|Year : 2016 | Volume
| Issue : 2 | Page : 105-110
The effect of different concentrations of water soluble azadirachtin (neem metabolite) on Streptococcus mutans compared with chlorhexidine
Amit R Kankariya, Alok R Patel, Sanket S Kunte
Department of Pediatric and Preventive Dentistry, Bharti Vidyapeeth Dental College and Hospital, Pune, Maharashtra, India
|Date of Web Publication||14-Apr-2016|
Dr. Amit R Kankariya
Bharti Vidyapeeth Dental College and Hospital, Pune, Maharashtra
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Despite advances in the development of anticaries chemotherapy, the newer agents are unable to control the initiation of dental caries. Research and development of natural antibacterial agents that are safe for the host as well as specific for oral pathogens is awaited. Neem tree extracts have been used for thousands of years for maintaining overall well-being. Chewing neem sticks in the morning is the most common indigenous method of cleaning the mouth in rural population. This has generated the interest of the dentists for the use of neem for controlling dental diseases. Aims: This study aims to evaluate the quantitative and qualitative effect of different concentrations of water soluble azadirachtin (neem metabolite) on Streptococcus mutans (S. mutans) against chlorhexidine. Materials and Methods: Plaque was collected from 30 children aged 8-12 years reporting to the Department of Pediatric and Preventive Dentistry, Bharti Vidyapeeth Dental College, Pune and transported to the laboratory. After incubation of the plates the inhibitory zones were noted and the diameter of the zone of inhibition was measured and recorded to check the inhibition of growth of S. mutans. For testing the bacterial survival, the biofilms were prepared and colony forming units (CFU) was enumerated using a digital colony counter. Statistical Analysis Used: Two-way analysis of variance (ANOVA) and Tukey's test. Results: The results show that there was no statistically significant difference in the inhibition of S. mutans between 40% concentration of water soluble azadirachtin and chlorhexidine. Conclusions: This study concluded that 40% water soluble azadirachtin is as effective as 0.2% chlorhexidine mouthrinse in reducing the S. mutans count in dental plaque. Hence, a water soluble formulation of azadirachtin may provide the maximum benefit to mankind to prevent dental caries.
Keywords: Neem, Streptococcus mutans, water soluble azadirachtin
|How to cite this article:|
Kankariya AR, Patel AR, Kunte SS. The effect of different concentrations of water soluble azadirachtin (neem metabolite) on Streptococcus mutans compared with chlorhexidine. J Indian Soc Pedod Prev Dent 2016;34:105-10
|How to cite this URL:|
Kankariya AR, Patel AR, Kunte SS. The effect of different concentrations of water soluble azadirachtin (neem metabolite) on Streptococcus mutans compared with chlorhexidine. J Indian Soc Pedod Prev Dent [serial online] 2016 [cited 2019 Aug 25];34:105-10. Available from: http://www.jisppd.com/text.asp?2016/34/2/105/180394
| Introduction|| |
Dental caries is an infectious oral disease that is prevalent across the world and is associated with various pathogenic microorganisms. Streptococcus mutans (S. mutans) is considered a crucial pathogen in the pathogenesis of dental caries. ,, Treating carious teeth in children with high caries incidence by just providing restorations does not cure the disease. With the increasing rate of dental caries, there is a global necessity of effective and economical product for caries prevention. Chemical agents such as fluoride and chlorhexidine which have been used to prevent dental caries for several decades are associated with some side effects such as staining of teeth and fluorosis. These drawbacks justify the need for further research and development of natural antibacterial agents that are safe for the host as well as specific for oral pathogens. But still, the discovery of such a Holy Grail antimicrobial agent is unknown to mankind. The need for affordable, effective, and nontoxic alternatives has led to the search for compounds from natural sources such as plants, which may overcome the high incidence of oral diseases.
Neem tree extracts have been used in the ayurveda tradition for thousands of years. The roots, bark, gum, leaves, fruit, seed kernels, and seed oil are all used in therapeutic preparations for both internal as well as topical use.  Modern science validates that neem has antimicrobial properties. Azadirachtin is a secondary metabolite present in the seeds of neem tree. It acts mainly as antifeedent and growth disruptor.  Currently available mouthrinses are available with an active ingredient in a mixture with organic solvents, viz., alcohol. A formulation of azadirachtin that can be stored for a long period of time and can be used with water has been reported. 
This study aims to evaluate the quantitative and qualitative effect of different concentrations of water soluble azadirachtin (neem metabolite) on S. mutans compared with chlorhexidine.
| Materials and Methods|| |
Dental plaque was collected from 30 children aged 8-12 years with Decayed, Missing, Filled, Teeth (DMFT or dmft) of 0-6, reporting to the Department of Pediatric and Preventive Dentistry, Bharti Vidyapeeth Dental College, Pune after obtaining written parental consent. Microbiological testing was performed in a microbiology laboratory (Rajiv Gandhi Institute of Biotechnology) in Pune to evaluate the antimicrobial effect of water soluble azadirachtin on S. mutans.
Method of collection of plaque sample
Children were made to sit comfortably on the chair. The teeth were isolated from buccal/lingual mucosa with cotton rolls during the entire sampling procedure. The subjects were refrained from oral hygiene procedures on the day of collection of plaque sample. The plaque was collected from six anterior teeth and transported into previously labeled sterile bottle containing 1 mL of transport medium (Thioglycollate broth).
The plaque samples were vortexed to uniformly mix the plaque and transport media using a cyclomixer (CM 101 - Sigma Scientific Instruments) [Figure 1]. By using inoculation loop (3 mm inner diameter) 10 μL of the sample was spread on mitis salivarius-bacitracin (MSB) agar [Figure 2] selective for S. mutans and incubated for 48 h at 37°C in an incubator [Figure 3] (Bio Technics India). After incubating the plates, the colonies with morphologic characteristics of S. mutans (0.5 mm raised convex undulated colonies of light blue color with rough margins, granular frosted glass appearance)  were identified.
A suspension of the pure culture was prepared in distilled water. A loop full of pure culture was inoculated in MSB broth and incubated overnight. A lawn culture of S. mutans was done on MSB agar from the overnight grown culture. Seven wells of 8 mm diameter were punched with a sterile cork borer aseptically. Azadirachtin was obtained from in-house facility of HiMedia Laboratories. The sample of water-soluble azadirachtin to be tested was prepared by diluting with distilled water to obtain the desired log concentrations (0.01 g/10 mL-0.05 g/10 mL distilled water). Then 10 μL of different concentrations of water-soluble azadirachtin to be tested were placed at the center of each of these wells. Similarly, 10 μL chlorhexidine in concentration of 0.2% was also incorporated in one of these wells. Distilled water was added to one well to kept as control. Then the MSB agar plates were incubated in aerobic conditions for 48 h at 37 ° C. After incubation of the plates the diameter of zone of inhibition was noted and measured by Vernier calliper (Labpro). Similarly, 30 trials were performed. Based on the area/diameter; inhibition zone was directly proportional to sensitivity.
For testing the bacterial survival, the biofilms were prepared in the 96-well microtiter plate by adding 100 μL "Brain-Heart Infusion Broth" along with the suspension of pure culture. These were then treated by different test samples for 2 min each and incubated for 24 h at 37° C. The suspensions from the overnight biofilms were collected in 2 mL of eppendorf R tube for dilution of the sample, and diluted using double dilution method so as to decrease its turbidity. Then 0.1 mL suspension from each tube was spread on MSB agar plates and incubated at 37 ° C for 48 h. CFU was enumerated using a digital colony counter. The results of zone of inhibition and the CFU count was then assessed statistically by using two-way ANOVA and pairwise comparison was done by using Tukey's test.
| Results and Observation|| |
The antibacterial activity of three test agents against S. mutans was assessed in the following groups: Positive control (Group I): 0.2% chlorhexidine and experimental group: Water soluble azadirachtin at five different concentrations, viz., 0.01 g/10 mL (Group II), 0.02 g/10 mL (Group III), 0.03 g/10 mL (Group IV), 0.04 g/10 mL (Group V), and 0.05 g/10 mL (Group VI).
Negative control (Group VII): Distilled water
The antibacterial activity of all test agents was assessed by using agar well diffusion method and measured using zones of inhibition (in mm). Qualitative analysis assessed by measuring the colony forming units (in hundreds). The results were statistically analyzed using two-way ANOVA. Distilled water was inactive in all tests, thus, this group was not subjected to statistical analysis. Pairwise comparison of zone of inhibition and the CFU count was done by using Tukey's test.
Comparison of mean zone of inhibition
From [Table 1] we can infer that the inhibition zone of S. mutans starts at a concentration of 10% with a mean zone of inhibition of 1.58 mm and at 20% concentration the mean inhibitory zone is found to be 4.08 mm that is far less than that of chlorhexidine (19.33 mm). The mean inhibition zone of S. mutans at a concentration of 30% is found to be 12.83 mm. This shows that there is noticeable action of water soluble azadirachtin that begins at 30% concentration. By 40% concentration of water soluble azadirachtin the mean inhibitory zone is 19.32 mm that is almost similar to that of 0.2% chlorhexidine. This shows that the action of 40% concentration of water soluble azadirachtin is as good as the effect of 0.2% chlorhexidine. Also, at 50% concentration of water soluble azadirachtin the mean zone of inhibition was found to be 20.13 mm. There is no difference between the mean values with increasing concentration and the zone of inhibition. By using ANOVA test P value > 0.05, therefore, there is no significant difference between mean zone of inhibition between that of 0.2% chlorhexidine and 40% and 50% water soluble azadirachtin [Table 2].
|Table 2: P value table for pair wise comparison of zone of inhibition by using Tukey's test |
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Comparison of mean colony forming unit count (in hundreds)
From [Table 3] we can infer that the mean CFU of S. mutans for distilled water is 147.57 CFU/mL. The colonies were decreased to the extent of 74.07 colonies after treating with 0.2% chlorhexidine. The CFU count for 10% water soluble azadirachtin and 20% water soluble azadirachtin was 140.13 CFU/mL and 132.40 CFU/mL, respectively. The CFU count for 30% water soluble azadirachtin showed noticeable decrease with a count of 95.47 CFU/mL. The mean CFU count for 40% and 50% water soluble azadirachtin was 75.40 CFU/mL and 75.63 CFU/mL, respectively, which was almost same as that of 0.2% chlorhexidine. By using ANOVA test P value > 0.05, therefore, there is no significant difference between mean CFU count of 0.2% chlorhexidine with respect to 40% and 50% water soluble azadirachtin [Table 4].
|Table 4: P value table for pair wise comparison of CFU by using Tukey's test |
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| Discussion|| |
Azadirachta indica popularly known as "Indian Neem" is well-known in India and its neighboring countries for more than 2,000 years as one of the most versatile medicinal plant having a wide spectrum of biological activity. Importance of neem tree has been recognized by United States National Academy of Sciences, where neem is entitled as "a tree for solving global problems." Neem finds its immense use in a number of products from cosmetics to agriculture and from pharmaceuticals to ayurveda. Although, literature search reveals that neem tree has multiple potential uses in dentistry, its application is limited in routine dental practice. 
The utilization of antimicrobial mouth rinses is considered as a useful adjunct to oral hygiene. Main classes that have been tested are antibiotics, metal ions, enzymes, plant extracts, and phenolic compounds. The efficacy of these antimicrobial agents depends on many factors like vehicle used, concentration of active agents, and duration of the treatment.
This study was undertaken to assess the antibacterial activity of different concentrations of water soluble azadirachtin on S. mutans involved in the process of causing dental caries.
Several studies have demonstrated the antimicrobial action of chlorhexidine on S. mutans. ,, The present study also demonstrates the antimicrobial action of chlorhexidine (mean inhibition diameter - 19.33 mm) on S. mutans. Chlorhexidine has the ability to bind to both soft and hard tissue surfaces. This property enables it to act over a long period after used. Its antibacterial action is due to an increase in cellular membrane permeability followed by coagulation of the cytoplasmic macromolecules. ,
In the present study, the water soluble azadirachtin has shown a maximum zone of inhibition at 40% and 50% concentration with no statistical difference between the above mentioned two concentrations [Table 2]. Similar study conducted by Prashanth et al. to evaluate the antimicrobial effects of the chewing sticks of neem and mango against the microorganisms like S. mutans, S. salivarius, S. mitis, S. sanguis concluded that mango extract, at 50% concentration, showed maximum zone of inhibition on S. mitis. Neem extract produced the maximum zone of inhibition on S. mutans at 50% concentration. In addition, other Streptococcus species that are involved in the development of dental caries such as S. salivarius, S. mitis, and S. sanguis are also inhibited by neem extracts. 
In the present study, chlorhexidine has shown significant reduction in the S. mutans count (mean value of 74.07 × 10 2 CFU/mL), this observation adds to the earlier study performed by Heffi et al. 
The mean CFU count for 10% and 20% water soluble azadirachtin was 140.13 CFU/mL and 132.40 CFU/mL, respectively, whereas 30% water soluble azadirachtin has reduced CFU count of S. mutans significantly (95.47 CFU/mL) but was less effective when compared to chlorhexidine. The mean CFU count for 40% and 50% water soluble azadirachtin was 75.40 CFU/mL and 75.63 CFU/mL, respectively, which was same as that of 0.2% chlorhexidine [Table 3]. Studies conducted on extracts of neem metabolite have shown that 50% neem metabolite is effective against S. mutans. 
A study related to the antibacterial effect of neem mouthwash against salivary levels of S. mutans and Lactobacillus acidophilus by Vanka et al. states that S. mutans was inhibited by neem mouthwashes as well as chlorhexidine; but lactobacillus growth was inhibited by chlorhexidine alone.  This means that neem can be used as an antimicrobial agent to prevent the initiation of dental caries and not for reversing incipient carious lesions.
The use of harvested chewing sticks after prolonged storage period is not advisable for oral hygiene measures because it was noted that Azadirachta indica chewing twigs were more susceptible to postharvest fungal overgrowth within 4 weeks of storage.  On the other hand water soluble azadirachtin has a shelf life of more than 2 years, thus, making it commercially acceptable.
In the present study, it was found that water soluble azadirachtin has the advantage of being a unique water soluble extract, when compared to other neem extracts which were formulated in various organic solvents, such as alcohol and ether, which reduced their shelf life. It is also cost effective as water is readily available as a universal solvent. Water soluble azadirachtin is highly thermostable even at 110°, compared to other neem seed kernel extracts that are considered to be thermolabile.  The above mentioned advantages of water soluble azadirachtin make it a unique and effective antimicrobial agent that can be used for the prevention of dental caries. The preparation of the water soluble azadirachtin is easy and can be instantly prepared. It can be used as an adjunct to oral hygiene measures on a day-to-day basis without any potential risks for caries prevention as well as periodontal problems.
Candida albicans and Enterococcus faecalis are the most predominant microorganisms recovered from root canals of teeth. Thorough debridement of an infected root canal and complete elimination of microorganisms are objectives of an effective endodontic therapy. Several in vitro studies have shown that neem leaf extract is a viable medicament against C. albicans, E. faecalis, and even their mixed state.  Further studies on water soluble azadirachtin can be undertaken to find out the effect on these microorganisms as its antioxidant and antimicrobial properties can make it a potential agent for root canal irrigation as an alternative to sodium hypochlorite.
According to the present study it can be stated that water soluble azadirachtin has got antimicrobial action on aerobic organisms. However, further studies have to be undertaken to conclude its effectiveness on different categories of aerobic and anaerobic microorganisms.
| Conclusion|| |
From the above study and discussion, it can be concluded that a 40% concentration of water soluble azadirachtin have potential antimicrobial action against the microorganism responsible for initiation of dental caries. This study on natural product to cure diseases may create a promising alternative to chlorhexidine. This study might as well open the possibilities of finding a new clinical and effective herbal remedy for prevention of dental caries. A drug-development program should be undertaken to find out the effect of water soluble azadirachtin on various other microorganisms present in the oral cavity.
As the taste of this powder is bitter a masking agent is required to mask the effect so as to obtain a better acceptability and patient compliance. Sachets of powders containing azadirachtin can be developed after extensive investigation of its bioactivity. These sachets can be used as a mouthrinse almost everywhere as water is readily available in every corner of the world. Once the neem tree extracts become the mainstay for the management of various oral diseases, it can be truly regarded as "aristha," which means "reliever of sickness" in Sanskrit.
I thank Dr. Rama Bhadekar, Head of Department, Microbial biotechnology, Rajiv Gandhi Institute of Biotechnology, Pune for helping me to conduct this study and providing me with continuous help and guiding me through the minor steps to be carried out during the entire procedure.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]