Enzymatic glycosylation of rooibos plant extract to improve stability of active compounds.

Rooibos (Aspalathus linearis), a South African native plant, is renowned for its remarkable antioxidant properties, largely driven by its rich flavonoid content, in particular aspalathin as predominant compound. Yet, the poor stability of this flavonoid sharply restricts rooibos applications. In nature, flavonoids are often found as glycosylated molecules. Inspired by this strategy, this study investigated enzymatic pathways to glycosylate four key rooibos flavonoids: aspalathin, rutin, isoorientin, and orientin, using sucrose as a renewable, abundant, and low-cost donor. Transglycosylases from Glycoside Hydrolases families 68 and 70 displayed an outstanding ability to glycosylate the four target flavonoids, both as pure compounds and, for first time, in a complex crude plant extract. Aspalathin and isoorientin were particularly well recognized, with complete conversion achieved by two-thirds of the screened enzymes from family 70. Selection of the biocatalyst and reaction conditions allows for tailored glycosylation, thereby providing control over the type and number of attached glycosyl units, linkage specificity and glycosylation site. Structural analyses of aspalathin monofructoside and mono- and diglucosides demonstrated the feasibility to improve aspalathin thermal stability, although with a loss in anti-scavenging activity. However, preliminary results indicate that glycosidic protection can be removed by enzymes found in the human digestive system or skin microbiota. Glycosylation of bioactive rooibos components is a promising strategy to stabilize these natural compounds, facilitating their incorporation into nutraceutical or cosmetic formulations.