Study On The Rule And Mechanism Of Degradation Of Ferulic Acid And Its Application In Drug Quality Control

As one of the most important parts of excellent Chinese traditional cultures, traditional Chinese medicine (TCM) was widely available and accepted during past hundreds of years, and nowadays more and more speciouses and forms were emerged prominently. However, there are several reports about adverse reaction and worse consequences for the past few years during the clinical application. They maybe caused by the unkown materials, even the products of transformation from the well-kown unstable materials. What's more, there are several kinds of unstable conpounds in the plant amedica or TCM, such as resveratrol, resveratrol glycoside and so on. And stability is one of principal factors in drug quality, which always caused great influence of the safety and effectivity of drug. Besides, a variety of drug went on sale was unstable, in some of which contained no exact researches. Therefore, study of the stability of them was still necessary after the registration of pharmaceuticals.Recently, a few researchers noticed ferulic acid (FA) was unstable in solution, but the study was not in detail, and then the stability had drawn our attentions. FA, which named 4-hydroxy-3-methoxy cinnamic acid, was obtained by both synthesis and extracting from TCMs. It generally serves pharmacological functions, such as anti-oxidation, anti-inflammatory, relieving pain and so on. In clinical, the injection of Sodium Ferulate (SF, sodium salt of FA) had been widely used to cure cardiovascular and cerebrovascular diseases. FA also widely existed in several herb frond as active or index substance, such as Rhizoma Chuanxiong, Radix Angelicae Sinensis, Resina Ferulae, Herba Equsieti Hiemalis and so on, which always took it as one of the marker compounds for their quality assessment.In this paper, FA was taken as an example of these components in TCM. Modern methods like LC, DAD, 1H-NMR, MS and prep-LC, were used in analysis, separation and identification of the degradation product. According to the investigation of stability testing, the rule and mechanism were furtherly discussed. All these may provide basic method and theory for stability study in the quality control. From the study, the conclusion can be reached that: (1) The HPLC method to analyse FA was established. Comparing the influence of a variety of environmental factors such as temperature, light, concentration and pH of solution, light is the most principal one of its stability. (2) Exposed solution of FA to light, the light degradation product was gained by column chromatograph and prep-LC. The production was verificated to be cis-FA by DAD, 1H-NMR and MS. (3) The concentrations of trans- and cis- FA were determined by HPLC using external reference method. The reaction of degradation belongs to 1-1 grade opposing reaction. The equilibrium constants at 4℃,23℃,40℃,60℃were calculated, then the activation energy was evaluated to be 20.51kJ·mol-1 and 35.68kJ·mol-1, respectively. (4) There is no acute toxicity by giving the mix of trans- FA and cis- FA to two groups mouse separately, according to the method in Chinese Pharmacopeia. (5) The rate of plasma protein binding determined by hollow fiber ultrafiltration was coincident with the one valuated by tradition equilibrium dialysis. The rate of plasma protein binding trans- FA and cis- FA were 23.8% and 31.0%, respectively. The difference of these may be caused by the distinction of polarity, but there were no significant differences by t-test.PART 1 Study on the Rule and Mechanism of Degradation of FA(1) Analyses FA and its degradation, simultaneously study the infulence factors of its stabiliityObjective: Compare the influence of a variety of environmental factors such as temperature, light, concentration and pH of solution and discuss the crucial factor of its stability.Methods: An HPLC method was applied to determine the contents of FA. The analysis was performed on a Hypersil ODS C18 column (4.6mm×150mm,5μm particles) with methanol: acetonitrile:1% acetic acid solution(15:10:75) as a mobile phase. The isocratic elution was 1.0 mL·min-1 at 320nm using a UV detector at room temperature. Fit the kinetic equations and calculate the degradation by determining the remained concentration in the solution.Results: The calibration curve of FA was shown to be linear over the range from 8.00 to 1.02×103μg·mL-1. The recovery of the method was more than 99.5%. Determine the remained content in the solution and calculate the average of their content, it well fit the first order among the equation of zero level, first order and second order. The stability was better in neutrality pH of the solution than in acid or alkaline environment. The rate constants of degradation raised follow the increase of the temperature and intensity of illumination. The rate seems hardly to be any.Conclusion: A simple and rapid method to determine the content of FA was developed. The results suggested that ferulic acid is sensitive to light rays. Comparing to the other environmental factors like temperature, concentration and pH of solution, light is the crucial factor of its stability.(2) Separation, identification and character study of main degradation product of FAObjective: Identify the structure of the main degradation product of FA, and study its character.Methods: Exposed solution of FA to light, separate the product by column chromatograph and prep-LC and verification it by DAD, 1H-NMR and MS.Results: The light degradation product gained was definite to be cis- FA, since these cues below: the m/z of the production was 193 illustrated by LC-MS; NMR analyses result confirmed that theδof H were just different from the ones surrounded to the double bond; the UV spectrum of absorption band was appearred to blue shift illustrated by the LC-DAD.Conclusion: The product of the main degradation of FA was identified to be cis-form, which formed as yellow oil, with purity higher than 95.0%.(3) Study the kinetics of FA's light transformation and approach the rule and mechanism of photoisomerizationObjective: Study the rule of FA transformation under light irradiation and reveal the mechanism of its photoisomerization.Methods: Simultaneously determine the contents of trans- FA and cis- FA by HPLC, reckon the rate constants of forward and backward reaction. Calculate the activation energy by Arrlenius empirical formula.Results: Both the forward and backward reaction fitted to the first order kinetic procession. The concentrations of trans- FA and cis- FA were determined by HPLC using external reference method. The reaction of degradation belongs to 1-1 grade opposing reaction. The equilibrium constants at 4℃,23℃,40℃,60℃were calculated, then the activation energy was evaluated to be 20.51kJ·mol-1 and 35.68kJ·mol-1. What's more, the isomerization under light was happened by overcoming the transition state in theory.Conclusion: A simple HPLC method to determine the contents of trans- FA and cis- FA simultaneously was established. Several kinetic parameters of this photoisomerization reaction like rate constants, equilibrium constants and activation energy were reckoned by both position of forward and backward. The rule and mechanism was coincident to isomerization reaction.PART 2 Initial researches of the abnormal toxicity and protein binding rate of cis- FA(1) Study of the abnormal toxicity of cis-FAObjective: To study the acute toxicity reaction of praeparatum of FA in which contained its degradation productMethods: Give FA mixed solution by the method of oral adminsreation and intravenous injection, test their abnormal toxicity according to the method in Chinese Pharmacopeia. Record the immediate reaction of two groups and observe their reaction in a week separately.Results: Intragastric administration and caudal vein injection administrat to two groups of mouse, record the immediately reaction and observe their reaction in a week, there was no death occurred.Conclusion: There is no acute toxicity by giving the mix of trans- FA and cis- FA to two groups (each with 10 mouses) separately, according to the method in Chinese Pharmacopeia.(2) Determination the rate of plasma protein binding determined of trans- FA and cis- FA by HPLC using hollow fiber centrifugalization ultrafiltrationObjective: Determine the rate of plasma protein binding determined of trans- FA and cis- FA, and compare the difference between their isomers.Methods: Determin the free concentration in plasma of these isomers by HPLC using hollow fiber centrifugalization ultrafiltration method, The analysis was performed on a Hypersil ODS C18 column (4.6mm×150mm,5μm particles) with methanol:acetonitrile:1% acetic acid solution(12:10:78) as a mobile phase. The isocratic elution was 1.0 mL·min-1 at 320nm using a UV detector at room temperature, and calculate the rate of plasma protein binding of them.Results: The calbration curves of trans- FA and cis- FA were shown to be linear over the ranges of 4.00×10-2~2.56μg·mL-1 and 3.10×10-2~1.94μg·mL-1, respectively. Mean recoveries were more than 95.1% and 94.1%. Solitude study the trans- FA, the rate of plasma protein binding was 24.3%. The rate of plasma protein binding trans- FA and cis- FA were 23.8% and 31.0%, respectively. The difference of these may be caused by the distinction of polarity, but there were no significant differences by t-test.Conclusion: The rate of plasma protein binding determined by hollow fiber centrifugalization ultrafiltration was coincident with the one valuated by tradition equilibrium dialysis. The method employed in present finding is easy to use within only one step, rapid since each sample involves treatment for a while, and economical as a sample pretreatment technique.