Based on the 4D Label-free protein qualitative analysis method and coomassie brilliant blue method quantitative determination of protein content to improve the quality standards of Xiangdan injection

Abstract

Xiangdan injection (XDI), as a common Chinese medicine injection (CMIs), is mainly used clinically to activate blood circulation, relieve blood congestion and dilate coronary arteries. However, allergic reactions occasionally occurred during the use of XDI, which could be related to the protein in XDI. Therefore, we conducted research on the commercial XDI and tested it according to the requirements of the Chinese Pharmacopoeia for CMIs. Firstly, 4D-Label-free quantitative proteomics technique was used to identify the samples of XDI and Salvia miltiorrhiza (SM), eleven identical proteins were found. The results indicated that XDI contained trace proteins which come from SM with a molecular weight range of 15-181KD. Then the qualitative experiments about proteins were carried out via the sulfosalicylic acid (SA) method and the tannic acid (TA) method, which were required in the Chinese pharmacopoeia. To our surprise, the qualitative experiments in the Chinese pharmacopoeia could not precise identify the proteins in XDI. Unexpectedly, the reason is that the excipient Tween 80 in XDI would cause a false-positive reaction. Therefore the proteins in XDI were inspected qualitatively and quantitatively after eliminating false-positive reactions. The protein content in XDI was determined by coomassie brilliant blue method. It was discovered that the protein concentration was 3.038 mg/ml, which would not generate a positive reaction. Finally the combination of qualitative and quantitative protein experiments provide an exclusive identification method for XDI, which could promote the quanlity standards of XDI and guarantee the clinic safety.