Silica gel 60 f254

Unless otherwise noted, all materials used in this study were purchased from commercial suppliers and used without further purification. Unless otherwise noted, work-up and purification procedures were performed with reagent-grade solvents under air. Analytical thin-layer chromatography (TLC) was performed using E. Merk silica gel 60 F₂₅₄ precoated plates (0.25 mm). The developed chromatogram was analyzed with a UV lamp (254 nm) and ethanolic ninhydrin. Flash column chromatography was performed with an Isolera Spektra instrument equipped with a Biotage® Sfär Silica HC D 10 g or Sfär Silica HC D 50 g cartridge. Preparative high performance liquid chromatography (HPLC) was performed on a Prominence HPLC system (Shimadzu) with a 5C18-AR-II column (#38150-41, Nacalai tesque). High-resolution mass spectrometry (HRMS) was measured using a Bruker micrOTOF II (ESI). Nuclear magnetic resonance (NMR) spectra were recorded on a JEOL ECS400 (¹H 400 MHz, ¹³C 100 MHz) spectrometer. Chemical shifts for ¹H NMR are expressed in parts per million (ppm) relative to CHCl₃ (δ 7.26 ppm) in CDCl₃, HOD (δ 4.79 ppm) in D₂O. Chemical shifts for ¹³C NMR are expressed in ppm relative to CDCl₃ (δ 77.19 ppm) in CDCl₃. Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet), coupling constant (Hz), and integration.

Melting points were determined on open glass capillaries using Gallenkamp apparatus and are uncorrected. Infrared spectra were measured on a Shimadzu FT-IR 8400s infrared spectrophotometer (cm⁻¹) using KBr disks, Faculty of Pharmacy, Cairo University, Cairo, Egypt. ¹H and ¹³C-NMR spectra were determined in DMSO-d₆ with Bruker Avance-400 spectrometer operating at 400 MHz and 100 MHz, respectively, at Applied Nucleic Acid Research Centre (ANARC), Faculty of Science, Zagazig University, Zagazig, Egypt. TLC was performed on 2.5 × 5 cm aluminum plates coated with silicagel 60F-254 (Merk, Germany) in an appropriate solvent. Span 60 was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Sodium taurocholate (STC) and sodium deoxycholate (SDC) were purchased from BASF Co. (Florham Park, NJ, USA). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N [methoxy(polyethylene glycol)-2000] (DSPE–mPEG-2000) was a generous gift from Lipoid GmbH (Ludwigshafen, Germany). Cholesterol was obtained from ITX Biomedicals (Santa Ana, CA, USA). Sodium hydroxide, potassium dihydrogen orthophosphate and absolute ethanol were purchased from El-Nasr Chemical Co., Cairo, Egypt. The Spectra/PoreVR dialysis membrane (12,000–14,000 molecular weight cut off) was purchased from Spectrum Laboratories Inc., Los Angeles, CA, USA. All chemicals and solvents were of HPLC grade and were used as received.

All manipulations and reactions were performed using standard Schlenk techniques. The starting materials, reagents, and solvents were obtained from commercial suppliers and were used without further purification. Thin-layer chromatography was performed with Merk silica gel 60 F₂₅₄ pre-coated glass sheets. Column chromatography was performed on Biotage Isolera One™ flash chromatography system and the eluting solvents are noted as a mixed solvent with given volume-to-volume ratios or as a percentage. Uncorrected melting points were measured using Optimelt Automated Melting Point System (Stanford Research Systems). ¹H and ¹³C NMR were measured on a 400 MHz Bruker Avance or a 500 MHz Agilent 500 NMR spectrometer. Chemical shifts and coupling constants are presented in parts per million (ppm) relative to Me₄Si and hertz (Hz), respectively, and the following abbreviations are used: s, singlet; d, doublet; dd, a doublet of doublets; t, triplet; m, multiplet. High-resolution mass spectra were performed on Waters ACQUITY UPLC BEH C18 1.7µ−Q-TOF SYNAPT G2-Si High Definition Mass Spectrometry.

The brown pigment degradation products were identified by hydrolysis of the pigment as described by Ellis and Griffiths 1974^{41 (link)} with slight modifications. Five milligrams of the dried pure brown pigment was added to 0.5 g of KOH in a 5 ml tightly sealed screw cap glass tube. The mixture was brought to boil on hot water bath (1 h) and the dark color residue thus formed was allowed to cool. To the dried residue 1.5 ml, distilled water was added and acidified (pH 2) with concentrated HCl and metabolites were extracted with diethyl ether and dried under rota evaporator (Heidolph, Germany) finally dissolved in HPLC grade methanol. To detect the indoles, the hydrolyzed fraction was run on TLC (Merk, Silica gel 60 F₂₅₄, 20×10 cm, 0.2 mm,) using a mixture of Chloroform: Methanol: Glacial acetic acid (9: 0.95: 0.05 v/v) as a solvent system and TLC plate was developed using indole-specific TLC reagent prepared as described by Ehmann et al.^{42 (link)}. Alkaline hydrogen peroxide oxidation of brown pigment was carriedout according to Ito et al. 2011 and LCMS analysis was performed as described in HRLC-MS.