|Year : 2019 | Volume
| Issue : 1 | Page : 39-45
Influence of plant extracts mixed with endodontic sealers on the growth of oral pathogens in root canal: An in vitro study
Sonali Saha1, Gurpreet Dhinsa2, Ujjala Ghoshal3, Amrin Nayani Fatima Afzal Hussain3, Soumyabrata Nag3, Aarti Garg4
1 Department of Pedodontics and Preventive Dentistry, Sardar Patel Post Graduate Institute of Dental and Medical Sciences, Lucknow, Uttar Pradesh, India
2 Department of Periodontology, Career Post Graduate Institute of Dental Sciences and Hospital, Lucknow, Uttar Pradesh, India
3 Department of Microbiology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
4 Department of Pedodontics and Preventive Dentistry, Jaipur Dental College, Jaipur, Rajasthan, India
|Date of Web Publication||25-Feb-2019|
Dr. Sonali Saha
Department of Pedodontics and Preventive Dentistry, Sardar Patel Post Graduate Institute of Dental and Medical Sciences, Raibarelly Road, Lucknow - 226 025, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Introduction: Microbes are considered as the primary etiological agents in endodontic diseases. Ways of reducing these agents are root canal debridement and antibacterial filling materials. One of the factors in determining the success of endodontic treatment previously was sealing root canals with materials possessing potent bactericidal effect. Due to cytotoxic reactions of sealers and their inability to eliminate bacteria completely from dentinal tubules, trend to use natural plants extracts have been introduced. Aim: To compare antimicrobial activity of endodontic sealers added to herbal extracts. Materials and Method: Three sealers mixed with three herbal extracts were evaluated against seven strains of bacteria at various time intervals using Agar Diffusion Test. The mean zones of inhibition were measured. Statistical Analysis: All statistical analysis was performed using the SPSS 15 statistical software version, Chicago. Intergroup comparison was evaluated using Kruskal Walls test along with Mann Whitney U test. The Intragroup comparison was evaluatd using Friedman test along with Wilcoxon test. Results: Statistically significant zones of bacterial growth inhibition were observed largest with Zinc Oxide Eugenol based sealer when mixed with Glycyrrhiza glabra (Licorice) followed in descending order by zinc oxide eugenol based sealer mixed with Tinospora cordifolia (Guduchi) and Mimusops elengi (Bakul) respectively. Conclusion: Zinc Oxide Eugenol based sealer with herbal extracts produced largest inhibitory zones followed in descending order by Resin based sealer and Calcium hydroxide along with three herbal extracts respectively.
Keywords: Antimicrobial efficacy, Bakul, Guduchi, Licorice, root canal sealers, root canal treatment
|How to cite this article:|
Saha S, Dhinsa G, Ghoshal U, Afzal Hussain AN, Nag S, Garg A. Influence of plant extracts mixed with endodontic sealers on the growth of oral pathogens in root canal: An in vitro study. J Indian Soc Pedod Prev Dent 2019;37:39-45
|How to cite this URL:|
Saha S, Dhinsa G, Ghoshal U, Afzal Hussain AN, Nag S, Garg A. Influence of plant extracts mixed with endodontic sealers on the growth of oral pathogens in root canal: An in vitro study. J Indian Soc Pedod Prev Dent [serial online] 2019 [cited 2019 May 26];37:39-45. Available from: http://www.jisppd.com/text.asp?2019/37/1/39/252861
| Introduction|| |
Root canal treatment aims to eliminate bacterial infection, which cannot be solely accomplished by chemomechanical preparation as the microorganism remains in the canal and dentinal tubules after instrumentation. Therefore, sealers with good sealing ability and antimicrobial activity are desired to eliminate residual microorganism. Root canal medicaments come in contact with periapical tissue; hence, the sealers must have minimal cytotoxic effects.
In spite of the use of herbs in medicinal field, less light has been shed for their use in the field of dentistry. Hence, the aim of the study was to evaluate the antimicrobial efficacy of root canal sealers added to herbal extracts against microbes found in the inflamed pulp.
| Materials and Methods|| |
The present study was conducted in the Department of Pedodontics and Preventive Dentistry, Sardar Patel Post Graduate Institute of Dental and Medical Sciences, in collaboration with the Department of Microbiology, Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGIMS), Lucknow, Uttar Pradesh (India).
In the present study, the antimicrobial efficacy of three commercially available root canal sealers of different bases (zinc oxide eugenol-based sealer, epoxy resin-based sealer, and calcium hydroxide-based sealer) when mixed with three herbal extracts, namely, Glycyrrhiza glabra (Licorice), Mimusops elengi (Bakul), and Tinospora cordifolia (Guduchi), was evaluated against seven strains of bacteria (aerobes, facultative, and obligate anaerobes) known to be common isolates in necrotic pulps and endodontic lesions using the agar diffusion test (ADT).
Previously, a pilot study was carried out in the same departments to overview the proper study design and to take care of the possible constraints during the main study.
Three commercially available root canal sealers of different bases used in this study were Endomethasone (zinc oxide eugenol-based sealer), AH Plus® (epoxy resin-based sealer), and Apexit Plus® (calcium hydroxide-based sealer) [Figure 1].
The sources of the sealer were as follows [Table 1].
Preparation of the sealers
The sealers were prepared in strict compliance with the manufacturer's recommendations.
Tested herbal extracts
Three herbal extracts used in this study were methanolic extracts of Licorice, Bakul, and Guduchi.
Procurement of the extract
The herbal plants and their extracts were procured from the National Botanical Research Institute, Lucknow.
The plant materials (roots of Licorice, bark of Bakul, and stem of Guduchi) were collected and shade dried. These dried parts of the plant were crushed and powdered using an electrical grinder. The powdered material (50 g of dry powder) was then extracted with 500 ml methanol.
Antibacterial activities of the sealers in combination with herbal extracts were evaluated against five aerobes and facultative anaerobes and two obligate anaerobes [Table 2].
|Table 2: Five aerobes and facultative anaerobes and two obligate anaerobes|
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The cultures of each strain of bacteria were obtained from the laboratory stock of the Department of Microbiology, SGPGIMS, Lucknow.
Growth conditions and bacterial culture
Staphylococcus aureus, Enterococcus faecalis, Escherichia More Details coli, and Pseudomonas aeruginosa were grown aerobically in brain–heart infusion (BHI) broth; S. ß haemoly were grown in Trypticase soy broth; and the obligate anaerobes (Bacteroides fragilis and Peptostreptococcus spp.) were grown in BHI containing hemin and menadione.
Preparation of the inoculums
For each bacterial strain inoculums were prepared by picking up four to five colonies with the help of a circular, previously sterilized loop of 4 mm internal diameter and dissolving them into respective test tubes containing 5 ml of 0.85% saline solution – to produce a turbidity of 0.5 on McFarland scale which corresponds to a concentration of 108 colony-forming units per milliliter. Petri dish More Detailses, 90 mm diameter, containing 4 mm thick Mueller-Hinton agar (MH; Difco Laboratories, Detroit, Michigan, USA) [Figure 2] were used for all the above bacterial strains except S. ß haemolyticus, for which blood agar plates were used [Figure 3].
|Figure 2: Culture plates showing zone of Inhibition on Mueller Hinton Agar Plates|
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|Figure 3: Culture plates showing zone of Inhibition on Blood Agar Plates|
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To ensure the even distribution of the inoculums, the respective bacterial dilutions were then swabbed evenly onto freshly prepared respective agar plates using the “lawn technique.” Each plate (for every individual bacterial strain) was evenly divided into three equal sections. In each section of each plate, wells of 6-mm diameter were created with the help of previously fabricated and sterilized copper wells. The three wells in each section were then filled with the three tested sealers mixed with three herbal extracts.
The inoculated plates with the tested medicaments were kept for 2 h at room temperature to allow the diffusion of the tested groups through the agar, as the ADT is influenced by the diffusibility of the material. The MH agar plates were incubated at 37°C. The blood agar plates inoculated with S. ß haemolyticus strain were incubated in a CO2 incubator (Jouan, Saint Herblain, France) in an atmosphere of 10% CO2. Plates with strict anaerobes were immediately placed into GasPak anaerobic jars (nitrogen [90%] and CO2[10%]).
The plates for facultative anaerobes were read at 24 h for the size of the zone of inhibition, while readings for strict anaerobes were carried out after 48 h. The whole experiment was repeated 6 times for each isolate, and the mean zone of inhibition was then calculated.
Measuring the size of zone of inhibition
Growth inhibitory zones around each tested medicaments were evidenced by lack of bacterial colonization (clearing of agar) adjacent to each group. The most uniform diameter segment of the zone of inhibition was measured, and 6 mm (diameter of the well) was extracted from the measurement as the cutoff value. All measurements above this value were considered, indicative of significant bacterial growth inhibition. Wider zones of inhibition were interpreted to indicate greater antimicrobial activity of the involved medicaments.
All statistical analyses were performed using the SPSS 15 statistical software version, IBM, USA. All the data were presented in tabular and bar diagram form. The intergroup comparison was evaluated using Kruskal–Wallis test along with Mann–Whitney U-test. The intragroup comparison was evaluated using Friedman test along with Wilcoxon signed-rank test. The confidence level of the study was proposed to be 95%; hence, P < 0.05 was considered statistically significant, P < 0.01 was considered highly statistically significant, and P < 0.001 was considered very highly statistically significant.
| Results|| |
[Graph 1] reveals the antibacterial efficacy of zinc oxide-based sealer (Endomethasone) combined with all three herbal extracts against seven microbial strains. It was seen that Endomethasone when mixed with Licorice showed the highest antimicrobial efficacy followed in descending order by its combination with Guduchi and Bakul, respectively. The highest zone of inhibition of Endomethasone mixed with Licorice was shown by S. aureus and E. coli followed by E. faecalis.
[Graph 2] reveals the antibacterial efficacy of resin-based sealer (AH Plus® Dentsply Germany) combined with all three herbal extracts against seven microbial strains. It was seen that AH Plus® mixed with Licorice had a higher antimicrobial efficacy than when mixed with Guduchi and Bakul. AH Plus® in combination with Licorice showed the highest zone of inhibition against E. coli followed by S. ß haemolyticus.
[Graph 3] reveals the antibacterial efficacy of calcium hydroxide sealer (Apexit Plus®) combined with all three herbal extracts against seven microbial strains. It was seen that Apexit Plus® mixed with Licorice showed the highest efficacy than when mixed with Bakul and Guduchi. The combination of Apexit Plus® with Licorice showed the highest zone of inhibition against S. aureus followed by E. coli.
| Discussion|| |
The objective of root canal treatment is to eliminate the bacteria by cleaning, shaping, and obturation of the root canal system. However, residual bacteria lead to endodontic failures. Therefore, root canal sealers with good sealing ability and antimicrobial activity with minimal cytotoxicity are desired to kill the surviving microorganisms. Because root canal medicaments can come in contact with periapical tissue, in addition to having good antibacterial ability, they must be biocompatible. In selecting the root canal medicaments, it is necessary to consider their therapeutic benefits against their potential cytotoxic effects. Ideal root canal medicaments should have strong antibacterial properties and minimal cytotoxic effect on the host tissues. It is well established that many metabolites produced by plant extracts such as tannins, terpenoids, alkaloids, and flavonoids provide a new source of antimicrobial substances that help in combating new developing drug-resistant pathogens. There is a lack of scientific reports that indicate the antimicrobial activities of herbal extracts when mixed with endodontic root canal sealers. Hence, this study was aimed at investigating the antimicrobial efficacy of endodontic sealers mixed with herbal extracts.
The most commonly used root canal sealers in endodontics depending on their composition are categorized as zinc oxide eugenol-based, epoxy resin–based, and calcium hydroxide-based root canal sealers.,,
To standardize the whole experimental process, great care had been taken regarding the inoculation density, proper incubation, careful reading of zone of inhibition, and repetition of the whole experiment six times.
Methanolic extracts of the three herbal plants were prepared as it was shown to have greater activity because more phytoconstituents are leached in out from when compared to ethanolic or aqueous extracts.
S. ß haemolyticus or facultative anaerobes as a group have been reported to be one of the most prevalent groups isolated from infected root canals. It has been studied in the present study, as it is importantly represents a standard against which the antibacterial action of a sealer mixed with herbal extracts should be studied.
E. faecalis, a Gram-positive coccus, was chosen as the test organism because it is a facultative anaerobe that is nonfastidious, easy to grow, and efficiently and rapidly colonizes dentinal tubules. It has a pathogenic role in chronic endodontic treatment failure, and its virulence may be related to the resistance to intracanal medicaments and an ability to survive in the root canal as a single organism without the support of other bacteria. It is considered to be the most resistant species in the oral cavity and the possible cause of failure of root canal treatment.
P. aeruginosa, a Gram-negative aerobic nonfermentative rod, is found in only 1.2% of infected root canals. This particular species is resistant to all antibiotics usually prescribed in endodontic infections. Therefore, successful chemotherapy of other microorganisms may favor its ingrowth. Hence, it was chosen as a test microorganism. Tronstad et al. reported that the flora of long-standing therapy/resistant lesions was frequently dominated by P. aeruginosa.
Other recovered microorganisms are Gram-negative bacteria, mainly Gram-negative rods such as E. coli. According to Gomes et al., E. faecalis and S. aureus have been considered to be the most resistant species in the oral cavity and possible cause for the failure of root canal treatment. However, by the time pulpal necrosis and a radiolucent periapical lesion develop, the root canal flora consists of strict (obligate) anaerobes such as Peptostreptococcus and Bacteroides species at the expense of facultative anaerobic species. Of the Bacteroides species, B. fragilis is commonly chosen as the representative test microorganism because it has been isolated from the infected root canals by several investigators, and it was easier to grow in the laboratory than Bacteroides melaninogenicus.
With few exceptions, most antibacterial tests have been performed using bacteria unassociated with endodontic infections. Because it has been shown that different bacteria may vary in their sensitivity to the same material, it was important to ensure that test bacteria selected were true endodontic pathogens.
The results of the present study showed that zinc oxide eugenol-based root canal sealer (Endomethasone) combined with Licorice showed the highest antimicrobial efficacy in descending order by Endomethasone mixed with Guduchi and Bakul. As reported by Steven and Grossman (1981), Endomethasone contains eugenol and formaldehyde. A gradual release of formaldehyde from the paraformaldehyde in the sealer (after setting) accounts for its high antibacterial effect over an extended period. However, Gerosa et al. (1995) stated that Endomethasone contains eugenol, thymol, and hydrocortisone, which was known as cytotoxic components. To overcome these drawbacks of cytotoxicity, endodontic sealers were mixed with biocompatible herbal extracts in the present study. When Endomethasone was used in combination with Licorice, the mixture showed the best antibacterial efficacy and a prominent zone of inhibition. The high antimicrobial efficacy of Licorice may be attributed to the various bioactive components identified from the roots of Licorice. Glycyrrhizin, which is a diglucuronide of glycyrrhetinic acid, is the active principle to which most, if not all, of the positive properties of the drug should be attributed. However, in spite of being a saponin, glycyrrhizin does not possess the most deleterious property of saponins, namely, that of inducing hemolysis of red blood cells. This saponin glycoside, glycyrrhizin, dose dependently inhibits the glucosyltransferase activity of mutans streptococci, which is involved in the formation of insoluble glucans required in biofilm formation. The mode of antibacterial action of saponins seems to involve membranolytic properties rather than simply altering the surface tension of the extracellular medium, thus being influenced by microbial population density. The flavonoid content of G. glabra is also a strong inhibitor of oxygen consumption in bacterial cells; the site of inhibition is thought to be between CoQ and cytochrome C in the bacterial respiratory electron transport chain.
The results of the present study showed that Licorice when combined with all three herbal extracts showed the highest antimicrobial efficacy followed in descending order by its combination with Guduchi and Bakul. The highest zone of inhibition of Licorice was shown by S. aureus and E. coli followed by E. faecalis. These findings were in accordance with the study conducted by Irani et al., Nitalikar et al., Lakshmi and Geetha, Nirmala and Selvaraj, and Rodino et al.,,,,
The results of the present study also revealed that the combination of Endomethasone and Guduchi showed a better antibacterial efficacy as compared to when mixed with Bakul. According to many researchers, Guduchi contains alkaloids, flavonoids, steroids, tannins, phenols, and saponins. Because of the presence of variety of active compounds, Guduchi showed a greater antibacterial efficacy in comparison to Bakul. On the other hand, Bakul only contains two components, flavonoids and tannins responsible for its antimicrobial efficacy.
The results of the study showed that resin-based sealer (AH Plus®) when used in combination with Licorice showed a maximum antibacterial efficacy followed in descending order by combination with Guduchi and Bakul, but lesser in comparison with the Endomethasone when mixed with three herbal extracts. The antibacterial efficacy of this sealer may be attributed to hexamethylenetetramine (methenamine) in its basic composition. Methenamine is a hydrophilic material and, in an acidic environment, is hydrolyzed to ammonia and formaldehyde. In an acidic environment, Methenamine being a hydrophilic material is hydrolysed to ammonia and formaldehyde. The release of formaldehyde during this process is responsible for its antimicrobial properties.
The results of the study showed that calcium hydroxide sealer (Apexit Plus®) when used in combination with Licorice showed a minimum antibacterial efficacy followed in descending order by combination with Bakul and Guduchi, respectively, but lesser in comparison with the Endomethasone and AH plus when mixed with three herbal extracts. Antibacterial activity of calcium hydroxide-based sealers is based on its ionic dissociation into calcium (Ca2+) and hydroxyl (OH−) ions causing an increase in pH (12.5). A pH > 9 may reversibly or irreversibly inactivate cellular membrane enzymes of the microorganism, resulting in a loss of biological activity of the cytoplasmic membrane.
In the present study, calcium hydroxide sealer (Apexit Plus®) when used in combination with Bakul showed a maximum antibacterial efficacy when compared to its combination with Guduchi. This may be due to the fact that calcium, which is an essential element for several life processes, is found to be highest in Bakul. This additive effect of the presence of calcium in both calcium hydroxide sealer and Bakul may be responsible for the better antimicrobial efficacy of calcium hydroxide-based sealer in combination with Bakul, rather than its combination with other two herbal extracts.
The results of our study also brought forward the fact that calcium hydroxide-based sealers used in combination with all three herbal extracts showed a minimum antimicrobial efficacy as compared to other tested combinations. Minimum antimicrobial effect with calcium hydroxide-based sealers might be explained by too slow release of hydroxyl ions during the duration of contact, and the release of hydroxyl ions from calcium hydroxide was not sufficient to inhibit the growth pH of many microorganisms. In addition, artificial media, mainly those containing blood, have a buffering ability that could provide a reduction in the high pH of calcium hydroxide, making it less effective. In clinical situations, buffer action of blood and tissue fluid may cause the same effects.
The present study was limited by some factors such as the efficacy of the extracts was observed for a short period of time. Besides, quantitative analysis of the extracts along with their qualitative screening would further assist to elucidate the active principles responsible for the antimicrobial activity. Further studies can be undertaken to explore the antimicrobial efficacy of endodontic sealers mixed with herbal extracts for a longer duration of time.
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]