Abstract
Context: Eucalyptus globulus Labill (Myrtaceae) is the principal source of eucalyptus oil in the world and has been used as an antiseptic and for relieving symptoms of cough, cold, sore throat, and other infections. The oil, well known as ‘eucalyptus oil’ commercially, has been produced from the leaves. Biological properties of the essential oil of fruits from E. globulus have not been investigated much.
Objective: The present study was performed to examine the antimicrobial activity of the fruit oil of E. globulus (EGF) and the leaf oils of E. globulus (EGL), E. radiata Sieber ex DC (ERL) and E. citriodora Hook (ECL) against multidrug-resistant (MDR) bacteria. Furthermore, this study was attempted to characterize the oils as well as to establish a relationship between the chemical composition and the corresponding antimicrobial properties.
Materials and methods: The chemical composition of the oils was analyzed by GLC-MS. The oils and isolated major components of the oils were tested against MDR bacteria using the broth microdilution method.
Results: EGF exerted the most pronounced activity against methicillin-resistant Staphylococcus aureus (MIC ~ 250 µg/ml). EGF mainly consisted of aromadendrene (31.17%), whereas ECL had citronellal (90.07%) and citronellol (4.32%) as the major compounds. 1,8-cineole was most abundant in EGL (86.51%) and ERL (82.66%).
Discussion and conclusion: The activity of the oils can be ranked as EGF > ECL > ERL ~ EGL. However, all the oils and the components were hardly active against MDR Gram-negative bacteria. Aromadendrene was found to be the most active, followed by citronellol, citronellal and 1,8-cineole.
Introduction
Increasing resistance of pathogens toward antibiotics presents a major threat to public health because it reduces the effectiveness of antibiotic treatment, which could lead to an increase in morbidity and mortality. Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) are the most important resistant pathogens among Gram-positive bacteria concerning nosocomial infections. Emergence of resistance in Gram-negative bacteria (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter) has also been documented. Many antibiotics that have frequently been used in the past have become less effective against these pathogens (CitationJones, 2001). Hence, there is an urgent need to find alternative antimicrobial agents for the treatment of resistant pathogenic microorganisms.
Many essential oils including those from Eucalyptus have been used in folk medicine throughout the world, and their medicinal properties have been investigated (CitationCoppen, 2002). Essential oils from Eucalyptus exhibit antibacterial, antifungal, analgesic and anti-inflammatory properties and have also been widely used in pharmaceutical, food, and cosmetics products (CitationRamezani et al., 2002; CitationSilva et al., 2003; CitationSartorelli et al., 2007).
Eucalyptus globulus Labill (Myrtaceae) is the principal source of eucalyptus oil in the world and has been used as an antiseptic and for relieving symptoms of cough, cold, sore throat and other infections (CitationVan Wyk and Wink, 2004; CitationKumar et al., 2007). The oil, well known as ‘eucalyptus oil’ commercially, has been produced from the leaves (CitationLis-Balchin et al., 1998). Furthermore, biological properties of the essential oil of fruits from E. globulus have not been much investigated, whereas the chemical composition of the fruit oil has been studied (CitationCimanga et al., 2002; CitationPereira et al., 2005). Instead of E. globulus, essential oils from E. radiata Sieber ex DC (Myrtaceae) and E. citriodora Hook (Myrtaceae) are widely used for aromatherapy. Eucalyptus radiata oil has also been shown to be useful for treating disorders of the respiratory system (CitationCoppen, 2002). Eucalyptus citriodora is another well-known eucalypt with antibacterial, antifungal, analgesic and anti-inflammatory properties (CitationLis-Balchin et al., 1998; CitationSilva et al., 2003). However, no report is available on its activity against multidrug-resistant bacteria.
In this investigation, we have examined the antimicrobial activity of the fruit oil of E. globulus and the leaf oils of E. globulus, E. radiata and E. citriodora against multidrug-resistant Gram-positive and Gram-negative bacteria. The major components of the oils (aromadendrene, 1,8-cineole, citronellal, and citronellol) were also evaluated. Furthermore, the chemical composition of the oils was reinvestigated by GLC-MS to characterize the oils as well as to establish a relationship between the chemical composition and the corresponding antimicrobial properties.
Materials and methods
Plant material
The fruits of E. globulus were kindly provided by Prof. Thomas Efferth. The identity of the plant has been authenticated by Dr. Wahyono at Department of Pharmacognosy, Gadjah Mada University, Indonesia, and the voucher specimen (P6868) was deposited at the Department of Biology, Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Germany.
Essential oils and monosubstances
The dried powder of fruits from E. globulus was subjected to hydrodistillation for 6 h using a Clevenger-type apparatus. After separation, the essential oil was kept in separate sealed vials at 4°C for further analysis. Essential oils of the leaves from E. globulus (Bergland-Pharma, Heimertingen, Germany), E. radiata (Primavera Life, Sulzberg, Germany) and E. citriodora (Primavera Life, Sulzberg, Germany) were obtained commercially. (+)-Aromadendrene (≥97% purity), (±)-citronellal (≥80% purity), (±)-citronellol (90–95% purity) were purchased from Fluka, Switzerland, and 1,8-cineole (99% purity) from Sigma-Aldrich (St. Louis, MO).
Microbial strains
The essential oils and their components were tested against multidrug-resistant Gram-positive and Gram-negative bacteria. Gram-positive bacteria: reference strain of MRSA (NCTC 10442), clinical isolates of MRSA (MR134/93, MR131/98, MR 2387/00, MR1150/93, MR 1000/93, MR635/93, MR1678/96, BL7127/98, and USA300), reference strain of VRE (ATCC 51299), and clinical isolates of VRE (VRE902247, VRE902251, VRE902316, and VRE 902267). Gram-negative bacteria: Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 700603, Acinetobacter baumannii ATCC BAA 747. All microorganisms were obtained from the Department of Infectious Diseases, Medical Microbiology and Hygiene, Heidelberg University, Germany. The strains were sub-cultured on Columbia 5% sheep blood agar (Becton Dickinson, Germany) 24 h prior to any antimicrobial test.
Minimal inhibitory concentration and minimal bactericidal concentration determination
The minimal inhibitory concentration (MIC) of the samples was determined by broth microdilution methods (Mulyaningsih et al., Citation2010a). Briefly, the samples were pipetted into 96-well microtiter plates in Mueller Hinton broth (Fluka, Switzerland) followed by a twofold serial dilution. An inoculum suspension was added to give a final concentration of 5 × 105 cfu/ml. After incubation at 37°C for 24 h, MIC was determined as the lowest concentration without bacterial growth (no color change in MTT assay). The minimal bactericidal concentration (MBC) was determined by subculturing 3 µl from each well without apparent microbial growth on Columbia 5% sheep blood agar and incubated at 36°C for 24 h. The lowest concentration without apparent microbial growth was taken as the MBC. The experiments were performed in duplicate and repeated twice.
Gas liquid chromatography/flame ionization detector
High resolution capillary gas liquid chromatography (GLC) was carried out on a Varian 3400 equipped with flame ionization detector (FID) detector and DB-5 column (30 m × 0.25 mm × 0.25 μm) (Ohio Valley, Marietta, GA). The operating conditions were as follows: carrier gas: helium with a flow rate of 2 ml/min, split ratio 1:20. The oven temperature was programmed with an initial temperature 40°C, 2 min isothermal, 300°C, 4°C/min, then 10 min isothermal. Injector and detector temperatures were set at 250°C and 300°C, respectively. PeakSimple® 2000 chromatography data system (SRI Instruments, Torrance, CA) was used for recording and integration of the chromatograms.
Gas liquid chromatography-mass spectrometry (GLC-MS) analysis
GLC-MS was carried out on a Hewlett-Packard gas chromatograph (GC 5890 II) equipped the same column as GLC (see above). Samples (2 µl) were injected in a split mode (split ratio, 1:15) with the carrier gas helium at a flow rate of 2 ml/min. The capillary column was coupled to a quadrupole mass spectrometer (SSQ 7000, Thermo-Finnigan, Bremen, Germany). The injector temperature was 250°C. Helium carrier gas flow rate was 2 ml/min. All the mass spectra were recorded with the following conditions: electron energy, 70 eV; ion source, 175°C. The oil components were identified by their retention time, retention indices relative to C8-C28 n-alkanes, computer matching with the Wiley Registry of Mass Spectral Data 8th edition, NIST Mass Spectral Library (December 2005) and by comparing their mass spectra with data already available in the literature and in our own database (CitationAdams, 2004; Mulyaningsih et al., Citation2010a).
Results and discussion
Chemical composition of the essential oils
The chemical composition of the fruit oil of E. globulus (EGF) was determined by GLC-MS (), in comparison to the leaf oils from E. globulus (EGL), E. radiata (ERL), and E. citriodora (ECL). About 65 compounds were unambiguously identified, representing 97.02–99.89% of the total oils. Monoterpenoids were predominantly found in the three leaf oils (EGL, ERL, and ECL), whereas sesquiterpenoids were more abundant in the fruit oil (EGF).
Table 1. Composition of the essential oil of fruits from Eucalyptus globulus and from Eucalyptus leaves.
EGF contained mainly aromadendrene (31.17%) followed by 1,8-cineole (14.55%), globulol (10.69%), and ledene (7.13%) (CitationMulyaningsih et al., 2010b). This result was in agreement with a previous study on EGF grown in Portugal (CitationPereira et al., 2005). However, another report on the EGF had found 1,8-cineole as the main compound (CitationBasias and Saxena, 1984). Some differences can occur in composition of oils from the same plant species probably due to genetic variation and different environmental factors (climate, harvesting seasons, geographical location) (CitationNishimura and Calvin, 1979; CitationPereira et al., 2005).
In contrast to EGF, 1,8-cineole (86.51%), α-pinene (4.74%), γ-terpinene (2.57%) and α-phellandrene (1.40%) were the main compounds in EGL, and aromadendrene was only present as a minor compound. EGL is well known to be a 1,8-Cineole-rich oil. The monoterpene 1,8-cineole has been used for medicinal, flavor and fragrance purposes. 1,8-cineole exhibits mosquito repellency (CitationKlocke, 1987), antitumor properties in rats, and anti-inflammatory activity (CitationSantos et al., 2004).
1,8-Cineole (82.66%) occurs as a major compound of ERL () followed by α-terpineol (7.03%), and α-pinene (3.68%). Eucalyptus radiata oil is sometimes preferred by aromatherapists, because its fragrance is more pleasant than EGL. This oil appears to be useful for treating disorders of the respiratory tract and is known with a high 1,8-cineole content of about 80% (CitationLis-Balchin et al., 1998). For medicinal purpose, British Pharmacopoeia, European Pharmacopoeia and Chinese Pharmacopoeia specify that eucalyptus oil must contain 1,8-cineole by not less than 70%. In the case of E. citriodora oil, it should contain at least 65% citronellal (CitationCoppen, 2002).
Neither aromadendrene nor 1,8-cineole was detected in ECL, but citronellal (90.07%) and citronellol (4.32%) were found to be major constituents. The high abundance of citronellal, the characteristic monoterpene of EGL (lemon-scented Eucalyptus), has been reported by previous authors (CitationSilva et al., 2003; CitationBatish et al., 2006). It was noteworthy that linalool, terpinen-4-ol, α-terpineol, piperiton, viridoflorol, and globulol were commonly found 1,8-cineole-rich oils, whereas the monoterpene α-pinene and γ-terpinene were present in all the oils.
Antimicrobial activity of the essential oils and the major components of the oils
The antimicrobial activities of the four essential oils and their major components (aromadendrene, 1,8-cineole, citronellal and citronellol) against multidrug-resistant bacteria are summarized in and . EGF exerted a powerful activity against MRSA strains with MIC values between 250 and 1000 µg/ml. All VRE strains were inhibited by EGF with MIC values of 500–1000 µg/ml. Against MRSA and VRE, the antimicrobial activity of aromadendrene was stronger than that of 1,8-cineole. It seems that the antimicrobial activity of EGF can be attributed to aromadendrene. This compound has a reactive exocyclic methylene group and a cyclopropane ring which can alkylate proteins and thereby disturb the conformation of proteins. Additionally, since the compound is highly lipophilic, it can disrupt the fluidity and permeability of biomembranes (CitationSikkema et al., 1995; CitationWink, 2008).
Table 2. The antimicrobial activity of the essential oils of Eucalyptus against multidrug-resistant bacteria.
Table 3. The antimicrobial activity of the major components of the Eucalyptus oils against multidrug-resistant bacteria.
In contrast to aromadendrene, the tested microorganisms were not inhibited by 1,8-cineole up to concentration 8000 µg/ml (CitationMulyaningsih et al., 2010b). This result was in agreement with previous studies (CitationInouye et al., 2001; CitationAridogan et al., 2002). The leaf oils with high 1,8-cineole contents (EGL and ERL) showed moderate activities against MRSA (MIC value of EGL: 2000 to >4000 µg/ml and of ERL: ≥4000 µg/ml). However, 1,8-cineole has been used as a flavor and fragrance purpose and for medicinal purposes. 1,8-Cineole works as a mosquito repellency (CitationKlocke, 1987) and shows antitumor activity in rats and anti-inflammatory properties (CitationSantos et al., 2004).
ECL which contains predominantly citronellal and citronellol exhibited anti-MRSA activity with MIC values ranging from 1000 to >4000 µg/ml. It was surprising that citronellal had lower activity than citronellol although it is more reactive because of its aldehyde group. Secondary metabolites with aldehyde groups normally possess good antibacterial activity through alkylation of amino group of proteins and DNA (CitationWink, 2008). Citronellol which bears a polar hydroxyl group may disturb protein conformation and cell membrane fluidity (CitationPelczar et al., 1988). The lower activity of citronellal might be due to its relatively high evaporation rate which would decrease the amount of citronellal in the assay (CitationLertsatitthanakorn et al., 2008). A number of studies had confirmed that citronellal was a weakly active monoterpene, whereas citronellol was evaluated to be more active than citronellal as an antifungal agent. A previous study reported a synergistic effect in a combination of citronellol and citronellal which would make the oil more active (CitationLow et al., 1974).
The four essential oils of Eucalyptus were hardly active against multidrug-resistant Gram-negative bacteria, except Acinetobacter baumannii (MIC of EGF, EGL, ERL, ECL was 1000, 2000, 1000, 2000 µg/ml, respectively). The low susceptibility of Gram-negative bacteria is probably due to the presence of an outer lipopolysaccharide membrane which acts a physical barrier to lipophilic compounds including essential oils (CitationVaara, 1992). Moreover, Tegos et al. reported that the low activity of lipophilic plant compounds might be due to MDR efflux through by ABC transporter (CitationTegos et al., 2002). Pseudomonas aeruginosa was reported to have a MexAB-OprM pump, whereas E. coli posses an AcrAB multidrug efflux pump (CitationMa et al., 1993). Multidrug efflux pumps were also reported from Enterobacteriaceae including efflux pumps that remove antibiotics as well as other lipophilic noxious substances from cells (CitationSchweizer, 2003). Acinetobacter baumannii is an important cause of nosocomial infections mainly affecting immunocompromised patients and shows an outstanding ability to rapidly evolve resistance to new antibiotics (CitationJoly-Guillou, 2005). Acinetobacter baumannii was more susceptible to the essential oils than the other tested bacteria probably due to the difference of its outer membrane from Enterobacteriaceae (CitationScott et al., 1976). Our results indicate that the Eucalyptus oils can be a good source of antibacterial agents particularly against A. baumannii.
Among the four oils tested, the antimicrobial activity of the oils can be ranked as EGF > ECL > ERL > EGL. Previous studies showed that E. globulus oil (91% 1,8-cineole) was less antibacterial than E. radiata (84% 1,8-cineole), whereas E. citriodora oil exhibited higher antifungal activity (CitationLis-Balchin et al., 1998; CitationRamezani et al., 2002). Regarding the activity of the major component of the oils, aromadendrene has shown the most active substance followed by citronellol, citronellal and 1,8-Cineole.
Our previous investigation demonstrated synergistic properties of combinations of aromadendrene and 1,8-cineole from the essential oil of EGF (CitationMulyaningsih et al., 2010b). For future work, it might be interesting to use the essential oils alone or in combinations with other antibacterial drugs to treat patients with nosocomial infections.
Conclusion
Our results indicate that the EGF oils are promising to be an antibacterial agent. The chemical composition of oils apparently determines the antimicrobial property. The major components will certainly contribute to the antimicrobial activity; however, minor components should also be considered because they can produce a synergistic, additive or even antagonistic interaction.
Acknowledgements
We are grateful to Prof. Dr. K. Heeg and Dr. Stefan Zimmermann for providing access to the lab facilities of the Department of Infectious Diseases, Medical Microbiology and Hygiene; and Astrid Backhaus for performing part of the GLC/FID experiments.
Declaration of interest
The authors report no declarations of interest.
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