Analysis of Nonclinical Safety and Corneal Pain-Inducing Potential of Amentoflavone (AMT) in Adult Zebrafish (Danio rerio).

Corneal pain affects the outer layer of the eye and can result from injuries, infections, autoimmune diseases, or dry eye syndrome. Amentoflavone (AMT), a natural biflavonoid used in traditional Chinese medicine, is known for its anti-inflammatory and neuroprotective properties. This study investigated the effects of AMT on the inhibition of corneal pain in adult zebrafish. Acute nociception was induced using formalin (cutaneous model) and hypertonic saline solution (corneal model). In a separate set of experiments, animals were pre-treated with naloxone, camphor, ruthenium red, capsazepine, L-NAME, methylene blue, ketamine, or amiloride to explore the mechanisms underlying AMT's antinociceptive effects. The results demonstrated that AMT exhibits corneal analgesic activity comparable to morphine, without causing sedation or motor impairment in zebrafish. AMT modulated antinociceptive responses through the TRPV1, ASIC, opioid, and nitrergic systems. In the in silico analysis, AMT showed lower binding energies compared to the antagonists naloxone and L-NAME, suggesting greater stability and potential efficacy. These findings support the potential of AMT as a candidate for the development of new treatments for corneal pain.