Integrative multi-omics profiling of geographical and organ-specific variations of bioactive constituents in Andrographis paniculata: UHPLC-QTOF-MS, MALDI-MSI and antioxidant activity

Andrographis paniculata , a medicinal economic value of crops, is extensively cultivated across subtropical regions of India and China, which contains ent -Labdane diterpenoid lactones ( ent -LDLs) represented by andrographolide (AD) as its core bioactive components responsible for anti-inflammatory, antimicrobial, and antioxidant activities. However, significant geographical variations in these components have led to inconsistent quality standards across regions, presenting challenges in overall quality control. This study comprehensively elucidated the distribution patterns and metabolic variations of core ent -LDLs in different geographical origins and organ tissues of A. paniculata through integrated UHPLC-QTOF-MS-GNPS analysis, MALDI-MSI, and chemometrics/machine learning approaches. Eight key differential ent -LDLs dominated by andrographolide (AD) and its derivatives were identified across geographical regions, with isoandrographolide (IAD, 14-hydroxyl epimer of andrographolide) being incorporated for the first time. Notably, we firstly discovered that the root tissue predominantly accumulates IAD rather than AD as its major ent -LDLs, preliminarily revealing tissue-specific metabolic accumulation patterns of bioactive ent -LDLs. Meanwhile, qRT-PCR analysis also confirmed the differential expression of ent -LDLs -associated genes ( CPS2 , GGPS , CPR2 , CPR4 , CYP71 and DXS1 ) in the roots, stems, and leaves. Furthermore, in vitro intervention studies using a pneumonia model demonstrated that these differential ent -LDLs alleviate LPS-induced pulmonary oxidative stress by suppressing ferroptosis in HPMEC cells through the Keap1-Nrf2/HO-1 pathway. These findings provide scientific evidence for quality improvement of A. paniculata and its potential high-value applications in pharmaceutical sectors for pneumonia prevention. • First quantitative revelation of seven critical differential ent -LDLs in A. paniculata using LC-MS. • Root-specific accumulation of ent -LDLs, particularly IAD, was first discovered in A. paniculata. • MALDI-MSI innovatively visualized spatial distribution of core constituents in A. paniculata. • AD prevents pulmonary inflammation by suppressing ferroptosis via Keap1-Nrf2/HO-1 pathway.