In vitro antimicrobial activity of a new series of 1,4-naphthoquinones

The antibacterial activity of a series of 1,4-naphthoquinones was demonstrated. Disk diffusion tests were carried out against several Gram-positive and Gram-negative bacteria. The compound 5-amino-8-hydroxy-1,4-naphthoquinone was the most effective, presenting inhibition zones measuring 20 mm against staphylococci, streptococci and bacilli at 50 µg/ml. Methicillin-resistant Staphylococcus aureus and several clinical isolates of this bacterium were also inhibited. Naphthazarin, 5-acetamido-8-hydroxy-1,4-naphthoquinone, and 2,3-diamino-1,4-naphthoquinone were the next most active compounds. The minimal inhibitory concentration of the active compounds was determined against S. aureus, ranging from 30 to 125 µg/ml. All compounds presented a minimal bactericidal concentration higher than 500 µg/ml, indicating that their effect was bacteriostatic. The EC50, defined as the drug concentration that produces 50% of maximal effect, was 8 µg/ml for 5-amino-8-hydroxy-1,4-naphthoquinone against S. aureus, S. intermedius, and S. epidermidis. These results indicate an effective in vitro activity of 5-amino-8-hydroxy-1,4-naphthoquinone and encourage further studies for its application in antibiotic therapy.

The modulatory effect of estradiol benzoate on superoxide dismutase activity in the developing rat brain

The sensitivity of copper,zinc (CuZn)- and manganese (Mn)-superoxide dismutase (SOD) to exogenous estradiol benzoate (EB) was investigated in Wistar rats during postnatal brain development. Enzyme activities were measured in samples prepared from brains of rats of both sexes and various ages between 0 and 75 days, treated sc with 0.5 µg EB/100 g body weight in 0.1 ml olive oil/100 g body weight, 48 and 24 h before sacrifice. In females, EB treatment stimulated MnSOD activity on days 0 (66.1%), 8 (72.7%) and 15 (81.7%). In males, the stimulatory effect of EB on MnSOD activity on day 0 (113.6%) disappeared on day 8 and on days 15 and 45 it became inhibitory (40.3 and 30.5%, respectively). EB had no effect on the other age groups. The stimulatory effect of EB on CuZnSOD activity in newborn females (51.8%) changed to an inhibitory effect on day 8 (38.4%) and disappeared by day 45 when inhibition was detected again (48.7%). In males, the inhibitory effect on this enzyme was observed on days 0 (45.0%) and 15 (28.9%), and then disappeared until day 60 when a stimulatory effect was observed (38.4%). EB treatment had no effect on the other age groups. The sensitivity of MnSOD to estradiol differed significantly between sexes during the neonatal and prepubertal period, whereas it followed a similar pattern thereafter. The sensitivity of CuZnSOD to estradiol differed significantly between sexes during most of the study period. Regression analysis showed that the sensitivity of MnSOD to this estrogen tended to decrease similarly in both sexes, whereas the sensitivity of CuZnSOD showed a significantly different opposite tendency in female and male rats. These are the first reports indicating hormonal modulation of antioxidant enzyme activities related to the developmental process.

A calcineurin inhibitory protein overexpressed in Down's syndrome interacts with the product of a ubiquitously expressed transcript

The Down's syndrome candidate region 1 (DSCR1) protein, encoded by a gene located in the human chromosome 21, interacts with calcineurin and is overexpressed in Down's syndrome patients. As an approach to clarifying a putative function for this protein, in the present study we used the yeast two-hybrid system to identify DSCR1 partners. The two-hybrid system is a method that allows the identification of protein-protein interactions through reconstitution of the activity of the yeast GAL 4 transcriptional activator. The gene DSCR1 fused to the GAL 4 binding domain (BD) was used to screen a human fetal brain cDNA library cloned in fusion with the GAL 4 activation domain (AD). Three positive clones were found and sequence analysis revealed that all the plasmids coded for the ubiquitously expressed transcript (UXT). UXT, which is encoded in human Xp11, is a 157-amino acid protein present in both cytosol and nucleus of the cells. This positive interaction of DSCR1 and UXT was confirmed in vivo by mating the yeast strain AH109 (MATaACE=Symbol>)expressing AD-UXT with the strain Y187 (MATalpha) expressing BD-DSCR1, and in vitro by co-immunoprecipitation experiments. These results may help elucidate a new function for DSCR1 and its participation in Down's syndrome pathogenesis.

A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity

A continuous assay using internally quenched fluorescent peptides with the general sequence Abz-peptidyl-(Dnp)P-OH (Abz = ortho-aminobenzoic acid; Dnp = 2,4-dinitrophenyl) was optimized for the measurement of angiotensin I-converting enzyme (ACE) in human plasma and rat tissues. Abz-FRK(Dnp)P-OH, which was cleaved at the Arg-Lys bond by ACE, was used for the enzyme evaluation in human plasma. Enzymatic activity was monitored by continuous recording of the fluorescence (lambdaex = 320 nm and lambdaem = 420 nm) at 37ºC, in 0.1 M Tris-HCl buffer, pH 7.0, with 50 mM NaCl and 10 µM ZnCl2. The assays can be performed directly in the cuvette of the fluorimeter and the hydrolysis followed for 5 to 10 min. ACE measurements in the plasma of 80 healthy patients with Hip-His-Leu and with Abz-FRK(Dnp)P-OH correlated closely (r = 0.90, P < 0.001). The specificity of the assay was demonstrated by the complete inhibition of hydrolysis by 0.5 µM lisinopril or captopril. Abz-FRK(Dnp)P-OH cleavage by ACE was monitored in rat lung, kidney, heart, and liver homogenates in the presence of a cocktail of inhibitors containing trans-epoxy-succinyl-L-leucylamido-(4-guanido)-butene, pepstatin, phenyl-methylsulfonyl fluoride, N-tosyl-L-phenylalanyl-chloromethyl ketone, and N-tosyl-lysyl-chloromethyl ketone to prevent undesirable hydrolysis. ACE activity in lung, heart and kidney homogenates, but not in liver homogenates, was completely abolished by 0.5 µM lisinopril or captopril. The advantages of the method are the procedural simplicity and the high sensitivity providing a rapid assay for ACE determinations.

Antibacterial activity of indole alkaloids from Aspidosperma ramiflorum

We evaluated the antibacterial activities of the crude methanol extract, fractions (I-V) obtained after acid-base extraction and pure compounds from the stem bark of Aspidosperma ramiflorum. The minimum inhibitory concentration (MIC) was determined by the microdilution technique in Mueller-Hinton broth. Inoculates were prepared in this medium from 24-h broth cultures of bacteria (10(7) CFU/mL). Microtiter plates were incubated at 37ºC and the MICs were recorded after 24 h of incubation. Two susceptibility endpoints were recorded for each isolate. The crude methanol extract presented moderate activity against the Gram-positive bacteria B. subtilis (MIC = 250 µg/mL) and S. aureus (MIC = 500 µg/mL), and was inactive against the Gram-negative bacteria E. coli and P. aeruginosa (MIC > 1000 µg/mL). Fractions I and II were inactive against standard strains at concentrations of <=1000 µg/mL and fraction III displayed moderate antibacterial activity against B. subtilis (MIC = 500 µg/mL) and S. aureus (MIC = 250 µg/mL). Fraction IV showed high activity against B. subtilis and S. aureus (MIC = 15.6 µg/mL) and moderate activity against E. coli and P. aeruginosa (MIC = 250 µg/mL). Fraction V presented high activity against B. subtilis (MIC = 15.6 µg/mL) and S. aureus (MIC = 31.3 µg/mL) and was inactive against Gram-negative bacteria (MIC > 1000 µg/mL). Fractions III, IV and V were then submitted to bioassay-guided fractionation by silica gel column chromatography, yielding individual purified ramiflorines A and B. Both ramiflorines showed significant activity against S. aureus (MIC = 25 µg/mL) and E. faecalis (MIC = 50 µg/mL), with EC50 of 8 and 2.5 µg/mL for ramiflorines A and B, respectively, against S. aureus. These results are promising, showing that these compounds are biologically active against Gram-positive bacteria.

Antimicrobial activity of the essential oil from Lippia sidoides, carvacrol and thymol against oral pathogens

Dental caries and periodontal disease are associated with oral pathogens. Several plant derivatives have been evaluated with respect to their antimicrobial effects against such pathogenic microorganisms. Lippia sidoides Cham (Verbenaceae), popularly known as "Alecrim-pimenta" is a typical shrub commonly found in the Northeast of Brazil. Many plant species belonging to the genus Lippia yield very fragrant essential oils of potential economic value which are used by the industry for the commercial production of perfumes, creams, lotions, and deodorants. Since the leaves of L. sidoides are also extensively used in popular medicine for the treatment of skin wounds and cuts, the objective of the present study was to evaluate the composition and antimicrobial activity of L. sidoides essential oil. The essential oil was obtained by hydro-distillation and analyzed by GC-MS. Twelve compounds were characterized, having as major constituents thymol (56.7%) and carvacrol (16.7%). The antimicrobial activity of the oil and the major components was tested against cariogenic bacterial species of the genus Streptococcus as well as Candida albicans using the broth dilution and disk diffusion assays. The essential oil and its major components thymol and carvacrol exhibited potent antimicrobial activity against the organisms tested with minimum inhibitory concentrations ranging from 0.625 to 10.0 mg/mL. The most sensitive microorganisms were C. albicans and Streptococcus mutans. The essential oil of L. sidoides and its major components exert promising antimicrobial effects against oral pathogens and suggest its likely usefulness to combat oral microbial growth.

Reduced plasma levels of angiotensin-(1-7) and renin activity in preeclamptic patients are associated with the angiotensin I- converting enzyme deletion/deletion genotype

The relationship between preeclampsia and the renin-angiotensin system (RAS) is poorly understood. Angiotensin I-converting enzyme (ACE) is a key RAS component and plays an important role in blood pressure homeostasis by generating angiotensin II (Ang II) and inactivating the vasodilator angiotensin-(1-7) (Ang-(1-7)). ACE (I/D) polymorphism is characterized by the insertion (I) or deletion (D) of a 287-bp fragment, leading to changes in ACE activity. In the present study, ACE (I/D) polymorphism was correlated with plasma Ang-(1-7) levels and several RAS components in both preeclamptic (N = 20) and normotensive pregnant women (N = 20). The percentage of the ACE DD genotype (60%) in the preeclamptic group was higher than that for the control group (35%); however, this percentage was not statistically significant (Fisher exact test = 2.86, d.f. = 2, P = 0.260). The highest plasma ACE activity was observed in the ACE DD preeclamptic women (58.1 ± 5.06 vs 27.6 ± 3.25 nmol Hip-His Leu-1 min-1 mL-1 in DD control patients; P = 0.0005). Plasma renin activity was markedly reduced in preeclampsia (0.81 ± 0.2 vs 3.43 ± 0.8 ng Ang I mL plasma-1 h-1 in DD normotensive patients; P = 0.0012). A reduced plasma level of Ang-(1-7) was also observed in preeclamptic women (15.6 ± 1.3 vs 22.7 ± 2.5 pg/mL in the DD control group; P = 0.0146). In contrast, plasma Ang II levels were unchanged in preeclamptic patients. The selective changes in the RAS described in the present study suggest that the ACE DD genotype may be used as a marker for susceptibility to preeclampsia.

The association of ACE gene D/I polymorphism with cardiovascular risk factors in a population from Rio de Janeiro

Our aim was to determine the frequencies of the angiotensin-converting enzyme (ACE) gene alleles D and I and any associations to cardiovascular risk factors in a population sample from Rio de Janeiro, Brazil. Eighty-four adults were selected consecutively during a 6-month period from a cohort subgroup of a previous large cross-sectional survey in Rio de Janeiro. Anthropometric data and blood pressure measurements, echocardiogram, albuminuria, glycemia, lipid profile, and ACE genotype and serum enzyme activity were determined. The frequency of the ACE*D and I alleles in the population under study, determined by PCR, was 0.59 and 0.41, respectively, and the frequencies of the DD, DI, and II genotypes were 0.33, 0.51, and 0.16, respectively. No association between hypertension and genotype was detected using the Kruskal-Wallis method. Mean plasma ACE activity (U/mL) in the DD (N = 28), DI (N = 45) and II (N = 13) groups was 43 (in males) and 52 (in females), 37 and 39, and 22 and 27, respectively; mean microalbuminuria (mg/dL) was 1.41 and 1.6, 0.85 and 0.9, and 0.6 and 0.63, respectively; mean HDL cholesterol (mg/dL) was 40 and 43, 37 and 45, and 41 and 49, respectively, and mean glucose (mg/dL) was 93 and 108, 107 and 98, and 85 and 124, respectively. A high level of ACE activity and albuminuria, and a low level of HDL cholesterol and glucose, were found to be associated with the DD genotype. Finally, the II genotype was found to be associated with variables related to glucose intolerance.

Association of angiotensin-converting enzyme activity and polymorphism with echocardiographic measures in familial and nonfamilial hypertrophic cardiomyopathy

Angiotensin-converting enzyme (ACE) activity and polymorphism contribute significantly to the prognosis of patients with cardiomyopathy. The aim of this study was to determine the activity and type of ACE polymorphism in patients with familial and nonfamilial hypertrophic cardiomyopathy (HCM) and to correlate these with echocardiographic measurements (echo-Doppler). We studied 136 patients (76 males) with HCM (69 familial and 67 nonfamilial cases). Mean age was 41 ± 17 years. DNA was extracted from blood samples for the polymerase chain reaction and the determination of plasma ACE levels. Left ventricular mass, interventricular septum, and wall thickness were measured. Mean left ventricular mass index, interventricular septum and wall thickness in familial and nonfamilial forms were 154 ± 63 and 174 ± 57 g/m² (P = 0.008), 19 ± 5 and 21 ± 5 mm (P = 0.02), and 10 ± 2 and 12 ± 3 mm (P = 0.0001), respectively. ACE genotype frequencies were DD = 35%, ID = 52%, and II = 13%. A positive association was observed between serum ACE activity and left ventricular mass index (P = 0.04). Logistic regression showed that ACE activity was twice as high in patients with familial HCM and left ventricular mass index ≥190 g/m² compared with the nonfamilial form (P = 0.02). No other correlation was observed between ACE polymorphisms and the degree of myocardial hypertrophy. In conclusion, ACE activity, but not ACE polymorphisms, was associated with the degree of myocardial hypertrophy in the patients with HCM.

Chemical characterization and antimicrobial activity of essential oils of salvia L. species

In this work, the essential oils of S. officinalis, S. sclarea, S. lavandulifolia and S. triloba were chemically analyzed by gas chromatography coupled to a mass spectrometry detector (GC/MSD), and their antimicrobial activity was tested against 10 microorganisms using the disk diffusion method and the Minimum Inhibitory Concentration (MIC) technique. The following major compounds were identified in the essential oils: α - and β-thujone, camphor and 1,8-cineole, except in S. sclarea, where linalool, linalyl acetate and α-terpineol were the major constituents. The antimicrobial activity showed significant differences (p < 0.05) only when obtained by the MIC method. Gram-positive microorganisms presented larger sensitivity for the essential oils. The lowest MIC was observed when Staphylococcus aureus was exposed to 2.31 mg.mL-1 of S. lavandulifolia essential oil, while the highest MIC value was obtained when Shigella flexneri was exposed to 9.25 mg.mL-1 of the same essential oil, thus demonstrating that this essential oil may be effective as a bacteriostatic agent against Gram-positive microorganisms.

Inhibitory effect of the essential oil from Cinnamomum zeylanicum Blume leaves on some food-related bacteria

Cinnamomum zeylanicum Blume, Lauraceae, has long been known for having many biological properties. This study aimed to identify the constituents of the essential oil from C. zeylanicum leaves using GC-MS and to assess its inhibitory effect on Salmonella enterica, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa based on MIC and MBC determination and kill-time study. Eugenol (73.27%) was the most prevalent compound in the essential oil followed by trans-β-cariophyllene (5.38%), linalool (3.31%), and alcohol cinamic acetate (2.53%). The results showed an interesting antibacterial activity of the oil with MIC ranging from 1.25 to 10 µL.mL-1. MBC values were in the range of 20 - 80 µL.mL-1. A concentration of 10 and 40 µL.mL-1 of the essential oil caused a fast and steady decrease in viable cell count (2 to 5 log cycles) of all assayed strains along 24 hours. A concentration of 40 µL.mL-1 of the oil provided a total elimination of the initial inocula of S. aureus after 2 hours. These results show the possibility of regarding the essential oil from C. zeylanicum leaves as alternative sources of antimicrobial compounds to be applied in food conservation systems.

In vitro and in vivo antifungal activity of natural inhibitors against Penicillium expansum

Penicillium expansum is the causative agent of apple blue mold. The inhibitory effects of the capsaicin derived from Capsicum spp. fruits and yeast Hansenula wingei against P. expansum were evaluated in an in vitro and in in vivo assay using Fuji apples. The minimum inhibitory concentration of capsaicin determined using the broth micro-dilution method was 122.16 µg mL-1. Capsaicin did not reduce blue mold incidence in apples. However, it was able to delay fungal growth in the first 14 days of the in vivo assay. The in vivo effect of the yeast Hansenula wingei AM2(-2), alone and combined with thiabendazole at low dosage (40 µg mL-1), on the incidence of apple diseases caused by P. expansum was also described. H. wingei AM2(-2) combined with a low fungicide dosage (10% of the dosage recommended by the manufacturer) showed the best efficacy (100%) up to 7 days of storage at 21 ºC, later showing a non-statistically different decrease (p > 0.05) after 14 (80.45%) and 21 days (72.13%), respectively. These results contribute providing new options for using antifungal agents against Penicillium expansum.

Peroxidase activity and sensory quality of ready to cook mixed vegetables for soup: combined effect of biopreservatives and refrigerated storage

Enzymatic senescence processes and browning of fresh cut vegetables negatively affect their sensory properties and nutritional value and finally result in the rejection of affected products by consumers. In order to prevent quality decay, the combined effects of natural antioxidants and storage temperature on peroxidase activity and sensory attributes (overall visual quality, browning and odor) of individual and mixed vegetables for soup (butternut squash, leek and celery) were evaluated. Fresh cut vegetables were treated with antioxidant solutions as tea tree essential oil (15 μl/mL), propolis extract (15 μl/mL) and gallic acid (2 mg/mL) and stored at optimal (5 °C) and abusive (15 °C) temperature for a maximum of 14 days. The application of natural preservatives, plus optimal storage conditions, exerted significant inhibitory effects in peroxidase activity of squash, celery and mixed vegetables throughout the storage. Furthermore, propolis treatment applied on mixed vegetables retarded browning appearance and preserved the visual quality for a longer period when compared to untreated product.

Antimicrobial activity of amurca (olive oil lees) extract against selected foodborne pathogens

The antimicrobial activity of a methanolic extract of amurca (olive oil lees) was determined against both Gram-positive (L. monocytogenes and S. aureus) and Gram-negative (E. coli O157:H7 and S. enteritidis) foodborne pathogens at 10 °C or 37 °C using microdilution and disk diffusion methods, and its relative activity was compared to selected antibiotics. Minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations of amurca extract ranged from 60 to 80 µl/ml at 37 °C after 24 h against all tested strains. At 10 °C, amurca was more inhibitory with MIC and MBC values of 40 and 60 µl/ml, respectively, after 7 d against tested strains. Amurca at 40 µl/ml reduced numbers of tested pathogens by 2.5 to 3.2 log10 CFU/ml at 10 °C after 7 d, but was not inhibitory at 37 °C after 24 h. Protein prepared from amurca was not antimicrobial. The relative antimicrobial activity (inhibition zone ratio) of 80 µl/ml amurca methanolic extract compared to chloramphenicol, erythromycin, gentamycin and tetracycline ranged from 0.36 to 1.0 against Gram-negative and from 0.45 to 2.0 against Gram-positive bacteria. In addition, amurca extract inhibited E. coli O157:H7 02-0628 and S. aureus 26127 which were resistant to tetracycline and chloramphenicol, respectively.

Antifungal and antimycotoxigenic activity of essential oils from Eucalyptus globulus, Thymus capitatus and Schinus molle

Abstract Essential oils (EO) of eucalyptus (Eucalyptus globulus L.), thymus (Thymus capitatus L.) pirul (Schinus molle L.) were evaluated for their efficacy to control Aspergillus parasiticus and Fusarium moniliforme growth and their ability to produce mycotoxins. Data from kinetics radial growth was used to obtain the half maximal inhibitory concentration (IC50). The IC50 was used to evaluate spore germination kinetic and mycotoxin production. Also, spore viability was evaluated by the MTT assay. All EO had an effect on the radial growth of both species. After 96 h of incubation, thymus EO at concentrations of 1000 and 2500 µL L–1 totally inhibited the growth of F. moniliforme and A. parasiticus, respectively. Eucalyptus and thymus EO significantly reduced spore germination of A. parasiticus. Inhibition of spore germination of F. moniliforme was 84.6, 34.0, and 30.6% when exposed to eucalyptus, pirul, and thymus EO, respectively. Thymus and eucalyptus EO reduced aflatoxin (4%) and fumonisin (31%) production, respectively. Spore viability was affected when oils concentration increased, being the thymus EO the one that reduced proliferation of both fungi. Our findings suggest that EO affect F. moniliforme and A. parasiticus development and mycotoxin production.