Potential of Indole-3-Carbinol compounds from broccoli (Brassica oleracea var. italica) as natural aromatase blockers: In silico prediction and in vivo studies

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 the downregulation of testosterone production in livestock. Therefore, an alternative method that does not affect the safety of livestock products is needed. Studies have shown that abnormal conversion from testosterone to estradiol (aromatization) causes high levels of estradiol in male animals. In this study, we analyzed the aromatase blocker activity of I3C extracted from the stem part of broccoli, which has beneficial effects on the reproductive system via unknown molecular mechanisms. Aim: This study aimed to explore 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 using Protox-3. Druglikeness was determined using Lipinski’s Rule of Five using SWISS ADME. Molecular docking simulations in PyRx 0.8 analyzed binding interactions with CYP19, CYP1A1, and CYP1B1, using LigPlot for amino acid interactions and PyMOL for visualization. Molecular dynamics simulations were used to assess the binding stability over time. Broccoli was freeze-dried, blended, and analyzed for I3C using thin-layer chromatography. Male Sprague-Dawley rats were used to measure CYP19 levels, and blood samples were analyzed using the ELISA method. Results: I3C met the criteria as a drug candidate, binding to CYP19, CYP1A1, and CYP1B1 at the active site with similar poses and good affinity of -5.8, -7.8, and -8.2, respectively. However, the binding affinity was not as strong as that of the control (Alpha-Naphthoflavone), and the formed bonds were unstable. Consistent with the in vivo results, the CYP19 concentrations in the letrozole and broccoli powder groups were not significantly different. Conclusion: I3C can influence CYP19 expression, suggesting its potential as an aromatase-blocking agent. However, further research is required to gain a comprehensive understanding of its effects.