CELINE MESSIER
EFFETS DES POLYPHENOLS DE PLANTES SUR LA CROISSANCE ET CERTAINS FACTEURS DE VIRULENCE DE CANDIDA ALBICANS
Mémoire présenté à la Faculté des études supérieures de l'Université Laval dans le cadre du programme des études en sciences dentaires pour l'obtention du grade de maître es sciences (M. Se.)
FACULTE DE MEDECINE DENTAIRE UNIVERSITÉ LAVAL QUÉBEC
2011
©Céline Messier, 2011 Résumé
Les buts de notre projet étaient d'étudier les effets de certains polyphenols de plantes sur la croissance, la survie, la formation du biofilm et la transition blastospore-hyphe de C. albicans, une levure responsable des candidoses humaines. Ce projet a permis de démontrer que deux polyphenols naturels de la réglisse, la licochalcone A et la glabridine, ainsi qu'un polyphenol synthétisé en laboratoire, le 4-hydroxycordoin, ont des propriétés intéressantes contre certains facteurs de virulence de C. albicans. La licochalcone A et la glabridine ont montré un effet fongicide et une action synergique avec le nystatin. La licochalcone A et le 4-hydroxycordoin ont démontré une capacité d'inhiber la formation du biofilm, alors que les trois polyphenols ont montré un effet inhibiteur sur la transition blastospore-hyphe de C. albicans. Ces molécules possèdent donc un potentiel pour le traitement ou la prévention d'infections fongiques buccales à C. albicans. 11
Avant-Propos
Je désire d'abord remercier mon directeur de mémoire, Dr Daniel Grenier, pour son appui, son temps et sa patience tout au long de mon projet. Je veux également mentionner l'aide reçue de mes collègues de laboratoire. D m'est aussi important de souligner l'influence de deux personnes, Dr Jean Barbeau et Mme Jacynthe Séguin, qui m'ont transmis une multitude de connaissances et leur intérêt pour la recherche en microbiologie buccale.
Contribution des auteurs
Premier article
Cet article a été soumis pour publication dans la revue «Critical Reviews in Food Science and Nutrition» sous la référence : Messier C, Epifano F, Genovese S, Grenier D, Beneficial properties of licorice for oral health - A review. Les Drs Epifano et Genovese ont contribué à la figure et au tableau synthèse de l'article, ainsi qu'à la section Description of the plant and chemical composition. Le Dr Grenier a rédigé la section Periodontal disease and licorice et a supervisé la rédaction de l'article. Le premier auteur a rédigé les sections suivantes : Abstract, Introduction, Dental caries and licorice, Oral candidosis and licorice, Recurrent aphtous ulceration and licorice, Conclusion et References.
Deuxième article
Cet article a été accepté pour publication dans la revue «Mycoses» sous la référence : Messier C, Grenier D, Effect of licorice compounds licochalcone A, glabridin and glycyrrhizic acid on growth and virulence properties of Candida albicans. Le Dr Grenier a supervisé la rédaction de l'article et a apporté ses commentaires et ses corrections. Le premier auteur a exécuté toutes les manipulations en laboratoire, procédé à l'analyse des résultats, exécuté les analyses statistiques, préparé les tableaux et figures et rédigé l'ensemble de l'article. Ill
Troisième article
Cet article a été publié dans la revue «Phytomedicine» sous la référence : Messier C, Epifano F, Genovese S, Grenier D, Inhibition of Candida albicans biofilm formation and yeast-hyphal transition by 4-hydroxycordoin. Les Drs Epifano et Genovese ont effectué la synthèse de la molécule 4-hydroxycordoin et ont préparé la figure 1. Le Dr Grenier a supervisé la rédaction de l'article et a apporté ses commentaires et ses corrections. Le premier auteur a réalisé toutes les manipulations en laboratoire, procédé à l'analyse des résultats, exécuté les analyses statistiques, préparé les figures 2 et 3 et rédigé l'ensemble de l'article. IV
Je dédie ce mémoire à ma famille Messier- Martin, amis et collègues qui m'ont soutenue tout au long de mes longues années d'étude. Table des matières Résumé i Avant-Propos ii Table des matières v Liste des tableaux vi Liste des figures vii Liste des abréviations viii CHAPITRE 1 : INTRODUCTION GÉNÉRALE 1 1 Pathologies buccales associées à C. albicans 1 1.1 Candidose buccale, non-prothétique 1 1.2 Stomatite prothétique 2 2 Traitement des candidoses buccales 2 3 Candida albicans 3 3.1 Généralités 3 3.2 Facteurs de virulence de C. albicans 4 3.2.1 Activités protéolytiques 5 3.2.2 Transition phénotypique ou «switch» 5 3.2.3 Capacité d'adhérence 6 3.2.4 Formation du biofilm 7 4 Polyphenols des plantes , 7 4.1 Généralités 7 4.2 Effets connus des polyphenols sur la santé buccale 9 4.3 Effets connus des polyphenols sur C. albicans 10 4.3.1 Effets connus des polyphenols de la réglisse sur C. albicans 11 Hypothèse de travail 12 Objectifs 12 Pertinence de la recherche 12 CHAPITRE 2 : Article 1 : Beneficial properties of licorice for oral health -A review 14 CHAPITRE 3 : Article 2 : Effect of licorice compounds licochalcone A, glabridin and glycyrrhizic acid on growth and virulence properties of Candida albicans 34 CHAPITRE 4 : Article 3 : Inhibition of Candida albicans biofilm formation and yeast- hyphal transition by 4-hydroxycordoin 53 CHAPITRE 5: CONCLUSION 67 Bibliographie 70 VI
Liste des tableaux Tableau 1 : Tableau 1 de l'article 1 : Composition chimique de la réglisse (Radix Glycyrrhizae) 32
Tableau 2 : Tableau 1 de l'article 2 : Valeurs des CMI et CMF (ug mL"1) des composés de la réglisse et du nystatin pour C. albicans 49
Tableau 3 : Tableau 2 de l'article 2 : Effets synergiques des composés de la réglisse et du nystatin sur l'inhibition de la croissance de C. albicans 50
Tableau 4 : Tableau 3 de l'article 2 : Effet cytotoxique de la licochalcone A, de la glabridine et de l'acide glycyrrhizique sur les cellules épithéliales buccales 51 Vil
Liste des figures Figure 1 : Microscopie en contraste de phase de C. albicans 4
Figure 2 : Figure 1 de l'article 1 : Feuillage et racine de Radix Glycyrrhizae (réglisse)....31
Figure 3 : Figure 1 de l'article 2 : Effets de la licochalcone A sur la formation du biofilm par C. albicans 52
Figure 4 : Figure 2 de l'article 2 : Effets de la licochalcone A (A) et de la glabridine (B) sur la transition blastospore-hyphe chez C. albicans 52
Figure 5 : Figure 1 de l'article 3 : Structure chimique du 4-hydroxycordoin 65
Figure 6 : Figure 2 de l'article 3 : Effets du 4-hydroxycordoin sur la formation de biofilm par C. albicans 65
Figure 7 : Figure 3 de l'article 3 : Effets du 4-hydroxycordoin sur la transition blastospore- hyphe chez C. albicans 66 vin
Liste des abréviations
C. albicans Candida albicans CMF Concentration minimale fongicide CMI Concentration minimale inhibitrice COX-2 Cyclooxygénase-2 EGCG Épigallocatéchine-3-gallate G. glabra Glycyrrhiza glabra G. uralensis Glycyrrhiza uralensis MMP Metalloproteinase matricielle Hg mL"1 Microgramme par millilitre P. gingivalis Porphyromonas gingivalis % Pourcentage Sap Secreted aspartyl protease SIDA Syndrome d'immunodéficience acquise S. mutans Streptoccocus mutons VEH Virus de l'immunodéficience humaine YNB Yeast Nitrogen Base YPD Yeast Peptone Dextrose CHAPITRE 1 : INTRODUCTION GENERALE
1 Pathologies buccales associées à C. albicans
1.1 Candidose buccale, non-prothétique La candidose buccale est une infection généralement causée par la levure C. albicans, laquelle fait souvent partie de la microflore commensale des cavités buccale et vaginale, et du tractus gastro-intestinal. La présence seule de ce mycète n'est pas suffisante pour causer la maladie. Il doit y avoir une certaine invasion des tissus par ce microorganisme, qui est souvent superficielle, et qui ne se produit que dans des circonstances favorables. D'autres sites communs de la candidose sont la peau, le vagin, le tractus gastro-intestinal, les voies urinaires et les poumons. Habituellement, la candidose buccale, également appelée muguet, demeure localisée, mais peut parfois s'étendre au pharynx, aux poumons ou à la circulation sanguine, avec un potentiel de complications fatales. Ces infections fongiques sont rarement retrouvées chez les sujets immunocompetents; elles affectent plutôt les nouveaux nés, les patients immunocompromis (ex. SIDA), les patients atteints de certaines maladies chroniques (ex. diabète, avitaminose) (Shafer et al., 1974) ou ceux qui prennent des corticostéroides en inhalation ou des antibiotiques systémiques (ADA guide, 2003). Le SIDA a créé une population de patients à risque de développer des candidoses buccales. En effet, le muguet est une des infections opportunistes la plus commune chez les sidéens (Ampel, 1996). Cette levure est également responsable d'un grand nombre d'infections systémiques et cardiovasculaires diagnostiquées dans les hôpitaux (Calderone 2002; Gudlaugsson et al, 2003). Les avancées de la médecine moderne ont permis de prolonger la durée de vie de certaines populations immunocompromises à risque de développer des infections à C. albicans.
Les manifestations cliniques de la candidose buccale consistent en des plaques blanchâtres légèrement surélevées, détachables, et laissant parfois apparaître une muqueuse inflammée. Ces plaques se retrouvent principalement au niveau de la langue et des muqueuses buccales, quoiqu'elles peuvent être également généralisées. Au niveau des commissures, une chélite angulaire peut apparaître, laquelle constitue une infection à levure qui cause la fissuration et l'inflammation des coins de la bouche. Les candidoses sont souvent asymptomatiques, quoique certains patients peuvent avoir une dysgueusie ou une sensation de brûlure dans la région affectée (Shafer et al., 1974).
1.2 Stomatite prothétique La stomatite prothétique se développe chez des porteurs de prothèses dentaires complète ou partielle et se définit comme une inflammation de la muqueuse palatine (localisée ou généralisée) en contact avec la prothèse avec ou sans hyperplasie tissulaire (Shafer et al., 1974). En 1970, Budtz-Jorgensen et Bertram ont démontré que les levures pouvaient être cultivées chez 90 % des patients ayant une stomatite prothétique mais chez seulement 40 % des patients qui étaient des porteurs sains de prothèse dentaire. Dagistan et coll. (2008) ont suggéré que les infections à levure ne sont pas le seul facteur prédisposant à la stomatite prothétique, mais qu'elles jouent cependant un rôle majeur dans cette pathologie. Une revue de littérature récente sur le sujet a révélé qu'il existe des résultats d'études contradictoires concernant le rôle des matériaux de regarnissage de prothèse, des propriétés de la salive et des interactions des levures et bactéries dans la stomatite prothétique (Pereira-Cenci et al., 2008).
2 Traitement des candidoses buccales Pour traiter les candidoses buccales non-reliées au port de prothèses dentaires, des agents antifongiques topiques et/ou systémiques doivent être prescrits. De plus, l'usage des corticostéroides en inhalation doit cesser ou alors le palais doit être protégé lors de l'inhalation. Pour traiter les stomatites prothétiques causées par C. albicans, il faut interdire le port de la prothèse durant la nuit, rebaser les prothèses mal adaptées, prescrire des antifongiques topiques à l'intrados de la prothèse el/ou systémiques dans lès cas plus sévères et renforcir les mesures d'hygiène (nettoyage de la prothèse et du palais). Pour les cas réfractaires au traitement, il peut être proposé de procéder à l'ablation des lésions au palais par électrochirurgie (Shafer et al, 1974). Les principaux fongiques utilisés dans le traitement des candidoses buccales proviennent des classes polyenes et azolés. Les polyenes, comme l'amphotéricine B et le nystatin, se lient de façon irréversible à l'ergostérol, une composante majeure de la membrane cytoplasmique de C. albicans. Ceci résulte en la formation de pores dans la membrane et entraîne la mort de la levure. L'amphotéricine B peut être administrée de façon topique ou systémique et son dosage est limité par sa néphrotoxicité (Ghannoum et al., 1999). L'ergostérol peut aussi être affecté par les azolés, tels que le fluconazole et le kétoconazole, via une inhibition de sa synthèse lors de l'interaction de ces molécules avec un enzyme dépendant du cytochrome P450 (Johnson et al., 1995). Ils sont utilisés de façon topique, mais aussi systémique dans le cas du fluconazole. Les souches de C. albicans des patients infectés au VIH présentent souvent une résistance à ce médicament. Le dosage des azolés est limité par leur hépatotoxicité (Rex et al., 1995). La résistance de C. albicans aux antifongiques semble impliquer certains de ses traits de virulence et ce sujet sera traité dans une autre section.
3 Candida albicans
3.1 Généralités La levure C. albicans, un membre de la microflore normale humaine, est retrouvée chez 60 % des adultes sains (Hellstein et al., 1993), 7.1 % des nouveaux-nés à leur jour de naissance et 96 % à l'âge de un mois (Russel et al, 1973). Elle peut parfois devenir un sérieux pathogène opportuniste chez les sujets immunocompromis, dont les patients atteints du diabète, du cancer, ayant subi une transplantation d'organes, ou contracté le SIDA. Parmi les levures pathogènes, C. albicans est l'agent causal prédominant des candidoses humaines (Kam et Xu, 2002). Cette levure est dimorphique; elle se présente sous forme de levure «blastospore» ou sous forme filamenteuse «hyphe». Des analyses d'isolats de patients montrant une candidose suggèrent que la majorité de ces infections sont causées par des souches commensales de la cavité orale, du tractus gastro-intestinal et du canal vaginal (Kim et al., 2002). C. albicans possède de nombreux facteurs de virulence qui constituent maintenant des cibles pour les nouveaux traitements antifongiques (Alksne et al., 2000). Figure 1 : Microscopie en contraste de phase de C. albicans. a) blastospores, b) hyphes.
Source : Laboratoire du Dr Jean Barbeau (Université de Montréal)
3.2 Facteurs de virulence de C. albicans Selon Soil (1992), les caractères phénotypiques de C. albicans qui ont déjà été considérés comme contribuant à sa virulence sont la formation d'hyphes, la sécrétion de proteases acides et de phospholipases, de même que l'adhésion sélective à l'épithélium. D'autres caractéristiques qui pourraient contribuer à sa virulence sont la capacité de neutraliser le système immunitaire, de résister aux agents antifongiques et de vivre de façon commensale dans plusieurs parties du corps chez l'humain sain. La transition morphologique de blastospore à hyphe, qui est réversible, peut affecter plusieurs de ces traits de virulence (Soil, 1992). Lo et coll. (1997) ont précédemment démontré que les mutants non filamenteux de C. albicans ne sont pas virulents dans un modèle animal de souris. 3.2.1 Activités protéolytiques C. albicans sécrète des aspartyls proteases (Saps) qui pourraient être responsables de son pouvoir d'invasion tissulaire. Les Saps sont des enzymes hydrolytiques provenant d'une famille de gènes et incluent 10 membres (Sapl à SaplO) de poids moléculaire variant de 35 à 50 kDa (Monod et al, 1998). Elles sont ancrées à la membrane cellulaire, incorporées dans la paroi cellulaire ou sécrétées dans le milieu extracellulaire. Leurs rôles principaux sont liés à l'assimilation des nutriments par la cellule, la pénétration et l'invasion des tissus, et l'évasion de la réponse immunitaire. Il a été démontré que différents gènes codant pour différentes Saps étaient activés selon le stade et le site d'infection (buccale ou vaginale) (Hube et al, 1994; De Bernardis et al., 1995; Schaller et al., 2003). Sapl, Sapl et Sap3 seraient importantes dans les dommages tissulaires et l'invasion de l'épithélium buccal, alors que Sap4, Sap5 et Sap6 joueraient un rôle dans les infections systémiques (Naglik et al., 1999). Sapl dégrade la matrice extracellulaire et les protéines de surface de l'hôte, telles que la kératine, le collagène et la murine, mais aussi les protéines du système de défense de l'hôte telles que l'IgA sécrétoire et la lactoferrine salivaire (Ruchel et al., 1983). Sap9 et SaplO auraient un rôle dans l'adhésion à l'épithélium, alors que les rôles de Sapl et SapS sont peu connus (Albrecht et al., 2006). En 1992, Soil a démontré que les souches qui sécrétaient le moins de proteases étaient les moins virulentes. Diverses études ont également établi une corrélation entre le niveau de proteases acides sécrétées in vitro et la pathogénicité des souches (Korting et al, 1999, Cauda et al, 1999; Ollert et al, 1995; Hube et al, 1994). Avant l'arrivée des agents antirétroviraux, 90 % des sujets infectés au VIH souffraient de candidose buccale (Ollert et al., 1995). L'utilisation de saquinavir et d'indinavir, des inhibiteurs de proteinases du VIH, a diminué la fréquence de ces infections buccales, puisque ces molécules avaient également un effet inhibiteur direct sur les Saps de C. albicans (Korting et al., 1999). Cauda et coll. (1999) ont démontré que les inhibiteurs des proteinases du VEH prévenaient directement la récurrence de la candidose buccale chez les patients infectés au VIH.
3.2.2 Transition phénotypique ou «switch» Certains auteurs ont avancé l'hypothèse que la virulence de C. albicans pourrait être reliée à la forme filamenteuse suite à une transition phénotypique de la forme blastospore à la forme hyphe (Jones et al, 1994; Slutsky et al., 1985). Ce phénomène de transition morphologique est connu sous l'appellation «switch» (Soil, 1992). Afin de pénétrer dans les tissus, les levures doivent premièrement adhérer à l'épithélium. Les cellules adhérentes produisent ensuite des hyphes dans les tissus, lesquels mènent à la formation de branches et libèrent des blastospores. Des différences d'adhérence entre les formes blastospores et hyphes ont été démontrées (Anderson et Odds, 1985). Radford et coll. (1994) ont étudié l'adhérence de C. albicans aux prothèses dentaires en tenant compte du phénomène de switch. Ils ont conclu que les hyphes provenant des colonies switchées sont plus adhérents à l'acrylique que les blastospores provenant des colonies non-switchées. C. albicans peut devenir résistante aux agents antifongiques par transition phénotypique. Une corrélation a été établie entre le niveau de résistance aux antifongiques de souches de C. albicans et leur capacité de former des hyphes en présence des antifongiques du groupe azole (Ha et ai, 1999). La plupart des souches de C. albicans colonisant la cavité buccale d'individus VIH positifs avaient une haute fréquence de switch, même s'ils n'avaient pas encore développé cliniquement un premier épisode de candidose buccale (Vargas et ai, 2000). Ces mêmes souches étaient déjà plus résistantes aux antifongiques que la majorité des souches commensales colonisant des individus sains. Plusieurs études suggèrent qu'il existerait une plus grande fréquence du phénotype hyphe de C. albicans dans les sites d'infection fongique (Soil etal, 1987; Hellstein etal., 1993; Jones etal, 1994).
3.2.3 Capacité d'adhérence La salive entière aurait un double effet sur l'adhésion de C. albicans à l'acrylique des prothèses dentaires. En effet, elle diminue l'adhésion des hyphes, mais augmente celle des blastospores. La salive ne semble pas réduire la production d'hyphes (Elguezabal et ai, 2008). Moura et coll. (2006) ont comparé l'adhésion de C. albicans à de l'acrylique polymérisé à la chaleur (méthode conventionnelle) et à de l'acrylique polymérisé au micro• onde, en présence ou absence de salive. Ds ont démontré que la salive diminue globalement l'adhérence de C. albicans à l'acrylique, peu importe son type de polymérisation. Leurs résultats ont aussi démontré que la porosité et l'énergie de surface des différents acryliques n'influencent pas l'adhésion de C. albicans. Par contre, He et coll. en 2006 ont rapporté que le type de polymérisation (à froid versus à chaud) de l'acrylique utilisé pour fabriquer (polymérisation à chaud) ou réparer (polymérisation à froid) la prothèse jouerait un rôle important dans l'adhésion à la prothèse de C. albicans, Candida glabrata et Candida krusei. L'acrylique polymérisé à chaud permettait une plus faible capacité d'adhésion de Candida par rapport à l'acrylique polymérisé à froid.
3.2.4 Formation du biofilm Les biomatériaux, tels que les stents, shunts, prothèses (dentaire, vocale, valve cardiaque et genou artificiel), lentille cornéenne, implant mammaire, tubes endotrachéaux, pacemakers et autres cathéters facilitent la colonisation par C. albicans et la formation subséquente de biofilms. Les biofilms sont des communautés hétérogènes de microorganismes organisés dans l'espace. Ils sont formés de cellules microbiennes entourées d'une matrice extracellulaire. Les infections associées aux biofilms sont difficiles à traiter, à cause de leur résistance aux antifongiques, notamment le fluconazole et l'amphotéricine B (Cao et al., 2008). Jabra-Rizk et coll. (2006) ont rapporté que les microorganismes sous forme de biofilm seraient de 50 à 500 fois plus résistants aux agents antimicrobiens que sous leur forme planctonique. Par examen microscopique, Ramage et coll. (2004) ont démontré la présence d'un biofilm contenant C. albicans sur des échantillons d'acrylique de prothèse de patients atteints de stomatite prothétique. Les isolats de C. albicans ont été capables de reproduire des biofilms in vitro. Ces biofilms ont montré une résistance accrue aux antifongiques. Les auteurs concluent donc que les biofilms à C. albicans ont un rôle à jouer dans la stomatite prothétique, et il est permis d'extrapoler que ces biofilms compromettent le traitement aux antifongiques.
4 Polyphenols des plantes
4.1 Généralités Les plantes contiennent toutes des composés phénoliques, qui sont pour la plupart des metabolites secondaires, en quantité et qualité inégales selon les stades physiologiques, les organes, les tissus et les espèces. Ils ont entre autre un rôle essentiel dans l'interaction de la plante avec son environnement; par exemple les pigments d'origine phénolique attirent les 8 insectes pour assurer la pollinisation. Les fruits, le vin rouge, le thé et le café sont les principales sources alimentaires de polyphenols, mais on en trouve aussi dans les céréales, légumes et légumineuses (Sarni-Manchado et Cheynier, 2006). Ces composés phénoliques donnent une qualité nutritionnelle et sensorielle (astringence, couleur, etc.) aux végétaux consommés par l'être humain et sont largement utilisés dans les industries agroalimentaire et pharmaceutique. Dans les domaines agroalimentaires, les polyphenols sont utilisés pour éviter le brunissement enzymatique, ce qui rend possible la transformation des produits végétaux. Les effets des polyphenols sur la santé des humains sont maintenant reconnus; certaines molécules exercent une action anticancer, antioxydante (retardent le vieillissement cellulaire), antiinflamatoire et antimicrobienne. Parmi les polyphenols les plus connus, il y a entre autre le resveratrol qui est une molécule isolée du raisin et bien connu pour être un agent anticancer naturel et un agent contre l'athérosclérose (Jung et al., 2007). Le thé est également reconnu pour ses propriétés antioxidantes et pourrait même contrôler certains types de cancers (Siddiqui et al., 2009). La curcumine a des propriétés inhibitrices intéressantes sur l'adipogenèse des adipocytes et sur l'obésité chez les rats (Ejaz et al., 2009). Des polyphenols ont été utilisés récemment pour stabiliser les matrices d'élastine tubulaire pour la synthèse de greffes vasculaires par ingénierie tissulaire (Chuang et al, 2009).
Tous les polyphenols ont une structure commune caractérisée par la présence d'un ou plusieurs cycles benzènes portant au moins une fonction hydroxyle. Les acides aminés aromatiques phenylalanine et tyrosine sont à l'origine de la formation de la plupart des polyphenols. La classe des phénols simples (Ce), tel que le catechol du thé, présente le squelette carboné le plus simple des polyphenols. Les stilbènes (C6-C2-C6) incluent les molécules comme le resveratrol du raisin. Les flavonoïdes (C15 : C6-C3-C6), retrouvés entre autre dans les fruits, légumes, et fleurs, sont un ensemble de milliers de molécules regroupées en une dizaine de classes, dont les flanovols (ex. quercétine), les anthocyanes qui donnent les pigments rouges ou bleus des fruits et des fleurs et les flavanes qui sont à l'origine des tannins. Ces derniers sont responsables de l'astringence de plusieurs légumes et fruits et ont une grande importance économique et écologique. Les tannins se divisent en deux classes: hydrolysables et condensés. Les tannins hydrolysables peuvent être dégradés par hydrolyse enzymatique ou chimique, et libèrent de l'acide gallique ou ellagique (partie phénolique). Ils sont retrouvés dans de nombreux arbres et arbustes, dont ceux du genre Rhus. Les tannins condensés sont résistants à l'hydrolyse et seules les attaques chimiques fortes peuvent les dégrader. Lors de traitement acide à chaud, ils se transforment en pigments rougeâtres. Es sont abondants dans des boissons fermentées ou non (cidre, vin, thé, etc.) et dans un grand nombre de fruits (pomme, fraise, etc.) (Sarni-Manchado et Cheynier, 2006).
4.2 Effets connus des polyphenols sur la santé buccale Les effets des polyphenols au niveau de la santé buccale ont surtout été étudiés dans le cas de la carie dentaire et la maladie parodontale. La carie dentaire résulte de la sécrétion d'acide par certaines bactéries, dont Streptoccocus mutans, entraînant une déminéralisation de l'émail et de la dentine. Certains polyphenols, dont l'épigallocatéchine gallate du thé vert (EGCG), le cacao non-fermenté, le thé vert entier et la graine de raisin rouge sont bactériostatiques et préviennent la sécrétion d'acide par S. mutans (Smullen et al., 2007). Les polyphenols du thé sont anticariogènes par leurs propriétés antimicrobiennes et non par une action de reminéralisation de l'émail (Li et al., 2004). L'extrait de thé oolong modifie entre autre les propriétés d'adhérence à l'émail de S. mutans (Matsymoto, 1999), un phénomène qui a également été observé avec les polyphenols du houblon (Tagashira et ai, 1997). L'extrait de thé oolong a permis de diminuer l'incidence de la carie chez les rats dans un modèle expérimental (Ooshima et al., 1993).
La maladie parodontale est caractérisée par la perte de tissus de support de la dent causée par l'action des ostéoclastes et métalloprotéinases matricielles (MMP) suite à la stimulation d'une série de réactions inflammatoires chez l'hôte par certaines bactéries pathogènes, dont Porphyromonas gingivalis, aggravant la destruction du parodonte. Selon Kou et coll. (2008), le houblon aurait des effets prometteurs pour la prévention ou l'atténuation des parodontites, puisqu'il serait un inhibiteur de la réponse inflammatoire induite par les vésicules de P. gingivalis, qui sont un important facteur de virulence de cette bactérie. Un groupe de chercheurs japonais a démontré une association inverse modeste entre la prise de 10 thé vert, un breuvage très populaire, et la maladie parodontale (Kushiyama et al, 2009). Une autre étude a rapporté que l'EGCG prévenait in vitro la résorption de l'os alvéolaire, qui est observée lors de la parodontite, en inhibant l'expression de la MMP-9 par les ostéoclastes de même que la formation des ostéoclastes (Yun et al, 2004). Chez les rats, la mangiférine de la mangue a réduit la perte de l'os alvéolaire en inhibant l'expression de la COX-2 ainsi que la migration et l'adhésion des leucocytes (Carvalho et al., 2009). Le ligament parodontal, qui se renouvelle continuellement, est la structure comprise entre l'os alvéolaire et le cément de la racine de la dent. Les fibroblastes du ligament parodontal permettent de maintenir en santé ce dernier. Les polyphenols du thé vert a eu un effet positif sur la prolifération de ces cellules dans un modèle in vitro, alors que les polyphenols de la pomme et du houblon ont eu des effets protecteurs contre P. gingivalis sur celles-ci (Inaba et al., 2005). Les composantes de la canneberge ont inhibé la production de molécules médiatrices de l'inflammation par les fibroblastes gingivaux humains (Bodet et al., 2007), ont protégé les macrophages et cellules épithéliales de la muqueuse buccale humaine contre certains parodontopathogènes (La et al., 2009) et ont inhibé la production de proteinases par certains pathogènes parodontaux (Bodet et al., 2006).
4.3 Effets connus des polyphenols sur C. albicans Plusieurs polyphenols ont démontré un pouvoir fongicide envers C. albicans, tel que le resveratrol, par accumulation de trehalose intracellulaire (Jung et al., 2007), le EGCG, par inhibition de la synthèse d'ergostérol en agissant sur le métabolisme du folate (Navarro- Martinez et al., 2006) et l'acide benzoique isolée du jus de canneberge qui inhibe totalement la croissance de C. albicans (Swartz et Merek, 1968). Une synergie entre le EGCG et les antifongiques azolés a été démontrée (Navarro-Martinez et al., 2006). Une étude réalisée par Hirasawa et coll. en 2004 a conclu que le EGCG augmente l'activité antifongique de l'amphotéricine B et du fluconazole sur les souches résistantes et non- résistantes de C. albicans. Il est permis de penser que ce polyphenol pourrait diminuer la quantité d'antifongiques nécessaires pour le traitement de mycoses, limitant ainsi les effets secondaires des antimycotiques. L'étude de Han (2007) a démontré que l'utilisation en combinaison d'un extrait de pépin de raisin et d'amphotéricine B permettait de réduire la dose requise d'amphotéricine B de plus de 75 %, à cause de l'effet synergique avec l'extrait 1] de pépin de raisin. Certains facteurs de virulence de C. albicans peuvent être influencés par les polyphenols, tel que l'extrait de feuille Streblus asper qui réduit la formation d'hyphes et l'adhérence de C. albicans aux cellules épithéliales (Taweechaisupapong et al., 2005). De plus, Taweechaisupapong et coll. (2006) ont démontré que ce même polyphenol réduit l'adhérence de C. albicans à l'acrylique de 20 %. Cette réduction d'adhérence serait due à un changement dans la paroi cellulaire de Candida. La formation du biofilm a également été inhibée jusqu'à 70 % par le baicalein provenant de l'herbe chinoise Scutellaria baicalensis (Cao et al. en 2008).
4.3.1 Effets connus des polyphenols de la réglisse sur C. albicans Plusieurs effets bénéfiques de certains polyphenols de la réglisse pour les infections buccales, incluant les candidoses, ont été rapportés. Le premier article scientifique du mémoire est une revue de la littérature exhaustive traitant de ce sujet. 12
Hypothèse de travail
Les polyphenols des plantes peuvent inhiber la croissance de C. albicans et affecter certains de ses facteurs de virulence.
Objectifs
1. Déterminer la concentration minimale inhibitrice et la concentration minimale fongicide de divers polyphenols pour C. albicans; 2. Étudier l'effet des polyphenols sur la formation de biofilm de C. albicans; 3. Investiguer la capacité des polyphenols à inhiber Ja transition blastospore-hyphe chez C. albicans.
Pertinence de la recherche
À cause du nombre grandissant de patients immunocompromis, tels que les diabétiques, les sidéens et les cancéreux, le traitement des infections fongiques buccales et systémiques devient problématique pour le praticien. Certaines souches de C. albicans sont de plus en plus résistantes aux antifongiques, qui eux-mêmes causent souvent des effets secondaires importants, d'où la nécessité d'identifier d'autres molécules pour inhiber la croissance ou les propriétés pathogènes de cette levure dimorphique. De plus, la stomatite prothétique associée à la présence de levures est souvent un défi à relever pour le spécialiste de la santé dentaire, puisque les traitements antifongiques ne sont pas efficaces dans tous les cas.
Le but de notre projet est d'étudier les effets de certains polyphenols sur la croissance, la survie, la formation de biofilm et la transition blastospore-hyphe de C. albicans. Nous croyons, suite à une analyse de la littérature, qu'il y a un grand potentiel d'identifier des 13 polyphenols qui seuls ou en association avec des antifongiques seraient fort utiles pour traiter les patients souffrant d'infection à C. albicans. De plus, un polyphenol réduisant la croissance et la formation du biofilm de C. albicans sur l'acrylique des prothèses dentaires pourrait être utilisé en trempage nocturne en prévention d'une stomatite prothétique. 14
CHAPITRE 2
Article 1: Soumis pour publication dans la revue Oral Diseases Beneficial properties of licorice for oral health - A review
C. Messier,1 F. Epifano,2 S. Genovese,2 D. Grenier1*
Groupe de Recherche en Écologie Buccale, Faculté de Médecine Dentaire, Université Laval, 2420 Rue de la Terrasse, Québec City, QC, Canada, G1V 0A6; 2Dipartimento di Science del Farmaco, Università G. D'Annunzio, Via Dei Vestini 31, 66013 Chieti Scalo, Chieti, Italy
Short title: Licorice and oral health
* Corresponding author. Mailing address: Groupe de Recherche en Écologie Buccale, Faculté de médecine dentaire, Université Laval, 2420 Rue de la Terrasse, Quebec City, Quebec, Canada, G1V 0A6 Phone: (418) 656-7341. Fax: (418) 656-2861. E-mail: [email protected] 15
Résumé
Les racines de Glycyrrhiza glabra et de Glycyrrhiza uralensis, connues communément sous le nom réglisse, ont été utilisées depuis des milliers d'années comme remède thérapeutique traditionnel. La réglisse contient plusieurs classes de metabolites secondaires, incluant les chalcones, les coumarins, les saponins et les flavonoïdes, auxquels certains bénéfices pour la santé humaine ont été associés. Des recherches récentes suggèrent que les extraits de réglisse et leurs ingrédients actifs possèdent un potentiel intéressant pour l'utilisation en suppléments en vue d'améliorer la santé buccale. Dans cet article, les effets des extraits de la réglisse et de leurs ingrédients actifs sur les pathogènes buccaux et la réponse immunodestructive de l'hôte, impliqués dans les maladies buccales, tels que la carie dentaire, la parodontite, la candidose et les ulcères aphteux récurrents, sont présentés. Les résultats des quelques études cliniques sur les bénéfices potentiels des éléments de la réglisse dans le maintien d'une bonne santé buccale sont discutés. 16
Abstract
The roots of Glycyrrhiza glabra and Glycyrrhiza uralensis, commonly known as licorice, have been used for thousands of years as a traditional herbal remedy. Licorice contains several classes of secondary metabolites, including chalcones, coumarins, saponins, and flavonoids, with which human health benefits have been associated. Recent research suggests that licorice extracts and their active ingredients possess a valuable potential for improving oral health. This paper reviews the effects of licorice extracts and their active ingredients on oral microbial pathogens and the host immunodestructive response involved in oral diseases such as dental caries, periodontitis, candidosis, and recurrent aphtous ulceration. It also summarizes results of the few clinical studies that investigated the potential beneficial effects of licorice constituents for preventing oral diseases. 17
INTRODUCTION Licorice root (Radix Glycyrrhizae) is obtained from perennial plants native to Mediterranean countries, central to southern Russia, and certain regions of Asia. Glycyrrhiza glabra L. and Glycyrrhiza uralensis Fisch. (Fam. Leguminosae) roots have been used for thousands of years as a traditional herbal remedy. Licorice contains several classes of secondary metabolites, including chalcones, coumarins, saponins, and flavonoids, with which numerous human health benefits have been associated (Nassiri Asl and Hosseinzadeh, 2008). For example, licorice has shown therapeutic properties for peptic ulcers (Aly et al., 2005). In vitro studies have associated part of this health benefit with the anti-Helicobacter pylori properties of a number of licorice-derived molecules, including glabridin and licochalcone A (Fukai et al., 2002). In Japan, a standardized licorice extract containing glycyrrhizin has been intravenously used to treat chronic hepatitis B and improve liver functions (Sato et al., 1996; Stickel and Schuppan, 2007). In recent years, licorice extracts and their active ingredients have been receiving increasing attention as cancer treatments. Licochalcone A possesses antiangiogenic activity against implanted colon cancer in mice, indicating that it shows promise for treating certain malignant tumors where angiogenesis is involved in their progression (Kim et al., 2010). Glabridin also inhibits human breast adenocarcinoma cells migration, invasion, and angiogenesis in vitro and has been proposed as a potential therapeutic molecule for treating cancer (Hsu et al., 2010). While serious side effects associated with licorice are infrequent, excess licorice consumption may cause hypertension (Touyz, 2009) due to the ability of glycyrrhizin to inhibit the conversion of precursor Cortisol to cortisone by suppressing 11-beta hydroxysteroid dehydrogenase (Quinkler and Stewart, 2003).
In recent decades, licorice has attracted the attention of dental researchers. This paper reviews the effects of licorice extracts and their active ingredients on oral microbial pathogens and the host immunodestructive response involved in oral diseases such as dental caries, periodontitis, candidosis, and recurrent aphtous ulceration. It also summarizes results of the few clinical studies that investigated the potential beneficial effects of licorice constituents for preventing oral diseases. 18
DESCRIPTION OF THE PLANT AND CHEMICAL COMPOSITION While the genus Glycyrrhiza includes about 30 species, the classic main botanical sources of Radix Glycyrrhizae, or licorice root, are G. glabra L. and G. uralensis Fisch. (Fam. Leguminosae) (Fig. 1). G. glabra is native to Mediterranean countries and certain regions of Asia. It is a herbaceous perennial shrub that grows up to 1 m in height and has 7 to 15- cm-long pinnate leaves with 9-17 leaflets. The licorice shrub has an extensive root system composed of a taproot and numerous runners. The taproot, which has been harvested for medicinal uses as far back as 6000 years, is soft and fibrous and has a bright yellow interior. It is 1.5 cm long and is subdivided into 3-5 subsidiary roots about 1.25 cm long from which the horizontal woody stolons arise. The stolons, which can reach 8 m in length, together with the taproot, are the source of commercial licorice. Roots break with a fibrous fracture, revealing the yellowish interior with a characteristic odor and sweet taste. G. glabra grows best in deep, fertile, well-drained soils, in full sun and is harvested in the autumn two to three years after planting. G uralensis is a perennial herbaceous plant native to the Urals, Siberia, and the steppes and semi-desertic regions of East Asia. The stems are 40-70 cm tall, erect, simple or branched, shortly pubescent and, like the entire plant, are covered with small brownish glandules, sometimes with an admixture of glandular spinules. The leaflets are oblong-elliptic or ovate and are produced in pairs of 4 to 6. The root system is similar to that of G. glabra.
Licorice is produced from the unpeeled, dried roots and stolons of G. glabra and G. uralensis. Phytochemically, both plants are among the most well studied and contain several classes of secondary metabolites, the most abundant being triterpene and steroidal saponins, flavonoids, isoflavonoids, chalcones, and coumarins as well as minor amounts of aurones, benzofurans, phenols, pterocarpans, and stilbenes. Table 1 lists the metabolites that have been identified to date in licorice extracts.
DENTAL CARIES AND LICORICE Over the past 30 years, dental caries has declined in high socioeconomic populations but has increased in low-income and fragile elderly populations. Dental caries is defined as tooth demineralization caused by acidogenic/aciduric bacteria in dental plaque, also known 19 as dental biofilm. Streptococcus mutans is the primary acidogenic bacterial species in dental biofilms, where it metabolizes exogenous dietary carbohydrates, including sucrose, and generates lactic acid as a by-product (Takahashi and Nyvad, 2008). The accumulation of lactic acid in the dental biofilm ultimately results in a localized drop in pH and subsequent demineralization of the tooth enamel, which marks the onset of dental decay.
While the anticariogenic properties of licorice have been known for over 30 years, few studies on this aspect have been published. Glycyrrhizin, a sweet-tasting compound (50 times sweeter than sucrose) and the main triterpenoid saponin glycoside in G. glabra, has been used in most studies on 5. mutans and dental caries. Segal et al. tested the effect of glycyrrhizin on the adherence properties and growth of 5. mutans and found that while the growth of 5. mutans was not affected in the presence of sucrose, its adherence to a glass surface was almost completely inhibited, even at low concentrations (0.5 to 1%) (Segal et al., 1985). A clinical trial using a split-mouth model to apply glycyrrhizin (in the absence of oral hygiene) showed that glycyrrhizin had a minor effect on controlling dental plaque formation after 3 to 4 days (Steinberg et al., 1989). Another pilot study showed that toothbrushing with a toothpaste containing 0.25% or 0.50% glycyrrhizin for up to 42 days had no effect on the plaque index (Goultschin et al., 1991). The authors suggested that the lack of effect may have been due to an insufficient concentration of glycyrrhizin or to a chemical incompatibility of glycyrrhizin with other ingredients in the toothpaste (Goultschin et al., 1991). A more recent in vivo study reported that toothbrushing three times a day with a gel containing 2.5-10% licorice extract (22% glycyrrhizin) for two weeks failed to reduce or change the composition of dental plaque (Sôderling et al., 2006) compared to a control gel containing 8% corn starch and 25% maltitol syrup. However, in an in vivo acid production test, the licorice-containing gel was shown to inhibit acid production (Sôderling et al., 2006). Gedaria et al. (1986) reported that glycyrrhizin increases fluoride uptake and protects enamel against demineralization.
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Glycyrrhizinic acid, the aglycon of glycyrrhizin, from G. glabra reduces enamel dissolution in vitro by inhibiting acid production by dental plaque (Edgar, 1977). Four additional compounds isolated from G. uralensis have been reported to have antibacterial activity 20 against S. mutans (He et al., 2006). The minimal inhibitory concentrations (MICs) for glycyrrhizol A, 6,8-diisoprenyl-5,7,4'-trihydroxyflavone, glycyrrhizol B, and gancaonin G are 1,2, 32, and 125 ug4nl, respectively (He et al., 2006). Recently, a sugar-free licorice (G. «ra/e/u/s^containing lollipop was developed and commercialized as a new approach for fighting tooth decay in young children who are at risk for dental caries (C3 Jian/Intelliherb Inc., Inglewood, CA). Peters et al., (2010) carried out a pilot study with the licorice root extract lollipop and showed that when used twice daily, it significantly reduces both number and relative percent of S. mutans in high-risk pre-school children.
PERIODONTAL DISEASE AND LICORICE Periodontal diseases are divided into two major types: gingivitis and periodontitis. Gingivitis is characterized by an inflammation limited to the unattached gingiva, whereas periodontitis is a progressive, destructive disease that affects all supporting tissues of the teeth, including the alveolar bone. The main pathogens associated with periodontitis include Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Aggregatibacter actinomycetemcomitans (Feng and Weinberg, 2006). These bacteria possess a variety of virulence characteristics that allow them to colonize subgingival sites, escape host defenses, and cause tissue damage (Feng and Weinberg, 2006). The persistence of the host immune response is also a determining factor in the progression of the disease (Garlet, 2010). In response to pathogenic bacteria, mucosal and immune cells secrete various inflammatory mediators and matrix métalloprotéinases (MMPs), which can modulate periodontal tissue destruction (Garlet, 2010).
Based on in vitro studies, licorice has been recently been proposed as a potential candidate for the development of a new natural therapy to treat or prevent periodontitis. First, a G. uralensis licorice extract was reported to inhibit both the growth and biofilm formation by P. gingivalis, a major etiologic agent of chronic periodontitis (Bergeron et al., 2008). Second, human macrophages pre-treated with a licorice extract prior to being stimulated with A. actinomycetemcomitans or P. gingivalis lipopolysaccharide (LPS) secrete significantly less pro-inflammatory cytokines (interleukin-ip, interleukin-6, interleukin-8 and tumor necrosis factor-a), indicating that the extract has an anti-inflammatory property 21
(Bodet et al., 2008). Licoricidin and licorisoflavan A, two major isoflavonoids of the licorice extract, have been shown to be responsible for the anti-inflammatory effect (La et al., 2010). Interestingly, both molecules also inhibit MMP-7, -8, and -9 secretion by LPS- stimulated macrophages (La et al., 2010). The inhibition of pro-inflammatory cytokine and
MMP secretion appears to be due to reduced activation of transcription factor NF-KB p65 (Bodet et al., 2008, La et al., 2010), which plays a key role in the inflammatory response (Biesalski, 2007).
Sasaki et al. (2010) reported that 18P-glycyrrhetinic acid suppresses the LPS- and RANKL- induced phosphorylation of NF-KB pl05 in vitro, which provides further support for the ability of licorice to modulate the inflammatory response. When administered either prophylactically or therapeutically in interleukin-10-deficient mice infected with a virulent strain of P. gingivalis, 18P-glycyrrhetinic acid markedly reduces alveolar bone loss. 18fi- Glycyrrhetinic acid appears to inhibit the severity of periodontitis in a mouse model by inactivating transcription factor NF-KB pl05 (Sasaki et al., 2010).
ORAL CANDIDOSIS AND LICORICE Oral candidosis is an opportunistic infection of the oral cavity caused by an overgrowth of Candida species, the most common being Candida albicans (McCullough and Savage, 2005; Samaranayake et al., 2009). This yeast-like fungus is a normal commensal microorganism in the mouth and generally causes no problems in healthy people. Oral candidosis is initiated when the host immune system is depressed by drug therapies or systemic diseases. Denture stomatitis, which can develop in complete or partial denture wearers, is an inflammation of the palatal mucosa that is in contact with the denture (localized or generalized) and can be associated with soft tissue hyperplasia (McCullough and Savage, 2005; Samaranayake et al., 2009). While the physiological status of the host is a critical factor governing the initiation of oral candidosis, the pathogenic potential of C. albicans is also of utmost importance. This pathogen has developed an effective range of virulence factors and strategies to colonize the host, overcome host immune defenses, and cause tissue damage (Yang, 2003). 22
Few studies have investigated the effect of licorice on C. albicans. Motsei et al. (2003) tested organic solvent extracts of G. glabra for their antifungal effect on C. albicans. The MICs for the ethanol extract were in the range of 0.5 to 2 mg/ml (Motsei et al., 2003). A more recent in vitro study showed that glabridin has potent activity against amphotericin B- resistant strains of C. albicans, with MIC values ranging from 31.25 to 0.125 ng/ml (Fatima et al., 2009). 18-f3 Glycyrrhetinic acid, another compound isolated from G. glabra, reduces the growth of C. albicans in a pH-dependent manner at relatively low concentrations (6.25 ng/ml) (Pellati et al., 2008). In a recent study, Messier and Grenier (2011) investigated the effects of two licorice polyphenolic compounds (licochalcone and glabridin) on the growth, killing, biofilm formation, and adherence of C. albicans. The MICs of both compounds were 6.25-12.5 ng/ml, while the minimal fungicidal concentration was 12.5 ng/ml for glabridin and 100 ng/ml for licochalcone. They also acted in synergy with nystatin to inhibit the growth of C. albicans. Biofilm formation was inhibited by 30-60% with 0.2 ng/ml of licochalcone and 75-80% with 2 ng/ml. Glabridin had no effect on biofilm formation. A strong inhibitory effect (60-100%) on the transition blastospore-hyphal form was observed with 100 ng/ml licochalcone and glabridin. These findings suggest that licochalcone and glabridin show promise as a therapeutic agents for treating oral C. albicans infections.
Glycyrrhizin improves the resistance of thermally injured mice against C. albicans infections by inducing CD4+ Thl cells that suppress cytokine production by burn- associated cells (Utsunomiya et al., 1999). Animal studies have shown that liquiritigenin, a licorice flavonoid, has immunomodulating activity and can protect mice against disseminated candidosis by acting on the CD4+ Thl immune response (Lee et al., 2009).
RECURRENT APHTOUS ULCERATION AND LICORICE Recurrent aphtous ulceration is a painful oral inflammatory condition characterized by one or several ulcers of the oral mucosa (Scully et al., 2003). This inflammatory condition may have various origins, including infections (Sun et al., 1996), allergies (Boulinguez et al., 2003), and other immune reactions (Natah et al., 2000). It is a common oral condition 23 diagnosed by dentists in patients who consult for pain when they speak, eat, or swallow (Miller and Ship, 1977).
Some reports on the beneficial effect of licorice on aphtous ulceration have been published. One showed that a mouthwash containing a deglycerinized licorice extract had a significant anti-aphtous effect (Das et al., 1989). Moghadamnia et al. (2008) reported that licorice bioadhesive hydrogel patches provide significant symptomatic pain relief. Licorice patches also significantly reduce the diameter of the inflammatory halo and necrotic center (Moghadamnia et al., 2008). Another clinical trial using a dissolving oral patch containing a licorice extract (CankerMelts®) provided similar results (Martin et al., 2008).
CONCLUSIONS Recent research suggests that licorice extracts and a number of compounds isolated from the extracts have potential for use as supplements for improving oral health. Some active principles such as glabridin, licoricidin, licorisoflavan A, licochalcone, and glycyrrhizin also possess anti-microbial and anti-inflammatory properties. Purified molecules or a licorice extract containing them can be incorporated into mouthrinses, toothpastes, gels, chewing gums, or patches to prevent and treat oral diseases, including dental caries, periodontal disease, oral candidosis, and aphtous ulcers. However, additional clinical studies are required to validate these benefits. 24
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Figure 1. Radix Glycyrrhizae (licorice) shrub and root. 32
Table 1. Chemical composition of licorice root (Radix Glycyrrhizae). Compounds in bold are present in both G. glabra and G. uralensis. Those in plain text are only present in G. glabra, while those in italics are only present in G. uralensis.
Category Compound Reference Aurones Licoagroaurone U et al., 2000
Benzofurans Gancanoin I, Licocoumarone Fukai et al., 2002; Kuroda et al., 2003
Chalcones* 1,2-Dihydroparatocarpin, Echinatin. Isoliquiritigenin, Bai et al., 2003; Chin et al., 2007; Fukai Isoliquiritin, Isoliquiritin apioside, Isoprenylchalcone, et al., 1989; Fukai et al., 1990; Fukai et Licoagrochalcone B, Licoagrochalcone C, Licoagrochalcone D, al., 1996; Fukai et al., 1998; Fukai et al., Licobichalcone Licochalcone A, Licochalcone B, Licochalcone 2002; Hatano et al„ 1991; Hatano et al., C, Licuraside, Neolicuraside, Paratocarpin B 2000; Jayaprakasam et al., 2009; Kinoshita et al., 2005; Kitagawa et al., 1994a; Kitagawa et al., 1994b; Kitagawa et al., 1994c; Kondo et al., 2007; Kuroda et al., 2003; Li et al., 2000; li et al., 2005; Mieting et al., 1989; Mitscher et al., 1980; Raggi et al., 1995; Tamir et al., 2001 ; Tanaka et al., 2001 ; Tsukamoto et al., 2005; Vaya et al., 1997; Wang et al., 1991;
Coumarins Gancaonol A, Gancaonol B, Glabrocoumarin, Glycycoumarin, Bai et al., 2003; Fukai et al., 1989; Fukai Glycyrin, Glycyrol, lsoglycycoumarin, Isoglycyrol, Isotrifoliol, et al., 1990; Fukai et al., 1996; Fukai et Kanzonol W, Licofuranocoumarin, Licopyranocoumarin, 3-0- al., 1998; Fukai et al., 2002; Hatano et Methylglycyrol, Neoglycyrol, Umbelliferone al., 1991; Hatano et al., 2000; Kinoshita et al., 2005; Kitagawa et al., 1994a; Kitagawa et al., 1994b; Kitagawa et al., 1994c; Kondo et al., 2007; Kuroda et al., 2003; U et al., 2000; U et al., 2005; Mitscher et al., 1980; Pan, 1999; Raggi et al., 1995; Tanaka et al., 2001; Tsukamoto et al., 2005; Wang et al., 1991 Flavonoids* 4,7-Dihydrostyflavone, 3-Hydroxyglabrol, Euchrenone as, Bai et al., 2003; Fukai et al., 1989; Fukai Gancaonin E, Gancaonin O, Gancanoin P, Gancanoin Q, et al., 1990; Fukai et al., 1998; Hatano et Glabrol. Glucoliquiritin apioside, Glychionide A, Glychionide al., 2000; Kitagawa et al., 1994a; B, Glycyridione A, Glynflavin B, Glynflavin K, Isoliquiritin, Kitagawa et al., 1994b; Kitagawa et al., Isoliquiritin apioside, Isoschaftoside, Kaempferol, Kaempferol 1994c; Li et al., 2000; U et al., 2005; 3-O-methyl ether, Kanzonol Y, Kanzonol Z, Licoagrodin, Mitscher et al., 1980; Pan, 1999; Licoflavone A, Licoflavonol, Liquiritigenin, Liquiritigenin 7,4- Tsukamoto et al., 2005 diglucoside, Liquiritin, Liquiritin apioside, 7-O-Methylluteone, Naringenin, Neoisoliquiritin, Pinocembrin, Prenyllicoflavone A, 3 '-Prenylnaringenin, Saxifragin, Shinflavanone, Sigmoidin B, Topazolin, Vicenin 33
Isoflavonoids j^fromorsin, Allolicoisoflavone B, Calycosin-7-O-glucoside, Fu et al., 2005; Fukai et al., 1989; Fukai * Dehydroglyasperine C, 3,4-Didehydroglabridin, et al., 1993; Fukai et al., 1996; Fukai et Dihydrolicoisoflavone, 6,8-Diisoprenyl-5,7',4'- al., 1998; Fukai et al., 2002; Hatano et trihydroxyisoflavone, 6.8-Diprenylorobol, Formononetin, al., 2000; He et al., 2006; Jayaprakasam Gancanoin B, Gancanoin C, Gancanoin G, Gancaonol C, et al., 2009; Kinoshita et al., 1976; Genistein, Glabrene, Glabridin, Glabroisoflavanone A, Kinoshita et al., 2005; Kitagawa et al., Glabroisoflavanone B, Glabrone, Glicoricone, Glisoflavanone, 1994c; Kiuchi et al., 1990; Kuroda et al., Glyasperin C, Glyasperin D, Glycyrrhisolflavone, Hispaglabridin 2003; Lam et al., 1992; Li et al., 2000; A, Hispaglabridin B, hoononin, Kanzonol H, Kanzonol I, Mitscher et al., 1980; Rauchensteiner et Kanzonol X, Licoagroside A, Licoagroside B, Licoricidin, al., 2005 Licoricone, Licorisoflavan A, Lupiwighteone, Y-O- Methoxylglabridin, 4'-0-Methylglabridin, Methylhispaglabridin B, 5-O-Methyllicoricidin, Ononin, Orobol, Phaseollinisoflavan, Semilicoisoflavone B, Vestitol, Wighteone, Wistin Phenols* 4-Hydroxyguaiacol apioside, Isotachioside, Tachioside Li et al., 2000; Tsukamoto et al., 2005
Pterocarpans Edudiol, Glycyrrhizol A, Glycyrrhizol B, Hemileiocarpin, Bai et al., 2003; Fukai et al., 1998; Fukai Medicatpin, Medicarpin 3-O-fi-D-glucopyranoside, 1- et al., 2002; He et al., 2006; Kitagawa et Methoxyficifolinol, 1-Methoxyphaseollidin, 1-Methoxyphaseolin, al., 1994c;Kiuchi etal., 1990 Shinpterocarpin Saponins Apioglycyrrhizin, j\raboglycyrrhizin, Glycyrrhizin, Licorice Hayashi et al., 2005; Kitagawa et al., saponin A3, Licorice saponin B2, Licorice saponin C2, Licorice 1994b; Kitagawa et al., 1994c; Kitagawa saponin D3, Licorice saponin E2, Licorice saponin F3, Licorice et al., 1994d; Kiuchi et al., 1990 saponin G2, Licorice saponin H2. Licorice saponin 12, Licorice saponin K2, Licorice saponin L3, Macedonoside A
Steroids ^-Sitosterol, Stigmasterol Wang et al., 1991; Zayed et al., 1964 Stilbenes Gancanoin R, Gancanoin S Fukai et al., 1991 Triterpenes P-Amyrin, 18-a-Glycyrrhetic acid, 18-[5-Glycyntietic acid, Andrisano et al., 1995; Elgamal and El- Glyuranolide, 24-Hydroxy-18-P-glycyrrhetic acid, 28- Tawil, 1975; Hayashi et al., 2004; Jia et Hydroxyglycyrrhetic acid, 18-a-Liquiritic acid, 18-p- Liquiritic al., 1989 acid * Glycosides and aglycones 34
CHAPITRE 3
Article 2: Accepté pour publication dans la revue Mycoses
Un résumé et une affiche scientifiques s'intitulant «Effects of Licochalcone and Glabridin on Candida Albicans Virulence Properties» ont été présentés au congrès de l'International Association for Dental Research, Barcelone juillet 2010.
Effect of licorice compounds licochalcone A, glabridin and glycyrrhizic acid on growth and virulence properties of Candida albicans
Céline Messier and Daniel Grenier Oral Ecology Research Group, Faculty of Dentistry, Université Laval, Quebec City, Quebec, Canada
Short title: Effect of licorice compounds on C. albicans
Key words: Candida albicans, licorice, anticandidal activity, biofilm, hyphae
Correspondence: Dr. Daniel Grenier, Oral Ecology Research Group, Faculty of Dentistry, Université Laval, 2420 de la Terrasse, Quebec City, Quebec, Canada, G1V 0A6. Tel: (418) 656-7341 Fax: (418) 656-2861. E-mail: Daniel .Grenier @ greb.ulaval.ca 35
Résumé Introduction: Candida albicans est l'agent causal prédominant de la candidose. Sa capacité à former des hyphes et un biofilm ont été suggérés comme étant des facteurs clés de virulence. Objectifs: Dans cette étude, nous avons étudié les effets de la licochalcone A, de la glabridine et de l'acide glycyrrhizique, des composantes majeures de la réglisse, sur la croissance, la formation du biofilm et la transition blastospore-hyphe de C. albicans. L'effet synergique de ces molécules avec le médicament antifongique nystatin a aussi été évalué. Méthodes: Les concentrations minimales inhibitrices (CMI) pour C. albicans ont été déterminées en utilisant des dilutions sérielles dans une microplaque. L'effet synergique avec le nystatin a été déterminé de façon similaire. L'effet des composantes de la réglisse sur la formation du biofilm a été évalué dans un essai en microplaque et une coloration au crystal violet. L'effet des molécules sur la transition blastospore-hyphe a été déterminé par observation microscopique. Résultats: La glabridine et la licochalcone A ont montré une activité antifongique contre C. albicans, alors que l'acide glycyrrhizique n'a pas eu d'effet. Une inhibition complète de croissance s'est produite avec des concentrations sous-inhibitrices de nystatin en présence de soit la glabridine ou la licochalcone A. La formation du biofilm a été inhibée de 35-60 % en présence de licochalcone A (0.2 pg mL"1). Un fort effet inhibiteur (>80 %) sur la formation d'hyphes a été observé avec la licochalcone A ou la glabridine (100 pg mL"1). Conclusions: La glabridine et la licochalcone A sont des agents antifongiques potentiels et pourraient agir en synergie avec le nystatin pour inhiber la croissance de C. albicans. La licochalcone A a eu un effet significatif sur la formation du biofilm, alors que la licochalcone A et la glabridine ont toutes deux empêché la transition blastospore-hyphe de C. albicans. Ces résultats suggèrent que la licochalcone A et la glabridine, des composantes majeures de la réglisse, ont un effet thérapeutique potentiel contre les infections buccales à C. albicans. 36
Summary Background: Candida albicans is the predominant causal agent of candidiasis. Its ability to form hyphae and biofilm has been suggested to be key virulence factors. Objectives: In this study, we investigated the effect of major licorice compounds licochalcone A, glabridin and glycyrrhizic acid on growth, biofilm formation and yeast- hyphal transition of C. albicans. The synergistic effect of licorice compounds with the antifungal drug Nystatin was also evaluated. Methods: Minimal inhibitory concentrations (MICs) for C. albicans were determined using a microplate dilution assay. The synergistic effect with Nystatin was determined similarly. The effect of licorice compounds on biofilm formation was evaluated using a microplate assay and crystal violet staining. The effect of licorice compounds on yeast-hyphal transition was determined by microscopic observation. The toxicity of licorice compounds towards oral epithelial cells was evaluated with a MTT assay. Results: Glabridin and licochalcone A showed antifungal activity on C. albicans while glycyrrhizic acid had no effect. Complete growth inhibition occurred with sub-inhibitory concentrations of Nystatin with either glabridin or licochalcone A. Biofilm formation was inhibited by 35-60% in the presence of licochalcone A (0.2 pg mL"1). A strong inhibitory effect (>80%) on hyphal formation was observed with licochalcone A or glabridin (lOOugmL1). Glabridin and licochalcone A at high concentrations showed toxicity towards oral epithelial cells. Conclusion: Glabridin and licochalcone A are potent antifungal agents and may act in synergy with Nystatin to inhibit growth of C. albicans. Licochalcone A has a significant effect on biofilm formation, while both licochalcone A and glabridin prevented yeast- hyphal transition in C. albicans. These results suggest a therapeutic potential of licochalcone A and glabridin for C. albicans oral infections. 37
Introduction Candida albicans is a member of the commensal human oral microflora and can be isolated in up to 60% of healthy adults [1]. Among pathogenic yeasts, C. albicans is the predominant causal agent of human candidiasis that affects primarily immunocompromised individuals and elderly patients [2]. Denture stomatitis develops among complete or partial denture wearers and consists in an inflammation of the palatal mucosa in direct contact with the denture, associated or not with soft tissue hyperplasia [3]. Budtz-Jorgensen and Bertram [4] reported that yeasts, including C. albicans, can be cultivated from 90% of patients with denture stomatitis compared to 40% of healthy denture wearers.
C. albicans possesses several virulence characteristics, which have been proposed to represent potential targets for new antimicrobial treatments to counteract the increased resistance of microorganisms to common antimicrobial drugs [5]. Many authors brought evidences that C. albicans virulence is related to the filamenteous form following a phenotypic transition, also known as switch, from blastospores to hyphae [6, 7]. Interestingly, a correlation was established between the level of antifungal drug resistance in C. albicans and its capacity to form hyphae in the presence of azole antifungal drugs [8]. Many studies have shown a much higher frequency of hyphae phenotype of C. albicans in infected sites [1, 6, 9]. Biofilms, which are heterogeneous communities of microorganisms growing on a solid substrate, are formed by C. albicans [10]. C. albicans organised in biofilms have been reported to be up to 1000 times more resistant to antifungal drugs compared to their planktonic form [11].
All plants contain polyphenols, which are characterized by the presence of one or many benzene cycle with at least one function hydroxyl. Previous studies have shown that polyphenols may exert numerous beneficial properties for humans, including anticancer, antioxidant, anti-inflammatory and antimicrobial activities [12]. Licorice root (Radix Glycyrrhizae) have been used for thousands of years as a traditional herbal remedy. Licorice contains several classes of secondary metabolites, including chalcones, coumarins, saponins, and flavonoids, to which human health benefits have been associated [13]. Licorice compounds have also been suggested to be beneficial for dental caries and 38 periodontal disease, through their anti-adhesion and anti-inflammatory properties [14, 15]. However, to the best of our knowledge, the effect of licorice compounds on C. albicans has been only poorly studied and limited to their antifungal effect [16-18].
The aim of this study was to investigate the effect of three major licorice compounds (licochalcone A, glabridin, glycyrrhizic acid) on growth, biofilm formation and yeast- hyphal transition by C. albicans. The synergy between the antifungal drug Nystatin and the licorice compounds was also tested. 39
Material and Methods C. albicans and culture conditions Two strains of C. albicans were used in this study: LAM-1 (isolated from blood of a patient with systemic candidiasis [19]) and ATCC 28366 (isolated from the human oral cavity [20]). The microorganisms were cultivated in Yeast Nitrogen Base (YNB) broth (BBL Microbiology Systems, Cokeysville, MD) containing 0.5% glucose (pH 7.0) under aerobic conditions at 37°C for 24 h. Cells were collected by centrifugation, washed twice with sterile physiologic saline (0.85% NaCl), concentrated ten times in saline and kept at 4°C until further use (for less than six days). This suspension of C. albicans contained the blastospore form, as determined by phase contrast microscopy. Cells were diluted in YNB containing 0.5% glucose (pH 7.0) to the appropriate concentration prior to perform experiments.
Licorice compounds Licorice-derived compounds tested were licochalcone A (Wako, Osaka, Japan), glabridin (Wako, Osaka, Japan) and glycyrrhizic acid (Sigma-Aldrich Canada, Oakville, Canada). All substances were prepared in 95% ethanol at a final concentration of 20 mg mL" and stored at 4°C protected from light, at least 24 h to allow sterilisation. Stock solutions were refreshed every two months.
Determination of minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) Briefly, 4 pL of each licorice compounds or Nystatin, a common antifungal drug used as control, was added to 96 uL of YNB broth + 0.5% glucose pH 7.0. Serial dilutions (1:2; final concentrations from 200 to 0.098 pg mL"1) were prepared in a flat-bottomed 96-well microplate, and then 100 pL of the yeast suspension (5 x IO4 cells mL"1 as determined with a Petroff-Hausser counting chamber) was added. The minimal inhibitory concentration (MIC), defined as the lowest concentration of compounds that completely inhibits the growth of C. albicans, was determined visually by the absence of turbidity after a 24-h incubation at 37°C under aerobic conditions. The minimal fungicidal concentration (MFC), 40 defined as the lowest concentration of compounds that killed C. albicans, was determined by spreading aliquots (10 pL) of each well showing no visible growth on Sabouraud- dextrose agar plates (BBL Microbiology Systems), which were incubated at 37°C for 24 h. MFC values were determined as the lowest concentration of licorice compounds at which no colony formation occurred. All assays were done in triplicate and three independent experiments were carried out.
Synergistic effect of Nystatin with licorice compounds The synergic effect of Nystatin (EMD Biosciences Inc., San Diego, CA) in association with either licochalcone A or glabridin was determined. In a microtitre plate, 50 pL of different concentrations of the licorice compounds (final concentration of 5 pg mL"1, 2.5 pg mL"1, 1.25 pg mL"1 and 0 pg mL"1 in YNB broth + 0.5% glucose) were mixed with 50 pL of Nystatin (final concentrations of 0.5 pg mL"1, 0.25 pg mL"1 and 0 pg mL"1) and 100 pL of the yeast suspension (5 x IO4 cells mL"1). Following a 24-h incubation at 37°C, MIC and MFC values were determined as above. Nystatin was prepared in 95% ethanol at a final concentration of 5 mg mL"1 and stored at 4°C for less than a week. Controls were C. albicans incubated without licorice compounds or Nystatin. Each experiment was done in triplicate and two independent experiments were performed.
Effect on biofilm formation One hundred pL of the yeast suspension (5 x IO4 cells mL"1) was added to the wells of a 96- well tissue culture plate (Sarstedt, Newton, NC) containing 100 [iL of 1:10 serial dilutions (200 to 0.0002 pg mL"1) of licorice compounds in YNB broth + 0.5% glucose pH 7.0. Control wells with no licorice compounds were also inoculated. After incubation for 24 h at 37°C under aerobic conditions, spent media and free-floating microorganisms were removed by aspiration and the wells were washed twice with 10 mM phosphate-buffered saline (PBS pH 7.2), stained with 0.02% crystal violet for 10 min, and then washed twice with PBS to remove unbound dye. After adding 100 pL of 95% ethanol into each well, the plate was shaken for 10 min to release the dye and the biofilm was quantified by measuring 41 the absorbance at 550 nm (A550). Assays were done in triplicate and three independent experiments were performed.
Effect on yeast-hyphal transition C. albicans at a final concentration of 2.5 x 105 cells mL"1 were incubated with licorice compounds at 200, 100 or 50 pg mL"1 in yeast peptone dextrose broth (YPD; BBL Microbiology Systems) pH 7.0 supplemented with 20% filter-sterilized heat-inactivated fetal bovine serum (FBS) at 37°C under aerobic conditions with constant shaking. Blastospore and hyphae forms were counted by observation under a phase contrast microscope, according to the criteria described by Taweechaisupapong et al. [21], at times 0, 2 and 4 h. Positive control consisted in C. albicans incubated in the presence of FBS as above but without licorice compounds. Assays were done in triplicate and two independent experiments were performed.
Effect on viability of oral epithelial cells Human oral epithelial cells GMSM-K, kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA), was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated FBS and 100 pg mL"1 of penicillin G-streptomycin, and were incubated at 37°C in an atmosphere of 5% CO2. Epithelial cells were seeded at a concentration of 4 x 105 cells mL"1 in 96-well microplates and cultured until confluence. Epithelial cells were treated with increasing concentrations (final) of licorice compounds 1 (0, 5, 10, 25, 50 pg/mL" ) and incubated for 2 h at 37°C in a 5% C02 atmosphere prior to determine the cell viability. Viability of epithelial cells was determined using a 3-[4, 5- dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium (MTT) colorimétrie assay (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's protocol. This assay measures mitochondrial dehydrogenase activity. Assays were performed in quadruplicate and repeated three times. 42
Statistical analysis The statistical analyses were performed using Student's t-test with a level of significance at P<0.05. 43
Results Of the three licorice compounds tested, only glabridin and licochalcone A were found to exert antifungal activity towards C. albicans. Glycyrrhizic acid did not show any effect at concentrations up to 200 pg mL"1. MIC and MFC values of glabridin and licochalcone A for the two strains of C. albicans are reported in Table 1. Insert Table 1 here.
Both glabridin and licochalcone A had MICs of 6.25 pg mL"1 and 12.5 pg mL"1 for C. albicans ATCC 28366 and LAM-1, respectively. For both strains, the MFC of glabridin was 12.5 pg mL"1 while that of licochalcone A was 100 pg mL"1. Data on the synergistic effect of Nystatin and the licorice compounds, both at subinhibitory concentrations, are reported in Table 2. Insert Table 2 here.
No growth for strain ATCC 28366 was observed with 0.25 pg mL"1 Nystatin combined with 1.25 pg mL"1 of either glabridin or licochalcone A. Similar concentrations of licochalcone A and Nystatin resulted in inhibition of strain LAM-1, while glabridin needed to be used at 2.5 pg mL"1 to observe growth inhibition in combination with 0.25 pg mL' of Nystatin. No fungicidal effect was observed with any of the combinations of Nystatin and the licorice compounds that caused growth inhibition of C. albicans.
Both strains of C. albicans formed a biofilm when cultivated in YNB broth containing 0.5% glucose (pH 7.0) under aerobic conditions. Biofilm staining with crystal violet gave values (A55o) of 0.60 ± 0.15 for LAM-1 and 0.91 ± 0.14 for ATCC 28366. Licochalcone A was the only molecule to show a significant inhibitory effect on biofilm formation by both strains of C. albicans. As reported in Fig. 1, biofilm formation was inhibited by 35 ± 22% for LAM-1 and 59 ± 10% for ATCC 28366 with licochalcone A at 0.2 pg mL"1. Increasing the concentration of licochalcone A at 2.0 pg mL"1 resulted in inhibition of 76 ± 14% for LAM-1 and 81 ± 7% for ATCC 28366. Concentrations as low as 0.02 pg mL"1 still had a significant inhibitory effect on biofilm formation by ATCC 28366 (25 ± 11%). The above 44 concentrations are lower than the MIC values, suggesting a true specific anti-biofilm effect for licochalcone A on C. albicans. Insert Figure 1 here.
Following aerobic incubation with constant shaking in the presence of 20% FBS, 100% hyphae were formed after 2 h and 4 h for LAM-1, whereas 89 ± 16% were formed for ATCC 28366 after 2 h and 100% after 4 h. As reported in Fig. 2A, no hyphae were formed in the presence of 200 pg mL"1 of licochalcone A after 2 and 4 h for both strains. Licochalcone A at a concentration of 100 pg mL"1 inhibited 79 ± 18% of hyphae formation at 2 h and 82 ± 11% at 4 h for LAM-1, while it completely prevented hyphae formation by ATCC 28366. No statistically significant inhibition of yeast-hyphal transition occurred with licochalcone A at 50 pg mL"1 for both strains. The effect of glabridin on yeast-hyphal transition is reported in Fig. 2B. A concentration of 200 pg mL"1 did not allow any hyphae formation in both strains of C. albicans. Glabridin at 100 pg mL"1 still caused a significant inhibition of yeast-hyphal transition for LAM-1 (86 ± 7% at 2 h) and for ATCC 28366 (93 ± 6% at 4 h). Insert Figures 2A and 2B here.
The cytotoxic effect of licochalcone A, glabridin and glycyrrhizic acid towards oral epithelial cells was investigated with a MTT test following a 2-h treatment. As reported in Table 3, while glycyrrhizic acid showed no toxicity at the highest concentration tested, licochalcone A and glabridin significantly reduced cell viability at concentrations 2.20 pg mL"1 and ^10 pg mL"1, respectively. Insert Table 3 here 45
Discussion C. albicans is the most important agent involved in human candidiasis [2] and is also strongly associated to denture stomatitis [4]. C. albicans possesses various virulence characteristics, such as the ability to form biofilm and to switch from yeast to hyphae form [7,10]. Both of these properties may represent potential targets for new antifungal treatments. Even if used for centuries in Chinese medicine, only few reports exist on the effect of licorice compounds on C. albicans. These studies have been limited to the effect of the compounds on growth of C. albicans. A Glycyrrhiza glabra L. extract [18], glabridin [16] as well as glycyrrhetinic acid [17] were found to inhibit the growth of C. albicans. In this study, we evaluated the effect of three major licorice compounds (licochalcone A, glabridin, glycyrrhizic acid) on growth/killing of C. albicans as well as on biofilm formation and yeast-hyphal transition. The synergistic antifungal activity between licorice compounds and Nystatin was also investigated.
Licochalcone A and glabridin showed an antifungal activity on C. albicans with MICs in the range of 6.25-12.5 pg mL"1. Licochalcone A and glabridin also demonstrated synergistic effects with Nystatin. Such a combination of the antifungal drug and the licorice compounds could be an interesting option to treat infections caused by highly antifungal resistant isolates of C. albicans or to reduce the cytotoxicity of Nystatin by reducing the amounts needed.
Interestingly, both licochalcone A and glabridin could also inhibit two key virulence properties of C. albicans. Licochalcone A had an inhibitory effect against C. albicans biofilm formation, even at a concentration (0.2 pg mL"1) much lower than the MIC values. This effect was more pronounced on ATCC 28366, which produced a more important biofilm than LAM-1 in the absence of licochalcone A. Considering that biofilm formation by C. albicans is critical for host surface colonization, infection development and antifungal drug resistance [10,11], such capacity of licochalcone A to prevent biofilm formation is of high therapeutic interest. Recently, Evensen and Braun [22] reported that 46 tea polyphenols also significantly decreased the capacity of C. albicans to grow and form biofilm.
Licochalcone A and glabridin also showed a capacity to inhibit hyphae formation from yeast cells. The inhibition caused by licochalcone A was found to be stronger than that associated to glabridin. The inhibition caused by glabridin was not observed at the MFC (12.5 pg mL"1), suggesting that the transition yeast-hyphal may occur prior to yeast killing. It was previously reported that the morphological switch from yeast to hyphal cells is important in many processes, such as biofilm formation [23]. Therefore, the high capacity of licochalcone A to efficiently inhibit yeast-hyphal transition may be associated to its ability to also prevent biofilm formation.
In conclusion, our study showed that licochalcone A and glabridin are potent antifungal agents and may act in synergy with Nystatin. They also exert inhibitory effect on hyphal formation. In addition, licochalcone A has also the capacity to inhibit the formation of biofilm by C. albicans. Such a property for these molecules to act as inhibitors of virulence represent alternative and innovative pathways of chemotherapy for pathogens that are resistant to classical antimicrobial agents. It is also likely that molecules that target the virulence process will not easily develop resistance. Except for the effect on hyphal formation, the beneficial properties of licochalcone and glabridin were obtained at concentrations that were non-toxic for oral epithelial cells. Therefore, our results suggest a therapeutic potential of licochalcone A and glabridin for C. albicans oral infections. Clinical studies to be performed should evaluate the beneficial effect of both licorice compounds, either applied topically or incorporated into oral hygiene products such as moutwash, for C. albicans infections.
Acknowledgements This study was supported by a grant from the Fondation de l'Ordre des Dentistes du Québec. We thank Vu Dang La for his technical assistance. 47
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Table 1. MIC and MFC values (pg mL"1) of licorice compounds and Nystatin for C. albicans.
> LAM-1 ATCC 28366
MIC MFC MIC MFC
Licochalcone A 12.5 100 6.25 100 Glabridin 12.5 12.5 6.25 12.5 Glycyrrhizic acid >200 >200 >200 >200 Nystatin 2.5 5.0 2.5 5.0 50
Table 2. Synergistic effect of licorice compounds and Nystatin on growth inhibition of C. albicans.
Glabridin or licochalcone A (pg mL" )
LAM-1 ATCC 28366 Nystatin (pg mL" ) 1.25 2.5 0 1.25 2.5 0 GG* GG GG GG GG GG 0.25 GG G/NG NGNG GG NGNG NGNG 0.5 GG G/NG NGNG GG NGNG NGNG
*, Result on glabridin/Result on licochalcone A. G: growth, NG: No growth. 51
Table 3. Cytotoxic effect of licochalcone A, glabridin and glycyrrhizic acid on oral epithelial cells. Assays were done in triplicate and three independent experiments were carried out. *, significantly different at P < 0.05 compared to the control (no licorice compounds).
Licorice compounds % Cell viability Concentration of licorice compounds (pg mL"1) 2 5 10 20
Licochalcone A 94 ±9 97 ±7 91 ±10 58 ±15* Glabridin 102 ±4 87 ±11 67 ±15* 43 ±18* Glycyrrhizic acid 98 ±7 102 ± 12 105 ±9 95 ±11 52
s 80 * I HI S I 40 S 20
20 2 0.2 Licochalcone A (ug mL'1)
Figure 1. Effect of licochalcone A on biofilm formation by C. albicans. A value of 100% was given to the biofilm formed in the absence of licochalcone A. *, significantly different at P < 0.05 compared to the control. .
Figure 2. Effect of licochalcone A (panel A) and glabridin (panel B) on yeast-hyphal transition by C. albicans. The % hyphae was determined by microscopic observation. *, significantly different at P < 0.05 compared to the control (no licorice compounds). 53
CHAPITRE 4
Article 3: Publié dans la revue Phytomedicine 18(2011) 380-383 Short communication
Inhibition of Candida albicans biofilm formation and yeast-hyphal transition by 4-hydroxycordoin
Céline Messier1, Francesco Epifano2, Salvatore Genovese2 and Daniel Grenier1* 'Groupe de Recherche en Écologie Buccale, Faculté de Médecine Dentaire, Université Laval, Quebec City, Quebec, Canada; 2Dipartimento di Science del Farmaco, Università G. D'Annunzio, Via Dei Vestini 31, 66013 Chieti Scalo, Chieti, Italy
Keywords: Candida albicans, stomatitis, biofilm, yeast-hyphal transition, 4- hydroxycordoin
Running title: Effect of 4-hydroxycordoin on C. albicans
The authors report no conflicts of interest related to this study Corresponding author: Dr. Daniel Grenier, Groupe de Recherche en Écologie Buccale, Faculté de Médecine Dentaire, Université Laval, 2420 rue de la Terrasse, Quebec City, Quebec, Canada, G1V 0A6. Fax: (418) 656-2861. E-mail: Daniel .Grenier® greb.ulaval.ca 54
RESUME
Candida albicans possède des traits distinctifs, tel que le dimorphisme et la formation du biofilm, qui sembleraient jouer un rôle clé dans la capacité d'invasion des tissus buccaux et dans la résistance aux mécanismes de défense de l'hôte et aux antifongiques. Dans cette étude, nous avons étudié les effets du 4-hydroxycordoin, un isopentenyloxychalcone naturel, sur la croissance, la formation du biofilm et la transition blastospore-hyphe de C. albicans. Des dilutions sérielles du 4-hydroxycordoin dans le milieu YNB ont été préparées dans des microplaques pour déterminer la concentration minimale inhibitrice (CMI) et les effets sur la formation du biofilm de deux souches de C. albicans. Le 4- hydroxycordoin à des concentrations atteignant 200 pg/mL n'a pas eu d'effet sur la croissance de C. albicans. Toutefois, la formation du biofilm était fortement inhibée (>85 %) par le 4-hydroxycordoin à 20 pglnL, alors que des concentrations allant de 50 à 200 pg/mL ont causé une inhibition significative de la transition blastospore-hyphe, tel que déterminé par observation microscopique. En conclusion, le 4-hydroxycordoin exerce des effets inhibiteurs sur deux importants facteurs de virulence de C. albicans : la formation du biofilm ou la transition blastospore-hyphe. Ceci suggère que le 4-hydroxycordoin pourrait posséder un potentiel thérapeutique contre les infections à C. albicans. 55
ABSTRACT
Candida albicans distinguishing features such as dimorphism and biofilm formation are thought to play a key role in oral tissue invasion and resistance to host defences and antifungal agents. In this study, we investigated the effect of 4-hydroxycordoin, a natural isopentenyloxychalcone, on growth, biofilm formation and yeast-hyphal transition of C. albicans. Serial dilutions of 4-hydroxycordoin in YNB medium were prepared in microplates to determine minimal inhibitory concentrations (MIC) and effects on biofilm formation for two strains of C. albicans. 4-hydroxycordoin at up to 200 pg/mL had no effect on growth of C. albicans. Biofilm formation was strongly inhibited (> 85%) by 4- hydroxycordoin at 20 pg/mL, while concentrations ranging from 50 to 200 pg/mL caused a significant inhibition of yeast-hyphal transition, as determined by microscopic observation. In conclusion, 4-hydroxycordoin exerts inhibitory effects on two important virulence factors of C. albicans: biofilm formation or yeast-hyphal transition. This suggests that 4- hydroxycordoin may have a therapeutic potential for C. albicans infections. 56
INTRODUCTION
Candida albicans is the predominant causal agent of human candidiasis (Lopez-Martinez, 2010). More specifically, denture stomatitis develops among complete and partial denture wearers and is defined as an inflammation of the palatal mucosa in contact with the denture, associated or not with soft tissue hyperplasia (Samaranayake et al., 2009). Budtz-Jorgensen and Bertram (1970) showed that yeasts, including C. albicans, can be isolated from 90% of patients with denture stomatitis compared to 40% of healthy denture wearers. C. albicans possesses various virulence attributes that may represent potential targets for new antifungal treatments. Many authors have suggested that the virulence of C. albicans could be attributed, at least in part, to the filamenteous form following a yeast-hyphal transition (Jones et al, 1994; Soil, 2002). A correlation was established between the degree of antifungal drug resistance of C. albicans strains and their capacity to form hyphae in the presence of azole antifungal drugs (Ha and White, 1999). Many studies reported that a higher frequency of hyphae phenotype of C. albicans exists in fungal infection sites (Soil et al., 1987; Hellstein et al., 1993; Jones et ai, 1994). Biofilms are heterogeneous communities of microorganisms entrapped in an extracellular matrix, which limits the penetration of antimicrobial drugs and antibodies. Consequently, biofilm infections are rather difficult to treat. Biofilm formation is an important property that helps C. albicans to cause many types of infections (Seneviratne et al., 2008). Candida biofilms have been reported to be 30 to 2 000 times more resistant to various antifungal agents compared to their planktonic counterparts (Hawser and Douglas, 1995).
Given the increased resistance of pathogenic microorganisms to currently used antibiotics and chemotherapeutics, there is a need for alternative prevention and treatment products for oral infections. Natural products derived from plants are considered as a significant source of new biologically active compounds. 4-hydroxycordoin is a natural isopentenyloxychalcone that represents a secondary metabolite not widespread in nature, like many other prenyloxyphenylpropanoids (Epifano et al., 2007). Few studies investigated the effects of 4-hydroxycordoin on growth and virulence properties of 57 pathogenic microorganisms. Avila et al. (2008) reported a modest antibacterial activity of 4-hydroxycordoin against Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. The objective of this study was to investigate the effect of 4- hydroxycordoin on growth, biofilm formation and yeast-hyphal transition of C. albicans. 58
MATERIALS AND METHODS
C albicans and culture conditions Two strains of C. albicans were used in this study: LAM-1 (isolated from blood of a patient with systemic candidiasis; Laçasse et ai, 1990) and ATCC 28366 (isolated from human oral cavity; Evans et al., 1974). The microorganisms were cultivated in yeast nitrogen base (YNB) broth (BBL Microbiology Systems, Cokeysville, MD) + 0.5% glucose pH 7.0 under aerobic conditions at 37°C for 24 h. Cells were collected by centrifugation, washed twice with sterile physiologic saline (0.85% NaCl), concentrated ten times in saline and kept at 4°C until further use (for less than 6 days). This C. albicans suspension contained the blastospore form, as determined by phase contrast microscopy. Cells were diluted in YNB + 0.5% glucose pH 7.0 to the appropriate concentration just before to perform experiments.
Synthesis of 4-hydroxycordoin 4-hydroxycordoin (Figure 1), (2E)-l-{2-hydroxy-4-[(3-methylbut-2-enyl)oxy]phenyl}-3- (4-hydroxyphenyl)prop-2-en-l-one), was chemically synthesized as previously reported (Genovese et ai, 2009). Briefly, commercially available 2,4-dihydroxyacetophenone was first alkylated selectively in position 4 with 3,3-dimethylallyl bromide in the presence of l,8-diazabicyclo[5.4.0]undec-7-ene as the base in acetone at room temperature. The resulting 2-hydroxy-4-isopentenyloxyacetophenone was coupled with 4- hydroxybenzaldehyde in aqueous ethanol in the presence of 60% KOH. The desired product was finally purified by crystallization from n-hexane. Its purity (> 99.7%) was assessed by gas chromatography-mass spectrometry. Stock solution of 4-hydroxycordoin was prepared in 95% ethanol at a final concentration of 20 mg/mL and stored at 4°C for at least 24 h to allow sterilisation. The stock solution was refreshed every two months.
Determination of minimal inhibitory concentration (MIC) Briefly, 4 pL of 4-hydroxycordoin was added to 96 pL of YNB broth + 0.5% glucose pH 7.0. Serial dilutions (1:2; final concentrations from 200 to 0.098 pg/mL) were prepared in a flat-bottomed 96-well microplate, and then 100 pL of the yeast suspension (5 x IO4 59 cells/mL as determined with a Petroff-Hausser counting chamber) was added. The MIC, defined as the lowest concentration that completely inhibits the growth of C. albicans, was determined visually by the absence of turbidity after a 24 h incubation at 37°C under aerobic conditions. Assays were done in triplicate and three independent experiments were carried out.
Effect on biofilm formation Biofilms were prepared in a flat-bottomed 96-well microplate as described above except that 1:10 serial dilutions were prepared (final concentrations from 200 to 0.0002 pg/mL). Following growth, supematants containing planktonic yeasts were removed, biofilms were washed twice with 10 mM phosphate-buffered saline (PBS pH 7.2), stained with 0.02% crystal violet for 10 min, and then washed twice with PBS to remove unbound dye. After adding 100 pL of 95% ethanol into each well, the plate was shaken for 10 min to release the dye and the biofilm was quantified by measuring the absorbance at 550 nm (A550). Assays were done in triplicate and three independent experiments were carried out.
Effect on yeast-hyphal transition In 1.5-mL microtubes, C. albicans at a final concentration of 25 x IO4 cells/mL were incubated with 4-hydroxycordoin at 200, 100 and 50 pg/mL in liquid yeast peptone dextrose (YPD; BBL Microbiology Systems) pH 7.0 containing 20% filter-sterilized heat- inactivated fetal bovine serum (FBS) at 37°C under aerobic conditions with constant shaking. Blastospore and hyphae were counted by observation under a phase contrast microscope, according to the criteria described by Taweechaisupapong et al. (2005), at times 0, 2 and 4 h. Positive control consisted in C. albicans incubated with FBS in the same conditions but without 4-hydroxycordoin. Assays were done in triplicate and two independent experiments were performed.
Statistical analysis The statistical analyses were performed using Student's t-test with a level of significance at P<0.05. 60
RESULTS AND DISCUSSION
The growth of both strains of C. albicans was not affected by the highest concentration tested (200 pg/mL) of 4-hydroxycordoin. The effect of 4-hydroxycordoin on biofilm formation by C. albicans was then tested in a microplate assay using crystal violet staining and determination of A550. As reported in Figure 2, both strains produced a significant biofilm (LAM-1: 0.735 ± 0.216; ATCC 28366: 0.980 ± 0.104). Almost complete inhibition of biofilm formation occurred when 4-hydroxycordoin was present in the culture medium at 20 pg/mL (96.7 ± 5.4% for LAM-1 and 87.5 ± 4.1% for ATCC 28366). A statistically significant reduction (approximately 30%) was still obtained with 4-hydroxycordoin at 2 pg/mL. No inhibition of biofilm formation was observed with 0.2 pg/mL.
Figure 3 reports the effect of 4-hydroxycordoin on FBS-induced hyphal formation in C. albicans. In the absence of 4-hydroxycordoin (positive control), 100% hyphae was observed for LAM-1 after 2 h, while for ATCC 28366, 88.8 ± 15.9% and 100% hyphae were observed after 2 and 4 h, respectively. At a concentration of 50 pg/mL, 4- hydroxycordoin inhibited at least 85.0% of hyphae formation while a concentration of 100 pg/mL inhibited at least 97% of hyphae formation for both strains.
C. albicans is the major etiologic agent in human candidiasis (Lopez-Martinez, 2010) and is thought to play an important role in denture stomatitis (Budtz-Jorgensen and Bertram, 1970; Samaranayake et al., 2009). C. albicans possesses various virulence factors, including the capacity to form biofilm, making the antifungal drugs less efficient. C. albicans has also the ability to form hyphae, facilitating the soft tissue invasion and allowing the microorganisms to hide from the host defense system. These virulence traits may represent new targets for anti-C albicans treatments due to increased resistance of numerous strains to conventional antifungal agents. In this study, we showed the ability of 4-hydroxycordoin to prevent biofilm formation and inhibit yeast-hyphal transition in C. albicans, while having no effect on growth. 61
4-hydroxycordoin is a natural chalcone that represents a secondary metabolite not widespread in nature (Epifano et al., 2007). To our knowledge, the effect of 4- hydroxycordoin on C. albicans has never been investigated. However, several synthetic chalcones have been previously tested for their anti-Candida activity, although none were evaluated for their effect on biofilm formation or yeast-hyphal transition. Batovska et al. (2007) studied 44 chalcones and found that all were moderately active for inhibiting growth of C. albicans, with MIC values around 62.5 pg/mL. In another study, Tsuchiya et al. (1994) showed that among different chalcone derivatives, 2,4,2'-trihydroxy-5'- methylchalcone had the most powerful antifungal effect against C. albicans. The authors brought evidence that the presence of hydroxyl groups at C-2, C-4 and C-2' in chalcone was essential to exert a fungicidal activity against C. albicans (Tsuchiya et al., 1994). Chemically-synthesized chalcones 2,4,2'-trihydroxy-5'-methylchalcone and 2,4,2'- trihydroxychalcone, effective on C. albicans, showed MIC values in the range of 37.5 to 100 pg/mL for Streptococcus mutans, Streptococcus sobrinus, Streptococcus cricetus, Streptococcus salivarius, Streptococcus sanguis, Streptococcus oralis, Streptococcus mitis, Streptococcus gordonii, Lactobacillus casei and S. aureus, which are bacteria often found in association with C. albicans (Sato et al., 1997).
Biofilm formation by microorganisms is a mechanism that allows them to become persistent colonisers, to resist clearance by the host immune system, to enhance their resistance to antibiotics and to exchange genetic materials (Donlan and Costerton, 2002). 4- hydroxycordoin was found to inhibit efficiently the formation of biofilm by C. albicans. Indeed, an inhibition of approximately 90% was obtained with 4-hydroxycordoin at 20 pg/mL, Additional natural products were also reported to prevent the formation of C. albicans biofilm. For instance, baicalein, one of the major flavonoids originally isolated from the roots of Scutellaria baicalensis Georgi, could reduce C. albicans biofilm formation (Cao et al, 2008). In addition, terpenes (Dalleau et al., 2008) as well as tea polyphenols, including epigallocatechin-3-gallatè, (Evensen and Braun, 2009) were also effective in reducing the formation of biofilm by C. albicans. On the contrary of 4- hydroxycordoin, the above substances also exert growth inhibition of C. albicans. 62
During tissue invasion, C. albicans transforms from blastospores into invasive hyphae forms (Jackson et ai, 2007). 4-hydroxycordoin was effective in blocking the yeast-hyphal transition in C. albicans. Inhibition of C. albicans hyphal formation by natural compounds has also been reported for epigallocatechin-3-gallate (Han, 2007) and thymol (Braga et ai, 2007).
In summary, we showed that 4-hydroxycordoin has no antifungal activity on C. albicans even at high concentrations, while at low concentrations it shows substantial inhibitory effect on biofilm formation and yeast-hyphal transition. Therefore, 4-hydroxycordoin may be a potent agent to prevent establishment of C. albicans in the oral cavity and inhibit soft tissue invasion by hyphal forms. Such a compound that targets the virulence properties of C. albicans offers the advantage that it will not give rise to antimicrobial resistance as observed with classical antimicrobial agents.
ACKNOWLEDGEMENTS This work was supported by the Ministère du Développement Économique de l'Innovation et de l'Exportation du Québec. 63
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Han, Y., 2007. Synergy anticandidal effect of epigallocatechin-(9-gallate combined with amphotericin B in a murine model of disseminated candidiasis and its anticandidal mechanism. Biol. Pharm. Bull. 30, 1693-1696. 64
Hawser, S.P., Douglas, L.J., 1995. Resistance of Candida albicans biofilms to antifungal agents in-vitro. Antimicrob. Agents Chemother. 39, 2128-2131.
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Jackson, B.E., Wilhelmus, K.R., Mitchell, B.M., 2007. Genetically regulated filamentation contributes to Candida albicans virulence during corneal infection. Microb. Pathol. 42, 88-93.
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Figure 1. Chemical structure of 4-hydroxycordoin.
4-hydroxycordoin i u 2 iuL )
Figure 2. Effect of 4-hydroxycordoin on biofilm formation by C. albicans. *, significantly different at P < 0.05 using a Student's t test. 66
\0ziglmL D 100 Mg/mL I 50 (ig/mL D 200 \iglmL 100
80
60
40
20-
LAM-1 ATCC 28366
Figure 3. Effect of 4-hydroxycordoin on yeast-hyphal transition by C. albicans. *, significantly different at P < 0.05 using a Student's t test. 67
CHAPITRE 5 : CONCLUSION
La levure C. albicans, un membre de la microflore indigène des cavités buccale et vaginale et du tractus gastro-intestinal chez l'humain, est retrouvée naturellement dans la cavité buccale chez 60 % des adultes sains (Hellstein et al., 1993). C. albicans est l'agent causal prédominant des candidoses humaines (Kam et Xu, 2002). Les manifestations cliniques de la candidose buccale consistent en des plaques blanchâtres légèrement surélevées, détachables, et laissant parfois apparaître une muqueuse inflammée (Shafer et al., 1974). Une autre maladie buccale est souvent associée à C. albicans, la stomatite prothétique, qui se développe chez des porteurs de prothèses dentaires complète ou partielle et consiste en une inflammation de la muqueuse palatine en contact avec la prothèse (localisée ou généralisée) avec ou sans hyperplasie tissulaire (Shafer et al., 1974). La présence seule de ce mycète n'est pas suffisante pour causer une maladie buccale; certains traits de virulence de C. albicans semblent jouer un rôle majeur dans l'établissement et la progression de la candidose, dont la formation d'hyphes et de biofilms. Certains auteurs soulèvent l'hypothèse que la virulence de C. albicans pourrait être reliée à la forme filamenteuse (Jones et al, 1994; Slutsky et al, 1985). De plus, les infections associées aux biofilms sont difficiles à traiter, à cause de leur résistance aux antifongiques topiques ou systémiques, qui sont à la base du traitement classique pour les infections fongiques (Cao et al., 2008), d'où la nécessité de découvrir de nouvelles molécules qui agissent sur ces facteurs de virulence. Les plantes représentent une importante source de nouvelles molécules à action thérapeutique, dont plusieurs ont déjà été reconnues comme ayant des effets contre certains pathogènes buccaux.
Notre projet de recherche avait trois objectifs principaux: i) déterminer la CMI et la CMF de divers polyphenols vis-à-vis C. albicans, ii) étudier l'effet des polyphenols sur la formation de biofilm par C. albicans et iii) investiguer la capacité des polyphenols d'inhiber la transition blastospore-hyphe chez C. albicans. 68
Les racines de G. glabra et de G. uralensis, connues communément sous le nom réglisse, ont été utilisées depuis des milliers d'années comme remède thérapeutique traditionnel. La réglisse contient plusieurs classes de metabolites secondaires, incluant les chalcones, les coumarins, les saponines et les flavonoïdes, auxquels certains bénéfices à la santé humaine ont été associés. Des recherches récentes suggèrent que les extraits de réglisse et leurs ingrédients actifs possèdent un potentiel intéressant pour l'utilisation en suppléments en vue d'améliorer la santé buccale. En effet, des extraits non-purifiés de G. glabra (Motsei et al., 2003) ainsi que la glabridine (Fatima et al, 2009) et l'acide 18-P-glycyrrhetinique (Pellati et al., 2008) ont démontré un effet fongicide in vitro contre C. albicans, alors que deux autres extraits de réglisse, la glycyrrhizine (Utsunomiya et al, 1999) et la liquiritigenine (Lee et al., 2009) auraient des propriétés immunomodulatrices et pourraient protéger contre les infections disséminées à C. albicans. Toutefois, très peu d'études in vitro et aucune étude in vivo n'ont été réalisées sur l'effet des polyphenols de la réglisse sur les différents facteurs de virulence de C. albicans. Nous avons donc choisi d'évaluer les effets de la licochalcone A, de la glabridine et de l'acide glycyrrhizique, trois composantes majeures de la réglisse, sur la croissance, la formation du biofilm et la transition blastospore-hyphe de C. albicans. Selon nos résultats, la glabridine et la licochalcone A semblent être prometteuses pour le traitement des infections buccales à C. albicans, puisque ces deux composantes de la réglisse ont des propriétés fongicides (à 100 pg/mL pour la licochalcone A et à 12.5 pg/mL pour la glabridine), synergiques avec le nystatin contre la croissance de C. albicans, et ils inhibent à plus de 80 % la transition blastospore- hyphe à une concentration de 100 pg/mL. En plus, la licochalcone A inhibe de 35-60 % la formation du biofilm de C. albicans à une concentration de 0.2 pg/mL.
Dans la deuxième partie de notre projet, nous avons étudié les effets du 4-hydroxycordoin, un isopentenyloxychalcone naturel, sur la croissance, la formation du biofilm et la transition blastospore-hyphe de C. albicans. L'effet du 4-hydroxycordoin n'a jamais été étudié sur C. albicans, mais une étude récente a rapporté son effet antimicrobien marqué sur trois pathogènes parodontaux majeurs : P. gingivalis, Fusobacterium nucleatum et Prevotella intermedia (Feldman et al., 2011). Le 4-hydroxycordoin à des concentrations 69
élevées (200 pg/mL) n'a pas eu d'effet sur la croissance de C. albicans. Cependant, la formation du biofilm était fortement inhibée (>85 %) par le 4-hydroxycordoin à de faibles concentrations (20 pg/mL), alors que des concentrations intermédiaires (50 à 100 pg/mL) ont causé une inhibition significative (>85 %) de la transition blastospore-hyphe. La molécule 4-hydroxycordoin exerce donc des effets inhibiteurs sur deux importants facteurs de virulence de C. albicans : la formation du biofilm ou la transition blastospore-hyphe. Ceci suggère que le 4-hydroxycordoin pourrait posséder un potentiel thérapeutique contre les infections à C. albicans.
En conclusion, notre étude a permis de démontrer que deux polyphenols naturels de la réglisse, la licochalcone A et la glabridine, ainsi qu'un polyphenol synthétisé en laboratoire, le 4-hydroxycordoin, ont des propriétés fongicide (licochalcone A et glabridine) ou d'inhibition de certains facteurs de virulence de C. albicans dont la formation du biofilm (licochalcone A et 4-hydroxycordoin) ou la transition blastospore-hyphe (licochalcone A, glabridine et 4-hydroxycordoin). Des recherches in vivo permettraient de valider leur utilisation comme agent antimicrobien topique en bouche ou en trempage nocturne pour les prothèses dans le traitement des infections buccales à C. albicans. 70
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