Luteolin

Rutin, quercetin, luteolin, apigenin, and luteolin-7-O-glucoside standards were obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). HPLC grade trifluoroacetic acid (TFA) and methanol were obtained from Aladdin Reagent Co., Ltd. (Shanghai, China). Ascrobic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ethylenediaminetetraacetic acid disodium salt (EDTA-2Na), sodium nitroprusside (SNP), potassium persulfate (K₂S₂O₈), and trolox were obtained from Sigma-Aldrich (St. Louis, MO, USA). Other chemicals used (analytical grade) were bought from Kemio Chemical Reagent Co., Ltd. (Tianjin, China).

Arctigenin (CAS: 7770-78-7, MW: 372.41, purity ≥98%), quercetin (CAS: 117-39-5, MW: 302.24, purity ≥97%), luteolin (CAS: 491-70-3, MW: 286.24, purity ≥98%), kaempferol (CAS: 520-18-3, MW: 286.24, purity ≥98%), rutin (CAS: 153-18-4, MW: 610.52, purity ≥98%), gallic acid (CAS: 149-91-7, MW: 170.12, purity ≥98%), and chlorogenic acid (CAS: 327-97-9, MW: 354.31, purity ≥98%) were purchased from Shanghai Yuanye Bio-technology Co. Ltd. (Shanghai, China). A Waters Acquity UPLC coupled with a Xevo G2-XS Q-TOF quadrupole mass spectrometer was used (Waters Co., Milford, MA, United States).

The reference standards of gallic acid (1), rutin (6), astragalin (12), and baicalin (15) were purchased from Chinese National Institute of Control of Pharmaceutical and Biological Products (Beijing, China). Chlorogenic acid (2) was received from Baoji Herbest Bio-Tech Co., Ltd. (Baoji, China). Caffeic acid (3), galuteolin (9), quercetin (13), and kaempferol (19) were provided by National Institutes for Food and Drug Control (Beijing, China). Forsythoside B (4), forsythoside I (5), forsythiaside A (8), (+)-phillyrin (16), and (+)-pinoresinol (20) were bought from Liangwei Bio-technology Co., Ltd. (Nanjing, China). p-Poumaric acid (7), ferulic acid (10), hesperidin (14), (−)-arctiin (17), luteolin (18), and (+)-phillygenin (21) were offered by Yuanye Bio-technology Co., Ltd. (Shanghai, China). (+)-Pinoresinol-4-O-β-D-glucoside (11) were supplied by Chengdu Chroma-Biotechnology Co., Ltd. (Chengdu, China). The purity of all standards was above 98% through liquid chromatography analysis. Figure S4 shows the chemical structures of the above twenty-one analytes. Merck offered Acetonitrile, Methanol and formic acid of chromatographic grade (Darmstadt, Germany). The deionized water was prepared by a Milli-Q water purification system (Millipore, Bedford, MA, USA).

Based on previous reports, 10 different batches of SHTL powders were separated using a ZORBAX SB-C18 column (4.6 × 250 mm, 5 μm, Agilent, Santa Clara, CA, USA) and analyzed for chemical fingerprints using high-performance liquid chromatography (HPLC, Shimadzu Corp., Nakagyo-ku, Kyoto, Japan) coupled with a diode array detector (DAD) (Shimadzu Corp.) [21 (link)]. Acetonitrile in water (solvent A) and 0.4% phosphoric acid in water (solvent B) constituted the mobile phase (Supplementary Table 1). The flow rate was 1.0 mL/min at 25°C, and the detection wavelength was set at 203 nm. Salidroside, chlorogenic acid, paeoniflorin, ferulic acid, luteoloside, ginsenoside Rg1, and luteolin were used as controls to analyze the retention time (Shanghai Yuanye Biotechnology Co., Ltd.; Shanghai, China).

Luteolin was purchased from Yuanye Biotechnology (Shanghai, China), dissolved in DMSO, and diluted in saline to the final concentration. Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Platelet-activating factor C-16 (PAF C-16) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) reagent kits were obtained from Jiancheng Bioengineering Institute (Nanjing, China). Quantitative real-time PCR kits were purchased from TaKaRa (Dalian, China). Detailed information on the primary antibodies used in our study is listed in Table 1. The anti-mouse and anti-rabbit secondary antibodies for western blot were purchased from LI-COR Biosciences (NE, USA). The secondary antibodies for immunohistochemistry were obtained from Servicebio (Wuhan, China). TdT-mediated dUTP nick end labeling (TUNEL) apoptosis assay kits were from Roche (Roche Ltd., Basel, Switzerland).

Luteolin (Cat#B20888, HPLC≥98%) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai, China) dissolved in DMSO (Solarbio, Beijing, China), and a 200 mM solution was prepared for storage. It was also diluted to a low concentration with RPMI-1640 medium (HyClone, Logan, UT, USA) as needed. Also cis-Diammineplatinum (II) dichloride (cisplatin, CIS; Sigma-Aldrich, St. Louis, MO, USA) was dissolved in physiological saline at a concentration of 3.33 mM(1 g/L) and stored until use.