Microencapsulation of fish liver oil by complex coacervation method with gelatin-arabic gum
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1 Microencapsulation of fish liver oil by complex coacervation method with gelatin-arabic gum Abstract Hiep X. Nguyen 1, Chien N. Nguyen 1,* 1 Department of Pharmaceutial Industry, Hanoi University of Pharmacy, Hanoi, 10000, Vietnam * Chiennnguyen@hup.edu.vn Fish liver oil was microencapsulated by complex coacervation with a gelatin arabic gum polymeric wall system. The effect of core/wall ratio, emulsification stirring speed, coacervation ph value, concentration of formaldehyde solution, ratio of gelatin/ arabic gum, weight of PVA, and microencapsules stabilizing time on the microcapsulation yield, encapsulation efficiency, particle size and morphology of microcapsules were investigated. The suitable conditions were determined and rational formulation were established. Keywords: Fish liver oil, complex coacervation, HPLC, extraction Introduction Microcapsule and microsphere have numerous advantages over tablet namely more stable bioavailabity amongst individuals since retention time of microcapsule is less fickle in intestinal tract. Microencapsulation especially takes active ingredients to undergo the metamorphosis from liquid state to solid form to be ultimately capsulated or tabletted [3], and also conceals both ingredients stinky odour and foul taste. Be based on that and the pharmaceutical project entitled Microencapsulation of fish liver oil by complex coacervation method with gelatin-arabic gum was excecuted with fish liver oil as drug model in order to investigate the effects of condition and formulation parameters on microencapsulation of fish liver oil. Materials, equipments and methods Materials Vitamin A palmitate, gelatin, gum arabic, fish liver oil, polyvinyl alcol (PVA): reach pharmaceutical standards. Other chemicals: reach analytical standards. Standard Vitamin A palmitate (500 mg/vial, 996,100 IU/g) is kindly provided by National Institute of Drug Quality Control. Equipments IKA RW 20 overhead digital stirrer, CHRIST Freeze Dryer, Refrigerated Centrifuge sigma 3 18K sartorius AG, Hettich Zentrifugen Centrifuge Rotina 46, IKA vortex Genius 3, Optical microscope Carl Zeiss, axiostar plus, High Performance Liquid Chromatography system (HPLC) Shimadzu, Scanning Electron Microscope (SEM) Hitachi S4800- NIHE 10000kV. Methods Preparation of microcapsules Microcapsules were prepared by complex coacervation method with a gelatin arabic gum polymeric wall system, analogous to the technique of Alavi Talab H. [1]. The incipient formula came as follows: 30g 12.5% gelatin solution was mixed with 30g 12.5% gum arabic solution at room temperature, stirring speed of 500rpm in 5 minutes (the first stirring stage). Added 4.2g fish liver oil, 0.5g PVA, 0.04g BHT (Butylated hydroxytoluene), and 10 ml distilled water to the mixture of gelatin solution and gum arabic solution, then continued agitating in 15 minutes, at room temperature and the same stirring speed as at the first stirring stage (the second stirring stage). Increased the mixture s temperature to 50 0 C and added 190ml distilled water, kept the stirring speed on par with that at the first stirring stage in 30 minutes (the third stirring stage). Adjusted the mixture s ph value to 4.5 by dropwise addition of 10% acetic acid solution at 40 0 C. Added 200ml distilled water and emulsified the mixture at stirring speed of 250rpm, temperature below 10 0 C in 60 minutes (the fourth stirring stage). Altered ph value of the mixture to 9.7 by 10% sodium hydroxide solution addition, then added 20ml 20% formaldehyde solution. Kept stirring at the speed of 250rpm, in 30 minutes, below 10 0 C to stablize the microcapsules. 22 hours later, centrifuged the mixture at the centrifugation speed of 4000rpm, at room temperature, in 15 minutes to collect microcapsules. After that the microcapsules were washed twice by 100ml distilled water below 10 0 C (centrifuged after each wash), vacuum filtered, and then washed once by solution of 25ml isopropanol and 25ml distilled water below 10 0 C [4]. The
2 obtained microcapsules were desiccated by freeze drying with the following parameters: temperature: C, pressure: 0.007mbar, pre-freeze time: 4 hours and freeze drying time: hours. Determination of microcapsules properties Size and shape of microcapsules Used an optical microscope, each sample of 50 microcapsules from 5% suspension of microcapsules in cold water; and utilized Scanning Electron Microscope (SEM). Vitamin A concentration Hydrolyzed fish liver oil samples (approximately equivalent to 1740IU Vitamin A) in fish liver oil material, microcapsules or standard Vitamin A palmitate by 1ml saturated potassium hydroxide solution, 2ml ethanol and 0.02 g BHT in an ultrasonic bath, in 30 minutes. Extracted Vitamin A once by 6ml n-hexane, washed n hexane extract by distilled water, evaporated n-hexane entirely to obtain solid deposit which was then dissloved in isopropanol. Diluted the resulted solution to the concentration range of 375IU/ml to 2350IU/ml. Ran these samples on a High Performance Liquid Chromatography system (HPLC) with the following parameters [2] : Supelco Discovery C8 Chromatographic column, 15cm x 4.6mm (5μm); Guard Column 2cm x 4.6mm (5μm); injected sample volume: 20μl; column temperature: 30 0 C; flow rate: 1.0ml/min; mobile phase: methanol:twice distilled water=95:5; detection wavelength: 325nm. Encapsulation Efficiency (EE) Encapsulation efficiency is percentage of Vitamin A in microcapsules in the total amount of Vitamin A in fish liver oil material used. Microcapsule Yield (MY) Calculated microcapsule yield by dividing the acquired microcapsules weight by the total weight of materials. Results and discussion Chromatograms of Vitamin A in HPLC analysis Ran HPLC with the Vitamin A treated samples to obtain chromatograms in image 1. The chromatograms gave a single, sharp and relatively symmetrical Vitamin A peak on a flat, little-noisy baseline with retention time of 2.6 min mau 200 m AU mau Minutes Minutes A B C Image 1: Chromatograms of the Vitamin A treated samples from fish liver oil microcapsules F D (A, the final formula, see below), standard Vitamin A palmitate (B) and fish liver oil material (C) Minutes The effects of core/wall ratio Prepared the formula F C11, altered core/wall ratio [the fish liver oil weight was 2.1g (F C12), 4.2g (F- C11) and 8.4g (F-C21)]. Properties of the resulted microcapsules were demonstrated in table 1.
3 Table 1: The effects of core/wall ratio on microcapsules properties F Core/wall ratio C (IU/g) Φ MC (μm) MY (%) EE (%) MC properties C12 1: ± Aggregation, hard to filter C11 1: ± Scattering, easy to filter C21 2: ± Aggregation, very hard to filter Annotation: C was Vitamin A concentration in microcapsules samples, Φ= diameter, MC=microcapsules. Table 1 indicated that when core/wall ratio varied from 1:2, 1:1, to 2:1, the microcapsules average size did not change (p>0.05), both encapsulation efficiency and microcapsule yield reached their maximums when the core/wall ratio was 1:2 (F- C12), and arrived at their minimums when the core/wall ratio was 2:1 (F-C21). These two fomulae produced aggregated and hard-to-filter microcapsules. On the other hand, microcapsules Table 2: The effects of emulsification stirring speed on microcapsules properties obtained by the formula core/wall ratio=1:1 (F- C11) were spherical, scattering and easy-to-filter. The effects of emulsification stirring speed Prepared the formula F C11, altered stirring speed at the first, second and third stages from 300rpm to 1000rpm. Properties of the resulted microcapsules were demonstrated in table 2. F v (v/ph) C (IU/g) Φ MC (μm) MY (%) EE (%) MC properties A X X Scattering, nonspherical, hard to filter B X X Scattering, nonspherical, hard to filter C ± Scattering, spherical, easy to filter D ± Scattering, spherical, easy to filter E ± Scattering, spherical, very easy to filter Annotation: X = undetermined As the emulsification stirring speed was 300rpm (F- A) and 400rpm (F-B), microcapsules were nonspherical, hard-to-filter. Be based on that and only encapsulation efficiency was investigated for these formulae. If the stirring speed increased from 500rpm to 1000rpm, then the microcapsules average size decreased correspondingly, EE and MY did not change significantly. In addition, the formulae with stirring speed of 500rpm (F-C) and 750rpm (F-D) created scattering microcapsules. EE of F-D was maximum. The effects of microencapsules stabilizing time Prepared the formula F D, altered the microencapsules stabilizing time from 8h, 15h to 22h. Properties of the resulted microcapsules were demonstrated in table 3. Table 3: The effects of the microencapsules stabilizing time on microcapsules properties F T (hours) C (IU/g) Φ MC (μm) MY (%) EE (%) MC properties T ± Aggregation, hard to filter T ± Scaterring, hard to filter D ± Scaterring, easy to filter
4 When the microcapsules stabilizing time changed from 8h to 15h and 22h, the microcapsules average size, microcapsule yeild and encapsulation efficiency did not change (p>0.05). As the stabilizing time was more than 15h, microcapsules were scattering and easy to filter. As a result, the microcapsules stabilizing time 22h was selected to serve the study. The effects of coacervation ph value Prepared the formula F D (the microcapsules stabilizing time was 22h), altered coacervation ph value from 3.0 to 5.0. Properties of the resulted microcapsules were demonstrated in table 4. Table 4: The effects of coacervation ph value on microcapsules properties F ph C (IU/g) Φ MC (μm) MY (%) EE (%) MC properties H X X Nonspherical, hard to filter H ± Scattering, spherical, easy to filter D ,13± Scattering, spherical, easy to filter H ± Scattering, spherical, easy to filter When the coacervation ph value was 3.02, the microcapsules clustered, nonspherical, hence only encapsulation efficiency was studied. When the coacervation ph value varied from 4.11; 4.50 to 5.06, the microcapsules average size changed insignificantly. The ph value 4.50 brought about the maximum microcapsule yield (81.94%), the maximum encapsulation efficiency (64.11%), easyto-filter microcapsules. Be based on that and the coacervation ph value 4.50 (F-D) was choosed. The effects of concentration of formaldehyde solution Prepared the formula F D (the coacervation ph value was 4.50), altered concentration of formaldehyde solution from 10%, 20% to 30%. Properties of the resulted microcapsules were demonstrated in table 5. Table 5: The effects of concentration of formaldehyde solution on microcapsules properties F C formaldehyde (% w/v) C (IU/g) Φ MC (μm) MY (%) EE (%) MC properties F ± Scattering, easy to filter D ± Scattering, easy to filter F ± Aggregation, hard to filter When the concentration of formaldehyde solution altered from 10%, 20% to 30%, the microcapsules average size changed insignificantly (p>0.05). The formula F-D (20% formadehyde) accounted for microcapsule yield and encapsulation effiency higher than F-F1 and lower than F-F3, but scattering microcapsules. The effects of ratio of gelatin/arabic gum (gel:gum) Prepared the formula F D (the concentration of formaldehyde solution was 20%), altered the gel: gum ratio: 3.75g:7.50g (1:2); 3.75g:3.75g (1:1); 7.50g:3.75g (2:1). Properties of the resulted microcapsules were demonstrated in table 6.
5 Hiep X. Nguyen et al. / Pharmaceutical Journal ISSN No 437 Vol. 52 (2012) 6-10 Table 6: The effects of gel:gum ratio on microcapsules properties F gel:gum C (IU/g) Φ MC (μm) MY (%) EE (%) MC properties M 1: X X Aggregation, nonspherical, very hard to filetr D 1:1 519, ± Scattering, spherical, easy to filter P 2:1 472, ± Aggregation, spherical, easy to filter When the gel:gum ratio was 1:2 (F-M), the product was nonspherical, cluster of polymers containing many fish liver oil droplets, encapsulation efficiency was lowest (58.89%). As the gel:gum ration was 2:1 (F-P), microcapsules were spherical with relatively equal size, but clustered with low MY and EE. When the gel:gum ratio was 1:1 (F-D), microcapsules were spherical, scattering, with relatively equal size and maximum encapsulation efficiency. The effects of weight of PVA Prepared the formula F D (the gel:gum ratio was 1:1), altered weight of PVA from 0.21g to 0.50g and 0.80g. Properties of the resulted microcapsules were demonstrated in table 7. Table 7: The effects of weight of PVA on microcapsules properties F PVA (g) C (IU/g) Φ MC (μm) MY (%) EE (%) MC properties P1 0, ± Scattering, easy to filter D 0, ± Scattering, easy to filter P3 0, ± Aggregation, easy to filter When the weight of PVA varied, the microcapsules average size changed insignificantly (p>0.05). As the weight of PVA increased from 0.21g (F-P1) to 0.80g (F-P3), microcapsule yield increased. Encapsulation efficiency reached its maximum (64.11%) when the weight of PVA was 0.50g (F-D) and its minimum (47.90%) when the weight of PVA was 0.21g (F-P1). Continuous augmentation of PVA weight to 0.80g (F-P3) did not lift the encapsulation efficiency, let alone made clustered, hard-to-filter microcapsules. F-D was selected as the final formula (F-D was similar to F-C11, only changed the emulsification stirring speed at the first, second and third stages to 750 rpm). The fundamental structure and size microcapsules were displayed in image A (optical microscope, 100X) B (SEM, magnification of 3000X) Image 2: Images of microcapsules (formula F-D) of 2.
6 Discussion Microencapsulation by complex coacervation method with gelatin-arabic gum is complicated, multi-stages, each stage affects the quality of microcapsules. In an alike study, Alavi et.al [1] demonstrated that as stirring speed increased, microcapsules average size decreased correspondingly. Besides, using 25% glutaraldehyde as the cross linking agent instead of formaldehyde brought the microcapsules more spherical shape, smooth surface with no obvious dents and narrower particle size distribution [1]. The project of Junyaprasert et. al [4] illustrated that the freeze drying method and the gel:gum ration=1:1 were appropriate to prepare Vitamin A palmitate microcapsules by complex coacervation method. Conclusion The study investigated the effects of some condition, formulation parameters on microencapsulation of fish liver oil by complex coacervation method, determined the suitable conditions and established rational formulation. References 1. Alavi T. H. et al. (2010), "Optimization of morphology and geometry of encapsulated Hypophthalmichthys molitrix oil", Iranian Journal of Fisheries Sciences, 9(2), p Vietnam Ministry of Health. (2009), Appendix 10.10: "Vitamin A quatitative analysis, Method 5: High Performance Liquid Chromatography (HPLC) method after Vitamin A extraction", Viet Nam pharmacopoeia IV, Hanoi, p. PL 190-PL James S. et al. (2007), "Microencapsulation Technology, Encyclopedia of Pharmaceutical Technology", Informa Healthcare, New York, 3(4), p Junyaprasert, V. B. et al. (2001), "Effect of Process Variables on the Microencapsulation of Vitamin A Palmitate by Gelatin-Acacia Coacervation", Drug Development and Industrial Pharmacy, 27(6), p
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