The extracts were prepared using different solvents: methanol for aerial parts of the plant and a mixture of ethanol and water (90:10) for roots. The multi-targets integrated fingerprinting for screening anti-diabetic compounds from a Chinese medicine Jinqi Jiangtang Tablet. Acarbose, a positive control, was used as an inhibitor for this experiment. Different concentrations of acarbose (0.5 to 12 g mL-1) were added to the enzymatic solution, and an enzyme assay was performed as mentioned above. The reagent has a dark orange colour and is stable for several days in room temperature. J Clin Chem Clin Biochem. It was described that t he activity of -amylase was detected by the DNS method in the legend of Fig 3d. Different parameters, such as enzyme concentration, substrate concentration (starch), preincubation time, incubation time, incubation temperature, and time in the water bath, were evaluated. FOIA Clipboard, Search History, and several other advanced features are temporarily unavailable. Schematic illustration of the workflow proposed for the determination of the glucose yield when pure starches were submitted to the action of amyloglucosidase. The variability test is conducted at three response levels with varied signal intensities.1515 Markossian, S.; Coussens, N. P.: Dahlin, J. L.; Sittampalam, G. S.; SLAS Discovery 2021, 26, 1241. Amylase Test. Validation of the method was performed according to international guidelines by analyzing linearity, precision, accuracy, robustness, plate uniformity, and signal variability.1414 Food and Drug Administration (USA); Bioanalytical method validation guidance for industry, US Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research and Center for Veterinary Medicine, 2018, available at https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf, accessed June 2022.https://www.fda.gov/files/drugs/publishe Master degree dissertation. Salvador, Bahia, Brazil, Tallapragada P, Dikshit R, Jadhav A, Sarah U (2017) Partial purification and characterization of amylase enzyme under solid state fermentation from Monascus sanguineus. Amber glass vials (15mm45mm8mm, e.g. This extract was the closest to the acarbose positive control. 5 ml of deionized water are added in each sample, followed by agitation. This method is based on the reaction of maltose with glucose oxidase (GOD) and the development of a red quinone. [Crossref]. R. P.; Mol. Acta Part A-Mol. official website and that any information you provide is encrypted [Crossref]. The -amylase assay was performed using Millers method, i.e., the DNS method. Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N.; de Sales, P. M.; de Souza, P. M.; Dartora, M.; Resck, I. S.; Simeoni, L. A.; Fonseca-Bazzo, Y. M.; de Oliveira-Magalhes P.; Silveira, D.; Moein, S.; Pimoradloo, E.; Moein, M.; Vessal, M.; Rahimzadeh, M.; Jahanshahi, S.; Moein, S.; Moein, M. R.; Valentina, P.; Ilango, K.; Chander, S.; Murugesan, S.; Paiva, R. V. N.; Chaves, A. C. T. A.; de Paula, V. F.; de Macedo, G. E. L.; Queiroz, R. F.; Yilmazer-Musa, M.; Griffith, A. M.; Michels, A. J.; Schneider, E.; Frei, B.; Szymaski, P.; Markowicz, M.; Mikiciuk-Olasik, E.; Markossian, S.; Coussens, N. P.: Dahlin, J. L.; Sittampalam, G. S.; Granados-Guzmn, G.; Castro-Ros, R.; de Torres, N. W.; Salazar-Aranda, R.; Satpathy, G.; Tyagi, Y. K.; Gupta, R. K.; Hou, W.; Li, Y.; Zhang, Q.; Wei, X.; Peng, A.; Chen, L.; Wei, Y.; Subramanian, R.; Asmawi, M. Z; Sadikun, A.; Wu, T.; Zhou, X.; Deng, Y.; Jing, Q.; Li, M.; Yuan, L.; Ha, K. S.; Jo, S. H.; Kang, B. H.; Apostolidis, E.; Lee, M. S.; Jang, H. D.; Kwon, Y. I.; Wang, Y.; Huang, S.; Shao, S.; Qian, L.; Xu, P.; Vadivel, V.; Nandety, A.; y Biesalski, H. K.; Tundis, R.; Loizzo, M. R.; Statti, G. A.; Menichini, F.; Iversen, P.; Beck, B.; Chen, Y.; Dere, W.; Devanarayan, V.; Eastwood, B. J.; Farmen, M. W.; Iturria, S. J.; Montrose, C.; Moore, R. A.; Weidner, J. R.; Sittampalam, G. S. In. Enzyme immobilization Preparation of alginate beads Alginate gel beads were prepared according to Awad et al. Epub 2018 Sep 19. Chem. The Pareto diagram (Figure 1) shows that some factors exhibit a significant effect on the response. Evolution of sensitivity (represented by the slope of the calibration curve) along time for the starch-iodine assay. Get this protocol in PDF format. Pol. The https:// ensures that you are connecting to the Int. HHS Vulnerability Disclosure, Help Place the vial in the block heater (or other alternative heating source) previously set at the target temperature of the study during the desired time of the assay (, Immediately stop the hydrolysis process by adding 750L of 1.0molL, Prepare the soluble starch standards as described in, Transfer 50L of the mixture (sample or standard), 150L of water, and 50L KI/I, Place the microplate in the plate reader, shake the plate (for 30s using the shaker function of the microplate reader) and read the absorbance at 580nm, at room temperature. The positive control used was acarbose (0.6 g mL-1). [Crossref], 4 Rahimzadeh, M.; Jahanshahi, S.; Moein, S.; Moein, M. R.; Iran. 2017, 109, 962. Glucose yield (average values from two determinations) measured by the described glucose oxidase/peroxidase assay that resulted from the hydrolysis of four different pure starches mediated by an amyloglucosidase from Aspergillus niger (PN: A9913, Sigma Aldrich). Standard deviation was calculated, and the relative standard deviation in terms of percentage was determined (Equation 2) to check the precision values on the experimentation day and other different days. 8600 Rockville Pike In the case of using a particular starch as a substrate, this factor should be studied in detail and the method should be adjusted accordingly. A total of 64 experiments were performed. In this case, the control experiment was used to monitor any glucose formation unrelated to the enzyme activity. Add slowly 30.0 gms sodium potassium tartrate tetrahydrate. Epub 2019 Aug 2. Amylase is also used in brewing and baking, as it helps to convert starch into sugar. The https:// ensures that you are connecting to the Plant extracts contain various phytochemicals, such as phenols, saponins, and alkaloids, that contribute to many observed pharmacologically relevant effects. It was experimentally established that the minimum concentration of acarbose that can be determined is 1 g mL-1. 17 Granados-Guzmn, G.; Castro-Ros, R.; de Torres, N. W.; Salazar-Aranda, R.; Curr. Screening 1999, 4, 67. phenol). The absorbance of the well was monitored at 505nm, and the value recorded was 0.681. 2, Fig. Later, the mixture was cooled, and 100 L of this solution was transferred to a 96-well plate and diluted with 100 L of water. The precision of the method was analyzed on the day of experimentation and on two different days. document.getElementById( "ak_js_1" ).setAttribute( "value", ( new Date() ).getTime() ); Assay of Salivary Amylase enzyme activity. Standard solutions of maltose (0-10 moles/l) are prepared in test tubes. Unit of Microbiology, Department of Biology, University of Patras, http://www.microbiology.biology.upatras.gr. J Genet Eng Biotechnol 15:8794, Zhao Y, Zhao Y, Zhang Z, Wei Y, Wang H, Lu Q, Li Y, Wei Z (2017) Effect of thermo-tolerant actinomycetes inoculation on cellulose degradation and the formation of humic substances during composting. 1% Starch. Rev. Bookshelf There are numerous methods used for the determination of amylase activity, of which 3,5-dinitrosalicylic acid (DNSA) assay is the most widely used. Those with normal plasma glucose had group I, those with impaired glucose tolerance had group II, and those who were diabetic had group III. The use of a microscale method offers several advantages such as 1) fewer chemicals needed, 2) reduction in material cost, 3) ease of disposal of the reaction mixture after experimentation, 4) reduction of the safety risk, and 5) multiple experiments can be performed at the same time. Cool and dilute with 10ml of distilled water. HCl 1.0molL1: add 8.8mL of HCl 37% (w/w) solution to 80mL of water, mix well, let cool down to room temperature and add water to a final volume of 100mL; keep at room temperature. Therefore, the objectives of this work were: (i) the consistent preparation of some reagents, especially the soluble starch and KI / I2 solution that generates I3, to improve batch to batch reproducibility and maximize the precision and accuracy of the of the starch-iodine method; (ii) the rescale of the activity assay, in order to minimize evaporation issues typical from microplate assays and to adapt it to different experimental requirements (in our particular case this was related with the US-assisted hydrolysis of starch); (iii) the adjustment of sensitivity by modifying the volumes of the starch-iodine microplate assay, to avoid precipitation and to minimize potential deviations to the Lamberts-Beer law of the absorbance measurements; and (iv) the measurement of glucose yield for amyloglucosidase through the glucose oxidase / peroxidase (GOPOD) method replicating the sample handling protocol used for measuring its activity, but using pure starches from different botanical sources instead of soluble starch. 12 Stevenson, R. A.; American Laboratory, 2011, available at https://www.americanlaboratory.com/913-Technical-Articles/19109-Bioassays-2011/, accessed June 2022. Dissolve 0.2M (2.7218 grams) of KH2PO4 in 100ml of distilled water to this solution add 0.5M (2.8053 grams) KOH drop by drop till the pH is set to 6.8. Hugo M. Oliveira and Margarida R. G. Maia thanks Fundao para a Cincia e a Tecnologia (FCT) for the post-doctoral grant SFRH/BPD/75065/2010 and SFRH/BPD/70176/2010, respectively. Universidade Federal da Bahia. Because acarbose is structurally similar to the substrate (starch), it competes for the active site on the enzyme. Hence, we kept the same volume ratio of the original method but we increased the assay to a total volume of 3.00mL according to our requirements, which were defined by the specifications of the US probe used to study the effect of US in the activity of amylolytic enzymes [7,8]. 0.5 ml of properly diluted (in acetic acid buffer solution; pH=4.9) crude enzyme are incubated for 15 min at T=40 C with 0.5 ml of soluble starch solution 1 % w/v. Analysis of results using the Tukey test ( = 0.05) indicated that the method is robust for two conditions, i.e., incubation temperature and preincubation time. This methodological approach can be used for measuring the activity of both alpha-amylase and amyloglucosidase [5]. Based upon the plasma glucose value, all subjects were divided into three groups. Visvanathan R, Jayathilake C, Liyanage R, Sivakanesan R. Food Chem. Maltoheptaoside hydrolysis with chromatographic detection and starch hydrolysis with reducing sugar analysis: Comparison of assays allows assessment of the roles of direct -amylase inhibition and starch complexation. Warm the assay buffer to room temperature before use. Unfortunately, to date, many published studies cannot be reproduced; most of them are nonreplicable or simply erroneous. Enzymatic reaction and determination of the enzymatic activity. One of the treatment methods used for postprandial hyperglycemia includes using potential therapeutic agents to inhibit -amylase activity. Then, we have partially purified these amylases employing three conventional methods: (a) cold acetone precipitation, (b) ion-exchange chromatography, and (c) gel filtration chromatography. The analytical protocol can also be conducted in mild conditions (room temperature), bypassing the heating steps and the protocol complexity that is present in the 2018, 14, 458. In this experiment, acarbose at a concentration of 2.5 g mL-1 was used to record the desired mean or mid signal. In this study, the IC50 was determined following the method described by Tundis et al., and an IC50 value of 0.6 g mL-1 was obtained.2626 Tundis, R.; Loizzo, M. R.; Statti, G. A.; Menichini, F.; Pharmazie 2007, 62, 473. In addition, the assay method could be applied to a wide variety of molecules, and the obtained results should be reproducible and achieve the same results every time.1010 White, R. E.; Annu. These solutions were added to an extract of J. dioica possessing zero inhibitory activity. When the enzymatic activity decreases, the formation of the product also decreases; therefore, the intensity of the color is reduced. Furthermore, all experimental conditions can be adapted according to the equipment available at each laboratory, transforming this method in a broadband analytical tool for screening alpha-amylase and amyloglucosidase activities. Without salivary amylase, we would not be able to digest carbohydrates properly and would not get the total nutritional value from these foods. Toxicol. Regarding reagents, we describe in detail the preparation of the solutions of soluble starch and triiodide (from the KI / I2 solution), which are key elements for the starch-iodine method. 6 This method, like the iodine procedure only measures ~-amylase activity and the results cannot be readily expressed in international units. Studies can not be readily expressed in international units glucose formation unrelated to the enzyme activity alpha-amylase! At a concentration of 2.5 g mL-1 was used as an inhibitor for experiment! Et al starch into sugar ; Castro-Ros, R. ; Iran like the iodine procedure only measures ~-amylase and. ; Jahanshahi, S. ; Moein, M. ; Jahanshahi, S. ; Moein, ;! ( Figure 1 ) shows that some factors exhibit a significant effect on the day of experimentation and two. 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