Coumarins from Angelica decursiva inhibit α-glucosidase activity and protein tyrosine phosphatase 1B

Accepted Manuscript Coumarins from Angelica decursiva inhibit α-glucosidase activity and protein tyrosine phosphatase 1B Md Yousof Ali, Susoma Jannat, Hyun Ah Jung, Hyong Oh Jeong, Hae Young Chung, Jae Sue Choi PII:

S0009-2797(16)30143-0

DOI:

10.1016/j.cbi.2016.04.020

Reference:

CBI 7659

To appear in:

Chemico-Biological Interactions

Received Date: 15 December 2015 Revised Date:

16 March 2016

Accepted Date: 11 April 2016

Please cite this article as: M.Y. Ali, S. Jannat, H.A. Jung, H.O. Jeong, H.Y. Chung, J.S. Choi, Coumarins from inhibit α-glucosidase activity and protein tyrosine phosphatase 1B, Chemico-Biological Interactions (2016), doi: 10.1016/j.cbi.2016.04.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Title: Coumarins from Angelica decursiva inhibit α-glucosidase activity and protein

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tyrosine phosphatase 1B

Running title: Anti-diabetic activities of coumarins from Angelica decursiva

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Authors:

Md. Yousof Ali1, Susoma Jannat1, Hyun Ah Jung2**, Hyong Oh Jeong3, Hae Young Chung3, Jae

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Sue Choi1*

Affiliations: 1

Department of Food and Life Science, Pukyong National University, Busan 608-737, Republic

of Korea

Department of Food Science and Human Nutrition, Chonbuk National University, Jeonju 561-

756, Republic of Korea

College of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea

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*Correspondence to:

Jae Sue Choi, Department of Food and Life Science, Pukyong National University, Busan 608737, Republic of Korea [Tel.: +82-51-629-5845; Fax: +82-51-629-5842; E-mail: [email protected]

**Co-corresponding author: Hyun Ah Jung, Department of Food Science and Human Nutrition, Chonbuk National University, Jeonju 561-756, Republic of Korea. Tel.: 82-63-2704882. Fax: 82-63-270-3854. E-mail: [email protected]. 1

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ABSTRACT In the present study, we investigated the anti-diabetic potential of six natural coumarins, 4-

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hydroxy Pd-C-III (1), 4′-methoxy Pd-C-I (2), decursinol (3), decursidin (4), umbelliferone 6carboxylic acid (5), and 2′-isopropyl psoralene (6) isolated from Angelica decursiva and evaluated their inhibitory activities against protein tyrosine phosphatase 1B (PTP1B), α-

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glucosidase, and ONOO‒-mediated protein tyrosine nitration. Coumarins 1-6 showed potent PTP1B and α-glucosidase inhibitory activities with ranges of IC50 values of 5.39–58.90 µM and

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65.29–172.10 µM, respectively. In the kinetic study for PTP1B enzyme inhibition, compounds 1, 5, and 6 were competitive, whereas 2 and 4 showed mixed type, and 3 displayed noncompetitive type inhibition. For α-glucosidase enzyme inhibition, compounds 1 and 3 exhibited good mixedtype, while 2, 5, and 6 showed noncompetitive and 4 displayed competitive type inhibition. Furthermore, these coumarins also effectively suppressed ONOO‒-mediated tyrosine nitration in

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a dose-dependent manner. To further investigate PTP1B inhibition, we generated a 3D structure of PTP1B using Autodock 4.2 and simulated the binding of compounds 1–6. Docking

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simulations showed that different residues of PTP1B interacted with different functional groups of compounds 1-6 through hydrogen and hydrophobic interactions. In addition, the binding

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energies of compounds 1–6 were negative, suggesting that hydrogen bonding may stabilize the open form of the enzyme and potentiate tight binding of the active site of PTP1B, thereby resulting in more effective PTP1B inhibition. These results demonstrate that the whole plant of A. decursiva and its coumarins are useful as potential functional food ingredients for the prevention and treatment of type 2 diabetes. Keywords: Angelica decursiva, coumarin, PTP1B, α-glucosidase, molecular docking simulation, enzyme kinetic study 2

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Abbreviations

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DMSO, dimethyl sulfoxide; IC50, inhibitory concentrations 50%; (PTP1B), protein tyrosine phosphatase 1B; DM, diabetes mellitus; ONOO‒ , nitrotyrosine; CDCl3, chloroform; pNPP, p-nitrophenyl phosphate; EDTA, ethylenediaminetetraacetic acid; pNPG, p-nitrophenyl α-D-

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glucopyranoside; DTT, dithiothreitol; TLC, thin layer chromatography

1. Introduction

Diabetes mellitus (DM) is a chronic metabolic disease characterized by hyperglycemia resulting from reduced insulin secretion and/or insulin resistance [1]. It is widely known that

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elevation of blood glucose caused by disruption of carbohydrate, protein, and fat metabolism can lead to diabetic complications in several organs and tissues, including eyes, kidneys, nerves, and blood vessels [2]. Several therapeutic approaches have been proposed, including inhibitors of

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PTP1B and α-glucosidase. Protein tyrosine phosphatases (PTPs) play a critical role in the

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regulation of a variety of cellular processes, such as growth, proliferation, differentiation, metabolism, immune response, cell-cell adhesion, and cell-matrix contacts [3]. PTP1B is a major non-transmembrane phosphotyrosine phosphatase in human tissues and is a known negative regulator of the insulin-stimulated signal transduction pathway [4]. Despite the identification of many potent compounds, a PTP1B-inhibiting drug has yet to reach the clinic, because of, these molecules still lack efficacy in vivo because they have weak oral bioavailability, poor membrane permeability and weak selectivity [5]. Therefore, it is still essential to search for new inhibitors 3

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with better safety and high efficacy. Modern medicine indicates that one of the most effective therapeutic approaches for controlling blood glucose level is to inhibit absorption of glucose by

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suppressing carbohydrate-hydrolyzing enzymes such as α-glucosidase [6–8]. In addition, nitrotyrosine is a product of ONOO‒ action and thus, the production of ONOO‒ can be indirectly inferred by the presence of nitrotyrosine residues [9]. Recently, much attention has been paid to

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the role of nitrotyrosine as a possible risk factor in diabetes, as increased levels of nitrotyrosine have been reported in the plasma of diabetic patients [10], and there is evidence that an acute

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increase in glycemia induces an increase in nitrotyrosine [11]. It has also been reported that postprandial hyperglycemia is accompanied by nitrotyrosine generation [12]. So, PTP1B, αglucosidase, and nitrotyrosine are therefore attractive targets in the development of new treatments for DM and other related metabolic syndromes.

Angelica decursiva Fr. et Sav (Umbelliferae) is a perennial herb and it is widely distributed in

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China, Japan, and Korea. This plant has been long used in traditional Korean medicine as an antitussive, analgesic, antipyretic, tumor suppressor, and cough remedy [13,14], while in

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traditional Chinese medicine, it is used as a remedy for thick phlegm, asthma, and upper respiratory tract infections [15–17]. This plant is a rich source of different types of coumarin

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derivatives which include nodakenin, nodakenetin, isorutarine, umbelliferone, umbelliferone 6carboxylic acid, 2'-isopropyl psoralene, cis-3′-acetyl-4′-angeloylkhellactone, 3′(R)-O-acetyl4′(S)-O-tigloylkhellactone, columbianadin, Pd-C-I, Pd-C-II, Pd-C-III, 4-hydroxy Pd-C-III, (+)decursidinol, decursin, and decursidin [13, 18–22], which have been reported to possess a wide range of biological activities, including anti-inflammatory, antioxidant, neuroprotective, antidiabetic, and anti-Alzheimer activities [13, 18–20, 22–24]. Despite the potentiality of different Angelica species including A. decursiva and its 4

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constituents (nodakenin, nodakenetin, 3′(R)-O-acetyl-4′(S)-O-tigloylkhellactone, isorutarine, umbelliferone, and cis-3′-acetyl-4′-angeloylkhellactone) as PTP1B and α-glucosidase weak

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inhibitors [18], there has been no detailed investigation into the possibility of developing antidiabetic drugs via enzyme kinetic and molecular docking evaluation. Therefore, as a part of our continuous research to identify potent anti-diabetic agents from A. decursiva, we isolated and

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investigated the activity of six coumarins against PTP1B, α-glucosidase, and nitrotyrosine. Enzyme kinetic analyses of the compounds 1-6 were also performed by using Dixon and

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Lineweaver-Burk plots in order to confirm the type of enzymatic inhibition and to propose guidelines for coumarins as anti-diabetic agents in discovering new drugs. Since there is currently no detailed information regarding the molecular interactions between compounds 1-6 and PTP1B, we performed molecular docking analysis and detailed enzyme kinetic analysis in

2. Materials and methods

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order to investigate the possibility of using compounds 1-6 as anti-diabetic drug candidates.

The 1H- and

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2.1. General experimental procedures

C-NMR spectra were acquired using a JEOL JNM ECP-400 spectrometer at

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400 and 100 MHz, respectively, in deuterated solvents chloroform (CDCl3). Column chromatography was conducted using silica gel 60 (70–230 mesh, Merck, Darmstadt, Germany), sephadex LH20 (20–100 µm, Sigma, St. Louis, MO, USA), and LiChroprep® RP-18 (40–63 lm, Merck). All TLC was conducted on pre-coated Merck Kieselgel 60 F254 plates (20 × 20 cm, 0.25 mm, Merck) and using 50% H2SO4 as a spray reagent.

2.2. Chemicals and reagents 5

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Yeast α-glucosidase, acarbose, p-nitrophenyl phosphate (pNPP), p-nitrophenyl α-Dglucopyranoside (pNPG), and ethylenediaminetetraacetic acid (EDTA) were purchased from

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Sigma-Aldrich. PTP1B (human recombinant) was purchased from Biomol International LP (Plymouth Meeting, PA, USA), and dithiothreitol (DTT) was purchased from Bio-Rad Laboratories (Hercules, CA, USA). All other chemicals and solvents used were purchased from

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E. Merck, Fluka, and Sigma-Aldrich, unless otherwise stated.

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2.3. Isolation of coumarins from A. decursiva

The coumarins 4-hydroxy Pd-C-III, 2'-isopropyl psoralene, decursidin and umbelliferone 6carboxylic acid were previously isolated and identified in our laboratory [13, 20, 22]. 4′-Methoxy Pd-C-I and decursinol was isolated from subfraction-4, and 5 respectively of dichloromethane fraction from A. decursiva. 4′-Methoxy Pd-C-I and decursinol were identified by spectroscopic

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evidence including 1H and 13C–NMR, as well as by comparison with spectral published data [25

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–27]. The structures of all isolated compounds are shown in Fig. 1.

2.4. Assay for PTP1B inhibition

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The PTP1B inhibitory activity was evaluated using pNPP Cui et al. [28]. In each well of a 96-well plate (each with a final volume of 100 µL), 40 µL of PTP1B enzyme [0.5 units diluted with a PTP1B reaction buffer containing 50 mM citrate (pH 6.0), 0.1 M NaCl, 1 mM EDTA, and 1 mM DTT] were added with or without sample dissolved in 10% DMSO. The plate was preincubated at 37°C for 10 min and then 50 µL of 2 mM pNPP in PTP1B reaction buffer was added. Following incubation at 37°C for 20 min, the reaction was terminated by the addition of 10 M NaOH. The amount of p-nitrophenyl produced after enzymatic dephosphorylation of pNPP 6

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was estimated by measuring the absorbance at 405 nm using a microplate spectrophotometer (Molecular Devices). The nonenzymatic hydrolysis of 2 mM pNPP was corrected for the

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measured increase in absorbance at 405 nm obtained in the absence of PTP1B enzyme. The inhibition percentage was obtained using the following equation: % inhibition = (Ac − As)/Ac ×100, where Ac is the absorbance of the control and As is the absorbance of the sample. Ursolic

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acid was used as a positive control.

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2.5. Assay for α-glucosidase inhibition

The enzyme inhibition study was performed spectrophotometrically using the procedure reported by Li et al. [29]. A total of 60 µL of reaction mixture containing 20 µL of 100 mM phosphate buffer (pH 6.8), 20 µL of 2.5 mM pNPG, and 20 µL of the sample dissolved in 10% DMSO, was added to each well followed by 20 µL of α-glucosidase [0.2 U/mL in 10 mM

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phosphate buffer (pH 6.8)]. The plate was incubated at 37°C for 15 min, and 80 µL of 0.2 M sodium carbonate solution was then added to stop the reaction. Immediately thereafter, the

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absorbance was recorded at 405 nm using a microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). The control contained the same reaction mixture except with an

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equivalent volume of phosphate buffer instead of any sample solution. Acarbose dissolved in 10% DMSO was used as a positive control. The inhibition percentage (%) was obtained using the same equation as in the PTP1B enzymatic assay.

2.6. Inhibition of ONOO– -mediated protein tyrosine nitration ONOO– -mediated protein tyrosine nitration was evaluated using the method of Aulak et al. [30], with slight modifications. Various concentrations of coumarins dissolved in 10% DMSO 7

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were added to 95 µL BSA (0.4 mg protein/mL) and mixed with 2.5 µL ONOO– (200 µM). After incubation with shaking at 37°C for 20 min, the sample was added to Bio-Rad gel buffer in a

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ratio of 1:1 and boiled for 5 min to denature the proteins. The total protein equivalent for the reactant was separated on 10% SDS-polyacrylamide minigel at 80 V for 30 min and 100 V for 1 h, and then transferred to a PVDF membrane at 80 V for 110 min using a wet transfer system

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(Bio-Rad). The membrane was immediately placed in a blocking solution (5% non-fat dry milk in TBS-Tween buffer (w/v), Bio-Rad TBS, and 0.1% Tween-20, pH 7.4) at room temperature for

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1 h. The membrane was washed three times (10 min each wash) in TBS-Tween buffer and incubated with a monoclonal anti-nitrotyrosine antibody (diluted 1:2,500 in TBS-Tween buffer with 5% non-fat dry milk) at 4°C overnight. After three more washes in TBST buffer (10 min and 5 min), the membrane was incubated with horseradish peroxidase-conjugated sheep antimouse secondary antibody diluted 1:2,000 in TBST buffer at room temperature for 1 h. After

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three washes in TBST buffer, antibody labeling was visualized using the Supersignal West Pico Chemiluminescent substrate (Pierce, Rockford, IL, USA) according to the manufacturer’s

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instructions, and then the membrane was exposed to x-ray film (Kodak, Rochester, NY, USA).

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Pre-stained blue protein markers were used for molecular weight determination.

2.7. Kinetic parameters of coumarins in both PTP1B and α-glucosidase inhibition– Lineweaver–Burk and Dixon plots In order to determine the kinetic mechanism, two kinetic methods using Lineweaver–Burk and Dixon plots were complementarily used [31–33]. Using Lineweaver–Burk and Dixon plots, PTP1B inhibition mode was determined at various concentrations of pNPP substrate (0.5, 1.0, and 2.0 mM) in the absence or presence of different test coumarin concentrations (0, 0.8, 4.0, and 8

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20.0 µM for 1 and 5; 0, 4.0, 20.0, and 100 µM for 2, 3, 4, and 6). Each enzymatic inhibition of test coumarins was evaluated by monitoring the effects of different concentrations of the

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substrates in the Dixon plots (single reciprocal plot). In addition, Lineweaver–Burk and Dixon plots for α-glucosidase inhibition of coumarins were also performed in the presence of different concentrations of substrate: 2.5, 1.25, and 0.625 mM pNPG. The test concentrations of the

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coumarins in the α-glucosidase kinetic analysis were as follows 0, 31.5, 62.5, and 125 µM for 3; 0, 62.5, 125, and 250 µM for 1, 2, 4, 5, and 6. Both enzymatic procedures consisted of the same,

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aforementioned PTP1B and α-glucosidase assay methods. The inhibition constants (Ki) were determined by interpretation of the Dixon plots, where the value of the x-axis implies -Ki.

2.8. Molecular docking simulation in PTP1B inhibition – Autodock 4.2 Potent, selective PTP1B inhibitor compound 23 using a linked-fragment strategy (PDB ID:

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1NNY) [34], a X-ray crystallographic structure obtained from RCSB Protein Data Bank website with a resolution of 2.40Å. This protein structure was regulated using X-ray diffraction method.

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The reported heteroatom compound 23 was removed, protein considered as ligand free. Water molecules also removed from protein structure for docking simulation using Accelrys Discovery

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Studio 4.1 (DS 4.1) (DS, http://www.accelrys.com; Accelrys, Inc. San Diego, CA, USA). Hydrogen atoms are added into protein using automated docking tool, AutoDock 4.2. In addition, compound 23 binding area of protein considered as most convenient region for ligand binding in docking simulation. The 3D structure of all tested compounds were drawn using Chemsketch 3.5 and automated docking simulation was performed using Autodock tools (ADTs) to assess the appropriate binding orientations and conformations of the ligand molecules with different protein inhibitors. A Lamarkian genetic algorithm method implemented in the program Autodock 4.2 9

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was employed. For docking calculations, Gasteiger charges were added by default and the rotatable bonds were set by the Autodock tools and all torsions were allowed to rotate. The grid

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maps were generated by Autogrid program where grid box size of 126 × 126 × 126 points with a default spacing of 0.375 Å between the grid points was executed covered almost the entire favorable protein binding site. The X, Y, Z center was 37.303, 30.97, 33.501, respectively. The

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docking protocol for rigid and flexible ligand docking consisted of 10 independent Genetic Algorithm (GA) and other parameters are used by defaults of ADTs. Binding aspect of PTP1B

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residues and their corresponding binding affinity score regarded as best molecular interaction. The results were visualized and analyzed using DS 4.1.

2.9. Statistics

All results are expressed as the mean ± SEM of triplicate samples. Statistical significance

3. Results

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was noted at p < 0.05.

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was analyzed using one-way ANOVA and Student’s t-test (Systat Inc., Evanston, IL, USA), and

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3.1. α-Glucosidase and PTP1B inhibitory activity of coumarins isolated from A. decursiva The anti-diabetic potential of coumarins 1-6 was evaluated via their inhibitory activities against PTP1B and α-glucosidase. The results of the enzyme inhibitory activities of coumarins are summarized in Table 1. Coumarins 1-6 exhibited potent inhibitory activities against αglucosidase with IC50 values of 77.30 ± 1.07, 89.19 ± 0.77, 65.29 ± 0.81, 79.09 ± 0.11, 172.10 ± 0.19, and 85.82 ± 0.99 µM, respectively, compared to the positive control, acarbose, which had an IC50 value of 183.29 ±1.02 µM. In addition, compounds 1, 2, 4, 5, and 6 showed promising 10

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inhibitory activities against PTP1B with IC50 values of 5.39 ± 0.19, 6.62 ± 0.77, 11.22 ± 0.39, 7.98 ± 0.91, and 10.78 ± 0.17 µM, respectively. The IC50 value of the positive control ursolic

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acid was 5.65 ± 0.29 µM. In contrast, compound 3 showed moderate inhibitory activities against

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PTP1B with an IC50 value of 58.90 ± 1.07 µM.

3.2. Enzyme kinetics in PTP1B and α-glucosidase inhibition

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As part of our continuous search for coumarin derivatives from A. decursiva as potent PTP1B inhibitors, the type of inhibition and inhibition constants (Ki) of coumarins 1–6 were investigated using Dixon and Lineweaver–Burk plots (Figure S1A-F and Figure S2A-F). Each line of inhibitors intersected at the xy-side, indicating mixed-type inhibitors, while the lines that penetrate the same point on the x-intercept represent noncompetitive inhibition and a shared y-

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intercept represents competitive inhibitors in the Lineweaver-Burk plots [31]. Therefore, coumarins 2 and 4 exhibited mixed-type inhibition and 3 displayed noncompetitive-type

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inhibition, while 1, 5, and 6 showed competitive-type PTP1B inhibition (Figure S2A-F). In addition, the Dixon plot is a common method for determining the type of enzyme inhibition and

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the dissociation or inhibition constant (Ki) for an enzyme–inhibitor complex, where the value at the x-axis implies -Ki [32, 33]. As shown in Figure S1A-F, the Ki values of 1–6 against PTP1B were 3.51, 9.63, 41.13, 26.38, 6.45, and 8.30 µM, respectively. Moreover, we also investigated the type of inhibition and inhibition constants (Ki) of coumarins 1–6 against α-glucosidase using Dixon and Lineweaver–Burk plots (Figure S3A-F and Figure S4A-F). Our results revealed that coumarin 4 exhibited competitive inhibition, 1 and 3 showed mixed-type inhibition, and 2, 5, and 6 displayed noncompetitive inhibition in the Lineweaver-Burk plot (Figure S4A-F). As shown in 11

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Figure S3A-F, the Ki values of 1–6 against α-glucosidase were 62.51, 108.51, 52.21, 27.11, 139.54, and 107.64 µM, respectively. As the Ki value represents the concentration needed to form

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an enzyme-inhibitor complex, a lower Ki value may manifest more effective inhibitors against PTP1B and α-glucosidase in the development of preventive and therapeutic agents.

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3.3. Inhibitory effect of the coumarins on ONOO–-mediated tyrosine nitration

In order to determine the inhibitory effect of the coumarins against ONOO–-induced tyrosine

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nitration, western blot analysis was performed using a 3-nitrotyrosine antibody. As shown in Fig. 2A, 2C and 2E, pretreatment with compounds 1, 2, and 4 at different concentrations (12.5–50 µM) resulted in strongly inhibited ONOO–-mediated tyrosine nitration in a concentrationdependent manner. However, pretreatment with 3, 5, and 6 at different concentrations (12.5–100

(Fig. 2B, 2D and 2F).

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µM) resulted in only moderate dose-dependent inhibition of ONOO−-mediated tyrosine nitration

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3.4. Molecular docking study of the inhibitory activity of coumarins 1-6 against PTP1B The molecular docking models of coumarins 1-6 and compound 23 are illustrated in Fig. 3A-

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G and Table 2. The ligand-enzyme complexes with coumarins 1-6/or compound 23 were stably posed in the same pocket of the PTP1B by Autodock 4.2. As illustrated in Fig. 3, the corresponding ligand interactions of 1 interacted with 10 active site amino acids namely PRO39, ASN90, THR91, CYS92, GLY93, ASP137, PHE135, ILE134, MET133, and GLU132 with four hydrogen bonds. The residues ILE134, PHE135, and GLY93 of the enzyme participated in hydrogen bonds interactions with the hydroxyl, ketone, and angeloyl groups of 1. In the case of 2 interacted with 13 active site amino acid residues namely ASP181, TRP179, PHE182, 12

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GLY183, CYS215, GLY218, ARG221, ALA217, GLY220, ILE219, GLN266, GLN262, and THR263 with five hydrogen bonds. Five residues of the enzyme, GLY183, CYS215, GLY218,

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ALA217, and ARG221, participated in five hydrogen-bonding interactions with the ketone, senecioyl groups and oxygen atom of 2. Regarding 3, interacted with 13 active site amino acid residues namely LYS116, PHE182, GLY183, ASP181, TRP179, ARG221, CYS215, GLN266,

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SER216, GLY220, GLY218, ILE219, and ALA217 with four hydrogen bonds. Three residues, GLY220, ARG221, and PHE182, of the enzyme participated in four hydrogen-bonding

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interactions, with the hydroxyl, ketone groups and oxygen atom of 3. For coumarin 4 interacted with 8 active site amino acid residues namely GLN127, PRO126, TRP125, LYS131, GLU132, MET133, ILE134, and PHE135 with two hydrogen bonds. Two residues of the enzyme, ILE134, and GLN127, participated in hydrogen bonds interactions with the senecioyl and ketone groups of 4. In case of 5 interacted with 7 active site amino acid residues namely LYS73, SER80,

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ARG79, GLN78, PRO206, SER205, and LEU204 with five hydrogen bonds. Three residues of the enzyme, LYS73, SER80, and LEU204, participated in five hydrogen-bonding interactions

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with the hydroxyl, ketone, and carboxylic groups of 5. With respect to 6 interacted with 13 active site amino acid residues namely ASP181, PRO180, TRP179, PHE182, GLY183, ARG221,

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GLN266, GLN262, GLY220, ILE219, ALA217, GLY218, and CYS215 with six hydrogen bonds. Five residues of the enzyme, CYS215, GLY218, ILE219, GLY220, and ARG221, participated in six hydrogen-bonding interactions with the ketone group and oxygen atom of 6. In contrast, compound 23 interacted with 22 active site amino acid residues namely TRP179, ARG221, CYS215, SER216, LYS120, ALA217, GLY218, ILE219, GLY220, GLN266, TYR46, VAL49, ASP48, THR263, GLN262, GLY259, MET258, ARG254, TYR20, ARG24, SER28, and ASP29 with 13 hydrogen bonds. Moreover, the binding energies of compounds 1-6 were 13

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negative (–6.21, –7.06, –6.89, –6.78, –5.73, and –6.65 kcal/mol, respectively, and -10.18 kcal/mol for compound 23), indicating that additional hydrogen bonding might stabilize the open

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form of the enzyme and potentiate tighter binding to the active site of PTP1B, resulting in more effective PTP1B inhibition. Our molecular docking results indicated that coumarins 1-6 bound

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tightly at the active site of PTP1B.

4. Discussion

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DM is a well-known metabolic disorder that is characterized by an abnormal postprandial increase in blood glucose level. The control of postprandial hyperglycemia is believed to be important in the treatment of DM. Postprandial hyperglycemia contributes to the development of macro and micro vascular complications associated with diabetes [35]. Therefore, one of the therapeutic approaches in DM is to reduce the demand for insulin by lowering the corresponding

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postprandial hyperglycemic levels [36]. Effective control of hyperglycemia is a prerequisite to control the incidence, progression, and severity of diabetic complications. However, single therapy eventually fails to control hyperglycemia-associated complications. Therefore, additional

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adjunct therapy, such as antioxidants and inhibitors of PTP1B and α-glucosidase, could be

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effective therapeutic options to attenuate the noxious effects of glucose. Research has shown that inhibition of the α-glucosidase enzyme located at the intestinal brush border of the intestine may play a role in the lowering of postprandial hyperglycemia [37, 38]. Furthermore, PTP1B inhibitors can be used to modulate insulin receptor phosphorylation and insulin therapy can reduce hyperglycemia and maintain normoglycemia [39]. Recently, coumarin derivatives have drawn much attention due to their availability, low toxicity, relative cheapness, presence in the diet, and multiple bioactivities [40, 41]. Dietary exposure to coumarin is quite significant, more 14

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than for other compounds, because it is widely found in fruits, vegetables, seeds, nuts, and higher plants. It is estimated that the average Western diet contains ~ 1 g/day, of mixed types of

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coumarin [41]. The uses of coumarin-containing higher plant extracts are also widely popular in Chinese medicine. Recently, we reported that coumarins from A. decursiva have significant antidiabetic activity through the inhibition of PTP1B, α-glucosidase, and RLAR [18]. As part of our

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continuing research to identify more active coumarins from A. decursiva, in the present study, we isolated coumarins 1-6 and evaluated their anti-diabetic potentials in terms of inhibition of

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PTP1B and α-glucosidase enzymes, which are involved in the pathogenesis of DM. Mammalian α-glucosidase are membrane-bound enzymes located in the brush border of the small intestine, where they catalyze the final step in the digestion of complex carbohydrates, thereby releasing absorbable monosaccharides from dietary sources [42]. Inhibition of αglucosidase leads to a decrease in the rate of glucose absorption and results in lowered

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postprandial blood glucose. It has further been shown that α-glucosidase inhibitors can prevent development of diabetes for people with impaired glucose tolerance and/or impaired fasting

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blood glucose [43, 44]. Several α-glucosidase inhibitors, such as acarbose and voglibose obtained from natural sources, can effectively control blood glucose levels after food intake and

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have been used clinically in the treatment of DM [45]. Acarbose is typically administered in combination with other anti-diabetic agents in a polypharmacological approach, but is associated with severe side effects such as abdominal pain, flatulence, and diarrhea [46, 47]. Therefore, it is necessary to search for alternatives that possess α-glucosidase inhibitory activity but without unacceptable side effects. In the present study, we found that the inhibitory potential of coumarins 1-6 against α-glucosidase was much stronger than that of acarbose, a clinically used α-glucosidase inhibitor. Therefore, coumarins 1-6 might be effective in controlling postprandial 15

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hyperglycemia by delaying the breakdown of polysaccharides. In addition, an enzyme kinetic study performed in the presence of varying substrate and inhibitor concentrations revealed that 1

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and 3 showed mixed type α-glucosidase inhibition, meaning these coumarins can bind to both the allosteric site of the free enzyme and to the enzyme/substrate complex, while 2, 5, and 6 displayed noncompetitive α-glucosidase inhibition because they bound to the free enzyme and

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inhibited the formation of the enzyme/substrate complex. On the other hand, coumarin 4 exhibited competitive inhibition against α-glucosidase and bound to the active site of the enzyme

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in order to prevent enzyme-substrate complex formation. Considering the above in vitro and kinetic results, we can conclude that compounds 1-6 have promising anti-diabetic activity through the inhibition of α-glucosidase. These new findings support previous research from our lab showing that coumarins from A. decursiva exhibited significant inhibitory activity against αglucosidase [18].

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PTP1B is a major nontransmembrane protein tyrosine phosphatase and an important factor in non-insulin-dependent DM. PTP1B is localized to the cytoplasmic face of the endoplasmic

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reticulum and is expressed ubiquitously, including in the classical insulin-targeted tissues such as liver, muscle, and fat [48]. Mounting evidence from biochemical, genetic, and pharmacological

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studies support a role for PTP1B as a negative regulator in both insulin and leptin signaling. PTP1B can associate with and dephosphorylate activated insulin receptor or insulin receptor substrates [49, 50]. So, it is not surprising that overproduction of this enzyme has been implicated in the onset of type II diabetes [51]. Recently, structure-based simulation of PTP1B inhibitors has been employed for developing novel therapeutic drugs with selectivity and cell permeability [52]. The main structural features of PTP1B have been well established and it is known to consist of 435 amino-acid residues, including residues 177–185, which are responsible 16

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for a flexible loop known as the WPD loop. The WPD loop includes the active site residues His214–Arg221. The α3 helix of PTP1B comprises residues Glu186– Glu200, the S loop

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includes residues Ser201–Gly209, and the α6 helix is defined by residues Ala264–Ile281 [53]. When the WPD loop is in the ‘‘open’’ conformation, the binding pocket of PTP1B is easily accessible to the substrate/inhibitor. After substrate binding, the WPD loop closes over the active

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site, forming a tight binding pocket for the substrate. Therefore, WPD loop closure is essential for the catalytic mechanism of PTP1B [54]. In addition, Wiesmann et al. [55] proposed a range

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of PTP1B inhibitor candidates that bind to a novel allosteric binding site 20 Å from the catalytic pocket. This allosteric site makes these inhibitors highly selective for PTP1B. The allosteric inhibitors might act by stabilizing a conformation that precludes the closure of the WPD loop. Thus, WPD loop closure plays an important role in the full catalytic activity of PTP1B [55]. In the present study we found that coumarins 1-6 showed strong PTP1B inhibitory activity

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compared with the positive control ursolic acid. Furthermore, we also investigated inhibition type using an enzyme kinetic study in the presence of varying substrate and inhibitor

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concentrations. These studies revealed that compounds 1, 5, and 6 competitively inhibit PTP1B, indicating that these compounds can bind to the active site of the PTP1B enzyme in order to

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prevent enzyme-substrate complex formation, whereas 2 and 4 exhibited mixed-type PTP1B inhibitors, indicating that 2 and 4 can bind to the allosteric site of the free enzyme or to the enzyme-substrate complex. In the case of coumarin 3, the results showed that it is a noncompetitive inhibitor that can bind to the enzyme-substrate complex. These findings are in support of our previous study showing that coumarins from A. decursiva exhibited significant inhibitory activity against PTP1B [18]. In order to confirm the inhibition mode of the PTP1B enzyme, we predicted the 3D structure 17

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of PTP1B using Autodock 4.2 to simulate the binding of coumarins 1-6 and compound 23, a well-scrutinized PTP1B inhibitor of the enzyme. Compound 23 is among the most potent

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nonpeptic PTP1B inhibitors reported to date [34]. The Autodock 4.2 docking program was used to predict the protein–ligand binding interactions. Currently, automated docking is widely used as an effective means of quickly and accurately prediction of biomolecular conformations and

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binding energies of protein–ligand complexes in molecular design. The molecular docking models of coumarins 1-6 and compound 23 are illustrated in Fig. 3A-G, and Table 2. The ligand–

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enzyme complexes with coumarins 1-6/or compound 23 were stably posed in the same pocket of the PTP1B by Autodock 4.2. As illustrated in Fig. 3, in the corresponding ligand interactions of 1 in the active site of PTP1B there are four hydrogen-bonding interactions with three important residues (ILE134 O–H, PHE135 O–H, and GLY93 C=O ) of the enzyme. In the case of 2, five important residues of the enzyme (GLY183 C=O, CYS215 C=O, GLY218 C=O, ALA217 C=O,

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and ARG221 O atom) participated in five hydrogen-bonding interactions. Moreover, for coumarin 3 three important residues of the enzyme (GLY220 C=O, ARG221 O atom, and

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PHE182 O–H) participated in four hydrogen-bonding interactions. In the case of 4, two important residues of the enzyme (ILE134 C=O, and GLN127 C=O) participated in two

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hydrogen-bonding interactions with the senecioyl and ketone groups of 4. Regarding 5, in the active site of PTP1B, three important residues (LYS73 C=O, SER80 O–H, and LEU204 C=O) participated in five hydrogen-bonding interaction with the hydroxyl, ketone, and carboxylic groups of 5. With respect to 6, five important residues (CYS215 C=O, GLY218 C=O, ILE219 C=O, GLY220 C=O, and ARG221 O atom) of the enzyme participated in six hydrogen-bonding interactions. Our findings of all negative binding energies for the tested coumarins indicated that additional hydrogen bonding might result in more effective PTP1B inhibition by stabilizing the 18

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open form of the enzyme and provide strong binding to the PTP1B active site. Taken together,

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the results indicate that compounds 1-6 have promising anti-diabetic properties.

5. Conclusion

In the present study, six coumarins were isolated from A. decursiva, and the results showed

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that these compounds were potential anti-diabetic candidates. The anti-diabetic activities of these compounds were associated with the inhibition of α-glucosidase, PTP1B, and ONOO‒-mediated

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tyrosine nitration. According to α-glucosidase and PTP1B kinetic analysis, coumarins 1-6 showed mixed, competitive, and noncompetitive types of inhibition, further these coumarins also effectively suppressed ONOO‒-mediated tyrosine nitration in a dose-dependent manner. Taken together with molecular docking data, the results of the present study suggest that coumarins 1-6 may be novel candidates for development as therapeutic agents for the treatment and prevention

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of diabetes by targeting PTP1B inhibition. The data also clearly suggest the potential of A. decursiva and its constituents for use in the development of therapeutic or preventive diabetic

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agents. Further in vivo and cellular-based studies are needed to help clarify the detailed

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mechanism of action of these compounds in the brain membrane and other organs.

Acknowledgments

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) and funded by the Ministry of Science, ICT & Future Planning (2014R1A1A3051684) and the Ministry of Education (2012R1A6A1028677).

Conflict of interest 19

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The authors declare that there are no conflicts of interest.

[1]

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Compounds

PTP1B

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Table 1. PTP1B and α-glucosidase inhibitory activities of coumarins from A. decursiva

α-glucosidase

IC50 (µM) a 5.39 ± 0.19

4′-Methoxy Pd-C-I (2)

6.62 ± 0.77 58.90 ± 1.07

Decursidin (4) Umbelliferone 6-carboxylic acid (5) 2′-Isopropyl psoralene (6) Ursolic acid b

89.19 ± 0.77

65.29 ± 0.81

11.22 ± 0.39

79.09 ± 0.11

7.98 ± 0.91

172.10 ± 0.19

10.78 ± 0.17

85.82 ± 0.99

5.65 ± 0.29

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Acarbose c a

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Decursinol (3)

77.30 ± 1.07

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4-Hydroxy Pd-C-III (1)

183.29 ± 1.02

The 50% inhibition concentration (µM) was calculated from a log–dose inhibition curve and is

Used as positive controls

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b,c

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expressed as the mean ± SEM from triplicate experiments.

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4′-Methoxy Pd-C-I (2)

–7.06

Decursinol (3)

–6.89

Decursidin (4)

–6.78

Umbelliferone 6-carboxylic acid (5)

2

GLN127, PRO126, TRP125, LYS131, GLU132, MET133, ILE134, PHE135

–5.73

5

LYS73, SER80, ARG79, GLN78, PRO206, SER205, LEU204

–6.65

6

ASP181, PRO180, TRP179, PHE182, GLY183, ARG221, GLN266, GLN262, GLY220, ILE219, ALA217, GLY218, CYS215

13

TRP179, ARG221, CYS215, SER216, LYS120, ALA217, GLY218, ILE219, GLY220, GLN266, TYR46, VAL49, AS P48, THR263, GLN262, GLY259, MET258, ARG254, TY R20, ARG24, SER28, ASP29

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LYS116, PHE182, GLY183, ASP181, TRP179, ARG221, CYS215, GLN266, SER216, GLY220, GLY218, ILE219, ALA217

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Compound 23

PRO39, ASN90, THR91, CYS92, GLY93, ASP137, PHE135, ILE134, MET133, GLU132 ASP181, TRP179, PHE182, GLY183, CYS215, GLY218, ARG221, ALA217, GLY220, ILE219, GLN266, GLN262, THR263

4

–10.18

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2′-Isopropyl psoralene (6)

5

Binding site residues

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4-Hydroxy Pd-C-III (1)

No. of H-bonds 4

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Autodock 4.2. Scores (Kcal/mol) –6.21

Test compounds

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Table 2. Binding sites and docking score of compounds 1-6 in PTP1B using the Autodock program

28

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Figures and captions

Fig. 1. Chemical structure of coumarins 1–6 isolated from Angelica decursiva

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Fig. 2. The inhibitory effect of the Angelica coumarins against ONOO‒-mediated tyrosine nitration. Mixtures of test samples, BSA, and ONOO‒ were incubated with shaking at 37oC for

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30 min. The reactant was resolved by electrophoresis in 10% polyacrylamide gel. Decursidin (A), decursinol (B), 4′-methoxy Pd-C-I (C), 2′-isopropyl psoralene (D), 4-hydroxy Pd-C-III (E), and umbelliferone 6-carboxylic acid (F) were used at the indicated concentrations

Fig. 3. Molecular docking models for PTP1B inhibition of compounds 1(A), 2 (B), 3 (C), 4 (D),

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5 (E), 6 (F), and 23 (G), showing interaction with neighboring residues through H-bonding

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(green color) in the PTP1B binding sites

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OH O O

O

O

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4-Hydroxy Pd-C-III (1)

4'-Methoxy Pd-C-I (2)

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HOO C HO

O

O

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Umbelliferone 6-carboxylic acid (5)

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Decursidin (4)

Fig. 1

Decursinol (3)

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O

2'-Isopropyl psoralene (6)

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B

-

ONOO- (200 µM)

+

12.5 +

25 +

50 +

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Nitrotyrosine → -

12.5

25

-

+

+

50 100 +

+

-

+

+

25

50

-

- 12.5 25 50 100

+

+

-

+

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-

+

+

+

+

12.5

25

50 100

+

+

+

F

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E

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Nitrotyrosine →

ONOO- (200 µM)

-

D

C

ONOO- (200 µM)

-

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Nitrotyrosine →

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A

-

-

12.5

25

50

-

+

+

+

+

Fig. 2

-

-

+

+

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B

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A

C D

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E

Fig 3

G

F

ACCEPTED MANUSCRIPT Highlights Biological screening resulted in the isolation and identification of the coumarins.




Enzyme kinetic and docking simulation studies of coumarin compounds.




Six coumarins as PTP1B and α-glucosidase inhibitors may be used in the treatment of DM.




Six coumarins can inhibits ONOO‒-mediated tyrosine nitration




Coumarins may be promising anti-diabetic candidates.

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