Post on 07-Aug-2018
8/20/2019 Haba et al IJP2014.pdf
1/8
Pharmaceutical nanotechnology
Rhamnolipids as emulsifying agents for essential oil formulations:Antimicrobial
effect
against
Candida
albicans
and
methicillin-resistantStaphylococcus
aureus
Ester Haba a, Samira Bouhdid c, Noelia Torrego-Solana a, A.M. Marqués a,M. José Espuny a, M. José García-Celma b, Angeles Manresa a,*aUnitat de Microbiologia, Facultat de Farmàcia, Universitat de Barcelona, Joan XXIII s/n, 08028 Barcelona, SpainbDepartment of Pharmacy and Pharmaceutical Technology, R+D Associated Unit to CSIC, Faculty of Pharmacy, University of Barcelona, Joan XXIII s/n, 08028
Barcelona, SpaincDépartement de Phytologie, Institut National des Plantes Médicinales et Aromatiques, Université Sidi Mohamed Ben Abdellah-Fès, Morocco
A
R
T
I
C
L
E
I
N
F
O
Article history:
Received 30 June 2014
Received in revised form 16 September 2014
Accepted 26 September 2014
Available online 28 September 2014
Keywords:
Essential oils
Rhamnolipids
Emulsions
Phase behaviour
Methicillin-resistant
A
B
S
T
R
A
C
T
This work examines the inuence of essential oil composition on emulsicationwith rhamnolipids and
their use as therapeutic antimicrobial agents against two opportunistic pathogens, methicillin-resistant
Staphylococcus aureus (MRSA)and Candida albicans.
Rhamnolipids, producedby 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. Ternaryphasediagramsweredesigned to evaluateemulsion stability,whichdiffereddepending
on theessential oil. The in vitro antimicrobial activity of the EOsalone and the emulsionswas evaluated.
The antimicrobial activity presentedby theessential oilsalone increasedwithemulsication. The surface
propertiesof rhamnolipidscontributeto thepositivedispersionof EOsand thus increase theiravailability
and antimicrobial activity against C. albicans and S. aureus. Therefore, rhamnolipid-based emulsions
represent a promising approach to the development of EO delivery systems.ã 2014 Elsevier B.V. All rights reserved.
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 compo-
nents, 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).
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* 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/ã 2014 Elsevier B.V. All rights reserved.
International Journal of Pharmaceutics 476 (2014) 134–141
Contents
lists
available
at
ScienceDirect
International
Journal
of
Pharmaceutics
journa l home page : www.e lsevier.com/loca te / i jpharm
mailto:amanresa@ub.edumailto:amanresa@ub.eduhttp://dx.doi.org/10.1016/j.ijpharm.2014.09.039http://dx.doi.org/10.1016/j.ijpharm.2014.09.039http://dx.doi.org/10.1016/j.ijpharm.2014.09.039http://dx.doi.org/10.1016/j.ijpharm.2014.09.039http://dx.doi.org/10.1016/j.ijpharm.2014.09.039http://dx.doi.org/10.1016/j.ijpharm.2014.09.039http://dx.doi.org/10.1016/j.ijpharm.2014.09.039http://dx.doi.org/10.1016/j.ijpharm.2014.09.039http://dx.doi.org/10.1016/j.ijpharm.2014.09.039http://dx.doi.org/10.1016/j.ijpharm.2014.09.039http://dx.doi.org/10.1016/j.ijpharm.2014.09.039http://dx.doi.org/10.1016/j.ijpharm.2014.09.039http://dx.doi.org/10.1016/j.ijpharm.2014.09.039http://dx.doi.org/10.1016/j.ijpharm.2014.09.039http://dx.doi.org/10.1016/j.ijpharm.2014.09.039http://dx.doi.org/10.1016/j.ijpharm.2014.09.039http://www.sciencedirect.com/science/journal/03785173http://www.elsevier.com/locate/ijpharmhttp://www.elsevier.com/locate/ijpharmhttp://www.elsevier.com/locate/ijpharmhttp://www.elsevier.com/locate/ijpharmhttp://www.elsevier.com/locate/ijpharmhttp://www.elsevier.com/locate/ijpharmhttp://www.sciencedirect.com/science/journal/03785173http://dx.doi.org/10.1016/j.ijpharm.2014.09.039http://dx.doi.org/10.1016/j.ijpharm.2014.09.039mailto:amanresa@ub.eduhttp://crossmark.dyndns.org/dialog/?doi=10.1016/j.ijpharm.2014.09.039&domain=pdf
8/20/2019 Haba et al IJP2014.pdf
2/8
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 antimicro-
bials (Gudiña 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 tochoose 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; Gudiña 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
oil-contaminated 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 baf ed 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, CaCl20.01, 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
(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 120C,
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 (LC–MS) 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 LC–MS. Rhamnolipid mixtures were separated and
identied by LC–MS using a Waters 2690 separation module
(Waters, Milford, MA, USA). Samples were injected (10 ml) into aC18 Spherisorb ODS2 150 4.6-mm column (Teknokroma, Sant
Cugat, Spain). The LC ow rate was 1 ml min1. An acetonitrile–
water gradient was used, starting with 30% acetonitrile for 2 min,
followed by a ramp of 30–100% 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 wasperformed using a Phoenix 20 syringe pump (Carlo Erba, Rodano,
Italy), since we observed a rise in sensitivity to rhamnolipids. The
LC ef uent 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 N-
ES 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 LC–MS (Haba et al., 2003b).
The
proportion
(%)
of
each
component
was
calculated
from
theareas obtained for each molecule of RL by LC–MS.
Equilibrium surface tension (g ST) and critical micelle concen-
tration (CMC) were measured at 25 C by the DuNoüy ring method
with a Krüss 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 (200–5 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.
E. Haba et al. / International Journal of Pharmaceutics 476 (2014) 134–141 135
8/20/2019 Haba et al IJP2014.pdf
3/8
2.3. Essential oils
The four EOs (supplied by Pranarôm, 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 Pranarôm 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%) forOEO; E-cinnamaldehyde (77.31%) for CEO; terpinen-4-ol (39.39%)
and g-terpinene (20.76%) for TTO; and linalool (30.60%) and linalylacetate (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 (Sadurní et al., 2005). Non-equilibrium phase diagramswere 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 dissolu-
tion 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 morephases. 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 0.5–90%. 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
aLeica 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 transferredfrom the second to the 11th well. The 12th well was considered as
the growth control, because no EO was added. We then added 50 mlof a microbial suspension to each well at a nal concentration of
approximately 105–106CFU/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 toeach 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(Sigma–Aldrich, 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 diametercylinders 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 24h, all plates wereexamined for any regionof growth inhibition, and the diameter (mm) of these regions was
measured (Bouhdid et al., 2008) All tests were performed in
triplicate. The emulsions wereprepared 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
toproduce biosurfactants (Abalos et al., 2004; Benincasa et al., 2004;
Haba et al., 2000; Mercadé 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–
MS–ES to be a mixture of eight homologues (RL8). As shown in
Table 1 most of the accumulated rhamnolipid (60.4%) were mono-
rhamnolipids: R 1-C8-C10; R 1-C10-C10; R 1-C10-C12; R 1-C10-C12:1Being
the major component R 1-C10-C10 (39.14%). The 39.6% of the mixture
were di-rhamnolipids: R 2-C8-C10; R 2-C10-C10; R 2-C10-C12; R 2-C10-
C12:1; being the main componentnt R 2-C10-C10 (19.18%). Accumu-
lation started soon after incubation and lasted until the end of the
process (96 h), whereas the minor homologues accumulated after
growth
ceased.
136 E. Haba et al. / International Journal of Pharmaceutics 476 (2014) 134–141
8/20/2019 Haba et al IJP2014.pdf
4/8
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
thesame 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 (27–32 mN/m) and HLB values (7–10) 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, anti-
adhesive and other biological applications (Cameotra and Makkar,
2004; Das et al., 2009; Fracchia et al., 2012; Gharaei-Fathabal,
2011; Gudiña 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
(Gudiña et al., 2013). By exploiting the excellent emulsicationbehaviour 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. 1a–d). 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; Marqués
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,
nomore 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, 1–20% RL and 50–95% water. Visual
inspection revealed thick, cream-coloured emulsions, most of
which presented creaming phenomena.With a low water concen-
tration and 5–20% 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, thedevelopment 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 watermade the emulsions
appear light and milky. Above this area, when TTO was in the range
2–5% 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
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 503 39,14 333,169, 119, 103
R 2-C10-C10 649 19,68 479, 169,163
R 2-C10-C12/R 2-C12-C10 677 10,73 507, 479, 197, 169, 163
R-C10-C12/R-C12-C10 531 8,41 361, 333,169, 163, 119, 103
R-C10-C12:1/R-C12:1-C10 529 9,88 333, 197, 169, 163, 119, 103
R 2-C
10-C
12:1 675 6,25 479, 195, 169, 103
R 2-C8-C10 621 2,91 451, 169, 141
R-C10-C8/R-C8-C10 475 3,01 305, 169, 163, 141, 119,103
E. Haba et al. / International Journal of Pharmaceutics 476 (2014) 134–141 137
8/20/2019 Haba et al IJP2014.pdf
5/8
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 carbox-
ylic 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
showeda 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 solubi-
lization 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. 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.
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.
138 E. Haba et al. / International Journal of Pharmaceutics 476 (2014) 134–141
8/20/2019 Haba et al IJP2014.pdf
6/8
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 methicillin-
resistant 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 ml1 against both of the microorganisms tested. Vancomy-cin hydrochloride inhibited MRSA at an MIC value of 0.125 mg ml1,while C. albicans was inhibited by amphotericin B at 0.065 mg ml1.
3.3.2. Antimicrobial effect of the emulsionsIn 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. 1a–d). 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
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
8/20/2019 Haba et al IJP2014.pdf
7/8
http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0195http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0195http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0190http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0190http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0185http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0185http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0185http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0180http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0180http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0175http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0175http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0175http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0170http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0170http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0170http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0165http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0165http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0165http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0160http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0160http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0160http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0155http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0155http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0155http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0150http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0150http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0145http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0145http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0145http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0140http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0140http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0140http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0140http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0135http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0135http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0135http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0135http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0130http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0130http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0130http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0125http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0125http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0125http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0120http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0120http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0115http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0115http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0110http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0110http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0110http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0105http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0105http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0105http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0105http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0100http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0100http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0100http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0095http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0095http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0095http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0090http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0090http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0090http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0085http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0085http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0085http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0085http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0080http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0080http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0075http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0075http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0075http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0070http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0070http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0070http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0065http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0065http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0065http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0060http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0060http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0060http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0060http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0055http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0055http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0055http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0050http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0050http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0045http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0045http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0045http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0040http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0040http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0040http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0035http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0035http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0035http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0035http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0030http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0030http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0030http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0030http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0025http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0025http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0025http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0020http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0020http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0015http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0015http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0015http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0010http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0010http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0010http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0010http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0005http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0005http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0005http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0005
8/20/2019 Haba et al IJP2014.pdf
8/8
Marqués, A.M., Pinazo, A., Farfan, M., Aranda, F.J., Teruel, J.A., Ortiz, A., Manresa, A.,Espuny, M.J., 2009. The physicochemical properties and chemical compositionof trehalose lipids produced by Rhodococcus erythropolis 51T7. Chem. Phys.Lipids 158, 110–117.
Mercadé, M.E., Manresa, A., Robert, M., Espuny, M.J., de Andrés, C., Guinea, J., 1993.Olive oil mill ef uent (OOME): new substrate for biosurfactant production.Bioresour. Technol. 43, 1–6.
Morais, G.G., Santos, O.D.H., Oliveira, W.P., Filho, P.A.R., 2008. Attainment of O/Wemulsions
containing
liquid
crystal
from
annatto
oil
(Bixa orellana), coffee oiland tea tree oil (Melaleuca alternifolia) as oily phase using HLB system andternary phase diagram. J. Disper. Sci. Technol. 29, 297–306.
Nitsche,
M.,
Siddhartha,
G.A.O.,
Contiero,
J.,
2005.
Ramnolipid
surfactants:
anupdate on the general aspects of these remarcable biomolecules. Biotechnol.Prog.
21,
1593–1600.Oliveira, J.S., Aguiar, T.A., Mezadri, H., dos Santos, O.D.H., 2011. Attainment of
hydrogel-thickened nanoemulsions with tea tree oil (Melaleuca alternifolia) andretinyl palmitate. Afr. J. Biotechnol. 10, 13014–13018.
Oussalah, M., Caillet, S., Saucier, L., Lacroix, M., 2007. Inhibitory effects of selectedplant
essential
oils
on
the
growth
of
four
pathogenic
bacteria: E. coli O157:H7
Salmonella Typhimurium, Staphylococcus aureus and Listeria monocytogenes.Food Control 18, 414–420.
Rodrigues, L., Banat, I.M., Teixeira, J., Oliveira, R., 2006. Biosurfactants: potentialapplications in medicine. J. Antimicrob. Chemother. 57, 609–618.
Rota, M.C., Herrera, A., Martínez, R.M., Sotomayor, J.A., Jordán, M.J., 2008.Antimicrobial
activity
and
chemical
composition
of Thymus vulgaris, Thymus
zygis and Thymus hyemalis essential oils. Food Control 19, 681–687.Sadurní, N., Solans, C., Azemar, N., García-Celma, M.J., 2005. Studies on the
formation of O/W nano-emulsions, by low-energy emulsication methods,suitable for pharmaceutical applications. Eur. J. Pharm. Sci. 26, 438–445.
Sharma,
N.C.,
Araujo,
M.W.,
Wu,
M.M.,
Qaqish,
J.,
Charles,
C.H.,
2010.
Superiority
of
an essential oil mouthrinse when compared with a 0.05% cetylpyridiniumchloride containing mouthrinse: a six-month study. Int. Dent. J. 60, 175–180.
Sikkema, J., de Bont, J., Poolman, B., 1994. Interactions of cyclic hydrocarbons withbiological membranes. J. Biol. Chem. 269, 8022–8028.
Stipcevic, T., Piljac, A., Piljac, G., 2006. Enhanced healing of full-thickness burnwounds using di-rhamnolipid. Burns 32, 24–34.
Sugumar, S., Ghosh, V., Nirmala, M.J., Mukherjee, A., Chandrasekaran, N., 2014.Ultrasonic emulsication of eucalyptus oil nanoemulsion: antibacterial activityagainst Staphylococcus aureus and wound healing activity in Wistar rats.Ultrason. Sonochem. 21, 1044–1049.
Suriyarak, S., Weiss, J., 2014. Cutoff Ostwald ripening stability of alkane-in-wateremulsion loaded with eugenol. Colloids Surf. A: Physicochem. Eng. Aspects 446,71–79.
Terjung, N., Lof er,M., Gibis, M., Hinrichs, J., Weiss, J., 2012. Inuence of droplet sizeon the ef cacy of oil-in-water emulsions loaded with phenolic antimicrobials.
Food
Funct.
3,
290–301.
Torrego-Solana, N., García-Celma, M.J., Garreta, A., Marqués, A.M., Diaz, P., Manresa,A.,
2014.
Rhamnolipids
obtained
from
a
PHA-negative
mutant
of Pseudomonas
aeruginosa 47T2DAD: composition and emulsifying behavior. J. Am. Oil Chem.Soc. 91, 503–511.
Unlu, M., Ergene, E., Unlu, G.V., Zeytinoglu, H.S., Vural, N., 2010. Composition:antimicrobial activity and in vitro cytotoxicity of essential oil from Cinnamo-mum zeylanicum Blume (Lauraceae). Food Chem. Toxicol. 48, 3274–3280.
Warnke, P.H., Becker, S.T., Podschun, R., Sivananthan, S., Springer, I.N., Russo, P.A.J.,Wiltfang, J., Fickenscher, H., Sherry, E., 2009. The battle against multi-resistantstrains: Renaissance of antimicrobial essential oils as a promising force to ghthospital-acquired infections. J. Cranio. Maxill. Surg. 37, 392–397.
Warnke, P.H., Lott, A.J.S., Sherry, E., Wiltfang, J., Podschun, R., 2013. The ongoingbattle
against
multi-resistant
strains
in-vitro
inhibition
of
hospital-acquiredMRSA, VRE, Pseudomonas, ESBL E. coli and Klebsiella species in the presence of plant-derived antiseptic oils. J. Cranio. Maxill. Surg. 41, 321–326.
Weiss, J., McClements, D.J., Davidson, P.M., 2014. Nanoscalar dispersion of antimicrobials: effect on food safety world food science. World Food Microbiol.16,
8–19.
Wu, J.-E., Lin, J., Zhong, Q., 2014. Physical and antimicrobial characteristics of thymeoil emulsied with soluble soybean polysaccharide. Food Hydrocolloids 39,144–150.
E. Haba et al. / International Journal of Pharmaceutics 476 (2014) 134–141 141
http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0200http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0205http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0210http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0215http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0220http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0225http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0230http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0235http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0240http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0240http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0240http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0240http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0240http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0240http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0240http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0240http://refhub.elsevier.com/S0378-5173(14)00694-2/sbref0240http://refh