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ORIGINAL RESEARCH Table of Contents   
Year : 2010  |  Volume : 21  |  Issue : 3  |  Page : 357-359
Evaluation of the antimicrobial activity of various concentrations of Tulsi (Ocimum sanctum) extract against Streptococcus mutans: An in vitro study


1 Department of Preventive and Community Dentistry, Bapuji Dental College and Hospital, Davangere - 577 004, Karnataka, India
2 Department of Pharmacognosy, Bapuji Pharmacy College, Davangere, Karnataka, India

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Date of Submission08-Aug-2009
Date of Decision21-Oct-2009
Date of Acceptance21-May-2010
Date of Web Publication29-Sep-2010
 

   Abstract 

Aim: To determine if Tulsi (Ocimum sanctum) extract has an antimicrobial activity against Streptococcus mutans and to determine which concentration of Tulsi (Ocimum sanctum) extract among the 15 concentrations investigated has the maximum antimicrobial activity.
Setting and Design: Experimental design, in vitro study, Lab setting.
Materials and Methods: Ethanolic extract of Tulsi was prepared by the cold extraction method. The extract was then diluted with an inert solvent, dimethyl formamide, to obtain 15 different concentrations (0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7% 8%, 9%, 10%) of the extract. 0.2% chlorhexidine was used as a positive control and dimethyl formamide was used as a negative control. The extract, along with the controls, was then subjected to microbiological investigation to determine which concentration among the 15 different concentrations of the extract gave a wider inhibition zone against Streptococcus mutans. The zones of inhibition were measured in millimeters using a vernier caliper.
Results: At the 4% concentration of Tulsi extract, a zone of inhibition of 22 mm was obtained. This was the widest zone of inhibition observed among all the 15 different concentrations of Tulsi that were investigated.
Conclusion: Tulsi extract demonstrated an antimicrobial property against Streptococcus mutans.

Keywords: Antimicrobial activity, Ocimum sanctum, Streptococcus mutans, Tulsi extract

How to cite this article:
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

How to cite this URL:
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 [serial online] 2010 [cited 2014 Nov 26];21:357-9. Available from: http://www.ijdr.in/text.asp?2010/21/3/357/70800
Dental caries is a disease of complex etiology. Microorganisms play an important role in the etiology of dental caries. There is substantial evidence that suggests that  Streptococcus mutans Scientific Name Search f the main culprit microorganisms responsible for dental caries. [1] Reducing their levels in the oral cavity would provide an additional rationale for the prevention of dental caries. The onus is on the dentist to come up with robust, innovative, effective, feasible and new strategies to manage the disease. One such strategy would be to verify the enormous wealth of medicinal plants. Finding healing powers in plants is an ancient idea. People on all continents have long applied poultices and imbibed infusions of hundreds of indigenous plants dating back to the pre-history eras. Currently, of the one-quarter to one-half of all pharmaceuticals dispensed in the United States having plant origins, very few are intended for use as antimicrobials. [2] Tulsi, scientifically known as Ocimum sanctum, is a time-tested premier medicinal herb. It is a plant of Indian origin, worshipped by the Hindus and used in Ayurvedic medicine since ancient times. It is one of the holiest and most sacred herbs grown widely in India. It is a herb that is bestowed with enormous antimicrobial substances and is used to treat a variety of illnesses ranging from diabetes mellitus, arthritis, bronchitis, skin diseases, etc. [3],[4],[5] Recent studies have also demonstrated significant anticancer properties of Ocimum sanctum. [6] Hence, it is also termed as the queen of herbs or the mother medicine of nature. Literature review reveals that the antimicrobial property of Tulsi has been tested against a variety of microorganisms like Candida albicans, Staphyloccus aureus, enteric pathogens, Klebisella,  Escherichia More Details coli and Proteus. [7] It has also demonstrated antigonorrheal efficacy against multiresistant strains of  Neisseria More Details gonorrhea and clinical isolates of beta lactamase-producing methicillin-resistant Staphylococcus aureus. [8],[9] The impetus for the study was the non-availability of literature about the antimicrobial activity of Tulsi against caries-causing microorganisms like Streptococcus mutans. Chlorhexidine is used as a gold standard against which other antimicrobial agents are compared. It has been studied extensively and is currently the most potent chemotherapeutic agent against Streptococcus mutans and dental caries. [10],[11],[12],[13] Consequently, chlorhexidine is also often used as a positive control for assessing the anticariogenic potential of other agents. [14] Therefore, an attempt is made to compare the antimicrobial activity of Tulsi with 0.2% chlorhexidine against Streptococcus mutans.


   Materials and Methods Top


Preparation of Tulsi extract

Tulsi leaves were obtained from courtyards and were dried in sunlight. The dried leaves were then powdered finely. Three hundred grams of finely powdered Tulsi was then macerated with 100% ethanol. It was then subjected to filtration with Whatman filter paper to obtain a clear filtrate. The filtrate so obtained was reduced at a low temperature of less than 60΀C to obtain a solid residue of Tulsi extract. From 300 g of Tulsi powder dissolved in 1 L of ethanol, 18 g of residue (extract) was obtained and thus the yield was 6% w/w.

Preparation of 15 different concentrations of Tulsi extract

One gram of extract was dissolved in 10 ml of dimethyl formamide to obtain a 10% concentration of extract. One milliliter of the extract was transferred to a sterilized test tube and labeled as 10%. The remaining 9 ml of the extract was then diluted further with dimethyl formamide to obtain 14 different concentrations (0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8%, 9%) of Tulsi extract. A total of 15 concentrations ranging from 0.5 to 10% were prepared. 0.2% chlorhexidine was used as a positive control and dimethyl formamide was used as a negative control.

Microbiological procedures

Pure strains of Streptococcus mutans (ATCC 890) were obtained from microbial type culture collection (MTCC), Chandigarh.

Medium used

Brain heart infusion agar was used as a medium.

The cup and plate method was used to determine the zone of inhibition. In this method, five circular wells that could incorporate five different volumes (10 μl, 20 μl, 30 μl, 50 μl, 75 μl) of the test agent (Tulsi extract) were cut in the agar plates using a template. Seventeen such plates were prepared and labeled, 15 for the 15 different concentrations of Tulsi extract (one plate each for one particular concentration) and two for the positive and negative controls, respectively. The extract, along with the controls, was transferred to the respective agar plates and these incubated aerobically at 37΀C for 48 h. The inhibition zones were measured using a vernier caliper.


   Results Top


No statistical tests were performed as they were not required and the data obtained were appraised observationally. At the 0.5% concentration, a minimum zone of inhibition of 12 mm was achieved at a high volume of 75 μl. At lower volumes, Streptococcus mutans was resistant to the action of Tulsi extract. Increasing the concentration further produced a zone of inhibition at a much lower volume of 30 μl and 50 μl, as shown in [Table 1]. An interesting result was obtained at the 4% concentration. At this particular concentration, even with the slightest volume of 10 μl, a zone of inhibition of 12 mm was achieved, and only at the 4% concentration was an inhibition zone seen with all the five volumes that were used. A maximum zone of inhibition of 22 mm was also achieved at the 4% concentration at a volume of 75 μl. However, a 22 mm inhibition zone was also seen at the 3% concentration at 75 μl. Increasing the concentration further above 4% did not produce any increase in the zone of inhibition. However, for 0.2% chlorhexidine (positive control), a much wider zone of inhibition of 24 mm was achieved at the lowest volume of 10 μl and a maximum inhibition zone of 34 mm was achieved at 75 μl, as shown in [Table 2]. Streptococcus mutans was resistant to the action of dimethyl formamaide (negative control).
Table 1 :Zones of inhibition for Tulsi extract


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Table 2 :Zones of inhibition for controls


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   Discussion Top


The chemical composition of Tulsi is highly complex, containing many nutrients and other biologically active compounds, the proportion of which may vary considerably between strains and even between the plants of the same field. Furthermore, the quantity of many of these constituents is affected by differing growing, harvesting, processing and storage conditions, which are not yet well understood. [15] Eugenol (l-hydroxy-2-methoxy-4-allylbenzene), the active constituent present in Ocimum sanctum, perhaps is largely responsible for the therapeutic potential of Tulsi. [3] The other important constituents include ursolic acid and carvacrol. The antimicrobial activity of Tulsi can be attributed to these constituents. In this study, we have used leaves of black Tulsi (Shyam Tulsi). Ethanol was used as a solvent because the essential oils in Tulsi are more soluble in alcohol when compared to distilled water. Dimethyl formamide, 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 Tulsi. In the present study, chlorhexidine was found to be more effective when compared to Tulsi extract. However, the well-known side-effect of Chlorhexidine, i.e staining of teeth and restoration, alteration of taste sensation and development of resistant microorganisms, may limit the long-term use of chlorhexidine. In comparison with herbal medicines, Tulsi is abundantly available, easily accessible, economically feasible and culturally acceptable and may possess minimal side-effects, hence it can be recommended for long-term use. The results of the current study could not be compared with that of other studies as this is the first study of its kind. Further, the composition of Tulsi extract at 4% might be such that it has a maximum antimicrobial potential, perhaps this needs to be confirmed by further studies.


   Conclusions Top


Tulsi extract demonstrated an antimicrobial activity against Streptococcus mutans. It has the maximum antimicrobial potential at the 4% concentration level.

 
   References Top

1.Balakrishnan M, Simmonds RS, Tagg JR. Dental caries is a preventable infectious disease. Aust Dent J 2000;45:235-45.  Back to cited text no. 1  [PUBMED]  [FULLTEXT]  
2.Cowan MM. Plant product as antimicrobial agents. Clin Microbiol Rev 1999;24:564-82.  Back to cited text no. 2      
3.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. 3  [PUBMED]    
4.Bhat M, Zinjarde SS, Bhargava SY, Kumar AR, Joshi BN. Antidiabetic Indian plants: A good source of potent amylase inhibitors. Evid Based Complement Alternat Med 2008 [In Press].  Back to cited text no. 4      
5.Viyoch J, Pisutthanan N, Faikreua A, Nupangta K, Wangtorpol K, Ngokkuen J. Evaluation of in vitro antimicrobial activity of Thai basil oils and their micro-emulsion formulas against Propionibacterium acnes. Int J Cosmet Sci 2006;28:125-33.  Back to cited text no. 5  [PUBMED]    
6.Magesh V, Lee JC, Ahn KS, Lee HJ, Lee EO, Shim BS, et al. Ocimum sanctum induces apoptosis in A549 lung cancer cells and suppresses the in vivo growth of Lewis lung carcinoma cells. Phytother Res 2009;23:1385-91.  Back to cited text no. 6  [PUBMED]  [FULLTEXT]  
7.Geeta, Vasudevan DM, Kedlaya R, Deepa S, Ballal M. Activity of Ocimum sanctum (the traditional Indian medicinal plant) against enteric pathogens. Indian J Med Sci 2001;55:434-8, 472.  Back to cited text no. 7      
8.Shokeen P, Bala M, Sing M, Tandon V. In vitro activity of eugenol, an active component from Ocimum sanctum, against multiresistant and susceptible strains of Neisseria gonorrhea. Int J Antimicrob Agents 2008;32:172-9.  Back to cited text no. 8      
9.Aqil F, Khan MS, Owais M, Ahmad I. Effect of certain bioactive plant extracts on clinical isolates of beta-lactamase producing methicillin resistant Staphylococcus aureus. J Basic Microbiol 2005;45:106-14.  Back to cited text no. 9  [PUBMED]  [FULLTEXT]  
10.Emilson CG. Outlook for hibitane in dental caries. J Clin Periodontol 1977a;4:136-43.  Back to cited text no. 10  [PUBMED]    
11.Loesche WJ. Antimicrobials can they be effective? In: Guggenheim B, editor. Cariology today. Zurich: Karger; 1984. p. 293-300.   Back to cited text no. 11      
12.Meurmann JH. Chemotherapy of Streptococcus mutans. Proc Finn Dent Soc 1986;82:305-11.  Back to cited text no. 12      
13.Kidd EA. Role of chlorhexidine in the management of dental caries. Int Dent J 1991;41:279-86.  Back to cited text no. 13  [PUBMED]    
14.Emilson CG. Potential efficacy of chlorhexidine against mutans streptococci and human dental caries. J Dent Res 1994;73:682-91.  Back to cited text no. 14  [PUBMED]  [FULLTEXT]  
15.Miller R, Miller S. Tulsi queen of herbs, India′s holy basil. Available from: http://www.nywellnessguide.com/nutrition/070410-TulsiHerbs.php [last accessed on 2007 Jul 26].  Back to cited text no. 15      

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Correspondence Address:
Pooja Agarwal
Department of Preventive and Community Dentistry, Bapuji Dental College and Hospital, Davangere - 577 004, Karnataka
India
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DOI: 10.4103/0970-9290.70800

PMID: 20930344

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    Tables

  [Table 1], [Table 2]

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