Thiamine HCl (B1), riboflavin (B2), nicotinamide (B3), nicotinic acid (another form of B3), pyridoxine HCl (B6), biotin (B7) and cobalamin (B12) were obtained from Sigma Chemical Co. Thiamine HCl, nicotinic acid, pyridoxine HCl, biotin, and cobalamin were all tested on larvae of C. teleta at 500 µM. nicotinic acid was also tested at 1 mM. riboflavin was tested on larvae of C. teleta at 10, 50, 100, 150, and 200 µM. nicotinamide was tested on larvae of C. teleta at 2, 3, 4, 5, 6, 7, and 8 µM. The concentrations tested for nicotinamide and riboflavin were based on preliminary pilot studies. The photo-degradation product of riboflavin, lumichrome, was obtained from Sigma Chemical and tested at concentrations of 100, 500, and 1000 µM. The pyridine receptor antagonists and agonists 4-acetylpyridine, pyrazinecarboxamide, and β-NAD were all purchased from Sigma Chemical Co. and tested in final concentrations as stated in results. The serotonin receptor antagonist ketanserin was purchased from Sigma Chemical Co. and prepared as a 10 mM stock solution in ethanol.
Riboflavin
Manufactured by Merck Group
Sourced in United States, Germany, India, Switzerland, Italy, United Kingdom, Japan, Macao, Sao Tome and Principe
Riboflavin, also known as vitamin B2, is a water-soluble vitamin that is commonly used in laboratory settings. It serves as a core component in various biological processes, including energy metabolism and cellular respiration. Riboflavin plays a crucial role as a cofactor for enzymes involved in the conversion of food into energy. This product is often used in research and analytical applications where its specific properties and functions are required.
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Lab products found in correlation
Nitroblue tetrazolium, by Merck Group (28 mentions)
Methionine, by Merck Group (26 mentions)
Ascorbic acid, by Merck Group (21 mentions)
Gallic acid, by Merck Group (19 mentions)
Methanol, by Merck Group (19 mentions)
L methionine, by Merck Group (18 mentions)
Sodium hydroxide (naoh), by Merck Group (14 mentions)
Hydrogen peroxide, by Merck Group (13 mentions)
Trichloroacetic acid, by Merck Group (13 mentions)
Quercetin, by Merck Group (12 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (12 mentions)
Acetonitrile, by Merck Group (11 mentions)
Thiobarbituric acid, by Merck Group (10 mentions)
Nicotinic acid, by Merck Group (10 mentions)
Folin ciocalteu reagent, by Merck Group (10 mentions)
Bovine serum albumin (bsa), by Merck Group (10 mentions)
Pantothenic acid, by Merck Group (9 mentions)
Taurine, by Merck Group (9 mentions)
Folic acid, by Merck Group (9 mentions)
Thiamine, by Merck Group (9 mentions)
216 protocols using riboflavin
1
Vitamin B Compounds Tested on C. teleta Larvae
2
Complete and Minimal Media Protocols
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YG (5 g/L yeast extract (Fisher Scientific, Pittsburg, PA, USA), 20 g/L D-glucose (Sigma-Aldrich, St. Louis, MO, USA) and 800 μL of a trace element solution [13 (link)]) was used as a complete medium. For strains carrying pyrG mutations it was supplemented with 10 mM uridine (Fisher Scientific, Pittsburg, PA, USA) and 8.9 mM uracil (Beantown Chemicals, Hudson, NH, USA). Because some batches of yeast extract are deficient in riboflavin, we added riboflavin (Sigma-Aldrich, St. Louis, MO, USA) to a final concentration of 2.5 μg/mL. Solid complete medium was YAG (YG + 15 g/L agar (Technova, Holister, CA, USA)). Solid minimal medium was 10 g/L D-glucose, 15 g/L agar, 2 mL/L of a 26% w/v MgSO4∙7H2O (Avantor, Radnor, PA, USA) solution, 800 μL/L trace element solution [13 (link)], 6 g/L NaNO3 (Fisher Scientific, Pittsburg, PA, USA), 0.52 g/L KCl (Sigma-Aldrich, St. Louis, MO, USA) and 1.52 g/L KH2PO4 (Sigma-Aldrich, St. Louis, MO, USA) (all values are final concentrations). The latter three salts were from a 10X stock salts solution adjusted to pH 6.0–6.5 with saturated KOH (Sigma-Aldrich, St. Louis, MO, USA), autoclaved separately from the other components, cooled to 60 °C and mixed with the solution containing the other components (100 mL + 900 mL) to give the stated final concentrations.
3
Photosensitizer Solutions Preparation
4
Comprehensive Methods for Cell Culture
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Dulbecco’s Modified Eagle Medium (DMEM), heat-inactivated low-endotoxin fetal bovine serum (FBS), and gentamicin purchased from Cambrex (Walkersville MD, USA) for tissue culture. All other cancer cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). Most of these following compounds (thiostrepton, ampicillin, dexamethasone, 2-methoxyestradiol, (−) riboflavin, ascorbic acid, amiloride-HCL, (−) Corey lactone, and quinine sulphate) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), and quercetin was purchased from Alfa Aesar (Johnson Matthey Co. Lancastor, UK). The 50% purified δ-tocotrienol fraction from annatto seeds was purchased from American River (Boston, MA, USA). RNeasy mini kit was purchased from QIAGEN Sciences (Germantown, MD, USA). DeltaGold (125 mg soft gels) from annatto seeds (composition 90% δ-tocotrienol + 10% γ-tocotrienol) was obtained as a gift by American River Nutrition, Inc. (Hadley, MA. USA). The hepatitis C antibody test kit was purchased from Sigma Chemical Co., St. Louis, USA. The second kit for diagnosing hepatitis C test is RNA (PCR) test. Pure total mRNA was obtained from the EDTA treated fresh whole blood using total RNA purification kit # 17200 (NORGEN Bioteck Corporation, Thorold, ON, Canada).
5
Quantitative Analysis of Vitamins and Compounds
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Thiamin hydrochloride, thiamin-monophosphate, thiamin-pyrophosphate, riboflavin, flavin adenine dinucleotide, riboflavin-dioxopyrimidine-13C4,15N2, perchloric acid, phosphoric acid and sodium hydroxide were purchased from Sigma-Aldrich (St. Louis, MO). Methanol, acetonitrile, water (all LC-MS grade), phosphoric acid and potassium phosphate dibasic were obtained from Fisher Scientific (Fair Lawn, NJ). Potassium ferricyanide (III) was purchased from Acros Organics (Geel, Belgium); caffeine-trimethyl-13C3 from Cambridge Isotope Laboratories (Andover, MA) and ammonium formate from Hampton Research (Aliso Viejo, CA). HPLC screw-cap amber vials and inserts were obtained from Supelco (Bellefonte, PA). LC-vial caps (PTFE/silicone) were purchased from Waters (Milford, MA) and Agilent Technologies (Santa Clara, CA), and ultrafree centrifugal filters Durapore® PVDF 0.1μm from Millipore (Billerica, MA).
6
Superoxide Dismutase Activity Assay
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According to the ability of SOD to inhibit the reduction of nitroblue tetrazolium (NBT; Sigma, Germany) by superoxide, the activity of SOD was determined. “For assay, 0.067 M potassium phosphate buffer, pH 7.8 was added to 0.1 M EDTA containing 0.3 mM sodium cyanide, 1.5 mM NBT and 0.1 mL of sample. Then, 0.12 mM riboflavin (Sigma, Germany) was added to each sample to initiate the reaction and was incubated for 12 min. The absorbance of samples was read on a Genesys 10 UV spectrophotometer (CECIL-2501, England) at 560 nm for 5 min. The amount of enzyme required to produce 50% inhibition was taken as 1 U and results were expressed as U/mg protein”.24 (link)
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According to the ability of SOD to inhibit the reduction of nitroblue tetrazolium (NBT; Sigma, Germany) by superoxide, the activity of SOD was determined. “For assay, 0.067 M potassium phosphate buffer, pH 7.8 was added to 0.1 M EDTA containing 0.3 mM sodium cyanide, 1.5 mM NBT and 0.1 mL of sample. Then, 0.12 mM riboflavin (Sigma, Germany) was added to each sample to initiate the reaction and was incubated for 12 min. The absorbance of samples was read on a Genesys 10 UV spectrophotometer (CECIL-2501, England) at 560 nm for 5 min. The amount of enzyme required to produce 50% inhibition was taken as 1 U and results were expressed as U/mg protein”.24 (link)
7
Antioxidant and DNA Protection Assays
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2,2-Diphenyl-1-picrylhydrazyl (DPPH), 6-hydroxyl-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 5,5-dimethyl-1-pyrroline N-oxide (DMPO), riboflavin, hydrogen peroxide (30%, H2O2), dimethyl sulfoxide (DMSO), 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH), fluorescein, Folin–-Ciocalteu’s phenol reagent, quercetin, gallic acid, chloroform-d, acetone-d6 and methanol-d were purchased from Sigma Aldrich (St. Louis, MO, USA). pBR322 plasmid DNA was purchased by Thermo Fisher Scientific (Waltham, MA, USA). Silica gel (230–400 mesh), NP F254 and RP-18 F254 TLC plates were obtained from Merck (Darmstadt, Germany). Spherical C18 100 Å reversed phase silica gel (particle size: 20–40 μm) was obtained from SILICYCLE (Ville de Québec, QC, Canada). Sodium hydroxide, ethylenediamine tetraacetic acid, disodium dehydrate (EDTA), iron (II) sulfate heptahydrate (FeSO4), sodium nitrite (NaNO2), sodium carbonate anhydrous (Na2CO3), aluminium chloride (AlCl3), methanol, acetone, ethylacetate, chloroform and n-hexane were purchased from Duksan Co. (Gyenggi, Korea).
8
Oxidative Stress Enzyme Assay
9
Glycation of Vitellogenin and Elongation Factor
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Vitellogenin in PBS solution (0.053 µM) was purchased from Biosense Laboratories AS (Bergen, NORWAY). Elongation factor solution (1.17 µM) was purchased from Abnova Corporation (Taipei City, Taiwan). These solutions were glycated with 0.1 mM ribose for 23 days at 37 °C. Riboflavin was purchased from Sigma (Tokyo, Japan) and dissolved in PBS at 0.1 mM. Each solution was measured by fluorescence spectrophotometry under the same conditions.
10
Isolation and Characterization of Metabolites from H. pustulata
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The following chemicals were used as received: Crystal Violet (CV, Anedra Tigre, Argentina); Fetal Bovine Serum (Greiner Bio-One, Frickenhausen, Germany); Sabouraud Dextrose Broth (SDB, Difco, Detroit, MI); Sabouraud dextrose agar (Difco, Detroit, MI), Phosphate Buffer Solution (PBS); dimethyl sulfoxide (DMSO, Merck Darmstadt, Germany); Calcofluor-White (Sigma-Aldrich Co, St Louis, MO, USA); MeOH (HPLC grade, Merck, Germany); Nitro Blue Tetrazolium (NBT, Sigma-Aldrich Co, St Louis, MO, USA); Methionine (Sigma-Aldrich); Riboflavin (Sigma-Aldrich Co, St Louis, MO, USA), Tiron (Sigma-Aldrich); Sodium azide (Sigma-Aldrich).
Deuterium oxide (99.9%) was purchased from Solvents Documentation Synthesis (Peypin, France). Deuterated PBS (D-PBS) was prepared by dissolving PBS powder in deuterium oxide.
1 and 2 were purified from benzene extracts of H. pustulata using a methodology described previously [10 (link),11 (link)]. They were unequivocally identified by their spectroscopic/spectrometric data (1H NMR, 13C NMR, IR, UV–Vis, MS) [10 (link)]. The purity was 93.6% ± 0.1% for 1 and 93.8 ± 0.1% for 2 , as established by HPLC analysis [13 (link)] (Fig 2 in S1 File ).
Deuterium oxide (99.9%) was purchased from Solvents Documentation Synthesis (Peypin, France). Deuterated PBS (D-PBS) was prepared by dissolving PBS powder in deuterium oxide.
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