Andrew L. Thomas

Determination of Neurotoxic Acetogenins in Pawpaw (Asimina triloba) Fruit by LC-HRMS

The concentrations of the neurotoxins, annonacin and squamocin, were determined in a lyophilized sample of the fruit pulp of the North American pawpaw (Asimina triloba) by LC coupled to high resolution mass spectrometry or LC-HRMS. The sample was extracted using dry methanol at 100 °C and 10 MPa pressure in a sealed container. The extraction of annonacin and squamocin was optimal at 100 °C with 7.72 and 0.162 mg/g, respectively, being found. Also, several isomers of annonacin and squamocin were separated and detected but not quantified.

Inhibition of microglial activation by elderberry extracts and its phenolic components

AIMS Elderberry (Sambucus spp.) is one of the oldest medicinal plants noted for its cardiovascular, anti-inflammatory, and immune-stimulatory properties. In this study, we investigated the anti-inflammatory and anti-oxidant effects of the American elderberry (Sambucus nigra subsp. canadensis) pomace as well as some of the anthocyanins (cyanidin chloride and cyanidin 3-O-glucoside) and flavonols (quercetin and rutin) in bv-2 mouse microglial cells. MAIN METHODS The bv-2 cells were pretreated with elderberry pomace (extracted with ethanol or ethyl acetate) or its anthocyanins and flavonols and stimulated by either lipopolysaccharide (LPS) or interferon-γ (IFNγ). Reactive oxygen species (ROS) and nitric oxide (NO) production (indicating oxidative stress and inflammatory response) were measured using the ROS detection reagent DCF-DA and the Griess reaction, respectively. KEY FINDINGS Analysis of total monomeric anthocyanin (as cyanidin 3-O-glucoside equivalents) indicated five-fold higher amount in the freeze-dried ethanol extract as compared to that of the oven-dried extract; anthocyanin was not detected in the ethyl acetate extracts. Elderberry ethanol extracts (freeze-dried or oven-dried) showed higher anti-oxidant activities and better ability to inhibit LPS or IFNγ-induced NO production as compared with the ethyl acetate extracts. The phenolic compounds strongly inhibited LPS or IFNγ-induced ROS production, but except for quercetin, they were relatively poor in inhibiting NO production. SIGNIFICANCE These results demonstrated differences in anti-oxidative and anti-inflammatory effects of elderberry extracts depending on solvents used. Results further identified quercetin as the most active component in suppressing oxidative stress and inflammatory responses on microglial cells.

Impact of Frozen Storage on the Anthocyanin and Polyphenol Contents of American Elderberry Fruit Juice

The effects of frozen storage on the anthocyanin and polyphenol content of elderberry fruit juice are investigated. Juice from three genotypes of American elderberry (Adams II, Bob Gordon, and Wyldewood) was screened for total phenolic (TP) and total monomeric anthocyanin (TMA) contents with spectrophotometric methods. The individual anthocyanin content (IAC) of the juice was tested by coupling solid phase extraction with ultraperformance liquid chromatography-tandem mass spectrometry. Juice samples were tested initially upon harvest and then again after 3, 6, and 9 months of frozen storage. Juice from the three different genotypes had significantly different TP, TMA, and IAC profiles initially (p < 0.05). The TP, TMA, and IAC contents of the juice from different genotypes were significantly affected (p < 0.05) by the frozen storage time, suggesting that both genotype and length of frozen storage time can affect the anthocyanin content of elderberry fruit juice.

Dietary Sutherlandia and elderberry mitigate cerebral ischemia-induced neuronal damage and attenuate p47phox and phospho-ERK1/2 expression in microglial cells.

Sutherlandia (Sutherlandia frutescens) and elderberry (Sambucus spp.) are used to promote health and for treatment of a number of ailments. Although studies with cultured cells have demonstrated antioxidative and anti-inflammatory properties of these botanicals, little is known about their ability to mitigate brain injury. In this study, C57BL/6 J male mice were fed AIN93G diets without or with Sutherlandia or American elderberry for 2 months prior to a 30-min global cerebral ischemia induced by occlusion of the bilateral common carotid arteries (BCCAs), followed by reperfusion for 3 days. Accelerating rotarod assessment at 24 h after BCCA occlusion showed amelioration of sensorimotor impairment in the mice fed the supplemented diets as compared with the ischemic mice fed the control diet. Quantitative digital pathology assessment of brain slides stained with cresyl violet at 3 days after ischemia/reperfusion (I/R) revealed significant reduction in neuronal cell death in both dietary groups. Immunohistochemical staining for ionized calcium-binding adapter molecule-1 demonstrated pronounced activation of microglia in the hippocampus and striatum in the ischemic brains 3 days after I/R, and microglial activation was significantly reduced in animals fed supplemented diets. Mitigation of microglial activation by the supplements was further supported by the decrease in expression of p47phox, a cytosolic subunit of NADPH oxidase, and phospho-ERK1/2, a mitogen-activated protein kinase known to mediate a number of cytoplasmic processes including oxidative stress and neuroinflammatory responses. These results demonstrate neuroprotective effect of Sutherlandia and American elderberry botanicals against oxidative and inflammatory responses to cerebral I/R.

Purification and Characterization of a Black Walnut (Juglans nigra) Allergen, Jug n 4

Tree nuts as a group cause a significant number of fatal anaphylactic reactions to foods. Walnuts (Juglans spp.) are one of the leading causes of allergic reactions to tree nuts in the U.S. and Japan. The purpose of this study was to purify and characterize potential food allergens from black walnut. Here, we report the isolation of the black walnuts allergen Jug n 4 (an 11S globulin) by ammonium sulfate precipitation, hydrophobic interaction, and size exclusion chromatography. Reducing SDS-PAGE analysis indicated that purified Jug n 4 consists of three major bands. N-Terminal sequencing data of these bands indicated that they were the results of a post-transcriptional protease cleavage of the mature protein at a site that consists of a known conserved protease recognition motif, NGXEET. Western blot experiments revealed that 32% of the sera from 25 patients with double-blind, placebo-controlled clinical walnut allergy contained IgE antibodies that recognized Jug n 4, indicating that it is a walnut allergen. Identifying this and additional allergens may facilitate the understanding of the allergenicity of seed storage proteins in tree nuts and their cross-reactivity.

Nut-yield variations and yield-diameter relationships in open-canopy black walnut trees in southern USA

Many landowners in the United States have little knowledge of the potential economic returns from agroforestry practices. Economic simulators for temperate agroforestry practices have been generated; yet, there are few data sets on yields of timber and other products to validate and refine such models. The objectives of this study were to characterize variations in nut yields among open canopy eastern black walnut (Juglans nigra L.) trees and apply this information to the development of predictive equations between tree diameter at breast height (DBH) and nut yields. Three data sets were analyzed that included results from Tennessee; Chetopa, Kansas; and Mt Vernon, Missouri. Tree-to-tree variation in nut yields was high within each data set, with coefficients of variation for nut yields typically exceeding 50%. Averaging nut yields over several consecutive years reduced coefficients of variation. Nearly half of the high nut producing trees exhibited an alternate, biennial nut bearing pattern. Trees with low average nut yields had either sporadic or irregular patterns of nut bearing. The regression coefficients for equations relating stem diameter and nut yields varied considerably. Averaging nut yields over consecutive years, and averaging stem diameter and nut yields over a number of trees increased regression coefficients of such equations. These results indicate that predicting nut yields of a tree stand over a several year-period will be easier than predicting yields for a specific tree in a specific year.

Peptidomics study of anthocyanin-rich juice of elderberry.

Biologically active peptides play a role in plant signaling and defense. Elderberry juice is known to contain a variety of anthocyanin compounds, a sub-set of polyphenols, which are responsible for the deep purple color of the juice. In this paper, we describe a method utilizing solid phase extraction (SPE) to remove anthocyanins from peptides. Liquid chromatography coupled with tandem mass spectrometry was used to separate and identify the peptides. The results showed that the use of SPE was an effective method to separate peptides from anthocyanins and other background compounds including high polyphenol content in the juice samples. More than 1000 peptides present in elderberry juice were successfully identified.

Discriminant Analyses of the Polyphenol Content of American Elderberry Juice from Multiple Environments Provide Genotype Fingerprint

The cultivation of American elderberry (Sambucus nigra subsp. canadensis) continues to increase as the use of this botanical has expanded. Elderberries contain a variety of polyphenols, including anthocyanins, which have purported health benefits. However, information is lacking regarding the impact of environmental, management, and genotypic factors on the quantity and type of polyphenols and anthocyanins produced. Quantification of 16 polyphenols including eight anthocyanins present in juice from three genotypes of American elderberry grown at two Missouri sites from 2013 to 2014 was performed. Large variances in anthocyanin and other polyphenol content were observed between the different harvest seasons, locations, and genotypes. Although specific phytochemical trends due to those factors were not apparent, a discriminant analysis was able to correctly identify 45 of 48 juice samples by genotype, based on their polyphenol profiles. This type of characterization could be beneficial in elderberry authentication studies and to help develop and document high-quality dietary supplement products with specific phytochemical contents.

Establishment Technique and Rootstock Impact ‘Chambourcin’ Grapevine Morphology and Production in Missouri

An evaluation of establishment techniques and rootstocks for ‘Chambourcin’ hybrid grape (Vitis sp.) was conducted 2009–12. Our objective was to evaluate four establishment methods and their interactions with grafted and ungrafted vines in terms of vine morphology and early fruit production under southwest Missouri conditions. The study was established in May 2009, as a factorial experiment comparing four establishment methods (open-trained without protection—two shoots, grow tube protected—two shoots, paperboard carton protected—two shoots, and fan-trained without protection—six shoots) across two vine types (own-rooted and grafted to ‘Couderc 3309’ hybrid grape rootstock). All vines in four of 12 field replicationswere destructively harvested near the conclusion of the first growing season, with leaf area and total vine dry matter determined. In years 3 and 4, yield, fruit composition, and vegetative growth were determined from the eight remaining replications. The fan training method increased leaf area and total vine dry matter compared with the other methods, but none of the establishment techniques affected fruit yield. Trunks that were tube protected had longer internodes, smaller diameter, and less dry matter, whereas both protection devices reduced glyphosate injury. Vine type (grafted and ungrafted) did not impact total leaf area or dry weight during the establishment year, but grafted vines had increased trunk and root shank dry weights compared with own-rooted vines. Grafted vines produced greater fruit yield in 2012. The fan training method required more labor to execute; although it was successful at increasing leaf area and root dry weight, it increased susceptibility to glyphosate injury and did not promote increased precocity or early fruit yield.

Performance of Late-Spring Forced Asparagus Rotated with Normal Spring Harvest Under Temperate Climate Conditions

Asparagus (Asparagus officinalis L.) is typically harvested for up to 6 weeks in spring, but cultural techniques can be used to extend the harvest. To force spear production beyond the normal harvest season, asparagus plants are allowed to grow undisturbed in spring and then are mowed later in the season, resulting in a flush of harvestable spears. Repetitive off-season forcing of asparagus does not appear to be a viable production practice in the temperate climate conditions of the Midwestern United States because the growing season is not long enough to permit adequate replenishment of energy reserves after even a brief forced harvest. An experiment was conducted in southwest Missouri to determine whether a system of rotating late-spring forced asparagus harvests with typical spring harvests would allow plants to recover sufficient vigor to establish a sustainable extended asparagus harvest regimen in the region. ‘Jersey Knight’ asparagus was planted in 1999 and harvested in eight different 3-year rotations involving late-spring forcing and normal spring harvest from 2001 to 2004. Though yield from normal spring harvests increased during the study, plants that were forced one or more times within a rotation produced less marketable yield and did not recover enough vigor to sustain a satisfactory harvest when subsequently rotated back to spring harvest. Initially, late-spring forced plants yielded roughly half that of normal spring-harvested plants. Though forced plants produced good, marketable spears, they were significantly smaller, with mean weight of 8.1 g compared with 10.3 g for normal spring-harvested spears. There appears to be a need for a longer period of recovery for forced asparagus plants under temperate conditions of the Midwestern United States.