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Design of microparticles containing natural antioxidants: Preparation, characterization and controlled release studies
Powder Technology 313 (2017) 287–292
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Design of microparticles containing natural antioxidants: Preparation, characterization and controlled release studies Joana Aguiar 1, Raquel Costa 1, F. Rocha, B.N. Estevinho ⁎, L. Santos LEPABE, Departamento de Engenharia Química, Faculdade de Engenharia da Universidade do Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal
a r t i c l e
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Article history: Received 18 December 2016 Received in revised form 26 February 2017 Accepted 3 March 2017 Available online 06 March 2017 Keywords: Carboxymethyl cellulose Microencapsulation Natural antioxidants Spray drying
a b s t r a c t Antioxidants are important compounds in the prevention of diseases like cancer, diabetes, cardiovascular, neurodegenerative diseases and premature aging, however they are very sensitive to the light, heat, oxygen and pH. In this study, caffeic acid (CAF), chlorogenic acid (CGA) and rosmarinic acid (RA), 3 natural antioxidants, were encapsulated by spray-drying using sodium carboxymethyl cellulose (Na-CMC) to overcome their limitations in industrial applications. Product yield values were around 40% and the encapsulation efficiency values were higher than 90%. Particles revealed a smooth spherical shape with mean diameter below 11 μm (considering volume size distribution). Microparticles loaded with CAF, CGA and RA were fully released after 45 min, 2 h and 4 h, respectively. Antioxidant activity was not compromised, highlighting the potential of spray-dried Na-CMC microparticles as carriers of natural antioxidants. © 2017 Elsevier B.V. All rights reserved.
1. Introduction According to recent studies, the excessive accumulation in the human body of products derived from oxygen and nitrogen reactions is responsible for premature aging and diseases like cancer, diabetes, cardiovascular and neurodegenerative diseases (Alzheimer and Parkinson) [1,2]. Antioxidants are a powerful tool to reduce the oxidative stress allowing the stopping or delay of a chain reaction through several mechanisms: reacting with free radicals, by depleting molecular oxygen, deactivating singlet oxygen, removing prooxidative metal ions, replenishing hydrogen to other antioxidants and absorbing UV light [3,4] and, furthermore, antioxidants can also be used as preservatives avoiding the oxidation of lipid ingredients [4]. Recently, interest in natural antioxidants is rising comparing to synthetic ones due to the consumer preference of organic and natural products with less additives and side effects [5,6]. However, natural antioxidants present some characteristics which may limit their industrial application such as sensitivity to heat, oxygen, pH and light [7,8] as well as unpleasant taste or smell, poor availability, high reactivity with other ingredients present in the product matrix and also high susceptibility to storage and processing conditions [9–11]. Microencapsulation may be used to overcome some of the restrictions of antioxidants and increase their applicability. Examples of final products where encapsulated antioxidants may be incorporated include cosmetic preparations [12], food products and supplements [13] and ⁎ Corresponding author. E-mail address: [email protected] (B.N. Estevinho). Equal participation as first author.
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http://dx.doi.org/10.1016/j.powtec.2017.03.013 0032-5910/© 2017 Elsevier B.V. All rights reserved.
food packaging [14]. In the microencapsulation technique, solid, liquid or gaseous compounds are surrounded by a coating material creating a microparticle that can be used in food, pharmaceutical, cosmetic, agrochemical and textile industries [12,15,16]. Some advantages of microencapsulation techniques include higher stability by isolating active ingredients in order to prevent their deterioration, delayed evaporation in case of a volatile core, controlled and targeted release of active ingredients and improved product esthetics and marketing perception, while maintaining core properties [17–21]. Microencapsulation is also able to mask core undesired properties such as undesirable taste, odor or activity and to reduce the amount of ingredients in formulation being a cost saving alternative [22,23]. There are various microencapsulation techniques available, although, spray-drying is one of the most used, due its simplicity, relatively low cost, flexibility, high stability of the final dried product (due to low moisture content), high volume reduction, ease of handling, transportation and storage of the particles [11]. It also allows a continuous operation and appropriate encapsulation of many heat-labile (low-boiling point) materials due to the lower temperatures the core material reaches [9,24–26]. Currently, substances such as antibiotics, medical ingredients, additives, vitamins and polyphenols, among others are encapsulated in large-scale using spraydrying [9,23,25,27]. In this work, 3 natural antioxidants (caffeic acid (CAF), chlorogenic acid (CGA) and rosmarinic acid (RA)) were selected, considering their aforementioned advantages for industrial applications, and microencapsulated in sodium carboxymethyl cellulose (Na-CMC). CAF and CGA are polyphenols mainly found in the coffee tree although other sources include fruits (e.g. apple, pear, berries, plum), vegetables (e.g. sweet potato, lettuce, spinach), black teas, soy beans and wheat
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[28,29]. RA is also a polyphenol, found in a wide variety of plants from the Lamiaceae family: oregano, rosemary, marjoram, clary sage, thyme, basil [30]. According to literature, there are several benefits related to these compounds besides their strong antioxidant activity: anti-inflammatory, anti-microbial and anti-viral properties, as well as prevention of diseases associated with oxidative stress (namely cardiovascular, cancer and neurodegenerative) [30–38]. On the other hand, carboxymethyl cellulose (CMC) is a water soluble anionic cellulose derivative with carboxymethyl substitution groups and has applications in a wide range of fields such as food, cosmetic, pharmaceutical, textiles, and detergents. Carboxymethyl cellulose is already widely used in cosmetic and personal products as emulsifier, stabilizer, film-former and thickener agent [39]. Carboxymethyl cellulose hydrogel swelling behavior is pH and ionic strength dependent due to the presence of electrostatic charges in the polymer network, originated from sodium ion and the carboxymethyl group [40]. Films formed using carboxymethyl cellulose have in general a moderate strength in aqueous solutions although such property is dependent on the degree of substitution and molecular weight [41,42]. The main purpose of this work was to perform the microencapsulation by spray drying of these three different antioxidant compounds (CAF, CGA and RA) using Na-CMC, considering the advantages of these natural antioxidants and also of the encapsulating agent. After encapsulation, the microparticles were characterized in terms of shape and size distribution, and controlled release studies were performed. The antioxidant capacity of the encapsulated acids was also evaluated. 2. Materials and methods 2.1. Chemicals Caffeic acid standard (Ref. C0625-2G) and chlorogenic acid standard (3-caffeoylquinic acid) were acquired from Sigma-Aldrich Chemical Co. (MO, USA). Rosmarinic acid (Ref. 536,954-5G) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Methanol was obtained from VWR International (Fontenay-sous-Bois, France). Carboxymethylcellulose sodium salt (viscosity 830 mPa·s (25 °C, 2% water)) was obtained from VWR International (Haasrode, Belgium) and methanol from VWR International (Fontenay-sous-Bois, France). ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) was purchased from AppliChem GmbH (Darmstadt, Germany), potassium persulfate was acquired from Panreac Química (Barcelona, Spain) and trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was obtained from Sigma-Aldrich Chemical Co. (MO, USA). Ethanol (96% purity) was obtained from AGA (Prior Velho, Portugal). Water was deionized in laboratory using Millipore™ water purification equipment (Massachusetts, USA). All the reagents used were of analytical grade purity.
The CAF calibration curve was linear from 0.25 to 15 mg/L and the linear regression was the following: Abs (AU) = 0.067 ± 0.001 [CAF] + 0.009 ± 0.006 (R2 = 0.999). Values obtained for limit of detection (LOD) and limit of quantification (LOQ) were, respectively, 0.29 and 0.96 mg/L. The CGA calibration curve was linear from 0.50 to 14 mg/L and the linear regression was the following: Abs (AU) = 0.0554 ± 0.0002 [CGA] − 0.003 ± 0.002 (R2 = 1.000). Values obtained for LOD and LOQ were, respectively, 0.10 and 0.34 mg/L. Finally, the RA calibration curve was linear from 1 to 15 mg/L and the linear regression was the following: Abs = 0.059 ± 0.003[RA] + 0.02 ± 0.03 (R2 = 1.000). Values obtained for LOD and LOQ were, respectively, 0.50 and 1.66 mg/L. The analytical method presented good intra-day and inter-day precision (coefficients of variation smaller than 5% except for the lowest concentrations) as well as great accuracy (recovery percentage close to 100%) for the antioxidants tested. 2.4. Preparation of microparticles by spray-drying Microparticles were prepared by spray-drying as described by [43–45]. Separate solutions of Na-CMC (10 g/L) and antioxidants (10 g/L for CAF and CGA, and 1 g/L for RA) were prepared in ultrapure water, at room temperature, under stirring. Before encapsulation, Na-CMC and antioxidant solution were mixed together under stirring, during 30 min, at room temperature. The antioxidant concentration in the feed solution was around 2%. The experimental conditions were previously optimized [46,47]: feed flow rate of 4 mL/min (15%), inlet temperature of 115 °C, air pressure of 6.0 bar, 100% aspiration rate and nozzle cleaner set to 3. The outlet temperature was around 60 °C. The powders were collected and stored in falcon tubes, wrapped in aluminum foil, and stored at 4 °C. 2.5. Microparticles characterization The size distribution of the microparticles was evaluated by laser granulometry using a Coulter Counter-LS 230 Particle Size Analyzer (Miami, FL, USA). For each experiment, a small sample of powder was suspended in ethanol before measurement. Samples were characterized by number and volume as an average of two runs of 60 s. Particles morphology was assessed by SEM analysis using a High resolution (Schottky) Environmental Scanning Electron Microscope with X-Ray Microanalysis and Electron Backscattered Diffraction analysis: Quanta 400 FEG ESEM/EDAX Genesis X4M. Samples were coated with Au/Pd thin film for 100 s and with a 15 mA current, by sputtering, using the SPI Module Sputter Coater equipment. 2.6. Controlled release studies
Weight measurements were performed with an analytical scale Mettler Toledo AG245 (Columbus, OH, USA). Quantification analysis of the antioxidants was accomplished using a spectrophotometer UV–Vis V-530 (Jasco), as well as the antioxidant activity assays. Microencapsulation was performed using a mini spray dryer BÜCHI B-290 (Flawil, Switzerland) with a standard 0.5 mm nozzle.
The controlled release studies were performed in ultrapure water (pH 5.6) by weighing 3 mg of each antioxidant powder into separate flasks (in duplicate) and then adding 4.5 mL of water. The release was performed at room temperature (20–25 °C), under low stirring and in the absence of light (by wrapping the flask with aluminum foil). Samples were taken every 0, 1, 2, 5, 10, 20, 30, 45 min and 1, 2, 4, 6, 24 h to evaluate the amount of antioxidant released using the UVspectrophotometry method.
2.3. Validation of analytical method
2.7. Antioxidant activity
CAF, CGA and RA were quantified by UV–Vis spectrometry (Jasco V-530 UV–Vis Spectrophotometer; Easton, USA) with detection at 313, 323 and 324 nm, respectively. Standard solutions of each antioxidant were analyzed in duplicate and three calibration curves were obtained. The main validation performance and reliability parameters of the analytical method for each antioxidant were determined.
The antioxidant activity was estimated in duplicate using the ABTS radical scavenging assay, as described by [48]. Samples consisted of Na-CMC microparticles loaded with CAF, CGA and RA at maximum release (after 4 h in aqueous solution) and also non-loaded microparticles of Na-CMC. The antioxidant activity of samples containing CAF, CGA and RA in free solution with the same amount as in the microparticles was
2.2. Equipment
J. Aguiar et al. / Powder Technology 313 (2017) 287–292 Table 1 Product yield values for CAF, CGA and RA encapsulated in Na-CMC by spray-drying. Sample
Product yield (%)
Na-CMC + CAF Na-CMC + CGA Na-CMC + RA
37.6 40.8 38.4
also assessed. Antioxidant activity was expressed in μM Trolox (SigmaAldrich) equivalents (TE), a synthetic analogue of vitamin E commonly used as antioxidant reference. 3. Results and discussion 3.1. Product yield The product yield of the encapsulation process relates the amount of powder that it is possible to recover comparing to the initial raw material used. Product yield values for the spray-dried microparticles are presented in Table 1. The results were quite satisfactory considering the employed method and scale used. Losses of the solid content may be explained by the significant small amount of used raw materials when compared to the equipment dimensions. Particles size may also contribute to solid losses since small microparticles are suctioned by the vacuum filter and therefore fail to be collected in the final powder. Similar results were found by Estevinho et al. [46] after producing microparticles containing β-galactosidase and using different biopolymers. They obtained product yields ranging from 36 to 59% (56% for Arabic gum, 48% for chitosan, 59% for modified chitosan, 36% for calcium alginate and 37% for sodium alginate). Also, Sansone et al. [49] encapsulated soybean extracts with sodium carboxymethyl cellulose and obtained a product yield of 65.3%, while Su et al. [50] obtained yield values ranging from 53 to 62% when microencapsulating Radix salvia
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miltiorrhiza using a mixture of gelatin and sodium carboxymethyl cellulose with different spray-drying conditions. 3.2. Microparticles characterization 3.2.1. SEM Microparticles loaded with CAF, CGA and RA were evaluated using scanning electron microscopy (Fig. 1). In all cases, a smooth surface and a spherical regular shape were observed. Similar results were obtained by other authors [43,46,49]. Studies performed by Sansone et al. found that Na-CMC spray-dried microparticles containing soybean extracts displayed small and spherical shape microparticles with no pores onto the surface able to promote the loss of the core material [49]. Spherical microparticles, with regular shape were also produced by Estevinho et al. [46] after encapsulation of β-galactosidase by spray-drying, using different biopolymers. However, the surface of the microparticles containing β-galactosidase presented different textural characteristics: very rough surface for particles formed with chitosan or Arabic gum while particles formed with calcium or sodium alginate or modified chitosan presented a very smooth surface [46]. Casanova et al. [43] prepared chitosan and modified chitosan microparticles loading rosmarinic acid using spray drying under the same operational conditions of this work. Modified chitosan microparticles presented a similar shape and surface comparing to Na-CMC microparticles while chitosan microparticles presented several wrinkles that were not observed in Na-CMC microparticles [43]. 3.2.2. Particle size distribution Particle size distribution analysis could be helpful to understand the behavior of the microparticles regarding the encapsulated agent controlled release and to infer about microparticles stability. The particle size can influence the texture and sensorial properties of the final product. Fig. 2 shows particle size distribution by volume and number. Table 2 shows the microparticles mean diameter for both distributions.
Fig. 1. SEM micrographs of Na-CMC microparticles loaded with RA (A), CGA (B) and CAF (C). Amplification of 10,000 times is presented in all micrographs (Beam intensity of 15.00 kV).
Fig. 2. Particle size distribution of the microparticles considering: A) volume distribution and B) number distribution.
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Table 2 Particle mean diameter by laser granulometry analysis. Sample
Na-CMC + CAF Na-CMC + CGA Na-CMC + RA
Table 3 Antioxidant activity of free and microencapsulated antioxidants.
Mean diameter (μm)
Sample
Differential number
Differential volume
0.41 ± 0.43 0.44 ± 0.44 0.36 ± 0.37
8.78 ± 9.62 8.37 ± 9.48 10.90 ± 12.18
Considering number distribution, Na-CMC microparticles loaded with CAF, CGA and RA presented an average size of 0.41, 0.44 and 0.36 μm, respectively (Table 2). On the other hand, considering volume distribution, the average size was in the micro-scale with average sizes of 8.78, 8.37 and 10.90 μm for CAF, CGA and RA, respectively. These results may suggest some polydispersity and/or aggregation of the particles. When considering industrial applications, size homogeneity of the microparticles is very important to guarantee consistency and similarity of the products, replicability and also to predict the microparticles behavior regarding their incorporation and release profile. However, the existence of polydispersity in the size of the microparticles is a characteristic of the spray drying methods [23]. On the other hand, the aggregation of the microparticles is undesirable in the final product. 3.3. Controlled release studies Protection and controlled release are the biggest advantages of microencapsulation. The controlled release allows the release under the desired conditions, improving compound effectiveness. Fig. 3 displays the obtained release profiles for the prepared Na-CMC microparticles, showing the release percentage (amount released at time t normalized by total released amount). In general, the results show similar release profiles for all the microparticles tested. Firstly, there is a sustained release of the active compound followed by a plateau level on the release profile, which means that the total release was achieved. A more detailed analysis suggests that CAF release is slightly faster than RA and CGA. Microparticles loaded with RA, CGA and CAF achieved total release after 4 h, 2 h and 45 min, respectively. However, 50% of the encapsulated acids was released between the first 5 and 10 min for all samples. Being a hydrophilic matrix-forming polymer, Na-CMC is able to swell in the presence of water forming a gel, and by this way release the core compounds. Particle size also influences controlled release. As previously seen, Na-CMC microparticles present small particle size and subsequently greater surface area to volume ratio, which favors the release. Similar results were found by Sansone et al. [49] while developing a sodium-carboxymethyl cellulose matrix by spray-drying to microencapsulate soy extracts, using different formulations and operating
CAF CGA RA
Antioxidant activity (μM TE) Free in solution
Encapsulated (after 4 h release in water)
176 ± 4 159 ± 9 208 ± 8
209 ± 6 207 ± 17 241 ± 2
μM TE: trolox equivalent.
conditions. The resulting microparticles were rapidly soluble in water and were able to release about 80–100% of the bioactive extract in 15–30 min [49]. The encapsulation of RA by spray drying was also performed by Casanova et al. [43] using chitosan and modified chitosan. Although chitosan microparticles showed similar release profiles as the ones presented in this work, the release of modified chitosan microparticles seemed faster than Na-CMC microparticles [43]. In another study performed by Zhang et al. [51], microparticles of chitosan and Na-CMC were prepared using bovine serum albumin as core material and emulsion phase separation method. Results evidenced that the combination of chitosan with Na-CMC slows the release of the protein due to the interaction of both polymers [51]. Another result that is possible to obtain from the release profiles is the encapsulation efficiency (EE) that measures the amount of antioxidant that was encapsulated. Free antioxidant (not encapsulated) is determined by measuring the amount of antioxidant right after dispersion of the microparticles in the solvent. EE values of 93% for microparticles loaded with CAF and 91% for both microparticles loaded with CGA and RA were obtained. Results show that the present method allows a successful encapsulation of these antioxidants within a Na-CMC matrix.
3.4. Antioxidant activity The antioxidant activity can be changed by the encapsulation process and/or interactions with the wall material. The results evidenced that RA exhibited a slightly greater antioxidant activity then CAF and CGA (Table 3). Furthermore, results showed a slight increase of the antioxidant activity for all of the compounds tested after the microencapsulation process. Interestingly, the same results were obtained by Carvalho et al. [52] regarding the sun protection factor of green coffee oil when encapsulated in corn syrup. The reasons for this increase are unknown, but the presence of Na-CMC might have contributed, leading to higher antioxidant activities. Also, it is well known that microencapsulation protects the antioxidant from degradation. In addition, several authors claim that after microencapsulation the core material still has
Fig. 3. Release profiles of Na-CMC microparticles in water.
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a proper antioxidant activity which is also confirmed by these results [52–54]. 4. Conclusion In this study, natural antioxidants (CAF, CGA and RA) were encapsulated by spray-drying using Na-CMC as encapsulating agent. The microparticles produced were characterized by their process product yield, particle size distribution (by laser granulometry), morphology (by scanning electron microscopy), controlled release in water and antioxidant activity (by ABTS assay). Product yield values were around 40%, for all samples. Size and morphology assessment revealed a smooth surface and spherical regular shape with mean diameter below 11 μm (considering volume size distribution). Controlled release profiles were similar for all the microparticles tested, however the total release was achieved with different times; after 45 min, 2 h and 4 h for microparticles loaded with CAF, CGA and RA, respectively. The encapsulation efficiency values were higher than 90% for all the microparticles. Antioxidant activity was determined for the different antioxidant microparticles prepared and the results showed higher values after the microencapsulation, proving that the microencapsulation process does not compromise the antioxidant capacity. The experiments performed during this work demonstrated the potential of Na-CMC spray-dried microparticles as natural antioxidants carriers, showing great encapsulation efficiency, total release ability and compounds stability without loss of antioxidant activity. Acknowledgments This work was financially supported by the projects POCI-01-0145FEDER-006939 — Laboratory for Process Engineering, Environment, Biotechnology and Energy — LEPABE and NORTE-01-0145-FEDER000005 – LEPABE-2-ECO-INNOVATION, funded by FEDER funds through COMPETE2020 — Programa Operacional Competitividade e Internacionalização (POCI) and Programa Operacional Regional do Norte (NORTE2020) and by national funds through FCT — Fundação para a Ciência e a Tecnologia, with the grant SFRH/BPD/73865/2010 (B. N. Estevinho). References [1] E. Stelmach, P. Pohl, A. Szymczycha-madeja, The content of Ca, Cu, Fe, Mg and Mn and antioxidant activity of green coffee brews, Food Chem. 182 (2015) 302–308, http://dx.doi.org/10.1016/j.foodchem.2015.02.105. [2] A. Yashin, Y. Yashin, J. Wang, B. Nemzer, Antioxidant and antiradical activity of coffee, Antioxidants 2 (2013) 230–245, http://dx.doi.org/10.3390/antiox2040230. [3] A.M. Pisoschi, A. Pop, The role of antioxidants in the chemistry of oxidative stress: a review, Eur. J. Med. Chem. 97 (2015) 55–74, http://dx.doi.org/10.1016/j.ejmech. 2015.04.040. [4] F.S. Shahidi, U.N. Wanasundara, Methods for measuring oxidative rancidity in fats and oils, Food Lipids—Chemistry, Nutrition and Biotechnology 2008, pp. 387–407 (doi:10.1201/9781420046649.ch14). [5] Y. Guan, Q. Chu, L. Fu, J. Ye, Determination of antioxidants in cosmetics by micellar electrokinetic capillary chromatography with electrochemical detection, J. Chromatogr. A 1074 (2005) 201–204, http://dx.doi.org/10.1016/j.chroma.2005.03.063. [6] M.E. Embuscado, Spices and herbs: natural sources of antioxidants — a mini review, J. Funct. Foods 18 (2015) 811–819, http://dx.doi.org/10.1016/j.jff.2015.03.005. [7] J. Chao, H. Wang, W. Zhao, M. Zhang, L. Zhang, Investigation of the inclusion behavior of chlorogenic acid with hydroxypropyl-β-cyclodextrin, Int. J. Biol. Macromol. 50 (2012) 277–282, http://dx.doi.org/10.1016/j.ijbiomac.2011.11.008. [8] G. Lozano-Vazquez, C. Lobato-Calleros, H. Escalona-Buendia, G. Chavez, J. AlvarezRamirez, E.J. Vernon-Carter, Effect of the weight ratio of alginate-modified tapioca starch on the physicochemical properties and release kinetics of chlorogenic acid containing beads, Food Hydrocoll. 48 (2015) 301–311, http://dx.doi.org/10.1016/j. foodhyd.2015.02.032. [9] S. Levi, V. Manojlovi, B. Bugarski, V. Nedovi, A. Kalusevic, An overview of encapsulation technologies for food applications, Proc. Food Sci. 1 (2011) 1816–1820, http:// dx.doi.org/10.1016/j.profoo.2011.09.266. [10] A. Poshadri, A. Kuna, Microencapsulation technology: a review, J. Res. ANGRAU 38 (2010) 86–102. [11] N. Wilson, N.P. Shah, Microencapsulation of vitamins, Int. Food Res. J. 14 (2007) 1–14.
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Design of microparticles containing natural antioxidants: Preparation, characterization and controlled release studies Joana Aguiar 1, Raquel Costa 1, F. Rocha, B.N. Estevinho ⁎, L. Santos LEPABE, Departamento de Engenharia Química, Faculdade de Engenharia da Universidade do Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal
a r t i c l e
i n f o
Article history: Received 18 December 2016 Received in revised form 26 February 2017 Accepted 3 March 2017 Available online 06 March 2017 Keywords: Carboxymethyl cellulose Microencapsulation Natural antioxidants Spray drying
a b s t r a c t Antioxidants are important compounds in the prevention of diseases like cancer, diabetes, cardiovascular, neurodegenerative diseases and premature aging, however they are very sensitive to the light, heat, oxygen and pH. In this study, caffeic acid (CAF), chlorogenic acid (CGA) and rosmarinic acid (RA), 3 natural antioxidants, were encapsulated by spray-drying using sodium carboxymethyl cellulose (Na-CMC) to overcome their limitations in industrial applications. Product yield values were around 40% and the encapsulation efficiency values were higher than 90%. Particles revealed a smooth spherical shape with mean diameter below 11 μm (considering volume size distribution). Microparticles loaded with CAF, CGA and RA were fully released after 45 min, 2 h and 4 h, respectively. Antioxidant activity was not compromised, highlighting the potential of spray-dried Na-CMC microparticles as carriers of natural antioxidants. © 2017 Elsevier B.V. All rights reserved.
1. Introduction According to recent studies, the excessive accumulation in the human body of products derived from oxygen and nitrogen reactions is responsible for premature aging and diseases like cancer, diabetes, cardiovascular and neurodegenerative diseases (Alzheimer and Parkinson) [1,2]. Antioxidants are a powerful tool to reduce the oxidative stress allowing the stopping or delay of a chain reaction through several mechanisms: reacting with free radicals, by depleting molecular oxygen, deactivating singlet oxygen, removing prooxidative metal ions, replenishing hydrogen to other antioxidants and absorbing UV light [3,4] and, furthermore, antioxidants can also be used as preservatives avoiding the oxidation of lipid ingredients [4]. Recently, interest in natural antioxidants is rising comparing to synthetic ones due to the consumer preference of organic and natural products with less additives and side effects [5,6]. However, natural antioxidants present some characteristics which may limit their industrial application such as sensitivity to heat, oxygen, pH and light [7,8] as well as unpleasant taste or smell, poor availability, high reactivity with other ingredients present in the product matrix and also high susceptibility to storage and processing conditions [9–11]. Microencapsulation may be used to overcome some of the restrictions of antioxidants and increase their applicability. Examples of final products where encapsulated antioxidants may be incorporated include cosmetic preparations [12], food products and supplements [13] and ⁎ Corresponding author. E-mail address: [email protected] (B.N. Estevinho). Equal participation as first author.
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http://dx.doi.org/10.1016/j.powtec.2017.03.013 0032-5910/© 2017 Elsevier B.V. All rights reserved.
food packaging [14]. In the microencapsulation technique, solid, liquid or gaseous compounds are surrounded by a coating material creating a microparticle that can be used in food, pharmaceutical, cosmetic, agrochemical and textile industries [12,15,16]. Some advantages of microencapsulation techniques include higher stability by isolating active ingredients in order to prevent their deterioration, delayed evaporation in case of a volatile core, controlled and targeted release of active ingredients and improved product esthetics and marketing perception, while maintaining core properties [17–21]. Microencapsulation is also able to mask core undesired properties such as undesirable taste, odor or activity and to reduce the amount of ingredients in formulation being a cost saving alternative [22,23]. There are various microencapsulation techniques available, although, spray-drying is one of the most used, due its simplicity, relatively low cost, flexibility, high stability of the final dried product (due to low moisture content), high volume reduction, ease of handling, transportation and storage of the particles [11]. It also allows a continuous operation and appropriate encapsulation of many heat-labile (low-boiling point) materials due to the lower temperatures the core material reaches [9,24–26]. Currently, substances such as antibiotics, medical ingredients, additives, vitamins and polyphenols, among others are encapsulated in large-scale using spraydrying [9,23,25,27]. In this work, 3 natural antioxidants (caffeic acid (CAF), chlorogenic acid (CGA) and rosmarinic acid (RA)) were selected, considering their aforementioned advantages for industrial applications, and microencapsulated in sodium carboxymethyl cellulose (Na-CMC). CAF and CGA are polyphenols mainly found in the coffee tree although other sources include fruits (e.g. apple, pear, berries, plum), vegetables (e.g. sweet potato, lettuce, spinach), black teas, soy beans and wheat
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[28,29]. RA is also a polyphenol, found in a wide variety of plants from the Lamiaceae family: oregano, rosemary, marjoram, clary sage, thyme, basil [30]. According to literature, there are several benefits related to these compounds besides their strong antioxidant activity: anti-inflammatory, anti-microbial and anti-viral properties, as well as prevention of diseases associated with oxidative stress (namely cardiovascular, cancer and neurodegenerative) [30–38]. On the other hand, carboxymethyl cellulose (CMC) is a water soluble anionic cellulose derivative with carboxymethyl substitution groups and has applications in a wide range of fields such as food, cosmetic, pharmaceutical, textiles, and detergents. Carboxymethyl cellulose is already widely used in cosmetic and personal products as emulsifier, stabilizer, film-former and thickener agent [39]. Carboxymethyl cellulose hydrogel swelling behavior is pH and ionic strength dependent due to the presence of electrostatic charges in the polymer network, originated from sodium ion and the carboxymethyl group [40]. Films formed using carboxymethyl cellulose have in general a moderate strength in aqueous solutions although such property is dependent on the degree of substitution and molecular weight [41,42]. The main purpose of this work was to perform the microencapsulation by spray drying of these three different antioxidant compounds (CAF, CGA and RA) using Na-CMC, considering the advantages of these natural antioxidants and also of the encapsulating agent. After encapsulation, the microparticles were characterized in terms of shape and size distribution, and controlled release studies were performed. The antioxidant capacity of the encapsulated acids was also evaluated. 2. Materials and methods 2.1. Chemicals Caffeic acid standard (Ref. C0625-2G) and chlorogenic acid standard (3-caffeoylquinic acid) were acquired from Sigma-Aldrich Chemical Co. (MO, USA). Rosmarinic acid (Ref. 536,954-5G) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Methanol was obtained from VWR International (Fontenay-sous-Bois, France). Carboxymethylcellulose sodium salt (viscosity 830 mPa·s (25 °C, 2% water)) was obtained from VWR International (Haasrode, Belgium) and methanol from VWR International (Fontenay-sous-Bois, France). ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) was purchased from AppliChem GmbH (Darmstadt, Germany), potassium persulfate was acquired from Panreac Química (Barcelona, Spain) and trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was obtained from Sigma-Aldrich Chemical Co. (MO, USA). Ethanol (96% purity) was obtained from AGA (Prior Velho, Portugal). Water was deionized in laboratory using Millipore™ water purification equipment (Massachusetts, USA). All the reagents used were of analytical grade purity.
The CAF calibration curve was linear from 0.25 to 15 mg/L and the linear regression was the following: Abs (AU) = 0.067 ± 0.001 [CAF] + 0.009 ± 0.006 (R2 = 0.999). Values obtained for limit of detection (LOD) and limit of quantification (LOQ) were, respectively, 0.29 and 0.96 mg/L. The CGA calibration curve was linear from 0.50 to 14 mg/L and the linear regression was the following: Abs (AU) = 0.0554 ± 0.0002 [CGA] − 0.003 ± 0.002 (R2 = 1.000). Values obtained for LOD and LOQ were, respectively, 0.10 and 0.34 mg/L. Finally, the RA calibration curve was linear from 1 to 15 mg/L and the linear regression was the following: Abs = 0.059 ± 0.003[RA] + 0.02 ± 0.03 (R2 = 1.000). Values obtained for LOD and LOQ were, respectively, 0.50 and 1.66 mg/L. The analytical method presented good intra-day and inter-day precision (coefficients of variation smaller than 5% except for the lowest concentrations) as well as great accuracy (recovery percentage close to 100%) for the antioxidants tested. 2.4. Preparation of microparticles by spray-drying Microparticles were prepared by spray-drying as described by [43–45]. Separate solutions of Na-CMC (10 g/L) and antioxidants (10 g/L for CAF and CGA, and 1 g/L for RA) were prepared in ultrapure water, at room temperature, under stirring. Before encapsulation, Na-CMC and antioxidant solution were mixed together under stirring, during 30 min, at room temperature. The antioxidant concentration in the feed solution was around 2%. The experimental conditions were previously optimized [46,47]: feed flow rate of 4 mL/min (15%), inlet temperature of 115 °C, air pressure of 6.0 bar, 100% aspiration rate and nozzle cleaner set to 3. The outlet temperature was around 60 °C. The powders were collected and stored in falcon tubes, wrapped in aluminum foil, and stored at 4 °C. 2.5. Microparticles characterization The size distribution of the microparticles was evaluated by laser granulometry using a Coulter Counter-LS 230 Particle Size Analyzer (Miami, FL, USA). For each experiment, a small sample of powder was suspended in ethanol before measurement. Samples were characterized by number and volume as an average of two runs of 60 s. Particles morphology was assessed by SEM analysis using a High resolution (Schottky) Environmental Scanning Electron Microscope with X-Ray Microanalysis and Electron Backscattered Diffraction analysis: Quanta 400 FEG ESEM/EDAX Genesis X4M. Samples were coated with Au/Pd thin film for 100 s and with a 15 mA current, by sputtering, using the SPI Module Sputter Coater equipment. 2.6. Controlled release studies
Weight measurements were performed with an analytical scale Mettler Toledo AG245 (Columbus, OH, USA). Quantification analysis of the antioxidants was accomplished using a spectrophotometer UV–Vis V-530 (Jasco), as well as the antioxidant activity assays. Microencapsulation was performed using a mini spray dryer BÜCHI B-290 (Flawil, Switzerland) with a standard 0.5 mm nozzle.
The controlled release studies were performed in ultrapure water (pH 5.6) by weighing 3 mg of each antioxidant powder into separate flasks (in duplicate) and then adding 4.5 mL of water. The release was performed at room temperature (20–25 °C), under low stirring and in the absence of light (by wrapping the flask with aluminum foil). Samples were taken every 0, 1, 2, 5, 10, 20, 30, 45 min and 1, 2, 4, 6, 24 h to evaluate the amount of antioxidant released using the UVspectrophotometry method.
2.3. Validation of analytical method
2.7. Antioxidant activity
CAF, CGA and RA were quantified by UV–Vis spectrometry (Jasco V-530 UV–Vis Spectrophotometer; Easton, USA) with detection at 313, 323 and 324 nm, respectively. Standard solutions of each antioxidant were analyzed in duplicate and three calibration curves were obtained. The main validation performance and reliability parameters of the analytical method for each antioxidant were determined.
The antioxidant activity was estimated in duplicate using the ABTS radical scavenging assay, as described by [48]. Samples consisted of Na-CMC microparticles loaded with CAF, CGA and RA at maximum release (after 4 h in aqueous solution) and also non-loaded microparticles of Na-CMC. The antioxidant activity of samples containing CAF, CGA and RA in free solution with the same amount as in the microparticles was
2.2. Equipment
J. Aguiar et al. / Powder Technology 313 (2017) 287–292 Table 1 Product yield values for CAF, CGA and RA encapsulated in Na-CMC by spray-drying. Sample
Product yield (%)
Na-CMC + CAF Na-CMC + CGA Na-CMC + RA
37.6 40.8 38.4
also assessed. Antioxidant activity was expressed in μM Trolox (SigmaAldrich) equivalents (TE), a synthetic analogue of vitamin E commonly used as antioxidant reference. 3. Results and discussion 3.1. Product yield The product yield of the encapsulation process relates the amount of powder that it is possible to recover comparing to the initial raw material used. Product yield values for the spray-dried microparticles are presented in Table 1. The results were quite satisfactory considering the employed method and scale used. Losses of the solid content may be explained by the significant small amount of used raw materials when compared to the equipment dimensions. Particles size may also contribute to solid losses since small microparticles are suctioned by the vacuum filter and therefore fail to be collected in the final powder. Similar results were found by Estevinho et al. [46] after producing microparticles containing β-galactosidase and using different biopolymers. They obtained product yields ranging from 36 to 59% (56% for Arabic gum, 48% for chitosan, 59% for modified chitosan, 36% for calcium alginate and 37% for sodium alginate). Also, Sansone et al. [49] encapsulated soybean extracts with sodium carboxymethyl cellulose and obtained a product yield of 65.3%, while Su et al. [50] obtained yield values ranging from 53 to 62% when microencapsulating Radix salvia
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miltiorrhiza using a mixture of gelatin and sodium carboxymethyl cellulose with different spray-drying conditions. 3.2. Microparticles characterization 3.2.1. SEM Microparticles loaded with CAF, CGA and RA were evaluated using scanning electron microscopy (Fig. 1). In all cases, a smooth surface and a spherical regular shape were observed. Similar results were obtained by other authors [43,46,49]. Studies performed by Sansone et al. found that Na-CMC spray-dried microparticles containing soybean extracts displayed small and spherical shape microparticles with no pores onto the surface able to promote the loss of the core material [49]. Spherical microparticles, with regular shape were also produced by Estevinho et al. [46] after encapsulation of β-galactosidase by spray-drying, using different biopolymers. However, the surface of the microparticles containing β-galactosidase presented different textural characteristics: very rough surface for particles formed with chitosan or Arabic gum while particles formed with calcium or sodium alginate or modified chitosan presented a very smooth surface [46]. Casanova et al. [43] prepared chitosan and modified chitosan microparticles loading rosmarinic acid using spray drying under the same operational conditions of this work. Modified chitosan microparticles presented a similar shape and surface comparing to Na-CMC microparticles while chitosan microparticles presented several wrinkles that were not observed in Na-CMC microparticles [43]. 3.2.2. Particle size distribution Particle size distribution analysis could be helpful to understand the behavior of the microparticles regarding the encapsulated agent controlled release and to infer about microparticles stability. The particle size can influence the texture and sensorial properties of the final product. Fig. 2 shows particle size distribution by volume and number. Table 2 shows the microparticles mean diameter for both distributions.
Fig. 1. SEM micrographs of Na-CMC microparticles loaded with RA (A), CGA (B) and CAF (C). Amplification of 10,000 times is presented in all micrographs (Beam intensity of 15.00 kV).
Fig. 2. Particle size distribution of the microparticles considering: A) volume distribution and B) number distribution.
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Table 2 Particle mean diameter by laser granulometry analysis. Sample
Na-CMC + CAF Na-CMC + CGA Na-CMC + RA
Table 3 Antioxidant activity of free and microencapsulated antioxidants.
Mean diameter (μm)
Sample
Differential number
Differential volume
0.41 ± 0.43 0.44 ± 0.44 0.36 ± 0.37
8.78 ± 9.62 8.37 ± 9.48 10.90 ± 12.18
Considering number distribution, Na-CMC microparticles loaded with CAF, CGA and RA presented an average size of 0.41, 0.44 and 0.36 μm, respectively (Table 2). On the other hand, considering volume distribution, the average size was in the micro-scale with average sizes of 8.78, 8.37 and 10.90 μm for CAF, CGA and RA, respectively. These results may suggest some polydispersity and/or aggregation of the particles. When considering industrial applications, size homogeneity of the microparticles is very important to guarantee consistency and similarity of the products, replicability and also to predict the microparticles behavior regarding their incorporation and release profile. However, the existence of polydispersity in the size of the microparticles is a characteristic of the spray drying methods [23]. On the other hand, the aggregation of the microparticles is undesirable in the final product. 3.3. Controlled release studies Protection and controlled release are the biggest advantages of microencapsulation. The controlled release allows the release under the desired conditions, improving compound effectiveness. Fig. 3 displays the obtained release profiles for the prepared Na-CMC microparticles, showing the release percentage (amount released at time t normalized by total released amount). In general, the results show similar release profiles for all the microparticles tested. Firstly, there is a sustained release of the active compound followed by a plateau level on the release profile, which means that the total release was achieved. A more detailed analysis suggests that CAF release is slightly faster than RA and CGA. Microparticles loaded with RA, CGA and CAF achieved total release after 4 h, 2 h and 45 min, respectively. However, 50% of the encapsulated acids was released between the first 5 and 10 min for all samples. Being a hydrophilic matrix-forming polymer, Na-CMC is able to swell in the presence of water forming a gel, and by this way release the core compounds. Particle size also influences controlled release. As previously seen, Na-CMC microparticles present small particle size and subsequently greater surface area to volume ratio, which favors the release. Similar results were found by Sansone et al. [49] while developing a sodium-carboxymethyl cellulose matrix by spray-drying to microencapsulate soy extracts, using different formulations and operating
CAF CGA RA
Antioxidant activity (μM TE) Free in solution
Encapsulated (after 4 h release in water)
176 ± 4 159 ± 9 208 ± 8
209 ± 6 207 ± 17 241 ± 2
μM TE: trolox equivalent.
conditions. The resulting microparticles were rapidly soluble in water and were able to release about 80–100% of the bioactive extract in 15–30 min [49]. The encapsulation of RA by spray drying was also performed by Casanova et al. [43] using chitosan and modified chitosan. Although chitosan microparticles showed similar release profiles as the ones presented in this work, the release of modified chitosan microparticles seemed faster than Na-CMC microparticles [43]. In another study performed by Zhang et al. [51], microparticles of chitosan and Na-CMC were prepared using bovine serum albumin as core material and emulsion phase separation method. Results evidenced that the combination of chitosan with Na-CMC slows the release of the protein due to the interaction of both polymers [51]. Another result that is possible to obtain from the release profiles is the encapsulation efficiency (EE) that measures the amount of antioxidant that was encapsulated. Free antioxidant (not encapsulated) is determined by measuring the amount of antioxidant right after dispersion of the microparticles in the solvent. EE values of 93% for microparticles loaded with CAF and 91% for both microparticles loaded with CGA and RA were obtained. Results show that the present method allows a successful encapsulation of these antioxidants within a Na-CMC matrix.
3.4. Antioxidant activity The antioxidant activity can be changed by the encapsulation process and/or interactions with the wall material. The results evidenced that RA exhibited a slightly greater antioxidant activity then CAF and CGA (Table 3). Furthermore, results showed a slight increase of the antioxidant activity for all of the compounds tested after the microencapsulation process. Interestingly, the same results were obtained by Carvalho et al. [52] regarding the sun protection factor of green coffee oil when encapsulated in corn syrup. The reasons for this increase are unknown, but the presence of Na-CMC might have contributed, leading to higher antioxidant activities. Also, it is well known that microencapsulation protects the antioxidant from degradation. In addition, several authors claim that after microencapsulation the core material still has
Fig. 3. Release profiles of Na-CMC microparticles in water.
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a proper antioxidant activity which is also confirmed by these results [52–54]. 4. Conclusion In this study, natural antioxidants (CAF, CGA and RA) were encapsulated by spray-drying using Na-CMC as encapsulating agent. The microparticles produced were characterized by their process product yield, particle size distribution (by laser granulometry), morphology (by scanning electron microscopy), controlled release in water and antioxidant activity (by ABTS assay). Product yield values were around 40%, for all samples. Size and morphology assessment revealed a smooth surface and spherical regular shape with mean diameter below 11 μm (considering volume size distribution). Controlled release profiles were similar for all the microparticles tested, however the total release was achieved with different times; after 45 min, 2 h and 4 h for microparticles loaded with CAF, CGA and RA, respectively. The encapsulation efficiency values were higher than 90% for all the microparticles. Antioxidant activity was determined for the different antioxidant microparticles prepared and the results showed higher values after the microencapsulation, proving that the microencapsulation process does not compromise the antioxidant capacity. The experiments performed during this work demonstrated the potential of Na-CMC spray-dried microparticles as natural antioxidants carriers, showing great encapsulation efficiency, total release ability and compounds stability without loss of antioxidant activity. Acknowledgments This work was financially supported by the projects POCI-01-0145FEDER-006939 — Laboratory for Process Engineering, Environment, Biotechnology and Energy — LEPABE and NORTE-01-0145-FEDER000005 – LEPABE-2-ECO-INNOVATION, funded by FEDER funds through COMPETE2020 — Programa Operacional Competitividade e Internacionalização (POCI) and Programa Operacional Regional do Norte (NORTE2020) and by national funds through FCT — Fundação para a Ciência e a Tecnologia, with the grant SFRH/BPD/73865/2010 (B. N. Estevinho). References [1] E. Stelmach, P. Pohl, A. Szymczycha-madeja, The content of Ca, Cu, Fe, Mg and Mn and antioxidant activity of green coffee brews, Food Chem. 182 (2015) 302–308, http://dx.doi.org/10.1016/j.foodchem.2015.02.105. [2] A. Yashin, Y. Yashin, J. Wang, B. Nemzer, Antioxidant and antiradical activity of coffee, Antioxidants 2 (2013) 230–245, http://dx.doi.org/10.3390/antiox2040230. [3] A.M. Pisoschi, A. Pop, The role of antioxidants in the chemistry of oxidative stress: a review, Eur. J. Med. Chem. 97 (2015) 55–74, http://dx.doi.org/10.1016/j.ejmech. 2015.04.040. [4] F.S. Shahidi, U.N. Wanasundara, Methods for measuring oxidative rancidity in fats and oils, Food Lipids—Chemistry, Nutrition and Biotechnology 2008, pp. 387–407 (doi:10.1201/9781420046649.ch14). [5] Y. Guan, Q. Chu, L. Fu, J. Ye, Determination of antioxidants in cosmetics by micellar electrokinetic capillary chromatography with electrochemical detection, J. Chromatogr. A 1074 (2005) 201–204, http://dx.doi.org/10.1016/j.chroma.2005.03.063. [6] M.E. Embuscado, Spices and herbs: natural sources of antioxidants — a mini review, J. Funct. Foods 18 (2015) 811–819, http://dx.doi.org/10.1016/j.jff.2015.03.005. [7] J. Chao, H. Wang, W. Zhao, M. Zhang, L. Zhang, Investigation of the inclusion behavior of chlorogenic acid with hydroxypropyl-β-cyclodextrin, Int. J. Biol. Macromol. 50 (2012) 277–282, http://dx.doi.org/10.1016/j.ijbiomac.2011.11.008. [8] G. Lozano-Vazquez, C. Lobato-Calleros, H. Escalona-Buendia, G. Chavez, J. AlvarezRamirez, E.J. Vernon-Carter, Effect of the weight ratio of alginate-modified tapioca starch on the physicochemical properties and release kinetics of chlorogenic acid containing beads, Food Hydrocoll. 48 (2015) 301–311, http://dx.doi.org/10.1016/j. foodhyd.2015.02.032. [9] S. Levi, V. Manojlovi, B. Bugarski, V. Nedovi, A. Kalusevic, An overview of encapsulation technologies for food applications, Proc. Food Sci. 1 (2011) 1816–1820, http:// dx.doi.org/10.1016/j.profoo.2011.09.266. [10] A. Poshadri, A. Kuna, Microencapsulation technology: a review, J. Res. ANGRAU 38 (2010) 86–102. [11] N. Wilson, N.P. Shah, Microencapsulation of vitamins, Int. Food Res. J. 14 (2007) 1–14.
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