Suppression of hepatitis C virus by the flavonoid quercetin is mediated by inhibition of NS3 protease activity.

Phytochemicals exert antiviral activity and may play a potential therapeutic role in hepatitis C virus (HCV) infection. In this work, we aimed to isolate NS3 inhibitors from traditional Indian medicinal plants that were found, in our earlier study, to inhibit HCV NS3 protease activity and to evaluate their potential to inhibit HCV replication. A potent inhibitory effect of NS3 catalytic activity was obtained with Embelia ribes plant extracts. Quercetin, a ubiquitous plant flavonoid, was identified as the active substance in the fractioned extract. It was found to inhibit NS3 activity in a specific dose-dependent manner in an in vitro catalysis assay. Quercetin inhibited HCV RNA replication as analysed in the subgenomic HCV RNA replicon system. It also inhibited HCV infectious virus production in the HCV infectious cell culture system (HCVcc), as analysed by the focus-forming unit reduction assay and HCV RNA real-time PCR. The inhibitory effect of quercetin was also obtained when using a model system in which NS3 engineered substrates were introduced in NS3-expressing cells, providing evidence that inhibition in vivo could be directed to the NS3 and do not involve other HCV proteins. Our work demonstrates that quercetin has a direct inhibitory effect on the HCV NS3 protease. These results point to the potential of quercetin as a natural nontoxic anti-HCV agent reducing viral production by inhibiting both NS3 and heat shock proteins essential for HCV replication.

Dried leaves of Rosmarinus officinalis as a treatment for streptococcosis in tilapia.

Dietary application of dried Rosmarinus officinalis (rosemary) leaves as a treatment for streptococcal infection was studied in tilapia, Oreochromis sp. Feeding with dried rosemary leaves significantly reduced mortality following infection with Streptococcus iniae: 44% mortality in the group fed 8% rosemary, similar to oxytetracycline treatment (43% mortality), and significantly lower than the control (65%). Dietary administration of 16% rosemary significantly reduced mortality because of Streptococcus agalactiae infection in 44 g fish (62% and 76% in 16% rosemary and control, respectively), but not in a similar experiment conducted with 5.5 g fish. The antibacterial effect of rosemary on S. iniae was studied. Activity of rosemary cultivar Israel was reduced during the winter, but there was no significant change in cultivars Oranit and Star. Storage of powdered rosemary leaves at 50 degrees C resulted in fourfold and eightfold higher MIC(24 h) values after 3 and 4.5 months, respectively. Storage at -20 degrees C, 4 degrees C and 25 degrees C and autoclaving (120 degrees C) each resulted in a twofold increase in MIC(24 h). Repeated exposures of S. iniae to rosemary did not affect minimal inhibitory concentration, suggesting no development of resistance to rosemary.

Anti-inflammatory effect of a Nuphar lutea partially purified leaf extract in murine models of septic shock.

ETHNOPHARMACOLOGICAL RELEVANCE: Various plant organs of Nuphar lutea (L.) SM. (Nymphaeaceae) are used in traditional medicine for the treatment of arthritis, fever, aches, pains and inflammation. The main purpose of this study was to determine the anti-inflammatory effect of Nuphar lutea leaf extract (NUP) in two septic shock models: (1) Survival of mice challenged with a lethal dose of LPS, determination of pro-inflammatory and anti-inflammatory cytokines in serum, as well as in peritoneal macrophages in cell culture. (2) The effect of NUP in a murine model of fecal-induced peritonitis. MATERIALS AND METHODS: NUP pre-treatment partially protected mice in two models of acute septic shock. We concluded that NUP is anti-inflammatory by inhibiting the NF-κB pathway, modulating cytokine production and ERK phosphorylation. RESULTS: A significant average survival rate (60%) of LPS lethally-challenged mice was achieved by pre-treatment with NUP. In addition, NUP pre-treatment reduced nuclear NF-κB translocation in peritoneal macrophages. The production of pro-inflammatory cytokines, TNF-α, IL-6 and IL-12, in the sera of LPS-treated mice or in the supernatants of peritoneal macrophages stimulated with LPS for 2-6 h was also decreased by NUP. Pre-treatment with NUP caused a significant increase in the anti-inflammatory cytokine IL-10. The NUP pre-treatment reduced and delayed mortality in mice with fecal-induced peritonitis. Our studies also revealed that NUP pre-treatment induced a dose-dependent phosphorylation of ERK in peritoneal macrophages. Since most of the reports about the anti-inflammatory effect of Nuphar lutea refer to rhizome and root powder and extracts, it is important to clarify the effectiveness of leaf extract as a source for such activity. CONCLUSION: NUP pre-treatment partially protected mice in two models of acute septic shock. We concluded that NUP is anti-inflammatory by inhibiting the NF-κB pathway, modulating cytokine production and ERK phosphorylation.

Cadmium accumulation in Allium schoenoprasum L. grown in an aqueous medium.

The ability of Allium schoenoprasum L. (chives) to accumulate and tolerate cadmium in aqueous Hoagland medium at 50microM and 250microM was tested under continuous growth or several successive harvests of shoots. After 28 days of continuous growth, chives accumulated the metal up to 0.2% and 0.5% of its dry weight, when grown in 50microM and 250microM, respectively. In experiments that the leaves were successively harvested every 16 days, there were no obvious stress symptoms after six harvests during a period of 96 days at 50microM Cd. At 250microM, after 64 days and four harvests, inhibition of growth occurred. In each treatment, a total of 1.2g kg(-1) DW and 2.4g kg(-1) DW was accumulated in the leaves, respectively. Total SH compounds concentration in leaf was found significantly higher by 3 and 7.4 times in plants treated with Cd at 50microM and 250microM in comparison to the control, respectively, while no difference in the concentration of glutathione (GSH+GSSG) was found. Thus, it is assumed that sulphur-containing compounds, yet unknown, are involved in defensive mechanisms against heavy metals in chives. The results presented, point to chives phytoremediation potential, but also on the potential risk in accumulation of heavy metals in a commonly edible plant.

Nuphar lutea: in vitro anti-leishmanial activity against Leishmania major promastigotes and amastigotes.

UNLABELLED: Several anti-leishmanial drugs of choice are of plant origin. Many of the available drugs against the disease are toxic and in certain cases parasite drug resistance is developed. The development of new compounds is urgently required. AIMS OF THE STUDY: To determine the leishmanicidal activity of the Nuphar lutea plant extract against Leishmania major in vitro. MATERIALS AND METHODS: The leishmanicidal activity of methanolic plant extract against L. major free living promastigotes and intracellular amastigotes was evaluated, using microscopic examinations and the enzymatic XTT assay. RESULTS: Methanolic extract of N. lutea was highly effective against both Leishmania promastigotes and L. amastigotes (IC(50)=2+/-0.12 microg/ml; ID(50)=0.65+/-0.02 3 microg/ml; LD(50)=2.1+/-0.096 microg/ml, STI=3.23). The extract at 1.25 microg/ml totally eliminated the intracellular parasites within 3 days of treatment. Also, a synergistic anti-leishmanial activity was demonstrated with N. lutea extract combined with the anti-leishmanial drug--paromomycin. The partially purified N. lutea active component was found to be a thermo-stable alkaloid(s) with no electrical charge and is resistant to boiling and to methanol, dichloromethane and xylene treatment. CONCLUSIONS: The present study suggests that N. lutea might be a potential source of anti-leishmanial compounds.

Cysteine proteases and acid phosphatases contribute to Tetrahymena spp. pathogenicity in guppies, Poecilia reticulata.

Systemic tetrahymenosis caused by the protozoan parasite Tetrahymena spp. is a serious problem in guppy (Poecilia reticulata) farms worldwide. There is no therapeutic solution for the systemic form of this disease. Guppies severely infected with Tetrahymena spp. were imported by a commercial ornamental fish farm and brought to our laboratory. Tetrahymena sp. (Tet-NI) was isolated and in vitro cultured. Isolates maintained in culture for different time periods (as reflected by different numbers of passages in culture) were analyzed-Tet-NI 1, 4, 5 and 6, with Tet-NI 1 being cultured for the longest period (about 15 months, 54 passages) and Tet-NI 6 for the shortest (2.5 months, 10 passages). Controlled internal infection was successfully achieved by IP injection with most isolates, except for Tet-NI 1 which produced no infection. The isolate Tet-NI 6 induced the highest infection rates in internal organs (80% vs. 50% and 64% for Tet-NI 4 and 5, respectively) and mortality rates (67% vs. 20% and 27% for Tet-NI 4 and 5, respectively, and 6.7% for Tet-NI 1). The correlation between pathogenicity and Tetrahymena enzymatic activity was studied. Electrophoretic analyses revealed at least two bands of gelanolytic activity in Tet-NI 4 and 5, three bands in Tet-NI 6, and no activity in Tet-NI 1. Total inhibition of gelanolytic activity was observed after pretreatment of Tet-NI 6 with E-64, a highly selective cysteine protease inhibitor. Using hemoglobin as a substrate, Tet-NI 6 had two bands of proteolytic activity and no bands were observed in Tet-NI 1. A correlation was observed between pathogenicity and acid phosphatase activities (analyzed by commercial fluorescence kit) for Tet-NI 1 and Tet-NI 6.

Antileishmanial activity in Israeli plants.

Leishmaniasis is a vector-borne disease caused by flagellated protozoan parasites of the genus Leishmania, which affects both humans and other mammals. Most of the available drugs against the disease are toxic and parasite resistance to some of the drugs has already developed. In the present study, the leishmanicidal activities of methanolic extracts of some Israeli plants have been evaluated in vitro, against the free-living promastigotes and intracellular amastigotes of Leishmania major. Of the 41 extracts examined, those of two plants (Nuphar lutea>Withania somnifera) were highly effective (with a maximum inhibitory effect of >50%), those of three other species (Pteris vittata>Smyrnium olusatrum>Trifolium clypeatum) were moderately effective (25%-50%) and another four extracts (Erodium malacoides>Hyparrhenia hirta>Thymelaea hirsuta>Pulicaria crispa) showed a marginal effect (15%-22%) against the parasites. Extracts of nine plant species therefore showed antileishmanial activity but only the extract of N. lutea, used at 1.25 microg/ml, eliminated all the intracellular parasites within 3 days of treatment, with no detectable toxicity to the host macrophages. The mean (S.D.) values recorded for the median inhibitory concentrations of this extract (IC50) against the promastigotes [2.0 (0.12) microg/ml] and amastigotes [0.65 (0.023) microg/ml] and the median lethal concentration (LD50) against macrophages [2.1 (0.096) microg/ml] were encouraging, giving a therapeutic selectivity index [LD50/IC50 for amastigotes)] of 3.23. The extract of N. lutea was, in fact, generally as effective as the paromomycin that was used as the 'gold standard' drug. These results indicate that N. lutea and probably also Withania somnifera might be potential sources of clinically useful, antileishmanial compounds.

Anti-malarial drug targets: screening for inhibitors of 2C-methyl-D-erythritol 4-phosphate synthase (IspC protein) in Mediterranean plants.

The recently discovered non-mevalonate pathway of isoprenoid biosynthesis serves as the unique source of terpenoids in numerous pathogenic eubacteria and in apicoplast-type protozoa, most notably Plasmodium, but is absent in mammalian cells. It is therefore an attractive target for anti-infective chemotherapy. The first committed step of the non-mevalonate pathway is catalyzed by 2C-methyl-D-erythritol 4-phosphate synthase (IspC). Using photometric and NMR spectroscopic assays, we screened extracts of Mediterranean plants for inhibitors of the enzyme. Strongest inhibitory activity was found in leaf extracts of Cercis siliquastrum.

Effect of the add-back process on the free amino acid pool of potatoes.

The major losses in nitrogen and free amino acids (FAA) of the potato during the add-back (A-B) process were in the pre-cooking and mash-mixing steps (Fig. 1). These losses were due to leaching of nitrogen compounds into the processing water and heating. The FAA pool of the potato is composed mainly of the amides (asparagine and glutamine), aspartic and glutamic acids (approx. 53%). These amino acids were degraded substantially during the add-back process. There was also a significant loss in valine, threonine, serine, leucine, phenylalanine, and arginine, but no change in gamma-aminobutyric acid (GABA).

Variability in the pattern of random amplified polymorphic DNA.

The random amplified polymorphic DNA (RAPD) technique is a simple method to detect DNA polymorphism. It is sensitive to reaction conditions. Small changes in the reactants' concentration cause variations in amplification products. Using DNA from Asparagus officinalis, Dactylis glomerata, Mercurialis annua and Escherichia coli, we examined variability in the amplification pattern associated with reaction constituents. An increase in the ratio of Taq DNA polymerase to DNA in the reaction increased the number of amplified fragments. Increasing the concentration of primer resulted in the amplification of low molecular weight DNA fragments, while lowering the concentration resulted in high molecular weight fragments. Subsets of amplified fragments required different concentrations of magnesium for their highest intensity. Mechanical shearing of DNA obtained by sonication led to reduction in amplification of a subset of products. Enzymatic fragmentation of DNA by restriction enzymes led to loss or gain of specific fragments, depending on the DNA, primer, and restriction enzyme. RAPD markers of pooled DNA of anonymous pedigree should be critically evaluated for frequent 'false positive' markers.

Screening South Indian medicinal plants for antifungal activity against cutaneous pathogens.

In this study, twenty-eight South Indian medicinal plants were screened for their anti-fungal activity against six species of fungi (Trichophyton mentagrophytes, T. rubrum, T. soudanense, Candida albicans, Torulopsis glabrata, and C. krusei). Three plant species extracts, Celastrus paniculatus, Eriodendron anfractuosum and Ficus glomerata showed inhibitory activity. An aqueous extract of galls of Terminalia chebula showed inhibitory effects on three dermatophytes (Trichophyton spp.) and three yeasts (Candida spp.). Seeds extract of T. chebula inhibited only the growth of T. glabrata. An aqueous extract of T. chebula showed inhibitory effects higher than those measured in ethanol extracts. It is therefore suggested that tannins are plausible candidates for the anti-dermatophytic effects of T. chebula. Chebulinic acid, a known tannin of T. chebula was tested and found not inhibitory, thus a search for the active compound is needed.

Cell transformation induced by hepatitis C virus NS3 serine protease.

Persistent infection with hepatitis C virus (HCV) may lead to hepatocellular carcinoma (HCC). It has been suggested that HCV-encoded proteins are directly involved in the tumorigenic process. The HCV nonstructural protein NS3 has been identified as a virus-encoded serine protease. To study whether HCV NS3 has oncogenic activity, nontumorigenic rat fibroblast (RF) cells were stably transfected with an expression vector containing cDNA for the NS3 serine protease (nucleotides 3356-4080). The NS3 serine protease activity was determined in the transfected cells. The transfected cells grew rapidly and proliferated serum independently, lost contact inhibition, grew anchorage independently in soft agar and induced significant tumour formation in nude mice. Cells transfected with an expression vector containing a mutated NS3 serine protease (serine 139 to alanine at the catalytic site) showed no transforming abilities; their growth was dependent on serum and they did not grow anchorage independently in soft agar. Moreover, cells transfected with the NS3 serine protease and treated with the chymotrypsin inhibitors TPCK and PMSF (a serine protease inhibitor) lost their transforming feature. These results suggest that the NS3 serine protease of HCV is involved in cell transformation and that the ability to transform requires an active enzyme.