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ORIGINAL ARTICLE
Year : 2018  |  Volume : 16  |  Issue : 1  |  Page : 75-77

Comparative evaluation of antibacterial effect of three commercially available herbal products against Streptococcus mutans: An In vitro Study


Department of Public Health Dentistry, A.J Institute of Dental Sciences, Mangalore, Karnataka, India

Date of Submission18-Sep-2017
Date of Acceptance14-Feb-2018
Date of Web Publication23-Mar-2018

Correspondence Address:
Dr. Sharanya Sreekumar
Department of Public Health Dentistry, A.J Institute of Dental Sciences, Mangalore, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jiaphd.jiaphd_131_17

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  Abstract 

Background: In recent years, the researches concerning the use of herbal products have been vastly analyzed to decrease the disease burden caused by dental caries. It has been found that certain phytochemicals present in the plants have certain inhibitory effect toward Streptococcus mutans which is currently the main causative organism for dental caries initiation. Hence, in the present study, certain commercial herbal products have been tested for their antibacterial effect. Aim: This study aims to compare the antibacterial action of three commercially available herbal products against S. mutans. Material and Methods: An in vitro study was conducted with commercially available herbal products. They were Aloe Barbadensis Miller (Aloe vera), Ocimum tenuiflorum (Thulasi), and Stevia rebaudiana (Stevia). The antimicrobial effectiveness (zone of inhibition) of herbal products was determined using agar well diffusion method. Pure strains of S. mutans MTCC 890 were procured from culture collection center, Imtech, Chandigarh. The culture was grown in Brain Heart Infusion Agar, and around 20 ml each was poured into sterile petri plates. Chlorhexidine (CHX) was taken as positive control, and distilled water was taken as negative control. Statistical analysis was done using SPSS version 17 software. Results: All herbal products were found to be having variable antimicrobial activity against S. mutans. The mean zone of inhibition after 24 h incubation measured for Stevia, Thulasi, and A. vera was 22.33 mm, 11 mm, and 0 mm, respectively. The mean zone of inhibition of positive control CHX was found to be 13.6 mm. Conclusion: The antibacterial effect shown by Stevia was superior when compared with CHX and Thulasi. The Thulasi have more inhibitory effect than A. vera, and A. vera fails to show any zone of inhibition against S. mutans when compared with other herbal products. Thus, Stevia product can be strongly recommended as a caries preventing agent after sufficient clinical trials by future research.

Keywords: Chlorhexidine, herbal products, Streptococcus mutans


How to cite this article:
Sreekumar S, Hegde VK. Comparative evaluation of antibacterial effect of three commercially available herbal products against Streptococcus mutans: An In vitro Study. J Indian Assoc Public Health Dent 2018;16:75-7

How to cite this URL:
Sreekumar S, Hegde VK. Comparative evaluation of antibacterial effect of three commercially available herbal products against Streptococcus mutans: An In vitro Study. J Indian Assoc Public Health Dent [serial online] 2018 [cited 2019 Nov 22];16:75-7. Available from: http://www.jiaphd.org/text.asp?2018/16/1/75/228295


  Introduction Top


Dental caries is an irreversible microbial disease of the calcified tissues of the teeth, characterized by demineralization of the inorganic portion and destruction of the organic substance of the tooth, which often leads to cavitation.[1] It was discovered that Streptococcus mutans was the main causative agent of dental caries. Mutans streptococci are the most cariogenic pathogens as they are highly acidogenic, producing short-chain carboxylic acids which dissolve hard tissues such as enamel and dentine. It can ferment most of the sugars and sugar alcohols present in food such as glucose, sucrose, lactose, trehalose, mannitol, sorbitol, and melibiose.[2] Several researches are being conducted with different herbal extracts to decrease the disease burden caused by dental caries.

Whole leaf components of Aloe vera are proposed to have direct antibacterial properties including anthraquinones and saponins, while polysaccharides have been attributed with indirect bactericidal activity through stimulation of phagocytic leukocytes to destroy bacteria.[3] Tulsi (Ocimum sanctum) has been used in teeth disorders. Dried powder of its leaves has been found to be very good for maintaining dental health, counteracting bad breath, and massaging the gums. The leaves contain an essential oil which has antibacterial, antifungal, and antiviral properties.[4]

Stevia is a green leafy plant that is native to South America. Whole or crushed Stevia leaves, or extract (either liquid or powder), or a refined version of the plant's isolated compounds can be used as a sweetener and they have medicinal value too.[5]

A study evaluated the antibacterial activity of S. rebaudiana leaves extracted using various solvents against  Escherichia More Details Coli, Bacillus Subtilis, Staphylococcus Aureus,  Salmonella More Details Typhi, and Vibrio Cholera, and it was found in the study that the acetone extract showed greater activity against Gram-positive bacteria than Gram-negative bacteria.[6]

In spite of its use in medicinal field, less light has been shed for the use of Stevia in dentistry. Therefore, this study was aimed to compare the antimicrobial effect of commercially available Stevia extract, A. vera, and Thulasi on caries causative microorganism, i.e. S. mutans in comparison with chlorhexidine (CHX).


  Materials and Methods Top


An in vitro study was conducted for comparing the antibacterial effectiveness of three commercially available herbal products against S. mutans. The commercially available herbal products were brought from local market in Mangalore City. The herbal products which were available as convenient in market were included in the study. The three commercially available products were Stevia liquid plant extract, Thulasi liquid plant extract, and A. vera powder. As per manufacturer specifications, the concentration of Stevia and Thulasi was found to be 50% (rest of it was distilled water) and A. vera dry powder was a 100% pure extract. To attain uniformity of 50% concentration of A. vera, the 50 mg of powder was diluted with 100 ml of distilled water. It was then centrifuged at 2000 rpm for 10 min and allowed to settle down. Then, supernatant solution was filtered using 0.45 mm membrane filter.

Laboratory procedures

All the laboratory procedures were done in Manipal laboratory, Manipal, Karnataka, India. CHX mouthwash was taken as positive control and distilled water was taken as the negative control. A. vera, Stevia, and Thulasi plant liquid extract were taken in a quantity of 75 μl. CHX of concentration 0.2% was taken in a quantity of 10 μl since it is used as positive control in this study.

Preparations of brain heart infusion agar

Brain heart infusion (5 g), tryptone (10 g), yeast extract (10 g), and glucose (10 g) were dissolved in 990 ml of distilled water. The pH was adjusted to 7.2 ± 0.5 and the volume was made up to 1000 ml. Finally, 15 g agar was added to the media and autoclaved at 121°C for 20 min.

Preparation of the inoculum

S. mutans MTCC 890 was procured from culture collection center, Imtech, Chandigarh. The culture was revived, maintained, and grown in brain heart infusion agar as per the instructions given by MTCC, Imtech. Loopful of 24-h old culture from the slants was transferred to sterile saline and mixed well to prepare a homogeneous inoculum.

Well diffusion method

The media was cooled to around 45°C–55°C, around 20 ml each was poured into sterile petri plates. One ml of the inoculum was immediately added to the plate, swirled for uniform distribution. Wells were bored using a sterile borer. The samples along with positive and negative controls were dispensed into the wells. Plates were incubated overnight at 30°C and observed after 24 h. Zones of inhibition were measured in millimeter by using vernier caliper. All the measurements were carried out by a single examiner who was blinded about the various test groups and concentration. The examiner repeated the measurements for three wells in each group.[7]

Statistical analysis

Descriptive statistics (mean and standard deviation) was tabulated using SPSS for Windows version 17 software (Chicago, IL, USA).


  Results Top


All herbal products were found to be having variable antimicrobial activity against S. mutans. Mean zone of inhibition after 24 h incubation of Stevia plant extract was 22.33 mm ± 12.8 (standard deviation), for Thulasi plant extract was 11 mm ± 6.35 (standard deviation), and no inhibition for A. vera aqueous extract. The mean zone of inhibition of positive control CHX mouthwash was found to be 13.6 mm ± 7.9 (standard deviation). A significant inhibitory effect was seen with extracts of Stevia (22.33 mm) against S. mutans compared with Thulasi and A. vera [Table 1]. The inhibitory effect of Thulasi (12.8 mm) was better than A. vera (0 mm). [Figure 1] shows the zone of inhibition of each sample against S. mutans. Abar diagram has been shown which clearly depicts the inhibitory effect of each herbal product [Figure 2].
Table 1: Mean zone of inhibition of the study samples against Streptococcus mutans

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Figure 1: The zone of inhibition of samples. T – Thulasi, S – Stevia, Ab – Chlorhexidine, Av – Aloe vera, Dw – Distilled water, and it is triplicated to ensure accuracy (I, II, III)

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Figure 2: The zone of inhibition of each sample against Streptococcus mutans

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  Discussion Top


Since ancient times, herbs and plants have been in use as a source of therapeutic compounds in traditional medicinal system. Plants have got a great attention of scientist for the development of alternative drugs to cure different diseases.[8]A. vera, Thulasi, and Stevia were used in the current study. According to the results of the present study, stevia plant extract has shown higher zone of inhibition against S. mutans compared to other herbal products, and Thulasi have shown a minimum amount of inhibition against S. mutans. CHX was considered as the positive control in this study. The bis-biguanide CHX, which has been studied extensively for over 25 years, is currently the most potent antimicrobial agent against mutans streptococci and dental caries.[5] Hence, only lower quantity of CHX is used in this study for comparing the effect of herbal products against S. mutans.

In a study conducted by Subramaniam et al., in 2012, they have compared pomegranate with A. vera extract in which pomegranate has shown superior effect compared to A. vera. They have also mentioned that A. vera extracts are highly technic sensitive and improper processing procedures lead to loss of its active ingredients. This may be the reason that in the present study aloe vera failed to show any inhibition against streptococcus mutans.[7]

Another study showing effect of alcoholic and aqueous extract of Stevia (S. rebaudiana) against S. mutans depicts that alcoholic Stevia extract has almost similar antibacterial effect as CHX, and this study results are in agreement with the present study.[5] In a study conducted by Mousumi, the antimicrobial properties of chloroform and methanol extract of Stevia were assessed and compared. They found that the antimicrobial effects of such materials were reliant on their concentration; however, Stevia extracts were observed to be more efficient in this regard, which is in accordance with the result of the present study.[9]

A study conducted by Sharma et al., 2015 with A. vera and Thulasi has shown that inhibitory activity of both against S. mutans is at higher concentrations, and the study have concluded that they both have synergistic effect against mutans.[4] Since the present study was carried out at a lower concentration of Thulasi (commercial product) that led it to lesser agreement with above mentioned study.

One of the limitations of this study is that this could have been conducted with pure extracts of herbal plants rather than commercial products for more accurate results. However, commercial herbal products were used in this study because most of the people use these products rather than noncommercial herbal products. An advantage of the present study is that herbal products are directly tested for its antibacterial activity without much manipulation in its concentration.


  Conclusion Top


The antibacterial effect shown by Stevia was superior when compared with CHX and Thulasi. Thulasi has more inhibitory effect than A. vera, and A. vera fails to show any zone of inhibition against S. mutans when compared to other herbal products. Thus, Stevia product can be strongly recommended as a caries preventing agent after sufficient clinical trials by future research.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
Rajendran R, Sivapathasundharam B. Shafer's Textbook of Oral Pathology. 6th ed. India: Elsevier; 2009.  Back to cited text no. 1
    
2.
Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiol Rev 1986;50:353-80.  Back to cited text no. 2
[PUBMED]    
3.
Nonong YH, Patawulandari S, Indriyanti R. Antibacterial test between Aloe vera and chlorhexidine based on the number of colony of Streptococcus mutans Atcc 25 175 in vitro. Int J Sci Res 2016;5:1379-85.  Back to cited text no. 3
    
4.
Sharma M, Dorwal R, Bhat KG, Kashyap N, Chandrashekhar P, Bagri S. Comparative evaluation of the antibacterial efficacy of the Aloe vera and Tulsi: An in vitro study. J Res Adv Dent 2015;4:170-5.  Back to cited text no. 4
    
5.
Ajagannanavar SL, Shamarao S, Battur H, Tikare S, Al-Kheraif AA, Al Sayed MS, et al. Effect of aqueous and alcoholic Stevia (Stevia rebaudiana) extracts against Streptococcus mutans and Lactobacillus acidophilus in comparison to chlorhexidine: An in vitro study. J Int Soc Prev Community Dent 2014;4:S116-21.  Back to cited text no. 5
    
6.
Jayaraman S, Manoharan MS, Illanchezian S.In vitro antimicrobial and antitumor activities of Stevia rebaudiana (Asteraceae) leaf extracts. Trop J Pharm Res 2008;7:1143-9.  Back to cited text no. 6
    
7.
Subramaniam P, Dwivedi S, Uma E, Babu KG. Effect of pomegranate and Aloe vera extract on Streptococcus mutans: An in vitro study. Dent Hypotheses 2012;3:99-105.  Back to cited text no. 7
  [Full text]  
8.
Available from: https://www.nhp.gov.in/introduction-and-importance- of-medicinal-plants-and-herbs_mtl. [Last accessed on 2017 Aug 27].  Back to cited text no. 8
    
9.
Mousumi D. Clonal propagation and antimicrobial activity of an endemic medicinal plant Stevia rebaudiana. J Med Plant Res 2008;2:45-51.  Back to cited text no. 9
    


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