The oral cavity acts as a primary route of entry to the human digestive system and the oral biofilm harbours a diverse group of microbial community. The diversity of microorganisms in the oral cavity is greater than any other location in the human body.1 Over 750 species of bacteria inhibits the oral cavity (~50% of which are yet to be identified) and a number of these are implicated in oral diseases.2,3 Rozkiewicz, et al.4 reported that the most common bacteria found in oral cavity are Streptococcus mitis, Streptococcus oral, Streptococcus sanguis, Streptococcus mutans, Streptococcus gordonii, Staphylococcus aureus, Staphylococcus epidermidis, Veillonella sp., Neisseria sicca, Fusobacteria sp., Actinomyces sp., Corynebacterium sp., Lactobacilli sp. and Prevotella sp. Microorganisms from the oral cavity may cause dental problems which includes dental caries (tooth decay),5 periodontitis (gum disease),6 endodontic (root canal) infections7 and formation of dental plagues.8 In India, dental caries affects 60-65% of the general population9 while the periodontal disease is estimated to occur in 50-90% of the population depending on age.10
Toothpaste removes stain caused by pigments contained in food and drinks11 and prevents bad breath.12 Triclosan is an antibacterial constituent in toothpaste used in the prevention of gum infections. Its antimicrobial and anti-inflammatory properties offer advantages in the management of periodontal diseases. Panagakos, et al.13 demonstrated the effect of triclosan/copolymer/ fluoride dentifrice on periodontitis, calculus, caries, whitening and stain removal, oral malodor, and on the oral microflora.
In recent times, it has been observed that many oral product manufacturers are formulating herbal or plant derived toothpastes. Studies showed that the leaf extracts of Adhatoda vasica Nees. improves gingival inflammation and reduces gum bleeding.14 Mimusops elengi L. has long been used in indigenous systems of medicine as a specific cure for diseases of the gums and teeth.15 Studies also demonstrated the anti-cancerous activity and suppression of oral squamous cell carcinoma by Curcuma longa16 Mentha spicata,17 Withania somnifera,18 Nigella sativa19 and Azadirachta indica.20 Feng, et al.21 reported that Camellia sinensis protects buccal cells from DNA damage caused by reactive oxygen species. Flower sap of Echinacea sp. and Aloe Vera stimulates oral immunity and treats gum wounds.22,23 Elettaria cardamomum disinfects the oral cavity and drives away bad breath.24 Antibacterial, antiviral, anti- inflammatory activity of Centella asiatica has been reported by Winston and Maimes 25. The natural origin of herbal products together with antibacterial, antiviral, anti-inflammatory activity found its way into dentistry.26,27 Thus, the present study aims to determine total bacterial count before and after brushing and evaluation of the antimicrobial efficacy of six different kinds of toothpaste against the isolated bacteria.
MATERIALS AND METHODS
Collection of saliva samples
Saliva samples were collected from 72 individuals of age group 20-22 years, who were not on medication or any antibiotic dosage for the past six months. Participants were randomly allocated into 6 groups, consisting of 12 individuals in each group and were given one of the following types of toothpastes: Dabur red, Babool, Himalaya, Close-up, Colgate and Pepsodent (Manufacture information are given in Table 1). All participants were advised to brush with the supplied toothpaste for 2 mins, twice daily, in the morning and in the evening during the test period of 10 days. They were instructed to use the same amount of toothpaste as they did usually. Pre-bushing and post-brushing saliva samples were collected from all participants on day 11 at approximately 9.00 a.m in sterile container, and immediately brought to the laboratory for isolation, enumeration and identification of oral bacteria. Similar process was repeated for all other brands of toothpastes in each group with a test period of 10 days, so as to minimize the error between groups and to reduce the likelihood of any carry over effects.
Toothpaste brands and their active ingredients.
|Colgate||Colgate-Palmolive India Limited||Calcium carbonate, sorbitol, sodium lauryl sulphate, silica, titanium dioxide, sodium silicate, flavor, carrageenan, sodium monofluorophosphate, sodium bicarbonate, benzyl alcohol, sodium saccharin, triclosan, in aqueous base.|
|Himalaya||The Himalaya Drug Company||Sorbitol, aqua, hydrated silica, glycerin, silica, sodium lauryl sulphate, bromelain, xanthan gum, titanium dioxide, flavor, sodium saccharin, sodium benzoate, potassium sorbate, papain, menthol, Salvadora persica stem extract, sodium citrate, Prunus amygdalus dulcis shell extract, Cinnamomum zeylanicum bark oil, Eugenia caryophyllus bud oil.|
|Pepsodent||Hindustan Unilever Limited||Calcium carbonate, water, sorbitol, sodium lauryl sulphate, hydrated silica, flavor, magnesium aluminium silicate, sodium monofluorophosphate, potassium nitrate, benzyl alcohol, sodium silicate, cellulose gum, triclosan, sodium saccharin|
|Close up||Hindustan Unilever Limited||Sorbitol, water, hydrated silica, sodium lauryl sulphate, cocamidopropylbetain, flavor, cellulose gum, sodium saccharin, sodium fluoride, zinc sulphate, sodium hydroxide|
|Babool||Dabur India limited||Calcium carbonate, sorbitol, water, silica, sodium lauryl sulphate, flavor containing clove oil, babul (Acacia arabica) extract, cellulose gum, xanthan gum, sodium silicate, sodium saccharin.|
|Dabur Red||Dabur India Limited||Clove oil, Pudina sativa, Tomar beej (Zanthoxylum alatum), Sunthi (Ginger)|
All procedures performed in this study were in accordance with the ethical standards of the institutional research committee. Instructions were given to all participants regarding brushing techniques, brushing time, amount of toothpaste, sampling procedure, sampling time etc. Formal consent was obtained from all individual participants included in the study.
Determination of total microbial count and identification of bacteria
Serial dilution of saliva samples was made up to 10-3 using sterile distilled water. 50 µl of the sample was inoculated on nutrient agar plates and incubated at 37oC for 24 h. The bacterial colonies were counted using a colony counter and were further sub-cultured to obtain a pure culture. The bacterial isolates were identified by colony morphology (shape, structure, colour, pattern, size) gram staining and biochemical tests (indole production, MR, VP, citrate utilization, starch hydrolysis and Triple Sugar Iron) following the standard procedure.28,29
Antibacterial efficacy of toothpaste
The effectiveness of different toothpaste against the isolated bacteria was studied by well diffusion method. Mueller-Hinton Agar (MHA) plates were prepared, and wells were made using a sterile syringe puncture. The cultured bacterial samples were spread onto the plates using a sterile spreader, and the wells were filled with toothpaste using micropipettes. The antibacterial activity was evaluated by measuring the zone of inhibition in millimetre.
RESULTS AND DISCUSSION
Reduction in total microbial count by toothpastes
The present study evaluates the effectiveness of different toothpaste by percentage microbial reduction. The total microbial reduction ranges from 42.83 to 57.40% (Table 2); where Dabur Red being the highest, followed by Close-up (56.42 %), Babool (53.44 %) and Himalaya (48.82 %). Toothpaste containing triclosan as active ingredients (i.e., Colgate and Pepsodent) were found to reduce 45.46 % and 42.83% of oral bacteria after brushing. Several researchers also reported that triclosan is retained in the oral cavity 12 h after brushing, thereby allowing prolonged control of oral bacteria that forms plaque, gingivitis and oral malodor.13,30 Oral rinses and toothpaste manufacturers have incorporated plant-derived antimicrobials into their formulations, thereby demonstrating efficacy in reducing oral bacteria similar to that of non-herbal toothpastes containing triclosan, sodium fluoride, sodium saccharin or other ingredients.
Percentage reduction of oral bacteria by different kinds of toothpaste.
|Toothpaste Brand||Reduction of Bacteria (in %)|
Identification of bacteria
The predominant isolates were identified as Staphylococcus sp., Streptococcus sp., Bacillus sp., Enterobacter sp., Corynebacterium sp., Micrococcus sp. and Klebsiella sp. These microflora on the enamel constantly adapts to changing environment within protective biofilms that predominate mucosal surface of the oral cavity,31 Bacillus and Corynebacterium sp. are frequently observed in the oral cavity,32 Staphylococcus aureus in the oral cavity and perioral region33 and Streptococcus mutans are reported to be one of the main opportunistic pathogens of dental caries.34 Klebsiella and related Enterobacteriaceae degrade proteinaceous substance resulting in bad breath.35 The oral cavity comprises of microflora that may exist as nonpathogens but may become opportunistic to cause various oral cavity associated infections.
Antibacterial efficacy of toothpaste
In most cases, brushing alone is inadequate to remove oral biofilms which cause periodontal disease.36,37 Therefore, the study attempts to find out the efficacy of different kinds of toothpaste on oral bacteria that harbours the oral cavity which is depicted in Table 3. Among all the tested toothpaste in-vitro, Pepsodent was more effective showing a significant zone of inhibition against most of the isolated bacteria. Staphylococcus sp. and Streptococcus sp. were found to be susceptible against all the tested brands of toothpaste. Prasanth38 observed that Triclosan, sorbitol, and sodium monofluoro phosphate has an inhibitory effect on oral pathogens Streptococcus mutans and Escherichia coli. Toothpaste containing fluoride is effective in reducing the adherence capability of Streptococcus mutans strains.39 Herbal toothpaste like Dabur red, Babool and Himalaya are however effective, and showed significant results against isolated bacteria. Chowdhury, et al.40 formulated toothpaste with active natural ingredients of clove oil, lemon peel, orange peel, banana peel etc. Laboratory test results showed that the toothpaste can whitens the teeth, kills germs and imparts freshness feel inside mouth. Natural toothpastes and plant extracts also showed significant results to cure various diseases of teeth like gingivitis,41,42 tooth decay,43 cavity,44 gum bleeding,45,46 bad breath47 and dental-caries48 as well as anti-cancerous properties. The present study reported the efficacy of herbal toothpaste in reducing oral bacterial flora, which is also evident by several studies.31-35 However, these findings may not correspond to actual in vivo potentialities and behaviour of the toothpaste.
Antibacterial efficacy of toothpaste.
|Isolated bacteria||Zone of inhibition (in mm)|
Present study showed that herbal toothpastes are capable of combating periodontal microorganisms, thereby preventing periodontal diseases. Several studies have proven the medicinal values of herbal products. However, the active antimicrobial ingredients and formulations of synthetic toothpaste demonstrate their effectiveness in dental care. This study suggests the use of herbal toothpaste over synthetic toothpaste as they are of natural origin and have significant antimicrobial properties. The study also suggests that efforts must be made to find plants and formulate herbal toothpaste capable of preventing or minimizing the effects of dental or oral diseases.
The authors sincerely acknowledge Institutional Biotech Hub and Bioinformatics Centre (funded by DBT, New Delhi) of Gurucharan College, Silchar, Assam to carry out the present study.
The authors declare no conflict of interest.
Oral bacteria cause infections leading to dental problems in humans. The aim of this study was to determine total bacterial count before and after brushing and to evaluate antimicrobial efficacy of six different kinds of toothpaste against the oral bacteria. The total microbial reduction after brushing ranged from 42.83 - 57.40 %, where Dabur Red being the highest. Predominant isolates were identified as Staphylococcus sp., Streptococcus sp., Bacillus sp., Enterobacter sp., Corynebacterium sp., Micrococcus sp. and Klebsiella sp. The active antimicrobial ingredients and formulations of synthetic toothpaste demonstrate their effectiveness in dental care and reduction in oral microbial load, probably due to the presence of triclosan, fluoride, and other ingredients.
Bacterial community variation in human body habitats across space and time.
Oral microbial communities in sickness and in health.
Trends in Microbiology.
Traditional medicinal plant extracts and natural products with activity against oral bacteria: Potential application in the prevention and treatment of oral diseases.
Evidence-Based Complementary and Alternative Medicine.
Bacterial composition in the supragingival plaques of children with and without dental caries.
Advances in Medical Sciences.
Microorganisms as risk indicators for periodontal disease.
Ecology of the root canal flora.
Journal of endodontics.
Dental bacterial plaques.
Journal of Clinical Periodontology.
Dental education and oral health problems in India.
Indian J Dent Educ.
Prevalence of periodontal diseases in India.
J Oral Health Comm Dent.
Efficacy of mouth rinses and toothpaste on tooth whitening.
Halitosis (breath odor).
Advanced oral antibacterial/anti-inflammatory technology: A comprehensive review of the clinical benefits of a triclosan/copolymer/fluoride dentifrice.
The Journal of Clinical Dentistry.
Effect of Adhatoda vasica massage in pyorrhoea.
International Journal of Crude Drug Research.
Bakula-a reputed drug of Ayurveda, its history, uses in Indian medicine.
Indian Journal Of History of Science.
Suppression of protein kinase C and nuclear oncogene expression as possible molecular mechanisms of cancer chemoprevention by apigenin and curcumin.
Journal of Cellular Biochemistry.
In vitro anticancer activity of extracts of Mentha Spp.
Against Human Cancer cells.
Antibacterial activities of Emblica officinalis and Coriandrum sativum against Gram negative urinary pathogens.
Pakistan Journal of Pharmaceutical Sciences.
Anticancer activities of Nigella sativa (black cumin).
African Journal of Traditional, Complementary and Alternative Medicines.
Chemopreventive potential of neem (Azadirachta indica) on 7, 12-dimethylbenz [a] anthracene (DMBA) induced hamster buccal pouch carcinogenesis.
Journal of Ethnopharmacology.
Black tea polyphenols, theaflavins, prevent cellular DNA damage by inhibiting oxidative stress and suppressing cytochrome P450 1A1 in cell cultures.
Journal of Agricultural And Food Chemistry.
Benefit-risk assessment of the squeezed sap of the purple coneflower (Echinacea purpurea) for long-term oral immunostimulation.
Wound healing, oral and topical activity of Aloe vera.
Journal of the American Podiatric Medical Association.
Indian Spices and Condiments as Natural Healers.
Jaico Publishing House.
Adaptogens: Herbs for strength, stamina, and stress relief.
(Inner Traditions/Bear and Co).
Current developments in the discovery and design of new drug candidates from plant natural product leads.
Journal of Natural Products.
A meta-analysis of six-month studies of antiplaque and antigingivitis agents.
The Journal of The American Dental Association.
Practical Handbook Of Microbiology.
Microbiology: A Laboratory Manual.
Zinc citrate/Triclosan: A new anti‐plaque system for the control of plaque and the prevention of gingivitis: Short‐term clinical and mode of action studies.
Journal of Clinical Periodontology.
The oral microbiota: Living with a permanent guest.
Dna And Cell Biology.
Mg with 25 wetland plant species from a paper mill contaminated site in North East India.
Environmental Science and Pollution Research.
Staphylococcus aureus and the oral cavity: An overlooked source of carriage and infection?
American Journal of Infection Control.
Presence of Streptococcus mutans in saliva and its relationship with dental caries: Antimicrobial susceptibility of the isolates.
Isolation of Enterobacteriaceae from the mouth and potential association with malodor.
Journal of Dental Research.
Oral hygiene products: Potential for harm to oral and systemic health?
Antimicrobial efficacy of different toothpastes and mouthrinses: An in vitro study.
Dental Research Journal.
Resistance of oral bacterial species to varied toothpastes effects.
Int J Engg Res and Sci and Tech.
Herbal toothpaste: A possible remedy for oral cancer.
J Nat Prod.
The efficacy of a herbal-based toothpaste in the control of plaque and gingivitis: A clinico-biochemical study.
Indian Journal of Dental Research.
Clinical and antibacterial effect of an anti-inflammatory toothpaste formulation with Scutellaria baicalensis extract on experimental gingivitis.
Clinical Oral Investigations.
In vitro effect of zingiber officinale extract on growth of Streptococcus mutans and Streptococcus sanguinis.
International Journal of Dentistry.
Effect of Triphala on dental bio-film.
Indian Journal of Science and Technology.
Formulation of herbal toothpaste from chamomile and myrrh, a preliminary clinical evaluation on bleeding gum.
Journal of Mazandaran University of Medical Sciences.
Evaluation of the effect of green tea extract on mouth bacterial activity in the presence of propylene glycol.
Jundishapur Journal Of Natural Pharmaceutical Products.