Silver-dendrimer nanocomposite as emerging therapeutics in anti-bacteria and beyond.

To develop next-generation nanomedicine, theranostic nanotherapeutic strategies are increasingly being emphasized. In recent years, it is observed that the effective lifetime of anti-bacterial and anti-cancer agent is diminishing, which undermines the economic incentives necessary for clinical development and therapeutic applications. Thus, novel formulations ought to not only kill drug resistant strains and cancerous cells but also inhibit their formation. Recently, metallic nanoparticles [for example- silver (Ag) nanoparticles] have been widely investigated for their biomedical applications. The so-called applications necessitate the inclusion of these nanoparticles inside polymeric matrices (for example- dendrimer) leading to chemical functionalization of the metallic nanoparticles. Silver and silver nanoparticles' antibacterial activity has already been well established over years. Dendrimers due to their homogeneous highly branched structure and uniform composition are perfectly suitable for the inclusion of silver nanoparticles [Ag NPs]. Recently, the increasing trend in the development of Ag-dendrimer nanocomposites is attributed to the excellent antibacterial activity of Ag as well as dendrimer's unique properties like variable functional terminal ends and potential antibacterial effect necessarily. This review provides an informative overview regarding the numerous aspects of bactericidal and other biomedical applications of Ag-dendrimer nanocomposites, particularly emphasizing analysis of existing research and prospective worth to the pharmaceutical sector in future.

[Anti-complement alkaloids from whole plants of Viola yedoensis].

Fifteen alkaloids were isolated from the 95% ethanol extract of the whole plants of Viola yedoensis by various column chromatographic techniques such as silica gel and Sephadex LH-20. Their structures were identified as neoechinulin A（1）,N-benzoyl-L-p-hydroxy-phenylalaninol（2）,aurantiamide acetate（3）,aurantiamide（4）,anabellamide（5）,trichosanatine（6）,indole-3-carboxylic acid methyl ester（7）,3-carboxyindole（8）,N-trans-feruloyl-tyramine（9）,paprazine（10）,7'-（3', 4'-dihydroxyphenyl）-N-[(4-methoxyphenyl)ethyl]propenamide（11）,cannabisin F（12）,N-(4-hydroxyphenethyl)octacosanamide（13）,N-(4-hydroxyphenethyl)hexacosanamide（14）and N-benzoyl-L-phenylalaninol（15）. All the compounds except 3 and 4 were isolated from this plant for the first time. These alkaloids exhibited anti-complement activity against the classical pathway(CP)and the alternative pathway(AP)with the CH50 and AP50 values ranging from 0.12 to 0.33 g•L⁻¹ and 0.22 to 0.50 g•L⁻¹, respectively. Preliminary mechanism study using complement-depleted sera showed that these alkaloids acted on different complement components in the complement activation cascade.

Anti-Complementary Components of Helicteres angustifolia.

A first phenalenon derivative with an acetyl side chain at C-8, 8-acetyl-9-hydroxy-3-methoxy-7-methyl-1-phenalenon (compound 1), and a pair of new sesquilignan epimers at C-7″ of hedyotol C and hedyotol D analogs, hedyotol C 7″-O-ß-d-glucopyranoside (compound 2) and hedyotol D 7″-O-ß-d-glucopyranoside (compound 3) were isolated from the aerial parts of Helicteres angustifolia together with nine known compounds (4-12). Their structures were elucidated on the basis of spectroscopic methods, including mass spectroscopy, and 1D and 2D nuclear magnetic resonance. Eleven isolates exhibited anti-complementary activity. In particular, compounds 4 and 5 exhibited potent anti-complementary activities against the classical and alternative pathways with CH50 values of 0.040 ± 0.009 and 0.009 ± 0.002 mM, and AP50 values of 0.105 ± 0.015 and 0.021 ± 0.003 mM, respectively. The targets of compounds 4 and 5 in the complement activation cascade were also identified. In conclusion, the anti-complementary components of H. angustifolia possessed chemical diversity and consisted mostly of flavonoids and lignans in this study.

Anticomplement monoterpenoid glucosides from the root bark of Paeonia suffruticosa.

Six new (1-6) and 19 known monoterpenoid glucosides were isolated from the root bark of Paeonia suffruticosa. The monoterpenoid glucosides 1, 2, 7, 10-19, and 22 exhibited anticomplement effects with CH50 and AP50 values ranging from 0.14 to 2.67 mM and 0.25 to 3.67 mM, respectively. In a mechanistic study, suffrupaeoniflorin A (1) interacted with C1q, C3, C5, and C9, while galloylpaeoniflorin (12) and galloyloxypaeoniflorin (19) acted on C1q, C3, and C5 components in the complement activation cascade.

Quantitative methylation analysis of multiple genes using methylation-sensitive restriction enzyme-based quantitative PCR for the detection of hepatocellular carcinoma.

DNA methylation is a promising biomarker for cancer. This study was aimed at investigating the methylation levels of multiple genes in hepatocellular carcinoma (HCC) and to identify a combination of methylation markers that would be useful for the diagnosis of HCC. The methylation status of a panel of nine tumor-associated genes (APC, GSTP1, RASSF1A, CDKN2A, SFRP1, RUNX3, SOCS1, Hint1, and HIC-1) in 8 normal liver tissues and 47 paired HCCs and non-tumorous tissues (NTs) was determined using a modified methylation-sensitive, restriction enzyme-based quantitative PCR (MSRE-qPCR) method. The methylation levels of six genes (APC, CDKN2A, GSTP1, RASSF1A, SFRP1 and RUNX3) were significantly higher in HCCs than in adjacent NTs (P<0.05). Although the AUC (area under the curve) for each individual gene was low to moderate (range: 0.576 to 0.835) according to receiver operator characteristic (ROC) curve analysis, the combination analysis of these six genes resulted in an increase of AUC of 0.954 with 85.1% sensitivity, 89.4% specificity, 88.9% positive predictive value, and 85.7% negative predictive value in discriminating HCC tissues from NT tissues. These results indicate that the analysis of a combination of these six methylated genes may be a promising method for the risk assessment and diagnosis of HCC.

Quantitative analysis of multiple methylated genes in plasma for the diagnosis and prognosis of hepatocellular carcinoma.

This study was aimed to evaluate the clinical value of plasma methylation analysis of a panel of four genes (APC, GSTP1, RASSF1A, and SFRP1), which was identified by our previous work, for the noninvasive diagnosis of hepatocellular carcinoma (HCC). The methylation status of these four genes in 150 plasma samples from 72 patients with HCC, 37 benign live diseases and 41 normal controls was detected with methylation-sensitive restriction enzymes-based quantitative PCR (MSRE-qPCR) method. The plasma methylation levels of APC, GSTP1, RASSF1A, and SFRP1 were significantly higher in HCCs than those in normal or benign controls (P<0.05). Although the area under the receiver-operation characteristic curve (AUC-ROC) for individual gene was moderate (range, from 0.800 to 0.881), the combination analysis of these four genes resulted in an increased AUC of 0.933 with 92.7% sensitivity, 81.9% specificity, 90.5% positive predictive value (PPV), and 87.2% negative predictive value (NPV) in discriminating HCC from normal control. The combination analysis also indicated an increased AUC of 0.877 when compared with individual gene (from 0.666 to 0.850) in discriminating HCC from benign control, and the consultant sensitivity, specificity, PPV, and NPV was 84.7%, 81.1%, 89.7%, and 73.2%, respectively. Patients with elevated plasma methylation levels of APC or RASSF1A showed poorer overall survival than those with low levels (P<0.05). Cox multivariate analysis demonstrated methylated RASSF1A in plasma to be an independent prognostic factor for overall survival (hazard ratio=3.262, 95% CI: 1.476-7.209, P=0.003). These data showed that quantitative analysis of multiple methylated genes in plasma may be a promising tool for noninvasive diagnosis of HCC; and methylated plasma RASSF1A appears to be a prognostic marker of HCC.

Correlation Study of Galectin-3 in Patients with an Ascending Aortic Aneurysm and Ventricular Remodeling Before and After Surgical Correction.

OBJECTIVE: The present study aims to observe the changes in galectin-3 (Gal-3) expression levels in patients with an ascending aortic aneurysm and ventricular remodeling and analyze Gal-3's correlation with ventricular remodeling. METHODS: A total of 102 patients with an ascending aortic aneurysm were included as the research subjects. Gal-3 expression levels in the peripheral blood of the patients were detected by an enzyme-linked immunosorbent assay before the operation and then three and six months after. The left ventricular ejection fraction (LVEF), left ventricular end-diastolic diameter (LVEDD), interventricular septal thickness, and left ventricular posterior wall thickness were recorded, and the left ventricular mass index (LVMI) was calculated. Changes in Gal-3 expression levels, LVMI, LVEF, and LVEDD were observed before and after surgery, and these changes were then analyzed. RESULTS: There were significant differences in Gal-3 expression levels, LVMI, and LVEDD before surgery and three months after (P < 0.001) but no significant difference in LVEF (P = 0.887). There were significant differences in Gal-3 expression levels, LVMI, LVEDD, and LVEF (P < 0.05) three and six months after surgery. Before surgery and three and six months after surgery, Gal-3 was positively correlated with LVMI and LVEDD (R = 0.697, R = 0.571, and R = 0.454, respectively), and a receiver operating characteristic curve found that Gal-3 was able to predict ventricular remodeling, with an area under the curve value of 0.721. CONCLUSION: Gal-3 expression levels are correlated with ascending aortic aneurysms combined with ventricular remodeling, which provides a reference value for predicting ventricular remodeling.

Flavonoids rather than alkaloids as the diagnostic constituents to distinguish Sophorae Flavescentis Radix from Sophorae Tonkinensis Radix et Rhizoma: an HPLC fingerprint study.

Sophorae Flavescentis Radix (Sophora flavescens Ait., SFR) and Sophorae Tonkinensis Radix et Rhizoma (S. tonkinensis Gapnep., STR) are two commonly used traditional Chinese medicines from Sophora (Leguminosae) plants, which are believed to possess similar bioactive components with entirely different clinical applications. In order to find out the characteristic chemical constituents potentially leading to the unique medicinal properties claimed for each of the two closely related TCMs, an HPLC fingerprint method was developed for analyses of the alkaloid and flavonoid constituents of SFR and STR, respectively, which were further evaluated and compared through similarity calculation and hierarchical clustering analysis (HCA). The results from the present study showed that the alkaloid fingerprints of the two herbs were similar, with many components co-existing in both drugs and various batches of samples from different species being mixed together in the HCA dendrogram. However, their flavonoid constituents were totally different with specific fingerprints being yielded for each herb, and further HCA analysis showed that the tested samples could almost be clearly divided into two groups based on their origins of species. The results from the present study indicated that the flavonoid constituents could serve as the differentially diagnostic constituents of SFR and STR and might potentially attributed to their distinct therapeutic effects.

Preparative separation and quantitative determination of two kaurenoic acid isomers in root barks of Acanthopanax gracilistylus.

The kaurenoic acid-type diterpenoids in Acanthopanacis Cortex have been reported to be the major active components. However, the diterpenoids are present as position isomers that exacerbate the challenges in obtaining standards compounds. Little work has been done on the quantitative analysis of the diterpenoids in the herb. In the present study, two diterpenoid isomers ent-16ßH,17-isovalerate-kauran-19-oic acid (1) and ent-16ßH,17-methyl butanoate-kauran-19-oic acid (2) with high purity were separated by analytical HPLC, followed by recrystallization in acetone. Furthermore, an HPLC-ELSD method was developed and validated for simultaneous determination of 1 and 2 in 9 batches of Acanthopanacis Cortex samples. The HPLC separation and quantification was achieved in 40 min using an Agela Promosil C18 column eluted with a gradient of water and acetonitrile. The calibration curves showed good linearity (r2 ≥ 0.999 9) within the test ranges. The LOD ranged from 0.407 2 to 0.518 0 µg and LOQ ranged from 1.018 0 to 1.295 0 µg. The precisions (%RSD) were within 1.47% for the two isomers. The recovery of the assay was in the range of 98.78%-99.11% with RSD values less than 2.76%. It is the first time to establish a quantitative HPLC method for the analysis of the bioactive kaurenoic acid isomers in the herb.

Iridoid glycosides from Harpagophytum procumbens D.C. (devil's claw).

Iridoid glycosides, harprocumbide A (6''-O-alpha-D-galactopyranosylharpagoside, 1) and harprocumbide B (6''-O-(cis-p-coumaroyl)-procumbide, 2) were isolated from the tubers of Harpagophytum prucumbens D.C., along with nine known iridoid glycosides 6-O-alpha-D-galactopyranosylharpagoside (3), and harpagoside (4), harpagide (5), 8-cinnamoylmyoporoside (6), 8-O-feruloylhapagide (7), procumbide (8), 6''-O-(p-coumaroyl)-procumbide (9), 8-O-(p-coumaroyl)-harpagide (10) and 8-O-(cis-p-coumaroyl)-harpagide (11). Compound 10 showed marginal inhibition activity against macrophages respiratory burst.

Regio- and stereo-selective hydroxylations of ingenane diterpenoids by Mortierella ramanniana and Gibberella fujikuroi.

The regio- and stereo-selective hydroxylations of two ingenane diterpenoids, 20-deoxyingenol (1) and 13-oxyingenol dodecanoat (2), by the filamentous fungi Mortierella ramanniana and Gibberella fujikuroi were investigated in the present study. Four undescribed metabolites (3-6) of substrate 1 and two undescribed metabolites (7 and 8) of substrate 2 were isolated. All the metabolites were identified as hydroxylated ingenane derivatives by extensive NMR and HR-ESI-MS data analyses. All the biotransformed compounds and the substrates were evaluated for their cytotoxicities against three human cancer cell lines, including human colon cancer Caco-2, breast cancer MCF-7, and adriamycin (ADM)-resistant MCF-7/ADM cell lines. All ingenane alcohols (1, and 3-6) displayed no significant cytotoxic activities. The substrate 13-oxyingenol dodecanoat (2) showed moderate cytotoxicity with IC50 values being 35.59 ± 5.37 µmol·L-1 (Caco-2), 24.04 ± 4.70 µmol·L-1 (MCF-7), and 22.24 ± 5.19 µmol·L-1 (MCF-7/ADM). However, metabolites 7 and 8 displayed no significant cytotoxicity. These results indicated that the hydroxylation at the C-13 aliphatic acid ester of substrate 2 can significantly reduce the cytotoxic activity.

Immobilized carbon nanotubes as matrix for MALDI-TOF-MS analysis: applications to neutral small carbohydrates.

In this work, we reported on the advantages of immobilized carbon nanotubes as a novel MALDI-matrix. Recently, carbon nanotubes have been reported to be an effective MALDI matrix for small molecules (Anal. Chem.2003, 75, 6191), as it can eliminate the interfering matrix peaks as well as form a web morphology to fully disperse the analyte and allow strong ultraviolet absorption for enhanced pulsed laser desorption and ionization. In our study, to overcome the problem that the carbon nanotube matrix may fly off from the target, a type of polyurethane adhesive, NIPPOLAN-DC-205, is introduced to immobilize carbon nanotubes on the target, which enables widespread application of carbon nanotubes as matrix for MALDI-MS analysis. At the same time, the properties of the carbon nanotubes as an efficient matrix remained after immobilization. The presence of NIPPOLAN-DC-205 increases the time for analysis at a particular desorption spot by minimizing the time-consuming search for "hot spots" and facilitating experiments such as post source decay (PSD) which need longer-lasting signals. Moreover, NIPPOLAN-DC-205 produces no interference peaks and can easily be cleaned with acetone. Fast evaporation technology may be used to enhance signal reproducibility in MALDI analysis using carbon nanotubes as matrix. Consequently, the applicability of the carbon nanotube as matrix for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of low molecular mass analytes is highly improved. The feasibility of the method employing polyurethane is demonstrated by comparison of the results produced from the carbon nanotube matrix with and without immobilization. In addition, neutral small carbohydrates, which are difficult to be ionized normally, can be cationized with high efficiency by MALDI-TOF-MS using the immobilized carbon nanotube matrix. The method was further applied to analyze peptides and detect urine glucose successfully.

[A study on quality standard for Herba Siegesbeckidae].

OBJECTIVE: To establish the qualitative and quantitative methods of Herba Siegesbeckiae. METHOD: A TLC method was used for qualitative identification and a HPLC analysis was applied for quantitative determination of Herba Siegesbeckiae with kirenol as the reference substances. RESULT: Chloroform-methanol-formic acid (25:5:1) as a mobile phase of TLC, the spot of kirenol can be easily detected; Methanol extracts of Herba Siegebeckiae were separated on a Polaris C18 column with acetonitrile-water (25:75) as mobile phase and kirenol was separated well. The average content of kirenol in Herba Siegebeckiae was 0.14%. A good linear relationship between the peak areas and injected amounts of kirenol in the range of 0.19-14.9 microg and the average recovery was 100.0% (RSD = 2.4%). CONCLUSION: The method can be used for qualitative identification and quantitation determination of Herba Siegesbeckiae.

[Identification of the fumatory Radix Bupleuri with sulfur by static headspace GC-MS].

OBJECTIVE: To identify whether Radix Bupleuri (Bupleurum chinense) was fumed with sulfur. METHOD: A static headspace GC-MS method was used to detect sulfur in the fumatory Radix Bupleuri, the authentic samples free of sulfur was detected as reference. RESULT: Sulfur was detected in six samples from nine samples collected in different locations. CONCLUSION: The method can be used to detect sulfur rapidly in the fumatory Radix Bupleuri with sulfur.

ING4 induces G2/M cell cycle arrest and enhances the chemosensitivity to DNA-damage agents in HepG2 cells.

The known members of inhibitor of growth (ING) gene family are considered as candidate tumor suppressor genes. ING4, a novel member of ING family, is recently reported to interact with tumor suppressor p53, p300 (a major component of histone acetyl transferase complexes), and p65(RelA) subunit of NF-kappaB. In this study, we investigated the cellular behaviors of HepG2 cells with exogenous ING4. Interestingly, the overexpression of ING4 negatively regulated the cell growth with significant G2/M arrest of cell cycle, and moreover, enhanced the cell apoptosis triggered by serum starvation in HepG2 cells. Furthermore, the exogenous ING4 could upregulate endogenous p21 and Bax in HepG2 cells, not in p53-deficient Saos-2 cells, suggesting that G2/M arrest induced by ING4 could be mediated by the increased p21 expression in a p53-dependent manner, although there is no significant increase of p53 expression in HepG2 cells. Moreover, HepG2 cells with exogenous ING4 could significantly increase cell death, as exposed to some DNA-damage agents, such as etoposide and doxorubicin, implying that ING4 could enhance chemosensitivity to certain DNA-damage agents in HepG2 cells.

Biotransformation of quinovic acid glycosides by microbes: direct conversion of the ursane to the oleanane triterpene skeleton by Nocardia sp. NRRL 5646.

[reaction: see text] Quinovic acid glycosides were microbially deglycosylated by a Nocardia sp. to their aglycon quinovic acid and its biogenetic counterpart, cincholic acid (3), via an unprecedented carbon skeleton rearrangement involving a methyl group migration. The structures of the metabolites were established by ESI-LC/MS and 2D-NMR techniques.

27-Nor-triterpenoid glycosides from Mitragyna inermis.

From the bark of Mitragyna inermis, two 27-nor-triterpenoid glycosides, named inermiside I (1) and II (2), were isolated and their structures determined based on extensive 2D-NMR and MS spectral analysis as 6-deoxy-beta-D-glucopyranosyl-[3-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl]-pyrocincholate and 6-deoxy-beta-D-glucopyranosyl-pyrocincholate, respectively. In addition, the known quinovic acid (6), 3-O-[beta-D-glucopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl]-quinovoic acid (3),beta-D-glucopyranosyl-[3-O-(beta-D-glucopyranosyl)]-quinoviate (4) and cytotoxic 3-O-(beta-D-6-deoxy-glucopyranosyl)-quinovic acid (5) were also isolated.

Establishment of an artificial beta-cell line expressing insulin under the control of doxycycline.

AIM: Artificial beta-cell lines may offer an abundant source of cells for the treatment of type I diabetes, but insulin secretion in beta-cells is tightly regulated in physiological conditions. The Tet-On system is a "gene switch" system, which can induce gene expression by administration of tetracycline (Tet) derivatives such as doxcycline (Dox). Using this system, we established 293 cells to an artificial cell line secreting insulin in response to stimulation by Dox. METHODS: The mutated proinsulin cDNA was obtained from plasmid pcDNA3.1/C-mINS by the polymerase chain reaction (PCR), and was inserted downstream from the promoter on the expression vector pTRE2, to construct a recombined expression vector pTRE2mINS. The promoter on pTRE2 consists of the tetracycline-response element and the CMV minimal promoter and is thus activated by the reverse tetracycline-controlled transactivator (rtTA) when Dox is administrated. pTRE2mINS and plasmid pTK-Hyg encoding hygromycin were co-transfected in the tet293 cells, which express rtTA stably. Following hygromycin screening, the survived cells expressing insulin were selected and enriched. Dox was used to control the expression of insulin in these cells. At the levels of mRNA and protein, the regulating effect of Dox in culture medium on the expression of proinsulin gene was estimated respectively with Northern blot, RT-PCR, and radioimmunoassay. RESULTS: From the 28 hygromycin-resistant cell strains, we selected one cell strain (tet293/Ins6) secreting insulin not only automatically, but in response to stimulation by Dox. The amount on insulin secretion was dependent on the Dox dose (0,10,100,200,400,800 and 1000 microg.L(-1)), the level of insulin secreted by the cells treated with Dox (1000 microg.L(-1)) was 241.0pU.d(-1).cell(-1) , which was 25-fold that of 9.7pU.d(-1).cell(-1) without Dox treatment. Northern blot analyses and RT-PCR further confirmed that the transcription of insulin gene had already been up-regulated after exposing tet293/Ins6 cells to Dox for 15 minutes, and was also induced in a dose-dependent manner. However, the concentration of insulin in the media did not increase significantly until 5 hours following the addition of Dox. CONCLUSION: Human proinsulin gene was transfected successfully and expressed efficiently in 293 cells, and the expression was modulated by tetracycline and its derivatives, improving the accuracy, safety, and reliability of gene therapy, suggesting that conditional establishment of artificial beta-cells may be a useful approach to develop cellular therapy for diabetes mellitus.

[Triterpenoid saponins from bark Mitragyna inermis].

OBJECTIVE: To study the constituents of Mitragyna inermis that have an anti-tumor activity on Bel-7402 of hepatic carcinoma. METHOD: The compounds were isolated by column chromatography on silica gel, ODS, Sephadex LH-20 separately and their structures were elucidated by chemical and spectral technology. RESULT: Three quinovic acid glycosides, quinovic acid 3 beta-beta-D-glucopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl-28-O-beta- D-glucopyranoside(I), quinovic acid 3 beta-O-beta-D-quinovopyranoside(II), quinovic acid 3 beta-O-beta-D-glucopyranoside(III), were obtained. CONCLUSION: I, II were isolated from Mitragyna for the first time.