FITOTE-03149; No of Pages 9 Fitoterapia xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Fitoterapia
3Q2 4 5 6 7
Quoc Hung Vo a,c, Phi Hung Nguyen a, Bing Tian Zhao a, Md Yousof Ali b, Jae Soo Choi b, Byung Sun Min a, Thi Hoai Nguyen c, Mi Hee Woo a,⁎ a b c
College of Pharmacy, Catholic University of Daegu, Gyeongsan 712-702, Republic of Korea Department of Food Science & Nutrition, Pukyong National University, Busan 608-737, Republic of Korea Faculty of Pharmacy, Hue University of Medicine and Pharmacy, Hue University, Hue City, Viet Nam
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Article history: Received 17 February 2015 Accepted in revised form 14 March 2015 Accepted 17 March 2015 Available online xxxx
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Keywords: Tradescantia spathacea Sw. Tradescantin Tradescantoside PTP1B inhibitors Type 2 diabetes
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1. Introduction
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Nowadays diabetes is a huge and growing problem. The most recent estimates in 2014 show that 387 million people are living with diabetes and this number is set to rise beyond 592 million in less than 25 years [1]. Type 2 diabetes (T2D), or noninsulin-dependent diabetes mellitus, is the most common type accounting for approximately 90% of the total cases among the three types of diabetes [2]. This type is characterized by a resistance to insulin, a peptide hormone produced by β-cells in the pancreas, which is responsible for glucose homeostasis [3,4]. The insulin signaling pathway is negatively regulated by protein tyrosine phosphatases, most notably, protein tyrosine phosphatase 1B (PTP1B) [4]. The overexpression of PTP1B has been shown to inhibit the increased expression of insulin in insulin-resistant states [5]. Furthermore, recent genetic evidence
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Inhibitors of protein tyrosine phosphatase 1B (PTP1B) are promising agents for the treatment of type 2 diabetes and obesity. The bioactivity-guided isolation led to the separation of two new compounds, (±)-tradescantin (13) and tradescantoside (16), along with fourteen known compounds (1–12, 14, and 15) from the aerial parts of Tradescantia spathacea Sw. (Commelinaceae). Their chemical structures were elucidated by spectroscopic methods as well as by comparing with those reported in the literature. The isolated compounds (1–16) were then examined for their inhibitory activity toward PTP1B. The results indicated that compounds 2, 6, 8, and 12 possessed potent inhibition with IC50 values of 7.82 ± 0.79, 6.80 ± 0.89, 4.55 ± 0.92, and 6.38 ± 0.14 μM, respectively. Kinetic study of compounds 2, 6, 8, 12, 13, and 16 was conducted and the structure– activity relationships of the isolated compounds (1–16) were also discussed herein. To the best of our knowledge, all the isolates were separated for the first time from this plant. © 2015 Published by Elsevier B.V.
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Protein tyrosine phosphatase 1B (PTP1B) inhibitory constituents from the aerial parts of Tradescantia spathacea Sw.
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journal homepage: www.elsevier.com/locate/fitote
⁎ Corresponding author. Tel.: +82 53 8503620. E-mail address: [email protected] (M.H. Woo).
has shown that PTP1B gene variants are associated with changes in insulin sensitivity [6]. At the genetic, molecular, biochemical, and physiological levels, PTP1B seems to be a promising drug target for the treatment of T2D and at-risk obese patients [7]. Natural products are rich sources of novel active agents for clinical uses [8]. Previous reports indicate that there are more than 1000 plant species being used to treat T2D all over the world [3] and various natural compounds display PTP1B inhibitory activity [9]. Tradescantia spathacea Sw. (Commelinaceae) is a herbal plant traditionally used as a functional food in Viet Nam for relieving cough and bleeding symptoms [10]. This plant has been proved to possess antitumoral, antigenotoxic, antimutagenic, antioxidant, and antibacterial activities [11–13]. Recently, Tan and co-workers [13] reported the identification of epigallocatechin, rhoeonin, peltatoside and rutin in the decoction of T. spathacea leaves, based on HPLC-DAD and MS data. However, to the best of our knowledge, the chemical constituents of this
http://dx.doi.org/10.1016/j.fitote.2015.03.017 0367-326X/© 2015 Published by Elsevier B.V.
Please cite this article as: Vo QH, et al, Protein tyrosine phosphatase 1B (PTP1B) inhibitory constituents from the aerial parts of Tradescantia spathacea Sw., Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.017
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The aerial parts of T. spathacea Sw. were collected from Hue city, Viet Nam in October 2012. A voucher specimen has been deposited at the College of Pharmacy, Catholic University of Daegu, Republic of Korea.
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2.2. General experimental procedures
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Melting points were determined on a Yanaco micro melting point apparatus. Optical 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 9423AQA2200E UV spectrophotometer. The NMR spectra were recorded in deuterated solvents on Bruker 250 MHz or Varian OXFORD-AS 400 MHz (Palo Alto, CA, USA) or Agilent Premium COMPACT NMR Magnet System 600 MHz instruments. 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 60 (Kieselgel 0.040–0.063 mm, 230–400 mesh, Merck) and/or reversed-phase silica gel (LiChroprep RP-18, 40–63 μm, Merck). Thin layer chromatography (TLC) tests were performed on Merck pre-coated TLC silica gel 60 F254 and/or TLC silica gel RP-18 F254S glass plates (0.25 mm), and visualized by heating after spraying with 10% H2SO4. MPLC was performed using Biotage Isolera One flash chromatography system. HPLC was performed using a Waters 600 Controller system, Waters 717 autosampler with an UV 2487 detector and YMC Pak ODS-A column (20 × 250 mm, 5 μm particle size, YMC Co., Ltd., Japan). HPLC solvents were purchased from Burdick & Jackson, USA. The (S)- and (R)-(−)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride were purchased from Sigma-Aldrich. All other chemicals and solvents were of analytical grade and used without further purification.
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2.3. Extraction and isolation
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The dried and grinded aerial parts of T. spathacea (7.0 kg) were extracted three times with 99.8% MeOH (10 L × 3 h/time) at 60 °C. The resulting solution was concentrated under reduced pressure to yield a residue (750 g). The MeOH extract was suspended in H2O (1.5 L) and partitioned successively with CH2Cl2 (6 × 1.0 L, 300 g), EtOAc (5 × 1.0 L, 40 g), n-BuOH (5 × 1.0 L, 60 g) and H2O-soluble fractions (250 g), respectively. Since the MeOH extract showed significant PTP1B inhibitory effect, its four extracts were subsequently tested for PTP1B inhibitory activity. Among them, the EtOAc and n-BuOH fractions showed good activity against PTP1B. These two extracts were then combined as their TLC patterns were quite similar after visualized. The combined fraction (100 g) was subjected to open flash column chromatography (10 × 23 cm, silica gel 230–400 mesh) eluting with gradient of CH2Cl2–MeOH (50:1 to 0:1) to
95 96 97 98 99 100 101 102
107 108 109 110 111 112 113 114 115 116 117 118 119 120 121
C
93 94
E
92
R
90 91
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88 89
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86 87
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84 85
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80 81
2.3.1. (±)-Tradescantin (13) Amorphous powder; [α]25 D 0 (c 0.2, MeOH); mp 200– 202 °C; UV (MeOH) λmax nm (log ε): 205 (5.29), 235 (5.21), 280 (5.03), 311 (4.96); IR (KBr) νmax cm−1: 3356, 1724, 1671, 1595, 1522; 1H NMR (Methanol-d4, 250 MHz) and 13C NMR (Methanol-d4, 62.5 MHz) data, see Table 1; HR-EI-MS m/z 240.0632 [M]+ (calcd. for C11H12O6, 240.0634).
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2.3.2. Tradescantoside (16) Amorphous powder; [α]25 D –46.8 (c 0.2, MeOH); mp 223– 228 °C; UV (MeOH) λmax nm (log ε): 205 (4.55), 246 (4.23), 293 (4.14), 324 (4.19); IR (KBr) νmax cm−1: 3291, 1689, 1597, 1515; 1H NMR (Methanol-d4, 600 MHz) and 13C NMR (Methanol-d4, 150 MHz), see Table 2; HR-ESI-MS (positive) m/z 515.1166 [M + Na]+ (calcd. for C23H24O12Na+, 515.1160); HR-ESI-MS (negative) m/z 491.1187 [M–H]− (calcd. for C23H23O− 12, 491.1195).
170 171
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2.1. Plant material
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2. Materials and methods
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afford seven subfractions (TD1–TD7). The fraction TD2 was chromatographed on a silica gel column using a stepwise gradient of n-hexanes:EtOAc mixture as a solvent system (9:1 to 4:1) to yield compounds 6 (~ 1 g) and 8 (75 mg) and four subfractions (TD2.1–TD2.4). The subfraction TD2.2 was applied for a reverse-phase column using MeOH:H2O (1:10 to 0:1) as a mobile phase to yield compound 7 (4 mg). The fraction TD3 was loaded on silica gel column eluted with a stepwise gradient of n-hexane:acetone mixture (4:1 to 1:1) to give three subfractions (TD3.1–TD3.2). TD3.1 was chromatographed on an open column using Sephadex LH-20 and eluted by 99.8% MeOH to yield compounds 1 (~1 g) and 14 (3 mg), respectively. TD3.2 was chromatographed by HPLC on a RP-C18 column using gradient of MeOH and 0.1% formic acid in H2O as a mobile phase (21% to 25%) to yield compounds 3 (5 mg), 9 (4 mg), 10 (9 mg), 11 (8 mg), 12 (70 mg), and 13 (7 mg). The fraction TD4 was subjected on MPLC column eluted with a gradient of CH2Cl2:MeOH mixture (40:1 to 0:1) to afford four subfractions (TD4.1–TD4.4). TD4.2 was further purified by an open column using Sephadex LH-20 (99.8% MeOH) and a RP-C18 column using gradient MeOH:H2O as a solvent system (1:10 to 0:1), respectively, to yield compound 2 (200 mg). The fraction TD6 was chromatographed on a reversedphase open column using RP-C18 eluted with a stepwise gradient of MeOH:H2O mixture (1:10 to 0:1) to afford four subfractions (TD6.1–TD6.4). TD6.1 was chromatographed on an open column using normal-phase silica gel eluted with a gradient of CH2Cl2:MeOH (5:1 to 0:1). The eluates was then purified with Sephadex LH-20 column using 99.8% MeOH as a mobile phase to yield compounds 4 (24 mg) and 5 (23 mg), respectively. Subfraction TD6.2 was chromatographed on an open column using Sephadex LH-20 (99.8% MeOH) and was then purified by HPLC on a RP-C18 column using gradient of MeOH and 0.1% formic acid in H2O as a solvent (32% to 35%) to yield compound 15 (10 mg). Finally, from subfraction TD6.4, compound 16 (8 mg) was isolated by using Sephadex LH-20 column eluted with 99.8% MeOH and silica gel column eluted with CH 2Cl2:MeOH mixture (5:1 to 0:1), respectively.
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plant have not been reported in detail. Therefore, in the interest of promoting drug discovery from natural sources, this research was conducted to identify bioactive compounds from the aerial parts of T. spathacea, focusing on PTP1B inhibitory activity.
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Please cite this article as: Vo QH, et al, Protein tyrosine phosphatase 1B (PTP1B) inhibitory constituents from the aerial parts of Tradescantia spathacea Sw., Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.017
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1 2
173.1 40.9
3 4 1′ 2′ 3′ 4′ 5′ 6′ OCH3
70.6 199.4 127.8 116.6 146.8 152.9 116.1 123.7 52.7
– 2.79 (dd, J = 4.3, 15.8) 2.50 (dd, J = 8.0, 15.8) 5.29 (dd, J = 4.3, 8.0) – – 7.37 (s) – – 6.77 (d, J = 8.3) 7.39 (d, J = 8.3) 3.61 (s)
2.4. Protein tyrosine phosphatase 1B inhibitory assay
180 181
The PTP1B inhibitory activity of tested compounds was evaluated using p-nitrophenyl phosphate (pNPP) as a substrate. To each of 96 wells (final volume of 100 μL), 40 μL of PTP1B enzyme in a buffer containing 50 mM citrate (pH 6.0), 0.1 M NaCl, 1 mM EDTA, and 1 mM dithiothreitol were added with or without samples dissolved in 10% DMSO. After being preincubated at 37 °C for 10 min, the plate was added 50 μL of 2 mM pNPP in PTP1B reaction buffer. Following incubation at 37 °C for 20 min, the reaction was terminated with the addition of 10 M NaOH. The amounts of p-nitrophenyl produced after enzymatic dephosphorylation from pNPP was estimated by measuring the absorbance at 405 nm using a microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). The nonenzymatic hydrolysis of 2 mM pNPP was corrected by measuring the increase in absorbance at 405 nm obtained in the absence of PTP1B enzyme. The percent inhibition (%) was calculated as (Ac − As) / Ac × 100%, where Ac is the absorbance of the control,
186 187 188 189 190 191 192 193 194 195
Table 2 1D and 2D NMR spectral data of compound 16 (600 MHz, methanol-d4). Position
δC
t2:4 t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20 t2:21 t2:22 t2:23 t2:24 t2:25 t2:26 t2:27
1 2 3 4 5 6 7 1′ 2′ 3′ 4′ 5′ 6′
124.3 120.1 146.4 153.2 116.9 127.4 170.1 104.0 74.9 77.6 71.9 75.9 64.9
1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ OCH3
U
N C
t2:3
O
t2:1 t2:2
R
R
196
E
184 185
C
179
128.0 112.2 149.5 150.7 116.7 124.3 147.3 115.5 169.4 56.7
δH (multiplicity, J in Hz) – 7.88 (d, J = 1.8) – – 6.92 (d, J = 8.4) 7.67 (dd, J = 1.8, 8.4) – 4.91 (d, J = 7.8) 3.58 (m) 3.56 (m) 3.48 (m) 3.79 (m) 4.61 (dd, J = 1.8, 12.0) 4.37 (dd, J = 7.2, 12.0) – 7.18 (d, J = 1.8) – – 6.85 (d, J = 8.4) 7.12 (dd, J = 1.8, 8.4) 7.63 (d, J = 15.6) 6.48 (d, J = 15.6) – 3.93 (s)
2.5. Kinetic analysis
199
The reaction mixture consisted of three different concentrations of pNPP used as a PTP1B substrate in the absence or presence of tested compounds [15]. The Michaelis–Menten constant (Km) and maximum velocity (Vmax) of PTP1B were determined by Lineweaver–Burk plots, and the inhibition constant (Ki) was calculated by Dixon plots using a SigmaPlot™ program (SPCC Inc., Chicago, IL).
200 201
2.6. Preparation of Mosher esters
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The experiment was conducted using a reported method [16,17]. To 0.5 mg of 13, 0.3 mL of CH2Cl2, 5 drops of pyridine, 0.5 mg of 4-(dimethylamino)pyridine, and 12 mg of (R)-(−)α-methoxy-α-(trifluoromethyl)phenylacetyl (MTPA) chloride were added sequentially. The mixture was stirred until being completely dissolved, then it was left at room temperature overnight and subsequently purified over a small column (0.6 × 6 cm) using silica gel (230–400 mesh) eluted with 3–4 mL of n-hexane:CH2Cl2 (1:2). The eluate was dried afterwards, CH2Cl2 (5 mL) was added, and the solution was washed using 1% NaHCO3 (5 mL × 3) and H2O (5 mL × 2); the washed solution was dried in vacuo to yield the (S)-MTPA ester derivative (13S) of 13. The treatment of 13 (0.5 mg) using (S)-(+)-MTPA chloride as mentioned above yielded the corresponding (R)-MTPA ester (13R).
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3. Results and discussion
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Sixteen compounds (1–16) were isolated from the aerial parts of T. spathacea Sw. using various chromatographic methods. Their chemical structures were shown in Fig. 1. Compound 13 was obtained as an amorphous powder with mp 200–202 °C. The molecular formula of 13 was determined as C11H12O6 from the molecular ion peak at m/z 240.0632 [M]+ (calcd. m/z 240.0634) obtained by HR-EI-MS. The IR spectrum of compound 13 indicated the presence of hydroxyl (3356 cm−1), ester (1724 cm−1) and ketone (1671 cm−1) groups as well as a substituted benzene ring (1595 and 1522 cm−1). Its UV spectrum showed absorption maxima at 205, 235, 280, and 311 nm. The 1H NMR spectrum of 13 displayed an ABX-type aromatic spin system at δH 7.39 (1H, d, J = 8.3 Hz, H-6′), 7.37 (1H, s, H-2′), and 6.77 (1H, d, J = 8.3 Hz, H-5′) with their corresponding carbons at δC 123.7 (C-6′), 116.6 (C-2′), and 116.1 (C-5′), respectively, assigned with the aid of HMQC spectroscopic data analysis. This observation suggested the presence of a 1,2,4-trisubstituted benzene ring which was also confirmed by the correlations observed in the HMBC experiment (Fig. 2). The connection between the ketone carbonyl at δC 199.4 (C-4) and the aromatic carbon at δC 127.8 (C-1′) was proved by the HMBC correlation from H-6′ (δH 7.39) to C-4 (δC 199.4). One aliphatic AMX-spin pattern at δH 5.29 (1H, dd, J = 4.3, 8.0 Hz, H-3), 2.79 (1H, dd, J = 4.3, 15.8 Hz, H-2a), and 2.50 (1H, dd, J = 8.0, 15.8 Hz, H-2b) suggested the presence of a partial structure of \CH2\CH(OH)\, which was confirmed by 1H–1H COSY correlation from H-3 to H-2. The carbinol group was adjacent to the ketone as observed by the
224
F
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15
R O O
δH (multiplicity, J in Hz)
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δC
D
Position
E
t1:3
182 183
and As is the absorbance of the sample. Ursolic acid was used as a 197 positive control [14]. 198
Table 1 1D and 2D NMR spectral data of compound 13 (250 MHz, methanol-d4).
T
t1:1 t1:2
3
Please cite this article as: Vo QH, et al, Protein tyrosine phosphatase 1B (PTP1B) inhibitory constituents from the aerial parts of Tradescantia spathacea Sw., Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.017
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cross peak from H-3 (δH 5.29) to C-1′ (δC 127.8) in the HMBC spectrum. The methylene group was linked with a methyl ester group inferred from the cross peaks of two methylenic protons [δH 2.79 (H-2a) and 2.50 (H-2b)] to ester carbonyl at δC 173.1 (C-1) in the HMBC spectrum. This linkage was further confirmed by the correlations between H-2 and C-4, between H-3 and C-1, and between the methyl protons at δH 3.61 (3H, s) and C-1 (173.1).
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252
C
Fig. 1. Structure of compounds 1–16 isolated from the combined EtOAc and n-BuOH extracts of Tradescantia spathacea Sw.
Fig. 2. 1H–1H COSY and 1H–13C key HMBC correlations of tradescantin (13).
Since the C-3 position of 13 is a chiral center, Mosher's method was applied to determine the absolute configuration [16,17]. Compound 13 was treated with (R)- and (S)-MTPA chloride to give (S)- and (R)-MTPA esters, 13S and 13R, respectively. The differences in the chemical shift of protons H-2a, H-2b, and H-3 were expected to be observed between the two MTPA esters of 13 (ΔδSR = δS − δR), which may reveal the absolute configuration at C-3. Interestingly, the 1H NMR data of 13S and 13R (see Table 3) showed the same pattern that the signals of H-3 were shifted downfield and were separated unambiguously into two at δH 6.41 (1H, dd, J = 4.2, 9.0 Hz) and δH 6.38 (1H, dd, J = 4.2, 9.0 Hz) with the approximate 1:1 ratio. Similarly, the signals of Hab-2 appeared as two sets of two double doublets at [δH 2.96 (1H, dd, J = 4.2, 16.8 Hz) and δH 2.90 (1H, dd, J = 9.0, 16.8 Hz)] and at [δH 2.92 (1H, dd, J = 4.2, 16.8 Hz) and δH 2.84 (1H, dd, J = 9.0, 16.8 Hz)]. This observation suggested that 13 was a racemic mixture which was further confirmed by [α]25 D = 0. Based on the above data,
Please cite this article as: Vo QH, et al, Protein tyrosine phosphatase 1B (PTP1B) inhibitory constituents from the aerial parts of Tradescantia spathacea Sw., Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.017
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t3:5
2a
t3:6
2b
t3:7
3
2.96 (dd, J = 4.2, 16.8 Hz) or 2.92 (dd, J = 4.2, 16.8 Hz) 2.90 (dd, J = 9.0, 16.8 Hz) or 2.84 (dd, J = 9.0, 16.8 Hz) 6.41 (dd, J = 4.2, 9.0 Hz) or 6.38 (dd, J = 4.2, 9.0 Hz)
2.92 (dd, J = 4.2, 16.8 Hz) or 2.96 (dd, J = 4.2, 16.8 Hz) 2.84 (dd, J = 9.0, 16.8 Hz) or 2.90 (dd, J = 9.0, 16.8 Hz) 6.38 (dd, J = 4.2, 9.0 Hz) or 6.41 (dd, J = 4.2, 9.0 Hz)
292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314
Fig. 4. 1H–1H key NOESY correlations of tradescantoside (16).
C
290 291
E
288 289
R
286 287
R
284 285
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282 283
N C
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compound 13 was established as (±)-methyl-4-(3′,4′dihydroxyphenyl)-3-hydroxy-4-oxobutanoate, a new natural product, and named (±)-tradescantin. Compound 16 was obtained as an amorphous powder. Its IR spectrum exhibited absorption bands at 3291 cm−1 (OH), 1689 cm−1 (COOH), 1597 cm−1 and 1515 cm−1 (aromatic ring), respectively. The UV spectrum of 16 showed absorption maxima at 205, 246, 293 and 324 nm. The HR-ESI-MS indicated a molecular formula of C23H24O12 from the ion peak observed at m/z 515.1166 [M + Na]+ (calcd. m/z 515.1160 for C23H24O12Na+) and at m/z 491.1187 [M–H]− (calcd. m/z 1 13 491.1195 for C23H23O− C NMR spectra of 16 12). The H and demonstrated signals of an anomeric proton and a carbon at δH 4.91 (1H, d, J = 7.8 Hz, H-1′) and at δC 104.0 (C-1′), respectively, a methylene group at δH 4.61 (1H, dd, J = 1.8, 12.0 Hz, H-6′a), 4.37 (1H, dd, J = 7.2, 12.0 Hz, H-6′b) and δC 64.9 (C-6′), a glucopyranosyl moiety from δH 3.48 to 3.79, with their corresponding carbons at δC 77.6 (C-3′), 75.9 (C-5′), 74.9 (C-2′), and 71.9 (C-4′). The coupling constant of the anomeric proton H-1′ (J = 7.8 Hz) indicated the β-configuration for this glucopyranosyl moiety. The presence of the β-D-glucopyranosyl residue was further supported by the analysis of NOESY experiment (Fig. 4) showing the correlations between H-1′, H-3′ and H-5′, and between H-2′ and H-4′. In addition, a trans double bond at [δH 7.63 (1H, d, J = 15.6 Hz, H-7″) and δC 147.3 (C-7″)], and [δH 6.48 (1H, d, J = 15.6 Hz, H-8″), δC 115.5 (C-8″)], an ABX aromatic spin system at δH 7.18 (1H, d, J = 1.8 Hz, H-2″), 7.12 (1H, dd, J = 1.8, 8.4 Hz, H-6″), and 6.85 (1H, d, J = 8.4 Hz, H-5″), and a methoxy group at [δH 3.93 (3H, s), δC 56.7], were the characteristic signals of a trans-feruloyl moiety. The feruloyl moiety was also evidenced by the HMBC correlations (Fig. 3) showing the cross peaks from methoxyl protons (δH 3.93) to C-3″ (δC 149.5), and from olefinic proton H-7″ (δH 7.63) to C-2″ (δC 112.2), C-6″ (δC 124.3), and C-9″ (δC 169.4). The presence of 3,4-dihydroxybenzoic acid residue was identified by the other ABX coupled aromatic system at δH 7.88 (1H, d, J = 1.8 Hz, H-2), 7.67 (1H, dd, J = 1.8, 8.4 Hz, H-6), and 6.92 (1H, d, J = 8.4 Hz,
U
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R O O
13R (δH)
Fig. 3. 1H–1H COSY and 1H–13C key HMBC correlations of tradescantoside (16).
H-5) in combination with the HMBC spectrum showing the cross peaks from H-2 to C-3, C-4, C-6, and C-7, from H-5 to C-1 and C-3, and from H-6 to C-2, C-4 and C-7. All the above observations suggested a structure similar to procumboside B which possessed 4-hydroxyphenol substituent instead of 3,4dihydroxybenzoic acid moiety [18]. Further analysis of the HMBC spectrum demonstrated the linkage of the methylenic protons (H-6′) of the sugar to the ester group (C-9″) of transferuloyl moiety. Another cross peak between H-1′ (4.91, d, J = 7.8 Hz) and C-3 (146.4) established the connection from the C-1′-OH of the sugar to the carbon C-3 of the 3,4dihydroxybenzoic acid moiety confirmed by the correlation between H-1′ and H-2, and by the absence of a correlation between H-1′ and H-5/H-6 in the NOESY experiment. Thus, compound 16 was determined as 3,4-dihydroxybenzoic acid 3-O[6′-(3″-methoxy,4″-hydroxycinnamoyl)-β-D-glucopyranoside], a new natural product, and named tradescantoside. Compound 16 was the derivative of protocatechuic acid occurring in rich quantity in various multiple fruits such as berries including raspberry, blueberry, mulberry, strawberry, cranberry, and
P
13S (δH)
D
Position
E
t3:4
F
Table 3 Characteristic 1H NMR data of Mosher esters (13S and 13R) of compound 13 (600 MHz, chloroform-d).
T
t3:1 t3:2 t3:3
5
315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334
Table 4 t4:1 PTP1B inhibitory activity of compounds 1–16 isolated from the aerial parts of t4:2 Tradescantia spathacea Sw. t4:3 Compound
IC50 (μM)a
t4:4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Ursolic acidb
15.13 ± 0.93 7.82 ± 0.79 25.65 ± 0.77 64.61 ± 0.39 40.20 ± 0.01 6.80 ± 0.89 33.83 ± 0.27 4.55 ± 0.92 N100 52.99 ± 1.93 68.16 ± 0.19 6.38 ± 0.14 17.62 ± 0.48 45.85 ± 0.59 41.83 ± 0.69 10.79 ± 1.04 2.80 ± 0.42
t4:5 t4:6 t4:7 t4:8 t4:9 t4:10 t4:11 t4:12 t4:13 t4:14 t4:15 t4:16 t4:17 t4:18 t4:19 t4:20 t4:21
a Results are expressed as IC50 values (μM) ± standard deviation of three experiments performed in triplicate. b Positive control.
t4:22 t4:23 t4:24
Please cite this article as: Vo QH, et al, Protein tyrosine phosphatase 1B (PTP1B) inhibitory constituents from the aerial parts of Tradescantia spathacea Sw., Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.017
Q.H. Vo et al. / Fitoterapia xxx (2015) xxx–xxx
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Fig. 5. Graphical determination of inhibition type for compounds 2, 6, 8, 12, 13, and 16 using Lineweaver–Burk plots was expressed as the mean reciprocal of initial velocity for n = 3 replicates at each substrate concentration (panels a, b, c, d, e, and f, respectively).
Please cite this article as: Vo QH, et al, Protein tyrosine phosphatase 1B (PTP1B) inhibitory constituents from the aerial parts of Tradescantia spathacea Sw., Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.017
Q.H. Vo et al. / Fitoterapia xxx (2015) xxx–xxx
2 6 8 12 13 16
12.16 4.45 7.15 4.06 18.36 8.03
Competitive Mixed Mixed Noncompetitive Mixed Mixed
350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385
4. Conclusions
C
348 349
E
346 347
R
344 345
R
342 343
O
340 341
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gooseberry, as well as in Loquat fruit, wine, honey, and soybean. Protocatechuic acid was reported to possess antibacterial, antiviral, antioxidant, antidiabetic, anticancer, anti-ageing, antifibrotic, and antiviral activities [19]. Its glucoside derivatives were found in the colleterial or sexual accessory glands of the cockroaches, Blatta orientalis L. and Periplaneta americana L. [20], and the aerial parts of Baccharis dracunculifolia DC. (dracunculifoside B) [21]. The fourteen known compounds were identified by comparing their spectroscopic data with those in the literature including (2R,3R)-2,3-dihydroxy-2-methylbutyrolactone (1) [22– 24], bracteanolide A (2) [25,26], 4-(3′,4′-dihydroxyphenyl) furan-2(5H)-one (3) [26], (S)-2-hydroxy-3-(4′-hydroxyphenyl) propanoic acid (4) [27], (R)-2-hydroxy-3-(4′-hydroxyphenyl) propanoic acid (5) [28,29], latifolicinin C (6), latifolicinin B (7), latifolicinin A (8) [30], protocatechuic acid (9) [31], 1-(3′,4′dihydroxyphenyl)-2-hydroxyethan-1-one (10), hydroxytyrosol (11) [32], oresbiusin A (12) [33], kaempferol (14) [34,35], and (6S,9R)-roseoside (15) [36,37]. As of 2012, according to the systematic review of Jiang et al., approximately 300 compounds with PTP1B inhibitory activity have been isolated and identified from about 79 species belonging to 70 genera. Most of the inhibitors are phenolic compounds accounting for more than 65% of total. The other groups are terpenes, steroids, N- or S-containing compounds, and several rare structures [9]. The in vitro inhibition study of the isolated compounds (1– 16) against PTP1B was conducted according to the reported method [14]. The results were shown in Table 4 with ursolic acid, the known PTP1B inhibitor, used as the positive control. All of the tested compounds, except compound 9, exhibited a dose-dependent inhibitory effect. Among these results, compound 8 showed the best inhibition, and compounds 2, 6, and 12 demonstrated a potent activity. Two new compounds, 13 and 16, together with compounds 1, 3, and 7 showed inhibitory effect with IC50 values of 17.62 ± 0.48, 10.79 ± 1.04, 15.13 ± 0.93, 25.65 ± 0.77, and 33.83 ± 0.27 μM, respectively. Compounds 4, 5, 10, 11, 14, and 15 displayed moderate activity. Compounds 2 and 3 were γ-butyrolactone derivatives and only differed at the C-5 position with or without hydroxyl group. However, the inhibitory activity of compound 2 was three times as strong as that of compound 3 (IC50 7.82 and 25.65 μM, respectively). This result suggested that the 5-hydroxyl group played an important role in the inhibition of this skeleton toward PTP1B activity. By comparing the two enantiomers, 4 and 5, it was found that the (R)-configuration (compound 5, IC50 40.20 μM) possessed the stronger effect than the (S)-configuration in 2-hydroxy-3-(4′-hydroxyphenyl)propanoic acid (compound 4, IC50 64.61 μM). Nevertheless, this difference may not be important since compounds 6 and 12 showed potent activity in the (S)-configuration (IC50 6.80 ± 0.89 and 6.38 ± 0.14 μM,
F
t5:4 t5:5 t5:6 t5:7 t5:8 t5:9
R O O
Inhibition type
P
Ki (μM)
D
Tested compounds
E
t5:3
335
respectively). It also can be deduced from the comparison that the presence of mono/dihydroxyphenyl might not significantly affect the inhibitory activity but the ester group possessed a decisive meaning for the effect. The important role of the ester functional group was confirmed by comparing IC50 values of compounds 1, 2, 6, 8, 12, 13, and 16 (ranging from 4.55 to 15.13 μM) with those of compounds 4, 5, 9, 10, 11, 14, and 15 (ranging from 33.83 to over 100 μM) which were not ester derivatives. The subsequent investigations were conducted to determine the inhibitory constants Ki using Dixon plots and the inhibitory mechanisms of the inhibitors which displayed good activity (compounds 2, 6, 8, 12, 13, and 16) using Lineweaver–Burk plots (Fig. 5). The results are shown in Table 5. Compound 12 was found to decrease the Vmax value but did not alter the Km value of PTP1B, suggesting that the inhibition type was noncompetitive toward pNPP (Fig. 5d) with Ki value of 4.06 μM. In contrast, an increase in the concentration of compound 2 did not affect the Vmax value but increased the Km value of PTP1B, indicating the competitive mode of compound 2 toward pNPP (Fig. 5a) [38] with Ki value of 12.16 μM. Compounds 6, 8, 13, and 16 were established as mixed-type inhibitors with Ki values ranging from 4.45 to 18.36 μM since they decreased the Vmax values while the Km values of PTP1B were increased (Fig. 5b, c, e, and f, respectively).
Table 5 Kinetic analysis of compounds 2, 6, 8, 12, 13, and 16.
T
t5:1 t5:2
7
386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411
Diabetes has become one of the most challenging health problems of modern society. Therefore, scientists have put much effort to develop drugs to treat or cure this metabolic disease, especially type 2 diabetes. The PTP1B inhibitors was considered as a promisingly therapeutic option, however, there are no novel drugs approved for clinical use to date, which encourages researchers to keep searching for PTP1B inhibitory agents. In our study, the bioassay-guided investigation of the aerial parts of T. spathacea Sw. resulted in the isolation of two new compounds, (±)-tradescantin (13) and tradescantoside (16), together with fourteen known compounds including three butyrolactone derivatives (1–3), nine phenolics (4–12), a flavonoid (14), and a glucoside (15) which possessed inhibitory effect toward PTP1B, except compound 9. Those results have revealed the potential utilization of T. spathacea Sw. in the discovery of PTP1B inhibitors for the treatment of type 2 diabetes. For the practical use, further studies should be conducted to confirm their cellular effects as well as to investigate their inhibitory effects in vivo.
412 413
Declaration of interest
431
414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430
The authors report no conflicts of interest. The authors alone 432 are responsible for the content and writing of this article. 433 Acknowledgments
434
This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No. 2010-0010549). We are grateful to the Korea Basic Science Institute (KBSI) for mass spectral measurements.
435 436
Please cite this article as: Vo QH, et al, Protein tyrosine phosphatase 1B (PTP1B) inhibitory constituents from the aerial parts of Tradescantia spathacea Sw., Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.017
437 438 439
442
A.1. (2R,3R)-2,3-Dihydroxy-2-methylbutyrolactone (1)
443 444
447 448
1 Colorless oil; [α]25 D –57.8 (c 0.4, MeOH); H NMR (Methanold4, 600 MHz) δ: 4.49 (1H, dd, J = 4.2, 10.2 Hz, H-4a), 4.20 (1H, dd, J = 1.8, 10.2 Hz, H-4b), 4.10 (1H, dd, J = 3.6, 1.2 Hz, H-2), 1.44 (3H, s, H-5); 13C NMR (Methanol-d4, 150 MHz) δ: 180.5 (C-1); 74.7 (C-2), 74.5 (C-3), 73.5 (C-4), 21.6 (C-5); EI-MS m/z 132 [M]+.
449
A.2. Bracteanolide A (2)
450
455 456
White amorphous powder; mp 240–242 °C; 1H NMR (Methanol-d4, 600 MHz) δ: 7.25 (1H, d, J = 2.4 Hz, H-2′), 7.23 (1H, dd, J = 2.4, 8.4 Hz, H-6′), 6.87 (1H, d, J = 8.4 Hz, H-5′), 6.49 (1H, s, H-5), 6.28 (1H, s, H-3); 13C NMR (Methanol-d4, 150 MHz) δ: 174.3 (C-2); 166.0 (C-4), 150.6 (C-4′), 146.9 (C-3′), 122.8 (C-1′), 122.5 (C-6′), 116.7 (C-5′), 116.1 (C-2′), 112.0 (C-3), 100.1 (C-5); EI-MS m/z 208 [M]+.
457
A.3. 4-(3′,4′-Dihydroxyphenyl)furan-2(5H)-one (3)
458
463
Amorphous powder; 1H NMR (Methanol-d4, 250 MHz) δ: 7.11 (1H, s, H-2′), 7.09 (1H, d, J = 6.3 Hz, H-6′), 6.91 (1H, d, J = 6.3 Hz, H-5′), 6.29 (1H, s, H-3), 5.33 (2H, s, H-5); 13C NMR (Methanol-d4, 62.5 MHz) δ: 177.7 (C-2), 167.9 (C-4), 150.9 (C-4′), 147.2 (C-3′), 122.9 (C-1′), 120.9 (C-6′), 116.8 (C-5′), 114.8 (C-2′), 109.8 (C-3), 73.1 (C-5).
464
A.4. (S)-2-Hydroxy-3-(4′-hydroxyphenyl)propanoic acid (4)
465
470 471
1 White needles; [α]25 H NMR D –10.6 (c 1.2, MeOH); (Methanol-d4, 250 MHz) δ: 6.96 (2H, d, J = 8.5 Hz, H-3′, H-5′), 6.58 (2H, d, J = 8.5, H-2′, H-6′), 4.15 (1H, dd, J = 4.5, 7.5 Hz, H-2), 2.89 (1H, dd, J = 4.0, 13.8 Hz, H-3a), 2.69 (1H, dd, J = 7.8, 13.8 Hz, H-3b); 13C NMR (Methanol-d4, 62.5 MHz) δ: 177.5 (C-1), 157.2 (C-4′), 131.7 (C-3′, C-5′), 129.7 (C-1′), 116.2 (C-2′, C-6′), 73.2 (C-2), 40.9 (C-3).
472
A.5. (R)-2-Hydroxy-3-(4′-hydroxyphenyl)propanoic acid (5)
473
478 479
1 White amorphous powder; [α]25 D +15.6 (c 0.6, MeOH); H NMR (Methanol-d4, 250 MHz) δ: 7.00 (2H, d, J = 8.3 Hz, H-3′, H-5′), 6.58 (2H, d, J = 8.5, H-2′, H-6′), 3.97 (1H, dd, J = 3.5, 8.3 Hz, H-2), 2.91 (1H, dd, J = 3.5, 14.0 Hz, H-3a), 2.61 (1H, dd, J = 8.3, 14.0 Hz, H-3b); 13C NMR (Methanol-d4, 62.5 MHz) δ: 181.1 (C-1), 156.8 (C-4′), 131.7 (C-3′, C-5′), 131.3 (C-1′), 116.0 (C-2′, C-6′), 75.1 (C-2), 41.7 (C-3).
480
A.6. Latifolicinin C (6)
481
Light yellow powder; mp 65–67 °C; [α]25 D –9.6 (c 0.4, MeOH); 1H NMR (Chloroform-d, 400 MHz) δ: 6.97 (2H, d, J = 8.4 Hz, H-3′, H-5′), 6.65 (2H, d, J = 8.4 Hz, H-2′, H-6′), 4.39 (1H, dd, J = 4.8, 6.8 Hz, H-2), 3.72 (3H, s, OCH3), 3.72 (1H, dd, J = 4.4, 14.0 Hz, H-3a), 2.84 (1H, dd, J = 6.8, 14.0 Hz, H-3b); 13C NMR (Methanol-d4, 100 MHz) δ: 175.9 (C-1), 155.0 (C-4′), 130.6 (C-3′, C-5′), 127.7 (C-1′), 115.6 (C-2′, C-6′), 71.7 (C-2), 52.6 (OCH3), 39.7 (C-3).
468 469
474 475 476 477
482 483 484 485 486 487 488
1 Syrup; [α]25 D –5.0 (c 0.1, MeOH); H NMR (Methanol-d4, 600 MHz) δ: 7.07 (2H, d, J = 8.4 Hz, H-3′, H-5′), 6.73 (2H, d, J = 8.4 Hz, H-2′, H-6′), 4.31 (1H, dd, J = 5.4, 7.2 Hz, H-2), 4.16 (2H, q, J = 7.2, H-1″), 2.97 (1H, dd, J = 5.4, 13.8 Hz, H-3a), 2.87 (1H, dd, J = 7.8, 13.8 Hz, H-3b), 1.25 (3H, t, J = 7.2 Hz, H-2″); 13C NMR (Methanol-d4, 150 MHz) δ: 175.6 (C-1), 157.3 (C-4′), 131.6 (C-3′, C-5′), 129.3 (C-1′), 116.2 (C-2′, C-6′), 73.5 (C-2), 62.1 (C-1″), 41.1 (C-3), 14.6 (C-2″).
490 491
D
P
R O
1 Syrup; [α]25 D –6.0 (c 0.3, MeOH); H NMR (Chloroform-d, 400 MHz) δ: 7.02 (2H, d, J = 8.4 Hz, H-3′, H-5′), 6.66 (2H, d, J = 8.4 Hz, H-2′, H-6′), 4.39 (1H, dd, J = 4.8, 6.4 Hz, H-2), 4.14 (2H, m, H-1″), 3.03 (1H, dd, J = 4.4, 14.0 Hz, H-3a), 2.87 (1H, dd, J = 6.4, 14.0 Hz, H-3b), 1.61 (2H, m, H-2″), 1.35 (2H, m, H-3″), 0.92 (3H, t, J = 7.6 Hz, H-4″); 13C NMR (Chloroform-d, 100 MHz) δ: 174.6 (C-1), 155.0 (C-4′), 130.8 (C-3′, C-5′), 128.0 (C-1′), 115.5 (C-2′, C-6′), 71.6 (C-2), 65.9 (C-1″), 39.8 (C-3), 30.7 (C-2″), 19.2 (C-3″), 13.8 (C-4″).
492 493 494 495 496 497
498 499 500 501 502 503 504 505 506 507
A.9. Protocatechuic acid (9)
508
Needle crystals; mp 219–221 °C; 1H NMR (Methanol-d4, 400 MHz) δ: 7.43 (1H, s, H-2), 7.42 (1H, d, J = 7.6 Hz, H-6), 6.80 (1H, d, J = 7.6 Hz, H-5); 13C NMR (Methanol-d4, 100 MHz) δ: 170.4 (COOH), 151.7 (C-4), 146.2 (C-3), 124.0 (C-6), 123.3 (C-1), 117.9 (C-5), 115.9 (C-2).
509
A.10. 1-(3′,4′-Dihydroxyphenyl)-2-hydroxyethan-1-one (10)
514
Amorphous powder; mp 178–181 °C; 1H NMR (Methanold4, 400 MHz) δ: 7.40 (1H, br s, H-2′), 7.39 (1H, m, overlap, H-5′), 6.84 (1H, d, J = 8.0 Hz, H-6′), 4.80 (2H, s, H-2); 13C NMR (Methanol-d4, 100 MHz) δ: 198.8 (C-1), 152.8 (C-4′), 146.8 (C-3′), 127.9 (C-1′), 122.5 (C-6′), 116.2 (C-5′), 115.5 (C-2′), 66.0 (C-2).
515
A.11. Hydroxytyrosol (11)
521
Syrup; 1H NMR (Methanol-d4, 400 MHz) δ: 6.68 (1H, d, J = 8.0 Hz, H-5′), 6.66 (1H, d, J = 1.6 Hz, H-2′), 6.53 (1H, dd, J = 1.6, 8.0 Hz, H-6′), 3.68 (2H, t, J = 7.2 Hz, H-1), 2.67 (2H, t, J = 7.2 Hz, H-2); 13C NMR (Methanol-d4, 100 MHz) δ: 146.3 (C-3′), 144.8 (C-4′), 131.9 (C-1′), 121.5 (C-6′), 117.2 (C-2′), 116.4 (C-5′), 64.8 (C-1), 39.8 (C-2).
522 523
A.12. Oresbiusin A (12)
528
Syrup; 1H NMR (Methanol-d4, 400 MHz) δ: 6.69 (1H, d, J = 7.6 Hz, H-5′), 6.68 (1H, br s, overlap, H-2′), 6.54 (1H, dd, J = 2.0, 8.0 Hz, H-6′), 4.31 (1H, dd, J = 5.2, 7.6 Hz, H-2), 3.67 (3H, s, OCH3), 2.91 (1H, dd, J = 5.2, 14.0 Hz, H-3a), 2.79 (1H, dd, J = 7.2, 14.0 Hz, H-3b); 13C NMR (Methanol-d4, 100 MHz) δ: 176.0 (C-1), 146.2 (C-3′), 145.2 (C-4′), 130.0 (C-1′), 121.9 (C-6′), 117.7 (C-2′), 116.3 (C-5′), 73.5 (C-2), 52.4 (OCH3), 41.3 (C-3).
529 530
T
C
E
R
466 467
R
461 462
O
459 460
C
453 454
N
451 452
489
A.8. Latifolicinin A (8)
U
445 446
A.7. Latifolicinin B (7)
F
Appendix A. NMR data of isolated compounds (except compounds 13 and 16)
E
440 441
Q.H. Vo et al. / Fitoterapia xxx (2015) xxx–xxx
O
8
Please cite this article as: Vo QH, et al, Protein tyrosine phosphatase 1B (PTP1B) inhibitory constituents from the aerial parts of Tradescantia spathacea Sw., Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.017
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A.14. (6S,9R)-Roseoside (15)
540 541
Appendix B. Supplementary data
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Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2015.03.017.
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References
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[1] IDF Diabetes Atlas. 6th, updated 2014 ed. Brussels, Belgium: International Diabetes Federation; 2013. [2] Day C. The rising tide of type 2 diabetes. Br J Diabetes Vasc Dis 2001;1(1): 37–43. [3] Coman C, Rugina OD, Socaciu C. Plants and natural compounds with antidiabetic action. Not Bot Horti Agrobo 2012;40(1). [4] Xue B, Kim YB, Lee A, Toschi E, Bonner-Weir S, Kahn CR, et al. Proteintyrosine phosphatase 1B deficiency reduces insulin resistance and the diabetic phenotype in mice with polygenic insulin resistance. J Biol Chem 2007;282(33):23829–40. [5] Ahmad F, Azevedo JL, Cortright R, Dohm GL, Goldstein BJ. Alterations in skeletal muscle protein-tyrosine phosphatase activity and expression in insulin-resistant human obesity and diabetes. J Clin Invest 1997;100(2): 449–58. [6] Elchebly M, Payette P, Michaliszyn E, Cromlish W, Collins S, Loy AL, et al. Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene. Science (New York, NY) 1999; 283(5407):1544–8. [7] Johnson TO, Ermolieff J, Jirousek MR. Protein tyrosine phosphatase 1B inhibitors for diabetes. Nat Rev Drug Discov 2002;1(9):696–709. [8] Newman DJ, Cragg GM. Natural products as sources of new drugs over the last 25 years. J Nat Prod 2007;70(3):461–77. [9] Jiang CS, Liang LF, Guo YW. Natural products possessing protein tyrosine phosphatase 1B (PTP1B) inhibitory activity found in the last decades. Acta Pharmacol Sin 2012;33(10):1217–45. [10] Do TL. Medicinal plants and drugs from Viet Nam (in Vietnamese). Hanoi, Viet Nam: Medical Publishing House; 2004. [11] Rosales-Reyes T, de la Garza M, Arias-Castro C, Rodríguez-Mendiola M, Fattel-Fazenda S, Arce-Popoca E, et al. Aqueous crude extract of Rhoeo discolor, a Mexican medicinal plant, decreases the formation of liver preneoplastic foci in rats. J Ethnopharmacol 2008;115(3):381–6. [12] González-Avila M, Arriaga-Alba M, De la Garza M, Del Carmen Hernández Pretelı́n M, Domı́nguez-Ortı́z MA, Fattel-Fazenda S, et al. Antigenotoxic,
[19] [20] [21]
[22]
[23]
[24]
C
E
R
R
O
554 555
N C
552 553
U
550 551
[18]
E
558
548 549
[16]
[17]
556 557
546 547
[15]
1
Colorless syrup; H NMR (Methanol-d4, 600 MHz) δ: 5.92 (1H, dd, J = 4.8, 15.6 Hz, H-8), 5.91 (1H, br s, H-4), 5.89 (1H, d, J = 15.0 Hz, H-7), 4.46 (1H, m, H-9), 4.38 (1H, d, J = 7.8 Hz, H-1′), 3.89 (1H, dd, J = 2.4, 12.0 Hz, H-6′a), 3.67 (1H, dd, J = 6.0, 12.0 Hz, H-6′b), 3.38 (1H, m, H-5′), 3.30 (1H, m, H-4′), 3.27 (1H, m, H-3′), 3.21 (1H, dd, J = 7.8, 9.0 Hz, H-2′), 2.56 (1H, d, J = 16.8 Hz, H-2a), 2.19 (1H, d, J = 16.8 Hz, H-2b), 1.96 (3H, d, J = 1.2 Hz, H-11), 1.33 (3H, d, J = 6.0 Hz, H-10), 1.08 (3H, s, H-12), 1.07 (3H, s, H-13); 13C NMR (Methanol-d4, 150 MHz) δ: 201.3 (C-3), 167.4 (C-5), 135.4 (C-8), 131.7 (C-7), 127.3 (C-4), 102.9 (C-1′), 80.2 (C-6), 78.3 (C-5′), 78.2 (C-3′), 77.4 (C-9), 75.4 (C-2′), 71.8 (C-4′), 63.0 (C-6′), 50.9 (C-2), 42.6 (C-1), 24.8 (C-13), 23.6 (C-12), 21.3 (C-10), 19.7 (C-11).
545
[14]
T
539
[13]
F
542 543
Yellow powder; 1H NMR (Methanol-d4, 400 MHz) δ: 8.10 (2H, d, J = 8.8 Hz, H-2′, H-6′), 6.92 (2H, d, J = 8.8 Hz, H-3′, H-5′), 6.41 (1H, d, J = 1.2 Hz, H-8), 6.20 (1H, d, J = 1.6 Hz, H-6); 13 C NMR (Methanol-d4, 100 MHz) δ: 177.6 (C-4), 165.7 (C-7), 162.7 (C-5), 160.7 (C-4′), 158.4 (C-9), 148.2 (C-2), 136.2 (C-3), 130.8 (C-2′, C-6′), 123.9 (C-1′), 116.5 (C-3′, C-5′), 104.7 (C-10), 99.4 (C-6), 94.6 (C-8).
R O O
537 538
antimutagenic and ROS scavenging activities of a Rhoeo discolor ethanolic crude extract. Toxicol in Vitro 2003;17(1):77–83. Tan J, Lim Y, Lee S. Antioxidant and antibacterial activity of Rhoeo spathacea (Swartz) Stearn leaves. J Food Sci Technol 2013:1–7. Choi JS, Nurul Islam M, Yousof Ali M, Kim EJ, Kim YM, Jung HA. Effects of C-glycosylation on anti-diabetic, anti-Alzheimer's disease and antiinflammatory potential of apigenin. Food Chem Toxicol 2014;64:27–33. Na M, Kim BY, Osada H, Ahn JS. Inhibition of protein tyrosine phosphatase 1B by lupeol and lupenone isolated from Sorbus commixta. J Enzyme Inhib Med Chem 2009;24(4):1056–9. Kim EJ, Suh KM, Kim DH, Jung EJ, Seo CS, Son JK, et al. Asimitrin and 4hydroxytrilobin, new bioactive annonaceous acetogenins from the seeds of Asimina triloba possessing a bis-tetrahydrofuran ring. J Nat Prod 2005; 68(2):194–7. Dale JA, Mosher HS. Nuclear magnetic resonance enantiomer regents. Configurational correlations via nuclear magnetic resonance chemical shifts of diastereomeric mandelate, O-methylmandelate, and α-methoxyα-trifluoromethylphenylacetate (MTPA) esters. J Am Chem Soc 1973; 95(2):512–9. Pathak A, Kulshreshtha DK, Maurya R. Chemical constituents of Bacopa procumbens. Nat Prod Res 2005;19(2):131–6. Kakkar S, Bais S. A review on protocatechuic acid and its pharmacological potential. ISRN Pharmacol 2014;2014:9. Kent PW, Brunet PCJ. The occurrence of protocatechuic acid and its 4-O-βD-glucoside in Blatta and Periplaneta. Tetrahedron 1959;7(3–4):252–6. Nagatani Y, Warashina T, Noro T. Studies on the constituents from the aerial part of Baccharis dracunculifolia DC. Chem Pharm Bull 2001;49(11): 1388–94. Kis K, Wungsintaweekul J, Eisenreich W, Zenk MH, Bacher A. An efficient preparation of 2-C-methyl-D-erythritol 4-phosphoric acid and its derivatives. J Org Chem 1999;65(2):587–92. Kobayashi S, Horibe M, Saito Y. Enantioselective synthesis of both diastereomers, including the α-alkoxy-β-hydroxy-β-methyl(phenyl) units, by chiral tin(II) Lewis acid-mediated. Tetrahedron 1994;50(32): 9629–42. De Pascual Teresa J, Hernández Aubanell JC, San Feliciano A, Miguel Del Corral JM. Saccharinic acid lactone from Astragalus lusitanicus Lam. (−)-2C-methyl-D-erythrono-1,4-lactone. Tetrahedron Lett 1980;21(14): 1359–60. Rasmussen S, Wolff C, Rudolph H. Compartmentalization of phenolic constituents in sphagnum. Phytochemistry 1995;38(1):35–9. Wang GJ, Chen SM, Chen WC, Chang YM, Lee TH. Selective inducible nitric oxide synthase suppression by new bracteanolides from Murdannia bracteata. J Ethnopharmacol 2007;112(2):221–7. Suzuki Y, Kosaka M, Shindo K, Kawasumi T, Kimoto-Nira H, Suzuki C. Identification of antioxidants produced by Lactobacillus plantarum. Biosci Biotechnol Biochem 2013;77(6):1299–302. Zeng QL, Wang HQ, Liu ZR, Li BG, Zhao YF. Facile synthesis of optically pure (S)-3-p-hydroxyphenyllactic acid derivatives. Amino Acids 2007;33(3): 537–41. Valls N, López-Canet M, Vallribera M, Bonjoch J. First total syntheses of aeruginosin 298-A and aeruginosin 298-B, based on a stereocontrolled route to the new amino acid 6-hydroxyoctahydroindole-2-carboxylic acid. Chem Eur J 2001;7(16):3446–60. Siddiqui BS, Perwaiz S, Begum S. Studies on the chemical constituents of the fruits of Cordia latifolia. Nat Prod Res 2006;20(2):131–7. Wang Q, Wang YF, Ju P, Luo SD. The phenolic components from Saurauia napaulensis. Nat Prod Res Dev 2008;20:641–3. Hua Y, Shong CZ, Wu ZK, Du ZZ, Wang YH. Chemical constituents of Clematis montana. Chin J Nat Med 2008;6(2):116–9. Zou Y, Tan C, Wang B, Jiang S, Zhu D. Phenolic compounds from Ranunculus chinensis. Chem Nat Comp 2010;46(1):19–21. Mabry T, Markham KR, Thomas MB. The NMR spectra of flavonoids. The systematic identification of flavonoids. Berlin Heidelberg: Springer; 1970 274–343. Markham KR, Ternai B, Stanley R, Geiger H, Mabry TJ. Carbon-13 NMR studies of flavonoids—III: Naturally occurring flavonoid glycosides and their acylated derivatives. Tetrahedron 1978;34(9):1389–97. Okamura N, Yagi A, Nishioka I. Studies on the constituents of Zizyphi Fructus. V. Structures of glycosides of benzyl alcohol, vomifoliol and naringenin. Chem Pharm Bull 1981;29(12):3507–14. Cui B, Nakamura M, Kinjo J, Nohara T. Chemical constituents of Astragali Semen. Chem Pharm Bull 1993;41(1):178–82. Rogers A, Gibon Y. Enzyme kinetics: theory and practice. In: Schwender J, editor. Plant metabolic networks. New York: Springer; 2009. p. 71–103.
P
A.13. Kaempferol (14)
D
536
9
[25] [26]
[27]
[28]
[29]
[30] [31] [32] [33] [34]
[35]
[36]
[37] [38]
Please cite this article as: Vo QH, et al, Protein tyrosine phosphatase 1B (PTP1B) inhibitory constituents from the aerial parts of Tradescantia spathacea Sw., Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.017
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Contents lists available at ScienceDirect
Fitoterapia
3Q2 4 5 6 7
Quoc Hung Vo a,c, Phi Hung Nguyen a, Bing Tian Zhao a, Md Yousof Ali b, Jae Soo Choi b, Byung Sun Min a, Thi Hoai Nguyen c, Mi Hee Woo a,⁎ a b c
College of Pharmacy, Catholic University of Daegu, Gyeongsan 712-702, Republic of Korea Department of Food Science & Nutrition, Pukyong National University, Busan 608-737, Republic of Korea Faculty of Pharmacy, Hue University of Medicine and Pharmacy, Hue University, Hue City, Viet Nam
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Article history: Received 17 February 2015 Accepted in revised form 14 March 2015 Accepted 17 March 2015 Available online xxxx
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Keywords: Tradescantia spathacea Sw. Tradescantin Tradescantoside PTP1B inhibitors Type 2 diabetes
34
1. Introduction
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Nowadays diabetes is a huge and growing problem. The most recent estimates in 2014 show that 387 million people are living with diabetes and this number is set to rise beyond 592 million in less than 25 years [1]. Type 2 diabetes (T2D), or noninsulin-dependent diabetes mellitus, is the most common type accounting for approximately 90% of the total cases among the three types of diabetes [2]. This type is characterized by a resistance to insulin, a peptide hormone produced by β-cells in the pancreas, which is responsible for glucose homeostasis [3,4]. The insulin signaling pathway is negatively regulated by protein tyrosine phosphatases, most notably, protein tyrosine phosphatase 1B (PTP1B) [4]. The overexpression of PTP1B has been shown to inhibit the increased expression of insulin in insulin-resistant states [5]. Furthermore, recent genetic evidence
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Inhibitors of protein tyrosine phosphatase 1B (PTP1B) are promising agents for the treatment of type 2 diabetes and obesity. The bioactivity-guided isolation led to the separation of two new compounds, (±)-tradescantin (13) and tradescantoside (16), along with fourteen known compounds (1–12, 14, and 15) from the aerial parts of Tradescantia spathacea Sw. (Commelinaceae). Their chemical structures were elucidated by spectroscopic methods as well as by comparing with those reported in the literature. The isolated compounds (1–16) were then examined for their inhibitory activity toward PTP1B. The results indicated that compounds 2, 6, 8, and 12 possessed potent inhibition with IC50 values of 7.82 ± 0.79, 6.80 ± 0.89, 4.55 ± 0.92, and 6.38 ± 0.14 μM, respectively. Kinetic study of compounds 2, 6, 8, 12, 13, and 16 was conducted and the structure– activity relationships of the isolated compounds (1–16) were also discussed herein. To the best of our knowledge, all the isolates were separated for the first time from this plant. © 2015 Published by Elsevier B.V.
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Protein tyrosine phosphatase 1B (PTP1B) inhibitory constituents from the aerial parts of Tradescantia spathacea Sw.
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journal homepage: www.elsevier.com/locate/fitote
⁎ Corresponding author. Tel.: +82 53 8503620. E-mail address: [email protected] (M.H. Woo).
has shown that PTP1B gene variants are associated with changes in insulin sensitivity [6]. At the genetic, molecular, biochemical, and physiological levels, PTP1B seems to be a promising drug target for the treatment of T2D and at-risk obese patients [7]. Natural products are rich sources of novel active agents for clinical uses [8]. Previous reports indicate that there are more than 1000 plant species being used to treat T2D all over the world [3] and various natural compounds display PTP1B inhibitory activity [9]. Tradescantia spathacea Sw. (Commelinaceae) is a herbal plant traditionally used as a functional food in Viet Nam for relieving cough and bleeding symptoms [10]. This plant has been proved to possess antitumoral, antigenotoxic, antimutagenic, antioxidant, and antibacterial activities [11–13]. Recently, Tan and co-workers [13] reported the identification of epigallocatechin, rhoeonin, peltatoside and rutin in the decoction of T. spathacea leaves, based on HPLC-DAD and MS data. However, to the best of our knowledge, the chemical constituents of this
http://dx.doi.org/10.1016/j.fitote.2015.03.017 0367-326X/© 2015 Published by Elsevier B.V.
Please cite this article as: Vo QH, et al, Protein tyrosine phosphatase 1B (PTP1B) inhibitory constituents from the aerial parts of Tradescantia spathacea Sw., Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.017
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The aerial parts of T. spathacea Sw. were collected from Hue city, Viet Nam in October 2012. A voucher specimen has been deposited at the College of Pharmacy, Catholic University of Daegu, Republic of Korea.
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2.2. General experimental procedures
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103 104
Melting points were determined on a Yanaco micro melting point apparatus. Optical 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 9423AQA2200E UV spectrophotometer. The NMR spectra were recorded in deuterated solvents on Bruker 250 MHz or Varian OXFORD-AS 400 MHz (Palo Alto, CA, USA) or Agilent Premium COMPACT NMR Magnet System 600 MHz instruments. 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 60 (Kieselgel 0.040–0.063 mm, 230–400 mesh, Merck) and/or reversed-phase silica gel (LiChroprep RP-18, 40–63 μm, Merck). Thin layer chromatography (TLC) tests were performed on Merck pre-coated TLC silica gel 60 F254 and/or TLC silica gel RP-18 F254S glass plates (0.25 mm), and visualized by heating after spraying with 10% H2SO4. MPLC was performed using Biotage Isolera One flash chromatography system. HPLC was performed using a Waters 600 Controller system, Waters 717 autosampler with an UV 2487 detector and YMC Pak ODS-A column (20 × 250 mm, 5 μm particle size, YMC Co., Ltd., Japan). HPLC solvents were purchased from Burdick & Jackson, USA. The (S)- and (R)-(−)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride were purchased from Sigma-Aldrich. All other chemicals and solvents were of analytical grade and used without further purification.
105
2.3. Extraction and isolation
106
The dried and grinded aerial parts of T. spathacea (7.0 kg) were extracted three times with 99.8% MeOH (10 L × 3 h/time) at 60 °C. The resulting solution was concentrated under reduced pressure to yield a residue (750 g). The MeOH extract was suspended in H2O (1.5 L) and partitioned successively with CH2Cl2 (6 × 1.0 L, 300 g), EtOAc (5 × 1.0 L, 40 g), n-BuOH (5 × 1.0 L, 60 g) and H2O-soluble fractions (250 g), respectively. Since the MeOH extract showed significant PTP1B inhibitory effect, its four extracts were subsequently tested for PTP1B inhibitory activity. Among them, the EtOAc and n-BuOH fractions showed good activity against PTP1B. These two extracts were then combined as their TLC patterns were quite similar after visualized. The combined fraction (100 g) was subjected to open flash column chromatography (10 × 23 cm, silica gel 230–400 mesh) eluting with gradient of CH2Cl2–MeOH (50:1 to 0:1) to
95 96 97 98 99 100 101 102
107 108 109 110 111 112 113 114 115 116 117 118 119 120 121
C
93 94
E
92
R
90 91
R
88 89
O
86 87
C
84 85
N
82 83
U
80 81
2.3.1. (±)-Tradescantin (13) Amorphous powder; [α]25 D 0 (c 0.2, MeOH); mp 200– 202 °C; UV (MeOH) λmax nm (log ε): 205 (5.29), 235 (5.21), 280 (5.03), 311 (4.96); IR (KBr) νmax cm−1: 3356, 1724, 1671, 1595, 1522; 1H NMR (Methanol-d4, 250 MHz) and 13C NMR (Methanol-d4, 62.5 MHz) data, see Table 1; HR-EI-MS m/z 240.0632 [M]+ (calcd. for C11H12O6, 240.0634).
163
2.3.2. Tradescantoside (16) Amorphous powder; [α]25 D –46.8 (c 0.2, MeOH); mp 223– 228 °C; UV (MeOH) λmax nm (log ε): 205 (4.55), 246 (4.23), 293 (4.14), 324 (4.19); IR (KBr) νmax cm−1: 3291, 1689, 1597, 1515; 1H NMR (Methanol-d4, 600 MHz) and 13C NMR (Methanol-d4, 150 MHz), see Table 2; HR-ESI-MS (positive) m/z 515.1166 [M + Na]+ (calcd. for C23H24O12Na+, 515.1160); HR-ESI-MS (negative) m/z 491.1187 [M–H]− (calcd. for C23H23O− 12, 491.1195).
170 171
F
2.1. Plant material
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2. Materials and methods
122 123
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72
afford seven subfractions (TD1–TD7). The fraction TD2 was chromatographed on a silica gel column using a stepwise gradient of n-hexanes:EtOAc mixture as a solvent system (9:1 to 4:1) to yield compounds 6 (~ 1 g) and 8 (75 mg) and four subfractions (TD2.1–TD2.4). The subfraction TD2.2 was applied for a reverse-phase column using MeOH:H2O (1:10 to 0:1) as a mobile phase to yield compound 7 (4 mg). The fraction TD3 was loaded on silica gel column eluted with a stepwise gradient of n-hexane:acetone mixture (4:1 to 1:1) to give three subfractions (TD3.1–TD3.2). TD3.1 was chromatographed on an open column using Sephadex LH-20 and eluted by 99.8% MeOH to yield compounds 1 (~1 g) and 14 (3 mg), respectively. TD3.2 was chromatographed by HPLC on a RP-C18 column using gradient of MeOH and 0.1% formic acid in H2O as a mobile phase (21% to 25%) to yield compounds 3 (5 mg), 9 (4 mg), 10 (9 mg), 11 (8 mg), 12 (70 mg), and 13 (7 mg). The fraction TD4 was subjected on MPLC column eluted with a gradient of CH2Cl2:MeOH mixture (40:1 to 0:1) to afford four subfractions (TD4.1–TD4.4). TD4.2 was further purified by an open column using Sephadex LH-20 (99.8% MeOH) and a RP-C18 column using gradient MeOH:H2O as a solvent system (1:10 to 0:1), respectively, to yield compound 2 (200 mg). The fraction TD6 was chromatographed on a reversedphase open column using RP-C18 eluted with a stepwise gradient of MeOH:H2O mixture (1:10 to 0:1) to afford four subfractions (TD6.1–TD6.4). TD6.1 was chromatographed on an open column using normal-phase silica gel eluted with a gradient of CH2Cl2:MeOH (5:1 to 0:1). The eluates was then purified with Sephadex LH-20 column using 99.8% MeOH as a mobile phase to yield compounds 4 (24 mg) and 5 (23 mg), respectively. Subfraction TD6.2 was chromatographed on an open column using Sephadex LH-20 (99.8% MeOH) and was then purified by HPLC on a RP-C18 column using gradient of MeOH and 0.1% formic acid in H2O as a solvent (32% to 35%) to yield compound 15 (10 mg). Finally, from subfraction TD6.4, compound 16 (8 mg) was isolated by using Sephadex LH-20 column eluted with 99.8% MeOH and silica gel column eluted with CH 2Cl2:MeOH mixture (5:1 to 0:1), respectively.
T
70 71
plant have not been reported in detail. Therefore, in the interest of promoting drug discovery from natural sources, this research was conducted to identify bioactive compounds from the aerial parts of T. spathacea, focusing on PTP1B inhibitory activity.
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Please cite this article as: Vo QH, et al, Protein tyrosine phosphatase 1B (PTP1B) inhibitory constituents from the aerial parts of Tradescantia spathacea Sw., Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.017
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1 2
173.1 40.9
3 4 1′ 2′ 3′ 4′ 5′ 6′ OCH3
70.6 199.4 127.8 116.6 146.8 152.9 116.1 123.7 52.7
– 2.79 (dd, J = 4.3, 15.8) 2.50 (dd, J = 8.0, 15.8) 5.29 (dd, J = 4.3, 8.0) – – 7.37 (s) – – 6.77 (d, J = 8.3) 7.39 (d, J = 8.3) 3.61 (s)
2.4. Protein tyrosine phosphatase 1B inhibitory assay
180 181
The PTP1B inhibitory activity of tested compounds was evaluated using p-nitrophenyl phosphate (pNPP) as a substrate. To each of 96 wells (final volume of 100 μL), 40 μL of PTP1B enzyme in a buffer containing 50 mM citrate (pH 6.0), 0.1 M NaCl, 1 mM EDTA, and 1 mM dithiothreitol were added with or without samples dissolved in 10% DMSO. After being preincubated at 37 °C for 10 min, the plate was added 50 μL of 2 mM pNPP in PTP1B reaction buffer. Following incubation at 37 °C for 20 min, the reaction was terminated with the addition of 10 M NaOH. The amounts of p-nitrophenyl produced after enzymatic dephosphorylation from pNPP was estimated by measuring the absorbance at 405 nm using a microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). The nonenzymatic hydrolysis of 2 mM pNPP was corrected by measuring the increase in absorbance at 405 nm obtained in the absence of PTP1B enzyme. The percent inhibition (%) was calculated as (Ac − As) / Ac × 100%, where Ac is the absorbance of the control,
186 187 188 189 190 191 192 193 194 195
Table 2 1D and 2D NMR spectral data of compound 16 (600 MHz, methanol-d4). Position
δC
t2:4 t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20 t2:21 t2:22 t2:23 t2:24 t2:25 t2:26 t2:27
1 2 3 4 5 6 7 1′ 2′ 3′ 4′ 5′ 6′
124.3 120.1 146.4 153.2 116.9 127.4 170.1 104.0 74.9 77.6 71.9 75.9 64.9
1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ OCH3
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N C
t2:3
O
t2:1 t2:2
R
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196
E
184 185
C
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128.0 112.2 149.5 150.7 116.7 124.3 147.3 115.5 169.4 56.7
δH (multiplicity, J in Hz) – 7.88 (d, J = 1.8) – – 6.92 (d, J = 8.4) 7.67 (dd, J = 1.8, 8.4) – 4.91 (d, J = 7.8) 3.58 (m) 3.56 (m) 3.48 (m) 3.79 (m) 4.61 (dd, J = 1.8, 12.0) 4.37 (dd, J = 7.2, 12.0) – 7.18 (d, J = 1.8) – – 6.85 (d, J = 8.4) 7.12 (dd, J = 1.8, 8.4) 7.63 (d, J = 15.6) 6.48 (d, J = 15.6) – 3.93 (s)
2.5. Kinetic analysis
199
The reaction mixture consisted of three different concentrations of pNPP used as a PTP1B substrate in the absence or presence of tested compounds [15]. The Michaelis–Menten constant (Km) and maximum velocity (Vmax) of PTP1B were determined by Lineweaver–Burk plots, and the inhibition constant (Ki) was calculated by Dixon plots using a SigmaPlot™ program (SPCC Inc., Chicago, IL).
200 201
2.6. Preparation of Mosher esters
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The experiment was conducted using a reported method [16,17]. To 0.5 mg of 13, 0.3 mL of CH2Cl2, 5 drops of pyridine, 0.5 mg of 4-(dimethylamino)pyridine, and 12 mg of (R)-(−)α-methoxy-α-(trifluoromethyl)phenylacetyl (MTPA) chloride were added sequentially. The mixture was stirred until being completely dissolved, then it was left at room temperature overnight and subsequently purified over a small column (0.6 × 6 cm) using silica gel (230–400 mesh) eluted with 3–4 mL of n-hexane:CH2Cl2 (1:2). The eluate was dried afterwards, CH2Cl2 (5 mL) was added, and the solution was washed using 1% NaHCO3 (5 mL × 3) and H2O (5 mL × 2); the washed solution was dried in vacuo to yield the (S)-MTPA ester derivative (13S) of 13. The treatment of 13 (0.5 mg) using (S)-(+)-MTPA chloride as mentioned above yielded the corresponding (R)-MTPA ester (13R).
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3. Results and discussion
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Sixteen compounds (1–16) were isolated from the aerial parts of T. spathacea Sw. using various chromatographic methods. Their chemical structures were shown in Fig. 1. Compound 13 was obtained as an amorphous powder with mp 200–202 °C. The molecular formula of 13 was determined as C11H12O6 from the molecular ion peak at m/z 240.0632 [M]+ (calcd. m/z 240.0634) obtained by HR-EI-MS. The IR spectrum of compound 13 indicated the presence of hydroxyl (3356 cm−1), ester (1724 cm−1) and ketone (1671 cm−1) groups as well as a substituted benzene ring (1595 and 1522 cm−1). Its UV spectrum showed absorption maxima at 205, 235, 280, and 311 nm. The 1H NMR spectrum of 13 displayed an ABX-type aromatic spin system at δH 7.39 (1H, d, J = 8.3 Hz, H-6′), 7.37 (1H, s, H-2′), and 6.77 (1H, d, J = 8.3 Hz, H-5′) with their corresponding carbons at δC 123.7 (C-6′), 116.6 (C-2′), and 116.1 (C-5′), respectively, assigned with the aid of HMQC spectroscopic data analysis. This observation suggested the presence of a 1,2,4-trisubstituted benzene ring which was also confirmed by the correlations observed in the HMBC experiment (Fig. 2). The connection between the ketone carbonyl at δC 199.4 (C-4) and the aromatic carbon at δC 127.8 (C-1′) was proved by the HMBC correlation from H-6′ (δH 7.39) to C-4 (δC 199.4). One aliphatic AMX-spin pattern at δH 5.29 (1H, dd, J = 4.3, 8.0 Hz, H-3), 2.79 (1H, dd, J = 4.3, 15.8 Hz, H-2a), and 2.50 (1H, dd, J = 8.0, 15.8 Hz, H-2b) suggested the presence of a partial structure of \CH2\CH(OH)\, which was confirmed by 1H–1H COSY correlation from H-3 to H-2. The carbinol group was adjacent to the ketone as observed by the
224
F
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15
R O O
δH (multiplicity, J in Hz)
P
δC
D
Position
E
t1:3
182 183
and As is the absorbance of the sample. Ursolic acid was used as a 197 positive control [14]. 198
Table 1 1D and 2D NMR spectral data of compound 13 (250 MHz, methanol-d4).
T
t1:1 t1:2
3
Please cite this article as: Vo QH, et al, Protein tyrosine phosphatase 1B (PTP1B) inhibitory constituents from the aerial parts of Tradescantia spathacea Sw., Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.017
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cross peak from H-3 (δH 5.29) to C-1′ (δC 127.8) in the HMBC spectrum. The methylene group was linked with a methyl ester group inferred from the cross peaks of two methylenic protons [δH 2.79 (H-2a) and 2.50 (H-2b)] to ester carbonyl at δC 173.1 (C-1) in the HMBC spectrum. This linkage was further confirmed by the correlations between H-2 and C-4, between H-3 and C-1, and between the methyl protons at δH 3.61 (3H, s) and C-1 (173.1).
N
253 254
U
252
C
Fig. 1. Structure of compounds 1–16 isolated from the combined EtOAc and n-BuOH extracts of Tradescantia spathacea Sw.
Fig. 2. 1H–1H COSY and 1H–13C key HMBC correlations of tradescantin (13).
Since the C-3 position of 13 is a chiral center, Mosher's method was applied to determine the absolute configuration [16,17]. Compound 13 was treated with (R)- and (S)-MTPA chloride to give (S)- and (R)-MTPA esters, 13S and 13R, respectively. The differences in the chemical shift of protons H-2a, H-2b, and H-3 were expected to be observed between the two MTPA esters of 13 (ΔδSR = δS − δR), which may reveal the absolute configuration at C-3. Interestingly, the 1H NMR data of 13S and 13R (see Table 3) showed the same pattern that the signals of H-3 were shifted downfield and were separated unambiguously into two at δH 6.41 (1H, dd, J = 4.2, 9.0 Hz) and δH 6.38 (1H, dd, J = 4.2, 9.0 Hz) with the approximate 1:1 ratio. Similarly, the signals of Hab-2 appeared as two sets of two double doublets at [δH 2.96 (1H, dd, J = 4.2, 16.8 Hz) and δH 2.90 (1H, dd, J = 9.0, 16.8 Hz)] and at [δH 2.92 (1H, dd, J = 4.2, 16.8 Hz) and δH 2.84 (1H, dd, J = 9.0, 16.8 Hz)]. This observation suggested that 13 was a racemic mixture which was further confirmed by [α]25 D = 0. Based on the above data,
Please cite this article as: Vo QH, et al, Protein tyrosine phosphatase 1B (PTP1B) inhibitory constituents from the aerial parts of Tradescantia spathacea Sw., Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.017
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t3:5
2a
t3:6
2b
t3:7
3
2.96 (dd, J = 4.2, 16.8 Hz) or 2.92 (dd, J = 4.2, 16.8 Hz) 2.90 (dd, J = 9.0, 16.8 Hz) or 2.84 (dd, J = 9.0, 16.8 Hz) 6.41 (dd, J = 4.2, 9.0 Hz) or 6.38 (dd, J = 4.2, 9.0 Hz)
2.92 (dd, J = 4.2, 16.8 Hz) or 2.96 (dd, J = 4.2, 16.8 Hz) 2.84 (dd, J = 9.0, 16.8 Hz) or 2.90 (dd, J = 9.0, 16.8 Hz) 6.38 (dd, J = 4.2, 9.0 Hz) or 6.41 (dd, J = 4.2, 9.0 Hz)
292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314
Fig. 4. 1H–1H key NOESY correlations of tradescantoside (16).
C
290 291
E
288 289
R
286 287
R
284 285
O
282 283
N C
280 281
compound 13 was established as (±)-methyl-4-(3′,4′dihydroxyphenyl)-3-hydroxy-4-oxobutanoate, a new natural product, and named (±)-tradescantin. Compound 16 was obtained as an amorphous powder. Its IR spectrum exhibited absorption bands at 3291 cm−1 (OH), 1689 cm−1 (COOH), 1597 cm−1 and 1515 cm−1 (aromatic ring), respectively. The UV spectrum of 16 showed absorption maxima at 205, 246, 293 and 324 nm. The HR-ESI-MS indicated a molecular formula of C23H24O12 from the ion peak observed at m/z 515.1166 [M + Na]+ (calcd. m/z 515.1160 for C23H24O12Na+) and at m/z 491.1187 [M–H]− (calcd. m/z 1 13 491.1195 for C23H23O− C NMR spectra of 16 12). The H and demonstrated signals of an anomeric proton and a carbon at δH 4.91 (1H, d, J = 7.8 Hz, H-1′) and at δC 104.0 (C-1′), respectively, a methylene group at δH 4.61 (1H, dd, J = 1.8, 12.0 Hz, H-6′a), 4.37 (1H, dd, J = 7.2, 12.0 Hz, H-6′b) and δC 64.9 (C-6′), a glucopyranosyl moiety from δH 3.48 to 3.79, with their corresponding carbons at δC 77.6 (C-3′), 75.9 (C-5′), 74.9 (C-2′), and 71.9 (C-4′). The coupling constant of the anomeric proton H-1′ (J = 7.8 Hz) indicated the β-configuration for this glucopyranosyl moiety. The presence of the β-D-glucopyranosyl residue was further supported by the analysis of NOESY experiment (Fig. 4) showing the correlations between H-1′, H-3′ and H-5′, and between H-2′ and H-4′. In addition, a trans double bond at [δH 7.63 (1H, d, J = 15.6 Hz, H-7″) and δC 147.3 (C-7″)], and [δH 6.48 (1H, d, J = 15.6 Hz, H-8″), δC 115.5 (C-8″)], an ABX aromatic spin system at δH 7.18 (1H, d, J = 1.8 Hz, H-2″), 7.12 (1H, dd, J = 1.8, 8.4 Hz, H-6″), and 6.85 (1H, d, J = 8.4 Hz, H-5″), and a methoxy group at [δH 3.93 (3H, s), δC 56.7], were the characteristic signals of a trans-feruloyl moiety. The feruloyl moiety was also evidenced by the HMBC correlations (Fig. 3) showing the cross peaks from methoxyl protons (δH 3.93) to C-3″ (δC 149.5), and from olefinic proton H-7″ (δH 7.63) to C-2″ (δC 112.2), C-6″ (δC 124.3), and C-9″ (δC 169.4). The presence of 3,4-dihydroxybenzoic acid residue was identified by the other ABX coupled aromatic system at δH 7.88 (1H, d, J = 1.8 Hz, H-2), 7.67 (1H, dd, J = 1.8, 8.4 Hz, H-6), and 6.92 (1H, d, J = 8.4 Hz,
U
278 279
R O O
13R (δH)
Fig. 3. 1H–1H COSY and 1H–13C key HMBC correlations of tradescantoside (16).
H-5) in combination with the HMBC spectrum showing the cross peaks from H-2 to C-3, C-4, C-6, and C-7, from H-5 to C-1 and C-3, and from H-6 to C-2, C-4 and C-7. All the above observations suggested a structure similar to procumboside B which possessed 4-hydroxyphenol substituent instead of 3,4dihydroxybenzoic acid moiety [18]. Further analysis of the HMBC spectrum demonstrated the linkage of the methylenic protons (H-6′) of the sugar to the ester group (C-9″) of transferuloyl moiety. Another cross peak between H-1′ (4.91, d, J = 7.8 Hz) and C-3 (146.4) established the connection from the C-1′-OH of the sugar to the carbon C-3 of the 3,4dihydroxybenzoic acid moiety confirmed by the correlation between H-1′ and H-2, and by the absence of a correlation between H-1′ and H-5/H-6 in the NOESY experiment. Thus, compound 16 was determined as 3,4-dihydroxybenzoic acid 3-O[6′-(3″-methoxy,4″-hydroxycinnamoyl)-β-D-glucopyranoside], a new natural product, and named tradescantoside. Compound 16 was the derivative of protocatechuic acid occurring in rich quantity in various multiple fruits such as berries including raspberry, blueberry, mulberry, strawberry, cranberry, and
P
13S (δH)
D
Position
E
t3:4
F
Table 3 Characteristic 1H NMR data of Mosher esters (13S and 13R) of compound 13 (600 MHz, chloroform-d).
T
t3:1 t3:2 t3:3
5
315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334
Table 4 t4:1 PTP1B inhibitory activity of compounds 1–16 isolated from the aerial parts of t4:2 Tradescantia spathacea Sw. t4:3 Compound
IC50 (μM)a
t4:4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Ursolic acidb
15.13 ± 0.93 7.82 ± 0.79 25.65 ± 0.77 64.61 ± 0.39 40.20 ± 0.01 6.80 ± 0.89 33.83 ± 0.27 4.55 ± 0.92 N100 52.99 ± 1.93 68.16 ± 0.19 6.38 ± 0.14 17.62 ± 0.48 45.85 ± 0.59 41.83 ± 0.69 10.79 ± 1.04 2.80 ± 0.42
t4:5 t4:6 t4:7 t4:8 t4:9 t4:10 t4:11 t4:12 t4:13 t4:14 t4:15 t4:16 t4:17 t4:18 t4:19 t4:20 t4:21
a Results are expressed as IC50 values (μM) ± standard deviation of three experiments performed in triplicate. b Positive control.
t4:22 t4:23 t4:24
Please cite this article as: Vo QH, et al, Protein tyrosine phosphatase 1B (PTP1B) inhibitory constituents from the aerial parts of Tradescantia spathacea Sw., Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.017
Q.H. Vo et al. / Fitoterapia xxx (2015) xxx–xxx
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b)
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2
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3
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3
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f)
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C
100 µM 20 µM 1400 4.0 µM 1200
-1
1/[S] (mM )
O
e)
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1/[S] (mM )
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C
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-1
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1/[S] (mM )
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U
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600 400
200
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0 0
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2 -1
1/[S] (mM )
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-2
-1
0 -1
1/[S] (mM )
Fig. 5. Graphical determination of inhibition type for compounds 2, 6, 8, 12, 13, and 16 using Lineweaver–Burk plots was expressed as the mean reciprocal of initial velocity for n = 3 replicates at each substrate concentration (panels a, b, c, d, e, and f, respectively).
Please cite this article as: Vo QH, et al, Protein tyrosine phosphatase 1B (PTP1B) inhibitory constituents from the aerial parts of Tradescantia spathacea Sw., Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.017
Q.H. Vo et al. / Fitoterapia xxx (2015) xxx–xxx
2 6 8 12 13 16
12.16 4.45 7.15 4.06 18.36 8.03
Competitive Mixed Mixed Noncompetitive Mixed Mixed
350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385
4. Conclusions
C
348 349
E
346 347
R
344 345
R
342 343
O
340 341
N C
338 339
U
336 337
gooseberry, as well as in Loquat fruit, wine, honey, and soybean. Protocatechuic acid was reported to possess antibacterial, antiviral, antioxidant, antidiabetic, anticancer, anti-ageing, antifibrotic, and antiviral activities [19]. Its glucoside derivatives were found in the colleterial or sexual accessory glands of the cockroaches, Blatta orientalis L. and Periplaneta americana L. [20], and the aerial parts of Baccharis dracunculifolia DC. (dracunculifoside B) [21]. The fourteen known compounds were identified by comparing their spectroscopic data with those in the literature including (2R,3R)-2,3-dihydroxy-2-methylbutyrolactone (1) [22– 24], bracteanolide A (2) [25,26], 4-(3′,4′-dihydroxyphenyl) furan-2(5H)-one (3) [26], (S)-2-hydroxy-3-(4′-hydroxyphenyl) propanoic acid (4) [27], (R)-2-hydroxy-3-(4′-hydroxyphenyl) propanoic acid (5) [28,29], latifolicinin C (6), latifolicinin B (7), latifolicinin A (8) [30], protocatechuic acid (9) [31], 1-(3′,4′dihydroxyphenyl)-2-hydroxyethan-1-one (10), hydroxytyrosol (11) [32], oresbiusin A (12) [33], kaempferol (14) [34,35], and (6S,9R)-roseoside (15) [36,37]. As of 2012, according to the systematic review of Jiang et al., approximately 300 compounds with PTP1B inhibitory activity have been isolated and identified from about 79 species belonging to 70 genera. Most of the inhibitors are phenolic compounds accounting for more than 65% of total. The other groups are terpenes, steroids, N- or S-containing compounds, and several rare structures [9]. The in vitro inhibition study of the isolated compounds (1– 16) against PTP1B was conducted according to the reported method [14]. The results were shown in Table 4 with ursolic acid, the known PTP1B inhibitor, used as the positive control. All of the tested compounds, except compound 9, exhibited a dose-dependent inhibitory effect. Among these results, compound 8 showed the best inhibition, and compounds 2, 6, and 12 demonstrated a potent activity. Two new compounds, 13 and 16, together with compounds 1, 3, and 7 showed inhibitory effect with IC50 values of 17.62 ± 0.48, 10.79 ± 1.04, 15.13 ± 0.93, 25.65 ± 0.77, and 33.83 ± 0.27 μM, respectively. Compounds 4, 5, 10, 11, 14, and 15 displayed moderate activity. Compounds 2 and 3 were γ-butyrolactone derivatives and only differed at the C-5 position with or without hydroxyl group. However, the inhibitory activity of compound 2 was three times as strong as that of compound 3 (IC50 7.82 and 25.65 μM, respectively). This result suggested that the 5-hydroxyl group played an important role in the inhibition of this skeleton toward PTP1B activity. By comparing the two enantiomers, 4 and 5, it was found that the (R)-configuration (compound 5, IC50 40.20 μM) possessed the stronger effect than the (S)-configuration in 2-hydroxy-3-(4′-hydroxyphenyl)propanoic acid (compound 4, IC50 64.61 μM). Nevertheless, this difference may not be important since compounds 6 and 12 showed potent activity in the (S)-configuration (IC50 6.80 ± 0.89 and 6.38 ± 0.14 μM,
F
t5:4 t5:5 t5:6 t5:7 t5:8 t5:9
R O O
Inhibition type
P
Ki (μM)
D
Tested compounds
E
t5:3
335
respectively). It also can be deduced from the comparison that the presence of mono/dihydroxyphenyl might not significantly affect the inhibitory activity but the ester group possessed a decisive meaning for the effect. The important role of the ester functional group was confirmed by comparing IC50 values of compounds 1, 2, 6, 8, 12, 13, and 16 (ranging from 4.55 to 15.13 μM) with those of compounds 4, 5, 9, 10, 11, 14, and 15 (ranging from 33.83 to over 100 μM) which were not ester derivatives. The subsequent investigations were conducted to determine the inhibitory constants Ki using Dixon plots and the inhibitory mechanisms of the inhibitors which displayed good activity (compounds 2, 6, 8, 12, 13, and 16) using Lineweaver–Burk plots (Fig. 5). The results are shown in Table 5. Compound 12 was found to decrease the Vmax value but did not alter the Km value of PTP1B, suggesting that the inhibition type was noncompetitive toward pNPP (Fig. 5d) with Ki value of 4.06 μM. In contrast, an increase in the concentration of compound 2 did not affect the Vmax value but increased the Km value of PTP1B, indicating the competitive mode of compound 2 toward pNPP (Fig. 5a) [38] with Ki value of 12.16 μM. Compounds 6, 8, 13, and 16 were established as mixed-type inhibitors with Ki values ranging from 4.45 to 18.36 μM since they decreased the Vmax values while the Km values of PTP1B were increased (Fig. 5b, c, e, and f, respectively).
Table 5 Kinetic analysis of compounds 2, 6, 8, 12, 13, and 16.
T
t5:1 t5:2
7
386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411
Diabetes has become one of the most challenging health problems of modern society. Therefore, scientists have put much effort to develop drugs to treat or cure this metabolic disease, especially type 2 diabetes. The PTP1B inhibitors was considered as a promisingly therapeutic option, however, there are no novel drugs approved for clinical use to date, which encourages researchers to keep searching for PTP1B inhibitory agents. In our study, the bioassay-guided investigation of the aerial parts of T. spathacea Sw. resulted in the isolation of two new compounds, (±)-tradescantin (13) and tradescantoside (16), together with fourteen known compounds including three butyrolactone derivatives (1–3), nine phenolics (4–12), a flavonoid (14), and a glucoside (15) which possessed inhibitory effect toward PTP1B, except compound 9. Those results have revealed the potential utilization of T. spathacea Sw. in the discovery of PTP1B inhibitors for the treatment of type 2 diabetes. For the practical use, further studies should be conducted to confirm their cellular effects as well as to investigate their inhibitory effects in vivo.
412 413
Declaration of interest
431
414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430
The authors report no conflicts of interest. The authors alone 432 are responsible for the content and writing of this article. 433 Acknowledgments
434
This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No. 2010-0010549). We are grateful to the Korea Basic Science Institute (KBSI) for mass spectral measurements.
435 436
Please cite this article as: Vo QH, et al, Protein tyrosine phosphatase 1B (PTP1B) inhibitory constituents from the aerial parts of Tradescantia spathacea Sw., Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.017
437 438 439
442
A.1. (2R,3R)-2,3-Dihydroxy-2-methylbutyrolactone (1)
443 444
447 448
1 Colorless oil; [α]25 D –57.8 (c 0.4, MeOH); H NMR (Methanold4, 600 MHz) δ: 4.49 (1H, dd, J = 4.2, 10.2 Hz, H-4a), 4.20 (1H, dd, J = 1.8, 10.2 Hz, H-4b), 4.10 (1H, dd, J = 3.6, 1.2 Hz, H-2), 1.44 (3H, s, H-5); 13C NMR (Methanol-d4, 150 MHz) δ: 180.5 (C-1); 74.7 (C-2), 74.5 (C-3), 73.5 (C-4), 21.6 (C-5); EI-MS m/z 132 [M]+.
449
A.2. Bracteanolide A (2)
450
455 456
White amorphous powder; mp 240–242 °C; 1H NMR (Methanol-d4, 600 MHz) δ: 7.25 (1H, d, J = 2.4 Hz, H-2′), 7.23 (1H, dd, J = 2.4, 8.4 Hz, H-6′), 6.87 (1H, d, J = 8.4 Hz, H-5′), 6.49 (1H, s, H-5), 6.28 (1H, s, H-3); 13C NMR (Methanol-d4, 150 MHz) δ: 174.3 (C-2); 166.0 (C-4), 150.6 (C-4′), 146.9 (C-3′), 122.8 (C-1′), 122.5 (C-6′), 116.7 (C-5′), 116.1 (C-2′), 112.0 (C-3), 100.1 (C-5); EI-MS m/z 208 [M]+.
457
A.3. 4-(3′,4′-Dihydroxyphenyl)furan-2(5H)-one (3)
458
463
Amorphous powder; 1H NMR (Methanol-d4, 250 MHz) δ: 7.11 (1H, s, H-2′), 7.09 (1H, d, J = 6.3 Hz, H-6′), 6.91 (1H, d, J = 6.3 Hz, H-5′), 6.29 (1H, s, H-3), 5.33 (2H, s, H-5); 13C NMR (Methanol-d4, 62.5 MHz) δ: 177.7 (C-2), 167.9 (C-4), 150.9 (C-4′), 147.2 (C-3′), 122.9 (C-1′), 120.9 (C-6′), 116.8 (C-5′), 114.8 (C-2′), 109.8 (C-3), 73.1 (C-5).
464
A.4. (S)-2-Hydroxy-3-(4′-hydroxyphenyl)propanoic acid (4)
465
470 471
1 White needles; [α]25 H NMR D –10.6 (c 1.2, MeOH); (Methanol-d4, 250 MHz) δ: 6.96 (2H, d, J = 8.5 Hz, H-3′, H-5′), 6.58 (2H, d, J = 8.5, H-2′, H-6′), 4.15 (1H, dd, J = 4.5, 7.5 Hz, H-2), 2.89 (1H, dd, J = 4.0, 13.8 Hz, H-3a), 2.69 (1H, dd, J = 7.8, 13.8 Hz, H-3b); 13C NMR (Methanol-d4, 62.5 MHz) δ: 177.5 (C-1), 157.2 (C-4′), 131.7 (C-3′, C-5′), 129.7 (C-1′), 116.2 (C-2′, C-6′), 73.2 (C-2), 40.9 (C-3).
472
A.5. (R)-2-Hydroxy-3-(4′-hydroxyphenyl)propanoic acid (5)
473
478 479
1 White amorphous powder; [α]25 D +15.6 (c 0.6, MeOH); H NMR (Methanol-d4, 250 MHz) δ: 7.00 (2H, d, J = 8.3 Hz, H-3′, H-5′), 6.58 (2H, d, J = 8.5, H-2′, H-6′), 3.97 (1H, dd, J = 3.5, 8.3 Hz, H-2), 2.91 (1H, dd, J = 3.5, 14.0 Hz, H-3a), 2.61 (1H, dd, J = 8.3, 14.0 Hz, H-3b); 13C NMR (Methanol-d4, 62.5 MHz) δ: 181.1 (C-1), 156.8 (C-4′), 131.7 (C-3′, C-5′), 131.3 (C-1′), 116.0 (C-2′, C-6′), 75.1 (C-2), 41.7 (C-3).
480
A.6. Latifolicinin C (6)
481
Light yellow powder; mp 65–67 °C; [α]25 D –9.6 (c 0.4, MeOH); 1H NMR (Chloroform-d, 400 MHz) δ: 6.97 (2H, d, J = 8.4 Hz, H-3′, H-5′), 6.65 (2H, d, J = 8.4 Hz, H-2′, H-6′), 4.39 (1H, dd, J = 4.8, 6.8 Hz, H-2), 3.72 (3H, s, OCH3), 3.72 (1H, dd, J = 4.4, 14.0 Hz, H-3a), 2.84 (1H, dd, J = 6.8, 14.0 Hz, H-3b); 13C NMR (Methanol-d4, 100 MHz) δ: 175.9 (C-1), 155.0 (C-4′), 130.6 (C-3′, C-5′), 127.7 (C-1′), 115.6 (C-2′, C-6′), 71.7 (C-2), 52.6 (OCH3), 39.7 (C-3).
468 469
474 475 476 477
482 483 484 485 486 487 488
1 Syrup; [α]25 D –5.0 (c 0.1, MeOH); H NMR (Methanol-d4, 600 MHz) δ: 7.07 (2H, d, J = 8.4 Hz, H-3′, H-5′), 6.73 (2H, d, J = 8.4 Hz, H-2′, H-6′), 4.31 (1H, dd, J = 5.4, 7.2 Hz, H-2), 4.16 (2H, q, J = 7.2, H-1″), 2.97 (1H, dd, J = 5.4, 13.8 Hz, H-3a), 2.87 (1H, dd, J = 7.8, 13.8 Hz, H-3b), 1.25 (3H, t, J = 7.2 Hz, H-2″); 13C NMR (Methanol-d4, 150 MHz) δ: 175.6 (C-1), 157.3 (C-4′), 131.6 (C-3′, C-5′), 129.3 (C-1′), 116.2 (C-2′, C-6′), 73.5 (C-2), 62.1 (C-1″), 41.1 (C-3), 14.6 (C-2″).
490 491
D
P
R O
1 Syrup; [α]25 D –6.0 (c 0.3, MeOH); H NMR (Chloroform-d, 400 MHz) δ: 7.02 (2H, d, J = 8.4 Hz, H-3′, H-5′), 6.66 (2H, d, J = 8.4 Hz, H-2′, H-6′), 4.39 (1H, dd, J = 4.8, 6.4 Hz, H-2), 4.14 (2H, m, H-1″), 3.03 (1H, dd, J = 4.4, 14.0 Hz, H-3a), 2.87 (1H, dd, J = 6.4, 14.0 Hz, H-3b), 1.61 (2H, m, H-2″), 1.35 (2H, m, H-3″), 0.92 (3H, t, J = 7.6 Hz, H-4″); 13C NMR (Chloroform-d, 100 MHz) δ: 174.6 (C-1), 155.0 (C-4′), 130.8 (C-3′, C-5′), 128.0 (C-1′), 115.5 (C-2′, C-6′), 71.6 (C-2), 65.9 (C-1″), 39.8 (C-3), 30.7 (C-2″), 19.2 (C-3″), 13.8 (C-4″).
492 493 494 495 496 497
498 499 500 501 502 503 504 505 506 507
A.9. Protocatechuic acid (9)
508
Needle crystals; mp 219–221 °C; 1H NMR (Methanol-d4, 400 MHz) δ: 7.43 (1H, s, H-2), 7.42 (1H, d, J = 7.6 Hz, H-6), 6.80 (1H, d, J = 7.6 Hz, H-5); 13C NMR (Methanol-d4, 100 MHz) δ: 170.4 (COOH), 151.7 (C-4), 146.2 (C-3), 124.0 (C-6), 123.3 (C-1), 117.9 (C-5), 115.9 (C-2).
509
A.10. 1-(3′,4′-Dihydroxyphenyl)-2-hydroxyethan-1-one (10)
514
Amorphous powder; mp 178–181 °C; 1H NMR (Methanold4, 400 MHz) δ: 7.40 (1H, br s, H-2′), 7.39 (1H, m, overlap, H-5′), 6.84 (1H, d, J = 8.0 Hz, H-6′), 4.80 (2H, s, H-2); 13C NMR (Methanol-d4, 100 MHz) δ: 198.8 (C-1), 152.8 (C-4′), 146.8 (C-3′), 127.9 (C-1′), 122.5 (C-6′), 116.2 (C-5′), 115.5 (C-2′), 66.0 (C-2).
515
A.11. Hydroxytyrosol (11)
521
Syrup; 1H NMR (Methanol-d4, 400 MHz) δ: 6.68 (1H, d, J = 8.0 Hz, H-5′), 6.66 (1H, d, J = 1.6 Hz, H-2′), 6.53 (1H, dd, J = 1.6, 8.0 Hz, H-6′), 3.68 (2H, t, J = 7.2 Hz, H-1), 2.67 (2H, t, J = 7.2 Hz, H-2); 13C NMR (Methanol-d4, 100 MHz) δ: 146.3 (C-3′), 144.8 (C-4′), 131.9 (C-1′), 121.5 (C-6′), 117.2 (C-2′), 116.4 (C-5′), 64.8 (C-1), 39.8 (C-2).
522 523
A.12. Oresbiusin A (12)
528
Syrup; 1H NMR (Methanol-d4, 400 MHz) δ: 6.69 (1H, d, J = 7.6 Hz, H-5′), 6.68 (1H, br s, overlap, H-2′), 6.54 (1H, dd, J = 2.0, 8.0 Hz, H-6′), 4.31 (1H, dd, J = 5.2, 7.6 Hz, H-2), 3.67 (3H, s, OCH3), 2.91 (1H, dd, J = 5.2, 14.0 Hz, H-3a), 2.79 (1H, dd, J = 7.2, 14.0 Hz, H-3b); 13C NMR (Methanol-d4, 100 MHz) δ: 176.0 (C-1), 146.2 (C-3′), 145.2 (C-4′), 130.0 (C-1′), 121.9 (C-6′), 117.7 (C-2′), 116.3 (C-5′), 73.5 (C-2), 52.4 (OCH3), 41.3 (C-3).
529 530
T
C
E
R
466 467
R
461 462
O
459 460
C
453 454
N
451 452
489
A.8. Latifolicinin A (8)
U
445 446
A.7. Latifolicinin B (7)
F
Appendix A. NMR data of isolated compounds (except compounds 13 and 16)
E
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Q.H. Vo et al. / Fitoterapia xxx (2015) xxx–xxx
O
8
Please cite this article as: Vo QH, et al, Protein tyrosine phosphatase 1B (PTP1B) inhibitory constituents from the aerial parts of Tradescantia spathacea Sw., Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.017
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A.14. (6S,9R)-Roseoside (15)
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Appendix B. Supplementary data
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Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2015.03.017.
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References
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[1] IDF Diabetes Atlas. 6th, updated 2014 ed. Brussels, Belgium: International Diabetes Federation; 2013. [2] Day C. The rising tide of type 2 diabetes. Br J Diabetes Vasc Dis 2001;1(1): 37–43. [3] Coman C, Rugina OD, Socaciu C. Plants and natural compounds with antidiabetic action. Not Bot Horti Agrobo 2012;40(1). [4] Xue B, Kim YB, Lee A, Toschi E, Bonner-Weir S, Kahn CR, et al. Proteintyrosine phosphatase 1B deficiency reduces insulin resistance and the diabetic phenotype in mice with polygenic insulin resistance. J Biol Chem 2007;282(33):23829–40. [5] Ahmad F, Azevedo JL, Cortright R, Dohm GL, Goldstein BJ. Alterations in skeletal muscle protein-tyrosine phosphatase activity and expression in insulin-resistant human obesity and diabetes. J Clin Invest 1997;100(2): 449–58. [6] Elchebly M, Payette P, Michaliszyn E, Cromlish W, Collins S, Loy AL, et al. Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene. Science (New York, NY) 1999; 283(5407):1544–8. [7] Johnson TO, Ermolieff J, Jirousek MR. Protein tyrosine phosphatase 1B inhibitors for diabetes. Nat Rev Drug Discov 2002;1(9):696–709. [8] Newman DJ, Cragg GM. Natural products as sources of new drugs over the last 25 years. J Nat Prod 2007;70(3):461–77. [9] Jiang CS, Liang LF, Guo YW. Natural products possessing protein tyrosine phosphatase 1B (PTP1B) inhibitory activity found in the last decades. Acta Pharmacol Sin 2012;33(10):1217–45. [10] Do TL. Medicinal plants and drugs from Viet Nam (in Vietnamese). Hanoi, Viet Nam: Medical Publishing House; 2004. [11] Rosales-Reyes T, de la Garza M, Arias-Castro C, Rodríguez-Mendiola M, Fattel-Fazenda S, Arce-Popoca E, et al. Aqueous crude extract of Rhoeo discolor, a Mexican medicinal plant, decreases the formation of liver preneoplastic foci in rats. J Ethnopharmacol 2008;115(3):381–6. [12] González-Avila M, Arriaga-Alba M, De la Garza M, Del Carmen Hernández Pretelı́n M, Domı́nguez-Ortı́z MA, Fattel-Fazenda S, et al. Antigenotoxic,
[19] [20] [21]
[22]
[23]
[24]
C
E
R
R
O
554 555
N C
552 553
U
550 551
[18]
E
558
548 549
[16]
[17]
556 557
546 547
[15]
1
Colorless syrup; H NMR (Methanol-d4, 600 MHz) δ: 5.92 (1H, dd, J = 4.8, 15.6 Hz, H-8), 5.91 (1H, br s, H-4), 5.89 (1H, d, J = 15.0 Hz, H-7), 4.46 (1H, m, H-9), 4.38 (1H, d, J = 7.8 Hz, H-1′), 3.89 (1H, dd, J = 2.4, 12.0 Hz, H-6′a), 3.67 (1H, dd, J = 6.0, 12.0 Hz, H-6′b), 3.38 (1H, m, H-5′), 3.30 (1H, m, H-4′), 3.27 (1H, m, H-3′), 3.21 (1H, dd, J = 7.8, 9.0 Hz, H-2′), 2.56 (1H, d, J = 16.8 Hz, H-2a), 2.19 (1H, d, J = 16.8 Hz, H-2b), 1.96 (3H, d, J = 1.2 Hz, H-11), 1.33 (3H, d, J = 6.0 Hz, H-10), 1.08 (3H, s, H-12), 1.07 (3H, s, H-13); 13C NMR (Methanol-d4, 150 MHz) δ: 201.3 (C-3), 167.4 (C-5), 135.4 (C-8), 131.7 (C-7), 127.3 (C-4), 102.9 (C-1′), 80.2 (C-6), 78.3 (C-5′), 78.2 (C-3′), 77.4 (C-9), 75.4 (C-2′), 71.8 (C-4′), 63.0 (C-6′), 50.9 (C-2), 42.6 (C-1), 24.8 (C-13), 23.6 (C-12), 21.3 (C-10), 19.7 (C-11).
545
[14]
T
539
[13]
F
542 543
Yellow powder; 1H NMR (Methanol-d4, 400 MHz) δ: 8.10 (2H, d, J = 8.8 Hz, H-2′, H-6′), 6.92 (2H, d, J = 8.8 Hz, H-3′, H-5′), 6.41 (1H, d, J = 1.2 Hz, H-8), 6.20 (1H, d, J = 1.6 Hz, H-6); 13 C NMR (Methanol-d4, 100 MHz) δ: 177.6 (C-4), 165.7 (C-7), 162.7 (C-5), 160.7 (C-4′), 158.4 (C-9), 148.2 (C-2), 136.2 (C-3), 130.8 (C-2′, C-6′), 123.9 (C-1′), 116.5 (C-3′, C-5′), 104.7 (C-10), 99.4 (C-6), 94.6 (C-8).
R O O
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antimutagenic and ROS scavenging activities of a Rhoeo discolor ethanolic crude extract. Toxicol in Vitro 2003;17(1):77–83. Tan J, Lim Y, Lee S. Antioxidant and antibacterial activity of Rhoeo spathacea (Swartz) Stearn leaves. J Food Sci Technol 2013:1–7. Choi JS, Nurul Islam M, Yousof Ali M, Kim EJ, Kim YM, Jung HA. Effects of C-glycosylation on anti-diabetic, anti-Alzheimer's disease and antiinflammatory potential of apigenin. Food Chem Toxicol 2014;64:27–33. Na M, Kim BY, Osada H, Ahn JS. Inhibition of protein tyrosine phosphatase 1B by lupeol and lupenone isolated from Sorbus commixta. J Enzyme Inhib Med Chem 2009;24(4):1056–9. Kim EJ, Suh KM, Kim DH, Jung EJ, Seo CS, Son JK, et al. Asimitrin and 4hydroxytrilobin, new bioactive annonaceous acetogenins from the seeds of Asimina triloba possessing a bis-tetrahydrofuran ring. J Nat Prod 2005; 68(2):194–7. Dale JA, Mosher HS. Nuclear magnetic resonance enantiomer regents. Configurational correlations via nuclear magnetic resonance chemical shifts of diastereomeric mandelate, O-methylmandelate, and α-methoxyα-trifluoromethylphenylacetate (MTPA) esters. J Am Chem Soc 1973; 95(2):512–9. Pathak A, Kulshreshtha DK, Maurya R. Chemical constituents of Bacopa procumbens. Nat Prod Res 2005;19(2):131–6. Kakkar S, Bais S. A review on protocatechuic acid and its pharmacological potential. ISRN Pharmacol 2014;2014:9. Kent PW, Brunet PCJ. The occurrence of protocatechuic acid and its 4-O-βD-glucoside in Blatta and Periplaneta. Tetrahedron 1959;7(3–4):252–6. Nagatani Y, Warashina T, Noro T. Studies on the constituents from the aerial part of Baccharis dracunculifolia DC. Chem Pharm Bull 2001;49(11): 1388–94. Kis K, Wungsintaweekul J, Eisenreich W, Zenk MH, Bacher A. An efficient preparation of 2-C-methyl-D-erythritol 4-phosphoric acid and its derivatives. J Org Chem 1999;65(2):587–92. Kobayashi S, Horibe M, Saito Y. Enantioselective synthesis of both diastereomers, including the α-alkoxy-β-hydroxy-β-methyl(phenyl) units, by chiral tin(II) Lewis acid-mediated. Tetrahedron 1994;50(32): 9629–42. De Pascual Teresa J, Hernández Aubanell JC, San Feliciano A, Miguel Del Corral JM. Saccharinic acid lactone from Astragalus lusitanicus Lam. (−)-2C-methyl-D-erythrono-1,4-lactone. Tetrahedron Lett 1980;21(14): 1359–60. Rasmussen S, Wolff C, Rudolph H. Compartmentalization of phenolic constituents in sphagnum. Phytochemistry 1995;38(1):35–9. Wang GJ, Chen SM, Chen WC, Chang YM, Lee TH. Selective inducible nitric oxide synthase suppression by new bracteanolides from Murdannia bracteata. J Ethnopharmacol 2007;112(2):221–7. Suzuki Y, Kosaka M, Shindo K, Kawasumi T, Kimoto-Nira H, Suzuki C. Identification of antioxidants produced by Lactobacillus plantarum. Biosci Biotechnol Biochem 2013;77(6):1299–302. Zeng QL, Wang HQ, Liu ZR, Li BG, Zhao YF. Facile synthesis of optically pure (S)-3-p-hydroxyphenyllactic acid derivatives. Amino Acids 2007;33(3): 537–41. Valls N, López-Canet M, Vallribera M, Bonjoch J. First total syntheses of aeruginosin 298-A and aeruginosin 298-B, based on a stereocontrolled route to the new amino acid 6-hydroxyoctahydroindole-2-carboxylic acid. Chem Eur J 2001;7(16):3446–60. Siddiqui BS, Perwaiz S, Begum S. Studies on the chemical constituents of the fruits of Cordia latifolia. Nat Prod Res 2006;20(2):131–7. Wang Q, Wang YF, Ju P, Luo SD. The phenolic components from Saurauia napaulensis. Nat Prod Res Dev 2008;20:641–3. Hua Y, Shong CZ, Wu ZK, Du ZZ, Wang YH. Chemical constituents of Clematis montana. Chin J Nat Med 2008;6(2):116–9. Zou Y, Tan C, Wang B, Jiang S, Zhu D. Phenolic compounds from Ranunculus chinensis. Chem Nat Comp 2010;46(1):19–21. Mabry T, Markham KR, Thomas MB. The NMR spectra of flavonoids. The systematic identification of flavonoids. Berlin Heidelberg: Springer; 1970 274–343. Markham KR, Ternai B, Stanley R, Geiger H, Mabry TJ. Carbon-13 NMR studies of flavonoids—III: Naturally occurring flavonoid glycosides and their acylated derivatives. Tetrahedron 1978;34(9):1389–97. Okamura N, Yagi A, Nishioka I. Studies on the constituents of Zizyphi Fructus. V. Structures of glycosides of benzyl alcohol, vomifoliol and naringenin. Chem Pharm Bull 1981;29(12):3507–14. Cui B, Nakamura M, Kinjo J, Nohara T. Chemical constituents of Astragali Semen. Chem Pharm Bull 1993;41(1):178–82. Rogers A, Gibon Y. Enzyme kinetics: theory and practice. In: Schwender J, editor. Plant metabolic networks. New York: Springer; 2009. p. 71–103.
P
A.13. Kaempferol (14)
D
536
9
[25] [26]
[27]
[28]
[29]
[30] [31] [32] [33] [34]
[35]
[36]
[37] [38]
Please cite this article as: Vo QH, et al, Protein tyrosine phosphatase 1B (PTP1B) inhibitory constituents from the aerial parts of Tradescantia spathacea Sw., Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.03.017
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