Neuronal apoptosis induced by oxidative stress is a major pathological process that occurs after cerebral ischemia-reperfusion. Calycosin-7-O-β-D-glucoside (CG) is a representative component of isoflavones in Radix Astragali (RA). Previous studies have shown that CG has potential neuroprotective effects. However, whether CG alleviates neuronal apoptosis through antioxidant stress after ischemia-reperfusion remains unknown. To investigate the positive effects of CG on oxidative stress and apoptosis of neurons, we simulated the ischemia-reperfusion process in vitro using an immortalized hippocampal neuron cell line (HT22) and oxygen-glucose deprivation/reperfusion (OGD/R) model. CG significantly improved cell viability and reduced oxidative stress and neuronal apoptosis. In addition, CG treatment upregulated the expression of SIRT1, FOXO1, PGC-1α, and Bcl-2 and downregulated the expression of Bax. In summary, our findings indicate that CG alleviates OGD/R-induced damage via the SIRT1/FOXO1/PGC-1α signaling pathway. Thus, CG maybe a promising therapeutic candidate for brain injury associated with ischemic stroke.

The aim of our research was to analyze and compare the pharmacokinetics of paeoniflorin, calycosin, calycosin-7-o-β-d-6-glucoside, and 6-gingerol in the blood and brain tissue of normal and cerebral ischemia-reperfusion injury rats by HPLC-MS/MS method. Methods: The blood and brain tissue samples of normal and middle cerebral artery occlusion (MCAO) rats were compared. The blood and brain tissue samples were collected by using microdialysis technique. The concentrations of paeoniflorin, calycosin, calycosin-7-o-β-d-6-glucoside, and 6-gingerol in blood and brain tissues were determined by the HPLC-MS/MS internal standard method. Results: Compared with the normal group, the model group after the administration of the Huangqi Guizhi Wuwu Decoction showed that C max blood , AUC 0-t blood , and AUC 0-inf blood of paeoniflorin were increased, CL blood , t 1/2 brain , and V brain of paeoniflorin were decreased; C maxblood , AUC 0-tblood , AUC 0-infblood , and average residence time (MRT brain) of calycosin-7-o-β-d-6-glucoside were decreased and the CL blood and C max brain of calycosin-7-o-βd-6-glucoside were increased; C max blood of calycosin was decreased, V blood and V brain of calycosin were increased; C max blood , AUC 0-t blood , AUC 0-inf blood , and MRT brain of 6-gingerol were decreased, CL blood of 6-gingerol was increased. Conclusion: This method is simple, rapid, and sensitive. It is suitable for the pharmacokinetic study of Huangqi Guizhi Wuwu Decoction in the blood and brain tissue of rats. Cerebral ischemia-reperfusion injury increased the content of paeoniflorin, calycosin, calycosin-7-o-β-d-6-glucoside, and 6-gingerol in the blood, affecting the clearance rate of paeoniflorin in the brain, the detention time of calycosin-7-o-β-d-6-glucoside and the 6-gingerol in the brain. In normal and cerebral ischemia-reperfusion rats, the content of paeoniflorin and 6-gingerol in the blood was higher than that in brain tissue, while the content of calycosin, calycosin-7-o-β-d-6-glucoside in the brain tissue was higher than that in blood, suggesting that calycosin and calycosin-7-o-β-d-6-glucoside have brain targeting properties.

Buyang Huanwu decoction (BHD), a popular formulation prescribed in traditional Chinese medicine (TCM) for the treatment of ischemic stroke, has been reported to have a potential role in promoting axonal regeneration. The purpose of the study was to screen and identify bioactive compounds from BHD using live PC12 cells coupled with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Using this approach, we successfully identified six bioactive components from BHD. These components have protective effects on oxygen-glucose deprivation/reperfusion (OGD/R) injury to PC12 cells. Furthermore, calycosin-7-d-glucoside (CG) and formononetin-7-O-β-d-glucoside (FG) could upregulate the protein expression of growth-associated protein 43 (GAP-43) and brain-derived neurotrophic factor (BDNF). This study suggests that living cells combined with HPLC-MS/MS can be used for the screening of active ingredients in TCMs.

Context Paeoniflorin (PF) and calycosin-7-glucoside (CG, Paeonia lactiflora Pall. extract) have demonstrated protective effects in ischaemic stroke. Objective To investigate the synergistic effects of PF + CG on ischaemia/reperfusion injury in vivo and in vitro . Materials and methods Male Sprague-Dawley rats were subjected to the middle cerebral artery occlusion/reperfusion (MCAO/R). After MCAO/R for 24 h, rats were randomly subdivided into 5 groups: sham, model (MCAO/R), study treatment (PF + CG, 40 + 20 mg/kg), LY294002 (20 mg/kg), and study treatment + LY294002. Males were given via intragastric administration; the duration of the in vivo experiment was 8 days. Neurologic deficits, cerebral infarction, brain edoema, and protein levels were assessed in vivo . Hippocampal neurons (HT22) were refreshed with glucose-free DMEM and placed in an anaerobic chamber for 8 h. Subsequently, HT22 cells were reoxygenated in a 37 °C incubator with 5% CO 2 for 6 h. SOD, MDA, ROS, LDH and protein levels were measured in vitro . Results PF + CG significantly reduced neurobehavioral outcomes (21%), cerebral infarct volume (44%), brain edoema (1.6%) compared with the MCAO/R group. Moreover, PF + CG increased p-PI3K/PI3K (4.69%, 7.4%), p-AKT/AKT (6.25%, 60.6%) and Bcl-2/BAX (33%, 49%) expression in vivo and in vitro , and reduced GSK-3β (10.5%, 9.6%) expression. In vitro , PF + CG suppressed apoptosis in HT22 cells and decreased ROS and MDA levels (20%, 50%, respectively). Conclusions PF + CG showed a synergistic protective effect against ischaemic brain injury, potentially being a future treatment for ischaemic stroke.

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