| Traditional Chinese Medicine(TCM) is traditional medicine in China and has unique effect on the clinical treatment through long-term practiceand studies. Since Aristolochic Acid Event, Safety of TCM caused widespread concern in the world, which has seriously hampered the development process of internationalization of TCM. Adverse reactions is accompanied by TCM simultaneously. From the first "Shen Nong’s Herbal Classic", TCM is divided into upper, middle and lower grades. Low-grade TCM refers to toxic medicine, which denies long-term use. Commonly toxic traditional Chinese medicine can be divided into five categories, namely alkaloids, toxic glycosides, toxic protein, animals and minerals. Toxic glycosides can be divided into cardiac glycosides, saponins and cyanogen glycosides. In order to ensure the safety of TCM, it need to establish standard limit of safety for toxic substances.Toxic medicine of Cyanide glycosides as a starting point for research. Cyanogenic glycosides, formed by condensation of cyanohydrin and sugar, are not only toxic ingredients, but also active ingredients in the TCM. The representative of cyanide glycosidesis is amygdalin, which is the active ingredient of Bitter almond, peach kernel and Yu Li Ren. Amygdalin main pharmacological activity generated by in vivo hydrolysis generates hydrogen cyanide, which can treat cough and asthma at the small dose, but large doses can lead to poisoning. In the Chinese Pharmacopoeia 2015, the content of amygdalin is not less than 3.0% in bitter almonds. Chinese patent medicine containing bitter almond are 66 species, in which Ting Bei capsules specifies the content of amygdalin is not less than 0.55 mg in each capsule(0.35 g). However, as endogenous toxic ingredient at the same time, safety limit of amygdalin is still blank.This thesis focuses on standard limit of safety for amygdalin of the cyanide glycosides medicine. Amygdalin generally form a pair of epimers, named D- and Lamygdalin, can be transformed into each other under certain conditions. D-amygdalin is present naturally and has a clear pharmacological activity, while the active mechanism of L-Amygdalin has not been reported. So we discussed the D- and Lamygdalin in vitro enzymatic conversion processes and toxigenic differences. We study the problems of existing form, content, transformation of amygdalin and detection of toxic metabolites hydrocyanic acid, which can provide scientific basis for establishment of standard limit of safety for amygdalin in Chinese patent medicine. This thesis is divided into four chapters.The first chapter is the introduction of TCM. we made a brief introduction of status and development of the process of internationalization of TCM. With frequent adverse reactions events, security has become a key issue to be solved in the development of traditional Chinese medicine. We presented an overview of the existing evaluation mechanisms of toxic substances. Secondly, it introduced the types toxic drugs, distribution, structure, metabolism in vivo and toxin-producing mechanism of cyanide glycosides. Since cyanide glycosides itself is both active ingredient and toxic ingredient, so the determination of cyanogenic glycosides in Chinese patent medicine is very important. Therefore, as the representative to amygdalin, test methods of its prototype and metabolites of HCN were summarized. These provided a reference basis for our further study. Finally, the study basis and the main contents were proposed.In the second chapter, based on in vitro enzymatic process of amygdalin epimers, activity differences of D- and L-amygdalin was preliminary assessment. First we systematic studied of the factors of D-amygdalin conversion to L-amygdalin. L-amygdalin was obtained by preparative liquid technologies, and its structure was identified and confirmed. On the basis of L-Amygdalin, we studied the enzymatic regularity of D- and L-Amygdalin in vitro. The results showed that basic conditions was one of the important factors of amygdalin epimerization. The optimal conversion conditions of L-Amygdalin was D-amygdalin in Na OH solution at p H 11 for 30 minutes. The mixture of amygdalin epimers was separated and purified. L-amygdalin was prepared with a purity of 99.7%, its structures was confirmed through NMR and mass spectrometry. In vitro enzymatic hydrolysis was found that there was significant differences of enzymatic toxin production process of D- and L-amygdalin. Under the same conditions, hydrolysis rate of D-amygdalin was L-amygdalin 7.7-fold. Amygdalin produced the efficacy in vivo by the enzyme to generate hydrogen cyanide, so different rate would lead to differences in exposure dose of hydrocyanic acid, 8 which may lead to difference in efficacy and toxicity of D- and L-amygdalin, Damygdalin of faster enzyme rate may play a bigger role in the efficacy.In third chapter, based on high performance liquid chromatography(HPLC), we measured the content of D- and L-amygdalin in cyanide glycosides medicine, focusing on content from different sources and forms of medicine, and its transformation of D-amygdalin. First, we established a sensitive and accurate HPLC method to measure the content of D- and L-amygdalin in Chinese patent medicine, and carried out related method validation. The results showed that linearity of D- and L-amygdalin were obtained in the range between 2 and 500 mg/m L(R2= 0.999), the limit of detection(LOD) was 0.8 mg/L, the limit of quantification(LOQ) is 2 mg/L. Precision, recovery, stability and repeatability were good, indicating that the method was stable and reliable. Then, by this method, 17 kinds of 42 batches of Chinese patent medicine, containing bitter almond, were determined in content of the D- and L-amygdalin and epimerization ratio. Results showed that the D-, L-amygdalin content and epimerization ratio ranges were 0.018%-1.158%, 0.003%-0.434% and 8.03%-53.35%, respectively. Based on the ratio of epimerization differences may result in different pharmacological and toxicity activity, research were carried out for Tongxuanlifei prescription. We compared D- and L-amygdalin content and epimerization ratio in different Tongxuanlifei formulations and Tongxuanlifei pills from different sources. Results found that formulations, processes, sources and other factors were likely to have an impact on amygdalin epimerization. Studies had shown that the formulation was an important factor for D- and L-amygdalin conversion.The more complex formulation, the higher the content of L-amygdalin. The results can provide references for process optimization related to Chinese patent medicine. Research results of content and the ratio of epimerization provided evidence for the safe limits of amygdalin in cyanide glycosides medicine. Finally, based on multi-stage mass spectrometry, we studied regularity of mass spectrometry fragmentation for different types of cyanogenic glycosides. Research shows that glycosylation and cyanohydrin C-O bond was easily cleaved in cyanide glycosides, which can form characteristic peaks of polysaccharides or monosaccharides. To summary regularity of mass spectrometry fragmentation, it provided reference for filter and structure identification of unknown cyanogenic glycosides. Experimental basis was provided for rapid screening strategy of cyanogenic glycosides.In fourth chapter, based on a complex matrix, we established two methods, surface-enhanced Raman spectroscopy(SERS) and gas chromatography(GC), to quickly detect hydrogen cyanide content in Chinese patent medicine extract. Cyanogenic glycosides may occur enzyme itself to produce HCN in process or oral medicine. This thesis was committed to developing rapid, simple and sensitive detection method for cyanide of Chinese patent medicine. SERS technology is rapid, sensitive, portable and has a great advantage in the field test. However in the complex matrices, SERS will be severely interfered with other substances. First, we designed an online purification enrichment equipment to solve the interference problems in complex matrices to measure CN-. To detect different concentrations of cyanide with online purification enrichment equipment, the linearity range of 0.1-2 mg/L was good(R2=0.997), and the limit of detection was 0.05 mg/L. Due to the Raman scattering intensity is easily affected by the parameters of the optical system, and the linear range is narrow. This thesis further developed GC method of more accurate quantitative to detect cyanide content in Chinese patent medicine, and its methodology was studied. Results showed that the linear range is 0.05-50 mg/L(R2=0.996), LOQ and LOD was 0.05 mg/L, 0.025 mg/L, respectively. By using these two methods, we measured the different types of cyanogenic glycosides medicine. The results showed that the cyanide was detected only in individual medicine. The results of SERS and GC were in agreement. By gas chromatography analysis results obtained, the highest of HCN content was 26.60 mg/L, which did not reach toxic dose. TCM is usually long-term use, So the long-term effects of low-dose toxicity can not be ignored. In addition, we found that Chinese patent medicines containing cyanide were pills, which may not result in complete inactivation of the enzyme in simple preparation process. For limited problem of amygdalin needs to integrate a number of factors to consider, including activity difference of D-amygdalin and L-amygdalin, conversion processing procedure and formulation process of TCM. |
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