Astragaloside 4

Seeds of A. mongholicus were purchased from AnGuo Chinese Herbal Medicine Base in HeBei Province of China. Astragaloside IV, cycloastragenol, calycosin-7-O-β-d-glucoside, calycosin, ononin, formononetin, scutellarin (IS, internal standard) and sodium chloride were purchased from Chengdu Must Bio-technology Co., LTD. (Chengdu, China). Sodium chloride (NaCl) was of analytical reagent grade. Water used for the UPLC–MS/MS analysis was prepared with a Milli-Q water purification system procured from Millipore (Milford, MA, USA). Acetonitrile (J & K Scientific Ltd. Beijing, China) was of HPLC grade. All other chemicals used in the method were of analytical grade.

Huperzine A was purchased from Shanghai Fudan Forward Pharmaceutical Co., Ltd. (Shanghai, China). Piracetam was obtained from Northeast General Pharmaceutical Factory (Shenyang, China). The In situ Cell Death Detection kit, peroxidase was from Roche (Basel, Switzerland). Protein lysis buffer for western blot was provided by Solarbio Science & Technology Co., Ltd. (Beijing, China). Antibodies directed to Bax (rabbit anti-rat; cat. no. 2772S; 1:800), c-Jun (mouse anti-rat; cat. no. 2315S; 1:500), Bcl-2 (rabbit anti-rat; cat. no. 2876S; 1:1,000), PKA C-α (rabbit anti-rat; cat. no. 4782S; 1:600), CREB (rabbit anti-rat; cat. no. 4820; 1:800), and phosphorylated CREB (p-CREB; rabbit anti-rat; cat. no. 4276; 1:500) were all from Cell Signaling Technology (Danvers, MA, USA). An antibody against β-actin (mouse anti-rat; cat. no. TA-09; 1:1,000) and secondary antibodies against peroxidase-conjugated goat anti-mouse IgG (cat. no. ZB-2305; 1:3,000) and peroxidase-conjugated goat anti-rabbit IgG (cat. no. ZB-2301; 1:3,000) were obtained from Zhongshan Jinqiao Biotechnology Co., Ltd. (Beijing, China). Notoginsenoside R1, astragaloside IV, amygdalin, platycodin D and ginsenoside Rg1 were purchased from Chengdu Must Biotechnology Co., Ltd. (Chengdu, China). Ginsenoside Rb1 was from National Institutes for Food and Drug Control (Beijing, China).