AUTHORS
Dr. Anurag Singhal, Dr. Anuraag Gurtu, Dr.Chandrawati Guha MDS
Department of Conservative Dentistry & Endodontics
Institute of Dental Sciences,
Bareilly UP.

CORRESPONDING AUTHOR
Dr. Sumit Mohan
C/O Dr. Chander Mohan
Professor & Head
Department of ENT Rohailkhand Medical College,
Bareilly UP. ABSTRACT

This in vitro study compares the antibacterial effectiveness of commonly used irrigants with herbal products like neem and green tea against Enterococcus faecalis. Calcium hydroxide was used as an intracanal medicament.
The herbal extracts were prepared and the samples were divided into five groups:

• GROUP I – Conventional irrigants (Sodium Hypochlorite and Chlorexidiene

Gluconate)

• GROUP II – Herbal irrigants (Green tea and Neem)
• GROUP III - Conventional irrigants + Calcium hydroxide
• GROUP IV - Herbal irrigants + Calcium hydroxide
• GROUP V – Control group (distilled water)

Antibacterial activities of the materials were evaluated against the Enterococcus faecalis. The culture was prepared and zones of inhibition were calculated. The results were tabulated. Under the limitations of this study it could be concluded that neem had significant antimicrobial action against Enterococcus faecalis alone and with calcium hydroxide.

KEY WORDS

Enterococcus faecalis, Neem, Green tea, Sodium hypochlorite, Chlorexidiene Gluconate, Calcium hydroxide, Zones of Inhibition

INTRODUCTION

The basis of success of endodontic treatment and retreatment depends on identifying and eliminating the causative factors in the development of apical periodontitis, so that healing can be achieved. The role of bacteria and their by products in the pathogenesis of apical periodontitis has been clearly established. (1)

It is evident that an infected root canal system is a unique niche for selective species of microorganisms. Results of studies have clearly shown that a variety of microorganisms reside in the root canal. Of them E. faecalis has been isolated from almost 29%-77% teeth undergoing endodontic procedures. (2) The reduction and elimination of bacteria and their by products should be given the utmost importance towards achieving a successful endodontic therapy. (3)

Endodontic therapy aims at removal of bacteria from the root canal space and to prevent re infection. (4) Chemical treatment of the root canal system can be arbitrarily divided into irrigants rinses and inter appointment medicaments. (5)

Although sodium hypochlorite has been the irrigant of choice for non surgical endodontic procedures, it has many deleterious effects if pushed beyond the apex. Chlorexidiene gluconate, a synthetic cationic bis-guanide is another commonly used disinfectant. But its activity is pH dependent and it is toxic to human periodontal ligament (PDL) cells.

The use of plants and plant products as medicines could be traced as far back as the beginning of human civilization. The earliest mention of medicinal use of plants in Hindu culture is found in “Rigveda”. Neem has been extensively used in ayurveda, unani and homoeopathic medicine and has become a cynosure of modern medicine. Tea is the second most commonly drank liquid on earth after water. It is known to possess anticariogenic and antibacterial properties.(14, 15)

Herbs like neem and green tea might have a potential use as irrigants as they have been found to possess antimicrobial and antifungal properties.(7,8,14,15)
The use of calcium hydroxide as an intracanal medicament has expanded in dentistry owning to its antimicrobial activity, tissue dissolving, ability, and inhibition of tooth resorption.(9)

The need for the study arose keeping in mind the inability of conventional irrigants to completely remove E.faecalis from the root canal system. Several mishaps during root canal irrigation have been described. These ranges from damage to the patient’s clothing, splashing the irrigant into the patient’s or operator’s eye, injection through the apical foramen, air emphysema, and allergic reactions to the irrigant. The deleterious effects of irrigants also stimulated the need for this study.

MATERIALS AND METHODS

Preparation of herbal extracts

Mature fresh Azadirachta indica (neem) leaves were collected. Leaves were washed in sterilized distilled water and weighed in a sterile disposable cup. 25 gms of fresh neem leaves were added to 50 ml of absolute ethanol. Mixture was macerated for 1-2 minutes and care was taken to prevent temperature rise beyond 45-50 C. Extract was filtered through muslin cloth for coarse residue. Extraction process was repeated again using coarse residue and 25 ml ethanol. Both the extracts were pooled together and filtered through fast filter paper. Alcohol part was removed from the extract on water bath till the volume was about 25 ml. Extract was ready and stored in airtight container.(8)

Commercially available green tea was used to prepare green tea extract. 25 gms of tea was added to 50 ml of water. Mixture was boiled for 1-2 minutes, cooled and stored in air tight container.

 

Fig A) Materials used in the study

Preparation of Calcium Hydroxide paste

Calcium hydroxide powder was mixed with sterilized saline in 1:1 ratio on a pad using a clean spatula. The mixture obtained was creamy in consistency.

The samples were divided into 5 groups:

GROUP I – Conventional irrigants
A) Sodium Hypochlorite
B) Chlorexidiene Gluconate
GROUP II – Herbal irrigants
A) Green tea
B) Neem
GROUP III - Conventional irrigants + Calcium hydroxide
GROUP IV - Herbal irrigants + Calcium hydroxide
GROUP V – Control group (distilled water)

TEST MICROORGANISMS AND GROWTH CONDITIONS

Antibacterial activities of the irrigants and intracanal medicamrnts were evaluated against the Enterococcus faecalis. The strain was obtained from the Department of Microbiology, Rohailkhand Medical College, Bareilly, Uttar Pradesh, India.

Bacteria were grown aerobically to late logarithmic or early stationary phase from frozen-stock cultures in brain-heart infusion (BHI). Bacterial suspensions were mixed with the respective groups equal volume. Distilled water served as a positive control.

After incubation at room temperature, the survival of bacterial in solution was assessed by 10 fold serial dilutions. The culture was prepared on TSA plates.
After incubation for 24-48 hours, at 37 degree centigrade, Colonies on plates were counted and colony forming units per ml (CFU/ ml) were calculated.
All the experiments were performed in triplicate.

RESULTS

The results indicate that under experimental conditions,

• GROUP IV where herbal irrigants were used in combination with calcium hydroxide had maximum antimicrobial activity against Enterococcus faecalis.
• Neem (Azadirachta indica) in combination with calcium hydroxide was

observed to have superior antibacterial efficacy than sodium hypochlorite
and calcium hydroxide.

• GROUP III had significant antimicrobial activity against Enterococcus faecalis.

Sodium hypochlorite calcium hydroxide combination had better
antibacterial efficacy in comparison to chlorexidiene calcium hydroxide
combination.

• No significant difference was observed in the antimicrobial activities of GROUP I and GROUP II Amongst the representatives of both groups, sodium hypochlorite and Azadirachta indica were found to possess superior bacterial inhibition properties.
• No antibacterial activity was observed in GROUP V (control group).
  
  

FIG II: ZONES OF INHIBITION

ABLE I
ZONES OF INHIBITION OF SAMPLES AGAINST ENTEROCOCCUS FAECALIS

GROUPS	ZONES OF INHIBITION (mm)
GROUP I (Conventional irrigants)	15.0
GROUP II (Herbal irrigants)	13.0
GROUP III (Conventional irrigants + Calcium hydroxide)	18.0
GROUP IV (Herbal irrigants + Calcium hydroxide)	20.0
GROUP V (Control)	00.0


TABLE II
ZONES OF INHIBITION OF IRRIGANTS AGAINST ENTEROCOCCUS FAECALIS



IRRIGANTS	ZONES OF INHIBITION (mm)
SODIUM HYPOCHLORITE	15.0
CHLOREXIDIENE	1I.0
NEEM	16.0
GREEN TEA	12.0
DISTILLED WATER	00.0


DISCUSSION

A strong correlation has been observed between apical periodontitis and the presence of bacteria in canals. If bacteria persist in the root canal system at the time of obturation, there is a higher risk of failure. (1,13) Enterococcus faecalis is the “star survivor” in the root canal. Even calcium hydroxide fails to eliminate it completely.2 This can be attributed to the fact that this bacteria has a proton pump which lowers the internal pH of the cell and maintains it. (11)

Sodium hypochlorite, although widely recommended has several undesirable characteristics such as tissue toxicity, risk of emphysema, allergic potential and disagreeable smell and taste.(8,12) Antibacterial activity of chlorexidiene is pH dependent. Moreover CHX has no capability to dissolve vital or necrotic tissue. In laboratory experiments, it has been demonstrated that CHX is highly cytotoxic to human periodontal ligament (PDL) cells and human fibroblasts via inhibition of protein synthesis.(6)

In dentistry, Azadirachta indica has been investigated, due to its antimicrobial potential against oral microorganisms especially those associated with gingivitis and periodontitis (8,14) Botelho et al (2008) and Behl et al in their experiments and trials concluded that Azadirachta indica is highly efficacious in the treatment of periodontal disease thus exhibiting its biocompatibility with human PDL fibroblasts.

The use of neem as an endodontic irrigant might be advantageous because it is a biocompatible antioxidant and thus not likely to cause the severe injuries to patients that might occur via NaOCl accidents. Neem acts on oral microflora because of its anti-adherence activity by altering bacterial adhesion. Nimbidin and nimbolide which are constituents of neem have been found to possess antibacterial and antifungal properties. These components cause lysis of the bacterial cell wall. (8,14)

The American Medical Association shows that green tea has excellent medicinal values.(15) It is also observed that green tea has antibacterial effect against Enterococcus faecalis. (7) Green tea polyphenols antioxidant potential is directly related to the combination of aromatic rings and hydroxyl groups that make up their structure, and is a result of binding and neutralization of free radicals by the hydroxyl groups leading to destruction and dissolution of bacterial cell wall.

CONCLUSION

As the global scenario is now changing towards the use of non toxic plant products that have traditional medicinal use, extensive research and developmental work therefore should be undertaken on neem and its products for their better economic and therapeutic utilization.

Under the limitations of this study, it was concluded that although conventional irrigants are more frequently used, have shortcomings in antibacterial efficacy. It was concluded that neem leaf and green tea extract has a significant antimicrobial effect against E. faecalis.

Microbial inhibition potential of neem leaf extract observed in this study opens perspectives for its use as an intracanal medication. It was also observed that neem calcium hydroxide combination had superior antimicrobial action against E. faecalis.

However, preclinical and clinical trials are needed to evaluate biocompatibility and safety before neem can conclusively be recommended as an intracanal irrigating solution, but in vitro observation of neem effectiveness appears promising.

References

References are available for request