Abstract
Background:
Oral infections and dental caries are still considered as serious public health problem and inflict a costly burden to health care services around the world especially in developing countries.Objectives:
In the present study, we evaluated the antibacterial activity of Capsella bursa-pastoris alone and also combined with Glycyrrhiza glabra against Streptococcus mutans, S. sanguis, Actinomyces viscosus, Enterococcus faecalis as oral pathogens.Materials and Methods:
The antimicrobial activities of an ethanol extract of C. bursa-pastoris alone and in combination with G. glabra were in vitro tested against six reference strains of oral pathogenic bacteria. The antimicrobial activities of the extracts were examined using disc diffusion method and the minimum inhibitory concentration (MIC) determined by both broth and Agar dilution methods and minimum bactericidal concentration (MBC) by broth dilution methods.Results:
In this study, C. bursa-pastoris extract showed good antibacterial activity against six bacteria in using in of the mentioned methods. No strain in this study showed resistance against this extract. Antibacterial activity of mixed extract including C .bursa-pastoris and G. glabra was evaluated and showed that mixed extract was more effective against all bacteria than any of the cases alone that indicate the synergistic effect between these two extracts.Conclusions:
C. bursa-pastoris and its mixture with G. glabra are suggested as appropriate candidates to control dental caries and endodontic infections.Keywords
Antibacterial Activity Capsella bursa-pastoris Glycyrrhiza glabra Oral Pathogen
2. Objectives
In the present study, we evaluated the antibacterial activity of C. bursa-pastoris and its mixture with G. glabra against oral pathogens.
3. Materials and Methods
3.1. Plant Material
3.1.1. Source, Collection and Identification
Total parts of C. bursa-pastoris and roots of G. glabra were collected from Garineh, a village near Neyshabour, Iran, in summer 2011. A voucher specimen was prepared and deposited at Research Institute of Plant Sciences Herbarium, Ferdowsi University of Mashhad, Iran.
3.1.2. Preparation of Extract
Different parts of C. bursa-pastoris (250 g) and roots of G. glabra (250 g) were dried at 25˚C and then powdered using a mechanical grinder separately. Each extraction was prepared using ethanol (80%, v/v) (Merck, Germany) for a period of 72 hours without using any heating procedure. The final volume of the filtrated mass was removed using a rotary vacuum evaporator (Heidolphlaborota 4000, Germany) at 40˚C to produce the concentrated extract, which was frozen and freeze-dried until the next use (2, 10). For preparation of mixed extract, equal amounts(2 mL) of the each extract (100 mg/mL) was thoroughly mixed in a sterile tube. So the concentration of each extract was 50 mg/mL in the mixed extract.
3.2. Antibacterial Activity
3.2.1. Microbial Strains
The microorganisms used in this study included Streptococcus mutans (PTCC 1683), S. sanguis (PTCC 1449), Actinomyces viscosus (PTCC 1202), Enterococcus faecalis (ATCC 29212) as oral pathogens and Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 29922) were used as controls. The bacterial strains were cultured in brain heart infusion (BHI) (Difco, MI, USA) under anaerobic condition in an anaerobic jar with Anaerocult A (Merk SA (Pty) Ltd), at 37˚C for 72 hours and subculturing was performed twice a week. The organisms suspensions were prepared by picking colonies from appropriately incubated agar cultures to sterile broth, to match a McFarland 0.5 turbidity standard (approximately 1.5 x 108 CFU/mL) (11).
4. Results
In vitro antibacterial activity of C. bursa-pastoris extract and the mixture of the C. bursa-pastoris and G. glabra extracts and also their potency were quantitatively and qualitatively assessed by determining the inhibition zone diameter and MIC as given in Tables 1-6. The analysis of C. bursa-pastoris extract showed positive inhibitory activity against six bacteria, in all methods. No strain in this study showed resistance to this extract. Results of antibacterial activity of these plants by agar diffusion method against six bacteria are shown in Tables 1 - 4.The inhibitory zone significantly increased in a dose dependent manner.
4.1. C. bursa-pastoris Extract
In agar dilution method MIC for S. aureus , A. viscosus , E. faecalis and S. sanguis were 15 mg/mL and for E. coli was 35 mg/mL. MIC for S. mutans was 12.5 mg/mL. E. coli demonstrated the greatest resistance to C. bursa-pastoris and appeared to be the most resistant bacterium (Table 5). The results of broth dilution method which are shown in Table 7 are consistent with the findings of the agar dilution method (Table 5).
4.2. Mixed Extract
In agar dilution method MIC for S. aureus , A. viscosus , E. faecalis and S. sanguis were 12.5 mg/mL and this amount for E. coli was 20 mg/mL. MIC for S. mutans was 10 mg/mL. E. coli demonstrated the greatest resistance to mixed extract and appeared to be the most resistant bacterium against C. bursa-pastoris extract (Table 5). In broth dilution method MIC for all of the bacteria were 12.5 mg/mL except E. coli that was 25 mg/mL (Table 6)
4.3. Chlorhexidine
For these microorganisms, MIC of chlorhexidine mouthwash in agar and broth dilution method was 0.0625 mg/mL except for E. coli that was 0.125 mg/mL (Tables 8, 9 and 10).
Antimicrobial Activity of C. bursa-pastoris Against Oral Microorganisms and Controls Inhibition Zones in Millimeter by Disk Diffusion Method
| Plant Extract a | Concentration, mg/mL | S. mutans | S. sanguis | A. viscosus | E. faecalis | S. aureus | E. coli |
|---|---|---|---|---|---|---|---|
| C. bursa-pastoris | 100 | 25.4 ± 0.8 | 21.6 ± 0.5 | 22.8 ± 0.4 | 17.8 ± 0.3 | 26.7 ± 0.3 | 23.6 ± 0.5 |
| 50 | 23 ± 0.0 | 20.3 ± 0.5 | 18.4 ± 0.4 | 16.3 ± 0.5 | 23.4 ± 0.4 | 20.8 ± 0.7 | |
| 25 | 20 ± 0.0 | 17.6 ± 0.3 | 14 ± 0.5 | 14.3 ± 0.5 | 21.6 ± 0.5 | 18 ± 0.0 | |
| 12.5 | 18.1 ± 0.2 | 15 ± 0.0 | 9.4 ± 0.0 | 11 ± 0.0 | 19.3 ± 0.5 | 14.7 ± 0.3 | |
| 6.25 | 16 ± 0.3 | 12.1 ± 0.2 | 8.2 ± 0.5 | 9 ± 0.0 | 15.3 ± 0.5 | 12.7 ± 0.3 | |
| 3.125 | 10.7 ± 1 | 8.2 ± 0.5 | - b | 8.2 ± 0.2 | 9.6 ± 0.3 | 11 ± 0.0 | |
| Negative Control | -b | -b | -b | -b | -b | -b |
Antimicrobial Activity of Mixed Extract Against Oral Microorganisms and Controls Inhibition Zones in Millimeter by Disk Diffusion Method.
| Plant Extracta | Concentration, mg/mL | S. mutans | S. sanguis | A. viscosus | E. faecalis | S. aureus | E. coli |
|---|---|---|---|---|---|---|---|
| Mixed extract | 100 | 29.2 ± 0.5 | 26.6 ± 0.5 | 26 ± 0.0 | 21.2 ± 0.5 | 29.7 ± 0.3 | 28.7 ± 0.5 |
| 50 | 27.4 ± 0.4 | 24.1 ± 0.2 | 23.2 ± 0.5 | 19.3 ± 0.5 | 28 ± 1 | 23 ± 0.0 | |
| 25 | 25 ± 0.0 | 21.8 ± 0.3 | 18 ± 1 | 17 ± 0.0 | 25.6 ± 0.5 | 21 ± 0.0 | |
| 12.5 | 22.6 ± 0.5 | 20.4 ± 0.4 | 14 ± 0.5 | 14.2 ± 0.5 | 22.2 ± 0.5 | 18 ± 1 | |
| 6.25 | 20.8 ± 0.3 | 16.8 ± 0.7 | 10.2 ± 0.5 | 13.1 ± 0.2 | 17.8 ± 0.3 | 15.2 ± 0.5 | |
| 3.125 | 14 ± 0.0 | 12 ± 0.0 | 8 ± 0.0 | 10 ± 0.0 | 13.4 ± 0.8 | 14 ± 0.0 | |
| Negative Control | -b | -b | -b | -b | -b | -b |
Antimicrobial Activity of C. bursa-pastoris Against Oral Microorganisms and Controls Inhibition Zones in Millimeter by Well Diffusion Method.
| Plant extracta | Concentration, mg/mL | S. mutans | S. sanguis | A. viscosus | E. faecalis | S. aureus | E. coli |
|---|---|---|---|---|---|---|---|
| C. bursa-pastoris | 100 | 26.3 ± 0.5 | 20.8 ± 0.3 | 23.9 ± 0.5 | 22.1 ± 0.2 | 27 ± 1 | 25 ± 1 |
| 50 | 23.3 ± 0.5 | 19.2 ± 0.2 | 18.8 ± 0.3 | 17 ± 0.0 | 22.3 ± 0.5 | 20.3 ± 0.4 | |
| 25 | 19.4 ± 0.5 | 17.8 ± 0.3 | 14 ± 0.0 | 14 ± 0.0 | 17.6 ± 0.5 | 18 ± 0.0 | |
| 12.5 | 16.3 ± 0.5 | 13.6 ± 0.5 | 11 ± 0.0 | 11 ± 0.3 | 15 ± 0.0 | 14.2 ± 0.2 | |
| 6.25 | 14 ± 0.0 | 9.8 ± 0.3 | 8.4 ± 0.0 | 9.3 ± 0.5 | 13 ± 0.0 | 11 ± 0.0 | |
| 3.125 | 10 ± 0.0 | 7.4 ± 0.5 | -b | 7.4 ± 0.5 | 10 ± 0.0 | 9.4 ± 0.3 | |
| Negative Control | -b | -b | -b | -b | -b | -b |
Antimicrobial Activity of Mixed Extract Against Oral Microorganisms and Controls Inhibition Zones in Millimeter using Well Diffusion Method
| Plant Extracta | Concentration, mg/mL | S. mutans | S. sanguis | A. viscosus | E. faecalis | S. aureus | E. coli |
|---|---|---|---|---|---|---|---|
| Mixed extract | 100 | 30.8 ± 0.3 | 27.1 ± 0.2 | 26.8 ± 0.3 | 22 ± 0.0 | 30 ± 1 | 29.4 ± 0.5 |
| 50 | 27 ± 0.0 | 25 ± 1 | 23 ± 0.0 | 20.1 ± 0.2 | 26.4 ± 0.0 | 24 ± 0.0 | |
| 25 | 25.4 ± 0.5 | 23.7 ± 0.3 | 18.1 ± 0.2 | 17.2 ± 0.2 | 21.8 ± 0.3 | 21.8 ± 0.3 | |
| 12.5 | 20 ± 0.0 | 19.2 ± 0.2 | 14.4 ± 0.5 | 15 ± 1 | 17.8 ±0.3 | 17.3 ± 0.3 | |
| 6.25 | 16.4 ± 0.5 | 14 ± 0.0 | 12.8 ± 0.3 | 13. 3± 0.5 | 15 ± 0.0 | 13 ± 1 | |
| 3.125 | 13 ± 0.0 | 11 ± 0.0 | 12 ± 1 | 11.2 ± 0.2 | 11.2 ± 0.2 | 11.6 ± 0.5 | |
| Negative Control | -b | -b | -b | -b | - b | -b |
Mean MIC (mg/mL) Results of C. bursa-pastoris Extract, C. bursa-pastoris Extract and Mixed Extract on Oral Microorganisms and Controls using Agar Dilution Method
| S. mutans | S. sanguis | A. viscosus | E. faecalis | S. aureus | E. coli | |
|---|---|---|---|---|---|---|
| C.bursa-pastoris Extract | ||||||
| MIC | 12.5 | 15 | 15 | 15 | 15 | 35 |
| C. bursa-pastoris Extract | ||||||
| MIC | 12.5 | 25 | 25 | 25 | 25 | 50 |
| MBC | 12.5 | 25 | 25 | 25 | 25 | 50 |
| Mixed Extract | ||||||
| MIC | 10 | 12.5 | 12.5 | 12.5 | 12.5 | 20 |
Mean MIC and MBC (mg/mL) Results of Mixed Extract on Oral Microorganisms and Controls using Broth Dilution Method
| Plant Extract | Concentration, mg/mL | S. mutans | S. sanguis | A. viscosus | E. faecalis | S. aureus | E. coli |
|---|---|---|---|---|---|---|---|
| Chlorhexidine | 2 | 26.2 ± 0.2 | 17.2 ± 0.2 | 23 ± 0.0 | 25.2 ± 0.2 | 26 ± 1 | 24 ± 0.2 |
| 1 | 22.2 ± 0.2 | 16 ± 0.0 | 17.7 ± 0.4 | 22.2 ± 0.1 | 23 ± 0.5 | 21.2 ± 0.0 | |
| 0.5 | 18 ± 0.0 | 15.2 ± 0.2 | 13.7 ± 1 | 17.4 ± 0.7 | 19 ± 0.0 | 18.4 ± 0.99 | |
| 0.25 | 14 ± 0.0 | 11.7 ± 0.99 | 11.7 ± 0.4 | 11 ± 0.0 | 15.4 ± 0.7 | 15 ± 0.0 | |
| 0.125 | 10 ± 0.0 | 10 ± 0.0 | 9.5 ± 0.7 | 8.2 ± 0.2 | 11.2 ± 0.2 | 12 ± 0.0 | |
| 0.625 | 8.5 ± 0.5 | - | 7.2 ± 0.2 | 6 ± 0.0 | 8 ± 1 | - | |
| Negative Control | - | - | - | - | - | - |
Antimicrobial Activity of the Chlorhexidine Against Oral Microorganisms and Controls Inhibition Zones in Millimeter using Disk Diffusion Method.
| Plant Extract | Concentration, mg/mL | S. mutans | S. sanguis | A. viscosus | E. faecalis | S. aureus | E. coli |
|---|---|---|---|---|---|---|---|
| Chlorhexidine | 2 | 26.2 ± 0.2 | 17.2 ± 0.2 | 23 ± 0.0 | 25.2 ± 0.2 | 26 ± 1 | 24 ± 0.2 |
| 1 | 22.2 ± 0.2 | 16 ± 0.0 | 17.7 ± 0.4 | 22.2 ± 0.1 | 23 ± 0.5 | 21.2 ± 0.0 | |
| 0.5 | 18 ± 0.0 | 15.2 ± 0.2 | 13.7 ± 1 | 17.4 ± 0.7 | 19 ± 0.0 | 18.4 ± 0.99 | |
| 0.25 | 14 ± 0.0 | 11.7 ± 0.99 | 11.7 ± 0.4 | 11 ± 0.0 | 15.4 ± 0.7 | 15 ± 0.0 | |
| 0.125 | 10 ± 0.0 | 10 ± 0.0 | 9.5 ± 0.7 | 8.2 ± 0.2 | 11.2 ± 0.2 | 12 ± 0.0 | |
| 0.625 | 8.5 ± 0.5 | - | 7.2 ± 0.2 | 6 ± 0.0 | 8 ± 1 | - | |
| Negative Control | - | - | - | - | - | - |
Antimicrobial Activity of the Chlorhexidine Against Oral Microorganisms and Controls Inhibition Zones in Millimeter using Well Diffusion Method
| Plant Extract | Concentration, mg/mL | S. mutans, Mean ± SD | S. sanguis, Mean ± SD | A. viscosus, Mean ± SD | E. faecalis, Mean ± SD | S. aureus, Mean ± SD | E. coli, Mean ± SD |
|---|---|---|---|---|---|---|---|
| Chlorhexidine | 2 | 28.2 ± 0.2 | 20 ± 0.0 | 22.2 ± 0.2 | 26 ± 0.0 | 26.8 ± 0.2 | 25 ± 0.2 |
| 1 | 23.9 ± 0.7 | 16.7 ± 0.4 | 18.2 ± 0.2 | 23.2 ± 0.1 | 24.6 ± 0.5 | 23 ± 0.0 | |
| 0.5 | 20.2 ± 0.2 | 14.2 ± 0.2 | 14.7 ± 0.4 | 18 ± 0.0 | 21 ± 0.0 | 22.2 ± 0.2 | |
| 0.25 | 14.4 ± 0.0 | 11.2 ± 0.2 | 12.5 ± 0.1 | 13 ± 0.2 | 16.7 ± 0.4 | 16.8 ± 0.0 | |
| 0.125 | 12 ± 0.0 | 9.7 ± 0.4 | 10.2 ± 0.2 | 10.8 ± 0.0 | 13.2 ± 0.2 | 12.8 ± 0.0 | |
| 0.625 | 9.4 ± 0.5 | - | 8 ± 0.0 | - | - | - | |
| Negative control | - | - | - | - | - | - |
Mean MIC (mg/mL) Results of Chlorhexidine extract on Oral Microorganisms and Controls using Agar Dilution Method
| S. mutans | S. sanguis | A. viscosus | E. faecalis | S. aureus | E. coli | |
|---|---|---|---|---|---|---|
| MIC | 0.0625 | 0.0625 | 0.0625 | 0.0625 | 0.0625 | 0.125 |
Mean MIC and MBC (mg/mL) Results of Chlorhexidine Extract on Oral Microorganisms and Controls using Broth Dilution Method
| S. mutans | S. sanguis | A. viscosus | E. faecalis | S. aureus | E. coli | |
|---|---|---|---|---|---|---|
| MIC, mg/mL | 0.0625 | 0.0625 | 0.0625 | 0.0625 | 0.0625 | 0.125 |
| MBC, mg/mL | 0.125 | 0.125 | 0.0625 | 0.125 | 0.125 | 0.125 |
Acknowledgements
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