A methanolic extract of Zanthoxylum bungeanum modulates secondary metabolism regulator genes in Aspergillus flavus and shuts down aflatoxin production

Abstract

<p> Aflatoxin B1 (AFB1) is a food-borne toxin produced by <em>Aspergillus flavus</em> and a few similar fungi. Natural anti-aflatoxigenic compounds are used as alternatives to chemical fungicides to prevent AFB1 accumulation. We found that a methanolic extract of the food additive <em>Zanthoxylum bungeanum</em> shuts down AFB1 production in <em>A. flavus</em>. A methanol sub-fraction (M20) showed the highest total phenolic/flavonoid content and the most potent antioxidant activity. Mass spectrometry analyses identified four flavonoids in M20: quercetin, epicatechin, kaempferol-3-O-rhamnoside, and hyperoside. The anti-aflatoxigenic potency of M20 (IC₅₀: 2–4 µg/mL) was significantly higher than its anti-proliferation potency (IC₅₀: 1800–1900 µg/mL). RNA-seq data indicated that M20 triggers significant transcriptional changes in 18 of 56 secondary metabolite pathways in <em>A. flavus</em>, including repression of the AFB1 biosynthesis pathway. Expression of <em>aflR</em>, the specific activator of the AFB1 pathway, was not changed by M20 treatment, suggesting that repression of the pathway is mediated by global regulators. Consistent with this, the Velvet complex, a prominent regulator of secondary metabolism and fungal development, was downregulated. Decreased expression of the conidial development regulators <em>brlA</em> and Medusa, genes that orchestrate redox responses, and GPCR/oxylipin-based signal transduction further suggests a broad cellular response to M20. <em>Z. bungeanum</em> extracts may facilitate the development of safe AFB1 control strategies. <br></p>