Printer Friendly

The in vitro antimicrobial activity of Cymbopogon essential oil (lemon grass) and its interaction with silver ions.

ABSTRACT

Background: It is well known that Cymbopogon (lemon grass) essential oil exhibits antimicrobial activity while the efficacy of silver ions as a disinfectant is equally well reported.

Hypothesis: The antimicrobial activity of CEO and [Ag.sup.+] and their synergistic combinations will be useful in improving the current treatment strategies for various infections.

Study design: In the present study, we determined the chemical composition and in vitro antimicrobial activity of six different Cymbopogon essential oils (CEO's) alone and in combination with silver ions ([Ag.sup.+]) against two Gram-positive (Staphylococcus aureus and Enterococcus faecalis), two Gram-negative (Escherichia coli and Moraxella catarrhalis) and two yeast species (Candida albicans and Candida tropicalis). The nature of potential interactions was determined by fractional inhibitory concentration indices (FICIs) for CEO's and [Ag.sup.+] calculated from microdilution assays and time-kill curves.

Results: Gas chromatography-mass spectrometry results confirmed the presence of nerol, geranial and geraniol as major volatile compounds. Minimum inhibitory concentration (MIC) values confirmed that all the tested pathogens are variably susceptible to both CEO's as well as [Ag.sup.+]. The MIC of CEO's and [Ag.sup.+] against all the tested pathogens ranged from 0.032 mg/ml to 1 mg/ml and 0.004 and 0.064 mg/ml respectively, whereas when assayed in combination the FICI values were drastically reduced to range between 0.258 and 2.186, indicating synergy, additive and indifferent interactions. The most prominent interaction was observed between Cymbopogon flexuosus essential oil and [Ag.sup.+] against C. albicans with 2FIC = 0.254. The synergistic interactions were further confirmed through the construction of isobolograms and time-kill plots. Transmission electron microscopy showed disturbance in the cell envelope upon the concomitant treatment of CEO's and [Ag.sup.+], which ultimately leads to cell death.

Conclusion: Results suggest that CEO's and [Ag.sup.+] when used in combination offers an opportunity to the formulation scientist to produce novel combinations acting synergistically in the continued quest to control important infectious pathogens.

Keywords:

Cymbopogon essential oil

Silver ions

Antimicrobial activity

Synergy

Introduction

Over the past 70 years, several antimicrobial agents have been discovered and synthesized by medicinal chemists to combat microbial pathogens including bacteria, fungi, viruses, parasites, etc. However, with the passage of time many pathogens have adapted to these drugs, leading to drug resistances. The incidences of antimicrobial resistance have steadily increased globally. Besides increasing the morbidity and mortality rates, resistance to antimicrobial agents has resulted in treatment failures and increased health care costs (Howard et al. 2003). This problem has ignited the interest of researchers and clinicians in the use of natural products as antimicrobial agents. Among the plethora of natural products used as antimicrobial agents, essential oils and metal ions gained interest because of their broad spectrum activity (Chopra 2007; Low et al. 2011). Several studies have demonstrated that essential oils have an appreciable antibiotic spectrum with multiple drug targets (Khan et al. 2010; Lu et al. 2012). Cymbopogon species are commonly used in folk medicines for the treatment of infectious diseases and several other diseases and disorders (Santin et al. 2009). Among all the inorganic metal ions, the antimicrobial properties of silver have been investigated most extensively (Guggenbichler et al. 1999). Since the 19th century, silver ions have been identified to be effective against a broad range of microorganisms and during the early 20th century these ions have been approved for use as antimicrobial agents (Hugo and Russell 1982; Chopra 2007). In addition, these ions are also used to control bacterial growth in a variety of medical applications, including dentistry, catheters, and the healing of burn wounds (Klasen 2000). However, with the discovery of new antibiotics the interest in [Ag.sup.+] has diminished but interest has recently resurfaced due to the growing concern of multidrug resistance.

In view of the lack of new classes of drugs emerging and the increase in antibiotic resistances, combination therapies might be considered a viable strategy, considering the popularity of a multiple microbial drug target approach (Mukherjee et al. 2005; Ahmad et al. 2010a). Among the numerous advantages of combinational strategies is a synergistic interaction, in which the antimicrobial activity is greater than the individual contribution of each agent. In the present study, we have evaluated the in vitro antimicrobial activity of CEO's against two Gram-positive, two Gram-negative and two yeast isolates. We have explored the possibility of synergistic interaction between CEO's and [Ag.sup.+] against a panel of microbes using broth microdilution assays, isobolograms and time-kill studies. We also generated images with transmission electron microscopy (TEM) to observe the possible mechanism of action of CEO/[Ag.sup.+] alone and in combination against different tested pathogens.

Materials and methods

Essential oils

Six Cymbopogon essential oils were obtained from a commercial supplier (Paranom, Belgium); Cymbopogon giganteus Chiov. (CgEO), Cymbopogon winterianus Jowitt ex Bor (CwEO), Cymbopogon flexuosus (Nees ex Steud.) W. Watson (CfEO), Cymbopogon martinii (Roxb.) W. Watson (CmEO), Cymbopogon nardus (L.) Rendle (CnEO) and Cymbopogon citratus (DC.) Stapf (CcEO).

Strains, media and chemicals

All strains were initially grown in Tryptone Soya Broth (TSB). Prior to assays, pure cultures from Tryptone Soya Agar (TSA) plates were sub-cultured and incubated for their respective incubation periods (bacteria for 24 h and fungi for 48 h) at 37 [degrees]C. All other chemicals and media were of analytical grade and were procured from Oxoid, England. DMSO, ciprofloxacin (CFL) and amphotericin B (AmB) and AgN03 (99.99%) was purchased from Sigma Fluke.

Inoculum preparation

All bacterial strains were initially sub-cultured aseptically on the TSA and single colonies of all the cultured bacteria were subsequently grown to exponential phase in TSB at 37 [degrees]C for 24 h and adjusted to a final density of 108 colony forming units (CFU/ml) by diluting fresh cultures and comparing with McFarland scale. Fungal cells were also cultured on TSA plates and incubated at 35 [+ or -] 2 [degrees]C for 48 h until white round colonies were observed. A fresh single colony of each fungal strain was then suspended in 5 ml of sterilized saline to yield a final concentration 106 CFU/ml, when compared with the McFarland solution.

Gas chromatography-mass spectrometry analysis

All six Cymbopogon essential oils (CEO's) were subjected to GC-MS analysis using a gas chromatograph coupled to a mass spectrometer and flame ionization detector (GCMS-FID) as described previously (de Rapper et al. 2013). The Agilent (6890N) GC system was equipped with a HP-Innowax polyethylene glycol column (60 m x 250 /cm i.d x 0.25 pm film thickness). The chemical components were identified by comparing mass spectra from the total ion chromatogram, and retention indices using NIST and Mass Finder GC-MS libraries.

Antimicrobial susceptibility

Determination of minimal inhibitory concentration (MIC)

The MICs of all six Cymbopogon essential oils and [Ag.sup.+] for bacteria and fungi were determined by the Clinical and Laboratory Standards Institute recommended broth microdilution methods M7-A6 and M27-A3, respectively (CLSI, 2003,2008). Essential oils and [Ag.sup.+] were diluted to yield a concentration of 8 mg/ml using 1% DMSO and sterile distilled water as diluents, respectively. The positive control ciprofloxacin (0.01 mg/ml) for bacteria and amphotericin B (0.1 mg/ml) for yeasts and the negative vehicle control (1% DMSO) were also included in every set of experiments. Media and culture controls were included to confirm the sterility and viability, respectively. The reference test organisms, from the initial densities were adjusted to obtain an approximate final inoculum size of 5 x 105 CFU/ml for bacteria and 1 x 103 CFU/ml for fungi, were then added to each well, at a volume of 0.1 ml. The microtitre plates were sealed with a sterile adhesive film to prevent any essential oil loss due to their inherent volatility. The microtitre plates were incubated under optimal conditions (37 [degrees]C for 24 h for bacteria and 37 [degrees]C for 48 h for yeasts). After incubation, 0.4 mg/ml of p-iodonitrotetrazolium violet solution (1NT) was added to each well (0.04 ml). Viable microorganisms interact with 1NT to create a color change from clear to a red-purple color. Thus, the lowest dilution with no color change was considered as the MIC for that CEO (de Rapper et al. 2013). All the results were calculated as a mean of the experiments done in duplicate.

Assessment of the FIC index

To determine the interaction of the essential oils with the [Ag.sup.+], microdilution assays were performed in 96-well microtitre plates as described previously (Ahmad et al. 2014). Briefly, CEO's and [Ag.sup.+] were added to the microtitre plates in 1:1 volumes together with 0.1 ml media and were serially diluted. To assess the interactions, the data obtained were further analyzed using the fractional inhibitory concentration index (FICI), which is based on the zero-interaction theory of Loewe additivity. The FICI was calculated as follows:

FCI = FICa + FICb = MICa in combination/MICa tested alone + MICb in combination/MICb tested alone

where MICa and MICb are the MICs of the CEO's and [Ag.sup.+] respectively. A FICI value was interpreted as synergy when FICI is [less than or equal to] 0.5 and antagonism when FICI is > 4. A FICI result between 0.5 and 1.0 was considered additive and a value between 1.0 and 4.0 was considered as indifferent (Van Vuuren and Viljoen 2011).

Varied ratio combinations and isobolograms

On the basis of the promising synergistic interactions between the CEO's and [Ag.sup.+] observed in the microdilution assays, isobolograms were constructed. Nine ratios (9:1; 8:2; 7:3; 6:4; 5:5; 4:6; 3:7; 2:8; and 1:9) of the CEO's and [Ag.sup.+] were mixed and thereafter the MIC values were determined for these combinations, as well as for the essential oils and [Ag.sup.+] independently. Isobolograms were plotted using GraphPad Prism, version 5 software, to present the mean MIC values of the combinations as ratios (Ahmad et al. 2014). The isobolograms were interpreted by examining the data points for each ratio in relation to the MIC values for the oils independently. All points between the 1.0:1.0 line and 4.0:4.0 line were classified as non-interactive. Points between the 0.5:0.5 and 1.0:1.0 line were interpreted as additive and points below or on the 0.5:0.5 line on the isobologram were interpreted as synergistic. Antagonism was identified as data points above the 4.0:4.0 line (Van Vuuren and Viljoen 2011).

Time-kill curves

In order to further confirm the possible synergy of the CEO's and [Ag.sup.+], killing assays were performed with one Gram-positive, one Gram-negative and one yeast strain, according to the standard protocol as per the guidelines of CLS1 (NCCLS 1999). Briefly, the cell suspension of 5 x [10.sup.6] CFU/ml was diluted 1:10 in media to yield a final inoculum concentration of 5 x 105 CFU/ml. Final concentrations of CEO and [Ag.sup.+] were % MIC values for each strain. Cultures (5 ml final volume) were incubated at 37 [degrees]C with agitation (200 rpm). At predetermined time points (0,2,4,8,12 and 24 h) 100 [micro]l aliquots were removed and transferred to Eppendorf tubes, centrifuged (4000 g at 4 [degrees]C for 1 min) and rinsed twice with 1 ml of sterile distilled water to obtain compound-free cells. Pellets were suspended in 0.1 ml of sterile distilled water and serially diluted. 20 p.1 was spread onto TSA plates and incubated at 37 [degrees]C for their respective incubation periods (until the colonies were seen on the plates) to determine the numbers of CFU/ml.

Transmission electron microscopy

To determine the mechanism of action of CEO's and [Ag.sup.+] in combination, TEM imaging was performed using CmEO and [Ag.sup.+] against E. coli, S. aureus and C. albicans. All the cells at the final concentration of 1 x [10.sup.6] CFU/ml were exposed to MIC values of CmEO and [Ag.sup.+] for 1 h with shaking at 37 [degrees]C. Post exposure, to determine the ultrastructural changes, cells were washed twice for 15 min in 0.1 M sodium phosphate buffer (pH 6.0) and fixed for 2 h in 2.5% (v/v) glutaraldehyde/formaldehyde in 0.075 M phosphate buffer (pH 7.4) at room temperature. Cells were then rinsed three times with the same buffer for a period of 10 min. The specimens were post-fixed with 0.5% (w/v) aqueous osmium tetroxide for 1 h and rinsed thrice with the same buffer for 10 min. The post-fixed specimens were dehydrated in a graded ethanol series (once in 30,50,70,80, and 95% and three times in 100% for 10 min each). All the specimens were then infiltrated with 50% quetol in ethanol for 1 h followed by an infiltration in 100% quetol for 4 h. All the samples were then polymerized at 60[degrees]C for 39 h. After polymerization ultrathin sections (0.1 [micro]m) were cut using a Reichert-Jung Ultracut E microtome (Vienna, Austria) and then transferred to a copper grid. Samples were stained for 10 min in 4% aqueous uranyl acetate followed by Reynolds' lead citrate for 2 min. Samples were washed three times in Milli-Q. water and dried by touching Whatman filter paper. Sections were examined with a Jeol (Tokyo, Japan) JEM-21 OOF transmission electron microscope at 120 kV.

Results

GC-MS analysis

Aromatic plants are known to be highly variable in terms of essential oil composition, therefore the composition of each studied essential oil was determined by GC-MS. The major compounds identified in each of the six oils are given in Table 1. From these results, it was observed that the oil composition of CgEO is qualitatively similar to previous reports (e.g. Bassole et al. 2011), however, quantitative differences were observed e.g. limonene (42% vs. 11%). Concerning CcEO, the chemical composition was also in congruent with that published by Bassole et al. (2011), who reported neral (34.6%) as the major constituent which is similar to our study (30.5%). Kpoviessi et al. (2014) reported the major component in CgEO to be trans-p-mentha-l(7),8-dien-2-ol (18.3%) which is similar to our results (16.2%). For CwEO, the qualitative composition compares favorably with that published by Gonsalves et al. (2010), however, quantitative differences were observed for citronellal, geraniol, citronellol and limonene. Interestingly, Malele et al. (2007) confirmed linalool (27%) as a lead constituent of CwEO, while in our study linalool was not detected. Aromatic plants are notoriously variable in their essential oil composition. Hence, it is imperative that studies reporting the biological properties of an essential oil are accompanied by a chemical fingerprint to document the specific chemotype which will allow the results to be appropriately discussed and provide a basis for future research.

Antimicrobial activity

The MIC values obtained for all the CEO's and [Ag.sup.+] as well as for the positive controls against the six microbial species are shown in Table 2. Evaluation of MIC data showed that both CEO's as well as [Ag.sup.+] were active against all the tested pathogens with MIC values ranging from 0.016 to 1.000 mg/ml for CEO's and 0.004-0.64 mg/ml for [Ag.sup.+]. The order of sensitivity on the basis of MIC values of the CEO's against all the pathogens tested was CcEO > CmEO > CwEO > CnEO > CfEO > CgEO. Sensitivity to [Ag.sup.+] to different pathogens was observed in the order of C. tropicalis (0.004 mg/ml) > C. albicans (0.008 mg/ml) > E. co/f (0.008 mg/ml) > M. catarrhalis (0.008 mg/ml) > E. faecalis (0.032 mg/ml) > S. aureus (0.064 mg/ml). Yeast strains are highly sensitive to both CEO's and [Ag.sup.+], but a difference in sensitivity was observed with the bacterial strains where Gram-positive pathogens were more sensitive to CEO's than Gram-negative pathogens while the Gram-negative pathogens were more sensitive to [Ag.sup.+] than Gram-positive pathogens. Differences in sensitivity by Gram-positive and Gram-negative bacteria against the hydrophobic EO's and hydrophilic [Ag.sup.+] are in agreement with previous findings (Low et al. 2011).

Susceptibility in combination with silver ions

The FICI values of CEO's combined with [Ag.sup.+] were calculated to determine their possible interactions against all the tested microorganisms using the microbroth dilution method as described previously (Ahmad et al. 2014). The data, shown in Table 2, for the six CEO's in combination with [Ag.sup.+] in a 1:1 ratio, indicates synergistic, additive or indifferent interactions, while no antagonistic interaction was observed. Of the 36 combinations produced (Table 2), the FICI values against all the tested pathogens ranged from 0.254 to 2.127. For all the combinations tested (n = 36), 33% were found to be synergistic, 42% additive and 25% indifferent (Table 2). The most pronounced synergistic interaction was observed between CfEO and [Ag.sup.+] against C. albicans (FICI = 0.254). Most of the synergistic interactions were observed between CmEO and [Ag.sup.+] (67%), while no synergistic interaction was observed between CwEO and [Ag.sup.+]. For 12 of the synergistic combinations observed in the 1:1 ratio, further in depth studies were carried out through the construction of isobolograms. For isobolograms, nine different ratios of CEO's and [Ag.sup.+] were blended and antimicrobial efficacies were determined (Fig. 1). From these isobolograms, it is evident that most of the combinations, regardless of the ratio, display synergistic interactions. The combinations of CnEO and [Ag.sup.+] against M. catarrhalis and CcEO and [Ag.sup.+] against E. faecalis showed synergistic interactions for all nine ratios examined. CmEO and CfEO in combination with [Ag.sup.+] against these pathogens (M. catarrhalis and E. faecalis, respectively) showed synergy for eight of the nine ratios assayed. In other CEO combinations with [Ag.sup.+], most of the ratios showed synergy while all other ratios exhibit additive effects.

Time-kill curves

The synergism observed using the broth microdilution assay and isobolograms were further confirmed by time-kill curves. As shown in Fig. 2, CEO's and [Ag.sup.+] at their respective % MIC values did not affect the growth of tested microorganisms. Both the agents alone at lower concentrations had a weak antimicrobial effect. In contrast, the combination of CEO's with [Ag.sup.+] showed potent antimicrobial activity. In the case ofE coli and C. albicans, after 6 h of incubation, the CmEO-[Ag.sup.+] combination yielded a 5.9-log-CFU/ml and 5.6-log-CFU/ml decrease compared with a CmEO treated alone (Fig. 2A and C). The decrease for S. aureus after 12 h was observed to be 5.3-log and 5.6-log in viable counts compared with the number of CFU/ml produced in CcEO and CfEO treatment, respectively (Fig. 2B). From these results, it can also be concluded that CmEO when combined with [Ag.sup.+] against E. coli and C. albicans, the end point was attained after 6 h of incubation while as for the other CEO's the end point was only reached after 12 h of incubation. These results are congruent with the FICI values obtained from the microbroth dilution assays.

Transmission electron microscopy

Transmission electron microscopy (TEM) revealed the possible mechanisms of action for the synergistic combinations of CEO's and [Ag.sup.+] by observing the morphological features of the tested strains. The untreated cells retained their normal morphology with intact cell wall and cell membranes (Fig. 3). Untreated E. coli cells showed normal morphology having small fimbriae on their membranes (Fig. 3(1a)). Control S. aureus cells retained their coccal shape (Fig. 3(2a)) and untreated yeast cells also showed intact cellular morphology with a bound plasma membrane and cell wall (Fig. 3(3a)). In contrast the cells which are exposed to CmEO alone and CmEO in combination with Ag1 appeared to undergo cell wall and membrane disruptions which results in the release of their cellular contents into the surrounding environment. The treated cells appeared to be damaged with aberrant morphology. Precipitates are clearly visible around the cells which are exposed either to CmEO alone or to CmEO in combination with [Ag.sup.+] (Fig. 3(1 c-d), (2c-d) and (3c-d)). The bacterial cells, on the other hand, exposed to [Ag.sup.+] alone showed an intact morphology which describes the involvement of the other target sites as a mechanism of antimicrobial action of [Ag.sup.+] (Fig. 3(1 b) and (2b)). Interestingly, in the case of C. albicans cells treated with [Ag.sup.+] alone, a complete separation of the cell membrane from the cell wall was observed which could be a cellular shrinkage (Fig. 3(3b)), which is a characteristic marker of apoptosis (Khan et al. 2014).

Discussion

Cymbopogon has been used as a folk medicine in tropical and subtropical regions and is known to possess pharmacological activity, including antimicrobial and disinfectant properties (Hanaa et al. 2012; Jeong et al. 2009). The oligodynamic effect of metals on biological systems (including the antimicrobial properties) was already described in 1893 by Nageli (Nageli 1893). Among all the metals, silver ions are known to be most efficient antimicrobial agent and have been used for many years in the medical field for antimicrobial purposes (Guggenbichler et al. 1999; Fu et al. 2006). Drug combination is now emerging as the therapy of choice to treat many infections e.g. tuberculosis, malaria etc. The main aim of combination therapy is to achieve synergy, reducing drug dosage and minimizing or delaying the onset of drug resistance (Van Vuuren and Viljoen 2011; Ahmad et al. 2014).

The differences in chemical compositions of the CEO, irrespective of qualitative and/or quantitative differences with the previously published papers, restrict the validity of these interesting biological results to the CEO's with similar chemical compositions. With main lead essential oil molecules identified as geraniol, geranial and neral as confirmed by GC-MS analysis; it has already been elucidated that these compounds exhibit high antimicrobial activities against various Gram-positive and Gram-negative bacteria, as well as against the yeast Candida (Jirovetz et al. 2007; Korenblum et al. 2013). The antimicrobial activity of CEO's may be attributed to the presence of these chemical components. The antimicrobial efficacies of CEO's and [Ag.sup.+] were determined against a panel of pathogens. These results depict differences in the chemical composition of the different species of CEO's which significantly influences the antimicrobial potency of the different essential oils. The MIC values for the CEO's against Gram-negative bacteria are comparatively higher than for the Gram-positives and yeasts. As already demonstrated, our results also showed that the activity of the agents against each microbe varies due to structural differences between the microorganisms. In addition, the permeability differences due to the hydrophobic (CEO's) and hydrophilic ([Ag.sup.+]) nature of agents across the cell membrane may influence the extent of their antimicrobial efficacies. The higher content of lipopolysaccharides in the outer membrane of the Gram-negative bacteria may hinder the penetration of the hydrophobic CEO's into the cells. Our results further demonstrate that Gram-negative bacteria are more susceptible to [Ag.sup.+] than Gram-positive bacteria, which are in alignment with previous findings (Kawahara et al. 2000). In the case of Gram-positive bacteria, it is hypothesized that the positively charged [Ag.sup.+] are trapped by the negatively charged peptidoglycans in the membrane, thus restricting their entry into the cell to reach their target. Gray et al. (2003) also reported this action due to the thin cell walls of Gram-negative bacteria, which may allow rapid absorption of [Ag.sup.+] ions into the cells.

Several reports have been published illustrating that the combination of natural products and silver ions collectively improve antimicrobial activity (Low et al. 2011). It has also been observed that safe and tolerant levels of silver in combination with essential oil components are effective in controlling bacterial infections (Ghosh et al. 2013). In the present work, we have focused on the combinations of the CEO's with [Ag.sup.+] and observed that combined agents showed an increase in antimicrobial activities as measured by FICI and time-kill curves. The mechanism of action was determined by TEM imaging of untreated and treated cells. Following the CEO's and [Ag.sup.+] treatment, the cell envelope ruptures resulting in the leakage of cellular contents which ultimately leads to cell death. Essential oils are already known for impairing membrane structures and functions and can bind to proteins and sterols and augment structural changes in the cell wall and membrane, leading to cell distortion and death (Khan et al. 2014). Silver ions, on the other hand, did not disrupt the cellular morphology and therefore the mechanisms for antimicrobial properties of [Ag.sup.+] are considered different from the membrane disintegrations. To achieve the efficient bactericidal activity of silver, the ions need to enter the bacterial cell to block DNA replication when DNA is in its condensed form and also deactivates vital enzymes of the cell (Huang et al. 2011). For the synergistic antimicrobial activity of CEO's and [Ag.sup.+], it is plausible to reason that the CEO's interact with the cell membranes resulting in a compromised and porous structure, thus altering the membrane integrity which increases the penetration of [Ag.sup.+] into the cells to reach their targets. The most prominent synergy (42%) was observed with the Gram-positive bacteria, consistent with these bacteria being considered to have a high intrinsic permeability barrier for [Ag.sup.+]. The Gram-negative and yeast species have intermediate and the lowest (although significant) degree of synergies due to the comparatively lower membrane permeability for [Ag.sup.+]. This dual killing effect on the microbes gives rise to the observed enhanced antimicrobial activity of CEO's and [Ag.sup.+] at the sub-MIC values.

The in vitro hemolytic assay is a possible screening tool to gauge in vivo toxicity to host cells (Ahmad et al. 2010b). Silver ions have no or negligible cytotoxicity and hemolytic activity to humans over a different range of concentrations which exceeds the MIC values determined in this study by a factor several times larger (Kawahara et al. 2000). CEO's have also been reported to possess negligible toxicity effect on humans (Olorunnisola et al. 2014) and some studies have shown the use of CEO for maize storage and herbal teas at prescribed concentrations (Leite et al. 1986; Fandohan et al. 2008). At higher concentrations, although, some biochemical disturbances were observed in some of the volunteers, however these changes did not exhibit any medical implication (Olorunnisola et al. 2014).

To our knowledge, this is the first attempt to study the synergistic interaction between CEO's and silver ions. CEO's may prove to be a potent phytotherapeutic and/or combination agent with silver ions. Since the CEO's concentration effective in vitro is achievable in vivo, the combination of this agent with [Ag.sup.+] represents an attractive prospect for the development of new strategies to target opportunistic pathogenic microorganisms, and should be investigated further using in vivo models. With this synergistic combinatorial approach many antimicrobial agents may find even broader therapeutic applications, especially in the formulation of environmentally friendly cleaning agents, sanitizers and herbal gels.

ARTICLE INFO

Article history:

Received 30 December 2014

Revised 28 March 2015

Accepted 13 April 2015

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgments

We are grateful to Dr. G. Kamatou for the GC-MS analysis. We are also thankful to the Tshwane University of Technology and National Research Foundation (SARChl grant 86923) in South Africa for financial support.

References

Ahmad, A., Khan, A., Manzoor, N., Khan, L.A., 2010, Evolution of ergosterol biosynthesis inhibitors as fungicidal against Candida. Microb. Pathog 48, 35-41.

Ahmad, A., Khan, A., Manzoor, N., Khan, L.A., 2010. In vitro synergy of eugenol and methyleugenol with fluconazole against clinical Candida isolates. J. Med. Microbiol. 59, 1178-1184.

Ahmad, A., Van Vuuren, S., Viljoen, A., 2014. Unravelling the complex antimicrobial interactions of essential oils-the case of Thymus vulgaris (Thyme). Molecules 19, 2896-2910.

Bassole, I.H., Lamien-Meda, A., Bayala, B., Obame, L.C., Ilboudo, A.J., Franz, C., Novak, J., Nebie, R.C., Dicko, M.H., 2011. Chemical composition and antimicrobial activity of Cymbopogon citratus and Cymbopogon giganteus essential oils alone and in combination. Phytomedicine 18, 1070-1074.

Chopra, 1., 2007. The increasing use of silver-based products as antimicrobial agents: a useful development or a cause for concern? J. Antimicrob. Chemother. 59, 587-590.

CLSI--Clinical and Laboratory Standards Institute, 2003, sixth ed Methods For Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, Approved Standard M7-A6, National Committee for Clinical Laboratory Standards, Fort Wayne. IN, USA.

CLSI--Clinical and Laboratory Standards Institute, 2008. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeast, Approved Standard M27-A3, Clinical and Laboratory Standards Institute Standards, Fort Wayne, IN, USA, p. 25.

de Rapper, S., Kamatou, G., Viljoen, A., Van Vuuren, S., 2013. The in vitro antimicrobial activity of Lavandula angustifolia essential oil in combination with other aromatherapeutic oils. Evid. Based Complement. Altern. Med. 852049, 10.

Fandohan, P., Gnonlonfin, B., Laleye, A.. Gbenou, J.D., Darboux, R., Moudachirou, M., 2008. Toxicity and gastric tolerance of essential oils from Cymbopogon citratus, Ocimum gratissimum and Ocimum basilicum in Wistar rats. Food Chem. Toxicol 46, 2493-2497.

Fu, J., Ji, J., Fan, D., Shen, J., 2006. Construction of antibacterial multilayer films containing nanosilver via layer-by-layer assembly of heparin and chitosan-siiver ions complex. J. Biomed. Mater. Res. A 79, 665-674.

Ghosh, I.N., Patil. S.D., Sharma, T.K., Srivastava, S.K., Pathania, R., Navani, N.K., 2013. Synergistic action of cinnamaldehyde with silver nanoparticles against spore-forming bacteria: a case for judicious use of silver nanoparticles for antibacterial applications. Int.J. Nanomed. 8, 4721-4731.

Gonsalves, T.B., de Sousa, E.O., Rodrigues. F.F.G., da Costa, J.G.M., 2010. Chemical composition and antibacterial evaluation of the essential oil from Cymbopogon winterianus Jowitt (Gramineae). JEOBP 13, 426-431.

Gray, J.E., Norton, P.R., Alnouno, R.. Marolda, C.L, Valvano, M.A., Griffiths, K., 2003. Biological efficacy of electroless-deposited silver on plasma activated polyurethane. Biomaterials 24, 2759-2765.

Guggenbichler, J.P., Boswald, M., Lugauer, S., Krall, T., 1999. A new technology of micro-dispersed silver in polyurethane induces antimicrobial activity in central venous catheters. Infection 27, 16-23.

Hanaa, A.R.M., Sallam, Y.I., El-Leithy, A.S., Aly, S.A., 2012. Lemongrass (Cymbopogon citratus) essential oil as affected by drying methods. Ann. Agric. Sci. 57, 113-116.

Howard, D.H., Scott 2nd, R.D., Packard, R., Jones, D., 2003. The global impact of drug resistance. Clin. Infect. Dis. 36. S4-S10.

Huang, L., Dai, T., Xuan, Y., Tegos, G.P., Hamblin, M.R., 2011. Synergistic combination of chitosan acetate with nanoparticle silver as a topical antimicrobial: efficacy against bacterial burn infections. Antimicrob. Agents Chemother. 55, 3432-3438.

Hugo, W.B., Russell, A.D., 1982. Types of antimicrobial agents, in: Russell, A.D., Hugo, W.B., Ayliffe, G.A.J. (Eds.), Principles and Practice of Disinfection, Preservation and Sterilisation. Blackwell Scientific Publications, Oxford, UK, pp. 8106.

Jeong, M.R., Park, P.B., Kim, D.H., Jang, Y.S., Jeong, H.S., Choi, S.H., 2009. Essential oil prepared from Cymbopogon citrates exerted an antimicrobial activity against plant pathogenic and medical microorganisms. Mycobiology 37, 48-52.

Jirovetz, L., Buchbauer, G., Schmidt, E., Stoyanova, A.S., Denkova, Z., Nikolova, R., Geissler, M., 2007. Purity, antimicrobial activities and olfactoric evaluations of geraniol/nerol and various of their derivatives. J. Essent. Oil Res. 19, 288-291.

Kawahara, K., Tsuruda, K., Morishita, M., Uchida, M., 2000. Antibacterial effect of silver-zeolite on oral bacteria under anaerobic conditions. Dent. Mater. 16, 452-455.

Khan, A., Ahmad, A., Akhter, F., Yousuf, S., Xess, I., Khan, LA, Manzoor, N., 2010. Ocimum sanctum essential oil and its active principles exert their antifungal activity by disrupting ergosterol biosynthesis and membrane integrity. Res. Microbiol 161, 816-823.

Khan, A., Ahmad, A., Khan, L.A., Manzoor, N., 2014. Ocimum sanctum (L.) essential oil and its lead molecules induce apoptosis in Candida albicans. Res. Microbiol 165, 411-419.

Klasen, H.J., 2000. Historical review of the use of silver in the treatment of burns. I. Early uses. Burns 26, 117-130.

Korenblum, E., Regina de Vasconcelos Goulart, F., de Almeida Rodrigues, I., Abreu, F., Lins, II., Alves, P.B., Blank, A.F., Valoni, E., Sebastidn, G.V., Alviano, D.S., Alviano, C.S., Seldin, L., 2013. Antimicrobial action and anti-corrosion effect against sulfate reducing bacteria by lemongrass (Cymbopogon citratus) essential oil and its major component, the citral. AMB Express 3, 44.

Kpoviessi, S., Bero, J., Agbani, P., Gbaguidi, F., Kpadonou-Kpoviessi, B., Sinsin, B., Accrombessi, G., Freddrich, M., Moudachirou, M., Quetin-Leclercq, J., 2014. Chemical composition, cytotoxicity and in vitro antitrypanosomal and antiplasmodial activity of the essential oils of four Cymbopogon species from Benin. J Ethnopharmacol 151, 652-659.

Leite, J.R., Seabra, M.L, Maluf, E., Assolant, K., Suchecki, D., Tufik, S.. Klepacz, S., Calil, H.M., Carlini, E.A., 1986. Pharmacology of lemongrass (Cymbopogon citratus Stapf). 111. Assessment of eventual toxic, hypnotic and anxiolytic effects on humans. J. Ethnopharmacol 17, 75-83.

Low, W.L., Martin, C., Hill, D.J., Kenward, M.A., 2011. Antimicrobial efficacy of silver ions in combination with tea tree oil against Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans. Int. J. Antimicrob. Agents 37, 162-165.

Lu. J.J., Pan, W., Hu, Y.J., Wang, Y.T., 2012. Multi-target drugs: the trend of drug research and development. PLoS One 7, e40262.

Malele, R.S., Mwangi, J.W., Thoithi, G.N., Kibwage, I.O., Ldpez, M.L., Zunino, M.P.. Ldpez, A.G., Zygadlo, J.A., Oliva, M.M., Demo, M.S., 2007. Essential oil of Cymbopogon winterianus Jowitt from Tanzania: composition and antimicrobial activity. JEOBP10, 83-87.

Mukherjee, P.K., Sheehan, D.J., Hitchcock, CA., Ghannoum, MA., 2005. Combination treatment of invasive fungal infections. Clin. Microbiol. Rev. 18, 163-194.

Nageli, K.W., 1893. Ober oligodynamische Erscheinungen in lebenden Zellen. Neue Denkschr. Allgemein. Schweiz. Gesellsch. Ges. Naturweiss. Bd XXXIII Abt 1.

NCCLS, 1999. Methods for Determining Bactericidal Activity of Antimicrobial Agents: Approved Guideline M26-A. National Committee for Clinical Laboratory Standards, Wayne, PA, p. 19.

Olorunnisola, S.K., Asiyanbi, H.T., Hammed, AM., Simsek, S., 2014. Biological properties of lemongrass: an overview. Int. Food Res.J. 21, 455-462.

Santin, M.R., dos Santos, A.O., Nakamura, C.V., Filho, B.P.D., Ferreira, I.C.P., Ueda-Nakamura, T., 2009. In vitro activity of the essential oil of Cymbopogon citratus and its major component (citral) on Leishmania amazonensis. Parasitol. Res. 105, 1489-1496.

Van Vuuren, S., Viljoen, A., 2011. Plant-based antimicrobial studies--methods and approaches to study the interaction between natural products. Planta Med 77, 1168-1182.

Aijaz Ahmad (3), Alvaro Viljoen (a, b), *

(a) Department of Pharmaceutical Sciences, Tshwane University of Technology, Private BagX680, Pretoria 0001, South Africa

(b) Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia

* Corresponding author at: Department of Pharmaceutical Sciences, Tshwane University of Technology. Private Bag X680, Pretoria 0001, South Africa. Tel.: +2712 382 6373.

E-mail address: viljoenam@tut.ac.za (A. Viljoen).

http://dx.doi.org/10.1016/j.phymed.2015.04.002

Table 1.
Chemical composition of different Cymbopogon essential
oils.

Compound                            RRI     %

Cymbopogon giganteus (CgEO)
Limonene                            1194   11.8
ris-p-Mentha-2,8-dien-l-ol          1613   21.3
1,3,8-p-Menthatriene                1664   17.8
Carveol (isomer)                    1713   8.8
Carvone                             1741   3.3
trans-p-Mentha-1 (7),8-dien-2-ol    1802   16.2
6-Acetoxy-p-mentha-l(7),8-diene     1808   5.6
Carveol (isomer)                    1839   3.1
Total                                      88.9

Cymbopogon winterianus (CwEO)
Limonene                            1194   3.6
Isopulegol                          1340   1.3
Citronellal                         1482   36.7
[beta]-Elemene                      1591   2.1
Citronellyl acetate                 1662   2.4
Bicyclo-sesquiphellandrene          1723   2.1
Citronellol                         1765   13.1
Cadinene                            1808   4.9
Geraniol                            1822   21.8
Elemol                              2087   2.9
Eugenol                             2188   1.5
Total                                      92.4

Cymbopogon flexuosus (CfEO)
Limonene                            1194   1.8
Linalyl acetate                     1563   2.1
[beta]-Caryophyllene                1596   1.0
Neral                               1689   32.9
Geranial                            1740   46.1
Cadinene                            1808   3.5
Geraniol                            1822   6.1
Total                                      93.5

Cymbopogon martinii (CmEO)
[lambda]-Terpinene                  1242   1.5
Linalool                            1541   3.1
[beta]-Caryophyllene                1596   1.9
Nerol                               1800   8.9
Geraniol                            1822   79.7
Total                                      95.1

Cymbopogon nardus (CnEO)
Tricydene                           1005   1.0
[alpha]-Pinene                      1016   1.2
Camphene                            1057   7.5
Limonene                            1194   7.5
Z-[beta]-Ocimene                    1232   1.9
E-[beta]-Ocimene                    1250   1.0
Citronellal                         1482   3.7
[beta]-Elemene                      1591   0.9
[beta]-Caryophyllene                1596   1.9
Citronellyl acetate                 1662   0.9
[alpha]-Farnesene                   1665   3.9
[alpha]-Terpineol                   1701   1.4
Borneol                             1702   5.9
Geranyl acetate                     1758   7.7
Cadinene                            1763   1.2
Citronellol                         1765   3.1
Geraniol                            1822   25.9
Geranyl butyrate                    1893   1.3
Methyl isoeugenol                   2021   7.3
Elemol                              2087   1.4
Total                                      86.6

Cymbopogon dtratus (CcEO)
Limonene                            1194   5.1
6-Methyl-5-hepten-2-one             1339   1.3
Linalyl acetate                     1563   1.2
[beta]-Caryophyllene                1596   1.9
Neral                               1689   30.5
Geranial                            1740   42.1
Geranyl acetate                     1758   4.7
Cadinene                            1808   1.2
Geraniol                            1822   7.1
Total                                      95.1

Table 2
Minimum inhibitory concentrations and fractional inhibitory
concentration index of Cymbopogon essential oils and silver ions.

Essential    MIC (mg/ml)                  E. coli    M. catarrhalis
oils                                      ATCC8739   ATCC 23246

CcEO         MIC (mg/ml) alone            0.063      0.250
             [MIC.sub.CcEO] (mg/ml) in    0.063      0.016
             combination
             [MIC.sub.Ag+] (mg/ml) in     0.500      0.500
             combination
             FICI                         0.563      0.516
             Interpretation               ADD        ADD
CmEO         MIC (mg/ml) alone            0.125      0.125
             [MIC.sub.CmEO] (mg/ml) in    0.016      0.016
             combination
             [MIC.sub.Ag+] (mg/ml) in     0.250      0.250
             combination
             FICI                         0.266      0.266
             Interpretation               SYN        SYN
CwEO         MIC (mg/ml) alone            0.500      0.500
             [MIC.sub.CwEO] (mg/ml) in    0.016      0.008
             combination
             [MIC.sub.Ag+] (mg/ml) in     1.000      0.500
             combination
             FICI                         1.016      0.508
             Interpretation               IND        ADD
CnEO         MIC (mg/ml) alone            0.125      0.250
             [MIC.sub.CnEo] (mg/ml) in    0.016      0.008
             combination
             [MIC.sub.Ag+] (mg/ml) in     0.250      0.250
             combination
             FICI                         0.266      0.258
             Interpretation               SYN        SYN
CfEO         MIC (mg/ml) alone            1.000      1.000
             [MIC.sub.cfEo] (mg/ml) in    0.008      0.008
             combination
             [MIC.sub.Ag+] (mg/ml) in     1.000      1.000
             combination
             FICI                         1.008      1.008
             Interpretation               IND        IND
CgEO         MIC (mg/ml) alone            1.000      0.500
             [MIC.sub.CgEO] (mg/ml) in    0.008      0.016
             combination
             [MIC.sub.ag+] (mg/ml) in     1.000      1.000
             combination
             FICI                         1.008      1.016
             Interpretation               IND        IND
[Ag.sup.+]   MIC alone                    0.008      0.008
Positive
  controls
  (a)        0.0005                       0.001      0.0005

Essential    MIC (mg/ml)                  S. aureus    E. faecalis
oils                                      ATCC126000   ATCC29212

CcEO         MIC (mg/ml) alone            0.125        0.125
             [MIC.sub.CcEO] (mg/ml) in    0.128        0.064
             combination
             [MIC.sub.Ag+] (mg/ml) in     0.250        0.250
             combination
             FICI                         0.378        0.314
             Interpretation               SYN          SYN
CmEO         MIC (mg/ml) alone            0.125        0.063
             [MIC.sub.CmEO] (mg/ml) in    0.500        0.254
             combination
             [MIC.sub.Ag+] (mg/ml) in     0.977        0.500
             combination
             FICI                         1.477        0.754
             Interpretation               ADD          ADD
CwEO         MIC (mg/ml) alone            0.125        0.125
             [MIC.sub.CwEO] (mg/ml) in    0.500        0.252
             combination
             [MIC.sub.Ag+] (mg/ml) in     0.977        0.984
             combination
             FICI                         1.477        1.236
             Interpretation               IND          IND
CnEO         MIC (mg/ml) alone            0.250        0.250
             [MIC.sub.CnEo] (mg/ml) in    0.128        0.064
             combination
             [MIC.sub.Ag+] (mg/ml) in     0.500        0.500
             combination
             FICI                         0.628        0.564
             Interpretation               ADD          ADD
CfEO         MIC (mg/ml) alone            0.500        0.250
             [MIC.sub.cfEo] (mg/ml) in    0.064        0.032
             combination
             [MIC.sub.Ag+] (mg/ml) in     0.500        0.250
             combination
             FICI                         0.564        0.282
             Interpretation               ADD          SYN
CgEO         MIC (mg/ml) alone            0.500        0.250
             [MIC.sub.CgEO] (mg/ml) in    0.032        0.032
             combination
             [MIC.sub.ag+] (mg/ml) in     0.250        0.250
             combination
             FICI                         0.282        0.282
             Interpretation               SYN          SYN
[Ag.sup.+]   MIC alone                    0.064        0.032
Positive
  controls
  (a)        0.0005                       0.001        0.001

Essential    MIC (mg/ml)                  C. albicans   C. tropicalis
oils                                      ATCC 10231    ATCC201380

CcEO         MIC (mg/ml) alone            0.063         0.016
             [MIC.sub.CcEO] (mg/ml) in    0.063         0.063
             combination
             [MIC.sub.Ag+] (mg/ml) in     0.500         0.500
             combination
             FICI                         0.563         0.563
             Interpretation               ADD           ADD
CmEO         MIC (mg/ml) alone            0.063         0.032
             [MIC.sub.CmEO] (mg/ml) in    0.032         0.032
             combination
             [MIC.sub.Ag+] (mg/ml) in     0.250         0.250
             combination
             FICI                         0.282         0.282
             Interpretation               SYN           SYN
CwEO         MIC (mg/ml) alone            0.125         0.063
             [MIC.sub.CwEO] (mg/ml) in    0.032         0.063
             combination
             [MIC.sub.Ag+] (mg/ml) in     0.500         1.000
             combination
             FICI                         0.532         1.063
             Interpretation               ADD           IND
CnEO         MIC (mg/ml) alone            0.125         0.063
             [MIC.sub.CnEo] (mg/ml) in    0.032         0.032
             combination
             [MIC.sub.Ag+] (mg/ml) in     0.500         0.500
             combination
             FICI                         0.532         0.532
             Interpretation               ADD           ADD
CfEO         MIC (mg/ml) alone            0.250         0.063
             [MIC.sub.cfEo] (mg/ml) in    0.004         0.127
             combination
             [MIC.sub.Ag+] (mg/ml) in     0.250         2.000
             combination
             FICI                         0.254         2.127
             Interpretation               SYN           IND
CgEO         MIC (mg/ml) alone            0.125         0.125
             [MIC.sub.CgEO] (mg/ml) in    0.016         0.016
             combination
             [MIC.sub.ag+] (mg/ml) in     0.500         0.500
             combination
             FICI                         0.516         0.516
             Interpretation               ADD           ADD
[Ag.sup.+]   MIC alone                    0.008         0.004
Positive
  controls
  (a)        0.0005                       0.001

(a) Controls are ciprofloxacin for bacteria and amphotericin
B for the yeasts.
COPYRIGHT 2015 Urban & Fischer Verlag
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2015 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:Phytomedicine
Author:Ahmad, Aijaz; Viljoen, Alvaro
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Jun 15, 2015
Words:6924
Previous Article:Detection of pyrrolizidine alkaloids in German licensed herbal medicinal teas.
Next Article:Synergistic mutual potentiation of antifungal activity of Zuccagnia punctata Cav. and Larrea nitida Cav. extracts in clinical isolates of Candida...
Topics:

Terms of use | Privacy policy | Copyright © 2019 Farlex, Inc. | Feedback | For webmasters