Antimicrobial Efficacy of Plant Extracts Combined with Endodontic Sealer on the Growth of Oral Pathogen - An In Vitro Study

Introduction

Microorganisms and their byproducts are the main agents causing endodontic disease [1]. Thus, destruction of these microorganisms in the root canal system is the major goal of endodontic treatment. Effective performance in all phases determines the success of endodontic treatment. Even after thorough chemo-mechanical preparation, micro-organisms still remain in the canal due to the complicated root canal anatomy including accessory canals, isthmus and fines which are inaccessible and the aim of root canal treatment cannot be accomplished completely [2]. Therefore, it is desirous to use sealers with a good sealing ability and antimicrobial activity to eliminate these residual microorganisms [3].

However, controversies related to antibacterial effects of sealers on common isolated bacteria in infected teeth as well as their degree of cytotoxicity , increased antibiotic resistant strains and side effects caused by synthetic drugs has made researchers to look for herbal alternatives [4, 5].

The usage of plants and plant products as medicines is outlined way back from beginning of human civilization. Medicinal plants are rich in Secondary metabolites and essential oils which are of therapeutic importance to humans. Several advantages of using medicinal plants are that they are safe, economical, less toxic, easily available, good shelf life and less chances of developing microbial resistance [6, 7].

Cinnamon also known as C. zeylanicum has gained popularity in recent time due its antibacterial activity. Vanillic, caffeic, gallic, protocatechuic, p coumaric, and ferulic acids present in the extracts are the ones which exhibit antibacterial action. It has shown effectiveness against S. mutans and E. faecalis organisms [8]. It has also known to show a wide spectrum of action on gram positive and gram negative organism. The C. zeylanicum bark is rich in cinnamaldehyde (50.5%), which has been proven to be active against many pathogenic gram-positive and gram-negative bacteria. Ali, et al. [3] reported cinnamaldehyde as the active agent to inhibit the growth of both antibiotic-sensitive and- resistant strains of Helicobacter pylori [3, 9].

Ginger belongs to Zingiberaceae family and it has also been used as a medicine from Vedic days due to its antibacterial and antifungal properties. Maekawa, et al. evaluated the antimicrobial efficacy of Glycolic propolis, ginger extract, Ca(OH)2, CHX and their combinations against E. faecalis, C. albicans, Escherichia coli and endotoxins in root canals. The ginger extract used in this study, was the dehydrated rhizome of Zingiber officinale, which contains flavonoids, gingerol, shogaol, zingerone, paradol and volatile oil [10].

Liquorice (Glycyrrhiza Glabra) commonly known as Atimaturam is the most commonly used flavouring agent in kampo medicines. The compound glycyrrhizin (or glycyrrhizic acid), found in liquorice, has antiviral, antimicrobial, anti-inflammatory, and anti- carcinogenic. Liquorice extract is efficient in reducing pain and promotes healing in apthous ulcer [11, 12, 13].

Though there are various uses of herbs in medicinal field, less studies have been done in the field of dentistry.

Hence, the aim of the study was to evaluate and to compare the antimicrobial efficacy of root canal sealer mixed with herbal extracts against oral pathogen.

Methodology

The present in vitro microbiological study was conducted in Department of Conservative dentistry and Endodontics, KVG Dental College and Hospital, in collaboration with Department of Microbiology, KVG Medical College and Hospital and KVG Ayurveda Medical College, Sullia. In the present study antimicrobial efficacy of three herbal extracts Glycyrrhiza glabra, Zingiber officinale, Cinnamon zeylanicum in combination with Zinc oxide eugenol based sealer (Pulp canal sealer EWT) was evaluated against Enterococcus faecalis using agar diffusion test.

Results

Group III- showed maximum zone of inhibition against the tested organism with mean value of 19 at70:30 concentration, followed by group II with mean value of 14.55 at 90:10 concentration, group with mean value 13.8 at 60:40 concentration and group IV with mean value 10 (Table 2, Figure 2).

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Intergroup comparison of mean zone of inhibition of E. faecalis showed statistically significant results (Table 3). Intragroup comparison of mean zone of inhibition of E. faecalis showed statistically significant results in group III and group IV (Table 4, Figure 2).

Statistical analysis: Post HOC Tukey test. * Statistically significant if P<0.05. Table 4: Intragroup Comparison of Mean Zones of Inhibition of Enterococcus faecalis.

Discussion

Elimination of bacteria by cleaning, shaping, and obturation of the root canal system is the objective of root canal treatment. So the root canal sealers with good sealing ability and antimicrobial activity with low toxic effects, which are desirous to kill residual microorganisms inside the canal are recommended [5]. Hypersensitivity, immune suppression, and allergic reactions are some of the adverse effects associated with antimicrobial agents, and the continuous evolution of bacterial resistance has necessitated the researchers to search for medicinal plant alternatives as antimicrobial agents [8].

Medicinal plants are rich sources of bioactive compounds such as alkaloids, flavonoids, and phenolic compounds. Owing to their ability to combat new developing drug resistant pathogens, for herbal preparations it is always a welcome in present day science [4]. There are insufficient scientific reports that indicate the antimicrobial activities of herbal extracts mixed with endodontic root canal sealers. Hence, the aim of the study was to evaluate and compare the antimicrobial efficacy of endodontic sealer mixed with herbal extracts.

The most commonly used root canal sealers in endodontics are zinc-oxide-eugenol-based sealer. As it has antimicrobial activity against E. faecalis, S. mutans, E. coli and S. aureus. Hashieh, et al. (1999) studied the useful effects of eugenol and stated that the amount of eugenol released in the periapical area immediately after placement was 104 and falls to 10-6 after 24 hrs, reaching zero after one month. Within these concentrations it is said to have anti-inflammatory and analgesic properties. Considering these valid points, the present study was designed to evaluate whether there is any improvement in the antibacterial activity by combining the herbal extracts with ZOE [5]. So in this study pulp canal sealer EWT (zinc oxide eugenol based sealer) composed of Powder: Zinc oxide, silver, Liquid: Eugenol, Balsam, Canada was taken.

Methanolic extracts were shown to have a greater activity compared to ethanolic or aqueous extracts because more phytoconstituents are leached out from it when compared to ethanolic extracts. Hence, methanolic extracts of three herbal plants were prepared as test medicaments [4, 15].

Enterococcus faecalis (a gram-positive coccus) was chosen as the test organism because it is a facultative anaerobe that is the most resistant species in the oral cavity and the possible cause of failure of root canal treatment. Sundqvist, et al. has quoted that 38% of failed root canal systems were contaminated with E. faecalis. Therefore, it might be advantageous if the sealer exerts some antimicrobial activity as the last element in treatment regimen [2, 10, 13].

Antimicrobial efficacy of any natural or synthetic agent can be evaluated by Broth dilution method, Agar dilution method, Disc diffusion method, Cup plate method or Agar well diffusion method and Ditch plate method.8However, agar well diffusion method was used in the present study as it depends upon the diffusion of the tested material to such an extent that growth of the added microorganism is prevented entirely in a zone around the well containing the test material [5].

The result of the present study showed that zinc oxide eugenol based root canal sealer (pulp canal sealer EWT) combined with Cinnamon showed highest antimicrobial efficacy in descending order followed by ginger and sealer combination and liquorice and sealer combination.

The high antimicrobial efficacy of cinnamon combination in group III may be attributed due to the presence of vanillic, caffeic, gallic, protocatechuic, p coumaric, and ferulic acids in the extract. It has shown effectiveness against S. mutans and E. faecalis organisms [6, 8]. The bark is rich in cinnamaldehyde (50.5%), which has been proven to be active against many pathogenic gram-positive and gram- negative bacteria. Compared to plain ZOE, more antibacterial activity was noticed when ZOE is mixed with herbal extracts. This results were in accordance with study by Cinthura C, et al. [10] Gupta Wadhwa, et al. [16] Sabarathinam, et al. [6] Veerale Panchal et al. [8], Gupta, et al. [17] have done done studies on with cinnamon zylanicum as irrigant, intracanal medicant and have obtained the similar result.

Group II sample exhibited better antibacterial activity compared to group I and group IV ,this may be due to presence of, the pungent substances namely gingerol, shogaol, zingerone, paradol and volatile oilin it, studies tell that The most effective antimicrobial constituent was found to be citral inhibiting growth of both gram-negative and gram-positive bacteria [15].

Group I sample showed better result than group group IV due to the presence of liquorice. Glycyrrhizin, a diglucuronide, is the active principle showing better antibacterial effect as compared to ZOE sealer alone [12].

Zinc oxide (Pulp canal sealer–powder) component was resistant and eugenol (pulp canal sealer-liquid) exhibited antibacterial activity in this study. Zinc oxide Eugenol based sealer showed least inhibition zone.Amongst the plain extract which was tested for antibacterial sensitivity Cinnamon showed maximum inhibition zone followed by ginger and liquorice.

The antimicrobial efficacy of any test agent depends upon its extent of diffusion capacity through the solid agar medium which will show the inhibitory zones. The variability in the inhibition zones noticed in the present study can be attributed to the formulations used that provided a compatible mix with zinc oxide eugenol. Studies have also been done with ethanolic or methanolic extract forms of herbs and has shown that these alterations can either increase or decrease the actual antimicrobial activity of these formulations. Another reason could be the type of organism selected. In the present study, lyophilized forms were used whereas in the earlier studies the organisms have been isolated directly from the root canals [18].

Conclusion

On the basis of the results, observations, and statistical analysis, the following conclusions could be drawn: Zinc- oxide-eugenol-based root canal sealer (pulp canal sealer EWT) when mixed with the three tested herbal extracts, showed maximum zone of inhibition for Cinnamon zylanicum followed by, Zingiber officinale and then Glycyrrhiza glabra. However the synergic effect needs to be further evaluated for clinical application.