Cien. Inv. Agr. 37(1):75-84. 2010

RESEARCH PAPER

 

The antifungal effect of six commercial extracts of Chilean propolis on Candida spp.

 

Christian L. Herrera^1,2, Marysol Alvear³, Leticia Barrientos^1,4, Gloria Montenegro⁵, and Luis A. Salazar ^1,4

¹Departamento de Ciencias Básicas, and ²Departamento de Ciencias Preclínicas, Facultad de Medicina, ³Departamento de Ciencias Químicas, Facultad de Ingeniería, Ciencias and Administración, and ⁴Núcleo de Desarrollo Científco-Tecnológico en Biorecursos, Universidad de La Frontera. Casilla 54-D, Temuco, Chile. ⁵Facultad de Agronomía e Ingeniería Forestal, Pontifcia Universidad Católica de Chile, Casilla 306-22, Santiago, Chile.

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Abstract

Propolis has been used in traditional medicine for many centuries because of its benefcial health properties, including its antimicrobial capacity. Prosthesis stomatitis affects a signifcant percentage of users of removable dentures; Candida albicans is the most common fungal species associated with the development of this pathology. Thus, the objectives of this study were: a. To evaluate the antifungal activity of six commercial propolis extracts against Candida spp. that was isolated from the oral cavity of removable dentures users, and b. To determine chemical characteristics of the propolis extracts evaluated. Among the results, we note that these concentrations of polyphenols varied between 9 ± 0.3 and 85 ± 2.1 mg mL^-1. Chromatographic analysis was able to detect 35 compounds, among which were caffeic acid, myricetin, quercetin, kaempferol, apigenin, pinocembrin, galangin, and caffeic acid phenyl ester (CAPE). All strains tested were inhibited by the liquid extracts of propolis. The MID ranged between 1:40 and 1:1280, and the MIC for C. albicans ranged from 197 µg mL^-1 to 441 µg mL^-1. From the results obtained in this investigation, we can conclude that all propolis extracts evaluated are capable of inhibiting the development of Candida spp. However, they show signifcant differences in the concentration of polyphenols present and in antifungal activity.

Key words: Antifungal activity, Candida albicans, propolis.

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Resumen

El propóleo ha sido utilizado por la medicina tradicional desde hace muchos siglos debido a sus propiedades benéfcas para la salud, entre las que destaca su capacidad antimicrobiana. La estomatitis subprotésica, afecta a un porcentaje importante de usuarios de prótesis dental removible, siendo Candida albicans la especie fúngica más común asociada al desarrollo de esta patología. Así, los objetivos de este estudio fueron: a) evaluar la actividad antifúngica de seis extractos de propóleos comerciales sobre cepas de Candida spp. aisladas de la cavidad oral de usuarios de prótesis dental removible, y b) determinar algunas características químicas de los extractos de propóleos utilizados. Entre los resultados obtenidos, podemos señalar que éstos mostraron concentraciones de polifenoles que variaron entre 9 ± 0,3 y 85 ± 2,1 mg/mL. El análisis cromatográfco permitió detectar 35 compuestos, entre los cuales se logró identifcar la presencia de ácido cafeico, miricetina, quercetina, kaempferol, apigenina, pinocembrina, galangina y ácido cafeico fenil éster (CAPE). Todas las cepas de Candida spp. evaluadas fueron inhibidas por los seis extractos líquidos de propóleos, observándose que la DIM varió entre 1/40 y 1/1280, y la CIM para C. albicans varió entre 197 µg/mL y 441 µg/ mL. A partir de los resultados obtenidos en esta investigación podemos concluir que todos los propóleos evaluados son capaces de inhibir el desarrollo de Candida spp., sin embargo, éstos muestran importantes diferencias en la concentración de los polifenoles presentes y en la actividad antifúngica.

Palabras claves: Actividad antifúngica, Candida albicans, propóleos.

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Introduction

Propolis has been used in traditional medicine for centuries; however, its benefcial properties for human health have been explained scientifcally only in recent decades.

It is well known that propolis composition shows great variability, both qualitatively and quantitatively, and its characteristics depend on botanical and geographical origin (Hegazi et al., 2000; Marcucci et al., 2000; Majiene et al., 2004; Peña, 2008). Salomao et al. (2004) states that the main components of an ethanolic xtract of Brazilian propolis are aromatic acids, unlike Bulgarian propolis, which showed a predominance of favonoids.

This variable composition might be responsible for the differences observed in the biological activity detected in propolis with different origins (Cafarchia et al., 1999; Kujumgiev et al., 1999; velikova et al., 2000). However, it is noteworthy to say that chemically different propolis, in some cases, shows similar biological activity (Salo-mao et al., 2004). Thus, beyond the activity of the individual components, the result is the consequence of a synergistic effect, which results in propolis having diverse pharmacological properties (Banskota et al., 2001; Kujumgiev et al., 1999). Currently, there is considerable scientifc evidence showing not only their important role in the infammatory response and cancer development but also their antioxidant activity and antimicrobial properties (Banskota et al., 2001; Burdock, 1998; Sforcin, 2007).

The antifungal activity of propolis has been spe-cifcally evaluated against different fungi. The genera Aspergillus (Aly and Elewa, 2007), Candida (Santos et al., 2005; Silici et al., 2005; Ol-iveira et al., 2006; Silici and Koc, 2006; Ghasem et al., 2007; Quintero-Mora et al., 2008;), Trichopyton (Koc et al., 2005; Siqueira et al., 2008), Trichosporon (Oliveira et al., 2006), Rhodotorula (Silici et al., 2005) and others have been analyzed regarding their susceptibility to propolis or to some of its components. These and other analyses have determined that pinocembrin, galangin and pinobanksin are the predominant compounds in the studied propolis (Hegazi et al., 2000; Uzel et al., 2005; Quiroga et al., 2006).

Yeasts of the genus Candida are part of the mi-crobial fora of human beings; the skin and the mucosal membranes of airways, gastrointestinal and genito-urinary tract serve as their habitat. This genus may be found in 20% to 70% of the population. However, the incidence has increased due to the use of dentures, xerostomia, endocrine disorders, use of multiple antibiotics, use of immunodepressants, use of antineopla-sics and other factors (Gonsalves et al., 2007).

Oral candidiasis is defned as an infection of the mucous membrane of the oral cavity caused by yeasts of the genus Candida. In users of removable dentures, C albicans is the most common species associated with the development of sub-prosthestic stomatitis, the infammation of oral mucous in contact with prosthesis. Dar-Odeth et al. (2003) found that C. albicans is responsible for 72% of the cases of subprosthestic stomatitis; although a variety of other species of this genus may also be responsible for this pathology (Pereira et al., 2008).

In recent years, the use of propolis has spread due not only to its benefcial properties but also to the emergence of propolis-supplemented food and cosmetics (Banskota et al., 2001). Although the consumption of alcoholic and non-alcoholic extracts of propolis has become popular in Chile, knowledge of the characteristics of these products is limited, unlike in other countries. According to different data bases, the study of the characteristics, composition and properties of Chilean propolis is restricted to a mere 12 scien-tifc articles in the last ten years, and some type of biological activity has been evaluated only in four of them (valcic et al., 1998; Valcic et al., 1999; Astudillo et al., 2000; Montenegro et al., 2000; Montenegro et al., 2001a; Montenegro et al., 2001b; Muñoz et al., 2001a; Muñoz et al., 2001b; Kumazawa et al., 2004; Montenegro et al., 2004; Russo et al., 2004; Hernández et al., 2005). Consequently, in addition to evaluating its botanical origin and chemical characteristics, valcic et al. (1999) assessed its antimicrobial activity against Mycobacterium avium, M. tuberculosis and Staphylococcus aureus. Astudillo et al. (2000) evaluated its antioxidant activity, and Kumasawa et al. and russo et al. (2004) evaluated its antioxidant and antitumoral activity, respectively.

Finally, with regard to the aforementioned studies, and to address the lack of studies evaluating the activity of the Chilean propolis against Candida spp., the objectives of the present study were: a. To evaluate the antifungal activity of six commercial extracts of propolis on Candida spp. strains that were isolated from the oral cavity of removable dental prosthesis users and b. To determine some of the chemical characteristics of the propolis extracts.

Materials and methods

Propolis samples

Six liquid extracts of propolis (mother tincture) were purchased from drugstores and stores of natural products in Temuco, Chile.

Microorganisms

The strains of Candida spp. used in this study were isolated from the oral cavity of removable dental prosthesis users from Temuco, Chile. The study protocol was approved by the Ethics Committee of the Universidad de La Frontera, and all subjects gave written, informed consent according to the basic principles of biomedical investigation enumerated in the Helsinki Declaration.

The isolation and identifcation of the micro-bial species were conducted in the Laboratory of Molecular Biology and Pharmacogenetics of Universidad de La Frontera, Temuco, Chile. For C. albicans, identifcation was made using CHROMagar Candida (Laboratorios Lin-san S.A., Chile), the germinal tube formation test and Polymerase Chain reaction (PCr). The primers used and conditions of reaction were previously described by Baquero et al. (2002). Identifcation by PCR of C. albicans strains is shown in Figure 1. In addition, the identifcation of C. dubliniensis, which presents phenotypical characteristics similar to that of C. albicans, was evaluated by monitoring development at 45°C in Sabouraud dextrose agar (Sharlau, Spain) (Pinjon et al., 1998), and by PCR-restriction fragment length polymorphism (PCR-RFLP), using the restriction endonuclease BlnI (Fermentas, Lithuania) (Mirhendi et al., 2005). Other species of the genus Candida were identifed through the use of CHROMagar Candida.

Total polyphenols

The Folin-Ciocalteu method was used to determine the total polyphenol content of the extracts evaluated (Singleton et al., 1999). Briefy, each extract was diluted 1:10 in 70% ethanol and then 1:10 in distilled water. Subsequently, 40 µl of this dilution was mixed with 560 µl of distilled water, 100 µl of the Folin-Ciocalteu reagent (Merck, Germany) and 300 µl of 7.5% sodium carbonate (w/v). The absorbance was measured at 760 nm after 2 h of incubation at room temperature. The concentrations were calculated from a calibration curve and were expressed in mg mL^-1 equivalent to the mixture of the pinocembrin:galangin standards in a 2:1 ratio (Popova et al., 2007). All measurements were made three times.

Chromatographic analysis

High performance liquid chromatographic (HPLC) analysis was made on an HPLC system (Merck-Hitachi model L-4200) equipped with a pump (model L-6200), a UV-visible detector and a Sphere Column Heater (Phenomenex Ter-ma model TS-130). The separation was made in an RP-18 column (12.5 x 0.4 cm, particle size 5 µm) (Merck, Germany), which separates at 25ºC using a mixture of formic acid 5% in water (A) and methanol (B) as mobile phase. The separation of the compounds was carried out by an isocratic-0 to 10 min-run, with the mixture 70% A and 30% B, followed by a gradient up to 100% B at 70 min. The compounds were detected at 290 nm, with 0.001 sensitivity; the injection volume was 10 µL. The identifcation of the phenolic compounds was made using the following standards: myricetin, kaempferol, quercetin, caffeic acid, galangin, pinocembrin, apigenin, caffeic acid phenyl ester (CAPE) and resveratrol (Sigma, USA).

Antifungal activity of propolis

The minimum inhibitory dilution (MID) and the minimum inhibitory concentration (MIC) were determined by the broth microdilution method following the standards of the National Committee for Clinical Laboratory Standards (NCCLS, 1997) with some modifcations. The culture medium used for this test was RPMI-1640 broth with L-glutamine, which was supplemented with glucose at 2% and buffered with morpholine-propanosulphonic acid (MOPS) at pH 7.0 (Silva et al., 2003).

The yeast suspensions were prepared in sterile physiological serum at a concentration of 1.5 x 10⁶ cfu mL^-1. The absorbance of this suspension was determined by spectrophotometry and was compared to the MacFarland standard. This suspension was diluted (1:50) in sterile physiological serum and then in the culture medium (1:20) to obtain a fnal concentration of 0.75 x 10³ cfu mL^-1 in the culture plate.

The commercial extracts of propolis were diluted serially in the culture plate to obtain dilutions varying from 1:10 to 1:2560 and to obtain ethanol proportions varying from 7 to 0.03%, respectively. Positive, negative and solvent controls were conducted for all conditions. Plates were incubated for 48 h at 37°C, and the results were determined by the presence or absence of microorganism development in a specifc propolis dilution. The MICs were determined as the lowest propolis concentration where a microbial development was not observed, and the MID was determined as the lowest propolis dilution where a microbial development was not observed.

Statistical analysis

Averages, standard deviations, and maximum and minimum values were calculated when necessary. Analysis of variance (ANOVA) was used for comparing the chosen level of statistical signifcance and was p < 0.05. The data analysis was made using the program Graph-Pad Prism, version 3.0 (Graphpad Software, San Diego, CA, USA).

Results

Microorganisms

For the evaluation of antifungal activity of the propolis extracts, a total of 21 isolates were used: C. albicans (18), C. tropicalis (2) and C. glabrata (1) . The PCR identifcation of C. albi-cans is shown in Figure 1.

Total polyphenols

The propolis extracts evaluated showed considerable differences in concentration of total polyphenols, with almost a 10-fold difference between the highest and the lowest concentration obtained. In addition, no relation between the concentrations determined in this study and the propolis concentration declared by the manufacturers was found (Table 1).

Chromatographic analysis

The chromatographic analysis of the propolis samples detected 35 compounds in sample I (Figure 2), which was the largest number of compounds detected. On the other hand, only seven compounds were detected in sample II. regardless of these differences, all the samples had similar chromatographic patterns, with pinocembrin being one of the predominant compounds in these propolis extracts.

Antifungal activity

It was determined that all propolis samples inhibited the development of the tested strains of Candida spp. However, there were important differences in extract dilutions that showed inhibition of Candida spp. (Table 2). For C. albi-cans, a 1:320 dilution had the highest MID with propolis I, while a 1:40 dilution of propolis II and v produced the highest MID. A similar result was obtained with C. tropicalis. Also, in the case of C. glabrata, the propolis with the highest and lowest activity was the same, but the inhibition was observed at higher dilutions.

In the case of C. albicans, when comparing the respective concentrations for dilutions inhibiting development, signifcant differences were observed in propolis extracts Iv and vI, which were active at lower concentrations, compared with propolis extracts I, III and v, which were active at higher concentrations.

Discussion

The evaluation of the biological activity of propolis, particularly its antifungal activity, has been studied previously (Sawaya et al., 2002; Santos et al., 2005; Silici and Kut-luca, 2005; Silici et al., 2005; Oliveira et al., 2006; Ghasem et al., 2007; Quintero-Mora et al., 2008). However, the information obtained in this research is needed in Chile because of unique propolis characteristics and the dependence of its chemical/biological characteristics on its botanical origin.

Bankova (2005) states that, as the concentration and activity of the components of different propolis extracts are unknown, it is necessary to consider each extract as an active principle in itself. On this basis, not only were the concentrations of total polyphenols in propolis extracts determined, but important differences in the concentrations of active compounds were also found; this is relevant because the concentration, in part, establishes the degree of the extracts’s biological activity. This relevance was evident when the samples with signifcant differences expressed in MID (propolis I and II: 1/320 and 1/40, respectively) showed no difference when compared to the MIC (propolis I and II: 287 ± 93 and 224±74 µg mL^-1, respect ively).

Chromatographic analysis detected at least 35 compounds in propolis I and identifed caffeic acid, myricetin, quercetin, kaempferol, apigenin, pinocembrin, galangin and caffeic acid phenyl ester (CAPE). regardless of the differences in the number of compounds detected among the samples analyzed, the chromatographic profles exhibited great similarities. Differences were mainly associated with compound concentration differences.

The activity of propolis on C. albicans has been evaluated by different methodologies (e.g., agar dilution, agar diffusion, macro and microdilu-tion broth). Due to variations both in the methodologies used and in the modes of determining the concentration of active substances, comparing the results from different studies becomes diffcult. For example, throught broth microdi-lution, Oliveira et al. (2006) showed that a concentration of 50 µg mL^-1 of total favonoids was able to inhibit the development of 67 onycho-mycosis fungal species; specifcally, the MIC₉₀ on C. albicans was 25 µg mL^-1. These results are different from those informed by Sawaya et al. (2002), who determined that the MIC on C. albicans varied between 6 and 12 µg mL^-1 using agar dilution. In addition, these researchers could not obtain satisfactory results by broth macrodilution and agar diffusion.

In our study, it was observed that all propolis samples evaluated were able to inhibit the development of Candida spp., although differences were observed regarding the MIC; ranging between 278 µg mL^-1 for C. albicans and 117 µg mL^-1 for C. glabrata.

Currently, subprosthesis stomatitis arises as a real problem for an important number of users of removable dentures. Infection by C. albicans is clearly identifed as one of the main predisposing factors in its development (Baena-Monroy et al., 2005; de Resende et al., 2006; Bilhan et al., 2008). The evaluated propolis demonstrated antifungal activity; but there is a new possibility for Chilean propolis to be used in the elaboration of products that prevent and/or treat subprosthesis stomatitis. An experience of this kind, conducted in Brazil showed that patients with subprosthesis stomatitis and treated with an ethanolic extract of propolis obtained a similar effect when compared with a group of patients treated with nystatin; however, the number of patients considered in that study was small (Santos et al., 2005).

Although many products in the Chilean market are supplemented with or based on propolis, the results obtained in this research show that the extracts of propolis studied were not equivalent in relation to the concentration of total polyphenols. These results show the need for regulation both in determining the geographical and botanical origin of propolis as well as in determining the chemical characterization of extracts. There fore, a clear standardization of characteristics of this product and knowledge of its composition are needed to extrapolate from composition to particular biological properties. These practices would not only add value to the national propolis, but it would also allow the population to choose and use products with known characteristics, as appointed by Peña (2008).

In summary, all of the propolis samples evalu- ated in this study have the ability to inhibit the development of Candida spp. since their qualitative characteristics are similar; however, they show important differences both in the concen-tration of polyphenols present and in antifungal activity.

Acknowledgements

This study was supported by grants from Dirección de Investigación y Desarrollo (DIDUFRO INI 110605) Universidad de La Frontera, and CONICYT (FONDEF D05I-10021), Chile.

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Received 7 April 2009. Accepted 28 May 2009.

Corresponding author: lsalazar@ufro.cl