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Antimicrobial Efficacy of Three Essential Oils against Decaying Cedar Wood Isolates



Fadoua Bennouna, Yassir Lekbach, Moulay Sadiki, Soumya El Abed, Saad Ibnsouda Koraichi and Mohammed Lachkar
 
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ABSTRACT

Background and Objective: The use of traditional methods as synthetic chemical products to protect cedar wood raises concerns because of the potential negative impact of these products on the public health and environment. The aim of this study was to investigate the chemical composition of three essential oils: Mentha pulegium, Rosmarinus officinalis and Cananga odorata and their antimicrobial effect against six fungi and two bacteria causing degradation of cedar wood. Materials and Methods: Determination of the chemical composition of essential oils was conducted using gas chromatography-mass spectrometry (GC-MS) (Trace GC Ultra). The minimal inhibitory concentrations and minimal bactericidal/fungicidal concentrations were also determined using the broth microdilution assays. Results: The GC/MS analysis of oils studied showed that menthone and pulegone were the major components of Mentha pulegium essential oil, camphene and α-humulene were the major ones in Rosmarinus officinalis, whereas linalool and geranyl acetate were the major components for Cananga odorata. Results also showed potent antifungal activity against all fungi tested, with MICs ranging from 0.0625-0.25% for Mentha pulegium, 0.25-1% for Rosmarinus officinalis and from 0.5-1% for Cananga odorata. An important antibacterial activity was shown against the two bacteria tested with MICs ranging from 0.125-2%. Conclusion: This study suggested that these essential oils may be used as an alternative of traditional methods used for wood protection.

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Fadoua Bennouna, Yassir Lekbach, Moulay Sadiki, Soumya El Abed, Saad Ibnsouda Koraichi and Mohammed Lachkar, 2018. Antimicrobial Efficacy of Three Essential Oils against Decaying Cedar Wood Isolates. Research Journal of Microbiology, 13: 119-126.

DOI: 10.3923/jm.2018.119.126

URL: https://scialert.net/abstract/?doi=jm.2018.119.126
 
Received: February 23, 2018; Accepted: August 12, 2018; Published: November 03, 2018



INTRODUCTION

Wood is a complex biopolymer composed of cellulose, hemicelluloses and lignin. It is used as raw material for paper industries but also as material in built constructions. In Morocco, cedar wood was used for building historical monuments of different imperial cities including the old medina of Fez. However, moisture effect promotes its deterioration with bacteria and fungi and caps its outdoor applications1. The growth of various wood degrading biological organisms is frequently associated with aesthetical degradation2,3, biodeterioration and reduction of wood durability4,5 and thus lead to a health risk by fungal mycotoxins6. Different techniques were used previously to improve the lifetime of cedar wood and boost its resistance to decay including the use of biological, physical and chemical means7. These traditional methods as the use of toxic chemicals provide certainly a decay resistance but have environmental effects. Recently, environmental concerns required the use of non-biocidal solutions for wood protection. Thus, the search to develop solutions with good environmental profile and antimicrobial assets is undeniably the priority for the researchers of material sciences. Among natural resources that can be used to prevent wood biodeterioration, essential oils and medicinal and aromatic plant extracts known by their important antimicrobial activity for one hand and for the other hand for their wealth in eco-friendly natural molecules.

Several studies have reported the strong antimicrobial activity of essential oils and their components against a broad range of pathogenic microorganisms8-10. However, few studies reported the antibacterial and antifungal activities against bacterial and fungal strains causing degradation of cedar wood11-13. Thus, the purpose of this research was to investigate firstly the chemical composition of three essential oils (Mentha pulegium, Rosmarinus officinalis and Cananga odorata) and secondly, to examine their antimicrobial activity against bacteria and fungi associated with deterioration of historical wood.

MATERIALS AND METHODS

The present study was conducted during the year 2016-2017. The essential oil extraction, the composition of essential oils and the antimicrobial activities have taken six months.

Plant material and extraction of essential oils: The leaves of Mentha pulegium and Rosmarinus officinalis were harvested in the region of Sefrou located in the Middle Atlas (Morocco) and the flowers of Cananga odorata (generally known as Ylang-Ylang) were harvested from the Comoros Islands. The plants were shade dried at room temperature with ventilation then subjected to hydrodistillation with Clevenger-type apparatus. The obtained oils were dried with anhydrous sodium sulfate and stored in a refrigerator at 4°C.

Essential oils analysis: Chemical composition of tested oils was determined using gas chromatography-mass spectrometry (GC-MS) (Trace GC Ultra). The analysis were carried out using a Trace GC Ultra gas chromatograph equipped with anapolar capillary TR-5 column (60 m× 0.32 mm ID, 0.25 μm film thickness) coupled to a mass detector (MS Quadrupole). Helium was used as carrier gas at a (1.2 mL min1). Oven temperature was held at 300°C and the set conditions were: initial temperature of 40°C/2 min, then increasing (5°C min1) until 280°C and held for 10 min; a 10:1 split ratio mode and an injector temperature of 220°C. The ionization energy was 70 eV. Retention Indices (RI) were calculated using a homologous series of n-alkanes C8-C18 (Sigma-Aldrich, St Louis, MO). Compounds were identified by comparing their RI and mass spectra with those of main components from literature and the NIST (National Institute of Standards and Technology) Library.

Microbial cultures: The following microbial strains were selected for their ability to deteriorate cedar wood: Two Gram-positive bacteria of the genus Bacillus (B. safensis and B. subtilis) and six fungi (Penicillium commune (PDLd”), Penicillium expansum, Penicillium crustosum, Penicillium commune (PDLd10), Thielavia hyalocarpa and Aspergillus niger). The strains were isolated from cedar wood decayed from an old house in the old medina of Fez and identified in our laboratory12,14,15. They were stored at -18°C in 20% glycerol and subcultured as follows: Bacteria were subcultured in Luria-Bertani (LB) agar and incubated at 37°C for 24 h and fungi in Malt-Extract (EM) agar medium at 25°C for 10 days. After the incubation period, bacterial inoculum was prepared in physiologic saline solution and adjusted to a final concentration of 2.10 6 Colony-Forming Unit mL1 (CFU mL1) for each bacterium under study. Likewise, the fungal spores were collected by scraping the culture surface using sterile physiologic saline solution with 1% Dimethylsulfoxyde (DMSO). The spore suspensions were concentrated by centrifugation at 7000 rpm for 15 min at 4°C to a final concentration of 2.10 4 spores mL–1 16.

Antimicrobial activity tests: The Minimum Inhibitory Concentration (MIC) corresponds to the lowest concentration of antibacterial agent that inhibits the bacterial growth17. It was determined using the broth microdilution assay18,19 with some modifications. The 96-well plate was prepared by dispensing into each well (from the second to the 12th well) 50 μL of Mueller Hinton Broth (MHB) for antibacterial assay and Malt Extract Broth (MEB) for antifungal activity supplemented with bacteriological agar (0.15% w/v). Essential oils were dissolved in the suitable culture medium with 0.15% of agar according to the test carried out. A serial dilution of these latter was realized until the 11th well in such a way that the final concentration was between 8-0.00781% (v/v) for Mentha pulegium and Rosmarinus officinalis and between 4-0.0081% (v/v) for Cananga odorata. The 12th well was considered as growth control. At the end, 50 μL of bacterial/spores suspension previously prepared were added to each well. Plates were incubated at 37°C for 20 h for antibacterial tests and for 48 h at 25°C for antifungal tests. A further incubation for 2 h was realized after addition of 5 μL of resazurin to determine the MIC of essential oils against bacteria tested18. Bacterial growth is revealed by reduction of blue dye resazurin to pink resorufin. The minimum bactericidal/fungicidal concentration (MBC/MFC) is defined as being the lowest concentration of the essential oil giving negative subcultures after incubation for 24 h at 37°C for bacterial strains and at 28°C for 72 h for fungi. It was determined by spot inoculating 5 μL from negative wells on LB/EM plates.

RESULTS AND DISCUSSION

Essential oils composition: Total 100% of the volatiles compounds of Mentha pulegium essential oil were identified as shown in Table 1.94.43% being oxygenated monoterpenes, principally represented by menthone and pulegone representing 60.56 and 15.77%, respectively of the total essential oil. About 1.53% of the constituents were represented by monoterpene hydrocarbons with a single compound which is the α-pinene. Sesquiterpenes hydrocarbons were represented by β-caryophyllene with a percentage of 4.04%. The chemical composition of Mentha pulegium essential oil was the subject of many investigations in different countries like Portugal with menthone as major component20, Morocco21-24, Tunisia25, Algeria26, India27, Spain28 and Iran29. The major constituent was pulegone in all these studies. However, the quantities were different from one to another study because of the geographical source collection and seasons growing conditions30.

For Rosmarinus officinalis essential oil, the identified volatiles were 99.55% and major constituents were represented by sesquiterpene hydrocarbons (39.06%) principally α-humulene which represents 25% followed by monoterpene hydrocarbons (28.11%), the main volatile constituent being camphene presents 21.22%. Oxygenated monoterpenes were 23.87% and oxygenated sesquiterpenes only 3%. Similar statements were reported in other studies. However, the amount achieved is greater than those found31-33. The chemical composition of the essential oil varies according to the plants origin. Derwich et al.34 have shown α-pinene (18.25%) as the major component of Rosmarinus officinalis essential oil collected from the region of Atlas median (Tafersoust), Morocco. The 1,8-cineol was the major constituent of Rosmarinus officinalis essential oil collected from Crete (Greece) with a percentage of 88.9% 35. Pintore et al.36 reported the presence of α-pinene, verbenone and bornyl acetate of rosemary oils from Sardinia and Corsica and Mata et al.37 reported the presence of verbenone (35.4%) of rosemary oil from Portugal.

The oxygenated fraction of Cananga odorata essential oil represents 67.71% of the total identified volatiles, principally constituted by 1,8-cineol, linalool, geranyl acetate, cinnamyl acetate, isoeugenol, methyleugenol, caryophyllene oxide, γ-cadinol, δ-cadinol, δ-dodecalactone, benzyl benzoate and benzyl salicylate. Geranyl acetate and linalool were shown to be main components present in oxygenated fraction with a percentage of 14.51 and 13.81%, respectively that are responsible for the floral smell of Ylang-Ylang. The hydrocarbon fraction of Ylang-Ylang oil represented 30.77% of the volatiles identified and consisted mainly of sesquiterpenes and monoterpenes. Current results were comparable with those found by Stashenko et al.38 for Colombian Ylang-Ylang flower sessential oil with an appearance of linalool in higher concentration, which is more than twice our reported value from Comoros Ylang-Ylang essential oil. Stashenko et al.38,39 have shown also the presence of 1,8-cineole, geranyl acetate, cinnamyl acetate, α-pinene, α-ylangene, β-caryophyllene and other compounds with different concentrations. These variations could be explained by the extraction method, extraction time, the origin and the flower maturity. Other studies have shown the chemical composition of leaves of Cananga odorata grown in Australia(β-caryophyllene 34.2%, sabinene 19.6%, α-humulene 6.8%, α-pinene 6%) and Cameroon (β-caryophyllene 26.3%, α-pinene 14%, germacrene D 11.7%)40,41.

Antibacterial activity: The MICs obtained in the antimicrobial test are shown in Table 2. The data showed that the three essential oils possess a strong antimicrobial activity against bacterial strains studied since MIC values were between 2 and 0.125%.

Table 1: Chemical composition of Mentha pulegium, Rosmarinus officinalis and Cananga odorata essential oils
RI: Retention index

Table 2: Determination of MIC values of Mentha pulegium, Rosmarinus officinalis and Cananga odorata essential oils against bacterial strains tested
MPE: Mentha pulegium essential oil, ROE: Rosmarinus officinalis essential oil, COE: Cananga odorata essential oil, +: Presence of growth, -: Absence of growth, *Not done; positive control: Bacterial suspensions and Mueller-Hinton Broth supplemented with agar (0.15% w/v)

The antibacterial effect of Mentha pulegium essential oil is eight times higher than that obtained for the essential oil of Rosmarinus officinalis and six times higher than that obtained for Cananga odorata essential oil with a MIC values lie between 0.125 and 0.25%, thus presenting an interesting potential as antimicrobial substance in cedar wood preservation. Mentha pulegium essential oil exhibits a bacteriostatic effect against B. subtilis with a MFC value of 8% and a ratio MBC/MIC value of 32 higher than four42.

As expected, bacterial strains tested were found susceptible to the three essential oils. These bacteria (Gram-positive) contain a layer of lipids in their cell wall, which help the passage of essential oils. Indeed, B. safensis showed a similar susceptibility for Rosmarinus officinalis and Cananga odorata essential oils. The Minimum Bactericidal Concentration (MBC) against B. safensis and B. subtilis was higher than 8% for Rosmarinus officinalis and Cananga odorata essential oils.

The essential oils evaluated in this study displayed a variable degree of antibacterial activity against the two bacterial strains tested. Regarding this concern, a number of studies on M. pulegium essential oil have already been published20,22-26,43. According to these reports, the latter has shown a high bacteriostatic and bactericidal activity against pathogenic microorganisms: MIC<0.5 μL mL1 and MBC = 0.5 μL mL1 for Enterococcus faecium22, Cherrat et al.23 have also reported it to have a strong antimicrobial activity against B. subtilis with value of MIC = 1 μL mL1 and MBC = 8 μL mL1. Hajlaoui et al.25 have also concluded that M. pulegium essential oil has a broader spectrum of antimicrobial activity (MIC = 0.62 μL mL1 and MBC>0.5 μL mL1 for Bacillus cereus, MIC = 2.5 μL mL1 and MBC = 0.312 μL mL1 for Enterococcus faecalis).

Pintore et al.31 and Zaouali et al. 36 have revealed a low to moderate antimicrobial activity for Rosmarinus officinalis essential oil against the range of bacteria tested with values of MIC lie between 2.4 and 4 mg mL1 and between 1.25-10 μL mL1, respectively. For Cananga odorata essential oil, few studies have been conducted on its antibacterial activity. However, a good antimicrobial activity has been reported against Staphylococcus aureus, Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae and Pseudomonas aeruginosa41,44.

The antibacterial activity is attributed not only to the major components of essential oil but also to the minor components that may have a synergistic effect. Gill et al.45 and Mourey and Canillac46 have shown that the whole essential oils have better antibacterial activity than the major components mixed. Interestingly, it can be seen from Table 1 that oxygenated monoterpenes present a high percentage for M. pulegium essential oil (94.43%) therefore an important antimicrobial activity compared to R. officinalis and C. odorata essential oil (23.87 and 33.96%, respectively). In addition, several studies have shown that the essential oils possessing the strongest antibacterial properties were rich in phenolic compounds47,48. So, the presence of phenolic compounds especially hydroxyl groups position played an important role in antimicrobial activity49.

Antifungal activity: The three essential oils studied show an antifungal activity as reflected by the MIC values obtained. The MICs determined for all fungal strains tested in this study oscillated between 0.0625 and 1% (v/v) (Table 3). Strong antifungal activity was recorded for Mentha pulegium essential oil with a MIC values lie between 0.0625 and 0.25% (v/v), followed by those of Rosmarinus officinalis and Cananga odorata essential oil. Mentha pulegium essential oil has a fungicidal activity against all fungal strains studied with a MFC values ranged from 2 and 4% (Table 4). Thus, a similar susceptibility was shown for P. commune (PDLd”), P. commune (PDLd10) and A. niger with a MIC value of 0.125% (v/v). Rosmarinus officinalis and Cananga odorata essential oil presented the same effect against P. commune (PDLd”) (MIC of 1% (v/v)), P. expansum and P. crustosum (MIC of 0.5% (v/v)). Similar studies have shown the antifungal activity of some essential oils including that of Zyani et al.50 who reported the important activity of Origanum compactum, Eugenia caryophyllata and Ocimum basilicaum essential oils against Thielavia hyalocarpa, Penicillium commune, Penicillium chrysogenum, Penicillium expansum and Cladosporium cladosporioides. Soidrou et al.11 have found that Comorian essential oils isolated from Piper capense, Piper borbonense and Vetiveria zizanoides have a strong fungicidal activity against fungi decay wood (Gloeophyllum trabeum, Poria placenta, Coniophora puteana and Coriolus versicolor).

Several authors have attributed the antifungal activity of essential oils to their major phenolic components9. Hassan et al.51 have shown the important antifungal activity of the carvacrol against P. expansum in a lower concentration (MIC = 0.625% (v/v)). The antifungal activity of the same component against A. niger, A. flavus, P. citrinum and P. chrysogenum was studied (MIC = 50, 100, 150 and 125 μg mL1, respectively)52.

Table 3: Determination of MIC values of Mentha pulegium, Rosmarinus officinalis and Cananga odorata essential oils against fungal strains tested
MP: Mentha pulegium essential oil, RO: Rosmarinus officinalis essential oil, CO: Cananga odorata essential oil, +: Presence of growth, -: Absence of growth, *Not done, positive control: Bacterial suspensions and Malt Extract Broth supplemented with agar (0.15% w/v)

Table 4: Determination of MFC values of Mentha pulegium, Rosmarinus officinalis and Cananga odorata essential oils against fungal strains tested
MP: Mentha pulegium essential oil, RO: Rosmarinus officinalis essential oil, CO: Cananga odorata essential oil

Menthone, borneol and other components have exhibited a good antifungal activity against Botrytis cinerea53 and linalool exhibited an effect on S. cepivorum and F. oxysporum54.

The essential oils studied could be used as a non-biocidal solution to be applied on cedar wood surface to ensure better protection against fungi and bacteria causing its degradation and thus a conservation of our cultural heritage while respecting the environment.

CONCLUSION

Mentha pulegium essential oil showed a strong antibacterial activity followed by Cananga odorata and Rosmarinus officinalis. It presented also an important antifungal activity against all fungal strains studied which was five times higher than that obtained for Cananga odorata essential oil and three times higher than that obtained for the essential oil of Rosmarinus officinalis. These results supported the use of these essential oils as natural product to prevent wood biodeterioration, thus providing an alternative to the use of synthetic chemical products.

SIGNIFICANCE STATEMENTS

This study discovers the antimicrobial effect of Mentha pulegium, Rosmarinus officinalis and Cananga odorata essential oils that can be beneficial for the protection of cedar wood against microorganisms causing it degradation and thus the preservation of our cultural heritage. This study will help the researcher to uncover the critical areas of essential oils efficacy against decaying cedar wood microorganisms that many researchers were not able to explore. Thus, a new theory on the antibacterial, antifungal and preservative effects of theses biological molecules may be arrived at.

ACKNOWLEDGMENTS

The authors would like to acknowledge the support and technical assistance of Interface Regional University Center (University Sidi Mohamed Ben Abdellah, Fez) and National Center for Scientific and Technical Research (CNRST-Rabat).

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