Chrysin

Trans-RSVT was purchased from Caymen Chemicals. Apigenin, chrysin, luteolin, and quercetin were purchased from Alfa Aesar. Myricetin, piceatannol, and pterostilbene were purchased from Enzo Life Sciences. Genistein and doxycycline were purchased from Tokyo Chemical Industry. All drugs were dissolved in DMSO.

Cells were grown in a customized formulation of SFFD (50:50 DMEM:Ham’s nutrient mix F12 with 15 mM HEPES) culture medium (GIBCO custom formula, F #00-5136 EL) purchased from GIBCO BRL (Grand Island, NY, USA). Culture media was supplemented with defined fetal bovine serum (FBS; Cat #SH30070) acquired from Hyclone Laboratories (GE Healthcare Life Sciences, South Logan, UT, USA). Human TGF-β1 was obtained from R&D Systems (Minneapolis, MN, USA; Cat #100-B-001). Chrysin was acquired from Acros Organics (Thermo Fisher Scientific, Somerset, NJ, USA; Cat #AC11032). Alfa Aesar was the vendor for cyclosporine A (Tewksbury, MA, USA; Cat #J63191). Collagen was determined with the Sircol™ Collagen Assay Kit (Accurate Chemical and Scientific, Westbury, NY, USA; Cat #S1000). Multispecies InstantOne™ ELISA kits to measure Akt (phospho/total) (Cat #85-86046-11) and GSK-3β (phospho/total) (Cat #85-86173-11) were purchased from Invitrogen (Carlsbad, CA, USA). Blood urea nitrogen (BUN) (Cat #2050) and creatinine (Cat #0430) were measured using kits from Stanbio Labs (Boerne, TX, USA). Antibodies used for Western blotting and immunofluorescence are listed in Table 1.

Berberine hydrochloride was purchased from TCI Co., Ltd. (Tokyo, Japan). Chrysin was obtained from Acros Organics (Geel, Belgium). Chrysophanol was purchased from Sigma-Aldrich (St. Louis, MI, USA). Vascular endothelial growth factor (VEGF) was bought from B & D Systems (Minneapolis, MN, USA). Rhubarb (dried stem and root from Rheum palmatum LINN) was a product of Da Rong Co., Ltd. (Tao Yuan City, Taiwan) (batch number: DK1070932). Scutellaria root (dried root from Scutellaria baicalensis Georgi) was the product of He Kang Chinese Medicine Co., Ltd. (New Taipei City, Taiwan) (batch number: 0704). Phellodendron bark (dried bark from Phellodendron amurense Ruprecht) was the product of Jin Rong Co., Ltd. (New Taipei City, Taiwan) (batch number: AG80303). Coptidis rhizome (dried rhizome of Coptis chinensis Franch) was the product of Fu Ji Co., Ltd. (Kaohsiung City, Taiwan) (batch number: FG0013). Further identification analysis of these plant-based materials was done by HPLC, as described below. The raw herb materials were ground into fine powder using a coffee grinder (Model: ECG3003S, Electrolux, New Taipei City, Taiwan). The milled powder was further sieved using ultrafine 100 mesh stainless steel filter and stored in a dry cabinet. The recipes of two different types of “San Huang Powder” are listed in Table 1.

Formic acid, p-coumaric acid, ferulic acid, isoferulic acid, 3,4-dimethoxycinnamic acid, and prenyl caffeate were purchased from Sigma-Aldrich (L'Isle d'Abeau Chesnes, Saint-Quentin-Fallavier, France). Caffeic acid and chrysin were obtained from Acros Organics (Geel, Belgium). Galangin was purchased from Extrasynthese (Genay, France). Pinocembrin and pinobanksin-3-acetate were isolated from the DCM extract of propolis.