Chemical and botanical characterization of Chilean propolis and biological activity on cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus

Brazilian Journal of Microbiology 44, 2, 577-585 (2013) ISSN 1678-4405 Copyright © 2013, Sociedade Brasileira de Microbiologia www.sbmicrobiologia.org.br Research Paper Chemical and botanical characterization of Chilean propolis and biological activity on cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus Leticia Barrientos1,2, Christian L. Herrera1, Gloria Montenegro3, Ximena Ortega3, Jorge Veloz1, Marysol Alvear4, Alejandro Cuevas1, Nicolás Saavedra1, Luis A. Salazar1,2 1 Centro de Biología Molecular & Farmacogenética, Scientific and Technological Bioresource Nucleus, Universidad de La Frontera, Temuco, Chile. 2 Centro de Excelencia en Estudios Genéticos e Inmunológicos, Universidad de La Frontera, Temuco, Chile. 3 Departamento de Ciencias Vegetales, Facultad de Agronomía e Ingeniería Forestal, Pontificia Universidad Católica de Chile, Santiago, Chile. 4 Departamento de Ciencias Químicas y Recursos Naturales, Facultad de Ingeniería, Ciencias y Administración, Universidad de La Frontera, Temuco, Chile. Submitted: June 2, 2012; Approved: September 10, 2012. Abstract Propolis is a non-toxic natural substance with multiple pharmacological properties including anticancer, antioxidant, fungicidal, antibacterial, antiviral, and anti-inflammatory among others. The aim of this study was to determine the chemical and botanical characterization of Chilean propolis samples and to evaluate their biological activity against the cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus. Twenty propolis samples were obtained from beekeeping producers from the central and southern regions of Chile. The botanical profile was determined by palynological analysis. Total phenolic contents were determined using colorimetric assays. Reverse phase HPLC and HPLC-MS were used to determine the chemical composition. The minimum inhibitory concentration (MIC) was determined on S. mutans and S. sobrinus. All propolis samples were dominated by structures from native plant species. The characterization by HPLC/MS, evidenced the presence of quercetin, myricetin, kaempferol, rutine, pinocembrin, coumaric acid, caffeic acid and caffeic acid phenethyl ester, that have already been described in these propolis with conventional HPLC. Although all propolis samples inhibited the mutans streptococci growth, it was observed a wide spectrum of action (MIC 0.90 to 8.22 mg mL-1). Given that results it becomes increasingly evident the need of standardization procedures, where we combine both the determination of botanical and the chemical characterization of the extracts. Research conducted to date, describes a promising effectiveness of propolis in the prevention of caries and other diseases of the oral cavity, making it necessary to develop studies to identify and understand the therapeutic targets or mechanisms of molecular action of the various compounds present on them. Key words: propolis, botanical characterization, chemical characterization, Streptococcus mutans, Streptococcus sobrinus. Introduction Among the natural products that have received attention recently, we draw attention on propolis, a resinous sub- stance collected by bees (Apis mellifera) from buds, shoots and wounds of various plant species and mixed with mandible secretions for use in construction, maintenance and protection of their hives (Burdock, 1998). It has over 300 Send correspondence to L.A. Salazar. Centro de Biología Molecular & Farmacogenética, Scientific and Technological Bioresource Nucleus, Universidad de La Frontera, Av. Francisco Salazar 01145, Casilla 54-D, Temuco, Chile. E-mail: . 578 compounds, among which polyphenols (flavonoids, phenolic acids and their esters), terpenoids, steroids, sugars and amino acids have been detected in raw propolis, but its composition is qualitatively and quantitatively variable, depending on the vegetation at the site from which it was collected and the collection season (Koo et al., 1999; Bankova, 2005; Tosi et al., 2007; Valencia et al., 2012). The main constituents of propolis in Europe, China, and North America are flavonoids and phenolic acid esters (Bankova et al., 2000; Chen et al., 2004; Lotti et al., 2010). Brazilian propolis contains, principally, diterpenes, lignans, p-coumaric acid derivatives, sesquiterpenes and acetophenones (Bankova, 2005; Piccinelli et al., 2005). Propolis is a non-toxic natural substance with multiple pharmacological properties including cytostatic, hepato-protective, antioxidant and anti-inflammatory (Burdock, 1998; Kujumgiev et al., 1999; Borrelli et al., 2002; Russo et al., 2002). It is also considered as an alternative in the treatment and prevention of many infectious diseases, since it displays a wide range of antimicrobial activity against a variety of bacteria, fungi, parasites and virus (Bankova et al., 1995; Kujumgiev et al., 1999; Sforcin et al., 2000; Orsi et al., 2005). Due to this wide range of biological activities, propolis is used in food industry, cosmetology, and complementary medicine products. These observations emphasize the need to extend our knowledge about the chemical and biological characterization of propolis, which would aid with the appropriate use of this natural product in human health (Valencia et al., 2012). Dental caries are known to be one of the most prevalent and costly oral infectious diseases worldwide (Dye et al., 2007); it is a multifactorial infectious disease in which diet, nutrition, microbial infection, and host response all play important roles. Dental plaque is a typical bacterial biofilm that contains mutans streptococci and other oral bacteria and their products. Among them, Streptococcus mutans and S. sobrinus have been recognized to be the mayor causative agent of dental caries in humans (Loesche, 1986; Smith, 2002). Some naturals compounds based in tea (HamiltonMiller, 2001), cranberries (Steinberg et al., 2004), cacao (Osawa et al., 2001), herbal extracts (Limsong et al., 2004) and propolis (Bankova et al., 1995; Koo et al., 2000, 2002a, 2002b; Duarte et al., 2006;), have shown inhibition of biofilm and caries development in some species of mutans streptococci, being propolis and its polyphenolic compounds, the most studied. The biofilm formation is vital for the progression of dental caries and thus, inhibition of this factor is one of the strategies currently used to prevent this disease (Xiao et al., 2007). In view of the foregoing considerations the aim of the present study was to determine both the chemical and botanical characterization and to determine the biological activity on mutans streptococci of 20 propolis samples obtained in central and southern Chile. Barrientos et al. Materials and Methods Preparation of ethanolic and methanolic extracts of propolis (EEP) Twenty propolis samples were obtained from various beekeeping producers from the central and southern region (Valparaíso, Metropolitana, Libertador Bernardo O’Higgins and La Araucanía Regions), C (...truncated)