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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.

We investigated the anti-inflammatory effect of the hexane fraction from Orostachys japonicus (OJH) in LPS (lipopolysaccharide)-stimulated RAW 264.7 cells. Pretreatment with OJH dose-dependently reduced the cellular NO (nitric oxide) concentration and also inhibited expression of iNOS (inducible nitric oxide synthase) protein and mRNA. By the prevention of IκBα (inhibitory factor kappa B alpha) phosphorylation and degradation, OJH inhibited LPS-induced NF-κB (nuclear factor-kappa B) activation. OJH had no effect on the LPS-induced phosphorylation of ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), or p38, whereas it attenuated the phosphorylation of Akt in a dose-dependent manner. In addition, OJH suppressed the LPS-induced expression of LPS receptors CD14 and TLR4 (toll like receptor 4). These results suggest that OJH may interrupt LPS-induced pro-inflammatory cascades through inhibition of NF-κB and Akt activation.

In this study, powder of Orostachys japonicus A. Berger (O. japonicus) was extracted with 95% ethyl alcohol and fractionated using a series of organic solvents, including n-hexane (hexane), dichloromethane (DCM), ethylacetate (EtOAc), n-butanol (BuOH), and water (H2O). We investigated the anti-inflammatory effects of these O. japonicus extracts on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Their effects on the expression of inflammatory mediators and transcription factors were analyzed by Western blotting. DCM fraction significantly inhibited formation of reactive oxygen species (ROS) as well as nitric oxide (NO) in LPS-stimulated RAW 264.7 cells. Phosphorylation of the pro-inflammatory transcription factor complex nuclear factor-kappa B (NF-κB) p65 and expression of inducible nitric oxide synthase (iNOS), one of its downstream proteins, were also suppressed by DCM fraction. These effects were regulated by upsteam proteins in the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/Akt (PI3K/Akt) signaling pathways. Taken together, our data suggest that O. japonicus could be used as a potential source for anti-inflammatory agents. [BMB reports 2011; 44(6): 399-404]