Daniela Sales Alviano

Screening of chemical composition, antimicrobial and antioxidant activities of Artemisia essential oils

The chemical composition of essential oils isolated from aerial parts of seven wild sages from Western Canada -Artemisia absinthium L., Artemisia biennis Willd., Artemisia cana Pursh, Artemisia dracunculus L., Artemisia frigida Willd., Artemisia longifolia Nutt. and Artemisia ludoviciana Nutt., was investigated by GC-MS. A total of 110 components were identified accounting for 71.0-98.8% of the oil composition. High contents of 1,8-cineole (21.5-27.6%) and camphor (15.9-37.3%) were found in Artemisia cana, A. frigida, A. longifolia and A. ludoviciana oils. The oil of A. ludoviciana was also characterized by a high content of oxygenated sesquiterpenes with a 5-ethenyltetrahydro-5-methyl-2-furanyl moiety, of which davanone (11.5%) was the main component identified. A. absinthium oil was characterized by high amounts of myrcene (10.8%), trans-thujone (10.1%) and trans-sabinyl acetate (26.4%). A. biennis yielded an oil rich in (Z)-beta-ocimene (34.7%), (E)-beta-farnesene (40.0%) and the acetylenes (11.0%) (Z)- and (E)-en-yn-dicycloethers. A. dracunculus oil contained predominantly phenylpropanoids such as methyl chavicol (16.2%) and methyl eugenol (35.8%). Artemisia oils had inhibitory effects on the growth of bacteria (Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis), yeasts (Candida albicans, Cryptococcus neoformans), dermatophytes (Trichophyton rubrum, Microsporum canis, and Microsporum gypseum), Fonsecaea pedrosoi and Aspergillus niger. A. biennis oil was the most active against dermatophytes, Cryptococcus neoformans, Fonsecaea pedrosoi and Aspergillus niger, and A. absinthium oil the most active against Staphylococcus strains. In addition, antioxidant (beta-carotene/linoleate model) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities were determined, and weak activities were found for these oils.

Biological Activities of a-Pinene and β-Pinene Enantiomers

The antimicrobial activities of the isomers and enantiomers of pinene were evaluated against bacterial and fungal cells. The agar diffusion test showed that only the positive enantiomers of the a- and β-isomers of pinene were active. The minimal inhibitory concentration (MIC) and minimal microbicidal concentration (MMC) of these monoterpenes were also determined, confirming that the positive enantiomers exhibited microbicidal activity against all fungi and bacteria tested with MICs ranging from 117 to 4,150 µg/mL. However, no antimicrobial activity was detected with the negative enantiomers up to 20 mg/mL. Time-kill curves showed that (+)-a-pinene and (+)-β-pinene were highly toxic to Candida albicans, killing 100% of inoculum within 60 min. By contrast, the bactericidal effect occurred after 6 h in methicillin-resistant Staphylococcus aureus (MRSA). In combination with commercial antimicrobials, ciprofloxacin plus (+)-a-pinene or (+)-β-pinene presented synergistic activity against MRSA whereas an indifferent effect against all fungi was detected when amphotericin B was combined with the positive enantiomers of pinene. The potential of (+)-a-pinene and (+)-β-pinene to inhibit phospholipase and esterase activities was also evaluated, and the best inhibition results were obtained with Cryptococcus neoformans. C. albicans biofilm formation was prevented with the MIC concentration of (+)-a-pinene and twice the MIC value of (+)-β-pinene. Finally, the cytotoxicity of the positive enantiomers of pinene to murine macrophages was evaluated, and 250 µg/mL of (+)-a-pinene and (+)-β-pinene reduced the cell viability to 66.8% and 57.7%, respectively.The antimicrobial activities of the isomers and enantiomers of pinene were evaluated against bacterial and fungal cells. The agar diffusion test showed that only the positive enantiomers of the α- and β-isomers of pinene were active. The minimal inhibitory concentration (MIC) and minimal microbicidal concentration (MMC) of these monoterpenes were also determined, confirming that the positive enantiomers exhibited microbicidal activity against all fungi and bacteria tested with MICs ranging from 117 to 4,150 µg/mL. However, no antimicrobial activity was detected with the negative enantiomers up to 20 mg/mL. Time-kill curves showed that (+)-α-pinene and (+)-β-pinene were highly toxic to Candida albicans, killing 100% of inoculum within 60 min. By contrast, the bactericidal effect occurred after 6 h in methicillin-resistant Staphylococcus aureus (MRSA). In combination with commercial antimicrobials, ciprofloxacin plus (+)-α-pinene or (+)-β-pinene presented synergistic activity against MRSA whereas an indifferent effect against all fungi was detected when amphotericin B was combined with the positive enantiomers of pinene. The potential of (+)-α-pinene and (+)-β-pinene to inhibit phospholipase and esterase activities was also evaluated, and the best inhibition results were obtained with Cryptococcusneoformans. C. albicans biofilm formation was prevented with the MIC concentration of (+)-α-pinene and twice the MIC value of (+)-β-pinene. Finally, the cytotoxicity of the positive enantiomers of pinene to murine macrophages was evaluated, and 250 µg/mL of (+)-α-pinene and (+)-β-pinene reduced the cell viability to 66.8% and 57.7%, respectively.

Melissa officinalis L. essential oil: antitumoral and antioxidant activities

Melissa officinalis L (lemon balm) is a traditional herbal medicine used widely as a mild sedative, spasmolytic and antibacterial agent. This paper focuses on the analysis of the chemical composition and the biological activities of M. officinalis essential oil obtained under controlled harvesting and drying conditions. An in‐vitro cytotoxicity assay using MTT indicated that this oil was very effective against a series of human cancer cell lines (A549, MCF‐7, Caco‐2, HL‐60, K562) and a mouse cell line (B16F10). This oil possessed antioxidant activity, as evidenced by reduction of 1,1‐diphenyl‐2‐picryl‐hydrazyl (DPPH). These results pointed to the potential use of M. officinalis essential oil as an antitumoral agent.

In vitro antioxidant potential of medicinal plant extracts and their activities against oral bacteria based on Brazilian folk medicine

BACKGROUND AND OBJECTIVES This study aims to determine antibacterial activities of Cocos nucifera (husk fiber), Ziziphus joazeiro (inner bark), Caesalpinia pyramidalis (leaves), aqueous extracts and Aristolochia cymbifera (rhizomes) alcoholic extract against Prevotella intermedia, Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mutans and Lactobacillus casei. The antioxidant activity and acute toxicity of these extracts were also evaluated. MATERIAL AND METHODS The plant extracts antibacterial activity was evaluated in vitro and the minimal inhibitory concentration (MIC) was determined by the broth micro-dilution assay. The bacterial killing kinetic was also evaluated for all extracts. In addition, the antibacterial effect of the extracts was tested in vitro on artificial oral biofilms. The acute toxicity of each extract was determined in according to Lorke [Lorke D. A new approach to practical acute toxicity testing. Arch Toxicol 1983;54:275-87] and the antioxidant activity was evaluated by DPPH photometric assay [Mensor LL, Menezes FS, Leitão GG, Reis AS, Santos TC, Coube CS, et al. Screening of Brazilian plants extract for antioxidant activity by the use of DPPH free radical method. Phytother Res 2001;15:127-30]. RESULTS MIC and the bactericidal concentrations were identical, for each evaluated extract. However, microbes of artificial biofilms were less sensitive to the extracts than the planktonic strains. A. cymbifera extract induced the highest bactericidal effect against all tested bacteria, followed by C. nucifera, Z. joazeiro and C. pyramidalis extracts, respectively. All extracts showed good antioxidant potential, being C. nucifera and C. pyramidalis aqueous extracts the most active ones. CONCLUSION In conclusion, all oral bacteria tested (planktonic or in artificial biofilms) were more susceptible to, and rapidly killed in presence of A. cymbifera, C. pyramidalis and C. nucifera than Z. joazeiro extracts, respectively. Thus, these extracts may be of great interest for future studies about treatment of oral diseases, considering their potent antioxidant activity and low toxicity.

Melanin from Fonsecaea pedrosoi Induces Production of Human Antifungal Antibodies and Enhances the Antimicrobial Efficacy of Phagocytes

ABSTRACT Fonsecaea pedrosoi is a fungal pathogen that produces melanin. The functions of melanin and its possible influence in the protective immunological response during infection by F. pedrosoi are not known. In this work, treatment of F. pedrosoi mycelia with proteases and glycosidases followed by a denaturing agent and hot concentrated acid left a black residue. Scanning electron microscopy demonstrated that this processed melanized residue resembled very closely the intact mycelium in shape and size. Melanin particles were also isolated from culture fluids of conidia or sclerotic forms of F. pedrosoi. Secreted melanins were reactive with sera from infected human patients, suggesting that F. pedrosoi synthesizes melanin in vivo. The antibodies against melanin were purified from patients’ sera and analyzed by indirect immunofluorescence. They reacted with sclerotic cells from patients’ lesions as well as with sclerotic bodies cultivated in vitro, conidia, mycelia, and digested residues. Treatment of F. pedrosoi with purified antibodies against melanin inhibited fungal growth in vitro. The interaction of F. pedrosoi with phagocytes in the presence of melanin resulted in higher levels of fungal internalization and destruction by host cells, which was accompanied by greater degrees of oxidative burst. Taken together, these results indicate that melanin from F. pedrosoi is an immunologically active fungal structure that activates humoral and cellular responses that could help the control of chromoblastomycosis by host defenses.

Identification of sialic acids on the cell surface of Candida albicans

The cell-surface expression of sialic acids in two isolates of Candida albicans was analyzed by thin-layer and gas chromatography, binding of lectins, colorimetry, sialidase treatment and flow cytofluorimetry with fluorescein-labeled lectins. N-acetylneuraminic acid (NANA) was the only derivative found in both strains of C. albicans grown in a chemically defined medium. Its identification was confirmed by mass spectrometry in comparison with an authentic standard. The density of sialic acid residues per cell ranged from 1. 6x10(6) to 2.8x10(6). The surface distribution of sialic acids over the entire C. albicans was inferred from labeling with fluorescein-Limulus polyphemus and Limax flavus agglutinins and directly observed by optical microscopy with (FITC)-Sambucus nigra agglutinin (SNA), abrogated by previous treatment of yeasts with bacterial sialidase. Sialidase-treated yeasts generated beta-galactopyranosyl terminal residues that reacted with peanut agglutinin. In C. albicans N-acetyl-neuraminic acids are alpha2,6- and alpha2,3-linked as indicated by yeast binding to SNA and Maackia amurensis agglutinin. The alpha2,6-linkage clearly predominated in both strains. We also investigated the contribution of sialic acids to the electronegativity of C. albicans, an important factor determining fungal interactions in vivo. Adhesion of yeast cells to a cationic solid phase substrate (poly-L-lysine) was mediated in part by sialic acids, since the number of adherent cells was significantly reduced after treatment with bacterial sialidase. The present evidence adds C. albicans to the list of pathogenic fungi that synthesize sialic acids, which contribute to the negative charge of fungal cells and have a role in their specific interaction with the host tissue.

Characterization of Gordonia sp. strain F.5.25.8 capable of dibenzothiophene desulfurization and carbazole utilization

A dibenzothiophene (DBT)-degrading bacterial strain able to utilize carbazole as the only source of nitrogen was identified as Gordonia sp. F.5.25.8 due to its 16S rRNA gene sequence and phenotypic characteristics. Gas chromatography (GC) and GC–mass spectroscopy analyses showed that strain F.5.25.8 transformed DBT into 2-hydroxybiphenyl (2-HBP). This strain was also able to grow using various organic sulfur or nitrogen compounds as the sole sulfur or nitrogen sources. Resting-cell studies indicated that desulfurization occurs either in cell-associated or in cell-free extracts of F.5.25.8. The biological responses of F.5.25.8 to a series of mutagens and environmental agents were also characterized. The results revealed that this strain is highly tolerant to DNA damage and also refractory to induced mutagenesis. Strain F.5.25.8 was also characterized genetically. Results showed that genes involved in desulfurization (dsz) are located in the chromosome, and PCR amplification was observed with primers dszA and dszB designed based on Rhodococcus genes. However, no amplification product was observed with the primer based on dszC.

The latex obtained from Hancornia speciosa Gomes possesses anti-inflammatory activity.

AIM OF THE STUDY Hancornia speciosa Gomes (Apocynaceae) is a tree that is widely distributed throughout Brazil. Its latex is collected and used extensively to treat acne, warts, diseases related to bursitis, and inflammation. In this work, we describe the anti-inflammatory effects of the latex. MATERIALS AND METHODS The latex from Hancornia speciosa (0.06-1.3mg/kg, p.o.) and the reference drug acetylsalicylic acid (ASA, 200mg/kg, p.o.) were evaluated in analgesia (formalin-induced licking, acetic acid-induced contortions, and hot plate) and inflammation models (formalin-induced licking, paw oedema, and subcutaneous air pouch, with measurement of cell migration, exudate volume, protein extravasations, nitric oxide, prostaglandin E2, TNF-α, and IL-6, and expression of the enzymes inducible nitric oxide synthase and cyclooxygenase 2). RESULTS The latex from Hancornia speciosa significantly inhibited the number of writhings and the time that the animal spent licking the formalin-injected paw (second phase). Doses of 0.1-1.3mg/kg latex reduced carrageenan-induced rat paw oedema. However, only the highest doses (0.6 and/or 1.3mg/kg) reduced the oedema induced by bradykinin, histamine, and serotonin. The latex also inhibited inflammation induced by subcutaneous carrageenan injection, cell migration, exudate volume, protein extravasations, increased levels of inflammatory mediators (nitric oxide, prostaglandin E2, TNF-α, and IL-6) produced in the pouch, and increased expression of the enzymes nitric oxide synthase and cyclooxygenase 2. CONCLUSIONS Our results indicate that the latex obtained from Hancornia speciosa demonstrates significant anti-inflammatory activity through the inhibition of nitric oxide, PGE2, and cytokine production, thus confirming the popular use of this plant as an anti-inflammatory agent.

Inhibitory activity of Paenibacillus polymyxa SCE2 against human pathogenic micro-organisms

Paenibacillus polymyxa strain SCE2 was shown to inhibit the growth of different potential human pathogenic bacterial strains and fungi in vitro. To determine the genetic characterization of this antimicrobial substance, strain SCE2 was transformed with plasmid pTV32(Ts), a delivery vector for Tn917‐lac. After transposition, four mutants were shown to have lost their capability to inhibit Micrococcus sp. and Staphylococcus aureus RN450, but they continued to inhibit the growth of Corynebacterium fimi NCTC7547 and Escherichia coli HB101. Hybridization experiments using the DNA of the four mutants digested with different endonucleases and pTV32(Ts) as a probe showed that the place of insertion of Tn917‐lac in the chromosome was the same in mutants 4 and 36 and in mutants 31 and 59, but different between these pairs. It is thought possible that more than one antimicrobial substance is being produced by strain SCE2.

Conventional Therapy and Promising Plant-Derived Compounds Against Trypanosomatid Parasites

Leishmaniasis and trypanosomiasis are two neglected and potentially lethal diseases that affect mostly the poor and marginal populations of developing countries around the world and consequently have an important impact on public health. Clinical manifestations such as cutaneous, mucocutaneous, and visceral disorders are the most frequent forms of leishmaniasis, a group of diseases caused by several Leishmania spp. American trypanosomiasis, or Chagas disease, is caused by Trypanosoma cruzi, a parasite that causes progressive damage to different organs, particularly the heart, esophagus, and lower intestine. African trypanosomiasis, or sleeping sickness, is caused by Trypanosoma brucei and is characterized by first presenting as an acute form that affects blood clotting and then becoming a chronic meningoencephalitis. The limited number, low efficacy, and side effects of conventional anti-leishmania and anti-trypanosomal drugs and the resistance developed by parasites are the major factors responsible for the growth in mortality rates. Recent research focused on plants has shown an ingenious way to obtain a solid and potentially rich source of drug candidates against various infectious diseases. Bioactive phytocompounds present in the crude extracts and essential oils of medicinal plants are components of an important strategy linked to the discovery of new medicines. These compounds have proven to be a good source of therapeutic agents for the treatment of leishmaniasis and trypanosomiasis. This work highlights some chemotherapeutic agents while emphasizing the importance of plants as a source of new and powerful drugs against these widespread diseases.