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.

Canadian boreal pulp and paper feedstocks contain neuroactive substances that interact in vitro with GABA and dopaminergic systems in the brain.

Pulp and paper wood feedstocks have been previously implicated as a source of chemicals with the ability to interact with or disrupt key neuroendocrine endpoints important in the control of reproduction. We tested nine Canadian conifers commonly used in pulp and paper production as well as 16 phytochemicals that have been observed in various pulp and paper mill effluent streams for their ability to interact in vitro with the enzymes monoamine oxidase (MAO), glutamic acid decarboxylase (GAD), and GABA-transaminase (GABA-T), and bind to the benzodiazepine-binding site of the GABA(A) receptor (GABA(A)-BZD). These neuroendocrine endpoints are also important targets for treatment of neurological disorders such as anxiety, epilepsy, or depression. MAO and GAD were inhibited by various conifer extracts of different polarities, including major feedstocks such as balsam fir, black spruce, and white spruce. MAO was selectively stimulated or inhibited by many of the tested phytochemicals, with inhibition observed by a group of phenylpropenes (e.g. isoeugenol and vanillin). Selective GAD inhibition was also observed, with all of the resin acids tested being inhibitory. GABA(A)-BZD ligand displacement was also observed. We compiled a table identifying which of these phytochemicals have been described in each of the species tested here. Given the diversity of conifer species and plant chemicals with these specific neuroactivities, it is reasonable to propose that MAO and GAD inhibition reported in effluents is phytochemical in origin. We propose disruption of these neuroendocrine endpoints as a possible mechanism of reproductive inhibition, and also identify an avenue for potential research and sourcing of conifer-derived neuroactive natural products.

Extracts from hardwood trees used in commercial paper mills contain biologically active neurochemical disruptors.

Following on our discovery that pulp and paper mill effluents can interact with, and disrupt, various neurotransmitter receptors and enzymes important to fish reproduction, we tested wood and bark extracts of 14 Eastern North American hardwood trees used in pulp and paper production. Radioligand binding to neurotransmitter receptors, including the dopamine-2 receptor (D2), the gamma aminobutyric acid receptor A (GABA(A)), N-methyl-D-aspartic acid (NMDA) receptor, and muscarinic cholinergic receptor (mACh-R), were significantly changed following in vitro incubations with many but not all extracts. Activities of neurotransmitter-related enzymes monoamine oxidase (MAO), GABA-transaminase (GABA-T), acetylcholinesterase (AChE) and glutamic acid decarboxylase (GAD) were also significantly altered. Butternut wood extracts and the isolated compound juglone significantly inhibited the enzymatic activities of MAO and GAD which we suggest may be part of a mechanism that may negatively affect fish reproduction. Besides giving credence to the hypothesis that neuroactive compounds in pulp and paper effluent may originate in the trees used by mills, the results reported here also indicate important neuropharmacological activities in hardwoods which may help identify new sources of biologically active natural products.