INTRODUCTION
Infections of the oral cavity have a broad microbial etiological profile, which varies according to the specific ecosystem in each part of the mouth, causing a range of clinical pictures with differing frequencies and seriousness1,2. Among these infections, odontogenic infections alone account for 7% to 10% of the total antibiotic therapy used in populations, with some cases requiring a combination of treatments and medications for resolution. Moreover, several studies have shown that oral infections can be a risk factor leading to the onset, development and progression of systemic diseases3. All of this, added to the problem of the increasing bacterial resistance to antimicrobial agents, creates a pressing need to find new antimicrobial strategies4.
Within the microbial etiology of infections of the oral cavity, the pathogenic flora consists mainly of Streptococcus and Staphylococcus species, as well as a series of opportunistic microorganisms2. Streptococcus mutans has often been associated to the onset and progression of dental caries –a disease with high repercussion in the oral cavity– as well as other infections of odontogenic origin5. Staphylococcus aureus is present in abscesses of odontogenic origin, being the second most important microorganism following the viridans group of Streptococci, and appearing recurrently in different infectious lesions of the oral cavity6. Escherichia coli, which is a very important microorganism of the family Enterobacteriaceae, is also often found in infections of the oral cavity and has great capacity to develop resistance to antimicrobial agents7. Enterococcus faecalis is associated to root canal infections, as well as being recognized for its broad resistance to different antimicrobial agents8. Finally, Candida albicans is an opportunistic fungus, and due to its long persistence time in the tissues that it infects, it is closely associated to denture-related stomatitis and different types of candidiasis which are difficult to treat9.
The particular behavior of many microbial species involved in infections of the oral cavity, and the difficulty to treat some of them, have led to increasing interest in the search for and development of new natural plant-based antimicrobial agents10-12 Around the world, many different plant species have been used as sources of natural medicines for the treatment of diseases 10-12. In vitro studies have found some raw extracts from plant species to be potentially useful in controlling multidrug resistance13. The plant Anacardium excelsum (family Anacardiaceae), known in the Andean region by the common name Caracoli, has promising potential for antimicrobial activity. It is a gigantic tree that grows along rivers in tropical zones of the Americas below 1300 m above sea level. Its wood is used commercially for carpentry and canoe building14. Within the Anacardium family, Anacardium occidentale L. and Anadenanthera macrocarpa are known to have anticariogenic activity15, while Anacardium occidentale L.16 and Anacardium microcarpum17 are known to have antibacterial activity on chemoresistant strains of S. aureus. In 2011, Celis et al.18 reported the antimicrobial activity of extracts and fractions from the species Anacardium excelsum against Bacillus subtillis and Staphylococcus aureus, suggesting that such extracts and fractions may have a potential role in controlling progression and development of dental caries and other odontogenic infections. However, the microbial action of A. excelsum extracts has not yet been explored against the microbial pathogens S. mutans, S. aureus, E. coli, E. faecalis and C. albicans, despite their importance as the cause of a range of infections of the oral cavity.
The aim of this study was to determine the phytochemical nuclei and the antimicrobial effect of Anacardium excelsum leaf and stem extracts, and of fractions derived from the leaf extract, against reference ATCC strains of S. mutans, S. aureus, E. coli, E. faecalis and C. albicans.
MATERIAL AND METHODS
Collection of plant material
Anacardium excelsum was collected from the Quindío Botanical Garden, located in the municipality of Calarcá-Quindío (Colombia) at an elevation of 1500 m above sea level. The plant species was characterized by a botanical expert, who collected and classified the material. For authentication of the plant material collected, a specimen was sent to the herbarium at the University of Quindío, where it was identified as Anacardium excelsum.
Extraction
Anacardium excelsum leaves and stems were processed in the laboratory of the School of Basic Sciences at the Antonio Nariño University, Armenia site (Colombia). Approximately 3000 g of each dried plant material was weighed at ambient temperature and ground in a hammer mill. Then it was macerated at ambient temperature in ethanol:water (7:3), for 15 days. The solvent mixture was retrieved for recirculation using a low-pressure rotary evaporator. The extracts were weighed, their yield percentage calculated, and finally labeled and stored at ambient temperature.
Fractionation
Fractionation of the hydroalcoholic extract of A. excelsum leaves only was performed with extraction by percolation. To do so, 150 g of the hydroalcoholic extract of A. excelsum leaves was weighed and resuspended in the (7:3) ethanol:water system until a homogenous solution was obtained. This extract was absorbed on silica gel flash 100G (Millipore™, Merck KGaA - Darmstadt, Germany) until a fine powder was obtained. For fractionation of the extract, silica 60G (Millipore™, Merck KGaA - Darmstadt, Germany) was used as an extraction base, and solvents with different polarities were used, beginning with the lowest polarity and moving to the highest (ethyl acetate, acetone, ethanol and water). The fractions were dried in a low pressure rotary evaporator. Their yields were calculated and then they were labeled and stored at ambient temperature. Finally, the extracts and fractions were eluted in different solvent systems using thin-layer chromatography (TLC), for which they were resuspended in the appropriate solvents. The phytochemical nuclei present in these fractions of A. excelsum leaves were identified using thin layer chromatography and spraying the plate with specific reagents to identify the most relevant nuclei.
Chromatography
Primary fractionation of the hydroalcoholic extract of A. excelsum leaves was followed by thin layer chromatography using as stationary phase 1 mmthick TLC plates with silica gel 60 F254 (Millipore™, Merck KGaA - Darmstadt, Germany) on aluminum. Elution was performed with a solvent system at different polarities. The plates were developed using 254 nm short-wave and 365 nm long-wave light. The phytochemical nuclei were identified by thin layer spray with specific reagents for alkaloids, flavonoids, terpenes, phenols and coumarins (Table 1).
Evaluation of the antimicrobial activity of extracts and fractions. Study strains
The A. excelsum leaf and stem extracts, and the fractions from the hydroalcoholic leaf extract were evaluated on the reference strains Streptococcus mutans ATCC 25175, Staphylococcus aureus ATCC 35548, Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 19433 and Candida albicans ATCC 10231. These were lyophilized strains, preserved by freezing at -70°C at the Microbiology Laboratory of the Center for Dental Research of the Pontificia Universidad Javeriana. The microorganisms were reconstituted and made viable in 5 mL of brain heart infusion (BHI) broth and incubated for 24 hours at 37 °C under anaerobic conditions (H2:CO2:N2 10:10:80). Then, for isolation and viability and purity testing, they were plated on BHI agar (Brain- Heart Infusion Agar) and incubated for 1-3 days at 37 °C under anaerobic conditions (H2:CO2:N2 10:10:80). Finally, the colonies grown on BHI agar were reconfirmed using Gram stain and biochemical tests.
Well-diffusion method
Antibacterial activity was identified using the agar well diffusion method on Mueller-Hinton agar, as described in Dobner et al.19. Suspensions of each fresh, viable bacterial strain were prepared in isotonic saline solution and adjusted to 0.5 on the McFarland scale. Each bacterial suspension was immediately swabbed on Mueller-Hinton agar, following the Kirby-Bauer technique20. After plating all the bacteria on Mueller-Hinton Agar, a 0.5 cm Pasteur pipette was used to make wells (distributed evenly on the agar). Then, 30 μL of the extracts, fractions, and positive and negative controls were placed in their corresponding wells. For each fraction (ethyl acetate, acetone, ethanol and water), concentrations of 2, 5, 10, 20 and 40 mg/ml were evaluated to determine the lowest concentration that inhibits bacterial growth. Negative control was 1% dimethyl sulfoxide (DMSO) and positive controls were 150 ug/ml vancomycin and 0.12% chlorhexidine. All tests were performed in duplicate and incubated at 37 °C for 24-48 hours. Following the incubation period, the diameters of the zones of inhibition were measured in millimeters and the two values averaged.
RESULTS
Chromatographic analysis
Table 2 shows the phytochemical nuclei studied in the fractions obtained from the hydroalcoholic extract of Anacardium excelsum leaves. Of the phytochemical nuclei studied, in the ethyl acetate (Fig. 1) and acetone fractions, only presence of phenols and tannins, as well as triterpene-type terpenes and steroidal terpenes, was identified. For the ethanol fraction, evaluation showed the presence of phenolic-type compounds and absence of alkaloids, flavonoids, terpenes and coumarins. In the fraction with highest polarity (aqueous), only presence of alkaloids, phenolic-type compounds, tannins and triterpene-type terpenes was found. Coumarins and flavonoid-type compounds were not found in any of the four fractions studied.
Antimicrobial activity of extracts
Table 3, parts A and B, show the antimicrobial activity of the A. excelsum extracts and fractions on the five microorganisms used in the study. Leaf extract had antimicrobial activity on S. mutans, S. aureus, E. faecalis and C. albicans, from concentrations of 10 mg/mL to 40 mg/ml, with zones of inhibition of 9 to 11 mm. None of the leaf extract concentrations evaluated inhibited E. coli. None of the stem extract concentrations evaluated inhibited S. mutans, E. coli or C. albicans, though they did inhibit E. faecalis at concentrations of 10 mg/mL to 40 mg/ml, with zones of inhibition of 11 mm; and S. aureus only at 40 mg/mL, with a zone of inhibition of 10 mm.
Antimicrobial activity of leaf extract fractions
Based on its antimicrobial activity on these bacteria, the leaf extract was fractionated and the antimicrobial activity of the resulting fractions assessed. The fractions with greatest inhibitory activity were ethyl acetate and acetone, which inhibited all study microorganisms as from concentrations of 10 mg/ ml with zones of inhibition ranging from 9 to 20 mm (Table 3 and Fig. 2). The ethyl acetate fraction inhibited all microorganisms at concentrations of 10, 20 and 40 mg/ml with zones of inhibition of 9 to 15 mm. The acetone fraction inhibited S. mutans, E. faecalis and C. albicans at concentrations of 10, 20 and 40 mg/ml with zones of inhibition of 10 to 14 mm, and inhibited S. aureus and E. coli only at concentrations of 20 and 40 mg/ml, with zones of inhibition of 9 to 20 mm. In general, the other two fractions (ethanol and aqueous) showed less activity on the 5 microorganisms evaluated. Table 3 also shows the inhibition results produced by the positive controls (chlorhexidine 0.12% and vancomycin 150 ug/ml) and negative control (DMSO 1%).
DISCUSSION
Due to its wide range of plant biodiversity, and favored by its geographic location, Colombia holds great promise for the discovery and development of new substances with pharmacological potential10. It is well known that secondary metabolites derived from plant species have shown therapeutic action against various diseases21. Thus, different plant species have been studied to determine the presence of substances with pharmacological activity, including, among others, Berberis goudotii, Isertia laevis, Borrichia frutences, Sarcocephalus coadunatus, Elaeagia utilis and Stevia rebaudiana, with the aim of broadening the antimicrobial arsenal used to treat diseases of interest to public health, such as infectious diseases of the oral cavity11, 12, 22, 23. The current study evaluated the antimicrobial activity of hydroalcoholic extracts of leaves and stems, as well as fractions derived from the leaf extract, of the plant species Anacardium excelsum, which is endemic to Colombia. Celis et al.18 conducted in vitro studies which showed that A. excelsum extracts inhibited the growth of Gram-positive bacteria such as Staphylococcus aureus and Bacillus subtillis, but showed no activity against Gram-negative bacteria such as Escherichia coli and Salmonella18. The results of the current study clearly showed that of the two extracts evaluated, leaf extract had more antimicrobial activity on the microorganisms evaluated at concentrations of 10, 20 and 40 mg/ ml. For this reason, the leaf extract was fractionated using four solvents (ethyl acetate, acetone, ethanol and water) and concentrations of 2 mg/ml, 5 mg/ ml, 10 mg/ml, 20 mg/ml and 40 mg/ml. The ethyl acetate fraction had the greatest antimicrobial activity, followed by the acetone fraction and, to a lesser degree, the ethanol and water fractions. The ethyl acetate and acetone fractions had antimicrobial activity at concentrations of 10, 20 and 40 mg/ml. Outstanding was the high inhibition of the ethyl acetate fraction at concentrations of 10, 20 and 40 mg/ml on C. albicans, E. faecalis and S. aureus with zones of inhibition of 13.5 to 15 mm, and lower inhibition on S. mutans and E. coli, with zones of inhibition of 9 mm to 11 mm. In general, the microorganism least inhibited by the 4 fractions was E. coli, in agreement with Celis et al.18.
In the current study, the two most active fractions (ethyl acetate and acetone) obtained from A. excelsum leaves behaved chemically in the same way, with compounds only of phenolic type, tannins, triterpene-type terpenes and steroidal-type terpenes. Celis et al.18 suggest that the antimicrobial activity of these two fractions against the study microorganisms was due to the presence of these phytochemical nuclei. Celis et. al 18 identified that the compounds 2- (1,1-dimethylethyl) -4- (1,1,3,3-tetramethylbutyl) phenol and 2,2’-methylenebis (6- (1,1-dimethylethyl) -4-ethylphenol, characterized by gas chromatography– mass spectrometry (GC/MSD), present in mediumpolarity fractions of A. excelsum, had powerful antimicrobial and antiseptic properties, and thus, excellent antimicrobial activity due to their biological potential18.
The current study found presence of terpenes and phenols in the ethyl-acetate and acetone fractions.
Other studies have found that terpenes are important components that produce antimicrobial activity in species of the family Rubiaceae, and that phenols and phenolic acids are the main components in plants with antimicrobial activity24, 25.
Urrea et al.14 assessed the antibacterial activity of extracts from A. excelsum, and using gas chromatography, identified the compounds oleic acid, octadecanoic acid; 9-octadecenoic acid; 2-methyl-3(Z), 13(Z)-octadecadienol; 2-hydroxy-1- (hydroxymethyl)ethyl-9(Z), 12(Z)-octadecadienoate; 6(Z)-octadecenoic acid; 9(Z)-octadecenal and 7(Z),11(E)-hexadecadienal acetate; 1-isopropyl-4- methyl-benzene; 4-isopropenyl-1-methylcyclohexil acetate and 3-pentadecylphenol, with chemical characteristics that generated strong antimicrobial potential14. The results of the current study showed, in low polarity fractions (ethyl acetate and acetone), the presence of phytochemical nuclei which may contain the compounds described by Urrea et al.14 and show the antimicrobial activity presented against the study microorganisms. Moreover, the ability of these compounds to provide protection to plants has been clearly demonstrated, thus, it is necessary to continue with the chemical characterization of the phytochemical nuclei in order to identify and characterize substances with potential antimicrobial activity.
The antimicrobial activity of A. excelsum found in the current study on reference ATTC strains of S. mutans, S. aureus, E. coli, E. faecalis and C. albicans, which are microorganisms which have been demonstrated to by highly pathogenic in different infectious processes of the oral cavity2, 3, makes it clear that A. excelsum (Caracolí) leaves are a potential source of chemical compounds with antibacterial activity. It is thus necessary to conduct further studies to elucidate the action mechanism of these extracts and fractions, providing information on the content of secondary metabolites with antibacterial and antifungal activity, which –after evaluating pharmacological safety– could be used in the future as antimicrobial agents for infectious processes of the oral cavity.
To conclude, (1) the hydroalcoholic extract of A. excelsum leaves and the ethyl acetate and acetone fractions obtained from the hydroalcoholic extract at concentrations of 10 to 40 mg/ml had the greatest antimicrobial activity against S. mutans ATCC 25175, S. aureus ATCC 35548, E. coli ATCC 25922, E. faecalis ATCC 19433 and C. albicans ATCC 10231; and (2) the evaluation of the phytochemical nuclei in the ethyl acetate and acetone fractions showed compounds of phenolic type, triterpenetype terpenes and steroidal-type terpenes, which might explain the antimicrobial activity observed.