Cassaine diterpenoid glycosides from the seeds of Erythrophleum fordii Oliv. and their antiviral and anti-inflammatory activities.

Seven new cassaine diterpenoids (1-7) along with four known ones (8-11) were isolated from the seeds of Erythrophleum fordii Oliv. (Leguminosae). Their chemical structures were elucidated by extensive spectroscopic data interpretation and chemical methods. Compound 1 is a rare unsymmetrical dimer, which is formed by the linking of another cassaine diterpenoid acid glycoside to the 6-hydroxyl group of the sugar unit in a cassaine amide glycoside through an ester bond. Compound 2 is a cassaine diterpenoid acid derivative featuring an unusual Z double bond at C-13 and C-15. The in vitro antiviral and anti-inflammatory activities of 1-11 were evaluated. The results showed that compounds 1, 2 and 3 showed significant antiviral activities against human respiratory syncytial virus (RSV) with IC50s of 6.3, 7.8, and 9.4 µM, respectively. Compound 9 significantly suppressed the expression of nuclear factor-kappa B (NF-κB) with an IC50 value of 2.6 µM.

Total Saponins of Panax notoginseng Activate Akt/mTOR Pathway and Exhibit Neuroprotection in vitro and in vivo against Ischemic Damage.

OBJECTIVE: To reveal the neuroprotective effect and the underlying mechanisms of a mixture of the main components of Panax notoginseng saponins (TSPN) on cerebral ischemia-reperfusion injury and oxygen-glucose deprivation/reoxygenation (OGD/R) of cultured cortical neurons. METHODS: The neuroprotective effect of TSPN was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, flow cytometry and live/dead cell assays. The morphology of dendrites was detected by immunofluorescence. Middle cerebral artery occlusion (MCAO) was developed in rats as a model of cerebral ischemia-reperfusion. The neuroprotective effect of TSPN was evaluated by neurological scoring, tail suspension test, 2,3,5-triphenyltetrazolium chloride (TTC) and Nissl stainings. Western blot analysis, immunohistochemistry and immunofluorescence were used to measure the changes in the Akt/mammalian target of rapamycin (mTOR) signaling pathway. RESULTS: MTT showed that TSPN (50, 25 and 12.5 µ g/mL) protected cortical neurons after OGD/R treatment (P<0.01 or P<0.05). Flow cytometry and live/dead cell assays indicated that 25 µ g/mL TSPN decreased neuronal apoptosis (P<0.05), and immunofluorescence showed that 25 µ g/mL TSPN restored the dendritic morphology of damaged neurons (P<0.05). Moreover, 12.5 µ g/mL TSPN downregulated the expression of Beclin-1, Cleaved-caspase 3 and LC3B-II/LC3B-I, and upregulated the levels of phosphorylated (p)-Akt and p-mTOR (P<0.01 or P<0.05). In the MCAO model, 50 µ g/mL TSPN improved defective neurological behavior and reduced infarct volume (P<0.05). Moreover, the expression of Beclin-1 and LC3B in cerebral ischemic penumbra was downregulated after 50 µ g/mL TSPN treatment, whereas the p-mTOR level was upregulated (P<0.05 or P<0.01). CONCLUSION: TSPN promoted neuronal survival and protected dendrite integrity after OGD/R and had a potential therapeutic effect by alleviating neurological deficits and reversing neuronal loss. TSPN promoted p-mTOR and inhibited Beclin-1 to alleviate ischemic damage, which may be the mechanism that underlies the neuroprotective activity of TSPN.

PHMH, a diarylheptanoid from Alpinia officinarum attenuates VEGF-induced angiogenesis via inhibition of the VEGFR-2 signaling pathway.

The rhizome of Alpinia officinarum Hance, a popular spice used as a condiment in China and Europe, has various reported bioactivities, including anticancer, anti-inflammatory and antioxidant effects. However, its anti-angiogenic activity has not previously been reported. In this study, a diarylheptanoid was isolated from Alpinia officinarum and identified as 1-phenyl-7-(4-hydroxy-3-methoxyphenyl)-4E-en-3-heptanone (PHMH). We demonstrated that PHMH exerts anti-angiogenic activity both in vitro and in vivo. PHMH inhibited vascular endothelial growth factor (VEGF)-induced viability, migration, invasion and tube formation in human umbilical vein endothelial cells (HUVECs) in vitro, and also suppressed VEGF-induced sprout formation of rat aorta ex vivo. Furthermore, PHMH was found to block VEGF-induced vessel formation in mice and suppress angiogenesis in both zebrafish and chorioallantoic membrane models. Mechanistic studies indicated that PHMH inhibited VEGF-induced VEGF receptor-2 (VEGFR-2) auto-phosphorylation and resulted in the blockage of VEGFR-2-mediated signaling cascades in HUVECs, including the Akt/mTOR, ERK1/2, and FAK pathways. Our findings provide new insights into the potential application of PHMH as a therapeutic agent for anti-angiogenesis.

Antiviral activity of ethanol extract of Lophatherum gracile against respiratory syncytial virus infection.

ETHNOPHARMACOLOGICAL RELEVANCE: Lophatherum gracile, an important medicinal plant, is used traditionally in the treatment of cough associated with lung heat and inflammation. In this study, an ethanol extract of L. gracile (DZY) was shown to inhibit respiratory syncytial virus (RSV) infection and RSV-induced inflammation in vitro and in vivo. These findings provide a strong and powerful support for the traditional use of L. gracile in the treatment of RSV-related diseases. AIM OF THE STUDY: To determine the anti-RSV activities of DZY and its ingredients, and explore the relationship between RSV infection and inflammation. MATERIALS AND METHODS: DZY was extracted from L. gracile and its major ingredients were determined by high-performance liquid chromatography (HPLC). RSV-infected HEp-2 and RAW264.7 cell models were established to assess the inhibitory effect of DZY on RSV replication and nitric oxide (NO) production in vitro. Three-week-old BALB/c mice challenged intranasally with RSV were used to establish RSV-infected animal mode. The mice were respectively administered DZY at high-, middle-, and low-dose in different groups. The anti-RSV activity of DZY was evaluated by detecting viral load, lung lesion, CD4+ and CD8+ T cell population, and interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ expression in the lung tissue. RESULTS: In HEp-2 cell line, DZY effectively inhibited RSV infection in a dose-dependent manner with IC50 values of 20 µg/mL against RSV (Long strain) and IC50 values of 25 µg/mL against RSV (A2 strain). The anti-RSV activity of DZY was mainly determined by isoorientin, swertiajaponin, 3, 5-di-caffeoylquinic acid, and 3, 4-di-caffeoylquinic acid. Moreover, DZY suppressed NO production induced by RSV in vitro. In vivo, oral administration of DZY significantly reduced the viral load and ameliorated lesions in the lung tissue. A probable antiviral mechanism was mediated by slightly improving the ratio of CD4+/CD8+ T cells and inhibiting the mRNA and protein expression of IL-1ß, TNF-α, and IFN-γ. CONCLUSIONS: (1) DZY exhibits anti-RSV activities both in vitro and in vivo. (2) RSV infection can trigger a series of inflammatory reactions; thus, ameliorating inflammation is helpful to control the course of disease caused by RSV. These findings provide the rationale and scientific evidence behind the extensive use of L. gracile in traditional medicine for the treatment of diseases potentially caused by RSV.

Small molecule inhibits respiratory syncytial virus entry and infection by blocking the interaction of the viral fusion protein with the cell membrane.

Antiviral drug development against respiratory syncytial virus (RSV) is urgently needed due to the public health significance of the viral infection. Here, we report the anti-RSV activity of a small molecule, (1S,3R,4R,5R)-3,4- bis{[(E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,5-dihydroxycyclohexane-1-carboxylic methyl ester (3,4-DCQAME) or 3,4- O-Dicaffeoylquinic acid methyl ester, which can be isolated from several plants of traditional Chinese medicine. We showed for the first time that compound 3,4-DCQAME potently inhibits RSV entry and infection. In vitro, 3,4-DCQAME can interact with F(ecto), the ectodomain of RSV fusion (F) protein. In cultured cells, the compound can block the interaction of F(ecto) protein with the cellular membrane and inhibit viral fusion during RSV entry, leading to inhibition of viral gene expression and infection. In RSV-infected mice that were treated with 3,4-DCQAME, we observed a reduction of RSV-induced pathologic changes and substantial inhibition of viral infection and growth in the lung tissues. Our results provide the first direct evidence of the anti-RSV activity of 3,4-DCQAME. Furthermore, these results suggest that 3,4-DCQAME represents a promising lead compound for anti-RSV therapeutic development.-Tang, W., Li, M., Liu, Y., Liang, N., Yang, Z., Zhao, Y., Wu, S., Lu, S., Li, Y., Liu, F. Small molecule inhibits respiratory syncytial virus entry and infection by blocking the interaction of the viral fusion protein with the cell membrane.

Rearranged Phloroglucinol-Monoterpenoid Adducts from Callistemon rigidus.

Callisretones A (1) and B (2), two rearranged phloroglucinol-monoterpenoid adducts featuring an unprecedented isopropylcyclopenta[b]benzofuran backbone, together with their postulated biosynthetic precursors (3-9), were isolated from Callistemon rigidus. The previously assigned absolute configurations of viminalins H (7), L (8), and N (9) were revised and unequivocally established by X-ray diffraction data. A putative biosynthetic pathway toward callisretones A and B involving the rearrangement of the terpenoid motif is proposed. In addition, 1 and 2 showed inhibitory effects on nitric oxide production with IC50 values of 15.3 ± 1.0 and 17.7 ± 1.1 µM, respectively.

Watsonianone A from Rhodomyrtus tomentosa Fruit Attenuates Respiratory-Syncytial-Virus-Induced Inflammation In Vitro.

Respiratory syncytial virus (RSV) is one of the most common respiratory pathogens. Immoderate inflammation plays a great role in causing RSV-induced diseases. In the present study, watsonianone A, isolated from the fruit of Rhodomyrtus tomentosa (Ait.) Hassk, was found to show a good inhibitory effect on RSV-induced NO production, with a half-maximal inhibitory concentration of 37.2 ± 1.6 µM. Enzyme-linked immunosorbent assay and fluorescence quantitative polymerase chain reaction analyses indicated that watsonianone A markedly reduced both mRNA and protein levels of tumor necrosis factor α, interleukin 6, and monocyte chemoattractant protein 1 in RSV-infected RAW264.7 cells. Mechanistically, watsonianone A inhibited nuclear factor κB (NF-κB) activation by suppressing IκBα phosphorylation. Further analysis revealed that watsonianone A activated the thioredoxin system and decreased intracellular reactive oxygen species (ROS) levels, which are closely associated with NF-κB activation in RSV-infected cells. These results reveal that watsonianone A can attenuate RSV-induced inflammation via the suppression of ROS-sensitive inflammatory signaling.

Sesquiterpene lactones from Elephantopus mollis and their anti-inflammatory activities.

Seven sesquiterpene lactones, 8-O-methacryloylelephanpane, 2,4-bis-O-methyl-8-O-methacryloylelephanpane, 4-O-ethyl-8-O-methacryloylelephanpane, 8-O-methacryloylisoelephanpane, 2-O-demethyltomenphantopin C, molephantin A, molephantin B, along with ten known ones, were isolated from Elephantopus mollis (Asteraceae). Their structures were elucidated by extensive analysis of spectroscopic data (IR, UV, HRESIMS, 1D and 2D NMR). The isolates were evaluated for their anti-inflammatory activities on LPS-stimulated RAW 264.7 cells, and all tested compounds exhibited potent anti-inflammatory effects with IC50 values of 0.57 ± 0.17 to 14.34 ± 1.61 µM, except that compound tomenphantopin C exhibited moderate anti-inflammatory activity with IC50 values of 59.97 ± 1.53 µM.

EM23, A Natural Sesquiterpene Lactone from Elephantopus mollis, Induces Apoptosis in Human Myeloid Leukemia Cells through Thioredoxin- and Reactive Oxygen Species-Mediated Signaling Pathways.

Elephantopus mollis (EM) is a traditional herbal medicine with multiple pharmacological activities. However, the efficacy of EM in treating human leukemia is currently unknown. In the current study, we report that EM23, a natural sesquiterpene lactone isolated from EM, inhibits the proliferation of human chronic myeloid leukemia (CML) K562 cells and acute myeloid leukemia (AML) HL-60 cells by inducing apoptosis. Translocation of membrane-associated phospholipid phosphatidylserines, changes in cell morphology, activation of caspases, and cleavage of PARP were concomitant with this inhibition. The involvement of the mitochondrial pathway in EM23-mediated apoptosis was suggested by observed disruptions in mitochondrial membrane potential. Mechanistic studies indicated that EM23 caused a marked increase in the level of reactive oxygen species (ROS). Pretreatment with N-acetyl-L-cysteine, a ROS scavenger, almost fully reversed EM23-mediated apoptosis. In EM23-treated cells, the expression levels of thioredoxin (Trx) and thioredoxinreductase (TrxR), two components of the Trx system involved in maintaining cellular redox homeostasis, were significantly down-regulated. Concomitantly, Trx regulated the activation of apoptosis signal-regulating kinase 1 (ASK1) and its downstream regulatory targets, the p38, JNK, and ERK MAPKs. EM23-mediated activation of ASK1/MAPKs was significantly inhibited in the presence of NAC. Furthermore, tumor necrosis factor alpha (TNF-α)-mediated activation of nuclear factor-κB (NF-κB) was suppressed by EM23, as suggested by the observed blockage of p65 nuclear translocation, phosphorylation, and reversion of IκBα degradation following EM23 treatment. Taken together, these results provide important insights into the anticancer activities of the EM component EM23 against human CML K562 cells and AML HL-60 cells.

EM23, a natural sesquiterpene lactone, targets thioredoxin reductase to activate JNK and cell death pathways in human cervical cancer cells.

Sesquiterpene lactones (SLs) are the active constituents of a variety of medicinal plants and found to have potential anticancer activities. However, the intracellular molecular targets of SLs and the underlying molecular mechanisms have not been well elucidated. In this study, we observed that EM23, a natural SL, exhibited anti-cancer activity in human cervical cancer cell lines by inducing apoptosis as indicated by caspase 3 activation, XIAP downregulation and mitochondrial dysfunction. Mechanistic studies indicated that EM23-induced apoptosis was mediated by reactive oxygen species (ROS) and the knockdown of thioredoxin (Trx) or thioredoxin reductase (TrxR) resulted in a reduction in apoptosis. EM23 attenuated TrxR activity by alkylation of C-terminal redox-active site Sec498 of TrxR and inhibited the expression levels of Trx/TrxR to facilitate ROS accumulation. Furthermore, inhibition of Trx/TrxR system resulted in the dissociation of ASK1 from Trx and the downstream activation of JNK. Pretreatment with ASK1/JNK inhibitors partially rescued cells from EM23-induced apoptosis. Additionally, EM23 inhibited Akt/mTOR pathway and induced autophagy, which was observed to be proapoptotic and mediated by ROS. Together, these results reveal a potential molecular mechanism for the apoptotic induction observed with SL compound EM23, and emphasize its putative role as a therapeutic agent for human cervical cancer.

Antiviral benzofurans from Eupatorium chinense.

Eleven benzofuran dimers, (+)-dieupachinins A-E, (-)-dieupachinins A-E and dieupachinin F, a benzofuran trimer trieupachinin A, as well as seven known compounds were isolated from the roots of Eupatorium chinense. The enantiomers of racemates dieupachinins A-E were separated by chiral HPLC. The structures with absolute configurations were elucidated on the basis of spectroscopic data, X-ray diffraction analysis, and circular dichroism experiments. The isolated compounds were evaluated for their in vitro antiviral activities against respiratory syncytial virus (RSV).

Tangeretin from Citrus reticulate Inhibits Respiratory Syncytial Virus Replication and Associated Inflammation in Vivo.

Human respiratory syncytial virus (RSV) is a common pathogen that causes pneumonia and bronchiolitis in infants and young children. Our previous study showed that tangeretin from Citrus reticulate possessed potent in vitro anti-RSV effects comparable to that of ribavirin. Therefore, in this study, we investigated the in vivo anti-RSV activity of tangeretin in 3-week-old male BALB/c mice. A plaque reduction assay and fluorescence quantitative polymerase chain reaction (FQ-PCR) showed that tangeretin inhibited RSV replication in the lung of mice. Moreover, a luminex assay indicated tangeretin relieved RSV-induced lung inflammation by attenuating interleukin (IL)-1ß secretion. Possible anti-inflammatory mechanisms of tangeretin were preliminarily explored using a RSV-infected macrophage model. A FQ-PCR, enzyme-linked immunosorbent assay (ELISA), and luciferase assay revealed that tangeretin inhibited RSV-induced inflammation by suppressing nuclear factor-κB (NF-κB) activation. This study demonstrates that tangeretin inhibited RSV replication and RSV-induced lung inflammation in vivo and may be useful in preventing and treating RSV infections and inflammation.

Four new triterpenes and triterpene glycosides from the leaves of Ilex latifolia and their inhibitory activity on triglyceride accumulation.

Two new triterpenes (1 and 2) and two new triterpene glycosides (3 and 4), along with six known triterpenes (5-10) were isolated from the leaves of Ilex latifolia. The structures of new compounds were elucidated on the basis of NMR, HR-MS, and X-ray diffraction analysis. Compounds 4 and 5 showed potent inhibitory activity on oleic acid/palmitic acid induced triglyceride accumulation on HepG2 cells.

Triterpenoid saponins from the rhizomes of Anemone flaccida and their inhibitory activities on LPS-induced NO production in macrophage RAW264.7 cells.

A new ursane-type triterpenoid saponin, flaccidoside IV (1), and three new oleanane-type triterpenoid saponins, flaccidosides V-VII (2-4), along with 17 known saponins (5-21), were isolated from the rhizomes of Anemone flaccida. The structures of the new triterpenoid saponins were determined based on spectroscopic analyses and chemical methods. All the isolated saponins were tested for their inhibitory activities on lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophages, and several bisdesmosidic oleanane-type triterpenoid saponins (2, 7, and 10) showed significant inhibitory activities, which indicated they had potential anti-inflammatory activities under their noncytotoxic concentrations in vitro.

Antiviral activity of polymethoxylated flavones from "Guangchenpi", the edible and medicinal pericarps of citrus reticulata 'Chachi'.

The present study found that the supercritical fluid extract of "Guangchenpi" possessed in vitro antiviral activity against respiratory syncytial virus (RSV). Bioassay-guided isolation and identification of this extract led to obtain five active polymethoxylated flavones (1-5). Cytopathic effect (CPE) reduction assay exhibited that tangeretin (2) and nobiletin (3), two major polymethoxylated flavones in the extract, possessed better anti-RSV effect comparable to the positive control ribavirin. Plaque reduction assay revealed that tangeretin dose-dependently inhibited RSV-induced plaque formation on the HEp-2 cells. This polymethoxylated flavone mainly affected the intracellular replication of RSV, and it also could inhibit RSV entry into the HEp-2 cells. Further investigations with quantitative real-time PCR and confocal and Western blot assays indicated that tangeretin downregulated the expression of RSV phosphoprotein (P protein). Results suggest the potential application of the supercritical fluid extract of "Guangchenpi" and tangeretin in the treatment and the prevention of RSV infection.

New ursane-type triterpenoid saponins from the stem bark of Schefflera heptaphylla.

Phytochemical investigation on the stem bark of Schefflera heptaphylla led to the isolation of five new ursane-type triterpenoid saponins (1-5). Their structures were determined on the basis of spectroscopic and chemical methods. It is noteworthy in this study that the genins of all compounds are reported for the first time. All compounds isolated from this plant were evaluated for their inhibitory activities on lipopolysaccharide-induced nitric oxide production in RAW264.7 cells, and compounds 2 and 5 showed weak anti-inflammatory activities under their non-cytotoxic concentrations.

Triterpenoid saponins from the roots of Clematis uncinata.

Eight new bisdesmosidic triterpenoid saponins, clematiunicinosides A-H (1-8), along with eleven known ones (9-19), were isolated from the roots of Clematis uncinata. Their structures were elucidated on the basis of spectroscopic analysis and chemical evidence. All the isolated saponins were tested for their cytotoxic activities on human caski cervical cancer (Caski) cells, and compounds 13, 17 and 19 exhibited inhibitory effect on Caski cells.

Chemical constituents from the roots and stems of Erycibe obtusifolia and their in vitro antiviral activity.

Three new quinic acid derivatives, 4-O-caffeoyl-3-O-sinapoylquinic acid methyl ester (1), 5-O-caffeoyl-4-O-syringoylquinic acid methyl ester (2), and 4-O-caffeoyl-3-O-syringoylquinic acid methyl ester (3), as well as four new coumarin glycosides, 7-O-(3-O-sinapoyl-ß-D-glucopyranosyl)-6-methoxycoumarin (12), 7-O-(6-O-sinapoyl-ß-D-glucopyranosyl)-6-methoxycoumarin (13), 7-O-(2-O-sinapoyl-ß-D-glucopyranosyl)-6-methoxycoumarin (14), and 7-O-(6-O-syringoyl-ß-D-glucopyranosyl)-6-methoxycoumarin (15), together with eight known compounds (4-11) were isolated from the roots and stems of Erycibe obtusifolia. Their structures were elucidated on the basis of spectroscopic analysis and chemical evidence. All the compounds were screened for their in vitro antiviral activity against respiratory syncytial virus with a cytopathic effect reduction assay. Among them, the di-O-caffeoyl quinates 8-11 displayed a potent in vitro anti-respiratory syncytial virus effect.

Triterpenoid saponins from the stem barks of Schefflera heptaphylla.

Nine new triterpenoid saponins, including four ursane-type triterpenoid saponins named heptursosides A-D (1-4), four oleanane-type triterpenoid saponins named heptoleosides A-D (5-8), and one dammarane-type triterpenoid saponin, heptdamoside A (9), along with two known saponins, asiaticoside D (10) and scheffoleoside B (11), were isolated from the stem barks of Schefflera heptaphylla. Their structures were determined on the basis of spectroscopic analysis and chemical methods. It is noteworthy in this study that the aglycone of 1-6 is reported for the first time, and to the best of our knowledge, this is the first report for the presence of the tetracyclic triterpenoid saponin from Schefflera. All the saponins were evaluated for their inhibitory effects on lipopolysaccharide-induced nitric oxide production in RAW264.7 cells, and 2, 6, 7, and 10 showed anti-inflammatory activities under their noncytotoxic concentrations.

Arenobufagin, a bufadienolide compound from toad venom, inhibits VEGF-mediated angiogenesis through suppression of VEGFR-2 signaling pathway.

Angiogenesis is crucial for carcinogenesis and other angiogenic processes. Arenobufagin, one of the major components of toad venom, is a traditional Chinese medicine used for cancer therapy. It inhibits cell growth in several cancer cell lines. However, little is known about arenobufagin's anti-angiogenic activity. In this study, we showed that arenobufagin inhibited vascular endothelial growth factor (VEGF)-induced viability, migration, invasion and tube formation in human umbilical vein endothelial cells (HUVECs) in vitro. Arenobufagin also suppressed sprouting formation from VEGF-treated aortic rings in an ex vivo model. Furthermore, we found that arenobufagin blocked angiogenesis in a matrigel plugs assay. Computer simulations suggested that arenobufagin interacted with the ATP-binding sites of VEGFR-2 by docking. In addition, arenobufagin inhibited VEGF-induced VEGFR-2 auto-phosphorylation and suppressed the activity of VEGFR-2-mediated signaling cascades. Taken together, our findings demonstrate that arenobufagin is a specific inhibitor of VEGF-mediated angiogenesis.