Skullcapflavone 2

The dried and pulverised biomass samples were extracted with 50 mL of analytical-grade methanol for 2 h at 78 ± 2 °C under a reflux condenser. After extraction, the obtained extracts were filtered through a paper filter, transferred to crystallisers, and left at room temperature to allow the solvent to evaporate. The remains were dissolved in 4 mL of HPLC-grade methanol (Merck) and subjected to HPLC. Qualitative and quantitative HPLC with diode array detection (DAD) was performed as described previously [20 (link),66 (link)]. Compounds were identified based on their retention times and UV spectra, as well as by comparison with reference substances (27 flavonoids, 19 phenolic acids, benzoic and cinnamic acids, 2 phenylethanoid glycosides). The reference standards used for the HPLC analyses of skullcap-specific flavonoids were baicalein, baicalin, scutellarin, wogonin (ChromaDex, Irvine, CA, USA), chrysin, scutellarein, wogonoside (Sigma–Aldrich Co., Saint Louis, MO, USA), oroxylin A and skullcapflavone II (ChemFaces Biochemical Co., Wuhan, China). The verbascoside was purchased form ChromaDex, Irvine, CA, USA.

Skullcapflavone II (5-hydroxy-2-[2-hydroxy-6-methoxyphenyl]-6,7,8-trimethoxychromen-4-one) was purchased from ChemFaces (Wuhan, Hubei, China). Antibodies against phospho-ERK, ERK2, p38, and NF-κB p65 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against phospho-NF-κB p65 (Ser536), phospho-JNK, JNK, phospho-p38, phospho-c-Jun, and c-Jun were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-phospho-tyrosine antibody (clone 4G10) was purchased from Millipore (Billerica, MA, USA). Anti-collagen type I cleavage-site antibody was purchased from ImmunoGlobe (Himmelstadt, Germany). Anti-GAPDH antibody was purchased from AbClone (Seoul, Korea). Horseradish peroxidase-conjugated goat anti-mouse IgG and rabbit IgG were obtained from KOMA Biotech (Seoul, Korea). Anti-MMP-1 and pro-collagen α1(I) N-propeptide (pN-ColIα1) antibodies were a gift from Dr. Chung, J. H. (Seoul National University College of Medicine, Republic of Korea) [53 (link)]. Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) and rhodamine-conjugated phalloidin were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Plant compounds skullcapflavone II (1), nobiletin (2), and tangeretin (3) were purchased from ChemFaces Biochemical Co., Wuhan, China. Baicalein (4) and wogonin (5) were obtained from Sigma Aldrich. Purities of the compounds were ≥98%. Chlorpromazine, verapamil, rifampicin, ethidium bromide, isoniazid, and phosphate-buffered saline (PBS) in tablets (0.01 M phosphate buffer, 0.0027 M KCl, 0.14 M NaCl and 0,05% Tween 80, pH = 7.4) were purchased from Sigma Aldrich (Poole, UK).
All compounds were dissolved either in dimethyl sulfoxide (DMSO) or in sterile deionized water.