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ORIGINAL ARTICLE
Year : 2022  |  Volume : 20  |  Issue : 4  |  Page : 398-402

Antibacterial efficacy of Trachyspermum ammi Oil against Porphyromonas gingivalis, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans: An in vitro study


1 Professor, Department of Public Health Dentistry, Bapuji Dental College and Hospital, Davangere, Karnataka, India
2 Intern, Bapuji Dental College and Hospital, Davangere, Karnataka, India

Date of Submission20-Sep-2021
Date of Decision17-Mar-2022
Date of Acceptance29-Aug-2022
Date of Web Publication19-Dec-2022

Correspondence Address:
Puja C Yavagal
Department of Public Health Dentistry, Bapuji Dental College and Hospital, Affiliated to Rajiv Gandhi University of Health Sciences, Davangere - 577 004, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jiaphd.jiaphd_170_21

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  Abstract 


Background: Ajwain (Trachyspermum ammi) oil is used widely because of its nutritional value, antioxidant and antibacterial activity against various microorganisms, but there are no studies that relate the antibacterial activity of Ajwain oil against periodontal pathogens. Aim: To evaluate and compare the minimum inhibitory concentration (MIC) and zone of inhibition of T. ammi (Ajwain seed) oil and 0.2% chlorhexidine gluconate against Porphyromonas gingivalis, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans through serial tube dilution and disc diffusion methods. Materials and Methods: An in vitro study was conducted. Ajwain oil (100%) and chlorhexidine solution (0.2%) were purchased. Blood agar media with hemin, Vitamin K and Kanamycin, crystal violet erythromycin blood agar, and thioglycolate broth with 1% horse serum were used to culture P. gingivalis, F. nucleatum, and A. actinomycetemcomitans, respectively. The MIC and minimum bactericidal concentration of test agents were assessed using the serial dilution method, and the zone of inhibition was assessed by the Agar disc diffusion method. Results: Ajwain oil had the ability to exert antibacterial activity against P. gingivalis, F. nucleatum, and A. actinomycetemcomitans at a MIC of 0.4, 0.4, and 0.8 μg/ml, respectively. Ajwain oil exerted bactericidal effect against P gingivalis, F. nucleatum, and A. actinomycetemcomitans at a minimum concentration of 0.4, 0.4, and 6.25 μg/ml, respectively, and bacteriostatic effect at a minimum concentration of 0.2, 0.2, and 3.12 μg/ml, respectively. At 50% μg/ml concentration, Ajwain oil exhibited a higher zone of inhibition compared to chlorhexidine against P. gingivalis, F. nucleatum, and A. actinomycetemcomitans. Conclusion: Ajwain oil demonstrated antibacterial effect against periodontal pathogens in vitro.

Keywords: Aggregatibacter actinomycetemcomitans, Ajwain oil, chlorhexidine, Fusobacterium nucleatum, Porphyromonas gingivalis


How to cite this article:
Yavagal PC, Rajeshwar SS. Antibacterial efficacy of Trachyspermum ammi Oil against Porphyromonas gingivalis, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans: An in vitro study. J Indian Assoc Public Health Dent 2022;20:398-402

How to cite this URL:
Yavagal PC, Rajeshwar SS. Antibacterial efficacy of Trachyspermum ammi Oil against Porphyromonas gingivalis, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans: An in vitro study. J Indian Assoc Public Health Dent [serial online] 2022 [cited 2023 Feb 2];20:398-402. Available from: https://www.jiaphd.org/text.asp?2022/20/4/398/364018




  Introduction Top


Although periodontal disease is profoundly preventable, it stays a noteworthy public health problem. Periodontal diseases are inflammatory and destructive diseases of the dentogingival complex associated with specific periodontal pathogens inhabiting periodontal pockets. Although it is considered that the disease has multifactorial etiology, some specific Gram-negative microorganisms in the subgingival plaque biofilm play a major role in the initiation and progression of periodontitis and other microorganisms have been implicated as predominant species in the disease process.[1] The “red complex” organism Porphyromonas gingivalis, is a Gram-negative, nonmotile, saccharolytic rod-like obligate anaerobe, related to the initiation and progression of the periodontal disease.[1] It is strongly associated with severe chronic periodontitis. Other microorganisms that have been implicated as predominant species in the periodontal disease process are Aggregatibacter actinomycetemcomitans, a nonmotile, Gram-negative rod, which is strongly associated with destructive periodontal lesions and Fusobacterium nucleatum, a Gram-negative bacilli, obligate anaerobe associated commonly with refractory periodontitis.[1]

The main periodontal disease treatment target remains the effective reduction of the pathogenic microorganisms in plaque biofilm, mostly by mechanical means augmented by chemicals, such as antibiotics and antiseptics. Anitmicrobials are known to cause antimicrobial resistance and the emergence of uncommon infections probably due to inappropriate or widespread overuse.[2] This has spurred scientists on the research for plant-based antimicrobial agents. Herbal medicine is the major stay of about 75%–80% of the world population, mainly in developing countries, for primary health care because of better cultural acceptability, better compatibility, and few side effects. India has well-recorded and well-practiced knowledge of traditional herbal medicine. Despite tremendous development of allopathic medicine, they are found to have some side effects. The World Health Organization encourages, recommends, and promotes traditional/herbal remedies in national health-care programs because the plant-based therapeutics are natural products, nonnarcotic, easily bio-degradable, pose minimum environmental hazards, have less adverse effects which are easily available and affordable too.[3]

Trachyspermum ammi, or Ajwain is a commonly found herb in India, seeds of which are used as a spice. It has therapeutic activity against multiple gastrointestinal ailments and bronchial problems. The oil extracted from seeds of T. ammi from the family Apiaceae has shown considerable antibacterial, antiviral, antifungal antitussive, anti-inflammatory, and analgesic effects, as well as antioxidant, and antitumor activities.[4] Despite its widespread use, in many areas of Allopathic and Ayurvedic medicine, its exact mode of action in the field of dentistry is less explored. Recently, an in vitro study revealed the antimicrobial property of Ajwain oil against caries-causing micro-organisms (Streptococcus mutans, Streptococcus oralis, Lactobacillus acidophilus, Lactobacillus fermentum), and Candida albicans.[5] The antimicrobial effect of Ajwain is largely attributed to high concentrations of polyphenols present in the seeds. Another study pointed out the inhibitory effects of Ajwain essential oil against Escherichia coli (E. coli), Klebsiella, and Staphylococcus aureus, which are considered endodontic pathogens.[6] Literature search revealed no study evaluating the antibacterial property of Ajwain essential oil against periodontal pathogens. Hence, an in vitro study was planned to evaluate the antibacterial efficacy of Ajwain seed oil against P. gingivalis, F. nucleatum, and A. actinomycetemcomitans. The study tested the null hypothesis that there was no antimicrobial effect of Ajwain seed oil against P. gingivalis (ATCC-33277), F. nucleatum, and A. actionomycetemcomitans (ATCC 29523).


  Materials and Methods Top


The objective of this study was to evaluate and compare the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and zone of inhibition of 100% T. ammi (Ajwain seed) oil and 0.2% chlorhexidine gluconate against P. Gingivalis, F. nucleatum and A. actinomycetemcomitans through serial tube dilution and disc diffusion methods.

Agents tested for antimicrobial property used:

  • T. ammi (Ajwain oil), 100% natural, Steam distilled by Deve Herbes, New Delhi, India. Product Code-FASDEVE-HERBES GREE868051BBE7EAD3
  • Chlorhexidine gluconate 0.2%. (Hexidine Mouth wash, ICPA Health Products Ltd., India).


Cultivation of microorganisms

Stock cultures of periodontal pathogens (P. gingivalis, A. actinomycetemcomitans, and F. nucleatum) used in this study were obtained from the Department of Microbiology, Maratha Mandal Dental College, Belgaum. Blood agar media with hemin, Vitamin K, and Kanamycin was used to culture P. gingivalis, and crystal violet erythromycin blood agar was used to culture F. nucleatum, and thiyoglycollate broth with 1% horse serum was used to culture A. actinomycetemcomitans

Minimum inhibitory concentration procedure

Nine dilutions of each interventional solution were done with thioglycolate broth to check MIC. In the initial tube, 20 μl of test solution was added to the 380 μl of thioglycolate broth. For dilutions, 200 μl of thioglycolate broth was added into the next nine tubes separately. Then from the initial tube, 200 μl was transferred to the first tube containing 200 μls of thioglycolate broth. This was considered 10−1 dilution. From 10−1 diluted tube, 200 μl was transferred to the second tube to make 10−2 dilution. The serial dilution was repeated up to 10−9 dilution for each interventional agent. From the maintained stock cultures of required organisms, 5 μl was taken and added to 2 ml of thioglycolate broth. In each serially diluted tube, 200 μl of the above culture suspension was added. By following this serial dilution, the concentrations of the test agents achieved were as follows: 100, 50, 25, 12.5, 6.5, 3.12, 1.6, 0.8, 0.4, and 0.2 μL/ml. The tubes were incubated for 48–72 h in the anaerobic jar at 37°C. After the incubation, the MIC values were determined by visual inspection of the tubes. In each series of tubes, the last tube with clear supernatant was considered without any growth and taken as MIC value. Turbidity in the MIC tube indicated growth of the bacteria implying that the bacteria were resistant to the test agent [Figure 1], [Figure 2], [Figure 3].
Figure 1: MIC testing against P. Gingivalis. MIC: Minimum inhibitory concentration, P. gingivalis: Porphyromonas gingivalis

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Figure 2: MIC testing against F. Nucleatum. MIC: Minimum inhibitory concentration, F. nucleatum: Fusobacterium nucleatum

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Figure 3: MIC testing against A actinomycetemcomitans. MIC: Minimum inhibitory concentration, A. actinomycetemcomitans: Aggregatibacter actinomycetemcomitans

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Minimum bactericidal concentration test

From the minimum inhibitory dilution tubes, first three or five tubes were plated (which was sensitive in MIC) and incubated for 24 h and assessed for colony counts. MBC was done to assess the bacteriostatic or bactericidal effect of the test agent against the organism. The absence and presence of growth indicated bactericidal and bacteriostatic effects, respectively. For facultative anaerobes, tubes were incubated at 37°C for 48–72 h in carbon dioxide jar. For strict anaerobes, tubes were incubated in anaerobic jars for 48–72 h. The organisms grown from the control tube were then compared with the organism grown from the MIC test tubes. All the microbiological examinations were performed by a microbiologist who was not involved in the study and who was blinded to the testing agents. The agents T. ammi oil and chlorhexidine bottle labels were masked, covered and coded before sending for microbiological analysis. The statistician was also blinded to test agents.

The test was read as follows:

  1. Similar number of colonies – indicating bacteriostatic activity only
  2. Reduced number of the colonies – indicating a partial or slow bactericidal activity
  3. No growth – if the whole inoculum has been killed.


Agar diffusion procedure

Inoculum preparations

The bacterial colonies were transferred from the plates to the brain heart infusion (BHI) Broth with a sterilized straight nichrome wire. The turbidity was visually adjusted with BHI broth to equal that of a 0.5 MacFarland unit turbidity standard that was freshly prepared. Alternatively, the suspension was standardized with a photometric device.

Inoculation of the agar plate

After adjusting the inoculum to a 0.5 MacFarland unit turbidity standard, a sterile cotton swab was dipped into the inoculum and rotated against the wall of the tube above the liquid to remove excess inoculum. The entire surface of blood agar plate was swabbed three times, rotating plates approximately 60° between streaking to ensure even distribution. The inoculated plate was allowed to stand for at least 3 min but no longer than 15 min before punching the wells in the agar plate. A hollow tube of 5 mm diameter was taken and heated. It was pressed on the inoculated agar plate and removed immediately after making a well in the plate. Similarly, three wells were made on each plate. Ajwain oil and chlorhexidine were added into the respective wells on each plate. The plates were incubated within 15 min of compound application for 18–24 h at 37°C anaerobically. The plates were read only if the lawn of growth was confluent or nearly confluent. The diameter of the inhibition zone (IZD) was measured to the nearest whole millimeter by holding the calipers [Figure 4].
Figure 4: Disc diffusion test. P. gingivalis: Porphyromonas gingivalis, F. nucleatum: Fusobacterium nucleatum A. actinomycetemcomitans: Aggregatibacter actinomycetemcomitans

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  Results Top


The serial dilutions of 100% ajwain oil and 0.2% chlorhexidine solution with turbidity response to P. Gingivalis, F. nucleatum and A. actinomycetemcomitans is summarized in [Table 1]. Ajwain oil had the ability to exert antibacterial activity against P. gingivalis, F. nucleatum and A. actinomycetemcomitans at a MIC of 0.4, 0.4, and 0.8 μg/ml, respectively. Chlorhexidine had the ability to exert antibacterial activity against P. gingivalis, F. nucleatum, and A. actinomycetemcomitans at a MIC of 0.2, 0.4, and 0.2 μg/ml, respectively.
Table 1: Results of minimum inhibitory concentration test

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Results of the MBC test indicated that Ajwain oil exerted bactericidal effect against P. gingivalis, F. Nucleatum, and A. actinomycetemcomitans at a minimum concentration of 0.4, 0.4, and 6.25 μg/ml, respectively, and bacteriostatic effect against P gingivalis, F. Nucleatum and A actinomycetemcomitans at a minimum concentration of 0.2, 0.2 and 3.12 μg/ml, respectively. Chlorhexidine exerted bactericidal effect against P. gingivalis at all concentrations tested and at a minimum concentration of 0.4 μg/ml against F. Nucleatum and A. actinomycetemcomitans. At 0.2 μg/ml, it exhibited bacteriostatic effect against F. Nucleatum and A. actinomycetemcomitans [Table 2]. Results of the Disc diffusion test indicate that, at 50% μg/ml concentration of test agents Ajwain oil exhibited a higher zone of inhibition compared to Chlorhexidine against P. gingivalis, F. nucleatum and A. actinomycetemcomitans [Table 3] and [Figure 4].
Table 2: Results of minimum bactericidal concentration test

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Table 3: Results of disc diffusion test

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  Discussion Top


Even though periodontitis is thought to have a multifactorial etiology, some specific Gram-negative microorganisms in the subgingival plaque biofilm play a key role in the disease's initiation and progression.[1] The main treatment goal of the periodontal disease remains the effective reduction of pathogenic microorganisms in plaque biofilm, which is accomplished primarily through mechanical means and chemicals such as antibiotics and antiseptics. Chlorhexidine, listerine, and cetylpyridinium chloride are the most widely used supplementary chemotherapeutic drugs for periodontitis.[5] However, there have been reports of side effects associated with these drugs such as tooth and restoration discoloration, burning sensation of the oral mucosa, taste alterations, mucosal irritation, parotid enlargement, and bacterial drug resistance. In this regard, herbal medicine could serve as a safe and economical alternative to combat periodontal diseases. T. ammi is one such herb that has long been used in Ayurveda for general health and well-being. It is a widely used spice in India which belongs to the Apiaceae family and is also known as Ajowan or Bishop's Weed. It is utilized as a primary source of thymol. The oil extracted from seeds of T. ammi has shown considerable antibacterial, antioxidant, antiviral, antifungal, antitussive, and anti-inflammatory properties.[7] Hence, it was planned to evaluate the antibacterial efficacy of Ajwain seed oil against P. gingivalis, F. nucleatum, and A. actinomycetemcomitans, which are implicated as predominant organisms causing periodontal disease.[1] The serial dilution method and Agar diffusion tests were used to test the MIC and zone of inhibition, respectively which are considered standard methods to assess the in vitro antimicrobial property of a drug.[8]

Results of the study indicated that Ajwain oil had the ability to exert antibacterial activity against P. gingivalis, F. nucleatum and A. actinomycetemcomitans at a MIC of 0.4, 0.4 and 0.8 μg/ml respectively. At 50% μg/ml concentration, Ajwain oil exhibited a higher zone of inhibition compared to Chlorhexidine against P. gingivalis, F. nucleatum and A. actinomycetemcomitans. On the literature search, the authors could not find studies which have tested the antibacterial activity of Ajwain oil against periodontal pathogens; hence, a comparison of study results with similar studies could not be made. However, an in-vitro study result by Mood et al. exhibited potent antibacterial activities of Ajwain essential oil (AEO) against Klebsiella, E. coli and S. aureus.[6] A study by Omidpanah et al. showed potent antibacterial properties of T ammi oil against S. aureus, E. coli, Pseudomonas aeruginosa, and Enterococcus faecalis. Thymol, p-cymene, and γ-terpinene were the major active components of the oil. All the tested bacteria were sufficiently suppressed by the oil with IZD diameters ranging from 26.6 to 50.3 mm with MIC of <0.02 μL/ml. Thymol inhibited the growth of the bacteria at a concentration of 100 mg/mL.[9] An in-vitro study by Dadpe et al., determined the antibacterial efficacy of T. ammi oil against five oral S. mutans, S. oralis, L. acidophilus, L. fermentum, and C. albicans. Results revealed that T. ammi oil moderately inhibited bacterial growth with mean MIC s of 250, 125, 250, 125, and 250 μg/ml, respectively. The MIC and minimum bactericidal values were higher as compared to chlorhexidine gluconate, and it was statistically significant.[5] Antibacterial activity of T ammi oil could be due to the hydrophobicity nature of oil components, which enable them to partition lipids of the bacterial cell membrane and mitochondria. While partitioning increases permeability from bacterial cells that could result in leakage of ions and other cell contents with or without loss of cell viability, depending on the exit ratio of critical molecules and ions.[10],[11] Strongest antibacterial activity of essential oil is correlated with a high percentage of phenolic compounds such as carvacrol, eugenol, and thymol.[10] Evidence suggests that phenolic chemicals disrupt cytoplasmic membranes, electron flow, active transport, proton motive force, and cause the contents of bacterial cells to coagulate.[12] The limitation of the present study was that the Ajwain oil tested was not freshly prepared but was the commercial product, which claimed to be 100% pure organic Ajwain oil. Hence, the study results should be considered with caution. The efficacy of Ajwain oil needs to be tested on biofilm models. If the antibacterial property is established, it should be further tested by conducting in-vivo studies.


  Conclusion Top


T. ammi oil (ajwain oil) demonstrated antibacterial effect against periodontal pathogens, namely P gingivalis, F nucleatum, and A. actinomycetemcomitans in vitro, which was comparable to the antibacterial activity of chlorhexidine.

Financial support and sponsorship

Trial is funded by Rajiv Gandhi University of Health Sciences, Bangalore, Karnataka 560041, India under Student short-term research grant for the year 2020, Reg No: UG20DEN216.

Conflicts of interest

There are no conflicts of interest.



 
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Popova C, Dosseva-Panova V, Panov V. Microbiology of periodontal diseases. A review. Biotechnol Biotechnol Equip 2013;27:3754-9.  Back to cited text no. 1
    
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Slots J. Low-cost periodontal therapy. Periodontol 2000 2012;60:110-37.  Back to cited text no. 2
    
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Sandhya S, Sudhakar K, Banji D, Rao KN. Pharmacognostical standardization of Borassus Flabellifer root. Ann Biol Res 2010;1:85-94.  Back to cited text no. 3
    
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Moein MR, Zomorodian K, Pakshir K, Yavari F, Motamedi M, Zarshenas MM. Trachyspermum ammi (L.) Sprague: Chemical composition of essential oil and antimicrobial activities of respective fractions. J Evid Based Complementary Altern Med 2015;20:50-6.  Back to cited text no. 4
    
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Dadpe MV, Dhore SV, Dahake PT, Kale YJ, Kendre SB, Siddiqui AG. Evaluation of antimicrobial efficacy of Trachyspermum ammi (Ajwain) oil and chlorhexidine against oral bacteria: An in vitro study. J Indian Soc Pedod Prev Dent 2018;36:357-63.  Back to cited text no. 5
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6.
Mood BS, Shafaghat M, Metanat M, Saeidi S, Sepehri N. The inhibitory effect of ajowan essential oil on bacterial growth. Int J Infect 2014;1:e19394.  Back to cited text no. 6
    
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Khan R, Zakir M, Khanam Z, Shakil S, Khan AU. Novel compound from Trachyspermum ammi (Ajowan caraway) seeds with antibiofilm and antiadherence activities against Streptococcus mutans: A potential chemotherapeutic agent against dental caries. J Appl Microbiol 2010;109:2151-9.  Back to cited text no. 7
    
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Balouiri M, Sadiki M, Ibnsouda SK. Methods for in vitro evaluating antimicrobial activity: A review. J Pharm Anal 2016;6:71-9.  Back to cited text no. 8
    
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Omidpanah S, Vazirian M, Hosseinkhani F, Hadjiakhondi A, Pirali M, Hamedani AM. Antibacterial activity of essential oil of Trachyspermum ammi (L.) Sprague ex Turrill against isolated and standard bacteria. Am J Essent Oils Nat Prod 2016;4:5-11.  Back to cited text no. 9
    
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Lambert RJ, Skandamis PN, Coote PJ, Nychas GJ. A study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol. J Appl Microbiol 2001;91:453-62.  Back to cited text no. 10
    
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Naz S, Arshad Javaid NA, Shoaib A. Antibacterial activity of essential oils of Trachyspermum ammi (L.) Sprague and Ocimum basilicum L. Against Acidovorax sp. Int J Biol Biotechnol 2014;11:671-5.  Back to cited text no. 11
    
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Sikkema J, de Bont JA, Poolman B. Mechanisms of membrane toxicity of hydrocarbons. Microbiol Rev 1995;59:201-22.  Back to cited text no. 12
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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