Estimation of Oleanolic Acid by HPTLC and HPLC Methods in Successive Leaf Extracts of Leucas aspera and Tridax procumbens and their In vitro Anti-Inflammatory Activity

Abstract

Aims: The study was started with the goal of quantifying the oleanolic acid from consecutive leaf extracts of Leucas aspera and Tridax procumbens using a marker oleanolic acid by utilizing HPTLC and HPLC techniques, as well as to perform an evaluation of their antiinflammatory activity.
 Place and Duration of Study: The study was carried out between October 2022 and June 2023 in the Department of Pharmaceutical Analysis and Pharmacognosy, Sri Ramakrishna Institute of Paramedical Sciences, College of Pharmacy, Coimbatore-44, Tamil nadu, India.
 Methodology: The extraction of leaves is done using successive extractions by Continuous hot percolation method using soxhlet extractor. Petroleum ether, ethyl acetate and methanol fractions for which the phytochemical analysis were conducted. Standardization of oleanolic acid was performed by using HPTLC and HPLC techniques. Quantification of oleanolic acid in the two plants sequential leaf extracts were done. In vitro study of antiinflammatory activity was performed by Xanthine oxidase inhibitory activity in the ethyl acetate fraction.
 Results: Alkaloids, glycosides, terpenoids, steroids, flavonoids, saponins, carbohydrates, and proteins were all found in the two plant extracts by using phytochemical screening. In HPTLC method, petroleum ether, ethyl acetate and methanol leaf extracts of Leucas aspera and Tridax procumbens were developed in suitable mobile phase of toluene: ethyl acetate: formic acid (7:3:0.2%v/v/v) followed by derivatizing with anisaldehyde sulphuric acid derivatizing agent and scanned under 530nm. HPLC of standard marker and successive leaf extracts of Leucas aspera and Tridax procumbens were carried out using methanol: 25mM phosphate buffer (pH-3) in the ratio of 90:10% v/v at flow rate of 1ml/min and chromatograms were recorded at 202nm. Xanthine oxidase inhibitory activity of combined leaf extracts of ethyl acetate showed IC50 value of 0.026μg/ml. 
 Conclusion: Standardization of oleanolic acid was conducted by HPTLC and HPLC methods and linearity were found to be 0.9964 and 0.9998 respectively. Quantification of oleanolic acid in successive leaf extracts of the two plants were conducted. In vitro study using xanthine oxidase inhibitory activity showed that combined extracts of ethyl acetate fractions exhibited better antiinflammatory property than the individual extracts of the selected plants.