Rooibos (Aspalathus linearis) is a legume originally from Africa. Rooibos can come “unfermented” (green rooibos) or “fermented” (rooibos). The latter is obtained through an oxidation process controlling temperature and time. Apart from their different organoleptic properties, “fermented” and “unfermented” products also present different chromatographic profiles, with the dihydrochalcone aspalathin being the major antioxidant affected after processing.

In the present study, a commercial green rooibos powdered extract standardized to dihydrochalcones (aspalathin, nothofagin) and flavones (orientin, isorientin, vitexin…), together with the corresponding volatile fingerprint (safranal, damascenone…), was evaluated in a zebrafish model. The experimental groups were “control” (fed with commercial standard feed) and “Treatment” (fed with the standard feed including the green rooibos extract) groups. The protocol was performed twice, at 2 and 4 months post the start of ingestion, as follows: Each animal was individually placed at the bottom of a quadrangular glass aquarium (20x8x18 cm; length x width x depth; swimming volume: 3.5 L). The fish were filmed (1920 x 1080 px) for 6 min. The 1st minute was considered the time required to acclimate to the new environment, and the behavior analysis was considered for the remaining 5 min. Individual swimming activity was analyzed using Noldus Ethovision® tracking software, generating a virtual grid dividing the tank into two areas (upper and lower).

A standard diet including 0.05% green rooibos extract may modulate the behavior of zebrafish after 4 months of its continuous ingestion. This change in swimming patterns is associated with a state of lower anxiety in the zebrafish model. These results are supported by the trend towards lower latency to the first entry at the top of the tank.