John T. Arnason

Essential Oils in Insect Control: Low-Risk Products in a High-Stakes World

In recent years, the use of essential oils (EOs) derived from aromatic plants as low-risk insecticides has increased considerably owing to their popularity with organic growers and environmentally conscious consumers. EOs are easily produced by steam distillation of plant material and contain many volatile, low-molecular-weight terpenes and phenolics. The major plant families from which EOs are extracted include Myrtaceae, Lauraceae, Lamiaceae, and Asteraceae. EOs have repellent, insecticidal, and growth-reducing effects on a variety of insects. They have been used effectively to control preharvest and postharvest phytophagous insects and as insect repellents for biting flies and for home and garden insects. The compounds exert their activities on insects through neurotoxic effects involving several mechanisms, notably through GABA, octopamine synapses, and the inhibition of acetylcholinesterase. With a few exceptions, their mammalian toxicity is low and environmental persistence is short. Registration has been the main bottleneck in putting new products on the market, but more EOs have been approved for use in the United States than elsewhere owing to reduced-risk processes for these materials.

AN IN VITRO EVALUATION OF HUMAN CYTOCHROME P450 3A4 INHIBITION BY SELECTED COMMERCIAL HERBAL EXTRACTS AND TINCTURES

Serial dilutions of 21 commercial ethanolic herbal extracts and tinctures, and 13 related pure plant compounds have been analyzed for their in vitro cytochrome P450 3A4 (CYP3A4) inhibitory capability via a fluorometric microtitre plate assay. Roughly 75% of the commercial products and 50% of the pure compounds showed significant inhibition of CYP3A4 metabolite formation. For each herbal product and pure compound exhibiting dose-dependency, the inhibition values were used to generate median inhibitory concentration (IC50) curves using linear regression. Among the commercial extracts, Hydrastis canadensis (goldenseal), Hypericum perforatum (St. Johns wort), and Uncaria tomentosa (cats claw) had the lowest IC50 values at < 1% full strength, followed by Echinacea angustifolia roots, Trifolium pratense (wild cherry), Matricaria chamomilla (chamomile), and Glycyrrhiza glabra (licorice), which had IC50 values ranging from 1%-2% of full strength. Dillapiol, hypericin, and naringenin had the lowest IC50 values among the pure plant compounds at < 0.5 mM; dillapiol was the most potent inhibitor at 23.3 times the concentration of the positive CYP3A4 inhibitor ketoconazole. Utilizing high-throughput screening methodologies for assessing CYP3A4 inhibition by natural products has important implications for predicting the likelihood of potential herbal-drug interactions, as well as determining candidates for further in-depth analyses.

Efficacy of essential oil of Ocimum basilicum L. and O. gratissimum L. applied as an insecticidal fumigant and powder to control Callosobruchus maculatus (Fab.) (Coleoptera: Bruchidae)

Essential oils from sweet basil, Ocimum basilicum, and African basil, O. gratissimum, (Labiatae) grown in Guinea were obtained by steam distillation. Following exposure of newly emerged adult beetles (Callosobruchus maculatus) to 12h of fumigation using pure essential oils at a dose of 25&mgr;l/vial, 80% mortality was recorded for O. basilicum, 70% for O. gratissimum and 0% in the control. A significant difference was observed between the responses of males and females with males exhibiting greater sensitivity. When 1g of aromatized powder was applied to adults, a 50% lethal concentration at 48h was found to be 65&mgr;l/g for O. basilicum and 116&mgr;l/g of O. gratissimum oils. The essential oils from the two plant species exhibited a significant effect both on the egg hatch rate and on the emergence of adults. The egg hatch rate was reduced to 3% with O. basilicum and 15% with O. gratissimum using an essential oil concentration of 30&mgr;l, whereas the egg hatch rate for the control was 95%. When compared with the control (97%), adult emergence dropped to 0% with O. basilicum and to 4% with O. gratissimum. Storage bioassays were run to assess the long-term effect of powders aromatized with essential oils of Ocimum. Complete protection was observed over 3 months starting at a dose of 400&mgr;l in the case of both oils. From a germination test, it was concluded that aromatized powders have no significant effect on the seed germination rate. After 5d, a rate of 88% germination was seen in seeds treated with aromatized powder and protected from insects, compared with 97% for untreated seeds that were not exposed to insects.

Insecticidal defenses of Piperaceae from the neotropics.

Insecticidal and growth-reducing properties of extracts of 14 species of American neotropical Piperaceae were investigated by inclusion in diets of a polyphagous lepidopteran, the European corn borer,Ostrinia nubilalis. Nutritional indices suggested most extracts acted by postdigestive toxicity.Piper aduncum, P. tuberculatum, andP. decurrens were among the most active species and were subjected to bioassay-guided isolation of the active components. Dillapiol was isolated from the active fraction ofP. aduncum, piperlonguminine was isolated fromP. tuberculatum, and a novel neolignan fromP. decurrens. The results support other studies on Asian and AfricanPiper species, which suggest that lignans and isobutyl amides are the active defence compounds in this family.

A review of Piper spp. (Piperaceae) phytochemistry, insecticidal activity and mode of action

The tropical plant family Piperaceae has provided many past and present civilizations with a source of diverse medicines and food grade spice. The secondary plant compounds that produce these desired qualities function also as chemical defenses for many species in the genus Piper. The compounds with the greatest insecticidal activity are the piperamides. Many studies have shown the effectiveness of Piper spp. extracts for the control of stored products pests and recently studies from our laboratory group have tested the extracts of Piper. nigrum, P. guineense and P. tuberculatum against insect pests of the home and garden. These results and those from investigations that examined the biochemical and molecular modes of action of the piperamides singly or in combination will be the focus of this review. The conclusions of our current work with Piperaceae are that Piper extracts offer a unique and useful source of biopesticide material for controlling small-scale insect out-breaks and reducing the likelihood of resistance development when applied as a synergist with other botanical insecticides such as pyrethrum.

Stimulation of AMP-activated protein kinase and enhancement of basal glucose uptake in muscle cells by quercetin and quercetin glycosides, active principles of the antidiabetic medicinal plant Vaccinium vitis-idaea

Several medicinal plants that stimulate glucose uptake in skeletal muscle cells were identified from among species used by the Cree of Eeyou Istchee of northern Quebec to treat symptoms of diabetes. This study aimed to elucidate the mechanism of action of one of these products, the berries of Vaccinium vitis idaea, as well as to isolate and identify its active constituents using a classical bioassay-guided fractionation approach. Western immunoblot analysis in C2C12 muscle cells revealed that the ethanol extract of the berries stimulated the insulin-independent AMP-activated protein kinase (AMPK) pathway. The extract mildly inhibited ADP-stimulated oxygen consumption in isolated mitochondria, an effect consistent with metabolic stress and the ensuing stimulation of AMPK. This mechanism is highly analogous to that of Metformin. Fractionation guided by glucose uptake activity resulted in the isolation of ten compounds. The two most active, quercetin-3-O-glycosides, enhanced glucose uptake by 38-59% (50 muM; 18 h treatment) in the absence of insulin. Quercetin aglycone, a minor constituent, stimulated uptake by 37%. The quercetin glycosides and the aglycone stimulated the AMPK pathway at concentrations of 25-100 muM, but only the aglycone inhibited ATP synthase in isolated mitochondria (by 34 and 79% at 25 and 100 muM, respectively). This discrepancy suggests that the activity of the glycosides may require hydrolysis to the aglycone form. These findings indicate that quercetin and quercetin 3-O-glycosides are responsible for the antidiabetic activity of V. vitis crude berry extract mediated by AMPK. These common plant products may thus have potential applications for the prevention and treatment of insulin resistance and other metabolic diseases.

Dehydrodimers of Ferulic Acid in Maize Grain Pericarp and Aleurone: Resistance Factors to Fusarium graminearum.

ABSTRACT The relationship between the primary cell wall phenolic acids, dehydrodimers of ferulic acid, and maize grain resistance to Fusarium graminearum, the causal agent of gibberella ear rot, was investigated. Concentrations of dehydrodimers of ferulic acid were determined in the pericarp and aleurone tissues of five inbreds and two hybrids of varying susceptibility and in a segregating population from a cross between a resistant and susceptible inbred. Significant negative correlations were found between disease severity and diferulic acid content. Even stronger correlations were observed between diferulic acid and the fungal steroid ergosterol, which is an indicator of fungal biomass in infected plant tissue. These results were consistent over two consecutive field seasons, which differed significantly for temperature and rainfall during pollination, the most susceptible stage of ear development. No correlation was found between the levels of these phenolics and deoxynivalenol levels. This is the first report of in vivo evidence that the dehydrodimers of ferulic acid content in pericarp and aleurone tissues may play a role in genotypic resistance of maize to gibberella ear rot.

Effects of traditionally used anxiolytic botanicals on enzymes of the γ-aminobutyric acid (GABA) systemThis article is one of a selection of papers published in this special issue (part 1 of 2) on the Safety and Efficacy of Natural Health Products.

In Canada, the use of botanical natural health products (NHPs) for anxiety disorders is on the rise, and a critical evaluation of their safety and efficacy is required. The purpose of this study was to determine whether commercially available botanicals directly affect the primary brain enzymes responsible for gamma-aminobutyric acid (GABA) metabolism. Anxiolytic plants may interact with either glutamic acid decarboxylase (GAD) or GABA transaminase (GABA-T) and ultimately influence brain GABA levels and neurotransmission. Two in vitro rat brain homogenate assays were developed to determine the inhibitory concentrations (IC50) of aqueous and ethanolic plant extracts. Approximately 70% of all extracts that were tested showed little or no inhibitory effect (IC50 values greater than 1 mg/mL) and are therefore unlikely to affect GABA metabolism as tested. The aqueous extract of Melissa officinalis (lemon balm) exhibited the greatest inhibition of GABA-T activity (IC50 = 0.35 mg/mL). Extracts from Centella asiatica (gotu kola) and Valeriana officinalis (valerian) stimulated GAD activity by over 40% at a dose of 1 mg/mL. On the other hand, both Matricaria recutita (German chamomile) and Humulus lupulus (hops) showed significant inhibition of GAD activity (0.11-0.65 mg/mL). Several of these species may therefore warrant further pharmacological investigation. The relation between enzyme activity and possible in vivo mode of action is discussed.

LOSS OF ENEMY RESISTANCE AMONG INTRODUCED POPULATIONS OF ST. JOHN'S WORT (HYPERICUM PERFORATUM)

The Evolution of Increased Competitive Ability (EICA) hypothesis predicts that introduced plants should lose enemy resistance and in turn evolve increased size or fecundity. We tested the first prediction of this hypothesis by growing introduced North American and native European genotypes of St. Johns Wort ( Hypericum perforatum )i n common gardens in the state of Washington, USA, and in Girona, Spain. In both gardens we measured levels of hypericin and pseudohypericin (and in Washington, hypericide)— compounds known to be toxic to generalist pathogens and herbivores. In a third common garden, in Spain, we experimentally manipulated native pathogen pressure (by treating plants with fungicides) and quantified how pathogen resistance varied between North Amer- ican and European genotypes. North American St. Johns Wort had lower levels of hypericin than European conspecifics in common gardens in Washington and Spain. North American plants also produced less hypericide (in Washington) and pseudohypericin (in Spain) than did European plants. In Spain, individuals were attacked by three generalist pathogens: Colletotrichum sp. (Coe- lomycetes), Alternaria sp. (Hyphomycetes), and Fusarium oxysporum(Hyphomycetes). A higher percentage of individuals from North American populations were infected by path- ogens and died from pathogen attack compared to European genotypes. Infection also appeared to reduce plant size and fecundity; these negative effects were similar in magnitude for North American and European genotypes. Taken together, results indicate that introduced St. Johns Wort has lost enemy resistance. However, contrary to EICA, current and previous results indicate that these changes have not been associated with an increase in plant size

A consensus ethnobotany of the Q’eqchi’ Maya of Southern Belize

An ethnobotany study in collaboration with Q’eqchi’ Maya healers of Southern Belize led to a collection of 169 medicinal plant species, belonging to 67 different families. The data show the use of a majority of species from primary or secondary semi-evergreen rainforests of Southern Belize, rather than weedy species. The medicinal uses of the plants were grouped into 17 usage categories. The frequency of use for each plant and the informant consensus factor for each usage category reveals a consensus among the healers on the use of plant species as well as on the diseases treated. These results suggest a well-defined medicinal tradition.ResumenUne étude ethnobotanique en collaboration avec les guérisseurs Mayas Q’eqchi’ du Sud du Belize a mené à la récolte de 169 espèces de plantes médicinales appartenant à 67 families différentes. Les données montrent l’utilisation d’une majorité d’espèces provenant des forêts tropicales humides primaires ou secondaires du Sud du Belize, plutôt que de mauvaises herbes. Les utilisations médicinales de ces plantes ont été classées dans 17 catégories d’usage. La fréquence d’utilisation de chaque plante et le facteur de consensus calculé pour chaque catégorie d’usage révèle un haut degré de consensus parmi les guérisseurs aussi bien pour l’utilisation des plantes que pour les maladies traitées par ces dernières. Ces résultats suggèrent une tradition médicinale bien définie.