Inhibition of gingipains and Porphyromonas gingivalis growth and biofilm formation by prenyl flavonoids

Abstract

Background and objective: Porphyromonas gingivalis is considered a major pathogen of chronic periodontitis, which also may be implicated with systemic diseases such as atherosclerosis. Secreted cysteine proteases, gingipains Rgp and Kgp, are essential for P. gingivalis virulence. Some polyphenols and flavonoids are known to inhibit gingipain activity and interfere with biofilm formation by P. gingivalis. Many bioactive compounds have been isolated from Epimedium species, but availability of these compounds on gingipains and P. gingivalis is still unclear. Therefore, the aim of this study was to evaluate natural products from medical plants to develop a new therapeutic agent against periodontal disease.

Material and methods: Prenylated flavonoids were isolated from Epimedium species plant using column chromatographies. The inhibitory effect of the prenylated flavonoids against protease activity of gingipains were examined using purified gingipains and fluorogenic substrates. Anti-P. gingivalis activity was evaluated to analyze planktonic growth and biofilm formation in brain heart infusion medium in the presence of the prenylated flavonoids.

Results: We isolated 17 prenylated flavonoids (Limonianin, Epimedokoreanin B, etc.) from Epimedium species. We found that some prenylated flavonoids inhibited gingipain activity in a non-competitive manner with K_i values at μm order. The prenylated flavonoids also hindered growth and biofilm formation of P. gingivalis, in a manner independent of gingipain inhibition by the compounds.

Conclusion: The results indicated an inhibitory effect of the prenylated flavonoids against P. gingivalis and would provide useful information for future development of periodontitis treatment that suppresses gingipains, P. gingivalis growth and biofilm formation.

Keywords: Porphyromonas gingivalis growth; biofilm; gingipains; prenylated flavonoids.

Figure 1. Structure of flavonoids from Epimedium species

Compound 1, Icariin; 2, Icariside I; 3, Icariside II; 4, Icarisoside A; 5, Epimedoside C; 6, Limonianin; 7, 8–5’ diprenyl apigenin; 8, Epimedokoreanin B; 9, Neophellamuretin; 10, 8-prenyl luteolin; 11, Broussonol D; 12, Anhydroicaritin; 13, Euchresta flavanone A; 14, Sagittatoside A; 15, Korepimeside A; 16, Desmethylicaritin; 17, Epimedokoreanin C.

Figure 2. Inhibition of Rgp and Kgp by prenylated flavonoids isolated from Epimedium species (A) and selected flavonoids (B)

Rgp and Kgp (each at 1.67 nM final concentration) were preincubated with 10 µM of prenylated flavonoid and residual enzyme activity was measured. The enzyme activity in the absence of flavonoid was taken as 100%. EC, Epicatechin; ECg. Epicatechin gallate; EGC, Epigallocatechin; EGCg, Epigallocatechin gallate. All values are expressed as average ± standard error in triplicate assay. Asterisk (P < 0.05) and double asterisk (P < 0.01) indicates significant difference of the values versus control values.

Figure 3. Determination of the mode of inhibition of RgpB (A) and Kgp (B) by Epimedokoreanin B (compound 8)

The velocity (v) of the reaction was determined at the substrate concentration [S] in the range from 20 µM to 100 µM in the presence of various concentrations (0–10 µM) of Epimedokoreanin B and the ratio of [S]/v for each inhibitor concentration [I] was plotted (the

Figure 4. Inhibitory effect of prenylated flavonoids on growth and biofilm formation of P. gingivalis

(A–C) P. gingivalis growth in the presence of prenylated flavonoids at 12.5 µM is shown by measuring absorbance at 620 nm different time points. Results were similar in each of three independent experiments and representative data are shown as means ± SD in a triplicate assay. (D) Biofilm formation (absorbance at 492 nm after staining with safranin) was assessed after a 24 h-culture in the presence of a compound at 1.25 µM. All the compounds at 1.25 M had no effect on growth of P. gingivalis. Results were similar in three independent experiments and representative data are shown. Results are expressed as means ± SD in a triplicate assay. Data were statistically analyzed by one-way analysis of variance (ANOVA) followed by the Dunnett’s multiple comparison test. Asterisks indicate significant difference (P < 0.05) from values obtained in the absence of flavonoids.