Development and validation of HPTLC method for the quantitative estimation of Eugenol in Piper betle leaves

Abstract

Piper betle is a medicinally important aromatic plant widely used in traditional systems of medicine and known for its rich content of phenolic compounds, particularly eugenol, which contributes significantly to its pharmacological properties. The present study aimed to develop and apply a simple, rapid and reliable High-Performance Thin Layer Chromatography (HPTLC) method for the quantitative estimation of eugenol in Piper betle leaf extract. Shade-dried leaves were subjected to ethanolic extraction, followed by preliminary phytochemical screening to assess the presence of major secondary metabolites. Quantitative analysis was performed using HPTLC densitometry on silica gel 60 F₂₅₄ plates with an optimized mobile phase, and detection was carried out at 254 nm. Eugenol was identified in the extract by comparing its retardation factor (Rf) and UV spectral characteristics with those of a reference standard. The method exhibited excellent linearity over the selected concentration range, with a high correlation coefficient (R = 99.95%) and low coefficient of variation (1.56%), indicating good precision and reproducibility. The eugenol content in the ethanolic leaf extract of Piper betle was quantified as 10.53% (w/w). The developed HPTLC method proved to be cost-effective, robust and suitable for routine analysis, offering a practical approach for quality control and standardization of Piper betle and eugenol-containing herbal formulations. The findings further highlight the potential of Piper betle leaves as a valuable natural source of eugenol for pharmaceutical and nutraceutical applications.