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Microencapsulation of brucea javanica oil: Characterization, stability and optimization of spray drying conditions
Accepted Manuscript Microencapsulation of brucea javanica oil: Characterization, stability and optimization of spray drying conditions Liandong Hu, Jianli Zhang, Qiaofeng Hu, Na Gao, Shaocheng Wang, Yongbing Sun, Xiaoning Yang PII:
S1773-2247(16)30214-3
DOI:
10.1016/j.jddst.2016.09.008
Reference:
JDDST 244
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 26 May 2016 Revised Date:
2 September 2016
Accepted Date: 25 September 2016
Please cite this article as: L. Hu, J. Zhang, Q. Hu, N. Gao, S. Wang, Y. Sun, X. Yang, Microencapsulation of brucea javanica oil: Characterization, stability and optimization of spray drying conditions, Journal of Drug Delivery Science and Technology (2016), doi: 10.1016/j.jddst.2016.09.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Microencapsulation of brucea javanica oil: Characterization, stability and optimization of spray drying conditions
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Liandong Hu1, *, Jianli Zhang1, Qiaofeng Hu1, Na Gao 1, Shaocheng Wang1, Yongbing Sun 2, Xiaoning Yang3
School of Pharmaceutical Sciences & Key Laboratory of Pharmaceutical Quality Control of
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Hebei Province, Hebei University. No.180, WuSi Road, Baoding, 071002
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Jiangxi University of Traditional Chinese Medicine, Nanchang 330006, China
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PR China
Tianjin Hemay Bio-Tech Co.,Ltd, Tianjin, PR China
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*Correspondence to Author: Liandong Hu
School of Pharmaceutical Sciences & Key Laboratory of Pharmaceutical Quality Control of
[email protected] (L. Hu)
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Email:
PR China
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Hebei Province, Hebei University. No.180, WuSi Road, Baoding, 071002
Tel: +86 312-5971107
Keywords
Arabic gum, Gelatin, Brucea javanica oil, Complex coacervation, Microencapsulation, Spray drying.
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ACCEPTED MANUSCRIPT Abstract
The aim of this work was to obtain the optimized formulation for brucea javanica oil (BJO) microencapsulation by spray drying with high yield and evaluate their resistance to oxidation
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through the stability test. The parameters of spray drying such as inlet air temperature, feed flow rate and drying air flow speed were optimized using a Box–Behnken design. Moisture content and process yield were analyzed as responses. The spray-dried microcapsules were
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characterized by SEM, DSC, FT-IR and XRD. At the optimal conditions, the encapsulation
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efficiency (EE) reached a maximum of 82.9% (w/w) and loading capacity (LC) reached 10.56% (w/w). The study of oxidative stability showed that microcapsules were stable against oxidation. The methyl thiazolyl tetrazolium (MTT) assay demonstrated that microcapsulation
1. Introduction
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did not influence the anticancer activity of BJO.
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Brucea javanica oil (BJO) is the fatty oil extracted from dried ripe fruit of simaroubaceae plant brucea javanica, it has been widely used as an anticancer active ingredients in the
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treatment of tumors, including lung, esophageal, gastrointestinal, bladder, liver and kidney cancers [1,2]. Furthermore, BJO has detoxification, anti-inflammatory and antimalarial activities [3]. The main activity components of BJO are triglycerides and other saturated and unsaturated fatty acids such as oleic, linoleic and stearic acids [4]. BJO can improve the safety of chemotherapy process, decrease toxicity and significantly reduce the adverse reaction [5]. But BJO is easily to be decomposed during storage because of its thermodynamic instability. And due to a series of unsaturated fatty acids in BJO, it can
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be easily oxidative deteriorated and producing undesirable flavors. So the utilization of BJO has been largely restricted and it is necessary to develop a new form of BJO to overcome its shortcoming.
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Microencapsulation technology refers to using natural or synthetic polymer materials as capsule wall materials, packing the solid or liquid materials in it to form microcapsules. The technology can prevent oxidation by forming an impermeable barrier to avoid oxygen
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diffusion [6,7]. After microencapsulation, the liquid oil can be transferred into solid powder
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to overcome the instability, and enhance its value in certain specialised application. It can also mask or preserve flavors, reduce volatilization, and protect sensitive effective components from degradation. Therefore, the storage properties can be greatly improved by microencapsulation technology.
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Complex coacervation is a kind of physical-chemical process that has been widely used to form microcapsules. Arabic gum and alginate are the common used encapsulating wall
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materials for complex coacervation [8]. In most cases, complex coacervation occurs when mixing oppositely charged polyelectrolyte solutions for shell formation around an active core
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under the effect of one of the following factors: change of pH or temperature, addition of a non-solvent or electrolyte compound, that forms two-phase coacervates which precipitate due to repulsion of the solvent. The microcapsules exhibit excellent controlled release characteristics and good resistance to high temperature and high humidity. It has been widely used in food, pharmaceuticals and other industries [9,10]. Spray drying is a widely used technique to convert a liquid state into a powder form. The quality of spray-dried microcapsule is quite dependent on processing parameters of the spray
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dryer and properties of the composition in the feed solution. The objective of this work was to investigate the combined effect of processing variables and wall material ratio on the quality of microencapsulated BJO by spray drying. Response surface methodology (RSM)
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was used to verify the effect of operating parameters on the yield and moisture content of BJO microcapsule powders obtained via spray drying. In addition, the physicochemical
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properties of the optimized microcapsules were also studied.
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2. Materials and methods 2.1. Materials
Arabic gum was purchased from Damao Chemical Reagent Co. (Tianjin, China). Gelatin (type B) was obtained from Yongda Chemical Reagent Development Center (Tianjin, China).
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Acetic acid was provided by Jinfeng Chemical Co. Ltd. (Tianjin, China). Sodium hydroxide was purchased from North Tianyi Chemical Reagent Co. (Tianjin, China). Glycerol was
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obtained from Meilin Industry and Trade Co. Ltd. (Tianjin, China). Glutaraldehyde 50% water solution was provided by Kemiou Chemical Reagent Co. Ltd. (Tianjin, China). BJO
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was made by YaoDa Pharmceutical Co. (Shenyang, China). Double-distilled water was used for the preparation of all solution. All other chemicals used in this study were of analytical grade.
2.2. Preparation of microcapsules BJO microcapsules were prepared by complex coacervation. 600 mL arabic gum solution (5.0%, w/v) and 600 mL gelatin solution (5.0%, w/v) were prepared respectively and mixed
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together with constantly agitating at 40 ºC overnight. The viscosity of gelatin solution (5.0%, w/v) was 5.7 mPa·s at 40 ºC. Then BJO (10g) and glycerol (10g) were added into the mixture gradually and stirring for 2 min at 5000 rpm with a high-speed dispersing machine (FJ200,
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Shanghai Specimen Company) for homogenization. Glycerol, as a hydrogen donor, could decrease biopolymer collision rate and increase biopolymer aggregation to promote hydrogen bond formation. In this preparation of microcapsules, glycerol was used to promote the
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complexation between arabic gum and gelatin. The rotate speed was tuned to 400 rpm in a
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water bath at 40 ºC and acetic acid (20%, v/v) was used for acidification. The weight ratios of BJO to arabic gum/gelatin were from 1:2 to 1:8. After stirring for 40 min, the temperature was cooled to 10 ºC by ice bath. Next, glutaraldehyde was added to harden the microcapsules and sodium hydroxide (20%, v/v) was used for adjusting the pH to 8.0. The suspension was
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kept stirring at 10 ºC for 3 h, and then spray dried in experimental spray drier (YC-015, Yacheng experimental Co. Ltd., Shanghai, China). The dried powders were collected and
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stored in the hermetically sealed glass bottles for further study.
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2.3. Experimental design
Box–Behnken design was used to evaluate and optimize the effects of the processing parameters of the spray dryer on the product yield and the moisture content using Design-Expert software (version 7.1; Stat-Ease, Inc., Minneapolis, Minnesota). In the applied design, three factors within 17 runs were studied. The independent and dependent variables were listed in Table 1 along with their low, medium and high levels. The independent variables were the inlet temperature (A), feed flow rate (B), air flow speed (C). The
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dependent response factor variables measured were the yield of the product (Y1) and moisture content (Y2).
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Table 1
2.4. Morphology of microcapsules
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The external morphology and shape of spray dried powders were observed by scanning
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electron microscope (SEM) at an accelerating voltage of 25 kV. The samples were fixed directly on an aluminum stub using a double-sided adhesive tape and then coated with a thin layer of platinum using an ion sputtering coater. SEM images showed the microstructure of
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the powders with magnifications of 2000×.
2.5. Characterization of microcapsules
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2.5.1. Fourier transform infrared spectroscopy (FTIR) The chemical composition of the microcapsules was characterized by FTIR. Arabic gum,
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BJO, gelatin and BJO microcapsules were recorded in a spectral range of 400-4000 cm−1 by FT-IR instrument (8400S, Shimadzu, Japan).
2.5.2. Differential scanning calorimetry (DSC) Thermodynamics property analysis of microcapsules was measured by differential scanning calorimeter (DSC822E, Perkin-Elmer, America). 5 mg microcapsule powders were accurately weighed and placed in an aluminium pan using a sealed empty pan as reference.
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2.5.3. X-ray diffraction (XRD)
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The crystallinity of powders was assessed by X-ray diffractometer (Y2000, Dandong ray industrial equipment Company, China). The powders were placed in the sample slot and then pressed with frosted glass to present a good surface texture. XRD patterns were recorded over
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2θ ranging from 10º to 40º.
2.5.4. Encapsulation efficiency (EE) and loading capacity (LC)
The total BJO amount in microcapsules [11,12] was quantified as follows: 3 g of dried microcapsules were accurately weighed and extracted with 50 mL petroleum ether under
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ultrasonic condition for 15 min. The extract process repeated three times and the extraction was collected. The solvent was filtered through a Buchner funnel and evaporated using a
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rotary evaporator to remove the petroleum ether. The total oil content was then gravimetrically calculated.
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Surface oil [11,13] was measured as follows: 5 g microcapsules were placed in a beaker containing 10 mL petroleum ether. The mixture was shaken gently for 3 min without microcapsule destruction. The solvent was filtered through a Buchner funnel and evaporated using a rotary evaporator and then weighted to obtain the weight of surface oil. EE and LC of microcapsules were calculated using the following equation (1) and (2) respectively: EE (%) = (Total amount of oil - surface oil)/Total amount of oil × 100
(1)
LC (%) = Total amount of oil/Weight of microcapsules × 100
(2)
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2.5.5. Product yield Yield was calculated as ratio of the weight of powders obtained by spray drying in
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collecting vessel and the weight of all solids (including wall and core materials) in the emulsion, expressed as percentage. Any powders adhering to the walls of drying chamber or cyclone were not considered. Product yield was determined using the following equation:
2.5.6. Moisture content (MC)
(3)
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Yield (%) = Mass of solid in the collecting vessel/Mass of solid in the feed × 100
The MC of the samples was determined by drying at the temperature of 105 °C in an oven
2.5.7. Bulk density
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until a constant weight was obtained.
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For determination of bulk density, the BJO microencapsule powder was gently poured into a 10 mL graduated cylinder. The bulk density value was determined by the ratio of mass of
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the powder and the volume occupied in the cylinder.
2.5.8. Oxidative stability
The oxidative stability of BJO microencapsules was tested during storage at 30 ºC for 6 weeks. Peroxide value (PV) was expressed as milliequivalents (meq) hydroperoxide per kg of oil to evaluate the extent of oil oxidation. In brief, BJO microencapsule was added into 30 mL chloroform-glacial acetic acid (1:1, v/v) mixture solution by shaking vigorously until the
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followed by
the addition of 1 mL saturated potassium iodine test solution and shaking for 0.5 min. After diluted with water the solution was titrated using sodium thiosulfate titrating solution (0.01
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mol/L) and starch indicator was served for indicate the end-point. The blank test was also performed.
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2.6. In vitro cytotoxicity
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The cytotoxicity of BJO microcapsules and blank microcapsules was performed using the MTT method. The Hela tumor cells were cultured in RPMI-1640 containing 10% fetal bovine serum at 37 °C in an incubator containing 0.5% CO2. Then, BJO microcapsules at oil concentrations of 40, 20, 10, 5, and 2.5 µg/mL were added into each of the three 96-well
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microtiter plates. Blank microcapsules without BJO were used as negative control group. After 24 h, 20 µL of MTT was added and then the cells were incubated for 4 h, the culture
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medium was then removed and 150 µL dimethyl sulfoxide was added to each well to dissolve the formazan crystals. The absorbance was investigated by Synergy HT reader (BioTek,USA)
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at 570 nm.
2.7. Physical stability
Physical stability is a crucial issue in formulating microcapsules. The dried microcapsules were sealed in glass bottles and stored at 4 °C in a refrigerator. The stability study was performed after a storage time of 3 months. Samples were withdrawn to measure MC, EE and LC.
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2.8. Statistical analysis Statistical analysis was conducted by using the students’ t-test with SPSS® 12.0 for
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Windows. Differences were considered statistically significant at p < 0.05.
3. Results and discussion
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3.1. Experimental design and optimization of spray-dried formulation
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During initial trials of spray drying, the ratios of BJO to arabic gum/gelatin were 1:2, 1:3, 1:4, 1:5, 1:6 and 1:8. The spray-dried BJO microcapsules in the ratio 1:2 to 1:5 were sticky and presented higher MC. When the ratio is less than 1: 6, BJO microcapsules were not sticky and presented lower MC. BJO was not completely encapsulated in the arabic gum and gelatin
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matrix from the ratio of 1:2 to 1:5. When the concentration of wall materials was excessive, the probability of collision of the wall material with opposite charge was greatly increased,
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the phenomenon of wall crosslinking was serious, the BJO content of prepared particles would be reduced. In order to optimize parameters of spray drying, the ratio of 1:6 was
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selected.
Box–Behnken designs have been applied toward the optimization of formulations and processing parameters. Table 2 showed the effect of independent variables on the product yield and MC in terms of a regression model. An experimental design of 17 runs was generated for three factors at three levels to identify the optimum process parameter levels. The independent variables and responses were related using polynomial equation with
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statistical analysis. A second-order polynomial equation was used to express the response variables as a function of the independent variables as follows: (4)
Y2 = 4.42-0.41A+0.54B-0.55C-0.025AB+0.25AC-0.20BC-0.30A2-0.03B2+0.13C2
(5)
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Y1 = 36.08+0.025A-1.10B+1.87C-0.22AB-0.33AC+1.42BC-1.85A2-1.00B2+0.50C2
Where Y1 represents the product yield, Y2 represents the moisture content. A, B, C are inlet
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Table 2
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air temperature, the feed flow rate, air flow speed, respectively.
Table 3
3.2. Response surface analysis
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3.2.1. Effect of drying conditions on yield from spray drying
Analysis of variance (ANOVA) was performed to evaluate the significance of the
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coefficients of the quadratic polynomial models (Table 3). The estimated regression coefficients for yield and moisture content with their corresponding R2, p value of regression
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were also shown in Table 3. The coefficients of multiple determinations, R2, of 0.9653 and 0.8471 all indicate that the models fit the experiment data points. For any of the terms in the models, a small p value would indicate a more significant effect on the respective response variables. The linear terms of feed flow rate and air flow speed (p < 0.01), quadratic terms of inlet temperature and feed flow rate (p < 0.01), and interactive terms of feed flow rate and air flow speed (p < 0.01) had a significant effect on the product yield. Fig. 1a-1c represented the effect of different independent variables on the product yield. As observed in Fig. 1a and 1b,
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the product yield increased with increasing inlet temperature first and then a slight reduction in the yield was observed for high levels of inlet temperature. Increasing temperatures led to higher process yield, which can be attributed to the greater efficiency of heat and mass
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transfer processes occurring when higher inlet air temperatures were used [14]. Fig. 1c showed the effect of feed flow rate and air flow speed on product yield. The use of a low level of air flow speed and the increase in the feed flow rate decreased the yield. When the air
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flow speed was at a higher value, the feed flow rate had no significant effect on product yield.
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As seen from Fig. 1b and 1c, the product yield significantly increased with increasing air flow speed. It could be concluded that the air flow speed had positive effects on the product yield.
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Figure 1
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3.2.2. Effect of drying conditions on moisture content of the powder The linear terms of inlet temperature, feed flow rate and air flow speed had a significant
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effect (p < 0.05) on the moisture content. Fig. 2 showed the effects of inlet temperature, feed flow rate and air flow speed on the MC of BJO. Fig. 2a and 2b showed the MC of the sample decreased as the inlet temperature increased from 140 to 160 ºC. Increasing the inlet air temperature accelerated the moisture evaporation rate, resulting in a lower MC. The reduction was due to the high inlet temperature causing a large temperature gradient between the atomized feed and the drying air. As observed in Fig. 2a and 2c, the effect of feed flow rate in the range studied was statistically significant. As the feed flow rate increased, the MC
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increased linearly over all the investigated ranges. Drying air flow speed had a negative effect on moisture content. The higher the air flow speed, the lower the MC.
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Figure 2
By superimposing the graphs, an optimum spray-drying process i.e. inlet temperature level
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of 151.3 °C, feed flow rate of 1.32 mL/min and air flow speed of 80 L/min was
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recommended. To verify the validity of the design, the responses of the optimized parameters were evaluated (Table 4) to ensure the product yield and MC. And the observed values of the prepared optimized spray-dried formulation were mostly similar with predicted values. It was also desirable to evaluate the other properties of the spray-dried powders including
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bulk density, EE and LC, PV and particle morphology.
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Table 4
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3.3. Morphology of microcapsules
The SEM morphology showed the diameter of spray-dried microcapsules was in the range of 2-6 µm, and no holes or cracks were found in the surface of microcapsules. The microcapsules at the proportion of 1:6 presented a clearly outline with smooth surface and the microcapsules had spherical shape and regular distribution (Fig. 3). However it was noted that most of the microcapsules were aggregated, probably due to the spray-drying process. This phenomenon had also been observed for other polymeric systems such as cashew gum
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3.4. Characterization of microcapsules 3.4.1. Fourier transform infrared spectroscopy (FTIR)
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Figure 3
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Infrared spectra of BJO, arabic gum, gelatin and microcapsules were shown in Fig. 4. BJO
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showed characteristic peaks corresponding to the vibrations C-H alkanes stretching (double signal at 2950 cm-1-2800 cm-1) and C=C stretch of alkenes at 1747 cm-1. The band at 1163 cm-1 corresponded to the symmetrical stretching of C-O-C structure. FTIR spectrum of gelatin and arabic gum revealed the presence of characteristic functional group at
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approximately 3400 cm-1 for the amine groups. The peaks that appeared with low intensity at approximately 2925 cm-1 from gum arabic were characteristic of carboxylic groups. The
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FTIR spectrum of gelatin and arabic gum both showed a strong characteristic peaks at 2360 cm-1. The intensity of the most characteristic bands for the BJO decreased in the
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microcapsules, this clearly revealed that the BJO was encapsulated into microcapsules. The FTIR spectroscopy indicated that there was no significant interaction between the arabic gum–gelatin complex and BJO. Figure 4
3.4.2. Differential Scanning Calorimetry (DSC) Fig. 5 showed the DSC data corresponding to gelatin, arabic gum and gelatin/arabic
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gum-loaded BJO microcapsules. The DSC curve of arabic gum showed a very broad endothermic peak in the range 70-120 °C and could be related to the melting and partial thermal decomposition of the complex polysaccharide consisting of several sugar units
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present as branches and sub-branches [16]. The gelatin presented an endothermic peak at around 80 ºC owing to the amorphous phase overlapped with the evaporation of the structural retained moisture. Another small endothermic peak was visible at around 220 °C because of
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the melting of crystalline fraction. DSC thermograms of arabic gum/gelatin-loaded BJO
evaporation of structural water.
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microcapsules showed an endothermic peak in the range of 50-110 °C owing to the
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Figure 5
3.4.3. X-ray diffraction (XRD)
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Crystallographic structure of arabic gum, gelatin and arabic gum/gelatin-loaded BJO microcapsules were determined by XRD and presented in Fig. 6. The diffractogram of arabic
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gum presented a crystalline characteristic peak at 2θ=18º. Gelatin powder showed a wide peak in Fig. 6b at 2θ around 15º and 30º as previously reported. Some authors found that the gelatin had a-helix and triple-helical structure which led to the crystallographic form observed in XRD pattern [17], and the interpenetrating between carbonyl and amine groups formed the inter-chain hydrogen bonds [18]. While for arabic gum/gelatin loaded BJO microcapsules, sharp new peaks appeared at 2θ of 18.8º, 26.8º, 31.0º and 36.0º were found (Fig. 6c), we speculated the new peaks above could be contributed to the salt
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formed (sodium acetate) during the process. In the preparation of BJO microcapsules, acetic acid (20%, v/v) was used for acidification and sodium hydroxide (20%, v/v) was used for adjusting pH. The salt of sodium acetate could form during the process and it
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dissolved in the BJO microcapsules suspension. When the suspension was dried by spray drier, sodium acetate was also spray dried and adsorbed on the dried solid surface of BJO microcapsules powders. Previously published articles [19, 20] reported that
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crystalline sodium acetate showed the PXRD peaks at 2θ of 18.8º, 26.8º, 30.5º and 36.0º.
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These results also confirmed our judgment and sodium acetate formed during the encapsulation process.
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Figure 6
3.4.4. MC, Bulk density, EE and LC
Table 5 showed the MC, bulk density, EE and LC of microcapsules with BJO to arabic
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gum/gelatin at the weight ratio of 1:6. The statistical results indicated that there was no
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significant change in the MC (p > 0.05). The MC of BJO was stable after storage for three months. Bulk density of powders was affected by chemical composition, particle size and moisture content as well as by processing and storage conditions [21]. The density of the particles slightly increased with increased storage, however this change was insignificant. After 3 months of storage, the prepared microcapsules showed good physical stability. MC, EE and LC of microcapsules did not have much change (Table 5), indicating a greater stability of the microcapsules.
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Table 5
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3.4.5. Peroxide value PV variations of BJO in spray dried microcapsules were shown in Fig. 7. At time zero, BJO showed a low level of oxidation, only 12.52 meq peroxide/kg oil. After one week
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storage, BJO presented higher peroxide concentration, reaching values of 22.8 meq
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peroxide/kg oil. The PV increased significantly at almost 165 meq peroxide/kg oil after 6 weeks of storage. The BJO encapsulated in microcapsules had higher oxidative levels (PV=17.1 meq peroxide/kg oil) at day 0, this may be attributed to the oxidation during the preparation of microcapsules by spray drying process. But after 6 weeks of storage, the BJO
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encapsulated in microcapsules showed a lower oxidative increase, reaching values of 34.8 meq peroxide/kg oil. The PV value in the BJO was significantly higher (p < 0.05) than the
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microencapsulated oil after 6 weeks of storage. The relatively lower PV value demonstrated that BJO was well protected in microcapsules, the oxidative stability of the
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microencapsulated oil was better than the oil without microencapsulation. This proved that the process of microencapsulation could protect oil from oxidation caused by the external environment.
Figure 7
3.5. Cell cytotoxicity
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The MTT assay of spray-dried BJO microcapsules and pure BJO on the viability of Hela cells was shown in Fig. 8. Blank microcapsules showed little toxicity to Hela cells with the cell viability more than 90%, confirmed the safety of the carrier (data not shown). Pure BJO
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and BJO microcapsules all showed a dose-dependent cytotoxicity effect against Hela cells. And the high concentration had a better effect on cancer cells. The values of MTT assay showed no significant different (p > 0.05) between the pure BJO and the spray-dried BJO
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microcapsules. This study demonstrated that microcapsulated BJO did not influence the
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anticancer activity of BJO.
Figure 8
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4. Conclusions
In this study BJO microcapsules could successfully be prepared by complex coacervation
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using arabic gum and gelation as coating materials. The spray drying conditions were optimized using MC and process yield as responses. Furthermore, other characteristics of
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dried microcapsules were confirmed by FTIR, DSC and XRD experiments. Results indicated that the best conditions for obtaining spray-dried BJO microcapsules were inlet air temperature of 151.3 ºC, a feed flow rate of 1.32 mL/min and a drying air flow speed of 80 L/min. The prepared microcapsules had good stability against oxidation, with a maximum EE of 82.9% and maximum loading capacity of 10.56% at the oil/wall materials ratio of 1:6. The microcapsules showed a small particle size and spherical shape with smooth surface. MTT analysis also verified that the encapsulated BJO maintained the anticancer
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activity of BJO after spray drying. Therefore, successfully prepared BJO microcapsules might have interesting properties and potential for applications in pharmaceutical and
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nutraceutical industries.
Acknowledgments
This work was supported by the Medical and Engineering Science Research Center of
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Hebei University (No. BM201109), Hebei Provincial Natural Science Foundation of
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China—Shijiazhuang Pharmaceutical Group (CSPC) Foundation (No. H2013201274) and the Top Young Talents Program of Hebei Province.
Declaration of interest
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The authors declare no competing financial interest.
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M. Jimenez, H.S. García, C.I. Beristain, Spray-dried encapsulation of Conjugated Linoleic Acid (CLA) with polymeric matrices, J. Sci. Food Agric. 14 (2006) 2431-2437.
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conditions on oil extraction efficiency, and β-carotene and lycopene contents, J. Food Eng. 4 (2013)
R.V. Tonon, C. Brabet, M.D. Hubinger, Influence of process conditions on the physicochemical properties of açai (Euterpe oleraceae Mart.) powder produced by spray drying, J. Food Eng. 3 (2008) 411-418. E.F.D. Oliveira, H.C.B. Paula, R.C.M.D. Paula, Alginate/cashew gum nanoparticles for essential oil
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[15]
encapsulation, Colloids & Surfaces B Biointerfaces. 1 (2014) 146-151. [16]
S.I. Ahmad, N. Mazumdar, S. Kumar, Functionalization of natural gum: An effective method to prepare
[17]
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iodine complex, Carbohydr. Polym. 1 (2013) 497-502.
C.S. Ki, D.H. Baek, K.D. Gang, K.H. Lee, I.C. Um, Y.H. Park, Characterization of gelatin nanofiber prepared from gelatin–formic acid solution, Polymer. 14 (2005) 5094-5102.
[18]
S. Rivero, M.A. García, A. Pinotti, Correlations between structural, barrier, thermal and mechanical properties of plasticized gelatin films, Innovative Food Science & Emerging Technologies. 2 (2010) 369-375.
[19]
A.N. Ngo, M.J.M. Ezoulin, J.B. Murowchick, A.D. Gounev, B.B.C. Youan, Sodium Acetate Coated (2015) 367-383.
[20]
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Tenofovir-Loaded Chitosan Nanoparticles for Improved Physico-Chemical Properties, Pharm. Res. 2 X. Gu, S. Qin, X. Wu, Y. Li, Y. Liu, Preparation and thermal characterization of sodium acetate trihydrate/expanded graphite composite phase change material, J Therm Anal Calorim. 2 (2016) 831-838.
C.I. Beristain, H.S. Garcı ́A, E.J. Vernon-Carter, Spray-dried Encapsulation of Cardamom ( Elettaria
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cardamomum ) Essential Oil with Mesquite ( Prosopis juliflora ) Gum, Lebensmittel-Wissenschaft und-Technologie. 6 (2001) 398-401.
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[21]
ACCEPTED MANUSCRIPT Table 1 Experimental parameters for Box–Behnken Design. Table 2 Experimental runs, independent variables, and measured responses of the Box–Behnken Design. of
regression
coefficients
calculated
for
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Table 3 Analysis of variance microencapsulation of BJO.
Table 4 Composition, predicted, and observed responses of the optimized spray-dried formulation.
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Table 5 Characterization of microcapsules with BJO to arabic gum/gelatin at the weight ratio of 1:6.
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Table 1 Experimental parameters for Box–Behnken Design.
Factors
Process parameters
Independent variables Inlet temperature (°C) Feed flow rate (mL/min) Air flow speed (L/min) Dependent variables Yield (%) Moisture Content (%)
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Y1 Y2
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A B C
Levels
Low
Medium
High
(-1)
(0)
(+1)
140 1.0 50
150 1.5 65
160 2.0 80
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Y1
Y2
Inlet temperature (°C)
Feed flow rate (mL/min)
Air flow speed (L/min)
Yield (%)
Moisture Content (%)
140 140 160 150 140 160 150 160 150 150 150 160 150 150
1.5 1.0 1.0 1.5 1.5 1.5 1.5 2.0 2.0 1.0 1.0 1.5 2.0 1.5
50 65 65 65 80 50 65 65 50 80 50 80 80 65
32.9 34.2 34.6 36.2 36.4 33.7 36.7 31.8 30.8 37.5 35.7 35.9 38.3 35.6
5.3 4.4 3.5 4.4 3.9 4.1 4.6 3.8 6.3 3.2 4.1 3.7 4.6 4.2
150 140 150
1.5 2.0 1.5
35.8 32.3 36.1
4.5 4.8 4.4
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F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 F16 F17
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Run
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A
65 65 65
ACCEPTED MANUSCRIPT of
regression
coefficients
calculated
for
p-value
Moisture Content Coefficient p-value
A
0.025
0.9071
-0.41
0.0281
B
-1.10
0.0011
0.54
0.0088
C
1.87
0.0001
-0.55
0.0079
AB
-0.22
0.4667
-0.025
0.9092
AC
-0.33
0.3030
0.25
0.2754
BC
1.42
0.0018
-0.20
0.3755
-1.85
0.0003
-0.30
0.1919
-1.00
0.0098
-0.03
0.9907
2
A
2
B C
2
0.50 36.08
R2
0.9653
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Constant
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Independent variable
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Yield Coefficient
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Table 3 Analysis of variance microencapsulation of BJO.
0.1243
0.13
0.5555
0.0090
4.42
0.0000
0.8471
ACCEPTED MANUSCRIPT Table 4 Composition, predicted, and observed responses of the optimized spray-dried formulation. Values
Responses
Predicted Observed values values
Inlet temperature (°C)
151.3
Yield (%)
38.2%
39.0%
Feed flow rate (mL/min)
1.32
Moisture Content (%)
3.85%
3.92%
Air flow speed (L/min)
80
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Variables
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Bulk density (g/cm3)
EE%
LC%
0 day
3.92
0.30
82.9
10.56
3 months
3.96
0.32
82.7
10.51
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Storage time
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(a)
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(b)
Fig. 1. Response surface plots for product yield. (A) inlet temperature, (B) feed flow rate, (C) air flow speed.
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(a)
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(c)
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(b)
Fig. 2. Response surface plots showing the effects of (A) inlet temperature, (B) feed flow rate, (C) air flow speed on the moisture content.
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Fig. 3. SEM image of microcapsules at the weight ratio of BJO to arabic gum/gelatin of 1:6.
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Fig. 4. FTIR spectra of (a) BJO, (b) microcapsules with BJO to arabic gum/gelatin at the weight ratio of 1:6, (c) arabic gum, (d) gelatin.
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Fig. 5. DSC thermograms of (a) gelatin, (b) arabic gum and (c) microcapsules with BJO to arabic gum/gelatin at the weight ratio of 1:6.
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Fig. 6. X-ray diffraction patterns of (a) arabic gum, (b) gelatin and (c) microcapsules with BJO to arabic gum/gelatin at the weight ratio of 1:6.
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Fig. 7. Oxidative stability of BJO and BJO microcapsules evaluated by peroxide value method. Values were expressed as mean values ± SD of three independent experiments and error bars were the standard deviations of the values.
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Fig. 8. The MTT assay of spray-dried BJO microcapsules and pure BJO on the viability of Hela cells. Values were expressed as mean values ±SD of three independent experiments and error bars were the standard deviations of the values.
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Highlights •The brucea javanica oil microcapsules were successfully prepared by complex coacervation. •A Box–Behnken design was used to optimize the spray drying process. •The microcapsules were characterized by FT-IR, DSC and XRD. •The storage and oxidative stability of brucea javanica oil was enhanced by microencapsulation.
S1773-2247(16)30214-3
DOI:
10.1016/j.jddst.2016.09.008
Reference:
JDDST 244
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 26 May 2016 Revised Date:
2 September 2016
Accepted Date: 25 September 2016
Please cite this article as: L. Hu, J. Zhang, Q. Hu, N. Gao, S. Wang, Y. Sun, X. Yang, Microencapsulation of brucea javanica oil: Characterization, stability and optimization of spray drying conditions, Journal of Drug Delivery Science and Technology (2016), doi: 10.1016/j.jddst.2016.09.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Microencapsulation of brucea javanica oil: Characterization, stability and optimization of spray drying conditions
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Liandong Hu1, *, Jianli Zhang1, Qiaofeng Hu1, Na Gao 1, Shaocheng Wang1, Yongbing Sun 2, Xiaoning Yang3
School of Pharmaceutical Sciences & Key Laboratory of Pharmaceutical Quality Control of
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1
Hebei Province, Hebei University. No.180, WuSi Road, Baoding, 071002
3
Jiangxi University of Traditional Chinese Medicine, Nanchang 330006, China
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2
PR China
Tianjin Hemay Bio-Tech Co.,Ltd, Tianjin, PR China
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*Correspondence to Author: Liandong Hu
School of Pharmaceutical Sciences & Key Laboratory of Pharmaceutical Quality Control of
[email protected] (L. Hu)
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Email:
PR China
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Hebei Province, Hebei University. No.180, WuSi Road, Baoding, 071002
Tel: +86 312-5971107
Keywords
Arabic gum, Gelatin, Brucea javanica oil, Complex coacervation, Microencapsulation, Spray drying.
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ACCEPTED MANUSCRIPT Abstract
The aim of this work was to obtain the optimized formulation for brucea javanica oil (BJO) microencapsulation by spray drying with high yield and evaluate their resistance to oxidation
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through the stability test. The parameters of spray drying such as inlet air temperature, feed flow rate and drying air flow speed were optimized using a Box–Behnken design. Moisture content and process yield were analyzed as responses. The spray-dried microcapsules were
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characterized by SEM, DSC, FT-IR and XRD. At the optimal conditions, the encapsulation
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efficiency (EE) reached a maximum of 82.9% (w/w) and loading capacity (LC) reached 10.56% (w/w). The study of oxidative stability showed that microcapsules were stable against oxidation. The methyl thiazolyl tetrazolium (MTT) assay demonstrated that microcapsulation
1. Introduction
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did not influence the anticancer activity of BJO.
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Brucea javanica oil (BJO) is the fatty oil extracted from dried ripe fruit of simaroubaceae plant brucea javanica, it has been widely used as an anticancer active ingredients in the
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treatment of tumors, including lung, esophageal, gastrointestinal, bladder, liver and kidney cancers [1,2]. Furthermore, BJO has detoxification, anti-inflammatory and antimalarial activities [3]. The main activity components of BJO are triglycerides and other saturated and unsaturated fatty acids such as oleic, linoleic and stearic acids [4]. BJO can improve the safety of chemotherapy process, decrease toxicity and significantly reduce the adverse reaction [5]. But BJO is easily to be decomposed during storage because of its thermodynamic instability. And due to a series of unsaturated fatty acids in BJO, it can
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be easily oxidative deteriorated and producing undesirable flavors. So the utilization of BJO has been largely restricted and it is necessary to develop a new form of BJO to overcome its shortcoming.
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Microencapsulation technology refers to using natural or synthetic polymer materials as capsule wall materials, packing the solid or liquid materials in it to form microcapsules. The technology can prevent oxidation by forming an impermeable barrier to avoid oxygen
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diffusion [6,7]. After microencapsulation, the liquid oil can be transferred into solid powder
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to overcome the instability, and enhance its value in certain specialised application. It can also mask or preserve flavors, reduce volatilization, and protect sensitive effective components from degradation. Therefore, the storage properties can be greatly improved by microencapsulation technology.
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Complex coacervation is a kind of physical-chemical process that has been widely used to form microcapsules. Arabic gum and alginate are the common used encapsulating wall
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materials for complex coacervation [8]. In most cases, complex coacervation occurs when mixing oppositely charged polyelectrolyte solutions for shell formation around an active core
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under the effect of one of the following factors: change of pH or temperature, addition of a non-solvent or electrolyte compound, that forms two-phase coacervates which precipitate due to repulsion of the solvent. The microcapsules exhibit excellent controlled release characteristics and good resistance to high temperature and high humidity. It has been widely used in food, pharmaceuticals and other industries [9,10]. Spray drying is a widely used technique to convert a liquid state into a powder form. The quality of spray-dried microcapsule is quite dependent on processing parameters of the spray
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dryer and properties of the composition in the feed solution. The objective of this work was to investigate the combined effect of processing variables and wall material ratio on the quality of microencapsulated BJO by spray drying. Response surface methodology (RSM)
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was used to verify the effect of operating parameters on the yield and moisture content of BJO microcapsule powders obtained via spray drying. In addition, the physicochemical
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properties of the optimized microcapsules were also studied.
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2. Materials and methods 2.1. Materials
Arabic gum was purchased from Damao Chemical Reagent Co. (Tianjin, China). Gelatin (type B) was obtained from Yongda Chemical Reagent Development Center (Tianjin, China).
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Acetic acid was provided by Jinfeng Chemical Co. Ltd. (Tianjin, China). Sodium hydroxide was purchased from North Tianyi Chemical Reagent Co. (Tianjin, China). Glycerol was
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obtained from Meilin Industry and Trade Co. Ltd. (Tianjin, China). Glutaraldehyde 50% water solution was provided by Kemiou Chemical Reagent Co. Ltd. (Tianjin, China). BJO
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was made by YaoDa Pharmceutical Co. (Shenyang, China). Double-distilled water was used for the preparation of all solution. All other chemicals used in this study were of analytical grade.
2.2. Preparation of microcapsules BJO microcapsules were prepared by complex coacervation. 600 mL arabic gum solution (5.0%, w/v) and 600 mL gelatin solution (5.0%, w/v) were prepared respectively and mixed
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together with constantly agitating at 40 ºC overnight. The viscosity of gelatin solution (5.0%, w/v) was 5.7 mPa·s at 40 ºC. Then BJO (10g) and glycerol (10g) were added into the mixture gradually and stirring for 2 min at 5000 rpm with a high-speed dispersing machine (FJ200,
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Shanghai Specimen Company) for homogenization. Glycerol, as a hydrogen donor, could decrease biopolymer collision rate and increase biopolymer aggregation to promote hydrogen bond formation. In this preparation of microcapsules, glycerol was used to promote the
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complexation between arabic gum and gelatin. The rotate speed was tuned to 400 rpm in a
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water bath at 40 ºC and acetic acid (20%, v/v) was used for acidification. The weight ratios of BJO to arabic gum/gelatin were from 1:2 to 1:8. After stirring for 40 min, the temperature was cooled to 10 ºC by ice bath. Next, glutaraldehyde was added to harden the microcapsules and sodium hydroxide (20%, v/v) was used for adjusting the pH to 8.0. The suspension was
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kept stirring at 10 ºC for 3 h, and then spray dried in experimental spray drier (YC-015, Yacheng experimental Co. Ltd., Shanghai, China). The dried powders were collected and
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stored in the hermetically sealed glass bottles for further study.
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2.3. Experimental design
Box–Behnken design was used to evaluate and optimize the effects of the processing parameters of the spray dryer on the product yield and the moisture content using Design-Expert software (version 7.1; Stat-Ease, Inc., Minneapolis, Minnesota). In the applied design, three factors within 17 runs were studied. The independent and dependent variables were listed in Table 1 along with their low, medium and high levels. The independent variables were the inlet temperature (A), feed flow rate (B), air flow speed (C). The
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dependent response factor variables measured were the yield of the product (Y1) and moisture content (Y2).
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Table 1
2.4. Morphology of microcapsules
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The external morphology and shape of spray dried powders were observed by scanning
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electron microscope (SEM) at an accelerating voltage of 25 kV. The samples were fixed directly on an aluminum stub using a double-sided adhesive tape and then coated with a thin layer of platinum using an ion sputtering coater. SEM images showed the microstructure of
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the powders with magnifications of 2000×.
2.5. Characterization of microcapsules
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2.5.1. Fourier transform infrared spectroscopy (FTIR) The chemical composition of the microcapsules was characterized by FTIR. Arabic gum,
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BJO, gelatin and BJO microcapsules were recorded in a spectral range of 400-4000 cm−1 by FT-IR instrument (8400S, Shimadzu, Japan).
2.5.2. Differential scanning calorimetry (DSC) Thermodynamics property analysis of microcapsules was measured by differential scanning calorimeter (DSC822E, Perkin-Elmer, America). 5 mg microcapsule powders were accurately weighed and placed in an aluminium pan using a sealed empty pan as reference.
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2.5.3. X-ray diffraction (XRD)
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The crystallinity of powders was assessed by X-ray diffractometer (Y2000, Dandong ray industrial equipment Company, China). The powders were placed in the sample slot and then pressed with frosted glass to present a good surface texture. XRD patterns were recorded over
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2θ ranging from 10º to 40º.
2.5.4. Encapsulation efficiency (EE) and loading capacity (LC)
The total BJO amount in microcapsules [11,12] was quantified as follows: 3 g of dried microcapsules were accurately weighed and extracted with 50 mL petroleum ether under
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ultrasonic condition for 15 min. The extract process repeated three times and the extraction was collected. The solvent was filtered through a Buchner funnel and evaporated using a
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rotary evaporator to remove the petroleum ether. The total oil content was then gravimetrically calculated.
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Surface oil [11,13] was measured as follows: 5 g microcapsules were placed in a beaker containing 10 mL petroleum ether. The mixture was shaken gently for 3 min without microcapsule destruction. The solvent was filtered through a Buchner funnel and evaporated using a rotary evaporator and then weighted to obtain the weight of surface oil. EE and LC of microcapsules were calculated using the following equation (1) and (2) respectively: EE (%) = (Total amount of oil - surface oil)/Total amount of oil × 100
(1)
LC (%) = Total amount of oil/Weight of microcapsules × 100
(2)
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2.5.5. Product yield Yield was calculated as ratio of the weight of powders obtained by spray drying in
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collecting vessel and the weight of all solids (including wall and core materials) in the emulsion, expressed as percentage. Any powders adhering to the walls of drying chamber or cyclone were not considered. Product yield was determined using the following equation:
2.5.6. Moisture content (MC)
(3)
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Yield (%) = Mass of solid in the collecting vessel/Mass of solid in the feed × 100
The MC of the samples was determined by drying at the temperature of 105 °C in an oven
2.5.7. Bulk density
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until a constant weight was obtained.
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For determination of bulk density, the BJO microencapsule powder was gently poured into a 10 mL graduated cylinder. The bulk density value was determined by the ratio of mass of
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the powder and the volume occupied in the cylinder.
2.5.8. Oxidative stability
The oxidative stability of BJO microencapsules was tested during storage at 30 ºC for 6 weeks. Peroxide value (PV) was expressed as milliequivalents (meq) hydroperoxide per kg of oil to evaluate the extent of oil oxidation. In brief, BJO microencapsule was added into 30 mL chloroform-glacial acetic acid (1:1, v/v) mixture solution by shaking vigorously until the
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followed by
the addition of 1 mL saturated potassium iodine test solution and shaking for 0.5 min. After diluted with water the solution was titrated using sodium thiosulfate titrating solution (0.01
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mol/L) and starch indicator was served for indicate the end-point. The blank test was also performed.
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2.6. In vitro cytotoxicity
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The cytotoxicity of BJO microcapsules and blank microcapsules was performed using the MTT method. The Hela tumor cells were cultured in RPMI-1640 containing 10% fetal bovine serum at 37 °C in an incubator containing 0.5% CO2. Then, BJO microcapsules at oil concentrations of 40, 20, 10, 5, and 2.5 µg/mL were added into each of the three 96-well
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microtiter plates. Blank microcapsules without BJO were used as negative control group. After 24 h, 20 µL of MTT was added and then the cells were incubated for 4 h, the culture
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medium was then removed and 150 µL dimethyl sulfoxide was added to each well to dissolve the formazan crystals. The absorbance was investigated by Synergy HT reader (BioTek,USA)
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at 570 nm.
2.7. Physical stability
Physical stability is a crucial issue in formulating microcapsules. The dried microcapsules were sealed in glass bottles and stored at 4 °C in a refrigerator. The stability study was performed after a storage time of 3 months. Samples were withdrawn to measure MC, EE and LC.
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2.8. Statistical analysis Statistical analysis was conducted by using the students’ t-test with SPSS® 12.0 for
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Windows. Differences were considered statistically significant at p < 0.05.
3. Results and discussion
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3.1. Experimental design and optimization of spray-dried formulation
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During initial trials of spray drying, the ratios of BJO to arabic gum/gelatin were 1:2, 1:3, 1:4, 1:5, 1:6 and 1:8. The spray-dried BJO microcapsules in the ratio 1:2 to 1:5 were sticky and presented higher MC. When the ratio is less than 1: 6, BJO microcapsules were not sticky and presented lower MC. BJO was not completely encapsulated in the arabic gum and gelatin
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matrix from the ratio of 1:2 to 1:5. When the concentration of wall materials was excessive, the probability of collision of the wall material with opposite charge was greatly increased,
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the phenomenon of wall crosslinking was serious, the BJO content of prepared particles would be reduced. In order to optimize parameters of spray drying, the ratio of 1:6 was
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selected.
Box–Behnken designs have been applied toward the optimization of formulations and processing parameters. Table 2 showed the effect of independent variables on the product yield and MC in terms of a regression model. An experimental design of 17 runs was generated for three factors at three levels to identify the optimum process parameter levels. The independent variables and responses were related using polynomial equation with
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statistical analysis. A second-order polynomial equation was used to express the response variables as a function of the independent variables as follows: (4)
Y2 = 4.42-0.41A+0.54B-0.55C-0.025AB+0.25AC-0.20BC-0.30A2-0.03B2+0.13C2
(5)
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Y1 = 36.08+0.025A-1.10B+1.87C-0.22AB-0.33AC+1.42BC-1.85A2-1.00B2+0.50C2
Where Y1 represents the product yield, Y2 represents the moisture content. A, B, C are inlet
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Table 2
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air temperature, the feed flow rate, air flow speed, respectively.
Table 3
3.2. Response surface analysis
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3.2.1. Effect of drying conditions on yield from spray drying
Analysis of variance (ANOVA) was performed to evaluate the significance of the
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coefficients of the quadratic polynomial models (Table 3). The estimated regression coefficients for yield and moisture content with their corresponding R2, p value of regression
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were also shown in Table 3. The coefficients of multiple determinations, R2, of 0.9653 and 0.8471 all indicate that the models fit the experiment data points. For any of the terms in the models, a small p value would indicate a more significant effect on the respective response variables. The linear terms of feed flow rate and air flow speed (p < 0.01), quadratic terms of inlet temperature and feed flow rate (p < 0.01), and interactive terms of feed flow rate and air flow speed (p < 0.01) had a significant effect on the product yield. Fig. 1a-1c represented the effect of different independent variables on the product yield. As observed in Fig. 1a and 1b,
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the product yield increased with increasing inlet temperature first and then a slight reduction in the yield was observed for high levels of inlet temperature. Increasing temperatures led to higher process yield, which can be attributed to the greater efficiency of heat and mass
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transfer processes occurring when higher inlet air temperatures were used [14]. Fig. 1c showed the effect of feed flow rate and air flow speed on product yield. The use of a low level of air flow speed and the increase in the feed flow rate decreased the yield. When the air
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flow speed was at a higher value, the feed flow rate had no significant effect on product yield.
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As seen from Fig. 1b and 1c, the product yield significantly increased with increasing air flow speed. It could be concluded that the air flow speed had positive effects on the product yield.
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Figure 1
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3.2.2. Effect of drying conditions on moisture content of the powder The linear terms of inlet temperature, feed flow rate and air flow speed had a significant
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effect (p < 0.05) on the moisture content. Fig. 2 showed the effects of inlet temperature, feed flow rate and air flow speed on the MC of BJO. Fig. 2a and 2b showed the MC of the sample decreased as the inlet temperature increased from 140 to 160 ºC. Increasing the inlet air temperature accelerated the moisture evaporation rate, resulting in a lower MC. The reduction was due to the high inlet temperature causing a large temperature gradient between the atomized feed and the drying air. As observed in Fig. 2a and 2c, the effect of feed flow rate in the range studied was statistically significant. As the feed flow rate increased, the MC
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increased linearly over all the investigated ranges. Drying air flow speed had a negative effect on moisture content. The higher the air flow speed, the lower the MC.
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Figure 2
By superimposing the graphs, an optimum spray-drying process i.e. inlet temperature level
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of 151.3 °C, feed flow rate of 1.32 mL/min and air flow speed of 80 L/min was
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recommended. To verify the validity of the design, the responses of the optimized parameters were evaluated (Table 4) to ensure the product yield and MC. And the observed values of the prepared optimized spray-dried formulation were mostly similar with predicted values. It was also desirable to evaluate the other properties of the spray-dried powders including
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bulk density, EE and LC, PV and particle morphology.
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Table 4
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3.3. Morphology of microcapsules
The SEM morphology showed the diameter of spray-dried microcapsules was in the range of 2-6 µm, and no holes or cracks were found in the surface of microcapsules. The microcapsules at the proportion of 1:6 presented a clearly outline with smooth surface and the microcapsules had spherical shape and regular distribution (Fig. 3). However it was noted that most of the microcapsules were aggregated, probably due to the spray-drying process. This phenomenon had also been observed for other polymeric systems such as cashew gum
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3.4. Characterization of microcapsules 3.4.1. Fourier transform infrared spectroscopy (FTIR)
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Figure 3
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Infrared spectra of BJO, arabic gum, gelatin and microcapsules were shown in Fig. 4. BJO
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showed characteristic peaks corresponding to the vibrations C-H alkanes stretching (double signal at 2950 cm-1-2800 cm-1) and C=C stretch of alkenes at 1747 cm-1. The band at 1163 cm-1 corresponded to the symmetrical stretching of C-O-C structure. FTIR spectrum of gelatin and arabic gum revealed the presence of characteristic functional group at
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approximately 3400 cm-1 for the amine groups. The peaks that appeared with low intensity at approximately 2925 cm-1 from gum arabic were characteristic of carboxylic groups. The
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FTIR spectrum of gelatin and arabic gum both showed a strong characteristic peaks at 2360 cm-1. The intensity of the most characteristic bands for the BJO decreased in the
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microcapsules, this clearly revealed that the BJO was encapsulated into microcapsules. The FTIR spectroscopy indicated that there was no significant interaction between the arabic gum–gelatin complex and BJO. Figure 4
3.4.2. Differential Scanning Calorimetry (DSC) Fig. 5 showed the DSC data corresponding to gelatin, arabic gum and gelatin/arabic
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gum-loaded BJO microcapsules. The DSC curve of arabic gum showed a very broad endothermic peak in the range 70-120 °C and could be related to the melting and partial thermal decomposition of the complex polysaccharide consisting of several sugar units
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present as branches and sub-branches [16]. The gelatin presented an endothermic peak at around 80 ºC owing to the amorphous phase overlapped with the evaporation of the structural retained moisture. Another small endothermic peak was visible at around 220 °C because of
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the melting of crystalline fraction. DSC thermograms of arabic gum/gelatin-loaded BJO
evaporation of structural water.
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microcapsules showed an endothermic peak in the range of 50-110 °C owing to the
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Figure 5
3.4.3. X-ray diffraction (XRD)
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Crystallographic structure of arabic gum, gelatin and arabic gum/gelatin-loaded BJO microcapsules were determined by XRD and presented in Fig. 6. The diffractogram of arabic
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gum presented a crystalline characteristic peak at 2θ=18º. Gelatin powder showed a wide peak in Fig. 6b at 2θ around 15º and 30º as previously reported. Some authors found that the gelatin had a-helix and triple-helical structure which led to the crystallographic form observed in XRD pattern [17], and the interpenetrating between carbonyl and amine groups formed the inter-chain hydrogen bonds [18]. While for arabic gum/gelatin loaded BJO microcapsules, sharp new peaks appeared at 2θ of 18.8º, 26.8º, 31.0º and 36.0º were found (Fig. 6c), we speculated the new peaks above could be contributed to the salt
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formed (sodium acetate) during the process. In the preparation of BJO microcapsules, acetic acid (20%, v/v) was used for acidification and sodium hydroxide (20%, v/v) was used for adjusting pH. The salt of sodium acetate could form during the process and it
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dissolved in the BJO microcapsules suspension. When the suspension was dried by spray drier, sodium acetate was also spray dried and adsorbed on the dried solid surface of BJO microcapsules powders. Previously published articles [19, 20] reported that
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crystalline sodium acetate showed the PXRD peaks at 2θ of 18.8º, 26.8º, 30.5º and 36.0º.
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These results also confirmed our judgment and sodium acetate formed during the encapsulation process.
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Figure 6
3.4.4. MC, Bulk density, EE and LC
Table 5 showed the MC, bulk density, EE and LC of microcapsules with BJO to arabic
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gum/gelatin at the weight ratio of 1:6. The statistical results indicated that there was no
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significant change in the MC (p > 0.05). The MC of BJO was stable after storage for three months. Bulk density of powders was affected by chemical composition, particle size and moisture content as well as by processing and storage conditions [21]. The density of the particles slightly increased with increased storage, however this change was insignificant. After 3 months of storage, the prepared microcapsules showed good physical stability. MC, EE and LC of microcapsules did not have much change (Table 5), indicating a greater stability of the microcapsules.
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Table 5
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3.4.5. Peroxide value PV variations of BJO in spray dried microcapsules were shown in Fig. 7. At time zero, BJO showed a low level of oxidation, only 12.52 meq peroxide/kg oil. After one week
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storage, BJO presented higher peroxide concentration, reaching values of 22.8 meq
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peroxide/kg oil. The PV increased significantly at almost 165 meq peroxide/kg oil after 6 weeks of storage. The BJO encapsulated in microcapsules had higher oxidative levels (PV=17.1 meq peroxide/kg oil) at day 0, this may be attributed to the oxidation during the preparation of microcapsules by spray drying process. But after 6 weeks of storage, the BJO
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encapsulated in microcapsules showed a lower oxidative increase, reaching values of 34.8 meq peroxide/kg oil. The PV value in the BJO was significantly higher (p < 0.05) than the
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microencapsulated oil after 6 weeks of storage. The relatively lower PV value demonstrated that BJO was well protected in microcapsules, the oxidative stability of the
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microencapsulated oil was better than the oil without microencapsulation. This proved that the process of microencapsulation could protect oil from oxidation caused by the external environment.
Figure 7
3.5. Cell cytotoxicity
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The MTT assay of spray-dried BJO microcapsules and pure BJO on the viability of Hela cells was shown in Fig. 8. Blank microcapsules showed little toxicity to Hela cells with the cell viability more than 90%, confirmed the safety of the carrier (data not shown). Pure BJO
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and BJO microcapsules all showed a dose-dependent cytotoxicity effect against Hela cells. And the high concentration had a better effect on cancer cells. The values of MTT assay showed no significant different (p > 0.05) between the pure BJO and the spray-dried BJO
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microcapsules. This study demonstrated that microcapsulated BJO did not influence the
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anticancer activity of BJO.
Figure 8
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4. Conclusions
In this study BJO microcapsules could successfully be prepared by complex coacervation
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using arabic gum and gelation as coating materials. The spray drying conditions were optimized using MC and process yield as responses. Furthermore, other characteristics of
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dried microcapsules were confirmed by FTIR, DSC and XRD experiments. Results indicated that the best conditions for obtaining spray-dried BJO microcapsules were inlet air temperature of 151.3 ºC, a feed flow rate of 1.32 mL/min and a drying air flow speed of 80 L/min. The prepared microcapsules had good stability against oxidation, with a maximum EE of 82.9% and maximum loading capacity of 10.56% at the oil/wall materials ratio of 1:6. The microcapsules showed a small particle size and spherical shape with smooth surface. MTT analysis also verified that the encapsulated BJO maintained the anticancer
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activity of BJO after spray drying. Therefore, successfully prepared BJO microcapsules might have interesting properties and potential for applications in pharmaceutical and
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nutraceutical industries.
Acknowledgments
This work was supported by the Medical and Engineering Science Research Center of
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Hebei University (No. BM201109), Hebei Provincial Natural Science Foundation of
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China—Shijiazhuang Pharmaceutical Group (CSPC) Foundation (No. H2013201274) and the Top Young Talents Program of Hebei Province.
Declaration of interest
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The authors declare no competing financial interest.
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[21]
ACCEPTED MANUSCRIPT Table 1 Experimental parameters for Box–Behnken Design. Table 2 Experimental runs, independent variables, and measured responses of the Box–Behnken Design. of
regression
coefficients
calculated
for
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Table 3 Analysis of variance microencapsulation of BJO.
Table 4 Composition, predicted, and observed responses of the optimized spray-dried formulation.
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Table 5 Characterization of microcapsules with BJO to arabic gum/gelatin at the weight ratio of 1:6.
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Table 1 Experimental parameters for Box–Behnken Design.
Factors
Process parameters
Independent variables Inlet temperature (°C) Feed flow rate (mL/min) Air flow speed (L/min) Dependent variables Yield (%) Moisture Content (%)
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Y1 Y2
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A B C
Levels
Low
Medium
High
(-1)
(0)
(+1)
140 1.0 50
150 1.5 65
160 2.0 80
ACCEPTED MANUSCRIPT Table 2 Experimental runs, independent variables, and measured responses of the Box–Behnken Design. B
C
Y1
Y2
Inlet temperature (°C)
Feed flow rate (mL/min)
Air flow speed (L/min)
Yield (%)
Moisture Content (%)
140 140 160 150 140 160 150 160 150 150 150 160 150 150
1.5 1.0 1.0 1.5 1.5 1.5 1.5 2.0 2.0 1.0 1.0 1.5 2.0 1.5
50 65 65 65 80 50 65 65 50 80 50 80 80 65
32.9 34.2 34.6 36.2 36.4 33.7 36.7 31.8 30.8 37.5 35.7 35.9 38.3 35.6
5.3 4.4 3.5 4.4 3.9 4.1 4.6 3.8 6.3 3.2 4.1 3.7 4.6 4.2
150 140 150
1.5 2.0 1.5
35.8 32.3 36.1
4.5 4.8 4.4
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F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 F16 F17
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Run
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A
65 65 65
ACCEPTED MANUSCRIPT of
regression
coefficients
calculated
for
p-value
Moisture Content Coefficient p-value
A
0.025
0.9071
-0.41
0.0281
B
-1.10
0.0011
0.54
0.0088
C
1.87
0.0001
-0.55
0.0079
AB
-0.22
0.4667
-0.025
0.9092
AC
-0.33
0.3030
0.25
0.2754
BC
1.42
0.0018
-0.20
0.3755
-1.85
0.0003
-0.30
0.1919
-1.00
0.0098
-0.03
0.9907
2
A
2
B C
2
0.50 36.08
R2
0.9653
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Constant
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Independent variable
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Yield Coefficient
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Table 3 Analysis of variance microencapsulation of BJO.
0.1243
0.13
0.5555
0.0090
4.42
0.0000
0.8471
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Responses
Predicted Observed values values
Inlet temperature (°C)
151.3
Yield (%)
38.2%
39.0%
Feed flow rate (mL/min)
1.32
Moisture Content (%)
3.85%
3.92%
Air flow speed (L/min)
80
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Variables
ACCEPTED MANUSCRIPT Table 5 Characterization of microcapsules with BJO to arabic gum/gelatin at the weight ratio of 1:6. MC%
Bulk density (g/cm3)
EE%
LC%
0 day
3.92
0.30
82.9
10.56
3 months
3.96
0.32
82.7
10.51
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Storage time
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(a)
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(c)
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(b)
Fig. 1. Response surface plots for product yield. (A) inlet temperature, (B) feed flow rate, (C) air flow speed.
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(a)
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(c)
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(b)
Fig. 2. Response surface plots showing the effects of (A) inlet temperature, (B) feed flow rate, (C) air flow speed on the moisture content.
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Fig. 3. SEM image of microcapsules at the weight ratio of BJO to arabic gum/gelatin of 1:6.
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Fig. 4. FTIR spectra of (a) BJO, (b) microcapsules with BJO to arabic gum/gelatin at the weight ratio of 1:6, (c) arabic gum, (d) gelatin.
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Fig. 5. DSC thermograms of (a) gelatin, (b) arabic gum and (c) microcapsules with BJO to arabic gum/gelatin at the weight ratio of 1:6.
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Fig. 6. X-ray diffraction patterns of (a) arabic gum, (b) gelatin and (c) microcapsules with BJO to arabic gum/gelatin at the weight ratio of 1:6.
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Fig. 7. Oxidative stability of BJO and BJO microcapsules evaluated by peroxide value method. Values were expressed as mean values ± SD of three independent experiments and error bars were the standard deviations of the values.
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Fig. 8. The MTT assay of spray-dried BJO microcapsules and pure BJO on the viability of Hela cells. Values were expressed as mean values ±SD of three independent experiments and error bars were the standard deviations of the values.
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Highlights •The brucea javanica oil microcapsules were successfully prepared by complex coacervation. •A Box–Behnken design was used to optimize the spray drying process. •The microcapsules were characterized by FT-IR, DSC and XRD. •The storage and oxidative stability of brucea javanica oil was enhanced by microencapsulation.