Persistence of anticancer activity in berry extracts after simulated gastrointestinal digestion and colonic fermentation

Abstract

Fruit and vegetable consumption is associated at the population level with a protective effect against colorectal cancer. Phenolic compounds, especially abundant in berries, are of interest due to their putative anticancer activity. After consumption, however, phenolic compounds are subject to digestive conditions within the gastrointestinal tract that alter their structures and potentially their function. However, the majority of phenolic compounds are not efficiently absorbed in the small intestine and a substantial portion pass into the colon. We characterized berry extracts (raspberries, strawberries, blackcurrants) produced by in vitro-simulated upper intestinal tract digestion and subsequent fecal fermentation. These extracts and selected individual colonic metabolites were then evaluated for their putative anticancer activities using in vitro models of colorectal cancer, representing the key stages of initiation, promotion and invasion. Over a physiologically-relevant dose range (0-50 µg/ml gallic acid equivalents), the digested and fermented extracts demonstrated significant anti-genotoxic, anti-mutagenic and anti-invasive activity on colonocytes. This work indicates that phenolic compounds from berries undergo considerable structural modifications during their passage through the gastrointestinal tract but their breakdown products and metabolites retain biological activity and can modulate cellular processes associated with colon cancer.

Figure 1. The cytotoxic effects of various types of berry extracts on HT29 cells.

(A) Effects of IVD berry extract, (B) fermented berry extract at different dilutions in growth medium, (C) individual polyphenols- tyrosol (T), 3-(3′-hydroxyphenyl)propionic acid (HPA), 4-hydroxybenzoic acid (HBA), 4′-hydroxyphenylacetic acid (HAA) at 1, 5 or 10 µg/mL concentrations. Data presented is mean of 3 independent experiments + SD. One-way ANOVA and Dunnett T test, * p<0.05, significance is compared to media control (0 ug/mL) for A & C and against control fermentate in B. Phenol levels for 1∶10 dilution of fermented extract were raspberry 15.5, strawberry 13.9 and blackcurrant 12.4 µg/mL GAE.

Figure 2. The anti-genotoxic effects of various types of berry extracts on HT29 cells.

(A) Effects of IVD berry extract, (B) berry fermentates at a 1∶10 dilution after 24 hr pre-incubation on DNA damage in HT29 cells challenged with 75 µM H₂O₂ (phenol levels for berry fermentates were raspberry 15.5, strawberry 13.9, blackcurrant 12.4 µg/mL GAE), (C) individual polyphenols – tyrosol (T), 3-(3′-hydroxyphenyl)propionic acid (HPA), 4-hydroxybenzoic acid (HBA), 4′-hydroxyphenylacetic acid (HAA) at 1, 5 or 10 µg/mL concentrations. Data is presented as mean of 3 independent experiments + SD. One-way ANOVA and Dunnett T test, * p<0.05, significance is compared to media control (0 µg/mL) for A & C and against control fermentate in B. Bonferroni 4-way comparison, bars with different letters are significantly different from each other p<0.05.

Figure 3. The anti-mutagenic effects of various types of berry extracts against fecal water-induced frameshift mutations in HT29 (G17 neo) cells.

(A) Effects of IVD berry extracts, (B) berry fermentates. Data presented as mean relative mutation frequency of 3 independent experiments + SD. One-way ANOVA and Dunnett T test, * p<0.05, significance is compared to fecal water control for A and against control fermentate FW in B. Phenol levels for berry fermentates were raspberry 15.5, strawberry 13.9 and blackcurrant 12.4 µg/mL GAE.

Figure 4. The anti-invasive effects of various types of berry extracts on HT115 cells.

(A) Effects of IVD berry extracts, (B) berry fermentates. Mean of 3 independent experiments + SD, One-way ANOVA and Dunnett T test, * p<0.05, significance is compared to media control (0 µg/mL) for A and against control fermentate in B. Values are expressed as % cell invasion normalised to control. Berry extracts had no effect on migration of HT115 cells (data not shown). Phenol levels for berry fermentates were raspberry 15.5, strawberry 13.9, blackcurrant 12.4 µg/mL GAE.