Cytotoxicity and characterization of secondary metabolites obtained from Anagallis arvensis L

PASTORIZA, A.C.; SOBERÓN, J.R.; SGARIGLIA, M.A.

Tucumán, Argentina

Congreso; IV JOINT MEETING OF THE BIOLOGY SOCIETIES OF ARGENTINA; 2020

TUCUMAN BIOLOGY ASSOCIATION

Anagallis arvensis L. is traditionally used with medicinal purposes as antifungal, antiviral, healing, anti-inflammatory, sedative, expectorant, and diuretic. In previous works we validated the antifungal activity of the ethanolic extract against Candida albicans strains, and the bioactive metabolites were partially characterized. The aims of this work were to optimize the obtaining of the complete triterpenic fraction, analyze the composition of present metabolites, and evaluate its cytotoxicity. Triterpenic fraction of A. arvensis (FTA) was obtained by precipitation with acetone, and purified by silica gel column chromatography, composition profiles were analyzed by TLC on silica gel revealed with sulfuric panisaldehyde. Ultra-high pressure liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) was used to analyze FTA. Cytotoxicity was evaluated on human lymphocytes isolated from peripheral blood (HLPBs), cultured in complete RPMI 1640 medium, at 37°C 5% CO2 conditions, under activation or not by treatment with lipopolysaccharide (LPS). It was determined: cellular metabolic activity, measuring the reduction of 3- (4,5 dimethylthiazolyl-2) 2,5 diphenyl tetrazolium bromide (MTT 2.5 mg/mL) on ELISA reader at 550 nm; and membrane integrity, by exclusion of Trypan blue dye observed and counted on Neubauer chamber under optic microscope. FTA was tested for concentrations in the 0?100 µg/mL range. All experiments were performed in duplicate and statistically analyzed accepting α = 5%. FTA presented significant cytotoxicity at 100 μg/mL (less than 75% viability). However, at concentrations lower than 50 μg/mL, a viability ≥ 75% was reached, comparable to the viability control of the cell model tested. The UHPLC-MS/MS experiments allowed the identification of nine triterpenic glycosides, derived from oleanolic acid: Methylanagallosaponin I; Anagallisin A, B, C and D; Anagallosaponin II; Anagalloside A; monomethoxylated Anagalloside A and 3 Beta-[2-O-beta-D-Glucopyranosyl-4-O-[2-O-beta-D-xylopyranosyl-3-O-(3-O-beta-D-glucopyranosyl-beta-D-glucopyranosyl)-beta-D-glucopyranosyl]-alphaL-arabinopyranosyloxy]-13,28-epoxyoleanano-16 alpha, 30-diol. These last two were informed for the first time for A. arvensis. These results show that FTA could be a viable alternative for medicinal use and the information obtained is being used in anti-inflammatory activity studies by our research group