Ilka M. Vasconcelos

Compositional and nutritional attributes of seeds from the multiple purpose tree Moringa oleifera Lamarck

Moringa oleifera Lam is a multipurpose tree cultivated to use as a vegetable, for spice, for cooking and cosmetic oil and as a medicinal plant. Owing to the use of its seeds as food and as a clarifying agent of turbid water some nutritional and antinutritional characteristics were studied. The mature seeds contained 332.5 g crude protein, 412.0 g crude fat, 211.2 g carbohydrate and 44.3 g ash per kg dry matter. The essential amino acid profile compared with the FAO/WHO/UNU scoring pattern requirements for different age groups showed deficiency of lysine, threonine and valine. The content of methionine + cysteine (43.6 g kg−1 protein), however, was exceptionally higher and close to that of human milk, chicken egg and cows milk. The seed extract agglutinated rabbit erythrocytes but did not show trypsin inhibitor and urease activities. Feeding rats with a diet containing the seed meal showed loss of appetite, impaired growth, lower NPU and enlargement of stomach, small intestine, caecum + colon, liver, pancreas, kidneys, heart and lungs and atrophy of thymus and spleen in comparison with rats fed on an egg-white diet. The results indicated that consumption of M oleifera raw mature seeds should be viewed with some caution until suitable processing methods are developed to abolish the yet unknown adverse factors.

Composition, toxic and antinutritional factors of newly developed cultivars of Brazilian soybean (Glycine max)

Five different recently released Brazilian soybean cultivars (Bays, BR-10, Rio Balsas, Serido and Tropical) were compared for their proximate analyses and presence of antinutritional or toxic factors. As expected, the seeds are rich in proteins, varying from 360·7 to 485·4 g kg−1 flour, and they also have a high amount of fat (from 183·0 to 215·3 g kg−1 flour). Crude extracts from seeds of Bays, BR-10, Serido and Tropical were highly toxic to mice within 1–12 h, depending on the administration route (intraperitoneal, intramuscular or subcutaneous) and dose used while Rio Balsas was not. These acute effects were very similar to those produced by the soytoxin, a neurotoxin that has been recently purified from the commercial soybean sold in Brazil. The amount of trypsin inhibited in the presence of crude extracts ranged from 28·5 to 62·5 g kg−1 flour. Urease was also present and the seed lectin agglutinated preferentially rabbit erythrocytes. A heat treatment at 92°C for 1 min destroyed completely the toxic activity while the haemagglutinating and trypsin inhibitor activities were abolished within 5 min. At these conditions urease was still active. Due to its high protein content, lack of soytoxin, and low levels of trypsin inhibitor, lectin, and urease it is suggested that Rio Balsas could be an alternative for breeding programmes aimed to improve the nutritional quality of soybeans. ©1997 SCI

Composition and nutritional properties of seeds from Pachira aquatica Aubl, Sterculia striata St Hil et Naud and Terminalia catappa Linn.

Abstract The seeds of three wild plants (Pachira aquatica, Sterculia striata and Terminalia catappa) were analyzed to establish their chemical compositions and nutritional properties in order to investigate the possibility of using them for human and/or animal consumption. Proximate analyses showed that they have high amounts of protein and oil. However, they are deficient in various essential amino acids but P. aquatica seeds have tryptophan, threonine and phenylalanine + tyrosine contents higher than those reported for human milk, chicken egg and cows milk. Haemagglutinating and trypsin inhibitor activities were found to be present in the seeds of P. aquatica and T. catappa but absent in S. striata. Coincidentally the rats fed on S. striata diet gained slightly in weight and presented alterations in the key internal organs which were less drastic throughout the 10-day test period. On the other hand, the rats fed on T. catappa diet maintained their body weight but suffered from stomah, small intestine and pancreas hypertrophy as well as spleen atrophy. Five out of six rats fed on P. aquatica diet died within 6–8 days. The remaining rat experienced enlargement of the stomach, liver, pancreas, kidneys, heart and lungs and had spleen atrophy when compared with the same organs of rats fed on egg-white diet. Hypertrophy of the pancreas and kidneys was very marked and these organs nearly doubled in dry weight in comparison with those of the egg-white control group, demonstrating that the raw seed of P. aquatica are highly toxic when fed to rats even at a meal protein concentration half that of S. striata or T. catappa, which were better tolerated by the experimental animals.

Nutritional study of two Brazilian soybean (Glycine max) cultivars differing in the contents of antinutritional and toxic proteins

The research was conducted with two different recently released Brazilian soybean cultivars (Rio Balsas and Bays) to evaluate whether there is any correlation between the different levels of antinutritional and/or toxic proteins in the cultivars and their nutritive value as sources of protein for monogastric animals (rats). Furthermore, it is discussed, for the first time, the role of the dietary soyatoxin on the performance of rats fed on diets containing soyatoxin-rich (cv. Bays) and soyatoxin-free (cv. Rio Balsas) soybean cultivars. Feeding rats with diets containing raw soybean cultivars showed a lower growth rate, net protein utilization and digestibility, a much higher dry matter and nitrogen excretion and macroscopic alterations in internal organs when compared to rats fed on egg-white protein. The nutritional parameters measured for the diet based on raw Bays cultivar were poorer than those of the diet prepared with Rio Balsas. In the raw soybeans, trypsin inhibitor and lectin, and urease to a lesser extent, significantly affected at different fashion the soybean protein utilization. Heating treatment of the Bays seeds increased the growth rate, NPU, in vivo protein digestibility and practically eliminated or attenuated all the organ alterations observed. This study might be helpful in the choice of safe and nutritious soybean cultivars.

A lactose specific lectin from the sponge Cinachyrella apion: purification, characterization, N-terminal sequences alignment and agglutinating activity on Leishmania promastigotes.

Crude extract from the sponge Cinachyrella apion showed cross-reactivity with the polyclonal antibody IgG anti-CvL (Cliona varians lectin) and also a strong haemagglutinating activity towards human erythrocytes of all ABO groups. Thus, it was submitted to acetone fractionation, IgG anti-deglycosylated CvL Sepharose affinity chromatography, and Fast Protein Liquid Chromatography (FPLC-AKTA Purifier) gel filtration on a Superose 6 10/300 column to purify a novel lectin. C. apion lectin (CaL) agglutinated all types of human erythrocytes with preference for papainized type A erythrocytes. The haemagglutinating activity is independent of Ca2+, Mg2+ and Mn2+ ions, and it was strongly inhibited by the disaccharide lactose, up to a minimum concentration of 6.25 mM. CaL molecular mass, determined by FPLC-gel filtration on a Superose 12 10/300 column and SDS gel electrophoresis, was approximately 124 kDa, consisting of eight subunits of 15.5 kDa, assembled by hydrophobic interactions. The lectin was heat-stable between 0 and 60 degrees C and pH-stable. The N-terminal amino acid sequence of CaL was also determined and a blast search on amino acid sequences revealed that the protein showed similarity only with a silicatein. Leishmania chagasi promastigotes were agglutinated by CaL and this activity was abolished by lactose, indicating that lactose receptors could be presented in this parasite stage. These findings are indicative of the potential biotechnological application of CaL as diagnostic of pathogenic protozoa.

Isolation, characterization and cloning of a cDNA encoding a new antifungal defensin from Phaseolus vulgaris L. seeds

The PvD1 defensin was purified from Phaseolus vulgaris (cv. Pérola) seeds, basically as described by Terras et al. [Terras FRG, Schoofs HME, De Bolle MFC, Van Leuven F, Ress SB, Vanderleyden J, Cammue BPA, Broekaer TWF. Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds. J Biol Chem 1992;267(22):15301-9], with some modifications. A DEAE-Sepharose, equilibrated with 20mM Tris-HCl, pH 8.0, was initially utilized for the separation of peptides after ammonium sulfate fractionation. The basic fraction (the non-retained peak) obtained showed the presence of one unique band in SDS-Tricine gel electrophoresis with a molecular mass of approximately 6kDa. The purification of this peptide was confirmed after a reverse-phase chromatography in a C2/C18 column by HPLC, where once again only one peak was observed and denominated H1. H1 was submitted to N-terminal sequencing and the comparative analysis in databanks revealed high similarity with sequences of different defensins isolated from other plants species. The N-terminal sequence of the mature defensin isolated was used to produce a degenerated primer. This primer allowed the amplification of the defensin cDNA by RT-PCR from mRNA of P. vulgaris seeds. The sequence analysis of the cloned cDNA, named PVD1, demonstrated 314bp encoding a polypeptide of 47 amino acids. The deduced peptide presented high similarity with plant defensins of Vigna unguiculata (93%), Cicer arietinum (95%) and Pachyrhizus erosus (87%). PvD1 inhibited the growth of the yeasts, Candida albicans, Candida parapsilosis, Candida tropicalis, Candida guilliermondii, Kluyveromyces marxiannus and Saccharomyces cerevisiae. PvD1 also presented an inhibitory activity against the growth of phytopathogenic fungi including Fusarium oxysporum, Fusarium solani, Fusarium lateritium and Rizoctonia solani.

Osmotin purified from the latex of Calotropis procera: biochemical characterization, biological activity and role in plant defense.

A protein, similar to osmotin- and thaumatin-like proteins, was purified from Calotropis procera (Ait.) R.Br latex. The isolation procedure required two cation exchange chromatography steps on 50mM Na-acetate buffer (pH 5.0) CM-Sepharose Fast Flow and 25 mM Na-phosphate buffer (pH 6.0) Resource-S, respectively. The protein purity was confirmed by an unique N-terminal sequence [ATFTIRNNCPYTIWAAAVPGGGRRLNSGGTWTINVAPGTA]. The osmotin (CpOsm) appeared as a single band (20,100 Da) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as two spots in two-dimensional electrophoresis (pI 8.9 and 9.1). Both polypeptides were further identified by mass spectrometry as two osmotin isoforms with molecular masses of 22,340 and 22,536 Da. The CpOsm exerted antifungal activity against Fusarium solani (IC₅₀=67.0 μg mL⁻¹), Neurospora sp. (IC₅₀=57.5 μg mL⁻¹) and Colletotrichum gloeosporioides (IC₅₀=32.1 μg mL⁻¹). However, this activity was lost when the protein was previously treated with a reducing agent (DTT, Dithiothreitol) suggesting the presence of disulfide bounds stabilizing the protein. The occurrence of osmotin in latex substantiates the defensive role of these fluids.

Purification and physicochemical characterization of a cotyledonary lectin from Luetzelburgia auriculata.

A lectin was purified from the cotyledons of Luetzelburgia auriculata (Fr. All) Ducke by affinity chromatography on agarose-N-acetyl-D-galactosamine. The lectin is a potent agglutinin for rabbit erythrocytes, reacts with human red cells, but is inactive against cow, sheep, and goat erythrocytes. Hemagglutination of rabbit erythrocytes was inhibited by either 0.39 mM N-acetyl-neuraminic acid or N-acetyl-D-galactosamin, 12.5 mM D-lactose or D-melibiose, 50 mM D-galactose or raffinose. Its hemagglutinating activity was lost at 80 degrees C, 5 min, and the activation energy required for denaturation was 104.75 kJ mol(-1). Chromatography on Sephadex G-100, at pH 7.6, showed that at this hydrogenic ionic concentration the native lectin was a homotetramer (123.5 kDa). By denaturing SDS-PAGE, LAA seemed to be composed of a mixture of 29 and 15 kDa polypeptide subunits. At acidic and basic pHs it assumed different conformations, as demonstrated by exclusion chromatography on Superdex 200 HR 10/30. The N-terminal sequence of the 29 kDa band was SEVVSFSFTKFNPNQKDII and the 15 kDa band contained a mixture of SEVVSFSFTKFNPNQKDII and KFNQIVAVEEDTDXESQPQ sequences, indicating that these bands may represent full-length and its endogenous fragments, respectively. The lectin is a glycoprotein having 3.2% neutral carbohydrate, with a pI of 5.8, containing high levels of Asp+Asn and Glu+Gln and hydroxy amino acids, and low amount or absence of sulfur amino acids. Its absorption spectrum showed a maximum at 280 nm and a epsilon (1%) x (1cm) of 5.2. Its CD spectrum was characterized by minima near 228 nm, maxima near 196 nm and a negative to positive crossover at 210 nm. The secondary structure content was 6% alpha-helix, 8% parallel beta-sheet, 38% antiparallel beta-sheet, 17% beta-turn, 31% unordered and others contribution, and 1% RMS (root mean square). In the fluorescence spectroscopy, excitation of the lectin solution at 280 nm gave an emission spectrum in the 285-445 nm range. The wavelength maximum emission was in 334.5 nm, typical for tryptophan residues buried inside the protein.

Purification of a novel antibacterial and haemagglutinating protein from the purple gland of the sea hare, Aplysia dactylomela Rang, 1828

Physicochemical characterisation and antibacterial and haemagglutinating properties of a new protein isolated from purple fluid of the Aplysia dactylomela are reported. The purification procedure consisted basically of ammonium sulphate fractionation, ion exchange, exclusion molecular and hydrophobic interaction chromatography. The highly purified protein, designated dactylomelin-P, is a single chain protein of 60,000 Da by SDS-polyacrylamide gel electrophoresis and 56,200 Da by gel filtration on calibrated Superose column at pH 7.5 and contains less than 0.05% of its weight in neutral carbohydrates. Dactylomelin-P has two biological activities, antibacterial and haemagglutinating. The antibacterial action is bacteriostatic but not bactericidal. The haemagglutinating activity is preferentially against rabbit erythrocytes. The glycoprotein fetuin was able to abolish the haemagglutinating activity but not the antibacterial one even when used at concentrations 10 fold higher. This is the first time that a chimeroprotein is described in the purple fluid of sea hares, which may be involved in the chemical defence mechanism of these organisms.

Study of the antiproliferative potential of seed extracts from Northeastern Brazilian plants

This study assessed the antiproliferative and cytotoxic potential against tumor lines of ethanolic seed extracts of 21 plant species belonging to different families from Northeastern Brazil. In addition, some underlying mechanisms involved in this cytotoxicity were also investigated. Among the 21 extracts tested, the MTT assay after 72 h of incubation demonstrated that only the ethanolic extract obtained from Myracrodruon urundeuva seeds (EEMUS), which has steroids, alkaloids and phenols, showed in vitro cytotoxic activity against human cancer cells, being 2-fold more active on leukemia HL-60 line [IC(50) value of 12.5 (9.5-16.7) μg/mL] than on glioblastoma SF-295 [IC(50) of 25.1 (17.3-36.3) μg/mL] and Sarcoma 180 cells [IC(50) of 38.1 (33.5-43.4) μg/mL]. After 72h exposure, flow cytometric and morphological analyses of HL-60-treated cells showed that EEMUS caused decrease in cell number, volume and viability as well as internucleosomal DNA fragmentation in a dose-dependent way, suggesting that the EEMUS triggers apoptotic pathways of cell death.