HPTLC Method Development and Validation for Simultaneous Determination of Beta-Sitosterol and Oleanolic Acid in Eclipta alba

Author: Arpan Chakraborty, Arka Bhattacharjee, Baishakhi Mondal, Manas Chakraborty, Goutam Mukhopadhyay and Alpana Majumder

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Abstract

Eclipta alba, a medicinal plant of the Asteraceae family, is widely recognized and extensively used in traditional medical systems such as Ayurveda, Siddha, Homeopathy, Unani, Chinese, and folk medicine. It contains significant phytochemical components like triterpenes, flavonoids, coumestans, steroids, saponins, and polypeptides. Eclipta alba is a key ingredient in various herbal and Ayurvedic formulations, including Indulekha Bringha oil and Liv.52 Gnx pill. The objective of this study was to develop a validated HPTLC method for the simultaneous quantification of beta-sitosterol and oleanolic acid in E. alba. Challenges may include optimizing separation and detection parameters. The method was validated following ICH guidelines. In the developed method, the stationary phase employed was silica gel 60 F254, while the mobile phase consisted of a mixture of ethyl acetate, toluene, and formic acid (6:5:0.1 v/v/v). Derivatization with anisaldehyde sulfuric acid resulted in the formation of compact bands. The calibration curves for beta-sitosterol and oleanolic acid exhibited high correlation coefficients (r2) of 0.9966 and 0.9945, respectively, indicating a good linear relationship within the concentration range of 450-1800 ng/spot and 400-1850 ng/spot based on the area. The method's precision, accuracy, robustness, and selectivity were evaluated. The LOD and LOQ for β-sitosterol were 107.94 ng/spot and 327.12 ng/spot, respectively. For oleanolic acid, the LOD was 128.81 ng/spot and the LOQ was 356.64 ng/spot. Overall, this HPTLC approach proved to be effective, straightforward, accurate, and reproducible. The study provides a reliable method for measuring the levels of oleanolic acid and beta-sitosterol in Eclipta alba. This method can be used to ensure the quality of Eclipta alba products and to guide their therapeutic use.

Keywords

Eclipta alba, β-sitosterol, Oleanolic acid, HPTLC, Validation

Conclusion

The developed HPTLC technique enables the quantitative analysis of β-sitosterol and oleanolic acid in E. alba entire plant material. With RSD values of 2%, the method demonstrates satisfactory accuracy. The recovery percentages for β-sitosterol (100.28–100.36%) and oleanolic acid (99.87–100.19%) further confirm the efficiency and reliability of the method. In addition, the fingerprint profiling of chromatograms obtained from E. alba extracts can be used for comparing and evaluating commercial samples of the whole plant or specific portions. Compared to HPLC, HPTLC offers advantages such as shorter processing times, lower sample requirements, optimized extractions using cost-effective chemicals, and smaller mobile phase volumes. This quick, easy, and sensitive HPTLC procedure serves as a valuable quality control tool for assessing the aerial portion of E. alba.

References

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How to cite this article

Arpan Chakraborty, Arka Bhattacharjee, Baishakhi Mondal, Manas Chakraborty, Goutam Mukhopadhyay and Alpana Majumder (2023). HPTLC Method Development and Validation for Simultaneous Determination of Beta-Sitosterol and Oleanolic Acid in Eclipta alba. Biological Forum – An International Journal, 15(5): 1344-1351.