Hplc grade acetonitrile and water

The LDD-2614 and LDD-2633 (used as an internal standard, IS) compound were synthesized at the Gwangju Institute of Science and Technology (Gwangju, Korea). HPLC grade acetonitrile and water were purchased from Honeywell Burdick and Jackson (Muskegon, MI, USA), and ethyl acetate was supplied from J.T. Baker (Avantor Performance Materials, Center Valley, PA, USA). Analytical reagent grade dimethyl sulfoxide (DMSO, purity ≥98%) and formic acid (purity ≥98%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Distilled water was purified with a Millipore Milli-Q system (Bedford, MA, USA). All other chemicals and reagents were of analytical grade.

Calibration curves for each standard were made using six concentrations (3.125 to 100 μg/mL). GE and GG03 were filtered through 0.22 μm membrane filters (Woongki Science Co., Ltd., Seoul, Korea) and a 10 μL aliquot of each extract solution in 80% MeOH (10.0 mg/mL) was injected into the HPLC system. Formic acid was purchased from Sigma. HPLC-grade acetonitrile and water were obtained from Honeywell Burdick and Jackson Inc. (Muskegon, MI, USA). HPLC analysis was achieved using a Waters 600S (Waters, Milford, MA, USA) with a Waters 2487 UV detector (254 nm). The column was a Shimpack Gist (4.6 × 250 mm, particle size: 3 µm, Shimadzu Co., Kyoto, Japan). The mobile phase consisted of 0.1% Formic acid in water (solvent A) and acetonitrile (solvent B), which were eluted at a flow rate of 0.4 mL/min with the following gradient elution with concentration of solvent 30% (0.01 min), 30% (5 min), 55% (10 min), 55% (13 min), 80% (20 min), 80% (23 min), 100% (30 min), 100% (60 min). The quantitative analysis was replicated three times.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) was purchased from Sigma–Aldrich (Saint Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), minimum essential medium alpha (α-MEM), and fetal bovine serum (FBS) were purchased from Welgene Bioscience (Daegu, Korea). Primary antibodies for iNOS, COX-2, HO-1, and EGFR rabbit polyclonal antibodies were obtained from Cell Signaling Technology Inc. (Danvers, MA, USA). The enhanced chemiluminescence (ECL) Western blotting detection system was obtained from Advansta Inc. (San Jose, CA, USA). The HPLC-grade acetonitrile and water were obtained from Honeywell-Burdick and Jackson (Muskegon, MI, USA). The TNF-α and IL-6 ELISA (enzyme-linked immunosorbent assay) kits were purchased from R&D Systems (Minneapolis, MN, USA). Lipopolysaccharide isolated from P. gingivalis (PG-LPS) was purchased from InvivoGen (San Diego, CA, USA). Alizarin Red S and tin protoporphyrin IX (SnPP) were obtained Sigma–Aldrich (St. Louis, MO, USA). n-Hexane, ethyl acetate, n-butanol, chloroform, and MeOH were purchased from Daejung Chemicals and Metals CO. LTD. (Siheung, Korea).