Determination of Tobacco Alkaloid Enantiomers Using Reversed Phase UPLC/MS/MS

Nʹ-Nitrosonornicotine (NNN), a carcinogenic tobacco-specific Nʹ-nitrosamine (TSNA), is on the FDA list of harmful and potentially harmful constituents (HPHCs). Nornicotine, a product of the demethylation of nicotine, is the immediate alkaloid precursor for NNN formation. Nicotine, nornicotine and NNN are optically active. The accumulation of the isomers of nicotine, nornicotine, and NNN impacts their biological activity. In this paper, we report the determination of tobacco alkaloid enantiomers (including nicotine, nornicotine, anabasine, and anatabine) in samples of different tobacco lines using a reversed phase ultra-performance liquid chromatography-tandem mass spectrometer (UPLC/MS/MS) method. Current method demonstates excellent detection capability for all alkaloid enantiomers, with correlation coefficients (r2) \u3e 0.996 within their linear dynamic ranges. The limit of detection (LOD) and limit of quantitation (LOQ) of all analytes are less than 10 ng/mL and 30 ng/mL, respectively. In addition, their recovery and coefficient of variation (CV%) are within 100–115% and 0.2–3.7%, respectively. The method validated in this paper is simple, fast, and sensitive for the quantification of alkaloid enantiomers in tobacco leaf and has been applied to investigations of tobacco alkaloid enantiomer ratios in different tobacco lines and tobacco products

Tall Fescue Seed Extraction and Partial Purification of Ergot Alkaloids

Many substances in the tall fescue/endophyte association (Schedonorus arundinaceus/Epichloë coenophiala) have biological activity. Of these compounds only the ergot alkaloids are known to have significant mammalian toxicity and the predominant ergot alkaloids are ergovaline and ergovalinine. Because synthetically produced ergovaline is difficult to obtain, we developed a seed extraction and partial purification protocol for ergovaline/ergovalinine that provided a biologically active product. Tall fescue seed was ground and packed into several different sized columns for liquid extraction. Smaller particle size and increased extraction time increased efficiency of extraction. Our largest column was a 114 × 52 × 61 cm (W × L × D) stainless steel tub. Approximately 150 kg of seed could be extracted in this tub. The extraction was done with 80% ethanol. When the solvent front migrated to bottom of the column, flow was stopped and seed was allowed to steep for at least 48 h. Light was excluded from the solvent from the beginning of this step to the end of the purification process. Following elution, ethanol was removed from the eluate by evaporation at room temperature and the resulting syrup was freeze-dried. About 80% recovery of alkaloids was achieved with 18-fold increase in concentration of ergovaline. Initial purification of the dried product was accomplished by extracting with hexane/water (6:1, v/v). The aqueous fraction was extracted with chloroform, the aqueous layer discarded, after which the chloroform was removed with a resulting 20-fold increase of ergovaline. About 65% of the ergovaline was recovered from the chloroform residue for an overall recovery of 50%. The resultant partially purified ergovaline had biological activities in in vivo and in vitro bovine bioassays that approximate that of synthetic ergovaline

Mitigation of Ergot Vasoconstriction by Clover Isoflavones in Goats (\u3cem\u3eCapra hircus\u3c/em\u3e)

Ergot alkaloids produced by a fungal endophyte (Epichloë coenophiala; formerly Neotyphodium coenophialum) that infects tall fescue (Lolium arundinaceum) can induce persistent constriction of the vasculature in ruminants, hindering their capability to thermo-regulate core body temperature. There is evidence that isoflavones produced by legumes can relax the vasculature, which suggests that they could relieve ergot alkaloid-induced vasoconstriction and mitigate the vulnerability to severe heat stress in ruminants that graze tall fescue. To test if isoflavones can relieve alkaloid-induced vasoconstriction, two pen experiments were conducted with rumen-fistulated goats (Capra hircus) to determine with ultrasonograpy if isoflavones can (1) promote vascular compliance by countering alkaloid-induced vasoconstriction and (2) relieve already imposed alkaloid-induced vasoconstriction. Goats were fed ad libitum chopped orchardgrass (Dactylis glomerata)–timothy (Phleum pratense) hay prior to conducting the experiments. Measures of carotid and interosseous luminal areas were obtained pre- (baseline) and post-ruminal infusions in both experiments with goats being fed the hay, and for blood flow rate in the carotid artery in Experiment 2. Responses to infusion treatments were evaluated as proportionate differences from baseline measures. Peak systolic velocity, pulsatility index, and heart rate were measured on the last day on treatment (DOT) in Experiment 1, and on all imaging sessions during Experiment 2. For Experiment 1, rumens were infused with ground toxic fescue seed and isoflavones in Phase A and with only the toxic seed in Phase B. The infusion treatments were switched between phases in Experiment 2, which employed a fescue seed extract having an ergot alkaloid composition equivalent to that of the ground seed used in Experiment 1. During Experiment 1, luminal areas of carotid and interosseous arteries in Phase A did not deviate (P \u3e 0.1) from baselines over 1, 2, 3, and 4 DOT, but the areas of both declined linearly from baselines over 1, 2, 3, and 4 DOT in Phase B. By 6, 7, and 8 DOT in Experiment 2, luminal areas of the arteries and flow rate declined from baselines with infusions with the only seed extract in Phase A, but luminal areas and flow rate increased over 4, 5, and 6 DOT with the additional infusion of isoflavones. Peak systolic velocity and heart rate were not affected by treatment in either experiment, but were highest when infused with only ergot alkaloids in both experiments. Treatment with isoflavones was demonstrated to relax the carotid and interosseous arteries and reduce resistance to blood flow. Results indicate that isoflavones can relax persistent vasoconstriction in goats caused by consumption of ergot alkaloids, and mitigate the adverse effect that ergot alkaloids have on dry matter intake

Warming Reduces Tall Fescue Abundance but Stimulates Toxic Alkaloid Concentrations in Transition Zone Pastures of the U.S.

Tall fescue pastures cover extensive acreage in the eastern half of the United States and contribute to important ecosystem services, including the provisioning of forage for grazing livestock. Yet little is known concerning how these pastures will respond to climate change. Tall fescue\u27s ability to persist and provide forage under a warmer and wetter environment, as is predicted for much of this region as a result of climate change, will likely depend on a symbiotic relationship the plant can form with the fungal endophyte, Epichloë coenophiala. While this symbiosis can confer environmental stress tolerance to the plant, the endophyte also produces alkaloids toxic to insects (e.g., lolines) and mammals (ergots; which can cause fescue toxicosis in grazing animals). The negative animal health and economic consequences of fescue toxicosis make understanding the response of the tall fescue symbiosis to climate change critical for the region. We experimentally increased temperature (+3°C) and growing season precipitation (+30% of the long-term mean) from 2009-2013 in a mixed species pasture, that included a tall fescue population that was 40% endophyte-infected. Warming reduced the relative abundance of tall fescue within the plant community, and additional precipitation did not ameliorate this effect. Warming did not alter the incidence of endophyte infection within the tall fescue population; however, warming significantly increased concentrations of ergot alkaloids (by 30-40%) in fall-harvested endophyte-infected individuals. Warming alone did not affect loline alkaloid concentrations, but when combined with additional precipitation, levels increased in fall-harvested material. Although future warming may reduce the dominance of tall fescue in eastern U.S. pastures and have limited effect on the incidence of endophyte infection, persisting endophyte-infected tall fescue will have higher concentrations of toxic alkaloids which may exacerbate fescue toxicosis

Expression of a Constitutively Active Nitrate Reductase Variant in Tobacco Reduces Tobacco-Specific Nitrosamine Accumulation in Cured Leaves and Cigarette Smoke

Burley tobaccos (Nicotiana tabacum) display a nitrogen-use-deficiency phenotype that is associated with the accumulation of high levels of nitrate within the leaf, a trait correlated with production of a class of compounds referred to as tobacco-specific nitrosamines (TSNAs). Two TSNA species, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN), have been shown to be strong carcinogens in numerous animal studies. We investigated the potential of molecular genetic strategies to lower nitrate levels in burley tobaccos by overexpressing genes encoding key enzymes of the nitrogen-assimilation pathway. Of the various constructs tested, only the expression of a constitutively active nitrate reductase (NR) dramatically decreased free nitrate levels in the leaves. Field-grown tobacco plants expressing this NR variant exhibited greatly reduced levels of TSNAs in both cured leaves and mainstream smoke of cigarettes made from these materials. Decreasing leaf nitrate levels via expression of a constitutively active NR enzyme represents an exceptionally promising means for reducing the production of NNN and NNK, two of the most well-documented animal carcinogens found in tobacco products

Effects of red clover isoflavones on tall fescue seed fermentation and microbial populations \u3ci\u3ein vitro\u3c/i\u3e

Negative impacts of endophyte-infected Lolium arundinaceum (Darbyshire) (tall fescue) are responsible for over $2 billion in losses to livestock producers annually. While the influence of endophyte-infected tall fescue has been studied for decades, mitigation methods have not been clearly elucidated. Isoflavones found in Trifolium pratense (red clover) have been the subject of recent research regarding tall fescue toxicosis mitigation. Therefore, the aim of this study was to determine the effect of ergovaline and red clover isoflavones on rumen microbial populations, fiber degradation, and volatile fatty acids (VFA) in an in vitro system. Using a dose of 1.10 mg × L-1, endophyte-infected or endophyte-free tall fescue seed was added to ANKOM fiber bags with or without 2.19 mg of isoflavones in the form of a control, powder, or pulverized tablet, resulting in a 2 × 3 factorial arrangements of treatments. Measurements of pH, VFA, bacterial taxa, as well as the disappearance of neutral detergent fiber (aNDF), acid detergent fiber (ADF), and crude protein (CP) were taken after 48 h of incubation. aNDF disappearance values were significantly altered by seed type (P = 0.003) and isoflavone treatment (P = 0.005), and ADF disappearance values were significantly different in a seed × isoflavone treatment interaction (P ≤ 0.05). A seed × isoflavone treatment interaction was also observed with respect to CP disappearance (P ≤ 0.05). Eighteen bacterial taxa were significantly altered by seed × isoflavone treatment interaction groups (P ≤ 0.05), eight bacterial taxa were increased by isoflavones (P ≤ 0.05), and ten bacterial taxa were altered by seed type (P ≤ 0.05). Due to the beneficial effect of isoflavones on tall fescue seed fiber degradation, these compounds may be viable options for mitigating fescue toxicosis. Further research should be conducted to determine physiological implications as well as microbiological changes in vivo

Effects of Red Clover Isoflavones on Tall Fescue Seed Fermentation and Microbial Populations \u3cem\u3eIn Vitro\u3c/em\u3e

Negative impacts of endophyte-infected Lolium arundinaceum (Darbyshire) (tall fescue) are responsible for over $2 billion in losses to livestock producers annually. While the influence of endophyte-infected tall fescue has been studied for decades, mitigation methods have not been clearly elucidated. Isoflavones found in Trifolium pratense (red clover) have been the subject of recent research regarding tall fescue toxicosis mitigation. Therefore, the aim of this study was to determine the effect of ergovaline and red clover isoflavones on rumen microbial populations, fiber degradation, and volatile fatty acids (VFA) in an in vitro system. Using a dose of 1.10 mg × L-1, endophyte-infected or endophyte-free tall fescue seed was added to ANKOM fiber bags with or without 2.19 mg of isoflavones in the form of a control, powder, or pulverized tablet, resulting in a 2 × 3 factorial arrangements of treatments. Measurements of pH, VFA, bacterial taxa, as well as the disappearance of neutral detergent fiber (aNDF), acid detergent fiber (ADF), and crude protein (CP) were taken after 48 h of incubation. aNDF disappearance values were significantly altered by seed type (P = 0.003) and isoflavone treatment (P = 0.005), and ADF disappearance values were significantly different in a seed × isoflavone treatment interaction (P ≤ 0.05). A seed × isoflavone treatment interaction was also observed with respect to CP disappearance (P ≤ 0.05). Eighteen bacterial taxa were significantly altered by seed × isoflavone treatment interaction groups (P ≤ 0.05), eight bacterial taxa were increased by isoflavones (P ≤ 0.05), and ten bacterial taxa were altered by seed type (P ≤ 0.05). Due to the beneficial effect of isoflavones on tall fescue seed fiber degradation, these compounds may be viable options for mitigating fescue toxicosis. Further research should be conducted to determine physiological implications as well as microbiological changes in vivo

Rumen and Serum Metabolomes in Response to Endophyte-Infected Tall Fescue Seed and Isoflavone Supplementation in Beef Steers

Fescue toxicosis impacts beef cattle production via reductions in weight gain and muscle development. Isoflavone supplementation has displayed potential for mitigating these effects. The objective of the current study was to evaluate isoflavone supplementation with fescue seed consumption on rumen and serum metabolomes. Angus steers (n = 36) were allocated randomly in a 2 × 2 factorial arrangement of treatments including endophyte-infected (E+) or endophyte-free (E−) tall fescue seed, with (P+) or without (P−) isoflavones. Steers were provided a basal diet with fescue seed for 21 days, while isoflavones were orally administered daily. Following the trial, blood and rumen fluid were collected for metabolite analysis. Metabolites were extracted and then analyzed by UPLC-MS. The MAVEN program was implemented to identify metabolites for MetaboAnalyst 4.0 and SAS 9.4 statistical analysis. Seven differentially abundant metabolites were identified in serum by isoflavone treatment, and eleven metabolites in the rumen due to seed type (p \u3c 0.05). Pathways affected by treatments were related to amino acid and nucleic acid metabolism in both rumen fluid and serum (p \u3c 0.05). Therefore, metabolism was altered by fescue seed in the rumen; however, isoflavones altered metabolism systemically to potentially mitigate detrimental effects of seed and improve animal performance

The gut microbiome in atherosclerotic cardiovascular disease

The gut microbiota may play a role in cardiovascular diseases. Here, the authors perform a metagenome-wide association study on stools from individuals with atherosclerotic cardiovascular disease and healthy controls, identifying microbial strains and functions associated with the disease

Promotion of flavonoid biosynthesis in leaves and calli of ornamental crabapple (Malus sp.) by high carbon to nitrogen ratios

Flavonoids are secondary metabolites that play important roles in plant physiology. Despite numerous studies examined the effects of available carbon (C) or nitrogen (N) on flavonoid biosynthesis, the mechanism of C/N interactive effects on flavonoid metabolism is still unclear. In this study, we analyzed the composition of flavonoids and the expression levels of flavonoid-related genes in leaves and calli of crabapple (Malus spp.) cultivars with different leaf colors grown on media with different C/N ratios. Our results show that high C/N ratios induce anthocyanin pigmentation in leaves of the ever-red cultivar ‘Royalty’ and the spring-red cultivar ‘Prairifire’, as well as in three types of calli derived from the ever-green cultivar ‘Spring Snow’, but not in the leaves of the ever-green cultivar ‘Flame’. This phenomenon therefore correlated with anthocyanin content in these different samples. In addition, high C/N ratios in the growth media resulted in an increase in the concentration of flavones and flavonols in the leaves of the three crabapple cultivars. The transcript levels of the general flavonoid pathway genes [from chalcone synthase (CHS) to uridine diphosphate (UDP)-glucose: flavonoid 3-O-glycosyltransferase (UFGT) and flavonol synthase (FLS)] increased in response to high C/N ratios, and this in turn was correlated with the concentration of anthocyanin, flavone and flavonol in the leaves and calli. Expression of the late flavonoid/anthocyanin biosynthetic genes, anthocyanidin synthase (ANS), UFGT and FLS in particular, was more strongly influenced by C/N ratios than other structural genes, and the increased expression of the structural genes under high C/N ratios coincided with a coordinated increase in transcript levels of a MYB transcription factor, MYB10. These results are likely to be useful for future generation of plants with an optimized flavonoid/anthocyanin content or desirable organ coloration