Inhibitory effects of sword bean extract on alveolar bone resorption induced in rats by Porphyromonas gingivalis infection.

BACKGROUND: The domesticated legume, Canavalia gladiata (commonly called the sword bean), is known to contain canavanine. The fruit is used in Chinese and Japanese herbal medicine for treating the discharge of pus, but its pharmacological mechanisms are still unclear. OBJECTIVES: This study examined the effect of sword bean extract (SBE) on (i) oral bacteria and human oral epithelial cells in vitro, and (ii) the initiation and progression of experimental Porphyromonas gingivalis-induced alveolar bone resorption in rats. MATERIAL AND METHODS: A high-performance liquid chromatography/ultraviolet method was applied to quantitate canavanine in SBE. By assessing oral bacterial growth, we estimated the minimum inhibitory concentration and minimum bactericidal concentration of SBE, canavanine, chlorhexidine gluconate (CHX) solution. The cytotoxicity of SBE, canavanine, CHX, leupeptin and cystatin for KB cells was determined using a trypan blue assay. The effects of SBE, canavanine, leupeptin and cystatin on Arg-gingipain (Rgp) and Lys-gingipain (Kgp) were evaluated by colorimetric assay using synthetic substrates. To examine its effects on P. gingivalis-associated periodontal tissue breakdown, SBE was orally administered to P. gingivalis-infected rats. RESULT: Sword bean extract contained 6.4% canavanine. SBE and canavanine inhibited the growth of P. gingivalis and Fusobacterium nucleatum. The cytotoxicity of SBE, canavanine and cystatin on KB cells was significantly lower than that of CHX. Inhibition of Rgp with SBE was comparable to that with leupeptin, a known Rgp inhibitor, and inhibition of Kgp with SBE was significantly higher than that with leupeptin at 500 µg/mL ( p < 0.05). P. gingivalis-induced alveolar bone resorption was significantly suppressed by administration of SBE, with bone levels remaining comparable to non-infected animals ( p < 0.05). CONCLUSION: The present study suggests that SBE might be effective against P. gingivalis-associated alveolar bone resorption.

Alkali-resistant bacteria in root canal systems.

The aim of this study was to isolate and identify alkali-resistant bacteria from the dentin of infected root canals. Bacteria from homogenized dentin powder made up from infected root canal walls from human teeth were cultured on buffer-enriched Brain Heart Infusion agar supplemented with 4% sheep blood (BHI-blood agar), adjusted to pH 7.0, 9.0 or 10.0. Incubation took place for 7 days at 37 degrees C in an anaerobic glove box. Bacterial strains selected according to colony and morphology were subcultured in buffer-enriched BHI broth adjusted to pH 9.0, 10.0 or 11.0 to confirm their growth as alkali-resistant bacteria. Polymerase chain reaction amplification using specific primer sets and 16S rDNA sequence analysis was performed for identification of alkali-resistant isolates. In the present study, 37 teeth extracted from 37 patients were used for preparation of the dentin powder samples. Bacteria were detected in 25 samples when standard BHI-blood agars (pH 7.0) were used. Of these, 29 strains from 15 samples were alkali resistant, 25 strains growing at pH 9.0 and 4 at pH 10.0. The alkali-resistant strains included Enterococcus faecium (10 strains) and Enterococcus faecalis (2 strains), Enterobacter cancerogenus (1 strains), Fusobacterium nucleatum (1 strains), Klebsiella ornithinolytica (2 strains), Lactobacillus rhamnosus (2 strains), Streptococcus anginosus (2 strains), Streptococcus constellatus (3 strains), and Streptococcus mitis (2 strains). Three strains were also identified as bacteria of genus Firmicutes or Staphylococcus at the genus level. The present study showed that many bacterial species in infected root canal dentin were alkali-resistant at pH 9.0 and/or pH 10.0, and belonged mainly to the genus Enterococcus.

Functional assessment of proliferating hepatocytes stimulated by hepatic stimulatory substance in ascorbic acid biosynthetic enzyme-deficient rats.

The functional ability of hepatic stimulatory substance (HSS)-stimulated proliferating hepatocytes was investigated by intrasplenic and/or intraportal transplantation in ascorbic acid (AsA) biosynthetic enzyme-deficient (ODS-od/od) rats that die of osteogenic disorders unless there is AsA supplementation. HSS was extracted from regenerating porcine livers. Hepatocytes isolated from the livers of congeneic ODS-+/+ rats that are capable of synthesizing AsA were transplanted into the spleen (Sp-HTx) and/or the portal vein (Pv-HTx) of ODS-od/od rats. The recipients were divided into eight groups as follows: HSS-untreated groups [group Ia, sham-operated, HTx(-); group IIa, Sp-HTx; group IIIa, Pv-HTx; and group IVa, Sp- and Pv-HTx], HSS-treated groups [group Ib, HSS only; group IIb, Sp-HTx + HSS; group IIIb, Pv-HTx + HSS; and group IVb, Sp- and Pv-HTx + HSS]. The recipients were given a diet and water containing AsA for 6 weeks after HTx, and AsA supplementation was then halted. The average bromodeoxyuridine (BrdU) labeling index (LI) and hepatocyte-occupied ratio in the spleen (H/S ratio) of HSS-treated rats were significantly higher than those of HSS-untreated rats. All the rats in HSS-untreated groups and group Ib died by 8 weeks after the cessation of AsA. In HSS-treated groups IIb, IIIb, and IVb, the survival rates were 60%, 50%, and 80%, respectively, at 16 weeks after HTx. The average serum AsA level of the surviving rats in groups IIb, IIIb, and IVb was significantly higher than that in HSS-untreated groups. These results indicate that HSS treatment induced rapid proliferation of transplanted hepatocytes in the spleen and the portal vein, and that these proliferating hepatocytes synthesized AsA and improved the survival rate of ODS-od/ od rats.