(325b) Micronization of Phytosterols Using Carbon Dioxide-Expanded Ethanol: Phase Behavior, Processing and Characterization

Phytosterols have received growing attention in recent years due to their demonstrated health benefits in terms of serum cholesterol reduction. Approval of the use of phytosterols in foods in the European Union, United States and other countries led to the introduction of numerous food products formulated with phytosterols. However, there are formulation issues because the phytosterols are not soluble in water and hardly soluble in fats and oils. In addition, the physicochemical structural characteristics of phytosterols have a significant influence on their efficacy as they compete with cholesterol for inclusion into the dietary mixed micelles during food digestion to inhibit cholesterol absorption [1]. It is desirable to have micro/nano particles of increased surface area for enhanced efficacy. The DELOS (depressurization of an expanded liquid organic solution) process, where CO₂ acts as a co-solvent under high pressure, has been used previously for the micronization of different molecular actives but not for phytosterols [2,3]. Successful development of this process requires knowledge of the phase behavior of the bioactive in the CO₂-expanded solvent; however, such information is lacking for phytosterols in CO₂-expanded ethanol. Therefore, the objectives of this study were to investigate the phase behavior of phytosterols in CO₂-expanded ethanol, to utilize the DELOS process for micronization of phytosterols and to characterize the particles obtained under different processing conditions.

The phase behavior of the phytosterol+ethanol+CO₂ mixture was studied at 25 and 35^oC and 100 bar using a variable volume view cell to observe the disappearance of solid particles and thus the transition from the two-phase to the single-phase region. Then, the DELOS process was applied at 25 and 35^oC and 100 bar with a starting solution of different CO₂ concentrations (X_CO2=0.44-0.77, solute-free basis) and supersaturation ratios (β=0.6-1.5). The particles obtained upon depressurization were collected by off-line filtration and characterized using different techniques, including scanning electron microscopy (SEM), differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), and laser diffraction particle size analysis.

The phase diagram established for the phytosterol+ethanol+CO₂ mixture demonstrated that CO₂ acts as a co-solvent at X_CO2 of less than approximately 0.8 with a substantially increased solubility of phytosterols relative to the ideal dilution line and as an antisolvent at higher X_CO2 levels. The phase boundary shifted upwards with an increase in temperature from 25 to 35^oC, leading to higher solubility of phytosterols in CO₂-expanded ethanol at 35^oC. The DELOS process resulted in the formation of phytosterol particles upon rapid and uniform subcooling that occurs during depressurization of the CO₂-expanded solution. The temperature drop upon depressurization increased with an increase in X_CO2 reaching 84^oC at X_CO2 of 0.77. The particle yield also increased with X_CO2 and initial supersaturation levels. The median particle size based on volumetric particle size distribution dropped from 188 µm with a broad distribution to 2.4-6.5 µm with a narrow distribution after the DELOS process. XRPD analysis showed that all samples obtained under different DELOS conditions crystallized into the same crystalline form or polymorph. Based on SEM micrographs, the particles had plate-like morphology. DSC analysis exhibited a melting peak at 138.5^oC, corresponding to pure β-sitosterol. The findings demonstrate that the micronization of phytosterol particles was achieved successfully using the DELOS process; however, further research is needed to demonstrate the physiological functionality of these particles.

References:

[1]        V Piironen, DG Lindsay, TA Miettinen, J Toivo, AM Lampi, J. Sci Food Agric. 80 (2000) 939-966.

[2]        N Ventosa, S Sala, J Veciana, J Supercrit Fluids26 (2003) 33-45.

[3]        S Sala, E Elizondo, E Moreno, T Calvet, MA Cuevas-Diarte, N Ventosa, J Veciana, Cryst Growth & Design 10 (2010) 1226-1232.