Identification and quantification of sesquiterpenes, alkaloids, flavonoids and coumarins in Artemisia annua L: Potential artemisinin activity-enhancing compounds.

Artemisia annua L. (qinghao) is renowned for its antimalarial properties, primarily attributed to artemisinin. However, the compounds that can enhance the antimalarial activity of artemisinin have not been fully explored. Here, we performed untargeted metabolomics using UHPLC-QTOF-MS/MS to characterize 103 chemical constituents in A. annua, including 31 sesquiterpenes (11 artemisinin-type compounds first discovered in QH), 24 alkaloids (21 first discovered in QH), 36 flavonoids, 8 coumarins, and 4 other types of compounds. Quantitative analysis revealed dynamic seasonal variations in these metabolites, with artemisinin content significantly higher in May and June than April. Intriguingly, April-collected samples exhibited superior antimalarial efficacy, suggesting the presence of active ingredients that enhance the efficacy of artemisinin. Heatmap and bioactivity correlation analyses identified seven candidate efficacy-enhancing active ingredients (compounds 3, 15, 18, 69, 87, 90 and 100) that were significantly enriched in April samples, and verified the potent enhance effect of compounds 69 and 90 with artemisinin. These findings validate sesquiterpenes, alkaloids, flavonoid and coumarins co-occurrence may enhance artemisinin's antimalarial activity. Our study establishes a metabolic foundation for optimizing A. annua harvesting and developing novel antimalarial formulations.