Rhein and Emodin anthraquinones of Cassia fistula leaves: HPTLC concurrent estimation, green synthesis of bimetallic ZnO-CuO NPs and anticancer activity against Panc-1 and OVCAR-3 cancer cells

A validated HPTLC method was developed for simultaneous quantification of rhein and emodin: two anthraquinones with diverse bioactivities in Cassia fistula (C. fistula) leaves extract, where concentrations of the measured compounds were calculated as 3.6 and 1.3 mg/g of plant extract for rhein and emodin, respectively. Moreover, rhein and emodin were incorporated into bimetallic zinc oxide-copper oxide nanoparticles (ZnO-CuO NPs) by a novel, safe, and environmentally friendly technique employing gum Arabic and gamma radiations. As indicated by HR-TEM, the formulated ZnO-CuO NPs were semi-spherical, with sizes ranging from 8.5 to 70.0 nm and average diameter of 22.27 ± 1.6 nm. They maintained a zeta potential of -25.2 mV at pH 7.2 because of Gum Arabic’s negative charge. Furthermore, C. fistula crude extract, rhein, emodin, and the synthesized ZnO-CuO nanoparticles were evaluated for their antiproliferative effect on two cancer cell lines (Panc-1; pancreatic cancer and OVCAR-3; ovarian cancer) and WISH normal cells. Both rhein and emodin showed promising cytotoxic effects on the tested cancer cells. However, their nano-loaded form exhibited potent cytotoxicity and enhanced selectivity towards Panc-1 and OVCAR-3 cancerous cells. Emodin-conjugated ZnO-CuO NPs displayed the lowest IC₅₀ value (0.64 ± 0.01 μg/mL on Panc-1 cells) compared to doxorubicin. Consequently, it was selected for further investigation. Annexin V/PI staining disclosed that emodin-conjugated ZnO-CuO NPs exerts its cytotoxic potential on Pan1 cells via apoptosis triggering since it increased the population of apoptotic cells by 8.3-fold change compared to untreated ones. Emodin-conjugated ZnO-CuO NPs has altered the apoptotic cascade in Panc-1 cells by causing overexpression of the proapoptotic genes while restricting the expression of Bcl-2 the antiapoptotic gene as indicated by RT-PCR.