2 aminofluorene

Negative, including blank sterility controls, and positive controls were included in the testing of microPEA, both in the presence and absence of S9 to verify spontaneous mutation frequencies and strain‐specific responses to known mutagens. Positive control compounds without S9 mix were as follows: sodium azide (Sigma Aldrich St. Louis, MO, USA) for TA1535, ICR 191 (Sigma Aldrich) for TA97a; 3‐methylmethane sulfonate (Sigma Aldrich) for TA100 and TA102; and, 4‐nitroquinoline‐N‐oxide (Acros Organics, Geel, Belgium) for TA98. Positive controls with S9 mix were as follows: 2‐aminoanthracene (Sigma Aldrich) for TA1535; 2‐aminofluorene (Sigma Aldrich) for TA97a, TA98, and TA100; and, danthron (Sigma Aldrich) for TA102.

The Ames test was performed in accordance with a previous study (Maron & Ames, 1983) and guidance of Organization for Economic Cooperation and Development (OECD) Guideline for the testing of chemicals #471: Bacterial reverse mutation test (OECD, 1997a). Dimethyl sulfoxide was used as the negative control while 4‐nitroquinoline‐N‐oxide, sodium azide, mitomycin C, 9‐aminoacridine, 2‐aminoanthracene, benzo [a] pyrene, and 2‐aminofluorene (Sigma‐Aldrich) were used as positive controls. In brief, 100 μl of Salmonella typhimurium strains TA98, TA100, TA102, TA1535, and TA1537 (MolTox Inc.) was exposed to hispidin‐enriched P. linteus mycelia at the highest dose of 5,000 µg/plate and four doses of 313, 625, 1,250, and 2,500 µg/plate as the dose range in the presence and absence of metabolic activation. The mixtures were added to 2 ml of molten top agar containing 0.5 mM of histidine/biotin, poured on the surface of minimal glucose agar plates, and then incubated for 48 hr at 37°C prior to revertant colonies counting. Each concentration of the test substance was set up in triplicates. A positive result is considered when there is a dose‐dependent increase of revertant colonies in at least one of the tester strains without cytotoxicity and a twofold increase of spontaneous revertants over the negative control.

The Ames test was conducted to evaluate the mutagenicity of the lyophilized powder with or without S9 activation in accordance with the preparation method of S9 in Salmonella typhimurium/Mammals Microsomal Enzyme Test, Organisation for Economic Co-operation and Development (OECD) Test Guideline (No. 471, 2020). Four strains of Salmonella typhimurium, TA97a, TA98, TA100, and TA1535, and one strain of Escherichia coli WP2urvA were used in this assay (Molecular Toxicology, Boone, NC, USA). Five dosages of lyophilized powder at 0.3125, 0.625, 1.25, 2.5, and 5 mg/plate were prepared for analysis. The sterile water was used as the negative control. The five positive control groups comprised 50 μg/plate of dexon (sodium p-dimethylamino-benzenediazo sulfonate, Chem Service, West Chester, PA, USA) and 1 μg/plate of methyl methanesulfonate (Fu Chen Chemical Reagents, Tianjin, China) for the −S9 comparisons. Ten (10) μg/plate of 2-aminofluorene (Sigma–Aldrich, St. Louis, MO, USA), 50 μg/plate of 1,8-dihydroxyanthraquinone (Sigma–Aldrich, St. Louis, MO, USA), and 200 μg/plate of cyclophosphamide (TCI, Tokyo, Japan) were comprised for the +S9 comparisons. Incubation was performed at 37 ± 1 °C for 48 h, and the number of colonies was counted in triplicate.

The coumarin derivative LP4C was synthesized according to the method reported previously [19 (link)]. The infrared spectra of LP4C were determined by a spectrophotometer (Bruker Fourier, Ettlingen, Germany). ¹H Nuclear Magnetic Resonance (NMR) spectrum, ¹³C NMR spectrum, DEPT-135 NMR spectrum, ¹H-¹H COSY, Heteronuclear Single Quantum Coherence (HSQC) and Heteronuclear Multiple-Bond Correlation (HMBC) characterization data were obtained using the spectrometer (Varian Inova-500, CA, USA), which is shown in Supplementary Figure S1 and Table S2. Samples of 4-nitrobenzene-1,2-diamine, mitomycin C, 2-aminofluorene and 1,8-dihydroxyanthraquinone were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Emu fatty tissue was obtained from Guangdong Xinji Emu Industry Co., Ltd. (Jiangmen, China). The β-carotene, gallic acid, Folin–Ciocalteau reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), cytochalasin B, penicillin GNa, ethyl methanesulfonate, 2-aminofluorene, trypsin-EDTA, 4-nitro-1,2-phenylenediamine monohydrochloride, 1,8-dihydroxyanthraquinone, mitomycin C, 4-nitroquinoline-N-oxide, linoleic acid, tertbutyl hydroquinone (TBHQ), cyclophosphamide monohydrate, and Giemsa were obtained from Sigma-Aldrich (Sigma Aldrich Trading Co., Ltd., Steinheim, Germany). Corn oil was obtained from Yihai (Guangzhou) Grain and Oil Industry Co., Ltd. (Guangzhou, China). The malondialdehyde (MDA) assay kit was obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). All the reagents used were of analytical grade.