|Year : 2022 | Volume
| Issue : 3 | Page : 266-272
Evaluating the antibacterial efficacy and minimal bactericidal concentration (MBC) of three different herbal extracts on recalcitrant endodontic pathogens - An in vitro study
S Rajakumar1, MP Revanth2, Anand Kasi1, P Sujitha1
1 Department of Pediatric and Preventive Dentistry, SRM Kattankulathur Dental College, Chennai, India
2 Department of Public Health Dentistry, Ragas Dental College and Hospital, Uthandi, Chennai, India
|Date of Submission||05-Jan-2022|
|Date of Decision||31-Mar-2022|
|Date of Acceptance||28-Apr-2022|
|Date of Web Publication||28-Jun-2022|
Dr. S Rajakumar
Department of Pediatric and Preventive Dentistry, SRM Kattankulathur Dental College and Hospital, Potheri
Source of Support: None, Conflict of Interest: None
Aim: To evaluate the antibacterial efficacy, minimal inhibitory concentration, and minimal bactericidal concentrations of different herbal extracts against endodontic pathogens. Materials and Methods: The Microbiological samples were taken using sterile paper points from the root canals undergoing retreatment and samples were transferred into cultural plates containing selective media and incubated for 24hrs for the specified bacterial growth. The methanolic extracts of plant were obtained and stored in capped bottles at 4°C until use. The susceptibility of endodontic bacteria to the various plant extracts at different concentrations was analysed using disc diffusion method by measuring the diameter of the inhibition zones. Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) of the extracts were assessed to determine the efficient plant concentration against the resistant endodontic bacteria. Triplicates of all the tests were carried out. The average of three experiments were subjected to statistical analysis. Data was analysed using one way ANOVA using SPSS version 20.0 (Armonk. NY, USA) to find out the zone of inhibition of herbal extracts against microbiota. Results: Clitoria Ternatae exhibits significance (P ≤ 0.05) and higher inhibitory zones (30.33 ± 1.528, 30.43 ± 0.577, 20.33 ± 1.528) against all three micro pathogens. ClitoriaTernatea at its minimal concentration of 102 mg/ml is better against all the pathogens, the least being Veronica Officinalis (425 mg/ml). Conclusions: ClitoriaTernatea, showed better bactericidal action under lower concentrations against all pathogens followed by Altheae officinalis and Veronica officinalis.
Keywords: Antimicrobial agents, herbal medicine, plant extracts, root canal irrigants
|How to cite this article:|
Rajakumar S, Revanth M P, Kasi A, Sujitha P. Evaluating the antibacterial efficacy and minimal bactericidal concentration (MBC) of three different herbal extracts on recalcitrant endodontic pathogens - An in vitro study. J Int Oral Health 2022;14:266-72
|How to cite this URL:|
Rajakumar S, Revanth M P, Kasi A, Sujitha P. Evaluating the antibacterial efficacy and minimal bactericidal concentration (MBC) of three different herbal extracts on recalcitrant endodontic pathogens - An in vitro study. J Int Oral Health [serial online] 2022 [cited 2022 Dec 2];14:266-72. Available from: https://www.jioh.org/text.asp?2022/14/3/266/348424
| Introduction|| |
The complete eradication of root canal microbes is essential for endodontic therapy to be successful. Narayanan et al. and Gomes et al, proved that the presence of various microbiota is the major deterrent in root canal therapy and progression of dental caries to periapical lesions. Some specific interradicular species are Enterococcus faecalis, Streptococcus mutans, Staphylococcus aureus, which are considered the commonest causes of failure in 95% of permanent root canal therapy and 30% of primary tooth pulpectomy. The gelatinase-producing ability of these microbiota aids in the survival of infection for a prolonged time period and resistance to the various antimicrobial agents. Recuperation of infected teeth from such pathogens is critical to avoid post-endodontic re infection and refractory periapical lesions. This can be accomplished using appropriate irrigation techniques and antibiotic prophylaxis.
According to Sundqvist et al., the antagonistic connections between bacteria create a unique habitat in the root canal leading to persistent infection. This led to the development of newer antimicrobial agents, which are essential in containment of root canal infections. In the meantime, the emergence of multidrug-resistant (MDR) pathogenic strains has gained widespread concern and poses a severe public health hazard.
This aids researchers in the search for a novel antibacterial agent. However, previous clinical trials have shown that pathogens develop rapid resistance to newer agents at a very short time period. Thus, need to find a alternative strategy would be a prime objective for the containment of antibiotic resistant pathogens. Herbal and plant extracts are one such desirable and satisfactory alternative to synthetic drugs.
Plants are the exclusive source of potent antibacterial compounds. Several advantages of using medicinal plants are, economical, less toxic, good shelf life and less chances of developing microbial resistance. Isolation of those extracts and characterization of a active phytochemical compound offers promising sources of newer antibacterial agents with specific as well as general activities providing an effective medicinal alternative to treat various oral infections, including root canal recalcitrant infections. The medicinal plants According to the World Health Organization(WHO), are valued as a multifarious drug and are a good substitute that can be effective in the treatment of diverse endodontic pathogens. But the vast potentiality of many herbal compounds against oral and endodontic pathogens are still unexplored.
Clitoriaternatea is an herbaceous perennial plant in the Fabaceae family that is recognized for its fodder and medicinal values. Antibacterial activity has been observed for the flavonol glycoside and cliotides of C.ternatea. Althaea officinalis is an annual plant of the Malvaceae family, containing phytochemicals, capable of acting as antibiotics to kill both gram positive, gram negative bacteria and also cocci., Veronica officinalis, a Plantaginaceae genus with approximately 450 known species, grows in both temperate and hemisphere regions and possesses both chemotaxonomic and anti-microbial properties.
Considering the advantages of herbal medicines for their broad antimicrobial spectrum, non-cytotoxic properties and the development of MDR of infectious diseases against synthetic antibiotics, the use of plant extracts derived from medicinal herbs seems to be the way forward. Therefore, a detailed investigation of three uniquely chosen plant extracts has been assessed for their minimal and potential bactericidal properties under various concentrations.
| Materials and Methods|| |
Microbial sample collection
The microbiological samples were obtained and cultured from one healthy individual who visited Endodontics (SRM KDC) for re-treatment. The infected tooth was identified using visual and radiographic examination, the canal orifice was widened using 21 mm file (Dentsply Wave one Gold Reciprocating File-Medium, USA) to allow easy entry of paper points deep into the canal. The microbiological samples were taken with a sterile absorbent paper point (Dentsply Wave one Gold Paper Points – Medium, USA) put into the canal, left for 30 seconds, and then transferred to a test tube containing 2 mL of brain heart infusion (BHI) broth. Later, the samples were emptied into a vortex mixer (RemiVM-100,Thane-Mumbai) for specimen shaking, followed by transfer of samples into the culture plates and incubated for 24 hrs containing selective media for the growth of specified bacteria. We extracted three of the most commonly prevailing endodontic species like Enterococcus faecalis, Streptococcus mutants and Staphylococcus aureus as study samples.
Plant extracts and concentrations
Clitoriaternatea, Veronica officinalis, Althaea officinalis (marshmallow) are the plants used in this study. All three species of whole plants were obtained from diverse locations in and around Chennai. Its identity was confirmed by comparing with herbarium specimens and authenticated by The Botanical Survey of India (BSI), Agricultural University, Coimbatore. The plants obtained were airdried and powdered. The maceration of dry powder weighing 1000 gms was done in 100 mL of methanol for 24 h, using a Soxhlet apparatus (Kesari scientific chemicals, Chintadripet). The methanolic crude extract was obtained by filtering the contents using filter paper, No. 1(Whatman,Able Brewing, USA). The extract was later evaporated using a rotatory evaporator (BUCHI Rotary Evaporator R-110, USA) and a final concentration of 850 mg was achieved. Until further use, the plant extracts were filled and stored in capped bottles at 4°C. Later, stock solution of the extract was prepared by double serial dilution using 10% dimethyl sulfoxide (DMSO) for the required concentration of 850 (100%), 637.5 (75%), 425 (50%), 212.5 (25%), 102 (12.5%) mg/ml, to evaluate MIC and MBC.,
Antimicrobial susceptibility assay
To determine the zone of inhibition, the Muller–Hinton Agar (MHA) was used for bacterial media preparation following disk-diffusion agar method reported by Bauer et al. (1966). The bacteria were cultured for 24 h on an MHA medium with a suspension equal to 0.5 McFarland, which corresponds to 108 colony forming units (CFU/mL). The culture media was made by dissolving 9.5 g of MHA in 250 mL of distilled water. The resultant amber colour solution is thoroughly mixed and boiled with repeated agitation to completely dissolve the powder, yielding a clear to slightly opalescent gel. After that, the medium was autoclaved for 15 minutes at 15 pounds of pressure at 121ºC. The sterile media is allowed to cool to room temperature in a laminar flow hood (KEWAUNEE, Bengaluru). Following that, 25 mL of media is pipetted and transferred into Petri plates, where it solidifies. After solidification, the sterile discs were prepared and placed on the surface of infected plates with bacteria by sterile forceps at the correct distance from each other, 10 ml of diluted extracts with various concentrations (100%, 75%, 50%, 25%, 12.5%) were poured on the discs with a sampler (Mettler-Toledo, Mumbai). Plates were stored for incubation in a biochemical oxygen demand (BOD) incubator (SIMA Labs, New Delhi) at 37°C for 24h. After 24h, the diameters of inhibition zones were measured. The readings were performed by measuring the inhibition halo around the disc in millimetres, using a calibrated calliper (Mitutoyo, Japan). All the tests were carried out in triplicate. The average of three experiments has been used to calculate the results. The following is the measurement scale and interpretation criteria: The zone of inhibition at 20 mm is strongly inhibitory, while the zone of inhibition between 20-12 mm is moderately or mildly inhibitory and the zone of inhibition below 12 mm is No inhibitory zone (Rota et al., 2008). Strains that inhibited a zone greater than 12 mm were considered for the MIC and MBC.
Determination of minimal inhibitory concentration (MIC)
The lowest of concentration that shows no visible microbial growth and no turbidity after 24 hrs of incubation are considered to be MIC of a plant extract. Broth micro-dilution assays using MHA were used to determine MICs of the extracts against microbial strains according to the Clinical and Laboratory Standard Institutes (CLSI). MIC was carried out in a sterile 96-well plate. 50 μL of different concentrations of prepared plant extracts (ranging from 850 to 102 mg/ml) were poured into respective wells containing 50 μL of BHI liquid medium, to which 0.5 mL of standardized bacteria suspension (1.5*105 CFU/ML) was added. To determine the bacterial growth, Resazurin (Otto Chemie Pvt Ltd-India) was added to each well of the microtiter plate and was incubated at 37ºC for 24 hrs. The wells containing bacterial growth turn pink, while that do not have bacterial growth remains blue. The MIC for each extract under different concentrations was determined in triplicate. MIC is obtained for the first well with no turbidity, claiming no bacterial growth.
Determination of minimum bactericidal concentration (MBC)
A 20 mL dilution of each plant extract where the bacterium is inhibited at a minimal concentration was taken from a test tube and the contents were inoculated in Petri dishes containing 30 mL of BHI agar and incubated for 24 hours at 35.5°C. The first dilution in which no bacterial (99% no growth) growth was observed during this period was considered as the MBC.
SPSS version 20.0 was used to analyse the data collected (Armonk. NY, USA). The mean and standard deviation values were tabulated, and a one-way analysis of variance (ANOVA) was used to determine the bacterial inhibition zones between the groups. The study’s confidence level was proposed to be 95%, so P < 0.05 was considered statistically significant.
| Results|| |
The present study shows that all the plant extracts are sensitive and exert anti-microbial properties against the endodontic pathogens. As stated in [Table 1], the inhibition zone of all three herbal extracts shows descending results from higher to lower concentrations. C.ternatae exhibits significance (P ≤ 0.05) and higher inhibitory zones (30.33 ± 1.528, 30.43 ± 0.577, 20.33 ± 1.528) against all three micro pathogens. [Figure 1] represents the image of different zones of inhibition of plants under different concentration against endodontic microbes.
|Table 1: Comparision of zone of inhibition of herbal extracts against microbiota between groups|
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|Figure 1: Zone of inhibition of different plant concentration against tested endodontic pathogens|
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[Table 2] shows C. Ternatea at its minimal concentration of 102 mg/ml is better against all the pathogens, the least being V. officinalis (425 mg/ml). Among all the tested herbals, C. ternatea gave the most consistent results against all pathogens, followed by A. officinalis and V. officinalis.
| Discussion|| |
The main rationale of the herbal medicinal plants is to eradicate pathogens from the radicular part of tooth and inhibit its recontamination through a variety of mechanisms., This can be accomplished by inhibiting cell wall synthesis, interfering with cell membrane permeability, causing membrane disruption, altering cellular components, and inducing cell mutation. Most plant extracts studied before were found to have a maximal inhibitory effect on oral micro pathogens like Gram-positive and Gram-negative bacteria.
S.mutans are endogenous bacteria considered to be the most major cause of dental caries and subsequent demineralization of tooth structure. In primary endodontic infection, S. mutans and S.aureus are frequently isolated and also been linked to increased post-surgical discomfort and swelling. The complex ecologic environment within the root canals of both primary and permanent tooth roots provides conditions that are suitable for the growth of E.faecalis species. Regardless of the fact that the root canal is deprived of nutrients, E. faecalis can sustain on dentinal fluid serum components and use local sources of energy even in a well-debrided root canal and proliferate rapidly. Alongside, Methanolic extracts were found to be more active than ethanolic or aqueous extracts because more phytoconstituents are leached out of them on comparing with ethanolic extracts. As a matter of fact, methanolic extracts of three herbal plants were prepared as test medicaments. Thus, this study is aimed at analyzing the antimicrobial efficacy of three different medicinal plants such as Clitoria ternatea, Althaea offinalis and Veronica officinalis extracts against three different endodontic pathogens like S. mutans, and S. aureus. E. faecalis.
Against the designated endodontic pathogens, all three plant extracts demonstrated substantial sensitivity and antibacterial activity. At 850 mg, Clitoriaternatea showed the maximum zone of inhibition against S.mutants and S.aures in the disc diffusion method [Table 1]. The inhibition rate decreased as the extract concentration decreased, but under all concentrations it exhibited potential antibacterial properties. It also exerted strong antimicrobial activity against E.feacalis, showing a inhibition zone ranging between the stronger with 20.33 ± 1.528(850 mg) to the moderate with 12.86 ± 0.687(102 mg). The C. ternatae extract, even at the minimum concentration, has an inhibition zone above 12 mm according to Rota et al. (2008) guidelines for the zone of inhibition, which was compared with the National Committee for Clinical Laboratory Standards (NCCLS). This study revealed that the extract is efficacious against the oral micropathogens at a minimal concentration with a MIC of 102 mg and MBC of 212.5 mg [Table 2] against all organisms and was statistically significant when compared with other agents like A.officinalis and V.offinalis (P ≤ 0.05).
The phytochemical constituents of this plant extract revealed a variety of primary and secondary metabolites, including flavonoids, glycosides, pentacyclic triterpenoids, and phytosterols, which were responsible for inhibiting bacterial cell division and increasing membrane permeability, ultimately leading to cell death. “Finotin,” a phytochemical protein isolate from C. ternatea, has shown significant antibacterial activity. It is possible that this substance must have been responsible for increased antimicrobial activity against all three-gram positive bacteria by adhering to N-acetyl glucosamine found in the bacterial cell wall, inhibiting bacterial growth by producing hydrogen peroxide, demonstrating its efficacy even at low concentrations.
Our study result was consistent and is in accordance with studies done by Priyadarshini et al. and Rao et al,, where the plant extracts inhibited gram-positive bacteria to the greatest extent showing maximum zone of inhibition. C.ternatea extract was tested against both gram-positive and gram-negative bacteria in a study done by Kamilla et al. and Anand et al., it was found that both the strains were highly sensitive at minimal concentration, which is in concordance with the finding of our study results.
In this investigation, A.officinalis was found to have antibacterial action against E. faecalis, S.mutans, and S.aures. However, a noticeable antibacterial activity is observed only against S.mutans and S.aureus with a minimum zone of inhibition (19.00 ± 2.000 and 12.67 ± 0.577 respectively) at 212.5 mg. Whereas zone of inhibition (14.27 ± 0.577) for E.feacalis was seen only at a higher concentration of 425 mg with MBC at 637.5 mg. In this study, A.offinalis inhibited S.mutants and S.aures more potently than E.feacalis, possibly because the major anthocyanin components of the plant extract interfered with bacterial adhesion to tooth surfaces and affected the enzymatic activity of cariogenic bacteria. This proves its effectiveness against S.mutans and S.aures, but failed against E.feacalis because it possesses serine protease and collagen binding properties, that make it more resistant to anthocyanin component of the plant extract and thus it resisted destruction. This statement is in accordance with an evaluation done by Stuart et al, Ferrazzano et al. and Haghgoo et al.,, Based on the obtained results, the inhibition rate decreased as the concentration decreased and shows nil effect on the lowest concentration of 102 mg. Our study result was in accordance with a study done by Rezae et al and Zarei et al. indicating that Marshmallow extracts exert antimicrobial effects against gram-positive bacteria.,
V.offinalis also exerted considerable antimicrobial property against tested pathogens. However, it is resistant (R) against E. faecalisand S. aureus at lower concentrations. The MBC for S.aureus is 637.5 mg and showed no bactericidal effect against E.feacalis. However, shown potential inhibition against S.mutans, this sensitivity may be due to cell membrane permeability and cell wall structure. The lower degree of microbial activity and decreased efficacy of V. officinalis may be due to the fact that during the extraction processes, all or some of the active components of the concentrates may be inactivated, or the concentration levels of the active ingredients may differ depending on geographical location and cultivation process. There are no studies found to be in resemblance with our study because our study is the first antimicrobial report on the medicinal plant V. offinalis species.
The antimicrobial efficacy of any test agents depends upon its extent of diffusion capacity through the solid agar medium which will show the inhibitory zones. It is undeniable that there are some complexities involved in laboratory methanolic extraction of the test agents (herbals) used in this study, these alterations might have caused variation in the bioactive chemical components of the test agents and can either increase or decrease the actual antimicrobial activity and diffusion capacity showing variations in the inhibitory zones between the plant extracts. Another reason could be the preparation of test organisms. The organisms in this study were isolated directly from the root canals, whereas in previous studies lyophilized forms were used. Furthermore, it is necessary to formulate analytical methodologies that can standardise plant extract in order to ensure the efficacy of the chemical compounds extracted and their effective incorporation in the dental field. In addition, for more consistent results, more complex antimicrobial methodologies to better simulate oral conditions, such as microsomes biofilm or in vivo biofilm assays, should be investigated. Besides which, the longevity of the antimicrobial and biological activity of the plant extracts was not determined in this study; perhaps establishing the mechanisms of action and interaction of the material with cells and microorganisms could elucidate better the presented results.
| Conclusion|| |
We conclude that the extracts of C. ternatea and A. offinalis showed varying degrees of antimicrobial activity on the microorganisms tested, suggesting these plant extracts could be efficiently used as endodontic irrigants to treat recalcitrant endodontic pathogens. However, in vivo, and clinical experiments are needed to additional evaluate the cytotoxicity of these plant extracts and further, research is required in drug development program and to identify the other active compounds of the plant extracts which is responsible for the biological activity and future studies on synergistic effects are also recommended.
MIC– Minimal inhibitory concentration, MBC – Minimal bactericidal concentration, R – Resistant, C.ternatea – Clitoriaternatea, A.officinalis – Altheaeoffinalis, V.offcinalis – Veronica offinalis.
We acknowledge the immense help of botanical society of India, Coimbatore and Life Teck Research laboratory, Chennai for their active contribution. We are also grateful for all the authors / editors / publishers of all those articles and journals referred and cited in the references of this manuscript.
Financial support and sponsorship
This study was self-funded by the authors.
Conflicts of interest
There are no conflicts of interest.
RKS and RMP contributed in study conception, design, intellectual content, data acquisition and analysis, data interpretation, and manuscript writing and review. AK and SP contributed to the literature search, date acquisition, data analysis and manuscript revision. All the authors approved the manuscript for final submission.
Ethical policy and institutional review board statement
This Research Received the Approval from Scientific Review Board, SRM Medical College Hospital And Research Centre (SRM MCH and RC) On 23/09/2021, Ethical Clearance Number: 2925/IEC/2021.
Patient declaration of consent
Data availability statement
Available on request (DR.S.RAJAKUMAR/[email protected])
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[Table 1], [Table 2]