Inhibitory Effect of Selected Guaianolide and Germacranolide Sesquiterpene Lactones on Nitric Oxide Production.

Trilobolide and its analogues belong to the guaianolide type of sesquiterpene lactones, which are characteristic and widely distributed within the families Asteraceae and Apiaceae. Certain guaianolides are receiving continuously increasing attention for their promising sarco-endoplasmic reticulum Ca2+-ATPase (SERCA)-inhibitory activity. However, because of their alkylation capabilities, they are generally toxic. Therefore, the search for compounds with significant immunobiological properties but with decreased cytotoxicities suitable for use in immune-based pharmacotherapy is ongoing. Therefore, we extended our previous investigation of the immunobiological effects of trilobolide to a series of structurally related guaianolides and germacranolides. To evaluate the relationship, we tested a series of selected derivatives containing α-methyl lactone or exomethylene lactone ring. For a wider comparison, we also included some of their glycosidic derivatives. We assessed the in vitro immunobiological effects of the tested compounds on nitric oxide (NO) production, cytokine secretion, and prostaglandin E2 (PGE2) release by mouse peritoneal cells, activated primarily by lipopolysaccharide (LPS), and evaluated their viability. The inhibitory effects of the apparently most active substance, 8-deoxylactucin, seem to be the most promising.

Anti-inflammatory germacrane-type sesquiterpene lactones from Vernonia sylvatica.

Nine new germacranolides, sylvaticalides A-H (1-9), and three known analogues (10-12) were isolated from the aerial part of Vernonia sylvatica. Their structures were established using comprehensive spectroscopic analysis, including high-resolution electrospray ionization mass spectroscopy (HR-ESI-MS) and 1D and 2D nuclear magnetic resonance (NMR) spectra. Their absolute configurations were determined by X-ray diffraction experiments. The anti-inflammatory activities of all isolated compounds were assessed by evaluating their inhibitory effects on the nuclear factor kappa B (NF-κB) pathway, which was activated by lipopolysaccharide (LPS)-stimulated human THP1-Dual cells, and the interferon-stimulated gene (ISG) pathway, activated by STING agonist MSA-2 in the same cell model. Compounds 1, 2 and 6 showed inhibitory effects on the NF-κB and ISG signaling pathways, with IC50 values ranging from 4.12 to 10.57 µmol·L-1.

Germacrane-type sesquiterpenes from Pilea cavaleriei Levl. subsp. cavaleriei.

The first systematic investigation of germacrane-type sesquiterpenes from Pilea cavaleriei Levl. subsp. cavaleriei was conducted. Eleven undescribed germacrane analogues named cavalinols A-K were identified. Their planar structures were determined by extensive analysis of 1D and 2D NMR spectroscopic data, and the absolute configurations were further determined by X-ray single crystal diffraction, Mosher method, and time dependent density functional theory (TDDFT) electron circular dichroism (ECD) calculation, with the aid from DFT NMR calculation and NOESY experiment. Except for the common 10-memebered ring, ten new compounds contained a p-coumaroyl sidechain connected to C-8 of the nucleus skeleton. All the isolated compounds were screened for anti-inflammatory activity in LPS stimulated RAW 264.7 cells, and compounds 5 and 6 showed moderate activity.

Germacrone, isolated from Curcuma wenyujin, inhibits melanin synthesis through the regulation of the MAPK signaling pathway.

Abnormal melanin synthesis causes hyperpigmentation disorders, such as chloasma, freckles, and melanoma, which are highly multiple and prevalent. There were few reports on the anti-melanogenic effect of Curcuma wenyujin Y.H. Chen et C. Ling, and the bioactive compound has not been elucidated as well. The study aims to investigate the anti-melanogenic effect of C. wenyujin, and identify the bioactive compound, and further explore its underlying mechanism. Our results showed that the Petroleum ether fraction extracted from C. wenyujin rhizome had a significant anti-melanogenic effect, and germacrone isolated from it was confirmed as the major bioactive compound. To our data, germacrone significantly inhibited tyrosinase (TYR) activity, reduced melanosome synthesis, reduced dendrites formation of B16F10 cells, and melanosome transport to keratinocytes. Moreover, germacrone effectively decreased the hyperpigmentation in zebrafish and the skin of guinea pigs in vivo. Western-blot analysis showed that germacrone down-regulated the expression of TYR, TRP-1, TRP-2, Rab27a, Cdc42, and MITF proteins via the activation of the MAPK signaling pathway. Taken together, germacrone is an effective bioactive compound for melanogenesis inhibition. Our studies suggest that germacrone may be considered a potential candidate for skin whitening.

Germacrone Protects against NF-κB-Mediated Inflammatory Signaling, Apoptosis, and Retinal Ganglion Cell Survival in a Rat Glaucoma Model.

Retinal ischemia-reperfusion (I/R) is a pathological phenomenon that causes cellular destruction in several ocular disorders, so there is a need for novel possible neuroprotective drugs. Researchers have reported numerous neuroprotective effects of Germacrone (GM). Therefore, this study aimed to elucidate the underlying processes of GM that may contribute to glaucoma development. 40 healthy rats underwent retinal ischemia-reperfusion (I/R) damage. The animals were divided into control, I/R-induced, GM-1d, and GM-7d. After 7 days of I/R, mice were sacrificed and retinal tissue removed. An enzyme-linked immunosorbent assay (ELISA) was used to assess retinal Malondialdehyde (MDA) and 8-OHdG levels after oxidative injury. The Fluro-Gold (FG) labelling assay counted retinal ganglion cells (RGC) before and after labelling. DNA from retinal tissue RNA was amplified. Western blotting and real-time qRT-PCR were utilised to assess Bax, Casapses-3, Bcl-2, retinal NF-kB, and COX-2 expression. Retinal cell apoptotic mediator expression was measured by a TUNEL assay. Retinal I/R damage increases ganglion cell death. Long-term GM treatment (GM-7d) reduced NF-κB activation and raised COX-2 expression, which suggests antioxidant potential. TUNEL-positive apoptotic cells were reduced in long-term GM-treated rats. In GM-treated retinas, the Bax-Bcl-2 ratio was identical to the control group and significantly different from the I/R group. GM reduces I/R-induced retinal cell damage by inhibiting RG cell death. Seven days after GM therapy, histology showed retinal tissue loss. NF-κB signaling and intrinsic mitochondrial apoptosis are possible mechanisms that may be attenuated by GM and are attributed to a retinal protective effect.

Germacrone alleviates breast cancer-associated osteolysis by inhibiting osteoclastogenesis via inhibition of MAPK/NF-κB signaling pathways.

Bone is one of the most frequent sites for metastasis in breast cancer patients. Bone metastasis significantly reduces the survival time and the life quality of breast cancer patients. Germacrone (GM) can serve humans as an anti-cancer and anti-inflammation agent, but its effect on breast cancer-induced osteolysis remains unclear. This study aims to investigate the functions and mechanisms of GM in alleviating breast cancer-induced osteolysis. The effects of GM on osteoclast differentiation, bone resorption, F-actin ring formation, and gene expression were examined in vitro. RNA-sequencing and Western Blot were conducted to explore the regulatory mechanisms of GM on osteoclastogenesis. The effects of GM on breast cancer-induced osteoclastogenesis, and breast cancer cell malignant behaviors were also evaluated. The in vivo efficacy of GM in the ovariectomy model and breast cancer bone metastasis model with micro-CT and histomorphometry. GM inhibited osteoclastogenesis, bone resorption and F-actin ring formation in vitro. Meanwhile, GM inhibited the expression of osteoclast-related genes. RNA-seq analysis and Western Blot confirmed that GM inhibited osteoclastogenesis via inhibition of MAPK/NF-κB signaling pathways. The in vivo mouse osteoporosis model further confirmed that GM inhibited osteolysis. In addition, GM suppressed the capability of proliferation, migration, and invasion and promoted the apoptosis of MDA-MB-231 cells. Furthermore, GM could inhibit MDA-MB-231 cell-induced osteoclastogenesis in vitro and alleviate breast cancer-associated osteolysis in vivo human MDA-MB-231 breast cancer bone metastasis-bearing mouse models. Our findings identify that GM can be a promising therapeutic agent for patients with breast cancer osteolytic bone metastasis.

Two Undescribed Germacrane-Type Sesquiterpenoids from Salvia cavaleriei var. simplicifolia Stib. and Their Anti-Alzheimer's Disease Activity.

Two undescribed germacrane-type sesquiterpenoids, salcasins A (1) and B (2), together with three known compounds (3-5) were isolated and identified from the whole plant of Salvia cavaleriei var. simplicifolia Stib. The structures of the undescribed compounds were elucidated on the basis of spectroscopic methods, such as HR-ESI-MS, 1D and 2D NMR data. The relative configurations of 1 and 2 were established by analyzing their NOESY spectra as well as by 13C NMR calculations with DP4+ probability analyses. The absolute configurations of 1 and 2 were determined by comparing experimental and calculated ECD spectra. Furthermore, the in vivo anti-Alzheimer's disease activities of 1-5 were evaluated using Caenorhabditis elegans AD pathological model. Among all isolated compounds, salcasin A (1) significantly delayed AD-like symptoms of worm paralysis, which may be a potential anti-AD candidate agent.

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.

Germacrane-type sesquiterpenoids from the flowers of Chrysanthemum indicum with hepatoprotective activity.

Two new germacrane-type sesquiterpenoids, chrysanthemolides A (1) and B (2), and four known germacrane-type sesquiterpenoids, hanphyllin (3), 3ß-hydroxy-11α,13-dihydro-costunolide (4), costunolide (5), and 6,7-dimethylmethylene-4-aldehyde-1ß-hydroxy-10(15)-ene-(4Z)-dicyclodecylene (6), were isolated and identified from the flowers of Chrysanthemum indicum. The structures of the new compounds were elucidated via high resolution electrospray ionization mass spectrometry (HR-ESI-MS), 1D and 2D nuclear magnetic resonance (NMR) spectra and electronic circular dichroism (ECD). Meanwhile, all the isolates were tested for their hepatoprotective activity in tert-butyl hydroperoxide (t-BHP) injured AML12 cells. Compounds 1, 2, and 4 showed significant protective effects at 40 µM, comparable with the positive control resveratrol at 10 µM. As the most potent one, compound 1 was chosen for further studies. Compound 1 dose-dependently increased the viability of t-BHP-injured AML12 cells. Furthermore, compound 1 decreased reactive oxygen species accumulation, while increased glutathione level, heme oxygenase-1 level and superoxide dismutase activity, through anchoring in the binding site of Kelch domain of the Kelch-like ECH-associated protein 1 (Keap1) to promote the dissociation of nuclear factor erythroid 2-related factor 2 from Keap1 and translocation to nuclei. In summary, germacrane-type sesquiterpenoids from C. indicum might be further developed to protect liver against oxidative damage.

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.

Germacrane-Type Sesquiterpene Lactones from Achillea millefolium L. and Their Anti-Inflammatory Activity.

Six undescribed germacrane-type sesquiterpene lactones, millefoliumons A-F, and two known analogs were isolated from the ethyl acetate fraction of the whole plant of Achillea millefolium L. growing in Xinjiang, China. The structures of these compounds were fully elucidated by their 1D and 2D nuclear magnetic resonance (NMR), and high resolution mass (HR-ESI-MS) spectral data, and comparison with literatures. The absolute configurations of millefoliumons A-F were confirmed by experimental and calculated electronic circular dichroism data (ECD), and 13 C-NMR calculations and DP4+ probability analysis. All compounds displayed the approximate tendency to inhibit the nitric oxide (NO) release in lipopolysaccharide (LPS)-induced BV2 cells.

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.

SCP-7, a germacrane-type sesquiterpene lactone derivative, induces ROS-mediated apoptosis in NSCLC cells in vitro and in vivo.

Scabertopin (SCP), an abundant germacrane-type sesquiterpene lactone (SLC) isolated from Elephantopus scaber, was selected as a reference compound for modification and evaluation as anticancer agents for non-small cell lung cancer (NSCLC) treatment. All derivatives (SCP-1-SCP-13) except for SCP-3 showed potential inhibitory effect (IC50 5.2-9.7 µM) against A549 cells. The most promising compound SCP-7 also showed good cytotoxic activity against another two NSCLC cell lines (H1299 and H460), with IC50 value of 4.4 and 8.9 µM, respectively. Furthermore, SCP-7 could induce apoptotic cell death that was associated with the increased reactive oxygen species (ROS) generation, the loss of mitochondrial membrane potential, Bcl-2 family proteins modulation, caspases-3 and PARP cleavage. In addition, SCP-7 also inhibited cell growth by increasing Bax expression and reducing the Ki-67 positive cells in vivo, but there were no obvious toxic and side effects on internal organs. Mechanistically, PharmMapper, molecular docking and Western blot analysis revealed that SCP-7 might interact with the epidermal growth factor receptor (EGFR) and inhibit its expression in lung cancer cells. Together, above results suggest further effective application of SCP-7 as a potential anti-tumor agent in the treatment of NSCLC.

Germacrone alleviates okadaic acid-induced neurotoxicity in PC12 cells via M1 muscarinic receptor-mediated Galphaq (Gq)/phospholipase C beta (PLCß)/ protein kinase C (PKC) signaling.

Alzheimer's disease (AD) is a neurodegenerative disorder with prominent individual morbidity and mortality among elderly people. Germacrone (Germ) has been reported to exert dominant protective roles in multiple human diseases, and neurological diseases are also included. The intention of this paper is to determine the impacts of Germ on okadaic acid (OA)-treated PC12 cells and confirm the hidden regulatory mechanism. First, PC12 cells were induced by OA in the absence or presence of Germ. Cell counting kit-8 assay was to monitor cell proliferation. Western blot was to test the protein levels of cholinergic muscarinic M1 receptor (CHRM1), Galphaq (Gq), phospholipase C beta (PLCß) and protein kinase C (PKC). The levels of reactive oxygen species (ROS) and other oxidative stress markers were evaluated using corresponding kits. ELISA was used to estimate the levels of AD markers. RT-qPCR was used to examine the mRNA levels of beta-site amyloid-precursor-protein-cleaving enzyme 1 (BACE-1) and apolipoprotein E (APOE). The results uncovered that Germ enhanced the proliferation of OA-insulted PC12 cells, elevated the protein level of CHRM1 and activated the Gq/PLCß/PKC signaling. Moreover, after OA-induced PC12 cells were administered with Germ, insufficiency of CHRM1 impeded cell proliferation, enhanced oxidative stress and neuron injury and inactivated the Gq/PLCß/PKC signaling. Furthermore, the addition of Gq inhibitor UBO-QIC, PLCß inhibitor U73122 or PKC inhibitor Go6983 reversed the enhanced proliferation, the reduced oxidative stress and neuron injury in OA-treated PC12 cells caused by Germ. Collectively, Germ modulated M1 muscarinic receptor-mediated Gq/PLCß/PKC signaling, thereby alleviating OA-induced PC12 cell injury.

Semisynthesis and Non-Small-Cell Lung Cancer Cytotoxicity Evaluation of Germacrane-Type Sesquiterpene Lactones from Elephantopus scaber.

Two series of germacrane-type sesquiterpene lactones were produced by semisynthetic modulation of scaberol C, which was prepared by a standard chemical transformation from an Elephantopus scaber extract. Their inhibition activities against non-small-cell lung cancer cells were screened, and preliminary structure-activity relationships were also established. Among them, monomeric analog 1u and dimeric analog 3d exhibited superior anti-non-small-cell lung cancer cytotoxic potencies with IC50 values of 4.3 and 0.7 µM against A549 cells, respectively, and were more active than cisplatin and the standard sesquiterpene lactones, parthenolide and scabertopin. Further studies revealed that compounds 1u and 3d cause G2/M phase arrest and induce apoptosis through the activation of mitochondrial pathways in A549 cells. Collectively, the results obtained suggest that compounds 1u and 3d are promising anti-non-small-cell lung cancer lead compounds.

A new highly oxygenated germacranolide from Carpesium nepalense var. lanatum (C.B.Clarke) Kitam.

A new highly oxygenated germacranolide, carcerlane A (1), together with four known highly oxygenated germacranolides (2-5), was isolated from an ethanol extract of the whole plant of Carpesium nepalense var. lanatum (C.B.Clarke) Kitam. The structures were determined by HRESIMS and extensive analysis of their spectroscopic data including IR, 1 D and 2 D NMR spectra. To our best knowledge, it was the first time to report the phytochemical investigation on this plant. The anti-Alzheimer's disease (AD) activities of 1-5 were evaluated using Caenorhabditis elegans AD pathological model. All the tested compounds showed that they have the anti-AD bioactivities of delaying worms paralysis.

Germacrane sesquiterpenes from leaves of Eupatorium chinense inhibit protein tyrosine phosphatase.

Three new germacrane-type sesquiterpene lactones (1-3) were isolated alongside seven known related congeners (4-10) from the leaves of Eupatorium chinense L. (Compositae). The planar structures of 1-3 were elucidated by their spectroscopic data, including 1D and 2D NMR spectra. The relative and absolute configurations of 1-3 were determined using NOESY experiments and electronic circular dichroism analyses. Compounds 1, 4, 5, and 7 inhibited protein tyrosine phosphatase (PTP) 1B activity with IC50 values of 25, 11, 28, and 24 µM, respectively. Among these, compound 4 exhibited an inhibitory effect on T-cell PTP (TCPTP) with an IC50 value of 25 µM. To our knowledge, this is the first study demonstrating the PTP inhibitory activity of the germacrane sesquiterpenes. The results show that compound 4 acts as an inhibitor of both PTP1B and TCPTP.

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.