Apigenin 7 o glucuronide

The methanol extract of the stem and leaves of Guettarda speciosa Linn. (MGS; voucher #: FBM224–095) was obtained from the International Biological Material Research Center at the Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea. Fingerprinting MGS was conducted with Agilent 1200 series high-performance liquid chromatographic (HPLC) system. MGS (15 μl in methanol) was injected onto a column (CapcellPAK MGII C18, Shiseido, Japan, 4.6 × 150 mm ID, 3 μm). The column temperature was 35 °C, and the flow rate was 0.5 mL/min. Samples were eluted in a gradient of 0.1% formic acid and incremental acetonitrile in water. Standards and samples were detected at wavelengths of 254 ~ 320 nm. Index chemicals, including 3-O-caffeoylquinic acid, quercetin 3-O-galactoside, quercetin 3-O-glucoside, 3,4-di-O-caffeoylquinic acid, apigenin 7-O-glucuronide, and quercetin rutinoside, were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chemstation software (Agilent Corporation, Germany) was used for data acquisition. The chemical constituents of MGS were identified by comparing their retention times to those of standard chemicals under identical analysis conditions and the UV spectra.

For the chemical analysis, HCl (analytical reagent grade) (a.r.), 37%, NaOH (≥97.0%) LC–MS grade water, and a 3kDa PES membrane filter were ordered from VWR International, Radnor, USA. The AccQ-Tag Ultra derivatization reagent kit, a mixture of standard proteinogenic amino acids, the AccQ-tag Ultra eluent A and AccQ-tag Ultra eluent B were purchased from Waters (Waters, Milford, MA, USA). The gradient grade methanol (MetOH) (≥99.9%) and standards of phytochemical compos: nicotinamide (≥98%), nicotinic acid (a. s.), biotin (≥99%), riboflavin (a. s.), liquiritigenin (≥97% (HPLC), formononetin (a. s.), chlorogenic acid (analytical standard), neochlorogenic acid (≥98%), chryptochlorogenic acid (a. s.), syringaldehyde (98%), sulforaphane (≥90% synthetic, liquid), sinapic acid (≥98%), scopoletin (analytical standard), apigenin (≥95.0%); apigenin-7-O-glucuronide (primary reference standard); luteolin (≥98%); quercetin (≥95.0%); isoquercitrin (a. s.); naringenin (≥95.0%); genkwanin (≥98%); kaempferol (≥97.0% (HPLC); isoliquiritigenin (a. s.), and ferulic acid (USP reference standard) were obtained from Sigma-Aldrich (Darmstadt, Germany).

Lipopolysaccharide (LPS; Escherichia coli O55:B5), dimethyl sulfoxide (DMSO), scutellarin, apigenin-7-O-glucuronide, rosmarinic acid, trifluoroacetic acid (TFA), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), bicinchoninic acid (BCA) kit, and cresyl-violet (C-V) powder were purchased from Sigma (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), penicillin-streptomycin (P/S) mixture, and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, NJ, USA). Enzyme-linked immunosorbent assay (ELISA) kits for measuring NO, TNF-α, and IL-6 were purchased from R&D Systems (Minneapolis, MN, USA). Primary and secondary antibodies were purchased from Cell Signaling (Danvers, MA, USA). Tertiary antibodies (avidin-biotin complex kit) and 3,3′-diaminobenzidine (DAB) were purchased from Vector Laboratories (Burlingame, CA, USA). Cell lysis buffer was purchased from iNtRON (PRO-PREP™; Seongnam, Korea). The cell compartment kit was purchased from QIAGEN (Qproteome; Hilden, Germany). An enhanced chemiluminescence kit was purchased from Merck (Immobilon™; Darmstadt, Germany).