Plantago major of the plantain family has been observed as a wound-healing plant species on account of its ursolic acid (UA) content. The study's objectives were twofold: to prepare UA gels from P. major and validate the analytical method using TLC-densitometer. Here, vacuum and open column chromatography were used to isolate UA from the methanol extract of P. major. Afterward, data on the compound's physical properties and infrared (IR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and Nuclear Magnetic Resonance ( 1 H NMR, 13 C NMR) results were analyzed. Carbomer and hydroxy propyl methyl cellulose (HPMC) were used as gelling agents to make the UA gel. To validate the analytical method, TLC-densitometer was performed using TLC silica gel 60 F254 plates (the stationary phase) and a mixture of toluene: ethyl acetate: formic acid (8:2:0.1, the developing solvent). The results showed that the UA gel was successfully prepared using both gelling agents and that its physical properties (i.e., visual forms, viscosity, and flow behavior) and pH values were maintained throughout the 90-day accelerated stability test (40±2°C; 75±5% RH). In addition, the method validation results indicated compliance with relevant parameters: linearity (r=0.998), limit of detection (4.55 ng/spot) and limit of quantification (15.17 ng/spot), recovery (95.04-116.82%), intraday precision (RSD=1.24-1.96%) and interday precision (RSD=6.29%). Overall, the UA gel is stable for 90 days (accelerated stability test), and the TLC-densitometry proposed in the research can be used to identify and measure the chemical content of a gel preparation.
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