The genus Ainsliaea, belonging to the family Asteraceae, is primarily distributed in Southeast Asia. To date, 70 species of this genus have been identified worldwide with 44 species found in China.^[1] Phytochemical studies have revealed that sesquiterpenoids are characteristic chemical constituents of Ainsliaea species. Numerous chemical compounds, such as triterpenoids, sesquiterpenoids, flavonoids, and phenylpropanoids, have been isolated from Ainsliaea species.^[2,3] Among these, sesquiterpenoids are considered the main active components, exhibiting significant antitumor and anti-inflammatory activities.^[4-6] Guaianolide sesquiterpenoids, as important secondary metabolites of natural products, have attracted considerable interest from researchers in natural product and medicinal chemistry due to their fascinating structures and diverse biological activities.^[7-10]

Eight compounds were isolated from Ainsliaea glabra. Among them, one is a previously undescribed guaianolide sesquiterpenoid (1), and seven are known compounds 2~8 (Figure 1). The known compounds were identified as eleganin (2),^[11] isolipidiol (3),^[12] α-cyclocostunolide (4),^[13] taraxinic acid (5),^[14] ainsliaea acid B (6),^[15] taraxinic acid β-D-glucopyranosyl ester (7),^[16] and germacra-7aH-1(10) E,4-Z,11(13)-trien-12,8a-olide-15-oic acid (15→1)-b-D- glu-cop-yranosyl ester (8).^[17]

Ainsglaolide A (1) was obtained as white amorphous powder, and gave an elemental formula C₁₅H₁₈O₄ from the (＋)-HRESIMS ion at m/z 263.1274 [M＋H]^＋, requiring 7 unsaturations. The IR spectrum revealed the existence of carbonyl (1770, 1725 cm^－1). The interpretation of the 1D NMR date (Table 1) indicated distinctive signals for one terminal vinyl group (δ_C 139.0, 120.6), and two carbonyl groups (δ_C 173.0, 170.0). The rest of resonances were determined by the ¹³C NMR and distortionless enhancement by polarization transfer (DEPT) spectra as being one methyl, four methylenes, five methines (including one oxygenated), and one quaternary carbon. Based on the ¹H-¹H COSY correlations (Figure 2), one structural fragment of H-5/H-6/H-7/H₂-8/H₂-9/H-10/H-1/H₂-2/H₂-3 could be derived. Subsequently, the five-membered ring and the seven-membered ring were fused together at C-1 and C-5 by the HMBC correlations (Figure 2) from H₃-14 (δ_H 1.49) to C-3 (δ_C 37.7), C-4 (δ_C 90.0) and C-5 (δ_C 50.8). Compound 1 was a guaianolide sesquiterpene, as evidenced by HMBC correlations from H₂-13 (δ_H 6.21, 5.50) to C-7 (δ_C 44.8), C-11 (δ_C 139.0) and C-12 (δ_C 170.0), and the chemical shift of C-6 (δ_C 81.0). In the previous structural elucidation, compound 1 was found to possess two carbonyl groups, one double bond, and three cyclic systems. Based on degree of unsaturation analysis, it was deduced that the compound must contain an additional ring system. The characteristic chemical shift of C-4 at δ_C 90 indicates an oxidized quaternary carbon. The HMBC correlations observed between H-10 (δ_H 2.67) and C-4 (δ_C 90.0)/C-15 (δ_C 173.0) establish the formation of a six-membered lactone ring through ether linkage between C-4 and C-15. Notably, this configuration represents the sole chemically viable position for con-structing such a lactone ring. The structural assignment was further corroborated by excellent agreement between experimental mass spectrometric data and computational simulations. Consequently, the planar structure of 1 could be determined.

The NOE cross-peaks (Figure 2) of H-5/H₃-14/H-1/H- 10/H-7 indicated these protons to be α-oriented and the six-membered lactone ring is β-oriented. Additionally, by contrasting theoretical and experimental electrostatic circular dichroism (ECD) curves (Figure 3), its absolute configuration (1R,4S,5S,6S,7S,10S) was verified. As a result, the relative configuration of 1 was established as shown (Figure 1).

To assess the anti-inflammatory potential of the comp-ounds, we employed J774A.1 cells and employed lipopolysaccharide (LPS) and Nigericin as agonists to activate the NLRP3 inflammasome. MCC950, also recognized as CRID3, a potent cytokine release inhibitor and NLRP3 inflammasome antagonist, served as our positive control. Notably, compound 1 demonstrated a substantial reduction in lactate dehydrogenase (LDH) release in a dose-dependent manner (Figure 4A). Specifically, the IC₅₀ values for com-pounds 1 and 3 were determined to be (1.86±0.2) and (2.90±0.3) μmol/L, respectively (Figure 4B). Additionally, compound 1 exhibited dose-dependent inhibition of LDH release, further confirming its inhibitory effect (Figure 4C). Further, compound 1 inhibited IL-1β level in a dose-de- pendent manner, ranging from 1.25 μmol/L to 10 μmol/L (Figure 4D).

Eight sesquiterpenes were isolated from Ainsliaea glabra. Among them, a new guaianolide sesquiterpenoid, named ainsglaolide A (1), and seven known sesquiterpenoids 2~8 were identified. Compound 1 was a novel guaianolide sesquiterpenoid featuring a rare six-membered lactone ring formed between C-4 and C-15. Subsequent anti-inflammatory activity assessments of the isolated compounds revealed that compound 1 exhibited a potent inhibitory effect on NLRP3 inflammasome activation with an IC₅₀ value of 1.86 μmol/L. Our results demonstrated that compound 1 dose-dependently suppressed LPS and nigericin-induced NLRP3 inflammasome activation in J774A.1 cells. This finding underscores compound 1 as a promising candidate for the treatment of diseases associated with hyperinflammatory responses. This compound provides a new solution for the treatment of diseases induced by excessive inflammatory response.