Exploring effects of Baoyuan capsule and its active compound on modulating mitochondrial function and improving neurogenesis in post-stroke recovery : from phytochemical analysis to pharmacological studies

Abstract

Stroke is a focal brain parenchymatous damage caused by poor blood supply. Ischemic stroke is the major type of stoke in the world and the disability adjusted life years caused by ischemic stroke is increasing in the stroke patients between the age of 20 and 64 years. With unsatisfactory therapeutic outcomes of stroke recovery, this study is trying to find a potential therapeutic strategy from Chinese Medicine to fill this gap. Inspired of traditional Chinese Medicine (TCM) and neurogenesis, we proposed a potential patented TCM formula BaoYuan Capsules (BYC) for post stroke treatment through regulating neurogenesis.

Before clinical application of TCM formulae, phytochemical analysis is one of the major concerns need to be addressed. With abundant chemical structures, complex TCM formulae preparations and different pharmacokinetic parameters of the chemical ingredients, it is extremely important to establish the systematic quality assessment methods. In this study, a rapid and efficient matrix solid phase dispersion (MSPD) method was developed for BYC sample preparation. Sensitive, accurate and precise qualitative and quantitative analysis were performed to clarify the chemical information in the BYC by using Qtrap and QTOF MS. According to the quantitative results of ginsenosides concentration in BYC, this research also adopts response surface methodology (RSM) to optimize extraction parameters. The results indicate that MSPD-MS-RSM could be a powerful and efficient method for performing accurately phytochemical analysis of complex TCM production preparations.

Neurogenesis is an essential therapeutic target for post stroke recovery. In pharmacological studies, this study applied transient middle cerebral artery occlusion (MCAO) induced ischemic stroke mice model to test the effects of BYC. BYC attenuated neurological functional impairment in post-stroke mice through improving neurogenesis in subventricular zone (SVZ) and subgranular zone (SGZ). Meanwhile, BYC up-regulated oxidative phosphorylation associated enzymes in hippocampus of post stroke mice. The in vitro C17.2 cell differentiation experiments suggest that BYC could promote C17.2 differentiation through regulating mitochondrial function and PI3K/Akt pathway.

Finally, combination of qualitative identification results and neurogenesis promotion activity of BYC, this investigation also screened out an active compound CO1 from BYC that improved ESCs induction into the NSCs. CO1 treatment prolonged the mitosis period of differentiating ESCs in G2/M phase, meanwhile, significantly improved the mitochondrial function of ESCs. After treated with CO1 for 14 days, the NSCs exhibited higher differentiation potential with the enhanced expression of doublecortin (DCX) and βIII tubulin (Tuj1) and neurosphere formation, increased mitochondrial function and enlarged mitochondria. Moreover, the effects of CO1 on fibroblasts from mitochondrial dysfunction patients were also investigated. The results revealed that CO1 improved ATP generation and enhanced the activity and expression of mitochondrial complexes in the complex IV mutated fibroblasts.

In summary, combination of MSPD, Qtrap-MS, QTOF-MS and RSM could be a novel efficient and systematic strategy for quality control of TCM formulae. BYC could ameliorate neurological functions in post stroke via increasing mitochondrial functions and neurogenesis. CO1, active compound from BYC, could be used for promoting stem cells differentiation promotion and mitochondrial function.