Mulberry (Morus spp.; Moraceae), whose leaves represent indispensable food for silkworms, has been widely cultivated in China and Japan since early times. On the other hand, the root bark of mulberry (Mori Radicis Cortex) has been used in the traditional Chinese medicine as antiphlogistic, diuretic, antitussive, expectorant, antiheadache, and antipyretic. 1)Many novel compounds, regarded biogenetically as "DielsAlder-type adducts" of dehydroprenyl phenols and chalcone derivatives, have been isolated from several Morus species.2,3) Recently, albanol A 4) (mulberrofuran G 5) ) (1) and several other related compounds, isolated from the stem bark of M. australis, have been reported to show potent cytotoxic activities against four human cancer cell lines, lung (A549), stomach (BGC 823), colon (HCT-8), and ovarian (A2780) cancer cell lines, by means of the thiazolyl blue tetrazolium bromide (MTT) assay.3) In the course of our search for potential bioactive compounds from natural sources, 6,7) we were especially interested to undertake the investigation on the mechanisms of cytotoxic action of the "Diels-Alder-type adducts". In this study, we have isolated albanol A (1) and mulberofuran Q (2) from the root bark of M. alba, and have evaluated their cytotoxic activity against human leukemia (HL60) and human melanoma (CRL1579) cells. In addition, we have studied the apoptosis-inducing activity of albanol A (1), which exhibited potent cytotoxicity against HL60 cells, and the mechanisms of apoptotic cell death in HL60. Results and DiscussionAlbanol A (1) and mulberofuran Q (2) (Fig. 1) were isolated from the bark extract of Morus alba L. with ordinaland reversed-phase column chromatography and subsequent preparative HPLC. Compounds 1 5) and 2 8) were identified by MS and 1 H-NMR comparison with the literature data. On evaluation of these compounds for their cytotoxicities on HL60 and CRL1579 cell lines, compound 1 exhibited potent activity against both HL60 (IC 50 1.7 mM) and CRL1579 (9.8 mM) cell lines which were almost comparable with those of a reference compound, cisplatin (Table 1).Compound 1 was then evaluated for its inducing activity of early apoptosis on HL60 cells. Exposure of the membrane phospholipid, phosphatidylserine, to the external cellular environment is one of the earliest markers of apoptotic cell death.9) Annexin V is a calcium-dependent phospholipidbinding protein with high affinity for phosphatidylserine expressed on the cell surface. Propidium iodide (PI) does not enter whole cells with intact membranes and was used to differentiate between early apoptotic (Annexin V possitive, PI negative), late apoptotic (Annexin V, PI double positive), or necrotic cell death (Annexin V negative, PI positive). The proportion of early apoptotic HL60 cells (lower right) was significantly increased after 6 h of incubation with 1 (30 mM) Albanol A (1), isolated from the root bark extract of Morus alba (mulberry), was evaluated for the cytotoxic and apoptosis-inducing activities in human leukemia (HL60) cells, and ...

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