Effects of a chitin-binding vicilin from Enterolobium contortisiliquum seeds on bean bruchid pests (Callosobruchus maculatus and Zabrotes subfasciatus) and phytopathogenic fungi (Fusarium solani and Colletrichum lindemuntianum)

Chitin-binding vicilin from Enterolobium contortisiliquum seeds was purified by ammonium sulfate followed by gel filtration on Sephacryl 300-SH and on Sephacryl 200-SH. The vicilin, called EcV, is a dimeric glycoprotein composed of 1.03% carbohydrates and a Mr of 151 kDa, consisting of two subunits of Mr of 66.2 and 63.8 kDa. The EcV homogeneity was confirmed in a PAGE where it was observed to be a unique acid protein band with slow mobility in this native gel. E. contortisiliquum vicilin (EcV) was tested for anti-insect activity against C. maculatus and Zabrotes subfasciatus larvae and for phytopathogenic fungi, F. solani and C. lindemuntianum. EcV was very effective against both bruchids, producing 50% mortality for Z. subfasciatus at an LD50 of 0.43% and affected 50% of the larvae mass with an ED50 of 0.65%. In artificial diets given to C. maculatus, 50% of the larvae mass was affected with an ED50 of 1.03%, and larva mortality was 50% at LD50 of 1.11%. EcV was not digested by midgut homogenates of C. maculatus and Z. Subfasciatus until 12 h of incubation, and at 24 h EcV was more resistant to Z. subfasciatus larval proteases. The binding to chitin present in larvae gut associated to low EcV digestibility could explain its lethal effects. EcV also exerted an inhibitory effect on the germination of F. solani at concentrations of 10 and 20 microg mL-1. The effect of EcV on fungi is possibly due to binding to chitin-containing structures of the fungal cell wall.     

In vivo bioinsecticidal activity toward Ceratitis capitata (fruit fly) and Callosobruchus maculatus (cowpea weevil) and in vitro bioinsecticidal activity toward different orders of insect pests of a trypsin inhibitor purified from tamarind tree (Tamarindus indica) seeds

A proteinaceous inhibitor with high activity against trypsin-like serine proteinases was purified from seeds of the tamarind tree (Tamarindus indica) by gel filtration on Shephacryl S-200 followed by a reverse-phase HPLC Vidac C18 TP. The inhibitor, called the tamarind trypsin inhibitor (TTI), showed a Mr of 21.42 kDa by mass spectrometry analysis. TTI was a noncompetitive inhibitor with a Ki value of 1.7 x 10(-9) M. In vitro bioinsecticidal activity against insect digestive enzymes from different orders showed that TTI had remarkable activity against enzymes from coleopteran, Anthonomus grandis (29.6%), Zabrotes subfasciatus (51.6%), Callosobruchus maculatus (86.7%), Rhyzopertha dominica(88.2%), and lepidopteron, Plodia interpuncptella (26.7%), Alabama argillacea (53.8%), and Spodoptera frugiperda (75.5%). Also, digestive enzymes from Diptera, Ceratitis capitata (fruit fly), were inhibited (52.9%). In vivo bioinsecticidal assays toward C. capitata and C. maculatus larvae were developed. The concentration of TTI (w/w) in the artificial seed necessary to cause 50% mortality (LD50) of larvae was 3.6%, and that to reduce mass larvae by 50.0% (ED50) was 3.2%. Furthermore, the mass C. capitata larvae were affected at 53.2% and produced approximately 34% mortality at a level of 4.0% (w/w) of TTI incorporated in artificial diets.     

Insecticidal and antifungal activity of a protein from Pouteria torta seeds with lectin-like properties

This paper describes the purification and characterization of a novel protein from the seeds of Pouteria torta (family Sapotaceae). The protein was purified by a combination of gel filtration, ion-exchange, and reverse phase chromatographies. SDS-PAGE of the purified protein resulted in a single protein band of 14 kDa in the presence and absence of DTT. The lectin-like activity of pouterin was best inhibited by glycoproteins such as fetuin, asialofetuin, heparin, orosomucoid, and ovoalbumin. Pouterin inhibited the growth of the fungi Fusarium oxysporum and Colletotrichum musae and of the yeast Saccharomyces cerevisiae. The incorporation of pouterin into an artificial diet (final concentration = 0.12%, w/w) caused 50% mortality in larvae of the insect Callosobruchus maculatus, whereas 0.08% pouterin produced an ED50.     

A Kunitz-type inhibitor of coleopteran proteases, isolated from Adenanthera pavonina L. seeds and its effect on Callosobruchus maculatus

The cowpea weevil Callosobruchus maculatus is one of the major pests of Vigna unguiculata cowpea. Digestion in the cowpea weevil is facilitated by high levels of cysteine and aspartic acid proteinases. Plants synthesize a variety of molecules, including proteinaceous proteinase inhibitors, to defend themselves against attack by insects. In this work, a trypsin inhibitor (ApTI) isolated from Adenanthera pavonina seeds showed activity against papain. The inhibition of papain by ApTI was of the noncompetitive type, with a K(i) of 1 microM. ApTI was highly effective against digestive proteinases from C. maculatus, Acanthoscelides obtectus (bean weevil), and Zabrotes subfasciatus (Mexican bean weevil) and was moderately active against midgut proteinases from the boll weevil Anthonomus grandis and the mealworm Tenebrio molitor. In C. maculates fed an artificial diet containing 0.25% and 0.5% ApTI (w/w), the latter concentration caused 50% mortality and reduced larval weight gain by approximately 40%. The action of ApTI on C. maculatus larvae may involve the inhibition of ApTI-sensitive cysteine proteinases and binding to chitin components of the peritrophic membrane (or equivalent structures) in the weevil midgut.     

Proteolytic digestive enzymes and peritrophic membranes during the development of Plodia interpunctella (Lepidoptera: Piralidae): targets for the action of soybean trypsin inhibitor (SBTI) and chitin-binding vicilin (EvV)

The digestive system of P. interpunctella was characterized during its larval development to determine possible targets for the action of proteinaceous enzyme inhibitors and chitin-binding proteins. High proteolytic activities using azocasein at pH 9.5 as substrate were found. These specific enzymatic activities (AU/mg protein) showed an increase in the homogenate of third instar larvae, and when analyzed by individual larvae (AU/gut), the increase was in sixth instar larvae. Zymograms showed two bands corresponding to those enzymatic activities, which were inhibited by TLCK and SBTI, indicating that the larvae mainly used serine proteinases at pH 9.5 in their digestive process. The presence of a peritrophic membrane in the larvae was confirmed by chemical testing and light microscopy. In a bioassay, P. interpunctella was not susceptible to the soybean trypsin inhibitor, which did not affect larval mass and mortality, likely due to the weak association with its target digestive enzyme. EvV (Erythrina velutina vicilin), when added to the diet, affected mortality (LD50 0.23%) and larval mass (ED50 0.27%). This effect was associated with EvV-binding to the peritrophic membrane, as seen by immunolocalization. EvV was susceptible to gut enzymes and after the digestion process, released an immunoreactive fragment that was bound to the peritrophic matrix, which probably was responsible for the action of EvV.     

Identification of an alpha-amylase inhibitor from Pterodon pubescens with ability to inhibit cowpea weevil digestive enzymes

Cowpea seeds (Vigna ungiculata) are widely cultivated by poor farmers in Latin America and Africa and are often severely damaged by the cowpea weevil Callosobruchus maculatus. A proteinaceous inhibitor of cowpea weevil digestive enzymes, PpAI, was purified from white sucupira seeds (Pterodon pubescens) and biochemically characterized in this study. Proteins were extracted from seeds and precipitated with ammonium sulfate at 100% saturation. This fraction was applied onto a Red-sepharose CL-6B column, and the retained peak showed 70% inhibitory activity toward larval C. maculatus digestive alpha-amylases. The retained peak was then purified using an analytical reversed-phase HPLC column. Purified PpAI showed 65% inhibitory activity against larval C. maculatus enzymes. Enzymatic assays also showed that the purified P. pubescens inhibitor was unable to reduce the activity of mammalian alpha-amylases, suggesting specificity toward insect enzymes. Moreover, artificial seeds containing PpAI were able to reduce larval weight by 36% and cause 55% mortality. Mass spectrometry and SDS-PAGE analyses indicated that PpAI showed a molecular mass of approximately 5.0 kDa. This alpha-amylase inhibitor, coming from a native Cerrado plant, could be used to construct a genetically engineered cowpea with enhanced resistance against weevil pests.     

Insecticidal activity of Arum maculatum tuber lectin and its binding to the glycosylated insect gut receptors

Mannose binding approximately 50 kDa homotetrameric lectin, purified from edible Arum maculatum tuber, was analyzed through SDS-PAGE and studied for its agglutination property using rabbit erythrocytes. Cross reactivity of the purified lectin was verified through western blot using Colocasia esculantum(Family, Araceae) tuber lectin antibody. The insecticidal activity of Arum maculatum tuber lectin (ATL) was tested against two economically important sucking pests, Lipaphis erysimi and Aphis craccivora, in an artificial diet. The LC(50) values for L. erysimi and A. craccivora were determined to be 21 microg/mL and 16 microg/mL, respectively. Addition of alpha-d-mannose in ATL-supplemented diet reduced the aphid mortality. Two major receptor proteins of ATL (approximately 40 kDa and approximately 35 kDa) were detected from the brush border membrane vesicle (BBMV) protein of L. erysimi and A. craccivora guts, respectively, using ligand-binding assay. Alpha-d-Mannose was found to be a deterrent to such binding of ATL to the BBMV receptors.     

Purification and characterization of a trypsin inhibitor from Plathymenia foliolosa seeds

A novel trypsin inhibitor (PFTI) was isolated from Plathymenia foliolosa (Benth.) seeds by gel filtration chromatography on a Sephadex G-100, DEAE-Sepharose, and trypsin-Sepharose columns. By SDSPAGE, PFTI yielded a single band with a M(r) of 19 kDa. PFTI inhibited bovine trypsin and bovine chymotrypsin with equilibrium dissociation constants (K(i)) of 4 x 10(-8) and 1.4 x 10(-6) M, respectively. PFTI retained more than 50% of activity at up to 50 degrees C for 30 min, but there were 80 and 100% losses of activity at 60 and 70 degrees C, respectively. DTT affected the activity or stability of PFTI. The N-terminal amino acid sequence of PFTI showed a high degree of homology with various members of the Kunitz family of inhibitors. Anagasta kuehniella is found worldwide; this insect attacks stored grains and products of rice, oat, rye, corn, and wheat. The velvet bean caterpillar (Anticarsia gemmatalis) is considered the main defoliator pest of soybean in Brazil. Diatraea saccharalis, the sugar cane borer, is the major pest of sugar cane crops, and its caterpillar-feeding behavior, inside the stems, hampers control. PFTI showed significant inhibitory activity against trypsin-like proteases present in the larval midguts on A. kuehniella and D. saccharalis and could suppress the growth of larvae.     

selective production and characterization of levan by Bacillus subtilis (Natto) Takahashi

To meet the industrial need of an efficient microbial method for increased levan production, Bacillus subtilis (natto) Takahashi, a commercial natto starter for preparing fermented soybeans (natto), was used to produce levan. After cultivation for 21 h, 40-50 mg of levan mL(-1) was produced in medium containing 20% (w/w) sucrose, which was approximately 50% yield on available fructose. The product consisted of two fractions with different molecular masses (1794 and 11 kDa), which were easily separated by fractionation using an ethanol gradient. The products were well characterized by GPC, 13C NMR, and 1H NMR. The various sugars and concentrations, initial pH, fermentation temperature, and agitation speed affected the levan production by B. subtilis (natto) Takahashi. Takahashi strain is the most efficient levan-producing strain among all of the B. subtilis strains tested and, as previously reported, it produced the highest yield of levan in the least time (21 h) under the common cultivation condition.     

Lutein and zeaxanthin in leafy greens and their bioavailability: olive oil influences the absorption of dietary lutein and its accumulation in adult rats

This study determined the lutein level in various green leafy vegetables (GLVs) and the influence of olive and sunflower oils on the postprandial plasma and eye response of dietary lutein in adult rats, previously induced with lutein depletion (LD). Fresh GLVs (n = 35) were assessed for lutein (L) and its isomer zeaxanthin (Z) levels by high-performance liquid chromatography and liquid chromatography-mass spectrometry. Among GLVs analyzed, Commelina benghalensis L. contained a higher level of L + Z (183 mg/100 g dry wt) and was used as a lutein source for feeding studies. Rats with LD were fed a diet containing powdered C. benghalensis (2.69 mg lutein/kg diet) with either olive oil (OO group), sunflower oil (SFO group), or groundnut oil (GNO group) for 16 days. The L + Z levels of the OO group were markedly (p > 0.05) higher than those of SFO and GNO groups, in plasma (37.6 and 40.9%) and eyes (22.7 and 30.8%), respectively. These results suggest that oleic acid or OO can be used as a suitable fat source to modulate the absorption of dietary lutein to manage age-related macular degeneration.     

Efficiency of mannose-binding plant lectins in controlling a homopteran insect,the red cotton bug

Yield losses of different crops due to the attack of various classes of insects are a worldwide problem. Sucking type homopteran pests causing damage to many crop species are not controlled by commonly known insecticidal proteins, namely, Bacillus thuringiensis delta-endotoxin (Bt). This study describes the purification of mannose-binding lectins from three different monocotyledonous plants (Allium sativum, Colocasia esculenta, and Diffenbachia sequina) and their effects on a homopteran insect, the red cotton bug. All of them had a detrimental effect on the growth and development of the insect, where A. sativum bulb lectin showed the highest mortality of all, in particular. The same bulb lectin not only affected the growth and fecundity of the insect but also imparted drastic changes in the color, weight, and size, even on the second generation of the insects which have been reared on artificial diet supplemented with a sublethal dose of the lectin. Thus, this finding opens up a possibility of using this lectin as an important component in crop management.     

Influence of feeding soybean oil on conjugated linoleic acid content in beef

Forty-eight steers were used to study the influence of feeding soybean oil (SO) on the conjugated linoleic acid (CLA) content of beef. Steers were fed either a control diet containing 954 g/kg of dry matter (DM) corn-based concentrate (CTL) or a control diet supplemented with SO at 20 (SO2) or 40 g/kg (SO4) of diet DM for 105 days. Adipose tissue samples were collected from the M. longissimus dorsi (LD) and from the M. semitendinosus (ST) on days 0 and 63 of the experiment. Adipose and muscle tissue samples were collected from the LD and ST immediately after slaughter. Feeding 40 g/kg of DM as SO increased the proportions of trans-C(18:1) in beef lipid as compared to CTL and SO2 treatments. The C(18:2) cis-9, trans-11 isomer of CLA as a proportion of total fat was not different in adipose and muscle across treatments. Supplementing SO increased C(18:2) trans-10, cis-12 CLA in adipose tissue of the LD. Supplementing high-grain finishing diets with SO is not an effective strategy to enhance the C(18:2) cis-9, trans-11 isomer of CLA in beef.     

Procyanidin-rich extract from grape seeds prevents cataract formation in hereditary cataractous (ICR/f) rats

Antioxidants such as vitamin C, vitamin E, and carotenoids have been reported to prevent the progression of experimentally induced cataracts. However, little is known of the effect of procyanidins, a powerful antioxidant, on cataract formation. This paper investigates the anticataract activity of grape seed extract (GSE, which contains 38.5% procyanidins) in hereditary cataractous rats (ICR/f rats). The ICR/f rats were fed a standard diet containing 0 or 0.213% GSE [0.082% procyanidins in the diet (w/w)] for 27 days. The GSE significantly prevented and postponed development of cataract formation by evaluation of slit lamp observations of the rats' eyes. Lens weight and malondialdehyde concentration in the lens and plasma cholesteryl ester hydroperoxide (ChE-OOH) level induced by CuSO4 were significantly lower in the GSE group compared with the control group. The rats were also fed for 14 days either the diet containing 0.085% procyanidin dimer to tetramer fraction (0.085% as the procyanidins), the diet containing 0.090% procyanidin pentamer to heptamer fraction (0.085% as the procyanidins), or the diet containing 0.093% procyanidin oligomers more than decamer fraction (0.085% as the procyanidins). The ChE-OOH levels in the procyanidin pentamer to heptamer and procyanidin oligomers more than decamer groups were significantly lower than in the procyanidin dimer to tetramer group. These results suggested that procyanidins and their antioxidative metabolites prevented the progression of cataract formation by their antioxidative action. The larger molecular procyanidins in the GSE might contribute this anticataract activity.     

Synthesis and biological evaluation of new analogues of the active fungal metabolites N-(2-methyl-3-oxodecanoyl)-2-pyrroline and N-(2-methyl-3-oxodec-8-enoyl)-2-pyrroline.

To evaluate the effect of simplifying the beta-ketoamide system present in active isolated metabolites from Penicillium brevicompactum (2 and 3) on the activity, new analogues with a monocarbonylic amide functionality have been obtained. In this way, the insecticidal and fungicidal activities have been improved in relation to the natural products taken as lead molecules. Thus, two of the synthetic analogues (5a and 5b) showed very important insecticidal activities against third-instar nymphs of Oncopeltus fasciatus Dallas, with acute LD(50) values of 3.0 and 1.5 microg/cm(2), respectively. Moreover, some analogues showed good levels of fungicidal activity against a wide range of commercially important and taxonomically diverse fungi; remarkably, compound 7c has proved to be highly active against Colletotrichum gloesporoides and Colletotrichum coccodes, with ED(50) values of 2.04 and 11.7 microg/mL, respectively.     

Commercial Phaseolus vulgaris extract (starch stopper) increases ileal endogenous amino acid and crude protein losses in the growing rat

The effect of a commercial Phaseolus vulgaris extract (PVE, starch stopper) on ileal and fecal endogenous protein losses was studied. Growing rats were fed for 14 days a protein-free diet containing PVE at a nutritional concentration of 0% (PF1), 0.4% (PF2), or 1.1% PVE (PF3) or 1.1% autoclaved PVE (PF4). An indigestible marker (TiO(2)) was included in each diet. Ileal endogenous amino acid (AA) losses were significantly higher (P < 0.05) in PF3 (20% higher than in PF1), except for Pro, Gly, Ala, and His. Endogenous ileal N losses were 22% higher in PF3 than in PF1. Endogenous fecal AA and N losses were all significantly higher (P < 0.05) in PF3. Starch digestibility ( approximately 100%), food intake (single daily meal, d10-23), and body weight loss were not significantly different among the groups. PVE, at 1.1% of the diet, not only was ineffective in reducing starch digestibility but also led to increased ileal endogenous N losses, possibly due to the antinutritional factors (trypsin inhibitor, lectin) present in the PVE.     

A semi-pilot-scale procedure for isolating and purifying soybean (Glycine max) lectin

Availability of gram quantities of purified soybean lectin (SBL) to scientists will foster discovery of novel biomedical applications of the lectin and provide the opportunity to investigate the antinutritional effects of SBL in soybean-consuming food animals and poultry. Therefore, a semi-pilot-scale procedure for isolating and purifying SBL was designed. Defatted soyflour was extracted overnight with 0.9% NaCl at 4 degrees C. The extract obtained was filtered (0.45 microm membrane) and subjected to affinity chromatography using a column containing N-acetyl-D-galactosamine resin that is specific for SBL. Bound SBL was eluted off the column with 0.14 M galactose solution. The eluent was ultrafiltered (30 kDa), and the resulting solution (SBL and water) was freeze-dried. Electrophoretic analysis and hemagglutination assay revealed that the freeze-dried SBL was similar to Sigma-grade SBL in purity and activity (35 and 33 HU/mg protein, respectively). The procedure yielded 141 mg of SBL/100 g of soyflour.     

Synthesis, identification, and acute toxicity of some N-alkyl derivatives of 3,4-methylenedioxyamphetamine

A series of N-alkyl derivatives of 3,4-methylenedioxyamphetamine (MDA) was prepared in an effort to characterize these potential drugs of abuse. These secondary amines were synthesized via reductive amination of the corresponding ketone with alkylamines. The ultraviolet absorption spectra for these compounds produced almost equally intense absorbance at 234 and 285 nm. The compounds were separated by liquid chromatography using reverse phase (C18) procedures with a ternary mobile phase mixture. Toxicity studies showed all of the compounds to have LD50 values similar to N-methyl MDA (MDMA).     

Usage of Herbal (Thyme and Chicory) Waste as an Organic Substrate in Cucumber Production

This experiment was conducted in a greenhouse to study the effect of herbal waste substrate as local substrate in cucumber (Cucumis sativus cv. Zomorod) production. Treatments included thyme: perlite (T50:PR50); thyme: perlite (T25:PR75); thyme, perlite: peat moss (T25:PR25:P50); chicory: perlite (C50:PR50); chicory: perlite (C25:PR75); chicory: perlite: peat moss (C25:PR25:P50), and perlite (100) as control. The experiment was conducted based on a completely randomized design with 5 replications. The results showed that fruit number and fruit weight were more (about 54% and 65%, respectively) in C25:PR25:P50 treatment than that in the control. Also, the highest leaves number, shoot length, shoot fresh and dry weights, and root fresh and dry weights were observed in this treatment (C25:PR25:P50). The highest K, Ca, Mg, Mn, and Fe contents were obtained in C50:PR50 treatment. Zn and Cu significantly increased in C25:PR25:P50 treatment. In general, substrate containing chicory was more effective than substrate containing thyme. As comparison among all the substrates, substrate containing chicory, C25:PR25:P50 had a significant effect on growth and yield, and C50:PR50 was more effective than the other substrate on nutrient element concentrations.     

Amelioration of obesity and glucose intolerance in high-fat-fed C57BL/6 mice by anthocyanins and ursolic acid in Cornelian cherry (Cornus mas)

Much attention has been focused on food that may be beneficial in preventing diet-induced body fat accumulation and possibly reduce the risk of diabetes and heart disease. Cornelian cherries (Cornus mas) are used in the preparation of beverages in Europe and also to treat diabetes-related disorders in Asia. In this study, the most abundant bioactive compounds in C. mas fruits, the anthocyanins and ursolic acid, were purified, and their ability to ameliorate obesity and insulin resistance in C57BL/6 mice fed a high-fat diet was evaluated. Mice were initially fed a high-fat diet for 4 weeks and then switched to a high-fat diet containing anthocyanins (1 g/kg of high-fat diet) and ursolic acid (500 mg/kg of high-fat diet) for an additional 8 weeks. The high-fat diet induced glucose intolerance, and this was prevented by anthocyanins and ursolic acid. The anthocyanin-treated mice showed a 24% decrease in weight gain. These mice also showed decreased lipid accumulation in the liver, including a significant decrease in liver triacylglycerol concentration. Anthocyanin and ursolic acid treated mice exhibited extremely elevated insulin levels. Both treatments, however, showed preserved islet architecture and insulin staining. Overall, these data suggest that anthocyanins and ursolic acid purified from C. mas fruits have biological activities that improve certain metabolic parameters associated with diets high in saturated fats and obesity.