The study of chemical components in Qishiwei Zhenzhu pills and its anti-apoptotic mechanism in cerebral ischemic based on LC-MS and network pharmacology.

Ethnopharmacological Relevance: Qishiwei Zhenzhu pills are one of the most representative precious treasure proprietary medicines and have been used for nearly 500 years in clinical practice in Chinese. This medicine can prevent diseases and play a certain role in fighting altitude sickness with cerebral ischemia.

Aim Of The Study: This study used LC-MS to analyse the chemical constituents of Qishiwei Zhenzhu pills, which laid a foundation for the improvement of the quality standard and the basic research of pharmacodynamics substances. This study aims to reveal the mechanism of Qishiwei Zhenzhu pills on cerebral ischemia from the perspective of the inflammatory and apoptotic pathway.

Materials And Method: UPLC-Q-TOF-MS was used to analyse the chemical constituents of Qishiwei Zhenzhu pills qualitatively. HPLC-QQQ-MS was used to analyse the contents of Qishiwei Zhenzhu pills quantitatively. The therapeutic target and pathway of Qishiwei Zhenzhu pills in the treatment of ischemic stroke were predicted on the basis of network pharmacology. On the basis of the MCAO rat model, the cerebral infarction rate (TTC staining) and the number of Nissl bodies (toluidine blue staining) were measured, the pathological morphologies of cortex and hippocampus were observed (HE staining), and the mRNA levels were determined by RT-PCR. The protein expressions of Bax, Bcl-2, and Caspase3 in the ischemic brain tissue of rats were determined using the WB method.

Results: A total of 42 chemical constituents, including 11 triterpenoids, 10 flavonoids, 8 organic acids and their derivatives, 4 diterpenoids, 4 tannins, 2 steroids, and 3 other components, were identified from Qishiwei Zhenzhu pills by UPLC-Q-TOF-MS. HPLC-QQQ-MS results found that among the 16 different batches, the content difference between the two batches of preparations with the national drug approval number was small and that the quality was stable. However, significant differences were observed among the preparations of nine medical institutions. Network pharmacology study found that the effect of Qishiwei Zhenzhu pills might be related to the AGE-rage and tumour necrosis factor signalling pathways. Qishiwei Zhenzhu pills could improve the neurobehavioral abnormalities of MCAO rats, reduce the rate of cerebral infarction, improve the pathological changes in the hippocampal area of brain tissue, and increase the number of Nissl body in the brain tissue. Qishiwei Zhenzhu pills tended to reduce the mRNA transcription levels of Bax, Caspase-3, p65, c-fos and VEGF-A and increase the expression of Bcl-2 and MAPK8 mRNA. Moreover, the Bax protein expression tended to decrease, and the bcl-2 protein expression tended to increase.

Conclusions: A total of 42 chemical components were qualitatively identified from Qishiwei Zhenzhu pills, and 16 chemical components from 16 batches were determined. These components improved the quality standard level of Qishiwei Zhenzhu pills and provided reference for the later exploration of its pharmacodynamics substance basis. The protective mechanism of Qishiwei Zhenzhu pills against ischemic stroke might be related to the downregulation of the apoptosis factor caspase-3.