Antibacterial potential of the Cistus incanus L. phenolics as studied with use of thin-layer chromatography combined with direct bioautography and in situ hydrolysis

• Published in: Journal of Chromatography A Vol. 1534; pp. 170 - 178
• Main Authors: Móricz, Ágnes M., Szeremeta, Dariusz, Knaś, Magdalena, Długosz, Ewa, Ott, Péter G., Kowalska, Teresa, Sajewicz, Mieczysław
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 26-01-2018
• Subjects: 4-Aminobenzoic Acid - chemistry; Acylated flavonoid glycosides; Anti-Bacterial Agents - chemistry; Anti-Bacterial Agents - isolation & purification; Anti-Bacterial Agents - pharmacology; Chromatography, Thin Layer - methods; Cistus - chemistry; Cistus - metabolism; Flavonoid aglycons; Flavonoids - chemistry; Glucose - analysis; Glucose - chemistry; Glycosides - chemistry; Gram-Negative Bacteria - drug effects; Gram-Positive Bacteria - drug effects; Hairy rockrose (Cistus incanus L.); Hydrolysis; In situ acidic hydrolysis; Multi-development and two-dimensional HPTLC; Paraffin - chemistry; Phenols - chemistry; Phenols - isolation & purification; Phenols - pharmacology; Plant Extracts - chemistry; Silicon Dioxide - chemistry; Spectrometry, Mass, Electrospray Ionization; Spectrophotometry, Ultraviolet; Thin-layer chromatography–direct bioautography; Hairy rockrose (Cistus incanus L.); Flavonoid aglycons; In situ acidic hydrolysis; Multi-development and two-dimensional HPTLC; Acylated flavonoid glycosides; Thin-layer chromatography–direct bioautography
• ISSN: 0021-9673; 1873-3778
• DOI: 10.1016/j.chroma.2017.12.056

•Antibacterial profiling of flavonoid fraction of 11 C. incanus samples using TLC-DB.•Apigenin, kaempferide and kaempferol glycosides as antibacterial components.•MD- and 2D-HPTLC combined with in situ liberation of glucose.•Enhanced sensitivity of glucose detection by use of paraffin after PABA.•Flavonoid glycosides in the fraction of aglycones obtained by traditional protocol.
The main aim of this study was to detect and identify antibacterial components of fraction I derived from eleven commercial C. incanus herbal teas. Fraction I obtained by a well-established phytochemical protocol of a multi-step extraction was expected to contain flavonoid aglycons alone. Antibacterial profile of fraction I was demonstrated by means of thin-layer chromatography – direct bioautography (TLC-DB) using a Gram positive B. subtilis and a Gram negative A. fischeri strain. Six chromatographic zones of fraction I exhibited a well pronounced antibacterial potential. In qualitative terms, a good agreement was observed among chromatographic fingerprints and the corresponding bioautograms of the eleven samples. The compounds isolated from the six zones were analyzed by HPLC- diode array detector (DAD)-electrospray ionization (ESI)-MS. High numerical m/z values valid for certain constituents of these isolates suggested that some selected antibacterial components are, unexpectedly, flavonoid glycosides. In order to confirm this suggestion, three independent HPTLC methods (multi-development on amino phase and two two-dimensional developments on silica gel phase) were devised to in situ hydrolyze flavonoid glycosides and then separate and visualize the liberated glucose and some other building blocks of the zones’ components. Additionally, the sensitivity of glucose detection with p-aminobenzoic acid reagent was enhanced by paraffin. In that way, the presence of the kaempferol glycosides (and not only the aglycones alone) in fraction I was confirmed. Beside kaempferol, p-coumaric acid as a building block unit was shown by HPLC-DAD-MS analysis of the hydrolyzed isolates. Results proved apigenin, kaempferide and acylated kaempferol glycosides (cis- and trans-tiliroside and their conjugates with p-coumaric acid) to be antibacterial components of fraction I. Because isomers of the coumaric acid conjugated tiliroside were detected only in fraction I and not in the crude C. incanus extract, they are regarded as artifacts produced through fractionation.