Tripterygium wilfordii Hook. f. is a well-known traditional Chinese medicine used to treat autoimmune diseases. Sesquiterpene pyridine alkaloids (SPAs) are a major class of components found in this herb that have piqued the interest of researchers due to their complex and diverse structures as well as significant biological activities. In this study, ten new SPAs, wilfordatine A–J (1–10), were isolated from the roots of T. wilfordii, along with ten known analogues (11–20). Their structures were primarily elucidated by extensive 1D and 2D NMR spectroscopic analysis. To search for more immunosuppressive ingredients related to the clinical efficacy of T. wilfordii, the total alkaloids (TA) and compounds 4, 5, and 9–16 were tested for their inhibitory effects on nuclear factor-kappa B (NF-κB) pathway in Lipopolysaccharide (LPS) induced HEK293/NF-κB-Luc cells. Among them, TA, compounds 5, 11, and 16 showed potent immunosuppressive activity, with IC₅₀ values of 7.25 μg/mL, 8.75 μM, 0.74 μM, and 15.66 μM, respectively, and no influence on the cell viability at a concentration of 100 μg/mL (TA) or 100 μM (5, 11, and 16). Accordingly, TA, 5, 11, and 16, especially 11, were identified as promising candidates for further investigation into their potential use as immunosuppressive agents.

Sesquiterpene pyridine alkaloids (SPAs), as a main class of components in Tripterygium wilfordii Hook. f., possess a variety of bioactivities, such as immunosuppressive, insecticidal, and anti-tumor activities. SPAs can be structurally classed into four subtypes: wilfordate-, evoninate-, iso-wilfordate-, and iso-evoninate types. Our previous study unveiled ten new wilfordate-type SPAs, named wilfordatine A–J, isolated from the roots of Tripterygium wilfordii Hook. f., several of which exhibited significant immunosuppressive activities. As an extension and augmentation of the previous findings, we have now isolated one new iso-wilfordate-type SPA, wilfordatine K (1), alongside three new iso-evoninate-type SPAs, wilfordatines L–N (3–5), and six known analogs. Their structures were characterized by the extensive use of 1D and 2D NMR spectroscopic analysis, as well as HRMS data. Interestingly, compounds 4 and 6 were found to exhibit potent inhibitory effects on the nuclear factor-kappa B (NF-κB) pathway in lipopolysaccharide (LPS)-induced HEK293/NF-κB-Luc cells, with IC₅₀ values of 1.64 μM and 9.05 μM, respectively. Notably, these two compounds had no influence on the cell viability at a concentration of 100 μM. Consequently, they hold significant promise as potential anti-inflammatory candidates for further exploration and development.

Characterising chemical and biological differences among different tissues of medicinal plants is important for understanding their tissue‐specific characteristics and medicinal efficacy. The root xylem (RX) and root bark (RB) of Tripterygium wilfordii have historically been used differently; however, the basis underlying their differential utilisation remains insufficiently characterised. In this study, complementary analytical measurements were integrated to compare the chemical and biological differences between the two root tissues across multiple dimensions. Ion fingerprinting revealed substantial overlap in the detected chemical features, with 81.09% of the ion features in the RX also detectable in the RB. Despite this high proportion of shared constituents, multivariate statistical analysis clearly separated the RX and RB, indicating significant differences in their overall ion abundance patterns. Mass spectrometry imaging further resolved tissue‐specific distribution patterns, showing that alkaloids were mainly localised in the phloem, triterpenoids were enriched in the periderm, whereas diterpenoids exhibited broader distributions across the root tissues. Targeted quantitative analysis provided direct evidence for the differential abundance of metabolites, showing that multiple bioactive constituents, including triptolide, triptonide and celastrol, were present at considerably higher levels in the RB than in the RX. Biological evaluation further demonstrated that, compared with the RX, the RB exhibited stronger inhibition of lipopolysaccharide‐induced nitric oxide production, albeit with higher cytotoxicity. Taken together, the complementary measurements confirmed that, although the RX and RB share a large number of chemical features, they differ markedly in overall metabolite abundance and spatial distribution, ultimately exhibiting distinct biological responses. This study established a multidimensional analytical framework for resolving tissue‐specific chemical variation in complex medicinal plant materials and demonstrates its utility for integrating chemical profiling, spatial mapping, targeted quantification and biological evaluation.

Investigation of the roots of Tripterygium wilfordii Hook. f. resulted in the isolation of nine sesquiterpene pyridine alkaloids (SPAs), including four previously undescribed compounds 1–4. The structures of all compounds were elucidated by extensive spectroscopic analysis, including NMR and HRESIMS. In particular, compound 1 was found to possess an unprecedented cage-like ether moiety, representing the first report of such a structural feature within this class of alkaloids. All isolated compounds were evaluated for their anti-inflammatory activity using a lipopolysaccharide (LPS)-induced RAW264.7 macrophage model. Compounds 1, 2, and 4 exhibited significant inhibition of nitric oxide (NO) production, with IC₅₀ values of 7.14 ± 1.89, 8.55 ± 0.37, and 14.76 ± 0.39 μM. Furthermore, compounds 1 and 2 suppressed the secretion of key pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β, in the same cellular model. These results not only enhanced the structural diversity of SPAs identified from T. wilfordii, but also highlight their potential as promising anti-inflammatory lead compounds.