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Table of Contents   
ORIGINAL RESEARCH  
Year : 2018  |  Volume : 29  |  Issue : 5  |  Page : 641-645
Antimicrobial Efficacy of Acacia nilotica, Murraya koenigii L. Sprengel, Eucalyptus hybrid, Psidium guajava extracts and their combinations on Fusobacterium nucleatum and Porphyromonas gingivalis


1 Department of Public Health Dentistry, JSS Dental College and Hospital, Jagadguru Sri Shivarathreeshwara University, Mysore, Karnataka, India
2 Department of Public Health Dentistry, Institute of Dental Sciences, Bhubaneshwar, Odisha, India
3 Center for Scientific Research and Development, People's University, Bhopal, Madhya Pradesh, India
4 Department of Orthodontics, JSS Dental College and Hospital, Jagadguru Sri Shivarathreeshwara University, Mysore, Karnataka, India

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Date of Web Publication2-Nov-2018
 

   Abstract 

Background: The herbal extracts have been effectively tried in the treatment and prevention of many oral diseases. Aim: The aim is to assess the antimicrobial efficacy of Acacia nilotica, Murraya koenigii L. Sprengel, Eucalyptus hybrid, Psidium guajava extracts and their combinations on Fusobacterium nucleatum (Fn) and Porphyromonas gingivalis (Pg). Materials and Methods: The extraction process was carried out by Soxhlet apparatus using ethanol as solvent. The combinations of the four plant extracts were prepared by combining an equal quantity of 10% solution of each of the four plant extracts. The antimicrobial efficacy testing of the plant extracts and their combinations on Fn and Pg was performed using agar well diffusion method. Columbia 5% of sheep blood agar plates were used for antimicrobial efficacy testing under anaerobic conditions. The qualitative assay was carried out to identify the various phytochemical constituents. Dimethyl sulfoxide and 0.2% chlorhexidine acted as negative and positive controls, respectively. The mean diameter of inhibition zone between different categories was compared using one-way analysis of variance. Results: All the individual plant extracts and their double, triple, and quadruple combinations were effective in inhibiting the growth of these bacteria. However, 0.2% chlorhexidine produced the highest mean diameter of inhibition zone. Conclusion: The plant extracts in combinations offer enhanced antimicrobial efficacy due to their synergistic action besides slowing the development of bacterial resistance. Hence, these extracts in combinations could be used tried as effective alternates to chlorhexidine.

Keywords: Acacia nilotica, Eucalyptus hybrid, Fusobacterium nucleatum, Murraya koenigii L. Sprengel, Porphyromonas gingivalis, Psidium guajava

How to cite this article:
Chandra Shekar B R, Nagarajappa R, Jain R, Singh R, Suma S, Thakur R. Antimicrobial Efficacy of Acacia nilotica, Murraya koenigii L. Sprengel, Eucalyptus hybrid, Psidium guajava extracts and their combinations on Fusobacterium nucleatum and Porphyromonas gingivalis. Indian J Dent Res 2018;29:641-5

How to cite this URL:
Chandra Shekar B R, Nagarajappa R, Jain R, Singh R, Suma S, Thakur R. Antimicrobial Efficacy of Acacia nilotica, Murraya koenigii L. Sprengel, Eucalyptus hybrid, Psidium guajava extracts and their combinations on Fusobacterium nucleatum and Porphyromonas gingivalis. Indian J Dent Res [serial online] 2018 [cited 2018 Dec 15];29:641-5. Available from: http://www.ijdr.in/text.asp?2018/29/5/641/244942

   Introduction Top


Dental plaque is defined clinically as a structured, resilient, yellow-grayish material that adheres tenaciously to the intraoral hard surfaces, including removable and fixed prosthesis.[1] Dental plaque as a potential risk factor in the etiology of periodontal diseases has been well documented in medical literature. The complete removal of plaque from the dento-gingival region is an established method of preventing gingivitis and periodontitis.[2],[3],[4]

Although over 500 bacterial species comprise of plaque, colonization follows a regimented pattern with adhesion of initial colonizers to the enamel salivary pellicle followed by secondary colonization through inter-bacterial adhesion. A variety of adhesins and molecular interactions underlie these adhesive interactions and contribute to plaque development. This can eventually lead to diseases such as caries and periodontal disease.[5]

“Home care” with tooth brushing and other mechanical cleansing procedures is the most dependable means of controlling plaque. However, mechanical plaque control is not properly practiced by most individuals. Adjunctive chemical plaque control using chlorhexidine and antibiotics have also been recommended in addition to mechanical plaque control. However, the additional effects of adjunctive antibiotic therapy are small, and topical chlorhexidine therapy is not without side effects.[6] This necessitates the development of strategies that can act against the plaque bacteria.

The herbal extracts have been effectively tried in the treatment and prevention of many oral diseases.[7] The previous studies have demonstrated the antimicrobial efficacy of Acacia nilotica, Murraya koenigii L. Sprengel, Eucalyptus hybrid and Psidium guajava extracts on dental caries and primary plaque colonizers.[8],[9],[10] The assessment of the antimicrobial efficacy of these extracts on secondary and tertiary plaque colonizers will offer an effective strategy to prevent periodontal diseases. In this background, the present study was undertaken to assess the antimicrobial efficacy of A. nilotica, M. koenigii L. Sprengel, E. hybrid, P. guajava extracts and their combinations on Fusobacterium nucleatum (Fn)and  Porphyromonas gingivalis Scientific Name Search > Pg).


   Materials and Methods Top


Study design and setting

This in vitro study was conducted over a period of 6 months from December 2013 to May 2014 at Center for Scientific Research and Development, People's University, Bhopal. The study protocol was approved by the Research Advisory Committee.

Preparation of plant extracts

The branches of four plants were collected from the surrounding areas, identified and authenticated by a taxonomist. The branches were thoroughly washed using water treated with reverse osmosis. Healthy leaves were separated from these branches and shade dried over a period of 3–4 weeks at room temperature. The dried leaves were hand crushed separately to obtain coarse powder, and subsequently, the fine powder was prepared using mixer grinder. The fine powder was stored in airtight plastic bottles, labeled, and stored in refrigerator at 4°C till further use.

The extraction process was carried out by Soxhlet apparatus using ethanol as solvent. The dried leaf extract was added to a required quantity of dimethyl sulfoxide (DMSO) to obtain the working concentration of the extract (i.e.,100 mg/ml). The combinations of the four plant extracts were prepared by combining an equal quantity of 10% solution of each of the four plant extracts.

Antimicrobial efficacy testing

The antimicrobial efficacy testing of the plant extracts and their combinations on Fn and Pg was done using agar well diffusion method. Columbia 5% sheep blood agar plates were used for antimicrobial efficacy testing under anaerobic conditions. The diameter of the inhibition zone was measured at three different planes on the undersurface of the agar plate using a transparent scale. DMSO and 0.2% chlorhexidine acted as negative and positive controls, respectively. The experiment was carried out in duplicate sets. The six readings were used to compute the mean diameter of inhibition zones for each extract and their combinations against these bacteria.

Photochemical constituents assay

The assessment of various phytochemical constituents such as alkaloids (using Mayer's reagent and Dragendorff's reagent), anthraquinones (Borntrager's test), terpenoids (Salkowski's test), saponins (Froth and emulsion test), flavonoids (Shinoda and alkaline reagent tests), tannins (Ferric chloride and lead acetate tests), and cardiac glycosides (Legal test and Keller–Killani test) was done using a qualitative assay.[11]

Statistical analysis

The data were entered into a personal computer, and statistical analysis was performed using SPSS version 20 (IBM, Chicago USA). The mean diameter of inhibition zone between different categories was compared using one-way analysis of variance and Tukey's post hoc test. The statistical significance was fixed at 0.05.


   Results Top


The details of the four plants used in the present study are denoted in [Table 1]. The details of bacteria (Fn and Pg) used in this study are denoted in [Table 2]. The mean diameter of inhibition zone produced by each plant extract and their double, triple, and quadruple combinations against Fn and Pg are denoted in [Table 3].
Table 1: Details of four plants used in the present study

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Table 2: Details of bacteria used in the present study

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Table 3: Antimicrobial efficacy of plant extracts and their combinations on Fusobacterium nucleatum and Porphyromonas gingivalis

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All the individual plant extracts and their double triple and quadruple combinations were effective in inhibiting the growth of these bacteria. However, 0.2% chlorhexidine produced the highest mean diameter of inhibition zone compared to plant extracts and their combinations against both of these bacteria. DMSO failed to inhibit the growth of both of these bacteria.

Among the various plant extracts and their combinations, the double combination of A. nilotica and E. hybrid produced the maximum zone of inhibition against Fn (14.42 ± 0.67), whereas M. koenigii L. Sprengel produced the highest zone of inhibition on Pg (15.33 ± 0.82). However, the post hoc test revealed a significant difference between 0.2% chlorhexidine and all other plant extracts as well as their combinations with no significant difference between other categories.

The qualitative assay of phytochemical constituents in A. nilotica, revealed the presence of anthraquinones, flavonoids, tannins, and cardiac glycosides. M. koenigii L. Sprengel contained tannins and cardiac glycosides. E. hybrid demonstrated the presence of terpenoids, saponins, flavonoids, tannins, and cardiac glycosides. P. guajava contained anthraquinones, terpenoids, flavonoids, tannins, and cardiac glycosides [Table 4].
Table 4: Results of qualitative assay of phytochemical constituents in plant extracts

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   Discussion Top


Dental plaque is the principal etiologic factor in periodontal disease. The accumulation of dental plaque with no intervention or oral hygiene methods causes gingivitis which on further progression becomes periodontitis. Mechanical plaque control and use of chemotherapeutic agents are established methods to treat periodontal disease. The major limitation of these chemotherapeutic agents is the toxicity and development of resistant microorganisms. The search for newer and safer chemotherapeutic agents continues in an attempt to overcome the limitations of mechanical debridement and adverse effects of chemotherapeutic agents. Natural phytochemical isolated from plants are considered an effective alternative to synthetic chemicals.[12] This study assessed the antimicrobial efficacy of A. nilotica, M. koenigii L. Sprengel, E. hybrid, P. guajava extracts and their combinations on Fn (secondary plaque colonizer) and Pg (tertiary plaque colonizer).

Clark et al.[13] in their study while assessing the efficacy of Acacia arabica gum on the growth and protease activities of periodontopathic bacteria found the extract to inhibit the growth of Pg. The authors concluded that the extract could be of clinical value to inhibit these bacteria similar to the results of the present study. The phytochemical constituents such as alkakoids, saponins, cardiac glycosides, tannins, flavonoids, and anthraquinones in A. nilotica may be responsible for the antimicrobial action of plant extract.[14]

Nagata et al.[15] in their study on the inhibitory effects of macrocarpals on the biological activity of Pg and other periodontopathic bacteria found macrocarpals A, B, C derived from eucalyptus demonstrated the antibacterial activity against periodontopathic bacteria. Among tested bacteria, Pg displayed the greatest sensitivity to macrocarpals. The study concluded that eucalyptus leaf extracts may be useful as a potent preventative of periodontal disease. Takarada et al.[16] while assessing the antibacterial efficacies of essential oils against oral pathogens found eucalyptus oil to exert an inhibitory effect on various oral bacteria that included Pg, Actinobacillus actinomycetemcomitans, Fn, Streptococcus mutans, and Streptococcus sobrinus. These results were similar to the results of the present study. M. koenigii L. Sprengel contains sterols, alkaloids, and flavonoids, which may be responsible for the antimicrobial action.[17] Ramesh et al.[18] in their study on the salivary and tongue coating pH on chewing household herbal leaves found curry leaf extracts as home remedies that create an oral environment unfavorable for microbes. Ravi and Divyashree[12] in their review on P. guajava as an adjunct in the treatment of periodontal disease concluded that the extract has an excellent antibacterial and antiplaque action and hence, may be considered a good adjunct to the mainstream periodontal treatment. Quercetin derived from P. guajava was shown to exhibit excellent antibacterial actions against periodontal pathogens such as Aggregatibacter actinomycetemcomitans, Pg, Prevotella intermedia, Fn.[19],[20] Guava extract has demonstrated in vitro antiplaque actions by inhibiting growth, adherence, and co-aggregation of dental plaque bacteria. Guava extracts may inhibit plaque development without disrupting homeostasis of the oral cavity.[21] The combinations of plants extracts used in the present study also inhibited the growth of Fn and Pg. The present study is the first of its kind assessing the antimicrobial efficacy of the combinations of A. nilotica, M. koenigii L. Sprengel, E. hybrid, P. guajava extracts on Fn and Pg. Hence, our results could not be compared with previously published literature.


   Conclusions Top


The individual plant extracts and their combinations were effective in inhibiting the growth of Fn and Pg. Although 0.2% chlorhexidine produced a significantly higher zone of inhibition compared to all the plant extracts and their combinations, these extracts could be considered as potential antiplaque and anticaries alternates. The plant extracts in combinations offer enhanced antimicrobial efficacy due to their synergistic action besides slowing the development of bacterial resistance. Hence, these extracts in combinations could be used tried as effective alternates to chlorhexidine. However, the in vivo studies on humans are to be conducted to validate these in vitro results.

Acknowledgment

We sincerely thank the management of People's University, Bhopal, Madhya Pradesh for their kind permission and co-operation in completing this research project.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

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Beck JD, Koch GG, Rozier RG, Tudor GE. Prevalence and risk indicators for periodontal attachment loss in a population of older community-dwelling blacks and whites. J Periodontol 1990;61:521-8.  Back to cited text no. 1
    
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Menon L, Ramamurthy J. New vistas in plaque control. IOSR J Dent Med Sci 2014;13:64-8.  Back to cited text no. 2
    
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Axelsson P, Lindhe J, Wäseby J. The effect of various plaque control measures on gingivitis and caries in schoolchildren. Community Dent Oral Epidemiol 1976;4:232-9.  Back to cited text no. 3
    
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Horowitz HS. The prevention of oral disease. Established methods of prevention. Br Dent J 1980;149:311-8.  Back to cited text no. 4
    
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Rosan B, Lamont RJ. Dental plaque formation. Microbes Infect 2000;2:1599-607.  Back to cited text no. 5
    
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Sugano N. Biological plaque control: Novel therapeutic approach to periodontal disease. J Oral Sci 2012;54:1-5.  Back to cited text no. 6
    
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Taheri JB, Azimi S, Rafieian N, Zanjani HA. Herbs in dentistry. Int Dent J 2011;61:287-96.  Back to cited text no. 7
    
8.
Chandrashekar BR, Nagarajappa R, Singh R, Thakur R. An in vitro study on the antimicrobial efficacy of ten herbal extracts on primary plaque colonizers. J Young Pharm 2014;6:33-9.  Back to cited text no. 8
    
9.
Chandra Shekar BR, Nagarajappa R, Singh R, Thaku R. Antimicrobial efficacy of the combinations of Acacia nilotica, Murraya koenigii L. Sprengel, Eucalyptus hybrid and Psidium guajava on primary plaque colonizers. J Basic Clin Pharm 2014;5:115-9.  Back to cited text no. 9
    
10.
Chandrashekar BR, Nagarajappa R, Singh R, Thakur R. Antimicrobial efficacy of the combinations of Acacia nilotica, Murraya koenigii L. Sprengel, Eucalyptus hybrid, and Psidium guajava on primary plaque colonizers: An in vitro comparison between hot and cold extraction process. J Indian Soc Periodontol 2015;19:174-9.  Back to cited text no. 10
    
11.
Deshpande SN, Kadam DG. Phytochemical analysis and antibacterial activity of Acacia nilotica against Streptococcus mutans. Int J Pharm Pharm Sci 2013;5:236-8.  Back to cited text no. 11
    
12.
Ravi K, Divyashree P. Psidium guajava: A review on its potential as an adjunct in treating periodontal disease. Pharmacogn Rev 2014;8:96-100.  Back to cited text no. 12
    
13.
Clark DT, Gazi MI, Cox SW, Eley BM, Tinsley GF. The effects of Acacia arabica gum on the in vitro growth and protease activities of periodontopathic bacteria. J Clin Periodontol 1993;20:238-43.  Back to cited text no. 13
    
14.
Dabur R, Gupta A, Mandal TK, Singh DD, Bajpai V, Gurav AM, et al. Antimicrobial activity of some Indian medicinal plants. Afr J Tradit Complement Altern Med 2007;4:313-8.  Back to cited text no. 14
    
15.
Nagata H, Inagaki Y, Yamamoto Y, Maeda K, Kataoka K, Osawa K, et al. Inhibitory effects of macrocarpals on the biological activity of Porphyromonas gingivalis and other periodontopathic bacteria. Oral Microbiol Immunol 2006;21:159-63.  Back to cited text no. 15
    
16.
Takarada K, Kimizuka R, Takahashi N, Honma K, Okuda K, Kato T, et al. Acomparison of the antibacterial efficacies of essential oils against oral pathogens. Oral Microbiol Immunol 2004;19:61-4.  Back to cited text no. 16
    
17.
Vats M, Singh H, Sardana S. Phytochemical screening and antimicrobial activity of roots of Murraya koenigii (Linn.) Spreng. (Rutaceae). Braz J Microbiol 2011;42:1569-73.  Back to cited text no. 17
    
18.
Ramesh G, Nagarajappa R, Madhusudan AS, Sandesh N, Batra M, Sharma A, et al. Estimation of salivary and tongue coating pH on chewing household herbal leaves: A randomized controlled trial. Anc Sci Life 2012;32:69-75.  Back to cited text no. 18
    
19.
Shu Y, Liu Y, Li L, Feng J, Lou B, Zhou X, et al. Antibacterial activity of quercetin on oral infectious pathogens. Afr J Microbiol Res 2011;5:5358-61.  Back to cited text no. 19
    
20.
Geoghegan F, Wong RW, Rabie AB. Inhibitory effect of quercetin on periodontal pathogens in vitro. Phytother Res 2010;24:817-20.  Back to cited text no. 20
    
21.
John NR, Gala VC, Sawant CS. Inhibitory effects of plant extracts on multi-species dental biofilm formation in-vitro. Int J Pharm Bio Sci 2013;4:487-95.  Back to cited text no. 21
    

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Correspondence Address:
Dr. B R Chandra Shekar
Department of Public Health Dentistry, JSS Dental College and Hospital, Jagadguru Sri Shivarathreeshwara University, Mysore, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.IJDR_52_17

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