Hydroalkoholischer Extrakt aus Sauerampferkraut (Rumex acetosa L.) zur Prophylaxe von Mundschleimhauterkrankungen und Periodontitis: Hemmung der bakteriellen Adhäsion und zentraler Virulenzfaktoren von Porphyromonas gingivalis

Zusammenfassung

Sauerampferkraut aus den oberirdischen Teilen von Rumex acetosa L. wird traditionell gegen entzündliche Erkrankungen der Mundhöhle verwendet. Die vorliegende Studie untersuchte den Einfluss eines Proanthocyanidin-angereicherten hydroalkoholischen Extraktes (RA1) auf die Adhäsion von Porphyromonas gingivalis, eines Bakteriums, welches ein zentraler Mitverursacher der chronischen und aggressiven Parodontitis darstellt. Ein weiteres Ziel der Untersuchungen war die Identifizierung von bioaktiven Leitstrukturen mit antiadhäsiver Aktivität und die Benennung der für die antiadhäsiven Effekte verantwortlichen molekularen Mechanismen. Der vollständig quantifizierte Extrakt RA1 (5-15 µg/ml) reduzierte die bakterielle Adhäsion von P. gingivalis an humanen KB-Mundschleimhautzellen in vitro dosisabhängig bis zu 90 %. Dies wurde sowohl in einem durchflusszytometrischen Test als auch mittels Konfokaler Laser Scanning Mikroskopie belegt. Die In-vitro-Datensätze konnten auch in einem In-situ-Modell an Buccalschleimhaut der Maus bestätigt werden. Die Kombination phytochemischer Methoden mit funktionellen Adhäsionsassays identifizierte für die aus RA1 isolierten Naturstoffen 1 bis 15 Epicatechin-3-O-gallat-(4β,8)-epicatechin-3’-O-gallat (syn. Procyanidin B2-di-gallat, Verbindung 8) als stark antiadhäsives Proanthocyanidin, welches als funktionelle Leitsubstanz des Extraktes angesehen wird. Struktur-Wirkungsbeziehungen zeigten, dass eine mindestens einfache Galloylierung von Flavan-3-olen für eine antiadhäsive Wirkung notwendig ist. Die Aktivität der Verbindungen wird durch Trihydroxylierung im B-Ring und durch Oligomerisierung weiter gesteigert.

Als molekularer Angriffspunkt für diese Wirkstoffe wurde der bakterielle Virulenzfaktor Arginin-Gingipain identifiziert, ein zentraler Virulenzfaktor von P. gingivalis, der neben der bakteriellen Adhäsion des Pathogens an der Wirtszelle auch proteolytischen Abbau von Wirtsproteinen, Modulation der immunologischen Wirtsabwehr und Reifung weiterer bakterieller Virulenzfaktoren induziert.

Aus diesen Untersuchungen zeigt sich ein ausreichendes Potenzial von Sauerampferextrakt zur weiteren präklinischen und klinischen Entwicklung zu zytoprotektiven Mundhygieneprodukten.

Abstract

Hydroalcoholic extracts from Rumex acetosa L. for prophylaxis of periodontitis: Inhibition of the bacterial adhesion and virulence factors of Porphyromonas gingivalis

The aerial parts of Rumex acetosa L. have been used in traditional European medicine for inflammatory diseases of the mouth epithelial tissue. The following study aimed to investigate the influence of a proanthocyanidin-enriched extract from R. acetosa against the adhesion of Porphyromonas gingivalis, a pathogen strongly involved in chronic and aggressive periodontitis. A further goal was to define the bioactive lead structures responsible for a potential antiadhesive activity and to characterize the underlying molecular mechanisms of the antiadhesive effects.

An extract of R. acetosa (RA1) with a defined mixture of flavan-3-ols, oligomeric proanthocyanidins and flavonoids, was used. Its impact on P. gingivalis adhesion to KB cells was studied by flow cytometry, confocal laser scanning microscopy and in situ adhesion assay using murine buccal tissue. RA1 and its compounds 1 to 15 were further investigated for additional effects on gingipain activity, hemagglutination and gene expression by RT-PCR.

RA1 (5 to 15 µg/mL) reduced P. gingivalis adhesion in a dose-dependent manner to about 90 %. Galloylated proanthocyanidins were confirmed to be responsible for this antiadhesive effect with epicatechin-3-O-gallate-(4β,8)-epicatechin-3’-O-gallate (syn. procyanidin B2-di-gallate) being the lead compound. Ungalloylated flavan-3-ols and oligomeric proanthocyanidins were inactive. RA1 and the galloylated proanthocyanidins strongly interact with the bacterial virulence factor Arg-gingipain, while the corresponding Lys-gingipain was hardly influenced. RA1 inhibited also hemagglutination.

In conclusion, the proanthocyanidin-enriched extract RA1 and its main active constituent procyanidin B2-di-gallate protect cells from P. gingivalis infection by inhibiting bacterial adhesion to the host cell. RA1 and procyanidin B2-di-gallate appear to be promising candidates for future cytoprotective preparations for oral mouth care products.

Schlüsselwörter

Rumex acetosa L. - Sauerampferkraut - Adhäsion - Zytoprotektion - Parodontitis - Porphyromonas gingivalis - Proanthocyanidine

Keywords

Rumex acetosa L - Adhesion - Cytoprotection - Periodontitis - Porphyromonas gingivalis - Proanthocyanidins

• Literatur

• 1 Beikler T, Flemmig TF. Oral biofilm-associated diseases: trends and implications for quality of life, systemic health and expenditures. Periodontol 2000 2011; 55: 87-103
• 2 Benakanakere M, Kinane DF. Innate cellular responses to the periodontal biofilm. Front Oral Biol 2012; 15: 41-55
• 3 Teles R, Teles F, Frias-Lopez J et al. Lessons learned and unlearned in periodontal microbiology. Periodontol 2000 2013; 62: 95-162
• 4 Darveau RP, Tanner A, Page RC. The microbial challenge in periodontitis. Periodontol 2000 1997; 14: 12-32
• 5 Rescala B, Rosalem W, Teles RP et al. Immunological and microbiological profiles of chronic and aggressive periodontitis subjects. J Periodontol 2010; 81: 1308-1316
• 6 Machtei EE, Dunford R, Hausmann E et al. Longitudinal study of prognostic factors in established periodontitis patients. J Clin Periodontol 1997; 24: 102-109
• 7 Haffajee AD, Socransky SS, Smith C, Dibart S. Relation of baseline microbial parameters to future periodontal attachment loss. J Clin Periodontol 1991; 18: 744-750
• 8 Koziel J, Mydel P, Potempa J. The link between periodontal disease and rheumatoid arthritis: an updated review. Curr Rheumatol Rep 2014; 16: 408-415
• 9 Atanasova KR, Yilmaz Ö. Looking in the Porphyromonas gingivalis cabinet of curiosities: the microbium, the host and cancer association. Mol Oral Microl 2014; 29: 55-66
• 10 Beikler T, Prior K, Ehmke B, Flemmig TF. Specific antibiotics in the treatment of periodontitis - a proposed strategy. J Periodontol 2004; 75: 169-175
• 11 Heitz-Mayfield LD, Trombell L et al. A systematic review of the effect of surgical debridement vs non-surgical debridement for the treatment of chronic periodontitis. J Clin Periodontol 2002; 29: 92-102
• 12 Palaska I, Papathanasiou E, Theoharides TC. Use of polyphenols in periodontal inflammation. Eur J Pharmacol 2013; 720: 77-83
• 13 Feldmann M, Grenier D. Cranberry proanthocyanidins act in synergy with licochalcone A to reduce Porphyromonas gingivalis growth and virulence properties and to suppress cytokine secretion by macrophages. J Appl Microbiol 2012; 113: 438-447
• 14 Löhr G, Beikler T, Podbielski A et al. Polyphenols from Myrothamnus flabellifolia Welw. inhibit in vitro adhesion of Porphyromonas gingivalis and exert anti-inflammatory cytoprotective effects in KB cells. J Clin Periodontol 2011; 38: 457-469
• 15 Bonifait L, Grenier D. Cranberry polyphenols: potential benefits for dental caries and periodontal disease. J Can Dent Assoc 2010; 76: a130
• 16 Bodet C, Chandad F, Grenier D. Cranberry components inhibit interleukin-6, interleukin-8 and prostaglandin E production by lipopolysaccharide-activated gingival fibroblasts. Eur J Oral Sci 2007; 115: 64-70
• 17 Amano A. Molecular interaction of Porphyromonas gingivalis with host cells: implication for the microbial pathogenesis of periodontal disease. J Periodontol 2003; 74: 90-96
• 18 Cutler CW, Kalmar JR, Genco CA. Pathogenic strategies of the oral anaerobe, Porphyromonas gingivalis . Trends Microbiol 1995; 3: 45-51
• 19 Okuda K, Yamamoto A, Vaito Y et al. Purification and properties of hemagglutinin from culture supernatant of Bacteroides gingivalis . Infect Immun 1986; 54: 659-665
• 20 Fitzpatrick RE, Wijeyewickrema C, Pike RN. The gingipains: scissors and glue of the periodontal pathogen, Porphyromonas gingivalis . Fut Microbiol 2009; 4: 471-487
• 21 Chen T, Nakayama K, Belliveau L, Duncan M. P. gingivalis gingipains and adhesion to epithelial cells. Infect Immun 2001; 69: 3048-3056
• 22 Travis J, Potempa J. Bacterial proteinases as targets for the development of second-generation antibiotics. Biochim Biophys Acta 2000; 1477: 35-50
• 23 Lantz MS, Allen RD, Duck LW et al. Identification of P. gingivalis components that mediate its interactions with fibronectin. J Bacteriol 1991; 173: 4263-4270
• 24 Pike RN, Potempa J, McGraw W et al. Characterization of the binding activities of proteinase-adhesin complexes from P. gingivalis . J Bacteriol 1996; 178: 2876-2882
• 25 Nakayama K, Yoshimura F, Kadowaki T, Yamamoto K. Involvement of arginine-specific cysteine proteinase (Arg-gingipain) in fimbriation of Porphyromonas gingivalis . J Bacteriol 1996; 178: 2818-2824
• 26 Socransky SS, Haffajee AD, Cugini MA et al. Microbial complexes in subgingival plaque. J Clin Periodontol 1998; 25: 134-144
• 27 Umeda M, Tominaga Y, He T et al. Microbial flora in the acute phase of periodontitis and the effect of local administration of minocycline. J Periodontol 1996; 67: 422-427
• 28 Yao ES, Lamont RJ, Leu SP, Weinberg A. Interbacterial binding of pathogenic and commensal oral bacterial species. Oral Microbiol Immunol 1996; 11: 35-41
• 29 Nilius AM, Spencer SC, Simonson LG. Stimulation of in vitro growth of Treponema denticola by extracellular growth factors produced by Porphyromonas gingivalis . J Dent Res 1993; 72: 1027-1031
• 30 Gescher K, Hensel A, Hafezi W et al. Oligomeric proanthocyanidins from Rumex acetosa L. inhibit the attachment of Herpes simplex virus type-1. Antivir Res 2011; 89: 9-18
• 31 Derksen A, Hensel A, Hafezi W et al. 3-O-galloylated procyanidins from Rumex acetosa L. inhibit the attachment of influenza A virus. PLoS One 2014; 9: 11008-9
• 32 Bicker A, Petereit F, Hensel A. Proanthocyanidins and a phloroglucinol derivative from Rumex acetosa L. Fitoterapia 2009; 80: 483-495
• 33 International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use; ICH Harmonized Tripartite Guideline; Validation of Analytical Procedures: Text and Methodology Q2(R1); Finalised Guidelines: October 1994/November 1996
• 34 Chen Z, Potempa J, Polanowski A et al. Purification and characterization of a 50-kDa cysteine proteinase (Gingipain) from Porphyromonas gingivalis . J Biol Chem 1992; 267: 18896-18901
• 35 Kadowaki T, Nakayama K, Yoshimura F et al. Arg-gingipain acts as a major processing enzyme for various cell surface proteins in Porphyromonas gingivalis . J Biol Chem 1998; 273: 29072-29076
• 36 Yokoyama K, Sugano N, Shimada T et al. Effects of egg yolk antibody against P. gingivalis gingipains in periodontitis patients. J Oral Sci 2007; 49: 201-206
• 37 Miyachi K, Ishihara K, Kimizuka R, Okuda K. Arg-gingipain A DNA vaccine prevents alveolar bone loss in mice. J Dental Res 2007; 86: 446-450
• 38 Schmuch J, Beckert S, Brandt S et al. Extract from Rumex acetosa L. for prophylaxis of periodontitis: Inhibition of bacterial in vitro adhesion and of Gingipains of Porphyromonas gingivalis by epicatechin-3-O-gallate-(4β-8]-epicatechin-3-O-gallate (procyanidin-B2-di-gallate) PLoS One 2015; 10: e0120130