Using different natural origin carriers for development of epigallocatechin gallate (EGCG) solid formulations with improved antioxidant activity by PGSS-drying

Abstract

Epigallocatechin gallate (EGCG) is the catechin with the highest antioxidant activity present in green tea. Nevertheless, due its low bioavailability, it is necessary to develop EGCG formulations capable of improving its stability resulting in increased bioavailability and thus higher biological activity of this catechin (e.g. antioxidant activity). The purpose of this work was the formulation of EGCG using three distinct natural origin carriers, namely OSA-starch, soybean lecithin and β-glucan, by particles from gas saturated solution drying (PGSS-drying). Non-cytotoxic solid formulations of EGCG in the range of micrometers and encapsulation efficiencies up to 80.5% were obtained. An improved antioxidant activity (AA) determined by the oxygen radical absorbance capacity (ORAC) method was obtained for all formulations. Furthermore, lecithin:EGCG and β-glucan:EGCG presented higher cellular antioxidant activity (Caco-2 cells) values than free EGCG at the same concentrations tested (around 1.5-fold higher values). Moreover, the solid formulations presented preservation of the AA over 4 months, and an improved storage stability in comparison with non-encapsulated EGCG over 48 h at 328 K in the absence of light (accelerated storage). These results show that PGSS-drying conditions enabled the preservation of the bioactive properties of catechin, allowing the formulation of solid particles with enhanced features.

---

1. Introduction

Green tea is an excellent source of health-promoting polyphenols, such as catechins, with epigallocatechin gallate (EGCG) being the most potent and abundant catechin present in this beverage.¹ Its antioxidant activity is at least 100 fold higher than vitamin C, being highly effective as an antimicrobial, antidiabetic or anticarcinogenic agent.² Due to its features, EGCG is highly attractive for use in the food, pharmaceutical and cosmetic industry in a micro-/nanoparticle form.^3,4 Nevertheless, its application can be hindered due to the chemical instability of the catechin. EGCG easily undergoes oxidation in the presence of light, high temperature and neutral or basic solutions, leading to inefficient systemic delivery and thus poor bioavailability.^1,5,6 One way to overcome this problem and to protect EGCG from unfavorable environmental conditions is the encapsulation of the catechin into micro and nanosystems in order to retain EGCG's structural integrity.^7–9

Different carrier materials and techniques have been used to encapsulate this polyphenol.^2,10 For instance, Zou and co-authors have prepared EGCG nanoliposomes by an ethanol injection method combined with dynamic high-pressure microfluidization, providing higher catechin's chemical stability during in vitro digestion.⁶ Gómes-Mascaraque and colleagues encapsulated EGCG into electrosprayed gelatin submicroparticles, while Sanna and co-authors encapsulated catechins-rich white tea extract into polymeric nanoparticles composed by poly(ε-caprolactone) and alginate by nanoprecipitation.^7,11 Spray drying was also already used for the encapsulation of EGCG with carbohydrates with the preservation of catechin antioxidant properties.^9,12,13 This technique could have some limitations such as the potential instability of materials sensitive to high temperatures. Furthermore, some carrier materials, such as polyethylene glycols, cannot be processed with this method due to their low melting temperatures.¹⁴ PGSS® (particles from gas saturated solutions)-drying method represents an attractive alternative to create microparticles with controlled particle size, avoiding thermal degradation under an inert atmosphere (CO₂, without oxygen).¹⁵ In this technique, an aqueous solution is mixed with supercritical carbon dioxide, using a static mixer, at determined temperature and pressure. In this mixer some water would be extracted by CO₂, being some supercritical fluid partially mixed in the liquid solution; then, the biphasic mixture is expanded through a capillary nozzle to atmospheric pressure into a tower. This will cause the formation of fine droplets that will be dried very fast leading to the production of fine particles. In order to ensure a complete removal of water it is necessary that the temperature conditions in the spray tower are above the dew line of temperature-composition phase equilibrium diagram of water and carbon dioxide.^14–18 Meterc and co-authors already used this technique with success for drying an aqueous green tea extract.¹⁹ Nevertheless, the co-precipitation with carriers for the production of EGCG-loaded delivery systems with enhanced features has not been investigated yet, up to the authors' knowledge.

Thus, the aim of the present work is to study for the first time the encapsulation of EGCG in three natural origin carrier materials, namely modified n-octenyl succinate anhydride (OSA) starch, soybean lecithin and barley β-glucan, by PGSS-drying technique. OSA is a modified amphiphilic starch which was already used for the encapsulation and delivery of compounds with distinct polarities.^20,21 Lecithin is composed by a mixture of naturally occurring phospholipids, mostly phosphatidylcholine, that is available from soy or eggs. Phospholipids are amphiphilic molecules with antioxidant properties that are able to rearrange themselves as liposomes, which are interesting carrier materials for the delivery of hydrophobic/hydrophilic compounds.^22–24 β-Glucans are soluble fibers mostly present in cereals (barley, oat), constituted by linear polysaccharides of glucose units, connected by (1 → 3) or (1 → 4)-beta linkages. These carbohydrates are recognized for their therapeutic effects on coronary heart disease, diabetes and hypercholesterolemia, and have been used as encapsulating agents.^25–28 Product analysis was performed in this work, which included morphology, particle size, structural characterization, encapsulation efficiency, EGCG release profiles, antioxidant activity (chemical and cell-based assay) and cytotoxicity. The stability of EGCG incorporated into the natural origin matrices was also studied by measuring its antioxidant activity after 4 months at ambient storage and after 48 h of accelerated storage (stored at 328 K in the absence of light for 48 h).

2. Materials and methods

2.1. Materials

Teavigo® (EGCG ≥ 90% purity) was kindly supplied by DSM Nutritional Products (Leon, Spain). Modified OSA-starch refined from waxy maize was kindly provided by Ingredion Germany GmbH. Soybean lecithin (97% phospholipids) was obtained from Glama-Sot (SOTYA, Madrid, Spain). Glucagel™ (barley β-glucan; purity 78%, MW: 125–140 kDa) was kindly supplied by DKSH (France). CO₂ (99.5% purity) was supplied by Carburos Metálicos (Spain). Chemicals used for antioxidant activity assay were: 2′,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) from Sigma-Aldrich (St Quentin Fallavier, France) and disodium fluorescein (FL) was from TCI Europe (Antwerp, Belgium). Reagents used for phosphate buffer solution (PBS) preparation included sodium chloride (NaCl), potassium chloride (KCl) and monopotassium phosphate (KH₂PO₄) from Sigma-Aldrich (St Quentin Fallavier, France) and sodium phosphate dibasic dehydrate (Na₂HPO₄·₂H₂O) from Riedel-de-Haën (Seelze, Germany). For sterilization experiments Tryptone Soya Broth (TSB) and Tryptone Soya Agar (TSA) were purchased from Oxoid (Hampshire, England). All cell culture media and supplements namely, RPMI 1640, Fetal Bovine Serum (FBS), Penicillin-Streptomycin (PS) and trypsin/EDTA were obtained from Invitrogen (Paisley, UK). For cell-based assays phosphate buffered saline (PBS) powder, 2′,7′-dichlorofluorescin diacetate (DCFH-DA) and quercetin were obtained from Sigma-Aldrich (St. Louis, USA). CellTiter 96® AQueous One Solution Cell Proliferation Assay was obtained from Promega (Wisconsin, USA) and DMSO was obtained from Carlo Erba Reagents Srl (Milan, Italy).

2.2. Particles from gas saturated solutions (PGSS)-drying

The aqueous solutions of EGCG with each carrier (OSA-starch, lecithin or β-glucan) were processed using PGSS-drying technique in order to co-precipitate the materials; the chemical structures of EGCG and each carrier material are represented in Fig. 1.

Fig. 1 Chemical structures of (a) epigallocatechin gallate, (b) OSA-starch, (c) lecithin (main component: chemical name of the included structure) and (d) barley (1–3, 1–4)-β-glucans.

Fig. 2 presents a flow diagram of the PGSS-drying equipment, being this system previously described²⁹.

Fig. 2 Flow diagram of the PGSS-drying equipment.

Briefly, CO₂ was fed by a membrane pump (Milton Roy, 10 kg CO₂/h) and preheated using electrical resistances before introducing it into the static mixer filled with 4 mm glass beads, where it was mixed with the aqueous solution of EGCG with each natural carrier material at high pressure and temperature. The flow rate of CO₂ was determined with a Coriolis flow meter (Sensor MICRO Motion Elite CMF010 NB, Transmitter MICRO Motion Elite RFT91) with an accuracy of ±0.01 kg h⁻¹, and temperature after the electrical preheater was measured with a Pt100 thermoresistance with an accuracy of ±0.1 K and controlled using a PID controller. The aqueous solution was pumped to the static mixer using a milton royal pump (maximum flow 0.67 L h⁻¹). Temperature before and after the static mixer was measured using Pt100 thermoresistances, and pressure in the static mixer was measured with a DESIN TPR-10 digital pressure meter (DESIN Instruments) with an accuracy of ±0.05 MPa. Expansion of the gas-saturated solution into the spraying tower at atmospheric pressure was accomplished using a capillary nozzle (Spraying Systems, nozzle diameter 500 μm). Temperature in the spray tower was also determined with a Pt100 probe. After the PGSS-drying process, the system was depressurized and particles were collected.

In order to compare the influence of the carrier materials in the co-precipitation and encapsulation of EGCG, the EGCG and carriers' concentrations in the aqueous solution, 5 g L⁻¹ and 15 g L⁻¹ respectively, were fixed. The conditions in the co-precipitation process, namely, temperature in the static mixer (398 K), temperature in the spray tower (343 K), pressure of the system (9.5 MPa) and gas to product ratio (30) were chosen and fixed based on previous experiments.^14,29

2.3. Product characterization

2.3.1. Morphology. The morphology of the particles was analysed and imaged by Scanning Electron Microscopy (SEM) using a scanning electron microscope model JEOL JSM. Particles of representative samples were gold sputtered in an argon atmosphere at room temperature before examination.

2.3.2. Particle size and particle size distribution. The particle size of the samples was measured by a Laser Diffraction (LD) equipment model Malvern Mastersizer 2000 (aqueous and dry state). For measurement of particle size at aqueous conditions, particles were dispersed in ultrapure water (Milli-Q® Water) and the determination of the hydrodynamic diameter was carried out after a gentle rotation of the suspension container in order to obtain a better dispersion of the particles. A Scirocco 2000 dry disperser (Malvern Instruments) was used for particle size measurements at dry state. In this work, particle size measurements are reported as volume distribution and defined as d_0.5, being the final result the average from 3 measurements. The span value is also reported, that is, the ratio between d_0.5 and (d_0.9–d_0.1); span values near to 1 represent narrow PSD.

2.3.3. Structural characterisation. Infrared spectra of the samples and milled pure materials were recorded on a Bruker ALPHA FT-IR apparatus equipped with a Platinum ATR module including a diamond crystal. The spectra in the range from 4000 to 400 cm⁻¹ were the average of 64 scans at a resolution of 2 cm⁻¹. The ATR signal was transformed to transmittance and the obtained spectra was normalised after the baseline correction.

2.3.4. Encapsulation efficiency (EE). For the determination of encapsulation efficiency, 10 mg of particles were suspended in 30 mL of methanol (MeOH), vortexed and left in ultrasound treatment for 15 min. MeOH has a high dielectric constant and dipole moment, allowing higher recovery of EGCG compared with other solvents.¹² The samples were filtered and HPLC analysis of EGCG was performed in accordance with a method previously reported.³⁰ A Waters 515 HPLC pump, equipped with In-Line Degasser AF (Waters), 717 plus Autosampler (Waters) and 2487 Dual Absorbance detector (Waters) were used. Chromatographic separation was carried out with a Symmetry C18 Column (5 μm, 4.6 mm × 150 mm, Waters) coupled with a Bio-Sil C18 precolumn (5 μm, 4.6 mm × 30 mm, Bio-Rad) with mobile phase of 0.1% trifluoroacetic acid in deionised water (pH 2.0) and methanol at a volume ratio of 75 : 25 at 298 K. The detection wavelength was set at 280 nm and the flow rate was 0.8 mL min⁻¹, being the sample injection volume of 20 μL. The run time for the assay was 15 min and the retention time for EGCG was 10.3 min (a typical HPLC chromatogram for the quantification of EGCG is presented as Fig. 1 of ESI†). The calibration curve was linear within the range of 1.5–150 μg mL⁻¹ (R² = 0.9984) using water : methanol at a volume ratio of 80 : 20 solution as solvent. Each sample was prepared and analyzed three times.

2.3.5. Evaluation of antioxidant activity of EGCG-loaded particles by oxygen radical absorbance capacity (ORAC) method. ORAC assay was carried out by a modified method for the FL800 microplate fluorescence reader (Bio-Tek Instruments, Winooski, VT, USA), as described by Serra and co-authors.³¹ This assay measures the ability of the antioxidant species in the sample to inhibit the oxidation of disodium fluorescein (FL) catalyzed by peroxyl radicals generated from AAPH (2,2′-azobis-2-methyl-propanimidamide, dihydrochloride). A suspension was prepared by adding an amount of EGCG-loaded particles in methanol (1 mg particles per mL MeOH), and further vortexed. After 15 minutes in an ultrasound bath, the suspension was filtered with a 0.45 μm filter and the MeOH media was used for further analysis (dilutions with PBS). Each sample was analysed in triplicated; the results were presented as μMol Trolox equivalent (TE) per g of particle and g of EGCG.

2.3.6. In vitro evaluation of drug release kinetics. A sample of particles was suspended in 50 mL of dissolution medium (phosphate buffer solution, pH = 6.8). Samples were stirred at 155 rpm and maintained at a temperature of 310 K. Aliquots (2 mL) were withdrawn at predetermined time intervals (5 min, 15 min, 30 min, 60 min, etc.) and the same volume of fresh medium was added to the suspension. All the samples were filtered and diluted with phosphate buffer solution. The concentration of the drug was determined using a UV-Visible Spectrophotometer (UV-Vis Genesys10uv spectrometer; Thermo Spectronic, λ = 276). Calibration was obtained by using standard samples with concentrations between 5 and 40 μg mL⁻¹. Each analysis was performed in triplicate.

2.3.7. In vitro cell-based assays.
2.3.7.1. Sterilization. Free EGCG and EGCG-loaded particles were sterilized using the protocol described by Li and co-workers with some modifications.³² Briefly, samples were put directly in contact with UV irradiation during 15 h at room temperature in a Biological Safety Cabinet (Nuaire, USA). To further confirm sterility, EGCG and EGCG-loaded particles were incubated in TSB at 310 K for 24 hours and then samples were plated in petri dishes containing TSA for more 24 hours at 310 K.
2.3.7.2. Cell culture. The human colon carcinoma Caco-2 were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany). Caco-2, originally obtained from human colon adenocarcinoma, undergoes in culture a process of spontaneous differentiation that leads to the formation of a monolayer with many small intestinal functions. This cell line is particularly useful because express several morphological and functional characteristics of the mature enterocyte (Sambuy et al. 2005) and also it is well recognized as cell model for antioxidant activity determination.^33,34 Caco-2 cells were routinely grown in a standard medium: RPMI 1640 supplemented with 10% (v/v) of inactivated FBS and 1% (v/v) of PS. Stock cells were maintained as monolayers in 80 cm² culture flasks. Cells were sub cultured every week at a split ratio of 1 to 4 by treatment with trypsin/EDTA (0.25%) and incubated at 310 K in a 5% CO₂ humidified atmosphere. For cytotoxicity and cellular antioxidant activity experiments, cells were used between passages 30 and 40. Caco-2 cells were seeded at a density of 2 × 10⁴ cells per well in 96-well plates and the medium was changed every 48 h. The experiments were performed using completely confluent (differentiated) cells (after ∼96 hours).
2.3.7.3. Cytotoxicity assay. For cytotoxicity experiments PBS was added to EGCG and EGCG-loaded particles to a final concentration of 500 μg EGCG per mL and placed in an ultrasound bath during 4 minutes. After dissolution, EGCG and EGCG-loaded particles were diluted in RPMI medium supplemented with 0.5% FBS to a final concentration of 25 and 50 μg EGCG per mL and were added to Caco-2 cells in triplicate. Also a control group with 10% (v/v) of PBS was performed. Following the incubation period (4 h at 310 K in a 5% CO₂ humidified atmosphere) samples were removed, cells were rinsed with PBS and 100 μL of a CellTiter 96® AQueous One Solution Cell Proliferation Assay reagent (MTS) was added to each well and left to react for 2 hours (16.6% v/v of MTS in cell culture media). The quantity of formazan produced was measured spectrophotometrically at 490 nm in a microplate reader (EPOCH, Bio-Tek, USA) and is directly proportional to the number of living cells in culture. Results were expressed in terms of percentage of cellular viability relative to a group control (cells only with RPMI medium). Experiments were performed in triplicate in three independent assays.
2.3.7.4. Cellular antioxidant activity. Cellular Antioxidant Activity (CAA) was carried out by the method previously described modified for Caco-2 cell line.³⁵ First, EGCG and EGCG loaded particles were dissolved in PBS and incubated in an ultrasonic bath during 10 minutes, to a stock solution of 500 μg EGCG per mL. After reaching confluence, Caco-2 cells were washed twice with PBS and triplicate wells were treated for 1 h with 100 μL of different concentrations of EGCG or EGCG-loaded particles (1–8 μg EGCG per mL) plus 25 μM of DCFH-DA diluted in PBS. Then, the medium was removed and replaced by PBS containing 600 μM of AAPH. The 96-well microplate was placed into a fluorescence reader (FL800, Bio-Tek Instruments, Winooski, VT, USA) at 310 K. Emission at 530 ± 25 nm was measured after excitation at 485 ± 20 nm every 5 min for 1 h. Each plate included triplicate control and blank wells: control wells contained cells treated with DCFH-DA and oxidant (AAPH); blank wells contained cells treated with DCFH-DA without oxidant. In addition, carriers (starch, lecithin and β-glucan) were also tested for CAA. Quercetin, previously dissolved in DMSO (20 mM) was used as standard (1.25–10 μM) for the calibration curve.

CAA of samples was quantified as follow: after blank and initial fluorescence subtraction, the area under the curve for fluorescence versus time was integrated to calculate the CAA value at each concentration of the sample:

where is the integrated area under the sample fluorescence versus time curve and is the integrated area of the control curve. The median effective concentration (EC₅₀) was determined for the sample from the median effect plot of log(f_a/f_u) versus log(dose), where f_a is the fraction affected (CAA unit) and f_u is the fraction unaffected (1-CAA unit) by the treatment. The EC₅₀ values (μg EGCG per mL) were stated as mean ± SD for triplicate sets of data obtained from the same experiment. EC₅₀ were converted to CAA values, expressed as micromoles of quercetin per g of EGCG (μmol QE/g EGCG), using the mean EC₅₀ value of quercetin from three independent experiments (2.47 ± 0.26 μM).

2.4. Storage stability of the EGCG-loaded solid formulations

The antioxidant activity of EGCG and EGCG-loaded particles was measured by the method previously described after 4 months of solid formulations' production; the particles were stored at ambient temperature and absence of light.

For accelerated storage stability assay, the same samples were stored at 328 K (bellow the melting point of carriers) in a forced-air oven (WTC FD-400, Binder) in the absence of light for 48 h.^36,37 After this period, the antioxidant activity was measured, by the ORAC method described previously. Each analysis was performed in triplicated.

2.5. Statistical analysis

All data are expressed as means ± standard errors (SD) and individual experiments were performed at least in triplicate. The statistical analysis was done using SigmaStat 3.0® software. All values were tested for normal distribution and equal variance. When homogeneous variances were confirmed, data were analyzed by One Way Analysis of Variance (ANOVA) coupled with the Tukey's post-hoc analysis to identify means with significant differences (p value of p ≤ 0.001 was considered significant).

3. Results and discussion

3.1. Particles characterization

3.1.1. Morphology, particle size and particle size distribution. Three natural carrier materials, namely OSA-starch, lecithin and β-glucan, were co-precipitated with EGCG by PGSS-drying. The particle size, EE and antioxidant properties of EGCG-loaded particles are listed in Table 1, while SEM images are shown in Fig. 3.

Table 1 Summary of experimental results; natural carrier : EGCG mass ratio of 3 : 1

Experiment	Aqueous state		Dry state		Load (% w/w)	EE (%)	μmol TE per g particle
	d0.5 (μm)	Span	d0.5 (μm)	Span
OSA-starch:EGCG	37.0	22.0	2.0	17.1	20.1 ± 0.5	80.5 ± 2.3	1990 ± 99
Lecithin:EGCG	47.1	12.0	8.1	40.9	18.9 ± 0.6	75.8 ± 3.1	1950 ± 43
β-Glucan:EGCG	479.8	2.1	20.9	21.3	19.4 ± 0.2	77.4 ± 0.9	1738 ± 69

---

Fig. 3 SEM micrographs at 2000× and 5000× magnification of EGCG-loaded (a and a′) OSA-modified starch particles, (b and b′) lecithin particles and (c and c′) β-glucan particles.

During PGSS-drying process, the main mechanism for the atomization of the solution and the subsequent formation of particles is the saturation of the solution with carbon dioxide, and the sudden vaporization and expansion of the gas during the depressurization in the nozzle.¹⁴ As visible in Fig. 3, particles obtained with OSA-starch and lecithin show higher amount of agglomerates composed by partially fused spheres. These morphology was also obtained by Esther and co-authors while formulating β-carotene with soybean lecithin by the same technique.¹⁴ Nevertheless, the spherical structural unit appears to be smaller than the one of β-glucan's particles. None of the dry particles obtained show cracks in their surface, being in accordance with previous works.^14,29,38 Laser diffraction measurements revealed lower particle size results in dry state (low d_0.5 values), in comparison with particle sizes in aqueous state, confirming the aggregation of these materials in aqueous media. OSA-starch was the carrier that resulted in EGCG-loaded particles with lower d_0.5, both in the solid and liquid state. EGCG-loaded particles in aqueous suspension revealed a bimodal size distribution for OSA-starch and lecithin carrier materials, possibly representing single and aggregated particles. These samples also presented high span values in comparison with β-glucan due to higher polydispersity. β-Glucan particles present unimodal size distribution with d_0.5 of 480 μm probably due to high agglomeration in cold water. The particle size distributions of the particles formed with the different encapsulating materials in both dry and aqueous state are present in Fig. 2 of ESI.†

3.1.2. Encapsulation efficiency and antioxidant activity. Results regarding the loading of epigallocatechin gallate for the different encapsulating materials are shown in Table 1. Encapsulation efficiencies were relatively high (above 75%) for all the natural origin carriers tested. Similar EE were observed by Salgado and co-authors while using lecithin and β-glucan for the encapsulation of resveratrol, another polyphenol, by PGSS-drying.²⁹ Slightly higher EE values were observed for OSA-starch and β-glucan; this could be due to the fact that EGCG interact with sugar molecules through the formation of hydrogen bonds.^39,40 During PGSS-drying process, lower drying temperature is used allowing the protection of EGCG from oxidation since it is processed in an inert atmosphere of carbon dioxide.²⁹ To determine the possible loss of antioxidant activity during the drying and precipitation process, the EGCG-loaded particles were assayed for their antioxidant properties using ORAC method. The ORAC assay measures the capacity of an antioxidant for scavenging the peroxyl radicals (ROO˙) via hydrogen atom transfer. The ORAC results (μmol TE per g particle) are shown in Table 1. OSA-starch and lecithin samples presented ORAC values almost near 2000 μmol TE per g particle, while β-glucan sample showed slightly lower ORAC values (1738 ± 69 μmol TE per g particle).

Nevertheless, if we analyze the ORAC values presented as μmol TE per g EGCG, presented in Fig. 4, it is possible to verify that all the PGSS-drying samples present higher antioxidant activity than the pure EGCG, which ORAC value was in good agreement with the literature results (7329 μmol TE per g EGCG)⁴¹.

Fig. 4 Results of ORAC antioxidant activity tests presented as antioxidant activity per unit mass of EGCG (*** refers to statistical differences of EGCG-loaded samples in relation with EGCG; p < 0.001).

Probably these higher antioxidant values are due to some synergistic effect between the natural matrices and EGCG. Nevertheless, from all the carriers used, only lecithin showed some antioxidant activity (187 μmol TE per g lecithin). The ORAC results for the EGCG-loaded particles produced demonstrates that PGSS-drying method preserved the strong original antioxidant properties of EGCG, resulting in delivery systems with enhanced properties.

3.1.3. ATR-FTIR. FTIR was used to verify whether EGCG was successfully incorporated in the natural-based carriers and their chemical interactions. Fig. 5 shows the infrared spectrum of unprocessed EGCG, unprocessed OSA-starch, unprocessed lecithin, unprocessed β-glucan and the corresponding EGCG-loaded particles.

Fig. 5 FTIR spectra of (a) unprocessed EGCG, (b) unprocessed OSA-starch, (c) EGCG-loaded OSA-starch particles, (d) unprocessed lecithin, (e) EGCG-loaded lecithin particles, (f) unprocessed β-glucan and (g) EGCG-loaded β-glucan particles.

It is possible to verify that epigallocatechin gallate presents its signature peaks at wavenumbers at 825 cm⁻¹ (C–H alkenes), 1039 cm⁻¹ (–C–O alcohols), 1097 cm⁻¹ (aromatic ring), 1148 cm⁻¹ (C–OH alcohols) 1346 cm⁻¹ (C–O alcohols), 1518/1528/1544 cm⁻¹ (aromatic semicircle ring stretch), 1618 cm⁻¹ (aromatic quadrant ring stretch), 1692 cm⁻¹ (carbonyl) and 3358 cm⁻¹ (O–H groups).^6,11 Overall, all the EGCG-loaded particles present the specific EGCG bands, which are not present in the spectrum of pure carriers. These results confirm that catechin is incorporated in the carrier's matrix through physical interaction without the formation of chemical bonds, as already verified by other authors^3,7,12.

3.2. In vitro cell-based assays: cytotoxicity and cellular antioxidant activity

The incidence of UV irradiation during 15 hours directly into the EGCG and EGCG-loaded particles showed to be an effective sterilization method, as confirmed by the absence of microorganism's growth in TSA plates (data not show). This sterilization step allowed the evaluation of cytotoxicity and cellular antioxidant activity (CAA) of samples without the interference of microorganism's contamination.

The cytotoxicity of EGCG and EGCG-loaded particles were evaluated in the human colon adenocarcinoma model Caco-2 cell line by using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS). This compound is bio-reduced by viable cells into a colored formazan product that is soluble in tissue culture medium, which is measured at 490 nm. The results obtained show that EGCG and EGCG-loaded particles did not show cytotoxicity in the range of concentrations tested (25–50 μg EGCG per mL) relatively to the control (100% of cell viability) after 4 hours of incubation (Fig. 6).

Fig. 6 Cell viability (percentage relative to control – cells with culture medium) in Caco-2 cells after incubation with pure EGCG and EGCG-loaded particles during 4 h.

These results indicate that free and EGCG-loaded particles have no effect in Caco-2 cell viability which ensures the safety of these samples to be tested for cellular antioxidant activity (CAA).

CAA assay is a more biologically relevant method than the popular chemical antioxidant assays because it accounts for some aspects of uptake, metabolism and location of antioxidant compounds within the cells.³⁵ According with the oxidation mechanism, the nonpolar DCFH-DA is taken up by cells through passive diffusion and deacetylated by cellular esterases to form polar 2′,7′-dichlorofluorescin (DCFH), which is trapped within the cells. Then peroxyl radicals (ROO˙), generated by AAPH after entering the cells, oxidized DCFH to fluorescent dichlorofluorescein (DCF). Therefore, a higher level of oxidation of DCFH causes a higher level of fluorescence intensity.^35,42 The EC50 and CAA values of EGCG and EGCG-loaded particles are presented in Table 2.

Table 2 EC₅₀ (μg EGCG per ml) and CAA (μmoL QE/g EGCG) values for the inhibition of peroxyl radical-induced DCFH oxidation by EGCG and EGCG loaded particles (mean ± SD, n = 3)

	EC50 (μg EGCG per ml)	CAA (μmol QE/g EGCG)
a Refers to statistical differences of EGCG-loaded samples in relation with EGCG; p < 0.001.
EGCG	2.39 ± 0.25	1.03 ± 0.11
OSA-starch:EGCG	2.29 ± 0.13	1.08 ± 0.06
Lecithin:EGCG	1.64 ± 0.15^a	1.51 ± 0.13^a
β-Glucan:EGCG	1.69 ± 0.10^a	1.46 ± 0.09^a

---

The results clearly demonstrated that lecithin:EGCG and β-glucan:EGCG had higher CAA values than free EGCG at the same concentrations tested (statistical differences; p < 0.001). These EGCG-loaded particles exhibited 1.5 and 1.4-fold higher values of CAA (1.51 ± 0.13 and 1.46 ± 0.09 μmol QE/g EGCG, respectively) than free EGCG (1.03 ± 0.11 μmol QE/g EGCG). On the other hand the incubation of starch:EGCG system on Caco-2 cells did not significantly improve the CAA value comparing with free EGGC value. Furthermore, pure carriers were also tested for CAA and the results demonstrated that the three materials (starch, lecithin and β-glucan) do not present antioxidant activity, indicating that the carriers alone are not responsible for the antioxidant activity in Caco-2 cells. In this way, higher CAA values of lecithin:EGCG and β-glucan:EGCG suggest that lecithin and β-glucan promote the delivery and cellular uptake of EGCG and can be further used as ideal delivery systems to increase catechin's antioxidant activity.

In a previous study, the CAA of free and encapsulated EGCG in caseinophosphopeptide (CPP) and chitosan (CS) was also quantified, using the hepatocellular carcinoma HepG2 cell line.⁴³ The EC50 values reported were 15.25 μg mL⁻¹ and 12.60 μg mL⁻¹ for free and encapsulated EGCG, respectively. The EGCG-loaded CS–CPP nanoparticles exhibited 1.2-fold higher value of CAA suggesting that the EGCG-loaded system play an important role in the improvement of the cellular antioxidant activity of the nanoencapsulated EGCG.⁴³

3.3. EGCG release

The cumulative release of EGCG from the natural-based carriers in phosphate buffer solution (pH = 6.8) is represented in Fig. 7.

Fig. 7 Drug release profiles of the particles produced by PGSS-drying (phosphate buffer solution, pH = 6.8).

A fast release of EGCG was verified for both polysaccharide matrices, namely OSA-starch and β-glucan, with around 50% content of encapsulated polyphenol released after 5 min of experiment and the total EGCG's release after 15 min from the beginning of the assay. This could be explained by the hydrophilic nature of these carriers that facilitates the hydration, swelling and dissolution of the matrix, leading to a fast release of the encapsulated agent.⁴⁴ On the other hand, and as expected, the amphiphilic nature of lecithin provided a more controlled release of EGCG after an initial burst release, which could lead to a lower dose requirement due to less plasma fluctuations.^2,3,6

3.4. Stability

The antioxidant activities from pure EGCG and EGCG-loaded particles obtained under normal storage conditions (4 months) and after 48 h at accelerated storage conditions (328 K; 48 h) are shown in Fig. 8.

Fig. 8 Results of ORAC antioxidant activity tests presented as antioxidant activity per unit mass of EGCG after 4 months of ambient storage and after the same period plus 48 of accelerated storage (** refers to statistical differences between samples after 4 month of ambient storage and the same period plus 48 h of accelerated storage; p = 0.05).

Comparing with Fig. 4, ORAC values per gram of EGCG remained constant after 4 months at ambient temperature in all the samples tested, without significant effect from the encapsulating agent. Nevertheless, after 48 h of accelerated storage conditions, pure EGCG showed a slightly decrease in the antioxidant activity. In fact, the encapsulation of EGCG by all the natural matrices showed a protective function against EGCG degradation, since EGCG-loaded particles retained the ORAC values. Li and co-authors verified that EGCG loaded in coated serum albumin degraded faster than pure EGCG at 333 K, attributing this result to the high surface area/mass ratio of EGCG-loaded nanoparticles (186 to 300 nm), which were more susceptible to oxidation.³⁶ In our work this was not observed, probably due to the micrometer size of our samples.

4. Conclusions

The formulation of epigallocatechin gallate using three distinct carrier materials, namely n-octenyl succinate anhydride starch, soybean lecithin and barley β-glucan, was investigated in this work through PGSS-drying technique. Non-cytotoxic microparticles with encapsulation efficiencies of EGCG up to 80.5% were obtained. EGCG loaded-particles showed slightly higher storage stability and higher antioxidant activity than free EGCG. Moreover, β-glucan and lecithin were the carriers able to improve the intracellular activity of EGCG, whether due to an increase of the catechin's stability in aqueous media or increased permeability through cell membranes. β-Glucan carrier can be used for obtaining faster release of EGCG comparing to lecithin, which provides a more sustained release of the catechin.

The results obtained in this work showed that PGSS-drying technique is a suitable method for the production of solid formulations of EGCG, with maintenance of the structure of catechin and increased antioxidant activity. The particles produced could be applied in pharmaceutical, cosmetic, food or cosmeceutical industries, in line with consumer concerns and regulatory demands since the use of organic solvents for its production was avoided and the carriers used were from natural origins.

Acknowledgements

This work was supported by Marie Curie Industry-Academia Partnerships and Pathways (European Commission) through the WineSense project (FP7-PEOPLE-2012- IAPP-612608). V. S. S. Gonçalves is grateful for the doctoral grant SFRH/BD/77350/2011 from Fundação para a Ciência e Tecnologia (FCT) and A. A. Matias also acknowledge the FCT for her FCT Investigator Starter Grant (IF/00723/2014). This work was also supported by FCT through Grant No. PEst-OE/EQB/LA0004/2011. S. Rodríguez-Rojo acknowledges the Ministerio de Economía y Competitividad and Universidad de Valladolid for her Juan de la Cierva fellowship (JCI-2012-14992). iNOVA4Health – UID/Multi/04462/2013, a program financially supported by Fundação para a Ciência e Tecnologia/Ministério da Educação e Ciência, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement is also acknowledged.

References

1. A. Rashidinejad, E. J. Birch, D. Sun-Waterhouse and D. W. Everett, Food Chem., 2014, 156, 176–183.
2. S. Dang, S. Gupta, R. Bansal, J. Ali and R. Gabrani, in Free Radicals in Human Health and Disease, ed. V. Rani and U. C. V. Yadav, Springer, India, 2015, pp. 397–415.
3. Q. Lu, D.-C. Li and J.-G. Jiang, J. Agric. Food Chem., 2011, 59, 13004–13011.
4. J. Li and X. Wang, Food Chem., 2015, 168, 566–571.
5. Q. Ru, H. Yu and Q. Huang, J. Agric. Food Chem., 2010, 58, 10373–10381.
6. L. Zou, S. Peng, W. Liu, L. Gan, W. Liu, R. Liang, C. Liu, J. Niu, Y. Cao, Z. Liu and X. Chen, Food Res. Int., 2014, 64, 492–499.
7. V. Sanna, G. Lubinu, P. Madau, N. Pala, S. Nurra, A. Mariani and M. Sechi, J. Agric. Food Chem., 2015, 63, 2026–2032.
8. M. Yao, D. J. Mcclements and H. Xiao, Current Opinion in Food Science, 2015, 2, 14–19.
9. N. Fu, Z. Zhou, T. B. Jones, T. T. Y. Tan, W. D. Wu, S. X. Lin, X. D. Chen and P. P. Y. Chan, Int. J. Pharm., 2011, 413, 155–166.
10. P. V. Gadkari and M. Balaraman, Food Bioprod. Process., 2015, 93, 122–138.
11. L. G. Gómez-Mascaraque, J. M. Lagarón and A. López-Rubio, Food Hydrocolloids, 2015, 49, 42–52.
12. I. Peres, S. Rocha, J. Gomes, S. Morais, M. C. Pereira and M. Coelho, Carbohydr. Polym., 2011, 86, 147–153.
13. J. F. P. S. Gomes, S. Rocha, M. do Carmo Pereira, I. Peres, S. Moreno, J. Toca-Herrera and M. a N. Coelho, Colloids Surf., B, 2010, 76, 449–455.
14. E. De Paz, Á. Martín and M. J. Cocero, J. Supercrit. Fluids, 2012, 72, 125–133.
15. M. Gomes, D. Santos and A. Meireles, in Conventional and Advanced Food Processing Technologies, ed. S. Bhattacharya, John Wiley & Sons, Ltd, First, 2015, pp. 249–265.
16. Á. Martín, H. M. Pham, A. Kilzer, S. Kareth and E. Weidner, Chemical Engineering and Processing: Process Intensification, 2010, 49, 1259–1266.
17. S. Varona, A. Martín and M. J. Cocero, Ind. Eng. Chem. Res., 2011, 50, 2088–2097.
18. Á. Martín and E. Weidner, J. Supercrit. Fluids, 2010, 55, 271–281.
19. D. Meterc, M. Petermann and E. Weidner, J. Supercrit. Fluids, 2008, 45, 253–259.
20. M. Fathi, Á. Martín, D. J. McClements and D. Julian, Trends Food Sci. Technol., 2014, 39, 18–39.
21. V. S. S. Gonçalves, S. Rodríguez-Rojo, E. De Paz, C. Mato, Á. Martín and M. J. Cocero, Food Hydrocolloids, 2015, 51, 295–304.
22. L. Bouarab, B. Maherani, A. Kheirolomoom, M. Hasan, B. Aliakbarian, M. Linder and E. Arab-Tehrany, Colloids Surf., B, 2014, 115, 197–204.
23. P. C. Suriyakala, N. Satheesh Babu, D. Senthil Rajan and L. Prabakaran, Int. J. Pharm. Pharm. Sci., 2014, 6, 6–9.
24. S. Hoeller, A. Sperger and C. Valenta, Int. J. Pharm., 2009, 370, 181–186.
25. D. Charalampopoulos, R. Wang, S. S. Pandiella and C. Webb, Int. J. Food Microbiol., 2002, 79, 131–141.
26. P. J. Wood, J. Cereal Sci., 2007, 46, 230–238.
27. K. Raemdonck, T. F. Martens, K. Braeckmans, J. Demeester, S. C. De Smedt and S. C. De Smedt, Adv. Drug Delivery Rev., 2013, 65, 1123–1147.
28. Y. Wang, J. Liu, F. Chen and G. Zhao, J. Agric. Food Chem., 2013, 61, 4533–4538.
29. M. Salgado, S. Rodríguez-Rojo, F. M. Alves-Santos and M. J. Cocero, J. Food Eng., 2015, 165, 13–21.
30. L. Gao, G. Liu, X. Wang, F. Liu, Y. Xu and J. Ma, Int. J. Pharm., 2011, 404, 231–237.
31. A. T. Serra, A. a. Matias, R. F. M. Frade, R. O. Duarte, R. P. Feliciano, M. R. Bronze, M. E. Figueira, A. de Carvalho and C. M. M. Duarte, J. Funct. Foods, 2010, 2, 46–53.
32. L. Li, K. Y. Y. Mak, J. Shi, C. W. W. H. C. H. Leung, C. M. M. Wong, C. W. W. H. C. H. Leung, C. S. K. S. K. Mak, K. Y. Y. Chan, N. M. M. M. M. Chan, E. X. X. Wu and P. W. T. W. T. Pong, Microelectron. Eng., 2013, 111, 310–313.
33. R. H. Liu and J. Finley, J. Agric. Food Chem., 2005, 53, 4311–4314.
34. Y. Sambuy, I. De Angelis, G. Ranaldi, M. L. Scarino, A. Stammati and F. Zucco, Cell Biol. Toxicol., 2005, 21, 1–26.
35. K. L. Wolfe and R. H. Liu, J. Agric. Food Chem., 2007, 55, 8896–8907.
36. Z. Li, J. Ha, T. Zou and L. Gu, Food Funct., 2014, 5, 1278.
37. P. Robert, V. Torres, P. García, C. Vergara and C. Sáenz, LWT--Food Sci. Technol., 2015, 60, 1039–1045.
38. S. Varona, S. Rodríguez Rojo, Á. Martín, M. J. Cocero, A. T. Serra, T. Crespo and C. M. M. Duarte, Ind. Crops Prod., 2013, 42, 243–250.
39. I. Peres, S. Rocha, M. D. C. Pereira, M. Coelho, M. Rangel and G. Ivanova, Carbohydr. Polym., 2010, 82, 861–866.
40. R. Gao, H. Liu, Z. Peng, Z. Wu, Y. Wang and G. Zhao, Food Chem., 2012, 132, 1936–1943.
41. M. K. Roy, M. Koide, T. P. Rao, T. Okubo, Y. Ogasawara and L. R. Juneja, Int. J. Food Sci. Nutr., 2010, 61, 109–124.
42. K. K. Adom and R. H. Liu, J. Agric. Food Chem., 2005, 53, 6572–6580.
43. B. Hu, Y. Ting, X. Zeng and Q. Huang, J. Agric. Food Chem., 2013, 61, 875–881.
44. V. S. S. Gonçalves, P. Gurikov, J. Poejo, A. Matias, S. Heinrich, C. M. M. Duarte and I. Smirnova, Eur. J. Pharm. Biopharm., 2016, 107, 160–170.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra13499h

---