Influence of the Growing Region on the Phytochemical Composition and Antioxidant Properties of North American Cranberry Fruit (Vaccinium macrocarpon Aiton).

The impact of the growth environment on the production of health-promoting phytochemicals in cranberry fruit (Vaccinium macrocarpon Aiton) is not well established despite increased production worldwide. We investigated the secondary metabolite composition among the cranberry fruit of nine cultivars produced in two major coastal North American growing regions that differ in climate. Using 1H NOESY NMR to generate metabolic fingerprints, principal component analysis revealed variation between the two regions and identified likely contributing metabolites. Triterpenoids ursolic and oleanolic acid, as well as citric and malic acids, were quantified using 1H qNMR, and anthocyanins and flavonols were determined by HPLC-DAD. Total proanthocyanidins (PACs), total soluble phenolics, and DPPH free-radical scavenging antioxidant activity were also evaluated. Across all cultivars, anthocyanins, flavonols, and total phenolic content were significantly higher in West Coast fruit than East Coast fruit, correlating with a regional trend of higher antioxidant activity in fruit grown on the West Coast. The opposite trend was observed for triterpenoids and organic acids, which were significantly higher across cultivars in East Coast fruit. These trends persisted over two growing seasons. The study demonstrates that climate plays an important role in the production of antioxidant and anti-inflammatory phytochemicals in cranberry plants.

Application of 1 H-NMR-based metabolomics to the analysis of cranberry (Vaccinium macrocarpon) supplements.

INTRODUCTION: Commercial cranberry supplements provide a low-sugar alternative to juices and sweetened fruit consumed for health benefits, but their phytochemical composition and associated biological activity varies depending on the source material and post-harvest processing. Proton nuclear magnetic resonance (1 H-NMR) is a rapid and environmentally friendly method of generating metabolic profiles of plant materials that may be used to authenticate cranberry products. OBJECTIVE: The 1 H NMR-based chemometrics were used to characterise variations in metabolic profiles of cranberry supplements in comparison to a whole cranberry powder reference standard. MATERIALS AND METHODS: The secondary metabolite profiles of nine commercial cranberry supplements were compared to a whole cranberry powder reference standard, using 1 H-NMR with Bruker AssureNMR software and principal component analysis (PCA). Content of selected triterpenoids and organic acids was determined by quantitative NMR. Total proanthocyanidins and anthocyanins were determined by established methods. RESULTS: PCA of 1 H-NMR spectra showed overlap between the cranberry standard and three supplements, but most products varied substantially in metabolic profile. Metabolites contributing to the observed variance include citric acid and cranberry peel constituents ursolic acid, oleanolic acid and hyperoside. Ursolic, oleanolic, citric, quinic and malic acids were readily determined by quantitative 1 H-NMR in the whole cranberry standard, but were below detection limits in many supplements. Proanthocyanidin and flavonoid content in several products was minimal or below detection limits. CONCLUSION: The 1 H-NMR chemometrics found significant variation in composition of characteristic cranberry metabolites among commercial preparations, reinforcing the need for reliable industry standards.

Performance of new 400-MHz HTS power-driven magnet NMR technology on typical pharmaceutical API, cinacalcet HCl.

After years towards higher field strength magnets, nuclear magnetic resonance (NMR) technology in commercial instruments in the past decade has expanded at low and high magnetic fields to take advantage of new opportunities. At lower field strengths, permanent magnets are well established, whereas for midrange and high field, developments utilize superconducting magnets cooled with cryogenic liquids. Recently, the desire to locate NMR spectrometers in nontypical NMR laboratories has created interest in the development of cryogen-free magnets. These magnets require no cryogenic maintenance, eliminating routine filling and large cryogen dewars in the facility. Risks of spontaneous quenches and safety concerns when working with cryogenic liquids are eliminated. The highest field commercially available cryogen-free NMR magnet previously reported was at 4.7 T in 2013. Here we tested a prototype cryogen-free 9.4-T power-driven high-temperature-superconducting (HTS) magnet mated to commercial NMR spectrometer electronics. We chose cinacalcet HCl, a typical active pharmaceutical ingredient, to evaluate its performance towards structure elucidation. Satisfactory standard 1D and 2D homonuclear and heteronuclear NMR results were obtained and compared with those from a standard 9.4-T cryogenically cooled superconducting NMR instrument. The results were similar between both systems with minor differences. Further comparison with different shims and probes in the HTS magnet system confirmed that the magnet homogeneity profile could be matched with commercially available NMR equipment for optimal results. We conclude that HTS magnet technology works well providing results comparable with those of standard instruments, leading us to investigate additional applications for this magnet technology outside a traditional NMR facility.

Comparison of Flow Injection MS, NMR, and DNA Sequencing: Methods for Identification and Authentication of Black Cohosh (Actaea racemosa).

Flow injection mass spectrometry and proton nuclear magnetic resonance spectrometry, two metabolic fingerprinting methods, and DNA sequencing were used to identify and authenticate Actaea species. Initially, samples of Actaea racemosa from a single source were distinguished from other Actaea species based on principal component analysis and soft independent modeling of class analogies of flow injection mass spectrometry and proton nuclear magnetic resonance spectrometry metabolic fingerprints. The chemometric results for flow injection mass spectrometry and proton nuclear magnetic resonance spectrometry agreed well and showed similar agreement throughout the study. DNA sequencing using DNA sequence data from two independent gene regions confirmed the metabolic fingerprinting results. Differences were observed between A. racemosa samples from four different sources, although the variance within species was still significantly less than the variance between species. A model based on the combined A. racemosa samples from the four sources consistently permitted distinction between species. Additionally, the combined A. racemosa samples were distinguishable from commercial root samples and from commercial supplements in tablet, capsule, or liquid form. DNA sequencing verified the lack of authenticity of the commercial roots but was unsuccessful in characterizing many of the supplements due to the lack of available DNA.

NMR flow tube for online NMR reaction monitoring.

In this paper we describe the development of a 5 mm NMR flow tube that can be used in a standard 5 mm NMR probe, enabling the user to conduct experiments on flowing samples or, more specifically, on flowing reaction mixtures. This enables reaction monitoring or kinetic experiments to be conducted by flowing reaction mixtures from a reaction vessel to detection in the coil area of the NMR, without the need for a specialized flow NMR probe. One of the key benefits of this flow tube is that it provides flexibility to be used across a range of available spectrometers of varying magnetic field strengths with a standard 5 mm probe setup. The applicability of this flow tube to reaction monitoring is demonstrated using the reaction of p-phenylenediamine and isobutyraldehyde to form the diimine product.

Distinguishing Vaccinium species by chemical fingerprinting based on NMR spectra, validated with spectra collected in different laboratories.

A method was developed to distinguish Vaccinium species based on leaf extracts using nuclear magnetic resonance spectroscopy. Reference spectra were measured on leaf extracts from several species, including lowbush blueberry (Vaccinium angustifolium), oval leaf huckleberry (Vaccinium ovalifolium), and cranberry (Vaccinium macrocarpon). Using principal component analysis, these leaf extracts were resolved in the scores plot. Analysis of variance statistical tests demonstrated that the three groups differ significantly on PC2, establishing that the three species can be distinguished by nuclear magnetic resonance. Soft independent modeling of class analogies models for each species also showed discrimination between species. To demonstrate the robustness of nuclear magnetic resonance spectroscopy for botanical identification, spectra of a sample of lowbush blueberry leaf extract were measured at five different sites, with different field strengths (600 versus 700 MHz), different probe types (cryogenic versus room temperature probes), different sample diameters (1.7 mm versus 5 mm), and different consoles (Avance I versus Avance III). Each laboratory independently demonstrated the linearity of their NMR measurements by acquiring a standard curve for chlorogenic acid (R(2) = 0.9782 to 0.9998). Spectra acquired on different spectrometers at different sites classifed into the expected group for the Vaccinium spp., confirming the utility of the method to distinguish Vaccinium species and demonstrating nuclear magnetic resonance fingerprinting for material validation of a natural health product.

Distinguishing Ontario ginseng landraces and ginseng species using NMR-based metabolomics.

The use of (1)H-NMR-based metabolomics to distinguish and identify unique markers of five Ontario ginseng (Panax quinquefolius L.) landraces and two ginseng species (P. quinquefolius and P. ginseng) was evaluated. Three landraces (2, 3, and 5) were distinguished from one another in the principal component analysis (PCA) scores plot. Further analysis was conducted and specific discriminating metabolites from the PCA loadings were determined. Landraces 3 and 5 were distinguishable on the basis of a decreased NMR intensity in the methyl ginsenoside region, indicating decreased overall ginsenoside levels. In addition, landrace 5 was separated by an increased amount of sucrose relative to the rest of the landraces. Landrace 2 was separated from the rest of the landraces by the increased level of ginsenoside R(b1). The Ontario P. quinquefolius was also compared with Asian P. ginseng by PCA, and clear separation between the two groups was detected in the PCA scores plot. The PCA loadings plot and a t-test NMR difference plot were able to identify an increased level of maltose and a decreased level of sucrose in the Asian ginseng compared with the Ontario ginseng. An overall decrease of ginsenoside content, especially ginsenoside R(b1), was also detected in the Asian ginseng's metabolic profile. This study demonstrates the potential of NMR-based metabolomics as a powerful high-throughput technique in distinguishing various closely related ginseng landraces and its ability to identify metabolic differences from Ontario and Asian ginseng. The results from this study will allow better understanding for quality assessment, species authentication, and the potential for developing a fully automated method for quality control.

Quantification of chlorogenic acid and hyperoside directly from crude blueberry (Vaccinium angustifolium) leaf extract by NMR spectroscopy analysis: single-laboratory validation.

A single-laboratory-validated NMR spectroscopy method was established for determining the quantity of chlorogenic acid and hyperoside from crude extract material of blueberry leaves of the species Vaccinium angustifolium var. laevifolium House. The calibration curve of chlorogenic acid showed a highly linear regression, R = 0.99998. NMR spectroscopy identification and quantification of the constituents directly from the mixture, within the error of HPLC-diode array detector analysis, were determined as 7.53 mM chlorogenic acid (64.0 mg chlorogenic acid/g powdered leaf) and 0.77 mM hyperoside (8.58 mg hyperoside/g powdered leaf). The LOD was calculated to be 0.01 mM and the LOQ 0.01 mM by the 9 min and 13 s NMR spectroscopy experiment utilized. The assay showed no significant interference from different field strengths, extraction mesh size, gravimetric scale precision, NMR spectroscopy tube type, pulse program, amount of starting dry material, or day-to-day operation. The robustness of NMR spectroscopy as a means of definitively monitoring chlorogenic acid and hyperoside content directly from crude extracts was demonstrated by Youden statistical analysis.

Metabolomics of microliter hemolymph samples enables an improved understanding of the combined metabolic and transcriptional responses of Daphnia magna to cadmium.

Omic technologies offer unprecedented opportunities to better understand mode(s)-of-toxicity and downstream secondary effects by providing a holistic view of the molecular changes underlying physiological disruption. Crustacean hemolymph represents a largely untapped biochemical resource for such toxicity studies. We sought to characterize changes in the hemolymph metabolome and whole-body transcriptome to reveal early processes leading to chronic toxicity in the indicator species, Daphnia magna, after 24-h sublethal cadmium exposure (18 µg/L, corresponding to 1/10 LC(50)). We first confirmed that metabolites can be detected and identified in small volumes (∼3-6 µL) of D. magna hemolymph using Fourier transform ion cyclotron resonance mass spectrometry and NMR spectroscopy. Subsequently, mass spectrometry based metabolomics of hemolymph identified disruption to two major classes of metabolites: amino acids and fatty acids. These findings were compared to differentially expressed genes identified by a D. magna 44k oligonucleotide microarray, which included decreased levels of digestive enzymes and increased expression of cuticle proteins and oxidative stress response genes. The combination of metabolic and transcriptional changes revealed through KEGG pathway analysis and gene ontology, respectively, enabled a more complete understanding of how cadmium disrupts nutrient uptake and metabolism, ultimately resulting in decreased energy reserves and chronic toxicity.

Ultrahigh-resolution (1)H-(13)C HSQC spectra of metabolite mixtures using nonlinear sampling and forward maximum entropy reconstruction.

To obtain a comprehensive assessment of metabolite levels from extracts of leukocytes, we have recorded ultrahigh-resolution 1H-13C HSQC NMR spectra of cell extracts, which exhibit spectral signatures of numerous small molecules. However, conventional acquisition of such spectra is time-consuming and hampers measurements on multiple samples, which would be needed for statistical analysis of metabolite concentrations. Here we show that the measurement time can be dramatically reduced without loss of spectral quality when using nonlinear sampling (NLS) and a new high-fidelity forward maximum-entropy (FM) reconstruction algorithm. This FM reconstruction conserves all measured time-domain data points and guesses the missing data points by an iterative process. This consists of discrete Fourier transformation of the sparse time-domain data set, computation of the spectral entropy, determination of a multidimensional entropy gradient, and calculation of new values for the missing time-domain data points with a conjugate gradient approach. Since this procedure does not alter measured data points, it reproduces signal intensities with high fidelity and does not suffer from a dynamic range problem. As an example we measured a natural abundance 1H-13C HSQC spectrum of metabolites from granulocyte cell extracts. We show that a high-resolution 1H-13C HSQC spectrum with 4k complex increments recorded linearly within 3.7 days can be reconstructed from one-seventh of the increments with nearly identical spectral appearance, indistinguishable signal intensities, and comparable or even lower root-mean-square (rms) and peak noise patterns measured in signal-free areas. Thus, this approach allows recording of ultrahigh resolution 1H-13C HSQC spectra in a fraction of the time needed for recording linearly sampled spectra.

Rapid analysis of protein backbone resonance assignments using cryogenic probes, a distributed Linux-based computing architecture, and an integrated set of spectral analysis tools.

Rapid data collection, spectral referencing, processing by time domain deconvolution, peak picking and editing, and assignment of NMR spectra are necessary components of any efficient integrated system for protein NMR structure analysis. We have developed a set of software tools designated AutoProc, AutoPeak, and AutoAssign, which function together with the data processing and peak-picking programs NMRPipe and Sparky, to provide an integrated software system for rapid analysis of protein backbone resonance assignments. In this paper we demonstrate that these tools, together with high-sensitivity triple resonance NMR cryoprobes for data collection and a Linux-based computer cluster architecture, can be combined to provide nearly complete backbone resonance assignments and secondary structures (based on chemical shift data) for a 59-residue protein in less than 30 hours of data collection and processing time. In this optimum case of a small protein providing excellent spectra, extensive backbone resonance assignments could also be obtained using less than 6 hours of data collection and processing time. These results demonstrate the feasibility of high throughput triple resonance NMR for determining resonance assignments and secondary structures of small proteins, and the potential for applying NMR in large scale structural proteomics projects.