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ORIGINAL ARTICLE
Year : 2020  |  Volume : 9  |  Issue : 2  |  Page : 98-102

Terminalia arjuna – A possible alternative to commercial mouthwashes, against periodontopathic bacteria: An in vitro study


1 Department of Periodontics and Implantology, CKS Theja Institute of Dental Sciences and Research, Tirupathi, Andhra Pradesh, India
2 Department of Prosthodontics Crown and Bridge and Implantology, CKS Theja Institute of Dental Sciences and Research, Tirupathi, Andhra Pradesh, India

Date of Submission08-Jun-2020
Date of Decision12-Jun-2020
Date of Acceptance15-Jun-2020
Date of Web Publication18-Jul-2020

Correspondence Address:
Dr. Ravindra Reddy Nagi Reddy
Department of Periodontics and Implantology, CKS Theja Institute of Dental Sciences and Research, Tirupathi, Andhra Pradesh - 517 501
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JDRNTRUHS.JDRNTRUHS_81_20

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  Abstract 


Background and Aims: Periodontitis is irreversible plaque-mediated damage to gums and its supporting structures. Plaque is a niche of complex organisms forming biological associations for their attachment and sustenance, forming the basis for periodontal diseases. There is a vast diversity in oral microbiota, depending on the site. Dentition creates natural surfaces and barriers for the diversification of bacteria. For instance, periodontal bacteria differ between supra and subgingival tissues. The plaque mounted on the subgingival area chiefly houses gram-negative anaerobes such as Treponema denticola, Tannerella forsythia, and Porphyromonas gingivalis. The current study aims to compare and contrast the antimicrobial potential of Terminalia arjuna with commercially available mouth rinses against clinical isolates of periodontal bacteria based on the well-diffusion method.
Materials and Methods: Preparation of Terminalia arjuna bark extract was done. For the detection of periodontal bacteria, Porphyromonas gingivalis, and Treponema denticola, polymerase chain reaction (PCR) was used. The well-diffusion method was used to test the antimicrobial activity.
Results: The aqueous extract of Terminalia arjuna showed minimum zone of inhibition of 23.33 ± 0.577 for Porphyromonas gingivalis and 24.33 ± 0.577 for Treponema denticola while the commercially available mouthwashes M1, M2, M3showed 29.33 ± 0.577, 29.33 ± 0.577, 24.33 ± 0.577, respectively towards Porphyromonas gingivalis and 29.66 ± 0.577, 27.33 ± 0.577, 25.66 ± 0.577, respectively towards Treponema denticola The results depicted nearly equal efficacy of Terminalia arjuna aqueous extract similar to those of commercial mouthwashes against the test bacteria.
Conclusion: Antibacterial tests of Terminalia arjuna bark extracts showed promising results even at low concentrations. Hence, it can be an alternative to commercially available mouthwashes, which suffer from having numerous drawbacks ranging from burning sensation while in use to yellowing of teeth surfaces. Herbal medicines are considered to be much safer than other synthetic formulations and possess wide acceptance by people. Hence, this study forms a basis for future studies in this area.

Keywords: Paper points, periodontitis, Porphyromonas gingivalis, Tannerella forsythia, Terminalia arjuna


How to cite this article:
Dandu SS, Sravanthi G, Mohammed S, Narahari S, Sistla SL, Reddy RR. Terminalia arjuna – A possible alternative to commercial mouthwashes, against periodontopathic bacteria: An in vitro study. J NTR Univ Health Sci 2020;9:98-102

How to cite this URL:
Dandu SS, Sravanthi G, Mohammed S, Narahari S, Sistla SL, Reddy RR. Terminalia arjuna – A possible alternative to commercial mouthwashes, against periodontopathic bacteria: An in vitro study. J NTR Univ Health Sci [serial online] 2020 [cited 2020 Aug 8];9:98-102. Available from: http://www.jdrntruhs.org/text.asp?2020/9/2/98/289899




  Introduction Top


Periodontitis is persistent across the globe by being the most frequent reason for tooth loss among adults. It generally affects gums and their structures, leading to permanent damage if left unattended at the proper time. The patient undergoes severe pain and discomfort and may also need cosmetic restorations based on its severity.[1] Hence, any clinician needs to control and restore the normalcy in the tissue.[2]

Insufficient and inefficient oral hygiene practices are considered to be the root cause of gum inflammations as the latter is due to plaque accumulation, and it needs mechanical methods for removal. Toothbrushes, dental floss, oral irrigators, conventional interdental brushes, and wooden interdental aids are few among others that are regularly used for plaque removal. However, these tools are found to be ineffective in the complete removal of plaque. Hence, modern medicine has come up with a variety of mechanical apparatus to clean the plaque.

Despite the agreed process of mechanical cleaning, a user might have less access to all surfaces of teeth, such as beneath and in-between the teeth with any of the tools mentioned earlier. It causes a hindrance to the manual rupture of plaque among people who are unwilling to do so. In such cases, mouthwash can be useful in reducing plaque accumulation and thereby prevent gum diseases.

Chlorhexidine gluconate is a widely studied oral disinfectant being used to contain plaque. Other antibacterial agents used for plaque control include phenols, quaternary ammonium compounds, and ammonium salts. However, these mouthwashes cannot reach the subgingival niche and hence cannot be a substitute for manual cleaning. Nevertheless, they can act as useful accompaniment in the process of regular brushing.

Earlier studies have supported the use of plant extracts in improving periodontal health by decreasing the bacterial load.[3],[4] India is a long-known practitioner of alternative medicines such as Ayurveda, Siddha, homeopathy, and naturopathy in treating several ailments. Most of these gained their popularity with the notion of having less to nil side effects among the users. Traditional practitioners and researchers have explored several medicinal plants, which are currently in use. However, there are many more whose activity has not been investigated.[5] With the advent of globalization, medicinal plants have also gained popularity among the western world, and recent years have witnessed natural-product biosynthesis.[6]

One such plant is Terminalia arjuna, whose potentiality against periodontal pathogens is measured in the following study.

Arjuna (Terminalia arjuna)

T. arjuna (Combretaceae) is known to decrease cholesterol, reduce hypertension, reduce blot clots, and prevent coagulation of blood. It was also found to be effective against several bacteria and fungi. Phytoconstituents such as triterpenoids were found to effectively treat cardiovascular diseases, while tannins and flavonoids were found to have anticancer properties.

Its leaves, bark, and fruits are used in extracting active compounds such as ethyl gallate, gallic acid, arjunolic acid, ellagic acid, and flavones, which are investigated for their antibacterial properties and hence can be used against periodontal bacteria.[7],[8]


  Materials and Methods Top


Plant extract preparation

Plants were selected from in and around Tirupati, and taxonomical identification of plants was made by Sri Venkateswara University's Department of Botanical Sciences, Andhra Pradesh, India. Disease-free plants and their parts were carefully selected for the study. Ethics committee approval obtained date 15-08-2019.

The bark of T. arjuna was surface cleaned under running water. It was further washed with autoclaved double-distilled water to remove any more impurities or surface pathogens. Upon air drying at room temperature, it was grounded into powder under sanitized conditions. About 10 mg/mL air-dried powder of the respective plant parts were macerated with 100 mL sterile distilled water and blended for 10 min to obtain a homogenous mixture. This was filtered through a muslin cloth, and the filtrate was centrifuged for 30 min at 4000 rotations per minute (RPM). The supernatant of the centrifuged mixture was passed through the Whatman No. 1 filter paper to obtain a pure solution that served as the aqueous extract of the plant.[4] It was refrigerated at 4°C for future use.[4]

Patient selection

The subjects diagnosed with chronic periodontitis were selected from the Department of Periodontics, CKS Theja College, Tirupati, Andhra Pradesh, India. Three periodontitis male patients (aged 30-40 years) were recruited in the study based on a gingival index ≥1, periodontal pocket ≥5 mm, and clinical attachment loss ≥2 mm. Subjects who fulfilled the inclusion/exclusion criteria were made clear about their voluntary participation in the study. Necessary consent was obtained in writing as per the approved guidelines from the institutional ethics committee (IEC). Ethical clearance for the study was obtained from the IEC [PR 139/IEC/CKSTEJA/2019] 15, March 2019.

Sample collection and bacterial preparation

Sterile cotton swabs were used to pick up supragingival plaque. The subgingival plaque was collected using sterile paper points by probing them deep into periodontal pockets using tweezers. These were left 20 s and then were transferred to vials (one vial per patient) containing 2 mL VMGA III transport medium.[9]

Within 48 h of sampling, they were processed for culturing and polymerase chain reaction (PCR) detection of P. gingivalis (P.g) and Tannerella forsythia (T.f) (formerly Bacteroides forsythias). Culturing and molecular identification was made based on previous studies.[10],[11],[12] Briefly, P.g and T.f were recovered from  Brucella More Details blood agar (Brucella agar supplemented with 5% defibrinated horse blood, 5 mg/mL hemin, and 10 mg/mL vitamin K). The Brucella blood agar plates were placed in anaerobic jars filled with combined gas (85% N2, 10% H2, and 5% CO2) at 37°C for 7 to 10 days. The identity of bacteria was established using PCR.

Detection using a polymerase chain reaction

Clinical samples were analyzed for the presence of P. gingivalis and T. forsythia. DNA extraction was carried out by using a commercially available DNA isolation technique (Medox). The previously described [13],[14] primer pairs for P. gingivalis (5'-AGG CAG CTT GCCATA CTG CG-3' and 5'-ACT GTT AGC AAC TACCGATGT-3') and T. forsythia (5'-TCA CTA TTG TGT CTC GCTG -3' and 5'- TCT CTC CGA TTG TGG -3') were used for PCR amplification.

Collection of mouth rinses

Three commercially available mouth rinses with different chemical formulations were labeled as M1, M2, and M3. Mouth rinse M1 chemically composed of chlorhexidine gluconate (0.2% w/v), sodium flouride IP (0.05% w/v), zinc chloride IP (0.09% w/v); mouth rinse M2 composed of essential oils, namely, eucalyptol 0.092%, menthol 0.042%, thymol 0.064%, methyl salicylate 0.06%; mouth rinse M3 contained glycerin, ethyl alcohol, sorbital, propylene glycol, polysorbate 20, sodium benzoate, sodium flouride, cetylperidinium quinoline yellow flavor in aqueous base. Standard ciprofloxacin (CF) antibiotic (HIMEDIA) dissolved in 1 mL of autoclaved, deionized double distilled water was taken as control.

Screening for antimicrobial activity

Microbial samples were transformed into standard suspensions by tube 0.5 McFarland standard (equivalent turbidity 1 × 106 CFU/mL).[4] This suspension was inoculated onto Wilkins Charlgren agar medium and Muller Hinton agar medium for determination of minimum inhibitory concentration as per CLSI-2019 standards. Few 6 mm wells were made on the culture plates before inoculation. About 40 mg/mL concentration of plant extract and 40 μL of each mouthwash and 40 μL of 40 mg/mL CF as control were dispensed into wells. After incubation at 37°C under standard culture conditions, they were examined for antimicrobial activity using inhibitory zones formed around the test samples. HI-Antibiotic zone scale (HI-MEDIA) was used to determine the diameter (mm) of inhibition zones.

Statistical analysis

Data analysis was carried out by one way ANOVA using SPSS software. The significance level of a “P” value of less than 0.005 (P < 0.005) was considered highly significant


  Results Top


The antimicrobial efficacy of plant extract and mouth rinses with CF taken as control is shown in [Table 1]. [Table 2] and [Table 3] denote significance “P” values on comparison on plant extracts against mouthwashes.
Table 1: Antimicrobial Potency of Mouth Rinses and Plant Extract

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Table 2: “P” Value for P.g.

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Table 3: “P” Value for T.f

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The aqueous extract of the T. arjuna showed minimum zone of inhibition of 23.33 ± 0.577 for P.g and 24.33 ± 0.577 for T.f while the commercially available mouthwashes M1, M2, M3 showed 29.33 ± 0.577, 29.33 ± 0.577, 24.33 ± 0.577, respectively towards P.g and 29.66 ± 0.577, 27.33 ± 0.577, 25.66 ± 0.577, respectively towards T.f. The results depicted nearly equal efficacy of T. arjuna aqueous extract similar to those of commercial mouthwashes against the test bacteria.


  Discussion Top


Plant and plant products have been a reservoir for many antibacterial agents. Prolonged use of synthetic drugs always had side effects and a chance for a microbe to develop resistance. Hence, traditional medicine using medicinal plants has gained its importance in treating several ailments.[15]

As a substitute, herbal extracts, which are generally devoid of sugar, artificial colors, and sweeteners such as saccharine or stannous fluoride which could give yellow stain to teeth on prolonged use, can be used.

In the present study, plant extracts, i.e., T. arjuna bark were tested for its antimicrobial activity against periodontal pathogens by comparing three types of mouthwash taking CF as control. Inclusion of P. g and T. f in this study is based on the literature that relates these microorganisms to periodontal infections.[16] The methodology of this study followed standard agar dilution tests, and the design of the study is in agreement with similar other antimicrobials.[17] The agar diffusion method may be used as a qualitative parameter of antimicrobial activity.

Aqueous extract of T. arjuna showed minimum inhibitory concentration (MIC) against P. g and T. f at 40 mg/mL. While three commercial mouth rinses with different compositions had MIC at 40 μL. This could be because of three main reasons: (1) Organic compounds may have more readiness towards the polarity of bacterial constituents rather than those of aqueous extract, (2) The amount of active compound or the concentration of the active compound in the crude extract might have been insufficient for the dose employed for the analysis, (3) As the test sample is an aqueous extract, there could be several other compounds along with active components, which might have shown an adverse effect or could even have interfered with the active compound's action potential.[4]

However, the results obtained were consistent with earlier studies of Alam et al., Ramya et al., and Phatak et al., which detailed broad range antibacterial activity possessed by bark and organic extracts of T. arjuna.[2],[19] Literature also suggests the presence of several other medicinal properties such as antiatherogenic, antineoplastic, antioxidant, antidysenteric, anti-inflammatory, antipyretic, and diuretic. It has also been linked with the treatment of cardiovascular diseases.[20]

Tannins, arjuonic acid, arjunogenin, arjunetine, and arjunolone were few among the identified active constituents of bark, which were found to exhibit antimicrobial effects.[21] The presence of these compounds might have brought the desired results in the current study. Yet, the antimicrobial activity of T. arjuna has been scantily explored,[22],[23],[24] and works against periodontal pathogens are none. Hence, the present research gains its importance.

The study demonstrates the sensitivity of periodontal microbes towards the bark extract of T. arjuna, indicating its potential to be a natural substitute for commercial mouthwashes. Further testing using purified active compounds might yield better results and lay stepping stones towards clinical analysis for safe therapeutic utilization.


  Conclusion Top


These results will helpin vivo trials searching for herbal alternatives to treat periodontal diseases. Much more in-depth investigations will be needed to purify the active compounds, which will defend against periodontal microbiota when integrated into oral care products.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Holt SC, Ebersole JL. Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia: The 'red complex,' a prototype polybacterial pathogenic consortium in periodontitis. Periodontol 2000 2005;38:72-122.  Back to cited text no. 16
    
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    Tables

  [Table 1], [Table 2], [Table 3]



 

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