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ISSN : 1226-2587(Print)
ISSN : 2288-9507(Online)
Journal of the Society of Cosmetic Scientists of Korea Vol.39 No.3 pp.225-231

아스타잔틴을 포함하는 나노에멀젼의 안정성 향상을 위한 신규 Glyceryl Ester 이용 캡슐화

김동명, 홍원기, 공수성, 이언엽
한국콜마 색조화장품 연구소

Novel Encapsulation with New Glyceryl Ester Vesicle Enhances Stability of Nanoemulsion Containing Astaxanthin

Dong Myung Kim, Weon Ki Hong, Soo Sung Kong, Un Yep Lee

Kolmar Korea, 183-5 Dodang-dong, Wonmi-gu, Bucheon-si, Gyeonggi-do, Korea
주 저자 (e-mail:
(Received April 23, 2013; Revised May 1, 2013; Accepted July 8, 2013)


Oil in water nanoemulsion of astaxanthin was prepared by high pressure homogenization. The emulsifying conditionsincluding emulsifier type, concentration and astaxanthin concentration were optimized. Stability of nanoemulsionwas measured using zetasizer, freeze-fracture scanning electron microscope (FF-SEM), particle analyzer and colorimeter.The mean diameter of the dispersed particles containing astaxanthin ranged from 160 to 190 nm. Size distribution wasunimodal and extended from 40 to 200 nm. The nanoemulsion prepared by glyceryl citrate/lactate/linoleate/oleate hadsmaller particle size and narrow size distribution. Stable incorporation of astaxanthin in nanoemulsion was performedand checked using high performance liquid chromatography (HPLC), freeze-fracture scanning electron microscope(FF-SEM). Physical stability of nanoemulsion was not significantly changed during storage at both light and thermalcondition for a month with zeta potential value of -41 mV meaning stable colloid.

1. Introduction

 In recent years, researchers have further shown that carotenoids posses certain health benefiting functions which might be helpful in the prevention in health disorders[ 1]. Especially in cosmetic industry, butylated hydroxyl toluene (BHT), acerora and tocopherol are the conventionally used antioxidant for improving formulation stability and durability. Astaxanthin (3, 3’-dihydroxy- β-β’-carotene-4-4’-dione) is a ketocarotenoid, used as a preferred pigment in aqua-culture feeds. Due to high antioxidant activity[2,3], it can be used as a potential prophylactic agent against skin cancer and a possible chemopreventive agent. Despite the availability of synthetic astaxanthin, astaxanthin from natural sources still received more interest due to its greater antioxidant activity and stability[4].

 However, like most carotenoids, astaxanthin is a highly unsaturated molecule and thus it is highly sensitive to high temperature, light, and oxidative conditions which may promote the isomerization of astaxanthin into cis form which possesses less activity than their corresponding trans configuration[5]. In additions, genericity of use in the food and cosmetic field are not much enough because of following reasons. Low solubility, color change and Photostability are of that[6]. One of the approaches that can be used to improve the solubility and bioavailability of carotenoids such asβ-carotene is to incorporate them in the fine particles of oil-in water (O/W) emulsions. But systemic study with active incorporation into a nanoemulsion using carotenoids especially, astaxanthin, has not been proposed. Besides, physicochemical influence of emulsifier concentration, homogenation condition, temperature as well as variety of co-antioxidants on astaxanthin incorporated nanoemulsion was not examined.

 Furthermore, the effect of astaxanthin nano emulsion during storage which is very important parameter of emulsions has not been studied thoroughly.

 Therefore, we report the stability evaluation of astaxanthin nanoemulsion prepared by high pressure homogenization under various conditions using new vesicle, glyceryl citrate/lactate/linoleate/oleate and it is thought be very useful for cosmetic industry.

2. Materials & Experiments

2.1. Extraction of astaxanthin

 Astaxanthin were collected from Hamatococcus pulvialis (Honghao Chemical Co., Ltd, China). The astaxanthin was specially extracted with a continuous-flow super critical-carbon dioxide extraction apparatus (Lab SFE, Supercritical labs, Korea).

2.2. Preparation of astaxanthin nanoemulsion

 Oil-in-water (O/W) nanoemulsion prepared using liquid paraffin (seojin chemical, Korea), Ceramide (Doosan biotech, Korea), cholesterol as dispersed phase and glyceryl citrate/lactate/linoleate/oleate (Imwitor 375, Sasol, Germany), ester of mono- and diglycerides of unsaturated edible fatty acids with citric acid and lactic acid, hydrogenated lecithin (Lipoid 100 - 3, Lipoid GMBH, Germany), polysorbate 60 (Croda Korea, Korea), ethanol (Korea alcohol, Korea), propylene glycol (SKC Chem, Korea), deionized water as continuous phase. Prescription of formulation for astaxanthin nanoemulsion was mentioned on table 1. The continuous and dispersed phases were each heated to 70 ~ 75 ℃ and then mixed to form pre-mix. The pre-mix was then homogenized using homomixer (T.K Homo mixer, Osaka, Japan) for 5 min to pre-emulsify, and then, high-pressure homogenization (Microfluidics, Panda, Italy) was performed at 1,000 bar.

Table 1. Prescription of Formulation for Astaxanthin Nanoemulsion

2.3. Analysis of astaxanthin concentration

 Standard astaxanthin and astaxanthin in the extract were analyzed by a high-performance liquid chromatography (HPLC, Agilent 1,200, and Japan) series, equipped with a diode array detector. The standard and extract solutions were diluted with dichloromethane/methanol (1 : 3, v/v) and injected through the auto sample and separated with a reversed-phase C18-YMCA Carotenoid column (5 μm, 4.6⋅250 mm) at 25 ℃. Gradient elution was performed. The detection wavelength was kept at 474 nm. The gradient elution condition was shown in table 2.

Table 2. Condition of Elution

2.4. Identification and analytical study of particle size and distribution

 The average particle size and size distribution of nanoemulsion were determined by dynamic light scattering using Mastersizer (Mastersizer 2,000 Hydro, Malvern Instrument, and Worcestershire, UK). The particle size of the emulsions was described by mean diameter and size distribution was described by volume per diameter and size distribution graph .

2.5. Evaluation of emulsion stability

 The emulsion stability of the nano emulsion was evaluated using particle size analyzer, zeta potential (Zetasizer 3000HS, Malvern, UK), change of C, E value were carried out by colorimeter (Croma Meter CR200, Minolta, Japan), the nanoemulsions were sampled immediately after the preparation and divided into three groups which store at 5 ℃, Cycle (-5 ℃ ~ 45 ℃), for four weeks, Light irradiated by suntester (Suntest CPS+, Atlas, USA) /

 Stability of nanoemulsion was evaluated according to the potential difference by zetasizer as well as change of size and color for certain period of time.

 The zeta potential was calculated from the following equation:


 Zeta potential is a scientific term for electro kinetic potential in colloidal systems. Zeta potential is electric potential in the interfacial double layer at the location of the slipping plane versus a point in the bulk fluid away from the interface. The significance of zeta potential is that its value can be related to the stability of colloidal dispersions. When the potential is low, attraction exceeds repulsion and the dispersion will break and flocculate. So, colloids with high zeta potential (approximately over ± 40) are electrically stabilized whereas colloids with low zeta potentials tend to coagulate or flocculate.

2.6. Encapsulation morphology by electron microscopy

 The measurement of astaxanthin incorporation status was carried out by freeze-fracture scanning electron microscope (FF-SEM, JEM1400, and Jeol, Japan) and transmission electron microscope (TEM, H-7600, and Hitachi, Japan). A small amount of sample was immersed into liquid ethane which was immediately frozen in liquid nitrogen, and morphology checked using FF-SEM with propane gas.

3. Results & Discussion

3.1. Effect of the emulsifier type and homogenization pass times on the particle size parameter of nanoemulsion

 Unimodal nanoemulsion sized about 170 nm was observed when used glyceryl citrate/lactate/linoleate/oleate as an emulsifier. but broad and larger size distribution of nanoemulsion was observed in the use of lecithin which may has much contents of phosphatidyl cholin.

3.2. Effect of the astaxanthin extract concentration on the particle size parameter in the astaxanthin nano emulsion

 Astaxanthin incorporated nanoemulsion was investigated according to the amount of astaxanthin concentration. Particle distribution on different vesicles between new vesicle, glyceryl citrate/lactate/linoleate/oleate and normal hydrogenated lecithin was studied. Tendency for unimodal particle size distribution was observed in the use of new vesicle, glyceryl citrate/lactate/linoleate/oleate compared to that of hydrogenated lecithin. Size distribution rate dependant on concentration from 1 to 5.5% and change of size distribution after storage under light and thermal condition were observed in figure 2 and 3. Also unimodal particle size can also make diluted solution transparent even though incorporated maximum 5.5% in Figure 4.

Figure 1. Particle size distribution on vesicle.

Figure 2. Particle size distribution on astaxanthin concentration.

Figure 3. Particle size distribution on astaxanthin concentration under storage condition.

Figure 4. Comparison of the clarity of astaxanthin emulsions made with glyceryl ester, with increasing astaxanthin concentration. (a) 1%, (b) 2%, (c) 4%, (d) 5.5%, (e) Astaxanthin 1% + FITC Dextran.

3.3. Observation of color change

 Colour change valued as E and C were also observed in Figure 5, The degree of which depended on the concentration of astaxanthin. Figure 5 shows the colour change of astaxanthin nanoemulsion under the light, and heat. Also it shows the result after the storage at cycle condition from 5 to 45 ℃. The nanoemulsions with glyceryl ester tended to show good colour stability with concentration of astaxanthin in the range of 1 ~ 4%. However, the nanoemulsion with hydrogenated lecithin shows serious colour change with concentration of astaxanthin at 2% or higher.

Figure 5. color change of astaxanthin nanoemulsion on vesicle concentration under storage condition.

3.4. Detection of astaxanthin in the nanoemulsion by HPLC

 The results from Table 3 show that the astaxanthin content of the nanoemulsions remained at > 90% after these physical stresses indicating the robustness of the nanoemulsion’s ability to retain the incorporated astaxanthin under typical potentially destabilizing physical stresses.

Table 3. Analytical Data of Astaxanthin

3.5. Observation of zeta potential

 One of the methods frequently used in the cosmetic stability is measurement of potential difference called zeta potential. Zeta potential was measured for stability check of nanoemulsion. Stable astaxanthin incorporation was predicted by figure valued over -10 mV of zeta potential. Zeta potential (mV) value was measured initial -47.2 mV to –52.5 mV after 1months, even measured -48.7 mV after light irradiation for 12 hours. But as it is shown in Table 4, lecithin derived nanoemulsion was found increase of particle size which means unstable.

Table 4. Zeta Potential Measurement of The Nanoemulsions on Vesicle under Storage Condition

3.6. Nano emulsion morphology by FF-SEM

 The FF-SEM observation of the astaxanthin nanoemulsions was made immediately after the emulsion was prepared. Resultant micrographs are presented in the Figure 5 and 6. Particles appear spherical or multi-layer shape which is able to check well incorporation of astaxanthin as you see in the Figure 6 but different result was observed on the nanoemulsion made by hydrogenated lecithin shown in Figure 7.

Figure 6. FF-SEM of astaxanthin contains nanoemulsion in glyceryl citrate/lactate/linoleate/oleate.

Figure 7. FF-SEM of astaxanthin contains nanoemulsion in hydrogenated lecithin.

4. Conclusion

 In this study, new vesicle, glyceryl citrate/lactate/linoleate/ oleate, was used as emulsifier instead of using conventional hydrogenated lecithin originated from bean or egg. New vesicle also helps emulsion stabilization by forming surface layer strong resulting in better stabilization of astaxanthin incorporated nanoemulsion, transparency and emulsion stability against storage condition. Nanoemulsion prepared in this study was sized about 160 ~190 nm and -40 mV for zeta potential. Especially, combination of ethyl alcohol makes the emulsion easy penetration when spreading on the skin by increasing flexibility and intended for convenient use with low viscosity emulsion. Due to the superior property against oxidation, astaxanthin easily causes color change, strange odor by light and heat. In terms of using cosmetic industry, using lots of astaxanthin causes sticky texture too. By integrated investigation of astaxanthin in this study it is said that it carries weight with resolving above problem of astaxanthin.

 In this study, we enhanced nanoemulsion with astaxanthin showed excellent light and thermal stability on the new vesicle. We anticipate that this study provides cosmetic industry the way how astaxanthin can be used as an active cosmeceutical ingredient in skin care production having the properties of anti-wrinkle, anti-aging and h2umectancy.


 This study was performed with World Class 300 R&D support project (Kolmar Korea, Development of functional herbal cosmetics using native plants for bulk purification technology, No. 10043192) from Ministry of Knowledge Economy.  


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