*3.3. Chromatographic and Spectroscopic Techniques*

Polyamide 6S, Silica Gel 60 (60–120 mesh), and Sephadex LH-20 (Riedel-de Haën AG, Seelze, Germany) were used for column chromatography (CC). Medium-pressure liquid

chromatography (MPLC) was performed using Reveleris Prep System set (Buchi, Flawel, Switzerland) with a UV-ELSD detector, a C-18 flash column (FP ID C18, 35–45 μM, 40 g). Celite No. 545 from Wako (Japan) was used for loading sample. Analytical pre-coated Silica Gel 60 F245 plates (NP-TLC), preparative reversed-phase silica gel 60 RP-18 F254S (RP18-PTLC) thin layer chromatography plates (Merck, Germany), and preparative normal phase silica Gel 70 FM (NP-PTLC) thin layer chromatography plates (Wako, Japan) were used for the final purification of compounds. Thin layer chromatography (TLC) plates were visualized under UV light at (254 and 365 nm) and sprayed with 10% MeOH-H2SO4 reagent, followed by heating for 2–3 min. Methanol used for extraction in CC was of analytical grade. Methanol and formic acid for MS and HPLC analyses were of HPLC grade. HPLC analysis was employed using an Agilent 1220 Infinity LC system, equipped with ELSD detector, a binary solvent delivery system, and an autosampler and connected to YMC column (5 μM, 4.6 × 150 mm, Japan). Aqueous formic acid (0.1%) and acetonitrile were used as mobile phases A and B, respectively, with the total flow rate at 1.0 mL/min for 35 min.

Detection of UV absorption of isolated compounds was done using a Shimadzu ultraviolet–visible (UV–Vis) 1601 recording spectrophotometer (P/N 206-67001, Kyoto, Japan) over the range of 190–500 nm was used for all measurements. Path length of cuvettes used was 1 cm. Manipulation of spectra was performed using UVProbe 2.42 software.

Optical rotation was measured on a Jasco DIP-370 polarimeter.

The NMR 1D and 2D spectra were recorded in CD3OD, using TMS as internal standard, and chemical shift values were recorded in δ ppm on a Bruker DRX 600 NMR spectrometer. Sample was completely dried to remove any residual solvent, resuspended in 600 μL deuterated methanol (CD3OD), and centrifuged prior to NMR analysis.

The HR-ESI-MS was acquired on an Agilent 6545 Q-TOF LC–MS system with dual electrospray ionization (ESI) (Santa Clara, CA, USA) in negative ionization mode, as it is more sensitive for the detection of phenolics, due to their acidic nature making it easier for them to lose protons. IR was recorded on an FTIR-6700 (JASCO, Tokyo, Japan). The sample was ground with KBr in a ratio of (1:10); the mixture is then pressed in disc form and placed into the sample hold, and the IR spectrum was run.
