Total PhGs were extracted from H. Cistanche as previously described (25). The air-dried stem of H. Cistanche was powdered and extracted by percolation with 80% EtOH. The percolate was evaporated under reduced pressure, followed by re-suspension in an appropriate amount of H₂O₂ (100 µmol/l). The mixture was isolated on an SP-825 macroporous resin column (Mitsubishi Chemical, Tokyo, Japan) and eluted with 0, 30, 50, 70 and 90% EtOH in water. To obtain the PhG-rich fraction, the 30–50% EtOH eluents were concentrated and dried under reduced pressure. Ultraviolet (UV) spectrophotometry was performed to determine the total PhGs. The content of echinacoside and acteoside was determined by high-pressure liquid chromatography according to a previous protocol (26). A Hypersil ODS-2 column (4.6×250 mm, 5 µm; Dalian Elite Analytical Instruments, Co., Ltd., Dalian, China) was used and maintained at room temperature. The mobile phases were methyl cyanides and water containing 0.4% phosphoric acid (v/v; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The flow rate was 0.75 ml/min and the wavelength was set to 333 nm.

The PC12 rat pheochromocytoma cell line was provided by Dr He Chunhui, the Medical School, Xinjiang Medical University (Urumqi, China). Cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (Sangon Biotech Co. Ltd., Shanghai, China), 100 U/ml penicillin and 100 U/ml streptomycin in an incubator at 37°C containing 5% CO₂ and 95% air. Upon reaching 80% confluence, the cells were treated with 0.25% trypsin and passaged.

In order to eliminate the interference of the drug itself on the growth of PC12 cells, a toxicity experiment was performed. In brief, PC12 cells (3×10⁴ cells/ml) were seeded in 96-well plates at 100 µl/well and incubated at 37°C overnight. After discarding the supernatant, 200 µl complete DMEM was added in the blank group, while cells in the intervention groups were treated by PhGs at various doses (5, 25, 50, 75, 100, 125, 150, 175 and 200 µg/ml). Following incubation of the cells at 37°C for 48 h, 20 µl MTT solution (Sigma-Aldrich; Merck KGaA) was added to each well. After incubation for 4 h, the supernatant was discarded and 150 µl dimethylsulfoxide was added per well, followed by agitation for 10 min. The optical density (OD) values at 490 nm were detected using an ELISA plate reader.

Aβ_1–42 peptide purchased from Bioss Biotech (Beijing, China) was dissolved in water (100 µg/ml). Subsequently, the mixture was incubated at 37°C for 4 days and stored at 4°C prior to use.

PC12 cells were seeded in 96-well plates (3x10⁴ cells in 100 µl per well). After culture for 24 h for adherence, 50 µl Aβ_1–42 at various final concentrations (0, 0.25, 0.5, 1, 1.5 or 2 µM) dissolved in serum-free DMEM was added, followed by incubation for 24, 48, 72 or 96 h. Cell viability was evaluated by an MTT assay. The optimal Aβ_1–42 concentration was 0.5 determined to be µM.

PC12 cells (3x10⁴ cells per well) were treated with various doses of PhGs (0, 0.5, 5, 25 or 50 µg/ml) in the presence of 0.5 µM Aβ_1–42 for 24 h. Cell viability was evaluated by an MTT assay.

PC12 cells were plated seeded in 96-well plates (3x10⁴ cells in 100 µl per well). After culture for 24 h for adherence, 100 µl H₂O₂ at various final concentrations (0, 25, 50, 100, 200, 300, 400 and 500 µM) dissolved in DMEM with or without PBS (0.01 mol/l) was added, followed by incubation for 24 h. Cell viability was evaluated by an MTT assay. The optimal H₂O₂ concentration and solvent was determined to establish the in vitro model of AD.

PC12 cells (3x10⁴ cells per well) were treated with various doses of PhGs (0, 0.5, 5, 25 and 50 µM). After culture for 24 h for adherence, PC12 cells were treated with 100 µl H₂O₂ dissolved in DMEM with PBS in the presence of PhGs for 24 h. The cell viability was evaluated by an MTT assay.

Cell injury was assessed through measuring the LDH activity in the supernatant of PC12 cells using an LDH kit according to the manufacturer's protocol (cat. no. 20150604; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). In brief, double-distilled H₂O, 0.2 µmol/ml pyruvic acid, matrix buffer and coenzyme I buffer were added in sequence at 48 h after drug treatment. After incubation at 37°C for 15 min, 2,4-dinitro-phenylhydrazine was added. Subsequently, 250 µl of a 0.4 M NaOH solution was added to each well. The supernatant was collected after incubation for 30 min at room temperature. The absorbance at 450 nm was then measured with a microplate reader.

MDA was measured in the supernatant of PC12 cells using commercial kit (cat. no. 20150604; Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocol In brief, dehydrated alcohol and other reagents were added in order, followed by incubation in a water bath at 95°C for 40 min. The mixture was centrifuged at 1,006 × g and 25°C for 10 min after cooling. The supernatant was then used to determine the MDA content. Absorbance was subsequently measured with a microplate reader at 532 nm.

PC12 cells were seeded at a density of 3x10⁴ cells/well in 96-well plates (100 µl/well). Cells were incubated with drugs including echinacoside (cat. no. 111670-200503; National Institutes for Food and Drug Control, Beijing, China) and acteoside (cat. no. 111530-200505; National Institutes for Food and Drug Control) at various concentrations (0.5, 25 and 50 µg/ml). Subsequently, the cells were treated with Aβ_1–42 or H₂O₂ for 24 h and the cell viability was measured by an MTT assay.

Values are expressed as the mean ± standard deviation. Student's t-test was used for inter-group comparisons. Statistical analyses were performed using SPSS 18.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The UV spectra of the PhGs extracted as well as standard solutions of echinacoside and acteoside were recorded, and the results showed that the UV spectra were consistent (Fig. 1). UV spectrophotometry showed that the PhG content was 87.6%. The HPLC results showed that the contents of echinacoside and acteoside were 37.7 and 17.8%, respectively (Fig. 2).

Compared with the blank group, PhG at 75, 100, 125, 150, 175 and 200 µg/ml had a significant inhibitory effect on PC12 cells (P<0.05), while PhG at 5, 25 and 50 µg/ml showed low toxicity on PC12 cells, and the cell viability was >80% (Fig. 3). Thus, PhGs at the concentration of 5, 25 and 50 µg/ml was used for treating PC12 cells in subsequent experiments due to not affecting the cell viability.

Compared with that in the control group, the cell viability in the 0.5 µM Aβ_1–42 injury group was 63% (P<0.05). The cell viability was decreased by Aβ_1–42 in a concentration-dependent manner, and the viability was <50% in the 1, 1.5 and 2 µM Aβ_1–42 injury groups (Fig. 4). Thus, treatment with 0.5 µM Aβ_1–42 for 48 h was determined to be the optimal condition for establishing the in vitro AD model.

The activity of PC12 cells treated with 0.5 µM Aβ_1–42 in the presence of safe doses of PhGs (5, 25 and 50 µg/ml) for 24 h was also determined. Compared with the model group (P<0.01), PhGs showed a significant neuroprotective effect on PC12 cells. The cell viability was rescued by PhGs in a dose-dependent manner (Fig. 5).

The viability of PC12 cells treated with 25 µM H₂O₂ dissolved in DMEM with PBS was 56.43%. The viability of PC12 cells treated with 200 µM H₂O₂ dissolved in DMEM without PBS was 71.64% (Table I). Thus, PC12 cells treated with 25 µM H₂O₂ dissolved in DMEM with PBS was the selected as the optimal condition for establishing the AD model.

Compared with the control group, the cell viability in the model group was 48.8% (P<0.05). Compared with the model group, PhGs had a significant neuroprotective effect on PC12 cells. The cell viability was dose-dependently increased by PhGs, and the viability of PC12 cells treated with PhGs at concentrations of 5, 25 and 50 µg/ml was 54, 57 and 64%, respectively (Table II).

Compared with the control group, the LDH content of the supernatant of injured PC12 cells was increased, which was inhibited by PhGs in a concentration-dependent manner. This result indicated that PhGs have a significant neuroprotective effect on PC12 cells (Fig. 6).

Compared with the control group, the MDA content in the supernatant of injured PC12 cells was increased, which was inhibited by PhGs in a concentration-dependent manner. This result indicated that PhGs have a significant neuroprotective effect on PC12 cells (Fig. 7).

Compared with the model group, treatment with acteoside significantly increased the viability of Aβ_1–42-injured PC12 cells in a dose dependent manner. PhGs and echinacoside also significantly increased the viability of Aβ_1–42-injured PC12 cells at all concentrations tested (Fig. 8A).

Compared with the model group, acteoside significantly increased the viability of PC12 cells treated with H₂O₂. PhGs also increased the viability of PC12 cells treated with H₂O₂, while the effect was not significant at concentrations of 5 and 25 µg/ml (Fig. 8B). In addition, echinacoside increased the cell viability at 25 µg/ml.

In conclusion, acteoside, PhGs and echinacoside exerted significant neuroprotective effects on PC12 cells subjected to injury with Aβ_1–42 or H₂O₂.