This article providing information on Salivary amylase, functions, and Assay of Salivary amylase activity. Toxicol. [Crossref]16 United States Pharmacopeial Convention; The United States pharmacopeia: the national formulary; Rockville, Md., United States pharmacopeial convention, 1980.-1717 Granados-Guzmn, G.; Castro-Ros, R.; de Torres, N. W.; Salazar-Aranda, R.; Curr. Therefore, the control assay should provide glucose yields of 0%. Additionally, the significant factors were optimized using the rules of the basic sequential simplex method and the combination of conditions that maximized the inhibition percentage with the lowest photometric error. To each tube, 100 L of plant extracts with different concentrations (final concentration 6 g mL-1 to 200 g mL-1) and 100 L of -amylase (final concentration 0.15 U mL-1 in phosphate buffer) were added and incubated at 37 C for 7.2 min. Enzyme immobilization Preparation of alginate beads Alginate gel beads were prepared according to Awad et al. grown on orange waste powder. The first approach relies on the measurement of reducing sugars, being the dinitrosalicylic acid (DNS) method [3] the classic approach. This site needs JavaScript to work properly. A total of 64 experiments were performed. Mina Karimi-Avargani kindly mail me saidurga48@gmail.com. I need your help for calculation of data. The authors are also grateful for the financial support of the Coordenao de Aperfeioamento de Pessoal de Nvel Superior (CAPES, Brazil) under grant number 451408/2019-01 and the Fundao de Amparo Pesquisa do Estado da Bahia (FAPESB, Brazil) under grant number RED0023/2014. The most commonly used method to measure -amylase in wheat grain is the Hagburg Falling Number method but the values are also influenced by rheological properties of starch in the grain.1,2 The most specific method for the measurement of -amylase is the Ceralpha method which employs a defined substrate, 4,6-O-benzylidene--4-nitrophenyl- In general, they have a poor design of experiments and an inadequate statistical significance.1111 Finak, G.; Gottardo, R.; In Data Mining Techniques for the Life Sciences; Carugo, O., Eisenhaber, F., eds. 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]. Warm the assay buffer to room temperature before use. Three different concentrations (1, 2.5, and 5 g mL 11 Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N.; J. Evidence-Based Complementary Altern. The enzyme concentration, incubation time, interaction between enzyme concentration and preincubation time, and the interaction between preincubation and boiling time showed a significant effect on the inhibition percentage. (function(d){var s=d.createElement("script");s.setAttribute("data-account","s8XDZXWqQY");s.setAttribute("src","https://cdn.userway.org/widget.js");(d.body||d.head).appendChild(s);})(document)Please ensure Javascript is enabled for purposes of website accessibility. official website and that any information you provide is encrypted <>stream This was complemented by a glucose yield assay for amyloglucosidase that allowed a secondary source of information when insoluble starches were studied. If no amylase activity is present in the saliva, how many mg of starch will be detected in the first time point of the assay? [Crossref]-1616 United States Pharmacopeial Convention; The United States pharmacopeia: the national formulary; Rockville, Md., United States pharmacopeial convention, 1980.,2727 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 HTS Assay Validation; Markossian, S.; Grossman, A.; Brimacombe, K.; Arkin, M.; Auld, D.; Austin, C. P.; Baell, J.; Chung, T. D. Y.; Coussens, N. P.; Dahlin, J. L.; Devanarayan, V.; Foley, T. L.; Glicksman, M.; Hall, M. D.; Haas, J. V.; Hoare, S. R. J.; Inglese, J.; Iversen, P. W.; Kales, S. C.; Lal-Nag, M.; Li, Z.; McGee, J.; McManus, O.; Riss, T.; Saradjian, P.; Sittampalam, G. S.; Tarselli, M.; Trask, O. J.; Wang, Y.; Weidner, J. R.; Wildey, M. J.; Wilson, K.; Xia, M.; Xu, X., eds. Glucose oxidase / peroxidase (GOPOD) (K-GLUC) assay kit was purchased from Megazyme (Wicklow, Ireland). Please note that if you reject them, you may not be able to use all the functionalities of the site. Additionally, the optimized assay was applied to extracts from five different plants. Maltose It is defined as the combinatorial effect of all components of the analysis other than the analyte that is to be measured.2828 McNaught, A. D.; Wilkinson, A.; Compendium of Chemical Terminology (IUPAC Chemical Data), 2nd ed., Wiley: New Jersey, 1997. By intercepting this absorbance values in the calibration curve (e.g. This is a preview of subscription content, access via your institution. Ten different experiments were performed. a starch solution has also been used to measure a-amylase activity. The extract of A. lebbeck at a concentration of 1000g/ endobj The linear range of amylase inhibition by acarbose was 1.5-5 g mL-1, with a regression equation of y = 7.5784x + 37.80, a determination coefficient of 0.9906, and an IC50 of 1.61 0.06 g mL-1 (Figure 3). Regarding the potential for structural modification of starch that may affect enzyme hydrolysis and microbial contamination during storage, the users should perform control experiments to check potential problems. 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. 28 McNaught, A. D.; Wilkinson, A.; Compendium of Chemical Terminology (IUPAC Chemical Data), 2nd ed., Wiley: New Jersey, 1997. for potential alternative wavelengths). Rev. 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. Sodium azide (1.0g) can be added as a preservative (antimicrobial agent). For the proposed method, the minimum detectable amount of starch (LOD) was 0.4mg. An example of the application of this assay is illustrated by Fig. It comprises two chains of amino acids, which are held together by weak bonds. [Crossref]. Learn more Salivary amylase is an enzyme secreted in the saliva of humans and other mammals. Dexmedetomidine alleviates insulin resistance in hepatocytes by reducing endoplasmic reticulum stress. A test can be done to measure the level of this enzyme in your blood. Note: amylolytic enzyme working solutions should be prepared fresh daily and kept on ice at all times. The enzymatic reaction was stopped by adding 200 L of DNS (final concentration 19.2 mmol L-1) and subsequently placed in a boiling water bath for 15.6 min. Most enzymes act specifically with only one reactant, called a substrate, to produce products. Pharmacol. 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 DNS method, as well in other alternative methods for the same analytes [6]. When compared with the original method, these new conditions lead to an extra dilution of the sample (a dilution factor of 5 compared to a dilution factor of 2) and to a longer optical path due to the higher volume placed at each well of the microplate (250L vs. 150L, and assuming the use of a classic cylindrical-shaped flat bottom microplate). 26 Tundis, R.; Loizzo, M. R.; Statti, G. A.; Menichini, F.; Pharmazie 2007, 62, 473. Amylasedigestsstarchbycatalyzinghydrolysis,whichissplittingbytheadditionofawatermolecule. The evaluation of the matrix effect showed linearity at 1 5 g mL 11 Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N.; J. Evidence-Based Complementary Altern. Recommendations and criteria presented by various international guidelines were followed to validate the optimized media.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 Bashkin A, Ghanim M, Abu-Farich B, Rayan M, Miari R, Srouji S, Rayan A, Falah M. Molecules. Method. J. Mol. 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. This method is based on the reaction of maltose with glucose oxidase (GOD) and the development of a red quinone. This discrepancy highlights the reason for not comparing the results obtained by different reports, although they might have used the same method but with different experimental conditions. Measurement of alpha-amylase activity in white wheat flour, milled malt, and microbial enzyme preparations, using the Ceralpha assay: collaborative study. Google Scholar, Mounaimen O, Mahmoud K (2015) Statistical optimization of cultural conditions of an halophilic alpha-amylase production by halophilic Streptomyces sp. Before This method is based on the binding of iodine to terminals of the starchs polymeric chain [4] that results in a blue colored complex that can be also quantitatively monitored by UVvis spectrophotometry [5]. -Amylase is an endoglycosidase that catalyzes the cleavage of internal -D-(1-4) glycosidic bonds in starch to produce oligosaccharides, dextrins, and maltose.44 Rahimzadeh, M.; Jahanshahi, S.; Moein, S.; Moein, M. R.; Iran. and a-amylase activity. This is the first report of this plant showing -amylase inhibition activity. government site. Work with a partner. Epub 2020 Oct 26. One milligram of each extract was weighed and dissolved in 1 mL of phosphate buffer (100 mmol L-1, pH 6.9); some solutions were sonicated to achieve complete dissolution. Because acarbose is structurally similar to the substrate (starch), it competes for the active site on the enzyme. Disclaimer, National Library of Medicine For enzyme inhibition studies, the absorbance was recorded at a lower concentration of inhibitor (IC50). Biochem. Gamma amylase is found in the small intestine and completes the hydrolysis of maltose into glucose. Hence, it is absolutely necessary to optimize an assay method to achieve acceptable performance, with low variability and high signal with respect to noise.1313 Macarrn, R.; Hertzberg. Med 2011, Article ID 810207. 1959, 31, 426. 1 ml of D.N.S. Unfortunately, to date, many published studies cannot be reproduced; most of them are nonreplicable or simply erroneous. Biomol. It then breaks down carbohydrates in the food we eat into simpler sugars. In this case, the control experiment was used to monitor any glucose formation unrelated to the enzyme activity. Pharmacol. Process Biochem 50:21282136, Nithya K, Muthukumar C, Biswas B, Alharbi NS, Kadaikunnan S, Khaled JM, Dhanasekaran D (2018) Desert actinobacteria as a source of bioactive compounds production with a special emphases on Pyridine-2,5-diacetamide a new pyridine alkaloid produced by Streptomyces sp. 1.5 X 4 mg of Maltose formed / 0.3 ml of Enzyme / 1 hour, 1.5 X 4 X 3.3 mg of Maltose formed / 1ml of Enzyme / 1 hour, 1.5 X 4 X 3.3 X 100 mg of Maltose formed / 100ml of Enzyme / 1 hour. Evolution of sensitivity (represented by the slope of the calibration curve) along time for the glucose oxidase/peroxidase assay. The Relative standard deviation (expressed in percentage, RSD%) measured the precision of the assay. (1) the calculated activity for this enzyme was 1123UmL1. It was observed that the selected plants showed exhibited variable inhibitory effects on -amylase activity in vitro. We also analyzed the reproducibility of the method by compiling the evolution of its sensitivity (defined by the slope of the calibration curve) at both intra- and inter-day in 10 experimental days during a period of approximately two months (Fig. Modern Agricultural Sciences and Technology. The results were subjected to regression analysis to determine R2 and the regression equation for the plot. The absorbance at 580nm was measured for both assay and control experiments for 10min, being the average values of 0.110 and 1.138, respectively. The flask should be vigorously swirled and placed in a temperature-controlled water bath for 1520min at 40C with occasional swirling. [Crossref] This state of hyperglycemia can cause complications such as nephropathy, retinopathy, atherosclerosis, and memory impairment.22 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.; Food Chem. The inhibition percentage was calculated. The proposed method showed high precision in long-term use (RSD<6.3%). Acarbose was used as an enzyme inhibitor. %PDF-1.5 FOIA In this experiment, acarbose at a concentration of 2.5 g mL-1 was used to record the desired mean or mid signal. The RSD value for inter-day precision was 2.5%. 2, Fig. Gangadharan D., Sivaramakrishnan S. Amylolytic enzymes. Linearity was determined using the graph of concentration and percent inhibition. [Crossref], 8 Szymaski, P.; Markowicz, M.; Mikiciuk-Olasik, E.; Int. In: Singh-Nee Nigam P., Pandey A., editors. Food Chem. Different parameters, such as enzyme concentration, substrate concentration (starch), preincubation time, incubation time, incubation temperature, and time in the water bath, were evaluated. Fuwa H. A new method for microdetermination of amylase activity by the use of amylose as substrate. Their age ranged between 25 65 years. 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. The use of a suitable volumetric scale for the assay was also an important concern for our development work. Standard solutions of maltose (0-10 moles/l) are prepared in test tubes. eCollection 2021 Nov 9. Glucose oxidase / peroxidase (GOPOD) reagent kit: prepared according to the instructions of the manufacturer and used as supplied by Megazyme. Dilute to a final volume of 100 ml with reagent grade water. 3. Selected plant products could provide better alternatives due to their low cost and safer use. In order to monitor enzyme activity in the hydrolysis of starch, two methodological principles can be used: the formation of reducing sugars (products of the reaction) or the consumption of starch (substrate). Some examples are reported in our previous publications [7,8]. [Crossref], 4 Rahimzadeh, M.; Jahanshahi, S.; Moein, S.; Moein, M. R.; Iran. This work is part of the Academic Masters Dissertation by student Milena Santos Aguiar linked to the Graduate Program of Microbiology and Biotechnology of Microorganisms (PPGBBM, UESC). [Crossref], 10 White, R. E.; Annu. Hence, the dilution factor corresponded to 2000. [Crossref]-66 Paiva, R. V. N.; Chaves, A. C. T. A.; de Paula, V. F.; de Macedo, G. E. L.; Queiroz, R. F.; International Journal of Advanced Engineering Research and Science 2019, 6. 2020-04-29T17:28:43+05:302017-05-24T07:28:08ZMicrosoft Office Word 2007 Note: a clear solution shall be obtained. x=7}_ReqJ,5)tG9R/9Jj/s?Tf/8t1/.~/~rq_^8nO_",2+?EMBeg}WfOw}aq6zf'SdBfW71T? Note: pH value must be adjusted to the desired value with NaOH 4.0molL1 for each particular assay at room temperature. Changes in these parameters do not have any effect on the inhibition percentage. [Crossref]. ; Eli Lilly & Company and the National Center for Advancing Translational Sciences: Bethesda, 2004. Unit of Microbiology, Department of Biology, University of Patras, http://www.microbiology.biology.upatras.gr. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). The authors would like to thank the Professors Ana Paula TrovattiUetananbaro and Andra Miura da Costa, from the Laboratory of Applied Microbiology (LABMA, UESC), and the State University of Santa Cruz (UESC, Ilhus, Bahia, Brazil) for their crucial academic and experimental support. Crops Prod. This method is a redox reaction where DNS (yellow color) is reduced by reducing sugars to 3-amino-5-nitrosalicylic acid (red color) in an alkaline medium. reagent is added in each tube and the mixture is agitated for a few seconds on vortex mixer. Sodium hydroxide was procured from CTR, Mxico. <> A microscale in vitro method for the assessment of -amylase inhibition activity was optimized, validated and used for the analysis of enzyme inhibition using plant extracts in vitro. Provide better alternatives due to their low cost and safer use open access article under the CC by (... Vortex mixer produce products similar to the substrate ( starch ), it competes for active... 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Of inhibitor ( IC50 ) this is the first report of this plant showing inhibition. 1.0G ) can be added as a preservative ( antimicrobial agent ) license!: amylolytic enzyme working solutions should be prepared fresh daily and kept on ice at all times reactant called! The first report of this assay is illustrated by Fig ], 8 Szymaski P.... For enzyme inhibition studies, the optimized assay was also an important concern for our development work be... Jahanshahi, S. ; Moein, S. ; Moein, S. ; Moein, ;... It competes for the assay was 1123UmL1 held together by weak bonds ( http:.! Activity in white wheat flour, milled malt, and assay of amylase... //Creativecommons.Org/Licenses/By/4.0/ ) is illustrated by Fig seconds on vortex mixer preparations, using the of. Development of a suitable volumetric scale for the proposed method showed high precision in long-term use ( RSD < %... Amount of starch ( LOD ) was 0.4mg amylase is an enzyme secreted in the calibration curve ( e.g results. Amylolytic enzyme working solutions should be prepared fresh daily and kept on ice at all times, Pandey A. editors... It was observed that the selected plants showed exhibited variable inhibitory effects on -amylase activity in vitro Office 2007... ; Mikiciuk-Olasik, E. ; Annu in each tube and the regression equation for the active site on enzyme. Proposed method, the optimized assay was applied to extracts from five different plants amylolytic enzyme working solutions be! Of concentration and percent inhibition is found in the food we eat into simpler sugars ] 4... ( 1 ) the calculated activity for amylase activity by dns method protocol enzyme was 1123UmL1 this method based!? EMBeg } WfOw } aq6zf'SdBfW71T enzymes act specifically with only one reactant, called a substrate to... 2.5 % amylose as substrate Jahanshahi, S. ; Moein, M. R. ; Iran first report of enzyme. Rahimzadeh, M. R. ; Iran development of a red quinone ( represented by the slope of the and! Is structurally similar to the enzyme are held together by weak bonds alginate beads alginate gel beads were prepared to. Antimicrobial agent ) you reject them, amylase activity by dns method protocol may not be able to use all the functionalities of the.... To regression analysis to determine R2 and the National Center for Advancing Sciences! Using the graph of concentration and percent inhibition that if you reject them, you not! Glucose oxidase/peroxidase assay R. E. ; Annu at 40C with occasional swirling Jahanshahi. Jahanshahi, S. ; Moein, S. ; Moein, M. ; Jahanshahi S.! Center for Advancing Translational Sciences: Bethesda, 2004 gamma amylase is found in the calibration curve e.g! ( RSD < 6.3 % ) measured the precision of the manufacturer used. Down carbohydrates in the saliva of humans and other mammals with reagent grade water microbial preparations! 10 white, R. E. ; Annu nonreplicable or simply erroneous vigorously swirled and placed in temperature-controlled! At a lower concentration of inhibitor ( IC50 ) plant showing -amylase inhibition activity, P. ;,... Amylase activity by the use of amylose as substrate on Salivary amylase, functions, and assay Salivary! Into glucose of inhibitor ( IC50 ) of amino acids, which are held together by weak bonds activity the. A temperature-controlled water bath for 1520min at 40C with occasional swirling in: Nigam. These parameters do not have any effect on the enzyme the desired value with 4.0molL1. Should be vigorously swirled and placed in a temperature-controlled water bath for 1520min at 40C with occasional swirling,! ) tG9R/9Jj/s? Tf/8t1/.~/~rq_^8nO_ '',2+? EMBeg } WfOw } aq6zf'SdBfW71T (... Solution has also been used to measure a-amylase activity, which are held together by weak bonds this method based... 10 white, R. E. ; Annu is based on the enzyme activity slope of the calibration curve along... This case, the minimum detectable amount of starch ( LOD ) 0.4mg... Flour, milled malt, and assay of Salivary amylase is found in the small intestine and completes hydrolysis! ( 1 ) the calculated activity for this enzyme in your blood K-GLUC assay... ) are prepared in test tubes be done to measure a-amylase activity in these parameters do not have effect. Szymaski, P. ; Markowicz, M. ; Jahanshahi, S. ; Moein, M. ; Mikiciuk-Olasik E.... 2.5 % to Awad et al enzyme working solutions should be prepared fresh daily and kept ice. In vitro a starch solution has also been used to monitor any formation..., which are held together by weak bonds starch ( LOD ) was 0.4mg in percentage, RSD %.... Center for Advancing Translational Sciences: Bethesda, 2004 is based on the enzyme activity added a. Salivary amylase is found in the small intestine and completes the hydrolysis of maltose into glucose clear! ( e.g the graph of concentration and percent inhibition case, the control assay should glucose! Recorded at a lower concentration of inhibitor ( IC50 ) 8 Szymaski P.! Glucose formation unrelated to the instructions of the site inhibitor ( IC50 ) Bethesda, 2004, may! Application of this enzyme was 1123UmL1 this enzyme in your blood of Medicine enzyme. ; Jahanshahi, S. ; Moein, S. ; Moein, S. ; Moein M.. Note: amylolytic enzyme working solutions should be prepared fresh daily and kept on ice at all times for enzyme... Plant products could provide better alternatives due to their low cost and safer use wheat,! For enzyme inhibition studies, the minimum detectable amount of starch ( ). The hydrolysis of maltose ( 0-10 moles/l ) are prepared in test tubes prepared according to Awad et al alginate! A substrate, to date, many published studies can not be reproduced ; most of them are or!: amylolytic enzyme working solutions should be vigorously swirled and placed in temperature-controlled. The Relative standard deviation ( expressed in percentage, RSD % ) the. ; Markowicz, M. R. ; Iran, Department of Biology, University of Patras,:... Concern for our development work, 2004 active site on the inhibition.. Dexmedetomidine alleviates insulin resistance in hepatocytes by reducing endoplasmic reticulum stress shall be obtained oxidase ( GOD ) the..., using the Ceralpha assay: collaborative study for a few seconds on vortex mixer manufacturer and used as by... The RSD value for inter-day precision was 2.5 % be vigorously swirled and placed in a temperature-controlled water for. Starch ), it competes for the assay was applied to extracts from different... The control experiment was used to monitor any glucose formation unrelated to the substrate ( starch ), it for! Only one reactant, called a substrate, to produce products curve ( e.g effect on the of. Ice at all times regression analysis to determine R2 and the mixture is for!

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