Production of new ent-hardwickiic acid derivatives by microbial transformation and their antifungal activity.

Ent-hardwickiic acid is the major compound of Copaifera pubiflora Benth oleoresin traditionally used in Brazilian folk medicine as an antimicrobial agent. Microbial transformation of ent-hardwickiic by Cunninghamella elegans ATCC 10028b resulted in two and five antifungal derivatives (four new ones) produced in the Czapek modified and Koch's K1 media, respectively. The derivatives were isolated and their structures were determined by spectral analysis, namely 1D/2D NMR and HR-ESIMS. All compounds were tested for cytotoxic and antifungal activities and they were not cytotoxic to the tested cell lines, but all derivatives showed fungicidal activity against Candida glabrata and Candida krusei, which have emerged as resistant to fluconazole. One of the yet unreported biotransformation products displayed the strongest activity with minimum fungicidal concentration values smaller than the other compounds, including fluconazole.

Insights into the Serum Metabolic Adaptations in Response to Inspiratory Muscle Training: A Metabolomic Approach Based on 1H NMR and UHPLC-HRMS/MS.

Inspiratory muscle training (IMT) is known to promote physiological benefits and improve physical performance in endurance sports activities. However, the metabolic adaptations promoted by different IMT prescribing strategies remain unclear. In this work, a longitudinal, randomized, double-blind, sham-controlled, parallel trial was performed to investigate the effects of 11 weeks (3 days·week-1) of IMT at different exercise intensities on the serum metabolomics profile and its main regulated metabolic pathways. Twenty-eight healthy male recreational cyclists (30.4 ± 6.5 years) were randomized into three groups: sham (6 cm·H2O of inspiratory pressure, n = 7), moderate-intensity (MI group, 60% maximal inspiratory pressure (MIP), n = 11) and high-intensity (HI group, 85-90% MIP, n = 10). Blood serum samples were collected before and after 11 weeks of IMT and analyzed by 1H NMR and UHPLC-HRMS/MS. Data were analyzed using linear mixed models and metabolite set enrichment analysis. The 1H NMR and UHPLC-HRMS/MS techniques resulted in 46 and 200 compounds, respectively. These results showed that ketone body metabolism, fatty acid biosynthesis, and aminoacyl-tRNA biosynthesis were upregulated after IMT, while alpha linolenic acid and linoleic acid metabolism as well as biosynthesis of unsaturated fatty acids were downregulated. The MI group presented higher MIP, Tryptophan, and Valine levels but decreased 2-Hydroxybutyrate levels when compared to the other two studied groups. These results suggest an increase in the oxidative metabolic processes after IMT at different intensities with additional evidence for the upregulation of essential amino acid metabolism in the MI group accompanied by greater improvement in respiratory muscle strength.

Bioactive compounds from Vellozia pyrantha A.A.Conc: A metabolomics and multivariate statistical analysis approach.

The chemical composition of V. pyrantha resin (VpR) and fractions (VpFr1-7 and VpWS) were assessed by LC-MS and NMR. Twenty-eight metabolites were identified, including 16 diterpenoids, seven nor-diterpenoids, one fatty acid, one bis-diterpenoid, one steroid, one flavonoid, and one triterpenoid. The pharmacological potential of VpR, VpFr1-7, and isolated compounds was assessed by determining their antioxidant, antimicrobial, and cytotoxic activities. VpFr4 (IC50 = 205.48 ± 3.37 µg.mL-1) had the highest antioxidant activity, whereas VpFr6 (IC50 = 842.79 ± 10.23 µg.mL-1) had the lowest. The resin was only active against Staphylococcus aureus (MIC 62.5 µg.mL-1) and Salmonella choleraesius (MIC and MFC 500 µg.mL-1), but fractions were enriched with antibacterial compounds. V. pyrantha resin and fractions showed great cytotoxic activity against HCT116 (IC50 = 20.08 µg.mL-1), HepG2 (IC50 = 20.50 µg.mL-1), and B16-F10 (12.17 µg.mL-1) cell lines. Multivariate statistical analysis was used as a powerful tool to pinpoint possible metabolites responsible for the observed activities.

Polyketide- and Terpenoid-Derived Metabolites Produced by a Marine-Derived Fungus, Peroneutypa sp.

Bioassay-guided investigation of the EtOAc-soluble extract of a culture of the marine-derived fungus Peroneutypa sp. M16 led to the isolation of seven new polyketide- and terpenoid-derived metabolites (1, 2, 4-8), along with known polyketides (3, 9-13). Structures of compounds 1, 2, and 4-8 were established by analysis of spectroscopic data. Absolute configurations of compounds 1, 2, 4, 6, 7, and 8 were determined by the comparison of experimental ECD spectra with calculated CD data. Compound 5 exhibited moderate antiplasmodial activity against both chloroquine-sensitive and -resistant strains of Plasmodium falciparum.

The potential of compounds isolated from Xylaria spp. as antifungal agents against anthracnose

ABSTRACT Anthracnose is a crop disease usually caused by fungi in the genus Colletotrichum or Gloeosporium. These are considered one of the main pathogens, causing significant economic losses, such as in peppers and guarana. The current forms of control include the use of resistant cultivars, sanitary pruning and fungicides. However, even with the use of some methods of controlling these cultures, the crops are not free of anthracnose. Additionally, excessive application of fungicides increases the resistance of pathogens to agrochemicals and cause harm to human health and the environment. In order to find natural antifungal agents against guarana anthracnose, endophytic fungi were isolated from Amazon guarana. The compounds piliformic acid and cytochalasin D were isolated by chromatographic techniques from two Xylaria spp., guided by assays with Colletotrichum gloeosporioides. The isolated compounds were identified by spectrometric techniques, as NMR and mass spectrometry. This is the first report that piliformic acid and cytochalasin D have antifungal activity against C. gloeosporioides with MIC 2.92 and 2.46 µmol mL-1 respectively. Captan and difenoconazole were included as positive controls (MIC 16.63 and 0.02 µmol mL-1, respectively). Thus, Xylaria species presented a biotechnological potential and production of different active compounds which might be promising against anthracnose disease.

The potential of compounds isolated from Xylaria spp. as antifungal agents against anthracnose.

Anthracnose is a crop disease usually caused by fungi in the genus Colletotrichum or Gloeosporium. These are considered one of the main pathogens, causing significant economic losses, such as in peppers and guarana. The current forms of control include the use of resistant cultivars, sanitary pruning and fungicides. However, even with the use of some methods of controlling these cultures, the crops are not free of anthracnose. Additionally, excessive application of fungicides increases the resistance of pathogens to agrochemicals and cause harm to human health and the environment. In order to find natural antifungal agents against guarana anthracnose, endophytic fungi were isolated from Amazon guarana. The compounds piliformic acid and cytochalasin D were isolated by chromatographic techniques from two Xylaria spp., guided by assays with Colletotrichum gloeosporioides. The isolated compounds were identified by spectrometric techniques, as NMR and mass spectrometry. This is the first report that piliformic acid and cytochalasin D have antifungal activity against C. gloeosporioides with MIC 2.92 and 2.46µmolmL-1 respectively. Captan and difenoconazole were included as positive controls (MIC 16.63 and 0.02µmolmL-1, respectively). Thus, Xylaria species presented a biotechnological potential and production of different active compounds which might be promising against anthracnose disease.

Mate extract as feed additive for improvement of beef quality.

Mate (Ilex paraguariensis A.St.-Hil.) is generally recognized as safe (GRAS status) and has a high content of alkaloids, saponins, and phenolic acids. Addition of mate extract to broilers feed has been shown to increase the oxidative stability of chicken meat, however, its effect on beef quality from animals supplemented with mate extract has not been investigated so far. Addition of extract of mate to a standard maize/soy feed at a level of 0.5, 1.0 or 1.5% w/w to the diet of feedlot for cattle resulted in increased levels of inosine monophosphate, creatine and carnosine in the fresh meat. The content of total conjugated linoleic acid increased in the meat as mate extract concentration was increased in the feed. The tendency to radical formation in meat slurries as quantified by EPR spin-trapping decreased as increasing mate extract addition to feed, especially after storage of the meat, indicating higher oxidative stability. Mate supplementation in the diet did not affect animal performance and carcass characteristics, but meat from these animals was more tender and consequently more accepted by consumers. Mate extract is shown to be a promising additive to feedlot diets for cattle to improve the oxidative stability, nutritive value and sensory quality of beef.

Unusual 2(1H)-pyrazinones isolated from a culture of a Brazilian marine-derived Streptomyces sp.

Four new secondary metabolites, giovaninones A-D (1-4), were isolated from an ethyl acetate extract of a culture of a marine-derived Streptomyces strain designated SS99BA-2. Chemical analysis was completely conducted in a coupled automated LC-SPE system with the use of a cryogenic NMR probehead and HRMS. The application of this system to identify, purify and elucidate all the structures is described.

Use of diffusion-ordered NMR spectroscopy and HPLC-UV-SPE-NMR to identify undeclared synthetic drugs in medicines illegally sold as phytotherapies.

The informal (and/or illegal) e-commerce of pharmaceutical formulations causes problems that governmental health agencies find hard to control, one of which concerns formulas sold as natural products. The purpose of this work was to explore the advantages and limitations of DOSY and HPLC-UV-SPE-NMR. These techniques were used to identify the components of a formula illegally marketed in Brazil as an herbal medicine possessing anti-inflammatory and analgesic properties. DOSY was able to detect the major components present at higher concentrations. Complete characterization was achieved using HPLC-UV-SPE-NMR, and 1D and 2D NMR analyses enabled the identification of known synthetic drugs. These were ranitidine and a mixture of orphenadrine citrate, piroxicam, and dexamethasone, which are co-formulated in a remedy called Rheumazim that is used to relieve severe pain, but it is prohibited in Brazil because of a lack of sufficient pharmacokinetic and pharmacodynamic information.

Crude to leads: a triple-pronged direct NMR approach in coordination with docking simulation.

The screening of compounds that bind to the target of interest (specific proteins) plays a vital role in drug discovery. Usually, the identification of biologically active compounds is done from a library of structurally known compounds. However, we successfully illustrate here, that NMR techniques including saturation transfer difference (STD), transfer nuclear Overhauser spectroscopy (TrNOESY) and STD-TOCSY (total correlation spectroscopy) in combination with separation methods not only enable the rapid and comprehensive screening of active components, but also their unequivocal structural characterization. Furthermore, a time saving for the recognition of leads is also possible with this application. To probe the binding studies, a hydroethanolic fraction of crude extract (1 mg) from natural product (Rauia resinous) was used for the initial assessment with BSA protein. The docking simulation was performed with BSA in the region of Thr190, Arg198, Arg217, Trp213, Arg256, Ala290 and Tyr451 to further refine the active compound towards the leads. Docking results mimic binding as identified by STD, Tr-NOESY and STD-TOCSY. Isovetexine-2-rhamnosoide (2) was found to be most active through group epitope mapping results as well as the docking simulation with relative free energy of -7.2770. This experiment provided excellent results through the direct NMR screening method. Using Bovine Serum Albumin as a reference, we illustrate that this approach offers an excellent way for the first hand detection of the active constituents/inhibitors from natural remedies used in folk medicinal treatments.

Production of 5-hydroxy-7-methoxy-4-methylphthalide in a culture of Penicillium crustosum.

The chemical reactions carried out by microorganisms have been used as a tool in modern chemistry. This paper reports the production of mycophenolic acid and a new phthalide by the endophytic fungus Penicillium crustosum obtained from coffee seeds. The fungus was cultivated in a liquid medium for a period of seven days and after that the culture medium was divided into four treatments: A, B, C and D, to which different organic substances were added. Treatment A was maintained as the control to evaluate the occurrence of biotransformation. Organic acids were added to the culture media of treatments B (ferulic and quinic acids) and C [cinnamic and 3,4-(methylenedioxy) cinnamic acids], and caffeine was added in the treatment D. All these organic compounds were dissolved in DMSO, and the fermentation was maintained for more 13 days, totalizing 20 days. Mycophenolic acid was isolated from the culture with no added acids (treatment A). Mycophenolic acid and a new phthalide, 5-hydroxy-7-methoxy-4-methylphthalide were isolated from treatments B and C, and mycophenolic acid and caffeine (added to the culture medium) were isolated from treatment D. The structures were determined by NMR techniques and confirmed by MS and MS/MS techniques.

Chemical composition and biological activities of soldiers of the Brazilian termite species, Nasutitermes macrocephalus (Isoptera: Natutitermitinae).

The defensive secretion of the frontal gland from termite soldiers is a mixture of monoterpenes, sesquiterpenes and diterpenes, the latter being the most representative. Analyses of the dichloromethane extract from soldiers of the Brazilian termite, Nasutitermes macrocephalus (Silvestri, 1903) (Isoptera, Nasutitermitinae), described for the first time, allowed to identify the presence of two monoterpenes (alpha-pinene and limonene) and two sesquiterpenes (beta-trans-caryophyllene and gamma-selinene) [corrected] by GC-EIMS, and the isolation of one rippertane and six trinervitane diterpenes by RP-HPLC. The chemical structures of the purified compounds were elucidated by interpretation of their spectroscopic data (1D and 2D NMR, EIMS, HRESIMS, and specific optical rotation) and the complete unequivocal assignment of the 3a-hydroxy-trinervita-1(15),8(19)-dien-2-one (6) was included in this paper, to complement the lack of information in the literature. Antibacterial, antifungal and cytotoxicity against cancer cell lines activities were evaluated. In particular, the compounds 2alpha,3beta-dihydroxytrinervita-l(15),8(19)-diene (2) and 3alpha-hydroxy-15-rippertene (7) exhibited the better activities against the clinically isolated Gram-positive bacterium methicillin-resistant Staphylococcus aureus BMB 9393, both with a MIC value of 31.2 microg mL(-1). This is the first description of a rippertane diterpene (7) as an antibacterial agent.

Phytochemical characterization and antinociceptive effect of Lippia gracilis Schauer.

Lippia gracilis Schauer is an aromatic plant widely found in Northeastern Brazil. The leaf infusions or decoctions and alcoholic macerate are used for some inflammatory diseases and headache. This paper reports the isolation of naringenin by semi-preparative liquid chromatography from the methanolic extract of L. gracilis (ELg) and the evaluation of the analgesic and anti-inflammatory activities of this extract by measuring nociception through acetic acid, formalin, and hot-plate tests in carrageenan-induced inflammation in mice. Following oral administration, ELg (100, 200, and 400 mg/kg) significantly reduced the number of writhes in the writhing test and the time of paw licks in both phases of the formalin test when compared to the control group animals. Mice treated with ELg did not exhibit any behavioral alteration during the hot plate and rota-rod tests, suggesting non-participation of the supraspinal components in the modulation of pain by ELg and no motor abnormality. The oral administration of 400 mg/kg of ELg produced an anti-inflammatory effect on peritonitis induced by carrageenan. These effects can be associated with a decrease of inflammatory mediator synthesis by compounds of ELg, such as naringenin, which has anti-inflammatory action as already described.

Saturated ceramides from the sponge Dysidea robusta.

Chemical investigation of the crude extract of a marine sponge Dysidea robusta led to the isolation of an inseparable mixture of saturated ceramides. These were identified from spectroscopic data as well as by hydrolysis followed by LC-MS analysis of the sphingosine moieties.

Classification of commercial Catuaba samples by NMR, HPLC and chemometrics.

For over a century, Catuaba has been used in Brazilian folk medicine as an aphrodisiac even though the identity of the plant material employed is often uncertain. The species recommended by the Brazilian Pharmacopeia is Anemopaegma arvense (Bignoniaceae), but many other plants, regionally known as Catuaba, are commercialised. Frequently, the quality control of such a complex system is based on chemical markers that do not supply a general idea of the system. With the advent of the metabolomics approach, a global analysis of samples becomes possible. It appears that (1)H-NMR is the most useful method for such application, since it can be used as a wide-spectrum chemical analysis technique. Unfortunately, the generated spectra is complex so a possible approach is to look at the metabolite profile as a whole using multivariate methods, for example, by application of principal component analysis (PCA). In the present paper, we describe for the first time a proton high-resolution magic angle spinning nuclear magnetic resonance ((1)H-HR-MAS NMR) method coupled with PCA for the metabolomic analysis of some commercial Catuaba samples, which provided a reduction in the time required for such analysis. A comparative study of HPLC, HR-MAS and liquid-NMR techniques is also reported.

A pyrimidine-beta-carboline and other alkaloids from Annona foetida with antileishmanial activity.

Bioassay-guided fractionation of the bark extract of Annona foetida afforded a new antileishmanial pyrimidine-beta-carboline alkaloid, N-hydroxyannomontine (1), together with the previously reported annomontine (2), O-methylmoschatoline (3), and liriodenine (4). The structure of compound 1 was established on the basis of extensive 1D and 2D NMR and MS analyses. This is the third reported pyrimidine-beta-carboline-type alkaloid and is particularly important for Annona genus chemotaxonomy. In addition, all compounds exhibit in vitro antileishmanial activity against promastigote forms of Leishmania braziliensis. Compounds 2 and 4 showed better activity than compounds 1 and 3 against L. braziliensis. Compound 2 was not active against L. guyanensis.

Coumarin glycoside from Cissus sicyoides.

From the aerial parts of Cissus sicyoides, a new coumarin glycoside 5,6,7,8-tetrahydroxycoumarin-5beta-xylopyranoside was obtained together with known coumarin sabandin, two flavonoids kaempferol 3-rhamnoside and quercetin 3-rhamnoside and two steroids, sitosterol and 3beta-O-beta-D-glucopyranosylsitosterol. The structure of compounds was elucidated by spectral analyses.