Steppogenin suppresses tumor growth and sprouting angiogenesis through inhibition of HIF-1α in tumors and DLL4 activity in the endothelium.

BACKGROUND: Hypoxia is a characteristic feature of many solid tumors. As an adaptive response to hypoxia, tumor cells activate hypoxia-inducible factor-1α (HIF-1α). Under hypoxic conditions, angiogenesis mediated by HIF-1α is involved in the growth and metastasis of tumor cells. During the angiogenic process, differentiated tip endothelial cells (ECs) characterized by high expression of DLL4 promote angiogenic germination through filopodia. Inhibitors of HIF-1α or DLL4 have been widely studied PURPOSE: We tried to find inhibitors targeting both HIF-1α and DLL4 in tumor which have not yet been developed. STUDY DESIGN: In this study, we examined a natural compound that inhibits sprouting angiogenesis and tumor growth by targeting both HIF-1α and DLL4 under hypoxic conditions. METHODS: After examining cell viability of 70 selected natural compounds, we assessed the effects of compounds on HIF-1α and DLL4 transcriptional activity using a dual-luciferase reporter assay. Western blot analysis, immunofluoresecnt assay and real-time qPCR were performed to identify expression of proteins, such as HIF-1α and DLL4, as well as HIF-1α target genes under hypoxic conditions. In vitro angiogenesis assay and in vivo allograft tumor experiment were performed to investigate inhibition of tumor growth through anti-angiogenic activity. RESULTS: Among these compounds, steppogenin, which is extracted from the root bark of Morus alba l, respectively inhibited the transcriptional activity of HIF-1α under hypoxic conditions in HEK293T cells and vascular endothelial growth factor (VEGF)-induced DLL4 expression in vascular ECs in a dose-dependent manner. In tumor cells and retinal pigment epithelial cells, steppogenin significantly suppressed HIF-1α protein levels under hypoxic conditions as well as VEGF-induced DLL4 expression in ECs. Furthermore, steppogenin suppressed hypoxia-induced vascular EC proliferation and migration as well as VEGF-induced sprouting of EC spheroids. CONCLUSION: These results suggest that the natural compound steppogenin could potentially be used to treat angiogenic diseases, such as those involving solid tumors, because of its dual inhibition of HIF-1α and DLL4.

In Vitro and In Vivo Antibacterial Activity of Serratamid, a Novel Peptide-Polyketide Antibiotic Isolated from Serratia plymuthica C1, against Phytopathogenic Bacteria.

A new hybrid non-ribosomal peptide-polyketide antibiotic (serratamid) for phytoprotection was isolated from the ethyl acetate layer of tryptic soy agar culture of the soil bacterium Serratia plymuthica C1 through bioassay-guided fractionation. Its chemical structure was elucidated using instrumental analyses, such as mass and nuclear magnetic resonance spectrometry. Serratamid showed antibacterial activity against 15 phytopathogenic bacteria, with minimum inhibitory concentration (MIC) values ranging from 0.244 to 31.25 µg/mL. In vitro, it displayed strong antibacterial activity against Ralstonia solanacearum and four Xanthomonas spp., with MIC values (0.244-0.488 µg/mL) superior to those of streptomycin sulfate, oxolinic acid, and oxytetracycline. Further, serratamid and the ethyl acetate layer of S. plymuthica C1 effectively reduced bacterial wilt caused by R. solanacearum on tomato seedlings and fire blight caused by Erwinia on apple fruits in a dose-dependent manner. These results suggest that serratamid is a promising candidate as a potent bactericide for controlling bacterial diseases.

Anti-Inflammatory Effect of Flavonoids from Brugmansia arborea L. Flowers.

Brugmansia arborea L. (Solanaceae), commonly known as "angel's trumpet," is widely grown in North America, Africa, Australia, and Asia. It has been mainly used for ornamental purposes as well as analgesic, anti-rheumatic, vulnerary, decongestant, and anti-spasmodic materials. B. arborea is also reported to show anti-cholinergic activity, for which many alkaloids were reported to be principally responsible. However, to the best of our knowledge, a phytochemical study of B. arborea flowers has not yet been performed. Four flavonol glycosides (1-4) and one dihydroflavanol (5) were for the first time isolated from B. arborea flowers in this study. The flavonoids showed significant antioxidant capacities, suppressed nitric oxide production in lipopolysaccharide (LPS)-treated RAW 264.7 cells, and reduced inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) protein production increased by LPS treatment. The contents of compounds 1-4 in n-BuOH fraction were determined to be 3.8 ± 0.9%, 2.2 ± 0.5%, 20.3 ± 1.1%, and 2.3 ± 0.4%, respectively, and that of compound 5 in EtOAc fraction was determined to be 12.7 ± 0.7%, by HPLC experiment. These results suggest that flavonol glycosides (1-4) and dihydroflavanol (5) can serve as index components of B. arborea flowers in standardizing anti-inflammatory materials.

Flavonoids from Chionanthus retusus (Oleaceae) Flowers and Their Protective Effects against Glutamate-Induced Cell Toxicity in HT22 Cells.

The dried flowers of Chionanthus retusus were extracted with 80% MeOH, and the concentrate was divided into EtOAc, n-BuOH, and H2O fractions. Repeated SiO2, octadecyl SiO2 (ODS), and Sephadex LH-20 column chromatography of the EtOAc fraction led to the isolation of four flavonols (1-4), three flavones (5-7), four flavanonols (8-11), and one flavanone (12), which were identified based on extensive analysis of various spectroscopic data. Flavonoids 4-6 and 8-11 were isolated from the flowers of C. retusus for the first time in this study. Flavonoids 1, 2, 5, 6, 8, and 10-12 significantly inhibited NO production in RAW 264.7 cells stimulated by lipopolysaccharide (LPS) and glutamate-induced cell toxicity and effectively increased HO-1 protein expression in mouse hippocampal HT22 cells. Flavonoids with significant neuroprotective activity were also found to recover oxidative-stress-induced cell damage by increasing HO-1 protein expression. This article demonstrates that flavonoids from C. retusus flowers have significant potential as therapeutic materials in inflammation and neurodisease.

Lanceoleins A-G, hydroxychalcones, from the flowers of Coreopsis lanceolata and their chemopreventive effects against human colon cancer cells.

Seven new chalcones, lanceolein A-G (compounds 5 and 7-12), as well as five known chalcones (1-4 and 6), were isolated from the methanolic extract of Coreopsis lanceolata flowers. The chemical structures of 5 and 7-12 were determined on the basis of spectroscopic data interpretation. All compounds inhibited the production of nitrite oxide (NO) induced by LPS in RAW264.7 macrophage cells. Also, compounds 1-6 showed moderated cytotoxicity against human colon cancer cell lines, while compounds 7-12 hardly showed the cytotoxicity. Especially, compounds 2, 5, and 6 exhibited a little higher cytotoxicity on HCT15 cells, with IC50 values of 43.7 ±â€¯2.17 µM, 35.6 ±â€¯0.24 µM, and 47.9 ±â€¯1.18 µM, respectively. In the Tali assay, compounds 2 and 5 increased the numeral of apoptotic cells. These compounds also significantly promoted the expression of apoptotic proteins including PARP and caspase-3.

Glycerides isolated from the aerial parts of Malva verticillata cause immunomodulation effects via splenocyte function and NK anti-tumor activity.

A preliminary study revealed that a 10 µg/mL n-BuOH fraction of Malva verticillata aerial parts significantly enhanced splenocyte proliferation and induced significant enhancement of natural-killer (NK) cell activity against tumor cells (YAC-1). This study was initiated to identify the principal components that exhibited these activities, and four glycerides were isolated through repeated SiO2 and ODS column chromatography. Structures of compounds 1-4 were determined to be (2S)-1-O-palmitoyl glyceride, (2S)-1-O-stearoyl glyceride, (2S)-1-O-linolenoyl glyceride, and (2S)-1,2-di-O-linoleoyl glyceride, respectively. Compounds 1-3 showed potential immune-enhancing activity in murine splenocyte and natural-killer (NK) cells at 10 µM. In contrast, compound 4 showed weak activity, indicating the monoacyl glycerides (1-3) are more effective than diacyl glyceride (4). Also, the longer the carbon number of the fatty acid in monoacyl glyceride, the better the activity, and the monoacyl glyceride including an unsaturated fatty acid (3) is more effective than the glycerides including the saturated fatty acids (1-2).

Glycosyl glycerides from the aerial parts of Malva verticillata and their chemopreventive effects.

A new glycosyl glyceride (5) along with twelve known ones (1-4 and 6-13) including two sulfoquinovosyl glycerides (1 and 2) were isolated from the aerial parts of Malva verticillata. Based on several spectroscopic methods, compound 5 was identified to be (2S)-1-O-ß-d-galactopyranosyl-3-O-isostearoyl glyceride, and named malvaglycolipid A. Compounds 1 and 2 contained a unique sugar, (6-deoxy-6-sulfo)-α-d-glucopyranose, which very rarely occurs in natural sources. This is the first report for the isolation of compounds 1 and 2 from natural sources and the structure determination using NMR experiment. It was also of note that no glycosyl glyceride has previously been isolated from the family of Malvaeae. Most glycosyl glycerides showed cytotoxicity to HepG2, AGS, HCT-15, and A549 human cancer cells. Especially, compounds 1, 2, and 11 exhibited significant cytotoxicity to AGS cells, with IC50 values of 33.7 ±â€¯0.64 µM, 11.1 ±â€¯0.07 µM, and 10.6 ±â€¯0.10 µM, respectively. The n-BuOH fraction and compounds 1, 2, and 11 increased the number of apoptotic cells in the Tali assay and had a significant effect on the levels of proteins related to apoptosis including PARP, caspase-3, Bcl-2, Bax, and ß-actin.

New Lignans from the Flower of Forsythia koreana and Their Suppression Effect on VCAM-1 Expression in MOVAS Cells.

Six lignans including two new lignans were obtained as the principal components of the Forsythia koreana flowers via silica gel (SiO2 ), octadecyl SiO2 (ODS) as well as Sephadex LH-20 column chromatography. In addition to two new lignans, named koreanaside A ((7R,8S,7'R,8'S)-7,7'-diepoxy-5'-hydroxy-3,3'-dimethoxylignan 4-O-ß-d-glucopyranoside) and koreanaside B ((7R,8S,7'S,8'R)-7,9'-epoxy-9,5',7'-trihydroxy-3,3'-dimethoxylignan 4-O-ß-d-glucopyranoside), four known lignans were identified to be (+)-phylligenin, (-)-epipinoresinol, pinoresinol, and tinosposide A. The structures and absolute configurations of koreanasides A and B were established by means of analysis of spectroscopic data (NMR, IR, FAB-MS, and CD), whereas the structures of known lignans were identified by comparison their NMR and MS values with those in the reported literature. Their chemical structures including configuration were established by means of analysis of spectroscopic data (NMR, IR, FAB-MS, and CD) but also comparison of their NMR and MS values with those in the reported literature. This is the first article for isolation of six lignans of F. koreana flowers. Koreanasides A and B showed high radical scavenging activity with oxygen radical absorbance capacity (ORAC) values of 0.97 ± 0.01 and 1.02 ± 0.01, respectively. Koreanaside A also prohibited expressing VCAM-1 in MOVAS cells with 80.5% at 25 mg/mL.

Isolation and Quantification of Ginsenoside Rh23, a New Anti-Melanogenic Compound from the Leaves of Panax ginseng.

A new ginsenoside, named ginsenoside Rh23 (1), and 20-O-ß-d-glucopyranosyl-3ß,6α,12ß,20ß,25-pentahydroxydammar-23-ene (2) were isolated from the leaves of hydroponic Panax ginseng. Compounds were isolated by various column chromatography and their structures were determined based on spectroscopic methods, including high resolution quadrupole/time of flight mass spectrometry (HR-QTOF/MS), nuclear magnetic resonance (NMR) spectroscopy, and infrared (IR) spectroscopy. To determine anti-melanogenic activity, the change in the melanin content in melan-a cells treated with identified compounds was tested. Additionally, we investigated the melanin inhibitory effects of ginsenoside Rh23 on pigmentation in a zebrafish in vivo model. Compound 1 inhibited potent melanogenesis in melan-a cells with 37.0% melanogenesis inhibition at 80 µM and also presented inhibition on the body pigmentation in zebrafish model. Although compound 2 showed slightly lower inhibitory activity than compound 1, it also showed significantly decreased melanogenesis in melan-a cell and in zebrafish model. These results indicated that compounds isolated from hydroponic P. ginseng may be used as new skin whitening compound through the in vitro and in vivo systems. Furthermore, this study demonstrated the utility of MS-based compound 1 for the quantitative analysis. Ginsenoside Rh23 (1) was found at a level of 0.31 mg/g in leaves of hydroponic P. ginseng.