Improvement Of Colla Corii Asini’s Quality Standards

Colla Corii Asini, also known as donkey-hide gelation, is praised as the "three treasures of traditional Chinese medicine" with ginseng and pilose antler. It is a common tonic Chinese medicine having the effects of nourishing yin, moisturizing lung, stanching bleeding and enriching blood. In recent years, the market of Colla Corii Asini is disordered and the qualities of products are good and bad mixed together, due to the absence of effective analytical method for the judgement of Colla Corii Asini quality. Focusing on the determination of heavy metal Cr(Ⅵ) content in Colla Corii Asini, the reduction ability of organic compounds in food, and the determination of molecular weight distribution of Colla Corii Asini, the determination method on Cr(Ⅵ) was established, the kinetics of Cr(Ⅵ) reduction with15typical organic compounds were studied, and gel permeation chromatograph method for the molecular weight distribution of Colla Corii Asini was established. This work provides information for the judgement of the Colla Corii Asini quality, and provides the experimental data and theory reference for the Cr(Ⅵ) reduction mechanism. The main research content and results are as below.By investigating different alkaline extraction solutions (sodium hydroxide solution and alkaline phosphate buffer solution) and different treatments of Colla Corii Asini (muffle furnace burning, adjusting pH value to alkaline after nitrolysis by nitric acid and precipitating proteins by acetonitrile), using the recovery of Cr(Ⅵ) as the quota,140mmol/L NaOH solution was employed as the Cr(Ⅵ) extraction solution and pre-treatment column was employed to online remove the organic compounds. Then, an online analytical determination method for Cr(Ⅵ) in Colla Corii Asini was established using Ion Chromatography-post-column derivatization-UV detector method. The method validation specificity, linear correlation, linear range, limit of detection, limit of quantification, precision, stability, and recovery were carried out. The results showed that this method had good specificity, linear correlation, precision and stability, and low limits of detection and quantification. Moreover, the recovery satisfied the requirement. Finally, the Cr(VI) content of ten Colla Corii Asini samples from different factories were determined. The results showed that only one Colla CoriiAsini sample’s Cr(VI) content was much higher than the limit of quantification, and the Cr(VI) contents in other Colla Corii Asini samples were all lower than the limit of quantification. Moreover, the Cr(VI) ratios in total chromium contents for all Colla Corii Asini samples were small.15kinds of typical organic compounds in food were selected. The data of Cr(VT) reduction with them were determined by the diphenylcarbazide spectrophotometry at different pH values (3.0,5.0, and7.0) and temperatures (25℃,37℃, and50℃). The results indicated that L-ascorbic acid, L-cysteine hydrochloride and L-methionine had relatively strong reduction activities for Cr(VI), while Cr(Ⅵ) hardly underwent the reduction with other12kinds of organics within5hours. Cr(Ⅵ) was completely reduced by L-ascorbic acid in a short period of time, and completely reduced by L-cysteine hydrochloride within60minutes, but reduced for23%by L-methionine after5hours. Therefore, the sequence of the reduction activity of the three organic compounds was L-ascorbic acid>L-cysteine hydrochloride>L-methionine. The values of pH had a great effect on the reduction kinetics. As pH value increased, the reduction with L-cysteine hydrochloride was enhanced, while the reduction with L-methionine was decreased, and even decreased to none. The kinetics data of Cr(Ⅵ) reduction with L-methionine were fitted by a zero-order kinetics equation at a pH of3.0, and the apparent activation energy was obtained as35.5kJ·mol-1. In addition, the kinetics data of Cr(Ⅵ) reduction with L-cysteine hydrochloride at the pH of3.0,5.0and7.0were fitted by a first-order kinetics equation, and the apparent activation energies of the reduction were obtained as34.4,45.3,34.9kJ·mol-1, respectively.The effects of different columns, mobile phases, pH value of mobile phases, mass fractions of Colla Corii Asini samples on chromatograms of Colla Corii Asini molecular weight distribution were investigated by gel permeation chromatograph (GPC). Through comparing the GPC chromatograms, the condition for determining Colla Corii Asini molecular weight distribution was obtained as below:the column is Waters Ultrahydrogel500column; the mobile phase is0.024M phosphate buffer solution (pH value=6.9)+0.02%sodium azide (mass fraction); the mass fraction of Colla Corii Asini sample is0.2%; the flow rate of mobile phase is0.5mL/min; the column temperature is45℃; and the injection volume is50μL. Afterwards, the method validations on specificity, precision, stability and so on were carried out. The result showed that the method was specific, precise and stable, which could be employed to determine the molecular weight distribution of Colla Corii Asini. Finally, the molecular weight distributions of eleven Colla Corii Asini samples from different factories were determined, and the results showed that protein molecular weight distributions in Colla Corii Asini from different factories were very different.