Isoorientin

Isoorientin (HPLC purity ≥ 98%), LiCl, and LPS (from Escherichia coli 0111:B4) were purchased from Sigma (HPLC purity ≥ 98%, St. Louis, MO, USA). MK-2206 was purchased from Selleck Chemicals (Houston, Texas, USA). Monoclonal antibodies against p-GSK3β, GSK3β, p-ERK1/2, ERK1/2, COX-2, NF-κBp65, IκB-α, HO-1, Nrf2, ZO-1, and occludin were purchased form Cell Signaling Technology (Danvers, MA, USA). The antibody against GAPDH was obtained from TransGen Biotech (Beijing, China). The horseradish peroxidase- (HRP-) conjugated anti-mouse and anti-rabbit IgG were purchased from MultiSciences (Hangzhou, China). Mouse TNF-α, IL-1β, and IL-6 ELISA detection kits were obtained from eBioscience (San Diego, CA, USA). ReverTra Ace qPCR RT Master Mix with gDNA Remover and SYBR® Green Realtime PCR Master Mix were purchased from Toyobo Co., Ltd. (Japan).

Chromatographic analysis of ELH was performed with a Hitachi HPLC system, and data were processed using EZchrom Elite software (Lachrom Elite; Hitachi High-Technologies Co., Tokyo, Japan). Separation was performed with a Phenomenex Luna C₁₈ column (Part No. 00G-4252-E0, 5 μm particle size, 100 Å, 4.6 × 250 mm, Phenomenex Co., Torrance, CA, USA) at 25 °C, and the injection volume was 3 μL. Gradient elution was performed using solvent A (1% aqueous acetic acid, v/v) and solvent B (100% acetonitrite); the gradient flow was as follows: 0–15 min with 14.5% B, 15–35 min with 14.6% B, 35–45 min with 100% B, and 45–60 min with 14.5% B. The flow rate was 1 mL/min, and HPLC chromatograms were obtained using a UV detector at 190–400 nm. Standard samples including isoorientin and p-coumaric acid (Sigma) were dissolved in methanol at 100 ppm, and the ELH sample was dissolved in methanol at 20 mg/mL.

Standards of the phenolic compounds apigenin, caffeic acid, catechin, chlorogenic acid, epicatechin, gallic acid, hesperidin, hyperoside, isoorientin, isoquercetin, iso-vitexin, kaempferol, naringenin, orientin, procyanidines B1 + B3, and procyanidine B2, quercetin, quercitrin, rutin, vitexin, and the internal standard probenecid were purchased from Sigma–Aldrich (St. Louis, MO, USA). Methanol (LC-MS grade, ≥99.9%) was obtained from Riedel de Haën (Seelze, Germany). Formic acid (LC-MS grade, 99%) was purchased from VWR (Leuven, Belgium). Pure water was obtained from a Milli-Q purification system (Millipore, Bedford, MA, USA).

Standard compounds mangiferin, isoorientin, and isovitexin were purchased from Sigma-Aldrich (Steinheim, Germany). Swertiamarin, gentiopicrin, and sweroside were purchased from Cfm Oscar Tropitzsch (Marktredwitz, Germany).

Luteolin, orientin, isoorientin, vitexin, isovitexin, D-glicose, gallic acid, Cremophor® EL (polyoxyl 35 castor oil), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), Bothrops jararaca snake venom, bovine fibrinogen, bovine serum albumin, ascorbic acid, pyrocatechol violet and ferrozine were purchased from Sigma-Aldrich (St. Louis, MO, USA). All reagents of sodium dodecilsuphate polyacrylamide gel electrophoresis were purchased from GE Healthcare (Piscataway, NJ, USA). All reagents used for cell culture procedures were purchased from Cultilab (Campinas, SP, Brazil). All other reagents and solvents used were of analytical grade. The water used was purified by reverse osmosis.

The chemical analysis was performed using the Prominence HPLC system (Shimadzu, Kyoto, Japan) equipped with a solvent delivery pump (LC-20AD), autosampler (SIL-20AC), and ELS detector (ELSD-LTII). For the analysis, samples were extracted from milled sugarcane tops using Speed Extractor E-916 (Buchi AG, Uster, Switzerland) with ethanol/water (80:20, v/v). The extracts were evaporated, freeze-dried, and filtered before use. ZORBAX SB-C18 reversed-phase columns (250 × 4.6 mm, 3.5 μm, Agilent, Santa Clara, CA, United States) were used and the column thermostat was maintained at 40°C. The mobile phase consisted of: A. formic acid/water (10:90, v/v), B. acetonitrile/methanol (50:50, v/v) with a 0−100% gradient for 40 min. Chromatography was carried out in gradient mode, using a flow rate of 1.0 mL/min, with detection wavelength at 328 nm. Four concentrations of pure compounds were prepared: 0.2, 0.3, 0.4, and 0.5 μg/mL, as external standards. Each injection volume was 10 μL. Pure chemical compounds of 3-O-caffeoylquinic acid (3-CQA), 5-O-caffeoylquinic acid (5-CQA) and 3-O-feruloylquinic acid (3-FQA) were purchased from the Nagara Science (Gifu, Japan). Isoorientin (ISO, chemically defined as luteolin-6-C-glucoside) was purchased from Sigma-Aldrich (St. Louis, MO, United States). Presented chemical structures were drawn in MarvinSketch (ChemAxon, Budapest, Hungary) software.