Abstract:
Marigold flowers is a rich source of lutein, a type of xanthophylls that has been demonstrate to reduce the risk of ocular diseases and provide protective effect against cardiovascular diseases and stroke, etc. However, to exhibit some of these pharmaceutical properties, the compound should be of high purity. Liquid chromatography has widely been used to effectively separate lutein from other impurities for analytical purposes. However, only few reports were found on larger preparative scale. The first part of this work therefore aims to develop a protocol for preparative scale liquid chromatography purification of free lutein derived from marigold flowers. With silica gel as a stationary and mixture of hexane:ethyl acetate at 70:30 volume ratio (10 ml/min flow rate) as a mobile phase, as high as 97.1 % purity lutein could be obtained. Unfortunately, other than the purity issue, one other limitation to the applicability of the compound is its poor water solubility. This causes low oral and dermal bioavailability. To reduce this problem, supercritical anti-solvent (SAS) micronization of lutein particles were investigated using carbon dioxide (CO2) as anti-solvent. In the second part of this work, because their binary phase equilibrium data with CO2 are available in literature, the possibility to use dichloromethane (DCM) and ethanol as solvents in which lutein was dissolved prior to the SAS experiments was investigated. The reduction in size of lutein from 202.3 µm of unprocessed lutein to 1.58 µm and 902 nm could be achieved by SAS technique using DCM and ethanol, respectively. In addition, the last part of this work aims to demonstrate the feasibility of SAS micronization of lutein dissolved in mixture of hexane and ethyl acetate (70:30 v/v). The solubility of the SAS micronized lutein particles in aqueous solution was found to increase significantly from being almost insoluble to having approximately 20% solubility. Success in SAS micronization in hexane:ethyl acetate solvent mixture, which was used as a mobile phase for the chromatographic purification, implies that a step of solvent evaporation from the eluted samples and re-dissolving the dried sample into another organic solvent can be omitted. By this, not only the process cost can be reduced, the degradation of lutein during complicated processing steps can also be minimized.
