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Orostachys japonicus is a popular traditional medicinal herb used in Asian countries. This study is focused on evaluating its role in lipid and glucose metabolism in cell and animal models to establish the plant as an anti-obesity and antidiabetic herb. A butanol fraction of O. japonicus was used in the study. The lipid production was evaluated by the Oil Red O technique while the expression of adipogenic markers by Western blotting and RT-PCR using 3T3-L1 preadipocyte. The effect on glucose uptake activity was evaluated in C2C12 myoblast cells. The animal study was carried out in C57BL mice to evaluate anti-obesity activity using the high-fat diet model. The evaluation of serum lipid, blood glucose, adipogenic and fibrosis markers in the liver, and fat deposition in the liver and adipose tissue (by histology) of mice was conducted. Butanol fraction of O. japonicus significantly inhibited the lipid production in the 3T3-L1 cells and reduced the expression of PPARγ, C/EBPα, SREBP-1c and aP2. It enhanced glucose uptake in insulin-resistant C2C12 myoblast cells. It reduced body weight, triglycerides, and blood glucose in the obese mice. It significantly inhibited lipid accumulation in the liver and adipose tissue of obese mice along with suppression of expression of adipogenic and fibrosis markers in the liver. In summary, supporting the previous results, this study helped to establish the potent anti-obesity, antidiabetic, and liver-protecting effect of the butanol fraction of O. japonicus. [ABSTRACT FROM AUTHOR]

Acne vulgaris is a common skin disease characterized by increased sebum production, inflammation, and Cutibacterium acnes (CA: formerly Propionibacterium acnes) hyperproliferation in pilosebaceous follicles. This study evaluated the efficacy of FRO, a formula composed of fermented Rhus verniciflua Stokes and Orostachys japonicus, against acne pathogenesis via antimicrobial assessment and an in vitro analysis. Stimulated model cells treated with hormones, CA, or lipopolysaccharide (LPS) were designed based on the characteristics of acne pathogenesis, including inflammation and sebum hypersecretion. High-performance liquid chromatography, disc diffusion, MTS, and western blotting assays were used to examine potential anti-acne effects. FRO was determined to contain phenolics such as gallic acid, fisetin, quercetin, and kaempferol. FRO exerted antimicrobial activity against CA and inhibited reactive oxygen species production that was otherwise increased by LPS or CA in HaCaT cells. Additionally, FRO exerted anti-inflammatory effects by inhibiting iNOS, TNF-α, IL-6, p-STAT-3, and p-NF-κB, which were previously upregulated by LPS or CA in THP-1 and HaCaT cells. FRO inhibited lipogenesis induced by steroid hormones and CA by decreasing FAS and SREBP-1 levels in sebocytes. Additionally, FRO down-regulated the androgen receptor, 5α-reductase, SREBP-1, and FAS levels, which were upregulated by steroid hormone in LNCaP cells. Taken together, our findings suggest that FRO alleviates acne by inhibiting the growth of CA, inflammation, and excess sebum and could be used for functional cosmetics or acne treatments. [ABSTRACT FROM AUTHOR]

Objective: To determine the bioactive compounds of Orostachys japonicus (O. japonicus) (Maxim.) A. Berger extraction by different solvents (70% ethanol or water) and to evaluate the in vitro antioxidant and anti-inflammatory activities. Methods: Total polyphenol, flavonoid, and anthocyanin contents in O. japonicus extract were measured. Antioxidant activities were determined by DPPH, ABTS, and superoxide radical-scavenging ability assays. Anti-inflammatory activities were evaluated by nitric oxide production, tumor necrosis factor-α, and interleukin-6 expression techniques. Results: Extraction with 70% ethanol yielded the highest total polyphenol (60.03 mg/g dry weight) and flavonoid (55.50 mg/g dry weight) contents. The total anthocyanin contents in 70% ethanol and water extracts were 57.25 and 91.71 mg/g dry weight, respectively. The 70% ethanol extract also showed stronger antioxidant activity than the water extract. Antioxidant activity and reducing power increased with the increasing concentration of O. japonicus extract. O. japonicus extract at 0-400 μg/mL did not affect the growth of RAW 264.7 cells, whereas dose-dependent inhibition of nitric oxide, tumor necrosis factor-a, and interleukin-6 production was observed. Conclusions: O. japonicus inhibits oxidative and inflammatory reactions with potentially positive health-related effects.

Objective: To investigate the effect of Orostachys (O.) japonicus, a perennial herbaceous plant of the Family Crassulaceae, on biofilm formed by methicillin-resistant Staphylococcus aureus (MRSA). Methods: Powdered O. japonicus was extracted by 95% methanol, concentrated, and then, systematically fractionated with n-hexane, dichloromethane (DCM), ethyl acetate (EtOAc), n-butanol, and H2O according to polarity. Among them, the flavonoid-rich EtOAc fraction demonstrated the highest antibacterial activity and was used in this study. Using the biofilm inhibition assay, cell-surface attachment assay, confocal laser scanning microscopy, latex agglutination assay, and real time qRT-PCR, we examined whether the EtOAc fraction inhibited the formation of MRSA biofilm. Results: The EtOAc fraction exhibited distinct activity against biofilm formation and cell-surface attachment of MRSA up to 1 mg/mL through down-regulating the expression of mecA gene and the production and agglutination of penicillin-binding protein 2a as solidly observed in biofilm inhibition assay, cell-suface attachment assay, confocal laser scanning microscopy, latex agglutination assay, and real time qRT-PCR analysis. Conclusions: These results suggest that O. japonicus could be utilized as a potential resource for the development of new anti-biofilm formation of MRSA and antibacterial agents in the future.

Orostachys japonicus (O. japonicus) was extracted with ethanol (EtOH) and sequentially separated with organic solvents, including n‐hexane (Hex), dichloromethane (DCM), ethyl acetate (EtOAc), n‐butanol (BuOH), and water (H2O). All the fractions were confirmed for anti‐inflammatory activity in an inflammatory condition. The DCM fraction showed the highest anti‐inflammatory ability. Here, we examined the effect of DCM fraction and investigated the intracellular signaling pathways in LPS‐stimulated RAW 264.7 macrophage cells. The DCM fraction significantly inhibited the mRNA levels of pro‐inflammatory mediators and cytokines including iNOS, COX‐2, IL‐1β, IL‐2, IL‐6, and IP‐10 in LPS‐stimulated cells. Also, the treatment of DCM fraction excellently reduced the expression of the proteins of AP‐1 (phospho‐c‐Jun and phospho‐c‐Fos) and phospho‐IRF3 as transcription factors. As a result, it suppressed LPS‐induced inflammatory mediator and cytokines via inhibition of transcription factors. In conclusion, our data demonstrated that DCM fraction has a strong anti‐inflammatory activity that improves the inflammatory state. [ABSTRACT FROM AUTHOR]

Objective: To investigate the anti-colon cancer effects of ethylacetate fraction from Orostachys japonicus (O. japonicus) on HT-29 cancer cells. Methods: The viability of HT-29 cells was assayed by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) method. Apoptosis induction and cell cycle inhibition were confirmed by fluorescein isothiocyanate and propidium iodide staining using flow cytometry. Morphological changes in the nucleus were observed, using a fluorescence microscope with 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining. The expression levels of the upstream and downstream proteins involved in the anti-cancer mechanism were confirmed by Western blotting. Results: After treating HT-29 cells with different concentrations of ethylacetate fraction from O. japonicus, the viability of cells decreased in a concentration-dependent manner, while apoptosis induction and apoptotic body formation increased. Cell cycle analysis showed that the arrest occurred at the sub-G1 and S phase. Among the upstream and downstream proteins involved in anti-cancer activity, the level of B cell lymphoma-2 decreased, and the bcl-2-associated x protein increased. The level of pro-caspase-3, pro-caspase-8, and pro-caspase-9 decreased, while the level of cleaved-caspase-3, cleaved-caspase-8, and cleaved-caspase-9 increased. Moreover, the phosphorylation, that is, activation of extracellular signal regulated kinase 1/2, Jun-N-terminal kinase, and p38 increased. Conclusions: Combining the above results, it is thought that the survival of HT-29 cells is suppressed by ethylacetate fraction from O. japonicus through mitochondrial regulation-induced caspase cascade activation, induction of apoptosis and cell cycle arrest.

Targeted therapy at the molecular level is important for pancreatic cancer treatment. This study looked over the anticancer activity of Orostachys japonicus in a human pancreatic cancer cell line, PANC‐1. An ethyl acetate fraction containing quercetin, kaempferol, and flavonol glycosides from O. japonicus (OJE) exhibited significant anticancer activity against the PANC‐1. OJE activated caspase‐3, caspase‐8, and caspase‐9, leading to the induction of both intrinsic and extrinsic apoptosis pathways. It also inhibited cyclin D1, cyclin B1, and cyclin‐dependent kinase 4, representing cell cycle arrest at both G1/S and G2/M phases. In addition, OJE phosphorylated MAPKs such as p38, JNK, and ERK, which are important upstream signaling factors in apoptosis and arrest of cell cycle inducing system. In conclusion, OJE effectively exerted antipancreatic cancer activity via induction of apoptosis directed by both intrinsic and extrinsic pathways and arrest of cell cycle regulated at both G1/S and G2/M stages, which is activated by MAPKs, p38, JNK, and ERK. [ABSTRACT FROM AUTHOR]

Orostachys japonicus (O. japonicus) is utilized as a traditional medicine for patients with various diseases. This study investigated the effect of the ethyl acetate fraction from O. japonicus extract (OJE) on the growth inhibition of OVCAR‐3 human ovarian cancer cells demonstrated to inhibit cell growth and arrest the cell cycle in OVCAR‐3 cells by blocking the sub‐G1 phase and decreasing cyclin E1/CDK2 expression. Cell cycle arrest was connected to the increased expression of the cell cycle regulating factors p53 and p21. Apoptosis was initiated through the intrinsic pathway by up‐regulating the expression of the Bcl‐2/Bax ratio and down‐regulating the expression of pro‐caspase‐3. Furthermore, OJE treatment elicited p‐p38 activation and p‐ERK1/2 inhibition. In conclusion, our results demonstrated that OJE reduced the growth of OVCAR‐3 human ovarian cancer cells mediated by arrest of the cell cycle and regulation of MAPK signaling pathways. Orostachys japonicus (Crassulaceae), a perennial herb as a medicinal plant, has been used as a treatment of folk medication. In this study, we investigated the effect of OJE (ethyl acetate fraction from O. japonicus) on cell proliferation as well as its apoptotic pathway and cell cycle progression in the OVCAR‐3 human ovarian cancer cell line. [ABSTRACT FROM AUTHOR]

Objective: To investigate the anti-colon cancer effects of ethylacetate fraction from Orostachys japonicus (O. japonicus) on HT-29 cancer cells. Methods: The viability of HT-29 cells was assayed by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) method. Apoptosis induction and cell cycle inhibition were confirmed by fluorescein isothiocyanate and propidium iodide staining using flow cytometry. Morphological changes in the nucleus were observed, using a fluorescence microscope with 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining. The expression levels of the upstream and downstream proteins involved in the anti-cancer mechanism were confirmed by Western blotting. Results: After treating HT-29 cells with different concentrations of ethylacetate fraction from O. japonicus, the viability of cells decreased in a concentration-dependent manner, while apoptosis induction and apoptotic body formation increased. Cell cycle analysis showed that the arrest occurred at the sub-G1 and S phase. Among the upstream and downstream proteins involved in anti-cancer activity, the level of B cell lymphoma-2 decreased, and the bcl-2-associated x protein increased. The level of pro-caspase-3, pro-caspase-8, and pro-caspase-9 decreased, while the level of cleaved-caspase-3, cleaved-caspase-8, and cleaved-caspase-9 increased. Moreover, the phosphorylation, that is, activation of extracellular signal regulated kinase 1/2, Jun-N-terminal kinase, and p38 increased. Conclusions: Combining the above results, it is thought that the survival of HT-29 cells is suppressed by ethylacetate fraction from O. japonicus through mitochondrial regulation-induced caspase cascade activation, induction of apoptosis and cell cycle arrest. [ABSTRACT FROM AUTHOR]

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.

Tight junctions (TJs) are a mode of cell-to-cell adhesion in epithelial or endothelial cells, and serve as a physical barrier to maintenance of homeostasis in body by controlling paracellular transport. Claudins are the most important molecules of the TJs, but paradoxically these proteins are frequently over-expressed in cancers and their overexpression is implicated in the invasive potential of cancer. Hence, we investigated the effects of flavonoids extracted from Orostachys japonicus A. Berger (FEOJ) on TJs and the expression of claudins as well as cancer invasion along with in LnCaP human prostate cancer. FEOJ suppressed cancer cell motility and invasiveness at the concentrations where FEOJ did not show anti-proliferative activity. FEOJ increased transepithelial electrical resistance (TER) associated with tightening TJs, and suppressed expression of claudin proteins. Furthermore, FEOJ suppressed the activities of MMP-2 and -9 in a dose-dependent manner, which came from the activation of tissue inhibitor of metalloproteinases (TIMPs) by FEOJ. FEOJ suppressed migration and invasion by suppressing PI3K/Akt signaling pathway. Taken together, this study suggest that FEOJ suppresses cancer migration and invasion by tightening TJs through the suppression of claudin expression, and by suppressing MMPs in LnCaP human prostate cancer cells, which at least in part results from the suppression of PI3K/Akt signaling pathway. [ABSTRACT FROM AUTHOR]