A GC-MS Protocol for the Identification of Polycyclic Aromatic Alkaloids from Annonaceae.

The Annonaceae are an old family of flowering plants belonging to the order Magnoliales, distributed mainly in tropical regions. Numerous Annonaceae species find ethnobotanical use for curing a broad range of diseases, among them cancer and infections by diverse pathogens. Hence, bioactive natural products from Annonaceae have received considerable interest in drug development. Beyond cytotoxic acetogenins, unique aporphine-derived polycyclic aromatic alkaloids are characteristic constituents of Annonaceae. Among them are unique tri- and tetracyclic aromatic alkaloids like azafluorenones, diazafluoranthenes, azaanthracenes, and azaoxoaporphines. The complex substitution pattern of these alkaloids represents a major challenge in structure elucidation of isolated natural products. Based on a broad spectrum of alkaloids available from our previous work, we present a GC-MS protocol for the identification of over 20 polycyclic aromatic alkaloids from Annonaceae. This collection of data will contribute to the future identification of the metabolite patterns of extracts from Annonaceae as an important source of novel bioactive secondary metabolites.

The Putative Caloric Restriction Mimetic Resveratrol has Moderate Impact on Insulin Sensitivity, Body Composition, and the Metabolome in Mice.

SCOPE: Data on resveratrol-(trans-3,5,4'-trihydroxystilbene)-induced caloric-restriction-(CR)-mimicking effects in mice receiving a high-fat diet (HFD) are contradictory. It is hypothesized that this can possibly stem from different bioactivities of resveratrol (RSV) microbial metabolites. METHODS AND RESULTS: C57BL/6Rj mice are fed an ad-libitum HFD supplemented with RSV or its metabolites, dihydroresveratrol (DHR) and lunularin (LUN) (≈28 mg (dihydro)stilbene kg-1 mouse per day). A 40% CR group was included in the study. While CR mice show robust changes in bodyweight and composition, hormone levels and mRNA expression, slight changes are found (more muscle, less adipose tissue) in body composition, leptin, and insulin levels in RSV-supplemented mice compared to ad libitum controls. LUN hardly and DHR does not change the hormone levels measured. Metabolome analysis of serum shows changes in CR mice but only slight, if any, changes in RSV-, DHR-, or LUN-supplemented mice compared to the controls. Evaluating the capability of RSV and its metabolites to inhibit carbohydrate-hydrolyzing enzymes in vitro, it is found that RSV reduced α-glucosidase activity to a stronger extent than DHR and LUN. CONCLUSION: Decelerated carbohydrate breakdown by RSV may have contributed to the moderate impact of dietary RSV on mouse insulin sensitivity (lowered fasting and post-glucose-bolus insulin levels).

Phytotherapy Adds to the Therapeutic Armamentarium for the Treatment of Mild-To-Moderate Lower Urinary Tract Symptoms in Men.

BACKGROUND: Practice guidelines hardly recommend herbal extracts for male lower urinary tract symptoms (LUTS). However, many patients are unsatisfied with first-line synthetic drugs and often prefer herbal medicines because of good tolerability. To improve the decision-making process, which should consider the patients' expectations, it is crucial to reflect on the role of phytotherapy in the treatment of LUTS. We (panel experts) reflected on current guideline recommendations and real practice across various European countries and debated the potential role of plant extracts with a focus on pumpkin seed soft extract investigated over 12 months in two randomised placebo-controlled trials. SUMMARY: Most guidelines give no clear recommendations on phytotherapy due to the heterogeneity of clinically investigated extracts. Nevertheless, plant extracts are prescribed to patients with mild-to-moderate LUTS. Also, self-medicating patients often handle their complaints with herbal products. Many patients aim to avoid synthetic drugs for fear of sexual functional side effects and a negative impact on their quality of life. For the elderly, vasoactive comedications might become an issue. When taking plant extracts, patients experience an acceptable symptomatic relief similar to that achieved with synthetics but without side effects. Key Messages: In shared decision-making for purely symptomatic treatment, a low risk of side effects takes priority. We propose to consider patient preferences in the treatment of mild-to-moderate LUTS in men with a low risk of disease progression. We found a consensus that pumpkin seed soft extract adds to the therapeutic armamentarium for patients who cannot or do not want to apply synthetic drugs.

Comparison of Strategies for the Determination of Sterol Sulfates via GC-MS Leading to a Novel Deconjugation-Derivatization Protocol.

Sulfoconjugates of sterols play important roles as neurosteroids, neurotransmitters, and ion channel ligands in health and disease. In most cases, sterol conjugate analysis is performed with liquid chromatography-mass spectrometry. This is a valuable tool for routine analytics with the advantage of direct sterol sulfates analysis without previous cleavage and/or derivatization. The complementary technique gas chromatography-mass spectrometry (GC-MS) is a preeminent discovery tool in the field of sterolomics, but the analysis of sterol sulfates is hampered by mandatory deconjugation and derivatization. Despite the difficulties in sample workup, GC-MS is an indispensable tool for untargeted analysis and steroid profiling. There are no general sample preparation protocols for sterol sulfate analysis using GC-MS. In this study we present a reinvestigation and evaluation of different deconjugation and derivatization procedures with a set of representative sterol sulfates. The advantages and disadvantages of trimethylsilyl (TMS), methyloxime-trimethylsilyl (MO-TMS), and trifluoroacetyl (TFA) derivatives were examined. Different published procedures of sterol sulfate deconjugation, including enzymatic and chemical cleavage, were reinvestigated and examined for diverse sterol sulfates. Finally, we present a new protocol for the chemical cleavage of sterol sulfates, allowing for simultaneous deconjugation and derivatization, simplifying GC-MS based sterol sulfate analysis.

Resveratrol, lunularin and dihydroresveratrol do not act as caloric restriction mimetics when administered intraperitoneally in mice.

Resveratrol as well as caloric restriction were shown to extend lifespan in some model organisms and may possibly delay onset of ageing-related diseases in humans. Yet, resveratrol supplementation does not always extend lifespan of animal models or improve health status of humans. Because of interindividual differences in human microbiota, resveratrol metabolite production in the gut differs. While some individuals produce lunularin and dihydroresveratrol in their gut, others produce dihydroresveratrol only. Therefore, we addressed the question whether these metabolites differ in their biological impact on ageing and intraperitoneally injected 13-month-old C57BL/6JRj mice on an ad-libitum (AL) HFD with resveratrol, dihydroresveratrol or lunularin (24 mg/kg bodyweight; 3 times/week). Compared to mice injected with vehicle (AL-control), resveratrol and dihydroresveratrol did not change bodyweight and had no impact on insulin or glucose levels while lunularin slightly reduced feed intake and bodyweight gain. CR-mice showed lowered cholesterol, insulin and leptin levels, elevated adiponectin and phosphorylated AMPK levels in liver as well as increased transcription of Pck1 and Pgc1α when compared to the AL-control. In contrast, injections with the test substances did not change these parameters. We therefore conclude that in our model, resveratrol, lunularin and dihydroresveratrol did not act as CR mimetics.

Determination by GC-IT/MS of phytosterols in herbal medicinal products for the treatment of lower urinary tract symptoms and food products marketed in Europe.

A method for the determination of phytosterols in herbal medicinal products for the treatment of lower urinary tract symptoms and food products is described here. Using a convenient sample preparation protocol and sensitive gas chromatography ion trap mass spectrometry analysis, ten different sterols, among them five Δ(7)-phytosterols as typical constituents of pumpkin seed preparations, could be identified and quantified. This protocol was applied to the analysis of 31 marketed products, from which seven were raw materials.

A convenient cellular assay for the identification of the molecular target of ergosterol biosynthesis inhibitors and quantification of their effects on total ergosterol biosynthesis.

Increasing resistance of clinically relevant fungi is causing major problems in anti-mycotic therapy. Particularly for immunosuppressed patients fungal infections are of concern and increasing resistance against clinically used antimycotic drugs is hampering successful treatment. In the search for new antifungals ergosterol biosynthesis still is the most prominent target. However, several pitfalls in the bioactivity testing of such substances remain. Two of the major drawbacks certainly are the membrane association of most enzymes participating in ergosterol biosynthesis, and the difficulty to selectively associate growth inhibitory effects with the target pathway (ergosterol biosynthesis). Here we describe a GC-MS based cellular assay for target identification and selective potency determination of test components. In the qualitative part of the assay GC-MS analysis of cell lysates allows target identification by analysis of the changes in the sterol pattern. The quantitative part of the assay makes use of 13C-acetate feeding combined with GC-MS analysis allowing the selective quantification of a compound's effect on total ergosterol biosynthesis. The described cellular assay was analytically and biologically validated and used to characterize the novel ergosterol biosynthesis inhibitor JK-250.

Crototropone, a new tropone derivative from Croton zehntneri.

Crototropone (3-hydroxy-5,6-dimethoxy-2-methylcyclohepta-2,4,6-trien-1-one) was isolated from roots of Croton zehntneri. The structure was established by spectroscopic methods.

Saludimerines A and B, novel-type dimeric alkaloids with stereogenic centers and configurationally semistable biaryl axes.

The first biarylic bis-morphinanedienone alkaloids, saludimerines A (3a) and B (3b), isolated from a tree of Croton flavens (Euphorbiaceae) are described. These naturally occurring dimers of the known alkaloid salutaridine are joined together via a rotationally hindered biaryl axis, giving rise to atropo-diastereomers that are configurationally stable at room temperature but slowly interconvert in methanolic solution within several days. Their structures were established by spectroscopic methods and by partial synthesis, which was achieved by a highly atropo-diastereoselective biomimetic oxidative coupling of the monomeric precursor, salutaridine. Their axial configurations were elucidated by circular dichroism (CD) investigations, which succeeded despite the fact that the two atropo-diastereomers exhibit near-identical CD spectra. This remarkable phenomenon was rationalized by quantum chemical CD calculations. The configurational assignment of saludimerines A (3a) as P-axial and B (3b) as M was corroborated by atropisomer-specific NOE interactions between protons of the one molecular half with nuclei in the other.

1-Substituted beta-carboline-3-carboxylates with high affinities to the benzodiazepine recognition site.

Naturally occurring derivatives of beta-carboline-3-carboxylic acid bearing acetyl or vinyl groups at C-1 were prepared by Pd-catalyzed cross-coupling reactions of methyl 1-chloro-beta-carboline-3-carboxylate with appropriate organostannanes. Esters with chloro or acetyl groups at C-1 showed high affinity for the brain benzodiazepine recognition site. Thus, in contrast to 1-alkyl and 1-aryl analogs, these beta-carboline-3-carboxylates with electron-withdrawing substituents at C-1 show high affinities.

A new approach towards ikimine A analogues.

An analogon of the alkylpyridine alkaloid ikimine A (1) was prepared in six steps starting from undec-10-ynoic acid. A key step in this synthesis was a Sonogashira coupling of the alkyne and 3-iodopyridine, followed by hydrogenation of the alkyne, reduction of the ester to the primary alcohol and oxidation to the corresponding aldehyde. This aldehyde was converted to the ikimine A analogon with O-methyl hydroxylamine hydrochloride. This product and the intermediate alkylpyridines were tested in the agar diffusion assay for antibacterial and antifungal activities.