Allelic exchange at the endogenous genomic locus in Plasmodium falciparum proves the role of dihydropteroate synthase in sulfadoxine-resistant malaria.

We have exploited the recently developed ability to trans- fect the malaria parasite Plasmodium falciparum to investigate the role of polymorphisms in the enzyme dihydropteroate synthase (DHPS), identified in sulfadoxine-resistant field isolates. By using a truncated form of the dhps gene, specific mutations were introduced into the endogenous gene by allelic replacement such that they were under the control of the endogenous promoter. Using this approach a series of mutant dhps alleles that mirror P.falciparum variants found in field isolates were found to confer different levels of sulfadoxine resistance. This analysis shows that alteration of Ala437 to Gly (A437G) confers on the parasite a 5-fold increase in sulfadoxine resistance and addition of further mutations increases the level of resistance to 24-fold above that seen for the transfectant expressing the wild-type dhps allele. This indicates that resistance to high levels of sulfadoxine in P.falciparum has arisen by an accumulation of mutations and that Gly437 is a key residue, consistent with its occurrence in most dhps alleles from resistant isolates. These studies provide proof that the mechanism of resistance to sulfadoxine in P.falciparum involves mutations in the dhps gene and determines the relative contribution of these mutations to this phenotype.     

Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase of isolates from the Amazon region of Brazil

Since the late 1970s pyrimethamine-sulfadoxine (PS; FansidarTM Hoffman-LaRoche, Basel) has been used as first line therapy for uncomplicated malaria in the Amazon basin. Unfortunately, resistance has developed over the last ten years in many regions of the Amazon and PS is no longer recommended for use in Brazil. In vitro resistance to pyrimethamine and cycloguanil (the active metabolite of proguanil) is caused by specific point mutations in Plasmodium falciparum dihydrofolate reductase (DHFR), and in vitro resistance to sulfadoxine has been associated with mutations in dihydropteroate synthase (DHPS). In association with a proguanil-sulfamethoxazole clinical trial in Brazil, we performed a nested mutation-specific polymerase chain reaction to measure the prevalence of DHFR mutations at codons 50, 51, 59, 108 and 164 and DHPS mutations at codons 436, 437, 540, 581 and 613 at three sites in the Brazilian Amazon. Samples from two isolated towns showed a high degree of homogeneity, with the DHFR Arg-50/Ile-51/Asn-108 and DHPS Gly-437/Glu-540/Gly-581 mutant genotype accounting for all infections in Peixoto de Azevedo (n = 15) and 60% of infections in Apiacás (n = 10), State of Mato Grosso. The remaining infections in Apiacás differed from this predominant genotype only by the addition of the Bolivia repeat at codon 30 and the Leu-164 mutation in DHFR. By contrast, 17 samples from Porto Velho, capital city of the State of Rond?nia, with much in- and out-migration, showed a wide variety of DHFR and DHPS genotypes.     

Origin and Evolution of Sulfadoxine Resistant Plasmodium falciparum

The Thailand-Cambodia border is the epicenter for drug-resistant falciparum malaria. Previous studies have shown that chloroquine (CQ) and pyrimethamine resistance originated in this region and eventually spread to other Asian countries and Africa. However, there is a dearth in understanding the origin and evolution of dhps alleles associated with sulfadoxine resistance. The present study was designed to reveal the origin(s) of sulfadoxine resistance in Cambodia and its evolutionary relationship to African and South American dhps alleles. We sequenced 234 Cambodian Plasmodium falciparum isolates for the dhps codons S436A/F, A437G, K540E, A581G and A613S/T implicated in sulfadoxine resistance. We also genotyped 10 microsatellite loci around dhps to determine the genetic backgrounds of various alleles and compared them with the backgrounds of alleles prevalent in Africa and South America. In addition to previously known highly-resistant triple mutant dhps alleles SGEGA and AGEAA (codons 436, 437, 540, 581, 613 are sequentially indicated), a large proportion of the isolates (19.3%) contained a 540N mutation in association with 437G/581G yielding a previously unreported triple mutant allele, SGNGA. Microsatellite data strongly suggest the strength of selection was greater on triple mutant dhps alleles followed by the double and single mutants. We provide evidence for at least three independent origins for the double mutants, one each for the SGKGA, AGKAA and SGEAA alleles. Our data suggest that the triple mutant allele SGEGA and the novel allele SGNGA have common origin on the SGKGA background, whereas the AGEAA triple mutant was derived from AGKAA on multiple, albeit limited, genetic backgrounds. The SGEAA did not share haplotypes with any of the triple mutants. Comparative analysis of the microsatellite haplotypes flanking dhps alleles from Cambodia, Kenya, Cameroon and Venezuela revealed an independent origin of sulfadoxine resistant alleles in each of these regions.     

Antifolates can have a role in the treatment of Plasmodium vivax

Plasmodium vivax is a serious health concern in many regions and is sometimes inadvertently treated with sulfadoxine-pyrimethamine (SP). Mutations in the genes that encode dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) confer resistance to pyrimethamine and sulfadoxine, respectively. Numerous studies have examined the prevalence and diversity of mutations in P. vivax dhfr and some have assessed the relationship between dhfr genotype and clinical or in vitro response to pyrimethamine. Other studies have examined the impact of dhps genotype on response to sulfadoxine. These data indicate that, under certain circumstances, SP could be a valuable tool in the fight against P. vivax.     

Sulfadoxine resistance in the human malaria parasite Plasmodium falciparum is determined by mutations in dihydropteroate synthetase and an additional factor associated with folate utilization

Sulfadoxine/pyrimethamine (Fansidar) is widely used in Africa for treating chloroquine-resistant falciparum malaria. To clarify how parasite resistance to this combination arises, various lines of Plasmodium falciparum were used to investigate the role of naturally occurring mutations in the target enzyme, dihydropteroate synthetase (DHPS), in the parasite response to sulfadoxine inhibition. An improved drug assay was employed to identify a clear correlation between sulfadoxine-resistance levels and the number of DHPS mutations. Moreover, tight linkage was observed between DHPS mutations and high-level resistance in the 16 progeny of a genetic cross between sulfadoxine-sensitive (HB3) and sulfadoxine-resistant (Dd2) parents. However, we also demonstrate a profound influence of exogenous folate on IC₅₀ values, which, under physiological conditions, may have a major role in determining resistance levels. Importantly, this phenotype does not segregate with dhps genotypes in the cross, but shows complete linkage to the two alleles of the dihydrofolate reductase ( dhfr ) gene inherited from the parental lines. However, in unrelated lines, this folate effect correlates less well with DHFR sequence, indicating that the gene responsible may be closely linked to dhfr , rather than dhfr itself. These results have major implications for the acquisition of Fansidar resistance by malaria parasites.     

Prevalence of molecular markers of Plasmodium falciparum drug resistance in Dakar, Senegal

ABSTRACT: BACKGROUND: As a result of the widespread resistance to chloroquine and sulphadoxinepyrimethamine, artemisinin-based combination therapy (ACT) (including artemetherlumefantrine and artesunate-amodiaquine) has been recommended as a first-line antimalarial regimen in Senegal since 2006. Intermittent preventive treatments with antimalarial drugs based on sulphadoxine-pyrimethamine are also given to children or pregnant women once per month during the transmission season. Since 2006, there have been very few reports on the susceptibility of Plasmodium falciparum to antimalarial drugs. To estimate the prevalence of resistance to several anti-malarial drugs since the introduction of the widespread use of ACT, the presence of molecular markers associated with resistance to chloroquine and sulphadoxine-pyrimethamine was assessed in local isolates at the military hospital of Dakar. METHODS: The prevalence of genetic polymorphisms in genes associated with anti-malarial drug resistance, i.e., Pfcrt, Pfdhfr, Pfdhps and Pfmdr1, and the copy number of Pfmdr1 were evaluated for a panel of 174 isolates collected from patients recruited at the military hospital of Dakar from 14 October 2009 to 19 January 2010. RESULTS: The Pfcrt 76 T mutation was identified in 37.2% of the samples. The Pfmdr1 86Y and 184 F mutations were found in 16.6% and 67.6% of the tested samples, respectively. Twenty-eight of the 29 isolates with the 86Y mutation were also mutated at codon 184. Only one isolate (0.6%) had two copies of Pfmdr1. The Pfdhfr 108 N/T, 51I and 59R mutations were identified in 82.4%, 83.5% and 74.1% of the samples, respectively. The double mutant (108 N and 51I) was detected in 83.5% of the isolates, and the triple mutant (108 N, 51I and 59R) was detected in 75.3%. The Pfdhps 437 G, 436 F/A and 613 S mutations were found in 40.2%, 35.1% and 1.8% of the samples, respectively. There was no double mutant (437 G and 540E) or no quintuple mutant (Pfdhfr 108 N, 51I and 59R and Pfdhps 437 G and 540E). The prevalence of the quadruple mutant (Pfdhfr 108 N, 51I and 59R and Pfdhps 437 G) was 36.5%. CONCLUSIONS: Since 2004, the prevalence of chloroquine resistance had decreased. The prevalence of isolates with high-level pyrimethamine resistance is 83.5%. The prevalence of isolates resistant to sulphadoxine is 40.2%. However, no quintuple mutant (Pfdhfr 108 N, 51I and 59R and Pfdhps 437 G and 540E), which is associated with a high level of sulphadoxine-pyrimethamine resistance, has been identified to date. The resistance to amodiaquine remains moderate.     

Novel biologically active bibenzyls from Bauhinia saccocalyx Pierre

Four new bibenzyls, bauhinols A-D (1-4), together with the two known bibenzyls 5 and 6, were isolated from the roots of Bauhinia saccocalyx, and their structures were elucidated by analyses of spectroscopic data. Bauhinol A (1) exhibits significant cytotoxicity towards NCI-H187 (small-cell lung cancer), BC (breast cancer), and KB (oral-cavity cancer) cell lines, with IC50 values of 2.7-4.5 microg/ml. Bauhinol B (2) is cytotoxic against NCI-H187 (IC50 = 1.1 microg/ml) and BC (IC50 = 9.7 microg/ml) cell lines, but inactive toward the KB cell line (at 20 microg/ml). Compound 2 also is mildly antifungal towards Candia albicans (IC50 = 28.9 microg/ml). Bibenzyl 6 is active against NCI-H187 (IC50 = 14.1 microg/ml) and BC (IC50 = 4.0 microg/ml) cells, but inactive (at 20 microg/ml) toward the KB cell line. Compounds 1, 2, and 6 show mild antimycobacterial activities, with MIC values of 25-50 microg/ml, but are inactive at 20 microg/ml against the K1 malarial parasite strain (Plasmodium falciparum). While bauhinol A (1) is inactive against cyclooxygenase 1 (COX-1) and cyclooxygenase 2 (COX-2), compounds 2 and 6 inhibit both COX-1 and COX-2, with IC50 values comparable to those of the standard drug, aspirin (Table 3).     

In vitro cytotoxic activity of Salsola oppositifolia Desf. (Amaranthaceae) in a panel of tumour cell lines

The aim of the present study was to evaluate for the first time the in vitro cytotoxic activity of fractions and isolated flavonols from Salsola oppositifolia Desf. (Amaranthaceae). The n-hexane fraction demonstrated an effective cytotoxic activity on the large lung carcinoma and amelanotic melanoma cell lines with IC50 values of 19.1 microg/ml and 24.4 microg/ml, respectively. Also the dichloromethane fraction exhibited cytotoxic activity against COR-L23 (IC50 30.4 microg/ml) and C32 (IC50 33.2 microg/ml) cells, while the EtOAc fraction demonstrated a selective cytotoxic activity against MCF-7 cells (IC50 67.9 microg/ml). The major active constituents of this fraction were isorhamnetin-3-O-glucoside (1) and isorhamnetin-3-O-rutinoside (2), which showed an interesting activity against the cell line MCF-7 with IC50 values of 18.2 and 25.2 microg/ml, respectively. Compound 2 exhibited a strong activity against the hormone-dependent prostate carcinoma LNCaP cell line with an IC50 of 20.5 microg/ml. Constituents of S. oppositifolia were identified by GC-MS and NMR analyses.     

Sensitivity of mango anthracnose pathogen, Colletotrichum gloeosporioides, to the fungicide prochloraz in Taiwan

In order to monitor the sensitivity of the mango anthracnose fungus, Colletotricthum gloeosporioides, to the eradicative imidazole fungicide prochloraz, a total of 43 mango orchards were surveyed throughout the Tainan area, covering a 4000 ha region of mango plantations. These orchards were recognized as having undergone higher prochloraz application. A subpopulation, 55 isolates in total, collected from Wufeng, Taichung, served as a baseline population since no fungicide was ever used in this mango plantation. A total of 545 isolates were surveyed, and it was found that the IC50s (50% inhibitory concentration) fell within a range of 0.009-0.1554 microg/ml. No significant resistance was found in the field even with higher frequency of prochloraz application. One orchard (Yujing (Wu)) located in the Yujing area known had a higher frequency of prochloraz application, the IC50s were between 0.0204 - 0.1554 microg/ml. The average IC50 was 0. 0766 microg/ml, which was about five times higher than the baseline population (0.015 microg/ml). A significant t test indicated that these two sub-populations were different at p = 0.01. The results indicated that the dose-response of C. gloeosporioides to prochloraz shifted slightly toward higher IC50 over time. A further survey using 10 microg/ml as the threshold dosage was conducted, and the results of 1375 isolates collected throughout this region showed that no isolate could survive at this dosage. Knowing that the registered dosage for field use is 83.3 microg/ml, based on above results, the author concluded that there was no sign of prochloraz resistance in mango plantations 13 years after prochloraz registration in Taiwan.     

Chemopreventive effects of minor dietary constituents in common foods on human cancer cells

Epidemiological evidence has suggested that vegetables and fruits may have a role in cancer prevention. The aim of the present study was to examine the anti-proliferative activity of ten related pure compounds from common vegetables and fruits. Studies were conducted on a series of carcinoma cells derived from eight human organs. The results show that linalool possessed the strongest activity against nine carcinoma cells, and that baicalein and luteolin also exhibited a broad spectrum of anti-proliferative activities. Among them, linalool showed the strongest activity against carcinoma of the cervix (IC50: 0.37 microg/ml), stomach (IC50: 14.1 microg/ml), skin (IC50: 14.9 microg/ml), lung (IC50: 21.5 microg/ml) and bone (IC50: 21.7 microg/ml). As for the flavonoids, luteolin exhibited the strongest activity against carcinoma of the stomach (IC50: 7.1 microg/ml), cervix (IC50: 7.7 microg/ml), lung (IC50: 11.7 microg/ml) and bladder (IC50: 19.5 microg/ml), whereas baicalein possessed the strongest anti-proliferative activity against carcinoma of the cervix (IC50: 9.8 microg/ml), stomach (IC50: 16.1 microg/ml) and skin (IC50: 19.5 microg/ml). The present study indicates that linalool possessed the strongest activity against a broad spectrum of carcinoma cells, especially cervical carcinoma cells, suggesting that linalool and flavonoids are partially responsible for the cancer prevention of common vegetables and fruits.     

Anti-protozoal and plasmodial FabI enzyme inhibiting metabolites of Scrophularia lepidota roots

The ethanolic root extract of Scrophularia lepidota, an endemic plant of the Turkish flora, has been investigated for its anti-protozoal and inhibitory effect towards plasmodial enoyl-ACP reductase (FabI), a key enzyme of fatty acid biosynthesis in Plasmodium falciparum. Chromatographic separation of the extract yielded 10 iridoids (1-10), two of which are new, and a known phenylethanoid glycoside (11). The structures of the new compounds were determined as 3,4-dihydro-methylcatalpol (8) and 6-O-[4'-O-trans-(3,4-dimethoxycinnamoyl)-alpha-L-rhamnopyranosyl]aucubin (scrolepidoside, 9) by spectroscopic means. The remaining metabolites were characterized as catalpol (1), 6-O-methylcatalpol (2), aucubin (3), 6-O-alpha-L-rhamnopyranosyl-aucubin (sinuatol, 4), 6-O-beta-D-xylopyranosylaucubin (5), ajugol (6), ajugoside (7), an iridoid-related aglycone (10) and angoroside C (11). Nine isolates were active against Leishmania donovani, with the new compound 9 being most potent (IC50 6.1 microg/ml). Except for 4, all pure compounds revealed some trypanocidal potential against Trypanosoma brucei rhodesiense (IC50 values 29.3-73.0 microg/ml). Only compound 10 showed moderate anti-plasmodial (IC50 40.6 microg/ml) and FabI enzyme inhibitory activity (IC50 100 microg/ml). 10 is the second natural product inhibiting the fatty acid biosynthesis of Plasmodium falciparum.     

Analysis in Escherichia coli of Plasmodium falciparum dihydropteroate synthase (DHPS) alleles implicated in resistance to sulfadoxine

Mutations in Plasmodium falciparum dihydropteroate synthase have been linked to resistance to the antimalarial drug, sulfadoxine, which competes with the dihydropteroate synthase substrate, p-aminobenzoate. In an effort to evaluate the role of these mutations in a simple model system, we have expressed six relevant alleles of the P. falciparum dihydropteroate synthase gene in Escherichia coli. When each construct was produced in a dihydropteroate synthase disrupted E. coli strain that required thymidine, the thymidine requirement was lost, indicating heterologous complementation had occurred. In the presence of sulfadoxine, the growth of the strain with the wild-type dihydropteroate synthase allele was inhibited while those containing each of the five mutant alleles grew, indicating that these mutations can confer sulfadoxine resistance in E. coli. When tested against twelve additional 'sulfa' drugs a variety of responses were obtained. All strains were resistant to sulfadiazine, but the wild-type allele conferred sensitivity to all other sulfa drugs. Three alleles conferred resistance to dapsone, a drug that is to be targetted for a new regime of malaria treatment in Africa. All mutant alleles remained sensitive to sulfachloropyridazine and sulfacetamide. These results suggest new drugs that could be tried for effective malaria treatment.     

Efficacy and safety of a new vaginal contraceptive antimicrobial formulation containing high molecular weight poly(sodium 4-styrenesulfonate)

Host cell infection by sexually transmitted disease (STD)-causing microbes and fertilization by spermatozoa may have some mechanisms in common. If so, certain noncytotoxic agents could inhibit the functional activity of both organisms. High molecular mass poly(sodium 4-styrenesulfonate) (T-PSS) may be one of these compounds. T-PSS alone (1 mg/ml) or in a gel (2% or 5% T-PSS) completely prevented conception in the rabbit. Contraception was not due to sperm cytotoxicity or to an effect on sperm migration. However, T-PSS inhibited sperm hyaluronidase (IC(50) = 5.3 microg/ml) and acrosin (IC(50) = 0.3 microg/ml) and caused the loss of acrosomes from spermatozoa (85% maximal loss by 0.5 microg/ml). T-PSS (5% in gel) also reduced sperm penetration into bovine cervical mucus (73% inhibition by 1 mg gel/ml). T-PSS (5% in gel) inhibited human immunodeficiency virus (HIV; IC(50)= 16 microg gel/ml) and herpes simplex viruses (HSV-1 and HSV-2; IC(50) = 1.3 and 1.0 microg gel/ml, respectively). The drug showed high efficacy against a number of clinical isolates and laboratory strains. T-PSS (5% in gel) also inhibited Neisseria gonorrhea (IC(50) < 1.0 gel/ml) and Chlamydia trachomatis (IC(50) = 1.2 microg gel/ml) but had no effect on lactobacilli. These results imply that T-PSS is an effective functional inhibitor of both spermatozoa and certain STD-causing microbes. The noncytotoxic nature should make T-PSS safe for vaginal use. T-PSS was nonmutagenic in vitro and possessed an acute oral toxicity of >5 g/kg (rat). Gel with 10% T-PSS did not irritate the skin or penile mucosa (rabbit) and caused no dermal sensitization (guinea pig). Vaginal administration of the 5% T-PSS gel to the rabbit for 14 consecutive days caused no systemic toxicity and only mild (acceptable) vaginal irritation. T-PSS in gel form is worthy of clinical evaluation as a vaginal contraceptive HIV/STD preventative.     

Improved breadth and potency of an HIV-1-neutralizing human single-chain antibody by random mutagenesis and sequential antigen panning

Several human monoclonal antibodies can neutralize a range of human immunodeficiency virus type 1 (HIV-1) primary isolates but their potency and related ability to suppress generation of HIV-1 escape mutants is significantly lower than the activity of antiretroviral drugs currently in clinical use. Recently, a human Fab, X5, was identified and found to neutralize primary isolates from different clades. Further improvement of the potency and breadth of HIV-1 neutralization by this antibody could be critical for its potential use in the treatment of HIV-1-infected patients. However, increasing potency of an antibody by selection from libraries may lead to a decrease in the breadth of neutralization. In an attempt to solve this problem, we subjected a random mutagenesis library of the scFv X5 to sequential rounds of selection on non-homologous HIV-1 envelope glycoproteins (Envs) dubbed sequential antigen panning (SAP). By using SAP, we identified two scFv antibodies, m6 and m9, that were tested with a panel of 33 diverse primary HIV-1 infectious isolates in an assay based on a reporter cell-line expressing high levels of CD4, CCR5 and CXCR4. The IC(50) was less than 50 microg/ml for 21 (m6) and 19 (m9) out of 29 isolates from group M (subtypes A-C, F, G and CRF-01AE) and one isolate from group N; three isolates from group O were not significantly inhibited at 50 microg/ml. The average IC(50) values for the two antibodies were significantly (p<0.001, n=29) lower compared to scFv X5. Their inhibitory activity does not appear to be related to the HIV-1 subtype, coreceptor usage or the disease stage. m9 inhibited infection of peripheral blood mononuclear cells by the primary isolates JRCSF, 89.6 and BR020 with IC(90) of 4, 6 and 25 microg/ml, respectively; for a single-round infection by pseudovirus, the IC(90) for JRSCF, 89.6, YU2 and HXBc2 was 15, 5, 15 and 5 microg/ml, respectively. In these two assays the IC(90) for m9 was, on average, two- to threefold lower than for scFv X5. These results demonstrate that both the potency and the breadth of HIV-1 neutralization of one of the few known potent broadly cross-reactive human monoclonal antibodies, scFv X5, could be improved significantly. However, only experiments in animal models and clinical trials in humans will show whether these new scFvs and the approach for their identification have potential in the development of prophylactics and therapeutics for HIV-1 infections.     

Anti-inflammatory and spasmolytic activity of extracts from Droserae herba.

An ethanolic extract of Drosera madagascariensis inhibited human neutrophil elastase with an IC50 of 9.4 microg/ml. The naphthoquinones present in the extract were not responsible for this effect, but flavonoids like quercetin (IC50 0.8 microg/ml), hyperoside (IC50 0.15 microg/ml) and isoquercitrin (IC50 0.7 microg/ml) contributed to inhibition of the enzyme. In guinea-pig ileum the extract (0.5-1 mg/ml) induced a spasmolytic effect via affecting cholinergic M3 receptors and histamine H1 receptors, respectively. At contractile prostanoid receptors of guinea-pig trachea the Drosera extract was not effective.     

Free radical scavenging and metal chelation by Tinospora cordifolia,a possible role in radioprotection

Aqueous extract of T. cordifolia inhibited Fenton (FeSO4) reaction and radiation mediated 2-deoxyribose degradation in a dose dependent fashion with an IC50 value of 700 microg/ml for both Fenton and radiation mediated 2-DR degradation. Similarly, it showed a moderate but dose dependent inhibition of chemically generated superoxide anion at 500 microg/ml concentration and above with an IC50 value of 2000 microg/ml. Aqueous extract inhibited the formation of Fe2+-bipiridyl complex and formation of comet tail by chelating Fe2+ ions in a dose dependent manner with an IC50 value of 150 microg/ml for Fe2+-bipirydyl formation and maximally 200 microg/ml for comet tail formation, respectively. The extract inhibited ferrous sulphate mediated lipid peroxidation in a dose-dependent manner with an IC50 value of 1300 microg/ml and maximally (70%) at 2000 microg/ml. The results reveal that the direct and indirect antioxidant actions of T. cordifolia probably act in corroboration to manifest the overall radioprotective effects.     

High-performance liquid chromatographic assay for the determination of sulfadoxine and N-acetyl sulfadoxine in plasma from patients infected with sensitive and resistant Plasmodium falciparum malaria

A reversed-phase high-performance liquid chromatographic method using a mobile phase of acetonitrile-methanol-trifluoroacetic acid-water (16.1:7.2:0.1:76.6, v/v/v/v) at a flow rate of 1.0 ml min(-1) on a LiChrospher RP-18 column with UV (254 nm) detection has been developed for the separation of sulfadoxine and its metabolite N-acetyl sulfadoxine in plasma. No interferences due to endogenous compounds or common antimalarial drugs were noticed. The limit of detection for sulfadoxine and N-acetyl sulfadoxine was 0.01 microg ml(-1) with a signal-to-noise ratio of 5:1 while the limit of quantification was 2.5 microg ml(-1). Intra-day mean relative standard deviations (RSD's) for sulfadoxine and N-acetyl sulfadoxine were 2.6 and 2.8%, respectively, while mean inter-day RSD's for sulfadoxine and N-acetyl sulfadoxine were 2.4 and 2.8%, respectively. Extraction recoveries averaged 90.6% for sulfadoxine and 86.9% for N-acetyl sulfadoxine. The method was applied for the assay of sulfadoxine and its metabolite N-acetyl sulfadoxine in plasma from Plasmodium falciparum malaria patients. Mean plasma sulfadoxine concentrations on day 2 (51 h) from samples collected from sensitive and resistant P. falciparum patients treated with three tablets of Fansidar were 62.8 and 60.5 microg ml(-1), respectively. Mean ratio of N-acetyl sulfadoxine to sulfadoxine was 9.1% for responders and 13.9% for non-responders which revealed that higher amounts of the metabolite N-acetyl sulfadoxine were present in non-responders. The method described should find an application in the therapeutic monitoring of malaria patients.     

Antioxidant effects of an aqueous extract of Ilex paraguariensis

In this work we investigate the antioxidant properties of an aqueous extract prepared from an infusion of Ilex paraguariensis (Aquifoliaceae) using free radical-generating systems. The extract inhibited the enzymatic and nonenzymatic lipid peroxidation in rat liver microsomes in a concentration-dependent fashion, with IC(50) values of 18 microg/ml and 28 microg/ml, respectively. The extract also inhibited the H(2)O(2)-induced peroxidation of red blood cell membranes with an IC(50) of 100 microg/ml and exhibited radical scavenging properties toward superoxide anion (IC(50) = 15 microg/ml) and 2,2-diphenyl-1-picrylhydrazyl radical. In the range of concentrations used, the extract was not a scavenger of the hydroxyl radical. Our results suggest that ingestion of extracts of Ilex paraguariensis could contribute to increase the antioxidant defense of an organism against free radicals attack.     

Anti-herpesvirus activities of Pseudomonas sp. S-17 rhamnolipid and its complex with alginate

The rhamnolipid biosurfactant PS-17 and its complex with the polysaccharide alginate, both produced by the Pseudomonas sp. S-17 strain, were studied for their antiviral activity against herpes simplex virus (HSV) types 1 and 2. They significantly inhibited the herpesvirus cytopathic effect (CPE) in the Madin-Darby bovine kidney (MDBK) cell line. The investigations were carried out according to the CPE inhibition assay protocol. The suppressive effect of the compounds on HSV replication was dose-dependent and occurred at concentrations lower than the critical micelle concentration of the surfactant. The 50% inhibitory concentration (IC50) of rhamnolipid PS-17 was 14.5 microg/ml against HSV-1 and 13 microg/ml against HSV-2. The IC50 values of the complex were 435 microg/ml for HSV-1 and 482 microg/ml for HSV-2. The inhibitory effects of the substances were confirmed by measuring the infectious virus yields with the multicycle virus growth experimental design as well: deltalog CCID50 of 1.84-2.0 against the two types of herpes simplex viruses by rhamnolipid PS-17 (20 microg/ml), and a strong reduction of the HSV-2 virus yield under the effect of the alginate complex at a concentration of 450 microg/ml. The results indicate that rhamnolipid PS-17 and its alginate complex may be considered as promising substances for the development of anti-herpetic compounds.     

Bioactive deoxypreussomerins and dimeric naphthoquinones from Diospyros ehretioides fruits: deoxypreussomerins may not be plant metabolites but may be from fungal epiphytes or endophytes

Deoxypreussomerin derivatives, palmarumycins JC1 (1) and JC2 (2), and two dimeric naphthoquinones, isodiospyrin (3) and its new derivative isodiospyrol A (4), were isolated from dried fruits of Diospyros ehretioides. Structures of the isolated compounds were elucidated by spectroscopic analyses. Palmarumycins were not found in the extract of freshly collected fruits; however, they were present in dried fruit extract. The absence of palmarumycins in fresh fruits of D. ehretioides, together with the chemotaxonomic point of view, we proposed that palmarumycins JC1 (1) and JC2 (2) are more likely to be fungal metabolites, i.e., endophytes or epiphytes. The isolation of palmarumycins 1 and 2 from dried D. ehretioides fruits could be reproducible; both plant samples collected in the years 2002 and 2004 provided the same result, and, therefore, symbiont fungal strains should be specific to the plant host, D. ehretioides, and they can grow on the fruits during drying the sample. Palmarumycin JC1 (1) did not exhibit antimalarial, antifungal, antimycobacterial, and cytotoxic activities. Palmarumycin JC2 (2) exhibited antimalarial (IC50 4.5 microg/ml), antifungal (IC50 12.5 microg/ml), antimycobacterial (MIC 6.25 microg/ml), and cytotoxic (IC50 11.0 microg/ml for NCI-H187 cell line) activities. In our bioassay systems, isodiospyrin (3) did not exhibit antimycobacterial, antifungal, antimalarial, and cytotoxic activities. Isodiospyrol A (4) exhibited antimalarial (IC50 2.7 microg/ml) and antimycobacterial (MIC 50 microg/ml) activities, but was inactive towards Candida albicans. Compound 4 also exhibited cytotoxicity against BC cells (IC50 12.3 microg/ml), but not towards KB and Vero cell lines.