GC-MS and LC-TOF-MS profiles, toxicity, and macrophage-dependent in vitro anti-osteoporosis activity of Prunus africana (Hook f.) Kalkman Bark.

Osteoporosis affects millions of people worldwide. As such, this study assessed the macrophage-dependent in vitro anti-osteoporosis, phytochemical profile and hepatotoxicity effects in zebrafish larvae of the stem bark extracts of P. africana. Mouse bone marrow macrophages (BMM) cells were plated in 96-well plates and treated with P. africana methanolic bark extracts at concentrations of 0, 6.25, 12.5, 25, and 50 µg/ml for 24 h. The osteoclast tartrate-resistant acid phosphatase (TRAP) activity and cell viability were measured. Lipopolysaccharides (LPS) induced Nitrite (NO) and interleukin-6 (IL-6) production inhibitory effects of P. africana bark extracts (Methanolic, 150 µg/ml) and ß-sitosterol (100 µM) were conducted using RAW 264.7 cells. Additionally, inhibition of IL-1ß secretion and TRAP activity were determined for chlorogenic acid, catechin, naringenin and ß-sitosterol. For toxicity study, zebrafish larvae were exposed to different concentrations of 25, 50, 100, and 200 µg/ml P. africana methanolic, ethanolic and water bark extracts. Dimethyl sulfoxide (0.05%) was used as a negative control and tamoxifen (5 µM) and dexamethasone (40 µM or 80 µM) were positive controls. The methanolic P. africana extracts significantly inhibited (p < 0.001) TRAP activity at all concentrations and at 12.5 and 25 µg/ml, the extract exhibited significant (p < 0.05) BMM cell viability. NO production was significantly inhibited (all p < 0.0001) by the sample. IL-6 secretion was significantly inhibited by P. africana methanolic extract (p < 0.0001) and ß-sitosterol (p < 0.0001) and further, chlorogenic acid and naringenin remarkably inhibited IL-1ß production. The P. africana methanolic extract significantly inhibited RANKL-induced TRAP activity. The phytochemical study of P. africana stem bark revealed a number of chemical compounds with anti-osteoporosis activity. There was no observed hepatocyte apoptosis in the liver of zebrafish larvae. In conclusion, the stem bark of P. africana is non-toxic to the liver and its inhibition of TRAP activity makes it an important source for future anti-osteoporosis drug development.

In Vitro Antiosteoporosis Activity and Hepatotoxicity Evaluation in Zebrafish Larvae of Bark Extracts of Prunus jamasakura Medicinal Plant.

Osteoporosis is one of the main health problems in the world today characterized by low bone mass and deterioration in bone microarchitecture. In recent years, the use of natural products approach to treat it has been in the increase. In this study, in vitro antiosteoporosis activity and hepatotoxicity of P. jamasakura bark extracts were evaluated. Methods. Mouse bone marrow macrophage (BMM) cells were incubated with tartrate-resistant acid phosphate (TRAP) buffers and p-nitrophenyl phosphate and cultured with different P. jamasakura bark extracts at concentrations of 0, 6.25, 12.5, 25, and 50 µg/ml in the presence of the receptor activator of nuclear factor kappa-Β ligand (RANKL) for 6 days. The osteoclast TRAP activity and cell viability were measured. Nitric oxide (NO) assay was conducted using murine macrophage-like RAW 264.7 cells treated with P. jamasakura ethanolic and methanolic bark extracts at concentrations of 0, 6.25, 12.5, 25, 50, 100, and 200 µg/ml. For hepatotoxicity assessment, zebrafish larvae were exposed to P. jamasakura bark extracts, 0.05% dimethyl sulfoxide as a negative control, and 5 µM tamoxifen as a positive control. The surviving larvae were anesthetized and assessed for hepatocyte apoptosis. Results. TRAP activity was significantly inhibited (p < 0.001) at all concentrations of P. jamasakura extracts compared to the control treatment. At 50 µg/ml, both ethanolic and methanolic extracts of P. jamasakura exhibited significant (p < 0.01) BMM cell viability compared to the control treatment. P. jamasakura ethanolic and methanolic extracts had significant inhibitory (p < 0.01) effects on lipopolysaccharide (LPS)-induced NO production at 200 µg/ml and exhibited significant (p < 0.01) and (p < 0.05) stimulative effects, respectively, on RAW 264.7 cell viability. No overt hepatotoxicity was observed in the liver of zebrafish larvae in any of the treatments. Conclusion. The TRAP activity of P. jamasakura bark gives a foundation for further studies to enhance future development of antiosteoporosis drug.

Identification of new arylsulfide derivatives as anti-melanogenic agents in a zebrafish model.

A series of aryl sulfide derivatives was synthesized and evaluated for their anti-melanogenic activities. Several compounds, including 3e, 3i and 3q exhibited good anti-melanogenic activities. Among the derivatives, compound 3i showed good inhibitory effects against melanin synthesis and showed no toxicity in reconstituted human eye and skin tissues.

Efficacy and safety of a novel topical agent for gallstone dissolution: 2-methoxy-6-methylpyridine.

BACKGROUND: Although methyl-tertiary butyl ether (MTBE) is the only clinical topical agent for gallstone dissolution, its use is limited by its side effects mostly arising from a relatively low boiling point (55 °C). In this study, we developed the gallstone-dissolving compound containing an aromatic moiety, named 2-methoxy-6-methylpyridine (MMP) with higher boiling point (156 °C), and compared its effectiveness and toxicities with MTBE. METHODS: The dissolubility of MTBE and MMP in vitro was determined by placing human gallstones in glass containers with either solvent and, then, measuring their dry weights. Their dissolubility in vivo was determined by comparing the weights of solvent-treated gallstones and control (dimethyl sulfoxide)-treated gallstones, after directly injecting each solvent into the gallbladder in hamster models with cholesterol and pigmented gallstones. RESULTS: In the in vitro dissolution test, MMP demonstrated statistically higher dissolubility than did MTBE for cholesterol and pigmented gallstones (88.2% vs. 65.7%, 50.8% vs. 29.0%, respectively; P < 0.05). In the in vivo experiments, MMP exhibited 59.0% and 54.3% dissolubility for cholesterol and pigmented gallstones, respectively, which were significantly higher than those of MTBE (50.0% and 32.0%, respectively; P < 0.05). The immunohistochemical stains of gallbladder specimens obtained from the MMP-treated hamsters demonstrated that MMP did not significantly increase the expression of cleaved caspase 9 or significantly decrease the expression of proliferation cell nuclear antigen. CONCLUSIONS: This study demonstrated that MMP has better potential than does MTBE in dissolving gallstones, especially pigmented gallstones, while resulting in lesser toxicities.

A novel anti-melanogenic agent, KDZ-001, inhibits tyrosinase enzymatic activity.

BACKGROUND: The demand for anti-melanogenic agents is increasing due to the unwanted side effects of current treatments. To find an effective anti-melanogenic agent, we used zebrafish as a whole animal model for phenotype-based drug and cosmetic discovery screening. OBJECTIVES: The aim of this study was to identify and explore a small molecule that could be used for skin-whitening cosmetics. METHODS: Using zebrafish embryos, we examined the effects of 1000 compounds on zebrafish development and pigmentation. Pigmentation production was assessed by tyrosinase (TYR) enzymatic activity and melanin contents. Pigmentation marker expression in the human melanoma cell line HMV-II was analyzed by western blot. We also tested reconstituted human skin tissue and analyzed KDZ-001 with computational molecular modeling. RESULTS: We identified three compounds that affected the pigmentation of developing melanophores in zebrafish. Among them, we identified KDZ-001, a novel anti-melanogenic agent, which strongly inhibits melanin synthesis in the developing melanophores of zebrafish, HMV-II cells, and reconstituted human skin with no toxicity. We found that KDZ-001 directly inhibits TYR enzymatic activity. Notably, computational molecular modeling of KDZ-001 suggested that its interaction with copper ions in the active site of TYR is essential for melanin synthesis, further demonstrating that KDZ-001 mainly acts as a TYR inhibitor to synthesize melanin. CONCLUSION: KDZ-001 inhibits melanin synthesis and has a potential for use in skin-whitening cosmetics.

Expression of miRNA-122 Induced by Liver Toxicants in Zebrafish.

MicroRNA-122 (miRNA-122), also known as liver-specific miRNA, has recently been shown to be a potent biomarker in response to liver injury in mammals. The objective of this study was to examine its expression in response to toxicant treatment and acute liver damage, using the zebrafish system as an alternative model organism. For the hepatotoxicity assay, larval zebrafish were arrayed in 24-well plates. Adult zebrafish were also tested and arrayed in 200 mL cages. Animals were exposed to liver toxicants (tamoxifen or acetaminophen) at various doses, and miRNA-122 expression levels were analyzed using qRT-PCR in dissected liver, brain, heart, and intestine, separately. Our results showed no significant changes in miRNA-122 expression level in tamoxifen-treated larvae; however, miRNA-122 expression was highly induced in tamoxifen-treated adults in a tissue-specific manner. In addition, we observed a histological change in adult liver (0.5 µM) and cell death in larval liver (5 µM) at different doses of tamoxifen. These results indicated that miRNA-122 may be utilized as a liver-specific biomarker for acute liver toxicity in zebrafish.

In vitro and in vivo Bone-Forming Activity of Saururus chinensis Extract.

Bone is maintained by osteoclast-mediated resorption and osteoblast-mediated formation. Recently, anti-osteoporotic activity of Saururus chinensis extract (SCE) and anti-osteoclastogenic activity of its components have been reported, but the effect of SCE on bone formation has not been studied well. Therefore, in this study, we investigated whether Saururus chinensis SCE exhibits in vitro osteogenic and in vivo bone-forming activity. extract strongly enhanced the bone morphogenetic protein (BMP)-2-stimulated induction of alkaline phosphatase, an early phase biomarker of osteoblast differentiation, in bi-potential mesenchymal progenitor C2C12 cells. In vitro osteogenic activity of SCE was accompanied by enhanced expression of BMP-2, BMP-4, BMP-7 and BMP-9 mRNA. In addition, a pharmacological inhibition study suggested the involvement of p38 activation in the osteogenic action of SCE. Moreover, the BMP dependency and the involvement of p38 activation in the osteogenic action of SCE were confirmed by the treatment of noggin, an antagonist of BMP. Saururus chinensis extract also exhibited to induce runt-related transcription factor 2 activation at the high concentration. Furthermore, the in vivo osteogenic activity of SCE was confirmed in zebrafish and mouse calvarial bone formation models, suggesting the possibility of its use for bone formation. In conclusion, we suggested that in vivo anti-osteoporotic activity of SCE could be because of its dual action in bone, anti-osteoclastogenic and anabolic activity.