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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: [email protected] (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:[email protected]:[email protected]://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:[email protected]://crossmark.dyndns.org/dialog/?doi=10.1016/j.ijpharm.2014.09.039&domain=pdf


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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


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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


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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



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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


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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


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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.

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