Search

As a complement to vaccines, small-molecule therapeutic agents are needed to treat or prevent infections by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants, which cause COVID-19. Affinity selection-mass spectrometry was used for the discovery of botanical ligands to the SARS-CoV-2 spike protein. Cannabinoid acids from hemp ( Cannabis sativa ) were found to be allosteric as well as orthosteric ligands with micromolar affinity for the spike protein. In follow-up virus neutralization assays, cannabigerolic acid and cannabidiolic acid prevented infection of human epithelial cells by a pseudovirus expressing the SARS-CoV-2 spike protein and prevented entry of live SARS-CoV-2 into cells. Importantly, cannabigerolic acid and cannabidiolic acid were equally effective against the SARS-CoV-2 alpha variant B.1.1.7 and the beta variant B.1.351. Orally bioavailable and with a long history of safe human use, these cannabinoids, isolated or in hemp extracts, have the potential to prevent as well as treat infection by SARS-CoV-2.

Affinity selection-mass spectrometry, which includes magnetic microbead affinity selection-screening (MagMASS), is ideal for the discovery of ligands in complex mixtures that bind to pharmacological targets. Therapeutic agents are needed to prevent or treat COVID-19, which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Infection of human cells by SARS-CoV-2 involves binding of the virus spike protein subunit 1 (S1) to the human cell receptor angiotensin converting enzyme-2 (ACE2). Like antibodies, small molecules have the potential to block the interaction of the viral S1 protein with human ACE2 and prevent SARS-CoV-2 infection. Therefore, a MagMASS assay was developed for the discovery of ligands to the S1 protein. Unlike previous MagMASS approaches, this new assay used robotics for 5-fold enhancement of throughput and sensitivity. The assay was validated using the SBP-1 peptide, which is identical to the ACE2 amino acid sequence recognized by the S1 protein, and then applied to the discovery of natural ligands from botanical extracts. Small molecule ligands to the S1 protein were discovered in extracts of the licorice species, Glycyrrhiza inflata . In particular, the licorice ligand licochalcone A was identified through dereplication and comparison with standards using HPLC with high-resolution tandem mass spectrometry.

As a starting point for drug discovery, affinity selection-mass spectrometry (AS-MS) is ideal for the discovery of lead compounds from chemically diverse sources such as botanical, fungal and microbial extracts. Based on binding interactions between macromolecular receptors and ligands of low molecular mass, AS-MS enables the rapid isolation of pharmacologically active small molecules from complex mixtures for mass spectrometric characterization and identification. Unlike conventional high-throughput screening, AS-MS requires no radiolabels, no UV or fluorescent chromophores, and is compatible with all classes of receptors, enzymes, incubation buffers, cofactors, and ligands. The most successful types of AS-MS include pulsed ultrafiltration (PUF) AS-MS, size exclusion chromatography (SEC) AS-MS, and magnetic microbead affinity selection screening (MagMASS), which differ in their approaches for separating the ligand-receptor complexes from the non-binding compounds in mixtures. After affinity isolation, the ligand(s) from the mixture are characterized using high resolution UHPLC-MS and tandem mass spectrometry. Based on these elemental composition and structural data, the identities of the lead compounds are determined by searching on-line databases for known natural products and by comparison with standards. The structures of novel natural products are determined using a combination of spectroscopic techniques including two-dimensional NMR and MS., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Main Conclusion: Metabolites in Rafflesia-infected and non-infected Tetrastigma were compared which may have applications in Rafflesia propagation. Benzylisoquinoline alkaloids, here reported for the first time in Vitaceae, were abundant in non-infected shoots and may be a form of defense. In Rafflesia-infected shoots, oxylipins, which mediate immune response, were elevated. Endemic to the forests of Southeast Asia, Rafflesia (Rafflesiaceae) is a genus of holoparasitic plants producing the largest flowers in the world, yet completely dependent on its host, the tropical grape vine, Tetrastigma. Rafflesia species are threatened with extinction, making them an iconic symbol of plant conservation. Thus far, propagation has proved challenging, greatly decreasing efficacy of conservation efforts. This study compared the metabolites in the shoots of Rafflesia-infected and non-infected Tetrastigma loheri to examine how Rafflesia infection affects host metabolomics and elucidate the Rafflesia infection process. Results from LC-MS-based untargeted metabolomics analysis showed benzylisoquinoline alkaloids were naturally more abundant in non-infected shoots and are here reported for the first time in the genus Tetrastigma, and in the grape family, Vitaceae. These metabolites have been implicated in plant defense mechanisms and may prevent a Rafflesia infection. In Rafflesia-infected shoots, oxygenated fatty acids, or oxylipins, and a flavonoid, previously shown involved in plant immune response, were significantly elevated. This study provides a preliminary assessment of metabolites that differ between Rafflesia-infected and non-infected Tetrastigma hosts and may have applications in Rafflesia propagation to meet conservation goals., (© 2021. The Author(s).)

A new isoflavonoid, xanthocerin J, along with previously described xanthocerin A, were isolated from a methanol extract of aerial parts of a traditional American Indian herb, Amorpha canescens Pursh (Asteraceae). The structures of these compounds were characterized using mass spectrometry and NMR based on an isolation protocol using magnetic microbead affinity selection screening (MagMASS) for ligands to the estrogen receptor alpha (ERα). These compounds bound to ERα from an active fraction that exhibited dose-dependent antiestrogenic activity in the in vitro Ishikawa assay. However, these compounds did not exhibit antiestrogenic activity in the cell-based Ishikawa assay. Xanthocerin A and J may exhibit synergistic or additive activity with other compounds found in A. canescens which needs further exploration. This work highlights the potential of A. canescens as a prospect for the future discovery of compounds for women's health related to estrogen pathways., Competing Interests: Declaration of Competing Interest The authors declare that they have no known personal relationships or competing financial interest that could influence the work reported in this paper.

Background: Curcuminoids from turmeric rhizome have significant health benefits but low bioavailability., Objectives: To assess the pharmacokinetics of a novel natural turmeric dried colloidal suspension compared with 4 other turmeric formulations (including a standardized extract) at their respective recommended dosages., Methods: Thirty healthy men and women (18 to 45 y old) were enrolled in a randomized, open-labeled, crossover trial, and sequentially consumed single oral doses of standard turmeric extract (1500 mg), liquid micellar preparation (1000 mg), piperine-curcuminoid combination (1515 mg), phytosome formulation (1000 mg), or the dried colloidal suspension (300 mg). Eleven blood samples were obtained over 24 h, plasma was extracted with or without deconjugation with β-glucuronidase or sulfatase, and ultra-high-pressure liquid chromatography/tandem MS was used to quantify the 3 parent curcuminoids and 12 metabolites. Classical pharmacokinetics parameters were derived., Results: The total AUC values of unconjugated curcuminoids were highly variable within participants, with no significant differences between formulations. However, the AUC values for total curcuminoids (including all metabolites) showed significant product effects. Indeed, the micellar preparation delivered higher levels of total curcuminoids than any other formulation (8540 ng·h/mL), reaching significance when compared with the dried colloidal suspension and standard extract (6520 and 5080 ng·h/mL, respectively). After dose normalization, both micellar and dried colloidal formulations showed significantly higher AUC levels than the standard extract (respectively 136 and 72.9, compared with 3.7 ng·h/mL/mg). Total curcuminoid absorption levels were also significantly higher for the dried colloidal suspension when compared with either piperine or phytosome formulations. Interestingly, no significant differences were observed between the piperine-curcuminoid combination and the standard extract. No serious adverse events were reported., Conclusions: The administration of a low dose of the novel natural dried colloidal suspension provided high unconjugated and conjugated curcuminoid absorption, with significant beneficial differences when compared with the high dose of standard extract.This trial was registered at clinicaltrials.gov as NCT03621865., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition.)

Neonatal AAV9-gene therapy of the lysosomal enzyme galactosylceramidase (GALC) significantly ameliorates central and peripheral neuropathology, prolongs survival, and largely normalizes motor deficits in Twitcher mice. Despite these therapeutic milestones, new observations identified the presence of multiple small focal demyelinating areas in the brain after 6-8 months. These lesions are in stark contrast to the diffuse, global demyelination that affects the brain of naive Twitcher mice. Late-onset lesions exhibited lysosomal alterations with reduced expression of GALC and increased psychosine levels. Furthermore, we found that lesions were closely associated with the extravasation of plasma fibrinogen and activation of the fibrinogen-BMP-SMAD-GFAP gliotic response. Extravasation of fibrinogen correlated with tight junction disruptions of the vasculature within the lesioned areas. The lesions were surrounded by normal appearing white matter. Our study shows that the dysregulation of therapeutic GALC was likely driven by the exhaustion of therapeutic AAV episomal DNA within the lesions, paralleling the presence of proliferating oligodendrocyte progenitors and glia. We believe that this is the first demonstration of diminishing expression in vivo from an AAV gene therapy vector with detrimental effects in the brain of a lysosomal storage disease animal model. The development of this phenotype linking localized loss of GALC activity with relapsing neuropathology in the adult brain of neonatally AAV-gene therapy-treated Twitcher mice identifies and alerts to possible late-onset reductions of AAV efficacy, with implications to other genetic leukodystrophies., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Extracts of milk thistle, Silybum marianum (L.) Gaertn., are used as dietary supplements for their hepatoprotective, anti-inflammatory, and anti-tumor activities., Objective: An assay based on UHPLC-MS/MS was developed and validated for the quantitative analysis of six major milk thistle flavonolignans extracted from human serum., Methods: Ethyl acetate containing 0.1% formic acid was used to extract flavonolignans from human serum. A 10-min UHPLC-MS/MS method using selected reaction ion monitoring was developed for measuring extracts for silybin A, silybin B, isosilybin A, isosilybin B, silychristin, and silydianin., Results: The quantitative method was validated with respect to selectivity, specificity, accuracy, linearity, precision, LOD, and LLOQ. Extraction efficiency for the quality control standards at LLOQ, low, medium, and high concentrations ranged between 81% and 109%, and the calibration curves were linear (R2 > 0.997) for all flavonolignans. The method precision was determined using coefficients of variation, which were <15%. The method accuracy was assessed using percent relative error which was <15%., Conclusions: The UHPLC-MS/MS assay is fast, precise, sensitive, selective, accurate, and useful for the analysis of milk thistle flavonolignans in human serum., Highlights: The UHPLC-MS/MS assay is suitable for rapid quantitative analysis of milk thistle flavonolignans in human serum., (© AOAC INTERNATIONAL 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Superwarfarins are second-generation long-acting anticoagulant rodenticides that can cause unintended human and wildlife toxicity due, in part, to their prolonged half-lives. Commercially available superwarfarin rodenticides are synthesized as racemates with two asymmetric carbons, producing four stereoisomers. To support studies of human plasma half-lives of individual superwarfarin stereoisomers, a method was developed based on LC-MS/MS to separate and quantify stereoisomers of the commercially important superwarfarins bromadiolone, difenacoum and brodifacoum. Human plasma samples were prepared using protein precipitation and centrifugation. Chiral-phase HPLC separation was carried out on-line with tandem mass spectrometric quantitative analysis of the eluting stereoisomers using selected-reaction monitoring with positive ion electrospray on a triple quadrupole mass spectrometer. All four stereoisomers of each superwarfarin were resolved within 12.5 min with calibration curves spanning 2-3 orders of magnitude and lower limits of quantitation between 0.87 and 2.55 ng/mL. This method was used to determine the half-lives of superwarfarin stereoisomers in plasma from patients who had inhaled synthetic cannabinoid products contaminated with superwarfarins. These data may be used to guide the development of safer next generation anticoagulant rodenticides stereoisomers., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Invented to address the high-throughput screening (HTS) demands of combinatorial chemistry, affinity selection-mass spectrometry (AS-MS) utilizes binding interactions between ligands and receptors to isolate pharmacologically active compounds from mixtures of small molecules and then relies on the selectivity, sensitivity, and speed of mass spectrometry to identify them. No radiolabels, fluorophores, or chromophores are required. Although many variations of AS-MS have been devised, three approaches have emerged as the most flexible, productive, and popular, and they differ primarily in how ligand-receptor complexes are separated from nonbinding compounds in the mixture. These are pulsed ultrafiltration (PUF) AS-MS, size exclusion chromatography (SEC) AS-MS, and magnetic microbead affinity selection screening (MagMASS). PUF and SEC AS-MS are solution-phase screening approaches, and MagMASS uses receptors immobilized on magnetic microbeads. Because pools of compounds are screened using AS-MS, each containing hundreds to thousands of potential ligands, hundreds of thousands of compounds can be screened per day. AS-MS is also compatible with complex mixtures of chemically diverse natural products in extracts of botanicals and fungi and microbial cultures, which often contain fluorophores and chromophores that can interfere with convention HTS. Unlike conventional HTS, AS-MS may be used to discover ligands binding to allosteric as well as orthosteric receptor sites, and AS-MS has been useful for discovering ligands to targets that are not easily incorporated into conventional HTS such as membrane-bound receptors., (© 2020 John Wiley & Sons, Ltd.)

The importance of real-time, quantitative toxicology data available for physicians treating poisoned patients was illustrated during the 2018 outbreak in Illinois of severe coagulopathy caused by inhaling illicit synthetic cannabinoids products contaminated with commercially-available brodifacoum, difenacoum, and bromadiolone, three potent, long-acting anticoagulant rodenticides (LAARs). Identification and quantification of these life-threatening toxins in blood samples of hospitalized patients required toxicology testing with liquid chromatography-tandem mass spectrometry (LC-MS/MS) that was not available in clinical laboratories of hospitals at the time of the outbreak. This highly-sensitive, quantitative assay can provide critical information to guide patient care during and after hospitalization, including identification of offending LAARs, estimates of the ingested dose, and dosage and discontinuation of oral vitamin K 1 therapy after hospital discharge once plasma LAARs concentrations decreased to a safe level (<10 ng/mL). Accordingly, we propose an action plan to enable treating physicians to quantify plasma concentrations of several LAARs simultaneously in poisoned patients. It involves rapid (<15 min), sensitive, and validated LC-MS/MS methods developed, tested and validated in our laboratory. This will allow treating physicians to request quantitative plasma LAARs testing, report test results in the patient's hospital discharge summary, and recommend regular monitoring of plasma LAARs concentrations in the outpatient setting., Competing Interests: Disclosure statement No potential conflict of interest was reported by the authors. The views expressed in this article are those of the authors and do not necessarily reflect the position or policy of the Department of Veterans Affairs or the United States government.

Standard of care follow-up therapy for patients poisoned by long-acting anticoagulant rodenticides (LAARs) is daily high-dose (up to 100 mg per day) oral vitamin K1 (VK 1 ) for weeks to months to over a year. The availability of CLIA-certified quantitative testing for plasma LAAR concentrations can now assist health care providers in determining when to safely discontinue VK 1 therapy. We present estimates of treatment duration required to reach safe concentrations (< =10ng/ml) using serial measurements of plasma brodifacoum (BDF, a potent LAAR) concentrations obtained from patients poisoned after inhaling synthetic cannabinoids containing BDF. We fit the data to zero-order (linear) and first-order (exponential) curves, the latter to account for enterohepatic circulation of BDF. The results show that estimates of therapy duration are significantly longer when exponential clearance is assumed. Accordingly, we recommend that plasma BDF concentrations be monitored simultaneously with international normalization ratio (INR) during follow-up of poisoned patients, and that concentrations be determined after VK 1 therapy is discontinued to document persistence of safe concentrations., Competing Interests: Disclosure of interest The authors report no conflict of interests.

Extracts of red clover ( Trifolium pratense L.), containing estrogenic isoflavones like genistein and daidzein and the proestrogenic isoflavones formononetin and biochanin A, are used by women as dietary supplements for the management of menopausal symptoms. Although marketed as a safer alternative to hormone therapy, red clover isoflavones have been reported to inhibit some cytochrome P450 (CYP) enzymes involved in drug metabolism. To evaluate the potential for clinically relevant drug-red clover interactions, we tested a standardized red clover dietary supplement (120 mg isoflavones per day) for interactions with the pharmacokinetics of four FDA-approved drugs (caffeine, tolbutamide, dextromethorphan, and alprazolam) as probe substrates for the enzymes CYP1A2, CYP2C9, CYP2D6, and CYP3A4/5, respectively. Fifteen peri- and postmenopausal women completed pharmacokinetic studies at baseline and 2 weeks after consuming red clover. The averaged pharmacokinetic profiles of probe substrates in serum showed no significant alterations and no changes in the areas under the curve (AUC) over 96 h. Subgroup analysis based on the demographic characteristics (BMI, menopausal status, race, and age) also showed no differences in AUC for each probe substrate. Analysis of red clover isoflavones in serum showed primarily conjugated metabolites that explain, at least in part, the red clover pharmacokinetic safety profile.

Background: An outbreak of synthetic cannabinoid (SC)-associated coagulopathy and bleeding in Illinois, USA was determined to be due to inhalation of SC contaminated with brodifacoum (BDF), difenacoum (DiF), and bromadiolone (BDL), highly potent long-acting anticoagulant rodenticides (LAARs). Treatment with high-dose vitamin K1 (VK1) prevented mortality; however, plasma LAAR levels were not measured risking recurrence of coagulopathy and bleeding due to premature discontinuation. The goal of this study was to determine if plasma LAAR levels were reduced following standard of care treatment to normalize coagulopathy. Methods: Blood samples were collected from a cohort of 32 patients, and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis used to quantify plasma LAAR levels including enantiomers. Results: BDF was detected in 31 samples; 30 also contained DiF and 18 contained BDL. Initial plasma levels were 581 ± 87, 11.0 ± 1.9, and 14.9 ± 5.9 ng/mL for BDF, DiF, and BDL, respectively (mean ± SE). At discharge plasma, BDF levels remained elevated at 453 ± 68 ng/mL. Plasma half-lives for BDF, DiF, and BDL were 7.5 ± 1.3, 7.2 ± 1.9, and 1.8 ± 0.3 days, respectively. The half-life for trans-BDF enantiomers (5.7 ± 0.8 days) was shorter than for cis-enantiomers (7.6 ± 1.9 days). BDF half-lives were shorter, and coagulopathy normalized faster in patients receiving intravenous VK1 as compared to oral VK1. Patients prescribed VK1 at discharge had fewer re-admittances. Conclusions: These results demonstrate that plasma LAAR levels at discharge were elevated in poisoned patients despite normal coagulation, and that the route of VK1 administration affected LAAR pharmacokinetics and INR normalization. We propose plasma LAAR levels and coagulation be monitored concomitantly during follow-up of patients with LAAR poisoning. KEY POINTSIn patients treated with high-dose vitamin K1 for LAAR poisoning, plasma levels remained 40-fold above safe levels upon discharge from hospital.LAAR half-lives, normalization of coagulopathy, and readmittances were reduced by treatment with intravenous vitamin K1.

Background: Extracts of red clover (Trifolium pratense L.) containing estrogenic and pro-estrogenic isoflavones are used in dietary supplements primarily for the management of menopausal symptoms in women., Objective: A UHPLC-MS/MS assay was developed and validated for the quantitative analysis of the six major red clover isoflavones in dietary supplements and in human serum in support of clinical trials., Methods: Enzymatic deconjugation of isoflavone glucuronides and sulfate conjugates in human serum specimens was carried out followed by protein precipitation. Isoflavones in red clover dietary supplements were acid hydrolyzed to release aglycons from glycosides. UHPLC separations (< 4 min) were combined with MS/MS using collision-induced dissociation, selective reaction monitoring and deuterated internal standards to measure biochanin A, formononetin, daidzein, genistein, irilone, and prunetin., Results: The method was validated with respect to selectivity, specificity, accuracy, linearity, precision, LOD, and LOQ. The calibration curves for all analytes were linear (R2 > 0.998). The mean recovery for low-, medium- and high-quality control standards ranged between 80% and 108%. The precision of the method was assessed using coefficients of variation, which were <15%., Conclusions: The UHPLC-MS/MS method is fast, precise, sensitive, selective, accurate, and applicable to the quantitative analysis of red clover isoflavones in different matrices., Highlights: This validated UHPLC-MS/MS assay is applicable to the rapid quantitative analysis of red clover isoflavones in human serum and in dietary supplements., (© AOAC INTERNATIONAL 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Superwarfarins, second-generation long-acting anticoagulant rodenticides, are 4-hydroxycoumarin analogues of warfarin that contain a large hydrophobic side chain. These compounds contain two chiral centers and are synthesized for commercial use as two pairs of diastereomer., Objective: To support studies of superwarfarin pharmacokinetics and other efforts to improve clinical care for poisoning victims, a quantitative assay was developed for the measurement of diastereomer of bromadiolone, difenacoum, flocoumafen, brodifacoum, and difethialone in human plasma., Method: Based on ultrahigh-pressure liquid chromatography-tandem mass-spectrometry (UHPLC-MS/MS), this method was validated according to U.S. Food and Drug Administration (FDA) guidelines. Sample preparation involved simple protein precipitation followed by reversed phase UHPLC, which resolved all five pairs of cis/trans diastereomer in less than 10 min. Superwarfarins were measured using negative ion electrospray followed by selected-reaction monitoring on a triple quadrupole mass spectrometer., Results: Calibration curves covered 3-4 orders of magnitude with linear regression coefficients of >0.999. The lower limits of quantitation were from 0.013 to 2.41 ng/mL, and intra-day and inter-day accuracy and precision coefficients of variation were <12%., Conclusions: A 10-min UHPLC-MS/MS assay was developed and validated for the separation and quantitative analysis of the pairs of diastereomer of five superwarfarins in human plasma., Highlights: This method was used to identify and measure superwarfarins and their cis/trans diastereomers in plasma obtained from patients treated for coagulopathy following consumption of contaminated synthetic cannabinoid products., (© AOAC INTERNATIONAL 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Botanical dietary supplements produced from hops ( Humulus lupulus ) containing the chemopreventive compound xanthohumol and phytoestrogen 8-prenylnaringenin are used by women to manage menopausal symptoms. Because of the long half-lives of prenylated hop phenols and reports that they inhibit certain cytochrome P450 enzymes, a botanically authenticated and chemically standardized hop extract was tested for Phase I pharmacokinetic drug interactions. Sixteen peri- and postmenopausal women consumed the hop extract twice daily for 2 weeks, and the pharmacokinetics of tolbutamide, caffeine, dextromethorphan, and alprazolam were evaluated before and after supplementation as probe substrates for the enzymes CYP2C9, CYP1A2, CYP2D6, and CYP3A4/5, respectively. The observed area under the time-concentration curves were unaffected, except for alprazolam which decreased 7.6% (564.6 ± 46.1 h·μg/L pre-hop and 521.9 ± 36.1 h·μg/L post-hop; p -value 0.047), suggesting minor induction of CYP3A4/5. No enzyme inhibition was detected. According to FDA guidelines, this hop dietary supplement caused no clinically relevant pharmacokinetic interactions with respect to CYP2C9, CYP1A2, CYP2D6, or CYP3A4/5. The serum obtained after consumption of the hop extract was analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry to confirm compliance. Abundant Phase II conjugates of the hop prenylated phenols were observed including monoglucuronides and monosulfates as well as previously unreported diglucuronides and sulfate-glucuronic acid diconjugates.

Utilizing the distinct HMBC cross-peak patterns of l ower- f ield r ange (LFR; 11.80-14.20 ppm) hydroxyl singlets, presented NMR methodology characterizes flavonoid metabolomes both qualitatively and quantitatively. It enables simultaneous classification of the structural types of 5-OH flavonoids and biogenetically related 2'-OH chalcones, as well as quantification of individual metabolites from 1 H NMR spectra, even in complex mixtures. Initially, metabolite-specific LFR 1D 1 H and 2D HMBC patterns were established via literature mining and experimental data interpretation, demonstrating that LFR HMBC patterns encode the different structural types of 5-OH flavonoids/2'-OH chalcones. Taking advantage of the simplistic multiplicity of the H,H-uncoupled LFR 5-/2'-OH singlets, individual metabolites could subsequently be quantified by peak fitting quantitative 1 H NMR (PF-qHNMR). Metabolomic analysis of enriched fractions from three medicinal licorice ( Glycyrrhiza ) species established proof-of-concept for distinguishing three major structural types and eight subtypes in biomedical applications. The method identified 15 G. uralensis (GU) phenols from the six possible subtypes of 5,7-diOH (iso)flav(an)ones with 6-, 8-, and nonprenyl substitution, including the new 6-prenyl-licoisoflavanone ( 1 ) and two previously unknown compounds ( 4 and 7 ). Relative (100%) qNMR established quantitative metabolome patterns suitable for species discrimination and plant metabolite studies. Absolute qNMR with combined external and internal (solvent) calibration (ECIC) identified and quantified 158 GU metabolites. HMBC-supported qHNMR analysis of flavonoid metabolomes ("flavonomics") empowers the exploration of structure-abundance-activity relationships of designated bioactivity. Its ability to identify and quantify numerous metabolites simultaneously and without identical reference materials opens new avenues for natural product discovery and botanical quality control and can be adopted to other flavonoid- and chalcone-containing taxa.

To evaluate the potential for interactions between botanical dietary supplements and drug metabolism, Phase I clinical pharmacokinetics studies are conducted using an oral cocktail of probe substrates of cytochrome P450 (CYP) enzymes. A sensitive, specific, and fast ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for determination of caffeine (probe of CYP1A2), tolbutamide (probe of CYP2C9), dextromethorphan (probe of CYP2D6), and alprazolam (probe of CYP3A4/5) in human serum. Stable isotope-labelled analogs were used as internal standards, and sample preparation involved only rapid protein precipitation and centrifugation. The method of standard addition was used for the measurement of caffeine, because commercially available pooled human serum contains caffeine. Out of 18 lots of pooled human serum tested, caffeine was detection in all lots, alprazolam was detected in 13 lots, 8 lots contained dextromethorphan, and no tolbutamide was detected. Only serum prepared from the blood of select individuals was determined to be drug-free. The analytical method was validated with respect to linearity, accuracy and precision, recovery, stability, and matrix effects. The calibration curves were linear over the range of 25-12,000 ng/mL for caffeine, 75-36,000 ng/mL for tolbutamide, 0.05-30 ng/mL for dextromethorphan, and 0.1-60 ng/mL for alprazolam. The intra-assay and inter-assay coefficients of variation (%CV) and %Bias were <13 % (<17 % at the lower limit of quantitation). The recovery of each probe substrate ranged from 84.2%-98.5 %. All analytes were stable during sample storage and handling. Matrix effects were minimized by using stable isotope-labeled internal standards. The method was successfully applied to clinical studies investigating the pharmacokinetic alterations of probe substrates caused by chronic consumption of botanical dietary supplements., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Procyanidins are polymeric flavan-3-ones occurring in many plants with antioxidant and other beneficial bioactivities. They are composed of catechin and epicatechin monomeric units connected by single carbon-carbon B-type linkages or A-type linkages containing both carbon-carbon and carbon-oxygen-carbon bonds. Their polymeric structure makes analysis of procyanidin mixtures always difficult. Evaluation of procyanidins according to degree of polymerization (DP) using high-performance liquid chromatography (HPLC) is time-consuming and at best has resolved polymeric families up to DP-17. To expedite studies of procyanidins, the utility of positive ion electrospray ion mobility-mass spectrometry (IM-MS) was investigated for the rapid separation and characterization of procyanidins in mixtures. Applying IM-MS to analyse structurally defined standards containing up to five subunits, procyanidins could be resolved in less than 6 ms not only by degree of polymerization but also by linkage type. A-type procyanidins could be resolved from B-type and both could be at least partially resolved from mixed-type procyanidins of the same DP. IM-MS separated higher order procyanidins with DP of at least 24 from extracts of cranberry. As DP increased, the abundances of multiply-charged procyanidins also increased. During IM-MS of ions of similar m/z, the ion drift times decreased inversely with increasing charge state. Therefore, IM-MS was shown to separate mixtures of procyanidins containing at least 24 interconnected subunits in less than 16 ms, not only according to DP, but also according to linkage type between subunits and charge state., (© 2019 John Wiley & Sons, Ltd.)

As in drug discovery and development, mass spectrometry has become essential at all stages for establishing the safety and efficacy of botanical dietary supplements. Applications of mass spectrometry to the development of botanical dietary supplements include preclinical studies of the mechanisms of action ( e.g ., proteomic target identification and validation); identification of active natural products using high resolution tandem mass spectrometry; chemical standardization using UHPLC-MS/MS; and studies of metabolism, absorption and toxicity of active compounds using high resolution and UHPLC-MS/MS. Clinical applications of mass spectrometry include evaluation of the potential for drug-botanical interactions; investigation of the pharmacokinetics of active compounds; and quantitative analysis of biomarkers of efficacy during Phase I and II and clinical trials of safety and efficacy of botanical dietary supplements., Competing Interests: The author declares no conflict of interest

Occurring in hops ( Humulus lupulus ) and beer as a racemic mixture, (2 R ,2 S )-8-prenylnaringenin (8-PN) is a potent phytoestrogen in hop dietary supplements used by women as alternatives to conventional hormone therapy. With a half-life exceeding 20 h, 8-PN is excreted primarily as 8-PN-7- O -glucuronide or 8-PN-4'- O -glucuronide. Human liver microsomes and 11 recombinant human UDP-glucuronosyltransferases (UGTs) were used to catalyze the formation of the two oxygen-linked glucuronides of purified (2 R ) - 8 - PN and (2 S ) - 8-PN, which were subsequently identified using mass spectrometry and nuclear magnetic resonance spectroscopy. Formation of (2 R )- and (2 S )-8-PN-7- O -glucuronides predominated over the 8-PN-4'- O- glucuronides except for intestinal UGT1A10, which formed more (2 S )-8-PN-4'- O -glucuronide. (2 R )-8-PN was a better substrate for all 11 UGTs except for UGT1A1, which formed more of both (2 S )-8-PN glucuronides than (2 R )-8-PN glucuronides. Although several UGTs conjugated both enantiomers of 8-PN, some conjugated just one enantiomer, suggesting that human phenotypic variation might affect the routes of metabolism of this chiral estrogenic constituent of hops.

Botanical dietary supplements for women's health are increasingly popular. Older women tend to take botanical supplements such as hops as natural alternatives to traditional hormone therapy to relieve menopausal symptoms. Especially extracts from spent hops, the plant material remaining after beer brewing, are enriched in bioactive prenylated flavonoids that correlate with the health benefits of the plant. The chalcone xanthohumol (XH) is the major prenylated flavonoid in spent hops. Other less abundant but important bioactive prenylated flavonoids are isoxanthohumol (IX), 8-prenylnaringenin (8-PN), and 6-prenylnaringenin (6-PN). Pharmacokinetic studies revealed that these flavonoids are conjugated rapidly with glucuronic acid. XH also undergoes phase I metabolism in vivo to form IX, 8-PN, and 6-PN. Several hop constituents are responsible for distinct effects linked to multiple biological targets, including hormonal, metabolic, inflammatory, and epigenetic pathways. 8-PN is one of the most potent phytoestrogens and is responsible for hops' estrogenic activities. Hops also inhibit aromatase activity, which is linked to 8-PN. The weak electrophile, XH, can activate the Keap1-Nrf2 pathway and turn on the synthesis of detoxification enzymes such as NAD(P)H-quinone oxidoreductase 1 and glutathione S-transferase. XH also alkylates IKK and NF-κB, resulting in anti-inflammatory activity. Antiobesity activities have been described for XH and XH-rich hop extracts likely through activation of AMP-activated protein kinase signaling pathways. Hop extracts modulate the estrogen chemical carcinogenesis pathway by enhancing P450 1A1 detoxification. The mechanism appears to involve activation of the aryl hydrocarbon receptor (AhR) by the AhR agonist, 6-PN, leading to degradation of the estrogen receptor. Finally, prenylated phenols from hops are known inhibitors of P450 1A1/2; P450 1B1; and P450 2C8, 2C9, and 2C19. Understanding the biological targets of hop dietary supplements and their phytoconstituents will ultimately lead to standardized botanical products with higher efficacy, safety, and chemopreventive properties.

Botanical-derived dietary supplements have widespread use in the general population. The complex and variable nature of botanical ingredients and reports of adverse responses have led to concern for negative human health impacts following consumption of these products. Toxicity testing of the vast number of available products, formulations, and combinations is not feasible due to the time and resource intensive nature of comprehensive testing. Methods are needed to assess the safety of a large number of products via more efficient frameworks. Identification of toxicologically-active constituents is one approach being used, with many advantages toward product regulation. Bioassay-guided fractionation (BGF) is the leading approach used to identify biologically-active constituents. Most BGF studies with botanicals focus on identifying pharmacologically-active constituents for drug discovery or botanical efficacy research. Here, we explore BGF in a toxicological context, drawing from both efficacy and poisonous plant research. Limitations of BGF, including loss of mixture activity and bias toward abundant constituents, and recent advancements in the field (e.g., biochemometrics) are discussed from a toxicological perspective. Identification of active constituents will allow better monitoring of market products for known toxicologically-active constituents, as well as surveying human exposure, two important steps to ensuring the safety of botanical dietary supplements., (Copyright © 2018. Published by Elsevier Ltd.)

Women are increasingly using botanical dietary supplements (BDS) to reduce menopausal hot flashes. Although licorice ( Glycyrrhiza sp.) is one of the frequently used ingredients in BDS, the exact plant species is often not identified. We previously showed that in breast epithelial cells (MCF-10A), Glycyrrhiza glabra (GG) and G. inflata (GI), and their compounds differentially modulated P450 1A1 and P450 1B1 gene expression, which are responsible for estrogen detoxification and genotoxicity, respectively. GG and isoliquiritigenin (LigC) increased CYP1A1 , whereas GI and its marker compound, licochalcone A (LicA), decreased CYP1A1 and CYP1B1 The objective of this study was to determine the distribution of the bioactive licorice compounds, the metabolism of LicA, and whether GG, GI, and/or pure LicA modulate NAD(P)H quinone oxidoreductase (NQO1) in an ACI rat model. In addition, the effect of licorice extracts and compounds on biomarkers of estrogen chemoprevention ( CYP1A1 ) as well as carcinogenesis ( CYP1B1 ) was studied. LicA was extensively glucuronidated and formed GSH adducts; however, free LicA as well as LigC were bioavailable in target tissues after oral intake of licorice extracts. GG, GI, and LicA caused induction of NQO1 activity in the liver. In mammary tissue, GI increased CYP1A1 and decreased CYP1B1 , whereas GG only increased CYP1A1 LigC may have contributed to the upregulation of CYP1A1 after GG and GI administration. In contrast, LicA was responsible for GI-mediated downregulation of CYP1B1 These studies highlight the polypharmacologic nature of botanicals and the importance of standardization of licorice BDS to specific Glycyrrhiza species and to multiple constituents., (©2018 American Association for Cancer Research.)

Rationale: Preclinical studies in the search for treatments for several neurodegenerative diseases have identified lanthionine ketimine (LK) and its monoethyl ester derivative (LKE) as potential candidates. An ultrahigh-pressure liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) assay was developed to evaluate bioavailability by measuring these compounds in mouse serum, whole blood and brain tissue., Methods: Following administration of LKE to mice for 3 days in chow at 300 ppm, the animals were sacrificed, and LKE was extracted from serum, whole blood and brain tissues through protein precipitation using cold methanol. To enhance chromatographic separation and electrospray ionization, LK was methylated using diazomethane. Separations were carried out using C 18 reversed-phase UHPLC, and quantitative measurements were obtained using on-line triple-quadruple mass spectrometry with positive ion electrospray ionization, collision-induced dissociation and selected reaction monitoring. Tolbutamide was used as internal standard., Results: LKE showed good recovery ranging from 77-90% in serum and 82-88% in brain tissue. An eight-point standard curve ranging from 0.005 to 4.6 μM was linear (R 2 0.998). The average LKE detected in mouse serum was 277.42 nM, while the concentration in whole blood was 38 nM. Neither LK nor LKE was detected in brain tissues., Conclusions: A rapid quantitative method to measure LKE in mouse serum, whole blood and brain tissues using UHPLC/MS/MS was developed and validated following FDA guidelines. This method is suitable for bioavailability and pharmacokinetic studies., (© 2018 John Wiley & Sons, Ltd.)

Although procyanidins constitute a unique class of polymeric plant secondary metabolites with a variety of biological properties including potent antioxidant activity, structure determination has been challenging, and structures of many complex procyanidins remain uncertain. To expedite the characterization of procyanidins, negative ion matrix-assisted laser desorption ionization high-energy collision-induced dissociation tandem time-of-flight (MALDI-ToF/ToF) mass spectra of 20 isolated procyanidins containing catechin and epicatechin subunits with degrees of polymerization up to five were obtained and evaluated. Structurally significant fragmentation pathways of singly charged, deprotonated molecules were identified representing quinone methide, heterocyclic ring fission, and retro-Diels-Alder fragmentation. The interpretation of the tandem mass spectra for sequencing A-type, B-type, mixed-type, linear, and branched procyanidins is explained using specific examples of each.

Postmenopausal women are increasingly using botanicals for menopausal symptom relief due to the increased breast cancer risk associated with traditional estrogen therapy. The deleterious effects of estrogens are associated with estrogen receptor (ER)α-dependent proliferation, while ERβ activation could enhance safety by opposing ERα effects. Three medicinal licorice species, Glycyrrhiza glabra ( G. glabra), G. uralensis, and G. inflata, were studied for their differential estrogenic efficacy. The data showed higher estrogenic potency for G. inflata in an alkaline phosphatase induction assay in Ishikawa cells (ERα) and an estrogen responsive element (ERE)-luciferase assay in MDA-MB-231/β41 breast cancer cells (ERβ). Bioassay-guided fractionation of G. inflata led to the isolation of 8-prenylapigenin (3). Surprisingly, a commercial batch of 3 was devoid of estrogenic activity. Quality control by MS and qNMR revealed an incorrect compound, 4'- O-methylbroussochalcone B (10), illustrating the importance of both structural and purity verification prior to any biological investigations. Authentic and pure 3 displayed 14-fold preferential ERβ agonist activity. Quantitative analyses revealed that 3 was 33 times more concentrated in G. inflata compared to the other medicinal licorice extracts. These data suggest that standardization of G. inflata to 3 might enhance the safety and efficacy of G. inflata supplements used for postmenopausal women's health.

We report a global adeno-associated virus (AAV)9-based gene therapy protocol to deliver therapeutic galactosylceramidase (GALC), a lysosomal enzyme that is deficient in Krabbe's disease. When globally administered via intrathecal, intracranial, and intravenous injections to newborn mice affected with GALC deficiency (twitcher mice), this approach largely surpassed prior published benchmarks of survival and metabolic correction, showing long-term protection of demyelination, neuroinflammation, and motor function. Bone marrow transplantation, performed in this protocol without immunosuppressive preconditioning, added minimal benefits to the AAV9 gene therapy. Contrasting with other proposed pre-clinical therapies, these results demonstrate that achieving nearly complete correction of GALC's metabolic deficiencies across the entire nervous system via gene therapy can have a significant improvement to behavioral deficits, pathophysiological changes, and survival. These results are an important consideration for determining the safest and most effective manner for adapting gene therapy to treat this leukodystrophy in the clinic., (Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

α-Synuclein aggregation has been linked to Gaucher's disease (GD) and Krabbe's disease (KD), lysosomal conditions affecting glycosphingolipid metabolism. α-Synuclein pathology has been directly attributed to the dysregulation of glycosphingolipids in both conditions, specifically to increased galactosylsphingosine (psychosine) content in the context of KD. Furthermore, the gene (GALC) coding for the psychosine degrading enzyme galactosylceramidase (GALC), has recently been identified as a risk loci for Parkinson's disease. However, it is unknown if changes in psychosine metabolism and GALC activity in the context of the aging human brain correlate with Parkinson's disease. We investigated psychosine accumulation and GALC activity in the aging brain using fresh frozen post-mortem tissue from Parkinson's (PD, n = 10), Alzheimer's (AD, n = 10), and healthy control patients (n = 9), along with tissue from neuropsychiatric patients (schizophrenia, bipolar disorder and depression, n = 15 each). An expanded mutational analysis of PD (n = 20), AD (n = 10), and healthy controls (n = 30) examined if PD was correlated with carriers for severe GALC mutations. Psychosine content within the cerebral cortex of PD patients was elevated above control patients. Within all patients, psychosine displayed a significant (p<0.05) and robust regional distribution in the brain with higher levels in the white matter and substantia nigra. A mutational analysis revealed an increase in the incidence of severe GALC mutations within the PD patient population compared to the cohorts of Alzheimer's patients and healthy controls tested. In addition to α-synuclein pathology identified in the KD brain, control patients identified as GALC mutational carriers or possessing a GALC pathogenic variant had evidence of α-synuclein pathology, indicating a possible correlation between α-synuclein pathology and dysregulation of psychosine metabolism in the adult brain. Carrier status for GALC mutations and prolonged exposure to increased psychosine could contribute to α-synuclein pathology, supporting psychosine metabolism by galactosylceramidase as a risk factor for Parkinson's disease.

Procyanidins are polyphenols abundant in dietary fruits, vegetables, nuts, legumes, and grains with a variety of chemopreventive biological effects. Rapid structure determination of these compounds is needed, notably for the more complex polymeric procyanidins. We review the recent developments in the structure elucidation of procyanidins with a focus on mass spectrometric approaches, especially liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matrix-assisted laser desorption ionization (MALDI) MS/MS.

Purpose: We aimed to test the hypothesis that doxorubicin (DOX) survives thermal ablative heating in an ex vivo model of combined transarterial chemoembolization (TACE) and thermal ablation., Methods: Fresh porcine psoas major muscle (3 samples, 15×10×3 cm) was submerged in aqueous DOX solution (60 µg/mL, 0.1 M) for 24 hours to passively saturate tissue. DOX-infused tissue was then dried and treated with microwave ablation (MWA) using a 2.45 GHz antenna at 65 W for 2, 5, and 10 minutes. Ablations were repeated in triplicate (9 total). Tissue was then sampled at both ablated and unablated control sites, and DOX concentration was quantified via ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS), with samples analyzed in triplicate. Tissue DOX levels in ablation and control groups were compared using one-way ANOVA., Results: Homogeneous DOX uptake into porcine tissue was evident in all three samples. Mean DOX concentration in unablated tissue was 8.0±2.2 µg/mL. MWA was technically successful in all 9 procedures (100%), with tissue heating to 95-100°C. Mean tissue DOX concentration showed progressive reduction with increasing ablation time, measuring 6.7±1.3, 4.9±0.9, and 4.8±1.3 µg/mL in MWA-treated tissue after 2, 5, and 10 minutes, respectively. Differences in tissue DOX levels between unablated tissue and MWA groups were statistically significant (P < 0.001)., Conclusion: Contrary to the initial hypothesis, tissue DOX concentration progressively decreased after MWA of longer ablation times. These results suggest that TACE followed by ablation may result in lower intratumoral DOX than would otherwise be anticipated for TACE alone.

The metabolism of the chemoprevention agent licochalcone A, which is a chemopreventive chalcone found in abundance in the licorice species Glycyrrhiza inflata, was investigated using human liver microsomes and human hepatocytes combined with analysis using high performance liquid chromatography-mass spectrometry (LC-MS). Five oxygenated phase I metabolites of licochalcone A were formed by human liver microsomes, including a catechol on the A-ring, two intramolecular cyclization products following epoxidation of the exocyclic alkene at position 5 of the B-ring, and two dioxygenated products. Nine phase II monoglucuronides of licochalcone A and its oxygenated phase I metabolites were formed during incubation with human hepatocytes. These included (E)-licochalcone A-4-glucuronide, (E)-licochalcone A-4'-glucuronide, (Z)-licochalcone A-4-glucuronide, glucuronic acid conjugates of all of the monooxygenated phase I metabolites, and glucuronides of the licochalcone catechol after methylation by catechol-O-methyl transferase. In addition, human hepatocytes formed one sulfate conjugate and one glutathione conjugate of licochalcone A. The structures of all major metabolites were determined using a combination of accurate mass measurement, LC-tandem mass spectrometry, LC-UV, nuclear magnetic resonance, and comparison with standards. The cytochrome P450 enzymes and UDP-glucuronosyltransferases responsible for the formation of the major metabolites were identified. Based on in vitro hepatic clearance calculations, licochalcone A is predicted to be metabolized primarily by phase II conjugation reactions. Graphical abstract Phase I and II metabolism of licochalcone A from the licorice species Glycyrrhiza inflata by human liver microsomes and hepatocytes determined using LC-MS/MS, LC-UV and NMR.

The potential of licorice dietary supplements to interact with drug metabolism was evaluated by testing extracts of three botanically identified licorice species (Glycyrrhiza glabra L., Glycyrrhiza uralensis Fish. ex DC. and Glycyrrhiza inflata Batalin) and 14 isolated licorice compounds for inhibition of 9 cytochrome P450 enzymes using a UHPLC-MS/MS cocktail assay. G. glabra showed moderate inhibitory effects against CYP2B6, CYP2C8, CYP2C9, and CYP2C19, and weak inhibition against CYP3A4 (testosterone). In contrast, G. uralensis strongly inhibited CYP2B6 and moderately inhibited CYP2C8, CYP2C9 and CYP2C19, and G. inflata strongly inhibited CYP2C enzymes and moderately inhibited CYP1A2, CYP2B6, CYP2D6, and CYP3A4 (midazolam). The licorice compounds isoliquiritigenin, licoricidin, licochalcone A, 18β-glycyrrhetinic acid, and glycycoumarin inhibited one or more members of the CYP2C family of enzymes. Glycycoumarin and licochalcone A inhibited CYP1A2, but only glycycoumarin inhibited CYP2B6. Isoliquiritigenin, glabridin and licoricidin competitively inhibited CYP3A4, while licochalcone A (specific to G. inflata roots) was a mechanism-based inhibitor. The three licorice species commonly used in botanical dietary supplements have varying potential for drug-botanical interactions as inhibitors of cytochrome P450 isoforms. Each species of licorice displays a unique profile of constituents with potential for drug interactions., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Supplements with estrogenic activities are intensively investigated as potential alternatives for the treatment of menopausal symptoms. These investigations include studies on their safety regarding potential breast cancer risks. Therefore, the aim of this study was to assess whether or not a standardized hops (Humulus lupulus) extract, containing 0.42% of the estrogenic flavanone, 8-prenylnaringenin, would stimulate growth of methyl-nitrosourea (MNU) induced mammary cancer in ovariectomized (OVX) Sprague-Dawley (SD) rats or would impact on the proliferative activity within the normal mammary gland of Wistar rats. To induce tumorigenesis SD-rats received an intraperitoneal injection of 50mg/kg body weight of MNU on postnatal days PND 50 and 52. 28days later animals were OVX or were SHAM operated (positive control) and randomly allocated and maintained for 140days on either a phytoestrogen-free placebo diet (SHAM and negative control) or on the hops fortified diet. For the investigations in the normal mammary gland young adult Wistar rats were bilaterally OVX and randomly allocated to a control group fed to a phytoestrogen-free diet, or to a diet supplemented either with E 2 -benzoate or the hops extract. As a major result, the tumor incidence was 15% (3 tumors totally) in OVX controls, whereas it was 85% (39 tumors totally) in SHAM operated positive controls. No tumors were detectable in the hops group. In addition, no estrogenic activity of the hops extract was detectable in uterus and liver of these animals. In investigations on the normal mammary gland, no impact of hops extract on the expression of estrogen dependent proliferation markers or of progesterone receptor became apparent. In conclusion, the lack of growth stimulation of MNU-induced breast cancer in OVX SD-rats and the lack of stimulation proliferative events in the normal mammary gland of OVX Wistar rats by standardized hops extracts provides an important piece of evidence regarding the safety of these extracts in the management of menopausal symptoms., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Hops (Humulus lupulus (L.)) dietary supplements are of interest as herbal remedies to alleviate menopausal symptoms, such as hot flushes, depression and anxiety. So far, the evidence regarding estrogenic and related properties of hops preparations has been considered insufficient for a market authorization for menopausal indications., Purpose: The study aims to investigate a chemically standardized hops extract regarding its safety in the uterus, as wells as its efficacy to prevent bone loss in the ovariectomized rat model., Study Design/methods: Female Wistar rats were ovariectomized and divided into a control group receiving phytoestrogen-free diet, a group treated with E 2 benzoate (0.93 mg/kg body weight/d) and a group treated with the standardized hops extract (60 mg/kg body weight/d) for 8 weeks. Micro-computed tomography of the tibiae and vertebrae, as wells as histological changes in the uterus and tibia were analyzed., Results: Neither uterotrophic nor proliferative effects were observed in the endometrium in response to the oral 8-week administration of the hops extract. However, site-dependent skeletal effects were observed. The hops extract significantly decreased the number of osteoclasts in the tibial metaphysis and prevented reduction of the trabecular thickness that resulted from estradiol depletion. In contrast, the hops extract did not prevent the ovariectomy-induced micro-architectural changes in the lumbar vertebra. Certain parameters (e.g. thickness and number of trabeculae) were even found to be below the values determined in the ovariectomized control group., Conclusion: Taken together, the results provide evidence for the safety of the standardized hops extract and point to a weak bone type-specific, protective effect on bone loss following estradiol depletion., (Copyright © 2017. Published by Elsevier GmbH.)

Rationale: Phenethylamides are a large group of naturally occurring molecules found both in the plant and animal kingdoms. In addition, they are used as intermediates for the synthesis of pharmaceutically important dihydro- and tetrahydroisoquinolines. To enable efficient characterization of this class of molecules, a detailed mass spectrometric fragmentation study of a broad series of analogs was carried out., Methods: The test compounds were synthesized using standard methods for amide bond formation. Low-energy high-resolution tandem mass spectra were acquired on a hybrid quadrupole/time-of-flight mass spectrometer using positive ion electrospray ionization., Results: A total of 26 analogs were investigated in the study. Fragmentation of phenethylamides was found to proceed via intermediate ion-neutral complexes. The complexes can break down via multiple pathways including dissociation, proton transfer, Friedel-Crafts acylation, and single electron transfer. The relative contribution of each of these pathways strongly depends on the structure of the coupling amine and acid., Conclusions: A general scheme for the fragmentation of phenethylamides was developed. This study further extends the knowledge base of the ion-neutral complex by discovering Friedel-Crafts acylation as a novel reaction. The strong influence of minor structural modifications on the fragmentation patterns highlights the importance of testing many analogs in order to fully predict a fragmentation pattern of a particular class of molecules., (Copyright © 2017 John Wiley & Sons, Ltd.)

In an effort to explore the residual complexity of naturally occurring chalcones from the roots of Glycyrrhiza inflata (Fabaceae), two new licochalcone A (LicA) derivatives were isolated as trace metabolites from enriched fractions. Both constituents contain a dihydrofuran moiety linked to carbons C-4 and C-5 of the retrochalcone core. Compound 1 (LicAF1) represents a new chemical entity, whereas compound 2 (LicAF2) has previously been reported as a Lewis acid catalyzed rearrangement of LicA. Evaluation of chirality revealed that both dihydrofuran derivatives existed as a mixture of R and S enantiomers. Interestingly, when solutions were exposed to sunlight, both dihydrofuran retrochalcones, initially isolated as trans isomers, were found to rapidly isomerize yielding trans and cis isomers. Analysis of the 1D 1 H NMR spectra of the photolysis products revealed the presence of two sets of proton resonances ascribed to each of the geometric isomers. An up-field shift of all proton resonances arising from the cis isomer was observed, suggesting that anisotropic shielding effects were introduced through an overall perturbation of the 3-dimensional structure upon photoisomerization. Similar up-field shifts were observed in the 13 C spectrum of the cis isomer, except for the CO, C-α, and C-6 carbons, which experienced downfield shifts. Analogous NMR results were observed for LicA. Hence, the results presented herein encompass the isolation and full characterization of LicAF analogs 1 and 2, and observations of their trans-to-cis photoisomerization through the systematic analysis of their NMR spectra., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Botanical dietary supplements contain multiple bioactive compounds that target numerous biological pathways. The lack of uniform standardization requirements is one reason that inconsistent clinical effects are reported frequently. The multifaceted biological interactions of active principles can be disentangled by a coupled pharmacological/phytochemical approach using specialized ("knock-out") extracts. This is demonstrated for hops, a botanical for menopausal symptom management. Employing targeted, adsorbent-free countercurrent separation, Humulus lupulus extracts were designed for pre- and postmenopausal women by containing various amounts of the phytoestrogen 8-prenylnaringenin (8-PN) and the chemopreventive constituent xanthohumol (XH). Analysis of their estrogenic (alkaline phosphatase), chemopreventive (NAD(P)H-quinone oxidoreductase 1 [NQO1]), and cytotoxic bioactivities revealed that the estrogenicity of hops is a function of 8-PN, whereas their NQO1 induction and cytotoxic properties depend on XH levels. Antagonization of the estrogenicity of 8-PN by elevated XH concentrations provided evidence for the interdependence of the biological effects. A designed postmenopausal hop extract was prepared to balance 8-PN and XH levels for both estrogenic and chemopreventive properties. An extract designed for premenopausal women contains reduced 8-PN levels and high XH concentrations to minimize estrogenic while retaining chemopreventive properties. This study demonstrates the feasibility of modulating the concentrations of bioactive compounds in botanical extracts for potentially improved efficacy and safety.

Background: Previous studies have uncovered heightened prostatic susceptibility to hormone-induced neoplasia from early-life exposure to low-dose bisphenol A (BPA). However, significant data gaps remain that are essential to address for biological relevance and necessary risk assessment., Objectives: A complete BPA dose-response analysis of prostate lesions across multiple prostatic lobes was conducted that included internal BPA dosimetry, progression to adenocarcinoma with aging and mechanistic connections to epigenetically reprogramed genes., Methods: Male neonatal Sprague-Dawley rats were briefly exposed to 0.1 to 5,000 μg BPA/kg BW on postnatal days (PND) 1, 3, and 5. Individual prostate lobes plus periurethral prostatic ducts were evaluated at 7 mo or 1 y of age without or with adult testosterone plus estradiol (T+E) to promote carcinogenesis. DNA methylation of five genes was quantified by bisulfite genomic sequencing in d-200 dorsal prostates across BPA doses. Serum free-BPA and BPA-glucuronide were quantitated in sera of individual PND 3 pups collected 1 hr postexposure utilizing ultra-high-pressure tandem mass spectrometry (UHPLC-MS-MS)., Results: The lowest BPA dose initiated maximal hormonal carcinogenesis in lateral prostates despite undetectable free BPA 1 hr postexposure. Further, prostatic intraepithelial neoplasia (PIN) progressed to carcinoma in rats given neonatal low-dose BPA with adult T+E but not in rats given adult T+E alone. The dorsal and ventral lobes and periurethral prostatic ducts exhibited a nonmonotonic dose response with peak PIN, proliferation and apoptotic values at 10–100 μg/kg BW. This was paralleled by nonmonotonic and dose-specific DNA hypomethylation of genes that confer carcinogenic risk, with greatest hypomethylation at the lowest BPA doses., Conclusions: Developmental BPA exposures heighten prostate cancer susceptibility in a complex dose- and lobe-specific manner. Importantly, elevated carcinogenic risk is found at doses that yield undetectable serum free BPA. Dose-specific epigenetic modifications of selected genes provide a mechanistic framework that may connect early-life BPA to later-life predisposition to prostate carcinogenesis. https://doi.org/10.1289/EHP1050.

To overcome limiting factors in mass spectrometry-based screening methods such as automation while still facilitating the screening of complex mixtures such as botanical extracts, magnetic microbead affinity selection screening (MagMASS) was developed. The screening process involves immobilization of a target protein on a magnetic microbead using a variety of possible chemistries, incubation with mixtures of molecules containing possible ligands, a washing step that removes non-bound compounds while a magnetic field retains the beads in the microtiter well, and an organic solvent release step followed by LC-MS analysis. Using retinoid X receptor-α (RXRα) as an example, which is a nuclear receptor and target for anti-inflammation therapy as well as cancer treatment and prevention, a MagMASS assay was developed and compared with an existing screening assay, pulsed ultrafiltration (PUF)-MS. Optimization of MagMASS involved evaluation of multiple protein constructs and several magnetic bead immobilization chemistries. The full-length RXRα construct immobilized with amylose beads provided optimum results. Additional enhancements of MagMASS were the application of 96-well plates to enable automation, use of UHPLC instead of HPLC for faster MS analyses, and application of metabolomics software for faster, automated data analysis. Performance of MagMASS was demonstrated using mixtures of synthetic compounds and known ligands spiked into botanical extracts. Graphical Abstract ᅟ.

Rationale: Electrospray mass spectrometry methods for the analysis of phosphatidylcholines (PCs) routinely include ammonium acetate or ammonium formate in the mobile phase. In an effort to justify and optimize the use of these additives, we investigated possible functions of ammonium compounds in the ionization of PCs., Methods: Because PCs contain a quaternary amine, the role of ammonium in neutralizing the negatively charged phosphate group was investigated by using deuterated ammonium acetate, adjusting the pH, varying the organic solvent composition, and by comparing the additives ammonium acetate, ammonium formate and ammonium bicarbonate. Seven PC standards were measured ranging from lyso 1-palmitoyl-sn-glycero-3-phosphocholine to 1,2-dieicosapentaenoyl-sn-glycero-3-phosphocholine as well as a mixture of PCs in a krill oil dietary supplement., Results: Under all conditions tested, aqueous acetonitrile provided more abundant formation of protonated PCs than did aqueous methanol. Regardless of the mobile phase composition and electrospray ion source parameters, no [M + NH 4 ] + ions were detected. Adding deuterated ammonium acetate to the mobile phase failed to form deuterated PCs, indicating that ammonium is not the source of the proton that neutralizes the phosphate negative charge. Instead, water was the source of the proton as deuterated water resulted in the formation of [M + D] + ions. Addition of organic acids, ammonium formate, ammonium acetate, or ammonium bicarbonate to the mobile phase did not enhance and in most cases suppressed PC ionization., Conclusions: Ammonium compounds and organic acids can suppress ionization of PCs when using an aqueous acetonitrile mobile phase during electrospray. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Extracellular vesicles (EVs) are membrane nanovesicles of diverse sizes secreted by different cell types and are involved in intercellular communication. EVs shuttle proteins, nucleic acids, and lipids that reflect their cellular origin and could mediate their biological function in recipient cells. EVs circulate in biological fluids and are considered as potential biomarkers that could be used to analyze and characterize disease development, course and response to treatment. EVs exhibit specific distribution of glycolipids and membrane organization, but little is known about the biological significance of this distribution or how it could contribute to pathological conditions such as multiple sclerosis (MS). We provide the first description of sulfatide composition in plasma-derived EVs by ultra-high-performance liquid chromatography tandem mass spectrometry. We found that EVs of different sizes showed C16:0 sulfatide but no detectable levels of C18:0, C24:0, or C24:1 sulfatide species. Small EVs isolated at 100,000 × g-enriched in exosomes-from plasma of patients with MS showed a significant increase of C16:0 sulfatide compared with healthy controls. Nanoparticle tracking analysis showed that the particle size distribution in MS plasma was significantly different compared with healthy controls. Characterization of small EVs isolated from MS plasma showed similar protein content and similar levels of exosomal markers (Alix, Rab-5B) and vesicular marker MHC class I (major histocompatibility complex class I) compared with healthy controls. Our findings indicate that C16:0 sulfatide associated with small EVs is a candidate biomarker for MS that could potentially reflect pathological changes associated with this disease and/or the effects of its treatment. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

To expedite the identification of active natural products in complex mixtures such as botanical extracts, a magnetic microbead affinity selection screening (MagMASS) procedure was developed. This technique utilizes target proteins immobilized on magnetic beads for rapid bioaffinity isolation of ligands from complex mixtures. A MagMASS method was developed and validated for 15-lipoxygenase. As a proof of concept, several North American prairie plants used medicinally by Native Americans were extracted with MeOH and screened. A hit from an extract of Proserpinaca palustris, also known as mermaid weed, was flagged for further characterization using high-resolution tandem mass spectrometry, dereplication, and identification using XCMS online. Through the application of high-resolution product ion tandem mass spectrometry, comparison with natural product databases, and confirmation using standards, the hit was identified as quercitrin, which is a known inhibitor of 15-lipoxygenase. The overall workflow of MagMASS is faster and more amendable to automation than alternative methods designed for screening botanical extracts or complex mixtures of combinatorial libraries.

Defined as the roots and underground stems of principally three Glycyrrhiza species, Glycyrrhiza glabra L., Glycyrrhiza uralensis Fish. ex DC., and Glycyrrhiza inflata Batalin, licorice has been used as a medicinal herb for millennia and is marketed as root sticks, powders, and extracts. Identity tests described in most pharmacopeial monographs enabled the distinction of Glycyrrhiza species. Accordingly, an ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay using the method of standard addition was developed to quantify 14 licorice components (liquiritin, isoliquiritin, liquiritin apioside, isoliquiritin apioside, licuraside, liquiritigenin, isoliquiritigenin, glycyrrhizin, glycyrrhetinic acid, glabridin, glycycoumarin, licoricidin, licochalcone A, and p-hydroxybenzylmalonic acid), representing several natural product classes including chalcones, flavanones, saponins, and isoflavonoids. Using this approach, G. glabra, G. uralensis, and G. inflata in a variety of forms including root powders and extracts as well as complex dietary supplements could be differentiated and chemically standardized without concerns due to matrix effects.

As with human beings, dogs suffer from the consequences of cancer. We investigated the potential of a formulation comprised of resveratrol, ellagic acid, genistein, curcumin and quercetin to modulate biomarkers indicative of disease prevention. Dog biscuits were evaluated for palatability and ability to deliver the chemopreventive agents. The extent of endogenous DNA damage in peripheral blood lymphocytes from dogs given the dietary supplement or placebo showed no change. However, H2O2-inducible DNA damage was significantly decreased after consumption of the supplement. The expression of 11 of 84 genes related to oxidative stress was altered. Hematological parameters remained in the reference range. The concept of chemoprevention for the explicit benefit of the canine is compelling since dogs are an important part of our culture. Our results establish a proof-of-principle and provide a framework for improving the health and well-being of "man's best friend".