## 4 Discussion  
(-)-Spirobrassinin is a unique secondary metabolite found exclusively in plants belonging to the Brassicaceae family. It has been investigated for its potential medical applications in cancer treatment, as it has demonstrated inhibitory effects on cancer cell proliferation [31]. Brassicaceae plants are known to be rich sources of natural products and have the potential to provide natural herbicides [32]. Certain isothiances derived from Brassicaceae plants have been shown to inhibit the germination and growth of other plants [33]. In general, allelochemicals exert their toxic effects on plants by targeting processes such as photosynthesis, respiration, and amino acid biosynthesis in receptor plants [34]. However, the specific mode of action and binding sites of (-)-Spirobrassinin have not been reported thus far. In this study, we assessed the binding energies of (-)-Spirobrassinin to the receptors, and these energies were found to be below \(-5\) kcal/mol. To validate these findings, verification experiments were conducted using _L. sativa_ Linn. as the receptor plant for these target proteins. ELISA tests were performed, confirming the reliability of the molecular docking results. The increase in PPO activity may indicate a resistance or adaptive phenomenon. This is similar to weeds developing herbicide resistance, which is a plant's adaptive ability to specific chemicals, resulting in previously effective herbicides no longer being effective [35; 36]. Moreover, the balance of phytohormones in plants was disrupted, as indicated by the accumulation of abscisic acid and gibberellins. These disruptions resulted in phenotypic changes, such as root shortening [37]. The abnormal increase in the content of abscisic acid and gibberellins may be attributed to the interference of (-)-Spirobrassinin as exogenous compounds, which disrupt the normal binding of abscisic acid and gibberellins to their target proteins [38]. This disruption leads to an abnormal increase in the levels of abscisic acid and gibberellins. On the other hand, gibberellins play a vital role in regulating plant germination and flowering processes, promoting bud elongation, and facilitating the transition to flowering [39]. The increase in gibberellin content may also be a self-regulatory response of plants to cope with external compound stress [40]. These findings provide evidence that supports the credibility of the molecular docking results, indicating their reliability. Traditional methods of searching for active sites in small molecules can be time-consuming and resource intensive. However, by employing molecular docking and computer-based calculations, we can predict and validate binding sites more efficiently. This approach significantly reduces the reliance on natural product resources and minimizes the time and cost involved, thereby overcoming challenges associated with the synthesis and low yield of natural products. We need to acknowledge the limitations of this method, as it is restricted to proteins with known crystal structures. However, Fpocket successfully identified the binding pocket of \(\alpha\)-amylase for acarbose [8]. In the case of (-)-Spirobrassinin, further studies on its mode of action using innovative methods can expedite its development and application. In the final part of our study, we investigated the potential of (-)-Spirobrassinin as a photosynthetic inhibitor-type herbicide. Due to the significant time and resource requirements for synthesis and experimental testing, we focused on computer simulations and growth inhibition assays using _C. vulgaris_. We performed a docking of (-)-Spirobrassinin to the active site of PSBD1 using the highest-resolution protein structure available, specifically the protein structure of _Thermostichus vulcanus_ photosystem II with properly positioned quinones [41]. Throughout the 50 ns simulation, the RMSD values remained around 1.5 A, indicating a stable and favorable binding of (-)-Spirobrassinin as a potential molecule for inhibiting photosystem II.