Antimicrobial activity of Antrodia camphorata extracts against oral bacteria

Abstract

Antrodia camphorata (A. camphorata) is a unique, endemic and extremely rare mushroom species native to Taiwan, and both crude extracts of and purified chemical compounds from A. camphorata have been reported to have a variety of significant beneficial effects, such as anti-tumor and anti-inflammatory activity. However, reports on the effects of A. camphorata against dental pathogens have been limited. Oral health is now recognized as important for overall general health, including conditions such as dental caries, periodontal disease and rheumatoid arthritis. Streptococcus mutans (S. mutans) and Porphyromonas gingivalis (P. gingivalis) are the most common bacteria associated with dental plaque and periodontopathic diseases, respectively. Thus, our study examined the ability of five various crude extracts of A. camphorata to inhibit the growth of dental bacteria and anti-adherence in vitro. Among the extracts, the ethanol, ethyl acetate and chloroform extracts exhibited the lowest MICs against P. gingivalis and S. mutans (MIC = 4∼16 µg/mL). The MIC of the aqueous extract was greater than 2048 µg/mL against both P. gingivalis and S. mutans. In vitro adherence of S. mutans was significantly inhibited by the addition of either the ethyl acetate extract or chloroform extract (MIC = 16∼24 µg/mL), while the ethanol extract (MIC = 32∼64 µg/mL) exhibited moderate inhibitory activity. Based on the result of this study, the ethyl acetate and chloroform extracts of A. camphorata may be good candidates for oral hygiene agents to control dental caries and periodontopathic conditions.

Figure 1. Diagram of separation and chemical analysis of A. camphorata extracts.

(A) A schematic representation of a liquid-liquid extraction and bioassay process for A. camphorata. (B) HPLC chromatogram of three reference standards of A. camphorata with corresponding retention times at λ = 245 nm, 4,7-dimethoxy-5-methyl- l,3-benzodioxole (AC-1) (retention time = 42.3 min); dehydrosulphurenic acid (AC-2) (retention time = 54.7 min); dehydroeburicoic acid (AC-3) (retention time = 78.1 min). Separation was performed in a Merck (50995) LiChrospher RP-18 column (4.6 mm×250 mm, 5 µm), at a flow rate of 1 mL/min. The mobile phase consisted of A (water) and B (100% acetonitrile): 0–90 min, 100–0% A and 0–100% B.(C) Quantitative analysis of three standards in five A. camphorate extracts: chloroform (CHCl₃), ethyl acetate (EA), 95% ethanol (EtOH), 50% ethanol (EtOH), and H₂O.

Figure 2. Determination of the antibacterial activity of A. amphorata extracts and chlorhexidine against P. gingivalis (Figure 2A and Figure 2B) and S. mutans (Figure 2C and Figure 2D).

Serial doses of extracts were added to the bacterial cultures in 96-well plates and incubated for 24 hours. Bacterial growth was determined by measuring the optical density of the cultures at 600 nm. The data are reported as the means±SD of triplicate wells. *P<0.05.

Figure 3. Cytotoxicity of different extracts (50% ethanol (EtOH), 95% ethanol (EtOH), ethyl acetate (EA) or chloroform (CHCl₃) of A. camphorata against HGF cells.

HGF cells were treated with extracts at various doses for 24 hours. Untreated cells were used as a control. Cell viability was assessed by an MTT assay. Six samples were analyzed in each group. The values represent the means±SE.* means significant between treatment and control. (P<0.001).