Chlorogenic acid

Emodin, genipin, chlorogenic acid, cimigenoside, and ginsenoside Rb1 were purchased from ChemFaces (Hubei, China). Imiquimod (IMQ) was obtained from 3M Health Care (Leicestershire, England). High glucose Dulbecco's modified Eagle's medium (DMEM) was acquired from WelGENE Inc. (Gyeongsangbuk, Korea). Fetal bovine serum (FBS) and antibiotics were supplied by Invitrogen Inc. (Carlsbad, CA, USA). U0126, SB202190, SP600125, cryptotanshinone, and wortmannin were obtained from Sigma-Aldrich (St. Louis, MO, USA). Human CXCL8, CXCL10, and mouse IFN-γ, IL-1β, IL-6 ELISA kits were purchased from KOMA Biotech Inc. (Seoul, Korea). The human CCL20 ELISA kit was bought from R&D system (Minneapolis, MN, USA). The antibodies for phosphorylated extracellular signal-regulated kinase (p-ERK), phosphorylated c-Jun N-terminal kinase (p-JNK), phosphorylated p38 (p-p38), phosphorylated signal transducer and activator of transcription 3 (p-STAT3), phosphorylated Akt (p-Akt), phosphorylated mammalian target of rapamycin (p-mTOR), ERK, p38, JNK, STAT3, Akt, and mTOR were obtained from Cell Signaling Technology (Danvers, MA, USA). The antibodies against keratin 16 (K16) and K17 were procured from Abcam (Cambridge, MA, USA). HRP-conjugated anti-β-actin was supplied by Sigma-Aldrich (St. Louis, MO, USA).

Geniposide (CFN98261, purity 98.9%), geniposidic acid (CFN98337, 98.9%), chlorogenic acid (CFN99116, 99.5%), genipin (CFN99142, 99.6%), and gardenoside (CFN90237, 99.4%) were purchased from ChemFaces Biochemical Co. Ltd (Wuhan, China). The chemical structures of these five commercial standards are shown in Figure 1(a). Levels of these five components in GFE were determined using an HPLC 1290 system (Agilent, Santa Clara, CA, USA) at the Korea Basic Science Institute (Seoul). Extracted samples (10 µl) were separated on an Extend C18 column (2.1 × 150 mm, 5 µm, Agilent), which was operated at 25°C and a flow rate of 0.5 ml/min using a dilute phosphoric acid (A; 0.4%) and acetonitrile (B) gradient, as follows: 5 to 20% (B) over 20 min followed by equilibration for 5 min. genipin, geniposidic acid, Geniposide, and gardenoside were detected at a UV wavelength of 238 nm and chlorogenic acid was detected at 330 nm. Compounds in GFE were quantified using standard calibration curves prepared using the commercial standards.

Rutin and chlorogenic acid were purchased from ChemFaces Biochemical (Wuhan, China), and kaempferol-3-O-Rutinoside was obtained from Biopurify Phytochemicals (Chengdu, China), respectively. These compounds were analyzed for a purity of ≥98.0% by HPLC. The HPLC grade acetonitrile and water of HPLC grade (J. T. Baker Chemical, Phillipsburg, NJ, USA), and TFA reagent (Sigma-Aldrich) were purchased.