Effect of Aquilaria sinensis (Lour.) Spreng leaf tea extract on zebrafish larvae. (A) Treatment of zebrafish larvae at 5 dpf with A. sinensis leaf tea extract. (B) Zebrafish larvae exposed to six concentrations: 0.0 g/L (control), 2.0 g/L, 4.0 g/L, 8.0 g/L, 16.0 g/L, and 32.0 g/L (n = 90). The number of dead larvae was counted between 6 and 10 dpf. (C) The survival ratio of zebrafish larvae was calculated and plotted as Kaplan-Meier curves (n = 90). (D) LC₅₀ of A. sinensis leaf extract (n = 90). Here, Aslt represents the A. sinensis leaf extract.

Effect of Aquilaria sinensis leaf tea on the sleep of zebrafish with sleep disturbance. (A) The schematic diagram shows sleep disturbance treatment. Sleep disturbance was performed for three consecutive days between 23:00 and 03:00 (ZT14-ZT18) from 5 dpf to 8 dpf. (B) Zebrafish larvae (5 dpf) were extract treated with six concentrations of A. sinensis leaf tea (0.00, 0.50, 0.75, 1.00, 1.25, and 1.50 g/L; n = 48) and placed in a behavior-tracking system. (C,D) Sleep and waking activity traces (±SEM) of zebrafish larvae treated with (A)sinensis leaf tea. The gray box indicates sleep disturbance time. (300 lux light on 23:00–03:00) (n = 48). (E) Quantification of total sleep across day time (9:00–23:00), sleep disturbance time (23:00–03:00), and night time (03:00–09:00) on 6 dpf (n = 48). (F) Sleep bout number between 23:00–03:00 (n = 48). (one-way ANOVA, Tukey’s post hoc test, Different lowercase letters indicate significant differences between various concentrations).

Transcriptome sequencing reveals changes in gene expression in zebrafish larvae exposed to Aquilaria sinensis leaf tea with sleep disturbance. (A) The schematic diagram shows the treatment and sample collection time of A. sinensis leaf tea-treated larvae under sleep disturbance conditions. The transcriptome of untreated larvae (0.00 g/L; Control) and larvae treated with 1.50 g/L tea (Treatment) and collected at 3:00 (ZT 18) on the third day of treatment. (B) The Volcano map displays the transcriptome sequencing results. (C) Heatmap of 1643 differentially expressed genes (p-value ≤0.05, log2FC ≥ 1.2). (D) GO enrichment analysis of the DEGs. (E) KEGG enrichment analysis showed that many genes were enriched in the NOD-like receptor. (F) The clustered heatmap of GO enrichment analysis, such as the regulation of the immune system process, myeloid leukocyte migration, granulocyte migration, neutrophil migration, leukocyte chemotaxis, and leukocyte migration.

Inflammatory response interacts with the NOD-like receptor signaling pathway from DEG between control and 1.50 g/L Aquilaria sinensis leaf tea treatment groups and expression levels of sleep-related genes. (A) String analysis of DEGs associated with immune response. The thicker the lines between the proteins, the stronger the interaction. (B) Six immune-related genes were identified from String analysis. (C) Heatmap shows the expression of six immune-related genes under sleep disturbance conditions in DEGs. (D) FPKM of sleep-related genes between control and treatment group (one-way ANOVA, Tukey’s post hoc test. Data are shown as mean ± standard deviations. n.s. indicates no significance, * indicates significance at p < 0.05).

A. sinensis tea influences immune response and sleep gene on zebrafish sleep disturbed. (A–G) qRT-PCR validation of seven differentially expressed genes between the treatment group and the control group. (H) The neutrophil image of 5 dpf Tg(mpo:GFP) under normal condition (n = 30), (I) sleep disturbed condition (SD; n = 30), and (J) sleep disturbed condition with 1.50 g/L A. sinensis leaf tea treatment (n = 30) (one-way ANOVA, Tukey’s post hoc test. Data are shown as mean ± standard deviations. *: p < 0.05, **: p < 0.01, and ***: p < 0.001).

Analysis of metabolites in Aquilaria sinensis leaf tea and the gene targets of flavonoids. (A) Among 1800 metabolites were detected, 335 types of flavonoids accounted for the highest proportion. (B) The content of flavonoids obtained accounted for 21.54% in all metabolites, higher than other substances. (C) Flavonoid and target gene network (Green represents target sites and orange represents metabolites of A. sinensis).

Eupatilin and quercetin, the potential active ingredients of Aquilaria sinensis leaf tea, improve the sleep of zebrafish with light-induced sleep disturbance. (A) 3D chemical structure of eupatilin based on PubChem database. (B) Molecular docking of eupatilin with Cd40. (C–D) Sleep and waking activity traces (±SEM) of zebrafish larvae treated with eupatilin. The gray box indicates sleep disturbance time (300 lux light on 23:00–03:00) (n = 48). (E) Quantification of total sleep across day time (9:00–23:00), sleep disturbance time (23:00 to 03:00), and night without sleep disturbance time (03:00–09:00) on 6 dpf in eupatilin treated and control groups (n = 48). (F) Quantification of sleep bout on sleep disturbance time between control and eupatilin treated groups (n = 48). (G) 3D chemical structure of quercetin based on PubChem database. (H) Molecular docking of quercetin with Il1b. (I–J) Sleep and waking activity traces (±SEM) of zebrafish larvae treated with quercetin (n = 48). (K) Quantification of total sleep across day time (9:00–23:00), sleep disturbance time (23:00 to 03:00), and night without sleep disturbance time (03:00–09:00) in quercetin treated and control groups on 6 dpf (n = 48). (L) Quantification of sleep bout on sleep disturbance time between control and quercetin-treated groups (n = 48). Lowercase letters indicate significant differences (one-way ANOVA, Tukey’s post hoc test. Data are shown as mean ± standard deviations. p < 0.05).