Disinfectant activities of extracts and metabolites from Baccharis dracunculifolia DC.

The concern regarding the harm caused by biocides to human health has been increasing over the years, making the natural products an alternative to less toxic and more efficient biocides. Therefore, this paper reports the investigation of the disinfectant potential of extracts and isolated compounds from Baccharis dracunculifolia. For this purpose, extracts of aerial parts (BD-C), tricomial wash (BD-L) and roots (BD-R) of B. dracunculifolia were obtained by maceration. The extracts were submitted to different chromatographic techniques, including high-speedy countercurrent chromatography (HSCCC) furnishing nine isolated compounds. The extracts and isolated compounds were evaluated regarding their antimicrobial activity by the broth microdilution method, according to the Clinical and Laboratory Standards Institute, and regarding their sanitizing activity according to Standard Operating Procedure No. 65·3210·007 (INCQS, 2011), developed by the National Institute for Quality Control in Health (INCQS) - Oswaldo Cruz Foundation (FIOCRUZ). In the antimicrobial evaluation the BD-C extract showed minimum inhibitory concentration (MIC) values of 200 and 100 µg/ml against Staphylococcus aureus and Tricophyton mentagrophytes, respectively. BD-L extract showed MIC value of 200 µg/ml against S. aureus. The isolated compounds caffeic acid (MBC 2·22 µmol l-1 ), ferulic acid (MBC 2·06 µmol l-1 ) and baccharin (MBC 0·27 µmol l-1 ) showed significant inhibitory activity against S. aureus. All B. dracunculifolia isolated compounds were active with exception of aromadrendin-4´-O-methyl-ether for T. mentagrophytes. Additionally, isosakuranetin was active against Salmonella choleraesuis (MIC 1·4 µmol l-1 ). Regarding the sanitizing activity, the hydroalcoholic solution containing 0·2% of B. dracunculifolia extract in 40°GL ethanol was effective in eliminating the microbial contamination on all carrier cylinders and against all microorganisms evaluated in the recommended exposure time of 10 min. Therefore, B. dracunculifolia has potential for the development of sanitizing agents to be used in hospitals, food manufactures and homes.

A New Antileishmanial Preparation of Combined Solamargine and Solasonine Heals Cutaneous Leishmaniasis through Different Immunochemical Pathways.

Little has been done during the past 100 years to develop new antileishmanial drugs. Most infected individuals live in poor countries and have a low cash income to be attractive targets to pharmaceutical corporations. Two heterosidic steroids, solamargine and solasonine, initially identified as major components of the Brazilian plant Solanum lycocarpum, were tested for leishmanicidal activity. Both alkaloids killed intracellular and extracellular Leishmania mexicana parasites more efficiently than the reference drug sodium stibogluconate. A total of 10 µM each individual alkaloid significantly reduced parasite counts in infected macrophages and dendritic cells. In vivo treatment of C57BL/6 mice with a standardized topical preparation containing solamargine (45.1%) and solasonine (44.4%) gave significant reductions in lesion sizes and parasite counts recovered from lesions. Alkaloids present different immunochemical pathways in macrophages and dendritic cells. We conclude that this topical preparation is effective and a potential new and inexpensive treatment for cutaneous leishmaniasis.

Δ-lactam derivative from thermophilic soil fungus exhibits in vitro anti-allergic activity.

From cultures of thermophilic soil fungus Humicola grisea var thermoidea, a δ-lactam derivative (3-(2-(4-hydroxyphenyl)-2-oxoethyl)-5,6-dihydropyridin-2(1H)-one) that displayed anti-allergic activity was isolated, which was predicted by in silico computational chemistry approaches. The in vitro anti-allergic activity was investigated by ß-hexosaminidase release assay in rat basophilic leukaemia RBL-2H3 cells. The δ-lactam derivative exhibited similar anti-allergic activity (IC(50) = 18.7 ± 6.7 µM) in comparison with ketotifen fumarate (IC(50) = 15.0 ± 1.3 µM) and stronger anti-allergic activity than azelastine (IC(50) = 32.0 µM). Also, the MTT cytotoxicity assay with RBL-2H3 cells showed that δ-lactam does not display cytotoxicity at concentrations lower than 50 µM. This study suggests that the δ-lactam derivative has the potential to be used as a lead compound in the development of anti-allergic drugs for clinical use in humans.

Inhibiting effect of Dorstenia asaroides extracts on cariogenic properties of Streptococcus mutans.

The aim of this study was to examine the effects of Dorstenia asaroides extracts on cariogenic properties of the most cariogenic bacteria, Streptococcus mutans. Hexane (HFr), ethyl-acetate (EFr) and chloroform (CFr) extracts obtained from D. asaroides rhizomes were submitted to chemical analyses, Minimal Inhibitory Concentrations (MIC), glycolysis assay and S. mutans 12-h-old initial biofilms. Chemical characterization showed that all the extracts present furanocoumarins. The MIC values were 80 (HFr and CFr) and 50 µg/mL (EFr). Acid production by S. mutans cells was significantly disrupted by HFr (12.5 mg/mL), EFr (at 2.5; 6.25 and 12.5 mg/mL) and CFr (at 2.5, 6.25 and 12.5 mg/mL) (p < 0.01). Topical applications of HFr, EFr and CFr significantly reduced the colony forming units of S. mutans biofilms compared with those treated with control group in order to 20, 30 and 25% respectively (p < 0.01). The results of the present study suggest that rhizomes of D. asaroides had inhibitory effects on cariogenic properties of S. mutans.

The effect of the dibenzylbutyrolactolic lignan (-)-cubebin on doxorubicin mutagenicity and recombinogenicity in wing somatic cells of Drosophila melanogaster.

The dibenzylbutyrolactolic lignan (-)-cubebin was isolated from dry seeds of Piper cubeba L. (Piperaceae). (-)-Cubebin possesses anti-inflammatory, analgesic and antimicrobial activities. Doxorubicin (DXR) is a topoisomerase-interactive agent that may induce single- and double-strand breaks, intercalate into the DNA and generate oxygen free radicals. Here, we examine the mutagenicity and recombinogenicity of different concentrations of (-)-cubebin alone or in combination with DXR using standard (ST) and high bioactivation (HB) crosses of the wing Somatic Mutation And Recombination Test in Drosophila melanogaster. The results from both crosses were rather similar. (-)-Cubebin alone did not induce mutation or recombination. At lower concentrations, (-)-cubebin statistically reduced the frequencies of DXR-induced mutant spots. At higher concentrations, however, (-)-cubebin was found to potentiate the effects of DXR, leading to either an increase in the production of mutant spots or a reduction, due to toxicity. These results suggest that depending on the concentration, (-)-cubebin may interact with the enzymatic system that catalyzes the metabolic detoxification of DXR, inhibiting the activity of mitochondrial complex I and thereby scavenging free radicals. Recombination was found to be the major effect of the treatments with DXR alone. The combined treatments reduced DXR mutagenicity but did not affect DXR recombinogenicity.

The role of seasonality on the inhibitory effect of Brazilian green propolis on the oxidative metabolism of neutrophils.

The reactive oxygen species (ROS) produced by neutrophils are involved in the pathogenesis of several diseases, for which the intake of antioxidants could benefit patients either as a prophylactic or therapeutic treatment. Propolis is among the known antioxidants, and its chemical composition may vary under the influence of seasonality, which may interfere in its biological properties. This work evaluates the role of seasonality on the production of some important compounds of propolis samples produced monthly from November 2001 through October 2002 as well as the effect of these samples on the oxidative metabolism of stimulated neutrophils, by using both luminol and lucigenin to produce chemiluminescence (CLlum and CLluc, respectively). The cytotoxicity of the most active extracts to neutrophils was also investigated. The inhibitory effect of the propolis samples varied significantly during the studied period for both assays (3.4 ± 1.1 to 16.0 ± 1.1 µg/mL for CLlum and 6.2 ± 2.0 to 30.0 ± 5.0 µg/mL for CLluc), which was also observed in the quantitative profile of the main analyzed compounds (aromadendrin-4'-methyl ether, artepillin C, and baccharin). This effect started to become more prominent during the fall and, among all the studied extracts, the one obtained in May displayed the highest inhibitory effect on CL production (3.4 ± 1.1 µg/mL for CLlum and 6.2 ± 2.0 µg/mL for CLluc). The HPLC qualitative profiles of the extracts of propolis samples were quite similar, but there was a huge variation in terms of quantitative profile. It seems that aromadendrin-4'-methyl ether and baccharin play an essential role in the antioxidant activity, while artepillin C is not very important for this effect. The extracts presenting the highest antioxidant activity were produced in May, June, and August, and they did not display cytotoxicity at 25 µg/mL; quercetin, used as control, was not toxic to neutrophils at 8.5 µg/mL.

In vitro cytotoxic activity of Baccharis dracunculifolia and propolis against HEp-2 cells.

Baccharis dracunculifolia is the most important vegetal source of propolis in southeast Brazil, and researchers have been investigating its biological properties. Propolis is a complex resinous hive product collected by bees from several plants, showing a very complex chemical composition. It has been employed since ancient times due to its therapeutic properties, such as antimicrobial, anti-inflammatory, antioxidant, immunomodulatory and antitumour activities, among others. The goal of this work was to compare the cytotoxic action of B. dracunculifolia, propolis and two isolated compounds (caffeic and cinnamic acids) on human laryngeal epidermoid carcinoma (HEp-2) cells in vitro. These cells were incubated with different concentrations of each variable, and cell viability was assessed by the crystal violet method. Lower concentrations of B. dracunculifolia (extract and essential oil), propolis, as well as caffeic and cinnamic acids, showed no cytotoxic activity against HEp-2 cells. On the other hand, elevated concentrations (50 and 100 µg per 100 µL) exerted a cytotoxic action, and propolis showed a more efficient action than its vegetal source and isolated compounds. Further investigation is still needed in order to explore the potential of these variables as antitumour agents and to understand their mechanisms of action.

Evaluation of the genotoxic potential of Austroplenckia populnea (Reiss) Lundell chloroform fraction from barkwood extract in rodent cells in vivo.

The genotoxic effect of the Austroplenckia populnea chloroform fraction from barkwood extract was tested in vivo on peripheral blood cells of Swiss mice with the comet assay (SCGE), and the clastogenic effect was investigated on peripheral blood cells of Swiss mice and bone marrow cells of Wistar rats, with the micronucleus and chromosome aberrations tests. The animals were treated by gavage with 3 concentrations of the extract: 300, 600 and 900 mg.kg-1. Peripheral blood cells of Swiss mice were collected 4 and 24 hours after the treatment to the SCGE assay and 48 and 72 hours to the micronucleus test. Bone marrow cells of Wistar rats were collected 24 hours after the treatment to the micronucleus and chromosome aberration tests. The results showed that the A. populnea chloroform fraction induced an increase in the average number of DNA damage in peripheral blood cells at the three concentrations tested, but this increase was not statistically significant. In the micronucleus and chromosome aberrations test, no significant increase was observed in the mean number of micronucleated polychromatic erythrocytes (MNPCE) of Swiss mice or MNPCE or chromosome aberrations for the rat bone marrow cells, for any of the tested doses. Our findings enable us to conclude that by the comet assay, A. populnea chloroform fraction from barkwood extract showed no genotoxic effects, and by the micronucleus and chromosome aberration tests, the extract fraction showed no clastogenic/aneugenic effects on the rodent cells tested.

Lemongrass effects on IL-1beta and IL-6 production by macrophages.

Cymbopogon citratus has been widely recognised for its ethnobotanical and medicinal usefulness. Its insecticidal, antimicrobial and therapeutic properties have been reported, but little is known about its effect on the immune system. This work aimed to investigate the in vivo effect of a water extract of lemongrass on pro-inflammatory cytokine (IL-1beta and IL-6) production by macrophages of BALB/c mice. The action of lemongrass essential oil on cytokine production by macrophages was also analysed in vitro. The chemical composition of the extract and the oil was also investigated. Treatment of mice with water extract of lemongrass inhibited macrophages to produce IL-1beta but induced IL-6 production by these cells. Lemongrass essential oil inhibited the cytokine production in vitro. Linalool oxide and epoxy-linalool oxide were found to be the major components of lemongrass water extract, and neral and geranial were the major compounds of its essential oil. Taken together, these data suggest an anti-inflammatory action of this natural product.

In vitro antileishmanial, antiplasmodial and cytotoxic activities of phenolics and triterpenoids from Baccharis dracunculifolia D. C. (Asteraceae).

Baccharis dracunculifolia (Asteraceae), the most important plant source of the Brazilian green propolis (GPE), displayed in vitro activity against Leishmania donovani, with an IC(50) value of 45 microg/mL, while GPE presented an IC(50) value of 49 microg/mL. Among the isolated compounds of B. dracunculifolia, ursolic acid, and hautriwaic acid lactone showed IC(50) values of 3.7 microg/mL and 7.0 microg/mL, respectively. Uvaol, acacetin, and ermanin displayed moderate antileishmanial activity. Regarding the antiplasmodial assay against Plasmodium falciparum, BdE and GPE gave similar IC(50) values (about 20 microg/mL), while Hautriwaic acid lactone led to an IC(50) value of 0.8 microg/mL (D6 clone).

Anti-poliovirus activity of Baccharis dracunculifolia and propolis by cell viability determination and real-time PCR.

AIMS: The aim of this work was to evaluate the antiviral activities of Baccharis dracunculifolia (extract and essential oil), propolis and some isolated compounds (caffeic and cinnamic acids) against poliovirus type 1 (PV1) replication in HEp-2 cells. METHOD: Three different protocols (pre-, simultaneous and post-treatments) were used to verify the effect of addition time of the variables on PV1 replication by crystal violet method and relative viral RNA quantification by real-time PCR for analysing in which step of virus replication the variables could interfere. CONCLUSIONS: Data revealed that the B. dracunculifolia showed the best antiviral activity percentage in the simultaneous treatment, as well as lower relative viral quantification by real-time PCR. Variables might block partially the viral entry within cells, affect the steps of viral cycle replication into cells, or lead to RNA degradation before the virus entry into cells or after their release to the supernatant. SIGNIFICANCE AND IMPACT OF THE STUDY: Baccharis dracunculifolia is the most important botanical source of the south-eastern Brazilian propolis, and its potential for the development of new phytotherapeutic medicines has been investigated. Propolis is commonly used for its antimicrobial and immunomodulatory activities. Nevertheless, B. dracunculifolia and propolis effects on PV1 have not been investigated yet.

In vitro and in vivo effects of clove on pro-inflammatory cytokines production by macrophages.

Biological properties of clove have been reported, but little is known about its effect on the immune system. This work was aimed to investigate the effect in vivo of a water-soluble part of hydroalcoholic extract of clove on pro-inflammatory cytokines (IL-1beta and IL-6) production by macrophages of BALB/c mice. The action of the essential oil of clove on the production of these cytokines macrophages was also investigated in vitro. The chemical compositions of the extract and of the oil were also investigated. Treatment of mice with water extract of clove was found to inhibit macrophages to produce both IL-1beta and IL-6. The essential oil of clove also inhibited the production of these cytokines in vitro. Eugenol was found to be the major component of the clove extract and essential oil, and probably is the causative agent of cytokine inhibition. Taken together, these data suggest an anti-inflammatory action of this spice.

Evaluation of piper cubeba extract, (-)-cubebin and its semi-synthetic derivatives against oral pathogens.

The activities of the crude ethanol extract from Piper cubeba seeds, (-)-cubebin and its semi-synthetic derivatives were evaluated against oral pathogens. The crude ethanol extract was more active against Streptococcus salivarius (MIC value of 80 microg/mL). (-)-Cubebin displayed MIC values ranging from 0.20 mm for Streptococcus mitis to 0.35 mm for Enterococcus faecalis. The natural product (-)-cubebin and its semi-synthetic derivative (-)-hinokinin displayed bacteriostatic activity at all evaluated concentrations, as well as fungicidal activity against Candida albicans at 0.28 mm. The O-benzyl cubebin derivative showed fungistatic and fungicidal effects against C. albicans at 0.28 mm and 0.35 mm, respectively. Also, the other dibenzylbutyrolactone derivatives [(-)-6,6'-dinitrohinokinin and (-)-O-(N,N-dimethylaminoethyl)-cubebin] displayed bacteriostatic and fungistatic effects at the evaluated concentrations. Moreover, the semi-synthetic derivative (-)-6,6'-dinitrohinokinin was the most active compound against all the evaluated microorganisms. Therefore, it may be suggested that the presence of the carbonyl group at C-9 plus the introduction of polar groups in the aromatic rings improve the antimicrobial activity of dibenzylbutyrolactone compounds.

Anti-inflammatory and antinociceptive activities of extract, fractions and populnoic acid from bark wood of Austroplenckia populnea.

Austroplenckia populnea (Reiss) Lund is a Brazilian plant from "cerrado", which belongs to Celastraceae family, popularity know as "marmelinho-do campo, mangabeira-brava, mangabarana, vime and maria-mole". This plant is used in folk medicine to treat dysenteries and inflammatory disorders, such as rheumatism. Austroplenckia populnea bark hydroalcoholic crude extract, and its hexane, chloroform and ethyl acetate fractions, obtained by partition, as well as the isolated populnoic acid were investigated for their anti-inflammatory (carrageenan, dextran and histamine-induced rat paw oedema, histamine-induced increase in vascular permeability, and granulomatous tissue induction) and analgesic activities (writhing and hot plate tests). The ED(50) (oral) of the crude extract for the inhibition of carragenan-induced rat paw oedema assay was determined to be 200 mg/kg, which was also used in the assays with the extract and its fractions in all other experiments. Populnoic acid was administered in the dose of 50 mg/kg. Crude extract, hexane and chloroform fractions (200 mg/kg), and indomethacin (10 mg/kg) inhibited significantly (p<0.05) the formation of the carrageenan-induced rat paw oedema, measured in third hour of experiment (peak of oedema formation) by 43.2%, 37.3%, 31.1% and 59.3%, respectively. There was a significant reduction (p<0.05) in dextran-induced rat paw oedema in all groups, while in the assay using histamine as the oedematogenic agent, only the groups treated with populnoic acid (50 mg/kg) and cyproheptadine (10 mg/kg) displayed significant reduction (p<0.05). The populnoic acid and cyproheptadine reduced the peak of oedema formation (1st hour) by 41.3% and 34.7%, respectively. Only for the groups treated with populnoic acid (50 mg/kg) and cyproheptadine (10 mg/kg) it was observed a significant (p<0.05) reduction in histamine-induced increase in vascular permeability (44.8% and 80.3%, respectively). Granulomatous tissue formation was significantly inhibited (p<0.05) by both hexane fraction (46.0%) and dexamethasone (66.2%). In the analgesic assays, the crude extract and its hexane and chloroform fractions, as well as indomethacin diminished significantly the number of writhings (p<0.05) by 69.6%, 47.2%, 44.8% and 62.8%, respectively. On the other hand, none assayed sample displayed significant result in the hot plate test. Based on the obtained results it is suggested that extracts of Austroplenckia populnea bark and populnoic acid display anti-inflammatory activity, supporting its folkloric use to treat inflammatory disorders.

Analgesic and anti-inflammatory activity of a crude root extract of Pfaffia glomerata (Spreng) Pedersen.

The anti-inflammatory and antinociceptive effects of the crude hydroalcoholic extract (PE) of Pfaffia glomerata roots was assessed in the carrageenan-induced rat paw edema at the doses of 100, 200 and 300 mg/kg, using different animal models. An anti-inflammatory dose effect response correlation of r=0.997 and Y=11.67x+0.02 was found. At the same doses, the extract-inhibited acetic acid-induced writhing in mice, but no dose response correlation was found. Oral administration of 100 mg/kg of PE and 0.5 mg/kg of dexamethazone inhibited by 29 and 61%, the granulomatous tissue formation (p>0.05), respectively. These results indicate the potential of this plant extract to treat chronic inflammation. At the assayed doses no significant activity was found in the hot plate test, as well as in the cell migration-induced by carrageenan.

Effect of Brazilian green propolis on the production of reactive oxygen species by stimulated neutrophils.

The activity of a crude ethanol extract of green propolis and its fractions obtained by partition with hexane, chloroform and n-butanol was assessed on luminol- and lucigenin- enhanced chemiluminescence (CL) produced by rabbit neutrophils (PMNs) stimulated with particles of serum-opsonized zymosan (OZ). The total production of reactive oxygen species (ROS) by PMNs was measured by the luminol-enhanced CL (LumCL) assay and the production of the superoxide anion (O2*-) by the lucigenin-enhanced CL (LucCL) assay. All evaluated propolis samples had inhibitory effect on the LumCL and LucCL, which was concentration dependent. The n-butanol and chloroform fractions displayed the highest inhibitory effect on the LumCL produced by PMNs stimulated with OZ, in comparison with both the ethanol extract and the hexane fraction. Besides, the hexane fraction was the one which presented the highest effect for the LucCL assay. Some isolated compounds from both n-butanol and chloroform fractions were also assessed, including kaempferide, isosakuranetin, aromadendrine-4'-methyl-ether and 3-prenyl-p-coumaric acid. Kaempferide presented the highest inhibitory effect on the LumCL in comparison with the other compounds. Moreover, under the conditions assessed, the studied green propolis samples and isolated compounds were not toxic to the rabbit PMNs.

Analgesic and anti-inflammatory activities evaluation of (-)-O-acetyl, (-)-O-methyl, (-)-O-dimethylethylamine cubebin and their preparation from (-)-cubebin.

The anti-inflammatory and antinociceptive effects of the acetylated (2), methylated (3) and aminated (4) derivatives of cubebin (1), obtained by its reaction with acetic anhydride, methyl iodide and dimethylethylamine chloride, respectively, were investigated, using different animal models. The compound (2) was the most effective anti-inflammatory one in the carrageenin-induced paw edema in rats and was the only one which showed dose-response correlation for this assay with r = 0.993 and Y = 64.58x + 0.22. Besides, compounds (2) and (4) were more effective than cubebin in inhibiting acetic acid-induced writhing in mice, producing dose-response correlation with doses of 10, 20 and 30 mg/kg, respectively. Regarding the hot plate and the cell migration tests in rats, none of the four tested compounds showed activity. Overall, the results showed that the acetylation and amination of cubebin were efficient in enhancing its analgesic activity, as well as its anti-inflammatory activity.

Evaluation of the trypanocidal and leishmanicidal in vitro activity of the crude hydroalcoholic extract of Pfaffia glomerata (Amarathanceae) roots.

Three different concentrations (1, 10 and 50 microg/ml) of lyophilized hydroalcoholic crude extract of Pfaffia glomerata roots were assayed in vitro against strains of Trypanosoma cruzi (Y) and Leishmania braziliensis. It was observed that P. glomerata hydroalcoholic extract was relatively active within the tested concentrations for L. (V) braziliensis, but inactive against T. cruzi. Despite the fact that both protozoans belong to the Trypanosomatidae family, we suggest that the difference observed for activity should be related to the biological differences between the two parasite species.

Anti-inflammatory activity of cubebin, a lignan from the leaves of Zanthoxyllum naranjillo Griseb.

Cubebin, a dibenzylbutyrolactone lignan isolated from the crude hexane extract of the leaves of Zanthoxyllum naranjillo, showed a significant anti-inflammatory activity by using the paw edema induced by carrageenin in rats, but did not provide a significant reduction in the cell migration for the acute carrageenin-induced inflammatory reaction in the peritoneal cavity of rats. Neither was it effective in reducing the edema induced by dextran nor the edema induced by histamine. It partially reduced the edema induced by serotonin. Moreover, it significantly reduced the edema induced by prostaglandin PGE2 and the number of writhings induced by both acetic acid and PGI2 in mice. Therefore, it may be suggested that the mechanism of action of cubebin is similar to that observed for most of the non-steroidal drugs.

Evaluation of the trypanocidal activity of lignans isolated from the leaves of Zanthoxylum naranjillo.

Seven lignans isolated from the hexane extract of the leaves of Zanthoxyllum naranjillo (Rutaceae) were tested in both in vitro and in vivo assays against two strains of Trypanosoma cruzi (Bolivia and Y). Of the seven assayed lignans, three were inactive [(+)-sesamin, (+)-piperitol-4'-O-gamma, gamma-dimethylallyl ether and (-)-3,4-dimethoxy-3',4'-demethylenedioxycubebin] and three were partially active in the in vitro assays [(-)-hibalactone, (-)-kaerophylin and (-)-cubebin]. Healthy animals developed the disease after injection of the tested infected blood samples containing these three compounds. One compound [(-)-methylpluviatolide] was highly effective in the in vitro assay and healthy animals injected with the tested samples did not develop the disease. Moreover, only (-)-methylpluviatolide was highly active against the bloodstream forms of both strains of T. cruzi for the in vivo assay. However, it was not active against the tissue forms of the parasite.