Caucasian Gentiana Species: Untargeted LC-MS Metabolic Profiling, Antioxidant and Digestive Enzyme Inhibiting Activity of Six Plants

Abstract

The members of Gentiana genus are widely distributed in the Caucasus region where they are used as phytoremedies, but they still have not been studied for their chemical composition and bioactivity. High-performance liquid chromatography with diode array and electrospray triple quadrupole mass detection (HPLC-DAD-ESI-QQQ-MS) was used to investigate metabolites of herb and roots of six gentians (Gentiana asclepiadea, G. cruciata, G. gelida, G. paradoxa, G. pneumonanthe, G. septemfida) grown in the Caucasus. In total, 137 compounds were found including three carbohydrates, 71 iridoid glycosides (mostly loganic acid), loganin, swertiamarin, gentiopicroside and sweroside derivatives, 40 flavones C-, O-, C,O-glycosides (such as luteolin, apigenin, chrysoeriol, and acacetin derivatives), two phenolic O-glycosides, five hydroxycinnamates, eight xanthones, and seven triterpene glycosides. Most of these compounds were identified in gentian samples for the first time. Quantitative differences were found in levels of seven iridoid glycosides, nine glycosylflavones, and two xanthones obtained by HPLC-DAD assay. The gentian extracts were evaluated for their radical-scavenging properties against DPPH and superoxide anion radicals, lipid peroxidation inhibition, and α-amylase/α-glycosidase inhibition. The herb extracts showed higher activity than root extracts. Positive correlations were found between the content of quantified phenolics and antioxidant and digestive enzymes inhibiting activity. The findings presented in our work suggest that the Caucasian gentians are a good source of bioactive phytocompounds with antioxidant and antidiabetic potential.

Keywords: Gentiana; LC-MS profile; amylase/glycosidase inhibition; antioxidant activity; flavone glycosides; iridoid glycosides; xanthones.

Figure 1

Caucasian gentians studied in present work: Gentiana asclepiadea (a), G. cruciata (b), G. gelida (c), G. paradoxa (d), G. pneumonanthe (e), G. septemfida (f).

Figure 2

(a) HPLC-ESI-MS chromatogram of G. septemfida root extract in SIM-mode {m/z 375 for loganic acid (18), m/z 389 for loganin (25), m/z 419 for swertiamarin (33), m/z 401 for gentiopicroside (38), m/z 403 for sweroside (40; isomeric 44)}. (b,c) Mass spectra of compound 38 in negative and positive ionization mode. (d) UV spectra of compounds 18, 38 and 40.

Figure 3

(a,b) HPLC-ESI-MS chromatogram of G. gelida root extract in SIM-mode: {m/z 797 for gelidoside (rindoside, 131, a); m/z 781 for trifloroside (132, b)}. (c) UV spectrum of compounds 132. (d,e) Mass spectrometric fragmentation of compounds 131 (d) and 132 (e). (f) MSⁿ spectra of compound 132.

Figure 4

(a) HPLC-ESI-MS chromatogram of G. gelida herb extract in SIM-mode (m/z 315). (b, c) Mass spectra (negative ionization) of compounds 11 (2,3-dihydroxybenzoic acid 2-O-glucoside) and 16 (2,3-dihydroxybenzoic acid 3-O-glucoside), respectively. (d) UV spectra of compounds 11 and 16.

Figure 5

(a) HPLC-ESI-MS chromatogram of G. asclepiadea herb extract in SIM-mode (m/z 421, 583). (b, c) Mass spectra (negative ionization) of compounds 49 (mangiferin) and 29 (mangiferin-7-O-glucoside), respectively. (d) UV spectra of compounds 29, 49, and 50.

Figure 6

(a) HPLC-ESI-MS chromatogram of G. gelida herb extract in SIM-mode (m/z 771). (b, c) UV spectra of compounds 36 and 73. (d) MSⁿ spectra (negative ionization) of compound 36. Lut-C-Hex-O-Hex-O-Hex—zone of isomeric luteolin-C-hexoside-O-hexoside-O-hexosides, Lut-C-Hex-O-Hex-O-Caf—zone of isomeric luteolin-C-hexoside-O-hexoside-O-caffeates.

Figure 7

(a) HPLC-ESI-MS chromatogram of G. gelida herb extract in SIM-mode (m/z 755). (b) UV spectra of compounds 47 and 68. (c) MS and MS² spectra (negative ionization) of compound 68. Api-C-Hex-O-Hex-O-Hex—apigenin-C-hexoside-O-hexoside-O-hexoside (isovitexin-2″,4″-di-O- glucoside), Api-C-Hex-O-Hex-O-Caf—zone of isomeric apigenin-C-hexoside-O-hexoside-O-caffeates.

Figure 8

(a,b,c) HPLC-ESI-MS chromatograms of G. asclepiades herb extract in SIM-mode (positive ionization; m/z 941, 779 (oleanolic acid glycosides; a), 939, 777 (dehydrooleanolic acid glycosides; b), 763, 617 (desoxyoleanolic acid glycosides; c)). (d) MSⁿ spectra (positive ionization) of compound 84.

Figure 9

HPLC-DAD chromatograms (210 nm) of G. asclepiadea herb extract before (a) and after (b) pre-chromatographic reaction with DPPH radicals and G. gelida roots extract before (c) and after (d) pre-chromatographic reaction with DPPH radicals. Zone of compounds numbered as follows: i—loganic acid; ii—swertiamarin; iii—isoorientin-2″-O-glucoside; iv—gentiopicroside; v—mangiferin; vi—isovitexin-2″-O-glucoside; vii—isoorientin; viii—saponarin; ix—isovitexin; x—luteolin-7-O-glucoside; xi—apigenin-7-O-glucoside; xii—gentiopicroside-6″-O-glucoside; xiii—gelidoside (rindoside); xiv—trifloroside. The numbers demonstrate the percentage of peak area reduction after pre-chromatographic reaction with DPPH radicals.

Figure 10

Correlation graphs between phenolic compound content in gentian extracts (mg/g) and their bioactivity. (a) DPPH—2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (as mg of trolox per gram of dry extract weight; correlation equation y = 2.07·x − 38.48, r = 0.9752); O₂^-—superoxide anion-radical scavenging activity (as mg of quercetin per gram of dry extract weight; correlation equation y = 0.84·x − 20.21, r = 0.9736); LPA—lipid peroxidation inhibition activity (as mg of caffeic acid per g of dry extract weight; correlation equation y = 0.76·x − 14.74, r = 0.9232). (b) αAIP—α-amylase inhibitory potential (as mg of acarbose per g of dry extract weight; correlation equation y = 1.86·x − 54.73, r = 0.9739); αGIP—α-glycosidase inhibitory potential (as mg of acarbose per gram of dry extract weight; correlation equation y = 1.29·x + 5.08, r = 0.9849).