Inhibition of host- and bacteria-derived proteinases by natural anthocyanins.

BACKGROUND AND OBJECTIVES: Host- and bacteria-derived proteinases are considered to play critical roles in periodontitis progression. This study investigated the ability of a blackcurrant extract and its major anthocyanins (cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside and delphinidin-3-O-rutinoside) to inhibit the activity of matrix metalloproteinases (MMPs), neutrophil elastase and periodontopathogen (Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola) proteinases. MATERIAL AND METHODS: Enzyme inhibition was detected using fluorometric and colorimetric assays after incubating blackcurrant extract and its major anthocyanins (at concentrations of 6.25, 12.5, 25 and 50 µg/mL) with MMPs, elastase or bacterial proteinases, along with their specific substrates. Substrate degradation was recorded every hour for up to 4 h. RESULTS: The blackcurrant extract (50 µg/mL) inhibited all proteinases tested. MMP-1 and MMP-9 were significantly inhibited by pure anthocyanins at concentrations ranging from 6.25 to 50 µg/mL. Elastase activity was inhibited by cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside in the range of 6.25-50 µg/mL and by delphinidin-3-O-rutinoside at 50 µg/mL. P. gingivalis, T. forsythia and T. denticola proteinases were also significantly inhibited by pure anthocyanins. In all cases, enzyme inhibition was time-dependent. CONCLUSION: Our study showed that a blackcurrant extract and its major anthocyanins were able to inhibit the activity of host- and bacteria-derived proteinases. This suggests that such natural compounds may represent promising agents for use in adjunctive treatments for periodontitis.

Cranberry proanthocyanidins inhibit MMP production and activity.

Matrix metalloproteinases (MMPs) produced by resident and inflammatory cells in response to periodontopathogens play a major role in periodontal tissue destruction. Our aim was to investigate the effects of A-type cranberry proanthocyanidins (AC-PACs) on: (i) the production of various MMPs by human monocyte-derived macrophages stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS), and (ii) the catalytic activity of recombinant MMP-1 and MMP-9. The effects of AC-PACs on the expression of 5 protein kinases and the activity of nuclear factor-kappa B (NF-kappaB) p65 in macrophages stimulated with LPS were also monitored. Our results indicated that AC-PACs inhibited the production of MMPs in a concentration-dependent manner. Furthermore, the catalytic activity of MMP-1 and MMP-9 was also inhibited. The inhibition of MMP production was associated with reduced phosphorylation of key intracellular kinases and the inhibition of NF-kappaB p65 activity. AC-PACs thus show potential for the development of novel host-modulating strategies to inhibit MMP-mediated tissue destruction during periodontitis.

Naringenin inhibits human osteoclastogenesis and osteoclastic bone resorption.

BACKGROUND AND OBJECTIVE: Naringenin, a naturally occurring flavonoid, possesses a wide range of pharmacological properties. The purpose of this study was to investigate the effect of naringenin on human osteoclastogenesis and osteoclastic bone resorption. MATERIAL AND METHODS: Naringenin was tested in a human osteoclastogenesis model using primary osteoclast precursor cells activated by receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) for 6 days. Osteoclastogenesis was assessed by determining the number of tartrate-resistant acid phosphatase (TRAP)-stained multinuclear cells, while the secretion of factors involved in osteoclastogenesis was assessed using enzyme-linked immunosorbent assays. The effect of naringenin on bone resorption was investigated using an OsteoAssay human bone plate coupled with an immunoassay to evaluate the release of helical peptide 620-633 from the alpha1 chain of type I collagen. RESULTS: Naringenin was non-toxic at the highest concentration used (50 microg/ml). Naringenin (10, 25 and 50 microg/ml) significantly inhibited osteoclastogenesis (by 29 +/- 5, 57 +/- 8 and 96 +/- 1%, respectively). Naringenin also markedly inhibited the secretion of interleukin (IL)-1alpha (by 59%), IL-23 (by 87%) and monocyte chemoattractant protein-1 (by 58%). Lastly, naringenin (10, 25 and 50 microg/ml) significantly decreased the release of helical peptide 620-633, an indicator of bone resorption activity (by 44 +/- 0.5, 73 +/- 0.5 and 86 +/- 1%, respectively). CONCLUSIONS: Naringenin can inhibit human osteoclastogenesis and osteoclastic bone resorption. It thus holds promise as a therapeutic or preventive agent for bone-related diseases such as periodontitis.

Naringenin has anti-inflammatory properties in macrophage and ex vivo human whole-blood models.

BACKGROUND AND OBJECTIVE: Periodontitis is a chronic inflammatory disease of bacterial etiology, affecting tooth-supporting tissues. The host inflammatory response to periodontopathogens, notably the high and continuous production of cytokines, is considered a major factor causing the local tissue destruction observed in periodontitis. The aim of the present study was to investigate the effect of naringenin, a major flavanone in grapefruits and tomatoes, on the lipopolysaccharide-induced pro-inflammatory cytokine production by host cells, using two different models. MATERIAL AND METHODS: The effect of naringenin was characterized using macrophages stimulated with the lipopolysaccharide of either Aggregatibacter actinomycetemcomitans or Escherichia coli and using whole blood stimulated with A. actinomycetemcomitans lipopolysaccharide, in the presence or absence of naringenin. Lipopolysaccharide-induced interleukin-1 beta, interleukin-6, interleukin-8 and tumor necrosis factor-alpha production by macrophages and whole-blood samples treated with naringenin were evaluated using an enzyme-linked immunosorbent assay. Changes in the phosphorylation states of macrophage kinases induced by A. actinomycetemcomitans lipopolysaccharide and naringenin were characterized by immunoblot screening. RESULTS: Our results clearly indicated that naringenin is a potent inhibitor of the pro-inflammatory cytokine response induced by lipopolysaccharide in both macrophages and in whole blood. Naringenin markedly inhibited the phosphorylation on serines 63 and 73 of Jun proto-oncogene-encoded AP-1 transcription factor in lipopolysaccharide-stimulated macrophages. CONCLUSION: The results from the present study suggest that naringenin holds promise as a therapeutic agent for treating inflammatory diseases such as periodontitis.