Densitometric Validation and Optimisation of Polyphenols in Ocimum sanctum Linn by High Performance Thin-layer Chromatography.

Introduction: Ocimum sanctum Linn (Sanskrit: Tulasi; family: Libiaceae), popularly known as holy basil or Ocimum teinufolium, is found throughout the semitropical and tropical parts of India. In Ayurveda, Tulasi has been well known for its therapeutic potentials.

Objective: To optimise and develop a standard method to quantify seven polyphenols simultaneously by HPTLC.

Methods: A three-level factor Box-Behnken statistical design was used for optimisation, where extraction time (min), temperature (°C) and methanol:water ratio (% v/v) are the independent variables with polyphenols as the dependent variable. The separation was archived on a silica-gel 60 F254 HPTLC plate using toluene:ethyl acetate:formic acid:methanol (3:3:0.8:0.2 v/v) as the mobile phase. Densitometric analysis of polyphenols was carried out in the absorbance mode at 366 nm.

Results: The quantification of polyphenols was carried out based on peak area with a linear calibration curve at concentration ranges of 60-240, 20-200, 100-1600, 40-200, 200-1400, 10-160, 200-1400, 100-5000 ng/band for caffeic acid, ellagic acid, rutin, kaempferol, catechin, quercetin, eupalitin and epicatechin respectively. The method was validated for peak purity, precision, accuracy, limit of detection (LOD) and quantification (LOQ). Method specificity was confirmed using the retention factor value and visible spectra correlation of marker compounds.

Conclusions: A validated HPTLC method was newly developed for simultaneous quantification of seven polyphenols in an Ayurvedic preparation of O. sanctum. The proposed method is simple, precise, specific, accurate, cost-effective, less time consuming and has the ability to separate the polyphenols from other constituents.