# Decomposition of Flavonols in the Presence of Saliva
## 3 Results and Discussion
### Stability of Flavonols in Saliva Solution  
Figure 3 presents the loss of the starting amount as percent of remaining kaempfer, quercetin, and isorhamneetin (Figure 3a) as well as myricetin (Figure 3b) for a single representative after 6 h of incubation with saliva. The levels of the examined compounds were significantly different after 6 h of incubation. Kaempfer was the most stable compound in saliva during this time (about 73% left). Quercetin and isorhamneetin were less stable than kaempfer, and the degree of their degradation was 52% and 59%, respectively. Myricetin was the least stable flavonol under these conditions, and its concentration was at trace levels at the end of the experiment. As can be readily seen, myricetin showed rapid degradation and disappeared after 90 min of incubation. The general order of stability of examined flavonoids was as follows for all saliva samples collected from five human volunteers: kaempfer \(>\) isorhamneetin \(>\) quercetin \(>>\) myricetin. Generally, the decomposition rate of flavonoids increases with an increasing number of hydroxyl groups attached to the B-ring. A similar order of degradation of these flavonoids was observed in H\({}_{2}\)O\({}_{2}\) solution. These results are in good agreement with observations of [PERSON] et al. for ultraviolet radiation A (UVA) [29]. During the incubation of quercetin with saliva solution, the decomposition of quercetin into compounds with \(m\)/\(z\) 305 and \(m\)/\(z\) = 317 was observed. These two peaks were identified as the intermediate products of quercetin oxidation (depsides) and their formation under oxidative conditions has already been described [28; 29; 33]. During further incubation, depsides decomposed with a simultaneous appearance of another peak with the retention time 4.41 min, mass spectrum, and SRM pair (153/109) characteristic for protocatechuic acid (Figure 4). In contrast to the incubation of quercetin in the presence of H\({}_{2}\)O\({}_{2}\) mixture, the formation of 2,4,6-trihydroxybenzoic acid as a degradation product was not observed. Figure 2: Degradation profile of quercetin and its main products formed during the incubation with H\({}_{2}\)O\({}_{2}\) solution. As mentioned above, saliva was proven to contain phenolics after consumption of phenolics-rich beverages, and these compounds have been found to persist in oral cavity up to 300 min, despite a constant salivary flow [21]. Our research shows that this time is sufficient for the partial decomposition of kaempfer, quercetin, and isorhamnetin and complete decomposition of myricetin. To check if detected compounds may be considered as a dead-end product, we decided to incubate analyzed flavonoids for further 18 h. After 24 h of incubation, no additional compounds were detected as degradation products of the analyzed flavonoids. However, further decomposition of kaempfer, quercetin, and isorhamnetin was observed. The levels of remaining flavonoids calculated as average amount for five volunteers was as follow: 55.42% \(\pm\) 8.12 of kaempfer, 23.17% \(\pm\) 5.48 of quercetin, and 39.37% \(\pm\) 11.11 of isorhamnetin. This indicates that, even after long time of incubation, the order of stability of flavonoids remains the same. Since it is known that the stability of flavonoids strongly depends on the pH, we controlled it during the experiment. For all solutions, pH fluctuation ranged from 6.96 \(\pm\) 0.10 to 7.06 \(\pm\) 0.15 at the beginning and from 6.98 \(\pm\) 0.12 to 7.39 \(\pm\) 0.09 at the end of the experimental period. Several studies have reported that saliva can hydrolyze flavonoids glycosides, hence we checked four quercetin glycosides: rutin (quercetin 3-rhamoglucoside), quercetin (quercetin 3-rhamoside), hyperoside (quercetin 3-galactoside), and spiraeoside (quercetin 4'-glucose). Preliminary studies showed that all of the analyzed glycosides were stable in the presence of saliva.