Pc12 cells

UPLC-grade acetonitrile was purchased from Merck (Darmstadt, Germany). Formic acid was purchased from Sigma-Aldrich (Mo, USA). Leucine enkephalin was obtained from Waters Corporation (Milford, MA, USA). Distilled water was obtained from Watson's Food & Beverage (Guangzhou, China).
The PC12 cells were purchased from Jiangsu KeyGEN BioTECH Corp., Ltd (Nanjing, China). Ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, ginsenoside Rg1, notoginsenoside R1, 20(S)-notoginsenoside R2, notoginsenoside Fa, gypenosides A, and gypenosides XLIX were purchased from Chengdu Must Bio-Technology Co., Ltd. (Chengdu, China). The 20(S)-ginsenoside Rb2, galantamine HBr, and berberine were purchased from the National Institutes for Food and Drug Control (Beijing, China). The structure of these compounds is shown in Figure 2. Dulbecco's modified Eagle medium (DMEM) and phosphate buffer saline (PBS) were purchased from Jiangsu KeyGEN BioTECH Corp., Ltd. (Nanjing, China). Fetal bovine serum (FBS), trypsin, and dimethyl sulfoxide (DMSO) were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Amyloid β-protein fragment 25–35 was purchased from Sigma-Aldrich (St. Louis, MO, USA). YZG was produced by Guangxi Wanshoutang Pharmaceutical Co., Ltd., and formulated by water into suspension.

PC12 cells were purchased from the NanJing KeyGen Biotech Co. Ltd. (Nanjing, China). These cells were cultivated in high-glucose DMEM supplemented with 10% FBS, 100 U/mL of penicillin and 100 µg/mL of streptomycin at 37 °C under a humidified 5% CO₂ atmosphere. Baicalein was dissolved in DMSO to prepare a 5 mM stock solution and diluted with serum-free media before use. PC12 cells were seeded into a 96-well plate at the density of 2 × 10 ⁴ cells/well (100 μL) and cultured for 24 h. Next, the medium was changed, and the cells were treated with baicalein at 0, 1, 10, 20, 40, and 80 µM. Each concentration had six replicate wells, and the culture was continued for 24 h. To determine the effect of baicalein on cell viability, 20 µL of MTT (5 mg/mL) was added to each well. After 4 h, the resulting formazan crystals were dissolved by adding 150 μL of DMSO for 10–15 min. The absorption (O.D.) of each hole at 570 nm wavelength was measured using an enzyme-labelled instrument (Varioskan LUX; Thermo Fisher Scientific, Waltham, MA, USA). Cell viability was expressed as the percentage of the control.

The PC12 cells were obtained from the KeyGEN Biotech Co., Ltd. (Nanjing, China). The cells were routinely maintained in medium containing 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 5% horse serum, penicillin (100 U/mL), and streptomycin (100 mg/mL) at 37 °C in a humidified incubator containing 5% CO₂. Culture medium was changed every other day. After 24 h, cells were pretreated in the absence or presence of fucoidan (100, 200, and 400 μg/mL) and cultured for 24 h, followed by incubating with Aβ25–35 (25 μM) + d-Gal (10 mM) for an additional 48 h.