2016 Volume 64 Issue 1 Pages 21-26
The coacervation between gelatin and sodium alginate for ginger volatile oil (GVO) microencapsulation as functions of mass ratio, pH and concentration of wall material and core material load was evaluated. The microencapsulation was characterized by scanning electron microscopy (SEM), Fourier transform infrared (FT-IR), and thermal gravimetric analysis (TGA). SEM and FT-IR studies indicated the formation of polyelectrolyte complexation between gelatin and sodium alginate and successful encapsulation of GVO into the microcapsules. Thermal property study showed that the crosslinked microparticles exhibited higher thermal stability than the neat GVO, gelatin, and sodium alginate. The stability of microencapsulation of GVO in a simulated gastric and an intestinal situation in vitro was also studied. The stability results indicated that the release of GVO from microcapsules was much higher in simulated intestinal fluid, compared with that in simulated-gastric fluid.
Ginger (Zingiber officinale ROSCOE), typically cultivated in tropical and subtropical countries, such as China, Nigeria, India, Jamaica, Australia and Haiti, is widely used in food and pharmacy industry on account of its culinary and medicinal properties.1,2) Its rhizomes and the obtained extracts, especially polyphenol compounds (6-gingerol, 8-gingerol, 10-gingerol and its derivatives) have a strong antioxidant activity,3,4) which has been confirmed in many literatures. Ginger volatile oil (GVO) is one of the extracts of ginger, which can be used in folk medicine for pharmacological activities including anti-inflammatory, anti-tumor, analgesic and antibacterial effect.5,6) However, environmental factors, such as temperature, pH, oxygen, UV and X-ray, affect the stability of GVO.7) Microencapsulation technique has recently been utilized to solve these problems.
Microencapsulation is considered as the technology for packaging solids, liquids or gaseous materials in miniature, which can release the contents at controlled rates under specific conditions.8) Thereby, it can be used to protect the sensitive core materials from its outside environment (e.g. heat, moisture, air and light) to extend its shelf-life, which contributes to reducing its reactivity.9) To choose a suitable encapsulating agent is an important task, which depends on the properties of core material and the characteristics of final product.10)
Biopolymers, such as polysaccharides and proteins, can be used to create food-grade microcapsules, which is one of the most promising approaches.11) Gelatin is biocompatible, edible biodegradable, and soluble at the body temperature and therefore it is an ideal material for food and pharmaceutical applications.12,13) It is an amphoteric protein and is positively charged below its isoelectric point. Sodium alginate, a natural polysaccharide, has been used in various food applications.14,15) At low pH, sodium alginate, having negative charges can form polyelectrolyte complex with gelatin. Thus gelatin–alginate complex microcapsules are prepared by emulsification crosslinking.16,17)
The objectives of the present work were to examine the influence of mass ratio, pH and concentration of wall materials and core material load on coacervation, to characterize the microscopic morphology of the microspheres, and to study the thermal stability and the release in vitro of the microcapsules.
Laiwu gingers (Zingiber officinale ROSCOE) were obtained from the local market. Gelatin B and sodium alginate were purchased from Aladdin (China) and food grade. Calcium chloride, ethanol, sodium citrate and hydrochloric acid were purchased from Sigma-Aldrich (Shanghai, China) and analytical grade. The double-distilled water was used throughout the experiment.
MethodsPreparation of GVOThe air-dried roots of ginger were ground in an attrition mill and sieved with a 60 to 80 mesh wire screen. The supercritical CO2 fluid extraction procedure was performed at the pressure of 25 MPa and temperature of 40°C and flow of 30 kg/h for CO2 and total processing time of 100 min. The GVO was collected in amber vials.5)
Polyelectrolyte Complexation and MicroencapsulationPolyelectrolyte complexation for preparing microencapsules was carried out by an oil-in-water (o/w) emulsification and crosslinking method. Gelatin-B solution (0.5–3.0% (w/v)) and sodium alginate solution (0.5–3.0% (w/v)) were prepared separately. GVO was dissolved in gelatin-B solution under continuously stirring condition at 40±2°C to form an emulsion. Sodium alginate solution (0.5–3.0% (w/v)) was added to the beaker dropwise with a magnetic stirrer. The glacial acetic acid solution (2.5% (v/v)) was used to bring the pH of the mixture to 3.2–4.0. After 60 min, the microcapsules were hardened by cooling down the temperature of the system to 5–10°C and adding the solution of calcium chloride. The microcapsules were filtered and washed with distilled water. They were further washed with ethanol to remove oil of the surface of microcapsules and freeze-dried. The microcapsules were stored in a glass bottle in refrigerator.18)
Calibration Curve of GVOA calibration curve of GVO is required to determine the encapsulation efficiency of the microcapsules. GVO–ethanol solution (5–50 µg/100 mL) was prepared, respectively. The solution was scanned by using UV visible spectrophotometer from 200 to 400 nm. The results showed that the maximum absorbance value was at 232 nm. The calibration curve was obtained by recording and plotting the absorbance values of different concentrations at 232 nm.19) From the calibration curve, the concentration of GVO microcapsules was obtained by determining the absorbance value.
Encapsulation EfficiencyGVO microcapsules were accurately weighed, dissolved in ethanol and grounded in a mortar, to which sodium citrate was added to broke microcapsules. The mixture was transferred with precaution to an erlenmeyer flask containing 100 mL of ethanol and kept for 60 min with continuous stirring to dissolve the GVO in the microcapsules. The solution was allowed to settle down, filtered, collected and transferred with precaution to a volumetric flask containing 100 mL of ethanol. The GVO inside the microcapsules was analyzed at 232 nm employing UV spectrophotometer.20) The loading efficiency (%) was calculated by using the calibration curve and the following formula.
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In vitro drug release study was carried out in simulated gastric fluid (0.1 N HCl, pH 1.2) and simulated intestinal fluid (phosphate buffer, pH 6.8) at 37°C.21) Two grams microcapsules were added to 50 mL simulated gastric fluid and simulated intestinal fluid respectively. Both the mixture was incubated at 37±0.5°C under continuously stirring (100 rpm) condition with the sample collecting 10, 20, 30, 60, 120, 180, 240 and 300 min. The treated samples were centrifuged and the collected supernatant was analyzed for determining GVO content released from microcapsules. To maintain a constant volume, the dissolution medium was returned to the container to 50 mL. Ten milliliters ether and 10 mL petroleum ether were added to the collected samples and shaken (100 rpm) for 3 min. The mixture was removed to separating funnel and settled down to collect the supernatant. The solvent in the supernatant was evaporated by using rotary evaporation instrument at no more than 30°C and accurately weighed.22) All treatments were triplicate.
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The detailed morphology and sizes of the GVO microcapsules were studied at room temperature by employing SEM (Model JSM-6380LV, JEOL, Japan) at an accelerated voltage of 20 kV. The microcapsules were covered with a thin layer of gold to conduct thermal and electricity.
Fourier Transform Infrared Spectroscopy (FT-IR)Gelatin, sodium alginate, polyelectrolyte complex of gelatin and sodium alginate and GVO microcapsules were grounded with KBr in a FT-IR spectrophotometer (Model VECTOR-220, Bruker, Germany), respectively. FT-IR spectra were recorded in the range of 4000–400 cm−1.
Thermal Gravimetric Analysis (TGA)Thermal properties of the samples were studied by employing TGA. TGA thermograms of gelatin, sodium alginate, gelatin–sodium alginate microcapsules and GVO microcapsules were recorded by using a thermogravymetric analyzer (Model TGAQ500, TA, U.S.A.) in the temperature range of 20–300°C at a heating rate of 10°C/min in a nitrogen atmosphere.
Polyelectrolyte complexation involves reaction between two oppositely charged polymers to yield a polymer rich and polymer poor region.23,24) The polyelectrolyte complex formation is dependent on many factors, such as mass ratio, polymer concentration, pH of the solution, temperature, reaction time and so on.25) In order to optimize the conditions for the formation of polyelectrolyte complex of gelatin/sodium alginate, the turbidity at 400 nm and relative viscosity of the supernatant solution were determined. The optimum condition of coacervation between alginate and gelatin was the point where both the absorbance and relative viscosity of the supernatant reached the minimum.
Influence of Mass Ratio on Complex CoacervationThe influence of different gelatin concentration in alginate–gelatin mixture on supernatant viscosity and absorbance were shown (Fig. 1a). With the increase of gelatin concentration, the viscosity decreased in the mass ratio range 1 : 0.5–1 : 2, while the absorbance exhibited the opposite trend. This is due to the presence of unreacted alginate in the supernatant. The minimum relative viscosity and minimum absorbance of the supernatant appeared at when the alginate–gelatin mass ratio was 1 : 6. At this mass ratio, both gelatin and alginate probably reacted maximum to form coacervation.
The different mass ratio (a), pH (b), polymer concentration (c), temperature (d) and reaction time (e) on supernatant viscosity and absorbance were shown.
The coacervation between gelatin and sodium alginate can occur only when the pH of the solution is lower than the isoelectric point of gelatin. Therefore, in this study, effect of variation of pH on polyelectrolyte complexation was carried out in the pH range 3.2–5.0 (Fig. 1b). The obvious change in relative viscosity and absorbance of the supernatant was found to appear at pH 4.0–5.0. This was due to that it was close to the isoelectric point of gelatin and decreased the ionization degree hence polyelectrolyte complexation. At pH 3.2, the absorbance was increasing and the polyelectrolyte complexation decreased, because of the molecular structure of alginate broke and protonated in acid condition.26)
Influence of Polymer Concentration on Complex CoacervationThe plot of absorbance and relative viscosity against polymer concentration was presented (Fig. 1c). Effect of polymer concentration in this study was varied from 0.5–3.0%, which is due to the excellent capacity of the two polymers for forming gels. In the range studied, polymer concentration had practically no effect on the viscosity and absorbance and hence on polyelectrolyte complex formation. The results indicated that the polyelectrolyte complexation in this study was completed.
Influence of Temperature on Complex CoacervationIn this study, effect of variation of temperature on polyelectrolyte complexation was carried out in the temperature range 20–60°C. The relative viscosity and absorbance of the supernatant were found to progressively decrease with the increase of temperature (Fig. 1d). This implied that polyelectrolyte complexation was more easily occur in higher temperature.
Influence of Reaction Time on Complex CoacervationThe effect of variation of reaction time on complex coacervation was plotted (Fig. 1e). The complexation between the two polymers was higher with the increase of reaction time. After 60 min, reaction time had practically no effect on the turbidity and absorbance. This indicated that the interaction between gelatin and sodium alginate tended to maximum.
Preparation of GVO MicrocapsulesEffect of pH Employed to Produce MicrocapsulesThe effect of variation of pH on the encapsulation efficiency and surface morphology of the microcapsules were shown (Figs. 2a, 3a). With increase of pH, the encapsulation efficiency increased initially, reached maximum and then decreased. Apparently, the shapes were more regular in pH 3.8 and pH 3.5 compared with others. Therefore microcapsules prepared in pH 3.5 could successfully improve the encapsulation efficiency.
The different pH (a), mass ratio (b), polymer concentration (c), core–wall ratio (d) on encapsulation efficiency of GVO microcapsules were shown.
SEM of GVO microcapsules prepared in pH 3.5 (a), mass ratio of 1 : 6 (b), polymer concentration of 1.0% (c), core–wall ratio 2 : 1 (d) were at 50 magnification. The e, f were core–wall ratio 2 : 1 at 100, 200 magnification, respectively.
The mass ratio of sodium alginate/gelatin of the mixing solution was varied from 1 : 4 to 1 : 8. The influence of variation of mass ratio on encapsulation efficiency and surface morphology were separately depicted (Figs. 2b, 3b). Encapsulation efficiency was found to increase initially and reach a maximum value and leveled off finally with the increase of the percentage of gelatin. For a higher gelatin concentration (1 : 8 and 1 : 7) more irregular aggregates were formed by capturing adjacent microspheres. Therefore, the mass ratio of 1 : 6 was chosen to prepare GVO microcapsules.
Effect of Polymer Concentration Employed to Produce MicrocapsulesPolymer concentration had strong impact on encapsulation efficiency and surface morphology of the microcapsules (Figs. 2c, 3c). The maximum encapsulation efficiency occurred when the polymer concentration was 1.0%. The encapsulation efficiency increased due to more and more polymers would be available to encapsulate the GVO and thereby encapsulation efficiency increased. When the polymer concentration was higher than 1.0%, the excess polymer would enhance the thickness of the microcapsule and then agglomerate. The dissolution of GVO decreased in determination of encapsulation efficiency. It was also observed from SEM photographs clearly. Therefore, the optimum polymer concentration was 1.0%.
Effect of Core–Wall Ratio Employed to Produce MicrocapsulesGVO microcapsules were prepared using four different amounts of core–wall ratio, i.e., 1 : 3, 1 : 2, 1 : 1 and 2 : 1, while all the other factors were kept constant. With the increase of core–wall ratio the maximum encapsulation efficiency occurred at the core–wall ratio of 1 : 1 (Fig. 2d). The increase in encapsulation efficiency could be due to more and more GVO available. It was found that the encapsulation efficiency decreased with the increase of core–wall ratio. This may be due to the presence of excess GVO, which could be observed from SEM photographs (Figs. 3d–f), especially core–wall ratio of 2 : 1.
GVO Release StudiesIn vitro, release rates of GVO was determined at pH 1.2 and 6.8 (Fig. 4). Initially, slower release rates were observed in both simulated fluid due to the diffusion through the swollen rubbery matrix. At longer time, drug release was much higher than the initial release by diffusing. There was a moderated burst effect in both cases, reached maximum and leveled off finally. Moreover, the release at pH=1.2 was less compared with that of at pH=6.8, which were 50.02 and 63.50%, respectively. It might be explained by considering the tendency of complexation and decomplexation between gelatin and sodium alginate at lower and higher pH.27)
Drug release study was carried out in simulated gastric fluid (0.1 N HCl, pH 1.2) and simulated intestinal fluid (phosphate buffer, pH 6.8) at 37°C.
Because GVO was sensitive to heat, thermal property study was carried out from 20 to 300°C (Fig. 5). The weight loss pattern of sodium alginate (Fig. 5a) was similar to gelatin’s (Fig. 5b). However, the weight loss of sodium alginate after decomposition was less than that of gelatin.28) GVO (Fig. 5d) showed higher weight loss compared with crossslinked microparticles (Fig. 5c) and GVO microcapsules (Fig. 5e). It resulted from GVO sensitivity to heat. The decomposition type and weight loss pattern of crosslinked microparticles were also similar to that of GVO microcapsules. And the crosslinked microparticles and GVO microcapsules exhibited higher thermal stability compared with gelatin, sodium alginate and GVO. The higher thermal stability of crosslinked microparticles might be due to the lower chance of elimination of small molecules like CO2 and CO with the formation of crosslinking, which acted as an infusible support and provided thermal resistance to the microcapsules.20,29,30)
The different profiles were presented sodium alginate (a), gelatin (b), gelatin–alginate complex coacervate (c), GVO (d), and GVO microcapsules (e), respectively.
Gelatin has positive charge at acidic pH due to presence of amino groups, while sodium alginate has free carboxyl group that imparts negative charge to these molecules. During complex coacervation carboxyl groups in polysaccharides such as sodium alginate interact with amino groups in protein such as gelatin to form amides. The formation of amide can be analyzed using FT-IR spectra.
The FT-IR spectra of gelatin, sodium alginate and the prepared microcapsules are depicted (Fig. 6). FT-IR spectrum of gelatin (Fig. 6a) revealed the presence of characteristic functional group of amino group at 3435 cm−1 (N–H stretching). In the spectrum of gelatin the other notable peaks were observed at 3078 cm−1 (C–H stretching of olefins), 2925 cm−1 (C–H stretching of alkanes) and 1649 cm−1 (amide-I, CO and CN stretching). Other peaks were observed at 1031 and 1334 cm−1 due to C–H bending and C–N stretching of amines respectively.31) The FT-IR spectrum (Fig. 6b) of sodium alginate showed notable peaks at 3452, 2166, and 1031 cm−1 for functional groups O–H, C–O and C–H, respectively. The following peaks were also observed at 1619, 1417 cm−1, which were assigned due to carboxylate salt C=O asymmetric stretching and symmetric stretching respectively.32) FT-IR spectrum of microcapsules (Fig. 6c) exhibited that the characteristic peak of amide appeared at 1633 cm−1, while that of gelatin was at 1649 cm−1. This indicated that the carboxyl group of sodium alginate interacted with the amino group of gelatin. The disappearance of peaks at 1334–1031 cm−1 confirmed the existence of electrostatic interaction between the polymers and the formation of coacervation between gelatin and sodium alginate.
The different profiles were presented gelatin (a), sodium alginate (b) and gelatin–alginate complex coacervate (c).
Gelatin and sodium alginate can form polyelectrolyte complex to encapsulate GVO. The results indicated that mass ratio, pH, temperature and reaction time had strong impact on complex coacervation. The optimum conditions of encapsulating GVO were as follows: sodium alginate to gelatin of 1 : 6, polymer concentration of 1.0%, pH of 3.5, and core material to wall of 1 : 1. The release of GVO depended on pH as for that the release of GVO was much higher in simulated intestinal fluid than in simulated gastric fluid. TGA study revealed that crosslinked microparticles improved the thermal stability of GVO. FT-IR study indicated the formation of polyelectrolyte complex between gelatin and sodium alginate.
This work was financially supported with funds provided by Hebei Province and School Science and Technology Cooperation Development Fund Project. The author wishes to thank the Modern Analysis Center for SEM, FT-IR analysis.
The authors declare no conflict of interest.
