*2.5. YGS Treatment Shows Better Climbing Index for Aβ<sup>42</sup> Flies*

Figure 4 shows a quantified climbing index of mCD8 and Aβ42 flies at 1, 5, and 10 days after eclosion under sham and YGS treatments. Our results show a quantified climbing index of Aβ<sup>42</sup> flies at 5 and 10 days after eclosion, and under YGS treatment was significantly greater than those Aβ<sup>42</sup> flies under sham treatment (*N* = 30 for each group, *p* < 0.01–0.05, Figure 4). In other words, YGS treatment should be able to enhance the climbing index for Aβ<sup>42</sup> flies.

**Figure 4.** Climbing index of mCD8 and Aβ<sup>42</sup> flies at 1, 5, 10 days after eclosion under sham and YGS treatments. The quantified climbing index of Aβ<sup>42</sup> flies at 5 and 10 days after eclosion under YGS treatment was significantly greater than those Aβ<sup>42</sup> flies under sham treatment (*N* = 30 for each group). In other words, YGS treatment should be able to enhance the climbing index for Aβ<sup>42</sup> flies. Values are mean ± SEM (\*\* *p* < 0.01, \* *p* < 0.05, two-way ANOVA followed by a Student–Newman– Keuls multiple comparisons post-test). Abbreviations: YGS, Yi-Gan-San; Aβ, amyloid-beta; SEM, standard error of the mean; ANOVA, analysis of variance.

### *2.6. YGS Treatment Shows Greater Green Fluorescent Protein (GFP) Fluorescence in the External Eyes for GFP-Aβ<sup>42</sup> Flies*

We modeled GFP fluorescence in Aβ42-expressing flies and observed that it was more sensitive and suitable for analyzing Aβ<sup>42</sup> toxicity. As shown in Figure 5, GFP fluorescence in the external eyes of GFP-Aβ<sup>42</sup> flies was weaker than those of GFP-WT flies. When GFP-Aβ<sup>42</sup> flies were treated with 0.1% and 1% YGS treatments, GFP fluorescence in the external eyes was stronger than those of GFP-Aβ<sup>42</sup> flies with sham treatment (Figure 5A). We quantified and then compared that GFP fluorescence in the external eyes of GFP-mCD8 flies was significantly stronger than those of GFP-Aβ<sup>42</sup> flies with sham, 0.1% and 1% YGS treatments (*N* = 30 for each group, *p* < 0.01, Figure 5B); while GFP fluorescence in the external eyes of GFP-Aβ<sup>42</sup> flies with 0.1% and 1% YGS treatments was significantly stronger than those of GFP-Aβ<sup>42</sup> flies with sham treatment (*N* = 30 for each group, *p* < 0.01, Figure 5B). In addition, GFP fluorescence in the external eyes of GFP-Aβ<sup>42</sup> flies with 1% YGS treatment was significantly stronger than those of GFP-Aβ<sup>42</sup> flies with 0.1% YGS treatment (*N* = 30 for each group, *p* < 0.05, Figure 5B). The results revealed that GFP-Aβ<sup>42</sup> flies showed increasing GFP fluorescence in the external eyes with an increasing dose of YGS treatment.

**Figure 5.** Eye GFP expressions of mCD8-GFP and Aβ42-GFP flies under sham and YGS treatments. (**A**) GFP expressions of external eyes in mCD8-GFP (normal control) flies under sham and YGS treatments were obviously greater than those of Aβ42-GFP under sham and YGS treatments, while GFP expressions of external eyes in the Aβ42-GFP flies under YGS treatment were obviously greater than those of Aβ42-GFP flies under sham treatment. (**B**) Completeness of external eyes in mCD8-GFP flies under sham and YGS treatments was obviously better than those of Aβ42-GFP under sham and YGS treatments, while completeness of external eyes in the Aβ42-GFP flies under YGS treatment was obviously better than those of Aβ42-GFP flies under sham treatment. (**C**) Quantified GFP fluorescence of external eyes of mCD8-GFP flies under sham and YGS treatments were significantly greater than those of Aβ42-GFP under sham and YGS treatments, while quantified GFP expressions of external eyes in the Aβ42-GFP flies under YGS treatment was significantly greater than those of Aβ42-GFP flies under sham treatment (*N* = 30 for each group). Values are mean ± SEM (\*\*\* *p* < 0.001, two-way ANOVA followed by a Student–Newman–Keuls multiple comparisons post-test). Abbreviations: YGS, Yi-Gan-San; Aβ, amyloid-beta, GFP, green fluorescent protein; SEM, standard error of the mean; ANOVA, analysis of variance.
