USE OF LECYTHIS PISONIS OIL IN COSMETIC CREAMS 241 potassium iodide solution and 30 ml of water were added. Thereafter, the mixture became yellow and was titrated with a standard solution of 0.1 N sodium thiosulfate until the yellowish color was not perceptible anymore. At this point, on adding 0.5 mL of starch indicator solution, the solution turned blue and titration with a standard solution of 0.1 N sodium thiosulfate was continued until the blue color disappeared. The peroxide index was calculated and expressed as meq KOH/100 g sample. Also, the lipids a nd fatty acids in the oil and formulations were determined, where the fat was extracted from the samples according to the Bligh–Dyer method (10). Briefl y, 2.5 g of the sample was mixed with a (10:20:8) solution of chloroform, methanol, and milli-Q water. The mixture was shaken and centrifuged at 900 rpm for 30 min, and then 10 mL of chloroform and 10 mL of aqueous solution of 1.5% sodium sulfate were added and shook again for 2 min. Then, the chloroform phase containing the fat was collected and fi ltered. After the removal of the solvent by evaporation with under-reduced pressure, the total fat content was determined by gravimetry. The fatty acids ex tracted from the sample were converted to fatty acid methyl esters (FAMEs) according to Joseph & Ackman (11), using 10% BF3 in methanol. Briefl y, 15 mg of the extracted fat was added to 1.5 mL of 0.5 M sodium hydroxide in methanol in a capped tube. The closed tubes were placed in a boiling water bath for 5 min. After cooling at room temperature, 2 mL of 10% BF3 in methanol was added, and the tubes were returned to boiling for 30 min. After another cooling step, 1 mL of isooctane was added and shook for 30 s. Finally, 5 mL of saturated sodium chloride solution was added to the tubes and shaken. The isooctane phase (containing FAMEs) was collected. As control, 1 mg·mL-1 methyl trioctanoate (C23:0Me) was added to the samples, to allow the correction of FAME quan- tifi cation due to variability in extraction, analytical instrument, or solvent evaporation. The resulting FAMEs w ere injected into a Shimadzu GC-2014 gas chromatograph (GC) with a fl ame ionization detector (FID) and an HP-INNOWax (Agilent, Santa Clara, CA) capillary column (50 m × 0.20 mm i.d. × 0.20 μm). The chromatographic conditions were as follows. The injector was operated at 250°C in split mode (1:10) for 1.0 min. The nitrogen drag gas fl ow was 1.25 mL/min, and the detector temperature was 260°C. The oven temperature gradient program was as follows: an initial temperature of 150°C, which was increased at 10°C/min to 260°C, where it was held for 9 minutes. A standard solution of FAMEs (GLC-85, Nu-check) was injected into the GC-FID system under the same conditions as for the samples. All analyses were performed in triplicate. The fatty acids were quantifi e d according to Visentainer (12) using the FAME areas in the chromatograms the areas were corrected with the theoretical correction factors (TCFs) and the conversion factors for fatty acids to FAME obtained using internal standards. To quantify the metals contain ed in the oil, nuts, arils, shells, and oil were digested with 10% nitric acid (65% purity, Sigma-Aldrich, Darmstadt, Germany) at 150°C using a Marconi digester (model MA 851, Marconi Equipamentos para Laboratório LTDA, Piracicaba, Brazil) and fi ltered through a 0.22-μm fi lter (Jet Biofi l, Guangzhou, China). Flame atomic absorption spectrophot ometry quantifi cation was performed in triplicate, using an atomic absorption spectrophotometer (iCE 3000, AA05141602 v.1.30, Thermo Scientifi c, Waltham, MA) and an atomizer with an air/acetylene burner and a hollow cathode lamp (Photron PTY. Ltd., Narre Warren, Australia) as a source of radiation to determine target elements. The calibration curve ranges for each analyte were as follows: Fe (1–15 mg/L), Na (1–15 mg/L), and Pb (10–40 mg/L). The chemicals were analytical
JOURNAL OF COSMETIC SCIENCE 242 grade, standard solutions (Specsol®, Quimlab Produtos de Química Fina Ltd., Jacareí, Brazil) and acetylene (99.5% purity, Sigma-Aldrich). COLORIMETRIC CELL VIABILITY TEST FOR THE OIL Cytotoxicity was evaluated in vitro u sing L929 fi broblasts (ATCC® CRL-6364™, Manassas, VA) and HaCat keratinocytes (BCRJ code: 0341) seeded on 96-well microplates at a cell density of 0.7·105 cells/mL in a fi nal volume of 200 μL per well and using the colorimet- ric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method (13), analyses were carried out in triplicate. The cells were exposed to concentrations of 1,000, 500, 250, 125, 62.5, and 31.2 μg/mL of L. pisonis oil. The oil was mixed with dimethyl sulfoxide (DMSO) to have a fi nal concentration of 0.5% of DMSO in the well. The dilu- tions of the oil/DMSO were made with the cell medium. Cell viability was determinated in relation to the negative control (0.5% DMSO), the limit of nontoxicity of DMSO, considered 100% of surviving cells, and the results are expressed as the mean ± standard deviation. CREAM OF L. PISONIS OIL The cream base was p repared in a commercial pharmacy (Farmácia Biomédica Manipulação, Vila Velha, Brazil) under supervision by the authors, according to the composition in Table I. The cream was separated in lots for the tests and fi ve different oil formulations were prepared for the assays: F0 (cream base), F1 (1% oil), F2 (5% oil), F3 (10% oil), and control (oil). EVALUATION OF THE FORMULATION CHARACTERISTICS A centrifugation test was performed, in triplica te for each formula except for the control group (oil), before and after the stability test according to the National Health Surveillance Agency (ANVISA) (14), with modifi cations. For each sample, 5 g was transferred to a tube and centrifuged at 3,000 g for 30 min and observed if any separation of phases occurs. Also, to evaluate the type of emulsion, fi ve drops of Scarlet Biedrit solution were added to the Table I Composition of the Cream Base for L. pisonis Oil Cream (3,000 g) Ingredient Function Amount Cetearyl alcohol/sodium cetearyl sulfate Self-emulsifying wax 360 g Isodecyl oleate Emollient 150 g Propylene glycol Solvent 150 g Phenoxyethanol + parabens Preservative 15 g Disodium EDTA Preservative 4.5 g Imidazolidinyl urea Antimicrobial preservative 4.5 g Demineralized water Vehicle 2,316 mL Cupuaçu essence Essence
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