Jundishapur J Nat Pharm Prod.2012;7(3):117-122. DOI: 10.

17795/jjnpp-5261

Pharmaceutical Products
Jundishapur Journal of Natural

www.jjnpp.com

Preparation and Characterization of Liposomes Containing Essential

Oil of Eucalyptus camaldulensis Leaf
Eskandar Moghimipour 1, 2, Nasrin Aghel 2, Ali Zarei Mahmoudabadi 3, Zahra Ramezani 1,
Somayeh Handali 1 *
1 Nanotechnology Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IR Iran
2 Medicinal Plant Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IR Iran
3 Infectious and Tropical Diseases Research Center, and Department of Medical Mycology, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IR Iran

A R T I C LE I N FO AB S TR AC T

Article type: Background: The increased incidence of fungal resistance has necessitated the need to
Original Article search for new antifungal agents.
Objective: The main objectives of the present study were to investigate the effectiveness
Article history: of the essential oil of Eucalyptus camaldulensis on dermatophytes growth and to
Received: 22 Apr 2012 formulate and characterize a liposomal gel loaded with the essential oil.
Revised: 10 Jun 2012 Materials and Methods: The essential oil extracted from the leaves of E. camaldulensis
Accepted: 13 Jun 2012 was analyzed by GC-MS. The antifungal activity of this essential oil was determined
against Microsporum canis, M. gypseum, Trichophyton rubrum and T. verrucosum, using
Keywords: the well diffusion method. Liposomes were prepared by the freeze-thaw method
Eucalyptus camaldulensis and evaluation of size distribution was performed using a particle size analyzer. The
Essential Oil liposomal gel was prepared using ‘hydroxethyl cellulose (HEC) as the gelling agent. The
Liposomes rheologic characteristics were determined by a Brookfield viscometer.
Antifungal Activity Results: The results showed that the minimum inhibitory volume of the essential oil was
0.125 ml and 95 ± 0.57% of the essential oil was successfully entrapped in the liposomes.
The main constituents of the essential oil detected by GC-MS were; phenol, 1, 8 cineole,
limonene, alcohol, pinene and terpinen. Results of particle size determination showed a
wide range from 40.5 to 298 nm for the different formulations. No significant thixotropy
was observed in the rheogram of the formulated liposomal gel.
Conclusion: Liposomal gel formulation of the essential oil may lead to improved
antifungal activity.
Published by DocS. 2012. cc 3.0.

Implication for health policy/practice/research/medical education:

This study is going to investigate the effectiveness of the essential oil of Eucalyptus camaldulensis on dermatophytes growth

Please cite this paper as:

Moghimipour E, Aghel N, Mahmoudabadi AZ, Ramezani Z, Handali S. Preparation and Characterization of Liposomes Containing
Essential Oil of Eucalyptus camaldulensis Leaf. Jundishapur J Nat Pharm Prod. 2012:7(3);117-22.

* Corresponding author: Somayeh Handali, Nanotechnology Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IR Iran. Tel: +98-
9161147998, Fax: +98-6113738381, Email: handali_s81@yahoo.com
© 2012 School of Pharmacy, Ahvaz Jundishapur University of Medical Sciences; Published by DocS.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
 Moghimipour E et al. Liposomes Containing Essential Oil of Eucalyptus camadulensis Leaf

1. Background from 60º C to 190º C with 5º C̸ min, from 190º C to 270º C

for 30º C min, and finally kept at 270º C for approximately
In recent years, the incidence of infections caused by
5 min, so that the total analysis time was about 34 min.
The total ion chromatogram of Eucalyptus essential oil
dermatophytes has increased considerably. Essential oils
are natural substances extracted from plants with proven
antifungal effects. Eucalyptus camaldulensis (Myrtaceae)
was analyzed by the GC-MS head space. To acquire this
chromatogram, a GC vial contain Eucalyptus essential oil
is a plant indigenous to eastern Australia and Tasmania
was put in the head space cavity and after shaking it for
which is also cultivated in southern Europe and many
approximately 3 minutes at 150° C, 25 ml of volatile com-
other regions of the world (1). Essential oil of Eucalyp-
pounds from the above sample was injected into the GC
tus is a colorless or pale yellow liquid (1) that contains;
running the above mentioned program.
cineol, terpenes, sesquiterpenes, aromatic aldehydes,

3.4. Antifungal Activity

phenols, pinene and limonene (1). Eucalyptus oil is fre-
quently used for the treatment of coughs and nasopha-
ryngeal infections; it can also be used as an antiseptic and Freeze-dried sealed glass ampoules of microorganisms
expectorant. It has been shown that an ethanolic leaf ex- were obtained from the Persian Type Culture Collection
tract of this plant has a marked fungicidal effect against (PTCC), Iranian Research Organization for Science and
dermatophytes (2). Due to a lack of stability of most of Technology, Tehran, Iran. The microorganisms were; Mi-
the essential oils, new methods have been developed to crosporum canis PTCC no. 5069, M. gypseum PTCC no. 5070,
improve their stability, among these is the encapsula- Trichophyton rubrum PTCC no. 5143 and T. verrucosum
tion of the essential oils in liposomes (3). Liposomes are PTCC no. 5056. These microorganisms were activated on
vesicles composed of concentric phospholipid bilayers Sabouraud dextrose broth (SDB) and then cultured on
(4). Due to their capability to deliver slow drug release, Sabouraud dextrose agar (SDA) for 21 days to obtain ad-
cutaneous targeting and extended transdermal delivery equate growth. For determination of the antifungal activ-
of drugs, liposomes have been reported to be promising ity, a well diffusion method was utilized. Each plate was
drug carriers for antimicrobial therapy (3). inoculated with 50 µL of the fungal suspension. Various
serial dilutions of the essential oil were prepared and 50
2. Objectives µL of each serial dilution transferred to plates and incu-
bated at room temperature for 3 weeks. The Minimum
The main objectives of the present study were to investi-
Inhibitory Concentration (MIC) was determined to be
gate the effectiveness and encapsulation of the essential
oil E. camaldulensis, and to formulate and characterize a
the lowest concentration of the essential oil that did not
show any viable growth after 3 weeks of incubation (5).
liposomal gel loaded with the essential oil. Some param-

3.5. Preparation of Liposomes

eters such as; vesicle size, entrapment efficacy and stabil-
ity of the formulation were also investigated.
Liposomes were prepared using a freeze-thaw meth-
3. Materials and Methods od. Soya lecithin and cholesterol (1:1) were dissolved in
chloroform and methanol (100 mg/ml). The solvent was

3.1. Plant Materials

removed by rotary evaporation under vacuum. Then
phosphate buffer saline (pH 7.4) and essential oil (0.25%)
The leaf of E. camaldulensis were collected from Ahvaz were added and mixed in a vortex for 5 min. The solution
(Iran), and indentified in the Department of Pharmacog- was frozen in ice-ethanol or acetone (5-10 min) and left
nosy, Faculty of Pharmacy, Ahvaz Jundishapur University to thaw at room temperature. The freeze-thaw cycle was
of Medical Sciences. repeated three times and then the samples were centri-
fuged at 10 000х g for 30 min (6-9).
3.2. Preparation of Essential Oil
500 g of fresh leaf samples of the plant were cut into 3.6. Determination of Encapsulation Efficiency
small pieces. Then, the essential oil was extracted using Cineol, the major component of E. camaldulensis oil, was
a distillator for 3 h and stored in a refrigerator for future chosen as an index for determination of encapsulation
use. efficacy. It was assayed according to the method previ-
ously described by British Pharmacopeia (determination
3.3. GC- MS Analysis of Essential Oil of cineol). After the samples were centrifuged, the super-
To identify the main constituents of the essential oil, natant was removed and the liposomes were treated with
its GC-MS analysis was performed on GC 7890A equipped Triton X-100 to disrupt them. Then 3 g of the substance
with MS 5975C detector and HP-5ms capillary column (30 was added to 2.10 g of melted o-cresol, this was allowed
m × 0.25 m, 0.25 µm) (Agilent Company, USA). The initial to cool by stirring continuously. Then the mixture was re-
column temperature was set at 60º C, then increased melted and allowed to cool again. The highest tempera-
ture at which the mixture froze was then recorded (10).

118 Published by Docs. © AJUMS 2012 Jundishapur J Nat Pharm Prod. 2012;7(3)
Liposomes Containing Essential Oil of Eucalyptus camadulensis Leaf Moghimipour E et al.

3.7. Measurement of Liposome Size the corresponding Newtonian and non-Newtonian equa-
tions.
The diameter of the liposomes before and after homog-

3.11. Stability Testing

enization (for 15 min) was determined using a particle
sizer, Qudix, ScatterO Scope I system (Korea).
The stability of the vesicle dispersions was monitored
3.8. Scanning Electron Microscopy (SEM) after 1 and 3 months storage in a refrigerator. At certain
time intervals, the liposomes were evaluated for their en-
The specimens were dried under light and coated with
capsulation efficiency and size distribution.
a silver layer. Then they were examined photographically

3.12. Statistical Analysis of Data

by a scanning electron microscope (Leo 1455 VP, Germany,
magnification 10 000x).
The data were reported as mean ± SD and frequently as
3.9. Preparation of Liposomal Gel percentages. For all of the analyses, the statistical signifi-
cance was assessed at a level of 0.05 and a t-test was used
Hydroxyethylcellulose (HEC) 5 g, was slowly added to a
to compare the averages.
PBS buffer solution (pH 7.4), and stirred constantly with

4. Results
a paddle stirrer. After the addition of the full amount of
solid material, the gels were allowed to swell under mod-
erate stirring for at least 4 h to achieve maximum volume The yield of the essential oil of E. camaldulensis was 2%
and transparency. Finally 0.4 g of the liposomes was add- v/w. GC-MS analysis of the essential oil showed that it con-
ed (11). tained; phenol, 1.8 cineol limonene, alcohol, pinene and
terpinene (Figure 1a, b). The minimum inhibitory concen-
3.10. Determination of Rheological Properties trations of the essential oil for all of the micro-organisms
were 0.125 ml. The results showed that 95 ± 0.57% of the
The rheologic behavior of the sample was determined
essential oil was successfully encapsulated in the lipo-
using a Brookfield viscometer (model DV-I with No. 34
somes (n = 3). The results of the size monitoring showed
spindle). The viscosity of the samples was determined at
the effectiveness of freezing time on the size of the par-
0.3, 0.6, 1.5, 3, 6, 12 and 30 rpm for 1 min at room tempera-
ticles. There was a significant increase in the particle size
ture. The results were plotted as a rheogram and their
of the liposomes. There was also a significant rise in the
rheologic behavior was determined by fitting these on
polydispersity of the particles (Table 1). Homogenization

Table 1. Effect of Freezing Time on the Particle Size of Liposome (n = 3)

Formulation Freezing time, min Particle size, nm Polydispersity index (PDI)

F1 4 157.66 ± 0.57 2.5 ± 0.0
F2 8 158.66 ± 0.57 2.58 ± 0.14
F3 10 286.33 ± 2.88 2.66 ± 0.14
F4 45 36430 ± 1.25 1.41 ± 0.94
F5
> 60 290000 ± 6.65 2.05 ± 0.42

Table 2. Results of Particle Size and Encapsulation of Essential Oil of Eucalyptus camaldulensis Before and After Storage
Characterization Before storage After storage
Particle size (nm) 157.66 ± 0.57 156.33± 1.15 a
Encapsulation (percent) 95 ± 0.57 94 ± 0.57 a
a
P > 0.05

Abundance
TIC:MOGHIMIPOUR6.D\data.ms
b 7500000
7000000
6500000
6000000
5500000
5000000
4500000
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
Time 5.00 10.00 15.00 20.00 25.00 30.00

Figure 1. a) Total Ion Chromatogram of GC-MS Analysis of Essential Oil (1 µl injection in split mode). b) Head Space GC-MS Analysis of Essential Oil as
Described in the Text.

Jundishapur J Nat Pharm Prod. 2012;7(3) Published by Docs. © AJUMS 2012 119
 Moghimipour E et al. Liposomes Containing Essential Oil of Eucalyptus camadulensis Leaf

Intensity Distribution Intensity Distribution

100 100
Distribution (%, intensity)

Distribution (%, intensity)

90 90
80 80
70 70
60 60
a b
50 50
40 40
30 30
20 20
10 10
0 0
1e-10 1e-9 1e-8 1e-7 1e-6 1e-5 1e-4 1e-3 1e-10 1e-9 1e-8 1e-7 1e-6 1e-5 1e-4 1e-3
Diameter (m) Diameter (m)

Figure 2. a) Particle Size Distribution of Non-homogenized Liposomes Containing Essential Oil of Eucalyptus camaldulensis. b) Particle Size Distribution
of Liposomes Containing Essential Oil of Eucalyptus camaldulensis After Homogenization

0.57%) and showed no significant size change (P > 0.05).

Results of particle size and encapsulation before and af-
ter storage are shown in Table 2.

5. Discussion
The development of fungal resistance to presently
available antifungal agents has necessitated the need to
search for new antifungal agents. Essential oils possess
a wide spectrum of biological activity in several fields,
from food chemistry to pharmaceutics. However, most
essential oils are biologically instable, poorly soluble in
water and they are distributed ineffectively to the target
sites. New methods have been developed in order to im-
Figure 3. SEM Image of Freeze-thaw Liposomes Containing Essential Oil of prove their stability, among these is the use of liposome
Eucalyptus camaldulensis (Magnification 10000x). bilayers to encapsulate the oil (3).
The compounds are revealed in the total ion chromato-
gram (TIC) shown in Figure 1a. When a split injection of
the Eucalyptus essential oil is performed on the specified
12

column, a broad peak appears around retention times 11

10
to 14 min. In order to simplify the spectra and distinguish
the compounds more precisely, a head space injection
was also performed and its TIC was recorded (Figure 1b is
8

a less crowded chromatogram). This allowed better esti-

RBM

6 asending curve
desending curve
mation of the compounds present in the essential oils,
which then became the mechanism used in the library
search, by comparing the two TIC (Figure 1a and Figure 1b)
4

2 RT of 4.37, 5.397, 5.984, 6.970, 7.90, 12.957, 13.256 that are

related to α-pinene, gamma-pinene, α-terpinene gamma-
0 terpinene linalool, and phenols respectively. The results
0 10 20 30 40 50 60 70 80 obtained by GC-MS analysis of the essential oil showed
Torque (dyne/cm)
that it contained phenol, 1, 8 cineol, limonene, alcohol,
Figure 4. Rheogram of Liposomes Containing Essential Oil of E. camaldu- pinene and terpinene. It had previously been reported
lensis at Room Temperature. that the major components of the essential oil of E. ca-
maldulensis were; ethanol (25.36%), eucalyptol (13.73%),
time was also significantly affected by the liposomal par- β-caryophyllene (11.55%) and carvacrol (9.05%) (12). In
ticle size, as indicated in Figure 2a, b. another study, the composition of this plant consisted
The SEM image of the liposomes containing essential oil of 1, 8 cineol (64%), α- pinene (9.6%), myrcenol (7.4%) and
of E. camaldulensis, confirmed the formation of spherical γ-terpinene (7%) (13).
particles and also monodispersity of particles, as shown The results of the present study confirmed the antifun-
in Figure 3. Rheological inspection of the formulations gal properties of the essential oil from E. camaldulensis on
showed mild pseudoplastic to Newtonian behavior with dermatophytes. Several previous studies have investigat-
no significant thixotropy (Figure 4). The results of the sta- ed the antibacterial and antifungal properties of E. camal-
bility study showed good incorporation efficiency of the dulensis. It has been shown that the essential oil of the E.
essential oil after one and three months of storage (94 ± camaldulensis leaf and E. globulus leaf effectively inhibits

120 Published by Docs. © AJUMS 2012 Jundishapur J Nat Pharm Prod. 2012;7(3)
Liposomes Containing Essential Oil of Eucalyptus camadulensis Leaf Moghimipour E et al.

the growth of S. aureus and E. coli. (14). In another report, fects than conventional formulations (19).
methanolic extract of E. camaldulensis had been formu- This study concluded that the presence of E.camaldulensis
lated as an antidermatophytic cream preparation (15). in liposomes may effectively enhance its stability and the
Another study showed the antitermitic activity of oils of entrapped oil remains stable for an extended period of
E. camaldulensis leaf against Coptotermes formosanus, it time. Liposomal gel formulation of essential oils may
was demonstrated that the termiticidal mechanism was also lead to improved and better antifungal activity.
due to inhibition of acetylcholinesterase activity (16).
In the preparation of the liposomes, the length of the Acknowledgment
freezing period affected particle size. With a time of more
The work was financially supported by the Nanotech-
than 60 min, particle size of the vesicles increased, while
nology Research Center, Ahvaz Jundishapur University of
in shorter time frames, particle size of the liposomes
decreased significantly (Table 1). So it is suggested that
Medical Sciences, Ahvaz, Iran, grant NO. N-01.

Financial Disclosure
the time of freezing is an important parameter in creat-
ing the size of the liposome. With more than 60 min of
freezing time, the particle size distribution shows poly- None declared.
dispersity and aggregation, while there is a decrease in
polydispersity due to shorter freezing times. Accord- Funding/Support
ing to our data, after homogenization the particle size The work was financially supported by the Nanotech-
of the vesicles decreased to 40.5-298 nm. The results of nology Research Center, Ahvaz Jundishapur University of
one study showed that poloxamers P338 and P407 inhib- Medical Sciences, Ahvaz, IR Iran
ited the particle growth observed during the freeze-thaw
cycle for egg PC MLVs dispersed in 1.0 M NaCl, probably References
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122 Published by Docs. © AJUMS 2012 Jundishapur J Nat Pharm Prod. 2012;7(3)