Assessment of Anticholinergic and Antidiabetic Properties of Some Natural and Synthetic Molecules: an In Vitro and In Silico Approach.

INTRODUCTION: In this study, it was aimed to determine the in vitro and in silico effects of some natural and synthetic molecules on acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and α-glucosidase enzymes. BACKGROUND: Alzheimer's disease (AD) and Type II diabetes mellitus (T2DM), which are considered amongst the most important diseases of today's world. However, the side effects of therapeutic agents used in both diseases limit their use. Therefore, it is important to develop drugs with high therapeutic efficacy and better pharmacological profile. OBJECTIVE: This study sets out to determine the related enzyme inhibitors used in the treatment of AD and T2DM, which are considered amongst the most important diseases of today's world. METHODS: In the current study, the in vitro and in silico effects of dienestrol, hesperetin, L-thyroxine, 3,3',5-Triiodo-L-thyronine (T3) and dobutamine molecules on AChE, BChE and α-glycosidase enzyme activities were investigated. RESULTS: All the molecules showed an inhibitory effect on the enzymes. The IC50 and Ki values of the L-Thyroxine molecule, which showed the strongest inhibition effect for the AChE enzyme, were determined as 1.71 µM and 0.83±0.195 µM, respectively. In addition, dienestrol, T3 and dobutamine molecules showed a more substantial inhibition effect than tacrine. Dobutamine molecule showed the most substantial inhibition effect for BChE enzyme, and IC50 and Ki values were determined as 1.83 µM and 0.845±0.143 µM, respectively. The IC50 and Ki values for the hesperetin molecule, which showed the strongest inhibition for the α-glycosidase enzyme, were determined as 13.57 µM and 12.33±2.57 µM, respectively. CONCLUSION: According to the results obtained, it may be said that the molecules used in the study are potential inhibitor candidates for AChE, BChE and α-glycosidase.

Enzyme Inhibition and Antioxidant Activities of Asparagus officinalis L. and Analysis of Its Phytochemical Content by LC/MS/MS.

In the study, water, ethanol, methanol, dichloromethane, and acetone extracts of Asparagus officinalis L. were obtained by maceration. DPPH⋅, ABTS⋅+ , FRAP, and CUPRAC methods determined the antioxidant capacities of all extracts. Moreover, the in vitro effects of extracts on acetylcholinesterase (AChE), butyrylcholinesterase (BChE), carbonic anhydrase (CA)-I, CA-II and α-Glycosidase were investigated. At a 10 µg/ml concentration, the extract with the highest Fe3+ reduction capacity was ethanol (AE), and the extract with the highest Cu2+ reduction capacity was acetone (AA). AE for AChE (IC50 =21.19 µg/ml) and α-Glycosidase (IC50 : 70.00 µg/ml), methanol (AM) for BChE (IC50 =17.33 µg/ml), CA-I and II (IC50 =79.65 and 36.09 µg/ml, respectively) showed the most potent inhibition effect. The content analysis of acetone extract was performed with LC/MS-MS, the first three phytochemicals found most were p-Coumaric acid, rutin, and 4-hydroxybenzoic acid (284.29±3.97, 135.39±8.19, and 102.06±5.51 µg analyte/g extract, respectively).

Screening inhibitory effects of selected flavonoids on human recombinant aldose reductase enzyme: in vitro and in silico study.

Aldose reductase (AR) is the first enzyme of the polyol pathway that has physiological importance under hyperglycaemic conditions. The article has been focussed on AR enzyme inhibition by selected compounds. For this purpose, the in vitro inhibitory effects of various compounds on commercially available recombinant human AR (rAR) enzyme activity were investigated. The IC50 values of compounds on rAR inhibition effect were found for 6-hydroxy flavone, syringic acid, diosmetin, 6-fluoroflavone, 7-hydroxy-4'-nitroisoflavone, myricetin as 2.05, 2.97, 15.75, 16.1, 49.5, and 63 µM, respectively. 6-Hydroxy flavone and syringic acid competitively inhibited rAR with respect to the NADPH with Ki values 0.509 ± 0.036 and 0.842 ± 0.012 µM. In addition, docking studies were performed to evaluate the potential enzyme binding positions of the compounds. Our in vitro and in silico results indicated that the 6-hydroxy flavone may be a good lead compound in the development of AR inhibitors to prevent diabetic complications.

Enhancing bactericidal strategy with selected aromatic compounds: in vitro and in silico study.

ß-Lactamases are enzymes that catalyze the hydrolysis of the ß-lactam ring, resulting in loss of function in ß-lactam antibiotics. In this report, the inhibition of ß-lactamase enzyme by group-based selected aromatic compounds, especially containing flavone ring, was investigated in vitro and in silico. For this purpose, the inhibitory effects of 7-hydroxy-4'-nitroisoflavone, myricetin, formononetin, 3-methylflavone-8-carboxylic acid, 6-fluoroflavone and caffeic acid on ß-lactamase from Bacillus cereus enzyme activity were investigated. IC50 values of these compounds were determined to range from 53 to 346 mM. 7-hydroxy-4'-nitroisoflavone, formononetin and myricetin inhibited the enzyme with the Ki values of 27.65 ± 4.22, 58.92 ± 12.83 and 67.42 ± 5.77 µM, respectively. To evaluate the potential enzyme binding positions of the active compounds, docking studies were performed. In addition to kinetic and in silico studies, the synergistic effects of the compounds with penicillin on Klebsiella pneumoniae (ATCC 700603) and Escherichia coli (ATCC 35128) were tested in vitro. Our results indicated that 7-hydroxy-4'-nitroisoflavone, 3-methylflavone-8-carboxylic acid and myricetin that combined with penicillin completely precluded the bacterial growth.Communicated by Ramaswamy H. Sarma.

Evaluation of the effects of Cyclotrichium niveum on brain acetylcholinesterase activity and oxidative stress in male rats orally exposed to lead acetate.

Cyclotrichium niveum is an endemic plant for Turkey and it appears to have in vitro antioxidant and acetylcholinesterase inhibition properties. To the best of our knowledge, there has been no study on the in vivo effects of this plant. Therefore, the purpose of this study was to evaluate the effects of C. niveum on lead (Pb)-acetate-induced potential alterations in brain acetylcholinesterase activity, as well as oxidative stress in male rats. The rats were randomly assigned to control, Pb-acetate, C. niveum and Pb-acetate+ C. niveum groups. Pb-acetate was provided in drinking water (500 ppm), and C. niveum was administered via orogastric gavage (4 ml/kg) for 30 days. The acetylcholinesterase activity in the brain significantly decreased only in the Pb-acetate group. The malondialdehyde level significantly increased, and the reduced glutathione activity decreased in the Pb-acetate group. The reduced glutathione and glutathione-S-transferase activities of the C. niveum group were higher than the control group. No Pb was detected on a ppb level in the brain tissue of the control and C. niveum groups, while it was detected in the brains of the rats in the Pb-acetate and Pb-acetate+ C. niveum groups (185+8.98 ppb and 206+56.65 ppb, respectively). The data collected in this study suggested that C. niveum may reduce inhibition of brain AChE activity and oxidative stress against Pb-acetate-induced alterations in the brain of male rats.

Inhibitory properties of some heavy metals on carbonic anhydrase I and II isozymes activities purified from Van Lake fish (Chalcalburnus Tarichi) gill.

In this study, CA I and II isoenzymes were purified from Van Lake fish gills by using Sepharose-4B-L-tyrosine-sulfanilamide affinity chromatography and to determine the effects of some metals on the enzyme activities. For purified CA I isoenzyme, yield, specific activity, and purification fold were obtained as 42.07%, 4948.12 EU/mg protein, and 116.61 and for CA II isoenzyme, 7%, 1798.56 EU/mg protein, and 42.38 respectively. Activity of CA was determined by measuring "CO2-hydratase activity". Purity control was checked by SDS-PAGE. In vitro inhibitory effect of Cu2+, Ag+, Cd2+, Ni2+ metal ions, and arsenic (V) oxide were also examined for both isozymes activities. Whereas Cu2+, Ag+, Cd2+, and Ni2+ ions showed inhibitory effects on both isozymes, arsenic (V) oxide showed activation effect. IC50 values were calculated by drawing activity %-[I] graphs for metal ions exhibiting inhibitory effects. IC50 values were determined as 3.39, 6.38, 13.52, and 206 µM for CA I isozyme and 6.16, 20.29, 46, and 223 µM for CA II isozyme respectively.

Role of geraniol against lead acetate-mediated hepatic damage and their interaction with liver carboxylesterase activity in rats.

In this study, the effect of geraniol (50 mg/kg for 30 d), a natural antioxidant and repellent/antifeedant monoterpene, in a rat model of lead acetate-induced (500 ppm for 30 d) liver damage was evaluated. Hepatic malondialdehyde increased in the lead acetate group. Reduced glutathione unchanged, but glutathione S-transferase, glutathione reductase, as well as carboxylesterase activities decreased in geraniol, lead acetate and geraniol + lead acetate groups. 8-OhDG immunoreactivity, mononuclear cell infiltrations and hepatic lead concentration were lower in the geraniol + lead acetate group than the lead acetate group. Serum aspartate aminotransferase and alanine aminotransferase activities increased in the Pb acetate group. In conclusion, lead acetate causes oxidative and toxic damage in the liver and this effect can reduce with geraniol treatment. However, we first observed that lead acetate, as well as geraniol, can affect liver carboxylesterase activity.

Pyruvate kinase activators as a therapy target: a patent review 2011-2017.

INTRODUCTION: It is well known that cancer cells have an altered metabolism both to meet the energy needs and to provide initial molecules for the synthesis of macromolecules. To cope with the new metabolic state, different forms of certain enzymes are expressed in extreme amounts. These enzymes are seen as very attractive targets to deal with cancer. Pyruvate kinases isoenzyme M2 (PKM2) is a key enzyme that determines whether glucose is used for energy or synthesis of biosynthetic molecules. The dimeric form of PKM2 main form in several cancer cells serves the formation of synthetic precursors required for the cell growth and proliferation from glycolytic intermediates. AREAS COVERED: This article reviews appropriate publications on PKM2 activators from the points of view of synthesis and biological activities between 2011-2017. Herein, based on the chemical structure, PKM2 activators are classified into sulfonamide, phenolic, carboxamide and pyridopyrimidinone derivatives. EXPERT OPINION: PKM2 activation inhibits cell growth and proliferation by decreasing a number of biomolecules required for cell building. Therefore; PKM2 activators are considered as an ideal drug for or the treatment of many cancer pathogens. It is necessary to discover new, more active and selective compounds for PKM2 activation.

Compounds from Terminalia mantaly L. (Combretaceae) Stem Bark Exhibit Potent Inhibition against Some Pathogenic Yeasts and Enzymes of Metabolic Significance.

Background: Pathogenic yeasts resistance to current drugs emphasizes the need for new, safe, and cost-effective drugs. Also, new inhibitors are needed to control the effects of enzymes that are implicated in metabolic dysfunctions such as cancer, obesity, and epilepsy. Methods: The anti-yeast extract from Terminalia mantaly (Combretaceae) was fractionated and the structures of the isolated compounds established by means of spectroscopic analysis and comparison with literature data. Activity was assessed against Candida albicans, C. parapsilosis and C. krusei using the microdilution method, and against four enzymes of metabolic significance: glucose-6-phosphate dehydrogenase, human erythrocyte carbonic anhydrase I and II, and glutathione S-transferase. Results: Seven compounds, 3,3'-di-O-methylellagic acid 4'-O-α-rhamnopyranoside; 3-O-methylellagic acid; arjungenin or 2,3,19,23-tetrahydroxyolean-12-en-28-oïc acid; arjunglucoside or 2,3,19,23-tetrahydroxyolean-12-en-28-oïc acid glucopyranoside; 2α,3α,24-trihydroxyolean-11,13(18)-dien-28-oïc acid; stigmasterol; and stigmasterol 3-O-ß-d-glucopyranoside were isolated from the extract. Among those, 3,3'-di-O-methylellagic acid 4'-O-α-rhamnopyranoside, 3-O-methylellagic acid, and arjunglucoside showed anti-yeast activity comparable to that of reference fluconazole with minimal inhibitory concentrations (MIC) below 32 µg/mL. Besides, Arjunglucoside potently inhibited the tested enzymes with 50% inhibitory concentrations (IC50) below 4 µM and inhibitory constant (Ki) <3 µM. Conclusions: The results achieved indicate that further SAR studies will likely identify potent hit derivatives that should subsequently enter the drug development pipeline.

In Vivo Effects of Naringenin and Lead on Rat Erythrocyte Carbonic Anhydrase Enzyme.

OBJECTIVES: Carbonic anhydrase (CA) enzyme catalyses the reversible reactions of CO2 with water and takes part in metabolically important events such as systemic acid-base regulation and respiration. In this study, in vivo effects of lead, which is a heavy metal and to which living beings are exposed by different ways, with naringenin, a flavanone, were investigated. MATERIALS AND METHODS: For this purpose, four different rat groups were established and one of them was chosen as the control group. The other three groups were given lead, naringenin and lead+naringenin substances to analyze the changes in the CA enzyme of rat erythrocytes. RESULTS: The research findings showed that the enzyme activity in the control group was higher than that in the other groups. The naringenin group showed the highest inhibition effect, while the lead group showed the lowest inhibition. CONCLUSION: Therefore, it can be said that naringenin is a strong inhibitor of the CA enzyme.

Purification of glucose-6-phosphate dehydrogenase and glutathione reductase enzymes from the gill tissue of Lake Van fish and analyzing the effects of some chalcone derivatives on enzyme activities.

Glucose-6-phosphate dehydrogenase (G6PD) and glutathione reductase (GR) are metabolically quite important enzymes. Within this study, these two enzymes were purified for the first time from the gills of Lake Van fish. In the purifying process, ammonium sulfate precipitation and 2',5'-ADP Sepharose 4B affinity column chromatography techniques for glucose-6-phosphate dehydrogenase, temperature degradation and 2',5'-ADP Sepharose 4B affinity column chromatography for glutathione reductase enzyme were used. The control of the enzyme purity and determination of molecular weight were done with sodium dodecyl sulfate polyacrylamide gel electrophoresis. K(M) and V(max) values were determined with Lineweaver-Burk plot. Besides, the effects of some chalcone derivatives on the purified enzymes were analyzed. For the ones showing inhibition effect, % activity-[I] figures were drawn and IC50 values were determined. K(i) value was calculated by using Cheng-Prusoff equation.

Inhibitory and Activating Effects of Some Flavonoid Derivatives on Human Pyruvate Kinase Isoenzyme M2.

Pyruvate kinase isoenzyme M2 (PKM2) is expressed excessively in many different cancer types and it plays an important role in the control of glucose metabolism. Thus, it is evaluated as an important target in the development of medication for cancer. The flavonoids comprise a large group of natural products with variable phenolic structures and occur mainly in plants. They are of great interest due to their biological properties. In this study, the effects of various flavonoid derivatives on the PKM2 enzyme activity were analyzed in vitro. The flavonoid derivatives 1 and 2 showed inhibition effect with IC50 values of <60 µM. IC50 values of compounds 3-8 were calculated as 134, 415, 145, 163, 295 µM, and 3.5 mM, respectively. The molecules 9-12 showed an activation effect with values of AC50 of less than 90 µM. The IC50 values of the derivatives 13-17 were calculated as 115, 150, 200, 221, and 275 µM, respectively. The results show that catechin derivatives can be probably used as lead compounds for the design of PKM2 enzyme activators and inhibitors.

Characterization and Purification of Glutathione S-Transferase from the Liver and Gill Tissues of Agri Balik Lake Trout Salmo trutta labrax and the Effects of Heavy Metal Ions on Its Activity.

Glutathione S-transferase (GST) from the liver and gill tissues of Agri Balik Lake Trout (also known as Black Sea Trout) Salmo trutta labrax was characterized and purified, and the toxic effects of some heavy metal ions on the enzyme's activity were analyzed. Liver GST was purified 930 times, resulting in 56% yield using glutathione-agarose affinity chromatography and a specific activity of 60.87 endotoxin units (EU)/mg protein. Using the same procedure, gill GST was purified 576 times, resulting in a 60% yield and specific activity of 46.8 EU/mg protein. The purity check of the purified enzymes was determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Optimal pH, ionic strength, and stable pH were found for each tissue, and separate KM and Vmax values were determined for reduced glutathione and 2,4-dinitrochlorobenzene substrates. Heavy metal ions that have toxic effects on living organisms and are known to contribute to environmental pollution were selected, and their in vitro effects on enzyme activity were analyzed. The IC50 values and Ki constants of those metal ions showing an inhibitory effect on GST activity were determined.

Examination of changes in enzyme activities of erythrocyte glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in rats given Naringenin and Lead acetate.

In our study, controlled experimental groups were performed by giving substances Lead acetate, Naringenin and Naringenin + Lead acetate to rats in vivo conditions Changes in the glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) enzyme activities in erythrocytes of rats in these groups were compared to the Control group. An inhibition significant degree for G6PD enzyme activity was observed in all groups when compared to the Control group (p < 0.001). While inhibition significant degree for 6PGD enzyme activity was observed in Lead acetate groups (p < 0.001), no significant effect was observed in the Naringenin and Naringenin + Lead acetate groups (p > 0.05). In addition, lead levels in the groups of rats were determined using an inductively coupled plasma mass spectrometer (ICP-MS) device. As a result of measurements by the ICP-MS device, lead levels were found as an average of 42.9 ± 2.51, 36.71 ± 1.13, 172.16 ± 9.63, and 95.07 ± 5.87 ppm in the Control, Naringenin, Lead acetate and Naringenin + Lead acetate groups, respectively. Our results were shown that Naringenin has protective effects on the Lead acetate induced oxidative stress erythrocytes in rat.

Purification and characterization of glucose 6-phosphate dehydrogenase enzyme from rainbow trout (Oncorhynchus mykiss) liver and investigation of the effects of some metal ions on enzyme activity.

Glucose 6-phosphate dehydrogenase (d-glucose 6-phosphate: NADP(+) oxidoreductase, EC 1.1.1.49; G6PD) is a key enzyme that is localized in all mammal tissues, especially in cytoplasmic sections and that catalyzes the first step of pentose phosphate metabolic pathway. In this study, G6PD enzyme was purified 1444-fold with a yield of 77% from rainbow trout liver using 2',5'-ADP-sepharose-4B affinity chromatography. Moreover, a purity check of the enzyme was performed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Some characteristic features like optimal pH, stable pH, optimal temperature and optimal ionic strength were determined for the purified enzyme. In addition to this, in vitro effects of ions like silver nitrate (Ag(+)), thallium sulphate (TI(+)), cobalt (II) nitrate (Co(2+)) and arsenic (V) oxide (As(5+)) on enzyme activity were researched. Half-maximal inhibitory concentration (IC50) values of Ag(+), Co(2+) and As(5+) metal ions, which showed an inhibitory effect, were found to be 0.0044, 0.084 and 4.058 mM, respectively; and their inhibition constants (K i) were found to be 0.0052 ± 0.00042, 0.087 ± 0.015700 and 4.833 ± 1.753207 mM, respectively. Tl(+) not exhibited inhibitory effect on the enzyme activity.

Investigation of the effects of some phenolic compounds on the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase from human erythrocytes.

Polyphenols are the important compounds that have various bioactivities. They constitute vital active agents of not only daily diet but also natural medicines that are used traditionally. It is generally considered that they are safe because they are natural. In some conducted studies, different negative effects of these compounds were mentioned. Twelve phenolic compounds have been assayed to determine the effect of inhibition on glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) enzymes activity. For in vitro studies, the enzymes were purified from human erythrocytes using 2',5'-ADP Sepharose 4B affinity chromatography. Naringenin, caffeic acid, ellagic acid, ferulic acid, and sinapic acid against two enzymes, hesperidin and polydatin, only on G6PD activity and chrysin solely against 6PGD showed inhibitory effect. Chlorogenic acid, p-coumaric acid, and syringic acid did not exhibit an effect on the activity of the two enzymes.

Purification and characterization of carbonic anhydrase from sheep kidney and effects of sulfonamides on enzyme activity.

Carbonic anhydrase (CA, EC: 4.2.1.1) was purified from sheep kidney by affinity chromatography on a Sepharose 4B-tyrosine-sulfanilamide column. By means of two consecutive procedures, the enzyme (sCA) was purified 227.61-fold with a yield of 60.75%, and a specific activity of 838.89U/mg proteins. The optimum temperature, ionic strength and pH were determined to be 35°C, 20mM and 8.5, respectively. The molecular weight determined by SDS-PAGE was found to be 29kDa. The kinetic parameters, KM and Vmax values were determined for the 4-nitrophenyl acetate (p-NpA) hydrolysis reaction. Some sulfonamides were tested as inhibitors against the purified CAs enzyme. The Ki constants for benzenesulfonamide (1), sulfanilamide (2), mafenide (3), 4-(2-aminoethyl) benzenesulfonamide (4), 4-methyl-benzenesulfonamide (5), 2-bromo-benzenesulfonamide (6), naphthalene-2-sulfonamide (7), 4-amino-6-chlorobenzene-1,3-disulfonamide (8) and saccharin (9) were in the range 1.348-69.31µM.

Effects of some metal ions on rainbow trout erythrocytes glutathione S-transferase enzyme: an in vitro study.

Glutathione S-transferase enzyme (GST) (EC 2.5.1.18) was purified from rainbow trout erythrocytes, and some characteristics of the enzyme and effects of some metal ions on enzyme activity were investigated. For this purpose, erythrocyte glutathione S-transferase enzyme which has 16.54 EU/mg protein specific activities was purified 11,026-fold by glutathione-agarose affinity chromatography with a yield of 59%. Temperature was kept under control (+4°C) during purification. Enzyme purification was checked by performing SDS-PAGE. Optimal pH, stable pH, optimal temperature, and K(M) and Vmax values for GSH and 1-chloro-2, 4-dinitrobenzene (CDNB) were also determined for the enzyme. In addition, IC50 values, Ki constants and the type of inhibition were determined by means of Line-Weaver-Burk graphs obtained for such inhibitors as Ag(+); Cd(2+), Cr(2+) and Mg(2+).

An analysis of expression patterns of genes encoding proteins with catalytic activities.

BACKGROUND: In situ hybridization (ISH) is a powerful method for visualizing gene expression patterns at the organismal level with cellular resolution. When automated, it is capable of determining the expression of a large number of genes. RESULTS: The expression patterns of 662 genes that encode enzymes were determined by ISH in the mid-gestation mouse embryo, a stage that models the complexity of the adult organism. Forty-five percent of transcripts encoding metabolic enzymes (n = 297) showed a regional expression pattern. A similar percentage was found for the 190 kinases that were also analyzed. Many mRNAs encoding glycolytic and TCA cycle enzymes exhibited a characteristic expression pattern. The annotated expression patterns were deposited on the Genepaint database and are retrievable by user-defined queries including gene name and sites of expression. CONCLUSION: The 662 expression patterns discussed here comprised gene products with activities associated with catalysis. Preliminary analysis of these data revealed that a significant number of genes encoding housekeeping functions such as biosynthesis and catabolism were expressed regionally, so they could be used as tissue-specific gene markers. We found no difference in tissue specificity between mRNAs encoding housekeeping functions and those encoding components of signal transduction pathways, as exemplified by the kinases.