Abstract
Background:
Testosterone is a vital androgen hormone responsible for sex differentiation, producing male sex characteristics, spermatogenesis, and fertility. Abnormalities in testosterone levels, leading to reduced weight gain and reproductive capacity, pose a significant problem for livestock farmers. Although synthetic testosterone injection is a general strategy to restore hormone levels, it leads to a downregulation of testosterone production in livestock. Therefore, an alternative method that does not affect the safety of livestock products is needed. Studies showed that the abnormal conversion process from testosterone to estradiol (aromatization) causes high estradiol in male animals. Here, we analyzed the aromatase inhibitory activity of I3C extracted from the stem part of broccoli, which has beneficial effects on the reproductive system by unknown molecular mechanisms.

Aim:
This study explores the potential of I3C as an aromatase blocker using in silico and in vivo approaches.

Methods:
Biological activity was predicted using PASS Online and toxicity was assessed via Protox-3. Drug-likeness was determined through Lipinski's Rule of 5 using SWISS ADME. Molecular docking simulations in PyRx 0.8 analyzed binding interactions with CYP19, CYP1A1, and CYP1B1, with LigPlot used for amino acid interactions and PyMOL for visualization. Molecular dynamics simulations assessed binding stability over time. Broccoli was freeze-dried, blended, and analyzed for I3C via Thin Layer Chromatography. Male Sprague Dawley rats were used to measure CYP19 levels, and blood samples were analyzed using the ELISA method.

Results:
I3C meets the criteria as a drug candidate, binding to CYP19, CYP1A1, and CYP1B1 at the active site with similar poses and good affinities of -5.8, -7.8, and -8.2, respectively. However, the binding affinity was not as strong as the control (Alpha-Naphthoflavone), and the formed bonds were unstable. Consistent with the in vivo results, the CYP19 concentration in the letrozole and broccoli powder treatment was not significantly different.

Conclusion:
I3C can influence CYP19, suggesting its potential as an aromatase-blocking agent. However, further research is required to gain a more comprehensive understanding of its effects.

Key words: Aromatase blocker, Broccoli, Estradiol, Indole-3-carbinol, Testosterone