PHYTOTHERAPY RESEARCH Phytother. Res. 29: 1540–1548 (2015) Published online 14 July 2015 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5411

Chemical Constituents of Euonymus alatus (Thunb.) Sieb. and Their PTP1B and α-Glucosidase Inhibitory Activities Su-Yang Jeong,1 Phi-Hung Nguyen,1 Bing-Tian Zhao,1 Md Yousof Ali,2 Jae-Sue Choi,2 Byung-Sun Min1 and Mi-Hee Woo1* 1

College of Pharmacy, Catholic University of Daegu, Gyeongsan 712-702, Korea Department of Food Science & Nutrition, Pukyong National University, Busan 608-737, Korea

2

Phytochemical study on the corks of Euonymus alatus resulted in the isolation of a novel 3hydroxycoumarinflavanol (23), along with ten triterpenoids (1–10), ten phenolic derivatives (11–20), and two flavonoid glycosides (21 and 22). Their structures were determined by extensive 1D and 2D-nuclear magnetic resonance spectroscopic and mass spectrometry data analysis. Furthermore, their inhibitory effects against the protein tyrosine phosphatases 1B (PTP1B) and α-glucosidase enzyme activity were evaluated. Compounds 6, 7, 9, 15, 19, and 23 were non-competitive inhibitors, exhibiting most potency with IC50 values ranging from 5.6 ± 0.9 to 18.4 ± 0.3 μM, against PTP1B. Compound 3 (competitive), compounds 5 and 15 (mixed-competitive) displayed potent inhibition with IC50 values of 15.1 ± 0.7, 23.6 ± 0.6 and 14.8 ± 0.9 μM, respectively. Moreover, compounds 15, 20, and 23 exhibited potent inhibition on α-glucosidase with IC50 values of 10.5 ± 0.8, 9.5 ± 0.6, and 9.1 ± 0.5 μM, respectively. Thus, these active ingredients may have value as new lead compounds for the development of new antidiabetic agents. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: Euonymus alatus; PTP1B inhibitors; α-glucosidase; flavanol; triterpenoids.

Diabetes mellitus, long regarded as a disease of minor consequence to world health, occupies now as one of the main causes of serious maladies in the 21st century. The number of people with diabetes anticipate rising from current estimate of 150–220 million in 2010, and 300 million in 2025 (Zimmet et al., 2001). Lifestyle patterns in industrialized societies comprise an increasing availability and ingestion of high-caloric food in the prevalence of a sedentary lifestyle. These factors are emerging as the fundamental causes of this fast-spread ‘epidemic’ (Friedman, 2003). Type-2 diabetes (DM2) is characterized by a resistance of insulin-sensitive tissues, such as muscle, liver, and fat, to insulin action. Although the mechanism of the insulin resistance is unknown, it is tightly associated with obesity. Approximately threequarters of obese individuals will develop DM2 (Hossain et al., 2007). This metabolic disorder accounts for 90% of the global DM incidents and plays a predisposing role in cardiovascular diseases from which about 18 million people die annually. Protein tyrosine phosphatases (PTPs) are involved in the down regulation of cellular signal transduction mediated by receptor tyrosine kinases such as insulin receptor and epidermal growth factor receptor (Burke and Zhang, 1998). Protein tyrosine phosphatases 1B (PTP1B), a member of the PTP family, is thought to function as a negative regulator of insulin signal transduction. PTP1B directly interacts with activated insulin receptor or insulin receptor substrate-1 (IRS-1) to dephosphorylate phosphotyrosine residues, resulting in down regulation of * Correspondence to: Mi Hee Woo, College of Pharmacy, Catholic University of Daegu Gyeongsan 712-702, Korea. E-mail: [email protected]

Copyright © 2015 John Wiley & Sons, Ltd.

insulin action (Goldstein et al., 2000). PTP1B knockdown mice show enhanced insulin sensitivity in glucose and insulin tolerance tests, indicating that PTP1B is a major player in the modulation of insulin sensitivity (Elchebly et al., 1999; Klaman et al., 2000). Increased expression of PTP1B in adipose tissue and muscle of obese humans and rodents is thought to be related to insulin resistance (Wu et al., 2001), whereas the increased insulin sensitivity from weight loss is accompanied by reduced PTP1B activity (Ahmad et al., 1997). PTP1B overexpression in rat primary adipose tissues and 3T3/L1 adipocytes has been shown to decrease insulinsensitive Glut4 translocation (Chen et al., 1999) and insulin receptor and IRS-1 phosphorylation (Venable et al., 2000), respectively. As with the insulin signaling pathway, the leptin signaling pathway can be attenuated by PTPs, and there is compelling evidence that PTP1B is also involved in this process. Therefore, it has been suggested that compounds that reduce PTP1B activity or expression levels cannot only be used for treating type 2 diabetes but also obesity (Moller, 2001). Although, there have been a number of reports on the design and development of the PTP1B inhibitors, including ertiprotafib, trodusquemine (He et al., 2014), difluoromethylene phosphonates, 2-carbomethoxybenzoic acids, 2-oxalylaminobenzoic acids, and several lipophilic compounds (Andersen et al., 2000; Bialy and Waldmann, 2003; Burke et al., 1994; Liljebris et al., 2002; Zhang and Zhang, 2007). However, new types of PTP1B inhibitors with suitable pharmacological properties still remain to be discovered. Euonymus alatus (Thunb.) Sieb. (Celastraceae), known as ‘gui-jun woo’ in Korea, was used in folk medicine for regulating blood circulation, relieving pain, Received 24 December 2014 Accepted 13 June 2015

CHEMICAL CONSTITUENTS OF EUONYMUS ALATUS (THUNB.) SIEB.

eliminating stagnant blood, and treating dysmenorrhea in oriental countries (Park et al., 2005). E. alatus has been reported to have several biological properties, such as anticancer and antioxidative effects, preventing hyperglycemia and hyperlipidemia induced by high-fat diet in Institute for Cancer Reaserch (ICR) mice (Park et al., 2005), attenuating lipopolysaccharide (LPS)-induced NF-κB activation via IKKβ inhibition in RAW 264.7 cells (Oh et al., 2011), inhibiting α-glucosidase in vitro and in vivo (Lee et al., 2007), and stimulating insulin secretion (Huang, 1998). Despite the number of study on preventing obesity-related DM2 effects of E. alatus, there are no reports on the PTP1B inhibitory activity by the extract and the chemical constituents isolated from E. alatus. Thus, the total extract of E. alatus was phytochemically investigated, using several chromatographic methods and an in vitro assay on PTP1B enzyme activity. As the result, ten triterpenoids (1–10), ten cinnamic acid derivatives (11–20), and two flavonoid glycosides (21–22), along with a novel flavanol (23) were separated and structurally identified (Fig. 1). Furthermore, the isolated compounds (1–23) were evaluated for their inhibitory effects on PTP1B enzyme activity. Moreover, Sttucture-Activity Relationship

1541

(SAR) and a kinetic study were further performed to investigate their inhibition modes. In addition, the inhibitory effects against α-glucosidase enzyme of these isolates were also investigated in vitro. In this report, the purification, structural determination, and antidiabetic properties of the isolated compounds (1–23) are discussed.

MATERIALS AND METHODS General experimental procedure. The optical rotations were determined on a JASCO DIP-1000 digital polarimeter (JASCO Corp., Tokyo, Japan) using a 100 mm glass microcell. UV spectra were recorded in MeOH using a Shimadzu UV spectrometer (Shimadzu Corp., Kyoto, Japan). Infrared spectra (IR) were recorded on a Nicolet 6700 FT-IR spectrometer (Thermo electron Corp., Tokyo, Japan). The nuclear magnetic resonance (NMR) spectra were recorded in methanol-d4 (CD3OD) on Varian Oxford-AS 400 MHz instrument (Palo Alto, CA, USA), with tetramethylsilane as the internal standard, at the Department of Pharmacy,

Figure 1. Chemical structures of compounds 1–23 isolated from the corks of Euonymus alatus Sieb. Copyright © 2015 John Wiley & Sons, Ltd.

Phytother. Res. 29: 1540–1548 (2015)

1542

S.-Y. JEONG ET AL.

Catholic University of Daegu, Korea. All mass spectrometric experiments were performed on a Quattro II mass spectrometer at Korea Basic Science Institute Daegu Center (KBSC, Daegu, Korea). Silica gel (63–200 μm particle size) and RP-C18 (40–63 μm particle size) for column chromatography were from Merck (Darmstadt, Germany). Thin-layer chromatography (TLC) was carried out on pre-coated Merck Silica Gel 60 F254 and RP-C18 F254 plates from Merck. HPLC was carried out using a Gilson system (Middleton, WI, USA) with a UV detector and an Optima Pak C18 column (10 × 250 mm, 10 μm particle size, RS Tech Corporation, Seoul, Korea). HPLC solvents were obtained from Fisher Scientific Korea Ltd. (Seoul, Korea). All other reagents were of the highest analytical grade.

Plant material. The corks of E. alatus were collected in July 2006 from the Palgong mountain in Gyeongsangbukdo, Korea. The material was confirmed taxonomically by Professor Byung Sun Min and a voucher specimen (CUDP 200602) has been deposited at the College of Pharmacy, Catholic University of Daegu, Korea.

Extraction and isolation. The corks of E. alatus (3.5 kg) were extracted four times with MeOH using reflux system for 10 h. The solvent soluble extract was concentrated under reduced pressure to yield black syrup (211.4 g). This MeOH-extract was then subjected onto a silica gel column chromatography (6.0 × 60 cm; 63–200 μm particle size) by using a gradient solvent system of CH2Cl2:MeOH (20:1 → 0:1) to yield six combined fractions (EA-1–EA-6) according to their TLC profiles. Fraction 2 (EA-2) was further chromatographed on a silica gel column, using a gradient solvent system of CH2Cl2:MeOH with increasing polarity, to afford ten sub-fractions (EA-2.1–EA-2.10). The sub-fraction EA-2.3 was re-chromatographed on a silica gel column with CH2Cl2:MeOH as solvent system (gradient) to afford compounds 5 (75.2 mg) and 6 (98.1 mg), respectively. Sub-fraction EA-2.4 was also re-chromatographed on a silica gel column with nhexane:CH2Cl2 as solvent system (gradient), yielded compounds 2 (169.6 mg) and 9 (9.5 mg), separately. Compound 7 (73.7 mg) was purified from sub-fraction EA-2.5 by using an open column chromatography with an isocratic solvent system of n-hexane:CH2Cl2. Subfraction EA-2.6 was also re-chromatographed on a silica gel column with CH2Cl2:MeOH as solvent system (gradient), yielded compounds 1 (267.9 mg) and 20 (13.1 mg), respectively. Compound 4 (16.2 mg) was purified from sub-fraction EA-2.7 by using an open column chromatography eluting with a gradient solvent system of CH2Cl2:MeOH. Sub-fraction EA-2.8 was also re-chromatographed on a silica gel column with nhexane:CH2Cl2 as solvent system (gradient), yielded compounds 3 (29.9 mg) and 10 (59.2 mg), separately. Compound 8 (293.7 mg) was purified from sub-fraction EA-2.9 by using an open column chromatography with an isocratic solvent system of n-hexane:CH2Cl2. Fraction 4 (EA-4) was further chromatographed on an open silica gel column, eluting with n-hexane:CH2Cl2 (gradient) and then CH2Cl2:MeOH (gradient) with increasing polarity, to afford five sub-fractions (EA-4.1–EA-4.5). Copyright © 2015 John Wiley & Sons, Ltd.

Compound 14 (73.7 mg) was purified from sub-fraction EA-4.1 by using an open reverse-phase (RP-C18, 40–63 μm particle size) column chromatography with an isocratic solvent system of MeOH:H2O (50:50). Purification of sub-fraction EA-4.2 by an open reversephase (RP-C18, 40–63 μm particle size) column chromatography, eluting with a gradient solvent system of MeOH:H2O, resulted in the isolation of compounds 12 (15.9 mg) and 18 (16.2 mg), respectively. Compounds 11 (11.9 mg) and 17 (15.3 mg) were also purified from sub-fraction EA-4.3 by open RP-C18 column using a solvent system of MeOH:H2O (15:85, gradient). Fraction 5 (EA-5) was also chromatographed on an open silica gel column (SiO2, 40–63 μm particle size), eluting with a gradient solvent system of n-hexane: CH2Cl2 and then CH2Cl2:MeOH with increasing polarity, to afford five sub-fractions (EA-4.1–EA-4.5) according to their TLC profiles. Compounds 16 (21.4 mg) and 22 (12.8 mg) were purified from sub-fraction EA-5.2 by using an open reverse-phase (RP-C18, 40–63 μm particle size) column chromatography, eluting with an isocratic solvent system of MeOH:H2O (20:80). Compounds 15 (41.4 mg) and 19 (22.0 mg) were also purified from sub-fraction EA-5.3 by using an open reverse-phase (RP-C18, 40–63 μm particle size) column chromatography, eluting with a gradient solvent system of MeOH: H2O (15:85–55:45). Purification of sub-fraction EA-5.4 by an open reverse-phase (RP-C18, 40–63 μm particle size) column chromatography, eluting with a gradient solvent system of MeOH:H2O (5:95–50:50), resulted in the isolation of compounds 21 (36.2 mg), 13 (12.7 mg), and 23 (10.2 mg), respectively. Euonymalatus (23). Yellowish amorphous powder; [α] 25 15.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε): D 220 (1.30), 256 (1.28), 272 (1.55); IR vmax (KBr) 3235 (OH), 1679 (C=O), 1610 (C=C) cm 1; CD (MeOH) [θ] 26.2, [θ]231 +10.5, [θ]280 8.6, [θ]330 +4.0; MS 212 (FAB) m/z (rel. int.): 425 [M+H]+; MS (HR-FAB) m/z 425.0870 [M + H]+ (calcd. for C22H16O9H, 425.0873); 1 H and 13C NMR data, see Table 1.

Protein tyrosine phosphatase 1B inhibitory assay. Protein tyrosine phosphatase (human recombinant) was purchased from BIOMOL@ International LP, Plymouth Meeting, PA, USA, and the inhibitory activities of the tested samples were evaluated using p-nitrophenyl phosphate (p-NPP) as substrate (Nguyen et al., 2011). Briefly, to each well (final volume 110 μL) was added 2 mM p-NPP and PTP1B (final concentration of 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 sample. The plate was preincubated at 37°C for 10 min, and then 50 μL of p-NPP in buffer were added. Following incubation at 20°C for 20 min, the reaction was terminated with the addition of 10 M NaOH. The amount of p-nitrophenyl produced after enzymetic dephosphorylation was estimated by measuring the absorbance at 405 nm using a VERSA max microplate reader (Molecular Devices, Sunnyvale, CA, USA) microplate reader. The non-enzymatic hydrolysis of 2 mM p-NPP was corrected by measuring the increase in absorbance at 405 nm obtained in absence of PTP1B enzyme. The inhibition (%) was Phytother. Res. 29: 1540–1548 (2015)

CHEMICAL CONSTITUENTS OF EUONYMUS ALATUS (THUNB.) SIEB.

Table 1. 1H (400 MHz) and 13C (100 MHz) nuclear magnetic resonance spectroscopic data for compound 23 measured in CD3OD Euonymalatus (23) Position 1 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″

δH (ppm, J in Hz)

5.09 (1H, d, J = 6.0) 4.22 (1H, m) 2.85 (1H, dd, J = 4.8, 16.8) 2.74 (1H, dd, J = 6.4, 16.8) 6.42 (1H, s)

6.79 (1H, d, J = 8.4) 6.81 (1H, d, J = 8.4)

6.89 (1H, br s)

7.64 (1H, s) 8.41 (1H, s)

δC (ppm, type)

83.0 (CH) 67.6 (CH) 25.7 (CH2) 157.7 (C) 96.5 (CH) 131.6 (C) 101.7 (C) 154.2 (C) 106.4 (C) 131.9 (C) 119.3 (CH) 116.5 (CH) 146.5 (C) 146.6 (C) 114.7 (CH) 164.1 (C) 154.3 (C) 115.1 (CH) 131.6 (C) 113.8 (CH) 152.4 (C) 146.4 (C)

calculated as: (Ac–As)/Ac*100%, where Ac is the absorbance of the control and As, the absorbance of the sample.

Determination of the inhibition mode of active compounds. To determine the kinetic mode of inhibition of isolated PTP1B inhibitors, Lineweaver–Burk and Dixon plot analyses were performed (Nguyen et al., 2010). This kinetic study was carried out in the presence and absence of inhibitors with various concentrations of p-NPP as the substrate. The mode of inhibition of the test compounds was assessed on the basis of the inhibitory effect on Km (dissociation constant) and Vmax (maximum reaction velocity) of the enzyme determined using a Lineweaver–Burk plot, which is the double reciprocal plot of the enzyme reaction velocity (V) versus the substrate (p-NPP) concentration (1/V versus 1/[p-NPP]).

α-Glucosidase inhibitory assay. The enzyme inhibition study was carried out spectrophotometrically using the reported procedure (Li et al., 2005). Briefly, 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 p-NPG, and 20 μL of the sample [test concentration ranging from 31.25 to 125 μg/mL dissolved in 10% dimethylsulfoxide (DMSO)] was added to each well, followed by 20 μL α-glucosidase [0.2 U/mL in 10 mM 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 Copyright © 2015 John Wiley & Sons, Ltd.

1543

was added to stop the reaction. The absorbance was immediately recorded at 405 nm using a microplate spectrophotometer (molecular devices). The negative control was the same reaction mixture containing an equivalent volume of phosphate buffer instead of the sample solution. Acarbose dissolved in 10% DMSO was used as a positive control. The percent inhibition (%) was obtained by the following equation: % inhibition = (Ac As)/Ac × 100, where Ac is the absorbance of the control and As is the absorbance of the sample.

Statistical analysis. All data are represented as means ± SD of at least three independent experiments performed in triplicate assays.

RESULTS AND DISCUSSION The total methanol extract of the corks of E. alatus was subjected into several repeated column chromatography (including open silica gel and RP-C18) to afford ten triterpenes (1–10), ten phenolic derivatives (11–20), two flavonoid glycosides (21 and 22), and a novel 3-hydroxycoumarinflavanol (23) (Fig. 1). By interpretation of physicochemical and spectroscopic data, together with comparison with reported literatures, their chemical structures were determined to be β-sitosterone (1) (Gaspar and Neves, 1993), β-sitosterol (2) (Su et al., 2009), 24R-methyllophenol (3) (Akihisa and Matsunoto, 1987), α-spinasterol (4) (Kim, 1999), arborinone (5) (Akihisa et al., 1992), lupeone (6) (Dai et al., 2006), lupeol (7) (Fuchino et al., 1995), epi-lupeol (8) (De Souza et al., 2001), taraxerol (9) (Lee and Jeong, 1992), germanicol (10) (Koul et al., 2000), benzoic acid (11) (Arora et al., 2005), p-hydroxybenzoic acid (12) (Mitase et al., 1984), 3,4-dihydroxybenzoic acid (13) (Mitase et al., 1984), vanillic acid (14) (Mitase et al., 1984), p-propoxybenzoic acid (15) (Ullah et al., 1999), p-coumaric acid (16) (Durust et al., 2001), caffeic acid (17) (Durust et al., 2001), ferulic acid (18) (Durust et al., 2001), 1-feruloyl-β-D-glucoside (19) (Gokhale, 2011), tetradecyl (E)-ferulate (20) (Das and Kashinatham, 1997), naringin (21) (Lewinsohn et al., 1986), and 7,4′-dihydroxy-8-Cglucoxylisoflavone (22) (Ingham et al., 1986). To the best of our knowledge, compound 23 is a new compound, and compounds 3 and 10 were isolated for the first time from this plant. Compound 23 was isolated as a white powder. The FABMS spectrum of 23 gave a [M+H]+ peak at m/z 425. The high resolution fast-atom bombardment mass spectroscopy (HR-FAB-MS) gave a positive ion peak at m/z 425.0870 for the [M+H]+, which corresponded to the molecular formula of C22H17O9 (calcd. 425.0873). Its IR spectrum exhibited absorptions at 3235 (OH), 1679 (C=O) and 1610 (C-O) cm 1. The occurrence of a flavan-3-ol skeleton in the molecule could be deduced from the 1H and 13C NMR (Table 1). The 1 H NMR signals at δH 5.09 (1H, d, J = 6.0 Hz, H-2), 4.22 (1H, m, H-3), 2.85 (1H, dd, J = 4.8, 16.8, H-4a), and 2.74 (1H, dd, J = 6.4, 16.8, H-4b), together with corresponding carbons at δC 83.0 (C-2), 67.6 (C-3), and 25.7 (C-4), were characteristic of a flavan-3-ol skeleton (Porter and Harborne, 1994). In the 1H and 13C NMR Phytother. Res. 29: 1540–1548 (2015)

1544

S.-Y. JEONG ET AL.

spectra of compound 23, the proton signals at δH 6.81 (1H, d, J = 8.4, H-3′), 6.79 (1H, d, J = 8.4, H-2′), and 6.89 (1H, br s, H-6’), and the corresponding carbons at δC 131.9 (C-1), 119.3 (C-2), 116.5 (C-3), 146.5 (C-4), 146.6 (C-5), and 114.7 (C-6), implied the presence of a 1′,4′,5′trisubstituted ring B. Moreover, analysis of HMQC, COSY, and HMBC NMR spectra of compound 23 led to the elucidation of a partial structure [Partial (B)] of 23 as a (+)-catechin analogue (Benavides et al., 2006) (Fig. 2 and Supporting Information). In addition, the 1H and 13C NMR spectra of compound 23 showed two aromatic singlet protons at δH 7.64 (1H, s, H-4″) and 8.41 (1H, s, H-6″), a lactone carbon at δC 164.1 (C-2″), three aromatic oxygenated carbons at δC 154.3 (C-3″), 152.4 (C-7″), and 146.4 (C-8″). Additionally, three quaternary aromatic carbons at δC 101.7 (C-8), 112.8 (C-7), 131.6 (C-5″), and two tertiary carbons at δC 115.1 (C-4″) and 113.8 (C-6″) were also observed. When analysis of the HMBC experiment, the correlations were found between the aromatic singlet proton at δH 8.41 (H-6″) and C-8″ (δC 146.4), and between H-4″ (δH 7.64) and the lactone carbonyl carbon at δC 164.1 (C-2″), oxygenated quaternary carbon at δC 154.3 (C-3″), 146.4 (C-8″), and aromatic quaternary carbon at δC 131.6 (C-5″). In the COSY spectrum, a cross peak between H-4″ (δH 7.64, 1H, s) and H-6″ (δH 8.41, 1H, s) was observed (see Supporting Information). The aforementioned data obtained indicated a structural skeleton of a 3-hydroxycoumarin (Bailly et al., 2004) [Fig. 2, Partial (A)]. Thus, compound 23 is proposed to be a structural combination of a flavan-3-ol skeleton and a 3-hydroxycoumarin derivative. Indeed, the HMBC correlations from H-6″ (δH 8.41, 1H, s) of the coumarin-skeleton [Partial (A)] to C-7 (δC 112.8), C-8 (δC 101.7), and C-9 (δC 154.2) of the catechin-skeleton [Partial (B)] established the linkage from C-6″ to C-8 (Fig. 2). In addition, the proton singlet at δH 6.42 (H-6) showed HMBC correlations to C-7″ (δC 152.4), C-7 (δC 112.8), C-8 (δC 101.7), C-5 (δC 157.7), and C-10 (δC 106.4), which strongly demonstrated the linkage chain of C-6―C-7―C-7″ (Fig. 2). The flavan-3-ols have two stereocenters and therefore four possible diastereomers, namely, (2R,3S)-2,3-trans, (2S,3R)-2,3-trans, (2R,3R)-2,3-cis, and (2S,3S)-2,3-cis exist (Slade et al., 2005). In compound 23, the 1H NMR signals of H-2 appeared at 5.09 (d, J = 6.0 Hz), H-3 at 4.22 (m, H-3), H-4a at 2.85 (dd, J = 4.8 and 16.8 Hz), and H-4b at 2.74 (dd, J = 6.4 and 16.8 Hz), respectively. A negative optical rotation value at 15.0 (c 0.1, MeOH) was observed for compound 23. The

1

1

1

13

Figure 2. H– H COSY (bold lines) and H– C key HMBC correlations (arrow) for euonymalatus (23). Copyright © 2015 John Wiley & Sons, Ltd.

aforementioned observations were characteristic of a 2,3-trans-flavan-3-ol skeleton (Donovan et al., 1999; Wilhelm, 2008). Moreover, the circular dichroism spectrum (Supporting Information) of compound 23 exhibited negative Cotton effects at 212 and 280 nm and positive Cotton effects at 231 and 330 nm, indicating an absolute configuration at C-2 and C-3 to be 2R,3S (Slade et al., 2005). Thus, compound 23 (euonymalatus) was elucidated as 5,4′,5′-trihydroxy-[3″,8″-dihydroxycoumarin]-(6″,7″:7,8)-(2R,3S)-flavan-3-ol, which is a novel 3-hydroxycoumarinflavanol. The PTP1B inhibitory activity of the isolated compounds (1–23) were evaluated using p-NPP as a substrate described previously (Nguyen et al., 2011), and the result is shown in Table 2. RK-682 was used as a positive control in this assay and displayed an IC50 value of 4.5 ± 0.1 μM (Hamaguchi et al., 1995; Cui et al., 2010). Among the isolates, compounds 6, 7, 15, and the new compound 23 exhibited most potency with IC50 values of 13.7 ± 2.1, 5.6 ± 0.9, 14.8 ± 0.9, and 13.4 ± 0.2 μM, respectively. Compounds 3, 15, and 19 displayed significant activity with IC50 values of 15.1 ± 0.7, 14.8 ± 0.9, and 18.4 ± 0.3 μM, respectively. While compounds 8–10, and 14 showed moderate effect with IC50 values of 28.4 ± 1.2, 21.9 ±v2.1, 25.7 ± 1.5, and 29.9 ± 1.5 μM, and the other compounds were weak activity (IC50 >30 μM). To determine the inhibition mode of the active compounds, the inhibitory properties of several appropriate concentrations of the active compounds (3, 5–7, 9, 15, 19, and 23) were evaluated at various concentrations of p-NPP (Nguyen et al., 2010; Na et al., 2009). The Sigma plot program (SPCC Inc., Chicago, IL) was used to analyze both the double reciprocal Lineweaver–Burk plot, which is the most straightforward means of diagnosing an inhibitor model and the Dixon plot by plotting 1/v as a function of inhibitor [I] for each substrate concentration. Figure 3 shows the results of the Lineweaver–Burk plot analysis [Fig. 3(A)], and Dixon plot analysis [Fig. 3(B)] for compounds 3, 5, 9, 15, 19, and 23, respectively. The Ki values were determined from the x-axis value at which the lines intersect. Table 1 lists the Ki values for the active compounds 3, 5, 9, 15, 19, and 23 as 17.5, 26.8, 20.8, 13.6, 18.5, and 10.3 μM, respectively. In this study, 24R-methyllophenol (3) showed competitive inhibition mode because the pattern of straight lines with intersecting y intercepts [Fig. 3(A)] is characteristic of competitive inhibitors, indicating that 3 may directly bind to the active binding site of the enzyme to inhibit the activity of PTP1B enzyme. The lupane-type triterpenes have been reported to possess a wide range of bioactivities that include antiinflammatory, antiviral, antimicrobial, antioxidant, antitumor, antiangiogenic, and antimalarial effects. Lupenone (6) and lupeol (7) showed non-competitive inhibition of PTP1B. Furthermore, both kinetic experiments showed that compounds 9, 19, and 23 exhibited non-competitive inhibition, because the Vmax values decreased with increasing concentration without changing the Km for the substrate, and the lines intersected at a value of 1/[S] under zero on the x-axis [at 1/(intensity/min) = 0] [Fig. 3(A)]. This is indicated that in allosteric inhibition, compounds 9, 19, and 23 may bind to the enzyme substrate complex or interact with a specific binding site distinct from the active site of the enzyme (Wiesmann et al., 2004). In contrast, increasing the substrate concentrations resulted in a series of lines that did not intersect Phytother. Res. 29: 1540–1548 (2015)

1545

CHEMICAL CONSTITUENTS OF EUONYMUS ALATUS (THUNB.) SIEB.

Table 2. Inhibitory effects of isolated compounds (1–23) on protein tyrosine phosphatases 1B (PTP1B) and α-glucosidase enzyme activities α-glucosidase

PTP1B inhibitory activity Compound

IC50, μMa

Ki value

Inhibition type

IC50, μMa

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 RK-682b Acarboseb

>30 >30 15.1 ± 0.7 >30 23.6 ± 0.6 13.7 ± 2.1 5.6 ± 0.9 28.4 ± 1.2 21.9 ± 2.1 27.5 ± 1.5 >30 >30 >30 29.9 ± 1.5 14.8 ± 0.9 >30 >30 >30 18.4 ± 0.3 >30 >30 >30 13.7 ± 0.2 4.5 ± 0.1 —

—c — 17.5 — 26.8 — — — 20.8 — — — — — 13.6 — — — 18.5 — — — 10.3 — —

— — Competitive — Mixed-competitive Non-competitive Non-competitive — Non-competitive — — — — — Mixed-competitive — — — Non-competitive — — — Non-competitive — —

>200 >200 183.1 ± 1.2 >200 >200 >200 >200 133.1 ± 0.9 >200 >200 >200 >200 >200 >200 10.5 ± 0.8 >200 >200 >200 169.7 ± 1.5 9.5 ± 0.6 >200 75.6 ± 0.7 9.1 ± 0.5 — 124.2 ± 0.4

a

Results are expressed as IC50 values (μM), determined by regression analyses, and expressed as the means ± SD of three replicates. Positive control. c Data not identified. b

both on the y-axis and x-axis in the Lineweaver–Burk plot [Fig. 3(A)] and the x-axis in Dixon plots [Fig. 3 (B)], confirming that compounds 5 and 15 serve as mixed-competitive inhibitors. This is an interesting inhibition mode, because the inhibitors not only bind to the enzyme substrate complex or interact with a specific binding site distinct from the active site but may also bind to the active binding site of the enzyme to allosteric inhibit the enzyme activity. When investigated, the chemical structure (Fig. 1) and the PTP1B activity (Table 2) relationship, it was found that the lupane-type triterpenes (compounds 5–8) showed strong activity on PTP1B. In this type, the orientation and the substitution of functional groups at C-3 might play an importance to the inhibitory effect of these compounds. Indeed, lupeol (7) with a 3-β-hydroxyl moiety displayed potential activity with an IC50 value of 5.6 ± 0.9 μM, while epi-lupeol (8) bearing a 3-α-OH group showed moderate activity (IC50 value 28.4 ± 1.2 μM). However, in the tetracyclic triterpenes (compounds 9 and 10), the location of the double bond is thought to play an important role in regulating the activity of these triterpenoids. Taraxarane-type (compound 9) was found to possess stronger activity (IC50 value of 21.9 ± 2.1 μM) than oleanane-type (compound 10, IC50 27.5 ± 1.5 μM). Moreover, in the steroids (compounds 1–4), compound 3 (cholestane-type), with an additional methyl moiety attached at C-4, displayed stronger activity (IC50 value 15.1 ± 0.7 μM) than the Copyright © 2015 John Wiley & Sons, Ltd.

other compounds 1, 2, and 4 (ergostane-type, IC50 value >30 μM). To our knowledge, the oleanane-type has been reported as PTP1B inhibitor; this is the first report that the taraxarane-type (8), steroids (1–4), and phenolics (11–14) have been investigated for their PTP1B inhibitory effects. In addition, some of the cinnamic acid derivatives have recently been reported as PTP1B inhibitors. o-hydroxycinnamic acid and p-hydroxycinnamic acid exhibited PTP1B inhibitory effects with IC50 values of 137.74 ± 13.20 and 168.40 ± 17.69 μM, respectively (Adisakwattana et al., 2013). However, p-propoxy benzoic acid (15) and 1-feruloyl-β-D-glucoside (19) showed potencies with IC50 values of 14.8 ± 0.9 and 18.4 ± 0.3 μM, respectively. The use of α-glucosidase inhibitors is considered to be an effective strategy in the treatment of diabetes. Postprandial hyperglycemia plays an important role in the development of diabetes mellitus Type II and the resulting complications. One another therapeutic approach to treat postprandial hyperglycemia is to retard the cleavage of glucose from disaccharide via inhibition of α-glucosidase in the digestive organs (Tewari et al., 2003). α-Glucosidase is a glucosidase that acts on 1,4-α bonds, breaking down starch and disaccharides into single α-glucose. Hence, inhibition of α-glucosidase leads to delay the absorption of carbohydrates by the gut in the small intestine, and thus have an effect on lowering postprandial blood glucose and insulin levels. In this regard, all isolated compounds were evaluated Phytother. Res. 29: 1540–1548 (2015)

1546

S.-Y. JEONG ET AL.

Figure 3. Graphical determination of inhibition type for the isolated compounds 3, 5, 9, 15, 19, and 23. (A) Lineweaver–Burk plots for the inhibition of compounds 3, 5, 9, 15, 19, and 23 on the protein tyrosine phosphatases 1B-catalyzed hydrolysis of p-nitrophenyl phosphate. (B) Dixon plots of compounds 3, 5, 9, 15, 19, and 23 used for determining the inhibition constant Ki. Ki values were determined from the negative x-axis value at the point of the intersection of the three lines. Data are expressed as the mean reciprocal of initial velocity for n = 3 replicates at each substrate concentration.

for their inhibitory effects on α-glucosidase activity using acarbose as positive control. Among them, compounds 15, 20, and 23 showed potential inhibitory effects, as ten times more potent than the positive control, with IC50 values of 10.5 ± 0.8, 9.5 ± 0.6, and 9.1 ± 0.5 μM, respectively. Compound 22 displayed an IC50 value of 75.6 ± 0.7 μM, whereas compounds 3, 8, and 19 possessed IC50 values of 183.1 ± 1.2, 133.1 ± 0.9, and 169.7 ± 1.5 μM, respectively. In this study, acarbose exhibited an IC50 value of 124.2 ± 0.4 μM. Copyright © 2015 John Wiley & Sons, Ltd.

CONCLUSIONS The herbal medicines have been extensively used to prevent and heal various inflammatory problems for over a millennium in oriental countries. According to the oldest Korean medical book (Dong-ui-bo-gam), E. alatus (Thunb.) Sieb., is called ‘Wi Mo’ or ‘Gui Jun Woo’ and introduced to be beneficial in alleviating ‘Boong-roo’ (uterine bleeding), ‘Dae-ha’ (metritis), Phytother. Res. 29: 1540–1548 (2015)

CHEMICAL CONSTITUENTS OF EUONYMUS ALATUS (THUNB.) SIEB.

and ‘Eo-hyeol’(stagnant blood) (Oh et al., 2011; Huang, 1998). Recently, many researchers have reported that E. alatus exhibited several pharmacological effects including anticancer and antioxidant effects, preventing hyperglycemia and hyperlipidemia induced by high-fat diet in ICR mice (Park et al., 2005), attenuating LPS-induced NF-κB activation via IKKβ inhibition in RAW 264.7 cells (Oh et al., 2011), inhibiting αglucosidase in vitro and in vivo, and stimulating insulin secretion (Lee et al., 2007). However, phytochemical research has not been studied to investigate the chemical components underlying the beneficial antidiabetic effect of E. alatus extracts. Thus, our results reveal that triterpenoids (1–10) and phenolics (11–20) are the main ingredients of the corks of E. alatus and that they may

1547

be the compounds responsible for the potential antidiabetic properties of E. alatus.

Acknowledgements This research was supported by research grants from the National Research Foundation of Korea (Basic Science Research Program; 2009–0067369). We are grateful to Korea Basic Science Institute (KBSI) for mass spectrometric measurements.

Conflict of Interest The authors have declared that there is no conflict of interest.

REFERENCES Adisakwattana S, Pongsuwan J, Wungcharoen C, Yibchok-anun S. 2013. In vitro effects of cinnamic acid derivatives on protein tyrosine phosphatase 1B. J Enzyme Inhib Med Chem 28: 1067–1072. Ahmad F, Considine RV, Bauer TL, Ohannesian JP, Marco CC, Goldstein BJ. 1997. Improved sensitivity to insulin in obese subjects following weight loss is accompanied by reduced protein tyrosine phosphatase in adipose tissue. Metabolism 46: 1140–1145. 13 Akihisa T, Matsunoto T. 1987. C-NMR spectra of sterols and triterpene alcohols. Yukagak 36: 301–319. Akihisa T, Yamamoto K, Tamura T, et al. 1992. Triterpenoid ketones from lingnania chungii Mcclure: arborinone, friedelin and glutinone. Chem Pharm Bull 40: 789–791. Andersen HS, Iversen LF, Jeppensen CB, et al. 2000. 2(Oxalylamino)benzoic acid is a general competitive inhibitor of protein tyrosine phosphatases. J Biol Chem 275: 7101–7108. Arora KK, Parakashareddy J, Pedireddi VR. 2005. Pyridine mediated supramolecular assemblies of 3,5-dinitro substituted benzoic acid, benzamide and benzonitrile. Tetrahedron 61: 10793–10800. Bailly F, Maurin C, Teissier E, Vezina H, Cotelle P. 2004. Antioxidant properties of 3-hydroxycoumarin derivatives. Bioorg Med Chem 12: 5611–5618. Benavides A, Montoro P, Bassarello C, Piacente S, Pizza C. 2006. Catechin derivatives in Jatropha macrantha stems: characterisation and LC/ESI/MS/MS quali–quantitative analysis. J Pharm Biomed Anal 40: 639–647. Bialy L, Waldmann H. 2003. Inhibitors of protein PTP1B inhibitors as potential therapeutics in the treatment of type 2 diabetes and obesity. Expert Opin Investig Drugs 12: 223–233. Burke TR, Zhang ZY. 1998. Protein–tyrosine phosphatases: structure, mechanism, and inhibitor discovery. Biopolymers 47: 225–241. Burke TR, Kole HK, Roller PP. 1994. Potent inhibition of insulin receptor dephosphorylation by a hexamer peptide containing the phosphotyrosyl mimetic F2Pmp. Biochem Biophys Res Commun 204: 129–134. Chen H, Cong LN, Li Y, et al. 1999. A phosphotyrosyl mimetic peptide reverses impairment of insulin-stimulated translocation of GLUT4 caused by overexpression of PTP-1B in rat adipose cells. Biochemistry 38: 384–389. Cui L, Lee HS, Ndinteh DT, et al. 2010. New prenylated flavones from Erythrina abyssinica with protein tyrosine phosphatase 1B (PTP1B) inhibitory activity. Planta med 76: 713–718. Dai Z, Wang F, Wang GL, Lin RC. 2006. Studies on chemical constituents of Balanophora spicata. China J Chinese Materia Medica 31: 1798–1800. Das B, Kashinatham A. 1997. Studies on phytochemicals: Park XVII-phenolics from the roots of Jatropha gossypifolia. Indian J Chem 36B: 1077–1078. De Souza ADL, Da Rocha AFI, Pingeiro MLB, et al. 2001. Constitutes quimicos de Gustavia augusta L. Quim Nova 24: 439–422. Donovan JL, Luthria DL, Stremple P, Waterhouse AL. 1999. Analysis of (+)-catechin, ( )-epicatechin and their 3’- and 4’O-methylated analogs. A comparison of sensitive methods. J Chromatogr B Biomed Sci Appl 726: 277–283. Copyright © 2015 John Wiley & Sons, Ltd.

Durust N, Ozden S, Umur E, Durust Y, Ducukislamoglu M. 2001. The isolation of carboxylic acids from the flowers of Delphinium formosum. Turk J Chem 25: 93–97. Elchebly M, Payette P, Michaliszyn E, et al. 1999. Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phophatase-1B gene. Science 283: 1544–1548. Friedman JM. 2003. A war on obesity, not the obese. Science 299: 856–858. Fuchino H, Satoh T, Tanaka N. 1995. Chemical evaluation of Betula species in Japan. I. Constituents of Betula ermanii. Chem Pharm Bull 43: 1937–1942. Gaspar EMM, Neves HJC. 1993. Steroidal constituents from mature wheat straw. Phytochemistry 34: 523–527. Gokhale KM. 2011. New method for synthesis of 3-(4-hydroxy-3methoxyphenyl) prop-2-enoic acid and 1-feruloyl-β-D-glucose. Int J Pharm Phytopharm Res 1: 17–22. Goldstein BJ, Bitter-Kowalczyk A, White MF, Harbeck MJ. 2000. Tyrosine dephosphorylation and deactivation of insulin receptor substrate-1 by protein tyrosine phosphatase 1B. Possible facilitation by the formation of a ternary complex with the GRB2 adaptor protein. Biol Chem 275: 4283–4289. Hamaquchi T, Sudo T, Osada H. 1995. RK-682, a potent inhibitor of tyrosine phosphatase, arrested the mammalian cell cycle prograssion at G1phase. FEBS Lett 372: 54–58. He RJ, Yu ZH, Zhang RY, Zhang ZY. 2014. Protein tyrosine phosphatases as potential therapeutic targets. Acta Pharm Sin 35: 1227–1246. Hossain P, Kawar B, El Nahas M. 2007. Obesity and diabetes in the developing world – a growing challenge. N Engl J Med 356: 213–215. Huang KC. 1998. The Pharmacology of Chinese Herbs, 2nd edn. CRC Press: New York. Ingham JL, Markham KR, Dziedzic SZ, Pope GS. 1986. Puerarin 6″-O-β-apiofuranoside, a C-glycosylisoflavone O-glycoside from Pueraria mirifica. Phytochemistry 25: 1772–1775. Kim YH. 1999. Isolation of acylated steryl glycosides from the legumes of Albizzia julibrissin. Kor J Pharmacogn 30: 290–294. Klaman LD, Boss O, Peroni OD, et al. 2000. Increased energy expenditure, decreased adiposity, and tissue-specific insulin sensitivity in protein tyrosine phosphatase 1B-deficient mice. Mol Cell Biol 20: 5479–5489. Koul S, Razdan TK, Andotra CS, et al. 2000. Koelpinin-A, B and C – three triterpenoids from Koelpinia linearis. Phytochemistry 53: 305–309. Lee IR, Jeong HK. 1992. Isolation of triterpenoid and phenylpropanoid from Codonopsis ussuriensis. Arch Pharm Res 15: 289–291. Lee SK, Hwang JY, Song JH, et al. 2007. Inhibitory activity of Euonymus alatus against alpha-glucosidase in vitro and in vivo. Nutr Res Pract 1: 184–188. Lewinsohn E, Berman E, Mazur Y, Gressel J. 1986. Glucosylation of exogenous flavanones by grapefruit (Citrus paradisi) cell vultures. Phytochemistry 25: 2531–2535. Li T, Zhang XD, Song YW, Liu JW. 2005. A microplate-based screening method for alpha-glucosidase inhibitors. Chin J Clin Pharmacol Ther 10: 1128–1134. Liljebris C, Larsen SD, Ogg D, Palazuk BJ, Bleasedale JE. 2002. Investigation of potential bioisosteric replacements for the carboxyl groups of peptidomimetic inhibitors of protein tyrosine Phytother. Res. 29: 1540–1548 (2015)

1548

S.-Y. JEONG ET AL.

phosphatase 1B: identification of a tetrazole-containing inhibitor with cellular activity. J Med Chem 45: 1785–1798. Mitase T, Fugushima S, Akitama Y. 1984. Studies on the constituents of Hedysarum polybotrys Hand.-Mazz. Chem Pharm Bull 32: 3267–3270. Moller DE. 2001. New drug targets for type 2 diabetes and the metabolic syndrome. Nature 414: 821–827. Na MK, Kim BY, Osada H, Ahn JS. 2009. Inhibition of protein tyrosine phosphatase 1B by lupeol and lupenone isolated from Sorbus commixta. J Enzyme Inhib Med Chem 24: 1056–1059. Nguyen PH, Nguyen TNA, Kang KW, et al. 2010. Prenylated pterocarpans as bacterial neuraminidase inhibitors. Bioorg Med Chem 18: 3335–3344. Nguyen PH, Dao TT, Kim JY, et al. 2011. New 5-deoxyflavonoids and their inhibitory effects on protein tyrosine phosphatase 1B (PTP1B) activity. Bioorg Med Chem 19: 3378–3383. Oh BK, Mun JH, Seo HW, et al. 2011. Euonymus alatus extract attenuates LPS-induced NF-κB activation via IKKβ inhibition in RAW 264.7 cells. J Ethnopharmacol 134: 288–293. Park SH, Ko SK, Chung SH. 2005. Euonymus alatus prevents the hyperglycemia and hyperlipidemia induced by high-fat diet in ICR mice. J Ethnopharmacol 102: 326–335. Porter LJ, Harborne JB. 1994. In the Flavonoids – Advances in Research Since 1986. Chapman & Hall: London. Slade D, Ferreira D, Marais JPJ. 2005. Circular dichroism, a powerful tool for the assessment of absolute configuration of flavonoids. Phytochemistry 66: 2177–2215.

Copyright © 2015 John Wiley & Sons, Ltd.

Su K, Gong M, Zhou J, Deng S, Iribarren AM, Pomilio AB. 2009. Components of Bauhinia candicans. Int J Chem 1: 77–82. Tewari N, Tiwari VK, Mishra RC, et al. 2003. Synthesis and bioevaluation of glycosyl ureas as alpha-glucosidase inhibitors and their effect on mycobacterium. Bioorg Med Chem 11: 2911–2922. Ullah N, Ahmad S, Malik A. 1999. Phenylpropanoid Glycosides from Daphne oleoides. Chem Pharm Bull 47: 114–115. Venable CL, Frevert EU, Kim YB, et al. 2000. Overexpression of protein tyrosine phosphatase 1B in adipocytes inhibits insulin-stimulated phosphoinositide 3-kinase activity without altering glucose transport or Akt/protein kinase B activation. J Biol Chem 275: 18318–18326. Wiesmann C, Barr KJ, Kung J, et al. 2004. Allosteric inhibition of protein tyrosine phosphatase 1B. Nat Struct Mol Biol 11: 730–737. Wilhelm, A 2008. Ms Thesis, University of the Free State Bloemfontein. Wu X, Hoffstedt J, Deeb W, et al. 2001. Depot-specific variation in protein tyrosine phosphatases activities in human omental and subcutaneous adipose tissue: a potential contribution to differential insulin sensitivity. J Clin Endocrinol Metab 86: 5973–5980. Zhang S, Zhang ZY. 2007. PTP1B as a drug target: recent developments in PTP1B inhibitor discovery. Drug Discov Today 12: 373–381. Zimmet P, Alberti KG, Shaw J. 2001. Global and societal implications of the diabetes epidemic. Nature 414: 782–787.

Phytother. Res. 29: 1540–1548 (2015)