Purification and Identification of the Antistiffness Factor. ∗

Abstract

In a previous study ( 1 ) evidence was presented that the infra-red absorption spectrum of the antistiffness factor obtained from sugar cane resembled the spectrum of stigmasterol more closely than any other spectrum of a large series of steroid compounds investigated. Purification and characterization of the natural antistiffness factor in the earlier work was accomplished by solvent partition and spectrophotometric analysis. The present paper is a continuation of this work.

Counter-current distribution was performed in a 24-plate Craig apparatus ( 2 ), a methanol-isooctane two-phase system being utilized( 3 ). Solvent partition was applied to a purified concentrate† from sugar cane, the migration of the material being followed by dry weight determinations and ultra-violet absorption characteristics. On the basis of both the latter analytical technics, the concentrate was divided into 3 fractions, A, B, and C. Infrared analysis of each fraction ascertained the relative purity and aided in characterization of each fraction.

The infra-red absorption data indicated that fractions A and C were free of each other and that fraction A probably was a sterol. The sterol nature of fraction A was further demonstrated by a positive Liebermann-Burchard reaction. Since fraction A gave also a mild, positive Rosenheim color reaction, the presence of some diene impurity was suggested and this was supported by the ultra-violet absorption spectrum of fraction A, which at high concentrations was nearly identical to those of ergosterol, 7-dehydrocholes-terol and other Δ^5,7-diene steroids( 4 ). It was not concluded by Ross, van Wagtendonk and Wulzen( 5 ), despite low extinction coefficients which they obtained, that the similar absorption spectrum of their antistiffness factor was due to a diene impurity.

The infra-red absorption spectrum of fraction A was utilized in screening a large series of steroid compounds, efforts being concentrated on stigmasterol and ergosterol derivatives of the dihydroergosterol type because of spectral similarities.