Compound Dan Zhi tablet attenuates experimental ischemic stroke via inhibiting platelet activation and thrombus formation.

BACKGROUND: Compound Dan Zhi tablet (DZT) is a commonly used traditional Chinese medicine formula. It has been used for the treatment of ischemic stroke for many years in clinical. However, its pharmacological mechanism is unclear. PURPOSE: The aim of the current study was to understand the protective effects and underlying mechanisms of DZT on ischemic stroke. METHODS: Fifteen representative chemical markers in DZT were determined by ultra-performance liquid chromatography coupled with tandem quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). The protective effect of DZT against ischemic stroke was studied in a rat model of middle cerebral artery occlusion (MCAO), and the mechanism was further explored through a combination of network pharmacology and experimental verification. RESULTS: Quantitative analysis showed that the contents of phenolic acids, furan sulfonic acids, tanshinones, flavonoids, saponins and phthalides in DZT were calculated as 7.47, 0.788, 0.627, 0.531 and 0.256 mg/g, respectively. Phenolic acids were the most abundant constituents. Orally administered DZT (1.701 g kg-1) significantly alleviated the infarct size and neurological scores in MCAO rats. The network analysis predicted that 53 absorbed active compounds in DZT-treated plasma targeted 189 proteins and 47 pathways. Ten pathways were associated with anti-platelet activity. In further experiments, DZT (0.4 and 0.8 mg mL-1) markedly inhibited in vitro prostaglandin G/H synthase 1 (PTGS1) activity. DZT (0.4 and 0.8 mg mL-1) significantly inhibited in vitro platelet aggregation in response to ADP or AA. DZT (113 and 226 mg kg-1, p.o.) also produced a marked inhibition of ADP- or AA-induced ex vivo platelet aggregation with a short duration of action. DZT decreased the level of thromboxane A2 (TXA2) in MCAO rats. In the carrageenan-induced tail thrombosis model and ADP-induced acute pulmonary thromboembolism mice model, DZT (113 and 226 mg kg-1, p.o.) prevented thrombus formation. Importantly, DZT (113 and 226 mg kg-1, p.o.) exhibited a low bleeding liability. CONCLUSION: DZT protected against cerebral ischemic injury. The inhibition of TXA2 level, platelet aggregation and thrombosis formation might involve in the protective mechanism.

The potential DPP-4 inhibitors from Xiao-Ke-An improve the glucolipid metabolism via the activation of AKT/GSK-3ß pathway.

Dipeptidyl Peptidase-4 (DPP-4) is a specific enzyme hydrolyzing the incretin hormone glucagon like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) to reduce insulin secretion, meanwhile DPP-4 inhibitors play an important role in diabetic therapy. In present study, 14 potential inhibitors were screened with an inhibition over 50% on DPP-4 activity from Xiao-Ke-An formula (XKA) and 12 of them exhibited a dose-dependently inhibitory effect at concentrations of 5-50 µmol/l. We found 10 DPP-4 inhibitors restrained differentiation of 3T3-L1 pre-adipocytes as well as reducing the triglycerides and total cholesterol content in 3T3-L1 adipocytes. Furthermore, 7 DPP-4 inhibitors promoted the glucose consumption in insulin-resistance BNL CL.2 cells. Thereinto, ginsenoside Rk1 up-regulated the protein kinase B (AKT) and glycogen synthase kinase-3 (GSK-3ß) phosphorylation expression, while kukoamine B and coptisine hydrochloride obviously increased the phosphorylation of AKT protein and columbamine, panaxadiol, ginsenoside Ro, timosaponin AI significantly promoted the phosphorylation of GSK-3ß protein. It's our first effort to confirm those seven compounds could serve as DPP-4 inhibitors to attenuate DPP-4 activities, accompanied with the ability to adjust glucolipid metabolism. Moreover, activating the AKT/GSK-3ß signaling pathway to ameliorate insulin resistant may be the anti-diabetic mechanism of XKA.

Hybrid multidimensional data acquisition and data processing strategy for comprehensive characterization of known, unknown and isomeric compounds from the compound Dan Zhi Tablet by UPLC-TWIMS-QTOFMS.

The compound Dan Zhi Tablet (DZT), a reputable traditional Chinese medicine prescription, is widely used for the treatment of ischemic stroke in clinic. However, its systematic chemical constituents have rarely been elucidated, which hampers its quality evaluation, the study of bioactive constituents and the mechanism of action interpretation. In this study, we developed a combination of multidimensional data acquisition and data processing strategy with the aim to globally and comprehensively identify the chemical constituents in DZT based on UPLC-TWIMS-QTOFMS. First, multidimensional acquisition modes (MSE, Fast DDA and HDMSE) were performed on UPLC-TWIMS-QTOFMS. Second, targeted characterizations of the known compounds and their analogues present in DZT were carried out on the basis of the corresponding commercial standards or Mass2Motifs. Third, untargeted identification of unknown compounds in DZT was performed by extracting shared Mass2Motifs from the raw fragmentation spectra. Finally, the coeluting isomers were characterized using a precursor and/or product ion mobility. Consequently, 202 compounds were detected from DZT: 29 of them were unambiguously identified by comparison with reference compounds, 29 unknown compounds were discovered in specific medicinal materials, and ten pairs of coeluting isomers, which could not be distinguished using conventional MSE or Fast-DDA, were resolved using HDMSE only. This strategy was successfully used for the rapid and global identification of complex compounds including known, unknown and coeluting isomeric compounds in DZT and provided helpful chemical information for further quality control, pharmacology and active mechanism research on DZT.

A Novel and Practical Chromatographic "Fingerprint-ROC-SVM" Strategy Applied to Quality Analysis of Traditional Chinese Medicine Injections: Using KuDieZi Injection as a Case Study.

Fingerprinting is widely and commonly used in the quality control of traditional Chinese medicine (TCM) injections. However, current studies informed that the fingerprint similarity evaluation was less sensitive and easily generated false positive results. For this reason, a novel and practical chromatographic "Fingerprint-ROC-SVM" strategy was established by using KuDieZi (KDZ) injection as a case study in the present article. Firstly, the chromatographic fingerprints of KDZ injection were obtained by UPLC and the common characteristic peaks were identified with UPLC/Q-TOF-MS under the same chromatographic conditions. Then, the receiver operating characteristic (ROC) curve was used to optimize common characteristic peaks by the AUCs value greater than 0.7. Finally, a support vector machine (SVM) model, with the accuracy of 97.06%, was established by the optimized characteristic peaks and applied to monitor the quality of KDZ injection. As a result, the established model could sensitively and accurately distinguish the qualified products (QPs) with the unqualified products (UPs), high-temperature processed samples (HTPs) and high-illumination processed samples (HIPs) of KDZ injection, and the prediction accuracy was 100.00%, 93.75% and 100.00%, respectively. Furthermore, through the comparison with other chemometrics methods, the superiority of the novel analytical strategy was more prominent. It indicated that the novel and practical chromatographic "Fingerprint-ROC-SVM" strategy could be further applied to facilitate the development of the quality analysis of TCM injections.