New Isoquinoline Alkaloids from Paraphaeosphaeria sporulosa F03, a Fungal Endophyte Isolated from Paepalanthus planifolius.

As part of our continuing efforts to discover new bioactive compounds from endophytic fungal sources, we have investigated the extract of the Paraphaeosphaeria sporulosa F03 strain. The study led to the isolation of four new 3-methyl-isoquinoline alkaloids (1:  - 4: ) and four known polyketides (5:  - 8: ). The structures of compounds 1:  - 4: were elucidated by 1D and 2D NMR experiments and HRMS analysis. The absolute configuration of 4: was determined by comparison of its experimental electronic circular dichroism spectrum with calculated data. Compounds 1:  - 4: exhibited antifungal activity with minimal inhibitory concentration values ranging from 6.25 - 50 µg/mL against six Candida species but they did not present any cytotoxic activity against the human tumor cell lines A549 (lung), MCF-7 (breast), and HepG2 (hepatocellular). In addition, compound 4: exhibited antiplasmodial activity in the low micromolar range (IC50 = 4 µM).

Syngonanthus nitens (Bong.) Ruhland Derivatives Loaded into a Lipid Nanoemulsion for Enhanced Antifungal Activity Against Candida parapsilosis.

BACKGROUND: Vaginal infections caused by non-albicans species have become common in women of all age groups. The resistance of species such as Candida parapsilosis to the various antifungal agents is a risk factor attributed to these types of infections, which instigates the search for new sources of active compounds in vulvovaginal candidiasis (VCC) therapy. OBJECTIVE: This study evaluated the antifungal activity of Syngonanthus nitens Bong. (Ruhland) derivatives and employed a lipid nanoemulsion as a delivery system.' METHODS: In this study, a lipid nanoemulsion was employed as a delivery system composed of Cholesterol (10%), soybean phosphatidylcholine: Brij 58 (1: 2) and PBS (pH 7.4) with the addition of 0.5% of a chitosan dispersion (80%), and evaluated the antifungal activity of S. nitens Bong. (Ruhland) derivatives against planktonic cells and biofilms of Candida parapsilosis. By a biomonitoring fractionation, the crude extract (EXT) and one fraction (F2) were selected and incorporated into a lipid nanoemulsion (NL) composed of cholesterol (10%), a 1:2 mixture of soybean phosphatidylcholine:polyoxyethylene -20- cetyl ether (10%), and phosphate buffer solution (pH 7.4) with a 0.5% chitosan dispersion (80%). The NL presented a diameter size between 50-200 nm, pseudoplastic behavior, and positive charge. The EXT and five fractions were active against planktonic cells. RESULTS AND DISCUSSION: The incorporation of EXT and F2 into the NL increased antifungal activity and enhanced the anti-biofilm potential. This study classified the use of an NL as an important tool for the administration of S. nitens derivatives in cases of infections caused by this C. parapsisilosis. CONCLUSION: This work concluded that S. nitens derivatives were important sources of active molecules against C. parapsilosis and the use of a lipid nanoemulsion was an important tool to promote more effective F2 release and to improve the antifungal activity aiming the control of C. parapsilosis infections.

Chitosan-laponite nanocomposite scaffolds for wound dressing application.

The pivotal issue of skin regeneration research is the development of effective biomaterials that exhibit biological activities as fungicide and bactericide, combining simple and low cost manufacturing technologies. In this context, nanocomposite scaffolds based on chitosan (Ch)/Laponite (Lap) were produced by using different concentrations of Lap via freeze-drying process for potential application in skin regeneration. The influence of Lap concentration on the scaffold properties was evaluated. The prepared scaffolds were characterized by x-ray diffraction (XRD), scanning electron microscopy (SEM), porosity, swelling capacity, and mechanical analyses. The results revealed that the scaffolds exhibited a porous architecture, besides the increase in the clay content, leads to an increase in the porosity, an improvement of mechanical strength, and a decrease of swelling capacity. In vitro tests were also carried out to evaluate the biocompatibility of the materials, such as bioadhesion, antibacterial activity, viability, and cell adhesion. Viability and cell adhesion demonstrated that all scaffolds were not cytotoxic and the fibroblast cells readily attached on the surface of the scaffolds. Thereby, the results suggested that the nanocomposite scaffolds are biomaterials potentially useful as wound dressings.

New Benzaldehyde and Benzopyran Compounds from the Endophytic Fungus Paraphaeosphaeria sp. F03 and Their Antimicrobial and Cytotoxic Activities.

Three new benzaldehyde derivatives, sporulosaldeins A - C (1: -3: ), and 3 new benzopyran derivatives, sporulosaldeins D - F (4: -6: ), were discovered from an endophytic fungus, Paraphaeosphaeria sp. F03, which was isolated from Paepalanthus planifolius leaves. Compounds 1: -6: were elucidated by 1- and 2-dimensional nuclear magnetic resonance experiments and high-resolution mass spectrometry analysis. The absolute configuration of compound 5: was determined through the comparison of experimental and calculated electronic circular dichroism data. Compounds 1: -6: were found to exhibit antifungal activity with minimum inhibitory concentration (MIC) values of 7.8 - 250 µg/mL and racemic mixture of compound 6: exhibited weak cytotoxicity against MCF-7 and LM3 with IC50 values of 34.4 and 39.2 µM, respectively.

Antibacterial Activity of the Non-Cytotoxic Peptide (p-BthTX-I)2 and Its Serum Degradation Product against Multidrug-Resistant Bacteria.

Antimicrobial peptides can be used systemically, however, their susceptibility to proteases is a major obstacle in peptide-based therapeutic development. In the present study, the serum stability of p-BthTX-I (KKYRYHLKPFCKK) and (p-BthTX-I)2, a p-BthTX-I disulfide-linked dimer, were analyzed by mass spectrometry and analytical high-performance liquid chromatography (HPLC). Antimicrobial activities were assessed by determining their minimum inhibitory concentrations (MIC) using cation-adjusted Mueller-Hinton broth. Furthermore, biofilm eradication and time-kill kinetics were performed. Our results showed that p-BthTX-I and (p-BthTX-I)2 were completely degraded after 25 min. Mass spectrometry showed that the primary degradation product was a peptide that had lost four lysine residues on its C-terminus region (des-Lys12/Lys13-(p-BthTX-I)2), which was stable after 24 h of incubation. The antibacterial activities of the peptides p-BthTX-I, (p-BthTX-I)2, and des-Lys12/Lys13-(p-BthTX-I)2 were evaluated against a variety of bacteria, including multidrug-resistant strains. Des-Lys12/Lys13-(p-BthTX-I)2 and (p-BthTX-I)2 degraded Staphylococcus epidermidis biofilms. Additionally, both the peptides exhibited bactericidal activities against planktonic S. epidermidis in time-kill assays. The emergence of bacterial resistance to a variety of antibiotics used in clinics is the ultimate challenge for microbial infection control. Therefore, our results demonstrated that both peptides analyzed and the product of proteolysis obtained from (p-BthTX-I)2 are promising prototypes as novel drugs to treat multidrug-resistant bacterial infections.

C-terminal Lysine-Linked Magainin 2 with Increased Activity Against Multidrug-Resistant Bacteria.

Due to the growing problem of antibiotic-resistant microorganisms, the development of novel antimicrobial agents is a very important challenge. Dimerization of cationic antimicrobial peptides (cAMPs) is a potential strategy for enhancing antimicrobial activity. Here, we studied the effects of magainin 2 (MG2) dimerization on its structure and biological activity. Lysine and glutamic acid were used to synthesize the C- and N-terminal dimers of MG2, respectively, in order to evaluate the impact of linker position used to obtain the dimers. Both MG2 and its dimeric versions showed a random coil structure in aqueous solution. However, in the presence of a structure-inducing solvent or a membrane mimetic, all peptides acquired helical structure. N-terminal dimerization did not affect the biological activity of the peptide. On the other hand, the C-terminal dimer, (MG2)2K, showed antimicrobial activity 8-16 times higher than that of MG2, and the time required to kill Escherichia coli was lower. The enhanced antimicrobial activity was related to membrane permeabilization. (MG2)2K was also more active against multidrug-resistant bacteria of clinical origin. Overall, the results presented here demonstrate that C-terminal lysine-linked dimerization improve the activity of MG2, and (MG2)2K can be considered as a potential antimicrobial agent.

A Nanostructured Lipid System as a Strategy to Improve the in Vitro Antibacterial Activity of Copper(II) Complexes.

The aim of this study was to construct a nanostructured lipid system as a strategy to improve the in vitro antibacterial activity of copper(II) complexes. New compounds with the general formulae [CuX2(INH)2]·nH2O (X = Cl(-) and n = 1 (1); X = NCS(-) and n = 5 (2); X = NCO(-) and n = 4 (3); INH = isoniazid, a drug widely used to treat tuberculosis) derived from the reaction between the copper(II) chloride and isoniazid in the presence or absence of pseudohalide ions (NCS(-) or NCO(-)) were synthesized and characterized by infrared spectrometry, electronic absorption spectroscopy, electron paramagnetic resonance (EPR) spectroscopy, elemental analysis, melting points and complexometry with 2,2',2'',2'''-(Ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA). The characterization techniques allowed us to confirm the formation of the copper(II) complexes. The Cu(II) complexes were loaded into microemulsion (MEs) composed of 10% phase oil (cholesterol), 10% surfactant [soy oleate and Brij(®) 58 (1:2)] and 80% aqueous phase (phosphate buffer pH = 7.4) prepared by sonication. The Cu(II) complex-loaded MEs displayed sizes ranging from 158.0 ± 1.060 to 212.6 ± 1.539 nm, whereas the polydispersity index (PDI) ranged from 0.218 ± 0.007 to 0.284 ± 0.034. The antibacterial activity of the free compounds and those that were loaded into the MEs against Staphylococcus aureus ATCC(®) 25923 and Escherichia coli ATCC(®) 25922, as evaluated by a microdilution technique, and the cytotoxicity index (IC50) against the Vero cell line (ATCC(®) CCL-81(TM)) were used to calculate the selectivity index (SI). Among the free compounds, only compound 2 (MIC 500 µg/mL) showed activity for S. aureus. After loading the compounds into the MEs, the antibacterial activity of compounds 1, 2 and 3 was significantly increased against E. coli (MIC's 125, 125 and 500 µg/mL, respectively) and S. aureus (MICs 250, 500 and 125 µg/mL, respectively). The loaded compounds were less toxic against the Vero cell line, especially compound 1 (IC50 from 109.5 to 319.3 µg/mL). The compound 2- and 3-loaded MEs displayed the best SI for E. coli and S. aureus, respectively. These results indicated that the Cu(II) complex-loaded MEs were considerably more selective than the free compounds, in some cases, up to 40 times higher.

Synthesis and characterization of an antibacterial and non-toxic dimeric peptide derived from the C-terminal region of Bothropstoxin-I.

Infectious diseases are among the leading global causes of death, increasing the search for novel antibacterial agents. Among these, biologically active peptides are an excellent research tool. Using solid-phase peptide synthesis (SPPS), this work aimed to synthesize the peptide derived from the C-terminal region of Bothropstoxin-I (BthTX-I) (p-BthTX-I, sequence: KKYRYHLKPFCKK), and its disulfide-linked dimeric form, obtained via air oxidation (p-BthTX-I)2. Two other peptides were synthesized to evaluate the dimerization effect on antimicrobial activity. In both sequences, the cysteine (Cys) residue was replaced by the serine (Ser) residue, differing, however, in their C-terminus position. The antimicrobial activity of the peptides against gram-negative (Escherichia (E.) coli) and gram-positive (Staphylococcus (S.) aureus) bacteria and yeast (Candida (C.) albicans) was evaluated. Interestingly, only peptides containing the Cys residue showed antimicrobial activity, suggesting the importance of Cys residue and its dimerization for the observed activity. Apparently, p-BthTX-I and (p-BthTX-I)2 did not promote lysis or form pores and were not able to interact with membranes. Furthermore, they neither showed antifungal activity against C. albicans nor toxicity against erythrocytes, epithelial cells, or macrophages, indicating a potential specificity against prokaryotic cells.

The effect of a minor constituent of essential oil from Citrus aurantium: the role of ß-myrcene in preventing peptic ulcer disease.

The monoterpene ß-myrcene has been widely used in cosmetics, food and beverages, and it is normally found in essential oil from citrus fruit. The aim of this study was to investigate the anti-ulcer effects of ß-myrcene on experimental models of ulcers that are induced by ethanol, NSAIDs (non-steroidal anti-inflammatory drugs), stress, Helicobacter pylori, ischaemia-reperfusion injury (I/R) and cysteamine in order to compare with the essential oil of Citrus aurantium and its major compound limonene. The results indicate that the oral administration of ß-myrcene at a dose of 7.50mg/kg has important anti-ulcer activity with significantly decreased gastric and duodenal lesions as well as increased gastric mucus production. The results showed treatment with ß-myrcene caused a significant increase in mucosal malondialdehyde level (MDA), an important index of oxidative tissue damage. The ß-myrcene was also endowed with marked enhancement of antioxidant enzyme activity from GR system as evidenced by the decreased activity of superoxide dismutase (SOD) and increased levels of glutathione peroxidase (GPx), glutathione reductase (GR), and total glutathione in gastric tissue. Our results also shown that treatment with ß-myrcene is not involved with thioredoxin reductase (TrxR) activity. Our results reveal, for the first time, the importance of ß-myrcene as an inhibitor of gastric and duodenal ulcers and demonstrate that an increase in the levels of gastric mucosa defence factors is involved in the anti-ulcer activity of ß-myrcene.

Phenolic compounds in leaves of Alchornea triplinervia: anatomical localization, mutagenicity, and antibacterial activity.

Phenolic compounds are produced by secretory idioblasts and hypodermis, and by specialized cells of the epidermis and chlorenchyma of leaves of Alchornea triplinervia. Phytochemical investigation of these leaves led to the isolation of the known substances quercetin, quercetin-7-O-beta-D-glucopyranoside, quercetin-3-O-beta-D-glucopyranoside, quercetin-3-O-beta-D-galactopyranoside, quercetin-3-O-alpha-L-arabinopyranoside, amentoflavone, brevifolin carboxylic acid, gallic acid, and methyl gallate from the methanolic extract, and stigmasterol, campesterol, sitosterol, lupeol, friedelan-3-ol, and friedelan-3-one from the chloroform extract. In studies of antibacterial activity and mutagenicity, the methanolic extract showed promising activity against Staphylococcus aureus (MIC = 62.5 microg/mL) and was slightly mutagenic in vitro and in vivo at the highest concentrations tested (1335 mg/kg b.w.).