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10 protocols using chlorogenic acid

1

Analytical Standards for Phytochemicals

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Acetonitrile and methanol were of HPLC grade and purchased from Roth GmbH (Karlsruhe, Germany). Reference compounds (crocin, safranal, apigenin, rutin, caffeic acid, ferulic acid, chlorogenic acid, gallic acid) were purchased from ChromaDex (Santa Ana, CA), Sigma-Aldrich (Saint Louis, MO), HWI Analytik GmbH, and Roth GmbH (Karlsruhe, Germany).
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2

HPLC Analysis of Phenolic Compounds

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HPLC grade methanol, acetonitrile, tetrahydrofuran and isopropanol were purchased from VWR International (Mississauga, ON, Canada). HPLC grade phosphoric acid and acetic acid were purchased from VWR International. The primary grade reference standards chlorogenic acid (purity: 93.9%), rutin (purity: 89.3%), quercetin (purity: 93.4%) and isoquercetin (purity: 93.2%) were purchased from Chromadex (Irvine, CA, USA). Water was deionized using a Barnstead water purification system (Fisher Scientific, Ottawa, ON, Canada). Stock solutions of the reference standards were prepared at 1000 µg/mL. Each day mixed calibration solutions ranging from 0.5 to 200 µg/mL were prepared.
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3

HPLC Analysis of Bioactive Compounds

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In order to determine the bioactive compound present in the plant extract, a standardized HPLC method was utilized, which has been described in the USP 30-NF25 Pharmacopoeia [49 ]. The HPLC system used consisted of an Agilent 1200 chromatogram equipped with a quaternary pump, DAD, thermostat, degas system, and autosampler (Agilent Tehnologies, Boblingen. Germany). A C18 Zorbax XDB column (250 mm × 4.6 mm; 5 µm) was employed, and the eluents were composed of 0.1% phosphoric acid (A) and acetonitrile (B), with a linear gradient of 10% B for 13 min, 22% B for 1 min, 40% B for 3 min, and 10% B for 1 min. The flow rate was set to 1.5 mL/min and the column temperature was maintained at 35 °C. The DAD system was set to detect wavelengths of 310 nm, 335 nm, and 360 nm simultaneously. The standards used in the study were purchased from Chromadex (Wesel, Germany) and included E-resveratrol, Z-resveratrol, caffeic acid, chlorogenic acid, cinnamic acid, ellagic acid, vanillin, gallic acid, ferulic acid, astragalin, isorhamnetin, kaempferol, scutellarin, rutoside, and quercetin. All assays were run in triplicate, and the results were expressed as mean ± SD.
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4

HPLC Analysis of Phenolic Compounds

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The acetonitrile and methanol were of HPLC grade and were purchased from Roth GmbH (Karlsruhe, Germany). The reference compounds (chlorogenic acid, caffeic acid, ferulic acid, t-cinnamic acid, mangiferin, kaempferol, tectoridin, iristectorigenin B, nigricin, irigenin, isoorientin, genistein-7-glucoside, and biochanin A) were purchased from ChromaDex (Santa Ana, CA, USA), Sigma-Aldrich (Saint Louis, MO, USA), HWI ANALYTIK GmbH, and Roth GmbH (Karlsruhe, Germany). All other chemicals were of analytical grade.
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5

Characterization of Red Leaf Lettuce

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The following standards were purchased: sinapic acid, hippuric acid, caffeic acid, quercetin 3-glucoside (all ≥98% purity, Sigma, St. Louis, MO), chlorogenic acid (primary standard grade, ≥98% purity, Chromadex, Irvine, CA), and cyanidin 3-O-β-glucopyranoside (≥97% purity, Polyphenols, Norway). BeadBug™ Microtube Homogenizer and 3.0 mm zirconium beads were purchased from Benchmark Scientific (Edison, NJ).
RSL looseleaf variety NFR, was provided by Shamrock Seed Co. (Salinas, CA) and green leaf lettuce (GL) variety Crispa (Andy Boy, Salinas, CA) were purchased from the grocery store (Stop and Shop, Highland Park, NJ). NFR plant and seed vouchers were made and deposited at the Chrysler Herbarium and ATCC [4 (link),12 (link)]. Outer leaves and tops of RSL lettuce heads were used as they have the highest levels of phenolics. Lettuce leaves were rinsed, lyophilized and homogenized in a Vitamix Professional 500 Blender (Cleveland, OH) to a fine powder. Dried RSL and GL powders were used for phytochemical extraction, nutritional analyses, and rodent diet formulations.
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6

Analytical UHPLC of Botanical Compounds

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Formic acid, methanol and LCMS grade acetonitrile were purchased from MERCK (Malaysia). HPLC grade water was prepared from distilled water using a Milli-Q-system (Millipore, MA,USA) and was used during analytical UHPLC analysis. Standards used were rosmarinic acid (Chromadex, CA, USA), caffeic acid, chlorogenic acid, eupatorin and sinensetin (Extrasynthase, Genay, France). All of other solvents and chemicals used in this study were analytical grade. Stock and working standards were prepared by dissolving these analytes in 100% methanol. The standard solutions stored at 4°C were stable for at least 3 months.
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7

Cytochrome P450 Inhibition Kinetics Assay

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Inhibition and kinetic reactions were carried out in Black costar 96-well plates bought from Thermo Fischer Scientific (Pittsburgh, PA, USA). α-naphthoflavone, furafylline, sulphaphenazole, and miconazole were purchased from Sigma-Aldrich (St. Louis, MO, USA). Vivid® Blue screening kits and vivid® substrates, 7-benzyl-oxymethyloxy-3-cyanocoumarin, (BOMCC-cat. no. P2975) and 7-ethoxy-methloxy-3-cyanocoumarin (EOMCC-cat. no. P3024) were obtained from Life Technologies, (Grand Island, NY, USA). CYP2C19 (cat. no. P2864), CYP2C9 (cat. no. P2861), and CYP1A2 (cat. no. P2863) blue screening kit included baculosome (respective isozymes and NADPH-P450 reductase); a regeneration system (glucose-6-phosphate, glucose-6-phosphate dehydrogenase) and NADP+ were used for the study. Standards for epicatechin, catechin, chlorogenic acid, caffeic acid, and p-coumaric acid for identification and quantification of constituents of herbal extracts were purchased from Chromadex (Wesel, Germany). Ultra-Pure double distilled and deionized water was obtained from Milli-Q plus water purification system (Millipore, Bedford, MA, USA). All reagents were of analytical grade.
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8

Quantification of Phenolic Acids in Plants

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Methanol, acetonitrile, and acetic acid, all HPLC grade were purchased from J.T. Baker (Phillispsburg, NJ). Chlorogenic acid and caffeic acid standards were purchased from Chromadex (Irvine, CA). Neo‐chlorogenic and cryptoChlorogenic acids were purchased from PhytoLab (Vestenbergsgreuth, Germany). All standards were prepared as stock solutions at 1.00 mg/mL in methanol and stored at −20°C until use. HPLC‐grade water was obtained using a Millipore system (Billerica, MA).
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9

Analytical Characterization of Bioactive Compounds

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Analytical- and chromatographic-grade chemicals and solvents were used for this study: acetonitrile, methanol, glacial acetic acid, erythrodiol, maslinic, oleanolic acids from Sigma-Aldrich (GmbH, Karlsruhe, Germany); uvaol, betulin, corosolic acids, ursolic acid from Carl Roth (Karlsruhe, Germany); polyphenols (gallic acid, ellagic acid, neochlorogenic acid, chlorogenic acid, oenothein B, rutin, hyperoside, isoquercitrin, quercitrin, quercetin, guaijaverin, afzelin) were purchased from ChromaDex (Santa Ana, CA, USA), Sigma-Aldrich (Saint Louis, MO, USA), and Hwi Analytik (Rülzheim, Germany); all solvents used were of HPLC grade. Water was obtained using a Mili-Q purification system (Millipore, Burlington, MA, USA). Also, other reagents were used, such as sodium carbonate (Sigma-Aldrich, Scnelldorf, Germany), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), aluminium chloride, hexaethylenetetraamine, acetic acid obtained from Sigma-Aldrich (Buchs, Switzerland); potassium persulfate from Alfa Aesar (Karlsruhe, Germany); Trolox (98%) was received from Fluka Chemika (Buchs, Switzerland).
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10

Quantitative Analysis of Phytochemicals

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Chemicals and reagents: Syringin, eleutheroside E, isofraxidin, hyperoside, rutin, kaempferol, oleanolic acid, and L-phenylalanine were purchased from the Chinese National Institute of Control of Pharmaceutical and Biological Products (Beijing, China). Syringic acid, vanillic acid, p-hydroxybenzoic, protocatechuic acid, cinnamic acid, gallic acid, ferulic acid, chlorogenic acid, caffeic acid, p-coumaric acid, luteolin, genistein, and quercetin were purchased from ChromaDex Inc. (Santa Ana, CA, USA). The water used for UPLC-MS/MS was prepared by a Milli-Q (Millipore, Bedford, MA, USA). Acetonitrile (J&K Scientific Ltd., Beijing, China) was HPLC grade. All other chemicals used in the research were of analytical grade.
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