Antimicrobial efficacy of Andrographis paniculata, sodium hypochlorite, and their combination with diode laser on endodontic pathogen—Enterococcus faecalis—An in vitro study

KG Malavika; Rajakumar Sekar; Kavitha Ramar

doi:10.4103/jioh.jioh_39_22

ORIGINAL RESEARCH

Year : 2022 | Volume : 14 | Issue : 3 | Page : 260-265

Antimicrobial efficacy of Andrographis paniculata, sodium hypochlorite, and their combination with diode laser on endodontic pathogen—Enterococcus faecalis—An in vitro study

KG Malavika, Rajakumar Sekar, Kavitha Ramar
Department of Pediatric and Preventive Dentistry, SRM Kattankulathur Dental College, Chennai, Tamil Nadu, India

Date of Submission	04-Feb-2022
Date of Decision	11-Apr-2022
Date of Acceptance	29-Apr-2022
Date of Web Publication	28-Jun-2022

Correspondence Address:
Dr. Rajakumar Sekar
Department of Pediatric and Preventive Dentistry, SRM Kattankulathur Dental College, Chennai, Tamil Nadu
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/jioh.jioh_39_22

Abstract

Aim: To evaluate and compare the antimicrobial efficacy of Andrographis paniculata and sodium hypochlorite with diode laser on endodontic pathogen Enterococcus faecalis. Materials and Methods: An in vitro study in which 40 freshly extracted single rooted anterior teeth with no root caries, apical fractures are collected. A sample size was calculated using G*Power Software. All the teeth are decoronated and prepared by using ProTaper Gold (DENTSPLY) rotary file system. The apex is then sealed and waterproofed by applying nail varnish. Precultured inoculum of E. faecalis strain is inoculated and then teeth are randomly divided into four groups with 10 teeth in each group; each group is irrigated with 0.5% of A. paniculata, 3% of sodium hypochlorite, laser-activated 3% sodium hypochlorite, and laser-activated 0.5% A. paniculata, respectively. Pre- and postirrigation colonial count is obtained. Statistical analysis of all the data was analyzed using SPSS 20.0 (Armonk, NY, USA); Analysis of Variance (ANOVA) was performed for comparing variation between groups and multiple comparison tests performed for intergroup comparison. Results: The reduction of colony count postirrigation of about 96% in group 4, 90% in group 3, 48% in group 1 with a least reduction of about 24% in group 2 is observed (P = 0.000). Conclusion: All four irrigation methods included in this study proved to be effective against E. faecalis, but A. paniculata with diode laser came off as an equally efficient method of disinfection compared to sodium hypochlorite with diode laser combination and highly efficient than plain A. paniculata and sodium hypochlorite.

Keywords: Andrographis paniculata, Diode Laser, E. faecalis, Laser-activated Irrigants, Sodium Hypochlorite

How to cite this article:
Malavika K G, Sekar R, Ramar K. Antimicrobial efficacy of Andrographis paniculata, sodium hypochlorite, and their combination with diode laser on endodontic pathogen—Enterococcus faecalis—An in vitro study. J Int Oral Health 2022;14:260-5

How to cite this URL:
Malavika K G, Sekar R, Ramar K. Antimicrobial efficacy of Andrographis paniculata, sodium hypochlorite, and their combination with diode laser on endodontic pathogen—Enterococcus faecalis—An in vitro study. J Int Oral Health [serial online] 2022 [cited 2023 Dec 9];14:260-5. Available from: https://www.jioh.org/text.asp?2022/14/3/260/348423

Introduction

The success of the endodontic therapy depends on good shaping and obturation, but the most challenging objective is the eradication of microbial colonies from the complicated root canals.^[1] The residual microorganisms play a key role in the development and persistence of endodontic infections. Enterococcus faecalis is most frequently encountered to be the common cause for the failure in 95% of the permanent teeth root canal treatment and 30% of the primary teeth pulpectomy.^[2]

E. faecalis is characterized by its ability to withstand adverse conditions such as alkaline environment and a lack of nutrients encountered in the root canals for extended periods of time.^[1],[2] The formation of oral biofilm and also the gelatinase-producing ability of E. faecalis aid in survival for longer period of time.^[1],[2],[3] So it is highly desirable that endodontic irrigants should have tissue dissolution properties along with an ability to penetrate into the areas that cannot be cleansed with mechanical instrumentation besides serving in the debridement of root canal system to eliminate the undesired root canal microbes.^[2],[3],[4] Sodium hypochlorite is the most popular irrigant and considered a gold standard owing to its efficiency of removing organic constituents and remarkable tissue-dissolving ability.^[5] Sodium hypochlorite exhibits antimicrobial properties by chloramination reaction and high pH. But the irritation to periapical tissues developed the need for discovering newer irrigants with less toxic and more penetrating efficacy.^[6]

Several natural products especially extracts obtained from plants have been studied for their antimicrobial properties, and few sources proved to be efficient as an irrigant against endodontic pathogens. Andrographis paniculata also known as Green Chiretta besides being antipyretic, anti-inflammatory, and immunostimulatory also exhibited antimicrobial properties.^[7],[8],[9] The acetone and methanol extract of leaves has found to be showing greater inhibitory effect on the growth of E. faecalis in few studies.^[10] Even in the absence of E. faecalis, the biofilm formed itself has the ability to cause reinfection of root canal-treated teeth.

Latest studies done by changing the physical properties of irrigants by heating or vibrating showed an enhanced dissolution of organic substances from inaccessible anatomy such as isthmus and accessory canals; also removal of debris was very efficient.^[11],[12] Moreover, recent studies on diode laser and its use in root canal therapy surged the contemporary era,^[6] particularly because of its high penetration depth of 940 nm into the dentinal tubules, and antimicrobial properties^[13] exhibited by generating cavitation effect by producing pressure waves at a velocity of 20 ms.^[2],[13] Latest strategies involve the active enhancement of irrigation solutions by laser energy, which simulates both heat and vibration, which found to increase the endodontic treatment outcome by effective eradication of microbes that yields the success of the therapy.^[5] Especially photodynamically activated irrigants show much effective reduction of bacterial load.^[14]

Thus the aim of the study is to evaluate and compare the antimicrobial efficacy of A. paniculata and sodium hypochlorite and their combination with diode laser on endodontic pathogen E. faecalis. The null hypothesis is that A. paniculata with diode laser or as alone did not have significant effect on E. faecalis.

Materials and Methods

This is an in vitro study done in two locations: study sample preparation was done in SRM Kattankulathur Dental College and Hospitals, Chennai, whereas the microbial inoculation and colony counting were done in Life Tech Research Laboratory, Chennai between June 15, 2021 and August 5, 2021.

Inclusion and exclusion criteria

The collection of extracted single-rooted teeth with intact root structure without any root resorption was included in the study. The teeth that are severely damaged, whose roots are resorbed, and those with erosion, abrasion, or internal resorption and abnormal morphology were excluded.

Teeth collection

Forty freshly collected single-rooted anterior teeth with no root caries, apical fractures are collected and kept in hydrogen peroxide to remove debris, and the teeth are washed thoroughly and dried in sunlight for 4 h.^[1] The dried samples are stored in normal saline until use. A sample size was calculated using G*Power Software, α err prob = 0.05 Power (1−β err prob) = 0.8.

Obtaining extract of Andrographis paniculata

Fifty grams of freshly collected A. paniculata leaf was obtained from Annai Ayurveda Pharmacy, Chennai. Leaves were cleaned and washed with water and were then dried and pulverized to a powdered form. The powder is then added in 1:1 ratio with 90% ethanol and mixed and soaked with continuous stirring for 6 h and left for 12 h without stirring and was later filtered by distillation under pressure and concentrated with a rotary evaporator to a concentration of 0.5%.^[8]

Obtaining sodium hypochlorite

Powdered sodium hypochlorite is obtained from Sri Kailash Commercial Market, Chennai, and added in a ratio of 3:10 with water to obtain 3% sodium hypochlorite solution as per manufacturer recommendation.

Isolation of microorganism

The precultured inoculum of E. faecalis strain, Microbial Type Culture Collection and Gene Bank (MTCC) 2729 (source-infected root canals), is obtained from Pondicherry Centre for Biological Sciences, which was cultured in blood–heart infusion broth (BHI-Merck) at 37°C for 18 h in an atmosphere of 5% CO₂. The validation was done by gram staining of a single isolated colony under microscope. Colonies were carried through inoculum tube and inoculated in BHI broth, incubation cycle was done at 37°C for 24 h, and changes in turbidity were recorded.^[2],[5],[13]

Preparation of teeth sample

All the collected teeth were kept in hydrogen peroxide to remove organic debris, and ultrasonic scaling was done to remove hard debris. The teeth were then decoronated by using diamond disc bur up to the level of cemento–enamel junction, and canals were prepared by Crown Down technique using ProTaper Gold (DENTSPLY) rotary file system in sequence of S_X, S₁, S₂, F₁, F₂ up to the working length of 15 mm kept 0.5 mm short of apex for standardization. The working length was determined by taking radio visio graphy (RVG) after placing 10-size K file with rubber stopper placed at cemento enamel junction (CEJ) as a reference point. The apex is then sealed with GIC TYPE 2 (GC GOLD LABEL, Japan) and waterproofed by applying nail varnish. All the teeth are then sterilized in autoclave at 121°C at 15 lbs for 30 min to ensure the canals are free from all microorganisms.^[1],[3],[4]

Randomization of teeth sample

The samples are divided randomly into four groups with 10 teeth in each group:

Group 1—to be irrigated with 0.5% extract of A. paniculata,

Group 2—to be irrigated with 3% sodium hypochlorite,

Group 3—to be irrigated with diode laser-activated 3% sodium hypochlorite,

Group 4—to be irrigated with diode laser-activated 0.5% A. paniculata.

Inoculation of organism

The organisms were centrifuged at 10,000 rpm for 5 min and then mixed with saline and standardized to a value of 3 × 10⁶ cells/mL using visible spectrophotometer. And 10 mL of microbial suspension in BHI broth was inoculated through micropipette with sterile needle into the canals by laminar flow technique. After allowing for an incubation period of another 24 h, colonial count before irrigation was obtained by harvesting bacteria from canals by adsorbent paper points and streaking in culture plates and allowing for colony formation and testing the colony-forming unit (CFU) using digital counter.^[2],[6],[13]

Irrigation

After obtaining CFUs, the samples are transported from Life Tech Research Laboratory to SRM Kattankulathur Dental College by segregating and placing in sterile microtubes containing nutrient broth, and irrigation procedure is then started.

Group 1 is irrigated with A. paniculata by using a 30-gauge needle by placing it in canals unto 0.5 mm short of working-length irrigation done repeatedly every 5 min with overall contact time of 15 min utilizing around 15 mL of solution.^[15]

Group 2 is irrigated with 3% sodium hypochlorite by using a 30-gauge side-vented needle by placing it in canals unto 0.5 mm short of working length. Irrigation done repeatedly every 5 min with overall contact time of 15 min utilizing around 15 mL of solution.^[15]

Group 3 is irrigated by placing 0.5 mL sodium hypochlorite in the canals by using a needle and agitating the solution by passing the diode laser pulses at a wavelength of 940 nm at output power of 3.5 W with a pulse duration of 0.5 ms and a pulse interval of 0.2 ms intermittently for 10 s with 10 s interval for 30 s.^[15]

Group 4 is to be irrigated by placing 0.5 mL A. paniculata in the canals by using a needle and agitating the solution by passing the diode laser pulses at a wavelength of 940 nm at output power of 3.5 W with a pulse duration of 0.5 ms and a pulse interval of 0.2 ms intermittently for 10 s with 10 s interval for 30 s.^[15]

Bacteriological analysis

After irrigation completed, samples are kept in sterile collecting tubes containing the BHI broth transport medium and sent for colonial count and postirrigation CFU count obtained by taking samples as before in wet absorbent paper points and placing in BHI agar and incubating for another 24 h. The CFU is obtained for each sample calculated with digital counter and sent for statistical analyses and verified.

Statistical analysis

All the data were analyzed using SPSS 20.0 (Armonk NY, USA). Analysis of Variance (ANOVA) was performed for comparing variation between groups and multiple comparison test performed for intergroup comparison. The pre- and postirrigation intergroup analysis was done using paired t test. The mean CFU between each group was compared using one-way ANOVA.

Results

The evaluation of samples was done based on pre- and postirrigation changes in the CFUs of E. faecalis, and predominant changes from baseline value of around 4.2–5.4 × 10³ to around 0.05–0.03 × 10³ were noted, and also intergroup comparisons revealed significant changes.

[Table 1] represents the pre- and postcolonial count, which depicts the reduction of colony count postirrigating of about 96% in group 4, 90% in group 3, 48% in group 1, with least reduction of about 24% in group 2.

Table 1: Mean value of CFUs of E. faecalis from pre- and postirrigation samples

Click here to view

[Table 2] depicts intergroup comparison, which shows that there was a significant difference between group 4 (P = 0.000) and other two groups except group 3. Even though group 3 and group 4 did not give a significant difference (P = 0.078), based on colonial reduction, group 4 is highly efficient than group 3 against E. faecalis. Significant results were derived on comparing groups 1, 2, and 3 (P = 0.000).

Table 2: Comparison of mean CFUs from pre- and postirrigation samples of all four experimental groups

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Discussion

The major hurdle for endodontic irrigation is the pathogens and their toxins residing in the complex anatomy of root canals. E. faecalis is the common pathogen associated with endodontic failures as it defends the chemomechanical preparation and also has the ability to cause monoinfection of dental root canal. It solely colonizes the canals without any other organism for support.^[16]

Kayaoglu and Ørstavik reported the entire root canal can be contaminated by E. faecalis within 48 h as it can penetrate up to depth of 300 μm.^[16] But a study done by Betancourt et al. showed that 24 h old biofilm is itself an optimal study subject.^[17] Hence, in the current study, samples were contaminated for 24 h prior to irrigation, and inoculation was standardized to 3 × 10³ in accordance with a study by Varman et al.^[7]

In this study, the efficacy of four different irrigants has been challenged against E. faecalis. The most widely used sodium hypochlorite has been proved to eliminate these organisms by acting as an organic and fat solvent that degrades fatty acids; furthermore, it forms hypochlorous acid (HOCl⁻) and hypochlorite ions (OCl⁻) that lead to amino acid degradation and hydrolysis. It also releases chloramines that in turn impede cell metabolism.^[2] A study by Estrela et al. has shown that 5.25% sodium hypochlorite solution is more effective in a shorter period of time but because of its cytotoxic property which at this high concentration if extruded beyond apex may lead to swelling and pain.^[18] In this study, 3% sodium hypochlorite is used considering the studies by Rahimi et al. and Mithra et al., which showed 3% sodium hypochlorite inhibited E. faecalis and also was most effective when used in combination with other irrigants such as lasers and 2% chlorhexidine.^[6] A demonstration given by Kayaoglu and Ørstavik reveals several E. faecalis have tolerance to sodium hypochlorite (NaOCl) and suggested a use of 0.5% NaOCl with contact time of 30 min continuously to obtain complete bacterial elimination.^[16] Considering the fact that cytotoxicity is usually dose- and time-dependent, we have used 3% NaOCl irrigation done repeatedly every 5 min with overall contact time of 15 min utilizing around 15 mL of solution.^[19]

A recent incidence of the development of resistance against chemical antimicrobials among these bacteria and also propagation of multidrug resistant strains has led to the search that has been spread across to natural sources for defence. One such herbal source named A. paniculata was found to be effective against E. faecalis in studies done by Ravi et al.^[6] The mechanism by which it acts against E. faecalis is mentioned by Sule et al. study, which revealed that the phytochemical constituents such as diterpenoid and flavonoids in this herb are biological response modifiers and also potent antimicrobial as well as antioxidant.^{[5],[7-10],[20],[21]} In addition, Radha et al. observed that extracts of A. paniculata had great inhibitory effects against E. faecalis; also the tannins in these were potent antifungal agents too.^[5] Thus, in this study, we utilized 0.5% extract of A. paniculata, which was in accordance with Varman et al.

The penetrating property and the ability of E. faecalis to survive entombment interiorly in dentinal tubules paved way for revolution in endodontic therapy when lasers came to play a role in irrigation.^[1] Diode laser was used as endodontic disinfection by Asnaashari et al. as they significantly access the impermeable areas in the dentinal tubules and exhibit bactericidal effect. Further studies by Iandolo et al. and Arikatla et al. revealed that the combination of temperature and vibration together enhanced the dissolution effect of irrigants.^{[3],[11],[12],[22]} Even though many studies used exclusively lasers for irrigation,^[23] its charring ability has questioned its usage directly in canals. Shahriar et al. in their study used laser-activated irrigants as they were more effective than when using them separately.^[21] Moreover, the time consumption of the activation of irrigants by heating those ex vivo and the risk of apical extrusion when using ultrasonic activation are all overcome by laser-activated irrigants.^[2]

In this study, photoacoustic streaming with subablative power in a pulsative mode was used at a wavelength of 940 nm at output power of laser-activated irrigant 3.5 W with a pulse duration of 0.5 ms and a pulse interval of 0.2 ms intermittently for 10 s with 10 s interval for 30 s by placing the fiber tip 5 mm short of apex and agitation done by circulating movement of tip from apical to coronal, which was found to be the optimum method for irrigation in Castelo et al. (2021) study.^[17],[23]

In the current study, the laser-activated irrigant technique was applied with both 0.5% A. paniculata and 3% NaOCl, which gave more or less similar results when compared with each other, which is not significant (P > 0.05) but showed significant difference compared with other two groups (P < 0.001), whereas studies done by Yavagal et al. show laser-activated irrigant-NaOCl as the most efficient irrigant comparing with other chemical techniques,^[13] but considering the fact laser-activated irrigant-0.5% A. paniculata has so far not been used as a irrigant to the best of our knowledge, this study provides with a novel and potent irrigant. This method of using laser-activated irrigants creates microcavitation effect, which causes subsequent implosion of irrigant bubbles that owes to the removal of a smear layer from dentinal wall, thereby exposing the dentinal tubules that lead to propulsion of irrigant solutions deep into dentinal tubules, thus acting on bacterial colonies.^[2],[3],[11]

Further, the results of this study showed that laser-activated irrigant 0.5% extract of A. paniculata was highly efficient than plain 0.5% A. paniculata and 3% sodium hypochlorite and equally efficient to laser-activated irrigant 3% sodium hypochlorite. Thus, laser-activated irrigant 0.5% A. paniculata proves to be potent alternative to laser-activated irrigant 3% NaOCl. Further, the sole use of 0.5% A. paniculata proved to be highly efficient than 3% NaOCl irrigation. Moreover A. paniculata is more biocompatible as it lacks the risk of tissue injury even if extruded, which is the major drawback of NaOCl.

Even though the study shows a significant efficacy of A. paniculata, a sample size taken is minimum and the time of colony maturation is only kept to 24 h; the efficacy of even more matured colonies in a large sample has to be done to analyze the efficacy of A. paniculata with laser combination. Further, the colony count obtained in this study is by swab-and-streak method; more microscopic evaluations can give a clear idea on the colonies present deep inside tubules.

More studies have to be done with varying concentrations of A. paniculata solely as well in combination with lasers to examine its efficacy elaborately and also including teeth with complex canal morphology such as mesial canals of molars to determine the actual potential of these irrigating techniques.

Conclusion

All four techniques included in this study proved to be effective against E. faecalis, but A. paniculata with diode laser came off as an equally efficient method of disinfection comparing to sodium hypochlorite with diode laser combination and highly efficient than plain A. paniculata and sodium hypochlorite. However, for clinical application, in vivo studies have to be conducted. Further studies altering concentration of A. paniculata and using different wavelengths and parameters of diode laser have to be done in teeth with more torturous morphology.

Acknowledgement

We acknowledge the help of Dr. A. V. Raghavendra Rao, MBBS, MD, assistant professor, Vinayaka Mission’s Kirupananda Variyar Medical College, Dr. P. Kennedy Kumar, MD, associate professor, Sri Ramachandra Medical College and Research Institute, in suggestion for procuring the microbiology sample, and Dr. Seethalakshmi Illanchezian, the director of Life Teck Research Centre, for providing laboratory facility to conduct a vital part of the research.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Authors contributions

MKG: conceptualization ideas, methodology, investigation, resources, data curation, and writing—original draft; RKS: conceptualization ideas, methodology, validation, formal analysis, investigation, writing—review and editing, supervision, and project administration; KR: methodology, validation, formal analysis, writing—review and editing, and supervision.

Ethical policy and institutional review board statement

Not applicable.

Patient declaration of consent

Not applicable.

Data availability statement

Data are available on reasonable request.

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