Cytotoxic and genotoxic effects of tambjamine D, an alkaloid isolated from the nudibranch Tambja eliora, on Chinese hamster lung fibroblasts

Marine organisms have been shown to be potential sources of bioactive compounds with pharmaceutical applications. Previous chemical investigation of the nudibranch Tambja eliora led to the isolation of the alkaloid tambjamine D. Tambjamines have been isolated from marine sources and belong to the family of 4-methoxypyrrolic-derived natural products, which display promising immunosuppressive and cytotoxic properties. Their ability to intercalate DNA and their pro-oxidant activity may be related to some of the biological effects of the 4-methoxypyrrolic alkaloids. The aim of the present investigation was to determine the cytotoxic, pro-oxidant and genotoxic properties of tambjamine D in V79 Chinese hamster lung fibroblast cells. Tambjamine D displayed a potent cytotoxic effect in V79 cells (IC50 1.2 microg/mL) evaluated by the MTT assay. Based on the MTT result, V79 cells were treated with different concentrations of tambjamine D (0.6, 1.2, 2.4 and 4.8 microg/mL). After 24h, tambjamine D reduced the number of viable cells in a concentration-dependent way at all concentrations tested, assessed by the trypan blue dye exclusion test. The hemolytic assay showed that the cytotoxic activity of tambjamine D was not related to membrane disruption (EC50>100 microg/mL). Tambjamine D increased the number of apoptotic cells in a concentration-dependent manner at all concentrations tested according to acridine orange/ethidium bromide staining, showing that the alkaloid cytotoxic effect was related to the induction of apoptosis. MTT reduction was stimulated by tambjamine D, which may indicate the generation of reactive oxygen species. Accordingly, treatment of cells with tambjamine D increased nitrite/nitrate at all concentrations and TBARS production starting at the concentration corresponding to the IC50. Tambjamine D, also, induced DNA strand breaks and increased the micronucleus cell frequency as evaluated by comet and micronucleus tests, respectively, at all concentrations evaluated, showing a genotoxic risk induced by tambjamine D.     

Bromelain purification through unconventional aqueous two-phase system (PEG/ammonium sulphate)

This paper focuses on the feasibility of unconventional aqueous two-phase systems for bromelain purification from pineapple processing waste. The main difference in comparison with conventional systems is the integration of the liquid–liquid extraction technique with fractional precipitation, which can decrease the protein content with no loss of biological activity by removing of unwanted molecules. The analysis of the results was based on the response surface methodology and revealed that the use of the desirability optimisation methodology (DOM) was necessary to achieve higher purification factor values and greater bromelain recovery. The use of DOM yielded an 11.80-fold purification factor and 66.38 % biological activity recovery using poly(ethylene glycol) (PEG) with a molar mass of 4,000, 10.86 % PEG concentration (m/m) and 36.21 % saturation of ammonium sulphate.     

Laccase production in bioreactor scale under saline condition by the marine-derived basidiomycete Peniophora sp. CBMAI 1063

Laccase production in saline conditions is still poorly studied. The aim of the present study was to investigate the production of laccase in two different types of bioreactors by the marine-derived basidiomycete Peniophora sp. CBMAI 1063. The highest laccase activity and productivity were obtained in the Stirred Tank (ST) bioreactor, while the highest biomass concentration in Air-lift (AL) bioreactor. The main laccase produced was purified by ion exchange and size exclusion chromatography and appeared to be monomeric with molecular weight of approximately 55 kDa. The optimum oxidation activity was obtained at pH 5.0. The thermal stability of the enzyme ranged from 30 to 50 °C (120 min). The Far-UV Circular Dichroism revealed the presence of high β-sheet and low α-helical conformation in the protein structure. Additional experiments carried out in flask scale showed that the marine-derived fungus was able to produce laccase only in the presence of artificial seawater and copper sulfate. Results from the present study confirmed the fungal adaptation to marine conditions and its potential for being used in saline environments and/or processes.     

Evaluation of sugar cane hemicellulose hydrolyzate for cultivation of yeasts and filamentous fungi

Sugar cane bagasse hemicellulosic fraction submitted to hydrolytic treatment with 100 mg of sulfuric acid per gram of dry mass, at 140°C for 20 min, was employed as a substrate for microbial protein production. Among the 22 species of microorganisms evaluated, Candida tropicalis IZ 1824 showed TRS consumption rate of 89.8%, net cell mass of 11.8 g L⁻¹ and yield coefficient (Y_x/s) of 0.50 g g⁻¹. The hydrolyzate supplemented with rice bran (20.0 g L⁻¹), P₂O₅ (2.0 g L⁻¹) and urea (2.0 g L⁻¹) provided a TRS consumption rate of 86.3% and a cell mass of 8.4 g L⁻¹. At pH 4.0 cellular metabolism was inhibited, whereas at pH 6.0 the highest yield was obtained. The presence of furfural (2.0 g L⁻¹) hydroxymethylfurfural (0.08 g L⁻¹) and acetic acid (3.7 g L⁻¹) in the hydrolyzate did not interfere with cultivation at pH 6.0. Received 25 October 1996/ Accepted in revised form 10 March 1997     

Analgesic and anti-inflammatory activities of a fraction rich in oncocalyxone A isolated from Auxemma oncocalyx.

In the present work we studied the antinociceptive and antiedematogenic effects of a quinone fraction (QF) isolated from the heartwood of Auxemma oncocalyx Taub. The major constituent of QF, which represented around 80% of this fraction, was a terpenoid quinone named oncocalyxone A (1). Results show that QF (10 and 30 mg/kg body wt., i.p.) significantly inhibited paw edema induced by carrageenan at the second, third, and fourth hours. The effect was dose-dependent and long lasting, and QF was less effective orally. An antiedematogenic effect was also demonstrated in the dextran-induced paw edema. In this model, however, QF was somewhat less potent. QF (1 and 5 mg/kg body wt., i.p.) inhibited acetic acid-induced abdominal contractions in mice in a dose-dependent manner. In addition, QF (5 and 10 mg/kg body wt., i.p.) inhibited only the second phase (inflammatory) in the formalin test, and showed no effect in the hot-plate test in mice. The antinociceptive activity of QF was predominantly peripheral and independent of the opioid system. The observed effects of QF are, at least in part, probably due to the presence of oncocalyxone A (1).     

Selective recovery of xylanase from Penicillium janthinellum using BDBAC reversed micelles

A xylanase was removed from crude extract of the fungus Penicillium janthinellum under optimized conditions: 0.10M phosphate buffer, pH 7.0, 0.2 M BDBAC (N-benzyl-N-dodeceyl-N-bis (2-hydroxyethyl) ammonium chloride), 7.5% hexanole, 30°C and an agitation time of 1 minute. At 1.42 mg per ml protein concentration, 73% of the xylanase activity was recovered and a 7-fold enrichment factor was obtained. The enzyme had a molecular weight (MW) of 20.1 kDa and the isoelectric point (PI) revealed the presence of two protein bands with a PI of 6.0 and 6.5. The optimum pH and optimum temperature were 4.2 and 50°C, respectively. The low pH differential between the aqueous medium and the protein PI seemed to influence the xylanase transportation into the reversed micelles.