Isolation and identification of root-inhibiting compounds from corn gluten hydrolysate

Interest has centered on the use of plantderived compounds as natural herbicides, and they are considered to represent an environmentally sound approach to weed control. Corn gluten hydrolysate, found to have a growth-regulating effect on the root system of germinating grass seeds, has been suggested as a natural herbicide. A protocol was developed to extract, isolate, and identify the root-inhibiting compounds from corn gluten hydrolysate aqueous solution and a perennial ryegrass (Lolium perenne L.). A Petri dish bioassay was used to test the root-inhibiting activity. Five bioactive dipeptides were isolated by using Sephadex G-15 gel filtration, solid-phase extraction, and C18 reversed-phase high-performance liquid chromatography procedures. The five dipeptides were glutaminyl-glutamine, alaninyl-asparagine, alaninyl-glutamine, glycinyl-alanine, and alaninyl-alanine. Their root-inhibiting activity on perennial ryegrass was demonstrated in Petri dish bioassays.Journal Paper No. J-15597 of Iowa Agriculture and Home Economics Experiment Station, Ames, IA Project No. 3149     

Herbicidal activity of hydrolyzed corn gluten meal on three grass species under controlled environments

US Patent No. 5,030,268 discloses that corn gluten meal, the protein fraction of corn (Zea mays L.) grain, can be used as a natural preemergence herbicide. However, corn gluten meal is insoluble in water, and this characteristic renders it difficult to apply as a herbicide. To seek a watersoluble material with more potent herbicidal activity, the phytotoxicity of various samples derived from corn gluten meal and other related crop materials were evaluated by using three different grass species under controlled environments. Greenhouse and growth chamber bioassays showed that the sample of enzymatically hydrolyzed corn gluten meal was more herbicidally active than the corn gluten itself and was highly water soluble. Gluten hydrolysate prepared with bacterial source proteinase had the greatest inhibitory activity to the root growth of germinating seeds. This water-soluble material derived from corn gluten meal had a growth-regulating effect on the root system and can be used as a natural herbicide.Journal Paper No. J-15609 of Iowa Agriculture and Home Economics Experiment Station, Ames, IA Project No. 3149.     

Production of hydrolysate with antioxidative activity by enzymatic hydrolysis of extruded corn gluten

Hydrolysate of extruded corn gluten with higher solubility and antioxidative property was prepared. Extrusion and starch removal of corn gluten were applied as pretreatment before enzymatic hydrolysis by Alcalase. The amylase hydrolysis of starch at 70°C for 3 h resulted in the removal of the starch from the extruded corn gluten. The best hydrolysis results can be obtained by conducting the hydrolysis at 60°C with water addition 20 g/g protein, enzyme addition 0.048 Ansen units/g protein, pH 8.5, and 120 min. Degree of hydrolysis of extruded and nonextruded corn gluten reached 39.54 and 31.16%, respectively, under the optimal condition. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the optimal hydrolysate revealed that proteolysis of extruded corn gluten was more extensive than proteolysis of its counterpart which was not subjected to extrusion. The molecular weight of the peptides in the optimal hydrolysate was mainly over 3,710–660 Da as determined by gel filtration chromatography. The hydrolysates displayed good solubility and antioxidative activity. The separation profile of the hydrolysate on an ion exchange chromatography of Q-Sepharose Fast Flow showed that many kinds of peptides had antioxidative effect. A new peptide with antioxidative activity was purified, and its amino acid sequence was Phe-Pro-Leu-Glu-Met-Met-Pro-Phe, which was identified by Q-TOF2 mass spectrometry.     

Identification of feeding stimulants in corn roots for western corn rootworm (Coleoptera: Chrysomelidae) larvae

Using a bioassay-driven approach, we have isolated and identified a blend of compounds from the roots of germinating corn, Zea mays L., that serve as feeding stimulants for neonate western corn rootworm larvae, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae). The active blend is a combination of simple sugars (30:4:4 mg/ml glucose:fructose:sucrose in the corn root) plus at least one of the free fatty acids in germinating corn roots (2:5 mg/ml oleic acid:linoleic acid in the corn root). When an extract of germinating corn was partitioned into an ethyl acetate fraction and an aqueous fraction, full feeding occurred only when the two fractions were recombined, indicating that the phagostimulant was comprised of both polar and nonpolar components. Gas chromatography-mass spectrometry analysis of root extracts from germinating corn seedlings revealed a blend of 20 compounds from a variety of chemical classes, including small sugars, diacids, amino acids, inorganic compounds, and free fatty acids. When the major components were tested in feeding bioassays, the sugars and lipids were shown to be essential for feeding by larvae, but the two classes of compounds were only effective when combined. The sugars alone elicited feeding by only 40% of larvae, but the percentage of larvae feeding was increased significantly with the addition of linoleic acid (91.7% larvae feeding) or oleic acid (85.8% larvae feeding). The amino acids alone were not essential elements for feeding by western corn rootworm larvae.     

Observations on root hair production by lucerne,maize and perennial ryegrass grown in a sandy loam

Summary Root hair production by young plants of lucerne, maize and perennial ryegrass grown in a sandy loam was assessed by examining roots growing at a soil-glass interface. Results are given for the percentage frequency distribution of root hair lengths and the numbers of root hairs produced per mm root. The mean lengths of root hairs observed on lucerne, maize and perennial ryegrass roots were 0.35, 0.90 and 1.12 mm respectively. Lucerne produced an average of 105 root hairs per mm of root, whereas maize produced 161 and perennial ryegrass produced 88. The total length of root hairs per mm length of root was estimated to be 37, 146 and 99 mm for lucerne, maize and perennial ryegrass resp. Letcombe Laboratory     

Corn Gluten Hydrolysate Affects the Time-Course of Metabolic Changes Through Appetite Control in High-Fat Diet-Induced Obese Rats

This study first investigated the effects of corn gluten hydrolysate (CGH) (1.5 g/day) administration for 7 days on appetite-responsive genes in lean Sprague-Dawley (SD) rats. In a second set of experiments, the metabolic changes occurring at multiple time points over 8 weeks in response to CGH (35.33% wt/wt) were observed in high-fat (HF, 60% of energy as fat) diet-fed SD rats. In lean rats, the hypothalamus neuropeptide-Y and proopiomelanocortin mRNA levels of the CGH group were significantly changed in response to CGH administration. In the second part of the study, CGH treatment was found to reduce body weight and perirenal and epididymal fat weight. CGH also prevented an increase in food intake at 2 weeks and lowered plasma leptin and insulin levels in comparison with the HF group. This reduction in the plasma and hepatic lipid levels was followed by improved insulin resistance, and the beneficial metabolic effects of CGH were also partly related to increases in plasma adiponectin levels. The Homeostasis Model of Assessment - Insulin Resistance (HOMA-IR), an index of insulin resistance, was markedly improved in the HF-CGH group compared with the HF group at 6 weeks. According to the microarray results, adipose tissue mRNA expression related to G-protein coupled receptor protein signaling pathway and sensory perception was significantly improved after 8 weeks of CGH administration. In conclusion, the present findings suggest that dietary CGH may be effective for improving hyperglycemia, dyslipidemia and insulin resistance in diet-induced obese rats as well as appetite control in lean rats.     

Isolation and identification of potential cancer chemopreventive agents from methanolic extracts of green onion (Allium cepa)

Phase II xenobiotic metabolizing enzymes confer amelioration of risk arising from potentially carcinogenic chemicals derived both endogenously, and exogenously, from food and the environment. In this study, efforts were made to isolate and identify potentially cancer preventive constituents from methanolic extracts of green onion (Allium cepa) directed by the quinone reductase (QR) induction bioassay using murine hepatoma (Hepa 1c1c7) cells. Crude methanolic extracts of green onion tissue were solvent-partitioned, and subsequently fractionated by flash chromatography, thin layer chromatography and high pressure preparative liquid chromatography to afford pure QR-inducing isolates. Multiple isolates were found active at inducing QR. One newly identified compound, 5-hydroxy-3-methyl-4-propylsulfanyl-5H-furan-2-one (3), and four known compounds: 5-(hydroxymethyl) furfural (1), acetovanillone (2), methyl 4-hydroxyl cinnamate (4) and ferulic acid methyl ester (5), were isolated and identified as active agents.     

Preparation of corn gluten hydrolysate with angiotensin I converting enzyme inhibitory activity and its solubility and moisture sorption

This study attempted to prepare a hydrolysate of corn gluten containing powerful ACE inhibitory activity and higher solubility, which could be used as a physiologically functional food material. The digestion of starch at 80 °C resulted in more removal of the starch from the corn gluten than that at 60 °C. More than 95% reducing sugars as enzymic digests of starch was removed by washing three-times. As for the thermal treatment effect on the increase of degree of hydrolysis (DH), degree of increase of protein content (slope) was 6.30 mg/ml min at 100 °C, 4.40 mg/ml min at 80 °C and 2.29 mg/ml min without heat treatment. After 45 min of heat treatment the increased ratio of protein content was greater than those by other heat treatments. After the pretreatment of corn gluten, hydrolysis with Flavourzyme, among six commercial proteases, resulted in the production of the hydrolysate with the highest ACE inhibitory activity, and with Protamax, the second highest ACE inhibitory activity was obtained. Flavourzyme hydrolysate of corn gluten at the 4% level was easily and completely soluble between pH 2 and 9. Water sorption of the hydrolysate slowly increased up to 0.8 of water activity and greatly increased at higher than 0.8 of water activity.     

IDENTIFICATION OF THE CYTOKININ ISOPENTENYLADENINE IN A STRAIN OF ARTHRONEMA AFRICANUM (CYANOBACTERIA)

Isopentenyladenine was isolated from a strain of the cyanobacterium Arthronema africanum (Schwabe et Simons) Komárek et Lukavský using a combination of biological and chemophysical techniques, including the soybean callus bioassay, cation exchange resin chromatography, paper chromatography, HPLC, and GC-MS. Positive identification of the major biologically active compound was achieved.     

Chickpea wilt: identification and toxicity of 8-O-methyl-fusarubin from Fusarium acutatum

Fusarium acutatum was isolated from wilting chickpea plants in Pakistan. Filtrates from cultures grown on a defined liquid medium caused permanent wilting of chickpea cuttings and killed cells, isolated enzymically from healthy plants, in a bioassay. Toxic activity was retained by a cyano solid phase extraction cartridge and the toxin was isolated by elution from the cartridge in acetonitrile and Si-gel thin layer chromatography of the eluate. Analytical HPLC of the compound on a cyano column with diode array detection gave a single peak with a homogeneous spectrum and lambda(max) at 224 and 281 nm. NMR and mass spectral studies showed that the toxin was 8-O-methyl-fusarubin. The pure compound caused permanent wilting of chickpea cuttings and the LD50 value in the cell bioassay was 327 ng/ml.     

Water Use Characteristics of Dactylis glomerata L., Lolium perenne L. and L. multiflorum Lam. Plants

Cocksfoot (Dactylis glomerata L.). perennial ryegrass (Loliumperenne L.) and Italian ryegrass (L. multiflorum Lam.) plantswere grown on deep (75–95 cm) columns of soil in glasshousesand growth rooms with and without irrigation. The species inwhich growth declined least rapidly after water had been withheldwere those which transpired most slowly. During early establishmentin the glasshouse cocksfoot transpired least because of slowroot growth. In the growth room, when root systems were deeperand denser, perennial ryegrass transpired least because of lowleaf water conductance. Results are discussed in relation to(a) drought resistance in the three species; (b) breeding forincreased drought resistance through modifying root distributionand leaf water conductance; and (c) the use of isolated soilcolumns in water relations studies. Dactylis glomerata L., Lolium perenne L., Lolium multiflorum Lam., cocksfoot, perennial ryegrass, Italian ryegrass, transpiration, roots, leaf water conductance     

Dihydroconiferyl alcohol — A cell division factor from Acer species

A compound that stimulated growth of soybean callus was isolated from spring sap of sycamore (Acer pseudoplatanus L.). Insufficient compound was isolated to permit it to be characterised. A compound with identical properties was isolated from commercial maple syrup, the concentrated spring sap of Acer saccharum L. The compound was identified as 3-(3-methoxy-4-hydroxyphenyl)-propan-1-ol (dihydroconiferyl alcohol, DCA). DCA was also active in the tobacco callus and radish leaf senescence assays, but was inactive in four other tests for cytokinin activity. DCA acted synergistically with kinetin to promote soybean callus growth. It is concluded that DCA has properties distinct from those of purine cytokinins.Abbreviation DCA dihydroconiferyl acohol - GC gas chromatography - IAA indole-3-acetic acid - iP isopentenyladenine - [9R]iP isopentenyladenosine - LC liquid chromatography - MS mass spectrometry - NAA 1-napthylacetic acid - TLC thinlayer chromatography - TMSi trimethylsilyl     

大金发藓和小蛇苔化学他感作用的生物测定

以绿豆、油菜和萝卜种子为生测材料,以大金发藓（Polytrichum commune Hedw.）和小蛇苔(Conocephalum japonicum(Thunb.) Grolle.)的水提取液为处理液,测定了二种苔藓植物提取液在三种浓度下对三种作物种子萌发率、萌发指数、萌发势、种子活力、幼苗根重和鲜重和根长等指标的影响。发现绿豆是理想的生测材料,种子活力指数、幼苗鲜重、根重和根长是理想的生测指标;二种苔藓植物的水提取液表现出类似于激素样的作用方式,对种子萌发和幼苗生长,表现为低浓度下促进、高浓度时抑制的化学他感效应。     

Purification and identification of adipogenesis inhibitory peptide from black soybean protein hydrolysate

Adipogenesis inhibitory peptide was isolated and identified from black soybean (Rhynchosia volubilis Lour.) hydrolysate. An adipogenesis inhibitor was purified using consecutive methods including: ultrafiltration (MWCO; 3 and 10kDa), gel filtration chromatography (Superdex Peptide 10/300 GL column), and reverse-phase high-performance liquid chromatography (microBondapak C(18) column). Also, the adipogenesis inhibition effect of the purified peptide was measured by observation of droplet of 3T3-L1 adipocyte by Oil Red O staining in the highest active fraction in each step. The peptide was shown to inhibit the differentiation of the 3T3-L1 pre-adipocyte, which was confirmed by morphological study. The adipogenesis inhibitory peptide was purified 71.43-fold from black soybean hydrolysate throughout a five-step purification procedure. The adipogenesis inhibitor was identified to be a tripeptide, Ile-Gln-Asn, having an IC(50) value of 0.014 mg protein/ml. Furthermore, the synthetic tripeptide (Ile-Gln-Asn) exhibited the similar adipogenesis effects to the purified peptide. Thus, these results showed the potential anti-obesity effect of the purified peptide through control of adiposity.     

The primary structure identification of a corn peptide facilitating alcohol metabolism by HPLC-MS/MS

The aim of this study is to identify the primary structure of corn peptides (CPs) with a facilitating alcohol metabolism effect. Corn protein was hydrolyzed by Alcalase first. The hydrolysate, crude corn peptides (CPs), was then fractionated through ultrafiltration technology. The primary structure of a peptide from the fraction (Mm<5kDa) was identified by HPLC-MS/MS, coupled with the peptide sequence retrieval using the MS-MS online database. The amino acid sequence of the peptide was determined as Q-L-L-P-F, and the pentapeptide was synthesized by Fmoc solid-phase peptide synthesis (SPPS) method. Its ability to facilitate alcohol metabolism was evaluated in vivo. Results showed that the synthetic peptide (10mg/kg) had a higher ability to eliminate alcohol in vivo compared to the mixed peptides (Mm<5kDa, 200mg/kg). In conclusion, the pentapeptide Q-L-L-P-F has a potent ability in facilitating alcohol metabolism, and this pentapeptide is the main bioactive component in the mixed peptides obtained from corn.     

Isolation of Zeanic Acid,a Natural Plant Growth-regulator from Corn Steep Liquor and Its Chemical Structure

Corn steep liquor markedly inhibited the growth of the grass, alfalfa, as previously reported. The active principles from corn steep liquor were isolated. From the acidic fraction of corn steep liquor, white needles and yellow needles were isolated. The compound of white needles, which inhibited the germination of alfalfa seeds at the concentrations above 500 ppm, was found to be identical with ferulic acid known as germination inhibitor of plant seeds. The compound of yellow needles did not inhibit the germination of alfalfa seeds, but stimulated the growth of the plant. It has a structure related to β-acid which has been isolated from rice-bran and has been identified as 2,6-dihydroxycinchoninic acid. This active substance is a new quinoline, 2,8-dihydroxycinchoninic acid and was named zeanic acid.     

Occurrence of aflatoxin producing strains of Aspergillus flavus link in stored corn

Twenty-five samples of moldy corn were examined for the presence of aflatoxin using thin-layer chromatography and the duckling bioassay. In addition, seven aflatoxigenic strains ofAspergillus flavus Link were isolated from these corn samples.     

Partial purification of hatching activity for Globodera rostochiensis from potato root diffusate

The potential of several chromatographic methods for isolating hatching factors for potato cyst nematodes from potato root diffusate was investigated using a bioassay based on emergence of juveniles from cysts. Gel filtration provided an overall estimate of molecular weight of 437 Da for the hatching activity and ion exchange chromatography indicated that at least 60% of the recovered activity was anionic in nature. Material less polar than the hatching activity could be removed by passing potato root diffusate through a reversed-phase Sep-Pak C₁₈ cartridge and the elutant showed 83.3 ± 4.4% (mean of 32 cysts) of the initial hatching activity. High performance liquid chromatography (HPLC) with a reversed phase, C₁₈ column and gradient elution (0–80% CH₃OH in water) confirmed that much of the hatching activity was polar and that it was not retained by this method of separation. A weak anion exchange resin achieved slight retention of much of the hatching activity and an ion pairing reagent lowered the polarity sufficiently to allow some retention in subsequent reversed phase HPLC on a C_IS column. Both ion exchange and ion pairing HPLC suggested that hatching activity was not chromatographed as a single compound and indicated that fractions able to influence the nucleolus of the nucleus within the dorsal pharyngeal gland cell did not always show hatching activity.     

Biosynthesis of Territrems by Aspergillus terreus

Different radioactive precursors were added to 8-day potato-dextrose liquid cultures of Aspergillus terreus 23-1. Territrems were isolated from chloroform extracts of the cultures at day 14 and purified by thin-layer chromatography and high-pressure liquid chromatography. The territrem B obtained was treated with alkaline hydrogen peroxide, and 3, 4, 5-trimethoxy benzoic acid was isolated from an ethyl acetate extract of the reaction mixture and purified by thin-layer chromatography and high-pressure liquid chromatography. By comparison of the specific radioactivities of territrem B and its cleaved aromatic product (disintegrations per minute per micromole of compound), it was demonstrated that the radioactivity of territrem B was located mainly on its aromatic moiety when [U-C]shikimate, l-[methyl-C]methionine, and l-[methyl-H]methionine were precursors; however, the radioactivity of territrem B was located mainly on its nonaromatic moiety when [2-C]mevalonate was the precursor. Mevinolin, a specific inhibitor of beta-hydroxyl beta-methyl glutaryl coenzyme A reductase, was shown to inhibit production of territrems by A. terreus 23-1. When [U-C]acetate was used as a precursor, mevinolin inhibited the incorporation of radioactive carbon into territrem but mevinolin did not inhibit incorporation of radioactive carbon from [2-C]mevalonate into territrem.     

Decreased severity of ryegrass mosaic virus after passage through resistant perennial ryegrass

After exposure to infection in the field, the proportion of plants showing distinct symptoms of ryegrass mosaic virus (RMV) was less in perennial than in Italian ryegrass. The perennial ryegrass cv. Mascot had a smaller proportion of plants with symptoms than cv. S.23. Far milder symptoms were induced in test plants by RMV from naturally infected perennial ryegrass plots than from Italian ryegrass plots. Within perennial ryegrass, RMV from cv. Mascot caused milder symptoms than that from cv. S.23. Severe RMV isolated from Italian ryegrass cv. Trident (RMVT) became milder after one passage through cv. Mascot, although not as mild as RMV obtained from field plots of cv. Mascot (RMVM). Families from two highly resistant perennial ryegrass clones and two randomly selected clones of cv. S.23 crossed in all possible combinations varied in symptom severity when inoculated with RMVT but not when inoculated with RMVM. Families inoculated with RMVT also yielded virus which varied in the severity of symptoms induced in test plants, families with severe symptoms yielding severer virus. Thus, much of the variation in the resistance of these clones could be attributed to infection with RMV of differing severity. Resistance was controlled by several genes which were mainly additive in their effect.