RediSep® Rf Reversed-phase C18 was used for low-pressure CC and silica gel 60 RP18 F254 (E. Merck, Darmstadt, Germany) for TLC on glass (Merck, Rahway, NJ, USA). Dimethylsulfoxide (DMSO), adenosine 5′-diphosphate sodium salt (ADP), and Sephadex® LH-20 were obtained from Sigma Aldrich (St. Louis, MO, USA). 1D (1H, 13C) and 2D NMR (HSQC, HMBC, COSY and NOESY) spectra were obtained using a Bruker Ascend 600 (600 MHz for 1H and 150 MHz for 13C) in deuterated chloroform (CDCl3; Sigma-Aldrich). All solvents used were of HPLC grade quality and obtained commercially from Sigma-Aldrich. The following drugs used for vascular reactivity experiments were also purchased from Sigma-Aldrich: D-glucose, KCl, CaCl2, (S)-(−)-BAY-K-8644, ACh, and PE. Reversed-phase C18 was used for low-pressure column chromatography and silica gel 60 RP18 F254 (Merck) for TLC on glass (Merck). TLC plates were sprayed with a saturated solution of ceric sulfate in 65% sulfuric acid (Sigma-Aldrich) and heated to 120 °C.
Rp 18 f254
Manufactured by Merck Group
Sourced in Germany, United States, China
RP-18 F254S is a type of reversed-phase silica gel thin-layer chromatography (TLC) plate. It is used for the separation, identification, and quantification of various organic compounds. The plate is coated with a layer of silica gel modified with n-octadecylsilyl (C18) functional groups, which provide a non-polar stationary phase. The plate also contains a fluorescent indicator (F254S) that allows for the visualization of separated compounds under ultraviolet (UV) light.
Automatically generated - may contain errors
Lab products found in correlation
Kieselgel 60 f254, by Merck Group (24 mentions)
Silica gel 60 f254, by Merck Group (21 mentions)
Kieselgel 60, by Merck Group (20 mentions)
Sephadex lh 20, by GE Healthcare (12 mentions)
Lichroprep rp 18, by Merck Group (9 mentions)
Silica gel, by Merck Group (8 mentions)
Sephadex lh 20, by Cytiva (7 mentions)
Combiflash rf, by Teledyne (7 mentions)
P 2000 polarimeter, by Jasco (7 mentions)
Sephadex lh 20, by Merck Group (7 mentions)
P 1020 polarimeter, by Jasco (6 mentions)
Rp 18, by Merck Group (6 mentions)
Diaion hp 20, by Mitsubishi (5 mentions)
Silica gel 60, by Merck Group (5 mentions)
Cosmosil 75c18 opn, by Nacalai Tesque (5 mentions)
Microtof q 2 mass spectrometer, by Bruker (5 mentions)
Uv 1800 spectrophotometer, by Shimadzu (5 mentions)
Jms 700, by JEOL (4 mentions)
Dip 370, by Jasco (4 mentions)
Eca 600 spectrometer, by JEOL (4 mentions)
151 protocols using rp 18 f254
1
Compound Separation and Characterization
2
Reversed-phase TLC of T. brownii Leaves
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Reversed-phase thin-layer chromatography aluminum-backed silica plates (RP-18F 254, 20 × 20 cm, Merck, Darmstadt, Germany) were used to fractionate the ethyl acetate extract of T. brownii leaves. Ellagic acid (E-2250, Sigma-Aldrich, Gillingham, UK), gallic acid (G-7384, Sigma-Aldrich, China), apigenin (Extrasynthese 286 Genay, France), corilagin (Sigma-Aldrich, Darmstadt, Germany), luteolin (Sigma-Aldrich, Darmstadt, Germany) and quercetin (Merck Art. 7546, 285 Darmstadt, Germany) were used as reference pure compounds, since many of them were previously known from Terminalia. A total of 20 µL of extract and compounds at 50 mg/mL and 5 mg/mL, respectively, was applied equidistantly 1.5 cm from the bottom of the TLC plate. The plate was developed in a mobile phase of methanol: water: orthophosphoric acid (50:50:1, v:v:v). The spots were visualized using a Camag Video documentation System (Camag Reprostar 3 TLC Visualizer) and two UV wavelengths at 366 and 254 nm.
The developed TLC plates (RP-18 F254s Merck, Darmstadt, Germany) containing the ethyl acetate extract of the T. brownii leaves were in situ subjected to qualitative antioxidant analysis using 0.2% w/v of the 2,2-Diphenyl-1-picrylhydrazyl reagent (DPPH, Sigma-Aldrich D9132-1G, Schnelldorf, Germany). All compounds with antioxidant properties were revealed with yellow color in visible light.
The developed TLC plates (RP-18 F254s Merck, Darmstadt, Germany) containing the ethyl acetate extract of the T. brownii leaves were in situ subjected to qualitative antioxidant analysis using 0.2% w/v of the 2,2-Diphenyl-1-picrylhydrazyl reagent (DPPH, Sigma-Aldrich D9132-1G, Schnelldorf, Germany). All compounds with antioxidant properties were revealed with yellow color in visible light.
3
Nuclear Magnetic Resonance Spectroscopy Protocol
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Nuclear magnetic resonance (NMR, H, 400 MHz; 13C, 100 MHz) spectra were recorded on a JHA-LAA 400 WB-FT spectrometer (Jeol Co., Tokyo, Japan), the chemical shifts are presented as ppm with tetramethylsilane as an internal standard. TLC was carried out on pre-coated silica gel 60 F254 (0.25 mm, Merck; Darmstadt, Germany) and RP-18 F254S (0.25 mm, Merck Co.). Column chromatography (CC) was carried out on a BW-820MH silica gel, Wakosil C-300 silica gel (40–63 µm) (Wako Chem. Co., Osaka, Japan). Medium pressure liquid chromatography (MPLC) was performed on LiChroprep RP-18 (size A and B; Merck Co.).
4
Structural Characterization of Natural Products
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NMR spectra (1D and 2D) were recorded using a JEOL JNM ECP-400 spectrometer (Tokyo, Japan) (400 MHz for 1H and 100 MHz for 13C). HMQC and HMBC experiments were optimized for 1JCH = 140 Hz and nJCH = 8 Hz, respectively. Optical rotations were recorded using a Jasco p-2000 digital polarimeter (Tokyo, Japan). HPLC (YOUNGLIN-YL9100, Younglin, Anyang, Korea) separation was performed on YMC-Pack ODS-A column (20 × 150 mm, 5 μm, and 12 nm) with a flow rate of 5 mL/min and the solvents used for HPLC were analytical grade. ESI-MS data were obtained using a Q-TOF micro-LC-MS/MS instrument (Waters, Milford, MA, USA) at Korea University, Seoul, Korea. TLC was performed on Kieselgel 60 F254 (1.05715; Merck, Darmstadt, Germany) or RP-18 F254s (Merck) plates. Spots were visualized by spraying with 10% aqueous H2SO4 solution followed by heating. Column chromatography (CC) was performed on silica gel (Kieselgel 60, 70-230 mesh and 230-400 mesh, Merck) and YMC octadecyl-functionalized silica gel (C18). These procedures were based on our previous report [13 (link)].
5
Tyrosinase Inhibition Assay Protocol
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NMR experiments were conducted on an ECA500 (JEOL, Tokyo, Japan), with the chemical shift referenced to the residual solvent signals, using methanol-d4 and DMSO-d6 as a solvent. TLC analysis was performed on silica-gel 60 F254 and RP-18 F254S plates (both 0.25 mm layer thickness, Merck, Darmstadt, Germany). Compounds were visualized by dipping plates into 10% (v/v) H2SO4 reagent and then air heat treated at 300 °C for 15 s. Silica gel (Merck 60A, 70–230 or 230–400 mesh ASTM) and reversed-phase silica gel (YMC Co., ODS-A 12 nm S-150, S-75 μm) were used for open column chromatography. Tyrosinase (T3824), kojic acid (K3125) and l -tyrosine (T3754) were purchased from Sigma-Aldrich (ST. Louis, Mo, USA). UV–vis and fluorescence spectra were measured by TECAN infinite 200 PRO® spectrophotometer (Zurich, Switzerland).
6
Chromatographic Isolation and Structural Elucidation
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Column chromatography (CC) was performed on a silica gel (70–230 and 230–400 mesh ASTM, Merck, Kenilworth, NJ, USA), Sephadex LH-20 (GE Healthcare, Chicago, IL, USA), and Diaion HP-20 (Mitsubishi Chemical Co., Tokyo, Japan). High-performance liquid chromatography (HPLC) was performed using the Gilson purification system, with a YMC ODS-A column (250 × 20.0 mm i.d., 5.0 μm, YMC Co., Tokyo, Japan). Thin-layer chromatography (TLC) was performed on silica gel 60 F254 and RP-18 F254S plates (Merck). The nuclear magnetic resonance (NMR) spectra were obtained using a JEOL 500 MHz spectrometer, using tetramethylsilane as the internal standard, and the chemical shifts were expressed as δ values. The organic solvents that were used for the chromatographic separations and extractions were distilled before use.
7
Analytical Characterization of Compounds
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Optical rotation was measured using a JASCO P-2000 polarimeter (Tokyo, Japan). The NMR spectra were acquired on a 400 MHz Agilent NMR spectrometer (DD2, Santa Clara, CA, USA) using CDCl3. The HRESIMS was performed on an Agilent 6220 Accurate-Mass TOF LC/MS system. Silica gel (230–400 mesh, Merck KGaA, 64271 Darmstadt, Germany) and RP-18 (YMC gel ODS-A, 12 nm, S-150 μm, YMC Co., Ltd., Kyoto 600-8106, Japan). Thin Layer Chromatography (TLC) analysis was performed on silica gel 60 F254 (0.2 mm thickness, Merck KGaA, 64271 Darmstadt, Germany) and RP-18 F 254S (Merck KGaA, 64271 Darmstadt, Germany) plates by visualization under UV light at 254 nm, 365 nm, and 10% (v/v) of sulfuric acid followed by heating. Preparative HPLC was performed using an Acme 9000 system (Young Lin, Anyang, Korea) equipped with a YMC-Pack Pro C18 column (5 μm, 250 × 20 mm i.d.).
8
Chromatographic Purification and Characterization
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Column chromatography were performed using silica gel (Kieselgel 60, 70–230, and 230–400 mesh, Merck, Darmstadt, Germany); Sephadex LH-20 (GE Healthcare, Uppsala, Sweden); and C-18 (ODS-A 12 nm S-150 and S-75 μm; YMC Co., Dinslaken, Germany) resins. Thin-layer chromatography (TLC) was performed using precoated silica gel 60 F254 and RP-18 F254S plates (both 0.25 mm, Merck). Spots in TLC were visualized by spraying with 10% aqueous H2SO4 solution, followed by heating at 300 °C dried air. Nuclear magnetic resonance (NMR) spectra were recorded using the JEOL ECA 500 spectrometer (1H, 500 MHz and 13C, 125 MHz) (Tokyo, Japan). AUDA (10007927), soluble epoxide hydrolase (10011669), and 3-phenyl-cyano(6-methoxy-2-naphthalenyl) methyl ester-2-oxiraneacetic acid (PHOME) (10009134) were purchased from Cayman (Cayman, Ann Arbor, MI, USA).
9
Chromatographic Purification and HPLC Analysis
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Column chromatography (CC), flash chromatography (FC), and thin layer chromatography (TLC) were performed on polyamide SC6 (50–160 µm, Macherey-Nagel GmbH and Co., Düren, Germany), silica gel 60 (45–63 µm, Molar Chemicals, Halásztelek, Hungary), and silica gel 60 F254 or RP-18 F254S (250 µm, Merck Co., Darmstadt, Germany), respectively. TLC was used at each chromatographic step to monitor the separation with a solvent system of toluene–ethyl acetate–formic acid (5:4:1, v/v/v), and the spots were detected under UV light (λ1 = 254 nm, λ2 = 365 nm) and daylight after spraying with 5% H2SO4 in ethanol followed by heating. HPLC analysis was performed on a Jasco 2010 series instrument equipped with a Jasco PU-2080 quaternary pump, a vacuum degasser, an AS-2055Plus intelligent autosampler and a Jasco MD-2010 Plus photodiode array detector (Jasco Co., Tokyo, Japan). Melting points were determined on a Boetius apparatus (VEB Analytik Dresden, Dresden, Germany). Organic solvents used for TLC, FC and CC (analytical grade) were purchased from Sigma-Aldrich (Budapest, Hungary), and HPLC solvents were purchased from Avantor Performance Materials (Gliwice, Poland). Papaverine was purchased from Takeda Pharma Ltd. (Budapest, Hungary).
10
Analytical Characterization of Isolated Compounds
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Optical rotation values were obtained using a JASCO P-2000 polarimeter (JASCO International Co. Ltd., Tokyo, Japan). IR spectra were acquired using a Nicolet 6700 FT-IR spectrometer (Thermo Electron Corp., Waltham, MA, USA). NMR spectra were recorded on an Advance 500 MHz spectrometer (Bruker, Billerica, MA, USA). HRESIMS data were obtained using an Agilent 6530 Q-TOF (Agilent Technologies, Inc., Santa Clara, CA, USA). Column chromatography (CC) was performed with silica gel (63–200 μm particle size, Zeochem, Lake Zurich, Switzerland) and RP-C18 (75 μm particle size, Nacalai Tesque, Kyoto, Japan). For TLC analysis, RP-18 F254S and silica gel 60 F254 plates, from Merck (Darmstadt, Germany) were used. A Gilson HPLC system equipped with an Optima Pak C18 column (10 × 250 mm, 10 μm particle size; RS Tech, Seoul, Korea) was used for the purification of compounds, with a flow rate of 2 mL/min and UV detection at 205 and 254 nm. Industrial-grade solvents (Daejung Chemical, Siheung, Korea) were used for preprocessing, including extraction and fractionation, and analytical-grade solvents (Daejung Chemical) were used for purification of the compounds.
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