
Periodontal diseases such as periodontitis, periodontal tissue destruction, and loss of alveolar bone are related with Gram-negative anaerobic bacteria. Basis on epidemiological study,
Mushrooms have been used for tea and nutritional food due to their special fragrance and texture.7 Furthermore, some mushrooms are applied as alternative medicine in Northeast Asia.8 Especially,
Despite advances in dental treatment, the number of patients with periodontitis is increasing, and the number of patients with peri-implantitis after implant surgery is also increasing.. Therefore, the purpose of present study was to investigate antimicrobial activity of soluble- and insoluble-extract from shiitake against periodontopathogens and to evaluate cytotoxicity of the soluble- and the insoluble-extract.
The susceptibility assay of periodontopathogens for shiitake extracts was performed according to the method suggested by Clinical Laboratory Standard Institute.16 Each specific medium (180 μl) was dispensed into 96-well plate (SPL lifescience, Pocheon, Korea), and each extract solution (180 μl) was dispensed into the 12th row well of a 96-well plate. The extract was performed 2-fold serial dilution to the 11th column with multi-micropipette. The cultured periodontopathogens were counted with bacterial counting chamber (Marienfeld, Lauda-Konigshöfen, Germany), and its concentration was adjusted at 3 × 106 cells/ml using the respective media. The bacterial suspension (20 μl) was inoculated into prepared well, and the bacteria-inoculated plate was incubated at 37°C under aerobic condition (H2 5%, CO2 10%, N2 85%) for 48 h. The bacterial growth was measured using optical density at a 660 nm wavelength by a spectrophotometer (Biotek, Winooski, USA).
Human gingival fibroblasts (HGF-1; CRL-2014) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Hyclone), penicillin (100 U/ml), and streptomycin sulfate (100 μg/ml). The HGFs were plated in 100 mm diameter cell culture dish (SPL Lifescience) and subcultured two times. The cells were detached using trypsin-EDTA (Welgene Co., Gyeongsan, Korea), and the cell number was counted by a cell counting chamber (Marienfeld). 1 × 105 cells were seeded into 96-well plate and grown to 90% confluence. After starvating for 4 h, the cells were treated with each shiitake extract for 12 h. The spent medium was removed, and fresh medium (100 μl) was inoculated. After incubating for 1 h in a CO2 incubator, 10 μl of CCK-8 solution (Dojindo Laboratories, Mashiki, Kumamoto, Japan) was added into each well of the plate to measure cell viability, and the cells were incubated for 2 h in the incubator. After adding 1% sodium dodecyl sulfate (SDS) solution, the optical density of the well was measured at 450 nm wavelength by a microplate reader (Biotek).
Statistical analysis was processed by IBM SPSS statistics ver. 23 software (IBM, Armonk, USA). Data was analyzed by the non-parametric Kruskal-Wallis and Mann-Whitney test. Statistical significance was defined by P value of less than 0.05.
Antimicrobial effect of the extracts from shiitake mushroom on periodontopathogens such as
A cytotoxicity test was evaluated using the cell line of HGF-1 to examine what kind of effect it has on cells at a concentration showing antibacterial activity. When HGF-1 was treated with the extract at a concentration in the range of 0.625 - 5 mg/ml for 12 h, both extracts showed significant cytotoxicity for the cell above the concentration of 2.5 mg/ml (
Periodontitis is a chronic inflammation of the gingiva with polymicrobial ecology. In the ecology,
The antimicrobial activity of the extracts from shiitake mushroom showed against
Finally, the cytotoxicity test was performed using HGF-1 for the extracts. The acetone extract in the concentration showing antimicrobial activity did not show cytotoxicity. When most of the components of the acetone extract may be predicted aroma molecules, these molecules are known to be good for health such as antioxidation and anti-cancer effect. 24,25 Since the acetone extract is also a lipid, it may have a good effect at moderate concentrations, but may show cytotoxicity at high concentrations. Furthermore, the acetone extract showed cytotoxicity from 4 h in high concentration as 2.5 mg/ml. Therefore, considering the length of the extract stays in the mouth, the extract may not adversely affect the oral cavity even at high concentrations in the end.
The extracts from shiitake mushroom have the antimicrobial activity against periodontopathogens. Detailly, the acetone extract from shiitake mushroom has stronger antimicrobial activity than the water extract. Also, the acetone extract did not show cytotoxicity in the concentration showing antimicrobial activity. Basis on these results, the shiitake mushroom may help to prevent and treat periodontitis.
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