Enzymatic Hydrolysis of Rutin: Evaluation of Kinetic Parameters and Anti-Proliferative, Mutagenic and Anti-Mutagenic Effects

Abstract

The bioavailability of glucoside flavonoids is influenced by the nature of the sugar, glucosides being absorbed faster than rhamnoglucosides, for example. One strategy to enhance the bioavailability is enzymatic hydrolysis. In this study, some kinetic parameters of hesperidinase-mediated hydrolysis of rutin were evaluated using an UHPLC/QTOF-MS^E analysis of the products of a bioconversion reaction. The resulting hydrolyzed rutins (after 4, 8 and 12 h of reaction) were submitted to anti-proliferative and Cytokinesis-Block Micronucleus (CBMN) assays in CHO-K1 cells. In the hesperidinase-mediated hydrolysis, the final concentration of quercetin-3-O-glucoside (Q3G) was directly proportional to the rutin concentration and inversely proportional to the reaction time. At an anti-proliferative concentration (2.5 μg/mL), hydrolyzed rutin derivatives did not show a mutagenic effect, except for the sample with a higher content of Q3G (after 4 h of the enzymatic hydrolysis of rutin). Moreover, the higher Q3G content in hydrolyzed rutin protected the CHO-K1 cells 92% of the time against methyl methanesulfonate-induced mutagenic damage. These results suggested that the anti-mutagenic effect of hydrolyzed rutin might be related to antioxidant and cell death induction. Presenting a good lipophilicity/hydrophilicity ratio, together with antioxidant and anti-mutagenic activities, the hesperidinase-mediated hydrolyzed rutin seemed to be a promisor raw material for the development of food supplements.

Keywords: genotoxicity test; hesperidinase; quercetin-3-O-glucoside.

Figure 1

Hydrolysis of rutin mediated by commercial hesperidinase. The commercial hesperidinase from Penicillium sp. acts both as α-l-rhamnosidase and β-d-glucosidase, affording quercetin 3-O-glusoside (Q3G) and quercetin, respectively.

Figure 2

Kinetics of the enzymatic hydrolysis of rutin and Q3G formation. The incubation time was 4 (■), 8 (●) and 12 (▲) h.

Figure 3

UPLC-MS analysis of hydrolyzed rutin. Chromatograms of hydrolyzed rutin after 4 h (a) and 12 h (b) of hesperidinase treatment; peak 1: rutin, peak 2: quercetin-3-glucoside and peak 3: quercetin; (c) representative mass spectrum acquired in negative ion mode.

Figure 4

Anti-proliferative profile of rutin, hydrolyzed rutin 01 (after 4 h of reaction), hydrolyzed rutin 02 (after 8 h of reaction), hydrolyzed rutin 03 (after 12 h of reaction) and quercetin against the CHO-K1 cell line after 4 + 20 h (a) or 48 h (b) of exposure. Sample concentration range: 0.25 to 250 μg/mL.