双黄补煎液对三种常见牙周致病厌氧菌体外抑菌活性的研究
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摘要
目的：牙周炎是由菌斑微生物引起的感染性疾病，菌斑细菌是牙周炎发生的始动因子，其特征为多种条件致病菌混合感染的炎症。当前研究表明牙龈紫质单胞菌是牙周炎证据充分的致病菌，而中间普氏菌和核梭杆菌则是与牙周炎中度相关的致病菌。各种牙周致病菌可通过各种酶类，毒素及代谢产物作用于牙周组织，造成牙周组织的破坏和牙周炎的发生, 所以牙周炎治疗成功的关键是菌斑控制，除常规牙周基础治疗之外，抗菌药物的全身或局部应用也是治疗的重要环节。全身及局部应用抗菌药物对于各种牙周致病菌均有很好的抑制作用，但由于全身用药可引起全身菌群失调，胃肠道反应等诸多副作用，所以牙周局部用药是当前牙周炎药物治疗研究的重点。中草药在我国研究应用历史悠久，许多种中草药均有比较明显的抑菌杀菌作用，同时中草药有资源丰富，价廉易得，毒副作用低等诸多优点，所以开发安全有效的牙周局部中药制剂治疗牙周炎越来越引起人们的重视。双黄补缓释药条是一种中药牙周局部缓释剂，其主要成分为黄连，黄芩和骨碎补。在临床应用中发现双黄补缓释药条可有效改善牙周炎的临床症状，控制炎症；能够降低牙周致病杆菌在牙周炎发病部位的存在比例，使其龈下微生物的组成向健康部位的菌群组成转
变。本研究主要采用液体二倍稀释法和打孔琼脂扩散法来体外检测中药双黄补煎液及其主要成分黄连、黄芩和骨碎补煎液对牙龈紫质单胞菌，中间普氏菌和核梭杆菌等三种牙周致病厌氧菌的抑菌活性。
方法：将黄连100g，黄芩50g和骨碎补50g混合后加水8倍浸泡30min，缓慢加热至103℃保持45min后，将药液用100目筛过滤。再将药渣重新加水，加热至103℃保持45min后，100目筛过滤除渣。合并两次滤液加热浓缩成为浓度为1:1（相当于每毫升药液含生药1g）的双黄补煎液200ml。用孔径为0.22μm的Millex-GP滤菌器过滤除菌。应用同样方法制备黄连煎液，黄芩煎液和骨碎补煎液，浓度均为1:1。实验菌株采用牙龈紫质单胞菌Pg ATCC33277，中间普氏菌Pi ATCC25611和核梭杆菌Fn ATCC10953，均为冻干粉保存。将实验菌株接种于斯氏血液琼脂培养基和脑心浸汤在厌氧环境下复苏。采用液体二倍稀释法来体外检测双黄补煎液及其主要成分黄连煎液、黄芩煎液和骨碎补煎液对牙龈紫质单胞菌，中间普氏菌和核梭杆菌三种牙周致病菌的抑菌活性。选择用斯氏肉汤液体培养基在试管内将中药煎液由高到底依次稀释为1:2 , 1:4 , 1:8 , 1:16 , 1:32 , 1:64 , 1:128 , 1:256 , 1:512 , 1:1024共10个药物浓度。同时设立2支仅加斯氏肉汤的试管分别作为阳性对照管和阴性对照管。配置厌氧菌菌悬液并用比浊仪校正浓度，将已校正浓度的厌氧菌菌悬液依次加入阳性对照管和抗菌药物实验管，最终每管菌浓度为1×106CFU/ml，37℃厌氧培养48h。观测其最小抑菌浓度（minimal inhibitory
concentration, MIC）。采用打孔琼脂扩散法，使用2%碘甘油为对照组，双黄补煎液为实验组，观察两种药液对牙龈紫质单胞菌，中间普氏菌和核梭杆菌的体外抑菌活性。制备4个斯氏血液琼脂培养平板，采用直径为6mm的自制打孔器打孔，每个培养平板上各打5孔，实验组与对照组各2个培养平板，每组各10孔。配置厌氧菌菌悬液，校正浓度为1.5×108CFU/ml。将已制备好的菌悬液均匀涂布于平板上，每孔内加入抗菌药物100μl，37℃厌氧培养48h。利用卡尺测量抑菌环直径。采用配对t检验的方法进行统计学处理。
结果：对于牙龈紫质单胞菌，双黄补煎液、黄连煎液、黄芩煎液和骨碎补煎液的最小抑菌浓度分别为1:128, 1:256, 1:8和1:8；对于中间普氏菌，双黄补煎液、黄连煎液、黄芩煎液和骨碎补煎液的最小抑菌浓度分别为1:128, 1:512, 1:8和1:8；对于核梭杆菌，双黄补煎液、黄连煎液、黄芩煎液和骨碎补煎液的最小抑菌浓度分别为1:128, 1:256, 1:16和1:16。双黄补煎液对牙龈紫质单胞菌、中间普氏菌和核梭杆菌的抑菌环直径分别为17~19mm、 20~22mm和22~24mm, 而对照组2%碘甘油的抑菌环直径分别为15~17mm、17~19mm和18~20mm，统计学处理结果显示双黄补煎液对牙龈紫质单胞菌、中间普氏菌和核梭杆菌的抑菌作用均比2%碘甘油药液效果更好（P<0.001）。
结论：1 双黄补煎液在体外对牙龈紫质单胞菌、中间普氏菌和核梭杆菌三种牙周厌氧致病菌均有比较明显的抑菌活性。
2 在双黄补煎液的三种组成成分中，黄连在体外对牙龈紫质单胞菌，中间普氏菌和核梭杆菌三种牙周厌氧致病菌显示出了明显的抑菌活性，其中对中间普氏菌的抑菌活性最为明显；而黄芩与骨碎补在本实验中也表现出一定的抑菌活性。
3 双黄补煎液在体外对牙龈紫质单胞菌、中间普氏菌和核梭杆菌三种牙周厌氧致病菌的抑菌活性均比2%碘甘油药液更明显。
Objective: Periodontal diseases are considered as infection of the periodontium due to subgingival pathgens. The characteristic is the infection of opportunistic pathgens. Porphyromonas gingivalis (Pg), Provotella intermedins (Pi) and Fuso-bacterium nucleartum (Fn) are believed very important for the emergence of periodontal diseases, They can produce lots of virulence factors such as endotoxin, proteas, ammonia, indole, etc. These virulence factors can destrust periodontium and caus periodontal diseases. The control of plaque is the key to periodontal therapy. There were widely study in the use of antibiotics and antimicrobials in the therapy. Systemically or locally administered bacteriocidal drugs had significant effects on inhibiting periodontopathogens. But the Systemical use of antibiotics and antimicrobials for a long time would lead to unpleasant side effects such as dysbacteriosis, the reaction of gastrointestinal tract. Therefore, the locally administered bacteriocidal drug in the therapy was more and more important. Chinese herbal medicine have been used for a long time. Many Chinese herbal medicines have exhibited strong inhibition on periodontopathogens. So, there was
increasing emphasis on researching safe and effective Chinese herbal medicines locally administered in periodontal pockets. Shuanghuangbu was a new double-layered local Chinese herbal delivery device, which mainly contained Rhizoma coptidis, Radix scutellariae and Rhizoma drynariae. We have found the Shuanghuangbu could significantly improve the clinical symptom of periodontitis and might change the composition of subgingival microflora at the disease sites to that at the healthy sites. The purpose of this study was using the liquid media two-fold dilution method and the digging holes agar diffusion method to research the effect of Shuanghuangbu decoction, Rhizoma coptidis decoction, Radix scutellariae decoction and Rhizoma drynariae decoction on Porphyromonas gingivalis, Provotella intermedins and Fusobacterium nucleartum.
Methods: A mixture of Rhizoma coptidis, Radix scu-tellariae and Rhizoma drynariae was soaked in water for 30 minutes , boiled for 45 minutes and filtered; The residue was reboiled with water and refiltered. All decoctions were mixed, heated and concentrated to 1:1 Shuanghuangbu decoction ( 1ml Shuanghuangbu decoction made from 1g of raw material ). 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. These periodontopathogens were cultured on Schaedler broth agar culture medium or in Brain-heat infusion broth. 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 and Rhizoma drynariae decoction on Porphyromonas gingivalis, Provotella intermedins and Fusobacterium nucleartum. 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. Two test-tubes that only contained Schaedler broth were used as a positive control and a negative control, respectively. Periodontopathic bacteria to be used in the MIC test was added to the test test-tubes and the positive control test-tube, finally, the bacteria concentration of each test-tube must became to 1×106 CFU/ml. The test-tubes were then incubated anaerobically at 37℃ for 48hr. The MIC was taken as the lowest concentration of the drugs 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 a control drug. Made four Schaedler broth bloo
变。本研究主要采用液体二倍稀释法和打孔琼脂扩散法来体外检测中药双黄补煎液及其主要成分黄连、黄芩和骨碎补煎液对牙龈紫质单胞菌，中间普氏菌和核梭杆菌等三种牙周致病厌氧菌的抑菌活性。
方法：将黄连100g，黄芩50g和骨碎补50g混合后加水8倍浸泡30min，缓慢加热至103℃保持45min后，将药液用100目筛过滤。再将药渣重新加水，加热至103℃保持45min后，100目筛过滤除渣。合并两次滤液加热浓缩成为浓度为1:1（相当于每毫升药液含生药1g）的双黄补煎液200ml。用孔径为0.22μm的Millex-GP滤菌器过滤除菌。应用同样方法制备黄连煎液，黄芩煎液和骨碎补煎液，浓度均为1:1。实验菌株采用牙龈紫质单胞菌Pg ATCC33277，中间普氏菌Pi ATCC25611和核梭杆菌Fn ATCC10953，均为冻干粉保存。将实验菌株接种于斯氏血液琼脂培养基和脑心浸汤在厌氧环境下复苏。采用液体二倍稀释法来体外检测双黄补煎液及其主要成分黄连煎液、黄芩煎液和骨碎补煎液对牙龈紫质单胞菌，中间普氏菌和核梭杆菌三种牙周致病菌的抑菌活性。选择用斯氏肉汤液体培养基在试管内将中药煎液由高到底依次稀释为1:2 , 1:4 , 1:8 , 1:16 , 1:32 , 1:64 , 1:128 , 1:256 , 1:512 , 1:1024共10个药物浓度。同时设立2支仅加斯氏肉汤的试管分别作为阳性对照管和阴性对照管。配置厌氧菌菌悬液并用比浊仪校正浓度，将已校正浓度的厌氧菌菌悬液依次加入阳性对照管和抗菌药物实验管，最终每管菌浓度为1×106CFU/ml，37℃厌氧培养48h。观测其最小抑菌浓度（minimal inhibitory
concentration, MIC）。采用打孔琼脂扩散法，使用2%碘甘油为对照组，双黄补煎液为实验组，观察两种药液对牙龈紫质单胞菌，中间普氏菌和核梭杆菌的体外抑菌活性。制备4个斯氏血液琼脂培养平板，采用直径为6mm的自制打孔器打孔，每个培养平板上各打5孔，实验组与对照组各2个培养平板，每组各10孔。配置厌氧菌菌悬液，校正浓度为1.5×108CFU/ml。将已制备好的菌悬液均匀涂布于平板上，每孔内加入抗菌药物100μl，37℃厌氧培养48h。利用卡尺测量抑菌环直径。采用配对t检验的方法进行统计学处理。
结果：对于牙龈紫质单胞菌，双黄补煎液、黄连煎液、黄芩煎液和骨碎补煎液的最小抑菌浓度分别为1:128, 1:256, 1:8和1:8；对于中间普氏菌，双黄补煎液、黄连煎液、黄芩煎液和骨碎补煎液的最小抑菌浓度分别为1:128, 1:512, 1:8和1:8；对于核梭杆菌，双黄补煎液、黄连煎液、黄芩煎液和骨碎补煎液的最小抑菌浓度分别为1:128, 1:256, 1:16和1:16。双黄补煎液对牙龈紫质单胞菌、中间普氏菌和核梭杆菌的抑菌环直径分别为17~19mm、 20~22mm和22~24mm, 而对照组2%碘甘油的抑菌环直径分别为15~17mm、17~19mm和18~20mm，统计学处理结果显示双黄补煎液对牙龈紫质单胞菌、中间普氏菌和核梭杆菌的抑菌作用均比2%碘甘油药液效果更好（P<0.001）。
结论：1 双黄补煎液在体外对牙龈紫质单胞菌、中间普氏菌和核梭杆菌三种牙周厌氧致病菌均有比较明显的抑菌活性。
2 在双黄补煎液的三种组成成分中，黄连在体外对牙龈紫质单胞菌，中间普氏菌和核梭杆菌三种牙周厌氧致病菌显示出了明显的抑菌活性，其中对中间普氏菌的抑菌活性最为明显；而黄芩与骨碎补在本实验中也表现出一定的抑菌活性。
3 双黄补煎液在体外对牙龈紫质单胞菌、中间普氏菌和核梭杆菌三种牙周厌氧致病菌的抑菌活性均比2%碘甘油药液更明显。
Objective: Periodontal diseases are considered as infection of the periodontium due to subgingival pathgens. The characteristic is the infection of opportunistic pathgens. Porphyromonas gingivalis (Pg), Provotella intermedins (Pi) and Fuso-bacterium nucleartum (Fn) are believed very important for the emergence of periodontal diseases, They can produce lots of virulence factors such as endotoxin, proteas, ammonia, indole, etc. These virulence factors can destrust periodontium and caus periodontal diseases. The control of plaque is the key to periodontal therapy. There were widely study in the use of antibiotics and antimicrobials in the therapy. Systemically or locally administered bacteriocidal drugs had significant effects on inhibiting periodontopathogens. But the Systemical use of antibiotics and antimicrobials for a long time would lead to unpleasant side effects such as dysbacteriosis, the reaction of gastrointestinal tract. Therefore, the locally administered bacteriocidal drug in the therapy was more and more important. Chinese herbal medicine have been used for a long time. Many Chinese herbal medicines have exhibited strong inhibition on periodontopathogens. So, there was
increasing emphasis on researching safe and effective Chinese herbal medicines locally administered in periodontal pockets. Shuanghuangbu was a new double-layered local Chinese herbal delivery device, which mainly contained Rhizoma coptidis, Radix scutellariae and Rhizoma drynariae. We have found the Shuanghuangbu could significantly improve the clinical symptom of periodontitis and might change the composition of subgingival microflora at the disease sites to that at the healthy sites. The purpose of this study was using the liquid media two-fold dilution method and the digging holes agar diffusion method to research the effect of Shuanghuangbu decoction, Rhizoma coptidis decoction, Radix scutellariae decoction and Rhizoma drynariae decoction on Porphyromonas gingivalis, Provotella intermedins and Fusobacterium nucleartum.
Methods: A mixture of Rhizoma coptidis, Radix scu-tellariae and Rhizoma drynariae was soaked in water for 30 minutes , boiled for 45 minutes and filtered; The residue was reboiled with water and refiltered. All decoctions were mixed, heated and concentrated to 1:1 Shuanghuangbu decoction ( 1ml Shuanghuangbu decoction made from 1g of raw material ). 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. These periodontopathogens were cultured on Schaedler broth agar culture medium or in Brain-heat infusion broth. 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 and Rhizoma drynariae decoction on Porphyromonas gingivalis, Provotella intermedins and Fusobacterium nucleartum. 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. Two test-tubes that only contained Schaedler broth were used as a positive control and a negative control, respectively. Periodontopathic bacteria to be used in the MIC test was added to the test test-tubes and the positive control test-tube, finally, the bacteria concentration of each test-tube must became to 1×106 CFU/ml. The test-tubes were then incubated anaerobically at 37℃ for 48hr. The MIC was taken as the lowest concentration of the drugs 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 a control drug. Made four Schaedler broth bloo
引文
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22 Jeffcoat MK, Bray KS, Ciancio SG, et al. A djunctive use of a subgingival controlled-release chlorhexidine chip reduces probing depth and improves attachment level compared with scaling and root planing alone. J Periodontol, 1998, 69(9): 989～997
23 Polson AM, Garrentt S, Stoller NH, et al. Multi-center comparative evaluation of subgingival deliveried sanguinarine and doxycycline in treatment of periodontitis Ⅱ Clinical results. J Periodontol, 1997, 68(2): 119～126
24 陈莉丽 ,主编. 口腔厌氧菌与牙周病. 北京:人民卫生出版社, 1998. 6, 196～207
25 吴贻谷, 宋立人, 主编. 中华本草. 北京:人民卫生出版社, 1996. 11, 528～532
26 姜广水．黄连提取物对牙周致病菌的抑制作用．山东医药，2000，40（18）: 41
27 周晓红．黄芩研究进展．河北中医药学报，2000，15（3）: 31
28 高学敏，主编. 中药学[M]. 北京：人民卫生出版社，1991, 345
29 汤亚玲，谭红，叶玲等. 黄芩对牙龈卟啉单胞菌生长、形态影响的体外实验．牙体牙髓牙周病学杂志，
2002，12（12）: 655～657
30 Tsao TF, Newman MG, Kwok YY, et al．Effect of Chinese and western antimicrobiol agents on selected oral bacteria. J Dent Res，1982，61(9)：1103～1106
31 吴贻谷, 宋立人, 主编. 中华本草. 北京:人民卫生出版社, 1996. 11, 226～227
32 王志儒. 用放射性同位素45Ca对中草药骨碎补治疗骨伤原理探讨. 北京中医学院学报, 1980,19: 13
33 丁继华. 骨碎补对骨性关节炎影响的实验的研究. 中国中医骨伤科杂志, 1989, 5(3): 3
34 严梅桢，高晓山，刘林祥. 黄连与黄芩、甘草配伍前后对金黄色葡萄球菌生长抑制作用的观察. 中国中药杂志 1998 ，23（6）：375～377
35 野口卫, 主编. 汉方制剂分析技术（胡宝华译）[M]. 北京：人民卫生出版社，1986，39
36 林似兰，赵陆华，吴智南. 大黄、黄连、黄柏、黄芩在复方汤剂中的反应研究-配伍变化对有效成分溶出率的影响. 中草药 , 1989 , 20(6): 10
37 黄泰康. 中药汤剂研究概论 . 中草药 , 1986, 17(2): 35
38 乔梁，彭嘉柔，濮训生等. 黄连-黄芩对煎煮液沉淀物的分析. 中国中药杂志, 1999，24(6): 352～353
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16 杨禾．常用局部缓释剂及其在牙周病治疗中的应用．国外医学口腔分册，2001, 28(4): 224～226
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18 Lowenguth RA, Caton J, Clin I, et al. Evaluation of periodontal treatment using controlled-release tetracyline fibers: microbiological response. J Periodontol, 1995, 66(8): 700～707
19 Noyan U, Yilmaz S, Kuru B, et al. A clinical and microbiogical evaluation of systemic and local metronidazole delivery in adult periodontitis patients. J Clin Periodontol, 1997, 24(3): 158～165
20 van Steenberghe D, Bercy P, Kohl J, et al. Subgingival minocyclin hydrochloride ointment in moderate to severe chronic adult periodontitis: randomized, doudleblind, vehicle-controlled, muti-center study. J Periodontol, 1993,
64(7):637～644
21 Soskolne WA, Heasman PA, Stabholz A, et al. Sutstained local delivery of chlorhexidine in the treatment of periodontitis: A multi-center study. J Periodontol, 1997, 68(1): 32～38
22 Jeffcoat MK, Bray KS, Ciancio SG, et al. A djunctive use of a subgingival controlled-release chlorhexidine chip reduces probing depth and improves attachment level compared with scaling and root planing alone. J Periodontol, 1998, 69(9): 989～997
23 Polson AM, Garrentt S, Stoller NH, et al. Multi-center comparative evaluation of subgingival deliveried sanguinarine and doxycycline in treatment of periodontitis Ⅱ Clinical results. J Periodontol, 1997, 68(2): 119～126
24 陈莉丽 ,主编. 口腔厌氧菌与牙周病. 北京:人民卫生出版社, 1998. 6, 196～207
25 吴贻谷, 宋立人, 主编. 中华本草. 北京:人民卫生出版社, 1996. 11, 528～532
26 姜广水．黄连提取物对牙周致病菌的抑制作用．山东医药，2000，40（18）: 41
27 周晓红．黄芩研究进展．河北中医药学报，2000，15（3）: 31
28 高学敏，主编. 中药学[M]. 北京：人民卫生出版社，1991, 345
29 汤亚玲，谭红，叶玲等. 黄芩对牙龈卟啉单胞菌生长、形态影响的体外实验．牙体牙髓牙周病学杂志，
2002，12（12）: 655～657
30 Tsao TF, Newman MG, Kwok YY, et al．Effect of Chinese and western antimicrobiol agents on selected oral bacteria. J Dent Res，1982，61(9)：1103～1106
31 吴贻谷, 宋立人, 主编. 中华本草. 北京:人民卫生出版社, 1996. 11, 226～227
32 王志儒. 用放射性同位素45Ca对中草药骨碎补治疗骨伤原理探讨. 北京中医学院学报, 1980,19: 13
33 丁继华. 骨碎补对骨性关节炎影响的实验的研究. 中国中医骨伤科杂志, 1989, 5(3): 3
34 严梅桢，高晓山，刘林祥. 黄连与黄芩、甘草配伍前后对金黄色葡萄球菌生长抑制作用的观察. 中国中药杂志 1998 ，23（6）：375～377
35 野口卫, 主编. 汉方制剂分析技术（胡宝华译）[M]. 北京：人民卫生出版社，1986，39
36 林似兰，赵陆华，吴智南. 大黄、黄连、黄柏、黄芩在复方汤剂中的反应研究-配伍变化对有效成分溶出率的影响. 中草药 , 1989 , 20(6): 10
37 黄泰康. 中药汤剂研究概论 . 中草药 , 1986, 17(2): 35
38 乔梁，彭嘉柔，濮训生等. 黄连-黄芩对煎煮液沉淀物的分析. 中国中药杂志, 1999，24(6): 352～353