A Metabolomic and HPLC-MS/MS Analysis of the Foliar Phenolics, Flavonoids and Coumarins of the Fraxinus Species Resistant and Susceptible to Emerald Ash Borer.

The Emerald Ash Borer (EAB), Agrilus planipennis, Fairmaire, an Asian invasive alien buprestid has devastated tens of millions of ash trees (Fraxinus spp.) in North America. Foliar phytochemicals of the genus Fraxinus (Oleaceae): Fraxinus pennsylvanica (Green ash), F. americana (White ash), F. profunda (Bush) Bush. (Pumpkin ash), F. quadrangulata Michx. (Blue ash), F. nigra Marsh. (Black ash) and F. mandshurica (Manchurian ash) were investigated using HPLC-MS/MS and untargeted metabolomics. HPLC-MS/MS help identified 26 compounds, including phenolics, flavonoids and coumarins in varying amounts. Hydroxycoumarins, esculetin, esculin, fraxetin, fraxin, fraxidin and scopoletin were isolated from blue, black and Manchurian ashes. High-throughput metabolomics revealed 35 metabolites, including terpenes, secoiridoids and lignans. Metabolomic profiling indicated several upregulated putative compounds from Manchurian ash, especially fraxinol, ligstroside, oleuropin, matairesinol, pinoresinol glucoside, 8-hydroxypinoresinol-4-glucoside, verbenalin, hydroxytyrosol-1-O-glucoside, totarol and ar-artemisene. Further, dicyclomine, aphidicolin, parthenolide, famciclovir, ar-turmerone and myriocin were identified upregulated in blue ash. Principal component analysis demonstrated a clear separation between Manchurian and blue ashes from black, green, white and pumpkin ashes. The presence of defensive compounds upregulated in Manchurian ash, suggests their potential role in providing constitutive resistance to EAB, and reflects its co-evolutionary history with A. planipennis, where they appear to coexist in their native habitats.

Antioxidant Activity of the Lignins Derived from Fluidized-Bed Fast Pyrolysis.

A challenge in recent years has been the rational use of forest and agriculture residues for the production of bio-fuel, biochemical, and other bioproducts. In this study, potentially useful compounds from pyrolytic lignins were identified by HPLC-MS/MS and untargeted metabolomics. The metabolites identified were 2-(4-allyl-2-methoxyphenoxy)-1-(4-hydroxy-3-methoxyphenyl)-1-propanol, benzyl benzoate, fisetinidol, phenyllactic acid, 2-phenylpropionic acid, 6,3'-dimethoxyflavone, and vanillin. The 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (DPPH), trolox equivalent antioxidant capacity (TEAC), and total phenolics content (TPC) per gram of pyrolytic lignin ranged from 14 to 503 mg ascorbic acid equivalents, 35 to 277 mg trolox equivalents, and 0.42 to 50 mg gallic acid equivalents, respectively. A very significant correlation was observed between the DPPH and TPC (r = 0.8663, p ≤ 0.0001), TEAC and TPC (r = 0.8044, p ≤ 0.0001), and DPPH and TEAC (r = 0.8851, p ≤ 0.0001). The polyphenolic compounds in the pyrolytic lignins which are responsible for radical scavenging activity and antioxidant properties can be readily profiled with HPLC-MS/MS combined with untargeted metabolomics. The results also suggest that DPPH, TEAC, and TPC assays are suitable methods for the measurement of antioxidant activity in a variety of pyrolytic lignins. These data show that the pyrolytic lignins can be considered as promising sources of natural antioxidants and value-added chemicals.

Antimicrobial activity of natural products from the flora of Northern Ontario, Canada.

CONTEXT: The number of multidrug resistant (MDR) microorganisms is increasing and the antimicrobial resistance expressed by these pathogens is generating a rising global health crisis. In fact, there are only a few antimicrobial agents left that can be used against MDR bacteria and fungi. OBJECTIVE: In this study, the antimicrobial activities of selected natural products from the flora of Northern Ontario against selected microorganisms are reported. MATERIALS AND METHODS: Plants were collected from Sault Ste. Marie, Ontario, Canada, and ethanol extracts were prepared using EtOH:H2O (1:1, v/v). Fungal cultures used in this study were Candida albicans ATCC 10231 and Schizosaccharomyces octosporus. Bacterial cultures employed included Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Mycobacterium phlei ATCC 11758, and Streptococcus lactis ATCC 19435. The microplate resazurin assay was used to screen for antimicrobial activity. RESULTS: Extracts of four plant species Chimaphila umbellata L. (Pyrolaceae), Betula papyrifera Marshall (Betulaceae), Rhus typhina L. (Anacardiaceae), and Fraxinus pennsylvanica Marshall (Oleaceae), and six compounds (gallic acid, ethyl gallate, caffeic acid, sinapic acid, gentisic acid, and chlorogenic acid) demonstrated antibacterial or antifungal activities with MICs ranging from 62.5 to 1000 µg/mL, respectively, for a chemical fraction of an extract from Betula papyrifera against the bacterium S. aureus. DISCUSSION AND CONCLUSION: The present study has shown that certain plant extracts and select fractions and standard chemical compounds exhibit antimicrobial effects. Prince's Pine, Chimaphila umbellate, White Birch, Betula papyrifera, Staghorn Sumac, Rhus typhina, and Green Ash, Fraxinus pennsylvanica were the principal extracts exhibiting notable antibacterial and/or antifungal activities; while gallic acid, ethyl gallate, and caffeic acid demonstrated antibacterial activities and sinapic acid, gentisic acid, and chlorogenic acid demonstrated antifungal activities.

Inhibition of insect glutathione S-transferase (GST) by conifer extracts.

Insecticide synergists biochemically inhibit insect metabolic enzyme activity and are used both to increase the effectiveness of insecticides and as a diagnostic tool for resistance mechanisms. Considerable attention has been focused on identifying new synergists from phytochemicals with recognized biological activities, specifically enzyme inhibition. Jack pine (Pinus banksiana Lamb.), black spruce (Picea mariana (Mill.) BSP.), balsam fir (Abies balsamea (L.) Mill.), and tamarack larch (Larix laricina (Du Roi) Koch) have been used by native Canadians as traditional medicine, specifically for the anti-inflammatory and antioxidant properties based on enzyme inhibitory activity. To identify the potential allelochemicals with synergistic activity, ethanol crude extracts and methanol/water fractions were separated by Sephadex LH-20 chromatographic column and tested for in vitro glutathione S-transferase (GST) inhibition activity using insecticide-resistant Colorado potato beetle, Leptinotarsa decemlineata (Say) midgut and fat-body homogenate. The fractions showing similar activity were combined and analyzed by ultra pressure liquid chromatography-mass spectrometry. A lignan, (+)-lariciresinol 9'-p-coumarate, was identified from P. mariana cone extracts, and L. laricina and A. balsamea bark extracts. A flavonoid, taxifolin, was identified from P. mariana and P. banksiana cone extracts and L. laricina bark extracts. Both compounds inhibit GST activity with taxifolin showing greater activity compared to (+)-lariciresinol 9'-p-coumarate and the standard GST inhibitor, diethyl maleate. The results suggested that these compounds can be considered as potential new insecticide synergists.

Antioxidant polyphenols in almond and its coproducts.

Antioxidant efficacy of defatted almond whole seed, brown skin, and green shell cover extracts was evaluated by monitoring inhibition of human low-density lipoprotein (LDL) oxidation, inhibition of DNA scission, and metal ion chelation activities. The total phenolic contents of ethanolic extracts of brown skin and green shell cover of almond were 10 and 9 times higher than that of the whole seed, respectively. Brown skin extract at 50 ppm effectively inhibited copper-induced oxidation of human LDL cholesterol compared to whole seed and green shell cover extracts, which reached the same level of efficacy at 200 ppm. Green shell cover extract at 50 ppm level completely arrested peroxyl radical-induced DNA scission, whereas 100 ppm of brown skin and whole seed extracts was required for similar efficiencies. All three almond extracts exhibited excellent metal ion chelation efficacies. High-performance liquid chromatographic (HPLC) analysis revealed the presence of quercetin, isorhamnetin, quercitrin, kaempferol 3-O-rutinoside, isorhamnetin 3-O-glucoside, and morin as the major flavonoids in all extracts.

Methyl gallate is a natural constituent of maple (Genus Acer) leaves.

Methyl gallate was found in ethanolic extracts of red maple (Acer rubrum L.), silver maple (A. saccharinum L.) and sugar maple (A. saccharum Marsh) leaves, but more was present in methanolic extracts. The increased amount of methyl gallate in methanolic extracts was accompanied by a disappearance of m-digallate. It is concluded that only some of the methyl gallate detected in methanolic extracts is an artefact as a result of methanolysis of m-digallate. Its presence in ethanolic extracts is evidence that it is also a natural constituent of maple leaves.

Sustained production of the labile pheromone component, (Z,Z)-6,9-heneicosadien-11-one, from a stable precursor for monitoring the whitemarked tussock moth.

The principal sex pheromone component of the whitemarked tussock moth (WMTM), Orgyia leucostigma, was recently identified as (Z,Z)-6,9-heneicosadien-11-one (Z6Z9-11-one-21Hy). However, it is thermally unstable and quickly degrades under field conditions so that baited traps are effective for only one night. We have developed a solution to this problem that combines two techniques: (1) the use of a stable pheromone precursor, (Z,Z)-6,9-heneicosadien-11-one ethylene ketal, which is hydrolyzed to the dienone by an acidic aqueous solution (2% p-toluenesulfonic acid in 35% aqueous sorbitol), and (2) use of a small, off-the-shelf, autonomous pump (the Med-e-Cell Infu-disktrade mark) to deliver the precursor continuously to a suitable substrate where it is converted rapidly into the attractive dienone pheromone component. The pump and hydrolysis substrate fit inside sticky traps and because generation and release of pheromone is continuous, the instability of the pheromone is not an issue. In electroantennogram bioassays, dose-dependent responses were obtained with 1 to 1000 ng of hydrolyzed ketal on filter paper, but no response was obtained to 1000 ng of the ketal itself. In wind tunnel bioassays, males were attracted to lures emitting the dienone pheromone component generated from 0.1 to 100 ng of the hydrolyzed ketal. Field tests in 2004 and 2005 showed that sticky traps fitted with the pump delivering the ketal (0.1-1 microg/microL in heptane) at 10 microL/hr to a cotton pad soaked with the hydrolyzing solution were attractive to male WMTM. No moths were caught in controls or traps baited with (Z)-6-heneicosen-11-one. An average of 0.51 moths per trap night was caught over an 18-night period in 2005. The results represent a first step toward developing a sensitive and practical monitoring tool for the WMTM by using a ketal precursor of its unstable dienone pheromone component.

(Z,Z)-6,9-heneicosadien-11-one, labile sex pheromone of the whitemarked tussock moth, Orgyia leucostigma.

The whitemarked tussock moth (WMTM), Orgyia leucostigma (J. E. Smith), is a major pest of coniferous and deciduous trees in eastern Canada. Chemical identification of its sex pheromone depended primarily on GC-EAD and HPLC analysis, with confirmation of behavioral activity by wind tunnel and field tests. We identified (Z,Z)-6,9-heneicosadien-11-one (Z,Z-6,9-ket) at 4-5 ng/female as the only essential sex pheromone component. Also detected in female extracts were (Z)-6-heneicosen-11-one (Z6-ket) at 2.5 ng/female, (Z,E)-6,8-heneicosadien-11-one (Z,E-6.8-ket) at about 0.5 ng/female, and a trace amount of (Z,E)-6,9-heneicosadien-11-one. Traps containing as little as 1 microg of Z,Z-6,9-ket attracted males at low population levels, indicating it is a potent sex attractant. Traps baited with Z6-ket attracted few males, and in windtunnel bioassays it was at least 100-fold less attractive to males than Z,Z-6,9-ket. No improvement in trap catch occurred with the addition of Z6-ket in various binary mixtures with Z,Z-6,9-ket, including the female ratio, and a ternary mixture of Z,Z-6.9-ket, Z6-ket, and Z,E-6,8-ket in the 9:5:1 ratio detected in females was no better than Z,Z-6,9-ket alone. We attribute the presence of Z,E-6,8-ket and Z,E-6,9-ket in female extracts to the spontaneous and rapid stereospecific isomerization of Z,Z-6,9-ket at room temperature. Male flight began at sunset but peaked during the second half of the night.

A flavanone and two phenolic acids from Chrysanthemum morifolium with phytotoxic and insect growth regulating activity.

Leaves of Chrysanthemum morifolium cv. Ramat were extracted sequentially with hexane, ethyl acetate, and methanol. The methanol fraction, when incorporated into artificial diet was found to reduce the growth of cabbage looper (Trichoplusia ni Hubner) larvae at concentrations between 500 and 5000 ppm of diet. Fractionation of the methanol extract on a Sephadex column yielded five fractions, three of which reduced the weight of larvae relative to the control. One fraction was analyzed using high performance liquid chromatography (HPLC) and found to contain three main constituents. These compounds were purified using a combination of gel permeation chromatography on Sephadex LH20 and HPLC, and analyzed by 1H and 13CNMR as well as undergoing chemical and physical analyses. The compounds were identified as: 1, chlorogenic acid (5-O-caffeoylquinic acid); 2, 3,5-O-dicaffeoylquinic acid; and 3, 3', 4',5-trihydroxyflavanone7-O-glucuronide (eriodictyol7-O-glucuronide). At concentrations between 100 to 1000 ppm these compounds reduced both growth and photosynthesis of Lemna gibba L. with the order of efficacy being: flavanone > chlorogenic acid > 3,5-O-dicaffeoylquinic acid. Furthermore, when incorporated separately into artificial diet these compounds, at 10 to 1000 ppm, enhanced or reduced growth of the cabbage looper (Trichoplusia ni) and gypsy moth (Lymantria dispar L.).