Unraveling the complexity of complex mixtures by combining high-resolution pharmacological, analytical and spectroscopic techniques: antidiabetic constituents in Chinese medicinal plants.

Medicinal plants have been widely used as (poly)pharmacological remedies and constitute a rich source for antidiabetic drug discovery. In the present study, forty medicinal plant samples collected in China were tested for inhibitory activity against α-glucosidase, α-amylase, and protein-tyrosine phosphatase 1B (PTP1B). Crude ethyl acetate extracts of Dioscorea bulbifera L., Boehmeria nivea Gaudich, Tinospora sagittata Gagnep. and Persicaria bistorta (L.) Samp. showed dual inhibitory activity towards α-glucosidase and PTP1B, and were chosen for further investigation. Subsequent dual high-resolution α-glucosidase/PTP1B profiling or triple high-resolution α-glucosidase/α-amylase/PTP1B profiling combined with HPLC-HRMS and NMR spectroscopy led to the identification of 28 metabolites with one or more bioactivities. Among these, three new phenanthrenes were identified from D. bulbifera, including one new biphenanthrene (10) exhibiting promising dual inhibitory activity towards α-glucosidase and PTP1B with IC50 values of 2.08 ± 0.19 and 3.36 ± 0.25 µM, respectively. Two triterpenoids and one fatty acid from B. nivea and T. sagittata as well as some commercially available fatty acids showed strong PTP1B inhibitory activity with IC50 values in the range of 4.89 ± 0.38 to 53.77 ± 4.20 µM.

High-dose naloxone, an experimental tool uncovering latent sensitisation: pharmacokinetics in humans.

BACKGROUND: Naloxone, an opioid receptor antagonist, is used as a pharmacological tool to detect tonic endogenous activation of opioid receptors in experimental pain models. We describe a pharmacokinetic model linking naloxone pharmacokinetics to its main metabolite after high-dose naloxone infusion. METHODS: Eight healthy volunteers received a three-stage stepwise high-dose i.v. naloxone infusion (total dose 3.25 mg kg-1). Naloxone and naloxone-3-glucuronide (N3G) plasma concentrations were sampled from infusion onset to 334 min after infusion discontinuation. Pharmacokinetic analysis was performed using non-linear mixed effect models (NONMEM). The predictive performances of Dowling's and Yassen's models were evaluated, and target-controlled infusion simulations were performed. RESULTS: Three- and two-compartment disposition models with linear elimination kinetics described the naloxone and N3G concentration time-courses, respectively. Two covariate models were developed: simple (weight proportional) and complex (with the shallow peripheral volume of distribution linearly increasing with body weight). The median prediction error (MDPE) and wobble for Dowling's model were -32.5% and 33.4%, respectively. For Yassen's model, the MDPE and wobble were 1.2% and 19.9%, respectively. CONCLUSIONS: A parent-metabolite pharmacokinetic model was developed for naloxone and N3G after high-dose naloxone infusion. No saturable pharmacokinetics were observed. Whereas Dowling's model was inaccurate and over-predicted naloxone concentrations, Yassen's model accurately predicted naloxone pharmacokinetics. The newly developed covariate models may be used for high-dose TCI-naloxone for experimental and clinical practice. CLINICAL TRIALS REGISTRATION: NCT01992146.

Synthesis of C-Glucosylated Octaketide Anthraquinones in Nicotiana benthamiana by Using a Multispecies-Based Biosynthetic Pathway.

Carminic acid is a C-glucosylated octaketide anthraquinone and the main constituent of the natural dye carmine (E120), possessing unique coloring, stability, and solubility properties. Despite being used since ancient times, longstanding efforts to elucidate its route of biosynthesis have been unsuccessful. Herein, a novel combination of enzymes derived from a plant (Aloe arborescens, Aa), a bacterium (Streptomyces sp. R1128, St), and an insect (Dactylopius coccus, Dc) that allows for the biosynthesis of the C-glucosylated anthraquinone, dcII, a precursor for carminic acid, is reported. The pathway, which consists of AaOKS, StZhuI, StZhuJ, and DcUGT2, presents an alternative biosynthetic approach for the production of polyketides by using a type III polyketide synthase (PKS) and tailoring enzymes originating from a type II PKS system. The current study showcases the power of using transient expression in Nicotiana benthamiana for efficient and rapid identification of functional biosynthetic pathways, including both soluble and membrane-bound enzymes.

Characterization of midazolam metabolism in locusts: the role of a CYP3A4-like enzyme in the formation of 1'-OH and 4-OH midazolam.

1. The metabolism of midazolam was investigated in vivo in locusts in order to evaluate the presence of an enzyme with functionality similar to human CYP3A4/5. 2. Hydroxylated metabolites of midazolam identical to human metabolites were detected in locusts and the apparent affinities (Km values) were in the same range as reported in humans (in locusts: 7-23 and 33-85 µM for the formation of the 1'-OH and 4-OH metabolites, respectively). 3. The formation of hydroxylated metabolites could successfully be inhibited by co-administration of ketoconazole, a known CYP3A4/5 inhibitor. 4. Besides phase I metabolites, a number of conjugated metabolites were detected using high-resolution mass spectrometry. The most abundant metabolites detected were structurally identified by (1)H NMR as two N-glucosides. NMR analysis strongly suggested that the glycosylation occurred at the two nitrogens (either one in each case) of the imidazole ring. 5. Distribution of midazolam and the glucose conjugates were successfully measured using desorption electrospray mass spectrometry imaging revealing time-dependent changes in distribution over time. 6. In conclusion, it appears that an enzyme with functionality similar to human CYP3A4/5 is present in locusts. However, it appears that conjugation with glucose is the main detoxification pathway of midazolam in locusts.

Expanding the Landscape of Diterpene Structural Diversity through Stereochemically Controlled Combinatorial Biosynthesis.

Plant-derived diterpenoids serve as important pharmaceuticals, food additives, and fragrances, yet their low natural abundance and high structural complexity limits their broader industrial utilization. By mimicking the modularity of diterpene biosynthesis in plants, we constructed 51 functional combinations of class I and II diterpene synthases, 41 of which are "new-to-nature". Stereoselective biosynthesis of over 50 diterpene skeletons was demonstrated, including natural variants and novel enantiomeric or diastereomeric counterparts. Scalable biotechnological production for four industrially relevant targets was accomplished in engineered strains of Saccharomyces cerevisiae.

Quadruple high-resolution α-glucosidase/α-amylase/PTP1B/radical scavenging 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 root bark of Morus alba L.

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

Recreational drug use at a major music festival: trend analysis of anonymised pooled urine.

OBJECTIVE: The spread of new psychoactive substances (NPS) has expanded rapidly in the last decade. The complexity of the pharmacological effects of NPS challenges the traditional treatment guidelines, and information of the emergence of new arrivals is valuable. Our knowledge on the actual range of recreational drugs used and NPS available in Denmark is limited as identification is possible only when consumers become patients in the healthcare system or through drug seizures. We aimed to detect classical recreational drugs and NPS in the urine of music festival attendees and evaluate if the use of NPS could have been predicted by comparing study data with drug seizure data from the previous year published by European and Danish health authorities. METHODS: In a cross-sectional study, 44 urine samples were collected from three urinals at Roskilde Festival 2016-the largest Danish music festival. Two urinals were placed at music stages with late-night concerts, and one urinal was placed at a camp site. Samples were prepared using enzymatic hydrolysis followed by cationic and anionic solid phase extraction, and analysed using ultra performance liquid chromatography-high-resolution time-of-flight mass spectrometry (UPLC-HR-TOF-MS). Data were processed using an in-house library of 467 target substances, including legal and illegal drugs and metabolites. Urine drug-screening immunoassays were also evaluated and results were compared to UPLC-HR-TOF-MS results. RESULTS: In total, 77 drugs, including metabolites, were qualitatively identified in the 44 urine samples. The recreational drugs identified were amphetamine (n = 30), cocaine (n = 44), MDA (n = 40), MDMA (n = 44), THC-COOH (n = 19) and ketamine (n = 17). No NPS were identified. Sample testing using the urine drug-screening immunoassays showed presence of cocaine (n = 27), methamphetamine/MDMA (n = 4), THC (n = 7), "Spice" (n = 7) and methylphenidate (n = 1). These discrepancies might be caused by differences in cut-off values between the analytical methods, limited specificity or cross-reactivity of the urine drug-screening immunoassays compared to UPLC-HR-TOFMS results. CONCLUSION: Widespread uses of classical recreational drugs were identified in pooled urine samples. The prevalence of NPS was not as comprehensive as expected based on the European and Danish health authorities reports on illegal drugs. Urine drug-screening immunoassays results are advised to be confirmed by chromatographic bioanalysis.

Potential of Polygonum cuspidatum Root as an Antidiabetic Food: Dual High-Resolution α-Glucosidase and PTP1B Inhibition Profiling Combined with HPLC-HRMS and NMR for Identification of Antidiabetic Constituents.

The worldwide increasing incidence of type 2 diabetes has fueled an intensified search for food and herbal remedies with preventive and/or therapeutic properties. Polygonum cuspidatum Siebold & Zucc. (Polygonaceae) is used as a functional food in Japan and South Korea, and it is also a well-known traditional antidiabetic herb used in China. In this study, dual high-resolution α-glucosidase and protein-tyrosine phosphatase 1B (PTP1B) inhibition profiling was used for the identification of individual antidiabetic constituents directly from the crude ethyl acetate extract and fractions of P. cuspidatum. Subsequent preparative-scale HPLC was used to isolate a series of α-glucosidase inhibitors, which after HPLC-HRMS and NMR analysis were identified as procyanidin B2 3,3″-O-digallate (3) and (-)-epicatechin gallate (5) with IC50 values of 0.42 ± 0.02 and 0.48 ± 0.0004 µM, respectively, as well as a series of stilbene analogues with IC50 value in the range from 6.05 ± 0.05 to 116.10 ± 2.04 µM. In addition, (trans)-emodin-physcion bianthrone (15b) and (cis)-emodin-physcion bianthrone (15c) were identified as potent PTP1B inhibitors with IC50 values of 2.77 ± 1.23 and 7.29 ± 2.32 µM, respectively. These findings show that P. cuspidatum is a potential functional food for management of type 2 diabetes.

Molecular Hybridization of Potent and Selective γ-Hydroxybutyric Acid (GHB) Ligands: Design, Synthesis, Binding Studies, and Molecular Modeling of Novel 3-Hydroxycyclopent-1-enecarboxylic Acid (HOCPCA) and trans-γ-Hydroxycrotonic Acid (T-HCA) Analogs.

γ-Hydroxybutyric acid (GHB) is a neuroactive substance with specific high-affinity binding sites. To facilitate target identification and ligand optimization, we herein report a comprehensive structure-affinity relationship study for novel ligands targeting these binding sites. A molecular hybridization strategy was used based on the conformationally restricted 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) and the linear GHB analog trans-4-hydroxycrotonic acid (T-HCA). In general, all structural modifications performed on HOCPCA led to reduced affinity. In contrast, introduction of diaromatic substituents into the 4-position of T-HCA led to high-affinity analogs (medium nanomolar Ki) for the GHB high-affinity binding sites as the most high-affinity analogs reported to date. The SAR data formed the basis for a three-dimensional pharmacophore model for GHB ligands, which identified molecular features important for high-affinity binding, with high predictive validity. These findings will be valuable in the further processes of both target characterization and ligand identification for the high-affinity GHB binding sites.