Bioprofiling of Salvia miltiorrhiza via planar chromatography linked to (bio)assays, high resolution mass spectrometry and nuclear magnetic resonance spectroscopy

Abstract

An affordable bioanalytical workflow supports the collection of data on active ingredients, required for the understanding of health-related food, superfood and traditional medicines. Targeted effect-directed responses of single compounds in a complex sample highlight this powerful bioanalytical hyphenation of planar chromatography with (bio)assays. Among many reports about biological properties of Salvia miltiorrhiza Bunge root (Danshen) and their analytical methods, the highly efficient direct bioautography (DB) workflow has not been considered so far. There was just one TLC-acetylcholinesterase (AChE) method with a poor zone resolution apart from our two HPTLC-DB studies, however, all methods were focused on the nonpolar extracts of Danshen (tanshinones) only. The current study on HPTLC-UV/Vis/FLD-(bio)assay-HRMS, followed by streamlined scale-up to preparative layer chromatography (PLC)-¹H-NMR, aimed at an even more streamlined, yet comprehensive bioanalytical workflow. It comprised effect-directed screening of both, its polar (containing phenolics) and nonpolar extracts (containing tanshinones) on the same HPTLC plate, the biochemical and biological profiling with four different (bio)assays and elucidation of structures of known and unidentified active compounds. The five AChE inhibitors, salvianolic acid B (SAB), lithiospermic acid (LSA) and rosmarinic acid (RA) as well as cryptotanshinone (CT) and 15,16-dihydrotanshinone I (DHTI) were confirmed, but also unidentified inhibitors were observed. In the polar extracts, SAB, LSA and RA exhibited free radical scavenging properties in the 2,2-diphenyl-1-picrylhydrazyl assay. CT, DHTI and some unidentified nonpolar compounds were found active against Gram-positive Bacillus subtilis and Gram-negative Aliivibrio fischeri (LOD 12 ng/band for CT, and 5 ng/band for DHTI). For the first time, the most multipotent unidentified active compound zone in the B. subtilis, A. fischeri and AChE fingerprints of the nonpolar Danshen extract was identified as co-eluted band of 1,2-dihydrotanshinone and methylenetanshinquinone in the ratio of 2:1.

Keywords: Direct bioautography (DB); HPTLC; High resolution mass spectrometry (HRMS); NMR; Preparative layer chromatography (PLC); Salvia miltiorrhiza root (Danshen).