j o u r n a l o f o r a l b i o l o g y a n d c r a n i o f a c i a l r e s e a r c h 5 ( 2 0 1 5 ) 7 e1 0
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/jobcr
Original Article
Antibacterial activity of clove, gall nut methanolic and ethanolic extracts on Streptococcus mutans PTCC 1683 and Streptococcus salivarius PTCC 1448 Mirsasan Mirpour a,*, Zohreh Gholizadeh Siahmazgi a, Masoumeh Sharifi Kiasaraie b a b
Microbiology Department, Islamic Azad University Lahijan Branch, Iran Chemistry Department, Islamic Azad University Rasht Branch, Iran
article info
abstract
Article history:
Introduction: Antimicrobial compounds from herbal sources have good therapeutic poten-
Received 23 November 2014
tial. In this study, the antibacterial effects of clove and gall nut, methanolic and ethanolic
Accepted 7 February 2015
extractions, were evaluated for their effect on Streptococcus mutans PTCC 1683 and Strep-
Available online 25 February 2015
tococcus salivarius PTCC 1448, as both the two cause oral diseases. Method: The clove and gall nut methanolic and ethanolic extracts were prepared and
Keywords:
antibacterial activity was evaluated for S. mutans and S. salivarius in the base of inhibition
Clove
zone diameter using agar diffusion method. In this part minimum inhibitory concentration
Gall nut extracts
(MIC) and minimal bactericidal concentration (MBC) were assessed.
Antibacterial effects
Results: These extracts showed effective antibacterial activity on bacteria. Antibacterial activity of Methanolic extract of clove was more than that of ethanolic extract, and ethanolic extracts of gall nut had antibacterial activity more than that of methanolic extracts. MIC and MBC results for clove methanolic extract were 1.5 mg/ml and 3 mg/ml for S. mutans and 6.25 mg/ml and 12.5 mg/ml for S. salivarius, respectively. These results for clove ethanolic extracts were 12.5 mg/ml and 25 mg/ml for S. mutans and 25 mg/ml and 50 mg/ml for S. salivarius, respectively. MIC and MBC results for gall nut methanolic extract were 25 mg/ml and 50 mg/ml for S. mutans and 12.5 mg/ml and 25 mg/ml for S. salivarius, respectively. These results for gall nut ethanolic extracts were 3.1 mg/ml and 6.2 mg/ml for S. mutans and 25 mg/ml and 50 mg/ml for S. salivarius, respectively. Discussion: The results showed effective antibacterial activity using clove and gall nut methanolic extracts. If other properties such as tolerance of tissue can also be studied, these extracts can be used as a mouthwash. Copyright © 2015, Craniofacial Research Foundation. All rights reserved.
* Corresponding author. Tel.: þ98 9121964387 (mobile). E-mail address: [email protected] (M. Mirpour). http://dx.doi.org/10.1016/j.jobcr.2015.02.002 2212-4268/Copyright © 2015, Craniofacial Research Foundation. All rights reserved.
8
1.
j o u r n a l o f o r a l b i o l o g y a n d c r a n i o f a c i a l r e s e a r c h 5 ( 2 0 1 5 ) 7 e1 0
Introduction
Identification and use of medicinal plants is as old as human life. Before the 19th century the use of natural resources, mainly plants were the main source of medicines.1 Rapid development of chemistry and the decline of natural resources caused the new synthetic chemicals to be used instead of the herbal medicines. In spite that even today, many scientists in different fields are using herbal medicines.2 Due to the high capacity of medicinal plants and its widespread use in traditional medicine, it seems that finding of the effective plant's extract against pathogenic agents is feasible. Due to increasing oral infections and no existence of medications without side effects, in this study we have attempted to identify plants effective against oral pathogens. Antimicrobial materials in dental therapy, in either oral or topical form, are frequently used to reduce the oral microbial flora before gingival surgery, during healing and after surgery.3 Certain elderly patients or those prone to microbial endocarditis, immunodeficiency, diabetic and blood diseases, are committed to the use of antimicrobial substances. With extending scopes for mouthwash, it seems necessary to assess materials with minimal side effects and desired therapeutic effects. The aim of this study is to find new medicinal plant extracts to replace chemicals that have many side effects.4 In this study, the effect of ethanolic and methanolic extracts of cloves and gall nut on to standard strains of bacteria Streptococcus mutans PTCC 1683 and Streptococcus salivarius PTCC 1448 were evaluated as they cause oral infections.
2.
Materials and methods
2.1.
Microorganisms
Two bacterial strains S. mutans PTCC 1683 and S. salivarius PTCC 1448 were purchased from Iranian Research Organization for Science and Technology (IROST). These bacteria were cultured on selective culture media in sterile condition.
2.2.
Chemistry
For this part, macerations method was used. 10 g of the powdered plant material were soaked in 100 ml solvent (Ethanol or Methanol) and shaken for 24 h. The extracts were then filtered using Whatman No.1 filter paper along with 2 g sodium sulfate to remove the trace of water in the filtrate. The filtrate was concentrated by vacuum distillation. The extracts were exposed under UV ray for sterilization.
2.2.1.
Gas chromatography/mass spectroscopy (GC/MS)
The constituent of the extracts were identified by gas chromatography (model Hewlett-Packard-GC6890) which was combined with mass spectrometry (Mass 5973N). The column was HP-5MS (30 nm 0.25 nm internal diameters, 0.25 mm film thickness) and Helium (2 ml/min) was used as a carrier gas. Samples were injected while the injection temperature was 250 C. The temperature of capillary column was 60 C for 3 min and then programmed to 250 C at 8 C/min and held for
10 min. The identification of compounds was performed by comparing their retention indices and mass spectra with Wiley 275 library installed on the instrument and NIST Mass Spectral.
2.3.
Antimicrobial activity
The effect of ethanol and methanol extracts of two plants, gall nut and cloves were performed using the disc diffusion method, according to the KirbyeBauer. The bacterial suspension was prepared using 0.5 McFarland standard's tube. It was cultured on Muller Hinton agar plates (Merck's) and used with discs impregnated with the compound. The plates were incubated for 24 h at 37 C. After the incubation period, inhibition zone was measured in centimeter. This study was performed 3 times for each extracts and bacterium. A blank disc, ethanol disc, methanol disc and Chlorhexidine disc was used as a control.
2.3.1.
Minimum inhibitory concentration (MIC)
A pure culture of a single microorganism was grown in Mueller-Hinton broth. The culture was standardized using standard microbiological 0.5 McFarland standard's tube. The better the microbial culture, more were the test results reproducible. The antimicrobial agent was diluted a number of times, usually 1:1, through a sterile diluent (Mueller-Hinton broth). After the antimicrobial agent was diluted, a volume of the standardized inoculum, equal to the volume of the diluted antimicrobial agent, was added to each dilution vessel, bringing the microbial concentration to approximately 75,000,000 cells/ml. The inoculated, serially diluted antimicrobial agent was incubated at an appropriate temperature for the test organism for a pre-set period, usually 18 h. After incubation, the series of dilution vessels was observed for microbial growth, usually indicated by turbidity and/or a pellet of microorganisms in the bottom of the vessel. The last tube in the dilution series that did not demonstrate growth corresponded with the MIC of the antimicrobial agent.
2.3.2.
Minimum bactericidal concentration (MBC)
A pure culture of a specified microorganism was diluted in growth-supporting broth (Muller Hinton Broth) to a concentration of 0.5 McFarland standard's tube. A stock dilution of the antimicrobial test substance was made at approximately 100 the level of the expected MIC. Further 1:1 dilutions were made in test tubes or 96 well microtiter plates. The dilutions, going from least concentrated to most concentrated, were inoculated with equal volumes of the specified microorganism. A positive and negative control tube or well was included for every test microorganism to demonstrate adequate microbial growth over the course of the incubation period and media sterility, respectively. An aliquot of the positive control was plated and used to establish a baseline concentration of the microorganism used. The tubes or microtiter plates were then incubated at the appropriate temperature and duration. Turbidity indicated growth of the microorganism. MIC was lowest where no growth was visually observed. To determine the MBC, the dilution representing the MIC and at least two of the more concentrated test product dilutions were plated and enumerated to determine viable CFU/ml. The MBC is the lowest concentration that
9
j o u r n a l o f o r a l b i o l o g y a n d c r a n i o f a c i a l r e s e a r c h 5 ( 2 0 1 5 ) 7 e1 0
Table 1 e Antibacterial activity of clove and gall nut. Microorganism
Plant Clove
Gall nut
Standard
Solvent
S. mutans S. salivarius
Methanol
Ethanol
Methanol
Ethanol
Chlorhexidine
IZ 24 ± 0.4 AI ¼ 1.6 IZ 17 ± 0.99 AI ¼ 1.4
IZ 20 ± 0.29 AI ¼ 1.3 IZ 12 ± 0.37 AI ¼ 1
IZ 24 ± 0.72 AI ¼ 1.6 IZ 14 ± 0.57 AI ¼ 1.1
IZ 10 ± 049 AI ¼ 0.6 IZ 7 ± 0.14 AI ¼ 0.58
IZ: 15 IZ: 12
IZ: Inhibition Zone (in mm). AI: Activity Index ¼ IZ of test sample/IZ of standard. Values are mean of triplicate reading (mean ± S.D.).
demonstrated a pre-determined reduction (such as 99.9%) in CFU/ml when compared to the MIC dilution.
3.
Results
3.1.
Antibacterial activity of clove and gall nut
The antibacterial activity of clove and gall nut extracts were tested using different bacteria and the results are listed in Table 1. It was found that the methanolic extract of clove and gull nut were potentially active against S. mutans PTCC 1683 and S. salivarius PTCC 1448 with zones of inhibition ranging from 14 to 24 mm. These range for ethanolic extracts were from 7 to 20 mm whereas, the inhibition zone for Chlorhexidine as control was 15 mm. These showed that the extracts were very effective on bacteria.
3.2.
MICs and MBCs of clove and gull nut extracts
The MIC and MBC values of the ethanolic and methanolic extracts of clove and gull nut for S. salivarius PTCC 1448and S. mutans PTCC 1683 are showed in Table 2. The best MIC and MBC against S. mutans PTCC 1683 were 1.5 and 3 for methanolic extract of clove respectively, and against S. salivarius PTCC 1448 were 6.25 and 12.5 for methanolic extract of clove, respectively.
3.3.
Clove and gall nut components
Chemical composition of these plants were specified using GC/MS (Table 3) that to be comparable to other studies.5 The
result showed that both methanolic and ethanolic clove extract mainly contain Eugenol, Eugenyl Acetate and b-Caryophyllene. As shown in Table 3, some substances are same in the base of results, the best antibacterial activities were achieved using clove methanolic extract and gall nut ethanolic extract. Hence, common components of these two extracts are important.
4.
Discussion
In recent years many studies have been conducted to evaluate the antimicrobial effects of essential oils and extracts, indicating the ability of these compounds to inhibit the growth of pathogenic and spoilage microorganisms in food.6 Since, these compounds are natural and in many cases containing other existing health compounds, it has been emphasized for protecting of human health.7 Overuse of antibiotics has caused increasing resistance to drugs. Also, indiscriminate use of antibiotics is often associated with adverse effects on the human body. Since some plants with antimicrobial effects have been recorded in pharmacopoeia of the drug in Iran, it can be used to deal with some specific pathogenic microbes and safe to replace with some antibiotics.8 In a study conducted on two plant extracts used in traditional medicine in Iran, they showed antibacterial activity against Streptococcus salivarius PTCC 1448 and Streptococcus mutans PTCC 1683 Extracts. Reduced colony growth was observed compared with the control drug (p < 0.05). Shoji et al showed that the aqueous and methanol extracts of this plant contain saponins and flavonoids. However, the need to further
Table 2 e MICs and MBCs of clove and gull nut extracts. Microorganism
Plant Clove
Gall nut Solvent
S. mutans S. salivarius
Methanol (mg/ml)
Ethanol (mg/ml)
Methanol (mg/ml)
Ethanol (mg/ml)
MIC:1.5 MBC: 3 MIC: 6.25 MBC: 12.5
MIC: 12.5 MBC: 25 MIC: 25 MBC: 50
MIC: 25 MBC: 50 MIC: 12.5 MBC: 25
MIC:3.1 MBC: 6.2 MIC: 25 MBC: 50
10
j o u r n a l o f o r a l b i o l o g y a n d c r a n i o f a c i a l r e s e a r c h 5 ( 2 0 1 5 ) 7 e1 0
Table 3 e Chemical composition of methanolic and ethanolic extracts of clove and gall nut. Components
Extracts Clove
Gall nut
Methanolic extract Ethanolic extract Methanolic extract Ethanolic extract 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
2-Furancarboxaldehyde Chavicol Eugenol 1,2,3-Trihydroxybenzene (Pyrogallol) b-Caryophyllene a-Humulene Eugenyl Acetate Caryophyllene Oxide Palmitic acid Linoleic acid Oleic acid Stearic acid n-Hexyl butyrate 5-(hydroxymethyl)-2-urancarboxaldehyde Psoralene Oroselone Lidocaine 2,3-dihydro-3,5-dihydroxy-6-methylpyran-4-one Cinnamaldehyde Thymol Carvacrol 4-vinyl-2-methoxy-phenol Quinic acid
✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
studies to identify the active agents responsible in other biological and pharmacological activities of this plant is necessary. Also, other antimicrobial activity of plant extracts, essential oil and other areas of native species from view of the chemical composition of contents and their clinical efficacy must be investigated.9 Plants contain phytochemicals that have pronounced antimicrobial activity. This underlies the antiquity of herbs to improve oral hygiene and prevent tooth decay, gum disease and periodontitis. The neem stick is an underestimated tool for dental hygiene which is beginning to be explored in controlled clinical studies.10
Conflicts of interest All authors have none to declare.
references
1. Andrews JM. Determination of minimum inhibitory concentrations. J Antimicrob Chemother. 2001;48(suppl 1):5e16.
✓ ✓ ✓ ✓
✓ ✓
✓
✓ ✓ ✓ ✓ ✓ ✓
✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
✓ ✓ ✓ ✓ ✓ ✓ ✓
2. Ajaybhan P, Chauhan N, Chauhan A. Evaluation of antimicrobial activity of six medicinal plants against dental pathogens. Rep Opin. 2010;2:37e42. 3. Gupta Ch, P, Garg A, Prakash Dh, Goyal S, Gupta S. Comparative study of cinnamon oil & clove oil on some oral microbial. Pharmacologyonline. 2011;2:45e49. 4. Johnson DB, Shringi BN, Patidar DK, Chalichem NSS, Javvadi AK. Screening of antimicrobial activity of alcoholic & aqueous extract of some indigenous plants. Indo-Global J Pharm Sci. 2011;1:186e193. 5. Memmou F, Mahboub R. Composition of essential oil from fresh flower of clove. J Sci Res Pharm. 2012;1:33e35. 6. Gaetti-Jardim Jr E, Landucci LF, Arafat OKK, et al. Antimicrobial activity of six plant extracts from the Brazilian savanna on periodontal pathogens. Int J Odontostomat. 2011;5:249e256. 7. Packia Lekshmi NCJ, Sowmia N, Viveka S, Brindha Raja, Jeeva S. The inhibiting effect of Azadirachtaindica against dental pathogens. Asian J Plant Sci Res. 2012;2:6e10. 8. Ghasemi-Pirbalouti A, Jahanbazi P, Enteshari S, et al. Antimicrobial activity of some Iranian medicinal plants. Arch Biol Sci Belgrade. 2010;62:633e641. 9. Al-Zubaydi SR, Al-Hmdany MA, Raesan SJ. Antibacterial effect of some medicinal plant extracts against some pathogenic bacterial strains. J Duhok Univ. 2009;12:244e249. 10. Silva NCC, Fernandes Ju´nior A. Biological properties of medicinal plants: a review of their antimicrobial activity. J Venom Anim Toxins Incl Trop Dis. 2010;16:402e413.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/jobcr
Original Article
Antibacterial activity of clove, gall nut methanolic and ethanolic extracts on Streptococcus mutans PTCC 1683 and Streptococcus salivarius PTCC 1448 Mirsasan Mirpour a,*, Zohreh Gholizadeh Siahmazgi a, Masoumeh Sharifi Kiasaraie b a b
Microbiology Department, Islamic Azad University Lahijan Branch, Iran Chemistry Department, Islamic Azad University Rasht Branch, Iran
article info
abstract
Article history:
Introduction: Antimicrobial compounds from herbal sources have good therapeutic poten-
Received 23 November 2014
tial. In this study, the antibacterial effects of clove and gall nut, methanolic and ethanolic
Accepted 7 February 2015
extractions, were evaluated for their effect on Streptococcus mutans PTCC 1683 and Strep-
Available online 25 February 2015
tococcus salivarius PTCC 1448, as both the two cause oral diseases. Method: The clove and gall nut methanolic and ethanolic extracts were prepared and
Keywords:
antibacterial activity was evaluated for S. mutans and S. salivarius in the base of inhibition
Clove
zone diameter using agar diffusion method. In this part minimum inhibitory concentration
Gall nut extracts
(MIC) and minimal bactericidal concentration (MBC) were assessed.
Antibacterial effects
Results: These extracts showed effective antibacterial activity on bacteria. Antibacterial activity of Methanolic extract of clove was more than that of ethanolic extract, and ethanolic extracts of gall nut had antibacterial activity more than that of methanolic extracts. MIC and MBC results for clove methanolic extract were 1.5 mg/ml and 3 mg/ml for S. mutans and 6.25 mg/ml and 12.5 mg/ml for S. salivarius, respectively. These results for clove ethanolic extracts were 12.5 mg/ml and 25 mg/ml for S. mutans and 25 mg/ml and 50 mg/ml for S. salivarius, respectively. MIC and MBC results for gall nut methanolic extract were 25 mg/ml and 50 mg/ml for S. mutans and 12.5 mg/ml and 25 mg/ml for S. salivarius, respectively. These results for gall nut ethanolic extracts were 3.1 mg/ml and 6.2 mg/ml for S. mutans and 25 mg/ml and 50 mg/ml for S. salivarius, respectively. Discussion: The results showed effective antibacterial activity using clove and gall nut methanolic extracts. If other properties such as tolerance of tissue can also be studied, these extracts can be used as a mouthwash. Copyright © 2015, Craniofacial Research Foundation. All rights reserved.
* Corresponding author. Tel.: þ98 9121964387 (mobile). E-mail address: [email protected] (M. Mirpour). http://dx.doi.org/10.1016/j.jobcr.2015.02.002 2212-4268/Copyright © 2015, Craniofacial Research Foundation. All rights reserved.
8
1.
j o u r n a l o f o r a l b i o l o g y a n d c r a n i o f a c i a l r e s e a r c h 5 ( 2 0 1 5 ) 7 e1 0
Introduction
Identification and use of medicinal plants is as old as human life. Before the 19th century the use of natural resources, mainly plants were the main source of medicines.1 Rapid development of chemistry and the decline of natural resources caused the new synthetic chemicals to be used instead of the herbal medicines. In spite that even today, many scientists in different fields are using herbal medicines.2 Due to the high capacity of medicinal plants and its widespread use in traditional medicine, it seems that finding of the effective plant's extract against pathogenic agents is feasible. Due to increasing oral infections and no existence of medications without side effects, in this study we have attempted to identify plants effective against oral pathogens. Antimicrobial materials in dental therapy, in either oral or topical form, are frequently used to reduce the oral microbial flora before gingival surgery, during healing and after surgery.3 Certain elderly patients or those prone to microbial endocarditis, immunodeficiency, diabetic and blood diseases, are committed to the use of antimicrobial substances. With extending scopes for mouthwash, it seems necessary to assess materials with minimal side effects and desired therapeutic effects. The aim of this study is to find new medicinal plant extracts to replace chemicals that have many side effects.4 In this study, the effect of ethanolic and methanolic extracts of cloves and gall nut on to standard strains of bacteria Streptococcus mutans PTCC 1683 and Streptococcus salivarius PTCC 1448 were evaluated as they cause oral infections.
2.
Materials and methods
2.1.
Microorganisms
Two bacterial strains S. mutans PTCC 1683 and S. salivarius PTCC 1448 were purchased from Iranian Research Organization for Science and Technology (IROST). These bacteria were cultured on selective culture media in sterile condition.
2.2.
Chemistry
For this part, macerations method was used. 10 g of the powdered plant material were soaked in 100 ml solvent (Ethanol or Methanol) and shaken for 24 h. The extracts were then filtered using Whatman No.1 filter paper along with 2 g sodium sulfate to remove the trace of water in the filtrate. The filtrate was concentrated by vacuum distillation. The extracts were exposed under UV ray for sterilization.
2.2.1.
Gas chromatography/mass spectroscopy (GC/MS)
The constituent of the extracts were identified by gas chromatography (model Hewlett-Packard-GC6890) which was combined with mass spectrometry (Mass 5973N). The column was HP-5MS (30 nm 0.25 nm internal diameters, 0.25 mm film thickness) and Helium (2 ml/min) was used as a carrier gas. Samples were injected while the injection temperature was 250 C. The temperature of capillary column was 60 C for 3 min and then programmed to 250 C at 8 C/min and held for
10 min. The identification of compounds was performed by comparing their retention indices and mass spectra with Wiley 275 library installed on the instrument and NIST Mass Spectral.
2.3.
Antimicrobial activity
The effect of ethanol and methanol extracts of two plants, gall nut and cloves were performed using the disc diffusion method, according to the KirbyeBauer. The bacterial suspension was prepared using 0.5 McFarland standard's tube. It was cultured on Muller Hinton agar plates (Merck's) and used with discs impregnated with the compound. The plates were incubated for 24 h at 37 C. After the incubation period, inhibition zone was measured in centimeter. This study was performed 3 times for each extracts and bacterium. A blank disc, ethanol disc, methanol disc and Chlorhexidine disc was used as a control.
2.3.1.
Minimum inhibitory concentration (MIC)
A pure culture of a single microorganism was grown in Mueller-Hinton broth. The culture was standardized using standard microbiological 0.5 McFarland standard's tube. The better the microbial culture, more were the test results reproducible. The antimicrobial agent was diluted a number of times, usually 1:1, through a sterile diluent (Mueller-Hinton broth). After the antimicrobial agent was diluted, a volume of the standardized inoculum, equal to the volume of the diluted antimicrobial agent, was added to each dilution vessel, bringing the microbial concentration to approximately 75,000,000 cells/ml. The inoculated, serially diluted antimicrobial agent was incubated at an appropriate temperature for the test organism for a pre-set period, usually 18 h. After incubation, the series of dilution vessels was observed for microbial growth, usually indicated by turbidity and/or a pellet of microorganisms in the bottom of the vessel. The last tube in the dilution series that did not demonstrate growth corresponded with the MIC of the antimicrobial agent.
2.3.2.
Minimum bactericidal concentration (MBC)
A pure culture of a specified microorganism was diluted in growth-supporting broth (Muller Hinton Broth) to a concentration of 0.5 McFarland standard's tube. A stock dilution of the antimicrobial test substance was made at approximately 100 the level of the expected MIC. Further 1:1 dilutions were made in test tubes or 96 well microtiter plates. The dilutions, going from least concentrated to most concentrated, were inoculated with equal volumes of the specified microorganism. A positive and negative control tube or well was included for every test microorganism to demonstrate adequate microbial growth over the course of the incubation period and media sterility, respectively. An aliquot of the positive control was plated and used to establish a baseline concentration of the microorganism used. The tubes or microtiter plates were then incubated at the appropriate temperature and duration. Turbidity indicated growth of the microorganism. MIC was lowest where no growth was visually observed. To determine the MBC, the dilution representing the MIC and at least two of the more concentrated test product dilutions were plated and enumerated to determine viable CFU/ml. The MBC is the lowest concentration that
9
j o u r n a l o f o r a l b i o l o g y a n d c r a n i o f a c i a l r e s e a r c h 5 ( 2 0 1 5 ) 7 e1 0
Table 1 e Antibacterial activity of clove and gall nut. Microorganism
Plant Clove
Gall nut
Standard
Solvent
S. mutans S. salivarius
Methanol
Ethanol
Methanol
Ethanol
Chlorhexidine
IZ 24 ± 0.4 AI ¼ 1.6 IZ 17 ± 0.99 AI ¼ 1.4
IZ 20 ± 0.29 AI ¼ 1.3 IZ 12 ± 0.37 AI ¼ 1
IZ 24 ± 0.72 AI ¼ 1.6 IZ 14 ± 0.57 AI ¼ 1.1
IZ 10 ± 049 AI ¼ 0.6 IZ 7 ± 0.14 AI ¼ 0.58
IZ: 15 IZ: 12
IZ: Inhibition Zone (in mm). AI: Activity Index ¼ IZ of test sample/IZ of standard. Values are mean of triplicate reading (mean ± S.D.).
demonstrated a pre-determined reduction (such as 99.9%) in CFU/ml when compared to the MIC dilution.
3.
Results
3.1.
Antibacterial activity of clove and gall nut
The antibacterial activity of clove and gall nut extracts were tested using different bacteria and the results are listed in Table 1. It was found that the methanolic extract of clove and gull nut were potentially active against S. mutans PTCC 1683 and S. salivarius PTCC 1448 with zones of inhibition ranging from 14 to 24 mm. These range for ethanolic extracts were from 7 to 20 mm whereas, the inhibition zone for Chlorhexidine as control was 15 mm. These showed that the extracts were very effective on bacteria.
3.2.
MICs and MBCs of clove and gull nut extracts
The MIC and MBC values of the ethanolic and methanolic extracts of clove and gull nut for S. salivarius PTCC 1448and S. mutans PTCC 1683 are showed in Table 2. The best MIC and MBC against S. mutans PTCC 1683 were 1.5 and 3 for methanolic extract of clove respectively, and against S. salivarius PTCC 1448 were 6.25 and 12.5 for methanolic extract of clove, respectively.
3.3.
Clove and gall nut components
Chemical composition of these plants were specified using GC/MS (Table 3) that to be comparable to other studies.5 The
result showed that both methanolic and ethanolic clove extract mainly contain Eugenol, Eugenyl Acetate and b-Caryophyllene. As shown in Table 3, some substances are same in the base of results, the best antibacterial activities were achieved using clove methanolic extract and gall nut ethanolic extract. Hence, common components of these two extracts are important.
4.
Discussion
In recent years many studies have been conducted to evaluate the antimicrobial effects of essential oils and extracts, indicating the ability of these compounds to inhibit the growth of pathogenic and spoilage microorganisms in food.6 Since, these compounds are natural and in many cases containing other existing health compounds, it has been emphasized for protecting of human health.7 Overuse of antibiotics has caused increasing resistance to drugs. Also, indiscriminate use of antibiotics is often associated with adverse effects on the human body. Since some plants with antimicrobial effects have been recorded in pharmacopoeia of the drug in Iran, it can be used to deal with some specific pathogenic microbes and safe to replace with some antibiotics.8 In a study conducted on two plant extracts used in traditional medicine in Iran, they showed antibacterial activity against Streptococcus salivarius PTCC 1448 and Streptococcus mutans PTCC 1683 Extracts. Reduced colony growth was observed compared with the control drug (p < 0.05). Shoji et al showed that the aqueous and methanol extracts of this plant contain saponins and flavonoids. However, the need to further
Table 2 e MICs and MBCs of clove and gull nut extracts. Microorganism
Plant Clove
Gall nut Solvent
S. mutans S. salivarius
Methanol (mg/ml)
Ethanol (mg/ml)
Methanol (mg/ml)
Ethanol (mg/ml)
MIC:1.5 MBC: 3 MIC: 6.25 MBC: 12.5
MIC: 12.5 MBC: 25 MIC: 25 MBC: 50
MIC: 25 MBC: 50 MIC: 12.5 MBC: 25
MIC:3.1 MBC: 6.2 MIC: 25 MBC: 50
10
j o u r n a l o f o r a l b i o l o g y a n d c r a n i o f a c i a l r e s e a r c h 5 ( 2 0 1 5 ) 7 e1 0
Table 3 e Chemical composition of methanolic and ethanolic extracts of clove and gall nut. Components
Extracts Clove
Gall nut
Methanolic extract Ethanolic extract Methanolic extract Ethanolic extract 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
2-Furancarboxaldehyde Chavicol Eugenol 1,2,3-Trihydroxybenzene (Pyrogallol) b-Caryophyllene a-Humulene Eugenyl Acetate Caryophyllene Oxide Palmitic acid Linoleic acid Oleic acid Stearic acid n-Hexyl butyrate 5-(hydroxymethyl)-2-urancarboxaldehyde Psoralene Oroselone Lidocaine 2,3-dihydro-3,5-dihydroxy-6-methylpyran-4-one Cinnamaldehyde Thymol Carvacrol 4-vinyl-2-methoxy-phenol Quinic acid
✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
studies to identify the active agents responsible in other biological and pharmacological activities of this plant is necessary. Also, other antimicrobial activity of plant extracts, essential oil and other areas of native species from view of the chemical composition of contents and their clinical efficacy must be investigated.9 Plants contain phytochemicals that have pronounced antimicrobial activity. This underlies the antiquity of herbs to improve oral hygiene and prevent tooth decay, gum disease and periodontitis. The neem stick is an underestimated tool for dental hygiene which is beginning to be explored in controlled clinical studies.10
Conflicts of interest All authors have none to declare.
references
1. Andrews JM. Determination of minimum inhibitory concentrations. J Antimicrob Chemother. 2001;48(suppl 1):5e16.
✓ ✓ ✓ ✓
✓ ✓
✓
✓ ✓ ✓ ✓ ✓ ✓
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