Metabolomic approaches to explore chemodiversity in seeds of guaraná (Paullinia cupana) using UPLC-QTOF-MSE and NMR analysis.

The growing interest in health and well-being has spurred the evolution of functional foods, which provide enhanced health benefits beyond basic nutrition. Guaraná seeds (Paullinia cupana) have been widely studied and used as a functional food due to their richness in caffeine, phenolic compounds, amino acids, and other nutrients. This has established guaraná as a significant food supplement, with Brazil being the largest producer of the world. This study aims to propose a set of analytical methods to chemically evaluate fifty-six different guaraná clones, from the Guaraná Germplasm Active Bank, to accommodate the diverse requirements of the food industry. Metabolomic approaches were employed, in which a non-target metabolomic analysis via UPLC-QTOF-MSE led to the annotation of nineteen specialized metabolites. Furthermore, targeted metabolomics was also used, leading to the identification and quantification of metabolites by NMR. The extensive data generated were subjected to multivariate analysis, elucidating the similarities and differences between the evaluated guaraná seeds, particularly concerning the varying concentration levels of the metabolites. The metabolomics approach based on the combination of UPLC-QTOF-MSE, NMR and chemometric tools provided sensitivity, precision and accuracy to establish the chemical profiles of guaraná seeds. In conclusion, evaluating and determining the metabolic specificities of different guarana clones allow for their application in the development of products with different levels of specific metabolites, such as caffeine. This caters to various purposes within the food industry. Moreover, the recognized pharmacological properties of the annotated specialized metabolites affirm the use of guarana clones as an excellent nutritional source.

GC/MS and 2D NMR-based approach to evaluate the chemical profile of hydroalcoholic extract from Agaricus blazei Murill and its anti-inflammatory effect on human neutrophils.

ETHNOPHARMACOLOGICAL RELEVANCE: Agaricus blazei Murill (AbM) is one of the main mushrooms used for medicinal purposes. The use of AbM in the preparation of teas is widespread mainly in Asian countries, while in Brazil it is used as a functional food to combat inflammatory diseases and cancer. AIM OF THE STUDY: The main focus of this study was the characterization of the chemical profile of the hydroalcoholic extract of Agaricus blazei Murill (AbE), as well as the evaluation of its cytotoxic and anti-inflammatory potential using human neutrophils. MATERIALS AND METHODS: The extract was prepared by dynamic maceration using a mixture of ethanol and water (70/30, v v-1) as solvent. The chemical profile characterization was carried out by 2D NMR and GC-MS techniques. The cytotoxicity of AbE was evaluated through studies of hemolytic potential, cell viability and membrane integrity. The anti-inflammatory activity was analyzed by a PMA-induced neutrophil degranulation assay. RESULTS: Chemical analysis of AbE revealed the presence of 28 metabolites in its composition, with mannitol as the major compound. AbE at 1-200 µg mL-1 and mannitol at 4-160 µg mL-1, showed low hemolytic and cytotoxic potential against human red blood cells and neutrophils. Furthermore, both were able to significantly reduce the release of myeloperoxidase. CONCLUSIONS: These results indicate that AbE is a promising natural product to be incorporated into pharmaceutical dosage forms intended for the adjuvant treatment of inflammatory diseases.

Metabolism Characterization and Chemical and Plasma Stability of Casearin B and Caseargrewiin F.

Oral preparations of Casearia sylvestris (guacatonga) are used as antacid, analgesic, anti-inflammatory, and antiulcerogenic medicines. The clerodane diterpenes casearin B and caseargrewiin F are major active compounds in vitro and in vivo. The oral bioavailability and metabolism of casearin B and caseargrewiin F were not previously investigated. We aimed to assess the stability of casearin B and caseargrewiin F in physiological conditions and their metabolism in human liver microsomes. The compounds were identified by UHPLC-QTOF-MS/MS and quantified by validated LC-MS methods. The stability of casearin B and caseargrewiin F in physiological conditions was assessed in vitro. Both diterpenes showed a fast degradation (p < 0.05) in simulated gastric fluid. Their metabolism was not mediated by cytochrome P-450 enzymes, but the depletion was inhibited by the esterase inhibitor NaF. Both diterpenes and their dialdehydes showed a octanol/water partition coefficient in the range of 3.6 to 4.0, suggesting high permeability. Metabolism kinetic data were fitted to the Michaelis-Menten profile with KM values of 61.4 and 66.4 µM and Vmax values of 327 and 648 nmol/min/mg of protein for casearin B and caseargrewiin F, respectively. Metabolism parameters in human liver microsomes were extrapolated to predict human hepatic clearance, and suggest that caseargrewiin F and casearin B have a high hepatic extraction ratio. In conclusion, our data suggest that caseargrewiin F and casearin B present low oral bioavailability due to extensive gastric degradation and high hepatic extraction.

Gastroprotective effect of hydroalcoholic extract from Agaricus blazei Murill against ethanol-induced gastric ulcer in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: The use of mushrooms in medicine is quite old and the first report about the use of genus Agaricus in treatment of ulcers occurred in Byzantine period. This mushroom is widely consumed as food, tea, food supplements, as well as nutraceutical and cosmeceutical applications, being cultivated and appreciated in several countries such as Brazil, Korea, Japan and China. AIM OF THE STUDY: This study aimed to characterize the chemical profile and the potential gastroprotective effect of hydroalcoholic extract from Agaricus blazei Murill (HEAb). MATERIALS AND METHODS: The extract was chemically characterized by elemental analysis, UPLC-QTOF-MSE, Nuclear Magnetic Resonance (NMR) and high-performance liquid chromatography (HPLC) techniques to elucidate the metabolites present in the extract. The quantification of phenolic compounds and the in vitro antioxidant activities were performed and the gastroprotective effect of this extract was evaluated against ethanol-induced gastric ulcer model. HEAb was administered by gavage at 5, 25 and 50 mg kg-1 and N-acetylcysteine at 300 mg kg-1 (positive control). Furthermore, the pathways of nitric oxide (NO), Cyclic Guanylate Monophosphate (cGMP), prostaglandins (PGs) and the involvement of ATP-sensitive K+ Channels were modulated. RESULTS: Mannitol, malic acid, pyroglutamic acid, L-agaritine and L-valine were putatively identified by UPLC-QTOF-MSE in HEAb. In addition, it was possible to identify mannitol by the intense signals in the NMR spectra, being still quantified as the main compound in the extract by HPLC. The contents of total phenols and flavonoids corroborated with the good antioxidant activity of HEAb. This study observed that HEAb at 25 and 50 mg kg-1 had gastroprotection effect demonstrated by the reduction of histopathological parameters and the reduction of mastocytosis in the stomach of mice. CONCLUSIONS: In this study was possible to conclude that HEAb has gastroprotective effect related to the involvement of NO and PG pathways in the ethanol-induced gastric ulcer model in mice.

Microfiltered red-purple pitaya colorant: UPLC-ESI-QTOF-MSE-based metabolic profile and its potential application as a natural food ingredient.

Complete characterization of microfiltered red-purple pitaya colorant (MRPPC) and its potential applications in foods is described. Using sensorial analysis, products that use carmine or beetroot dye as a food colorant in their formulations were compared. The effect of storage under refrigeration on the microbiological, physicochemical, and chemical changes of MRPPC were evaluated. The results showed that UPLC-ESI-QTOF-MSE was effective for the simultaneous determination of twenty metabolites, putatively identified as carbohydrates, flavonoids, and betalains. The MRPPC was shown to have microbiological and physicochemical stability through twelve weeks of storage, and chemometric analyses efficiently distinguished the metabolic profile in each storage period. Sensory analysis revealed that the MRPPC was useful as a food colorant in yogurt, where it improved color quality without affecting aroma and other characteristics. These results indicate that MRPPC is promising food ingredient as a natural red-purple colorant.

Metabolic profile of pitaya (Hylocereus polyrhizus (F.A.C. Weber) Britton & Rose) by UPLC-QTOF-MSE and assessment of its toxicity and anxiolytic-like effect in adult zebrafish.

Pitaya is a Cactacea with potential for economic exploitation, due to its high commercial value and its functional components - such betalains, oligosaccharides and phenolic compounds. Although the biological activities of pitaya have been studied using in vivo and in vitro models (anti-inflammatory and antiproliferative activities, as example), its anxiolytic-like effect is still unexplored. Therefore, the aim of this work was to perform a characterization of pulp and peel of pitaya (Hylocereus polyrhizus (F.A.C. Weber) Britton & Rose) using UPLC-QTOF-MSE, and to assess its toxicity and anxiolytic-like effect in adult zebrafish (Danio rerio). The results showed 16 and 15 compounds (in pulp and peel, respectively), including maltotriose, quercetin-3-O-hexoside, and betalains, putatively identified by UPLC-QTOF-MSE. Thus, pitaya pulp and peel showed no toxicity in both models tested (Vero cell lines and zebrafish model, LC50 ˃ 1 mg/mL); and a significant anxiolytic activity, since the treated fish reduced the permanence in the clear zone (Light & Dark Test) compared to that in the control, exhibiting anxiolytic-simile effect of diazepam. However, these effects were reduced by pre-treatment with the flumazenil suggesting that the pulp and peel of pitaya are anxiolytics agents mediated via the GABAergic system. These findings suggested that H. polyrizhus has the potential of developing an alternative plant-derived anxiolytic therapy. In addition, pitaya peel (which is a waste in the food industry) should be regarded as a valuable product, which has the potential as an economic value-added ingredient for anxiety disorders.

Yacon syrup reduces postprandial glycemic response to breakfast: A randomized, crossover, double-blind clinical trial.

Yacon is a root rich in fructooligosaccharides (FOS), which act as prebiotics. Numerous studies have shown promising results in the technological aspects of producing yacon syrup. However, uncertainties exist concerning whether yacon syrup can modulate postprandial glucose and lipid profiles. In order to assess the effect of yacon syrup on postprandial glucose, insulin and triglyceride (TG) responses, a randomized, crossover, double-blind clinical intervention with 40 women (20 normal weight and 20 grade I obese) was performed. Participants underwent two-arms of intervention with at least a one-week wash-out period between visits. On each intervention day, after 12 h of fasting, an aliquot of blood was collected. For intervention A, volunteers consumed breakfast +40 g of placebo, whereas for intervention B, participants consumed breakfast +40 g of yacon syrup (14 g of FOS). Blood samples were drawn at 15, 30, 45, 60, 90, and 120 min. Glucose and insulin concentrations were lowered after yacon syrup intake as compared to placebo at following times: 30 min for glucose and 15, 30 and 45 min for insulin. In conclusion, yacon syrup has a postprandial decreasing effect glucose and insulin concentrations in adult women. This effect was not evident for triglyceride concentration. Clinical trial registry: RBR-33wf46. Available in: http://www.ensaiosclinicos.gov.br/rg/RBR-33wf46/.

UPLC-HRMS and NMR applied in the evaluation of solid-phase extraction methods as a rational strategy of dereplication of Phyllanthus spp. aiming at the discovery of cytotoxic metabolites.

The classical approach to drug discovery from natural products (NP's) requires strenuous and complex purification steps for the isolation and structural elucidation. Modern strategies as dereplication aim to accelerate the identification of known compounds present in a crude or partially purified extract. In this work, we investigated the influence of the solid-phase extraction (Oasis, Plexa, and Agilent C18 cartridges with and without organic modifiers) chemical profile obtained by UPLC-QTOF-MS and NMR and cytotoxicities of aqueous extracts from Phyllanthus niruri and P. amarus. Our results showed differences between the SPE cartridges and the mass recovered. P. niruri showed higher mass recovery than P. amarus indicating a higher amount of secondary metabolites. The UPLC-QTOF-MS analysis revealed that P. niruri crude extract presents higher contents of phenolic compounds than P. amarus. According to NMR analysis, P. niruri contained more tyrosine, corilagin, and glycosidic residues while P. amarus, presented higher content of ellagic acid. The different stationary phases, as well as mobile phases for exploratory SPE, enabled the exploitation of the different chemical functionalities within the Phyllanthus species. The SPE (MeOH:H2O 70:30 with C18 cartridges) samples showed greater in vitro cytotoxicity than the crude extracts, with IC50 ranging from 8.01 to 94.92 µg mL-1 against the tumor lines tested. The solid phase extraction allowed the concentration of molecules with desirable physicochemical characteristics, which might increase the hit of therapeutically useful substances.

Metabolomic profile of Schinopsis brasiliensis via UPLC-QTOF-MS for identification of biomarkers and evaluation of its cytotoxic potential.

Schinopsis brasiliensis is a plant typically found in the caatinga biome (northeastern Brazil). Its leaves and bark have been used for the treatment of health dysfunctions such as cough, influenza, diarrhea, throat inflammation, and sexual impotence. However, there is a lack of knowledge regarding the chemical composition and pharmacological activities of this plant. High-performance liquid chromatography coupled to high-resolution mass spectrometry (UPLC-QTOF-MSE) allowed the partial identification of 33 compounds, including isomers from leaf, branch, and bark samples, with 16 compounds reported for the first time (corilagin, chlorogenic acid, and quercetin derivatives) in S. brasiliensis. Principal component analysis efficiently distinguished the respective parts of the plant. Orthogonal partial least squares discriminatory analysis, together with the variable importance in projection and S-Plot graphs were used to identify 23 biomarker compounds associated with cytotoxic activity against a colorectal cancer cell line.

Chemical profiling of guarana seeds (Paullinia cupana) from different geographical origins using UPLC-QTOF-MS combined with chemometrics.

Paullinia cupana, commonly known as guarana, is an Amazonian fruit whose seeds are used to produce the powdered guarana, which is rich in caffeine and consumed for its stimulating activity. The metabolic profile of guarana from the two largest producing regions was investigated using UPLC-MS combined with multivariate statistical analysis. The principal component analysis (PCA) showed significant differences between samples produced in the states of Bahia and Amazonas. The metabolites responsible for the differentiation were identified by orthogonal partial least squares discriminant analysis (OPLS-DA). Fourteen phenolic compounds were characterized in guarana powder samples, and catechin, epicatechin, B-type procyanidin dimer, A-type procyanidin trimer and A-type procyanidin dimer were the main compounds responsible for the geographical variation of the samples.

Essential Oil of Cymbopogon nardus (L.) Rendle: A Strategy to Combat Fungal Infections Caused by Candida Species.

BACKGROUND: The incidence of fungal infections, especially those caused by Candida yeasts, has increased over the last two decades. However, the indicated therapy for fungal control has limitations. Hence, medicinal plants have emerged as an alternative in the search for new antifungal agents as they present compounds, such as essential oils, with important biological effects. Published data demonstrate important pharmacological properties of the essential oil of Cymbopogon nardus (L.) Rendle; these include anti-tumor, anti-nociceptive, and antibacterial activities, and so an investigation of this compound against pathogenic fungi is interesting. OBJECTIVE: The aim of this study was to evaluate the chemical composition and biological potential of essential oil (EO) obtained from the leaves of C. nardus focusing on its antifungal profile against Candida species. METHODS: The EO was obtained by hydrodistillation and analyzed by gas chromatography-mass spectrometry (GC-MS). Testing of the antifungal potential against standard and clinical strains was performed by determining the minimal inhibitory concentration (MIC), time-kill, inhibition of Candida albicans hyphae growth, and inhibition of mature biofilms. Additionally, the cytotoxicity was investigated by the IC50 against HepG-2 (hepatic) and MRC-5 (fibroblast) cell lines. RESULTS: According to the chemical analysis, the main compounds of the EO were the oxygen-containing monoterpenes: citronellal, geranial, geraniol, citronellol, and neral. The results showed important antifungal potential for all strains tested with MIC values ranging from 250 to 1000 µg/mL, except for two clinical isolates of C. tropicalis (MIC > 1000 µg/mL). The time-kill assay showed that the EO inhibited the growth of the yeast and inhibited hyphal formation of C. albicans strains at concentrations ranging from 15.8 to 1000 µg/mL. Inhibition of mature biofilms of strains of C. albicans, C. krusei and C. parapsilosis occurred at a concentration of 10× MIC. The values of the IC50 for the EO were 96.6 µg/mL (HepG-2) and 33.1 µg/mL (MRC-5). CONCLUSION: As a major virulence mechanism is attributed to these types of infections, the EO is a promising compound to inhibit Candida species, especially considering its action against biofilm.

Antimicrobial activity of Miconia species (Melastomataceae).

This work evaluated the antimicrobial activity of the methanol and chloroform extracts of the leaves of Miconia cabucu, Miconia rubiginosa, and Miconia stenostachya using the disc-diffusion method. The results obtained showed that the methanol extracts of the leaves of M. rubiginosa and M. stenostachya and the chloroform extract of the leaves of M. cabucu presented antimicrobial activity against the tested microorganisms.