Original Paper Received: May 20, 2013 Accepted: November 12, 2013 Published online: March 6, 2014
Caries Res 2014;48:353–360 DOI: 10.1159/000357225
Systematic Screening of Plant Extracts from the Brazilian Pantanal with Antimicrobial Activity against Bacteria with Cariogenic Relevance F.L. Brighenti b M.J. Salvador c Alberto Carlos Botazzo Delbem d Ádina Cleia Bottazzo Delbem d M.A.C. Oliveira a C.P. Soares e L.S.F. Freitas a C.Y. Koga-Ito a, f
a
Department of Biosciences and Oral Diagnosis, Universidade Estadual Paulista (UNESP), São José dos Campos, Department of Orthodontics and Pediatric Dentistry, Araraquara Dental School, Universidade Estadual Paulista (UNESP), Araraquara, c Department of Plant Biology, Institute of Biology, Universidade Estadual de Campinas (UNICAMP), Campinas, d Department of Pediatric Dentistry and Public Health, Araçatuba Dental School, Universidade Estadual Paulista (UNESP), Araçatuba, e Laboratory of Cell and Tissue Biology, Universidade do Vale do Paraíba (UNIVAP), São José dos Campos, and f Department of Environment Engineering, Universidade Estadual Paulista (UNESP), São José dos Campos, Brazil
b
Key Words Biological agents · Dental caries · Prevention and control
Abstract This study proposes a bioprospection methodology regarding the antimicrobial potential of plant extracts against bacteria with cariogenic relevance. Sixty extracts were obtained from ten plants – (1) Jatropha weddelliana, (2) Attalea phalerata, (3) Buchenavia tomentosa, (4) Croton doctoris, (5) Mouriri elliptica, (6) Mascagnia benthamiana, (7) Senna aculeata, (8) Unonopsis guatterioides, (9) Allagoptera leucocalyx and (10) Bactris glaucescens – using different extraction methods – (A) 70° ethanol 72 h/25 ° C, (B) water 5 min/100 ° C, (C) water 1 h/55 ° C, (D) water 72 h/25 ° C, (E) hexane 72 h/25 ° C and (F) 90° ethanol 72 h/25 ° C. The plants were screened for antibacterial activity at 50 mg/ml using the agar well diffusion test against Actinomyces naeslundii ATCC 19039, Lactobacillus acidophilus ATCC 4356, Streptococcus gordonii ATCC 10558, Streptococcus mutans ATCC 35688, Streptococcus sanguinis ATCC 10556, Streptococcus sobrinus ATCC 33478 and Streptococcus mitis ATCC 9811. The active extracts were test
© 2014 S. Karger AG, Basel 0008–6568/14/0485–0353$39.50/0 E-Mail [email protected] www.karger.com/cre
The utmost importance of the scientific value of the biodiversity in Brazil is widely known. This country’s vast and extensive territory consists of a great diversity of biomes. To date, the number of known or recorded Brazilian biota is estimated to be 200,000 species, which represents about Cristiane Yumi Koga-Ito Department of Environmental Engineering (UNESP) Av. Engenheiro Francisco José Longo, 777 São José dos Campos, SP 12245-000 (Brazil) E-Mail cristiane @ fosjc.unesp.br
Downloaded by: NYU Medical Center Library 128.122.253.212 - 10/19/2014 3:39:11 AM
ed to determine their minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), cytotoxicity and chemical characterization. Forty-seven extracts (78%) were active against at least one microorganism. Extract 4A demonstrated the lowest MIC and MBC for all microorganisms except S. gordonii and the extract at MIC concentration was non-cytotoxic. The concentrated extracts were slightly cytotoxic. Electrospray ionization with tandem mass spectrometry analyses demonstrated that the extract constituents coincided with the mass of the terpenoids and phenolics. Overall, the best results were obtained for extraction methods A, B and C. The present work proved the antimicrobial activity of several plants. Particularly, extracts from C. doctoris were the most active against bacteria involved in dental caries disease. © 2014 S. Karger AG, Basel
Materials and Methods Selection of Plants The species used in the study were selected from two local books [Pott and Pott, 1994, 2000] considering (1) the availability of the plant on a large scale, (2) its location, ease of access, and the amount of leaf mass available, and (3) the lack of studies on activity against oral bacteria. The ten plants selected were all spermatophytes (they produce seeds) and angiosperms (flowering plants), with the exception of Salvinia auriculata (a seedless, floating Pteridophyta that reproduces via spores) (table 1). As the diversity of the Pantanal is dependent mainly on its hydrologic cycles, the extracts were prepared from plants collected during the flood period (October to March). The sampling was performed at Corumbá, MS, Brazil (latitude 19°0′42.95′′ south; longitude 57°40′22.99′′ west). Only healthy leaves were used. After being harvested, the leaves were dried, stabilized at 38 ° C and powdered in a knife mill [Brighenti et al., 2008]. The plants were identified by a botanist, and a voucher specimen was deposited in the herbarium at the Universidade Estadual de Campinas (UNICAMP), São Paulo, Brazil.
Preparation of Extracts In order to use biological sources with maximum efficiency, it is necessary to optimize the extraction methods used to obtain the active substances. The bioactive compounds that are extracted de-
354
Caries Res 2014;48:353–360 DOI: 10.1159/000357225
Table 1. Families and species selected for the present study
Family
Species
Annonaceae
Unonopsis guatterioides (A. DC.) R. E. Fr.
Arecaceae
Allagoptera leucocalyx (Drude) Kuntze Bactris glaucescens Drude Attalea phalerata Mart. ex Spreng.
Fabaceae
Senna aculeata (Benth.) H.S. Irwin & Barneby
Euphorbiaceae
Croton doctoris S. Moore Jatropha weddelliana Baillon
Combretaceae
Buchenavia tomentosa (Mart.) Eichler
Malpighiaceae
Mascagnia benthamiana (Gries.) W.R. Anderson
Melastomataceae Mouriri elliptica Mart
Table 2. Solvents, temperatures and times used to extract the bioactive substances from leaves
Code Solvent
Time
Temperature 25°C
Processing
A
70° ethanol 72 h
solvent evaporation/ freeze dry
B
water
5 min 100°C
filter sterilized/ freeze dry
C
water
1h
55°C
filter sterilized/ freeze dry
D
water
72 h
25°C
filter sterilized/ freeze dry
E
hexane
72 h
25°C
solvent evaporation
F
90° ethanol 72 h
25°C
solvent evaporation
pend on the polarity of the solvent used. Hence, the extracts were prepared with 20 g leaves/400 ml solvent under the conditions described in table 2. All extracts were filtered using qualitative filter papers to remove the plant powder. Aqueous extracts were filtered again using the Sterifil Aseptic System (Millipore) and the following filters: glass fiber (AP20, Millipore) and mixed cellulose ester filters (pore sizes 0.45 and 0.22 μm). Organic solvents (ethanol and hexane) were evaporated under reduced pressure (60 ° C/120 rpm for hexane and 80 ° C/200 rpm for ethanol). The aqueous and hydroalcoholic extracts were then re-suspended in brain heart infusion (BHI) broth. Extracts obtained from organic solvents (hexane and ethanol) were re-suspended in BHI-DMSO (95:5 v/v) and sonicated just before use. Extraction efficiency was determined by assessing the change in weight of the vials after freeze-drying or evaporating the sol
Brighenti/Salvador/Delbem/Delbem/ Oliveira/Soares/Freitas/Koga-Ito
Downloaded by: NYU Medical Center Library 128.122.253.212 - 10/19/2014 3:39:11 AM
10% of the world’s total known number of species [Alho, 2008]. The wetland known as the Pantanal is remarkable for its cycles of droughts and floods, which define the biodiversity in this area [Junk et al., 2006]. Pott et al. [2011] reported that the number of spermatophyte species that it shelters is nearly 2,000. Due to this great richness, plants with medicinal potential are one of the main resources to be explored in this region. However, the biological activity of these plants has not yet been scientifically studied. Dental caries is a multifactorial disease of microbial origin that still constitutes one of the main challenges in dentistry. The primary bacteria related to dental caries are Streptococcus spp., Lactobacillus spp. and Actinomyces spp. These bacteria are able to adhere to the enamel’s surface and to metabolize carbohydrates from the diet, producing acids as a final metabolite [van Houte, 1980; de Soet et al., 2000; Rupf et al., 2006; Thomas et al., 2008]. The use of natural products for the control of oral microbiota is seen as an alternative therapeutic option and shows promising results [Jeon et al., 2011]. Thus, the aim of this study was to propose a successful bioprospection methodology regarding the antimicrobial potential of plant extracts against bacteria with cariogenic relevance. To achieve this purpose, a systematic screening was carried out using ten plant species from the Brazilian Pantanal, six different extraction methods and seven different microorganisms with cariogenic relevance.
vents. Yield percentage was calculated by considering the original amount of ground leaves used (20 g) to be 100%. The different extraction methods were compared according to Eloff [1998] with modifications. The following parameters were considered: extraction efficiency, ease of removing the solvent, number of active plants (considering the inhibition halo to be >11 mm), chemical hazard, and solubility in water, which is directly related to the ease of incorporating the extract into an oral formulation as most oral formulations are aqueous. To each parameter, a 10-point scale score (1 being the worst outcome and 10 being the best outcome) was attributed to the extraction method. The parameters also received a weight based on a judgment on the importance of the parameter [Eloff, 1998]. For all antimicrobial experiments, BHI broth with and without 5:95 v/v DMSO and 1,200 μg/ml chlorhexidine digluconate were used as negative and reference drug controls, respectively. Bacterial Strains and Growth Conditions The following reference strains were used: Actinomyces naeslundii ATCC 19039, Lactobacillus acidophilus ATCC 4356, Streptococcus gordonii ATCC 10558, Streptococcus mutans ATCC 35688, Streptococcus sanguinis ATCC 10556, Streptococcus sobrinus ATCC 33478 and Streptococcus mitis ATCC 9811. Cultures were kept in BHI broth supplemented with 20% glycerol at –20 ° C. The best growing conditions at 37 ° C for each microorganism were determined in a pilot study as follows: A. naeslundii: oxygenfree atmosphere generated by AnaeroGen (Oxoid), 48 h; L. acidophilus: aerobiosis, 24 h; Streptococcus spp.: 5% CO2, 48 h. Fresh cultures were obtained by streaking the microorganisms in BHI agar and incubating them according to the conditions described above. To obtain working suspensions, the bacterial colonies were re-suspended in 0.9% NaCl solution. The bacterial concentration was set to 106 CFU/ml using a spectrophotometer. All experiments were performed in duplicate.
Primary Screening for the Bioactivity of the Extracts The primary bioactivity of the extracts was determined by using double-layer agar well diffusions [Salvador et al., 2002; Bruschi et al., 2006]. Briefly, 20 ml of BHI agar (50 ° C) and 200 μl of the working suspension were poured onto a previously set layer of 40 ml BHI agar (Petri plates with a diameter of 15 cm). Fifty microliters of the extracts (50,000 μg/ml) were applied to the wells (diameter 5.0 mm). A 2-hour period was allowed to promote the diffusion of the extracts before incubating the plates. After incubation for 24 h at 37 ° C, the inhibition halo (mm) was measured. The data were analyzed with descriptive statistics.
Cytotoxicity Assay of the Most Active Extract Human epithelial cells isolated from oral mucosa (KB cells; ATCC CCL-17) provided by the Adolfo Lutz Institute Culture Collection were kept in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum at 5% CO2 and 37 ° C. Semi-confluent cells (3 × 104 cells) were added to 24-well plates in triplicate for 24 h. The concentrations of the extract used were 1, 2 or 4 times the highest MIC values obtained for the tested microorganisms. After incubation for 24 h, mitochondrial activity was assessed using the tetrazolium salt reduction method (MTT assay) [Sletten and Dahl, 1999]. Untreated cells and latex were used as negative and positive controls, respectively. The percentage of mitochondrial activity was calculated considering the negative control to be 100%. The extracts were classified as severely, moderately or slightly cytotoxic [Sletten and Dahl, 1999]. The data showed normal distributions and were analyzed by ANOVA followed by Tukey’s test using GraphPad Prism 3.2. The significance level was set at 5%.
Chemical Characterization of the Most Active Extract An electrospray ionization with tandem mass spectrometry (ESI-MS/MS) profile was obtained by diluting the extract in 50% (v/v) chromatographic methanol, 50% (v/v) deionized water and 0.5% ammonium hydroxide (Merck, Darmstadt, Germany). The sample was analyzed with direct infusion electrospray ionization in positive and negative modes using a Quadrupole Mass Spectrometer (100 ° C source temperature, 3.0 kV capillary tension, 30 V cone tension). A structural analysis of single ions in the mass spectra from the extract was performed by ESI-MS/MS. The ion with the m/z of interest was selected and submitted to 15–45 eV collisions with argon. The collision gas pressure was optimized to produce extensive fragmentation of the ion under investigation. The constituents were identified by comparing their ESI-MS/MS fragmentation spectra to the fragmentation spectra of authentic standard samples and literature data [Craveiro and Silveira, 1982; Maciel et al., 1998; Zhang et al., 2001; Martinsen et al., 2010; Motta et al., 2011; Pascoal et al., 2011; Silva et al., 2011].
Results
Evaluation of Antibacterial Activity The antibacterial activity of the extracts was determined by their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using the broth microdilution method in 96-well plates [Salvador et al., 2002]. A bacterial inoculum was prepared by adding 0.5 ml of the working suspension to 9.5 ml of BHI broth. Next, 10 μl of the bacterial inoculum was added to the wells containing two-fold serial dilutions (100–50,000 μg/ml) of the extracts. After incubation for 24 h at 37 ° C, a subculture in BHI agar was prepared in order to evaluate bacterial growth. MIC was defined as the lowest concentration that inhibited bacterial growth while MBC was defined as
Extraction efficiency varied from 1.49 to 67.22% of the initial mass used. Aqueous extracts B and C (5 min/100 ° C and 1 h/55 ° C) yielded the highest extraction efficiencies followed by the hydroalcoholic extract. The hexanic extracts yielded the lowest extraction efficiency (fig. 1). Inhibition haloes for the tested microorganisms demonstrated that of the 60 extracts tested, 47 (78%) demonstrated activity against at least one microorganism. The microorganisms that were affected by the fewest number of active extracts were S. mutans and L. acidophilus, while A. naeslundii and S. mitis were the most susceptible micro-
Bioprospection Studies and Antibacterial Extracts
Caries Res 2014;48:353–360 DOI: 10.1159/000357225
355
Downloaded by: NYU Medical Center Library 128.122.253.212 - 10/19/2014 3:39:11 AM
the lowest concentration that killed 99.9% of the bacteria. The data were analyzed with descriptive statistics. The most active extract was selected for chemical characterization and cytotoxicity analyses.
Extraction efficiency (% of dry weight)
70 60 50
70º ethanol 72 h/25ºC Water 5 min/100ºC Water 1 h/55ºC Water 72 h/25ºC Hexane 72 h/25ºC 90º ethanol 72 h/25ºC
40 30 20 10
J. w
0 edd
elli
s x a a a a es ns ca o sa ori an aly ra t eat ian pti oid sce ent . doct ale co c cul am eri au elli h l t m a u h . t p g t e . o l a C t S M S. B. A. B. gu ben U. M.
Plants
Fig. 1. Extraction efficiency (% dry weight) of ten plant species using six different extraction methods.
Table 3. Comparison of the different extraction methods
Extraction efficiency Ease of solvent removal Number of active plants (inhibition haloes >11 mm) Chemical hazard Solubility in water
Weight
A
B
C
D
E
F
3.0 3.0 5.0 2.0 4.0
6.4 21.0 23.6 18.0 32.0
14.1 18.0 13.6 20.0 36.0
14.3 18.0 13.6 20.0 36.0
10.6 18.0 4.3 20.0 36.0
1.1 30.0 5.7 10.0 20.0
2.8 24.0 22.9 16.0 28.0
101
102
102
89
67
94
Total
organisms. Extraction methods A (70° ethanol 72 h/25 ° C) and B (water 5 min/100 ° C) produced antimicrobial activity for all tested plants against at least one microorganism, while extraction method E (hexane 72 h/25 ° C) produced the smallest number of active extracts. When considering the breakpoint for selecting the extracts for MIC/MBC evaluation (inhibition haloes >11 mm), only extraction method A (70° ethanol 72 h/25 ° C) produced antimicrobial activity in all of the tested plants. The only plant that showed activity against all microorganisms was Croton doctoris (extraction methods A, E, and F) (online suppl. material 1; for all online suppl. material, see www. karger. com/doi/10.1159/000357225).
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Caries Res 2014;48:353–360 DOI: 10.1159/000357225
Extracts that produced inhibition haloes >11 mm for at least one microorganism were selected for MIC/MBC tests. Of the 39 extracts tested, only three, all from the C. doctoris plant, were active against all of the microorganisms tested. The hydroalcoholic extract of C. doctoris showed the lowest MIC and MBC for the majority of the microorganisms tested, and it was therefore selected for chemical analysis and cytotoxicity assay. For this extract, MIC values varied from
Caries Res 2014;48:353–360 DOI: 10.1159/000357225
Systematic Screening of Plant Extracts from the Brazilian Pantanal with Antimicrobial Activity against Bacteria with Cariogenic Relevance F.L. Brighenti b M.J. Salvador c Alberto Carlos Botazzo Delbem d Ádina Cleia Bottazzo Delbem d M.A.C. Oliveira a C.P. Soares e L.S.F. Freitas a C.Y. Koga-Ito a, f
a
Department of Biosciences and Oral Diagnosis, Universidade Estadual Paulista (UNESP), São José dos Campos, Department of Orthodontics and Pediatric Dentistry, Araraquara Dental School, Universidade Estadual Paulista (UNESP), Araraquara, c Department of Plant Biology, Institute of Biology, Universidade Estadual de Campinas (UNICAMP), Campinas, d Department of Pediatric Dentistry and Public Health, Araçatuba Dental School, Universidade Estadual Paulista (UNESP), Araçatuba, e Laboratory of Cell and Tissue Biology, Universidade do Vale do Paraíba (UNIVAP), São José dos Campos, and f Department of Environment Engineering, Universidade Estadual Paulista (UNESP), São José dos Campos, Brazil
b
Key Words Biological agents · Dental caries · Prevention and control
Abstract This study proposes a bioprospection methodology regarding the antimicrobial potential of plant extracts against bacteria with cariogenic relevance. Sixty extracts were obtained from ten plants – (1) Jatropha weddelliana, (2) Attalea phalerata, (3) Buchenavia tomentosa, (4) Croton doctoris, (5) Mouriri elliptica, (6) Mascagnia benthamiana, (7) Senna aculeata, (8) Unonopsis guatterioides, (9) Allagoptera leucocalyx and (10) Bactris glaucescens – using different extraction methods – (A) 70° ethanol 72 h/25 ° C, (B) water 5 min/100 ° C, (C) water 1 h/55 ° C, (D) water 72 h/25 ° C, (E) hexane 72 h/25 ° C and (F) 90° ethanol 72 h/25 ° C. The plants were screened for antibacterial activity at 50 mg/ml using the agar well diffusion test against Actinomyces naeslundii ATCC 19039, Lactobacillus acidophilus ATCC 4356, Streptococcus gordonii ATCC 10558, Streptococcus mutans ATCC 35688, Streptococcus sanguinis ATCC 10556, Streptococcus sobrinus ATCC 33478 and Streptococcus mitis ATCC 9811. The active extracts were test
© 2014 S. Karger AG, Basel 0008–6568/14/0485–0353$39.50/0 E-Mail [email protected] www.karger.com/cre
The utmost importance of the scientific value of the biodiversity in Brazil is widely known. This country’s vast and extensive territory consists of a great diversity of biomes. To date, the number of known or recorded Brazilian biota is estimated to be 200,000 species, which represents about Cristiane Yumi Koga-Ito Department of Environmental Engineering (UNESP) Av. Engenheiro Francisco José Longo, 777 São José dos Campos, SP 12245-000 (Brazil) E-Mail cristiane @ fosjc.unesp.br
Downloaded by: NYU Medical Center Library 128.122.253.212 - 10/19/2014 3:39:11 AM
ed to determine their minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), cytotoxicity and chemical characterization. Forty-seven extracts (78%) were active against at least one microorganism. Extract 4A demonstrated the lowest MIC and MBC for all microorganisms except S. gordonii and the extract at MIC concentration was non-cytotoxic. The concentrated extracts were slightly cytotoxic. Electrospray ionization with tandem mass spectrometry analyses demonstrated that the extract constituents coincided with the mass of the terpenoids and phenolics. Overall, the best results were obtained for extraction methods A, B and C. The present work proved the antimicrobial activity of several plants. Particularly, extracts from C. doctoris were the most active against bacteria involved in dental caries disease. © 2014 S. Karger AG, Basel
Materials and Methods Selection of Plants The species used in the study were selected from two local books [Pott and Pott, 1994, 2000] considering (1) the availability of the plant on a large scale, (2) its location, ease of access, and the amount of leaf mass available, and (3) the lack of studies on activity against oral bacteria. The ten plants selected were all spermatophytes (they produce seeds) and angiosperms (flowering plants), with the exception of Salvinia auriculata (a seedless, floating Pteridophyta that reproduces via spores) (table 1). As the diversity of the Pantanal is dependent mainly on its hydrologic cycles, the extracts were prepared from plants collected during the flood period (October to March). The sampling was performed at Corumbá, MS, Brazil (latitude 19°0′42.95′′ south; longitude 57°40′22.99′′ west). Only healthy leaves were used. After being harvested, the leaves were dried, stabilized at 38 ° C and powdered in a knife mill [Brighenti et al., 2008]. The plants were identified by a botanist, and a voucher specimen was deposited in the herbarium at the Universidade Estadual de Campinas (UNICAMP), São Paulo, Brazil.
Preparation of Extracts In order to use biological sources with maximum efficiency, it is necessary to optimize the extraction methods used to obtain the active substances. The bioactive compounds that are extracted de-
354
Caries Res 2014;48:353–360 DOI: 10.1159/000357225
Table 1. Families and species selected for the present study
Family
Species
Annonaceae
Unonopsis guatterioides (A. DC.) R. E. Fr.
Arecaceae
Allagoptera leucocalyx (Drude) Kuntze Bactris glaucescens Drude Attalea phalerata Mart. ex Spreng.
Fabaceae
Senna aculeata (Benth.) H.S. Irwin & Barneby
Euphorbiaceae
Croton doctoris S. Moore Jatropha weddelliana Baillon
Combretaceae
Buchenavia tomentosa (Mart.) Eichler
Malpighiaceae
Mascagnia benthamiana (Gries.) W.R. Anderson
Melastomataceae Mouriri elliptica Mart
Table 2. Solvents, temperatures and times used to extract the bioactive substances from leaves
Code Solvent
Time
Temperature 25°C
Processing
A
70° ethanol 72 h
solvent evaporation/ freeze dry
B
water
5 min 100°C
filter sterilized/ freeze dry
C
water
1h
55°C
filter sterilized/ freeze dry
D
water
72 h
25°C
filter sterilized/ freeze dry
E
hexane
72 h
25°C
solvent evaporation
F
90° ethanol 72 h
25°C
solvent evaporation
pend on the polarity of the solvent used. Hence, the extracts were prepared with 20 g leaves/400 ml solvent under the conditions described in table 2. All extracts were filtered using qualitative filter papers to remove the plant powder. Aqueous extracts were filtered again using the Sterifil Aseptic System (Millipore) and the following filters: glass fiber (AP20, Millipore) and mixed cellulose ester filters (pore sizes 0.45 and 0.22 μm). Organic solvents (ethanol and hexane) were evaporated under reduced pressure (60 ° C/120 rpm for hexane and 80 ° C/200 rpm for ethanol). The aqueous and hydroalcoholic extracts were then re-suspended in brain heart infusion (BHI) broth. Extracts obtained from organic solvents (hexane and ethanol) were re-suspended in BHI-DMSO (95:5 v/v) and sonicated just before use. Extraction efficiency was determined by assessing the change in weight of the vials after freeze-drying or evaporating the sol
Brighenti/Salvador/Delbem/Delbem/ Oliveira/Soares/Freitas/Koga-Ito
Downloaded by: NYU Medical Center Library 128.122.253.212 - 10/19/2014 3:39:11 AM
10% of the world’s total known number of species [Alho, 2008]. The wetland known as the Pantanal is remarkable for its cycles of droughts and floods, which define the biodiversity in this area [Junk et al., 2006]. Pott et al. [2011] reported that the number of spermatophyte species that it shelters is nearly 2,000. Due to this great richness, plants with medicinal potential are one of the main resources to be explored in this region. However, the biological activity of these plants has not yet been scientifically studied. Dental caries is a multifactorial disease of microbial origin that still constitutes one of the main challenges in dentistry. The primary bacteria related to dental caries are Streptococcus spp., Lactobacillus spp. and Actinomyces spp. These bacteria are able to adhere to the enamel’s surface and to metabolize carbohydrates from the diet, producing acids as a final metabolite [van Houte, 1980; de Soet et al., 2000; Rupf et al., 2006; Thomas et al., 2008]. The use of natural products for the control of oral microbiota is seen as an alternative therapeutic option and shows promising results [Jeon et al., 2011]. Thus, the aim of this study was to propose a successful bioprospection methodology regarding the antimicrobial potential of plant extracts against bacteria with cariogenic relevance. To achieve this purpose, a systematic screening was carried out using ten plant species from the Brazilian Pantanal, six different extraction methods and seven different microorganisms with cariogenic relevance.
vents. Yield percentage was calculated by considering the original amount of ground leaves used (20 g) to be 100%. The different extraction methods were compared according to Eloff [1998] with modifications. The following parameters were considered: extraction efficiency, ease of removing the solvent, number of active plants (considering the inhibition halo to be >11 mm), chemical hazard, and solubility in water, which is directly related to the ease of incorporating the extract into an oral formulation as most oral formulations are aqueous. To each parameter, a 10-point scale score (1 being the worst outcome and 10 being the best outcome) was attributed to the extraction method. The parameters also received a weight based on a judgment on the importance of the parameter [Eloff, 1998]. For all antimicrobial experiments, BHI broth with and without 5:95 v/v DMSO and 1,200 μg/ml chlorhexidine digluconate were used as negative and reference drug controls, respectively. Bacterial Strains and Growth Conditions The following reference strains were used: Actinomyces naeslundii ATCC 19039, Lactobacillus acidophilus ATCC 4356, Streptococcus gordonii ATCC 10558, Streptococcus mutans ATCC 35688, Streptococcus sanguinis ATCC 10556, Streptococcus sobrinus ATCC 33478 and Streptococcus mitis ATCC 9811. Cultures were kept in BHI broth supplemented with 20% glycerol at –20 ° C. The best growing conditions at 37 ° C for each microorganism were determined in a pilot study as follows: A. naeslundii: oxygenfree atmosphere generated by AnaeroGen (Oxoid), 48 h; L. acidophilus: aerobiosis, 24 h; Streptococcus spp.: 5% CO2, 48 h. Fresh cultures were obtained by streaking the microorganisms in BHI agar and incubating them according to the conditions described above. To obtain working suspensions, the bacterial colonies were re-suspended in 0.9% NaCl solution. The bacterial concentration was set to 106 CFU/ml using a spectrophotometer. All experiments were performed in duplicate.
Primary Screening for the Bioactivity of the Extracts The primary bioactivity of the extracts was determined by using double-layer agar well diffusions [Salvador et al., 2002; Bruschi et al., 2006]. Briefly, 20 ml of BHI agar (50 ° C) and 200 μl of the working suspension were poured onto a previously set layer of 40 ml BHI agar (Petri plates with a diameter of 15 cm). Fifty microliters of the extracts (50,000 μg/ml) were applied to the wells (diameter 5.0 mm). A 2-hour period was allowed to promote the diffusion of the extracts before incubating the plates. After incubation for 24 h at 37 ° C, the inhibition halo (mm) was measured. The data were analyzed with descriptive statistics.
Cytotoxicity Assay of the Most Active Extract Human epithelial cells isolated from oral mucosa (KB cells; ATCC CCL-17) provided by the Adolfo Lutz Institute Culture Collection were kept in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum at 5% CO2 and 37 ° C. Semi-confluent cells (3 × 104 cells) were added to 24-well plates in triplicate for 24 h. The concentrations of the extract used were 1, 2 or 4 times the highest MIC values obtained for the tested microorganisms. After incubation for 24 h, mitochondrial activity was assessed using the tetrazolium salt reduction method (MTT assay) [Sletten and Dahl, 1999]. Untreated cells and latex were used as negative and positive controls, respectively. The percentage of mitochondrial activity was calculated considering the negative control to be 100%. The extracts were classified as severely, moderately or slightly cytotoxic [Sletten and Dahl, 1999]. The data showed normal distributions and were analyzed by ANOVA followed by Tukey’s test using GraphPad Prism 3.2. The significance level was set at 5%.
Chemical Characterization of the Most Active Extract An electrospray ionization with tandem mass spectrometry (ESI-MS/MS) profile was obtained by diluting the extract in 50% (v/v) chromatographic methanol, 50% (v/v) deionized water and 0.5% ammonium hydroxide (Merck, Darmstadt, Germany). The sample was analyzed with direct infusion electrospray ionization in positive and negative modes using a Quadrupole Mass Spectrometer (100 ° C source temperature, 3.0 kV capillary tension, 30 V cone tension). A structural analysis of single ions in the mass spectra from the extract was performed by ESI-MS/MS. The ion with the m/z of interest was selected and submitted to 15–45 eV collisions with argon. The collision gas pressure was optimized to produce extensive fragmentation of the ion under investigation. The constituents were identified by comparing their ESI-MS/MS fragmentation spectra to the fragmentation spectra of authentic standard samples and literature data [Craveiro and Silveira, 1982; Maciel et al., 1998; Zhang et al., 2001; Martinsen et al., 2010; Motta et al., 2011; Pascoal et al., 2011; Silva et al., 2011].
Results
Evaluation of Antibacterial Activity The antibacterial activity of the extracts was determined by their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using the broth microdilution method in 96-well plates [Salvador et al., 2002]. A bacterial inoculum was prepared by adding 0.5 ml of the working suspension to 9.5 ml of BHI broth. Next, 10 μl of the bacterial inoculum was added to the wells containing two-fold serial dilutions (100–50,000 μg/ml) of the extracts. After incubation for 24 h at 37 ° C, a subculture in BHI agar was prepared in order to evaluate bacterial growth. MIC was defined as the lowest concentration that inhibited bacterial growth while MBC was defined as
Extraction efficiency varied from 1.49 to 67.22% of the initial mass used. Aqueous extracts B and C (5 min/100 ° C and 1 h/55 ° C) yielded the highest extraction efficiencies followed by the hydroalcoholic extract. The hexanic extracts yielded the lowest extraction efficiency (fig. 1). Inhibition haloes for the tested microorganisms demonstrated that of the 60 extracts tested, 47 (78%) demonstrated activity against at least one microorganism. The microorganisms that were affected by the fewest number of active extracts were S. mutans and L. acidophilus, while A. naeslundii and S. mitis were the most susceptible micro-
Bioprospection Studies and Antibacterial Extracts
Caries Res 2014;48:353–360 DOI: 10.1159/000357225
355
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the lowest concentration that killed 99.9% of the bacteria. The data were analyzed with descriptive statistics. The most active extract was selected for chemical characterization and cytotoxicity analyses.
Extraction efficiency (% of dry weight)
70 60 50
70º ethanol 72 h/25ºC Water 5 min/100ºC Water 1 h/55ºC Water 72 h/25ºC Hexane 72 h/25ºC 90º ethanol 72 h/25ºC
40 30 20 10
J. w
0 edd
elli
s x a a a a es ns ca o sa ori an aly ra t eat ian pti oid sce ent . doct ale co c cul am eri au elli h l t m a u h . t p g t e . o l a C t S M S. B. A. B. gu ben U. M.
Plants
Fig. 1. Extraction efficiency (% dry weight) of ten plant species using six different extraction methods.
Table 3. Comparison of the different extraction methods
Extraction efficiency Ease of solvent removal Number of active plants (inhibition haloes >11 mm) Chemical hazard Solubility in water
Weight
A
B
C
D
E
F
3.0 3.0 5.0 2.0 4.0
6.4 21.0 23.6 18.0 32.0
14.1 18.0 13.6 20.0 36.0
14.3 18.0 13.6 20.0 36.0
10.6 18.0 4.3 20.0 36.0
1.1 30.0 5.7 10.0 20.0
2.8 24.0 22.9 16.0 28.0
101
102
102
89
67
94
Total
organisms. Extraction methods A (70° ethanol 72 h/25 ° C) and B (water 5 min/100 ° C) produced antimicrobial activity for all tested plants against at least one microorganism, while extraction method E (hexane 72 h/25 ° C) produced the smallest number of active extracts. When considering the breakpoint for selecting the extracts for MIC/MBC evaluation (inhibition haloes >11 mm), only extraction method A (70° ethanol 72 h/25 ° C) produced antimicrobial activity in all of the tested plants. The only plant that showed activity against all microorganisms was Croton doctoris (extraction methods A, E, and F) (online suppl. material 1; for all online suppl. material, see www. karger. com/doi/10.1159/000357225).
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Caries Res 2014;48:353–360 DOI: 10.1159/000357225
Extracts that produced inhibition haloes >11 mm for at least one microorganism were selected for MIC/MBC tests. Of the 39 extracts tested, only three, all from the C. doctoris plant, were active against all of the microorganisms tested. The hydroalcoholic extract of C. doctoris showed the lowest MIC and MBC for the majority of the microorganisms tested, and it was therefore selected for chemical analysis and cytotoxicity assay. For this extract, MIC values varied from
