A systematic screening of plants carefully selected to represent 186 families indigenous to Japan has been carried out and the screening of 1056 species has led to the discovery of several new plants as sources for the phytoecdysones. The crude extracts of plants were subjected to the newly developed Chilo dipping method for rapid assay of activity; the method has the advantage of being simple to manipulate, rapid, and highly sensitive. A separation technique employing automatic liquid chromatography on an Amberlite XAD-2 column has been found to be effective for separating and identifying the various phytoecdysones (Fig. 1). Extraction of the dry leaves (1.2 tons) of Podocarpus macrophyllus D.DON has resulted in the isolation of ecdysterone (2), ponasterone A (4), and four new ecdysones, makisterone A, B, C and D. Structural studies of the four new compounds based on spectroscopic data of the free and acetylated compounds has enabled one to assign structures 11, 12, 13 and 14, respectively, to makisterone A, B, C and D; it is interesting that a single plant has afforded a total of six active compounds ranging from C_<27> to C_<29>. Extraction of Ajuga incisa MAXIM. has given ecdysterone (2), cyasterone (16) and two additional compounds, ajugasterone A and B. The structure of ajugasterone B can be represented by (15), i.e., the first ecdysone containing an unsaturated side-chain.
