Key Takeaways & Executive Findings
- •• • Compounds 1–6 exhibited potent cytotoxicity against HepG2 cells with IC50 values ranging from 2.61 to 13.94 μmol/L, demonstrating significant anti-hepatocellular carcinoma potential. Compound 6 (japonicone X) was the most active (IC50 = 2.61 μmol/L), outperforming the positive control doxorubicin (IC50 = 0.82 μmol/L) by a factor of ~3.2, yet still within a clinically relevant range for lead optimization. • • The new compound 1 (inujaponolide T) showed an IC50 of 3.82 ± 0.21 μmol/L, while compound 3 (inujaponolide U) had an IC50 of 13.94 ± 0.28 μmol/L. The 3.6-fold difference in potency between these two new dimers highlights the impact of structural variations, particularly the 1,10-seco modification in compound 3, on cytotoxic activity. • • A preliminary structure-activity relationship analysis revealed a positive correlation between the number of acetoxy substituents and anti-tumor activity. This suggests that acetoxy groups contribute to the cytotoxic effects, providing a strategic handle for synthetic or semi-synthetic optimization to enhance potency. • • Colony formation assays confirmed that compounds 1 and 3 inhibit HepG2 cell viability and reduce colony numbers in a dose-dependent manner (P < 0.001 vs. DMSO control), indicating suppression of clonogenic survival, a key metric for evaluating long-term anti-proliferative effects and potential to prevent tumor recurrence.
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Abstract
The inflorescences of Inula japonica Thunb. (Asteraceae) are a traditional Chinese medicine used for treating cough, phlegm, and vomiting. Sesquiterpenoid dimers, formed via Diels-Alder, hetero-Diels-Alder, [2+2] cycloaddition, or radical coupling, exhibit potent anti-inflammatory, neuroprotective, and antitumor activities. However, their low natural abundance and structural complexity hinder isolation and development. This study isolated six sesquiterpenoid dimers from the ethyl acetate fraction of a 90% ethanol extract of I. japonica using multiple chromatographic techniques. Their structures were elucidated by HRESIMS, NMR, IR, UV, and calculated NMR/ECD. Compounds 1 (inujaponolide T) and 3 (inujaponolide U) are new: a eudesmane-guaiane dimer and a 1,10-seco-eudesmane-guaiane dimer, respectively. The other four were identified as inujaponolide E (2), inujaponolide D (4), inujaponolide I (5), and japonicone X (6). In vitro anti-hepatocellular carcinoma activity was evaluated against HepG2 cells using MTT and colony formation assays. All compounds exhibited potent cytotoxicity with IC50 values of 2.61–13.94 μmol/L; compound 6 was most active (IC50 = 2.61 μmol/L). A preliminary structure-activity relationship indicated a positive correlation between the number of acetoxy substituents and antitumor activity. Compounds 1 and 3 inhibited cell viability and reduced colony formation in a dose-dependent manner. These findings expand the chemical diversity of I. japonica and provide a basis for developing these dimers as anti-hepatocellular carcinoma lead compounds.
1. Introduction
Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality worldwide, with limited therapeutic options due to high recurrence rates and resistance to conventional chemotherapeutics. Existing systemic treatments, such as sorafenib and lenvatinib, offer modest survival benefits but are hampered by toxicity and acquired resistance. Natural products, particularly sesquiterpenoid dimers from Inula japonica, have emerged as a promising source of novel anti-HCC agents. These dimers, formed via Diels-Alder or radical coupling, exhibit potent cytotoxicity against various cancer cell lines, yet their low natural abundance and complex structures have impeded systematic development. The current study addresses this bottleneck by isolating and characterizing six sesquiterpenoid dimers, including two new compounds, from I. japonica, and evaluating their anti-HCC activity.
Prior efforts to develop sesquiterpenoid dimers as anticancer leads have been stalled by challenges in isolation, structural elucidation, and supply. For instance, japonicone A, a eudesmane-guaiane dimer, showed remarkable activity against multiple cancer cell lines (IC50 0.001–1.620 μg/mL) but its scarcity in planta limits preclinical evaluation. The present work employs a combination of chromatographic techniques and advanced spectroscopic methods (HRESIMS, NMR, ECD) to isolate and determine the absolute configurations of two new dimers, inujaponolide T (1) and inujaponolide U (3), alongside four known analogues. The in vitro anti-HCC activity was assessed using MTT and colony formation assays, revealing IC50 values in the low micromolar range (2.61–13.94 μmol/L) and a structure-activity relationship linking acetoxy substitution to enhanced potency. These findings provide a chemical and pharmacological foundation for further development of these dimers as anti-HCC leads.
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YU Hang, LIU Guiyou, YU Jinzheng, ZHAO Yongxue, HUANG Mingsi, JIN Haixiang, SU Xuan, LI Qianzi, CHEN Mingxuan, HU Dongbao, DONG Wei (2026). Two New Sesquiterpenoid Dimers from Inula japonica and Their In Vitro Anti-Hepatocellular Carcinoma Activity. Chinese Traditional and Herbal Drugs. https://doi.org/10.7501/j.issn.0253-2670.2026.16.20261603
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Frequently Asked Questions
What is the mechanism of action for the observed cytotoxicity of compounds 1 and 3 against HepG2 cells?
The study did not elucidate the specific molecular mechanism; however, the dose-dependent inhibition of cell viability and colony formation (P < 0.001) suggests interference with cell proliferation and survival pathways. Future work will focus on identifying cell death induction and target engagement.
How do the IC50 values of the new compounds compare to existing chemotherapeutics like doxorubicin?
Compound 1 (IC50 = 3.82 μmol/L) and compound 3 (IC50 = 13.94 μmol/L) are less potent than doxorubicin (IC50 = 0.82 μmol/L) but still exhibit strong cytotoxicity. The most active compound, 6 (IC50 = 2.61 μmol/L), is within a 3.2-fold range of doxorubicin, indicating potential for further optimization.
What are the scalability challenges for isolating these sesquiterpenoid dimers from natural sources?
The low natural abundance of sesquiterpenoid dimers in Inula japonica necessitates large-scale extraction and multiple chromatographic steps, resulting in low yields. This bottleneck could be addressed through synthetic biology or total synthesis, but cost parity with plant extraction remains unproven.
Is the observed structure-activity relationship (SAR) between acetoxy groups and potency statistically robust?
The SAR is preliminary, based on six compounds. While a positive trend exists, the sample size is small and confounded by other structural differences. Further analogues with controlled acetoxy substitution are needed to validate this correlation.
What are the potential toxicity risks of these compounds given their potent cytotoxicity?
No selectivity data against normal hepatocytes were provided. The IC50 values are in the low micromolar range, which may pose toxicity risks. Future studies should assess selectivity indices using normal liver cell lines (e.g., LO2) to evaluate therapeutic windows.
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