Antianxiety effects of nutrient combinations. (A) Representative trace map of the zebrafish behavioral tests (n = 10). The moving speed <4, 4–20, and >20 mm/s were marked in green, black, and red lines, respectively. (B) Moving distance of zebrafish in each group (n = 10). (C) Cortisol production level of zebrafish in each group (zebrafish in three wells were combined and measured as one sample, n = 3). (D) The cortisol production level of zebrafish in each group was plotted against the distance measured in the behavioral test. n = 3. Data in panels (B–D) were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared with anxiety model. In panel (B), Stachyose + PS128 (83.3 + 500 μg/ml) (#) vs. WP + Th (14.2 + 33.3 μg/ml) (#), P = 0.003. Data were collected from three biological replicates.

Antioxidant effects of nutrient combinations. (A) The representative images of yolk sac fluorescence in each group. (B) The fluorescence intensity of the yolk sac in each group (n = 10). Data were presented as mean ± SEM. *P < 0.05, ***P < 0.001 compared with OS model. Data were collected from three biological replicates.

Central neuroprotective effect of nutrient combinations. (A) The representative fluorescence images of central neural apoptosis in zebrafish. (B) The quantification of fluorescence intensity of central neural apoptosis in each group. n = 10, **P < 0.01, ***P < 0.001. (C) The oxidative stress (from Figure 2B, value was normalized to the control) was plotted against the central neural apoptosis level (normalized to the control), (r = 0.897, P = 0.003). Data in panels (B,C) were presented as mean ± SEM. **P < 0.01, ***P < 0.001 compared with NSI model. Data were collected from three biological replicates.

Memory-improving effects of nutrients. (A) The lines represented the average moving speed of zebrafish per minute under light and in dark environments for 20 min (n = 12). (B) The difference in velocity in light (0–5 and 10–15 min) and dark (5–10 and 15–20 min) was measured (n = 12). (C) The histogram of zebrafish’s average movement velocity in each cycle (5 min per cycle, n = 12). (D) The fluorescence intensity of AChE in each group (n = 6). (E) The intensity of AChE was plotted against the AUC of the mean velocity from panel (A) (n = 6, r = –0.836, P = 0.009). Data in panels (B,C) were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared with AD model; ^##P < 0.001, ^###P < 0.001 compared with WP + CP (14.2 + 25 μg/ml). Data were collected from three biological replicates.

The effects of nutrient combinations were universal and related. (A) Results of all the effects from the test groups were consolidated and presented in bar chat. The four models, including anxiety, OS, NSI and AD, were shown as “Model” for short. Data were normalized to the control (value of which was normalized to 1) of each test and presented as mean ± SEM. (B) The effects tested from test groups were plotted against each other. The r-value and P-value of each plot are shown in panel (B).