Separation and quantification of lupeol in Hygrophila schulli by high-performance thin-layer chromatography


Lupeol is the main active constituent of Hygrophila schulli (HS) plant. A simple, reliable and reproducible method of quantification is needed for the quality control of HS. Therefore, high-performance thin-layer chromatography (HPTLC) method was established for determining lupeol in HS roots and aerial parts. The laboratory isolated and identified (by UV, FT-IR, 1H- and 13C-NMR and MS) lupeol from HS roots was used to develop the proposed method. The method was validated in terms of linearity, limits of detection and quantification, stability, precision, accuracy and robustness studies. Aluminum-backed HPTLC plates precoated with silica gel 60F254 were used as the stationary phase and benzene‒chloroform‒methanol (93:5.75:1.25, V/V) as the optimized mobile phase. Post-chromatographic derivatization was done with anisaldehye‒H2SO4 reagent, and the chromatograms were scanned using CAMAG TLC Scanner III at 540 nm. The linear regression analysis data for the calibration plots of lupeol showed good linearity relationship with y = 7.1841x + 751.42 (r2 = 0.9979) regression equation in the concentration range of 200–1000 ng band−1 (RF = 0.43 ± 0.02). Mean ± SD values of slope and intercept were 7.1840 ± 0.067 and 751.42 ± 45.50, respectively. The stability study suggested that lupeol in chloroform was stable within 24 h at room temperature (%RSD = 1.07). The average percentage recovery of lupeol from the petroleum ether extracts of HS roots and aerial parts was 98.66 ± 0.65 and 98.77 ± 0.39, respectively, with a % w/w content 5.02 ± 0.23 and 0.39 ± 0.11% w/w on dry weight basis. The limits of detection and quantification were found to be 17.85 and 54.11, respectively.

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The authors gratefully acknowledge Sophisticated Analytical Instrument Facility (SAIF), Punjab University, Chandigarh, India, for providing the results on spectral analysis (1H- and 13C-NMR, EIMS) of lupeol.

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BG had designed the work, isolated and identified lupeol from HS roots. PA and PL assisted to perform the experiment while DK and NK evaluated the data and helped to prepare this article.

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Correspondence to Balu Ghule.

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Ghule, B., Agrawal, P., Lal, P. et al. Separation and quantification of lupeol in Hygrophila schulli by high-performance thin-layer chromatography. JPC-J Planar Chromat (2021).

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  • Hygrophila schulli
  • HPTLC validation
  • Lupeol
  • 1H- and 13C-NMR