Year : 2008 | Volume
: 19 | Issue : 1 | Page : 26--28
In vitro evaluation of antibacterial activity of an herbal dentifrice against Streptococcus mutans and Lactobacillus acidophilus
Yogesh Kumar Vyas1, Maheep Bhatnagar2, Kanika Sharma3,
1 Department of Microbiology, Darshan Dental College and Hospital, Loyara, Udaipur - 313 011, Rajasthan, India
2 Department of Zoology, University College of Science, ML Sukhadia University, Udaipur - 313 011, Rajasthan, India
3 Department of Botany, University College of Science, ML Sukhadia University, Udaipur - 313 011, Rajasthan, India
Department of Botany, University College of Science, ML Sukhadia University, Udaipur - 313 011, Rajasthan
Antibacterial activity of a herbal dentifrice Arodent™ against Streptococcus mutans and Lactobacillus acidophilus was evaluated using Colgate® as standard. Both bacterial strains were isolated from the oral cavity on selective media and identified by standard methods. The antibacterial activity was assayed by cup-well method. The bacterial lawn of facultative anaerobe S. mutans was established between two layers of agar under microaerophilic conditions. Five and a half millimeters and 10 mm zones of inhibition were produced by Arodent against S. mutans and L. acidophilus , respectively, under microaerophilic conditions. On the other hand, the standard dentifrice Colgate produced 5.83 mm and 10.17 mm zones of inhibition against S. mutans and L. acidophilus , respectively, under microaerophilic condition. The results suggest that Arodent is an effective antibacterial herbal dentifrice.
|How to cite this article:|
Vyas YK, Bhatnagar M, Sharma K. In vitro evaluation of antibacterial activity of an herbal dentifrice against Streptococcus mutans and Lactobacillus acidophilus.Indian J Dent Res 2008;19:26-28
|How to cite this URL:|
Vyas YK, Bhatnagar M, Sharma K. In vitro evaluation of antibacterial activity of an herbal dentifrice against Streptococcus mutans and Lactobacillus acidophilus. Indian J Dent Res [serial online] 2008 [cited 2021 Feb 27 ];19:26-28
Available from: https://www.ijdr.in/text.asp?2008/19/1/26/38928
Dental caries is a localized, transmissible infectious process that ends up in the destruction of hard dental tissue. It results from the accumulation of plaque on the surface of teeth and biochemical activities of complex micro-communities. Streptococcus mutans is one of the main opportunist pathogen of dental caries,  which plays a central role in fermenting carbohydrates that results in acid production, leading to the corrosion of tooth enamel. In addition, other microflora like Lactobacillus species are also associated with active caries lesions. Lactobacillus acidophilus is the most common.  Poor oral hygiene is one of the reasons for accumulation of these microbes and their harmful activities. The common method for maintaining good oral hygiene is brushing the teeth with dentifrices that have antimicrobial properties and can prevent the degradation of tooth enamel. Synthetic dentifrices commonly used contain chemical agents, which are known to produce harmful side effects on prolonged use.  Hence, dentifrices that contain extracts of medicinal plants and herbs are becoming popular. One such dentifrice is Arodent™ Gum and Dental paste (IPSA), an herbal formulation that is supposed to possess antiseptic, cariostatic, antibacterial, desensitizing, and gum-tightening properties. It has been claimed to be effective in the prevention of bleeding from gums, pyorrhoea, toothache, as prophylaxis against dental caries, inflamed gums with hemorrhage, and foul smell (as per information given on dentifrice cover). The present study was, therefore, undertaken to evaluate the antibacterial activity of Arodent against cariogenic organisms S. mutans and L. acidophilus .
The main objectives of the study were:
a. To evaluate the antibacterial activity of Arodent against S. mutans and L. acidophilus .
b. To evaluate the effects of Arodent as compared to a standard dentifrice.
Materials and Methods
Streptococcus mutans and L. acidophilus were isolated from excavated carious tooth material and saliva samples (collected from the Department of Conservative Dentistry and Endodontics) on selective media and identified by standard methods.  Pure cultures of S. mutans developed in Todd-Hewitt meat infusion broth and L. acidophilus in tomato juice broth, respectively, at 37° C, 24 h, were taken as inoculum.  The dentifrices Arodent (herbal synthetic) and Colgate® (Calcium and Minerals) (chemical synthetic) were obtained from local market. The composition of these dentifrices is given in [Table 1]. Both dentifrices (2.0 gm) were dissolved in 10 ml sterile distilled water to give 200 mg ml -1 concentration of stock solution, respectively. Half a milliliter of this stock containing 100 mg dentifrice was used for the assay of antibacterial activity. The assay was performed by cup-well method. 
A bacterial lawn of S. mutans was developed by spreading it over the culture medium (surface culture) as well as by agar double layer. , An agar double layer was prepared by first pouring 30 ml of molten Mutans-Sanguis agar (HiMedia) in a petridish and allowing it to solidify. Ten milliliters of molten Mutans-Sanguis agar (45° C) containing 0.1 ml bacterial suspension (0.5 McFarland turbidity standard) was then poured on the first layer and further allowed to solidify. The thickness of both layers was approximately 3 mm and 1 mm, respectively.  S. mutans as well as L. acidophilus lawn was developed on Mutans-Sanguis agar and tomato juice agar, respectively, by spreading 0.1 ml bacterial suspension (0.5 McFarland turbidity standard) with a sterile glass spreader. Sixteen-millimeter-diameter wells were made in the medium with the help of a sterile steel borer and filled with 0.5 ml of the stock solution of dentifrices. S. mutans and L. acidophilus were incubated at 37° C, 24 h, under microaerophilic conditions (5-10% CO 2 in candle jar); anaerobic jar was not used for this purpose. The antibacterial activity was measured as size of zone of inhibition (in millimeter). Three replicates were maintained for each dentifrice, and the experiment was repeated thrice.
A uniform lawn of S. mutans was established in even layer together with a uniform lawn by agar double layer under microaerophilic conditions, and a well-defined zone of inhibition, which could be measured accurately, was observed without anaerobic incubation. The results obtained with respect to antibacterial activity are given in [Table 2]. The development of clear area around the well after 24 h indicates antibacterial activity. The test dentifrice Arodent produced 5.5 mm and 10 mm zones of inhibition against S. mutans and L. acidophilus , respectively, under microaerophilic conditions. On the other hand, the standard dentifrice Colgate produced 5.83 mm and 10.17 mm zones of inhibition against S. mutans and L. acidophilus , respectively, under microaerophilic conditions. The medium plus bacterial culture was taken as positive control.
The main objective of this study was to investigate the effect of Arodent against cariogenic bacteria S. mutans and L. acidophilus .
The Kirby-Bauer method is the method of choice for antimicrobial sensitivity test,  in which the bacterial lawn is prepared by carpet culture. However, a uniform lawn of S. mutans was established by surface culture (spread culture) only under anaerobic conditions (80% N 2 + 10% CO 2 + 10% H 2 ) in an anaerobic jar  but under aerobic conditions. To culture facultative anaerobic S. mutans under microaerophilic/aerobic conditions in the absence of an anaerobic jar, the agar double layer technique is a standard method. Cury et al .  and Gomes et al .  used the pour plate technique to culture S. mutans for antibacterial activity test.
In the present investigation, as in previous reports, the strain of S. mutans was cultured by the agar double layer technique, and Mutans-Sanguis agar, which contains sucrose as 5% concentration, was used as the medium in order to simulate the oral conditions. Dental caries is caused by the combined effects of plaque accumulation and complex microbial communities, especially S. mutans ,  which is the first important colonizer of tooth surface in people who consume large amounts of sugary foods.  Under this culture method, the S. mutans strain could grow as a uniform lawn, although incubation was done under aerobic as well as microaerophilic conditions.
Hence, any antimicrobial agent is considered effective if the size of inhibition zone produced by it measures 2 mm or more.  Since the zones of inhibition produced by Arodent against S. mutans and L. acidophilus measured 5.5 mm and 10 mm, respectively, under microaerophilic conditions, it can be said that this dentifrice possesses good antibacterial activity. The zone of inhibition is comparable with that of a standard dentifrice against both bacteria. The antibacterial activity of Colgate against both bacteria could be due to the presence of fluoride and triclosan,  but studies have shown that prolonged use of fluoride may result in adverse effects such as fluorosis of teeth, skeletal fluorosis, and destruction of epithelial layer of intestine. 
On the other hand, the herbal dentifrice Arodent contains extracts of several plants. The antimicrobial activity of plant extracts is due to the presence of secondary metabolites, such as alkaloids, flavonoids, phenolics, polyphenols, quinines, flavones, flavonols, tannins, terpenoids, lectins, polypeptides, proanthocynidins, tannins, and quercetin.  Plant extracts provide protection by immune stimulation and do not have any known side effects.  Thus, we can say that Arodent is a safe and effective antibacterial dentifrice.
Summary and Conclusions
On the basis of results obtained, it can be said that culturing S. mutans in between two layers of agar is a good method of growing this facultative anaerobe under microaerophilic conditions for antimicrobial assay. The test dentifrice Arodent possesses good antibacterial activity against both cariogenic bacteria S. mutans and L. acidophilus , and its antibacterial activity is comparable to the standard dentifrice Colgate.
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