Dmitri Sitnikov, PhD: No relevant financial relationship(s) with ineligible companies to disclose.
Objectives: The objective was to replace the laborious detection of bio-active compounds in complex samples used by a bio-directed fractionation (BDF). We hypothesized that the bioactive compounds could be detected and identified in cells co-incubated with complex plant extracts without fractionation.
Methods: Plant extracts were added to a media of in-vitro cell cultures in parallel with correspondent negative and positive controls. The incubation was followed by the extraction of cell metabolites and their analysis by LC-MS (liquid chromatography-mass spectrometry). The detection of phytochemicals was based on a statistical analysis of the differential abundance of plant metabolites between exposed and non-exposed cells. Putative compound identification was achieved by high-resolution MS methods (Orbitrap Fusion) including fragmentation and comparison to standard analytes. Functional annotations were assigned using an in-house blockchain application, that integrated experimental results, PubChem, and David databases. In addition, fractionated bio-actives were introduced to cell cultures for the confirmation/exploration of functional annotations by RNA analysis.
Results: The method resulted in the detection and annotation of at least 34 bio-active compounds in a rosemary extract and was accomplished in 3 weeks. Carnosol, rosmarinic acid, carnosic acid were identified among the ten most abundant chemicals; two were putatively identified and five remained unidentified. Functional annotations of the four isolated (fractionated) compounds by blockchain and RNA analysis indicated their anti-inflammatory, antioxidation, and cell junction enhancement effects. In addition, the method matches the high-throughput capacity of LC-MS and was successfully used in a separate experiment for the simultaneous profiling of bioactive compounds in 4 plant ingredients.
Conclusions: We have developed and implemented the high-throughput method for the detection, identification, and annotation of multiple bioactive chemicals in complex samples. The new method outperformed the BDF discovery method several-fold.
