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 copolymer 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 the polymers separated cell debris, proteins and other comtaminants partitioned to the lower Reppal phase and ecdysone and 20-hydroxyecdysone partitioned to the upper UCON phase. The UCON phase was isolated and subjected to a temperature increase to 56°C which induced phase separation between UCON and water. Ecdysone and 20-hydroxyecdysone partitioned 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 ecdysteroids were obtained in this phase at 56°C with yields higher than 80%. With 20% ethanol in the primary system recovery was 88.7% for ecdysone and 91.2% for 20-hydroxyecdysone. Results indicate that aqueous two-phase partitioning coupled with temperature-induced phase separation is a quick, easy and inexpensive bench-top technique for extracting and purifying ecdysteroids from raw material. This technique can also be readily up-scaled for commercial use.