Reactivation of peroxidase activity in human saliva samples by polyphenols.

OBJECTIVES: The enzyme lactoperoxidase (LPO), which is released into several body fluids like saliva, is an essential part to maintain the oral bacterial homeostasis by catalysing the oxidation of thiocyanate (SCN-) to hypo-thiocyanite (-OSCN). The formation of unreactive redox intermediates (like Compound II) leads to a decreased pseudo-halogenating enzyme activity, which is associated with a higher risk for oral infections. According to former studies with bovine LPO selected flavonoids were tested in respect to their potential to reactivate the enzymatic activity in a more physiological, human salivary system. DESIGN: Saliva samples from healthy donors were collected and characterized by using several gel staining methods and immunoblotting. Afterwards kinetic measurements were performed by applying the TNB-assay to evaluate the pseudo-halogenating salivary peroxidase (SAPX) activity. The measurements were performed in the presence of excess H2O2 to simulate pro-inflammatory conditions. Moreover selected flavonoids or an ethanolic extract of Tormentillae rhizoma were applied to test their regenerating effect on the LPO-derived -OSCN production. RESULTS: Despite the complex protein composition of the collected saliva samples, an SAPX-derived pseudo-halogenating activity could be identified. The -OSCN regenerating effects of the tested polyphenols were completely comparable to previous in vitro experiments with bovine LPO. Thus, we could show that phenolic substances are suitable to regenerate the peroxidase activity in human saliva samples after H2O2-induced inactivation. CONCLUSION: The studies provide new insights into the effect of pharmaceutical relevant polyphenols on salivary peroxidase activity and thus, suggest this enzyme as a new target for the prevention and therapy of oral inflammatory diseases.

Flavonoids as promoters of the (pseudo-)halogenating activity of lactoperoxidase and myeloperoxidase.

In this study several flavonoids were tested for their potential to regenerate the (pseudo-)halogenating activity (hypothiocyanite formation) of the heme peroxidases lactoperoxidase (LPO) and myeloperoxidase (MPO) after hydrogen peroxide-mediated enzyme inactivation. Several flavonoid subclasses with varying hydroxylation patterns (especially of the flavonoid B-ring) were examined in order to identify structural properties of efficient enzyme regenerators. Kinetic parameters and second-order rate constants were determined. A 3',4'-dihydroxylated B-ring together with C-ring saturation and hydroxylation were found to be important structural elements, which strongly influence the flavonoid binding and oxidizability by the LPO/MPO redox intermediates Compounds I and II. In combination with docking studies these results allow an understanding of the differences between flavonoids that promote the hypothiocyanite production by LPO and MPO and those that inhibit this enzymatic reaction.