Graphical abstract

A novel functional drink with nutraceutical properties was formulated from the aqueous extracts of Ilex guayusa, and Vernonanthura patens leaves, and cocoa husks. This combination presents bioactive compounds, such as phenolic compounds, with antioxidant properties of pharmacological interest. However, it is known that the interaction of herbal extracts compounds may cause adverse toxic effects on human health. To evaluate this functional drink's innocuity, we estimated the acute oral toxicity in experimental mice. The toxicological study was carried out following the FASEB Statement of Principles for the Use of Animals in Research and Education. Female albino mice were treated with a single oral dose (2000 mg/kg of body weight) of the aqueous extract of the functional drink's two formulations. In addition, changes in behavior, body weight, food and water consumption, and histopathological studies were evaluated. No signs of adverse toxicity and mortality were observed after a single oral dose of 2000 mg/kg b.w. Likewise, there were no significant body and organ weight changes, food and water consumption behavior, and no histopathological changes were observed in the main organs evaluated. In conclusion, this functional drink can be categorized as “non‐toxic,” according to the Globally Harmonized Classification System (GHS), making it a potent antioxidant beverage with high nutritional value.

Liver diseases are considered a severe public health problem involving disorders that alter liver function. Since the liver plays an essential role in the metabolism and detoxification of substances that enter the organism, it is susceptible to damage caused by xenobiotic agents such as viruses, alcohol, and drugs. Acetaminophen is an over‐the‐counter drug widely used as an antipyretic and analgesic. It is safe at therapeutic doses, but it is easily overused, causing severe liver diseases. In a previous study, our investigation group developed a functional drink from the aqueous extracts of Ilex guayusa, Vernonanthura patens, and cocoa husks. This combination presented various bioactive compounds with antioxidant properties that could help reduce oxidative stress in pathologic conditions. For the above mentioned, the study's objective was to evaluate the hepatoprotective effect of the beverage through an in vivo test. Male mice were treated with two formulations (pre‐formulated and microencapsulated) of the functional drink in three concentrations for seven days as a preventive oral treatment. After that, mice were induced to liver damage at a single intraperitoneal dose of acetaminophen (250 mg/kg of body weight). The evaluation was based on the liver damage biomarkers, ALT, and AST; anatomopathological studies were also evaluated. The results showed that the best formulation as an oral pre‐treatment was the pre‐formulated. The pre‐formulated beverage attenuated the injury caused by the acetaminophen by inhibiting serum ALT and AST activities and reducing liver macro and microscopic liver alterations. The successful doses were at 250 and 1000 mg/kg b.w. The microencapsulated did not present adequate liver protection at any doses evaluated because it has no statistical difference with the toxic model group. In conclusion, the present study demonstrated for the first time that the functional drink effectively prevented acetaminophen‐induced liver injury in vivo. Our data indicated that the pre‐formulated was the most effective pre‐treatment to attenuate the hepatic injury. These results suggested that this beverage possessed antioxidative activity that likely contributed to its protective effect against acetaminophen liver damage. *Corresponding Author: Orellana‐Manzano Andrea, email: akorella@espol.edu.ec