Secondary Metabolites in Ramalina terebrata Detected by UHPLC/ESI/MS/MS and Identification of Parietin as Tau Protein Inhibitor

Abstract

Liquid chromatography coupled with mass spectrometry is an outstanding methodology for fast analysis of phenolic compounds in biological samples. Twenty two compounds were quickly and accurately identified in the methanolic extract of the Antarctic lichen Ramalina terebrata for the first time using ultra high pressure liquid chromatography coupled with photodiode array detector and high resolution mass spectrometry (UHPLC-PDA-Q/Orbitrap/MS/MS). In addition, the extract and the four compounds isolated from this species were tested for the inhibitory activity of tau protein aggregation, which is a protein involved in Alzheimer's disease (AD). All compounds showed null activity with the exception of parietin, which it was able to inhibit aggregation process of tau in a concentration range between 3 µg/mL (10 µM) to 28 µg/mL (100 µM). In addition, we show how parietin interact with tau (306)VQIVYK(311) hexapeptide inside of the microtubule binding domain (4R) with the help of molecular docking experiments. Finally, the constituents present in the methanolic extract could possibly contribute to the established anti-aggregation activity for this extract and this in-depth analysis of the chemical composition of R. terebrata could guide further research into its medicinal properties and potential uses.

Keywords: Alzheimer’s disease; Ramalina; UHPLC/MS; docking; lichens; parietin; tau protein.

Figure 1

Structures of compounds 1–4 isolated from Antartic lichen R. terebrata.

Figure 2

Tau aggregation process inhibited by both Ramalina extract and parietin. Black and grey bars represent positive control (aggregation) and inhibition respectively. A paired samples-t-test was conducted in order to compare control (aggregation) and tau inhibitors (Ramalina extract and parietin). There was a significant difference for both Ramalina extract t (4) = 25, p < 0.05 and parietin t (4) = 3.223, p < 0.05 (data are represented as Mean ± SEM).

Figure 3

(A) Docking of parietin in protonation state P1 in the interface of two poly-³⁰⁶VQIVYK³¹¹ hexapeptide zippers (a,c-left). Hydrogen bounds (HBs) between parietin and lysine residues are indicted as broken lines in the perpendicular view (b,d-right); (B) Docking of parietin in protonation state P2 in the interface of two poly-³⁰⁶VQIVYK³¹¹ hexapeptide zippers (a,c-left). Hydrogen bounds (HBs) between parietin and lysine residues are indicated as broken lines in the perpendicular view (b,d-right).