| Objective: Periodontitis is a common disease with the incidence of more than 80%. It is the main reason of clinical for adults tooth loss. Periodontitis is a disease caused by many factors, for example, plaque, bacteria and some metabolism productions. As the disease developing, periodontium is destructed , attachment is lost, periodontal pocket goes deepen , tooth becomes mobile and even lost. Therefore, it is the key for periodontal therapy to eliminate inflammations, promote the destroyed or absorbed alveolar bone regeneration and regain normal dental function. Nowadays, routine clinical treatment remains as basic periodontal treatment and supplemented with systematically or locally administered antibacterial medicine. But systematical use of antibiotics for a long time would lead to unpleasant side effects such as dysbacteriosis, the reaction of gastrointestinal tract. The relatively low effective concentration of antibiotics exists in periodontal pocket and persists for a short sustained period. The preparations of locally administered periodontal medicine are mouthrinse, films, softcream, paste, glue. The effective components are mainly metronidazole or tetracycline. Although locally administered medicine can avoid systematic side effects, the preparations still have shortcomings, such as unconvenient and so frequent usage, short effective period and drug-resistance. There is no ideal complex preparation which can not only eliminate inflammations and inhibit bacteria but also promote periodontal regeneration upto now. Recently, herbal medicine plays an important role in the periodontal treatment. Scholars found that Radix scutellariae could inhibit 11 kinds of bacteria such as porphyromonas gingivalis, actionbacillus actionmycetemcomitans and provotellaintermedins, etc. Rhizoma coptidis could inhibit periodontal bacteria. Researchers also found that Rhizoma drynariae could promote proliferation and attachment to root surface of cultured human gingival fibroblasts. Nowadays, the research on the local use of Chinese herbal medicine still remains in medical effects of single medicine, whereas complex prescription and its pharmaceutical mechanisms are still unknown. This study used diagnosis based on overall analysis of patients' condition, mixed Rhizoma coptidis, Radix scutellariae and Rhizona drynariae in the reasonable proportion, then made sustained-release preparation. We evaluated Shuanghuangbu sustained-release preparation's to eliminate inflammation, inhibit bacteria and promote periodontal regeneration in vivo and in vitro, and observed the activity of inhibiting on bacteria, animal experiment, cell culture and clinical application. This research provided experimental evidence in developing effective and practical Chinese herbal preparation on periodontal treatment. Methods 1 The dissolution rate of Shuanghuangbu sustained-release preparation in vitro Shuanghuangbu sustained-release preparation and Shuanghuangbu preparation was made respectively. They both contained Rhizoma coptidis, Radix scutellariae and Rhizona drynariae. Sets of standard solution of Baicalin and Beberine Hydrochloride were compounded and their peak chromatogram of at 280nm and 270nm separately, then their regression formulas could be figured out. The tests of precision and recovery were taken to the standard solution of Baicalin and Beberine Hydrochloride. HPLC method blasket stirring method was used to determin the contents and the dissolution rate of them. Shuanghuangbu sustained-release preparation was weighted accuratelly and put in a flask contained distilled water. The stirring speed was 30r/min(37±0.5)℃. Samples were taken out respectively at different time. The dissolution rates of Baicalin and Beberine Hydrochloride was determined and the curves were designed about relation of dissolution rate and time. 2 The release of Shuanghuangbu sustained-release preparation in vivo Seven patients (male 3,female 4) with moderate to severe periodontitiswere enrolled. The subjects had at least one periodontal pocket with probing depth of 4mm to 9mm and bleeding on probing gently on each side of the mouth. Sixteen sites were divided into experiment and control groups. One week after the initial the rapy, we put Shuanghuangbu sustained-release preparation and Shuanghuangbu preparation respectively in periodontal pocket, then collected GCF sample at different time to determine the concentration of Beberine Hydrochloride and Baicalin in GCF. 3 The study of Shuanghuangbu sustained-release preparation in experimental regeneration of periodontium Thirty-two teeth of four beagle dogs were randomly divided into the experiment group and the control group. Shuanghuangbu sustained-release preparation was applied to periodontal defect in the experimental group and no disposition of the defect for the control group. The specimens were obtained from one month to three months postoperatively. In addition, two teeth of every dog were chosen as the normal group and did not give any disposition. The following histological observations and measurements were made: ①the histological manifestation of periodontal tissue and root resorption; ②the measurements of the buccal and lingual surfaces of each root: experimental defect (ED), new bone formation (NB), new cementum formation (NC) and the length of junctional epithelium (JE); ③the expression of BMP2,IGF1,bFGF and FN. 4 The effects of Shuanghuangbu on prolioferation, ALP activity and protein synthesis of periodontal ligament cells 1) Shuanghuangbu decoction preparation: A mixture of Rhizoma coptidis, Radix scutellariae and Rhizoma drynariae was soaked in water, boiled twice and filtered, made the concentration of 1g/ml Shuanghuangbu decoction. And then filtered sterilization, diluted to final concentrations respectively, modified the pH to 7.0～7.1, divided and stored at 4℃. 2) Cell culture: Human periodontal ligament cells were isolated from the mid-root of 2 premolars extracted due to orthodontic therapy from 1 adolescent patient (12 years of age) with healthy periodontium. Theperiodontal ligament tissue was mechanically removed under sterile conditions by scraping the middle third of the root surface with a sharp blade, according to the method of Situzhenqiang. Using the second passage of cells to identify. 3) ALP activity assay and total protein content measurement: The fifth passage cells were incubated in 96 wells plate, cultured 24 hours, randomizely divided into experiment group and control group, added decoctions to each well, using Triton X-100 to destroy the cellmembranes, then using PNPP method and Commasie brilliant blue stain to measure the ALP activity and protein content. 4) Scanning electron microscopy(SEM): The fifth passage cells were incubated in 6 wells plate, cultured 24 hours, randomizely divided into experiment group and control group, added decoctions to each well, washed twice ,fixed in cold glutaraldehyde, post-fixed in osmium tetroxide, dehydrated in an ethanol series, critical-point dried, coated with gold, then observed. 5) Transmission electron microscopy(TEM): Both the experimental group and control group were fixed in glutaraldehyde, post-fixed in osmium tetroxide, dehydrated in an ethanol series, embedded in Epoxy resin, then observed. 5 The susceptibility of three periodontopathic bacteria to Shuanghuangbu decoction in vitro A mixture of Rhizoma coptidis, Radix scutellariae and Rhizoma drynariae was soaked in water, boiled and filtered. Decoctions were mixed, heated and concentrated to 1:1 Shuanghuangbu decoction. The Millex-GP filter was used to filter sterilization the Shuanghuangbu decoction. Rhizoma coptidis decoction, Radix scutellariae decoction and Rhizoma drynariae decoction were made in the same methods. Porphyromonas gingivalis, Provotella intermedins and Fusobacterium nucleartum were chosen as the experimental bacteria. The liquid media two-fold dilution method was used to measure the minimum inhibitory concentration (MIC) of Shuanghuangbu decoction, Rhizoma coptidis decoction, Radix scutellariae decoction andRhizoma drynariae decoction. Schaedler broth was used to dilute 2-fold the Chinese herbal medicines decoction in the test-tubes. The final concentration of the drugs ranged from 1:2 to 1:1024. Periodontopathic bacteria to be used in the MIC test were added to the experimental test-tubes and the positive control test-tube. finally, the bacteria concentration of each test-tube must reach 1×106 CFU/ml. The test-tubes were then incubated anaerobically for 48hr. The MIC was taken as the lowest concentration of the medicine that completely inhibited growth. The digging holes agar diffusion method was used to measure the diameter of inhibition ring of Shuanghuangbu decoction on Porphyromonas gingivalis, Provotella intermedins and Fusobacterium nucleartum, 2% organidin was chosen as a control medicine. 6 Clinical study of Shuanghuangbu sustained-release preparation in chronic periodontitis Thirty-one healthy adults with moderate to advanced chronic periodontitis (male 15, female 16) participated in this study. The subjects had at least one periodontal pocket with probing depth of 5mm to 9 mm and bleeding on probing gently on each side of the mouth. This was a randomized, split mouth design study. The 120 target sites were randomized by left-side and right-side into one of the two groups (the test group and the control group). Subsequently, each patient was scheduled for two appointments for the full-mouth supra-gingival scaling and oral hygiene instruction. At baseline all target sites were taken gingival crevicular fluid (GCF) for measuring the levels of alkine phosphatase (ALP), aspartate aminotransferase (AST) and lactate dehydyogenase (LDH), and subgingival plaque was sampled from each of target sites for determining the percent of spirochetes (S%). Three clinical parameters including probing depth, attachment loss and bleeding on probing were examined for each site. At baseline all the patients received subgingival scaling and root planning. The test group was followed by 4-time applications of Shuanghuangbu sustained-release preparation with three-day interval for totally two weeks, and no medicine was applied in the control group. On day 14, the examination was the same as the procedure of baseline, On day 41, thetarget sites were examined based on three clinical parameters and taken subgingival plaque. Results 1 The dissolution rate of Shuanghuangbu sustained-release preparation in vitro Baicalin had a good linearity(r=0.9997) in the range of 4.59μg/ml～114.80μg/ml. Beberine Hydrochloride had a good linearity(r=0.9999) in the range of 2.52μg/ml～50.40μg/ml. Addition to 5 batches and 3 batches which were determined, the content of Baicalin can not be less than 1.63% and the content of Beberine Hydrochloride can not be less than 0.564%. Baicalin of Shuanghuangbu sustained-release preparation released early, quickly and its releasing continuous time was short, the whole releasing time was fewer 3 hours than that of Shuanghuangbu preparation. Beberine Hydrochloride of Shuanghuangbu sustained-release preparation released lately, slowly and its releasing continuous time was longer, the whole releasing time more was 35 hours than that of Shuanghuangbu preparation. 2 The release of Shuanghuangbu sustained-release preparation in vivo A half hour after the Shuanghuangbu sustained-release preparation was put into the pocket, the concentration of Beberine Hydrochloride and Baicalin in GCF was 303.41μg/ml and 168.55μg/ml; 2.5h later, the concentration of the two ingredients decreased rapidly to 15.99μg/ml and 3.00μg/ml, When it came to the trial group, the concentration of Beberine Hydrochloride and Baicalin in GCF was 1177.03μg/ml and 1474.40μg/ml respectively, a half hour after Shuanghuangbu sustained-release preparation was applied; 24 hours after the application, the concentration of Beberine Hydrochloride in GCF was 142.79μg/ml ,while it of Baicalin dropped to 2.12μg/ml; 96 hours after the preparation was used, the concentration of Beberine Hydrochloride was 9.32μg/ml. 3 The study of Shuanghuangbu sustained-release preparation in experimental regeneration of periodontium Histological observation: Inflammatory cells were not observed inperiodontal tissue of experimental group and substantial bone and cementum regeneration was observed from the apical defect to coronal. Periodontal tissue had many osteoblast cells and cementoblast cells. There were many blood vessels and no obviously fibrous tissue in periodontium. There were a large number of inflammatory cells in connective tissue of control group. New bone and cementum and periodontal ligament was not obviously observed in control group. Root resorption was observed in this group. Result of histometric measurements: Height of NB and NC in experiment group on one month and three months were obviously larger than that in control group respectively. The length of JE in experimental group was less than that in control group on one month after surgery; but there was no significant difference in the two groups on three months after surgery. Expression of cytokines in periodontal tissue: The positive rates of BMP2 in gingival tissue and periodontal tissue of the experimental group on one month were larger than those of the control group and the normal group; the positive rate of BMP2 was enhanced in the experimental group on three months than that of the control group and the normal group; the two positive areas of the control group were larger than those of the normal group on one month; but there was no significant difference between the control group and the normal group on three months. The positive rates of IGF1 in gingival tissue and periodontic tissue of the experiment group on one month and on three months were obviously larger than those of the control group. There was no significant difference of the positive rate of bFGF between the experiment group and control group. There was expression of FN only in the experiment group. 4 ALP activity and protein content were measured by PNPP method and Commasie brilliant blue stain,there were significant differences between the experimental group and control group. Under the SEM, the quantity of cells in the experiment group was lager than in the control group. Under the TEM, the experiment group exhibited more number of bigger cells than that of the control group, Bundles ofmicrofilaments were uniformly distributed throughout the cytoplasm. Free ribosomes and rough endoplasmic reticulum cisternae were interspersed throughout the cytoplasm. Mitochondria appeared normal and abundant in quantity. 5 The susceptibility of three periodontopathic bacteria to Shuanghuangbu in vitro Against Porphyromonas gingivalis, Provotella intermedins and Fusobacterium nucleartum, the diameter of inhibition ring of Shuanghuangbu decoction was 17～19mm, 20～22mm and 22～24mm; while the diameter of inhibition ring of 2% organidin was 15～17mm, 17～19mm and 18～20mm. Against Porphyromonas gingivalis, MICs of Shuanghuangbu decoction, Rhizoma coptidis decoction, Radix scutellariae decoction and Rhizoma drynariae decoction was 1:128, 1:256, 1:8 and 1:8 respectively; against Provotella intermedins, MICs of Shuanghuangbu decoction, Rhizoma coptidis decoction, Radix scutellariae decoction and Rhizoma drynariae decoction was 1:128, 1:512, 1:8 and 1:8 respectively; against Fusobacterium nucleartum, MICs of Shuanghuangbu decoction, Rhizoma coptidis decoction, Radix scutellariae decoction and Rhizoma drynariae decoction was 1:128, 1:256, 1:16 and 1:16, respectively. 6 Clinical Study of Shuanghuangbu sustained-release preparation in Chronic Periodontitis At baseline, there was no statistically significant differences between the experiment and the control group in clinical parameters, therthe levels of three enzymes in GCF and the percent of spirochetes. The results of experiment and control group showed a decrease in PD and AL on day 14 and 41. On day 14 and 41 there was a significant decrease in BOP(+)% in the expere was no significant difference among imental group compared to that at baseline. Whereas BOP(+)% on day 14,41 and baseline showed no statistically significant differences in the control group. On day 14 the percent of coccoid cells increased significantly, and the percent of spirochetes and rods was significantly less in the both groups. The results of experiment group exhibitedgreater differences than the control group. At the 41st days S% maintained the level of day 14 in the experiment group, whereas S% in the control group rebounded. On 14 days the levels of GCF-AST, GCF-ALP and GCF-LDH were significantly lower than baseline in the two groups, and the decrease of the levels of the three enzymes was greater in the experiment group than the control group. Conclusions 1 Shuanghuangbu sustained-release preparation was consist of two parts which were fast releasing layer and slow releasing layer, which can release slowly in periodontal pockets and maintained the effective concentration above 4 days to inhibit bacteria in periodontal pockets. The release of Beberine Hydrochloride was more slowly than that of Baicalin. So it was a good Chinese herbal medicine local sustained-release preparation. 2 Shuanghuangbu sustained-release preparation can inhibit inflammation effectively and restraint migration of junctional epithelium evidently. Shuanghuangbu sustained-release preparation also can promote the new bone and cementum formation obviously and can increase the expression of BMP2 and IGF1 in the periodontal tissue, and stimulate periodontal ligament cells (PDL-C) to differentiate into metaplastic osteoblasts, and enhance the regeneration of alveolar bone. 3 Shuanghuangbu stimulated the proliferation, ALP activity and protein synthesis significantly. 4 Shuanghuangbu decoction had showed obvious effect on inhibiting Porphyromonas gingivalis, Provotella intermedins and Fusobacterium nucleartum in vitro. 5 Shuanghuangbu sustained-release preparation can significantly improve the clinical symptom of periodontitis and change the composition of subgingival microflora at the disease sites to that at healthy sites.
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