The Efficacy of Commercially Available Herbal Dentifrices in Comparison with Conventional Dentifrices against Two Common Oral Microbes: An In vitro Study.

BACKGROUND: Mechanical plaque removal is considered the gold standard for controlling and prevention of plaque and gingivitis. The present study was conducted to evaluate the efficacy of commercially available herbal dentifrices in comparison with conventional dentifrices against two of the common oral microbes, namely Streptococcus mutans and Candida albicans.
MATERIALS AND METHODS: To determine the antimicrobial properties of individual dentifrices, the zone of inhibition on the blood agar plates was measured and also disc diffusion method was used for testing the antibacterial properties for which a thin paper disc containing the antimicrobial agent was applied on a culture of bacteria grown on agar media. Each dentifrice was tested in 50% concentration (1:1 dilution [50% w/w] using sterile deionized distilled water/half strength) and 100% concentration (full strength). The blood agar plates were then incubated aerobically at 37°C. The measurement of zone of inhibition was done after 24-48 and 72 h on the underside of the Petri dishes using Vernier calipers.
RESULTS: At 50% concentration, Regimen B2 showed the maximum zone of inhibition which was closely followed by Regimen A1 for S. mutans, whereas at 100% concentration, Regimen A1 showed better effects. For C. albicans, it was observed that Dabur red toothpaste showed the significantly higher zone of inhibition at 50% concentration when the concentration was made 100% and also Dabur red toothpaste showed the maximum zone of inhibition.
CONCLUSION: This concluded that the herbal dentifrices are equally and sometimes better than the conventional ones. Copyright:

INTRODUCTION

Mechanical plaque removal is considered the gold standard for controlling and prevention of plaque and gingivitis. Toothbrushing is usually underestimated and also commonly not performed in the right way, hence to improve the efficacy of mechanical tooth cleaning procedures; antimicrobial agents are added to dentifrices.[1] Few examples of these agents are chlorhexidine, triclosan, essential oils, and fluorides, but we can see more inclination of consumers toward herbal products rather than the chemical ones. Dentifrices labeled as “herbal” are formulated from natural derivatives. For example, in herbal toothpaste, the fluoride is derived from fluorspar, instead of synthesized abrasive, the abrasive system is ground calcium carbonate (chalk), the carrageenan is used as a thickener which is derived from seaweed, sweetener xylitol, and an extract from birch tree is used instead of saccharin. Antimicrobial properties are claimed by many herbal dentifrices. These claims are usually based on the result of experiments on a single ingredient showing antimicrobial properties in a Petri dish, but the efficacy of the final dentifrices depends on factors such as degradation of the active ingredient during manufacture. There is a lack of research which proves the efficacy of dentifrices containing a combination of herbal components.[123]

Hence, we undertook this in vitro study to evaluate the efficacy of commercially available herbal dentifrices in comparison with conventional dentifrices against two of the common oral microbes, namely Streptococcus mutans and Candida albicans.

The aim of this study was to evaluate the antimicrobial efficacy of four conventional and herbal dentifrices against two of the common oral microorganisms and to compare the antimicrobial efficacy of herbal dentifrices with conventional dentifrices.

MATERIALS AND METHODS

The present in vitro study was conducted to evaluate the efficacy of commercially available herbal and conventional dentifrices against two of the common oral microorganisms.

Dentifrices tested were Himalaya herbals dental cream (Regimen A1) and Dabur red toothpaste (Regimen A2) under herbal category, and Colgate super shakti dental cream (Regimen B1) and Pepsodent complete germicheck toothpaste (Regimen B2) were the conventional ones included in the study. Microorganisms taken into account were S. mutans and C. albicans. was done from Microbial Type Culture Collection (MTCC) and Gene Bank, Institute of Microbial Technology, Chandigarh [India]. The ampules of freeze-dried form of the microbes were kept in the refrigerator at 4°C.

Recovering feasible growth from freeze-dried form of microbes

Preparation of nutrient broth was done as per manufacturers' instructions. To the prepared nutrient broth, freeze dried form of microbes were added, under strict sterile conditions, these test tubes were then incubated at 37°C for 24 h. The concentration of dentifrices tested were 50% and 100%. For 50% concentration sterile deionized distilled water/half strength was used. Growth of microbes was confirmed by the turbidity in the test tube. This turbidity was compared with McFarland 0.5 turbidity standard.[3] Microbial species confirmation was done by inoculating blood agar plates with the test organisms, and these were incubated aerobically and anaerobically at 37°C for 24 h. After 24 h, the particular species was confirmed by the types of hemolysis and colony morphology. Blood agar plates with 5% defibrinated sheep blood were used in the study as a growth medium for microorganisms.

To determine the antimicrobial properties of individual dentifrices, the zone of inhibition on the blood agar plates were measured and also disc diffusion method was used for testing the antibacterial properties for which a thin paper disc containing the antimicrobial agent was applied on a culture of bacteria grown on agar media.

Each dentifrice was tested in 50% concentration (1:1 dilution [50% w/w] using sterile deionized distilled water/half strength) and 100% concentration (full strength). The blood agar plates were then incubated aerobically at 37°C.

Measurement of zone of inhibition

The measurement of zone of inhibition was done after 24, 48, and 72 h on the underside of the Petri dishes using Vernier calipers. The area where visible growth had been inhibited if that is obvious is defined as the zone of inhibition, but if it was not that clearly defined, then the measurement was made to the point of 80% inhibition of growth.[34]

The diameter of filter disc was deducted from the whole of the diameter of the zone of inhibition giving the actual zone of inhibition around the filter disc.[4]

Statistical analysis

For statistical analysis, mean and standard deviation were calculated. One-way analysis of variance test was used for multiple group comparisons, followed by Tukey's post hoc for group-wise comparisons. P < 0.05 was considered statistically significant.

RESULTS

We evaluated the antimicrobial efficacy by disc diffusion method on blood agar plates, inoculated with two species of microorganisms consisting of S. mutans and C. albicans.

The zone of inhibition was observed at 24, 48, and 72 h and was measured using Vernier calipers. The result showed the following observations.

Tables 1 and 2 reveal the effect of dentifrices in 50% concentration and 100% concentration and control on S. mutans at 24, 48, and 72 h.

Table 1

Effect of dentifrices in 50% concentration and control on Streptococcus mutans at 24, 48, and 72 h

Dentifrices	Zone of inhibition (mm), mean±SD
	24 h	48 h	72 h
Regimen A1	2.36±0.11	2.55±0.11	2.57±0.11
Regimen A2	1.91±0.05	1.98±0.05	2.14±0.11
Regimen B1	3.74±0.13	3.78±0.16	3.78±0.16
Regimen B2	2.58±0.11	2.62±0.11	2.98±0.11
P	0.0000	0.0000	0.0000

SD: Standard deviation

Table 2

Effect of dentifrices in 100% concentration and control on Streptococcus mutants at 24, 48, and 72 h

Dentifrices	Zone of inhibition (mm), mean±SD
	24 h	48 h	72 h
Regimen A1	4.92±0.55	5.11±0.55	5.12±0.55
Regimen A2	2.77±0.05	3.28±0.09	3.34±0.13
Regimen B1	4.32±0.27	4.41±0.22	4.44±0.22
Regimen B2	4.48±0.05	4.52±0.07	4.52±0.07
P	0.000	0.000	0.000

SD: Standard deviation

The herbal dentifrice group Regimen A1 was compared with Dabur red tooth paste. For the preparation having 50% concentration it was observed that the maximum zone of inhibition was with Regimen A1 at 24, 48, and 72 hours.

In the non-Herbal group Regimen B1 and Pepsodent complete germicheck toothpaste were compared. At 50% concentration and after 24, 48, and 72 h the maximum zone of inhibition was observed with Colgate super shakti dental cream.

It was found that Regimen B1 was most effective among the other tested and it was highly statistically significant with P = 0.000.

Table 2 shows the effects of dentifrices in 100% concentration on Streptococcus mutants at 24, 48, and 72 h. It was observed that among the herbal tooth dentifrices tested, the maximum zone of inhibition was observed with Regimen A1, and with those nonherbal, it was observed with Pepsodent complete germicheck tooth. When all the four dentifrices were compared, Regimen A1 was found to have the maximum zone of inhibition. It was found that Regimen A1 was the most effective among the other tested and it was highly statistically significant with P = 0.000. It was also observed that the mean and standard deviation values of 24, 48, and 72 h in all the four dentifrices when compared with that of Tukey's post hoc were ≥0.05, and hence they were not statistically significant.

Table 3 shows the effect of dentifrices in 50% concentration and control on C. albicans at 24, 48, and 72 h. It was observed that among the herbal ones, Dabur red toothpaste was most effective in this concentration, and when nonherbal dentifrices were compared, Regimen B1 was found to be slightly better. When all the four dentifrices were compared, it was found that Dabur red toothpaste showed the maximum zone of inhibition.

Table 3

Effect of dentifrices in 50% concentration and control on Candida albicans at 24, 48, and 72 h

Dentifrices	Zone of inhibition (mm), mean±SD
	24 h	48 h	72 h
Regimen A1	3.30±0.27	3.84±0.22	3.86±0.24
Regimen A2	6.90±0.27	7.42±0.11	7.44±0.09
Regimen B1	3.28±0.38	4.94±0.88	4.98±0.84
Regimen B2	3.14±0.22	4.60±0.82	4.70±0.76
P	0.000	0.000	0.000

SD: Standard deviation

It was found that Dabur red toothpaste was the most effective among the other tested and it was highly statistically significant with P = 0.000. It was also observed that the mean and standard deviation values of 24, 48, and 72 h in all the four dentifrices when compared with that of Tukey's post hoc were ≥0.05, and hence they were not statistically significant.

Table 4 shows the effect of dentifrices in 100% concentration and control on C. albicans at 24, 48, and 72 h. When we compared the herbal toothpastes, we found that Dabur red toothpaste was most effective in this concentration for C. albicans. When nonherbal dentifrices were compared, Pepsodent complete germicheck tooth showed the maximum zone of inhibition. When all the four dentifrices were compared, it was found that Dabur red toothpaste showed the maximum zone of inhibition among all the four dentifrices tested.

Table 4

Effect of dentifrices in 100% concentration and control on Candida albicans at 24, 48, and 72 h

Dentifrices	Zone of inhibition (mm), mean±SD
	24 h	48 h	72 h
Regimen A1	4.54±0.07	4.44±0.18	4.56±0.09
Regimen A2	9.16±0.60	10.48±0.88	10.54±0.87
Regimen B1	4.12±0.5	665.54±0.22	56±0.24
Regimen B2	7.46±1.86	7.94±1.75	8.19±1.48
P	0.000	0.000	0.000

SD: Standard deviation

DISCUSSION

Individuals' overall health has a strong correlation with the oral health, as the oral cavity serves as the entry point for nutritional, diet, and respiratory elements, and it is also more prone to numerous microbial populations,[4] as it provides a conducive environment for their growth. Chlorhexidine, triclosan, essential oils, fluorides, and herbal extracts are added in several oral hygiene products including dentifrices to fight and inhibit the growth of this microorganisms.[456]

Among the dentifrices tested in the present study, Regimens A1, A2, B1, and B2 were fluoridated dentifrices.

S. mutans and C. albicans were the microorganisms selected for the present study because of their implicaton in oral diseases. It has been observed by many studies that the predominant organisms which form about 50% of the total colony-forming units are from genus Streptococcus and that is irrespective of the age of the plaque.[7]

Clarke JK from England in the year 1942 isolated a Streptococcus species that was seen to be predominant in many human carious lesions and he named this species as S. mutans. Since then, till date, this species is one of the most heavily investigated agents for dental caries.[789]

C. albicans grows aerobically at 25°C–37°C on almost all common culture media including blood agar.[101112]

The antimicrobial properties of the dentifrices were determined by the measurement of zone of inhibition on blood agar plates. Testing methods included the disc diffusion method of Cormican et al.[4]

There are many in vivo studies available which have shown that the herbal dentifrices were as effective as conventional dentifrices and these studies are in unison with the results we obtained from our study.[131415]

There are studies which differ in opinion with the above results; according to one such study, the dentifrices containing extract of calendula were less effective against oral microbes compared to a dentifrice containing 1100 ppm sodium fluoride.[16]

Our results showed that at 50% concentration, Pepsodent complete germicheck tooth showed the maximum zone of inhibition which was closely followed by Regimen A1 for S. mutans, whereas at 100% concentration, Regimen A1 showed better effects.

For C. albicans, it was observed that Dabur red toothpaste showed the significantly higher zone of inhibition at 50% concentration when the concentration was made 100% and also Dabur red toothpaste showed the maximum zone of inhibition. This indicates that the herbal dentifrices are no less than the conventional ones and are equally and sometimes better than the conventional ones.

CONCLUSION

There are many dentifrices available in the market, and the discussion which is better herbal or conventional has always been the point of debate. Hence, we conducted a study comparing two herbal and two conventional dentifrices, namely Regimens A1, A2, B1, and B2, respectively.

We compared the above dentifrices for their efficacy against two microorganisms S. mutans and C. albicans and found that the herbal dentifrices were equally effective in inhibition of S. mutans, but for C. albicans, an herbal dentifrice Dabur red toothpaste was better than the conventional ones.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.