concentrations of Sal B and its degradation products in process samples of degradation reaction.NMR pulse program was set as noesyprl d,probe temperature was set at 303 K,the flow rate of nitrogen was set at 535 L/h,scan numbers was set at 32,relaxation delay time was set at 15 s and total analysis time was about 11 mininutes.Degradation kinetic runs have been carried out to systematically investigate the effects of reaction temperatures,initial pH values and initial concentration of Sal B on degradation kinetic constants.The kinetic studies indicated that initial pH values had great impact on degradation rate constant.Arrhenius equation was obtained for degradation rate constant as a function of temperature,and half-life at 25 ℃ was predicted to be 98.57 days in a phosphate buffer solution at an initial pH value of 7.53.The results showed that the initial pH value,heating temperature and heating times were key parameters affecting the stability of Sal B.2.A novel Q-NMR method was proposed and validated for monitoring the concentration of LA and salvianolic acid A(Sal A)in process samples of degradation reaction.Degradation products of LA were tentatively structural elucidated with the help of high performance liquid chromatography tandem mass spectrometry(HPLC-MSn).A possible degradation pathway of LA was proposed.NMR pulse program was set as noesyprld,probe temperature was set at 293 K,the flow rate of nitrogen was set at 400 L/h,scan numbers was set at 32,relaxation delay time was set at 15 s and total analysis time was about 10 mininutes.The results indicated that between initial pH values 4.75 to 6.95,Sal A was the primary degradation product of LA.In this research,apparent degradation kinetic constants for LA degradation,LA transform to Sal A and further degradation of Sal A were all achieved in one run and correlation coefficients of fitting were higher than 0.98.3.HPLC preparation combined with high performance liquid chromatography-mass spectrometry(HPLC-MS)method was employed to study degration mechanism of Sal B.Seven products of Sal B were purified using preparative HPLC and five of which were structurally elucidated with the help of HPLC-MS and NMR.Process samples of degradation reaction under different initial pH values were analyzed using HPLC coupled with high resolution mass spectrometry and HPLC-MSn method.Degradation products of Sal B were tentatively structurally elucidated.Subsequently,a possible degradation pathway of Sal B was proposed and part of the pathway was verified.The results indicated that the degradation pathway of Sal B differed greatly under different initial pH values of degradation solution.Sal B could easily degrade to Salvianolic acid E(Sal E)through ring opening of benzofuran under initial pH value of 7.0,however under the initial pH value of 4.0 it trended to generate LA through ester bond hydrolysis.Degradation intermediates Sal E and LA would undergo further degradation.The results showed that the initial pH value was key parameters influencing the degration mechanism of Sal B.4.A Sal B degradation kinetic model was established.Q-NMR spectra of degradation process samples of Sal B were pretreated to obtain accurate time-concentration data of Sal B and its degradation products using line fitting function of MestReNova.Kinetic constants for degradation products of Sal B were fitted to data obtained from kinetic runs and the degradation pathway of Sal B.Subsequently,a kinetic model of Sal B degradation was obtained.