Zygophyllaceae R.Br. is a family with a high level of variability in plasticity that results in many conflicts about its taxa ranking and authenticating processes. Five selected Egyptian species belonging to genera, Fagonia, Tribulus, and Zygophyllum were applied for a comparative chemical and molecular study to assess their range of variations. The plants were evaluated for their DPPH radical scavenging activity. LC-ESI-MS/MS profiling of the aqueous methanol extracts of the investigated plants and GNPS molecular networking tentatively identified 174 compounds belonging to various chemical classes. Flavonoids are predominant, and myricetin derivatives were reported for the first time from Fagonia glutinosa. Diethyl ether extracts analysis with GC/FID detected 10 unsaturated and 13 saturated fatty acids. T. terrestris possessed the largest concentration of unsaturated fatty acids (up to 76.39%) whereas Z. simplex had the highest concentration of the saturated ones (76.82%). DNA fingerprinting was performed using ISSR markers and variations with 75.32% polymorphism were detected. Chemical and molecular data suggest the distinction of Tribulus sp. from the other studied taxa and grouping Fagonia close to Zygophyllum. Moreover, the DPPH assay showed weak to moderate radical scavenging activity at 500 µg/ml with cell viability ranging from 63.5% to 90.1%.
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