Formulation and Evaluation of Herbal Toothpaste against Biofilm Producing Staphylococcus aureus

Shreeti Mishra; Andrea Pereira Kolla; Rakhi Bajpai; Chitranshu Pandey; Varaprasad Kolla*

doi:10.52228/NBW-JAAB.2023-5-1-7

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Formulation and Evaluation of Herbal Toothpaste against Biofilm Producing Staphylococcus aureus

Author(s): Shreeti Mishra¹, Andrea Pereira Kolla², Rakhi Bajpai³, Chitranshu Pandey⁴, Varaprasad Kolla*⁵

Email(s): ¹shreeti123mishra@gmail.com, ²annpereira@gmail.com, ³bajpai.rakhi683@gmail.com, ⁴chitranshupandeyniu@gmail.com, ⁵naidu.prasad@gmail.com

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⁵

Published In: Volume - 5, Issue - 1, Year - 2023

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Cite this article:
Shreeti Mishra, Andrea Pereira Kolla, Rakhi Bajpai, Chitranshu Pandey, Varaprasad Kolla (2023) Formulation and Evaluation of Herbal Toothpaste against Biofilm Producing Staphylococcus aureus. NewBioWorld A Journal of Alumni Association of Biotechnology,5(1):37-44.

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NewBioWorld A Journal of Alumni Association of Biotechnology (2023) 5(1):37-44

RESEARCH ARTICLE

Formulation and Evaluation of Herbal Toothpaste against Biofilm Producing Staphylococcus aureus

Shreeti Mishra¹, Andrea Pereira Kolla², Rakhi Bajpai³, Chitranshu Pandey⁴, Varaprasad Kolla⁵*

¹Ajjenkya DY Patil University, Pune, Maharashtra, India

²Seth Phoolchand Agrawal Smriti College, Nawaparra, Chhattishgarh, India

³ITM University, Naya Raipur, Chhattishgarh, India

⁴MRD Life Sciences, Lucknow, Uttar Pradesh, India

⁵Amity University, Raipur, Chhattishgarh, India

Author’s Email- ¹shreeti123mishra@gmail.com; ²annpereira@gmail.com; ³bajpai.rakhi683@gmail.com; ⁴chitranshupandeyniu@gmail.com; ⁵naidu.prasad@gmail.com

*Corresponding Author Email- naidu.prasad@gmail.com

ARTICLE INFORMATION

ABSTRACT

Article history:

Received

30 May 2023

Received in revised form

17 June 2023

Accepted

19 June 2023

Keywords:

Herbs;

Phytochemicals;

Biofilm;

Staphylococcus aureus;

Eucalyptus;

Orange peel;

Tomato peel

Dental and oral hygiene is essential for overall health and well-being. Tooth brush and dental floss help remove food debris and plaque, while tooth paste and mouth wash are a key part of oral hygiene as they reduce bacterial growth. The use of chemical agents in tooth paste formulations are effective but can have undesirable side effects. The use of herbal extracts instead can be equally effective due to the presence of phytochemicals which render antibacterial and antiseptic properties, with no side effects. In this study we have formulated toothpaste using the extracts of orange peel, tomato peel and Eucalyptus leaves. The extracts were positive for phytochemicals like terpenoids, flavonoids and tannins. The formulated tooth paste demonstrated antibacterial and biofilm degrading activity against Staphylococcus aureus isolated from dental plaques. On comparison with commercially available toothpastes, the herbal formulation was observed to be more effective against the biofilm forming Staphylococcus aureus.

Introduction

Tooth is an important part of the body responsible for chewing food. In case of bad oral hygiene many dental problems can occur such as dental caries, periodontitis and gingivitis. The most important and common problem is accumulation of plaque and germs (Pitts et al. 2017). Dental plaque forms structurally & functionally organized biofilms with species rich microbial community on a tooth surface (Marsh 2004; Socransky et al. 2002). The types of bacteria inhabiting dental plaque is defined by the diet, nutrition and immunity of an individual and hence can vary. In case of non-removal, microbial plaque becomes harder and becomes tartar which cannot be easily removed by brushing. Bacterial activities at the surface of tooth cause gingival irritation and immune response against this which results in inflammation. The tissues near the tooth become extremely thin and sensitive to heat, cold in cases like periodontal disease. Dental caries is a disease induced by dental plaque (Grigalauskiene et al. 2015). Gingivitis if left untreated can lead to periodontal disease, jawbone erosion, pus extraction from gingival and spontaneous falling of the tooth (Axelsson et al. 2015). Oral hygiene is hence very important and should be maintained (Tadin et al. 2022).

For the control of plaque and gingivitis many mechanical methods were earlier used which include tooth brushes, dental floss, mouth rinses and however as per study effective mechanical plaque removal methods are performed by only less than one third of the population. Another method for controlling plaque and oral diseases is the use of toothpastes which is a gel dentifrice used to brush the teeth regularly to maintain oral hygiene. Toothpaste can be synthetic or herbal. Chemical agents like triclosan, chlorohexidine and fluoride and many such chemical formulations were tried in dentifrices (Barnes et al. 2010; George et al. 2009; Habashneh et al. 2017). Of these Chlorohexidine is considered a gold standard and is useful amongst various areas of dentistry including oral surgery, periodontics, and even general dentistry (Deus and Ouanounou 2022). However long-term usage of chemicals like triclosan and chlorhexidine showed certain undesirable side effects such as discolouration of the teeth, parotid swelling, oral mucosal lesions, dental fluorosis and altered taste, thus increasing the limitations on the safety of its long term usage (Barnes et al. 2010; Bossù et al. 2019; de Oliveira et al. 2008). Considering the harmful effects of chemical in the synthetic toothpaste the choice for herbal oral products have increased.

DOI: 10.52228/NBW-JAAB.2023-5-1-7

Herbal ingredients have several benefits. They have anti-bacterial and anti-septic properties and give a cooling effect to mouth (Grace et al. 2015; Chatterjee et al. 2021; Srivastava et al. 2022). Herbal toothpaste contains molecules such as phenols, alkaloids, glycosides which make toothpaste more effective against different group of bacteria and protect the mouth from infection (Sherikar et al. 2015). Further, no side effects or adverse reaction were observed with the use of herbal toothpaste (Garg et al. 2023).

Previous studies have reported the use of neem leaves, guava leaves, cinnamon bark, camphor and honey in herbal toothpaste formulations (Deshmukh et al. 2015).Earlier studies have tested herbal ingredients like aloe vera, turmeric, miswak, chamomile,pudina leaves, pomegranate peel, lemon peel, amalaka, babul, orange peel, banana peel and have observed that aloe vera, turmeric, miswak, orange peel have antibacterial property (Chowdhury et al. 2017; Deshmukh et al. 2017; Ghelichli 2014; Shende et al. 2018; Gupta et al. 2021). Amalaka, guava and chamomile have anti-inflammatory property (Deshmukh et al. 2017; Doijad et al. 2018; Ghelichli 2014; Dubey et al. 2022). Pudina leaf has germicidal property (Chowdhury et al. 2012). Pomegranate peel, banana peel has antimicrobial propertyand lemon peel has good abrasive property (Sekar and Abdullah 2016; Sekar and Ariffin 2016).

The main reason to promote herbal products is the awareness and tendency of consumers to “go natural”. They are not tested on animals, carry no side-effects, use no animal products, are vegan friendly, contain no added artificial colours or flavours and are also based on various invitro and invivo studies.

In our herbal toothpaste formulation, we have used orange peel, Eucalyptus leaves, Jungle jalebi fruit, Mango leaf and tomato peel. Orange peel is an immune- enhancer, has the ability to fight viral and bacterial infections (Grosso et al. 2013). Extract from Eucalyptus leaveshave anti-inflammatory and antimicrobial properties (Ragul et al. 2018), and have been effective in both toothpastes as well as in oral rinse on dental plaque and sulcus bleeding (Nagata et al. 2008). In Mango the presence of tannins and resin contributes to its astringent effect on mucous membrane which helps in protecting enamel by forming layer on it (Mahant and Thakur 2020). Tomato, has antioxidant properties, it contains pigment lycopene which is effective in treatment of oral cavity diseases such as oral cancer and precancerous lesions (Mehta and Lycopene et al. 2012). The fruit of Jungle jalebihas antibacterial activity against pathogenic bacteria Staphylococcus aureus, Klebsiella pneumonia and Escherichia coli (Singh et al. 2016).

Materials and methods

Sample collection and extract preparation

Plant parts were washed with tap water and then wiped with 70% ethanol. The desired plant part was finely chopped and dried at room temperature for 2-3 days. After drying, the plant was ground to powderin a mortar and pestle.

The powder was dissolved in various solvents (Acetone, Distilled water, Hexane, Benzene, propanol, DMSO, Chloroform, Methanol, Ether) and incubated for 24 hours in dark place. After incubation the extract was filtered with the help of a filter paper. The filtrate was dried in a hot air oven. After evaporation DMSO was added for preservation.

Sample	Botanical name	Plant part
Tomato	Solanum lycopersicum	Peel
Orange	Citrus X sinensis	Peel
Eucalyptus	Eucalyptus globules	Leaf
Mango	Mangifera indica	Leaf
Jungle Jalebi	Pithecellobium dulce	Fruit

Bacteria isolation

Samples from plaque were collectedin saline. 20µl of samples were serially diluted in 9ml of saline water. 40µl of the last three serially diluted tubes were spread on nutrient agar plates. 3 individual coloniesfrom were isolated from each plate and streaked nutrient agar plates and pure culture were maintained.

Characterization and identification of bacteria

The identification bacteria isolated from plaques was done based on morphology, Gram’s staining, endospore staining and biochemical characterization by IMViC, oxidase, coagulase tests and fermentation of sugars and the Analytical profile index (API). (Muhammad S et al. 2020)

Screening for biofilm production

75ml of Nutrient Agar Medium (NAM) was prepared and autoclaved. 15ml media was poured in each Petri plate. After the media solidified 45µl of sample was spread in the NAM plates. The cover slip coated with 20% plasma was placed on the spread Petri plates. The plates were incubated at 37̊C. (Walker J et al. 2012)

Antibiotic Sensitivity Test (AST)

NAM was prepared, poured into petriplates and allowed to solidify. 45µl of bacterial sample was spread on the NAM plates. Wells were prepared with the help of micro tips. Each extract was loaded in the respective well and incubated for 24hours at 37̊C. After incubation, results were obtained as zone of inhibition and were measured with a scale.

Biofilm degradation by plant extract

Biofilms were produced on coverslips. These coverslips were then placed on a slide. Then the cover slip was filled with extract and incubated for 1-2 days.

Phytochemical analysis

The extract was first dissolved in methanol and incubated for 1hour at room temperature and then filtered. The filtrate was then used for phytochemical analysis as per the standard procedure (Alfalluos et al. 2017).

Formulation

Orange peel, eucalyptus leaves and tomato peel were dried and ground to a powder. Sodium bicarbonate, sodium lauryl sulphate, honey, coconut oil, clove oil, salt and herbal ingredient were added in a mortar and mixed well until it formed a paste consistency. The composition of the herbs and chemicals used in toothpaste preparation is as follows:

Plant extract

Ingredients

Orange peel -0.3gm, Eucalyptus leaf - 0.1gm, Tomato peel - 0.4gm

Composition of chemicals

Ingredient

Sodium bicarbonate – 0.9gm, Sodium lauryl sulphate – 1.5gm, Honey – 1 spoon, Salt – 0.1gm, Coconut oil – 5 drops, Clove oil – 5 drops

Physical examination

Formulated toothpaste was evaluated for the following parameters:

Colour was visually checked.

Odour was tested by smelling the product.

Taste was checked manually by tasting the formulation.

The smoothness was tested by rubbing the paste formulation between the fingers.

Composition

All the ingredients used complied with the Indian standards. Toothpaste was not composed of mono or disaccharide such as sucrose or fermentable carbohydrate.

10gm of toothpaste was mixed with 10ml water, boiled and then cooled to make a suspension. The pH was determined using pH meter.

Foam ability

The foam ability of formulated toothpaste was evaluated by taking small amount of toothpaste with water in measuring cylinder. Initial volume was noted and then shaken 10 times. The final volume of was noted.

Spread ability

1gm of formulated paste was placed on the slide. The toothpaste was placed like sandwich between two slides for 5min to expel air and to provide a uniform film of paste between the slides. Excess of the paste was scrapped off from the edges. The top slide was then subjected to pull with the help of string attached to a hook and time required by the top slide to cover a particular distance was noted.

Comparative study

Nutrient agar plates were prepared. The bacterial culture was spread on the agar and wells were made. The formulated toothpaste and marketed ones were poured in the respective wells and incubated for 24 hours.The Zone of Inhibition (ZOI) of each sample was measured using a scale and compared.

Results

Different herbs like tomato (Solanum lycopersicum), orange (Citrus X sinensis), Mango (Mangifera indica), Eucalyptus (Eucalyptus globules) and Jungle jalebi (Pithecellobium dulce) were collected for the preparation of herbal toothpaste, extracts of these herbs were prepared in various solvents. Micro-organisms were isolated from plaques. The isolated bacterium was capable of producing a biofilm (Figure 1). The biofilm forming bacterium was then biochemically characterized (Table 1, Figure 2 and Figure 3) and based on morphology, cuture characters, API was identified as Staphylococcus aureus. The Antimicrobial susceptibility test (AST) of these extracts were performed and analysed on the isolated biofilm producing Staphylococcus aureus isolated from plaques.

Table- 1: Biochemical characters of plaque bacteria

TESTS	PLAQUE BACTERIA
Gram stating	Gram positive
Endospore test	Positive (Figure 3)
Catalase test	Positive
Mannitol test	Positive
Gram nature	Positive cocci (Figure 2)
Coagulase	Positive

Figure- 1: Slide view of biofilm production

Figure- 2: Gram positive bacteria

Figure- 3: Endospore positive

Extracts of tomato peel, jungle jalebi fruit and orange peel extracted in acetone showed a Zone of Inhibition (ZOI) of 16.5 mm, 16.5 mm and 27.5 mm respectively (Table 2).

The biofilm degrading capability of the extracts of tomato, orange and jungle jalebi in acetone were checked. These extracts together degrade the biofilm produced in a test tube (Figure 4).

Figure- 4: Test tube view of biofilm degradation

Phytochemical analysis of the extract was done. Terpenoid, Flavonoid, Tannin and Steroid were present in 3 extracts apart from other phytochemicals (Table 3).

Tooth Paste Formulation

The herbal toothpaste was formulated by mixing the selected herbs as per the formulation protocol. The physical parameters of the formulation were observed (Table 4).

Table-2: ZOI of the extracts in different solvents

Zone of inhibition
S.No	Extract	Hexane	Acetone	Ether	DW	Methanol	Benzene	Chloroform
1	Tomato peel	-	16.5mm	-	14mm	-	-	-
2	Mango leaf	-	-	-	-	-	-	-
3	Orange peel	-	27.5mm	-	-	-	-	-
4	Jungle jalebi fruit	-	16.5mm	-	-	-	-	-
5	Eucalyptus leaf	17.5 mm	20.5 mm	17 mm	-	16.5 mm	19.5 mm	18.5 mm

Table-3: Phytochemicals test of orange peel, tomato peel and Eucalyptus

Tests	Orange Peel	Eucalyptus	Tomato Peel
Terpenoid	Positive	Positive	Positive
Flavonoid	Positive	Positive	Positive
Phlobatannin	Positive	Negative	Negative
Tannin	Positive	Positive	Positive
Leucoanthocyanin	Negative	Negative	Negative
Coumarin	Negative	Positive	Positive
Steroid	Positive	Positive	Positive
Fatty acid	Positive	Positive	Negative

Table- 4: Physical parameters of the formulated toothpaste

A Comparative Study

S.No.	Parameter	Observation
1	Color	Muddy Orange
2	Odour	-
3	Taste	sweetish
4	Smoothness	Slightly smooth
5	pH	6.5
6	Foam ability	Good
7	Spread ability	Average

The AST against Staphylococcus aureus was done with commercially available toothpastes Colgate, Dabur Red & Dantkanti. The AST of each of the toothpastes were compared with the new formulation against the isolated Staphylococcus aureus. The ZOI of Dant kanti was 27.5 mm and the new formulation was 24.5 mm (Figure 5).

Figure-5: AST of different toothpastes.

A. AST of Formulated herbal toothpaste against Staphylococcus aureus

B. AST of Dabur Red against Staphylococcus aureus

C. AST of Colgate against Staphylococcus aureus

D. AST of Dant Kanti against Staphylococcus aureus

Discussion

Plaque formation is a most common problem faced by people all over the world. Untreated Plaque formation can lead to dental caries which one of the most common oral infections. Many chemical and herbal remedies have already been tried and tested. Chemical remedies have been effective but come along with many side effects on long term usage. Herbal remedies have been equally effective with better acceptance and no side effects as they are natural. The presence of a variety of phytochemicals in herbal extracts such as tannins, terpenoids, flavonoids, saponinsconfer antimicrobial properties to the extracts and they are able to kill or inhibit the growth of different bacterial communities.

Previous studies have shown that the extract of C. sinensis has antimicrobial and anti-biofilm activities against S. aureus and P. aeruginosa (Baba et al. 2018; Zubir 2020). Extracts from other citrus species have also demonstrated anti-microbial activity against Prevotella intermedia, Porphyromonas gingivalis, Bacillus subtilis, Shigella flexineri, Staphylococcus epidermidis (Hussain et al. 2015; Zubair 2020). Our results indicate that orange peel extract in acetone has antibacterial activity against biofilm forming isolates of S. aureus from dental plaque with a ZOI of 27.5 mm. Phytochemical like tannins, flavonoids, terpenoids have been identified in the extract prepared in acetone. The anti-microbial activity in the extracts is due to the presence of phytochemicals like tannin which is capable of inhibiting protein synthesis by forming reversible complexes with proteins rich in proline, flavonoids which exhibit anti-inflammatory, anti-microbial, cytostatic and antioxidant properties (Alams et al. 2005; Hodek et al. 2002; Shimda et al. 2006). The individual effect of the orange peel extract on biofilm degradation was not checked. However, the extract of orange, along with Eucalyptus and tomato can degrades the biofilim.

Many studies have reported the antibacterial activity of Eucalyptus against oral and other pathogens. Extracts of Eucalyptus prepared in a wide variety of solvents have abundant phytochemicals which contribute to its antimicrobial activity. It has been observed that the effect of eucalyptus in maintaining oral hygiene is comparative or equivalent to chlorohexidine (Amabye et al. 2016; Asimuddin et al. 2017; Nagpal et al. 2010; Ragul et al. 2018). In our study extracts were prepared in acetone have terpenoids, tannins, flavonoids, coumarin, steroid. These extracts showed a zone of inhibition of 20.5mm against S. aureus. The antibacterial activity of Eucalyptus against S. aureus is in agreement with other studies (Asimuddin et al. 2017). The best antimicrobial activity of Eucalyptus extract was observed in acetone (ZOI of 20.5mm) compared to methanol (ZOI -16.5mm).

Tomato peel and extracts due to the high presence of biologically active compounds have been shown to inhibit the growth of foodborne pathogenic bacteria. The pigment lycopene present in tomato has a very high singlet oxygen quenching capacity and plays an important role in the treatment of oral diseases like leukoplakia, oral submucous fibrosis, lichen planus, oral squamous cell carcinoma, and also prevents the destruction of periodontal tissues (Gupta et al. 2015). The acetone extracts of tomato in our study have of terpenoids, flavonoids, tannins, coumarin and steroid. Extracts in acetone showed a better antibacterial activity.

The combined anti-biofilm activity of the extracts with the best anti-bacterial activity was tested and we observed the degradation of the biofilm with the combined extracts.

The toothpaste formulation was made selecting the acetone extracts of orange peel, tomato peel and eucalyptus which showed the best antibacterial activity by well-diffusion method, against the biofilm producing S. aureus isolated from plaque.

A comparative study of the prepared formulation with the commercially available Colgate, Dabur red and Dantkanti, revealed that the anti-bacterial activity of the formulation (ZOI 24.5mm) against S. aureus was almost equivalent to Dant Kanti (ZOI 27.5mm).

Conclusion

The formulated tooth paste contains herbal ingredients that are active against biofilm forming S. aureus isolated from dental plaques. The extracts of the herbs used contain phytochemicals which contribute to their anti-microbial activity. The formulated herbal tooth paste is equivalent to an even better that some of the available commercial preparations.

Conflict of Interest

The authors declare that there is no conflict of interest.

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