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Inhibiting effect of Dorstenia asaroides extracts on cariogenic properties of Streptococcus mutans
Anaerobe 18 (2012) 31e36
Contents lists available at SciVerse ScienceDirect
Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Clinical microbiology
Inhibiting effect of Dorstenia asaroides extracts on cariogenic properties of Streptococcus mutans C.E.M. D’Angelis a, c, M.F. Leite a, J.P.B. Sousa a, L. Alonso a, A.C.M. Polizello a, M. Groppo Jr. b, C.P. Aires a, J.K. Bastos a, A.C.C. Spadaro a, * a
Department of Physics and Chemistry, Faculty of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo-USP, Av. do Café s/n, Monte Alegre, 14040-903 Ribeirão Preto, São Paulo, Brazil Department of Biology, Faculty of Philosophy, Sciences and Languages of Ribeirão Preto, University of São Paulo-USP, Brazil c Pitágoras Integrated School of Montes Claros, Montes Claros, Minas Gerais, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 July 2011 Received in revised form 16 November 2011 Accepted 22 December 2011 Available online 10 January 2012
The aim of this study was to examine the effects of Dorstenia asaroides extracts on cariogenic properties of the most cariogenic bacteria, Streptococcus mutans. Hexane (HFr), ethyl-acetate (EFr) and chloroform (CFr) extracts obtained from D. asaroides rhizomes were submitted to chemical analyses, Minimal Inhibitory Concentrations (MIC), glycolysis assay and S. mutans 12-h-old initial biofilms. Chemical characterization showed that all the extracts present furanocoumarins. The MIC values were 80 (HFr and CFr) and 50 mg/mL (EFr). Acid production by S. mutans cells was significantly disrupted by HFr (12.5 mg/mL), EFr (at 2.5; 6.25 and 12.5 mg/mL) and CFr (at 2.5, 6.25 and 12.5 mg/mL) (p < 0.01). Topical applications of HFr, EFr and CFr significantly reduced the colony forming units of S. mutans biofilms compared with those treated with control group in order to 20, 30 and 25% respectively (p < 0.01). The results of the present study suggest that rhizomes of D. asaroides had inhibitory effects on cariogenic properties of S. mutans. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Antimicrobial Biofilm Streptococcus mutans Dorstenia asaroides
1. Introduction Streptococcus mutans has been strongly implicated as the principal etiological agent in human dental caries [1] although other additional acidogenic microorganisms may be involved [2]. The virulence of this microorganism has been attributed to their acidogenic and aciduric properties which play a critical role in the pathogenesis of dental caries [3,4]. Mutans streptococci catabolise multiple fermentable dietary carbohydrates, and also carry out glycolysis at low biofilm pH values in the oral cavity [5] promoting tooth demineralization. Therefore, antimicrobial substances against these bacteria or those that affect acid production could play an important role in preventing the formation of dental plaque and caries. Plants are an important possible source of new bioactive compounds to disrupt bacteria virulence because they may serve as alternatives to the commonly used chemicals for dental biofilm [6]. It is well established that many of the metabolites produced by plants present properties against oral pathogens [7e11]. Several Dorstenia species have been extensively used in traditional medicine. Dorstenia, a herbaceous genus rich in furanocoumarins, belongs to the family Moraceae and is found throughout Africa, besides Central and South America. The * Corresponding author. Tel.: þ55 16 3602 4215; fax: þ55 16 3633 1936. E-mail address: [email protected] (A.C.C. Spadaro). 1075-9964/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2011.12.020
pharmacological data on the genus Dorstenia are limited, but it is known that species of the genus popularly known in Brazil as “Carapiá” are used due to its medicinal qualities [12e14]. In Brazilian folk medicine, the species D. asaroides is used for infections, but its antimicrobial properties have not been well established yet. Thus, as herbal medicine can be a source for new therapeutics and considering the evidences of biological activities of the Dorstenia genus should be also considered, the aim of this work was to investigate the possible effects of D. asaroides on cariogenic properties of S. mutans. 2. Materials and methods 2.1. Bacterial strain The bacterial strain used was S. mutans ATCC 25175. The culture was stored at 70 C in Brain Heart Infusion (Oxoid) containing 20% (w/v) glycerol. 2.2. Plant material Samples belonging to D. asaroides were collected in Rio Pardo de Minas, Minas Gerais State, Brazil. A voucher specimen was deposited in the Herbarium of Department of Biology (Herbarium SPFR) at
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C.E.M. D’Angelis et al. / Anaerobe 18 (2012) 31e36
Faculty of Philosophy, Sciences and Languages of Ribeirão Preto, University of São Paulo, under the record SPFR 12262. Rhizome (1000 g) was air dried at 40 C, grounded and extracted in ethanol (4 L) during 3 days with a daily exchange of solvent. The solvent was removed under reduced pressure to yield the crude extract (70 g). 2.3. Chemical characterization The crude extract was treated with 400 mL of n-hexane at room temperature to allow the chemical fractioning based on the polarity gradient of such solvents as hexane (HFr), ethyl-acetate (EFr) and chloroform (CFr). Each extract concentration was obtained under reduced pressure and dissolved in dimethylsulphoxide (DMSO). These extracts were analyzed by gas chromatography/mass spectrometry GCeMS; Shimadzu GC/MS e QP2010 gas chromatograph with automatic sampler AOC e 20Si at split mode (1:60), using a fused-silica capillary column (DB-5, 30 m 0.25 mm, coating thickness 0.25 mm) programmed from 70 to 250 C at 3 C/min, with hydrogen (1.30 mL/min) as the carrier gas. The products were identified by their typical retention times and also the electron ionization spectra as well by peaks available in the spectrometric electronic library Wiley 7.0. 2.4. Antimicrobial activity assay Minimal inhibitory concentrations (MIC) of D. asaroides extracts was determined based on a microdilution method in 96 multi-well microtiter plates as previously described by Eloff [15]. Briefly, S. mutans was cultured overnight at 37 C on Brain Heart Infusion (BHI) broth plus 1% glucose and adjusted to a final density of 104 cfu/mL. The cell suspension was inoculated in microplates containing a dilution series of HFr, EFr and CFr (concentrations ranging from 10 to 120 mg/ mL), chlorhexidine digluconate 0.06% (positive control; concentrations ranging from 0.05 to 120 mg/mL) or no treatment (negative control). The triplicate plates were incubated at 37 C for 24 h. As an indicator of bacterial growth, 50 mL of 0.2 mg/mL nitroblue tetrazolium (NBT) was added to the wells and incubated at 37 C for 30 min. The lowest concentration of the compound showing no colour change was taken as its minimal inhibitory concentration. The colourless tetrazolium salt acts as an electron acceptor and it is reduced to a red-coloured formazan product by biologically active organisms [15]. Where bacterial growth was inhibited, the solution in the well remained clear after incubation with NBT. Triplicates were made for each concentration of the tested extracts. 2.5. Glycolysis assay The effects of D. asaroides extracts on glycolysis were evaluated through the potentiometric measurement of pH from dense cell suspensions (5 mg cell-dry-weight mL1) as described previously by Leitão et al. [16]. Briefly, cells of S. mutans from suspension cultures were harvested and resuspended in 6.0 mmol/L phosphate buffer, pH 6.9, containing 0.865 g/L (w/v) NaCl, 0.625 g/L (w/v) KCl, 0.125 g/L (w/v) MgCl2 6H2O, 0.072 g/L (w/v) CaCl2$2H2O, 0.326 g/L (w/v) KH2PO4, and 0.803 g/L (w/v) K2HPO4 supplemented with 10.0 mmol/L glucose as a bacterial substrate and one of the following solutions was added: extract stock solution of D. asaroides (HFr, EFr, CFr) previously dissolved in (DMSO)eH2O (1:1, v/v; final concentrations ranged from 0.625 to 12.5 mg/mL); chlorhexidine digluconate 0.06% (positive control). The negative control test (no inhibition) consisted of S. mutans aliquots resuspended in the same buffer solution supplemented with 10.0 mmol/L glucose. A thin-bulb analytical electrode (model 8103, Orion) was placed in the cell suspension, which was warmed to 37 C, and the glycolytic production of acid was monitored during a 30-min time period by measuring pH fall with a pH meter (model 710 A, Orion).
Fig. 1. Initial adherence of Streptococcus mutans to glass slide surface.
The pH curves (pH versus time) were built from a plot of the average values of triplicate assays, and their integrated area values were used to estimate the inhibitory activity of extracts at each concentration tested, considering that no acid production was observed in the positive control (i.e., absence of pH decay), and the negative control pH curve represented no inhibition of the acid production. Inhibition curves (percentage of inhibition versus concentration) were plotted, and 50% of the inhibitory concentrations (IC50; the concentration of extract required to inhibit acidogenic activity by 50%) were calculated from non-linear regression sigmoidal curves using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, California, U.S.A.). 2.6. Initial biofilm formation Since the adhesion of bacteria to the surface represents the initial step in the pathogenesis of dental plaque formation and subsequent caries development [17], the anti-binding effects of the D. asaroides gain importance by a possible prevention of specific adherence mechanisms and subsequent biofilm formation. Thus, considering that a previous test showed that counts of S. mutans in the biofilms did not differ between 12 and 24 h (Fig. 1), the biofilms were grown undisturbed for 12 h to allow initial adherence. The slides were clamped vertically to a sterile steel rack [18], immersed in a beaker containing BHI broth (1% glucose e w/v) with an overnight culture of S. mutans, and incubated at 37 C for up to 12 h with slow constant agitation. At this point (12 h old), the biofilms were treated with D. asaroides fractions (HFr, EFr or CFr; concentrations ranged from 1.25 to 12.5 mg/mL) or Phosphate Buffered Saline (PBS: 0.8% NaCl, 0.02% KCl, 0.144% Na2HPO4, and 0.024% NaH2PO4, pH 7.4). The biofilms were exposed to the treatments for 3 min under agitation, rinsed in a sterile PBS solution and removed manually by scrubbing the surface with a sterile cotton swab for 1 min [19]. Swabs were transferred to tubes containing 10 mL of PBS, and 100 mL serial decimal dilutions were plated on BHI agar. Plates in triplicate were incubated at 37 C under microaerophylic conditions for 24 h. 2.7. Statistical analysis Statistical analysis was performed using two-way ANOVA, assuming equality of variance with Bonfferoni’s post hoc test for Table 1 MIC results from Dorstenia asaroides extracts. Microorganism
Extracts (mg/mL) HFr
EFr
CFr
S. mutans 25175
50
50
80
MIC values for chlorhexidine (positive control) were 0.93 mg/mL.
C.E.M. D’Angelis et al. / Anaerobe 18 (2012) 31e36
33
H Fr 7.5
120
Inhibition (%)
100
7.0
6.5
**
80 ***
60 40
***
1, 25
2, 5
6, 25
Concentration (mg/mL)
Negative Control
5.5
12 ,5
0
6.0
PC
pH
20
12 5mg/mL 6. 5mg/mL 2. mg/mL
5.0
1 25mg/mL Positive Control
4.5 0
5
10
15
20
25
30
Time (min)
7.5
E Fr
120 100
Inhibition (%)
7.0
6.5
80 ***
60
***
40
***
5 62
25 1,
0,
5
85
25
2,
1,
12,5mg/mL
Concentration (mg/mL)
6,25mg/mL
5.5
6,
PC
Negative Control
,5
0
6.0
12
pH
20
2,5mg/mL 1.875mg/mL 1,25mg/mL
5.0
0,25 mg/mL Positive Control
4.5 0
5
10
7.5
15 Time (min)
20
25
30
C Fr
120
Inhibition (%)
100
7.0
6.5
***
80 60 40 20
***
5 0, 62
1, 25
2, 5
25 6,
,5 12
6.0
PC
pH
0
Concentration (mg/mL)
Negative Control 12 5mg/mL
5.5
6. 5mg/mL 2. mg/mL 1 25mg/mL
5.0
4.5
0.6 5mg/mL Positive Control
0
5
10
15 Time (min)
20
25
30
Fig. 2. Inhibitory effects of HFr, EFr and CFr of Dorstenia asaroides. Significant difference between positive control (total inhibition) and treatment with extracts: **p < 0.01; ***p < 0.001.
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pairwise comparisons and Tukey’s Multiple Comparison Test. The results are presented as the mean SEM. Results with p < 0.01 were considered statistically significant. 3. Results 3.1. Chemical characterization Chemical characterization showed that all the extracts present furanocoumarins. HFr contained fatty acids, esters, campesterol, triterpenic alcohols, psoralen and bergapten; EFr and CFr only psoralen and bergapten. The structures of these compounds were determined by comparison with spectral data reported in the literature. 3.2. Antimicrobial activity assay The MIC values were 80 (HFr and CFr) and 50 mg/mL (EFr) for S. mutans. Chlorhexidine showed MIC value of 0.93 mg/mL (Table 1). 3.3. Glycolytic assay Cariogenicity of S. mutans can be related directly to its capacity to produce acids from glycolysis. The influence of different extracts of D. asaroides on glycolytic pH drop by S. mutans cells in the presence of glucose is shown in Fig. 2. The acid production by S. mutans cells was significantly disrupted by HFr (12.5 mg/mL), EFr (at 2.5; 6.25 and 12.5 mg/mL) and CFr (at 2.5, 6.25 and 12.5 mg/mL) (p < 0.01). When comparing the pH values of the tested samples to the one chlorhexidine digluconate was used as positive control, the inhibitory effect of tested extracts could mostly be considered as important (Fig. 2, bar graph). IC50 values of the three extracts were represented by curves with different slopes (Fig. 3). HFr, EFr and CFr exhibited IC50 of 2.32, 1.67 and 0.89 mg/mL, respectively. 3.4. Biofilm formation Finally, we assessed the effects of D. asaroides extracts on initial adherence to surface by S. mutans on glass slide. Topical applications of HFr, EFr and CFr significantly reduced the colony forming units of S. mutans biofilms compared with those treated with control group (PBS) in order to 20, 30 and 25% respectively (p < 0.01).
HFr
100
EFr CFr
4. Discussion Natural products are still major sources of innovative therapeutic agents which could be useful for the development of alternative or adjunctive anticaries therapies [20]. It has long been acknowledged by Brazilian popular medicine that D. asaroides exhibit antimicrobial properties. However, the antimicrobial activity of this plant against oral microorganisms had never been investigated. The antibacterial activities of D. asaroides extracts could be associated to the presence of furanocoumarins compounds like psoralen and bergapten that were identified for all tested fractions. Antimicrobial effects of psoralen and bergapten have been reported for other plants of Moraceae family [21] and it is suggested that furanocoumarins possess a variety of biological effects including antimicrobial and antifungal activities [6,21e23]. In addition, furanocoumarins are contemplating to combat periodontopathogenic bacteria as an alternative to the systemic application of antibacterial drugs used in the treatment of oral diseases [24]. Future studies should be conducted in order to investigate the efficacy of these compounds in dentistry. The present study showed that extracts obtained from rhizomes of D. asaroides showed a potential activity against S. mutans at low concentrations, showing maximum MIC values of 80 mg/mL. Plant extracts that are active in a concentration lower than 100 mg/mL could have a good antimicrobial potential, since the active compounds can be isolated and used at lower concentrations [25]. S. mutans has generally been regarded as a primary etiologic agent of dental caries [1]. The leading role of S. mutans is determined mainly by its acid production [26] which can lead to the demineralization of the adjacent dental enamel, promoting dental caries. Thus, acid production is an important virulence property of S. mutans that deserves attention when evaluating natural products for their potential ability to prevent dental caries. In this study, the D. asaroides extracts reduced significantly the level of acid production from S. mutans in suspension. The influence of the extracts on glycolytic pH drop by S. mutans indicates the following order of potency: CFr > EFr > HFr. In fact, CFr extract is composed uniquely by psoralen and bergapten. Microorganisms attached to surfaces are known to present a more distinct behaviour than cells in suspension (planktonic state) [27]. S. mutans cells can attach initially to surfaces through sucrose-independent mechanisms mediated primarily by adhesins present on the bacterial cell surface in order to initiate biofilm formation. However, the inhibitory effect of tested extracts reduced attached bacteria at maximum 30%. Probably, differences could be expected if the experimental biofilm were formed in presence of sucrose and saliva proteins, what should be further evaluated. 5. Conclusion
Inhibition (%)
75
The results of the present study suggest that rhizomes of D. asaroides had inhibitory effects on cariogenic properties of S. mutans. Although these preliminary observations are promising, additional studies are needed to elucidate the mechanisms by which these agents impair virulence factors of cariogenic streptococci.
50
25
Acknowledgements 0 0
2
4
6
8
10
12
14
Concentration (mg/mL) Fig. 3. IC50 values of Dorstenia asaroides extracts. (HFr ¼ 2.3; EFr ¼ 1.7; CFr ¼ 0.9).
We thank Prof. Dr. Norberto Peporine Lopes from Organic Chemistry Laboratory, Department of Physics and Chemistry, Faculty of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo-USP, Brazil for the phytochemical analysis and Pierre Alexandre dos Santos and Marley Garcia Silva for the technical assistance.
C.E.M. D’Angelis et al. / Anaerobe 18 (2012) 31e36
35
Appendix
Fig. 1. Location of the samples in 96 multi-well microtiter plates. AeC: Dorstenia’s extract; D: No treatment (no inhibition, negative control). E: chlorhexidine digluconate (positive control). F: Sterility control of BHI (no inoculation).
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Fig. 2. Minimal inhibitory concentrations (MIC) of Dorstenia asaroides extracts. Photograph of microtiter wells with different treatments after nitroblue tetrazolium (NBT) incubation: a) chloroform fraction; b) Ethyl-acetate fraction; c) Hexanic fraction.
References [1] Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiol Rev 1986;50:353e80.
[2] ten Cate JM. Biofilms, a new approach to the microbiology of dental plaque. Odontology 2006;94(1):1e9. [3] Bowden GH, Hamilton IR. Survival of oral bacteria. Crit Rev Oral Biol Med 1998;9(1):54e85. [4] Sansone C, Van Houte, Joshipura K, Kent R, Margolis HC. The association of mutans streptococci and non-mutans streptococci capable of acidogenesis at a low pH with dental caries on enamel and root surfaces. J Dent Res 1993;72: 508e16. [5] Belli WA, Marquis RE. Adaptation of Streptococcus mutans and Enterococcus hirae to acid stress in continuous culture. Appl Environ Microbiol 1991;57: 1134e8. [6] Allaker RP, Douglas CW. Novel anti-microbial therapies for dental plaquerelated diseases. Int J Antimicrob Agents 2009;33:8e13. [7] Toi CS, Cleaton-Jones P. The effect of traditional African food mixtures on growth, pH and extracellular polysaccharide production by mutans streptococci in vitro. Anaerobe 2006;12:99e105. [8] Duarte S, Gregoire S, Singh AP, Vorsa N, Schaich K, Bowen WH, et al. Inhibitory effects of cranberry polyphenols on formation and acidogenicity of Streptococcus mutans biofilms. FEMS Microbiol Lett 2006;257: 50e6. [9] Gregoire S, Singh AP, Vorsa N, Koo H. Influence of cranberry phenolics on glucan synthesis by glucosyltransferases and Streptococcus mutans acidogenicity. J Appl Microbiol 2007;103:1960e8. [10] Karthikeyan R, Amaechi BT, Rawls HR, Lee VA. Antimicrobial activity of nanoemulsion on cariogenic Streptococcus mutans. Arch Oral Bio 2011;56: 437e45. [11] Badet C, Quero F. The in vitro effect of manuka honeys on growth and adherence of oral bacteria. Anaerobe 2011;17:19e22. [12] Iwu MM, Jackson JE, Tally JD, Klayman DL. Evaluation of plant extracts for antileishmanial activity using a mechanism-based radiorespirometric microtechnique (RAM). Planta Med 1992;58(5):436e41. [13] Tsopmo A, Tene M, Kamnaing P, Ngnokam D, Ayafor JF, Sterner O. Geranylated flavonoids from Dorstenia poinsettifolia. Phytochem 1998;48: 345e8. [14] Zapata-Sudo G, Mendes TCF, Kartnaller MA, Fortes TO, Freitas NFB, Kaplan MAC, et al. Sedative and anticonvulsant activities of methanol extract of Dorstenia arifolia in mice. J Ethnopharmacol 2010;130:9e12. [15] Eloff JN. A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Med 1998;64: 711e3. [16] Leitão DPS, Filhom AAS, Polizello ACM, Bastos JK, Spadaro ACC. Comparative evaluation of in-vitro effects of Brazilian green propolis and Baccharis dracunculifolia extracts on cariogenic factors of Streptococcus mutans. Biol Pharm Bull 2004;27:1834e9. [17] Gibbons RJ. Bacterial adhesion to oral tissues: a model for infectious diseases. J Dental Res 1989;68:750e60. [18] Minei CC, Gomes BC, Ratti RP, D’Angelis CEM, De Martinis ECP. Influence of peroxyacetic acid and nisin and coculture with Enterococcus faecium on Listeria monocytogenes biofilm formation. J Food Prot 2008;71: 634e8. [19] Chae MS, Schraft H. Comparative evaluation of adhesion and biofilm formation of different Listeria monocytogenes strains. Int J Food Microbiol 2000;62: 103e11. [20] Jeon JG, Rosalen PL, Falsetta ML, Koo H. Natural products in caries research: current (limited) knowledge, challenges and future perspective. Caries Res 2011;45(3):243e63. [21] Cowan MM. Plant products as antimicrobial agents. Clin Microbiol Rev 1999; 12(4):564e82. [22] Smith ML, Gregory P, Bafi-Yeboa NFA, Arnason JT. Inhibition of DNA polymerization and antifungal specificity of furanocoumarins present in traditional medicines. Photochem Photobiol 2004;79:506e9. [23] Heinke R, Franke K, Porzel A, Wessjohann LA, Ali NAA, Schmidt J. Furanocoumarins from Dorstenia foetida. Phytochem 2011;72:929e34. [24] Meisel P, Kocher T. Photodynamic therapy for periodontal diseases: state of the art. J Photochem Photobiol B 2005;79(2):159e70. [25] Holetz FB, Pessini GL, Sanches NR, Cortez DAG, Nakamura CV, Dias Filho BP. Screening of some plants used in the Brazilian folk medicine for the treatment of infectious diseases. Mem Inst Oswaldo Cruz 2002;97: 1027e31. [26] Bender GR, Thibodeau EA, Marquis RE. Reduction of acidurance of streptococcal growth and glycolysis by fluoride and gramicidin. J Dent Res 1985;64: 90e5. [27] Lewis K. Riddle of biofilm resistance. Antimicrob Agents Chemother 2001; 45(4):999e1007.
Contents lists available at SciVerse ScienceDirect
Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Clinical microbiology
Inhibiting effect of Dorstenia asaroides extracts on cariogenic properties of Streptococcus mutans C.E.M. D’Angelis a, c, M.F. Leite a, J.P.B. Sousa a, L. Alonso a, A.C.M. Polizello a, M. Groppo Jr. b, C.P. Aires a, J.K. Bastos a, A.C.C. Spadaro a, * a
Department of Physics and Chemistry, Faculty of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo-USP, Av. do Café s/n, Monte Alegre, 14040-903 Ribeirão Preto, São Paulo, Brazil Department of Biology, Faculty of Philosophy, Sciences and Languages of Ribeirão Preto, University of São Paulo-USP, Brazil c Pitágoras Integrated School of Montes Claros, Montes Claros, Minas Gerais, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 July 2011 Received in revised form 16 November 2011 Accepted 22 December 2011 Available online 10 January 2012
The aim of this study was to examine the effects of Dorstenia asaroides extracts on cariogenic properties of the most cariogenic bacteria, Streptococcus mutans. Hexane (HFr), ethyl-acetate (EFr) and chloroform (CFr) extracts obtained from D. asaroides rhizomes were submitted to chemical analyses, Minimal Inhibitory Concentrations (MIC), glycolysis assay and S. mutans 12-h-old initial biofilms. Chemical characterization showed that all the extracts present furanocoumarins. The MIC values were 80 (HFr and CFr) and 50 mg/mL (EFr). Acid production by S. mutans cells was significantly disrupted by HFr (12.5 mg/mL), EFr (at 2.5; 6.25 and 12.5 mg/mL) and CFr (at 2.5, 6.25 and 12.5 mg/mL) (p < 0.01). Topical applications of HFr, EFr and CFr significantly reduced the colony forming units of S. mutans biofilms compared with those treated with control group in order to 20, 30 and 25% respectively (p < 0.01). The results of the present study suggest that rhizomes of D. asaroides had inhibitory effects on cariogenic properties of S. mutans. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Antimicrobial Biofilm Streptococcus mutans Dorstenia asaroides
1. Introduction Streptococcus mutans has been strongly implicated as the principal etiological agent in human dental caries [1] although other additional acidogenic microorganisms may be involved [2]. The virulence of this microorganism has been attributed to their acidogenic and aciduric properties which play a critical role in the pathogenesis of dental caries [3,4]. Mutans streptococci catabolise multiple fermentable dietary carbohydrates, and also carry out glycolysis at low biofilm pH values in the oral cavity [5] promoting tooth demineralization. Therefore, antimicrobial substances against these bacteria or those that affect acid production could play an important role in preventing the formation of dental plaque and caries. Plants are an important possible source of new bioactive compounds to disrupt bacteria virulence because they may serve as alternatives to the commonly used chemicals for dental biofilm [6]. It is well established that many of the metabolites produced by plants present properties against oral pathogens [7e11]. Several Dorstenia species have been extensively used in traditional medicine. Dorstenia, a herbaceous genus rich in furanocoumarins, belongs to the family Moraceae and is found throughout Africa, besides Central and South America. The * Corresponding author. Tel.: þ55 16 3602 4215; fax: þ55 16 3633 1936. E-mail address: [email protected] (A.C.C. Spadaro). 1075-9964/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2011.12.020
pharmacological data on the genus Dorstenia are limited, but it is known that species of the genus popularly known in Brazil as “Carapiá” are used due to its medicinal qualities [12e14]. In Brazilian folk medicine, the species D. asaroides is used for infections, but its antimicrobial properties have not been well established yet. Thus, as herbal medicine can be a source for new therapeutics and considering the evidences of biological activities of the Dorstenia genus should be also considered, the aim of this work was to investigate the possible effects of D. asaroides on cariogenic properties of S. mutans. 2. Materials and methods 2.1. Bacterial strain The bacterial strain used was S. mutans ATCC 25175. The culture was stored at 70 C in Brain Heart Infusion (Oxoid) containing 20% (w/v) glycerol. 2.2. Plant material Samples belonging to D. asaroides were collected in Rio Pardo de Minas, Minas Gerais State, Brazil. A voucher specimen was deposited in the Herbarium of Department of Biology (Herbarium SPFR) at
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C.E.M. D’Angelis et al. / Anaerobe 18 (2012) 31e36
Faculty of Philosophy, Sciences and Languages of Ribeirão Preto, University of São Paulo, under the record SPFR 12262. Rhizome (1000 g) was air dried at 40 C, grounded and extracted in ethanol (4 L) during 3 days with a daily exchange of solvent. The solvent was removed under reduced pressure to yield the crude extract (70 g). 2.3. Chemical characterization The crude extract was treated with 400 mL of n-hexane at room temperature to allow the chemical fractioning based on the polarity gradient of such solvents as hexane (HFr), ethyl-acetate (EFr) and chloroform (CFr). Each extract concentration was obtained under reduced pressure and dissolved in dimethylsulphoxide (DMSO). These extracts were analyzed by gas chromatography/mass spectrometry GCeMS; Shimadzu GC/MS e QP2010 gas chromatograph with automatic sampler AOC e 20Si at split mode (1:60), using a fused-silica capillary column (DB-5, 30 m 0.25 mm, coating thickness 0.25 mm) programmed from 70 to 250 C at 3 C/min, with hydrogen (1.30 mL/min) as the carrier gas. The products were identified by their typical retention times and also the electron ionization spectra as well by peaks available in the spectrometric electronic library Wiley 7.0. 2.4. Antimicrobial activity assay Minimal inhibitory concentrations (MIC) of D. asaroides extracts was determined based on a microdilution method in 96 multi-well microtiter plates as previously described by Eloff [15]. Briefly, S. mutans was cultured overnight at 37 C on Brain Heart Infusion (BHI) broth plus 1% glucose and adjusted to a final density of 104 cfu/mL. The cell suspension was inoculated in microplates containing a dilution series of HFr, EFr and CFr (concentrations ranging from 10 to 120 mg/ mL), chlorhexidine digluconate 0.06% (positive control; concentrations ranging from 0.05 to 120 mg/mL) or no treatment (negative control). The triplicate plates were incubated at 37 C for 24 h. As an indicator of bacterial growth, 50 mL of 0.2 mg/mL nitroblue tetrazolium (NBT) was added to the wells and incubated at 37 C for 30 min. The lowest concentration of the compound showing no colour change was taken as its minimal inhibitory concentration. The colourless tetrazolium salt acts as an electron acceptor and it is reduced to a red-coloured formazan product by biologically active organisms [15]. Where bacterial growth was inhibited, the solution in the well remained clear after incubation with NBT. Triplicates were made for each concentration of the tested extracts. 2.5. Glycolysis assay The effects of D. asaroides extracts on glycolysis were evaluated through the potentiometric measurement of pH from dense cell suspensions (5 mg cell-dry-weight mL1) as described previously by Leitão et al. [16]. Briefly, cells of S. mutans from suspension cultures were harvested and resuspended in 6.0 mmol/L phosphate buffer, pH 6.9, containing 0.865 g/L (w/v) NaCl, 0.625 g/L (w/v) KCl, 0.125 g/L (w/v) MgCl2 6H2O, 0.072 g/L (w/v) CaCl2$2H2O, 0.326 g/L (w/v) KH2PO4, and 0.803 g/L (w/v) K2HPO4 supplemented with 10.0 mmol/L glucose as a bacterial substrate and one of the following solutions was added: extract stock solution of D. asaroides (HFr, EFr, CFr) previously dissolved in (DMSO)eH2O (1:1, v/v; final concentrations ranged from 0.625 to 12.5 mg/mL); chlorhexidine digluconate 0.06% (positive control). The negative control test (no inhibition) consisted of S. mutans aliquots resuspended in the same buffer solution supplemented with 10.0 mmol/L glucose. A thin-bulb analytical electrode (model 8103, Orion) was placed in the cell suspension, which was warmed to 37 C, and the glycolytic production of acid was monitored during a 30-min time period by measuring pH fall with a pH meter (model 710 A, Orion).
Fig. 1. Initial adherence of Streptococcus mutans to glass slide surface.
The pH curves (pH versus time) were built from a plot of the average values of triplicate assays, and their integrated area values were used to estimate the inhibitory activity of extracts at each concentration tested, considering that no acid production was observed in the positive control (i.e., absence of pH decay), and the negative control pH curve represented no inhibition of the acid production. Inhibition curves (percentage of inhibition versus concentration) were plotted, and 50% of the inhibitory concentrations (IC50; the concentration of extract required to inhibit acidogenic activity by 50%) were calculated from non-linear regression sigmoidal curves using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, California, U.S.A.). 2.6. Initial biofilm formation Since the adhesion of bacteria to the surface represents the initial step in the pathogenesis of dental plaque formation and subsequent caries development [17], the anti-binding effects of the D. asaroides gain importance by a possible prevention of specific adherence mechanisms and subsequent biofilm formation. Thus, considering that a previous test showed that counts of S. mutans in the biofilms did not differ between 12 and 24 h (Fig. 1), the biofilms were grown undisturbed for 12 h to allow initial adherence. The slides were clamped vertically to a sterile steel rack [18], immersed in a beaker containing BHI broth (1% glucose e w/v) with an overnight culture of S. mutans, and incubated at 37 C for up to 12 h with slow constant agitation. At this point (12 h old), the biofilms were treated with D. asaroides fractions (HFr, EFr or CFr; concentrations ranged from 1.25 to 12.5 mg/mL) or Phosphate Buffered Saline (PBS: 0.8% NaCl, 0.02% KCl, 0.144% Na2HPO4, and 0.024% NaH2PO4, pH 7.4). The biofilms were exposed to the treatments for 3 min under agitation, rinsed in a sterile PBS solution and removed manually by scrubbing the surface with a sterile cotton swab for 1 min [19]. Swabs were transferred to tubes containing 10 mL of PBS, and 100 mL serial decimal dilutions were plated on BHI agar. Plates in triplicate were incubated at 37 C under microaerophylic conditions for 24 h. 2.7. Statistical analysis Statistical analysis was performed using two-way ANOVA, assuming equality of variance with Bonfferoni’s post hoc test for Table 1 MIC results from Dorstenia asaroides extracts. Microorganism
Extracts (mg/mL) HFr
EFr
CFr
S. mutans 25175
50
50
80
MIC values for chlorhexidine (positive control) were 0.93 mg/mL.
C.E.M. D’Angelis et al. / Anaerobe 18 (2012) 31e36
33
H Fr 7.5
120
Inhibition (%)
100
7.0
6.5
**
80 ***
60 40
***
1, 25
2, 5
6, 25
Concentration (mg/mL)
Negative Control
5.5
12 ,5
0
6.0
PC
pH
20
12 5mg/mL 6. 5mg/mL 2. mg/mL
5.0
1 25mg/mL Positive Control
4.5 0
5
10
15
20
25
30
Time (min)
7.5
E Fr
120 100
Inhibition (%)
7.0
6.5
80 ***
60
***
40
***
5 62
25 1,
0,
5
85
25
2,
1,
12,5mg/mL
Concentration (mg/mL)
6,25mg/mL
5.5
6,
PC
Negative Control
,5
0
6.0
12
pH
20
2,5mg/mL 1.875mg/mL 1,25mg/mL
5.0
0,25 mg/mL Positive Control
4.5 0
5
10
7.5
15 Time (min)
20
25
30
C Fr
120
Inhibition (%)
100
7.0
6.5
***
80 60 40 20
***
5 0, 62
1, 25
2, 5
25 6,
,5 12
6.0
PC
pH
0
Concentration (mg/mL)
Negative Control 12 5mg/mL
5.5
6. 5mg/mL 2. mg/mL 1 25mg/mL
5.0
4.5
0.6 5mg/mL Positive Control
0
5
10
15 Time (min)
20
25
30
Fig. 2. Inhibitory effects of HFr, EFr and CFr of Dorstenia asaroides. Significant difference between positive control (total inhibition) and treatment with extracts: **p < 0.01; ***p < 0.001.
34
C.E.M. D’Angelis et al. / Anaerobe 18 (2012) 31e36
pairwise comparisons and Tukey’s Multiple Comparison Test. The results are presented as the mean SEM. Results with p < 0.01 were considered statistically significant. 3. Results 3.1. Chemical characterization Chemical characterization showed that all the extracts present furanocoumarins. HFr contained fatty acids, esters, campesterol, triterpenic alcohols, psoralen and bergapten; EFr and CFr only psoralen and bergapten. The structures of these compounds were determined by comparison with spectral data reported in the literature. 3.2. Antimicrobial activity assay The MIC values were 80 (HFr and CFr) and 50 mg/mL (EFr) for S. mutans. Chlorhexidine showed MIC value of 0.93 mg/mL (Table 1). 3.3. Glycolytic assay Cariogenicity of S. mutans can be related directly to its capacity to produce acids from glycolysis. The influence of different extracts of D. asaroides on glycolytic pH drop by S. mutans cells in the presence of glucose is shown in Fig. 2. The acid production by S. mutans cells was significantly disrupted by HFr (12.5 mg/mL), EFr (at 2.5; 6.25 and 12.5 mg/mL) and CFr (at 2.5, 6.25 and 12.5 mg/mL) (p < 0.01). When comparing the pH values of the tested samples to the one chlorhexidine digluconate was used as positive control, the inhibitory effect of tested extracts could mostly be considered as important (Fig. 2, bar graph). IC50 values of the three extracts were represented by curves with different slopes (Fig. 3). HFr, EFr and CFr exhibited IC50 of 2.32, 1.67 and 0.89 mg/mL, respectively. 3.4. Biofilm formation Finally, we assessed the effects of D. asaroides extracts on initial adherence to surface by S. mutans on glass slide. Topical applications of HFr, EFr and CFr significantly reduced the colony forming units of S. mutans biofilms compared with those treated with control group (PBS) in order to 20, 30 and 25% respectively (p < 0.01).
HFr
100
EFr CFr
4. Discussion Natural products are still major sources of innovative therapeutic agents which could be useful for the development of alternative or adjunctive anticaries therapies [20]. It has long been acknowledged by Brazilian popular medicine that D. asaroides exhibit antimicrobial properties. However, the antimicrobial activity of this plant against oral microorganisms had never been investigated. The antibacterial activities of D. asaroides extracts could be associated to the presence of furanocoumarins compounds like psoralen and bergapten that were identified for all tested fractions. Antimicrobial effects of psoralen and bergapten have been reported for other plants of Moraceae family [21] and it is suggested that furanocoumarins possess a variety of biological effects including antimicrobial and antifungal activities [6,21e23]. In addition, furanocoumarins are contemplating to combat periodontopathogenic bacteria as an alternative to the systemic application of antibacterial drugs used in the treatment of oral diseases [24]. Future studies should be conducted in order to investigate the efficacy of these compounds in dentistry. The present study showed that extracts obtained from rhizomes of D. asaroides showed a potential activity against S. mutans at low concentrations, showing maximum MIC values of 80 mg/mL. Plant extracts that are active in a concentration lower than 100 mg/mL could have a good antimicrobial potential, since the active compounds can be isolated and used at lower concentrations [25]. S. mutans has generally been regarded as a primary etiologic agent of dental caries [1]. The leading role of S. mutans is determined mainly by its acid production [26] which can lead to the demineralization of the adjacent dental enamel, promoting dental caries. Thus, acid production is an important virulence property of S. mutans that deserves attention when evaluating natural products for their potential ability to prevent dental caries. In this study, the D. asaroides extracts reduced significantly the level of acid production from S. mutans in suspension. The influence of the extracts on glycolytic pH drop by S. mutans indicates the following order of potency: CFr > EFr > HFr. In fact, CFr extract is composed uniquely by psoralen and bergapten. Microorganisms attached to surfaces are known to present a more distinct behaviour than cells in suspension (planktonic state) [27]. S. mutans cells can attach initially to surfaces through sucrose-independent mechanisms mediated primarily by adhesins present on the bacterial cell surface in order to initiate biofilm formation. However, the inhibitory effect of tested extracts reduced attached bacteria at maximum 30%. Probably, differences could be expected if the experimental biofilm were formed in presence of sucrose and saliva proteins, what should be further evaluated. 5. Conclusion
Inhibition (%)
75
The results of the present study suggest that rhizomes of D. asaroides had inhibitory effects on cariogenic properties of S. mutans. Although these preliminary observations are promising, additional studies are needed to elucidate the mechanisms by which these agents impair virulence factors of cariogenic streptococci.
50
25
Acknowledgements 0 0
2
4
6
8
10
12
14
Concentration (mg/mL) Fig. 3. IC50 values of Dorstenia asaroides extracts. (HFr ¼ 2.3; EFr ¼ 1.7; CFr ¼ 0.9).
We thank Prof. Dr. Norberto Peporine Lopes from Organic Chemistry Laboratory, Department of Physics and Chemistry, Faculty of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo-USP, Brazil for the phytochemical analysis and Pierre Alexandre dos Santos and Marley Garcia Silva for the technical assistance.
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35
Appendix
Fig. 1. Location of the samples in 96 multi-well microtiter plates. AeC: Dorstenia’s extract; D: No treatment (no inhibition, negative control). E: chlorhexidine digluconate (positive control). F: Sterility control of BHI (no inoculation).
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Fig. 2. Minimal inhibitory concentrations (MIC) of Dorstenia asaroides extracts. Photograph of microtiter wells with different treatments after nitroblue tetrazolium (NBT) incubation: a) chloroform fraction; b) Ethyl-acetate fraction; c) Hexanic fraction.
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