| BackgroundH. pylori, a helix-shaped, Gram-negative, microaerophilic, curved rod bacterium, which was cultured from intact areas of antral mucosa of human bionsv specimen in patient with chronic active gastritis by Warren and Marshall in 1983. This bacterium revoluted the gastroenterology in the past twenty years, it has been known to us as one of the human pathogens with highest prevalence around the world, attracine many scientists to make research. Recently years, H. pylori were also sperated and cultured from dental plaque and saliva respectively by Krajden and Ferguson. Oral H. pylori have become another investigative hot spot. Firstly, many ways, such as the rapid urease test, PCR, cultivation and so on, were used to detect bacteria from dental plaque, saliva, faeces, vomitus and various oral lesions. Secondly, many studies demonstrate oral H. pylori has a relationship to halitosis, glossitis, periodontitis. burning mouth syndrome, recurrent aphthous stomatitis, dental caries and other oral cavity diseases. Thirdly, H. pylori can inhabit in both oral cavity and stomach of the same person, they may be infected from each other. Ferguson, though soluble-protein electrophoresis and PCR-restriction endonuclease analysis (PCR-REA), confirmed that H. pylori from oral cavity and stomach in a patient was homologous. Fourthly, the reinfected H. pylori strain of some patients was homologous as last infection, it may be one of reasons H. pylori recur after eradication. Some foreign scholars found that patients infected with previous homologous H. pylori after one years' eradication by random primer PCR. Fifthly, people with poor health in oral cavity or dental patients have a lower H. pylori eradication rates. Last, some authors confirmed the current triple therapy can remove gastric H. pylori, but cannot remove oral H. pylori. It may have a link to the special "biofilm" in dental plaque.The point that human beings were the natural H. pylori infection resource, the stomach was the main habitation, were recognized by most scholars. The separation and cultivate of H. pylori from oral cavity suggest oral cavity is another habitation of H. pylori. First, H. pylori as one of the numerous microbes in the oral cavity, affecting whole or partial oral tiny ecosystem, can promote some oral disease occurrence and development. Second, oral H. pylori may be a important factor of gastric H. pylori infection and recurrence. So, we think, inhibiting the growth of oral H. pylori can make benefits for both oral and stomach. The fact that H. pylori increased resistance to antibiotic and the effect of clearing H. pylori with oral antibiotics is not ideal, suggests that the therapy way of clearing oral H. pylori is different from traditional triple therapy eradication gastric H. pylori. In China, traditional Chinese medical science with abundant herbal medicices and theroy is an indispensable part of medical care in people's life. Chinese herbal medicines are important raw materials of new drug development, have their advantages over Western drugs, including a lower price, a lower toxicity, less adverse reactions and less resistance. Therefore, we tried to screen a Chinese traditional medicine formula to produce toothpaste, buccal tablets or other oral care products, applying locally in oral cavity to inhibit the growth of oral H. pylori and maintain the balance of microecological environment, with effects of antibacterial, antiphlogistion, clear heat, detoxification, hemostatic, and pain relief, can prevent and cure gingivitis, periodontitis and other periodontal inflammtory diseases. Moreover, application locally in oral cavity cooperate with triple therapy, make the current H. pylori eradication of prevention and cure to be more perfect, so as to reduce the recurrence rates and stomach H. pylori.This research consists of three parts:the first, separating and cultivating H. pylori from oral cavity in college students and analysing its correlation with gingivitis, testing the drug susceptibility of seven antibiotics to separated oral H. pylori strains and discussing the drug mechanisms; The second part, screening the drug susceptibility of 50 kinds of traditional Chinese medicine to standard H. pylori strains and discussing medicinal properties, meridian tropism and efficacy of some traditional Chinese medicine with strong anti-H. pylori activity; The third part, screening three kinds of traditional Chinese medicine with a strong anti-H. pylori activity to prouduce a formula which can inhabit the growth of H. pylori and keep oral cavity health.Part I A investigation of oral Helicobacter pylori and its correlation with gingivitis and antibiotic drug susceptibilityObjective1. To survey H. pylori infection rate in the general population2. To analyse its correlaton betweem H. pylori infection rate and gingivitis.3. To test the drug susceptibility of metronidazole, clarithromycin, amoxicillin, furazolidone, levofloxacin, gentamicin and erythromycin seven antibiotics to separated oral H. pylori strainsMethods1. Population, college students from foshan institute of technology, ages range from 18 to 25, had obtained the informed consent (1)Questionnaire check exclude the following:①smoking;②systemic diseases, like diabetes, hemorrhagic disease, upper respiratory tract infection, etc.;③received periodontal therapy, antibiotics or nonsteroidal anti-inflammatory drug within the half-year;④obvious flossing and serious alveolar bone absorption, extensive caries, teeth displacement and same serious oral diseases; (2) General oral examination:experience clinical dentist asked the patient complaints, history, self-conscious symptom, checking depth of periodontal bag (PD), bleeding index (SBI) and gum index (GI) ruled out serious periodontal inflammatory disease, periodontitis was also excluded. Finally, subjects with or without gingivitis were separated.2. Specimen collection:A sterile curette removed supra- and sub-gingival plaque from regions of incisors, canines, premolars and molars, 1ml saliva also collected, fully mixed transferred to 2ml of phosphate buffered saline.3. Bacteria vaccination, isolation, cultivation, subcultivation, identification and save: 0.1ml fluid specimen was vaccinated in selective Columbia solid culture medium under a micro-aerobic environment. After 3 days, with macroscopic observation colonies, picked suspicious colonies, subcultivated, smear, gram stain, microscopically observation mycelium form, and had biochemical tests including urease experiment, touching enzyme experiment, oxidase experiment. Indentified stains were preserved in improved brinell broth glycerin liquid medium.4. Statistical method:The relationship between gingivitis and H. pylori positive rate was analyzed with stratified analysis chi-square (SPSS13.0) analysis.5. Antibiotic susceptibility experiment:Using Kirby-Bauer disc diffusion, the susceptibility of metronidazole, clarithromycin, amoxicillin, furazolidone, levofloxacin, gentamicin, erythromycin seven kinds of common antibiotics to oral separated H. pylori strains was tested. The bacteriostatic annulus radius was measured with vernier gauges. The antimicrobial susceptibility test criteria to judge the sensitive, intermediary and drug-resistant was referred to China pharmaceutical and biological products appraisal institute. Results1. Total 356 college students were enrolled in the survey, including 152 boys,204 girls.30.9%(110/356) students suffered from gingivitis.2.356 mixed samples from dental plaque and saliva of subjects were obtained.48 (13.5%,48/356) H. pylori strains were separated, cultivated, identified and preserved. H. pylori infection rates in subjects with gingivitis were 24.5 percent (28/110). H pylori infection rates in subjects without gingivitis were 8.5 percent (21/246). Subjects with gingivitis had a significantly higher H. pylori infection rate than that without gingivitis x,2= 16.7, P= 0.000 (P< 0.05).3. Of boys group, subjects with gingivitis had a H. pylori infection rate of 20.8% (11/53), subjects without gingivitis had a H. pylori infection rate of 7.1%(7/99), Subjects with gingivitis had a significantly higher H. pylori infection rate than that without gingivitis,χ2= 6.192, P= 0.013 (P< 0.05).4. Of girls group, subjects with gingivitis had a H. pylori infection rate 28.1%(16/57), subjects without gingivitis had a H. pylori infection rate of 9.5%(14/147). Subjects with gingivitis had a significantly higher H. pylori infection rate than that without gingivitis, x 2= 11.263, P= 0.001 (P< 0.05).5. The drug resistance rate of Metronidazole, clarithromycin, amoxicillin, furazolidone, levofloxacin, gentamicin and erythromycin's resistance was 83.3%(40/48),33.3%(16/48),14.6% (7/48),6.3%(3/48),29.2%(14/48),52.1%(25/48),27.1%(13/48), respectivelyConclusion1. Oral cavity is the habitant of H. pylori, under certain circumstances, gingivitis may provide a more suitable microenvironment, oral H. pylori may a promoted condition of gingivitis. But their specific relationship and mechanism need further research.2. Oral H. pylori have a high drug resistance rate to metronidazole, clarithromycin, amoxicillin, furazolidone, levofloxacin, gentamicin and erythromycin. PartⅡScreening test for anti-H. pylori activity of traditional Chinese herbal medicinesObjective:To evaluate the anti-H. pylori activity of 50 traditional Chinese herbal medicines in order to provide the primary evidence for their use in clinical practice.Methods:A susceptibility test of water extract from 50 selected traditional Chinese herbal medicines for in vitro H. pylori Sydney strain 1 was performed with broth dilution method. Anti-H. pylori activity of the selected Chinese herbal medicines was evaluated according to their minimum inhibitory concentration (MIC).Results:The water extract from Rhizoma Coptidis, Radix Scutellariae and Radix isatidis could significantly inhibit the H. pylori activity with their MIC less than 7.8 mg/mL, suggesting that traditional Chinese herbal medicines have anti-inflammatory and antibacterial effects and can thus be used in treatment of H. pylori infection.Conclusion:The major components and antibacterial mechanism of Rhizoma Coptidis, Radix Scutellariae and Radix isatidis were analyzed and discussed. Combining their disvantages of a lower price, a low toxicity and less adverse reactions, less resistance, Rhizoma Coptidis, Radix Scutellariae and Radix isatidis are the potential sources for the synthesis of new drugs against H. pylori.PartⅢscreening a Chinese medicine formula for inhibiting oral helicobacter pyloriObjective:To develop a preliminary anti-H. pylori traditional Chinese medicine formula which can inhibit the growth and reproduction of oral H. pylori though applying in oral cavity for long-term, so as to maintain the balance of oral ecology environment and keep oral health.Methods:Based MICs of traditional Chinese medicines, three kinds of compatible herbs in Chinese medicine with strong anti-H. pylori activity in vitro were chosed. MIC50,MIC75 and MIC90 were tested of oral separation H. pylori strains. According ing orthogonal design method with three different drug factors and three different concentrations levels,9 kinds of mixed liquid composed three different drugs with different concentrations levels were tested.Results:Radix scutellariae, Honeysuckle and Dandelion three kinds of Chinese medicines have strong anti-H. pylori activity in vitro. And these three have a good compatiblity in drug property and efficacy of Chinese medicine. Anti-H. pylori activity in vitro was strengthened after mixed these three.Conclusion:The formula consisted of Radix scutellariae, Honeysuckle, and Dandelion has a strengthened activity in anti-H. pylori and anti-inflammatory, also has functions of improving symptoms. This study provided a theoretical basis for clinical use in Chinese medicine and Western medicine. |
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