Comparing the Antimicrobial efficacy of Dentifrice containing Rosmarinus officinalis and Fluoride containing Dentifrice - An In vitro study

Indumathy Pandiyan¹, Jayashri Prabakar², Meignana Arumugham Indiran³,

Pradeep Kumar Rathinavelu⁴, Srisakthi Doraikannan⁵, Leelavathi. L⁶, S. Rajeshkumar⁷

¹Post-graduate Student, Department of Public Health Dentistry, Saveetha Dental College and Hospital, Chennai.

²Senior Lecturer, Department of Public Health Dentistry, Saveetha Dental College and Hospital, Chennai.

³Professor and Head of the Department, Department of Public Health Dentistry,

Saveetha Dental College and Hospital, Chennai.

⁴Professor, Department of Public Health Dentistry, Saveetha Dental College and Hospital, Chennai.

⁵Reader, Department of Public Health Dentistry, Saveetha Dental College and Hospital, Chennai.

⁶Senior Lecturer, Department of Public Health Dentistry, Saveetha Dental College and Hospital, Chennai.

⁷Associate Professor, Department of Pharmacology, Saveetha Dental College and Hospital, Chennai.

*Corresponding Author E-mail: indupandiyan1995@gmail.com

ABSTRACT:

Background: Dental biofilm is still a concern for dental professionals, given that it has been correlated with the aetiology of oral diseases. Herbal medicine or plant derived extracts are considered to be safer and exhibits less or no adverse effects in comparison with chemically synthesized medicines. Hence, herbal medicine and their extract can be used as an adjuvant in dental disease treatment. One among the various plant extract exhibiting antimicrobial activity against oral microorganisms is Rosemarinus officinalis is an aromatic plant belonging to lamiaceae family originating from Mediterranean region. Therefore the present study was designed to assess the antimicrobial efficacy of Rosmarinus officinalis and Fluoride containing dentifrices against streptococcus aureus, Enterococcus Faecalis and Candida albicans. Materials and Method: Rosemary leaves were procured from the markets of South India and were powdered to prepare the aqueous and ethanolic extract of Rosmarinus Officinalis. Both the extracts of Rosmarinus Officinalis and Fluoride containing dentifrice were subjected to antimicrobial analysis. The antimicrobial activity was evaluated using Agar well diffusion method. The diameters of the zones of inhibited growth were measured to the nearest whole millimetre. Data was entered in Microsoft excel spread sheet and analysed using SPSS software (version 23). Descriptive statistics were expressed by means of percentage. Results: The antimicrobial activity of Ethanolic extract of Rosmarinus officinalis at 100 conc. showed zone of inhibition of 20mm, 22mm, 17mm against Streptococcus mutans, Enterococcus faecalis and Candida albicans respectively. Hence the Rosemary toothpaste had good antimicrobial property when compared to fluoride toothpaste. Conclusion: Based on the results of the present study, it can be concluded that, rosemary containing dentifrice exhibited good antimicrobial properties when compared with fluoride dentifrice and it can be serve as an excellent replacement of several commercial available dentifrices.

KEYWORDS: Antimicrobial; dentifrice; fluoride; herbal; Rosemarinus officinalis.

INTRODUCTION:

In spite of the tremendous progress and growth in the development of medical science, plants and herbs continue to be an important source of drugs in many countries. During the past two decades reliability and usage of herbs and herbal related product has become of increasing importance, due to the side effects and complications of many chemical and synthetic medicines¹.Almost 25% of drugs are derived from plants and many other are formed from prototype compounds isolated from plant species². Herbal medicine is based on the idea that plants contain natural substances that can promote health and assuage illness. There could be direct side effects, allergic reactions, effects from contaminants and/or interactions with drugs and other herbs. Studies should be conducted to examine the side effects considering the interaction with other herbs and modern drugs³. Recently the use of herbal preparations in remedies for various medical conditions have been rapidly increasing especially in India⁴. Sesbania grandiflora L. (Fabaceae) is popularly known as “Basna” is an ornamental plant and is found in plains of western Himalayas to Sri Lanka. Sesbania grandiflora is a folk remedy for bruises, catarrh, dysentry, eyes, fevers, headaches, small pox, sores, sore throat and stomatitis⁵. The aqueous and ethanolic extract of S. lappa roots significantly inhibited the growth of microorganisms as compared to standard bactericide and fungicide drugs. Diethyl ether fraction exhibited prominent fungicidal activity against Candida albicans⁶.

Oral health can be defined as being multidimensional in nature, including physical, psychological, emotional, and social domains that are integral to overall health and well-being⁷. Over 750 species of bacteria inhabit the oral cavity (50% of which are yet to be identified) and a number of these are implicated in oral diseases⁸. Dental caries remains the most prevalent and costly oral infectious disease worldwide. The prevalence of dental caries is generally estimated at the ages of 5, 12, 15, 35–44 and 65–74 years for global monitoring of trends and international comparisons. As per the WHO Oral Health Surveillance 1992, the DMFT index in 12-year-old Indian is 0.89. Streptococcus mutans, a major etiological agent of human dental caries, lives primarily on the tooth surface in biofilms. The ability of S. mutans to bind and colonize the tooth surface is the result of many cell surface-localized and secreted factors and is significantly influenced by the availability of dietary sucrose^9.

The Periodontal diseases are one of the major causes of tooth loss in India. Periodontal disease is mostly linked with anaerobic Gram-negative rods such as A. actinomycetemcomitans, P. gingivalis, Tannerella forsythus, Bacteroides, Prevotella and Fusobacterium species. These periodontopathogens are frequently isolated from periodontal pockets of patients with periodontitis¹⁰. Toothpaste is the common term used to describe preparations that are used with a toothbrush with the purpose to clean and polish the teeth. Tooth brush has two objectives: to help the toothbrush in cleaning the tooth surface and to provide a therapeutic effect. The therapeutic effect may have an anti-plaque or anti-inflammatory basis when the nature of the agents is antimicrobial¹¹. Nowadays many herbal types of toothpaste are commercially available in markets as an agent for brushing.

R. officinalis, is a plant belonging to the family Lamiaceae and was originated from the Mediterranean region. However, it could be found all over the world. It is a perennial and aromatic plant, shrub-shaped with branches full of leaves, having a height of up to two meters and green leaves that exude a characteristic fragrance. It is also known that Rosmarinus officinalis may have beneficial pharmacological effects, protecting the tissues from the damaging effects of free radicals as reactive oxygen species¹². Dentifrices which are labelled as “natural” do not include ingredients such as synthetic sweeteners, artificial colours, preservatives, additives, or synthetic flavours and fragrances^13. The term “herbal” on the label implies that most of these dentifrice’s active ingredients are plant-based¹⁴. Plant based dentifrices shows minimal or no side effects. Hence, our study is framed to assess the antimicrobial effects in rosemary so that it can be incorporated in the dentifrice without harming the humans.

MATERIALS AND METHODS:

Study design: Invitro study

Preparation of aqueous plant extract (refer Figure 1): Rosemary leaves were obtained from the markets of South India and were powdered using a mixer grinder. To the prepared 1 gram of powder 100 ml of distilled water is added and heated in a heating mantle for 60^oC to 80^oC for 10 minutes until the bubbles appeared. The solution was then filtered using a funnel and a Whatman filter paper and collected in a conical flask to obtain the plant extract. Then the plant extract was transferred to an air tight container and refrigerated overnight.

Figure 1: Aqueous extract of Rosmarinus officinalis

Preparation of Ethanolic plant extract (refer Figure 2):

To the prepared 5gram of rosemary powder 50ml of ethanol is added and it is kept in a refrigerator.

Figure 2: Ethanolic extract of Rosemarinus officinalis

Preparation of Rosmarinus officinalis toothpaste using standard toothpaste formulation:

Composition, function and quantity of the formulation of the toothpaste containing Rosemarinus officinalis (100g of product) were done using products of Calcium carbonate(abrasive)- 35g, Sorbitol(humectant)- 15g, Carboxymethylcellulose(agglutinant)- 0.7g, Carbopol 940(agglutinant)-0.3g, Sodium lauryl sulphate (foaming agent)-1.0g, Saccharin (sweetener)- 0.5g, Sodium benzoate (conservative)-0.1g, Menthol (flavouring agent)-1.0g, Distilled water (diluent). The above mentioned formulations is mixed with 9g of rosemary extract and triturated well until a paste consistency is formed¹⁵. The parameters were checked to obtain the marketed herbal formulation. The formulated toothpaste and marketed toothpaste kept for long period of time to test any change in appearance. pH of the formulated herbal toothpaste was determined using pH meter (Elico LI 120 pH meter). 1 gram of toothpaste kept in 100 ml of beaker to that 10 ml of water is added stir vigorously and make a mixture. The prepared dentifrice was tested for antibacterial property.

Antimicrobial activity using Agar Well Diffusion Method:

Media preparation:

100ml of Mueller Hinton agar (for Streptococcus mutans and Enterococcus faecalis) and 20ml of Rose bengal agar (for Candida albicans) was prepared, sterilized and poured onto the petriplates. The plates were allowed for solidification.

Swabbing:

After solidification, the respective plates were swabbed with the oral pathogens of Streptococcus mutans, Candida albicans and Enterococcus faecalis which was isolated from saliva sample and identified in microbiology lab.

Well formation:

After swabbing, three wells on each plate were formed using a gel puncher. To those three wells, Rosemary extract solution was loaded in the concentration range of 25µL, 50µL and 100µL. The plates were then incubated at 37^oC for 24h and after incubation, the zone of inhibition was measured and calculated.

Measurement of Zone of Inhibition (refer Figure 3, 4 and 5):

The test plates were held in front of a desk lamp, and the zones were measured with a ruler held against the back of the petri plate. The diameters of the zones of inhibited growth were measured to the nearest whole millimetre. The antimicrobial activity of Rosemary incorporated toothpaste against Streptococcus aureus, Enterococcus faecalis, Candida albicans under two formulations of Rosemary toothpaste were assessed.

Statistical Analysis:

Data was entered in Microsoft excel spread sheet and analysed using SPSS software (version 23). Descriptive statistics were expressed by means of percentage.

Figure 3: Zone of inhibition of Aqueous and ethanolic extract of Rosmarinus officinalis against S.mutans

Table 1: Antimicrobial property of dentifrice containing Rosmarinus officinalis (aqueous extract, ethanolic extract) and fluoride containing dentifrice

Conc in µl	Aqueous extract of *Rosmarinus officinalis* Toothpaste (in mm)			Ethanolic extract of *Rosmarinus officinalis* Toothpaste (in mm)			Commercial Toothpaste (in mm)
Conc in µl	*S. mutans*	*E. faecalis*	*C. albicans*	*S. mutans*	*E. faecalis*	*C. Albicans*	*S. Mutans*	*E. Faecalis*	*C. Albicans*
25 µl	12	11	9	20	13	12	18	15	14
50 µl	16	15	14	22	17	18	21	16	18
100 µl	18	18	17	25	20	24	22	18	20
Ab	30	30	40	22	30	32	25	26	15

DISCUSSION:

Natural products are a valuable and unexplored source of potentially effective antimicrobials, with low levels of toxicity. Plants and plant products have been used extensively throughout history to treat medicinal problems¹⁶. Recent advances in analytical technology, particularly those related to phytotherapy, have created a new era of anti-plaque therapies and natural products^17,18. In this sense, plant extracts and their compounds appear as an alternative for the treatment of periodontal diseases in view of the fact that pharmacological research evoked the antibacterial and anti-inflammatory activities of rosemary. Liza Barreto et al¹⁹analysed in vitro the antimicrobial potential of seven dentifrices containing medicinal herbs and concluded that most of the dentifrices exhibited antimicrobial activity against the analysed microorganisms. Furthermore, according to Liza-Barreto et al plant extracts incorporated into dentifrice formulae add to the action of fluorine, thereby improving (and increasing) the therapeutic properties of the product. Studies reported that rosemary extract may act in the synthesis of anti-inflammatory leukotrienes in intact poly morphonuclear cells. In addition, it can inhibit 5-lipoxygenase, cyclooxygenase-2 (CPX-2) and the mobilization of intracellular calcium ions in mice with inflamed skin²⁰.

A number of authors have conducted clinical trials to assess the effectiveness of toothpastes containing herbs, as well as, there are reports in the scientific literature of researches with mouthwashes which contained the rosemary in its composition^20-23. Nascimento et al. (2000) proposed the association between antibiotics and plant extracts for resistant bacteria, indicating the occurrence of synergism, allowing ineffective antibiotics to exert action against bacteria^24.

Silva et al also confirmed the antimicrobial power of rosemary against oral bacteria in their research. These authors used common bacteria from the oral cavity (Streptococcus mitis, Streptococcus sanguis, Streptococcus sobrinus, Streptococcus mutans and Lactobacillus casei) in planktonic form²⁵. These findings were corroborated by Freires et al. who conducted a systematic review on the antimicrobial power of the essential oils of medicinal plants, including rosemary²⁶. In our present study it is seen that, at 100µl concentration, ethanolic extract of Rosmarinus offcinalis toothpaste has zone of inhibition of 25mm, 20mm, 24mm against S. mutans, E. faecalis, C.albicans respectively. At the concentration of 50µl, zone of inhibition for ethanolic extract of Rosmarinus officinalis toothpaste was 22mm and 17mm against S. mutans, E. faecalis respectively, which is actually greater than the commercially available tooth paste which showed 21mm and 16mm zone of inhibition against S. mutans, E. faecalis respectively.

Komiyama et al carried out an in vitro assessment of the effect of commercially available dentifrices in Brazil in the context of S. mutans growth, using a methodology similar to the present study. Those authors concluded that dentifrices containing triclosan or a combination of herbal extracts were the most effective for inhibiting the growth of this bacterium²⁷. Gupta et al²⁸evaluated the anti-plaque efficacy of a commercially available herbal dentifrice compared to conventional fluoride toothpaste and found a significant reduction in mean plaque index scores in subjects of both groups.The present study showed antibacterial activity of commercial toothpaste with zone of inhibition 22mm, 18mm and 20m against S. mutans, E. faecalis, C.albicans respectively. Currently, some plants are being researched to fight infections that affect the oral cavity, mainly caused by the presence of bacterial biofilms²⁹. Takarada et al³⁰ reported that rosemary essential oil showed inhibitory effect on the adherence of S. mutans and inhibitory activity against the growth of Gram-negative bacteria (A. comitans, P. gingivalis and F. nucleatum). In the present study, the best results with the dentifrice containing the ethanolic extract of rosemary occurred for the pathogens S. mutans, E. faecalis and C. albicans, for which antimicrobial activity of the rosemary-based dentifrice proved its efficacy.

CONCLUSION:

We suggest that the search for alternative therapies represents the main goal of research into the properties of plant extracts. Nevertheless, the in vitro method is a well-established technique which is commonly used for evaluating the antimicrobial efficacy of chemicals before in vivo testing. Findings from this study suggests that Rosmarinus officinalis incorporated toothpaste exhibits antimicrobial activity against Streptococcus mutans, Candida albicans and Enterococcus faecalis and therefore, can be used as an better novel alternative to commercially available dentifrice.

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Received on 08.07.2020 Modified on 30.08.2020

Research J. Pharm. and Tech. 2021; 14(7):3651-3656.

DOI: 10.52711/0974-360X.2021.00631