Assessment of Cytotoxic and Antioxidant Effects of Rosmarinus officinalis and Fluoride containing Dentifrice - An In vitro Study

S. Sushanthi¹, Jayashri Prabakar²*, Meignana Arumugham Indiran³,

R. Pradeep Kumar³, Srisakthi Doraikannan⁴, Leelavathi L.⁴, S. Rajesh Kumar⁵

¹Post-Graduate Student, Saveetha Dental College, Saveetha University, Saveetha Institute of Medical and Technical Science, Chennai, India.

²Senior Lecturer, Department of Public Health Dentistry, Saveetha Dental College, Saveetha University, Saveetha Institute of Medical and Technical Science, Chennai, India.

³Professor, Department of Public Health Dentistry, Saveetha Dental College, SIMATS, Chennai- 77, India.

⁴Reader, Department of Public Health Dentistry, Saveetha Dental College, SIMATS, Chennai- 77, India.

⁵Department of Pharmacology, Saveetha Dental College, SIMATS, Chennai- 77, India.

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

ABSTRACT:

Background: Rosmarinus officinalis (rosemary) is a medicinal plant native to the Mediterranean region and cultivated around the world. This medicinal plant possesses antimicrobial and antioxidant properties. It has been proved that naturally occurring active ingredients of herbal medicinal plants shows least adverse effects and can act as effective oral hygiene aid for the control of common oral diseases without the need for synthetically derived or chemical agents. Hence, the present study invitro was designed to assess the cytotoxic and antioxidant effects of rosemary so that it can be used as an effective plaque control measures against various oral diseases. Materials and Methods: Rosemary leaves were procured from the markets of South India and were powdered to prepare the aqueous and ethanolic plant extract. The plant extracts were then subjected for cytotoxic and antioxidant analysis. Cytotoxic effects were evaluated using Brine shrimp lethality assay and antioxidant effects using DPPH radical scavenging assay. After the evaluation of antioxidant and cytotoxic property, rosemary-based toothpaste using herbal tooth formulation was prepared. Results: The results show that 5,10 and 15µl conc. all the nauplii fishes were alive in rosemary toothpaste whereas 60% of fishes were dead. Rosemary toothpaste have good antioxidant property when compared to fluoride toothpaste. Descriptive statistics were used. Conclusion: From the above findings, rosemary containing dentifrice has very less cytotoxicity and good antioxidant properties when compared with fluoride dentifrice and it can be served as an excellent replacement of several commercially available dentifrices.

KEYWORDS: Antioxidant, Cytotoxic, Dentifrice, Fluoride, Rosemary.

INTRODUCTION:

Since prehistoric times, people have exploring the nature, particularly, medicinal plants in search of new drugs¹. Herbal medicines are employed in a wide variety of health-related applications ranging from treatment of common colds to treatment of cancer². Plants are the main source of natural antioxidants, including several edible species³. Plant products have been part of phytomedicines since time immemorial⁴. A large number of medicinal plants and their purified constituents have shown beneficial therapeutic potentials⁵. The World Health Organization calculated that 80% of the population of developing countries relies on traditional medicines which is mostly plant drugs for their primary health care needs⁶. Cleaning of teeth regularly maintains the oral health condition of the teeth and gums. Plaque is the important etiological factor for origin of tooth decay, bad breath and gingival/periodontal diseases causing to loss of teeth⁷, so we need an agent which removes plaque. Toothpaste is a paste or gel to be used along with a toothbrush to conserve and enhance oral health condition and aesthetics⁸. Brushing with toothpaste is the most regular form of dental disease prevention in many countries around the world⁹.

Rosmarinus officinalis is a medicinal plant native to the Mediterranean region and grown all around the world. It is a perennial and aromatic plant, shrub with full branches or leaves, growing with a maximum height of 2 meters and with green leaves which has fragrance.

Excluding its therapeutic purpose, it is also used as condiment and food preservative too. It belongs to the family Lamiaceae and it is commonly used in folk medicine¹⁰. Scientifically, it has antispasmodic, anti-inflammatory, antimicrobial properties. Rosemary also used as preservative and spice in cooking and medicinal plant widely¹¹.

Many phytocompounds having pharmacological activities may be extracted from extracts of rosemary. These phytocompounds have caffeic acid, carnosic acid, rosmarinic acid rosmadial, rosemanol¹². This plant have several pharmacological actions and they include

1. Ability to reduce the effect of asthma, atherosclerosis, peptic ulcer etc

2. Antioxidant and anti-inflammatory activity of rosmarinic acid¹³.

3. Myocardial blood pressure reduction with rosmarinic acid¹⁴.

4. Ability to reduce anticancer and antiproliferative actions¹⁵.

The extract can be acquired from roots, leaves, stems, seeds, bark. Fresh and dried extracts can be used for study but still dried samples are best when compared to fresh samples¹⁶. Many studies make us to think that the antimicrobial property of rosemary have the potential to treat oral diseases. Rosemary leaves also economically available at the market and hence this can have easy access of purchase.

Diseases associated with oral cavity prevalent worldwide can be reduced by herbal toothpaste and the side effects are very less when compared with marketed toothpaste. Recently herbal dentifrice is increasing in markets as they have very fewer side effects, favorable for oral cavity and reduce tooth decay.

According to the World Health Organization (WHO), 80 percent of the people solely rely on herbal medicine for their primary needs¹⁷. In the developed and developing countries over 25% of the medicines are purely based on herbs and its derivatives. Many herbal extracts have been continuously used in dentistry as tooth cleansing agent. The use of herbal medicines is widely increasing day by day across the world. Although only few of the herbal medicines have been approved for their excellent medicinal properties. The effectiveness of basically herbal dentifrice is due to the presence of antimicrobial activity. Antioxidant principles from natural resources are multifactored in their multitude/magnitude of activities and give more scope in correcting the imbalance through regular intake of good diet¹⁸. The brine shrimp lethality bioassay has been used routinely in the primary screening of the crude extracts to assess the toxicity towards brine shrimp¹⁹. There is a great deal of interest in edible plants that contain antioxidants and health promoting phytochemicals, in view of their health implications²⁰. The elevating amount of natural material reimbursed into dental materials, as well as the paucity of studies in the literature depicting about the safety and effectiveness of these products, tells us the need for more studies of this type. In addition to this paucity of data, scientific evidence related to the therapeutic properties of rosemary²¹ suggest the creation of rosemary-based toothpaste for clinical assessments. Thus the aim of the study is to evaluate the cytotoxic effects and antioxidant effects of Rosmarinus officinalis based dentifrice which in turn enables this dentifrice to be safely used by the general population for the prevention of oral disease and promoting oral health.

OBJECTIVE:

1. To determine the cytotoxic effects of Rosmarinus officinalis-based dentifrice and fluoride containing dentifrice.

2. To determine the antioxidant effects of Rosmarinus officinalis-based dentifrice and fluoride containing dentifrice.

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 1gram of powder 100ml 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 ethanolic plant extract (refer Figure 2):

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

Figure 2: ethanolic extract of Rosmarinus officinalis

Preparation of Rosmarinus officinalis toothpaste using herbal toothpaste formulation:

Composition, function and quantity of the formulation of the toothpaste containing Rosmarinus officinalis.(100g of product)²²

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 (flavoring agent) 1.0g

Rosemary extract 9g

Distilled water (diluent) q.s.

The above -mentioned formulations is mixed with rosemary extract and triturated well until a paste consistency is formed. 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 Elico LI 120 pH meter. 1gram of toothpaste kept in 100ml of beaker to that 10ml of water is added stir vigorously and make a mixture. The prepared dentifrice was tested for cytotoxicity using Brine Shrimp lethality assay and antioxidant effect using DPPH radical scavenging assay.

CYTOTOXIC EFFECTS:

Setup preparation:

The artemia tank was filled with 6 liters of distilled water. To that 50 grams of iodine free salt was added and mixed well using a spatula. 2 capsules containing 15 grams of Brine Shrimp eggs were added to the tank and left undisturbed for 5 minutes for proper soaking in salt water²³.

After that, airline tip was placed inside the artemia tank and the aeration level was increased to maximum level. After 24 hours of incubation, the nauplii’s hatch out from the brine shrimp eggs, and observed using stereomicroscope. 6 tubes were taken and filled with 3ml of artificial sea water. To that 10 nauplii’s (refer Figure 3 and 4) were added in all test tubes.

Figure 3: Microscopic picture of nauplii

Figure 4: nauplii in a petridish

Rosemary toothpaste was loaded in the concentration range of 5ul , 10ul, 15ul, 30ul, 50ul . A control tube was prepared by adding 3ml of artificial sea water 10 nauplii. The tubes were kept for 24 hours of incubation. After incubation, the live and dead nauplii’s were counted and percentage death was calculated. This test is also done for rosemary aqueous extract, ethanolic extract and also for fluoride containing dentifrice.

Number of dead nauplii

Percentage Death =------------------------------------------------------ × 100

Number of dead nauplii-number of live nauplii

ANTI-OXIDANT ACTIVITY:

DPPH(2,2-diphenyl-1-picryl-hydrazyl-hydrate) radical scavenging assay was performed to monitor the antioxidant potential of plant crude extract. DPPH (oxidized form) is a stable lipophilic free radical, nitrogen-centered with purple color. The antioxidant can donate an electron to DPPH radical and the change in absorbance at 517nm will follow. Color will change to pale yellow gradually.

Test group: 10µL, 20µL, 30µL, 40µL and 50µL of the was taken in 5 test tubes (refer Figure 5) respectively. To each test tube 1ml of DPPH (2,2-diphenyl-1-picrylhydrazyl) was added. 1990µL, 1980µL, 1970µL, 1960µL and 1950µL of 50% methanol solution was added to the test tube containing 10µL, 20µL, 30µL, 40 µL and 50µL of rosemary toothpaste respectively. This test is also done for rosemary aqueous extract, ethanolic extract and fluoride containing dentifrice.

Control group: 1mL of DPPH was added to 2 mL of methanol solution. Standard group: Ascorbic acid was used as standard

The test tubes were incubated in a dark cupboard for around 20 minutes. Absorbance was measured at 517 nm in UV Spectrophotometer.

% inhibition was calculated using the following formula:

Control Absorbance-Sample Absorbance

% of inhibition = --------------------------------------------------------- × 100

Control Absorbance

Table 1: Cytotoxicity of dentifrice containing Rosmarinus officinalis, aqueous extract, ethanolic extract and fluoride containing dentifrice

Conc in µl	Viable nauplii				Percentage death of nauplii
Conc in µl	Aqueous extract	Ethanolic extract	Rosemary toothpaste	Fluoride toothpaste	Aqueous extract	Ethanolic extract	Rosemary toothpaste	Fluoride toothpaste
5 µl	10	9	10	4	0%	10%	0%	60%
10 µl	10	7	10	4	0%	30%	0%	60%
15 µl	10	6	10	4	0%	40%	0%	60%
30 µl	10	4	4	0	0%	60%	60%	100%
50 µl	9	4	2	0	10%	60%	80%	100%

Figure 6: Antioxidant activity of dentifrice containing Rosmarinus officinalis and fluoride containing dentifrice.

DISCUSSION:

Natural products are precious and undiscovered source of effective antimicrobials, with less amount of toxicity. Current researches in analytical technology, particularly those related to phytotherapy, have introduced a new era of anti-plaque therapies and natural products²⁴. In this sense, plant extracts appear as an effective replacement for the treatment of periodontal diseases in view of the fact that pharmacological research induce the antibacterial and anti- inflammatory activities of rosemary. Some studies suggest that rosemary extract may involve in the synthesis of anti- inflammatory leukotrienes in intact polymorphonuclear cells. In addition, it can also inhibit 5-lipoxygenase, cyclooxygenase-2 (CPX-2) and the mobilization of intracellular calcium ions in mice with inflamed skin²⁵.

A number of authors have done clinical trials to find out the effectiveness of dentifrice containing herbs, as well as, there are reports in the scientific literature of researches with mouthwashes which includes the rosemary in its composition^12,13,26.

Rosemary contains terpenoids, flavonoids, phenols and essential oils. Each of these compounds has its own pharmacological properties¹⁸, making it a precious raw material for therapeutic products, such as the toothpaste produced for the present study. The good performance of this experimental toothpaste, in relation to gingival bleeding and plaque reduction, was due to the previously proven anti-inflammatory and antimicrobial therapeutic properties of rosemary²⁷. The experimental toothpaste is efficient of decreasing biofilm and gingival bleeding. The reduction of biofilm has also been exhibited in vivo by Rasooli et al.²⁸, who utilized the essential oil of rosemary and explained its use in new anti-caries treatment protocols.

According to Silva et al.¹¹, the antimicrobial power of rosemary against oral bacteria is explained. These authors used specific group of bacteria from the oral cavity (Streptococcus mitis, Streptococcus mutans and Lactobacillus casei) in planktonic form. These findings were collaborated by Freires et al.²⁹ who conducted a systematic review on the antimicrobial power of the essential oils of medicinal plants which includes rosemary.

In the present study it was seen that for the dentifrice containing Rosmarinus officinalis, at 5µL concentration there was a death of 0% of nauplii at 5µL, 10µL and 15 µL, there was a death of 60% of nauplii at 30µL and at 50µL there was a death of 80% of nauplii. Percentage of inhibition of DPPH free radicals (anti-oxidant activity) was 51.7% at 10µL concentration, 53.5% at 20µL, 58.4% at 30µL, 61.3% at 40µL and 62.6% at 50µL.

As the concentration increased the cytotoxicity and anti-oxidant activity of the dentifrice containing Rosmarinus officinalis increased. The highest percentage of death of nauplii was at 50µL concentration of the dentifrice containing Rosmarinus officinalis whereas for fluoride containing dentifrice at 30µL and 50µL concentration, there was 100% death of nauplii. The values for anti-oxidant property of the dentifrice containing Rosmarinus officinalis was found to be mostly same as that of standard values whereas for fluoride containing dentifrice percentage inhibition values was found to be in negative for 10µL (-46.5%) and 20µL (-30%). According to Rafie et al, chemical compounds present in the Rosemary are known to be strong antioxidants hence the fluoride toothpaste formulation lack the antioxidant which is naturally present in Rosemary30. According to Hendel N et al, rosemary methanolic extract exhibited high radical scavenging activity (11.741±0.004µg/ml) which is close to that of synthetic tested antioxidants³¹. According to Ledder et al.³² plant extracts reimbursed into toothpaste formulation add to the action of other components, improving the efficiency of product.

CONCLUSION:

Therefore, a toothpaste such as the one produced in the present study, which contained extract of a medicinal plant with therapeutic properties (rosemary), is scientifically relevant and can be classified as a significant technological innovation. The experimental toothpaste containing Rosmarinus officinalis, had very less cytotoxicity and good antioxidant properties when compared with conventional fluoridated toothpaste. Therefore, it is a viable alternative to the synthetic products used in the field of dentistry.

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Received on 08.07.2020 Modified on 10.08.2020

Research J. Pharm. and Tech. 2021; 14(7):3779-3784.

DOI: 10.52711/0974-360X.2021.00654