Patrícia da Silva Melo

Violacein: properties and biological activities.

The violet pigment violacein is an indole derivative, isolated mainly from bacteria of the genus Chromobacterium, which exhibits important antitumoural, antimicrobial and antiparasitary properties. Furthermore, the formulation of violacein in different polymeric carriers developed so far offers alternative approaches to overcoming physiological barriers and undesirable physicochemical properties in vivo, thus improving its efficacy.

Violacein and its β-cyclodextrin complexes induce apoptosis and differentiation in HL60 cells

Violacein, a pigment isolated from Chromobacterium violaceum, has been reported to have multiple biological activities including in vitro antitumor effects. Certain anticancer agents are known to induce apoptosis in human tumor cell lines. In this work, our aim was to investigate the effectiveness of violacein/beta-cyclodextrin (beta-CD)-containing systems to produce lethal effects in the human promyelocytic leukemia cell line HL60. Using the MTT tetrazolium reduction test, IC(50) for the inclusion complexes (1:1 and 1:2 violacein:beta-CD molar ratios) and violacein alone were less than 1 microM. Violacein and violacein/beta-CD complexes were able to induce NBT reduction. Moreover, by using the Feulgen reaction, all the compounds were found to trigger apoptosis in HL60 cells, inducing around 35% of DNA fragmentation, as analyzed through the diphenylamine assay. In addition, caspases seem to play an important role in the activation of the executioner phase of apoptosis induced by violacein and its derivatives.

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.

Antitumoral activity of L-ascorbic acid-poly- D,L-(lactide-co-glycolide) nanoparticles containing violacein

It has been demonstrated that tumoral cells have a higher uptake of ascorbic acid compared to normal cells. This differential characteristic can be used as a way to improve the specificity of antitumoral compounds if combined with polymeric drug delivery systems. The aim of this study was to prepare, characterize and evaluate the antitumoral activity of poly- D,L-(lactide-co-glycolide) 50:50 loading the antitumoral compound violacein and capped with L-ascorbic acid. Nanoparticles were prepared using the nanoprecipitation method and morphologically characterized by scanning electron microscopy (SEM). The average diameter and Zeta potential were determined by photon correlation spectroscopy method (PCS), and assays were carried out to determine the content of ascorbic acid and in vitro drug release kinetics. The antitumoral activity of this system was also evaluated against HL-60 cells by tetrazolium reduction assay. Nanoparticles with size distribution between 300–400 nm and strong negative outer surface (−40 mV) were obtained by this method. Analysis of ascorbic acid content showed that this compound was mainly localized on the external surface of nanoparticles. Violacein loading efficiency was determined as 32% ± 1% and this drug was gradually released from nanoparticles at different rates depending on the composition of the release media. In addition, this system was observed to be 2 × more efficient as an antitumoral compared with free violacein.

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.

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.

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.

Development of a Sustained-release System for Nitric Oxide Delivery using Alginate/Chitosan Nanoparticles

Free radical nitric oxide (NO) has been known to interact with various physiological processes, such as wound repair processes and control of vascular tone. However, NO is an unstable molecule and the development of NO delivery systems that enhance its stability has also been studied. In this work, alginate/chitosan nanoparticles have been studied as a drug delivery system of the S- nitrosoglutathione (GSNO) as NO donor. For this, glutathione, GSH, the GSNO precursor, was encapsulated in alginate/chitosan nanoparticles. The presence of GSNO was confirmed by UV spectra at 336 nm. Alginate/chitosan nanoparticles with negative and posi- tive surface charges were obtained by increasing the chitosan amount. The encapsulation efficiency (EE) relied on the nanoparticle zeta potential, obtaining 80% of EE for positive particles. The NO release from GSNO showed that polymeric nanoparticles lead to the stabi- lization of GSNO decomposition, at physiological temperature. Moreover, this system did not exhibit cytotoxicity for fibroblast V79 cells up to the maximum concentration tested (18 μmolL -1 ). These results showed that alginate/chitosan nanoparticles are interesting particles to encapsulate NO donors for biomedical applications where NO might have a therapeutic effect.

Cytotoxicity of derivatives from dehydrocrotonin on V79 cells and Escherichia coli

New derivatives from dehydrocrotonin (DHC, compound I), with the same anti-ulcerogenic properties but less toxicity were synthesised by reducing the cyclohexenone moiety of DHC with NaBH(4) (compound II), by reducing the cyclohexenone and lactone moieties with LiAlH(4) (compound III) and by transforming the lactone moiety into an amide (compound IV) using dimethylamine. The cytotoxicity of these derivatives from DHC was assayed on V79 fibroblast cell line. Three independent endpoints for cytotoxicity were evaluated; namely, the nucleic acid content (NAC), tetrazolium reduction (MTT) and neutral red uptake (NRU). IC(50) values of 540 and 350 microM were obtained for compound II in the NRU and NAC tests, respectively. Compound III was less toxic than the other DHC derivatives (IC(50)=1800 microM) on V79 cells based on NAC assay. Compound IV showed an IC(50) ranging from 350 to 600 microM based on the three endpoints evaluated. The three compounds were less toxic on V79 cells than DHC. DHC, compounds II, III and IV did not change the respiration rate of Escherichia coli on the acute toxicity assay.

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.