Deok-Seon Ryu

Anti-inflammatory effects of dichloromethane fraction from Orostachys japonicus in RAW 264.7 cells: Suppression of NF-κB activation and MAPK signaling

ETHNOPHARMACOLOGICAL RELEVANCE Orostachys japonicus A. Berger (O. japonicus) is known to reduce the risk of many diseases. AIM OF THE STUDY We investigated the anti-inflammatory effects of the dichloromethane (DCM) fraction from O. japonicus (OJD) in LPS-stimulated RAW 264.7 cells. MATERIALS AND METHODS NO was measured using the Griess method. Key pro-inflammatory cytokines and mediators including IL-1β, TLR4, iNOS, and COX-2; 2 important pro-inflammatory transcription factors, NF-κB p65 and IκBα; and MAPKs such as ERK1/2, JNK, and p38 were analyzed by Western blotting. RESULTS OJD significantly inhibited NO production, IL-1β, TLR4, iNOS, and COX-2 expression in LPS-stimulated cells. Additionally, it inhibited LPS-induced NF-κB p65 activation via inhibition of IκBα phosphorylation. Furthermore, phosphorylation of p38 and JNK was suppressed by OJD in a dose-dependent manner in the LPS-stimulated RAW 264.7 cells. CONCLUSIONS Our data suggest that OJD inhibits the inflammatory response via suppression of NF-κB activation and MAPK signaling.

Effects of polysaccharides derived from Orostachys japonicus on induction of cell cycle arrest and apoptotic cell death in human colon cancer cells

Crude Orostachys japonicus polysaccharide extract (OJP) was prepared by hot steam extraction. Polysaccharides (OJPI) were separated from OJP by gel filtration chromatography and phenol-sulfuric acid assay. The average molecular weight of the OJPI was 30-50 kDa. The anti-proliferative effect of OJPI on HT-29 human colon cancer cells was investigated via morphology study, cell viability assay, apoptosis assay, cell cycle analysis, and cDNA microarray. OJPI inhibited proliferation and growth of HT29 cells and also stimulated apoptosis in a dose- and time-dependent manner. In cell cycle analysis, treatment with OJPI resulted in a marked increase of cells in the G0 (sub G1) and G2/M phases. To screen for genes involved in the induction of cell cycle arrest and apoptosis, the gene expression profiles of HT-29 cells treated with OJPI were examined by cDNA microarray, revealing that a number of genes were up- or down-regulated by OJPI. Whereas several genes involved in anti-apoptosis, cell proliferation and growth, and cell cycle regulation were down-regulated, expression levels of several genes involved in apoptosis, tumor suppression, and other signal transduction events were up-regulated. These results suggest that OJPI inhibits the growth of HT-29 human colon cancer cells by various apoptosis-aiding activities as well as apoptosis itself. Therefore, OJPI deserve further development as an effective agent exhibiting anticancer activity.

Orostachys japonicus induces apoptosis and cell cycle arrest through the mitochondria-dependent apoptotic pathway in AGS human gastric cancer cells

We investigated the anticancer mechanisms of the ethylacetate (EtOAc) fraction from Orostachys japonicus in human gastric cancer (AGS) cells. Flow cytometric analysis revealed that the number of total apoptotic cells following treatment with the EtOAc fraction increased in a dose-dependent manner. In the cell cycle analyses, the EtOAc fraction increased the peak in the sub-G1, indicating apoptosis, and in the G₂/M phases in a dose-dependent manner. In the RT-PCR analysis, the expression of cyclin-dependent kinase 1 (CDK 1) and cyclin B1 decreased in a dose- and time-dependent manner. The results of western blotting revealed that the protein levels of p53, cytochrome c, and cleaved caspase-3, -8 and -9 proteins increased and those of B cell lymphoma-2 (bcl-2) and pro-caspase-3, -8 and -9 proteins decreased in a dose- and time-dependent manner, whereas the levels of bcl-2-associated x protein (bax) remained unchanged. Furthermore, the changes in the levels of pro-caspase-3, -8 and -9 and cleaved caspase-3, -8 and -9 were abolished by the pan-caspase inhibitor Z-VAD-FMK. In addition, phosphorylation of p38 and JNK increased in a time-dependent manner. These results, for the first time, provide an understanding of the potential anticancer activity of the O. japonicus, which functions through the induction of apoptosis and cell cycle arrest.

Anti-cancer activity of the ethylacetate fraction from Orostachys japonicus for modulation of the signaling pathway in HepG2 human hepatoma cells

The molecular mechanisms of the ethylacetate (EtOAc) fraction from Orostachys japonicus (OJE) (including gallic acid, kaempferol, and quercetin) for anti-cancer activities in HepG2 cells were investigated. Apoptosis was detected using morphological observation of nuclear changes and investigation of phosphatidylserine exposure at the cytoplasmic membrane using FITC-Annexin V/PI staining. Activities of the apoptotic factors bcl-2, bax, cytochrome c, pro-caspase-3, 8, and 9, as well as MAPKs levels, were measured using western blotting. Some morphologic features of apoptosis were identified using confocal microscopy. OJE caused the expression of apoptotic proteins to change, as evidenced by an increased bax/bcl-2 ratio and increased expression of cytochrome c, and decreased expressions of pro-caspase-3, 8, and 9. After HepG2 cells were exposed to OJE, expressions of p-JNK and p-ERK1/2 increased in a dose dependent manner. OJE exhibits anti-cancer activity via apoptosis induction through a mitochondria dependent signaling pathway in HepG2 cells.

Acute oral toxicity of the ethyl acetate fraction of Orostachys japonicus in mice

Abstract Context: Orostachys japonicus (Crassulaceae) is referred to as Wa-song in Korea. It is used as an anti-inflammatory, antifebrile, hemostatic, and anti cancer agent, and as an antidote. Objective: The purpose of this study was to evaluate the acute toxicity of the ethyl acetate fraction of O. japonicus (OJE) after the oral administration in Balb/c mice of both sexes. Materials and methods: Mice were oral administered a single doses of 500, 1000, and 2000 mg/kg of body weight and were monitored for 14 d. Biochemical parameters [aspartate amino transferase (AST), alanine amino transferase (ALT), alkaline phosphatase (ALP), total protein (TP), globulin (GB), total cholesterol (TC), triglyceride (TG), blood urea nitrogen (BUN), and creatinine (CR)] and histopathological examination of liver were performed. Results and conclusion: No animals died and no toxic changes were observed in clinical signs, body weight, and organ weight. The LD50 of orally administered OJE was higher than 2000 mg/kg/d in both sexes. No toxicological findings were found in biochemical parameters. In histophathological examination, neutrophilic infiltration was observed at a dose of 2000 mg/kg group in both sexes. These finding suggest that oral administration of OJE does not produce acute toxicity. Therefore, these results could provide satisfactory preclinical evidence of safety to launch clinical trials on standardized formulation of OJE to be a biohealth product.