Anxiety induced by excess mental or physical stress is deeply involved in the onset of human psychiatric diseases such as depression, bipolar disorder, and panic disorder. Recently, Kampo medicines have received focus as antidepressant drugs for clinical use because of their synergistic and additive effects. Thus, we evaluated the anxiolytic activity of Ninjinyoeito (NYT) using neuropeptide Y-knockout (NPY-KO) zebrafish that exhibit severe anxiety responses to acute stress. Adult NPY-KO zebrafish were fed either a 3% NYT-supplemented or normal diet (i.e., the control diet) for four days and were then examined via behavioral tests. After short-term cold stress (10 °C, 2 s) was applied, control-fed NPY-KO zebrafish exhibited anxiety behaviors such as freezing, erratic movement, and increased swimming time along the tank wall. On the other hand, NYT-fed NPY-KO zebrafish significantly suppressed these anxiety behaviors, accompanied by a downregulation of tyrosine hydroxylase levels and phosphorylation of extracellular signal-regulated kinases in the brain. To understand the responsible component(s) in NYT, twelve kinds of herbal medicines that composed NYT were tested in behavioral trials with the zebrafish. Among them, nine significantly reduced freezing behavior in NPY-KO zebrafish. In particular, Schisandra fruit induced the most potent effect on abnormal zebrafish behavior, even in the lower amount (0.3% equivalent to NYT), followed by Atractylodes rhizome and Cinnamon bark. Subsequently, four lignans uniquely found in Schisandra fruit (i.e., gomisin A, gomisin N, schizandrin, and schizandrin B) were investigated for their anxiolytic activity in NPY-KO zebrafish. As a result, schizandrin was identified as a responsible compound in the anxiolytic effect of NYT. These results suggest that NYT has a positive effect on mental stress-induced anxiety and may be a promising therapeutic for psychiatric diseases.

过度的精神或身体压力引起的焦虑与抑郁症、双相情感障碍和恐慌症等人类精神疾病的发生密切相关。近日，坎普药物因其协同和相加作用而成为临床使用的抗抑郁药。因此，我们使用对急性压力表现出严重焦虑反应的神经肽 Y 基因敲除 (NPY-KO) 斑马鱼评估了 Ninjinyoeito (NYT) 的抗焦虑活性。成年 NPY-KO 斑马鱼被喂食 3% NYT 补充剂或正常饮食（即对照饮食）四天，然后通过行为测试进行检查。在施加短期冷应激 (10 °C, 2 s) 后，对照喂养的 NPY-KO 斑马鱼表现出焦虑行为，如冻结、不稳定的运动和沿着水箱壁的游泳时间增加。另一方面，纽约时报喂养的 NPY-KO 斑马鱼显着抑制了这些焦虑行为，伴随着大脑中酪氨酸羟化酶水平的下调和细胞外信号调节激酶的磷酸化。为了了解 NYT 中的相关成分，在斑马鱼的行为试验中测试了构成 NYT 的 12 种草药。其中，九种显着降低了 NPY-KO 斑马鱼的冷冻行为。特别是，五味子果实对异常斑马鱼的行为产生了最有效的影响，即使用量较低（相当于 NYT 的 0.3%），其次是白术根茎和肉桂皮。随后，研究了五味子果实中独特的四种木酚素（即五味子 A、五味子 N、五味子甲素和五味子乙素）在 NPY-KO 斑马鱼中的抗焦虑活性。因此，五味子素被确定为 NYT 抗焦虑作用的重要化合物。这些结果表明 NYT 对精神压力引起的焦虑有积极作用，可能是治疗精神疾病的一种有前途的疗法。