LC/MS/MS and GC/MS/MS metabolic profiling of Leontodon hispidulus, in vitro and in silico anticancer activity evaluation targeting hexokinase 2 enzyme.

Leontodon hispidulus Boiss is a wild annual plant growing in Egypt. The present study aims for the first time, to evaluate the phytochemical profile of the main secondary metabolites of the optimized ethanolic extract of the plant using Quadrupole Time-of-Flight Liquid chromatography-mass spectrometry and Gas chromatography-mass spectrometry. It also aims to assess the anticancer activity of its different fractions against the prostate carcinoma cell line. Moreover, an in-silico docking study was performed using the Hexokinase-two enzyme. LC-qToF-MS analysis revealed the tentative identification of 36 phenolic compounds including the glycosides of (luteolin, quercetin, kaempferol, apigenin, isorhamnetin, and daidzein), coumarines (esculin, esculetin, and daphnetin), and phenolic acids (chlorogenic, caffeic, quinic, P-coumaric, and rosmarinic). GC-MS/MS analysis revealed the presence of 18 compounds where palmitic acid, myristic acid, alpha-amyrin, and beta-amyrin were the major ones. The cytotoxic activity results revealed that methylene chloride and ethyl acetate fractions showed the highest cytotoxic activity against the PC3 cell line, with IC50 values of 19, and 19.6 µg/ml, respectively. Interestingly, the docking study demonstrated that apigenin-7-O-glucoside, luteolin-7-O-glucoside, kaempferol-3-O-glucuronide, quercetin-4'-O-glucoside, esculin, rosmarinic acid, chlorogenic acid, and α-amyrin exhibited high affinity to the selected target, HEK-2 enzyme.

Application of Box-Behnken design for optimization of phenolics extraction from Leontodon hispidulus in relation to its antioxidant, anti-inflammatory and cytotoxic activities.

To the best of our knowledge, there have been no phytochemical studies concerning the wild plant Leontodon hispidulus Boiss. (Asteraceae). Optimization of the green extraction process of the plant aerial parts, identification of main phenolic compounds, evaluation of antioxidant, anti-inflammatory and anticancer activities of the optimized extract have been carried out. HPLC-analysis was performed using 95% ethanolic extract. 3-Level Box-Behnken Design was applied for optimization of extraction yield and total phenolic content using 3-factors (ethanol/water ratio, material/solvent ratio and extraction time). Antioxidant, anticancer and anti-inflammatory activities were evaluated by ABTS-assay, prostate and cervical carcinoma human cell lines and carrageenan-induced rat paw edema model, respectively. HPLC-analysis showed the presence of quercetin, rutin, kaempferol, chlorogenic and ρ-coumaric acids. Increasing both ethanol/water ratio and material/solvent ratio decreased the yield, while, it increased by prolongation of the extraction time. High material/solvent ratio increased the phenolic content. The optimized extract showed high total phenolic content (104.18 µg/mg) using 201 ml of 74.5% ethanol/water at 72 h and good biological activities. Antioxidant activity was found to be 41.89 mg Trolox-equivalent/gm, with 80% free radicals inhibition. For anti-inflammatory activity, 100 mg/kg of the extract inhibited the edema in rats by 83.5% after 4 h of carrageenan injection as compared to 81.7% inhibition by indomethacin. Prostate carcinoma cell line was more sensitive to the anticancer activity of the extract than cervical carcinoma cell line (IC50 = 16.5 and 23 µg/ml, respectively). The developed extraction procedure proved to be efficient in enriching the extract with phenolic compounds with promising anticancer, anti-inflammatory and antioxidant activities.