Encapsulation of Bioactive Compounds from Aloe Vera Agrowastes in Electrospun Poly (Ethylene Oxide) Nanofibers.

2.1. Materials Fresh Aloe Vera leaves from three-year old plants having a weight around 700–1000 g and a length of 50–70 cm were supplied by Las Coronas (Carnota, Sevilla, Spain). The tip, base and spikes of the leaves were removed and the epidermis was carefully separated from the inner gel using a sharp knife. The resulting Aloe Vera skin was intensively washed with distilled water and cut into small pieces. Then, it was freeze-dried with a Telstar Lyoquest −55 PLUS (Terrassa, Barcelona, Spain) and ground using a ZM 200 high-speed rotatory mill (Restch, Hann, Germany). Particles passing through a 1.0 mm sieve were used to ensure the homogeneity of the sample. Poly(ethylene oxide) (PEO, Mw ≈ 500,000 Da), absolute ethanol (99.8%), sodium acetate, 2,2-diphenyl-1-picrylhydrazyl (DPPH), glacial acetic acid, hydrochloric acid, 2,4,6-tripyridyl-s-triazine (TPTZ), ferric chloride hexahydrate, potassium persulphate, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were purchased from Sigma Aldrich (Madrid, Spain). Distilled-deionized water from a Millipore Milli-Q ultrapure water system was used (18.2 MΩ·cm at 25 °C). 2.2.

Preparation of Aloe Vera Extract

Microwave-assisted extraction method was performed to obtain Aloe Vera extract (AVE) by following a procedure previously optimized. A Milestone Flexiwave (Milestone srl, Sorisole, Italy) was used in open vessel mode. 1.5 g of freeze-dried Aloe Vera skin powder were mixed with 50 mL of an ethanolic solution (80%, v / v ) in a round-bottom flask. Then, the sample was heated in the microwave oven at 40 °C for 20 min under magnetic stirring (400 rpm). After the extraction, the supernatant was collected and stored overnight at −20 °C to remove possible interferences by precipitation. Subsequently, the supernatant was separated by centrifugation and ethanol was evaporated under reduced pressure. Finally, the Aloe Vera extract (AVE) was freeze-dried and stored at −20 °C in darkness until further use. 2.3. Preparation of Electrospun Nanofibers PEO (5 wt%) was dissolved in milli-Q water under magnetic stirring for 24 h. PEO/AVE mixtures were prepared in order to obtain nanofibers with antioxidant activity by electrospinning. PEO and AVE concentrations were selected based on preliminary experiments considering the morphology of obtained nanofibers. AVE was previously dissolved in an ethanolic solution (80%, v / v ) and it was added to PEO solution at different concentrations (0, 5, 10, and 20 wt% of AVE, with respect to the polymer content). All solutions were stirred in closed vials for 4 h in the dark to avoid any detrimental light effect. The electrospinning process was carried out at room temperature with a homemade apparatus composed of a digitally controlled KDS-100-CE syringe pump (KD Scientific Inc, Holliston, MA, USA), a high voltage power supply (Spellman, SLM50P300, Hauppauge, NY, USA), a circular aluminium fixed collector and a 10.0 mL glass syringe. Different combinations of applied voltage, flow rate and distance between the needle (20 G, i.d. = 0.6 mm) and the collector were tested to select the optimal process parameters. Final optimized conditions were 16 kV, 0.5 mL h −1 , and 15 cm, respectively. Experiments were performed at 25 ± 2 °C and 40–50% relative humidity. After the electrospinning process, the obtained mats were carefully separated from the collector and conveniently stored until further analysis. 2.4. Characterization of Electrospun Nanofibers 2.4.1. Scanning Electron Microscopy (SEM) SEM (Zeiss Leo Supra 35, Cambridge, UK) was employed to examine the morphology of PEO/AVE nanofibers. The fibres were mounted on aluminium stubs and then coated with an Au layer (≈5 nm) by sputtering (argon atmosphere, 25 mA, 7 × 10 −4 Bar, 120 s) prior to SEM analysis. The average fibre diameter (AFD) of each fibrous mat was calculated by means of ImageJ software, considering at least 100 random points from the SEM images at 30k× magnification level. The results were reported as mean ± standard deviation. 2.4.2. Fourier Transform Infrared Spectroscopy (FTIR) FTIR spectra of pure components and PEO/AVE nanofibers were recorded using an infrared spectrophotometer in ATR mode (ATR-FTIR-4600 Jasco, Oklahoma City, OK, USA) in the 4000–600 cm −1 range (spectral resolution 4 cm −1 , 32 scans). 2.4.3.

Thermal Analysis

Thermal properties of PEO/AVE electrospun nanofibers were evaluated by thermogravimetric analysis (TGA). TGA was carried out using a Mettler Toledo TGA/SDTA 851e equipment (Schwarzenbach, Switzerland). Approximately 5 mg of each sample were heated from room temperature up to 800 °C at 10 °C min −1 under nitrogen atmosphere (50 mL min −1 ). The onset temperature at 1% weight loss, temperature of maximum degradation and amount of residue at 800 °C were determined. Analyses were performed in triplicate. 2.4.4. Antioxidant Activity Antioxidant activity (AO) of PEO/AVE electrospun nanofibers was determined, in triplicate, by using three different methods: DPPH (2,2-diphenyl-1-picrylhydrazyl), FRAP (ferric reducing antioxidant power) and ABTS (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) assays. DPPH Radical Scavenging Method The DPPH scavenging activity was determined according to the method proposed by Aytac et al. [ 74 ] with some modifications, dissolving 1 mg of each PEO/AVE nanofiber sample in 2 mL of 10 −4 mol L −1 freshly prepared DPPH ethanolic solution (80%, v / v ). The absorbance was measured at 517 nm during 5 h using a Biomate-3 UV/Vis spectrophotometer (Thermospectronic, Mobile, AL, USA). The scavenging activity (%) was calculated as percentage of inhibition following Equation (1), where A control and A sample represent the absorbance values of the DPPH solution with, and without, the presence of the sample, respectively. 0.1 mL of AVE ethanolic solutions (80%, v / v ) at different concentrations were mixed with 2 mL of the freshly prepared DPPH solution and the absorbance was measured following the same procedure. The inhibition of DPPH free radicals was also compared with a Trolox calibration curve (10–250 mg kg −1 ; 6 points, R 2 = 0.9991), expressing results as µmol of Trolox equivalents per mg of nanofiber: (1) AO activity ( % ) = 100 × A control − A sample A control . FRAP Method The FRAP assay was determined according to Benzie and Strain [ 75 ]. The FRAP reagent was prepared mixing 0.3 mol L −1 acetate buffer (pH = 3.6), 10 mmol L −1 TPTZ made up in 40 mmol L −1 HCl and 20 mmol L −1 of aqueous FeCl 3 at a 10:1:1 ( v / v / v ) ratio. Then, 1 mg of the PEO/AVE nanofiber samples was dissolved in 3 mL of the freshly prepared FRAP reagent pre-heated at 37 °C. The absorbance was measured at 593 nm during 5 h. 0.1 mL of AVE ethanolic solutions (80%, v / v ) at different concentrations were also mixed with 3 mL of the FRAP working solution and the absorbance was measured analogously. Trolox was used as standard for preparing the calibration curve (10–250 mg kg −1 ; 6 points, R 2 = 0.9988) and results were expressed as µmol of Trolox equivalents per mg of nanofiber. ABTS Radical Scavenging Method The ABTS assay was performed according to Quispe et al. [ 25 ]. The ABTS radical cation was produced by mixing the ABTS solution (7 mM) with 2.45 mM potassium persulfate in a 1:1 ratio and allowing the mixture to stand in the dark at room temperature for 12 h. The ABTS working solution was obtained by diluting with aqueous ethanol (80%, v / v ) to a final absorbance of 1.00 ± 0.01 at 734 nm. 1 mg of the PEO/AVE nanofiber samples was dissolved in 3 mL of the freshly prepared working ABTS solution and the absorbance was measured during 5 h. 0.1 mL of AVE ethanolic solutions (80%, v / v ) at different concentrations were also mixed with 3 mL of the ABTS working solution and the absorbance was measured following the same procedure. The AO activity (%) was calculated analogously to the DPPH method using Equation (1). The inhibition of ABTS radicals was also compared with a Trolox calibration curve (10–250 mg kg −1 ; 6 points, R 2 = 0.9995), expressing results as µmol of Trolox equivalents per mg of nanofiber. 2.4.5. Encapsulation Efficiency The encapsulation efficiency (EE) of AVE in the mat of fibres obtained after the electrospinning process was calculated according to Equation (2): (2) EE ( % ) = 100 × amount of AVE calculated from AO assay initial amount of AVE used in the polymer formulation considering the AVE content determined by the antioxidant assays and the AVE amount used in the polymer formulations. The measurements were performed in triplicate. 2.5.

Statistical Analysis

Statistical analysis of results was performed with Statgraphics Centurion XVI statistical software. An analysis of variance (ANOVA) was carried out. Differences between average values were assessed based on the Tukey test at a confidence level of 95% ( p < 0.05).

2.1. Materials Fresh Aloe Vera leaves from three-year old plants having a weight around 700–1000 g and a length of 50–70 cm were supplied by Las Coronas (Carnota, Sevilla, Spain). The tip, base and spikes of the leaves were removed and the epidermis was carefully separated from the inner gel using a sharp knife. The resulting Aloe Vera skin was intensively washed with distilled water and cut into small pieces. Then, it was freeze-dried with a Telstar Lyoquest −55 PLUS (Terrassa, Barcelona, Spain) and ground using a ZM 200 high-speed rotatory mill (Restch, Hann, Germany). Particles passing through a 1.0 mm sieve were used to ensure the homogeneity of the sample. Poly(ethylene oxide) (PEO, Mw ≈ 500,000 Da), absolute ethanol (99.8%), sodium acetate, 2,2-diphenyl-1-picrylhydrazyl (DPPH), glacial acetic acid, hydrochloric acid, 2,4,6-tripyridyl-s-triazine (TPTZ), ferric chloride hexahydrate, potassium persulphate, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were purchased from Sigma Aldrich (Madrid, Spain). Distilled-deionized water from a Millipore Milli-Q ultrapure water system was used (18.2 MΩ·cm at 25 °C).

DPPH Radical Scavenging Method The DPPH scavenging activity was determined according to the method proposed by Aytac et al. [ 74 ] with some modifications, dissolving 1 mg of each PEO/AVE nanofiber sample in 2 mL of 10 −4 mol L −1 freshly prepared DPPH ethanolic solution (80%, v / v ). The absorbance was measured at 517 nm during 5 h using a Biomate-3 UV/Vis spectrophotometer (Thermospectronic, Mobile, AL, USA). The scavenging activity (%) was calculated as percentage of inhibition following Equation (1), where A control and A sample represent the absorbance values of the DPPH solution with, and without, the presence of the sample, respectively. 0.1 mL of AVE ethanolic solutions (80%, v / v ) at different concentrations were mixed with 2 mL of the freshly prepared DPPH solution and the absorbance was measured following the same procedure. The inhibition of DPPH free radicals was also compared with a Trolox calibration curve (10–250 mg kg −1 ; 6 points, R 2 = 0.9991), expressing results as µmol of Trolox equivalents per mg of nanofiber: (1) AO activity ( % ) = 100 × A control − A sample A control .

FRAP Method The FRAP assay was determined according to Benzie and Strain [ 75 ]. The FRAP reagent was prepared mixing 0.3 mol L −1 acetate buffer (pH = 3.6), 10 mmol L −1 TPTZ made up in 40 mmol L −1 HCl and 20 mmol L −1 of aqueous FeCl 3 at a 10:1:1 ( v / v / v ) ratio. Then, 1 mg of the PEO/AVE nanofiber samples was dissolved in 3 mL of the freshly prepared FRAP reagent pre-heated at 37 °C. The absorbance was measured at 593 nm during 5 h. 0.1 mL of AVE ethanolic solutions (80%, v / v ) at different concentrations were also mixed with 3 mL of the FRAP working solution and the absorbance was measured analogously. Trolox was used as standard for preparing the calibration curve (10–250 mg kg −1 ; 6 points, R 2 = 0.9988) and results were expressed as µmol of Trolox equivalents per mg of nanofiber.

ABTS Radical Scavenging Method The ABTS assay was performed according to Quispe et al. [ 25 ]. The ABTS radical cation was produced by mixing the ABTS solution (7 mM) with 2.45 mM potassium persulfate in a 1:1 ratio and allowing the mixture to stand in the dark at room temperature for 12 h. The ABTS working solution was obtained by diluting with aqueous ethanol (80%, v / v ) to a final absorbance of 1.00 ± 0.01 at 734 nm. 1 mg of the PEO/AVE nanofiber samples was dissolved in 3 mL of the freshly prepared working ABTS solution and the absorbance was measured during 5 h. 0.1 mL of AVE ethanolic solutions (80%, v / v ) at different concentrations were also mixed with 3 mL of the ABTS working solution and the absorbance was measured following the same procedure. The AO activity (%) was calculated analogously to the DPPH method using Equation (1). The inhibition of ABTS radicals was also compared with a Trolox calibration curve (10–250 mg kg −1 ; 6 points, R 2 = 0.9995), expressing results as µmol of Trolox equivalents per mg of nanofiber.