Phenolic Compounds of Reynoutria sp. as Modulators of Oral Cavity Lactoperoxidase System

Abstract

Lactoperoxidase (LPO) together with its (pseudo)halogenation cycle substrates, H₂O₂ and thiocyanate ions oxidized to hypothiocyanite ions, form one of the main systems involved in antimicrobial defense within the oral cavity. In bacterial diseases such as dental caries, lactoperoxidase is oxidized to a form known as Compound II, which is characterized by its inability to oxidize SCN^-, resulting in a decreased generation of antimicrobial products. Reynoutria sp. rizome extracts, due to their high polyphenol content, have been tested as a source of compounds able to regenerate the antimicrobial activity of lactoperoxidase through converting the Compound II to the native LPO state. In the presented study, acetone extracts of R. japonica, R. sachalinensis, and R. x bohemica, together with their five fractions and four selected polyphenols dominating in the studied in extracts, were tested toward lactoperoxidase reactivating potential. For this purpose, IC50, EC50, and activation percentage were determined by Ellman's method. Furthermore, the rate constants for the conversion of Compound I-Compound II and Compound II-native-LPO in the presence of extracts, extracts fractions, and selected polyphenols were determined. Finally, the ability to enhance the antimicrobial properties of the lactoperoxidase system was tested against Streptococcus mutans. We proved that Reynoutria sp. rhizome is the source of lactoperoxidase peroxidation cycle substrates, which can act as activators and inhibitors of the antimicrobial properties of that system. The presented study shows that the reactivation of lactoperoxidase could become a potential therapeutic target in prevention and treatment support in some infectious oral diseases.

Keywords: Reynoutria; Streptococcus mutans; dental caries; lactoperoxidase; lactoperoxidase reactivators; peroxidase; polyphenols; stopped-flow spectroscopy.

Figure 1

Relationship between the initial rate of OSCN^– formation and the concentration of hydrogen peroxide. The green triangles indicate the OSCN^– formation initial reaction rate, derived from the DTNB oxidation in the presence of just hydrogen peroxide (without the presence of LPO). The blue squares indicate the sum of the H₂O₂ effect and the enzymatic reaction. The enzyme activity-related reaction rate was calculated by subtracting the H₂O₂-dependent rate from the sum rate value and was marked with a red circle.

Figure 2

(A): IC₅₀ of acetone extracts from the rhizome of Reynoutria sp. and individual fractions obtained with various solvents. Black lines indicate the standard error of the quadruple marking of each fraction. (B): Dose–response curves (the Hill model) showing the activating effect of (-)-epicatechin and resveratrol in relation to the pseudo(halogenation) activity of LPO. (C): Dose–response curves (the Hill model) showing the inhibitory effect of (-)-epicatechin, resveratrol, and vanicoside B on the pseudo(halogenation) activity of LPO.

Figure 3

The dependence curve of observed OSCN^– formation rate in the presence of successive dilutions of the studied extract showing both inhibitory and activating effects (extract of diethyl ether fraction from R. japonica).

Figure 4

(A): Spectral changes accompanying the spontaneous generation of Compound II from Compound I. First spectrum (red) was obtained 200 ms after mixing LPO with hydrogen peroxide and corresponds to Compound I. Last spectrum (green) was registered after 5 s of incubation; complete generation of Compound II can be observed. (B): After addition of organic substrates, rapid reduction of Compound II (red) to native LPO (green) is observed. Typical kinetic trace reflecting the reaction between (-)-epicatechin and Compound I or Compound II with exponential fit (blue line) is shown at (C,D), respectively. Experimental conditions: 15°C; (A,C) LPO 1 µM, H₂O₂ 1 µM; (B,D) LPO 1 µM, H₂O₂ 0.75 µM, (-)-epicatechin 7 µM.

Figure 5

Plots of k_obs vs. concentration of selected polyphenols for formation (A) and decay (B) of Compound II of LPO. Experimental conditions: 0.02 M phosphate buffer pH = 6, 15°C; (A) (LPO) = 1 µM, (H₂O₂) = 1 µM; (B) (LPO) = 1 µM, (H₂O₂) = 0.75 µM.

Figure 6

Green line with dots represents concentration-dependent toxicity against S. mutans by lactoperoxidase substrates (H₂O₂ and SCN^– ions) and is expressed as time difference to reach half of log phase between the sample containing substrates and sample containing S. mutans only. Similarly, blue line with triangles represents the toxicity dependent on substrates and products of lactoperoxidase system generated in situ. Red line with squares is the difference between the whole LPO system toxicity and substrates-only toxicity, which corresponds to the toxicity dependent on the LPO products. Positive value of that parameter indicates an antimicrobial effect of the system, while negative value indicates protection against the toxicity of the substrates.

Figure 7

Examples of growth curves recorded spectrophotometrically at 600 nm. The green line shows the growth of S. mutans in the presence of substrates of the LPO system (H₂O₂ and SCN^– ions). The blue line shows the growth in the presence of the complete LPO system (H₂O₂–SCN–LPO). The black line shows microbial growth in the presence of the complete LPO system and 12 µM (-)-epicatechin. Vertical lines indicate the time taken by the sample containing the activated LPO system and the LPO system to reach half the logarithmic growth phase in the graph. The red line shows microbial growth in the presence of the complete LPO system and 760 µM (-)-epicatechin.

Figure 8

Dose–response curves (the Hill model) for the analyzed polyphenols and extracts that showed activating or inhibiting antimicrobial effect on the LPO system. Abbreviations: Epi, (-)-epicatechin; Res, resveratrol; RB, R. x bohemica; DCM, dichloromethane; Et2O, diethyl ether.