Research On Purifying Pig Pulmonary Surfactant By Supercritical Carbon Dioxide Extraction

Pulmonary surfactant (PS) is a complex lipoprotein mainly consisting of phospholipids and specific proteins . Lack of PS causes a severe respiratory disease named respiratory distress, which is the cause of high morbidity and mortality, particularly in pre-term infants. Replacement therapy with exogenous pulmonary surfactant material is the most effective path for the treatment of RDS and can be also used in other pulmonary diseases. Previously the preparation of natural PS is complicated and expensive. So it is seldom used on clinic. It is necessary to found a simple, efficient and safe method for isolation of pulmonary surfactant.Supercritical fluid extraction (SFE) is a new separation technique. Compared with conventional methods, it is rapid, automation, easy to control and seldom uses organic solvent. Supercritical carbon dioxide extraction (SFE-COa) can be especially applicable for the material which is easy to be oxygenated and decomposed, such as phospholipid.The present research relates to a process for the purification of pig pulmonary surfactant by means of extraction and separation of undesired components consisting of lipidic substances of a low polar power. Now it has been found that the procedure with supercritical fluids can advantageously be used for the purification of PS if the extractive step is preceded by dispersing the material on a high surface area inert solid, to increase as much as possible the surface area of the material to extract, therefore promoting the permeation of the material itself with the solvent, increasing its extractive power. This method included a few steps: at first the crude PPS was obtained from bronchoaveloar lavage fluid (BALF), and dispersed said crude PPS on aninert carrier material, such as glass beads or diatomaceous earth, then undesired components consisting of lipidic substances of a low polar power were extracted and separated by SFE-CO2, finally the mixture is removed from the extractor, and filtered with suction to separate diatomaceous earth.The best condition to extract low polar substances was found, which was under presser 30Mpa, temperature 50 C, 1g crude PPS with 0.2ml ethanol as modifier, static extraction 20 minute, dynamic extraction 4 hour, the weight ratio of diatomaceous earth to crude PPS to extract is 1.5:1, With orthogonal experiment design, we found the efficiency of extraction was improved when pressure or dynamic extraction time was increased. But dosage of modifier and temperature hadn't remarkable effect on it.The chemical composition of the production was determined. Phospholipids were found to be the main component, composing (91.33 ?1.58%),The concentration of glyceride, cholesterol and protein was 0.72 + 0.11%, 0.66+0.10%, 2.36+0.51% respectively. It was be found that the production contain six kinds of phospholipids by by thin-layer chromatography and High Performance Liquid Chromatograph, among witch phosphatidylcholine (PC) was the chief substance (81.42?.35%).Surface activity of the production reduced minimal surface tension to less than lOmN/m, showing that the novel purification procedure does not affect the biological properties in any way.