Citrinin, an inhibitor of cholesterol synthesis.

A compound active against sterol biosynthesis was isolated from the culture filtrate of the fungus Prthium ultimum IAM 6073 and identified as citrinin which had been isolated as an antibiotic.') The sequence of reactions by which cholesterol is formed from acetyl-CoA is one of the most complex biosynthetic pathways in eukaryotic cells. In every vertebrate species so far studied, potent activity in cholesterol synthesis can be detected in the liver which seems to be the sole organ supplying plasma cholesterol. Because of the importance of the plasma cholesterol level in atherosclerosis, several approaches have been made to control it by decreasing cholesterol synthesis in the liver.2~4) The inhibition of endogenous cholesterol synthesis could lead to a lowering of its level in the plasma. This communication describes inhibition by citrinin of cholesterol and ergosterol synthesis and its hypocholesterolemic activity in rats. The enzymatic synthesis of cholesterol was assayed by measuring the radioactive nonsaponifiable products derived from 14C-acetate in a rat liver enzyme system by the method of KNAUSS et al.5) Culture filtrates of microorganisms including fungi, bacteria, and actinomycetes were tested for inhibitory activity in this assay system, and Pvthium ultimum was selected as one of the producers of an inhibitor. The fungus was cultured aerobically in a medium containing 2.0% glucose, 2% peptone and 0.3 % corn steep liquor in a 30-liter fermentor for 4 days. The culture filtrate was adjusted to pH 2, and the active compound was extracted with benzene. The extract was concentrated in vacuo, resulting in the formation of yellowish crystals of the active compound. The compound was recrystallized from hot chloroform. Calcd.: C 62.39, H 5.64, O 31.97 Found: C 62.39, H 5.58, O 32.03 The chemical structure of 4,6-dihydro-8-hydroxy-3,4, 5-trimethyl 6 oxo3 H-2-benzopyran-7 carboxylic acid was suggested by n.m.r. spectrum. The identity with citrinin was proven by comparison with authentic samples (kindly supplied by Dr. S. UDAGAWA, National Institute of Hygienic Science, Tokyo). As shown in Fig. 1, citrinin strongly inhibited cholesterol synthesis from 14C-acetate. The concentration at which cholesterol synthesis was inhibited by 50 % was about 8.5 mcg/ml (3.4 x 10-5M). Under the same conditions, 14C-acetate incorporation into fatty acid fraction was also reduced by citrinin but to a lesser extent. The concentration of the inhibitor required for 50 inhibition of fatty acid synthesis was approximately 50 mcg/ml. Citrinin was also inhibitory in the sterol synthesizing system of the yeast Saccharomyces cerevisiae in which 14C-acetate incorporation into nonsaponifiable lipids was determined as described by KAWAGUCHI (Fig. 1).e) At a concentration of 100 mcg/ml, the inhibitor reduced the sterol synthesis by approximately 50%. For studies of hypocholesterolemic effect of