High-Resolution Î±-Amylase Assay Combined with High-Performance

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Article

High-resolution #-amylase assay combined with high-performance liquid chromatography-solid-phase extraction-nuclear magnetic resonance spectroscopy for expedited identification of #-amylase inhibitors – proof of concept and #-amylase inhibitor in cinnamon Leyla Okutan, Kenneth T. Kongstad, Anna K. Jäger, and Dan Staerk J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5047283 • Publication Date (Web): 04 Nov 2014 Downloaded from http://pubs.acs.org on November 8, 2014

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Journal of Agricultural and Food Chemistry

Manuscript for Journal of Agricultural and Food Chemistry

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High-resolution α-amylase assay combined with high-performance liquid

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chromatography−solid-phase extraction−nuclear magnetic resonance

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spectroscopy for expedited identification of α-amylase inhibitors – proof

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of concept and α-amylase inhibitor in cinnamon

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Leyla Okutan, Kenneth T. Kongstad, Anna K. Jäger, and Dan Staerk*

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Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of

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Copenhagen, Copenhagen, Denmark

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*

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Sciences, University of Copenhagen, Copenhagen, Denmark. Tel. +45 3533 6177; fax: +45 3533 6001.

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E-mail address: [email protected]

Corresponding author at: Department of Drug Design and Pharmacology, Faculty of Health and Medical

ACS Paragon Plus Environment

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ABSTRACT

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Type 2 diabetes affects millions of people worldwide, and new improved drugs or functional foods

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containing selective α-amylase inhibitors are needed for improved management of blood glucose.

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In this study the development of a microplate-based high-resolution α-amylase inhibition assay

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with direct photometric measurement of α-amylase activity is described. The inhibition assay is

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based on porcine pancreatic α-amylase with 2-chloro-4-nitrophenyl-α-D-maltotriose as substrate;

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resulting in a stable, sensitive and cheap inhibition assay as requested for high-resolution purposes.

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In combination with HPLC−HRMS−SPE−NMR, this provides an analytical platform that allows

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simultaneous chemical and biological profiling of α-amylase inhibitors in plant extracts. Proof-of-

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concept with an artificial mixture of six compounds - of which three are known α-amylase

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inhibitors - showed that the high-resolution α-amylase inhibition profiles allowed detection of

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submicrogram amounts of the α-amylase inhibitors. Furthermore, the high-resolution α-amylase

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inhibition assay/HPLC−HRMS−SPE−NMR platform allowed identification of cinnamaldehyde as

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the α-amylase inhibitor in cinnamon (Cinnamomum verum Presl.).

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KEYWORDS: Type 2 diabetes, high-resolution α-amylase assay, microplate,

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HPLC−HRMS−SPE−NMR

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INTRODUCTION

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Diabetes is a chronic metabolic disorder - estimated to affect 382 million people worldwide in 2013,

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causing 5.1 million deaths and amounting to global health expenditures of 548 billion USD, i.e., 11

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% of all health care expenditures.1 It is estimated that 592 million people will be affected by

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diabetes in 2035, i.e., an increase of 55 % in 23 years, and public health expenditures are expected

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to increase to 627 billion USD. Type 2 diabetes (T2D) is characterized by an insufficient response

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to the pancreatic production of insulin, leading to elevated and highly fluctuating blood glucose

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levels. This is associated with development of severe late complications such as atherosclerosis,

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cardiac dysfunction, retinopathy, neuropathy and nephropathy.2

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One way of maintaining lower and more stable blood glucose is by inhibiting the carbohydrate

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digestive enzyme α-amylase (α-1,4-glucan-4-glucanohydrolases; E.C. 3.2.1.1). α-Amylase is one

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of the major secretory products of the pancreas (5-6 %), and the enzyme catalyses the cleavage of

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α-D-1,4 glycosidic bonds of amylose and amylopectin in the lumen of the small intestine.1,3 This

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results in formation of, e.g., maltose, maltotriose and a number of α-(1-6) and α-(1-4) oligoglucans,

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which are subsequently cleaved to absorbable glucose by α-glucosidases.1 Dietary starch are rapidly

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cleaved after eating a meal, which leads to postprandial hyperglycaemia for diabetics. Eichler and

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coworkers have shown that the activity of pancreatic α-amylase in the small intestine correlates

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with the increase in post-prandial glucose levels.4 This finding supports the hypothesis of α-

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amylase as an important target for management of type 2 diabetes, and Acarbose, a non-selective α-

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amylase and α-glucosidase inhibitor, is available on the market despite several side effects such as

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abdominal pain, flatulence and diarrhea.5 It has been suggested that the side effects are caused by

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excessive inhibition of pancreatic α-amylase; resulting in bacterial fermentation of undigested

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carbohydrates in the colon.6


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Several studies have shown α-amylase inhibitory activity of spices, fruits and vegetables,7 which

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emphasize the importance of plants as sources of new α-amylase inhibitors or as sources of new

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functional food, i.e., food with health-promoting effects beyond their nutritional value. In both

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instances, individual bioactive constituents responsible for the α-amylase inhibitory activity must

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be identified and characterized. Bioassay-guided fractionation has for decades been the preferred

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method of choice in natural products research, but this method is labor-intensive and requires

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lengthy and repeated isolation and separation processes followed by assessment of bioactivity of

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individual fractions. Therefore, the recent combination of high-resolution bioactivity profiling8 with

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the hyphenated high-performance liquid chromatography−solid-phase extraction−nuclear magnetic

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resonance spectroscopy,9,10 has proven a very promising and effective new technology for targeting

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analysis towards bioactive constituents only. The resulting high-resolution

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bioassay/HPLC−SPE−NMR technology platform has already proven efficient for identification of

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monoamine oxidase-A inhibitors,11 antioxidants,12-14 fungal membrane H+ ATPase inhibitors,15 and

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α-glucosidase inhibitors16,17 direct from crude extracts of medicinal plants, endophytic fungi and

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food.

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For high-resolution α-amylase inhibition profiling of plant extracts, a sensitive, stable and cheap

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assay performed in microplates is needed. Most studies reported so far have been based on

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colorimetric measurements of reducing sugars by reaction with dinitrosalicylic acid (DNS method)

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or blue starch-iodine complexes formed between iodine and starch polymers.18,19 Both methods use

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cheap reagents, but are experimentally time-consuming and relying on secondary chemical

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processes to occur (i.e., reaction with DNS and formation of starch-iodine complexes). Direct

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assessment of α-amylase activity can be performed by colorimetric measurement of the formation

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of chromophores like 2-chloro-4-nitrophenolate and p-nitrophenolate by cleavage of substrates like

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2-chloro-4-nitrophenyl-α-maltotrioside (CNP-G3) and p-nitrophenyl-α-D-maltoheptaoside (PNP-


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G7), respectively.19,20 The 2-chloro-4-nitrophenyl chromophore hydrolyzes 10 times faster than the

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corresponding 4-nitrophenyl group, and α-amylase assays based on the former are therefore more

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sensitive.20,21 In this work the development of a high-resolution microplate-based α-amylase

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inhibition assay using 2-chloro-4-nitrophenyl-α-D-maltotrioside as substrate is described. The aim

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is to implement a stable, cheap and sensitive α-amylase assay suitable for fast and efficient high-

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resolution α-amylase profiling in combination with HPLC−SPE−NMR for analysis of α-amylase

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inhibitors in plant extracts with presumed antidiabetic effect.

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EXPERIMENTAL SECTION Chemicals. α-Amylase type VI - B (E.C. 3.2.1.1, from porcine pancreas, lyophilized powder), 2-

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chloro-4-nitrophenyl-α-D-maltotrioside (CNP-G3), sodium chloride, sodium azide, dimethyl

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sulfoxide (≥99.5%), Acarbose, (-)-epicatechin, brucine, luteolin, myricetin, coumarin, trolox and

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HPLC grade acetonitrile were purchased from Sigma-Aldrich (St. Louis, MO). Calcium acetate and

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formic acid were purchased from Merck (Darmstadt, Germany), HPLC-grade methanol for

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extraction of cinnamon was from Fischer Scientific (Leicester, UK), and methanol-d4 (99.80% D,

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less than 0.03% water) was from Euriso-top (Saint-Aubin Cedex, France). Cinnamon

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(Cinnamomum verum Presl) was purchased from Urtekram (Mariager, Denmark). Water was

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purified by 0.22 µm membrane filtration and deionization. (Millipore, Billerica, MA).

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Extraction and sample preparation. The stock solution of the artificial mixture consisted of 5

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g/L brucine, 5 g/L coumarin, 2.5 g/L trolox, 0.875 g/L luteolin, 0.875 g/L myricetin and 1.25 g/L

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(−)-epicatechin in a 1:1 mixture of HPLC solvent A and B, vide infra. Crude methanol extract of

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cinnamon was prepared by extracting 1 g of ground material with 10 mL of solvent at room

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temperature overnight. The extract was filtered (45 µm filter fitted to a 10 mL syringe) and


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evaporated to dryness. The dry extracts were reconstituted in phosphate buffer containing 10 %

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DMSO to a concentration of 20 mg/mL. Tenfold dilutions were performed 12 times for IC50

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determinations.

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Development of α-amylase assay. The α-amylase assay was performed in 96-well microplates

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using a final volume of 200 µL. Unless otherwise stated, experiments were performed with

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phosphate buffer (0.1 mM) containing 0.02% NaN3 and adjusted to pH 6.0 with 2.0 M phosphoric

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acid. Stock solutions of α-amylase and CNP-G3 in phosphate buffer were prepared at

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concentrations of 2.0 U/mL and 10.0 mM, respectively. All wells were added 175 µL buffer and 5

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µL enzyme solution (final well concentration 0.05 U/mL), and the microplate was subsequently

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incubated for 10 min. The reaction was started by addition of 20 µL substrate solution (final well

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concentration 1.0 mM), whereafter the absorbance was measured at 405 nm every third min. for 30

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min. Preincubation, incubation and absorbance measurements were performed with a Multiscan FC

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microplate photometer with built-in incubator (Thermo Scientific, Waltham, MA) coupled to SkanIt

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version 2.5.1 software for data acquisition. Cleavage rates were obtained by plotting the absorbance

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values as function of time using Microsoft Excel 2010 for slope calculations. Optimal

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concentrations of Ca2+, Cl- and DMSO as well as optimal pH and temperature were assessed by

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measuring enzyme activities for the following array of parameter changes (in addition to above-

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described protocol): sodium chloride: 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mM; calcium

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acetate (using 60 mM sodium chloride): 0, 0.5, 1.0, 2.0, and 2.5 mM; DMSO (using 60 mM sodium

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chloride and 1 mM calcium acetate): 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 % v/v; temperature: 25,

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28, 31, 34, 37, and 40 °C; pH: 5.0, 5.5, 5.7, 6.0, 6.3, 6.7, 7.0, and 7.5 (using 2.0 M phosphoric acid

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or 2.0 M NaOH for adjustment of buffer). Stability of the α-amylase enzyme was assessed for a

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period of 8 weeks. Stock solution of 20 U/mL enzyme in buffer (60 mM sodium chloride, 1 mM


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calcium acetate, pH 6.0) was dispersed in 600 µL aliquots and stored at −18 °C. The first

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measurement at day 0 was performed on a non-frozen enzyme solution; whereas the remaining

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measurements were performed using thawed stock solutions diluted to 2.0 U/mL as above.

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Enzymatic activity of α-amylase was subsequently assessed 3 times per week for 2 weeks and then

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once a week for additional 6 weeks. For comparison of kinetic vs. endpoint measurements (using 5

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% DMSO, 60 mM sodium chloride, 1 mM calcium acetate, and pH 6.0), absorbance was measured

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at 405 nm every third min. for 30 min after addition of substrate. Subsequently pH was adjusted to

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12 by addition of 13 µL 4 M NaOH solution and the absorbance was measured for an additional 30

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min.

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HPLC separations and high-resolution α-amylase inhibition chromatograms. HPLC

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separations were performed on an Aglient 1200 series instrument (Santa Clara, CA) consisting of a

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G1311A quaternary pump, a G1322A degasser, a G1316A thermostatted column compartment, a

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G1315C photodiode-array detector, a G1367C high-performance auto sampler, and a G1364C

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fraction collector, all controlled by Agilent ChemStation ver. B.03.02 software. Analyses were

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performed at 40 °C on a 150 × 4.6 mm i.d. Phenomenex Luna C18(2) reversed-phase column (3 µm

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particle size, 100 Å pore size) with a flow rate of 0.5 mL/min. HPLC solvent A consisted of water-

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acetonitrile 95:5 added 0.1 % formic acid and solvent B consisted of acetonitrile-water 95:5 added

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0.1 % formic acid. For high-resolution α-amylase inhibition profiling, 10 µL of the artificial

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mixture was separated using the following gradient elution profile: 0 min., 5% B; 20 min., 70% B;

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23 min., 70% B; 24 min., 5% B; 35 min., 5 % B. The column eluate from 7.5 to 20 min. was

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fractionated into one 96-well microplate (Sterilin Limited, Aberbargoed Caerphilly, United

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Kingdom) using the 8 wells in column 12 as blank controls, yielding a resolution of 7 points per

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min. The microplates were subsequently evaporated to dryness using a SPD121P Savant SpeedVac

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concentrator (Thermo Scientific, Waltham, MA) equipped with an OFP400 Oil Free Pump and a


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RVT400 Refrigerated Vapor Trap. α-Amylase cleavage rates (kinetic measurements) were

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measured for each well as described above (using 5 % DMSO, 60 mM sodium chloride, 1 mM

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calcium acetate, pH 6.0), and inhibition was calculated using the formulae: % inhibition =

Slopeblank − Slopesample Slopeblank

∙ 100%

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Inhibition for each well was plotted at its respective retention time as an α-amylase inhibition

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profile underneath the HPLC chromatogram.

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Validation of HPLC method for separation of artificial mixture. The HPLC method

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described above was used for the quantification of individual constituents in the artificial mixture

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and validation of linearity, sensitivity (limit of detection, LOD and limit of quantification, LOQ)

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and precision (intra-day and inter-day). The linearity was assessed using regression analysis of

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standard curves (254 nm) obtained from the injection of 10 µL of a complex mixture containing

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brucin, coumarin, trolox, luteolin, myricetin and epicatechin. Thus, a stock solution containing 5

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g/L brucine, 5 g/L coumarin, 2.5 g/L trolox, 0.875 g/L luteolin, 0.875 g/L myricetin and 1.25 g/L

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(−)-epicatechin in a 1:1 mixture of HPLC solvent A and B, was diluted two-fold 12 times with the

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same solvent mixture. The HPLC calibration curves were constructed plotting the peak area (A)

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against the concentration of each standard solution (c) of each compound. The data were used for

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determining limit of detection (LOD: S/N=3) and limit of quantification (LOQ: S/N=10). Precision

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of the method was determined after injection of 10 µL of the artificial mixture containing 200, 150

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and 100 mg/L of each compound. The analysis was performed in quadruplicate on day 1 (intraday

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precision) and for 5 additional days (interday precision). Results are expressed in % RSD (=

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SD/average) of the instruments.

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HPLC-HRMS-SPE-NMR analysis. Chromatographic separation of a 19.8 mg/mL solution of

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crude extract of cinnamon was performed using an Agilent 1200 system (Santa Clara, CA)


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comprising a photodiode-array detector, an auto sampler, a quaternary pump, a degasser, and a

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thermostatted column compartment; using the same column, solvent composition, temperature, and

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gradient profile as described above. A small proportion (1%) of the HPLC eluate was directed to a

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micrOTOF-Q II mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) equipped with an

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electrospray ionization interface. Mass spectra were acquired in positive-ion mode, using drying

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temperature of 200 °C, capillary voltage of 4100 V, nebulizer pressure of 2.0 bar, and drying gas

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flow of 7 L/min. A solution of sodium formate clusters was automatically injected in the beginning

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of each run to enable internal mass calibration. The rest of the HPLC eluate was directed to the

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photo-diode array detector, and subsequently to a Prospekt 2 SPE-unit (Spark Holland, Emmen, The

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Netherlands). Cumulative SPE trappings of individual analytes were performed for five consecutive

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injections (10 µL/injection) using absorption-threshold at 254 nm) to trigger analyte trapping.

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Chromatography, peak trapping and analyte transfer from the SPE unit were controlled with HyStar

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ver. 3.2 software (Bruker Biospin GmbH, Rheinstetten, Germany). The HPLC eluate was diluted

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with Milli-Q water at a flow rate of 1.0 mL/min. prior to trapping on 10 × 2 mm i.d. Resin GP

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(general purpose, 5-15 µm, spherical shape, polydivinyl-benzene phase) SPE cartridges from Spark

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Holland (Emmen, The Netherlands). After drying for 45 min with pressurized nitrogen gas, analytes

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were automatically eluted into 1.7-mm o.d. NMR tubes (96 position tube racks) with methanol-d4

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using a Gilson Liquid Handler controlled by PrepGilson software Version 1.2 (Bruker Biospin

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GmbH, Rheinstetten, Germany). NMR experiments were performed with a Bruker Avance III

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system (1H operating frequency of 600.13 MHz) equipped with a Bruker SampleJet sample changer

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and a cryogenically cooled gradient inverse triple-resonance 1.7 mm TCI probe-head (Bruker

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Biospin) optimized for 1H and 13C observation.. Icon NMR (version 4.2, Bruker Biospin) was used

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for controlling automated acquisition of NMR data and processing of NMR data was performed

200

using Topspin (version 3.0, Bruker Biospin). All NMR spectra were recorded in methanol-d4 at 300


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K. 1H and 13C chemical shifts were referenced to the residual solvent signal (δ 3.31 and δ 49.00,

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respectively). One-dimensional 1H NMR spectrum was acquired in automation (temperature

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equilibration to 300 K, optimization of lock parameters, gradient shimming, and setting of receiver

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gain) with 30°-pulses, 3.66 s inter-pulse intervals, 64k data points and multiplied with an

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exponential function corresponding to line-broadening of 0.3 Hz prior to Fourier transform. Phase-

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sensitive DQF-COSY and NOESY spectra were recorded using a gradient-based pulse sequence

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with a 20 ppm spectral width and 2k x 512 data points (processed with forward linear prediction to

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1k data points). Multiplicity-edited HSQC spectrum was acquired with the following parameters:

209

spectral width 20 ppm for 1H and 200 ppm for 13C, 2k x 256 data points (processed with forward

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linear prediction to 1k data points), and 1.0 s relaxation delay. HMBC spectrum was optimized for

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n

212

ppm for 13C, 2k x 128 data points (processed with forward linear prediction to 1k data points), and

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1.0 s relaxation delay.

JC,H = 8 Hz and acquired using the following parameters: spectral width 20 ppm for 1H and 240

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RESULTS AND DISCUSSION

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This work describes development and optimization of a direct colorimetric α-amylase inhibitory

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assay based on cleavage of 2-chloro-4-nitrophenyl-α-D-maltotrioside (CNP-G3), its implementation

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in microplates for high-resolution inhibition profiling, and its combination with

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HPLC−HRMS−SPE−NMR for fast and efficient identification of α-amylase inhibitors in medicinal

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plant and functional food extracts with a presumed anti-diabetic effect. Porcine α-amylase and

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CNP-G3 was chosen as enzyme and substrate, respectively, in the current work because they are

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relatively cheap compared to human α-amylase and maltopentaoside/maltoheptaoside substrates –

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an important factor for the use of high-resolution α-amylase inhibition assays as a routine method in

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natural products and functional food research.


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Development and optimization of α-amylase bioassay with 2-chloro-4-nitrophenyl-α α-D-

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maltotrioside substrate. Development and optimization of the α-amylase inhibition assay using 2-

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chloro-4-nitrophenyl-α-D-maltotrioside as substrate was based on the assay previously

228

described.20,21 The optimization and microplate-based adaptation ensures a sensitive, robust and

229

reproducible setup that is suitable for high-resolution α-amylase inhibition profiling and further

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coupling with HPLC−HRMS−SPE−NMR. Initial experiments were performed with 0.1 mM

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phosphate buffer (pH 6.0) containing 0.02% NaN3, and 0.05 U/mL α-amylase (final well

232

concentration). NaN3 was added as a preservative, and furthermore it has been reported that NaN3 in

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the buffer contributes to improvement of the enzyme activity as well.20 After 10 min. incubation,

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CNP-3G (1.0 mM) was added to start the reaction, whereafter the absorbance was measured at 405

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nm every third min. for 30 min to obtain cleavage rates. Chloride ions are necessary for activation

236

of the enzyme´s hydrolyzing property,22 and sodium chloride was therefore added to the phosphate

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buffer in concentrations of 0-100 mM. As seen in Fig. 1A, the maximal enzyme activity was

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reached at 60 mM NaCl, and no further increase in enzyme activity was observed at higher

239

concentrations. Likewise, calcium is required to stabilize and activate the enzyme,23 and therefore

240

calcium acetate was added to the buffer in concentrations of 0-2.5 mM. As seen in Fig. 1B, maximal

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enzyme activity was achieved at a calcium acetate concentration of 1.0 mM. Thus, further

242

experiments were performed with 0.1 mM phosphate buffer containing 60 mM sodium chloride, 1.0

243

mM calcium acetate and 0.02 % NaN3.

244

The microplate-based α-amylase inhibition assay used in a HPLC microfractionation setting

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requires subsequent evaporation of the HPLC solvent to allow use of high amount of organic

246

solvent in the elution solvent. Subsequent dissolution in the above-described buffer containing

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DMSO improves dissolution of lipophilic constituents, but high amounts of DMSO are known to

248

interfere with enzymes. Thus, the activity of α-amylase was assessed with DMSO concentrations in


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the range 0-20% as seen in Fig. 2A. Maximum enzyme activity was achieved at 10% DMSO, i.e., a

250

twofold increase in activity compared to no DMSO in the buffer; and DMSO did not decrease

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enzyme activity of porcine α-amylase dramatically at the tested concentrations. This confirms the

252

robustness of porcine α-amylase in presence of DMSO as previously described.24

253

The enzyme activities at different temperatures were assessed to determine the optimal incubation

254

temperature, but also to assure enzyme stability during preincubation as well as incubation. As seen

255

in Fig. 2B, enzyme activity increased significantly from 25 °C to 34°C, with only a slight decrease

256

at 37 °C and 40 °C. The enzymatic cleavage rate at 37 °C remained constant during the 30 min of

257

incubation, and 37 °C was therefore chosen as optimal temperature for all further experiments.

258

The highest cleavage rate of CNP-G3 with porcine α-amylase was observed at pH 6 (Fig. S1

259

Supporting Information), which is comparable with previous findings of pH optimum for pancreatic

260

and salivary human α-amylase.20 All results are based on cleavage rates, i.e., the slopes of the

261

absorbance values plotted against measurement times, which i) makes it possible to monitor the

262

linearity of the reaction during the entire incubation time, and ii) prevents the need for subtracting

263

background absorbance for analytes that absorbs at 405 nm. However, we also explored the use of

264

endpoint measurements after 30 min., especially because the increased absorbance of 2-

265

chlorophenol under alkaline conditions might increase the sensitivity of the assay. In Fig. S2 in

266

Supporting Information, the endpoint measurements at pH 5-7.5, i.e., absorbance measurements at

267

30 min., are compared with absorbance measurements after basifying all wells to pH 12 after 30

268

min. It is clear that adjusting pH to 12 yields an increase in absorbance; with the relative increase

269

being highest at incubation performed at low pH values. For pH 6 the adjustment to pH 12 gives an

270

increase of approximately 20%, but given the potential problems with background absorbance of

271

analytes from HPLC microfractionation, the use of cleavage rates are preferred and used for the

272

remainder of this work. A further warning of using endpoint measurements after adjusting to pH 12


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is the autohydrolysis of CNP-G3 as shown in Fig. S3 in Supporting Information. Finally, the

274

stability of α-amylase stored at −18 °C as ready-to-use aliquots of enzyme solution were assessed as

275

a means of avoiding repeated freeze-thaw cycles of crude enzyme. Thus aliquots corresponding to

276

the amount of enzyme needed for one 96-well microplate were prepared, and the enzyme activity of

277

these aliquots were measured for 8 weeks. Fig. S4 in Supporting Information shows that the enzyme

278

had maximum activity within the first two weeks, whereafter the enzyme activity decreased. Thus,

279

it is unfortunately not advisable to store porcine α-amylase in solution. With the above-described

280

conditions, (−)-epicatechin, myricetin and luteolin showed IC50 values of 2,300 ± 45 µM, 37 ± 4.7

281

µM, and 33 ± 8.9 µM, respectively.

282

Validation of HPLC method. A mixture of three compounds with α-amylase inhibitory activity,

283

i.e., (−)-epicatechin (2), myricetin (3) and luteolin (4), and three compounds without α-amylase

284

inhibitory activity, i.e., brucine (1), coumarin (5) and trolox (6), was prepared. A reversed-phase

285

HPLC separation that allowed baseline separation of 1-6 in the retention time range 7.5 to 20 min.

286

was developed. The HPLC method was validated by determining linearity, sensitivity (limit of

287

detection, LOD and limit of quantification, LOQ) and precision (intra-day and inter-day). The data

288

are given in Table 1, and demonstrates a satisfactory performance of the HPLC method to be used

289

for high-resolution α-amylase inhibition profiling.

290

Microplate-based high-resolution α-amylase inhibition profiles (proof-of-concept with

291

artificial mixture). Using the above-described HPLC method for separation of 1-6 (see Fig. 3), the

292

HPLC eluate from 7.5 to 20 min. was fractionated into 88 wells of a 96-well microplate. The

293

HPLC-solvent was subsequently evaporated, and the α-amylase inhibition assay was performed for

294

all wells as described above. The α-amylase cleavage rate of each well was calculated as percent

295

inhibition (using the average of the last column of the microplate as blank control), and the results


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were plotted underneath the HPLC trace at 254 nm as well as the trace at 230 nm. The resulting

297

high-resolution biochromatogram with a resolution of 7 data points per min. is shown in Fig. 4.

298

From the figure it is evident that there is a perfect retention time match between the peaks of (−)-

299

epicatechin (2), myricetin (3) and luteolin (4) in the HPLC chromatogram and the bioactivity peaks

300

in the high-resolution α-amylase inhibition profile – in agreement with these compounds possessing

301

α-amylase inhibitory activity. The weak peak for (−)-epicatechin in the biochromatogram is in

302

agreement with the high IC50 value of 2,300 µM for 2 compared to the IC50 values of 37 µM and 33

303

µM for 3 and 4, respectively. Likewise, there are no observable bioactivity peaks corresponding

304

with peaks for brucine (1), coumarin (5) and trolox (6) in the HPLC chromatogram. Thus, this

305

proof-of-concept experiment shows that the high-resolution α-amylase inhibition profile is an

306

excellent tool for pinpointing individual constituents with α-amylase inhibitory activity – and

307

thereby focusing subsequent dereplication or full structure elucidation towards the bioactive peaks

308

only. The peaks in the high-resolution α-amylase inhibition profile are marginally broader than the

309

corresponding peaks in the HPLC chromatogram, which might cause problems with closely eluting

310

peaks. However, if needed, the resolution of the biochromatogram can be increased by fractionating

311

into two 96-well plates and subsequently combine the results from α-amylase inhibition assaying of

312

both microplates. However, one should be aware of the lower sensitivity of the α-amylase

313

inhibition assay that evidently will follow from fractionating one HPLC peak into more wells in the

314

microplate.

315

High-resolution α-amylase inhibition coupled with HPLC−HRMS−SPE−NMR for analysis of

316

cinnamon extract. Cinnamon has previously been shown to reduce serum glucose, triglyceride,

317

LDL cholesterol, and total cholesterol for type 2 diabetics,25 and successive extraction with polar to

318

nonpolar solvent showed 50% inhibition of the relative activity of porcine pancreatic α-amylase in


14

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319

vitro at a concentration of 0.06 g/mL isopropanol extract.26 The fractionation might however

320

concentrate the active constituent(s), and we therefore wanted to investigate crude extract of

321

cinnamon. Crude methanol extract was prepared, and the IC50 value of the crude extract was 0.200

322

± 0.061 µg/mL. A total of 25 µg of crude methanol extract were subjected to analytical-scale

323

reversed-phase HPLC, and the eluate from 11-27 min. was fractionated into 88 wells (resolution =

324

5.5 data points per min.). After evaporation to dryness, the α-amylase inhibition of each well was

325

determined and plotted at their respective retention time to give the high-resolution

326

biochromatogram shown in Fig. 5. It is clearly seen that only the major peak at Rt = 22 min. showed

327

α-amylase inhibitory activity. Subsequent HPLC−HRMS−SPE−NMR analysis showed the major

328

peak to be cinnamaldehyde (see Supporting Information Table S1 for 1H NMR data assignment and

329

Fig. S5-S10 for annotated MS, 1H NMR, and 2D COSY, NOESY, HSQC, and HMBC spectra

330

obtained in the HPLC−HRMS−SPE−NMR mode).

331

In conclusion, a microplate-based high-resolution α-amylase inhibition assay that is compatible

332

with HPLC−HRMS−SPE−NMR was developed. The proof-of-concept study with an artificial

333

mixture as well as the investigation of crude methanol extract allowed direct identification of α-

334

amylase inhibitors by observing correlations between peaks in the high-resolution α-amylase

335

inhibition profile and the HPLC chromatogram

336 337

ASSOCIATED CONTENT

338

Supporting Information.

339

Fig. S1-S4 showing details from bioassay optimization, Fig. S5-S10 showing annotated MS, 1H

340

NMR, and 2D COSY, NOESY, HSQC, and HMBC spectra of cinnamaldehyde, and Table S1

341

showing 1H NMR data of cinnamaldehyde. This material is available free of charge via the Internet

342

at http://pubs.acs.org.


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343 344

AUTHOR INFORMATION

345

Corresponding Author

346

* Phone: +45 3533 6177. Fax: +45 3533 6001. Email: [email protected].

347

Author Contributions

348

The manuscript was written through contributions of all authors. All authors have given approval to

349

the final version of the manuscript.

350 351

Funding

352

HPLC equipment used for high-resolution bioassay profiles was obtained via a grant from The

353

Carlsberg Foundation. The 600 MHz HPLC−HRMS−SPE−NMR system used in this work was

354

acquired through a grant from 'Apotekerfonden af 1991', The Carlsberg Foundation, and the Danish

355

Agency for Science, Technology and Innovation via the National Research Infrastructure funds.

356 357

Notes

358

The authors declare no competing financial interest.

359 360 361

REFERENCES (1)

85-3. Online version at http://www.idf.org/diabetesatlas. Assessed july 24, 2014.

362 363

(2)

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Marcovecchio, M. L., Lucantoni, M., Chiarelli, F. Role of chronic and acute hyperglycemia in the development of diabetes complications. Diabetes Technol. Ther. 2011, 13, 389-394.

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International Diabetes Federation. IDF Diabetes Atlas, 6th edition, 2013. ISBN: 2-930229-

(3)

Whitcomb, D.C; Lowe, M.E. Human pancreatic digestive enzymes. Dig. Dis. Sci. 2007, 52, 1-17.


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(4)

Eichler, H.G.; Korn, A; Gasic, S; Pirson, W; Businger, J. The effect of a new specific α-

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amylase inhibitor on post-prandial glucose and insulin excursions in normal subjects and

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Type 2 (non-insulin-dependent) diabetic patients. Diabetologia 1984, 26, 278-281.

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Fujisawa, T; Ikegami, H; Inoue, K.; Kawabata, Y; Ogihara, T. Effect of two alpha-

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glucosidase inhibitors, voglibose and acarbose, on postprandial hyperglycemia correlates

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with subjective abdominal symptoms. Metab. Clin. Exp. 2005, 54, 387-390.

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pharmacokinetic properties and therapeutic potential. Drugs 1988, 3, 214-243.

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Clissold, S.P; Edwards, C. Acarbose. A preliminary review of its pharmacodynamics and

(7)

Etxeberria, U; de la Garza, A.L.; Campión, J; Martínez, J.A.; Milagro, F.I. Antidiabetic

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effects of natural plant extracts via inhibition of carbohydrate hydrolysis enzymes with

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emphasis on pancreatic alpha amylase. Expert Opin. Ther. Targets 2012, 16, 269-297.

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revisited: A 1536 well high resolution screening assay. Anal. Chem. 2009, 81, 5460-5466

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Giera, M.; Heus, F.; Janssen, L.; Kool, J.; Lingeman, H.; Irth, H. Microfractionation

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Sprogøe, K.; Stærk, D.; Jäger, A.K.; Adsersen, A.; Hansen, S.H.; Witt, M.; Landbo, A.-K.

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R.; Meyer, A.S.; Jaroszewski, J.W. Targeted natural product isolation guided by HPLC-

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SPE-NMR: constituents of Hubertia species. J. Nat. Prod. 2007, 70, 1472-1477.

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(10) Sprogøe, K.; Stærk, D.; Ziegler, H.L.; Jensen, T.H.; Holm-Møller, S.B.; Jaroszewski, J.W.

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Combining HPLC-PDA-MS-SPE-NMR with circular dichroism for complete natural

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product characterization in crude extracts: levorotatory gossypol in Thespesia danis. J. Nat.

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Prod. 2008, 71, 516-519.

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(11) Grosso, C.; Jäger, A.K.; Staerk, D. Coupling of a high-resolution monoamine oxidase-A

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inhibitor assay and HPLC-SPE-NMR for advanced bioactivity profiling of plant extracts.

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Phytochem. Anal. 2013, 24, 141-147.


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(12) Agnolet, S.; Wiese, S.; Verpoorte, R.; Staerk, D. Comprehensive analysis of commercial

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willow bark extracts by new technology platform: Combined use of metabolomics, high-

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performance liquid chromatography-solid-phase extraction-nuclear magnetic resonance

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spectroscopy and high-resolution radical scavenging assay. J. Chromatogr., A 2012, 1262,

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130-137.

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(13) Wiese, S.; Wubshet, S.G.; Nielsen, J.; Staerk, D. Coupling HPLC-SPE-NMR with a

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microplate-based high-resolution antioxidant assay for efficient analysis of antioxidants in

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food - Validation and proof-of-concept study with caper buds. Food Chem. 2013, 141, 4010-

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4018.

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(14) Wubshet, S.G.; Nyberg, N.T.; Tejesvi, M.V.; Pirttilä, A.M.; Kajula, M.; Mattila, S.; Staerk,

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D. Targeting high-performance liquid chromatography-high-resolution mass spectrometry-

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solid-phase extraction-nuclear magnetic resonance analysis with high-resolution radical

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scavenging profiles - bioactive secondary metabolites from the endophytic fungus

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Penicillium namyslowskii. J. Chromatogr., A 2013, 1302, 34-39.

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(15) Kongstad, K. T.; Wubshet, S.G.; Johannesen, A.; Kjellerup, L.; Winther, A.-M.L.; Jäger,

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A.K.; Staerk, D. High-resolution screening combined with HPLC–HRMS–SPE–NMR for

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identification of fungal plasma membrane H+-ATPase inhibitors from plants. J. Agric. Food

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Chem. 2014, 62, 5595-5602.

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(16) Schmidt, J.S.; Lauridsen, M.B.; Dragsted, L.O.; Nielsen, J.; Staerk, D. Development of a

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bioassay-coupled HPLC-SPE-ttNMR platform for identification of α-glucosidase inhibitors

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in apple peel (Malus × domestica Borkh.). Food Chem. 2012, 135, 1692-1699.

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(17) Wubshet, S. G.; Schmidt J. S.; Wiese S.; Staerk D. High-Resolution Screening Combined

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with HPLC-HRMS-SPE-NMR for Identification of Potential Health-Promoting Constituents


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in Sea Aster and Searocket - New Nordic Food Ingredients. J. Agric. Food Chem. 2013, 61,

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8616-8623.

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(18) Xiao, Z.; Storms, R.; Tsang, A. A quantitative starch-iodine method for measuring alphaamylase and glucoamylase activities. Anal. Biochem. 2006, 351, 146-148. (19) Soor, S.K.; Hincke M.T. Microplate reader-based kinetic determination of α-amylase

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activity: Application to quantitation of secretion from rat parotid acini. Anal. Biochem. 1990,

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188, 187-191.

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(20) Winn-Deen, E.S; David, H; Sigler, G; Chavez, R. Development of a direct assay for αamylase. Clin. Chem. 1988, 34, 2005-2008. (21) Gella, F.J.; Gubern, G.; Vidal, R.; Canalias, F. Determination of total and pancreatic α-

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amylase in human serum with 2-chloro-4-nitrophenyl-α-D-maltotrioside as substrate. Clin.

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Chim. Acta 1997, 259, 147-160.

425 426 427

(22) Wakim, J.; Robinson, M.; Thoma, J.A. The active site of porcine-pancreatic alpha-amylase: Factors contributing to catalysis. Carbohydr. Res. 1969, 10, 487-503. (23) Buisson, G.; Duée, E.; Haser, R.; Payan, F. Three dimensional structure of porcine

428

pancreatic alfa-amylase at 2.9 Å resolution. Role of calcium in structure and acticity. EMBO

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J. 1987, 6, 3909-3916.

430

(24) Tao, B.Y.; Reilly, P.J.; Robyt, J.F. Detection of a covalent intermediate in the mechanism of

431

action of porcine pancreatic alpha-amylase by using 13C nuclear magnetic resonance.

432

Biochim. Biophys. Acta 1989, 995, 214-220

433 434

435 436

(25) Khan, A.; Safdar, M.; Khan, M.M.A.; Khattak, K.N.; Anderson, R.A. Cinnamon improves glucose and lipids of people with type 2 diabetes. Diabetes Care 2003, 26, 3215-3218. (26) Ponnusamy, S.; Ravindran, R.; Zinjarde, S.; Bhargava, S.; Kumar, A.R. Evaluation of traditional Indian antidiabetic medicinal plants for human pancreatic amylase inhibitory


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effect in vitro. Evid.-based Complement. Altern. Med. 2010, 2011, 1-10.

438 439

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441

FIGURE CAPTIONS

442 443

Figure 1. A: Relative enzyme activity at different chloride ion concentrations. The enzyme activity

444

in buffer containing 60 mM Cl- is normalized to 100 %. B: Relative enzyme activities at calcium ion

445

concentrations in the range 0-2.5 mM. Data represent mean ± standard deviation of three replicate

446

measurements.

447 448

Figure 2. A: Relative enzyme activities at different DMSO concentrations. The enzyme activity in

449

buffer containing 0 % DMSO is normalized to 100 %. B: Relative enzyme activities at different

450

temperatures. The enzyme activity at 25 °C normalized to 100 %. Data represent mean ± standard

451

deviation of three replicate measurements.

452 453

Figure 3. Structures of α-amylase inhibitors (−)-epicatechin (2), myricetin (3) and luteolin (4), and

454

inactive constituents brucine (1), coumarin (5) and trolox (6) used for proof-of-concept of the

455

microplate-based high-resolution α-amylase inhibition profiling developed in this work.

456 457

Figure 4. High-resolution biochromatogram of α-amylase inhibitors (−)-epicatechin (2), myricetin

458

(3) and luteolin (4) as well as inactive constituents brucine (1), coumarin (5) and trolox (6). Black

459

line: UV trace at 254 nm. Grey line: UV trace at 230 nm. Red line: high-resolution α-amylase

460

inhibition profile.

461 462

Figure 5. High-resolution biochromatogram of crude methanol extract of cinnamon. Black line: UV

463

trace at 254 nm. Red line: high-resolution α-amylase inhibition profile.


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TABLES Table 1. Parameters for validation of the HPLC method for the reference compounds stated: precision (intraday and interday), linearity and sensitivity (limit of detection [LoD] and limit of quantification [LoQ]).

Compound

Amount oncolumn (µg)

Brucine

2.00

Precision (% RSD) Intraday Interday precisiona precisiona 0.10

2.46

(−)-Epicatechin

0.28

1.29

Myricetin

0.099

1.41

Luteolin

0.076

1.92

Coumarin

0.71

1.10

Trolox

0.21

5.45

0.45

5.24

(−)-Epicatechin

0.32

2.55

Myricetin

0.51

3.05

Luteolin

0.34

2.78

Coumarin

0.041

1.65

Trolox

0.45

3.79

0.99

2.75

(−)-Epicatechin

0.23

1.09

Myricetin

0.27

1.12

Luteolin

0.27

0.78

Coumarin

0.60

1.12

Trolox

1.64

3.12

Brucine

Brucine

a

1.50

1.00

Sensitivity (µg) Linearityb A = 1987.2c + 395.11 r = 0.997 A = 442.38c - 5.4712 r = 0.9998 A = 6198.5c + 182.56 r = 0.9968 A = 7168.5c + 10.307 r = 0.9999 A = 4266.7c - 8.6232 r = 0.9999 A = 221.02c + 51.112 r = 0.999

LoD

LoQ

0.37

1.22

0.15

0.49

0.012

0.040

0.0053

0.018

0.039

0.13

0.79

2.64

n = 4, b A = peak areas; c = concentration of reference compound; r = correlation coefficient


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Figure 1. 52x15mm (600 x 600 DPI)



Figure 2. 50x14mm (600 x 600 DPI)


Page 24 of 28

Page 25 of 28


Figure 3. 111x138mm (600 x 600 DPI)



Figure 4. 57x39mm (600 x 600 DPI)


Page 26 of 28

Page 27 of 28


Figure 5. 75x62mm (600 x 600 DPI)



Table of Contents Graphic 46x26mm (600 x 600 DPI)


Page 28 of 28

High-Resolution Î±-Amylase Assay Combined with High-Performance

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