In vitro antitumor effect of a lignan isolated from Combretum fruticosum, trachelogenin, in HCT-116 human colon cancer cells.

The use of natural products in therapeutics has been growing over the years. Lignans are compounds with large pharmaceutical use, which has aroused interest in the search for new drugs to treat diseases. The present study evaluated the cytotoxicity of (-)-trachelogenin, a dibenzylbutyrolactone type lignan isolated from Combretum fruticosum, against several tumor and non-tumor cell lines using the MTT assay and its possible mechanism of action. (-)-Trachelogenin showed IC50 values ranging of 0.8-32.4µM in SF-295 and HL-60 cell lines, respectively and IC50 values >64µM in non-tumor cell lines. (-)-trachelogenin persistently induced autophagic cell death, with cytoplasmic vacuolization and formation of autophagosomes mediated by increasing LC3 activation and altering the expression levels of Beclin-1.

Comparative study of the antihypertensive effects of hexane, chloroform and methanol fractions of essential oil of Alpinia zerumbet in rats Wistar / Estudo comparativo do efeito anti-hipertensivo das frações hexânixa, clorofórmica e metanólica do óleo essencial da Alpinia zerumbet

ABSTRACT The aim of this study was to characterize components of the EOAz and its hexane (HFEOAz), chloroform (CFEOAz) and methanol (MFEOAz) fractions, and its antihypertensive effect. EOAz was extracted from leaves by hydrodistillation. Aliquot was subjected to selective desorption with silica gel column and eluted with hexane, chloroform and methanol. The components of the EOAz and fractions were analyzed by gas chromatography coupled with mass spectrometry and nuclear magnetic resonance spectroscopy of hydrogen. Experiments of vascular reactivity were performed with isolated aortic rings of male Wistar rats. Antihypertensive effect was evaluated in hypertensive rats submitted to the inhibition of synthesis of nitric oxide. Blood pressure was measured indirectly by tail plethysmography. MFEOAz showed the lowest EC50 (150.45 µg/mL), 1,8-cineole (27.81%) and terpinen-4-ol (57.35%) as main components. Single administration by nasogastric tube of EOAz, fractions and captopril significantly reduced the blood pressure of hypertensive rats, when compared to animals of the negative control group with distilled water. In conclusion, the potency of the MFEOAz was higher than that of EOAz and other fractions. The antihypertensive effect of EOAz and fractions was similar, higher than the negative control and lower than that of captopril.

RESUMO O objetivo deste estudo foi caracterizar os componentes do óleo essencial das folhas de Alpinia zerumbet (OEAz) e suas frações hexânica (FHOEAz), clorofórmica (FCOEAz) e metanólica (FMOEAz), e seu efeito anti-hipertensivo. OEAz foi extraído das folhas por hidrodestilação. Uma alíquota foi submetida à desadsorção seletiva com coluna de gel de sílica e eluída com hexano, clorofórmio e metanol. Os componentes do OEAz e fracções foram analisadas por cromatografia gasosa acoplada à detector de massa e por espectros de ressonância magnética nuclear de hidrogênio. Experimentos de reatividade vascular foram realizados com anéis aórticos isolados de ratos Wistar machos. Efeito anti-hipertensivo foi avaliado em ratos hipertensos submetidos à inibição da síntese de óxido nítrico. A pressão arterial foi medida indiretamente por pletismografia de cauda. FMOEAz mostrou a menor CE50 (150,45 μg/mL), 1,8-cineol (27,81%) e terpinen-4-ol (57,35%) como componentes principais. A administração em dose única por sonda nasogástrica de OEAz, frações e captopril reduziu significativamente a pressão arterial de ratos hipertensos, quando comparados aos animais do grupo controle negativo com água destilada. Em conclusão, a potência da FMOEAz foi maior que a do OEAz e outras frações. O efeito anti-hipertensivo de OEAz e frações foi semelhante, maior do que o controle negativo e menor do que o captopril.

Structure-mutagenicity relationship of kaurenoic acid from Xylopia sericeae (Annonaceae).

Kaurane diterpenes are considered important compounds in the development of new highly effective anticancer chemotherapeutic agents. Genotoxic effects of anticancer drugs in non-tumour cells are of special significance due to the possibility that they induce secondary tumours in cancer patients. In this context, we evaluated the genotoxic and mutagenic potential of the natural diterpenoid kaurenoic acid (KA), i.e. (-)-kaur-16-en-19-oic acid, isolated from Xylopia sericeae St. Hill, using several standard in vitro and in vivo protocols (comet, chromosomal aberration, micronucleus and Saccharomyces cerevisiae assays). Also, an analysis of structure-activity relationships was performed with two natural diterpenoid compounds, 14-hydroxy-kaurane (1) and xylopic acid (2), isolated from X. sericeae, and three semi-synthetic derivatives of KA (3-5). In addition, considering the importance of the exocyclic double bond (C16) moiety as an active pharmacophore of KA cytotoxicity, we also evaluated the hydrogenated derivative of KA, (-)-kauran-19-oic acid (KAH), to determine the role of the exocyclic bond (C16) in the genotoxic activity of KA. In summary, the present study shows that KA is genotoxic and mutagenic in human peripheral blood leukocytes (PBLs), yeast (S. cerevisiae) and mice (bone marrow, liver and kidney) probably due to the generation of DNA double-strand breaks (DSB) and/or inhibition of topoisomerase I. Unlike KA, compounds 1-5 and KAH are completely devoid of genotoxic and mutagenic effects under the experimental conditions used in this study, suggesting that the exocyclic double bond (C16) moiety may be the active pharmacophore of the genetic toxicity of KA.

The antitumoral, trypanocidal and antileishmanial activities of extract and alkaloids isolated from Duguetia furfuracea.

The alkaloid extract and five alkaloids isolated from subterranean stem bark of Duguetia furfuracea (Annonaceae) were investigated for the following activities: antitumoral, trypanocidal and leishmanicidal. Dicentrinone showed weak cytotoxicity, but it had the strongest leishmanicidal activity (IC(50) 0.01 microM). Duguetine and duguetine beta-N-oxide caused considerable antitumoral activity in every cell lines evaluated, although duguetine was more active against trypomastigote forms (IC(50) 9.32 microM) than other alkaloids tested.

Cell cycle arrest induced by pisosterol in HL60 cells with gene amplification.

The leukemia cell line HL60 is widely used in studies of the cell cycle, apoptosis, and adhesion mechanisms in cancer cells. We conducted a focused cytogenetic study in an HL60 cell line, by analyzing GTG-banded chromosomes before and after treatment with pisosterol (at 0.5, 1.0, and 1.8 microg/ml), a triterpene isolated from Pisolithus tinctorius, a fungus collected in the Northeast of Brazil. Before treatment, 99% of the cells showed the homogeneously staining region (HSR) 8q24 aberration. After treatment with 1.8 microg/ml pisosterol, 90% of the analyzed cells lacked this aberration. We further performed a pulse test, in which the cells treated with pisosterol (0.5, 1.0, and 1.8 microg/ml) were washed and re-incubated in the absence of pisosterol. Only 30% of the analyzed cells lacked the HSR 8q24 aberration, suggesting that pisosterol probably blocks the cells with HSRs at interphase. No effects were detected at lower concentrations. At the highest concentration examined (1.8 microg/ml), pisosterol also inhibited cell growth, but this effect was not observed in the pulse test, reinforcing our hypothesis that, at the concentrations tested, pisosterol probably does not induce cell death in the HL60 line. The results found for pisosterol were compared with those for doxorubicin. Cells that do not show a high degree of gene amplification (HSRs and double-minute chromosomes) have a less aggressive and invasive behavior and are easy targets for chemotherapy. Therefore, further studies are needed to examine the use of pisosterol in combination with conventional anti-cancer therapy.

Genotoxic effects of tanshinones from Hyptis martiusii in V79 cell line.

The genotoxic effect of two tanshinones isolated from roots of Hyptis martiussi Benth (Labiatae) was studied using V79 (Chinese hamster lung) cells by the alkaline comet assay and micronucleus test. Tanshinones were incubated with the cells at concentrations of 1, 3, 6 and 12 microg/mL for 3 h. Tanshinones were shown to be quite strongly genotoxic against V79 cells at all tested concentrations. The data obtained provide support to the view that tanshinones has DNA damaging activity in cultured V79 cells under the conditions of the assays.

In vivo growth-inhibition of Sarcoma 180 by piplartine and piperine, two alkaloid amides from Piper.

Piplartine {5,6-dihydro-1-[1-oxo-3-(3,4,5-trimethoxyphenyl)-2-propenyl]-2(1H)pyridinone} and piperine {1-5-(1,3)-benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]piperidine} are alkaloid amides isolated from Piper. Both have been reported to show cytotoxic activity towards several tumor cell lines. In the present study, the in vivo antitumor activity of these compounds was evaluated in 60 female Swiss mice (N = 10 per group) transplanted with Sarcoma 180. Histopathological and morphological analyses of the tumor and the organs, including liver, spleen, and kidney, were performed in order to evaluate the toxicological aspects of the treatment with these amides. Administration of piplartine or piperine (50 or 100 mg kg(-1) day(-1) intraperitoneally for 7 days starting 1 day after inoculation) inhibited solid tumor development in mice transplanted with Sarcoma 180 cells. The inhibition rates were 28.7 and 52.3% for piplartine and 55.1 and 56.8% for piperine, after 7 days of treatment, at the lower and higher doses, respectively. The antitumor activity of piplartine was related to inhibition of the tumor proliferation rate, as observed by reduction of Ki67 staining, a nuclear antigen associated with G1, S, G2, and M cell cycle phases, in tumors from treated animals. However, piperine did not inhibit cell proliferation as observed in Ki67 immunohistochemical analysis. Histopathological analysis of liver and kidney showed that both organs were reversibly affected by piplartine and piperine treatment, but in a different way. Piperine was more toxic to the liver, leading to ballooning degeneration of hepatocytes, accompanied by microvesicular steatosis in some areas, than piplartine which, in turn, was more toxic to the kidney, leading to discrete hydropic changes of the proximal tubular and glomerular epithelium and tubular hemorrhage in treated animals.

Quebrachitol (2-O-methyl-L-inositol) attenuates 6-hydroxydopamine-induced cytotoxicity in rat fetal mesencephalic cell cultures.

Naturally occurring plant substances have the potential to prevent oxidative damage in various pathophysiological conditions including neurodegenerative disorders. Recent findings indicate that impaired energy metabolism plays a prominent role in neurodegeneration. The present study investigated whether quebrachitol (2-O-methyl-L-inositol) (QCT), a sugar like natural compound that was suggested to have both antioxidant and membrane stabilization activity prevents the cytotoxic effect of 6-hydroxydopamine (6-OHDA, 200 microM) on cultured rat fetal mesencephalic cells. While QCT (0.1-100 microg/ml) produced no effect per se on cell viability as measured in the 3[4,5-dimethylthiazole-2il]-2,5-diphenyltetrazolium bromide (MTT) test, it offered concentration-related protection against cell death induced by 6-OHDA. In addition, QCT demonstrated an antioxidant activity against 6-OHDA-induced oxidative stress as evidenced by reduced formation of nitrite-nitrate and thiobarbituric acid-related substances. Fluorescence microscopy using acridine orange/ethidium bromide double staining further affirmed the absence of 6-OHDA (200 microM)-induced morphological changes characteristic of apoptosis/necrosis in cultures pretreated with QCT (100 microg/ml). Also, results of tyrosine hydroxylase immunoreactivity indicated that 6-OHDA induces cell death in mesencephalic cultures affecting both TH+ positive and TH- negative (TH+ and TH-, respectively) and QCT pretreatment protects them from cell death, in a non-specific manner. Our data indicate that QCT has a cytoprotective role due, at least in part, to an antioxidant and free radical scavenging mechanism. Furthermore, the study suggests that inositol compounds might serve as leads in developing drugs for the treatment of various neurodegenerative disorders.

In vivo growth-inhibition of Sarcoma 180 by piplartine and piperine, two alkaloid amides from Piper

Piplartine {5,6-dihydro-1-[1-oxo-3-(3,4,5-trimethoxyphenyl)-2-propenyl]-2(1H)pyridinone} and piperine {1-5-(1,3)-benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]piperidine} are alkaloid amides isolated from Piper. Both have been reported to show cytotoxic activity towards several tumor cell lines. In the present study, the in vivo antitumor activity of these compounds was evaluated in 60 female Swiss mice (N = 10 per group) transplanted with Sarcoma 180. Histopathological and morphological analyses of the tumor and the organs, including liver, spleen, and kidney, were performed in order to evaluate the toxicological aspects of the treatment with these amides. Administration of piplartine or piperine (50 or 100 mg kg-1 day-1 intraperitoneally for 7 days starting 1 day after inoculation) inhibited solid tumor development in mice transplanted with Sarcoma 180 cells. The inhibition rates were 28.7 and 52.3 percent for piplartine and 55.1 and 56.8 percent for piperine, after 7 days of treatment, at the lower and higher doses, respectively. The antitumor activity of piplartine was related to inhibition of the tumor proliferation rate, as observed by reduction of Ki67 staining, a nuclear antigen associated with G1, S, G2, and M cell cycle phases, in tumors from treated animals. However, piperine did not inhibit cell proliferation as observed in Ki67 immunohistochemical analysis. Histopathological analysis of liver and kidney showed that both organs were reversibly affected by piplartine and piperine treatment, but in a different way. Piperine was more toxic to the liver, leading to ballooning degeneration of hepatocytes, accompanied by microvesicular steatosis in some areas, than piplartine which, in turn, was more toxic to the kidney, leading to discrete hydropic changes of the proximal tubular and glomerular epithelium and tubular hemorrhage in treated animals.

Genotoxicity evaluation of kaurenoic acid, a bioactive diterpenoid present in Copaiba oil.

Copaiba oil extracted from the Amazon traditional medicinal plant Copaifera langsdorffii is rich in kaurenoic acid (ent-kaur-16-en-19-oic acid), a diterpene that has been shown to exert anti-inflammatory, hypotensive, and diuretic effects in vivo and antimicrobial, smooth muscle relaxant and cytotoxic actions in vitro. This study evaluated its potential genotoxicity against Chinese hamster lung fibroblast (V79) cells in vitro, using the Comet and the micronucleus assays. Kaurenoic acid was tested at concentrations of 2.5, 5,10, 30 and 60 microg/mL. The positive control was the methylmethanesulfonate (MMS). The duration of the treatment of V79 cells with these agents was 3h. The results showed that unlike MMS, kaurenoic acid (2.5, 5, and 10 microg/mL) failed to induce significantly elevated cell DNA damage or the micronucleus frequencies in the studied tests. However, exposure of V79 cells to higher concentrations of kaurenoic acid (30 and 60 microg/mL) caused significant increases in cell damage index and frequency. The data obtained provide support to the view that the diterpene kaurenoic acid induces genotoxicity.

Amburoside A, a glucoside from Amburanacearensis, protects mesencephalic cells against 6-hydroxydopamine-induced neurotoxicity.

This study evaluates the potential neuroprotective properties of amburoside A, a glucoside isolated from Amburana cearensis, on rat mesencephalic cell cultures exposure to the neurotoxin 6-hydroxydopamine (6-OHDA). The parameters determined were cell viability by the 3[4,5-dimethylthiazole-2-il]-2,5-diphenyltetrazolium bromide (MTT) method, nitric oxide (NO) and free radical formation by the measurement of nitrite concentration and thiobarbituric acid reacting substance (TBARS) formation as an indication of cellular lipid peroxidation. The results showed that AMB was less effective as a curative agent in the MTT assay, since its addition after 6-OHDA did not reverse the neurotoxin's effect, except at the highest concentration (AMB, 100 microg/ml). Similarly, the higher nitrite levels observed after exposure of the cells to 6-OHDA were only partially reversed by AMB, at this highest concentration. However, when AMB (0.5, 1, 10 and 100 microg/ml) was added before the toxin, it appeared to protect neuronal cells against 6-OHDA toxicity in a concentration-dependent manner, as shown by MTT assay. AMB also prevented free radical formation indicated by the increased nitrite concentration induced by 6-OHDA. Cells exposed to 6-OHDA showed a 3.4 times increase in TBARS concentration as compared to controls, and this effect was inhibited from 24% up to 64% by AMB (0.1-100 microg/ml), indicative of a neuroprotective effect. In conclusion, we show that AMB, acting as an antioxidant compound, presents a significant neuroprotective effect, suggesting that this compound could provide benefits as a therapeutic agent in neurodegenerative disease such as Parkinson's.

Oncocalyxone A from Auxemma oncocalyx lacks genotoxic activity in phytohemagglutinin-stimulated lymphocytes.

Inadequate doses or prolonged chemotherapy can be cytotoxic or genotoxic to cancer patients, increasing the risk for the development of a second cancer, particularly acute leukemia. The association between therapeutic and genotoxic properties of oncocalyxone A (Onco A), make cytotoxic tests (mitotic index and chromosomal aberrations) fundamental in the accompaniment of the effects of this active compound. Therefore, the aim of the present study was to determine the genotoxic action of Onco A in vitro, during different phases of the cell cycle, utilizing primary cultures of lymphocytes of healthy individuals. The results showed that Onco A is cytotoxic during the cell cycle phases G1, G1/S, and S, however, not in G2. Onco A did not demonstrate a genotoxic effect in any of the cell cycle phases at the concentration studied. It is concluded that during the period of exposure, this active substance inhibits DNA synthesis and consequently cell division. Therefore, the absence of such genotoxicity for Onco A in the tests performed in this study provides important information in regard to the therapeutic use of this agent. Further studies are necessary to better understand the molecular mechanism of action of Onco A.

The cytotoxic and embryotoxic effects of kaurenoic acid, a diterpene isolated from Copaifera langsdorffii oleo-resin.

In this work, we studied the effects of kaurenoic acid, a diterpene isolated from the oleo-resin of Copaifera langsdorffii in developing sea urchin (Lytechinus variegatus) embryos, on tumor cell growth in microculture tetrazolium (MTT) test and on mouse and human erythrocytes in hemolysis assay. Continuous exposure of embryos to kaurenoic acid starting immediately after fertilization inhibited the first cleavage (IC(50): 84.2 microM) and progressively induced embryo destruction (IC(50): 44.7 microM and < 10 microM for blastulae and larvae stages, respectively). In MTT assay, kaurenoic acid at a concentration of 78 microM produced growth inhibition of CEM leukemic cells by 95%, MCF-7 breast and HCT-8 colon cancer cells by 45% each. Further, kaurenoic acid induced a dose-dependent hemolysis of mouse and human erythrocytes with an EC(50) of 74.0 and 56.4 microM, respectively. The destruction of sea urchin embryos, the inhibition of tumor cell growth and the hemolysis of mouse and human erythrocytes indicate the potential cytotoxicity of kaurenoic acid.

Oncocalyxones A and C, 1,4-anthracenediones from Auxemma oncocalyx: comparison with anticancer 1,9-anthracenediones.

Oncocalyxones A and C are 1,4-anthracenediones isolated from Auxemma oncocalyx (Boraginaceae) that have been shown to be cytotoxic to tumor cells in vitro. The present study compared the cytotoxicity of these compounds with that of two conventional anticancer agents doxorubicin and mitoxantrone, both 1,9-anthracenediones, in a panel of human tumor cell lines. The effect on cell growth was examined using an MTT microtiter assay in two leukemia lines, five solid tumor lines of different histological origin, and two multidrug-resistant sublines of a lung tumor line. The oncocalyxones showed much lower potency than the 1,9-anthracenediones, but were similarly more cytotoxic to leukemia cells compared to solid tumor lines. However, in the multidrug-resistant cells with 10 to 500 times decreased sensitivity to doxorubicin, the cytotoxicity of oncocalyxones A and C was only modestly reduced by about twofold, 1,4-Anthracenediones may be a promising novel class of chemotherapeutic agents effective against multidrug resistant tumors.

Antiproliferative effects of compounds derived from plants of Northeast Brazil.

Ten compounds derived from plants indigenous to Northeast Brazil were examined for antiproliferative effects on human cells in vitro. The effects of these phytochemicals on cell growth were determined by the MTT microtitre assay with 3-day continuous drug exposure. Three human cell lines were used: CEM leukaemia, SW1573 lung tumour and CCD922 normal skin fibroblasts. Four active compounds were found with IC(50) values less than 10 microg/mL in the two cancer cell lines. Oncocalyxones A and C, both 1,4-anthracenediones from Auxemma oncocalyx (Boraginaceae), showed cytotoxicity with mean IC(50) values of 0.8-2, 7-8 and 12-13 microg/mL against CEM, SW1573 and CCD922, respectively. One diterpene and one flavonoid, both from Egletes viscosa (Compositae), were also active. 12-Acetoxy-hawtriwaic acid lactone was cytotoxic with mean IC(50) values of 6, 10 and 10 microg/mL, respectively. 4,5-Dihydroxy-3,3,7, 8-tetramethoxy flavone (ternatin) was only growth-inhibitory with mean IC(50) values of 2, 1 and 10 microg/mL, respectively. These four most active compounds were examined further for their effects on DNA integrity and on DNA synthesis. All but ternatin caused substantial DNA damage and marked inhibition of 5-bromo-2'-deoxyuridine incorporation within 24 h. This study demonstrated the antiproliferative activity of four novel phytochemicals, three of which are DNA-reactive and inhibit DNA synthesis. Further studies are warranted to evaluate these compounds for antitumour potential.

Evaluation of the purified fraction of Wilbrandia (c.f.) verticillata for antitumour activity.

Cucurbitacins are known to produce cytotoxic and anticancer activities. Two novel norcucurbitacin glucosides (Wv1 and Wv2) have recently been isolated from a purified fraction obtained from the rhizome of Wilbrandia verticillata. The present study evaluates the cytotoxic and antitumour activities of these norcucurbitacins. We have found a regular cytotoxicity in KB cells (Cy50 = 12 micrograms/ml) as well as a significant inhibition in the Walker 256 carcinosarcoma growth (approximately 75%).

Evaluation of the purified fraction of Wilbrandia (c. f. ) verticillata for antitumour activity

Cucurbatacins are known to produce cytotoxic and anticancer activities. Two novel norcucurbitacin glucosides (Wvl and Wv2) have recently been isolated from a purified fraction obtained from the rhizome of Wilbrandia verticillata. The present study evaluates the cytotoxic and anti-tumour activities of the norcucurbitacins. We have found a regular cytotoxicity in KB cells (Cy50 = 12µg/ml) as well as a significant inhibition in the Walker 256 carcinosarcoma growth (approximately 75%).