Isosteviol Derivative Inhibits Osteoclast Differentiation and Ameliorates Ovariectomy-Induced Osteoporosis.

NC-8 (ent-16-oxobeyeran-19-N-methylureido) is an isosteviol-derived analogue with multiple biological effects, including anti-inflammation and anti-bacterial activities and inhibition of HBV viral surface antigen gene expression. In this study, we explored the effects of NC-8 on the formation of osteoclasts from RAW 264.7 cells. We found that NC-8 exerts the novel effect of inhibiting osteoclast-like cell formation. Our experiments showed that RANKL-induced ERK, p38, and JNK phosphorylation were inhibited by NC-8. An ovariectomy-induced osteoporosis animal model was used to examine the protective effects of oral treatment with NC-8. Serum analysis was used to examine markers of osteoblasts, osteoclasts, and renal and hepatic function in rats. Micro CT scanning and histological analysis were used to measure bone loss in ovariectomized rats. Oral administration of NC-8 effectively decreased excess bone resorption and significantly antagonized trabecular bone loss in ovariectomized rats. Serum analysis of C-terminal telopeptide of type-I collagen, an osteoclast marker, also showed that NC-8 administration inhibited excess bone resorption. Furthermore, serum analysis showed that renal and liver function were not affected by these doses of NC-8 during long-term treatment. Our results demonstrate that NC-8 inhibits osteoclast differentiation and effectively ameliorates ovariectomy-induced osteoporosis.

Dose-dependent pharmacokinetics and tentative identification of urine metabolites from an isosteviol derivative with anti-hepatitis B activity in rats.

Compound 1 (ent-16-oxobeyeran-19-N-methylureido) is a semisynthetic isosteviol derivative that shows anti-hepatitis B virus activity in Huh7 cells by affecting viral DNA transcription and the Toll-like receptor 2/nuclear factor-κB signaling pathway. Thus, the pharmacokinetics and metabolite identification were studied as part of the discovery and development process of compound 1. The pharmacokinetics was evaluated after administration to rats at an intravenous dose of 2 mg/kg, and oral doses of 2, 5 and 10 mg/kg. Plasma concentrations were determined using LC-MS/MS. Moreover, urine samples from rats dosed at 10 mg/kg were scanned for metabolites using UPLC-QTOF-MS/MS. Results for the intravenously administered dose of 2 mg/kg showed that the area under the concentration-time curve was 65,223.31 ± 4269.80 ng min/mL, and the systemic clearance was 0.031 ± 0.0021 L/min. Oral pharmacokinetic parameters were dose-dependent, showing nonproportional increases in the oral AUCs with respective values of 4371.62 ± 3084.81, 22,472.75 ± 9103.33 and 135,141.83 ± 38,934.03 ng min/mL for 2, 5 and 10 mg/kg. The bioavailability was low at 1.5% for 2 mg/kg dose, and at 1.1% for both 5 and 10 mg/kg doses. Metabolites excreted in the urine indicate possible N-oxidation and glucuronide conjugation.

Development and validation of an LC-MS/MS method for quantification of NC-8 in rat plasma and its application to pharmacokinetic studies.

ent-16-Oxobeyeran-19-N-methylureido (NC-8) is a recently synthesized derivative of isosteviol that showed anti-hepatitis B virus (HBV) activity by disturbing replication and gene expression of the HBV and by inhibiting the host toll-like receptor 2/nuclear factor-κB signaling pathway. To study its pharmacokinetics as a part of the drug development process, a highly sensitive, rapid, and reliable liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for determining NC-8 in rat plasma. After protein precipitation extraction, the chromatographic separation of the analyte and internal standard (IS; diclofenac sodium) was performed on a reverse-phase Luna C18 column coupled with a Quattro Ultima triple quadruple mass spectrometer in the multiple-reaction monitoring mode using the transitions, m/z 347.31 â†’ 75.09 for NC-8 and m/z 295.89 â†’ 214.06 for the IS. The lower limit of quantitation was 0.5 ng/mL. The linear scope of the standard curve was between 0.5 and 500 ng/mL. Both the precision (coefficient of variation; %) and accuracy (relative error; %) were within acceptable criteria of <15%. Recoveries ranged from 104% to 113.4%, and the matrix effects (absolute) were non-significant (CV ≤ 6%). The validated method was successfully applied to investigate the pharmacokinetics of NC-8 in male Sprague-Dawley rats. The present methodology provides an analytical means to better understand the preliminary pharmacokinetics of NC-8 for investigations on further drug development.

Class 1 integrons and plasmid-mediated multiple resistance genes of the Campylobacter species from pediatric patient of a university hospital in Taiwan.

BACKGROUND: The Campylobacter species usually causes infection between humans and livestock interaction via livestock breeding. The studies of the Campylobacter species thus far in all clinical isolates were to show the many kinds of antibiotic phenomenon that were produced. Their integrons cause the induction of antibiotic resistance between bacterial species in the Campylobacter species. RESULTS: The bacterial strains from the diarrhea of pediatric patient which isolated by China Medical University Hospital storage bank. These isolates were identified by MALDI-TOF mass spectrometry. The anti-microbial susceptibility test showed that Campylobacter species resistant to cefepime, streptomycin, tobramycin and trimethoprim/sulfamethoxazole (all C. jejuni and C. coli isolates), ampicillin (89% of C. jejuni; 75% of C. coli), cefotaxime (78% of C. jejuni; 100% of C. coli), nalidixic acid (78% of C. jejuni; 100% of C. coli), tetracycline (89% of C. jejuni; 25% C. coli), ciprofloxacin (67% of C. jejuni; 50% C. coli), kanamycin (33% of C. jejuni; 75% C. coli) and the C. fetus isolate resisted to ampicillin, cefotaxime, nalidixic acid, tetracycline, ciprofloxacin, kanamycin by disc-diffusion method. The effect for ciprofloxacin and tetracycline of the Campylobacter species was tested using an E-test. The tet, erm, and integron genes were detected by PCR assay. According to the sequencing analysis (type I: dfr12-gcuF-aadA2 genes and type II: dfrA7 gene), the cassette type was identified. The most common gene cassette type (type I: 9 C. jejuni and 2 C. coli isolates; type II: 1 C. coli isolates) was found in 12 class I integrase-positive isolates. CONCLUSIONS: Our results suggested an important information in the latency of Campylobacter species with resistance genes, and irrational antimicrobial use should be concerned.

Synthesis of C-4-Substituted Steviol Derivatives and Their Inhibitory Effects against Hepatitis B Virus.

ent-13-Hydroxykaur-16-ene-19-N-butylureide (6) was one of 33 synthesized C-4-substituted steviol derivatives that were evaluated for their effects on hepatitis B virus (HBV) surface antigen (HBsAg) secretion. The IC50 (16.9 µM) and SI (57.7) values for inhibiting HBV DNA replication of compound 6 were greater than those of the reference compound, lamivudine (3-TC; IC50: 107.5 µM; SI: 22.0). Thus, the anti-HBV mechanism of 6 was investigated, and it specifically inhibited viral gene expression and reduced viral DNA levels, as well as potently attenuated all of the viral promoter activity of HBV-expressing Huh7 cells. Examination of cellular signaling pathways found that 6 inhibited the activities of the nuclear factor (NF)-κB- and activator protein (AP)-1 element-containing promoters, but had no effects on AP-2 or interferon-stimulated response element (ISRE)-containing promoters in HBV-expressing cells. Meanwhile, it significantly eliminated NF-κB and extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) signaling-related protein levels and inhibited their phosphorylation in HBV-transfected Huh7 cells. The inhibitory potency of 6 against HBV DNA replication was reversed by cotransfecting the NF-κB p65 expression plasmid. Using the MAPK-specific activator anisomycin also reversed the inhibitory effect of 6 on viral DNA replication. The present findings suggest that the anti-HBV mechanism of 6 is partly mediated through the NF-κB and MAPK signaling pathways.

The complete mitochondrial genome of Squalidus argentatus (Cypriniformes, Cyprinidae).

In the present study, the complete mitogenome sequence of the Cyprinid fish Squalidus argentatus (Cypriniformes, Cyprinidae) was determined. The mitogenome, consisting of 16,609 base pairs (bp), was characterized by the typical vertebrate mitochondrial gene arrangement, including 13 protein-coding, 22 transfer RNAs, 2 ribosomal RNAs genes and a noncoding control region. Control region (CR), of 931 bp in length, was located between tRNA(Pro) and tRNA(Phe). The overall base composition of S. argentatus was 30.45% for A, 16.70% for G, 25.62% for T and 27.24% for C, with a slight AT bias of 56.07%.

Climatic Stress during Stand Development Alters the Sign and Magnitude of Age-Related Growth Responses in a Subtropical Mountain Pine.

The modification of typical age-related growth by environmental changes is poorly understood, In part because there is a lack of consensus at individual tree level regarding age-dependent growth responses to climate warming as stands develop. To increase our current understanding about how multiple drivers of environmental change can modify growth responses as trees age we used tree ring data of a mountain subtropical pine species along an altitudinal gradient covering more than 2,200 m of altitude. We applied mixed-linear models to determine how absolute and relative age-dependent growth varies depending on stand development; and to quantify the relative importance of tree age and climate on individual tree growth responses. Tree age was the most important factor for tree growth in models parameterised using data from all forest developmental stages. Contrastingly, the relationship found between tree age and growth became non-significant in models parameterised using data corresponding to mature stages. These results suggest that although absolute tree growth can continuously increase along tree size when trees reach maturity age had no effect on growth. Tree growth was strongly reduced under increased annual temperature, leading to more constant age-related growth responses. Furthermore, young trees were the most sensitive to reductions in relative growth rates, but absolute growth was strongly reduced under increased temperature in old trees. Our results help to reconcile previous contrasting findings of age-related growth responses at the individual tree level, suggesting that the sign and magnitude of age-related growth responses vary with stand development. The different responses found to climate for absolute and relative growth rates suggest that young trees are particularly vulnerable under warming climate, but reduced absolute growth in old trees could alter the species' potential as a carbon sink in the future.

Synthesis and anti-hepatitis B virus activity of C4 amide-substituted isosteviol derivatives.

A series of novel isosteviol derivatives having C4-amide substituents were synthesized in order to test for antiviral effects against the hepatitis B virus (HBV) in vitro. Among them, IN-4 [N-(propylcarbonyl)-4α-amino-19-nor-ent-16-ketobeyeran] (5) exhibited inhibitory activity against secretion of HBsAg and HBeAg as well as inhibition of HBV DNA replication. Therefore, the mechanism of its antiviral activity was further analyzed using HBV-transfected Huh7 cells. Exposure to IN-4 produced minimal inhibitory effects on viral precore/pregenomic RNA expression. However, expression levels of the 2.4/2.1-kb preS/major S RNA of the viral surface gene significantly decreased, along with intracellular levels of HBV DNA. A promoter activity analysis demonstrated that IN-4 significantly inhibited viral X, S, and preS expression levels but not viral core promoter activities. In particular, IN-4 was observed to significantly inhibit HBV gene regulation by disrupting nuclear factor (NF)-κB-associated promoter activity. In addition, the nuclear expression of p65/p50 NF-κB member proteins was attenuated following IN-4 treatment, while cytoplasmic IκBα protein levels were enhanced. Meanwhile, IN-4 was observed to inhibit the binding activity of NF-κB to putative DNA elements. Furthermore, transfection of a p65 expression plasmid into Huh7 cells significantly reversed the inhibitory effect of IN-4 on HBV DNA levels, providing further evidence of the central role of NF-κB in its antiviral mechanism. It is therefore suggested that IN-4 inhibits HBV by interfering with the NF-κB signaling pathway, resulting in downregulation of viral gene expression and DNA replication.

Design, synthesis, and bioevaluation of paeonol derivatives as potential anti-HBV agents.

Hepatitis B virus (HBV) is a causative reagent that frequently causes progressive liver diseases, leading to the development of acute, chronic hepatitis, cirrhosis, and eventually hepatocellular carcinoma (HCC). Despite several antiviral drugs including interferon-α and nucleotide derivatives are approved for clinical treatment for HBV, critical issues remain unresolved, e.g., low-to-moderate efficacy, adverse side effects, and resistant strains. In this study, novel Paeonol-phenylsulfonyl derivatives were synthesized and their antiviral effect against HBV was evaluated. The experimental results indicated that these compounds process significant antiviral potential, including the inhibition of viral antigen expression and secretion, and the suppression of HBV viral DNA replication. Among compounds synthesized in this research, compound 2-acetyl-5-methoxyphenyl 4-methoxybenzenesulfonate (7f) had the most potent inhibitory activity with IC50 value of 0.36 µM, and high selectivity index, SI (TC50/IC50) 47.75; which exhibited an apparent inhibition effect on viral gene expression and viral propagation in cell culture model. So, we believe our compounds could serve as reservoir for antiviral drug development.

Anti-viral effect of a compound isolated from Liriope platyphylla against hepatitis B virus in vitro.

The compound LPRP-Et-97543 was isolated from Liriope platyphylla roots and was observed to have potential anti-viral effects in HepG2.2.15 cells against hepatitis B virus (HBV). The antiviral mode was further clarified, and the HBV-transfected Huh7 cells were used as the platform. During viral gene expression, LPRP-Et-97543 treatment had apparent effects on the viral precore/pregenomic and S/preS RNA. Promoter activity analysis demonstrated that LPRP-Et-97543 significantly reduced Core, S, and preS but not X promoter activities. Further examination showed that putative signaling pathways were involved in this inhibitory effect, indicating that NF-κB may serve a putative mediator of HBV gene regulation with LPRP-Et-97543. In addition, the nuclear expression of p65/p50 NF-κB member proteins was attenuated with LPRP-Et-97543 and augmented cytoplasmic IκBα protein levels but without affecting the expression of these proteins in HBV non-transfected cells during treatment. Moreover, LPRP-Et-97543 reduced the binding activity of NF-κB protein to CS1 element of HBV surface gene in a gel retardation analysis and inhibited CS1 containing promoter activity in HBV expressed cells. However, HBV transfection significantly enhances CS1 containing promoter activity without compound treatment in cells. Finally, transfection of the p65 expression plasmid significantly reversed the inhibitory effect of LPRP-Et-97543 on the replicated HBV DNA level in HBV positive cells. In conclusion, this study suggests that the mechanism of HBV inhibition by LPRP-Et-97543 may involve the feedback regulation of viral gene expression and viral DNA replication by HBV viral proteins, which interferes with the NF-κB signaling pathway.