C18 column

Nine polyphenolic standards were used in HPLC analysis for their detection and quantification in P. wallachiana fractions [77 (link)]. Fractions (1 mg/mL concentration) were filtered with the help of a Membrane filter (0.45 µm) and analyzed on a Perkin Elmer HPLC system equipped with a LC 295 UV/VIS detector, binary LC pump, and a reverse phase C18 column (4.6 mm × 250 mm, 5 µm). Solvent A (acetonitrile) and solvent B (distilled water/acetic acid, 99:1 v/v, pH 3.30 ± 0.1) were used in combination to serve as a mobile phase. Linear gradient mobile phase with a flow rate of 1 mL/min and 20 µL injection volume of the sample was employed with detector setting at 285 nm and 370 nm for phenolics and flavanoids, respectively. Gallic acid, epicatechin, catechin, trans-ferulic acid, and trans-p-coumaric acid were used as phenolic standards (Λ max: 285 nm). The conditions of gradient program used for phenolic acid separation were 20% A (5 min), 20% A (5 min), 80% A (10 min), 20% A (5 min). Flavonoids standards (Λ max: 370 nm) used were Rutin, Myrecitin, Quercitin and Kaempferol, and flavanoids were separated using the program: 20% A (5 min), 20% A (5 min), 80% A (7 min), 20% A (8 min). The analytes were identified by comparing the Rt (retention time), and spike samples with polyphenolic standards and subsequent quantification of phenolic compounds was determined.

All reagents and solvents, including dichloromethane, chloroform, methanol, hexane, ethyl acetate, and butanol, were purchased from Sigma Aldrich. The plant extracts were concentrated using a Buchi Rotavapor R-300. Column chromatography was performed using silica gel (35–75 mesh and 200–425 mesh), diol silica gel (75–200 mesh), silica gel C-18 reversed-phase (35–75 mesh), and sephadex LH-20. TLC analyses were carried out using Analtech glass precoated Si gel plates and precoated reversed-phase Si gel plates. Spots were detected on TLC under UV light or iodine vapors chamber. IR spectra were recorded on a Jasco FT/IR 4200 spectrometer. HPLC was performed on a Perkin Elmer Series 200 instrument equiped with a Kromasil C-18 Column (250 × 4.6 mm, 5 μm, flow rate: 1 mL/min). NMR data were recorded on a Bruker NMR spectrometer operating at 400 MHz for ¹H-NMR and 100 MHz for ¹³C-NMR. All ¹H-NMR and ¹³C-NMR chemical shifts were referenced to residual CHCl₃ in the deuterated solvent (7.26 ppm for ¹H-NMR and 77.0 ppm for ¹³C-NMR).