Zebrafish obesogenic test (ZOT) identifies seven anti-obese natural products (NPs). (a) Experimental design for ZOT. (b) Short term high-fat diet (HFD) increased the amount of visceral adipose tissue (VAT). Right panel indicates typical images of the control and HFD groups. Red indicates Nile Red (NR)-stained VAT. sb; swimming bladder, vat; visceral adipose tissue. Left panel indicates quantification of NR stained area. Values presented are means. Error bars indicate SD. n = 5, ** p < 0.01, Student’s t-test. (c) ZOT result. Dotted lines indicate 1 and 0.6 ratio to HFD alone group (HFD). NPs with asterisks were administered at lower volumes (10 µg/mL) because of their toxicity. The others were administered at 100 µg/mL in the breeding water. Values presented are means. Error bars indicate SD. n = 5–10, * p < 0.05, ** p < 0.01 vs. control, one-way ANOVA with Bonferroni–Dunn multiple comparison. nd indicates not determined because of high toxicity (NP18). (d) Typical image of effective NPs in (c).

Mouse 3T3-L1 preadipocyte differentiation assay identified ten lipid-lowering NPs. (a) Experimental design for 3T3-L1 assay. (b) Result of 3T3-L1 screening. NPs without data (not determined: ND) were due to inability to extract compounds with ethanol extraction. Values presented are means. Error bars indicate SD. n = 8, * p < 0.05, ** p < 0.01 vs. control l, one-way ANOVA with Bonferroni–Dunn multiple comparison. (c) Representative images of differentiated 3T3-L1 cells with the same NPs as in Figure 1d. Red indicates lipid accumulation. (d) Venn diagram analysis of the effective NPs in both the ZOT (Figure 1) and 3T3-L1 cell-based assay. * While NP01 (globin digested peptides [GDP]) could not be evaluated in the 3T3-L1 assay, NP02 (GDP with magnesium) reduced lipid accumulation in 3T3-L1 cells. Thus, we categorized NP01 as effective in both assays.

NP administration in adult diet-induced obese (DIO) zebrafish. (a) Experimental design for DIO-zebrafish experiment. (b) Body weight increases in DIO-zebrafish with or without NPs. (c–e) Plasma triglycerides (c), total cholesterol (d), and fasting blood glucose (e) at the end of the experiment. Values presented are means. Error bars indicate SD. n = 5–10, * p < 0.05, ** p < 0.01 vs. control, one-way analysis of variance (ANOVA) with Bonferroni–Dunn multiple comparison.