Extraction Of Essential Oil From Asarum And Evaluation Of Its Antibacterial Activity Against Periodontal Pathogens

Periodontitis is a chronic infection-induced inflammatory disease characterized by the destruction of the supporting structures of the teeth and is the most important cause of tooth loss among adults. Chronic periodontitis is initiated by an accumulation of anaerobic bacteria in the subgingival sites and is particularly associated with Fusobacterium nucleatum, Prevotella intermedia and Porphyromonas gingivalis. At present, there is less published information about the natural plant against Periodontitis. Asarum heterotropoides var. Mandshuricum, a traditional Chinese medicine, is a perennial herb endemic to China. It has a widely medicinal value such as analgesic, anti-tussive, anti-inflammatory, anti-allergic and antioxidant activity.Extraction of oil from Asarum was carried out using a conventional heat-reflux extraction system. Testing the effect of water to raw material ratio, particle size and extraction time on the extraction yield of essential oils based on the single-factor test. Then these factors were examined using an orthogonal L9(3)3test design. The influence on the extraction yield of essential oil decreased in the order:water to raw material ratio> extraction time> particle size according to the R values observerd. The optimum extraction conditions were water to raw material ratio of17, a particle size of2.5<D95≤3.8mm and an extraction time of2h. The major constituents of the essential oil analyzed by gas chromatography/mass spectrometry (GC-MS) were methyleugenol,3,5-dimethoxytoluene and safrole.The minimum inhibitory concentrations of essential oil against the three periodontal pathogens (Fusobacterium nucleatum, Prevotella intermedia, Porphyromonas gingivalis) and Streptococcus sanguinis were tested by broth-dilutions method and spread plate method. The minimum inhibitory concentration and minimum bactericidal concentration of oil were0.01and0.02%against Fusobacterium nucleatum,0.04and0.08%against Prevotella intermedia,0.005and0.005%against Porphyromonas gingivalis and0.04%and0.32%against Streptococcus sanguinis. Next is to evaluation of the inhibitory effects of the essential oil from Asarum on alveolar bone loss in mice. Experimental group and negative control group both were infected with109colony-forming units of live F. nucleatum in100μL of PBS with2%carboxymethylcellulose by gavage at2-day intervals. Subsequently, the mice in the experimental group were given2%Asarum oil5times at3-day intervals while the mice in the negative control group only received2%carboxymethylcellulose in PBS. Mice in the blank control group were neither infected with F. nucleatum nor treated with essential oil. The results show that mice challenged with F. nucleatum alone showed obvious bone loss (P<0.01) during the experimental period reflecting the effects of F. nucleatum on periodontal disease. By contrast, mice received essential oil after F. Nucleatum inoculation showed a marked decrease (P<0.05) in bone loss.Evaluation of the Asarum safety in vivo, the mice were given20%Asarum oil in100μL for20days, and then making liver and kidney pathological slices. The result showed that experimental group and the blank control group were not obvious different.In conclusion, the essential oil had obvious antibacterial activity against three periodontal pathogens (Fusobacterium nucleatum, Prevotella intermedia, Porphyromonas gingivalis). Infected mice treated with essential oil showed significant reductions in bone resorption confirming the effectiveness of the essential oil against periodontal disease-causing F.nucleatum. Asarum oil could be a natural therapeutic agent for the treatment of periodontitis.