Antioxidant Potential and Chemical Evaluation of Extracts from Endophytic Fungus Lasiodiplodia sp.

Antioxidants are compounds that can eliminate free radicals and are known to prevent cell damage and health disorders, in turn, improving the quality of life of human beings. This study aims to evaluate the presence of antioxidants in ethyl acetate (EtOAc) extracts from Lasiodiplodia endophyte, which was previously isolated and cultivated by our research group, given its ability to produce a variety of metabolites with different chemical and biological properties. The antioxidant activities were determined using ABTS+●, DPPH and FRAP. Total phenolic content (TPC) and total flavonoid content (TFC) were evaluated, as well as the NMR metabolomics of extracts. All extracts showed promising antioxidant activity, and optimal results were achieved between the sixth and eleventh days of fungus cultivation. Furthermore, one of the extracts showed no in vivo toxicity against Galleria mellonella for all tested concentrations. The 1H-NMR spectra showed that there are two distinct groups of molecules present in EtOAc extracts obtained from Lasiodiplodia cultivation, and the data corroborate the results concerning antioxidant activity for TPC and TFC. This suggests that it is possible to monitor the chemical profile of fungal extracts using NMR, and to correlate compounds within days of fungus cultivation, along with their antioxidant activities.

Characterisation of Sclerocarya birrea (marula) seed oil and investigation of the geographical origin by applying similarity calculations, differential NMR and hierarchical cluster analysis.

INTRODUCTION: The marula fruit is an important indigenous African fruit since various commercial products are produced from the pulp and the seed oil. The increased demand requires methods for authentication, quality control and determination of geographical origin. OBJECTIVE: The study aimed to establish a fast and reliable method for characterisation and authentication of marula seed oil. Furthermore, to identify marker compounds that can distinguish marula seed oils from other commercial oils and indicate regional differences. MATERIALS AND METHODS: Metabolic profiling of 44 commercial marula seed oils was performed using proton nuclear magnetic resonance (1 H NMR). For rapid classification similarity calculations were compared with principal component analysis. Differential NMR was used to determine marker compounds. RESULTS: Marula seed oil was found to be similar to macadamia and olive oils and was distinguished from these oils by the detection of minor components. Marula seed oil is differentiated from the other two oils by the absence of α-linolenic acid, relatively high levels of monoglycerides and diglycerides, and an approximately 1:1 ratio of 1,2- and 1,3-diglycerides. When comparing marula seed oils from various regions using hierarchical cluster analysis, clustering of the marula seed oils from Namibia and Zimbabwe was observed and was related to the quantities of linoleic acid and monoglycerides and diglycerides. Some samples displayed deviations in their composition which might indicate adulteration or contamination during the production process. CONCLUSION: The study demonstrates the potential of NMR as a tool in the quality control of marula seed oil. This technique requires very little sample preparation, circumvents derivatisation of the oil components with fast run-times. In addition, samples with chemical profiles that differ from the general signature profile can easily be identified.

Antifungal activity of Allamanda polyantha seed extract and its iridoids promote morphological alterations in Cryptococcus spp.

Cryptococcosis, caused by Cryptococcus spp., is an invasive fungal infection of the central nervous system, associated with high mortality, affecting mainly immunocompromised patients. Due to the development of resistance to the current therapy, there is an urgent need for less toxic and more effective antifungal agents. In this study, we describe the antifungal activity against Cryptococcus spp. of an aqueous seed extract from Allamanda polyantha (ASEAP) and two iridoids, plumieride and plumieridine, isolated from this extract with an antifungal activity. The capsule formation and the morphological alterations were evaluated using fluorescent microscopy. The cytotoxic activity was also investigated. The minimal inhibitory concentration (MIC) values of ASEAP for Cryptococcus gattii were 70 and 36 µg/ml (for the R265 and R272 strains, respectively) and 563 µg/ml for Cryptococcus neoformans H99. ASEAP inhibited C. neoformans H99 capsule formation, an important virulence factor, and decreased the cell body size for both the C. gattii strains. H99 cells also presented morphological alterations, with defects in bud detachment and nuclear fragmentation. Plumieride and plumieridine presented higher MIC values than ASEAP, indicating that other compounds might contribute to antifungal activity and/or that combination of the compounds results in a higher antifungal activity.

Similarity and differential NMR spectroscopy in metabolomics: application to the analysis of vegetable oils with 1H and 13C NMR.

INTRODUCTION: In NMR based metabolomics there is a need for tools to easily compare spectra and to extract the maximum of information from the data. OBJECTIVES: The calculation of similarity and performing differential NMR spectroscopy provides important additional information for classification and validation in metabolomics experiments. METHODS: From 13 different vegetable oils samples were analysed by 1H and 13C NMR. The similarity between spectra was calculated and differential NMR spectroscopy was used to discover marker compounds. RESULTS: The similarity between the individual spectra was calculated for the spectra of all samples. The similarity was used to verify and improve the alignment. For vegetable oils which showed a high similarity, e.g. chia seed oil and linseed oil, differential NMR spectroscopy was used to discover marker compounds. CONCLUSIONS: The calculation of similarity is an important tool to reveal variability between samples and spectra and can be used to verify data sets and improve alignment or binning procedures. With differential spectroscopy marker compounds are easily discovered. The methods can be seen as an important addition to the routine procedures of metabolomics experiments.

Anti-Leishmania effect of icetexanes from Salvia procurrens.

BACKGROUND AND PURPOSE: Leishmaniasis is a globally prevalent vector-borne disease caused by parasites of the genus Leishmania. The available chemotherapeutic drugs present problems related to efficacy, emergence of parasite resistance, toxicity and high cost, justifying the search for new drugs. Several classes of compounds have demonstrated activity against Leishmania, including icetexane-type diterpenes, previously isolated from Salvia and other Lamiaceae genera. Thus, in this study, compounds of Salvia procurrens were investigated for their leishmanicidal and immunomodulatory activities. METHODS: The exudate of S. procurrens was obtained by rapidly dipping the aerial parts in dichloromethane. The compounds were isolated by column and centrifugal planar chromatography over silica gel. The effects on L. amazonensis growth, survival, membrane integrity, reactive oxygen species (ROS) generation, mitochondrial membrane potential and cytotoxicity of the compounds towards human erythrocytes, peripheral blood mononuclear cells and macrophages were evaluated. The effects on intracellular amastigote forms, nitric oxide (NO) and TNF-α production were also investigated. RESULTS: The exudate from the leaves afforded the novel icetexane 7-hydroxyfruticulin A (1) as well as the known demethylisofruticulin A (2), fruticulin A (3) and demethylfruticulin A (4). The compounds (1-4) were tested against promastigotes of L. amazonensis and showed an effective inhibition of the parasite survival (IC50 = 4.08-16.26 µM). In addition, they also induced mitochondrial ROS production, plasma membrane permeability and mitochondrial dysfunction in treated parasites, and presented low cytotoxicity against macrophages. Furthermore, all diterpenes tested reduced the number of parasites inside macrophages, by mechanisms involving TNF-α, NO and ROS. CONCLUSION: The results suggest the potential of 7-hydroxyfruticulin A (1) as well as the known demethylisofruticulin A (2),fruticulin A (3) and demethylfruticulin A (4) as candidates for use in further studies on the design of anti-leishmanial drugs.

Differential NMR and chromatography for the detection and analysis of adulteration of vetiver essential oils.

Adulteration of essential oils is a common and serious problem. Adequate and fast methods are required to establish the authenticity and purity. GC-MS, 1H and 13C NMR were compared in combination with similarity calculations as well as differential spectroscopy and chromatography for the authentication and determination of purity of vetiver essential oils. In the investigation of eight commercial oils 1H and 13C NMR adequately detected all six adulterants encountered in four of the commercial samples, while GC-MS was not able to detect adulteration with vegetable oils. A great advantage of the combined use of similarity calculation and NMR is that the authenticity can be verified without the need of concomitant measurement of a standard sample. The calculation can be carried out with a registered reference spectrum, in the case of 1H NMR acquired using the same magnetic field, but in the case of 13C NMR the spectrum can be obtained with another magnetic field. 1H NMR has the advantage of high speed, and the results can be obtained within minutes. 13C NMR was found to be superior in its ability to provide unequivocal identification of eventual adulterants, with differential spectroscopy revealing all signals of the adulterants.

Towards a holistic view of tablet quality, an extensive study on paracetamol tablets with nuclear magnetic resonance using similarity calculations, differential NMR and hierarchical cluster analysis.

The 1H NMR spectra obtained from 56 different paracetamol tablets were thoroughly investigated to analyse and quantify besides paracetamol, the excipients and eventual minor components present in the formulations. In the NMR spectra the amide-iminol tautomerism of paracetamol was observed, with the iminol form present at a quantity of only 0.80% of the amide form. Furthermore, seventeen different components of the tablets were identified, ranging from major excipients like starch and polyvinylpyrrolidone (PVP) to minor components like different parabens, sorbate and triacetin. The similarity from all spectra in relation to each of the other spectra was calculated and based on the similarity table a hierarchical cluster analysis (HCA) was performed. Each of the components of the DMSO extracts of the tablets was quantified, providing a component table, which also was used for HCA. The quantitative analysis of paracetamol was determined to have a precision of 0.2% using the residual solvent signals as internal standard. The HCA of the similarity data and the component table were compared with the HCAs obtained by analysis of the crude NMR data. This clearly shows the limitations of multivariate data analysis and the strength of similarity calculations combined with differential NMR, especially in relation to the analysis of trace components. In fact, the spectrum is not a series of unrelated variables, but a superposition of a limited number of component spectra, and the quantities of these components were determined.

The anti-immobility effect of hyperoside on the forced swimming test in rats is mediated by the D2-like receptors activation.

The crude extracts of HYPERICUM species native to South Brazil showed analgesic and antidepressant-like effects in rodents. The chemical characterization of these species revealed that they are rich in flavonoids and phloroglucinol derivatives. In the present study a detailed investigation was performed on the activities of hyperoside (HYP), a common flavonoid in the genus HYPERICUM. Hyperoside was obtained from the aerial parts of H. CAPRIFOLIATUM by chromatographic procedures. Mice treated with single doses (10, 20 and 40 mg/kg i.p.) did not present signs of toxicity or weight loss. At 20 and 40 mg/kg i.p. the mice exploratory behavior in the open field test was reduced. At 20 mg/kg i. p. the pentobarbital sleeping time increased, but not the sleeping latency. No activity was found on the hot-plate (10 and 20 mg/kg i.p.) or in the acetic acid-induced writhing test (20 and 40 mg/kg p.o.). Nevertheless, an antidepressant-like effect in the forced swimming test in mice and rats was observed (HYP 10 and 20 mg/kg i.p. in mice; HYP 1.8 mg/kg/day p.o. in rats). The antidepressant-like effect in rats was prevented by the administration of sulpiride (50 mg/kg i.p.) a D2 antagonist. In conclusion, hyperoside was found to present a depressor effect on the central nervous system as well as an antidepressant-like effect in rodents which is, at least in part, mediated by the dopaminergic system.

Anthraquinones from the bark of Senna macranthera.

2-acetyl physcion (2-acetyl-1,8-dihydroxy-6-methoxy-3-methyl-9,10-anthraquinone, 2), a rare anthraquinone, was isolated from Senna macranthera var. nervosa (Vogel) H.S. Irwin & Barneby (Fabaceae). The chemical structure was elucidated and all (1)H and (13)C NMR chemical shifts were assigned by NMR one- ((1)HNMR, {(1)H}-(13)CNMR, and APT-(13)CNMR) and two (COSY, NOESY, HMQC and HMBC) dimensional of this natural compound. Furthermore, the minor anthraquinones chrysophanol (3), chrysophanol-8-methyl ether (4) and physcion (5) were characterized by GC-MS analysis. The occurrence of the anthraquinones 3-5 confirms that S. macranthera is a typical representative of the genus Senna.

Rapid and accurate verification of drug identity, purity and quality by 1H-NMR using similarity calculations and differential NMR.

The illegal trade in counterfeit and fake drugs is a worldwide multi-billion dollar industry, not only generating enormous economic losses, but health problems for the general population, through direct toxicity, treatment failure and the increased generation of antibiotic resistance. Techniques for high-throughput testing of suspect medicines are needed to face the challenges of the problem. In this study we show that with nuclear magnetic resonance spectroscopy (NMR) drug compliance can be verified in a few minutes, providing data on drug identity, purity and quality without the necessity to develop a specific methodology and using a direct extraction with deuterated solvent. The evaluation of the data is facilitated by similarity calculations and differential NMR spectroscopy. The viability and limitations of this method were assessed, with the application on five different drugs, namely sertraline hydrochloride, alprazolam, vitamin D3, enalapril maleate and paracetamol, in which the individual dosage quantity of the active ingredient ranged from 750 mg down to 0.25 mg. The appropriate sample weight, solvent and internal standard were determined for each drug and quantification was carried out by choosing the most adequate NMR signals for each compound and the internal standard. With the method the accuracy of the quantification is somewhat sacrificed for increased speed in the analysis, but the measurements offer excellent precision and offer the possibility of external calibration. Spectral similarity calculations and differential NMR spectroscopy were used to compare different generic brands and detect eventual undeclared constituents and contaminants. In one brand of alprazolam tablets the undeclared constituent tristearin was found, while in paracetamol tablets the contaminant para-aminophenol was found at a level above the allowed by the legislation. The applicability and limitations of the method are discussed.

Application of NMR in plant metabolomics: techniques, problems and prospects.

The present state-of-the-art of NMR in plant metabolomics is reviewed. Attention is paid to the different practical aspects of the application of NMR. The sample preparation, the measurement of the spectrum, quantitative aspects and data analysis are discussed. Each stage has its specific problems, which are pointed out and recommendations are made.

Can soil phosphorus availability in tropical forest systems be increased by nitrogen-fixing leguminous trees?

Understanding the role of N-fixing leguminous trees for phosphorus (P) cycling in highly weathered tropical soils is relevant for the conservation of natural forests as well as the sustainable management of agroforests and forest plantations with low P input in the Brazilian Atlantic Forest region. We hypothesized that N-fixing leguminous trees can increase the availability of soil P by exploiting different P sources without causing a depletion of soil organic P due to efficient biogeochemical cycling, but empirical evidence remains scarce. For this purpose, 31P nuclear magnetic resonance spectroscopy (31P NMR) was used for quantifying soil P forms and the Hedley sequential extraction to determine soil P fractions. The studied sites were forestry systems with leguminous trees: mixed forest plantations with different proportions of fast-growing N-fixing leguminous trees; pure plantations, and agroforestry systems with leguminous trees. The results show that all N-fixing leguminous trees and N mineral fertilization positively affected the concentrations of available soil P in relation to the control treatments. There were increases of all P fractions through cycling in all forest sites. 31P NMR spectra clearly identified and quantified that a large amount of phosphomonoesters followed by phosphodiesters in the form of DNA, as well as high reserves of Pi species (ortho-P and pyrophosphate) in the first eleven years of growth at pure plantations, mixed plantations or agroforests. The relations between both ortho-P and DNA with the resin-Pi, NaHCO3-Pi and NaOH-Pi fractions suggest that both analysis methods provide complementary information about the soil P transformations. Thus, the paper highlights the importance of the use of different N-fixing leguminous tree species under different environmental conditions, production systems and management practices for recovering heavily degraded areas, which may be a suitable strategy through efficient management of P in highly weathered tropical soils in the Brazilian Atlantic Forest biome.

Metabolomic fingerprinting of Cissampelos sympodialis Eichler leaf extract and correlation with its spasmolytic activity.

ETHNOPHARMACOLOGICAL RELEVANCE: Cissampelos sympodialis Eichler (Menispermaceae) is popularly used in northeastern Brazil for the treatment of respiratory diseases such as bronchitis and asthma. Despite many pre-clinical pharmacological studies, the compounds mediating the anti-asthma activity of polar extracts of Cissampelos sympodialis leaves have not been definitively identified. AIM OF THE STUDY: Aim of the study: The aim of the study was to investigate the correlation between the bioactivity of polar extracts prepared from the leaves of C. sympodialis and the chemical composition using a 1H-NMR-based metabolomics approach. MATERIAL AND METHODS: The metabolic profile of the leaf polar extract during different phenological stages of the plant was investigated using 1H NMR spectroscopy while simultaneously screening for spasmolytic activity using guinea-pig tracheal preparations. The content of the alkaloids previously implicated in the bioactivity of Cissampelos sympodialis was determined by HPLC. RESULTS: PCA analysis of the 1H NMR data discriminated the extracts from different plant phenological stages. The contents of the major alkaloids decrease (from 2 ± 0.32 µg/mL for warifteine and 1 ± 0.14 µg/mL for methylwarifteine) to undetectable levels from 90 (CsL90 extract) and 120 (CsL120) days onwards for warifteine and methylwarifteine, respectively. All six extracts relaxed the trachea pre-contracted with carbachol, but the CsF210 extract was more potent (EC50 = 74.6 ± 7.9 µg/mL) compared to both CsL90 extracts and CsL180 in the presence of functional epithelium. PLS regression analysis of 1H-NMR spectral data demonstrated that the spasmolytic activity was better correlated with signals for flavonol derivatives. CONCLUSIONS: Our data challenge the idea that warifteine and methylwarifteine mediate the spasmolytic activity of the polar extract of C. sympodialis leaves.

Synthesis and stereochemistry-activity relationship of small bacteriocin, an autoinducer of the symbiotic nitrogen-fixing bacterium Rhizobium leguminosarum.

The four stereoisomers of small bacteriocin, an autoinducer of the symbiotic nitrogen-fixing bacterium Rhizobium leguminosarum, were synthesized via a versatile methodology for 3'-hydroxyacyl homoserine lactones based on the Nagao asymmetric aldol reaction. The synthetic isomers were much less effective at inhibiting the growth of R. leguminosarum RBL5523 than the natural isomer, showing the importance of stereochemistry for activity.

Comprehensive analysis of polar and apolar constituents of butter and margarine by nuclear magnetic resonance, reflecting quality and production processes.

The separation of butter or margarine into polar (soluble in water) and apolar fractions (soluble in chloroform) and subsequent analysis of these fractions by (1)H NMR permits a comprehensive analysis of its constituents. In the polar fraction the preservatives benzoic and sorbic acid, the organic acids citric, lactic, butyric, acetic, and formic acid, and, furthermore, the carbohydrate lactose were quantified. In the apolar fraction the conjugated linoleic acid (CLA) rumenic acid, diglycerides, and linoleic acid were quantified. Rumenic acid is a characteristic component of ruminant fats and was found in all butter samples. The levels varied between 0.50 and 1.08%. Ten brands of Brazilian butter were investigated as was one brand from Norway. Also, two brands of margarine were investigated for comparison. A large variation in especially polar constituents was found between the butter samples, revealing the presence of preservatives in five brands of butter from Brazil, remarkable because these additives are legally not allowed. Furthermore, the levels of organic acids and lactose permitted conclusions about the production process and quality; for example, the presence of higher levels of free butyric acid indicate lipolysis, leading to a lower quality, and low levels of lactose indicate that after churning the residual milk fluids have been removed by an additional washing step in the production process.

Two-Phase Extraction for Comprehensive Analysis of the Plant Metabolome by NMR.

Metabolomics is the area of research, which strives to obtain complete metabolic fingerprints, to detect differences between them, and to provide hypothesis to explain those differences [1]. But obtaining complete metabolic fingerprints is not an easy task. Metabolite extraction is a key step during this process, and much research has been devoted to finding the best solvent mixture to extract as much metabolites as possible.Here a procedure is described for analysis of both polar and apolar metabolites using a two-phase extraction system. D2O and CDCl3 are the solvents of choice, and their major advantage is that, for the identification of the compounds, standard databases can be used because D2O and CDCl3 are the solvents most commonly used for pure compound NMR spectra. The procedure enables the absolute quantification of components via the addition of suitable internal standards. The extracts are also suitable for further analysis with other systems like LC-MS or GC-MS.

Cyathenosin A, a spiropyranosyl derivative of protocatechuic acid from Cyathea phalerata.

Cyathenosin A, a spiropyranosyl derivative of protocatechuic acid was isolated from the stem pith of Cyathea phalerata Mart. Its structure was determined by MS, 1D and 2D NMR spectroscopic analyses and confirmed by single crystal X-ray analysis. Cyathenosin A is the first example of a naturally occurring compound containing a spirocyclic orthoester pyranosidic structure.

Herpes virus inhibitory substances from Hypericum connatum Lam., a plant used in southern Brazil to treat oral lesions.

Hypericum connatum (Guttiferae) is used in southern Brazil in the treatment of lesions in the mouth, often related to acute herpetic gingivo-stomatitis. The chemical investigation of the plant revealed the presence of phloroglucinol derivatives and flavonoids. From the n-hexane extract of the aerial parts a phloroglucinol derivative, hyperbrasilol B, was isolated, while the methanolic extract afforded four flavonoids: amentoflavone, hyperoside, guaijaverine and luteoforol. The crude methanolic extract and fractions (n-hexane, dichloromethane and methanol) as well as the isolated compounds were tested for antiviral activity against herpes simplex viruses (HSV). Among the tested samples, luteoforol was the most active inhibiting the cytopathic effect (CPE) and reducing the viral titer of HSV-1 DNA viral strains KOS and VR733 (ATCC).

Combining molecular and bioprocess techniques to produce poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with controlled monomer composition by Burkholderia sacchari.

Biopolymers as polyhydroxyalkanoates (PHA) composed by different co-monomers 3-hydroxybutyrate and 3-hydroxyhexanoate [P(3HB-co-3HHx)] has attracted interest since its properties are similar to low density polyethylene. Burkholderia sacchari produces this copolymer with a very low 3HHx molar fraction, about 2 mol%. B. sacchari mutant (unable to produce polymer) was engineered to host PHA biosynthesis genes (phaPCJ) from Aeromonas sp. In addition, a two-step bioprocess to increase biopolymer production was developed. The combination of these techniques resulted in the production of P(3HB-co-3HHx) with 3HHx content up to 20 mol%. The PHA content was about 78% of dry biomass, resulting in PHA volumetric productivities around 0.45gl-1h-1. The P(3HB-co-3HHx) containing 20 mol% of 3HHx presented an elongation at brake of 945%, higher than reported before for this PHA composition. Here we have described an approach to increase 3HHx content into the copolymer, allowing the precise control of the 3HHx molar fractions.

Revision of the structures of citrifolinin A, citrifolinoside, yopaaoside A, yopaaoside B, and morindacin, iridoids from Morinda citrifolia L. and Morinda coreia Ham.

[Structure: see text] Citrifolin A was revised to dehydromethoxygaertneroside (2). Citrifolinoside and yopaaoside A were found to be identical, and their structures were revised to the new structure dehydroepoxymethoxygaertneroside (5). Yopaaoside B was revised to citrifolinoside A (9), and morindacin was revised to borreriagenin (11).