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ORIGINAL RESEARCH
Year : 2021  |  Volume : 12  |  Issue : 3  |  Page : 118-123

Antimicrobial Efficacy of Herbex and Himalaya Herbal Toothpastes: An In Vitro Experimental Study


1 Department of Pediatric Dentistry, School of Dentistry, Shahid Beheshti University of Medical Sciences, Tehran, Iran
2 Department of Dental Biomaterials, School of Dentistry, Shahid Beheshti University of Medical Sciences, Tehran, Iran
3 Dental Research Center, School of Dentistry, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Date of Submission12-Sep-2021
Date of Decision17-Mar-2021
Date of Acceptance02-May-2021
Date of Web Publication2-Nov-2021

Correspondence Address:
Parastoo Iranparvar
School of Dentistry, Daneshjoo Blvd, Evin, Shahid Chamran Highway, postal code 1983963113
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/denthyp.denthyp_37_21

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  Abstract 


Introduction: Recently, toothpastes containing herbal antimicrobial ingredients have gained popularity due to their reduced side effects. This study aimed to compare the antimicrobial efficacy of two herbal toothpastes and a nonherbal type on pathogens responsible for caries and periodontal disease. Materials and Methods: Full and 1:3 concentrations of two herbal toothpastes (Himalaya® and Herbex®) and a nonherbal type as the positive control (Crest® cavity protection) were prepared. Sterile distilled water was considered as the negative control. Streptococcus mutans, Streptococcus sobrinus, Lactobacillus casei, and Actinobacillus actinomycetemcomitans (A.a) were cultivated on agar plates and incubated after adding toothpaste preparations. The diameter of the inhibition zone was measured in millimeters. Two-way analysis of variance and Tukey Post-hoc tests were applied at P < 0.05. Results: The mean margin diameter was higher in full concentration than the diluted 1:3 formula for all examined toothpastes (P < 0.001). All three toothpaste types exerted a significant antimicrobial effect compared to the negative control (P < 0.05). The antimicrobial effect of Herbex® on S. sobrinus was significantly lower than the positive control, and it was significantly less effective against S. mutans compared to Himalaya (P < 0.05). Furthermore, the efficacy of Himalaya® on L. casei and A.a was significantly lower than the positive control (P < 0.05). No statistically significant differences were observed in other pair comparisons. Conclusion: Considering the observed efficacy of herbal toothpastes against cariogenic bacteria and periopathogens, they potentially qualify as complementary agents for self-care oral hygiene procedures.

Keywords: Actinobacillus actinomycetemcomitans, anti-bacterial agents, herbal, Lactobacillus casei, propolis, Streptococcus mutans, Streptococcus sobrinus, toothpastes


How to cite this article:
Biria M, Rezvani Y, Yadegari Z, Rahmati MH, Iranparvar P. Antimicrobial Efficacy of Herbex and Himalaya Herbal Toothpastes: An In Vitro Experimental Study. Dent Hypotheses 2021;12:118-23

How to cite this URL:
Biria M, Rezvani Y, Yadegari Z, Rahmati MH, Iranparvar P. Antimicrobial Efficacy of Herbex and Himalaya Herbal Toothpastes: An In Vitro Experimental Study. Dent Hypotheses [serial online] 2021 [cited 2021 Dec 3];12:118-23. Available from: http://www.dentalhypotheses.com/text.asp?2021/12/3/118/329761




  Introduction Top


Dental caries and periodontal problems are considered the most prevalent oral diseases worldwide that are initiated by various microorganisms present in dental plaque biofilm.[1] Some of these microorganisms, both acidogenic and aciduric (such as Streptococcus and Lactobacillus species), are associated with enamel demineralization and dental caries.[2] Dental plaque may also contain gram-negative bacteria responsible for periodontal diseases.[3]

Plaque-related oral diseases are mostly preventable and can be more effectively controlled locally than systematically due to their localized nature. Toothbrushing, with toothpaste containing antimicrobial agents, is the most popular method of prevention. The antimicrobial properties of toothpastes are attributed to various chemical or herbal agents that can directly inhibit plaque formation. Recently, toothpastes containing herbal antimicrobial agents have gained popularity due to reduced side effects.[4] Propolis is the active ingredient in some herbal toothpastes such as Herbex®, which exerts anti-inflammatory, antioxidant, and antimicrobial effects owing to flavonoids and phenolic acid in its composition.[5] Other herbal antibacterial agents with an ancient background, such as miswak extract, pomegranate extract, neem tree extract, and Babool extract, are considered as the active ingredients in toothpastes such as Himalaya.[6]

Considering the increasing popularity of herbal toothpastes, and the limited research regarding their efficacy, the current study aimed to evaluate the antimicrobial effects of two herbal toothpastes (Herbex® and Himalaya®) compared to a nonherbal type (Crest® cavity protection) on S. mutans, S. sobrinus, L. casei, and Actinobacillus actinomycetemcomitans (A.a). Furthermore, to evaluate the effect of toothpaste concentration on its efficacy, both full and diluted formulas were investigated.


  Materials and Methods Top


In the present in vitro study, two herbal toothpastes, that is, Herbex® (Dr Jahangir Pharmacy Lab, Iran) and Himalaya® (Himalaya tray company, India), were selected for investigation of their antimicrobial effects. For this purpose, a nonherbal, food and drug administration (FDA) approved toothpaste with a proven antimicrobial effect (Crest® cavity protection, Procter & Gamble, Weybridge, UK) was considered the positive control and deionized sterile distilled water (Daroupakhsh Co., Tehran, Iran) as the negative control. The composition of examined toothpaste types is given in [Table 1]. The study was conducted following the proper institutional ethics approval (0310/5987).
Table 1 Composition of toothpastes investigated in the study

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Toothpaste sample preparation

Empty sterile microtubes were weighed using a calibrated scale with a precision of 0.0001 (Sartorius, Germany). The full-strength toothpaste samples were prepared by collecting 150 mg of each toothpaste into the microtube using the same scale. In order to prepare a diluted 1:3 concentration, 150 mg of each toothpaste was mixed with 450 mL of deionized sterile distilled water for 2 minutes using a Vortex mixer (Techne, South Korea).

Bacteria collection and culture

S. mutans American Type Culture Collection (ATCC 35668), L. Casei (ATCC 39392), A.a (ATCC No V99), and S. sobrinus (ATCC 27607) were collected from regional culture collection, transferred to appropriate culture media, and incubated at 37 ± 0.5°C for 24 to 72 hours. Examined microorganisms, their culture media, and incubation time are listed in [Table 2].
Table 2 List of examined microorganisms and their culture media

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Concentrations of 1.5 × 108 colony forming units per milliliters (CFU/mL) (equal to 0.5 McFarland turbidity standard) were prepared from the existing broths using a spectrophotometer (solutions of 0.5 McFarland have absorption of 0.08–0.1 at a wavelength of 600 nm in spectrophotometer). Solutions were then transferred to their specialized culture media using sterile cotton swabs and cultivated by the lawn technique.

The sample size was calculated to be 24 culture plates (i.e., six plates for each microorganism) based on a previous study (power = 80%, α = 5%).[7] Each plate was divided into four equal sections, and wells with a diameter of 5 mm were prepared in each section. Fifty microliters of each preparation (including the positive and negative control) were then transferred into each well. In order to assess the antimicrobial effects by agar diffusion method,[8] the plates were incubated at 37 ± 0.5°C (Memmert, Germany) for 48 hours using anaerobic jar for growth induction of L. casei and A.a. The diameter of inhibition zone around the wells (if present) was measured by an experienced microbiologist who was blinded to the toothpaste type in each sample, using a ruler with divisions of 1 mm [Figure 1].
Figure 1 Examples of inhibition zones in four sections of each plate (negative control, positive control, Herbex®, Himalaya®)

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Statistical tests

Numerical data were presented as the mean and standard deviation amounts. A two-way analysis of variance (ANOVA) was carried out to determine the effect of toothpastes’ type and concentration on growth inhibition. Tukey post-hoc test was also used for intergroup comparisons. Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) software version 22 (IBM, Chicago, US) (α = 0.05) (confidence interval = 95%).


  Results Top


Inhibition zone was not observed in wells containing deionized sterile distilled water, indicating the absence of antimicrobial effects in negative control; however, all three toothpaste types exerted a significant antimicrobial effect compared to the negative control (P < 0.05).

The mean values of inhibition zones in the study groups are presented in [Table 3]. Two-way ANOVA test revealed a significant difference between the antimicrobial effects of the three toothpaste types on S. mutans (P = 0.025), S. sobrinus (P = 0.038), L. casei (P = 0.018), and A.a (P = 0.022). Furthermore, for all toothpaste types, the mean margin diameter was higher for full concentration than the diluted 1:3 formula (P < 0.001) [Figure 2]. The interaction effect of toothpaste type and concentration was not significant on S. mutans (P = 0.61), S. sobrinus (P = 0.27), L. casei (P = 0.31), and A.a (P = 0.54).
Table 3 Diameter of inhibition zone for microorganisms at full and 1:3 concentrations of toothpastes (mm)

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Figure 2 Mean diameter of inhibition zone for examined microorganisms (group 1: Crest, group 2: Himalaya, group 3: Herbex) (condition 1: diluted 1/3 concentration, condition 2: full concentration)

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As presented in [Table 4], Tukey post-hoc test revealed that the antimicrobial effect of Herbex® on S. sobrinus was significantly lower than the positive control (P = 0.031). Furthermore, Herbex® was significantly less effective against S. mutans compared to Himalaya® (P = 0.020). The efficacy of Himalaya® on L. casei and A.a was significantly lower than the positive control (P = 0.020 and 0.044, respectively). No statistically significant differences were observed in other pair comparisons.
Table 4 Comparative antibacterial effects of examined toothpastes

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  Discussion Top


In recent years, the addition of herbal extracts to toothpastes has been gaining increasing attention. Active ingredients in herbal products prevent the colonization of oral bacteria on tooth surfaces by penetrating the biofilm and preventing plaque accumulation.[9] However, a few studies have evaluated the efficacy of herbal toothpastes on oral microflora. This study aimed to observe the antimicrobial effects exerted by natural components in two herbal toothpastes compared to a nonherbal type, and antimicrobial effects were observed in all examined toothpastes.

In the present study, S. mutans, S. sobrinus, and L. casei were selected because an increase in the proportion of these microorganisms in dental plaque is highly associated with caries initiation and progression.[2] A.a was also chosen because of its many virulence factors responsible for the pathogenesis and rapid progression of periodontal diseases.[3]

Crest® cavity protection was considered as the nonherbal positive control with proven antimicrobial effects, containing sodium fluoride as the primary antibacterial agent acting by various mechanisms (i.e., inhibition of glycolysis and acidogenesis, reinforcement of enamel structure) and sodium lauryl sulfate with antimicrobial and antiplaque effects.[9],[10]

In the present study, Himalaya® was less effective against L. casei and A.a compared to the positive control; however, its efficacy on S. mutans and S. sobrinus was similar to the positive control. The antibacterial effects of Himalaya® have been shown in previous studies and are attributed to its herbal ingredients such as miswak extract, pomegranate extract, Babool extract, and neem extract.[7],[11],[12],[13],[14] Miswak extract (Salvadora persica) contains sulfated compounds and isothiocyanate, which exerts antibacterial properties. It also contains trimethylamine, which prevents dental plaque accumulation by reducing surface adhesion.[11] Pomegranate extract prevents bacterial adhesion and biofilm formation, though it cannot destroy the already formed biofilm. The efficacy of pomegranate extract on oral bacteria such as S. mutans can be attributed to the presence of lactic acid (roughly 40%) in its formula.[7],[12],[15] Finally, neem leaf extract’s efficacy against S. mutans, considering the absence of bacterial resistance and its good availability, makes it a potentially suitable ingredient for herbal toothpastes.[13]

Herbex® (containing propolis) was also effective against cariogenic microorganisms in this study, although its effect on S. mutans was significantly lower than Himalaya®, and it was significantly less effective against S. sobrinus compared to the positive control. Antimicrobial effects of propolis on cariogenic bacteria have been shown in previous studies by Moshin et al.,[16] Yu et al., [17] and Bertolini et al., [18] and it is attributed to the presence of phenolic esters and flavonoids (i.e., pinocembrin, caffeic acid, baicalin, naringin, galangin). Pinocembrin is proven to be effective against Streptococcus species.[19] Apigenin is another agent in propolis inhibiting the activity of glycosyltransferase enzyme in streptococcus.[20] Furthermore, the high antibacterial efficacy of propolis against some anaerobic oral bacteria, such as periodontal microorganisms, has also been observed previously.[19] In the present study, the mean diameter of the inhibition zone for the toothpaste containing propolis (i.e., Herbex®) against A.a was high and comparable to the positive control (i.e., Crest®), which is consistent with the results of studies by Agarwal et al. and Akca et al. regarding the effect of propolis on periopathogens.[21],[22]Since the toothpastes are diluted by saliva in the oral cavity, their 1:3 concentrations were also evaluated in the present study. It was shown that all toothpastes were more effective in full concentration than the 1:3 diluted formula, which was consistent with the results of previous studies.[8],[10]

In the present study, observations were made by the agar diffusion method, which is a widely used method in the preliminary evaluation of the antimicrobial effects of toothpastes in vitro.[8],[9],[13] However, a limitation of the present study was its in vitro design, overlooking the impact of the oral environment’s specific characteristics on the study results; therefore, further in vivo studies are recommended in order to be able to draw more definite conclusions.


  Conclusion Top


Within the limitations of this study, it can be concluded that due to the antimicrobial efficacy of herbal toothpastes on oral bacteria responsible for caries and periodontal diseases, they potentially qualify as complementary agents for self-care oral hygiene procedures.

Acknowledgement

None.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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[PUBMED]  [Full text]  
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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