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Protein tyrosine phosphatase 1B (PTP1B) is an intracellular enzyme responsible for deactivation of the insulin receptor, and consequently acts as a negative regulator of insulin signal transduction. In recent years, PTP1B has become an important target for controlling insulin resistance and type 2 diabetes. In the present study, the ethyl acetate extract of leaves of Miconia albicans (IC50 = 4.92 µg/mL) was assessed by high-resolution PTP1B inhibition profiling combined with HPLC-HRMS-SPE-NMR for identification of antidiabetic compounds. This disclosed eleven PTP1B inhibitors, including five polyphenolics: 1-O-(E)-caffeoyl-4,6-di-O-galloyl-ß-d-glucopyranose (2), myricetin 3-O-α-l-rhamnopyranoside (3), quercetin 3-O-(2″-galloyl)-α-l-rhamnopyranoside (5), mearnsetin 3-O-α-l-rhamnopyranoside (6), and kaempferol 3-O-α-l-arabinopyranoside (8) as well as eight triterpenoids: maslinic acid (13), 3-epi-sumaresinolic acid (14), sumaresinolic acid (15), 3-O-cis-p-coumaroyl maslinic acid (16), 3-O-trans-p-coumaroyl maslinic acid (17), 3-O-trans-p-coumaroyl 2α-hydroxydulcioic acid (18), oleanolic acid (19), and ursolic acid (20). These results support the use of M. albicans as a traditional medicine with antidiabetic properties and its potential as a source of PTP1B inhibitors.

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In this paper, quadruple high-resolution α-glucosidase/α-amylase/PTP1B/radical scavenging profiling combined with HPLC-HRMS-SPE-NMR were used for studying the polypharmacological properties of crude root bark extract of Morus alba L. This species is used as an anti-diabetic principle in many traditional treatment systems around the world, and the crude ethyl acetate extract of M. alba root bark was found to inhibit α-glucosidase, α-amylase and protein-tyrosine phosphatase 1B (PTP1B) with IC50 values of 1.70 ±â€¯0.72, 5.16 ±â€¯0.69, and 5.07 ±â€¯0.68 µg/mL as well as showing radical scavenging activity equaling a TEAC value of (3.82 ±â€¯0.14) × 104 mM per gram extract. Subsequent investigation of the crude extract using quadruple high-resolution α-glucosidase/α-amylase/PTP1B/radical scavenging profiling provided a quadruple biochromatogram that allowed direct correlation of the HPLC peaks with one or more of the tested bioactivities. This was used to target subsequent HPLC-HRMS-SPE-NMR analysis towards peaks representing bioactive analytes, and led to identification of a new Diels-Alder adduct named Moracenin E as well as a series of Diels-Alder adducts and isoprenylated flavonoids as potent α-glucosidase and α-amylase inhibitors with IC50 values in the range of 0.60-27.15 µM and 1.22-69.38 µM, respectively. In addition, these compounds and two 2-arylbenzofurans were found to be potent PTP1B inhibitors with IC50 values ranging from 4.04 to 21.67 µM. The high-resolution radical scavenging profile also revealed that almost all of the compounds possess radical scavenging activity. In conclusion the quadruple high-resolution profiling method presented here allowed a detailed profiling of individual constituents in crude root bark extract of M. alba, and the method provides a general tool for detailed mapping of bioactive constituents in polypharmacological herbal remedies.

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This work describes an analytical platform based on semi-high-resolution antileishmanial profiling combined with hyphenation of high-performance liquid chromatography - high-resolution mass spectrometry - solid-phase extraction - nuclear magnetic resonance spectroscopy, i.e., semiHR-antileishmanial assay/HPLC-HRMS-SPE-NMR. The platform enables fast pinpointing of HPLC peaks representing Leishmania tropica inhibitors in complex matrices, with subsequent structural identification of targeted inhibitors. Active analytes were cumulatively trapped on SPE cartridges and the structures elucidated by analysis of NMR spectra obtained in the HPLC-HRMS-SPE-NMR mode. This led to the identification of six known compounds 2,4,6-trihydroxyacetophenone-2-O-ß-D-glucopyranoside (1), lalioside (2), luteolin-4'-O-ß-D-glucopyranoside (3), apigenin-4'-O-ß-D-glucopyranoside (4), luteolin (5), and apigenin (6). IC50 of the active compounds were determined with luteolin being the most potent inhibitor with an IC50 value of 4.15 µg/ml. The platform proved to be an efficient method for the identification of L. tropica inhibitors.

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Solanum americanum is one of the most prominent species used to treat type 2 diabetes in Guatemala. In our ongoing efforts to find antidiabetic and antioxidative compounds from natural sources, an ethyl acetate extract of this medicinal herb was investigated using dual high-resolution α-glucosidase/radical scavenging inhibition profiling. The high-resolution biochromatograms obtained by this technique were used to target subsequent structural elucidation by HPLC-HRMS-SPE-NMR analysis towards the bioactive constituents. This led to identification of 4-hydroxybenzoic acid (1) and 3-indolecarboxylic acid (6) associated with radical scavenging activity, and the amide alkaloids N-trans-p-coumaroyloctopamine (3), N-trans-p-feruloyloctopamine (4), N-trans-p-coumaroyltyramine (8) and N-trans-p-feruloyltyramine (9) correlated with α-glucosidase inhibitory activity as well as radical scavenging activity. Further analysis revealed a new lactone, methyl 5-ethyl-4-hydroxy-5-methyl-2-oxotetrahydro-2H-pyran-4-carboxylate (7) and a new steroid with a rare F ring (11). Corchorifatty acid B (12) was reported for the first time in the Solanaceae family. Their structures were elucidated by extensive use of 1D and 2D NMR spectroscopy as well as HRMS analysis.

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α-Glucosidase inhibitors decrease the cleavage- and absorption rate of monosaccharides from complex dietary carbohydrates, and represent therefore an important class of drugs for management of type 2 diabetes. In this study, a defatted ethyl acetate extract of Eremanthus crotonoides leaves with an inhibitory concentration (IC50) of 34.5 µg/mL towards α-glucosidase was investigated by high-resolution α-glucosidase inhibition profiling combined with HPLC-HRMS-SPE-NMR. This led to identification of six α-glucosidase inhibitors, namely quercetin (16), trans-tiliroside (17), luteolin (19), quercetin-3-methyl ether (20), 3,5-di-O-caffeoylquinic acid n-butyl ester (26) and 4,5-di-O-caffeoylquinic acid n-butyl ester (29). In addition, nineteen other metabolites were identified. The most active compounds were the two regioisomeric di-O-caffeoylquinic acid derivatives 26 and 29, with IC50 values of 5.93 and 5.20 µM, respectively. This is the first report of the α-glucosidase inhibitory activity of compounds 20, 26, and 29, and the findings support the important role of Eremanthus species as novel sources of new drugs and/or herbal remedies for treatment of type 2 diabetes.

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Crude chloroform, ethanol and acetone extracts of nineteen seaweed species were screened for their antioxidant and α-glucosidase inhibitory activity. Samples showing more than 60% α-glucosidase inhibitory activity, at a concentration of 1 mg/ml, were furthermore investigated using high-resolution α-glucosidase inhibition profiling combined with high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy (HR-bioassay/HPLC-HRMS-SPE-NMR). The results showed Ascophyllum nodosum and Fucus vesicolosus to be rich in antioxidants, equaling a Trolox equivalent antioxidant capacity of 135 and 108 mM Troloxmg(-1) extract, respectively. HR-bioassay/HPLC-HRMS-SPE-NMR showed the α-glucosidase inhibitory activity of A. nodosum, F. vesoculosus, Laminaria digitata, Laminaria japonica and Undaria pinnatifida to be caused by phlorotannins as well as fatty acids - with oleic acid, linoleic acid and eicosapentaenoic acid being the most potent with IC50 values of 0.069, 0.075 and 0.10 mM, respectively, and showing a mixed-type inhibition mode.

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Type 2 diabetes (T2D) constituted 90% of the global 387 million diabetes cases in 2014. The enzyme protein-tyrosine phosphatase 1B (PTP1B) has been recognized as a therapeutic target for treatment of T2D and its adverse complications. With the aim of accelerating the investigation of complex natural sources, such as crude plant extracts, for potential PTP1B inhibitors, we have developed a bio-analytical platform combining high-resolution PTP1B inhibition profiling and high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy, i.e., HR-bioassay/HPLC-HRMS-SPE-NMR. Human recombinant PTP1B enzyme was used for the microplate-based PTP1B inhibition assay, which was optimized for pH and substrate concentration to be compatible with rate measurements within the 10 min incubation time. Subsequently, analytical-scale HPLC-based microfractionation followed by colorimetric microplate-based PTP1B bioassaying enabled construction of a high-resolution inhibition profile corresponding to the HPLC profile. The high-resolution PTP1B inhibition profiling was validated using an artificial mixture of known PTP1B inhibitors and non-inhibiting compounds as negative controls. Finally, a proof-of-concept study with a real sample was performed using crude ethyl acetate extract of the phytochemically hitherto unexplored plant Eremophila lucida. This led to the identification of the first viscidane type diterpene, i.e., 5-hydroxyviscida-3,14-dien-20-oic acid (9) as PTP1B inhibitor with an IC50 value of 42.0 ± 5.9 µM. In addition, a series of flavonoids, i.e., luteolin (1), dinatin (3a), tricin (3b), 3,6-dimethoxyapigenin (4), jaceidin (5), and cirsimaritin (6) as well as a cembrene diterpene, (3Z, 7E, 11Z)-15-hydroxycembra-3,7,11-trien-19-oic acid (8), were also identified for the first time from E. lucida.

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Inhibition of the necrotizing hyaluronidase, phospholipase A2 and protease enzymes in four snake venoms by crude water and ethanol extracts of 88 plant species used against snakebites in traditional Chinese medicine was measured. High-resolution hyaluronidase inhibition profiles were constructed for the 22 plants showing highest hyaluronidase inhibition, and the results were used to guide subsequent structural analysis towards specific hyaluronidase inhibitors. Structural analysis was performed by high-performance liquid chromatography, high-resolution mass spectrometry, solid-phase extraction and nuclear magnetic resonance spectroscopy, i.e., HPLC-HRMS-SPE-NMR. This allowed identification of four non-tannin inhibitors, i.e., lansiumamide B (6) from Clausena excavata Burm.f., myricetin 3-O-ß-D-glucopyranoside (7) from Androsace umbellata (Lour.) Merr., and vitexin (8) and 4',7-dihydroxy-5-methoxyflavone-8-C-ß-D-glucopyranoside (9) from Oxalis corniculata L. Absolute configuration of 2,3-dihydroxy-N-methyl-3-phenyl-N-[(Z)-styryl]propanamide (1) was determined using the Mosher method, which revealed two enantiomers, i.e., (2S,3R)-2,3-dihydroxy-N-methyl-3-phenyl-N-[(Z)-styryl]propanamide and (2R,3S)-2,3-dihydroxy-N-methyl-3-phenyl-N-[(Z)-styryl]propanamide with a ratio of 7:3.

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In this work, development of a new microplate-based high-resolution profiling assay using recombinant human aldose reductase is presented. Used together with high-resolution radical scavenging and high-resolution α-glucosidase assays, it provided the first report of a triple aldose reductase/α-glucosidase/radical scavenging high-resolution inhibition profile - allowing proof of concept with Radix Scutellariae crude extract as a polypharmacological herbal drug. The triple bioactivity high-resolution profiles were used to pinpoint bioactive compounds, and subsequent structure elucidation was performed with hyphenated high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy. The only α-glucosidase inhibitor was baicalein, whereas main aldose reductase inhibitors in the crude extract were baicalein and skullcapflavone II, and main radical scavengers were ganhuangemin, viscidulin III, baicalin, oroxylin A 7-O-glucuronide, wogonoside, baicalein, wogonin, and skullcapflavone II.

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In our ongoing efforts of finding natural fungicides to fight food and feed spoilage during production and storage, the antifungal potential of Ghanaian Uvaria chamae P. Beauv. was investigated, with emphasis on plant metabolites targeting the fungal plasma membrane (PM) H(+)-ATPase. Ethyl acetate extract of U. chamae was subjected to high-resolution fungal PM H(+)-ATPase inhibition screening followed by structural elucidation by high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy (HPLC-HRMS-SPE-NMR). This led to identification of a series of uncommon o-hydroxybenzylated flavanones and chalcones, i.e., chamanetin (8), isochamanetin (9), isouvaretin (10), uvaretin (11), dichamanetin (12), and diuvaretin (15). Preparative-scale isolation of the active metabolites allowed determination of IC50 values for inhibition of the PM H(+)-ATPase, and growth inhibition of Saccharomyces cerevisiae and Candida albicans. These revealed a strong correlation between o-hydroxybenzyl substituents and PM H(+)-ATPase activity, with dichamanetin being the most potent compound, but showing moderate activity in the fungal growth inhibition assays.

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Type 2 diabetes (T2D) is an endocrine metabolic disease with a worldwide prevalence of more than 8%, and an expected increase close to 50% in the next 15-20years. T2D is associated with severe and life-threatening complications like retinopathy, neuropathy, nephropathy, and cardiovascular diseases, and therefore improved drug leads or functional foods containing α-glucosidase inhibitors are needed for management of blood glucose. In this study, leaves of Myrcia palustris were investigated by high-resolution α-glucosidase inhibition profiling combined with HPLC-HRMS-SPE-NMR. This led to identification of casuarinin, myricetin 3-O-ß-d-(6″-galloyl)galactopyranoside, kaempferol 3-O-ß-d-galactopyranoside, myricetin, and quercetin as α-glucosidase inhibitors. In addition, four acetylated ellagic acid rhamnosides, i.e., 4-O-(2″,4″-O-diacetyl-α-l-rhamnopyranosyl)ellagic acid, 4-O-(2″,3″-O-diacetyl-α-l-rhamnopyranosyl)ellagic acid, 4-O-(3″,4″-O-diacetyl-α-l-rhamnopyranosyl)ellagic acid, and 4-O-(2″,3″,4″-O-triacetyl-α-l-rhamnopyranosyl)ellagic acid were identified.

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Bulbs and leaves of 35 Allium species and cultivars bought or collected in 2010-2012 were investigated with multivariate data analysis, high-resolution α-glucosidase inhibition assays and HPLC-HRMS-SPE-NMR with the aim of exploring the potential of Allium as a future functional food for management of type 2 diabetes. It was found that 30 out of 106 crude extracts showed more than 80% inhibition of the α-glucosidase enzyme at a concentration of 40mg/mL (dry sample) or 0.4g/mL (fresh sample). High-resolution α-glucosidase biochromatograms of these extracts allowed fast identification of three analytes with α-glucosidase inhibitory activity, and subsequent HPLC-HRMS-SPE-NMR experiments allowed identification of these as N-p-coumaroyloctopamine, N-p-coumaroyltyramine, and quercetin. The distribution of these three compounds was mapped for all samples by HPLC-ESI-HRMS. Unsupervised principal component analysis of samples from 2012 indicated that a major difference between fresh material and dried material is the increased amount of quercetin, a known α-glucosidase inhibitor.

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The high-resolution radical scavenging profile of an extract of the endophytic fungus Penicillium namyslowskii was used to target analysis by high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy, i.e., HPLC-HRMS-SPE-NMR, for identification of anti-oxidative secondary metabolites. This revealed the two chromatographic peaks with the highest relative response in the radical scavenging profile to be griseophenone C and peniprequinolone. The HPLC-HRMS-SPE-NMR analysis was performed in the tube-transfer mode using a cryogenically cooled NMR probe designed for 1.7mm NMR tubes. To further explore the potential of the above HPLC-HRMS-SPE-NMR platform for analysis of endophytic extracts, six peaks displaying no radical scavenging activity were also analyzed. This allowed unambiguous identification of six metabolites, i.e., dechlorogriseofulvin, dechlorodehydrogriseofulvin, griseofulvin, dehydrogriseofulvin, mevastatin acid, and mevastatin. The high mass sensitivity of the 1.7mm cryogenically cooled NMR probe allowed for the first time acquisition of direct detected (13)C NMR spectra of fungal metabolites, i.e., dechlorogriseofulvin and griseofulvin, directly from crude extract via HPLC-HRMS-SPE-NMR. Dechlorodehydrogriseofulvin was reported for the first time from nature.

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