Method For Determination Of Activity Research Articles

Methods for Determination of Antimicrobial Activity of Essential Oils In Vitro-A Review.

Essential oils (EOs) have been gaining popularity in the past decades among researchers due to their potential to replace conventional chemicals used in the fight against pests, pathogenic and spoilage microbes, and oxidation processes. EOs are complex mixtures with many chemical components, the content of which depends on many factors-not just the plant genus, species, or subspecies, but also chemotype, locality, climatic conditions, phase of vegetation, method of extraction, and others. Due to this fact, there is still much to study, with antimicrobial effect being one of the key properties of EOs. There are many methods that have been frequently used by researchers for in vitro evaluation; however, although the research has been going on for decades, an internationally accepted standard is still missing. Most of methods are based on time-proven standards used for the testing of antibiotics. Due to the specific properties of EOs and their components, such as volatility and hydrophobicity, many modifications of these standard procedures have been adopted. The aim of this review is to describe the most common methods and their modifications for the testing of antimicrobial properties of EOs and to point out the most controversial variables which can potentially affect results of the assays.

Optimizing Polyphenol Content and Extraction Methods for Antioxidant Constituents from <i>Portulaca oleracea</i>: Comparing Reflux and Maceration Methods with Various Solvents

Background: The pursuit of developing effective drugs as antioxidants can be traced back to herbal ingredients, including purslane (Portulaca oleracea). The potential of purslane as a medicinal herb can be maximized by selecting the most suitable extraction method and type of solvent. Objective: To assess the levels of total phenolic compounds, total flavonoids, and radical scavenging capacity in P. oleracea obtained through various extraction methods and solvents. Methods: This study combines maceration and reflux methods with 96% ethanol, 80% ethanol, 96% methanol, and 80% methanol solvents to extract total phenolics, flavonoids, and radical scavenging activity from purslane. The folin-ciocalteu method was employed for measuring phenolic content, the AlCl3 method for flavonoid content, and the DPPH method for radical scavenging activity determination. Results: The reflux method using 80% methanol produced the highest total phenolic content, 5.15 ± 0.07 mg GAE/g DW. The maceration method using the same solvent yielded the highest total flavonoid content, 5.74 ± 0.29 mg QE/g DW. Both extraction methods showed similar radical scavenging activity, 1.10 ± 0.003 mg AAE/g DW for maceration and 1.07 ± 0.04 mg AAE/g DW for reflux. Conclusion: The extraction method and solvent significantly impact the total phenolic and flavonoid content produced by P. oleracea.

Qualitative and quantitative protease activity tests based on protein degradation in three-dimensional structures

The pattern of the activity of proteases is related to distinct physiological states of living organisms. Often activity changes of a certain protease can be assigned to a specific disease. Hence, they are useful biomarkers and a simple and fast determination method of their activity could be a valuable tool for the efficient monitoring of numerous diseases. Here, two different methods for the qualitative and quantitative determination of protease activity are demonstrated using the model system of proteinase K. The first test system is based on a protein-modified and colored 3D silica structure that changes color when exposed to the enzyme. This method has also been used for the detection of matrix metallo-protease 2 (MMP2) with gelatine as protease substrate on the plates. The second detection system uses the decrease in the voltammetric signal of a cytochrome c/DNA multilayer electrode after incubation with a protease to quantitatively determine its proteolytic activity. While activities down to 0.15 U/ml can be detected with the first method, the second one provides detection limits of about 0.03U/ml (for proteinase K.) The functionality of both systems can be demonstrated and ways for further enhancement of sensitivity have been elucidated.

PROPOSAL OF THE METHOD FOR QUANTITATIVE DETERMINATION OF PROTEOLYTIC ACTIVITY OF A PANCREATIN REFERENCE STANDARD

Relevance. Pancreatin is a polyenzyme drug with lipolytic, amylolytic and proteolytic activity, and is prescribed as a replacement therapy for exocrine pancreatic insufficiency. Modern requirements for quality control of drugs involve the use of highly specific and highly sensitive methods of analysis using reference standards (RS).For RS certification it is necessary to establish the values of the certifiable characteristic. For enzyme preparations the certifiable characteristic is catalytic activity. In this connection for certification of pancreatin RS it is necessary to establish the value of its catalytic ac-tivity.The analysis of the proteolytic activity of pancreatin consists in performing a proteolysis reaction and quantifying the reaction products. The most common methods used for this purpose are those in which the quantitative content of proteolysis products is determined by measuring the optical density of the solution using tyrosine as a standard. It should be noted that the value of proteolytic activity established in this way is relative, since the measured optical density actually corresponds to a mixture of tyrosine and tryptophan. The absolute method for determining proteolytic activity is the Kjeldahl method for analyzing fragmented peptide nitrogen. Aim. Propose the method for quantitative determination of the proteolytic activity of a pancreatin RS. Material and methods. The object of the study was a pharmaceutical substance pancreatin. Quantitative determination of proteolytic activity in pancreatin substance samples was performed according to the proposed method by determining protein nitrogen by the Kjeldahl method after the en-zymatic reaction. Results. As a result of testing the samples of pancreatin substance using the proposed method of quantitative analysis of proteolytic activity, we ob-tained reliable results, indicating the high sensitivity, accuracy and reliability of this method. Conclusions. The present study proposed the method for quantitative determination of the proteolytic activity of pancreatin substance and evaluated the acceptability of its use in the quality control of a pancreatin RS. Conflict of interest. The authors declare that they have no obvious and potential conflicts of interest related to the publication of this article.

Green-emitting carbon dots as a "turn on" fluorescence bio-probe for highly sensitive and selective detection of lipase in human serum.

Enzyme activity assays play a crucial role in numerous fields, including biotechnology, the food industry, and clinical diagnostics. Lipases are particularly important enzymes due to their widespread use in lipid metabolism and esterification reactions. Here, we present a pioneering method for the sensitive and selective determination of lipase activity using green carbon dots (G-CDs) for first time. G-CDs are a fascinating class of carbon nanomaterials with unique optical properties and biocompatibility, making them ideal candidates for enzyme activity assays. This approach eliminates the need for traditional fluorophores or chromogenic substrates, reducing costs, fast response time (1 min), and environmental impact with a quantum yield (QY) of 7.42%. As designed, the G-CDs fluorescent probe turn-on demonstrated a reliable linear detection range from 0 to 9 mg/mL under ideal conditions, with detection limit of 0.01 mg/mL and limit of quantification (LOQ) of 0.045 mg/mL, respectively. Furthermore, the G-CDs system was thoroughly evaluated in human serum samples, showing recoveries ranging from 100.0 to 105.0%. These findings highlight the promising applicability of the G-CDs probe for lipase detection, yielding highly favorable results.

Antioxidant Status in the Blood of Psychosocially Stressed Rats Treated with Honey

Abstract Linden honey represents a unique honey variety valued for its nutritional benefits, distinctive taste and aroma. Phenols, polyphenols, flavonoids, ascorbic acid and phenolic acids in honey have antioxidant activities. This study aimed to investigate the effects of linden honey on the activities of antioxidant enzymes catalase (CAT) and glutathione peroxidase (GPx), as well as on the concentration of malondialdehyde (MDA) in individually housed animals. The investigated parameters were quantified using spectrophotometric method for determination of enzyme activities and MDA concentration in the blood. We found that treatment with linden honey in the socially isolated animals significantly increased the enzyme activities of CAT and GPx, and significantly decreased the concentration of MDA. The modulation of CAT and GPx activities in socially isolated animals treated with linden honey may be very important for understanding the role of honey in the capacity of antioxidant defense system to increase and maintain its stability in psychosocial stress conditions. Our results may be important in biomedical research for understanding the role of honey in the amelioration of oxidative stress.

Methods for Determination of Antimicrobial Activity of Bacteriocins of Lactic Acid Bacteria

Methods for Determination of Antimicrobial Activity of Bacteriocins of Lactic Acid Bacteria

Validation of a newly adopted chemical method for determination of 226Ra and 228Ra activities in water and shrimp samples

Validation of a newly adopted chemical method for determination of 226Ra and 228Ra activities in water and shrimp samples

Simple and rapid colorimetric method for determination of erythrocyte and plasma cholinesterase activities and comparison with the standard Ellman’s method

Simple and rapid colorimetric method for determination of erythrocyte and plasma cholinesterase activities and comparison with the standard Ellman’s method

Modification and Validation of a Laboratory Method for Determination of L-Asparaginase Activity in Patient Blood Serum

Modification and Validation of a Laboratory Method for Determination of L-Asparaginase Activity in Patient Blood Serum

Mid-infrared spectroscopy coupled with chemometrics for quantitative determination of biomaterial activity

In view of the complicated operation and time-consuming of traditional detection methods for biomaterial activity, a method based on the mid-infrared (MIR) spectroscopy coupled with chemometrics methods was proposed to achieve the non-destructive and rapid determination of the biomaterial activity. The spectral preprocessing methods were applied to reduce the noise and scattering effects in the spectral analysis, which can effectively improve the performance of biomaterial activity determination model. The characteristic wavelengths of the preprocessed spectrum were extracted by the successive projections algorithm (SPA). And the determination performance of different regression algorithms for biomaterial activity was compared and discussed. Results demonstrated that the model of SPA combined with partial least squares regression (PLSR), which exhibited better potential for the determination of biomaterial activity with higher coefficient of determination (R2) and lower root mean square error of prediction (RMSEP). The R2 acquired by the optimal model for three biomaterials are 0.9622, 0.9949, and 0.9936, respectively. And the RMSEP were 0.0611, 0.0186, and 0.0193, respectively. Therefore, the SPA-PLSR established by MIR is expected to replace the traditional methods for quantitative determination of biomaterial activity.

ALPHA-AMYLASE AND METHODS FOR DETERMINATION OF ITS ACTIVITY USED AT CLINICAL LABORATORY

The quality improvement of medical care requires the developing of clinical laboratory patient support. To achieve the effective results need a searching, developing and applying in practice new, with improved properties, methods for estimation analytes, including common ones, such as α-amylase activity. This article presents a review of the main materials published over the past few decades. It includes a description of the structure of α-amylase and its main isoenzymes; its role in human physiology and pathology; diagnostic significance of the α-amylase isoenzymes activity determination in clinical laboratory diagnostics. The principles of the most common methods of α-amylase activity determination are presented, and their important characteristics are compared. The method based on the substrate GalG2CNP using has the best analytical characteristics for the developing reagent kits for measuring the total α-amylase and its pancreatic isoenzyme activities, which can be used in clinical diagnostic laboratories.

A Combined Study on Optimization, In Silico Modeling, and Genetic Modification of Large Scale Microbial Cellulase Production.

Cellulase is a biocatalyst that hydrolyzes cellulosic biomass and is considered a major group of industrial enzymes for its applications. Extensive work has been done on microbial cellulase but fungi are considered a novel strain for their maximum cellulase production. Production cost and novel microbial strains are major challenges for its improvement where cheap agro wastes can be essential sources of cellulose as substrates. The researcher searches for more cellulolytic microbes from natural sources but the production level of isolated strains is comparatively low. So genetic modification or mutation can be employed for large-scale cellulase production before optimization. After genetic modification than in silico molecular modeling can be evaluated for substrate molecule's binding affinity. In this review, we focus not only on the conventional methods of cellulase production but also on modern biotechnological approaches applied to cellulase production by a sequential study on common cellulase-producing microbes, modified microbes, culture media, carbon sources, substrate pretreatment process, and the importance of optimum pH and temperature on fermentation. In this review, we also compare different cellulase activity determination methods. As a result, this review provides insights into the interrelationship between the characteristics of optimizing different culture conditions, genetic modification, and in silico enzyme modeling for the production of cellulase enzymes, which may aid in the advancement of large-scale integrated enzyme manufacturing of substrate-specific enzymes.

Assay for ADAMTS-13 Activity with Flow Cytometric Readout.

A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13) is a metalloprotease that regulates the size of circulating von Willebrand factor (vWF) multimers. Severe lack of ADAMTS-13 activity [<10% of normal (0.1 IU/mL)] leads to thrombotic thrombocytopenic purpura (TTP), a specific type of thrombotic microangiopathy (TMA). Timely determination of plasma ADAMTS-13 activity is essential to discriminate TTP from other types of TMA with respect to adequate treatment. Identification of the minimal substrate motif for ADAMTS-13 within the A2 domain of vWF (vWF73) as well as the generation of monoclonal antibodies (mAbs) that specifically recognize the ADAMTS-13 cleavage site enabled the development of a variety of methods for determination of plasma ADAMTS-13 activity. In order to further extend the range of analytical platforms applicable for quantitative determination of plasma ADAMTS-13 activity, a specific, vWF/mAb-based assay with flow cytometric readout was developed and validated. Basic assay characteristics include a total assay time of 80 to 90 min, a near linear dynamic range from 0.005 (lower limit of quantification) to 0.2 IU/mL, and intra- and interassay coefficients of variation below 5 and 30% at input plasma ADAMTS-13 activities of 0.015 and ≤0.050 IU/mL, respectively. When compared to the results obtained with a commercially available quantitative ADAMTS-13 activity ELISA, analysis of 18 plasma samples obtained from patients with suspected TTP revealed full agreement of results with respect to the clinical 0.1 IU/mL TTP threshold. Based on these data, it is assumed that the described assay principle can be successfully transferred to virtually all laboratories that have a flow cytometer available.

Rapid, real-time sucrase characterization: Showcasing the feasibility of a one-pot activity assay

Sucrases can modify numerous carbohydrates, and short-chain oligosaccharides produced by the unique transfructosylation activity of levansucrases are promising candidates for the growing sugar substitute market. These compounds could counteract the increasing number of diseases associated with the consumption of high-calorie sugars. Thus, there is great interest in the characterization of novel levansucrases. The commonly used method for sucrase activity determination is to quantify d-glucose released in the sucrose-splitting reaction. This is usually done in a discontinuous mode, i.e., several samples taken from the sucrase reaction are applied to a separately performed d-glucose determination (e.g., GOPOD assay). Employing the newly isolated levansucrase LevSKK21 from Pseudomonas sp. KK21, the feasibility of a one-pot sucrase characterization was investigated by combining sucrase reaction and GOPOD-based d-glucose determination into a single, continuous assay (Real-time GOPOD). The enzyme was characterized with respect to kinetic parameters, ion dependency, pH value, and reaction temperature in a comparative approach employing Real-time GOPOD and HPLC. High data consistency for all investigated enzyme parameters demonstrated that current processes for sucrase characterization can be considerably accelerated by the continuous assay while maintaining data validity. However, the assay was not applicable at acidic pH, as decolorization of the quinoneimine dye formed during the GOPOD reaction was observed. Overall, the study presents valuable data on the potentials of real-time sucrase activity assessment for an accelerated discovery and characterization of interesting enzymes such as the hereby introduced levansucrase LevSKK21. Progress in sucrase discovery will finally foster the development of health-promoting sucrose substitutes.

Fast Electrochemical Measurement of Laccase Activity for Monitoring Grapes’ Infection with Botrytis cinerea

Grapes’ infection with the fungi Botrytis cinerea is one of the major causes of economic loss in the winemaking sector worldwide. The laccase activity of grapes is considered an appropriate indicator of this type of fungal infection, and enzymatic activity higher than 3 U/mL indicates a high risk of irreversibly damaged grape must due to enzymatic browning. This work describes a fast test for the measurement of laccase activity based on a dual optical and electrochemical detection method. A paper sensor impregnated with the enzymatic substrate dye 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) provides a semi-quantitative optical measurement. While the paper sensor can be used independently, when combined with a screen-printed electrode and amperometry measurements, it enables the quantitative detection of laccase activities down to 0.4 U/mL in only 5 min. The method was applied for monitoring the artificial infection of white, rosé, and red grapes with different strains of Botrytis cinerea. The results were confirmed by parallel analysis using the spectrophotometric method of laccase activity determination based on syringaldazine. The influence of the fungal strain and type of grape on laccase activity levels is reported. The demonstrated robustness, simplicity, and versatility of the developed method make it ideal for application on-site in the vineyard or at grape processing points.

Biological activities and in vitro digestion characteristics of glycosylated α-lactalbumin prepared by microwave heating: Impacts of ultrasonication

The research focuses on application of ultrasonic-assisted glycosylation in food protein modification. Biological activities and in vitro digestion characteristics of the glycosylated α-lactalbumin (UMGLA) and α-lactalbumin (UMLA) prepared by ultrasonic pretreatment and microwave heating at 85 °C for 2 h were investigated mainly through the browning degree, free amino group, reducing sugar and surface hydrophobicity, in vitro digestibility and degree of hydrolysis, total antioxidant capacity, α-glucosidase inhibitory activity, xanthine oxidase inhibitory activity determination methods. The results showed that ultrasonic pretreatment could effectively promote the occurrence of glycosylation reaction, reduce in vitro digestibility and degree of hydrolysis of glycosylated α-lactalbumin and enhance biological activities of glycosylated α-lactalbumin digestive products. Furthermore, after 60 min of ultrasound treatment, the total antioxidant capacity and α-glucosidase inhibitory activity of glycosylated α-lactalbumin digested products were increased by 76.52 ± 0.02% and 12.42 ± 0.04%, respectively, compared with α-lactalbumin without ultrasound treatment. In conclusion, ultrasonic-assisted glycosylation had positive effects on improving the biological activities of in vitro digestion product of α-lactalbumin. It provides a theoretical basis for ultrasonic-assisted glycosylation in the modification field of food proteins.

Assay for Protealysin-like Protease Inhibitor Activity.

Here, we present the first quantitative method for the activity analysis of protealysin-like protease (PLP) inhibitors. This approach is based on a previously developed method for protealysin activity determination by hydrolysis of internally quenched fluorescent peptide substrate 2-aminobenzoyl-L-arginyl-L-seryl-L-valyl-L-isoleucyl-L-(ε-2,4-dinitrophenyl)lysine. In this protocol, we significantly reduced enzyme concentration and introduced some minor modifications to decrease variation between replicates. The protocol was validated using emfourin, a novel proteinaceous metalloprotease inhibitor. Data obtained demonstrates that the developed assay method is an affordable approach for characterizing and screening various PLP inhibitors. Graphical abstract.

Electrochemical Analysis of Metabolites as a Method for Cytochromes P450 Activity Determination

The review deals with the electrochemical methods for determination of metabolites of cytochromes P450 catalyzed reactions. We have focused on the electrochemical determination of metabolites of drugs and some endogenous compounds. We have reviewed bielectrode systems for determination of cytochrome P450 activity, where one electrode serves as a matrix for enzyme immobilization and a source of electrons for heme iron ion reduction and initialization of the catalytic reaction towards a substrate and the second one is being used for quantification of the products formed by their electrochemical oxidation. Such systems allow one to elude additional steps of separation of reaction substrates and products. The review also includes discussion of the ways to increase the analytical sensitivity and decrease the limit of detection of the investigated metabolites by chemical modification of electrodes. We demonstrate the possibilities of these systems for cytochrome P450 kinetics analysis and the perspectives of their further improvement, such as increasing the sensitivity of metabolite electrochemical determination by modern electrode modificators, including carbon-based, and construction of devices for automatic monitoring of the products.

Research Progress of Nattokinase Quality and Safety Evaluation

Nattokinase is a serine protease produced by Bacillus natto in the process of fermenting natto. It has physiological functions such as dissolving blood clots. This article briefly summarizes the thrombolytic function and mechanism of nattokinase, focusing on the research progress of nattokinase activity determination methods and other quality indicators and safety evaluation. The research direction of nattokinase product development and quality and safety is prospected, in order to provide a theoretical basis for the standardization of natto and its products (including nattokinase products).