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ORIGINAL ARTICLE
Year : 2019  |  Volume : 37  |  Issue : 1  |  Page : 87-91
 

Estimation of salivary pH and viability of Streptococcus mutans on chewing of Tulsi leaves in children


1 Department of Pedodontics and Preventive Dentistry, A B Shetty Memorial Institute of Dental Sciences, Nitte University, Mangalore, Karnataka, India
2 Intern, A B Shetty Memorial Institute of Dental Sciences, Nitte University, Mangalore, Karnataka, India

Date of Web Publication25-Feb-2019

Correspondence Address:
Dr. Nikita V Lolayekar
Department of Pedodontics and Preventive Dentistry, A B Shetty Memorial Institute of Dental Sciences, Nitte University, Mangalore, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JISPPD.JISPPD_91_17

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   Abstract 


Background: The current concepts of dental caries focus on cariogenic bacteria such as Streptococcus mutans fermenting carbohydrates to form organic acids, which cause a drop in pH, resulting in demineralization of the tooth surface.[l] Studies show that Tulsi has broad-spectrum antimicrobial activity.[2] Hence, this study aimed at estimating the change in salivary pH and viability of S. mutans on chewing of Tulsi leaves, in children. Aims: This study aimed to estimate the change in salivary pH and viability of S. mutans on chewing of Tulsi leaves. Materials and Methods: The study was carried out on thirty children aged 9–12 years. Oral prophylaxis was performed prior to sample collection. Three samples were collected per child, one before and two after chewing of Tulsi leaves. The change in salivary pH and viability of S. mutans was assessed. Statistical Analysis: The obtained data were analyzed using Friedman test and Wilcoxon's test. The level of significance was set at P < 0.05. Results: The results obtained showed no significant difference in the pH values obtained from the samples, but there was a significant difference in the S. mutans' colony counts. Conclusion: Based on the results obtained from this study, it may be concluded that Tulsi (Ocimum sanctum) has the potential to be developed into an antimicrobial agent against cariogenic bacteria, specifically S. mutans.


Keywords: Dental caries, pH, saliva, Streptococcus mutans, Tulsi


How to cite this article:
Lolayekar NV, Kadkhodayan SS. Estimation of salivary pH and viability of Streptococcus mutans on chewing of Tulsi leaves in children. J Indian Soc Pedod Prev Dent 2019;37:87-91

How to cite this URL:
Lolayekar NV, Kadkhodayan SS. Estimation of salivary pH and viability of Streptococcus mutans on chewing of Tulsi leaves in children. J Indian Soc Pedod Prev Dent [serial online] 2019 [cited 2019 Mar 26];37:87-91. Available from: http://www.jisppd.com/text.asp?2019/37/1/87/252864





   Introduction Top


In children, the initiation of dental caries is attributed to the effects of Streptococcus mutans on the tooth surface.[1] In the presence of fermentable carbohydrates, organic acids such as lactic, formic, and acetic acids are formed which coincide with a drop in plaque pH, resulting in demineralization of the tooth surface.[2] Currently, there is a need for alternate prevention and treatment options that are safe, effective, and economical, which has led to the search for alternative products such as natural phytochemicals isolated from plants.[3] Tulsi (Ocimum sanctum) is one such aromatic shrub used in Ayurveda, whose benefits have just begun to be confirmed by modern sciences.[4] The rationale behind this study was the nonavailability of scientific literature about the effect of Tulsi on the salivary pH and viability of S. mutans.

Objectives

To evaluate:

  1. The change in salivary pH on chewing of Tulsi leaves
  2. The viability of S. mutans on chewing of Tulsi leaves.



   Materials and Methods Top


Sample size

The sample size was calculated to be 30.

Sample selection

Patients visiting the department of pedodontics and preventive dentistry, who fulfilled the inclusion and exclusion criteria, were selected for the study. After explaining the procedure, informed consent from the parents as well as assent from the children were obtained. Ethical clearance was obtained prior to starting the study. The duration of the study was 6 months.

Inclusion criteria

  • Children between 9 and 12 years of age.


Exclusion criteria

  • Children with any medical disorders
  • Children with dental caries
  • Children who are mouth breathers
  • Children with any periodontal problems.


The following materials and armamentarium were used in the study:

Armamentarium

  1. Screening instruments: Sterile mouth mirrors and sterile explorers
  2. For saliva sample collection: Sterile sample containers (30 ml)
  3. Ultrasonic scalers.


Materials

  1. Freshly picked Tulsi leaves (Rama Tulsi)
  2. For pH estimation: pH strips
  3. For S. mutans' viability estimation: Mutans-Sanguis agar.


Miscellaneous

  1. Disposable latex gloves
  2. Disposable mouthmasks
  3. Disposable surgical headcaps.


Sample collection

The children were asked to refrain from consuming water or food 1 h prior to the procedure. The same examiner collected all the three samples per child on the same day.

  • Step 1: Oral prophylaxis: Scaling was performed prior to collection of saliva. The absence of plaque was confirmed by thorough examination of the oral cavity using a mouth mirror and probe
  • Step 2: Collection of baseline saliva (Sample 1). Unstimulated whole saliva was collected in a sterile container
  • Step 3: Chewing of Tulsi leaves. Four to five fresh Tulsi leaves were given to the children. The children were asked to chew the leaves for 5 min after which they were asked to thoroughly rinse and expectorate
  • Step 4: Collection of saliva (Sample 2). The stimulated whole saliva was collected in a sterile container immediately after chewing the Tulsi leaves
  • Step 5: Collection of saliva (Sample 3). Unstimulated whole saliva was collected in a sterile container 30 min after chewing the Tulsi leaves.


Collection of unstimulated saliva

The child was seated with his/her head bent slightly down. The child was asked not to swallow or move his/her lips or tongue during the procedure. The saliva was allowed to accumulate in the mouth for 2 min after which the patient was asked to expectorate the collected saliva into the sample container provided. The first expectoration was discarded as it may have been contaminated with debris.[5] The collected sample was stored at 4°C.

Methodology

Evaluation of salivary pH

Evaluation of salivary pH in all the three samples of each child was done using pH strips immediately after collection of the samples to minimize any time-based salivary pH changes. The change in the color in the pH strips indicated the salivary pH.

Test for  Streptococcus mutans Scientific Name Search n saliva

The samples were delivered to the laboratory in a cooler bag within an hour of collection. The samples were processed as soon as they were brought to the laboratory. They were streaked onto Mutans-Sanguis agar plates and incubated aerobically at 37°C for 24 h. The colonies were identified by colony morphology, Gram staining, and standard biochemical tests. The results were drawn based on the number of colonies.

Statistical analysis

The values obtained were tabulated and subjected to statistical analysis. The statistical analysis was done using IBM SPSS Version 22 software (International Business Machines Corp., USA). The data were analyzed using Friedman test and Wilcoxon's test. The level of significance was set at P < 0.05.


   Results Top


The results of the study conducted were as follows:

  1. The measurements of salivary pH of the three samples in thirty children are presented in [Table 1]. There was no statistically significant difference (P > 0.05) in the salivary pH between the three sample groups
  2. The measurements of the S. mutans colonies of the three samples in thirty children are presented in [Table 2]. The Logs of the colony counts were considered during the analysis. There was a statistically significant (P < 0.05) difference in the colony count seen in the following order: Sample 3 had the least count followed by Sample 2 and Sample 1 which had the highest count
  3. [Table 3] shows the pair-wise differences in the colony counts. Wilcoxon's signed rank test was used for this analysis. A significant difference (P < 0.05) was observed in the samples.
Table 1: Mean, median, and standard deviation of salivary pH

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Table 2: Mean, median, and standard deviation of logs of colony-forming units

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Table 3: Differences in colony units

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   Discussion Top


Despite the advances in various fields of medicine, oral infections and dental caries are still considered as serious public health problems and are a major burden to health-care services around the world, especially in developing countries.[6]

The principal cause of dental caries is well understood; the consumption of easily fermentable carbohydrates (in practice usually sucrose) stimulates the growth of oral microbes, most notably S. mutans. Due to the absence of oxygen and the species' general fermentative metabolism, growth on sucrose leads to the formation of organic acids. Even though the produced amounts of acid are small, they are produced very locally and reduce the pH in the bacterium's microenvironment. Since S. mutans can directly adhere to the tooth's hydroxyl-apatite (HA) matrix, the pH of the tooth surface may be easily reduced to below the critical pH of HA demineralization (pH 5.5).[7]

O. sanctum (also Tulsi, tulasī, or Holy Basil) is an aromatic plant in the family Lamiaceae that is native throughout the old-world tropics and widespread as a cultivated plant and an escaped weed. Tulsi is cultivated for religious and medicinal purposes and for its essential oil. It is widely known across South Asia as a medicinal plant, used as an herbal tea, and commonly used in Ayurveda.[8] The medicinal properties of Tulsi have been studied in hundreds of scientific studies including in vitro, animal, and human experiments. These studies reveal that Tulsi has a unique combination of actions such as antibacterial, antiviral, antifungal, antiprotozoal, antimalarial, and anthelminthic, to name a few.[4]

Within Ayurveda, Tulsi is known as “The Incomparable One,” “Mother Medicine of Nature,” and “The Queen of Herbs,” and is revered as an “elixir of life” that is without equal for both its medicinal and spiritual properties.[9] Tulsi has been revered in India over 5000 years, as a healing balm for body, mind, and spirit, and is known to bestow an amazing number of health benefits. Three varieties of Tulsi available are Rama or Light Tulsi (O. sanctum), Shyama or Dark Tulsi (O. sanctum), and Vana Tulsi (Ocimum Gratissimum).[8] In this study, we used the Rama Tulsi leaves.

Chaurasia[10] stated that using Tulsi leaves can effectively treat common oral infections. Chewing of the leaves of the plant helps in proper maintenance of oral hygiene. Strong antibacterials such as carracrol and terpene and sesquiterpene β-caryophylline are present in Tulsi leaves. The Food and Drug Administration has approved the antibacterials present in Tulsi as food additive. Ramesh et al., 2014,[3] stated that >99% of germs and bacteria in the mouth that cause dental cavities, plaque formation, halitosis, etc., are destroyed by Tulsi, thus making it an excellent mouth freshener and an oral disinfectant. Agarwal et al.[11] in their study reported 4% concentration of Tulsi extract, when Tulsi leaves were obtained from courtyard, had maximum antimicrobial potential against S. mutans. This is why we decided to ask our volunteers to chew the fresh Tulsi leaves in our study.

The normal pH of saliva is considered to be around 6.8. If the pH becomes higher, it indicates that our body can respond easily to strong stimuli.[12] However, the salivary buffer effect in unstimulated saliva is sparsely documented.[2] Larsen et al.[13] emphasized that the buffering capacity of unstimulated saliva varies so much that single measurements are not reliable for caries prediction. This is why we collected three salivary samples in the present study; before chewing the leaves, immediately after, and 30 min later. In the present study, on evaluating the change in salivary pH on chewing of Tulsi leaves, the most notable changes in pH were observed immediately after chewing, but were stabilized after 30 min. Even though there was no statistically significant difference (P > 0.05) seen in the salivary pH between the three sample groups, on an average, the pH remained alkaline in the range of 6–8. It may also be noted that the pH did not drop below the critical pH in any of the samples collected.

Similarly, Willershausen et al.[14] in their study observed a shift in the salivary pH values to the alkaline range in the herbal extract group, which demonstrates the usefulness of herbal extracts as an adjunct in routine prevention programs. In a randomized controlled trial by Ramesh et al.,[3] the most notable changes in salivary pH were observed immediately and 30 min after chewing the herbal leaves, which included Tulsi leaves. However, the mechanism of action is not clearly described and there is no specific information in literature about the influence of herbal leaves on pH. The increase in salivary pH in these studies can be explained by the fact that chewing herbal leaves stimulates salivation that increases the saliva's bicarbonate concentration,[3],[14] Thus, in the present study, chewing of Tulsi leaves may have increased the salivary flow rate, which in turn led to a slight increase in pH, promoting remineralization and buffer capacity and subsequently could help in the prevention of caries.

On testing the viability of S. mutans on chewing of Tulsi leaves in our study, there was a statistically significant difference (P < 0.05) in the colony count seen among all the three samples collected, with the least number of S. mutans in the salivary sample collected after 30 min. Past literature shows that most studies evaluating the antibacterial efficacy of Tulsi against S. mutans have been done in vitro using Tulsi extracts or commercial Tulsi preparations, unlike in our study, which was done in vivo asking the volunteers to chew fresh Tulsi leaves.

The antibacterial principle in the leaves studied may probably be attributed to one or more of the following phytochemicals previously identified: essential oils, flavonoids, terpenes, and terpenoids.[3] Eugenol (l-hydroxyl-2-methoxy-4-allylbenzene), the active constituent present in O. sanctum, perhaps is said to be largely responsible for the therapeutic potential of Tulsi,[15] the other constituents being ursolic acid and carvacrol.[11] Even though it is not possible to attribute the antibacterial activity observed to any particular phytochemical group, it is of paramount significance that the plant extracts have exhibited a strong inhibition of the growth, acid production, sucrose-induced adherence, and glucan-induced aggregation of S. mutans.[16]


   Conclusion Top


Previously done phytochemical screening and antimicrobial activity tests of some medicinal plants such as Tulsi against oral flora have proven that the maximum numbers of secondary metabolites found in these plants have the potential for development of antimicrobial agents against oral microorganisms, for use in toothpaste, mouthwash, etc., for preventing and treating oral infections.

According to the methodology applied in this study, we could conclude the following:

  • Tulsi has an antimicrobial effect on S. mutans present in the saliva, without causing any known systemic side effects
  • Chewing of Tulsi leaves has no significant effect on the salivary pH levels
  • Based on the results obtained from this study, it may be concluded that Tulsi (O. sanctum) has the potential to be developed into an antimicrobial agent against cariogenic bacteria, specifically S. mutans.


However, further detailed studies with a larger sample size and for a longer duration need to be done to confirm these beneficial effects of chewing Tulsi leaves.

Suggestions

Factors to be considered while using Tulsi leaves in vivo are as follows:

  1. Tulsi has a highly complex chemical composition. Different strains of Tulsi may show variations in the proportion of the nutrients and other biologically active compounds present in the plant. Difference in growing, harvesting, processing, and storage of the leaves results in a considerable change in the quantity of the constituents.[17] Thus, the results of every study might vary depending on these factors
  2. Groppo et al.[18] have reviewed the safety and possible side effects caused by natural products used in dentistry, wherein they suggest that there is a need of caution while using herbal medicines and there is a possibility of adverse interactions between the herbal formulations and conventional drugs.


Acknowledgment

This study was done in collaboration with the Central Research Laboratory, Nitte University, Mangalore.

Financial support and sponsorship

This study was financially supported by the Indian Council of Medical Research (ICMR) under the Short Term Studentship STS (2015) program.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiol Rev 1986;50:353-80.  Back to cited text no. 1
    
2.
Lenander-Lumikari M, Loimaranta V. Saliva and dental caries. Adv Dent Res 2000;14:40-7.  Back to cited text no. 2
    
3.
Ramesh G, Nagarajappa R, Madhusudan AS, Sandesh N, Batra M, Sharma A, et al. Estimation of salivary and tongue coating pH on chewing household herbal leaves: A randomized controlled trial. Anc Sci Life 2012;32:69-75.  Back to cited text no. 3
    
4.
Cohen MM. Tulsi-Ocimum sanctum: A herb for all reasons. J Ayurveda Integr Med 2014;5:251-9.  Back to cited text no. 4
[PUBMED]  [Full text]  
5.
Mohamed R, Campbell JL, Cooper-White J, Dimeski G, Punyadeera C. The impact of saliva collection and processing methods on CRP, IgE, and myoglobin immunoassays. Clin Transl Med 2012;1:19.  Back to cited text no. 5
    
6.
Sandhu KS, Gupta N, Gupta P, Arora V, Mehta S. Caries protective foods: A futurist perspective. Int J Adv Health Sci 2014;1:21-5.  Back to cited text no. 6
    
7.
Forssten SD, Björklund M, Ouwehand AC. Streptococcus mutans, caries and simulation models. Nutrients 2010;2:290-8.  Back to cited text no. 7
    
8.
Singh E, Sharma S, Dwivedi J, Sharma S. Diversified potentials of Ocimum sanctum Linn (Tulsi): An exhaustive survey. J Nat Prod Plant Resour 2012;2:39-48.  Back to cited text no. 8
    
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Singh N, Hoette Y, Miller R. Tulsi: The mother medicine of nature. Int Inst Herbal Med 2010;2:28-47.  Back to cited text no. 9
    
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Chaurasia A. Tulsi– A promising herb in dentistry. J Oral Med Oral Surg Oral Pathol Oral Radiol 2015;1:21-3.  Back to cited text no. 10
    
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Agarwal P, Nagesh L, Murlikrishnan. Evaluation of the antimicrobial activity of various concentrations of Tulsi (Ocimum sanctum) extract against Streptococcus mutans: An in vitro study. Indian J Dent Res 2010;21:357-9.  Back to cited text no. 11
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12.
Gupta B, Kumar VN, Mallaiah S. Assessment of antimicrobial activity of various concentrations of commercially available Tulsi (Ocimum sanctum) powder against Streptococcus Mutans. Open J Dent Oral Med 2013;1:19-24.  Back to cited text no. 12
    
13.
Larsen MJ, Jensen AF, Madsen DM, Pearce EI. Individual variations of pH, buffer capacity, and concentrations of calcium and phosphate in unstimulated whole saliva. Arch Oral Biol 1999;44:111-7.  Back to cited text no. 13
    
14.
Willershausen B, Gruber I, Hamm G. The effects of herbal agents on dental plaque and sulcus bleeding index. J Clin Dent. 1991; 2:75-8.  Back to cited text no. 14
    
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Prakash P, Gupta N. Therapeutic uses of Ocimum sanctum Linn (Tulsi) with a note on eugenol and its pharmacological actions: A short review. Indian J Physiol Pharmacol 2005;49:125-31.  Back to cited text no. 15
    
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Walsh LJ. Microbiology: Biofilms. The Royal Australian College of Dental Surgeons Incorporated 64 Castlereagh Street, Sydney, NSW 2000: School of Dentistry, The University of Queensland Brisbane, Australia; 2009.  Back to cited text no. 16
    
17.
Miller R, Miller S, TulsiQueen of Herbs, India's Holy Basil. Available from: http://www.nywellnessguide.com/nutrition/070410-TulsiHerbs.php. [Last accessed on 2015 Sep 23].  Back to cited text no. 17
    
18.
Groppo FC, Bergamaschi Cde C, Cogo K, Franz-Montan M, Motta RH, de Andrade ED, et al. Use of phytotherapy in dentistry. Phytother Res 2008;22:993-8.  Back to cited text no. 18
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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