橘皮提取物对口腔常见微生物抑制作用的研究
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摘要
龋病、牙周病是与牙菌斑生物膜相关的细菌感染性疾病。龋病的发病原因主要是菌斑中的细菌利用碳水化合物产酸、导致牙齿脱矿,继发侵入牙髓和根尖周围组织；牙周病的发病是菌斑中的致炎因子导致的牙周组织直接损害和间接免疫病理损害。研究发现,龋病长期不治疗,可以遗留慢性菌斑,成为牙源性病灶,使大量细菌滋生,蔓延至临近组织器官或通过血液／淋巴液循环传播到远处的组织器官,导致慢性直接和间接损害,例如细菌性心内膜炎、肾炎、关节炎等。慢性口腔细菌感染还可以引起面部临近的眼、耳、鼻等感染。最近研究发现,龋病和牙周病作为牙源性病灶还可以影响胚胎发育。
变形链球菌是龋病和牙周病的始动因子,它首先附着在牙齿硬组织表面,产酸引起牙齿硬组织脱矿,继发其它细菌黏附,形成致病菌斑；金黄色葡萄球菌、白色葡萄球菌是口腔化脓感染及手术感染的主要致病因素；白假丝酵母菌是引发口腔院内交叉感染的重要原因,也是口腔修复体周围炎、口腔黏膜疾病、颌面部感染的主要病因。经口内途径的外科手术后感染等都是由口腔病原生物感染所引起。口腔中的微生物除细菌、真菌外,由唾液中或可检出HIV、HSV、EBV以及CMV等多种病毒。
目前控制口腔感染主要使用化学制剂、抗生素、无机抗菌剂等方法。随着大量免疫抑制剂、广谱抗生素的应用,普遍开展整形外科、正畸技术；广泛进行放疗、化疗、器官移植,口腔细菌耐药和继发感染病例不断增加,口腔菌群失调的机会增加。目前,国际上对口腔感染控制的研究热点主要集中在两个方面,一是研究解决细菌耐药和口腔微生态系菌群失调问题,解决化学药物的毒性问题；二是控制细菌对牙齿的附着,防止菌斑形成。
本研究的目的是探究天然植物成分(橘皮提取物OPE)对口腔主要致病病原生物变形链球菌、金黄色葡萄球菌、白色葡萄球菌白假丝酵母菌的生长抑制作用；对变形链球菌对硬组织粘附作用过程的影响；检验OPE对单纯疱疹病毒的抑制作用,同时检验其细胞毒性作用。为开发新型、天然、无毒副作用的口腔局部抑菌剂、预防和控制口腔疾病特别是龋病和牙周病的发生和发展提供依据。
方法
一.将橘皮提取物按不同比例进行稀释,加入到含有细菌的培养基内,恒温37℃条件下培养24h,测量抑菌环直径,检验其对变形链球菌、金黄色葡萄球菌、白色葡萄球菌、白假丝酵母菌的生长抑制作用。
二.利用细菌附着物洗脱液培养菌落记数方法,检测不同浓度的橘皮提取物对变形链球菌在光滑玻璃表面附着作用的抑制效应。
三.利用扫描电镜观察橘皮提取物对变形链球菌附着形态的影响。
四.利用扫描电镜观察橘皮提取物对未确定菌种粘附形态的影响。
五.检验橘皮提取物对293细胞的毒性作用。
六.采取MTT方法检测橘皮提取物对单纯疱疹病毒的灭活作用。
结果
一.橘皮提取物稀释1600倍仍对变形链球菌生长有抑制作用、稀释6400倍对金黄色葡萄球菌生长有抑制作用、稀释12800倍对白色葡萄球菌生长有抑制作用、稀释800倍对白假丝酵母菌的生长有抑制作用,抑菌作用强度与药物浓度有关。
二.橘皮提取物稀释25600倍仍对变形链球菌的黏附有抑制作用。
三.取橘皮提取物组和对照组变形链球菌附着玻片,在扫描电子显微镜下可见明显的细菌形态学改变。结果表明,橘皮提取物有抑制变形链球菌对光滑玻璃表面黏附的作用。
四.在扫描电子显微镜下可见,橘皮提取物可以抑制未知杆菌对光滑玻璃表面的黏附。
五.橘皮提取物有低度细胞毒性作用。
六.橘皮提取物对单纯疱疹病毒有灭活作用。
结论
OPE对口腔常见微生物变形链球菌、金黄色葡萄球菌、白色葡萄球菌、白假丝酵母菌有抑菌作用；可以抑制变形链球菌等细菌对光滑玻璃表面的粘附；对单纯疱疹病毒有灭活作用。橘皮提取物在有效抑菌及抗病毒浓度未见明显细胞毒性反应。而且使用低浓度橘皮提取物对细菌仅有抑制其生长增殖和附着的作用,而不会全面杀死细菌,从而能够有效维持口腔内的微生态平衡。因此,橘皮提取物可作为一种天然、高效、安全的新型口腔局部抑菌剂、在口腔疾病特别是龋病和牙周病的预防和控制中发挥重要作用。
Purpose:
Dental caries is a multifactorial, chronic infectious disease, which mainly caused by dental plaque. The cariogenic bacteria such as Streptococcus mutans can metabolize carbohydrates whose products can lead to demineralization. Dental plaque, a complex and dynamic microbial ecosystem, plays a vital role on the formation of caries and periodontal disease. Strategies to control and prevent dental caries are mostly based on antiplaque or antimicrobial agents. Considering the bacteria resistance, more and better strategies for the prevention of caries and periodontal disease are needed.
In this study, we investigate the anti-bacteria effects of orange peel extracts (OPE) by the main pathogenic microorganisms found in mouth, such as Streptococcus mutans, Staphylococcus aureus, Staphylococcus albus,Candida albican and herpes simples virus-1. Moreover, we observed the anti-adhesive effect of Streptococcus mutans and an unidentified bacterium on the smooth glass. Another purpose of my study is to analyze the cell-toxicity of the OPE, which can prove that the application of OPE is a new, natural and safety bacteriostatic agent on the prevention and control of oral diseases.
Methods:
1. Dilute the OPE in different proportion, add it in the bacterio-culture medium, and culture at 37℃for 24 hours. Then measure the diameter of bacteriostasis ring and analyze its inhibition effect on the growth of Streptococcus mutans, Staphylococcus aureus, Staphylococcus albus and Candida albican.
2. Make use of colony forming unit (CFU) to examine the inhibition effect of adhesiveness of Streptococcus mutans to the smooth glass rod in OPE with different concentration.
3. Use scanning electron microscope (SEM) to observe the effect of the OPE on the adhesiveness of Streptococcus mutans and an unidentified bacterium.
4. Examine the toxicity of the OPE to human 293 cells.
5. Adopt MTT method to examine the effect of the OPE on herpes simples virus-typel (HSV-!).
Results:
1. The orange peel extracts (OPE) diluted by 1600 times can inhibit growth of Streptococcus mutans; diluted by 6400 times,it can inhibit growth of Staphylococcus aureus; diluted by 12800 times, it can inhibit growth of Staphylococcus albus; diluted by 800 times,it can inhibit growth of Candida albican. The inhibition effect is related to the different concentration of the OPE.
2. After diluted by 25600 times the OPE still can inhibit adhesion of Streptococcus mutans. Under the scanning electron microscope, the different adhesive forms of inhibition effect of adhesive attraction of Streptococcus mutans can be obsereved. This result shows that the OPE can inhibit the adhesive ability of Streptococcus mutans and an unidentified bacterium to smooth glass. The inhibition effects are related to the concentration of the OPE.
3. OPE has shown a very low toxicity to human 293 cells.
4. The OPE can inhibit the pathogenicity of herpes simples virus-type1.
Conclusion:
Orange peel extracts have anti-bacterial effect on main oralpathogenic microorganisms and they can inhibit the adhesion of Streptococcus and an unidentified bacterium to smooth glass. Besides, the OPE have the anti-virus effect on herpes simples virus-type 1. The original fluid has shown a very low toxicity to human 293 cells.The application of OPE is a new, natural and safety bacteriostatic agent on the prevention and control of oral diseases.
变形链球菌是龋病和牙周病的始动因子,它首先附着在牙齿硬组织表面,产酸引起牙齿硬组织脱矿,继发其它细菌黏附,形成致病菌斑；金黄色葡萄球菌、白色葡萄球菌是口腔化脓感染及手术感染的主要致病因素；白假丝酵母菌是引发口腔院内交叉感染的重要原因,也是口腔修复体周围炎、口腔黏膜疾病、颌面部感染的主要病因。经口内途径的外科手术后感染等都是由口腔病原生物感染所引起。口腔中的微生物除细菌、真菌外,由唾液中或可检出HIV、HSV、EBV以及CMV等多种病毒。
目前控制口腔感染主要使用化学制剂、抗生素、无机抗菌剂等方法。随着大量免疫抑制剂、广谱抗生素的应用,普遍开展整形外科、正畸技术；广泛进行放疗、化疗、器官移植,口腔细菌耐药和继发感染病例不断增加,口腔菌群失调的机会增加。目前,国际上对口腔感染控制的研究热点主要集中在两个方面,一是研究解决细菌耐药和口腔微生态系菌群失调问题,解决化学药物的毒性问题；二是控制细菌对牙齿的附着,防止菌斑形成。
本研究的目的是探究天然植物成分(橘皮提取物OPE)对口腔主要致病病原生物变形链球菌、金黄色葡萄球菌、白色葡萄球菌白假丝酵母菌的生长抑制作用；对变形链球菌对硬组织粘附作用过程的影响；检验OPE对单纯疱疹病毒的抑制作用,同时检验其细胞毒性作用。为开发新型、天然、无毒副作用的口腔局部抑菌剂、预防和控制口腔疾病特别是龋病和牙周病的发生和发展提供依据。
方法
一.将橘皮提取物按不同比例进行稀释,加入到含有细菌的培养基内,恒温37℃条件下培养24h,测量抑菌环直径,检验其对变形链球菌、金黄色葡萄球菌、白色葡萄球菌、白假丝酵母菌的生长抑制作用。
二.利用细菌附着物洗脱液培养菌落记数方法,检测不同浓度的橘皮提取物对变形链球菌在光滑玻璃表面附着作用的抑制效应。
三.利用扫描电镜观察橘皮提取物对变形链球菌附着形态的影响。
四.利用扫描电镜观察橘皮提取物对未确定菌种粘附形态的影响。
五.检验橘皮提取物对293细胞的毒性作用。
六.采取MTT方法检测橘皮提取物对单纯疱疹病毒的灭活作用。
结果
一.橘皮提取物稀释1600倍仍对变形链球菌生长有抑制作用、稀释6400倍对金黄色葡萄球菌生长有抑制作用、稀释12800倍对白色葡萄球菌生长有抑制作用、稀释800倍对白假丝酵母菌的生长有抑制作用,抑菌作用强度与药物浓度有关。
二.橘皮提取物稀释25600倍仍对变形链球菌的黏附有抑制作用。
三.取橘皮提取物组和对照组变形链球菌附着玻片,在扫描电子显微镜下可见明显的细菌形态学改变。结果表明,橘皮提取物有抑制变形链球菌对光滑玻璃表面黏附的作用。
四.在扫描电子显微镜下可见,橘皮提取物可以抑制未知杆菌对光滑玻璃表面的黏附。
五.橘皮提取物有低度细胞毒性作用。
六.橘皮提取物对单纯疱疹病毒有灭活作用。
结论
OPE对口腔常见微生物变形链球菌、金黄色葡萄球菌、白色葡萄球菌、白假丝酵母菌有抑菌作用；可以抑制变形链球菌等细菌对光滑玻璃表面的粘附；对单纯疱疹病毒有灭活作用。橘皮提取物在有效抑菌及抗病毒浓度未见明显细胞毒性反应。而且使用低浓度橘皮提取物对细菌仅有抑制其生长增殖和附着的作用,而不会全面杀死细菌,从而能够有效维持口腔内的微生态平衡。因此,橘皮提取物可作为一种天然、高效、安全的新型口腔局部抑菌剂、在口腔疾病特别是龋病和牙周病的预防和控制中发挥重要作用。
Purpose:
Dental caries is a multifactorial, chronic infectious disease, which mainly caused by dental plaque. The cariogenic bacteria such as Streptococcus mutans can metabolize carbohydrates whose products can lead to demineralization. Dental plaque, a complex and dynamic microbial ecosystem, plays a vital role on the formation of caries and periodontal disease. Strategies to control and prevent dental caries are mostly based on antiplaque or antimicrobial agents. Considering the bacteria resistance, more and better strategies for the prevention of caries and periodontal disease are needed.
In this study, we investigate the anti-bacteria effects of orange peel extracts (OPE) by the main pathogenic microorganisms found in mouth, such as Streptococcus mutans, Staphylococcus aureus, Staphylococcus albus,Candida albican and herpes simples virus-1. Moreover, we observed the anti-adhesive effect of Streptococcus mutans and an unidentified bacterium on the smooth glass. Another purpose of my study is to analyze the cell-toxicity of the OPE, which can prove that the application of OPE is a new, natural and safety bacteriostatic agent on the prevention and control of oral diseases.
Methods:
1. Dilute the OPE in different proportion, add it in the bacterio-culture medium, and culture at 37℃for 24 hours. Then measure the diameter of bacteriostasis ring and analyze its inhibition effect on the growth of Streptococcus mutans, Staphylococcus aureus, Staphylococcus albus and Candida albican.
2. Make use of colony forming unit (CFU) to examine the inhibition effect of adhesiveness of Streptococcus mutans to the smooth glass rod in OPE with different concentration.
3. Use scanning electron microscope (SEM) to observe the effect of the OPE on the adhesiveness of Streptococcus mutans and an unidentified bacterium.
4. Examine the toxicity of the OPE to human 293 cells.
5. Adopt MTT method to examine the effect of the OPE on herpes simples virus-typel (HSV-!).
Results:
1. The orange peel extracts (OPE) diluted by 1600 times can inhibit growth of Streptococcus mutans; diluted by 6400 times,it can inhibit growth of Staphylococcus aureus; diluted by 12800 times, it can inhibit growth of Staphylococcus albus; diluted by 800 times,it can inhibit growth of Candida albican. The inhibition effect is related to the different concentration of the OPE.
2. After diluted by 25600 times the OPE still can inhibit adhesion of Streptococcus mutans. Under the scanning electron microscope, the different adhesive forms of inhibition effect of adhesive attraction of Streptococcus mutans can be obsereved. This result shows that the OPE can inhibit the adhesive ability of Streptococcus mutans and an unidentified bacterium to smooth glass. The inhibition effects are related to the concentration of the OPE.
3. OPE has shown a very low toxicity to human 293 cells.
4. The OPE can inhibit the pathogenicity of herpes simples virus-type1.
Conclusion:
Orange peel extracts have anti-bacterial effect on main oralpathogenic microorganisms and they can inhibit the adhesion of Streptococcus and an unidentified bacterium to smooth glass. Besides, the OPE have the anti-virus effect on herpes simples virus-type 1. The original fluid has shown a very low toxicity to human 293 cells.The application of OPE is a new, natural and safety bacteriostatic agent on the prevention and control of oral diseases.
引文
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34. Munson, M. A., A. Banerjee, T. F. Watson, et al. Molecular analysis of the microflora associated with dental caries [J]. J. Clin. Microbiol, 2004,42:3023-3029.
35. Siqueira, J. F., Jr., and I. N. Ro^c,as. Positive and negative bacterial associations involving Dialister pneumosintes in primary endodontic infections [J]. J. Endod,2003,29:438-441.
36. Siqueira, J. F., Jr., and I. N. Ro^c,as. Polymerase chain reaction-based analysis of microorganisms associated with failed endodontic treatment [J]. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod, 2004,97:85-94.
37. Siqueira, J. F., Jr., I. N. Ro^c,as, and A. S. Rosado. Investigation of bacterial communities associated with asymptomatic and symptomatic endodontic infections by DGGE fingerprinting approach [J]. Oral Microbiol. Immunol,2004,19:363-370.
38.汪振华,王金玉,袁祥民.固定桥对口腔微生态系影响的研究现状[J].国外医学口腔医学分册,2005,32(2)：159-161.
39.白丽,申元英,王晶,等.健康人群口腔酵母茵的分离及菌种分布[J].中国微生态学杂志,2005,17(2)： 111-2.
40.郝玉庆,周学东.抗菌肽及其对口腔微生物作用研究进展[J].国外医学口腔医学分册,2004,31(5)：346-348.
41.徐红云,林一南,范群.口臭症口腔微生态学的研究[J].中国微生态学杂志,2003,15(6)：340-1.
42.刘慧颍,马国武.种植体周围炎研究进展[J].中国微生态学杂志,2005,17(2)：158-9.
43. 凌均 ,陈罕.龋病病因微生物学研究和龋病分子诊断[J].牙体牙髓牙周病学杂志,2004,14(1)：1-3.
44.Orallo F, Alvarez E, Basaran H,et al. Comparative study of the vasorelaxant activity, superoxide-scavenging ability and cyclic nucleotide phosphodiesterase-inhibitory effects of hesperetin and hesperidin[J]. Naunyn Schmiedebergs Arch Pharmacol,2004,370(6):452-63.
45.Pehlivan M, Hazinedaroglu SM, Kayaoglu HA,et al. The effect of diosmin hesperidin on intestinal ischaemia--reperfusion injury[J]. Acta Chir Belg, 2004,104(6):715-8.
46.Cvetnic Z, Vladimir-Knezevic S. Antimicrobial activity of grapefruit seed and pulp ethanolic extract [J]. Acta Pharm, 2004,54(3):243-50.
47.Neves RN, Carvalho F, Carvalho M,et al. Protective activity of hesperidin and lipoic acid against sodium arsenite acute toxicity in mice[J]. Toxicol Pathol, 2004,32(5):527-35.
48.Jung UJ, Lee MK, Jeong KS, et al. The hypoglycemic effects of hesperidin and naringin are partly mediated by hepatic glucose-regulating enzymes in C57BL/KsJ-db/db mice [J]. J Nutr,2004,134(10):2499-503.
49.Matsumoto H, Ikoma Y, Sugiura M, et al. Identification and quantification of the conjugated metabolites derived from orally administered hesperidin in rat plasma[J]. J Agric Food Chem,2004,52(21):6653-9.
50.李泽洪,陈丽、陈敏珊,植物提取物添加剂对口腔微生物抑制研究及应用[J].牙膏工业,2000,2：6.
51.雷培琪,尹才渊,肖建春.桔皮油对大鼠虱、豚鼠螨的杀 虫效果观察[J].四川动物,2004,20(3)：162-163.
52.欧壮益,桔皮苷的药理作用[J].中药材,2002,25(7) ： 533-535.
53.汤建国,汪秋安,单杨.从柑橘皮中超声提取橘皮甙[J].精细化工,2004,21(30)：171-2.
54.赵榆林,杨亚玲,林文添等.分光光度法测定橘皮中橙皮苷含量[J].光谱实验,2004,20(3)：353-5.
55.赵建颖,邓宇.微波提取叶黄素的研究[J].食品添加剂,2004,25(8)：121-3.
56.林伸二,橘皮提取物抑制黑色素形成[J].国外医学中医中药分册,2002,24(6)：368.
57.吴永魁,田慧英.橘皮提取物的毒性研究[J].中兽医医药杂志,1992,3,4-6.
58.吴永魁,张祚新,杨永胜.橘皮素已酰酯毒性研究[J].中兽医医药杂志,2003,4：12-3.
59.张祚新,吴永魁,蔡辉,等.橘皮衍生物对离体痒螨及临床疗效观察[J].中兽医医药杂志,2002,2：5-6.
60.贾冬英,姚开河,何强,等.橙皮苷的高效液相色谱定量分析[J].华西药学杂志,2001,16(60)：444-6.
1. Rassameemasmaung S,Sirikulsathean A,Amornchat C, et al. Effects of herbal mouthwash containing the pericarp extract of Garcinia mangostana L on halitosis, plaque and papillary bleeding index [J]. J Int Acad Periodontol,2007,9(1):19-25
2. Shu M,Morou-Bermudez E, Suarez-Perez E, The relationship between dental caries status and dental plaque urease activity [J]. Oral Microbiol Immunol,2007,22(1):61-6.
3. Bots CP, Brand HS, Poorterman JH,et al. Oral and salivary changes in patients with end stage renal disease (ESRD):a two year follow-up study[J]. Br Dent J,2007,202(2):E3.
4. Heidari E, Dickinson C, Wilson R.,et al. Verifiable CPD paper:oral health of remand prisoners in HMP Brixton, London[J].Br Dent J, 2007,27:202(2):E1.
5. Kinane DF, Mark Bartold P. Clinical relevance of the host responses of periodontitis [J].Periodontol 2000,2007,43:278-93.
6. Mahanonda R, Pichyangkul S. Toll-like receptors and their role in periodontal health and disease [J]. Periodontol 2000,2007,43:41-55.
7. Boutaga K, Savelkoul PH, Winkel EG,et al. Comparison of subgingival bacterial sampling with oral lavage for detection and quantification of periodontal pathogens by real-time polymerase chain reaction [J]. J Periodontol,2007,78(1):79-86.
8..Krigar DM, Kaltschmitt J, Krieger JK, et al. Two subgingival plaque-sampling strategies used with RNA probes [J].J Periodontol, 2007,78(1):72-8.
9. Wong L, Sissons CH. Human dental plaque microcosm biofilms: effect of nutrient variation on calcium phosphate deposition and growth [J].Arch Oral Biol,2007,52(3):280-9.
10. Bae K,Jun EJ, Lee SM, et al.Effect of water-soluble reduced chitosan on Streptococcus mutans, plaque regrowth and biofilm vitality [J].Clin Oral Investig,2006,10(2):102-7.
11. Stahl J, Zandona AF. Rationale and protocol for the treatment of non-cavitated smooth surface carious lesions [J].Gen Dent,2007, 55(2):105-11.
12. Miguel Bresc 6 Salinas, Noelia Costa Riu, Leonardo Berini Ayt es, et al. Antibiotic susceptibility of the bacteria causing odontogenic infections [J]. Med Oral Patol Oral Cir Bucal,2006, 11:E70-5.
13. Khemaleelakul S, Baumgartner JC, Pruksakorn S. Identification of bacteria in acute endodontic infections and their antimicrobial susceptibility [J]. Oral Surg Oral Med Oral Radiol Oral Pathol Endod,2002,94:746-55.
14. Baumgartner JC, Xia T. Which antibiotics susceptibility of bacteria associated with endodontic abscesses [J]. J Endodont,2003,29:44-7.
15. Khalil Boutaga, Arie Jan van Winkelhoff, Christina M. J. E, et al. Considerations in Evaluating the Applicability of Universal Detection of Oral Pathogens [J]. J of clinical microbiology,2004,42(9): 4414-4415.
16. Boutaga, K., A. J. van Winkelhoff, C. M. Vandenbroucke-Grauls, et al. Comparison of real-time PCR and culture for detection of Porphyromonas gingivalis in subgingival plaque samples [J]. J. Clin. Microbiol,2003,41:4950-4954.
17. Rocas IN, Siqueira JF Jr, Aboim MC, et al.. Denaturing gradient gel electrophoresis analysis of bacterial communities associated with failed endodontic treatment [J]. Oral Surg Oral Med Oral Pathol Oral Radio Endod,2004,98(6):741-9.
18. Pia T. Sunde, Ingar Olsen, Ulf B. Go bel,et al. Fluorescence in situ hybridization (FISH) for direct visualization of bacteria in periapical lesions of asymptomatic root-filled teeth [J].Microbiology,2003, 149:1095-1102.
19. Leys, E. J., Lyons, S. R., Moeschberger, et al. Association of Bacteroides forsythus and a novel Bacteroides phylotype with periodontitis [J]. J Clin Microbiol,2002,40:821-825.
20. Sunde, P. T., Olsen, I., Debelian, G. J.& Tronstad, L.Microbiota of periapical lesions refractory to endodontic therapy [J]. J Endod, 2002.28:304-310.
21. A. Villedieu, M. L. Diaz-Torres, A. P. Roberts, et al. Genetic Basis of Erythromycin Resistance in Oral Bacteria [J].Antimicrobial agents and chemotherapy,2004,48(6):2298-2301.
22. Villedieu, A., M. L. Diaz-Torres, N. Hunt, et al. Prevalence of tetracycline resistance genes in oral bacteria [J]. Antimicrob. Agents Chemother,2003.47:878-882.
23. S.K.Filoche,M.Zhe,C.D.Wu. In situ Biofilm Formation by Multi-species Oral Bacteria under Flowing and Anaerobic conditions [J]. J Dent Res,2004,83(10):802-806.
24. Rickard AH, Gilbert P, High NJ, etal. Bacterial coaggregation:an integral process in the development of multi-species biofilms[J]. Trends Microbiol,2003,11:94-100.
25. Stoodley P, Sauer K, Davies Dg.et al. Biofilms as complex differentiated communities [J]. Ann Rev Microbiol,2002,56:187-209.
26. Tanaka M, Yamamoto Y, Kuboniwa M, et al. Contribution of periodontal pathogens on tongue dorsa analyzed with real-time PCR to oral malodor [J]. Microbes Infect,2004,6:1078-1083.
27. Dewhirst FE, Paster BJ. Diversity of bacterial populations on the tongue dorsa of patients with halitosis and healthy patients [J]. J Clin Microbiol,2003,41:558-563.
28. Li J, Helmerhorst EJ, Leone CW, et al. Identification of early microbial colonizers in human dental biofilm [J]. J Appl Microbiol, 2004,97:1311-1318.
29. Mager DL, Ximenez-Fyvie LA, Haffajee AD, et al. Distribution of selected bacterial species on intraoral surfaces [J]. J Clin Periodontol, 2003,30:644-654.
30. Tsute Chen, Kevin Abbey, Wen-jie Deng, et al. The bioinformatics resource for oral pathogens [J]. Nucleic Acid Research,2005,33(1):734-40.
31. Duncan,M.J. Genomics of oral bacteria [J].Crit. Rev. Oral Biol.Med, 2003,14:175-187.
32. Chen,T., Hosogi,Y., Nishikawa K, A., et al. Comparative whole-genome analysis of virulent and avirulent strains of Porphyromonas gingivalis [J]. J. Bacteriol,2004,186:5473-5479.
33. J. F. Siqueira, Jr.* I. N. Ro^c,as. Uncultivated Phylotypes and Newly Named Species Associated with Primary and Persistent Endodontic Infections [J]. J. Clin. Microbiol,2005,43(7):3314-3319.
34. Munson, M. A., A. Banerjee, T. F. Watson, et al. Molecular analysis of the microflora associated with dental caries [J]. J. Clin. Microbiol, 2004,42:3023-3029.
35. Siqueira, J. F., Jr., and I. N. Ro^c,as. Positive and negative bacterial associations involving Dialister pneumosintes in primary endodontic infections [J]. J. Endod,2003,29:438-441.
36. Siqueira, J. F., Jr., and I. N. Ro^c,as. Polymerase chain reaction-based analysis of microorganisms associated with failed endodontic treatment [J]. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod, 2004,97:85-94.
37. Siqueira, J. F., Jr., I. N. Ro^c,as, A. S. Rosado. Investigation of bacterial communities associated with asymptomatic and symptomatic endodontic infections by DGGE fingerprinting approach [J]. Oral Microbiol. Immunol,2004,19:363-370.
38.汪振华.王金玉,袁祥民.固定桥对口腔微生态系影响的研究现状[J].国外医学口腔医学分册,2005,32(2)：159-161.
39.白丽,申元英,王晶,等.健康人群口腔酵母茵的分离及菌种分布[J].中国微生态学杂志,2005,17(2)： 111-2.
40.郝玉庆综述周学东审校.抗菌肽及其对口腔微生物作用研究进展[J].国外医学口腔医学分册,2004,31(5)：346-348.
41.徐红云,林一南,范群.口臭症口腔微生态学的研究[J].中国微生态学杂志,2003,15(6)：340-1.
42.刘慧颍,马国武.种植体周围炎研究进展[J].中国微生态学杂志,2005,17(2)：158-9.
43.凌均,陈罕.龋病病因微生物学研究和龋病分子诊断[J].牙体牙髓牙周病学杂志,2004,14(1)：1-3.44.
44.张冬梅,赵戬,潘亚萍.龈下微生物检测方法研究的现状[J].牙体牙 髓牙周病学杂志,2004,14(3)：166-8.
45.刘慧颍,马国武.种植体周围炎研究进展[J].中国微生态学杂志,2005,17(2)：158-9.
1. Orallo F, Alvarez E, Basaran H,et al. Comparative study of the vasorelaxant activity, superoxide-scavenging ability and cyclic nucleotide phosphodiesterase-inhibitory effects of hesperetin and hesperidin [J]. Naunyn Schmiedebergs Arch Pharmacol,2004,370(6):452-63.
2. Pehlivan M, Hazinedaroglu SM, Kayaoglu HA,et al. The effect of diosmin hesperidin on intestinal ischaemia--reperfusion injury[J]. Acta Chir Belg,2004, 104(6):715-8.
3. Cvetnic Z, Vladimir-Knezevic S. Antimicrobial activity of grapefruit seed and pulp ethanolic extract [J]. Acta Pharm,2004,54(3):243-50.
4. Neves RN, Carvalho F, Carvalho M,et al. Protective activity of hesperidin and lipoic acid against sodium arsenite acute toxicity in mice [J]. Toxicol Pathol, 2004,32 (5):527-35.
5. Jung UJ, Lee MK, Jeong KS,et al. The hypoglycemic effects of hesperidin and naringin are partly mediated by hepatic glucose-regulating enzymes in C57BL/KsJ-db/db mice [J]. J Nutr,2004,134(10):2499-503.
6. Matsumoto H, Ikoma Y, Sugiura M, Yano M, Identification and quantification of the conjugated metabolites derived from orally administered hesperidin in rat plasma [J]. J Agric Food Chem,2004,52(21):6653-9.
7.李泽洪,陈丽,陈敏珊,植物提取物添加剂对口腔微生物抑制研究及应用[J]. 牙膏工业,2000,2：6.
8.雷培琪,尹才渊,肖建春.桔皮油对大鼠虱、豚鼠螨的杀虫效果观察[J].四川动物,2004,20(3)：162-163.
9.欧壮益,桔皮苷的药理作用[J].中药材,2002,25(7)：533-535.
10.汤建国,汪秋安,单杨.从柑橘皮中超声提取橘皮甙[J].精细化工,2004.21(30)： 171-2.
11.赵榆林,杨亚玲,林文添等.分光光度法测定橘皮中橙皮苷含量[J].光谱实验,2004,20(3)：353-5.
12.赵建颖,邓宇.微波提取叶黄素的研究[J].食品添加剂,2004,25(8)：121-3.
13.林伸二,橘皮提取物抑制黑色素形成[J].国外医学中医中药分册,2002,24(6)：368.
14.吴永魁,田慧英.橘皮提取物的毒性研究[J].中兽医医药杂志,1992,3：4-6.
15.吴永魁,张祚新,杨永胜.橘皮素已酰酯毒性研究[J].中兽医医药杂志,2003,4：12-3.
16.张祚新,吴永魁,蔡辉,等.橘皮衍生物对离体痒螨及临床疗效观察[J].中兽医医药杂志,2002,2：5-6.
17.贾冬英,姚开河,何强,等.橙皮苷的高效液相色谱定量分析[J].华西药学杂志,2001,16(60)：444-6.
2. Shu M,Morou-Bermudez E, Suarez-Perez E.The relationship between dental caries status and dental plaque urease activity [J]. Oral Microbiol Immunol,2007,22(1):61-6.
3. Bots CP, Brand HS, Poorterman JH,et al. Oral and salivary changes in patients with end stage renal disease (ESRD):a two year follow-up study [J]. Br Dent J,2007,202(2):E3
4. Heidari E, Dickinson C, Wilson R.,et al. Verifiable CPD paper:oral health of remand prisoners in HMP Brixton, London [J].Br Dent J, 2007,202(2):E1.
5. Kinane DF, Mark Bartold P. Clinical relevance of the host responses of periodontitis [J].Periodontol 2000,2007,43:278-93.
6. Mahanonda R, Pichyangkul S. Toll-like receptors and their role in periodontal health and disease[J]. Periodontol 2000,2007,43:41-55.
7. Boutaga K, Savelkoul PH, Winkel EG,et al. Comparison of subgingival bacterial sampling with oral lavage for detection and quantification of periodontal pathogens by real-time polymerase chain reaction [J]. J Periodontol,2007,78(1):79-86.
8..Krigar DM, Kaltschmitt J, Krieger JK, et al. Two subgingival plaque-sampling strategies used with RNA probes [J].J Periodontol, 2007,78(1):72-8.
9. Wong L, Sissons CH. Human dental plaque microcosm biofilms:effect of nutrient variation on calcium phosphate deposition and growth [J].Arch Oral Biol,2007,52(3):280-9.
10. Bae K,Jun EJ, Lee SM, et al. Effect of water-soluble reduced chitosan on Streptococcus mutans, plaque regrowth and biofilm vitality [J].Clin Oral Investig,2006,10(2):102-7.
11.Stahl J, Zandona AF. Rationale and protocol for the treatment of non-cavitated smooth surface carious lesions [J].Gen Dent,2007, 55(2):105-11.
12. Miguel Bresc 6 Salinas, Noelia Costa Riu, Leonardo Berini Ayt es, et al. Antibiotic susceptibility of the bacteria causing odontogenic infections [J]. Med Oral Patol Oral Cir Bucal,2006,11:E70-5.
13. Khemaleelakul S, Baumgartner JC, Pruksakorn S. Identification of bacteria in acute endodontic infections and their antimicrobial susceptibility [J]. Oral Surg Oral Med Oral Radiol Oral Pathol Endod,2002,94:746-55.
14. Baumgartner JC, Xia T. Which antibiotics susceptibility of bacteria associated with endodontic abscesses [J]. J Endodont,2003,29:44-7.
15. Khalil Boutaga, Arie Jan van Winkelhoff, Christina M. J. E, et al. Considerations in Evaluating the Applicability of Universal Detection of Oral Pathogens [J].J of clinical microbiology,2004,42(9): 4414-4415.
16. Boutaga K, A. J. van Winkelhoff, C. M. Vandenbroucke-Grauls, et al.. Comparison of real-time PCR and culture for detection of Porphyromonas gingivalis in subgingival plaque samples [J]. J. Clin. Microbiol,2003,41:4950-4954.
17. Rocas IN, Siqueira JF Jr, Aboim MC, et al.. Denaturing gradient gel electrophoresis analysis of bacterial communities associated with failed endodontic treatment [J]. Oral Surg Oral Med Oral Pathol Oral Radio Endod,2004,98(6):741-9.
18. Pia T. Sunde, Ingar Olsen, Ulf B. Go bel,et al. Fluorescence in situ hybridization (FISH) for direct visualization of bacteria in periapical lesions of asymptomatic root-filled teeth [J].Microbiology,2003,149: 1095-1102.
19. Leys, E. J., Lyons, S. R., Moeschberger, et al. Association of Bacteroides forsythus and a novel Bacteroides phylotype with periodontitis[J].J Clin Microbiol,2002,40:821-825.
20. Sunde, P. T., Olsen, I., Debelian, G. J.& Tronstad, L.Microbiota of periapical lesions refractory to endodontic therapy [J]. J Endod,2002.28:304-310.
21. A. Villedieu, M. L. Diaz-Torres, A. P. Roberts,et al. Genetic Basis of Erythromycin Resistance in Oral Bacteria [J]. Antimicrobial agents and chemotherapy,2004,48(6):2298-2301.
22. Villedieu, A., M. L. Diaz-Torres, N. Hunt, et al. Prevalence of tetracycline resistance genes in oral bacteria [J]. Antimicrob. Agents Chemother,2003.47:878-882.
23. S.K.Filoche,M Zhe,C.D.Wu. In situ Biofilm Formation by Multi-species Oral Bacteria under Flowing and Anaerobic conditions [J]. J Dent Res,2004,83(10):802-806.
24. Rickard AH, Gilbert P, High NJ, et al. Bacterial coaggregation:an integral process in the development of multi-species biofilms [J]. Trends Microbiol,2003,11:94-100.
25. Stoodley P, Sauer K, Davies DG, Costerton JW. Biofilms as complex differentiated communities [J]. Ann Rev Microbiol,2002,56:187-209.
26. Tanaka M, Yamamoto Y, Kuboniwa M,et al. Contribution of periodontal pathogens on tongue dorsa analyzed with real-time PCR to oral malodor [J]. Microbes Infect,2004,6:1078-1083.
27. Dewhirst FE, Paster BJ. Diversity of bacterial populations on the tongue dorsa of patients with halitosis and healthy patients [J]. J Clin Microbiol,2003,41:558-563.
28. Li J, Helmerhorst EJ, Leone CW, etal. Identification of early microbial colonizers in human dental biofilm [J]. J Appl Microbiol, 2004,97:1311-1318.
29. Mager DL, Ximenez-Fyvie LA, Haffajee AD, et al.Distribution of selected bacterial species on intraoral surfaces [J]. J Clin Periodontol, 2003,30:644-654.
30. Tsute Chen, Kevin Abbey, Wen-jie Deng, et al. The bioinformatics resource for oral pathogens [J].Nucleic Acid Research,2005,33(1):734-40.
31. Duncan,M.J. Genomics of oral bacteria [J].Crit. Rev. Oral Biol.Med, 2003,14,:175-187.
32. Chen, T., Hosogi,Y., Nishikawa,K,A.et al. Comparative whole-genome analysis of virulent and avirulent strains of Porphyromonas gingivalis [J]. J. Bacteriol,2004,186:5473-5479.
33. J. F. Siqueira, Jr.* I. N. Ro"c,as. Uncultivated Phylotypes and Newly Named Species Associated with Primary and Persistent Endodontic Infections [J]. J. Clin. Microbiol,2005,43(7):3314-3319.
34. Munson, M. A., A. Banerjee, T. F. Watson, et al. Molecular analysis of the microflora associated with dental caries [J]. J. Clin. Microbiol, 2004,42:3023-3029.
35. Siqueira, J. F., Jr., and I. N. Ro^c,as. Positive and negative bacterial associations involving Dialister pneumosintes in primary endodontic infections [J]. J. Endod,2003,29:438-441.
36. Siqueira, J. F., Jr., and I. N. Ro^c,as. Polymerase chain reaction-based analysis of microorganisms associated with failed endodontic treatment [J]. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod, 2004,97:85-94.
37. Siqueira, J. F., Jr., I. N. Ro^c,as, and A. S. Rosado. Investigation of bacterial communities associated with asymptomatic and symptomatic endodontic infections by DGGE fingerprinting approach [J]. Oral Microbiol. Immunol,2004,19:363-370.
38.汪振华,王金玉,袁祥民.固定桥对口腔微生态系影响的研究现状[J].国外医学口腔医学分册,2005,32(2)：159-161.
39.白丽,申元英,王晶,等.健康人群口腔酵母茵的分离及菌种分布[J].中国微生态学杂志,2005,17(2)： 111-2.
40.郝玉庆,周学东.抗菌肽及其对口腔微生物作用研究进展[J].国外医学口腔医学分册,2004,31(5)：346-348.
41.徐红云,林一南,范群.口臭症口腔微生态学的研究[J].中国微生态学杂志,2003,15(6)：340-1.
42.刘慧颍,马国武.种植体周围炎研究进展[J].中国微生态学杂志,2005,17(2)：158-9.
43. 凌均 ,陈罕.龋病病因微生物学研究和龋病分子诊断[J].牙体牙髓牙周病学杂志,2004,14(1)：1-3.
44.Orallo F, Alvarez E, Basaran H,et al. Comparative study of the vasorelaxant activity, superoxide-scavenging ability and cyclic nucleotide phosphodiesterase-inhibitory effects of hesperetin and hesperidin[J]. Naunyn Schmiedebergs Arch Pharmacol,2004,370(6):452-63.
45.Pehlivan M, Hazinedaroglu SM, Kayaoglu HA,et al. The effect of diosmin hesperidin on intestinal ischaemia--reperfusion injury[J]. Acta Chir Belg, 2004,104(6):715-8.
46.Cvetnic Z, Vladimir-Knezevic S. Antimicrobial activity of grapefruit seed and pulp ethanolic extract [J]. Acta Pharm, 2004,54(3):243-50.
47.Neves RN, Carvalho F, Carvalho M,et al. Protective activity of hesperidin and lipoic acid against sodium arsenite acute toxicity in mice[J]. Toxicol Pathol, 2004,32(5):527-35.
48.Jung UJ, Lee MK, Jeong KS, et al. The hypoglycemic effects of hesperidin and naringin are partly mediated by hepatic glucose-regulating enzymes in C57BL/KsJ-db/db mice [J]. J Nutr,2004,134(10):2499-503.
49.Matsumoto H, Ikoma Y, Sugiura M, et al. Identification and quantification of the conjugated metabolites derived from orally administered hesperidin in rat plasma[J]. J Agric Food Chem,2004,52(21):6653-9.
50.李泽洪,陈丽、陈敏珊,植物提取物添加剂对口腔微生物抑制研究及应用[J].牙膏工业,2000,2：6.
51.雷培琪,尹才渊,肖建春.桔皮油对大鼠虱、豚鼠螨的杀 虫效果观察[J].四川动物,2004,20(3)：162-163.
52.欧壮益,桔皮苷的药理作用[J].中药材,2002,25(7) ： 533-535.
53.汤建国,汪秋安,单杨.从柑橘皮中超声提取橘皮甙[J].精细化工,2004,21(30)：171-2.
54.赵榆林,杨亚玲,林文添等.分光光度法测定橘皮中橙皮苷含量[J].光谱实验,2004,20(3)：353-5.
55.赵建颖,邓宇.微波提取叶黄素的研究[J].食品添加剂,2004,25(8)：121-3.
56.林伸二,橘皮提取物抑制黑色素形成[J].国外医学中医中药分册,2002,24(6)：368.
57.吴永魁,田慧英.橘皮提取物的毒性研究[J].中兽医医药杂志,1992,3,4-6.
58.吴永魁,张祚新,杨永胜.橘皮素已酰酯毒性研究[J].中兽医医药杂志,2003,4：12-3.
59.张祚新,吴永魁,蔡辉,等.橘皮衍生物对离体痒螨及临床疗效观察[J].中兽医医药杂志,2002,2：5-6.
60.贾冬英,姚开河,何强,等.橙皮苷的高效液相色谱定量分析[J].华西药学杂志,2001,16(60)：444-6.
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1. Orallo F, Alvarez E, Basaran H,et al. Comparative study of the vasorelaxant activity, superoxide-scavenging ability and cyclic nucleotide phosphodiesterase-inhibitory effects of hesperetin and hesperidin [J]. Naunyn Schmiedebergs Arch Pharmacol,2004,370(6):452-63.
2. Pehlivan M, Hazinedaroglu SM, Kayaoglu HA,et al. The effect of diosmin hesperidin on intestinal ischaemia--reperfusion injury[J]. Acta Chir Belg,2004, 104(6):715-8.
3. Cvetnic Z, Vladimir-Knezevic S. Antimicrobial activity of grapefruit seed and pulp ethanolic extract [J]. Acta Pharm,2004,54(3):243-50.
4. Neves RN, Carvalho F, Carvalho M,et al. Protective activity of hesperidin and lipoic acid against sodium arsenite acute toxicity in mice [J]. Toxicol Pathol, 2004,32 (5):527-35.
5. Jung UJ, Lee MK, Jeong KS,et al. The hypoglycemic effects of hesperidin and naringin are partly mediated by hepatic glucose-regulating enzymes in C57BL/KsJ-db/db mice [J]. J Nutr,2004,134(10):2499-503.
6. Matsumoto H, Ikoma Y, Sugiura M, Yano M, Identification and quantification of the conjugated metabolites derived from orally administered hesperidin in rat plasma [J]. J Agric Food Chem,2004,52(21):6653-9.
7.李泽洪,陈丽,陈敏珊,植物提取物添加剂对口腔微生物抑制研究及应用[J]. 牙膏工业,2000,2：6.
8.雷培琪,尹才渊,肖建春.桔皮油对大鼠虱、豚鼠螨的杀虫效果观察[J].四川动物,2004,20(3)：162-163.
9.欧壮益,桔皮苷的药理作用[J].中药材,2002,25(7)：533-535.
10.汤建国,汪秋安,单杨.从柑橘皮中超声提取橘皮甙[J].精细化工,2004.21(30)： 171-2.
11.赵榆林,杨亚玲,林文添等.分光光度法测定橘皮中橙皮苷含量[J].光谱实验,2004,20(3)：353-5.
12.赵建颖,邓宇.微波提取叶黄素的研究[J].食品添加剂,2004,25(8)：121-3.
13.林伸二,橘皮提取物抑制黑色素形成[J].国外医学中医中药分册,2002,24(6)：368.
14.吴永魁,田慧英.橘皮提取物的毒性研究[J].中兽医医药杂志,1992,3：4-6.
15.吴永魁,张祚新,杨永胜.橘皮素已酰酯毒性研究[J].中兽医医药杂志,2003,4：12-3.
16.张祚新,吴永魁,蔡辉,等.橘皮衍生物对离体痒螨及临床疗效观察[J].中兽医医药杂志,2002,2：5-6.
17.贾冬英,姚开河,何强,等.橙皮苷的高效液相色谱定量分析[J].华西药学杂志,2001,16(60)：444-6.