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 Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 1  |  Issue : 1  |  Page : 20-23

An In-vitro Evaluation of the Effect of Anti-candidal Herb (Olive) on Streptococcus Mutans


1 Department of Community Dentistry, Government Dental College and Hospital, Aurangabad, Maharashtra, India
2 Department of Public Health Dentistry, K.M. Shah Dental College and Hospital, Vadodara, India
3 Department of Public Health Dentistry, Goenka Research Institute of Dental Sciences, Gandhinagar, Gujarat, India

Date of Web Publication17-Jun-2015

Correspondence Address:
Harshal Prakash Bafna
Department of Community Dentistry, Government Dental College and Hospital, Aurangabad, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2393-8692.158905

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  Abstract 

Aim: The aim was to evaluate the antimicrobial effect of olive extracts on Streptococcus mutans in in-vitro conditions. Materials and Methods: An in-vitro experimental study was conducted in a laboratory setting. Ethanolic extract of olive was prepared separately by cold maceration technique. The extract was then diluted with an inert solvent, dimethylformamide, to obtain five different concentrations (2%, 4%, 6%, 8%, and 10%) of each. 0.2% chlorhexidine was used as a positive control and dimethylformamide was used as a negative control. The different extracts, along with controls, were then subjected to microbiological investigation to determine, which gave a wider zone of inhibition against S. mutans. The zone of inhibition was measured in millimeters. Results: Olive extract presented the largest zone of inhibition of 33 mm at the concentration of 8%. Conclusion: Ethanolic extract of olive demonstrated antimicrobial activity against S. mutans.

Keywords: Antimicrobial, olive, Streptococcus mutans


How to cite this article:
Bafna HP, Ajith Krishnan C G, Kalantharakath T, Kalyan P, Arhi RP. An In-vitro Evaluation of the Effect of Anti-candidal Herb (Olive) on Streptococcus Mutans . Indian J Oral Health Res 2015;1:20-3

How to cite this URL:
Bafna HP, Ajith Krishnan C G, Kalantharakath T, Kalyan P, Arhi RP. An In-vitro Evaluation of the Effect of Anti-candidal Herb (Olive) on Streptococcus Mutans . Indian J Oral Health Res [serial online] 2015 [cited 2019 Jul 21];1:20-3. Available from: http://www.ijohr.org/text.asp?2015/1/1/20/158905


  Introduction Top


The practitioners of the traditional system of medicine treat about 80% of patients in India, 85% in Burma and 90% in Bangladesh. [1] Medicinal plants rich in secondary metabolites (potential sources of drugs) and essential oils are of the important advantage claimed for their therapeutic uses in various ailments besides being safe, economical, effective and easily available. [1]

Moreover in today's world consumer and producer alike have become highly conscious about the health benefits of food leading to value added products in health sector and discovery of "functional food" that encompasses all edible items having a health-promoting and/or disease-preventing property beyond the primary function of providing nutrients. Some of these have already been used in successful management of both general and oral disease conditions such as bronchitis, bronchial asthma, skin diseases, oral thrush, oral cancer, periodontal diseases, etc.

Several plants such as olive, ginger, tulsi, garlic, and turmeric have been used as neutraceuticals in treatment of oral candidiasis by local and systemic routes. [2],[3],[4],[5],[6] When used locally or systemically they also affect the oral bacterial flora, some having proved their action against bacteria like Streptococcus mutans, the main causative organism for dental caries.

Lack of sufficient data on effect of olive needs to be accounted against S. mutans owing to their day to day use. The aim of present study was to comparatively evaluate the antimicrobial effect of olive extract on S. mutans in in-vitro conditions.


  Materials and Methods Top


Prestudy procedure

Preparation of olive extract

Fifty grams of sun-dried finely powdered olive fruits were macerated with 150 ml of 100% ethanol and then subjected to filtration with Whatman filter paper to obtain a clear filtrate. The filtrate so obtained was reduced in a borosilicate glass beaker at a low temperature of <40°C with the help of a soxhlet extraction unit (heating mantle) Macro Scientific Works (MSW) - 436 of MAC MSW Limited., to obtain semi solid residue of olive extract. From 50 g of powder dissolved in 150 ml of ethanol, 2 g of residue extract was obtained, so the yield was 2% w/w.

Preparation of different concentrations of olive extract

Two grams of extract was dissolved in 20 ml of dimethylformamide to obtain 10% concentration of the extract, which was used as a stock solution. Subsequent serial dilution of the stock solution with dimethylformamide was done to obtain 2%, 4%, 6%, 8% and 10% concentration of stock solution of olive extract.

Furthermore, 1% of the extract of the stock solution was used as a starting point, followed by other concentrations as mentioned above for minimum inhibitory concentration (MIC) determination.

Controls

Control of 0.2% chlorhexidine was used as a positive control, a gold standard for comparison and dimethylformamide was used a negative control to rule out its effect on S. mutans.

Collection of micro-organisms

Microbial type culture collection (MTCC) strain number 497 was obtained from MTCC and Gene Bank, Chandigarh. The strain belonged to genus Streptococcus while the species was mutans that is, S. mutans, was used for the study purpose.

Preparation of culture media

The brain heart infusion agar powder (special infusion agar) for in-vitro diagnostics, M211, was obtained from HiMedia Laboratories Limited, Mumbai, Maharashtra, India. Fifty-two grams of this powder was suspended in 1000 ml of distilled water. It was then boiled to dissolve the medium completely and then sterilized by autoclaving at 15 lbs pressure and 121°C for 15 min. The pH of the agar was maintained at 7.4 at 25°C. The media was then mixed well and poured into petri-dishes. The process of making culture media was carried out as per the instructions provided by the manufacturer.

Streptococcus mutans MTCC was then added to nutrient broth which was incubated at 37°C for 24 h. It was sub-cultured onto nutrient agar plate and incubated at 37°C for 24 h. The inoculum for antimicrobial activity was prepared by adjusting the density of organism to approximately 10 8 colony forming units/ml with the help of 0.5 Mcfurland opacity standards. Then it was inoculated on agar plate by lawn culture method. The growth conditions were aerobic as specified by Gene bank, Chandigarh.

Determining microbic sensitivity

Determination of microbic sensitivity mainly can be done by two methods, that is, dilution methods and diffusion methods. Ditch plate diffusion method was used in the present study as it has been proven to be more suitable for research purpose. [7] In this method, ditches were made in petri-dishes by using a punch. These ditches were filled with the equal amount of olive extract. Six plates were used for six different concentrations. Chlorhexidine and dimethylformamide were used as controls. Plates were then incubated at 37°C for 48 h, after which zone of inhibition were measured.

Minimum inhibitory concentration was determined by broth dilution method and values were determined by visual inspection of tubes. In the series of tubes, the last tube with a clear supernatant was considered to be without any growth and taken as MIC value.

Study procedure

Antimicrobial susceptibility testing

The ditch plate method was used to test the antimicrobial activity. Ditches were prepared on agar plates with the help of the punch having 6 mm diameter and 50 μl of each concentration were introduced in each.

The plates were left for 1 h at room temp and then incubated at 37°C for 48 h and later examined for zone of inhibition.

The zone of inhibition was measured with the help of Hi Antibiotic Zone scale from HiMedia Laboratories Limited, Mumbai, Maharashtra, India, which is certified by Indian Standard Organisation and World Health Organisation Goods Manufacturing Practices. [8] The scale used was of high quality, standardized, efficient and easy to use with high reproducibility of observations.

Statistical analysis

Statistical analysis was performed using SPSS 15.0 (SPSS Inc) windows evaluation trail version release 15.0.0, September 6, 2006, USA. The effect of olive was compared with chlorhexidine using unpaired Student's t-test.


  Results Top


The present study was conducted to assess the efficacy of ethanolic extracts of olive on S. mutans. The zones of inhibition were measured with the help of HiAntibiotic Zone scale from HiMedia Laboratories Limited, Mumbai, Maharashtra, India. These zones were measured at the end of 48 h.

[Table 1] shows the zone of inhibition of various concentrations of ethanolic extracts of olive.
Table 1: Zones of inhibition of different extract olive

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The zone of inhibition did not form for 1% concentration while it was observed at 2% and above of the stock solution indicating that the MIC of olive was 2% concentration.

[Table 2] shows zone of inhibition of the positive and negative controls.
Table 2: Zones of inhibition of controls

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[Table 3] shows results for one-sample Kolmogorov-Smirnov test for normality. The results showed that data were normally distributed in all the two groups, that is, chlorhexidine and olive.
Table 3: One-sample Kolmogorov - Smirnov test for normality of data

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[Table 4] shows results for unpaired t-test. The test was conducted to compare the effect of olive extract with chlorhexidine. There was a nonsignificant difference between the means of zones of inhibition of the two.
Table 4: Comparison of effect of olive and chlorhexidine on Streptococcus mutans using unpaired t-test


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  Discussion Top


A current review of the available epidemiological data from many countries clearly indicates that there is a marked increase in the prevalence of dental caries. [9] This increase in dental caries signals a pending public health crisis. Although there are differences of opinion regarding the cause of this global dental caries increase, the remedy could be shifting to functional food.

Functional foods have been introduced into the corporate mainstream owing to continuously increasing health care costs, an expanding aging population arising out of increased life expectancy, rising consumer awareness about health aspects of foods and food regulations and above all an increased level of education and literacy. [10]

Olive (Olea europaea) has been commonly used as anti candidal herb [2],[3],[6] and also have been suggested to have antibacterial action. The polyphenols, oleanolic acid and secoiridoid glycosides contained in olive leaves and fruits have a broad antimicrobial activity. The flavonoid polyphenols in olive oil are natural antioxidants that contribute to a bitter taste, astringency, and resistance to oxidation and host beneficial effects from healing sunburn to lowering cholesterol, blood pressure, and risk of coronary disease. [11],[12] Owing to these properties the aqueous extract of olive have already shown antimicrobial activity against pathogens like Staphylococcus aureus ATCC 43300, and S. aureus MU 40,  Escherichia More Details coli and Klebsiella pneumoniae CCM 2318. [13] In the present study, different concentrations of ethanolic olive extract were used of which, 8% ethanolic olive extract showed highest antimicrobial activity against S. mutans with maximum zone of inhibition of 33 mm at 50 μl. At higher concentrations the zone of inhibition did not increase further.

In the present study, ethanol was used as a solvent because the essential oils in olive are more soluble in alcohol when compared with distilled water. Dimethylformamide, an inert solvent, was used to dilute the extract to neutralize the effect of alcohol, which itself is an antiseptic, attributing the result solely to olive, respectively. [14]

The volume of extract to be used for the study was determined randomly since this study was the first of its kind where the in-vitro effect of ethanolic extract of olive was evaluated against S. mutans, furthermore no attempt had been done to recommend precise amount of drug to be used for antimicrobic sensitivity tests [7] and also the objective of studying the efficacy of olive extract was achieved with a single volume.

 
  References Top

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Prakash P, Gupta N. Therapeutic uses of Ocimum sanctum Linn (Tulsi) with a note on eugenol and its pharmacological actions: A short review. Indian J Physiol Pharmacol 2005;49:125-31.  Back to cited text no. 1
    
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Candida - A Blessing in Disguise. Available from: . [Last accessed on 2012 Mar 12].  Back to cited text no. 2
    
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Atai Z, Atapour M, Mohseni M. Inhibitory effect of Ginger extract on Candida albicans. Am J Appl Sci 2009;6:1067-9.  Back to cited text no. 3
    
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Yob NJ, Jofrry SM, Affandi MM, Teh LK, Salleh MZ, Zakaria ZA. Zingiber zerumbet (L.) Smith: A review of its ethnomedicinal, chemical, and pharmacological uses. Evid based complement Alternat Med 2011;2011:543216.  Back to cited text no. 4
    
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Esimone CO, Okoye FB, Odimegwu DC, Nworu CS, Oleghe PO, Ejogha PW. In vitro antimicrobial evaluation of lozenges containing extract of garlic and ginger. Int J Health Res 2010;3:105-10.  Back to cited text no. 5
    
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Martins CV, da Silva DL, Neres AT, Magalhães TF, Watanabe GA, Modolo LV, et al. Curcumin as a promising antifungal of clinical interest. J Antimicrob Chemother 2009;63:337-9.  Back to cited text no. 6
    
7.
Branch A, Chabbert Y, Ericsson H, Garrod L, Gause G, Walter A, et al. Standardization of methods for conducting microbic sensitivity tests - Second Report of Expert Committee on Antibiotics. World Health Organ Tech Rep Ser 1961;210:10-16.  Back to cited text no. 7
    
8.
HiMedia Laboratories Pvt. Limited (India). Available from: . [Last accessed on 2013 Feb 13].  Back to cited text no. 8
    
9.
Bagramian RA, Garcia-Godoy F, Volpe AR. The global increase in dental caries. A pending public health crisis. Am J Dent 2009;22:3-8.  Back to cited text no. 9
    
10.
Kubo A, Lunde CS, Kubo I. Antimicrobial activity of the olive oil flavor compounds. J Agric Food Chem 1995;43:1629-33.  Back to cited text no. 10
    
11.
Chemical Composition of Olive Oil. Available from: . [Last accessed on 2013 Feb 12].  Back to cited text no. 11
    
12.
Upadhyay RK. Screening of antibacterial activity of six plant essential oils against pathogenic bacterial strains. Asian J Med Sci 2010;2:152-8.  Back to cited text no. 12
    
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Agarwal P, Nagesh L, Murlikrishnan. Evaluation of the antimicrobial activity of various concentrations of Tulsi (Ocimum sanctum) extract against Streptococcus mutans: An in vitro study. Indian J Dent Res 2010;21:357-9.  Back to cited text no. 13
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14.
Agarwal P, Nagesh L, Murlikrishnan. Evaluation of the antimicrobial activity of various concentrations of Tulsi (Ocimum sanctum) extract against Streptococcus mutans: An in vitro study. Indian J Dent Res 2010;21:357-9.  Back to cited text no. 14
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  [Table 1], [Table 2], [Table 3], [Table 4]



 

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