| ObjectivePeriodontal disease is a common ailment and frequently occurring illness in oral cavity.It is a bacterial infection disease occurring in periodontally supporting tissue, and it is themost important reason that causes pathological teeth-losing in adults. Suspiciousperiodontally pathogenic bacteria is the main causative agent of periodontal disease, and theinitiation factor to the chronically destructive disease in dental capsule. The chief way toprevent and cure periodontal disease is to eliminate the causative agent and obstruct itsformation and development. For instance, Curettage around gum or drug treatment cancontrol the bacterial plaque. For many years, many scholars have done considerable studyon the cause, treatment and precaution from the perspective of Chinese Medicine. Andsince 1980s, the use of naturally occurring drugs in preventing and treating oral disease hasincreasingly drawn the attention of the scholars both abroad and at home.This study shows the clinical and experimental research with respect to theprescriptional extract from Chinese crude drug, which is based on the empirical formularecommended by Professor Gan Zuwang, a famous TCM doctor. And a survey onperiodontal health-status has been made on the basis of Basic Approaches to the Survey ofPeriodontal Health by the WHO.To gain a profound understanding of the situation of periodontal health, so as to collectbasic information,and further demonstrate that searching for and improving the medicinethat can prevent and cure periodontal disease is very meaningful. To discover the functionof prescription extract on reducing bacterial plaque and find a proper medication method. To examine the bactericidal effect of the exact on relative pathogenic bacteria. To observethe influence of the exact with different density on the multiplication of HPLF and on thechange of total protein level. And to observe its effect on the form and theultramicrostructure of the HPLF. All these will help provide experimental data for the studyof preventing and treating mechanism for periodontal disease from the aspect of ChineseMedicine.MethodsThis study includes the following three parts: experimental research, clinical researchand epidemiologic research.1.Experimental researchThe prescriptional Exact: It is based on the empirical formula recommended byProfessor Gan Zuwang, which includes honeysuckble, herba, rhizome and phragmitis. Thecrude drug is from the pharmaceutical division of the Jiangsu Provincial Hospital of TCM.The crude drug in 5 times amount of time is extracted three times. All the colature isfiltered, then the 90% ethanol is added, and the precipitation is removed. And the clearsupernatant liquid is chosen, and is condensed to 1 g/ml, which is the stock solution that isneeded in the study. And the stock solution is prepared for use after being in boiling waterfor 10 minutes.Experimental strain and the nutrient medium: They are of international standard,which include Porphyromonas gingivalis （ATCC 33277）, Fusobacterium nucleatum （ATCC25586）, and Actinobacillus actinomycetemcomitans （ATCC Y4）. They are cultivated byserial subcultivation with profession anaerobian medium. Streptococcus mutans（NCTCIngbritt） is from serial subcultivation with saliva mudium.Cell Culture: Take a bicuspid tooth for a youngster between 10 to 16 who has amisshapen problem but has no periodontal disease or odontosphacelism, and put it in thecooled DMEM mudium which has in it penicillin of 100U/ml and phytomycin of 100μg/ml.In the germ free condition, on the clean working table, one third of the tissue ofalveolodental ligament in the dental root is scraped and cut into small pieces of 1 mm×1 mm×1mm, then displayed in a culture flask of 25ml volume. Put into the flask 2 ml DMEMnutrient medium including 20% serum, 100U/ml penicillin and phytomycin 100μg/ml, andplace it into CO2 incubato, with a fine condition of 37℃and 5%CO2, for development. After 4 hours, reverse the flask for development. When cell emigration appears, the culturefluid is changed every 3 other days. When the flask bottom is full of cells, trypsinization,the density of 0.25%, helps its serial subcultivation. Those cells from the 4th generation tothe 10th generation, which are negative in ceratin and positive in vimentin, are used in theexperiment.The preparation of the drug extract with different density: Dilute a lg/ml stocksolution with DMEM medium of 5% serum, into 0g/ml（control group）, 1×10-7g/ml,1×10-6g/ml、1×104g/ml、1×10-4g/ml、1×10-3g/ml、1×10-2g/ml, and develop them in differentconditions.The operation of extraorgan sensitivity test: the TSA plate cultivation is adopted, anaerobicculture （90%N2 5%CO2 37℃） lasted 18 hours. Reanimate the germs survived and inoculatethem at agar plate. Pick out a single coenobium from its surface and suspend it a respectiveliquid medium for cultivation （one 1mm coenobium per ml）. When the density comes upto 105-6CFU/ml, it is ready for use.Two sorts of sample liquid are imbibed. The final liquid is from the two-fold dilutionto the stock solution （1:1, 1:2,1:4......）. In the extraorgan sensitivity test, microamountliquid dilution is taken to test minimal bactericidal concentration （MBC）.MBC Test: by using aseptic inoculation loop, pick out some culture in the microwellplate that cannot be seen by naked eyes. And inoculate them in the respective plates, andput them in proper conditions for incubation. After 48 hours, observe them again, MBC isthe minimum drug density where no bacteria grow.MIT method tests cell multiplication: Cells from the 5th generation, digested byparenzyme of 0.25%, are inoculated in 96 shadow mask at the density of 1×105/ml, 100μlper mask. After 24 hours, remove the previous culture fluid. And then put into themconditioned medium 200μl.8 masks for each level of density. After cultivated for one day,three days, and five days respectively, put into each mask MTT （Smg/ml） 20μl. Aftercultivated for four hours under saturation condition of 37℃, 5%CO2, remove the culturefluid, put into each mask DMSO150μl. Vibrate them for ten minutes, and test its OD atbiocatalyst scale meter, with the wave length 490nm. The nutrient medium with only 5%blood serum is the control group.The staining method of Coomassie brilliant blue tests cell total protein: Cells from the5th generation, digested by parenzyme of 0.25%, are inoculated in 96 shadow mask at thedensity of 1×105/ml, 100μl per mask. After 24 hours, remove the previous culture fluid. And then put into them conditioned medium 200μl(excluding 1×10-2g/ml). 5 masks foreach level of density. After cultivated for one day, three days, and five days respectively,put into each mask 100μl0.1%TritonX-100. Vibrate them for 30 minutes, and shift them toanother 96 shadow mask. Put into each mask the liquid of Coomassie brilliant blue 200μl,vibrate them for 10 minutes, and test the OD with spectro photometer at the wave length of595nm. The nutrient medium with only 5% blood serum is the control group.Ultramicrostructure: Cells from the 5th generation, digested by parenzyme of 0.25%,are inoculated into a new culture flask. After 24 hours, remove the culture fluid. Put into itconditioned medium at 1×10-3g/m for 5 days. 5%DMEM Medium with no drug extract isthe control group. On the 5th day, digest it with 0.25%parenzyme. And collect it in somecentrifuge tubes, centrifugalize them for 10 minutes at the speed of 1000r/min. Thenremove the clear supernatant liquid. Fix the cell aggregate with 2% glutaral for 2 hours,dehydrate it at room temperature with series acetone. Embed it with EPON812, observe itwith transmission electron microscope after extra thin section.2. Clinical researchPrescriptional Extract is prepared into the following 4 groups: low-drug-density group（0.2g/ml, diluted with distilled water）, high-drug-density group （0.4g/ml, diluted withdistilled water）, and positive control group （half the density of KouTai）, and negativecontrol group （distilled water）. Double blind method is utilized for experimentalobservation. Those who provide collutory don’t join for the check. The workers who arecarrying out the periodontal index inspection are the trained stomatological doctors. Andthose who provide children with collutory and supervise them are the kindergarten teachers.Before the experiment, stomatological doctors inspect the periodontal index of each child.And then the teachers arrange for them to rinse the mouth twice a day for two weeks. Eachtime, 10ml is used for mouthwash, and 2 minutes later, clean the mouth. One hour after thelast mouthwash, stomatologicaI doctors inspect the periodontal index again. In the wholeprocess of the experiment, the children don’t change their daily life habit. Test theperiodontal index, including the plaque index （PLI）, and the gingival index （GI）3. Epidemiologic researchOn the basis of the surveying method in the Basic Approaches to the Survey ofPeriodontal Health by the WHO, this research involves the following features asmulti-stage, stratification, equal capacity, and random sampling. 1584 people in JiangsuProvince are divided into two age-groups of 35-44 and 65-74. Half of them are men and from countryside.Result1.The drug extract has bactericidal effect on every sort of bacterium in theexperiment. It’s MBC value on porphyrin unit-cell bacterium, actinobacillusactinomycetem comitans, fusobacterium nucleatum and streptococcus mutans arerespectively 7.8125,3.90625,7.8125 and 7.8125 mg/ml2.The drug extract can noticeably improve the generation of HPLF at the density ofbetween 1×10-7g/ml and 1×10-3g/ml. Compared with the control group, it has significantdifference. And it functions the best at 1×10-3g/ml. At the density of between 1×10-4g/mland 1×10-3g/ml, it can noticeably increase the composition of the total protein ofcollagenoblast. Compared with the control group, it has significant difference. Within thisrange, the function of drug extract shows a clear density and time-reliance effect.3.Compared with the control group, the endocytoplasmic reticulum and the bioblast ofthe cells in the experimental group show a clear increase.4.The use of gargarism that has such ingredients as honeysuckble and Tokyo violetherb can help reduce the periodontal index, which shows highly significant difference,compared with the control group.5.（1）The detection rate and average segment of periodontal health of the citizens in therural area and the city are only 0.70% and 0.50.67.74% and 1.78 gingiva bleeding. 93.93%and 4.59 calculus dentalis. 32.07% and 0.56 for shallow periodontal pocket; 7.46% and0.10 for deep periodontal pocket.（2） The detection rate and average segment of periodontal pocket in the age group of65-74 is higher than that in the age group of 35-44 （P〈0.01 ）（3） The detection rate and average segment of the periodontal attachment whosedeprivation scores are 2, 3 and 4 in the age group of 65-74 is higher than that in the agegroup of 35-44 （P〈0.01）.Conclusion1. The prescriptional Exact at the density of 7.8125 mg/ml can effectively killperidontal bacterium without causing damage to the regional peridontally eubiosis 2. The prescriptional Exact can help the generation and protein synthesis of HPLF.3. The prescriptional Exact can promote corpuscular metabolism and proteinaceouscomposition, which has the same result as the test in cell multiplication experiment and thetotal protein test.4. The prescriptional collutory of honeysuckble and Tokyo violet herb can effectivelyremove Tokyo violet herb, and shows antibiosis and antiphlogosis.5. Antiphlogosis is a common and frequently encountered ailment. Calculus dentalis isanother important issue that the dental hygiene is faced with. So that it is an obligation tointensify the prevention of periodontal disease, to offer dental hygiene service incommunity and to increase dental health of all the citizens, and further demonstrate thatthere is a great prospect to search for the medicine that can prevent and cure periodontaldisease and put the medicine into treatment. |
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