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This study aimed to investigate the major metabolites of Tolypocladium sinense fermentation broth (TSFB) and evaluate its in vitro antioxidant activity, as well as its ability to alleviate hydrogen peroxide (H2O2)-induced oxidative stress in mouse hippocampal neuronal cells (HT22). Ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) was employed for untargeted metabolomic analysis of TSFB. An antioxidant evaluation system combined with Western blotting was used to assess the antioxidant effects of TSFB and its influence on the expression levels of superoxide dismutase 1 (SOD1), nuclear factor erythroid 2-related factor 2 (Nrf-2), superoxide dismutase 2 (SOD2), and heme oxygenase-1 (HO-1). The results revealed that detected 2745 metabolites from TSFB, primarily composed of lipids and lipid-like molecules, organic acids and derivatives, and organoheterocyclic compounds. The dominant bioactive constituents included sphingosine, citric acid, and phytosphingosine, which exhibited the highest relative peak areas. In vitro antioxidant assays demonstrated that TSFB possessed strong free radical scavenging capacity. In the oxidative stress cell model, the results showed that both low and high doses of TSFB treatment significantly increased cell survival rates (P<0.001). Meanwhile, oxidative stress markers such as MDA content, LDH levels, and relative ROS levels were significantly reduced (P<0.05), while the antioxidant capacity indicator GSH-Px levels were significantly elevated (P<0.05). Additionally, the high-dose TSFB group notably increased SOD levels (P<0.01). Both low- and high-dose TSFB treatment groups significantly upregulated the protein relative expression of Nrf-2, HO-1, and SOD1 (P<0.05), and the high-dose group further significantly increased SOD2 protein relative expression (P<0.001). This study systematically analyzed the major metabolites and bioactive components of TSFB, elucidating its antioxidant mechanisms and highlighting its potential therapeutic applications.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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