Antimicrobial effect of herbal dentifrices: An in vitro study.

AIM: This study was taken up to compare the antimicrobial effect of few herbal dentifrices against cariogenic organism such as Streptococcus mutans and Lactobacillus acidophilus.
MATERIALS AND METHODS: This study was an in vitro model using the well method of microbial culture. Colgate total was used as the positive control and distilled water as the negative control. Dentifrices were prepared in 1:1 dilution using sterile distilled water. The standard strains were inoculated and incubated for 4 h. They were then lawn cultured. Wells were made using a standard template, and the dentifrices were placed in these wells Kruskal-Wallis test and Mann-Whitney test were used for statistical analysis.
RESULTS: In case of S. mutans, the maximum antimicrobial effect among the six dentifrices was shown by Babool followed by Colgate Herbal. For L. acidophilus, the antimicrobial zone exhibited by all the six dentifrices were similar to the positive control.
CONCLUSIONS: Babool and Colgate Herbal have more inhibitory effect against S. mutans than the other dentifrices of the group. Dabur Red, Colgate Herbal, and Himalaya are efficient against L. acidophilus.

From time immemorial herbs have been used as an oral hygiene aid in India. India being the birthplace of natural sciences such as Ayurveda, Naturopathy, and Siddha. The uses of these herbs are not new in India. Since ancient times, Ayurveda recommends tender twigs of neem as effective dentifrice, and it has proven credential for the same.[1]

Dentifrices aid in cleaning and polishing tooth surfaces and are available in the form of pastes, gels, and powders.[2] With the changing trends, these herbs have been incorporated into dentifrices, which make them more palatable and easier to use. Many herbal dentifrices claim to have antibacterial action, although there has a dearth of studies to investigate these claims.[3]

Dental plaque is a host associated biofilm, which is made up of a plethora of microorganisms. The strongest correlation between Streptococcus mutans and human caries is for fissure caries, but its relation to other types of caries is also substantial.[2] Lactobacilli are the pioneer organism in the progression of carious lesion deep into the enamel and dentin and are also implicated in root caries.[4] As these dentifrices are becoming increasingly popular, it is the responsibility of the dental practitioners to ensure that they have adequate knowledge about the efficacy of these products. Since there is a dearth of studies related to these herbal dentifrices, this study was taken up to compare the antimicrobial effect of few herbal dentifrices available in the Indian market against S. mutans and Lactobacilli acidophilus.

Materials and Methods

The study was designed as an in vitro model using the well method of microbial culture. We used Colgate Total as the positive control due to its multi benefits effects and because it is one of the toothpaste to get the Food and Drug Administration's clearance for its ability to prevent plaque, gingivitis, and caries.[5] We used sterile distilled water as the negative control. Six dentifrices were chosen which were readily available in the retail market. The microorganisms chosen were S. mutans and L. acidophilus. The standard strains were obtained from Microbial Type Culture Collection (MTCC) Chandigarh, MTCC no. 497 (S.mutans), MTCC no. 227 (L. acidophilus). The standard strains were revived on the following medium. Dentifrices were prepared in 1:1 dilution using sterile distilled water. The standard strains were inoculated into the respective broth and incubated for 4 h. The inoculums were prepared and adjusted to 0.5 McFarland turbidity standard according to National Committee on Clinical Laboratory Standard guidelines The suspension of S. mutans, L. acidophilus standard strains were lawn cultured on Trypticase Soy Agar and Tomato Juice Agar respectively, using sterile cotton swab [Figure 1]. Wells were made using a sterile template [Figure 2]. The following six dentifrices with brand names were Neem, Vicco Vajradanti, Himalaya Herbal, Colgate Herbal, Dabur Red, and Dabur Babool. 100 μl of the 1:1 diluted dentifrices were dropped into each well. The culture plates were then incubated aerobically at 37°C for 48 h. The diameter of zone of inhibition was measured in millimeters for 24 h and 48 h [Figures 3–5]. The test was done in triplicate.

Figure 1

Lawn culture of organisms

Figure 2

Well preparation using sterile template

Figure 3

Antimicrobial zones of inhibition on a Streptococcus culture

Figure 5

Antimicrobial zones of inhibition on a Lactobacillus culture

Antimicrobial zones of inhibition on a Streptococcus mutans culture

Results

The positive control Colgate Total had the maximum inhibition of 38.33 mm and 39.33 mm after 24 h and 48 h, respectively against S. mutans. The negative control water had nil resistance against the same. The herbal dentifrices had the resistance against the same organism after 24 h and 48 h as follows Neem (16.66 mm and 16.66 mm), Vicco Vajradanti (15.6 mm and 15.6 mm), Himalaya Herbal (16.66 mm and 17.33 mm), Colgate Herbal (18 mm and 18 mm), Dabur Red (17.33 m and 18 mm), and Dabur Babool (19 mm and 19 mm). The maximum efficacy was exerted by Babool (19 mm and 19 mm) followed by Colgate Herbal (18 mm and 18 mm).

The efficacy of the same products against L. acidophilus was found to be after 24h and 48 h as follows neem (23 mm and 24 mm), Vicco Vajradanti (22 mm and 22 mm), Himalaya Herbal (24 mm an 24 mm), Colgate Herbal (23 mm and 25 mm), Dabur Red (24 mm and 25 mm), and Dabur Babool (22 mm and 22 mm). The maximum efficacy after 24 h was maximum for Dabur (24 mm) and Himalayas (24 mm) [Graph 1] but after 48 h maximum inhibition was by Dabur and Colgate Herbal (25 mm and 25 mm), respectively [Figure 6]. Kruskal–Wallis test and Mann–Whitney test were used for statistical analysis.

Graph 1

Comparison of the zones of inhibition shows that the zones are maximum for Babool and Colgate Herbal for 24 h and 48 h

Figure 6

Antimicrobial zones of inhibition on a Lactobacillus culture

Discussion

Interest in mouthrinses and toothpastes based on plant extracts has increased recently.[6] These herbal dentifrices in the present days contain varied ingredients from neem, camomile, babul, meswalk. Numerous studies have proven the antimicrobial properties of herbs like neem, whose extracts have shown to alter bacterial adhesion and the ability of streptococci to colonize the tooth surface.[17] In vivo studies have proved that, the use of German chamomile mouthwash twice daily for 4 weeks, could reduce both the plaque accumulation and gingival inflammation.[8] Mathur et al. studied mint leaves and found the highest inhibitory effect was observed against S. mutans.[9] thus providing scientific evidence for the use of mint in herbal dentifrices.

The question as to the claims of antimicrobial properties can only be validated by both in vivo and in vitro studies using these dentifrices. Hence, this study was done to evaluate the in vitro antimicrobial properties of six herbal dentifrices and also to compare their antimicrobial effect with a nonherbal, fluoridated dentifrice against S. mutans and L. acidophilus.

Any herb is considered to have antimicrobial efficacy if the zone of inhibition is 2 mm or more.[10] In our study, the minimum zone diameter was found to be 15.6 mm. The in vitro study on various herbal dentifrices available in California by Lee et al., 2004 have shown that some of the dentifrices have consistent antimicrobial activity, against S. mutans, Streptococcus sanguis, Actinomyces viscosus, and Candida albicans while some showed nil inhibition.[2] In contrast in the present study, all the dentifrices were having an effective zone of inhibition. The results were statistically significant for S. mutans [Graph 2].

Graph 2

Comparison of the zones of inhibition shows that the zones are maximum for Dabur and Himalaya for 24 h and 48 h

Jacob George et al. studied the in vivo efficacy of Colgate Herbal in comparison with a conventional toothpaste in the control of plaque and gingivitis and found no significant difference between the two.[8] Sudha patil et al conducted an in vivo study on Himalaya Herbal dental cream and a fluoridated toothpaste.[9] Both the dentifrice were equally efficacious against S. mutans. In this study, Himalaya Herbal Dental Cream did not show a substantial antimicrobial activity against S. mutans.

Fluoride, the key agent in defending against caries, works through topical mechanisms like inhibition of demineralization of hard tissue, increasing remineralization and inhibition of certain bacterial enzymes.[9] In the current study, fluoride was present in the control dentifrice and two other herbal dentifrice. On the other hand, all the other herbal dentifrices were nonfluoridated. Hence, the antimicrobial activity shown by these dentifrices with herbal ingredients is commendable.

Several potentially active natural molecules against cariogenic streptococci have been identified, including flavan-3-ols, flavonols, flavones, terpenoids, and oligomeric and polymeric proanthocyanidins.[11] Natural products remain a largely unexplored source of effective and nontoxic anti-biofilm molecules.[12]

In the present study, Dabur and Colgate Herbal had maximum resistance against L. acidophilus, which is more than the positive control and is statistically significant (P < 0.05). This statement is in contradiction to Mohankumar et al. 2013 which states that herbal toothpastes are equally efficient like the conventional paste.[13]

When dentifrices are used in the oral cavity, it should be able to penetrate the biofilm matrix.[14] The time for which the tooth is exposed to the dentifrice is limited in the oral cavity. Since the present study is done in vitro the results obtained may not be the same in vivo. Therefore, more similar research works are suggested to be done using various herbal combinations in vitro and in vivo to highlight the goodness of nature and its healing properties.

Conclusion

Dental caries being the most common microbial disease affecting the oral cavity can be prevented by simple oral cleansing methods. The dentifrices with antimicrobial ingredients have more efficacy against the cariogenic organisms. There is an increase in the number of people opting for herbal dentifrices and use of native herbal products could be a more economical and effective option compared to a conventional nonherbal toothpaste. In our study, the herbal dentifrices are more efficient against the lactobacillus organisms at par with the conventional paste and less efficient to the S. mutans when compared to the nonherbal toothpaste. Further research on these dentifrices in vivo could provide an insight into the antimicrobial properties of the commercially available herbal dentifrices.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.