Screening natural product extracts for potential enzyme inhibitors: protocols, and the standardisation of the usage of blanks in α-amylase, α-glucosidase and lipase assays
Tóm tắt
Enzyme assays have widespread applications in drug discovery from plants to natural products. The appropriate use of blanks in enzyme assays is important for assay baseline-correction, and the correction of false signals associated with background matrix interferences. However, the blank-correction procedures reported in published literature are highly inconsistent. We investigated the influence of using different types of blanks on the final calculated activity/inhibition results for three enzymes of significance in diabetes and obesity; α-glucosidase, α-amylase, and lipase. This is the first study to examine how different blank-correcting methods affect enzyme assay results. Although assays targeting the above enzymes are common in the literature, there is a scarcity of detailed published protocols. Therefore, we have provided comprehensive, step-by-step protocols for α-glucosidase-, α-amylase- and lipase-inhibition assays that can be performed in 96-well format in a simple, fast, and resource-efficient manner with clear instructions for blank-correction and calculation of results. In the three assays analysed here, using only a buffer blank underestimated the enzyme inhibitory potential of the test sample. In the absorbance-based α-glucosidase assay, enzyme inhibition was underestimated when a sample blank was omitted for the coloured plant extracts. Similarly, in the fluorescence-based α-amylase and lipase assays, enzyme inhibition was underestimated when a substrate blank was omitted. For all three assays, method six [Raw Data - (Substrate + Sample Blank)] enabled the correction of interferences due to the buffer, sample, and substrate without double-blanking, and eliminated the need to add substrate to each sample blank. The choice of blanks and blank-correction methods contribute to the variability of assay results and the likelihood of underestimating the enzyme inhibitory potential of a test sample. This highlights the importance of standardising the use of blanks and the reporting of blank-correction procedures in published studies in order to ensure the accuracy and reproducibility of results, and avoid overlooked opportunities in drug discovery research due to inadvertent underestimation of enzyme inhibitory potential of test samples resulting from unsuitable blank-correction. Based on our assessments, we recommend method six [RD − (Su + SaB)] as a suitable method for blank-correction of raw data in enzyme assays.
Tài liệu tham khảo
Hopkins AL, Groom CR. The druggable genome. Nat Rev Drug Discov. 2002;1(9):727–30.
Copeland RA. Why enzymes as drug targets? Evaluation of enzyme inhibitors in drug discovery: a guide for medicinal chemists and pharmacologists. 2nd ed. Hoboken: Wiley; 2013. p. 1–23.
Copeland RA, Harpel MR, Tummino PJ. Targeting enzyme inhibitors in drug discovery. Expert Opin Ther Targets. 2007;11(7):967–78.
Seiler N. Thirty years of polyamine-related approaches to cancer therapy. Retrospect and prospect. Part 1. Selective enzyme inhibitors. Curr Drug Targets. 2003;4(7):537–64.
Scatena R, Bottoni P, Pontoglio A, Mastrototaro L, Giardina B. Glycolytic enzyme inhibitors in cancer treatment. Expert Opin Investig Drugs. 2008;17(10):1533–45.
McFarlane SI, Kumar A, Sowers JR. Mechanisms by which angiotensin-converting enzyme inhibitors prevent diabetes and cardiovascular disease. Am J Cardiol. 2003;91(12):30–7.
Messerli FH, Bangalore S, Bavishi C, Rimoldi SF. Angiotensin-converting enzyme inhibitors in hypertension. J Am Coll Cardiol. 2018;71(13):1474–82.
Chiasson J-L, Josse RG, Gomis R, Hanefeld M, Karasik A, Laakso M. Acarbose for prevention of type 2 diabetes mellitus: the STOP-NIDDM randomised trial. Lancet. 2002;359(9323):2072–7.
Van de Laar FA, Lucassen PLBJ, Akkermans RP, Van de Lisdonk EH, Rutten GEHM, Van Weel C. Alpha glucosidase inhibitors for type 2 diabetes mellitus. Cochrane Database Syst Rev. 2005;2:CD003639.
Youdim MBH, Edmondson D, Tipton KF. The therapeutic potential of monoamine oxidase inhibitors. Nat Rev Neurosci. 2006;7(4):295.
Finberg JPM, Rabey JM. Inhibitors of MAO-A and MAO-B in Psychiatry and Neurology. Front Pharmacol. 2016;7:340.
Mehta M, Adem A, Sabbagh M. New acetylcholinesterase inhibitors for Alzheimer’s disease. Int J Alzheimers Dis. 2012;2012:728983.
Rolinski M, Fox C, Maidment I, McShane R. Cholinesterase inhibitors for dementia with Lewy bodies, Parkinson’s disease dementia and cognitive impairment in Parkinson’s disease. Cochrane Database Syst Rev. 2012;2012(3):CD006504.
Luo S, Gill H, Dias DA, Li M, Hung A, Nguyen LT, et al. The inhibitory effects of an eight-herb formula (RCM-107) on pancreatic lipase: enzymatic, HPTLC profiling and in silico approaches. Heliyon. 2019;5(9):e02453.
Tucci SA, Boyand EJ, Halford JCG. The role of lipid and carbohydrate digestive enzyme inhibitors in the management of obesity. Diabetes Metab Syndr Obes. 2010;3:125–43.
Agarwal P, Gupta R. Alpha-amylase inhibition can treat diabetes mellitus. Res Rev J Med Health Sci. 2016;54:1–8.
Sellami M, Louati H, Kamoun J, Kchaou A, Damak M, Gargouri Y. Inhibition of pancreatic lipase and amylase by extracts of different spices and plants. Int J Food Sci Nutr. 2017;68(3):313–20.
Buchholz T, Melzig MF. Medicinal plants traditionally used for treatment of obesity and diabetes mellitus—screening for pancreatic lipase and α-amylase inhibition. Phytother Res. 2016;30(2):260–6.
Rajan L, Palaniswamy D, Mohankumar SK. Targeting obesity with plant-derived pancreatic lipase inhibitors: a comprehensive review. Pharmacol Res. 2020;155:104681.
Tundis R, Loizzo M. Natural products as alpha-amylase and alpha-glucosidase inhibitors and their hypoglycaemic potential in the treatment of diabetes: an update. Mini Rev Med Chem. 2010;10:315–31.
Dias DA, Urban S, Roessner U. A historical overview of natural products in drug discovery. Metabolites. 2012;2(2):303–36.
Cragg GM, Grothaus PG, Newman DJ. New horizons for old drugs and drug leads. J Nat Prod. 2014;77(3):703–23.
Newman DJ, Cragg GM. Natural products as sources of new drugs from 1981 to 2014. J Nat Prod. 2016;79(3):629–61.
Beutler JA. Natural Products as a Foundation for Drug Discovery. Curr Protoc Pharmacol. 2009;46(1):9–11.
Lahlou M. The Success of natural products in drug discovery. Pharmacol Pharm. 2013;4(3A):17–31.
Shen B. A New Golden Age of Natural Products Drug Discovery. Cell. 2015;163(6):1297–300.
Qurratul A, Ashiq U, Jamal RA, Saleem M, Mahroof-Tahir M. Alpha-glucosidase and carbonic anhydrase inhibition studies of Pd(II)-hydrazide complexes. Arabian J Chem. 2017;10(4):488–99.
Molecular Probes Inc. EnzChek Ultra Amylase Assay Kit (E33651). 2006. https://www.thermofisher.com/order/catalog/product/E33651. Accessed 10 Feb 2018.
Goddard JP, Reymond JL. Enzyme assays for high-throughput screening. Curr Opin Biotechnol. 2004;15(4):314–22.
Reymond JL, Fluxa VS, Maillard N. Enzyme assays. Chem Commun. 2009;1:34–46.
Lankatillake C, Huynh T, Dias DA. Understanding glycaemic control and current approaches for screening antidiabetic natural products from evidence-based medicinal plants. Plant Methods. 2019;15(1):105.
Granados-Guzman G, Castro-Rios R, Waksman de Torres N, Salazar-Aranda R. Optimization and validation of a microscale in vitro method to assess alpha-glucosidase inhibition activity. Curr Anal Chem. 2018;14(5):458–64.
Beneyton T, Wijaya IP, Postros P, Najah M, Leblond P, Couvent A, et al. High-throughput screening of filamentous fungi using nanoliter-range droplet-based microfluidics. Sci Rep. 2016;6:27223.
Held P. Enzymatic digestion of polysaccharides. Part 1: monitoring polymer digestion and glucose production in microplates: BioTek Instruments, Inc. 2012. https://www.biotek.com/assets/tech_resources/Cellulosic_App_Note_Part_I.pdf. Accessed 15 Aug 2020.
Koyama K, Hirao T, Toriba A, Hayakawa K. An analytical method for measuring alpha-amylase activity in starch containing foods. Biomed Chromatogr. 2013;27(5):583–8.
Thermo Fisher Scientific Inc. Detecting Peptidases and Proteases—Section 10.4. 2020. https://www.thermofisher.com/au/en/home/references/molecular-probes-the-handbook/enzymesubstrates/detecting-peptidases-and-proteases.html. Accessed 16 Aug 2020.
Buchholz T, Melzig MF. Polyphenolic compounds as pancreatic lipase inhibitors. Planta Med. 2015;81(10):771–83.
Ortiz-Miranda S, Ji R, Jurczyk A, Aryee KE, Mo S, Fletcher T, et al. A novel transgenic mouse model of lysosomal storage disorder. Am J Physiol Gastrointest Liver Physiol. 2016;311(5):G903–19.
Jahn M, Zerr A, Fedorowicz FM, Brigger F, Koulov A, Mahler HC. Measuring lipolytic activity to support process improvements to manage lipase-mediated polysorbate degradation. Pharm Res. 2020;37(6):118.
Li X, Zhong X, Wang X, Li J, Liu J, Wang K, et al. Bioassay-guided isolation of triterpenoids as α-glucosidase inhibitors from Cirsium setosum. Molecules. 2019;24(10):1844.
Di Sotto A, Locatelli M, Macone A, Toniolo C, Cesa S, Carradori S, et al. Hypoglycemic, antiglycation, and cytoprotective properties of a phenol-rich extract from waste peel of Punica granatum L. Var. Dente di cavallo DC2. Molecules. 2019;24(17):3103.
Ostberg-Potthoff JJ, Berger K, Richling E, Winterhalter P. Activity-guided fractionation of red fruit extracts for the identification of compounds influencing glucose metabolism. Nutrients. 2019;11(5):1166.
Olaokun OO, Alaba AE, Ligege K, Mkolo NM. Phytochemical content, antidiabetes, anti-inflammatory antioxidant and cytotoxic activity of leaf extracts of Elephantorrhiza elephantina (Burch.) Skeels. S Afr J Bot. 2020;128:319–25.
Popović BM, Blagojević B, Ždero Pavlović R, Mićić N, Bijelić S, Bogdanović B, et al. Comparison between polyphenol profile and bioactive response in blackthorn (Prunus spinosa L.) genotypes from north Serbia-from raw data to PCA analysis. Food Chem. 2020;302:125373.
Yilmazer-Musa M, Griffith AM, Michels AJ, Schneider E, Frei B. Grape seed and tea extracts and catechin 3 gallates are potent inhibitors of alpha-amylase and alpha-glucosidase activity. J Agric Food Chem. 2012;60(36):8924–9.
Auld DS, Coassin PA, Coussens NP, Hensley P, Klumpp-Thomas C, Michael S, et al. Microplate Selection and Recommended Practices in High-throughput Screening and Quantitative Biology. In: Assay Guidance Manual. Bethesda: Eli Lilly & Company and the National Center for Advancing Translational Sciences. 2020.
Simeonov A, Davis MI. Interference with Fluorescence and Absorbance. In: Assay Guidance Manual. Bethesda: Eli Lilly & Company and the National Center for Advancing Translational Sciences. 2018
Thorne N, Auld DS, Inglese J. Apparent activity in high-throughput screening: origins of compound-dependent assay interference. Curr Opin Chem Biol. 2010;14(3):315–24.
Chlorophylls Gross J. Pigments in Vegetables. Boston: Springer; 1991. p. 3–74.
Carotenoids Gross J. Pigments in Vegetables. Boston: Springer; 1991. p. 75–278.
Azeredo HMC. Betalains: properties, sources, applications, and stability—a review. Int J Food Sci Technol. 2009;44(12):2365–76.
Mlodzinska E. Survey of plant pigments: molecular and environmental determinants of plant colors. Acta Biol Crac Ser Bot. 2009;51(1):7–16.
Glover BJ, Martin C. Anthocyanins. Curr Biol. 2012;22(5):R147–50.
Moon KM, Kwon EB, Lee B, Kim CY. Recent trends in controlling the enzymatic browning of fruit and vegetable products. Molecules. 2020;25(12):2754.
Nicolas JJ, Richard-Forget FC, Goupy PM, Amiot MJ, Aubert SY. Enzymatic browning reactions in apple and apple products. Crit Rev Food Sci Nutr. 1994;34(2):109–57.
Toledo L, Aguirre C. Enzymatic browning in avocado (Persea americana) revisited: history, advances, and future perspectives. Crit Rev Food Sci Nutr. 2017;57(18):3860–72.
Geremias-Andrade IM, Rocheto AC, Gallo FA, Petrus RR. The shelf life of standardized sugarcane juice stored under refrigeration. Food Sci Technol. 2020;40(1):95–101.
Roshchina VV, Kuchin AV, Yashin VA. Application of autofluorescence for analysis of medicinal plants. Int J Spectrosc. 2017;2017:1–8.
Duval R, Duplais C. Fluorescent natural products as probes and tracers in biology. Nat Prod Rep. 2017;34(2):161–93.
Donaldson L. Autofluorescence in plants. Molecules. 2020;25(10):2393.
Muller SM, Galliardt H, Schneider J, Barisas BG, Seidel T. Quantification of Forster resonance energy transfer by monitoring sensitized emission in living plant cells. Front Plant Sci. 2013;4:413.
Müller MG, Georgakoudi I, Zhang Q, Wu J, Feld MS. Intrinsic fluorescence spectroscopy in turbid media: disentangling effects of scattering and absorption. Appl Opt. 2001;40(25):4633–46.
Hach. What is the difference between a reagent blank and a sample blank? 2019. https://support.hach.com/app/answers/answer_view/a_id/1000699/loc/en_US#__highlight. Accessed 28 Apr 2020.
Földesi B. Guide to enzyme unit definitions and assay design. 2019. https://www.biomol.com/resources/biomol-blog/guide-to-enzyme-unit-definitions-and-assay-design. Accessed 16 Aug 2020.
Yang X, Kong F. Evaluation of the in vitro α-glucosidase inhibitory activity of green tea polyphenols and different tea types. J Sci Food Agric. 2016;96(3):777–82.
Özek G, Özbek MU, Yur S, Göger F, Arslan M, Özek T. Assessment of endemic cota fulvida (Asteraceae) for phytochemical composition and inhibitory activities against oxidation, α-amylase, lipoxygenase, xanthine oxidase and tyrosinase enzymes. Rec Nat Prod. 2019;13(4):333–45.
Tan Y, Chang SKC, Zhang Y. Comparison of α-amylase, α-glucosidase and lipase inhibitory activity of the phenolic substances in two black legumes of different genera. Food Chem. 2017;214:259–68.
Tunsaringkarn T, Rungsiyothin A, Ruangrungsi N. α-Glucosidase inhibitory activity of Thai mimosaceous plant extracts. J Health Res. 2008;22(1):29–33.
Cardullo N, Muccilli V, Pulvirenti L, Cornu A, Pouysegu L, Deffieux D, et al. C-glucosidic ellagitannins and galloylated glucoses as potential functional food ingredients with anti-diabetic properties: a study of alpha glucosidase and alpha-amylase inhibition. Food Chem. 2020;313:126099.
Rubilar M, Jara C, Poo Y, Acevedo F, Gutierrez C, Sineiro J, et al. Extracts of Maqui (Aristotelia chilensis) and Murta (Ugni molinae Turcz.): sources of antioxidant compounds and alpha-Glucosidase/alpha-Amylase inhibitors. J Agric Food Chem. 2011;59(5):1630–7.
Podsedek A, Majewska I, Redzynia M, Sosnowska D, Koziolkiewicz M. In vitro inhibitory effect on digestive enzymes and antioxidant potential of commonly consumed fruits. J Agric Food Chem. 2014;62(20):4610–7.
Sosnowska D, Podsędek A, Redzynia M, Kucharska AZ. Inhibitory effect of black chokeberry fruit polyphenols on pancreatic lipase – Searching for most active inhibitors. J Funct Foods. 2018;49:196–204.
Yuan Y, Zhang J, Fan J, Clark J, Shen P, Li Y, et al. Microwave assisted extraction of phenolic compounds from four economic brown macroalgae species and evaluation of their antioxidant activities and inhibitory effects on alpha-amylase, alpha-glucosidase, pancreatic lipase and tyrosinase. Food Res Int. 2018;113:288–97.
Hao W, Wang M, Lv M. The inhibitory effects of Yixing Black Tea extracts on A-Glucosidase. J Food Biochem. 2017;41(1):e12269.
Jacobson RL, Schlein Y. Phlebotomus papatasi and Leishmania major parasites express α-amylase and α-glucosidase. Acta Trop. 2001;78(1):41–9.
Khalil AT, Ovais M, Ullah I, Ali M, Jan SA, Shinwari ZK, et al. Bioinspired synthesis of pure massicot phase lead oxide nanoparticles and assessment of their biocompatibility, cytotoxicity and in vitro biological properties. Arabian J Chem. 2020;13(1):916–31.
Sun L, Warren FJ, Gidley MJ. Soluble polysaccharides reduce binding and inhibitory activity of tea polyphenols against porcine pancreatic α-amylase. Food Hydrocoll. 2018;79:63–70.
Dou F, Xi M, Wang J, Tian X, Hong L, Tang H, et al. α-Glucosidase and-amylase inhibitory activities of saponins from traditional Chinese medicines in the treatment of diabetes mellitus. Die Pharmazie-Int J Pharm Sci. 2013;68(4):300–4.
Awosika TO, Aluko RE. Inhibition of the in vitro activities of α-amylase, α -glucosidase and pancreatic lipase by yellow field pea (Pisum sativum L.) protein hydrolysates. Int J Food Sci Technol. 2019;54(6):2021–34.
Hamdani SS, Khan BA, Ahmed MN, Hameed S, Akhter K, Ayub K, et al. Synthesis, crystal structures, computational studies and α-amylase inhibition of three novel 1,3,4-oxadiazole derivatives. J Mol Struct. 2020;1200:127085.
Phuwapraisirisan P, Puksasook T, Jong-Aramruang J, Kokpol U. Phenylethyl cinnamides: a new series of alpha glucosidase inhibitors from the leaves of Aegle marmelos. Bioorg Med Chem Lett. 2008;18(18):4956–8.
Ansari P, Afroz N, Jalil S, Azad SB, Mustakim MG, Anwar S, et al. Anti-hyperglycemic activity of Aegle marmelos (L.) corr. is partly mediated by increased insulin secretion, α-amylase inhibition, and retardation of glucose absorption. J Pediatr Endocrinol Metab. 2017;30(1):37–47.
Beidokhti MN, Andersen MV, Eid HM, Sanchez Villavicencio ML, Staerk D, Haddad PS, et al. Investigation of antidiabetic potential of Phyllanthus niruri L. using assays for α-glucosidase, muscle glucose transport, liver glucose production, and adipogenesis. Biochem Biophys Res Commun. 2017;493(1):869–74.
Guillen Quispe YN, Hwang SH, Wang Z, Zuo G, Lim SS. Screening in vitro targets related to diabetes in herbal extracts from Peru: identification of active compounds in hypericum laricifolium juss by offline high performance liquid chromatography. Int J Mol Sci. 2017;18(12):2512.
Okoli CO, Obidike IC, Ezike AC, Akah PA, Salawu OA. Studies on the possible mechanisms of antidiabetic activity of extract of aerial parts of Phyllanthus niruri. Pharm Biol. 2011;49(3):248–55.
Ranilla LG, Kwon YI, Apostolidis E, Shetty K. Phenolic compounds, antioxidant activity and in vitro inhibitory potential against key enzymes relevant for hyperglycemia and hypertension of commonly used medicinal plants, herbs and spices in Latin America. Bioresour Technol. 2010;101(12):4676–89.
Luo S, Lenon GB, Gill H, Hung A, Dias DA, Li M, et al. Inhibitory effect of a weight-loss Chinese herbal formula RCM-107 on pancreatic alpha-amylase activity: enzymatic and in silico approaches. PLoS ONE. 2020;15(4):e0231815.
He Q, Lv Y, Yao K. Effects of tea polyphenols on the activities of α-amylase, pepsin, trypsin and lipase. Food Chem. 2007;101(3):1178–82.
Ramirez G, Zavala M, Perez J, Zamilpa A. In vitro screening of medicinal plants used in Mexico as antidiabetics with glucosidase and lipase inhibitory activities. Evid Based Complement Alternat Med. 2012;2012:701261.
Gondoin A, Grussu D, Stewart D, McDougall GJ. White and green tea polyphenols inhibit pancreatic lipase in vitro. Food Res Int. 2010;43(5):1537–44.
Lordan S, Smyth TJ, Soler-Vila A, Stanton C, Ross RP. The alpha-amylase and alpha-glucosidase inhibitory effects of Irish seaweed extracts. Food Chem. 2013;141(3):2170–6.
Herrera T, Navarro del Hierro J, Fornari T, Reglero G, Martin D. Inhibitory effect of quinoa and fenugreek extracts on pancreatic lipase and α-amylase under in vitro traditional conditions or intestinal simulated conditions. Food Chem. 2019;270:509–17.
Zhang B, Deng Z, Ramdath DD, Tang Y, Chen PX, Liu R, et al. Phenolic profiles of 20 Canadian lentil cultivars and their contribution to antioxidant activity and inhibitory effects on alpha-glucosidase and pancreatic lipase. Food Chem. 2015;172:862–72.
Di L, Kerns EH. Biological assay challenges from compound solubility: strategies for bioassay optimization. Drug Discov Today. 2006;11(9–10):446–51.
Johnston PA, Johnston PA. Cellular platforms for HTS: three case studies. Drug Discov Today. 2002;7(6):353–63.
Lundholt BK, Scudder KM, Pagliaro L. A simple technique for reducing edge effect in cell-based assays. J Biomol Screen. 2003;8(5):566–70.
Lau CY, Zahidi AAA, Liew OW, Ng TW. A direct heating model to overcome the edge effect in microplates. J Pharm Biomed Anal. 2015;2015(102):199–202.
Clarkson JR, Cui ZF, Darton RC. Protein denaturation in Foam: II. Surface activity and conformational change. J Coll Interface Sci. 1999;1999(215):333–8.
Clarkson JR, Cui ZF, Darton RC. Effect of solution conditions on protein damage in foam. Biochem Eng J. 2000;2000(4):107–14.
Bisswanger H. Enzyme assays. Perspect Sci. 2014;1(1–6):41–55.
Nakai M, Fukui Y, Asami S, Toyoda-Ono Y, Iwashita T, Shibata H, et al. Inhibitory effects of oolong tea polyphenols on pancreatic lipase in vitro. J Agric Food Chemi. 2005;53(11):4593–8.
Gorovits B, Roldan MA, Baltrukonis D, Cai CH, Donley J, Jani D, et al. Anti-drug antibody assay validation: improved reporting of the assay selectivity via simpler positive control recovery data analysis. AAPS J. 2019;21(5):76.