*2.4. Bioactivity Evaluation of DGS Based on Zebrafish Model In Vivo*

A zebrafish assay was performed according to the schematic diagram (Figure 5A). As shown in Figure 5B, zebrafish treated with DGS for 3 days demonstrated mortality and abnormalities at concentrations ≥ 100 µg/mL. The LD50 of DGS was 753.7 µg/mL and the 5% lethality was 478 µg/mL. These results suggest that efficacy assessment of DGS at concentrations ≤ 100 µg/mL can be used for the evaluation of the efficacy of angiogenesis.

**Figure 5.** DGS promoted the angiogenesis of zebrafish: (**A**) Schematic diagram of the zebrafish experiment. (**B**) The lethal curve of DGS (**C**) Fluorescent images of the ISV of the zebrafish. The images of a'–f' were partial enlargements of images a–f. Scale bar: 200 µm. (**D**) Fluorescent image of the MSIV of the zebrafish. The images of g'–k' were partial enlargements of images g–k. Scale bar: 200 µm. (**E**) Effect of DGS on the length of ISV in zebrafish. (**F**) The effect of DGS on the sprouting of SIV in zebrafish. (**G**) Effect of DGS on the growth of the MSIV in zebrafish. Values are expressed as the mean <sup>±</sup> SEM (n = 10). #### *<sup>p</sup>* < 0.0001 vs. Control, \* *<sup>p</sup>* < 0.05 vs. Model, \*\* *<sup>p</sup>* < 0.01 vs. Control, \*\*\* *p* < 0.01 vs. Control, \*\*\*\* *p* < 0.0001 vs. Control.

Transgenic Tg (flk1a: EGFP) zebrafish embryos were used to explore the proangiogenic effects of DGS. The findings indicated that PTK787 significantly inhibited the formation of intersegment vessels (ISVs). Most vessels were incomplete and discontinuous after PTK787 treatment (Figure 5B,D). The ISVs length in the model was 323.3 ± 38.42 µm, which was significantly lower than that of the control (1017 ± 58.96 µm; *p* < 0.0001). After treatment with CoQ10 as a positive drug, the total ISV length reached 730.0 ± 59.77 µm, which was significantly higher than that of the model group (*p* < 0.0001). The lengths of ISVs after treatment with DGS at concentrations of 10, 25, and 40 µg/mL were 610.0 ± 85.08 µm (*p* < 0.01), 793.7 ± 23.71 µm (*p* < 0.0001), and 654.3 ± 70.81 µm (*p* < 0.0001), respectively. Concerning the number of vascular sprouting, the number was significantly increased in the CoQ10 and DGS-treated groups. In conclusion, DGS was able to significantly promote the growth of the inhibited vessels.

To further investigate the proangiogenic effect of DGS on normal blood vessels, the effects were assessed using the main subintestinal veins (MSIV) of the zebrafish. As depicted in Figure 5C,F, the length of MSIV in the zebrafish treated with CoQ10 for 24 h was 688.2 ± 22.13 µm, which was significantly higher compared with that of the control (628.0 ± 22.13 µm; *p* < 0.05). After treatment with DGS at concentrations of 5, 10, and 20 µg/mL, the lengths of MSIV were 708.0 ± 18.22 µm (*p* < 0.01), 771.5 ± 22.72 µm (*p* < 0.0001), and 775.5 ± 23.62 µm (*p* < 0.0001), respectively.
