Anomeric Proton Research Articles

Adaptive mechanism of the marine bacterium Pseudomonas sihuiensis-BFB-6S towards pCO2 variation: Insights into synthesis of extracellular polymeric substances and physiochemical modulation

Marine bacteria can adapt to various extreme environments by the production of extracellular polymeric substances (EPS). Throughout this investigation, impact of variable pCO2 levels on the metabolic activity and physiochemical modulation in EPS matrix of marine bacterium Pseudomonas sihuiensis - BFB-6S was evaluated using a fluorescence microscope, excitation-emission matrix (EEM), 2D-Fourier transform infrared correlation spectroscopy (2D-ATR-FTIR-COS), FT-NMR and TGA-DSC. From the results at higher pCO2 levels, there was a substantial reduction in EPS production by 58–62.8 % (DW). In addition to the biochemical composition of EPS, reduction in carbohydrates (8.7–47.6 %), protein (7.1–91.5 %), and lipids (16.9–68.6 %) content were observed at higher pCO2 levels. Functional discrepancies of fluorophores (tyrosine and tryptophan-like) in EPS, speckled differently in response to variable pCO2. The 2D-ATR-FTIR-COS analysis revealed functional amides (CN, CC, CO bending, -NH bending in amines) of EPS were preferentially altered, which led to the domination of polysaccharides relevant functional groups at higher pCO2. 1H NMR analysis of EPS confirmed the absence of chemical signals from H-C-COOH of proteins, α, β anomeric protons, and acetyl group relevant region at higher pCO2 levels. These findings can contribute new insights into the influence of pCO2 on the adaptation of marine microbes in future ocean acidification scenarios.

Molecular Profiling of Peanut under Raw, Roasting, and Autoclaving Conditions Using High-Resolution Magic Angle Spinning and Solution 1H NMR Spectroscopy.

Higher rates of peanut allergy have been observed in countries that commonly roast peanuts prior to consumption. Despite the importance of understanding the role of thermal processing in allergy and on peanut composition, studies toward generating signatures that identify molecular contents following processing are scant. Here, we identified spectral signatures to track changes and differences in the molecular composition of peanuts under raw, roasted, and high-pressure and high-temperature autoclaved conditions. We analyzed both the solid flesh of the seed and solutions derived from soaking peanuts using High-Resolution Magic Angle Spinning (HR-MAS) and solution 1H Nuclear Magnetic Resonance (NMR) spectroscopy, respectively. The NMR spectra of intact peanuts revealed triglycerides as the dominant species, assigned on the basis of multiplets at 4.1 and 4.3 ppm, and corresponding defatted flours revealed the presence of sugars. Sucrose assigned based on a doublet at 5.4 ppm (anomeric proton), and triglycerides were the most abundant small molecules observed, with little variation between conditions. Soaked peanut solutions were devoid of lipids, and their resulting spectra matched the profiles of defatted peanuts. Spectral signatures resulting from autoclaving differed strikingly between those from raw and roasted peanuts, with considerable line-broadening in regions corresponding to proteins and amino-acid side chains, from 0.5 to 2.0 ppm and 6.5 to 8.5 ppm. Taken together, by using complementary NMR methods to obtain a fingerprint of the molecular components in peanuts, we demonstrated that autoclaving led to a distinct composition, likely resulting from the hydrolytic cleavage of proteins, the most important molecule of the allergic reaction.

Elucidation of the O-antigen structure of Escherichia coli O93 and characterization of its biosynthetic genes.

The structure of the O-antigen from the international reference strain Escherichia coli O93:-:H16 has been determined. A nonrandom modal chain-length distribution was observed for the lipopolysaccharide, a pattern which is typical when long O-specific polysaccharides are expressed. By a combination of (i) bioinformatics information on the gene cluster related to O-antigen synthesis including putative function on glycosyl transferases, (ii) the magnitude of NMR coupling constants of anomeric protons, and (iii) unassigned 2D 1H, 13C-HSQC, and 1H,1H-TOCSY NMR spectra it was possible to efficiently elucidate the structure of the carbohydrate polymer in an automated fashion using the computer program CASPER. The polysaccharide also carries O-acetyl groups and their locations were determined by 2D NMR experiments showing that ~½ of the population was 2,6-di-O-acetylated, ~¼ was 2-O-acetylated, whereas ~¼ did not carry O-acetyl group(s) in the 3-O-substituted mannosyl residue of the repeating unit. The structure of the tetrasaccharide repeating unit of the O-antigen is given by: →2)-β-d-Manp-(1→3)-β-d-Manp2Ac6Ac-(1→4)-β-d-GlcpA-(1→3)-α-d-GlcpNAc-(1→, which should also be the biological repeating unit and it shares structural elements with capsular polysaccharides from E. coli K84 and K50. The structure of the acidic O-specific polysaccharide from Cellulophaga baltica strain NN015840T differs to that of the O-antigen from E. coli O93 by lacking the O-acetyl group at O6 of the O-acetylated mannosyl residue.

Sulfated polysaccharide-rich extract from <i>Navicula incerta</i>: physicochemical characteristics, antioxidant activity, and anti-hemolytic property

<abstract> <p>A sulfated polysaccharide from <italic>Navicula incerta</italic> (SPNi) was extracted, and its physicochemical characteristics, antioxidant activity, and anti-hemolytic property were investigated. The polysaccharide yield was 4.8% (SPNi weight/biomass dry weight). Glucose, galactose, mannose, and xylose were the primary sugars. The sulfate content and <italic>Mw</italic> values were 0.46% and 45 kDa, respectively. The FT-IR spectrum showed characteristic bands at 3276,1079,1255, and 820 cm<sup>−1</sup>, related to -OH, C-O-C, S=O, and C-O-S stretching vibration. The <sup>1</sup>H-NMR analysis revealed signals of anomeric protons, indicating the presence of CH<sub>2</sub>-O and CH-O groups. SPNi registered ferric-reducing antioxidant power (up to 1.47 µmol TE/g) and 54% anti-radical activity on ABTS<sup>+</sup>. This polysaccharide registered 90% hemolysis inhibition achieving integrity of the erythrocyte membrane. The results indicate that SPNi could be a candidate for biotechnology applications where antioxidant activity and hemolysis inhibition are required.</p> </abstract>

Best Conditions for the Production of Natural Isopentyl Acetate (Banana Aroma) from Cheese Industry Waste: An Experimental Precursor Approach

In some fermentation systems, whey components (lactose, proteins and minerals) can produce isopentyl acetate (IA). An analysis of the best conditions for IA production with Kluyveromyces marxianus was developed in this work. The experiment design was two-factor and three-level design based on a response surface methodology (RSM) using Design-Expert® software. The analysis of anomeric protons by nuclear magnetic resonance (1H-NMR) showed 81.25% of β lactose content. This characteristic favored the production of IA. The maximum output (Mp) of IA, determined by gas chromatography, was 9.52 g/L (p < 0.05). The central composite design (CCD) was used to perform the factor analysis. Results showed that concentrations of 0.03 (g/L) ammonium sulphate and 0.3 (v/v) of isoamyl alcohol are the best conditions for a maximum rate of IA production. The production of IA can reduce the discharge of whey, allowing its reuse and revaluation.

Physicochemical Characterization of an Exopolysaccharide Produced by Lipomyces sp. and Investigation of Rheological and Interfacial Behavior.

The present study aimed to evaluate the rheological and interfacial behaviors of a novel microbial exopolysaccharide fermented by L. starkeyi (LSEP). The structure of LSEP was measured by LC-MS, 1H and 13C NMR spectra, and FT-IR. Results showed that the monosaccharide composition of LSEP was D-mannose (8.53%), D-glucose (79.25%), D-galactose (7.15%), and L-arabinose (5.07%); there existed the anomeric proton of α-configuration and the anomeric carbon of α- and β-configuration; there appeared the characteristic absorption peak of the phosphate ester bond. The molecular weight of LSEP was 401.8 kDa. The water holding capacity (WHC, 2.10 g/g) and oil holding capacity (OHC, 12.89 g/g) were also evaluated. The results of rheological properties showed that the aqueous solution of LSEP was a non-Newtonian fluid, exhibiting the shear-thinning characteristics. The adsorption of LSEP can reduce the interfacial tension (11.64 mN/m) well and form an elastic interface layer at the MCT–water interface. Such functional properties make LSEP a good candidate for use as thickener, gelling agent, and emulsifier to form long-term emulsions for food, pharmaceutical, and cosmetic products.

Production of Alginate from Persian Gulf Sargassum angustifolium Seaweeds: Novel Extraction and Characterization Methods

Background: Brown seaweeds contain polysaccharides, minerals, proteins, pigments, polyphenols, and fatty acids. Several of these compounds show a wide range of biological activities, such as anticoagulant, anti-tumor, antiviral, and anti-cancer effects. Objectives: This study was designed to evaluate the extraction, purification, and characterization of alginate from Sargassum angustifolium simultaneous with fucoidan extraction and the effect of this process on the structure and properties of alginate. Methods: The extraction of alginate from S. angustifolium was carried out using defatting with organic solvents mixture, treatment with acid-base solutions, and purification with absolute ethanol. The novel characterization of this compound was carried out by the Fourier transform infrared spectroscopy (FT-IR), FT-NMR, energy dispersive X-ray (EDX), and florescent spectrophotometry methods. Results: The fluorescent emission of alginate showed 66.54% removal of impurities, such as phenolic compounds. The FT-IR analysis showed the carboxyl and hydroxyl groups as significant signals in the alginate structure. By analyzing the anomeric protons and other aspects of 1H-NMR, M/G ratio, FG, FM, FGG, FMM, FMG (or FGM) were determined to be 0.61, 0.62, 0.38, 0.31, 0.07, and 0.31, respectively. The intrinsic viscosity and molecular weight of alginate were 0.9 dL/g and 41.53 kDa, respectively. Conclusions: The total amount of alginate from the residual S. angustifolium was 17% of dried seaweed. The structure elucidation of alginate was performed with the FT-IR, FT-NMR, and EDX methods.

Structural elucidation of the exopolysaccharide produced by Curvularia lunata isolate RJ01

Abstract. Jayus J, Akroman R, Nurhayati, Nugraha AS, Piluharto B, Seviour RJ. 2021. Structural elucidation of the exopolysaccharide produced by Curvularia lunata isolate RJ01. Biodiversitas 22: 2699-2705. An exopolysaccharide (EPS) was recovered from culture filtrates of the fungus Curvularia lunata isolate RJ01 prepared in Jember, Indonesia. Based on a prominent peak at 883 cm-1, FTIR analysis suggested it is a ?-D-glucan, a proposal confirmed by subsequent nuclear magnetic resonance (NMR) analysis which showed anomeric protons omit the signal at d4.37 and 4.8 ppm, and sugar proton signal in the region of 2.5 to 4.2 ppm. High-performance liquid chromatography (HPLC) analysis of the acid hydrolysates of this glucan revealed the presence of glucose (64%) and a mixture of galactose and mannose (36%). Digestion with (1-->3)-?- and (1-->6)-?-glucanase from Acremonium sp. IMI 383068 gave consistent linear products of (1-->3)-?-glycosidic linkages, since the glucan was digested by (1-->3)-?-glucanase only. The rheological behavior of aqueous EPS solution suggests it behaves as a pseudoplastic non-Newtonian fluid and thus has the potential for use as a thickening agent in foods. This study is the first report on the structural elucidation of EPS from C. lunata.

Anomeric Proton and Carbon (H1-C1) NMR Chemical Shifts of Antigenic Mannans Obtained from Pathogenic Yeast <i>Candida tropicalis</i>

On two dimensional maps of 1H-13C correlation spectroscopy (H-C COSY) analysis for the mannan of Candida tropicalis, nine cross peaks of anomeric proton and carbon were useful for the purpose of obtaining information on the chemical structure of this molecule. Namely, the mannans was comb-like structure constructed with the linear α-1,6-linked polymannnosyl backbone and several oligomannnosyl side chains composed of α-1,2-, α-1,3-, and β-1,2-linkages. Therefore, in the structural investigation of comb-like mannan, two-dimensional H-C COSY analysis is as useful as two-dimensional nuclear Hartmann-Hahn (HOHAHA) analysis.

Threshold of Thioglycoside Reactivity Difference Is Critical for Efficient Synthesis of Type I Oligosaccharides by Chemoselective Glycosylation

Synthesis of type I LacNAc (Galβ1 → 3GlcNAc) oligosaccharides usually suffers from low yields. We herein report the efficient synthesis of type I LacNAc oligosaccharides by chemoselective glycosylation. With 16 relative reactivity values (RRVs) measured thiotoluenyl-linked disaccharide donors and acceptors, chemoselective glycosylations were investigated to obtain optimal conditions. In these reactions, the RRV difference between the donors and acceptors had to be more than 6311 to obtain type I LacNAc tetrasaccharides in 72-86% yields, with minimal occurrence of aglycon transfer. The threshold of RRV difference was further applied to plan the synthesis of longer glycans. Because it is challenging to measure the RRVs of tetrasaccharides, anomeric proton chemical shifts were utilized to predict the corresponding RRVs, which consequently explained the outcome of glycosylations for the synthesis of type I LacNAc hexasaccharides. The result supported the idea that elongation of glycan chains has to proceed from the reducing to the nonreducing end for a better yield.

Exploration of bioactive prospects of a polysaccharide fraction from Termitomyces heimii against colorectal cancer and broad spectrum bacteria

Termitomyces heimii is a highly cherished wild edible mushroom in India for its pleasant delicacy, rich proximate compositions and profound ethno medicinal applications. A water soluble crude polysaccharide fraction (THP-I) was isolated from dried fruit bodies of T. heimii and purified by GPC. The 1H- NMR spectrum of THP-I exhibited anomeric protons within chemical shifts (δH) between 3.40 to 4.0, indicating that the compound possess –CH and –CH2 functional groups, which resonate with the oxygen atoms of the –OH group, confirmed it as a polysaccharide. THP-I exhibited the MIC values of 62.5 and 125 <mu>g/ml against Staphylococcus aureus and Shigella flexneri respectively. DPPH and FRAP assays showed effective radical scavenging potential by THP-I. In vitro cytotoxicity of THP-I was evaluated by MTT assay and showed notable cellular toxicity against HCT cells at a dose concentration 200 <mu>g/ml. It is first time reported herein that, THP-I has effectively diminished hyperplasia in 1, 2-dimethylhydrazine (DMH) induced colon cancer of Swiss albino rats along with sprouting of goblet cells. Through the present study it is evident that polysaccharide from T. heimii exhibited manifold bioactivities and therefore could be exploited further for its undiscovered potential applications.

Structural Determination of a New Cycloartane Glycoside from Asplenium ruprechtii.

We characterized a new cycloartane glycoside, herein known as aspleniumside F (1), along with five known compounds as kaempferol-3-O-[(6-O-(E)-feruloyl)-β-D-glucopyranosyl]-(1→2)-β-D-galacopyranoside (2), quercetin-3-O-[(6-O-(E)-feruloyl)-β-D-glucopyranosyl]-(1→2)-β-D-glucopyranoside (3), kaempferol-3-O-[(6-O-(E)-caffeoyl)-β-D-glucopyranosyl]-(1→2)-β-D-glucopyranoside (4), kaempferol-3-O-[(6-O-(E)-caffeoyl)-β-D-glucopyranosyl]-(1→2)-β-D-glucopyranosyl-7-O-β-D-glucopyranoside (5), and kaempferol-3-O-[(6-O-p-coumaroyl)-β-D-glucopyranosyl]-(1→2)-β-D-glucopyranosyl-7-O-β-D-glucopyranoside (6), from Asplenium ruprechtii Sa. Kurata, a folk medicine widely used to treat Thromboangiitis obliterans in China, Japan, and Korea. Based on spectroscopic, mainly 1D-, 2D-NMR and (+)-HR-ESI-MS, analyses as well as through comparisons with previous reports, its chemical structure was determined as 3β,24,30-tri-β-D-glucopyranosyl-23,25-dihydroxycycloartane (= (23R,24R)-3β,24-bis-(β-D-glucopyranosyloxy)-23,25-dihydroxy-9β-9,19-cyclolanostan-29-yl β-D-glucopyranoside). According to the 1 H coupling constant of anomeric protons and co-TLC of the acid hydrolysate with D-glucose, all three glycoside groups in 1 were revealed as β-D-glucopyranosyl. Furthermore, SOD-like antioxidant activity evaluation via IC50 of 12.43, 6.78, 9.12, 6.94 and 4.85 μM revealed that compounds 2-6 had bioactivity.

Structural determination and in vitro tumor cytotoxicity evaluation of five new cycloartane glycosides from Asplenium ruprechtii Sa. Kurata

Five new cycloartane glycosides, named aspleniumside A – E, were discovered and characterized by re-investigated the remaining extracts of the whole plant of Asplenium ruprechtii Sa. Kurata, a famous folk medicine for treating thromboangitis obliterans in China, Japan, and Korea. Compounds 3–5 possessed the 9,19-seco-cycloartane-9,11-en triterpene aglycone with 3,7(or 23),24,25,30-highly oxidized methylene, methylene or quaternary carbons, that was found in this species for the first time. The stereo-chemistry of all new compounds were fully discussed by extensive analysis of the 1D and 2D NMR data, and comparisons with those data of known compounds. 24R configuration was determined here which indicated the different growing areas of the same species could influence the secondary metabolic behavior, leading to the differences in chemical composition. All glycoside groups were determined as β-d-glucopyranosyl by 1H coupling constant of anomeric protons and co-TLC of the acid hydrolysate with d-glucose. All the cycloartane glycosides were evaluated against HL-60 and HepG2 cells for cytotoxicity, compounds 1–3, showed potential cytotoxicity with the IC50 in range of 18–60 μM, while the standard sorafenib showed IC50 value of 10.61 ± 0.43 and 13.43 ± 1.12 μM against HL-60 and HepG2, respectively. The results attained in this study indicated that cycloartane glycosides should be the cytotoxicity substance in A. ruprechtii Sa. Kurata, and had the potential to be developed as tumor cytotoxicity agent applied in clinic.

Assignment of anomeric protons and base 1H, 13C and 15N chemical shifts for 5_SL4

Assignment of anomeric protons and base 1H, 13C and 15N chemical shifts for 5_SL4

Optimising deproteinisation methods and effect of deproteinisation on structural and functional characteristics of flaxseed gum

SummaryIn the current study, deproteinisation rate (DPR) and carbohydrate loss ratio (CLR) in flaxseed gum (FSG) were compared as indexes by using four different deproteinised methods, that is sodium chloride (NaCl), trichloroacetic acid (TCA), hydrochloric acid (HCl) and calcium chloride (CaCl2). In addition, the effect of deproteinisation on physicochemical, functional and structural characteristics (FTIR, 1H NMR and SEM analysis) of deproteinised flaxseed gum (DFSG) was further examined. The results revealed that HCl and TCA methods had better ability of deproteinisation (97.83% and 96.21%) but resulted in higher CLR (27.66% and 24.19%), while CaCl2 method excelled over the NaCl method in DPR (94.7% and 88.6%) and CLR (4.90% and 4.21%), respectively. Flaxseed gum showed significant functional properties even after deproteinisation and exhibited resolved resonances for both ring and anomeric protons in 1H NMR spectra. The characteristic groups in FTIR spectra were retained after deproteinisation.

Resorcinosides A and B, Glycosylated Alkylresorcinols from a Marine-Derived Strain of the Fungus Penicillium janthinellum.

Two new glycosylated alkylresorcinols, resorcinosides A (1) and B (2), were isolated from a strain of the fungus Penicillium janthinellum derived from a marine sediment sample collected from Cu Lao Cham Island, Vietnam. The structures of 1 and 2 were established by interpretation of 1D and 2D NMR and high-resolution ESIMS data, and their absolute configurations were confirmed by the coupling constant of the anomeric proton, acid hydrolysis, subsequent HPLC analysis, Mosher's method, and quantum-mechanics-based computational analysis of NMR chemical shifts. The structure elucidation indicated that 1 and 2 are new alkylresorcinols with d-glucose, and 2 has an α-pyrone moiety attached to the aromatic ring. Compound 1 exhibited cytotoxic activity against the NUGC-3 cancer cell line with a GI50 value of 9.3 μM.

Facile rare-earth triflate-catalyzed esterification of cellulose by carboxylic anhydrides under solvent-free conditions

We report here the facile rare-earth triflate-catalyzed esterification of cellulose by carboxylic anhydrides, including acetic anhydride, at room temperature under solvent-free conditions. As a model, d-glucose was peraceylated using only equimolar amounts of acetic anhydrides against hydroxyl groups to give the expected 1,2,3,4,6-pentaacetyl α-d-glucopyranoside (crude yield 98%, 68% yield after recrystallization) under solvent-free condition. In the case of powdery microcrystalline cellulose, acetylation proceeded both in the absence of pyridine and solvent to give peracetylated cellulose, even in the bulk. From 1H NMR measurements, the degree of substitution (DS) was 3.0 (100% acetylation) and we concluded that Sc(OTf)3-catalyzed solvent-free esterification of cellulose had taken place. The number-average molecular weight (Mn), calculated using the 1H NMR intensity ratio based on the anomeric proton in the reducing terminus, was 1.7 × 104 (repeating unit of pyranose unit: 56).

KARAKTERISASI SENYAWA FENOLIK DARI BIJI BUAH RAMBUTAN (Nephelium lappaceum L.)

Rambutan seeds are one of the waste produced from rambutan that is potential to be used in the food and pharmaceutical fields because it is rich in nutrients and secondary metabolites. This research was carried out to characterize phenolic compounds from the ethyl acetate fraction by using chromatographic methods, phytochemical tests, and 1H-NMR analysis. The isolate was obtained as white powder with 6.2 mg and gave positive sign for phenolic by phytochemical test. 1H-NMR spectrum data (CD3OD, 500 MHz) showed the chemical shift characteristic of coumarin compounds at δH (ppm): 7.82 (1H, d, J= 8.3 Hz), 7.39 (1H, d, J= 7.55 Hz), 6.73 (1H, d, J= 8.05 Hz), and 5.61 (1H, d, J= 7.55 Hz), an anomeric proton at δH 4.8-5 ppm, the characteristics of the methyl group of L-rhamnose sugar at δH 1.29 ppm and other sugar protons at δH 3-4 ppm. Based on the 1H-NMR analysis and comparison with literature, a phenolic compound obtained from rambutan (Nephelium lappaceum L.) seeds is 7-O-β-L-rhamnose-8-hydroxycoumarin or 8-O-β-L-rhamnose-7-hydroxycoumarin.

Structures, NMR Spectroscopic Features, and Cytotoxic Properties of Oligomeric Hellinoyl (m-GO-m-GOG)-Type Ellagitannins from the Galls of Tamarix aphylla.

Ellagitannin oligomers are large molecules habitually showing complex NMR spectra that are sometimes misinterpreted and lead to incorrect structures. Understanding the NMR spectroscopic features of a group of ellagitannins would overcome these inadequacies. In this study, investigation of the galls of Tamarix aphylla led to the isolation of three new ellagitannin oligomers, phyllagallins T1 (1), T2 (2), and Q1 (3), a known monomer nilotinin M4 (4), four known dimers, nilotinins D7 (5) and D8 (6), hirtellin B (7), and tamarixinin A (8), and a simple phenolic, dehydrotrigallic acid (9). 1D and 2D NMR, HRESI-TOFMS, and ECD experiments show that compounds 1-8 are hellinoyl-type ellagitannins. The NMR spectroscopic features of this type of ellagitannins and the reasons for the abnormal upfield shifts of glucose anomeric proton and hellinoyl moiety proton signals are established considering the experimental results as well as quantum chemical calculation on a simple hellinoyl-type monomer, phyllagallin M2. Based on these results, the NMR assignments reported previously by a different research group for bracteatinin T1 and hirtellin T3 are revised. A cytotoxicity study against human oral squamous cell carcinoma cell lines (Ca9-22, HSC-2, and HSC-4) and human mesenchymal normal oral cells (HGF, HPC, and HPLF) showed cytotoxic effects with tumor-specificity higher than 5.2, 3.0, 1.6, and 2.0 for compounds 5, 2, 9, and 3, respectively.

Two-dimensional qNMR of anthraquinones in Frangula alnus (Rhamnus frangula) using surrogate standards and delay time adaption

In the absence of adequate reference material, a problem often encountered in natural product chemistry, we investigated the use of surrogate standards in two-dimensional qNMR for the quantification of anthraquinones in the bark of alder buckthorn (Frangula alnus). Using the integrals of cross signals in the HSQC spectrum obtained from commercial standards rutin and duroquinone and adapting the delays for the 1JCH coupling, we quantified the total amount of anthraquinones and anthraquinone glucosides, as well as the content of the value-determining glucofrangulins and frangulins. Thereby, duroquinone was used as an external standard to establish the calibration curve for the methylated anthraquinone scaffold, whereas calibration curves for the glycosides were obtained using the anomeric proton signals of the rutinose disaccharide. The method was validated for accuracy, precision, specificity, linearity and limit of quantitation and shows clear advantages over the method of the European Pharmacopeia, especially in terms of specificity and meaningfulness of the results. Apart from being a useful alternative in the quality control of alder buckthorn, the presented approach demonstrates, moreover, the versatility of sophisticated 2D measurements in quantitative NMR.