Phytochemical and Safety Evaluations of Finger Lime, Mountain Pepper, and Tamarind in Zebrafish Embryos

Abstract

Plants play a pivotal role in drug discovery, constituting 50% of modern pharmacopeia. Many human diseases, including age-related degenerative diseases, converge onto common cellular oxidative stress pathways. This provides an opportunity to develop broad treatments to treat a wide range of diseases in the ageing population. Here, we characterize and assess the toxicological effects of finger lime (Citrus australasica), mountain pepper (Tasmannia lanceolata), and small-leaved tamarind (Diploglottis australis) extracts. The characterization demonstrates that these Australian native plants have antioxidant potential and, importantly, they have high concentrations of distinct combinations of different antioxidant classes. Using zebrafish larvae as a high-throughput pre-clinical in vivo toxicology screening model, our experiment effectively discriminates which of these extracts (and at what exposure levels) are suitable for development towards future therapies. The LC50-96h for finger lime and tamarind were >480 mg/L, and 1.70 mg/L for mountain pepper. Critically, this work shows that adverse effects are not correlated to the properties of these antioxidants, thus highlighting the need for combining characterization and in vivo screening to identify the most promising plant extracts for further development. Thus, we present a high-throughput pre-clinical screening that robustly tests natural plant products to utilize the diversity of antioxidant compounds for drug development.

Keywords: antioxidant capacity; finger lime; mountain pepper; native Australian plants; phytochemical characterization; plant toxicity screening; tamarind; zebrafish.

Figure 1

Heatmap of the concentration of bioactive compounds (μg/g) in finger lime (FL), mountain pepper (MP), and tamarind (T).

Figure 2

Heatmap of the alterations observed in zebrafish embryos exposed during 96 h to the plant extracts. The mean of three replicates (n = 20/replicate) is reported for finger lime (A), mountain pepper (B), and tamarind (C).

Figure 3

Phenotypic assessment of zebrafish development for the whole organism exposed 96 h post-fertilization to different concentrations of finger lime (A,D), mountain pepper (B,E), and tamarind (C,F). Cumulative mortality observed at 96 h of exposure are shown in panel (A) for finger lime, (B) for mountain pepper, and (C) for tamarind. The mortality frequency observed at different experimental times of exposure is shown for finger lime (D), mountain pepper (E), and tamarind (F). The colours in (D–F) represent the age at which mortality was observed (in hours post-fertilization). Data show the mean ± SD, n = 60. Asterisks indicate statistical significance when compared to control groups: p < 0.001 (***).

Figure 4

Morbidity assessments of zebrafish development for the whole organism exposed at 96 h post-fertilization to different concentrations of finger lime (A), mountain pepper (B), and tamarind (C). In (C), bradycardia is represented for groups exposed to tamarind. Non-Cir. Tail = non-circulation (of blood) in the tail; Balance A = balance alteration. Colours in (A,B) represent the concentrations tested (mg/L). Data represent the mean ± SD, n = 60. Asterisks indicate statistical significance when comparing the exposed groups with the control groups: p < 0.05 (*) and p < 0.001 (***).

Figure 5

Developmental alterations observed in organisms exposed at 96 h post-fertilization to different concentrations of finger lime (A), mountain pepper (B), and tamarind (C) extracts. The alterations observed were hatching delay (Hatching D), pigmentation delay (Pig. D), developmental delay (Dev. D), yolk sac absorption delay (Yolk Sac AD), yolk sac edema (Yolk Sac E), and cardiac edema (Cardiac E). Bar colours represent the concentration tested in mg/L. Data represent the mean ± SD, n = 60. Asterisks indicate statistical significance when comparing the exposed groups with the control groups: p < 0.05 (*), p < 0.01(**) and p < 0.001 (***).

Figure 6

Malformations observed in zebrafish exposed at 96 h post-fertilization to different concentrations of finger lime and mountain pepper extracts. Each colour bar denotes the concentration tested in mg/L for finger lime (A) and mountain pepper (B). Malformations were mainly observed in the head, eyes, spine and tail. No statistically significant difference was observed when comparing the exposed and control groups for both finger lime and mountain pepper, and no malformations were observed for tamarind. Data represent the mean ± SD, n = 60.