PMA Induces Vaccine Adjuvant Activity by the Modulation of TLR Signaling Pathway
Figure 2
Isolation procedures of an active compound from Croton tiglium. (a) Column chromatography and HPLC.Components of the chloroform fraction from Croton tiglium were divided using column chromatography. The dried chloroform fraction was eluted on a silica gel column (5 × 40 cm; Merck, 63–200 μm particle size) with a solvent gradient of hexane/acetone (20 : 1 to 1 : 3 ratios) to yield ten fractions (Fr.1–Fr.10). Fr.8 showing the most potent activity was divided into 9 subfractions (Fr.81–Fr.89) using RP-C18 column with a solvent gradient of MeOH/H2O (1 : 2 to 10 : 1). Fr.87 was applied to 8 fractions (Fr.871–Fr.878) using Gilson HPLC system with OptimaPak C18 column (10 × 250 mm, 5 m particle size). For activity-guided fractionation, pNF-B reporter activities of each fraction were evaluated in HEK293T cells transiently transfected with pNF-B-SEAP and phTLR5. (b) HPLC comparison of compound 1 and PMA. An isolated compound 1 from Croton tiglium was analyzed by coinjection with PMA standard from Sigma Co. (St. Louis, USA) by a Gilson HPLC with the 321-pumps systems; UV/Vis-155; 234-autoinjector; an OptimaPak C18 column ( mm, particle size 5 μm), using a gradient of methanol and 0.1% formic acid in H2O as mobile phase. Detection was analyzed with two channels at 205 and 254 nm (blue line; 205 nm, red line; 254 nm). Solvent elution was carried out with a gradient of methanol and 0.1% formic acid in H2O as mobile phase, at a flow rate of 2 mL/min. PMA and compound 1 had the same retention time at 31.3 minute. (c) The chemical structure. The chemical structure of compound 1 was confirmed by 1H and 13C NMR spectra (Varian Unity Inova 600 MHz spectrometer) and MS data (Micromass, Wythenshawe, UK).
