Anticancer activity and concurrent analysis of ursolic acid, β-sitosterol and lupeol in three different Hibiscus species (aerial parts) by validated HPTLC method.

The genus Hibiscus contains about 275 species of flowering plants widely grown in the tropics and sub-tropics. The available literature revealed that several Hibiscus species exhibited excellent anticancer activity against several cancer cells like lung, breast, and liver. This motivated the authors to explore the anticancer property of other Hibiscus species (Hibiscus calyphyllus, H. deflersii and H. micranthus) along with development of a validated HPTLC method for the concurrent analysis of three anticancer biomarkers (ursolic acid, β-sitosterol and lupeol) in different Hibiscus species. The anticancer activity of various fractions (petroleum ether, toluene, dichloromethane, ethyl acetate and n-butanol) of all the Hibiscus species (aerial parts) were evaluated in vitro against HepG2 and MCF-7 cell lines using MTT assay. The HPTLC analysis was carried out using chloroform and methanol as mobile phase (97:3; v/v) on 20 × 10 cm glass-backed silica gel 60F254 plates and analyzed different phytoconstituents present in all fractions at λ = 575 nm wavelength. Of the tested fractions of H. calyphyllus, H. deflersii and H. micranthus, HdP (H. deflersii petroleum ether fraction) exhibited the most potent cytotoxic effect on HepG2 and MCF-7 (IC50: 14.4 and 11.1 μg/mL, respectively) cell lines. Using the developed HPTLC method a compact and intense peak of ursolic acid, β-sitosterol and lupeol were obtained at Rf = 0.22, 0.39 and 0.51, respectively. The LOD/LOQ (ng) for ursolic acid, β-sitosterol and lupeol were found as 42.30/128.20, 13.20/40.01 and 31.57/95.68, respectively in the linearity range 100-1200 ng/spot. The obtained result showed maximum presence of ursolic acid, β-sitosterol and lupeol (5.50, 11.85 and 7.47 μg/mg, respectively) in HdP which also supported its strong anticancer effect. Our data suggest that H. deflersii petroleum ether fraction (HdP) can be further subjected to the isolation of active cytotoxic phytoconstituents and establishment of their mechanism of action. The maiden developed HPTLC method for concurrent analysis of anticancer biomarkers may be further employed in the in process quality control of herbal formulation containing the said biomarkers.