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.

Alcoholic beverages from araçá-boi fruit: quantification of antioxidant compounds by NMR ERETIC2.

Fermentation is a preservation process responsible for increasing food product shelf life. In this context, alcoholic fermentation can add value to unconventional Amazon fruits, e.g., araçá-boi (Eugenia stipitata). This fruit has various antioxidant phenolic compounds with well-known nutraceutical properties. However, araçá-boi is still underexplored by food industry. This rationale led to investigate the influence of five commercial yeasts (Saccharomyces cerevisiae) and filtration process on chemical composition and antioxidant capacity of araçá-boi beverages. DPPH and Folin Ciocalteu assays were used to determine antioxidant capacity and total phenolic content. Organic compounds' contents were assessed by NMR-ERETIC2. In all beverages, ten compounds [tyrosol, sucrose, fructose, (α/ß)-glucose, ethanol, malic, citric, gallic, and succinic acids] were identified and quantified. The highest phenolic concentrations [gallic acid (390.0 µM) and tyrosol (380.0 µM)] were found in Biolievito Bayanus (BBA) beverage. The new BBA beverage was used for investigating filtration process influence on chemical composition and antioxidant responses. Alcoholic content (unfiltered: 13.9°GL and filtered: 12.7°GL), antioxidant responses, and total phenolic contents were influenced by filtration process. The yeast type and unfiltered process were determinant for chemical content and antioxidant capacity of beverages. These results might be useful to private sector and future production and commercialization of araçá-boi beverages.

Encapsulation of Piper aduncum and Piper hispidinervum essential oils in gelatin nanoparticles: a possible sustainable control tool of Aedes aegypti, Tetranychus urticae and Cerataphis lataniae.

BACKGROUND: The encapsulated essential oils (EOs) of Piper aduncum L. and Piper hispidinervum C. DC. in gelatin nanoparticles were evaluated against Aedes aegypti Linn., Tetranychus urticae Koch and Cerataphis lataniae Boisd. RESULTS: Encapsulation efficiency of the EOs was measured for absolute concentrations of 500 µg mL-1 (79.2 and 72.7%) and 1000 µg mL-1 (84.5 and 82.2%). The loaded nanoparticles were nearly spherical and well dispersed. The nanoparticles loaded with P. hispidinervum EO had an average size of 100 ± 2 nm, while the nanoparticles containing P. aduncum EO ranged from 175 ± 4 to 220 ± 4 nm. According to zeta potential analysis, the nanoparticles loaded with P. hispidinervum and P. aduncum EOs presented values around -43.5 ± 3 and -37.5 ± 2 mV respectively. The controlled release of EOs was described by the anomalous mechanism of Korsmeyer-Peppas. Both encapsulated EOs reached lethal dosages within 24 h of exposure and total mortality of the tested pests. CONCLUSION: The present work successfully developed gelatin-based nanoparticles that served as carriers for the EOs of P. aduncum and P. hispidinervum to be applied as a sustainable control tool of A. aegypti, T. urticae and C. lataniae. The developed loaded nanoparticles presented high encapsulation efficiency and EO concentration release higher than lethal dosages. This indicates that it is feasible to use gelatin-based nanoparticles loaded with P. aduncum and P. hispidinervum EOs to control the tested pests. © 2018 Society of Chemical Industry.

1H NMR spectroscopy as a tool to probe potential biomarkers of the drying-salting process: A proof-of-concept study with the Amazon fish pirarucu.

Dry-salted pirarucu (Arapaima gigas) plays an important cultural role in the Amazon region - South America. In this study, we explored the changes in the chemical composition of pirarucu meat following the drying-salting process via 1H NMR spectroscopy. Combining multivariate and univariate statistical analyses yielded a robust differentiation of metabolites involved in the process. VIP score (>1), p-value (<0.05), and AUC (>0.7) were considered to selecting compounds that had significant fluctuations in their contents along the process. Our results pointed out acetate, lactate, succinate, and creatinine as metabolites undergoing significant changes during the drying-salting process. Creatinine was not detected in fresh samples. The investigation of multiple components delves deeper into the molecular nuances of the salting-drying process's impact on fish meat, providing a more comprehensive understanding of the possible chemical transformations and how the matrix's quality control and nutritional aspects should be addressed.

Hypoglycemic effect of the dry leaf extract of Myrcia multiflora in streptozotocin-induced diabetic mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Myrcia multiflora (Lam) DC. is a medicinal plant used in folk medicine for diabetes control, mainly in the Brazilian Amazon. The leaves of this species has already demonstrated antidiabetic properties; however, in mice with type 2 diabetes (DM2), the cumulative effect of the consumption of the dry extract of M. multiflora leaves (Mm) has not yet been reported. AIM OF THE STUDY: To investigate the effect of the dry extract obtained from the infusion of the dried leaves of M. multiflora on the blood glucose levels of diabetic mice. MATERIALS AND METHODS: DM2 was induced in Swiss male mice by a single intraperitoneal injection of streptozotocin [150 mg/kg body weight (bw)]. The animals were divided into two control groups (healthy and diabetic without treatment) and three sample groups that received Mm (25 and 50 mg/kg bw) and acarbose (200 mg/kg bw) by gavage once daily for 28 days (D28). Additionally, biochemical parameters, thiobarbituric acid reactive species (TBARS) levels in the liver, and histopathological analyses of the kidneys and liver were performed. RESULTS: On the seventh day of treatment, a 74.7% reduction in glucose levels were observed in the group of diabetic animals treated with Mm (50 mg/kg bw) when compared to the beginning of the treatment. At D28, the hypoglycemic effect was maintained. The results of the biochemical and histopathological parameters and the TBARS levels suggest that this dry extract exerts nephro- and hepatoprotective effects. CONCLUSIONS: The findings demonstrate the potential that this extract has to inhibit the α-glucosidase enzyme, and it acts similarly to the positive control acarbose. Furthermore, this extract is nephro- and hepatoprotective. Therefore, this dry extract has the potential to be an adjuvant for DM2, which corroborates its use in folk medicine.

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.

Pedra-ume caá fruit: An Amazon cherry rich in phenolic compounds with antiglycant and antioxidant properties.

Species of Eugenia have been used as an antidiabetic natural source. Chemical, antioxidant and antiglycant screening of extracts from pedra-ume caá (Eugenia punicifolia) fruits were performed. 1H NMR assisted by non-supervised chemometric methods were employed for the evaluation of the chemical profiles which were distinguished according to the color of fruit maturation stages, as well as for pulp and seed fruit. Furthermore, 1H NMR fingerprint analysis of the crude extract allowed the identification of quercitrin and myricitrin, beside other nine compounds. The extracts of the yellow (YP) and green (GP) pulps presented higher antiglycant and antioxidant activities. Fresh juice from E. punicifolia was encapsulated in microcapsules produced with dextrose equivalent (DE) of 10, 20 or 30 as wall materials for the maintainment of their antioxidant and antiglycant properties. The more efficient retention of the bioactive compounds was found using the DE30. The Encapsulation Efficiency (EE) and the Retention Efficiency (RE) of this system was found around 89.7% and 97.6%, respectively. In addition, NMR spectra revealed the presence of flavonoids O-glycosylated (quercitrin and myricitrin) which might be related to the antiglycant and antioxidant activities. The YP presented larger content of quercitrin (117.6 ±â€¯0.4 mg per each 100 g of fresh fruit). Therefore, pedra-ume caá should be employed as an alternative nutraceutical source, as well as intherapeutic pourposes.

Tetraflavonoid and biflavonoids from Aristolochia ridicula.

Biflavones, a chalcone-flavone, and a tetraflavonoid with a new carbon skeleton were isolated from the leaves of Aristolochia ridicula. Their structures were determined by chemical derivatizations and spectrometric analyses.

Evaluation of insecticidal activity of diterpenes and lignans from Aristolochia malmeana against Anticarsia gemmatalis.

The insecticidal activity of hexane extracts from the roots and leaves of Aristolochia malmeana was evaluated against Anticarsia gemmatalis larvae by topical application. Extract from the roots was the most active and caused 50% mortality in larvae at 308.4 microg/microL. From this extract, a clerodane diterpene, (-)-kolavenic acid, and three lignans, (-)-kusunokinin, (-)-hinokinin, and (8 S,8' R,9 S)-cubebin, were isolated by chromatography and partition procedures and then evaluated for their insecticidal activities either individually or in pairs. (-)-Kusunokinin showed higher activity against A. gemmatalis (LD10=9.3, LD50=230.1 microg/microL) than the crude extract, and its activity was dose-dependent, whereas the other constituents did not exhibit any significant activity. Together with (-)-kusunokinin and (-)-hinokinin, (-)-copalic acid, (-)-2-oxokolavenic acid, (-)- ent-6-beta-hydroxy-copalic acid, (8 R,8' R,9 R)- and (8 R,8' R,9 S)-cubebins, (-)-fargesin, and (-)-phillygenin were isolated from the hexane extract of the leaves. The compounds were identified on the basis of spectroscopic analysis.

Chalcone-flavone tetramer and biflavones from Aristolochia ridicula.

Biflavones and a chalcone-flavone tetramer were isolated from the leaves of Aristolochia ridicula, together with proto-quercitol. Their structures were determined by spectroscopic methods.

Chemical characterization and antioxidant capacity of the araçá-pera (Psidium acutangulum): An exotic Amazon fruit.

The "araçá-pera" (Psidium acutangulum DC) is an exotic guava consumed by the Brazilian Amazon population. This paper describes 22 compounds from this fruit by UHPLC-HRMS and NMR methods, being one disaccharide, five monosaccharides, two organic acids, one trihydroxycinnamic acid glucopyranosyl, one tannine digalloyl glucopyranosyl, five triterpenoid acids, and six fatty acids. It also quantifies the level of ascorbic acid (AA) by HPLC-MS (74.32±1.23mg/100g of fresh fruit), and defines the chemical antioxidant activities by DPPH and ABTS+ assays (24.96±0.75, 90.57±0.63mg of vitamin C/100g fresh fruit, respectively), and a cell-based assays (76-100%). These results have shown that this exotic guava can be consumed as a nutraceutical ingredient, as well as be used in the production of functional foods in the Amazonian diet to prevent chronic and oxidative diseases.