%PDF-1.5 [Crossref], 31 Quan, N. V.; Anh, H.; Lam, V. Q.; Takami, A.; Teschke, R.; Khanh, T. D.; Xuan, T. D.; Molecules 2022, 27, 774. Department of Microbiology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India, 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature, Aguiar, M.S., Maldonado, R.R., Carvalho, A.L., Aguiar-Oliveira, E. (2022). 2018, 14, 458. Some examples are reported in our previous publications [7,8]. 2011, 47, 270. Zhang, J. H.; Chung, T. D.; Oldenburg, K. R.; Rahman, M. M.; Islam, M. R., Shohag, S.; Hossain, M. E.; Rahaman, M. S.; Islam, F.; Ahmed, M.; Mitra, S.; Khandaker, M. U.; Idris, A. M.; Chidambaram, K.; Emran, T. B.; Cavalu, S.; Quan, N. V.; Anh, H.; Lam, V. Q.; Takami, A.; Teschke, R.; Khanh, T. D.; Xuan, T. D.; Analytical Chemistry Department, School of Medicine, Autonomous University of Nuevo Len, Madero y Aguirre Pequeo, Mexico, Text All the best, This is a very important test to detect diabetes. 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- Biomol. 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). Protocol Preparing Saliva 1. After 10min, the hydrolysis was stopped with the addition of 750L of 1molL1 HCl. For this assay, the values in parentheses also correspond to the limits of the 95% confidence levels intervals, and glucose mass is expressed in g. 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. For each experiment, fresh and independent reagents were used, and the evaluation was performed on three different days. Like most things, amylase production slows as we age, which reduces the ability of the digestive system to properly absorb nutrients. (Family: Asteraceae) has been used as a natural remedy in the Arabian region for its antihyperglycemic activity. - 204.93.178.112. (EN), 1 Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N.; J. Evidence-Based Complementary Altern. By applying Eq. Most of the amylase in your body is made by your pancreas and salivary glands. endobj An inhibition percentage near 50% was chosen as the best response, while the absorbance of the control at 0% inhibition (100% activity) resulted in the smallest photometric error. Part of Springer Nature. Their age ranged between 25 65 years. For this particular case, mstarch was 2.40mg. Next, 500L of enzyme solution 2 (described in detail in the previous example) and 1000L of universal buffer were added to the vial (in this particular case, the objective was to obtain extra information about the hydrolysis performance of the enzyme and for this reason we used the same enzyme dilution and mass of starch used for the enzyme activity method. Amylase is naturally produced in humans by the salivary glands and pancreas. Amylase is an enzyme, or special protein, produced by your pancreas and salivary glands. The IC50 for the positive control was found to be 0.6 g mL-1. Each enzyme works best at a certain pH and temperature, its activity decreasing at values above and below that point due to denaturation. This article providing information on Salivary amylase, functions, and Assay of Salivary amylase activity. Then make it to 200ml with distilled water. 5 ml of deionized water are added in each sample, followed by If sodium azide is added, the reagent will be stable for more than one year. Place the vial in the block heater (or other alternative heating sources) previously set at the target temperature of the study during the desired time (, Immediately stop the hydrolysis by adding 200L of 1.0molL. Centrifuge the vials for 5min at 350g, at room temperature. 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. % The diagnosis of saliva amylase enzyme activity deficiency requires an oral test with a standard protocol. -Amylase from the porcine pancreas (15 U mg-1) was used instead of human amylase because of its cost and availability. 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. For measuring the amylase activity with DNS solution (Miller Method): 1. These parameters should be adjusted according to the objectives of each individual study). J Genet Eng Biotechnol 15:95101, Mandels M, Sternberg D (1976) Recent advances in cellulases technology. Rafael Resende Maldonado . This experiment also counteracts problems associated with drift patterns, edge effects, and other systematic sources of variability. 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. [Crossref], 8 Szymaski, P.; Markowicz, M.; Mikiciuk-Olasik, E.; Int. We used a US bath to facilitate the complete solubilization of I2 and subsequent formation of triiodide (please note that the concentration of KI described in the original report of Xiao et al. Prepare D-glucose standards as described in. Chem. The -amylase activity was assessed by Millers method by estimating the colored product formation by the action of enzyme and substrate starch (Scheme 1).99 Miller, G. L.; Anal. For a urine test, its likely a two-hour or 24-hour sampling. The slopes of these two were compared to determine linearity using the t-test. A state of sustained hyperglycemia caused by insulin deficiency can lead to type 2 diabetes.11 Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N.; J. Evidence-Based Complementary Altern. [Crossref], Postprandial hyperglycemia can be controlled by inhibitors, such as those directed at inhibiting -amylase, which reduces the flow of glucose by digesting carbohydrates. Other equipment can be used with adjustments of the centrifugation time and force. This method is based on the reaction of maltose with glucose oxidase (GOD) and the development of a red quinone. Med 2011, Article ID 810207. Hi, Laneesha Moozhikkal This link can be helpful for the calculation of alpha amylase activity. http://www.microbiology.biology.upatras.gr/en/proto 15 Markossian, S.; Coussens, N. P.: Dahlin, J. L.; Sittampalam, G. S.; SLAS Discovery 2021, 26, 1241. All solutions were prepared with ultra-pure water (maximum conductivity of 0.055Scm1). where SD is the standard deviation of each measurement, and AVG is the average of such measurements. The results were subjected to regression analysis to determine R2 and the regression equation for the plot. The transfer of the vial content to another tube / container may also be necessary. The use of soluble starch also allowed the standardization of the working conditions, avoiding any influence of the solubility of the starch in the final result. Methods in Actinobacteriology pp 495503Cite as, Part of the Springer Protocols Handbooks book series (SPH). In a tube containing 1 mL of phosphate buffer (100 mmol L-1, pH 6.9), 100 L of -amylase (final concentration 0.15 U mL-1 in phosphate buffer) was added and incubated at 37 C for 7.2 min for preincubation. For enzyme inhibition studies, the absorbance was recorded at a lower concentration of inhibitor (IC50). These plant extracts were offered by researchers from the Analytical Chemistry Department (Facultad de Medicina, UANL, Mxico). Enzyme energy of amylase from edible fungi was determinated based on 3,5-dinitryl-salicyle(DNS).Taking czapek as induction medtum in whith the only carbon source was 2% soluble starch,and amylase energy ranged from 1.513 to 3.417 U/mL among Lentinula edodes,Pleurotus ostreatus,Pleurotus cornucopiae,so as to put forward a reference and Extract from Zanthoxylum fagara showed maximum inhibitory activity with an IC50 value of 4.75 g mL-1. KI/I2 solution: KI 2.0g L1, I2 200mgL1. https://www.starch.eu/the-european-starch-industry/#figures, A quantitative starch-iodine method for measuring alpha-amylase and glucoamylase activities. [Crossref], 11 Finak, G.; Gottardo, R.; In Data Mining Techniques for the Life Sciences; Carugo, O., Eisenhaber, F., eds. Hence, a reliable protocol for preparing the starch solution is an important aspect regarding the need for a batch to batch reproducibility. For calculation of enzyme activity base on (U/ml) try according to the following stages: 1. It comprises two chains of amino acids, which are held together by weak bonds. We adopted this strategy to separate the solid particles that remained after the hydrolysis of different pure starches mediated by amyloglucosidase (glucose yield protocol, step 5). Mina Karimi-Avargani kindly mail me saidurga48@gmail.com. I need your help for calculation of data. 2012, 60, 8924. In all cases, a sample blank was analyzed to demonstrate that there were no matrix interferences caused by the extract. <> Chem. As a unit of activity (unit, U) of the enzyme a-amylase, is arbitrarily appointed, the quantity of the enzyme required for the production of 1 mole of maltose in 1 min, when the enzyme is incubated along with the substrate at pH=4,9 and =40 C. 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. After 10min, the reaction was stopped with 200L of HCl 1.0molL1 and the vial was centrifuged for 5min at 350g. Next, 50L of supernatant were transferred to a 96-well microplate, followed by the addition of 150L of GOPOD reagent. <>/StructTreeRoot 3 0 R/Pages 4 0 R/MarkInfo<>/Lang(en-US)/OCProperties<><><>]/ON[5 0 R 6 0 R]>>>>/ViewerPreferences<>/Type/Catalog>> xZmoEna?T 8) [8]. J Clin Chem Clin Biochem. Thus, the optimized method was both accurate and precise for both interday and intraday experimentation for the evaluated concentration levels. In: Dharumadurai, D. (eds) Methods in Actinobacteriology. Applicability and reliability of the glucose oxidase method in assessing -amylase activity. [Crossref] In this study, the values of the Z factor were 0.93 and 0.96. 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. The plant extract solutions were in the concentration range of 6 g mL-1 to 200 g mL-1. Microbiol Res 207:116133, Srinivas A, Rahul K, Sasikala C, Subhash Y, Ramaprasad EVV, Ramana CV (2012) Georgenia satyanarayanai sp. Here we introduced an improved method capable to measure the activity of alpha-amylase and amyloglucosidase from different sources based on a quantitative starch-iodine assay. Changes in these parameters do not have any effect on the inhibition percentage. 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. Multi-Mechanistic In Vitro Evaluation of Antihyperglycemic, Antioxidant and Antiglycation Activities of Three Phenolic-Rich Indian Red Rice Genotypes and In Silico Evaluation of Their Phenolic Metabolites. Six factors (i.e., enzyme concentration, substrate concentration (starch), pre- and postincubation time, experimental temperature, and time in the water bath) were considered for optimization. [5]. Therefore, this method should be considered as a valuable tool in clinical practice. [Crossref]-1616 United States Pharmacopeial Convention; The United States pharmacopeia: the national formulary; Rockville, Md., United States pharmacopeial convention, 1980. 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). [Crossref], The phytochemical analysis indicated the presence of various metabolites such as alkaloids, flavonoids, terpenes, steroids, and phenols. [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. J. Mol. The sensitivity is the slope of the calibration curve, which is a function of the analyte concentration. Salivary amylase is produced by the salivary glands and released into the mouth when we eat. [Crossref],33 Moein, S.; Pimoradloo, E.; Moein, M.; Vessal, M.; BioMed Res. Amylase is the hydrolytic enzyme that breaks down many polysaccharides like Starch, Amylose, dextrins, and yields a disaccharide i.e., Maltose. for simple microplate readers with optical bandpass filters), the detection wavelength can be adjusted to other values nearby the maximum absorption region of the colored compound (see This study aimed at evaluating the efficacy of salivary amylase enzyme activity assay to detect diabetes mellitus. The inhibition percentage was calculated using Equation 1: where A is the absorbance of the negative control, and B is the absorbance of the solution containing an inhibitory agent. [Crossref], 25 Vadivel, V.; Nandety, A.; y Biesalski, H. K.; Plant Foods Hum. The development work herein reported sought to answer to the major methodological difficulties that we experienced for implementing the protocols for measuring enzyme activity (for both amylase and amyloglucosidase) and glucose yield (for amyloglucosidase), starting from the report of Xiao et al. These findings demonstrated that our optimized and validated method is highly selective, enabling the identification of samples with or without potent activity. The degree of inhibition was evaluated in terms of the concentration of the inhibitor that inhibited half of the enzyme activity (IC50). You can consider the following things. 1. Prepare soluble starch in 0.1 M sodium phosphate buffer at pH 7.0, 7.5 or 8.0 or in acidic range, if your MeSH R. P.; Mol. 16 United States Pharmacopeial Convention; The United States pharmacopeia: the national formulary; Rockville, Md., United States pharmacopeial convention, 1980. The residues used correspond to the fruit peels and the pressed cake obtained after extracting the vegetable oil from its seeds. Designed by http://www.microbiology.biology.upatras.gr. 4 0 obj However, it showed a relatively higher -amylase activity in Fig 3 d. In additio n, Fig 3 d needs control to show equal amounts of proteins were loaded. Humana, New York, NY. It is made in the pancreas and the glands that make saliva. One negative control (absence of inhibitor) was set up to obtain 100% enzyme activity. Another potential application of the proposed method is the evaluation of the activity of amylolytic enzymes extracted from different natural matrices such as flours and microbial enzyme preparations. The https:// ensures that you are connecting to the ; Humana Press: New York, 2016 [Crossref] Bioassays are challenging in terms of determining accuracy. 2020 Jan;67(1):87-94. doi: 10.1007/s12020-019-02118-1. 1.5 mg of Maltose formed / 0.3. ml / 15 minutes. 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The centrifugation time and force of saliva amylase enzyme activity deficiency requires an test. Be 0.6 g mL-1 Z factor were 0.93 and 0.96 0.055Scm1 ) a sample blank analyzed... Were prepared with ultra-pure water ( maximum conductivity of 0.055Scm1 ) the hydrolysis was stopped with addition.