The antifungal activity of Trichoderma hamatum K01 to control Colletotrichum gloeosporioides C01 was recorded. Pathogenicity of C. gloeosporioides C01 on citrus leaves was confirmed by the Koch postulate. T. hamatum K01 inhibited the colony growth and conidia production of C. gloeosporioides C01 by 70.55 and 79.07%, respectively. Crude extracts from T. hamatum K01 expressed an antifungal activity against C. gloeosporioides C01. The crude TK01-MeOH showed the most potent inhibitory effect on the colony growth and conidia production at the ED₅₀ values of 273 and 355 ppm, respectively. Nanofiber TK01M gave the highest inhibition on the colony growth and conidia production at the ED₅₀ values of 13 and 3 ppm, respectively. Control mechanism was related to the major pyrone metabolite group (6-pentyl-2H-pyran-2-one), organic compounds (benzoic acid, hexadecane, tetracosane), fatty acids (palmitic acid, linoleic acid, tetradecanoic acid, pentadecanoic acid, hexadecenoic acid, ethyl ester, linoleic acid ethyl ester, and ethyl oleate), and sorbicillin from GC-MS analysis, which were produced by T. hamatum K01. It is reported for the first time to isolate secondary metabolite pyrone (6-pentyl-2H-pyran-2-one) from T. hamatum K01, and the first report of nanofibers constructed from T. hamatum K01 against C. gloeosporioides.

This study investigated ethanol-based extraction of total flavonoids from vine tea (Ampelopsis grossedentata) leaves and characterized their chemical composition and in vitro biological activities. Ultrasound-assisted and heat-reflux extraction conditions were first screened by single-factor experiments and then evaluated using unreplicated L9(3³) orthogonal designs. For heat reflux extraction, the solid-to-liquid ratio had the greatest effect on flavonoid yield among the factors evaluated. The highest observed yield (58.20 mg rutin equivalents [RE]/g) was obtained with 70% ethanol, a 1:10 solid-to-liquid ratio, and 60 min (TCY6), representing the best-performing condition within the L9 array. TCY3 and TCY9 were not additional optima; they were retained as high-yield heat reflux comparison extracts for subsequent biological evaluation. Because the L9 runs were not independently replicated, the selected condition should be regarded as provisional, pending confirmation with replicated extraction batches. UPLC-QTOF-MS/MS profiling generated 94 metabolite annotations, comprising 43 higher-confidence assignments confirmed by accurate-mass matching and comparison with authentic reference standards and 51 tentative assignments supported by high-resolution MS/MS fragmentation. Flavones, flavonols, and flavanones accounted for 25.53%, 18.09%, and 15.96% of the annotated features, respectively. The extracts exhibited strong antioxidant activity, as demonstrated by their DPPH and ABTS radical-scavenging activities and ferric-reducing antioxidant power. In addition, they inhibited the growth of Escherichia coli, Salmonella spp., and Staphylococcus aureus, with the strongest antibacterial activity observed against S. aureus. In lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages, TCY3, TCY6, and TCY9 significantly reduced malondialdehyde (MDA) levels while enhancing the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT) (p < 0.01). Furthermore, all three extracts significantly increased the level of interleukin-10 (IL-10) while decreasing the production of the pro-inflammatory interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) (p < 0.01). Collectively, total flavonoids extracted from vine tea leaves under the optimized conditions exhibited potent antioxidant, antibacterial, and anti-inflammatory activities, highlighting their potential as natural bioactive ingredients for functional food and pharmaceutical applications.