The beneficial effects of plant-rich diets and traditional medicines are increasingly recognized in the treatment of civilization diseases due to the abundance and diversity of bioactive substances therein. However, the important active portion of natural food or plant-based medicine is presently not under control. Hence, a paradigm shift from quality control based on marker compounds to effect-directed profiling is postulated. We investigated 68 powdered plant extracts (botanicals) which are added to food products in food industry. Among them are many plants that are used as traditional medicines, herbs and spices. A generic strategy was developed to evaluate the bioactivity profile of each botanical as completely as possible and to straightforwardly assign the most potent bioactive compounds. It is an 8-dimensional hyphenation of normal-phase high-performance thin-layer chromatography with multi-imaging by ultraviolet, visible and fluorescence light detection as well as effect-directed assay and heart-cut of the bioactive zone to orthogonal reversed-phase high-performance liquid chromato-graphy−photodiode array detection−heated electrospray ionization mass spectrometry. In the non-target, effect-directed screening via 16 different on-surface assays, we tentatively assigned more than 60 important bioactive compounds in the studied botanicals. These were antibacterials, estrogens, antiestrogens, androgens, and antiandrogens, as well as acetylcholinesterase, butyrylcholinesterase, α-amylase, α-glucosidase, β-glucosidase, β-glucuronidase, and tyrosinase inhibitors, which were on-surface heart-cut eluted from the bioautogram or enzyme inhibition autogram to the next dimension for further targeted characterization. This biological-physicochemical hyphenation is able to detect and control active mechanisms of traditional medicines or botanicals as well as the essentials of plant-based food. The array of 1,292 profiles (68 samples × 19 detections) showed the versatile bioactivity potential of natural food. It reveals how efficiently and powerful our natural food contributes to our homeostasis.

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