Cirsimaritin

Samples of the dried aerial part of Cirsium japonicum var. maackii (CJM) were obtained commercially from Imsil Herbal Medicine (Imsil‐gun, Jeollabuk‐do, Korea). The dried samples were mixed with 30% ethanol and the mixture was refluxed at 65°C for 3 hours. Total CJM extract was obtained as the filtrate after vacuum filtration at 25°C. Apigenin, cirsimarin and cirsimaritin were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Pro‐inflammatory cytokines (IL‐1β, IL‐6, IL‐17 and TNF‐α) were purchased from GenScript (Piscataway, NJ, USA). Mouse articular chondrocytes were treated with IL‐1β (1 ng/mL), IL‐6 (100 ng/mL), IL‐17 (10 ng/mL) or TNF‐α (50 ng/mL) and co‐treated with CJM extract (10, 50 or 100 μg/mL), Apigenin (10, 25 or 50 μmol/L), cirsimarin (10, 25 or 50 μmol/L) or cirsimaritin (10, 25 or 50 μmol/L) for 24 h before they were harvested.

Acetonitrile (hypergrade for LC–MS), formic acid (for LC-MS) and methanol (analytical grade) were purchased from Merck (Merck, Bulgaria). The authentic standards used for compound identification were obtained from Extrasynthese (Genay, France) for protocatechuic, syringic, vanillic gentisic, p-coumaric, m-coumaric, o-coumaric acid, quercetin, apigenin, luteolin, apigenin 7-O-glucoside, kaempferol 3-O-glucoside, isorhamnetin 3-O-glucoside, luteolin 7-O-glucoside and rutin, and kaempferol 3-O-rutinoside, homoorientin, isoquercitrin, hyperoside, nepetin. Caffeic acid, neoChlorogenic acid, 3,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, jaceosidin and hispidulin were supplied from Phytolab (Vestenbergsgreuth, Germany). Chlorogenic acid, isorhamnetin and cirsimaritin were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The phenolic compounds in S. officinalis leaves were determined by the HPLC system HP1100 (Agilent Technologies, Palo Alto, CA, USA) equipped with an autosampler, quaternary pump, and diode array detector DAD (Hewlett Packard 1050), using a column (Altima C18, 5 × 150 mm, 4.6 mm ID) and a guard column Altima C18, 5 mm (Alltech) according to Kim et al. [38 (link)]. The solvent system contained a gradient of A (Acetic acid 2.5%), B (Acetic acid 8%), and C (Acetonitrile). The 10 µL of solvent was injected at a flow rate of 1 mL min^−1, and separation was performed at 25 °C. The peaks of phenolic compounds (µg g⁻¹) were identified by comparing the results with library and external standards’ retention times and mass spectrums. The external standards such as naringin, hispidulin, cirsimaritin, luteolin, chrysin, and resveratrol were purchased from Sigma-Aldrich, St. Louis, MI, USA.