(-)-Spirobrassinin, a glucosinolate compound from Brassicaceae plants, has shown inhibitory effects on weeds. However, its specific target sites are not well explored. This study used molecular docking, molecular dynamics simulations, and biological experiments to investigate (-)-Spirobrassinin’s target sites. The inhibitory effects of (-)-Spirobrassinin were observed on various enzymes crucial for plant metabolic pathways, including dihydroxyacid dehydrogenase, 4-hydroxyphenylpyruvate dioxygenase, protoporphyrinogen oxidase, and acetolactate synthase. Additionally, it disrupts the metabolism of vital phytohormones, namely abscisic acid and gibberellin. Molecular dynamics simulations revealed stable interactions between (-)-Spirobrassinin and specific residues (Phe270 and Phe261) of the photosystem II D1, involving electrostatic, hydrophobic, and van der Waals forces. This study provides valuable insights into (-)-Spirobrassinin’s mode of action, highlighting its potential as a natural herbicide from Brassicaceae plants.

Brassica oleracea L. has strong allelopathic effects on weeds. However, the allelochemicals with herbicidal activity in B. oleracea L. are still unknown. In this study, we evaluated the activity of allelochemicals isolated from Brassica oleracea L. based on the germination and growth of model plant Lactuca sativa Linn., grass weed Panicum miliaceum, and broadleaf weed Chenopodium album. Additionally, we employed molecular docking to predict the binding of brassicanate A sulfoxide to herbicide targets. The results of this study showed that eight compounds with herbicidal activity were isolated from B. oleracea L., and the predicted results indicated that brassicanate A sulfoxide was stably bound to dihydroxyacid dehydratase, hydroxymethylpyruvate dioxygenase, acetolactate synthase, PYL family proteins and transport inhibitor response 1. This research provides compound sources and a theoretical foundation for the development of natural herbicides.