Alumina Column Chromatography Lab Report

Isolation of Ecdysterone from Sesuvium portulacastrum

As detailed in Figure1, Ecdysterone was isolated from Sesuvium portulacastrum using a sequential extraction process with Chloroform (CHCl3) followed by Methanol (MeOH), after which Alumina column chromatography of MeOH extract was performed. The different fractions were then eluted using varying proportions of CHCl3 and MeOH, resulting in MeOH: CHCl3 (20:80) bioactive fraction.

Thin-layer chromatography (TLC) analysis of MeOH: CHCl3 (20:80) fraction was carried out using a solvent system containing MeOH (15%) and Ethyl Acetate (85%). The TLC plates were visualized by spraying with vanillin- HCl reagent resulting in a UV sensitive band, which on heating, developed green color. The UV sensitive bands were purified using repetitive preparative TLC followed by crystallization. The identity of Ecdysterone was established by the following procedure: HPLC, with a Shimadzu LC-20, a Phenomenex C-18 reverse-phase Luna C18 which was used with a mobile phase of MeOH:Water (1:1) at 1.80 mL/min and the absorbance was monitored at 254 nm. Studies confirming the presence of a single peak of the isolated Ecdysterone, with a characteristic UV absorption at 246 nm were done using commercial standard Ecdysterone (Sigma) (Figure 2 A and B).

A linear wound was created on a monolayer of 3T3L1 cells and treated with or without Ecdysterone at a concentration of 100µM. Ecdysterone significantly enhanced wound healing activity in a time dependant manner when compared to the control (Figure 3A). Further studies with Ecdysterone at increasing concentrations demonstrated that Ecdysterone enhances wound healing in a dose dependent manner as well (Figure