Simultaneous determination of betulinic acid, β-sitosterol and lupeol in fruits, leaves, root and stem bark of Dillenia pentagyna Roxb. by a validated high-performance thin-layer chromatography method

In the present investigation, a simple and effective high-performance thin-layer chromatography (HPTLC) method was developed and validated for the simultaneous determination of three biologically active phytoconstituents, i.e. betulinic acid (BA), β-sitosterol (BS) and lupeol (LU) in fruits, leaves, root and stem bark of Dillenia pentagyna Roxb., a critically endangered and medicinally important species of Madhya Pradesh state of India. The separation was achieved on thin-layer chromatography (TLC) aluminum plates precoated with 0.2 mm layer of silica gel 60F254 in the mobile phase petroleum ether‒ethyl acetate‒acetonitrile (8.2:1.8:0.1, V/V). The plates were derivatized with anisaldehyde‒sulphuric acid reagent followed by heating at 105 °C on CAMAG hot plate for 5 min. Densitometric scanning of the plates was carried out using CAMAG TLC Scanner 4 equipped with winCATS software at λmax = 580 nm under tungsten lamp. The linear regression data for the calibration plots exhibited a satisfactory linear relationship with r2 = 0.995, 0.989 and 0.996 for BA, BS and LU, respectively. Accuracy of the method was checked by recovery study conducted at three different levels with the average recovery of 100.7, 101.67 and 101.99% for BA, BS and LU, respectively. The results showed the highest content of BA (0.920 ± 0.02%) and LU (0.369 ± 0.01%) in stem bark, while leaves contained the maximum amount of BS (1.555 ± 0.07%). The developed method may be used for the quality control of fruits, leaves, root and stem bark of D. pentagyna.