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Densitometric validation of phyllanthin, oleanolic acid, and betulinic acid in Phyllanthus maderaspatensis linn by high-performance thin layer chromatography

1 Department of Pharmacognosy and Phytochemistry, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India
2 Proteomic and Translational Research Lab, Centre for Medical Biotechnology, Amity Institute of Biotechnology, Amity University Uttar Pradesh, Noida, Uttar Pradesh, India

Correspondence Address:
Vidhu Aeri,
Hamdard University, New Delhi
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0973-1296.251402

Introduction: Phyllanthus maderaspatensis Linn. (Euphorbiaceae) is widely distributed in China, Southern India, South Africa, and Sri Lanka. In India, it is traditionally used for its hepatoprotective activity. Objective: To optimize and develop a simple and rapid high-performance thin layer chromatography method for simultaneous determination of phyllanthin, oleanolic acid, and betulinic acid in the P. maderaspatensis. Methods: Separation and quantification of markers were achieved by thin layer chromatography using mobile phase of hexane:ethyl acetate:glacial acetic acid (7:3:0.2 v/v/v) on precoated silica gel 60F254 aluminum plates and simultaneous densitometric determination of oleanolic acid and betulinic acid were performed after derivatization with anisaldehyde – sulfuric acid reagent in the reflection/absorbance mode at 356 nm and 530 nm while determination of phyllanthin was densitometric measurements of bands at 284 nm without derivatization. Result: The quantification of markers was carried out based on peak area with linear calibration curve at concentration ranges 50–400 for phyllanthin, 100–5000 ng/bands for oleanolic acid. Validation of the method was allotted supported the ICH guidelines in terms of peak purity, linearity, precision, robustness, accuracy, limit of detection, and quantification. The recovery studies were allotted to ascertain the sensitivity of the method and for system preciseness study indicated that no significant intra- and inter-day variation. The detection and quantification limits and were 10 and 50 ng/band for phyllanthin and 50 and 100 ng/band for oleanolic acid, respectively. Robustness was studied by introducing small change parameters such as mobile phase ratio and detection wavelength of analysis and therefore, the variance of peak areas were calculated. Conclusions: A validated HPTLC method was developed for simultaneous quantification of Phyllanthin, oleanolic acid and betulinic acid in P. maderaspatensis. The proposed method is rapid, simple, precise, specific, accurate, cost effective and has the ability to separate the lignan and triterpenoids from other constituents. Abbreviations Used: HPTLC: High-performance thin layer chromatography; RSD: Relative standard deviation; CV: Coefficient of variation; LOD: Limit of detection; LOQ: Limit of quantification; TLC: Thin layer chromatography; nm: Nanometer.


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