Metabolism of the mesoionic compound (MI-D) by mouse liver microsome, detection of its metabolite in vivo, and acute toxicity in mice.

The mesoionic derivative 4-phenyl-5-[4-nitrocinnamoyl]-1,3,4-thiadiazolyl-2-phenylamine chloride (MI-D) has antitumoral and anti-inflammatory effects. In this study, we present aspects of its metabolism and toxicity in mice. MI-D was metabolized in vitro by liver microsome, generating a main product with a much shorter retention time than MI-D in high-performance liquid chromatography (HPLC) analysis but with a spectrum similar to that of the original molecule. Mass spectrometry with electrospray ionization in positive mode analysis of the purified compound by HPLC indicated that the product of metabolism has four additional hydroxyl groups (m/z = 465) compared with MI-D (m/z = 401). The HPLC analyses of plasma and urine samples from mice treated with MI-D showed the presence of the metabolite product. The kinetic parameters K(m) (19.5 +/- 4.5 microM) and V(max) [1.5 +/- 0.4 units of fluorescence/(100 microg of microsomal protein/mL/s)] were estimated, confirming the metabolism of MI-D and indicating that the reaction follows Michaelis-Menten kinetics. Acute toxicity was established on the basis of an estimation of mean lethal dose (LD-50; 181.2 mg/kg) and histopathological analysis of animals that survived the LD-50 test. Abdominal adhesions, inflammatory foci, and formation of granulomas were observed. Altogether, the results contribute to the advancement of research in support of MI-D as a future chemotherapeutic drug.

Two galactomannan preparations from seeds from Mimosa scabrella (bracatinga): Complexation with oxovanadium(IV/V) and cytotoxicity on HeLa cells.

Two galactomannans, GALMAN-A and GALMAN-B, were isolated from seeds of Mimosa scabrella (bracatinga), with deactivation and exposure to native enzymes, respectively. They were treated with oxovanadium(IV) and oxovanadium(V), designated (VO(2+)/VO(3+)) to form GALMAN-A:VO(2+)/VO(3+) and GALMAN-B:VO(2+)/VO(3+) complexes, respectively. The potentiometric studies provided the binding constants for the complexes and the resulting complexed species were a function of pH. (51)V NMR spectra of GALMAN-A:VO(2+)/VO(3+) and GALMAN-B:VO(2+)/VO(3+) at pH 7.8 and at 30 degrees C indicated the occurrence of two types of complexes formed by oxovanadium ions and galactomannans. GALMAN-A:VO(2+)/VO(3+) and GALMAN-B:VO(2+)/VO(3+) caused loss of HeLa cells viability at concentrations of 50-200microg/mL. GALMAN-A:VO(2+)/VO(3+) exhibited low toxicity for 24h, although GALMAN-B:VO(2+)/VO(3+) was extremely toxic, since 50microg/mL was sufficient to decrease HeLa cell viability after 48h by 60%. GALMAN-A gave rise to a slight increase in cell proliferation after 48h at 100microg/mL, whereas GALMAN-B promoted a slight decrease at concentrations of 50-100microg/mL. GALMAN-A:VO(2+)/VO(3+) and GALMAN-B:VO(2+)/VO(3+) exhibited a significant decrease in cell proliferation after 48h, each reaching 60% inhibition at 5-10microg/mL. The complexes which caused this effect were at concentrations 10 times lower than the uncomplexed polymers.

Chemical and immunological modifications of an arabinogalactan present in tea preparations of Phyllanthus niruri after treatment with gastric fluid.

An arabinogalactan (AG) obtained from tea preparations of Phyllanthus niruri was previously investigated and presented immunological properties when tested with peritoneal mice macrophages. AG was now submitted to acidic and neutral gastric conditions using human gastric fluids and aq. HCl solution. Since the acidic procedures gave rise to the same free monosaccharidic composition, the acid hydrolyzate of AG at pH 2.00 was treated with ethanol to form insoluble (AG-P) and soluble fractions (AG-S). These were analyzed using (13)C NMR, HPSEC, and GC-MS for monosaccharide composition and methylation analyses. The results showed an intense partial degradation, including cleavages of the main chain. AG-S presented the monosaccharides released from the native polymer and some oligosaccharides as shown by methylation data. AG-P contained larger molecular fragments comprising the internal units from AG, which were not attacked by the hydrolysis condition. Both fractions were tested in peritoneal mice macrophages and remained active, promoting an increase of superoxide anion production of 2.0 and 2.3-fold, at 250 microg/mL, for AG-S and AG-P, respectively. When compared to AG, a slight diminished response was observed, revealing a structure-activity relation. The significance of the results is that most plant extracts are orally ingested and will reach the gastrointestinal tract before performing a biological function, so checking these changes is crucial to propose future clinical therapies based on the rational use of phytomedicine.

Chemical and biological properties of an arabinogalactan from Phyllanthusniruri.

Phyllanthus niruri is a well-known herb widely used medicinally in Asia, Africa, and South America. Aqueous extraction of the intact plant provided an acidic arabinogalactan, which was characterized chemically, and its effects on peritoneal macrophage activation were determined. Methylation analyses and (13)C NMR spectroscopy showed it to have a complex structure with a (1-->4)-linked beta-Galp main chain, substituted by rhamnose, galacturonic acid, arabinose, xylose, galactose, and glucose-containing side chains, with nonreducing end-units of arabinofuranose, xylopyranose, galactopyranose, and glucopyranose. In immunological studies, the arabinogalactan stimulated superoxide anion production, when tested using peritoneal macrophages of mice, but did not interfere with the nitric oxide pathway. Thus, traditional aqueous extraction methods, such as decoction and infusion, provide a major polysaccharide, which stimulates an intense biological response in macrophages: this could represent an interesting approach in phytotherapeutic treatments.

Effects of deltamethrin on functions of rat liver mitochondria and on native and synthetic model membranes.

Deltamethrin (DTM) is a synthetic pyrethroid insecticide used wideworld in agriculture, home pest control, protection of foodstuff, and disease vector control. It has widespread applications in Brazilian agriculture. The effects of DTM on mitochondrial respiratory parameters and on the organization of artificial and native membranes are described. DTM (200 nmol mg(-1) protein) on isolated liver mitochondria decreased oxygen consumption of both, state III and state IV, as well as the inner mitochondrial membrane potential (Deltapsi). Analysis of segments of the respiratory chain suggested that the DTM inhibition site is located between complex II and complex III. Mitochondrial swelling, energized or driven by the K+ diffusion potential using valinomycin, were partially inhibited by DTM (200 nmol mg(-1) protein). Fluorescence polarization of DPH and DPH-PA, probing the core and outer regions, respectively, of dimyristoylphosphatidylcholine (DMPC) and native mitochondrial membranes, indicated that DTM shifts the midpoint phase transition to lower values, besides broadening the phase transition. DTM decreased the lipid order of DMPC bilayers, at temperatures lower than the transition temperature and also caused a disordering effect on native membranes. However at temperatures above the transition temperature, the pesticide increased the rigidity of the membrane. These results suggest that DTM causes perturbations in lipid-lipid and lipid-protein interactions, interferes in transport mechanisms operating at the membrane level, and causes alterations of membrane permeability and mitochondrial enzyme activities. These effects could be associated with the toxicity of deltamethrin.

Induction of secretory and tumoricidal activities in peritoneal macrophages activated by an acidic heteropolysaccharide (ARAGAL) from the gum of Anadenanthera colubrina (Angico branco).

The immunomodulatory and anti-tumoral effects of an acidic heteropolysaccharide containing mainly galactose and arabinose (ARAGAL), isolated from the gum of the leguminous tree Anadenanthera colubrina (Angico branco) native to Brazil, were studied. It has been demonstrated that activation of mice peritoneal macrophages both in vivo and in vitro, increases phagocytic ability and anion superoxide production. In order to obtain further insights on the biological effects of ARAGAL, the capacity of eliciting peritoneal macrophages and tumor necrosis factor-alpha (TNF-alpha) production, and anti-tumoral effect against Sarcoma 180 (S-180), are now evaluated. Cell eliciting activity was observed in ARAGAL-treated animals in a dose dependent manner. Treatment of animals with 50, 100 or 200 mg/kg of ARAGAL increased peritoneal exudate cell (PEC) numbers by approximately 18, approximately 44 and approximately 88%, respectively. ARAGAL also increased 26-fold TNF-alpha production by peritoneal macrophages. Macrophages, treated in vitro for 18 h with ARAGAL, were able to kill Sarcoma 180 cells, as observed by their structures inside the macrophage cytoplasm. ARAGAL (100 mg/kg) showed anti-tumoral activity against S-180 in ascites or solid tumors, the tumoral inhibition being 63 and 38%, respectively. The results suggest a possible role as a BRM for ARAGAL.

Complexation of vanadium(V) oxyanions with hexopyranose- and mannopyranoseuronic acid-containing polysaccharides: stereochemical considerations.

Carbohydrates containing galactopyranosyl and mannopyranosyl units with vicinal cis-diols were treated with NaVO(3) in D(2)O, and complexation was determined by (51)V NMR spectroscopy. Me alpha-Galp, Me beta-Galp (3,4-cis-diols), and Me alpha-Manp (2,3-cis-diol) complexed, but Me beta-Manp barely did so. This low degree of complexation also occurred with a beta-mannan containing alternate (1-->3)- and (1-->4)-linkages and an alginate having beta-ManpA blocks. In contrast, branched alpha-mannans complexed readily, although the (51)V resonances for one with side chains terminated with alpha-Manp-(1-->3)-alpha-Manp-(1--> differed from another with only alpha-Manp-(1-->2)-alpha-Manp-(1--> groups. The anomeric configuration of Me alpha-Galp and Me beta-Galp, each with 3,4-cis-diols remote from C-1, gave rise to three (51)V signals of complexes with similar shifts and proportions. The shifts of a galactomannan with terminal alpha-Galp-(1-->2)-alpha-Manp- were the same as those with alpha-Galp-(1-->6)-beta-Manp- groups, but fewer complexes were formed with the former structure, probably due to greater steric crowding of the vanadate esters. Most of the complexes gave rise to a signal in the delta515 region, consistent with the dimeric trigonal-bipyramidal structure.

Effect of an acidic heteropolysaccharide (ARAGAL) from the gum of Anadenanthera colubrina (Angico branco) on peritoneal macrophage functions.

Brazilian flora are a source of interesting polysaccharides which, either in their native state or when submitted to structural modifications, might have potential applications as biological response modifiers (BRM). A complex acidic heteropolysaccharide, containing mainly galactose and arabinose (ARAGAL), isolated from the gum of the native leguminous tree Anadenanthera colubrina (Angico branco), was studied for its immunological properties on peritoneal exudate cells, namely their superoxide anion production, phagocytic activity, morphological alterations and percentage content of activated macrophages. Activation of macrophages showing increased cytoplasm, bright and large nuclei, various cytoplasmatic projections and spreading ability, was detected following in vitro cell exposure to ARAGAL or in cells obtained from treated animals. In vitro exposure to ARAGAL increased the occurrence of activated macrophages in a time- and a dose-dependent pattern, since approximately 82% of the cells were activated in the presence of 300 microg/ml of ARAGAL after 24 h of incubation and approximately 91% after 48 h. The occurrence of activated macrophages was also evident in cell preparations from ARAGAL-treated mice, their percentage showing a dose-dependent pattern. There were approximately 60, 75 and 75% following treatment with 100, 250 and 500 mg/kg of ARAGAL, respectively. A phagocytic assay showed that 25 microg/ml ARAGAL was sufficient to impose a maximum phagocytic ability, although this effect was dose-dependent. O(2)(-) production by macrophages from ARAGAL-treated mice was 70% higher than that of cells from untreated mice. Moreover, cells from treated mice responded to PMA, the effect being 25% higher than that of the control using untreated mice. These results thus suggest a possible role of ARAGAL from A. colubrina as a BRM.

Complexes of sodium vanadate(V) with methyl alpha-D-mannopyranoside, methyl alpha- and beta-D-galactopyranoside, and selected O-methyl derivatives: a 51V and 13C NMR study.

The hydroxyl group stereochemistry of complexation of sodium vanadate(V) with Me alpha-Manp, Me alpha- and beta-Galp and selected O-methyl derivatives in D(2)O was determined by 51V, 1D and 2D 13C NMR spectroscopy at pD 7.8. The 51V approach served to show the extent of complexation and the minimum number of esters formed. That of Me alpha-Manp gave rise mainly to a 51V signal at delta -515, identical with that of its 4,6-di-O-methyl derivative, which had only a 2,3-cis-diol exposed. The 13C NMR spectra contained much weaker signals of the complexes, but both glycosides showed strong C-2 and C-3 alpha-shifts of +17.3 and +10.8 ppm, respectively. As expected, Me 2,3-Me(2)-alpha-Manp, which contains a 4,6-diol, did not complex. Me Galp anomers and their derivatives showed more diversity in the structure of its oxyvanadium derivatives. Me alpha-Galp, with its 3,4-cis-diol, complexed to give rise to 51V signals at delta -495 (9%), -508 (10%), and -534 (4%). These shifts and proportions were maintained with Me beta-Galp and Me 6Me-alpha-Galp. 51V NMR spectroscopy showed that Me 3Me-beta-Galp, with its possibly available 4,6-diol, did not complex. Similarly, Me alpha-Galp+vanadate gave a 13C DEPT spectrum that did not contain an inverted signal at delta >71.4, as would be expected of a C-6 resonance suffering a strong downfield alpha-shift. Me 2,6-Me(2)-alpha-Galp, with a 3,4-cis-diol group, gave rise to two 51V signals of complexes at delta -492 (9%) and -508 (9%), showing more than one structure of oxyvanadium derivatives.

Activity of isosteviol lactone on mitochondrial metabolism.

Isosteviol lactone (LAC), a lactone derivative of the diterpenic acid isosteviol (ISO) was evaluated for its effect on the oxidative metabolism of mitochondria isolated from rat liver. In this model, LAC (1 mM) depressed the phosphorylation efficiency, as shown by the decreased respiratory control coefficient (RCC) and ADP/O ratio. LAC (1 mM) inhibited NADH oxidase (45%), succinate oxidase (34%) and promoted low-level inhibitions on succinate dehydrogenase (13%), succinate-cytochrome c oxide-reductase (23%), cytochrome c oxidase (10%), and NADH dehydrogenase (13%). Glutamate dehydrogenase was also a target for LAC, as it was 85% inhibited by 1 mM LAC. Cyclic voltammetry data showed that LAC, as well as ISO, does not undergo redox reactions under current experimental conditions. LAC (0.05-0.75 mM) inhibited the swelling dependent on the glutamate oxidation, 50% of the effect occurring at 0.5 mM LAC. Swelling supported by KNO(3) and valinomycin was also inhibited over all concentrations used of LAC and ISO, the effect being of a lower intensity for LAC, suggesting that the modification of the structure of ISO by lactonization diminished its interaction with the membrane. This could contribute to attenuation of the toxic effects described for ISO on mitochondrial function, such as those on respiratory chain enzymatic complexes and phosphorylating activity.

Effects of a lichen galactomannan and its vanadyl (IV) complex on peritoneal macrophages and leishmanicidal activity.

A galactomannan (GMPOLY) isolated from lichen Ramalina celastri was complexed with vanadyl ion (IV;VO) forming the complex GMPOLY-VO. Both GMPOLY and GMPOLY-VO diminished the superoxide anion production by macrophages triggered with PMA, the complex giving rise to this effect at concentrations 100 times lower than GMPOLY. Macrophages treated with GMPOLY enhanced the nitric oxide production (40%), this effect not being observed when interferon-gamma (IFN-gamma) or IFN-gamma plus lipopolysaccharide (LPS) were present. No effect on nitric oxide production was observed by treatment of macrophage with GMPOLY-VO. Both GMPOLY and GMPOLY-VO exhibited leishmanicidal effects on the amastigote form of Leishmania amazonesis, but only GMPOLY-VO inhibited the growth of promastigote form.

Effect of a soluble alpha-D-glucan from the lichenized fungus Ramalina celastri on macrophage activity.

An alpha-glucan from the lichen Ramalina celastri has previously been demonstrated to have cytotoxic effects against HeLa cells. This polysaccharide was studied using Sarcoma-180 cells as tumoral model, and its effects on peritoneal exudate cells, namely, hydrogen peroxide production, phagocytic activity and cell eliciting activity are evaluated. Tumors developing in animals treated with the glucan at a dose of 200 mg kg(-1), had a tumor size approximately 80% smaller than that of the control group, showing an impairment of tumor establishment. The polysaccharide was injected into mice not bearing a tumor and after 7, 15 and 30 days the cells were collected from the peritonea. The number of peritoneal cells increased approximately 130% 7 days after inoculation, and then gradually decreased. Hydrogen peroxide production was 75% greater 7 and 15 days after inoculation, on in vitro phorbol myristate acetate (PMA) triggering. Without PMA, the difference in hydrogen peroxide production was not significant. Phagocytic assays using fluorescent beads showed that the uptake increased 7 and 15 days after inoculation, when compared with the control. These results thus suggest a possible role of the R. celastri glucan as a biological response modifier (BRM).