Structure of rhamnoglucan, an unexpected alkali-stable polysaccharide extracted from Streptococcus mutans cell wall.

This study identified a rhamnose-containing cell wall polysaccharide (RhaCWP) in an alkaline extract prepared to analyze intracellular polysaccharides (IPS) from Streptococcus mutans biofilm. IPS was an 1,4-α-D-glucan with branchpoints introduced by 1,6-α-glucan while RhaCWP presented 1,2-α-L-and 1,3-α-L rhamnose backbone and side chains connected by 1,2-α-D-glucans, as identified by nuclear magnetic resonance (NMR) spectroscopy and methylation analyses. The MW of IPS and RhaCWP was 11,298 Da, as determined by diffusion-ordered NMR spectroscopy. Therefore, this study analyzed the chemical structure of RhaCWP and IPS from biofilm in a single fraction prepared via a convenient hot-alkali extraction method. This method could be a feasible approach to obtain such molecules and improve the comprehension of the structure-function relationships in polymers from S. mutans in future studies.

Cell wall polysaccharides of streptococci: A genetic and structural perspective.

The Streptococcus genus comprises both commensal and pathogenic species. Additionally, Streptococcus thermophilus is exploited in fermented foods and in probiotic preparations. The ecological and metabolic diversity of members of this genus is matched by the complex range of cell wall polysaccharides that they present on their cell surfaces. These glycopolymers facilitate their interactions and environmental adaptation. Here, current knowledge on the genetic and compositional diversity of streptococcal cell wall polysaccharides including rhamnose-glucose polysaccharides, exopolysaccharides and teichoic acids is discussed. Furthermore, the species-specific cell wall polysaccharide combinations and specifically highlighting the presence of rhamnose-glucose polysaccharides in certain species, which are replaced by teichoic acids in other species. This review highlights model pathogenic and non-pathogenic species for which there is considerable information regarding cell wall polysaccharide composition, structure and genetic information. These serve as foundations to predict and focus research efforts in other streptococcal species for which such data currently does not exist.

Precolumn derivatization LC-MS/MS method to simultaneous quantitation of 10 monosaccharides in rat plasma.

Monosaccharides are essential for maintaining the normal physiological functions of living organisms. Under disease states, metabolic disorders in vivo will inevitably affect the levels of monosaccharides, which brings the possibility of monosaccharides as a biomarker of some diseases. In this study, a method was developed and validated for simultaneously determining 10 monosaccharides (glucose, galactose, mannose, rhamnose, fucose, xylose, iduronic acid, glucuronic acid, N-acetylgalactosamine and N-acetylglucosamine) in SD rat plasma using liquid chromatography-tandem mass spectrometry. The method employed 1-phenyl-3-methyl-5-pyrazolone (PMP) as a derivatization reagent, considerably improved the chromatographic retention and ionization efficiency of monosaccharides. After protein precipitation of plasma samples, monosaccharides and isotope internal standards were derivatized and liquid-liquid extraction was performed to remove excess PMP. To achieve the baseline separation of several isomers, the resulting derivatives were chromatographed on a Bridged ethyl hybrid (BEH) Phenyl column using gradient elution with a total run time of 8 min. The method was linear within the range of 0.0100-5.00 µg/mL for rhamnose, 0.0500-25.0 µg/mL for fucose, xylose, iduronic acid, glucuronic acid, N-acetylgalactosamine and N-acetylglucosamine, 1.00-500 µg/mL for galactose, 10.0-5000 µg/mL for mannose, and 50.0-25,000 µg/mL for glucose. And the accuracy and precision verification of surrogate matrix samples and plasma samples met the required criteria. The method has been used successfully to study the effect of hepatic insufficiency on monosaccharide levels in rats. It was found that the concentration of glucuronic acid in SD rat plasma was abnormally increased in rats with liver injury.

Isolation, Purification, and Antioxidant Activities of Polysaccharides from Choerospondias axillaris Leaves.

The extraction, characterization and antioxidant activity of polysaccharides from Choerospondias axillaris leaves were investigated in the present study. Two purified polysaccharide fractions, CALP-1 and CALP-2, were isolated from crude Choerospondias axillaris leaf polysaccharides (CALP) by DEAE-52 cellulose chromatography and Sephadex G-100 column chromatography. The characteristics of CAL-1 and CALP-2 were determined by using High-performance Gel Permeation Chromatography (HPGPC), High-Performance Anion-Exchange Chromatography, HPAEC (HPAEC-PAD) and Fourier transform infrared spectroscopy (FTIR). CALP-1 with molecular weight of 11.20 KDa was comprised of Rhamnose, Arabinose, Galactose, Glucose, Xylose, Mannose and galacturonic acid in a molar ratio of 5.16:2.31:5.50:27.18:1.00:0.76:1.07. CAL-2 with molecular weight of 8.03 KDa consisted of Rhamnose, Arabinose, Galactose, Glucose, and galacturonic acid at a ratio of 1.38:3.63:18.84:8.28:1.45. FTIR revealed that CALP-1 and CALP-2 were acidic polysaccharides. The antioxidant activity of crude CALP, CALP-1 and CALP-2 was evaluated in vitro. The fraction CALP-2 was demonstrated to be of polysaccharide nature containing a large percentage of Galactose but no Xylose and Mannose. The antioxidant activity assays showed that CALP-1 and CALP-2 exhibited antioxidant and scavenging activities on hydroxyl and DPPH radicals in vitro. Compared with pure polysaccharide, crude CALP exhibited stronger anti-oxidant activities. These results will provide a better understanding of Choerospondias axillaris leaf polysaccharide and promote the potential applications of Choerospondias axillaris leaf polysaccharide in the pharmacological field and as a natural antioxidant.

Systematical Ingredient Investigations of Ficus tikoua Bur. Fruit and Immunoregulatory and Antioxidant Effects of Different Fractions.

Although the fruit of Ficus tikoua Bur. has been consumed by montanic people in China for centuries, its chemical and biological composition was still unclear. A series of comprehensive investigations on its chemical constituents and bioactivities were carried out for the first time. As a result, six compounds were isolated and identified as the main components in this fruit. GC-MS analysis of the lipid components demonstrated that Ficus tikoua Bur. fruit contains some wholesome constituents such as fatty acids, vitamins, triterpenoids, and phytosterols. The fatty acids are mainly composed of linolenic acid (61.27%) and linoleic acid (22.79%). Furthermore, this fruit contains a relative high content of crude protein (9.41 ± 0.03%), total amino acids (9.28%), and total polyphenols (0.86 ± 0.01 g/100 g). The analysis of monosaccharide composition showed that the total polysaccharide mainly consists of glucose, glucuronic acid, xylose, arabinose, mannose, galactose, galacturonic acid, and rhamnose. The polysaccharide, polyphenol, water, ethanol, and flavonoid extracts exhibited prominent antioxidant activity determined by ABTS, DPPH, and FRAPS methods. Meanwhile, the total polysaccharide exhibited significant immunomodulatory effect by enhancing the release of cytokines and expression of iNOS and COX-2 in RAW264.7 cells, significantly decreasing the expression of c-Jun and p65 proteins in the cytoplasm; increasing the translocation of c-Jun and p65 to the nucleus; and regulating the phosphorylation level of Akt, PI3K, and PDK1 in the PI3K/AKT signaling pathway. This study proved that the fruit of F. tikoua is a reliable source of functional food.

Acetylated Rhamnose Triterpenoid Saponins from Glechoma longituba Analyzed by LC-Q-TOFMS.

The aim of the present work is to isolate a series of triterpene derivatives with rhamnosyl linking acetyl groups from Glechoma longituba according to the structural characteristics of previously described triterpene saponins. The extract ion chromatography spectrum of the crude extract of G. longituba was detected and analyzed by HPLC-HR-ESI-MS to determine possible components, and these metabolites were traced and separated by combining high-resolution mass spectrometry and predicted liquid chromatography retention time. Three 11α, 12α-epoxypentacyclic oleanolic acid triterpene saponins (glechomanosides H-J) and one ursane triterpene aldehyde saponin with a C-28 aldehyde group were isolated from G. longituba. The structure of these compounds was confirmed by NMR and compared with those of previously characterized compounds. The strategy described in this report enables a rapid, reliable, and complete analysis of glycoside compounds containing different numbers of acetyl groups at different positions on the sugar.

Extraction optimization and structural characterization of pectin from persimmon fruit (Diospyros kaki Thunb. var. Rojo brillante).

In this work we have efficiently extracted and characterized pectin from different tissues of astringent (AS) and non-astringent (NAS) persimmon fruits (peel, pulp, whole fruit) for the first time. The highest pectin extraction (≥7.2%) was carried out at 80 °C, 120 min with 1.5% sodium citrate in peel of both AS and NAS persimmon samples. All persimmon pectins showed a molecular weight and galacturonic acid content upper than 328 kDa and 78%, respectively, indicating their suitability as food ingredient. Pectin extracted from AS pulp and peel tissues exhibited an enriched structure in rhamnose and arabinose, whereas the opposite behavior was observed in NAS persimmon whole fruit samples. Remarkably, both pulp tissues (AS and NAS) presented the highest levels of glucose and mannose, non-pectic carbohydrates. In addition, techno-functional assessment (zeta potential, particle size, apparent viscosity, gelation) showed the suitability of the persimmon pectins for a broad range of industrial applications.

Defects in Cell Wall Differentiation of the Arabidopsis Mutant rol1-2 Is Dependent on Cyclin-Dependent Kinase CDK8.

Plant cells are encapsulated by cell walls whose properties largely determine cell growth. We have previously identified the rol1-2 mutant, which shows defects in seedling root and shoot development. rol1-2 is affected in the Rhamnose synthase 1 (RHM1) and shows alterations in the structures of Rhamnogalacturonan I (RG I) and RG II, two rhamnose-containing pectins. The data presented here shows that root tissue of the rol1-2 mutant fails to properly differentiate the cell wall in cell corners and accumulates excessive amounts of callose, both of which likely alter the physical properties of cells. A surr (suppressor of the rol1-2 root developmental defect) mutant was identified that alleviates the cell growth defects in rol1-2. The cell wall differentiation defect is re-established in the rol1-2 surr mutant and callose accumulation is reduced compared to rol1-2. The surr mutation is an allele of the cyclin-dependent kinase 8 (CDK8), which encodes a component of the mediator complex that influences processes central to plant growth and development. Together, the identification of the surr mutant suggests that changes in cell wall composition and turnover in the rol1-2 mutant have a significant impact on cell growth and reveals a function of CDK8 in cell wall architecture and composition.

A quantitative LC-MS/MS approach for monitoring 2'-fluoro-2'-deoxy-D-glucose uptake in tumor tissue.

Purpose: Develop a quantitative LC-MS/MS method for FDG, FDG-monophosphate, glucose and glucose-monophosphate in mouse tumor models to assist in validating the use of [18F]FDG-positron emission tomography (PET) imaging for anticancer therapies in a clinical setting. Methodology/results: Analytes were isolated from tumors by protein precipitation and detected on a Sciex API-5500 mass spectrometer. Improved assay robustness and selectivity were achieved through chromatographic separation of FDG-monophosphate from glucose-monophosphate, selection of a unique ion transition and incorporation of stable isotope labeled internal standards. In a mouse JIMT-1 tumor model, FDG-monophosphate levels measured by LC-MS/MS correlated with [18F]FDG-PET imaging results. Conclusion: LC-MS/MS analysis of FDG-monophosphate accumulation in tumors is a cost-effective tool to gauge the translational potential of [18F]FDG-PET imaging as a noninvasive biomarker in clinical studies.

Structural elucidation and immuno-stimulatory activity of a novel polysaccharide containing glucuronic acid from the fungus Echinodontium tinctorium.

An immuno-stimulatory polysaccharide (EtISPFa) was purified from water extract of the fungus Echinodontium tinctorium. EtISPFa has an estimated weight average molecular weight (Mw) of 1354 kDa and is composed of glucose (66.2 %), glucuronic acid (10.1 %), mannose (6.7 %), galactose (6.4 %), xylose (5.6 %), rhamnose (3.1 %), fucose (1.8 %), and arabinose (0.2 %). It has multiple glycosidic linkages, with 3-Glcp (19.8 %), 4-GlcpA (10.8 %), 6-Glcp (10.7 %), and 3,6-Glcp (8.7 %) being the most prominent. NMR analysis showed that EtISPFa has a backbone containing mostly of 3-substituted ß-glucopyranose with 4-substituted glucopyranosyluronic acid. Short side chains consisting of an average of two ß-glycopyranose residues, connected through 1→6 linkages, are attached to the 6-position of about every 4th or 5th backbone glucose residue. EtISPFa is a novel glucuronic acid-containing ß-glucan capable of significantly inducing the production of cytokines IL-17, IL-16, MIP-2, G-CSF,GM-CSF, LIF, MIP-1α, MIP-1ß, and RANTES in vitro. EtISPFa should be further explored for its immuno-stimulatory activity in vivo.

96-Well Microtiter Plate Made of Paper: A Printed Chemosensor Array for Quantitative Detection of Saccharides.

Simple, rapid, and accurate detection methods for saccharides are potentially applicable to various fields such as clinical and food chemistry. However, the practical applications of on-site analytical methods are still limited. To this end, herein, we propose a 96-well microtiter plate made of paper as a paper-based chemosensor array device (PCSAD) for the simultaneous classification of 12 saccharides and the quantification of fructose and glucose among 12 saccharides. The mechanism of the saccharide detection relied on an indicator displacement assay (IDA) on the PCSAD using four types of catechol dyes, 3-nitrophenylboronic acid, and the saccharides. The design of the PCSAD and the experimental conditions for the IDA were optimized using a central composite design. The chemosensors exhibited clear color changes upon the addition of saccharides on the paper because of the competitive boronate esterification. The color changes were employed for the subsequent qualitative, semiquantitative, and quantitative analyses using an automated algorithm combined with pattern recognition for digital images. A qualitative linear discrimination analysis offered discrimination of 12 saccharides with a 100% classification rate. The semiquantitative analysis of fructose in the presence of glucose was carried out from the viewpoint of food analysis utilizing a support vector machine, resulting in clear discrimination of the various concentrations of fructose. Most importantly, the quantitative detection of fructose in two types of commercial soft drinks was also successfully carried out without sample pretreatments. Thus, the proposed PCSAD can be a powerful method for on-site food analyses that can meet the increasing demand from consumers for sensors of saccharides.

Structure, antioxidant and immunomodulatory activity of a polysaccharide extracted from Sacha inchi seeds.

In this study, a novel water-soluble polysaccharide (PVLP-1) was extracted and purified from Sacha inchi (Plukenetia volubilis L.) seeds and the structure, antioxidant and immunomodulatory activity of PVLP-1 were investigated. PVLP-1 (144 kDa) consisted of glucose (69.76%), mannose (14.86%), arabinose (10.53%), galactose (2.42%), ribose (1.23%), rhamnose (0.27%) and xylose (0.93%). PVLP-1 displayed characteristic polysaccharide bands in Fourier transform NMR spectra and infrared. The primary structure of PVLP-1 was a heteropolysaccharide with a backbone of (1 â†’ 6)-linked glucose, sidechains of (1 â†’ 4)-linked mannose, (1 â†’ 4)-linked glucose and (1 â†’ 3, 6)-linked mannose and a residue unit of →1)-linked arabinose as revealed the methylation analysis. PVLP-1 possessed good water-holding capacity (WHC), oil-holding capacity (OHC) and antioxidant capacities. Besides, PVLP-1 induced the proliferation of RAW264.7 cell and enhanced the expression of inflammatory cytokines IL-6, TNF-alpha(TNF-α) and IL-1 beta (IL-1ß). The present study indicated that PVLP-1 possessed immune-enhancing bioactivities and could be functional food or adjuvant drug to improve biological immunity of immunodeficiency diseases and hypoimmunity.

Characterization of a polysaccharide from Eupolyphaga sinensis walker and its effective antitumor activity via lymphocyte activation.

A polysaccharide (ESPS) purified from Eupolyphaga sinensis Walker by ion exchange chromatography and gel chromatography was investigated, including its structure characterization and antitumor activity. The results showed that ESPS was composed of rhamnose, fucose, arabinose, xylose, glucose, and galactose in a molar ratio of 7.4: 3.1: 13.9: 9.3: 39.7: 26.5, with the mean weight (Mw) of 2.14 × 104Da; the main chain of ESPS was mainly composed of → 4) - α - D - Glcp - (1 â†’ and → 3) - ß - D - Galp - (1 →, and the side chains were connected to the main chain through the O-6 atom of glucose and O-4 and O-6 atom of galactose. In addition, ESPS promoted the lymphocyte proliferation and inhibited liver cancer cells growth through enhancing lymphocyte activity in vitro, mainly NK cells. Moreover, ESPS markedly stimulated immunity in H22-bearing mice by increasing the spleen and thymus indices and effectively inhibited H22 cell growth in vivo. These data indicated that ESPS was a polysaccharide component possessing high anti-hepatocellular carcinoma activity, representing a potential immunotherapy candidate for the treatment of liver cancer.

Enzymatic hydrolysis of polysaccharide from Auricularia auricula and characterization of the degradation product.

An efficient enzymatic hydrolysis method was developed and optimized for the degradation of auricularia auricula polysaccharide (AAP) and the degradation product of AAP was characterized. Cellulase was used for the degradation of AAP. The yield of reducing sugar and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging rate were used as indices to optimize the enzymatic hydrolysis of AAP, based on response surface methodology (RSM). The resulting optimal enzymolysis conditions were as follows: enzyme dosage, 13,500 U/g; enzymolysis temperature, 50 °C; and pH, 4.2. Under these conditions, the actual yield of reducing sugar was 16.50 mg/mL and the DPPH radical scavenging rate was 87.97%. The degradation product of AAP (C-EAAP) was homogeneous and contained alpha and beta glycoside bonds, but did not contain protein or nucleic acid. The molecular weight of the degradation product was 5.94 × 105 Da. Monosaccharide composition analysis revealed that C-EAAP was composed of mannose (57.1%), glucuronic acid (10.0%), rhamnose (0.4%), glucose (22.5%), galactose (2.9%), xylose (6.0%), and fucose (1.1%). The antioxidant activity of the polysaccharide indicated that C-EAAP had better antioxidant activity than AAP. The scavenging rates of C-EAAP for hydroxyl radicals (·OH) and superoxide anion radicals (O2-·) were 1.65 and 1.90 times those of AAP.

Characterization of Micronutrients, Bioaccessibility and Antioxidant Activity of Prickly Pear Cladodes as Functional Ingredient.

The Opuntia ficus indica (L.) (OFI) is used as a nutritional and pharmaceutical agent in various dietary and value added products. This study underlines the possible use of native prickly pear cladode powder as a functional ingredient for health-promoting food production. To summarise, chemical characterization of polyphenols, minerals and soluble dietary fibre was performed; furthermore, the antioxidant activity and bioaccessibility of polyphenols and minerals were assessed. Eleven compounds between phenolic acids and flavonoids were identified, with piscidic acid and isorhamnetin derivatives being the most abundant. Opuntia's dietary fibre was mainly constituted of mucilage and pectin, and was composed of arabinose, galactose, glucose, mannose, rhamnose, and xylose sugars. The polyphenols' bioaccessibility was very high: piscidic acid at 200%, eucomic and ferulic acids >110% and flavonoids from 89% to 100%. The prickly pear cladode powder is also a source of minerals, as cations (calcium, sodium, potassium and magnesium) and anions (sulphate and chloride), with high magnesium bioaccessibilty (93%). OFI powder showed good capacity of radical scavenging measured by DPPH and ABTS methods, with 740 and 775 µmol Trolox/100 g OFI, respectively. Finally, the presented results allow the consideration of this natural product as a source of several essential nutrients, with a possible use in the food industry as a functional ingredient.

Chemical structure, chain conformation and rheological properties of pectic polysaccharides from soy hulls.

We obtained a new acidic soy hull polysaccharide (SHP-1) with a molecular weight (Mw) of 4.81 × 105 g/mol through ammonium oxalate and microwave assisted extraction. SHP-1 was mainly composed of galacturonic acid, galactose, rhamnose and arabinose (molar ratio = 46.59%:17.95%:14.77%:13.97%) with small amounts of fucose, glucose, mannose and xylose. The chemical structure was presumed to be of pectin-I type, consisting of 2/3 HGA and 1/3 RG-I. Furthermore, the rheological information and the chain morphology of SHP-1 were different in five solvents. Surfactant, salt and alkali solutions enhanced the solubility and flexibility of the polysaccharide, but the polysaccharide showed decreased fluidity under acidic conditions. The addition of ions and alkali increased the consistency coefficient of the solution, but the effect was far less than that of the cross-linking morphology. The structural and morphological information of purified SHP should aid in further study of its structure-function relationships and applications.

Nontoxic Glucomoringin-Isothiocyanate (GMG-ITC) Rich Soluble Extract Induces Apoptosis and Inhibits Proliferation of Human Prostate Adenocarcinoma Cells (PC-3).

The incidence of prostate cancer malignancy along with other cancer types is increasing worldwide, resulting in high mortality rate due to lack of effective medications. Moringa oleifera has been used for the treatment of communicable and non-communicable ailments across tropical countries, yet, little has been documented regarding its effect on prostate cancer. We evaluated the acute toxicity and apoptosis inducing effect of glucomoringin-isothiocyanate rich soluble extracts (GMG-ITC-RSE) from M. oleifera in vivo and in vitro, respectively. Glucomoringin was isolated, identified, and characterized using fundamental analytical chemistry tools where Sprague-Dawley (SD) rats, murine fibroblast (3T3), and human prostate adenocarcinoma cells (PC-3) were used for acute toxicity and bioassays experiments. GMG-ITC-RSE did not instigate adverse toxic reactions to the animals even at high doses (2000 mg/kg body weight) and affected none of the vital organs in the rats. The extract exhibited high levels of safety in 3T3 cells, where more than 90% of the cells appeared viable when treated with the extract in a time-dependent manner even at high dose (250 µg/mL). GMG-ITC-RSE significantly triggered morphological aberrations distinctive to apoptosis observed under microscope. These findings obviously revealed the putative safety of GMG-ITC-RSE in vivo and in vitro, in addition to its anti-proliferative effect on PC-3 cells.

Husk Tomato (Physalis ixocarpa Brot.) Waste as a Promising Source of Pectin: Extraction and Physicochemical Characterization.

Husk tomato (Physalis ixocarpa Brot. var. Rendidora) waste was evaluated as a source of specialized pectin, and pectin extracted from this waste was characterized physicochemically. Fruit was blanched for 10 or 15 min and extracted in 0.1 N HCl for 15 to 25 min. Extracted pectin was subjected to physicochemical analysis. For all extraction conditions, the percentage of anhydrogalacturonic acid exceeded 60%, indicating that husk tomato was a good source of pectin. The degree of esterification of pectin molecules was 63% to 91%. The amount of extracted pectin decreased with increasing extraction time. The apparent viscosity of husk tomato pectin showed the characteristic behavior of pseudoplastic fluids. Neutral sugars were identified, and the amounts of 6 sugars (fucose, rhamnose, arabinose, galactose, glucose, and xylose) were quantified. Sugars identified in husk tomato pectin and present in the Rhamnogalacturonan I region, arabinose, galactose, and rhamnose suggest a highly branched structure, which will influence its future applications. Molecular weight values were 542 to 699 kDa, exceeding molecular weight values reported for commercial citrus pectins from 134 to 480 kDa. The extraction process significantly (P < 0.05) influenced the physicochemical properties of pectin. Up to 19.8% from the total amount of pectin in the husk tomato was extracted by 10 min of blanching and 20 min of a more heat treatment. Our findings indicate that husk tomato can be a good alternative source of pectin having highly distinctive physicochemical characteristics.

Integrated Analytical and Statistical Two-Dimensional Spectroscopy Strategy for Metabolite Identification: Application to Dietary Biomarkers.

A major purpose of exploratory metabolic profiling is for the identification of molecular species that are statistically associated with specific biological or medical outcomes; unfortunately, the structure elucidation process of unknowns is often a major bottleneck in this process. We present here new holistic strategies that combine different statistical spectroscopic and analytical techniques to improve and simplify the process of metabolite identification. We exemplify these strategies using study data collected as part of a dietary intervention to improve health and which elicits a relatively subtle suite of changes from complex molecular profiles. We identify three new dietary biomarkers related to the consumption of peas (N-methyl nicotinic acid), apples (rhamnitol), and onions (N-acetyl-S-(1Z)-propenyl-cysteine-sulfoxide) that can be used to enhance dietary assessment and assess adherence to diet. As part of the strategy, we introduce a new probabilistic statistical spectroscopy tool, RED-STORM (Resolution EnhanceD SubseT Optimization by Reference Matching), that uses 2D J-resolved 1H NMR spectra for enhanced information recovery using the Bayesian paradigm to extract a subset of spectra with similar spectral signatures to a reference. RED-STORM provided new information for subsequent experiments (e.g., 2D-NMR spectroscopy, solid-phase extraction, liquid chromatography prefaced mass spectrometry) used to ultimately identify an unknown compound. In summary, we illustrate the benefit of acquiring J-resolved experiments alongside conventional 1D 1H NMR as part of routine metabolic profiling in large data sets and show that application of complementary statistical and analytical techniques for the identification of unknown metabolites can be used to save valuable time and resources.