Antibacterial activity of citronella oil solid lipid particles in oleogel against Propionibacterium acnes and its chemical stability

Abstract

Acne vulgaris is the most common skin disease. Normal skin flora, particularly Propionibacterium acnes, is a major cause of acne vulgaris. Bioactivity studies revealed that Java citronella oil (Cymbopogon winterianus Jowitt) appears to have potential for use in the development of topical anti-acne preparations. The aims of this study were twofold: first, to compare antimicrobial activity against P. acnes between two preparations, oleogel containing citronella oil and oleogel containing citronella oil-loaded solid lipid particles; second, to investigate the stability of their major components. The results demonstrated that the solid lipid particle preparation process caused a change to the main components of the citronella oil-loaded solid lipid particles. Nevertheless, when the preparations were kept at 40°C for 120 days, the anti-P. acnes activity of the oleogel containing the citronella oil-loaded solid lipid particles remained active, whereas the oleogel containing unencapsulated citronella oil was inactive by day 45. Moreover, a solid lipid wall provided protection from the volatility of the oil components of the oleogel and prolonged the release of citronella oil. In conclusion, topical anti-acne preparations containing citronella oil-loaded solid lipid particles could be good candidates for an alternative treatment for acne. Clinical trial on acne control efficacy would be needed for further study.

Full text

167
Introduction
In our previous works we found that Java type citronella
essential oil appears to be a good candidate for the
development of a topical anti-acne preparation [1];
however, the oil is easily oxidized, is rapidly volatile and
has poor water solubility. In order to overcome these
problems, solid lipid particles (SLPs) were used as carriers
of citronella oil. SLPs represent a promising carrier system
for pharmaceutical and cosmetic active ingredients because
they are made from naturally occurring lipids and hence
they are biocompatible and biodegradable. In addition,
their solid matrix protects loaded labile substances against
decomposition, is occlusive and prolongs the release of
the active ingredients [2, 3]. In this study, oleogel was used
as a dermatological base because it can be easily miscible
with citronella oil and sebum and the base itself has an
occlusive effect. These advantages would help to deliver
the active oil to the pilosebaceous gland where P. acnes
reside. Thus, the aims of this study were to compare
antimicrobial activity against P. acnes between oleogel
containing unencapsulated citronella oil and oleogel
containing citronella oil entrapped in SLPs. Stability of the
major components of citronella oil in both formulations
was also evaluated.
Materials and methods
Materials
Citronella grass oil was purchased from Thai China
Flavours & Fragrances Industry Co. (Thailand). Tryptic
casein soy agar was provided by Hispanlab (Spain) and
uid, thioglycollate broth, was obtained from Difco (USA).
International Journal of
Essential Oil Therapeutics
www.ijeot.com
Antibacterial activity of citronella oil solid lipid particles
in oleogel against Propionibacterium acnes and its
chemical stability
P. Lertsatitthanakorn
a
, S. Taweechaisupapong
b
, C. Aromdee
c
, W. Khunkitti
c
*
a
Faculty of Pharmacy, Mahasarakham University, Mahasarakham 44150, Thailand
b
Faculty of Dentistry, Khon Kaen University, Khon Kaen 40002, Thailand
c
Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand
Abstract
Acne vulgaris is the most common skin disease. Normal skin ora, particularly Propionibacterium acnes, is
a major cause of acne vulgaris. Bioactivity studies revealed that Java citronella oil (Cymbopogon winterianus
Jowitt) appears to have potential for use in the development of topical anti-acne preparations. The aims of
this study were twofold: rst, to compare antimicrobial activity against P. acnes between two preparations,
oleogel containing citronella oil and oleogel containing citronella oil-loaded solid lipid particles; second, to
investigate the stability of their major components. The results demonstrated that the solid lipid particle
preparation process caused a change to the main components of the citronella oil-loaded solid lipid
particles. Nevertheless, when the preparations were kept at 40°C for 120 days, the anti-P. acnes activity of
the oleogel containing the citronella oil-loaded solid lipid particles remained active, whereas the oleogel
containing unencapsulated citronella oil was inactive by day 45. Moreover, a solid lipid wall provided
protection from the volatility of the oil components of the oleogel and prolonged the release of citronella
oil. In conclusion, topical anti-acne preparations containing citronella oil-loaded solid lipid particles could
be good candidates for an alternative treatment for acne. Clinical trial on acne control efcacy would be
needed for further study.
Key words: Cymbopogon winterianus, citronella oil stability, acne, oleogel, solid lipid particles
EORC
e
rcrc
* Corresponding author.
E-mail address: watkhu@kku.ac.th
© Essential Oil Resource Consultants. All rights reserved.
International Journal of Essential Oil Therapeutics (2008) 2, 167-171

168
Cetyl alcohol was provided by Sigma-Aldrich (Germany).
Sodium dodecyl sulfate, cholesterol, squalene, lanolin,
stearic acid and the essential oil components, citronellal,
β-citronellol and geraniol, were purchased from Fluka
(Switzerland). Poloxamer 188 was a gift from BASF
(Germany). Light mineral oil was obtained from K. Science
Center and Medical, Thailand. Aerosil

was purchased
from Wacker HDK, Germany. Pork lard was obtained from
local suppliers in Thailand. All other chemicals were of
reagent grade.
Determination of citronella grass oil constituents by gas
chromatography/ mass spectrometry (GC/MS)
The chemical constituents of citronella grass oil were
identied by gas chromatograph mass spectrometer
(GCMS-QP 2010, Shimadzu

). Conditions were as follows:
Column: Rtx

-5 MS (30 m x 0.25 mm id. x 0.25 µm lm
thickness), Carrier gas: Helium. Oven temperature: 80-
230°C (10°C/min) fold for 1 min. Injection temperature:
270°C; Injection volume:1µL, split mode 1:50. Flow rate:
2 ml/min. MS conditions: positive ion detection, Interface
temperature: 250°C, Ion source: 0.98 kV, Scan Mode:
positive ion, full scan 35-550 m/z . The compounds were
identied by their linear retention indices and by comparing
their mass spectra with the NIST mass spectral libraries.
Some constituents were also compared with the synthetic
reference compounds (Fluka, Switzerland). The purity of
standard citronellal was ≥80-90%, standard geraniol was ≥
96.0% and standard β-citronellol was 90-95%.
To determine the quantity of citronella oil and reference
compounds in the preparations, ethanolic solution of
citronella oil (0.3-2.5 µl/ml
-1
) or the synthetic reference
compounds (0.1-1.0 µl/ml
-1
) were prepared and analyzed
using a gas chromatograph connected to a ame ionization
detector (FID) (GC 1850, Agilent

). A HP

-5 (30 m x
0.32 mm id. x 0.25 µm lm thickness) capillary column
was used. The injector temperature was 250°C, The oven
temperature was started at 100°C and held for 1 minute.
Temperature programming was increased from 100°C
to 220°C at 10°C/min
-1
and held for 1 min. The carrier
gas was nitrogen at a ow rate of 2 ml/min
-1
while the
split ratio was 1:10. The efuent was detected by FID at
280°C. A calibration curve was constructed by plotting
between concentrations of either reference compounds
or citronella oil and their peak area.
Citronella oil-loaded solid lipid particles
Preparation of solid lipid particles of citronella oil
Citronella grass oil SLPs were prepared using the hot
homogenization technique. Firstly, cetyl alcohol 6.5%
w/w, citronella grass oil 4.2% v/w and a mixed surfactants
solution containing 7.58%w/v poloxamer 188 and 7.58%
w/v sodium dodecyl sulfate 7.8 % w/w were accurately
weighed. Cetyl alcohol was melted at 65°C and citronella
oil was added and vigorously stirred for 1 min. The mixed
surfactant solution heated to 65°C was added to the
essential oil-lipid mixture and vigorously stirred for 1 min.
The dispersion was cooled down in an ice bath (4°C) for
30 min resulting in dispersion of the citronella oil SLPs.
The SLPs dispersion was freeze-dried using a freeze dryer
(Martin Christ, Germany). The SLPs were then analyzed for
citronella oil entrapment before being incorporated into
the oleogel. A sample of the preparation was extracted
with ethanol. The ltrate was analyzed for amount of
citronella oil and their major constituents by GC. The
freshly freeze-dried SLPs were kept at 40°C for 120 days
prior to morphologic investigation.
Characteristics of solid lipid particles
The morphology of both the freshly freeze-dried SLPs
containing citronella oil and the 120-day storage at 40°C
SLPs in lyophilized form was examined by scanning electron
microscopy (SEM; model JSM-6460 LV, Jeol

, Japan).
One hundred microcapsules per group were randomly
selected from the SEM monitor and the diameters of
three planes of each SLP were measured directly using
an automatic ruler provided in the SEM software. Some
microcapsules representing the freshly prepared SLPs and
the SLPs stored for 120 days were photographed. The
ultrastructure of cross-sectioned citronella grass oil SLPs
was investigated by an invert microscope (Nikon eclipse TS
100, Japan). Briey, lyophilized SLPs were transferred into
a at embedding mold containing pure embedding media,
namely Spurr’s resin

(Electron Microscopy Sciences,
USA). The molds were dried and polymerized at 45°C in a
hot air oven for 3 days. The resulting polymerized blocks
were then cut into thin sections and examined using an
invert microscope.
Preparation of oleogel containing citronella oil
Firstly, either citronella oil (6.5 ml) or the lyophilized SLPs,
having citronella oil equivalent to 6.5 ml, was added to light
mineral oil (80 g). Fumed silica (Aerosil

) 6.0 g was then
gradually added to increase the viscosity of the oil mixture.
Finally, light mineral oil was added to make 100 g and
mixed until a homogenous gel was obtained. A sample of
the preparation was extracted with ethanol and analyzed
for the amount of citronella oil and its major constituents
by GC.
Antibacterial activity of the preparations against P. acnes
Bacterial suspensions
P. acnes DMST 14916 was obtained from the culture
collection of the Department of Medical Sciences, Ministry
of Public Health, Thailand. Two colonies of P. acnes DMST
14916 from overnight cultures on Tryptic casein soy agar
were inoculated into 5 ml of uid thioglycollate broth and
incubated at 37 ºC for 72 h under anaerobic conditions to
give approximately 1 x 10
9
CFU/ml
-1
.
P. acne challenge test
500 µl of molten articial skin sebum modied from
Musial & Kubis, 2003, which consisted of pork lard (38%
w/w), stearic acid (24% w/w), lanolin (22% w/w), squalene
(12% w/w) and cholesterol (4% w/w) was added into a
test tube that resulted in the immediate solidication of
the sebum. A sample 0.76 g of each oleogel (equivalent
to 50 µl citronella grass oil) was accurately weighed. P.
acnes suspension (approximately 10
6
CFU/ml
-1
) was then
added. Finally, the total mixture was adjusted to 1.5 ml
with 1% Tween 80 in uid thioglycolate broth and stirred
well by a vortex mixer. The samples were kept at 37ºC
under anaerobic conditions. From each sample 0.1 ml
was collected for serial 10-fold dilution at 0 and 12 hours
of contact time. The sample was then plated on Tryptic
International Journal of Essential Oil Therapeutics (2008) 2, 167-171

169
casein soy agar and counted for survival bacteria after 72
hour incubation. The results were presented graphically
between log reduction of bacteria after 12 hours contact
time (log survival bacteria at 0 hour contact time - log
survival bacteria at 12 hour contact time) and the oleogel
storage time.
Evaporation rate of reference oil compounds in comparison
with citronella oil
Citronella oil and its major components consisting of
citronellol, citronellal and geraniol were kept at 50°C. The
oil and each compound were weighed at 30, 60, 90, 120, 150
and 180 minutes. The results were presented graphically
between mg of evaporated compound and time (minutes).
Data analysis
SPSS 11.5 for windows was used to perform statistical
analysis. The statistical difference between morphological
changes of SLPs before and after storage was analyzed
by Pair t-test. The composition changes of citronella
oil between the oleogel preparations were tested by
student t-test. P values of less than 0.05 were considered
statistically signicant.
Results
Characteristics of citronella oil loaded solid lipid particles
According to GC/MS, the main components of citronella
oil were citronellal (34.0% v/v), β-citronellol (11.1% v/v)
and geraniol (23.4% v/v). As shown in Table 1, the mean
particle sizes of freshly prepared citronella oil loaded-SLPs
are in the range of 70.83 to 82.20 µm, while the mean
particle size of 120 day-storage SLPs are in the range of
21.78 to 30.80 µm.
Table 1. Particle size of citronella grass oil SLPs directly
measured from SEM monitor (n = 100).
group diameter (µm)
freshly prepared citronella oil SLPs 77.62 ± 31.56
40°C, 120 day-storage citronella oil SLPs 26.61 ± 19.80*
* P-value <0.05 by Pair t-test
Morphology of the citronella SLPs revealed that the freshly
prepared SLPs had a spherical shape and possessed a rigid
wall with the oil enriched inside the particles, whereas that
of SLPs stored at 40°C for 120 days appeared to have an
irregular shape and a porous wall (Figures 1 and 2a, b).
Chemical stability of the key components of citronella oil in
oleogel
When unencapsulated citronella oil and the citronella SLPs
(equivalent to 6.5% v/w of citronella oil) were incorporated
into the oleogel base and kept at 40°C, the compositional
changes of the following components, citronellal,
β-citronellol, geraniol and elemol, were observed. On day
a) b)
Figure 1. Morphology of citronella grass oil SLP investigated by SEM.
a) Freshly prepared citronella grass oil SLP, magnication 1,000 ×.
b) 120 days-storage citronella grass oil SLP, magnication 3,000 ×.
a)

b)

Figure 2. Morphology of cross sectioned-citronella grass oil-loaded SLPs by invert microscope.
a) Freshly prepared citronella grass oil-loaded SLPs.
b) 120 days-storage at 40°C citronella grass oil–loaded SLPs.
International Journal of Essential Oil Therapeutics (2008) 2, 167-171


170
0, the amounts of the main components of unencapsulated
citronella oil in oleogel were as follows; citronellal was
1.9% v/w, geraniol was 1.27% v/w, β-citronellol was 0.73%
v/w and elemol was 0.19% v/w. As the preparation was
kept at 40°C for a longer period of time, the amount
of citronellal rapidly decreased whereas that of geraniol
and β-citronellol slowly decreased, while the amount of
elemol was practically stable (Figure 3a). The total amount
of these components had decreased by 31.5% v/w on day
120. However, the amount of the main components of
encapsulated citronella oil in oleogel was different from
that of unencapsulated citronella oil in oleogel. On day
0 the amounts of citronellal, geraniol, β-citronellol and
elemol were 0.24, 1.86, 0.07 and 1.41% v/w, respectively. It
was noticeable that by day 120, citronellal had dramatically
decreased to 0.13% v/w whereas β-citronellol and
geraniol had slightly decreased and elemol seemed to be
slightly increased (Figure 3b). The total amount of these
components had decreased by 4.96% v/w.
Antibacterial activity of the preparations against P. acnes
As shown in Figure 4, on day 0 the log reduction of P. acnes,
when exposed to oleogel containing citronella oil (6.52%
v/w), was 1.27. It decreased dramatically after 15 days
when stored at 40°C whereas oleogel containing citronella
oil entrapped in SLPs was 2.16 and slightly decreased to
1.62 on day 120.
Evaporation rate of reference oil compounds in comparison
with citronella oil
As demonstrated in Figure 5, evaporation rate of oil
components and citronella oil at 50°C was increased as
a function of time. The evaporation rate of citronella oil,
citronellal, β-citronellol and geraniol were 450.8, 254.5,
67.0 and 48.5 µg/min, respectively.
Discussion
The percentage of citronella oil entrapment in SLPs
appeared as 28.69% v/w [4]. Freeze-dried SLPs containing
citronella oil prepared by a simple hot homogenization
method gave particularly large microparticles. When
kept at 40°C for 120 days, the size of the microparticles
shrank signicantly by 65.89 ± 3.36% (p <0.05). This could
be due to the evaporation of volatile substances from
the SLPs causing porosity on the particle wall. Moreover,
the encapsulation process of citronella oil with hot
homogenization affected the compositional changes of
the citronella oil. The amount of citronellal decreased
dramatically while that of geraniol and elemol increased. In
order to explain this phenomenon, the evaporation rates
of the reference compounds of the main components
were carried out at 50°C. It might be possible that SLPs
of citronella oil prepared at 65°C cause citronellal to
evaporate rapidly. Nevertheless, the antibacterial activity
of the SLPs in oleogel was more effective against P. acnes
than that of citronella oil oleogel. This nding corresponds
to the total amount of the oil components in the SLPs
preparation, which was signicantly greater than that of
citronella oil in oleogel.
According to our previous studies, the minimum inhibitory
concentrations against P. acnes of citronellal, citronellol and
geraniol was 0.625, 0.0195 and 0.078% v/v, respectively, and
geraniol also had bactericidal effect at minimum bactericidal
concentration of 0.156% v/v [5]. Moreover, antibacterial
activity of elemol has been reported [6-8]. Therefore, the
antibacterial activity against P. acnes remained effective
even though the amount of the main components of
citronella oil in SLPs had changed. Since the targeting
site for acne control is located in sebaceous glands, the
particle size should be in the range of 3 to10 µm [9]. In
this study the SLPs were larger than 10 µm, suggesting
that the particles would remain on the skin surface. The
SLP characteristics revealed that their production gave oil-
enriched core microparticles, with an antibacterial activity
that was higher and longer than those of oleogel containing
citronella oil alone. Hence, its anti-P. acnes activity would
be due to the slow release of citronella oil from the SLPs.
These ndings were supportive of the statement that
solid lipid nanoparticles with a drug-enriched core lead to
sustained release [10].
In conclusion, it has been shown that the SLP production
process, particularly the processing temperature, could
affect the chemical stability of the citronella oil components.
Figure 3. The main components changes of citronella oil
in oleogel when stored at 40°C
unencapsulated citronella oil
% v/w of main components of citronella oil% v/w of main components of citronella oil
entrapped in SLP oleogel
citronella oil loaded-SLP


International Journal of Essential Oil Therapeutics (2008) 2, 167-171

171
Nevertheless, a solid lipid wall was able to protect against
the volatility of the oil components from oleogel and
prolonged the release of citronella oil. Therefore, oleogel
containing citronella oil SLPs might be an alternative choice
for acne control. However, in order to deliver citronella oil
into the sebaceous gland to improve anti acne efcacy, it
is important to reduce the SLPs’ size to the optimal range.
Furthermore, clinical study on acne control efcacy would
be needed.
Acknowledgements
The authors are thankful for the support of a Co-operative
Research Network (CRN) grant from the Pharmacy
Section of The Ministry of Education, Thailand and The
Graduate Research Fund of Khon Kaen University.
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log reduction (CFU/ml
-1
)
Figure 4. Log reduction of P. acnes after 12 hours
exposure to unencapsulated citronella and citronella
oil-loaded SLPs in oleogels (Mean ± S.D., n = 3).
mg of evaporated oil
Figure 5. Evaporation of reference oil compounds and
citronella oil at 50°C.

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International Journal of Essential Oil Therapeutics (2008) 2, 167-171