The Identity of this Lepidopteron

The Identity of this Lepidopteron

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I snapped this picture on a walk in the Pacific Northwest last week, and, being new to entomology, cannot manage to identify whether this is a moth or a butterfly.

It seems to have the coloring of a moth, yet it was out around 4-5 PM, with the sun still shining brightly. Its antenna look like a butterfly's, but its face (particularly the eyes) seems to be that of a moth. The wings are folded up like a butterfly (in another picture of the lepidoptera), but they look quite drab for a butterfly. Any help?

This looks like the Satyr Comma or Polygonia satyrus.

Characteristic of this species is a dark border near the tops of wings, fading near the bottom. They are common across the Western United States and Southern Canada.

For more information on this species, try this link and this link.

To differentiate between moths and butterflies you can look at the anntenae. Butterflies often have a bulb at the end whereas moth antennae are feathery or saw-edged.

This is an anglewing (genus Polygonia), but the hindwing submarginal spots are more consistent with the Zephyr Anglewing (P. gracilis zephyrus); see for several photographs of set specimens and living butterflies. For future reference on photographing this genus: showing the underside is often more useful than the upperside.

Antimicrobial peptides/proteins (AMPs) are a group of immune proteins that exhibit strong antibiotic properties against numerous infectious bacterial strains. They are evolutionarily conserved and present in every kingdom and phylum, ranging from prokaryotes to humans. We analyzed the transciptome from the larvae of Asian corn borer, Ostrinia furnacalis (Guenée), and identified several putative AMP transcripts, OfgLys5, OfgLys6, OfgLys10, OfgAtt, and OfgIID. OfgLys5, OfgLys6, and OfgLys10 are all highly homologous with c-type lysozymes, and OfgAtt shows significant identities with Lepidoptera attacin. The amino acid sequence of OfgLys5 and OfgLys6 possessed all conserved features critical for fundamental structure and function of c-type lysozyme, including the two catalytic sites, Glu 32 and Asp 50 . OfgAtt is a typical glycine-rich protein. The antimicrobial activity of O. furnacalis hemolymph increased significantly after injection with Escherichia coli, Micrococcus luteus, or Beauveria bassiana. OfgAtt, IDD, and Lys6 are expressed at low level prior to the challenge, but strongly induced against Gram-positive and negative bacteria, and fungi. Under the same inducement conditions, the transcripts of these three genes elevated most when fifth instar larvae were injected. Therefore, O. furnacalis larvae are induced to produce antimicrobial materials in the hemolymph after the infection, and increase of lysozyme and attacin may contribute to the antimicrobial activity.

Keywords: Ostrinia furnacalis Guenée, antimicrobial peptide, Lysozyme, Attacin


Production of agricultural crops are at risk due to the incidence of pests, especially weeds, pathogens and animal pests. Billions of dollars are spent globally each year for pest control and a large sum of money is lost due to inadequate control of pests. It is evident that the world food supply depends on effective protection of crops and animals from pests 1,2,3,4 . The chemical control of pests was efficacious and attractive during the forties and fifties of the last century. However, adverse effects of such chemicals quickly became apparent as evidenced by the accumulation of chemical pesticides in soil, water, air, agricultural products and in the body of animals. In addition, development of resistance in target organisms necessitated use of more selective and environmentally acceptable agents for pest control 5,6 . To fulfill the growing needs of human population and to prevent the environment, it is necessary to seek new, effective and environment-friendly ways of controlling pests. For instance, chitinases are being used to perturb structures containing chitin such as cuticle and peritrophic matrix (PM) that are essential for growth, development and survival of insects 7,8 . Cuticle is an extracellular matrix produced by most invertebrates, including insects. It is composed of chitin (a polymer of N-acetylgluosamine), a large assortment of proteins and lipids. Chitinases can help fungal and microbial pathogens to infect insect hosts by penetrating their cuticle. Additionally, chitinases affect the larval and adult PM that forms a chitinous tubular structure deposited by the gut epithelium 9,10 . Scanning electron microscopic examination has proved the ability of chitinases to perforate PM in midgut of cotton leaf worm larvae in-vitro at the concentration range of 0.1–10 μg/ml 11 .

All kinds of insects produce chitinases, which are essential for cuticle turnover and mobilization 12 . For instance, insects periodically shed their old exoskeleton and PM either continuously or periodically and resynthesize new ones 13 . Chitinases are among a group of proteins that insects use to digest the structural polysaccharide chitin in their exoskeletons and gut linings during the molting process 14 . Pesticidal activity is due to their ability to bind to chitin component of PM lining the insect gut and cuticle causing degradation of chitin containing matrices and death to the organisms 15,16 .

It is possible that the chitinase gene transfer technology will become as effective as the Bt gene transfer for the production of a pesticide free environment 17 . Alternatively, chitinase transgene may synergize the effect of Bt by decreasing the effective dose needed for insect control 18,19 . In fact, chitinase gene transfer technology may ultimately prove to be more important, since chitinase affects the growth and survival of both insect and fungal pathogens. It is generally assumed that plant chitinases, which belong to 18 glycosylhyrolases family, are effective against some insects when they are fed grain-based diets containing high concentrations of these enzymes 18 . Chitinase alone has been shown to significantly inhibit the insect feeding 20 . Crude chitinase preparations from B. circulans enhanced the toxicity of Bt kurstaki toward diamondback moth larvae 19 . Larvae of C. fumiferana died more rapidly when exposed to chitinase–Bt mixtures than when exposed to the enzyme or bacterium alone 20,21 . These studies have shown that insect chitinases, which belong to family 18 glycosylhydrolases, are good alternative for insect control.

Corn is considered the most important cereal crop after wheat and rice all over the world 22 . Corn borers (S. cretica, Ostrinia nubilalis, Chilo agamemnon) are serious insect pests in much of the corn growing areas around the world and responsible for significant loss of crop yield 23 . In addition, maize is susceptible to fungal diseases such as ear and stalk rot caused by Fusarium moniliforme, Helminthosporium spp., and Rhizoctonia zeae and late welt caused by Cephalosporium maydis 24 . The development of a reliable transformation system could facilitate the introduction of useful genes conferring resistance to fungal diseases and insects into agronomical important cultivars. The addition of chitinolytic enzyme genes, whose encoded proteins adversely affect insect pests and fungal pathogens, to the repertoire of other defense genes in plants should enhance the effectiveness of this type of biotechnological control strategy. To date, only a few studies have been reported that directly used insect-derived chitinases as biopesticides for the control of pests. Elmenofy 6 constructed a recombinant AcMNPV baculovirus expressing a group I chitinase from M. sexta under the control of the polyhedrin promoter. When the fourth instar larvae of M. sexta or S. frugiperda were injected with the recombinant virus, the chitinase was detectable in large amounts in the hemolymph. Liquefaction of infected S. frugiperda larvae occurred significantly earlier than when the insects were infected with a wild-type virus, indicating increased insecticidal activity 15 . A mixture of recombinant virus and purified recombinant protein were found to be more efficient in killing the ticks than recombinant virus and pure chitinase alone. Interestingly, even though purified recombinant chitinase has insecticidal effects. The objectives of this study were to isolate a chitinase gene from insects, to develop transgenic maize expressing chitinase and to test the transgenic lines against corn borer (S. cretica).

Cloning and characterization of piggyBac- like elements in lepidopteran insects

PiggyBac-like elements (PLE) are widespread in variety of organisms, however, few of them are active or have an intact transposon structure. To further define the distribution PLEs in Lepidoptera, where the original active piggyBac IFP2 was discovered, and potentially isolate new functional elements, a survey for PLEs by PCR amplification and Southern dot blots was performed. Two new PLEs, AyPLE and AaPLE, were successfully isolated from the noctuid species, Agrotis ypsilon and Argyrogramma agnate, respectively. These elements were found to be closely related to each other by sequence similarity, and by sharing the same 16 bp inverted terminal repeat sequences. The AyPLE1.1 and AaPLE1.1 elements are structurally intact having characteristic TTAA target site duplications, inverted terminal repeats and intact open reading frames encoding putative transposases with the presumed piggyBac DDD domains, which are features consistent with autonomous functional transposons. Phylogenetic analysis revealed that AyPLE1.1 and AaPLE1.1 cluster with another noctuid species element, HaPLE1.1, suggesting a common ancestor for the three types of PLEs. This contributes to our understanding of the distribution and evolution of piggyBac in Lepidoptera.

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The Identity of this Lepidopteron - Biology

The genetically modified (GM) Bt crops expressing delta-endotoxins from Bacillus thuringiensis provide protection against a wide range of lepidopteron insect pests throughout the growing season of the plant. Bt cotton is the only commercialized crop in India that is planted on an area of 7.6 million hectares. With the increase in development and commercialization of transgenic crops, it is necessary to develop appropriate qualitative and quantitative methods for detection of different transgenic events. The present study reports on the development of a decaplex polymerase chain reaction (PCR) assay for simultaneous detection of transgene sequences, specific transgene constructs, and endogenous stearoyl acyl desaturase (Sad1) gene in two events of Bt cotton, i.e., MON531 and MON15985. The decaplex PCR assay is an efficient tool to identify and discriminate the two major commercialized events of Bt cotton, i.e., MON531 and MON15985, in India. Real-time PCR assays were also developed for quantification of cry1Ac and cry2Ab genes being employed in these two events. The quantitative method was developed using seven serial dilutions containing different levels of Bt cotton DNA mixed with a non-Bt counterpart ranging from 0.01 to 100%. The results revealed that the biases from the true value and the relative standard deviations were all within the range of ±20%. The limit of quantification (LOQ) of the developed real-time PCR method has also been established up to 0.01%.

Chemical Structure and Heterogeneity Differences of Two Lignins from Loblolly Pine As Investigated by Advanced Solid-State NMR Spectroscopy
  • Kevin M. Holtman ,
  • Na Chen ,
  • Mark A. Chappell ,
  • John F. Kadla ,
  • Ling Xu , and
  • Jingdong Mao*

Advanced solid-state NMR was employed to investigate differences in chemical structure and heterogeneity between milled wood lignin (MWL) and residual enzyme lignin (REL). Wiley and conventional milled woods were also studied. The advanced NMR techniques included 13C quantitative direct polarization, various spectral-editing techniques, and two-dimensional 1H−13C heteronuclear correlation NMR with 1H spin diffusion. The 13C chemical shift regions between 110 and 160 ppm of two lignins were quite similar to those of two milled woods. REL contained much more residual carbohydrates than MWL, showing that MWL extraction more successfully separated lignin from cellulose and hemicelluloses than REL extraction REL was also of higher COO, aromatic C−C, and condensed aromatics but of lower aromatic C−H. At a spin diffusion time of 0.55 ms, the magnetization was equilibrated through the whole structure of MWL lignin, but not through that of REL, indicating that REL is more heterogeneous than MWL.

Development of a Competitive Indirect ELISA for the Determination of Lincomycin in Milk, Eggs, and Honey

Polyclonal antibodies to lincomycin (LIN) were developed in rabbit as a result of immunization with BSA−LIN conjugate. Periodate oxidizing of hapten was the common step of both immunogen synthesis and preparation of conjugated antigens for coating plates (homologous and heterologous). Several ELISA variants on a base of the different antigens immobilized on polystyrene were compared. Heterology of solid-phase antigens was provided with relative hapten clindamycin (CLIN) and ethylene- or hexanediamine as spacer arm between hapten and carrier. The spacer insertion yielded no desirable effect, whereas gelatin−CLIN assay variant showed better test characteristics in comparison with the homologous one, although insignificant (IC50 was 9.15 vs 18.3 ng mL−1). The detection limits of the developed test, being estimated as 0.43 ng mL−1 (milk) and 0.65 ng mL−1 (eggs), were sufficient to measure maximum residue levels for LIN in examined matrices. This value for honey was 1.9 ng mL−1 (1.3 μg kg−1). The assay sensitivity was enough to dilute milk, egg, and honey samples by 10−100 times to minimize matrix effect. The examination of matrix effect and simple ways of its overcoming are detailed in the paper. The developed assay showed 111% cross-reactivity with CLIN therefore, it is suitable for the determination of both lincosamides.

Preparative Isolation of Anthocyanins from Japanese Purple Sweet Potato (Ipomoea batatas L.) Varieties by High-Speed Countercurrent Chromatography
  • Elyana Cuevas Montilla ,
  • Silke Hillebrand ,
  • Daniela Butschbach ,
  • Susanne Baldermann ,
  • Naoharu Watanabe , and
  • Peter Winterhalter*

Purple-fleshed sweet potatoes (Ipomoea batatas L.) contain a very complex anthocyanin profile due to the presence of several non-, mono-, and diacylated glucosides of cyanidin and peonidin. In this study, the anthocyanin composition of four Japanese purple sweet potato cultivars (Chiran Murasaki, Tanegashima Murasaki, Naka Murasaki, and Purple Sweet) were investigated by HPLC-DAD and ESI-MSn analyses. The HPLC chromatograms of the different cultivars show a remarkable variation of the two major pigments, cyanidin-3-(6′′-caffeoylsophoroside)-5-glucoside and peonidin-3-(6′′-caffeoylsophoroside)-5-glucoside, respectively. According to this, they can be categorized into two groups on the basis of the peonidin/cyanidin ratio: the cultivars Chiran Murasaki and Purple Sweet showed a high content of peonidin derivatives (peonidin type), whereas the varieties Tanegashima Murasaki and Naka Murasaki were classified as cyanidin types. By means of high-speed countercurrent chromatography (HSCCC) the nonacylated 3-sophoroside-5-glucoside of cyanidin was isolated on a preparative scale. Furthermore, it was possible to isolate the monoacylated cyanidin-3-(6′′-caffeoylsophoroside)-5-glucoside as well as three diacylated major pigments, cyanidin-3-(6′′,6′′′-dicaffeoylsophoroside)-5-glucoside, cyanidin-3-(6′′-caffeoyl-6′′′-p-hydroxy-benzoylsophoroside)-5-glucoside, and peonidin-3-(6′′-caffeoyl-6′′′-p-hydroxybenzoyl-sophoroside)-5-glucoside. The purity and identity of the so-obtained pigments were confirmed by NMR measurements.

Analysis of Biliary Excretion of Icariin in Rats
  • Yu-Tse Wu ,
  • Chia-Wen Lin ,
  • Lie-Chwen Lin ,
  • Allen W. Chiu ,
  • Kuang-Kuo Chen , and
  • Tung-Hu Tsai*

Icariin is a bioactive herbal ingredient isolated from Epimedii Herba. This study evaluates the distribution of icariin in rats by microdialysis sampling and high-performance liquid chromatography with ultraviolet detection (HPLC-UV). Microdialysis probes were simultaneously placed in the jugular vein, brain striatum, and bile duct of each anesthetized rat for sampling after the administration of icariin (dose = 10 or 20 mg/kg) via the femoral vein. The role of P-glycoprotein (P-gp) on icariin distribution was assessed by pretreatment with cyclosporine (CsA, dose = 20 mg/kg). This study is the first report of the biliary excretion of icarin in rats, defined as the blood-to-bile distribution (k value), calculated by dividing the area under the concentration−time curve (AUC) of icariin in bile by that in blood (k = AUCbile/AUCblood). The k values were 19.0 ± 5.9 and 18.8 ± 3.8 at the doses of 10 and 20 mg/kg, respectively. The decreased biliary excretion of icariin due to pretreatment with CsA was evidenced by the reduced k values (18.8 ± 3.8 vs 9.9 ± 1.9, p = 0.005). This work demonstrates that biliary excretion is the major elimination pathway for icariin disposition and that transporters, such as P-gp, might be related to icariin’s biliary excretion.

Improvement of Total Lipid and Glycerophospholipid Recoveries from Various Food Matrices Using Pressurized Liquid Extraction
  • Li Zhou ,
  • Julie Le Grandois ,
  • Eric Marchioni* ,
  • Minjie Zhao ,
  • Saïd Ennahar , and
  • Françoise Bindler

The extraction of three major phospholipid (PL) classes contained in soybean, egg yolk, calf brain, and ox liver was investigated by means of two methods. The PL amounts were evaluated. A new method, based on pressurized liquid extraction (PLE), was applied for total lipids (TL), including PL, extraction and compared with a standard liquid extraction method, a modified Folch method. The three PL classes (phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylcholine (PC)) that were recovered in the obtained TL extracts were quantified using HPLC with an evaporative light-scattering detector (ELSD). Using the PLE method, a single extraction allowed a recovery of more than 94% of TL and 96% of each PL class. Two successive extractions could achieve a total recovery of the three studied PL classes. With the modified Folch method, 77−83% of TL, 80−91% of PE, 82−94% of PC, and no more than 78% of PI could be achieved from various food matrices after one extraction. Four successive extractions were necessary to recover the whole TL content and each PL class. Results indicate that PLE is a rapid and efficient lipid extraction system for the broad range of plant and animal tissues.

Development and Application of an HILIC-MS/MS Method for the Quantitation of Nucleotides in Infant Formula
  • Koichi Inoue* ,
  • Rutsuko Obara ,
  • Tomoaki Hino , and
  • Hisao Oka

A method for the quantitation of nucleotides (adenosine 5′-monophosphate (AMP), guanosine 5′-monophosphate (GMP), uridine 5′-monophosphate (UMP), cytidine 5′-monophosphate (CMP), and inosine 5′-monophosphate (IMP)) in infant formula was developed by hydrophilic interaction chromatography with tandem mass spectrometry (HILIC-MS/MS). The internal standards used (AMP-13C10, 15N5 GMP-13C10, 15N5 UMP-13C9, 15N2 CMP-13C9, 15N3) were prepared with centrifugal ultrafiltration (CUF). Data acquisition was achieved by using multiple reaction monitoring (MRM) of product ions of protonated molecules of the five nucleotides generated by the positive-ion ESI. HILIC conditions were performed with 30 mmol/L ammonium formate in water (pH 2.5, adjusted with formic acid) and methanol. The LOD and LOQ were 5−10 μg/mL and 10−30 μg/mL for standard solution, respectively. Recovery for intra- and interday assays ranged from 98.1 to 108.9% (RSD: 0.7−5.4%) spiked with three concentration levels (5, 25, and 250 μg/g powder infant formula). This method could be applied for the determination of nucleotides in infant formula samples. The detected concentrations of five nucleotides ranged from not detected (n.d.) to 278 μg/g powder infant formula. The total nucleotide level ranged from n.d. to 600.2 μg/g powder infant formula.

Purification, Identification, and Characterization of Methylcobalamin from Spirulina platensis
  • Anantharajappa Kumudha ,
  • Sagaya Selva Kumar ,
  • Munna Singh Thakur ,
  • Gokare Aswathanarayana Ravishankar , and
  • Ravi Sarada*

The present study reports methylcobalamin in Spirulina platensis using high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), microbiological assay, chemiluminescence assay, liquid chromatography−mass spectrometry (LC−MS), and tandem mass spectrometry (MS/MS). Extraction of vitamin B12 from S. platensis was carried out without using cyanide. Partial purification was achieved using Amberlite XAD-2 followed by elution with 80% (v/v) methanol. Activated charcoal facilitated removal of impurities in S. platensis extract and in further purification of vitamin B12. The purified fraction was identified to contain methylcobalamin as analyzed by HPLC and TLC. Authenticity of methylcobalamin was further confirmed by LC−MS and MS/MS. Quantitation of methylcobalamin in a test sample of S. platensis biomass was performed using microbiological assay and chemiluminescence assay and was found to be 38.5 ± 2 and 35.7 ± 2 μg/100 g of dry biomass, respectively.

Fast and Simple Droplet Sampling of Sap from Plant Tissues and Capillary Microextraction of Soluble Saccharides for Picogram-Scale Quantitative Determination with GC-MS
  • Yupeng Tan ,
  • Ke Li ,
  • Lei Hu ,
  • Shu Chen ,
  • Ying Gai* , and
  • Xiangning Jiang*

Soluble saccharides are very important metabolites of the life cycle and synthesis of structural polysaccharide components (cellulose, hemicellulose, pectin, etc.) of cell walls. A new method for droplet sampling of saps from tissues of organisms and manipulation routines in capillaries for extraction, derivation, and partitioning were developed for picogram-scale quantitative determination with gas chromatography−mass spectrometry (GC-MS). Five to ten microliters of sap was sampled with a glass capillary containing ribitol (internal standard). Subsequently, the analytes were acetylated with acetic anhydride and catalyzed by 1-methylimidazole. Finally, the soluble saccharides were qualitatively detected with GC-MS SIM (selective ion monitoring) mode. The linear ranges of the method were up to 1 × 10−6 mol/L and the theoretically lowest limits of detection (LOD, s/n ≥ 3) were up to 1 × 10−9 mol/L. The method is suitable and applicable to analysis of soluble monosaccharides in fresh tissues and other aqueous samples in wide fields of agriculture, food science, biological sciences, and even medical studies.

Bioactive Constituents
Phytochemicals from Acacia confusa Heartwood Extracts Reduce Serum Uric Acid Levels in Oxonate-Induced Mice: Their Potential Use as Xanthine Oxidase Inhibitors
  • Yu-Tang Tung ,
  • Chih-An Hsu ,
  • Chien-Shu Chen ,
  • Suh−Ching Yang ,
  • Chi-Chang Huang , and
  • Shang-Tzen Chang*

In this study, the antihyperuricemic effect of Acacia confusa heartwood extracts and their phytochemicals on potassium oxonate (PO)-induced acute hyperuricemia was investigated for the first time. All treatments at the same dosage (100 mmol/kg) were administered to the abdominal cavity of PO-induced hyperuricemic mice, and serum uric acid level was measured at 3 h after administration. In experimental mice, serum uric acid level was significantly suppressed by the administration of A. confusa heartwood extracts and their major phytochemicals, (−)-2,3-cis-3,4-cis-3,3′,4,4′,7,8-hexahydroxyflavan, (−)-2,3-cis-3,4-cis-4′-methoxy-3,3′,4,7,8-pentahydroxyflavan, melanoxetin, transilitin, and okanin, relative to the PO group. The direct inhibitory effect of these five compounds on xanthine oxidase (XOD) activity was examined using isothermal titration calorimetry (ITC). Among them, melanoxetin showed a more remarkable inhibitory effect on XOD activity than allopurinol, a clinical drug used for XOD inhibitor. To further understand the stereochemistry between XOD and melanoxetin (or allopurinol), structure-based molecular modeling was performed. Melanoxetin undergoes extended interactions in the hydrophobic region via the 3′,4′-dihydroxyphenyl moiety, thus accounting for its higher binding affinity to XOD than allopurinol. These results indicate that A. confusa heartwood extracts and their major phytochemicals exhibit strong XOD inhibitory effects, which reduce serum uric acid levels while inhibiting uric acid generation in purine metabolism.

Prediction of Extra Virgin Olive Oil Varieties through Their Phenolic Profile. Potential Cytotoxic Activity against Human Breast Cancer Cells
  • Jesús Lozano-Sánchez ,
  • Antonio Segura-Carretero* ,
  • Javier A. Menendez ,
  • Cristina Oliveras-Ferraros ,
  • Lorenzo Cerretani , and
  • Alberto Fernández- Gutiérrez

The aim of this work was to develop a rapid resolution liquid chromatography coupled to electrospray ionization time-of-flight mass spectrometry (RRLC-ESI-TOF-MS) method followed by tetrazolium salt (MTT)-based cell viability assays for qualitative and quantitative classification of extra virgin olive oil (EVOO) varieties by phenolic and other polar compound contents as well as for rapid characterization of putative cytotoxic activities against human cancer cells. Five different Spanish EVOO varieties were analyzed, and RRLC-ESI-TOF-MS method was applied for qualitative and quantitative identification of most important phenolic compounds. We finally employed MTT-based cell viability protocol to assess the effects of crude EVOO phenolic extracts (PEs) on the metabolic status of cultured SKBR3 human breast cancer cells. MTT-based cell viability assays revealed a wide range of breast cancer cytotoxic potencies among individual crude PE obtained from EVOO monovarietals. Remarkably, breast cancer cell sensitivity to crude EVOO-PEs was up to 12 times higher in secoiridoids enriched-PE than in secoiridoids-low/null EVOO-PE.

Plant-Growth Regulator, Imidazole-4-Carboxamide, Produced by the Fairy Ring Forming Fungus Lepista sordida
  • Jae-Hoon Choi ,
  • Nobuo Abe ,
  • Hidekazu Tanaka ,
  • Keiji Fushimi ,
  • Yoshifumi Nishina ,
  • Akio Morita ,
  • Yoshikazu Kiriiwa ,
  • Reiko Motohashi ,
  • Daisuke Hashizume ,
  • Hiroyuki Koshino , and
  • Hirokazu Kawagishi*

Rings or arcs of fungus-regulated plant growth occur often on the floor of woodlands, in agricultural areas, and in grasslands worldwide. These rings are commonly called “fairy rings”. A plant-growth regulating compound was isolated from a fairy ring forming fungus, Lepista sordida, and its chemical structure was identified as imidazole-4-carboxamide (ICA) by spectroscopic analyses including single-crystal X-ray diffraction techniques. ICA inhibited the growth of turfgrass and rice seedling. On the other hand, in a greenhouse experiment, this compound increased rice grain yield by 26% compared with control.

Oenothera paradoxa Defatted Seeds Extract and Its Bioactive Component Penta-O-galloyl-β- d -glucose Decreased Production of Reactive Oxygen Species and Inhibited Release of Leukotriene B4, Interleukin-8, Elastase, and Myeloperoxidase in Human Neutrophils
  • Anna K. Kiss ,
  • Agnieszka Filipek ,
  • Monika Czerwińska , and
  • Marek Naruszewicz*

In this study, we analyzed ex vivo the effect of an aqueous extract of Oenothera paradoxa defatted seeds on the formation of neutrophil-derived oxidants. For defining active compounds, we also tested lypophilic extract constituents such as gallic acid, (+)-catechin, ellagic acid, and penta-O-galloyl-β-d-glucose and a hydrophilic fraction containing polymeric procyanidins. The anti-inflammatory potential of the extract and compounds was tested by determining the release from activated neutrophils of elastase, myeloperoxidase, interleukin-8 (IL-8), and leukotriene B4 (LTB4), which are considered relevant for the pathogenesis of cardiovascular diseases. The extract of O. paradoxa defatted seeds displays potent antioxidant effects against both 4β-phorbol-12β-myristate-α13-acetate- and formyl-met-leu-phenylalanine-induced reactive oxygen species production in neutrophils with IC50 values around 0.2 μg/mL. All types of polyphenolics present in the extract contributed to the extract antioxidant activity. According to their IC50 values, penta-O-galloyl-β-d-glucose was the more potent constituent of the extract. In cell-free assays, we demonstrated that this effect is partially due to the scavenging of O2− and H2O2 oxygen species. The extract and especially penta-O-galloyl-β-d-glucose significantly inhibit elastase, myeloperoxidase IL-8, and LTB4 release with an IC50 for penta-O-galloyl-β-d-glucose of 17 ± 1, 15 ± 1, 6.5 ± 2.5, and around 20 μM, respectively. The inhibition of penta-O-galloyl-β-d-glucose on reactive oxygen species and especially on O2− production, myeloperoxidase, and chemoattractant release may reduce the interaction of polymorphonuclear leukocyte with the vascular endothelium and by that potentially diminish the risk of progression of atherosclerosis development.

Kinetic Study of the Quenching Reaction of Singlet Oxygen by Carotenoids and Food Extracts in Solution. Development of a Singlet Oxygen Absorption Capacity (SOAC) Assay Method
  • Aya Ouchi ,
  • Koichi Aizawa ,
  • Yuko Iwasaki ,
  • Takahiro Inakuma ,
  • Junji Terao ,
  • Shin-ichi Nagaoka , and
  • Kazuo Mukai*

A kinetic study of the quenching reaction of singlet oxygen (1O2) with eight kinds of carotenoids and α-tocopherol was performed in ethanol/chloroform/D2O (50:50:1, v/v/v) solution at 35 °C. The overall rate constants, kQ (= kq + kr, physical quenching + chemical reaction), for the reaction of carotenoids with 1O2 were measured, using the competition reaction method, where endoperoxide was used as a singlet oxygen generator, 2,5-diphenyl-3,4-benzofuran (DPBF) as an UV−vis absorption prove, and α-tocopherol as a standard compound. The rate constants, kQ (S) and kQ (t1/2), were determined by analyzing the first-order rate constant (S) and the half-life (t1/2) of the decay curve of DPBF with carotenoids, respectively, showing good accordance with each other. Similar measurements were performed for tomato and carrot extracts. From the results, a new assay method that can quantify the singlet oxygen absorption capacity (SOAC) of antioxidants, including carotenoids, α-tocopherol, and vegetable extracts, has been proposed.

Screening and Selection of High Carotenoid Producing in Vitro Tomato Cell Culture Lines for [ 13 C]-Carotenoid Production
  • Nancy J. Engelmann ,
  • Jessica K. Campbell ,
  • Randy B. Rogers ,
  • S. Indumathie Rupassara ,
  • Peter J. Garlick ,
  • Mary Ann Lila , and
  • John W. Erdman Jr., *

Isotopically labeled tomato carotenoids, phytoene, phytofluene, and lycopene, are needed for mammalian bioavailability and metabolism research but are currently commercially unavailable. The goals of this work were to establish and screen multiple in vitro tomato cell lines for carotenoid production, test the best producers with or without the bleaching herbicides, norflurazon and 2-(4-chlorophenyl-thio)triethylamine (CPTA), and to use the greatest carotenoid accumulator for in vitro 13C-labeling. Different Solanum lycopersicum allelic variants for high lycopene and varying herbicide treatments were compared for carotenoid accumulation in callus and suspension culture, and cell suspension cultures of the hp-1 line were chosen for isotopic labeling. When grown with [U]-13C-glucose and treated with CPTA, hp-1 suspensions yielded highly enriched 13C-lycopene with 45% of lycopene in the M+40 form and 88% in the M+35 to M+40 isotopomer range. To the authors’ knowledge this is the first report of highly enriched 13C-carotenoid production from in vitro plant cell culture.

Yeast α-Glucosidase Inhibition by Isoflavones from Plants of Leguminosae as an in Vitro Alternative to Acarbose
  • Chun Whan Choi ,
  • Yeon Hee Choi ,
  • Mi-Ran Cha ,
  • Dae Seok Yoo ,
  • Young Sup Kim ,
  • Gyu Hwan Yon ,
  • Kyung Sik Hong ,
  • Young Ho Kim* , and
  • Shi Yong Ryu*

In the course of searching for new classes of α-glucosidase inhibitors originated from natural resources, 11 kinds of isoflavones, i.e., medicarpin (1), formononetin (2), mucronulatol (3), (3R)-calussequinone (5), (3R)-5′-methoxyvestitol (6), tectorigenin (7), biochanin A (8), tuberosin (9), calycosin (10), daidzein (11), and genistein (12), as well as a flavone, liquritigenin (4), were isolated as active principles responsible for the yeast α-glucosidase inhibitory activity from two leguminous plant extracts, i.e., the heartwood extract of Dalbergia odorifera and the roots extract of Pueraria thunbergiana. Each components (1−12) demonstrated a significantly potent inhibition on yeast α-glucosidase in a dose dependent manner when the p-nitrophenyl-α-d-glucopyranoside was used as a substrate in vitro. The concentration required for 50% enzyme inhibition (IC50) were calculated as 2.93 mM (1), 0.51 mM (2), 3.52 mM (7) 0.35 mM (8), 3.52 mM (9), 0.85 mM (11), and 0.15 mM (12) when that of reference drug acarbose was evaluated as 9.11 mM, in vitro. However, isoflavone glycosides, i.e., puerarin (13), daidzin (14), formononetin-7-O-β-glucopyranoside (15), and genistin (16), exhibited a relatively poor inhibitory activity on yeast α-glucosidase as compared with the corresponding isoflavone (2, 11, 12), respectively

Phytotoxicity of Sarmentine Isolated from Long Pepper (Piper longum) Fruit
  • Huazhang Huang* ,
  • Christy M. Morgan ,
  • Ratnakar N. Asolkar ,
  • Marja E. Koivunen , and
  • Pamela G. Marrone

Discovery of novel natural herbicides has become crucial to overcome increasing weed resistance and environmental issues. In this article, we describe the finding that a methanol extract of dry long pepper (Piper longum L.) fruits is phytotoxic to lettuce (Lactuca sativa L.) seedlings. The bioassay-guided fractionation and purification of the crude extract led to isolation of sarmentine (1), a known compound, as the active principle. Phytotoxicity of 1 was examined with a variety of seedlings of field crops and weeds. Results indicated that 1 was a contact herbicide and possessed broad-spectrum herbicidal activity. Moreover, a series of sarmentine analogues were then synthesized to study the structure−activity relationship (SAR). SAR studies suggested that phytotoxicity of sarmentine and its analogues was specific due to chemical structures, i.e., the analogues of the acid moiety of 1 were active, but the amine and its analogues were inactive the ester analogues and amide analogues with a primary amine of 1 were also inactive. In addition, quantification of 1 from different resources of the dry P. longum fruits using liquid chromatography−mass spectrometry showed a wide variation, ranging from almost zero to 0.57%. This study suggests that 1 has potential as an active lead molecule for synthesized herbicides as well as for bioherbicides derived from natural resources.

Larvicidal Activity of Asarum heterotropoides Root Constituents against Insecticide-Susceptible and -Resistant Culex pipiens pallens and Aedes aegypti and Ochlerotatus togoi
  • Haribalan Perumalsamy ,
  • Kyu Sik Chang ,
  • Chan Park , and
  • Young-Joon Ahn*

We investigated the toxicity of (−)-asarinin, α-asarone, methyleugenol, pellitorine, and pentadecane identified in Asarum heterotropoides root to third instar larvae from insecticide-susceptible Culex pipiens pallens (KS-CP strain), Aedes aegypti, and Ochlerotatus togoi as well as field-collected C. p. pallens (DJ-CP colony), identified by polymerase chain reaction. Results were compared with those of two conventional mosquito larvicides: fenthion and temephos. Pellitorine (LC50, 2.08, 2.33, and 2.38 ppm) was 5.5, 10.8, and 25.6 times, 4.5, 11.6, and 24.7 times, and 6.9, 11.1, and 24.6 times more toxic than (−)-asarinin, α-asarone, and methyleugenol against susceptible C. p. pallens, A. aegypti, and O. togoi larvae, respectively. Pentadecane was least toxic. Overall, all the compounds were less toxic than either fenthion or temephos. However, these compounds did not differ in toxicity against larvae from the two Culex strains, even though the DJ-CP larvae exhibited high levels of resistance to fenthion (resistance ratio (RR), 1179), chlorpyrifos (RR, 1174), fenitrothion (RR, 428), deltamethrin (RR, 316), chlorfenapyr (RR, 225), and α-cypermethrin (RR, 94). This finding indicates that the isolated compounds and the pyrethroid, organophosphorus, and pyrrole insecticides do not share a common mode of action or elicit cross-resistance. A. heterotropoides root-derived materials, particularly (−)-asarinin and pellitorine, merit further study as potential mosquito larvicides for the control of insecticide-resistant mosquito populations in light of global efforts to reduce the level of highly toxic synthetic insecticides in the aquatic environment.

Protective Effects of Black Rice Bran against Chemically-Induced Inflammation of Mouse Skin
  • Sun Phil Choi ,
  • Sung Phil Kim ,
  • Mi Young Kang ,
  • Seok Hyun Nam* , and
  • Mendel Friedman*

We investigated the inhibitory effects of black rice (cv. LK1−3−6−12−1−1) bran against 12-O-tetradecanolylphorbol-13-acetate (TPA)-induced skin edema and 2,4-dinitrofluorobenzene (DNFB)-induced allergic contact dermatitis (ACD) in inflammatory mouse models. We also determined the effects of the bran extract on the following biomarkers: pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), eicosanoids leukotriene B4 (LTB4), and prostaglandin E2 (PGE2). Topical application of TPA to ears of CD-1 mice induced inflammation accompanied with substantial increase in TNF-α, IL-1β, IL-6, LTB4, and PGE2 levels and an elevation in intercellular adhesion molecule-1 (ICAM-1) gene expressions in ear skin tissues. Intraperitoneal injection of black rice bran extract prior to TPA application in mice significantly suppressed TPA-induced inflammation (edema) and induced a marked decrease in the production of TNF-α, IL-1β, IL-6, and LTB4. Feeding mice a standard diet with added 10% black rice bran also significantly suppressed DNFB-induced allergic contact dermatitis on the skin of the mice. By contrast, a nonpigmented brown rice bran extract did not inhibit the TPA-induced edema and failed to significantly suppress production of pro-inflammatory biomarkers (mediators). These in vivo findings further demonstrate the potential value of black rice bran as an anti-inflammatory and antiallergic food ingredient and possibly also as a therapeutic agent for the treatment and prevention of diseases associated with chronic inflammation.

Anticancer and Anti-inflammatory Effects of Cysteine Metabolites of the Green Tea Polyphenol, (−)-Epigallocatechin-3-gallate
  • Joshua D. Lambert* ,
  • Shengmin Sang ,
  • Jungil Hong , and
  • Chung S. Yang

(−)-Epigallocatechin-3-gallate (EGCG) has been shown to have cancer preventive activity in vitro and in vivo. We have previously shown that EGCG can undergo conjugation to cysteine to form 2′-cysteinyl-EGCG and 2′′-cysteinyl-EGCG. Studies of thiol-conjugated metabolites of methamphetamine indicate that such metabolites are not detoxified but retain biological activity. Here, we examined the growth inhibitory, pro-oxidant, and anti-inflammatory activities of the cysteine metabolites of EGCG. Both compounds dose-dependently inhibited the growth of colon cancer and intestinal cell lines. Both metabolites prevented aberrant arachidonic acid release and nitric oxide production by lipopolysaccharide-stimulated RAW264.7 cells. Under cell culture conditions, 2′′-cysteinyl-EGCG produced H2O2 at a faster rate than EGCG. The results of the present study show that cysteine conjugates of EGCG retain the growth inhibitory, anti-inflammatory, and pro-oxidant activities of EGCG in vitro and may play a role in disease prevention in vivo. These results remain to be confirmed in vivo.

Hispidulin Sensitizes Human Ovarian Cancer Cells to TRAIL-Induced Apoptosis by AMPK Activation Leading to Mcl-1 Block in Translation
  • Jung-Mu Yang ,
  • Chao-Ming Hung ,
  • Chen-Nan Fu ,
  • Jang-Chang Lee ,
  • Chi-Hung Huang ,
  • Muh-Hwa Yang ,
  • Chih-Li Lin ,
  • Jung-Yie Kao* , and
  • Tzong-Der Way*

Whether hispidulin, a flavone from traditional Chinese medicine, can modulate the anticancer effects of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), the cytokine currently in clinical trials was investigated. In the present study, we found that hispidulin potentiated the TRAIL-induced apoptosis in human ovarian cancer cells and converted TRAIL-resistant cells to TRAIL-sensitive cells. When examined for its mechanism, we found that hispidulin was highly effective in activation of caspases 8 and caspase 3 and consequent poly(ADP-ribose) polymerase (PARP) cleavage. Moreover, we found that hispidulin downregulated the expression of Mcl-1, Bcl-2, and Bcl-xL. Whereas the downregulation of Bcl-2 and Bcl-xL was less pronounced, the downregulation of Mcl-1 was quite dramatic and was time-dependent. This sensitization is controlled through the adenosine monophosphate (AMP)-activated protein kinase (AMPK), which is the central energy-sensing system of the cell. Interestingly, we determined that AMPK is activated upon hispidulin treatment, resulting in mammalian target of rapamycin (mTOR) inhibition leading to Mcl-1 decrease. Therefore, our results show a novel mechanism for the sensitization to TRAIL-induced apoptosis linking hispidulin treatment to Mcl-1 downexpression. In addition, this study provides a rationale for the combined use of death receptor (DR) ligands with AMPK activators or mTOR inhibitors in the treatment of human cancers.

Synthesis and Structure−Activity Relationship Study of Deoxybenzoins on Relaxing Effects of Porcine Coronary Artery
  • Tzy-Ming Lu* ,
  • Daih-Huang Kuo ,
  • Horng-Huey Ko , and
  • Lean-Teik Ng

Deoxybenzoins are potent antioxidants and tyrosinase inhibitors with potential to be developed as food preservatives and cosmetic ingredients. To explore the potential in cardiovascular protection, 25 polyphenolic deoxybenzoins were synthesized and evaluated for inhibitory effects on KCl-induced porcine coronary arterial contraction. The results revealed deoxybenzoins are significant inhibitors of KCl-induced arterial contraction. Among those synthesized, two-thirds of the deoxybenzoins exhibited moderate to good efficacy on relaxing contracted artery including 2,4-dihydroxydeoxybenzoin with EC50 = 3.30 μM (Emax = 100%, n = 7) and 2,4-dihydroxy-4′-methoxydeoxybenzoin EC50 = 3.70 μM (Emax = 100%, n = 5). Deoxybenzoins displayed an endothelium-dependent relaxing manner on the contracted artery the contractile responses of neither endothelium denuded nor L-NAME deactivated rings were inhibited. The structure−activity relationships of deoxybenzoin on arterial relaxing effects concluded that the 2,4-dihydroxylated deoxybenzoins presented a potential vascular relaxing pharmacophore, with favoring substitution on ring B in the order of H ≥ p-OMe > p-OH > o-OMe > m,p-diOMe ≥ m-OMe.

Antihyperglycemic Effect of a Caffeamide Derivative, KS370G, in Normal and Diabetic Mice
  • Yi-Chun Weng ,
  • Hsi-Lin Chiu ,
  • Yen-Chu Lin ,
  • Tzong-Cherng Chi ,
  • Yueh-Hsiung Kuo , and
  • Ming-Jai Su*

The antihyperglycemic actions of caffeamide derivatives, especially KS370G, in normal ICR, streptozotocin-induced diabetic (T1DM) and diet-induced diabetic (T2DM) mice were investigated in this study. Oral administration of the compound decreased the plasma glucose levels in both normal and diabetic mice, and appeared to be in a dose-dependent manner in normal and diet-induced type 2 diabetic mice. It was found that KS370G could stimulate the release of insulin in both normal and T2DM mice, and a dose of 1 mg per kg KS370G could significantly attenuate the increase of plasma glucose induced by an intraperitoneal glucose challenge test in normal and diabetic mice. Similar treatment with KS370G significantly increased glycogen content in both liver and skeletal muscle. Hence, the hypoglycemic effect of KS370G in normal and diabetic mice could be attributed to the stimulation of insulin release and the increase of glucose utilization.

Biofuels and Bioproducts Chemistry
Effects of Kernel Breakage and Fermentation on Corn Germ Integrity and Oil Quality

To investigate the ability of corn germ to withstand the fuel ethanol fermentation process without major damage to germ integrity and germ oil quality, five treatments were designed to explore degerming before fermentation (front-end) and after fermentation (tail-end), and the feasibility of breaking the kernel with minimum shear forces (wet-split). Germ from low-shear (wet-split) tail-end degerming maintained its integrity during the process. The wet-grind pretreatment caused 22% germ damage, and the subsequent fermentation caused 18% additional germ damage. The germ recovered after fermentation showed physical strength similar to that of those isolated by wet means before fermentation. The oils extracted from the tail-end germ fractions had the same low free fatty acid (FFA) content (2%) and similar low peroxide value (2 meq/kg) as those extracted at the front end. The good oil quality of the tail-end germ fraction was attributed to excellent germ integrity. The oil recovered after traditional dry-grind ethanol production was highly deteriorated, with 22% FFAs and 9 meq/kg peroxide value because the germ was broken into small pieces during dry grinding. So long as kernel-breakage or size-reduction pretreatments are conducted to retain intact germs or keep them in large pieces before fermentation, the germ can survive the cooking, starch hydrolysis, and yeast metabolism during the ethanol fermentation process. These findings lay a foundation for developing new degerming strategies where the germ can be isolated during or after fermentation, which could be easily integrated into the conventional dry-grind corn ethanol process.

Accelerated Solvent Extraction of Lignin from Aleurites moluccana (Candlenut) Nutshells
  • Andrew P. Klein ,
  • Evan S. Beach* ,
  • John W. Emerson , and
  • Julie B. Zimmerman

Lignin from candlenut shells was isolated using an ethanol−water accelerated solvent extraction method. Yields (based on Klason lignin) increased from about 14 to 33% as temperature increased from 100 to 195 °C and were also influenced by the amount of aqueous acid used to precipitate lignin from the extraction liquor. These yields were higher than could be obtained using a conventional dioxane−water acidolysis method. The resulting lignin was characterized by IR, 31P NMR, and 1H−13C HMQC NMR spectroscopic techniques. The lignin contained predominantly guaiacyl units, and both the total hydroxyl group content and phenolic hydroxyl group content were high.

Structural Characterization and Antioxidant Activity Evaluation of Lignins from Rice Husk
  • Anika Salanti ,
  • Luca Zoia* ,
  • Marco Orlandi ,
  • Fabiana Zanini , and
  • Graziano Elegir

In recent years, lignin and extractives from herbaceous plants and crops are receiving increasing attention for their renewability and large annual biomass stock. It is worth noting that only a few studies deal with the chemical characterization of rice husk, a side product of one of the most important crops with regard to human nutrition. Thus, in this study lignin from rice husk was isolated and characterized. Two different extraction procedures were optimized and tested: acidolysis and alkaline enzymatic (AE). The different lignins isolated were fully characterized by means of gravimetric, chromatographic (GPC), and spectroscopic (31P NMR, 2D-HSQC-NMR) analyses with the aim to compare yields, sample purity, and chemical properties, recognized as key parameters for future development. Notwithstanding the extraction procedure, the results highlighted that rice husk lignin is mainly formed by guaiacyl and p-hydroxyphenyl units. The acidolytic approach showed an appreciable lignin recovery and high purity, whereas the AE lignin sample was found to be rich in residual polysaccharides and oxidized functionalities. Moreover, different rice husk extracts, along with acidolysis lignin and AE lignin specimens, were assayed for their antioxidant activity by means of a DPPH radical scavenging test.

Chemical Aspects of Biotechnology/Molecular Biology
Plant-Produced Trastuzumab Inhibits the Growth of HER2 Positive Cancer Cells
  • Brittany M. Grohs ,
  • Yongqing Niu ,
  • Linda J. Veldhuis ,
  • Salma Trabelsi ,
  • Freydoun Garabagi ,
  • John A. Hassell ,
  • Michael D. McLean , and
  • J. Christopher Hall*

To study the agricultural production of biosimilar antibodies, trastuzumab (Herceptin) was expressed in Nicotiana benthamiana using the magnICON viral-based transient expression system. Immunoblot analyses of crude plant extracts revealed that trastuzumab accumulates within plants mostly in the fully assembled tetrameric form. Purification of trastuzumab from N. benthamiana was achieved using a scheme that combined ammonium sulfate precipitation with affinity chromatography. Following purification, the specificity of the plant-produced trastuzumab for the HER2 receptor was compared with Herceptin and confirmed by western immunoblot. Functional assays revealed that plant-produced trastuzumab and Herceptin have similar in vitro antiproliferative effects on breast cancer cells that overexpress HER2. Results confirm that plants may be developed as an alternative to traditional antibody expression systems for the production of therapeutic mAbs.

Β-Sitosterol Inhibits Cell Cycle Progression of Rat Aortic Smooth Muscle Cells through Increases of p21 cip1 Protein
  • Ming-Hsien Chien ,
  • Tong-Sheng Lee ,
  • Yu-Chih Liang , and
  • Wen-Sen Lee*

Abnormal proliferation of vascular smooth muscle cells (VSMCs) plays a central role in the pathogenesis of atherosclerosis. β-Sitosterol, an important phytosterol found in plant food, is known to exert antiatherosclerosis activity. However, the molecular mechanisms underlying β-sitosterol-induced antiproliferation of VSMCs were still not clear. This study demonstrated that β-sitosterol (1−20 μM) concentration-dependently inhibited proliferation of rat aortic smooth muscle cells (RASMCs) without cytotoxic effect. Flow cytometric analysis revealed that β-sitosterol arrested cell cycle progression through down-regulation of cyclin E and cyclin-dependent kinase (CDK)2 and up-regulation of p21cip1. In the β-sitosterol-treated RASMCs, the formation of the CDK2-p21cip1 complex was increased and the assayable CDK2 activity was decreased. Knockdown of the expression of p21cip1 gene prevented β-sitosterol-induced cell cycle arrest in RASMCs. In conclusion, β-sitosterol inhibited VSMC proliferation by increasing the levels of p21cip1 protein, which in turn inhibited the CDK2 activity, and finally interrupted the progress of the cell cycle.

Chemical Changes Induced by Processing/Storage
Ascorbic Acid Is the Only Bioactive That Is Better Preserved by High Hydrostatic Pressure than by Thermal Treatment of a Vegetable Beverage

Variations in levels of antioxidant compounds (ascorbic acid, total phenolics, and total carotenoids), total antioxidant capacity, and color changes in a vegetable (tomato, green pepper, green celery, onion, carrot, lemon, and olive oil) beverage treated by high hydrostatic pressure (HHP) were evaluated in this work. The effects of HHP treatment, four different pressures (100, 200, 300, and 400 MPa) and four treatment times for each pressure (from 120 to 540 s) were compared with those of thermal treatment (90−98 °C for 15 and 21 s). High pressure treatment retained significantly more ascorbic acid in the vegetable beverage than thermal treatment. However, no significant changes in total phenolics were observed between HHP treated and thermally processed vegetable beverage and unprocessed beverage. Color changes (a*, b*, L, chroma, h°, and ΔE) were less for pressurized beverage than thermally treated samples compared with unprocessed beverage.

Pressure and Temperature Effects on Degradation Kinetics and Storage Stability of Total Anthocyanins in Blueberry Juice
  • Roman Buckow* ,
  • Anja Kastell ,
  • Netsanet Shiferaw Terefe , and
  • Cornelis Versteeg

The degradation kinetics of total anthocyanins in blueberry (Vaccinium myrtillus) juice were studied during thermal processing by treatment at selected temperatures (60−121 °C) and combined high pressure−temperature processing (100−700 MPa, 40−121 °C). Anthocyanin stability was also studied for several of these treatments during storage at 4, 25, and 40 °C. Both pressure and temperature increased d, the degradation rate of total anthocyanins in blueberry juice, meaning that at constant temperature, anthocyanins were more rapidly degraded with increasing pressure. For example, 32% degradation of anthocyanins was observed after 20 min heating at 100 °C and atmospheric pressure, whereas at 100 °C and 600 MPa, approximately 50% of total anthocyanins were lost. Degradation of anthocyanins was significantly accelerated with increasing storage temperatures. Combined pressure−temperature treatment of pasteurized juice led to a slightly faster degradation of total anthocyanins during storage compared to heat treatments at ambient pressure. Degradation of anthocyanins was best described by a 1.4th-order reaction at all conditions investigated. A mathematical model describing the degradation of blueberry anthocyanins in juice as a function of pressure, temperature, and treatment time is presented.

Effects of Thermal Processing on the Enzyme-Linked Immunosorbent Assay (ELISA) Detection of Milk Residues in a Model Food Matrix

Food products and ingredients are frequently tested for the presence of undeclared allergenic food residues (including milk) using commercial enzyme-linked immunosorbent assays (ELISAs). However, little is understood about the efficacy of these kits with thermally processed foods. This study evaluated the performance of three milk ELISA kits with a model food processed by several methods. A model food (pastry dough squares) was spiked with nonfat dry milk at several concentrations. The pastry squares were processed by boiling (100 °C for 2 min), baking (190 °C for 30 min), frying (190 °C for 2 min), and retorting (121 °C for 20 min with 17 psi overpressure). Samples were analyzed with three commercial ELISA kits: Neogen Veratox Total Milk, ELISA Systems β-lactoglobulin, and ELISA Systems casein. The detection of milk residues depended upon the type of processing applied to the food and the specific milk analyte targeted by the ELISA kit. Poor recoveries were obtained in all processed samples (2−10% of expected values) using the β-lactoglobulin kit. Better recoveries were obtained in boiled samples (44 and 59%, respectively) using the total milk and casein kits. However, these kits performed poorly with baked (9 and 21%) and fried (7 and 18%) samples. Moderate recoveries were observed in retorted samples (23 and 28%). The decreased detection in processed samples is likely due to protein modifications, including aggregation and Maillard reactions, which affect the solubility and immunoreactivity of the antigens detected by the ELISA methods. The observed decreases in ELISA detection of milk are dramatic enough to affect risk-assessment decisions. However, a lower detection of milk residues does not necessarily indicate decreased allergenicity. These ELISA kits are not acceptable for all applications, and users should understand the strengths and limitations of each method.

Thermal Behavior of Soy Protein Fractions Depending on Their Preparation Methods, Individual Interactions, and Storage Conditions

Different soy protein isolates (SPI) and whey soy protein (WSP) samples were obtained from fresh and stored soybean flour. Some samples were subjected to a long, cold storage. DSC thermograms of SPI showed the two characteristic endotherms, corresponding to denaturation of β-conglycinin and glycinin. Low value of denaturation enthalpy and high glycinin denaturation temperature were related to a reduction of protein solubility of SPI. DSC thermograms of WSP also showed two characteristic endotherms, corresponding to Kunitz trypsin inhibitor and lectin. The methods and conditions of preparation and storage of WSP samples were factors that modified their thermal behavior. Some SPI−WSP mixtures (1:1) exhibited more complex thermograms and higher denaturation temperatures. Thermograms of SPI-denatured WSP mixtures showed that the thermal stabilization of soybean storage proteins was attributed to protein−protein interactions. The differences in the thermal behavior of single or mixed SPI and WSP could not be explained on the basis of mineral content.

Evaluation of Phenolic Profile and Antioxidant Properties of Pardina Lentil As Affected by Industrial Dehydration
  • Yolanda Aguilera ,
  • Montserrat Dueñas ,
  • Isabel Estrella ,
  • Teresa Hernández ,
  • Vanesa Benitez ,
  • Rosa M. Esteban , and
  • María A. Martín-Cabrejas*

This study presents the effects of soaking, cooking, and industrial dehydration treatments on phenolic profile and also on antioxidant properties in Pardina lentil. HPLC-PAC and HPLC-MS (ESI) analysis identified a total of 35 phenolic compounds in raw and processed lentil flours, corresponding to catechins and procyanidins (69% of the total of identified phenolic compounds), flavonols (17%), flavones, and flavanones (5%), and hydroxybenzoic and hydroxycinnamic compounds (5 and 4%, respectively). During the industrial process, catechins and procyanidins, flavonols, flavones, and flavanones decreased, while hydroxybenzoic compounds exhibited an important increase. In addition, raw lentils showed high values of the antioxidant activity (66.97 μmol Trolox/g) although the thermal processing promotes decreased, the levels of antioxidant activity were still relevant. Thus, the significant occurrence of bioactive phenolic compounds along with the interesting antioxidant capacity of dehydrated lentil flours make them useful for daily inclusion in the human diet as ready-to-use for special meals to specific populations.

Stability of Lutein in Wholegrain Bakery Products Naturally High in Lutein or Fortified with Free Lutein
  • El-Sayed M. Abdel-Aal* ,
  • J. Christopher Young ,
  • Humayoun Akhtar , and
  • Iwona Rabalski

Lutein is a yellow pigment found in common foods that promotes the health of eyes and skin and is associated with reduced risk of age-related macular degeneration and cataracts. In the present study, selected high-lutein wheat and corn were milled into wholegrain flours by two mills to improve flour uniformity. The high-lutein and lutein-fortified wholegrain flours were processed into breads, cookies, and muffins to study lutein stability during baking and subsequent storage. Lutein and its isomers were separated, identified, and quantified by LC-UV/vis and LC-MS following extraction with water-saturated 1-butanol. Baking resulted in a significant reduction in all-trans-lutein and the formation of cis-lutein and cis-zeaxanthin isomers. Subsequent storage at ambient temperature had a slight impact on the content of all-trans-lutein. Effects of processing were more pronounced in lutein-fortified products, and the degradation rate of lutein was influenced by concentration and baking recipe. Fortified cookies and muffins showed greater lutein reduction compared with bread. Despite the significant reduction in lutein, the fortified bakery products still possessed reasonable amounts per serving that would enhance daily intake and consumption of wholegrain foods.

Design of a Multifunctional Nanohybrid System of the Phytohormone Gibberellic Acid Using an Inorganic Layered Double-Hydroxide Material
  • Inas H. Hafez ,
  • Mohamed R. Berber* ,
  • Keiji Minagawa ,
  • Takeshi Mori , and
  • Masami Tanaka

To offer a multifunctional and applicable system of the high-value biotechnological phytohormone gibberellic acid (GA), a nanohybrid system of GA using the inorganic Mg−Al layered double-hydroxide material (LDH) was formulated. The ion-exchange technique of LDH was applied to synthesize the GA−LDH hybrid. The hybrid structure of GA−LDH was confirmed by different spectroscopic techniques. The nanohybrid size was described by SEM to be ∼0.1 μm. The GA−LDH nanohybrid structure was the key parameter that controlled GA properties. The layered molecular structure of LDH limited the interaction of GA molecules in two-dimensional directions. Accordingly, GA molecules did not crystallize and were released in an amorphous form suitable for dissolution. At various simulated soil solutions, the nanohybrids showed a sustained release process following Higuchi kinetics. The biodegradation process of the intercalated GA showed an extended period of soil preservation as well as a slow rate of degradation.

Chemical Composition of Foods/Feeds
Phenolic Compounds Responsible for the Superoxide Dismutase-like Activity in High-Brix Apple Vinegar
  • Kozo Nakamura* ,
  • Yasushi Ogasawara ,
  • Kiyoshi Endou ,
  • Seiji Fujimori ,
  • Masahiro Koyama , and
  • Hirofumi Akano

High-Brix apple vinegar (HBAV) with palatable drinking qualities has been developed using a greater amount of apple ingredients. In HBAV and in regular apple vinegar (RAV), constituents of 4 kinds of organic acids, 20 kinds of amino acids, 3 kinds of sugars, 4 kinds of minerals, and phenols were determined. These constituents, except for acetic acid, in HBAV are of higher abundance than in RAV. HBAV had a 7.1 times greater superoxide dismutase (SOD)-like activity compared with RAV. Those constituents, except for phenols, had very low SOD-like activity, and total phenol levels in HBAV were comparable to 181 mg of gallic acid equivalents/100 mL, which was 6.0 times more abundant than in RAV. Nine kinds of phenols including two kinds of hydroxycinnamates, two kinds of hydroxybenzoates, and five kinds of hydroxycinnamoyl quinates, originating from raw material were determined, but there were no ascorbic acid and flavonoids in HBAV. Chlorogenic acid, 4-p-coumaroylquinic acid, and caffeic acid were the three major phenols, and their content levels were 19.6, 13.5, and 0.76 mg in 100 mL of HBAV, respectively. Sum of contents of chlorogenic acid and the isomers was 24.0 mg/100 mL, and the percentage was 56.9% in the total identified phenols in HBAV. In RAV, only chlorogenic acid was determined as phenols, and the content was 3.1 mg/100 mL. SOD-like activities of the constituents of HBAV were obtained through high-accuracy assays using vinegar reconstitutions, and each contribution to the total SOD-like activity was found. As a result, 77.2% for all SOD-like activity of HBAV was reconstituted using the determined nine phenols and other constituents. Chlorogenic acids were the most effective, and the contribution to the total activity was 41.7%. The most abundant phenols, chlorogenic acids, were the most important contributors to the SOD-like activity. These SOD-active phenols originated from raw material and remained through the acetic acid fermentation processes. In the fermentation process of HBAV, the active constituents were well maintained, providing an advantage in the production of a phenol-rich product.

Phenolic Contents and Antioxidant Activities of Major Australian Red Wines throughout the Winemaking Process
  • Irine R. Ginjom ,
  • Bruce R. D’Arcy* ,
  • Nola A. Caffin , and
  • Michael J. Gidley

Three Australian red wine types (Shiraz, Cabernet Sauvignon, and Merlot) were analyzed for antioxidant activity and a range of phenolic component contents using various spectral methods. More than half of the total phenolic compounds were tannins, whereas monomeric anthocyanins and flavonols were present in much lesser amounts (<10%). The evolution of phenolic contents and the respective antioxidant activities in wine samples from all stages of winemaking showed progressive changes toward those of commercial wines. The antioxidant activity of the wines in DPPH and ABTS assays was positively correlated with total phenolic contents and tannins. Comparisons of the three wine varieties based on their individual phenolic component groups and antioxidant activities showed limited differences between the different varieties. However, when all of the variables were combined in a principal component analysis, variety differentiation was observed. The three varieties of red wines all contained similar and high concentrations of antioxidants despite differences in grape variety/maturity and winemaking process, suggesting that related health benefits would accrue from all of the red wines studied.

Elderberry (Sambucus nigra L.) Wine: A Product Rich in Health Promoting Compounds
  • Valentina Schmitzer* ,
  • Robert Veberic ,
  • Ana Slatnar , and
  • Franci Stampar

Color components, antioxidative potential, and total phenolic content were monitored in elderberry must and wine. Among individual phenolic compounds, quercetin and kaempferol compounds, phenolic acids, and anthocyanins were detected with high performance liquid chromatography coupled with mass spectrometry. Conventional enological parameters were measured in elderberry wine and compared to grape and other fruit wines. Elderberry wine has a moderate ethanol concentration, intense red coloration, and higher pH value compared to most red wines. Total phenolic content of elderberry must and wine ranged up to 2004.13 GAE L−1. Antioxidative potential of elderberry wine was in the range of red wine, and a tight correlation was detected between total phenolic content and antioxidative potential of elderberry wine. Anthocyanins were the most abundant phenolics in elderberry wine in tight correlation with color hue, and their content significantly decreased with aging. Similarly, a decrease in total phenolic content and antioxidative potential was determined after storage.

Crop and Animal Protection Chemistry
Herbicidal Activity of Cineole Derivatives

Essential oils and their constituents have potential as ecologically acceptable pesticides that may also have novel modes of action. In this work hydroxy and ester derivatives of the naturally occurring monoterpenoids 1,8-cineole 3, the main component in most eucalyptus oils, and 1,4-cineole 4 were prepared and their pre-emergence herbicidal activity against annual ryegrass (Lolium rigidum) and radish (Raphanus sativus var. Long Scarlet) investigated in laboratory-based bioassays. 1,8-Cineole, eucalyptus oil and all derivatives showed a dose-dependent herbicidal activity against annual ryegrass and radish with many of the derivatives showing improved herbicidal activity relative to 1,8-cineole and high-cineole eucalyptus oil. Increased activity of cineole ester derivatives compared to their associated hydroxy-cineole and carboxylic acid was not observed. No relationship between lipophilicity of the carboxylic acid portion of cineole ester derivatives and herbicidal activity was observed. The results indicate that these cineole derivatives could be environmentally acceptable herbicides.

Antimicrobial Activity of Lipophilic Avian Eggshell Surface Extracts
  • Olivier Wellman-Labadie* ,
  • Simon Lemaire ,
  • Karlheinz Mann ,
  • Jaroslav Picman , and
  • Maxwell T. Hincke

The avian eggshell cuticle is the waxy outermost layer of the mineralized eggshell in direct contact with the environment. In this study, lipophilic eggshell surface extracts from three domestic species were evaluated for their antimicrobial activity. Chicken and goose extracts demonstrated potent bactericidal activity against both Gram-positive and Gram-negative bacteria, while activity could not be detected for duck eggshell surface extracts. Using the chicken as a model species, evaluation of albumen, fecal material, and uropygial gland extracts eliminated these as a potential source of the observed activity. Results suggest that lipophilic components are incorporated into the egg during its formation and play a role in antimicrobial defense. This study represents the first successful extraction and evaluation of lipophilic antimicrobial components from the avian egg.

Environmental Chemistry
Stabilization of Kerosene/Water Emulsions Using Bioemulsifiers Obtained by Fermentation of Hemicellulosic Sugars with Lactobacillus pentosus
  • Oscar Manuel Portilla-Rivera ,
  • Ana María Torrado ,
  • José Manuel Domínguez , and
  • Ana Belén Moldes*

The results of the present study show that Lactobacillus pentosus can produce extracellular bioemulsifiers by utilizing hemicellulosic sugars from grape marc as a source of carbon. The effectiveness of these bioemulsifiers (LPEM) was studied by preparing kerosene/water (K/W) emulsions in the presence and absence of these emulsifiers. Various parameters such as relative emulsion volume (EV), stabilizing capacity (ES), viscosity, and droplet size of K/W emulsions were measured. The EV values for K/W emulsions stabilized by concentrated LPEM were approximately 74.5% after 72 h of emulsion formation, with ES values of 97%. These values were higher than those obtained with dodecyl sodium sulfate as emulsifier (EV = 62.3% and ES = 87.7%). Additionally, K/W emulsions stabilized by LPEM produced polydisperse emulsions containing droplets of radius between 10 and 40 μm, which were smaller than those obtained for K/W emulsions without LPEM (droplet radius = 60 −100 μm). Moreover, the viscosity values of the K/W emulsions without and with LPEM were approximately 236 and 495 cP, respectively.

Species-Dependent Degradation of Ciprofloxacin in a Membrane Anodic Fenton System

The anodic Fenton treatment method (AFT) has been successfully applied to the removal of ciprofloxacin (CIP), a widely used fluoroquinolone antibiotic, from aqueous solution. Degradation kinetics were found to be species dependent. At initial pH 3.2, CIP remained in its cationic form and the kinetics followed a previously developed AFT model. At an initial near-neutral pH, CIP speciation changed during the degradation, due to pH changes over the process, and no obvious model fit the data. Density functional theory (DFT) calculations indicated a protonated species-dependent reaction affinity toward hydroxyl radicals. A new model based on the AFT model with the addition of species distribution during the degradation was derived, and it was shown to describe the degradation kinetics successfully. Degradation of reference compounds further confirmed that the free carboxylic acid group, which contributes to the species changes, plays a key role in the observed degradation pattern. Furthermore, degradation of reference CIP−metal complexes confirmed that the formation of these complexes does not have a major effect on the degradation pattern. Optimization of CIP degradation was carried out at pH 3.2 with an optimal H2O2/Fe2+ ratio found between 10:1 and 15:1. Three degradation pathways based on mass spectrometry data were also proposed: (1) hydroxylation and defluorination on the aromatic ring (2) oxidative decarboxylation and (3) oxidation on the piperazine ring and dealkylation. By the end of the AFT treatment, neither CIP nor its degradation products were detected, indicating successful removal of antibacterial properties.

Arsenic Contamination of the Environment−Food Chain: A Survey on Wheat as a Test Plant To Investigate Phytoavailable Arsenic in Italian Agricultural Soils and as a Source of Inorganic Arsenic in the Diet
  • Francesco Cubadda* ,
  • Silvia Ciardullo ,
  • Marilena D’Amato ,
  • Andrea Raggi ,
  • Federica Aureli , and
  • Marina Carcea

Seven hundred and twenty-six samples of wheat grains from the majority of Italian agricultural areas were pooled into 141 composite samples, homogeneous with respect to geographical origin and wheat variety. The average arsenic concentration of the pooled samples was 9 ng g−1, with a range of 2−55 ng g−1 (dry weight basis). The spread of arsenic concentrations (coefficient of variation of 91%) was related to spatial variability associated with geochemical and environmental factors. Temporal variability was investigated by a 3-year longitudinal study on 7 wheat cultivars grown in 22 areas of central and northern Italy. Average year-to-year variation in arsenic levels was low, and the average of the coefficients of variation was 23%. These results show that mapping of phytoavailable arsenic in agricultural soils can be done by measuring arsenic concentration in representative samples of wheat grains. Arsenic speciation in the grain showed that As(III) and As(V) were the major As compounds, highlighting the importance of wheat as a source of inorganic arsenic in the diet.

Flavors and Aromas/Chemosensory Perception
Analysis, Occurrence, and Potential Sensory Significance of Five Polyfunctional Mercaptans in White Wines
  • Laura Mateo-Vivaracho ,
  • Julián Zapata ,
  • Juan Cacho , and
  • Vicente Ferreira*

A previously developed analytical method has been improved, validated and adapted for the analysis of 2-furfurylthiol (FFT), 4-methyl-4-mercapto-2-pentanone (MP), 3-mercaptohexyl acetate (MHA), 3-mercaptohexanol (MH) and benzylmercaptan (BM) in 136 white wines from different parts of the world. The overall uncertainty of the determinations was found to be around 20%, which was considered satisfactory given the low levels at which these compounds are found. The levels ranged from the method detection limits (0.5 0.6 2.0 8.0 and 0.5 ng/L for FFT, MP, MHA, MH and BM, respectively) to 225 87.9 591 7255 and 131 ng/L, which implies that nearly all of them can reach more than 100 Odor Units in some wines. The levels are significantly linked to both the grape variety (with the exception of FFT) and to the origin (in the case of Sauvignon Blanc samples), however, the range of variation within groups are so large that clear clusters could not be observed. Different sensory tests carried out on white wine models showed that all these compounds, even at low concentration, play an outstanding role on the aroma of wine, contributing to fruity, fresh and green notes. In some wines they are at concentrations high enough to act as genuine impact compounds.

Food Chemistry/Biochemistry
Comparison on Characterization of Longan (Dimocarpus longan Lour.) Polyphenoloxidase Using Endogenous and Exogenous Substrates
  • Jian Sun* ,
  • Ezhen Zhang ,
  • Liangxiong Xu ,
  • Zhichun Li ,
  • Zhenxing Wang , and
  • Changbao Li

Longan polyphenoloxidase (PPO) was extracted and partially purified using ammonium sulfate precipitation and dialysis. The PPO characterizations were compared using endogenous substrate (−)-epicatechin and exogenous substrate catechol. The optimal pH and optimal temperature for the PPO activity were different when reacting with both substrates. The addition of ethylenediaminetetraacetic acid disodium salt into both substrate−enzyme systems exhibited the same lowest inhibition of the PPO activity. l-Ascorbic acid and l-cysteine were the best inhibitors to endogenous substrate−enzyme system, while l-ascorbic acid and glutathione were most effective inhibitors to exogenous substrate−enzyme system. Cupric (Cu2+), ferric (Fe3+), and ferrous (Fe2+) ions accelerated the enzymatic-catalyzed reactions of both substrates. Kinetic analysis indicated that longan PPO strongly bound endogenous substrate but possessed a higher catalytic efficiency to exogenous substrate, and moreover, (−)-epicatechin was determined as the optimal substrate for longan PPO. This study is useful to exactly illuminate the enzymatic-catalyzed browning mechanism of postharvest longan fruit.

Histaminol: Identification and HPLC Analysis of a Novel Compound in Wine
  • Matteo Bordiga* ,
  • Fabiano Travaglia ,
  • Monica Locatelli ,
  • Marco Arlorio , and
  • Jean Daniel Coïsson

Histaminol, a minor histamine metabolite originating from imidazole acetaldehyde, has been detected in a food matrix as complex as wine. The standard molecule was synthesized, and subsequently the chemical structure was confirmed by ESI-MS and NMR measurements. The development, optimization, and in-house validation of a HPLC-DAD chromatographic method for the quantitative determination of histaminol in wine are described and discussed. The expanded uncertainty (U(k=2)) of the procedure was estimated as 11.06%. Twenty commercial Italian wine samples were selected. All samples (16 red and 4 white wines) were analyzed after a C-18 SPE cartridge fractionation procedure. The content of this alcohol was in the range of 0.289−1.094 mg/L (minimum and maximum values were obtained for Nero d’Avola vintage 2007 and Barolo vintage 1969, respectively).

Protein Aggregation in White Wines: Influence of the Temperature on Aggregation Kinetics and Mechanisms
  • Marie Dufrechou ,
  • Francois-Xavier Sauvage ,
  • Benoit Bach , and
  • Aude Vernhet*

High temperatures (typically 80 °C) are widely used to assess wine stability with regard to protein haze or to study mechanisms involved in their formation. Dynamic light scattering experiments were performed to follow aggregation kinetics and aggregate characteristics in white wines at different temperatures (30−70 °C). Aggregation was followed during heating and cooling to 25 °C. Results were coupled with the study of the time−temperature dependence of heat-induced protein aggregation. At low temperature (40 °C), aggregation developed during heating. Colloidal equilibria were such that attractive interactions between species led to the rapid formation of micrometer-sized aggregates. At higher temperatures (60 and 70 °C), enhanced protein precipitation was expected and observed. However, high temperatures prevented aggregation, which mainly developed during cooling. Depending on the wine, cooling induced the formation of sub-micronic metastable aggregates stabilized by electrostatic repulsions, or the rapid formation of micrometer-sized aggregates, prone to sedimentation.

Comprehensive Study of the Evolution of Gas−Liquid Partitioning of Aroma Compounds during Wine Alcoholic Fermentation
  • Sumallika Morakul ,
  • Violaine Athes ,
  • Jean-Roch Mouret , and
  • Jean-Marie Sablayrolles*

Calculating the gas−liquid partitioning of aromatic molecules during winemaking fermentation is essential to minimize the loss of aroma and to optimize the fermentation conditions. In this study, the effect of the main fermentation parameters on the partition coefficients (ki) of higher alcohols (2-methylpropan-1-ol and 3-methyl butan-1-ol) and esters (ethyl acetate, 3-methyl-1-butyl acetate, and 2-ethyl hexanoate) was assessed. The values of ki were first determined in synthetic media simulating must and wine. They varied considerably with both the hydrophobicity of the compound and the composition of the medium. Then, the effect of temperature on ki was quantified. The absence of any effect of gas composition was also established by replacing air with CO2. Finally, the impact of CO2 stripping was assessed by running specific fermentations in which the rate of CO2 production was kept constant by perfusion with assimilable nitrogen. These fermentations showed that in contrast to temperature and must composition, CO2 stripping did not change the gas−liquid partitioning of higher alcohols and esters.

Synthesis of 7-Hydroperoxycholesterol and Its Separation, Identification, and Quantification in Cholesterol Heated Model Systems
  • Gislaine C. Nogueira ,
  • Bruna Z. Costa ,
  • Antonio E. M. Crotti , and
  • Neura Bragagnolo*

7-Hydroperoxycholesterol is considered to be an intermediate compound of the cholesterol oxidation path as the first product formed when cholesterol is oxidized by triplet oxygen. However, there is a limitation on cholesterol mechanism studies because of the lack of 7-hydroperoxycholesterol analytical standard due to its low stability. To verify the formation of hydroperoxides in cholesterol model systems heated at 140, 180, and 220 °C, 7α-hydroperoxycholesterol was synthesized by cholesterol photooxidation followed by rearrangement at room temperature in chloroform. Its structure was confirmed on the basis of 13C NMR and mass spectra obtained by APCI-LC-MS. The synthesized compound was also used as standard for the quantification of 7-hydroperoxycholesterol as the sum of 7α- and 7β-hydroperoxycholesterol. The results demonstrated that 7-hydroperoxycholesterol is the first compound formed when the temperature is lower (140 °C). However, the concentration of the 7-hydroperoxycholesterol depends on the temperature and time of exposure: the higher the time, the higher the amount of 7-hydroperoxycholesterol at lower temperatures, and the lower the time, the lower the amount of 7-hydroperoxycholesterol at higher temperatures (180 and 220 °C). By the formation of 7-hydroperoxycholesterol, the known cholesterol oxidation mechanism in three phases (initiation, propagation, and termination) could be confirmed once at lower temperatures, the stage of cholesterol oxidation is at initiation, at which hydroperoxide formation predominates.

Molecular Nutrition
Influence of Processing on the Allergenic Properties of Pistachio Nut Assessed in Vitro
  • Reihaneh Noorbakhsh ,
  • Seyed Ali Mortazavi ,
  • Mojtaba Sankian ,
  • Fakhri Shahidi ,
  • Soheila J. Maleki ,
  • Leila Roozbeh Nasiraii ,
  • Reza Falak ,
  • Hamid Reza Sima , and
  • AbdolReza Varasteh*

Pistachio (Pistacia vera) is a tree nut that has been reported to cause IgE-mediated allergic reactions. This study was undertaken to investigate the distinctions between different cultivars of pistachio nut and the influence of different processing on the IgE-binding capacity of whole pistachio protein extracts. The influence of different processes on allergenicity was investigated using competitive inhibition ELISA and Western blotting assays. The Western blotting results of extracts from pistachio cultivars showed no marked difference among them. The IgE-binding capacity was significantly lower for the protein extract prepared from steam-roasted than from raw and dry-roasted pistachio nuts. The results of sensory evaluation analysis and hedonic rating proved no significant differences in color, taste, flavor, and overall quality of raw, roasted, and steam-roasted pistachio nut treatments. The most significant finding of the present study was the successful reduction of IgE-binding by pistachio extracts using steam-roast processing without any significant changes in sensory quality of product.

In Vitro Bioconversion of Polyphenols from Black Tea and Red Wine/Grape Juice by Human Intestinal Microbiota Displays Strong Interindividual Variability
  • Gabriele Gross ,
  • Doris M. Jacobs* ,
  • Sonja Peters ,
  • Sam Possemiers ,
  • John van Duynhoven ,
  • Elaine E. Vaughan , and
  • Tom van de Wiele

Dietary polyphenols in tea and wine have been associated with beneficial health effects. After ingestion, most polyphenols are metabolized by the colonic microbiota. The current study aimed at exploring the interindividual variation of gut microbial polyphenol bioconversion from 10 healthy human subjects. In vitro fecal batch fermentations simulating conditions in the distal colon were performed using polyphenols from black tea and a mixture of red wine and grape juice. Microbial bioconversion was monitored by NMR- and GC-MS-based profiling of diverse metabolites and phenolics. The complex polyphenol mixtures were degraded to a limited number of key metabolites. Each subject displayed a specific metabolite profile differing in composition and time courses as well as levels of these metabolites. Moreover, clear differences depending on the polyphenol sources were observed. In conclusion, varying metabolite pathways among individuals result in different metabolome profiles and therefore related health effects are hypothesized to differ between subjects.

Effects of Protocatechuic Acid on Trans Fat Induced Hepatic Steatosis in Mice
  • Wen-Hu Liu ,
  • Chun-Che Lin ,
  • Zhi-Hong Wang ,
  • Mei-Chin Mong , and
  • Mei-Chin Yin*

The effects of protocatechuic acid (PCA) on hepatic activity and/or mRNA expression of lipogenic enzymes and sterol regulatory element-binding proteins (SREBPs) in mice fed a trans fatty acid (TFA)-rich diet were examined. PCA at 1, 2, or 4% was provided for 10 weeks. Results showed that TFA diet significantly enhanced hepatic activity and mRNA expression of fatty acid synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, stearoyl-CoA desaturase-1, and SREBP-1c (P < 0.05) however, the intake of PCA significantly diminished the activity and mRNA expression of these lipogenic factors and decreased hepatic lipid accumulation (P < 0.05). TFA diet significantly increased hepatic levels of TFA and pro-inflammatory cytokines (P < 0.05). However, PCA intake significantly lowered hepatic content of 18:1 trans and 18:2 trans, as well as reduced the level of test cytokines (P < 0.05). These results indicate that PCA is a potent agent for attenuating TFA-induced hepatic steatosis.

Toxicology in Agriculture and Food
National Study of Exposure to Pesticides among Professional Applicators: An Investigation Based on Urinary Biomarkers
  • Shelley A. Harris* ,
  • Paul J. Villeneuve ,
  • Charlene D. Crawley ,
  • James E. Mays ,
  • Roger A. Yeary ,
  • Kirk A. Hurto , and
  • John D. Meeker

Epidemiologic studies of pesticides have been subject to important biases arising from exposure misclassification. Although turf applicators are exposed to a variety of pesticides, these exposures have not been well characterized. This paper describes a repeated measures study of 135 TruGreen applicators over three spraying seasons via the collection of 1028 urine samples. These applicators were employed in six cities across the United States. Twenty-four-hour estimates (μg) were calculated for the parent compounds 2,4-D, MCPA, mecoprop, dicamba, and imidacloprid and for the insecticide metabolites MPA and 6-CNA. Descriptive statistics were used to characterize the urinary levels of these pesticides, whereas mixed models were applied to describe the variance apportionment with respect to city, season, individual, and day of sampling. The contributions to the overall variance explained by each of these factors varied considerably by the type of pesticide. The implications for characterizing exposures in these workers within the context of a cohort study are discussed.

Binding of Oxytetracycline to Bovine Serum Albumin: Spectroscopic and Molecular Modeling Investigations
  • Zhenxing Chi ,
  • Rutao Liu* ,
  • Yue Teng ,
  • Xiaoyan Fang , and
  • Canzhu Gao

The residue of the widely used veterinary drug oxytetracycline (OTC) in the environment (e.g., animal food, soils, surface water, and groundwater) is potentially harmful. Knowledge of its binding to proteins contributes to the understanding of its toxicity in vivo. This work establishes the binding mode of OTC with bovine serum albumin (BSA) under physiological conditions by spectroscopic methods and molecular modeling techniques. The inner filter effect was eliminated to get accurate data (binding parameters). On the basis of the thermodynamic results and site marker competition experiments, it was considered that OTC binds to site II (subdomain IIIA) of BSA mainly by electrostatic interaction. Furthermore, using the BSA model established with CPHmodels, molecular docking and some other molecular modeling methods were applied to further define that OTC interacts with the Arg 433, Arg 436, Ala 429, and Pro 516 residues of BSA. In addition, UV−visible absorption, synchronous fluorescence, and circular dichroism (CD) results showed that the binding of OTC can cause conformational and some microenvironmental changes of BSA. The work provides accurate and full basic data for clarifying the binding mechanisms of OTC with BSA in vivo and is helpful for understanding its effect on protein function during its transportation and distribution in blood.

Copper Uptake Is Differentially Modulated by Phenylalanine Ammonia-lyase Inhibition in Diploid and Tetraploid Chamomile
  • Jozef Kováčik* ,
  • Bořivoj Klejdus ,
  • Josef Hedbavny , and
  • Jerzy Zoń

The effect of phenylalanine ammonia-lyase (PAL) inhibition by 2-aminoindane-2-phosphonic acid (AIP) in copper-exposed diploid and tetraploid chamomile (Matricaria chamomilla) roots has been studied in a short-term experiment (24 h). Cu evoked stronger induction of PAL activity and accumulation of soluble phenols, flavonols (quercetin and kaempferol), and lignin in diploid plants, whereas AlCl3-reactive flavonoids and phenolic acids did not differ with respect to ploidy. Amounts of hydrogen peroxide and superoxide also preferentially increased in diploid. Surprisingly, PAL activity was restored in both +AIP cultivars, being inversely correlated with the accumulation of free phenylalanine. Notwithstanding this, total soluble phenols and flavonols were more depleted in Cu+AIP diploid roots. Soluble proteins decreased in response to Cu, and AIP had no effect. Among free amino acids, proline increased more visibly in Cu+AIP diploid, suggesting that this could be a protective mechanism in conditions with depleted content of phenols. Decrease in potassium content was ploidy-independent, calcium increased in all Cu variants, and Fe increased in Cu-exposed tetraploid. Shoot Cu content did not differ in Cu-exposed cultivars, but diploid roots contained more Cu. AIP decreased root Cu but increased shoot Cu amounts in diploid, whereas tetraploid plants did not exhibit similar responses. These data indicate that inhibition of root phenolic metabolism by AIP was effective enough, allowing Cu to accumulate in diploid shoots. The present findings are discussed in the context of available data about AIP effects and with respect to the role of phenols in metal uptake.

Comparative Effects of Cellulose and Soluble Fibers (Pectin, Konjac Glucomannan, Inulin) on Fecal Water Toxicity toward Caco-2 Cells, Fecal Bacteria Enzymes, Bile Acid, and Short-Chain Fatty Acids

The aim of this study was to compare the effects of cellulose and three soluble dietary fibers, pectin, konjac glucomannan (KGM), and inulin, on the cytotoxicity and DNA damage of fecal water-treated Caco-2 cells, a human colon adenocarcinoma cell line, and to investigate the fecal components that potentially modulate the fecal toxicity, that is, bacterial enzymes, bile acids, and short-chain fatty acids. Six-week-old BALB/cJ mice were randomly allocated to consume an AIN-93 diet that contained no dietary fiber (fiber-free) or 5% (w/w) cellulose, pectin, KGM, and inulin for 3 weeks. Feces were collected during days 18−21. Fecal waters were co-incubated with Caco-2 cells to determine the cytotoxicity and DNA damage. In addition, the fecal bacterial enzymes, bile acids, and short-chain fatty acids were determined. Results indicated that all fiber diets similarly increased the survival rate (%) of fecal water-treated Caco-2 cells as compared with the fiber-free diet. The inhibition of fecal water-induced DNA damage in Caco-2 cells was greater for the pectin and inulin diets than for the cellulose and KGM diets. In contrast, cellulose exerted the greatest inhibitory effect on the fecal β-glucuronidase activity. Cellulose and all soluble dietary fibers reduced the secondary bile acid concentrations in the fecal water, but only soluble fibers increased the fecal concentrations of short-chain fatty acids, as compared with no fiber. Therefore, this study suggests that all dietary fibers substantially reduced the fecal water toxicity, which is associated with decreased secondary bile acid levels by all fibers, reduced fecal β-glucuronidase activity by cellulose, and increased short-chain fatty acid levels by soluble dietary fibers.

Infinity's Reflection

(If you are interested in Ecology read this)

(Information From Visit To Flutter: Butterflies and Moths In Art and Science exhibit @ the Bell Museum)

This paper is about Lepidopteron—better known as butterflies and moths. My paper will go into some of the basic characteristics about these insects, their life cycles, mating habits and some conclusions in respect to our relationship with them. It is interesting to note that there are 170,000 known species of moths and butterflies, while with primates there are only 233. Their name comes from Greek with Lepis meaning scale (their wings have scales) and ptoron meaning flight.

Lepidopteron have six legs with some degree of difference in positioning. Some have a set of legs which are much shorter and close to the head making it look like they only have four. The two basic groupings o Lepidopteron are Butterflies and Moths. Basic differences are: Moths are nocturnal, much duller in color, their wings overlap and, whereas most butterflies rest with their wings closed above their bodies, moths rest with their wings horizontally spread. There is also a variety of butterflies called Skippers—given that name because their flight has jumpier movements. They too tend to be duller in color.

Lepidopteron drink their food through a tube like proboscis, though some have mandibles to crunch pollen. Males have a need for salt and they will take it in through mud and dung left by birds. In adult life, many eat very little and mostly sugary liquids.

The color patterns which we see are actually composed of the scales on their wings. These scales are interlocked by protein molecules and are hinged or hooked to their wings, so that they keep beat with their wings. They also have compound eyes, which remind one of pixels on a computer. They can see all the colors we do, but they also see ultra-violet light, which I will go into more later.

There are four life cycle stages of the Lepidopteron. The adult attaches eggs with glue like substance (usually to the underside of leaves). She does not care for the eggs, but places them near a food source. They can lay hundreds of eggs and will do so either singularly or in groups with a lot being dependent on how abundant a food source there is in the area. When the caterpillar emerges from the egg it has a voracious appetite and feeds and feeds on leafy substances till it grows enough to where it needs to shed its skin. Caterpillars do this 4-5 times and each shedding is called an Instar. The last time has the most significant hormonal changes with the eating pattern slowing down. The caterpillar then releases a silky substance which it hangs from and the metamorphosis begins. During this time, that the pupae is forming, the caterpillar goes through the radical change to become an adult and finally pushes out of the pupae producing a new adult lepidopteron.

Lepidopteron actually have an anti-freeze like hormone which slows development in the cold– Diapause. Most significantly this occurs in the pupae stage. Monarchs cannot take northern winters and actually migrate to places like Mexico and California. When it gets too hot for them they come back up north. On cool summer days, Lepidopteron often lay in the sun to warm up their muscles before flight. The mating process of Lepidopteron is fascinating. Some will mate immediately coming out of the pupae others like the Monarch will wait three or four days. Others will be injected by the male right into the pupae and come out ready to lay eggs. Still others wait for months. Some during the mating process will stay together overnight or a full sixteen hours with the male eating while they are joined together. Their entire body has sense and smell detectors, so they can easily detect pheromones, as well as on the female antennae. The detection of ultraviolet rays is said to also help to identify the males of the species.

Ultraviolet light also is a protective mechanism to detect reflections and movement. The many sense and smell detectors on their bodies are protection bright color patterns, as with other insects, can be a warning against toxicity. Monarchs eat milk weed to make themselves toxic to predators. Mimicry blends them in with one’s environment camouflaging them. Owl butterflies actually show what looks like the face of an owl, while mating and keeps predators owls feed on away, but he worst enemies to Lepidopteron—aside from man—are insects that attack them in the egg and pupae stages.

Conclusion: Because caterpillars ravage leaves (often on crops), chemicals are used against them, but recently, through DNA, it has been proven butterflies emerged earlier in history and plants depended on pollination through them. Therefore, we are controlling one aspect, while harming pollination. Also, if we consider the males’ dependence on mud and droppings from birds for salt, we again can be exposing them to chemicals through polluted lands and what birds eat. Considering the mystery of the disappearing bees, what about the Lepidopteron? Are we creating, as with other things, chemical resistant mites, etc., which will destroy them and create further hindrances to the pollination process?

Molecular characterization and genetic organization of the inhibitor of apoptosis gene (iap-5) region of the Pieris rapae granulovirus

Pieris rapae granulovirus (PiraGV) is a baculovirus pathogenic to the insect P. rapae (Pieridae, Lepidoptera). Though being known for decades, information on the genetic organization of this virus remains limited. In an effort to characterize this virus, an 11.8 kb BamHI restriction fragment that harbors the inhibitor of apoptosis gene (iap-5) was sequenced. Our results indicate that this region contains important genes such as dnapol, lef-3, lef-9, and dnaligase that are involved in transcription and replication of the virus. The gene content and synteny in this region are highly conserved among granulovirus genomes. Phylogenetic analysis showed that PiraGV genes are more closely related to the Choristoneura occidentalis granulovirus (ChocGV) than other characterized granulovirus (GVs).

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Butterfly Garden

Butterflies, moths, and skippers are some of the most beautiful of all insects. Their striking appearance adds both color and activity to the most pleasing of landscapes. They may be observed more easily and closely than other species of wildlife. Moths expand the enjoyment time of your garden because they are active primarily during the night, while butterflies and skippers are active during the day.

Butterflies, moths, and skippers belong to the Lepidoptera Order and are instrumental in pollinating plants some specific to a single plant species. Lepidopteron should be conserved and managed as they are an essential component of both the animal food chain and the reproductive process of plants.

Basic Needs of the Butterfly and Larvae

Sun: Locate garden in a sunny spot that is sheltered from the wind. This helps to warm the butterflies. A butterfly's body temperature needs to be above 86° before they can fly.

Plants on Which to Lay Eggs

Called host plants, these plants are eaten by the caterpillars after the eggs hatch. Host plants include trees, shrubs, vines, perennials and annuals. Each type of butterfly eats specific host plants.

Eastern Swallowtail: parsley, dill, fennel

Monarch: Butterfly Milkweed

Gulf Fritillary: Passion Flower

Nectar Plants

Adult butterflies use nectar from specific flowers as an energy source. Not all flowers are attractive to butterflies. Adult butterflies like nectar plants in groups of color at different levels, it's easier for butterflies to key in on.

  • Butterfly Weed – Asclepias tuberosa
  • Honeysuckle Vine – ‘John Clayton’
  • Purple Cone Flower – Echinacea purpurea
  • Lantana – Lantana camara
  • Goldenrods – Solidago spp.
  • Asters - Aster spp.
  • Pentas – Pentas lanceolata
  • Glossy Abelia – Abelia x grandiflora
  • Blazing Star – Liatris spp.
  • Bee Balm – Monarda spp.
  • Rudbeckia – ‘Goldstrum’

No Pesticides

Pesticides can kill caterpillars and butterflies. Identify the source of the plant problem before using chemicals. Non-chemical methods are available to combat most common plant pests.


Dušan Žitňan , Ivana Daubnerová , in Handbook of Hormones , 2016

Synthesis and Release

Gene, mRNA, and Precursor

In the moth Bombyx mori , mRNA of burs (burs-α) has 483 nt that encode a 160-aa precursor containing a 20-aa signal peptide and a mature protein of 140 aa residues pburs (burs-β) mRNA has 611 nt and encodes 137-aa precursor that consists of a signal peptide of 24 aa residues and a mature peptide of 113 aa residues.

Distribution of mRNA

Bursicon is expressed in the CNS in a specific segmental network of neurons named 27/704. In the larval CNS of the moth Manduca sexta, bursicon expression is restricted to the subesophageal ganglion (SG), thoracic ganglia, and first abdominal ganglion. Pharate pupae and pharate adults show expression of this heterodimer in all ganglia [4] . In Drosophila larvae, expression of a bursicon heterodimer is confined to abdominal neuromeres 1–4 the additional neurons in the ventral nerve cord express only burs. In pharate adults, bursicon is produced by 2 neurons in the SG and 14 abdominal neurons [5,6] .

Hemolymph and Tissue Concentration

Tissue: Crab Callinectes sapidus, 36 pmol per animal [7] .

20 pM during the molt cycle and

Regulation of Synthesis and Release

In larvae of D. melanogaster, bursicon appears to be released in two temporal waves, the first prior to the onset of ecdysis and the second immediately after ecdysis onset. In adults, bursicon is also released in two consecutive waves. Initial bursicon secretion from two neurons in the SG subsequently elicits its secondary release from a cluster of abdominal neurons, which controls expansion, hardening, and pigmentation of the cuticle and wings [5–7] .

Detection and characterization of recombinant DNA expressing vip3A-type insecticidal gene in GMOs—standard single, multiplex and construct-specific PCR assays

Vegetative insecticidal protein (Vip), a unique class of insecticidal protein, is now part of transgenic plants for conferring resistance against lepidopteron pests. In order to address the imminent regulatory need for detection and labeling of vip3A carrying genetically modified (GM) products, we have developed a standard single PCR and a multiplex PCR assay. As far as we are aware, this is the first report on PCR-based detection of a vip3A-type gene (vip-s) in transgenic cotton and tobacco. Our assay involves amplification of a 284-bp region of the vip-s gene. This assay can possibly detect as many as 20 natural wild-type isolates bearing a vip3A-like gene and two synthetic genes of vip3A in transgenic plants. The limit of detection as established by our assay for GM trait (vip-s) is 0.1%. Spiking with nontarget DNA originating from diverse plant sources had no inhibitory effect on vip-s detection. Since autoclaving of vip-s bearing GM leaf samples showed no deterioration/interference in detection efficacy, the assay seems to be suitable for processed food products as well. The vip-s amplicon identity was reconfirmed by restriction endonuclease assay. The primer set for vip-s was equally effective in a multiplex PCR assay format (duplex, triplex and quadruplex), used in conjunction with the primer sets for the npt-II selectable marker gene, Cauliflower mosaic virus 35S promoter and nopaline synthetase terminator, enabling concurrent detection of the transgene, regulatory sequences and marker gene. Further, the entire transgene construct was amplified using the forward primer of the promoter and the reverse primer of the terminator. The resultant amplicon served as a template for nested PCR to confirm the construct integrity. The method is suitable for screening any vip3A-carrying GM plant and food. The availability of a reliable PCR assay method prior to commercial release of vip3A-based transgenic crops and food would facilitate rapid and efficient regulatory compliance.

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