Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry Identification of Metabolites in Winterberry Fruit Putatively Associated with Natural Disease Resistance to Diaporthe ilicicola.

Winterberry holly (Ilex verticillata) is an ornamental plant popularly used in landscape design and sold as cut branches for fall and winter seasonal decoration. Latent fruit rot of winterberry is an emerging disease caused by the fungus Diaporthe ilicicola, which can result in up to 100% crop loss. Diaporthe ilicicola infects open flowers in spring, but symptom onset does not occur until the end of the growing season when the fruit is fully mature. This study was conducted to identify compounds displaying significant variation in abundance during fruit maturation and that may be putatively associated with natural disease resistance observed when the fruit is immature. Winterberry 'Sparkleberry' fruits collected at four timepoints during the 2018 and 2019 seasons were extracted in methanol and analyzed using high resolution ultra-high performance liquid chromatography-tandem mass spectrometry. The results showed a distinct separation of metabolic profiles based on fruit phenological stage. The top 100 features that were differentially expressed between immature and mature fruit were selected from both electrospray ionization (ESI) (-) and ESI (+) datasets for annotation. Eleven compounds shown to decrease throughout the season included cinnamic acids, a triterpenoid, terpene lactones, stilbene glycosides, a cyanidin glycoside, and a furopyran. Nine compounds shown to accumulate throughout the season included chlorogenic acid derivatives, hydrolysable tannins, flavonoid glycosides, and a triterpene saponin. Future research will further confirm the exact identity of the compounds of interest and determine whether they are biologically active toward D. ilicicola or I. verticillata. The results could inform breeding programs, chemical management programs, and novel antifungal compound development pipelines.

Integrating genomics and multiplatform metabolomics enables metabolite quantitative trait loci detection in breeding-relevant apple germplasm.

Apple (Malus × domestica) has commercial and nutritional value, but breeding constraints of tree crops limit varietal improvement. Marker-assisted selection minimises these drawbacks, but breeders lack applications for targeting fruit phytochemicals. To understand genotype-phytochemical associations in apples, we have developed a high-throughput integration strategy for genomic and multiplatform metabolomics data. Here, 124 apple genotypes, including members of three pedigree-connected breeding families alongside diverse cultivars and wild selections, were genotyped and phenotyped. Metabolite genome-wide association studies (mGWAS) were conducted with c. 10 000 single nucleotide polymorphisms and phenotypic data acquired via LC-MS and 1 H NMR untargeted metabolomics. Putative metabolite quantitative trait loci (mQTL) were then validated via pedigree-based analyses (PBA). Using our developed method, 519, 726 and 177 putative mQTL were detected in LC-MS positive and negative ionisation modes, and NMR, respectively. mQTL were indicated on each chromosome, with hotspots on linkage groups 16 and 17. A chlorogenic acid mQTL was discovered on chromosome 17 via mGWAS and validated with a two-step PBA, enabling discovery of novel candidate gene-metabolite relationships. Complementary data from three metabolomics approaches and dual genomics analyses increased confidence in validity of compound annotation and mQTL detection. Our platform demonstrates the utility of multiomic integration to advance data-driven, phytochemical-based plant breeding.

Avocado consumption enhances human postprandial provitamin A absorption and conversion from a novel high-ß-carotene tomato sauce and from carrots.

Dietary lipids have been shown to increase bioavailability of provitamin A carotenoids from a single meal, but the effects of dietary lipids on conversion to vitamin A during absorption are essentially unknown. Based on previous animal studies, we hypothesized that the consumption of provitamin A carotenoids with dietary lipid would enhance conversion to vitamin A during absorption compared with the consumption of provitamin A carotenoids alone. Two separate sets of 12 healthy men and women were recruited for 2 randomized, 2-way crossover studies. One meal was served with fresh avocado (Persea americana Mill), cultivated variety Hass (delivering 23 g of lipid), and a second meal was served without avocado. In study 1, the source of provitamin A carotenoids was a tomato sauce made from a novel, high-ß-carotene variety of tomatoes (delivering 33.7 mg of ß-carotene). In study 2, the source of provitamin A carotenoids was raw carrots (delivering 27.3 mg of ß-carotene and 18.7 mg of α-carotene). Postprandial blood samples were taken over 12 h, and provitamin A carotenoids and vitamin A were quantified in triglyceride-rich lipoprotein fractions to determine baseline-corrected area under the concentration-vs.-time curve. Consumption of lipid-rich avocado enhanced the absorption of ß-carotene from study 1 by 2.4-fold (P < 0.0001). In study 2, the absorption of ß-carotene and α-carotene increased by 6.6- and 4.8-fold, respectively (P < 0.0001 for both). Most notably, consumption of avocado enhanced the efficiency of conversion to vitamin A (as measured by retinyl esters) by 4.6-fold in study 1 (P < 0.0001) and 12.6-fold in study 2 (P = 0.0013). These observations highlight the importance of provitamin A carotenoid consumption with a lipid-rich food such as avocado for maximum absorption and conversion to vitamin A, especially in populations in which vitamin A deficiency is prevalent. This trial was registered at clinicaltrials.gov as NCT01432210.

The timing of bacterial mesophyll infection shapes the leaf chemical landscape.

Chemistry in eukaryotic intercellular spaces is shaped by both hosts and symbiotic microorganisms such as bacteria. Pathogenic microorganisms like barley-associated Xanthomonas translucens (Xt) swiftly overtake the inner leaf tissue becoming the dominant microbial community member during disease development. The dynamic metabolic changes due to Xt pathogenesis in the mesophyll spaces remain unknown. Genomic group I of Xt consists of two barley-infecting lineages: pathovar translucens (Xtt) and pathovar undulosa (Xtu). Xtu and Xtt, although genomically distinct, cause similar water-soaked lesions. To define the metabolic signals associated with inner leaf colonization, we used untargeted metabolomics to characterize Xtu and Xtt metabolism signatures associated with mesophyll growth. We found that mesophyll apoplast fluid from infected tissue yielded a distinct metabolic profile and shift from catabolic to anabolic processes over time compared to water-infiltrated control. The pathways with the most differentially expressed metabolites by time were glycolysis, tricarboxylic acid cycle, sucrose metabolism, pentose interconversion, amino acids, galactose, and purine metabolism. Hierarchical clustering and principal component analysis showed that metabolic changes were more affected by the time point rather than the individual colonization of the inner leaves by Xtt compared to Xtu. Overall, in this study, we identified metabolic pathways that explain carbon and nitrogen usage during host-bacterial interactions over time for mesophyll tissue colonization. This foundational research provides initial insights into shared metabolic strategies of inner leaf colonization niche occupation by related but phylogenetically distinct phyllosphere bacteria. IMPORTANCE: The phyllosphere is a habitat for microorganisms including pathogenic bacteria. Metabolic shifts in the inner leaf spaces for most plant-microbe interactions are unknown, especially for Xanthomonas species in understudied plants like barley (Hordeum vulgare). Xanthomonas translucens pv. translucens (Xtt) and Xanthomonas translucens pv. undulosa (Xtu) are phylogenomically distinct, but both colonize barley leaves for pathogenesis. In this study, we used untargeted metabolomics to shed light on Xtu and Xtt metabolic signatures. Our findings revealed a dynamic metabolic landscape that changes over time, rather than exhibiting a pattern associated with individual pathovars. These results provide initial insights into the metabolic mechanisms of X. translucens inner leaf pathogenesis.

Transcriptomics and Metabolomics Reveal Tomato Consumption Alters Hepatic Xenobiotic Metabolism and Induces Steroidal Alkaloid Metabolite Accumulation in Mice.

SCOPE: Tomato consumption is associated with many health benefits including lowered risk for developing certain cancers. It is hypothesized that tomato phytochemicals are transported to the liver and other tissues where they alter gene expression in ways that lead to favorable health outcomes. However, the effects of tomato consumption on mammalian liver gene expression and chemical profile are not well defined. METHODS AND RESULTS: The study hypothesizes that tomato consumption would alter mouse liver transcriptomes and metabolomes compared to a control diet. C57BL/6J mice (n = 11-12/group) are fed a macronutrient matched diet containing either 10% red tomato, 10% tangerine tomato, or no tomato powder for 6 weeks after weaning. RNA-Seq followed by gene set enrichment analyses indicates that tomato type and consumption, in general, altered expression of phase I and II xenobiotic metabolism genes. Untargeted metabolomics experiments reveal distinct clustering between control and tomato fed animals. Nineteen molecular formulas (representing 75 chemical features) are identified or tentatively identified as steroidal alkaloids and isomers of their phase I and II metabolites; many of which are reported for the first time in mammals. CONCLUSION: These data together suggest tomato consumption may impart benefits partly through enhancing detoxification potential.

Effect of extrusion process conditions on extrudates enriched with carotenoids encapsulated by different methods using gum arabic and vegetable fat as carriers.

Bioactive compounds into extruded foods enhance their nutritional value but they are heat and shear labile and prone to oxidation. This study was aimed to examine the impacts of distinct encapsulation methods on the stability of carotenoids under typical extrusion conditions. The study presents innovative encapsulation methods and investigates the protection efficacy of carotenoids degradation, as well as the effects on the physicochemical characteristics of carotenoid-rich products. Thus, spray drying, spray chilling, and their combination were compared based on their ability to protect carotenoids. Processing temperatures were 110 °C and 140 °C, and shear rates 500 and 2000 1/s. Carotenoid retention was determined, ß- and α-carotene retention ranged from 17 to 44 % and 18 to 48 %, respectively. Upon storage at room temperature, the carotenoid content was stable for 15 days, followed by a marked reduction after 30 days. Extrudates enriched microparticles produced by spray chilling and the combined methods exhibited higher carotenoid protection during storage. They also showed better quality attributes, notably bulk density, high water absorption index, color properties, and carotenoid retention. These findings suggest that encapsulation can protect carotenoids during extrusion, and the protection can be tailored to optimize the attributes of the final products.

Steroidal alkaloid biosynthesis is coordinately regulated and differs among tomatoes in the red-fruited clade.

The tomato (Solanum spp.) clade of Solanaceae features a unique assortment of cholesterol-derived steroidal alkaloids. However, little quantitative data exists reporting the profile and concentration of these alkaloids across diverse fruit germplasm. To address the lack of knowledge regarding the chemical diversity, concentration, and genetic architecture controlling tomato steroidal alkaloids, we quantitatively profiled and genotyped two tomato populations representing diversity in the red-fruited clade. We grew 107 genetically diverse fresh market, processing, landrace, and wild tomato in multiple environments. Nine steroidal alkaloid groups were quantified using ultra-high performance liquid chromatography tandem mass spectrometry. The diversity panel and a biparental population segregating for high alpha-tomatine were genotyped to identify and validate quantitative trait loci (QTL) associated with steroidal alkaloids. Landraces and wild material exhibited higher alkaloid concentrations and more chemical diversity. Average total content of steroidal alkaloids, often dominated by lycoperoside F/G/esculeoside A, ranged from 1.9 to 23.3 mg 100 g-1 fresh wt. across accessions. Landrace and wild cherry accessions distinctly clustered based on elevated concentrations of early or late-pathway steroidal alkaloids. Significant correlations were observed among alkaloids from the early and late parts of the biosynthetic pathway in a species-dependent manner. A QTL controlling multiple, early steroidal alkaloid pathway intermediates on chromosome 3 was identified by genome-wide association studies (GWAS) and validated in a backcross population. Overall, tomato steroidal alkaloids are diverse in the red-fruited clade and their biosynthesis is regulated in a coordinated manner.

Carotenoid extraction and analysis from blood plasma/serum.

Accurate and sensitive methods to quantify carotenoids in blood plasma/serum are the basis for assessing carotenoid intake and associating physiological effects. This chapter introduces carotenoid chemistry, an overview of carotenoid food sources, and current knowledge of carotenoid absorption and metabolism, along with factors that affect these processes. We also detail a commonly used method to extract plasma/serum carotenoids using liquid-liquid extraction and analysis by high performance liquid chromatography coupled with diode array detection (HPLC-DAD). A spreadsheet to aid in this quantitative analysis can be found at www.github.com/CooperstoneLab/carotenoid-analysis.

Eggs Improve Plasma Biomarkers in Patients with Metabolic Syndrome Following a Plant-Based Diet-A Randomized Crossover Study.

Plant-based (PB) diets are considered a healthy dietary pattern; however, eggs are not always included in this dietary regime. We hypothesized that the addition of two eggs per day would increase HDL cholesterol as well as plasma lutein, zeaxanthin and choline in individuals with metabolic syndrome (MetS). In this randomized controlled crossover intervention, we recruited 30 participants (49.3 ± 8 y) with MetS who followed a PB diet for 13 weeks. A registered dietitian advised all subjects on food selection and followed them through the intervention to ensure compliance. Participants underwent a 2-week washout with no eggs or spinach (a source of dietary lutein and zeaxanthin) and were randomly allocated to consume spinach (70 g) with either two eggs (EGG) or the equivalent amount of egg substitute (SUB) for breakfast for 4 weeks. After a 3-week washout, they were allocated the alternate breakfast. A total of 24 participants (13 women/11 men) finished the intervention. Plasma lipids, glucose, insulin, anthropometrics, plasma lutein, zeaxanthin, choline and trimethylamine oxide (TMAO) were assessed at baseline and the end of each intervention. When we compared individuals consuming the EGG versus the SUB breakfast, we observed a lower body weight (p < 0.02) and a higher HDL cholesterol (p < 0.025) after the EGG diet. There were no differences in plasma LDL cholesterol, triglycerides, glucose, insulin, or blood pressure. The number of large HDL particles measured by NMR was higher after EGG (p < 0.01) as compared to SUB. Plasma choline was higher in both treatments (p < 0.01) compared to baseline (8.3 ± 2.1 µmol/L). However, plasma choline values were higher in EGG (10.54 ± 2.8 µmol/L) compared to SUB (9.47 ± 2.7 µmol/L) p < 0.025. Both breakfasts increased plasma lutein compared to baseline (p < 0.01), while plasma zeaxanthin was only increased in the egg intervention (p < 0.01). These results indicate that consuming a plant-based diet in combination with whole eggs increases plasma HDL cholesterol, choline and zeaxanthin, important biomarkers in subjects with MetS.

Comparison between Egg Intake versus Choline Supplementation on Gut Microbiota and Plasma Carotenoids in Subjects with Metabolic Syndrome.

We previously demonstrated that intake of three eggs/d for 4 weeks increased plasma choline and decreased inflammation in subjects with metabolic syndrome (MetS). The purpose of the current study was to further explore the effects of phosphatidylcholine (PC) provided by eggs versus a choline bitartrate (CB) supplement on the gut microbiota, trimethylamine N-oxide (TMAO) formation, and plasma carotenoids lutein and zeaxanthin in MetS. This randomized, controlled crossover clinical trial included 23 subjects with MetS. Following a washout period of 2 weeks without consuming any choline-containing foods, subjects were randomly allocated to consume either three eggs/d or a CB supplement for 4 weeks (both diets had a choline equivalent of 400 mg/day). DNA was extracted from stool samples to sequence the 16S rRNA gene region for community analysis. Operational taxonomic units (OTUs) and the α-diversity of the community were determined using QIIME software. Plasma TMAO, methionine, betaine, and dimethylglycine (DMG) were quantified by stable isotope dilution liquid chromatography with tandem mass spectrometry. Plasma carotenoids, lutein, and zeaxanthin were measured using reversed-phase high-performance liquid chromatography. There were significant increases in plasma lutein and zeaxanthin after egg intake compared to the baseline or intake of CB supplement (p < 0.01). In contrast, TMAO was not different between treatments compared to the baseline (p > 0.05). Additionally, while diet intervention had no effects on microbiota diversity measures or relative taxa abundances, a correlation between bacterial biodiversity and HDL was observed. Following egg intake, the observed increases in plasma lutein and zeaxanthin may suggest additional protection against oxidative stress, a common condition in MetS.

Short-Term Tomato Consumption Alters the Pig Gut Microbiome toward a More Favorable Profile.

Diets rich in fruits and vegetables have been shown to exert positive effects on the gut microbiome. However, little is known about the specific effect of individual fruits or vegetables on gut microbe profiles. This study aims to elucidate the effects of tomato consumption on the gut microbiome, as tomatoes account for 22% of vegetable consumption in Western diets, and their consumption has been associated with positive health outcomes. Using piglets as a physiologically relevant model of human metabolism, 20 animals were assigned to either a control or a tomato powder-supplemented diet (both macronutrient matched and isocaloric) for 14 days. The microbiome was sampled rectally at three time points: day 0 (baseline), day 7 (midpoint), and day 14 (end of study). DNA was sequenced using shotgun metagenomics, and reads were annotated using MG-RAST. There were no differences in body weight or feed intake between our two treatment groups. There was a microbial shift which included a higher ratio of Bacteroidota to Bacillota (formerly known as Bacteroidetes and Firmicutes, respectively) and higher alpha-diversity in tomato-fed animals, indicating a shift to a more desirable phenotype. Analyses at both the phylum and genus levels showed global microbiome profile changes (permutational multivariate analysis of variance [PERMANOVA], P ≤ 0.05) over time but not with tomato consumption. These data suggest that short-term tomato consumption can beneficially influence the gut microbial profile, warranting further investigation in humans. IMPORTANCE The composition of the microorganisms in the gut is a contributor to overall health, prompting the development of strategies to alter the microbiome composition. Studies have investigated the role of the diet on the microbiome, as it is a major modifiable risk factor contributing to health; however, little is known about the causal effects of consumption of specific foods on the gut microbiota. A more complete understanding of how individual foods impact the microbiome will enable more evidence-based dietary recommendations for long-term health. Tomatoes are of interest as the most consumed nonstarchy vegetable and a common source of nutrients and phytochemicals across the world. This study aimed to elucidate the effect of short-term tomato consumption on the microbiome, using piglets as a physiologically relevant model to humans. We found that tomato consumption can positively affect the gut microbial profile, which warrants further investigation in humans.

Identification and assessment of alleles in the promoter of the Cyc-B gene that modulate levels of ß-carotene in ripe tomato fruit.

Novel diversity may be mined from databases and de novo sequencing, but functional characterization remains a limiting step to identifying new alleles. Classical breeding approaches augmented by marker-assisted selection offer a means to rapidly assess the function of new variation in coding or regulatory regions to modulate traits. We used the Cyc-B gene (B) of tomato (Solanum lycopersicum L.) for a proof of concept because of its role in the production of ß-carotene, a provitamin A carotenoid with importance to human nutrition. We measured carotenoid content in vintage and contemporary varieties and the profiles had a range of ß-carotene from 0.2 to 4.06 mg 100 g-1 fresh weight. We characterized variation in B from 84 sequences recovered from public databases and from an additional 29 high ß-carotene tomato, S. galapagense S. C. Darwin & Peralta, and S. cheesmaniae (L. Riley) Fosberg accessions. Thirteen unique haplotypes across 1600 bp of sequence 5' to the first ATG were identified with 11 occurring in high ß-carotene accessions we sequenced, and additional haplotypes were identified in public data. Phylogenetic analysis suggested that the alleles in high ß-carotene varieties were derived from wild species. Association analysis suggested two single nucleotide polymorphisms (SNPs) as the most likely causes of high ß-carotene, presumably through their influence on transcription of B that is elevated in ripening fruit. A marker-assisted backcross breeding scheme leveraging SNPs for background genome selection was used to rapidly develop germplasm resources containing different alleles of B in a uniform genetic background. Evaluation demonstrated that distinct promoter haplotypes function as different alleles that can be used to modulate the levels of ß-carotene in tomato.

A High-Throughput Extraction and Analysis Method for Steroidal Glycoalkaloids in Tomato.

Tomato steroidal glycoalkaloids (tSGAs) are a class of cholesterol-derived metabolites uniquely produced by the tomato clade. These compounds provide protection against biotic stress due to their fungicidal and insecticidal properties. Although commonly reported as being anti-nutritional, both in vitro as well as pre-clinical animal studies have indicated that some tSGAs may have a beneficial impact on human health. However, the paucity of quantitative extraction and analysis methods presents a major obstacle for determining the biological and nutritional functions of tSGAs. To address this problem, we developed and validated the first comprehensive extraction and ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) quantification method for tSGAs. Our extraction method allows for up to 16 samples to be extracted simultaneously in 20 min with 93.0 ± 6.8 and 100.8 ± 13.1% recovery rates for tomatidine and alpha-tomatine, respectively. Our UHPLC-MS/MS method was able to chromatographically separate analytes derived from 18 tSGA peaks representing 9 different tSGA masses, as well as two internal standards, in 13 min. Tomato steroidal glycoalkaloids that did not have available standards were annotated using high resolution mass spectrometry as well as product ion scans that provided fragmentation data. Lastly, we utilized our method to survey a variety of commonly consumed tomato-based products. Total tSGA concentrations ranged from 0.2 to 3.4 mg/serving and represent some of the first reported tSGA concentrations in tomato-based products. Our validation studies indicate that our method is sensitive, robust, and able to be used for a variety of applications where concentrations of biologically relevant tSGAs need to be quantified.

Concentrations of the Opium Alkaloids Morphine, Codeine, and Thebaine in Poppy Seeds are Reduced after Thermal and Washing Treatments but are Not Affected when Incorporated in a Model Baked Product.

Limited information exists on the effectiveness of potential treatments to reduce levels of opium alkaloids that may be present in seeds from poppy (Papaver somniferum L.). Poppy seeds containing morphine at relatively lower (14.7 mg kg-1) and higher (210.0 mg kg-1) concentrations were subjected to dry heat and steam treatments, water washing, and baking. Sample extracts were then analyzed using liquid chromatography-tandem mass spectrometry for the opium alkaloids morphine, codeine, and thebaine. The results indicated that thermal treatment promoted opium alkaloid degradation in poppy seed samples, with a 50% loss of morphine observed after 30-40 min at 200 °C. Water washing reduced concentrations of opium alkaloids in poppy seeds by approximately 50-80%, while steam treatment resulted in reduction of morphine in only one sample type. Importantly, baking had no significant effect on concentrations of opium alkaloids. Overall, these results indicate that opium alkaloids may not be significantly affected by baking or steam application and that poppy seeds may require water washing or extended thermal treatment to promote reduction of these compounds.

Black Raspberries and Protocatechuic Acid Mitigate DNFB-Induced Contact Hypersensitivity by Down-Regulating Dendritic Cell Activation and Inhibiting Mediators of Effector Responses.

Contact hypersensitivity (CHS) is the most common occupational dermatological disease. Dendritic cells (DCs) mediate the sensitization stage of CHS, while T-cells facilitate the effector mechanisms that drive CHS. Black raspberry (Rubus occidentalis, BRB) and BRB phytochemicals possess immunomodulatory properties, but their dietary effects on CHS are unknown. We examined the effects of diets containing BRB and protocatechuic acid (PCA, a constituent of BRB and an anthocyanin metabolite produced largely by gut microbes), on CHS, using a model induced by 2,4-dinitrofluorobenze (DNFB). Mice were fed control diet or diets supplemented with BRB or PCA. In vitro bone-marrow derived DCs and RAW264.7 macrophages were treated with BRB extract and PCA. Mice fed BRB or PCA supplemented diets displayed decreased DNFB-induced ear swelling, marked by decreased splenic DC accumulation. BRB extract diminished DC maturation associated with reduced Cd80 expression and Interleukin (IL)-12 secretion, and PCA reduced IL-12. Dietary supplementation with BRB and PCA induced differential decreases in IL-12-driven CHS mediators, including Interferon (IFN)-γ and IL-17 production by T-cells. BRB extracts and PCA directly attenuated CHS-promoting macrophage activity mediated by nitric oxide and IL-12. Our results demonstrate that BRB and PCA mitigate CHS pathology, providing a rationale for CHS alleviation via dietary supplementation with BRB or BRB derived anthocyanins.

Soy-tomato enriched diet reduces inflammation and disease severity in a pre-clinical model of chronic pancreatitis.

Chronic pancreatitis (CP) is a fibro-inflammatory syndrome in individuals who develop persistent pathological responses to parenchymal injury or stress. Novel therapeutic or dietary interventions that could lessen inflammation in this disease could significantly improve quality of life in patients with CP. Complex dietary foods like soy and tomatoes are composed of active metabolites with anti-inflammatory effects. Data from our group reports that bioactive agents in soy and tomatoes can reduce pro-inflammatory cytokines and suppressive immune populations. Additionally, our team has developed a novel soy-tomato juice currently being studied in healthy individuals with no toxicities, and good compliance and bioavailability. Thus, we hypothesize that administration of a soy-tomato enriched diet can reduce inflammation and severity of CP. C57BL/6 mice were injected intraperitoneally with 50 µg/kg caeurlein (7 hourly injections, twice weekly) for 6 weeks to induce CP. After 4 weeks of caerulein injections, mice were administered a control or a soy-tomato enriched diet for 2 weeks. Disease severity was measured via immunohistochemical analysis of pancreata measuring loss of acini, fibrosis, inflammation, and necrosis. Serum lipase and amylase levels were analyzed at the end of the study. Inflammatory factors in the serum and pancreas, and immune populations in the spleen of mice were analyzed by cytokine multiplex detection, qRT-PCR, and flow cytometry respectively. Infra-red (IR) sensing of mice was used to monitor spontaneous activity and distress of mice. Mice fed a soy-tomato enriched diet had a significantly reduced level of inflammation and severity of CP (p = 0.032) compared to mice administered a control diet with restored serum lipase and amylase levels (p < 0.05). Mice with CP fed a soy-tomato diet had a reduction in inflammatory factors (TNF-α, IL-1ß, IL-5) and suppressive immune populations (myeloid-derived suppressor cells; MDSC) compared to control diet fed mice (p < 0.05). Infra-red sensing to monitor spontaneous activity of mice showed that soy-tomato enriched diet improved total activity and overall health of mice with CP (p = 0.055) and CP mice on a control diet were determined to spend more time at rest (p = 0.053). These pre-clinical results indicate that a soy-tomato enriched diet may be a novel treatment approach to reduce inflammation and pain in patients with CP.

Profiling the impact of thermal processing on black raspberry phytochemicals using untargeted metabolomics.

Clinical and laboratory studies have implicated black raspberries (BRBs) and their associated phytochemicals in the modulation of several chronic diseases. Most research on the health benefits of BRBs is conducted using freeze-dried or otherwise minimally processed products, yet BRBs are typically consumed as thermally processed goods like jams and syrups. The objective of this work was to profile the chemical changes that result from thermal processing of BRB powder into a nectar beverage. Using an untargeted UHPLC-QTOF-MS metabolomics approach, key degradation products of anthocyanins were identified along with several other proposed phenolic degradants. The effects of processing on other key BRB compound groups, including ellagitannins, are also discussed. This work demonstrates the utility of an untargeted metabolomics approach in describing the chemistry of complex food systems and provides a foundation for future research on the impact of processing on BRB product bioactivity.

Analysis of Tomato Carotenoids: Comparing Extraction and Chromatographic Methods.

Background: Tomatoes (Solanum lycopersicum) are an economically and nutritionally important crop colored by carotenoids such as lycopene and ß-carotene. Market diversification and interest in the health benefits of carotenoids has created the desire in plant, food, and nutritional scientists for improved extraction and quantification protocols that avoid the analytical bottlenecks caused by current methods. Objective: Our objective was to compare standard and rapid extraction as well as chromatographic separation methods for tomato carotenoids. Method: Comparison was based on accuracy and the ability to discriminate between alleles and genetic backgrounds. Estimates of the contribution to variance in the presence of genetic and environmental effects were further used for comparison. Selections of cherry and processing tomatoes with varying carotenoid profiles were assessed using both established extraction and HPLC-diode array detector (HPLC-DAD) methods and rapid extraction and ultra-HPLC-DAD (UHPLC-DAD) protocols. Results: Discrimination of alleles in samples extracted rapidly (<5 min/sample) was similar to samples extracted using a standard method (10 min/sample), although carotenoid concentrations were lower due to reduced extraction efficiency. Quantification by HPLC-DAD (21.5 min/sample) and UHPLC-DAD (4.2 min/sample) were comparable, but the UHPLC-DAD method could not separate all carotenoids and isomers of tangerine tomatoes. Random effects modeling indicated that extraction and chromatographic methods explained a small proportion of variance compared with genetic and environmental sources. Conclusions: The rapid extraction and UHPLC-DAD methods could enhance throughput for some applications compared with standard protocols.

Metabolic Regulation of Glycolysis and AMP Activated Protein Kinase Pathways during Black Raspberry-Mediated Oral Cancer Chemoprevention.

Oral cancer is a public health problem with an incidence of almost 50,000 and a mortality of 10,000 each year in the USA alone. Black raspberries (BRBs) have been shown to inhibit oral carcinogenesis in several preclinical models, but our understanding of how BRB phytochemicals affect the metabolic pathways during oral carcinogenesis remains incomplete. We used a well-established rat oral cancer model to determine potential metabolic pathways impacted by BRBs during oral carcinogenesis. F344 rats were exposed to the oral carcinogen 4-nitroquinoline-1-oxide in drinking water for 14 weeks, then regular drinking water for six weeks. Carcinogen exposed rats were fed a 5% or 10% BRB supplemented diet or control diet for six weeks after carcinogen exposure. RNA-Seq transcriptome analysis on rat tongue, and mass spectrometry and NMR metabolomics analysis on rat urine were performed. We tentatively identified 57 differentially or uniquely expressed metabolites and over 662 modulated genes in rats being fed with BRB. Glycolysis and AMPK pathways were modulated during BRB-mediated oral cancer chemoprevention. Glycolytic enzymes Aldoa, Hk2, Tpi1, Pgam2, Pfkl, and Pkm2 as well as the PKA-AMPK pathway genes Prkaa2, Pde4a, Pde10a, Ywhag, and Crebbp were downregulated by BRBs during oral cancer chemoprevention. Furthermore, the glycolysis metabolite glucose-6-phosphate decreased in BRB-administered rats. Our data reveal the novel metabolic pathways modulated by BRB phytochemicals that can be targeted during the chemoprevention of oral cancer.

Storage conditions modulate the metabolomic profile of a black raspberry nectar with minimal impact on bioactivity.

Pre-clinical and clinical studies suggest black raspberries (BRBs) may inhibit the development of oral cancer. Lyophilized BRB powder is commonly used in these studies, but processed BRB products are more often consumed. The objective of this work was to understand how storage conditions influence the phytochemical profile and anti-proliferative activity of a BRB nectar beverage. Untargeted UHPLC-Q-TOF-MS based metabolomics analyses demonstrated that large chemical variation was introduced by storage above -20 °C over 60 days. However, minimal change in anti-proliferative activity was observed when stored nectar extracts were applied to SCC-83-01-82 premalignant oral epithelial cells. As proof of concept, cyanidin-3-O-rutinoside and its degradation product, protocatechuic acid, were administered in different ratios maintaining an equimolar dose, and anti-proliferative activity was maintained. This study shows the utility of metabolomics to profile global chemical changes in foods, while demonstrating that isolated phytochemicals do not explain the complete bioactivity of a complex food product.