Phytochemical screening and biological evaluation of Greek sage (Salvia fruticosa Mill.) extracts

Abstract

This study explores the influence of extraction solvents on the composition and bioactivity of Salvia fruticosa extracts. Ultrasound-assisted extraction with water, ethanol and their mixtures in variable proportions was used to produce four different extracts. An untargeted UPLC/MS‑based metabolomics was performed to discover metabolites profile variation between the extracts. In the analyzed samples, 2704 features had been detected, of which 95 were tentatively identified. The concentrations of the important metabolites, namely, caffeic acid, carnosic acid, carnosol, rosmarinic acid, salvianolic acid B and scutellarin, were determined, using UPLC-PDA methods. Rosmarinic acid was the dominant metabolite and antioxidant in all tested extracts, except the aqueous extract, in which scutellarin was the most abundant compound. The extracts and standards were examined for antioxidant activity and xanthine oxidase (XO) inhibitory activity. The most diverse in terms of chemical composition and rich in antioxidant compounds was 70% ethanolic extract and the strongest antioxidant was caffeic acid. All analyzed extracts showed the ability to inhibit XO activity, but the highest value was recorded for 30% ethanolic extract. Among tested standards, the most potent XO inhibitor was caffeic acid. The results suggest that the leaves of Greek sage are a source of natural XO inhibitors and may be an alternative to drugs produced by chemical synthesis.

Figure 1

Total ion chromatograms obtained by LC-Q-Orbitrap in negative mode (black) combined with chromatograms registered by UV–Vis detector at 270 nm (orange) (a), set with heat map representing the mean MS peak area value of the identified compounds in four different S. fruticosa extracts: SFH₂O–water extract; SF30–30% ethanol extract; SF70–70% ethanol extract; SF100–ethanol extract (b). For the identity of peaks, see Supplementary Table S1.

Figure 2

The antioxidant activity of standards (caffeic acid, scutellarin, salvianolic acid B, rosmarinic acid, carnosic acid, carnosol and trolox) and S. fruticosa extracts: SFH₂O–water extract; SF30–30% ethanol extract; SF70–70% ethanol extract; SF100–ethanol extract, tested in vitro with ABTS, DPPH and F–C reagents presented as plots showing the dependency curves of reagent reduced by tested standards (a) or extracts (b) and expressed as slopes of the curves equal to the milomoles of reagent reduced by 1 g of tested sample (c) set with the antioxidant profiles of extracts, registered at 734 nm after post-column derivatization with ABTS, with the main classes of antioxidants on the pie charts (d). For the identity of peaks, see Supplementary Table S1.

Figure 3

The examples of HPLC chromatograms at 285 nm of post-reaction mixtures containing (from top): xanthine; xanthine and xanthine oxidase (XO); xanthine, XO and inhibitor (a), which were the basis for preparing the plots representing the curves of XO activity in the presence of tested standards (caffeic acid, scutellarin, salvianolic acid B, rosmarinic acid, carnosic acid, carnosol and allopurinol) or S. fruticosa extracts (SFH₂O–water extract; SF30–30% ethanol extract; SF70–70% ethanol extract; SF100 – ethanol extract) (b), which were used to determine the parameter IC_50, meaning the micrograms of tested sample needed to reduce XO activity to 50% (c).