Establishment Of Characteristic Chemical Profile For Shikonins And Shikonofurans Based On UHPLC-QTRAP-MS/MS (MRM) Technology And Its Application In The Resource Quality Evaluation Of Medicinal Zicao

The Traditional Chinese Medicine "Zicao" is a valuable multi-species medicinal material, which is recorded in the "Chinese Pharmacopoeia". Shikonins and shikonofurans are the main antitumor components. However, complex sources and different varieties leaded to chaotic circulating market of medicinal Zicao. The efficacy was in doubt because of confused origins, complex specifications and various quality levels. The underlying reason of this situation is due to that the method for quality control of Zicao resource was not well established yet. In order to evaluate the quality and reveal the bioactive components for its efficacy, a reliable qualitative and quantitative analytical method must be established for comprehensive identification and specific characterization of shikonins and shikonofurans. The characteristic chemical profiling approach is an excellent way to address this issue. Therefore, three Zicao species including Arnebia euchroma (Royle) Johnst, Arnebia guttata Bunge and Lithospermum erythrorhizon Sieb.et Zucc were investigated using the above approach. The study was mainly focused on the establishment of characteristic chemical profile for shikonins and shikonofurans based on UHPLC-QTRAP-MS/MS (MRM) technology and its application in the resource quality evaluation of medicinal Zicao. This thesis is divided into five parts:1. Extraction, isolation and structural identification of the chemical constituents from medicinal Zicao22 compounds were obtained from the dichloromethane fraction and the chemical structures of the 20 ones among them were identified, including two new shikonindimers. These compounds were used as reference standards for subsequent studies.2. The systematic identification of shikonins and shikonofurans from medicinal Zicao with a two-step data mining strategyThe fragmentation rules of the standards in negative ionization mode were investigated and summarized, then the diagnostic product ions of all subtypes were determined. Besides, a two-step data mining strategy for rapid screening and systematic identification of shikonins and shikonofurans from the complex materials were proposed. As a result,58 compounds including 32 novel ones were identified from the pooled Zicao samples by applying this strategy. This study provided important scientific evidence for the subsequent research.3. The establishment of characteristic chemical profile for shikonins and shikonofurans from Zicao based on UHPLC-QTRAP-MS/MS (MRM) technology74 compounds were identified from the pooled Zicao samples by integrating different scanning modes of UHPLC-QTOF-MS/MS and UHPLC-QTRAP-MS/MS technologies. Meanwhile,100 pairs of characteristic MRM transitions were developed and their EPI spectra and retention time information were checked carefully. On top of that, the characteristic chemical profile of shikonins and shikonofurans from Zicao were established using UHPLC-QTRAP-MS/MS (MRM) technology, which provided an important basis for the quality evaluation of Zicao resources.4. The resource quality evaluation of medicinal Zicao based on the characteristic chemical profile for shikonins and shikonofuransThe characteristic chemical profile database of shikonins and shikonofurans were constructed at first. Then different Zicao samples were evaluated using principal component analysis. The results showed that the interspecies and intraspecies differences existed among three Zicao species.27 shikonins and shikonofuran compounds were identified by orthogonal partial least square analysis in the characteristic chemical profile-antitumor relationship study. In terms of the chemical components and antitumor activities, Lithospermum erythrorhizon is not inferior to Arnebia euchroma and Arnebia guttata. The underlying reason of the uneven quality for Zicao materials is probably not due to the species difference. The underlying reasons are yet to be determined in further studies.5. Simultaneous determination of 13 shikonins and shikonofurans in medicinal Zicao by HPLC-MSA new liquid chromatography-mass spectrometry method for the simultaneous determination of 13 shikonins and shikonofurans in medicinal Zicao was developed. The contents of shikonins and shikonofurans of 22 Zicao samples from the medicinal market were determined and their qualities were evaluated. The results clearly indicated that the method is specific, sensitive and accurate with a short analysis time, a wide linear range and a simple sample pretreatment, which is suitable for a large number of samples analyses. It can be used for the quality control of medicinal Zicao and its related preparations.