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ORIGINAL ARTICLE
Year : 2020  |  Volume : 12  |  Issue : 1  |  Page : 65-70  

Evaluation of the chemical composition and oral antimicrobial activity of the essential oil from the leaves of Pimenta pseudocaryophyllus (Gomes) landrum


Department of Industrial Biotechnology, Universidade Positivo, Curitiba, Paraná 81280-330, Brazil

Date of Web Publication3-Feb-2020

Correspondence Address:
Prof. Marilisa Carneiro Leão Gabardo
Universidade Positivo, Rua Professor Pedro Viriato Parigot de Souza, 5300, Cidade Industrial, Curitiba, Parana 81280.330
Brazil
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pr.pr_83_19

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   Abstract 


Background: Pimenta pseudocaryophyllus is a native species from Brazil, whose leaves are used in the traditional culture as medicinal plants, being reported with antimicrobial and antifungal properties. Objectives: The aim of this study is to analyze and characterize the chemical composition of the essential oil (EO) extracted from the leaves of P. pseudocaryophyllus, as well as to evaluate its potential for antimicrobial activity against pathogenic bacteria inhabiting the human oral cavity. Materials and Methods: The extraction was performed by steam distillation and the chemical composition was analyzed using gas chromatography-mass spectrometry (GC-MS). The antimicrobial potential of the EO against Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis was evaluated by agar well diffusion method. Different dilutions of the EO (10, 5, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078, and 0.039%, v/v) were tested against the micro-organisms in disk diffusion tests to evaluate the minimum inhibitory concentration. Results: The extraction yielded 0.65% of EO in relation to fresh leaves of P. pseudocaryophyllus. GC-MS analysis identified eugenol, eucalyptol, and limonene as the major compounds of its. The material showed antimicrobial activity, especially against E. faecalis, with moderate activity against S. aureus, both at concentrations from 1.25% to 10%. Conclusion: The results suggested that this EO presents potential of activity against the oral micro-organisms here evaluated.

Keywords: Antimicrobial activity, essential oil, eugenol, minimum inhibitory concentration, phytotherapy


How to cite this article:
Maranho LT, Rosas EC, Costa TA, Ribas JL, Baratto-Filho F, Gabardo MC. Evaluation of the chemical composition and oral antimicrobial activity of the essential oil from the leaves of Pimenta pseudocaryophyllus (Gomes) landrum. Phcog Res 2020;12:65-70

How to cite this URL:
Maranho LT, Rosas EC, Costa TA, Ribas JL, Baratto-Filho F, Gabardo MC. Evaluation of the chemical composition and oral antimicrobial activity of the essential oil from the leaves of Pimenta pseudocaryophyllus (Gomes) landrum. Phcog Res [serial online] 2020 [cited 2020 Nov 25];12:65-70. Available from: http://www.phcogres.com/text.asp?2020/12/1/65/277483



SUMMARY

  • Pimenta pseudocaryophyllus have been used for the treatment of many physical disorders
  • P. pseudocaryophyllus leaves are used as a medicinal plant, with reported antimicrobial and antifungal properties
  • Eugenol, eucalyptol, and limonene as the major compounds of this plant
  • This plant showed antimicrobial activity against  Enterococcus faecalis Scientific Name Search i>Staphlylococcus aureus.




Abbreviations Used: BA: Blood agar; BHI: Brain Heart Infusion; DMSO: Dimethyl sulfoxide; EO: Essential oil; MH: Mueller-Hinton Broth; GC-MS: Gas chromatography-mass spectrometry; MIC: Minimum inhibitory concentration.


   Introduction Top


In dentistry, a variety of micro-organisms present in the biofilm is found in both the oral cavity and the dental work environment. In the mouth, for example, Enterococcus faecalis is the bacteria identified as the main cause of failure in endodontic treatment and diverse types of micro-organisms may compose the microbiota of persistent apical lesions.[1],[2] In hospitals and dental clinics, the major problem with contamination is related to opportunistic bacteria, such as Pseudomonas aeruginosa and Staphylococcus aureus, commonly found in equipment, and that may represent a potential source of cross-contamination, resulting in many health problems.[3],[4],[5],[6] Biofilms formed by pathogenic bacteria are associated with a wide range of diseases, from device-related infections.[5],[7]

In this context, the indiscriminate use of antibiotics to eliminate pathogens plays an important role, associated with treatment ineffectiveness and the increase of persistent infections, as a consequence of antimicrobial resistance.[5],[8] Thus, alternative compounds may present potential activity against oral pathogenic micro-organisms. Studies with natural substances against E. faecalis[9],[10],[11] and P. aeruginosa[8],[11],[12],[13] were reported, and an interesting candidate for tests is Pimenta pseudocaryophyllus (Gomes) Landrum, which belongs to Myrtaceae family.

Myrtaceae comprises one of the largest and most important families of Brazilian flora, being their members predominantly found in the Atlantic Forest. The species of the genus Pimenta are noteworthy for medicinal interest and are recognized for their potential to produce essential oil (EO) with pharmacological effects.[14],[15]

The EO from P. pseudocaryophyllus has a complex chemical composition and includes phenylpropanoids, monoterpenes, aldehydes, and monoterpene alcohols.[14] Pharmacological properties were popularly propagated and attributed to this species by the consume of the infusion of their leaves in the form of teas, which is considered suitable for the treatment of flu, colds, arthritis, gonorrhea, bloody diarrhea, dysentery, fevers, and syphilis, besides it is used as digestive and regulator of menstruation. It also shows anthelmintic activities and displays an anti-inflammatory, antimicrobial, and antifungal potential.[14],[16],[17] In rats, low toxicity of the dry leaf extract of the P. pseudocaryophyllus, (E)-methyl isoeugenol chemotype was found.[18]

Hence, the aim of this study was to analyze and characterize the chemical composition of the EO extracted from the leaves of P. pseudocaryophyllus, as well as to evaluate its potential for antimicrobial activity against pathogenic bacteria inhabiting the human oral cavity.


   Materials and Methods Top


Sampling and material preparation

The sampling of P. pseudocaryophyllus was performed in an area of Mixed Ombrophilous Forest (Araucária Forest) located on private property in the city of Curitiba, Paraná, Brazil, around the geographical coordinates 25°C 35' 01.2” S and 49°C 15' 43.7” W, at an altitude of approximately 900 m. The material was collected with authorization from the Biodiversity Information and Authorization System-Sistema de Autorização e Informação em Biodiversidade (SISBIO/IBAMA), number 48580. The plants were taxonomically identified, and the species was confirmed with the website www.theplantlist.org. A dried sample was deposited in the institutional Herbarium as number 1457.

Leaves from different individual specimens were randomly collected [Figure 1] during April and May 2014. The material was conducted to the Botanical Laboratory and was visually sorted to discard foreign organic and inorganic materials, as well as dry leaves or leaves attacked by insects and/or fungi, resulting in 1200 g of leaves adequate for EO extraction.
Figure 1: Sample of Pimenta pseudocaryophyllus leaves

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Extraction of essential oil

The EO was extracted from fresh leaves (200 g) that were cut into pieces with scissors and subjected to 6 h of extraction by steam dragging in Clevenger-type equipment as modified by Wasicky.[19] This procedure was repeated on six different batches.

Chemical composition analysis of the essential oil

The EO obtained from the fresh leaves was analyzed by gas chromatography-mass spectrometry (GC-MS), adapted from the method of Matos et al.[20] Analyses were carried out on a HP chromatograph model 6890, under the following experimental conditions: capillary column HP 19091S-433 (5% phenyl-methyl-silicone [HP-5MS] with 30 cm × 250 cm i.d.); film thickness, 0.25 μm; initial column temperature, 40°C (3°C min−1) to 200°C (3°C min−1), 200°C–280°C (10°C min−1); injector temperature, 280°C, split ratio 200:1; carrier gas, helium (56 kPa); flow ratio, 0.8 mL/min; ionization energy, 70 eV; and injected volume, 0.2 μL of EO.

The evaluation of the chromatogram [Figure 2] was performed according to retention times and comparison with mass spectra in the library provided with the equipment (Using Aqc Method). Triplicates were performed.
Figure 2: Chromatogram obtained for the essential oil from fresh leaves of Pimenta pseudocaryophyllus

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Evaluation of antimicrobial activity

The antimicrobial activity was evaluated in a Microbiology Laboratory. The biological activity of the oil obtained from the leaves was tested on oral pathogenic micro-organisms. The agar well diffusion was the method elected, including test against oral micro-organisms and the minimum inhibitory concentration (MIC), assayed by the dilution method (macrodilution) in Mueller-Hinton (MH) Broth.[21] The inhibition of bacterial growth was confirmed by subsequent culture on blood agar (BA) plates according to the standard protocols.[22],[23],[24]

Agar well diffusion

Concentrations of 1.00, 0.75, 0.50, 0.25, and 0.00 g/L (v/v) of the EO diluted in MH agar were done, with nine replicates for each concentration.

Micro-organisms were commercially obtained on impregnated discs, which included E. faecalis (NEWPROV-0033), P. aeruginosa (NEWPROV-0053), and S. aureus (NEWPROV-0023). Suspensions of these micro-organisms were prepared in brain heart infusion (BHI) broth at 36°C for 24 h, which were adjusted according to the McFarland scale of 0.5 (equivalent to 1.5 × 108 cells/mL). Confirmation of inoculum concentration was obtained by spectrophotometer optical density analysis (Shimadzu, Kyoto, Japan) at 600 nm.

Next, 1 mL of each micro-organism suspension was inoculated in 140 mm diameter  Petri dish More Detailses filled with 50 mL of MH agar, using a sterile swab in the horizontal, vertical, and diagonal directions. Subsequently, nine equidistant perforations were drilled in the medium with a 5 mm diameter steel cylinder for the formation of the wells, which were filled with 20 μL of the respective concentrations of EO to be tested. Afterward, the plates were kept in the refrigerator for 30 min to avoid the volatilization of the oil and to perfuse in the agar. The plates were then kept in an incubator at 36°C for 24 h, followed by the analysis and measurement of halo inhibition using a caliper with a readability of 0.05 mm (Vonder, Curitiba, Brazil).

As positive controls, discs impregnated with chloramphenicol at 30 μg per disk for P. aeruginosa and S. aureus and vancomycin at 30 μg per disk for E. faecalis were used.

Data were subjected to Shapiro-Wilk and Levene tests. Assumptions of normality and homogeneity of the variances were made and were compared statistically by t-tests and analysis of variance, followed by Tukey's test at 5% of probability.

Broth dilution method for assessing minimum inhibitory concentration

P. aeruginosa (NEWPROV 0053), S. aureus (NEWPROV 0023), and E. faecalis (ATCC 29212), purchased in the lyophilized form, were reconstituted in BHI broth and incubated for 24 h at 35°C ± 2°C before use. For bacterial isolation, aliquots were inoculated on BHI agar plates and incubated in the same described conditions. After 24 h, four to five isolated colonies were selected and transferred to a tube containing 10 mL of MH broth. This suspension was adjusted to match the turbidity to that of a McFarland 0.5 standard solution (equivalent to 108 cells/mL for most pathogens).[22],[23],[24]

A 20% (v/v) solution of the EO from P. pseudocaryophyllus in dimethyl sulfoxide (DMSO) was used as a master solution to evaluate the antimicrobial activity. Nine doubling dilutions of this solution were prepared in DMSO from 10% (v/v) to 0.039% (v/v).[22],[23],[24]

An aliquot of 100 μL of each serial dilution was added to 1 mL of MH broth containing the equivalent of 108 cells/mL of each test micro-organism. To verify the effect of DMSO in the dilutions, two additional tubes were used as controls, one of them with only the test micro-organisms in MH broth with DMSO and the other without DMSO. The tubes were incubated at 35°C ± 2°C for 18, 24, 48, and 72 h. At each time point, the tubes were examined for turbidity, and the cultures were then inoculated with a platinum loop onto BA plates and incubated for 24 h to ascertain the MIC. The tests were performed in duplicates for each micro-organism tested.[22],[23],[24]


   Results Top


Chemical composition of the essential oil

The mean yield of EO extracted from fresh leaves of P. pseudocaryophyllus was 1.3 mL (±0.2 mL) for each 200 g of extracted fresh leaves.

The GC-MS analysis revealed 30 compounds in the sample, of which 28 were identified, as shown in [Table 1]. A qualitative diversity was observed in the chemical composition of the obtained essential, and the three major constituents found were eugenol (31.5%), eucalyptol (14.2%), and limonene (9.3%).
Table 1: Composition of the essential oil from fresh leaves of Pimenta pseudocaryophyllus

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Evaluation of antimicrobial activity

The diffusion tests in agar showed no formation of halos at the 0.00 g/L concentration for any of the treatments and also at the 0.25 g/L concentration in the presence of P. aeruginosa.

For the three tested bacteria, the halo was significantly higher in the control treatments, demonstrating the effectiveness of the antibiotic used in relation to the EO.

In S. aureus and E. faecalis, there was a significant reduction of the halo size at the 0.25 g/L concentration and at the 0.75 g/L and 0.50 g/L concentrations, the halo remained statistically equal, with these means being lower than those recorded at the 1.00 g/L concentration. In E. faecalis, the means of the halo size at concentrations of 1.00, 0.75, and 0.50 g/L were significantly higher than that observed at 0.25 g/L [Table 2].
Table 2: Mean±standard deviation referring to the inhibition halo sizes formed in three species of bacteria submitted to five concentrations of essential oil and in the presence of antibiotic (control)

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At concentrations of 1.00, 0.75, and 0.50 g/L, the size of the halos formed in the presence of the three bacteria differed statistically, being higher in S. aureus. At the 0.25 g/L concentration, the inhibition halo was significantly higher in S. aureus than in E. faecalis. The statistical comparison between the control treatments showed that the halo formed in S. aureus was higher than in E. faecalis and P. aeruginosa [Table 2].

[Table 3] describes the results of the MIC tests. There was no inhibition of P. aeruginosa at any of the dilutions tested. For S. aureus, after 48 h of incubation, there was a moderate inhibition of bacterial growth in the dilutions corresponding to the following concentrations: 10%, 5%, 2.5%, and 1.25%. After 72 h, there was complete inhibition of bacterial growth at 10%, with moderate growth at the other concentrations (5%, 2.5%, and 1.25%). There was complete growth inhibition of E. faecalis after 48 h of incubation at 10, 5, and 2.5% and moderate growth at 1.25%. After 72 h, there was total inhibition at 10%, 5%, 2.5%, and 1.25%, demonstrating the antimicrobial activity of the EO from P. pseudocaryophyllus.
Table 3: Minimum inhibitory concentration of the essential oil from fresh leaves of Pimenta pseudocaryophyllus against different micro-organisms

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   Discussion Top


This study aimed to analyze and characterize the chemical composition of the EO extracted from the fresh leaves of P. pseudocaryophyllus and to evaluate its antimicrobial activity against S. aureus, P. aeruginosa, and E. faecalis, micro-organisms commonly found in the mouth and biofilms.

The EO extracted presented a yield of 0.65% in relation to the fresh leaves. A value of 1.0% yield was reported to EO from fresh leaves of the same species[25] and the difference could be related to the geographic origin of the samples. Other study found 1.9% and 2.3% yield of EO from dried leaves of P. pseudocaryophyllus.[26] In this case, the difference observed is due to the previous dehydration of the leaves, besides the sample origin.

Analysis of the composition of the EO from fresh leaves of P. pseudocaryophyllus identified components that match those previously found in other species of the family Myrtaceae.[27]

A previous pharmacognostic study analyzed the oil from the leaves of P. pseudocaryophyllus using various methods such as phytochemical screening, colorimetric reactions, and protein precipitation, which revealed the presence of phenolic compounds, tannins, and flavonoids.[16]

The chemical analyzes in the present work identified eugenol (31.5%) as the predominant compound, as reported by other studies with P. pseudocaryophyllus.[15],[25] Eucalyptol (14.2%) and limonene (9.3%) are also found in considerable amount in the present work. Together, the three major components identified in the extracted EO totalized 55% of the sample. Overall, satisfactory antimicrobial activities against the micro-organisms studied were observed.

Authors reported that eugenol and 4-methyl-eugenol were the major constituents of the EO from the leaves of P. pseudocaryophyllus and that eugenol is responsible for most of the antiseptic and antimicrobial activities attributed to the species.[15] In a later study, others concluded that eugenol represented the main component in the EO of Tynanthus micranthus as well as P. pseudocaryophyllus and that both of these oils possessed antimicrobial and antifungal activities.[25] These authors also suggested that eugenol could be useful as an antimicrobial agent in agriculture, the food industry and in the preparation of pharmaceutical products. In dentistry, eugenol is used in the manufacture of dentifrice because of its powerful potential as an antimicrobial agent.[25]

The antimicrobial activity of plant extracts can be measured by sequential dilution of the test substances to identify the lowest concentration that inhibits the growth of micro-organisms. In sensitivity tests using the broth dilution method, the MIC is the lowest concentration of the test antimicrobial substance that prevents visible growth of the micro-organism.[21]

Although the disk diffusion test is widely used, it is not considered the most appropriate method to measure antimicrobial activity. During the incubation period, the antimicrobial agent diffuses out of the disk impregnated with the antimicrobial solution into the culture medium. If an inhibition zone is formed around the disk, the test agent material is scored as effective; however, the result may be affected by the diffusion capacity of the agent in the culture medium.[28]

The oil from P. pseudocaryophyllus inhibited the growth of S. aureus at the higher concentrations (10%) and longer incubation times tested (72 h), while moderate inhibition was also noted at the lower concentrations (5.0%, 2.5%, and 1.25%) and times (48 h/72 h). In this study, there was no inhibition of P. aeruginosa at any of the concentrations of the tested oil. In contrast, a study demonstrated similar antimicrobial activities against both P. aeruginosa (ATCC 9027) and S. aureus (ATCC 6538) when EO from the leaves of two specimens of P. pseudocaryophyllus collected from different sites were tested.[15]

The reasons for choosing P. aeruginosa and S. aureus as test organisms in this study are based on the presence of these two bacterial species in the oral cavity and because they display a great capacity for biofilm formation.[3],[4],[5],[6] They are commonly found in moist environments and even in dental equipment, thus, representing a potential source of cross-contamination.[3],[4] The presence of biofilms formed by P. aeruginosa and S. aureus in fragments of dental handpieces have been reported.[3]

Water collected from 33 samples of dental apparatus from private clinics, public services, and a College of Dentistry were examined and P. aeruginosa was found in 21.2% of these samples.[4] This observation deserves more attention from dental professionals because it represents a risk of cross-infections in clinics. P. aeruginosa is known as an opportunist bacterium since it can invade the bloodstream, resulting in sepsis, which is a serious and potentially fatal complication.[28]

Another opportunist bacterium, S. aureus, may be transmitted through disrupted skin barriers or during invasive surgical procedures, posing a major problem in hospitals and dental clinics. The evaluation of the presence of S. aureus on different surfaces from the School of Clinical Dentistry demonstrated the presence of this micro-organism in 34% of the samples tested, suggesting that the surfaces became contaminated during the dental treatment.[6] These findings highlight the importance of biosecurity designed to minimize cross-contamination between the oral health team and patients in the dental clinic.

In this study, the EO from P. pseudocaryophyllus inhibited the growth of E. faecalis, confirming its antimicrobial potential. These results are particularly important since this bacterium is a common contaminant in endodontic treatments and displays resistance. In addition, this bacteria forms biofilm that can adhere to the inside of root canals and its presence has been noted in persistent periapical infections.[1],[2] Moreover, E. faecalis can tolerate long periods of nutritional restriction and withstand extremes of salinity and pH, with the ability to remain viable in the walls of the root canal for up to 12 months after dental procedures.[29] The organism features several virulence factors that modulate the expression of genes in unfavorable conditions, increasing its adherence and resistance to antimicrobials and rendering this bacterium a potential risk to oral health.[30]

The antimicrobial activity of natural substances against P. aeruginosa[9],[10],[11] and S. aureus[9],[11],[12],[13] has been investigated. The action of the EO from P. pseudocaryophyllus against E. faecalis illustrates the importance of evaluating the antimicrobial effects of medicinal plants as a support for dental treatments. In Addition, the development of a formulation for intracanal medications and irrigators to assist in the treatment and blocking of the action of virulence factors is essential to prevent bacterial adherence and invasion of the oral mucosal tissues.

The variable effect on the studied micro-organisms may be explained by the fact that the inhibitory concentration of EO required for each bacterial species is different. The greater sensitivity of Gram-positive compared to Gram-negative bacteria may be related to differences in the bacterial cell wall structure, which hinders the efficacy of the EO for Gram-negative bacteria.[31] Many plants with potential antimicrobial activity have been shown to be effective only against strains of Gram-positive bacteria.[32],[33] Researchers have postulated that the outer membrane of Gram-negative bacteria could act as a barrier against the active principles found in medicinal plants.[34] However, other studies have verified antimicrobial activity against both Gram-positive and Gram-negative bacteria.[35],[36]


   Conclusion Top


EO from fresh leaves of P. pseudocaryophyllus presented as the three major components eugenol, eucalyptol, and limonene. Although complementary studies are necessary, these compounds SE may be related to the observed antimicrobial activity against certain micro-organisms present in the human oral cavity and suggest the biological potential of this species of the Myrtaceae Family.

Acknowledgements

The authors are grateful to the Director of the Second Integrated Center for Air Defense and Air Traffic Control (Segundo Centro Integrado de Defesa Aérea e Controle de Tráfego Aéreo-CINDACTA II), as well as the Head of the Clinical Analysis Laboratory, for providing the facilities of the Microbiology Laboratory for the conduction of this study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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