Ring B substitution pattern and glycosylation strongly affect the stability of flavonols towards in vitro digestion

In vitro digestion models are frequently used to study the bioaccessibility and stability of plant constituents in food and medicinal plants [1]. Varying degrees of stability towards in vitro digestion have been reported for flavonols. Moreover, flavonols possessing catechol or pyrogallol substitution in ring B have been reported to be unstable in cell culture media or buffer solutions frequently used in pharmacological in vitro assays [2], [3].

In this study, the stability of flavonols with phenol, catechol and pyrogallol substitution in ring B and different position and degree of glycosylation ([Fig. 1]) should be assessed in an in vitro digestion protocol based on the INFOGEST consensus method [4]. Flavonoid levels in the different digestion phases were determined by HPLC-UV.

Fig. 1 Flavonols subjected to in vitro digestion. 1: kaempferol; 2: kaempferol-3-O-β-D-glucoside (astragalin); 3: kaempferol-7-O-β-D-glucoside (populnin); 4: kampferol-3-O-β-D-rutinoside (nicotiflorin); 5: quercetin; 6: quercetin-3-O-β-D-glucoside (isoquercitrin); 7: quercetin-7-O-β-D-glucoside (quercimetrin); 8: qercetin-3-O-α-L-rhamnoside (quercitrin); 9: quercetin-3-O-β-D-glucuronide (miquelianin); 10: quercetin-3- O-β-D-rutinoside (rutin); 11: myricetin; 12: myricetin-3 O-α-L-rhamnoside (myricitrin); 13: myricetin-3-O-β-D-glucuronide.

While all flavonoids were found to be stable in the oral and gastric digestive phase, stability towards intestinal phase digestion was found to be strongly dependent on ring B substitution. While kaempferol and its glycosides remained unaffected, the levels of quercetin were reduced to 26.8% of a quercetin control solution prepared in ethanol. The observed reduction was obviously caused by enzymes or bile, since quercetin was stable when incubated in the intestinal phase buffer only. Myricetin was undetectable after incubation in intestinal phase buffer with and without enzymes and bile, indicating that its decline is due to instability in intestinal phase buffer. For the respective glycosides, lower degrees of reduction were observed, indicating that glycosylation generally enhanced the stability of quercetin and myricetin towards intestinal phase digestion.

• References

• 1 Alminger M, Aura AM, Bohn T. et al. In vitro models for studying secondary plant metabolite digestion and bioaccessibility. Compreh Rev Food Sci Food Saf 2014; 13: 413-436
• 2 Xiao J, Hoegger P. Stability of Dietary Polyphenols under the Cell Culture Conditions: Avoiding Erroneous Conclusions. J Agric Food Chem 2015; 63: 1547-1557
• 3 Cao H, Hoegger P, Aroo R, Xiao J. Flavonols with a catechol or pyrogallol substitution pattern on ring B readily form stable dimers in phosphate buffered saline at four degrees Celsius. Food Chem 2020; 311: 125902
• 4 Minekus M, Alminger M, Alvito P. et al. A standardised static in vitro digestion method suitable for food – an international consensus. Food Funct 2014; 5: 1113-1124