Cytotoxic and DNA-Damaging Effects of Aronia melanocarpa, Cornus mas, and Chaenomeles superba Leaf Extracts on the Human Colon Adenocarcinoma Cell Line Caco-2

Abstract

Aronia melanocarpa, Cornus mas, and Chaenomeles superba leaf extracts contain large amounts of bioactive compounds-mainly polyphenols, which possess many health benefits including anti-cancer properties. Here, we investigate the biological effects of A. melanocarpa, C. mas, and C. superba leaf extracts on the human colon adenocarcinoma cell line Caco-2. The antiproliferative activity of the extracts was measured using the MTT assay. The most cytotoxic extract was C. mas (IC₅₀ = 0.60%). The extracts caused morphological changes in the Caco-2 cells, including partial detachment of cells, necrotic cells, chromatin condensation, cytoplasmic vacuolization, cell nuclei lysis, and nucleus fragmentation. The DNA damage in the Caco-2 cells after exposure to the leaf extracts was measured using the alkaline comet assay. The extracts increased DNA damage in a concentration dependent manner. However, at lower non-cyto- and non-genotoxic (IC₀) concentrations the extracts induced DNA repair in Caco-2 cells after exposure to hydrogen peroxide. In conclusion, the results of these studies suggest that A. melanocarpa, C. mas and C. superba leaf extracts can show anticancer activity. However, further research is required on the mechanisms of anti-cancer activity by these extracts, with the application of more advanced and wide-ranging techniques including in vivo experiments.

Keywords: Aronia melanocarpa; Caco-2; Chaenomeles superba; Cornus mas; DNA repair; cytotoxicity; genotoxicity; polyphenols.

Figure 1

Cytotoxicity of Aronia melanocarpa, Chaenomeles superba, and Cornus mas leaf extracts in Caco2 cells estimated using the MTT assay after 72 h of exposure. Each data point represents the mean of the absorbance values for cells from four individual wells (±SD).

Figure 2

Caco-2 cells stained using the May–Grünwald–Giemsa method after exposure to leaf extracts (A,B—control cells (not exposed to extracts); C,D—Aronia melanocarpa (2.50%); E—Cornus mas (0.31%); F—Cornus mas (0.63%); G,H—Chaenomeles superba (0.63%)).

Figure 3

Typical images of DAPI-stained comets exposed to 0.63% leaf extracts: A—Aronia melanocarpa; B—Cornus mas, C—Chaenomeles superba. Magnification 200× (Nikon Eclipse, Japan).

Figure 4

DNA damage in Caco-2 cells after exposure to different concentrations of Aronia melanocarpa, Chaenomeles superba, and Cornus mas leaf extracts expressed as the mean percentage of DNA in the comet tail according to the alkaline comet assay. Fifty cells were analysed for each treatment. Data from two independent experiments. Error bars denote SEM. (*)—statistically significant differences compared to the control (ANOVA (p < 0.05).

Figure 5

Histograms illustrating the distribution of endogenous DNA damage in Caco-2 cells exposed to Aronia melanocarpa, Cornus mas and Chaenomeles superba leaf extracts at concentrations of 0.01%, 0.02%, 0.04%, 0.08%, 0.16%, and 0.63%.

Figure 6

Efficiency of DNA repair in Caco-2 cells exposed to different IC₀ concentrations (0.04% and 0.08%) of Aronia melanocarpa, Cornus mas, and Chaenomeles superba leaf extracts. DNA repair was measured at 0 min, after 60 and 120 min of incubation. The number of cells analysed for each time-interval was 50. Error bars denote S.E.M. (*) statistically significant differences from the positive control (ANOVA (p < 0.05)).