ORIGINAL ARTICLE Antimicrobial Efficacy of Different


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ORIGINAL ARTICLE
Antimicrobial Efficacy of Different Commercially Available Herbal vs Chemical-based Dentifrices on Escherichia coli: An In Vitro Study
Saummya Singh1, Ashok K Agarwal2, Rashmi Bansal3, Deepika Verma4, Anuraag Gurtu5, Aishwarya Roy6
Abstr ac t​
Introduction: Toothbrushing aids in control over cariogenic microorganisms. Various chemical and herbal ingredients are added to dentifrices for antimicrobial efficacy. But sparse knowledge regarding the ingredients used in herbal dentifrices and their efficacy compared to chemical dentifrices is present. So, various herbal dentifrices are evaluated and compared for their antimicrobial efficacy against Escherichia coli. Materials and methods: Pure cultures of Escherichia coli (ATCC 25922) were subcultured on Müeller-Hinton broth at 37°C for 24 hours. The nutrient agar plates were inoculated with 0.5 mL of 24-hour broth culture. Dilutions of the selected dentifrices were prepared and evaluated by agar-well diffusion method. The agar plates were then incubated. The diameter of obtained zones of inhibition was measured. The antimicrobial efficacy of the dentifrices was considered to be directly proportional with the size of obtained zone of inhibition. Statistical analysis: The obtained results were tabulated and subjected to statistical analysis using one-way analysis of variance (ANOVA) and independent t test. Results: Amongst the herbal combinations, maximum zone of inhibition was observed with the dentifrices containing akarkara, neem, babool, tomar, pudina, lavang, pippli, vajradanti, bakul, vidang, haldi, pilu, majuphal, and meswak. Amongst chemical-based dentifrices, triclosan and sodium fluoride containing dentifrice depicted maximum zone of inhibition against E. coli. On comparing antimicrobial efficacy of herbal vs chemical dentifrices, it was observed that there was no significant difference between them (p > 0.05). Conclusion: Herbal dentifrices are more effective than chemical dentifrices and possess no side-effects. So, many lesions of oral cavity can be treated by herbal dentifrices. Keywords: Agar-well diffusion method, Antimicrobial efficacy, Chemical dentifrices, E. coli, Herbal dentifrices. Journal of Dental Sciences and Oral Rehabilitation (2019): 10.5005/jp-journals-10086-1216

I n t ro d u c t i o n​
Dental caries is an irreversible microbial disease of the calcified tissues of the teeth, characterized by the demineralization of the inorganic portion and destruction of the organic substance of the tooth, which often leads to cavitation. Its prevalence varies from 50 to 60% in India.1 Apart from the chemical and nutritive measures, mechanical measures like toothbrushing, oral prophylaxis, and mouth rinsing are incorporated for control over caries causing microorganisms.2
Dental plaque is specific, but highly variable framework which results from the establishment of microbes onto the tooth surfaces and restorations, etc., and is made up of salivary components like mucin, debris, microbes, and desquamated epithelial cells, etc., embedded in gelatinous extracellular matrix. Microorganism Escherichia coli is a pathogenic microbe responsible for the advancement of dental caries. One of the main reasons for growth of E. coli and its harmful activities is poor oral hygiene.3
A characteristic feature of plaque is the resistance to removal by physiologic and oral cleansing procedures such as saliva and tongue movement but it is found to be removable by toothbrushing.2 The incorporation of dentifrice helps to reduce the surface tension on tooth surface, thereby reducing adherence of plaque. A number of chemical agents like triclosan, sodium fluoride, sodium monofluorophosphate, calcium sodium phosphosilicate, chlorhexidine gluconate, and amine fluoride, etc., have been advocated in dentifrices.

1,3,5,6Department of Conservative Dentistry and Endodontics, Institute of Dental Sciences, Bareilly International University, Bareilly, Uttar Pradesh, India 2Department of Pediatrics, Rohilkhand Medical College and Hospital, Bareilly International University, Bareilly, Uttar Pradesh, India 4Department of Microbiology, Rohilkhand Medical College and Hospital, Bareilly International University, Bareilly, Uttar Pradesh, India
Corresponding Author: Rashmi Bansal, Department of Conservative Dentistry and Endodontics, Institute of Dental Sciences, Bareilly International University, Bareilly, Uttar Pradesh, India, Phone: +91 9927107555, e-mail: [email protected]
How to cite this article: Singh S, Agarwal AK, Bansal R, et al. Antimicrobial Efficacy of Different Commercially Available Herbal vs Chemical-based Dentifrices on Escherichia coli: An In Vitro Study. J Dent Sci Oral Rehab 2019;10(2):43–47.
Source of support: Nil
Conflict of interest: None
But it is observed that though such formulations bear good antimicrobial properties, they also possess several side effects like occurrence of dental fluorosis and staining of teeth, etc.4 In a study, triclosan was even reported to be carcinogenic.5
Hence, the current societal desire is relying on “Being Natural” with the use of natural phytochemicals present in plants. These herbal formulations are often cheaper, locally available, and they do not need preservatives, alcohol, colors, or flavors for their

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons. org/licenses/by-nc/4.0/), which permits unrestricted use, distribution, and non-commercial reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Antimicrobial efficacy of Herbal dentifrices on E. coli

antimicrobial activity, which makes them further more appealing.6 But a lacuna regarding the ingredients used in herbal dentifrices and their efficacy compared to that of chemical dentifrices is present.
Antimicrobial activity is generally tested by assays based on disk diffusion method,1 agar-well diffusion method,7,8 linear regression method,9 measuring minimum inhibitory concentration,10 or salivary streptococcus count11,12 via in vivo11 and in vitro10,13 studies. Amongst these, the most readily available method which is easy to execute and time savvy is agar-well diffusion method.
Hence, this in vitro study was aimed to evaluate antimicrobial efficacy of various herbal formulations-based dentifrices against various chemical agents-based dentifrices on caries progressing microorganism E. coli by agar-well diffusion method. A null hypothesis was assumed that there will not be any significant difference between antimicrobial efficacy of various herbal and chemical combinations. The significance level was set as p < 0.05.

Materials and​Me thods​
The study was carried out in the Department of Conservative Dentistry and Endodontics, Institute of Dental Sciences, and the Department of Microbiology, Rohilkhand Medical College and Hospital, Bareilly, Uttar Pradesh, India. The ethical clearance was obtained from the Institutional Research Board Committee, Institute of Dental Sciences, Bareilly.

Herbs and Chemicals in Dentifrices
Various herbal (group I)- and chemical (group II)-based dentifrices were purchased from local markets in Bareilly, Uttar Pradesh, India, for this study (Table 1). Only those samples with a date of manufacture within the last 6 months were selected for the present study for the purpose of standardization.

Preparation of Broth
Pure culture of Escherichia coli (ATCC 25922) was obtained from HiMedia Laboratories Pvt. Ltd., Mumbai, India. It was then subcultured in nutrient broth (HiMedia Laboratories Pvt. Ltd.) at

Table 1: Composition of different dentifrices

Groups

Herbal dentifrices

Group I IA (Himalaya Complete Care)
IB (Dabur Babool) IC (Dabur Red) ID (Dabur Meswak) IE (Patanjali Dantkanti)
IF (Himalaya HiOra)

Anar, babool, neem, triphala, ajwain, kababchini
Babool
Lavang, pudina, tomar, adrak
Meswak
Akarkara, neem, babool, tomar, pudina, lavang, pippli, vajradanti, bakul, vidang, haldi, pilu, majuphal
Pilu, triphala, babool, amra, lavang, nimba

Group II IIA (Colgate Strong Teeth) IIB (Metrogyl DG Gel)
IIC (Kidodent) IID (Elgydium)
IIE (Vantej) IIF (Amflor)

Triclosan, sodium fluoride Metronidazol, chlorhexidine gluconate Sodium monofluorophosphate Chlorhexidine gluconate, calcium carbonate Calcium sodiumphosphosilicate Amine fluoride

37°C for 24 hours. The identification of the strains was confirmed by standard biochemical and staining methods. Asepsis was maintained by performing the procedures on laminar air flow.
To standardize inoculum density, a BaSO4 turbidity standard (0.5 McFarland standard) was used and its density was verified using a spectrophotometer.
Preparation of Dilution
Each dentifrice (2.0 g) was mixed in pyrogen free distilled water (2 mL) to obtain 1:1 dilution. These dentifrices dilutions were transferred to sterile urine containers.
Agar-well Diffusion Assay
The antimicrobial activity of different dentifrices was determined by agar-well diffusion method. Nutrient agar plates were inoculated with 0.5 mL of 24-hour broth culture of E. coli. Four wells at equidistance were punched in each agar plate using sterile Durham’s tube (inner diameter of 7 mm). These wells were then filled with 0.1 mL each of the different dentifrice dilutions and pyrogen free distilled water (control). The Petri plates were then incubated at 37°C for 24 hours.
After the prescribed time of incubation, zones of inhibitions were observed around the wells and the diameters of these inhibition zones were measured (in mm) using graduated scale by Kirby–Bauer method.
Only those Petri plates were selected in which uniformly circular zones of inhibition was observed with a confluent lawn of growth. Petri plates with tear in agar during punching of holes or those with individual colonies of microorganisms were discarded.
The size of obtained zone of inhibition was considered to be directly related with the antimicrobial efficacy of the sample dentifrice. After one day, the intraobserver readings were verified and interobserver verification was done by a microbiologist. Kappa statistics was applied on the collected data and the coefficient of correlation was found to be in the range of 0.8–0.9 for inter- and intraobservers. All the correlation coefficient values were found to be reflecting high degree of concordance.
After the evaluation of zones of inhibition, the culture plates were collected in yellow-coded bags and sent to the biomedical waste management company for further disposal, wherein the waste was incinerated and the ashes were buried.
The results obtained were tabulated and subjected to statistical analysis.
Statistic al​Analysis​
Statistical analysis was performed using one-way analysis of variance (ANOVA) and independent t test using SPSS version 22.0. Significance level was kept at 95% interval (p < 0.05).
R e s u lt s​
Null hypothesis assumed in this study was rejected as there was significant difference among different dentifrices containing herbs and chemicals (Table 2).
Amongst herbal combinations, group IE dentifrice (Patanjali Dantkanti) containing herb combination: akarkara, neem, babool, tomar, pudina, lavang, pippli, vajradanti, bakul, vidang, haldi, pilu, and majuphal; and group ID dentifrice (Dabur Meswak) containing meswak depicted maximum mean zone of inhibition (30.0 mm) against E. coli (Fig. 1) while with the groups IA, IB, IC, and IF, no zones

44 Journal of Dental Sciences and Oral Rehabilitation, Volume 10 Issue 2 (July–December 2019)

Antimicrobial efficacy of Herbal dentifrices on E. coli

Table 2: Comparison of mean zone of inhibition (mm) of antimicrobial efficacy of herbal and chemical dentifrices against Escherichia coli by oneway ANOVA

Antibacterial efficacy of herbal dentifrices on Escherichia coli
Antibacterial efficacy of chemical dentifrices on Escherichia coli

Between groups Within groups Total Between groups Within groups Total

Sum of squares

df

6150.521

5

21.225

54

6171.746

59

3832.033

5

31.894

54

3863.927

59

Mean square 1230.104 0.393
766.407 0.591

F 3129.594
1297.610

Sig. p < 0.001*
p < 0.001*

*Mean difference is significant at the 0.05 level

Fig. 1: Observed zones of inhibition on herbal- and chemical-based dentifrices

Fig. 2: Mean zone of inhibition (mm) in Escherichia coli against herbal dentifrices

of inhibition were observed (Fig. 2). The difference in antimicrobial efficacy of herbal dentifrices was statistically significant.
Amongst chemical combinations, IIA dentifrice (Colgate Strong Teeth) containing combination of triclosan, sodium fluoride depicted maximum mean zone of inhibition (31.0 mm) against E. coli. This was followed by group IIB dentifrice containing combination of metronidazole and chlorhexidine which depicted a mean zone of inhibition of 10.5 mm, while with the groups IIC, IID, IIE, and IIF, no zones of inhibition were observed (Fig. 3). The difference in antimicrobial efficacy of chemical dentifrices was statistically significant (Table 2). The difference in antimicrobial efficacy of herbal dentifrices ID and IE as compared to that of chemical dentifrice IIA was not statistically significant (Table 3).

D i s c u s s i o n​
E. coli is gram-negative, rod-shaped, facultative anerobic microbe associated with active caries lesions. Studies have also shown the growth of E. coli in subgingival flora of aggressive and chronic periodontitis. Hence, there is association of E. coli to cariogenic biofilm.4
To prevent bacterial accumulation, toothbrushing is preferred. But it can remove only approximately 50% of the plaque, so additional means are required to control bacterial load.14,15 For this, incorporation of dentifrices containing chemical agents is advocated but they have certain side effects and microbial resistance to most antibiotics is also reported; therefore,

Journal of Dental Sciences and Oral Rehabilitation, Volume 10 Issue 2 (July–December 2019) 45

Antimicrobial efficacy of Herbal dentifrices on E. coli

Fig. 3: Mean zone of inhibition (mm) in Escherichia coli against chemical dentifrices

Table 3: Comparison of mean zone of inhibition (mm) of herbal dentifrice with different chemical dentifrices against Escherichia coli by independent t test

Escherichia coli

Mean

SD

IA to IIA

7.00

0.00

IA to IIB

7.00

0.00

IA to IIC

7.00

0.00

IA to IID

7.00

0.00

IA to IIE

7.00

0.00

IA to IIF

7.00

0.00

IB to IIA

7.00

0.00

IB to IIB

7.00

0.00

IB to IIC

7.00

0.00

IB to IID

7.00

0.00

IB to IIE

7.00

0.00

IB to IIF

7.00

0.00

IC to IIA

7.00

0.0

IC to IIB

7.00

0.0

IC to IIC

7.00

0.0

IC to IID

7.00

0.0

IC to IIE

7.00

0.0

IC to IIF

7.00

0.0

ID to IIA

28.20

1.44

ID to IIB

28.20

1.44

ID to IIC

28.20

1.44

ID to IID

28.20

1.44

ID to IIE

28.20

1.44

ID to IIF

28.20

1.44

IE to IIA

28.75

0.54

IE to IIB

28.75

0.54

IE to IIC

28.75

0.54

IE to IID

28.75

0.54

IE to IIE

28.75

0.54

IE to IIF

28.75

0.54

IF to IIA

7.00

0.00

IF to IIB

7.00

0.00

IF to IIC

7.00

0.00

IF to IID

7.00

0.00

IF to IIE

7.00

0.00

IF to IIF

7.00

0.00

*Mean difference is significant at 0.05 level

Mean

SD

28.79

1.76

9.35

0.67

7.00

0.00

7.00

0.00

7.00

0.00

7.00

0.00

28.79

1.76

9.35

0.67

7.00

0.00

7.00

0.00

7.00

0.00

7.00

0.00

28.79

1.76

9.35

0.67

7.00

0.00

7.00

0.00

7.00

0.00

7.00

0.00

28.79

1.76

9.35

0.67

7.00

0.00

7.00

0.00

7.00

0.00

7.00

0.00

28.79

1.76

9.35

0.67

7.00

0.00

7.00

0.00

7.00

0.00

7.00

0.00

28.79

1.76

9.35

0.67

7.00

0.00

7.00

0.00

7.00

0.00

7.00

0.00

p value 0.000* 0.000* – – – – 0.000* 0.000* – – – – 0.000* 0.000* – – – – 0.42 0.00* 0.00* 0.00* 0.00* 0.00* 0.95 0.00* 0.00* 0.00* 0.00* 0.00* 0.000* 0.000* – – – –

46 Journal of Dental Sciences and Oral Rehabilitation, Volume 10 Issue 2 (July–December 2019)

Antimicrobial efficacy of Herbal dentifrices on E. coli

herbal-based products are explored. A variety of conventionally used medicinal herbs have not yet been meticulously scrutinized against caries causing microorganisms.
It has also been concluded that agents with demonstrable in vitro antimicrobial activity may be effective against the same microorganisms in vivo, whereas agents without demonstrable in vitro antimicrobial activity are unlikely to exhibit in vivo antimicrobial activity.10
In the present study, the antimicrobial efficacy of dentifrices containing herbs was evaluated and compared with dentifrices containing chemicals against E. coli. It was observed that only the herbal dentifrices ID and IE exhibited antimicrobial efficacy against E. coli. The difference in antimicrobial efficacy of these dentifrices was not significant (p > 0.05). The principle components of ID containing meswak and IE containing herbs: akarkara, neem, babool, tomar, pudina, lavang, pippli, vajradanti, bakul, vidang, haldi, pilu, and majuphal have secondary metabolites such as alkaloids, flavonoids, polyphenols, and lectins, which are considered to be the sole reason of their antimicrobial efficacy.16
As per the chemical dentifrices, only the groups IIA and IIB exhibited antimicrobial efficacy against E. coli. The difference between the antimicrobial efficacy of these two was statistically significant (p < 0.05). Group IIA contains triclosan as the active ingredient while IIB contains metronidazole and chlorhexidine as the active ingredient. Triclosan is reported to have better antimicrobial efficacy against E. coli while with chlorhexidine, though it is considered to be the “Gold Standard” of oral antiseptics, but when used in dentifrices, its antibacterial effect may be influenced by other ingredients that may inhibit chlorhexidine action.17 Triclosan [5-chloro-2-(2,4-dichlorophenoxy)phenol] has been used for more than 30 years as a general antibacterial antifungal agent, and it is incorporated in formulation of dentifrices and mouthrinses. It has recently been suggested that triclosan blocks lipid biosynthesis by specifically inhibiting the enzyme enoyl-acyl carrier protein reductase (ENR).18 Systematic reviews of 6-months clinical studies have concluded that formulations containing triclosan and copolymer significantly improve plaque control and periodontal health.19 But it has certain side effects. Formulations containing amine fluoride have least efficacy against E. coli as the ingredient lacks antimicrobial activity.4
On comparing herbal dentifrices ID (Patanjali Dantkanti) and IE (Dabur Meswak) with chemical dentifrice IIA (Colgate Strong Teeth), there was no significant difference in their antimicrobial efficacy (p > 0.05). Hence, dentifrices with herbal formulations are equally effective. Further, the selection of dentifrice depends upon the oral hygiene status and caries index of the individual.
C o n c l u s i o n​
Dentifrices containing herbal combinations are more effective than chemical dentifrices. When evaluated in overall dimensions, the added advantage of being natural and causing no known side effects, as compared to chemical dentifrices tip the scales in favor of herbal dentifrices. The prevention of caries progression can be achieved by inhibiting growth of Escherichia coli. This can be combated by use of several ingredients like akarkara, neem, babool, tomar, pudina, lavang, pippli, vajradanti, bakul, vidang, haldi, pilu, majuphal, and meswak in herbal formulation-based dentifrices.

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ORIGINAL ARTICLE Antimicrobial Efficacy of Different