Heterologous production of the widely used natural food colorant carminic acid in <i>Aspergillus nidulans</i>

Abstract The natural red food colorants carmine (E120) and carminic acid are currently produced from scale insects. The access to raw material is limited and current production is sensitive to fluctuation in weather conditions. A cheaper and more stable supply is therefore desirable. Here we present the first proof-of-concept of heterologous microbial production of carminic acid in Aspergillus nidulans by developing a semi-natural biosynthetic pathway. Formation of the tricyclic core of carminic acid is achieved via a two-step process wherein a plant type III polyketide synthase (PKS) forms a non-reduced linear octaketide, which subsequently is folded into the desired flavokermesic acid anthrone (FKA) structure by a cyclase and a aromatase from a bacterial type II PKS system. The formed FKA is oxidized to flavokermesic acid and kermesic acid, catalyzed by endogenous A. nidulans monooxygenases, and further converted to dcII and carminic acid by the Dactylopius coccus C-glucosyltransferase DcUGT2. The establishment of a functional biosynthetic carminic acid pathway in A. nidulans serves as an important step towards industrial-scale production of carminic acid via liquid-state fermentation using a microbial cell factory

Characterization of Antileishmanial Compounds from Lawsonia inermis L. Leaves Using Semi-High Resolution Antileishmanial Profiling Combined with HPLC-HRMS-SPE-NMR

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

Potential antidiabetic phytochemicals in plant roots : a review of in vivo studies

Background: Medicinal plants are used to treat various disorders, including diabetes, globally in a range of formulations. While attention has mainly been on the aerial plant parts, there are only a few review studies to date that are focused on the natural constituents present in the plant roots with health benefits. Thus, the present study was performed to review in vivo studies investigating the antidiabetic potential of the natural compounds in plant roots. Methods: We sorted relevant data in 2001–2019 from scientific databases and search engines, including Web of Knowledge, PubMed, ScienceDirect, Medline, Reaxys, and Google Scholar. The class of phytochemicals, plant families, major compounds, active constituents, effective dosages, type of extracts, time of experiments, and type of diabetic induction were described. Results: In our literature review, we found 104 plants with determined antidiabetic activity in their root extracts. The biosynthesis pathways and mechanism of actions of the most frequent class of compounds were also proposed. The results of this review indicated that flavonoids, phenolic compounds, alkaloids, and phytosteroids are the most abundant natural compounds in plant roots with antidiabetic activity. Phytochemicals in plant roots possess different mechanisms of action to control diabetes, including inhibition of α-amylase and α-glucosidase enzymes, oxidative stress reduction, secretion of insulin, improvement of diabetic retinopathy/nephropathy, slow the starch digestion, and contribution against hyperglycemia. Conclusion: This review concludes that plant roots are a promising source of bioactive compounds which can be explored to develop against diabetes and diabetes-related complications. Graphical abstract: [Figure not available: see fulltext.

High-Resolution α‑Amylase Assay Combined with High-Performance Liquid Chromatography–Solid-Phase Extraction–Nuclear Magnetic Resonance Spectroscopy for Expedited Identification of α‑Amylase Inhibitors: Proof of Concept and α‑Amylase Inhibitor in Cinnamon

Type 2 diabetes affects millions of people worldwide, and new improved drugs or functional foods containing selective α-amylase inhibitors are needed for improved management of blood glucose. In this article the development of a microplate-based high-resolution α-amylase inhibition assay with direct photometric measurement of α-amylase activity is described. The inhibition assay is based on porcine pancreatic α-amylase with 2-chloro-4-nitrophenyl-α-d-maltotriose as substrate, which this gives a stable, sensitive, and cheap inhibition assay as requested for high-resolution purposes. In combination with HPLC–​HRMS–​SPE–​NMR, this provides an analytical platform that allows simultaneous chemical and biological profiling of α-amylase inhibitors in plant extracts. Proof-of-concept with an artificial mixture of six compoundsof which three are known α-amylase inhibitorsshowed that the high-resolution α-amylase inhibition profiles allowed detection of sub-microgram amounts of the α-amylase inhibitors. Furthermore, the high-resolution α-amylase inhibition assay/HPLC–​HRMS–​SPE–​NMR platform allowed identification of cinnamaldehyde as the α-amylase inhibitor in cinnamon (<i>Cinnamomum verum</i> Presl.)

Combined Use of High-Resolution α‑Glucosidase Inhibition Profiling and High-Performance Liquid Chromatography–High-Resolution Mass Spectrometry–Solid-Phase Extraction–Nuclear Magnetic Resonance Spectroscopy for Investigation of Antidiabetic Principles in Crude Plant Extracts

Type 2 diabetes is a metabolic disorder affecting millions of people worldwide, and new drug leads or functional foods containing selective α-glucosidase inhibitors are needed. Crude extract of 24 plants were assessed for α-glucosidase inhibitory activity. Methanol extracts of Cinnamomum zeylanicum bark, Rheum rhabarbarum peel, and Rheum palmatum root and ethyl acetate extracts of C. zeylanicum bark, Allium ascalonicum peel, and R. palmatum root showed IC₅₀ values below 20 μg/mL. Subsequently, high-resolution α-glucosidase profiling was used in combination with high-performance liquid chromatography–high-resolution mass spectrometry–solid-phase extraction–nuclear magnetic resonance spectroscopy for identification of metabolites responsible for the α-glucosidase inhibitory activity. Quercetin (<b>1</b>) and its dimer (<b>2</b>), trimer (<b>3</b>), and tetramer (<b>4</b>) were identified as main α-glucosidase inhibitors in A. ascalonicum peel, whereas (<i>E</i>)-piceatannol 3′-<i>O</i>-β-d-glucopyranoside (<b>5</b>), (<i>E</i>)-rhapontigenin 3′-<i>O</i>-β-d-glucopyranoside (<b>6</b>), (<i>E</i>)-piceatannol (<b>8</b>), and emodin (<b>12</b>) were identified as main α-glucosidase inhibitors in R. palmatum root

High-Resolution PTP1B Inhibition Profiling Combined with HPLC-HRMS-SPE-NMR for Identification of PTP1B Inhibitors from Miconia albicans

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