| Colla Corii Asini (namely E’jiao in Chinese), one of the most valuable traditionalChinese medicines which was firstly recorded in the Shennong’s Classic of MateriaMedica, According to the record in Chinese Pharmacopoeia (2010edition), the CollaCorii Asini was produced from dry (or fresh) skin of Equus asinus L.(donkey) bydecoction and concentration. However, the population of donkey decreased rapidly, thusmade the donkey skins became deficient and expensive. Herein, the E’jiao products inthe market are usually adulterated by skins from pig, cattle even horse, mule and hinny.And the adulteration is difficult to be identified. Therefore establish an effective qualityevaluation method for E’jiao products to distinguish the species of origin of gelatin is ofgreat significance, especially from the fraudaration of the horse hide gelatin or the mule(E. asinus×E. caballus) and hinny (E. caballus×E. asinus) hide gelatin, for they areall belong to equids and share similar characters in appearance and nature with eachother. The thesis contains four main parts, including8chapters.In the first part (the1stchapter), the progress on quality evaluation methods ofgelatin drugs was introduced; Next, the research progress on chemical composisions,pharmacological activities and quality control of E’jiao has been summarized; Finally,the significance and purpose of the present study was indicated.The second part (the chapter2-3), was the determination of contents of lowmolecule weight compounds. In the chapter2, the amino acids were determinedaccording to HPLC method listed in Chinese Pharmacopoeia (2010edition). The resultsshow that the content of amino acid in all of the E’jiao products (price various30-380RMB) collected from the market was complied with the specification in ChinesePharmacopoeia. In the chapter3, the nucleosides compounds were determined by anewly established RP-HPLC methods, based on the theory that the nucleosides andE’jiao might enhance the immunity of human. The results shows the variationcoefficient of the total amounts of nucleosides various greatly (RSD>40%) in E’jiaoprodued by different manufactors. Because of the producing processes as well as theskin material might lead to this big difference, so this method can not be used toevaluate the quality of E’jiao.The third part (chapter4-6), was focused on the analysis of protein or polypeptides.Firstly, capillary electrophoresis (chapter4) and HPLC-GFC (chapter5) were used for the analysis, but no satisfying result was obtained. Then the gel electrophoresis, whichis most widely used in protein analysis, was used in the present study. The SDS-PAGEresults show the molecule weight of seriously degraded gelatins were high andcontinuously distribute. So the enzyme hydrosis was used in the sample preparation forfurther analysis. While the hide gelatin (donkey, horse, mule, pig, cattle) enzymehydrolysis products were all share the similar characteristic in SDS-PAGE and2-DE.Thus, the analyses of protein are rather diffidult to be used for the quality evaluation ofE’jiao.In the fourth part (chapter7), DNA species specific identification was carried out.The easy conducted PCR method was developed based on the horse species specificprimer (SEQ-1&SEQ-2) which was designed from to the COI gene on mtDNA, coulddetect even microscale adulteration of horse (or mule) in E’jiao. And the primer GQ-1&GQ-2which was optimized based on SEQ sequences, might be used to the hideidentification.In conclusion, the hide gelatins share much in common with each other, the qualitycontrol system need to be established on series methods. The contamination of proteinand amino acid was firstly required, then the harmful elements should not exceed theprescribed limit; besides, the authenticity of the donkey hides needed to be verified. TheDNA species specific identification was the most effective method at present. |
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