Solid Lipid Microparticles (SLM) Containing Juniper Oil as Anti-Acne Topical
Carriers: Preliminary Studies
Elisabetta Gavini and Vanna Sanna
Dipartimento di Scienze del Farmaco, University of Sassari, Sassari, Italy
Reeta Sharma
Institute of Pharmacy—Faculty of Mathematics and Natural Sciences, University of Oslo, Oslo, Norway
Claudia Juliano and Marianna Usai
Dipartimento di Scienze del Farmaco, University of Sassari, Sassari, Italy
Mauro Marchetti
Sezione di Sassari, Istituto di Chimica Biomolecolare-CNR, Sassari, Italy
Jan Karlsen
Institute of Pharmacy—Faculty of Mathematics and Natural Sciences, University of Oslo, Oslo, Norway
Paolo Giunchedi
Dipartimento di Scienze del Farmaco, University of Sassari, Sassari, Italy
Solid lipid microparticles (SLM) were used as carriers of
juniper oil and proposed for the topical treatment of acne vulgare.
The formulations were obtained by the o/w emulsification
method. Compritol and Precirol were employed as lipidic
materials. Emulsions containing 1.5% (w/w) of lipophilic phase
(lipid and oil) and two different lipid to oil ratios (1:1 and 2:1)
were prepared. Blank particles were also prepared, as a com-
parison. The SLM were characterized in terms of encapsulation
efficiency, size, and morphology. The particle size stability in
aqueous dispersions was monitored over one month. Evaporation
of volatile compounds of oil from microparticles by weight loss
was investigated. The qualitative composition of Juniper oil
before and after the encapsulation process was determined by gas
chromatography (GC) and gas chromatography/mass spectrum
(GC/MS) analyses. The antimicrobial activity of the oil
encapsulated into the lipid microparticles against P. acnes was
studied as contact time assay and compared to the activity of the
oil not encapsulated. The emulsification method here described
was a good technique for the encapsulation of essential oils.
Percentage yields of production and encapsulation efficiencies
were higher for Compritol preparations than for these prepared
using Precirol. All preparations were characterized by similar
particle size distributions (dvs about 3–4 mm) regardless of lipid
type and lipid to oil ratios. Microscopy observations showed that
the microparticles in aqueous dispersions had almost spherical
shape, independently from their composition. The scanning
electron microscopy (SEM) analyses showed that when the
particles were dried, they had an irregular shape and a rough
surface. The SLM dispersions based on Compritol revealed
particle size stability over the investigated period of 30 days. In
contrast, an increase of the mean dimensions in the preparations
containing Precirol was observed. A low loss of volatile oil
compounds owing to evaporation from dry particles was found in
all preparations. This indicated that the microparticles were able
to substantially maintain the oil loaded inside their lipidic
structure, reducing its volatility. Some modifications of compo-
sition were found in the oil encapsulated in SLM with respect to
the juniper oil raw material, but these modifications did not
decrease the antibacterial activity of the oil. The SLM here
described are promising carriers for the development of anti-acne
topical formulations containing Juniper oil.
Keywords solid lipid microparticles, juniper oil, particle size
stability, antibacterial activity, GC-analysis
Received 27 January 2005, Accepted 20 April 2005.
Address correspondence to Paolo Giunchedi, Dipartimento di
Scienze del Farmaco, University of Sassari, via Muroni 23/a,
Sassari 07100, Italy; Fax: +39-079-228733; E-mail: pgiunc@
uniss.it
479
Pharmaceutical Development and Technology, 10:479–487, 2005
Copyright D 2005 Taylor & Francis Inc.
ISSN: 1083-7450 print / 1097-9867 online
DOI: 10.1080/10837450500299727
Order reprints of this article at www.copyright.rightslink.com
Phar
mac
euti
cal
Dev
elopm
ent
and T
echnolo
gy D
ow
nlo
aded
fro
m i
nfo
rmah
ealt
hca
re.c
om
by H
ealt
h S
cien
ce L
earn
ing C
tr o
n 0
6/0
9/1
2F
or
per
sonal
use
only
.
INTRODUCTION
Acne vulgaris is an inflammation of sebaceous glands
characterized by pustules and skin lesions. Various
bacteria, including chiefly P. acnes and S. epidermidis,
have been identified as the etiological agents of acne.[1,2]
Current clinical practice for mild to moderate
inflammatory acne frequently involves the topical use of
drugs that affect the main pathogenetic factors responsible
for the development of acne, i.e., hyperseborrhea, hyper-
keratosis, microbial colonization, and inflammation.[3–5]
The juniper berry oil is stated to exhibit a variety of
pharmacological effects such as diuretic, carminative, and
antioxidative;[6,7] it is also effective for acne and
congested skin for its antimicrobial and antiseptic
activities.[8] In recent studies the antifungal and antibac-
terial activities of juniper berry oil have been put into
evidence.[9,10]
Microencapsulation of essential oils is an important
technique that offers protection against oxidation of the
active components[11] and reduces volatility.[12] Further-
more, conversion of the oil to a powder allows easier
handling and preparing of formulations.
Recently, lipid particles have been employed in the
encapsulation of retinol, tocopherol,[13] vitamin-A,[14]
glucocorticoids,[15] and the sunscreen oxybenzone.[16]
Lipid nanoparticles have been also proposed as cosmetic
and dermatological carriers for topical application of
synthetic fragrances and volatile substances.[17] They are
capable of protecting unstable active compounds from
degradation and releasing the actives in a controlled
way.[15,18,19] Essential oil encapsulation into lipid par-
ticles appears attractive also because of the high affinity
of the oil for the lipid materials.
In this study, solid lipid microparticles (SLM) were
proposed as carriers for the topical application of juniper
oil in the treatment of acne.
The SLM-containing Juniper oil were obtained by an
oil in water (o/w) emulsification method. Compritol and
Precirol were employed as lipidic carrier materials. Blank
lipidic microparticles were also prepared as a comparison.
The SLM have been characterized in terms of encapsu-
lation efficiency, yield of production, size, and morphol-
ogy. The particle size stability of SLM was monitored for
one month. Evaporation of volatile compounds of oil from
microparticles was studied.
The commercial oil used as raw material and the
encapsulated oil in SLM were analyzed and compared in
terms of quality composition by GC and GC/MS analyses.
The antimicrobial activity of SLM was tested as contact
time assay and compared to the antimicrobial activity of
the free oil.
MATERIALS
Glycerol behenate (Compritol1 888 ATO) and
glycerol palmitostearate (Precirol1 ATO 5) were obtained
from Gattefosse (Saint-Priest, Cedex, France). The essen-
tial oil of Juniperus communis L (juniper oil) was provided
by Aboca (San Sepolcro, Firenze, Italy). Hexane was
purchased from Carlo Erba Reagenti (Roma, Italy) and
polyoxyethylene-sorbitan monooleat (Tween1 80) from
Sigma-Aldrich Chemie GmbH (Steinheim, Germany).
Propionibacterium acnes ATCC 6919 was obtained
from LGC Promochem (Sesto San Giovanni, Italy). Brain
heart infusion agar (BHA), defibrinated horse blood,
phosphate buffered saline (PBS, Dulbecco A; pH 7.3),
and Gas Generating Kit were purchased from Oxoid
(Basingstoke, United Kingdom).
METHODS
Solid Lipid Microparticles (SLM) Preparation
Solid lipid microparticles were prepared by oil in
water (o/w) emulsification using a method previously
described.[20]
Table 1
Compositions, mean particle sizes, yields of production and encapsulation efficiencies of SLM prepared
SLM Lipid
Lipid-to-oil
ratio
Particle size±SD
(dvs, mm)
Yield of
production±SD (%)
Encapsulation
efficiency±SD (%)
SL C Compritol 1:0 3.3±1.2 60.1±2.5 —
SL P Precirol 1:0 3.1±0.2 51.9±2.3 —
SL 1 Compritol 1:1 4.3±1.3 56.1±4.3 35.0±4.6
SL 2 Compritol 2:1 4.1±1.4 60.0±1.9 34.7±1.8
SL 3 Precirol 1:1 4.6±1.9 53.1±1.6 23.3±4.4
SL 4 Precirol 2:1 4.1±1.3 51.7±6.7 21.8±4.1
E. Gavini et al.480
Phar
mac
euti
cal
Dev
elopm
ent
and T
echnolo
gy D
ow
nlo
aded
fro
m i
nfo
rmah
ealt
hca
re.c
om
by H
ealt
h S
cien
ce L
earn
ing C
tr o
n 0
6/0
9/1
2F
or
per
sonal
use
only
.
Compritol and Precirol were chosen as lipidic
components. Table 1 reports the composition of SLM
microparticles. Emulsions consisting always of 1.5% w/
w of lipophilic phase (lipidic excipient and juniper oil)
in purified water were produced; the lipid-to-oil
weight ratios of 1:1 and 2:1 were used. Two batches of
blank SLM, constituted only by the lipidic excipients
(1.5% w/w) were prepared as a comparison.
Briefly, the lipidic excipients were melted (at a
temperature of 90°C for Compritol and 70°C for Precirol)
and the juniper oil was then slowly added. Distilled water
up to 100 g was heated to the same temperature and then
poured into the hot oily phase.
The emulsions obtained were homogenized by high
shear homogenization (Silverson SL2, Crami, Italy), at
6000 rev/min, for 5 min. The emulsions were then
cooled under magnetic stirring, at room temperature,
until solidification of the microparticles occurred. The
SLM obtained were collected by filtration and dried
at room temperature. Each preparation was carried out
in triplicate.
Yield of Production, Encapsulation Efficiency
and Particle Size
Percentage yield of production, Y%, of dry solid lipid
microparticles was calculated using the following formula:
Y% ¼ ðWa=WtÞ � 100% ½1�
where Wa was the actual amount of dry microparticles
obtained, and Wt was the theoretical amount of lipidic
materials (lipid and oil) introduced for each preparation.
Mean values of three preparations were used to state the
yield of production for every batch.
For the determination of the amount of essential oil
encapsulated it was not possible to distill the oil directly
from the particles, due to the high affinity of the oil to the
lipids. An extraction method of the oil from the lipidic
carrier material was performed using hexane. The extract
phase was then evaporated under vacuum at about 30°C;
the residue obtained was suspended in a suitable volume
of solution containing 0.15% w/v of Tween 80. The
surfactant was used to obtain the optimal dispersion of
the residue.
The suspension was distilled for 3 hr at a distillation
rate of 2–3 mL/min. The lighter than water, slightly
yellow, and limpid oil was then dried over anhydrous
sodium sulfate and stored in sealed containers under
refrigeration. Steam distillation was carried out using the
European Pharmacopoeia hydrodistillation apparatus
(Clevenger type).
Percentage oil encapsulation efficiency, E%, was
defined as follows:
E% ¼ ðW1=W2Þ � 100% ½2�
where W1 was the actual amount of oil encapsulated
in a known amount of microparticles and W2 was
the theoretical amount of oil introduced in the same
amount of particles. Each determination was carried out
in triplicate.
The mean particle size of SLM was determined by
Coulter Laser Diffractometry (Coulter LS 100Q laser
sizer, Beckman Coulter, Miami, FLA). The test was
carried out in triplicate on SLM before the drying process
and, after the filtration, on the dry microparticles
suspended in aqueous medium. The average particle size
was expressed as mean volume-surface diameter (dvs,
mm).[21]
Microscopy Observations
The morphology of SLM in aqueous dispersion was
examined by photomicrography with a stereomicroscope
(magnitude 6.4�10) (Zeiss MC 63, Zeiss, Germany)
connected to a digital camera (Zeiss M 35 Winder). The
morphology of the dry microparticles was examined using
a scanning electron microscope (SEM, Zeiss DSM 962,
Zeiss, Germany). Samples of particles were placed on
double-sided tapes, which had previously been secured on
aluminium stubs, and then analyzed after gold sputtering
at 20 kV acceleration voltage, under argon atmosphere.
Particle Size Stability of SLM
This test was performed on microparticles using the
Coulter Laser Diffraction method previously described to
investigate the possible aggregation of particles during a
defined time period and in relation to the possible
formulation of SLM into an aqueous medium. The
particle size stability was carried out both on the primary
dispersion (i.e., the aqueous medium, purified water, in
which the particles were formed) and on a redispersion
(i.e., the suspension of the dry particles, obtained by
filtration of the primary dispersion, into a solution
containing 0.15% w/v of Tween1 80. This concentration
of surfactant was used to obtain the optimal dispersion
of SLM).
The stability was monitored at day 1 and after 30
days, after storage of the samples in sealed glass vials at
room temperature. All experiments of particle size
stability were performed in triplicate (SD within 1.14).
481SLM-Containing Juniper Oil
Phar
mac
euti
cal
Dev
elopm
ent
and T
echnolo
gy D
ow
nlo
aded
fro
m i
nfo
rmah
ealt
hca
re.c
om
by H
ealt
h S
cien
ce L
earn
ing C
tr o
n 0
6/0
9/1
2F
or
per
sonal
use
only
.
In Vitro Evaporation of Volatile Oil Compounds
from Dry Particles
Evaporation of volatile oil components from the
particles was investigated in terms of percent weight loss.
The tests were carried out on oil loaded microparticles
(SL 1, SL 2, SL 3 and SL 4). A quantity of microparticles
containing 15.0 mg of oil was filled in glass vials and
stored in an oven at 32°C up to 48 hr. At selected time
intervals (from 1 to 6 times every hour and after 10, 24,
and 48 hr) the weight loss of the particles was measured.
Percentage weight loss, W%, was estimated accord-
ing to the equation:
W% ¼ ½ðWiÿWfÞ=Wi� � 100% ½3�
where Wi and Wf were the initial and final weight of
samples, respectively. Each experiment was performed in
triplicate and the mean percentage was calculated.
GC Analyses
For the evaluation of the composition of oil en-
capsulated in SLM, a distillation process of the oil from
the lipidic particles was carried out and then the oil was
analyzed by GC. In fact it was not possible to perform
direct GC analyses on SLM particles due to the
interference of the lipidic material with the oil.
Oil Distillation
The juniper oil encapsulated in SL 1 (chosen on the
basis of the results obtained previously) was extracted
from lipid material using n-hexane after breaking the
particles in a mortar. This extract was treated following
the procedure described earlier for the determination of
the encapsulation efficiency.
Oil Analyses
The commercial oil and the oil distilled from SL 1
microparticles were analyzed to compare their composi-
tion. Analyses were performed by GC and GC/MS.
The GC- four replicates of each sample were
analyzed by using a Hewlett-Packard Model 5890A GC
equipped with a flame ionization detector and fitted with
a 60 m�0.25 mm, thickness 0.25 mm AT-5 fused silica
capillary column (Alltech). Injection port and detector
temperature were 280°C. The column temperature was
programmed from 50°C to 135°C at 5°C/min (1 min),
5°C/min up 225°C (5 min), 5°C/min up 260°C, and held
for 10 min.
The samples (0.2 ml each), analyzed without dilution
with 2,6-dimethylphenol as internal standard, were
injected using a split/splitless automatic injector HP
7673 and using helium as the carrier gas. Measurements
of peak areas were performed with an HP workstation; the
threshold was set at 0, peak width at 0.02. The data
reported in Table 2 are the average of four GC injections.
The quantization of each compound was expressed as
absolute weight percentage using internal standard and
response factors. The detector response factors (RFs) were
determined for key components relative to 2,6-dimethyl-
phenol and assigned to other components on the basis of
functional group and/or structural similarity.[22]
The GC/MS analyses were carried out with a Hewlett
Packard G1800B-GCD System using the same conditions
and column described earlier. The column was connected
with the ion source of the mass spectrometer. Mass units
were monitored from 10 to 450 at 70 eV. The samples
(0.1 mL each), analyzed diluted in hexane, were injected
manually. The identification of compounds was based on
comparison with their Kovat’s Indexes (KI) and by
comparison with their mass spectra with those of
published data and HP libraries.[23,24]
Contact Time Assay
This assay was performed on pure oil (commercial
juniper oil), on oil distilled from SL 1 formulation, and on
SL 1 loaded microparticles. For this test, essential oils
were dissolved in PEG 200 at 100 mg/mL concentration.
Empty Compritol (SL C) microparticles were used as a
comparison. P. acnes was selected as bacterium to test the
potential anti-acne activity of SL 1.
Contact time was determined as the exposure time
required by fixed amount of oil to kill a standardized
microbial inoculum of P. acnes. Microorganisms in the
logarithmic phase of growth were suspended at a density
of 5�105–1�106 colony forming units (c.f.u.)/mL in 10
mL of PBS containing 2 mg/mL of the commercial oil or
distilled oil from SL 1 formulation, or a quantity of
microparticles corresponding to the same oil concentra-
tion. Two control tubes (bacteria suspended in PBS alone
at the same density and bacteria suspended in PBS+PEG
200) were included in each experiment. At time zero and
at regular intervals, 0.5 mL of suspension were removed,
subjected to serial 10-fold dilutions in PBS and seeded on
blood agar plates (BHA+10% horse blood) for P. acnes.
The number of viable microorganisms at each time was
evaluated counting colonies after anaerobic incubation for
24 hr at 35°C.
E. Gavini et al.482
Phar
mac
euti
cal
Dev
elopm
ent
and T
echnolo
gy D
ow
nlo
aded
fro
m i
nfo
rmah
ealt
hca
re.c
om
by H
ealt
h S
cien
ce L
earn
ing C
tr o
n 0
6/0
9/1
2F
or
per
sonal
use
only
.
Table 2
Some of the main components (%) of the pure juniper oil compared with those of the distilled oil from SL 1
Compoundsa KIb Commercial juniper oil (%) SL 1 (%)
a-thujene 931 1.91
a-pinene 939 22.75 0.16
Camphene 953 0.47
Sabinene 976 0.35
b-pinene 980 2.89
b-myrcene 991 11.88 0.36
a-phellandrene 1005 0.62
d-3-carene 1011 3.95
Para-cymene 1026 2.12 0.70
Limonene 1031 6.56
g-terpinene 1062 6.11
Terpinolene 1088 2.98 0.71
Terpinen-4-ol 1177 8.14 3.34
a-terpineol 1189 0.75 0.47
Merthyl citronellate 1261 0.53
Bornyl acetate 1285 1.22
2-undecanone 1291 0.32
a-cubebene 1351 0.87 3.91
a-copaene 1376 0.40 2.68
Geranyl acetate 1383 0.51
b-elemene 1391 0.85 6.30
Trans-a-ambrinol 1412 1.08
b-cedrene 1418 0.41
b-caryophyllene 1418 3.01 7.32
g-elemene 1433 3.21 4.33
a-caryophyllene 1454 3.22 6.27
9-epi-caryophyllene 1467 1.72
Germacrene D 1480 0.65 4.40
Epi-cubebol 1493 2.34
a-selinene 1494 2.65
Eudesma-4(14),11-diene 1494 0.45 2.43
a-muurolene 1499 2.78
g-cadinene 1513 0.65 3.62
d-cadinene 1524 0.61 5.11
a-cadinene 1538 0.72 1.18
Selina-3,7(11)-diene 1542 2.16 0.58
a-calacorene 1542 1.19
Sesquisabinene hydrate 1545 0.58
Germacrene B 1556 1.43
b-calacorene 1563 0.48
Caryophyllene alcohol 1568 3.15
Caryophyllene oxide 1581 2.06
Trans-b-elemenone 1600 1.94
Humulene epoxide II 1606 2.20
2-caren-4-ol 4.09
5-cedranone 1618 0.51
Cedr-8-(15)-en-alpha-ol 1644 1.97
a-muurolol 0.63
a-cadinol 1653 3.18 3.10
5-neo-cedranol 1677 0.59
Crysolide 1721 1.04
aCompounds are listed in elution order.bKovat Index.
483SLM-Containing Juniper Oil
Phar
mac
euti
cal
Dev
elopm
ent
and T
echnolo
gy D
ow
nlo
aded
fro
m i
nfo
rmah
ealt
hca
re.c
om
by H
ealt
h S
cien
ce L
earn
ing C
tr o
n 0
6/0
9/1
2F
or
per
sonal
use
only
.
RESULTS AND DISCUSSION
SLM Preparation and Characterization
The high shear homogenization used in the experi-
mental conditions previously described was a good
technique for the preparation of SLM: it was a one-step
process that involved simply the preparation of an
emulsion containing a lipophilic phase (lipid and oil)
and water.
Table 1 reported mean particle size, as dvs (mm),
percentage yields of production, and encapsulation
efficiencies of the SLM prepared. Particle size analyses
revealed no significant differences comparing the
different batches: oil empty microparticles were charac-
terized by dvs of about 3 mm, while for oil-loaded
particles the mean diameter was about 4 mm, independent
of their compositions.
As shown in Table 1, the yields of production of SLM
varied from 52% to about 60%; microparticles constituted
by Compritol showed the highest values compared to
those made of Precirol. The relatively lower yields of
production found in the case of Precirol preparations were
probably due to the consistency of the microparticles
observed during the filtering process, which was ‘‘wax
like,’’ with consequent sticking of the particles to the
filter and difficulty in their collection. Compritol micro-
particles did not have this consistency and consequently
the difficulty in the collection was not found. These two
behaviors could be due to the different chemical
composition of the two lipids used as carriers.
Relatively low values of encapsulation efficiency
were found for all SLM: as reported in Table 1 the
percentage of the oil encapsulated into microparticles
varied from 22% to about 35%. These values appeared to
depend on the lipid employed and on the lipid-to-oil ratio.
SLM containing Compritol always showed higher values
of encapsulation efficiency compared to Precirol prepara-
tions. As reported in literature, the chemical composition
of the lipid can influence determining drug incorpora-
tion.[25] In all cases, an increase in the encapsulation of oil
was obtained for batches having a lipid to oil ratio of 1:1 in
comparison with formulations with a 2:1 ratio.
Microscopy Observations
Shape and surface of microparticles were analyzed by
stereomicroscopy and SEM analyses (photos not
reported). Photomicrographies of SLM in aqueous
dispersions revealed that the particles for all the batches
prepared had an almost spherical shape, were well
separated, and did not form aggregates.
Particles after filtration and drying had an irregular
shape and a rough surface, characterized by numerous
lipidic slivers regardless of lipidic composition and lipid-
to-oil ratio.
Particle Size Stability of SLM
In Figure 1A–B the diagrams of mean particle sizes
(expressed as dvs, mm) of the primary SLM aqueous
dispersions containing the Compritol (SL 1, SL 2, and SL
C) and Precirol microparticles (SL 3, SL 4, and SL P)
were reported. The analyses were performed at two
different time periods (day 1 and day 30).
Right after production (day 1), the particle size for all
batches is of about 3–4 mm. The lipidic microparticles
based on Compritol were characterized by a good long-
term stability as the particle size of SLM dispersions
remained unchanged during 1 month (Figure 1A). A
different behavior was found for batches containing
Figure 1. Particle size stability of SLM primary dispersions
containing Compritol (a) and Precirol (b).
E. Gavini et al.484
Phar
mac
euti
cal
Dev
elopm
ent
and T
echnolo
gy D
ow
nlo
aded
fro
m i
nfo
rmah
ealt
hca
re.c
om
by H
ealt
h S
cien
ce L
earn
ing C
tr o
n 0
6/0
9/1
2F
or
per
sonal
use
only
.
Precirol: in this case a significant particle size increase
occurred (Figure 1B).
As reported by Jenning and Gohla,[26] large amounts
of glycerides like monoglycerides are possibly responsi-
ble for physical destabilization. The amount of mono-
glycerides for Precirol is higher than that for Compritol;
thus, this could result in unstable forms and particle
dimension growth taking place.
In both cases (Compritol and Precirol particles) the
presence of the oil did not influence particle stability, as
their behavior was similar both on oil-loaded and oil-
empty microparticles.
Figure 2A–B reported mean particle sizes (dvs, mm)
of redispersed dry microparticles in Tween 80 solution at
day 1 and day 30. Immediately after the redispersion
(day 1), the mean particle size of oil-loaded SL 1 and SL 2
resulted in about 7 mm while the dvs of particles in the
primary dispersion was 3–4 mm. On the contrary, the oil-
empty microparticles (SL C) maintained their original size.
After 30 days, the dvs values of SL 1 and SL 2
microparticles remained unchanged while SL C particle
size slightly increased from 3 mm to about 5 mm
(Figure 2A).
The redispersion of particles based on Precirol was
characterized at the day 1 by an increased dvs to 8–9 mm
in the case of the oil-loaded SLM (SL 3 and SL 4), and to
about 6 mm for the oil-empty microparticles SL P.
After 1 month SL 3 and SL 4 showed similar particle
size while SL P particles reveal an increased value of dvs
to approximately 9 mm (Figure 2B).
When the dry microparticles are redispersed, the
increase of particle size observed was probably due to
formation of small aggregates (hence leading to increased
mean values compared to the primary dispersion). This
was more evident for the oil-loaded SLM compared to the
oil-empty SLM, indicating that, in this case, the presence
of the oil promoted particle aggregation.
However the particle size values were stable during
the investigated period (1 month); it can be due to the
influence of surfactant in the aqueous medium used for
the redispersion.
In Vitro Evaporation of Volatile Oil Compounds
The evaporation tests were performed in order to
evaluate the loss of the volatile compounds of the oil. The
obtained profiles were illustrated in Figure 3.
A relatively low percentage evaporation of volatile
compounds was achieved for all formulations: from 12%
to about 3%.
Compritol seemed to preserve the evaporation of
volatile compounds more efficiently as both preparations
SL 1 and SL 2 showed low amounts of oil evaporated
(about 5% and 1.5%), dependent on the lipid-to-oil ratios.
Precirol preparation SL 3 (characterized by the lipid-
to-oil ratio 1:1) showed the highest loss (12%), while only
SL 4 microparticles had an oil loss of about 3% in 10 hr.
Figure 3. Evaporation profiles of volatile compounds from dry
microparticles upon storage at 32°C.
Figure 2. Particle size stability of redispersed dry micro-
particles containing Compritol (a) and Precirol (b).
485SLM-Containing Juniper Oil
Phar
mac
euti
cal
Dev
elopm
ent
and T
echnolo
gy D
ow
nlo
aded
fro
m i
nfo
rmah
ealt
hca
re.c
om
by H
ealt
h S
cien
ce L
earn
ing C
tr o
n 0
6/0
9/1
2F
or
per
sonal
use
only
.
GC Analyses
Thirty-one compounds were identified in the com-
mercial juniper oil used as raw material, representing
97.4% of the total oil. As shown in Table 2 a change in
the chemical composition was observed when juniper oil
was encapsulated into SL 1 microparticles. The analyses
showed that juniper oil raw material was dominated by
the monoterpenes a-pinene (22.75%), b-myrcene
(11.88%), limonene (6.56%), g-terpinene (6.11%), and
the alcohol terpinen-4-ol (8.14%). The juniper oil
recovered from SL 1 showed a decrease of the named
monoterpenes, in particular, the monoterpenes hydrocar-
bon disappeared completely and constituents such as b-
elemene, b-caryophyllene, a-caryophyllene, and d-cadi-
nene were present in higher values compared to the
commercial juniper oil. Moreover series of new com-
pounds were detected in the distilled essential oil from SL
1. Among the new compounds, the more representative
were oxygenated terpenes, such as 2-caren-4-ol and
caryophyllene alcohol.
The modification of oil composition (distilled from
SL 1) compared to the commercial juniper oil, could be
due to the different loading of the oil components into
the microparticles (the observed order of loading was:
b-pinene= limonene=gamma;-terpinene<a-pinene<bÿmircene<terpinen-4-ol) and/or to chemical reactions
(such as oxidation and isomerization) of the oil
encapsulated.[27]
Contact Time Assay
The results of the contact time tests were shown in
Figure 4. At a concentration of 2 mg/mL, the essential oils
were able to decrease microbial viability in a time-
dependent way. In particular, the commercial oil was able
to completely inactivate P. acnes in 1 hr while oil loaded
into microparticles killed 60% of P. acnes after 1.5 hr.
The oil obtained by distilling SL 1 microparticles
resulted as more effective on bacterial inocula: it killed
100% of P. acnes in only 30 min.
The killing effect of the microparticles can be
explained by the prolonged delivery rate of the oil from
microparticles. The more potent activity against P. acnes
of the distilled oil compared with the commercial oil
could be ascribed to the different composition of the oils.
CONCLUSIONS
The incorporation of juniper oil into SLM provided a
microparticulate system that can offer possible topical
applications. Both lipids used (Compritol and Precirol)
were able to entrap the oil, but higher values for yield of
production and encapsulation efficiency were achieved
for particles based on Compritol.
Long-term particle size stability of dispersions based
on Compritol was better compare to Precirol. Dry
particles redispersed in Tween 80 solution showed
stability in particle size over the investigated period for
both Compritol and Precirol formulations.
According to the results, Compritol can be proposed
as a lipidic carrier suitable for the preparation of topical
suspensions based on microparticles. The SL 1 particles
maintained the antibacterial activity against P. acnes. The
higher killing activity of the distilled oil for the micro-
particles could be related to the relatively higher fraction
of new oxygenate compounds of the distilled oil com-
pared to the commercial juniper oil shown by GC.
REFERENCES
1. Ashkenazi, H.; Malik, Z.; Harth, Y.; Nitzan, Y.
Eradication of Propionibacterium acnes by its
endogenic porphyrins after illumination with high
intensity blue light. FEMS Immunol. Med. Micro-
biol. 2003, 35 (1), 17–24.
2. Higaki, S. Lipase inhibitors for the treatment of acne.
J. Mol. Catal., B Enzym. 2003, 22 (5–6), 377–384.
3. Wolf, J.E., Jr.; Kaplan, D.; Kraus, S.J.; Loven, K.H.;
Rist, T.; Swinyer, L.J.; Baker, M.D.; Liu, Y.S.;
Czernielewski, J. Efficacy and tolerability of
combined topical treatment of acne vulgaris with
adapalene and clindamycin: a multicenter, random-
ized, investigator-blinded study. J. Am. Acad.
Dermatol. 2003, 49 (3), S211–S217.
4. Akhavan, A.; Bershad, S. Topical acne drugs:
review of clinical properties, systemic exposure,
and safety. Am. J. Clin. Dermatol. 2003, 4 (7), 473–
492.Figure 4. Effect of SL 1 and oil distilled from SL 1 compared
to commercial oil on bacteria P. acnes.
E. Gavini et al.486
Phar
mac
euti
cal
Dev
elopm
ent
and T
echnolo
gy D
ow
nlo
aded
fro
m i
nfo
rmah
ealt
hca
re.c
om
by H
ealt
h S
cien
ce L
earn
ing C
tr o
n 0
6/0
9/1
2F
or
per
sonal
use
only
.
5. Gollnick, H.; Schramm, M. Topical drug treatment
in acne. Dermatology 1998, 196 (1), 119–125.
6. Filipowicz, N.; Kaminski, M.; Kurlenda, J.; Asztem-
borska, M.; Ochocka, J.R. Antibacterial and antifun-
gal activity of juniper berry oil and its selected
components. Phytother. Res. 2003, 17 (3), 227–231.
7. Sanchez de Medina, F.; Gamez, M.J.; Jimenez, I.;
Jimenez, J.; Osuna, J.I.; Zarzuelo, A. Hypoglycemic
activity of juniper ‘‘berries.’’ Planta Med. 1994, 60
(3), 197–200.
8. Chatzopoulou, P.; de Haan, A.; Katsiotis, S.T.
Investigation on the supercritical CO2 extraction of
the volatile constituents from Juniper communis
obtained under different treatments of the ‘‘berries’’
(cones). Planta Med. 2002, 68, 827–831.
9. Angioni, A.; Barra, A.; Russo, M.T.; Coroneo, V.;
Dessi, S.; Cabras, P. Chemical composition of the
essential oils of Juniperus from ripe and unripe
berries and leaves and their antimicrobial activity. J.
Agric. Food Chem. 2003, 51 (10), 3073–3078.
10. Cosentino, S.; Barra, A.; Pisano, B.; Cabizza, M.;
Pirisi, F.M.; Palmas, F. Composition and antimicro-
bial properties of Sardinian Juniperus essential oils
against foodborne pathogens and spoilage micro-
organisms. J. Food Prot. 2003, 66 (7), 1288–1291.
11. Ribeiro, A.; Arnaud, P.; Frazao, S.; Venancio, F.;
Chaumeil, J.C. Development of vegetable extracts
by microencapsulation. J. Microencapsul. 1997, 14
(6), 735–742.
12. Chan, L.W.; Lim, L.T.; Heng, P.W.S. Microencap-
sulation of oils using sodium arginate. J. Micro-
encapsul. 2000, 17 (6), 757–766.
13. Wissing, S.A.; Muller, R.H. Cosmetic applications
for solid lipid nanoparticles (SLN). Int. J. Pharm.
2003, 254 (1), 65–68.
14. Jenning, V.; Gysler, A.; Schafer-Korting, M.; Gohla,
S.H. Vitamin A loaded solid lipid nanoparticles for
topical use: occlusive properties and drug targeting
to the upper skin. Eur. J. Pharm. Biopharm. 2000,
49, 211–218.
15. Maia, C.S.; Mehnert, W.; Schafer-Korting, M. Solid
lipid nanoparticles as drug carriers for topical gluco-
corticoids. Int. J. Pharm. 2000, 196 (2), 165–167.
16. Wissing, S.A.; Muller, R.H. Solid lipid nanopar-
ticles as carrier for sunscreens: in vitro release and
in vivo skin penetration. J. Control. Release 2002,
81 (3), 225–233.
17. Wissing, S.A.; Mader, K.; Muller, R.H. Solid lipid
nanoparticles (SLNTM) as a novel carrier system
offering prolonged release of the perfume Allure
(Chanel). Int. Symp. Control. Release Bioact. Mater.
2000, 27, 311–312.
18. Zur Muhlen, A.; Mehnert, W. Drug release and
release mechanism of prednisolone loaded solid
lipid nanoparticles. Pharmazie 1998, 53, 552.
19. Yang, S.C.; Lu, L.F.; Cai, Y.; Zhu, J.B.;
Liang, B.W.; Yang, C.Z. Body distribution in
mice of intravenously injected camptothecin
solid lipid nanoparticles and targeting effect on
brain. J. Control. Release 1999, 59 (3), 299–
307.
20. Sanna, V.; Kirschvink, N.; Gustin, P.; Gavini, E.;
Roland, I.; Delattre, L.; Evrard, B. Preparation and
in vivo toxicity study of solid lipid microparticles as
carrier for pulmonary administration. AAPS
PharmSciTech 2004, 5 (2), article 27.
21. Edmundson, I.C. Advances in Pharmaceutical
Sciences 2; Academic Press: London, 1967; 950.
22. Dugo, G.; Licandro, G.; Cotroneo, A.; Dugo, G.
Sulla genuinita delle essenze agrumarie. Nota I.
Caratterizzazione di essenze di limone Siciliano.
Essenze Deriv. Agrum. 1983, 53, 173.
23. Guenther, E. The Essential Oils. D; Van Nostrand
Company, Inc.: New York, 1952; 467.
24. NIST98 Library of Mass Spectra.
25. Muller, R.H.; Mader, K.; Gohla, S. Solid lipid
nanoparticles (SLN) for controlled drug delivery—a
review of the state of the art. Eur. J. Pharm.
Biopharm. 2000, 50, 161–177.
26. Jenning, V.; Gohla, S. Comparison of wax and
glyceride solid lipid nanoparticles (SLN1). Int. J.
Pharm. 2000, 196 (2), 219–222.
27. Bertolini, A.C.; Siani, A.C.; Grosso, C.R.F. Stability
of monoterpenes encapsulated in gum Arabic by
spray-drying. J. Agric. Food Chem. 2001, 49 (2),
780–785.
487SLM-Containing Juniper Oil
Phar
mac
euti
cal
Dev
elopm
ent
and T
echnolo
gy D
ow
nlo
aded
fro
m i
nfo
rmah
ealt
hca
re.c
om
by H
ealt
h S
cien
ce L
earn
ing C
tr o
n 0
6/0
9/1
2F
or
per
sonal
use
only
.
Top Related