Pharmaceutical nanotechnology
Unitat de Microbiologia, Facultat de Farmcia, Universitat de Barcelona, Joan XXIII s/n, 08028 Barcelona, Spain
Department of Pharmacy and Pharmaceutical Technology, R+D Associated Unit to CSIC, Faculty of Pharmacy, University of Barcelona, Joan XXIII s/n, 08028
Barcelona, Spain
c
Dpartement de Phytologie, Institut National des Plantes Mdicinales et Aromatiques, Universit Sidi Mohamed Ben Abdellah-Fs, Morocco
b
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 30 June 2014
Received in revised form 16 September 2014
Accepted 26 September 2014
Available online 28 September 2014
This work examines the inuence of essential oil composition on emulsication with rhamnolipids and
their use as therapeutic antimicrobial agents against two opportunistic pathogens, methicillin-resistant
Staphylococcus aureus (MRSA) and Candida albicans. Rhamnolipids, produced by Pseudomonas aeruginosa,
with waste frying oil as the carbon source, were composed of eight rhamnolipid homologues. The
rhamnolipid mixture was used to produce emulsions containing essential oils (EOs) of Melaleuca
alternifolia,Cinnamomum verum, Origanum compactum and Lavandula angustifolia using the titration
method. Ternary phase diagrams were designed to evaluate emulsion stability, which differed depending
on the essential oil. The in vitro antimicrobial activity of the EOs alone and the emulsions was evaluated.
The antimicrobial activity presented by the essential oils alone increased with emulsication. The surface
properties of rhamnolipids contribute to the positive dispersion of EOs and thus increase their availability
and antimicrobial activity against C. albicans and S. aureus. Therefore, rhamnolipid-based emulsions
represent a promising approach to the development of EO delivery systems.
2014 Elsevier B.V. All rights reserved.
Keywords:
Essential oils
Rhamnolipids
Emulsions
Phase behaviour
Methicillin-resistant
1. Introduction
Essential oils (EOs) are complex mixtures of volatile organic
molecules extracted from aromatic plants by different methods.
They are oil-like in nature, frequently characterized by a strong
fragrance (Morais et al., 2008) and may contain up to 100 components, mainly terpenes and phenylpropanoids (Hammer and
Carson, 2011). This structural diversity is responsible for the wide
variety of biological activities exhibited by EOs. Several authors
have reported that many EOs, especially those rich in phenols,
aldehydes and alcohols, are effective in inhibiting spoilage and
pathogenic microorganisms (Hammer and Carson, 2011). They
therefore represent a natural alternative to the synthetic
antimicrobials used in the cosmetic, food and pharmaceutical
industries (Lang and Buchbauer, 2012). Their effectiveness means
they can be used in small amounts (Hammer and Carson, 2011).
135
136
137
Table 1
Rhamnolipid homologues RL8 produced by P. aeruginosa 47T2. Relative abundances (%) were calculated from the pseudomolecular ion peak areas.
Rhamnolipid
homologues
Fragments (m/z)
R-C10-C10
R2-C10-C10
R2-C10-C12/R2-C12-C10
R-C10-C12/R-C12-C10
R-C10-C12:1/R-C12:1-C10
R2-C10-C12:1
R2-C8-C10
R-C10-C8/R-C8-C10
503
649
677
531
529
675
621
475
39,14
19,68
10,73
8,41
9,88
6,25
2,91
3,01
138
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.
C. albicans
MRSA S. aureus
0.0156
0.008
0.5
0.5
0.125
0.125
2
2
139
found with LEO. OEO was the most effective against C. albicans
(OEO > CEO > TTO), while the order of effectiveness against MRSA
was CEO > OEO > TTO (Table 3). In terms of emulsion systems, the
CEO emulsion was the most effective against both microorganisms,
with an inhibition zone of 42.8 mm and 24.2 mm for C. albicans and
MRSA, respectively, followed by the OEO and TTO emulsions. The
least effective emulsion was LEO (10.0 mm), i.e. CEOe > OEOe >
TTOe > LEOe.
According to Rota et al. (2008), the antimicrobial activity of EOs
can be classied into three levels, depending on the inhibition zone
diameter: weak activity (inhibition zone 12 mm), moderate
activity (12 mm < inhibition zone < 20 mm) and strong activity
(inhibition zone 20 mm) (Rota et al., 2008). Thus, RL8 emulsions
with OEO and CEO have strong activity against C. albicans and S.
aureus (MRSA), while the TTO emulsion has strong activity against
C. albicans and moderate activity against S. aureus (MRSA). The LEO
emulsion showed weak activity in both cases.
From the results obtained, we can conclude that the EOs used in
this study (OEO, TTO, CEO and LEO) could be used topically, since
the oil concentrations fall within the safe interval. Moreover, with
the inclusion of RL in category IV of the EPA classication, which
includes non-irritant products (Haba et al., 2003b), they could
yield some very useful but simple formulations.
The nature of the solubilizing agent is one of the physicochemical parameters that may affect the antimicrobial effect of EOs.
High-molecular-weight surfactants such as Tween 80 or Tween 20,
which are used in many in vitro antimicrobial assays, may
compromise the antimicrobial effect of EOs. This effect is due to
the possible encapsulation of oil in surfactant micelles that
prevents the agent from interacting with microorganisms (Hammer and Carson, 2011; Hammer et al., 1999b; Lang and Buchbauer,
2012). In the present study, we found that rhamnolipids (lowmolecular-weight surfactants) enhance the antimicrobial effect of
the EOs tested. In fact, RL micelles may transport EOs through the
aqueous phase to the surface of bacterial cells and thus improve
contact between the bacterial membrane and EO components. It is
generally accepted that the treatment of microorganisms by EOs
results in the impairment of membrane integrity and function
(Hammer and Carson, 2011). Thus, the hydrophobicity of EOs and
their components allows them to diffuse through the cell wall of
gram-positive bacteria and fungi and the outer membrane of gramnegative bacteria. They enter the phospholipid bilayer of the
bacterial cell membrane, which results in the alteration of the
physical properties of the membrane. Terpene accumulation leads
to the swelling of the lipid bilayer and hence a rise in membrane
uidity (Sikkema et al., 1994). Membrane damage is veried by a
collapsed membrane potential, inhibited respiration, potassium
leakage, loss of intracellular material, including DNA and proteins,
and ultrastructural alterations (Bouhdid et al., 2010; Carson et al.,
2002; Chami et al., 2005).
In recent years, emulsions and nanoemulsions have been
studied as carrier systems for hydrophobic antimicrobials such as
EO components (Dons et al., 2011; Sugumar et al., 2014 Wu et al.,
2014). The effectiveness of emulsions as hydrophobic drug carriers
seems to be affected by the emulsion's surfactant nature and
Table 3
Growth inhibition zone for the neat oil (EO), RL and emulsion (W/RL/EO). Inhibition zone diameters (mm) produced around the wells by adding 50 ml of the emulsion and the
amount of RL and EO equivalent to the one used in the emulsion. Values are means of three measurements. NI: indicates no inhibition.
EO Emulsion
W/RL/EO
OEO: 72.2/11.1/16.7
TTO: 71.8/2.8/25.3
CEO: 80.9/1.9/17.1
LEO: 78.7/8.5/12.8
RL
EO
Emulsion
RL
EO
Emulsion
10.0 0.0
9.0 0.0
NI
9.0 0.0
37.3 0.1
21.3 0.4
36.0 0.2
NI
39.3 0.3
27.3 0.3
42.8 0.1
10.0 0.0
10.0 0.0
9.0 0.0
NI
9.0 0.0
21.3 0.3
11.0 0.0
24.6 0.2
NI
22.3 0.1
15.2 0.0
24.2 0.1
10.0 0.0
140
Carson, C.F., Hammer, K.A., Riley, T.V., 2006. Melaleuca alternifolia (tea tree) oil: a
review of antimicrobial and other medicinal properties. Clin. Microbiol. Rev 19,
5062.
Carson, C.F., Mee, B.J., Riley, T.V., 2002. Mechanism of action of Melaleuca alternifolia
(tea tree) oil on Staphylococcus aureus determined by time-kill lysis, leakage,
and salt tolerance assays and electron microscopy. Antimicrob. Agents
Chemother. 46, 19141920.
Chami, N., Bennis, S., Chami, F., Aboussekhra, A., Remmal, A., 2005. Study of
anticandidal activity of carvacrol and eugenol in vitro and in vivo. Oral
Microbiol. Immun. 20, 106111.
Chandrasekaran, E.V., Bemiller, J.N., 1980. Constituent anlysis of glucosamonoglucans. In: Wrhiste, L., Wolfrom, M.L. (Eds.), Methods in Carbohydrate Chemistry.
Academic Press, NY, pp. 8996.
Charles, C.A., McGuire, J.A., Qaqish, J., Amini, P., 2013. Increasing antiplaque/
antigingivitis efcacy of an essential oil mouthrinse over time: an in vivo study.
Gen. Dent 61, 2328.
Das, P., Mukherjee, S., Sen, R., 2009. Antiadhesive action of a marine microbial
surfactant. Colloids Surface B 71, 183186.
de Rapper, S., Kamatou, G., Viljoen, A., van Vuuren, S., 2013. The in vitro
antimicrobial activity of Lavandula angustifolia essential oil in combination with
other aroma-therapeutic oils. J. Evid. Based Complement. Altern. Med. 2013,
124.
Dons, F., Annunziata, M., Sessa, M., Ferrari, G., 2011. Nanoencapsulation of essential
oils to enhance their antimicrobial activity in foods. LWT-Food Sci. Technol. 44,
19081914.
Dons, F., Annunziata, M., Vincensi, M., Ferrari, G., 2012. Design of nanoemulsionbased delivery systems of natural antimicrobials: effect of the emulsier. J.
Biotechnol. 159, 342350.
Edris, A.E., Malone, C.F.R., 2011. Alcohol-free delivery system carrying thyme
essential oil nanoparticles formulated via microemulsion technique. Adv. Sci.
Eng. Med. 3, 219225.
Fracchia, L., Cavallo, M., Martinotti, M., Banat, I.M., 2012. Biosurfactant and
bioemulsiers biomedical and related applications- present status and future
potentials. In: Ghista, D.N. (Ed.), Biommedical Science, Engineering and
Technology. Pub Inthec, pp. 325370.
Gaysinsky, S., Davidson, P.M., McClements, D.J., Weiss, J., 2008. Formulation and
characterization of phytophenol-carrying antimicrobial microemulsions. Food
Biophys. 3, 5465.
Gharaei-Fathabal, E., 2011. Biosurfactants in pharmaceutical industry: a minireview. Am. J. Drug Discov. Dev. 1, 5869.
Gudia, E., Rangarajan, V., Sen, R., Rodrges, L., 2013. Potential therapeutic
applications of biosurfactants. Trends Pharmacol. Sci. 34, 667675.
Gudia, E., Rocha, V., Teixeira, J., Rodrigues, L., 2010. Antimicrobial and antiadhesive
properties of a biosurfactant isolated from Lactobacillus paracasei ssp paracasei
A20. Lett. Appl. Microbiol. 50, 124419.
Haba, E., Espuny, M.J., Busquets, M., Manresa, A., 2000. Screening and production of
rhamnolipids by Pseudomonas aeruginosa 47T2 NCIB 40044 from waste frying
oils. J. Appl. Microbiol. 88, 379387.
Haba, E., Abalos, A., Juregui, O., Espuny, M.J., Manresa, A., 2003a. Use of liquid
chromatographymass spectroscopy for studying the composition and
properties of rhamnolipids produced by different strains of Pseudomonas
aeruginosa. J. Surfactants Deterg. 6, 155161.
Haba, E., Pinazo, A., Jauregui, O., Espuny, M.J., Infante, M.R., Manresa, A., 2003b.
Physicochemical characterization and antimicrobial properties of rhamnolipids
produced by Pseudomonas aeruginosa 47T2 40044. Biotechnol. Bioeng. 81,
316322.
Hammer, K.A., Carson, C.F., 2011. Antibacterial and antifungal activities of essential
oils. In: Thormar, H. (Ed.), Lipids and essential oils as Antimicrobial agents.
Wiley, UK, pp. 256306.
Hammer, K.A., Carson, C.F., Riley, T.V., 1999a. Antimicrobial activity of essential oils
and other plant extracts. J. Appl. Microbiol. 86, 985990.
Hammer, K.A., Carson, C.F., Riley, T.V., 1999b. Inuence of organic matter, cations and
surfactants on the antimicrobial activity of Melaleuca alternifolia (tea tree) oil in
vitro. J. Appl. Microbiol. 86, 446452.
Hyldgaard, M., Mygind, T., Meyer, R.L., 2012. Essential oils in food preservation:
mode of action, synergies, and interactions with food matrix components.
Front. Microbiol. 3, 1124. doi:http://dx.doi.org/10.3389/fmicb.2012.00012.
Kitamoto, D., Morita, T., Fukuoka, T., Masa-aki, K.T.I., 2009. Self assembling
properties of glycolipids biosurfactants and their potential applications. Curr.
Opin. Colloids Interface Sci. 14, 315328.
Kitamoto, D., Yanagishita, H., Shinbo, T., Nakane, T., Kamisawa, C., Nakahara, T., 1993.
Surface active properties antimicrobial activities of mannosylerythritol lipids as
biosurfactants produced by Candida antarctica. J. Biotechnol. 29, 9196.
Lang, G., Buchbauer, G., 2012. A review on recent research results (20082010) on
essential oils as antimicrobials and antifungals. A review. Flavour Frag. J. 27,
1339.
Lee, R., Balick, M.J., 2005. Sweet woodcinnamon and its importance as a spice and
medicine. EXPLORE 1, 6164.
Lovaglio, R.B., dos Santos, F.J., Jafelicci Junior, M., Contiero, J., 2011. Rhamnolipid
emulsifying activity and emulsion stability: pH rules. Colloids Surf. B 85,
301305.
Lowry, O.H., Rosebrought, N.J., Farr, A., Randall, R.J., 1951. Protein measurement with
the Folin phenol reagent. J. Biol. Chem. 139, 265274.
Mann, C.M., Markham, J.L., 1998. A new method for determining the minimum
inhibitory concentration of essential oils. J. Appl. Microbiol. 84, 538544.
141
Stipcevic, T., Piljac, A., Piljac, G., 2006. Enhanced healing of full-thickness burn
wounds using di-rhamnolipid. Burns 32, 2434.
Sugumar, S., Ghosh, V., Nirmala, M.J., Mukherjee, A., Chandrasekaran, N., 2014.
Ultrasonic emulsication of eucalyptus oil nanoemulsion: antibacterial activity
against Staphylococcus aureus and wound healing activity in Wistar rats.
Ultrason. Sonochem. 21, 10441049.
Suriyarak, S., Weiss, J., 2014. Cutoff Ostwald ripening stability of alkane-in-water
emulsion loaded with eugenol. Colloids Surf. A: Physicochem. Eng. Aspects 446,
7179.
Terjung, N., Lofer, M., Gibis, M., Hinrichs, J., Weiss, J., 2012. Inuence of droplet size
on the efcacy of oil-in-water emulsions loaded with phenolic antimicrobials.
Food Funct. 3, 290301.
Torrego-Solana, N., Garca-Celma, M.J., Garreta, A., Marqus, 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, 503511.
Unlu, M., Ergene, E., Unlu, G.V., Zeytinoglu, H.S., Vural, N., 2010. Composition:
antimicrobial activity and in vitro cytotoxicity of essential oil from Cinnamomum zeylanicum Blume (Lauraceae). Food Chem. Toxicol. 48, 32743280.
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-resistant
strains: Renaissance of antimicrobial essential oils as a promising force to ght
hospital-acquired infections. J. Cranio. Maxill. Surg. 37, 392397.
Warnke, P.H., Lott, A.J.S., Sherry, E., Wiltfang, J., Podschun, R., 2013. The ongoing
battle against multi-resistant strains in-vitro inhibition of hospital-acquired
MRSA, VRE, Pseudomonas, ESBL E. coli and Klebsiella species in the presence of
plant-derived antiseptic oils. J. Cranio. Maxill. Surg. 41, 321326.
Weiss, J., McClements, D.J., Davidson, P.M., 2014. Nanoscalar dispersion of
antimicrobials: effect on food safety world food science. World Food Microbiol.
16, 819.
Wu, J.-E., Lin, J., Zhong, Q., 2014. Physical and antimicrobial characteristics of thyme
oil emulsied with soluble soybean polysaccharide. Food Hydrocolloids 39,
144150.