PTP1B, α-glucosidase, and DPP-IV inhibitory effects for chromene derivatives from the leaves of Smilax china L.

Accepted Manuscript PTP1B, α-glucosidase, and DPP-IV inhibitory effects for chromene derivatives from the leaves of Smilax china L. Bing Tian Zhao, Duc Dat Le, Phi Hung Nguyen, Md Yousof Ali, Jae-Sue Choi, Byung Sun Min, Heung Mook Shin, Hae Ik Rhee, Mi Hee Woo PII:

S0009-2797(16)30135-1

DOI:

10.1016/j.cbi.2016.04.012

Reference:

CBI 7651

To appear in:

Chemico-Biological Interactions

Received Date: 13 October 2015 Revised Date:

16 March 2016

Accepted Date: 5 April 2016

Please cite this article as: B.T. Zhao, D.D. Le, P.H. Nguyen, M.Y. Ali, J.-S. Choi, B.S. Min, H.M. Shin, H.I. Rhee, M.H. Woo, PTP1B, α-glucosidase, and DPP-IV inhibitory effects for chromene derivatives from the leaves of Smilax china L., Chemico-Biological Interactions (2016), doi: 10.1016/ j.cbi.2016.04.012. 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.

ACCEPTED MANUSCRIPT Graphical abstract PTP1B, α-glucosidase, and DPP-IV inhibitory effects for chromene derivatives from the leaves of Smilax china L. Bing Tian Zhaoa, Duc Dat Lea, Phi Hung Nguyena, Md Yousof Alib, Jae-Sue Choib, Byung

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Sun Mina, Heung Mook Shinc, Hae Ik Rheed, Mi Hee Wooa,*

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ACCEPTED MANUSCRIPT PTP1B, α-glucosidase, and DPP-IV inhibitory effects for chromene derivatives from the leaves of Smilax china L.

Bing Tian Zhaoa, Duc Dat Lea, Phi Hung Nguyena, Md Yousof Alib, Jae-Sue Choib, Byung

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Sun Mina, Heung Mook Shinc, Hae Ik Rheed, Mi Hee Wooa,*

a

College of Pharmacy, Catholic University of Daegu, Gyeongsan 38430, Republic of Korea

b

Department of Food Science & Nutrition, Pukyong National University, Busan 48513,

Republic of Korea

Department of Physiology, College of Oriental Medicine,Dongguk University, Seoul 04620,

Republic of Korea d

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Department of Biotechnology, Kangwon National University, Chuncheon, Gangwon 24341,

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Republic of Korea

* Corresponding authors.

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E-mail address: [email protected] (M.H. Woo).

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ACCEPTED MANUSCRIPT ABSTRACT Two new flavonoids, bismilachinone (11) and smilachinin (14), were isolated from the leaves of Smilax china L. together with 14 known compounds. Their structures were elucidated using spectroscopic methods. The PTP1B, α-glucosidase, and DPP-IV inhibitory activities of

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compounds 1– –16 were evaluated at the molecular level. Among them, compounds 4, 7, and 10 showed moderate DPP-IV inhibitory activities with IC50 values of 20.81, 33.12, and 32.93 µM, respectively. Compounds 3, 4, 6, 11, 12, and 16 showed strong PTP1B inhibitory

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activities, with respective IC50 values of 7.62, 10.80, 0.92, 2.68, 9.77, and 24.17 µM compared with the IC50 value for the positive control (ursolic acid: IC50 = 1.21 µM).

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Compounds 2– –7, 11, 12, 15, and 16 showed potent α-glucosidase inhibitory activities, with respective IC50 values of 8.70, 81.66, 35.11, 35.92, 7.99, 26.28, 11.28, 62.68, 44.32, and 70.12 µM. The positive control, acarbose, displayed an IC50 value of 175.84 µM. In the kinetic study for the PTP1B enzyme, compounds 6, 11, and 12 displayed competitive inhibition with Ki values of 3.20, 8.56, and 5.86 µM, respectively. Compounds 3, 4, and 16

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showed noncompetitive inhibition with Ki values of 18.75, 5.95, and 22.86 µM, respectively. Molecular docking study for the competitive inhibitors (6, 11, and 12) radically corroborates the binding affinities and inhibition of PTP1B enzymes. These results indicated that the

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leaves of S. china L. may contain compounds with anti-diabetic activity.

Keywords: Smilax china L.; DPP-IV, PTP1B, α-Glucosidase inhibitor; Molecular docking

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ACCEPTED MANUSCRIPT 1. Introduction The use of traditional herbs for the treatment and management of chronic diseases is still a mainstay in developing countries. Among all the chronic diseases, the application of traditional medicine for diabetes mellitus (DM) is the most representative [1]. DM is a

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metabolic and chronic disease characterized by high blood sugar levels. It can be broadly categorized into diabetes mellitus type I (T1DM), diabetes mellitus type II (T2DM), gestational diabetes and diabetes of other etiology. T2DM accounts for about 90– –95% of all

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cases of diabetes [2– –6]. Therefore, there have been various studies to identify the T2DM mechanisms through which plant drugs bring about diabetic inhibition activities, such as the

1B (PTP1B) inhibitors,

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mechanisms of α-amylase inhibitors, α-glucosidase inhibitors, protein tyrosine phosphatase and dipeptidyl peptidase-4 (DPP-IV) inhibitors [7]. Recently,

PTP1B inhibition was reported as a promising target for the treatment of T2DM [8]. PTP1B has been implicated in the negative regulation of insulin receptor (IR) and insulin receptor substrate-1 (IRS-1) within the insulin-stimulated signal transduction pathway. Thus,

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pharmacological agents that inhibit PTP1B activity have the potential to augment and prolong insulin action for the treatment of T2DM [9– –11]. Nowadays, α-glucosidase inhibitors are widely used in the treatment of T2DM [12, 13]. The diabetic inhibitor agents used clinically,

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namely acarbose, voglibose, and miglitol, inhibit α-glucosidase competitively in the brush border of the small intestine, and consequently delay the hydrolysis of carbohydrates (starch

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and disaccharides), alleviating postprandial hyperglycemia [14– –16]. DPP-IV inhibitors were recently introduced as some of the newest diabetes drugs [17]. The DPP-IV enzyme is responsible for the rapid degradation of incretins, such as glucagon like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). GLP-1 secreted by intestinal L cells helps control blood sugar levels since it has important physiological functions, such as the stimulation of insulin secretion, inhibition of glucagon release, and delayed gastric emptying. Hence, inhibition of DPP-IV leads to the potentiation of endogenous GLP-1 and GIP, which have clearly established roles in glucose-dependent insulin secretion [18– –21]. 3

ACCEPTED MANUSCRIPT Smilax china L., a member of the Smilacaceae family, is distributed widely in tropical and temperate parts of the world, especially in East Asia [22]. It is a perennial, slightly woody climber with paired tendrils for climbing. The tuber of S. china L. is commonly used in

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traditional medicines for the treatment of furunculosis, tumors, and inflammation [23]. Pharmacological research has been reported indicating that the tuber of S. china L. possesses anti-inflammatory, anti-cancer, anti-microbial, and anti-tyrosinase activities [24, 25].

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However, all of the published studies were focused on the tuber of S. china L. There are only

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a few reports of experiments using the leaves of S. china L. Flavonoids, such as rutin, kaempferin, and kaempferitrin have been isolated from the leaves of S. china L [26], but there is no report on anti-diabetic activity by the chemical constituents isolated from the leaves of this plant [27, 28]. The aim of this study was to isolate the compounds from the leaves of this

activities.

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plant and to investigate their inhibitory effects on DPP-IV, PTP1B, and α-glucosidase enzyme

2. Materials and methods

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2.1. Plant materials

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The leaves of S. china L. were collected in September 2012 from Cho-lye mountain in Daegu, Republic of Korea. These materials were confirmed taxonomically by Professor Byung Sun Min, College of Pharmacy, Catholic University of Daegu, Korea. A voucher specimen, No. 201209, has been deposited at the College of Pharmacy, Catholic University of Daegu, Korea. 2.2. Instruments and reagents Melting points were determined on a Yanaco micro melting point apparatus. Optical

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ACCEPTED MANUSCRIPT rotations were measured on a JASCO DIP-370 digital polarimeter. IR spectra were measured on a Mattson Polaris FT/IR-300E spectrophotometer. UV spectra were measured on a Thermo 9423 AQA 2200E UV spectrophotometer. NMR spectroscopy was taken on a Varian Unity

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INOVA-400 spectrometer (USA), and chemical shifts are expressed as δ values using TMS as an internal standard. Low- and high-resolution EI-MS and FAB-MS data were collected on a Quattro II spectrometer. Open column chromatography was performed using silica gel

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(Kieselgel 60, 70-230 mesh and 230-400 mesh, Merck). TLC tests were performed on Merck

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precoated silica gel 60 F254 (EM5717) and/or RP-18 F254s glass plates (0.25mm), and were visualized by spraying with 10% H2SO4 and subsequent heating. p-Nitrophenyl phosphate (p-NPP), ethylenediaminetetraacetic acid (EDTA), and dipeptidyl peptidase-IV (DPP-IV) from porcine kidney, were purchased from Sigma-Aldrich. PTP1B (human recombinant) was

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purchased from Biomol International LP, and dithiothreitol (DTT) was purchased from Bio-Red laboratories. All other chemicals and solvents used were purchased from Merck, Fluka, and Sigma-Aldrich and analytical grade, unless otherwise stated.

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2.3. Extraction and isolation

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The fresh leaves of S. china L. (31.5 kg) were dried in the shade. The dried leaves of S. china L. (10.4 kg) were extracted with 70% EtOH at room temperature. The 70% EtOH extract was concentrated under reduced pressure to yield black syrup (2.5 kg). The concentrated 70% EtOH extract was suspended in H2O (6.0 L) and partitioned successively with n-hexane (4 × 3 L, 628.6 g), CH2Cl2 (4 × 2 L, 72.0 g), EtOAc (3 × 3 L, 104.0 g), n-BuOH (3 × 3 L, 341.1 g) and H2O-soluble fractions (1367.1 g), respectively. Among them EtOAc fraction showed the best activity against PTP1B. 5

ACCEPTED MANUSCRIPT The EtOAc fraction (104.0 g) was chromatographed over a silica gel column (15 × 35 cm) and eluted with CH2Cl2:MeOH:H2O (100:0:0.1 to 0:100:0.1) gradient. Twelve fractions (SM-Et-1 to SM-Et-12) were collected and grouped according to their similar TLC patterns.

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The fraction SM-Et-3 (2.6 g) was applied to a silica gel column chromatography using CH2Cl2:MeOH:H2O (5:1:0.1 to 0:100:0.1) gradient to afford 30 subfractions (SM-Et-3-1 to SM-Et-3-30). Subfraction SM-Et-3-26 (1.6 g) was chromatographed over a MCI gel CHP20

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column with MeOH:H2O as a stepwise gradient (1:1 to 1:0) to obtain 6 subfractions

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(SM-Et-3-26-1 to SM-Et-3-26-6). Fraction SM-Et-3-26-3 (30.0 mg) was chromatographed on RP-C18 column (HPLC) using MeOH:H2O (80:20) to yield compound 13 (19.6 mg). Subfraction SM-Et-3-26-5 (48.0 mg) was chromatographed on RP-C18 column (HPLC) using acetonitrile–H2O (40:60) to yield compounds 12 (13.0 mg) and 16 (5.0 mg), respectively.

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Subfraction SM-Et-4 (8.9 g) was chromatographed on a MCI gel CHP20 column with MeOH:H2O as stepwise gradient (1:1 to 1:0) to obtain 10 subfractions (SM-Et-4-1 to SM-Et-4-10). Fraction SM-Et-4-1 (3.5 g) was chromatographed on silica gel column using

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n-hexane:acetone (4:1 to 0:1) gradient to obtain 10 subfractions (SM-Et-4-1-1 to

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SM-Et-4-1-10), then the fraction SM-Et-4-1-9 was crystallized using MeOH to yield compound 11 (2.6 mg). Fraction SM-Et-4-7 (215.4 mg) was chromatographed on a silica gel column using n-hexane:acetone (3:1 to 0:1) gradient to yield compound 2 (40.4 mg). The fraction SM-Et-4-9 (312.7 mg) was chromatographed on a silica gel column using n-hexane:acetone (4:1 to 0:1) gradient to yield compound 3 (5.8 mg). Fraction SM-Et-7 (12.0 g) was chromatographed on a RP-C18 column using the MeOH:H2O mixture as a solvent system and eluted with a stepwise gradient (3:7 to 1:0). Sixteen subfractions (SM-Et-7-1 to 6

ACCEPTED MANUSCRIPT SM-Et-7-16) were collected and pooled according to their similar TLC patterns. SM-Et-7-9 (740.0 mg) was chromatographed on a silica gel column, using CH2Cl2–acetone (2:1) as a solvent system to yield compound 14 (16.6 mg). SM-Et-7-11 (518.0 mg) was

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chromatographed on a silica gel column with CH2Cl2:MeOH:H2O (95:5:1) to yield compound 5 (109.9 mg). SM-Et-7-14 (269.7 mg) was chromatographed on a silica gel column with CH2Cl2:MeOH:H2O (90:10:1) to yield compound 1 (180.0 mg). Fraction SM-Et-6 (1.1 g) was

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also chromatographed on a RP-C18 column, using the MeOH:H2O mixture as a solvent and

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eluted with a stepwise gradient (3:7 to 1:0) to obtain 13 subfractions (SM-Et-6-1 to SM-Et-6-13). Subfraction SM-Et-6-7 (73.0 mg) was chromatographed on a Sephadex LH-20 gel column using a solvent system of MeOH:H2O (1:1) to yield compound 16 (28.7 mg). Subfraction SM-Et-6-11 (73.0 mg) was isolated on RP-C18 column (HPLC) using

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acetonitrile:H2O (1:3) to yield compound 7 (16.8 mg). Fraction SM-Et-9 (7.1 g) was subjected to RP-C18 column chromatography to produce six subfractions, SM-Et-9-1 to SM-Et-9-6. Subfraction SM-Et-9-3 was purified by Sephadex LH-20 column chromatography

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with MeOH:H2O (1:1) to provide compounds 15 (51.4 mg) and 7 (75.5 mg), respectively. The

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fraction SM-Et-11 (9.4 g) was isolated on HP-20 column with MeOH:H2O (1:0 to 0:1) gradient to yield 7 subfractions (SM-Et-11-1 to SM-Et-11-7). The subfraction SM-Et-11-4 (4.0 g) was chromatographed on a RP-C18 column using MeOH:H2O mixture as a solvent system and eluted with a stepwise gradient (3:7 to 9:11) to afford compounds 8 (600.0 mg), 9 (300.8 mg), and 10 (10.5 mg), respectively. Subraction SM-Et-11-6 (1.1 g) was isolated on RP-C18 column using the acetonitrile:H2O (27:73) as solvent system to obtain compound 4 (65.0 mg). 7

ACCEPTED MANUSCRIPT 2.3.1. 7-Methoxy-4′,5-dihydroxyflavone-(3-O-7′′)-4′′′,5′′-dihydroxyflavone (11) ; [α]D27: +7.2° (c 0.001, MeOH); UV (MeOH)

Yellow amorphous powder; mp 225-226

λmax nm (log ε): 225 (4.82), 267 (4.76), 365 (4.64); IR (KBr) νmax cm-1: 3310 (OH), 1656

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(C=O), 1610 (C=C), 1452 (C-O); MS-HR-FAB m/z 553.1140 [M+H]+ (calcd. for 553.1135, C31H21O10); 1H-NMR (pyridine-d5, 600 MHz) δ: 8.04 (2H, d, J = 8.7 Hz, H-2′, 6′), 8.02 (2H, d, J = 8.7 Hz, H-2′′′, 6′′′), 7.39 (2H, d, J = 8.7 Hz, H-3′′′, 5′′′), 7.34 (2H, d, J = 8.7 Hz, H-3′, 5′),

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7.04 (1H, d, J = 1.3 Hz, H-8), 6.97 (1H, d, J = 1.9 Hz, H-8′′), 6.97 (1H, s, H-3′′), 6.81 (1H, d,

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J = 1.3 Hz, H-6′′), 6.76 (1H, d, J = 1.3 Hz, H-6), 3.89 (3H, s, OCH3); 13C-NMR (pyridine-d5, 150 MHz) δ: 181.9 (C-4), 181.5 (C-4′′), 164.8 (C-5), 164.0 (C-2), 162.6 (C-2′′), 161.9 (C-4′), 161.9 (C-5′′), 160.5 (C-4′′′), 157.6 (C-7), 157.3 (C-7′′), 153.7 (C-9′′), 152.7 (C-9), 128.0 (C-2′, 6′), 127.5 (C-2′′′, 6′′′), 125.0 (C-3), 124.3 (C-1′′′), 120.8 (C-1′), 115.9 (C-3′, 5′), 114.8 (C-3′′′,

(C-8′′), 57.0 (OCH3).

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5′′′), 106.4 (C-10), 105.2 (C-10′′), 103.8 (C-3′′), 102.8 (C-6′′), 98.9 (C-6), 93.7 (C-8), 90.8

2.3.2. 5,7-Dihydroxy-4′-methoxyisoflavone-2′-O-β-D-glucopyranoside (14)

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Yellow needle; mp 220-221

; [α]D27: –65.3° (c 0.001, DMSO); UV (MeOH) λmax nm

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(log ε): 260 (4.56); IR (KBr) νmax cm-1: 3566 (glycoside), 3370 (OH), 1651 (C=O), 1613 (C=C), 1071 (C-O); MS-HR-FAB m/z 463.1237 [M+H]+ (calcd. for 463.1240, C22H23O11); 1

H-NMR (pyridine-d5, 400 MHz) δ: 8.55 (1H, s, H-2), 7.64 (1H, d, J = 8.5 Hz, H-6′), 7.43

(1H, d, J = 2.5 Hz, H-3′), 6.80 (1H, dd, J = 2.5, 8.5 Hz, H-5′), 6.72 (1H, d, J = 2.1 Hz, H-8), 6.54 (1H, d, J = 2.1 Hz, H-6), 5.62 (1H, d, J = 7.8 Hz, H-1′′), 4.57 (1H, dd, J = 2.2, 11.9 Hz, H-6′′a), 4.35 (1H dd, J = 4.3, 7.8 Hz, H-6′′b), 4.31 (1H, t, J = 8.9 Hz, H-3′′), 4.24 (1H, m, H-2′′), 4.21 (1H, m, H4′′), 4.12 (1H, m, H-5′′), 3.72 (3H, s, OCH3); 13C-NMR (pyridine-d5, 8

ACCEPTED MANUSCRIPT 100 MHz) δ: 181.9 (C-4), 166.4 (C-7), 164.1 (C-5), 162.0 (C-4′), 159.1 (C-9), 157.9 (C-2′), 157.3 (C-2), 133.8 (C-6′), 120.4 (C-3), 113.8 (C-1′), 108.4 (C-5′), 106.5 (C-10), 103.5 (C-3′), 103.5 (C-1′′), 100.6 (C-8), 95.2 (C-6), 79.7 (C-5′′), 79.2 (C-3′′), 75.4 (C-2′′), 71.9 (C-4′′), 63.0

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(C-6′′), 55.9 (OCH3). 2.4. DPP-IV inhibitory assay

Porcine kidney DPP-1V (Sigma) activity was measured using Gly-Pro-p-nitroanilide as

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a substrate. To each 96-well (final volume: 200 µL) were added 0.75 mM Gly-Pro-p-nitroanilide and DPP-IV (1.1 Unit) in a buffer containing 500 mM Gly-NaOH (pH for 30 min, the reaction

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8.6), with or without test compounds. Following incubation at 37

was terminated with 10 M NaOH. The amount of p-nitroanilide produced was measured at 405 nm using an ELISA plate reader. The nonenzymatic hydrolysis of 0.75 mM Gly-Pro-p-nitroanilide was corrected by measuring the increase in absorbance at 405 nm obtained in the absence of DPP-IV enzyme. The DPP-IV inhibitory activity of each sample

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was calculated by the following equation: Inhibition ratio (%) = [1 − (Sample OD − Blank 2 OD)/(Control OD − Blank 1 OD)] × 100, where OD is absorbance at 405 nm.

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2.5. PTP1B inhibitory assay

PTP1B (human, recombinant) was purchased from BIOMOL Internation LP (USA) and

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the enzyme activity was measured using p-nitrophenyl phosphate (p-NPP) as a substrate. To each 96-well (final volume: 200 µL) were added 2 mM p-NPP and PTP1B (0.05-0.1 µg) in a buffer containing 50 mM citrate (pH 6.0), 0.1 M NaCl, 1 mM EDTA, and 1 mM dithiothreitol (DTT) with or without test compounds. Following incubation at 37

for 30 min, the reaction

was terminated with 10 M NaOH. The amount of p-nitroanilide produced was measured at 405 nm using an ELISA plate reader. The nonenzymatic hydrolysis of 2 mM p-NPP was corrected by measuring the increase in absorbance at 405 nm obtained in the absence of 9

ACCEPTED MANUSCRIPT PTP1B enzyme [29]. The PTP1B inhibitory activity of each sample was calculated by the following equation: Inhibition ratio (%) = [1 − (Sample OD − Blank 2 OD)/(Control OD − Blank 1 OD)] × 100, where OD is absorbance at 405 nm.

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2.6. α-Glucosidase inhibitory assay The α-glucosidase inhibition assay was performed as 20 µL of α-glucosidase (0.25 unit/mL, Sigma) was mixed with 65 µL of phosphate buffer (50 mM, pH 6.8) and 15 µL of

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individual inhibitors dissolved in DMSO, prior to pre-incubation at 37

for 10 min.

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Sequentially, 100 µL of p-nitrophenyl α-D-glucopyranoside (3 mM) as a substrate was added to the mixture. The reaction was performed at 37

for 30 min. α-Glucosidase activity was

determined by measuring release of p-nitrophenol from p-nitrophenyl α-D-glucopyranoside at 405 nm. Acarbose was used as a positive control, and all assays were conducted in triplicates

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[30]. The α-glucosidase inhibitory activity of each sample was calculated by the following equation: Inhibition ratio (%) = [1 − (Sample OD − Blank 2 OD)/(Control OD − Blank 1 OD)] × 100, where OD is absorbance at 405 nm.

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2.7. Kinetic analysis

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The reaction mixture consisted of different concentrations of p-NNP as a PTP1B substrate in the absence or presence of compounds. The assay which described with above data was fitted by nonlinear regression analysis according to a Michaclis-Menten kinetic model (Graphpad Prism version 4.02). 2.8. Molecular docking analysis The accurate prediction of protein (PTP1B)-ligand interaction geometries are essential for the success of the virtual-screening approaches. The Autodock vina 1.1.2 program was used 10

ACCEPTED MANUSCRIPT to estimate the conformation of protein-isolated compounds complex. The 3D structure of the protein target (NCBI protein ID: 1NNY for human AR) was used for the docking studies without further modification [31]. The ligand 3-({5-[(N-acetyl-3-{4-[(carboxycarbonyl) acid

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(2-carboxyphenyl)amino]-1-naphthyl}-L-alanyl)amino]pentyl}oxy)-2-naphthoic

(3-NNA) was found in the conformed crystal structure. And then, the control of ligand was docked back to the corresponding binding pocket, to reproduce the orientation and position of

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the inhibitor observed in the crystal structure. Subsequently, the active compounds were

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docked using optimal orientation of the docked for 3-NNA. The binding sites of all ligands were determined by Discovery Studio software (Ver. 4.1). Finally, we evaluated the inhibitory effect for potential active isolates on protein by molecular docking model. 3. Results and discussions 3.1. Phytochemical investigation

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In this study, the leaves of S. china L. were extracted with 70% EtOH, and the concentrated extract was partitioned in succession with n-hexane, methylene chloride, ethyl acetate,

and

n-butanol.

From

the

ethyl

acetate

fraction,

a

new

biflavonoid,

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4′-methoxy-5,7-dihydroxyflavone-(3-O-7′′)-4′′′,5′′,7′′-trihydroxyflavone (11), and a new isoflavone glucoside, 5,7-dihydroxy-4'-methoxyisoflavone-2'-O-β-D-glucopyranoside (14),

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along with 14 known compounds were isolated and characterized. The known compounds were determined to be kaempferol (1) [27], kaempferide (2) [32], morin (3) [34, 35], kaempferol 7-O-α-L-rhamnoside (4) [35], kaempferin (5) [36], quercetin 4′-O-β-D-glucoside (6) [37], vitexin (7) [38, 39], kaempferitrin (8) [33, 40], lepidoside (9) [41, 42], rutin (10) [33], partensein

(12)

[43,

44],

puerarin

(13)

[41,

42],

naringenin

(15)

[45],

and

1,3,6-trithydroxyxanthone (16) [46, 47], based on NMR spectroscopic data analysis and comparison with published literature (Fig. 1). Among these compounds, compounds 11 and 14 were new, named bismilachinone and smilachinin, respectively. Furthermore, compounds 11

ACCEPTED MANUSCRIPT 2, 3, 6, 7, 9, 12, 13, 15, and 16 were isolated for the first time from this plant. Compound 16 had been obtained previously from synthesis [46], but this was the first instance of isolation from a natural source. Compound 11 was obtained as a yellow amorphous solid. Its HR-FAB-MS spectrum

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contained an ion peak of [M+H]+ at m/z 553.1140 (calcd for C31H21O10+, 553.1135), suggested its molecular formula of C31H20O10. The UV spectrum showed absorption maxima at 267 and 365 nm, suggesting a flavone derivative [48]. The IR spectrum showed absorption bands at 3310 (OH), 1656 (C=O), 1610 (C=C) and 1452 (C-O) cm-1. The 1H-NMR spectrum of 11

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showed two sets of AA'BB' pattern splitting at δ 8.04 (2H, d, J = 8.7 Hz) and 7.34 (2H, d, J = 8.7 Hz), 8.02 (2H, d, J = 8.7 Hz) and 7.39 (2H, d, J = 8.7 Hz), which were assigned to H-2′, 6′

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and H-3′, 5′, and H-2′′′, 6′′′ and H-3′′′, 5′′′. In addition, four meta-aromatic proton signals were observed at δ 7.04 (1H, d, J = 1.3 Hz, H-8), 6.97 (1H, d, J = 1.9 Hz, H-8′′), 6.81 (1H, d, J = 1.3 Hz, H-6′′) and 6.76 (1H, d, J = 1.3 Hz, H-6). These data are in agreement with a flavonoid dimeric structure. In addition, the 1H-NMR spectrum of 11 showed the presence of one

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aromatic proton at δ 6.97 (1H, s, H-3′′), which provided a correlation with C-2′′ (δC 162.6) and indicated that one unit was flavone. The

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C-NMR spectrum of compound 11 showed 31

signals including eight carbon signals at δ 128.0 (C-2′, 6′), 127.5 (C-2′′′, 6′′′), 115.9 (C-3′, 5′),

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and 114.8 (C-3′′′, 5′′′), from ring B of the flavonoid dimeric structure, two ketone signals at δ 181.9 (C-4) and 181.5 (C-4′′), eleven oxygenated aromatic quaternary carbon signals at δ

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164.8 (C-5), 164.0 (C-2), 162.6 (C-2′′), 161.9 (C-4′), 161.9 (C-5′′), 160.5 (C-4′′′), 157.6 (C-7), 157.3 (C-7′′), 153.7 (C-9′′), 152.7 (C-9), and 125.0 (C-3), five aromatic tertiary carbons (C-6, 8, 6′′, 8′′, and 3′′), four aromatic quaternary carbons (C-10, 1′, 10′′, and 1′′′), and one methoxy carbon at δ 57.0 (OCH3). The HMBC spectrum of 11 showed heteronuclear long-range couplings of H-2′, 6′ with C-1′, H-2′′′, 6′′′ with C-1′′′, H-2′, 6′ with C-2, and H-2′′′, 6′′′ with C-2′′, which confirmed the ring B of both flavones. Furthermore, in the HMBC spectrum, the proton H-8′′ showed correlations with carbon at δ 125.0 (C-3). The data imply that carbon 3 (δc 125.0) from one flavonoid unit was involved in the inter-flavonoid linkage. Finally, the correlations for all carbons directly bonded to protons were established by 1H-1H COSY while 12

ACCEPTED MANUSCRIPT HMBC and HSQC confirmed the assignment of the proton and carbon frequencies. Thus, compound

11

was

characterized

as

7-methoxy-4′,5-dihydroxyflavone-(3-O-7′′)-4′′′,5′′-dihydroxyflavone, a new natural product, and named bismilachinone (Fig. 2 and Table 1).

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Compound 14 was obtained as a yellow needle. Its HR-FAB-MS spectrum yielded an ion peak of [M+H]+ at m/z 463.1237 (calcd for C22H23O11+, 463.1240), confirmed its molecular formula of C22H22O11. The UV spectrum showed an absorbance band at λmax (MeOH) 260 nm, which is characteristic of an isoflavone skeleton [49]. The IR spectrum

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showed absorption bands at 3566 (glycoside), 3370 (OH), 1651 (C=O), 1613 (C=C), 1071 (C-O) cm-1. A positive reaction with ferric chloride reagent indicated that the compound

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consisted of a hydroxyl-phenyl group, and the positive Molisch test also confirmed it to be a glycoside. In the 1H-NMR spectrum of 14, the characteristic resonance for H-2 of an isoflavone was observed at δ 8.55 (1H, s) [50]. This assignment was confirmed by long-range connectivities to the quaternary carbons at δ 181.9 (C-4), 159.1 (C-9), and 120.4 (C-3) in the

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HMBC spectrum. In addition, an ABX aromatic spin system was observed at δ 7.64 (1H, d, J = 8.5 Hz, H-6′), 7.43 (1H, d, J = 2.5 Hz, H-3′), and 6.80 (1H, dd, J = 2.5, 8.5 Hz, H-5′), suggesting that C-2′ and C-4′ were substituted. The ortho-aromatic proton signal H-6′ gave

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cross peaks with three carbon signals resonating at δc 120.4 (C-3), 157.9 (C-2′), and 162.0 (C-4′) in the HMBC spectrum. The meta-aromatic proton signal H-3′ also showed HMBC

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correlations with C-5′ (δC 108.4), C-1′ (δC 113.8), C-2′ (δC 157.9), C-4′ (δC 162.0), and ortho and meta-coupled proton H-5′ with C-1′ (δC 113.8). In addition, the 1H-NMR spectrum of 14 showed the presence of one anomeric proton at δ 5.62 (1H, J = 7.8 Hz, H-1′′) and one methoxy group at δ 3.72 (3H, s). The anomeric proton showed a correlation with C-2′ (δC 157.9), indicated that the sugar was attached at the C-2′ position. The methoxy protons correlated with C-4′ (δC 162.0), suggesting that methoxy was connected at C-4′. Moreover, two meta-aromatic protons of ring A from the isoflavone skeleton appeared as doublets at δ 6.72 (1H, d, J = 2.1 Hz, H-8) and 6.54 (1H, d, J = 2.1 Hz, H-6) in the 1H-NMR spectrum. The proton H-8 showed HMBC correlation with C-6 (δC 95.2) and C-10 (δC 106.5) while the 13

ACCEPTED MANUSCRIPT proton H-6 gave a cross peak with carbon at δC 100.6 (C-8). The anomeric proton at δ 5.62 (H-1′′) possessed a coupling constant (J value) of 7.8 Hz, suggesting a β-configuration for sugar [49]. The sugar protons appeared at δ 4.57 and 4.35 (2H, dd, J = 2.2, 11.9 Hz and dd, 4.3, 7.8 Hz, H-6′′), 4.31 (1H, t, J = 8.9 Hz, H-3′′), 4.24 (1H, m, H-2′′), 4.21 (1H, m, H-4′′),

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and 4.12 (1H, m, H-5′′). Also, in the HMBC spectrum, the proton H-3′′ showed correlations with two carbons at δ 71.9 (C-4′′) and 75.4 (C-2′′), the proton H-4′′ correlated with one carbon at δ 79.2 (C-3′′), and the proton H-2′′ correlated with C-1′′ (δC 103.5). The 13C-NMR spectrum indicated that compound 14 was an isoflavonoid glycoside with 22 carbons, including fifteen

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isoflavonoid carbons, one methoxy carbon, and six sugar carbons, with δ 103.5 being a characteristic signal for the C-1′′ of the β-D-glucose. Furthermore, the presence of the

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β-D-glucopyranosyl residue was further supported by the analysis of the NOESY experiment showing correlations between H-1ʹʹ, H-3ʹʹ, and H-5ʹʹ, and between H-4ʹʹ and H-6ʹʹ. The correlations for all carbons directly bonded to protons were established by 1H-1H COSY. HMBC, HMQC, and NOESY confirmed the assignment of the proton and carbon frequencies. compound

14

was

characterized

as

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Thus,

5,7-dihydroxy-4′-methoxyisoflavone-2′-O-β-D-glucopyranoside, a new natural product, and

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named smilachinin (Fig. 2 and Table 2).

3.2. Bioactivity assay

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The isolated compounds 1– –16 from the leaves of S. china L. were investigated for their enzyme inhibitory effects using in-vitro assays on DPP-IV, PTP1B, and α-glucosidase enzyme assays. The results are presented in Table 3

3.2.1. PTP1B inhibition assay All the isolates (1– –16) were assayed for their inhibitory activity against PTP1B using an in-vitro assay. Among the isolates, compounds 3, 4, 6, 11, 12, and 16 showed significant PTP1B inhibitory activities, with IC50 values of 7.62, 10.80, 0.92, 2.68, 9.77, and 24.17 µM, 14

ACCEPTED MANUSCRIPT respectively, comparing to these of positive control (ursolic acid, IC50: 1.21 ± 0.18 µM) [51]. Furthermore, compounds 5 and 8– –10, with a sugar moiety attached at the C-3 position, showed no inhibitory effects. However, compound 6, with a sugar moiety attached at C-4′, showed the strongest activity with an IC50 value of 0.92 µM. Compound 4, with a sugar

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moiety linked at C-7, displayed an IC50 value of 10.80 µM. However, compound 5, with a sugar moiety at C-3, exhibited no activity with IC50 > 100 µM. This finding suggests that the substituted position of a sugar moiety on the flavonol derivative plays an important role in the

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PTP1B inhibitory activity of these compounds. Furthermore, compounds 1 – 3, with kaempferol skeleton, inhibited PTP1B activity with IC50 values of 42.85, 67.06, and 7.62 µM,

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respectively. This result indicated that the decreasing activity was due to the exchange of one methoxy group to C-4′ in the B-ring, and the addition of one hydroxy group to C-2′ in the B-ring increased the PTP1B inhibitory activity. Finally, the new compound (14) showed moderate inhibitory activity with an IC50 value of 35.28 µM.

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3.2.2. α-Glucosidase inhibition assay

In the α-glucosidase inhibition assay, compounds 2– –7, 10– –13, 15, and 16 inhibited α-glucosidase enzyme with IC50 values ranging from 7.99 to 195.55 µM. Compounds 2– –7, 11,

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12, 15, and 16 showed potent inhibition, with IC50 values of 8.70, 81.66, 35.11, 35.92, 7.99, 26.28, 11.28, 62.68, 44.32, and 70.12 µM, respectively. Compounds 10 and 13 displayed

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moderate inhibitions with IC50 values of 194.45, and 195.55 µM, respectively, while the positive control, acarbose, displayed an IC50 value of 175.84 µM. Among the isolates, compound 2, with a kaempferol skeleton, showed a strong inhibitory effect. However, kaempferol (1) exhibited no activity with IC50 > 200 µM. It can be suggested that the methoxy groups linked at C-4' are responsible for the increase in the activity of the kaempferol skeleton. Furthermore, it was found that flavonoids substituted with two sugar moieties at C-3 and C-7 exhibited no activity. Compound 6, with a sugar moiety attached at C-4′, showed the strongest activity with an IC50 value of 7.99 µM. Interestingly, the same result was also reflected in the 15

ACCEPTED MANUSCRIPT PTP1B inhibitory activity of these compounds. From the above results, it can be suggested that the substitution at position C-4′ on the flavonoid derivative was the most important factor in the inhibition of PTP1B and α-glucosidase enzyme inhibitory activity.

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3.2.3. DPP-IV inhibition assay Compounds 4, 7, and 10 showed moderate inhibitory effects against DPP-IV enzyme activity with IC50 values of 20.81, 33.12, and 32.93 µM, respectively. Fan et al. reported that flavonoids including luteolin, apigenin, and kaempferol had strong DPP-IV activities with

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IC50 values of 0.12, 0.14, and 0.49 µM, respectively [52– – 55]. However, in our assay,

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kaempferol showed inhibitory activity with an IC50 value of 45.96 µM. This IC50 value was about 100 times weaker than the reported data. The significant difference with our result is attributed to the different substrate used in our assay. In our method, Gly-Pro-p-nitroanilide was used as the substrate; however, Gly-Pro-amino methyl coumarin (Gly-Pro-AMC) was used as the substrate in the reported paper. Hence, this result indicated that compounds 4, 7,

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and 10 can act as naturally occurring DPP-IV inhibitors.

3.3. Enzyme kinetic assay

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In our continued efforts to identify the type of PTP1B inhibition, we employed a Lineweaver-Burk plot (Fig. 3). The reciprocal of the rate of the reaction was plotted against

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the reciprocal of the substrate concentration to monitor the effect of the inhibitor on both Km and Vmax. The Ki value was confirmed by a Dixon plot, by plotting the reciprocal of the rate of reaction against the different concentrations of active compounds (Fig. 4). The kinetics studies of compounds 6, 11, and 12 revealed competitive inhibition toward to the substrate with p-NPP (Fig. 3 and Table 4). Figure 3 showed that the Vmax of jack bean urease was not affected in the presence of different concentrations of PTP1B inhibitory activity compounds 6, 11, and 12, whereas the Km increased, indicating a pure competitive type of inhibition. Furthermore, compounds 6, 11, and 12 displayed Ki values of 3.20, 8.56, and 5.86 µM, 16

ACCEPTED MANUSCRIPT respectively (Fig. 4 and Table 4). In contrast, the active compounds 3, 4, and 16 were established as mixed-type inhibitors since they reduced the Vmax values while the Km values of PTP1B increased (Fig. 3 and Table 4), with Ki values of 18.75, 5.95, and 22.86 µM,

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respectively (Fig. 4 and Table 4).

3.4. Molecular docking simulation of isolated compounds in inhibition of PTP1B

Based on the kinetics of enzyme inhibition results, we predicted the 3D structure of high competitive inhibitors 6, 11, and 12. As shown in Fig. 5, three PTP1B inhibitors (6, 11, and

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12) were bonded to PTP1B enzyme by H-bonds formation (green point line) on the optimized active sites of 3NNA. As summarized in Table 5, the compounds 6, 11, and 12 demonstrated

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negative binding energies as − 7.0, − 8.2, and − 7.2 kcal/mol, respectively. This information indicated that the ligands have high affinity on the active site of PTP1B enzyme, and compared with control of 3-NNY (− 7.2 kcal/mol). Therefore, the result obtained from the docking study shows that compounds 6, 11, and 12 possess great PTP1B inhibitory activity.

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In addition, the compound 6 was linked by 14 bonding sites with protein amino acid residues that are His60, Gln61, Glu62, Glu97, Trp100, Glu101, Lys103, Asp137, Thr138, Asn139, Asn162, Thr164, Thr165, and Glu167. Fifteen amino acid residues (His60, Gln61, Glu97, Trp100, Glu101,

EP

Lys103, Phe135, Glu136, Asp137, Thr138, Asn139, Leu140, Asn162, Thr164, and Thr165) from the active sites of PTP1B were formed with compound 11. In particular, the activated flavones of 6

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and 11 were conjugated with PTP1B enzyme, and both of these inhibitors have 10 common binding sites as His60, Gln61, Glu97, Trp100, Glu101, Lys103, Asp137, Thr138, Asn139, and Asn162. This result indicated that compounds 6 and 11 have similar skeleton parts. The isoflavone of compound 12 was correlated with amino acids residues on the active sites of protein like Lys73, Glu75, Glu76, Gln78, Arg79, Ser80, Ser203, Ser205, Pro206, His208, Gly209, and Pro210. The control of 3-NNA has different active binding sites (Tyr20, Gln21, Arg24, Tyr46, Asp48, Asn111, Lys116, Lys120, Trp179, Pro180, Asp181, Gly183, Ser216, Gly218, Gly220, Arg221, Ser222, Gln262, Thr263, Asp265, and Gln266) with compounds 6, 11, and 12. These

docking results could be helpful to find the crucial residues of PTP1B catalytic site and good 17

ACCEPTED MANUSCRIPT inhibitors.

4. Conclusion In conclusion, the inhibitory activities of the isolated compounds against DPP-IV,

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PTP1B, and α-glucosidase enzymes indicate that the leaves of S. china L. may have beneficial effects in the treatment of diabetes. However, further work will be necessary to definitively determine if the activity is sufficient to warrant evaluation of the compounds for clinical

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applications.

Acknowledgements

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The authors are grateful to S. H. Kim and collaborators at the Korea Basic Science Institute (Daegu) for measuring the mass spectra.

Conflict of interest

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The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

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Appendix A. Supplementary data

Supporting Information: IR, UV, 1H- and 13C-NMR, HMBC, 1H-1H COSY, HMQC and

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MS spectra for compounds 11 and 14. And the spectroscopic data of isolated compounds (except compounds 11 and 14).

18

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SC

Sin. 33 (2012) 1217–1245.

25

ACCEPTED MANUSCRIPT Table 1. 1H- and 13C-NMR spectral data for compound 11 Position

RI PT

SC

7.04 (1H, d, J = 1.3 Hz)

8.04 (1H, d, J = 8.7 Hz) 7.34 (1H, d, J = 8.7 Hz)

7.34 (1H, d, J = 8.7 Hz) 8.04 (1H, d, J = 8.7 Hz)

6.97 (1H, s)

TE D

162.6 103.8 181.5 161.9 102.8 157.3 90.8 153.7 105.2 124.3 127.5 114.8 160.5 114.8 127.5 57.0

6.76 (1H, d, J = 1.3 Hz)

M AN U

164.0 125.0 181.9 164.8 98.9 157.6 93.7 152.7 106.4 120.8 128.0 115.9 161.9 115.9 128

AC C

EP

1 2 3 4 5 6 7 8 9 10 1' 2' 3' 4' 5' 6' 1'' 2'' 3'' 4'' 5'' 6'' 7'' 8'' 9'' 10'' 1''' 2''' 3''' 4''' 5''' 6''' OCH3

δH (J in Hz)

δC

6.81 (1H, d, J = 1.3 Hz) 6.97 (1H, d, J = 1.9 Hz)

8.02 (1H, d, J = 8.7 Hz) 7.39 (1H, d, J = 8.7 Hz) 7.39 (1H, d, J = 8.7 Hz) 8.02 (1H, d, J = 8.7 Hz) 3.89 (3H, s)

26

ACCEPTED MANUSCRIPT Table 2. 1H- and 13C-NMR spectral data for compound 14 Position

δC

δH (J in Hz)

1 2 3 4 5 6 7 8 9 10 1ʹ 2ʹ 3ʹ 4ʹ 5ʹ 6ʹ 1ʹʹ 2ʹʹ 3ʹʹ 4ʹʹ 5ʹʹ

157.3 120.4 181.9 164.1 95.2 166.4 100.6 159.1 106.5 113.8 157.9 103.5 162.0 108.4 133.8 103.5 75.4 75.4 71.9 79.7

8.55 (1H, s)

RI PT

SC

M AN U

7.43 (1H. d, 2.5 Hz)

6.80 (1H, dd, J = 2.5, 8.5 Hz) 7.64 (1H, d, 8.5 Hz) 5.62 (1H, d, J = 7.8 Hz) 4.24 (1H, m) 4.31 (1H, t, J = 8.9 Hz)

4.12 (1H, m) 4.57 (1H, dd, J = 2.2, 11.9 Hz, H-6ʹʹa), 4.35 (1H dd, J = 4.3, 7.8 Hz, H-6ʹʹb) 3.72 (3H, s)

63.0

55.9

AC C

EP

OCH3

6.72 (1H, d, J = 2.1 Hz)

TE D

6ʹʹ

6.54 (1H,d, J = 2.1 Hz)

27

ACCEPTED MANUSCRIPT Table 3. Inhibitory effects of isolated compounds (1– –16) on DPP-IV, PTP1B and α-glucosidase enzyme activity DPP-IV inhibitory activity

PTP1B inhibitory activity IC50 (µM)a

α-glucosidase inhibitory activity

1 2 3 4 5 6 7 8 9 10 11

45.96 ± 1.52 >100 >100 20.81 ± 1.20 >100 >100 33.12 ± 1.01 >100 43.61 ± 0.97 32.93 ± 1.84 >100

42.85 ± 0.73 67.06 ± 0.82 7.62 ± 0.99 10.80 ± 0.03 >100 0.92 ± 0.19 >100 >100 >100 >100 2.68 ± 0.18

>200 8.70 ± 0.17 81.66 ± 0.93 35.11 ± 1.71 35.92 ±0.36 7.99 ± 0.48 26.28 ± 1.30 >200 >200 194.45 ± 4.10 11.28 ± 0.52

12 13

>100 >100

M AN U

SC

RI PT

Compounds

62.68 ± 1.01 195.55 ± 0.79

>100 35.28 ± 0.49 14 >100 63.25 ± 0.49 15 >100 24.17 ± 0.61 16 b 0.14 ± 0.03 linagliptin c 1.21 ± 0.18 ursolic acid acarbosed a The concentration (µM) exhibiting 50% inhibition.

>200 44.32 ± 1.02 70.12 ± 1.93 175.84 ± 1.69

b

Positive control for DPP-IV inhibitory activity.

d

EP

Positive control for PTP1B inhibitory activity. Positive control for α-glucosidase inhibitory activity.

AC C

c

TE D

9.77 ± 0.98 53.01 ± 1.29

28

ACCEPTED MANUSCRIPT Table 4. Kinetic analysis results of compounds 3, 4, 6, 11, 12, and 16 Tested compounds Ki (µM)

Inhibition type

18.75

noncompetitive

4

5.95

noncompetitive

6

3.20

competitive

11

8.56

competitive

12

5.86

competitive

16

22.86

RI PT

3

AC C

EP

TE D

M AN U

SC

noncompetitive

29

ACCEPTED MANUSCRIPT Table 5. Binding sites and docking score of compounds 6, 11, and 12 in PTP1B using the Autodock program Docking scoreb

Binding sitesa

Ligands

(kcal/mol)

His60, Gln61, Glu62, Glu97, Trp100, Glu101, Lys103, Asp137, Thr138, Asn139, Asn162, Thr164, Thr165, Glu167 His60, Gln61, Glu97, Trp100, Glu101, Lys103, Phe135, 11

Glu136, Asp137, Thr138, Asn139, Leu140, Asn162, Thr164, Thr165 Lys73, Glu75, Glu76, Gln78, Arg79, Ser80, Ser203, Ser205, Pro206, His208, Gly209, Pro210

− 8.2

− 7.2

SC

12

− 7.0

RI PT

6

Tyr20, Gln21, Arg24, Tyr46, Asp48, Asn111, Lys116, Lys120, Trp179, Pro180, Asp181, Gly183, Ser216,

Gly218, Gly220, Arg221, Ser222, Gln262, Thr263, Asp265, Gln266

a

M AN U

Control

− 7.2

AC C

EP

TE D

All amino acid residues located 5– –6 Å from the original 1NNY(enzyme)/3-NNA (ligand; control) complex in the Autodock program. b The empirical scoring function TM-score, which estimates the binding free energy of the ligand receptor complex.

30

ACCEPTED MANUSCRIPT 3' 4' OH

2' B

8 H3CO 7

O 2 A

9 10

5'

1'

3'''

6'

C

2'''

3

4'''OH

6 5 OH

4

8''

O

7''

O 6''

1''' O 2'' 9'' 10'' 3'' 4'' 5'' O

RI PT

OH

5''' 6'''

Bismilachinone (11)

Quercetin-4'-O-β-D-glucoside (6) Vitexin (7) Kaempferitrin (8) Lepidoside (9)

R8 R7

7

8 9

O

2

R7

OH OH OH OH O-Rham

OH OH OH OH OH

OH

H O-Rham O-Rham O-rutinosid e

R8

R2′

R3′

R4′

OH OH OH O-Rham OH

H H H H H

H H OH H H

H H H H H

OH OCH3

OH

OH

H

H

OH

O-Glu

OH OH OH

OH O-Rham O-Xyl

O-Glu H H

H H H

H H H

OH OH OH

OH

OH

H

H

OH

OH

TE D

Rutin (10)

R5

SC

Kaempferol (1) Kaemperide (2) Morin (3) Kaempferol 7-O-α-L-ranmnoside (4) Kaemperin (5)

R3

M AN U

Flavones

OH OH OH

Isoflavones

R5

R7

R8

R2′

R3′

R4′

Pratensein (12)

OH

OH

H

H

OH OCH3

Puerarin (13)

H

Smilachinin (14)

OH

R2' A 5 R5

10

3

1'

2'

R3'

4

3'

EP

6

C

B

O

6'

4'

5'

OH -Glu

H

H

OH

R4'

OH

H

O-Glu H OCH3

AC C

Isoflavones

Fig. 1. Structure of compounds 1– –16 isolated from the EtOAc fraction of S. china L.

31

ACCEPTED MANUSCRIPT

5' OH

O

5 6

4

3

10

8

2

O H

O

HO

6''

5''

4''

HMBC →

OH

O

2''

1''

OH 3''

OH

SC

COSY —

3'

1'

RI PT

7

4'

2'

9 HO

OCH 3

6'

Bismilachinone (11)

Smilachinin (14)

AC C

EP

TE D

M AN U

Fig. 2. The HMBC and 1H-1H COSY correlations of compounds 11 and 14.

32

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig. 3. Graphical determination of inhibition type for compounds 3, 4, 6, 11, 12, and 16 using

AC C

Lineweaver-Burk plots was expressed as the mean reciprocal of initial velocity for n = 3 replicates at each substrate concentration.

33

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

Fig. 4. Inhibition kinetics of compounds 3, 4, 6, 11, 12, and 16 were expressed in Dixon plots as the mean reciprocal of initial velocity for n = 3 replicates at each substrate concentration. The inhibition constant Ki value was determined from the x-axis value at the point of the intersection of the three lines.

34

ACCEPTED MANUSCRIPT

RI PT

(A)

AC C

EP

TE D

(C)

M AN U

SC

(B)

Fig. 5. Docked conformation of compounds 6 (A), 11 (B), and 12 (C), showing interaction with neighboring residues through H-bonding (green) in the PTP1B binding sites.

35

ACCEPTED MANUSCRIPT

Research highlights Sixteen chromene derivatives including two new compounds were isolated from the leaves of S. china L.

and DPP-IV enzymes. Compounds 6, 11, and 12 are competitive inhibition of PTP1B.

RI PT

Isolates (1– –16) were evaluated for their ability to inhibit activity of PTP1B, α-glucosidase,

The binding sites of PTP1B to compounds 6, 11, and 12 were studied using molecular

AC C

EP

TE D

M AN U

SC

docking.