NMR and multivariate methods: Identification of chemical markers in extracts of pedra-ume-caá and their antiglycation, antioxidant, and enzymatic inhibition activities.

INTRODUCTION: In Brazil, the plant group popularly known as "pedra-ume-caá" is used in folk medicine for the treatment of diabetes, and its raw material is commonly sold. OBJECTIVE: The aim of the study was to apply a method for chemical identification of extracts of dry pedra-ume-caá leaves using HPLC-high-resolution mass spectrometry (HRMS) and NMR and develop a multivariate model with NMR data to authenticate commercial samples. In addition, to evaluate the biological activities of the extracts. MATERIALS AND METHODS: Dry extracts of Myrcia multiflora, Myrcia amazonica, Myrcia guianensis, Myrcia sylvatica, Eugenia punicifolia leaves, and 15 commercial samples (sold in Manaus and Belém, Brazil) were prepared by infusion. All the extracts were analysed using HPLC-high-resolution mass spectrometry (HRMS), NMR, principal component analysis (PCA), and hierarchical cluster analysis (HCA). The antidiabetic effect of extracts was evaluated according to enzymatic inhibition. Their content of total phenols, cell viability, and antioxidant and antiglycation activities were also determined. RESULTS: HPLC-HRMS and NMR analysis of these extracts permitted the identification of 17 compounds. 1H NMR data combined with multivariate analyses allowed us to conclude that catechin, myricitrin, quercitrin, and gallic and quinic acids are the main chemical markers of pedra-ume-caá species. These markers were identified in 15 commercial samples of pedra-ume-caá. Additionally, only the extracts of M. multiflora and E. punicifolia inhibited α-glucosidase. All the extracts inhibited the formation of advanced glycation end products (AGEs) and showed free-radical-scavenging activity. These extracts did not present cytotoxicity. CONCLUSION: This study revealed the chemical markers of matrices, and it was possible to differentiate the materials marketed as pedra-ume-caá. Moreover, this study corroborates the potential of these species for treating diabetes.

Hypoglycemic effect and toxicity of the dry extract of Eugenia biflora (L.) DC. leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: The leaves of Eugenia biflora (Myrtaceae) are traditionally used by Amazonian populations for the control of diabetes. However, their chemical composition has not yet been described and pharmacological evidence has not been reported. OBJECTIVE: This study aimed to identify the chemical constituents and evaluate the hypoglycemic and toxic effect of the dry extract of the E. biflora leaves (DEEB). MATERIALS AND METHODS: DEEB, obtained by infusion, was analyzed using LC-HRMS and NMR, whose the catechin flavonoid was quantified using NMR. The antidiabetic effect of DEEB was evaluated according to its inhibition of the enzymes α-amylase and α-glucosidase, as well as the content of total phenols, free radical scavengingand antiglycation activities, and its in vitro cell viability. Oral maltose tolerance and chronic multiple dose tests (28 days) in streptozotocin-induced diabetic mice (STZ) were performed. The hypoglycemic effect and toxicity of this extract were evaluated in the multiple dose assay. Biochemical parameters, hemolysis, and levels of the thiobarbituric acid reactive species in the liver were investigated and histopathological analyses of the kidneys and liver were performed. RESULTS: Eight phenolic compounds were identified, with catechin (15.5 ± 1.7 mg g-1) being the majority compound and a possible chemical marker of DEEB. The extract showed inhibition activity of the enzyme α-glucosidase. Chronic administration of DEEB (50 mg/kg of body weight) reduced glucose levels in diabetic animals, similar to acarbose; however, DEEB (100 and 200 mg/kg bw) caused premature death of mice by D22 of the treatment. Our data indicate that one of the mechanisms of toxicity in DEEB may be related to the aggravation of oxidative stress in the liver. This histopathological study indicated that DEEB failed to minimize the progression of the toxicity of diabetes caused by STZ. CONCLUSIONS: This study demonstrated the hypoglycemic potential of E. biflora leaves. However, the prolonged use of this tea can be harmful to its users due to its considerable toxicity, which needs to be better investigated.

qNMR quantification of phenolic compounds in dry extract of Myrcia multiflora leaves and its antioxidant, anti-AGE, and enzymatic inhibition activities.

Myrcia multiflora (Lam.) DC. is often used in Brazilian folk medicine to control diabetes. Analysis using HPLC-HRMS and NMR of the dry extract from the infusion of leaves of this species revealed twelve phenolic compounds. Among these compounds, chlorogenic acid (1), 4-O-caffeoylquinic acid (2), corilagin (3), chebulagic acid (4), pedunculagin (5), quercetin-3-O-ß-2″-galloylglucoside (7), and kaempferol-3-O-rhamnoside (12) are described for the first time in this matrix. Furthermore, six compounds were quantified using qNMR. The compounds in the dry extracts are 3, 6 (myricetin-3-O-d-glucoside), 8 (myricitrin), 9 (hyperoside), 10 (guaijaverin) and 11 (quercitrin). These compounds may be considered chemical markers in this matrix. In addition, this extract presents activities of α-glucosidase inhibition (IC50 = 79.9 µg mL-1) and glycation in vitro (IC50 = 10.2 µg mL-1), in addition to antioxidant activity against DPPH and ABTS radicals (1,856.7 and 1,032.0 µmol TEq, respectively). This extract did not show significant cytotoxicity in human fibroblasts. Therefore, the enzymatic inhibition, anti-AGE (advanced glycation end-products) and antioxidant activities of Myrcia multiflora leaves corroborated its antidiabetic therapeutic potential and instigates future preclinical studies aimed at the treatment of diabetes mellitus and its complications.

The Protein-Protein Interaction Network Reveals a Novel Role of the Signal Transduction Protein PII in the Control of c-di-GMP Homeostasis in Azospirillum brasilense.

The PII family comprises a group of widely distributed signal transduction proteins ubiquitous in prokaryotes and in the chloroplasts of plants. PII proteins sense the levels of key metabolites ATP, ADP, and 2-oxoglutarate, which affect the PII protein structure and thereby the ability of PII to interact with a range of target proteins. Here, we performed multiple ligand fishing assays with the PII protein orthologue GlnZ from the plant growth-promoting nitrogen-fixing bacterium Azospirillum brasilense to identify 37 proteins that are likely to be part of the PII protein-protein interaction network. Among the PII targets identified were enzymes related to nitrogen and fatty acid metabolism, signaling, coenzyme synthesis, RNA catabolism, and transcription. Direct binary PII-target complex was confirmed for 15 protein complexes using pulldown assays with recombinant proteins. Untargeted metabolome analysis showed that PII is required for proper homeostasis of important metabolites. Two enzymes involved in c-di-GMP metabolism were among the identified PII targets. A PII-deficient strain showed reduced c-di-GMP levels and altered aerotaxis and flocculation behavior. These data support that PII acts as a major metabolic hub controlling important enzymes and the homeostasis of key metabolites such as c-di-GMP in response to the prevailing nutritional status.IMPORTANCE The PII proteins sense and integrate important metabolic signals which reflect the cellular nutrition and energy status. Such extraordinary ability was capitalized by nature in such a way that the various PII proteins regulate different facets of metabolism by controlling the activity of a range of target proteins by protein-protein interactions. Here, we determined the PII protein interaction network in the plant growth-promoting nitrogen-fixing bacterium Azospirillum brasilense The interactome data along with metabolome analysis suggest that PII functions as a master metabolic regulator hub. We provide evidence that PII proteins act to regulate c-di-GMP levels in vivo and cell motility and adherence behaviors.

Antidiabetic potential of Musa spp. inflorescence: a systematic review.

OBJECTIVES: Extracts of parts Musa spp. have been used for the treatment of various diseases in traditional medicine. Studies have shown that these extracts have hypoglycaemic properties. The aim of this work was to gather evidence on the antidiabetic effects of Musa spp. inflorescence. METHODS: A systematic review was conducted with searches in three electronic databases, along with manual searches. Studies evaluating the antidiabetic properties of extracts of flower or bract of the genus Musa (in vitro or in vivo) were included. KEY FINDINGS: Overall, 16 studies were found. The reported assays were of hypoglycaemic effects, oral glucose tolerance, inhibitory activities in carbohydrate metabolism and digestive enzymes, enhanced glucose uptake activity and popular use of the extract in patients with diabetes type 2. In vitro studies showed that use of the extract was associated with antidiabetic effects (e.g. increased glucose uptake and inhibition of carbohydrate digestion enzymes). In induced diabetic models, Musa spp. extracts showed dose-dependent glycaemic level reductions compared with pharmacological drugs (P < 0.05). SUMMARY: In general, promising results regarding antidiabetic activity were found for inflorescence of Musa spp., suggesting that this plant could represent a natural alternative therapy for treating diabetes mellitus type 2.

Chemometric quality inspection control of pyrantel pamoate, febantel and praziquantel in veterinary tablets by mid infrared spectroscopy.

This paper describes the development and validation of a new multivariate calibration method based on diffuse reflectance mid infrared spectroscopy for direct and simultaneous determination of three veterinary pharmaceutical drugs, pyrantel pamoate, praziquantel and febantel, in commercial tablets. The best synergy interval partial least squares (siPLS) model was obtained by selecting three spectral regions, 3715-3150, 2865-2583, and 2298-1733 cm(-1), preprocessed by first derivative and Savitzky-Golay smoothing followed by mean centering. This model was built with five latent variables and provided root mean square errors of prediction (RMSEP) equal or lower than 0.69 mg per 100 mg of powder for the three analytes. The method was validated according the appropriate regulations through the estimate of figures of merit, such as trueness, precision, linearity, analytical sensitivity, bias and residual prediction deviation (RPD). Then, it was applied to three different veterinary pharmaceutical formulations found in the Brazilian market, in a situation of multi-product calibration, since the excipient composition of these commercial products, which was not known a priori, was modeled by an experimental design that scanned the likely content range of the possible constituents. The results were verified with high performance liquid chromatography with diode array detection (HPLC-DAD) and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and were in agreement with the predicted values at 95% confidence level. The developed method presented the advantages of being simple, rapid, solvent free, and about ten times faster than the HPLC ones.