Colloids and Surfaces B: Biointerfaces 87 (2011) 180186

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Colloids and Surfaces B: Biointerfaces

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An improved method for the preparations of nanostructured lipid carriers

containing heat-sensitive bioactives
Loo Chew Hung a,b , Mahiran Basri a, , Bimo A. Tejo a , Rosnah Ismail b , Harrison Lau Lik Nang b ,
Hazimah Abu Hassan b , Choo Yuen May b
a
Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia
b
Malaysian Palm Oil Board, P.O. Box 10620, 50720 Kuala Lumpur, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: Heat-sensitive bioactive compounds such as -carotene and tocols, are widely used in the pharmaceutical
Received 29 November 2010 and cosmetic elds. Their chemical stability in delivery systems is one of the major concerns in the pro-
Received in revised form 1 May 2011 duction of nanostructured lipid carriers (NLCs). A previously established high-temperature high-pressure
Accepted 10 May 2011
homogenisation technique involved in the preparation of NLCs can cause degradation of heat-sensitive
Available online 17 May 2011
compounds. Therefore, a novel preparation process needs to be developed to minimise the degradation of
heat-sensitive active compounds during the preparation of NLCs. In this work, modied methods A and B
Keywords:
were designed to minimise the degradation of -carotene and tocols during the production of NLCs. These
-Carotene
Tocols
methods improved the chemical stability of heat-sensitive bioactive compounds (-carotene and tocols)
Nanostructured lipid carriers (NLC) signicantly compared to the previously established method. The physical stability of the formulation
Modied method was maintained throughout study duration.
Previously established method 2011 Elsevier B.V. All rights reserved.
Stability

1. Introduction known to be unstable in the presence of light, oxygen and heat

Palm oil, which is the richest source of carotenoids, contains 13
different types of carotenes consisting of - and -carotenes [1].
Carotenoids are widely used as natural colouring agents in food
industries and in cosmetic industries [2]. Carotenoids are lipid-
soluble antioxidants that have free-radical scavenging properties.
For example, the presence of low concentrations of -carotene
can quench the singlet oxygen effectively [3]. Topically applied -
carotene has been proven to have photoprotective effects making
this compound useful as an active component in sun-protection
products [4].
Palm oil is also a rich sources of tocols (vitamin E) [5], of which
tocotrienols are the main component accounting for 7280% of the
total vitamin E content in palm oil. -Tocotrienols has been shown
to have antioxidant activity that is approximately 4060 times
higher than -tocopherol [6]. In addition, -tocotrienol is shown
to have better absorption on topical application compared to -
tocopherol. The vitamin E family (tocols) of antioxidants (tocols)
have also been reported to protect skin against photoaging [6].
Most of the bioactive ingredients used in cosmetics are vitamins
that are inherently unstable over time. -Carotene and tocols are
Corresponding author. Tel.: +60 3 89467269; fax: +60 3 89435380.
E-mail address: mahiran@science.upm.edu.my (M. Basri).
[7,8] as these factors increase the rate of degradation of both com-
pounds. Most of the oxidised forms of the compounds found in the
cosmetic products are toxic to human cells [9,10]. The stability of
these compounds could be maintained by incorporating them into
nanostructured lipid carriers (NLCs) that could provide the labile
compounds with protection from degradation.
NLCs are very attractive in the cosmetic elds because they
provide an occlusive effect to the skin and therefore increase
skin hydration; they also enhance skin bioavailability of active
compounds and increase the physical stability of topical formu-
lations [11]. NLCs consisting of a solid lipid matrix with a certain
amount of liquid lipid are considered to be the new generation
of solid lipid nanoparticles [12]. NLCs are generally produced by a
high-temperature, high-pressure homogenisation technique. This
technique involves processing of a melted lipid blend of lipids (solid
lipid + liquid lipid + active compound) in a hot aqueous surfactant
solution [13].
The drawback of this homogenisation technique is that the
high heating temperature promotes degradation of the labile active
compounds. The heating temperature of 85 C for the previously
established method is too high [13], and the method exposes the
bioactives to high temperatures twice: during the heating of the
lipid phase and during the high-pressure homogenisation. The sec-
ond drawback of this method is that most surfactants have cloud
points lower than 85 C; therefore, the high temperatures may

0927-7765/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2011.05.019
 L.C. Hung et al. / Colloids and Surfaces B: Biointerfaces 87 (2011) 180186
Table 1
The compositions of NLC-1, NLC-2 and NLC-3.
NLC-1 NLC-2
Ingredients Compositions Ingredients
(%, w/w)
Oil phase Span 40 3.00 Span 40
Hydrogenated palm kernel 17.91 Hydrogenated palm kernel
and palm glycerides and palm glycerides
Isopropyl palmitate 1.99 Isopropyl palmitate
Palm-based phytonutrients 0.10 Palm-based phytonutrients
Water phase Tween 80 3.00 Tween 80
Phenonip 0.70 Phenonip
Water 73.30 Water
reduce the emulsifying capability of the surfactants. This effect
induces the instability to the NLC. The third drawback is that the
cooling rate for the lipid dispersion is too low because no cooling
agent is used.
In this work, we have successfully developed two new NLC
production methods that maintain the chemical stability of the
bioactives compounds and the physical stability of the NLCs.
2. Materials and methods
2.1. Materials
Span 40 (sorbitan monopalmitate) and Tween 80 (polysor-
bate 80) were obtained from Croda (Goole, England). Isopropyl
palmitate was purchased from Intermed Sdn. Bhd. (Kuala Lumpur,
Malaysia). Lipocire DM (hydrogenated palm kernel glycerides)
was purchased from Gattefoss Inc. (Toronto, Canada). A mixture
of -carotene and tocols was obtained from Malaysian Palm Oil
Board (Kuala Lumpur, Malaysia). Hexane, isopropyl alcohol and
tetrahydrofuran (THF) were purchased from Merck (Darmstadt,
Germany). -Tocopherol was purchased from SigmaAldrich Inc.
(St. Louis, MO). -Tocotrienol, -tocotrienol and -tocotrienol were
purchased from Calbiochem (San Diego, CA).
2.2. Preparation of nanostructured lipid carrier (NLC)
The NLC formulations were named NLC-1 (prepared by the
previously established method), NLC-2 (prepared by modied
method A) and NLC-3 (prepared by modied method B). NLC-1
was designed according to the previously described protocol [14],
with minor modications, i.e., the homogenisation speed and dura-
tion changed from 8000 rpm for 1 min as stated in the protocol
to 10,000 rpm for 2 min. Briey, the melted lipid phase contain-
ing solid lipid (hydrogenated palm kernel glycerides), liquid lipid
(isopropyl palmitate), lipophilic surfactant (Span 40) and bioac-
tive compounds (concentrated -carotene and tocols) were added
to aqueous hot hydrophilic surfactant (Tween 80). Both phases
(lipid and water phases) were heated to 85 C. The mixture was
homogenised with a Polytron PT 3100 homogeniser (Kinematica
Inc., Switzerland) at 10,000 rpm for 2 min. The hot pre-emulsion
was further homogenised at 85 C by a high-pressure homogeniser
with three cycles at 500 bar. The lipid dispersion was then cooled
at ambient conditions to room temperature.
For NLC-2 (produced by modied method A), the heating tem-
perature of the lipid phase containing the solid lipid and liquid
lipid was reduced to 60 C (versus 85 C for the previously estab-
lished method), which is 10 C higher than the melting point of the
solid lipid (50 C). This step is necessary to avoid recrystallisation
of the lipid phase. The water phase containing the surfactant was
181
NLC-3
Compositions Ingredients Compositions
(%, w/w) (%, w/w)
3.00 Span 40 3.00
17.91 Tween 80 3.00
1.99 Hydrogenated palm kernel and 17.91
palm glycerides
0.10 Isopropyl palmitate 1.99
Palm-based phytonutrients 0.10
3.00 Water (1st portion hot 70 C) 26.00
Phenonip 0.70
0.70 Water (2nd portion 25 C) 47.30
73.30
heated to 70 C to prevent recrystallisation of the lipid phase (85 C
for the previously established method). The bioactive compounds
(-carotene and tocols) were then added into the lipid phase, and
this was followed by the addition of the lipid phase immediately
into the water phase. The mixture of bioactive compounds, lipid
phase and water phase was homogenised at 10,000 rpm for 2 min
to obtain a smooth texture for the pre-emulsion. After homogenisa-
tion, the pre-emulsion obtained was rapidly cooled to 25 C. Then,
the pre-emulsion underwent high-pressure homogenisation for
three cycles at 500 bar and 55 C. To further minimise the loss of
heat-sensitive compounds, the NLC dispersion was cooled rapidly
in an ice bath to 25 C.
For NLC-3 (produced by modied method B), lipophilic and
hydrophilic surfactants were added into the lipid phase containing
the solid lipid and liquid lipid. The heating temperature for the lipid
phase was reduced to 60 C as compared to 85 C for the previously
established method, which is 10 C higher than the melting point
of the solid lipid (50 C) to avoid recrystallisation. The volume of
the water phase was halved (the water phase was not divided for
the previously established method and modied method A). The
volume of the rst water portion was adjusted to have the same
volume as the lipid phase. The volume of the second portion was
not adjusted.
The rst portion of the water was heated to the same tem-
perature as the production of NLC-2, which was 70 C, to prevent
recrystallisation of the lipid phase. The bioactive compounds were
then added into the lipid phase followed by the addition of the
rst hot-water portion immediately into the lipid phase. The mix-
ture of bioactive compounds, lipid phase, and water phase was
homogenised at 10,000 rpm for 2 min. During homogenisation, the
second water portion (25 C) was added slowly to the mixture to
form the pre-emulsion. The pre-emulsion then underwent high-
pressure homogenisation for three cycles at 500 bar and 55 C.
Finally, the NLC dispersion was cooled rapidly in an ice bath to 25 C.
The compositions of NLC-1, NLC-2 and NLC-3 are shown in Table 1.
All of the characterisation tests conducted on the NLC formulations
were performed in duplicate.
2.3. Particle size analysis
Particle size analyses of the pre-emulsions (emulsion before
high pressure homogenisation) were performed using a laser
diffraction particle analyser (Mastersizer 2000, Malvern Instru-
ments, UK) [14]. This instrument can measure particle sizes ranging
from 0.02 to 2000 m. Wet samples were prepared for the analy-
sis by diluting the pre-emulsions with deionised water. The pump
was set at 1645 rpm to mix the sample. The wet samples were then
added to the Hydro 2000S and segregated by ultrasound for few
seconds. Sample quantity was adjusted to obtain laser beam obscu-
182 L.C. Hung et al. / Colloids and Surfaces B: Biointerfaces 87 (2011) 180186
ration in the range of 1020%. The particle size of the pre-emulsions
was described by the cumulants mean diameter.
Particle size analyses for the NLCs were performed by photon
correlation spectroscopy (PCS) with a Malvern HPP5001 high-
performance particle sizer (Malvern Instruments Ltd.). In photon
correlation spectroscopy (PCS), the intensity uctuations of scat-
tered light arising from Brownian motion are measured. The size
distribution of the particles is measured by the StokesEinstein
equation. The mean particle size was obtained from the average of
ve measurements (number of runs = 10 and run duration = 30 s) at
an angle of 90 . All of the samples were diluted in distilled water to
weaken opalescence. The dispersant (water) has a refractive index
of 1.333 and a viscosity of 0.8905 cP at 25 C. The mean values for
all of the NLC formulations are reported.
2.4. Determination of -carotene content in NLCs
-Carotenes contain conjugated double bonds, and they can
absorb light and display unique patterns in the UVvisible spec-
trum [15]. The -carotene content was determined using a
UV-spectrophotometer (Hitachi U-2001 spectrophotometer). The
conjugations of 11 double bonds in -carotene molecule exhibit
a triplet absorbance peak with a maximum at 446 nm [16]. The
-carotene content of sample was determined spectrophotometri-
cally at 446 nm.
NLC (0.5 g) was weighed, and -carotene was extracted with
a mixture of 2 ml ethanol and 3 ml of n-hexane. The sample mix-
ture was then shaken, and the hexane phase was removed. The
extraction was repeated twice, and the removed hexane phases
were combined in a 25 ml volumetric ask [17]. The mixture was
then diluted with hexane to 25 ml. The absorbance of the solution at
446 nm in a 1 cm cuvette was measured against the blank (solvent).
The -carotene content was calculated using Eq. 1 [15].
A446 383 25
-carotenes content (ppm) =
100W
A446 is the absorbance of sample at 446 nm and W is the weight of
sample (in g).
The absorbance was converted to concentration (ppm) by multi-
plying by the universally accepted factor of 383 based on the molar
extinction coefcient of pure -carotene in organic solvents [16].
The mean values of the duplicates of each sample are reported.
2.5. Determination of tocols content in NLCs
Tocols from NLCs were extracted according to the procedure
as explained in Section 2.4. The solution was transferred into a
1.5 ml vial and closed tightly with a PTFE septum for HPLC anal-
ysis. A normal-phase Hewlett-Packard high-performance liquid
chromatograph (HPLC; Agilent 1100 series) with a Zorbax silica
column (4.6 mm 150 mm) and uorescence detector was used.
The mobile phase was hexane (93.9%), isopropyl alcohol (0.4%) and
tetrahydrofuran (5.7%) with ow rate of 1 ml/min. The detector was
set at to an excitation wavelength of 292 nm and emission wave-
length of 326 nm. Standard calibration curves for -tocopherol and
-, - and -tocotrienols were plotted. The quantication of the
major components of the tocols from sample (NLC) was performed
by comparing the peak areas of the components with those of the
standards [18]. Quantication of the tocols isomers was performed
using a ve-point external standard calibration.
2.6. Accelerated stability testing
The stability of the NLCs was investigated by maintaining the
formulation at room temperature (25 C), 45 C and 5 C for 3
months. The NLCs were also centrifuged for at 3500 rpm for 15 min.
Table 2
Particle size for pre-emulsion produced by a previously established method [13],
modied method A and modied method B. Data represent means SD (n = 2).
Pre-emulsion Particle size (nm)
Previously established method 3013.33 8.74
Modied method A 2590.00 4.24
Modied method B 2193.67 0.58
Three freezethaw cycles (1 cycle = 1st day at 5 C and 2nd day at
25 C) were performed to investigate the stability of the NLCs. The
phase separation of NLCs can be observed under polarised light.
3. Results and discussion
3.1. Particle size analysis
3.1.1. Pre-emulsion
Table 2 shows that the particle sizes of the pre-emulsion samples
produced by the modied methods were smaller than the pre-
emulsion samples produced by the previously established method.
The particle size of the pre-emulsion produced by the previously
established method was 3013.33 8.74 nm, whereas the particle
size of the pre-emulsions produced by modied method A was
2590.00 4.24 nm. The larger particle size for the pre-emulsion
produced by the previously established method might be due to
the higher production temperature (85 C), which is higher than the
cloud point of the emulsier used in this experiment, i.e., Tween 80
with cloud point of 72.6 C. Above the cloud point, the hydrophilic
group of the emulsier becomes dehydrated, and this may decrease
the hydration repulsion between them. At this point, the emulsier
cannot prevent aggregation of emulsion droplets, which results in
the formation of larger emulsion droplets [19].
Fig. 1 shows that the pre-emulsion produced by the previously
(1) established method has a broader particle size distribution com-
pared to the pre-emulsion produced by modied method B. The
span value of the pre-emulsion produced by the previously estab-
lished method is 1.75, and it is 0.85 for the pre-emulsion produced
by modied method B. The span value indicates the width of the
distributions, regardless of the median size [20]. The span value
indicates that the pre-emulsion produced by modied method B
has lower polydispersity as compared to the pre-emulsion pro-
duced by the previously established method.
This result indicates that the high temperature involved in
the production of the pre-emulsion in the previously established
method induces some of the small emulsion droplets to coalesce
to produce a broader particle size distribution. In addition to the
high-temperature effect, the cooling of the pre-emulsion after
homogenisation contributes to the narrow particle size distribu-
tion for the pre-emulsion produced by modied method B, which
agrees with the nding of Lin et al. [21].
The pre-emulsion produced by the previously established
method and modied method A have larger particle sizes and
Fig. 1. Particle size distribution of NLC-1 ( ), NLC-2 ( ) and NLC-3
( ).
 L.C. Hung et al. / Colloids and Surfaces B: Biointerfaces 87 (2011) 180186
Fig. 2. Emulsication mechanism when surfactants are introduced into the lipid phase.
Modied after Lin et al. [22].
broader particle size distribution than the pre-emulsion produced
by modied method B (Table 2 and Fig. 1). The span value of the
pre-emulsion produced by method A was 1.45. The initial location
of the surfactants greatly inuences the properties of the emulsion.
When the surfactants are initially placed into the lipid phase, this
yielded a very good emulsion with uniform particle size distribu-
tion. In contrast, the emulsions prepared by initially dispersing the
surfactants in the water phase produces a very unstable emulsion
with coarse droplets. A double emulsion is formed during the emul-
sication process by initially placing the surfactants in the lipid
phase; however, the double emulsion droplets are not observed
in the emulsion prepared by initially placing the surfactant in the
water phase [22].
Fig. 2 shows the emulsication mechanism involved in the
emulsion prepared by placing the surfactants in the lipid
phase. When hot water is initially added to the lipid phase
during homogenisation, part of the water is emulsied into
the lipid phase, forming water-in-oil (W/O) emulsions. The
hydrophile/lipophile/balance (HLB) value of the surfactants was 11
(blend of Span 40 and Tween 80 at a ratio of 50:50); therefore, the
initial W/O emulsion was not stable because this system favours
formation of the oil-in-water (O/W) emulsion. With continuous
mixing of the second part of water into the W/O emulsion dur-
ing homogenisation, the W/O emulsion mixes into excess water
to form a double emulsion (W/O/W). The surfactants migrate to
the water phase, leading the unstable larger droplets to break into
smaller droplets and produce the nal O/W emulsion [22].
Fig. 3. Emulsication mechanism when surfactants are introduced into the water phase.
183
Fig. 3 shows the mechanism involved in the formation of the
emulsion that was prepared by placing the surfactants in the water
phase. The lipid phase mixes into the aqueous phase in the pres-
ence of sufcient mechanical forces (homogenisation) to form the
O/W emulsion. The lipid droplets become smaller as homogeni-
sation continues, and the nal size of the O/W emulsion depends
on the intensity of homogenisation. With the phase inversion pro-
cess occurring during the emulsication, the droplets are more
easily, and with minimum mechanical agitation, reduced in size
to form smaller particles. The breaking of droplets in the emul-
sion prepared by initially placing surfactants into the water phase
is highly dependent on mechanical shear [22]. Pre-emulsions pro-
duced by the previously established method and modied method
A follow the mechanisms shown in Fig. 3. Both pre-emulsions have
larger and broader particle size distributions than the pre-emulsion
produced by modied method B.
3.1.2. NLC
Fig. 4 shows that the particle size of NLC-1 increased from the
rst month to third month, whereas the particle sizes for NLC-2
and NLC-3 remained constant for 3 months. This result is in agree-
ment with a stability study in which NLC-1 separated into two
phases on the second month. No separation was observed for NLC-2
and NLC-3 for 3 months at 45 C. In addition to the high produc-
tion temperature, temperature increases during the high-pressure
homogenisation might be a factor contributing to the instability.
The temperature rise is due to force-induced phenomena, such as a
184 L.C. Hung et al. / Colloids and Surfaces B: Biointerfaces 87 (2011) 180186
Fig. 4. Particle size for NLC-1 (produced by previously established method), NLC-
2 (produced by modied method A) and NLC-3 (produced by modied method B)
within 3 months. Data represent means SD (n = 2); % error < 15.20%.
combination of intense shear, cavitational and turbulent ow con-
ditions. These forces dissipate mechanical energy as heat during
high-pressure homogenisation [23].
Although high temperature facilitates the break-up of droplets
by lowering the viscosity, Laplace pressure and interfacial tension,
it may affect the nature of the emulsier and increase the colli-
sion frequency, which can lead to the droplets coalescing [20]. The
lower heating temperature and cooling step after the high-pressure
homogenisation process could be the main factor that enhances the
stability of the NLCs for modied methods A and B. High-pressure
homogenisation and the heat generated during homogenisation
increases the collision frequency of the emulsion droplets, which
might induce re-coalescence. Therefore, a fast cooling step is
incorporated after homogenisation to quench droplets in the emul-
sion system and reduce the collision frequency of the droplets
[21].
3.2. Chemical stability of -carotene and tocols in NLCs
3.2.1. -Carotene content
Table 3 shows that only 34.56 ppm of the -carotene was found
in NLC-1 on day 1, which was lower than the -carotene content
found in NLC-2 and NLC-3, i.e., 51.13 ppm and 53.97 ppm in NLC-
2 and NLC-3, respectively, for the same amount of concentrated
-carotene as was added to NLC-1. It is likely that the produc-
tion temperature decreased the chemical stability of -carotene
in the NLC-1. NLC-2 and NLC-3 are produced at a lower tempera-
ture compared to NLC-1. The concentration of -carotene in NLC-3
(53.97 ppm) was higher than NLC-2 (51.13 ppm). This difference
could be due to smaller temperature increases during homogenisa-
tion in the preparation of the pre-emulsion produced by established
method as compared to the pre-emulsion produced by modi-
ed method A. The second portion of water (25 C) added during
homogenisation in the production of the pre-emulsion using mod-
ied method B reduces the temperature, whereas no cooling agent
Table 3
-Carotene and tocols content in NLC-1 (produced by previously established
method), NLC-2 (produced by modied method A) and NLC-3 (produced by modied
method B) after 3-month storage at 25 C. Data represent means SD (n = 2).
Sample -Carotenes Tocols
1st day 90th day 1st day 90th day
NLC-1 34.12 0.62 34.12 0.62 27.02 0.34 18.28 0.34
NLC-2 51.22 0.12 51.22 0.12 41.88 0.18 43.00 0.16
NLC-3 53.80 0.24 53.80 0.24 44.29 0.16 45.47 0.33
(ice bath) was applied during the production of the pre-emulsion
using the previously established method.
Carotenoids are comprised of a system of conjugated double
bonds that make them susceptible to heat. The degradation rate of
carotenoids therefore increases with increasing temperature. The
effect of temperature on the degradation rate can be explained by
the Arrhenius equation [13]. When -carotene is heated to high
temperatures, degradation may occur and form several products,
including ionene, toluene, m-xylene and 2,6-dimethylnapthalene
[24]. The mechanism of degradation involves cyclisation and elim-
ination reactions with four-membered-ring intermediates. Fig. 5
shows the rearrangement of the eight-electron system to give the
degradation products [24].
3.2.2. Tocols content
Table 3 shows the changes in the concentration of tocols in
NLC-1, NLC-2 and NLC-3 during 90-day study storage at 25 C. This
result agrees with the results from Section 3.3, which indicates
that NLC-2 and NLC-3 have a greater ability to retain the valu-
able active ingredients than NLC-1 on day 1. The total contents of
tocols were 27.26 ppm, 42.01 ppm, 44.40 ppm in NLC-1, NLC-2 and
NLC-3, respectively. These results indicate that the modied meth-
ods can minimise the loss of the heat-sensitive bioactives during
the preparation of NLCs. The cooling system incorporated in the
production of NLC-2 and NLC-3 reduces the contact time for heat-
sensitive bioactives with high temperatures, and the excessive heat
generated during high-pressure homogenisation can be dissipated
faster.
For the 90-day storage stability study, 32.06% of the tocols were
degraded in NLC-1 by day 90, whereas no degradation was observed
in NLC-2 and NLC-3. The main factor in the degradation of tocols is
oxidation. Three steps are involved in oxidation: initiation, propa-
gation and termination. The presence of heat and light promotes the
formation of free radicals. These free radicals combine with oxygen
to cause the propagation step, and the oxidation progresses to the
termination step (chain reactions) [13]. Fig. 6 shows the oxidation
of -tocopherol.
The initial step of oxidation of -tocopherol is reversible. This
step results in the production of a phenoxyl radical that forms an
unstable oxidationreduction system with phenol. The second step
is irreversible and produces -tocopherylquinone. The reversible
step controls the rate of the irreversible oxidation of phenol [25].
When -tocopherylquinone is formed, the antioxidant capacity of
tocopherol is lost. The oxidation of -tocopherol can be prevented
by eliminating oxygen from the container and by storage at low
temperatures (<5 C). Therefore, chain reactions can be minimised,
and the oxidation can be inhibited [13].
Table 3 shows that the amount of degraded tocols was greater
than -carotene degradation in NLC-1. Tocols are stronger antioxi-
dants than -carotene because they have a smaller redox potential
compared to -carotene. The redox potentials of tocols and -
carotene are 0.48 V and 0.69 V, respectively [26,27]. The smaller
the redox potential, the greater the afnity of the antioxi-
dant to be oxidised and to scavenge the free radicals found in
the NLC.
Chemical instability of the lipid phase may contribute to the
physical instability of the emulsion. This instability could result
from many oxidised products generated during lipid oxidation
being surface active. These products may react with the surround-
ing interfacial membrane of droplets in such a way that leads to
droplet coalescence [19]. Thus, the chemical instability of the tocols
may contribute to the physical instability of NLC-1 as shown in
Fig. 4. This instability of NLC-1 highlights that the modied methods
can help to minimise the degradation of tocols during the produc-
tion of NLCs and enhance the long-term chemical stability of the
bioactives.
 L.C. Hung et al. / Colloids and Surfaces B: Biointerfaces 87 (2011) 180186 185

Fig. 5. Thermal degradation products of -carotene.

Modied after Wong [24].

3.3. Accelerated stability testing intensive and time consuming, which are uneconomical. Therefore,
accelerated stability testing was carried out to achieve quick and
Studying the long-term stability of cosmetic emulsions under reliable results. NLCs were exposed to extreme conditions over a
real-life conditions (room temperature, 25 C) can be labour period of time, and the changes in their physical appearances were

Fig. 6. Mechanism of degradation of -tocopherol.

Modied after Golumbic and Mattill [25].
186 L.C. Hung et al. / Colloids and Surfaces B: Biointerfaces 87 (2011) 180186
recorded. The ability of products to withstand 34 months of ele-
vated temperature testing and 34 freezethaw cycles is usually
indicative that the product would have an adequate shelf life [28].
NLC-1, NLC-2 and NLC-3 passed the centrifugation test, three
freezethaw cycles and 3-month storage at room temperature and
freezing temperature (5 C). However, NLC-1 was found to separate
into two layers on the second month at 45 C, whereas no separa-
tion was observed in NLC-2 and NLC-3. This result agrees with the
particle size study, which shows a sudden increase in particle size in
the second month for NLC-1 (Fig. 4). Neither crystallisation nor gel
formation was observed for NLC-1, NLC-2 and NLC-3 when stored
at low temperature (5 C). This nding suggests that the undesired
crystallisation of the lipid phase does not occur even at low temper-
atures (5 C). Crystallisation and gel formation of the NLC promotes
expulsion of the active compounds into water phase, which could
degrade them [29].
4. Conclusion
The steps involved in the preparation of NLCs greatly inuence
the chemical stability of heat-sensitive bioactive compounds and
the physical stability of NLCs. In this work, we have successfully
developed two methods that are suitable in the preparation of
heat-sensitive bioactive compounds, which enhance the chemical
stability of heat-sensitive bioactive compounds and the physical
stability of NLCs.
Acknowledgements
The authors would like to acknowledge Malaysian Palm Oil
Board Graduate Students Assistantship Scheme for the research
grant. The authors would also like to thank to Malaysian Palm Oil
Board and Universiti Putra Malaysia (Faculty Science and Institute
Bioscience) for the facilities used in the project. Special thanks to Dr.
Cornelia M. Keck and Professor Dr. Rainer H. Mller for the inspiring
workshop on untapped potential of lipid nanoparticles in cosmetics
and pharmaceuticals products.
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