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...
Crude extracts of Mexican medicinal plants were screened for trypanocidal activity against Trypanosoma cruzi, which is the etiological agent for Chagas' disease, one of the most serious protozoan diseases in Latin America. There were 43 kinds of methanolic and other organic extracts from 39 plants which were examined by the preliminary screening test to see immobilization of epimastigotes of T. cruzi in vitro. Eighteen of them showed activity at the concentration of 2 mg/ml after incubation for 2 h, while 13 showed activity at the concentration of 1 mg/ml after incubation for 48 h. Among them, the MeOH extract of roots of Aristolochia taliscana (Aristolochiaceae), locally known as "Guaco," immobilized all the epimastigotes even at lower concentration of 0.5 mg/ml (48 h). In order to identify principal compounds for this activity, the MeOH extract of Guaco was subjected to bioassay-guided fractionation. From the active fractions, four neolignans, eupomatenoid-7 (1), licarin A (2), eupomatenoid-1 (5) and licarin B (6), and two lignans, austrobailignan-7 (3) and fragransin E 1 (4) were isolated. Compounds 1-4 immobilized all the epimastigotes at the minimum concentration of 25-75 m mg/ml after incubation for 48 h, while compounds 5 and 6 were inactive. Corresponding concentration of gossypol, berberine chloride and harmine was 280 m mg/ml, 300 m mg/ml and Ͼ500 m mg/ml, respectively.
Physalis angulata L. (Solanaceae) is indigenous in tropicalAmerica 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.2) In the previous paper in this series, 3) we described the investigation of constituents in P. angulata based on the plant's trypanocidal activity against epimastigotes of Trypanosoma cruzi, the etiologic agent for Chagas' disease (American trypanosomiasis). Ten withanolides (1-10), including four new ones, were isolated from the active fraction. Trypanocidal activity of these withanolides against trypomastigotes, an infectious form of T. cruzi, was also estimated, as well as cytotoxic activity against human uterine carcinoma (HeLa) cells in vitro to examine the possibility of chemotherapeutic utility.Continuous study of the same plants resulted in the isolation of three new withanolides (11-13), designated physagulin L, M and N, respectively, together with known withanolide, physagulin D 4) (14) and flavonol glycoside, quercetin 3-O-rhamnosyl-(1→6)-galactoside (15).5) The structures of the known compounds were identified from their spectral data and confirmed by comparison with those published in the literature. In this paper we describe the structural elucidation of the three new withanolides and the trypanocidal activity of the isolates.Compound 11 has the molecular formula C 30 H 42 O 10 , one oxygen atom more than physagulin K (10).3) The 1 H-and 13 C-NMR spectra of 11 were similar to those of 10, except for signals in ring E, suggesting the same substitution pattern in rings A-D. In the 1 H-NMR spectrum, one olefinic methyl signal at d 2.04 (br s) and one hydroxymethylene signal at d 4.74 and 4.88 (d each, Jϭ12 Hz) were observed in 11, instead of two olefinic methyl signals in 10. In the 13 C-NMR spectrum of 11, the corresponding signals due to the olefinic methyl and hydroxymethylene groups were observed at d 20.0 and 56.2 accompanied with downfield shifts of C-24 (ϩ4.4 ppm) and C-25 (ϩ5.5 ppm) in comparison with those of 10. Therefore, it was suggested that one of the methyl groups at C-24 or C-25 was replaced with a hydroxymethylene group in 11. The methyl proton signal showed 3-bond connectivity with C-23 as well as C-25, and the hydroxymethylene proton signals with C-26 in the heteronuclear multiple-bond connectivity (HMBC) spectrum. Thus, the structure of 11 was elucidated to be (20R,22R)-15a-acetoxy5a ,6b ,14b ,17b ,27-pentahydroxy-1-oxo-witha-2,24-dienolide, and it is called physagulin L.The molecular formula of 12 was determined to be C 30 H 40 O 9 by HR-FAB-MS, one oxygen atom more than withaminimin (8).6) The 1 H-and 13 C-NMR spectra showed characteristic signals for a 1-one-2-ene system, 5a,6b-glycol moiety, and a 14a-hydroxy-15a-acetoxy-16-ene system. In fact, signals due to rings A-D were in good agreement with those of 8. As a part of the side chain, a carbon signal of hy- 1226 Vol. 54, No. 8 Trypanocidal Constituents in Plants 6. 1) Minor Withanolides from the Aerial Parts of Phy...
In the preceding paper in this series, 1) we described the identification of trypanocidal constituents in Guaco (Aristolochia taliscana, Aristolochiaceae) against epimastigotes of the rotozoan Trypanosoma cruzi, which is the etiologic agent of Chagas' disease (American trypanosomiasis). It is transmitted to humans by triatomine bugs or through blood transfusion. In the course of our search for trypanocidal constituents in plants, we have found that the MeOH extract of the stem bark of Garcinia subelliptica MERR. (Guttiferae) showed considerable activity.G. subelliptica (Guttiferae) is indigenous to the Ryukyu Islands and Taiwan. It is a large tree, growing up to 15-20 m high, and has been planted for use as a windbreak in the Yaeyama Islands of Japan.2) Its bark has been traditionally utilized to dye thread and cloth yellow.Many prenylated xanthones, garciniaxanthones A-E [3][4][5][6] and subelliptenones A-I, [7][8][9][10]
As a preliminary screening test for the trypanocidal activity against epimastigotes of Trypanosoma cruzi in vitro, we examined some crude extracts of Mexican medicinal plants. 2)T. cruzi is the etiological agent for Chagas' disease (American trypanosomiasis), one of the most serious protozoan diseases in Latin America. Then we started on identification of the principal compounds of the active extracts seeking new chemotherapeutic agents for Chagas' disease. Lignans 2) from Guaco (roots of Aristolochia taliscana) and xanthones 3) from Bari (stems of Calophyllum brasiliense) were isolated as active components against epimastigotes in vitro.T. cruzi exhibits three developmental stages: epimastigote in the insect gut; trypomastigote, an infectious form in the mammalian blood stream; and an amastigote, a proliferative form in mammalian cells. In this paper we describe the results of continuous preliminary screening tests for trypanocidal activity against trypomastigotes as well as against epimastigotes in some Mexican plants.The edible fruit of Persea americana MILL. (Lauraceae), avocado, is grown throughout the tropics worldwides. The MeOH extract of seeds of Persea americana also showed moderate activity in the screening test. Herein we also describe the identification of the active constituents in seeds of P. americana. MATERIALS AND METHODS Plant Materials and Preparation of Their ExtractsPlant materials were mainly purchased at a medicinal plant market in Guadalajara, Jalisco, and collected in the fields. Identification of the plants was done by M. A. Martinez-Alfaro, one of the co-authors. The voucher specimens were deposited in the Herbarium of Jardín Botánico del Instituto de Biologia, Universidad Nacional Autonoma de Mexico.The plants examined are listed in Table 1 including those referred to in ref. 2. Dried and powdered materials were extracted with MeOH or MeOH-CH 2 Cl 2 at room temperature overnight. In the case of fresh materials, chopped materials were soaked in MeOH and filtered. The residue was extracted again with MeOH. The solvent was concentrated in vacuo to give each extract.Cultivation of T. cruzi The strain of T. cruzi used in this study and the cultivation method of epimastigotes were the same as those described in the previous paper.2) The trypomastigotes were harvested from the culture supernatant of rhesus monkey kidney (LLC-MK2) cells infected with T. cruzi as described in the previous paper.4,5) LLC-MK-2 cells were maintained in DMEM containing 10% FBS at 37°C under 5% CO 2 . After inoculation of trypomastigotes into LLC-MK2 cells, the medium was changed to DMEM containing 10% NCS.Instruments and Reagents The instruments and reagents used in this study are the same as those described in the preceding paper. 1)Trypanocidal Assay The detailed assay procedure against epimastigotes was described in the previous paper.2) The activity was expressed as the MC 100 value (mg/ml or mM), which was defined as the minimum concentration at which all the epimastigotes were terminated after incubation fo...
We examined the antioxidant and antibacterial capacities of fukugetin and five flavonoids from Fukugi(Garcinia subelliptica) . Their capacities were compared with those of known structural analogs. Antioxidant capacities of the compounds were determined using different assays : radical scavenging capacity [2, 2-diphenyl-1-picrylhydrazyl ( DPPH ) , 2, 2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid ) (ABTS) ]; oxygen radical absorbance capacity(ORAC); and superoxide scavenging activity. All the compounds showed strong antioxidant capacity in all methods. In particular, fukugetin, a biflavonoid composed of the monomers naringenin and luteolin, showed highest antioxidant capacity among the compounds tested. The values were comparable to those of known antioxidants. In the DPPH and ABTS assays, the luteolin structure of fukugetin appeared to contribute to its antioxidant capacity. In the ORAC assay, the high antioxidant capacity of fukugetin resulted from the additive effect of naringenin and luteolin structures. Fukugetin also showed antibacterial capacity against some grampositive bacteria. The minimum inhibitory concentration estimated by disk diffusion method was 0.125 − 1 ! / ! , which is equivalent to known flavonoids such as kaempferol or quercetin. Our results showed the potential of fukugetin not only as an antioxidant but also as an antibacterial agent.
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