Comprehensive Chemical Profiling and Multidirectional Biological Investigation of Two Wild Anthemis Species (Anthemis tinctoria var. Pallida and A. cretica subsp. tenuiloba): Focus on Neuroprotective Effects

Abstract

Ethyl acetate (EA), methanol (MeOH), and aqueous extracts of aerial parts of Anthemis tinctoria var. pallida (ATP) and A. cretica subsp. tenuiloba (ACT) were investigated for their phenol and flavonoid content, antioxidant, and key enzyme inhibitory potentials. All extracts displayed antiradical effects, with MeOH and aqueous extracts being a superior source of antioxidants. On the other hand, EA and MeOH extracts were potent against AChE and BChE. Enzyme inhibitory effects against tyrosinase and α-glucosidase were observed, as well. We also studied Anthemis extracts in an ex vivo experimental neurotoxicity paradigm. We assayed extract influence on oxidative stress and neurotransmission biomarkers, including lactate dehydrogenase (LDH) and serotonin (5-HT), in isolated rat cortex challenged with K⁺ 60 mM Krebs-Ringer buffer (excitotoxicity stimulus). An untargeted proteomic analysis was finally performed in order to explore the putative mechanism in the brain. The pharmacological study highlighted the capability of ACT water extract to blunt K⁺ 60 mM increase in LDH level and 5-HT turnover, and restore physiological activity of specific proteins involved in neuron morphology and neurotransmission, including NEFMs, VAMP-2, and PKCγ, thus further supporting the neuroprotective role of ACT water extract.

Keywords: Anthemis; neurotransmission; oxidative stress; phytomedicine; proteomic.

Figure 1

(A): Relationship between total phenol content (TPC), total flavonoid content (TFC) and biological activities. (B,C): results of preliminary multivariate analysis with PCA (B: Percentage of explained variance and Eigen value per component, C: PCA sample plot on PC1 vs. PC2 and PC1 vs. PC3 respectively).

Figure 2

Supervised analysis with sPLS-DA. A: sPLS-DA samples plot with confidence ellipse plots considering the species as class membership criteria. B: Performance of the model (BER) for three prediction distances using 10 × 5-fold cross-validation. C: VIP score plot displaying the biological activities having highly contributed to the discrimination of both studied species. D: Factorial plan 1-2 of the sPLS-DA with confidence ellipse plots according to the extraction conditions as class membership criteria. E: The model performance per component for the three prediction distances using 5-fold cross-validation repeated 10 times. F: VIP score plot showing the biological activities outlining the difference between the three extraction conditions.

Figure 3

Seedling germination and growth of Canasta (C), Romana verde (RV) and Romana bionda (RB) seeds challenged with A. tinctoria and A. Cretica extracts. Results are expressed as root and hypocotyl (seedling) length ± SD at different concentrations and mean of GP after the fourth day since the sowing. (A): Effect of A. cretica water extract on seedling germination. (B): Effect of A. cretica ethyl acetate (EA) extract on seedling germination. (C): Effect of A. cretica water methanol (MeOH) on seedling germination. (D): Effect of A. tinctoria water extract on seedling germination. (E): Effect of A. tinctoria ethyl acetate (EA) extract on seedling germination. (F): Effect of A. tinctoria water methanol (MeOH) on seedling germination.

Figure 4

Effect of A. tinctoria (A. T.) and A. cretica (A. C.) extracts (100 µg/mL) on HypoE22 cell line viability (MTT test). Data are means ± SD of three experiments performed in triplicate.

Figure 5

Effect of A. tinctoria (A. T.) and A. cretica (A. C.) extracts (100 µg/mL) on serotonin (5-HT) turnover, expressed as 5HIIA/5-HT ratio. Turnover was evaluated on isolated rat cortex challenged with basal (K⁺ 3mM) and depolarizing stimuli (K⁺ 15 mM; K⁺ 60 mM). Data are means ± SD of three experiments performed in triplicate. ANOVA, p < 0.0001; post-hoc, * p < 0.05, *** p < 0.001 vs. K⁺ 60 mM control group.

Figure 6

Effect of A. tinctoria (A. T.) and A. cretica (A. C.) extracts (100 µg/mL) on lactate dehydrogenase (LDH) level, measured on isolated rat cortex challenged with basal (K⁺ 3mM) and depolarizing stimuli (K⁺ 15 mM; K⁺ 60 mM). Data are means ± SD of three experiments performed in triplicate. ANOVA, p < 0.0001; post-hoc, * p < 0.05 vs. K⁺ 60 mM control group.

Figure 7

Panel A: Untargeted proteomic analysis performed on rat cortex challenged with basal (K+ 3mM) and depolarizing stimuli (K⁺ 15 mM; K⁺ 60 mM). The activity of the detected proteins was calculated in comparison with the calibrator of the experiment (K⁺ 60 mM). Panel B: Untargeted proteomic analysis showing the effects of A. tinctoria and A. cretica water extracts (100 µg/mL) on rat cortex challenged with excitotoxicity depolarizing stimulus (K⁺ 60 mM). T The activity of the detected proteins was calculated in comparison with the calibrator of the experiment (K⁺ 60 mM). In subfigure A, it is showed that K⁺ 60 mM depolarizing stimulus downregulated NEFMs and upregulated VAMP-2 and PKCγ levels. On the other hand, as depicted in subfigure B, A. cretica water extract (100 µg/mL) was able to restore the activity of specific proteins involved in neuron morphology and neurotransmission, including NEFMs, VAMP-2, and PKCγ. After treating rat cortex with that A. cretica water extract, the activity of these proteins was similar to that measured after challenging the brain tissue with physiologic depolarizing stimulus (K⁺ 15 mM).

Figure 8

Protective effects induced by A. cretica and A. tinctoria extracts, as evidenced by the present pharmacological investigation.