Production of water soluble quercetin formulations by pressurized ethyl acetate-in-water emulsion technique using natural origin surfactants

Production of water soluble quercetin formulations by pressurized ethyl acetate-in-water 1 emulsion technique using natural origin surfactants 2 V.S.S. Gonçalvesa, b, c, S. Rodríguez-Rojoc, *, E. De Paz c, C. Mato c, A. Martín c, M.J. Cocero c 3 a Instituto de Tecnologia Química e Biologica Antonio Xavier, Universidade Nova de Lisboa, Av. 4 da República, 2780-157 Oeiras, Portugal 5 b iBET, Instituto de Biologia Experimental e Tecnologica, Apartado 12, 2780-901 Oeiras, 6 Portugal 7 c Escuela de Ingenierías Industriales, Universidad de Valladolid, C/Dr. Mergelina s/n, 47011 8 Valladolid, Spain 9 Abstract 10 Quercetin is a strong antioxidant flavonoid with several bioactive properties such as anti11 inflammatory and anticarcinogenic activities, becoming an interesting compound to be 12 incorporated into pharmaceutical, cosmetic or food products. However, these applications are 13 limited by the low bioavailability of this flavonoid. Quercetin is poorly soluble in aqueous 14 media, such as gastrointestinal fluids, being also degraded by gut flora. Thus, it is necessary the 15 development of quercetin’s formulations capable of improving its water solubility resulting in 16 increased bioavailability and thus higher biological activity of this compound. 17 The aim of the present work was the formulation of quercetin using three distinct natural 18 origin surfactants, namely OSA-starch, Lecithin and β-glucan, by precipitation from a 19 pressurized ethyl acetate-in-water emulsion. Formulations of quercetin with encapsulation 20 efficiencies up to near 76% and a micellar particle size in the range of nanometers were 21 obtained using lecithin. An improved antioxidant activity (3-fold higher per unit mass of 22 quercetin) was also observed in these formulations, demonstrating that lecithin is a good 23 emulsifier for the encapsulation of quercetin. Furthermore, the addition of glycerol as co24 solvent increased the colloidal stability of the suspension and the encapsulation efficiency 25 of the flavonoid. 26 27 Keywords: Quercetin, OSA-starch, Lecithin, β-glucan, Encapsulation, High Pressure Emulsion 28 Technique, Nanosuspension 29 30 1. Introduction 31 Quercetin (3,3',4',5,7-Pentahydroxyflavone), one of the most representative member of the 32 flavonoid family with high antioxidant activity, is commonly found in several fruits and 33 vegetables like onions, apples, grapes or strawberries, as well as in red wine or green/black tea 34 [1,2]. This compound has attracted the interest of the pharmaceutical and nutraceutical 35 industries due to its bioactive properties, such as anti-inflammatory, anti-proliferative and 36 neuroprotective effects [1,3]. In order to achieve quercetin plasma’s concentration above 10 37 mM required for obtaining pharmacological activity, the ingestion of quercetin-enriched foods 38 or supplements could not be enough due to the low bioavailability of this flavonoid [4]. The 39 low water solubility (2ppm at 25ºC to 60 ppm at 100 °C) allied with gastrointestinal 40 degradation limits quercetin’s biological effects in vivo [2,5]. There are two main approaches to 41 increase the bioavailability of this compound, whether by chemical modification or by the 42 development of colloidal quercetin delivery systems [6]. 43 Formulations of quercetin using different methods and distinct carrier materials have been 44 developed by several authors. Regarding polymers, Kumari and co-authors achieved a 45 controlled release of quercetin by its encapsulation into poly-D,L-lactide (PLA) nanoparticles 46 through solvent evaporation technique [7]. Wu et al. have produced quercetin-loaded 47 nanoparticles by a nanoprecipitation method using Eudragit® E and polyvinyl alcohol (PVA) as 48 carriers, obtaining a quercetin’s release 74-fold higher in comparison with the pure compound 49 [8]. Quercetin was also encapsulated in Pluronic F127 through supercritical antisolvent method 50 by Fraile and co-authors, enabling an improved dissolution behaviour of the compound in 51 simulated physiological fluids [9]. Cyclodextrins (CD), such as α-CD, β-CD or HP-β-CD, have also 52 been used for the encapsulation of quercetin using the freeze-drying or co-evaporation 53 method [10,11]. Barras and co-authors used lipids to encapsulate quercetin, being able to 54 increase its apparent aqueous solubility by a factor of 100 [6]. Besides solid lipid nanoparticles, 55 nanostructured lipid carriers and lipid nanoemulsions were produced using a high pressure 56 homogenizer for the encapsulation of quercetin by Aditya et al., achieving encapsulation 57 efficiencies above 90% [12]. Quercetin-loaded liposomes have also been produced and are 58 reported in the literature [1,13]. Inorganic materials can also be an option as carriers for the 59 encapsulation of quercetin and, recently, quercetin-loaded silica microspheres were developed 60 by Kim and co-authors using polyol-in-oil-in-water (P/O/W) emulsion and sol-gel methods to 61 improve the flavonoid’s stability as well as its properties [14]. 62 In 2012, de Paz and co-authors [15] developed a novel method for the encapsulation of 63 hydrophobic compounds, based on the production and processing of ethyl acetate-water 64 emulsions at high pressure and temperature. The authors achieved stable aqueous 65 suspensions of β-carotene with micellar particle sizes down to 400 nm and encapsulation 66 efficiencies up to 80%. Moreover, the type of emulsifier used had been shown to affect the 67 final properties of the suspension [16]. This process is an attractive alternative to the 68 conventional emulsion evaporation process, since it enables the acceleration of the mass 69 transfer kinetics to the time scales of the precipitation processes. This intensification of the 70 process allows an improved control over the precipitation, at the same time that the 71 exposition of the product to degrading high-temperature conditions is decreased. 72 This work presents the development of water soluble formulations of quercetin through 73 pressurized ethyl acetate-water emulsion technique. Ethyl acetate has been chosen as organic 74 solvent because it is a Generally Recognized as Safe (GRAS) solvent with low toxicity (Lethal 75 Dose LD50 in rats: 11.3 g/kg) and it can be safely used as a flavouring agent [17]. Three natural 76 origin surfactants, namely modified n-octenyl succinate anhydride (OSA) starch, soybean 77 lecithin and barley β-glucan, were used in this work for the encapsulation of quercetin. Starch 78 is the second most abundant biomass material present in nature being the most abundant 79 storage polysaccharide in plants [18,19]. However, natural starch is mainly hydrophilic, which 80 could limit its application in the encapsulation of hydrophobic compounds. Nevertheless, OSA 81 (modified amphiphilic starch) is capable to overcome this drawback, and was already used for 82 the encapsulation and delivery of compounds with distinct polarities[19]. Lecithin is a mixture 83 of naturally occurring phospholipids, mainly phosphatidylcholine, which is usually available 84 from sources such as soybeans or eggs. Phospholipids are amphiphilic molecules composed by 85 hydrophobic tails and hydrophilic heads, being capable to rearrange themselves as liposomes, 86 spherical and closed structures composed of lipid bilayers [20,21]. Liposomes are interesting 87 carrier materials for the delivery of hydrophobic/hydrophilic compounds, and since they have 88 affinity to cellular membranes, they are capable to increase the absorption of several drugs 89 [22]. Besides the good properties as encapsulating agent and emulsifier, lecithin is also known 90 for its antioxidant activity [23]. β-glucans are soluble fibers present in cereal grains, especially 91 in barley, constituted by linear polysaccharides of glucose units, connected by (1 → 3) or (1 → 92 4)-beta linkages. These carbohydrates are known for their therapeutic effects on coronary 93 heart disease, diabetes and hypercholesterolemia, and have been used as encapsulating 94 agents [24–27]. The influence of the main process parameters has been studied, namely the 95 effect of quercetin and emulsifier’s concentration, the effect of the flows of organic solvent, 96 suspension of quercetin and dissolution of emulsifier and also the organic to water ratio. By 97 comparing the results obtained with the three different emulsifiers, their roles on the 98 emulsion formation and quercetin’s encapsulation can be established. Product analysis 99 included particle size, encapsulation efficiency, antioxidant activity and structural 100 characterisation. 101 102 2. Materials and methods 103 2.1. Materials 104 Quercetin hydrate (Q) with a (purity≥95%) was purchased from Sigma-Aldrich. Ethyl Acetate 105 with a purity of 99.5% and glycerol were purchased from Panreac Química (Barcelona, Spain). 106 Modified OSA-starch refined from waxy maize was kindly provided by National Starch Group 107 (Hamburg, Germany). Soybean lecithin (97% phospholipids) was obtained from Glama-Sot 108 (SOTYA, Madrid, Spain). Glucagel™ (barley β-Glucan) was kindly supplied by DKSH France 109 (purity 78%, MW : 125 -140 kDa). 110 2.2. Precipitation from pressurized ethyl acetate-on-water emulsions 111 The equipment used in this work, already described by De Paz et al[15,16], is represented in 112 Fig. 1 with a schematic flow diagram. 113 (FIGURE 1) 114 Briefly, it consists of three small storages at ambient pressure, corresponding to the feed of 115 pure ethyl acetate (V-1), quercetin suspension in the same organic solvent (V-2) and the 116 aqueous solution of the emulsifier (V-3). The installation also counts with two piston pumps 117 GILSON 305 (maximum flow rate: 25 mL/min; flow rate control with an a accuracy of 0.1 118 mL/min) used to feed the aqueous dissolution of the emulsifier and the quercetin suspension 119 (pumps P-3 and P-2, respectively) and a piston pump JASCO PU-2080 plus (maximum flow rate: 120 10 mL/min; flow control with an a accuracy of 0.1 mL/min) used to feed the pure organic 121 solvent (pump P-1). An oven (KNK-2000-C series GAS CHROMATOGRAPH) is used to preheat 122 the organic solvent stream. 123 This process starts with the total dissolution of quercetin in hot and pressurized ethyl acetate, 124 where by increasing temperature, it is possible to increase the solubility of quercetin in this 125 solvent, which is around 1g/L at ambient conditions. In this work, a temperature, typically, 126 between 125-140ºC was used, keeping a constant pressure between 6.0 and 6.5 MPa in order 127 to maintain the solvent in liquid state. The dissolution is achieved by mixing a flavonoid’s 128 suspension in pressurized ethyl acetate at ambient temperature, with a stream of preheated 129 and pressurized ethyl acetate, using a Tmixer (M-1 in Fig. 1). In order to reduce the exposure 130 of quercetin to high temperatures, this hot and pressurized solution is mixed with the 131 ambient-temperature aqueous solution of emulsifier using the second T-mixer of the 132 equipment (M2). Therefore, the contact time between quercetin and the hot solvent is 133 reduced to less than two seconds, avoiding the degradation of the product. The contact of the 134 hot solution of quercetin with the aqueous solution in mixer M-2 causes the emulsification of 135 the organic solvent and the precipitation of quercetin by a combined antisolvent and cooling 136 effect. The last step is the removal of the organic solvent from the formed emulsion using a 137 rotary evaporator, in order to obtain an aqueous dispersion of quercetin particles stabilized by 138 the surfactant. 139 A typical experiment started with the preparation of an aqueous solution of emulsifier and a 140 suspension of quercetin in ethyl acetate, which were pre-heated and stirred in order to obtain 141 homogeneous mixtures. Afterwards, pumps were switched on and the pressure in the system 142 was fixed between 6.0 and 6.5 MPa. The oven was switched on to heat the organic solvent 143 until the desired temperature (140-150ºC). When the required temperature in mixer M-1 was 144 reached, the pure solvents used until this moment were replaced with the quercetin 145 suspension, in continuous agitation, and with the aqueous solution of the emulsifier. In all 146 experiments, a volume of approximately 25 mL of suspension containing quercetin was 147 processed. Afterwards, the organic solvent of the obtained emulsion was removed from the 148 sample using a rotary evaporator (BÜCHI 011-BÜCHI 461 Water Bath) under vacuum (-0.06, 149 60ºC and 110min), to eliminate the organic solvent. 150 2.3. Product characterization 151 2.3.1. Microscopy 152 An automated upright microscope system for life science research (Leica DM4000 B, Wetzlar, 153 Germany) was used to obtain microscopic images of the emulsions and suspensions. 154 2.3.2. Physical stability of emulsion 155 The physical stability of prepared emulsions was studied through the optical analyser 156 Turbiscan™ Classic on the production day, by measuring the variations in backscattering. 157 2.3.3. Particle size 158 The particle size of the micelles were measured by a Laser Diffraction (LD) equipment model 159 Malvern Mastersizer 2000 following a centrifugation step at 7800rpm, 5min, in order to 160 precipitate the crystals of quercetin that were not encapsulated. Suspensions were dispersed 161 in distilled water and measurement was carried out after a gentle rotation of the suspension 162 container in order to obtain a better dispersion of the micelles. In this work, particle size 163 measurements are reported as volume distribution and defined as the average diameter (d0.5). 164 2.3.4. Encapsulation efficiency (EE) 165 For the quercetin’s water suspensions stabilized with starch the Encapsulation Efficiency (EE) 166 was determined through Ultraviolet-visible spectrophotometry, while in the suspensions 167 stabilized with lecithin or β-glucan, due to the interference of these carrier materials on UV 168 assays, this parameter was determined by HPLC. 169 For UV-Vis measurements, the suspensions were previously centrifugated at 7800rpm (5min) 170 and diluted in water. The concentration of quercetin was determined using a UV-Visible 171 Spectrophotometer (Shimadzu UV-2550, λ= 375 nm). Calibration was obtained by using 172 standard samples with concentrations between 3 and 12μg/mL, using ethanol as solvent. 173 HPLC analysis of quercetin was performed in accordance with a method previously reported 174 [28]. A Waters 515 HPLC pump, equipped with In-Line Degasser AF (Waters), 717 plus 175 Autosampler (Waters) and 2487 Dual ʎ Absorbance detector (Waters) were used. 176 Chromatographic separation was carried out with a Symmetry C18 Column (5 µm, 4.6 mm X 177 150 mm, Waters) coupled with a Bio-Sil C18 precolumn (5 µm, 4.6 mm X 30 mm, Bio-Rad) with 178 mobile phase of acetonitrile/5% acetic acid solution (27:73, v/v) at 30 ◦C. The detection 179 wavelength was set at 373 nm and the flow rate was 1.0 mL/min. The calibration curve was 180 linear within the range of 25–200µg/mL (R2= 0.9997) using methanol:water (70:30) solution as 181 solvent. The quercetin suspension samples were detected by HPLC following centrifugation at 182 7800rpm (5min) and dilution in methanol. The run time for the assay was 13 min and the 183 retention time for quercetin was 8.32 min. 184 The absorbance is proportional to the amount of quercetin dispersed in solution which 185 corresponds to the encapsulated quercetin. Non-encapsulated quercetin can correspond 186 either to quercetin precipitated as big crystals and not stabilized in the suspension, or to 187 quercetin that has undergone degradation during the process. The encapsulation efficiency is 188 reported as the ratio between the amount of flavonoid dispersed in solution and total 189 quercetin amount in the initial feed. 190 2.3.5. Structural characterisation 191 Infrared spectra of the suspensions and pure materials were recorded on a Bruker ALPHA FT-IR 192 apparatus equipped with a Platinum ATR sampling module including a diamond crystal. The 193 suspensions were centrifuged at 13300rpm during 30min. The supernatant was removed and 194 the sample pellet’s was submitted to further evaporation of water at -0.09MPa and 35ºC for 195 48h. The pellet was then used to perform FTIR. The spectra in the range from 4000 to 400 cm-1 196 were the average of 64 scans at a resolution of 2cm-1. The ATR signal was transformed to 197 Transmittance and the obtained spectra was normalised after the baseline correction. 198 2.3.6. Cryo-TEM 199 Cryo-TEM analysis were performed through a method already described [29]. Briefly, 4μl of 200 sample was deposited on a rack C-Flat 1.2/1.3, which was previously hydrophilized by a plasma 201 cleaner. A blotting is performed on either side of the filter paper grid during 4s where, 202 afterwards, liquid ethane is introduced in order to freeze the samples, avoiding the formation 203 of crystals (Gatan Cryoplunge 3). Samples are maintained in liquid nitrogen until their transfer 204 to the holder (Gatan Cryotransfer 626). JEOL JEM-FS2200 HRP 200 kV TEM equipment with 205 electron filtering was used to perform the TEM images. 206 2.3.7. Stability of suspensions 207 One of the aqueous suspensions with the best relation between EE and final quercetin’s 208 concentration was chosen for stability examination. Some authors demonstrated that it’s 209 possible to increase the stability of lecithin, reducing the appearance of its agglomerates and 210 increase drug’s solubility by adding glycerol (G), as co-solvent, to aqueous lecithin dispersions 211 [30,31]. Thus, an equal suspension with 2.5% (v/v) of glycerol as co-solvent in the water phase 212 was prepared in order to compare the physicochemical properties of the suspension without 213 it. The stability examination was performed through the assessement of micellar particle size, 214 presence of crystals, quercetin’s concentration and antioxidant activity of the suspensions 215 after 75 days of storage in a refrigerated dark room (10ºC). 216 The assessment of antioxidant activity was performed through oxygen radical absorption 217 capacity (ORAC) assay, which was carried out by a method previously described [32]. This assay 218 measures the ability of the antioxidant species present in the sample to inhibit the oxidation of 219 disodium fluorescein (FL) catalysed by peroxyl radicals generated from AAPH. 220 important to remember that turbiscan only provides mean particle size value but not the 369 dispersion of this value, hence the differences between turbiscan’s values and optical 370 microscope images. 371 Regarding experiment E20, crystals of quercetin precipitated in the flask during the 372 evaporation of the emulsions, which was confirmed by optical microscopy. β-Glucan did not 373 seem to be a good carrier/emulsifier to encapsulate this flavonoid, and so, higher 374 concentrations of quercetin were not tested. The final particle of the quercetin-loaded 375 micelles was in the micrometer range. In the case of E20, the presence of quercetin’s crystals 376 contributed for the micrometer size, whereas in the samples E21-E23 the presence of carrier’s 377 agglomerates contributed to this particle size. Comparing E20 and E22, it is possible to see that 378 the addition of lecithin increased the particle size due to the formation of lecithin:β-glucan 379 complexes [41]. Nevertheless, the addition of more lecithin (E21 and E23) led to a reduction of 380 particle size, probably due to the formation of higher amount of small sized quercetin 381 encapsulated particles. 382 Regarding the encapsulation efficiency, it is possible to verify that comparing E20 and E22, the 383 addition of lecithin to the system led to a decrease of EE. The addition of lecithin led to the 384 complexation of the carriers, and their further precipitation. Perhaps there is a competition 385 between quercetin and lecithin for the complexation with β-glucan, resulting in less EE in E22 386 comparing with E20. 387 Nevertheless, as the amount of the lecithin increased in β-glucan:lecithin mixtures 388 (E22<E21<E23), so as increased the EE of the system. These values are in agreement with the 389 reduction of particle size in these samples, as discussed previously. Perhaps in experiment E23 390 there is a higher portion of free lecithin, not complexed with β-glucan, to interact with 391 quercetin, increasing its encapsulation. This fact reinforces the capacity of lecithin to 392 encapsulate quercetin 393 3.4. Stability 394 Experiment E17 was repeated with and without the inclusion of glycerol in order to evaluate 395 and compare their stability. For that purpose, the micellar particle size, presence of crystals, 396 quercetin’s concentration and antioxidant activity of the resulting suspensions were measured 397 after 75 days of storage. During this period, the aqueous solutions were stored in a 398 refrigerated room at temperatures around 10ºC and protected from light. The refrigeration 399 condition was chosen for the stability study since it is recommended as the storage condition 400 for the phospholipids [42]. Results are reported in Table 4. 401 (TABLE 4) 402 As presented in this table, the sample prepared with glycerol as co-solvent maintained 403 colloidal stability at the end of one month and a half, with no significant change in its particle 404 size, indicating good stability during this period. In contrast, the sample without glycerol nearly 405 tripled its size after the same period, being this value even higher after two months and a half 406 after its preparation. 407 (FIGURE 6) 408 Evaluating the shape of the size distribution, shown in Fig. 6, the distribution of the sample 409 freshly prepared have main peak in the nanometers range, corresponding to quercetin 410 particles that were successfully encapsulated, and other peaks at sizes above 1 µm possibly 411 corresponding to quercetin’s crystals and lecithin’s agglomerates. During storage, the peaks 412 above 1 µm are displaced to considerably higher particle sizes, indicating that these bigger 413 particles are becoming more agglomerated and further destabilizing the suspension due to 414 their higher size. The addition of a co-solvent is essential for an adequate stability of the 415 formulation, avoiding not only the agglomeration of lecithin, but also delaying the appearance 416 of quercetin’s crystals. In fact, in the sample prepared without glycerol, the appearance of 417 flavonoid’s crystals was observed shortly after fifteen days of preparation. Regarding 418 encapsulation efficiency, the addition of a co-solvent appeared to slightly enhance the 419 solubility of quercetin in aqueous media. Nevertheless, the stability of quercetin over storage 420 was similar in the two tested samples. 421 Table 4 also shows the results achieved by ORAC antioxidant assay for the two suspensions 422 over storage. The results are expressed as micromoles of Trolox equivalents per gram of 423 quercetin. Antioxidant activity of pure quercetin are lecithin was also measured in this work, 424 revealing values of 6586 and 2.5 μmol of TE/g of product, respectively. The probable formation 425 of hydrogen bonds between lecithin and quercetin resulted in a 3-fold increased antioxidant 426 activity per unit mass of quercetin for both suspensions, being these values nearly preserved 427 after 75 days of storage. 428 4. Conclusion 429 The formulation of quercetin using three distinct emulsifiers, namely n-octenyl succinate 430 anhydride starch, soybean lecithin and barley β-glucan, was investigated in this work through 431 pressurized ethyl acetate-in-water emulsions. Results showed that OSA-starch was not suitable 432 for the encapsulation of quercetin due to the low encapsulating efficiencies achieved (below 433 10%) and micellar particle sizes in the range of micrometers. Experiments carried out with 434 lecithin yielded the best results, with encapsulation efficiencies up to near 76% and particle 435 sizes in the sub-micrometer range. Moreover, the aqueous suspensions of quercetin stabilized 436 with this surfactant presented concentrations of the flavonoid up to 630ppm, being 315437 fold higher compared with solubility of pure quercetin in water. The formation of 438 hydrogen bonds between lecithin and quercetin could explain the good results obtained 439 with this emulsifier, further comprising improved antioxidant activity (3-fold higher per 440 unit mass of quercetin) due to a synergistic effect. Furthermore, the addition of lecithin to 441 the emulsions stabilized with β-glucan improved the encapsulation efficiency and particle 442 size of the micelles, reinforcing the capacity of lecithin to encapsulate quercetin. 443 The results obtained in this work showed that high pressure emulsion technique is a versatile 444 method for the production of water soluble formulations of quercetin, avoiding quercetin’s 445 degradation and increasing its solubility. Furthermore, the addition of glycerol as co-solvent 446 can increase the colloidal stability of the suspension and the encapsulation efficiency of 447 the flavonoid. 448 Acknowledgements 449 This work was supported by Marie Curie Industry-Academia Partnerships and Pathways 450 (European Commission) through the WineSense project (FP7-PEOPLE-2012IAPP-612608). 451 V.S.S. Gonçalves is grateful for the doctoral grant SFRH/BD/77350/2011 from Fundação 452 para a Ciência e Tecnologia. S. Rodríguez-Rojo acknowledges the Ministerio de Economía y 453 Competitividad and Universidad de Valladolid for her Juan de la Cierva fellowship (JCI454 2012-14992). 455 References 456 [1] A. Priprem, J. Watanatorn, S. Sutthiparinyanont, W. Phachonpai, S. Muchimapura, 457 Anxiety and cognitive effects of quercetin liposomes in rats., Nanomedicine. 4 (2008) 458 70–8. 459 [2] M.P. Souza, A.F.M. Vaz, M.T.S. Correia, M.A. Cerqueira, A.A. Vicente, M.G. Carneiro-da460 Cunha, Quercetin-Loaded Lecithin/Chitosan Nanoparticles for Functional Food 461 Applications, Food Bioprocess Technol. 7 (2013) 1149–1159. 462 [3] A.W. Boots, G.R.M.M. Haenen, A. Bast, Health effects of quercetin: from antioxidant to 463 nutraceutical., Eur. J. Pharmacol. 585 (2008) 325–37. 464 [4] M. Russo, C. Spagnuolo, I. Tedesco, S. Bilotto, G.L. Russo, The flavonoid quercetin in 465 disease prevention and therapy: facts and fancies., Biochem. Pharmacol. 83 (2012) 6– 466 15. 467 [5] K. Srinivas, J.W. King, L.R. Howard, J.K. Monrad, Solubility and solution thermodynamic 468 properties of quercetin and quercetin dihydrate in subcritical water, J. Food Eng. 100 469 (2010) 208–218. 470 [6] A. Barras, A. Mezzetti, A. Richard, S. Lazzaroni, S. Roux, P. Melnyk, et al., Formulation 471 and characterization of polyphenol-loaded lipid nanocapsules., Int. J. Pharm. 379 (2009) 472 270–7. 473 [7] A. Kumari, S.K. Yadav, Y.B. Pakade, B. Singh, S.C. Yadav, Development of biodegradable 474 nanoparticles for delivery of quercetin, Colloids Surf. B. Biointerfaces. 80 (2010) 184– 475 92. 476 [8] T.-H. Wu, F.-L. Yen, L.-T. Lin, T.-R. Tsai, C.-C. Lin, T.-M. Cham, Preparation, 477 physicochemical characterization, and antioxidant effects of quercetin nanoparticles., 478 Int. J. Pharm. 346 (2008) 160–8. 479 [9] M. Fraile, R. Buratto, B. Goméz, Á. Martín, M.J. Cocero, Enhanced Delivery of Quercetin 480 by Encapsulation in Poloxamers by Supercritical Antisolvent Process, Ind. Eng. Chem. 481 Res. 53 (2014) 4318–4327. 482 [10] T. Pralhad, K. Rajendrakumar, Study of freeze-dried quercetin-cyclodextrin binary 483 systems by DSC, FT-IR, X-ray diffraction and SEM analysis., J. Pharm. Biomed. Anal. 34 484 (2004) 333–9. 485 [11] M.L. Calabrò, S. Tommasini, P. Donato, D. Raneri, R. Stancanelli, P. Ficarra, et al., Effects 486 of alphaand beta-cyclodextrin complexation on the physico-chemical properties and 487 antioxidant activity of some 3-hydroxyflavones., J. Pharm. Biomed. Anal. 35 (2004) 365– 488 77. 489 [12] N.P. Aditya, A.S. Macedo, S. Doktorovova, E.B. Souto, S. Kim, P.-S. Chang, et al., 490 Development and evaluation of lipid nanocarriers for quercetin delivery: A comparative 491 study of solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), and lipid 492 nanoemulsions (LNE), LWT - Food Sci. Technol. 59 (2014) 115–121. 493 [13] N. Mignet, J. Seguin, G.G. Chabot, Bioavailability of polyphenol liposomes: a challenge 494 ahead., Pharmaceutics. 5 (2013) 457–71. 495 [14] Y.H. Kim, D.W. Lee, E.J. Jung, J.T. Bae, S.G. Lee, H.B. Pyo, et al., Preparation and 496 characterization of quercetin-loaded silica microspheres stabilized by combined 497 multiple emulsion and sol-gel processes young, Chem. Ind. Chem. Eng. Q. (2014) 10–10. 498 [15] E. de Paz, Á. Martín, A. Estrella, S. Rodríguez-Rojo, A.A. Matias, C.M.M. Duarte, et al., 499 Formulation of β-carotene by precipitation from pressurized ethyl acetate-on-water 500 emulsions for application as natural colorant, Food Hydrocoll. 26 (2012) 17–27. 501 [16] E. de Paz, Á. Martín, A. Bartolomé, M. Largo, M.J. Cocero, Development of water502 soluble β-carotene formulations by high-temperature, high-pressure emulsification and 503 antisolvent precipitation, Food Hydrocoll. 37 (2014) 14–24. 504 [17] W.I.R. Iemenschneider, H. Ag, F. Republic, Ullmann’s Encyclopedia of Industrial 505 Chemistry, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 2000. 506 [18] S.K. Nitta, K. Numata, Biopolymer-based nanoparticles for drug/gene delivery and 507 tissue engineering., Int. J. Mol. Sci. 14 (2013) 1629–54. 508 [19] M. Fathi, Á. Martín, D.J. McClements, Nanoencapsulation of food ingredients using 509 carbohydrate based delivery systems, Trends Food Sci. Technol. 39 (2014) 18–39. 510 [20] L. Bouarab, B. Maherani, A. Kheirolomoom, M. Hasan, B. Aliakbarian, M. Linder, et al., 511 Influence of lecithin-lipid composition on physico-chemical properties of 512 nanoliposomes loaded with a hydrophobic molecule., Colloids Surf. B. Biointerfaces. 513 115 (2014) 197–204. 514 [21] P.C. Suriyakala, S.B. N, S.R. D, L. Prabakaran, Phospholipids as versatile polymer in drug 515 delivery systems, Int. J. Pharm. Pharm. Sci. 6 (2014) 6–9. 516 [22] S. Hoeller, A. Sperger, C. Valenta, Lecithin based nanoemulsions: A comparative study 517 of the influence of non-ionic surfactants and the cationic phytosphingosine on 518 physicochemical behaviour and skin permeation., Int. J. Pharm. 370 (2009) 181–6. 519 [23] Y. Pan, R. V Tikekar, N. Nitin, Effect of antioxidant properties of lecithin emulsifier on 520 oxidative stability of encapsulated bioactive compounds., Int. J. Pharm. 450 (2013) 129– 521 37. 522 [24] D. Charalampopoulos, R. Wang, S.S. Pandiella, C. Webb, Application of cereals and 523 cereal components in functional foods: a review., Int. J. Food Microbiol. 79 (2002) 131– 524 41. 525 [25] P.J. Wood, Cereal β-glucans in diet and health, J. Cereal Sci. 46 (2007) 230–238. 526 [26] K. Raemdonck, T.F. Martens, K. Braeckmans, J. Demeester, S.C. De Smedt, 527 Polysaccharide-based nucleic acid nanoformulations, Adv. Drug Deliv. Rev. 65 (2013) 528 1123–47. 529 [27] Y. Wang, J. Liu, F. Chen, G. Zhao, Effects of molecular structure of polyphenols on their 530 noncovalent interactions with oat β-glucan., J. Agric. Food Chem. 61 (2013) 4533–8. 531 [28] L. Gao, G. Liu, X. Wang, F. Liu, Y. Xu, J. Ma, Preparation of a chemically stable quercetin 532 formulation using nanosuspension technology., Int. J. Pharm. 404 (2011) 231–7. 533 [29] L. Melanson, CryoplungeTM3 and Solarus® 950: a Perfect Duet for High Quality Frozen 534 Hydrated Specimen Preparations for CryoEM,, Microsc. Microanal. 15 (2009) 2–3. 535 [30] M. Płaczek, M. Sznitowska, J. Jacyna, The influence of co-solvents and surfactants on 536 physicochemical properties of aqueous lecithin dispersions ( WLD ) intended for 537 parenteral administration, in: 9th World Meet. Pharm. Biopharm. Pharm. Technol., 538 Lisbon, Portugal, 2014. 539 [31] D. Watrobska-Swietlikowska, M. Przybylo, J. Procek, M. Sznitowska, Distribution of 540 phosphatidylcholine into liposomalmicellar and aqueous phases of submicron 541 emulsions stabilized with lecithin and phospholipids, in: 9th World Meet. Pharm. 542 Biopharm. Pharm. Technol., Lisbon, Portugal, 2014. 543 [32] B. Ou, D. Huang, M. Hampsch-Woodill, J. a Flanagan, E.K. Deemer, Analysis of 544 antioxidant activities of common vegetables employing oxygen radical absorbance 545 capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays: a comparative 546 study., J. Agric. Food Chem. 50 (2002) 3122–8. 547 [33] S. Varona, Á. Martín, M.J. Cocero, Formulation of a natural biocide based on lavandin 548 essential oil by emulsification using modified starches, Chem. Eng. Process. Process 549 Intensif. 48 (2009) 1121–1128. 550 [34] S. Varona, A. Martn, M.J. Cocero, Liposomal Incorporation of Lavandin Essential Oil by 551 a Thin-Film Hydration Method and by Particles from Gas-Saturated Solutions, Ind. Eng. 552 Chem. Res. 50 (2011) 2088–2097. 553 [35] E. de Paz, Á. Martín, M.J. Cocero, Formulation of β-carotene with soybean lecithin by 554 PGSS (Particles from Gas Saturated Solutions)-drying, J. Supercrit. Fluids. 72 (2012) 125– 555 133. 556 [36] R.S. Nasibullin, T.I. Nikitina, Y. Afanas’eva, T.R. Nasibullin, L.V. Spirikhin, Complex of 557 3,5,7,3?,4?-pentahydroxyplavonol with phospatidylcholine, Pharm. Chem. J. 36 (2002) 558 492–495. 559 [37] M.F. Ramadan, Antioxidant characteristics of phenolipids (quercetin-enriched lecithin) 560 in lipid matrices, Ind. Crops Prod. 36 (2012) 363–369. 561 [38] M.F. Ramadan, Phenolipids : New Generation of Antioxidants with Higher 562 Bioavailability, Austin J. Nutr. Food Sci. 1 (2014) 2–3. 563 [39] Y. Li, Y. Yang, T. Bai, J. Zhu, Heat Capacity for the Binary System of Quercetin and 564 Poly(vinylpyrrolidone) K30, J. Chem. Eng. Data. 55 (2010) 5856–5861. 565 [40] Y. Zheng, A.H.L. Chow, Production and characterization of a spray-dried hydroxypropyl566 beta-cyclodextrin/quercetin complex., Drug Dev. Ind. Pharm. 35 (2009) 727–34. 567 [41] M. Veverka, T. Dubaj, J. Gallovič, V. Jork, E. Veverková, M. Mičušk, et al., Beta-glucan 568 complexes with selected nutraceuticals: Synthesis, characterization, and stability, J. 569 Funct. Foods. 8 (2014) 309–318. 570 [42] A.A. Date, M.S. Nagarsenker, S. Patere, V. Dhawan, R.P. Gude, P. a Hassan, et al., 571 Lecithin-based novel cationic nanocarriers (Leciplex) II: improving therapeutic efficacy 572 of quercetin on oral administration., Mol. Pharm. 8 (2011) 716–26. 573 574