RESUMEN
In this study, we have evaluated by HPLC-DAD, DLS and MALDI-TOF a synergic effect of the coexistence of two salivary-PRP fractions (basic-PRPs and acidic PRPs) on the interaction with flavanols. Results obtained showed noticeable enhancement of the interaction between (epi)catechin and PRPs when both types of proteins are blended. Up to 30 soluble aggregates have been tentatively identified with molecular weight from 4680 to 35,851. (epi)Catechins seem to bind preferentially bPRPs than aPRPs, although the medium size aggregates flavanol-bPRPs formed could favour the interaction with aPRPs giving rise to soluble mixed aggregates.
Asunto(s)
Flavonoides/farmacología , Proteínas Salivales Ricas en Prolina/farmacología , Vino/análisis , Sinergismo Farmacológico , Proteínas Salivales Ricas en Prolina/química , Sensación/efectos de los fármacos , SolubilidadRESUMEN
The interaction of tannins with salivary proteins is involved in astringency. This paper focussed on saliva lining oral mucosae, the mucosal pellicle. Using a cell-based model, the impact of two dietary tannins (EgC and EgCG) on the mucosal pellicle structure and properties was investigated by microscopic techniques. The role of basic Proline-Rich-Proteins (bPRPs) in protecting the mucosal pellicle was also evaluated. At low (0.05â¯mM) tannin concentration, below the sensory detection threshold, the distribution of salivary mucins MUC5B on cells remained unaffected. At 0.5 and 1â¯mM, MUC5B-tannin aggregates were observed and their size increased with tannin concentration and with galloylation. In addition, 3â¯mM EgCG resulted in higher friction forces measured by AFM. In presence of bPRPs, the size distribution of aggregates was greatly modified and tended to resemble that of the "no tannin" condition, highlighting that bPRPs have a protective effect against the structural alteration induced by dietary tannins.
Asunto(s)
Astringentes/farmacología , Mucina 5B/metabolismo , Proteínas Salivales Ricas en Prolina/farmacología , Taninos/farmacología , Astringentes/química , Astringentes/metabolismo , Catequina/análogos & derivados , Catequina/química , Catequina/metabolismo , Catequina/farmacología , Línea Celular , Película Dental/efectos de los fármacos , Película Dental/metabolismo , Dieta , Humanos , Microscopía de Fuerza Atómica , Microscopía Electrónica de Rastreo , Mucosa Bucal/efectos de los fármacos , Mucina 5B/farmacología , Agregado de Proteínas/efectos de los fármacos , Saliva/química , Proteínas Salivales Ricas en Prolina/metabolismo , Taninos/química , Taninos/metabolismoRESUMEN
Saliva contains hundreds of small proline-rich peptides most of which derive from the post-translational and post-secretory processing of the acidic and basic salivary proline-rich proteins. Among these peptides we found that a 20 residue proline-rich peptide (p1932), commonly present in human saliva and patented for its antiviral activity, was internalized within cells of the oral mucosa. The cell-penetrating properties of p1932 have been studied in a primary gingival fibroblast cell line and in a squamous cancer cell line, and compared to its retro-inverso form. We observed by mass-spectrometry, flow cytometry and confocal microscopy that both peptides were internalized in the two cell lines on a time scale of minutes, being the natural form more efficient than the retro-inverso one. The cytosolic localization was dependent on the cell type: both peptide forms were able to localize within nuclei of tumoral cells, but not in the nuclei of gingival fibroblasts. The uptake was shown to be dependent on the culture conditions used: peptide internalization was indeed effective in a complete medium than in a serum-free one allowing the hypothesis that the internalization could be dependent on the cell cycle. Both peptides were internalized likely by a lipid raft-mediated endocytosis mechanism as suggested by the reduced uptake in the presence of methyl-ß-cyclodextrin. These results suggest that the natural peptide may play a role within the cells of the oral mucosa after its secretion and subsequent internalization. Furthermore, lack of cytotoxicity of both peptide forms highlights their possible application as novel drug delivery agents.
Asunto(s)
Péptidos de Penetración Celular/metabolismo , Endocitosis/fisiología , Péptidos/metabolismo , Proteínas Salivales Ricas en Prolina/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Péptidos de Penetración Celular/farmacocinética , Péptidos de Penetración Celular/farmacología , Células Cultivadas , Medios de Cultivo/farmacología , Medio de Cultivo Libre de Suero/farmacología , Endocitosis/efectos de los fármacos , Fibroblastos/metabolismo , Citometría de Flujo , Encía/citología , Humanos , Microscopía Confocal , Péptidos/farmacocinética , Péptidos/farmacología , Proteínas Salivales Ricas en Prolina/farmacocinética , Proteínas Salivales Ricas en Prolina/farmacología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , beta-Ciclodextrinas/farmacologíaRESUMEN
Understanding astringency has focused on the interaction of tannins with the salivary proline-rich proteins (PRPs), although it remains unclear if other astringents precipitate the PRPs or how this interaction relates to sensory perceptions of astringency. We used 2 approaches to compare how distinct classes of astringent compounds interacted with the salivary PRPs and mucins. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis, we evaluated protein patterns and characterized the salivary proteins present in the supernatants and pellets of pooled saliva assayed with tannin, alum, and hydrochloric acid solutions. Tannins and alum precipitated many of the PRPs, but acid did not. Mucins were precipitated by both the acid and alum, but not by the tannins. From our research, it appears that the precipitation of salivary proteins may be involved in the mechanism of astringency, but the precipitation of PRPs is not requisite for the development of astringency. We also measured mucin and deoxyribonucleic acid content of expectorated solutions of astringents that panelists swished in their mouths to determine if astringency was associated with a loss of oral lubricating films.