The psychoactive effects of Bryophyllum pinnatum (Lam.) Oken leaves in young zebrafish

Abstract

Bryophyllum pinnatum (Lam.) Oken (BP) is a plant that is used worldwide to treat inflammation, infections, anxiety, restlessness, and sleep disorders. While it is known that BP leaves are rich in flavonoids, the extent of the beneficial and toxic effects of its crude extracts remains unclear. Although some neurobehavioral studies using leaf extracts have been conducted, none has examined the effects of water-extracted leaf samples. The zebrafish is a powerful animal model used to gain insights into the efficacy and toxicity profiles of this plant due to its high fecundity, external development, and ease of performing behavioral assays. In this study, we performed behavioral testing after acute exposure to different concentrations of aqueous extract from leaves of B. pinnatum (LABP) on larval zebrafish, investigating light/dark preference, thigmotaxis, and locomotor activity parameters under both normal and stressed conditions. LABP demonstrated dose-and time-dependent biphasic effects on larval behavior. Acute exposure (25 min) to 500 mg/L LABP resulted in decreased locomotor activity. Exposure to 300 mg/L LABP during the sleep cycle decreased dark avoidance and thigmotaxis while increasing swimming velocity. After sleep deprivation, the group treated with 100 mg/L LABP showed decreased dark avoidance and increased velocity. After a heating stressor, the 30 mg/L and 300 mg/L LABP-treated groups showed decreased dark avoidance. These results suggest both anxiolytic and psychoactive effects of LABP in a dose-dependent manner in a larval zebrafish model. These findings provide a better understanding of the mechanisms underlying relevant behavioral effects, consequently supporting the safe and effective use of LABP for the treatment of mood disorders.

Fig 1. LABP chemical profile characterization.

A. Overlapping of UV (ultraviolet) and EIC (extracted ions) chromatograms obtained from analysis of LABP analyzed by UPLC/DAD-ESI/HRMS/MS (electrospray ionization—positive ion mode). The blue line corresponds to the UV chromatogram. The purple, green and red lines correspond to the EIC. B. The table shows the compounds annotated following the peaks, retention times, molecular formulas, and masses.

Fig 2. Behavior effects after LABP acute exposure.

A. The schematic of the behavioral testing after LABP exposure (20, 25, or 60 min) in different concentrations (1, 3, 100, 300, or 500 mg/L). B. There was no significant difference in choice index between the control group and LABP treated groups. C. After 20 min of 3 mg/L LBP exposure decreased thigmotaxis significantly (p = 0.0082). The other groups did not show significant differences when compared to control. D. 100 and 300 mg/L LABP added 20 min before testing increased distance significantly (p = 0.0297 and p<0.0001, respectively). 500 mg/L LABP decreased the distance significantly after 25- and 60-min exposure both (p<0.0001 and p = 0.0285, respectively). E. 100 and 300 mg/L LABP added 20 min before testing increased velocity significantly (p = 0.0031 and p<0.0001, respectively). 500 mg/L LABP decreased velocity significantly after both 25- and 60-min exposure (p<0.0001 and p = 0.0254, respectively). Asterisks indicate statistical differences between groups (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). n = 24/group a-20 min; 28/group b-20 min; 28/group c-25 min; 50/group d-60 min.

Fig 3. Behavior effects after LABP exposure during the sleep cycle.

A. The schematic of the behavior test after LABP exposure during the sleep cycle. The larvae were kept on dark and LABP exposure from 10 pm-8 am. B. 300 mg/L LABP significantly increased the choice index when compared to control (p = 0.0410). C. Thigmotaxis significantly showed a reduction in the group treated with 300 mg/L LABP when compared to the control group (p = 0.0346). D. Distance was increased in 300 mg/L LABP but not significantly (p = 0.0870). E. LABP significantly increased velocity in both 100 and 300 mg/L concentrations (p = 0.0416; p<0.0001). Asterisks indicate statistical differences between groups (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). n = 30/group.

Fig 4. Behavior effects after sleep deprivation and LABP exposure.

A. The schematic of the behavior test after sleep deprivation + LABP exposure. The larvae were kept on light and LABP exposure during the sleep cycle. B. 100 mg/L LABP significantly increased the choice index when compared to control on light (p = 0.0438), while the normal control showed a significantly higher choice index when compared to control on light (p = 0.0009). C. Thigmotaxis did not show a significant difference between the groups. D. Distance was significantly decreased in control on light groups when compared to normal control (p = 0.0001). E. LABP treatment showed significantly high swimming velocity when compared to control on light (p = 0.0228), similar to normal control (p = 0.0015). Asterisks indicate statistical differences between groups (*p < 0.05, **p < 0.01, ***p < 0.001). n = 30/group.

Fig 5. Behavior effects after heat stressor and LABP exposure.

A. The schematic of the behavior test after the addition of a heat stressor + LABP exposure. B. 30 and 300 mg/L of hot LABP significantly increased the choice index when compared to hot BEW (p = 0.0473 and p = 0.0348, respectively). C. 30 mg/L of hot LABP significantly decreased the thigmotaxis when compared to hot BEW (p = 0.0314). D. There was a significant difference in distance between BEW at room temperature and hot BEW (p = 0.0334). E. There was no significant difference in velocity between the groups. Asterisks indicate statistical differences between groups (*p < 0.05). n = 30/group.