Survival of Plants During Short-Term BOA-OH Exposure: ROS Related Gene Expression and Detoxification Reactions Are Accompanied With Fast Membrane Lipid Repair in Root Tips.

For the characterization of BOA-OH insensitive plants, we studied the time-dependent effects of the benzoxazolinone-4/5/6/7-OH isomers on maize roots. Exposure of Zea mays seedlings to 0.5 mM BOA-OH elicits root zone-specific reactions by the formation of dark rings and spots in the zone of lateral roots, high catalase activity on root hairs, and no visible defense reaction at the root tip. We studied BOA-6-OH- short-term effects on membrane lipids and fatty acids in maize root tips in comparison to the benzoxazinone-free species Abutilon theophrasti Medik. Decreased contents of phosphatidylinositol in A. theophrasti and phosphatidylcholine in maize were found after 10-30 min. In the youngest tissue, α-linoleic acid (18:2), decreased considerably in both species and recovered within one hr. Disturbances in membrane phospholipid contents were balanced in both species within 30-60 min. Triacylglycerols (TAGs) were also affected, but levels of maize diacylglycerols (DAGs) were almost unchanged, suggesting a release of fatty acids for membrane lipid regeneration from TAGs while resulting DAGs are buildings blocks for phospholipid reconstitution, concomitant with BOA-6-OH glucosylation. Expression of superoxide dismutase (SOD2) and of ER-bound oleoyl desaturase (FAD2-2) genes were contemporaneously up regulated in contrast to the catalase CAT1, while CAT3 was arguably involved at a later stage of the detoxification process. Immuno-responses were not elicited in short-terms, since the expression of NPR1, POX12 were barely affected, PR4 after 6 h with BOA-4/7-OH and PR1 after 24 h with BOA-5/6-OH. The rapid membrane recovery, reactive oxygen species, and allelochemical detoxification may be characteristic for BOA-OH insensitive plants.

Curare alkaloids from Matis Dart Poison: Comparison with d-tubocurarine in interactions with nicotinic, 5-HT3 serotonin and GABAA receptors.

Several novel bisbenzylisoquinoline alkaloids (BBIQAs) have recently been isolated from a Matis tribe arrow poison and shown by two-electrode voltage-clamp to inhibit mouse muscle nicotinic acetylcholine receptors (nAChR). Here, using radioligand assay with Aplysia californica AChBP and radioiodinated α-bungarotoxin ([125I]-αBgt), we show that BBIQA1, BBIQA2, and d-tubocurarine (d-TC) have similar affinities to nAChR orthosteric site. However, a competition with [125I]-αBgt for binding to the Torpedo californica muscle-type nAChR revealed that BBIQAs1, 2, and 3 are less potent (IC50s = 26.3, 8.75, and 17.0 µM) than d-TC (IC50 = 0.39 µM), while with α7 nAChR in GH4C1 cells, BBIQA1 was less potent that d-TC (IC50s = 162 µM and 7.77 µM, respectively), but BBIQA2 was similar (IC50 = 5.52 µM). In inhibiting the Ca2+ responses induced by acetylcholine in Neuro2a cells expressing the mouse adult α1ß1εδ nAChR or human α7 nAChR, BBIQAs1 and 2 had similar potencies to d-TC (IC50s in the range 0.75-3.08 µM). Our data suggest that BBIQA1 and BBIQA2 can inhibit adult muscle α1ß1εδ nAChR by both competitive and noncompetitive mechanisms. Further experiments on neuronal α3ß2, α4ß2, and α9α10 nAChRs, expressed in Xenopus laevis oocytes, showed that similar potencies for BBIQAs1, 2, and d-TC. With α3ß2γ2 GABAAR currents were almost completely inhibited by d-TC at a high (100 µM) concentration, but BBIQAs1 and 2 were less potent (only 40-50% inhibition), whereas in competition with Alexa Fluor 546-α-cobratoxin for binding to α1ß3γ2 GABAAR in Neuro2a cells, d-TC and these analogs had comparable affinities. Especially interesting effects of BBIQAs1 and 2 in comparison with d-TC were observed for 5-HT3AR: BBIQA1 and BBIQA2 were 5- and 87-fold less potent than d-TC (IC50 = 22.63 nM). Thus, our results reveal that these BBIQAs differ from d-TC in their potencies towards certain Cys-loop receptors, and we suggest that understanding the reasons behind this might be useful for future drug design.

Interspecies-cooperations of abutilon theophrasti with root colonizing microorganisms disarm BOA-OH allelochemicals.

A facultative, microbial micro-community colonizing roots of Abutilon theophrasti Medik. supports the plant in detoxifying hydroxylated benzoxazolinones. The root micro-community is composed of several fungi and bacteria with Actinomucor elegans as a dominant species. The yeast Papiliotrema baii and the bacterium Pantoea ananatis are actively involved in the detoxification of hydroxylated benzoxazolinones by generating H2O2. At the root surface, laccases, peroxidases and polyphenol oxidases cooperate for initiating polymerization reactions, whereby enzyme combinations seem to differ depending on the hydroxylation position of BOA-OHs. A glucosyltransferase, able to glucosylate the natural benzoxazolinone detoxification intermediates BOA-5- and BOA-6-OH, is thought to reduce oxidative overshoots by damping BOA-OH induced H2O2 generation. Due to this detoxification network, growth of Abutilon theophrasti seedlings is not suppressed by BOA-OHs. Polymer coats have no negative influence. Alternatively, quickly degradable 6-hydroxy-5-nitrobenzo[d]oxazol-2(3H)-one can be produced by the micro-community member Pantoea ananatis at the root surfaces. The results indicate that Abutilon theophrasti has evolved an efficient strategy by recruiting soil microorganisms with special abilities for different detoxification reactions which are variable and may be triggered by the allelochemical´s structure and by environmental conditions.

6-Hydroxy-5-nitrobenzo[d]oxazol-2(3H)-one-A degradable derivative of natural 6-Hydroxybenzoxazolin-2(3H)-one produced by Pantoea ananatis.

Pantoea ananatis is a bacterium associated with other microorganisms on Abutilon theophrasti Medik. roots. It converts 6-hydroxybenzoxazolin-2(3H)-one (BOA-6-OH), a hydroxylated derivative of the allelochemical benzoxazolin-2(3H)-one, into 6-hydroxy-5-nitrobenzo[d]oxazol-2(3H)-one. The compound was identified by NMR and mass spectrometric methods. In vitro synthesis succeeded with Pantoea protein, with isolated proteins from the Abutilon root surface or with horseradish peroxidase in the presence of nitrite and H2O2. Nitro-BOA-6-OH is completely degraded further by Pantoea ananatis and Abutilon root surface proteins. Under laboratory conditions, 6-hydroxy-5-nitrobenzo[d]oxazol-2(3H)-one inhibits Lepidium sativum seedling growth whereas Abutilon theophrasti is much less affected. Although biodegradable, an agricultural use of 6-hydroxy-5-nitrobenzo[d]oxazol-2(3H)-one is undesirable because of the high toxicity of nitro aromatic compounds to mammals.

Constituents from the bark resin of Schinus molle

ABSTRACT A total of five terpenes was isolated from the bark resin of Schinus molle L., Anacardiaceae, and their structures were determined by spectroscopic techniques. Among these compounds the sesquiterpene hydrocarbon terebinthene showed significant growth inhibitory activity against human colon carcinoma HCT-116 cells. Furthermore, terebinthene and pinicolic acid (5) also showed antibacterial activity against Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 6633.

Coumarins of Loricaria ferruginea

ABSTRACT In the presented research we isolated and characterized compounds from Loricaria ferruginea (Ruiz & Pav.) Wedd., Asteraceae. To the best of our knowledge no data on any compounds from L. ferruginea have been published to this day. As main compounds of the hexane extract we found four known coumarins: 5,7-dimethoxycoumarin; 5,7,8-trimethoxycoumarin; 5-hydroxyobliquine and 5-methoxyobliquine. All the structures were determined by spectroscopic and spectrometric methods.

Benzoxazolinone detoxification by N-Glucosylation: The multi-compartment-network of Zea mays L.

The major detoxification product in maize roots after 24 h benzoxazolin-2(3H)-one (BOA) exposure was identified as glucoside carbamate resulting from rearrangement of BOA-N-glucoside, but the pathway of N-glucosylation, enzymes involved and the site of synthesis were previously unknown. Assaying whole cell proteins revealed the necessity of H2O2 and Fe(2+) ions for glucoside carbamate production. Peroxidase produced BOA radicals are apparently formed within the extraplastic space of the young maize root. Radicals seem to be the preferred substrate for N-glucosylation, either by direct reaction with glucose or, more likely, the N-glucoside is released by glucanase/glucosidase catalyzed hydrolysis from cell wall components harboring fixed BOA. The processes are accompanied by alterations of cell wall polymers. Glucoside carbamate accumulation could be suppressed by the oxireductase inhibitor 2-bromo-4´-nitroacetophenone and by peroxidase inhibitor 2,3-butanedione. Alternatively, activated BOA molecules with an open heterocycle may be produced by microorganisms (e.g., endophyte Fusarium verticillioides) and channeled for enzymatic N-glucosylation. Experiments with transgenic Arabidopsis lines indicate a role of maize glucosyltransferase BX9 in BOA-N-glycosylation. Western blots with BX9 antibody demonstrate the presence of BX9 in the extraplastic space. Proteomic analyses verified a high BOA responsiveness of multiple peroxidases in the apoplast/cell wall. BOA incubations led to shifting, altered abundances and identities of the apoplast and cell wall located peroxidases, glucanases, glucosidases and glutathione transferases (GSTs). GSTs could function as glucoside carbamate transporters. The highly complex, compartment spanning and redox-regulated glucoside carbamate pathway seems to be mainly realized in Poaceae. In maize, carbamate production is independent from benzoxazinone synthesis.

Curare Alkaloids: Constituents of a Matis Dart Poison.

A phytochemical study of dart and arrow poison from the Matis tribe led to the identification of D-(-)-quinic acid, L-malic acid, ethyldimethylamine, magnoflorine, and five new bisbenzyltetrahydroisoquinoline alkaloids (BBIQAs), 1-5. D-Tubocurarine could not be identified among these products. BBIQA (3) contains a unique linkage at C-8 and C-11'. All structures were characterized by a combination of NMR and HRESIMS data. The effects of Matis poison and individual BBIQAs (1-3) on rat muscle nAChR expressed in Xenopus oocytes have been investigated using the two-electrode voltage clamp technique.

Corrigendum to "Synthesis and Characterization of New Palladium(II) Thiosemicarbazone Complexes and Their Cytotoxic Activity against Various Human Tumor Cell Lines".

[This corrects the article DOI: 10.1155/2013/524701.].

Constituents of Corynaea crassa "Peruvian Viagra"

Abstract A phytochemical investigation of methanol and n-hexane extracts of tuber/roots of Corynaea crassa Hook. f., Balanophoraceae, led to the isolation and characterization of β-sitosterol, lupenone, β-amyrone, lupeol, and β-amyrine. Unusual complex 1:1 mixtures of lupenone/β-amyrone and lupeol/β-amyrine obtained from the extracts were identified by NMR and HR-MS experiments. The structure of the 1:1 lupenone/β-amyrone mixture was confirmed by X-ray analysis. These triterpene ketone derivatives, only distinguished either by 5- or 6-membered E ring, co-crystallize in one common unit cell in the solid state.

Discrimination of fennel chemotypes applying IR and Raman spectroscopy: discovery of a new γ-asarone chemotype.

Various vibrational spectroscopy methods have been applied to classify different fennel chemotypes according to their individual profile of volatile substances. Intact fennel fruits of different chemotypes could be successfully discriminated by attenuated total reflectance Fourier transform infrared (ATR-FTIR) and near infrared (NIR) spectroscopy. Solvent extracts (CCl4) of the considered fennel fruits showed characteristic fingerprints with marker bands related to the individual volatile components (trans-anethole, fenchone, estragole, piperitenone oxide, γ-asarone, limonene) for ATR-FTIR and FT-Raman spectroscopy. Especially νC═C and νC═O absorption bands contribute to the different spectral profiles. On the basis of hierarchical cluster analysis, the considered fennel accessions were classified according to gas chromatographic (GC) and vibrational spectroscopic data. Furthermore, even a discrimination of "sweet" and "bitter" fennel fruits, both belonging to the trans-anethole chemotype, could be successfully performed. All vibrational spectroscopical techniques used in this study are rapid and easy to apply. Hence, they allow different fennel chemotypes to be reliably distinguished and can also be used for on-site measurement in free nature.

¹H-qNMR for direct quantification of stachydrine in Leonurus japonicus and L. cardiaca.

(1)H-qNMR-spectroscopy was successfully applied to quantify the pharmacologically active alkaloid stachydrine ((2S)-1,1-dimethylpyrrolidinium-2-carboxylic-acid) in aerial parts of Leonurus japonicus (Leonuri herba, yimucao; Chin.Ph.2010, DAB2012) which are used in TCM and Kampo for the treatment of various gynaecological and cardiovascular disorders. Pharmacological publications on this betaine describe cardiovascular, hypotensive, and tissue-protective effects. However, its pharmacopeial analytics poses severe difficulties as it does not contain any chromophore suitable for HPLC-UV-detection. Nine samples from three countries were prepared as decoctions and freeze-dried. (1)H-NMR-spectra were recorded in D2O. The direct-quantitative (1)H-qNMR-procedure was carried out using the N-CH3-singlet at δ 3.03 ppm in comparison to the δ 6.18 ppm singlet of the two vinylic protons of maleic-acid, which was identified as a most favourable internal standard. The quantification limit of stachydrine was 0.44 mg/g drug material. Neither reference-compounds for calibration-curves nor sample-pre-purification was necessary. This protocol revealed stachydrine contents in the range from 0.09 up to 1.01% (w/w) for the tested yimucao samples. Furthermore, between 0.18 and 0.21% of stachydrine was found in the L. japonicus fruit-drug (Leonuri fructus, chongweizi; Chin.Ph.2010) which was examined for this constituent for the first time. In four co-investigated samples of the closely related and similarly used European herb Leonurus cardiaca Ph.Eur., even higher contents up to 1.55% were attested. The presented quantitative (1)H-qNMR-method was shown to be precise with respect to concentration, and yielded highly reproducible data in a series of inter-day repetitions. Methodically, (1)H-qNMR may be a powerful tool for quality assurance of stachydrine containing plants and herbal drugs, especially for industrial routine protocols.

Anthraquinone Content in Noni (Morinda citrifolia L.).

Noni has been used in traditional medicine and as food for thousands of years. While the fruits serve as food and internal medicine, leaves were traditionally used only topically. In recent years, concern regarding the possible content of anthraquinones in noni has led to scrutiny by the European Food Safety Authority. Little research existed on the content of anthraquinones in different noni preparations, with no information about the potential effect of harvest and preparation methods. Our research focused on lucidin, alizarin, and rubiadin, the most important anthraquinones from a health perspective. We found that the production process (fermentation/juice production versus drying/lyophilization) has no effect on the anthraquinone content. The source product, however, does have implications: noni fruit puree from which seeds had been removed as well as consumer products produced from such puree had no detectable amounts of any anthraquinones. Products that did contain seed or leaf material in all cases did contain partly significant amounts of anthraquinones. To alleviate safety concerns, we suggest that noni products, whether fermented or unfermented juice or powder, should be derived only from fully ripe noni fruits, and that any seed material needs to be removed during the production process.

Hünlich base: (re)discovery, synthesis, and structure elucidation after a century.

After almost 100 years, the structure of the product of the reaction between 2,4-diaminotoluene and formaldehyde was elucidated: derivative 3, which we call the Hünlich base, was synthesized on a multigram scale and its enantiomers were easily separated in preparative amounts. Furthermore, transformation of the NH2 groups to the corresponding bis-iodides and bis-azides is presented. The latter was also used for desymmetrization by click chemistry.

Synthesis and Characterization of New Palladium(II) Thiosemicarbazone Complexes and Their Cytotoxic Activity against Various Human Tumor Cell Lines.

The palladium(II) bis-chelate complexes of the type [Pd(TSC(1-5))2] (6-10), with their corresponding ligands 4-phenyl-1-(acetone)-thiosemicarbazone, HTSC(1) (1), 4-phenyl-1-(2'-chloro-benzaldehyde)-thiosemicarbazone, HTSC(2) (2), 4-phenyl-1-(3'-hydroxy-benzaldehyde)-thiosemicarbazone, HTSC(3) (3), 4-phenyl-1-(2'-naphthaldehyde)-thiosemicarbazone, HTSC(4) (4), and 4-phenyl-1-(1'-nitro-2'-naphthaldehyde)-thiosemicarbazone, HTSC(5) (5), were synthesized and characterized by elemental analysis and spectroscopic techniques (IR and (1)H- and (13)C-NMR). The molecular structure of HTSC(3), HTSC(4), and [Pd(TSC(1))2] (6) have been determined by single crystal X-ray crystallography. Complex 6 shows a square planar geometry with two deprotonated ligands coordinated to Pd(II) through the azomethine nitrogen and thione sulfur atoms in a cis arrangement. The in vitro cytotoxic activity measurements indicate that the palladium(II) complexes (IC50 = 0.01-9.87 µM) exhibited higher antiproliferative activity than their free ligands (IC50 = 23.48-70.86 and >250 µM) against different types of human tumor cell lines. Among all the studied palladium(II) complexes, the [Pd(TSC(3))2] (8) complex exhibited high antitumor activity on the DU145 prostate carcinoma and K562 chronic myelogenous leukemia cells, with low values of the inhibitory concentration (0.01 and 0.02 µM, resp.).

Cannabinoid receptor type 2 (CB2)-selective N-aryl-oxadiazolyl-propionamides: synthesis, radiolabelling, molecular modelling and biological evaluation.

BACKGROUND: The endocannabinoid system is involved in many physiological and pathological processes. Two receptors (cannabinoid receptor type 1 (CB1) and type 2 (CB2)) are known so far. Many unwanted psychotic side effects of inhibitors of this system can be addressed to the interaction with CB1. While CB1 is one of the most abundant neuroreceptors, CB2 is expressed in the brain only at very low levels. Thus, highly potent and selective compounds for CB2 are desired. N-aryl-((hetero)aromatic)-oxadiazolyl-propionamides represent a promising class of such selective ligands for the human CB2. Here, a library of various derivatives is studied for suitable routes for labelling with 18F. Such 18F-labelled compounds can then be employed as CB2-selective radiotracers for molecular imaging studies employing positron emission tomography (PET). RESULTS: By varying the N-arylamide substructure, we explored the binding pocket of the human CB2 receptor and identified 9-ethyl-9H-carbazole amide as the group with optimal size. Radioligand replacement experiments revealed that the modification of the (hetero)aromatic moiety in 3-position of the 1,2,4-oxadiazoles shows only moderate impact on affinity to CB2 but high impact on selectivity towards CB2 with respect to CB1. Further, we could show by autoradiography studies that the most promising compounds bind selectively on CB2 receptors in mouse spleen tissue. Molecular docking studies based on a novel three-dimensional structural model of the human CB2 receptor in its activated form indicate that the compounds bind with the N-arylamide substructure in the binding pocket. 18F labelling at the (hetero)aromatic moiety at the opposite site of the compounds via radiochemistry was carried out. CONCLUSIONS: The synthesized CB2-selective compounds have high affinity towards CB2 and good selectivity against CB1. The introduction of labelling groups at the (hetero)aromatic moiety shows only moderate impact on CB2 affinity, indicating the introduction of potential labelling groups at this position as a promising approach to develop CB2-selective ligands suitable for molecular imaging with PET. The high affinity for human CB2 and selectivity against human CB1 of the herein presented compounds renders them as suitable candidates for molecular imaging studies.

The effects of substituents and solvents on the ground-state π-electronic structure and electronic absorption spectra of a series of model merocyanine dyes and their theoretical interpretation.

Two series of new merocyanine dyes have been synthesised and the dependence of their electronic structure on substituents and solvents has been studied by NMR spectroscopy, by using both the NMR (13)C chemical shifts between adjacent C atoms in the polymethine chain and the (3)J(H,H) coupling constants for trans-vicinal protons. The widely used valence bond (VB) model based on two contributing structures cannot account theoretically for the observed alternating π-electron density in the polymethine chain. In addition, the prediction of zero-π-bond order alternation (or zero-bond length alternation) by this model is also incorrect. However, the results are consistent with the predictions of a qualitative VB model which considers the resonance of a positive charge throughout the whole polymethine chain. Based on this model and the Franck-Condon principle the effect of substituents and solvents on the fine structure of the electronic spectra of these dyes can be explained as vibronic transitions from the vibrational state v = 0 to v', where v is the vibrational quantum number of the totally symmetric C=C valence vibration of the polymethine chain in the electronic ground state and v' is that in the electronic excited state. In contrast, neither the effects of substituents or solvents on the electronic structure of merocyanines and their electronic spectra can be accounted for by the simple two state VB model.

Allium ursinum L.: bioassay-guided isolation and identification of a galactolipid and a phytosterol exerting antiaggregatory effects.

AIMS: We wanted to investigate the possible antithrom botic effects and elucidate the chemical identity of the active principles involved in inhibitory effects against adenosine diphosphate(ADP)-induced aggregation of human platelets by wild garlic, Allium ursinum L. METHODS: For this purpose, a bioassay-guided isolation procedure was used followed by spectrometric identification of pure active compounds. For the bioassay, blood was taken from healthy human volunteers and platelet-rich plasma was prepared for turbidimetric platelet aggregation tests. Platelet-rich plasma, stimulated with 20 µ mol/l of ADP, was treated with extracts of different polarities, fractions and isolated single compounds from A. ursinum. The extracts were investigated by thin-layer chromatography(TLC), HPLC, mass spectroscopy, electrospray ionization mass spectrometry (ESI-MS) and 1/2-dimensional (1)H/(13) C-nuclear magnetic resonance (NMR) spectroscopic techniques. RESULTS: Fresh A. ursinum leaves were extracted with ethanol, which was the potent form that effectively inhibited ADP-induced aggregation of human platelets. Thisethanolic extract was subjected to liquid-liquid partition. Whilst the aqueous phase, containing the moiety of cysteine sulphoxide and thiosulphinate derivatives, showed only weak activity on platelet aggregation, the ethyl-acetate and particularly the chloroform partitions showed the high estaggregation-inhibiting potency. Thus, in our bioassay, the effects of alliins/allicins could be neglected. The chloroform phase, possessing the strongest activity, was separated into 28 fractions by gradient-elution open column chromatography on silica gel. The most active fractions 11­17 were separated again, yielding 10 subfractions. This afforded 1,2-di-O-α-linolenoyl-3-O-ß-D-galactopyranosyl-sn-glycerol and ß-sitosterol-3-O-ß-D-glucopyranoside, the structures of which were determined by ESI-MS and 1/2-dimensional (1)H/(13) CNMR spectroscopic techniques. Furthermore, the minute amounts of volatile oil of A. ursinum leaves obtained by steam distillation according to Ph. Eur. could be evaluated asa third aggregation-inhibiting principle. CONCLUSION: In our study, for the first time, 2 active, non-sulphur-containing constituents of wild garlic, namely a galactolipid and a phytosterol,could be identified exhibiting inhibitory action on ADP-induced aggregation in human blood platelets. As a major constituent, the galactolipid, 1,2-di-O-α-linolenoyl-3-O-ß-D-galactopyranosyl-sn-glycerol, not yet found in Allium sp., appears as a new, highly useful marker substance for A. ursinum drugs, or their pharmaceutical or food preparations,as shown by our orientating TLC analyses.

Synthesis of peptides containing 5-hydroxytryptophan, oxindolylalanine, N-formylkynurenine and kynurenine.

ROS, continuously produced in cells, can reversibly or irreversibly oxidize proteins, lipids, and DNA. At the protein level, cysteine, methionine, tryptophan, and tyrosine residues are particularly prone to oxidation. Here, we describe the solid phase synthesis of peptides containing four different oxidation products of tryptophan residues that can be formed by oxidation in proteins in vitro and in vivo: 5-HTP, Oia, Kyn, and NFK. First, we synthesized Oia and NFK by selective oxidation of tryptophan and then protected the α-amino group of both amino acids, and the commercially available 5-HTP, with Fmoc-succinimide. High yields of Fmoc-Kyn were obtained by acid hydrolysis of Fmoc-NFK. All four Fmoc derivatives were successfully incorporated, at high yields, into three different peptide sequences from skeletal muscle actin, creatin kinase (M-type), and ß-enolase. The correct structure of all modified peptides was confirmed by tandem mass spectrometry. Interestingly, isobaric peptides containing 5-HTP and Oia were always well separated in an acetonitrile gradient with TFA as the ion-pair reagent on a C18-phase. Such synthetic peptides should prove useful in future studies to distinguish isobaric oxidation products of tryptophan.