The dichloromethane–methanol extracts of both hairy root lines, SaTRN12.2 (1) and SaTRN7.1 (2), were analyzed using HPLC by monitoring Scopoletin (1 ) absorbance at λ = 344 nm and sphaeralcic acid (3 ) at λ = 357 nm. Scopoletin (99% purity; Sigma–Aldrich Chemical) and sphaeralcic acid (95% purity) were identified by comparing their absorption spectra and their retention times (Scopoletin, 10.3 min, and sphaeralcic acid, 22.8 min). Sphaeralcic acid was purified from cells in a suspension of S. angustifolia and was identified by our group [18 (link),19 (link),20 (link)]. The concentration ratio for Scopoletin quantification ranged from 1.25 to 20 µg/mL, and for sphaeralcic acid from 2.5 to 40 µg/mL. The regression equation for Scopoletin was (y) = 165,407 (x) + 16,720, r2 = 0.9993, and for sphaeralcic acid it was (y) = 7381.9 (x) + 1362.2, r2 = 0.9998.
Scopoletin
Manufactured by Merck Group
Sourced in United States, Germany, Italy
Scopoletin is a fluorescent compound used in laboratory equipment and instrumentation. It serves as a standard or reference material for fluorescence-based analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Caffeic acid, by Merck Group (9 mentions)
Chlorogenic acid, by Merck Group (9 mentions)
Gallic acid, by Merck Group (9 mentions)
P coumaric acid, by Merck Group (7 mentions)
Quercetin, by Merck Group (7 mentions)
Methanol, by Merck Group (7 mentions)
Ferulic acid, by Merck Group (6 mentions)
Umbelliferone, by Merck Group (6 mentions)
Esculetin, by Merck Group (6 mentions)
Apigenin, by Merck Group (5 mentions)
Luteolin, by Merck Group (5 mentions)
Kaempferol, by Merck Group (5 mentions)
Catechin, by Merck Group (5 mentions)
Formic acid, by Merck Group (5 mentions)
Fraxetin, by Merck Group (4 mentions)
Rutin, by Merck Group (4 mentions)
Sucrose, by Merck Group (4 mentions)
Milli q water purification system, by Merck Group (4 mentions)
Hesperidin, by Merck Group (4 mentions)
Esculin, by Merck Group (4 mentions)
59 protocols using scopoletin
1
HPLC Analysis of Scopoletin and Sphaeralcic Acid
2
Phytochemical Profiling of Portulaca oleracea
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All reagents and solvents used in this study were of analytical reagent or HPLC grade and were purchased from Merck (Darmstadt, Germany). The raw leaves and sprouts of Portulaca oleracea were collected in the spontaneous state, in a region of southern Italy. Standards used to identify purslane antioxidant compounds were: caffeic acid; p-coumaric acid; scopoletin; ferulic acid; quercetin-3-O-rhamnoside; quercetin; apigenin; bergapten, all purchased from Sigma Aldrich (Milan, Italy), or were purified with an HPLC-UV/VIS working at 280 nm using the same chromatographic conditions described in the LC/MS/MS section from purslane leaves extracts. Fractions of the different compounds collected from HPLC were quickly freeze-dried, then dissolved in methanol at a concentration of 10 µg/mL and used for LC/MS/MS investigations. All the other reagents used were purchased from Sigma Aldrich (Milan, Italy).
3
NF-κB Signaling Assay with Triptolide and Scopoletin
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HEK-Blue-Null1 was derived from the human embryonic kidney-293 (HEK-293) cell line and obtained from Invivogen (San Diego, CA, USA). These cells stably express an optimized secreted embryonic alkaline phosphatase (SEAP) reporter gene preceded by the NF-κB promoter.
In a 96-well plate, 5.0 × 104 HEK-Blue-Null1 were incubated overnight at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Dreieich, Germany) supplemented with 2 mMl -glutamine, 10% (v/v) heat-inactivated fetal bovine serum (FBS), 50 units/mL penicillin, 50 µg/mL streptomycin (Gibco) and 100 µg/mL Normocin (Invivogen). Cell were treated with 1.6 nM triptolide (Invivogen) and varying concentrations of scopoletin (5 µM, 20 µM and 40 µM) (Sigma-Aldrich). The activation of NF-κB was induced by 100 ng/mL of TNF-α for 24 h. Then, 20 µL aliquots of cell culture were added to 180 µL of pre-warmed Quanti-Blue detection reagent (Invivogen) per well according to the manufacturer’s instructions. NF-κB activation was detected by measuring SEAP spectrophotometrically at 630 nm (Tecan Teader, Tecan Group Ltd., Maennedorf, Switzerland).
In a 96-well plate, 5.0 × 104 HEK-Blue-Null1 were incubated overnight at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Dreieich, Germany) supplemented with 2 mM
4
Screening of Bioactive Compounds
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Dipyridamole (Sigma‐Aldrich and LKT Labs), VCR, scopoletin, tazarotene, dihydroxyflavone (Sigma‐Aldrich), quetiapine fumarate (LKT Labs and Selleck Chemicals), hymecromone, esculin (Selleck Chemicals, Houston, TX, USA), chir 99021 (Cayman Chemical), harmaline, fisetin (Tokyo Chemical Industry, Thermo Fisher Scientific, USA), aurintricarboxylic acid (Calbiochem, Millipore Sigma), piperacillin sodium (Santa Cruz Biotechnology), and olmesartan medoxomil (Ark Pharm).
5
Scopoletin-Induced Scopolin Accumulation
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Five-day-old BY-2 cell suspension (3 ml) was treated with 15 µl of 0.1 M scopoletin (Sigma) in dimethyl sulfoxide to produce and accumulate scopolin in vacuole44 (link).
6
Antimicrobial Effects of Coumarins
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The antimicrobial activity of scopoletin and fraxetin (Sigma Aldrich) against single bacterial strains was assayed in liquid culture in 50% tryptic soy broth (TSB, 15g/L; Sigma Aldrich). scopoletin and fraxetin stocks were prepared in sterile DMSO (Sigma Aldrich) and stored at -80°C. Bacterial colonies were picked from TSA plates into liquid TSB and grown for 2–3 days at 25°C with 180 rpm agitation. Liquid cultures were subcultured by diluting 1:100 into fresh TSB and incubated for 1–2 h. In a clear flat-bottom 96-well microtiter plate, 100 μl of subculture were added to 100 μl of fresh TSB media supplemented with scopoletin or fraxetin for a final 50 μM concentration, or equivalent DMSO negative control. The microtiter plate was sealed with a clear adhesive film to prevent evaporation. Growth was monitored kinetically in a microplate reader (Infinite M200 PRO, Tecan) with 30 seconds of shaking followed by measurement of optical density (OD) at 600 nm in four locations per well every 30–60 min for 18–20 h. The OD in each experiment was expressed as the average of triplicate wells per condition. Relative growth (Figure 2 D) was calculated by dividing the average final OD measurement of each strain and indicated condition by the average OD in the coumarin-free control.
7
Murine Osteoclastogenesis Assay
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Dulbecco’s modified eagle’s media (DMEM), minimum essential medium alpha medium (α-MEM), D-glucose, and Alizarin red S dye were supplied by Sigma-Aldrich Chemical (St. Louis, MO, USA), as were all other reagents, unless specifically stated otherwise. Fetal bovine serum (FBS), trypsin–EDTA, and penicillin–streptomycin were obtained from BioWhittaker (San Diego, CA, USA). 3-(4, 5-Dimetylthiazol-yl)-diphenyl tetrazolium bromide (MTT) was purchased from DUCHEFA Biochemie (Haarlem, Netherlands). The recombinant murine sRANKL was purchased from PeproTech (Rocky Hill, NJ, USA). Scopoletin (Sigma-Aldrich Chemical, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO) for live culture with cells; the final culture concentration of DMSO was <0.5%.
8
Detailed Reagent Procurement Protocol
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Reagent grade ethanol (ETOH), methanol (MeOH), chloroform, ethyl acetate (EtOAc) and HPLC grade water were obtained from Merck KGaA, (Darmstadt, Germany). Flumazenil (Lanexat®), diazepam (Valium®) clonazepam (Klonopin™) were from Hoffmann-La Roche Inc., [3H]-methyl-flunitrazepam 60-85Ci/mmol were purchased from ARC American Radiolabeled Chemicals Inc. Saint Louis, MO, Trizma® Base 99.9% (Tris [Hydroxymethyl] aminomethane) Sigma-Aldrich Saint Louis, MO. HPLC standards, (rutin, quercetin, isorhamnetin, scopoletin, citric acid, kaempferol-3-O-rutinose, isorhamnetin-3-O-glucoside, atropine and scopolamine all standards with purity higher than 95% by HPLC) were purchased either from Sigma Aldrich (Saint Louis, Mo, USA), ChromaDex (Santa Ana, CA, USA), or Extrasynthèse (Genay, France).
9
Artemisia Bioactive Compound Extraction
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Artemisia samples were harvested in the years 1999 and 2000 and kindly provided by the TCM Hospital Bad Kötzting (Bad Kötzting, Germany). Artemisinin and scopoletin were used as standards. Artemisinin was isolated from A. annua, whereas scopoletin was obtained from Sigma-Aldrich (Taufkirchen, Germany). Three different batches of A. annua were purchased from Anguo Ruiqi Chinese Herbal Medicines Co., Ltd. (Anguo, China).
10
Calibration Curves for Natural Compounds
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Scopoletin (99% purity, Sigma-Aldrich, Mexico City, Mexico), tomentin (98% purity), and sphaeralcic acid (99% purity) calibration curves were performed using the HPLC system from a 1.0 mg/mL solution in high-purity methanol (Merck, Kenilworth, NJ, USA). Six serial dilutions were performed at concentrations of 1.25–20 µg/mL for Scopoletin, 0.625–20 µg/mL for tomentin, and 2.5–40 µg/mL for sphaeralcic acid. The chromatograms were analyzed at λ = 343 nm for tomentin and Scopoletin, and at λ = 357 nm for sphaeralcic acid. Each calibration curve was constructed by plotting the peak area ratio of the compound (y) versus the analyte concentrations (x). Calibration curves of tomentin, Scopoletin, and sphaeralcic acid were fitted using a linear square model (y) = m (x) + b using Microsoft Office Excel software 2010 with correlation values of ≥ 0.9995.
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