Spirostanol glucosides from the leaves of Cestrum laevigatum L.

Two new steroidal saponins, (25R)-spirost-5-ene-3ß,26ß-diol 3-O-α-L-rhamnopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 4)-[(1 → 2)-α-L-rhamnopyranosyl]-ß-D-glucopyranoside (1) and (25R)-spirost-6-ene-3ß,5ß-diol 3-O-α-L-rhamnopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 4)-[(1 → 2)-α-L-rhamnopyranosyl]-ß-D-glucopyranoside (2), along with the known diosgenin 3-O-α-L-rhamnopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 4)-ß-D-glucopyranoside (3), chonglouoside SL-5 (4) and Paris saponin Pb (5) were isolated from the leaves of Cestrum laevigatum. The structures of the compounds were determined using spectroscopic analyses including HRESI-MS, 1D and 2D NMR data, followed by comparison with data from the literature. Among them, two are particularly unique, compound 1 is the first (6)Δ-spirostanol saponin and compound 2 has an unusual C-26 hydroxyl in the (5)Δ-spirostanol skeleton. Antifungal testing showed a potent activity to formosanin C against Candida albicans and Candida parapsilosis. Evaluation of the cytotoxic activity indicated that compound 1 has a moderate activity against HL-60 and SF-295 cell lines, while compound 2 were active only against HL-60.

Evaluation of the genotoxicity of piplartine, an alkamide of Piper tuberculatum, in yeast and mammalian V79 cells.

The genus Piper belongs to the Piperaceae family, and includes species of commercial and medicinal importance. Chemical studies on Piper species resulted in the isolation of several biologically active molecules, including alkaloid amides, such as piplartine. This molecule, isolated from Piper tuberculatum, has significant cytotoxic activity against tumor cell lines, and presents antifungal, anti-platelet aggregation, anxiolytic, and antidepressant effects. In order to understand the biological properties of piplartine, this study investigated the genotoxicity and the induction of apoptosis by piplartine in V79 cells and its mutagenic and recombinogenic potential in Saccharomyces cerevisiae. Piplartine induced dose-dependent cytotoxicity in S. cerevisiae cultures in either stationary -- or exponential growth phase. In addition, piplartine was not mutagenic when cells were treated during exponential-growth phase and kept in buffer solution, but it increased the frequencies of point, frameshift, and forward mutations when cells were treated in medium during growth. Piplartine treatment induced DNA strand breaks in V79 cells, as detected by neutral and alkaline comet assay. In cell cycle analysis, piplartine induced G2/M cell cycle arrest, probably as a consequence of the DNA damage induced and repair. Moreover, piplartine treatment induced apoptosis in a dose-dependent manner, as observed by a decrease in mitochondrial membrane potential and an increase in internucleosomal DNA fragmentation. These data suggest that the DNA damage caused by piplartine induces G2/M cell cycle arrest, followed by apoptosis. Moreover, we suggest that cells surviving piplartine-induced DNA damage can accumulate mutations, since this alkaloid was mutagenic and recombinogenic in S. cerevisiae assays.

Piplartine induces inhibition of leukemia cell proliferation triggering both apoptosis and necrosis pathways.

Piplartine {5,6-dihydro-1-[1-oxo-3-(3,4,5-trimethoxyphenyl)-2-propenyl]-2(1H)pyridinone} is an alkaloid/amide component of Piper species. The purpose of the present study was to examine the antiproliferative effects of piplartine on human leukemia cell lines HL-60, K562, Jukart, and Molt-4 using the trypan blue exclusion method, as well as the effect of piplartine on DNA synthesis. The viability of all human leukemia cell lines were not affected by piplartine after 6 h, 9 h, and 12 h exposure, whereas a steady decline was seen after an exposure time of 24 h. The antiproliferative activity of piplartine seemed to be related to the inhibition of DNA synthesis, as revealed by the reduction of 5-bromo-2'-deoxyuridine (BrdU) incorporation after 24h of incubation. Piplartine-mediated reduction in cell number was associated with an increasing number of dead cells at a concentration of 10 microg/ml. These findings were corroborated by morphologic analysis. However, at the lowest concentration (2.5 microg/ml), piplartine-treated cells exhibited typical apoptotic morphological changes. The increase in caspase-3 activity was also observed in lysates of piplartine-treated cells (2.5 microg/ml). Our findings suggest that piplartine can suppress leukemia growth and reduce cell survival, triggering both apoptosis and/or necrosis, depending on the concentration used.

Antiproliferative effects of two amides, piperine and piplartine, from Piper species.

The present work evaluated the cytotoxicity of piplartine {5,6-dihydro-1-[1-oxo-3-(3,4,5-trimethoxyphenyl)-trans-2-propenyl]-2(1H)pyridinone} and piperine {1-[5-(1,3)-benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]piperidine}, components obtained from Piper species. The substances were tested for their cytotoxicity on the brine shrimp lethality assay, sea urchin eggs development, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay using tumor cell lines and lytic activity on mouse erythrocytes. Piperine showed higher toxicity in brine shrimp (DL50 = 2.8 +/- 0.3 microg/ml) than piplartine (DL50 = 32.3 +/- 3.4 microg/ml). Both piplartine and piperine inhibited the sea urchin eggs development during all phases examined, first and third cleavage and blastulae, but in this assay piplartine was more potent than piperine. In the MTT assay, piplartine was the most active with IC50 values in the range of 0.7 to 1.7 microg/ml. None of the tested substances induced hemolysis of mouse erythrocytes, suggesting that the cytotoxicity of piplartine and piperine was not related to membrane damage.

Spectral assignments of new diterpenes from Hyptis martiusii Benth.

The structural characterization of two new abietanes and a new spiro-fused tricyclic diterpene isolated from the roots of Hyptis martiusii is described. The first member of a new class of rearranged abietane diterpenoids designated martiusane was characterized by the use of 1D NMR and several 2D shift correlated NMR pulse sequences (1H,1H-COSY, HMQC, HMBC and NOESY). Unambiguous 1H and 13C chemical shift assignments for all compounds are reported.