Cholesterol-Lowering Effects of Asperidine B, a Pyrrolidine Derivative from the Soil-Derived Fungus Aspergillus sclerotiorum PSU-RSPG178: A Potential Cholesterol Absorption Inhibitor

Abstract

Isolated secondary metabolites asperidine B (preussin) and asperidine C, produced by the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178, were found to exhibit inhibitory effects against 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase and oxidative stress in an in vitro assay. Whether or not the known pyrrolidine asperidine B and the recently isolated piperidine asperidine C have lipid-lowering effects remains unknown. Thus, this study aimed to investigate the hypocholesterolemic effects of asperidines B and C and identify the mechanisms involved in using in vitro, ex vivo, and in vivo models. The results show that both compounds interfered with cholesterol micelle formation by increasing bile acid binding capacity, similar to the action of the bile acid sequestrant drug cholestyramine. However, only asperidine B, but not asperidine C, was found to inhibit cholesterol uptake in Caco-2 cells by up-regulating LXRα without changing cholesterol transporter NPC1L1 protein expression. Likewise, reduced cholesterol absorption via asperidine-B-mediated activation of LXRα was also observed in isolated rat jejunal loops. Asperidine B consistently decreases plasma cholesterol absorption, similar to the effect of ezetimibe in rats. Therefore, asperidine B, the pyrrolidine derivative, has therapeutic potential to be developed into a type of cholesterol absorption inhibitor for the treatment of hypercholesterolemia.

Keywords: LXRα; NPC1L1; asperidine B (preussin); cholesterol absorption; pyrrolidine derivative.

Figure 1

Structures of asperidine B (preussin) and asperidine C isolated from the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178.

Figure 2

Effects of asperidine B (preussin) and asperidine C on (A) micellar cholesterol particle size, (B) intermicellar cholesterol levels, and (C) bile acid binding capacity. Values are mean ± SEM (n = 3), * p < 0.05 vs. control.

Figure 3

Effect of asperidine B (preussin) and asperidine C on [³H]-cholesterol mixed micelle transport in intestinal Caco-2 cells. (A) Asperidine B and (B) asperidine C at 12.5–100 µM or 100 µM ezetimibe were co-incubated with 1 μCi/mL [³H]-cholesterol mixed micelles for 30 min at 37 °C (n = 5). (C) Viability of CacO-2 cells after exposure to asperidine B, asperidine C, or ezetimibe at the same concentrations as shown in (A,B). Caco-2 cells were pre-incubated with tested compounds for 3 h and incubated with an MTT reagent. Absorbance was measured at a wavelength of 570 nm and percentage cell viability was compared with control cells. Data are represented as mean ± SEM (n = 5); * p < 0.05, ** p < 0.01 vs. control.

Figure 4

Expression of NPC1L1 and LXRα in Caco-2 cells. Cells were incubated with 100 µM asperidines B and C for 3 h. (A) Membrane and cytosolic fractions expressing NPC1L1 were detected by electrophoresis and a Western blot analysis. Anti-alkaline phosphatase (ALP) and anti-β-actin antibodies were used as a membrane marker and loading control, respectively. A representative blot of NPC1L1 protein expression is shown in the top panel and quantification of relative NPC1L1/β-actin protein expression in each fraction is presented at the bottom. (B) Total RNA was extracted from Caco-2 cells and LXRα mRNA levels were determined using qPCR. Data are expressed as mean ± SEM from five separate experiments. Data are represented as mean ± SEM (n = 3); * p < 0.05 vs. control.

Figure 5

Effect of asperidines B and C, ezetimibe, LXRα activator (GW3965), and LXRα inhibitor (SR9238) on [³H]-cholesterol transport using ex vivo rat jejunal loops: (A) 100 µM asperidine B, asperidine C, or ezetimibe was co-injected in rat jejunal loops and incubated for 30 min; (B) 100 µM asperidine B, 1 µM LXRα activator (GW3965), 10 µM LXRα inhibitor (SR9238), a combination of GW3965 with either SR9238 or asperidine B, and a combination of SR9238 with asperidine B were incubated in a buffer containing [³H]-cholesterol mixed micelle for 30 min. The radioactivity accumulated in intestinal epithelium was then measured and expressed as fmol/mg protein. Values are mean ± SEM (n = 6), * p < 0.05 vs. control.

Figure 6

Effect of asperidine B or ezetimibe on plasma [³H]-cholesterol in rats. (A) The plasma [³H] cholesterol level was measured at 0, 4, 8, 12, 24, 32, and 48 h after oral administration with [³H]cholesterol (10 µCi/mL), with or without 31.75 µg/kg BW asperidine B or 10 mg/kg BW ezetimibe. (B) The total area under the curve from (A) was analyzed. Values are mean ± SEM (n = 4–6), * p < 0.05 vs. control.