Antimicrobial Efficacy of Azadirachta indica against Streptococcus mutans– An In vitro Study

 

Christy. S1, Dr. Nivedhitha. M. S2

1Postgraduate Student, Department of Conservative Dentistry and Endodontics, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, India.

2Professor and Head of the Department, Department of Conservative Dentistry and Endodontics,

Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, India.

*Corresponding Author E-mail: nivea16@hotmail.com

 

ABSTRACT:

Introduction: Dental caries is one of the most common chronic diseases among all age groups. The main bacterial agents in caries development are Streptococcus mutans. Reducing these microorganisms causes a significant decrease in dental caries. Plaque-induced caries is a local disease; therefore, local use of antimicrobial agents is more efficient than their systemic use. Streptococcus mutans is a Gram-positive, non-motile, non-spore forming, catalase negative, facultative anaerobic cocci bacterium commonly found in the human oral cavity, is a significant contributor to tooth decay. Azadirachta indica (neem) is known for its Indian medicinal value and demonstrated anti‑inflammatory, immune‑modulatory, antibacterial, antifungal, antiviral, antioxidant, and anti‑carcinogenic properties. The aim of this study is to evaluate the Antimicrobial efficacy of neem extract against Streptococcus mutans. Materials and methods: Neem leaf extract and 0.2% chlorhexidine was used to assess the antimicrobial efficiency. Agar well diffusion test was used to study the antimicrobial efficacy. Each well was loaded with 20, 40, 60, 80 and 100 µl of corresponding concentration of neem extract and calcium hydroxide was used. The plates were incubated for 24h at 37°C. The development of inhibition zone around the well was measured (diameter). The zone of inhibition was recorded, tabulated, and analyzed statistically with the help of IBM Statistical Package for the Social Sciences statistics version 20 using analysis of variance test. Results: Both neem extract and chlorhexidine showed well-defined and comparable zones of inhibition around their respective wells. Zone of inhibition of neem extract was significantly higher than chlorhexidine. Analysis of variance showed significant difference between zone diameters of neem leaf extract, and chlorhexidine against Streptococcus mutans (p < 0.05). Conclusion: From the present study, it can be concluded that neem leaf extract shows significant difference in zones of inhibition when compared with chlorhexidine.

 

KEYWORDS: Neem extract, Antimicrobial activity, Inhibition zone, MIC, MBC.

 

 


 

 

INTRODUCTION:

Dental caries is one of the most common human diseases that affect the vast majority of individuals among all age groups. Dental caries is defined as the localized destruction of tooth tissue by bacterial fermentation of dietary carbohydrates by Samaranayake (1). Among the cariogenic bacteria present in the oral cavity, the most common and most destructive bacteria responsible for dental caries are the Streptococcus mutans. The epidemiological longitudinal and cross sectional surveys showed that the Streptococcus mutans are the main etiology for human dental caries (2).

 

Streptococcus mutans is a Gram-positive, non-motile, non-spore forming, catalase negative, facultative anaerobic cocci bacterium commonly found in the human oral cavity. Streptococcus mutans can rapidly metabolize sugars to lactic acid and other organic acids, reducing pH and initializing enamel demineralization (3). Inhibiting the growth of the Streptococcus mutans in the oral cavity would lead to healthier teeth and gums. Manson and Aley also states that with the current soft civilized diet, teeth wear is slight or absent, thus encouraging bacterial deposition.

 

Neem (Azadirachta indica, A. Juss) is a tree which belongs to the Meliaceae family and is considered to be a holy medicinal tree found in India. It is also known as “Indian neem/ margosa tree” or “Indian Lilac” and “Persian Lilac”. It possesses a wide range of biological activities, such as anti-inflammatory, antimalarial, antimicrobial, antiviral, antifungal, antipyretic, antioxidant, analgesic, immunestimulant, anti-fertility, anti-acne, anti-hypoglycemic, anti-cancer and nematicidal properties (4). Neem is a common plant which is cultivated in various parts of India for religious and medicinal reasons. Neem is considered to be a “village dispensary” since every part of the tree including the leaves, bark, and seeds have medicinal properties. US National Academy of Sciences recognized the importance of neem tree in 1992 and entitled neem as “a tree for solving global problems”. The Neem is of particular interest to the field of dentistry for it has a long history treating teeth and gum problems. In rural areas of India, the twigs are used as toothbrush to prevent gingivitis (5-7).

 

The objective of this study is to determine the anti-bacterial effectiveness of the Neem leaves extract against Streptococcus mutans. The null hypothesis to be tested was that there was a difference in the antimicrobial efficacy of Azadirachta indica, and chlorhexidine against Streptococcus mutans.

 

MATERIALS AND METHODS:

In this study E. faecalis and C. albicans species was used to test the antimicrobial efficacy. The bacterial culture was prepared in sterile Luria broth and adjusted to an optical density to match the turbidity of a McFarland 0.5 scale. The sensitivity assay was done using Neem leaf (A. indica) extract and Chlorhexidine.

 

 

 

Preparation of Neem Extract:

Neem leaves were shade dried, powdered and stored in air-tight containers. They were soaked at room temperature for 24 hours, then filtered. Air dried Neem powder was repeatedly macerated with 500ml of 99% ethanol and filtered using whatman filter paper. The ethanol was evaporated and the extracts were concentrated using rotary flash evaporator and used in the assay (8-10).

 

 

Figure 1: Preparation of Ethanolic Extract of Neem Leaves

 

Agar Well Diffusion Method:

Sample Preparation: 10mg/ml of sample was provided for antimicrobial assay.

 

Procedure: Luria broth Agar (LBA) plates were inoculated with test organisms. The plates were evenly spread out. Then wells were prepared in the plates with a cork borer. Each well was loaded with 20, 40, 60, 80 and 100 µl of corresponding concentration of neem extract and calcium hydroxide was used. The plates were incubated for 24h at 37°C. The development of inhibition zone around the well was measured (diameter) and recorded.

 

 

 


Figure 2: Zone of inhibition for Streptococcus mutans for Neem Extract and Chlorhexidine

 


Recording of the zones of inhibition was done for all the culture plates and the results were analyzed statistically with the help of IBM Statistical Package for the Social Sciences statistics version 20 using analysis of variance test.

 


 

Table 1: Results for Zone of Inhibition for Streptococcus mutans

Source of variation

Sum of squares

Degree of freedom

Mean square

Variance ratio

(F)

Table value at 0.05 level of Significance

Between columns

260.1

1

257

6.00

5.99

Within columns

166.4

4

37

0.16

4.53

Total

426.5

6

 


DISCUSSION:

There is a growing interest throughout the oral health care profession in incorporating herbals in oral health care products. Extensive studies are being done to find plant based alternatives for oral health. Many plants have been reported to inhibit the growth of oral bacteria particularly Streptococcus mutans and control plaque and thus prevent caries (12).

 

Neem (A. indica A Juss, margosa tree) is a traditional plant in India, with medicinal properties in each part of the tree. It has antibacterial, antifungal, antiviral, antioxidant, anti-inflammatory, and immune‑stimulatory activity. The isoprenoid group (nimbin, nimbinin, nimbidinin, nimbolide and nimbidic acid) of constituents of neem has a broad range of therapeutic and antimicrobial effects suggesting its potential as an endodontic intracanal medicament (13,14).

 

The use of neem in reduction of dental plaque is advantageous because neem is an excellent antioxidant with a very high biocompatibility, and there is no risk of tissue toxicity with its use. Biocompatibility of neem to the human periodontal ligament fibroblasts has already been proved, and this is an important factor favoring its clinical application in endodontics (15). Because of the presence of nimbidin in neem, it has the bitter taste which is unpleasant for the patient. The bitter taste can be overcome by adding sweeteners to the neem extract (16,17).

 

It is known that a balance exists in a person’s oral microbial population. If this balance is lost, opportunistic microorganisms can proliferate, enabling the initiation of disease processes (18). Chlorhexidine is a cationic biguanide with broadspectrum antimicrobial action, whose effectiveness in decreasing the formation of dental plaque has been demonstrated in several clinical studies. The common side effects of Chlorhexidine as discoloration of mouth, increase of Tartar formation on the teeth, taste problems such as decreased taste or change in taste, tooth discoloration (19). The Brownish discoloration of teeth is known as Maillard reaction. The serious side effects of Chlorhexidine are mouth ulcer, white patches or sores inside the mouth or on the lips, swelling of salivary glands, signs of an allergic reaction which may include difficulty in breathing or swelling of face, lips, tongue and throat (20). Immediately after brushing using of chlorhexidine reacts with anions of the toothpaste and reduces the antimicrobial activity.

 

The isoprenoid group (nimbin, nimbinin, nimbidinin, azadirachtin, nimbolide and nimbidic acid) of constituents of neem has a broad range of therapeutic and antimicrobial effects suggesting its potential as an effective agent to reduce dental caries (21).

 

Azadirachtin the active component of Neem is of the phenol group and that phenols substances can destroy bacterial cell walls which will inevitably inhibit the growth of bacteria. The breakdown of cell wall disturbs osmotic pressure and leads to cell death (22).

 

 

 

The results of this study shows that the Neem leaves extract has antibacterial activity towards Streptococcus mutans. The inhibition area that is formed is larger for Neem leaves extract than chlorhexidine. The statistical analysis carried out using the ANOVA proves that the neem extract has significant influence on the growth of Streptococcus mutans (23).

 

These results also coincide with the results in the research of Satya and Pramularsih. Satya documented a study using Neem leaves to inhibit the growth of Bacillus subtilis and E. coli. Pramularsih on the other hand studied the influence of Neem leaves extract in the inhibition of Staphylococcus aureus and Salmonella typhi (24). Although the bacteria used in the both the experiment are different, all the results correspond with the theory that Azadirachtinthe active component of Neem is of the phenol group and that phenols substances can destroy bacterial cell walls which will inevitably inhibit the growth of bacteria.  The breakdown of cell wall disturbs osmotic pressure and leads to cell death (25,26).

 

Wolinsky et al in a study have reported that the active components from a bark containing neem stick have appeared to inhibit virulence factors of oral streptococci related with dental plaque formation.Prashant et al study showed aqueous neem stick extract showed significant zone of inhibition against S.mutans.Sajankumar in his study reported that aqueous neem stick extract showed greater zone of inhibition (27).

 

Geetha et al did a on essential oils and found that neem oil has greater zone of inhibition against S. mutans. Sri KaviSubramaniam et al reported in her study that aqueous neem leaf extract greater zone of inhibition against S. Mutans (28).

 

Vankaet al., (29) studied on acetone extract of Azadirachta indica showed maximum inhibitory activity against Streptococcus mutans with 22mm, whereas the chloroform and methanol extracts were with sensible inhibitory effect on all tested organisms. Azadirachta indica mouth wash is reported to inhibit growth of S. mutans and carious lesions.

 

The isolate Streptococcus salivarius was highly sensitive to the chloroform extracts (18mm) of A. indica and it gives comparable sensitivity pattern for all the other extracts (16mm) were given in table 2. Bhuaiyanet al., (30) made astudy on antibacterial property of crude neem bark on Streptococcus and were found that neem was effectivetowards dental diseases.

 

The pathogen Fusobacterium nucleatum was sensitive to water extract as well as ethanol extract (16mm) of Azardirachta indica followed by the other two extracts such as petroleum ether and chloroform. Khan et al (31) evaluated the antimicrobial properties of A. indica leaves against Micrococcus albus, Staphylococcus aureus, Proteus vulgaris and Pseudomonas aerogenosa causing dental carries. The anti-microbial activities of petroleum ether extract, chloroform extract and methanol extract of neem leaves are checked by disc diffusion method (32). All the leaf extracts exhibited significant inhibition. Comparative study of the results obtained from both the methods indicates that the chloroform Extract shows better antimicrobial activity.

 

Future experiments with a narrower range are necessary to determine the effective concentration of Neem leaves extract to inhibit the growth of Streptococcus mutans (32). In this experiment, the Neem leaves are extracted using Organic dissolvent such as ethanol is more effective because theoretically the active component belongs to the phenol group and phenols would dissolve better in organic dissolvent (33). The use of ethanol extractions may also be able to show the effective concentration of Neem leaves extract in the inhibition of Streptococcus mutans which could not be determined in this experiment.

 

CONCLUSION:

The antimicrobial potency of the neem extract is believed to be due to tannins, saponins, phenolic compounds, essential oils and flavonoids. This Invitro study gives us natural antimicrobial agents which can help us to control dental caries. The effects of the neem extracts may benefit if incorporated in tooth paste, mouth rinses and dental products to reduce plaque and dental caries. In vivo clinical testing is essential to conform in vitro results. More research into herbals in dentistry can give us simple, effective solutions to dental diseases.

 

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Received on 21.05.2019            Accepted on 23.06.2019           

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Tech.  2019; 9(3):149-153.

DOI: 10.5958/2231-5713.2019.00025.4