Xylodiol from Xylopia langsdorfiana induces apoptosis in HL60 cells

An atisane diterpene was isolated from Xylopia langsdorfiana St. Hilaire & Tulasne, Annonaceae, leaves, ent-atisane-7α,16α-diol (xylodiol). Preliminary study showed that xylodiol was cytotoxic and induced differentiation on human leukemia cell lines. However, the molecular mechanisms of xylodiol-mediated cytotoxicity have not been fully defined. Thus, we investigated the anti-tumor effect of xylodiol in human leukemia HL60 cell line. Xylodiol induced apoptosis and necrosis. HL60 cells treated with xylodiol showed biochemical changes characteristic of apoptosis, including caspases-8, -9 and -3 activation and loss of mitochondrial transmembrane potential (∆ Ψm). However, there was a condensation rather than swelling of mitochondria. Moreover, the formation of condensed mitochondria and the loss of ∆ Ψm occurred downstream of caspase activation. Cyclosporine A did not protect HL60 cells from the cytotoxic effects of xylodiol, suggesting that the loss of ∆ Ψm is a late event in xylodiol-induced apoptosis. Oxidative stress was involved in xylodiol-induced apoptosis. Thus, we conclude that activated caspases cleave cellular proteins resulting in mitochondrial damage leading to mitochondrial condensation, loss of ∆ Ψm and ROS release from the mitochondria. ROS can further induce and maintain a collapse of ∆ Ψm leading to cellular damage through oxidation of lipids and proteins resulting in apoptotic cell death.

Growth inhibition and pro-apoptotic activity of violacein in Ehrlich ascites tumor.

The continuing threat to biodiversity lends urgency to the need of identification of sustainable source of natural products. This is not so much trouble if there is a microbial source of the compound. Herein, violacein, a natural indolic pigment extracted from Chromobacterium violaceum, was evaluated for its antitumoral potential against the Ehrlich ascites tumor (EAT) in vivo and in vitro. Evaluation of violacein cytotoxicity using different endpoints indicated that EAT cells were twofold (IC(50)=5.0 microM) more sensitive to the compound than normal human peripheral blood lymphocytes. In vitro studies indicated that violacein cytotoxicity to EAT cells is mediated by a rapid (8-12h) production of reactive oxygen species (ROS) and a decrease in intracellular GSH levels, probably due to oxidative stress. Additionally, apoptosis was primarily induced, as demonstrated by an increase in Annexin-V positive cells, concurrently with increased levels of DNA fragmentation and increased caspase-2, caspase-9 and caspase-3 activities up to 4.5-, 6.0- and 5.5-fold, respectively, after 72 h of treatment. Moreover, doses of 0.1 and 1.0 microg kg(-1) violacein, administered intraperitoneally (i.p.) to EAT-bearing mice throughout the lifespan of the animals significantly inhibited tumor growth and increased survival of mice. In view of these results, a 35-day toxicity study was conducted in vivo. Complete hematology, biochemistry (ALT, AST and creatinine levels) and histopathological analysis of liver and kidney indicated that daily doses of violacein up to 1000 microg kg(-1) for 35 days are well tolerated and did not cause hematotoxicity nor renal or hepatotoxicity when administered i.p. to mice. Altogether, these results indicate that violacein causes oxidative stress and an imbalance in the antioxidant defense machinery of cells culminating in apoptotic cell death. Furthermore, this is the first report of its antitumor activity in vivo, which occurs in the absence of toxicity to major organs.

Diterpenes from Xylopia langsdorffiana inhibit cell growth and induce differentiation in human leukemia cells.

Two new diterpenes were isolated from stems and leaves of Xylopia langsdorffiana, ent-atisane-7alpha,16alpha-diol (xylodiol) and ent-7alpha-acetoxytrachyloban-18-oic acid (trachylobane), along with the known 8(17),12E,14-labdatrien-18-oic acid (labdane). We investigated their antitumour effects on HL60, U937 and K562 human leukemia cell lines. We found that xylodiol was the most potent diterpene in inhibiting cell proliferation of HL60, U937 and K562 cells, with mean IC50 values of 90, 80 and 50 microM, respectively. Based on the nitroblue tetrazolium (NBT) reduction assay, all the diterpenes were found to induce terminal differentiation in HL60 and K562 cells, with xylodiol being the most effective. NBT reduction was increased by almost 120% after 12 h exposure of HL60 cells to xylodiol at a concentration lower than the IC50 (50 microM). Thus, xylodiol inhibited human leukemia cell growth in vitro partly by inducing cell differentiation, and merits further studies to examine its mechanism of action as a potential antitumoural agent.

Cytotoxic activity of violacein in human colon cancer cells.

Several studies have shown that violacein, a purple pigment extracted from Chromobacterium violaceum, is capable to induce apoptosis in a variety of cancer cells, including those leukemia cell lines. Herein, we examined the effects of violacein on reactive oxygen species (ROS) production during the apoptotic colon cancer cell death. We demonstrate that violacein mediates ROS production followed by activation of Caspase-3, release of cytochrome c, and calcium release to citosol in Caco-2 cells. Moreover, presence of ROS scavengers such as N-acetyl-cysteine (NAC) diminishes ROS cytotoxicity induced by violacein in Caco-2 cells, indicating that violacein mediates cellular critical mechanisms in the triggering of apoptotic tumor cell death. These data also imply that violacein-induced ROS are collectively key mediators of mitochondrial membrane collapse, leading to cytochrome c release, and culminating in tumor apoptosis. Unlike in Caco-2 cells, violacein was incapable of increasing ROS levels in HT29 cells, suggesting the existence of violacein cell-type specific mechanisms. Those findings bring light to the violacein cytotoxic mechanism studies, indicating that oxidative stress play a role in the violacein-induced cytotoxicity.

ent-Trachylobane diterpenoids from Xylopia langsdorffiana.

Two new diterpenes of the ent-trachylobane type were isolated from the stems of Xylopia langsdorffiana, ent-7alpha-acetoxytrachyloban-18-oic acid (1) and ent-7alpha-hydroxytrachyloban-18-oic acid (2). The structures of these isolates were deduced by spectroscopic data interpretation. X-ray crystallography of 1 was used to confirm its structure. The cytotoxic activity of 1 against V79 fibroblasts and rat hepatocytes was investigated.

Comparative toxicity of effluents processed by different treatments in V79 fibroblasts and the algae Selenastrum capricornutum.

The efficacy of ozonation and of photocatalysis processing in the treatment of pulp mill ECF (elementary chlorine free) bleaching and textile effluents was evaluated by determining total organic carbon reduction (TOC) and the toxicity. The chronic toxicity of the effluents was evaluated by the ability to inhibit the growth of algae Selenastrum capricornutum. Cultured hamster V79 fibroblasts were used to assess the cytotoxicity of effluents submitted to different detoxification processes. Two endpoints were measured in V79 cells: 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) reduction and neutral red uptake (NRU). Both treatment processes were able to reduce the TOC, although ozonization was less effective for pulp mill ECF bleaching. The pulp mill ECF bleaching and textile effluents reduced the growth of S. capricornutum by 39% and 27%, respectively. However, at the highest concentration tested, the textile effluents treated by photochemical process for 60 min showed increased cytotoxicity in V79 cells compared to the untreated effluent when assessed by the NRU and MTT reduction assays (increases of 30% and 40%, respectively). Pulp mill ECF bleaching effluent treated by ozonization had a similar cytotoxicity to that of untreated effluent in the NRU assay. In contrast, the MTT reduction assay indicated that effluents treated with ozone were around 20% more cytotoxic than untreated effluents. These results show that cultured fibroblasts may be useful for studying cellular responses to pollutants and may be included in tests to monitor the efficiency of effluent detoxification processes.

Dehydrocrotonin and its beta-cyclodextrin complex: cytotoxicity in V79 fibroblasts and rat cultured hepatocytes.

Trans-dehydrocrotonin has antiulcerogenic and antitumor activities. A complex of beta-cyclodextrin with dehydrocrotonin was developed to improve the delivery of dehydrocrotonin. Complex in solid state was evaluated using X-ray diffraction (XRD), differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA) and scanning electron microscopy (SEM). X-ray diffraction and scanning electron microscopy studies showed that dehydrocrotonin exists in a semicrystalline state in the complexed form with beta-cyclodextrin. Differential scanning calorimetry studies showed the existence of a complex of dehydrocrotonin with beta-cyclodextrin. The thermal gravimetric analysis studies confirmed the differential scanning calorimetry results of the complex. Free dehydrocrotonin and the dehydrocrotonin/beta-cyclodextrin inclusion complex were assayed in freshly isolated rat hepatocytes and in V79 cells. Cytotoxicity was determined using nucleic acid content, methylthiazoletetrazolium (MTT) reduction and neutral red uptake assays. In all assays, there was a large reduction (3.5-16.1-fold) in the cytotoxicity of dehydrocrotonin in hepatocytes when complexed with beta-cyclodextrin, whereas for V79 cells the decrease in cytotoxicity was 1.7- and 1.87-fold for MTT reduction and nucleic acid content assays, respectively. The lower cytotoxicity of the dehydrocrotonin/beta-cyclodextrin complex compared to free dehydrocrotonin in rat hepatocytes and V79 cells suggests that such a complex may be useful for the administration of dehydrocrotonin in vivo.

Cytotoxicity of the phytosterol diosgenin and its derivatives in rat cultured hepatocytes and V79 fibroblasts.

In this work, the cytotoxic effects of some spirostane derivatives were examined in cultured hepatocytes and V79 fibroblasts using different viability assays. The derivatives were obtained by modifying the A and B rings of diosgenin. Diosgenin and its derivatives were more toxic in V79 fibroblasts (IC50 40-300 microM) than in hepatocytes (IC50 280-1000 microM). Inhibition of cytochrome P450IIIA in cultured hepatocytes by incubation with 1 mM cimetidine did not alter the toxicity of these compounds in these cells. These observations suggest that other pathways of detoxification may be involved in hepatocytes. In conclusion, the compounds studied merit investigation for their potential pharmacological and industrial applicability.

Dehydrocrotonin and its derivative, dimethylamide-crotonin induce apoptosis with lipid peroxidation and activation of caspases-2, -6 and -9 in human leukemic cells HL60.

A variety of stimuli can induce cells to undergo apoptosis, with one of the most reproducible inducers being mild oxidative stress following exposure to anticancer agents. Apoptosis involves events mediated by cysteine proteases (caspases) that are classified as initiators (-8, -9 and -12) or executors (-2, -3, -6 and -7). In this study, we examined the mechanisms of apoptosis induced by dehydrocrotonin (DHC), a diterpene lactone isolated from the Amazonian plant Croton cajucara, and its synthetic derivative, dimethylamide-crotonin (DCR), in human HL60 promyelocytic leukemia cells. Flow cytometric analysis of HL60 cells after treatment for 72 h showed that DCR- and DHC-induced apoptosis, with maximum cell death at a concentration of 250 microM for both compounds. DCR and DHC were effective in triggering the activation of caspases-2, -6 and -9. The level of reduced glutathione (GSH) decreased, whereas there was an increase in thiobarbituric acid-reactive substance (TBARS) production and in mitochondrial swelling. These effects on mitochondrial swelling, GSH content and lipid peroxidation were abolished by cyclosporine A, an inhibitor of the membrane permeability transition. The cytotoxicity of DHC and DCR was prevented by a high concentration of GSH (15 mM) in the culture medium. These results indicate that DCR and DHC produced apoptosis partly by oxidative stress-induced lipid peroxidation, which triggered the caspase cascade, that lead to apoptotic cell death in HL60 cells. Based on the pattern of caspase activation, on the increase in mitochondrial swelling and on the inhibitory action of cyclosporine A, we conclude that DCR and DHC triggered apoptosis in HL60 cells probably through cytochrome c release and apoptosome formation.

Effect of angiotensin II and losartan on the phagocytic activity of peritoneal macrophages from Balb/C mice.

Angiotensin II (AII), a product of rennin-angiotensin system, exerts an important role on the function of immune system cells. In this study, the effect of AII on the phagocytic activity of mouse peritoneal macrophages was assessed. Mice peritoneal macrophages were cultured for 48 h and the influence of different concentrations of AII (10(-14) to 10(-7) M) and/or losartan, 10(-16) to 10(-6) M), an AT1 angiotensin receptor antagonist, on phagocytic activity and superoxide anion production was determined. Dimethylthiazoldiphenyltetrazolium bromide reduction and the nucleic acid content were used to assess the cvtotoxicity of losartan. A stimulatory effect on phagocytic activity (P < 0.05) was observed with 10(-13) M and 10(-12 M) AII concentrations. The addition of losartan (up to10(-14) M) to the cell cultures blocked (P < 0.001) the phagocytosis indicating the involvement of AT1 receptors. In contrast, superoxide anion production was not affected by AII or losartan. The existence of AT1 and AT2 receptors in peritoneal macrophages was demonstrated by immunofluorescence microscopy. These results support the hypothesis that AII receptors can modulate murine macrophage activity and phagocytosis, and suggest that AII may have a therapeutic role as an immunomodulatory agent in modifying the host resistance to infection.

Natural killer cell activity and anti-tumour effects of dehydrocrotonin and its synthetic derivatives.

In this work, the anti-tumour properties of dehydrocrotonin and its derivatives were investigated in vitro and in vivo using the Ehrlich ascites tumour model. Treatment of Ehrlich ascites tumour-bearing mice with 20 mg/kg dehydrocrotonin for 4 days significantly increased survival, whereas administration of dehydrocrotonin derivatives was ineffective in affording protection. Compound IV exhibited little activity against Ehrlich tumour cells in vitro. Investigation of the effects of dehydrocrotonin treatment on total natural killer (NK) cell activity of tumour-bearing mice as a possible mechanism of dehydrocrotonin action in vivo revealed that this sesquiterpene lactone significantly improved NK cytotoxicity against YAC-1, a Moloney virus-induced mouse T-cell lymphoma of A/SN origin. As expected, tumour growth in non-treated mice markedly suppressed NK cell cytolysis. No effects on NK functional activity were observed in normal mice receiving dehydrocrotonin. In summary, only the natural compound exhibits anti-tumour efficacy and immunomodulatory actions in vivo, which may be related to its chemical structure.

Effect of angiotensin II and losartan on the phagocytic activity of peritoneal macrophages from Balb/C mice

Angiotensin II (AII), a product of rennin-angiotensin system, exerts an important role on the function of immune system cells. In this study, the effect of AII on the phagocytic activity of mouse peritoneal macrophages was assessed. Mice peritoneal macrophages were cultured for 48 h and the influence of different concentrations of AII (10-14 to 10-7 M) and/or losartan, 10-16 to 10-6 M), an AT1 angiotensin receptor antagonist, on phagocytic activity and superoxide anion production was determined. Dimethylthiazoldiphenyltetrazolium bromide reduction and the nucleic acid content were used to assess the cytotoxicity of losartan. A stimulatory effect on phagocytic activity (P < 0.05) was observed with 10-13 M and 10-12 M AII concentrations. The addition of losartan (up to10-14 M) to the cell cultures blocked (P < 0.001) the phagocytosis indicating the involvement of AT1 receptors. In contrast, superoxide anion production was not affected by AII or losartan. The existence of AT1 and AT2 receptors in peritoneal macrophages was demonstrated by immunofluorescence microscopy. These results support the hypothesis that AII receptors can modulate murine macrophage activity and phagocytosis, and suggest that AII may have a therapeutic role as an immunomodulatory agent in modifying the host resistance to infection.

Citotoxicidade do promotor de tumor e sua ação mitogênica sobre os linfócitos humanos / Cytotoxicity studies of okadaic acid on human lymphocytes and its mitogenic effect

Proteínas fosfatases são moléculas sinalizadoras que agem juntamente com as proteínas quinases para regular uma variedade de processos celulares fundamentais, bem como, crescimento celular, mitogênese, metabolismo, transcrição de gene, ciclo celular e resposta ao estresse e imune. O ácido okadáico é um potente e específico inibidor de proteína serina/treonina fosfatase (PP1 e PP2A). O objetivo deste estudo foi avaliar o efeito citotóxico do ácido okadáico na viabilidade de linfócitos humanos e sua ação mitogênica. Nos estudos de citotoxicidade avaliamos o efeito do ácido okadáico através dos seguintes parâmetros: redução do MTT (integridade mitocondrial), conteúdo total de proteínas (número de células) e atividade fosfatásica (metabolismo celular). O valor para redução do MTT e atividade fosfatase foram: 50nM e 100nM, respectivamente; não foi encontrado valor de IC50 para o conteúdo de proteína. A atividade fosfatásica não foi afetada pelo ácido okadáico (100nM) quando este composto foi adicionado no extrato celular. A proliferação de linfócitos foi estimulada em 25 porcento quando as células foram tratadas com o ácido okadáico durante o plaqueamento e na ausência da fitohemaglutinina (mitogêno). O estímulo máximo foi até 30 minutos. O ácido okadáico não foi citotóxico para os linfócitos. A ação mitogênica deste composto foi confirmada pelo conteúdo de proteína.

Relationship between phosphatase activity and cytotoxic effect of two protein phosphatase inhibitors, okadaic acid and pervanadate, on human myeloid leukemia cell line.

Protein phosphatases are signalling molecules that regulate a variety of fundamental cellular processes including cell growth, metabolism and apoptosis. The aim of this work was to correlate the cytotoxicity of pervanadate and okadaic acid on HL60 cells and their effect on the phosphatase obtained from these cells. The cytotoxicity of these protein phosphatase inhibitors was evaluated on HL60 cells using phosphatase activity, protein quantification and MTT reduction as indices. The major phosphatase presents in the cellular extract showed high activity (80%) and affinity (Km = 0.08 mM) to tyrosine phosphate in relation to p-nitrophenyl phosphate (pNPP)-(Km = 0.51 mM). Total phosphatase (pNPP) was inhibited in the presence of 10 mM vanadate (98%), 200 microM pervanadate (95%) and 100 microM p-chloromercuribenzoate (80%) but okadaic acid caused a slight increase in enzyme activity (25%). When the HL60 cells were treated with the phosphatase inhibitors (pervanadate and okadaic acid) for 24hours, only 20% residual activity was observed in presence of 200 microM pervanadate, whereas in the presence of okadaic acid this inhibitory effect was not observed. However, in respect to mitochondrial function, cell viability decreased about 80% in the presence of 100 nM okadaic acid. The total protein content was decreased 25% when the cells were treated with 100 nM okadaic acid in combination with 200 microM pervanadate. Our results suggest that both phosphatase inhibitors presented different mechanisms of action on HL60 cells. However, their effect on the cell redox status have to be considered.

Antiulcerogenic effect and cytotoxic activity of semi-synthetic crotonin obtained from Croton cajucara Benth.

Trans-dehydrocrotonin, the major diterpene isolated from the bark of Croton cajucara, has good antiulcerogenic activity which, however, is accompanied by toxic effects. On the basis of these results, a semi-synthetic crotonin, named 4SRC, was prepared to determine whether this substance has similar antiulcerogenic activity with lower or no toxicity. The natural crotonin was also isolated from the bark of C. cajucara but was not used due to the small amount obtained. The cytotoxic effect of semi-synthetic crotonin, expressed as cell viability, was assessed in (a) lung fibroblast cell line (V79) derived from Chinese hamsters, a system commonly used for cytotoxicity studies, and (b) rat hepatocytes isolated from male Wistar rats. After treatment, cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction (MTT reduction), total acid content and neutral red uptake assays. To evaluate V79 cell viability, different concentrations of semi-synthetic crotonin were incubated with the cells. To evaluate the antiulcerogenic effects of semi-synthetic crotonin (50, 100 and 200 mg/kg), we used the models of gastric ulcer induced by ethanol/HCl, stress, indomethacin/bethanechol, and ethanol in male Swiss mice and male Wistar rats. The substance had an IC(50)=500 microM in the neutral red uptake and MTT reduction tests and an IC(50)=200 microM in the nucleic acid content test. With regard to hepatocyte viability after treatment with semi-synthetic crotonin at different concentrations, semi-synthetic crotonin had an IC(50)=10-500 microM in the nucleic acid content and MTT reduction tests and an IC(50)=120 microM in the neutral red uptake test. In another experiment, V79 cells were incubated with the metabolites produced by hepatocytes treated with different concentrations of semi-synthetic crotonin. After a 4-h incubation, semi-synthetic crotonin had an IC(50)=500 microM in the MTT reduction and neutral red uptake tests and an IC(50)=370 microM in nucleic acid content test. The substance had significant antiulcerogenic activity in all models studied, suggesting the presence of a possible antisecretory effect combined with a cytoprotective effect. For this reason, the effect of semi-synthetic crotonin was also evaluated on biochemical parameters of gastric juice and gastric wall mucus, both obtained from pylorus-ligated mice. No significant differences were observed in these parameters between semi-synthetic crotonin-treated and control animals. The results obtained with semi-synthetic crotonin are promising, with a significant preventive effect against gastric ulcer induced by different agents. Our data also show that semi-synthetic crotonin was less toxic than dehydrocrotonin and that the cytotoxic effects decreases with the time that isolated hepatocytes were in culture.

Comparison of the gastroprotective effect of a diterpene lactone isolated from Croton cajucara with its synthetic derivatives.

The effect of three new derivatives from dehydrocrotonin (DHC-compound I) on gastric damage in different animal models including gastric ulceration induced by a necrotic agent and hypothermic restrained-stress was studied: compound II (produced by reducing the cyclohexenone moiety of DHC with NaBH(4)); compound III (produced by reducing the carbonyls with LiAlH(4)); and compound IV (produced by transforming the lactone moiety into an amide). Their structures were confirmed on the basis of chemical and physicochemical evidence. When previously administered (p.o.) at a dose of 100mg/kg, compound II significantly (P<0.01) reduced gastric injury induced by HCl/ethanol (78%) and indomethacin (88%) better than did reference compound I (48 and 43%, respectively). But the anti-ulcerogenic activity of compound II was completely abolished by the stress-induced ulcer. Reduction of carbonyls with LiAlH(4) (compound III) caused decreased activity, markedly when no protective effect in any of the models was applied (P>0.05). However, compound IV, in which the lactone moiety was changed into an amide, when administered at the same dose (100mg/kg, p.o.), was more effective. The presence of a lactone moiety or Michael acceptor is probably essential for the anti-ulcerogenic effect of these compounds.

Influence of protein phosphatase inhibitors on HL60 cells death induction by dehydrocrotonin.

Oxidative stress can be involved in several cellular responses, such as differentiation, apoptosis and necrosis. Dehydrocrotonin (DCTN, diterpene lactone) from Croton cajucara, Brazilian medicinal plant, slightly induced NBT-reducing activity. In presence of protein phosphatase inhibitors significant differentiation of HL60 cells was observed. Flow cytometry analysis demonstrated that apoptosis was induced when the cells were treated with okadaic acid (OKA) and plus trans-dehydrocrotonin (t-DCTN) this effect was two-fold increased. Unlike, when the cells were treated only with t-DCTN, necrosis was observed. On the other hand, the necrosis induced by t-DCTN could be due to oxidative stress, revealed by increase of GSH content. Therefore, this differentiation pathway involves the modulation of protein phosphatases and this inhibition promotes the t-DCTN action on apoptosis induction.

Warifteine and milonine, alkaloids isolated from Cissampelos sympodialis Eichl: cytotoxicity on rat hepatocyte culture and in V79 cells.

Two alkaloids were isolated from the leaves of Cissampelos sympodialis; a bisbenzylisoquinoline compound named warifteine and a novel 8,14-dihydromorphinandienone alkaloid named milonine. The cytotoxic effects of these alkaloids were assayed in cultured hepatocytes and V79 fibroblasts. Three independent endpoint assays for cytotoxicity in vitro were used: the nucleic acid content (NAC), tetrazolium reduction (MTT) and neutral red uptake (NRU). Milonine was less toxic than warifteine in both cell cultures. The IC50 values determined in the three different viability assays were around 100 and 400 microM after milonine treatment of V79 cells or hepatocytes. IC50 values ranging from 10 to 35 microM were obtained for warifteine in the viability tests evaluated in V79 cells and hepatocytes. Due to the similar cytotoxic effects detected on V79 cells and hepatocytes, probably warifteine and milonine induced toxic effects independent to the cytochrome P450. This hypothesis was corroborated by the results where Cimetidine (1.0 mM), a traditional cytochrome P450 inhibitor, did not protect the cells from the toxic action of warifteine or milonine. In conclusion, these alkaloids merit further investigations as potential novel pharmacological agents although milonine was less toxic than warifteine in the cells models investigated.

Evaluation of the antiulcerogenic activity of violacein and its modulation by the inclusion complexation with beta-cyclodextrin.

The effects of beta-cyclodextrin (betaCD) inclusion complexation on the ability of violacein to prevent gastric ulceration in mice were studied. Violacein-betaCD inclusion complexes were prepared in 1:1 and 1:2 molar ratios and analysed by differential scanning calorimetry and powder X-ray diffractometry. Violacein previously administered orally at 10 mg/kg significantly reduced indomethacin-induced gastric lesions, as well as 100 mg/kg of cimetidine (positive control). However, betaCD complexation in both molar ratios significantly potentiated the protective action of violacein. In the HCl--ethanol-induced gastric ulcer model, violacein and the 1:2 inclusion complex (10 mg/kg, p.o.) inhibited gastric damage by almost 85%, whereas a 63% reduction was observed for the positive control, lansoprazole, at 30 mg/kg. In contrast, treatment with the 1:1 inclusion complex resulted in almost total disappearance of the antiulcer activity in this model. No significant changes in stress-induced gastric injury were found. In addition, the 1:2 inclusion complex improved the antilipoperoxidant activity of violacein in rat liver cells exposed to t-butyl hydroperoxide, whereas the 1:1 complex was less active than violacein. In summary, the 1:2 betaCD inclusion complex has gastroprotective properties similar to or higher than that of violacein. An increase in mucosal defensive mechanisms and protection against peroxidative damage might be involved.

Comparative cytotoxicity of dimethylamide-crotonin in the promyelocytic leukemia cell line (HL60) and human peripheral blood mononuclear cells.

Dehydrocrotonin (DHC) is a diterpene lactone obtained from Croton cajucara (Sacaca). Dimethylamide-crotonin (DCR), a DHC derivative, has a similar inhibitory effect on leukemic HL60 cells than its parent compound evaluated by different endpoints of cytotoxicity. No cytotoxicity or morphological alterations associated with apoptosis were detected in human peripheral blood mononuclear cells (PBMC) after treatment with up to 400 micro M DCR in presence of phytohemaglutinin (5 micro g/ml). Based on morphological changes and the pattern of DNA fragmentation, DHC and DCR were found to induce apoptosis and terminal differentiation (assessed by nitro blue tetrazolium reduction) in HL60 cells, but these compounds did not show any toxic effect in PBMC. Thus, DCR and DHC inhibit HL60 cell growth in vitro partly by inducing apoptosis and cell differentiation, but does not cause serious damage to immune cells according to our experimental conditions.