STUDIES ON THE HEMORRHAGIC SWEET CLOVER DISEASE IV. THE ISOLATION AND CRYSTALLIZATION OF THE HEMORRHAGIC AGENT

1. The hemorrahgic agent is spoiled sweet clover hay (Melilotus alaba) has been isolatd in a pure state, m. p. 288-289°, from experimentally produced spoiled hays as well as form hyas that killed cattle in argicultural practice. 2. This substance has the empirical formula C12H12O6. It is optically inactive. There are two acidic hydroxyls in the molecule. The acidity of the pure substance falls between that of the phenols and the carboxylic acids. A crystalline dimethyl other C12H12O4(OCH2)2 with a melting point fo 168-170° (physiologically inactive) has been prepared by methylation with diazomethane. 3. In the pure state the substance has a low soulubility in the ordinary organic solvents. It is insoluble in acid media. Basic solvenets. and dilute alkali effect solution radily (salt formation). 4. The substance could not be characterized or identified on the basis of its behavior towards the usual identification reagents. Its occurrence in nature has not previosly been reproted. 5. 1.5 mg. of hte crystalline hemorrhagic agent cause approximately the same reduciton in the prothrombin level or activity of the plasma of standardized susceptible reabbits in 40 hours as 50 gm. of the standard spoiled hay sample. 6. Spoiled sweet cover hays produced experimentally from Melilotus alba and those relized in agricultural parctice contain approximately 0.003 per cent of the hemorrhagic agent on the dry substance basis. The over-all yield of the subsance in a fractionation scheme involving sixteen steps approximates 86 per cent of the quantity present.