A novel sesquiterpene lactone fraction from Eupatorium chinense L. suppresses hepatocellular carcinoma growth by triggering ferritinophagy and mitochondrial damage.

BACKGROUND: Hepatocellular carcinoma (HCC) is an aggressive tumor with limited treatment options, and it is the third leading cause of cancer-related deaths. Hence, novel therapeutic strategies are required to treat HCC. Eupatorium chinense L. is a traditional Chinese medicine (TCM) that can effectively neutralize heat and smoothen the flow of "Qi" through the liver. However, the anti-HCC effects of Eupatorium chinense L. remain unknown. PURPOSE: The present study investigated the anti-HCC effects and the underlying mechanisms of the electrophilic sesquiterpenes isolated from E. chinense L. (EChLESs) in the regulation of ferroptosis and apoptosis in HCC cells. STUDY DESIGN/METHODS: Cell viability was assessed by the MTT assay. Cell apoptosis was confirmed by flow cytometry and western blotting assay. Ferroptosis was assessed by flow cytometry, transmission electron microscopy, and western blotting assay. Ferritinophagy was detected by acridine orange staining and western blotting assay. Small interfering RNA of nuclear receptor coactivator 4 (NCOA4) was used to confirm the role of ferritinophagy in the therapeutic effect of EChLESs on HCC cells. A mouse xenograft model was constructed to determine the inhibitory effect of EChLESs on HCC in vivo. RESULTS: EChLESs induced apoptosis by disrupting mitochondrial membrane potential depolarization and mitochondrial reactive oxygen species. EChLESs induced ferroptosis as noted by a significant increase in mitochondrial disruption, lipid peroxidation, and intracellular iron level and decreased glutathione level. The apoptosis inhibitor Z-VAD-FMK and lipid reactive oxygen species scavenger ferrostatin 1 attenuated EChLESs-induced cell death. NCOA4-mediated ferritinophagy through autophagic flux was the crucial pathway for ferroptosis induced by EChLESs. NCOA4 knockdown alleviated EChLESs-induced cell death. EChLESs controlled the expression of NCOA4 at the transcriptional and post-transcriptional levels. In the in vivo experiment, EChLESs suppressed HCC growth in the xenograft tumor mouse model. CONCLUSION: EChLESs enhances cell apoptosis through mitochondrial dysfunction and ferroptosis through NCOA4-mediated ferritinophagy. Thus, Eupatorium chinense L. could be a potential TCM for treating HCC.

Alkyl-benzofuran dimers from Eupatorium chinense with insulin-sensitizing and anti-inflammatory activities.

Five new racemic alkyl-benzofuran dimers, (±)-dieupachinins I-M (1-5), were isolated from the root tubers of Eupatorium chinense, a well-known traditional Chinese medicine for the treatment of diphtheria in Guangdong province. The structures of these compounds, especially the first examples of 12,10'-epoxy dimer dieupachinin I (1), 12-nor-dimer dieupachinin J (2), and 12,12'-dinor-dimer dieupachinin K (3), were elucidated by spectroscopic data analysis. Chiral resolution were further carried out on a cellulose column by HPLC, and compounds 2-5 were successfully separated into two enantiomers, respectively. The absolute configurations of (+)-(2-5) and (-)-(2-5) were established by theoretical ECD calculation. All the compounds were evaluated for insulin-stimulated glucose uptake in C2C12 myotubes and (±)-dieupachinin I (1) exhibited the best activity. Compound 1 enhanced insulin-stimulated glucose uptake via activating the insulin receptor substrate 1/protein kinase B/glycogen synthase kinase-3ß signaling pathway. Moreover, all the isolates were tested for their nitric oxygen (NO) inhibitory effects in lipopolysaccharide-treated RAW264.7 macrophages, and compounds (±)-1, (±)-2, and (±)-4 showed promising inhibitory effects with IC50 values of 6.42 ± 1.85, 6.29 ± 1.94, and 16.03 ± 2.07 µM, respectively. (±)-Dieupachinin I (1) again dose-dependently suppressed LPS-induced expression of inducible NO synthase and nuclear translocation of p65.

Potentially toxic pyrrolizidine alkaloids in Eupatorium perfoliatum and three related species. Implications for herbal use as boneset.

INTRODUCTION: Pro-toxic dehydropyrrolizidine alkaloids are associated with liver disease in humans. The potential for long-term, low-level or intermittent exposures to cause or contribute to chronically-developing diseases is of international concern. Eupatorium perfoliatum is a medicinal herb referred to as boneset. While the presence of dehydropyrrolizidine alkaloids in some Eupatorium species is well-established, reports on Eupatorium perfoliatum are scant and contradictory. OBJECTIVE: To investigate the presence of dehydropyrrolizidine alkaloids in a survey of boneset samples and related alcoholic tinctures, and hot water infusions and decoctions. METHODS: Methanol, hot water or aqueous ethanol extracts of Eupatorium perfoliatum and three closely-related species were subjected to HPLC-ESI(+)MS and MS/MS analysis using three complementary column methods. Dehydropyrrolizidine alkaloids were identified from their MS data and comparison with standards. RESULTS: Forty-nine samples of Eupatorium perfoliatum were shown to contain dehydropyrrolizidine alkaloids (0.0002-0.07% w/w), the majority dominated by lycopsamine and intermedine, their N-oxides and acetylated derivatives. Alcoholic tinctures and hot water infusions and decoctions had high concentrations of the alkaloids. Different chemotypes, hybridisation or contamination of some Eupatorium perfoliatum samples with related species were suggested by the co-presence of retronecine- and heliotridine-based alkaloids. CONCLUSIONS: Sampling issues, low and high alkaloid chemotypes of Eupatorium perfoliatum or interspecies hybridization could cause the wide variation in dehydropyrrolizidine alkaloid concentrations or the different profiles observed. Concerns associated with dehydropyrrolizidine alkaloids provide a compelling reason for preclusive caution until further research can better define the toxicity and carcinogenicity of the dehydropyrrolizidine alkaloid content of Eupatorium perfoliatum. [Correction added on 12 July 2018, after first online publication: The 'Conclusions' section in the abstract has been added.].