- Email: [email protected]
Synergistic effect between clove oil and its major compounds and antibiotics against oral bacteria
archives of oral biology 56 (2011) 907–916
available at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
Synergistic effect between clove oil and its major compounds and antibiotics against oral bacteria Sang-Eun Moon a, Hye-Young Kim b, Jeong-Dan Cha b,* a
Department of Dental Hygiene, Kwangju Women’s University, Kwangju, South Korea Department of Dental Hygiene, College of Natural Sciences, Dongeui University, 995 Eomgwangno, Busan jin-gu, Busan 614-714, South Korea b
article info
abstract
Article history:
Background: Essential oils have been found to be antibacterial, antifungal, spasmolytic, and
Accepted 10 February 2011
antiplasmodial activity and therapeutic effect in cancer treatment. Objective and design: In this study, clove oil and its major compounds, eugenol and b-
Keywords:
caryophyllene were evaluated against oral bacteria, either alone or in combination with
Essential oil
ampicillin or gentamicin, via checkerboard and time kill assay.
Clove oil
Results: The antibacterial activity of the clove oil was higher than b-caryophyllene but was
Eugenol
similar to eugenol against all tested oral bacteria. Furthermore, the MIC and MBC were
Oral bacteria
reduced to one half-one sixteenth as a result of the combination of clove oil or eugenol with
Antibacterial activity
antibiotics. The synergistic interaction was verified by time kill studies using the clove oil or
Synergistic effect
eugenol with antibiotics. 60 min of treatment with MIC of the clove oil or eugenol with
Minimum inhibitory concentrations
ampicillin or gentamicin resulted in an increase in the rate of killing in units of CFU/mL to a
(MICs)
greater degree than was observed with alone.
Minimum bactericidal
Conclusion: The results suggest that the clove oil and eugenol could be employed as a natural
concentrations (MBCs)
antibacterial agent against cariogenic and periodontopathogenic bacteria. # 2011 Elsevier Ltd. All rights reserved.
1.
Introduction
Dental caries and periodontitis are common oral diseases caused by bacterial infection and the development of dental plaque.1 Cariogenic bacteria such as Streptococcus mutans and Streptococcus sobrinus as well as periodontopathogenic bacteria such as Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans have been identified as major causative agents of oral diseases.1,2 Corrective treatment for such infectious diseases requires the reduction and/or elimination of bacterial accumulations in the retentive sites on the top of the teeth (occlusal surfaces) and between teeth by daily toothbrushing and frequent dental cleanings or prophylaxys.3–6 Several antibacterial agents including, fluorides, phenol derivatives,
ampicillin, erythromycin, penicillin, tetracycline, and vancomycin have been used widely in dentistry to inhibit bacterial growth.6–10 However, excessive use of these chemicals can result in derangements of the oral and intestinal flora and cause side effects such as microorganism susceptibility, vomiting, diarrhoea and tooth staining.6,7,11 These problems necessitate further search for natural antibacterial agents that are safe for humans and specific for oral pathogens.12 Natural products have recently been investigated more thoroughly as promising agents to prevent oral diseases, especially plaquerelated diseases such as dental caries.12–16 Essential oils contain a complex mixture of odourous and volatile compounds from secondary plant metabolism, and are widely used in cosmetics as fragrance components, in the food industry as flavouring additives and in a variety of
* Corresponding author. Tel.: +82 51 890 2688; fax: +82 51 890 2623. E-mail address: [email protected] (J.-D. Cha). 0003–9969/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2011.02.005
908
archives of oral biology 56 (2011) 907–916
Table 1 – Antibacterial activity of clove oil and its main compounds, eugenol and b-caryophyllene against oral bacteria. Strains
MIC/MBC (mg/mL) Clove oil
S. mutans ATCC 25175 S. sanguinis ATCC 10556 S. sobrinus ATCC 27607 S. rattib KCTC 3294 S. criceti KCTC 3292 S. anginosus ATCC 31412 S. gordonii ATCC 10558 A. actinomycetemcomitans ATCC 43717 F. nucleatum ATCC 10953 P. intermedia ATCC 25611 P. gingivalis ATCC 33277 a b
0.2/0.8 0.4/0.8 0.2/0.8 0.8/1.6 0.4/1.6 0.8/1.6 0.1/0.2 0.8/1.6 0.1/0.2 0.1/0.2 0.1/0.1
Eugenol
b-Caryophyllene
0.1/0.2 0.4/0.8 0.2/0.4 0.8/1.6 0.4/0.8 0.4/0.8 0.1/0.1 0.4/0.8 0.1/0.2 0.1/0.2 0.1/0.1
Ampicillin a
1.6/3.2 1.6/3.2 12.8/12.8 6.4/12.8 3.2/12.8 6.4/12.8< 1.6/3.2 6.4/12.8 3.2/12.8 1.6/3.2 0.8/1.6
2/4 32/64 2/4 4/8 4/16 2/4 1/4 32/64 0.25/0.5 32/64 0.5/2
Gentamicin 8/16 8/32 4/16 8/16 8/32 16/32 4/8 2/4 16/32 0.5/2 256/512
MIC/MBC (mg/mL). Korean Collection for Type Cultures.
household products as scenting agents.17,18 The essential oils of many different plants have been previously tested in both in vitro and in vivo studies, as promising agents in the treatment of oral diseases and other infections.14,19–21 The essential oil extracted from the dried flower buds of clove, Eugenia caryophyllata L. Merr. & Perry (Myrtaceae), is used as a topical application to relieve pain and to promote healing and
also finds use in the fragrance and flavouring industries.22 The main compounds of the clove oil are phenylpropanoids such as eugenol (76.8%), followed by b-caryophyllene (17.4%), ahumulene (2.1%), and eugenyl acetate (1.2%). The biological activity of E. caryophyllata has been investigated on several microorganisms and parasites, including pathogenic bacteria, Herpes simplex and hepatitis C viruses.22,23 In addition to its
Table 2 – Synergistic effect of clove oil with ampicillin against oral bacteria. Strains
Agent
MIC/MBC
FICa
FICIb
Outcome
Alone
Combination
S. mutans ATCC 25175
Clove oil Ampicillin
0.2/0.8c 2/4d
0.05/0.1 0.5/1
0.25/0.125 0.25/0.25
0.5/0.375
Synergistic/synergistic
S. sanguinis ATCC 10556
Clove oil Ampicillin
0.4/0.8 32/64
0.1/0.4 8/16
0.25/0.5 0.25/0.25
0.5/0.75
Synergistic/additive
S. sobrinus ATCC 27607
Clove oil Ampicillin
0.2/0.8 2/4
0.05/0.1 0.5/1
0.25/0.125 0.25/0.25
0.5/0.375
Synergistic/synergistic
S. rattie KCTC 3294
Clove oil Ampicillin
0.8/1.6 4/8
0.2/0.4 1/2
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
S. criceti KCTC 3292
Clove oil Ampicillin
0.4/1.6 4/16
0.1/0.4 1/4
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
S. anginosus ATCC 31412
Clove oil Ampicillin
0.8/1.6 2/4
0.2/0.4 0.5/2
0.25/0.25 0.25/0.5
0.5/0.75
Synergistic/additive
S. gordonii ATCC 10558
Clove oil Ampicillin
0.1/0.2 1/4
0.025/0.05 0.25/0.5
0.25/0.25 0.25/0.125
0.5/0.375
Synergistic/synergistic
A. actinomycetemcomitans ATCC 43717
Clove oil Ampicillin
0.8/1.6 32/64
0.2/0.4 8/16
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
F. nucleatum ATCC 51190
Clove oil Ampicillin
0.1/0.2 0.25/0.5
0.025/0.05 0.062/0.125
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
P. intermedia ATCC 49049
Clove oil Ampicillin
0.1/0.2 32/64
0.025/0.05 8/16
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
P. gingivalis ATCC 33277
Clove oil Ampicillin
0.1/0.1 0.5/2
0.025/0.05 0.125/0.5
0.25/0.5 0.25/0.25
0.5/0.75
Synergistic/additive
a b c d e
The fractional inhibitory concentration (FIC). The fractional inhibitory concentration index (FICI). Clove oil: MIC/MBC (mg/mL). Ampicillin: MIC/MBC (mg/mL). Korean Collection for Type Cultures.
909
archives of oral biology 56 (2011) 907–916
antimicrobial, antioxidant, antifungal and antiviral activity, clove essential oil possesses antiinflammatory, cytotoxic, insect repellent and anaesthetic properties.21–23 Eugenol, the principal chemical component of clove oil from E. aromatica has been long known for its analgesic, local anaesthetic, antiinflammatory, and antibacterial effects.21,24,25 It is used in the form of a paste or mixture as dental cement, filler and restorative material.5,24 It belongs to the class of essential oils that is generally recognized as safe (GRAS) by the Food and Drug Administration.26 It is a known antibacterial agent against pathogens including Escherichia coli O157:H7, Listeria monocytogenes, Campylobacter jejuni, Salmonella enterica, Staphylococcus aureus, Lactobacillus sakei and Helicobacter pyroli and is reported to act primarily by disrupting the cytoplasmic membrane.24,27 Moreover, the hydrophobic nature of eugenol enables it to penetrate the lipopolysaccharide of the Gramnegative bacterial cell membrane and alters the cell structure, which subsequently results in the leakage of intracellular constituents.26 The clove oil and eugenol have not yet previously been biologically investigated for oral bacteria. In this study, the antimicrobial activities of clove oil and its major compounds, eugenol and b-caryophyllene against oral bacteria though several analyses were investigated.
2.
Materials and methods
2.1.
Preparation of antimicrobial agent
Clove oil was purchased by Hurbmall, Inc., Seoul, Korea. Eugenol and b-caryophyllene were supplied by Sigma–Aldrich Co., St. Louis, MO, USA. Stock solutions of 30% (v/v) clove oil, eugenol and b-caryophyllene were prepared in methanol and the concentrations required for the experiments were prepared from this stock solution.
2.2.
Bacterial strains
The oral bacterial strains used in this study were: S. mutans ATCC (American Type Culture Collection) 25175, Streptococcus sanguinis ATCC 10556, S. sobrinus ATCC 27607, Streptococcus ratti KCTC (Korean Collection for Type Cultures) 3294, Streptococcus criceti KCTC 3292, Streptococcus anginosus ATCC 31412, Streptococcus gordonii ATCC 10558, A. actinomycetemcomitans ATCC 43717, Fusobacterium nucleatum ATCC 10953, Prevotella intermedia ATCC 25611, and Porphylomonas gingivalis ATCC 33277. Brain-Heart Infusion broth supplemented with 1% yeast
Table 3 – Synergistic effect of clove oil with gentamicin against oral bacteria. Strains
Agent
MIC/MBC
FICa
FICIb
Outcome
Alone
Combination
S. mutans ATCC 25175
Clove oil Gentamicin
0.2/0.8c 8/16d
0.05/0.2 2/4
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
S. sanguinis ATCC 10556
Clove oil Gentamicin
0.4/0.8 8/32
0.1/0.2 2/4
0.25/0.25 0.25/0.125
0.5/0.375
Synergistic/synergistic
S. sobrinus ATCC 27607
Clove oil Gentamicin
0.2/0.8 4/16
0.05/0.1 1/4
0.25/0.125 0.25/0.25
0.5/0.375
Synergistic/synergistic
S. rattie KCTC 3294
Clove oil Gentamicin
0.8/1.6 8/16
0.2/0.4 4/4
0.25/0.25 0.5/0.25
0.75/0.5
Additive/synergistic
S. criceti KCTC 3292
Clove oil Gentamicin
0.4/1.6 8/32
0.1/0.2 2/8
0.25/0.125 0.25/0.25
0.5/0.375
Synergistic/synergistic
S. anginosus ATCC 31412
Clove oil Gentamicin
0.8/1.6 16/32
0.2/0.4 4/8
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
S. gordonii ATCC 10558
Clove oil Gentamicin
0.1/0.2 4/8
0.05/0.1 1/2
0.25/0.5 0.25/0.25
0.5/0.75
Synergistic/additive
A. actinomycetemcomitans ATCC 43717
Clove oil Gentamicin
0.8/1.6 2/4
0.2/0.4 0.5/1
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
F. nucleatum ATCC 51190
Clove oil Gentamicin
0.1/0.2 16/32
0.025/0.05 4/8
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
P. intermedia ATCC 49049
Clove oil Gentamicin
0.1/0.2 0.5/2
0.025/0.05 0.125/0.5
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
P. gingivalis ATCC 33277
Clove oil Gentamicin
0.1/0.1 256/512
0.025/0.025 32/128
0.25/0.25 0.125/0.25
0.375/0.5
Synergistic/synergistic
a b c d e
The fractional inhibitory concentration (FIC). The fractional inhibitory concentration index (FICI). Clove oil: MIC/MBC (mg/mL). Gentamicin: MIC/MBC (mg/mL). Korean Collection for Type Cultures.
910
archives of oral biology 56 (2011) 907–916
extract (Difco Laboratories, Detroit, MI) was used for all bacterial strains except P. intermedia and P. gingivalis. For P. intermedia and P. gingivalis, Brain-Heart Infusion broth containing hemin and menadione (Sigma) was used.
antibiotics in order to compare the sensitivity of clove oil, eugenol, and b-caryophyllene against test bacteria.
2.3. Minimum inhibitory concentration/minimum bactericidal concentration assay
The synergistic effects of clove oil, and eugenol, which exhibited the highest antimicrobial activity, and antibiotics were assessed by the checkerboard test as previously described.28 The antimicrobial combinations assayed included clove oil or eugenol plus ampicillin or gentamicin. Serial dilutions of two different antimicrobial agents were mixed in cation-supplemented Mueller-Hinton broth. After 24 h of incubation at 37 8C, the MIC was determined to be the minimal concentration at which there was no visible growth. The fractional inhibitory concentration index (FICI) is the sum of the FICs of each of the drugs, which in turn is defined as the MIC of each drug when it is used in combination divided by the MIC of the drug when it is used alone. The interaction was defined as synergistic if the FIC index was less than or equal to 0.5, additive if the FIC index was greater than 0.5 and less than or equal 1.0, indifferent if the FIC index was greater than 1.0 and less than or equal to 2.0, and antagonistic if the FIC index was greater than 2.0.28
The minimum inhibitory concentrations (MICs) were determined for clove oil and its main compounds, eugenol and bcaryophyllene by the broth dilution method, and were carried out in triplicate. The antibacterial activities were examined after incubation at 37 8C for 18 h (facultative anaerobic bacteria), for 24 h (microaerophilic bacteria), and for 1–2 days (obligate anaerobic bacteria) under anaerobic conditions. MICs were determined as the lowest concentration of test samples that resulted in a complete inhibition of visible growth in the broth. Following anaerobic incubation of MICs plates, the minimum bactericidal concentrations (MBCs) were determined on the basis of the lowest concentration of clove oil, eugenol, and b-caryophyllene that kills 99.9% of the test bacteria by plating out onto each appropriate agar plate. Ampicillin and gentamicin (Sigma) were used as standard
2.4.
Checkerboard dilution test
Table 4 – Synergistic effect of eugenol with ampicillin against oral bacteria. Strains
Agent
MIC/MBC
FICa
FICIb
Outcome
Alone
Combination
S. mutans ATCC 25175
Eugenol Ampicillin
0.1/0.2c 2/4d
0.025/0.05 0.5/1
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
S. sanguinis ATCC 10556
Eugenol Ampicillin
0.4/0.8 32/64
0.1/0.2 8/16
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
S. sobrinus ATCC 27607
Eugenol Ampicillin
0.2/0.4 2/4
0.05/0.1 0.5/1
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
S. rattie KCTC 3294
Eugenol Ampicillin
0.8/1.6 4/8
0.4/0.8 1/2
0.5/0.5 0.25/0.25
0.75/0.75
Additive/additive
S. criceti KCTC 3292
Eugenol Ampicillin
0.4/0.8 4/16
0.1/0.2 1/2
0.25/0.25 0.25/0.125
0.5/0.375
Synergistic/synergistic
S. anginosus ATCC 31412
Eugenol Ampicillin
0.4/0.8 2/4
0.1/0.2 0.5/1
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
S. gordonii ATCC 10558
Eugenol Ampicillin
0.1/0.1 1/4
0.0125/0.025 0.25/0.5
0.125/0.25 0.25/0.125
0.5/0.375
Synergistic/synergistic
A. actinomycetemcomitans ATCC 43717
Eugenol Ampicillin
0.4/0.8 32/64
0.1/0.2 8/16
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
F. nucleatum ATCC 51190
Eugenol Ampicillin
0.1/0.2 0.25/0.5
0.025/0.05 0.063/0.125
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
P. intermedia ATCC 49049
Eugenol Ampicillin
0.1/0.2 32/64
0.025/0.05 8/16
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
P. gingivalis ATCC 33277
Eugenol Ampicillin
0.1/0.1 0.5/2
0.025/0.025 0.125/0.25
0.25/0.25 0.25/0.125
0.5/0.375
Synergistic/synergistic
a b c d e
The fractional inhibitory concentration (FIC). The fractional inhibitory concentration index (FICI). Clove oil: MIC/MBC (mg/mL). Ampicillin: MIC/MBC (mg/mL). Korean Collection for Type Cultures.
911
archives of oral biology 56 (2011) 907–916
2.5.
Time-kill curves
Bactericidal activities of the drugs under study were also evaluated using time-kill curves on oral bacteria. Tubes containing Mueller-Hinton supplemented to which antibiotics had been added at the MIC were inoculated with a suspension of the test strain, giving a final bacterial count between 0.5 106 CFU/mL and 6.5 106 CFU/mL. The tubes were thereafter incubated at 37 8C in anaerobic chamber and viable counts were performed at 0, 0.5, 1, 2, 3, 4, 5, 6, 12 and 24 h after addition of antimicrobial agents, on agar plates incubated for up to 48 h in anaerobic chamber at 37 8C. Antibiotic carryover was minimized by washings by centrifugation and serial 10fold dilution in sterile phosphate-buffered saline, pH 7.3. Colony counts were performed in duplicate, and means were taken.
3.
Results
3.1.
Antibacterial activity
The antibacterial activities of oral bacteria to clove oil and its main compounds, eugenol and b-caryophyllene, and antibiotics, ampicillin and gentamicin are presented in Table 1,
respectively. The MIC/MBC for clove oil was found to be either 0.1/0.2 or 0.8/1.6 mg/mL on tested cariogenic bacteria and either 0.1/0.1 or 0.8/1.6 mg/mL on tested periodontopathogenic bacteria; for eugenol was found to be either 0.1/0.1 or 0.8/ 1.6 mg/mL on tested cariogenic bacteria and either 0.1/0.1 or 0.4/0.8 mg/mL on tested periodontopathogenic acteria; for bcaryophyllene, either 1.6/3.2 or 12.8/>128 on tested cariogenic bacteria and either 0.8/1.6 or 6.4/12.8 mg/mL on tested periodontopathogenic bacteria; for ampicillin, either 0.25/0.5 or 32/64 mg/mL; for gentamicin, either 0.5/2 or 256/512 mg/mL on tested all bacteria.
3.2.
Synergistic efeect
The synergistic effect of clove oil or eugenol with ampicillin or gentamicin in oral bacteria is presented in Tables 2–5, respectively. In combination with clove oil and ampicillin, the MIC was reduced 4-fold in all bacteria, producing a synergistic effect as defined by FICI 0.5 and the MBC was reduced 4-fold in S. mutans, S. sobrinus, and S. gordonii synergistic effect as defined by FICI 0.375, in S. ratti, S. criceti, A. actinomycetemcomitans, F. nucleatum, and P. intermedia by FICI 0.5, and additive effect in S. sanguinis, S. anginosus, P. gingivalis by FICI 0.75 (Table 2). The combination of gentamicin and clove oil resulted in the decrease in MIC/MBC for all
Table 5 – Synergistic effect of eugenol with gentamicin against oral bacteria. Strains
Agent
MIC/MBC
FICa
FICIb
Outcome
Alone
Combination
S. mutans ATCC 25175
Eugenol Gentamicin
0.1/0.2c 8/16d
0.05/0.05 2/8
0.5/0.25 0.25/0.5
0.75/0.75
Additive/additive
S. sanguinis ATCC 10556
Eugenol Gentamicin
0.4/0.8 8/32
0.1/0.2 2/4
0.25/0.25 0.25/0.125
0.5/0.375
Synergistic/synergistic
S. sobrinus ATCC 27607
Eugenol Gentamicin
0.2/0.4 4/16
0.05/0.1 1/4
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
S. rattie KCTC 3294
Eugenol Gentamicin
0.8/1.6 8/16
0.2/0.8 4/4
0.25/0.5 0.5/0.25
0.75/0.75
Additive/additive
S. criceti KCTC 3292
Eugenol Gentamicin
0.4/0.8 8/32
0.1/0.2 2/4
0.25/0.25 0.25/01.25
0.5/0.375
Synergistic/synergistic
S. anginosus ATCC 31412
Eugenol Gentamicin
0.4/0.8 16/32
0.1/0.2 4/8
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
S. gordonii ATCC 10558
Eugenol Gentamicin
0.1/0.1 4/8
0.025/0.025 1/2
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
A. actinomycetemcomitans ATCC 43717
Eugenol Gentamicin
0.4/0.8 2/4
0.1/0.2 0.5/1
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
F. nucleatum ATCC 51190
Eugenol Gentamicin
0.1/0.2 16/32
0.025/0.05 4/8
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
P. intermedia ATCC 49049
Eugenol Gentamicin
0.1/0.2 0.5/2
0.025/0.05 0.125/0.5
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
P. gingivalis ATCC 33277
Eugenol Gentamicin
0.1/0.1 256/512
0.025/0.025 64/64
0.25/0.25 0.25/0.125
0.5/0.375
Synergistic/synergistic
a b c d e
The fractional inhibitory concentration (FIC). The fractional inhibitory concentration index (FICI). Clove oil: MIC/MBC (mg/mL). Gentamicin: MIC/MBC (mg/mL). Korean Collection for Type Cultures.
912
archives of oral biology 56 (2011) 907–916
bacteria (Table 3). The FICI classified the combination of clove oil with gentamicin as synergistic for all bacteria except additive for S. ratti. In combination with eugenol and ampicillin, the MIC/MBC was reduced 4–8-fold in all tested bacteria, producing a synergistic as defined by FICI 0.375–0.5 except the additive of eugenol led to a reduction of a double dilution in S. ratti, and as defined by FICI 0.75 (Table 4). The combination of eugenol with gentamicin was resulted in the decrease in MIC for all tested bacteria, with the MIC of 0.0045– 1.25 mg/mL for gentamicin becoming 0.25–32 mg/mL and MBC reduced 4–8-fold in all tested bacteria except S. mutans and S. ratti. The FICI classified the combination of eugenol with
gentamicin as synergistic for all tested bacteria except additive in S. mutans and S. ratti (Table 5). The synergistic effect of clove oil or eugenol with ampicillin or gentamicin against oral bacteria was confirmed by time-kill curve experiments. The cultures of all bacteria, with a cell density of 1–5 105 CFU/mL, were exposed to MIC of clove oil or eugenol alone and with ampicillin or gentamicin. We observed that clove oil or eugenol with antibiotics resulted rate of killing increasing in CFU/mL at time dependent manner (Figs. 1–4). These results suggest that clove oil and eugenol completely reduce the reproducing capability of cariogenic and periodontopathogenic bacteria within 1 h of exposure.
Fig. 1 – Time-kill curves of MIC of clove oil alone and its combination with MIC of Amp or Gen against S. mutans, S. sanguinis, S. sobrinus, S. anginosus, S. criceti, and S. ratti. Bacteria were incubated with Clove oil (*), Clove oil + Amp (*), and Clove oil + Gen (!) over time. CFU, colony-forming units.
archives of oral biology 56 (2011) 907–916
913
Fig. 2 – Time-kill curves of MIC of clove oil alone and its combination with MIC of Amp or Gen against S. gordonii, A. actinomycetemcomitans, F. nucleatum, P. intermedia, and P. gingivalis. Bacteria were incubated with Clove oil (*), Clove oil + Amp (*), and Clove oil + Gen (!) over time. CFU, colony-forming units.
4.
Discussion
Many plant-derived medicines used in traditional medicinal systems have been recorded in pharmacopoeias as agents used to treat infections and a number of these have been recently investigated for their efficacy against oral microbial pathogens.14,16,29 The general antimicrobial activities of medicinal plants and plant products, such as essential oils, have been reviewed previously.14,21,27 The antibacterial activity showed that the clove oil and eugenol exhibited stronger activities than b-caryophyllene on tested oral bacteria. Moreover, the clove oil and eugenol showed strong activity against periodontopathogenic bacteria, F. nucleatum, P. intermedia, and P. gingivalis. The
MIC/MBC for clove oil was found for 0.1–0.8/0.2–1.6 mg/mL on tested cariogenic bacteria and 0.1–0.8/0.1–1.6 mg/mL on tested periodontopathogenic bacteria. In eugenol the MIC/MBC was found for 0.1–0.8/0.1–1.6 mg/mL on tested cariogenic bacteria and either 0.1–0.4/0.1–0.8 mg/mL on tested periodontopathogenic acteria. Many studies have proved the synergistic action of essential oil fractions from different plants with synthetic drugs as antifungal agents and as antibacterial agents.14,26,30 Early studies have clearly established that a number of substances had potential to be utilized in the dental industry, given their activity against cariogenic bacteria and those bacteria associated with periodontal diseases.15,31 Substances that exhibited activity included spice and herb extracts, such as cinnamon bark oil, papua-mace extracts and clove bud oil and
914
archives of oral biology 56 (2011) 907–916
Fig. 3 – Time-kill curves of MIC of eugenol alone and its combination with MIC of Amp or Gen against S. mutans, S. sanguinis, S. sobrinus, S. anginosus, S. criceti, and S. ratti. Bacteria were incubated with Eugenol (*), Eugenol + Amp (*), and Eugenol + Gen (!) over time. CFU, colony-forming units.
constituents of these extracts, such as cinnamic aldehyde and eugenol.22,23 In synergistic effect of clove oil or eugenol with ampicillin, the MIC and MBC were reduced 4–8-fold in most of tested bacteria, producing a synergistic effect as defined by FICI 0.5. The combination of gentamicin and clove oil or eugenol resulted in the decrease in MIC/MBC for all bacteria. The FICI classified the combination of clove oil or eugenol with gentamicin as synergistic for all bacteria except additive for S. mutans or S. ratti. Essential oils are also capable of enhancing the activity of chlorhexidine.20 When used in combination, the essential oils of cinnamon and manuka were able to significantly reduce the amount of chlorhexidine required to inhibit the growth of oral pathogens.15,20 Between 4- and 10-fold
reductions of the amount of chlorhexidine required to inhibit biofilm bacteria is observed when used in combination with cinnamon, manuka, and Leptospermum morrisonii oils.32 The relatively high antimicrobial activities of essential oils are most likely due to the presence of compounds with antimicrobial properties.14,19,32 A number of compounds present in relatively high concentrations in the essential oils are known to have antimicrobial properties.14,24 Eugenol, the main compound of clove oil is observed to have biological activities.22–24 In this study, eugenol, the major compound of clove oil was found to possess strong antibacterial activity against cariogenic (MIC 0.1–0.8 mg/ mL; MBC 0.2–1.6 mg/mL) and periodontopathogenic bacteria (MIC 0.1–0.8 mg/mL; MBC 0.1–1.6 mg/mL). Moreover, Synergistic
archives of oral biology 56 (2011) 907–916
915
Fig. 4 – Time-kill curves of MIC of eugenol alone and its combination with MIC of Amp or Gen against S. gordonii, A. actinomycetemcomitans, F. nucleatum, P. intermedia, and P. gingivalis. Bacteria were incubated with Eugenol (*), Eugenol + Amp (*), and Eugenol + Gen (!) over time. CFU, colony-forming units.
effect of clove oil or eugenol with antibiotic in oral bacteria was shown by reduced 4-16-fold. Therefore, the synergistic effects of these active chemicals with other constituents of the essential oil should be taken into consideration for the antimicrobial activity. Determination of rapidity and duration of antibacterial activity can be assessed by time-kill analysis.24 The synergistic interaction was verified by time kill studies using the clove oil or eugenol with antibiotics. 60 min of treatment with MIC of the clove oil or eugenol with ampicillin or gentamicin resulted in an increase in the rate of killing in units of CFU/mL to a greater degree than was observed with alone. The results suggest that combinations of clove oil or eugenol with antibiotics should be investigated further for
possible use in antibacterial products. Particularly, these may be useful in the future for the treatment of cariogenic and periodontopathogenic bacteria.
Acknowledgement We thank Prof. B. S. Kil, Division of Life Science, College of Natural Science, Wonkwang University for the confirmation of plant authenticity. Funding: None. Competing interests: None declared. Ethical approval: Not required.
916
archives of oral biology 56 (2011) 907–916
references
1. Loesche W. Dental caries and periodontitis: contrasting two infections that have medical implications. Infect Dis Clin Nth Am 2007;21:471–502. 2. Ekstrand KR, Bruun G, Bruun M. Plaque and gingival status as indicators for caries progression on approximal surfaces. Caries Res 1998;32:41–5. 3. Feres M, Figueiredo LC, Faveri M, Stewart B, de Vizio W. The effectiveness of a preprocedural mouthrinse containing cetylpyridinium chloride in reducing bacteria in the dental office. J Am Dent Assoc 2010;141:415–22. 4. Jardim JJ, Alves LS, Maltz M. The history and global market of oral home-care products. Braz Oral Res 2009;1:17–22. 5. Stefanovska E, Nakova M, Radojkova-Nikolovska V, Ristoska S. Tooth-brushing intervention programme among children with mental handicap. Bratisl Lek Listy 2010;111:299–302. 6. Van Strydonck DA, Timmerman MG, van der Velden U, van der Weijden GA. Plaque inhibition of two commercially available chlorhexidine mouthrinses. J Clin Periodontol 2005;32:305–9. 7. Bidault P, Chandad F, Grenier D. Risk of bacterial resistance associated with systemic antibiotic therapy in periodontology. J Can Dent Assoc 2007;73:721–5. 8. De Poi R. Chlorhexidine as an anticaries agent. Aust Dent J 2001;46:60. 9. Ivanova EN. The comparative efficacy of local anticaries agents. Stomatologiia 1990;69:60–1. 10. Maltz M, Jardim JJ, Alves LS. Health promotion and dental caries. Braz Oral Res 2010;1:18–25. 11. Claydon NC, Addy M, Adams G, Smith SR, Bosma ML, North M, et al. A comparison of two chlorhexidine gel brushing regimens and a conventional toothpaste brushing regimen for the development of tooth staining over a 6-week period. Int J Dent Hyg 2006;4:183–8. 12. Allaker RP, Douglas CW. Novel anti-microbial therapies for dental plaque-related diseases. Int J Antimicrob Agents 2009;33:8–13. 13. Badria FA, Zidan OA. Natural products for dental caries prevention. J Med Food 2004;7:381–4. 14. Cha JD, Jeong MR, Jeong SI, Moon SE, Kim JY, Kil BS, et al. Chemical composition and antimicrobial activity of the essential oils of Artemisia scoparia and A. capillaris. Planta Med 2005;71:186–90. 15. Palombo EA. Traditional medicinal plant extracts and natural products with activity against oral bacteria: potential application in the prevention and treatment of oral diseases. Evid Based Complement Alternat Med 2009;13, in press [Epub ahead of print]. 16. Park KM, You JS, Lee HY, Baek NI, Hwang JK, Kuwanon G. An antibacterial agent from the root bark of Morus alba against oral pathogens. J Ethnopharmacol 2003;84:181–5. 17. Go´mez-Estaca J, Lo´pez de Lacey A, Lo´pez-Caballero ME, Go´mez-Guille´n MC, Montero P. Biodegradable gelatin– chitosan films incorporated with essential oils as antimicrobial agents for fish preservation. Food Microbiol 2010;27:889–96.
18. Rubel DM, Freeman S, Southwell IA. Tea tree oil allergy: what is the offending agent? Report of three cases of tea tree oil allergy and review of the literature. Australas J Dermatol 1998;39:244–7. 19. Cha JD, Jeong MR, Jeong SI, Moon SE, Kil BS, Yun SI, et al. Chemical composition and antimicrobial activity of the essential oil of Cryptomeria japonica. Phytother Res 2007;21:295–9. 20. Haffajee AD, Roberts C, Murray L, Veiga N, Martin L, Teles RP, et al. Effect of herbal, essential oil, and chlorhexidine mouthrinses on the composition of the subgingival microbiota and clinical periodontal parameters. J Clin Dent 2009;20:211–7. 21. Pinto E, Vale-Silva L, Cavaleiro C, Salgueiro L. Antifungal activity of the clove essential oil from Syzygium aromaticum on Candida, Aspergillus and dermatophyte species. J Med Microbiol 2009;58:1454–62. 22. Chaieb K, Hajlaoui H, Zmantar T, Kahla-Nakbi AB, Rouabhia M, Mahdouani K, et al. The chemical composition and biological activity of clove essential oil, Eugenia caryophyllata (Syzygium aromaticum L. Myrtaceae): a short review. Phytother Res 2007;21:501–6. 23. Jirovetz L, Buchbauer G, Stoilova I, Stoyanova A, Krastanov A, Schmidt E. Chemical composition and antioxidant properties of clove leaf essential oil. J Agric Food Chem 2006;54:6303–7. 24. Devi KP, Nisha SA, Sakthivel R, Pandian SK. Eugenol (an essential oil) acts as an antibacterial agent against Salmonella typhi by disrupting the cellular membrane. J Ethnopharmacol 2010;130:107–15. 25. Yogalakshmi B, Viswanathan P, Anuradha CV. Investigation of antioxidant, anti-inflammatory, and DNA-protective properties of eugenol in thioacetamide-induced liver injury in rats. Toxicology 2010;268:204–12. 26. Hemaiswarya S, Doble M. Synergistic interaction of eugenol with antibiotics against Gram negative bacteria. Phytomedicine 2009;16:997–1005. 27. Gill AO, Holly RA. Disruption of Escherichia coli. Listeria monocytogenes and Lactobacillus sakei cellular membranes by plant oil aromatics. Int J Food Microbiol 2006;108:1–9. 28. Cha JD, Jeong MR, Jeong SI, Lee KY. Antibacterial activity of sophoraflavanone G isolated from the roots of Sophora flavescens. J Microbiol Biotechnol 2007;17:858–64. 29. Vermani A, Navneet. Prabhat. Screening of Quercus infectoria gall extracts as antibacterial agents against dental pathogens. Indian J Dent Res 2009;20: 337–9. 30. Mahboubi M, Bidgoli FG. Antistaphylococcal activity of Zataria multiflora essential oil and its synergy with vancomycin. Phytomedicine 2010;17:548–50. 31. Fine DH, Furgang D, McKiernan M, Tereski-Bischio D, RicciNittel D, Zhang P, et al. An investigation of the effect of an essential oil mouthrinse on induced bacteraemia: a pilot study. J Clin Periodontol 2010;37:840–7. 32. Filoche SK, Soma K, Sissons CH. Antimicrobial effects of essential oils in combination with chlorhexidine digluconate. Oral Microbiol Immunol 2005;20:221–5.
available at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
Synergistic effect between clove oil and its major compounds and antibiotics against oral bacteria Sang-Eun Moon a, Hye-Young Kim b, Jeong-Dan Cha b,* a
Department of Dental Hygiene, Kwangju Women’s University, Kwangju, South Korea Department of Dental Hygiene, College of Natural Sciences, Dongeui University, 995 Eomgwangno, Busan jin-gu, Busan 614-714, South Korea b
article info
abstract
Article history:
Background: Essential oils have been found to be antibacterial, antifungal, spasmolytic, and
Accepted 10 February 2011
antiplasmodial activity and therapeutic effect in cancer treatment. Objective and design: In this study, clove oil and its major compounds, eugenol and b-
Keywords:
caryophyllene were evaluated against oral bacteria, either alone or in combination with
Essential oil
ampicillin or gentamicin, via checkerboard and time kill assay.
Clove oil
Results: The antibacterial activity of the clove oil was higher than b-caryophyllene but was
Eugenol
similar to eugenol against all tested oral bacteria. Furthermore, the MIC and MBC were
Oral bacteria
reduced to one half-one sixteenth as a result of the combination of clove oil or eugenol with
Antibacterial activity
antibiotics. The synergistic interaction was verified by time kill studies using the clove oil or
Synergistic effect
eugenol with antibiotics. 60 min of treatment with MIC of the clove oil or eugenol with
Minimum inhibitory concentrations
ampicillin or gentamicin resulted in an increase in the rate of killing in units of CFU/mL to a
(MICs)
greater degree than was observed with alone.
Minimum bactericidal
Conclusion: The results suggest that the clove oil and eugenol could be employed as a natural
concentrations (MBCs)
antibacterial agent against cariogenic and periodontopathogenic bacteria. # 2011 Elsevier Ltd. All rights reserved.
1.
Introduction
Dental caries and periodontitis are common oral diseases caused by bacterial infection and the development of dental plaque.1 Cariogenic bacteria such as Streptococcus mutans and Streptococcus sobrinus as well as periodontopathogenic bacteria such as Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans have been identified as major causative agents of oral diseases.1,2 Corrective treatment for such infectious diseases requires the reduction and/or elimination of bacterial accumulations in the retentive sites on the top of the teeth (occlusal surfaces) and between teeth by daily toothbrushing and frequent dental cleanings or prophylaxys.3–6 Several antibacterial agents including, fluorides, phenol derivatives,
ampicillin, erythromycin, penicillin, tetracycline, and vancomycin have been used widely in dentistry to inhibit bacterial growth.6–10 However, excessive use of these chemicals can result in derangements of the oral and intestinal flora and cause side effects such as microorganism susceptibility, vomiting, diarrhoea and tooth staining.6,7,11 These problems necessitate further search for natural antibacterial agents that are safe for humans and specific for oral pathogens.12 Natural products have recently been investigated more thoroughly as promising agents to prevent oral diseases, especially plaquerelated diseases such as dental caries.12–16 Essential oils contain a complex mixture of odourous and volatile compounds from secondary plant metabolism, and are widely used in cosmetics as fragrance components, in the food industry as flavouring additives and in a variety of
* Corresponding author. Tel.: +82 51 890 2688; fax: +82 51 890 2623. E-mail address: [email protected] (J.-D. Cha). 0003–9969/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2011.02.005
908
archives of oral biology 56 (2011) 907–916
Table 1 – Antibacterial activity of clove oil and its main compounds, eugenol and b-caryophyllene against oral bacteria. Strains
MIC/MBC (mg/mL) Clove oil
S. mutans ATCC 25175 S. sanguinis ATCC 10556 S. sobrinus ATCC 27607 S. rattib KCTC 3294 S. criceti KCTC 3292 S. anginosus ATCC 31412 S. gordonii ATCC 10558 A. actinomycetemcomitans ATCC 43717 F. nucleatum ATCC 10953 P. intermedia ATCC 25611 P. gingivalis ATCC 33277 a b
0.2/0.8 0.4/0.8 0.2/0.8 0.8/1.6 0.4/1.6 0.8/1.6 0.1/0.2 0.8/1.6 0.1/0.2 0.1/0.2 0.1/0.1
Eugenol
b-Caryophyllene
0.1/0.2 0.4/0.8 0.2/0.4 0.8/1.6 0.4/0.8 0.4/0.8 0.1/0.1 0.4/0.8 0.1/0.2 0.1/0.2 0.1/0.1
Ampicillin a
1.6/3.2 1.6/3.2 12.8/12.8 6.4/12.8 3.2/12.8 6.4/12.8< 1.6/3.2 6.4/12.8 3.2/12.8 1.6/3.2 0.8/1.6
2/4 32/64 2/4 4/8 4/16 2/4 1/4 32/64 0.25/0.5 32/64 0.5/2
Gentamicin 8/16 8/32 4/16 8/16 8/32 16/32 4/8 2/4 16/32 0.5/2 256/512
MIC/MBC (mg/mL). Korean Collection for Type Cultures.
household products as scenting agents.17,18 The essential oils of many different plants have been previously tested in both in vitro and in vivo studies, as promising agents in the treatment of oral diseases and other infections.14,19–21 The essential oil extracted from the dried flower buds of clove, Eugenia caryophyllata L. Merr. & Perry (Myrtaceae), is used as a topical application to relieve pain and to promote healing and
also finds use in the fragrance and flavouring industries.22 The main compounds of the clove oil are phenylpropanoids such as eugenol (76.8%), followed by b-caryophyllene (17.4%), ahumulene (2.1%), and eugenyl acetate (1.2%). The biological activity of E. caryophyllata has been investigated on several microorganisms and parasites, including pathogenic bacteria, Herpes simplex and hepatitis C viruses.22,23 In addition to its
Table 2 – Synergistic effect of clove oil with ampicillin against oral bacteria. Strains
Agent
MIC/MBC
FICa
FICIb
Outcome
Alone
Combination
S. mutans ATCC 25175
Clove oil Ampicillin
0.2/0.8c 2/4d
0.05/0.1 0.5/1
0.25/0.125 0.25/0.25
0.5/0.375
Synergistic/synergistic
S. sanguinis ATCC 10556
Clove oil Ampicillin
0.4/0.8 32/64
0.1/0.4 8/16
0.25/0.5 0.25/0.25
0.5/0.75
Synergistic/additive
S. sobrinus ATCC 27607
Clove oil Ampicillin
0.2/0.8 2/4
0.05/0.1 0.5/1
0.25/0.125 0.25/0.25
0.5/0.375
Synergistic/synergistic
S. rattie KCTC 3294
Clove oil Ampicillin
0.8/1.6 4/8
0.2/0.4 1/2
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
S. criceti KCTC 3292
Clove oil Ampicillin
0.4/1.6 4/16
0.1/0.4 1/4
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
S. anginosus ATCC 31412
Clove oil Ampicillin
0.8/1.6 2/4
0.2/0.4 0.5/2
0.25/0.25 0.25/0.5
0.5/0.75
Synergistic/additive
S. gordonii ATCC 10558
Clove oil Ampicillin
0.1/0.2 1/4
0.025/0.05 0.25/0.5
0.25/0.25 0.25/0.125
0.5/0.375
Synergistic/synergistic
A. actinomycetemcomitans ATCC 43717
Clove oil Ampicillin
0.8/1.6 32/64
0.2/0.4 8/16
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
F. nucleatum ATCC 51190
Clove oil Ampicillin
0.1/0.2 0.25/0.5
0.025/0.05 0.062/0.125
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
P. intermedia ATCC 49049
Clove oil Ampicillin
0.1/0.2 32/64
0.025/0.05 8/16
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
P. gingivalis ATCC 33277
Clove oil Ampicillin
0.1/0.1 0.5/2
0.025/0.05 0.125/0.5
0.25/0.5 0.25/0.25
0.5/0.75
Synergistic/additive
a b c d e
The fractional inhibitory concentration (FIC). The fractional inhibitory concentration index (FICI). Clove oil: MIC/MBC (mg/mL). Ampicillin: MIC/MBC (mg/mL). Korean Collection for Type Cultures.
909
archives of oral biology 56 (2011) 907–916
antimicrobial, antioxidant, antifungal and antiviral activity, clove essential oil possesses antiinflammatory, cytotoxic, insect repellent and anaesthetic properties.21–23 Eugenol, the principal chemical component of clove oil from E. aromatica has been long known for its analgesic, local anaesthetic, antiinflammatory, and antibacterial effects.21,24,25 It is used in the form of a paste or mixture as dental cement, filler and restorative material.5,24 It belongs to the class of essential oils that is generally recognized as safe (GRAS) by the Food and Drug Administration.26 It is a known antibacterial agent against pathogens including Escherichia coli O157:H7, Listeria monocytogenes, Campylobacter jejuni, Salmonella enterica, Staphylococcus aureus, Lactobacillus sakei and Helicobacter pyroli and is reported to act primarily by disrupting the cytoplasmic membrane.24,27 Moreover, the hydrophobic nature of eugenol enables it to penetrate the lipopolysaccharide of the Gramnegative bacterial cell membrane and alters the cell structure, which subsequently results in the leakage of intracellular constituents.26 The clove oil and eugenol have not yet previously been biologically investigated for oral bacteria. In this study, the antimicrobial activities of clove oil and its major compounds, eugenol and b-caryophyllene against oral bacteria though several analyses were investigated.
2.
Materials and methods
2.1.
Preparation of antimicrobial agent
Clove oil was purchased by Hurbmall, Inc., Seoul, Korea. Eugenol and b-caryophyllene were supplied by Sigma–Aldrich Co., St. Louis, MO, USA. Stock solutions of 30% (v/v) clove oil, eugenol and b-caryophyllene were prepared in methanol and the concentrations required for the experiments were prepared from this stock solution.
2.2.
Bacterial strains
The oral bacterial strains used in this study were: S. mutans ATCC (American Type Culture Collection) 25175, Streptococcus sanguinis ATCC 10556, S. sobrinus ATCC 27607, Streptococcus ratti KCTC (Korean Collection for Type Cultures) 3294, Streptococcus criceti KCTC 3292, Streptococcus anginosus ATCC 31412, Streptococcus gordonii ATCC 10558, A. actinomycetemcomitans ATCC 43717, Fusobacterium nucleatum ATCC 10953, Prevotella intermedia ATCC 25611, and Porphylomonas gingivalis ATCC 33277. Brain-Heart Infusion broth supplemented with 1% yeast
Table 3 – Synergistic effect of clove oil with gentamicin against oral bacteria. Strains
Agent
MIC/MBC
FICa
FICIb
Outcome
Alone
Combination
S. mutans ATCC 25175
Clove oil Gentamicin
0.2/0.8c 8/16d
0.05/0.2 2/4
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
S. sanguinis ATCC 10556
Clove oil Gentamicin
0.4/0.8 8/32
0.1/0.2 2/4
0.25/0.25 0.25/0.125
0.5/0.375
Synergistic/synergistic
S. sobrinus ATCC 27607
Clove oil Gentamicin
0.2/0.8 4/16
0.05/0.1 1/4
0.25/0.125 0.25/0.25
0.5/0.375
Synergistic/synergistic
S. rattie KCTC 3294
Clove oil Gentamicin
0.8/1.6 8/16
0.2/0.4 4/4
0.25/0.25 0.5/0.25
0.75/0.5
Additive/synergistic
S. criceti KCTC 3292
Clove oil Gentamicin
0.4/1.6 8/32
0.1/0.2 2/8
0.25/0.125 0.25/0.25
0.5/0.375
Synergistic/synergistic
S. anginosus ATCC 31412
Clove oil Gentamicin
0.8/1.6 16/32
0.2/0.4 4/8
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
S. gordonii ATCC 10558
Clove oil Gentamicin
0.1/0.2 4/8
0.05/0.1 1/2
0.25/0.5 0.25/0.25
0.5/0.75
Synergistic/additive
A. actinomycetemcomitans ATCC 43717
Clove oil Gentamicin
0.8/1.6 2/4
0.2/0.4 0.5/1
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
F. nucleatum ATCC 51190
Clove oil Gentamicin
0.1/0.2 16/32
0.025/0.05 4/8
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
P. intermedia ATCC 49049
Clove oil Gentamicin
0.1/0.2 0.5/2
0.025/0.05 0.125/0.5
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
P. gingivalis ATCC 33277
Clove oil Gentamicin
0.1/0.1 256/512
0.025/0.025 32/128
0.25/0.25 0.125/0.25
0.375/0.5
Synergistic/synergistic
a b c d e
The fractional inhibitory concentration (FIC). The fractional inhibitory concentration index (FICI). Clove oil: MIC/MBC (mg/mL). Gentamicin: MIC/MBC (mg/mL). Korean Collection for Type Cultures.
910
archives of oral biology 56 (2011) 907–916
extract (Difco Laboratories, Detroit, MI) was used for all bacterial strains except P. intermedia and P. gingivalis. For P. intermedia and P. gingivalis, Brain-Heart Infusion broth containing hemin and menadione (Sigma) was used.
antibiotics in order to compare the sensitivity of clove oil, eugenol, and b-caryophyllene against test bacteria.
2.3. Minimum inhibitory concentration/minimum bactericidal concentration assay
The synergistic effects of clove oil, and eugenol, which exhibited the highest antimicrobial activity, and antibiotics were assessed by the checkerboard test as previously described.28 The antimicrobial combinations assayed included clove oil or eugenol plus ampicillin or gentamicin. Serial dilutions of two different antimicrobial agents were mixed in cation-supplemented Mueller-Hinton broth. After 24 h of incubation at 37 8C, the MIC was determined to be the minimal concentration at which there was no visible growth. The fractional inhibitory concentration index (FICI) is the sum of the FICs of each of the drugs, which in turn is defined as the MIC of each drug when it is used in combination divided by the MIC of the drug when it is used alone. The interaction was defined as synergistic if the FIC index was less than or equal to 0.5, additive if the FIC index was greater than 0.5 and less than or equal 1.0, indifferent if the FIC index was greater than 1.0 and less than or equal to 2.0, and antagonistic if the FIC index was greater than 2.0.28
The minimum inhibitory concentrations (MICs) were determined for clove oil and its main compounds, eugenol and bcaryophyllene by the broth dilution method, and were carried out in triplicate. The antibacterial activities were examined after incubation at 37 8C for 18 h (facultative anaerobic bacteria), for 24 h (microaerophilic bacteria), and for 1–2 days (obligate anaerobic bacteria) under anaerobic conditions. MICs were determined as the lowest concentration of test samples that resulted in a complete inhibition of visible growth in the broth. Following anaerobic incubation of MICs plates, the minimum bactericidal concentrations (MBCs) were determined on the basis of the lowest concentration of clove oil, eugenol, and b-caryophyllene that kills 99.9% of the test bacteria by plating out onto each appropriate agar plate. Ampicillin and gentamicin (Sigma) were used as standard
2.4.
Checkerboard dilution test
Table 4 – Synergistic effect of eugenol with ampicillin against oral bacteria. Strains
Agent
MIC/MBC
FICa
FICIb
Outcome
Alone
Combination
S. mutans ATCC 25175
Eugenol Ampicillin
0.1/0.2c 2/4d
0.025/0.05 0.5/1
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
S. sanguinis ATCC 10556
Eugenol Ampicillin
0.4/0.8 32/64
0.1/0.2 8/16
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
S. sobrinus ATCC 27607
Eugenol Ampicillin
0.2/0.4 2/4
0.05/0.1 0.5/1
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
S. rattie KCTC 3294
Eugenol Ampicillin
0.8/1.6 4/8
0.4/0.8 1/2
0.5/0.5 0.25/0.25
0.75/0.75
Additive/additive
S. criceti KCTC 3292
Eugenol Ampicillin
0.4/0.8 4/16
0.1/0.2 1/2
0.25/0.25 0.25/0.125
0.5/0.375
Synergistic/synergistic
S. anginosus ATCC 31412
Eugenol Ampicillin
0.4/0.8 2/4
0.1/0.2 0.5/1
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
S. gordonii ATCC 10558
Eugenol Ampicillin
0.1/0.1 1/4
0.0125/0.025 0.25/0.5
0.125/0.25 0.25/0.125
0.5/0.375
Synergistic/synergistic
A. actinomycetemcomitans ATCC 43717
Eugenol Ampicillin
0.4/0.8 32/64
0.1/0.2 8/16
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
F. nucleatum ATCC 51190
Eugenol Ampicillin
0.1/0.2 0.25/0.5
0.025/0.05 0.063/0.125
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
P. intermedia ATCC 49049
Eugenol Ampicillin
0.1/0.2 32/64
0.025/0.05 8/16
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
P. gingivalis ATCC 33277
Eugenol Ampicillin
0.1/0.1 0.5/2
0.025/0.025 0.125/0.25
0.25/0.25 0.25/0.125
0.5/0.375
Synergistic/synergistic
a b c d e
The fractional inhibitory concentration (FIC). The fractional inhibitory concentration index (FICI). Clove oil: MIC/MBC (mg/mL). Ampicillin: MIC/MBC (mg/mL). Korean Collection for Type Cultures.
911
archives of oral biology 56 (2011) 907–916
2.5.
Time-kill curves
Bactericidal activities of the drugs under study were also evaluated using time-kill curves on oral bacteria. Tubes containing Mueller-Hinton supplemented to which antibiotics had been added at the MIC were inoculated with a suspension of the test strain, giving a final bacterial count between 0.5 106 CFU/mL and 6.5 106 CFU/mL. The tubes were thereafter incubated at 37 8C in anaerobic chamber and viable counts were performed at 0, 0.5, 1, 2, 3, 4, 5, 6, 12 and 24 h after addition of antimicrobial agents, on agar plates incubated for up to 48 h in anaerobic chamber at 37 8C. Antibiotic carryover was minimized by washings by centrifugation and serial 10fold dilution in sterile phosphate-buffered saline, pH 7.3. Colony counts were performed in duplicate, and means were taken.
3.
Results
3.1.
Antibacterial activity
The antibacterial activities of oral bacteria to clove oil and its main compounds, eugenol and b-caryophyllene, and antibiotics, ampicillin and gentamicin are presented in Table 1,
respectively. The MIC/MBC for clove oil was found to be either 0.1/0.2 or 0.8/1.6 mg/mL on tested cariogenic bacteria and either 0.1/0.1 or 0.8/1.6 mg/mL on tested periodontopathogenic bacteria; for eugenol was found to be either 0.1/0.1 or 0.8/ 1.6 mg/mL on tested cariogenic bacteria and either 0.1/0.1 or 0.4/0.8 mg/mL on tested periodontopathogenic acteria; for bcaryophyllene, either 1.6/3.2 or 12.8/>128 on tested cariogenic bacteria and either 0.8/1.6 or 6.4/12.8 mg/mL on tested periodontopathogenic bacteria; for ampicillin, either 0.25/0.5 or 32/64 mg/mL; for gentamicin, either 0.5/2 or 256/512 mg/mL on tested all bacteria.
3.2.
Synergistic efeect
The synergistic effect of clove oil or eugenol with ampicillin or gentamicin in oral bacteria is presented in Tables 2–5, respectively. In combination with clove oil and ampicillin, the MIC was reduced 4-fold in all bacteria, producing a synergistic effect as defined by FICI 0.5 and the MBC was reduced 4-fold in S. mutans, S. sobrinus, and S. gordonii synergistic effect as defined by FICI 0.375, in S. ratti, S. criceti, A. actinomycetemcomitans, F. nucleatum, and P. intermedia by FICI 0.5, and additive effect in S. sanguinis, S. anginosus, P. gingivalis by FICI 0.75 (Table 2). The combination of gentamicin and clove oil resulted in the decrease in MIC/MBC for all
Table 5 – Synergistic effect of eugenol with gentamicin against oral bacteria. Strains
Agent
MIC/MBC
FICa
FICIb
Outcome
Alone
Combination
S. mutans ATCC 25175
Eugenol Gentamicin
0.1/0.2c 8/16d
0.05/0.05 2/8
0.5/0.25 0.25/0.5
0.75/0.75
Additive/additive
S. sanguinis ATCC 10556
Eugenol Gentamicin
0.4/0.8 8/32
0.1/0.2 2/4
0.25/0.25 0.25/0.125
0.5/0.375
Synergistic/synergistic
S. sobrinus ATCC 27607
Eugenol Gentamicin
0.2/0.4 4/16
0.05/0.1 1/4
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
S. rattie KCTC 3294
Eugenol Gentamicin
0.8/1.6 8/16
0.2/0.8 4/4
0.25/0.5 0.5/0.25
0.75/0.75
Additive/additive
S. criceti KCTC 3292
Eugenol Gentamicin
0.4/0.8 8/32
0.1/0.2 2/4
0.25/0.25 0.25/01.25
0.5/0.375
Synergistic/synergistic
S. anginosus ATCC 31412
Eugenol Gentamicin
0.4/0.8 16/32
0.1/0.2 4/8
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
S. gordonii ATCC 10558
Eugenol Gentamicin
0.1/0.1 4/8
0.025/0.025 1/2
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
A. actinomycetemcomitans ATCC 43717
Eugenol Gentamicin
0.4/0.8 2/4
0.1/0.2 0.5/1
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
F. nucleatum ATCC 51190
Eugenol Gentamicin
0.1/0.2 16/32
0.025/0.05 4/8
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
P. intermedia ATCC 49049
Eugenol Gentamicin
0.1/0.2 0.5/2
0.025/0.05 0.125/0.5
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
P. gingivalis ATCC 33277
Eugenol Gentamicin
0.1/0.1 256/512
0.025/0.025 64/64
0.25/0.25 0.25/0.125
0.5/0.375
Synergistic/synergistic
a b c d e
The fractional inhibitory concentration (FIC). The fractional inhibitory concentration index (FICI). Clove oil: MIC/MBC (mg/mL). Gentamicin: MIC/MBC (mg/mL). Korean Collection for Type Cultures.
912
archives of oral biology 56 (2011) 907–916
bacteria (Table 3). The FICI classified the combination of clove oil with gentamicin as synergistic for all bacteria except additive for S. ratti. In combination with eugenol and ampicillin, the MIC/MBC was reduced 4–8-fold in all tested bacteria, producing a synergistic as defined by FICI 0.375–0.5 except the additive of eugenol led to a reduction of a double dilution in S. ratti, and as defined by FICI 0.75 (Table 4). The combination of eugenol with gentamicin was resulted in the decrease in MIC for all tested bacteria, with the MIC of 0.0045– 1.25 mg/mL for gentamicin becoming 0.25–32 mg/mL and MBC reduced 4–8-fold in all tested bacteria except S. mutans and S. ratti. The FICI classified the combination of eugenol with
gentamicin as synergistic for all tested bacteria except additive in S. mutans and S. ratti (Table 5). The synergistic effect of clove oil or eugenol with ampicillin or gentamicin against oral bacteria was confirmed by time-kill curve experiments. The cultures of all bacteria, with a cell density of 1–5 105 CFU/mL, were exposed to MIC of clove oil or eugenol alone and with ampicillin or gentamicin. We observed that clove oil or eugenol with antibiotics resulted rate of killing increasing in CFU/mL at time dependent manner (Figs. 1–4). These results suggest that clove oil and eugenol completely reduce the reproducing capability of cariogenic and periodontopathogenic bacteria within 1 h of exposure.
Fig. 1 – Time-kill curves of MIC of clove oil alone and its combination with MIC of Amp or Gen against S. mutans, S. sanguinis, S. sobrinus, S. anginosus, S. criceti, and S. ratti. Bacteria were incubated with Clove oil (*), Clove oil + Amp (*), and Clove oil + Gen (!) over time. CFU, colony-forming units.
archives of oral biology 56 (2011) 907–916
913
Fig. 2 – Time-kill curves of MIC of clove oil alone and its combination with MIC of Amp or Gen against S. gordonii, A. actinomycetemcomitans, F. nucleatum, P. intermedia, and P. gingivalis. Bacteria were incubated with Clove oil (*), Clove oil + Amp (*), and Clove oil + Gen (!) over time. CFU, colony-forming units.
4.
Discussion
Many plant-derived medicines used in traditional medicinal systems have been recorded in pharmacopoeias as agents used to treat infections and a number of these have been recently investigated for their efficacy against oral microbial pathogens.14,16,29 The general antimicrobial activities of medicinal plants and plant products, such as essential oils, have been reviewed previously.14,21,27 The antibacterial activity showed that the clove oil and eugenol exhibited stronger activities than b-caryophyllene on tested oral bacteria. Moreover, the clove oil and eugenol showed strong activity against periodontopathogenic bacteria, F. nucleatum, P. intermedia, and P. gingivalis. The
MIC/MBC for clove oil was found for 0.1–0.8/0.2–1.6 mg/mL on tested cariogenic bacteria and 0.1–0.8/0.1–1.6 mg/mL on tested periodontopathogenic bacteria. In eugenol the MIC/MBC was found for 0.1–0.8/0.1–1.6 mg/mL on tested cariogenic bacteria and either 0.1–0.4/0.1–0.8 mg/mL on tested periodontopathogenic acteria. Many studies have proved the synergistic action of essential oil fractions from different plants with synthetic drugs as antifungal agents and as antibacterial agents.14,26,30 Early studies have clearly established that a number of substances had potential to be utilized in the dental industry, given their activity against cariogenic bacteria and those bacteria associated with periodontal diseases.15,31 Substances that exhibited activity included spice and herb extracts, such as cinnamon bark oil, papua-mace extracts and clove bud oil and
914
archives of oral biology 56 (2011) 907–916
Fig. 3 – Time-kill curves of MIC of eugenol alone and its combination with MIC of Amp or Gen against S. mutans, S. sanguinis, S. sobrinus, S. anginosus, S. criceti, and S. ratti. Bacteria were incubated with Eugenol (*), Eugenol + Amp (*), and Eugenol + Gen (!) over time. CFU, colony-forming units.
constituents of these extracts, such as cinnamic aldehyde and eugenol.22,23 In synergistic effect of clove oil or eugenol with ampicillin, the MIC and MBC were reduced 4–8-fold in most of tested bacteria, producing a synergistic effect as defined by FICI 0.5. The combination of gentamicin and clove oil or eugenol resulted in the decrease in MIC/MBC for all bacteria. The FICI classified the combination of clove oil or eugenol with gentamicin as synergistic for all bacteria except additive for S. mutans or S. ratti. Essential oils are also capable of enhancing the activity of chlorhexidine.20 When used in combination, the essential oils of cinnamon and manuka were able to significantly reduce the amount of chlorhexidine required to inhibit the growth of oral pathogens.15,20 Between 4- and 10-fold
reductions of the amount of chlorhexidine required to inhibit biofilm bacteria is observed when used in combination with cinnamon, manuka, and Leptospermum morrisonii oils.32 The relatively high antimicrobial activities of essential oils are most likely due to the presence of compounds with antimicrobial properties.14,19,32 A number of compounds present in relatively high concentrations in the essential oils are known to have antimicrobial properties.14,24 Eugenol, the main compound of clove oil is observed to have biological activities.22–24 In this study, eugenol, the major compound of clove oil was found to possess strong antibacterial activity against cariogenic (MIC 0.1–0.8 mg/ mL; MBC 0.2–1.6 mg/mL) and periodontopathogenic bacteria (MIC 0.1–0.8 mg/mL; MBC 0.1–1.6 mg/mL). Moreover, Synergistic
archives of oral biology 56 (2011) 907–916
915
Fig. 4 – Time-kill curves of MIC of eugenol alone and its combination with MIC of Amp or Gen against S. gordonii, A. actinomycetemcomitans, F. nucleatum, P. intermedia, and P. gingivalis. Bacteria were incubated with Eugenol (*), Eugenol + Amp (*), and Eugenol + Gen (!) over time. CFU, colony-forming units.
effect of clove oil or eugenol with antibiotic in oral bacteria was shown by reduced 4-16-fold. Therefore, the synergistic effects of these active chemicals with other constituents of the essential oil should be taken into consideration for the antimicrobial activity. Determination of rapidity and duration of antibacterial activity can be assessed by time-kill analysis.24 The synergistic interaction was verified by time kill studies using the clove oil or eugenol with antibiotics. 60 min of treatment with MIC of the clove oil or eugenol with ampicillin or gentamicin resulted in an increase in the rate of killing in units of CFU/mL to a greater degree than was observed with alone. The results suggest that combinations of clove oil or eugenol with antibiotics should be investigated further for
possible use in antibacterial products. Particularly, these may be useful in the future for the treatment of cariogenic and periodontopathogenic bacteria.
Acknowledgement We thank Prof. B. S. Kil, Division of Life Science, College of Natural Science, Wonkwang University for the confirmation of plant authenticity. Funding: None. Competing interests: None declared. Ethical approval: Not required.
916
archives of oral biology 56 (2011) 907–916
references
1. Loesche W. Dental caries and periodontitis: contrasting two infections that have medical implications. Infect Dis Clin Nth Am 2007;21:471–502. 2. Ekstrand KR, Bruun G, Bruun M. Plaque and gingival status as indicators for caries progression on approximal surfaces. Caries Res 1998;32:41–5. 3. Feres M, Figueiredo LC, Faveri M, Stewart B, de Vizio W. The effectiveness of a preprocedural mouthrinse containing cetylpyridinium chloride in reducing bacteria in the dental office. J Am Dent Assoc 2010;141:415–22. 4. Jardim JJ, Alves LS, Maltz M. The history and global market of oral home-care products. Braz Oral Res 2009;1:17–22. 5. Stefanovska E, Nakova M, Radojkova-Nikolovska V, Ristoska S. Tooth-brushing intervention programme among children with mental handicap. Bratisl Lek Listy 2010;111:299–302. 6. Van Strydonck DA, Timmerman MG, van der Velden U, van der Weijden GA. Plaque inhibition of two commercially available chlorhexidine mouthrinses. J Clin Periodontol 2005;32:305–9. 7. Bidault P, Chandad F, Grenier D. Risk of bacterial resistance associated with systemic antibiotic therapy in periodontology. J Can Dent Assoc 2007;73:721–5. 8. De Poi R. Chlorhexidine as an anticaries agent. Aust Dent J 2001;46:60. 9. Ivanova EN. The comparative efficacy of local anticaries agents. Stomatologiia 1990;69:60–1. 10. Maltz M, Jardim JJ, Alves LS. Health promotion and dental caries. Braz Oral Res 2010;1:18–25. 11. Claydon NC, Addy M, Adams G, Smith SR, Bosma ML, North M, et al. A comparison of two chlorhexidine gel brushing regimens and a conventional toothpaste brushing regimen for the development of tooth staining over a 6-week period. Int J Dent Hyg 2006;4:183–8. 12. Allaker RP, Douglas CW. Novel anti-microbial therapies for dental plaque-related diseases. Int J Antimicrob Agents 2009;33:8–13. 13. Badria FA, Zidan OA. Natural products for dental caries prevention. J Med Food 2004;7:381–4. 14. Cha JD, Jeong MR, Jeong SI, Moon SE, Kim JY, Kil BS, et al. Chemical composition and antimicrobial activity of the essential oils of Artemisia scoparia and A. capillaris. Planta Med 2005;71:186–90. 15. Palombo EA. Traditional medicinal plant extracts and natural products with activity against oral bacteria: potential application in the prevention and treatment of oral diseases. Evid Based Complement Alternat Med 2009;13, in press [Epub ahead of print]. 16. Park KM, You JS, Lee HY, Baek NI, Hwang JK, Kuwanon G. An antibacterial agent from the root bark of Morus alba against oral pathogens. J Ethnopharmacol 2003;84:181–5. 17. Go´mez-Estaca J, Lo´pez de Lacey A, Lo´pez-Caballero ME, Go´mez-Guille´n MC, Montero P. Biodegradable gelatin– chitosan films incorporated with essential oils as antimicrobial agents for fish preservation. Food Microbiol 2010;27:889–96.
18. Rubel DM, Freeman S, Southwell IA. Tea tree oil allergy: what is the offending agent? Report of three cases of tea tree oil allergy and review of the literature. Australas J Dermatol 1998;39:244–7. 19. Cha JD, Jeong MR, Jeong SI, Moon SE, Kil BS, Yun SI, et al. Chemical composition and antimicrobial activity of the essential oil of Cryptomeria japonica. Phytother Res 2007;21:295–9. 20. Haffajee AD, Roberts C, Murray L, Veiga N, Martin L, Teles RP, et al. Effect of herbal, essential oil, and chlorhexidine mouthrinses on the composition of the subgingival microbiota and clinical periodontal parameters. J Clin Dent 2009;20:211–7. 21. Pinto E, Vale-Silva L, Cavaleiro C, Salgueiro L. Antifungal activity of the clove essential oil from Syzygium aromaticum on Candida, Aspergillus and dermatophyte species. J Med Microbiol 2009;58:1454–62. 22. Chaieb K, Hajlaoui H, Zmantar T, Kahla-Nakbi AB, Rouabhia M, Mahdouani K, et al. The chemical composition and biological activity of clove essential oil, Eugenia caryophyllata (Syzygium aromaticum L. Myrtaceae): a short review. Phytother Res 2007;21:501–6. 23. Jirovetz L, Buchbauer G, Stoilova I, Stoyanova A, Krastanov A, Schmidt E. Chemical composition and antioxidant properties of clove leaf essential oil. J Agric Food Chem 2006;54:6303–7. 24. Devi KP, Nisha SA, Sakthivel R, Pandian SK. Eugenol (an essential oil) acts as an antibacterial agent against Salmonella typhi by disrupting the cellular membrane. J Ethnopharmacol 2010;130:107–15. 25. Yogalakshmi B, Viswanathan P, Anuradha CV. Investigation of antioxidant, anti-inflammatory, and DNA-protective properties of eugenol in thioacetamide-induced liver injury in rats. Toxicology 2010;268:204–12. 26. Hemaiswarya S, Doble M. Synergistic interaction of eugenol with antibiotics against Gram negative bacteria. Phytomedicine 2009;16:997–1005. 27. Gill AO, Holly RA. Disruption of Escherichia coli. Listeria monocytogenes and Lactobacillus sakei cellular membranes by plant oil aromatics. Int J Food Microbiol 2006;108:1–9. 28. Cha JD, Jeong MR, Jeong SI, Lee KY. Antibacterial activity of sophoraflavanone G isolated from the roots of Sophora flavescens. J Microbiol Biotechnol 2007;17:858–64. 29. Vermani A, Navneet. Prabhat. Screening of Quercus infectoria gall extracts as antibacterial agents against dental pathogens. Indian J Dent Res 2009;20: 337–9. 30. Mahboubi M, Bidgoli FG. Antistaphylococcal activity of Zataria multiflora essential oil and its synergy with vancomycin. Phytomedicine 2010;17:548–50. 31. Fine DH, Furgang D, McKiernan M, Tereski-Bischio D, RicciNittel D, Zhang P, et al. An investigation of the effect of an essential oil mouthrinse on induced bacteraemia: a pilot study. J Clin Periodontol 2010;37:840–7. 32. Filoche SK, Soma K, Sissons CH. Antimicrobial effects of essential oils in combination with chlorhexidine digluconate. Oral Microbiol Immunol 2005;20:221–5.