Pu 2089 plus

The chromatographic analyses of all prepared solutions and extracts were performed using an HPLC from Jasco that consisted of a quaternary piston pump (PU-2089 Plus), an autosampler (AS-2055 Plus), and an UV/Vis detector (UV-2070 Plus). The data treatment was performed using Chrompass software (Jasco, Tokyo, Japan, v.1.8.6.1). The elution conditions, being based on previous work, were as follows [21 (link)]: the stationary phase was a column Gemini C₁₈ from Phenomenex (150 mm × 4.6 mm, 3 μm), and the elution was performed in gradient mode using water and acetonitrile (0 min: 50% acetonitrile, 50% water; 20 min: 65% of acetonitrile, 35% water; 45 min: 100% of acetonitrile; 50 min: 50% of acetonitrile, 50% water; and 55 min: 50% of acetonitrile, 50% water); the mobile phase flow rate was 0.45 mL min⁻¹. The volume of the injected sample was 25 µL, and the wavelength of detection was set at 360 nm.
For the identification of carbonyl compounds, the extracts obtained using the fan assisted extraction procedure were analyzed using an HPLC-DAD-MS/MS system, model LTQ XL, from Thermo Scientific (Waltham, MA, USA). The same elution conditions as above-mentioned were used. The settings of the mass spectrum detector (see Supplementary Data S1) were adjusted via proper tuning with the hydrazones of the carbonyl compounds studied.

Fecal samples and cecum contents were collected 14 and 17 days after starting the pectin-supplemented diets, respectively. Each sample (20 mg) was suspended at a ratio of 1:50 (w/v) in MilliQ water, and disrupted using zirconia beads. After centrifugation at 10,000 × g for 10 min, SCFAs in the supernatant were measured using a YMC pack FA kit (YMC, Kyoto, Japan) and high-pressure liquid chromatography (HPLC) system (PU-2089 Plus, JASCO, Tokyo, Japan). The column was maintained at 50°C with a mobile phase consisting of acetonitrile-methanol-water (30:16:54 v/v/v, pH 4–5 adjusted by 0.1 M HCl) delivered at a flow rate of 0.5 mL/minute. Labeled SCFAs were detected at a wavelength of 400 nm using a UV/Visible HPLC detector (UV-2075 Plus, JASCO).

The determination of α-, β-, γ-, and δ-tocopherols and tocotrienols was performed according to ISO 9936 (2006), using high-performance liquid chromatography with fluorescence detection [46 ]. In brief, a JASCO HPLC system (JASCO International Co., Ltd., Tokyo, Japan) was used, consisting of a quaternary pump (PU-2089 Plus), an autosampler (AS-1555), and a fluorescence detector (FP-920). Separation was accomplished with a Pinnacle DB Silica column (250 mm × 4.6 mm i.d., 5 μm, Restek, USA) using isocratic elution with n-Hexane/1,4-Dioxane (97:3 v/v). The flow rate was set at 1.5 mL/min, and the injection volume was 20 μL. Analytical grade hexane was purchased from Merck and 1,4-dioxane was purchased from LAB-SCAN (Labscan International Ltd., Dublin, Ireland). The excitation and emission wavelength were set at 295 nm and 330 nm, respectively. The content of each tocol was calculated using the calibration factor of a standard solution of (±)-α-tocopherol (Merck, Darmstadt, Germany) and expressed in mg/kg.

NIH1 was dissolved in DMSO to obtain a stock solution 10 mM. Samples of this stock solution were diluted until 1 mM in cell-free complete culture medium and left incubating at 37 °C for up to three days. At selected time points (0, 24, 48, and 72 h) samples were collected, diluted at 0.1 mM with mobile phase ACN/H2O 40:60, and analyzed through HPLC reversed-phase conditions on a Phenomenex Jupiter C18 (150 × 4.6 mm I.D.) column, UV detection at λ = 302 nm and a flow rate of 1 ml/min. Analyses were performed on a liquid chromatograph model PU 2089 PLUS equipped with a 20 µl loop valve and linked to MD 2010 Plus UV detector (Jasco Europe, Lecco, Italy). Areas of NIH1 peak, identified by co-injection, were analyzed and their percentage reductions vs time were reported in the graph.

GLAP was synthesized according to a slightly modified method of Usui et al. [28 (link)]. In brief, glyceraldehyde (0.2 M) and N-acetyl-L-lysine (0.1 M) were dissolved in 0.2 M sodium phosphate buffer (pH 7.4), and incubated at 37°C for a week. The reaction mixture was filtered with a polyvinylidene difluoride membrane filter (0.22 mm, Millipore, Bedford, MA, USA), and then put on a C8 column on preparative reversed phase high-performance liquid chromatography (HPLC). HPLC was done with a quaternary gradient pump PU-2089 plus (JASCO Co. Ltd., Tokyo, Japan) and monitoring at 215 nm with the UV–VIS spectrophotometric detector UV-2075 plus (JASCO Co., Ltd. Tokyo, Japan) under the following conditions: Column: COSMOSIL 5C8-AR-300 column (250 × 20 mm I.D., Nacalai Tesque Inc., Kyoto, Japan). Elution: isocratic of 25 mM sodium phosphate buffer (pH 7.0) from 0 to 30 min and a linear gradient of 0-40% acetonitrile containing 25 mM sodium phosphate buffer (pH 7.0) from 30 to 60 min. Flow rate: 2 ml/min. Temperature: ambient.

The general chemicals were purchased from Fujifilm Wako Pure Chemical, the Tokyo Chemical Industry, Kanto Chemical or Aldrich. The target DNAs and RNAs were purchased from JBioS (Japan) (Table 1). The ¹H NMR spectra (400 MHz) were recorded by a Bruker 400 spectrometer. The ¹H NMR spectra (600 MHz) and ¹³C NMR spectra (150 MHz) were recorded by a Bruker AVANCE III 600 spectrometer. The high resolution electrospray mass analysis was performed by a Bruker MicrOTOFQ II. The HPLC purification was performed by a JASCO HPLC System (PU-2089Plus, UV-2075Plus, FP-2015Plus and CO-2065Plus). A reverse-phase C₁₈ column (COSMOSIL 5C₁₈-AR-II, Nacalai tesque, 4.6 × 250 mm or 10 × 250 mm for ligand purification). MALDI-TOF MS measurements were performed by a Bruker Autoflex speed instrument using a 3-hydroxypicolinic acid/diammonium hydrogen citrate matrix.