Artemongolins A-K, undescribed germacrane-guaiane sesquiterpenoid dimers from Artemisia mongolica and their antihepatoma activities.

Artemongolins A-K (1-11), which are undescribed sesquiterpenoid dimers, were obtained from Artemisia mongolica and characterized through comprehensive spectral data, including HRESIMS, IR, 1D and 2D NMR, and ECD calculations. The absolute configurations of compounds 1, 4, and 7 were undoubtedly determined by a single-crystal X-ray crystallography. Artemongolins A-K (1-11) featured a rare 5/7/5/5/5/10 hexacyclic system composed of a germacrene and a guaianolide by a fused 2-oxaspiro[4,4]nonane-1-one ring system. Antihepatoma evaluation against three human hepatoma cell lines demonstrated that the most active compounds 5 and 6 displayed inhibitory activity with IC50 values of 88.6 and 57.0 (HepG2), 59.1 and 26.4 (Huh7), and 67.5 and 32.5 (SK-Hep-1) µM, respectively.

Highly oxygenated germacrane-type sesquiterpenoids from the whole plant of Salvia cavaleriei H.Lév. and their biological activities.

The entire plant Salvia cavaleriei H.Lév. (Lamiaceae) is used as a traditional Chinese herbal medicine. Its leaves are edible, and the flowers can be soaked in water to make a health-care tea. In an effort to find natural bioactive chemical components, twelve undescribed germacrane-type sesquiterpenoids, salcavalins A-L, were isolated from the whole plant of S. cavaleriei and were identified as analogs. This is the first study to isolate highly oxygenated germacrane-type sesquiterpenoids from this plant. The structures of these undescribed compounds were elucidated by various spectroscopic methods, and their absolute configurations were confirmed by single-crystal X-ray diffraction analysis with Cu Kα radiation and electronic circular dichroism calculations. The biological activity of these undescribed compounds on the production of tumor necrosis factor-alpha in lipopolysaccharide induced NR8383 cells was evaluated, and salcavalins I and K showed anti-inflammatory activity to some extent. Salcavalins A-C, F and L were found to be neuroprotective with antiparkinsonic potential in a nematode (Caenorhabditis elegans) model. In addition, salcavalins F and I displayed marked phytotoxic activity against radish seeds at a low concentration of 50 ppm. Our findings provide scientific justification to show that bioactive sesquiterpenoids from the edible herb have anti-inflammatory in vitro, neuroprotective and phytotoxic activities.

Deoxyelephantopin, a germacrane-type sesquiterpene lactone from Elephantopus scaber, induces mitochondrial apoptosis of hepatocarcinoma cells by targeting Hsp90α in vitro and in vivo.

Hepatocellular carcinoma has been known as the most frequent subtype of liver cancer with a high rate of spread, metastases, and recurrence, also dismal treatment effects. However, effective therapies for HCC are still required. Nowadays, natural products have been known as a valuable source for drug discovery. In this research, 44 sesquiterpene lactones isolated from the Elephantopus scaber Linn. (Asteraceae) were tested by MTT assay for the antitumor activities. Deoxyelephantopin (DET) was found to exert significant cytotoxicity on HepG2 and Hep3B cells. Moreover, we found that DET treatment markedly reduced the growth of HCC cells in a concentration-dependent manner, which was better than sorafenib. Furthermore, DET induced mitochondrial dysfunction, oxidative stress, and cellular apoptosis. Additionally, we found that DET and sorafenib synergistically induced apoptosis and mitochondrial dysfunction in HCC cells. DET combined with sorafenib was also efficacious in tumor xenograft model. Molecular docking experiments revealed that DET had a potentially high binding affinity with Hsp90α. Moreover, Drug Affinity Responsive Target Stability assay suggested that DET could directly target Hsp90α. Additionally, the expression of Hsp90α was both decreased in vitro and in vivo. Altogether, this study revealed that DET might be a promising agent for HCC therapy by targeting Hsp90α.

Carpelipines C and D, Two Anti-Inflammatory Germacranolides from the Flowers of Carpesium lipskyi Winkl. (Asteraceae).

Two new germacranolides, carpelipine C (1) and carpelipine D (2), together with four known ones (3-6), were isolated from Carpesium lipskyi Winkl. flowers, a folk Tibetan herbal medicine with antipyretic-analgesic and anti-inflammatory effects. The chemical structures of new structure were illuminated by diversified spectroscopic and X-ray crystallographic analyses. Compounds 1 and 3 dramatically suppressed the synthesis of NO and decreased pre-inflammatory protein expression of iNOS and COX-2 in LPS-induced RAW264.7 cells. Furthermore, it was revealed that NF-κB/MAPK signaling pathway were involved in the anti-inflammatory process of 1 and 3, and their effects on reducing oxidative stress by activating Nrf2/HO-1 pathway were also measured. This article indicated that the traditional use of C. lipskyi to treat inflammatory diseases has a certain rationality.

Combinative treatment of Curdione and docetaxel triggers reactive oxygen species (ROS)-mediated intrinsic apoptosis of triple-negative breast cancer cells.

ABBREVIATIONS: TNBC: triple negative breast cancer; ROS: reactive oxygen species; NAC: N-acetyl-L-cysteine; DTX: docetaxel; MAPKs: mitogen-actived protein kinases; PI3K/Akt: phosphatidylinositol 3-kinases (PI3K) /Akt; NF-Κb: the nuclear factor κB (NF-κB).

Germacranolides from Carpesium divaricatum: Some New Data on Cytotoxic and Anti-Inflammatory Activity.

Carpesium divaricatum Sieb. & Zucc., a traditional medicinal plant used as an inflammation-relieving remedy, is a rich source of terpenoids. At least 40 germacrane-type sesquiterpene lactones, representatives of four different structural groups, were isolated from the plant. Cytotoxicity against cancer cells in vitro is the most frequently described biological activity of the compounds. However, little is known about the selectivity of the cytotoxic effect. The anti-inflammatory activity of the germacranolides is also poorly documented. The objective of the present study was to assess the cytotoxic activity of selected C. divaricatum germacranolides-derivatives of 4,5,8,9-tetrahydroxy-3-oxo-germacran-6,12-olide towards cancer and normal cell lines (including cells of different p53 status). Moreover, to assess the anti-inflammatory effect of the compounds, the release of four proinflammatory cytokines/chemokines (IL-1ß, IL-8, TNF-α and CCL2) by lipopolysaccharide-stimulated human neutrophils was measured by ELISA. The investigated sesquiterpene lactones demonstrated nonselective activity towards prostate cancer (Du145 and PC3) and normal prostate epithelial cells (PNT2) as well as against melanoma cells (A375 and HTB140) and keratinocytes (HaCaT). Cytotoxic activity against osteosarcoma cells was independent of their p53 status. In sub-cytotoxic concentrations (0.5-2.5 µM) the studied compounds significantly decreased cytokine/chemokine release by lipopolysaccharide-stimulated human leukocytes.

Germacranolide sesquiterpenes from Carpesium cernuum and their anti-leukemia activity.

In this study, three new germacranolide sesquiterpenes (1-3), together with six related known analogues (4-9) were isolated from the whole plant of Carpesium cernuum. Their structures were established by a combination of extensive NMR spectroscopic analysis, HR-ESIMS data, and ECD calculations. The anti-leukemia activities of all compounds towards three cell lines (HEL, KG-1a, and K562) were evaluated in vitro. Compounds 1-3 exhibited moderate cytotoxicity with IC50 values ranging from 1.59 to 5.47 µmol·L-1. Mechanistic studies indicated that 2 induced apoptosis by decreasing anti-apoptotic protein Bcl-2 and activating the caspase family in K562 cells. These results suggest that compound 2 is a potential anti-leukemia agent.

Germacrone improves liver fibrosis by regulating the PI3K/AKT/mTOR signalling pathway.

Liver fibrosis is a primary threat to public health, owing to limited therapeutic options. Germacrone (GM) has been shown to exert various curative effects against human diseases, including liver injury. The aim of this study was to investigate the pharmacological effects of GM in the pathophysiology of hepatic fibrosis and determine its potential mechanisms of action. A liver fibrosis rat model was established via carbon tetrachloride (CCl4 ) treatment, and LX-2 cells were stimulated with TGF-ß1. The effects of GM on liver fibrosis and its relationship with the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signalling pathway were investigated. In the CCl4 fibrosis-induced rat model, GM improved histological damage, inhibited the activity of hepatic α-smooth muscle actin and improved serum alanine aminotransferase and aspartate aminotransferase levels in a dose-dependent manner. GM potently inhibited hepatic stellate cells (HSCs) growth and epithelial-mesenchymal transition (EMT) progression, as reflected by the altered expression of proliferative (Ki-67, PCNA and cleaved caspase-3) and EMT-related (E-cadherin and vimentin) proteins. In TGF-ß1-stimulated LX-2 cells, GM significantly inhibited the survival and activation of HSCs and induced cell apoptosis. GM also suppressed the migration ability and reversed the EMT process in HSCs. Following GM treatment, the phosphorylation of the PI3K, AKT and mTOR proteins was reduced in the liver of CCl4 -treated rats and TGF-ß1-stimulated LX-2 cells, indicating that GM may attenuate hepatic fibrosis via the PI3K/AKT/mTOR signalling pathway. These outcomes highlight the anti-fibrotic effects of GM and suggest that it is a potential therapeutic agent for the treatment of liver fibrosis.

Germacrone alleviates neurological deficits following traumatic brain injury by modulating neuroinflammation and oxidative stress.

BACKGROUND: Germacrone (GM) is a terpenoid compound which is reported to have anti-inflammatory and anti-oxidative effects. However, its role in treating traumatic brain injury (TBI) remains largely unknown. METHODS: Male C57BL/6 mice were divided into the following groups: control group, TBI group [controlled cortical impact (CCI) model], CCI + 5 mg/kg GM group, CCI + 10 mg/kg GM group and CCI + 20 mg/kg GM group. GM was administered via intraperitoneal injection. The neurological functions (including motor coordination, spatial learning and memory abilities) and brain edema were measured. Nissl staining was used to detect the neuronal apoptosis. Colorimetric assays and enzyme linked immunosorbent assay (ELISA) kits were used to determine the expression levels of oxidative stress markers including myeloperoxidase (MPO), malondialdehyde (MDA) and superoxide dismutase (SOD), as well as the expressions of inflammatory markers, including tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6). Additionally, protein levels of Nrf2 and p-p65 were detected by Western blot assay. RESULTS: GM significantly ameliorated motor dysfunction, spatial learning and memory deficits of the mice induced by TBI and it also reduced neuronal apoptosis and microglial activation in a dose-dependent manner. Besides, GM treatment reduced neuroinflammation and oxidative stress compared to those in the CCI group in a dose-dependent manner. Furthermore, GM up-regulated the expression of antioxidant protein Nrf2 and inhibited the expression of inflammatory response protein p-p65. CONCLUSIONS: GM is a promising drug to improve the functional recovery after TBI via repressing neuroinflammation and oxidative stress.

Germacrone Reduces Cisplatin-Induced Toxicity of Renal Proximal Tubular Cells via Inhibition of Organic Cation Transporter.

Cisplatin is a widely used chemotherapy for solid tumors; however, its benefits are limited by serious nephrotoxicity, particularly in proximal tubular cells. The present study investigated the renoprotective effect and mechanisms of germacrone, a bioactive terpenoid compound found in Curcuma species on cisplatin-induced toxicity of renal cells. Germacrone (50 and 100 µM) attenuated apoptosis of human renal proximal tubular cells, RPTEC/TERT1 following treatment with 50 µM cisplatin and for 48 h. Co-treating RPTEC/TERT1 cells with cisplatin and germacrone significantly reduced cellular platinum content compared with cisplatin treatment alone. The effect of germacrone on organic cation transporter 2 (OCT2) which is a transporter responsible for cisplatin uptake was determined. Germacrone showed an inhibitory effect on OCT2-mediated methyl-4-phenylpyridinium acetate (3H-MPP+) uptake with IC50 of 15 µM with less effect on OCT1. The germacrone's protective effect on cisplatin-induced cytotoxicity was not observed in cancer cells; cisplatin's anti-cancer activity was preserved. In conclusion, germacrone prevents cisplatin-induced toxicity in renal proximal tubular cells via inhibition OCT2 transport function and reducing cisplatin accumulation. Thus germacrone may be a good candidate agent used for reducing cisplatin-induced nephrotoxicity.

Targeted isolation of cytotoxic germacranolide sesquiterpenes from Elephantopus scaber L. using small molecule accurate recognition technology.

Small molecule accurate recognition technology (SMART) is an emerging method for the rapid structural prediction of major constituents from crude extracts and fractions. In the present study, a targeted isolation of an Elephantopus scaber extract by SMART resulted in the obtention of 15 new (1-15) and five known germacranolide sesquiterpenes (16-20). Their structures were assigned by extensively analyzing HRESIMS, NMR, X-ray crystallographic analyses, modified Mosher's method results, and quantum chemical calculate electronic circular dichroism (ECD) spectra. All germacranolide sesquiterpenes were screened to determine their inhibitory effects with two hepatoma cell lines (HepG2 and Hep3B), and compounds 14, 16, 18, 19 and 20 showed significant cytotoxic activities against the HepG2 (IC50, 3.3-9.9 µM) and Hep3B (IC50, 4.5-8.6 µM) cell lines. Further study suggested that 18 can induce the apoptosis of hepatoma cells via mitochondrial dysfunction.

Co-delivery of miR-29b and germacrone based on cyclic RGD-modified nanoparticles for liver fibrosis therapy.

Hepatic stellate cells (HSCs) were activated and secreted excessive amounts of extracellular matrix (ECM) proteins during pathogenetic progress of liver fibrosis. Germacrone (GMO) and miR-29b can play an important role in inhibiting growth of HSCs and production of type I collagen. GMO and miR-29b were co-encapsulated into nanoparticles (NPs) based on poly(ethylene glycol)-block-poly(lactide-co-glycolide) (PEG-PLGA). Then, NPs were modified with cyclic RGD peptides (cRGDfK). cRGDfK is an effective ligand to bind integrin αvß3 and increase the targeting ability for fibrotic liver. GMO- and miR-29b-loaded NPs exhibited great cytotoxicity to activated HSCs and significantly inhibited production of type I collagen. Liver fibrosis model of mice was induced by administration of carbon tetrachloride. Great targeting ability was achieved in liver fibrotic mice treated with cRGD-modified NPs. Significant ant-fibrotic effects have been presented based on hematoxylin and eosin (H&E), Masson and Sirius Red staining results of liver tissues collected from mice treated with drug-loaded NPs. All these results indicate GMO- and miR-29b-loaded cRGD-modified NPs have the potential for clinical use to treat liver fibrosis.

Cytotoxic Activity of Germacrane-Type Sesquiterpene Lactones from Dimerostemma aspilioides.

The need for effective candidates as cytotoxic drugs that at the same time challenge cancer multidrug resistance encouraged a search for these in plants of central Argentina. Bioassay-guided fractionation of the cytotoxic extract from Dimerostemma aspilioides led to the isolation of the germacranolide tomenphantin A (1), along with three new analogues (2-4). These efficiently inhibited the proliferation of the leukemia cell lines K562 and CCRF-CEM and their resistant variants, Lucena 1 and CEM/ADR5000, respectively, with IC50 values ranging from 0.40 to 7.7 µM. The structures and relative configurations of compounds 1-4 were elucidated by analysis of the spectroscopic data, in particular NMR spectroscopy. The most active among these was compound 1 (IC50 = 0.40-5.1 µM), and, therefore, this was selected as a model for a mechanistic study, which revealed that its antiproliferative effect was mediated by cell cycle arrest in the G2/M phase followed by apoptosis. The activity of compound 1 was selective, given the absence of cytotoxicity toward peripheral blood mononuclear cells. The results show the potential of these compounds, and in particular of compound 1, as leads for the development of drug candidates to fight sensitive and resistant leukemia cells.

Germacrone Inhibits Cell Proliferation and Induces Apoptosis in Human Esophageal Squamous Cell Carcinoma Cells.

Germacrone, a natural 10-membered monocyclic sesquiterpene with three double bonds and a ketone, was isolated from the roots of traditional Chinese medicine Saussurea costus (SC). The pharmacological value and intrinsic mechanism of germacrone in the treatment of esophageal squamous cell carcinoma (ESCC) are still unclear. Therefore, in this study, we further explored the internal molecular mechanism by which germacrone exerts its antiproliferation and antimigration ability against ESCC. 3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assays showed that germacrone dose-dependently inhibited the proliferation of ESCC cells. Flow cytometry analysis (FACS) and wound healing experiments on germacrone treated ESCC cells showed that germacrone could induce apoptosis and inhibit the migration of ESCC cells in a dose-dependent manner. In the study on the mechanism of action of germacrone in antiesophageal cancer, we found that germacrone increased the ratio of Bax/Bcl-2 in the cytoplasm of ESCC, resulting in the activation of Caspase-9 and Caspase-3 and decreased the expression of Grp78, thereby reducing the inhibition of Caspase-12 and Caspase-7. In addition, we found that germacrone also inhibited STAT3 phosphorylation in a dose-dependent manner. In conclusion, we determined that germacrone exerted an antiesophageal effect through intrinsic apoptotic signaling pathways and by inhibiting STAT3 activity in ESCC cells.

Anti-H1N1 viral activity of three main active ingredients from zedoary oil.

Influenza virus is one of the most widespread infectious diseases in the world. It poses a serious public health threat to humans. With the emergence of drug-resistant virus strains, antiviral drugs are urgently needed to control virus transmission and disease progression. In this study, three main active substances-curcumol, curdione and germacrone-were isolated from the traditional Chinese medicine zedoary. They inhibited the replication of influenza A (H1N1) virus in a dose-dependent manner. After treatment with these compounds, the expression of viral protein and RNA synthesis were inhibited. In vivo, these compounds also reduced H1N1-induced lung damage and the load of virus in serum as well as whole blood cells. In a proteomic analysis, after treatment with germacrone, the expression of antiviral protein and the amount of intracellular virus were significantly reduced, further proving that germacrone can inhibit viral replication. Our experiments have shown that curcumol, curdione and germacrone can inhibit the replication of H1N1 virus; in particular, germacrone shows potential both in vitro and in vivo as a therapeutic drug.

Macromolecular Targets of Antiparasitic Germacranolide Sesquiterpenoids: An In Silico Investigation.

BACKGROUND: The parasitic protozoal infections leishmaniasis, human African trypanosomiasis, and Chagas disease are neglected tropical diseases that pose serious health risks for much of the world's population. Current treatment options suffer from limitations, but plantderived natural products may provide economically advantageous therapeutic alternatives. Several germacranolide sesquiterpenoids have shown promising antiparasitic activities, but the mechanisms of activity have not been clearly established. OBJECTIVE: The objective is to use in silico screening of known antiparasitic germacranolides against recognized protozoal protein targets in order to provide insight into the molecular mechanisms of activity of these natural products. METHODS: Conformational analyses of the germacranolides were carried out using density functional theory, followed by molecular docking. A total of 88 Leishmania protein structures, 86 T. brucei protein structures, and 50 T. cruzi protein structures were screened against 27 antiparasitic germacranolides. RESULTS: The in-silico screening has revealed which of the protein targets of Leishmania spp., Trypanosoma brucei, and Trypanosoma cruzi are preferred by the sesquiterpenoid ligands.

Glechomanamides A-C, Germacrane Sesquiterpenoids with an Unusual Δ8-7,12-Lactam Moiety from Salvia scapiformis and Their Antiangiogenic Activity.

Three new germacrane sesquiterpenoid-type alkaloids with an unusual Δ8-7,12-lactam moiety, glechomanamides A-C (1-3), and two pairs of 7,12-hemiketal sesquiterpenoid epimers (4a/b, 5a/b) were isolated from Salvia scapiformis. Their structures were elucidated by spectroscopic methods including HRESIMS, IR, UV, and 1D and 2D NMR and also confirmed by single-crystal X-ray diffraction analysis. The chemical transformation of compounds 1-5 in a solution environment was analyzed by 2D NMR spectroscopy. The aza acetallactams (1-3) were stable in organic solvent, while single crystals of the hemiacetal esters (4a/b, 5a/b) underwent a tautomeric equilibrium after being dissolved. Single crystals of 4a, 4b, and 5a were obtained for the first time as their naturally occurring forms. Glechomanamide B (2) exhibited antiangiogenic activity by suppression of vascular endothelial growth factor (VEGF)-induced tube formation through modulation of VEGF receptor 2 (VEGFR2)-mediated signaling pathways in human umbilical vascular endothelial cells (HUVECs). In addition, compound 2 also showed the significant suppression of mRNA expression associated with glycolysis and angiogenesis biomarkers in high glucose (30 mM)-induced HUVECs. These findings suggest that compound 2 might be a potential lead compound candidate for the management of diabetic retinopathy.

Antifungal activity of zedoary turmeric oil against Phytophthora capsici through damaging cell membrane.

Phytophthora capsici is a plant oomycete pathogen, which causes many devastating diseases on a broad range of hosts. Zedoary turmeric oil (ZTO) is a kind of natural plant essential oil that has been widely used in pharmaceutical applications. However, the antifungal activity of ZTO against phytopathogens remains unknown. In this study, we found ZTO could inhibit P. capsici growth and development in vitro and in detached cucumber and Nicotiana benthamiana leaves. Besides, ZTO treatment resulted in severe damage to the cell membrane of P. capsici, leading to the leakage of intracellular contents. ZTO also induced a significant increase in relative conductivity, malondialdehyde concentration and glycerol content. Furthermore, we identified 50 volatile organic compounds from ZTO, and uncovered Curcumol, ß-elemene, curdione and curcumenol with strong inhibitory activities against mycelial growth of P. capsici. Overall, our results not only shed new light on the antifungal mechanism of ZTO, but also imply a promising alternative for the control of phytophthora blight caused by P. capsici.

Germacrane Sesquiterpenoids as a New Type of Anticardiac Fibrosis Agent Targeting Transforming Growth Factor ß Type I Receptor.

A germacrane sesquiterpenoid library containing 30 compounds (2-31) was constructed by structural modification of a major component aristolactone (1) from the traditional Chinese medicine Aristolochia yunnanensis. Compound 11 was identified as a promising anticardiac fibrosis agent by systematic screening of this library. 11 could inhibit the expression of fibronectin (FN), α-smooth muscle actin (α-SMA), and collagens in transforming growth factor ß 1 (TGFß1)-stimulated cardiac fibroblasts at a micromolar level and ameliorate myocardial fibrosis and heart function in abdominal aortic constriction (AAC) rats at 5 mg/kg dose. Mechanistic study revealed that 11 inhibited the TGFß/small mother against decapentaplegic (Smad) signaling pathway by targeting TGFß type I receptor (IC50 = 14.9 ± 1.6 nM). The structure-activity relationships (SARs) study indicated that the unsaturated γ-lactone ring and oxidation of C-1 were important to the activity. These findings may provide a new type of structural motif for future anticardiac fibrosis drug development.

Daucovirgolides I-L, four congeners of the antimalarial daucovirgolide G from Daucus virgatus.

Repeated chromatographic purifications of aerial parts of the Tunisian plant Daucus virgatus led to the isolation of four new germacranolides, named daucovirgolides I-L (2-5), along with the Plasmodium transmission-blocking agent daucovirgolide G. The chemical structures of the new compounds were defined as mono- or di-angeloylated germacrane-type sesquiterpenoids by spectroscopic (mainly 1D and 2D NMR) and spectrometric methods (ESIMS). The low potency exhibited by daucovirgolides I-L further supports the observation that strict structural requirements do exist for the Plasmodium transmission blocking activity in the daucovirgolide series. In particular, the endocyclic double bond system seems to be crucial for bioactivity.