The survey was aimed at knowing the antimicrobial efficacy of the brands of toothpastes that are mostly used. As a result, nine toothpastes in three groups, purchased from different pharmacies in Hyderabad, Andhra Pradesh, India, were selected for assessment of their in vitro antimicrobial activities. This study was conducted in the Department of Microbiology, Sri Sai College of Dental Surgery, Vikarabad, Andhra Pradesh, India. The composition of these dentifrices is given in [Table/Fig-1].

Pure cultures of Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923) and Streptococcus mutans (ATCC 0266P) were cultured in Brain heart infusion broth (Hi-Media, India) at 37°C for 24 h while Candida albicans (laboratory strain) was cultured in Sabouraud’s dextrose broth for 48 hours.

Dentifrices solutions were made by mixing the calculated amount of selected toothpastes (2.0 gm) in measured volume (2 ml) of sterile pyrogen-free distilled water to attain 1:1 dilution; they were further diluted in sterile distilled water and four different dilutions of 1:2, 1:4, 1:8 and 1:16 were made.

The antimicrobial activity of different concentrations of the dentifrices was determined by modified agar well diffusion method [1]. In this method, Mueller hinton agar plates were inoculated with 0.5 mL of 24 h broth cultures of each isolate (Wilkins chalgren agar was used for Streptococcus mutans strain and Sabouraud’s dextrose agar was for yeast). After the plates were dry, five wells at equidistance were punched in each of the plates using sterile corkborer (8 mm). Then each well was filled with 0.2 mL of the dentifrice dilutions. The same amount of control was also introduced into the wells meant for it. The plates were incubated at 37°C for 24 h (48 h for Candida albicans). The antimicrobial activity was interpreted as the diameter of zones of inhibition (in mm). All the plates were made in triplicates.

Statistical Analysis was performed using Minitab Software by regression method. Level of statistical significance was assumed at p < 0.05.

Triclosan- containing Toothpastes: The results of this investigation showed that the zones of inhibition were statistically significant (p<0.05) for triclosan-formulation I against all the four test organisms [Table/Fig-2,3,4,5, and 6]. Formulation II did not possess significant antibacterial activity against Escherichia coli. Against Streptococcus mutans, toothpaste formulations III failed to demonstrate any significant activity.

[Table/Fig-2]:

Zone of inhibition produced by different toothpaste formulations at 24hrs against Escherichia coli at five different dilutions; zone of inhibitions by A) triclosan based toothpastes, B) herbal toothpastes, C) homeo toothpastes, D: by control)

Herbal Dentifrices: Ayurvedic products such as formulations IV, V and VI were found to have antimicrobial activities, although these were not statistically significant except for that of IV. However, against Streptococcus mutans, all three herbal products showed significant antimicrobial activity.

Homeo Dentifrices: Formulations VII, VIII and IX are homeo- based products and exhibited least effectiveness compared to the other test formulations. Among all the investigated homeo toothpastes, formulation VIII emerged as the significantly effective, against the tested microorganism, Streptococcus mutans. No zone of inhibition was observed for the control.

The biofilms by the oral microflora being the centre of caries and periodontal disease, it is of utmost importance to control these biofilms by mechanical debridement and use of adjunctive antimicrobials in toothpastes in prevention of plaque-mediated diseases [10]. Several clinical studies have demonstrated the inhibitory effects of antimicrobial dentifrice on oral bacteria and gingival [11]. Concerns regarding the increase in antibiotic resistance in microorganisms against triclosan [12–14] has promoted interest in the therapeutic use of non-conventional or alternative dentifrices and thus this study.

Among all the investigated triclosan-toothpastes, formulations I emerged as the most effective, based on the significant zone of microbial inhibition produced by it in agar well diffusion method, against all the four tested microorganisms. This might be due to the presence of triclosan and fluoride in its formulation. The results of this study were consistent with the previous reports by Yigit et al., and Deng et al., that fluoride combination is more effective than that of sodium monofluorophosphate [15,16]. Triclosan (5-Chloro-2(2, 4-dichlorophenoxy) phenol), a halogenated phenol has been used for more than 30 years as a non-ionic broad spectrum antimicrobial [1]. It is an active component in detergents, clothing, toothpastes and mouthwashes [17]. Many studies using triclosan as an anti-plaque agent were carried out [18] and have given good results. Study carried out by Jenkins et al., reported a significant reduction in total microbial count in saliva by 0.2% triclosan [19]. Systematic reviews by Davies et al., and Gunsolley et al., have concluded that formulations containing triclosan and copolymer significantly improve plaque control and periodontal health [20,21]. In another invitro study by Manupati Prasanth, triclosan showed a significant reduction in Candida albicans and Streptococcus mutans counts with antimicrobial activity even at higher dilution of 1:16 [1].

Next to triclosan, are herbal products. “Ayurveda” system has been used successfully for treating various systemic ailments in Indian medicine [2]. Using natural medicines to cure various diseases has become an increasing trend [1]. In recent years, a number of toothpaste preparations containing herbal ingredients have made significant contribution to dental prophylaxis in improving oral health. The popularity of herbs is due the antimicrobial and anti-inflammatory effects of their ingredients known as Phytochemicals [5].

In the present study, herbal formulations such as formulations IV, V and VI were found to have antimicrobial activities, although these were statistically significant only for formulation IV. This may be due to the synergistic interactions between the ingredients present in their formulations, which, however, need to be established [1]. The principle components of this toothpaste include Neem, Pudina, Long, Babool, Turmeric and Vajradanti. The presence of secondary metabolites such as alkaloids, flavonoids, polyphenols, and lectins in these ingredients are considered to be the sole reason of their antimicrobial efficacy [22]. Some of these ingredients were previously demonstrated and known to have antimicrobial activity [2,5]. Against Streptococcus mutans, all three herbal formulations showed significant antimicrobial activity (p<0.05). Many studies on anti-plaque activity of herbal base toothpaste have been reported [23,24]. A systematic review by Moran et al., concluded that herbal toothpastes had shown less anti-plaque activity than conventional pastes [25]. Our data are in consistent with a recent report by Manupati Prasanth which found herbal based products to be less effective compared to triclosan formulations [1].

With respect to homeo-based toothpastes, only formulation VIII has shown significant activity only against Streptococcus mutans. This may be due to the presence of kreosotum, Plantago major and calendula as major ingredients in its formulation. This observation contradicts the earlier information claiming kreosotum and plantago as efficient anticandidal ingredients but coincides the earlier experiments done by Stanisavljevic et al., Sharifa et al., and Kemal et al., Hoeletz, Kahyaolu et al., that demonstrated Plantago major extract to possess light antimicrobial activity against various bacteria including Escherichia coli [7–9,26,27].

This study have shown that antimicrobial activity of the toothpaste containing both triclosan and fluoride (1000ppm) as key ingredients showed a significant difference (p< 0.05) against all four tested microflora compared to that of with lower fluoride-concentration or sodium monofluorophosphate. Also, both the herbal toothpaste (having several Phytochemicals) and triclosan-fluoride (1000ppm) formulation have comparable antimicrobial properties. Therefore, this herbal dentifrice can be used as alternative to triclosan-based formulations. But, the results of this study might not be clinically applicable. Firstly, the agar diffusion test solely depends on the diffusing ability of the testing agents through the agar matrix. Secondly, formulations for topical dentifrices might not be equally active within limited exposure times under actual conditions in vivo. Nevertheless, invitro method is commonly used in screening the antimicrobial agents before invivo testing. Thus, dental professionals may recommend a dentifrice based on patients clinical conditions and possible susceptibilities.