Lychnophora pinaster in endangered campos rupestres: phenolic compounds and population ecogeography.

Lychnophora pinaster, known as arnica-mineira, is endemic to campos rupestres, at risk of extinction. The present study aimed to characterize the ecogeography and phenolic constituents of 11 L. pinaster populations collected in the mesoregions North, Jequitinhonha, Metropolitan of Belo Horizonte and Campos das Vertentes in the state of Minas Gerais, Brazil. Phenolic constituents were identified and quantified by Ultra-high performance liquid chromatography- mass spectrometry. Lychnophora pinaster occurs in sites at high altitude (700 and 1498 m), annual rainfall of up to 1455 m, soils with low fertility and predominantly loamy texture. Therefore, it can be considered tolerant to acidic soils, with low availability of nutrient. The most abundant substances in all populations were vitexin (18 - 1345 ng/g) and chlorogenic acid (60 - 767 ng/g). The 11 populations formed four groups in relation to the phenolic constituents, with group 1 consisting of the populations of the North Mesoregion (GM, OD) and Jetiquinhonha (DIMa), group 2, the Metropolitan of Belo Horizonte (SRM, NLSC, SM, RPS, CTRA), group 3, the North Mesoregion (ODMa and DI), and group 4, the Campos das Vertentes (CC). Among the populations, only those from the Metropolitan of Belo Horizonte showed correlation of soil properties with phenolic constituents.

Evaluation of the Antiproliferative Potential of Eugenia pyriformis Leaves in Cervical Cancer Cells.

Eugenia pyriformis, typically known as uvaia, ubaia, uvaieira, uvalha or uvalha-do-campo, is a plant representative of the Myrtaceae family. E. pyriformis decreased HeLa cells proliferation, can induce cell death and reduce cell migration that may be related to metastasis and induction of cell death by apoptosis in vitro assays. Its leaves are used in folk medicine for hypertension control, decreased cholesterol and uric acid, slimming, astringent, and digestive. In this work, the evaluation of the in vitro anticancer potential Cervical Cancer (HeLa cells) and phytochemical analysis in E. pyriformes was performed. It was possible to quantify phenolic compounds and total flavonoids and identify Chlorogenic acid, Quercetrin, and Myricitrin in this species. The crude extract and ethyl acetate fraction inhibited cell viability by 50 % in the dose of 44.42 µg/mL and 40.39 µg/mL, respectively. The induced effector caspase 3/7 activation, which results in apoptosis and the ethyl acetate fraction, decreases cell migration of cancer cell lines; it is responsible for the cleavage of several cellular proteins that will result in the classic phenotype of the apoptotic cell.

Melipona mondury produces a geopropolis with antioxidant, antibacterial and antiproliferative activities.

Geopropolis is a special type of propolis produced by stingless bees. Several pharmacological properties have been described for different types of geopropolis, but there have been no previous studies of the geopropolis from Melipona mondury. In this study, we investigated the antioxidant, antibacterial, and antiproliferative activities of M. mondury geopropolis, and determined its chemical profile. The antioxidant activity was determined using in vitro ABTS·+, ·DPPH, and ß-carotene/linoleic acid co-oxidation methods. The antibacterial activity was determined using a microdilution method with Pseudomonas aeruginosa, Staphylococcus aureus, and methicillin-resistant S. aureus. The antiproliferative effect was determined in tumor cell lines using the Alamar Blue assay. The chemical profile was obtained using UHPLC-MS and UHPLC-MS/MS. The butanolic fraction had the highest concentration of phenolic compounds and more potent antioxidant properties in all assays. This fraction also had bacteriostatic and bactericidal effects against all bacterial strains at low concentrations, especially S. aureus. The hexane fraction had the highest antiproliferative potential, with IC50 values ranging from 24.2 to 46.6 µg/mL in HL-60 (human promyelocytic leukemia cell) and K562 (human chronic myelocytic leukemia cell), respectively. Preliminary chemical analysis indicates the presence of terpenes and gallic acid in the geopropolis. Our results indicate the therapeutic potential of geopropolis from M. mondury against inflammatory, oxidative, infectious, and neoplastic diseases.

Detoxification of Atrazine by Endophytic Streptomyces sp. Isolated from Sugarcane and Detection of Nontoxic Metabolite.

Atrazine is still one of the most used agricultural pesticides worldwide and it has been recognized as a major contaminant of surface and ground water. The aims of this research were to isolate an endophytic microorganism from leaves of sugarcane, evaluate its ability to degrade atrazine, and investigate the formation of metabolites. By sequencing of the 16S rRNA gene, the endophytic isolate atz2 was identified as Streptomyces sp. The reduction in atrazine concentration by Streptomyces sp. atz2 was 98 % and UHPLC-MS/MS analyses showed the appearance of an unknown metabolite observed as m/z 311. Ecotoxicity tests with an aquatic organism, Daphnia similis, confirmed that this metabolite was nontoxic. This mechanism of detoxification of atrazine is different from the ones of other free-living microorganisms that inhabit the soil or rhizosphere. The results show new aspects of atrazine detoxification, highlighting a new role of endophytic bacteria in plants.

Multilayer microparticles for programmed sequential release of phenolic compounds from Eugenia stipitata: Stability and bioavailability.

A co-delivery system based on multilayer microparticles was developed and characterized for the sequential release of phenolic compounds (PCs) using different encapsulation processes (spray drying: SD and drying-chilling spray: SDC) and wall materials to improve the stability and bioavailability of PCs. Samples were characterized in terms of process yield (PY%), phenolic retention efficiency (PRE%), chemical structure and crystallinity (NMR, FTIR, DXR), thermal stability (DSC and FT-IR), anti-radical capacity (ORAC and ABTS) and in vitro digestion. PRE% of samples by SD were higher (p < 0.05) than SDC due to the formation of PCs from CRF (cará-roxo flour). NMR, FTIR, DXR confirmed the presence of key components and interactions for the formation of the advanced co-delivery system. The SDC particles showed crystalline regions by XRD and were stable at ∼47 °C. All samples showed good release of PC in the intestinal phase, and antiradical capacity that reached 23.66 µmol TE g-1.

Analysis of soluble lignin in sugarcane by ultrahigh performance liquid chromatography-tandem mass spectrometry with a do-it-yourself oligomer database.

Lignin is a polymer found in the cell wall of plants and is one of the main obstacles to the implementation of second-generation ethanol production because it confers the recalcitrance of the lignocellulosic material. The recalcitrance of biomass is affected by the amount of lignin, by its monomer composition, and the way the monomers are arranged in the plant cell wall. Analysis of lignin structure demands mass spectrometry analysis, and identification of oligomers is usually based on libraries produced by laborious protocols. A robust method to build a do-it-yourself lignin oligomer library was tested. This library can be built using commercially available enzymes, standards, and reagents and is relatively easy to accomplish. An ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the separation and characterization of monomers and oligomers was developed and was equally applicable to the synthetic lignin and to soluble lignin extracted from a sample of sugar cane.

The chemical composition and antioxidant activity of mandaçaia (melipona quadrifasciata) geopropolis varies more due to region than month of collection.

Stingless bees are responsible for pollinating up to 90% of Brazilian flora, so their study is of commercial and ecological importance. Stingless bees mix plant resins with wax and soil, forming geopropolis. Studies of the variability in composition and activity due to species, place and season of collection are lacking. Yield, total phenolic and flavonoid content, and antioxidant activity (via DPPH) of the extracts of fifteen M. quadrifasciata (Mandaçaia) geopropolis samples collected over a year in the State of São Paulo, Brazil, as well as two samples from different regions of the state of Minas Gerais in Brazil were compared. Composition was determined by ultra-high-resolution liquid chromatography and mass spectrometry, identifying 16 compounds. The month and region of collection affected the available plant resins and influenced their yield, composition and antioxidant capacity. Depending on the place of collection, M. quadrifasciata geopropolis is a promising natural source of antioxidant phenolic compounds.

Metabolic alterations in different developmental stages of Pilocarpus microphyllus.

Pilocarpine is an imidazole alkaloid that has been used for more than a century in glaucoma treatment. It is present in several species of the Pilocarpus genus (jaborandi), with its highest concentrations in P. microphyllus. In addition to pilocarpine, pilosine--an imidazole alkaloid without pharmacological use--is produced in high concentrations in mature plants. A metabolomic study was carried out on juvenile and mature plants to obtain information about pilocarpine metabolism at different developmental stages. Methanol-water and alkaloid extracts were analyzed by ¹H NMR and ESI-MS. Metabolic profiles from both techniques showed clear differences between various developmental stages. Intense signals in the aromatic region of the ¹H NMR spectrum and ions from pilosine and related alkaloids by ESI/MS were found only in extracts from mature plant. Two new imidazole alkaloids were identified by MS(n). Our results suggest that pilosine is produced exclusively in mature developmental stage, and juvenile plant material seems to be appropriate for further studies on pilocarpine biosynthesis.

Evidence of altitudinal gradient modifying genomic and chemical diversity in populations of Lychnophora pinaster Mart.

Lychnophora pinaster Mart. (Asteraceae) is endemic to the Brazilian Cerrado. It is distributed along the altitudinal gradient of the mountainous ranges of the state of Minas Gerais. This study aimed to evaluate the influence of altitude on the genetic diversity of L. pinaster populations and the effects of altitude and climatic factors on essential oil chemical composition. Essential oils from L. pinaster populations from the north (North 01, North 02, and North 03, 700-859 m) and the Metropolitan region of Belo Horizonte (MhBH 01 and MrBH 02, 1366-1498 m) were analyzed. SNP markers from L. pinaster in these regions and Campos das Vertentes (CV 01, CV 02, and CV 03, 1055-1292 m) were also analyzed. The main compounds in essential oils were 14-hydroxy-α-humulene (North 01 and North 03), cedr-8(15)-en-9-α-ol (North 02), 14-acetoxy-α-humulene (MrBH 01), and 4-oxo-15-nor-eudesman-11-ene (MrBH 02). Hierarchical cluster and heatmap analyses showed that the North and MrBH populations included five different groups, indicating the chemical composition of essential oils is distinct in each population. Furthermore, principal component analysis showed that higher altitudes (1366 m and 1498 m) in the MrBH influence the chemical composition of essential oils, and climatic factors determine the chemical composition in North region. The genetic diversity showed that most alleles are in Hardy-Weinberg equilibrium and imply high genetic variation and genetic polymorphisms between populations. Furthermore, the results of Mantel tests (R = 0.3861517; p = 0.04709529; R = 0.9423121; p = 0.02739726) also showed that higher altitude (>1360 m) shapes the genetic diversity at the MrBH. The genetic structure showed that higher altitudes (>1360 m) contribute to the structure of the MrBH populations, but not to North and CV populations. Therefore, the altitudinal ranges of Minas Gerais mountainous ranges determine the higher genetic and chemical diversity of L. pinaster populations.

Medicinal properties of Angelica archangelica root extract: Cytotoxicity in breast cancer cells and its protective effects against in vivo tumor development.

OBJECTIVE: Although Angelica archangelica is a medicinal and aromatic plant with a long history of use for both medicinal and food purposes, there are no studies regarding the antineoplastic activity of its root. This study aimed to evaluate the cytotoxicity and antitumor effects of the crude extract of A. archangelica root (CEAA) on breast cancer. METHODS: The cytotoxicity of CEAA against breast adenocarcinoma cells (4T1 and MCF-7) was evaluated by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Morphological and biochemical changes were detected by Hoechst 33342/propidium iodide (PI) and annexin V/PI staining. Cytosolic calcium mobilization was evaluated in cells staining with FURA-4NW. Immunoblotting was used to determine the effect of CEAA on anti- and pro-apoptotic proteins (Bcl-2 and Bax, respectively). The 4T1 cell-challenged mice were used for in vivo assay. RESULTS: Using ultra-high-performance liquid chromatography-mass spectrometry analysis, angelicin, a constituent of the roots and leaves of A. archangelica, was found to be the major constituent of the CEAA evaluated in this study (73 µg/mL). The CEAA was cytotoxic for both breast cancer cell lines studied but not for human fibroblasts. Treatment of 4T1 cells with the CEAA increased Bax protein levels accompanied by decreased Bcl-2 expression, in the presence of cleaved caspase-3 and cytosolic calcium mobilization, suggesting mitochondrial involvement in breast cancer cell death induced by the CEAA in this cell line. No changes on the Bcl-2/Bax ratio were observed in CEAA-treated MCF7 cells. Gavage administration of the CEAA (500 mg/kg) to 4T1 cell-challenged mice significantly decreased tumor growth when compared with untreated animals. CONCLUSION: Altogether, our data show the antitumor potential of the CEAA against breast cancer cells in vitro and in vivo. Further research is necessary to better elucidate the pharmacological application of the CEAA in breast cancer therapy.

HPLC-ESI-MS/MS of imidazole alkaloids in Pilocarpus microphyllus.

Pilocarpine, an important imidazole alkaloid, is extracted from the leaves of Pilocarpus microphyllus (Rutaceae), known in Brazil as jaborandi and used mainly for the treatment of glaucoma. Jaborandi leaves also contain other imidazole alkaloids, whose pharmacological and physiological properties are unknown, and whose biosynthetic pathways are under investigation. In the present study, a HPLC method coupled with ESI-MS(n) was developed for their qualitative and quantitative analysis. This method permits the chromatographic separation of the imidazole alkaloids found in extracts of jaborandi, as well as the MS/MS analysis of the individual compounds. Thus two samples: leaves of P. microphyllus and a paste that is left over after the industrial extraction of pilocarpine; were compared. The paste was found to contain significant amounts of pilocarpine and other imidazole alkaloids, but had a slightly different alkaloid profile than the leaf extract. The method is suitable for the routine analysis of samples containing these alkaloids, as well as for the separation and identification of known and novel alkaloids from this family, and may be applied to further studies of the biosynthetic pathway of pilocarpine in P. microphyllus.

Drought tolerance of sugarcane is improved by previous exposure to water deficit.

Under field conditions, plants are exposed to cycles of dehydration and rehydration during their lifespan. In this study, we hypothesized that sugarcane plants previously exposed to cycles of water deficits will perform better than plants that have never faced water deficits when both are subjected to low water availability. Sugarcane plants were grown in a nutrient solution and exposed to one (1WD), two (2WD) or three (3WD) water deficit cycles. As the reference, plants were grown in a nutrient solution without adding polyethylene glycol. Under water deficits, leaf gas exchange was significantly reduced in 1WD and 2WD plants. However, 3WD plants showed similar CO2 assimilation and lower stomatal conductance compared to the reference plants, with increases in intrinsic water-use efficiency. Abscisic acid concentrations were lower in 3WD plants than in 1WD plants. Our data revealed root H2O2 concentration as an important chemical signal, with the highest root H2O2 concentrations found in 3WD plants. These plants presented higher root dry matter and root:shoot ratios compared to the reference plants, as well as higher biomass production when water was available. Our data suggest that sugarcane plants were able to store information from previous stressful events, with plant performance improving under water deficits. In addition, our findings provide a new perspective for increasing drought tolerance in sugarcane plants under nursery conditions.

Effect of the consumption of green tea extract during pregnancy and lactation on metabolism of mothers and 28d-old offspring.

The objective was to investigate the effects of the maternal consumption of the green tea extract during pregnancy and lactation on mothers and offspring metabolism. The female Wistar rats, on the first day of pregnancy until the end of lactation, was divided into groups: MC- received water and ME- received green tea extract (400 mg/kg body weight/day), both ingested control diet. After lactation, at day 28th post-partum, the mothers and pups from each mother were euthanized and composed the groups: FC- pup from mother received water and FE- pup from mother received green tea extract. The ME group increased IL-10/TNF-α ratio and IL-1ß content in the mesenteric and IL-1ß content in retroperitoneal adipose tissues, and decreased catalase activity. The FE group decreased the retroperitoneal adipose tissue relative weight and SOD activity, but increased adiponectin, LPS, IL-10 and IL-6 content and IL-10/TNF-α ratio in retroperitoneal, IL-10 and TNF-α content in gonadal, and IL-6 content in mesenteric adipose tissues. In summary, the maternal consumption of green tea extract associated with control diet ingestion during pregnancy and lactation altered the inflammatory status of mothers and 28d-old offspring. These data elucidate the effects of green tea during pregnancy and lactation on maternal and offspring metabolism.

Electrochemical and spectroscopic characterization of the interaction between DNA and Cu(II)-naringin complex.

In this work the interaction of DNA and the flavonoid-transition metal complex (Cu(II)-naringin complex) is characterized. The interaction was evaluated by using electrochemical ssDNA- and dsDNA-based biosensors and the results were supported by UV, CD and (1)H NMR data. In the electrochemical method, changes in the oxidation peak of the guanine and adenine bases obtained by square wave voltammetry (SWV) showed evidence of the interaction. The variations of the spectroscopic characteristics of DNA and Cu(II)-naringin complex in aqueous medium demonstrated that the predominant interaction mode may be by intercalation. Cu(II)-naringin complex interacts to dsDNA probably via N(7) of guanine site.

Phenolic antioxidants identified by ESI-MS from Yerba maté (Ilex paraguariensis) and green tea (Camelia sinensis) extracts.

Aqueous extracts of green yerba maté (Ilex paraguariensis) and green tea (Camellia sinensis) are good sources of phenolic antioxidants, as already described in the literature. The subject of this study were organic extracts from yerba maté, both green and roasted, and from green tea. Their phenolic profiles were characterized by direct infusion electrospray insertion mass spectrometry (ESI-MS) and their free radical scavenging activity was determined by the DPPH assay. Organic extracts containing phenolic antioxidants might be used as natural antioxidants by the food industry, replacing the synthetic phenolic additives used nowadays. Ethanolic and aqueous extracts from green yerba maté, roasted yerba maté and green tea showed excellent DPPH scavenging activity (>89%). The ether extracts from green and roasted yerba maté displayed a weak scavenging activity, different from the behavior observed for the green tea ether extract. The main phenolic compounds identified in green yerba maté water and ethanolic extracts were: caffeic acid, quinic acid, caffeoyl glucose, caffeoylquinic acid, feruloylquinic acid, dicaffeoylquinic acid and rutin. After the roasting process two new compounds were formed: caffeoylshikimic acid and dicaffeoylshikimic acid. The ethanolic extracts from yerba maté, both roasted and green, with lower content of phenolic compounds (3.80 and 2.83 mg/mL) presented high antioxidant activity and even at very low phenolic concentrations, ether extract from GT (0.07 mg/mL) inhibited DPPH over 90%.

Synthesis and characterization of a metal complex containing naringin and Cu, and its antioxidant, antimicrobial, antiinflammatory and tumor cell cytotoxicity.

The antioxidant activity of flavonoids is believed to increase when they are coordinated with transition metal ions. However, the literature on this subject is contradictory and the outcome seems to largely depend on the experimental conditions. In order to understand the contribution of the metal coordination and the type of interaction between a flavonoid and the metal ion, in this study a new metal complex of Cu (II) with naringin was synthesized and characterized by FT-IR, UV-VIS, mass spectrometry (ESI-MS/MS), elemental analysis and 1H-NMR. The results of these analyses indicate that the complex has a Cu (II) ion coordinated via positions 4 and 5 of the flavonoid. The antioxidant, anti-inflammatory and antimicrobial activities of this complex were studied and compared with the activity of free naringin. The Naringin-Cu (II) complex 1 showed higher antioxidant, anti-inflammatory and tumor cell cytotoxicity activities than free naringin without reducing cell viability.

The screening of organic matter in mineral and tap water by UHPLC-HRMS.

It is highly desirable to screen for a large variety of organic compounds in water. Ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) can analyse semi-polar to polar organic compounds in tap and mineral water. The use of UHPLC-HRMS is well consolidated for lipidomic, metabolomic and proteomic studies; based on the detection of a very large number of compounds of a variety of organic functions. Water analysis is usually performed by gas chromatography -mass spectrometry (GC-MS) techniques that are efficient for volatile organic compounds. Therefore the use of UHPLC-HRMS as a screening method based on an untargeted omic approach for the analysis of organic compounds with polar functional groups is welcome. This UHPLC-HRMS method was developed and tested with tap water and mineral water. We analysed six different brands of mineral water from France, Spain, Norway and Brazil and tap water from Jandaia do Sul, PR, Brazil to test the approach and demonstrate how UHPLC-HRMS may be used as a screening method for water quality. Forty-seven different ions were observed in tap and mineral water and their intensities were submitted to principal component analysis (PCA) evaluation. Fifteen ions were identified comparing the mass spectrometric results to metabolomic and lipidomic libraries.

Impact of Air Frying on Cholesterol and Fatty Acids Oxidation in Sardines: Protective Effects of Aromatic Herbs.

The high temperatures used to fry fish may induce thermo-oxidation of cholesterol, forming cholesterol oxidation products (COPs). COPs have been associated to coronary heart diseases, atherosclerosis, and other chronic diseases. Air fryers are an alternative thermal process technology to fry foods without oil, and are considered a healthier cooking method. This study is the 1st to evaluate the formation of COPs and the degradation of polyunsaturated fatty acids (PUFAs) in air-fried sardine fillets. Furthermore, we evaluated the effect of fresh herbs added as natural antioxidants to sardines subjected to air frying. Parsley (Petroselinum crispum), chives (Allium schoenoprasum L.), and a mixture of both herbs (cheiro-verde) were added in quantities of 0%, 2%, and 4%. Air frying significantly decreased the content of essential PUFAs, and increased the levels of COPs from 61.2 (raw) to 283 µg/g (P < 0.05) in the control samples. However, the use of herbs as natural antioxidants proved to be effective reducing such levels of COPs in most samples. The addition of 4% of cheiro-verde in air-fried sardines presented the best protective effect against lipid oxidation. PRACTICAL APPLICATION: Fish is an important source of essential lipids. However, oxidized cholesterol products, which are formed during thermal processing, are potential hazards to human health. Air fryers present an alternative thermal process for frying food without oil, and this method of cooking is considered to be more convenient and healthier This study shows that the air frying increased the formation of cholesterol oxidation products and decreased the essential polyunsaturated fatty acids in sardine fillets. However, the lipid oxidation is significantly reduced by adding fresh herbs, such as parsley (Petroselinum crispum), chives (Allium schoenoprasum L.), or a mixture of both herbs (cheiro-verde) that are natural antioxidants.

Characterization of must and wine of six varieties of grapes by direct infusion electrospray ionization mass spectrometry.

Samples of must derived from six different varieties of grapes taken during the fermentation process, as well as the respective wine samples directly after the end of the malolactic fermentation, were analyzed by direct infusion negative ion mode electrospray ionization mass spectrometry (ESI-MS). Diagnostic ions for must were different from those of wine samples, although small variations for each of the grape varieties were also detected. The addition of unfermented must or sugar to wine could also be clearly detected. The spectra were acquired in a few minutes per sample, indicating that ESI-MS can be used for high-throughput analysis of samples and should prove useful for quality control during and after the fermentation process.

HPLC method for quantification and characterization of cholesterol and its oxidation products in eggs.

A new method was developed for the simultaneous determination of cholesterol and its oxidation products in eggs, using HPLC with UV and refractive index (RI) detectors, and HPLC interfaced with atmospheric pressure chemical ionization coupled to MS (HPLC-APCI-MS). The best conditions for direct saponification of the sample and extraction of the non-saponifiable material were defined using complete factorial designs with central points. The method showed accuracy and precision with a detection limit between 0.002 and 0.079 microg/g. The oxides cholest-5-ene-3beta,20alpha-diol and cholest-5-ene-3beta,25-diol identified by HPLC-UV-RI were not confirmed by HPLC-APCI-MS.