|Year : 2020 | Volume
| Issue : 2 | Page : 145-151
In vitro evaluation of antimicrobial effect of Myristica fragrans on common endodontic pathogens
Jyothsna Vittoba Setty1, Ila Srinivasan1, Roopashree Teeka Sathiesh2, Mamata Kale3, Vidyullatha Vittoba Shetty4, Salgundi Venkatesh5
1 Department of Pediatric and Preventive Dentistry, M. R. Ambedkar Dental College and Hospital, Bengaluru, Karnataka, India
2 Department of Pharmacognosy, Government College of Pharmacy, B. R. Ambedkar Medical College and Hospital, Bengaluru, Karnataka, India
3 Department of Microbiology, B. R. Ambedkar Medical College and Hospital, Bengaluru, Karnataka, India
4 Department of Forensic Medicine, Oxford Medical College, Bengaluru, Karnataka, India
5 Department of Drugs Control, Government of Karnataka, Bengaluru, Karnataka, India
|Date of Submission||08-May-2020|
|Date of Acceptance||20-Jun-2020|
|Date of Web Publication||28-Jun-2020|
Dr. Jyothsna Vittoba Setty
Department of Pediatric and Preventive Dentistry, M. R. Ambedkar Dental College and Hospital, Bengaluru - 560 005, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Complete elimination of microorganisms from the root canals is the important key for the successful endodontic treatment. Constant emergence of resistant strains and adverse effects of synthetic drugs has led to the search of effective herbal alternatives. Nutmeg (Myristica fragrans) is one such spice used for its various medicinal activities. Aims: To evaluate the antimicrobial effect of M. fragrans on common endodontic pathogens of primary tooth. Materials and Methods: Essential oil of nutmeg was extracted by hydrodistillation method, and its phytoconstituents were determined by thin-layer chromatography (TLC), high-performance TLC, and gas chromatography–mass spectrometry analysis. Minimum inhibitory concentration of essential oil against standard strains of common endodontic pathogens (Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Streptococcus mutans, Candida albicans, Lactobacillus casei, Actinomyces viscosus, Prevotella intermedia, and Porphyromonas gingivalis) was determined by serial tube dilution method. Results: Essential oil of M. fragrans was effective against all tested endodontic microorganisms. Discussion: The active components of essential oil of nutmeg such as myristicin, myristic acid, trimyristin, elemicin, and safrole have good antimicrobial activity and are effective against endodontic microorganisms. Conclusion: M. fragrans can be used as an effective medicament in the treatment of endodontic infections.
Keywords: Endodontic infections, minimum inhibitory concentration, Myristica fragrans, nutmeg
|How to cite this article:|
Setty JV, Srinivasan I, Sathiesh RT, Kale M, Shetty VV, Venkatesh S. In vitro evaluation of antimicrobial effect of Myristica fragrans on common endodontic pathogens. J Indian Soc Pedod Prev Dent 2020;38:145-51
|How to cite this URL:|
Setty JV, Srinivasan I, Sathiesh RT, Kale M, Shetty VV, Venkatesh S. In vitro evaluation of antimicrobial effect of Myristica fragrans on common endodontic pathogens. J Indian Soc Pedod Prev Dent [serial online] 2020 [cited 2020 Jul 13];38:145-51. Available from: http://www.jisppd.com/text.asp?2020/38/2/145/288220
| Introduction|| |
The success of an endodontic treatment depends on many factors, with the reduction or elimination of infection being the most important one. Studies have shown that some species of microorganisms found in root canals are resistant to routine therapy and cause persistent infections. Besides, the infections of primary teeth have extreme deleterious effect on developing permanent teeth. Microflora of teeth with persistence disease showed a high prevalence of Enterococci, Streptococci, and Candida,, which are capable of invading dentinal tubules. Staphylococcus aureus are capable of surviving for extended periods because of resistance to drying and temperature changes. Initial stages of irreversible pulpitis have shown the presence of organisms such as Enterococcus faecalis, Escherichia More Details coli, Streptococcus mutans, S. aureus, anaerobes, and Candida albicans, which were said to be found in persistent lesions. Bacteria of deep carious lesions,, namely, S. mutans, Actinomyces viscosus, and Lactobacillus casei, and bacteria of infected root canals,, namely, Porphyromonas gingivalis, Prevotella intermedia, and E. faecalis, are common causes of endodontic infections. The constant increase in antibiotic-resistant strains and side effects caused by synthetic drugs has prompted researchers to look for herbal alternatives. Studies have acknowledged the pharmacological actions of medicinal plants as a potential source of bioactive compounds. One such nature's wonder is nutmeg (Myristica fragrans). It is used as a spice in various dishes, as components of tea and soft drinks or mixed in milk and alcohol. The most important part of the plant in terms of its pharmacological activity and also in commerce is of course the dried kernel (seed), the nutmeg. In traditional medicine, nutmeg is sometimes used as a stomachic, stimulant, carminative as well as for intestinal catarrh and colic, to stimulate appetite, to control flatulence, and it has a reputation as an emmenagogue and abortifacient. Mace is widely used as a flavoring agent, hair dye, and folk medicine. M. fragrans also possess antipapillomagenic, anticarcinogenic, anti- inflammatory activity, antioxidant, antidiabetic, hepatoprotective and excellent antibacterial properties.
M. fragrans (both nutmeg and mace) is known to exhibit strong antimicrobial activity against animal and plant pathogens, food poisoning, and spoilage bacteria including Bacillus subtilis, E. coli, Saccharomyces cerevisiae, multidrug-resistant Salmonella More Details typhi, and Helicobacter pylori (Orabi et al., 1991; De et al., 1999; Dorman and Deans, 2000; Rani and Khullar, 2004; Mahady et al., 2005; and O'Mahony et al., 2005).M. fragrans extracts have shown to be very effective against oral microorganisms such as S. mutans. Vinothkumar et al. found M. fragrans extract to be effective against E. faecalis and C. albicans when used as an endodontic irrigant. Considering these beneficial effects, especially anti-inflammatory and antibacterial activities, the present study was intended to investigate the effect of M. fragrans against standard strains of endodontic pathogens in primary teeth.
| Materials and Methods|| |
Extraction of essential oil of Myristica fragrans
The essential oil of M. fragrans was extracted by Clavenger's method, of hydrodistillation. Nutmeg which is the kernel of M. fragrans was grounded into fine powder. The 500 ml distillation flask contained distilled water about 2/3 of its volume and 50 g of the powder. The operation proceeded by heating the flask using heating mantle. Heat was applied to the flask and the volatile oil was carried with the steam to a cold condenser. The lighter oil rises to the top of the separator. This process was carried out for about 12 h until the oil inside the water runs out and stops collecting. The volume of essential oils was determined from a calibrated trap. The essential oil collected was dried over anhydrous sodium sulfate, weighed, and stored in a sealed dark-colored vial at 4°C.
Phytochemical analysis of essential oil of nutmeg
Phytochemical analysis was done for the detection of the presence of different phytoconstituents in the essential oil of nutmeg using thin-layer chromatography (TLC), high-performance TLC (HPTLC), and gas chromatography and mass spectrometry (GC-MS) analysis.
Thin-layer chromatography and high-performance thin-layer chromatography fingerprint profile of nutmeg oil
TLC is a simple, quick, and inexpensive procedure that gives the chemist a quick answer as to how many components are there in a mixture. The TLC profile of essential oil of nutmeg, samples S1(high concentration) and S2(low concentration, 50%), was carried out on HPTLC silica gel 60 plates (5 cm × 10 cm dimensions) with toluene: ethyl acetate (9:1) as a mobile phase. After spraying with anisaldehyde–sulfuric acid reagent, major spots at value 0.32, 0.40, 0.55, 0.59, 0.63, 0.72, and 0.90 at 366 nm were seen.,,,,
HPTLC fingerprint profile was carried out of nutmeg oil in the instrument (CAMAG Linomat 5 “Linomat5_150710” S/N 150710 [1.00.12]). HPTLC fingerprint profile was determined to assure identity of the drug. For HPTLC, 7 μl of this nutmeg oil solution was applied to the chromatogram. Chromatography was performed on (5 × 10) aluminum-packed silica gel 60F254 HPTLC plate (Merck, Darmstadt, Germany). Before use, the plates were dried in an oven at 105°C for 5 min and samples were applied as 9 mm band by means of sample applicator (Camag linomat-5, Switzerland) equipped with a 100 μl microsyringe. The developing solvent was allowed to ascend to 90 mm with toluene: ethyl acetate (9:1) (V/V) as a mobile phase in a Twin Trough Chamber, previously saturated for 20 min by lining with thick Whatman filter paper at room temperature (27°C) and relative humidity of 37%.
After development of chromatogram, the plates were removed and completely dried in air at room temperature. Then, the spots produced were observed before and after derivatization with vanillin–sulfuric acid reagent. The images were documented by means of photodocumentation system (Camag Reprostar 3). The distance of each spot from the point of its application was measured and recorded. Rf value is calculated by dividing the distance travelled by the spots and the distance travelled by the front of the solvent system.,
Gas chromatography–mass spectroscopy analysis
Gas chromatography–mass spectroscopy, a hyphenated system, is a very compatible and the most commonly used technique for identification and quantification purpose. The unknown organic compounds in a complex mixture can be determined by interpretation and also by matching the spectra with reference spectra.
GC-MS analysis was carried out at Auriga Research Private Limited, Bengaluru, with the following protocols:
Preparation of test solution: 100 μl of essential oil of nutmeg was pipetted into a fresh vial containing anhydrous sodium sulfate. 1 ml of chloroform was added and mixed to make the stock solution. 500 μl of this was added to 500 μl of chloroform and mixed thoroughly and this solution was used for analysis.
GC-MS was carried out with Shimadzu GC MS-QP2010S equipped with a RTX-5MS column (inner diameter 0:25mm, length 30m, and the film thickness of 0.25 μm). The oven temperature was maintained at isothermal (80°C) for 2 min and increased to 250°C (5°C/min) and was maintained for 20 min. Injector and detector temperature at 290°C with helium carrier gas with the flow rate of 80 ml/min was used.
Evaluation of antimicrobial activity
Endodontic microorganisms included in the study were:
- Bacteria of deep carious lesions,, namely, S. mutans (MTCC497), A. viscosus (ATCC 10048), and L. casei (ATCC 334)
- Bacteria of infected root canals,, namely, P. gingivalis (ATCC 33277), P. intermedia (ATCC 25611), and E. faecalis (ATCC 29212)
- c. Microorganisms from persistent infection of primary teeth pulp, namely, E. faecalis (ATCC 33277), E. coli (25922), S. mutans (MTCC 497/ATCC 35668), S. aureus (ATCC 25923), and anaerobes – C. albicans (ATCC 90028).
Bacterial strain maintenance
The respective bacterial strains from the stock were revived. After overnight incubation at 37°C, isolated colonies were selected and the identities of the organisms were confirmed. Isolated colonies were transferred to sterile MHA (Mueller–Hinton agar) broth and once again incubated overnight. The growth concentration was adjusted to 5 × 105 organisms/ml using 0.5 McFarland's turbidity standard.,
Determination of minimum inhibitory concentration
The procedure was carried out for all the test organisms in triplicate. Stock solution of the test agent (essential oil of nutmeg) was made up in DMSO (dimethyl sulfoxide; Merck, Germany) to ensure complete solubilization. 200 μL of the MHA broth was added in each of ten MIC tubes per bacterial strain. In the first MIC tube containing 200 μL broth, 200 μL of stock was added. After mixing well, 200 μL was transferred to the second MIC tube. This was continued till the last (10th) tube. From the last tube, 200 μL of the final solution was discarded. By following this serial dilution, the concentrations of the aqueous extract achieved were the following: 500, 250,125, 62.5, 31.25, 16, 8, 4, 2, and 1 μg/ml.
To each of the ten such prepared MIC tubes with varying concentrations, 200 μL of the earlier prepared strain of organism was added such that the final volume per tube was 400 μL. For S. mutans, Muller broth with lysed horse blood was used and for C. albicans, RPMI media was used. The tubes were then incubated for 24 h 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 to be the one without any growth and taken as MIC value. Turbidity in the MIC tube indicated growth of the bacteria, implying that the bacteria are resistant to M. fragrans essential oil. Tests were done in triplicate and the data were tabulated.
| Results|| |
TLC showed major spots at value 0.32, 0.40, 0.55, 0.59, 0.63, 0.72, and 0.90 at 366 nm. HPTLC of nutmeg oil at lower concentration showed spots at Rf value of 0.52, 0.64, and 0.91, whereas at higher concentration showed spots at Rf value of 0.32, 0.40, 0.52, 0.59, 0.63, 0.72, and 0.90 [Figure 1] and [Figure 2].
|Figure 2: High-performance thin-layer chromatography fingerprint profile of Myristica fragrans|
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GC-MS analysis showed about 20 different chemical constituents [Table 1] and [Figure 3].
|Table 1: Constituents of essential oil of nutmeg (gas chromatography-mass spectrometry analysis)|
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|Figure 3: Phytochemical compounds of Essential oil of Nutmeg using gas chromatography–mass spectroscopy analysis|
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M. fragrans has shown to be effective against all the tested microorganism: E. coli, S. aureus, E. faecalis, S. mutans, C. albicans, L. casei, Actinomyces viscosus, P. intermedia, and P. gingivalis. It is highly effective against C. albicans, as no growth was seen even at 1 μg/ml concentration [Table 2].
|Table 2: Minimum inhibitory concentration of Myristica fragrans on endodontic pathogens|
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| Discussion|| |
For quality control of herbal products, TLC is the most versatile technique for the identification of botanical raw materials. HPTLC of essential oil of nutmeg at lower concentration showed 3 spots at Rf value of 0.52, 0.64, and 0.91, whereas at higher concentration showed 7 spots at Rf value of 0.32, 0.40, 0.52, 0.59, 0.63, 0.72, and 0.90 when the mobile phase of toluene: ethyl acetate (9:1) was used.,
Rf value of 0.9 indicates the presence of myristicin in the sample which is in accordance with the study by Al Jumaily EF, where a similar solvent system is used. Further Rf values cherry red, red, brown, violet, brick red, brown, and red spots seen in visible light are similar to those presented by Tripathi and Dwivedi and probably confirm the presence of myristicin, elemicin, eugenol, safrole, and hesperidin as explained by Sanghai et al.
GC-MS analysis showed about 20 constituents which are similar to those identified in other previous studies.,, The key component comprised monoterpenes (sabinene and α–pinene), terpene alcohol (terpinene-4-ol), and phenylpropenes (elemicin and safrole) and had important aromatic compounds such as myristicin, elemicin, safrole, and isoeugenol. Thus, in general, there are three constituent groups in the volatile components of nutmeg oil: hydrocarbons, oxygenated hydrocarbons, and aromatic hydrocarbons. Hydrocarbons mostly are of terpene compound; oxygenated hydrocarbons are composed mainly of compounds that contain alcohol, ketone, or ester, whereas the aromatic compounds, which are responsible for distinctive aroma of nutmeg, consist of aromatic ether compounds.,,,,,,
M. fragrans showed excellent antimicrobial effect against endodontic microorganism of primary tooth root canal.
M. fragrans (nutmeg and mace) is known to exhibit strong antimicrobial activity against animal and plant pathogens, food poisoning, and spoilage bacteria including Bacillus subtilis, E. coli, Saccharomyces cerevisiae, multidrug-resistant Salmonella typhi, and Helicobacter pylori (Orabi et al.,1991; De et al., 1999; Dorman and Deans, 2000; Rani and Khullar, 2004; Mahady et al., 2005; O'Mahony et al., 2005).,M. fragrans extracts have shown to be very effective against oral microorganisms such as S. mutans. 13,28 It selectively suppressed P. gingivalis growth and is a potent natural antibiofilm agent against oral primary colonizers Streptococcus sanguinis and A. viscosus.
The compounds present in M. fragrans essential oil such as myristicin, trimyristin, and myristic acid showed a different spectrum of activity against different microorganisms. Groups such as -COOH, -COOR, -NH2, or-SH have been suggested to be the functional groups responsible for antibacterial activity., Therefore, the presence of such groups in myristic acid and trimyristin also suggests that these compounds may act in a similar way, which may be responsible for their antibacterial activity. Studies have shown that the presence of an unsaturated side chain on the aromatic ring is responsible for antimicrobial activity. The presence of an unsaturated side chain on the aromatic ring of myristicin may be responsible for its antibacterial potential. The possible target of the compounds such as organic acids, esters, and aromatic compounds is the cell wall of the bacterium. This suggests that the target of trimyristin, myristic acid, and myristicin may be the cell wall of these bacteria. Macelignan of nutmeg is proven to be anticaries as it is very effective against S. mutans. Shinohara et al. determined that malabaricone C isolated from nutmeg irreversibly inhibited Arg-gingipain by 50% at a concentration of 0.7 μg/ml and selectively suppressed P. gingivalis growth., Anin vitro study by Yanti et al. determined that at 24 h of biofilm growth, S. mutans, A. viscosus, and S sanguis biofilms were reduced by up to 30%, 30%, and 38%, respectively, after treatment with μg/mL macelignan isolated from nutmeg for 5 min. Shafiei et al. showed a significant decrease in the bacterial concentration of Aggregatibacter actinomycetemcomitans and P. gingivalis with the ethyl acetate and ethanol extracts of flesh, seed, and mace of M. fragrans as determined by minimum bacterial concentration and minimum inhibitory concentration., Hence, M. fragrans can potentially be used as a natural antimicrobial agent in oral care products and thereby mitigates systemic toxicity and bacterial resistance otherwise caused by standard synthetic antibacterial drugs. It is interesting to note that compounds possessing analgesic and anti-inflammatory activities may also possess antibacterial activity. Nutmeg has been reported to have analgesic and anti-inflammatory potential,, and the antibacterial activity showed by essential oil of nutmeg in the present study supports the above fact.
| Conclusion|| |
Essential oil of M. fragrans has excellent antimicrobial activity against endodontic pathogens of primary teeth as shown by its minimum inhibitory activity. Hence, M. fragrans can be considered in various formulations used for the treatment of infectious pulpal conditions.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]