Inhibition of carcinogen induced biological responses with a coffee water-insoluble fraction and a model system melanoidin
Creators: name: Molund, Vincent Paul
Publisher: name: University of British Columbia
Year: 2010

description: Previous research studies have indicated that coffee brew and water extracts of heated brown food systems such as molasses, beef, prunes and raisins have an inhibitory effect on carcinogen-induced mutagenicity in the Ames Salmonella strains. A hypothesis to explain these observations is that carcinogens are adsorbed onto water-insoluble complexes in coffee brew and melanoidins in heat-treated foods. The present study was undertaken to characterize the water-insoluble fraction (WIF) from reconstituted instant coffee powder; to examine the genotoxic inhibitory effect of the WIF and browning reaction melanoidins; to determine the degree and type of binding of benzo(a)pyrene (BP) and afla-toxin B₁ (AFB₁) by the water-insoluble fraction; and to assess the effect of a model system melanoidin (MSM) on the inhibition of BP induction of aryl hydrocarbon hydroxylase (AHH) in the small intestine of rats. WIF was separated from reconstituted spray dried coffee by precipitation of particulate matter with ethanol at a 90% level and was further purified by resuspension of the precipitate in water and subsequent centrifugal sedimentation. The yield of WIF was about 3% of the instant coffee powder (1.4% moisture). From the non-metallic elemental analysis of WIF, the empirical formula C₄₇H₇₉O₄₁N, was determined. The ratio of C, H, and O atoms suggests the presence of carbohydrates and the nitrogen atom implies the presence of amino acids. The molecular weight of WIF was estimated to be around 200,000 as determined by a column chromatographic technique. A variety of inorganic elements were found in WIF, with potassium in the highest concentration Phenolic compounds, reductones and amino acids were found in WIF. Phenolic compounds were detected by a semiquantitative FeCl₃ colorimetric method which indicated that these compounds were present at a level of 4 mg of caffeic acid equivalent per 10 mg of WIF. The reductone content of WIF was about 140 mg ascorbic acid equivalent per g. Eleven amino acids were identified in the acid hydrolyzate of WIF. The major amino acids were: aspartic acid, glutamic acid, glycine, valine, isoleucine and histidine. Dubois et al. (1956) sugar analysis indicated that 56% of WIF consisted of carbohydrates. Analysis of acid-hydrolyzed WIF by paper chromatography, gas liquid chromatography and GLC-mass spectroscopy indicated that mannose, galactose, glucose and arabinose were the major monosaccharides. Gas liquid chromatography-mass spectrometry showed that the carbohydrates in WIF hydrolyzate were mostly simple hexose sugars with trace amounts of deoxy-sugar fragments, but no N-acetyl glucosamines were identified. The Ames Salmonella test was employed to assess the genotoxic inhibitory effect of coffee WIF and model system melanoidin (MSM) on benzo(a)-pyrene (BP), afiatoxin (AFB₁) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The reverse mutation frequencies were reduced in the presence of WIF and MSM over a range of dosages. Studies were conducted to examine the effect of pH on the binding ability of WIF with BP and AFB₁. A maximum binding of about 83% of BP by WIF in buffer at 37°C was achieved at pH 2.0, and approximately 63% binding of BP by WIF occurred at pH values ranging from 4 to 9. The binding of AFB₁ by WIF in buffers at pH values between 2 to 9 ranged from 47 to 55% at 37°C. A pH effect on afiatoxin B₁ binding to WIF was not apparent. Binding of BP by WIF in citrate buffer (pH 3.0) was examined by column chromatography using different eluants to try to determine the type of binding. The BP peak coincided with the WIF peak when citrate buffer was used (pH 3.0) with and without added NaCl, urea and mercaptoethanol. With SDS added to the citrate buffer eluant, WIF was broken down into smaller particles yet retaining most of the BP. Hydrophobic bonds are presumably involved in the binding of BP to WIF. The activities of aryl hydrocarbon hydroxylase (AHH) in the microsomes of the small intestine of rats fed diets with or without BP and with or without MSM were assessed. The AHH activity for rats on a diet containing both BP and MSM was significantly smaller than that for rats on a diet with BP and no MSM. Components of MSM presumably bind BP to the extent that less BP was available for induction of AHH activity.