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ORIGINAL ARTICLE
Year : 2015  |  Volume : 13  |  Issue : 4  |  Page : 517-520

Antimicrobial effectiveness of Neem (Azadirachta indica) and Babool (Acacia nilotica) on Streptococcus mutans: An in vitro study


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

Date of Web Publication7-Dec-2015

Correspondence Address:
R Pandya Sajankumar
Department of Public Health Dentistry, A. J. Institute of Dental Sciences, NH 17, Kuntikhana, Mangalore - 575 004, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2319-5932.171159

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  Abstract 

Introduction: There is an exponential growth in the field of herbal medicine because of their natural origin, easy availability, efficacy, and safety. Aim: To compare the antimicrobial effectiveness of Neem and Babool on Streptococcus mutans. Materials and Methods: An in vitro study was conducted to compare the effectiveness of 5%, 10%, and 50% of Neem and Babool aqueous extract with 5%, 10%, and 50% of 0.2% chlorhexidine mouthwash on S. mutans. The ditch plate method was used to test the antimicrobial activity. Ditches were prepared on blood agar plates with the help of punch having 7 mm diameter. The plates were left for 1 h at room temperature and then incubated at 37°C for 48 h and examined for zone of inhibition. Results: Inhibitory effect of 5% Neem is significantly better than 5% Babool and 5% chlorhexidine mouthwash (P < 0.05). At 10% and 50%, Neem and Babool are significantly better than chlorhexidine mouthwash (P < 0.05). Inhibitory effect of Babool increases as the concentration increases (P < 0.05). The inhibitory effect of 5% and 50% chlorhexidine mouthwash is better than 10% chlorhexidine mouthwash (P < 0.05). Conclusion: Aqueous extract of Neem exhibited the highest antimicrobial activity compared with Babool and chlorhexidine mouthwash.

Keywords: Antimicrobial, Babool (Acacia nilotica), Neem (Azadirachta indica), Streptococcus mutans


How to cite this article:
Sajankumar R P, Hegde V, Shetty PJ. Antimicrobial effectiveness of Neem (Azadirachta indica) and Babool (Acacia nilotica) on Streptococcus mutans: An in vitro study. J Indian Assoc Public Health Dent 2015;13:517-20

How to cite this URL:
Sajankumar R P, Hegde V, Shetty PJ. Antimicrobial effectiveness of Neem (Azadirachta indica) and Babool (Acacia nilotica) on Streptococcus mutans: An in vitro study. J Indian Assoc Public Health Dent [serial online] 2015 [cited 2019 Dec 5];13:517-20. Available from: http://www.jiaphd.org/text.asp?2015/13/4/517/171159


  Introduction Top


Oral hygiene measures have been practiced by different populations and cultures around the world. Since antiquity, medicinal plants are used in oral healthcare due to the presence of alkaloids, essential oils, resins, glycosides, steroids, tannins, and phenol. These plants are used to maintain oral hygiene because of its antibacterial effects.[1] Despite the widespread use of toothbrushes and toothpastes, natural methods of teeth cleaning using chewing sticks have been practiced for thousands of years in different part of the world.[2] Due to a wide biodiversity in India, a variety of twigs such as Neem (Azadirachtaindica), Babool (Acacianilotica), Mango (Mangiferaindica), and Guava (Psidiumguajava) are mainly used as tooth sticks.[2]

In the recent years, Neem has attracted worldwide prominence because of their medicinal values. Neem has been broadly used in Ayurveda, Unani, and Homoeopathic medicine and has become a center of admiration in modern medicine. Products made from Neem have been used in India for over two millennia for their medicinal properties. The extract of these plants inhibit the growth of oral pathogens, diminish the progress of dental plaque, and manipulate the adhesions of bacteria to the tooth surface.[3] Different cross-sectional and longitudinal studies have been done on the inhibitory effect of Neem and Babool extract on Streptococcusmutans (Microbial Type Culture Collection (MTCC-497), IMTECH, Chandigarh, India).[4]

In the modern world, commercially available mouthwashes are widely used along with toothbrushing and flossing procedures for preventing and controlling both supragingival plaque and gingivitis. Chlorhexidine which is an active ingredient of these mouthwashes is used as an antibiotic and disinfectant. In view of, the fact that chlorhexidine is sometimes associated with adverse side effects to the host including hypersensitivity and allergic reactions, staining of teeth, and unpleasant taste; it is of interest to develop antimicrobial aids from alternative sources such as medicinal plants for treatment of infectious diseases.[5] Studies show that lowering the concentration of chlorhexidine gluconate has the same therapeutic effect as the standard, and also there is decrease in the side effects. The local side effects of chlorhexidine gluconate are dose dependent and concentration dependent. In this context, a study was undertaken to ascertain the effect of Neem and Babool aqueous extract on S.mutans and to compare it with the effect of diluted chlorhexidine mouthwash.[6]

Aim

To compare the antimicrobial effectiveness of Neem and Babool on S.mutans.

Objectives

  • To compare the antimicrobial effectiveness of 5% of Neem and Babool with 5%, 10%, 50% of 0.2% chlorhexidine gluconate on S.mutans
  • To compare the antimicrobial effectiveness of 10% of Neem and Babool with 5%, 10%, 50% of 0.2% chlorhexidine gluconate on S.mutans
  • To compare the antimicrobial effectiveness of 50% of Neem and Babool with 5%, 10%, 50% of 0.2% chlorhexidine gluconate on S.mutans.



  Materials and Methods Top


An in vitro study was conducted to compare the effectiveness of 5%, 10%, and 50% of Neem and Babool aqueous extracts with 5%, 10%, and 50% of 0.2% chlorhexidine gluconate on S.mutans.

Preparation of Neem and Babool aqueous extract

Neem and Babool sticks were obtained from the local market, and the aqueous extract of Neem and Babool was prepared at Department of Microbiology, Vanbandhu College of Veterinary Science and Animal Husbandry, N.A.U, Navsari, Gujarat.

Two hundred grams each of Neem and Babool sticks was taken. The sticks were sun dried for 15 days before extract preparation. The sticks were ground to powder in a ball mill. The powder was weighed into 5 g, 10 g, and 50 g and transferred into sterile and dry screw capped bottles. In each bottle, 10 ml of distilled water was added and allowed to soak for 48 h. A solution was centrifuged at 2000 rpm for 10 min. The supernatant was filtered using 0.45 mm membrane filter. The aqueous extract was prepared at 5%, 10%, 50% concentration, respectively and stored at 20°C.[4]

Preparation of culture media

S.mutans was obtained from MTCC-497 Chandigarh, India. The capsule containing freeze-dried forms of microorganism were added to nutrient broth which was incubated at 37°C for 24 h. A sterile cotton swab was taken and dipped into the nutrient broth and then inoculated on the agar plates and incubated overnight at 37°C. The growth of S.mutans obtained on the agar plate and was transferred onto a blood agar plate for its enhanced growth.

Preparation of 0.2% chlorhexidine gluconate aqueous extract

The mouthwash was weighed into 5 ml, 10 ml, and 50 ml amounts and transferred into labeled bottles, to which 95 ml, 90 ml, and 50 ml of distilled water were added, respectively. The mixture was then shaken well.

Ditch plate method

The solid agar plate was punched with wells having a diameter of 7 mm, and the wells were filled with extracts of various concentration. Separate plates for Neem, Babool, and 0.2% chlorhexidine gluconate were used, with different concentrations. The plates were then incubated at 37°C for 48 h. After incubation, the zone of inhibition was measured in millimeters using Vernier calipers. Each extract was tested 5 times. Negative control does not yield any result as they were not involved in any of the tests conducted in the study. To make study cost-effective, negative control were not taken.

Statistical analysis

Kruskal–Wallis test, Mann–Whitney U-test, ANOVA, and Tukey honest significant difference (post-hoc) test were used, and data were analyzed using SPSS software (version 17.0) (IMTECH, Chandigarh, India). A P < 0.05 was considered as significant.


  Results Top


The mean zone of inhibition at 5% concentrations of Neem, Babool, and chlorhexidine was found to be 15.6 mm, 5.4 mm, and 11.6 mm, respectively. The mean zone of inhibition at 10% concentrations of Neem, Babool, and chlorhexidine was found to be 12.4 mm, 11.2 mm, and 8.2 mm, respectively. The mean zone of inhibition at 50% concentrations of Neem, Babool, and chlorhexidine was found to be 15.2 mm, 14.8 mm, and 12.6 mm, respectively [Table 1a].
Table 1a: Mean zone of inhibition of different products with varying concentration

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The inhibitory effect of 5% Neem is significantly better than 5% Babool and 5% chlorhexidine mouthwash (P < 0.05). At 10% and 50%, Neem and Babool are significantly better than chlorhexidine mouthwash (P < 0.05) [Table 1b].
Table 1b: Comparison of various products at different concentrations

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The mean zone of inhibition at 5%, 10%, and 50% concentrations of Neem was found to be 15.6 mm, 12.4 mm, and 15.2 mm, respectively. The mean zone of inhibition at 5%, 10%, and 50% concentrations of Babool was found to be 5.4 mm, 11.2 mm, and 14.8 mm, respectively. The mean zone of inhibition at 5%, 10%, and 50% concentrations of chlorhexidine mouthwash was found to be 11.6 mm, 8.2 mm, and 12.6 mm, respectively. The inhibitory effect of 5% and 50% Neem is significantly better than 10% Neem (P < 0.05). The inhibitory effect of Babool increases as the concentration increases (P < 0.05). The inhibitory effect of 5% and 50% chlorhexidine mouthwash is better than 10% chlorhexidine mouthwash (P < 0.05) [Table 2a] and [Table 2b].
Table 2a: Intragroup comparison of mean zone of inhibition at different concentrations

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Table 2b: Intragroup comparison of mean zone of inhibition at different concentrations (post-hoc)

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  Discussion Top


The present study was conducted to compare the effectiveness of Neem and Babool aqueous extract with chlorhexidine mouthwash on S.mutans. Beneficial effects of drugs derived from plants act as good source of antibiotics, antioxidants, and anti-inflammatory agents have been shown in various studies.[4]

In this study, 5% concentration of Neem aqueous extract showed maximum antimicrobial effect on S. mutans. The mean zone of inhibition at 5% concentration of Neem aqueous extract was found to be 15.6 mm. This is in contrast to a study conducted by Sharma et al.,[4] in which they found no antimicrobial effect on S. mutans at 5% concentration of Neem, whereas according to a study conducted by Prashant et al.,[7] the mean zone of inhibition was 2.4 mm.

In the present study, the mean zone of inhibition at 10% and 50% concentrations of Neem aqueous extract was found to be 12.4 mm and 15.2 mm, respectively. Similar results were found in study conducted by Sharma et al.[4] and Prashant et al.[7]

The antimicrobial activity of Neem may be attributed to bioactive compounds such as nimbidin nimbolide, mahmoodin, margolone, margolonone, and isomargolonone.[8] The bioactive compounds in Neem are important for various functions such as antibacterial, anti-inflammatory, antiarthritic, antipyretic, hypoglycemic, antigastric ulcer, and antifungal.[8]

In the present study, as the concentration of Babool aqueous extract increases, the antimicrobial effect of Babool increases. Similar results were found in a study conducted by Sharma et al.[4]

Babool includes hydrophilic compounds such as polyphenols, gums (polysaccharides), and tannis, which explains its antimicrobial activity. It is relatively high in bioactive secondary compound. Hence, there is increasing evidence to support this and more promising for drug discovery. Babool has other secondary compounds which are essential for variety of other functions, chief among these are diuretic (glucosides), nutraceutical (polysaccharide and gum), antioxidant (polyphenols), antidigestive disorder (saponins, tannins, and flavonoids), antiplasmodial (treptamine, tannins, organic acids), anticancer (triterpenoid and saponins).[4]

Limited number of studies has investigated the antimicrobial effect of Neem and Babool extracts against S.mutans at concentration of 5%, 10%, and 50%, respectively.

Chlorhexidine mouthwash is considered as the gold standard against which other antiplaque and antigingivitis agents are measured.[9] In the present study, chlorhexidine mouthwash is used as positive control. It has been proved in various studies that chlorhexidine mouthwash at 0.2% and 0.12% is a good antimicrobial agent; however, it is known to provoke an immediate hypersensitivity reaction and considered to be cytotoxic to human periodontal ligament cells.[10] In view of this, Neem and Babool extract might seem a suitable alternative.

There were a few limitations associated with this study as a lower concentration of chlorhexidine mouthwash was used; an effort could have been made to check the effectiveness of 0.12% and 0.2% of chlorhexidine mouthwash with Neem and Babool extract.

Recommendations

An in vivo studies need to be conducted to know the effectiveness of Neem at the same concentration.


  Conclusion Top


Aqueous extract of Neem exhibited highest antimicrobial activity compared with Babool and chlorhexidine mouthwash. Thus, an extensive research and development work should be undertaken on Neem and its products for their better economics and therapeutic utilization.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
Ravishankar P, Lakshmi T, Arvind Kumar S. Ethno-botanical approach for root canal treatment – An update. J Pharm Sci Res 2011;3:1511-9.  Back to cited text no. 1
    
2.
Pahlajani V, Pathak AD, Thadani J. Commonly used plants in India to maintain oral hygiene – A review. Natl J Med Dent Res 2013;1:22-5.  Back to cited text no. 2
    
3.
Packia Lekshmi NC, Sowmia N, Viveka S, Brindha JR, Jeeva S. The inhibiting effect of Azadirachta indica against dental pathogens. Asian J Plant Sci Res 2012;2:6-10.  Back to cited text no. 3
    
4.
Sharma A, Sankhla B, Parkar SM, Hongal S, Thanveer K, Ajithkrishnan CG. Effect of traditionally used Neem and Babool chewing stick (datun) on Streptococcus mutans: An in-vitro study. J Clin Diagn Res 2014;8:15-7.  Back to cited text no. 4
    
5.
Cortelli JR, de la Sotta Thénoux RE. The effect of mouthrinses against oral microorganisms. Braz Oral Res 2007;21:23-8.  Back to cited text no. 5
    
6.
Rath SK, Singh M. Comparative clinical and microbiological efficacy of mouthwashes containing 0.2% and 0.12% chlorhexidine. Dent Res J (Isfahan) 2013;10:364-9.  Back to cited text no. 6
    
7.
Prashant GM, Chandu GN, Murulikrishna KS, Shafiulla MD. The effect of mango and Neem extract on four organisms causing dental caries: Streptococcus mutans, Streptococcus salivavius, Streptococcus mitis, and Streptococcus sanguis: An in vitro study. Indian J Dent Res 2007;18:148-51.  Back to cited text no. 7
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8.
Biswas K, Chattopadhyay I, Banerjee RK, Bandyopadhyay U. Biological activities and medicinal properties of Neem (Azadirachta indica). Curr Sci 2002;82:1336-45.  Back to cited text no. 8
    
9.
Balagopal S, Arjunkumar R. Chlorhexidine: The gold standard antiplaque agent. J Pharm Sci Res 2013;5:270-4.  Back to cited text no. 9
    
10.
Houshmand B, Mahjour F, Dianat O. Antibacterial effect of different concentrations of garlic (Allium sativum) extract on dental plaque bacteria. Indian J Dent Res 2013;24:71-5.  Back to cited text no. 10
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    Tables

  [Table 1a], [Table 1b], [Table 2a], [Table 2b]



 

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