2015 Volume 21 Issue 4 Pages 503-507
This study was performed to investigate the physicochemical properties of lutein microencapsulation (LM) and to assess its bioavailability (BA). LM was prepared with advanced spray and starch-catching drying technology. Its physicochemical properties (characterization, storage stability and dissolution) and BA were evaluated. The BA study of LM was performed using SD rats following a single oral administration of lutein equivalent to 100 mg, and were compared with commercial reference sample (RS). LM was nearly sphericity, its mean particle size was 214.7 nm. It can be free-flowing. Solubility of lutein was increased and it could be directly dissolved in water to form a homogeneity of solution. The storage stability of lutein was enhanced under accelerated and long-term storage conditions. More than 85% lutein could be released from LM after 15 min during the dissolution test. The relative BA of lutein was 139.1% in comparison to RS. The results would be helpful to conduct the application of LM in food industry.
Lutein is a kind of important carotenoids with many valuable physiological functions. As reported, antioxidant ability and free radical scavenger enables lutein to be a excellent free radicals scavenger in human body (Roberts et al., 2009; Calvo, 2005), filtration of blue light capability makes lutein to be able to protect skin or eye from photo damage (Alves-Rdorigues et al., 2004), its coloration ability make lutein be a good choice to improve food and beverage color (QV et al., 2011). However, lutein is not synthesized in the body, dietary ingestion is the only source (Amar et al., 2003). Lutein is a lipophilic substance and poorly soluble in water, the application of lutein in food is limited due to its instability and water insolubility, and poorly soluble actives also could result in delivery problems (Müller and Keck, 2004), bioavailability (BA) of lutein is relatively lower due to the inherently poor aqueous solubility. Therefore, it is necessary to find a method to avoid these disadvantages.
Microencapsulation is one of promising methods for protecting food ingredients against deterioration, which has been widely used for stabilization of unstable compounds. Many studies reported that microencapsulation of lutein is able to improve the stability and water solubility, and controlling release of lutein (QV et al., 2011; Jin et at., 2009; Wang et al., 2012). But to the best of our knowledge, many studies have focused on the preparation of LM, little has been reported on the physicochemical properties and BA of LM. Physicochemical properties and BA are very important for the final application of LM product, such as particle shape and particle diameter are affected its flowability, poor flowability may influence its application, and reduction of particle size may enhance BA. Good stability and water-solubility enhances the dissolution velocity of LM, and thus enhanced its BA.
In this study, LM was manufactured with advanced spray and starch-catching drying technology, in which, lutein and dl-alpha-tocopherol were dissolved as oil phase; modified food starch, water and sucrose were dissolved as aqueous phase; then oil phase and aqueous phase were emulsified, and filtrated, spray granulation and corn starch catching, fluidized drying, sieving and packaging to obtain LM product. Physicochemical properties (characterization, storage stability and dissolution) of LM were evaluated. Furthermore, its BA was assessed in SD rats following a single oral administration of lutein equivalent to 100 mg, and were compared with that of reference sample (RS). The results could be provide some potential useful parameters for application of LM in food industry.
Materials LM was obtained from Zhejiang Medicine Co., Ltd. Xinchang Pharmaceutical Factory (ZMC, China). Commercial reference sample (RS, FloraGLO® Lutein 5% CWS/S-TG) was the similar product with LM, which was purchased from DSM Nutritional Products Ltd. (DSM, Switzerland). Methanol and TBME (tert butyl methyl ether) were chromatographically pure and other chemicals were analytical grade and were used without further purification.
Preparation of LM Compositions used for the preparation of LM are shown in Table 1. It was manufactured with advanced spray and starch-catching drying technology. The individual particles containing lutein were finely dispersed in the matrix of modified food starch and sucrose, coated with corn starch. dl-alpha-tocopherol was added as antioxidant. LM was stored in a closed container and protected from light, heat, oxygen and moisture.
| Component | Function | Amount (%) |
|---|---|---|
| Lutein | Core material | 5.0 |
| Modified food starch | Wall material | 38.0 |
| Sucrose | Filling material | 37.0 |
| Corn starch | Wall material | 18.0 |
| dl-alpha-tocopherol | Antioxidant | 2.0 |
| Total | 100 |
Characterization of LM The particle shape of LM were measured using scanning electronic microscopy (FEI, USA). Repose angle of LM was measured using FT-104 angle of repose tester (Ningbo rooko instrument Co., Ltd, China). Particle size of dispersion formed by addition of 80 mg of LM into 200 mL of distilled water under slight agitation was determined by Backman coulter LS-230 laser diffraction particle size analyzer (International Equipment Trading Ltd., USA). All measurements were performed in triplicates.
Storage stability of LM In order to investigate the chemical stability of lutein in the prepared microencapsulation, stability studies were performed at accelerated condition (40°C ± 2°C and 75% ± 5% relative humidity (RH)) and at long-term condition (25°C ± 2°C and 60% ± 5% RH), for a period of 36 months in stability chamber. The samples were analyzed at 0, 1, 2, 3, 6, 12, 18, 24 and 36 months. The amount of lutein was analyzed by UV spectrometry method mentioned in USP (U.S.Pharmacopeia) lutein preparation. All measurements were performed in triplicates. The physical stability in terms of appearance was also assessed for the same period of time at the same storage conditions.
Lutein dissolution study LM and RS were filled into the body of hard gelatin capsules to investigate lutein releasing from LM and RS using USP (U.S.Pharmacopeia) method. The capsules containing LM (20 mg as of lutein) were introduced into DT 600 dissolution tester (ERWEKA, Germany). Dissolution apparatus I was operated with 900 mL of distilled water as dissolution medium at 37°C ± 0.5°C with basket speed of 100 rpm. At 5, 10, 15, 20, 30, 45 and 60 min, an aliquot of 5 mL was collected, respectively, filtered and analyzed for the content of lutein by above-mentioned UV spectrometry method. 5 mL of fresh dissolution medium was replaced to compensate the loss due to sampling every time, and the sink condition was maintained throughout the dissolution study.
BA study in SD rats1) Animals SD rats weighing about 230 – 280 g were obtained from Experimental Animal Center of Zhejiang Academy of Medical Sciences (Zhejiang, China). The rats were housed in cages at a temperature between 20°C and 23°C with free access to water. All animals were handled in accordance with the principles and guidelines for the care and use of laboratory animals of National Institutes of Health, China.
2) Study design LM and RS was dissolved with distilled water to form the dispersion with the concentration of lutein at 100 mg/mL. Twelve SD rats (six males and six females) were randomized into two groups with six rats in each group. All experimental rats were fasted for 8 h prior to study with free access to water and weighed before dosing. LM or RS dispersion with lutein equivalent to 100 mg/kg was orally administered to SD rats after feeding 0.5 h. Blood samples were collected from fundus venous plexus at 0 (pre-dose), 1, 2, 4, 6, 8, 10, 12, 20, 26 and 32 h following oral administration. Blood samples were collected in pre-labeled centrifuge tubes containing heparin as an anticoagulant. Plasma samples was separated by centrifugation at 2000 ×g for 8 min.
3) HPLC-MS/MS analysis of lutein in SD rats plasma Lutein levels in plasma were measured by an Agilent 1290 infinity LC system/6460 triple quadrupole mass spectrometer equipped with an APCI source. Chromatographic colum was Agilent Eclipse Plus C18 (ϕ2.1 mm × L50 mm, particle diameter 1.8 µm) with column temperature of 35°C, flow rate of 0.45 mL/min and injection volume of 3 µL. The mobile phase was the mixture of methanol-0.1% formic acid aqueous solution(87:13,v:v). MS conditions were gas temperature of 300°C, APCI heater of 350°C, gas flow of 4 L/min and nebulizer of 206.84 kPa. APCI(+) ion pair was lutein of 551/175.2 and ergosterol of 379.3/69.3. Quantification was performed by applying internal calibration, ergosterol as internal standard. Validation of the HPLC/MS/MS assay was performed, the method has good specificity, the endogenous impurity in plasma has no interference with the determination of lutein. Linearity of lutein was investigated by constructing seven-point calibration curves at concentration range of 5.0 ng/mL–200 ng/mL. Calibration curves showed excellent linearnity with good coefficient (r = 0.997), LLOQ as 5.0 µg/L. The method was precise and accurate with coefficient of variations of less than 15%.
An aliquot of 100 µL plasma was used for extraction of lutein. 100 µL plasma, 5 µL of internal standard (500 ng/mL of ergosterol in methanol) and 100 µL of ethanol were added, agitated vigorously for 3 min for protein deposition, 800 µL of TBME was added, and agitated vigorously again for 5 min for extraction of lutein, followed by centrifugation at 4,400 ×g for 10 min at 25°C. The supernatant organic layer evaporated under nitrogen gas. The residue was then re-dissolved with 100 µL of mobile phase using vortex mixer followed by centrifugation at 4,400 ×g for 15 min at 25°C, and 3 µL of this solution was injected into LC-MS/MS for lutein content measurement.
4) Pharmacokinetic parameter analyses The plasma concentration of lutein versus time profile was analyzed The pharmacokinetic parameters were obtained by fitting the plasma concentration-time data to the non-compartmental model with DAS 2.0 software. Parameters included the time of maximum plasma concentration (Tmax), maximum measured concentration (Cmax), elimination half-life (t1/2z) and area under the concentration-time curve to terminal time (AUC(0–32)). Tmax, Cmax and t1/2z were read directly from the observed concentration versus time data. AUC(0–32) was calculated using the linear trapezoidal rule. The relative BA of LM to reference RS was calculated using the following equation:
 |
where DoseRS and DoseLM is administration dose of RS and LM, respectively.
Lutein is a poorly water-soluble compound that exhibits dissolution-rate-limited absorption. Microencapsulation can offer an improvement of lutein solubility in water, the rate and extent of lutein absorption were also improved. Hence, microencapsulation of lutein has been expected to enhance solubility and absorption of lutein because it forms nanosized dispersion in aqueous environment.
Characterization of LM Shape of LM was demonstrated by the scanning electronic microscopy image was nearly sphericity (Fig.1). The particle size and repose angle of LM were shown in Table 2. Repose angle is an index to classify the flowability (Xu et al., 2007), repose angel between of 30 – 45° is classified as free-flowing. Repose angle of LM was 31.8°, which can be free-flowing. The particle size of the solution formed by adding 80 mg of LM into 200 mL of distilled water under slight agitation was found to be 214.7 nm (Table 2), suggesting reasonable homogeneity of the dispersion. Particle size could be influence on the bioavailability, reduction of particle size may enhance BA (Meor Mohd Affandi et al., 2012). Smaller particle size will increase the surface area of lutein and improve its solubility behavior as well as its absorption. Nano-dispersion of LM in water solution suggested good stability and high BA of the prepared LM. The particle size distribution may result from the composition and drying technology.
SEM micrographs of LM (5.0 kV, 100×)
| Properties | LM | RS |
|---|---|---|
| LM characteristics | ||
| Particle shape | Sphericity | Sphericity |
| Repose angle (°) | 31.8 | 30.6 |
| Dispersion characteristicsa | ||
| Particle size (nm) | 214.7 | 222.6 |
All values were mean of three values.
Storage stability of LM Lutein is an unstable carotenoid, which can be easily oxidized, degraded (Shi et al., 1997; Henry et al., 1998). For this reason, chemical stability study was performed and the % mass lutein remaining in LM and RS stored at accelerated condition (40°C ± 2°C and 75% ± 5% RH) for a period of 12 months and long-term condition (25°C ± 2°C and 60% ± 5% RH) for a period of 36 months is presented in Fig. 2a and 2b, respectively. The chemical stability of lutein at the end of 36 months was not significantly different from the initial stability, and no significant degradation of the active from the initial value was found. The % of lutein remaining in LM was 95.6% after 12 months of storage at accelerated test and 97.1% after 36 months of storage at long-term test. The percentage change of lutein from the initial period was less than 5.0% at both storage conditions, suggesting good chemical compatibility of lutein with the formulated excipients. In terms of physical stability, LM showed good physical stability without any change of appearance at both storage conditions.
Chemical stability of lutein in LM and RS(40°C ± 2°C/75% ± 5% RH(a), 25°C ± 2°C/60% ± 5% RH(b))
Lutein dissolution study Release profiles of lutein from the capsules containing 20 mg of lutein are presented in Fig. 3. The release of lutein from LM and RS was similar. The dissolution of lutein from the LM capsule was 60.39% at 5 min, 85.61% at 15 min and 98.78% at the end of dissolution study, respectively. The aqueous solubility of lutein was greatly enhanced due to the high solubility of wall materials of microencapsulation and small mean particle size, which formed nano-dispersion after introduction to dissolution medium. Under the condition of dissolution test, the dissolution of lutein from LM was more than 85% accomplished within 15 min, which can be thought that bioavailability of LM was not restricted by dissolution behavior.
Lutein release profile from LM
BA study in SD rats The plasma concentration time profiles of lutein from LM and RS in SD rats following a single oral administration of lutein equivalent to 100 mg is shown in Fig. 4. The pharmacokinetic parameters of lutein following 100 mg oral dose of lutein from LM or RS were summarized in Table 3.
Rat plasma concentration-time profile of lutein after oral dose of 100 mg lutein from LM or RS (n = 6).
| Parameters | LM | RS |
|---|---|---|
| AUC (0–32) (mg/L*h) | 566.6 ± 203.6 | 407.4 ± 33.8 |
| Tmax (h) | 4.7 ± 3.0 | 6.0 ± 2.2 |
| t1/2z (h) | 13.5 ± 9.5 | 10.6 ± 4.0 |
| Cmax (mg/L) | 65.8 ± 38.9 | 36.8 ± 10.5 |
| Relative BA (%) | 139.1 | / |
Although there were no significant difference between the Cmax, Tmax, t1/2z and AUC(0–32) of LM and RS (P > 0.05), but Cmax of LM (65.8 ± 38.9 mg/L) was higher compared to RS (36.8 ± 10.5 mg/L), which clearly indicates the solubility and absorption of LM was superior to RS in vivo. Tmax was 4.7 ± 3.0 h when administered with 100 mg dose of lutein as LM, while it was 6.0 ± 2.2 h when administered with lutein as RS. The relatively early in Tmax of lutein from LM could be explained based on the faster rate of absorption, LM reached the Cmax relatively early compared to RS. t1/2z of LM (13.5 ± 9.5 h) was slightly prolonged compared to RS (10.6 ± 4.0 h), which indicates elimination of lutein obtained from LM was slower. AUC(0–32) of LM was higher compared to RS. Regarding the relative BA of lutein from LM, LM enhanced BA of lutein in comparison to RS, and the relative BA was 139.1%.
The second peak appeared in plasma concentration-time profile of both LM as well as RS, could be explained by the general characteristics of carotenoid absorption. After ingestion of β-carotene, a similar second plasma concentration peak were reported (Johnson and Russell, 1992). The early rise in circulation lutein concentrations is caused by the intestinal input, whereas hepatic secretion is the source of later increases. It is likely that lutein behave similar to beta-carotene.
Lutein is the main carotenoid with several essential biological functions, including antioxidation, protection against UV light effects, pigmentation, etc. Therefore, it has important applications in nutraceutical, food and feed industries. However, free lutein is sensitive to oxygen and light, so it is important to stabilize the lutein, and improve its stability and BA.
In this study, LM was prepared with advanced spray and starch-catching drying technology. Physicochemical properties (characterization, storage stability and dissolution) and BA of LM were evaluated. The stability of lutein was improved under storage conditions. The dissolution of lutein from LM was more than 85% accomplished after 15 min. The relative BA of lutein was 139.1% in comparison to commercial RS. In summary, LM could improve the stability, solubility and BA of lutein.
