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An improved method for the preparations of nanostructured lipid carriers containing heat-sensitive bioactives
Colloids and Surfaces B: Biointerfaces 87 (2011) 180–186
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
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 b
Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia Malaysian Palm Oil Board, P.O. Box 10620, 50720 Kuala Lumpur, Malaysia
a r t i c l e
i n f o
Article history: Received 29 November 2010 Received in revised form 1 May 2011 Accepted 10 May 2011 Available online 17 May 2011 Keywords: ˇ-Carotene Tocols Nanostructured lipid carriers (NLC) Modified method Previously established method Stability
a b s t r a c t Heat-sensitive bioactive compounds such as ˇ-carotene and tocols, are widely used in the pharmaceutical and cosmetic fields. Their chemical stability in delivery systems is one of the major concerns in the production of nanostructured lipid carriers (NLCs). A previously established high-temperature high-pressure homogenisation technique involved in the preparation of NLCs can cause degradation of heat-sensitive 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, modified methods A and B were designed to minimise the degradation of ˇ-carotene and tocols during the production of NLCs. These methods improved the chemical stability of heat-sensitive bioactive compounds (ˇ-carotene and tocols) significantly compared to the previously established method. The physical stability of the formulation was maintained throughout study duration. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Palm oil, which is the richest source of carotenoids, contains 13 different types of carotenes consisting of ˛- and ˇ-carotenes [1]. Carotenoids are widely used as natural colouring agents in food industries and in cosmetic industries [2]. Carotenoids are lipidsoluble antioxidants that have free-radical scavenging properties. For example, the presence of low concentrations of ˇ-carotene can quench the singlet oxygen effectively [3]. Topically applied ˇcarotene 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 72–80% of the total vitamin E content in palm oil. ˛-Tocotrienols has been shown to have antioxidant activity that is approximately 40–60 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. ˇ-Carotene and tocols are
∗ Corresponding author. Tel.: +60 3 89467269; fax: +60 3 89435380. E-mail address: [email protected] (M. Basri). 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.05.019
known to be unstable in the presence of light, oxygen and heat [7,8] as these factors increase the rate of degradation of both compounds. 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 fields 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 formulations [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 second drawback of this method is that most surfactants have cloud points lower than 85 ◦ C; therefore, the high temperatures may
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Table 1 The compositions of NLC-1, NLC-2 and NLC-3. NLC-1
Oil phase
Water phase
NLC-2
NLC-3
Ingredients
Compositions (%, w/w)
Ingredients
Compositions (%, w/w)
Ingredients
Span 40 Hydrogenated palm kernel and palm glycerides Isopropyl palmitate
3.00 17.91
3.00 17.91
Span 40 Tween 80
1.99
Span 40 Hydrogenated palm kernel and palm glycerides Isopropyl palmitate
Palm-based phytonutrients
0.10
Palm-based phytonutrients
0.10
Tween 80
3.00
Tween 80
3.00
Phenonip Water
0.70 73.30
Phenonip Water
0.70 73.30
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 (polysorbate 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 Gattefossé Inc. (Toronto, Canada). A mixture of ˇ-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 Sigma–Aldrich Inc. (St. Louis, MO). ˛-Tocotrienol, -tocotrienol and ı-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 modified method A) and NLC-3 (prepared by modified method B). NLC-1 was designed according to the previously described protocol [14], with minor modifications, i.e., the homogenisation speed and duration changed from 8000 rpm for 1 min as stated in the protocol to 10,000 rpm for 2 min. Briefly, the melted lipid phase containing solid lipid (hydrogenated palm kernel glycerides), liquid lipid (isopropyl palmitate), lipophilic surfactant (Span 40) and bioactive compounds (concentrated ˇ-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 modified method A), the heating temperature of the lipid phase containing the solid lipid and liquid lipid was reduced to 60 ◦ C (versus 85 ◦ C for the previously established 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
1.99
Hydrogenated palm kernel and palm glycerides Isopropyl palmitate Palm-based phytonutrients Water (1st portion hot – 70 ◦ C) Phenonip Water (2nd portion – 25 ◦ C)
Compositions (%, w/w) 3.00 3.00 17.91 1.99 0.10 26.00 0.70 47.30
heated to 70 ◦ C to prevent recrystallisation of the lipid phase (85 ◦ C for the previously established method). The bioactive compounds (ˇ-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 homogenisation, 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 modified 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 modified method A). The volume of the first water portion was adjusted to have the same volume as the lipid phase. The volume of the second portion was not adjusted. The first portion of the water was heated to the same temperature 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 first hot-water portion immediately into the lipid phase. The mixture 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 highpressure 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 Instruments, UK) [14]. This instrument can measure particle sizes ranging from 0.02 to 2000 m. Wet samples were prepared for the analysis 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-
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ration in the range of 10–20%. 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 highperformance particle sizer (Malvern Instruments Ltd.). In photon correlation spectroscopy (PCS), the intensity fluctuations of scattered light arising from Brownian motion are measured. The size distribution of the particles is measured by the Stokes–Einstein equation. The mean particle size was obtained from the average of five 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.
Table 2 Particle size for pre-emulsion produced by a previously established method [13], modified method A and modified method B. Data represent means ± SD (n = 2).
2.4. Determination of ˇ-carotene content in NLCs
3.1. Particle size analysis
ˇ-Carotenes contain conjugated double bonds, and they can absorb light and display unique patterns in the UV–visible spectrum [15]. The ˇ-carotene content was determined using a UV-spectrophotometer (Hitachi U-2001 spectrophotometer). The conjugations of 11 double bonds in ˇ-carotene molecule exhibit a triplet absorbance peak with a maximum at 446 nm [16]. The ˇ-carotene content of sample was determined spectrophotometrically at 446 nm. NLC (0.5 g) was weighed, and ˇ-carotene was extracted with a mixture of 2 ml ethanol and 3 ml of n-hexane. The sample mixture 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 flask [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 ˇ-carotene content was calculated using Eq. 1 [15].
3.1.1. Pre-emulsion Table 2 shows that the particle sizes of the pre-emulsion samples produced by the modified methods were smaller than the preemulsion 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 modified 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 emulsifier used in this experiment, i.e., Tween 80 with cloud point of 72.6 ◦ C. Above the cloud point, the hydrophilic group of the emulsifier becomes dehydrated, and this may decrease the hydration repulsion between them. At this point, the emulsifier 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 established method has a broader particle size distribution compared to the pre-emulsion produced by modified method B. The span value of the pre-emulsion produced by the previously established method is 1.75, and it is 0.85 for the pre-emulsion produced by modified 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 modified method B has lower polydispersity as compared to the pre-emulsion produced 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 distribution for the pre-emulsion produced by modified method B, which agrees with the finding of Lin et al. [21]. The pre-emulsion produced by the previously established method and modified method A have larger particle sizes and
ˇ-carotenes content (ppm) =
A446 × 383 × 25 100W
(1)
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 multiplying by the universally accepted factor of 383 based on the molar extinction coefficient of pure ˇ-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 analysis. A normal-phase Hewlett-Packard high-performance liquid chromatograph (HPLC; Agilent 1100 series) with a Zorbax silica column (4.6 mm × 150 mm) and fluorescence detector was used. The mobile phase was hexane (93.9%), isopropyl alcohol (0.4%) and tetrahydrofuran (5.7%) with flow rate of 1 ml/min. The detector was set at to an excitation wavelength of 292 nm and emission wavelength of 326 nm. Standard calibration curves for ␣-tocopherol and ˛-, - and ı-tocotrienols were plotted. The quantification 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]. Quantification of the tocols isomers was performed using a five-point external standard calibration.
Pre-emulsion
Particle size (nm) 3013.33 ± 8.74 2590.00 ± 4.24 2193.67 ± 0.58
Previously established method Modified method A Modified method B
Three freeze–thaw 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
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.
Fig. 1. Particle size distribution of NLC-1 ( ). (
), NLC-2 (
) and NLC-3
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Fig. 2. Emulsification mechanism when surfactants are introduced into the lipid phase. Modified after Lin et al. [22].
broader particle size distribution than the pre-emulsion produced by modified 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 influences 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 distribution. 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 emulsification 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 emulsification 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 emulsified 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 during 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 final O/W emulsion [22].
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 presence of sufficient mechanical forces (homogenisation) to form the O/W emulsion. The lipid droplets become smaller as homogenisation continues, and the final size of the O/W emulsion depends on the intensity of homogenisation. With the phase inversion process occurring during the emulsification, the droplets are more easily, and with minimum mechanical agitation, reduced in size to form smaller particles. The breaking of droplets in the emulsion prepared by initially placing surfactants into the water phase is highly dependent on mechanical shear [22]. Pre-emulsions produced by the previously established method and modified 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 modified method B. 3.1.2. NLC Fig. 4 shows that the particle size of NLC-1 increased from the first month to third month, whereas the particle sizes for NLC-2 and NLC-3 remained constant for 3 months. This result is in agreement 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 production 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
Fig. 3. Emulsification mechanism when surfactants are introduced into the water phase.
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(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 ˇ-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 elimination reactions with four-membered-ring intermediates. Fig. 5 shows the rearrangement of the eight-electron system to give the degradation products [24]. Fig. 4. Particle size for NLC-1 (produced by previously established method), NLC2 (produced by modified method A) and NLC-3 (produced by modified method B) within 3 months. Data represent means ± SD (n = 2); % error < 15.20%.
combination of intense shear, cavitational and turbulent flow conditions. 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 emulsifier and increase the collision 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 modified 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 emulsion system and reduce the collision frequency of the droplets [21].
3.2. Chemical stability of ˇ-carotene and tocols in NLCs 3.2.1. ˇ-Carotene content Table 3 shows that only 34.56 ppm of the ˇ-carotene was found in NLC-1 on day 1, which was lower than the ˇ-carotene content found in NLC-2 and NLC-3, i.e., 51.13 ppm and 53.97 ppm in NLC2 and NLC-3, respectively, for the same amount of concentrated ˇ-carotene as was added to NLC-1. It is likely that the production temperature decreased the chemical stability of ˇ-carotene in the NLC-1. NLC-2 and NLC-3 are produced at a lower temperature compared to NLC-1. The concentration of ˇ-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 homogenisation in the preparation of the pre-emulsion produced by established method as compared to the pre-emulsion produced by modified method A. The second portion of water (25 ◦ C) added during homogenisation in the production of the pre-emulsion using modified method B reduces the temperature, whereas no cooling agent
Table 3 ˇ-Carotene and tocols content in NLC-1 (produced by previously established method), NLC-2 (produced by modified method A) and NLC-3 (produced by modified method B) after 3-month storage at 25 ◦ C. Data represent means ± SD (n = 2). Sample
ˇ-Carotenes 1st day
NLC-1 NLC-2 NLC-3
34.12 ± 0.62 51.22 ± 0.12 53.80 ± 0.24
Tocols 90th day
1st day
90th day
34.12 ± 0.62 51.22 ± 0.12 53.80 ± 0.24
27.02 ± 0.34 41.88 ± 0.18 44.29 ± 0.16
18.28 ± 0.34 43.00 ± 0.16 45.47 ± 0.33
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 valuable 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 modified methods 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 heatsensitive 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, propagation 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 oxidation–reduction 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 ˇ-carotene degradation in NLC-1. Tocols are stronger antioxidants than ˇ-carotene because they have a smaller redox potential compared to ˇ-carotene. The redox potentials of tocols and ˇcarotene are 0.48 V and 0.69 V, respectively [26,27]. The smaller the redox potential, the greater the affinity of the antioxidant 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 surrounding 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 modified methods can help to minimise the degradation of tocols during the production of NLCs and enhance the long-term chemical stability of the bioactives.
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Fig. 5. Thermal degradation products of ˇ-carotene. Modified after Wong [24].
3.3. Accelerated stability testing Studying the long-term stability of cosmetic emulsions under “real-life” conditions (room temperature, 25 ◦ C) can be labour
intensive and time consuming, which are uneconomical. Therefore, accelerated stability testing was carried out to achieve quick and reliable results. NLCs were exposed to “extreme conditions” over a period of time, and the changes in their physical appearances were
Fig. 6. Mechanism of degradation of ˛-tocopherol. Modified after Golumbic and Mattill [25].
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recorded. The ability of products to withstand 3–4 months of elevated temperature testing and 3–4 freeze–thaw 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 freeze–thaw 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 separation 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 finding suggests that the undesired crystallisation of the lipid phase does not occur even at low temperatures (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 influence 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. Müller for the inspiring
workshop on untapped potential of lipid nanoparticles in cosmetics and pharmaceuticals products. References [1] Y.M. Choo, Palm Oil Developments: Carotenoids from Palm Oil, PORIM Publications, Kuala Lumpur, 1997. [2] C.K. Ooi, Palm Oil Tech. Bull. 1 (1998) 1–3. [3] C.S. Foote, R.W. Denny, J. Am. Chem. Soc. 90 (1968) 6233–6235. [4] C.M. Burgess, Cosmetic Dermatology, Springer, Heidelberg, 2005. [5] B. Fife, The Palm Oil Miracle, Piccadilly Books Ltd., New York, 2007. [6] J.J. Thiele, S.N. Hsieh, S. Ekanayake-Mudiyanselage, Dermatol. Surg. 31 (2005) 805–813. [7] J.B. Hutchings, Food Color and Appearance, Springer, New York, 1999. [8] R.R. Eitenmiller, W.O. Landen, Vitamin Analysis for the Health and Food Sciences, CRC Press, Florida, 1999. [9] J.S. Hurst, M.K. Saini, G.F. Jin, Y.C. Awasthi, F.J.G.M. Van Kuijka, Exp. Eye Res. 81 (2005) 239–243. [10] L. Cercaci, M.T. Rodriguez-Estrada, G. Lercker, E.A. Decker, Food Chem. 102 (2007) 161–167. [11] R.H. Müller, R.D. Peterson, A. Hommoss, J. Pardeike, Adv. Drug Delivery Rev. 59 (2007) 522–530. [12] F. Hu, S. Jiang, Y. Du, Y. Hong, Y. Ye, Z. Su, Int. J. Pharmaceut. 314 (2006) 83–89. [13] V. Teeranachaideekul, R.H. Müller, V.B. Junyaprasert, Int. J. Pharmaceut. 340 (2007) 198–206. [14] Malvern, HPPS Operators Guide, Malvern, England, 2003. [15] T.S. Tang, Eng. News 011 (1988) 18. [16] C.L. Chong, PORIM Bull. 15 (1987) 18–26. [17] Y. Yuan, Y.X. Gao, J. Zhao, L. Mao, Food Res. Int. 41 (2008) 61–68. [18] H.L.N. Lau, Y.M. Choo, A.N. Ma, C.H. Chuah, J. Food Eng. 84 (2008) 289–296. [19] D.J. McClements, Food Emulsions: Principles, Practices, and Techniques, CRC Press, Florida, 2005. [20] G.G. Palazolo, D.A. Sorgentini, M.C. Anon, JAOCS 81 (2004) 625–632. [21] P. Marie, J.M. Perrier-Cornet, P. Gervais, J. Food Eng. 53 (2002) 43–51. [22] T.J. Lin, K. Haruki, O. Hideaki, J. Soc. Cosmet. Chem. 26 (1975) 121–139. [23] A. Desrumaux, J. Marcand, Int. J. Food Sci. Technol. 37 (2002) 263–269. [24] D.W.S. Wong, Mechanism and Theory in Food Chemistry, Van Nostrand Reinhold, New York, 1989. [25] C. Golumbic, H.A. Matill, J. Biol. Chem. (1940) 535–541. [26] V.E. Kagan, J.P. Fabisiak, P.J. Quinn, Protoplasma 214 (2000) 11–18. [27] M. Kleinová, M. Hewitt, V. Brezová, J.C. Madden, M.T.D. Cronin, M. Valko, Gen. Physiol. Biophys. 26 (2007) 97–103. [28] D.F. William, W.H. Schmitt, Chemistry and Technology of the Cosmetics and Toiletries Industry, Chapman & Hall, London, 1996. ¯ Crystallization Processes in Fats and Lipid Systems, CRC Press, [29] N. Garti, K. Sato, New York, 2001.
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
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 b
Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia Malaysian Palm Oil Board, P.O. Box 10620, 50720 Kuala Lumpur, Malaysia
a r t i c l e
i n f o
Article history: Received 29 November 2010 Received in revised form 1 May 2011 Accepted 10 May 2011 Available online 17 May 2011 Keywords: ˇ-Carotene Tocols Nanostructured lipid carriers (NLC) Modified method Previously established method Stability
a b s t r a c t Heat-sensitive bioactive compounds such as ˇ-carotene and tocols, are widely used in the pharmaceutical and cosmetic fields. Their chemical stability in delivery systems is one of the major concerns in the production of nanostructured lipid carriers (NLCs). A previously established high-temperature high-pressure homogenisation technique involved in the preparation of NLCs can cause degradation of heat-sensitive 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, modified methods A and B were designed to minimise the degradation of ˇ-carotene and tocols during the production of NLCs. These methods improved the chemical stability of heat-sensitive bioactive compounds (ˇ-carotene and tocols) significantly compared to the previously established method. The physical stability of the formulation was maintained throughout study duration. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Palm oil, which is the richest source of carotenoids, contains 13 different types of carotenes consisting of ˛- and ˇ-carotenes [1]. Carotenoids are widely used as natural colouring agents in food industries and in cosmetic industries [2]. Carotenoids are lipidsoluble antioxidants that have free-radical scavenging properties. For example, the presence of low concentrations of ˇ-carotene can quench the singlet oxygen effectively [3]. Topically applied ˇcarotene 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 72–80% of the total vitamin E content in palm oil. ˛-Tocotrienols has been shown to have antioxidant activity that is approximately 40–60 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. ˇ-Carotene and tocols are
∗ Corresponding author. Tel.: +60 3 89467269; fax: +60 3 89435380. E-mail address: [email protected] (M. Basri). 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.05.019
known to be unstable in the presence of light, oxygen and heat [7,8] as these factors increase the rate of degradation of both compounds. 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 fields 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 formulations [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 second drawback of this method is that most surfactants have cloud points lower than 85 ◦ C; therefore, the high temperatures may
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Table 1 The compositions of NLC-1, NLC-2 and NLC-3. NLC-1
Oil phase
Water phase
NLC-2
NLC-3
Ingredients
Compositions (%, w/w)
Ingredients
Compositions (%, w/w)
Ingredients
Span 40 Hydrogenated palm kernel and palm glycerides Isopropyl palmitate
3.00 17.91
3.00 17.91
Span 40 Tween 80
1.99
Span 40 Hydrogenated palm kernel and palm glycerides Isopropyl palmitate
Palm-based phytonutrients
0.10
Palm-based phytonutrients
0.10
Tween 80
3.00
Tween 80
3.00
Phenonip Water
0.70 73.30
Phenonip Water
0.70 73.30
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 (polysorbate 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 Gattefossé Inc. (Toronto, Canada). A mixture of ˇ-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 Sigma–Aldrich Inc. (St. Louis, MO). ˛-Tocotrienol, -tocotrienol and ı-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 modified method A) and NLC-3 (prepared by modified method B). NLC-1 was designed according to the previously described protocol [14], with minor modifications, i.e., the homogenisation speed and duration changed from 8000 rpm for 1 min as stated in the protocol to 10,000 rpm for 2 min. Briefly, the melted lipid phase containing solid lipid (hydrogenated palm kernel glycerides), liquid lipid (isopropyl palmitate), lipophilic surfactant (Span 40) and bioactive compounds (concentrated ˇ-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 modified method A), the heating temperature of the lipid phase containing the solid lipid and liquid lipid was reduced to 60 ◦ C (versus 85 ◦ C for the previously established 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
1.99
Hydrogenated palm kernel and palm glycerides Isopropyl palmitate Palm-based phytonutrients Water (1st portion hot – 70 ◦ C) Phenonip Water (2nd portion – 25 ◦ C)
Compositions (%, w/w) 3.00 3.00 17.91 1.99 0.10 26.00 0.70 47.30
heated to 70 ◦ C to prevent recrystallisation of the lipid phase (85 ◦ C for the previously established method). The bioactive compounds (ˇ-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 homogenisation, 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 modified 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 modified method A). The volume of the first water portion was adjusted to have the same volume as the lipid phase. The volume of the second portion was not adjusted. The first portion of the water was heated to the same temperature 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 first hot-water portion immediately into the lipid phase. The mixture 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 highpressure 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 Instruments, UK) [14]. This instrument can measure particle sizes ranging from 0.02 to 2000 m. Wet samples were prepared for the analysis 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-
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ration in the range of 10–20%. 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 highperformance particle sizer (Malvern Instruments Ltd.). In photon correlation spectroscopy (PCS), the intensity fluctuations of scattered light arising from Brownian motion are measured. The size distribution of the particles is measured by the Stokes–Einstein equation. The mean particle size was obtained from the average of five 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.
Table 2 Particle size for pre-emulsion produced by a previously established method [13], modified method A and modified method B. Data represent means ± SD (n = 2).
2.4. Determination of ˇ-carotene content in NLCs
3.1. Particle size analysis
ˇ-Carotenes contain conjugated double bonds, and they can absorb light and display unique patterns in the UV–visible spectrum [15]. The ˇ-carotene content was determined using a UV-spectrophotometer (Hitachi U-2001 spectrophotometer). The conjugations of 11 double bonds in ˇ-carotene molecule exhibit a triplet absorbance peak with a maximum at 446 nm [16]. The ˇ-carotene content of sample was determined spectrophotometrically at 446 nm. NLC (0.5 g) was weighed, and ˇ-carotene was extracted with a mixture of 2 ml ethanol and 3 ml of n-hexane. The sample mixture 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 flask [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 ˇ-carotene content was calculated using Eq. 1 [15].
3.1.1. Pre-emulsion Table 2 shows that the particle sizes of the pre-emulsion samples produced by the modified methods were smaller than the preemulsion 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 modified 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 emulsifier used in this experiment, i.e., Tween 80 with cloud point of 72.6 ◦ C. Above the cloud point, the hydrophilic group of the emulsifier becomes dehydrated, and this may decrease the hydration repulsion between them. At this point, the emulsifier 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 established method has a broader particle size distribution compared to the pre-emulsion produced by modified method B. The span value of the pre-emulsion produced by the previously established method is 1.75, and it is 0.85 for the pre-emulsion produced by modified 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 modified method B has lower polydispersity as compared to the pre-emulsion produced 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 distribution for the pre-emulsion produced by modified method B, which agrees with the finding of Lin et al. [21]. The pre-emulsion produced by the previously established method and modified method A have larger particle sizes and
ˇ-carotenes content (ppm) =
A446 × 383 × 25 100W
(1)
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 multiplying by the universally accepted factor of 383 based on the molar extinction coefficient of pure ˇ-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 analysis. A normal-phase Hewlett-Packard high-performance liquid chromatograph (HPLC; Agilent 1100 series) with a Zorbax silica column (4.6 mm × 150 mm) and fluorescence detector was used. The mobile phase was hexane (93.9%), isopropyl alcohol (0.4%) and tetrahydrofuran (5.7%) with flow rate of 1 ml/min. The detector was set at to an excitation wavelength of 292 nm and emission wavelength of 326 nm. Standard calibration curves for ␣-tocopherol and ˛-, - and ı-tocotrienols were plotted. The quantification 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]. Quantification of the tocols isomers was performed using a five-point external standard calibration.
Pre-emulsion
Particle size (nm) 3013.33 ± 8.74 2590.00 ± 4.24 2193.67 ± 0.58
Previously established method Modified method A Modified method B
Three freeze–thaw 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
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.
Fig. 1. Particle size distribution of NLC-1 ( ). (
), NLC-2 (
) and NLC-3
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183
Fig. 2. Emulsification mechanism when surfactants are introduced into the lipid phase. Modified after Lin et al. [22].
broader particle size distribution than the pre-emulsion produced by modified 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 influences 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 distribution. 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 emulsification 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 emulsification 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 emulsified 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 during 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 final O/W emulsion [22].
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 presence of sufficient mechanical forces (homogenisation) to form the O/W emulsion. The lipid droplets become smaller as homogenisation continues, and the final size of the O/W emulsion depends on the intensity of homogenisation. With the phase inversion process occurring during the emulsification, the droplets are more easily, and with minimum mechanical agitation, reduced in size to form smaller particles. The breaking of droplets in the emulsion prepared by initially placing surfactants into the water phase is highly dependent on mechanical shear [22]. Pre-emulsions produced by the previously established method and modified 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 modified method B. 3.1.2. NLC Fig. 4 shows that the particle size of NLC-1 increased from the first month to third month, whereas the particle sizes for NLC-2 and NLC-3 remained constant for 3 months. This result is in agreement 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 production 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
Fig. 3. Emulsification mechanism when surfactants are introduced into the water phase.
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(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 ˇ-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 elimination reactions with four-membered-ring intermediates. Fig. 5 shows the rearrangement of the eight-electron system to give the degradation products [24]. Fig. 4. Particle size for NLC-1 (produced by previously established method), NLC2 (produced by modified method A) and NLC-3 (produced by modified method B) within 3 months. Data represent means ± SD (n = 2); % error < 15.20%.
combination of intense shear, cavitational and turbulent flow conditions. 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 emulsifier and increase the collision 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 modified 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 emulsion system and reduce the collision frequency of the droplets [21].
3.2. Chemical stability of ˇ-carotene and tocols in NLCs 3.2.1. ˇ-Carotene content Table 3 shows that only 34.56 ppm of the ˇ-carotene was found in NLC-1 on day 1, which was lower than the ˇ-carotene content found in NLC-2 and NLC-3, i.e., 51.13 ppm and 53.97 ppm in NLC2 and NLC-3, respectively, for the same amount of concentrated ˇ-carotene as was added to NLC-1. It is likely that the production temperature decreased the chemical stability of ˇ-carotene in the NLC-1. NLC-2 and NLC-3 are produced at a lower temperature compared to NLC-1. The concentration of ˇ-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 homogenisation in the preparation of the pre-emulsion produced by established method as compared to the pre-emulsion produced by modified method A. The second portion of water (25 ◦ C) added during homogenisation in the production of the pre-emulsion using modified method B reduces the temperature, whereas no cooling agent
Table 3 ˇ-Carotene and tocols content in NLC-1 (produced by previously established method), NLC-2 (produced by modified method A) and NLC-3 (produced by modified method B) after 3-month storage at 25 ◦ C. Data represent means ± SD (n = 2). Sample
ˇ-Carotenes 1st day
NLC-1 NLC-2 NLC-3
34.12 ± 0.62 51.22 ± 0.12 53.80 ± 0.24
Tocols 90th day
1st day
90th day
34.12 ± 0.62 51.22 ± 0.12 53.80 ± 0.24
27.02 ± 0.34 41.88 ± 0.18 44.29 ± 0.16
18.28 ± 0.34 43.00 ± 0.16 45.47 ± 0.33
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 valuable 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 modified methods 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 heatsensitive 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, propagation 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 oxidation–reduction 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 ˇ-carotene degradation in NLC-1. Tocols are stronger antioxidants than ˇ-carotene because they have a smaller redox potential compared to ˇ-carotene. The redox potentials of tocols and ˇcarotene are 0.48 V and 0.69 V, respectively [26,27]. The smaller the redox potential, the greater the affinity of the antioxidant 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 surrounding 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 modified methods can help to minimise the degradation of tocols during the production of NLCs and enhance the long-term chemical stability of the bioactives.
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Fig. 5. Thermal degradation products of ˇ-carotene. Modified after Wong [24].
3.3. Accelerated stability testing Studying the long-term stability of cosmetic emulsions under “real-life” conditions (room temperature, 25 ◦ C) can be labour
intensive and time consuming, which are uneconomical. Therefore, accelerated stability testing was carried out to achieve quick and reliable results. NLCs were exposed to “extreme conditions” over a period of time, and the changes in their physical appearances were
Fig. 6. Mechanism of degradation of ˛-tocopherol. Modified after Golumbic and Mattill [25].
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recorded. The ability of products to withstand 3–4 months of elevated temperature testing and 3–4 freeze–thaw 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 freeze–thaw 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 separation 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 finding suggests that the undesired crystallisation of the lipid phase does not occur even at low temperatures (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 influence 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. Müller for the inspiring
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