Development of gallic acid formazans as novel enoyl acyl carrier protein reductase inhibitors for the treatment of tuberculosis.

The enoyl acyl carrier protein reductase (InhA) of Mycobacterium tuberculosis (MTB) is an attractive target for developing novel antitubercular agents. A series of gallic acid formazans, were computationally designed and docked into the active site of InhA to understand their binding mode and potential to inhibit InhA. Nine compounds from the designed series were identified as potential InhA inhibitors, on the basis of good Glide score. These compounds were synthesized in the laboratory and evaluated for in vitro antitubercular activity against drug-sensitive and multi-drug resistant strains of MTB. Out of nine compounds, three compounds exhibited the most promising MIC of <2µM against the sensitive strain of MTB, H37Rv. The compounds were evaluated against five resistant strains of MTB. Most of the compounds exhibited activity superior to the standard, linezolid, against all these resistant strains. The mechanism of action of these compounds was concluded to be InhA inhibition, through InhA enzyme inhibition study. Insignificant cytotoxicity of these compounds was observed on RAW 264.7 cell line. Inactivity of all these compounds against gram positive and gram negative bacteria indicated their specificity against MTB. The compounds were further analyzed for ADME properties and showed potential as good oral drug candidates. The results clearly identified some novel, selective and specific InhA inhibitors against sensitive and resistant strains of MTB.

Facile Formation of Redox-Active Totally Organic Nanoparticles in Water by In Situ Reduction of Organic Precursors Stabilized through Aromatic-Aromatic Interactions by Aromatic Polyelectrolytes.

The formation of redox-active, totally organic nanoparticles in water is achieved following a strategy similar to that used to form metal nanoparticles. It is based on two fundamental concepts: i) complexation through aromatic-aromatic interactions of a water-soluble precursor aromatic molecule with polyelectrolytes bearing complementary charged aromatic rings, and ii) reduction of the precursor molecule to achieve stabilized nanoparticles. Thus, formazan nanoparticles are synthesized by reduction of a tetrazolium salt with ascorbic acid using polyelectrolytes bearing benzene sulfonate residues of high linear aromatic density, but cannot be formed in the presence of nonaromatic polyelectrolytes. The red colored nanoparticles are efficiently encapsulated in calcium alginate beads, showing macroscopic homogeneity. Bleaching kinetics with chlorine show linear rates on the order of tenths of milli-meters per minute. A linear behavior of the dependence of the rate of bleaching on the chlorine concentration is found, showing the potential of the nanoparticles for chlorine sensing.

Molecular size and origin do not influence the harmful side effects of hydroxyethyl starch on human proximal tubule cells (HK-2) in vitro.

BACKGROUND: Recently, clinical trials revealed renal impairment induced by hydroxyethyl starch (HES) in septic patients. In prior studies, we managed to demonstrate that HES accumulated in renal proximal tubule cells (PTCs). The related pathomechanism has not yet been discovered. To validate our hypothesis that the HES molecule itself is harmful, regardless of its molecule size or origin, we conducted a comprehensive study to elucidate the influences of different HES preparations on PTC viability in vitro. METHODS: Cell viability of human PTC was measured with a cytotoxicity assay, quantifying the reduction of tetrazolium salt to colored formazan. Experiments were performed by assessing the influence of different carrier solutions of HES (balanced, nonbalanced, culture medium), different average molecular weights (70, 130, 200 kDa), different origins (potato or corn derived), and various durations of incubation (2-21 hours). Furthermore, HES 130/0.4 was fractionated by ultrafiltration, and the impact on cell viability of average single-size fractions with <3, 3 to 10, 10 to 30, 30 to 50, 50 to 100, and >100 kDa was investigated. We also tested the possible synergistic effects of inflammation induced by tumor necrosis factor-α. RESULTS: All tested HES solutions, regardless of origin or carrier matrix, decreased cell viability in an equivalent, dose-dependent manner. Coincubation with tumor necrosis factor-α did not reduce HES-induced reduction of cell viability. Minor differences were detected comparing 70, 130, and 200 kDa preparations. Analysis of fractionated HES revealed that each fraction decreased cell viability. Even small HES molecules (10-30 kDa) were significantly deleterious. CONCLUSIONS: For the first time, we were able to show that only the total mass of HES molecules applied is responsible for the harmful impact on renal PTC in vitro. Neither molecular size nor their origin showed any relevance.

Cytotoxicity of gold nanoparticles.

Nanomaterials are now routinely used in technical as well as medical applications. The very physicochemical properties that favor nanomaterial application are the prime cause that these materials cannot be considered "generally safe." We are still far from predicting the toxicological profile of new nanoparticles, despite continuous attempts to establish a structure-function relation between the physical and chemical properties of nanoparticles and their interactions with biological systems. Herein, we summarize some basic concept to assess nanoparticle toxicity, death pathways, cell cycle, and oxidative stress in response to nanoparticle exposure of cells.

Bioassay-guided isolation of an alkaloid with antiangiogenic and antitumor activities from the extract of Fissistigma cavaleriei root.

Fissistigma cavaleriei (Levl) Rehd (Annonaceae) is used as a folklore medicine for treatment of inflammation, arthritis, and tuberculosis by Miao people in China. In the present study, the antiangiogenic activity of F. cavaleriei was investigated. The chorioallantoic membrane of the fertilized hen's egg (CAM assay) was used to determine antiangiogenic activity of the plant extract. Compound (1), a compound with antiangiogenic activity, was isolated by bioassay-guided fractionation from F. cavaleriei for the first time. The structure of compound (1) was elucidated on the basis of spectroscopic methods. Colorimetric COX (ovine) inhibitor screening assay was used to determine its inhibitory effect on COX-1 and COX-2. MTT and Sulforhodamine B assays were used to investigate its cytotoxic effects on tumor cell lines. As a result, compound (1) showed a selectively inhibiting effect on COX-2 and could inhibit the growth of tumor cells in vitro. The antitumor activity of compound (1) was further confirmed by the observation that compound (1) administration significantly inhibited the growth of S-180 cells in mice. Moreover, compound (1) was able to enhance the antitumor activity of doxorubicin in the mice bearing with S-180 cells while combined with doxorubicin. In conclusion, compound (1) is a multi-target molecule and further experimental investigations are needed to determine whether it can be used as a lead molecule for tumor treatment.

Parthenolide, an NF-κB inhibitor, suppresses tumor growth and enhances response to chemotherapy in gastric cancer.

AIM: This study evaluated the sesquiterpene lactone parthenolide, an inhibitor of transcription factor nuclear factor-kappaB (NF-κB), in the treatment of gastric cancer in vitro and in vivo. MATERIALS AND METHODS: In vitro, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to evaluate the effect of parthenolide on growth inhibition and chemosensitization to antitumor drugs of three gastric cancer cell lines (MKN-28, MKN-45 and MKN-74). Microarray analysis was performed to identify genes which were up- or down-regulated on the treatment of parthenolide. The isobologram analysis was introduced to evaluate the synergic effect of parthenolide on antitumor drugs. In vivo, the effect of parthenolide was investigated in a mouse peritoneal dissemination model with and without chemotherapy. RESULTS: Parthenolide significantly inhibited cell growth in three gastric cancer cell lines. The phosphorylation of NF-κB was down-regulated by the treatment of parthenolide. The synergic effect of parthenolide was confirmed in combination with paclitaxel and cisplatin. In the peritoneal dissemination model, parthenolide significantly suppressed the disseminated nodules as a single agent and also enhanced chemosensitivity to paclitaxel. Furthermore, the combined therapy of parthenolide and paclitaxel significantly contributed to prolonging the survival duration. CONCLUSION: The NF-κB inhibitor, parthenolide, may enhance chemosensitivity to paclitaxel in the treatment of patients with gastric cancer.

Evaluation of the effect of Thai breadfruit's heartwood extract on the biological functions of fibroblasts from wrinkles.

In previous studies, extract from Artocarpus incisus's heartwood (breadfruit tree) had antioxidant and antimelanogenic activities. Here, we investigated the extract's action on facial skin fibroblasts from wrinkled skin and nonwrinkled skin biopsies, particularly in the production of type I procollagen and metalloproteinase- 1 (MMP-1) and in the reorganization of collagen fibers. We found that the extract at a concentration of 50 microg/ml significantly enhanced percent viability and proliferation of wrinkled-skin fibroblasts. Flow cytometry showed that a 3.6-fold increased proportion of the wrinkled-skin fibroblasts were in their cell cycle S-phase, indicating increased proliferation. Type I procollagen synthesis by wrinkled-skin fibroblasts was augmented by the extract. Nonwrinkled-skin fibroblasts had higher synthesis and were unaffected by the extract. MMP-1 secretion was greater for wrinkled-skin fibroblasts, but the extract decreased its secretion for both fi broblasts samples. Fibroblasts were incorporated in collagen lattice disks. Lattices with nonwrinkled-skin fibroblasts contracted uniformly by 56% after a three-day culture and the extract had little effect. However, wrinkled-skin fi broblast lattices failed to show appreciable contractions (to 12% after three days). But remarkably, the extract conferred an ability of the wrinkled-skin fibroblast lattices to fully contract (to 53%). This shows that wrinkled-skin fi broblasts have the ability to reorganize collagen but that the extract can reactivate this latent potential. Our findings for the first time reveal that A. incisus's heartwood extract reversed the fibroblast deficiencies in the metabolism and reorganization of collagen and may underlie a wrinkle treatment.

Sesquiterpene lactones from Inula oculus-christi.

Inula oculus-christi L. (Compositae) extract was chromatographed and three sesquiterpene lactones ergolide, gaillardin and pulchellin C were isolated. The structures of these compounds were determined by analysis of their spectroscopic data, and their crystal structures were defined using X-ray crystallography; the isolation of ergolide and pulchellin C is reported for the first time from this species. These three compounds were evaluated for their in vitro cytotoxic activity against MDBK, MCF7 and WEHI164 cells; ergolide and gaillardin exhibited lower and significantly different IC50 values compared with pulchellin C (p<0.001).

Antimutagenic and anticarcinogenic effect of methanol extracts of Petasites japonicus Maxim leaves.

The methanol extract from the leaves of Petasites japonicus Maxim (PJ) was studied for its (anti-)mutagenic effect with the SOS chromotest and reverse mutation assay. The (anti-)carcinogenic effects were evaluated by the cytotoxicity on human cancer line cells and by the function and the expression of gap junctions in rat liver epithelial cell. PJ extracts significantly decreased spontaneous beta-galactosidase activity and beta-galactosidase activity induced by a mutagen, ICR, in Salmonella (S.) typhimurium TA 1535/pSK 1002. All doses of the extract (0.08-100 mg/plate) decreased the reversion frequency induced by benzo (alpha)pyrene (BaP) in S. typhimurium TA 98. It decreased not only the spontaneous reversion frequency but also that induced by BaP in S. typhimurium TA 100. PJ extract showed greater cytotoxic effects on human stomach, colon and uterus cancer cells than on other cancer cell types and normal rat liver epithelial cells. Dye transfers though gap junctions were significantly increased by PJ extracts at concentrations greater than 200 microg/mL and the inhibition of dye transfer by 12-O-tetradecanoylphorobol-13-acetate (TPA) was obstructed in all concentrations of PJ. PJ significantly increased the numbers of gap junction protein connexin 43, and increased the protein expression decreased by TPA in a dose-dependent manner. Based on these findings, PJ is suggested to contain antimutagenic and anticarcionogenic compounds.

Antimutagenic and anticarcinogenic effect of methanol extracts of Petasites japonicus Maxim leaves

The methanol extract from the leaves of Petasites japonicus Maxim (PJ) was studied for its (anti-)mutagenic effect with the SOS chromotest and reverse mutation assay. The (anti-)carcinogenic effects were evaluated by the cytotoxicity on human cancer line cells and by the function and the expression of gap junctions in rat liver epithelial cell. PJ extracts significantly decreased spontaneous beta-galactosidase activity and beta-galactosidase activity induced by a mutagen, ICR, in Salmonella (S.) typhimurium TA 1535/pSK 1002. All doses of the extract (0.08-100 mg/plate) decreased the reversion frequency induced by benzo (alpha)pyrene (BaP) in S. typhimurium TA 98. It decreased not only the spontaneous reversion frequency but also that induced by BaP in S. typhimurium TA 100. PJ extract showed greater cytotoxic effects on human stomach, colon and uterus cancer cells than on other cancer cell types and normal rat liver epithelial cells. Dye transfers though gap junctions were significantly increased by PJ extracts at concentrations greater than 200 microg/mL and the inhibition of dye transfer by 12-O-tetradecanoylphorobol-13-acetate (TPA) was obstructed in all concentrations of PJ. PJ significantly increased the numbers of gap junction protein connexin 43, and increased the protein expression decreased by TPA in a dose-dependent manner. Based on these findings, PJ is suggested to contain antimutagenic and anticarcionogenic compounds.

Salvia somalensis essential oil as a potential cosmetic ingredient: solvent-free microwave extraction, hydrodistillation, GC-MS analysis, odour evaluation and in vitro cytotoxicity assays.

Salvia somalensis Vatke, a wild sage native of Somalia, has been studied with the aim of assessing the potential cosmetic application of its essential oil, recovered from fresh aerial parts by solvent-free microwave extraction - SFME. To evaluate the efficiency and reliability of this eco-friendly procedure, the recovery of the essential oil was also processed by conventional hydrodistillation (HD) and the results compared. The essential oils obtained by both SFME and HD were analysed by gas chromatography-mass spectrometry using apolar and polar capillary columns. The essential oil recovered by SFME was submitted to an odour evaluation that revealed peculiar olfactive characteristics interesting in alcoholic male perfumery and body detergents.In vitro cytotoxicity assays were carried out using NCTC 2544 human keratinocytes as target cells. The oil displayed slight cytotoxic effects, which were three orders of magnitude lower than those found for sodium dodecyl sulphate positive control. The promising results in terms of chemical composition, scent and safety seem to indicate this essential oil as an interesting potential functional ingredient useful in a cosmetic context.

A new anticancer dihydroflavanoid from the root of Spiranthes australis (R. Brown) Lindl.

A new dihydroflavanoid was obtained from the root of Spiranthes australis (R. Brown) Lindl, a traditional Chinese medicinal herb. The structure was elucidated as (2S)-5,2',6'-trihydroxy-6-lavandulyl-4''-(gamma,gamma-dimethylallyl)-2'',2''-dimethylpyrano-[5'',6'' : 7,8]-flavanone by spectroscopic methods including UV, IR, HR-EI-MS, ESI-MS, 1D NMR and 2D NMR techniques, and subsequently, the anticancer activities of the compound to inhibit human cancer cells' growth including A549, BEL-7402, SGC-7901, MCF-7, HT-29, K562, and A498 cell lines by MTT method was evaluated in vitro.

Inhibitory effects of natural plants of Jeju Island on elastase and MMP-1 expression.

In order to search for new active cosmetic ingredients of natural origin, we screened about 60 plants collected from Jeju Island, which is located in the southernmost part of the Republic of Korea. We investigated their free radical scavenging activity, elastase inhibition activity, and reduction of MMP-1 mRNA expression for the development of anti-aging ingredients as raw materials for use in cosmetics. In the free radical scavenging capacity assay, 12 extracts, including Typha orientalis (seed) and Torreya nucifera (leaf), showed significant free radical scavenging activity (up to SC(50)<30 microg/ml). Among these extracts, Nymphaea tetragona (rhizome) extract showed the highest free radical scavenging activity (SC(50)=4.7 microg/ml). In the anti-elastase inhibition assay, seven extracts, including Typha orientalis (seed) and Persicaria hydropiper (whole plant), showed high inhibitory activity (>50% at 100 mug/ml). Among these extracts, Persicaria hydropiper (whole plant) extract showed the highest elastase inhibition activity (IC(50) = 46.7 mug/ml). In the MMP-1 expression assay using RT-PCR, Typha orientalis (seed), Pyrrosia hastata (root), and Capsicum annum (whole plant) showed slightly lower inhibition activity than EGCG, which was used as a control. Furthermore, four extracts, including Persicaria hydropiper (whole plant), Filipendula glaberrima (root), Nymphaea tetragona (root), and Camellia japonica (leaf), completely inhibited the expression of MMP-1 in human fibroblast cells. The results showed that four of the 60 plant extracts may hold potential for use as natural active ingredients for anti-aging cosmetics.

Assay of angiotensin I-converting enzyme-inhibiting activity based on the detection of 3-hydroxybutyric acid.

Hypertension and related diseases afflict millions of individuals worldwide, and many investigations of angiotensin I-converting enzyme (ACE) activity have been carried out. Most of these have used hippuryl-histidyl-leucine (HHL) as a substrate for ACE reaction with considerable interferences. Here we propose the use of a new substrate, 3-hydroxybutyrylglycyl-glycyl-glycine (3HB-GGG) for the screening of ACE inhibitors. Under the actions of ACE and aminoacylase, 3HB-GGG is cleaved into amino acids (Gly and Gly-Gly) and 3-hydroxybutyric acid (3HB). The assay conditions were optimized and applied to monitor the ACE inhibitory activity in terms of 3HB measured using an F-kit. Under the optimum assay parameters-ACE (0.2 U/ml) and aminoacylase (172 kU/ml) incubated with 3HB-GGG (3.4 mg/ml) at 37 degrees C for 30 min-the Gly-Gly and Gly cleaved from 3HB-GGG by enzymes was able to be identified, affirming the feasibility of substituting 3HB-GGG for the conventional substrate HHL. In addition, the current method was more sensitive, accurate, rapid, and convenient than the conventional method.

LC-MS characterization of efficacy substances in serum of experimental animals treated with Sophora flavescens extracts.

Anti-DHBV (duck hepatitis B virus) activity was found in the aqueous extracts of Sophora flavescens Ait. in vivo. Liquid chromatography/electrospray ionization ion trap mass spectrometry was applied to characterize the components in duck serum after oral administration of S. flavescens extract. Oxymatrine (1), sophoranol (2), sophoridine (3) and matrine (4) were identified in the serum. Further research on the four compounds was evaluated for their antiviral activity against HBV (hepatitis B virus) in cell culture. The results suggested that oxymatrine, sophoranol and matrine were the efficacy substances for anti-HBV activity in aqueous extracts of S. flavescens Ait.

Evaluation of antimicrobial activity of selected plant extracts by rapid XTT colorimetry and bacterial enumeration.

The aim of this study was to screen and evaluate the antimicrobial activity of indigenous Jordanian plant extracts, dissolved in dimethylsulfoxide, using the rapid XTT assay and viable count methods. XTT rapid assay was used for the initial screening of antimicrobial activity for the plant extracts. Antimicrobial activity of potentially active plant extracts was further assessed using the "viable plate count" method. Four degrees of antimicrobial activity (high, moderate, weak and inactive) against Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, respectively, were recorded. The plant extracts of Hypericum triquetrifolium, Ballota undulata, Ruta chalepensis, Ononis natrix, Paronychia argentea and Marrubium vulgare had shown promising antimicrobial activity. This study showed that while both XTT and viable count methods are comparable when estimating the overall antimicrobial activity of experimental substances, there is no strong linear correlation between the two methods.

Broccoli and watercress suppress matrix metalloproteinase-9 activity and invasiveness of human MDA-MB-231 breast cancer cells.

A high dietary intake of cruciferous vegetables has been associated with a reduction in numerous human pathologies particularly cancer. In the current study, we examined the inhibitory effects of broccoli (Brassica oleracea var. italica) and watercress (Rorripa nasturtium aquaticum) extracts on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cancer cell invasion and matrix metalloproteinase-9 activity using human MDA-MB-231 breast cancer cells. Aberrant overexpression of matrix metalloproteinases, including metalloproteinase-9, is associated with increased invasive potential in cancer cell lines. Our results demonstrate that extracts of broccoli and Rorripa suppressed TPA-induced MMP-9 activity and invasiveness in a concentration dependent manner as determined by zymographic analysis. Furthermore, fractionation of individual extracts followed by liquid chromatography mass spectroscopy analysis (LC-MS) revealed that the inhibitory effects of each vegetable were associated with the presence of 4-methysulfinylbutyl (sulforaphane) and 7-methylsulphinylheptyl isothiocyanates. Taken together, our data indicate that isothiocyanates derived form broccoli and Rorripa inhibit metalloproteinase 9 activities and also suppress the invasive potential of human MDA-MB-231 breast cancer cells in vitro. The inhibitory effects observed in the current study may contribute to the suppression of carcinogenesis by diets high in cruciferous vegetables.

Lysostaphin as a potential therapeutic agent for staphylococcal biofilm eradication.

The aim was to study the activity of lysostaphin in monotherapy or in combination with oxacillin, towards biofilms built by clinical and reference S. aureus and S. epidermidis strains in the wells of microplate, in the chambers of a LabTekII chamber slide or on the polyethylene catheter. MICs of oxacillin and lysostaphin for planktonic bacteria were determined according to the standards of NCCLS. BIC (Biofilm Inhibitory Concentration) was estimated by the MTT assay. The integrity of biofilm treated with antimicrobials was also examined: by staining with FITC and laser scanning fluorescence confocal microscopy and visually by TTC reduction assay. Despite the fact that susceptibility of planktonic cultures of 25 staphylococcal strains to lysostaphin action was various, we have demonstrated the effectiveness of lysostaphin in the treatment of biofilm, built not only on the flat surface of the microplates but also on catheter's surface. The synergistic effect of subBIC lysostaphin+oxacillin was observed for MSSA and MRSA biofilms but not for 1474/01 hVISA strain. Also BICOXA for S. epidermidis RP12 and A4c strains, but not for 6756/99 MRSE biofilms was reduced when lysostaphin was simultaneously used.

Immunoenhancing properties of Plantago major leaf extract.

Plantago major (PM), also known as plantain, is a weed found in temperate zones worldwide. PM leaves have been associated with various biological properties ranging from antiinflammatory, antimicrobial and antitumour to wound healing. However, its mechanism of action associated with boosting of the immune function remains to be elucidated. We found that endotoxin-free methanol extracts from PM leaves, at doses of 50, 100, 250, and 500 microg/mL, were associated with 4.4 +/- 1, 6 +/- 1, 12 +/- 0.4, and 18 +/- 0.4-fold increases of nitric oxide (NO) production, and increased TNF-alpha production (621 +/- 31, 721 +/- 36, 727 +/- 36, and 1056 +/- 52 U/mL, respectively) by rat peritoneal macrophages, in the absence of IFN-gamma or LPS. NO and TNF-alpha production by untreated macrophages was negligible. In addition, PM extracts potentiated Con A-induced lymphoproliferation (3- to 12-fold increases) in a dose-dependent fashion, compared with the effect of Con A alone. The regulation of immune parameters induced by plant extracts may be clinically relevant in numerous diseases including chronic viral infections, tuberculosis, AIDS and cancer.

Selenium stimulates estradiol production in bovine granulosa cells: possible involvement of nitric oxide.

Reduction in fertility is well known to be possibly related to selenium deficiencies, even if target organ for selenium action is, at present, unclear. The present study was aimed to examine whether selenium directly influences granulosa cells. Bovine granulosa cells from different size follicles were used to investigate the effect of selenium (5 ng/ml), with or without bovine follicle-stimulating hormone (bFSH) (100 ng/ml), on proliferation and steroidogenesis. In addition, we sought to determine if selenium modulates the production of nitric oxide, which is known to play an important role in ovarian activity. Our data demonstrate that selenium significantly (P < 0.001) stimulates the proliferation of the cells from small follicles; moreover, it further potentiates the stimulatory effect of the gonadotropin in the same cells. Furthermore, selenium significantly (P < 0.01) augments E2 output by cells from both kinds of follicles. bFSH increases E2 production (P < 0.01) by cells from large follicles, whereas it exerts a stimulatory (P < 0.01) effect only in the presence of selenium in the cells from the small ones. The production of nitric oxide is significantly increased (P < 0.001) by bFSH, but only in cells from small follicles. Selenium inhibits (P < 0.001) nitric oxide production in cells from both kinds of follicles and significantly decreases (P < 0.001) bFSH-induced nitric oxide production in cells from the small ones. We conclude that selenium acts on granulosa cells by modulating their proliferation and E2 synthesis; moreover, its effect could be mediated, at least in part, through an inhibition of nitric oxide.