Enzymatic bioautography on HPTLC: combined phytochemical and activity screening tool for quality assessment and in vitro cultivation bioprocess control for selected medicinal plants from the Balkan region
Wolfram, Evelyn; Bräm, Sarah; ; (2015). Enzymatic bioautography on HPTLC: combined phytochemical and activity screening tool for quality assessment and in vitro cultivation bioprocess control for selected medicinal plants from the Balkan region. Planta Medica, 81, 16. SL4B_05. Peer reviewed.
HPTLC is a tool of long term tradition in medicinal plants analysis. Separated compounds are fixed on the solid silica phase like a compound library. By direct performance of visualizable health relevant enzyme reactions on the dried plate, similarities and differences in the phytochemical and activity fingerprints can be identified.
In this work, enzymatic bioautographic inhibition assays, using xanthine oxidase (XOD) ; lipase  and acetylcholinesterase (AchE)  have been optimized and applied for screening to show challenges and pitfalls.
Merck HPTLC plates were used on CAMAG equipment. Flavonoid fingerprints, derivatized with Neu's/PEG reagent were detected at 366nm. Validated, robust procedures have been established. False positive results are avoided by detection at different wavelength. (...)
A binary answer for the activity of the separated zones appears visually on the HPTLC chromatogram. Application of positive controls on the silica layer as a concentration ladder allows for intensity benchmarking as a qualitative control activity equivalent.
It can be concluded, that bioautography offers a rapid and simple tool for screening of secondary metabolite profiles combined with enzymatic inhibition screening by HPTLC, either for quality control purposes or bioprocess control for in vitro cultivated medicinal plant biomass. Such assays can complement sophisticated assays to reduce the number of samples.
Acknowledgements: BSRP, grant No. IZEBZ0, 142989; DO2 – 1153