Selective disruption of wheat secondary metabolism by herbicide safeners

In wheat (Triticum aestivum L.), treatment with herbicide safeners enhances the expression of enzymes involved in pesticide detoxification and reduces crop sensitivity to herbicides. Since these same enzymes are involved in plant secondary metabolism, it was of interest to determine whether or not the safener cloquintocet mexyl perturbed phenolic metabolism in wheat seedlings. LC/ESI/MS analysis identified 14 phenolic substrates in the shoots of young wheat plants. Fragmentation imposed by collision induced dissociation identified specific C-glycosidic conjugates of 4′,5,7-trihydroxflavone (apigenin), 3′,4′,5,7-tetrahydroxyflavone (luteolin) and 3′-O-methylluteolin. Treatment of 7-day-old wheat shoots with cloquintocet mexyl resulted in an accelerated depletion of the conjugates of all three flavones, most notably with the glycosides of luteolin. In contrast, safener treatment caused the selective accumulation of 4′,5,7-trihydroxy-3′,5′-dimethoxyflavone (tricin) and the phenylpropanoid ferulic acid. Changes in phenolic content were associated with an increase in O-methyltransferase and C-glucosyltransferase activity toward flavonoid substrates as well as the classic enhancement of detoxifying glutathione transferases. Our results suggest that in addition to altering the capacity of wheat to metabolise herbicides and other xenobiotics, safeners can also cause a selective shift in the metabolism of endogenous phenolics.     

Chemical characterisation of the terpenoid constituents of the Algerian plant Launaea arborescens

Chemical investigation of endemic Algerian plant Launaea arborescens resulted in the isolation of a series of triterpenes and sesquiterpenes from both the aerial parts and roots. Five terpenoids have been chemically characterised by means of spectral methods mainly NMR techniques. In addition, the absolute stereochemistry at the chiral carbon in the side chain of 8-deoxy-15-(3′-hydroxy-2′-methyl-propanoyl)-lactucin-3′-sulfate (6) has been determined by comparison of the ¹H NMR spectra of Mosher derivatives of 6 with those of the corresponding MTPA esters of model compounds.     

Sequential O-methylation of tricetin by a single gene product in wheat

Flavonoid compounds are ubiquitous in nature. They constitute an important part of the human diet and act as active principles of many medicinal plants. Their O-methylation increases their lipophilicity and hence, their compartmentation and functional diversity. We have isolated and characterized a full-length flavonoid O-methyltransferase cDNA (TaOMT2) from a wheat leaf cDNA library. The recombinant TaOMT2 protein was purified to near homogeneity and tested for its substrate preference against a number of phenolic compounds. Enzyme assays and kinetic analyses indicate that TaOMT2 exhibits a pronounced preference for the flavone, tricetin and gives rise to three methylated enzyme reaction products that were identified by TLC, HPLC and ESI-MS/MS as its mono-, di- and trimethyl ether derivatives. The sequential order of tricetin methylation by TaOMT2 is envisaged to proceed via its 3′-mono- → 3′,5′-di- → 3′,4′,5′-trimethyl ether derivatives. To our knowledge, this is the first report of a gene product that catalyzes three sequential O-methylations of a flavonoid substrate.     

Structure and thermal stability of pyruvated carrageenans from the red alga Coccotylus truncatus

The composition, structure, and thermal stability of carrageenans from unattached Coccotylus truncatus (the Baltic Sea, Estonia) were investigated. The complex polysaccharide was characterized by ¹³C NMR and FTIR spectroscopy, ICP-OES and gel permeation chromatography methods. The main components of C. truncatus galactan are 3,6-anhydro-α-d-galactose-2-sulfate (30 ± 1.5%) and β-d-galactose-4-sulfate (45.3%), indicating a ι-carrageenan backbone. As the minor components, α-d-galactose-2,6-disulfate (12 ± 2%) from ν-carrageenan and 4′,6′-pyruvated β-d-galactopyranosyl residues (1.4%) from pyruvated α-carrageenan are found to be present, latter being responsible for the undersulfated nature of the galactan. The native polysaccharide with the average molecular weight of about 1500 kDa is highly susceptible to thermal degradation. The high-temperature treatment of this galactan gives products with 3,6-anhydro-α-d-galactose units predominantly at the reducing end. The carrageenan extraction from C. truncatus gives characteristically low yields (12-17%); weak gelling ability of the polysaccharides from this seaweed species (gel strength 30-40 g/cm²) does not depend significantly on extraction conditions.     

Galactans from Cryptonemia species. Part II: Studies on the system of galactans of Cryptonemia seminervis (Halymeniales) and on the structure of major fractions

Cryptonemia seminervis biosynthesizes a family of d,l-hybrid galactans based on the classical 3-linked β-d-galactopyranosyl→4-linked α-d- and α-l-galactopyranosyl alternating sequence (A-units→B-units) with major amounts of α-d- and α-l-galactose and 3,6-anhydro-d- and l-galactose and lesser percentages of 3,6-anhydro-2-O-methyl-l-galactose, 2-O-methyl-, 4-O-methyl- and 6-O-methylgalactoses. The dispersion of structures in this family is based on five structural factors, namely: (a) the amount and position of substituent groups as sulfate (major), pyruvic acid ketals, methoxyl and glycosyl side-chain (4-O-methyl galactopyranosyl and/or xylosyl); (b) the ratio galactose/3,6-anhydrogalactose in the B-units; (c) the ratio d,l-galactoses and d,l-3,6-anhydrogalactoses also in the B-units, (d) the formation of diads and (e) the sequence of the diads in the linear backbone. Considering these variables it is not unexpected to find in the fractions studied at least 18 structural units producing highly complex structures. Structural studies carried out in two major fractions (S2S-3 and S2S-4) showed that these galactans were formed mainly by β-d-galactopyranosyl 2-sulfate (20 and 11.9 mol %), β-d-galactopyranosyl 2-sulfate 4,6-O-(1′-carboxyethylidene) (8.9 and 6.0 mol %) and β-d-galactopyranosyl 2,6-sulfate (5.4 and 18.6 mol %), together with 3,6-anhydro-α-l-galactopyranosyl (11.4 and 7.3 mol %) and 3,6-anhydro-α-l-galactopyranosyl 2-sulfate (4.9 and 15.4 mol %) and minor quantities of 12-15 other structural units.Preparative alkaline treatment carried out on fraction (S2S-3) produced a quantitative formation of 3,6-anhydro α-l-galactopyranosyl units from precursor units (α-l-galactose 6-sulfate and α-l-galactose 2,6-sulfate). Kinetic studies on this 3,6-anhydro cyclization show a rate constant of 5.2 × 10⁴ s⁻¹ indicating diads of the type G→L6S/2,6S. Data from chemical, spectroscopic and kinetic studies suggest that, in S2S-3, the agaran block in the d,l-hybrid galactan is composed of the following diads: G(6R)→L6S/2,6S and G2S(P)(2,6S)→LA(2S)(2R)(2M) and the carrageenan block of G2S(P)→D(2S)(2,3S)(3S)(3,6S) in a molar ratio of agaran to carrageenan structures of ∼2:1.     

Efficient synthesis of 6′-sialyllactose, 6,6′-disialyllactose, and 6′-KDO-lactose by metabolically engineered E. coli expressing a multifunctional sialyltransferase from the Photobacterium sp. JT-ISH-224

We have previously reported the efficient conversion of lactose into 3′-sialyllactose by high cell density cultures of a genetically engineered Escherichia coli strain expressing the Neisseria meningitidis gene for α-(2→3)-sialyltransferase [Fierfort, N.; Samain, E. J. Biotechnol. 2008, 134, 261-265.]. First attempts to use a similar strategy to produce 6′-sialyllactose with a strain expressing α-(2→6)-sialyltransferase from the Photobacterium sp. JT-ISH-224 led to the production of a trisaccharide that was identified as KDO-lactose (2-keto-3-deoxy-manno-octonyllactose). This result showed that α-(2→6)-sialyltransferase was able to use CMP-KDO as sugar donor and preferentially used CMP-KDO over CMP-Neu5Ac. By reducing the expression level of the sialyltransferase gene and increasing that of the neuABC genes, we have been able to favour the formation of 6′-sialyllactose and to prevent the formation of KDO-lactose. However, in this case, a third lactose derivative, which was identified as 6,6′-disialyllactose, was also produced. Formation of 6,6′-disialyllactose was mainly observed under conditions of lactose shortage. On the other hand, when the culture was continuously fed with an excess of lactose, 6′-sialyllactose was almost the only product detected and its final concentration was higher than 30 g/L of culture medium.     

Mimulaxanthin—a new allenic xanthophyll from the petals of Mimulus guttatus

Besides neoxanthin and deepoxineoxanthin, the main xanthophylls in the petals of Mimulus guttatus, an additional pigment, mimulaxanthin, has been obtained; it is 3,3′–5,5′-tetrahydroxy-6′-hydro-7-dehydro-β-carotene. Its neoxanthin-like spectrum is not changed by acids. Two hydroxyl groups are acetylatable. The diacetate can be silylated to a di(trimethylsilyl)-ether, and with acidic alcohols, yields a monoether. With CHCl₃ plus HCl 18 defined reaction-products are obtained, one of which is 3,3′-di-hydroxy-7,8-didehydro-α-carotene (monadoxanthin). One of the LiAlH₄-reduction products of neoxanthin is identical with mimulaxanthin, whereas mimulaxanthin with LiAlH₄ gives an acetylenic derivative. These results have been confirmed by IR spectrometry. Mimulaxanthin is not an artefact formed during extraction and purification.     

Interconversions of different forms of vitamin B6 in tobacco plants

There are six different vitamin B₆ (VB₆) forms, pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), pyridoxal 5′-phosphate (PLP), pyridoxamine 5′-phosphate (PMP), and pyridoxine 5′-phosphate (PNP), of which PLP is the active form. Although plants are a major source of VB₆ in the human diet, and VB₆ plays an important role in plants, the mechanisms underlying the interconversions of different VB₆ forms are not well understood. In this study, in vitro tobacco plants were grown on Murashige and Skoog (MS) basal media supplemented with 100 mg/L of PM, PL or PN and the abundance of the different B₆ vitamers in leaf tissue was quantified by high performance liquid chromatography (HPLC). The total amount of VB₆ was about 3.9 μg/g fresh weight of which PL, PM, PN, PLP and PMP accounted for 23%, 14%, 37%, 20% and 6%, respectively. Tobacco plants contained a trace amount of PNP. Supplementation of the culture medium with any of the non-phosphorylated vitamers resulted in an increase in total VB₆ by about 10-fold, but had very little impact on the concentrations of the endogenous phosphorylated vitamers. Administration of either PM or PN increased their endogenous levels more than the levels of any other endogenous B₆ vitamers. PL supplementation increased the levels of plant PN and PM significantly, but not that of PL, suggesting that efficient conversion pathways from PL to PN and PM are present in tobacco. Additionally, maintenance of a stable level of PLP in the plant is not well-correlated to changes in levels of non-phosphorylated forms.     

Solvolysis of chenodeoxycholic acid sulfates

Chemical solvolysls of chenodeoxycholic acid sulfates was studied using 4 published methods. Quantitative recovery of chenodeoxycholic acid from the 3-sulfate was obtained with each method. However, only 2 methods yielded chenodeoxycholic acid after solvolysis of the 7-sulfate. In each instance a compound resembling lithocholic acid by GLC but identifiable as a derivative of chenodeoxycholic acid by mass spectrometry was obtained and represents a product formed during solvolysis. Failure to obtain adequate solvolysis of chenodeoxycholic acid 7-sulfate can lead to false identification of monohydroxy bile acids and apparent absence of the 7-sulfate and disulfate esters.     

Syntheses of chondroitin 4- and 6-sulfate pentasaccharide derivatives having a methyl beta-D-glucopyranosiduronic acid at the reducing end

The syntheses are reported of beta-D-GlcpA-(1-->3)-beta-D-GalpNAc-(1-->4)-beta-D-GlcpA-(1- ->3)-beta-D-GalpNAc-(1-->4)-beta-D-GlcpA-(1-->OMe), O-sulfonated at C-4 or C-6 of the aminosugar moieties, which represent structural elements of chondroitin 4- and 6-sulfate proteoglycans. Starting from a synthetic disaccharide glycosyl acceptor, the stepwise or blockwise construction of the sugar backbone with appropriate synthons led to a pentasaccharide tetraol, which was used as a common intermediate. Selective 6-O-sulfonation of this tetraol, followed by saponification, gave the 6-sulfate derivative, whereas selective 6-O-benzoylation, followed by O-sulfonation and saponification, afforded the 4-sulfate derivative as their sodium salts.     

Phytotoxic polyacetylenes from roots of Russian knapweed (Acroptilon repens (L.) DC.)

There are several factors thought to assist invasive weeds in colonization of ecosystems. One of these factors is allelopathy, the negative effect of chemicals produced by one plant on neighboring plants, frequently mediated through root exudates and other plant leachates. Acroptilon repens (Asteraceae) is one of the most invasive and ecologically threatening weed species in western North America. A bioassay-guided fractionation of the root extracts of this plant led to the isolation of five polyacetylenic compounds, of which one [5′-methoxy-1′-(5-prop-1-yn-1-yl-2-thienyl)-hexa-2′,4′-diyin-6′-yl acetate] was hitherto unknown. The structures of these compounds were elucidated on the basis of spectroscopic analysis (IR, ESIMS, ¹H, ¹³C NMR and 2D NMR). All of the compounds obtained, except 1-chloro-4-(5-penta-1,3-diyn-1-yl-2-thienyl)but-3-yn-2-ol, showed phytotoxic activity against Arabidopsis thaliana seedlings. The presence of 4′-chloro-1′-(5-penta-1,3-diyn-1-yl-2-thienyl)-but-2′-yn-3′-ol was detected in the root exudates of aeroponically grown A. repens plants. None of the polyacetylenes isolated in this study were found in Colorado soils collected between September 2006 and July 2007 in an A. repens colonized site. However, polyacetylene 5 in A. repens infested soil from Washington was found in June, 2007. Contrary to our previous report, the compound 7,8-benzoflavone (6) was not detected in root exudates, nor was it encountered in extracts of roots, aerial parts or infested soil. Since we could not repeat this work, the original report has been retracted [Stermitz, F.R., Bais, H.P., Foderaro, T.A., Vivanco, J.M., 2003. 7,8-Benzoflavone: a phytotoxin from root exudates of invasive Russian knapweed [A retraction]. Phytochemistry 64, 493-497.].     

Phenolic analogs of the N,C-coupled naphthylisoquinoline alkaloid ancistrocladinium A, from Ancistrocladus cochinchinensis (Ancistrocladaceae), with improved antiprotozoal activities

The first N,8′-coupled naphthylisoquinoline alkaloids with free phenolic OH groups, 4′-O-demethylancistrocladinium A and 6,4′-O-didemethylancistrocladinium A, have been isolated from the leaves and bark of the Vietnamese liana Ancistrocladus cochinchinensis, along with its known, non-phenolic parent compound, ancistrocladinium A, and four C,C-coupled representatives. The structure elucidation was achieved by chemical, spectroscopic, and chiroptical methods. The mono-phenolic alkaloid showed excellent activities in particular against the pathogen causing Chagas’ disease, Trypanosoma cruzi.     

Iridoids from Gentiana loureirii

Iridoid glycosides, 2′,3′,6′-tri-O-acetyl-4′-O-trans-p-(O-β-d-glucopyranosyl)coumaroyl-7-ketologanin (1), 2′-O-caffeoylloganic acid (2), 2′-O-p-hydroxybenzoylloganic acid (3), 2′-O-trans-p-coumaroylloganic acid (4), and 2′-O-cis-p-coumaroylloganic acid (5), were isolated from whole plants of Gentiana loureirii along with six known iridoids, 7-ketologanin (6), loganin (7), loganic acid (8), sweroside, boonein, and isoboonein, and three other known compounds. Their structures were elucidated by spectroscopic means and chemical correlations. The isolated iridoids were evaluated for antibacterial and antioxidant activities, but were either inactive or very weakly active.     

Antibacterial phenolic components from Eriocaulon buergerianum

Five phenolic components, 1,3,6-trihydroxy-2,5,7-trimethoxyxanthone (1), 7,3′-dihydroxy-5,4′,5′-trimethoxyisoflavone (2), toralactone-9-O-β-d-glucopyranoside (3), patuletin-3-O-[2-O-E-feruloyl-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranoside] (4), patuletin-3-O-[β-d-glucopyranosyl-(1 → 3)-2-O-E-caffeoyl-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranoside] (5), along with 19 known compounds were isolated from Eriocaulon buergerianum (Eriocaulaceae). Their structures were determined by spectroscopic and chemical methods. All 24 isolated compounds were tested against the pathogenic bacteria Staphylococcus aureus (ATCC 25923); as a result, 10 compounds were found to exhibit antibacterial activity with MICs ranging from 32 to 256 μg/ml.     

Synthesis and characterization of quaternized β-chitin

Water-soluble 2′-O-hydroxypropyltrimethylammoniumchitin chloride (2′-O-HTACCt) was prepared directly from β-chitin and 3-chloro-2-hydroxypropyltrimethylammonium chloride (CTA) in basic medium. The effect of alkali concentration, reaction temperature, reaction time, and dosage of CTA on yield and degree of substitution (DS) of 2′-O-HTACCt were studied. These quaternized chitin derivatives were characterized by FTIR and ¹H NMR spectroscopy, conductometric titration, and elemental analysis methods. Research results indicate that β-chitin can react directly with CTA to produce a water-soluble 2′-O-HTACCt derivative with a high DS. The optimal preparation conditions were determined to be 35-40 wt % (aq NaOH), 40 °C (reaction temperature), 6 h (reaction time), and 4 (molar ratio of CTA to β-chitin unit).     

Tyrosinase activity of Greyia flanaganii (Bolus) constituents

Hyper-pigmentation of the skin is a common problem that is prevalent in middle aged and elderly people. It is caused by over production of melanin. Tyrosinase is known to be the key enzyme in melanin production. Ethanolic extract of Greyia flanaganii leaves showed significant (P < 0.05) antityrosinase activity exhibiting the IC₅₀ of 32.62 μg/ml. The total extract was further investigated for its toxicity and effect on melanin production by melanocytes cells, and showed significant inhibition (P < 0.05) (20%) of melanin production at 6.25 μg/ml and low levels of cytotoxicity (IC₅₀ < 400 μg/ml). The amount of antioxidants necessary to decrease the initial DPPH absorbance by 50% (EC₅₀) by the total ethanolic extract was found to be 22.01 μg/ml. The effect of G. flanaganii against acne causing bacteria, Propionibacterium acnes, was investigated using microdilution assay. The MIC of the extract of G. flanaganii was found to be 250 μg/ml. Bioassay-guided fractionation led to the isolation of (3S)-4-hydroxyphenethyl 3-hydroxy-5-phenylpentanoate (1), 2′,4′,6′-trihydroxydihydrochalcone (2), 2′,6′,4-trihydroxy-4′-methoxydihydrochalcone (3), 2′,6′-dihydroxy-4′-methoxydihydrochalcone (4), 5,7-dihydroxyflavanone [(2S)-pinocembrin] (5), 2′,6′-dihydroxy-4′,4-dimethoxy dihydrochalcone (6) and (2R,3R)-3,5,7-trihydroxy-3-O-acetylflavanone (7). The isolated compounds were tested for their antioxidant, cytotoxicity, tyrosinase inhibition and antibacterial activities. Compound 2 exhibited significant (P < 0.05) antityrosinase activity exhibiting the IC₅₀ of 69.15 μM. The isolated compounds showed low toxicity of the cells with reduction of melanin content of the cells. All compounds tested showed good radical scavenging activity. These data indicates that G. flanaganii extract and its isolated phenolic constituents could be possible skin lightening agents.     

New class of acetylcholinesterase inhibitors from the stem bark of Knema laurina and their structural insights

Bioassay-guided extraction of the stem bark of Knema laurina showed the acetylcholinesterase (AChE) inhibitory activity of DCM and hexane fractions. Further repeated column chromatography of hexane and DCM fractions resulted in the isolation and purification of five alkenyl phenol and salicylic acid derivatives. New compounds, (+)-2-hydroxy-6-(10′-hydroxypentadec-8′(E)-enyl)benzoic acid (1) and 3-pentadec-10′(Z)-enylphenol (2), along with known 3-heptadec-10′(Z)-enylphenol (3), 2-hydroxy-6-(pentadec-10′(Z)-enyl)benzoic acid (4), and 2-hydroxy-6-(10′(Z)-heptadecenyl)benzoic acid (5) were isolated from the stem bark of this plant. Compounds (1-5) were tested for their acetylcholinesterase inhibitory activity. The structures of these compounds were elucidated by the 1D and 2D NMR spectroscopy, mass spectrometry and chemical derivatizations. Compound 5 showed strong acetylcholinesterase inhibitory activity with IC₅₀ of 0.573 ± 0.0260 μM. Docking studies of compound 5 indicated that the phenolic compound with an elongated side chain could possibly penetrate deep into the active site of the enzyme and arrange itself through π-π interaction, H-bonding, and hydrophobic contacts with some critical residues along the complex geometry of the active gorge.     

Acylated anthocyanins in inflorescence of spider flower (Cleome hassleriana)

Five anthocyanins, cyanidin 3-(2′′-(6′′′-caffeoyl-β-glucopyranosyl)-6′′-(E-p-coumaroyl)-β-glucopyranoside)-5-β-glucopyranoside, cyanidin 3-(2′′-(6′′′-E-sinapoyl-β-glucopyranosyl)-6′′-(E-p-coumaroyl)-β-glucopyranoside)-5-β-glucopyranoside, cyanidin 3-(2′′-(6′′′-feroyl-β-glucopyranosyl)-6′′-(E-p-coumaroyl)-β-glucopyranoside)-5-β-glucopyranoside, pelargonidin 3-(2′′-(6′′′-E-sinapoyl-β-glucopyranosyl)-6′′-(E-p-coumaroyl)-β-glucopyranoside)-5-β-glucopyranoside, and pelargonidin 3-(2′′-(6′′′-E-p-coumaroyl-β-glucopyranosyl)-6′′-(E-p-coumaroyl)-β-glucopyranoside)-5-β-glucopyranoside, together with five known anthocyanins have been identified in flowers of Cleome hassleriana Queen line. One monoacylated and four diacylated cyanidin 3-sophoroside-5-glucosides were identified as the main anthocyanins in flowers with mauve colouration, while a homologous glycosidic pattern based on pelargonidin was found in the five main anthocyanins from flowers with pink colouration. The anthocyanins identified in C. hassleriana share the same glycosidic pattern as anthocyanins isolated from the genera Raphanus, Brassica and Iberis in the sister family Brassicaceae.     

Chemical constituents from Anthemis wiedemanniana Fisch. & Mey.

The lipophilic extract of the aerial parts of Anthemis wiedemanniana Fisch. & Mey, a Turkish endemic species, was investigated. In addition to one new natural product, namely 3,4,4-trimethyl-6-carboxy-cyclohex-2-ene-1-one, five known sesquiterpene lactones, five methylated flavonoids, one simple phenolic derivative and one nor-isoprenoid were isolated. Among the sesquiterpene lactones, canin was found to be the main compound.