Sileshi G. Wubshet

From Retrospective Assessment to Prospective Decisions in Natural Product Isolation: HPLC-SPE-NMR Analysis of Carthamus oxyacantha

An extract of Carthamus oxyacantha (wild safflower) was investigated using two approaches: a traditional, nontarget fractionation by VLC and HPLC, and the hyphenated technique HPLC-PDA-HRMS-SPE-NMR followed by targeted isolation of selected constituents for inclusion in a screening library of pure natural products. While the nontarget fractionation involved considerable time spent on pursuing fractions containing well-known or undesired compounds, the hyphenated analysis was considerably faster and required less solvent and other consumables. The results were used to design and execute an optimized, HPLC-HRMS-guided, targeted isolation scheme aiming exclusively at a series of identified spiro compounds. Thus, HPLC-PDA-HRMS-SPE-NMR is a dereplication technique of choice, allowing economical acquisition of comprehensive data about compounds in crude extracts, which can be used for rational, prospective decisions about further isolation efforts. A total of 15 compounds were identified in the extract. Six spiro compounds, of which four have not previously been characterized, and tracheloside (a lignin glucoside) are presented with assigned 1H and 13C chemical shifts.

Coupling HPLC-SPE-NMR with a microplate-based high-resolution antioxidant assay for efficient analysis of antioxidants in food--validation and proof-of-concept study with caper buds.

This work describes the coupling of a microplate-based antioxidant assay with a hyphenated system consisting of high-performance liquid chromatography-solid-phase extraction-nuclear magnetic resonance spectroscopy, i.e., HPLC-SPE-NMR/high-resolution antioxidant assay, for the analysis of complex food extracts. The applicability of the microplate-based antioxidant assay for high-resolution screening of common food phenolics as well as parameters related to their trapping efficiency, elution behavior, and recovery on/from SPE cartridges are described. It was found that the microplate-based high-resolution antioxidant assay is an attractive and easy implementable alternative to direct on-line screening methods. Furthermore, it was shown that Resin SH and Resin GP SPE material are superior to RP C18HD for trapping of phenolic compounds. Proof-of-concept study was performed with caper bud extract, revealing the most important antioxidants to be quercetin, kaempferol, rutin, kaempferol-3-O-β-rutinoside and N(1),N(5),N(10)-triphenylpropenoyl spermidine amides. Targeted isolation of the latter, and comprehensive NMR experiments showed them to be N(1),N(10)-di-(E)-caffeoyl-N(5)-p-(E)-coumaroyl spermidine, N(1)-(E)-caffeoyl-N(5),N(10)-di-p-(E)-coumaroyl spermidine, N(10)-(E)-caffeoyl-N(1),N(5)-di-p-(E)-coumaroyl spermidine, and N(1),N(5),N(10)-tri-p-(E)-coumaroyl spermidine amides.

Targeting high-performance liquid chromatography–high-resolution mass spectrometry–solid-phase extraction–nuclear magnetic resonance analysis with high-resolution radical scavenging profiles—Bioactive secondary metabolites from the endophytic fungus Penicillium namyslowskii

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.

Maritime Halophyte Species from Southern Portugal as Sources of Bioactive Molecules

Extracts of five halophytes from southern Portugal (Arthrocnemum macrostachyum, Mesembryanthemum edule, Juncus acutus, Plantago coronopus and Halimione portulacoides), were studied for antioxidant, anti-inflammatory and in vitro antitumor properties. The most active extracts towards the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical were the methanol extracts of M. edule (IC50 = 0.1 mg/mL) and J. acutus (IC50 = 0.4 mg/mL), and the ether extracts of J. acutus (IC50 = 0.2 mg/mL) and A. macrostachyum (IC50 = 0.3 mg/mL). The highest radical scavenging activity (RSA) against the 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical was obtained in the ether extract of J. acutus (IC50 = 0.4 mg/mL) and H. portulacoides (IC50 = 0.9 mg/mL). The maximum total phenolic content (TPC) was found in the methanol extract of M. edule (147 mg gallic acid equivalents (GAE)/g) and in the ether extract of J. acutus (94 mg GAE/g). Significant decreases in nitric oxide (NO) production were observed after incubation of macrophages with lipopolysaccharide (LPS) and the chloroform extract of H. portulacoides (IC50 = 109 µg/mL) and the hexane extract of P. coronopus (IC50 = 98.0 µg/mL). High in vitro cytotoxic activity and selectivity was obtained with the ether extract of J. acutus. Juncunol was identified as the active compound and for the first time was shown to display selective in vitro cytotoxicity towards various human cancer cells.

Direct 13C NMR Detection in HPLC Hyphenation Mode: Analysis of Ganoderma lucidum Terpenoids

Solid phase extraction (SPE) was introduced as a crucial step in the HPLC-SPE-NMR technique to enable online analyte enrichment from which proton-detected NMR experiments on submicrogram amounts from complex mixtures were possible. However, the significance of direct-detected (13)C NMR experiments is indubitable in simplifying structural elucidations. In the current study, we demonstrated direct (13)C NMR detection of triterpenoids from a Ganoderma lucidum extract in hyphenation mode. The combined advantage of a cryogenically cooled probe, miniaturization, and multiple trapping enabled the first reported application of HPLC-SPE-NMR analysis using direct-detected (13)C NMR spectra. HPLC column loading, accumulative SPE trappings, and the effect of different elution solvents were evaluated and optimized. A column loading of approximately 600 μg of a prefractionated triterpenoid mixture, six trappings, and an acquisition time of 13 h resulted in spectra with adequate signal-to-noise ratios to detect all C-13 signals.

High-Resolution Screening Combined with HPLC-HRMS-SPE-NMR for Identification of Potential Health-Promoting Constituents in Sea Aster and Searocket—New Nordic Food Ingredients

Sea aster (Aster tripolium L.) and searocket (Cakile maritima Scop.), potential ingredients in the New Nordic Diet, were analyzed by high-resolution radical scavenging and high-resolution α-glucosidase inhibition assays. Results from the two bioactivity profiles were used to guide subsequent structural analysis toward constituents with potential health-promoting effects. Structural analysis was performed by high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction and automated tube transfer nuclear magnetic resonance spectroscopy, that is, HPLC-HRMS-SPE-ttNMR. High-resolution mass spectrometry together with detailed analysis of one- and two-dimensional proton detected NMR experiments enabled unambiguous assignment of the targeted analytes. This revealed a series of caffeoyl esters (1, 2, 5), flavonoid glycosides (3, 4, 6, 11-15), flavonoids (7-9), sinapate esters (10, 16, 17), and sinapinic acid (18) associated with radical scavenging and/or α-glucosidase inhibition. In vitro assays implemented in this study showed that sea aster holds potential as a future functional food ingredient for lowering postprandial blood glucose level for diabetics, but further investigations are needed to prove the effect in vivo.

HPLC-NMR revisited: using time-slice high-performance liquid chromatography-solid-phase extraction-nuclear magnetic resonance with database-assisted dereplication.

Time-based trapping of chromatographically separated compounds onto solid-phase extraction (SPE) cartridges and subsequent elution to NMR tubes was done to emulate the function of HPLC-NMR for dereplication purposes. Sufficient mass sensitivity was obtained by use of a state-of-the-art HPLC-SPE-NMR system with a cryogenically cooled probe head, designed for 1.7 mm NMR tubes. The resulting (1)H NMR spectra (600 MHz) were evaluated against a database of previously acquired and prepared spectra. The in-house-developed matching algorithm, based on partitioning of the spectra and allowing for changes in the chemical shifts, is described. Two mixtures of natural products were used to test the approach: an extract of Carthamus oxyacantha (wild safflower), containing an array of spiro compounds, and an extract of the endophytic fungus Penicillum namyslowski, containing griseofulvin and analogues. The database matching of the resulting spectra positively identified expected compounds, while the number of false positives was few and easily recognized.

Triple aldose reductase/α-glucosidase/radical scavenging high-resolution profiling combined with high-performance liquid chromatography–high-resolution mass spectrometry–solid-phase extraction–nuclear magnetic resonance spectroscopy for identification of antidiabetic constituents in crude extract of Radix Scutellariae

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.

High-resolution bioactivity profiling combined with HPLC–HRMS–SPE–NMR: α-Glucosidase inhibitors and acetylated ellagic acid rhamnosides from Myrcia palustris DC. (Myrtaceae)

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-β-d-(6″-galloyl)galactopyranoside, kaempferol 3-O-β-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.

Oxidation and cyclization of casbene in the biosynthesis of Euphorbia factors from mature seeds of Euphorbia lathyris L.

Significance Ingenol mebutate is a diterpene ester with a highly complex macrocyclic structure that has been approved for the treatment of actinic keratosis, a precondition of skin cancer. The current production of ingenol mebutate through plant extraction or chemical synthesis is inefficient and costly. Here, we describe the discovery of a biosynthetic route in Euphorbia lathyris L. (caper spurge) in which regio-specific oxidation of casbene is followed by an unconventional cyclization to yield jolkinol C, a probable key intermediate in the biosynthesis of macrocyclic diterpenes, including ingenol mebutate. These results can facilitate the biotechnological production of this high-value pharmaceutical and discovery of new biosynthetic intermediates with important bioactivities. The seed oil of Euphorbia lathyris L. contains a series of macrocyclic diterpenoids known as Euphorbia factors. They are the current industrial source of ingenol mebutate, which is approved for the treatment of actinic keratosis, a precancerous skin condition. Here, we report an alcohol dehydrogenase-mediated cyclization step in the biosynthetic pathway of Euphorbia factors, illustrating the origin of the intramolecular carbon–carbon bonds present in lathyrane and ingenane diterpenoids. This unconventional cyclization describes the ring closure of the macrocyclic diterpene casbene. Through transcriptomic analysis of E. lathyris L. mature seeds and in planta functional characterization, we identified three enzymes involved in the cyclization route from casbene to jolkinol C, a lathyrane diterpene. These enzymes include two cytochromes P450 from the CYP71 clan and an alcohol dehydrogenase (ADH). CYP71D445 and CYP726A27 catalyze regio-specific 9-oxidation and 5-oxidation of casbene, respectively. When coupled with these P450-catalyzed monooxygenations, E. lathyris ADH1 catalyzes dehydrogenation of the hydroxyl groups, leading to the subsequent rearrangement and cyclization. The discovery of this nonconventional cyclization may provide the key link to complete elucidation of the biosynthetic pathways of ingenol mebutate and other bioactive macrocyclic diterpenoids.