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Table of Contents
ORIGINAL ARTICLE
Year : 2020  |  Volume : 10  |  Issue : 2  |  Page : 51-55

Evaluation of antibacterial activity of Nigella sativa seed extract against Porphyromonas gingivalis and Prevotella intermedia


1 Department of Periodontology and Oral Implantology, Priyadarshini Dental College and Hospital, Thiruvallur, Tamil Nadu, India
2 Department of Paedodontics and Preventive Dentistry, Thai Mookambigai Dental College and Hospital, Chennai, Tamil Nadu, India
3 Department of Periodontology and Implantology, Indira Gandhi Institute of Dental Sciences, Puducherry, India

Date of Submission23-Nov-2019
Date of Acceptance12-Mar-2020
Date of Web Publication21-Aug-2020

Correspondence Address:
Dr. Paavai Ilango
90/1, Magizhchi Colony, Mogappair, Chennai - 600 037, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jid.jid_62_19

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   Abstract 


Context: Chemical plaque control agents and local drug delivery used in the management of periodontal therapy possess many adverse effects upon long-term usage. Hence, there is a search of herbal antibacterial agents with least adverse effects, which are being experimented to be used as an alternative to chemical agents. Nigella sativa is one such herb known for its antibacterial activity against many microorganisms. In this study, N. sativa is tested for its efficacy against significant periodontal pathogens. Aims: The study aims were to evaluate the antibacterial activity of N. sativa against significant periodontal pathogens, Porphyromonas gingivalis and Prevotella Intermedia, and to compare its efficacy with tetracycline. Materials and Methods: Methanolic extract of N. sativa seeds with three different concentrations of 12.5, 25, and 50 mg/ml was prepared, and the antibacterial activity of N. sativa seeds was tested against bacterial strains of P. gingivalis and P. intermedia in culture plates. Zones of inhibition of bacterial growth were measured in three different concentrations and compared with tetracycline. Statistical Analysis Used: The results were statistically analyzed using one-way analysis of variance, and P < 0.05 was considered statistically significant. Results: Although the results of N. sativa were slightly lesser than those of tetracycline (15.3 mm and 16.5 mm), the methanolic extract of N. sativa showed highly statistically significant (P < 0.001) antibacterial activity with zone of inhibition of 5.4 and 7.4 mm at 25 mg/ml and 9.4 and 10.1 mm at 50 mg/ml concentration against P. gingivalis and P. intermedia, respectively. Conclusions: N. sativa is found to possess antibacterial efficacy against periodontal pathogens such as P. gingivalis and P. intermedia. Hence, it can be used as a safe adjunct in periodontal therapy. Further clinical studies are required to check its efficacy clinically in patients with periodontitis.

Keywords: Antibacterial activity, Nigella sativa, periodontal pathogens, periodontitis, Porphyromonas gingivalis, Prevotella intermedia


How to cite this article:
Senthilnathan K, Ilango P, Abirami T, Vummidi VA, Mahalingam AP, Reddy VK. Evaluation of antibacterial activity of Nigella sativa seed extract against Porphyromonas gingivalis and Prevotella intermedia. J Interdiscip Dentistry 2020;10:51-5

How to cite this URL:
Senthilnathan K, Ilango P, Abirami T, Vummidi VA, Mahalingam AP, Reddy VK. Evaluation of antibacterial activity of Nigella sativa seed extract against Porphyromonas gingivalis and Prevotella intermedia. J Interdiscip Dentistry [serial online] 2020 [cited 2020 Sep 23];10:51-5. Available from: http://www.jidonline.com/text.asp?2020/10/2/51/292920




   Clinical Relevance to Interdisciplinary Dentistry Top


Plaque microbiota and local factors are considered as the major etiology of periodontitis. Eradication of plaque is the major goal of periodontal therapy. Though there are array of chemical plaque control agents, profound side effects were also found. Thus herbal alternatives are current trend in plaque control because of limited adverse effects compared to chemical agents. Nigella sativa extract is currently experimented in this study to test its efficacy against plaque microbiota to further elaborate its usage as prophylactic and adjunctive agent in periodontal therapy


   Introduction Top


Gingivitis and periodontitis are highly prevalent diseases of the oral cavity, while gingivitis is considered to be the initial stage of periodontal disease. Untreated gingivitis may result in more serious and destructive form of periodontal disease.[1],[2] Periodontitis is an inflammatory condition of periodontium, in which the bacterial invasion spreads beyond the gingiva and destructs the periodontal tissue, resulting in attachment loss.[3],[4],[5],[6] Recently, it is identified that periodontitis is in fact a host response mechanism to eradicate dental plaque from gingival sulcus.[7] The main etiology of periodontal disease is colonization of microorganisms, resulting in plaque formation. Although scaling and root planing is the primary treatment which involves removal of local infection resulting in the reduction of inflammation, use of adjuncts such as mouthwashes and local drug delivery along with surgical procedures in periodontal disease yields good results. Chlorhexidine is considered the “gold standard,” but it also possesses disadvantages such as staining of teeth, allergy, and altered taste sensation on long-term use.[8] Many new formulations of herbal mouthwashes and periodontal chips of herbal origin are in clinical trial phase to overcome the disadvantages of synthetic local delivery drugs.

Nigella sativa, commonly known as black cumin seeds, is an annual flowering plant in the family Ranunculaceae, described as “miracle herb of the century,” which is attributed to its beneficial antioxidant, antibacterial, antiepileptic, anti-inflammatory, and antitumor properties.[9],[10] Thymoquinone (TQ) and thymohydroquinone (THQ) are bioactive components of N. sativa, which have enhanced antibiotic action, which, in turn, increases the antibacterial potency.[11],[12] A study on TQ and THQ identified their antibacterial activity against both Gram-positive and Gram-negative bacteria.[13] Formulation of TQ-containing periodontal chip has been used and evaluated in chronic periodontitis patients, which showed significant improvement in mean plaque index, bleeding on probing, periodontal pocket depth, and clinical attachment loss after treatment.[14] Many studies used the active principle of N. sativa (TQ), which works by producing 5-lipoxygenase, while some of the studies have extracted the N. sativa seeds in different methods and tested their antibacterial activity against Gram-positive and few Gram-negative bacteria.[15],[16] Yet, the antibacterial activity of this agent against periodontal pathogens such as Tannerella forsythia, Treponema denticola, Porphyromonas gingivalis, and Prevotella intermedia has not been reported till date. The current study aims to detect the antibacterial effect of N. sativa seed extract against two significant periodontal pathogens, P. gingivalis and P. intermedia, and to compare its efficacy with tetracycline.


   Materials and Methods Top


Preparation of extract

N. sativa seeds were procured and ground into a fine powder using a blender. Methanolic extract of N. sativa seeds was prepared by percolation method as previously described by Salman et al.[17] Briefly, 100 g of N. sativa powder was soaked in 300 ml of methanol for 7 days, and then filtered using Whatman® No. 1 filter paper and evaporated using a rotary evaporation apparatus. The extract was further dried in an hot air oven at 50°C for 24 h and finally kept at 4°C until further testing. One gram of each extract was dissolved separately in 1 ml of 10% dimethyl sulfoxide (DMSO) to give a stock solution of 1000 mg/ml. Three different concentrations of 50, 25, and 12.5 mg/ml were prepared from the stock solution.

Bacterial strains and media

Laboratory strains of P. gingivalis (ATCC 3277) were grown for 3 h in Mueller–Hinton broth at 37°C. Density of the bacterial cell suspension should contain approximately 1 × 108 colony-forming unit (CFU/ml) and diluted with media to contain 105 CFU/ml. To use these microorganisms in the microdilution assay, the appropriate starting concentrations in the assay inoculum were determined from preliminary growth curve studies in the microtiter plates. P. gingivalis strain was grown in tryptic soy broth supplemented with 5-μg/ml hemin and vitamins for 72 h at 37°C in an anaerobic chamber. The bacterial cell suspension was adjusted to 106 CFU/ml. P. gingivalis and P. intermedia ATCC 25611 strains were revived in 5-mL thioglycollate broth and incubated for 8 days at 35°C under anaerobic conditions. Subsequently, they were seeded on blood agar and incubated for 48 h at 35°C under anaerobic conditions. To verify the strain morphology and purity, Gram's staining was performed on the isolated colonies.

Antimicrobial susceptibility testing

The ability of an antimicrobial agent or antibiotics to inhibit the growth of bacteriain vitro is measured by antimicrobial susceptibility test. A volume of 100 μl of bacterial suspension was spread on each nutrient agar plate. Different concentrations of the extract (12.5–50 mg/ml) (25 μl) were then used to impregnate in sterilized 6-mm blank discs. Ten percent DMSO-loaded discs were used as negative controls for the extract. All the impregnated discs were ensured to be fully dried in a 45°C incubator for 18–24 h prior to the application on bacteria. Tetracycline 30 μg was used as positive control for all strains. The discs which had been impregnated with the extract using sterile forceps were applied on the inoculated Muller–Hinton agar once they had completely dried. The discs were pressed gently to ensure uniform contact with the agar surface. Within 15 min of application, the plates were shifted to an anaerobic jar, which was kept in an incubator for 48 h. After incubation was complete, the plates were read only if the lawn of growth was confluent or nearly confluent. The diameter of inhibition zone around the discs was measured for the antibacterial activity. If present, their diameters were measured to the nearest whole millimeter with a ruler. All tests were carried out three times to ensure reliability, and the average of the three replicates for each concentration of extract and positive control was calculated. Percentage inhibition of growth of bacterial microorganism was calculated after subtracting control from the values of inhibition diameter using control as standard.

Statistical analysis

For statistical analysis of data, multiple comparisons were performed using one-way analysis of variance followed by the least significant difference test for post hoc analysis. Statistical significance was accepted at a level of P < 0.05. Data were analyzed using SPSS - Statistical Package of Social Sciences, (SPSS Statistics for Windows, Version 17.0, Chicago).


   Results Top


[Table 1] shows the antibacterial effects of N. sativa against periodontal pathogens such as P. gingivalis and P. intermedia with different concentrations of N. sativa seeds. In 12.5-mg/ml concentration, P. gingivalis did not show inhibition, while against P. intermedia, it showed 1.1 mm. N.sativa at a concentration of 25mg/ml showed a Zone of inhibition of 5.4mm against P.gingivalis and 10.1mm against P.intermedia. Similarly at 50mg/ml it showed a ZOI of 9.6mm for P.gingivalis and 10.1mm for P.intermedia. Inhibition of growth for positive control (tetracycline) was found to be 15.3 mm against P. gingivalis and 16.5 mm against P. intermedia. Ten percent DMSO-loaded discs, which is a negative control, did not show any zone of inhibition. Although the results of N. sativa were slightly lesser than those of tetracycline, it was found to possess highly significant antibacterial activity against P. gingivalis and P. intermedia.
Table 1: Antibacterial activity of NSE

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[Table 2] shows the mean of minimal inhibitory concentration (MIC) and maximum bactericidal concentration (MBC) results. The MIC and MBC for P. gingivalis and P. intermedia were found to be 25 and 50 mg/ml, respectively. [Figure 1] and [Figure 2] depict the culture plates showing the zone of inhibition of P. gingivalis and P. intermedia.
Table 2: Mean minimal inhibitory concentration and maximum bactericidal concentration values

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Figure 1: Porphyromonas gingivalis. Disc diffusion method showing zone of inhibition. (1) Negative control; (2) NSE (12.5 mg/ml); (3) NSE (25 mg/ml); (4) NSE (50 mg/ml); (5) Tetracycline. NSE = Nigella Sativa Extract

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Figure 2: Prevotella intermedia. (1) Negative control; (2) NSE (12.5 mg/ml); (3) NSE (25 mg/ml); (4) NSE (50 mg/ml); (5) tetracycline. NSE = Nigella Sativa Extract

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   Discussion Top


Periodontitis is a multifactorial disease in which microorganisms play a major role. Microorganisms involved in periodontitis are anaerobic bacteria. Chlorhexidine is the commonly used local drug delivery which yields good prognosis, but it is also inherent with many disadvantages such as allergic reactions, staining of teeth, and altered taste sensation.[8] Herbal research is considered a global alternative to overcome these side effects. Among researches on various herbal components, N. sativa is found to possess antibacterial activity against different Gram-positive and Gram-negative organisms. Although numerous studies have been done so far to identify the antibacterial activity of N. sativa,in vitro study against periodontal pathogens is not reported yet. In the presentin vitro study, we have evaluated the antimicrobial activity of N. sativa extract (methanolic extract) against periodontal pathogens such as P. gingivalis and P. intermedia and compared its efficacy with tetracycline. As tetracycline is a broad-spectrum antibiotic and commonly used in nonsurgical periodontal therapy, we compared the antibacterial efficacy of N. sativa with tetracycline.

The present study shows that N. sativa possesses highly significant bactericidal activity against P. gingivalis and P. intermedia, with MIC and MBC of 25 and 50 mg/ml, respectively. Although its zone of inhibition was slightly lesser compared to that of tetracycline (positive control), N. sativa in 50 mg/ml showed a significant zone of inhibition against P. gingivalis and P. intermedia. Hussain et al., in 2011, studied the inhibition of Staphylococcus aureus growth by N. sativa ground seeds from both Hadramout and Ethiopia. Although their inhibition of growth was lesser than that of positive control, results were significant in N. sativa ground seeds from Ethiopia: 20 mm and positive control (azithromycin): 22 mm.[15] Similarly, in our study, inhibition of zone of N. sativa was slightly lesser than positive control (tetracycline), and hence the results were significant against periodontal pathogens.

Several studies have been conducted to identify the antibacterial effect of N. sativa against Gram-positive organism.[16],[17],[18],[19] Morsi investigated the antibacterial effect of N. sativa (methanolic extract) at 100 mg/ml against S. aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, and Bacillus cereus, in which inhibition zone was found to be 9 mm in E. coli and 25 mm in S. aureus.[19]

Baydaa et al. (2012) compared the effect of black seed (N. sativa) oil ethanolic extract at 20% concentration to 0.2% chlorhexidine gluconate against Streptococcus mutans. N. sativa showed higher zone of inhibition than chlorhexidine.[11] Anotherin vitro study in 2009 used the bioactive components of N. sativa, TQ and THQ, to identify their antibacterial activity against E. coli, Salmonella typhimurium, Salmonella enteritidis, Shigella flexneri, P. aeruginosa, and S. aureus and their interaction with other antibiotics. S. aureus was found to be most susceptible with MIC 3 μg/ml to TQ, whereas S. flexneri was more susceptible to TQ and THQ.[13] Al-Bayaty et al. in 2013 used a biodegradable periodontal chip containing TQ, which showed a significant reduction in all the three plaque index, bleeding on probing, and periodontal pocket depth.[14] Although numerous studies have been conducted to identify the antibacterial efficacy of N. sativa against Gram-positive organisms, a study attributing to its antibacterial effect against periodontal pathogens has not been reported yet.

To the best of our knowledge, this study is the first to evaluate the antibacterial activity of N. sativa against periodontal Gram-negative pathogens. This preliminary study showed highly significant antibacterial activity of N. sativa against P. gingivalis and P. intermedia. Despite the fact of our results that N. sativa shows slightly lesser antibacterial effect than positive control, tetracycline can be used only therapeutically in nonsurgical therapy for periodontitis. Whereas N. sativa can be used both prophylactically and therapeutically, as it is easily available and a natural product consumed day to day with no reported side effects. Limitations of the present study include comparison with chlorhexidine as positive control, antibacterial activity at higher concentrations of N. sativa, and testing against other significant periodontal pathogens. Further clinical trials shall pave way for N. sativa, as a natural herbal alternative of chemical plaque control along with periodontal therapy.


   Conclusions Top


N. sativa possesses significant antibacterial activity against P. gingivalis and P. intermedia. It might be used as a safe therapeutic adjunct in periodontal therapy and also a prophylactic alternate for maintaining periodontal health.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Ezzat B, Fares AE. The effect of orally administered curcumin and/or ginger on Escherichia coli induced periodontitis in rats (histopathological, immunohistochemical, and stereomicroscopic study). Egypt Dent J 2014;60:1465-77.  Back to cited text no. 1
    
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Saravanan D, Ramkumar S, Vineetha K. Effect of oil pulling with sesame oil on plaque-induced gingivitis: A microbiological study. J Orofac Res 2013;3:175.  Back to cited text no. 2
    
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Visser MB, Ellen RP. New insights into the emerging role of oral spirochaetes in periodontal disease. Clin Microbiol Infect 2011;17:502-12.  Back to cited text no. 3
    
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Deo V, Bhongade ML. Pathogenesis of periodontitis: Role of cytokines in host response. Dent Today 2010;29:60-2, 64-6.  Back to cited text no. 4
    
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Armitage GC. Periodontal diagnoses and classification of periodontal diseases. Periodontol 2000 2004;34:9-21.  Back to cited text no. 5
    
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Ilango P, Mahalingam A, Parthasarathy H, Katamreddy V, Subbareddy V. Evaluation of TLR2 and 4 in chronic periodontitis. J Clin Diagn Res 2016;10:ZC86-9.  Back to cited text no. 6
    
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Garlet GP. Destructive and protective roles of cytokines in periodontitis: A re-appraisal from host defense and tissue destruction viewpoints. J Dent Res 2010;89:1349-63.  Back to cited text no. 7
    
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Flötra L, Gjermo P, Rölla G, Waerhaug J. Side effects of chlorhexidine mouth washes. Scand J Dent Res 1971;79:119-25.  Back to cited text no. 8
    
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Mendi A. Nigella sativa oil could induce osteogenic differentiation of dental pulp mesenchymal stem cells: Clinical nutrition for dentistry. Food Health 2018;4:19-24.  Back to cited text no. 9
    
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Al-Attass SA, Zahran FM, Turkistany SA. Nigella sativa and its active constituent thymoquinone in oral health. Saudi Med J 2016;37:235-44.  Back to cited text no. 10
    
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Norsharina I, Maznah I, Aied A, Ghanya AN. Thymoquinone rich fraction from Nigella sativa and thymoquinone are cytotoxic towards colon and leukemic carcinoma cell lines. J Med Plants Res 2011;5:3359-66.  Back to cited text no. 11
    
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Abd-Awn B, Al-Dhaher Z, Al-Dafaai R. The effect of black seed oil extracts on mutans streptococci in comparison to chlorhexidine gluconate (in vitro). J Bagh Coll Dent 2012;24:126-31.  Back to cited text no. 12
    
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Halawani E. Antibacterial activity of thymoquinone and thymohydroquinone of Nigella sativa L. and their interaction with some antibiotics. Adv Biol Res 2009;3:148-52.  Back to cited text no. 13
    
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Al-Bayaty FH, Kamaruddin AA, Ismail M, Abdulla MA. Formulation and evaluation of a new biodegradable periodontal chip containing thymoquinone in a chitosan base for the management of chronic periodontitis. J Nanomater 2013;2013:5.  Back to cited text no. 14
    
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Bakathir HA, Abbas NA. Detection of the antibacterial effect of Nigella sativa ground seeds with water. Afr J Tradit Complement Altern Med 2011;8:159-64.  Back to cited text no. 15
    
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Zuridah H, Fairuz AR, Zakri AH, Rahim MN.In vitro antibacterial activity of Nigella sativa against Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli and Bacillus cereus. Asian J Plant Sci 2008;7:331-3.  Back to cited text no. 16
    
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Salman MT, Khan RA, Shukla I. A study of Nigella sativa Linn. Seeds for antimicrobial activity against multidrug resistant clinical strains of Pseudomonas aeruginosa. Hippocratic J Unaru Med 2009;4:95-104.  Back to cited text no. 17
    
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Mashhadan NV, Rakhshandeh H. Antibacterial and antifungal effects of NS extracts against SA, Pseudomonas aeruginosa, and Candida albicans. Pak J Med Sci 2005;21:47-52.  Back to cited text no. 18
    
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Morsi NM. Antimicrobial effect of crude extracts of Nigella sativa on multiple antibiotics-resistant Bacteria. Acta Microbiol Pol 2000;49:63-74.  Back to cited text no. 19
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2]



 

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