NANOBERRIES FOR TOPICAL DELIVERY OF ANTIOXIDANTS 471 MATERIALS AND METHODS CHEMICALS Soybean phosphatidylcholine (SPC) (phospholipon 90 G, purity 90%) was a gift from Phospholipid/Natterman (Köln, Germany). NaChol, Sephadex G-50, butylated hidroxy- toluene (BHT), Folin–Ciocalteu reagent, gallic acid dimethyl-thiazolyltetrazolium bro- mide (MTT), linoleic acid, b-carotene, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich (St. Louis, MO). Tween 80 was purchased from Riedel-de Haën (Seelze, Germany). Other reagents were analytic grade from Anedra (Tigre, Argentina). Blueberries (Millenia, O’Neal, and Blue Crisp varieties) were donated by The Berry Store (San Pedro, Argentina). EXTRACTION OF POLYPHENOLS The varieties from V.myrtillus named Millenia, O’Neal, and Blue Crisp were harvested in November (springtime in the Southern hemisphere) 2010 at “The Berry Store” planta- tion in San Pedro, Argentina (latitude 33°45ʹ S, longitude 59°45ʹW, elevation 36 m) and maintained at −18°C. Before extraction, 25 g of each variety were defrozen at room tem- perature and grounded with the help of scissors. Crushed fruit from each variety was transferred to an Erlenmeyer and then, 150 ml of ethanol (96%) and HCl 0.1% were added. Erlenmeyers were submitted to bath sonication for 75 min at 25°C, 40 kHz. The resultant extracts were fi ltered under vacuum through Whatman No. 1 fi lters. Organic solvent was eliminated under rotary evaporation at 30°C, 120 rpm, and the water was eliminated by increasing vacuum and rotation rate to 250 rpm, up to constant weight. Residues were weighted and redissolved in ethanol-to-water 5:1 v/v, to a fi nal volume of 35 ml. The process rendered extracts of 715 mg of fresh fruit per milliliter. Three extracts were obtained by this process (38,39). Hydrodistillation was discarded as an extraction method after a 48-h distillation rendered no appreciable extract. DETERMINATION OF TOTAL POLYPHENOLIC COMPOUNDS Total polyphenolic compounds in each extract were measured by the method of the Folin–Ciocalteu reactive (40,41) using gallic acid as a standard. Aliquots containing 0.25 mg of each extract were mixed with 11.5 ml water and 0.25 ml Folin–Ciocalteu reactive under vigorous agitation. After 3 min, 0.75 ml of Na2CO3 (2%) was added. The samples were incubated for 2 h, and the tubes were periodically shaken at 15-min inter- vals. The same procedure was followed with gallic acid in the range from 0 to 0.25 mg to obtain a calibration curve. Absorbance of samples was then measured at 760 nm. DETERMINATION OF ANTHOCYANINS The content of total and monomeric anthocyanins of each extract was determined by the spectrophotometric pH differential method (42,43). Aliquots of each extract were added to two solutions of KCl 0.025 M and sodium acetate 0.4 M, respectively. The fi rst one

JOURNAL OF COSMETIC SCIENCE 472 was adjusted to pH 1.0, and the second one to pH 4.5 with HCl and their respective absorbance was determined at 520 and 700 nm in a Shimadzu UV–Vis 160-A spectro- photometer (Shimadzu Corp., Kyoto, Japan). For the calculations, cyanidin-3-glucoside (MW 449.2 ε= 26,900) was used as an absorbance standard (44). The anthocyanin con- tent was determined as: Anthocyanins (mg/l) = A × MW × Dilution Factor × 1,000/(ε × 1) The absorbance (A) for the monomeric anthocyanins was then calculated as: 510 700 510 700 ( )pH1.0 ( )pH4.5 A A A A A whereas for the total anthocyanins, it was calculated as: 510 700 ( )pH1.0 A A A PREPARATION OF NANOBERRIES: BLUEBERRY-LOADED ULTRADEFORMABLE LIPOSOMES (UL-B) Blueberry-loaded ultradeformable liposomes (UL-B) were prepared according to Cevc (21). Briefl y, UL composed of SPC and NaChol at 6:1 (w/w) ratio were prepared by mix- ing lipids from CHCl3 and CHCl3 : CH3OH (1:1, v/v) solutions, respectively, that were further fl ash-evaporated at 40°C in a round-bottom fl ask until all of the organic solvent was eliminated. The thin lipid fi lm was fl ushed with N2, and hydrated in a 7.65 mg of extract/ml solution of the ethanol extract of Millenia variety in 10 mM Tris–HCl buffer plus 0.9% (w/v) NaCl, pH 7.4 (Tris buffer) (extract dilution-to-Tris buffer 1:19), up to a fi nal concentration of 43 mg SPC/ml. The suspension was sonicated (45 min with a bath type sonicator 80W, 40 kHz) and extruded 12 times through two stacked 0.2 and 0.1 μm pore size polycarbonate fi lters using a 100 ml Thermobarrel extruder (Northern Lipids, Burnaby, Canada). After extrusion, the nonincorporated extract of Millenia variety was eliminated by gel fi ltration chromatography in a Shepadex G-50 column using the mini- column centrifugation method (45). Ultradeformability was tested allowing the UL-B suspension to pass through a 50 nm pore membrane under low pressure (less than 0.8 MPa) and comparing it with a conventional liposomal suspension (L) (46). Liposomal phospholipids were quantifi ed by a colorimetric phosphate micro assay (47). To quantify the content of extract of Millenia variety in liposomes, a calibration curve was prepared by recording the absorbance of aliquots at 583.5 nm of extract of Millenia variety in ethanol in the presence of phosphate, by diluting a phosphate salt to the same concentra- tion (50 mmol/ml) as in the phospholipid mix. Mean particle size and zeta potential of the UL-B were determined by dynamic light scattering with a Nanozetasizer (Malvern Instruments, Malvern, Worcestershire, UK). The antioxidant capacity retained in the UL-B was evaluated by the free radical scavenging as stated earlier. SPECTROSCOPIC ANALYSIS The UV–Vis spectra of the ethanolic extracts alone, in UL-B or in presence of phosphate salts at the same phosphate concentration than that of the liposomal suspension, were