UTILIZATION OF TEMPERATURE-INDUCED PHASE-SEPARATION FOR THE PURIFICATION OF ECDYSONE AND 20-HYDROXYECDYSONE FROM SPINACH

An aqueous solution of the ethylene oxide-propylene oxide random copol ymer UCON 50-HB-5100 was successfully used to extract ecdysone and 20- hydroxyecdysone from the common spinach plant, Spinacia oleracea. The UCON spinach extract was mixed with a hydroxypropyl starch Reppal PES 200 solution and allowed to form an aqueous two-phase system. After th e polymers separated cell debris, proteins and other comtaminants part itioned to the lower Reppal phase and ecdysone and 20-hydroxyecdysone partitioned to the upper UCON phase. The UCON phase was isolated and s ubjected to a temperature increase to 56 degrees C which induced phase separation between UCON and water. Ecdysone and 20-hydroxyecdysone pa rtitioned between the UCON phase and the water phase at concentrations determined by their degree of hydrophobicity. The less hydrophobic 20 -hydroxyecdysone had a greater affinity for the water-rich phase than did ecdysone. Due to the larger volume of the water phase both ecdyste roids were obtained in this phase at 56 degrees C with yields higher t han 80%. With 20% ethanol in the primary system recovery was 88.7% for ecdysone and 91.2% for 20-hydroxyecdysone. Results indicate that aque ous two-phase partitioning coupled with temperature-induced phase sepa ration is a quick, easy and inexpensive bench-top technique for extrac ting and purifying ecdysteroids from raw material. This technique can also be readily up-scaled for commercial use.