Kaempferol

RPMI 1640 medium (#SH30027.01), Dulbecco’s Modified Eagle’s Medium (DMEM, #SH30243.01), F-12 Kaighn’s Modification (#SH30526.01) medium, fetal bovine serum (FBS, #SH30084.3), 0.25% trypsin-EDTA, and penicillin-streptomycin (#SV30010) were purchased from Hyclone (South Logan, UT, USA). The human PD-1/PD-L1 Inhibitor Screening Assay Kit (#72005), human PD-1 (hPD-1) neutralizing antibody (#71120), and human PD-L1 (hPD-L1) neutralizing antibody (#71213) were purchased from BPS Bioscience (San Diego, CA, USA). Kaempferitrin (KI), kaempferol (KO), and kaempferol 7-O-rhamnoside (KR) were purchased from ChemFaces (Wuhan, Hubei, China). PD-1/PD-L1 Inhibitor C1 (M60312-2s) was purchased from Xcess Biosciences Inc. (San Diego, CA, USA). The PD-1/PD-L1 Blockade Bioassay Kit was purchased from Promega (Fitchburg, WI, USA).

All plant samples were collected from Goesan-gun (Chungcheongbuk-do, Republic of Korea) in 2015 and were immediately cleaned, ground, lyophilized, and stored at −20 °C before use. Lipase from porcine pancreas type II, 4-methylumbelliferyl oleate (4-MUO), and quercetin (≥95%) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Kaempferol (≥98.3%), myricitrin (≥98.3%), quercitrin (≥98.5%), and avicularin (≥99.3%) were purchased from ChemFaces Biochemical Co. (Wuhan, Hubei, China), and their purity was confirmed analytically by proton nuclear magnetic resonance (¹H-NMR) and HPLC. Methanol, n-hexane, and ethyl acetate (EtOAc) were purchased from Daejung Chemicals & Metals Co. (Siheung-si, Gyeonggi-do, Republic of Korea), and were of analytical grade. Water and acetonitrile purchased from J. T. Baker Co. (Phillipsburg, NJ, USA) were of high-performance liquid chromatography (HPLC) grade. The acetonitrile was dehydrated by molecular sieves 4 Å (Sigma-Aldrich Co.) and filtered through a membrane filter (0.45 µm) before use. All of the other chemicals were of extra-pure grade.

The standard compounds and reagents of resazurin sodium salt, dimethyl sulfoxide, ferulic acid, p-coumaric acid, 2-phenoxyethanol, acetylsalicylic acid, ibuprofen, acetaminophen, chlorhexidine, phosphate-buffered saline tablet were purchased from Sigma-Aldrich (Saint Louis, MO, USA). The standards of sakuranetin, pinobanksin, pinocembrin, pinostrombin, galangin, apigenin, chrysin, kaempferol were obtained from ChemFaces Biochemical (Wuhan, China). Ethyl alcohol was from Avantor Performance Materials (Gliwice, Poland). Ultrapure water (18.0 MΩ) was obtained with the Milli-Q Advantage A10 system (Millipore, Billerica, MA, USA). The absorbance of the reaction mixture in resazurin assays was measured using the SPARK^® multimode microplate reader (Tecan, Männedorf, Switzerland).