Isolation Of Pig Pulmonary Surfactant By Supercritical Carbon Dioxide Extraction And Analysis Of Its Chemical Composition

Pulmonary surfactant (PS) is a lipid/protein mixture containing primarily phospholipid which is a vital factor for normal respiration. Replacement therapy with pulmonary surfactant is the most effective path for the treatment of RDS and can also be used in other pulmonary diseases. Previously the preparation of natural PS is complicated and expensive. So natural PS 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-C02) can be especially applicable for the material which is easy to be oxygenated and decomposed, such as phospholipid. The authors extracted pulmonary surfactant from bronchoaveloar lavage fluid (BALF) of pig by SFE-C02. This method included three steps: at first BALF was centrifuged, then glyceride and cholesterol were isolated by SFE-C02, at last phospholipids were purified by SFE-C02. With orthogonal experiment design, the best condition to isolate -4- glyceride and cholesterol was selected, which was with pressure 45Mpa, temperature 50C, extraction time 5 hour. Extraction time is the main factor to improve extraction. The best condition to purified phosphlipids was under pressure 55Mpa, temperature 55 慍, with 1 .Oglg ethanol as modifier, static extraction 20 minute, dynamic extraction 3 hour. The efficiency of extraction was improved when pressure or dosage of modifier or static extraction time was increased. But dynamic extraction time hadn抰 remarkable effect on it, and the effect of temperature was multiple. The chemical composition of the production was determined by High Performance Liquid Chromatograph and other methods. Phospholipids were found to be the main component (89.76 ?2.0 1%), among which phosphatidylcholine was the chief substance (92.97 ?2.37%). The concentration of glyceride and cholesterol was 2. 44 ?0. 96%, 6. 57 ?1. 55% respectively. It didn抰 contain protein. The production meets the requirement for therapy. Therefore, this study is very useful for the preparation of natural surfactant.