Glucosyl hesperidin exhibits more potent anxiolytic activity than hesperidin accompanied by the attenuation of noradrenaline induction in a zebrafish model

**Background:** Hesperidin (Hes), a flavanone glycoside abundant in citrus fruits, has demonstrated anxiolytic and antidepressant-like activities in rodent models. However, its poor water solubility (0.002 g/100 g water) limits oral bioavailability and bioactivity. Glucosyl hesperidin (GHes) is a monoglucose conjugate of Hes produced via cyclodextrin glucanotransferase, with approximately 10,000-fold higher water solubility. Previous studies showed that GHes leads to a 3.7-fold greater area under the concentration-time curve for hesperetin in rat serum compared to Hes. Despite this, the anxiolytic effects of GHes had not been investigated. This study aimed to compare the anxiolytic activity of GHes and Hes using zebrafish (a validated model for anxiety research) and to explore underlying mechanisms.

**Methods:** Adult RIKEN WT zebrafish (6–12 months old, 1:1 male:female) were fed a control diet, 1% GHes diet, or 1% Hes diet for varying durations depending on the behavioral test. Behavioral assessments included: motility test (52 days feeding), novel tank test (31 days), black-white preference test (21 days), low water level stress test (14 days), and alarm substance exposure test (7 days). Swimming behavior was recorded and analyzed using Move-tr/2D software. After alarm substance exposure, brain monoamine levels (noradrenaline, dopamine, serotonin) were measured by HPLC. Protein expression (Th1, phospho-ERK, total ERK, CREB) was assessed by immunoblotting, and mRNA levels (c-fos, bdnf, trkb, nrf2, keap1) by real-time PCR. Statistical analyses used one-way ANOVA followed by Tukey's test.

**Key Results:** In the novel tank test, GHes significantly reduced freezing time (p < 0.05 vs control) and freezing frequency (p < 0.01 vs control), and increased total distance traveled (p < 0.01 vs control). Hes showed no significant effects. In the black-white preference test, GHes increased time in the white area (p < 0.01 vs control) and decreased time in the black area (p < 0.01 vs control), while Hes had no effect. Under low water level stress, GHes decreased freezing time in both 0–5 min (p < 0.01) and 5–10 min (p < 0.05) periods; Hes only reduced freezing in the 5–10 min period (p < 0.05). In the alarm substance test, GHes suppressed freezing time (p < 0.01) and frequency (p < 0.05), whereas Hes did not. GHes significantly reduced brain noradrenaline (p < 0.01) and dopamine (p < 0.01) levels, but not serotonin. GHes downregulated Th1 protein (p < 0.01), c-fos mRNA (p < 0.05), and phospho-ERK (p < 0.05). GHes upregulated CREB protein (3.0-fold increase, p < 0.05) and bdnf mRNA (1.5-fold increase, p < 0.05), while trkb, nrf2, and keap1 showed no significant changes. GHes and Hes both slightly increased daily food intake (p < 0.01 vs control).

**Clinical Implications:** This study demonstrates that glycosylation of hesperidin to form GHes significantly enhances its anxiolytic activity in a zebrafish model, likely due to improved water solubility and bioavailability. GHes suppressed anxiety-like behaviors across multiple stress paradigms (visual, physical, and chemical) more effectively than Hes. The mechanism involves attenuation of the noradrenaline/ERK/AP-1/TH1 pathway and activation of the CREB/BDNF pathway, both implicated in anxiety regulation. These findings suggest GHes could be developed as a functional food ingredient or nutraceutical for anxiety reduction. However, limitations include the use of zebrafish (differences in cranial nerves and gastrointestinal tract compared to mammals), lack of chronic stress testing, and unclear molecular targets. Future studies in mammalian models and clinical trials are needed to confirm efficacy and safety in humans.