Immobilized acetylcholinesterase in magnetic nanoparticles for in-line inhibition studies using a capillary electrophoresis system.

Enzyme assays can be performed with the capillary electrophoresis technique (CE) in many approaches, such as the immobilized enzyme micro-reactor. Acetylcholinesterase is a promising enzyme to be used when pursuing such a method, as it has already been explored in the proposal of similar methods of miniaturizing enzyme assays. The present work proposes a novel enzyme micro-reactor, based on the anchorage of the enzyme on magnetic nanoparticles of MnFe2O4, with chitosan and glutaraldehyde as the cross-linker in the capillary by means of an arrange of neodymium magnets. The calculated Km of the enzyme evaluated by this method was 1.12 mmol L-1, comparable to other studies in the literature that utilizes immobilized enzymes. Also, IC50 for neostigmine was assessed in 3 different micro-reactors, with an average of 29.42 ± 3.88 µmol L-1. In terms of the micro-reactor stability, it was possible to perform at least 25 experiments with assembled micro-reactor. The method was applied to hydroalcoholic extracts of 7 plant species. Plinia cauliflora had the best result, with 42.31 ± 6.81% of enzyme inhibition in a concentration of 100 mg L-1.

Effects of Myrcia pubipetala Miq (Myrtaceae) extract on innate inflammatory response.

Myrcia is a genus widespread in South America with many species presenting anti-inflammatory and biological properties. We investigated the anti-inflammatory activity of crude hydroalcoholic extract of Myrcia pubipetala leaves (CHE-MP) using macrophages (RAW 264.7), and the air pouch model in mice to evaluate leukocyte migration and mediator's release. Adhesion molecule expression, CD49 and CD18, was evaluated in neutrophils. In vitro, the CHE-MP significantly reduced nitric oxide (NO), interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF) levels in the exudate and the supernatant culture. CHE-MP did not present cytotoxicity and modulated the percentage of positive neutrophils for CD18 and its expression per cell, without modifying the expression of CD49, which corroborated with significantly reduced neutrophil migration to inflammatory exudate and subcutaneous tissue. Taken together, the data demonstrate that CHE-MP presents a potential activity on innate inflammatory.

Phenolic compounds of Eugenia involucrata (Myrtaceae) extracts and associated antioxidant and inhibitory effects on acetylcholinesterase and α-glucosidase.

Eugenia involucrata DC. (Myrtaceae), popularly known as "cereja-do-Rio-Grande", is a native tree from Brazil, popularly used as a hypoglycemiant. Crude hydroalcoholic extract (CHE) and fractions (insoluble (FI), dichloromethane (FDM), ethyl acetate (FEA) and butanol (FBu)) of leaves were assessed to determine the phenolic chemical composition by HPLC-ESI-MS/MS. 10 compounds were identified, being 7 new for this species: rutin, isoquercitrin, luteolin-7-O-rutinoside, mandelic acid, naringenin, luteolin-7-O-glucoside and salicylic acid. Extract and fractions showed inhibitory activity on acetylcholinesterase (AchE) enzyme (best result: IC50 = 44.19 µg mL-1, for FEA) and α-glucosidase (α-Glu) (best result: IC50 = 31.25 ± 0.15 µg mL-1, for CHE). The observed antioxidant and inhibitory activity on the AchE and α-Glu is due to, at least in part, the presence of phenolic compounds in the samples.

Phenolic profile by HPLC-ESI-MS/MS and enzymatic inhibitory effect of Bryophyllum delagoense.

Bryophyllum delagoense (Eckl. & Zeyh.) Druce, native to Madagascar, is popularly known as "abyssian cactus" and popularly used in folk medicine as an analgesic and healing agent. The study methodology was divided into the phytochemical study: fractionation and identification of phenolic compounds by HLPC-ESI-MS/MS from the methanolic extract (ME), and fractions (DCMF and EAF) of leaves of B. delagoense, and biological activity with acetylcholinesterase and α-glucosidase inhibition of extracts and fractions by in vitro enzymatic techniques. Twenty-seven phenolic compounds were identified, being the highest concentration of syringic acid (87.848 mg g-1). The DCMF fraction showed the best biological activity for inhibition of α-glucosidase enzyme (0.125 mg mL-1).

Anti-inflammatory activity of the epicuticular wax and its isolated compounds catechin and gallocatechin from Eugenia brasiliensis Lam. (Myrtaceae) leaves.

This study describes anti-inflamatory activity of epicuticular wax (ECW) extracted from leaves of Eugenia brasiliensis Lam., and its isolated compounds, catechin and gallocatechin in a pleurisy model in mice. The results for exudation and cell migration showed that the extract and isolated compounds from the plant were effective in inhibiting these inflammatory parameters. The ECW demonstrated a dose-dependent effect on the inhibition of cell migration. The results for NOx levels reveals that the isolated compounds from Eugenia brasiliensis were effective in inhibiting the production of this inflammatory mediator, however, ECW were not able in inhibiting the production of NO in the pleural cavity, at the tested doses. The results here obtained, helps to reinforce the scientifically hypothesis that E. brasiliensis has an important anti-inflammatory activity, and that this effect may be, at least in part, by the presence of the compounds catechin and gallocatechin.

Phenolic profile by HPLC-ESI-MS/MS of six Brazilian Eugenia species and their potential as cholinesterase inhibitors.

Eugenia genus is known for its phenolic metabolites, which may influence the progression of the Alzheimer Disease. This study aimed to evaluate the anticholinesterase effects of six Eugenia species from Brazil. Leaves and stems were submitted to maceration (methanol) and partitioned with dichloromethane and ethyl acetate (EtOAc). Samples were screened (200 µg mL-1) for the inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). HPLC-ESI-MS/MS analysis allowed the identification of twenty-eight phenolic compounds. Regarding the enzymatic activity, EtOAc fraction of E. mattosii exhibited the best results. Chemical and pharmacological aspects of seasonal E. mattosii extracts were evaluated. The extract from leaves collected in the winter was the most effective for AChE, and the extract from leaves collected in the spring was the most effective for BuChE. Correlating the enzymatic results with the chemical data, it was possible to associate these effects to isoquercitrin, quercetin, catechin, epicatechin, procatecuic acid and myricitrin content.

Dual electrophoretically-mediated microanalysis in multiple injection mode for the simultaneous determination of acetylcholinesterase and α-glucosidase activity applied to selected polyphenols.

Acetylcholinesterase (AChE) and α-glucosidase (α-glu) are key target enzymes in the search for novel strategies in the treatment of Alzheimer's disease and type II diabetes. Therefore, methods to assess the enzyme inhibition are of great value in the research field. Here is proposed a novel a dual electrophoretically-mediated microanalysis for the simultaneous determination of both enzymes' activity. In order to do so, the various solutions required for both assays were introduced in the capillary electrophoresis system using the multiple injections approach. Enzymatic kinetic parameters were tested, Km for AChE and α-glu were 3.81 and 0.43 mmol L-1. Ki values were 4.27 µmol L-1 for neostigmine (an AChE inhibitor) and 0.40 mmol L-1 for acarbose (an α-glu inhibitor). Results of IC50 (concentration for 50% of inhibition) were 5.11 ± 0.47 µmol L-1 and 0.58 ± 0,02 mmol L-1 for neostigmine and acarbose, respectively. All parameters (except for Ki of neostigmine) were comparable with the literature, indicating a good reliability of the proposed method to evaluate these enzymes activity. Total time analysis was approximately 10 min, being possible to perform around 12 enzymatic assays per hour, with low sample and reagent consumption, thus satisfying some of the principles of green chemistry. The method was applied to evaluate 10 phenolic compounds, of wich p-coumaric acid showed the best inhibitory activity for AChE (40.14 ± 4.75% at 10 mg L-1); and quercetin for α-glu (46.53 ± 4.90% at 10 mg L-1).

Kinetics Analysis of the Inhibitory Effects of Alpha-Glucosidase and Identification of Compounds from Ganoderma lipsiense Mycelium.

The studies on natural compounds to diabetes mellitus treatment have been increasing in recent years. Research suggests that natural components can inhibit alpha-glucosidase activities, an important strategy in the management of blood glucose levels. In this work, for the first time in the literature, the compounds produced by Ganoderma lipsiense extracts were identified and evaluated on the inhibitory effect of these on alpha-glucosidase activity. Four phenolic compounds were identified by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) to crude extract from G. lipsiense grown in red rice medium (RCE) and synthetic medium (SCE), being syringic acid identified in both extracts. Gas chromatography-mass spectrometry (GC-MS) analysis showed fatty acids and their derivatives, terpene, steroid, niacin, and nitrogen compounds to SCE, while RCE was rich in fatty acids and their derivatives. Both extracts demonstrated alpha-glucosidase inhibition (RCE IC50 = 0.269 ± 8.25 mg mL-1; SCE IC50 = 0.218 ± 9.67 mg mL-1), and the purified hexane fraction of RCE (RHEX) demonstrated the highest inhibition of enzyme (81.1%). Studies on kinetic inhibition showed competitive inhibition mode to RCE, while SCE showed uncompetitive inhibition mode. Although the inhibitory effects of RCE and SCE were satisfactory, the present findings identified some unpublished compounds to G. lipsiense in the literature with important therapeutic properties.

Determination of acetylcholinesterase and α-glucosidase inhibition by electrophoretically-mediated microanalysis and phenolic profile by HPLC-ESI-MS/MS of fruit juices from Brazilian Myrtaceae Plinia cauliflora (Mart.) Kausel and Eugenia uniflora L.

Alzheimer's disease and diabetes mellitus are contemporary diseases of great concern. Phenolic compounds are linked to several health benefits and could lead to novel strategies to combat these ailments. The objective of this study was to evaluate by electrophoretically-mediated microanalysis the potential inhibitory activity of the fruit juices from Plinia cauliflora ("jaboticaba") and Eugenia uniflora ("pitanga") toward acetylcholinesterase (AChE) and α-glucosidase, target enzymes in strategies for the treatment of these diseases. The phenolic profiles of the samples were also investigated. Jaboticaba and pitanga juices inhibited 85.90 ± 1.73 and 52.67 ± 1.24% of AChE activity at 5 mg mL-1, and 57.91 ± 2.60 and 69.47 ± 2.89% of α-glucosidase activity at 1 mg mL-1, respectively. Total phenolic content of the juices were 303.54 ± 28.28 and 367.00 ± 11.42 mgGA L-1, respectively. The observed inhibitory activity can be explained, at least in part, by the presence of the phenolic compounds.

Sorocea guilleminiana Gaudich.: Wound healing activity, action mechanisms, and chemical characterization of the leaf infusion.

ETHNOPHARMACOLOGICAL RELEVANCE: Sorocea guilleminina Gaudich. is a tree or shrub endemic to Brazil. Its leaves are used in Brazilian folk medicine for the healing of wounds, stomach problems, inflammation and as diuretic. The present study evaluates the activity and action mechanisms of the healing properties of the aqueous extract of S. guilleminiana leaves (AESg), in experimental models in vivo and in vitro, as well as performs a phytochemical analysis of the extract. MATERIALS AND METHODS: The AESg was prepared by infusion: Ten g of dry leaves powder in 1 L hot water, soaked for 15 min, filtered, lyophilized, and stored at -30 °C. Phytochemical analyses were realized by colorimetry and HPLC/ESI/MS. Its' in vitro cytotoxicity was evaluated on fibroblastic N3T3 cells. The potential of the wound healing activity in vivo was evaluated using excision and incision wound rat models, by histopathology of the injured skin along with the determination of nitric oxide, cytokines (IL-1ß, IL-10, and TNF-α), and antioxidant parameters (GSH, MPO and CAT). In vitro wound healing activity was also demonstrated in scratched N3T3 cells, by measuring the proliferation/migration rate. RESULTS: The phytochemical analysis of the AESg revealed a strong presence of polar compounds, especially flavonoids (4 majoritarian), as well as terpenes and/or sterols (2 majoritarian). The AESg showed no toxicity in the N3T3 cell line (IC50 > 800 µg/mL). Topical treatment with the AESg showed an increase (p < 0.05) in wound contraction with 2 mg/g cream on days 5 and 9 (43.56% and 6.70% increase, respectively), and with 50 mg/g on days 7 and 9 (10.88% and 7.91%, respectively), compared to the vehicle (non-ionic neutral cream). Topical application of AESg (2 or 50 mg/g non-ionic cream) in incised wounds caused an increase in the force necessary for the rupture of the wound when compared to the vehicle group. No changes in cytokines (IL-1ß, IL-10, or TNF-α) or NO accumulation was found with up to 50 mg/g AESg treatment. For antioxidant activity on the incision wound, an increase in GSH levels was denoted with the AESg use, at the lowest and highest dose (2 and 50 mg/g) by 75.86% and 61.20% respectively, when compared to the vehicle. Also, the CAT activity was accentuated by AESg at the highest dose (50 mg/g) by 85.87%. Finally, the AESg at all doses attenuated MPO activity significantly in the incision wound by 71.35%, 73.21%, 78.08%, respectively. In the scratch test on N3T3 cells, the treatment with AESg resulted also in an increase in fibroblast proliferation/migration rate, compared to the vehicle. CONCLUSION: AESg is not cytotoxic. The results confirm the popular use of the leaf infusion of S. guilleminiana for the treatment of cutaneous wounds, possibly by stimulating the proliferation of fibroblasts with a consequent deposition of collagen, fastening rearrangement of collagen fibers, and greater transformation into myofibroblasts, essential in the healing process. Preliminary chemical analyzes of AESg revealed the presence mainly of phenolic compounds, being salicylic acid, gallic acid, pinocembrin and isoquercitrin the majoritarian ones.

Bauhinia forficata link, a Brazilian medicinal plant traditionally used to treat cardiovascular disorders, exerts endothelium-dependent and independent vasorelaxation in thoracic aorta of normotensive and hypertensive rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Bauhinia forficata Link, commonly known as "cow's paw", is a native plant from South America. Its leaves are widely used in Brazilian folk medicine to treat diabetes and cardiovascular disorders. Although this species' biological potential has been extensively proven as an antidiabetic, anti-inflammatory and antioxidant agent, there is a lack of studies to evidence its action on the cardiovascular system. AIM OF THE STUDY: This study was designed to investigate the vascular effects of B. forficata leaves preparations and its majority compound kaempferitrin, as well as its aglycone form kaempferol, in rat aortic rings of normotensive (NTR) and hypertensive (SHR) rats. MATERIALS AND METHODS: Aorta rings from NTR and SHR precontracted with phenylephrine were exposed to cumulative concentrations of B. forficata extract, fractions (1-50 µg/mL) and compounds (0.001-0.3 µg/mL). The mechanisms involved in the vasorelaxant effect of ethyl-acetate plus butanol fraction (EAButF) were also evaluated. RESULTS: Although kaempferitrin is the most abundant compound found in both methanolic extract and EAButF, 24 minor phenolic compounds were identified in B. forficata leaves, including kaempferol. EAButF was the only with endothelium-dependent and independent vasorelaxant properties in both NTR and SHR. The incubation with L-NAME or ODQ completely blocked EAButF-induced vasorelaxation. On the other hand, the incubation with propranolol, atropine, indomethacin, glibenclamide or barium chloride did not change the vasorelaxant activity of EAButF (50 µg/mL). Nevertheless, the incubation with tetraethylammonium and 4-aminopyridine significantly influenced the EAButF activity. It was also shown that Ca2+ influx or efflux is not related to EAButF vasorelaxation potential. Kaempferitrin and kaempferol were also able to relax the rat aortic rings in 34.70% and 40.54%, respectively. CONCLUSIONS: This study shows, for the first time, the vasorelaxant effect of EAButF from B. forficata leaves, an effect that may be attributed to the modulation of vascular tone through nitric oxide/soluble guanylate cyclase pathway, and potassium channels. The bioactive kaempferitrin and kaempferol seem to be important for the effects observed with the fraction. Finally, preparations obtained from the leaves of B. forficata may be interesting candidates for new or complementary strategies regarding cardiovascular diseases.

Kinetic identification of phenolic compounds and potential production of caffeic acid by Ganoderma lipsiense in solid-state fermentation.

Basidiomycetes fungi have been utilized for the production of several compounds with bioactive properties, such as phenolic compounds. The present work quantified and identified the phenolic compounds produced in a kinetic study (63 days) and evaluated the antimicrobial activity from the extract obtained by Ganoderma lipsiense cultivation in solid-state fermentation using red rice. Phenolic compounds were identified by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) and caffeic acid content was measured by high-performance liquid chromatography with diode-array detection (HPLC-DAD). Caffeic and syringic acids were produced by G. lipsiense. In the control medium (red rice), the following compounds were identified: p-coumaric acid, salicylic acid, ferulic acid and vanillin. High concentrations of caffeic acid (0.977 µg g-1) were measured in 49 days. Antimicrobial activity was investigated against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus using a minimum inhibitory concentration (MIC) technique. Ganoderma lipsiense extract was only effective against P. aeruginosa. These data have proved to be satisfactory in the study of biosynthesis of caffeic acid and antibacterial compounds by G. lipsiense in solid-state fermentation with red rice.

Antioxidant and antidepressant-like effects of Eugenia catharinensis D. Legrand in an animal model of depression induced by corticosterone.

This work investigated the antioxidant and antidepressant-like effects of ethyl acetate extract from Eugenia catharinensis in mice treated with corticosterone (20 mg/Kg). The animals received saline or corticosterone (21 days) and, in the last 7 days, they were treated with the extract (50, 125, 200 or 250 mg/Kg) or vehicle. After 24 h, the mice were submitted to the open field and forced swimming tests, after which the hippocampus and cerebral cortex were removed. Our results showed that the extract decreased the immobility time of mice in the forced swimming test and that the extract was able to reverse the effect caused by corticosterone. Corticosterone pre-treatment generated oxidative stress, altering antioxidant enzymes in the nervous tissue. The extract increased the catalase and superoxide dismutase activities and reversed the effects of corticosterone. In the hippocampus, the extract increased superoxide dismutase activity and reversed the increase in catalase activity elicited by corticosterone. We propose that the effects elicited by the Eugenia catharinensis are dependent on the presence of phenolic compounds (gallic acid, protocatechuic acid, syringic acid, 4-hydroxy methylbenzoic acid, chlorogenic acid, salicylic acid, caffeic acid, vanillic acid, p-coumaric acid, isoquercetin, rutin, ferulic acid, aromadendrin, galangin and apigenin) in this extract, as demonstrated by HPLC-ESI-MS/MS.

Characterisation of phenolic compounds of the ethyl acetate fraction from Tabernaemontana catharinensis and its potential antidepressant-like effect.

This study evaluates the antidepressant-like effect and analysed the qualitative and quantitative 74 phenolic standards of ethyl acetate fraction from Tabernaemontana catharinensis leaves. Acute administration of fraction in mice reduced the immobility time in forced swimming and tail suspension tests confirming its antidepressant-like activity. The anti-immobility effect elicited by this fraction was prevented by the pretreatment of mice with PCPA (100 mg kg-1), ketanserin (5 mg kg-1), SCH 23,390 (0.05 mg kg-1) or yohimbine (1 mg kg-1). A sub effective dose of the fraction produced a synergistic effect with fluoxetine (5 mg kg-1). Chromatographic analysis identified 4-hydroxybenzoic and p-coumaric acids in the ethyl acetate fraction from T. catharinensis. Capillary electrophoresis presented 7.34 ± 0.02 mg g-1 of p-coumaric acid concentration in the fraction. Therefore, it is possible that antidepressant-like effect elicited by ethyl acetate fraction from T. catharinensis be dependent on the p-coumaric acid.

Hypolipemiant and antioxidant effects of Eugenia brasiliensis in an animal model of coconut oil-induced hypertriglyceridemia.

We investigated the effects of chronic administration of crude hydroalcoholic extract (CHE) and crude acetone extract (CAE) obtained from leaves of Eugenia brasiliensis species on hypertriglyceridemia and oxidative stress caused by the chronic administration of coconut oil. Rats received CHE or CAE (50, 100 or 150mg/kg, orally) for 30days, plus coconut oil (2mL, orally) or saline for 15th. Triglyceride levels, liver cell lipid accumulation, thiobarbituric acid reactive substances (TBA-RS), total sulfhydryl content and the activities of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were evaluated in the blood and liver of rats. Results showed that chronic administration of CHE or CAE was able to prevent hypertriglyceridemia and decrease the lipid droplets in liver cells, as well as the increase in TBA-RS, the reduction in total sulfhydryl content and CAT activity in the blood and prevent total or partial the increase in CAT and reduction in SOD and GSH-Px activities in the liver. These findings indicate that both extracts may have hypolipidemic and antioxidant effects.

Chemical composition and evaluation of the antimicrobial activity of the essential oil from leaves of Eugenia platysema.

This study describes the qualitative and quantitative chemical composition and evaluates the antibacterial activity of essential oil from Eugenia platysema leaves. Analysis by GC-FID and GC-MS allowed the identification of 22 compounds. Different from the other species of the Eugenia genus, the major compound found in the essential oil was the diterpene phytol (66.05%), being this the first report of the presence of this compound in the essential oils from Eugenia genus. The sesquiterpene elixene was the second most concentrated compound in the studied essential oil (9.16%). The essential oil from E. platysema was tested for its antibacterial activity against cell-walled bacteria and mollicute strains of clinical interest using the microdilution broth assay. The results showed that the essential oil of E. platysema was inactive until 1000 µg mL(-1) against tested bacteria.

Evaluation of seasonal chemical composition, antibacterial, antioxidant and anticholinesterase activity of essential oil from Eugenia brasiliensis Lam.

This study describes the seasonal composition and the antibacterial, antioxidant and anticholinesterase activity of the essential oil from Eugenia brasiliensis leaves. Analysis by using GC allowed the identification of 40 compounds. It was observed that the monoterpenes varied more (42%) than the sesquiterpenes (14%), and that the monoterpene hydrocarbons suffered the greatest variation throughout the year (64%). Major compounds were spathulenol in the spring (16.02 ± 0.44%) and summer (18.17 ± 0.41%), τ-cadinol in the autumn (12.83 ± 0.03%) and α-pinene (15.94 ± 0.58%) in the winter. Essential oils were tested for their antibacterial activity, and the best result was obtained from the autumn oil, with MIC = 500 µg mL(- 1) against Staphylococcus saprophyticus and Pseudomonas aeruginosa. Antioxidant activity was evaluated using DPPH, lipid peroxidation and iron-reducing power assays, as well as the anticholinesterase activity. Both tests showed a weak performance of the essential oils.