The Phytochemical Analysis of Vinca L. Species Leaf Extracts Is Correlated with the Antioxidant, Antibacterial, and Antitumor Effects

Abstract

The phytochemical analysis of Vinca minor, V. herbacea, V. major, and V. major var. variegata leaf extracts showed species-dependent antioxidant, antibacterial, and cytotoxic effects correlated with the identified phytoconstituents. Vincamine was present in V. minor, V. major, and V. major var. variegata, while V. minor had the richest alkaloid content, followed by V. herbacea. V. major var. variegata was richest in flavonoids and the highest total phenolic content was found in V. herbacea which also had elevated levels of rutin. Consequently, V. herbacea had the highest antioxidant activity followed by V. major var. variegata. Whereas, the lowest one was of V. major. The V. minor extract showed the most efficient inhibitory effect against both Staphylococcusaureus and E. coli. On the other hand, V. herbacea had a good anti-bacterial potential only against S. aureus, which was most affected at morphological levels, as indicated by scanning electron microscopy. The Vinca extracts acted in a dose-depended manner against HaCaT keratinocytes and A375 melanoma cells and moreover, with effects on the ultrastructure, nitric oxide concentration, and lactate dehydrogenase release. Therefore, the Vinca species could be exploited further for the development of alternative treatments in bacterial infections or as anticancer adjuvants.

Keywords: alkaloids; antibacterial; antioxidant; phenolic compounds; vincamine.

Figure 1

(a) HPLC-DAD chromatograms of the Vinca minor, V. major, V. major var. variegata, and V. herbacea plant extracts monitored at 230 nm. Chromatogram of analytical standards includes: (1) 3,4-dihydroxybenzoic acid, (2) chlorogenic acid, (3) 4-hydroxybenzoic acid, (4) caffeic acid, (5) syringic acid, (6) rutin, (7) p-coumaric acid, (8) isoquercitrin, (9) ferulic acid, (10) quercitrin, (11) myricetin, (12) berbamine, (13) vincamine, (14) jatrorrhizine, (15) quercetin, (16) palmatine, (17) berberine, (18) kaempferol, (19) vinblastine, and (20) galangin. The identified compounds in the studied Vinca species are marked in green in the upper chromatogram; (b) UV molecular absorption spectra as registered by the DAD detector for the representative standards from each main phytochemical group (grey-hydroxybenzoic acids, green-cinnamic acids, blue-flavonoids, and red-alkaloids).

Figure 2

Chemo-mapping of the major chromatographic peaks—phytoconstituents classification—based on spectral similarities for each studied extract using PCA. PCA was applied on exported UV DAD spectra, for each chromatographic peak. Shown here are the scatterplots of the scores for the first two principal components for (a) V. minor, (b) V. herbacea, (c) V. major, and (d) V. major var. variegata. The classified compounds are detailed in Table S1. Groups with high similarity are clustered in specific color for each phytoconstituent group or class (grey-hydroxybenzoic acids, green-cinnamic acids, blue-flavonoids, and red-alkaloids); (e) Total standard (vincamine for TAC, quercetin for TFC, cynnamic acid for TCaC and benzoic acid for TBzaC) equivalent content (%) for each extract for 230 nm chromatogram, after PCA classification. TAC-total alkaloid content, TFC-total flavonoid content, TCaC-total cinnamic acids content, TBzaC-total hydroxybenzoic acids content, Vm = V. minor, VM = V. major, VMv = V. major var. variegata, Vh = V. herbacea.

Figure 3

(a) Calibration curve of the proposed Dragendorff assay with vinblastine as a standard; (b) graphical correlation between Dragendorff TAC and HPLC calculated Total Vincamine Equivalents.

Figure 4

The antibacterial effect of Vinca minor (a), V. major (b), V. major var. variegata (c), and V. herbacea (d) leaf extracts against E. coli and S. aureus, assessed through the microdilutions method. The values represent the mean of at least three independent experiments ± standard error of the mean (s.e.m.); *** p < 0.0001, ** p < 0.001, * p < 0.05 according to one way ANOVA and Student’s t tests.

Figure 5

SEM micrographs of the untreated S. aureus strain, (a) and compared to the strains treated with (b) V. minor, (c) V. major, (d) V. major var. variegata, and (e) V. herbacea leaf extracts at Minimal Inhibitory Concentration (MIC).

Figure 6

In vitro cytotoxic effects of the Vinca plant extracts used against normal keratinocytes (HaCaT) for 48 h (a), and skin melanoma cells (A375) at 72 h (b), compared to a positive control (untreated cells) and a negative control (cells treated with Tween 20 at 2% concentration); *** p < 0.0001, ** p < 0.001, * p < 0.05 according to one way ANOVA and Student’s t tests.

Figure 7

In vitro analysis of LDH (a) and NO (b) release in normal keratinocytes (HaCaT) treated with the plant extracts for 48 h and in vitro analysis of LDH (c) and NO (d) release in skin melanoma cells (A375) treated with the Vinca leaf extracts for 72 h; LPS = lipopolysaccharides.

Figure 8

TEM micrographs of untreated A375 melanoma cells (a) and compared to the cells treated with (b) V. minor, (c) V. major, (d) V. major var. variegata, and (e) V. herbacea leaf extracts at IC₅₀ values. White triangle = mitochondria, black rhombs = vesicles/lysosomes, N = nucleus, n = nucleolus.