. 2020; 17(2): 148-158

High Performance Thin Layer Chromatographic method using DoE approach for estimation of phytochemicals in Moringa oleifera leaves extract

Asha Thomas1, Abhilash Kanakdhar1, Adinath Shirsat2, Sanjeevani Deshkar3, Lata Kothapalli1
1Department of Pharmaceutical Chemistry, Dr. D.Y. Patil Institute of Pharmaceutical Sciences & Research, Pimpri, Pune, India
2Peptide- Formulation Development (NDDS), Sun Pharmaceutical Industries Ltd, Vadodara, India
3Department of Pharmaceutics, Dr. D.Y. Patil Institute of Pharmaceutical Sciences & Research, Pimpri, Pune, India

INTRODUCTION: A systematic Design of Experiment (DoE) based sensitive, robust high performance thin layer chromatographic (HPTLC) method was established for simultaneous estimation of gallic acid (GA), quercetin (QT) and rutin (RT) from ethanolic and aqueous leaves extracts of Moringa oleifera.
METHODS: The chromatographic separation was carried on Merck TLC aluminum sheets of silica gel 60 F254 (10 X 10 cm) with mobile phase of toluene: ethyl acetate: methanol: formic acid (4.9: 4.1: 2: 0.5, v/v/v/v) with densitometric scanning at 300 nm. The Critical Method Parameters were initially identified by Regular Two Level Factorial design and further systematically optimized using Central Composite design, evaluating effect on selected Critical Analytical Attributes, retention factor (Rf) and Peak-area.

RESULTS: The Pareto charts, 3D response surface plots and polynomial equations for the generated models suggested significant influence of selected factors on responses of QT, GA and RT. Desirability and overlay plots employed provided appropriate solutions which were experimentally validated. Under the optimized conditions, the biomarkers were suitably resolved with Rf values of 0.64 ± 0.02, 0.80 ± 0.03 and 0.22 ± 0.02 for GA, QT, and RT respectively with wide linear dynamic range (200-1200 ng/band each), high accuracy (98.1-99.4 %) and intra- and inter-day precision (%RSD<2%).
DISCUSSION AND CONCLUSION: When employed for quantification of these biomarkers in Moringa oleifera extracts, the ethanolic and aqueous extracts exhibited higher content of QT (993.5µg/g and 832 µg/g respectively).The ethanolic extract showed larger amount of RT (701µg/g). In contrast, aqueous extract exhibited higher proportion of GA (591.1µg/g) compared to ethanolic extract (150µg/g).
Conclusions: This validated HPTLC method developed through DoE approach was successfully employed for quantification of GA, QT and RT from Moringa oleifera extracts and may also be extended for their simultaneous estimation in other herbal extracts thereby reducing time and may serve as a cost effective tool for analysis.


Keywords: DoE, Gallic acid, HPTLC, Moringa oleifera, Quercetin, Rutin


Asha Thomas, Abhilash Kanakdhar, Adinath Shirsat, Sanjeevani Deshkar, Lata Kothapalli. High Performance Thin Layer Chromatographic method using DoE approach for estimation of phytochemicals in Moringa oleifera leaves extract. . 2020; 17(2): 148-158

Corresponding Author: Asha Thomas, India


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