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.

Dose-Dependent Effects of Aloin on the Intestinal Bacterial Community Structure, Short Chain Fatty Acids Metabolism and Intestinal Epithelial Cell Permeability

“…The MIC values were unable to be determined likely because aloin was metabolized as noted previously (Che et al, 1991; Pogribna et al, 2008). We confirmed this observation by analyzing metabolism of aloin in Eubacterium cultures by HPLC (Lobbens et al, 2016) (Figure 2A). The HPLC analysis revealed disappearance of aloin-A and aloin-B (retention time was 10.30 and 8.50 min, respectively) and appearance of aloe-emodin (retention time was 22.1 min), which provides evidence that Eubacterium sp.…”

Dual concentration-dependent effect of ascorbic acid on PAP(248–286) amyloid formation and SEVI-mediated HIV infection

“…Ascorbic acid (AA) has been shown to have potential therapeutic effects against several neurodegenerative diseases such as AD, PD, Huntington's disease (HD) and ischemic stroke. 54,64,72,73 Furthermore, AA has been shown to interact with Aβ, stefin B, and insulin resulting in inhibition of their aggregation. 30,60 Human semen, which naturally contains SEVI amyloid, also contains a number of vitamins amongst which AA is in the highest concentration (0.4±0.1 mM).…”

“…[60][61][62][63][64] In addition to interaction with the β-sheet backbone of amyloids, AA has been shown to exert inhibition of amyloid fibril formation through its degradation products, such as ascorbate anions and dehydroascorbic acid. 72,73 The stability of AA varies with pH and incubation temperature, where self-degradation products are obtained within ~6 hours under physiological pH at 37 ˚C. 60,72 These degradation products were found to inhibit fibrillation and disrupt preformed mature amyloid fibrils of insulin, with an activity positively correlated with the degree of AA degradation.…”

“…60,72 These degradation products were found to inhibit fibrillation and disrupt preformed mature amyloid fibrils of insulin, with an activity positively correlated with the degree of AA degradation. 72,73 The metabolites of AA were shown to shield the electrostatic interactions between the β-sheet forming areas of protein monomers prone to form amyloid structures and to disrupt β-sheet stacking. 60 Since in our in vitro assay, effects on fibril formation were seen after 24 hours, we speculate that such degradation products might contribute to the amyloid inhibitory effects observed at concentrations of AA higher than its physiological concentration.…”

Prophylactic Aloe Components on Autoimmune Diseases: Barbaloin, Aloe-Emodin, Emodin, and Fermented Butyrate

“…In a case study, the inhibitory activity of barbaloin against insulin fibrillation was investigated. The degradation products of barbaloin formed over weeks of storage, in particular oxidation products, were able to significantly inhibit insulin-fibrillation [20] .…”