Antimicrobial Property of Herbal Toothpastes: An In-Vitro Analysis.

Manupati Prasanth¹* and Satya Narayana Ratha²

¹School of Microbiology, Mahatma Gandhi National Institute of Research and Social Action (M.G.N.I.R.S.A), Hyderabad, Andhra Pradesh, India.

²Alluri Seetharama Raju Centre for Tribal Studies, Mahatma Gandhi National Institute of Research and Social Action (M.G.N.I.R.S.A), Hyderabad, Andhra Pradesh, India.

*Corresponding Author E-mail: prasanthmanupati@gmail.com

ABSTRACT:

Oral care is an important component of personal hygiene. Meticulous plaque control, on a daily basis with toothbrush, is the most important step to achieve good oral health. Available dental-care measures have not been inadequate to deal with this problem. Consequently, incorporation of herbal extracts in the formulation of toothpastes is becoming a popular mode of plaque control. On the backdrop of this scenario, the present study is designed to evaluate antimicrobial properties and effect of six toothpastes claiming herbal ingradiants by using agar well diffusion method and to determine their suitability for regular usage. Six herbal dentifrices are tested for their anti-microbial activity against three oral pathogens, namely, Streptococcus mutans (MTCC 890), Lactobacillus acidophilus (MTCC *447) and Candida albicans (MTCC 854) by well agar diffusion assay. Anti-caries evaluation shows that herbal based dentifrice formulation A displayed the maximum zones of inhibition against all the organisms tested (p<0.05) as compared to all other dentifrice formulations. Hence, dentifrice formulation A has significant antimicrobial activity; and thus can be employed as an effective anti-plaque agent in the prevention of dental caries. The findings from this study may be of use to dental professionals.

KEYWORDS: Anti-microbial activity, Dental caries, Herbal based dentifrices, Zone of inhibition, Well agar diffusion assay

INTRODUCTION:

Oral health is central to our general well-being and quality of life, which connote healthy teeth, absence of oral-facial pain and oral soft-tissue lesions, etc. One cannot be entirely healthy without good oral health. Despite great amelioration in the management of global oral hygiene over the past several decades, problems still persist. Dental caries and diseases of the periodontium are among the most common afflictions of the humankind.

Dental caries (tooth decay) is an infection of the calcified tissues of the teeth, caused primarily by Streptococcus mutans ¹.Other microorganisms, like Lactobacillus acidophilus and Candida albicans act as catalysts. According to the World Health Organization (W.H.O), 60-90% of school children, worldwide, have dental cavities ². However, a marked decline in the pervasiveness of caries has been observed in developed countries. On the other hand, it is on the rise in developing countries³.

Diseases affecting the periodontal structures beyond the gingiva and leading to loss of connective tissue attachment are called periodontal diseases. Periodontal diseases are among the most prevalent diseases in mankind, afflicting about 30% of the adult population⁴. The prevalence and severity of periodontal diseases have been found to increase with age and is more common in males than females⁵. Majority of adults exhibit some degree of periodontal disease. Streptococci, Spirochetes and bacteroids are the pathogens responsible for the ailment.

Daily plaque removal with a toothbrush is an important component of most oral hygiene programs intended to prevent and treat plaque mediated diseases, dental caries and periodontal diseases. As all are not likely to follow the optimal oral hygiene regimen, synthetic chemotherapeutic agents appear as beneficial alternatives⁶. Consequently, despite being beneficial to some extent, entry of these synthetic chemical agents into the mouth have resulted in undesirable side effects⁷. Hence, the search for alternative products continues and plants (herbs) and naturally derived products (herbal supplements) isolated from plants used in traditional medicine are considered as good alternatives to synthetic chemicals.

Plants have been looked upon as important resource bases to combat serious diseases, from time immemorial. According to WHO (1993), 80% of the world’s population is dependent on the traditional medicine, and a major part of the traditional therapies involves the use of plant extracts or their active constituents⁸. Yet, a scientific study of plants, to determine their antimicrobial active components, is a comparatively new field. Development in alternative medicine research has led to many efficient mouthrinses and toothpastes based on plant extracts⁹. The scientific evidence supports the efficacy and safety of numerous herbs¹⁰. Therefore, the aim of this study is to see the efficacy of the selected six herbal dentifrices, against specific plaque mediated microorganisms by using agar well diffusion method.

MATERIALS AND METHODS:

Test microorganisms:

Pure cultures of Streptococcus mutans (MTCC 890), Lactobacillus acidophilus (MTCC *447) and Candida albicans (MTCC 854) were procured from Institute of Microbial Technology, Chandigarh, India. These were cultured on the specific media, procured from Himedia Laboratory Pvt. Ltd., Mumbai, India, recommended for different microorganisms such as Brain heart infusion agar (S. mutans), Lactobacillus MRS agar (L.acidophilus), Nutrient agar (C. albicans) and incubated aerobically at 37°C for 24 h while Candida albicans was incubated for 48 h.

Assortment of dentifrices:

Six different brands of herbal dentifrices, were randomly selected and procured from the local market. The particulars of the ingredients in each dentifrice formulation, as mentioned by the manufacturers, are noted (Table 1). The selected herbal dentifrice solutions were made by mixing the calculated amount of the dentifrices (2.0 gm) in a measured volume (2.0 ml) of the sterile pyrogen-free distilled water, to give 1:1 dilution. They were further diluted in sterile pyrogen-free distilled water and three different dilutions of 1:2, 1:4 and 1:8 were made. Nutrient agar, lactobacillus MRS agar and brain heart infusion agar plates were prepared to assess the antimicrobial activity of six herbal dentifrices against the pathogens. All other chemicals and reagents used are of analytical grade.

Antimicrobial assay:

The antimicrobial activity of each herbal dentifrice formulation is determined, using a modified agar well diffusion method¹¹. Growth from freshly subcultured isolates are suspended in 10 mL of sterile saline to obtain a turbidity of 0.5 McFarland standards. Nutrient agar, lactobacillus MRS agar and brain heart infusion agar plates were prepared and seeded with 0.5 mL of suspended cultures of each isolate (Nutrient agar was used for C. albicans, lactobacillus MRS agar for L.acidophilus, where as brain heart infusion agar was used for Streptococcus mutans strain). Then wells of 8 mm diameter, are punched into the plate surface with equal distance from each other. Each well was then filled with 0.2mL diluted dentifrice solutions, while the same amount of sterile distilled water was also added as a control. The plates are then incubated at 37°C for 24 h (48 h for yeast species). The antimicrobial activity is evaluated by measuring the diameter of zones of inhibition in mm (Figure 1). All the plates are made in triplicate, and the experiments repeated three times.

Table 1: Ingredients of various herbal dentifrices tested for antimicrobial potential.

Dentifrices	Ingredients as listed on the packages.
A	Dried Ginger 1.5mg, Black Pepper 1.5mg, Long Pepper 1.5mg, Harra 1.0mg, Amla 1.0mg, Liquirice 1.0mg, Karpoor 3.0mg, Clove 2.5mg.
B	Dadima (Punica granatum) 2.57mg,Tumburu (Zanthoxylum alatum) 1.80mg, Babbula (Acacia arabica) 1.71mg, Triphala 1.71mg, Vidanga (Embelia ribes) 1.71mg, Nirgundi (Vitex negundo) 1.14mg, Vaikranta bhasma 2mg, Nimba (Azadirachta indica) 1.44mg, Ajamoda satva 1mg, Sodium Benzoate IP, Bronopol IP, Saccharin Sodium IP 0.5%.
C	Dicalcium Phosphate Dihydrate, Sorbitol, Deionised Water, Sodium Lauryl Sulphate, Ganoderma Lucidum (Mushroom) Stem Extract, Propylene Glycol, Silica, Menta Piperita (Peppermint) Flavour, Chondrus Crispus (Carrageenan) Extract, Cellulose Gum, Sodium Benzoate, Sodium Saccharin, Menthol.
D	Herbal extracts 2.5% w/w (Maricha / Marica (Piper nigrum) 31.04g, Pippali (Piper longum) 31.04g, Shunthi (Zingiber officinale) 31.04g, Tejovati / Tomar (Zanthoxylum armatum) 31.04g)., Lavang / Laung oil (Syzygium aromaticum) 0.50g, Karpoor (Cinnamomum camphora) 0.50g, Pudina satva (Mentha spicata) 0.50g, Gairic Powder 1.80g, Sweetener (Sodium saccharin), Preservatives (Methyl paraben, Propyl paraben, Sodium benzoate), Excipient q.s.
E	Calcium Carbonate, Sorbitol, Silica, Sodium Lauryl Sulfate, Polyethylene Glycol, Flavor, Sodium Carboxy Methyl Cellulose, Sodium Silicate, Sodium Monofluorophosphate, Sodium Saccharin, Xanthan Gum, Pigment Green No. 7 (Cl 74260), Myrrh Tincture, Chamomile Tincture, Tea Tree Oil, Sage Oil, Eucalyptus, in aqueous base.
F	Cardamom Extract 0.50%, Ginger Extract 0.50%, Cinnamon Extract 0.50%, Neem Extract 0.50%, Mango Leaf Extract 0.50%, Camphor 0.01%, Alum 0.01%, Mango Flavour Q.S.

Figure 1: Zones of inhibition produced by herbal dentifrice formulation A at 24 h against the three tested microorganisms at four different dilutions. (A) Streptococcus mutans (B) Lactobacillus acidophilus (C) Candida albicans (C) Control

Statistical analysis:

Results are expressed as Mean ± Standard Deviation for illustration. Data are tested using a statistical package, namely, SPSS Windows version 19. Statistical analysis is based on Kruskal–Wallis test. A P-value, less than 0.05, is considered to be significant.

RESULTS:

Results of this preliminary in vitro study demonstrate that dentifrice formulation A is equally effective as formulations C and D, against Streptococcus mutans,  (p<0.015, Table 2) whereas, against L.acidophilus (p<0.014, Table 3) and C. albicans,  (p<0.012, Table 4), dentifrice formulation A displayed the maximum zones of inhibition as compared to all other tested dentifrice formulations. The mean values ± standard deviation of zones of inhibition are given for all the test organisms. Each experiment was repeated three times (n = 3).

DISCUSSION:

A majority of oral diseases exhibit multifactorial etiology ¹². The interplay of host, agent and environmental factors ultimately determine the oral health status of an individual. Dental caries and diseases of the periodontium are among the most prevalent chronic diseases worldwide. Poor oral hygiene is one of the key reasons for their prevalence and harmful activities. Therefore; plaque control is an important procedure involves the removal of microbial dental plaque biofilm and the prevention of its accumulation on the teeth and adjacent gingival surfaces to prevent tooth decay and periodontal disease. Most studies on antimicrobial activity of synthetic dentifrices have been focused on their potential to inhibit oral microflora. In particular, little information was found in recent literature research concerning about herbal dentifrices and their therapeutic values for prevention of dental caries, and other oral infections ^13,14. A number of toothpaste preparations containing herbal ingredients that may be beneficial by improving oral health have been developed in recent years.

In our study, among all the investigated dentifrices, dentifrice formulation A emerged as the most effective, based on the mean diameter of the zone of microbial inhibition produced by the dentifrices in agar well diffusion method, against all the three microorganisms tested. This might be due to the synergistic effect between the active ingredients of the formulation (Table 1). All plant extracts used in this product are known to have positive therapeutic effects, such as dried Ginger, the underground stem or rhizome of the plant Zingiber officinale has been used in Ayurvedic and Unani systems of medicine in Indian and Arabic herbal traditions since ancient times, to treat a wide array of ailments¹⁵. Ginger is one of the natural products having antimicrobial property against various human pathogens, including oral pathogens¹⁶. Black Pepper, has anti-inflammatory properties, which are effective in treating swollen gums¹⁷. Long Pepper, an analgesic¹⁸. Harra, it is also one of the widely used plants in Ayurvedic medicine. Few of the reported activities include antioxidant ¹⁹, anti-caries ²⁰, immunomodulatory²¹, anti-bacterial ²² and for wound healing²³. Amla, is a natural antioxidant with the richest source of vitamin C. It is antibacterial, and it has astringent properties²⁴. Liquorice, it is one of the most widely used herbs from throughout history of Ayurveda. It has been studied for its biological actions, including antioxidant, anti-genotoxic, anti-inflammatory, anti-microbial, anti-plaque, anti-caries²⁵. Cloves are the aromatic dried flower buds contains an anesthetic chemical compound called eugenol, which numbs nerves and controls pain. It also possesses anti-inflammatory, antiseptic and astringent properties²⁶. Next to the dentifrice formulation A, all other herbal dentifrices examined in the study, are found to have antimicrobial activities and these are statistically significant but less effective when compared to dentifrice formulation A, and the samples can be arranged in a descending order, of their effectiveness (Table 1).

It is known that a balance exists in each person’s oral microbial population. If this balance is gone astray, opportunistic microorganisms can multiply, enabling the initiation of disease processes. Therefore, the dentifrice formulation identified as having the maximum sensitivity to all three organisms at all concentrations and thus probably the strongest antimicrobial properties, may not be certainly superior to the other dentifrices. Because, the formulation used in vivo is likely to be diluted by saliva, the level to which antimicrobial properties are buffered or lost in dilution in vitro of interest ¹¹.

Table 2: Antimicrobial activity of herbal dentifrice formulations against Streptococcus mutans.

Dentifrice	N	Concentration tested	Mean	Standard Deviation	Chi- Square	P value
A	3	1:1	20.00	0.000	10.497	0.015**
		1:2	18.00	0.000
		1:4	15.00	1.000
		1:8	13.00	1.000
B	3	1:1	18.33	0.577	10.532	0.015**
		1:2	14.67	0.577
		1:4	12.33	0.577
		1:8	10.33	0.577
C	3	1:1	20.00	0.000	10.879	0.012**
		1:2	18.00	0.000
		1:4	15.67	0.577
		1:8	14.00	0.000
D	3	1:1	20.00	0.000	10.761	0.013**
		1:2	18.00	0.000
		1:4	14.67	0.577
		1:8	11.67	0.577
E	3	1:1	18.00	0.000	10.645	0.014**
		1:2	14.67	0.577
		1:4	12.33	0.577
		1:8	10.33	0.577
F	3	1:1	15.00	0.000	10.298	0.016**
		1:2	12.33	0.577
		1:4	10.33	0.577
		1:8	09.00	1.000

n = 3, ***P < 0.01, **P < 0.05, *P < 0.10.

Table 3: Antimicrobial activity of herbal dentifrice formulations against Lactobacillus- acidophilus.

Dentifrice	N	Concentration tested	Mean	Standard Deviation	Chi- Square	P value
A	3	1:1	21.67	0.577	10.645	0.014**
		1:2	19.67	0.577
		1:4	18.00	0.000
		1:8	14.33	0.577
B	3	1:1	20.00	0.000	10.879	0.012**
		1:2	18.00	0.000
		1:4	14.33	0.577
		1:8	12.00	0.000
C	3	1:1	19.67	0.577	10.761	0.013**
		1:2	18.00	0.000
		1:4	15.67	0.577
		1:8	14.00	0.000
D	3	1:1	19.67	0.577	10.761	0.013**
		1:2	18.00	0.000
		1:4	14.00	0.000
		1:8	11.33	0.577
E	3	1:1	18.00	0.000	10.879	0.012**
		1:2	16.00	0.000
		1:4	14.00	0.000
		1:8	11.67	0.577
F	3	1:1	18.00	0.000	10.761	0.013**
		1:2	16.00	0.000
		1:4	13.33	0.577
		1:8	11.67	0.577

n = 3, ***P < 0.01, **P < 0.05, *P < 0.10.

Table 4: Antimicrobial activity of herbal dentifrice formulations against Candida albicans.

Dentifrice	N	Concentration tested	Mean	Standard Deviation	Chi- Square	P value
A	3	1:1	22.00	0.000	11.000	0.012**
		1:2	20.00	0.000
		1:4	18.00	0.000
		1:8	14.00	0.000
B	3	1:1	20.33	0.577	10.645	0.014**
		1:2	18.33	0.577
		1:4	14.33	0.577
		1:8	12.00	0.000
C	3	1:1	20.00	0.000	10.761	0.013**
		1:2	18.00	0.000
		1:4	14.33	0.577
		1:8	12.33	0.577
D	3	1:1	20.00	0.000	10.761	0.013**
		1:2	18.00	0.000
		1:4	14.67	0.577
		1:8	12.67	0.577
E	3	1:1	20.00	0.000	10.879	0.012**
		1:2	18.00	0.000
		1:4	14.67	0.577
		1:8	12.00	0.000
F	3	1:1	20.00	0.000	10.879	0.012**
		1:2	18.00	0.000
		1:4	14.33	0.577
		1:8	12.00	0.000

n = 3, ***P < 0.01, **P < 0.05, *P < 0.10.

CONCLUSION:

This study shows significant differences in the ability of the various herbal based dentifrices to inhibit the growth of certain plaque microorganisms. Dentifrice formulation A emerged as the most effective toothpaste compared to all other formulations tested.

ACKNOWLEDGEMENT:

Sincere thanks Dr. N. Balakrishnan, Department of Biostatistics, National Institute of Nutrition, for carrying out the statistical analysis.

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Received on 23.05.2013 Modified on 08.06.2013

Accepted on 12.06.2013 ©A&V Publications All right reserved

Res. J. Pharmacology & P’dynamics. 6(1): Jan.-Mar. 2014; Page 30-35