Zebrafish obesogenic test identifies anti-adipogenic fraction in Moringa oreifera leaf extracts

Abstract

The zebrafish obesogenic test (ZOT) is a powerful tool for identifying anti-adipogenic compounds for in vivo screening. In our previous study, we found that Moringa oleifera (MO) leaf powder suppressed the accumulation of visceral adipose tissue (VAT) in ZOT. MO demonstrates a wide range of pharmacological effects; however, little is known about its functional constituents. To identify the anti-adipogenic components of MO leaves, we prepared extracts using different extraction methods and tested the obtained extracts and fractions using ZOT. We found that the dichloromethane extract and its hexane:EtOAc = 8:2 fraction reduced VAT accumulation in young zebrafish fed a high-fat diet. We also performed gene expression analysis in the zebrafish VAT and found that CCAAT/enhancer-binding protein beta and CCAAT/enhancer-binding protein delta (associated with early stages of adipogenesis) gene expression was downregulated after fraction 2 administration. We identified a new MO fraction that suppressed VAT accumulation by inhibiting early adipogenesis using the ZOT. Phenotype-driven zebrafish screening is a reasonable strategy for identifying bioactive components in natural products.

Keywords: diabetes; dyslipidemia; herbal medicine; natural products; visceral obesity.

FIGURE 1

CH₂Cl₂ extract reduces total visceral adipose tissue (VAT) in high‐fat diet‐fed zebrafish. (a) Experimental design of this study. (b) Preparation of MO extracts. Extracts were prepared using serial extraction with each solvent. (c) NR fluorescent intensities in the VAT were calculated. The Y‐axis indicates the ratio of NR staining before and after 48‐h treatment with each extract. Data are shown as the means ± standard deviation. n = 5, *p < .05 versus control, as calculated by using one‐way ANOVA. Reproducibility test results are displayed in Figure S2. Each extract was administered at 10 μg/ml. Phenylephrine (20 μM) was used as a positive control. (d) Representative images of control‐ and CH₂Cl₂ extract‐treated zebrafish

FIGURE 2

Fraction 2 of CH₂Cl₂ extract suppresses adipogenesis in zebrafish VAT. (a) Flow chart showing elution fractions obtained using silica gel chromatography. (b and c) Zebrafish obesogenic test. Fraction 2 (10 μg/ml) reduced the amount of VAT compared to control treatment (0.1% DMSO). Data are shown as the means ± standard deviation. n = 5, *p < .05 versus control, as calculated by using one‐way ANOVA. Reproducibility test results are provided in Figure S3. (d and e) qPCR analysis of early adipocyte differentiation markers CCAAT/enhancer‐binding protein beta (cebpb) and CCAAT/enhancer‐binding protein delta (cebpd; d) and late differentiation markers peroxisome proliferator‐activated receptor gamma (pparg) and CCAAT/enhancer‐binding protein alpha (cebpa; e). Data are shown as the means ± standard deviation. n = 5 or 6, **p < .01 versus control, as calculated by using the Student's t‐test

FIGURE 3

Subfractions of Fr. 2 suppress adipogenesis. (a) Fr. 2 was subfractionated into seven pools including washout. (b) The yield of each step. (c) Zebrafish obesogenic test. the 4th, 5th, and 6th fractions (10 μg/ml) reduced the amount of VAT. *p < .05 versus control, n = 5, error bars indicate standard deviation (SD). (d) In vitro adipogenesis assays using mouse preadipocyte 3T3‐L1 cells. Data are shown as the means ± SD. n = 8, *p < .05 versus control, as calculated by using one‐way ANOVA. (e) Representative images of (d). Red indicates mature adipocytes