Microbiological and Clinical Effects of a Proanthocyanidin-enriched Extract from Rumex acetosa in Periodontally Healthy Carriers of Porphyromonas gingivalis: a Randomized Controlled Pilot Study.

Rumex acetosa significantly inhibits the adhesion of Porphyromonas gingivalis (P. g.) to eukaryotic host cells in vitro. The objective of this randomized placebo-controlled pilot-trial was to analyze effects of a mouth rinse containing 0.8% (w/w) of a quantified proanthocyanidin-enriched extract from Rumex acetosa (RA1) on microbiological, clinical, and cytological parameters in systemically healthy individuals without history of periodontitis, harboring P. g. intraorally. 35 subjects received a supragingival debridement (SD) followed by mouth rinsing (3 times daily) with either RA1 mouth rinse solution (test) or placebo (control) for 7 days as adjunct to routine oral hygiene. Supragingival biofilm samples were taken at screening visit, baseline (BL), 2, 4, 7 and 14 days after SD. P. g. and 11 other oral microorganisms were detected and quantified by rtPCR. Changes in the oral microbiota composition of one test and one control subject were assessed via high throughput 16S rRNS gene amplicon sequencing. Approximal Plaque Index (API) and the modified Sulcular Bleeding Index (SBI) were assessed at BL, 7- and 14-days following SD. Brush biopsies were taken at BL and 14 d following SD. Intergroup comparisons revealed no significant microbiological, cytological, and clinical differences at any timepoint. However, a significant reduction in SBI at day 14 (p = 0.003) and API at day 7 (p = 0.02) and day 14 (p = 0.009) was found in the test group by intragroup comparison. No severe adverse events were observed. The results indicate that RA1 mouth rinse is safe but does not seem to inhibit colonization of P. g. or improve periodontal health following SD.

Microstructured Polymer System Containing Proanthocyanidin-Enriched Extract from Limonium brasiliense as a Prophylaxis Strategy to Prevent Recurrence of Porphyromonas gingivalis.

Periodontal diseases are a global oral health problem affecting almost 10% of the global population. Porphyromonas gingivalis is one of the main bacteria involved in the initiation and progression of inflammatory processes as a result of the action of the cysteine proteases lysin- and arginine-gingipain. Surelease/polycarbophil microparticles containing a lyophilized proanthocyanidin-enriched fraction from the rhizomes of Limonium brasiliense, traditionally named "baicuru" (ethyl acetate fraction), were manufactured. The ethyl acetate fraction was characterized by UHPLC by the presence of samarangenins A and B (12.10 ± 0.07 and 21.05 ± 0.44%, respectively) and epigallocatechin-3-O-gallate (13.44 ± 0.27%). Physiochemical aspects of Surelease/polycarbophil microparticles were characterized concerning particle size, zeta potential, entrapment efficiency, ethyl acetate fraction release, and mucoadhesion. Additionally, the presence of the ethyl acetate fraction-loaded microparticles was performed concerning potential influence on viability of human buccal KB cells, P. gingivalis adhesion to KB cells, gingipain activity, and P. gingivalis biofilm formation. In general, all Surelease/polycarbophil microparticles tested showed strong adhesion to porcine cheek mucosa (93.1 ± 4.2% in a 30-min test), associated with a prolonged release of the ethyl acetate fraction (up to 16.5 ± 0.8% in 24 h). Preincubation of KB cells with Surelease/polycarbophil microparticles (25 µg/mL) resulted in an up to 93 ± 2% reduced infection rate by P. gingivalis. Decreased activity of the P. gingivalis-specific virulence factors lysin- and arginine-gingipain proteases by Surelease/polycarbophil microparticles was confirmed. Surelease/polycarbophil microparticles decreased biofilm formation of P. gingivalis (97 ± 2% at 60 µg/mL). Results from this study prove the promising activity of Surelease/polycarbophil microparticles containing ethyl acetate fraction microparticles as a prophylaxis strategy to prevent the recurrence of P. gingivalis.

Smart drug delivery against Helicobacter pylori: pectin-coated, mucoadhesive liposomes with antiadhesive activity and antibiotic cargo.

The first step in the development of Helicobacter pylori pathogenicity is the receptor-mediated adhesion to the gastric epithelium. Inhibition of outer membrane proteins of H. pylori (e.g. BabA) by antiadhesive drugs will contribute to reduced recolonization and infection. Pectin from apple inhibits the BabA and LPS-mediated adhesion of H. pylori to human stomach cells. Pectin-coated liposomes with encapsulated amoxicillin were characterized for polydispersity, zeta potential, encapsulation efficiency, stability, and amoxicillin release. Coated liposomes did not influence the viability of AGS and HT29-MTX cells up to 100 µg/mL but exert cytotoxicity against H. pylori at 10 µg/mL. Pectin-coating of liposomes provoked direct interaction and subsequent binding of the particles to surface structures of H. pylori, and interaction with mucus from porcine stomach and mucus secreted by HT29-MTX cells. Laser scanning microscopy of H. pylori and AGS cells together with liposomes indicated co-aggregation. The mucoadhesive effect seems interesting as stomach cells are covered by a mucus layer. H. pylori is able to penetrate and cross the mucin rapidly to reach pH-neutral epithelium to escape the acidic environment, followed by interaction with epithelial cells. In summary, all experimental evidence is consistent with a specific interaction of pectin-coated liposomes with mucins and surface structures of H. pylori. As the coated liposomes show mucoadhesion to the negatively charged mucins, docking to stomach mucin, mucus penetration, and recognition of and adhesion to H. pylori, they can be considered a novel type of multifunctional drug carriers for local antibiotic therapy against H. pylori. KEY POINTS: • Smart, multifunctional mucoadhesive liposomes • Specific targeting against BabA/LPS of Helicobacter pylori • Inhibition of bacterial adhesion of H. pylori to human host cells • Release of antibiotic cargo.

Isolation and Quantification of Oligomeric and Polymeric Procyanidins in the Aerial Parts of St. John's Wort (Hypericum perforatum).

Proanthocyanidins (condensed tannins) constitute a class of oligomeric and polymeric polyphenols with flavan-3-ols as monomeric building blocks. Despite the high impact of proanthocyanidins, these polyphenols are mostly quantified by colorimetric methods or by chromatographic determination of the flavan-3-ols as cleavage products or low molecular oligomers as lead compounds. For St. John's wort (Hyperici herba) from Hypericum perforatum, a protocol for preparative isolation of oligomeric and polymeric proanthocyanidins from an acetone-water extract by chromatography on Sephadex®LH20 in combination with preparative high-performance liquid chromatography on the diol stationary phase was developed, yielding procyanidin reference clusters with a defined degree of polymerization from 3 to 10. Identity and purity of these clusters was proven by high-performance liquid chromatography (RP18 and diol phase) and mass spectrometry. For identification and quantification of proanthocyanidin clusters from St. John's wort, an ICH-Q2 (International harmonized guideline for analytical validation) validated high-performance liquid chromatography method with fluorimetric detection was developed using an acetone-water extract of the herbal material, purified by solid-phase extraction for the removal of naphthodianthrones. The method enabled the quantification of procyanidin clusters with a degree of polymerization from 2 to 10. Analysis of nine batches of Hyperici herba from different sources indicated a high variability of proanthocyanidin content in the range from 8 to 37 mg/g. In all of the batches investigated, the trimer cluster DP3 was the dominant proanthocyanidin (about 40 %), followed by DP 4 (about 15 %) and DP5 (about 12 %). Monitoring of procyanidin distribution during seasonal growth of fresh plants of H. perforatum indicated the highest proanthocyanidin content in young plants (about 50 mg/g) and a time-dependent decrease during the growing season to about 16 mg/g. The highest proanthocyanidin content was found in young leaves and flowers, while the fruits were proanthocyanidin-free; older parts of the stem and the herb had a lower proanthocyanidin content. From these data, it can be concluded that proanthocyanidins serve as part of the plant defense system in the reproductive organs.

Limonium brasiliense rhizomes extract against virulence factors of Porphyromonas gingivalis: Inhibition of gingipains, bacterial adhesion, and biofilm formation.

Periodontitis is clinically characterized by destruction of the tooth support system and tooth loss. Porphyromonas gingivalis (Pg) plays a dominant role in periodontitis. Fractions and isolated compounds from an acetone-water extract of the roots of Limonium brasiliense (Lb) were tested in vitro for their anti-adhesive capacity against Pg on human KB buccal cells, influence on gingipains, the main virulence factors of Pg, and biofilm formation. Fractions EAF and FLB7 (50 µg/mL) reduced the bacterial adhesion of Pg to KB cells significantly (63 resp. 70%). The proanthocyanidin samarangenin A inhibited the adhesion (72%, 30 µM), samarangenin B (71%, 20 µM), and the flavan-3-ol epigallocatechin-3-O-gallate (79%, 30 µM). Fraction AQF, representing hydrophilic compounds, reduced the proteolytic activity of Arginin-specific gingipain (IC50 12.78 µg/mL). Fractions EAF and FLB7, characterized by lipohilic constituents, inhibited Arg-gingipain (IC50 3 µg/mL). On Lysine-specific gingipain, AQF has an IC50 15.89, EAF 14.15, and FLB7 6 µg/mL. The reduced bacterial adhesion is due to a strong interaction of proanthocyanidins with gingipains. AQF, EAF, and FLB7 significantly inhibited biofilm formation: IC50 11.34 (AQF), 11.66 (EAF), and 12.09 µg/mL (FLB7). In silico analysis indicated, that the polyphenols act against specific targets of Pg, not affecting mammalian cells. Therefore, Lb might be effective for prevention of periodontal disease by influencing virulence factors of Pg.

Polyphenols from Myrothamnus flabellifolia Welw. inhibit in vitro adhesion of Porphyromonas gingivalis and exert anti-inflammatory cytoprotective effects in KB cells.

AIM: Identification of anti-adhesive plant extracts against cell surface binding of Porphyromonas gingivalis and underlying mechanisms; investigation of potential cytoprotective effects of anti-adhesive extract on KB cells. MATERIALS AND METHODS: Polyphenol-enriched extract, fully characterized concerning flavan-3-ols and oligomeric proanthocyanidins, from Myrothamnus flabellifolia (MF), traditionally used for periodontitis, was tested for inhibition of P. gingivalis-mediated adhesion to KB cells by flow cytometry, for influence on gingipain activity (protease assay), haemagglutination and by microarray analysis for effects on bacterial transcriptome. The influence of MF on P. gingivalis-induced cytokine gene expression was monitored by RT-PCR and IL-6 titres by ELISA. RESULTS: MF (100 µg/ml) reduced P. gingivalis adhesion/invasion about 50% by interacting with bacterial OMPs. As shown by RT-PCR, fimbrillin and Arg-gingipain encoding genes were up-regulated by MF. On the protein level, inhibition (70%) of Arg-gingipain activity was observed, while the corresponding Lys-gingipain was hardly influenced. MF also inhibited haemagglutination. While exposure to P. gingivalis resulted in an increased expression of inflammation-related genes in KB cells, pre-treatment of KB cells with MF evoked cytoprotective effects concerning IL-1ß, IL-6, IL-8 and TNF-α gene expression as well as IL-6 release rates. Compounds from the plant extract belonging to the class of proanthocyanidins were shown to be responsible for the observed effects and were characterized for their respective structural features. CONCLUSIONS: While being cytoprotective, MF exerts anti-adhesive effects against P. gingivalis. Thus, MF may be useful for the prevention of P. gingivalis-associated periodontal diseases.

Polyphenols in the prevention and treatment of periodontal disease: A systematic review of in vivo, ex vivo and in vitro studies.

Plant-derived polyphenols with antimicrobial and immunomodulatory characteristics appear to provide a variety of oral health benefits. Thus, the aim of the present study was to review the scientific literature to identify these effects of polyphenols on periodontal pathogens and inflammation. A MEDLINE search from 1st January 2013 to 18th January 2018 was performed to identify studies reporting polyphenol-containing plant extracts. Reports regarding pure compounds and essential oils, as well as effects on bacteria that are not defined as periodontal pathogens, were excluded. Thirty-eight studies matched the selection criteria. Studies on immunomodulatory effects included in vitro, ex vivo, and in vivo studies (n = 23), whereas studies reporting antibacterial effects against periodontal pathogens included only in vitro studies (n = 18). Three studies were included in both groups. The antibacterial effects were characterised by inhibition of bacterial growth, adhesion to oral cells, and enzymatic activity. Decreased secretion of pro-inflammatory and increased secretion of anti-inflammatory cytokines were demonstrated. Higher attachment levels, lower inflammation, and bone loss were reported by in vivo studies. Due to the high heterogeneity, it is difficult to draw clear conclusions for applicability; nevertheless, polyphenols have great potential as antimicrobial and immunomodulatory substances in the treatment and prevention of periodontal disease.

Flavan-3-ols and proanthocyanidins from Limonium brasiliense inhibit the adhesion of Porphyromonas gingivalis to epithelial host cells by interaction with gingipains.

Porphyromonas gingivalis is a pathogen strongly involved in chronic and aggressive forms of periodontitis. Natural products, mainly polyphenols, have been described for advanced treatment of periodontitis by inhibition of the bacterial adhesion of P. gingivalis to the epithelial host cells. An acetone:water extract (LBE) from the rhizomes of Limonium brasiliense (Boiss.) Kuntze was tested under in vitro conditions for potential antiadhesive effects against P. gingivalis to human KB cells and for inhibition of the proteolytic activity of gingipains, the main virulence factor of P. gingivalis. LBE≤100µg/mL had no cytotoxicity against the bacteria and did not influence the cell physiology of human epithelial KB cells. At 100µg/mL LBE reduced the adhesion of P. gingivalis to KB cells significantly by about 80%. LBE at 20µg/mL reduced the proteolytic activity of the arginin-specific Rgp gingipain by about 75%. Chemical profiling of LBE indicated the presence of gallic acid, epigallocatechin-3-O-gallate and samarangenins A and B as lead compounds. UHPLC by using MS and UV detection displays a suitable method for quality control of the extract for identification and quantification of the lead compounds.

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-(4ß→8)-epicatechin-3-O-gallate (procyanidin-B2-Di-gallate).

BACKGROUND: 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 extract against the adhesion of Porphyromonas gingivalis (P. 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. METHODOLOGY: 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. PRINCIPAL FINDINGS: 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-(4ß,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 silico docking studies indicated that epicatechin-3-O-gallate-(4ß,8)-epicatechin-3'-O-gallate interacts with the active side of Arg-gingipain and hemaglutinin from P. gingivalis; the galloylation of the molecule seems to be responsible for fixation of the ligand to the protein. 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.