Apple polyphenol extracts prevent damage to human gastric epithelial cells in vitro and to rat gastric mucosa in vivo

Abstract

Background: Fresh fruit and vegetables exert multiple biological effects on the gastrointestinal mucosa.

Aim: To assess whether apple extracts counteract oxidative or indomethacin induced damage to gastric epithelial cells in vitro and to rat gastric mucosa in vivo.

Methods: Apple extracts were obtained from freeze dried apple flesh of the "Annurca" variety. Cell damage was induced by incubating MKN 28 cells with xanthine-xanthine oxidase or indomethacin and quantitated by MTT. In vivo gastric damage was induced by indomethacin 35 mg/kg. Intracellular antioxidant activity was determined using the (2,2'-azinobis (3-ethylbenzothiazolin-6-sulfonate) method. Malondialdehyde intracellular concentration, an index of lipid peroxidation, was determined by high pressure liquid chromatography with fluorometric detection.

Results: (1) Apple extracts decreased xanthine-xanthine oxidase or indomethacin induced injury to gastric epithelial cells by 50%; (2) catechin or chlorogenic acid (the main phenolic components of apple extracts) were equally effective as apple extracts in preventing oxidative injury to gastric cells; and (3) apple extracts (i) caused a fourfold increase in intracellular antioxidant activity, (ii) prevented its decrease induced by xanthine-xanthine oxidase, (iii) counteracted xanthine-xanthine oxidase induced lipid peroxidation, and (iv) decreased indomethacin injury to the rat gastric mucosa by 40%.

Conclusions: Apple extracts prevent exogenous damage to human gastric epithelial cells in vitro and to the rat gastric mucosa in vivo. This effect seems to be associated with the antioxidant activity of apple phenolic compounds. A diet rich in apple antioxidants might exert a beneficial effect in the prevention of gastric diseases related to generation of reactive oxygen species.

Figure 1

Xanthine-xanthine oxidase (X-XO) dose dependently injured MKN 28 cells. Cells were incubated for two hours with XO (10–100 mU/ml) in the presence of X 1 mM or serum free medium (control). Mean (SD) from three separate experiments run in duplicate. *p<0.05 versus control.

Figure 2

Apple polyphenol extracts (APE) dose dependently prevented xanthine-xanthine oxidase (X-XO) induced damage to MKN 28 cells. MKN 28 cells were incubated with APE 10⁻⁷ to 10⁻⁴ M or with serum free medium (control) for three hours and then, after washing, with X (1 mM)-XO (50 mU/ml) or serum free medium for two hours. Mean (SD) from three separate experiments run in duplicate. *p<0.05 versus control; †p<0.05 versus X-XO.

Figure 3

Protective effect of apple polyphenol extracts (APE), catechin (CAT), or chlorogenic acid (CA) against xanthine-xanthine oxidase (X-XO) induced cell injury. Cells were incubated with serum free medium (control) or with APE (10⁻⁴ M), CAT (10⁻⁴ M), or CA(10⁻⁴ M) for three hours and then, after washing, with X (1 mM)-XO (50 mU/ml) or serum free medium (control) for two hours. Mean (SD) from three separate experiments run in duplicate. *p<0.05 versus control; †p<0.05 versus X-XO.

Figure 4

Effects of apple polyphenol extracts (APE) on total antioxidant intracellular activity and on lipid peroxidation in MKN 28 cells. (A) APE prevented xanthine-xanthine oxidase (X-XO) induced decrease in total antioxidant intracellular activity in MKN 28 cells. Cells were treated with serum free medium (control) or APE (10⁻⁴ M) for three hours and then with serum free medium or X (1 mM)-XO (50 mU/ml) for two hours. Mean (SD) of three separate experiments run in duplicate. *p<0.05 versus control; †p<0.05 versus X-XO. (B) APE prevented X-XO induced increase in cellular MDA in MKN 28 cells. Cells were treated with serum free medium (control) or APE (10⁻⁴ M) for three hours and then with serum free medium or X (1 mM)-XO (50 mU/ml) for two hours. Mean (SD) from three separate experiments run in duplicate. *p<0.05 versus control; †p<0.05 versus X-XO.

Figure 5

Apple polyphenol extracts (APE) prevent indomethacin induced injury to MKN 28 cells. Cells were treated with serum free medium (control) or APE (10⁻⁴ M) for three hours and then with serum free medium or indomethacin 2.5 mM or 5 mM for three hours. Mean (SD) from three separate experiments run in duplicate. *p<0.05 versus control; †p<0.05 versus indomethacin.

Figure 6

Apple polyphenol extracts (APE) prevented indomethacin induced injury to the rat gastric mucosa. Rats were pretreated with APE 10⁻⁴ M in drinking water or vehicle (control) and then with indomethacin 35 mg/kg body weight subcutaneously and sacrificed three hours later. (A) Macroscopic appearance of gastric mucosa following indomethacin treatment in control vehicle treated rats. (B) Macroscopic appearance of the gastric mucosa following indomethacin treatment in APE treated rats. (C) Normal rat gastric mucosa. (D) Histological appearance of rat gastric mucosa following indomethacin treatment in control vehicle treated rats, showing extensive superficial epithelial damage and necrosis of gastric glands. (E) Histological appearance of rat gastric mucosa following indomethacin treatment in APE treated rats showing reduction in the extent of both superficial epithelial damage and necrotic lesions in the glandular area.

Figure 7

Quantitation of macroscopic (A) and microscopic (B) injury in control vehicle treated and APE treated rats, prior to indomethacin injection. Mean (SD) from eight rats per group. *p<0.05 compared with controls.