Objective Probable mechanisms and active ingredients of the aged Liupao tea (ALPT) and green tea (GT) in preventing and treating neurodegenerative diseases were investigated.

Method Protein samples were prepared by mixing 1 mg·mL⁻¹ of amyloid-β 25-35 (Aβ_25-35) stock solution with 1 mg·mL⁻¹ of aqueous extract of GT and that of ALPT stored for 7 years in varied proportions to incubated at 37 ℃ for 7d to obtain solutions of 1 μg·mL⁻¹ Aβ_25-35 as well as Aβ_25-35/ALPT and Aβ_25-35/GT in the concentrations of 25 μg·mL⁻¹ and 50 μg·mL⁻¹. PC12 cells were cultured in a medium containing 10% fetal bovine serum and antibiotics with 5% CO₂ at 37 ℃. Survival rate of PC12 cells after 24 h of treatments was detected by MTT method. On the tea extracts, metabolomics analysis was performed using ultra-performance liquid chromatography-tandem mass spectrometry, conversion to SMILES format done by Pubchem, BBB permeant and bioavailability evaluated by SwissADME, pharmacological targets predicted by SwissTargetPrediction, and neurodegeneration-related targets searched in the GeneCard database.

Result The MTT cell viability assays indicated that both GT and ALPT mitigated Aβ_25-35-mediated cytotoxicity on PC12 cells at the optimally enhanced concentration of 50 g·mL⁻¹. Microscopically, PC12 cells in the control group maintained a polygonal or spindle shape, whereas Aβ_25-35-treated counterparts reduced in volume with abnormal morphology and sparse synaptic networks. The PC12s treated with 50 g·mL⁻¹ Aβ_25-35/ALPT were near-normal morphologically, indicating alleviation of Aβ_25-35 toxicity due to the presence of ALPT. The metabolomic analysis under VIP＞1, fold change ≥2, and fold change ≤0.5 as screening criteria identified 1310 differentially expressed metabolites between GT and ALPT. A network pharmacology analysis identified 58 active compounds from the GT-upregulated metabolites and 96 from the ALPT-upregulated metabolites. GT had 831 predicted targets, while ALPT had 911. The GeneCard database found 10431 targets related to neurodegenerative changes (NC). The intersection analysis showed 652 overlapping targets between GT and NC, and 710 between ALPT and NC. The network pharmacology analysis further indicated that GT and ALPT might attack neurodegenerative diseases by affecting the multiple core targets such as AKT1, TP53, and GAPDH as well as the mechanisms including PI3K-Akt signaling pathway. Notably, three compounds from ALPT, i.e., 2',7-dihydroxy-3',4'-dimethoxyisoflavan, N-benzoyltyramine, and 17-hydroxylinolenic acid, exhibited broader effects on gene expression, suggesting that the potential in combating neurodegenerative diseases by ALPT could be greater than by GT. In addition, high binding energies were observed in the molecular docking between these compounds and the core targets.

Conclusion Both GT and ALPT effectively mitigated the Aβ_25-35-induced damage in PC12 cells. However, ALPT was superior to GT in restoring cell morphology and modulated more differentially expressed metabolites and AD-related targets than did GT. The 2',7-Dihydroxy-3',4'-dimethoxyisoflavan, N-benzoyltyramine, and 17-hydroxylinolenic acid contained in ALPT that exhibited high binding affinities to the core targets such as AKT1, TP53, and GAPDH, as well as significant neuroprotective effects through multiple pathways including PI3K-Akt were identified as the compounds critical to the potential neuroprotective effect of ALPT. With that, it appeared that ALPT, more than GT, could be applicable for combating neurodegenerative diseases.