title: The Inhibitory Effect of Natural Products on Protein Fibrillation May Be Caused by Degradation Products--A Study Using Aloin and Insulin.
abstract: Protein fibrillation is the pathological hallmark of several neurodegenerative diseases and also complicates the manufacturing and use of protein drugs. As a case study, the inhibitory activity of the natural compound aloin against insulin fibrillation was investigated. Based on Thioflavin T assays, high-performance liquid chromatography and transmission electron microscopy it was found that a degradation product of aloin, formed over weeks of storage, was able to significantly inhibit insulin fibrillation. The activity of the stored aloin was significantly reduced in the presence of small amounts of sodium azide or ascorbic acid, suggesting the active compound to be an oxidation product. A high-performance liquid chromatography method and a liquid chromatography-mass spectrometry method were developed to investigate the degradation products in the aged aloin solution. We found that the major compounds in the solution were aloin A and aloin B. In addition, 10-hydroxy aloin and elgonica dimers were detected in smaller amounts. The identified compounds were isolated and tested for activity by means of Thioflavin T assays, but no activity was observed. Thus, the actual fibrillation inhibitor is an as yet unidentified and potentially metastable degradation product of aloin. These results suggest that degradation products, and in particular oxidation products, are to be considered thoroughly when natural products are investigated for activity against protein fibrillation.
authors.affiliation: Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Eva S
authors.initials: ES
authors.lastname: Lobbens
authors.affiliation: Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Vito
authors.initials: V
authors.lastname: Foderà
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Nils T
authors.initials: NT
authors.lastname: Nyberg
authors.affiliation: Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Andersen
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Anna K
authors.initials: AK
authors.lastname: Jäger
authors.affiliation: Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Lene
authors.initials: L
authors.lastname: Jorgensen
authors.affiliation: Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Marco
authors.initials: M
authors.lastname: van de Weert
mesh_terms: D000880:Anthraquinones / Q000737:chemistry; D052160:Benzothiazoles; D001688:Biological Products; D002626:Chemistry, Pharmaceutical; D002851:Chromatography, High Pressure Liquid; D004642:Emodin / Q000031:analogs & derivatives* / Q000737:chemistry; D005079:Excipients / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D005960:Glucosides / Q000737:chemistry; D007328:Insulin / Q000737:chemistry*; D046529:Microscopy, Electron, Transmission; D010084:Oxidation-Reduction; D055550:Protein Stability; D059748:Proteolysis; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013844:Thiazoles / Q000737:chemistry
chemical_list: D000880:Anthraquinones; D052160:Benzothiazoles; D001688:Biological Products; D005079:Excipients; D005456:Fluorescent Dyes; D005960:Glucosides; D007328:Insulin; D012996:Solutions; D013844:Thiazoles; C459214:elgonica dimer A; C009462:thioflavin T; D004642:Emodin; C006457:alloin