Enzyme Inhibition Method Research Articles

The potential of Endophytic Fungal Extract Isolated from Cinnamon (Cinnamomum burmannii) as Antidiabetic and Antioxidant

An increase in blood glucose levels in people with diabetes can cause an increase in free radicals, which can worsen the disease. Thus, drugs that have antidiabetic and antioxidant activities are needed. The cinnamon plant is high in antioxidants and has long been used as a source for a diabetes drug. The utilization of endophytic fungi isolated from cinnamon plants as antidiabetic and antioxidant has never been reported. This study aims to investigate the antidiabetic as well as antioxidant activity from the extract of endophytic fungi from the cinnamon plant. The antidiabetic activity was tested using the α-glucosidase enzyme inhibition method, while antioxidant activity was tested using the DPPH free radical scavenging method. Total phenol content was measured based on the Follin-Ciocalteu reagent reaction. All endophytic fungal extracts from the cinnamon leaves, twigs, flowers, and fruit have antidiabetic and antioxidant activity as well as high total phenol content. The three parameters measured showed a positive correlation. Endophytic fungal extract of Cb.D6 isolate derived from the leaf had the highest antidiabetic and antioxidant activity among the other isolates amounting to 92.41% and 90.28%, respectively. In addition, the total phenol content of Cb.D6 isolates was also the highest with 357.83 mg equivalent to gallic acid/g extract. Therefore, the endophytic fungal extract of Cb.D6 isolate has the potential to be developed as a source of the antidiabetic and antioxidant ingredients.

Physiopathology of multiple organ dysfunctions in severely monocrotophos-poisoned rabbits

ObjectiveAcute organophosphorus pesticides poisoning has a serious threat on people's health. This study aimed to investigate the pathogenesis and molecular mechanism of multiple organ dysfunction syndrome (MODS) in severely monocrotophos-poisoned rabbits. MethodsChinchilla rabbits were used to build the monocrotophos-poisoned animal model via subcutaneous abdominal injection. Acetylcholinesterase activity was determined using the dithiobisnitrobenzoic acid enzyme kinetics method, and the free organophosphorus (FOP) toxic substances content was analyzed using the enzyme inhibition method. The contents of tumor necrosis factor (TNF-α), interleukin 1-β (IL-β) and thromboxane B2 (TXB2) in the plasma and tissue homogenates were determined via radioimmunoassay. ResultsTwenty-four hours after exposure, in comparison to the plasma, blood cells and homogenates of various tissues, the bile had a significantly different FOP content (P < 0.05). In different phases, HE staining results confirmed that several degrees of pathological lesions (such as hemorrhage, edema, degeneration and necrosis) were detected in FOP poisoned rabbits. The TXB2 and TNF contents in plasma were significantly higher than those of the control (P < 0.05). Except for the intercostal muscle, all of the tissues had significantly higher TXB2 contents than the control. The TNF contents of the liver and lung and the IL-1β contents of the liver and kidney were significantly higher than those of the control (P < 0.05). ConclusionFOP stored in the gallbladder may play important role in enterohepatic circulation. In MODS rabbits, caused by OP poisoning, the TXB2 and TNF-α may play important role in inflammatory response and complement and coagulation systems respectively.

Effective improvement in performance of a miniature FIA-calorimetric biosensing system via denoising column addition and flow rate optimization

The noise reduction and heat-response improvement of a miniature FIA-calorimetric biosensing system were evaluated for the determination of enzyme activity. An innovative denoising column was designed in combination with the use of a mathematical moving average for effectively decreasing signal noise from 30mV to 0.2mV. Additionally, various flow rates were tested for a comprehensive analysis of the influence of the flow rate on the heat response of this system. As the flow rate rose in the 0.1–0.5mL/min range, the temperature sensitivity of the calorimetric system increased linearly from 0.8mV/K to 1.184mV/K, showing an improvement of 48%. However, the exothermic peak integral reached the highest point at the flow rate of 0.3mL/min, when the system was used to detect the heat release of hydrolysis reaction catalysed by chicken liver esterase. Thus, the flow rate of 0.4mL/min was finally employed in this system. The biosensor was successfully applied for the detection of malathion, where 0.01mg/L malathion was detected significantly. Thus, this biosensor could potentially be used for the determinations of inhibitors based on enzyme inhibition methods.

Quantitative assessment of intestinal first-pass metabolism of oral drugs using portal-vein cannulated rats.

To evaluate the impact of intestinal first-pass metabolism (Fg) by cytochrome P4503A (CYP3A) and uridine 5'-diphosphate-glucuronosyltransferases (UGT) on in vivo oral absorption of their substrate drugs. CYP3A and UGT substrates were orally administered to portal-vein cannulated (PV) rats to evaluate their intestinal availability (Fa · Fg). In the case of CYP3A substrates, vehicle or 1-aminobenzotriazole (ABT), a potent inhibitor of CYP enzymes, was pretreated to assess Fg separately from Fa (Enzyme-inhibition method). On the other hand, since potent inhibitors of UGT have not been identified, Fg of UGT substrate was calculated from total amount of metabolites generated in enterocytes (Metabolite-distribution method). After oral administration of CYP3A substrates in ABT-pretreated rats, the portal and systemic plasma concentrations of the metabolite were nearly the same, indicating almost complete inhibition of intestinal CYP3A-mediated metabolism. Using Enzyme-inhibition method, Fg of midazolam (1 mg/kg) was calculated as 0.71. Additionally, total amount of raloxifene-6-glucuronide generated in enterocytes after oral administration of raloxifene was estimated using Metabolite-distribution method and Fg of raloxifene (0.98 μmol/kg) was calculated as 0.21. PV rats enabled in vivo quantitative assessment of intestinal first-pass metabolism by CYP3A and UGT. This method is useful for clarifying the cause of low bioavailability.

Mobilized lipase enzymatic biosensor for the determination of Chlorfenvinphos and Malathion in contaminated water samples: A voltammetric study

Concentration levels of organophosphorus pesticides like Chlorfenvinphos and Malathion were determined by lipase enzyme inhibition method. The developed enzymatic method was purely based on in situ generation of p-Nitrophenol by enzymatic hydrolysis of p-Nitrophenyl Acetate. The production of this in situ generated p-Nitrophenol concentration was depending on the effective hydrolysis of p-Nitrophenyl Acetate by the lipase enzyme. This electrochemically active p-Nitrophenol gave an anodic oxidation peak at a potential of +0.024V versus SCE. The various parameters such as effective hydrolysis of substrate, substrate concentration and effect of pH were studied for lipase enzyme. A linear calibration for the Chlorfenvinphos and Malathion was obtained in the concentration ranges of 100–900μM and 100–900mM respectively with a correlation coefficient of 0.9894 and 0.9680 under the optimized conditions by following the incubation time of 25min. The electrochemical experiments were performed in 0.1M phosphate buffer solution (pH7.0) at room temperature. The limit of detection and limit of quantification values were found to be 84.45μM, 253.03mM and 281.5μM, 843.1mM respectively for Chlorfenvinphos and Malathion under optimized conditions. The developed liquid state lipase enzyme sensor could be applied for the analysis of water samples contaminated with pesticides. The developed method gives the information about the intensity of toxicity of pesticides.

Biosensor based on atemoya peroxidase immobilised on modified nanoclay for glyphosate biomonitoring

A biosensor based on atemoya peroxidase immobilised on modified nanoclay was developed for the determination of glyphosate by the enzyme inhibition method. The inhibitor effect of the biocide results in a decrease in the current response of the hydroquinone that was used as a phenolic substrate to obtain the base signal. The biosensor was constructed using graphite powder, multiwalled carbon nanotubes, peroxidase immobilised on nanoclay and mineral oil. Square-wave voltammetry was utilised for the optimisation and application of the biosensor, and several parameters were investigated to determine the optimum experimental conditions. The best performance was obtained using a 0.1molL−1 phosphate buffer solution (pH 7.0), 1.9×10−4molL−1 hydrogen peroxide, a frequency of 30Hz, a pulse amplitude of 50mV and a scan increment of 4mV. The glyphosate concentration response was linear between 0.10 and 4.55mgL−1 with a detection limit of 30μgL−1. The average recovery of glyphosate from spiked water samples ranged from 94.9 to 108.9%. The biosensor remained stable for a period of eight weeks.

Determination of organophosphorus pesticide residues in vegetables by an enzyme inhibition method using α-naphthyl acetate esterase extracted from wheat flour

The widespread use of organophosphorus pesticides (OPs) poses a great threat to human health and has made the detection of OP residues in food an important task, especially in view of the fact that easy and rapid detection methods are needed. Because OPs have inhibitory effects on the activity of α-naphthyl acetate esterase (ANAE) in plants, in this work we evaluated the possibility of detecting OPs in vegetables with ANAE extracted from commercial flour. The limits of detection (LODs) obtained for methamidophos, dichlorvos, phoxim, dimethoate, and malathion in lettuce samples with crude ANAE were 0.17, 0.11, 0.11, 0.96, and 1.70 mg/kg, respectively. Based on the maximum residue limits (MRLs) for OPs in food stipulated by Chinese laws which are 0.05, 0.20, 0.05, 1.00, and 8.00 mg/kg for methamidophos, dichlorvos, phoxim, dimethoate, and malathion, respectively, the esterase inhibition method with crude ANAE had sufficient sensitivity to detect the residues of dichlorvos, dimethoate, and malathion in lettuce, but it could not be used to guarantee the safety of the same samples if methamidophos or phoxim residue was present. The sensitivity of the method was improved by the use of esterase purified by ammonium sulfate salting-out. The LODs obtained for methamidophos and phoxim with purified esterase were lower than the MRLs for these OPs in food. This is a very promising method for the detection of OP residues in vegetables using crude or purified esterase because of its cheapness, sensitivity, and convenience.

효소 저해법을 이용한 Carbamate계 농약의 다성분 잔류분석법 개발

This study was carried out with the detection for multiresidue of the carbamate pesticide such as carbaryl and cabofuran by enzyme-inhibition method. The check time for determination of acetylcholinesterase(AChE) activity was selected at 60 sec. The AChE activity in chicken brain determined by the Ellman's method was <TEX>$162{\mu}$</TEX>mol/min/g protein. <TEX>$I_{50}$</TEX> for AChE by carbamate pesticide with wet kit was 0.169mg/L of carbaryl and 0.089mg/L of cabofuran, respectively. The incubation time for enzyme kit with substrate kit was 30min for determination of AChE activity. Enzyme kit with substrate kit was stable at <TEX>$4^{\circ}C\;and\;25^{\circ}C$</TEX> for 5 days. Limit detection concentration of carbaryl with dry kit for AChE was 0.05mg/L. The dry kit such as wet kit applied Enzyme-Inhibition(EI) method with AChE was confirmed the multi residue method to detect the carbamate pesticides.

Preparation and application of streptavidin magnetic particles

Two kinds of streptavidin magnetic particles, namely streptavidin GoldMag particles and streptavidin amino terminal particles were prepared by the methods of physical adsorption and covalent interaction respectively. The streptavidin coated on magnetic particle surface, crucial to many applications, was greatly influenced by the choice of the different buffer. Compared with Dynalbeads®M-270 streptavidin, the binding capacity for biotin of different streptavidin magnetic particles was determined by enzyme inhibition method, and the coupling capacity and activity of biotinylated oligonucleotide on their surface were also analyzed. The results indicated that the streptavidin GoldMag particle prepared by physical adsorption was stable in STE (NaCl-Tris-EDTA) buffer that was frequently used in nucleic acid hybridization and detection. The streptavidin amino terminal particles prepared by covalent interaction could be used both in STE buffer and PBS (phosphate buffered saline) buffer. The biotin binding capacity for 1 mg of streptavidin GoldMag particles and streptavidin amino terminal particles was 4950 and 5115 pmol respectively. The capacity of biotinylated oligonucleotide (24 bp) coupled on 1 mg of GoldMag and amino terminal magnetic particles was 2839 and 2978 pmol separately. These data were about 6–7 times higher than those of Dynabeads®M-270 streptavidin. The hybridization results with FITC-labeled complementary probe on magnetic particle surface demonstrated that the oligonucleotide coupled on streptavidin magnetic particles had high biological activity.

Enzyme Inhibition and Enzyme-Linked Immunosorbent Assay Methods for Carbamate Pesticide Residue Analysis in Fresh Produce

An acetylcholinesterase inhibition method was employed for detection of 21 carbamate pesticides in bananas, peaches, strawberries, and tomatoes. Each of these four agricultural commodities was spiked with 0.1 to 10 ppm of each of the 21 carbamates and individual detection levels were determined. Similar responses and detection limits were observed for all four produce when tested for a given carbamate. The detection levels ranged from 0.1 ppm for carbofuran and 3-hydroxycarbofuran to 6 ppm for promecarb and aldicarb sulfoxide. These results are generally at or below the tolerances established by the Environmental Protection Agency for these commodities. Positive samples from the enzyme inhibition screening were also analyzed with the carbaryl-specific enzyme-linked immunosorbent assay (ELISA) kit. The detection limits for carbaryl and carbofuran were 2.0 ppb and 8.0 ppb, respectively. The other carbamates did not exhibit cross-reactivity even at high ppb levels. Thus, the enzyme inhibition assay and ELISA are simple and fast screening procedures for the detection of carbamate pesticide residues in food commodities.

Further immunological study on the phylogenetic relationships among sea-urchins of the order echinoida

1. 1. In a previous study, I examined immunologically the phylogenetic relationships among 10 sea-urchin species belonging to the four families of the order Echinoida by the enzyme inhibition method with anti- Strongylocentrotus intermedius G6PD antibody (Matsuoka, 1980). The results suggested the close affinity between the two families, Strongylocentrotidae and Toxopneustidae of this order. 2. 2. In order to extend the previous immunological study, the immunological cross-reactions of the antibody to G6PDs from nine sea-urchin species collected thereafter were also examined by the enzyme inhibition method. 3. 3. The further immunological results thus obtained confirmed the suggestion that the family Strongylocentrotidae might be closely related to the family Toxopneustidae.

A semiautomated procedure for urinary D-glucaric acid using a centrifugal analyzer.

The most commonly used method for quantifying D-glucaric acid, the enzyme inhibition method, is a two-step procedure. In the first step of the present study, urine was boiled at acidic pH to convert glucaric acid to 1,4-glucarolactone, which inhibits beta-glucuronidase activity. In the second step, inhibition of beta-glucuronidase by the prepared urine was measured. This is an indirect measure of the D-glucaric acid concentration. The second step was adapted to a Cobas-Bio centrifugal analyzer. Values on a reference population obtained by the manual method were highly correlated with values obtained by the semiautomated method described here (r = 0.976, p = 0.25), and precision was comparable by the two methods. The throughput increased from 8 to 20 samples per day when the automated method was used.

Constant rate of evolution in the antigenicity of glucose 6-phosphate dehydrogenase revealed by the enzyme inhibition method

1. 1. Rabbit and chicken antisera against pure and impure rat glucose 6-phosphate dehydrogenase (G6PD) samples were prepared, and their inhibition potencies (RIP) were measured against G6PDs from 26 vertebrate and invertebrate species. 2. 2. The results demonstrated that the — logRIP values of antisera against G6PDs from various test species neatly correlate with paleontologically estimated divergence times between rat and the test species. This suggests that the antigenicity of G6PD has evolved at a constant rate. 3. 3. The correlation coefficients were evidently greater with chicken antisera than with rabbit antisera, but did not markedly differ between chicken antisera elicited to pure and impure rat G6PD samples. 4. 4. Based on the data obtained with chicken antisera, it is estimated that lineages which separated 250–300 million years ago differ in their G6PDs by one unit of the — logRIP.

Sensitive and rapid detection of paraoxon by thin-layer chromatography and strips using enzyme inhibition and Ellman's method

Sensitive and rapid detection of paraoxon by thin-layer chromatography and strips using enzyme inhibition and Ellman's method

Enzyme immunoassay and enzyme inhibition assay of methotrexate, with use of the centrifugal analyzer.

Methotrexate was determined by the homogeneous enzyme immunoassay (EMIT) with the Multistat and CentrifiChem centrifugal analyzers and by the enzyme inhibition assay with use of the Multistat centrifugal analyzer. With both methods, the standard curve extends from 0.2 to 2.0 mumol/L and there is no interference from bilirubin in concentrations up to 100 mg/L. Moderately lipemic samples do not interfere with the EMIT method, but lower the values obtained with the enzyme inhibition assay. Hemoglobin concentrations as great as 1 g/L do not affect results for methotrexate obtained by the enzyme inhibition assay. With the EMIT assay, methotrexate values are lowered in samples containing hemoglobin in concentrations exceeding 750 mg/L. With the EMIT assay, the following compounds in concentrations of 1 mmol/L do not interfere: leucovorin, 5-methyl-tetrahydrofolate, 5-fluorouracil, and 6-mercaptopurine. When folic acid (100 mumol/L) was added to a serum that did not contain methotrexate, a response equivalent to 0.15 mumol/L was obtained. Methotrexate is stable for five days in serum stored at 23, 4, or -20 degrees C. Within-run precision (CV) for the enzyme inhibition method ranged from 4.7 to 8.1% and for the EMIT assay from 2.5 to 6.2%. Methotrexate concentrations in the serum of children receiving high-dose therapy were compared by three methods: competitive protein binding, EMIT, and enzyme inhibition assays. The correlation coefficients averaged 0.95.

Enzymatic detection of carbamate pesticides on impregnated cellulose plates

The detection of carbamate pesticides by an enzyme-inhibition method on impregnated (ethylene glycol, triethylene glycol, beta,beta'-oxydipropionitrile) cellulose thin layer plates is described. The enzyme-substrate combination: pig liver esterase-indophenylacetate (chromogenic substrate) was the most sensitive. The following parameters concerning the sensitivity of the method were investigated: enzyme source, substrate, pH, inhibition time, inhibition temperature, thickness of the cellulose layer, stationary phase.

Serum α1, antitrypsin deficiency: a family study

Twenty members of a family, in which α1, antitrypsin deficiency is associated with both liver and lung disease, have been studied. Three individuals were shown to be Pi-type ZZ. One of these, the propositus, is a 7-yr-old girl who has had evidence of liver damage from 9 days of age. Her α1, antitrypsin value is 80 mg/100 ml (normal: 200–400). The remaining two ZZ members are both adults, with emphysema and normal liver function tests. Their α1, antitrypsin values are 40 and 55 mg/100 ml. Good correlation (r = 0.901) is obtained between the serum α1 antitrypsin levels determined by immunochemical and by enzyme inhibition methods. Either result was equally useful for differentiating the homozygous ab-normals from both the heterozygous (carrier) and the normal population. Three other, unrelated cases of slow-to-resolve childhood jaundice have been shown to be α1 antitrypsin deficient of the ZZ type.

Propanil Hydrolysis : Inhibition in Rice Plants by Diazinon and Carbaryl Translocated from the Soil

Abstract Greenhouse and laboratory experiments were designed to determine the effect of soil-applied carbaryl and diazinon on the phytotoxicity of foliage-sprayed propanil. A time-course study of the activity of propanil-hydrolyzing enzyme in rice indicated a slow increase in the rate of hydrolysis during early stages of growth, followed by a sharp increase for extracts of the 7-week-old samples. The rate of hydrolysis reached its maximum at harvest time (approximately 17 weeks). The enzyme activity of barnyard grass was not detected at different growth stages. The enzyme was inhibited by diazinon and strongly inhibited by carbaryl absorbed and translocated from the soil over a wide range of growth stages of rice. Rice plants were more susceptible at early stages of growth and this confirms the significant correlation between growth stage and enzyme activities. Analyses of tissue extracts by paper chromatography and an enzyme inhibition method agreed closely at all intervals. It seems probable that the inhibition rate of enzyme is related to the persistence and rate of translocation of carbaryl.

Gas-liquid and thin-layer chromatography of phorate, disulfoton and five of their oxidation products

Phorate, phorate sulfoxide, phorate sulfone, phoratoxon, phoratoxon sulfoxide, phoratoxon sulfone, disulfoton, disulfoton sulfoxide, disulfoton sulfone, disulfoton oxygen analog, disulfoton oxygen analog sulfoxide and disulfoton oxygen analog sulfone were resolved by gas-liquid chromatography with temperature programming on a column containing Chromosorb W AW-DMCS coated with 5% stabilized DEGS. The limit of detection of the organophosphorus compounds after thin-layer chromatography was determined for eleven detection methods. An enzyme inhibition method with 5-bromoindoxyl acetate as substrate was found to be the most sensitive method. Bromocresol Green-AgNO 3 reagent detected all the compounds at the sub-microgram level. Sixteen mobile phases and six sorbents were compared for the TLC resolution of the organophosphorus compounds. The mobile phase methanol-benzene (10:90) with the sorbent MN-Kieselgel G-HR gave the best resolution of phorate, disulfoton and their oxidation products.