| The therapeutic effects and toxic reaction of drugs depend on the prototypes and metabolites that entered the organisms, namely drug-derived ingredient. At present, the study of drug metabolism has become a hot issue in the medicinal field. Clarified the drug-derived ingredient of the drugs would not only contribute to elucidate the mechanism of drug metabolism, screen the candidate drugs, provide theoretical basis for the new drug research & development, but also helpful to improve the rationality and security of clinical use of drugs. In recent years, many researchs on the drug metabolism have been reported, but there is still no a clear and perfect protocol on the study of drug metabolism. In addition, the chemical ingredients variety, the metabolites unpredictability, the matrix complexity and the trace concentrations of most herb-related compounds which made mining and identification the prototypes and metabolites entered the organisms effectively became a significant challenge. In this research, an integrated research methodology of in vivo metabolites of herb was presented taking rhubarb as the carrier, using a hybrid linear ion trap/Orbitrap mass spectrometer and multiple post-acquisition data mining techniques. The main contents and results are presented below.1. The development of chemical ingredients profile of rhubarbBased on the correlative literatures and the global analysis of rhubarb, we summarized the main categories of chemical ingredients in rhubarb and the fragmentation behavior of each categoric Anthraquinones, anthranones, tannins, stilbenes, phenylbutanone, acylglucosides and Chromones are the mainly chemical ingredients in rhubarb. Among them, anthraquinones, anthranones and tannins preserved their basic parent structure during the fragmentation process, while stilbenes, phenylbutanone, acylglucosides and Chromones would eliminate their basic parent structure to produce characteristic neutral fragment ioa The characteristic ions (CIs) used to quick search and qualitative analysis were confirmed according to the fragmentation patterns of each categoric Developing the HPLC LTQ-Orbitrap data acquisition method by using dynamic exclusion and data depend scan to acquire the high resolution mass spectrometry data. Extraction of the CIs was performed in the multi-stage chromato grams of the rhubarb extract, combining the accurate mass and MSn fragmentations, a total of 274 ingredients were mined and iditified including 34 anthraquinones,84 anthranones,47 tannins,18 stilbenes,24 phenylbutanone,26 acylglucosides,24 Chromones and 15 other. By this step analysis, we preliminary established the CIs ([A-H]’) ued for the metabolism research and their characteristic fragmentation patterns, which provide a chemical foundation for the metabolism study of rhubarb.2. The metabolism study of the representative compoundsThe metabolic profiles of fourteen representative compounds were studied to provide helpful information for the metabolism study of rhubarb in vivo. The Metworks software was used to conduct a fully search of metabolites by setting a series of metabolic pathways. Next, we enriched the metabolites profiles of each representative compound by reading the retrieved literatures, then analysied the structure of metabolites according to the obtained accurate mass and fragment imformations. After that, a total 20 metabolites were observed in the bio fluids of the physcion,24 metabolities in chrysophanol,32 metabolities in emodin,35 metabolities in rhein,28 metabolities in atoe-emodin,34 metabolities in emodin-8-O-glucopyranoside,25 metabolities in aloe-emodin-8-O-glucopyranoside,20 metabolities in sennoside A,13 metabolities in sennoside B,6 metabolities in sennoside C,27 metabolities in gallic acid,49 metabolities in (+)-catechin,36 metabolities in (-)-epicatechin gallate, and 21 metabolities in polydatin. By analyzing the metabolic pathways of existing monomeric compounds, we can speculate the metabolic pathways of other categories compound, to confirm the CIs of each categories metabolite, which provide a metabolism foundation for the metabolism study of rhubarb.3. The development of drug-derived ingredient profile of rhubarb in ratA strategy used to effectively min and identifies herb-derived compounds in the biological samples after rhubarb-treated was developed:firstly, the prototypes were identified by matching their accurate mass, retention time and MSn fragments with the chemical ingredients profile of rhubarb; second, after extracting of CIs, deducting the prototypes and endogenic components, we can confirm the metabolites entered the body. The metabolites without any fragment information were identified by matching with the results of monomeric compounds metabolism or tentatively identified with their accurate mass. As a result, a tatol of 105 prototypes were detected, among them,11 prototypes were detected in the plasma samples,36 prototypes were detected in the urine samples, and all of them detected in the fece samples. There were 250 metabolites mined and identified, including 66 anthraquinone-type metabolites,21 anthranone-type metabolites,71 tannin-type metabolites,20 stilbene-type metabolites,20 phenylbutanone-type metabolites,29 acylglucoside-type metabolites,20 Chromone-type metabolites and 3 other-type metabolites. In this study, we employed the proposed method:from representative monomeric compounds to chemical ingredients profile of herbal, from the metabolism study of representative monomeric compounds and chemical ingredients profile of herbal to in vivo metabolite profile of herb to finish the metabolism study of rhubab, which would provie a reference for the metabolism research on other herbs and their preparations. |
