Calycosin

Qualitative analysis was carried out using LC-MS system (Shimadzu 8050). The standard reference materials were prepared: amygdalin, hydroxysafflor yellow A, calycosin, and digoxin (Yuanye Biotechnology Co., Ltd). For plasma samples, digoxin was added as an internal standard. 20 μL of formic acid was added to 200 μL of plasma samples. The samples were vortex-mixed for 10 s, and then vortex-mixed for 1 min and centrifuged for 15 min (13,000 r/min, 4°C) after being added with 800 μL of acetonitrile. The obtained supernatants were dried in a nitrogen dryer, and diluted with 10% acetonitrile water for 200 μL. They were vortex-mixed for 1 min, ultrasound for 2 min, and then centrifuged for 15 min (13,000 r/min, 4°C). The obtained supernatants were injected into the LC-MS for analysis. Brain tissue samples were homogenized, and 100 μL of formic acid was added to precipitate the protein. The acetonitrile solution was added at 1 : 4 volume for extraction, and then vortex-mixed for 1 min and centrifuged for 10 min (4,000 r/min, 4°C). The supernatant was filtrated with a 0.22 μm membrane and centrifuged for 15 min (13,000 r/min, 4°C). The obtained supernatants were dried in a nitrogen dryer, diluted with 10% acetonitrile water, and repeated the extraction above. The brain samples were also added to digoxin as the internal standard (Figures 1(a)–1(d)).

The production of YGYSG was undertaken in The First Clinical Medical College at Anhui University of Chinese Medicine. A Milli-Q water purification system (Millipore, Bedford, MA, USA) was employed to acquire deionized water. For mass spectrometry analysis, formic acid of LC-MS grade was purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile and methanol were LC-MS grade and obtained from Merck (Darmstadt, Germany). Overall, 27 chemical standards, including chikusetsusaponin IVa, chikusetsusaponin IV, ginsenoside Ro, 25R-inokosterone, β-ecdysone, acacetin, cymaroside, quercetin, gallic acid, oleanolic acid, hyperoside, chlorogenic acid, astragalin, kaempferol, cryptotanshinone, tanshinone II_A, rosmarinic acid, calycosin-7-O-β-D-glucoside, salvianolic acid B, astragaloside IV, formononetin-7-O-β-D-glycoside, calycosin, formononetin, scopoletin, ferulic acid, rutin, and narcissoside, were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China), and their purity levels exceeded 98% as determined through high-performance liquid chromatography (HPLC) analysis. Figure 2 displays the chemical configurations of each reference standard.

Calycosin (cat #B20846), bafilomycin A1 (Baf A1; cat #S17106), rapamycin (Rapa; cat #T54160), and MHY1485 (cat #S31626) were from Yuanye Bio-Technology Co. (Shanghai, China). β-glycerophosphate (cat #G9422) was from Sigma-Aldrich (Shanghai, China). LysoTracker Deep Red was from Beyotime. Self-quenched BODIPY FL conjugate of BSA (DQ-BSA; green; cat #ab286868) was from BioVision, Inc. (Milpitas, CA, USA). Antibodies against RUNX2, BMP2, OPN, Cathepsin B (CTSB), microtubule associated protein 1 light chain 3 beta 2 (LC3B), sequestosome 1 (p62), AMPK, p-AMPK (Thr172), mTOR, p-mTOR, ULK1, p-ULK1 (757), VAMP8, and SNAP29 were from Abcam (Cambridge, UK). Antibodies against SM22α, lysosome-associated membrane protein 2 (LAMP), p-S6K, and ribosomal protein S6 kinase (S6K) were from Cell Signaling Technology (Danvers, MA, USA). Antibodies against β-tubulin, β-actin, GAPDH, and STX17 were from Sino Biological, Inc. (Beijing, China).