1 8 dihydroxyanthraquinone

All experiments were performed 20 min following LP administration. Briefly, brains were dissected on an ice bed, wrapped in aluminum foil, snap frozen in liquid nitrogen and stored at −80°C until use. Tumor-bearing and cancer-free brain tissues in the frozen sections were separately collected using standard procedures [13 (link)] and used for HPLC analysis. Sample tissues were extracted with 416 μl methanol and 84 μl internal standard (IS, 1, 8-dihydroxyanthraquinone, 200 μg/ml) in a centrifuge tube. The tissue homogenates were vortexed for 5 min and centrifuged at 12000 rpm for 10 min at 4°C. The supernatant was then transferred to a clean tube. The residue was extracted twice more with 1 ml methanol by vigorous 5 min agitation, followed by centrifugation. The combined organic solvent of the supernatants was evaporated to a final volume of 400 μl, and subsequently placed in a sealed amber vial for HPLC analysis [11 (link)]. To improve the sensitivity and precision of quantification, we purified the samples and cited 1, 8-dihydroxyanthraquinone (Sigma-Aldrich) as the internal standard and trans-resveratrol (Sigma-Aldrich) as the standard for drawing a standard curve using previously described methods [13 (link)].

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.

DEN (Diethylnitrosamine, CAS No. 55-18-5), MNU (N-methyl-N-nitrosourea, CAS No. 684-93-5) and DHPN (Dihydroxy-di-N-propyl-nitrosamine, CAS No. 53609-64-6) were purchased from J & K Scientific Ltd., (Beijing, China). Resveratrol, dimethyl sulfoxide (DMSO), HPLC-grade acetonitrile, methanol, acetic acid, and 1,8-dihydroxyanthraquinone were purchased from Sigma-Aldrich (St. Louis, MO, USA).