Anthocyanins and their metabolism in the in vitro gastrointestinal tract

Anthocyanins are flavonoids which produce blue, purple red and intermediate colours in plants. They are present as glycosides with an anthocyanidin C6-C3-C6 skeleton, whose structure is dependent on pH. Anthocyanins are poorly detected from the biological fluids: plasma and urine. They may be poorly absorbed or they undergo changes in their structure. Nevertheless, large amounts of the ingested compounds are likely to enter the colon either within the matrix of the dietary source or through enterohepatic circulation. In vitro and in vivo studies have shown that colonic bacteria transform various flavonoids to smaller phenolic acids. However, there is very little information on bacterial transformations of anthocyanins. Aims of the study were to find out the release and changes of anthocyanins in the in vitro upper intestinal model and their bacterial metabolism in the colon model. Frozen berries, bilberry (Vaccinium myrtillus) and red raspberry (Rebus idaeus, v. Ottawa), were subjected to physiological conditions in the presence and absence of active or heat-inactivated alimentary enzymes (salivary amylase, pepsin, pancreatin and mucin) in successive stages of mouth, stomach and duodenum. Changes in stomach and duodenum were compared for their anthocyanin response by HPLC detected at 520 nm. Bacterial transformations of pure anthocyanins (cyanidin-3-glucoside and -rutinoside) isolated from red blum (Prunus domestica L.) and red grapes (Vitis vinifera L.) were studied in an in vitro colon model using human faecal microbiota as an inoculum. Metabolites were analysed and characterized by HPLC-DAS and LC-MS. They were identified by comparing their characteristics with those of available standards, and semi-quantified using the amount of substrate analysed from samples at initial timepoint. Anthocyanins gave a good response as flavylium cations in 520 nm in acidic conditions mimicking stomach and the detection was decreased dramatically in neutral to mildly basic conditions of duodenum indicating opening of the flavylium cation to quinoidal base. In the colon model in vitro cyanidin-3-rutinoside and cyanidin-3-glucoside were deglycosylated and formation of three low molecular weight metabolites were shown. The major metabolite at earlier time points was identified as protocatechuic acid. Furthermore, reconjugation of the aglycone with other groups, non-typical for dietary anthocyanins, was evident at the later timepoints. In conclusion, anthocyanins are poorly detected due to their pH dependent structural characteristics and their bioavailability is difficult to define on the basis of detection of flavylium cations only. Also, smaller molecular weight metabolites are formed due to bacterial actions in the colon, which involve the cleavage of glycosidic linkages, breakdown of the anthocyanidin heterocycle and reconjugation of the aglycone. Information about the health effects of the bacterial metabolites in general is scarce and therefore research directed to this area is necessary.