USE OF LECYTHIS PISONIS OIL IN COSMETIC CREAMS 243 formulations, followed by homogenization. Thereafter, 100 μL was transferred to a micro- scopic slide and analyzed on a Leica Galen III microscope (Leica, Wetzlar, Germany). The pH was evaluated according to ANVISA (14) and measured using a Gehaka digital pH meter (Gehaka, São Paulo, Brazil). The pH of L. pisonis oil was measured using pH test strips from MColorpHast® (Merck, Darmstadt, Germany). The viscosity was evaluated at room temperature (21 °C) with a Brookfi eld viscosimeter using spindle number 4. The applied rotation speeds were 5–11 rpm for the samples tested before the stability evaluation and 8–16 rpm for those tested after the stability evaluation. The results are expressed in centipoise (cP) (14). The spreadability assay was performed according to K norst (15), with modifi cations, for all formulations before and after the stability test. One gram of each sample was placed on the central spot of the acrylic plate, and another plate was placed aligned over the sample. On top, a calibrated weight of 200 g was placed for 2 min. The diameter of the sample spot was recorded in opposing directions, and the median diameter was calculated. A commercial moisture cream from Granado was used as a reference. For the organoleptic characteristics, all the formula tions were visually analyzed for changes in color, odor, and homogeneity throughout the study and after each test (16). The stability test was performed on all samples in tr iplicate, according to ANVISA (17). The samples in sterilized glass jars were subjected to different temperature conditions: frozen (-5°C ± 2°C) for 24 h, room temperature (15°–30°C) for 24 h, and heated in an oven (40°C) for 24 h. Three cycles of the stability test were performed. The stability of the samples was evaluated based on the organoleptic properties (16) and physical–chemical characteristics. MICROBIAL LIMIT TEST The microbiological safety was assessed on the basis of the 481 /99 Brazilian regulation for cosmetic products (18). Samples were randomly selected prior and after the stability test and artifi cially contaminated with different microorganisms for the challenge test. This test aims to determine the resistance of a product to microbial contamination, thereby refl ecting the effectiveness of its preservative system. The formulations were arti- fi cially contaminated via inoculation with a suspension of selected microorganisms at 0.5 on the McFarland scale, followed by a survival determination by the plate counting method at 0, 24 h, 48 h, 7 d, 14 d, 21 d, and 28 d. Escherichia coli ATCC 8739, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa CCCD P003, Candida albicans ATCC 10231, and Aspergillus niger ATCC 40067 were cultured in Müller–Hinton medium (Kasvi, São José dos Pinhais, Brazil) and then inoculated to 10% of cream for different formulations. STATISTICAL ANALYSIS The quantitative variables were expressed as means ± standard dev iations, and the data were submitted to Student’s t-test with a signifi cance level of p 0.05. For the quantifi ca- tion of fatty acids in the oils from different regions, data were analyzed by one-way anal- ysis of variance and Bonferroni post hoc tests. For the quantifi cation of fatty acids in the cream before and after the stability test, a t-test was performed. The qualitative variables were expressed in a descriptive manner.

JOURNAL OF COSMETIC SCIENCE 244 RESULTS AND DISCUSSION The L. pisonis nut oil was transparent and presented a light yello wish green color and a pleasant characteristic oil odor, in agreement with the literature (4,5,19). The average yield of the oil was 35%, which is similar to what is described for other natural oils used in cosmetics, such as tucumã oil (22%) and murumuru oil (34–46.2%) (20), indicating it is a considerable raw material to the development of cosmetics. Considering the fatty acid contents in L. pisonis oils from nuts obt ained in different regions of Brazil, they were similar to the one determined in Espírito Santo in which the most abundant fatty acids were oleic and linoleic acids, followed by palmitic acid (Table II). These fatty acids are desirable for moisturizing products used for skin and hair, such as cream, body oil, and emollient or massage purposes (20). As the physical–chemical characteristics of the nut oil, the acidity index (0.12 mg/g) and peroxide index (6 meq/kg), are in accordance with the ANVISA parameters (maximum of 0.6 mg KOH/g and below 15 meq/kg, respectively) (21), it is indicated that no hydro- lysis of free fatty acids occurred during the extraction or oxidation, demonstrating its good quality. Also, comparing with the L. pisonis oil from Amazonia, the one from Espírito Santo presented a lower acidity value (0.31 ± 0.01 mg/g) and a higher peroxide value (0.27 ± 0.02 meq/kg) (19). This variation may be due to the difference in the soil compo- sition that interfere in the oil constituents, where the Espírito Santo’s oil is more diverse than the one from Amazonia. The level of lead detected in the nuts, arils, and oil evaluated was below the allowable limit, indicating that the nuts are secure for consumption and use. The content of iron was 2.17 ± 0.47 mg/100 g in nuts and 1.12 ± 0.15 mg/100 g in arils. These values are comparable with those reported for Brazil nuts (2.3 mg/100 g) and coconut (1.8 mg/100 g) (22) and lower than those in L. pisonis nuts from the Minas Gerais region (7.0 mg/100 g) (5). For the oil, the lead level was 9.40 μg/g and similar to those found in sunfl ower, olive, canola, and soy oils (9.14–9.82 μg/g) (23). The sodium concentrations were 3.27 ± 0.62 mg/100 g in nuts, 6.62 ± 1.06 mg/100 g in arils, and 0.7 μg/g in oil. These values indicate that the tested L. pisonis oil is of high Table II Fatty Acid Contents in L. pisonis Oil Samples from Espírito Santo, Minas Gerais, Amazonia, and Piauí Fatty acids (%) Espírito Santo Minas Gerais (5) Amazônia (19) Piauí (4) Myristic (C14:0) 0.09 ± 0.1 — — 0.09 ± 0.01 Palmitic (C16:0) 12.20 ± 0.01 11.29 14.49 ± 0.24 14.17 ± 1.17 Palmitoleic (C16:1) 0.41 ± 0.02 0.34 0.21 ± 0.01 0.26 ± 0.08 Margaric (C17:0) — — — 0.08 ± 0.01 Stearic (C18:0) 6.99 ± 0.03 3.77 5.84 ± 0.04 8.09 ± 0.30 Oleic (C18:1 n9) 49.95 ± 0.02 41.37 38.82 ± 0.08 43.17 ± 3.04 Linoleic (C18:2 n6) 29.84 ± 0.01 42.86 39.93 ± 0.11 34.83 ± 4.47 Linolenic (C18:3 n3) 0.2 ± 0.03 0.24 0.24 ± 0.01 0.58 ± 0.06 Arachidic (C20:0) 0.18 ± 0.01 0.13 0.22 ± 0.01 — (C20:1 n9) 0.07 ± 0.01 — — — Cis-11-eicosenoic (C20:1) — — — 0.07 ± 0.01 Behenic (C22:0) 0.05 ± 0.01 — Tra — a Tr = percentage 0.1.