Article pubs.acs.org/JAFC
Holy Basil (Ocimum sanctum Linn.) Essential Oil Delivery to Swine Gastrointestinal Tract Using Gelatin Microcapsules Coated with Aluminum Carboxymethyl Cellulose and Beeswax Pakamon Chitprasert* and Polin Sutaphanit Biotechnology of Biopolymers and Bioactive Compounds Special Research Unit, Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, 50 Ngamwongwan Road, Chatuchak, Bangkok, Thailand 10900 ABSTRACT: Holy basil essential oil (HBEO) can be applied as a feed additive; however, its benefits depend on the available amount in the gastrointestinal tract. In this study, the physicochemical properties, including the release properties of three different microcapsules, HBEO-loaded gelatin microcapsules (UC), UC coated with aluminum carboxymethyl cellulose (CC), and UC coated with aluminum carboxymethyl cellulose−beeswax composite (CB), were compared. The encapsulation efficiency, HBEO content, and 2,2-diphenyl-2-picrylhydrazyl radical scavenging activity for the microcapsules were 95.4 ± 0.17%, 66.7− 67.7%, and 94.3−96.5%, respectively. Scanning electron microscopy and confocal laser scanning microscopy (CLSM) revealed nonuniform HBEO distributions in honeycomb-like networks in the microcapsules. An X-ray diffraction analysis determined that UC and CC microcapsules were amorphous, but CB microcapsules were semicrystalline. UV−vis spectrophotometer and CLSM analyses results determined that HBEO was released from CC and CB microcapsules in greater amounts than from UC microcapsules in simulated intestinal fluid. Therefore, the HBEO amount reaching the intestine can be controlled using the optimal encapsulation system. KEYWORDS: holy basil essential oil, gastrointestinal tract, microcapsule, carboxymethyl cellulose, beeswax
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INTRODUCTION Essential oils have been considered to be a potential alternative to antibiotic growth promoters (AGP) because they can act as antimicrobials, antioxidants, anti-inflammatories, and coccidiostatics. Essential oils can also serve as palatability, digestive, and immune enhancers.1−4 Without antibiotics, the routine use of essential oils can support gut health by reducing the levels of pathogenic bacteria, resulting in better animal performance.5 However, there is some evidence suggesting that essential oils produce inconsistent health-promoting effects presumably due to the small quantity available at target sites, particularly the ileum, cecum, and colon.6,7 Because essential oils can increase gastric retention time and improve protein digestion,6 the partial release of essential oils from microcapsules in the stomach is required. However, essential oils are lost during the passage through the gastrointestinal tract due to absorption in the stomach and the proximal small intestine.8 The remaining level of essential oils is less than minimal inhibitory concentrations or minimal bactericidal concentrations when they arrive at the distal parts of the intestine.9 Microencapsulation using a coacervation method may be a useful tool to achieve a controlled release of essential oils.10−14 Gelatin is a protein extracted from collagen. It contains large amounts of glycine arranged every third residue, proline, and 4hydroxyproline residues. It is considered an appropriate encapsulant for gastric release.15 However, gelatin microcapsules can undergo rapid dissolution and disintegration in the gastric fluids via the protease enzyme pepsin.16,17 Therefore, surface modification of the gelatin microcapsule by coating with materials possessing pepsin- and acid-resistant properties may be beneficial for the delayed release of essential oils in the © XXXX American Chemical Society
stomach, which would prolong contact between the essential oils and pathogenic bacteria in the intestine. Sodium carboxymethyl cellulose (SCMC) is a potential coating material for this purpose because it is gastric acid resistant and intestinally soluble. SCMC is a water-soluble cellulose-ether derivative consisting of β-linked glucopyranose residues with various levels of carboxymethyl substitution. Because SCMC is negatively charged in aqueous solutions due to its anionic carboxyl groups (pKa = 4.3) and gelatin is positively charged below its isoelectric point, gelatin microcapsules with SCMC coatings are achievable via electrostatic attractions.18 SCMC alone has been used for colon-targeted drug delivery.19 Due to the intrinsically hydrophilic nature of SCMC, cross-linking with multivalent cations was necessary to increase its hydrophobicity, resulting in delayed drug release.20 Another possible approach for increasing SCMC hydrophobicity is the fabrication of a composite material containing lipids. Several studies have reported improved water vapor transmission resistance in edible SCMC-based films by incorporating lipids.21−23 Among the lipidic materials, waxes, such as beeswax, carnauba wax, and microcrystalline wax, are the most favorable because they are primarily composed of long-chain fatty alcohols and alkanes, resulting in a strongly hydrophobic material.24 To the best of our knowledge, there have been no reports detailing the application of SCMC−wax composites for hydrophilic microcapsules coating. Wax alone is also commonly used as a release retardant in sustained release Received: April 24, 2014 Revised: November 2, 2014 Accepted: November 9, 2014
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HBEO.29 Briefly, the microcapsules (80 mg) were dissolved in dichloromethane to a final volume of 1 mL, and their concentration was calculated using the calibration curve shown in eq 1 (R2 = 0.998)
microsphere design due to its good stability at various pH and moisture levels.25,26 Drug-loaded beeswax microspheres demonstrated the absence of drug release at pH 1.2, proving the suitability for its use as a gastric-resistant material.27 The present research investigated the role of SCMC crosslinked with aluminum ions (AlCMC) and AlCMC−beeswax composites as the coating materials on the gelatin microcapsules for controlling essential oil release in simulated swine gastrointestinal fluid. Holy basil essential oil (HBEO) from Ocimum sanctum Linn., which has received significant attention as a natural substitute for AGP,28 was used in this study. It contains several secondary metabolites with the major components being 42.6% methyleugenol, 26.9% β-caryophyllene, 10.7% eugenol, and 6.00% β-elemene.29 The physicochemical properties of the microcapsules that were relevant to HBEO release and HBEO release kinetics were also examined.
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y = 1.8082x + 0.0662
(1)
where y is the HBEO concentration and x is the absorbance. Then, the encapsulation efficiency was calculated according to eq 2
encapsulation efficiency = (WO/ WI) × 100
(2)
where WO is the mass of HBEO in the microcapsule and WI is the initial mass of HBEO added for the encapsulation. The HBEO content was determined using eq 3
HBEO content = (WO/ WM) × 100
(3)
where WM is the mass of the microcapsule. Determination of Antioxidant Activity. Antioxidant activity was estimated using the 2,2-diphenyl-2-picrylhydrazyl (DPPH) radical assay as previously described with minor modifications.30 The microcapsule solution (1 mL) in dichloromethane was mixed with 1 mL of 0.2 mM DPPH radical solution in methanol. The mixture was then shaken vigorously and left to stand for 20 min in the dark. The decrease in absorbance was measured at 517 nm against a blank sample. All samples were analyzed in triplicate. The DPPH radical scavenging activity was calculated according to eq 4
MATERIALS AND METHODS
Chemicals. HBEO was supplied by Thai-China Flavours and Fragrances Industry Co., Ltd. (Phra Nakhon Si Ayutthaya, Thailand). Gelatin from porcine skin (300 Bloom, type A) and NaCMC (medium viscosity) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium dodecyl sulfate (SDS) and Tween 80 were obtained from Ajak Finechem (NSW, Australia). Glutaraldehyde was provided by Merck (Darmstadt, Germany). Stearic acid was obtained from Laboratory Reagents & Fine Chemicals (Mumbai, India). White beeswax (pharmaceutical grade) was purchased from Paramelt BV (Heerhugowaard, The Netherlands). Porcine bile extract, pancreatin, and lipase from porcine pancreas and pepsin from porcine gastric mucosa were purchased from Sigma (Hampshire, UK). All chemicals were of analytical grade. Preparation of HBEO-Loaded Gelatin Microcapsules. HBEOloaded gelatin microcapsules were prepared using simple coacervation via glutaraldehyde cross-linking.29 HBEO was dispersed into a gelatin solution (11.75% (w/v) gelatin and 0.1% (w/v) SDS) with stirring at 450 rpm and 40 °C for 25 min. Then, sodium sulfate solution (20% w/v) was added dropwise with continuous stirring at 550 rpm for phase separation. The temperature was subsequently decreased to 5 °C. Next, a glutaraldehyde solution consisting of 25% (v/v) glutaraldehyde, acetone, and distilled water was introduced into the mixture. This was followed by increasing the temperature to 40 °C with continuous stirring for 60 min. The microcapsules were washed twice with 0.3% (v/v) Tween 80 and were filtered through an 11 μm filtration Whatman membrane under vacuum. They were collected and dried at 60 °C until a constant dry weight was obtained. The finished microcapsules were stored in a hermetically sealed glass bottle at room temperature. Coating of HBEO-Loaded Gelatin Microcapsules with AlCMC and AlCMC−Beeswax Composites. The HBEO-loaded gelatin microcapsules (UC) were coated with AlCMC or AlCMC−beeswax (1:1, w/w) composites, respectively named CC and CB. For the CC microcapsules, 20 g of UC was added into 200 mL of 1% (w/v) NaCMC solution, which contained 1% (w/v) glycerol as a plasticizer and 1% (w/v) stearic acid as an emulsifier. The mixture was shaken at 100 rpm and 30 °C. This was followed by the addition of 200 mL of 0.05 M aluminum chloride solution as a cross-linking agent with continuous stirring. To prepare the composite coating, the NaCMC solution was mixed with melted beeswax with stirring at 1200 rpm. Next, 20 g of UC was transferred into 200 mL of the composite, and the mixture was shaken at 100 rpm and 30 °C. Finally, to obtain CB microcapsules, NaCMC cross-linking was performed as previously described. The CC and CB microcapsules were filtered under vacuum and were stored at 4 °C until further analysis. Determination of HBEO Content and Encapsulation Efficiency. The HBEO content was determined using a Thermo Spectronic Heλios γ UV−vis spectrophotometer (Cambridge, UK) at 289 nm, which is the wavelength of maximum absorbance of eugenol, the most powerful antioxidant and antimicrobial component of
DPPH radical scavenging activity = [A O − (AM − A S)]/A O × 100
(4)
where AO is the absorbance of the control, AM is the absorbance of the DPPH solution mixed with the samples, and AS is the absorbance of the samples without DPPH. Particle Size Analysis. The area-volume mean diameter (D[4,3]) was measured using a Mastersizer 2000 particle size analyzer (Malvern, Worcestershire, UK) equipped with a Hydro 2000 MU (A) wet dispersion unit and 0.3% Tween 80 as the dispersion medium. Scanning Electron Microscopy (SEM) Studies. The microcapsules were fixed using 2% (v/v) osmium tetroxide for 1 h and were rinsed twice with distilled water. Then, they were dehydrated using a graded ethanol series (30, 50, 70, 95, and 100% (v/v)) three times for 10 min. Preparation of a microcapsule cross section was performed by cutting the sample with a razor blade. The samples were consecutively dried using a critical point dryer and were fixed on an aluminum stub using double-sided adhesive tape. This was followed by gold coating using an ion sputtering coater (IB2, Giko Engineering, Japan). The microcapsule surface morphology was characterized using SEM (JSM5600, JEOL, Tokyo, Japan) at an accelerating voltage of 15 kV. Confocal Laser Scanning Microscopy (CLSM) Studies. Fluorescence imaging with CLSM (LSM 5 Pascal, Carl Zeiss GmbH, Jena, Germany) was used to visualize the HBEO distribution in gelatin. HBEO was labeled with Nile blue dye prior to encapsulation, and the microcapsules were stained with Rhodamine B dye to localize gelatin. All fluorescence pictures were obtained using a ×10 objective (Plan-Neofluar, Carl Zeiss) and a 0.3 numerical aperture. Confocal illumination of HBEO and gelatin was provided using a multiline argon laser (488 nm excitation) and a helium/neon laser (543 nm excitation), respectively. Fourier Transform Infrared Spectroscopy (FTIR) Studies. The FTIR spectra of UC, CC, CB, gelatin, beeswax, and HBEO were recorded using a TENSOR series FTIR spectrometer (Bruker Optics, Ettlingen, Germany) in ATR mode. The samples were scanned in transmission mode from 4000 to 500 cm−1 with a resolution of 4 cm−1. X-ray Diffraction (XRD) Studies. The crystal structures of UC, CC, CB, and beeswax were determined using an XRD diffractometer (Philips model X’Pert MPD, Almelo, The Netherlands) equipped with Cu Kα radiation (wavelength 1.5406 Å, 40 kV, and 30 mA). The samples were scanned along a 2θ range of 5−50° at a rate of 2.4°/min. Differential Scanning Calorimetry (DSC) Studies. The thermal properties of UC, CC, CB, the physical mixture of HBEO and gelatin powder, and beeswax as well as HBEO were determined using a DSCB
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1 STARe System (Mettler Toledo, Switzerland) calibrated with indium. Accurately weighed samples (5 mg) were placed in a 40 μL aluminum pan and heated at a programmed rate of 10 °C/min in the temperature range from −80 to 300 °C. HBEO Release under Simulated Swine Gastrointestinal Conditions Experiments. HBEO release studies were performed using a previously published method with slight modifications.31 One percent (w/v) microcapsules were added into simulated gastric fluids (SGF) consisting of 3 g/L NaCl, 1.1 g/L KCl, 0.15 g/L CaCl2, 0.6 g/L NaHCO3, 3 g/L SDS, 0.21 g/L pepsin, and 0.25 g/L lipase. The pH was adjusted to 6.0, 3.5, 3.0, 2.5, and 2.0 at 0, 5, 30, 120, and 180 min, respectively, using 1 M HCl. The experiment was performed at 39 ± 0.5 °C with a stirring speed of 100 rpm. The absorbance of the sample at 289 nm was measured at 0, 5, 10, 20, 30, 40, 60, 80, 100, 120, 180, 210, and 240 min, and the electrolytes were replaced with fresh solution. CLSM observations were performed to verify HBEO diffusion after 10 min of incubation in SGF. After 240 min, 1 mL of trypsin solution (2 mg/mL), 14 mL of bile solution (40 mg/mL), 7.5 mL of pancreatic solution, and 7.5 mL of small intestinal electrolyte solution (0.6 g/L KCl, 5.0 g/L NaCl, 0.23 g/L CaCl2·2H2O, and 3 g/L SDS) were mixed with SGF. Then, the pH of the simulated intestinal fluid (SIF) was increased to 5 and 6.5 at 240 and 300 min, respectively, by adding 1 M NaHCO3. A 1 mL sample was collected from the medium to measure absorbance, and the electrolytes were replaced with fresh SIF at predetermined time intervals for 4 h. The HBEO in vitro release data were analyzed to describe the release kinetics by fitting the data into various release kinetic models. The zero-order model describes a system in which the release rate is independent of concentration (eq 5). The first-order model describes a system in which the release rate is concentration dependent (eq 6). The Higuchi model (eq 7) describes the release from an insoluble matrix system as the square root of a time-dependent process based on Fickian diffusion. The Hixson and Crowell model (eq 8) describes the release by dissolution with a change in the surface area and the diameter of a particle. The Korsmeyer−Peppas model (eq 9) describes the release from a polymeric system. The kinetic model that best fits the release data was selected on the basis of the correlation coefficient (R2) value for each model Q = K 0t
port). Values above 0.85 indicate case II transport relating to polymer relaxation during gel swelling. Statistical Analysis. A completely randomized design was used in this experiment. The statistical analysis was performed using one-way ANOVA. The experiments were performed in triplicate. The data are presented as the mean value ± standard deviation. Statistical significance was evaluated using Duncan’s multiple-range test at the 95% confidence level.
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RESULTS AND DISCUSSION HBEO was encapsulated in gelatin using simple coacervation and was cross-linked with glutaraldehyde to serve as a health enhancers for swine-raising. To delay HBEO release in the gastrointestinal tract, the microcapsule was successively coated with AlCMC or AlCMC−beeswax composites. Due to the acid resistance of AlCMC and the highly hydrophobic nature of beeswax, a delayed HBEO release was possible. The AlCMC layer was formed on the surface of the microcapsule by dispersing the microcapsules in a SCMC solution. Complex coacervation between SCMC and gelatin occurred due to the electrostatic attraction between the negatively charged SCMC carboxyl group and the positively charged gelatin amide group.32 In addition to electrostatic attraction, the interaction between SCMC and gelatin was partially driven by hydrophobic interactions as well as hydrogen bond and van der Waals forces.33 To produce a stable layer in the water-insoluble AlCMC gel, SCMC was consecutively cross-linked with aluminum ions. For the AlCMC−beeswax composite coating, a homogeneous dispersion emulsification was performed comprising beeswax in SCMC. Then, the microcapsules were added into the emulsion and were subsequently cross-linked with AlCl3. The preparation of the emulsion composite film using the emulsification technique is simple and feasible. Additionally, the obtained film had adequate structural cohesion due to the long-chain polymer and hydrorepellency imparted by the lipids.22 The physicochemical properties of the microcapsules were characterized in subsequent experiments. Encapsulation Efficiency, HBEO Content, and Antioxidant Activity. The encapsulation efficiency of all samples was calculated on the basis of the HBEO weight in the microcapsule and the HBEO weight used initially. The encapsulation efficiency was found to be 95.4 ± 0.17%, which suggested that HBEO was effectively encapsulated using our simple coacervation procedure. UC, CC, and CB microcapsules contained 67.7 ± 0.16, 67.1 ± 0.13, and 66.7 ± 0.26% HBEO and exhibited 96.5 ± 0.20, 95.8 ± 0.18, and 94.3 ± 0.27% DPPH radical scavenging activity, respectively, which were significantly different for both values (p ≤ 0.05). The radical scavenging activities of the CC and CB microcapsules were slightly weaker than that of the UC microcapsules. This result may be attributed to the increased weight of the microcapsule and HBEO loss after coating. However, with regard to their high antioxidant activity, all microcapsule samples could be potentially useful as feed additives. Size and Surface Morphology. The microcapsule coating was also expected to have an influence on the size and surface morphology of the microcapsules. The volume moment mean diameter of the UC microcapsules was 392 μm, which was slightly smaller than those of the CC and CB microcapsules (407 and 414 μm, respectively). This increase in size indicates the presence of the coating layer on the outer surface of the microcapsule. In addition, the incorporation of beeswax, a long
(5)
where Q is the amount of HBEO released in time t and K0 is the zeroorder release constant. To study the release kinetics, the release data were plotted as a cumulative percentage of HBEO release versus time
log C = log C0 − (K /2.303)t
(6)
where K is the first-order rate constant, C0 is the initial concentration of HBEO, and C is the concentration of HBEO at time t. The HBEO release data were plotted as the log cumulative percentage of the HBEO remaining versus time Q = KHt 1/2
(7)
where KH is the Higuchi dissolution constant. The HBEO release data were plotted as the cumulative percentage of HBEO release and the square root of time
W01/3 − Wt1/3 = KHCt
(8)
where Wt is the amount of HBEO released in time t, W0 is the initial amount of HBEO in the microcapsule, and KHC is the rate constant. The HBEO release data were plotted using the cube root of the HBEO percentage remaining in a matrix and time
M t /M∞ = kt n
(9)
where Mt/M∞ is the fraction of the HBEO released at time t, k is the release rate constant, and n is the release exponent. The calculations of k and n were measured up to the initial 60% release of HBEO. The value of n characterizes the release mechanism of the HBEO. For spheres, values of n lower than 0.45 indicate a diffusion-controlled system, and values of n between 0.45 and 0.85 indicate both diffusioncontrolled release and swelling-controlled release (anomalous transC
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Figure 1. SEM images of UC, CC, and CB microcapsules: (a) whole UC; (b) whole CC; (c) whole CB; (d) external surfaces of UC; (e) external surfaces of CC; (f) external surfaces of CB; (g) inner edges of UC; (h) inner edges of CC; (i) inner edges of CB.
carbon chain molecule of C16−C9534 in the AlCMC matrix results in an increase in the coating thickness. SEM was used to characterize the internal and external surface morphology of the microcapsules. The UC microcapsules were almost spherical in shape (Figure 1a). After coating, the CC (Figure 1b) and CB (Figure 1c) microcapsules were more spherical. At high magnification, the UC microcapsule images revealed the highly porous sponge-like structure of the external surface (Figure 1d), and those of the CC (Figure 1e) and CB (Figure 1f) microcapsules were denser. The beeswax was markedly dispersed in the AlCMC matrix, resulting in a bumpy appearance. The cross-section view image showed a highly porous honeycomb-like internal morphology of the gelatin matrix (Figure 1g) as previously reported.35,36 A large number of micropores on the multiwalls of the microcapsule were also observed. The cross-section images of the CC (Figure 1h) and CB (Figure 1i) microcapsules showed the compact outer coating layer covering the honeycomb-like inner microcore and, with the presence of beeswax, the coating layer appeared to be thicker. FTIR Spectra. FTIR was used to confirm the successful coating and to reveal the interactions between each component that are possibly associated with the oil release properties. All of the characteristic peaks of gelatin (Figure 2a) and HBEO (Figure 2b) appeared in the UC microcapsule spectrum (Figure 2c). The NH stretching vibrations of the amide groups, representing the amide A, appeared at 3351 cm−1. The carbonyl CO stretching vibrations, with minor contributions from the out-of-phase CN stretching vibrations of the amide I, occurred at 1638 cm−1. The out-of-phase combination of the NH in-plane bending and the CN stretching vibrations of the amide II were at 1568 and 1455 cm−1. The peak at 1235 cm−1 belonged to the in-phase combination of the NH bending and the CN stretching vibrations of amide III. The characteristic peaks of the main components of HBEO, methyleugenol, eugenol, and caryophyllene, were also observed
Figure 2. FTIR spectra of (a) gelatin, (b) HBEO, (c) UC, (d) NaCMC solution mixed with glycerol, (e) CC, (f) beeswax, and (g) CB.
in the UC microcapsule spectrum. The asymmetric and symmetric CH stretching vibrations in −CH2− were observed at 2924 and 2852 cm−1. The peaks in the region of 1638−1513 cm−1 were assigned to the alkene/aromatic −C C− stretching vibrations. The CH2 scissor bends occurred at 1463 cm−1. The asymmetric and symmetric CH bending vibrations in −CH3 were observed at 1452 and 1368 cm−1. There was also C−H bending of the alkene/aromatic groups in the range of 994−554 cm−1. A comparison of the HBEO spectrum with the microcapsule spectrum revealed the chemical D
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stability of the encapsulated HBEO; therefore, its antioxidant and antimicrobial properties should remain intact. After coating the UC microcapsules with AlCMC, the presence of AlCMC was evaluated by comparing the FTIR spectra of UC (Figure 2c) and SCMC (Figure 2d) with CC (Figure 2e). The SCMC spectrum had a peak at 3328 cm−1 that was assigned to O−H stretching vibrations. The asymmetric and symmetric stretching vibrations of the aliphatic C−H were observed at 2916 and 2849 cm−1, respectively. The peak at 1638 cm−1 was attributed to the absorbed water molecules in cellulose. The bands at 1175, 1099, and 718 cm−1 indicated the C−O−C (glycosidic linkage) stretching vibrations. As the characteristic peaks of SCMC were observed in the spectrum of the CC microcapsules, successful coating was achieved. The beeswax incorporation into the AlCMC matrix as the coating material for the microcapsule was confirmed using FTIR. The beeswax FTIR spectrum (Figure 2f) had the characteristic peaks of its primary components, which consisted of a mixture of esters of fatty acids and fatty alcohols, longchain hydrocarbons, and free fatty acids. OH stretching was observed at 3244 cm−1. The peak at 2955 cm−1 was assigned to asymmetric CH stretching vibrations. The peaks at 2915 and 2848 cm−1 corresponded to the asymmetric and symmetric CH 2 stretching vibrations of the fatty acid chains, respectively. The CO stretching vibrations of the ester were observed at 1741 cm−1. The symmetric CH2 scissoring vibrations appeared at 1463 cm−1. The peak at 1378 cm−1 indicated the symmetric CH3 bending vibrations. The band at 1175 cm−1 was assigned to the CH2 wagging vibrations. The peak at 1099 cm−1 was due to the symmetric CC stretching vibrations. The band at 720 cm−1 represented the CH2 rocking vibrations of the long-chain hydrocarbons. The CB microcapsule spectrum contained most of the characteristic peaks of UC, AlCMC, and beeswax, indicating the successful coating of AlCMC−beeswax composites on the surfaces of the gelatin microcapsules. However, when compared with the CC spectrum, a significant decrease in the intensities of the aprroximately 3351 and 1638 cm−1 peaks was observed, indicating that beeswax may interfere with the electrostatic attractions between the Al3+ and COO− of SCMC. XRD Analysis. XRD was used to evaluate the effect of AlCMC and beeswax on the structure of the microcapsule. The UC XRD pattern revealed an amorphous structure due to the amorphous nature of HBEO and the β-sheet conformation of gelatin (Figure 3a). In addition, an examination of the amorphous pattern also possibly indicated that amorphous HBEO is molecularly dispersed in the amorphous gelatin matrix. For the CC microcapsules, a flat and broad contour was observed (Figure 3b), suggesting that the presence of AlCMC decreased the crystallinity of the gelatin microcapsule. This result is attributed to the significant hydrogen bonding interactions between AlCMC and gelatin. With the incorporation of beeswax, the CB microcapsules had a semicrystalline structure composed of both crystals and amorphous structures (Figure 3c). The crystalline nature of beeswax was detected at 2θ = 21° and 23° (Figure 3d) as previously observed.34,37 Therefore, the XRD pattern demonstrates the successful incorporation of beeswax in the AlCMC layer. As a result of the semicrystalline structure of the AlCMC−beeswax composite, the release characteristics of the CB microcapsules may be different from those of UC and CC, which requires further investigation.
Figure 3. XRD patterns of (a) UC, (b) CC, (c) CB, and (d) beeswax.
DSC Analysis. DSC was used to monitor the physical state and thermal behavior of the samples. When HBEO was physically mixed with gelatin, its thermal analysis indicated three endothermic phase changes at 121, 220, and 251 °C, signifying the gelatin melting, HBEO vaporization, and gelatin degradation, respectively (Figure 4a). The characteristic
Figure 4. DSC thermograms of (a) physical mixtures of HBEO and gelatin powder, (b) UC, (c) CC, and (d) CB.
thermogram of the UC microcapsules (Figure 4b) was noticeably different from that of the physical mixture. The first absorption peak was around −1 °C, attributed to the melting endotherm of HBEO. The next sharp endothermic peak at 143 °C was associated with the melting of the microcapsule. The shift of the melting peak toward a higher temperature (from 121 to 143 °C) indicated the effect of crosslinking on the physical properties of the gelatin. The final phase transition showed the decomposition of the microcapsule. However, the DSC profile of the microcapsule did not clearly show the HBEO vaporization peak. This result suggests that HBEO is highly dispersed in the amorphous gelatin. Another possibility for this result is that HBEO vaporization occurs simultaneously with the decomposition of the microcapsule in that temperature range. In addition, the lack of any visible E
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the CC microcapsules is slower. The slowest HBEO release rate was observed for the CB microcapsules. Because beeswax is highly hydrophobic, it has a low affinity for water. This potentially slows HBEO diffusion out of the microcapsules. In addition, the dispersed beeswax particle in the AlCMC matrix increases the diffusional path length of the HBEO, ions, and water, consequently hindering their transport across the composite layer as illustrated in Figure 6.
exothermic peak confirmed that there was no chemical interaction between HBEO and gelatin. A comparison of the absorbed energy during the decomposition process between the microcapsule and the physical mixture also revealed the effect of cross-linking. The DSC thermogram of the CC microcapsules (Figure 4c) was also composed of three endothermic events. The first peak, around −1 °C, corresponded to the melting temperature of HBEO. The sharp peak at 148 °C and the broad peak in the 250−280 °C temperature range represented the melting and decomposition of the microcapsule, respectively. The CC melting temperature (148 °C) was slightly higher than that of the UC microcapsules (143 °C), indicating the interaction between AlCMC and gelatin. Therefore, the DSC thermograms confirmed the FTIR spectrum data, demonstrating the presence of the intermolecular hydrogen bonds between AlCMC and gelatin. The presence of beeswax in the AlCMC network and its effect on the thermal behavior of the microcapsule was also determined. The DSC thermogram of the CB microcapsules (Figure 4d) showed four endothermic peaks with one peak at 60 °C, representing the beeswax melting peak.37 Moreover, the broadening of the melting peak and the decline in the melting temperature and melting enthalpy compared with the CC thermogram can be attributed to the colloidal beeswax due to its large surface-to-volume ratio properties. Release Characteristics of Microencapsulated HBEO in SGF. The previous physicochemical analysis suggested a possible role for the microcapsule in controlling HBEO release. Therefore, the final study examining in vitro release was conducted. All of the microcapsules had a two-step biphasic HBEO release that was characterized by an initial burst, followed by a sustained release (Figure 5). Initially, the UC
Figure 6. Schematic diagrams of SGF traversing (a) AlCMC and (b) AlCMC−beeswax composite coated on HBEO-loaded gelatin microcapsules.
Under simulated intestinal conditions, all of the microcapsules demonstrated sustained release. The physical and chemical disintegration of the UC microcapsules continuously occurred due to microcapsule swelling and gelatin hydrolysis by pancreatin and trypsin. AlCMC only experienced the physical disintegration induced by the polymer chain relaxation and swelling. In the CB microcapsules, the beeswax cannot retain its structural integrity because its ester bond is cleaved by the lipase found in pancreatin.42 The experimental results determined that the coating layer, particularly the AlCMC−beeswax composite, decreased the initial HBEO burst release in the SGF. The AlCMC−beeswax composite enhanced HBEO stability against acid denaturation and preserved antioxidant activity, which prevents free radicalinduced damage and suppresses oxidative stress. The cumulative amounts of HBEO released in the SGF were 77.9, 74.7, and 70.5%, and the amounts released in the SIF were 11.6, 15.8, and 21.1% for the UC, CC, and CB microcapsules, respectively. Because antimicrobial activity depends on concentration, the CB microcapsules should exhibit the strongest activity in the intestine. In our in vitro studies, HBEO demonstrated high antimicrobial activity against pathogenic bacteria found in the intestine of swine, Salmonella Typhimurium, Escherichia coli O157, and Staphylococcus aureus (unpublished data). In our experiment, all types of microcapsules, particularly CB, released HBEO at concentrations greater than its minimal inhibitory concentration (the lowest concentration of an antimicrobial that can inhibit the growth of a microorganism) in the intestine. Therefore, HBEO encapsulation is a method for achieving HBEO release at desirable sites in the intestinal tract, rather than being almost completely absorbed in the stomach and the proximal intestine within 2 h after oral administration.8 To understand the mode of HBEO release and determine a suitable release kinetic model, the HBEO release data were fitted to various kinetic models. The model that provided the highest correlation coefficient (R2) value was considered the optimal model for describing the release kinetic data. The results indicated that the in vitro HBEO release kinetics were best described by the Korsmeyer−Peppas model for all microcapsule preparations (Table 1). The HBEO release mechanism for the UC microcapsules followed the anomalous transport mechanism, which is controlled by diffusion and
Figure 5. HBEO release profiles of UC, CC, and CB microcapsules in simulated swine gastrointestinal fluids.
microcapsules exhibited the highest burst release, followed by the CC and CB microcapsules, respectively. For example, within the first hour of incubation, the UC microcapsules released 69% of HBEO compared with 61% for the CC microcapsules and 50% for the CB microcapsules. The SGF penetration into the microcapsule leads to chain relaxation, microcapsule swelling, and finally HBEO release. Moreover, pepsin degrades the amide linkage in the gelatin chain,38−40 resulting in microcapsule erosion and burst release. The HBEO release rate decreased after coating with AlCMC, which may be because AlCMC has a lower quantity of polar groups per monomeric unit.41 As a result, the passage of the SGF through F
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nonhomogeneous distribution of large amounts of HBEO throughout the gelatin matrix in the UC microcapsules before incubation. The difference in fluorescence intensity was attributed to the difference in the HBEO amounts, which are directly related to the gelatin pore sizes. CLSM detected the gelatin matrix (Figure 7b), but it was not able to provide information on the shape of the gelatin pores. Nevertheless, the image shown in Figure 7c confirmed the multicore structure of the microcapsule with distinct gelatin walls, as previously observed by SEM. In addition, HBEO was almost entirely entrapped inside the microcapsule; nonencapsulated oil was barely detected on the outer surface. The information obtained in this study can be used to indicate the protective ability of microcapsules and HBEO stability during SGF exposure. Panels d−f of Figure 7 show that HBEO was released from the UC microcapsules after incubation in the SGF for 10 min at pH 3 due to the degradation of the gelatin network by pepsin. Conversely, large amounts of HBEO were retained on the edge of the CC (Figure 7g−i) and CB microcapsules (Figure 7j−l); HBEO was not able to traverse the coated layers. These results confirmed that higher amounts of HBEO were released from the UC microcapsules. In summary, microcapsules for the controlled release of HBEO in swine gastrointestinal fluid were developed. The analysis of physicochemical properties confirmed the successful coating of AlCMC and AlCMC−beeswax composite on the gelatin microcapsule. The coating material, particularly the AlCMC−beeswax composite, inhibited HBEO burst release in the SGF and promoted HBEO release in the SIF of swine over an extended period of time. Therefore, the encapsulation method proposed in the present study is an effective process for improving HBEO efficacy for pathogen reduction in the distal region of the intestine.
Table 1. Correlation Coefficients, Rate Constants, and Release Exponents for Different HBEO Release Kinetic Models for UC, CC, and CB Microcapsules microcapsule formulations release kinetic model
UC
CC
CB
zero order
R2 K0
0.84 0.07
0.80 0.09
0.84 0.10
first order
R2 K
0.95 0.002
0.94 0.002
0.86 0.002
Higuchi
R2 KH
0.92 1.72
0.92 2.34
0.95 2.72
Hixson−Croswell
R2 KHC
0.90 0.002
0.90 0.002
0.89 0.002
Korsmeyer−Peppas
R2 k n
0.97 3.52 0.51
0.97 3.64 0.30
0.97 3.30 0.30
swelling. These results confirmed the initial HBEO burst release from the gelatin. However, the HBEO release mechanism of the CC and CB microcapsules showed Fickian diffusion, indicating a diffusion-controlled release system due to the chemical potential gradient. As HBEO diffused through the water-filled pores, because CC and CB were denser than UC, the diffusion rate of HBEO from the coated microcapsules was lower. Stability of Microencapsulated HBEO during SGF Exposure. CLSM was used to visualize and identify the location of HBEO in the microcapsule before and after exposure to SGF for 10 min. Figure 7a represents the
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AUTHOR INFORMATION
Corresponding Author
*(P.C.) Phone: 66-02-562-5085. Fax: 66-02579-4096. E-mail:
[email protected]. Funding
This research was cofunded by the Thailand Research FundMaster Research grants (TRF-MAG), No. MRGWI535S019, and Better Pharma Co., Ltd., Thailand. Notes
The authors declare no competing financial interest.
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REFERENCES
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Figure 7. CLSM images of microcapsules before and after exposure to SGF: (a−c) UC before exposure; (d−f) UC after exposure; (g−i) CC after exposure; (j−l) CB after exposure. HBEO was colored with Nile blue dye (a, d, g, and j). Gelatin was colored with Rhodamine B dye (b, e, h, and k). Red and green represent gelatin and HBEO, respectively (c, f, i, and l). G
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