In the previous paper in this series, 2) we described the identification of trypanocidal xanthones in Garcinia subelliptica against epimastigotes and trypomastigotes of the protozoan Trypanosoma cruzi, which is the etiologic agent of Chagas' disease (American trypanosomiasis). In the course of our search for trypanocidal constituents in plants, we have found that the MeOH extract of the aerial parts of Physalis angulata L. (Solanaceae) showed considerable activity.P. angulata is indigenous to tropical America and was naturalized in Japan in the Edo era. It is an annual small herb and has been traditionally utilized for antipyretic purposes in Japan.3) The isolation of ergostane-type steroidal compounds, physagulins A-G, 4-6) withangulatin A 7) and physalins, 8) has been reported from this plant.T. cruzi is transformed into three stages in its life cycle: epimastigote in the insect gut; trypomastigote, an infectious form in the mammalian blood stream; and amastigote, a proliferative form in mammalian cells. During the isolation procedure, trypanocidal activity was monitored against epimastigotes, because it is easy to cultivate them and they are not infectious. Ten withanolides (1-10) were isolated from the active fraction. To check the therapeutic possibility of these withanolides, trypanocidal activity against trypomastigotes, an infectious form, was examined. Furthermore, evaluation of the IC 50 value against epimastigotes was performed using Cell Counting Kit-8 in order to compare with cytotoxicity at the IC 50 value. In this paper we describe the structural elucidation of the four new withanolides (2, 5, 9, 10) and the trypanocidal activity of the isolates. The possibility of chemotherapeutic utility is also discussed.
MATERIALS AND METHODSPlant Materials P. angulata was cultivated in the medicinal plant garden of Fukuoka University and harvested in September 2002. The aerial parts were air-dried and powdered.Instruments and Reagents The instruments used in this study were a JEOL JNM-A500 for NMR spectra (500 MHz for 1 H-NMR, 125 MHz for 13 C-NMR), a JEOL JMS-HX 110 for MS spectra, a JASCO DIP 360 polarimeter for optical rotations, and a Labsystems Multiskan JX Microplate Reader for measuring the absorbance of a formazan product. The following materials were used for chromatography: Diaion HP-20 (a styrene polymer, Mitsubishi Chemical Industries, Ltd.), Silica gel 60N (Cica), Sephadex LH-20 (Pharmacia Fine Chemicals), and YMC gel (ODS, YMC Co. Ltd.). Newborn calf serum (NCS) was purchased from Nacalai Tesque. Dulbecco's modified Eagle's medium (DMEM) and RPMI 1640 medium were purchased from Sigma. Gentian violet, ketoconazole, and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Wako. A Cell Counting Kit-8 was purchased from Dojindo. Other reagents are the same as in the previous papers.
2,9)Cultivation of T. cruzi The strain of T. cruzi used in this study and the cultivation methods of epimastigotes and trypomastigotes were the same as those described in the previous p...