| Abstract|| |
Background: Streptococcus mutans, Candida albicans, and Enterococcus faecalis are the three oral microorganisms most commonly implicated in the causation of oral infections. All these oral microorganisms have shown resistant to routinely used antimicrobials. There is a need for an antimicrobial agent which is effective, safe, and economical. Zingiber officinale, commonly known as ginger is one such plant product which has been used from ancient time. It has been shown to possess promising inhibitory effect on many of the oral microorganisms. On review of dental literature, there was scarcity of studies which had tried to assess antimicrobial potential of ginger extract against S. mutans, E. faecalis, and C. albicans; hence, the present study was designed.
Aim: To evaluate the in vitro antimicrobial potential of 10% ginger extract against S. mutans, E. faecalis, and C. albicans.
Settings and Design: Laboratory setting and experimental design.
Materials and Methods: In the first part of the study, 10% ethanolic ginger extract was prepared in the laboratory of Pharmacy College. It was then subjected to microbiological assay to determine its zone of inhibition using Agar disk diffusion test and minimum inhibitory concentration (MIC) using serial broth dilution method against S. mutans, C. albicans, and E. faecalis.
Results: 10% ethanolic ginger extract showed: (a) Maximum zone of inhibition of 8 mm, 14 mm, and 11 mm against S. mutans, C. albicans, and E. faecalis respectively. (b) MIC of 1.25%, 2.5%, and 2.5% against S. mutans, C. albicans, and E. faecalis respectively.
Conclusion: 10% ethanolic ginger extract was found to possess antimicrobial potential against all the three pathogens used in the study.
Keywords: Antimicrobial potential, 10% ethanolic ginger extract, Zingiber officinale
|How to cite this article:|
Giriraju A, Yunus G Y. Assessment of antimicrobial potential of 10% ginger extract against Streptococcus mutans, Candida albicans, and Enterococcus faecalis: An in vitro study. Indian J Dent Res 2013;24:397-400
Streptococcus mutans (causative organism for dental caries), Candida albicans (causative organisms for candidiasis), and Enterococcus faecalis (causative agent for secondary root canal infection) have been considered very difficult to control as they have developed tolerance against various antimicrobial agents in routine use. This calls for an urgent need to explore novel bio-active compounds, which are safer and biodegradable. Ginger (Zingiber officinale) one such medicinal plant is having antimicrobial property against various human pathogens; however, less data is available on its antimicrobial potential against oral pathogens. Hence present study is an attempt to explore the antimicrobial potential of ginger against S. mutans, C. albicans and E. faecalis.
|How to cite this URL:|
Giriraju A, Yunus G Y. Assessment of antimicrobial potential of 10% ginger extract against Streptococcus mutans, Candida albicans, and Enterococcus faecalis: An in vitro study. Indian J Dent Res [serial online] 2013 [cited 2019 Jul 19];24:397-400. Available from: http://www.ijdr.in/text.asp?2013/24/4/397/118356
| Materials and Methods|| |
Color and appearance: Yellowish brown or light brown. Peeled rhizome with buff external surface showing longitudinal striations and occasional loose fibers, outer surface dark brown and, more or less covered with cork which shows conspicuous, narrow, longitudinal, and transverse ridges.
Tests for extraneous material:
- Odor: Aromatic
- Taste: Pungent
- Foreign matter: <1.0%
- Sand and silica: Absent
- Insect infestation: Nil
- Rodent contamination: Nil.
Alcohol soluble extractive value >6.0% w/w.
- Acid content: <8.0% w/w
- Acid insoluble ash: <1.0% w/w
- Moisture content: <12.0% w/w
- Volatile oil content: 2.0% w/w.
Water soluble extractive value >14.0% w/w.
Successive extractive value:
Identification of crude drug by high performance thin layer chromatography
- Petroleum ether extractive value: 2.2% w/w
- Chloroform extractive value: 3.9% w/w
- Methanol extractive value: 3.3% w/w.
Preparation of ethanolic ginger extract
- Sample: Z. officinale crude drug [gingerol]
- Solvent system: n-hexane:ether = 40:60
- Sample preparation: The powdered sample extracted with chloroform. The extracted concentrated in chloroform.
- Detection: Vanillin sulfuric acid
- Densitometer scan: 254 nm
- Inference: 6 - gingerol - 1.2% w/w by high performance thin layer chromatography.
First 500 g of fresh ginger was procured from the local market and was cleaned using the distilled water. The cleaned ginger was then minced into fine pieces and was suspended in a sterile jar containing 1000 ml of 70% ethanol. The suspended minced ginger in sterile jar was then subjected to process of cold maceration (continuous shaking at constant intervals of time) for 48 h. After 48 h, the suspended minced ginger was subjected to the process of filtration using sterile muslin cloth. During the filtration process, residue of ginger and the filtrate was obtained. The filtrate so obtained was placed over steam bath apparatus for 5 days to facilitate evaporation of ethanol content from the filtrate. After 5 days, dried extract (total yield was 25 g) was obtained, which was pulverized into fine powder using a mortar and pestle.
Preparation of 10% stock solution of ethanolic ginger extract
10 grams of 10% ethanolic ginger extract powder was dissolved in 100 ml of Dimethyl sulphoxide (an inert solvent) to obtain 10% ethanolic ginger extract.
The stock solution of 10% ethanolic ginger extract obtained was subjected to microbiological tests namely disk diffusion test (to determine zone of inhibition) and Serial broth dilution test (to determine minimum inhibitory concentration [MIC]) in order to determine the antimicrobial potential against S. mutans, E. faecalis, and C. albicans.
The standard strains of the organisms used in the study were S. mutans (ATCC 25175), C. albicans (ATCC 2091), and E. faecalis (ATCC 35550). Brain Heart Infusion (BHI) broth, Sabouraud Dextrose broth, sterile MIC tubes, Micropipettes and 10% stock solution of 10% ethanolic ginger extract were other instruments used in the study.
Determination of zone of inhibition using 10% ethanolic ginger extract
Agar disk diffusion method was used to determine zone of inhibition. Various volumes of 10% concentration ginger extract measuring 75 μl, 50 μl, 25 μl, 10 μl, and 5 μl were tested. Interpretation of diffusion results were carried out by noting the presence or absence of zone of inhibition around the wells.
Determination of MIC using ethanolic ginger extract
Procedure: Revival of organisms: The selected bacterial and candida strains were revived by plating on blood agar medium. After overnight incubation at 37°C, isolated colonies were selected and identities of the organisms were confirmed. Isolated colonies were then transferred to sterile BHI broth and Sabouraud dextrose broth for the bacterial and Candida strains respectively and once again incubated overnight. The growth concentration was adjusted to 10 5 organisms/ml by using 0.5 McFarland's turbidity standard. 
An ethanolic solution of 10% concentration was prepared from the ginger powder as the stock solution. A volume of 200 μl of the BHI broth was added into each of the ten MIC tubes per bacterial strain. For the Candida strain, 200 μl of the Sabouraud's broth was added in each of the ten MIC tubes. In the first MIC tube containing 200 μl broths, 200 μl of stock solution was added. After mixing well, 200 μl was transferred to the second MIC tube. This was continued till the last (10 th tube). From the last tube, 200 μl final solutions were discarded. By following this serial dilution, the concentration of ginger powder achieved was 10%, 5%, 2.5%, 1.25%, 0.62%, 0.31%, 0.15%, 0.07%, 0.03%, and 0.01% respectively.
To each of the 10 such prepared MIC tubes with various concentrations, 200 μl of the earlier prepared strains of S. mutans was added such that the final volume per tube was 400 μl. The procedure was repeated for the E. faecalis and C. albicans strains. The tube was then incubated for 24 h at 35°C.
After the incubation, the MIC values were determined by visual inspection of the tubes. In each series of tubes, the last tube with clear supernatant was considered to be without any growth and taken as MIC value. Turbidity in the MIC tube indicated growth of bacterial/Candida strain implying that the organisms were resistant to ethanolic ginger extract.
| Results|| |
[Table 1] shows the zone of inhibition of 10% ethanolic ginger extract against E. faecalis, S. mutans and C. albicans respectively. For E. faecalis, maximum zone of inhibition was 14 mm at 75 μl and minimum zone of inhibition was 10 mm at 25 μl when compared to positive control (4.2% Sodium hypochlorite), which had zone of inhibition of 16 mm. For S. mutans, maximum zone of inhibition was 8 mm at 75 μl and minimum zone of inhibition was 6 mm at 50 μl when compared to positive control (0.2% Chlorhexidine), which had zone of inhibition of 15 mm. For C. albicans, maximum zone of inhibition was 11 mm at 75 μl and minimum zone of inhibition was 9 mm at 50 μl when compared to positive control (Flucanazole), which had zone of inhibition of 14 mm.
|Table 1: Disc diffusion test [zone of inhibition] for 10% ethanolic ginger extract|
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[Table 2] shows MIC of 10% ethanolic ginger extract against E. faecalis, S. mutans and C. albicans respectively. MIC of 10% ethanolic ginger extract for E. faecalis was established at 2.5%, S. mutans at 1.25% and C. albicans at 2.5%.
|Table 2: MIC of 10% ethanolic ginger extract by serial broth dilution method|
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| Discussion|| |
"Spicy to the tongue, yet soothing to the digestive tract" are how herbalist Steven Foster describes the rhizome (or root) that's been prized for more than 4000 years. From its use in breads baked by ancient Greeks to ginger and spicy cuisine, ginger (Z. officinale) is a popular flavoring agent. Members of the Zingiberaceae family are important components in traditional medicine for the treatment of many diseases. Ginger's pungent components offer powerful anti-inflammatory and antioxidant activities, making it useful in arthritis, Alzheimer's, cancer, and cardiovascular disease. The active compound responsible for this effect is zingibain, an enzyme that counteracts inflammation. Ginger also has shown real promise in preventing blood platelets from clumping, helping to fight heart disease and stroke. Research suggests this root may protect nerve cells in the brain, potentially preventing Alzheimer's disease. Focus is currently on its anti-diabetic benefits. Ginger not only increases insulin level; however, it also helps lower blood pressure, cholesterol, and triglyceride levels. It may even help prevent many complications of diabetes. With antimutagenic properties, ginger also inhibits the development of cancer. Recent research points to specific protection against colon and ultraviolet B-related skin cancers. Ginger's phenol compounds help fight Helicobacter pylori, possibly preventing ulcers. Essential oils from this rhizome show antiviral benefits against antibiotic-resistant "bugs." Animal studies suggest its oil may even be effective in preventing liver damage from acetaminophen and other chemical toxins. ,,,,,,,,,
The active compounds contained in ginger are divided into two groups: volatile essential oils and fragrant or harsh phenol compounds.  Among these volatile essential components, which constitute gingerol and shagelol have been accounted for antimicrobial activity of ginger. 
In the present study, 10% ethanolic ginger extract showed antimicrobial activity against S. mutans exhibiting maximum zone of inhibition of 8 mm at 75 μl and MIC at 1.25% concentration. Similar result was obtained in a study conducted by Akihiro et al. where zone of inhibition of 8.2 mm was seen, supporting the findings of our present study. 
In the present study, 10% ethanolic ginger extract showed antimicrobial activity against C. albicans exhibiting maximum zone of inhibition of 11 mm at 75 μl and MIC at 2.5% concentration. Similar result was obtained in the study conducted by Atai et al., where zone of inhibition of 11.2 mm and MIC at 2% concentration was seen, supporting the findings of this present study.  In another study conducted by Joe et al., using 10% ethanolic ginger extract, the zone of inhibition of 8 mm was seen indicating the present study had better results.  The difference observed could be attributed to variations in the quality of ginger used, differences in the microbiological techniques used, variation in temperature and solvent used to prepare ginger extract.
In the present study, 10% ethanolic ginger extract showed antimicrobial activity against E. faecalis exhibiting maximum zone of inhibition of 14 mm at 75 μl and MIC at 2.5% concentration. Similar result was obtained in a study conducted by Rahman et al. where zone of inhibition of 12 mm was seen, supporting the findings of the present study. 
In the present study, positive control was used against all the three organisms in order to compare the antimicrobial efficacy of ethanolic ginger extract. 10% ethanolic ginger extract showed zone of inhibition of 8 mm against S. mutans when compared to positive control (0.2% Chlorhexidine solution) which showed zone of inhibition of 15 mm, indicating it was less potent than the positive control. 10% ethanolic ginger extract showed zone of inhibition of 11 mm against C. albicans when compared to positive control (Flucanazole) which showed zone of inhibition of 14 mm, indicating it was comparative to positive control. 10% ethanolic ginger extract showed zone of inhibition of 14 mm against E. faecalis when compared to positive control (4.2% NaOCl), which showed zone of inhibition of 16 mm, indicating it was comparative to positive control. This study was first of its kind where 10% ethanolic ginger extract along with positive control was used against all 3 microorganisms in order to compare the efficacy of ethanolic ginger extract.
| Conclusion|| |
10% ethanolic ginger extract showed good antimicrobial potential against S. mutans, E. faecalis and C. albicans. However, further studies are recommended at clinical and field setting to assess its practical and economic feasibility and to recommend its use in the clinical setting.
| Acknowledgment|| |
Authors would like to acknowledge:
- Dr. Kishore Bhat, Professor and Head and his faculty members, Department of Microbiology, Maratha Mandal Dental College and Hospital, Belgaum, for their assistance in Microbiological procedures during the study.
- Mr. Narayan Myskin, Professor and Mr. Praveen, Laboratory Technician, Department of Phamacognancy, Bapuji Pharmacy College, Davangere, for their assistance in preparation of extract.
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Department of Public Health Dentistry, Bapuji Dental College, Davangere, Karnataka
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2]