Evaluation of Concentration Dependent Antimicrobial Efficacy of Herbal And Non Herbal Dentifrices against Salivary Microflora – An In vitro Study

Published by Oriental Scientific Publishing Company © 2018 This is an Open Access article licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-nc-sa/4.0/ ), which permits unrestricted Non Commercial use, distribution and reproduction in any medium, provided the original work is properly cited. Evaluation of Concentration Dependent Antimicrobial Efficacy of Herbal And Non Herbal Dentifrices against Salivary Microflora – An In vitro Study

Dental plaque can be defined as the soft deposits that form the biofilm adhering to the tooth surface or other hard surfaces in the oral cavity, including removable and fixed restorations. 1 Microorganisms present in dental plaque cause dental caries and periodontal diseases. 2 Dental caries is a localized, transmissible infectious process that ends up in the destruction of hard dental tissue. It results from accumulation of plaque on the surface of the teeth and biochemical activities of complex micro-communities. Streptococcus mutans is one of the main opportunistic pathogens of dental caries, 3 which plays a central role in fermenting carbohydrates resulting in acid production, and leading to the demineralization of the tooth enamel. In addition, other microflora like Escherichia coli and Candida are also associated with active caries lesions. Candida albicans is the most common yeast isolated from the oral cavity. It is by far the fungal species most commonly isolated from infected root canals, showing resistance to intracanal medication. 4 Poor oral hygiene is one of the reasons for accumulation of these microbes and their harmful activities. Periodontal diseases are bacterial infections that affect the supporting structure of the teeth (gingival, cementum, periodontal membrane and alveolar bone). The hydrolytic enzymes, endotoxins, and toxic bacterial metabolites are involved in this disease. Gingivitis, an inflammatory condition of gum, is the most common form of periodontal disease. Serious forms of periodontal disease that affect the periodontal membrane and alveolar bone may result in tooth loss. Streptococci, Spirochetes and Bacteroides are found to be the possible pathogens responsible for the disease. 5 Biofilm formation is a natural process in the oral environment, but needs to be controlled through regular brushing in order to prevent the development of caries and periodontal diseases. 6 Tooth brushing with toothpaste is the most widely practiced form of oral hygiene in most countries. 7 Nevertheless, in many individuals, the customary oral hygiene method of tooth brushing is, by itself, usually insufficient over a long period to provide a level of plaque control consistent with oral health. 8 Hence, the addition of antimicrobial agents to toothpaste has been suggested as one possible method to improve the efficacy of mechanical tooth-cleaning procedures, 9, 10 aiding the control of dental plaque and preventing dental caries and periodontal diseases. 11,12 When these substances are added to oral products, they kill microorganisms by disrupting their cell walls and inhibiting their enzymatic activity. They prevent bacterial aggregation, slow multiplication and release endotoxins. [12][13][14] Several clinical studies have demonstrated the inhibitory effects of antimicrobial dentifrices, commonly known as tooth-pastes, on oral bacteria and gingiva. 8,10,15 Also, the antimicrobial agents in dentifrices can significantly reduce contamination of toothbrushes. 16,17 Fitzgerald, Jordan, and Achard in 1964 18 demonstrated that dental caries would not occur in the absence of microorganisms. Complete removal of microorganisms from the oral cavity is impossible, but reduction in microbial count may reduce the cariogenic effect. Oral cavity is an ecological niche, which contains 500-1000 different types of bacteria along with fungi, protozoa, and occasionally viruses. Oral microflora increases due to frequent intake of sucrose and carbohydrate and poor oral hygiene and decreases due to various preventive measures such as topical fluoride application, pit and fissure sealants, etc., the mechanical measures used in our daily routine, such as tooth brushing, dental flossing, etc., if complimented with a dentifrice having antimicrobial efficacy, can reduce the microbial count of the oral cavity on a daily basis and, thus, reduce the prevalence of dental caries and periodontal disease.
Routinely used synthetic dentifrices contain fluoride and triclosan, which are known to produce harmful side effects such as mucosal ulceration, circum-oral dermatitis, dental fluorosis, etc. on prolonged use. 19,20 Hence, despite the efficacy of many synthetic toothpaste formulations with antibacterial properties, 21,22 there is an increasing societal desire to rely on naturally occurring compounds for health care, which has also found its way into dentistry 23 . Consumers who gravitate towards using herbal products often view these products as being much safer. Their efficacy can be attributed to various properties such as anti-inflammatory, anti-microbial, astringent action, anti-diabetic, anti-fungal, analgesic and antiseptic properties. 24 herbs have been used in India and south Asia for thousands of years to clean and fight bacterial and fungal infections. 25 Herbal medicine has made significant contribution to modern medical practice as well 26 . The antimicrobial activity of the herbs is due to the presence of secondary metabolites such as alkaloids, flavonoids, polyphenols, and lectins 27 . This study focuses on evaluating the concentration dependent antimicrobial efficacy of 3 herbal dentifrices in comparison with a conventional non herbal dentifrice.

MATERIALS AND METHODS
Four toothpastes namely one non herbal toothpaste: Pepsodent Germicheck and three herbal tooth pastes: Lever -Ayush Anti Cavity Clove Oil, Dabur Red and Himalaya Sparkling White were selected for assessment of their in vitro antimicrobial activity. The products were collected from local market, Chennai. Approval was obtained from the Institution's Ethical Committee. Informed consent was obtained from the study participants.  Selection of patients Inclusion criteria 10 participants in the age group of 19-21 years with good general health following routine oral hygiene procedures and agreement to comply with the study visits were included in the study.

Exclusion criteria
Participants suffering from systemic diseases, currently using antibiotics, undergoing orthodontic treatment, using additional oral Hygiene aids other than toothbrush & toothpaste, having adverse habits like smoking, alcohol consumption were excluded from the Study.

Method of saliva collection and storage
The subjects were told to rinse with water; saliva was allowed to accumulate in the floor of the mouth for approximately two minutes and by asking the subject to spit in uricol container. By following the above mention method, 10 samples were collected in the early morning time. The samples were transported immediately to the laboratory.

Minimal inhibitory concentration of toothpastes
All toothpaste samples were diluted in saline and prepared series dilution. The concentrations considered -25%, 50% and 100%.

Antimicrobial assay
The antimicrobial activity of different concentrations of the dentifrices was determined by modified agar well diffusion method. In this method, nutrient agar plates were seeded with 0.5 ml collected saliva sample for 24h. The plates were allowed to dry for 1 h. A sterile 8 mm cork borer was used to cut one central and two wells at equidistance in each of the plates. Dentifrice dilutions at different concentrations were introduced into each of the three wells. The plates were incubated at 37ºc for 24 h. The antimicrobial activity was evaluated by measuring the zone of inhibition around the wells.

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From Table 1 and Figure 1, non -herbal tooth paste (Pepsodent Germicheck) showed zones of inhibition of 32 mm, 37 mm, and 37 mm in 25%, 50% and 100% concentrations respectively. Comparing the zones of inhibition across the varying concentrations of the different dentifrices it was observed that the antimicrobial efficacy was similar in herbal and non-herbal toothpastes and also in their different concentrations.

DISCUSSION
Microorganisms play a vital role in causation of dental caries. Various studies 18 have provided evidence of bacterial specificity in caries aetiology. Complete removal of microorganisms from the oral cavity is impossible, but their count can be reduced so that it becomes less cariogenic with the help of various preventive measures, for example, probiotics, antibiotics, fluorides, and oral hygiene aids. Various oral hygiene measures are available, such as tooth brushing, dental flossing, mouthwashes, dentifrices, etc., among which tooth brushing with dentifrice is the most commonly used. These mechanical measures are feasible, cost-effective, and can easily be used by children. Dentifrices are therapeutic mechanical aids which are available as tooth powder or toothpaste and aid in removal of plaque. Their antimicrobial effect has been proven in various studies. 28 In our study, four toothpastes namely one non herbal toothpaste (pepsodent germicheck) and three herbal tooth pastes (ayush clove oil, dabur red and himalaya sparling white) were selected for assessment of their in vitro antimicrobial activity.
In fluoridated dentifrice, fluoride is mostly available in the form of sodium fluoride or sodium monofluorophosphate. 29 Antimicrobial activity of fluoridated toothpastes occurs by interfering with the glucose transport, carbohydrate storage, extracellular polysaccharide formation and acid formation by oral streptococci. 15 T r i c l o s a n ( 2 , 4 , 4 ' -t r i c h l o r o -2'hydroxydiphenylether) is a phenolic agent with broad-spectrum antibacterial activity that disrupts bacterial cytoplasmic membranes by blocking fatty acid biosynthesis i.e., the enzyme enoyl-acyl carrier protein reductase (enr). 2,30,31,32 Although the commonly used and recommend toothpastes by WHO, ADA, FDI is the fluoride and triclosan containing, some of the constituents of these toothpastes have undesirable side effects like staining, abrasions and taste alterations. 13,25 Moreover, these pastes are not recommended in high fluoridated belt and also in children under 3 years of age as they could cause dental fluorosis, skeletal fluorosis and destruction of epithelial layer of intestine. 33 . Thus, the quest for a dentifrice which has less abrasive effect, less chemical agents, and more antimicrobial property made the researchers focus on age-old medicinal alternative "herbs." 29 The antimicrobial activity of plant extracts is due to the presence of secondary metabolites, such as alkaloids, flavonoids, phenolics, polyphenols, quinines, flavones, flavonols, tannins, terpenoids, lectins, polypeptides, proanthocynidins, tannins, and quercetin. 34 plant extracts provide protection by immune stimulation and do not have any known side effects. 35 clove oil has biological activities, such as antibacterial, antifungal, insecticidal and antioxidant properties, and is used traditionally as a savoring agent and antimicrobial material in food 36,37,38 . In addition, clove oil is used as an antiseptic in oral infections 39,40 . This essential oil has been reported to inhibit the growth of molds, yeasts and bacteria 41 . The high levels of eugenol contained in clove essential oil are responsible for its strong biological and antimicrobial activities. It is well known that both eugenol and clove essential oil phenolic compounds can denature proteins and react with cell membrane phospholipids changing their permeability and inhibiting a great number of gram-negative and gram-positive bacteria as well as different types of yeast. 42,43 Meswak herb is a rare, potent, priceless wonder herb that delivers incredible oral care benefits. It is scientifically proven to reduce tartar and plaque, fights germs and bacteria to keep the gum healthy, helps prevent tooth decay, eliminates bad breath, and ensures strong teeth. In sa. Persica (meswak), a natural component benzylisothiocyanate (bit) is present that acts as an inhibitor of bacterial growth and their acidic products. Also, the antibiotic effects found in meswak may prevent the attachment of bacteria. 44 the antibacterial effects of cinnamon is due to its active constituents such as cinnamaldehyde and eugenol. 45 the antibacterial activity and inhibition activity of ginger extracts could be attributed to zingiberene. Other components include ²-sesquiphellandrene, bisabolene and farnesene, which are sesquiterpenoids, and trace monoterpenoid fraction, (² sesquiphellandrene, cineol and citral) 46 . The principle active constituents of peppermint are the essential oils, which comprise about 1% of the herb. The oils are dominated by monoterpenes, mainly menthol, menthone and their derivatives (e.g., isomenthone, neomenthol, acetylmenthol, pulegone). These essential oils dilate blood vessels and inhibit bacteria. Especially menthol has a broad spectrum antibacterial activity 47 . The antibacterial activity of Carica papya may be attributed to the presence of bioactive compounds such as phenols, saponin, steriods, alkaloids and flavonoids 48 .
In our study, the antimicrobial efficacy was similar in herbal and non-herbal tooth pastes and also in their different concentrations. Based on another study by Mahesh R. K. et al, there is a trend to believe that the herbal toothpastes possess significant salivary glucose inhibitory activity and favor transient increase in salivary ph. 49 The anti-plaque and anti-gingival effects of herbal toothpaste, which were comparable to those of conventional toothpastes. [50,51,52,53,54] In a study by Vyas Y.K., Arodent, a herbal tooth paste possesses good antibacterial activity against both cariogenic bacteria S. mutans and L. acidophilus, and its antibacterial activity is comparable to the standard dentifrice Colgate. 55 Another study by Neha Bhati et al states that the three test dentifrices (tooth brushing using nonherbal fluoridated dentifrice, A. Vera containing dentifrice, and meswak containing dentifrice) have equal antimicrobial efficacy. 29 it can be inferred from the above results that the herbal dentifrice is as efficacious as the conventionally formulated dentifrice and could be used as an alternative for people interested in natural products.
Although the bacterial count had shown reduction in 15 days and remained the same over a period of 1 month (30 days), the prolonged effect on the bacterial count, as well as whether the reduced count is stabilized or the counts return back to baseline values need to be checked. Whether there is any bacterial resistance developing to these products also needs to be monitored carefully in further studies. 29 Herbal products had less potency as an antimicrobial agent in a study, but the tannins, flavonoids and lectins present in it definitely strengthens the periodontium and gingival tissue 56 . It can definitely be used in children upto 3 years of age to avoid fluoride toxicity as fluoride uptake upto 0.3-0.6ppm is sufficient for this age group which generally met through the dietary supplements and drinking water as specified by the AAPD guidelines 2013. 57

CONCLUSION
We can advocate herbal dentifrices, as there is a sudden surge in the concern over using chemical and non-herbal products. Thus, comparable properties with standard dentifrices make herbal dentifrices a viable option for plaque control.