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International Journal of Pharmaceutics 476 (2014) 134141

Contents lists available at ScienceDirect

International Journal of Pharmaceutics


journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical nanotechnology

Rhamnolipids as emulsifying agents for essential oil formulations:


Antimicrobial effect against Candida albicans and methicillin-resistant
Staphylococcus aureus
Ester Haba a , Samira Bouhdid c , Noelia Torrego-Solana a , A.M. Marqus a ,
M. Jos Espuny a , M. Jos Garca-Celma b , Angeles Manresa a, *
a

Unitat de Microbiologia, Facultat de Farmcia, Universitat de Barcelona, Joan XXIII s/n, 08028 Barcelona, Spain
Department of Pharmacy and Pharmaceutical Technology, R+D Associated Unit to CSIC, Faculty of Pharmacy, University of Barcelona, Joan XXIII s/n, 08028
Barcelona, Spain
c
Dpartement de Phytologie, Institut National des Plantes Mdicinales et Aromatiques, Universit Sidi Mohamed Ben Abdellah-Fs, Morocco
b

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 30 June 2014
Received in revised form 16 September 2014
Accepted 26 September 2014
Available online 28 September 2014

This work examines the inuence of essential oil composition on emulsication with rhamnolipids and
their use as therapeutic antimicrobial agents against two opportunistic pathogens, methicillin-resistant
Staphylococcus aureus (MRSA) and Candida albicans. Rhamnolipids, produced by Pseudomonas aeruginosa,
with waste frying oil as the carbon source, were composed of eight rhamnolipid homologues. The
rhamnolipid mixture was used to produce emulsions containing essential oils (EOs) of Melaleuca
alternifolia,Cinnamomum verum, Origanum compactum and Lavandula angustifolia using the titration
method. Ternary phase diagrams were designed to evaluate emulsion stability, which differed depending
on the essential oil. The in vitro antimicrobial activity of the EOs alone and the emulsions was evaluated.
The antimicrobial activity presented by the essential oils alone increased with emulsication. The surface
properties of rhamnolipids contribute to the positive dispersion of EOs and thus increase their availability
and antimicrobial activity against C. albicans and S. aureus. Therefore, rhamnolipid-based emulsions
represent a promising approach to the development of EO delivery systems.
2014 Elsevier B.V. All rights reserved.

Keywords:
Essential oils
Rhamnolipids
Emulsions
Phase behaviour
Methicillin-resistant

1. Introduction
Essential oils (EOs) are complex mixtures of volatile organic
molecules extracted from aromatic plants by different methods.
They are oil-like in nature, frequently characterized by a strong
fragrance (Morais et al., 2008) and may contain up to 100 components, mainly terpenes and phenylpropanoids (Hammer and
Carson, 2011). This structural diversity is responsible for the wide
variety of biological activities exhibited by EOs. Several authors
have reported that many EOs, especially those rich in phenols,
aldehydes and alcohols, are effective in inhibiting spoilage and
pathogenic microorganisms (Hammer and Carson, 2011). They
therefore represent a natural alternative to the synthetic
antimicrobials used in the cosmetic, food and pharmaceutical
industries (Lang and Buchbauer, 2012). Their effectiveness means
they can be used in small amounts (Hammer and Carson, 2011).

* Corresponding author. Tel.: +34 93 4024496; fax: +34 93 4024498.


E-mail address: amanresa@ub.edu (A. Manresa).
http://dx.doi.org/10.1016/j.ijpharm.2014.09.039
0378-5173/ 2014 Elsevier B.V. All rights reserved.

One of the most widely studied EO is that derived from


Melaleuca alternifolia, (Myrtaceae), also known as tea tree oil (TTO).
This oil is extracted by steam distillation from the leaves and
terminal branchlets of M. alternifolia and has been used
medicinally for many years. TTO has broad-spectrum in vitro
antimicrobial activity that is mainly attributed to the presence of
terpinen-4-ol and 1,8-cineole, which are both major components
of the oil. TTO is widely used as an antiseptic agent in denture-,
mouth- and hand-wash products. It is also added to topical
formulations for the treatment of cutaneous infections (Carson
et al., 2006; Charles et al., 2013; Sharma et al., 2010).
The EO extracted from Origanum compactum (Lamiaceae), an
endemic species growing in Morocco, has been reported to be
effective in vitro against a wide range of bacteria and fungi,
including pathogenic strains (Bouhdid et al., 2009; Oussalah et al.,
2007). This oil is mainly composed of carvacrol and thymol, which
are phenolic terpene isomers known for their antimicrobial
activity (Ahmad et al., 2011). Cinnamon (Cinnamomum zeylanicum
Blume, syn Cinnamomum verum) belongs to the Lauraceae family.
The EO extracted from cinnamon bark is widely used and has many
applications in perfumery and the food and pharmaceutical

E. Haba et al. / International Journal of Pharmaceutics 476 (2014) 134141

industries (Lee and Balick, 2005). This oil is rich in cinnamaldehyde


and eugenol and exerts several biological functions. Numerous
studies have reported that cinnamon bark oil effectively inhibits
the growth of bacteria and fungi (Bouhdid et al., 2010; Unlu et al.,
2010). Lavender (Lavandula angustifolia), a member of the
Lamiaceae family, produces an EO that is rich in linalyl
acetate and linalool. This oil has been reported to exhibit some
antimicrobial activity against pathogenic microorganisms (de
Rapper et al., 2013; Hammer et al., 1999a).
The industrial application of antimicrobial EOs and their
components is limited by their hydrophobicity, avouring
properties and high volatility (Hyldgaard et al., 2012); it is
therefore important to determine an appropriate formulation for
commercial applications. The pharmaceutical and food industries
have developed several approaches to deliver sensitive antimicrobials (Gudia et al., 2013). Recent studies have reported that some
delivery systems (i.e. emulsion) generally improve the activity of
antimicrobials from herbs and spices and help maintain their
stability (Gaysinsky et al., 2008). An emulsion consists of one liquid
dispersed in another immiscible liquid in the form of droplets, and
is usually stabilized by surfactants that assemble at the interface.
This system may and ensure dispersion of oil in the aqueous phase
where microorganisms are most likely to accumulate. To avoid
possible negative interactions with EO activity, it is important to
choose an appropriate surfactant for the emulsion, since some
surfactants have been found to compromise the antimicrobial
activity of TTO (Hammer et al., 1999b).
Rhamnolipids constitute one of the most important classes of
microbial surfactants and have shown excellent emulsifying
potential with a variety of compounds. They are suitable
candidates for industrial use, since their surface-active properties
remain stable over a broad pH range and when heated (Lovaglio
et al., 2011). Rhamnolipids exert antimicrobial activity against both
fungi and bacteria (Abalos et al., 2001; Haba et al., 2003a; Nitsche
et al., 2005). Their therapeutic applications have been the subject
of studies (Gharaei-Fathabal, 2011; Rodrigues et al., 2006) and
some interesting articles have recently been published (Fracchia
et al., 2012; Gudia et al., 2010).
To our knowledge, no information is available on the use of
rhamnolipids as emulsifying agents in EO formulations intended
for microbial inhibition. The objectives of this study were: (i) to
explore the emulsication behaviour of rhamnolipids with four
commercial EOs (O. compactum, M. alternifolia, C. zeylanicum and L.
angustifolia,) using ternary phase diagrams, and (ii) to assess the in
vitro antimicrobial activity of the emulsions against Staphylococcus
aureus (MRSA) and Candida albicans.
2. Materials and methods
2.1. Microorganisms and growth culture
Pseudomonas aeruginosa 47T2 NCIB 40,044, isolated from an oilcontaminated soil sample, was selected due to its capacity to
produce surface-active rhamnolipids from hydrophobic substrates.
After being grown on TSA (tripticase soy agar, Pronadisa, Barcelona,
Spain), the bacterial strain was maintained at 4  C and also
preserved in cryobilles (AES Chemunex S.A., Terrassa, Spain) at
20  C.
Experiments were carried out in 2-l bafed Erlenmeyer asks
containing 400 ml of medium with the following composition (g/l):
NaNO3 5, KH2PO4 2.0, K2HPO4 1.0, KCl 0.1, MgSO47H2O 0.5, CaCl2
0.01, FeSO47H2O 0.012 and yeast extract 0.01. We added 0.05 ml of a
trace element solution containing (g/l): H3BO3 0.26, CuSO45H2O
0.5, MnSO4H2O 0.5, MoNa2O42H2O 0.06 and ZnSO47H2O 0.7 to
this medium. Finally, 40 g/l of olive/sunower (50:50 v/v) waste
frying oil was used as a carbon source, containing oleic acid

135

(50.29%) and linoleic acid (34.23%) as the major components, and


stearic acid (7.70%) and palmitic acid (7.77%) in smaller quantities.
The medium components were sterilized separately at 120  C,
1 atm for 20 min. The initial pH of the medium was adjusted to 7.2.
A 2% (v/v) cell suspension in sterile saline (0.9% NaCl) of an
overnight culture on TSA (Pronadisa, Barcelona, Spain) was used as
the inoculum. Cultures were incubated at 30  C for 96 h on a
reciprocal rotary shaker at 150 rpm.
2.2. Rhamnolipid production and characterization
Microbial growth was calculated by measuring the protein
content of the cultures in accordance with the method described
by Lowry et al. (1951). Total rhamnolipid (RL) production was
measured as rhamnose by a specic colorimetric method
(Chandrasekaran and Bemiller, 1980). The RL content was
calculated by multiplying the rhamnose concentration by a factor
of 3.0, which represents the rhamnolipid/rhamnose calculated
using the puried product.
Rhamnolipids were recovered and puried. Cells were removed
from the culture by centrifugation (12,000  g) for 30 min.
Purication was carried out by adsorption chromatography
(Torrego-Solana et al., 2014).
Liquid chromatography-mass spectrometry (LCMS) was
performed in order to analyse the composition of the RL mixture,
and a 10-mg portion was resuspended in 1 ml of methanol and
analysed by LCMS. Rhamnolipid mixtures were separated and
identied by LCMS using a Waters 2690 separation module
(Waters, Milford, MA, USA). Samples were injected (10 ml) into a
C18 Spherisorb ODS2 150  4.6-mm column (Teknokroma, Sant
Cugat, Spain). The LC ow rate was 1 ml min 1. An acetonitrile
water gradient was used, starting with 30% acetonitrile for 2 min,
followed by a ramp of 30100% acetonitrile for 30 min, before
being left to stand for 5 min and then returned to the initial
conditions. Post-column addition of acetone at 200 ml/min was
performed using a Phoenix 20 syringe pump (Carlo Erba, Rodano,
Italy), since we observed a rise in sensitivity to rhamnolipids. The
LC efuent and acetone were mixed in a tee valve (Valco) and split
(1/50) before being introduced into the mass spectrometer. MS
was performed with a single quadrupole mass spectrometer, VG
Platform II (Micromass, Manchester, UK), equipped with a
pneumatically assisted electrospray (ES) source. Negative ion
mode was used (N-ES). Full scan data were obtained by scanning
from m/z 100 to 750 in centroid mode, using a scan duration of
2.0 s and an inter-scan time of 0.2 s. The working conditions for NES were as follows: dry nitrogen was heated to 80  C and
introduced into the capillary region at a ow rate of 400 l/h.
The capillary was held at a potential of 3.5 kV and the extraction
voltage was held at
80 V. Rhamnolipid homologues were
quantied from the molecular proportion of each of the
pseudomolecular ions calculated by LCMS (Haba et al., 2003b).
The proportion (%) of each component was calculated from the
areas obtained for each molecule of RL by LCMS.
Equilibrium surface tension (g ST) and critical micelle concentration (CMC) were measured at 25  C by the DuNoy ring method
with a Krss K9 tensiometer (Hamburg, Germany). The instrument
was calibrated against Milli-Q ultrapure distilled water (Millipore,
Billerica, MA, USA). Aqueous solutions with various concentrations
of biosurfactants (2005 mg/l) were obtained by successively
dilutions prepared by weight in Millipore ultrapure water for CMC
determination. To reach equilibrium, all sample solutions were
aged in appropriate cells at room temperature (25  C). The
platinum plate and all glassware used were cleaned in chromic
mixture. CMC was calculated from surface tension plot values
versus log surfactant concentration after reaching equilibrium
at 25  C.

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E. Haba et al. / International Journal of Pharmaceutics 476 (2014) 134141

2.3. Essential oils


The four EOs (supplied by Pranarm, Ghislenghien, Belgium)
used for this study were oregano essential oil (OEO) extracted from
O. compactum ower heads from Morocco, cinnamon essential oil
(CEO) extracted from the bark of C. zeylanicum from Sri Lanka, tea
tree oil (TTO) extracted from the leaves of M. alternifolia from
Australia, and lavender essential oil (LEO) extracted from ower
heads of L. angustifolia from France. Oils were commercialized and
chemotyped by Pranarm International (Ghislenghien, Belgium).
According to the supplier, the major components of the oils were:
carvacrol (37.77%), thymol (19.79%) and g-terpinene (17.01%) for
OEO; E-cinnamaldehyde (77.31%) for CEO; terpinen-4-ol (39.39%)
and g-terpinene (20.76%) for TTO; and linalool (30.60%) and linalyl
acetate (34.09%) for LEO). The GC and the physical characteristics of
the EOs are included (Supplementary information). All EOs were
used as supplied.
2.4. Determination of emulsifying properties of rhamnolipids in water/
oil mixtures
The emulsifying properties of puried RL were determined
following the titration method and represented in ternary phase
diagrams (Sadurn et al., 2005). Non-equilibrium phase diagrams
were performed at 25  C by stepwise addition of water to the RL
and oil mixture. Emulsions were prepared by weighing the
components up to 1 g as follows: the required concentrations of
RL were weighed in glass tubes, mixed with the required amount of
the hydrophobic component and vortexed until complete dissolution of the biosurfactant was achieved. The appropriate amount of
deionized water was then added slowly and the tube was tightly
stoppered and mixed vigorously for 1 min. The shaking time and
vortex speed were kept constant. Finally, the mixtures were kept in
a thermostatic bath at a constant temperature of 25  C for 24 h. The
emulsion boundaries were determined by visual inspection. The
samples were examined to determine if a clear single phase formed
immediately or phase separation occurred, or an opaque mixture
appeared, which would indicate the coexistence of two or more
phases. The optical anisotropic aspect was detected with crossed
polarizers.
The physical aspect of the mixtures was plotted on a ternary
phase diagram with three vertexes representing water, RL and oil
(W/RL/EO). The emulsion region was represented. The selected
hydrophobic components assayed in this study were the four EOs
in the range of 0.590%. The maximum amount of biosurfactant
was 20% (from 0.1% to 20%). The dots of emulsions on the phase
diagram were coded and the percentage of each component given
(%W/%RL/%EO); the starting points were: (75-20-5); (66-20-14);
(50-20-30); (34-20-46); (0-20-80); (0-10-90). The results obtained
with the different oils studied were compared and the emulsions
were also characterized by optical microscopy (Leica DM IL LED,
Leica Microsystems Barcelona, Spain). Images were acquired with a
Leica EC3 digital camera and the software Image.
2.5. Minimum inhibitory concentration (MIC) of essential oils and
rhamnolipids
First the antimicrobial activity, based on MICs, of the EOs and
rhamnolipids was determined; for C. albicans it was ATCC 10231
and for methicillin-resistant S. aureus (MRSA) it was ATCC
43300.
MICs were determined using the broth microdilution assay
(Bouhdid et al., 2009): rst, 50 ml of Mueller Hinton Broth (MHB,
Oxoid, Basingstoke, UK) or Sabouraud-dextrose broth (SDB, Oxoid,
Basingstoke, UK) supplemented with bacteriological agar (0.15% w/
v) was distributed from the second to the 12th well of a 96-well

polypropylene microtitre plate (Costar, Corning Incorporated,


Corning, NY, USA). A dilution of the EO was prepared to a nal
concentration of 4% in the corresponding medium. Then, 100 ml of
these suspensions were added to the rst test well of each
microtitre line, and then 50 ml of scalar dilution was transferred
from the second to the 11th well. The 12th well was considered as
the growth control, because no EO was added. We then added 50 ml
of a microbial suspension to each well at a nal concentration of
approximately 105106CFU/ml. The nal concentration of EO was
between 2% and 0.0019% (v/v). Plates were incubated at 37  C for
18 h. After incubation, 5 ml of resazurin (0.01% w/v) was added to
each well to assess active microbial growth. After further
incubation at 37  C for 2 h, the MIC was determined as the lowest
EO concentration that prevented a change in resazurin colour.
Active microbial growth was detected by reduction of blue dye
resazurin to pink resorun. A control was carried out to ensure that
the EO did not cause a colour change in the resazurin at the
concentrations tested. Experiments were performed in triplicate
and modal values were selected. The same method was followed
for the RL, although nal concentrations of rhamnolipids ranged
from 256 mg/ml to 0.0244 mg/ml. Vancomycin hydrochloride
(SigmaAldrich, Saint Louis, USA)) and amphotericin B (Sigma
Aldrich, Germany) were used as the control.
2.6. Antimicrobial activity of the emulsions by agar-well diffusion
assay
The antimicrobial activity of the selected emulsions and
individual components was assessed by agar-well diffusion. After
solidication of a basal layer (20 ml) of sterile Mueller-Hinton agar or
Sabouraud dextrose agar (Oxoid, Basingstoke, UK) in Petri dishes,
sterile 8-mm diameter cylinders were deposited on top. Then, 6 ml of
LB medium (Oxoid, Basingstoke, UK) containing 0.8% agar was
inoculated with a fresh culture of the microbial strain (nal
concentration 106 CFU/ml) and poured over the surface of the
medium. After solidication, the cylinders were pulled out and the
wells were lled with 50 ml of the chosen emulsions. After
incubation at 37  C for 24 h, all plates were examined for any region
of growth inhibition, and the diameter (mm) of these regions was
measured (Bouhdid et al., 2008) All tests were performed in
triplicate. The emulsions were prepared as described previously and
placed into the wells immediately after preparation. In addition, RL
and EOs were tested individually. The volumes applied to the wells
were equivalent to the concentration in the emulsions tested.
3. Results and discussion
3.1. Rhamnolipid characterization
In order to reduce the cost of rhamnolipid production, prolong
the life of the materials and therefore minimize the environmental
impact, edible oil waste was used for microbial conversion to
produce biosurfactants (Abalos et al., 2004; Benincasa et al., 2004;
Haba et al., 2000; Mercad et al., 1993).
After RL purication, a sticky, semi-solid, brown-coloured oil
product with 98% purity was obtained. The composition of the
surfactant produced by P. aeruginosa 47T2 was determined by LC
MSES to be a mixture of eight homologues (RL8). As shown in
Table 1 most of the accumulated rhamnolipid (60.4%) were monorhamnolipids: R1-C8-C10; R1-C10-C10; R1-C10-C12; R1-C10-C12:1 Being
the major component R1-C10-C10 (39.14%). The 39.6% of the mixture
were di-rhamnolipids: R2-C8-C10; R2-C10-C10; R2-C10-C12; R2-C10C12:1; being the main componentnt R2-C10-C10 (19.18%). Accumulation started soon after incubation and lasted until the end of the
process (96 h), whereas the minor homologues accumulated after
growth ceased.

E. Haba et al. / International Journal of Pharmaceutics 476 (2014) 134141

137

Table 1
Rhamnolipid homologues RL8 produced by P. aeruginosa 47T2. Relative abundances (%) were calculated from the pseudomolecular ion peak areas.
Rhamnolipid
homologues

Pseudo-molecular ion (m/z)

Relative abundance (%)

Fragments (m/z)

R-C10-C10
R2-C10-C10
R2-C10-C12/R2-C12-C10
R-C10-C12/R-C12-C10
R-C10-C12:1/R-C12:1-C10
R2-C10-C12:1
R2-C8-C10
R-C10-C8/R-C8-C10

503
649
677
531
529
675
621
475

39,14
19,68
10,73
8,41
9,88
6,25
2,91
3,01

333,169, 119, 103


479, 169,163
507, 479, 197, 169, 163
361, 333,169, 163, 119, 103
333, 197, 169, 163, 119, 103
479, 195, 169, 103
451, 169, 141
305, 169, 163, 141, 119,103

Rhamnolipids accumulated in the culture medium as a mixture


of different homologues whose nal composition appeared to
depend on the bacterial strain and substrate composition.
Although rhamnolipid properties are known to depend on the
distribution of their homologues, little is known about the
contribution of each individual homologue to the surface
properties of rhamnolipid mixtures. The new surfactant (RL8)
reduced the surface tension to 32.21 mN/m and the critical micelle
concentration (CMC) observed was 105 mg/l. The CMC value for
RL8 appeared to be slightly lower than that observed with a
rhamnolipid mixture of 11 homologues (108 mg/l) produced by the
same strain (Haba et al., 2003a). This could be due to the smaller
amount of unsaturated components of the surfactant produced in
the present study (9.88%) compared to the 13.87% reported
previously (Haba et al., 2003a). The effect of the presence of
unsaturated compounds was also observed in the case of strain LBI,
which contained up to 31% unsaturated carbon with a CMC of
120 mg/l (Benincasa et al., 2004), and also for the AT10 strain,
which contained up to 43.2% unsaturated compounds with a CMC
of 150 mg/l (Abalos et al., 2001). However, despite differences in
the composition of individual homologues and CMC values, surface
tension values (2732 mN/m) and HLB values (710) were in the
same range of hydrophobicity, and therefore the same range of
emulsication behaviour (Attwood and Florence, 1983); in the case
of RL8, an HLB of 8.13 was calculated, which favours O/W
microemulsions.
3.2. Emulsifying properties of RL47T2 in water/oil mixtures
Biosurfactants have been shown to have valuable biological
properties and several pharmaceutical (Gharaei-Fathabal, 2011)
and biomedical applications (Rodrigues et al., 2006; Stipcevic et al.,
2006). They have been reported to have antimicrobial, antiadhesive and other biological applications (Cameotra and Makkar,
2004; Das et al., 2009; Fracchia et al., 2012; Gharaei-Fathabal,
2011; Gudia et al., 2010; Haba et al., 2003b; Kitamoto et al., 1993).
The trend for using natural emulsifying agents in pharmaceutical
formulations favours the use of biosurfactants as an alternative to
their chemical counterparts, and these show promise for the future
(Gudia et al., 2013). By exploiting the excellent emulsication
behaviour of rhamnolipids in an attempt to broaden their range of
applications in therapeutic EO formulations, ternary systems were
plotted to study the emulsifying capacity of RL8 and one of the EOs
of O. compactum (OEO), C. zeylanicum (CEO), M. alternifolia (TTO)
and L. angustifolia (LEO) and water (Fig. 1ad). The ternary phase
diagrams provide in-depth knowledge about what a system may
offer in terms of the phase behaviour, depending on the relative
proportions of its components.
Despite the fact that the phase behaviour of water/surfactant/
oil systems at constant temperatures has been studied extensively
for pharmaceutical and cosmetic formulations (Attwood and
Florence, 1983), and for agricultural and food applications, the
literature provides little information on phase diagrams with

biosurfactants (Abalos et al., 2004; Kitamoto et al., 2009; Marqus


et al., 2009; Torrego-Solana et al., 2014).
OEO, TTO and LEO have a lower density than water; they are
immiscible in water and phase separation was apparent. Although
TTO is slightly soluble in water, some turbidity was observed in the
composition studied due to the low solubility (Morais et al., 2008).
In terms of the miscibility of RL8 with the EOs, RL8 was found to be
soluble in CEO and TTO. While rhamnolipids are soluble in water,
the lipophilic nature of EOs promotes phase separation. Since the
role of biosurfactants as emulsifying agents is to ensure the
dispersion of EOs in water, and low concentrations are needed, no
more than 20% of RL8 was added. Destabilization of emulsions
observed after few minutes of preparation in emulsions with TTO
and OEO could be attributed to Ostwald ripening. In oil-in-water
emulsions, the kinetics of Ostwald ripening depends on the
diffusion of the oil molecules across the aqueous phase separating
droplets (Suriyarak and Weiss, 2014). This destabilization process
is of particular importance for emulsions containing lipids with
appreciable water solubility (e.g. avours and essential oils).
Emulsions of essential oils, which consist of various terpenes, can
show distinct Ostwald ripening kinetics.
Oregano oil is dark brown in colour and, together with RL8,
produces the brownish colouration observed in some emulsions.
Examination of the ternary phase diagrams showed that, in the
OEO system (W/RL/OEO), two main areas with two different
behaviours were distinguished (Fig. 1a). In zone A, the emulsions
contained up to 40% OEO, 120% RL and 5095% water. Visual
inspection revealed thick, cream-coloured emulsions, most of
which presented creaming phenomena.With a low water concentration and 520% RL8, a different area (zone B), composed of more
than 50% emulsion and water separation at the bottom, was
observed; these emulsions were thick and dark brown in colour.
The emulsions were consistent in both areas and when observed
under optical microscopy multiple emulsions were visible (Fig. 2a).
This phenomenon has been described previously in emulsions of
rhamnolipids from strain AT10 and Casablanca crude oil, which is
also a complex substrate with more than 26% aromatic compounds
(Abalos et al., 2004).
TTO formulations of a monoolein/water system as a carrier for
terpinen-4-ol (Caboi et al., 2002) and more recently, the
development of hydrogel-thickened nanoemulsions with vitamin
A palmitate (Oliveira et al., 2011) have been reported, no other
information on the behaviour of TTO in biologically active
formulations was found. In the TTO system (W/RL/TTO), zone A
was smaller than in the OEO system (Fig. 1b). This zone extended
from 55% water to the water vertex, with up to 30% of the oil being
solubilized. In contrast to the OEO system, the maximum amount
of surfactant used was 9%. The excess of water made the emulsions
appear light and milky. Above this area, when TTO was in the range
25% and RL was over 9%, gels rather than emulsions were
observed. Birefringence was observed under polarized light for
some compositions with a water concentration ranging from 73%
to 93% and 5% TTO, that could be attributed to the formation of

138

E. Haba et al. / International Journal of Pharmaceutics 476 (2014) 134141

Fig. 1. The pseudo-phase diagrams show the emulsion areas for the OEO (a), TTO (b), CEO (c) and LEO (d) systems. White dots indicate monophasic compositions. The
intersection (black dots) between the dotted lines show the emulsion used to study the antimicrobial activity of the emulsions.

anisotropic structures, such as lamellar or hexagonal liquid


crystals. These results differ from those reported to obtain stable
emulsions with TTO/TS weight ratios (%) 15/10, 10/5, 15/5 and 20/
10, points outside the best-area boundaries found in this work. This
might be due to the surfactant used, a mixture of nonionic
surfactants (ethers), whereas rhamnolipids belong to the carboxylic esters in the anionic group (Morais et al., 2008).
In the case of the CEO system (W/RL/CEO), the emulsions were
not formed in the same way as the OEO or TTO systems. As shown
in Fig. 1c, despite the fact that RL8 was soluble in CEO, phase
separation was apparent in all compositions studied. Even at high
water concentrations a dark phase appeared at the bottom in
equilibrium with a uid emulsion. This behaviour could be
explained by the high percentage of E-cinnamaldehyde (77.31%),
which is barely soluble in water, as well as the low proportion of

oxygenated monoterpenes such as 1,8-cineole (0.16%), linalool


(2.8%) and a-terpineol (0.27%) in the oil (Edris and Malone, 2011).
Observation of the bottom phase under optical microscopy showed
a compact and homogeneous emulsion (Fig. 2b). In the LEO system
(W/RL/LEO), the best emulsions were observed when the EO
ranged between 0.7% and 1.6% and RL between 3% and 7.5%, and the
weight ratio for water was over 91% (Fig. 1d, zone A). Milky
emulsions with a thin creamy lm formed in the aqueous region. A
multiphase area (zone B) near the oil vertex formed from the EO/RL
axis to near 50% water.
EOs are multi-component systems, and therefore their solubilization and consequent emulsication result from the interaction
of all minor and major constituents, with each other and with the
surfactant at the interface layer (Edris and Malone, 2011).
Comparing the four systems studied, OEO produced the thickest

Fig. 2. Emulsions observed under optical microscopy. Emulsion A, formed from OEO, is an exemple of multiple emulsion W/OE/W. Emulsion B, would be an example of
emulsion formed from CEO, TTO or LEO.

E. Haba et al. / International Journal of Pharmaceutics 476 (2014) 134141


Table 2
Minimal inhibitory concentration MIC % (v/v). The values presented belong to each
essential oil against C. albicans and MRSA S. aureus. OEO and CEO presented same
antimicrobial activity against both microorganisms.
Essential oil

C. albicans

MRSA S. aureus

Oreganum oil (OEO)


Cinnamomum oil (CEO)
Tea tree oil (TTO)
Lavander oil (LEO)

0.0156
0.008
0.5
0.5

0.125
0.125
2
2

emulsions, while TTO and LEO produced light, milky emulsions


with less than 9% RL. For the OEO, TTO and LEO systems, the best
emulsions were formed when water was over 50% and EO content
was below 40%.
3.3. Antimicrobial effect
3.3.1. Minimum inhibitory concentration (MIC)
The EOs used were rst examined for their antimicrobial effect.
Specically, their minimum inhibitory concentrations (MIC) were
determined against two pathogens: C. albicans and methicillinresistant S. aureus (MRSA). The MICs were assayed in liquid
medium and a small amount (0.15%) of agar was added to prevent
phase separation (Mann and Markham, 1998). As shown in Table 2,
CEO and OEO showed the highest antimicrobial activity against
MRSA, with an MIC value of 0.125% (v/v), whereas the activity
against C. albicans revealed MIC values of 0.0156% and 0.008% (v/v)
for CEO and OEO, respectively. Regarding the other oils, TTO and
LEO inhibited C. albicans at an MIC value of 0.5% (v/v) and MRSA at
2% (v/v). These results are consistent with those obtained over
44 resistant clinical isolates and against signicant pathogenic
bacterial and fungal isolates from the oral cavity and skin (Warnke
et al., 2009; Warnke et al., 2013). The MICs of RL8 were higher than
256 mg ml 1 against both of the microorganisms tested. Vancomycin hydrochloride inhibited MRSA at an MIC value of 0.125 mg ml 1,
while C. albicans was inhibited by amphotericin B at 0.065 mg ml 1.
3.3.2. Antimicrobial effect of the emulsions
In order to prevent disaggregation, the antimicrobial activity of
EO emulsions was studied by means of the diffusion test, rather
than the dilution method. To our knowledge, this is the rst time
that rhamnolipids, low-molecular-weight biosurfactants, have
been used as an emulsifying agent in an EO formulation.
The emulsions tested all had a high EO content that was within
the therapeutic range. The following emulsions were selected
(% W/RL8/EO): OEO, 72.2/11.1/16.7; TTO, 71.8/2.8/25.3; CEO, 80.9/
1.9/17.1; and LEO, 78.7/8.5/12.8 (Fig. 1ad). Single components,
RL8 and EOs, were also tested as controls due to their inhibitory
effects at the concentrations used in the chosen emulsions
(Table 3). As shown, RL8 showed some inhibition (9.0 mm) at
2.8% and 8.5% (w/v) (Table 3) for both strains. This effect reached
10.0 mm when the concentration was increased to 11.1% (w/v).
The inhibition zone measured for EOs at the concentration used
in the emulsion ranged from 21.3 mm to 37.3 mm for C. albicans
and from 11.0 mm to 24.6 mm for MRSA. No inhibition effect was

139

found with LEO. OEO was the most effective against C. albicans
(OEO > CEO > TTO), while the order of effectiveness against MRSA
was CEO > OEO > TTO (Table 3). In terms of emulsion systems, the
CEO emulsion was the most effective against both microorganisms,
with an inhibition zone of 42.8 mm and 24.2 mm for C. albicans and
MRSA, respectively, followed by the OEO and TTO emulsions. The
least effective emulsion was LEO (10.0 mm), i.e. CEOe > OEOe >
TTOe > LEOe.
According to Rota et al. (2008), the antimicrobial activity of EOs
can be classied into three levels, depending on the inhibition zone
diameter: weak activity (inhibition zone 12 mm), moderate
activity (12 mm < inhibition zone < 20 mm) and strong activity
(inhibition zone 20 mm) (Rota et al., 2008). Thus, RL8 emulsions
with OEO and CEO have strong activity against C. albicans and S.
aureus (MRSA), while the TTO emulsion has strong activity against
C. albicans and moderate activity against S. aureus (MRSA). The LEO
emulsion showed weak activity in both cases.
From the results obtained, we can conclude that the EOs used in
this study (OEO, TTO, CEO and LEO) could be used topically, since
the oil concentrations fall within the safe interval. Moreover, with
the inclusion of RL in category IV of the EPA classication, which
includes non-irritant products (Haba et al., 2003b), they could
yield some very useful but simple formulations.
The nature of the solubilizing agent is one of the physicochemical parameters that may affect the antimicrobial effect of EOs.
High-molecular-weight surfactants such as Tween 80 or Tween 20,
which are used in many in vitro antimicrobial assays, may
compromise the antimicrobial effect of EOs. This effect is due to
the possible encapsulation of oil in surfactant micelles that
prevents the agent from interacting with microorganisms (Hammer and Carson, 2011; Hammer et al., 1999b; Lang and Buchbauer,
2012). In the present study, we found that rhamnolipids (lowmolecular-weight surfactants) enhance the antimicrobial effect of
the EOs tested. In fact, RL micelles may transport EOs through the
aqueous phase to the surface of bacterial cells and thus improve
contact between the bacterial membrane and EO components. It is
generally accepted that the treatment of microorganisms by EOs
results in the impairment of membrane integrity and function
(Hammer and Carson, 2011). Thus, the hydrophobicity of EOs and
their components allows them to diffuse through the cell wall of
gram-positive bacteria and fungi and the outer membrane of gramnegative bacteria. They enter the phospholipid bilayer of the
bacterial cell membrane, which results in the alteration of the
physical properties of the membrane. Terpene accumulation leads
to the swelling of the lipid bilayer and hence a rise in membrane
uidity (Sikkema et al., 1994). Membrane damage is veried by a
collapsed membrane potential, inhibited respiration, potassium
leakage, loss of intracellular material, including DNA and proteins,
and ultrastructural alterations (Bouhdid et al., 2010; Carson et al.,
2002; Chami et al., 2005).
In recent years, emulsions and nanoemulsions have been
studied as carrier systems for hydrophobic antimicrobials such as
EO components (Dons et al., 2011; Sugumar et al., 2014 Wu et al.,
2014). The effectiveness of emulsions as hydrophobic drug carriers
seems to be affected by the emulsion's surfactant nature and

Table 3
Growth inhibition zone for the neat oil (EO), RL and emulsion (W/RL/EO). Inhibition zone diameters (mm) produced around the wells by adding 50 ml of the emulsion and the
amount of RL and EO equivalent to the one used in the emulsion. Values are means of three measurements. NI: indicates no inhibition.
EO Emulsion
W/RL/EO
OEO: 72.2/11.1/16.7
TTO: 71.8/2.8/25.3
CEO: 80.9/1.9/17.1
LEO: 78.7/8.5/12.8

C.albicans ATCC 10,231

S. aureus ATCC 43300 (MRSA)

RL

EO

Emulsion

RL

EO

Emulsion

10.0  0.0
9.0  0.0
NI
9.0  0.0

37.3  0.1
21.3  0.4
36.0  0.2
NI

39.3  0.3
27.3  0.3
42.8  0.1
10.0  0.0

10.0  0.0
9.0  0.0
NI
9.0  0.0

21.3  0.3
11.0  0.0
24.6  0.2
NI

22.3  0.1
15.2  0.0
24.2  0.1
10.0  0.0

140

E. Haba et al. / International Journal of Pharmaceutics 476 (2014) 134141

droplet size (Dons et al., 2012; Terjung et al., 2012). In fact,


complex interactions between microorganisms and the emulsion
may take place. In some cases, reducing the size of the micelles
improves their interaction with the target microorganisms. In
other cases, when antimicrobials are strongly bound to the droplet
interfaces and the droplets do not interact directly with the
microbial cells, antimicrobials may no longer be released from the
micelle and therefore fail to inhibit microorganisms (Weiss et al.,
2014).
4. Conclusion
These results led us to suggest that formulations of the assayed
EOs could be used in the treatment of candidiasis and MRSA
infections as alternatives to well-established drugs. Furthermore,
we demonstrated that rhamnolipids are highly suitable surfactants
in the context of EO emulsication. Specically, their surface
properties promote the dispersion of EOs, thereby increasing their
availability and antimicrobial activity against C. albicans and MRSA.
Rhamnolipid-based emulsions present a promising avenue in the
development of EO delivery systems. Our study provides preliminary results for further research into the incorporation of
rhamnolipids in different EO-based formulations designed to
enhance the antimicrobial effect of EOs.
Acknowledgements
The nancial support of the Comisin Interministerial de
Ciencia y Tecnologia (CICYT), project CTQ 2010-21183-C02-01/PPQ
of the Ministerio de Educacin y Ciencia, Spain, and the Comissi
Interdepartamental de Recerca i Tecnologia (CIRIT), Generalitat de
Catalunya project 2014SGR534, is gratefully acknowledged.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
ijpharm.2014.09.039.
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