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Four new neolignans isolated from Eleutherococcus senticosus and their protein tyrosine phosphatase 1B inhibitory activity (PTP1B)
Fitoterapia 121 (2017) 58–63
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Four new neolignans isolated from Eleutherococcus senticosus and their protein tyrosine phosphatase 1B inhibitory activity (PTP1B)
MARK
Le Zhanga,1, Ban-Ban Lia,1, Hao-Ze Lia, Xiao Menga, Xin Lina, Yi-Yu Jianga, Jong-Seog Ahnb, Long Cuia,⁎ a b
College of Pharmacy, Beihua University, 3999 Binjiangdong Road, Jilin City, Jilin Province 132013, People's Republic of China Chemical Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Chungbuk 363-883, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords: Eleutherococcus senticosus PTP1B Obesity Neo-lignans
Four new compounds, erythro-7′E-4-hydroxy-3,3′-dimethoxy-8,5′-oxyneoligna-7′-ene-7,9-diol-9′-al (1), (7S,8S)4-hydroxy-3,1′,3′-trimethoxy-4′,7-epoxy-8,5′-neolign-9-ol (5), (7S,8S,7′E)-5-hydroxy-3,3′-dimethoxy-4′,7-epoxy8,5′-neolign-7′-ene-9,9′-diol (6) and (7S,8S,7′E)-5-hydroxy-3,3′,9′-trimethoxy-4′-7-epoxy-8,5′-neolign-7′-ene-9ol (7). Along with four known compounds (2–4, 8) were isolated from the EtOAc-soluble extract of Eleutherococcus senticosus. Their structures were elucidated on the basis of spectroscopic and physicochemical analyses. All the compounds were evaluated for in vitro inhibitory activity against PTP1B, VHR and PP1. Among them, compounds 1–4 and 6–8 were found to exhibit selective inhibitory activity on PTP1B with IC50 values ranging from 17.2 ± 1.6 to 32.7 ± 1.2 μM.
1. Introduction In physiologic condition, insulin and leptin are two crucial hormones for long-term energy storage. Insulin controls the pathways responsible for glucose uptake and lipogenesis, and leptin, that regulates food intake and energy expenditure. Resistance to insulin and leptin is the common hallmark of type 2 diabetes mellitus and obesity [1–3]. It is worth to mention that two pathologies are continuously increasing in prevalence worldwide. Insulin resistance is obvious in hepatocytes, skeletal muscle and adipocytes by defects of insulin receptor (IR) and post-receptor signaling, accompanied with the increase in expression or activity of protein tyrosine phosphatase 1B (PTP1B) [4]. PTP1B, a member of Protein tyrosine phosphatases (PTPs), is an enzyme that catalyze protein tyrosine dephosphorylate. Moreover, PTP1B has been considered as a negative regulator of insulin signaling. Although several PTPs such as PTP-α, SH2-domain-containing phosphotyrosine phosphatase (SHP2) and leukocyte antigen-related tyrosine phosphatase (LAR) have been found to implicate in the regulation of insulin signaling, there are a mass of evidences supporting PTP1B as the critical PTP-controlling insulin signaling pathway [5–6]. Overexpression of PTP1B inhibit the insulin receptor (IR) signaling cascade, whereas reducing the level of PTP1B, augments insulin-initiated signaling [7]. Furthermore, recent evidences have showed that leptin signaling pathway can be attenuated by PTPs and PTP1B also played a key role in
⁎
1
Corresponding author. E-mail address: [email protected] (L. Cui). These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.fitote.2017.06.025 Received 24 May 2017; Received in revised form 30 June 2017; Accepted 30 June 2017 Available online 01 July 2017 0367-326X/ © 2017 Elsevier B.V. All rights reserved.
this process [8–9]. Therefore, it suggested that the inhibitor of PTP1B can not only be used for the treatment of Type 2 diabetes but also obesity. Because of the severe situation that many current anti-diabetic and anti-obesity chemical agents have some limitations and even some severe adverse-effects, making effort to discover novel PTP1B inhibitors with suitable pharmacological properties is essential. Eleutherococcus senticosus is a shrub belonging to the Araliaceae, which is commonly distributed in China, Korea, Japan and Russia. It has been traditionally used as folk medicine for the treatment of rheumatism, diabetes, and hepatitis. The roots and stems of E. senticosus exhibit anti-inflammatory [10], anti-diabetic [11], hepatoprotective [12], anti-oxidant [13], and other such activities. Previous phytochemical and biological investigations found its roots and stem barks include lignans, triterpenoids, phenylpropanoids, flavonoids and diphenyl ethers [14]. During the course of our search for protein tyrosine phosphatase 1B (PTP1B) inhibitors from natural sources, E. senticosus was investigated. Four new neolignans were isolated from an EtOAc extract of the stems of E. senticosus along with four known compounds, and the evaluation of their PTP1B inhibitory activity.
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Table 1 1 H (500 MHz) and Position
13
1
C (125 MHz) NMR data of compounds 1 and 5–7 (δ in ppm, J in Hz).
a
δC 1 2 3 4 5 6 7 8 9a 9b 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 3-OCH3 1′-OCH3 3′-OCH3 9′-OCH3 a b c
134.6 112.1 148.4 147.2 115.6 121.1 74.3 86.0 62.5
5a δH
δC
7.12, d (1.5)
6.77, d (8.0) 6.91, dd (1.5,8.0) 4.92, d (6.0) 4.45, m 3.58, m 3.76,dd (4.0,12.0)
129.4 112.8 152.1 117.9 152.7 124.5 154.0 128.2 194.4 56.7
7.38, 7.58, 6.68, 9.64, 3.82,
56.9
3.94, s
7.22, s 7.22, s s d (15.5) dd (8.0,15.5) d (8.0) s
133.2 112.4 148.4 147.2 115.5 122.0 87.9 56.6 65.9 148.7 108.2 148.7 135.9 132.2 108.2
56.8 57.1 57.1
6b δH
δC
6.69, d (1.0)
6.64, 6.60, 4.41, 3.00, 4.01, 3.87,
d (8.0) dd (1.0,8.0) d (9.0) m m m
6.33, s
6.33, s
3.72, s 3.68, s 3.68, s
134.1 111.4 149.4 117.1 148.8 120.3 89.3 55.3 64.8
7c δH
δC
7.35, s 7.25, s 7.25, s 6.10 d (7.0) 4.00, m 4.27, m
132.4 112.2 145.5 149.4 131.2 116.5 130.6 129.5 63.6 56.4
7.35, 6.94, 6.60, 4.61, 3.68,
56.8
3.88, s
7.17, s
s d (15.5) dt (5.5,15.5) d (4.0) s
133.1 109.0 146.9 114.6 145.9 119.7 88.5 53.8 64.2
δH
6.92, s 6.90, s 6.90, 5.58, 3.62, 3.96, 3.92,
131.1 110.7 144.7 148.6 128.3 115.0 132.8 124.1 73.4 56.2
6.89, 6.56, 6.16, 4.08, 3.87
56.2 58.1
3.91 3.40
s d (7.0) m m d (5.0)
6.89, s
s d (16.0) dt (6.0,16.0) d (6.0)
NMR data were measured in acetone-d6. NMR data were measured in pyridine-d5. NMR data were measured in chloroform-d.
2. Experimental
and BuOH soluble fractions, respectively. A portion of the EtOAc-soluble fraction (81.0 g) was subjected to a silica gel column using a gradient of CH2Cl2/MeOH (from 100/1, 50/1, 30/1, 10/1, 5/1 to 1/1), and separated into 9 fractions (Fr.1–Fr.9). Fr.6 (39.5 g) was chromatographed over silica gel, eluted with a stepwise gradient of CH2Cl2/ MeOH (from 100/1, 50/1, 20/1, 10/1, 5/1, to 2/1) to afford 8 subfractions (Fr.6.1–Fr.6.8). Further purification of Fr.6.2 (11.8 g) was subjected to RP-18 column eluted with MeOH/H2O (20–100%) to get 24 fractions, after which the Fr.6.2.5 (511.5 mg) was subjected to the silica gel column and eluted with CH2Cl2/MeOH (from 100/0, 100/1, 50/1 to 25/1) to afford 9 fractions (Fr.6.2.5.1-Fr.6.2.5.9). Fr.6.2.5.6 (171.8 mg) was separated by HPLC, using a gradient solvent system of 35–40% MeOH in H2O over 70 min yielded compounds 1 (7.2 mg, tR = 51.0 min) and 3 (12 mg, tR = 48.6 min). Further purification of Fr.6.2.4 (251.7 mg) via semi-preparatory HPLC using an isocratic solvent system of 17% ACN in H2O over 120 min yielded compounds 2 (5.4 mg, tR = 85.2 min), 4 (13.9 mg, tR = 37.8) and 5 (21.6 mg, tR = 46.1 min). Fr.6.2.7 (736.9 mg) was subjected to the silica gel column using a gradient of CH2Cl2/MeOH (from 100/1, 50/1, 30/1, 10/1, 5/1 to 0/100), and separated into 12 fractions (Fr.6.2.7.1–Fr.6.2.7.12). Fr.6.2.7.2 (39.7 mg) was separated by HPLC, using an isocratic solvent system of 55% MeOH in H2O over 60 min yielded compound 6 (3.9 mg, tR = 30.0 min). In addition, Fr.6.2.7.1 (31.4 mg) via semi-preparatory HPLC using a gradient solvent system of 55% MeOH in H2O over 70 min yielded compound 7 (9.5 mg, tR = 61.3 min). Further purification of Fr.6.2.7.6 (131.7 mg) by semipreparative HPLC eluting with an isocratic solvent system of 45% MeOH in H2O over 65 min gave compound 8 (35.5 mg, tR = 41.9 min).
2.1. General Optical rotations were determined on a JASCO P-1020 polarimeter using a 100-mm glass microcell (JASCO, Tokyo, Japan). UV spectra were recorded in MeOH using a Shimadzu spectrometer (Shimadzu, Tokyo, Japan). IR spectra were recorded with a JASCO FT-IR 620 spectrophotometer (JASCO Corporation, Tokyo, Japan). Nuclear magnetic resonance (NMR) spectra were obtained from a Varian Unity Inova 500 MHz spectrometer (Varian Unity Inova, Phoenix, USA) using TMS as the internal standard. Mass spectra were obtained on a QTOF2 high resolution mass spectrometer (Micromass, Wythenshawe, UK). Column chromatography was conducted using silica gel 60 (200 μm particle size, Yantai Xinde Chemical Co., Ltd., Yantai, China) and RP-18 (150–63 μm particle size, Merck, Darmstadt, Germany). Thin-layer chromatography precoated TLC silica gel 60 F254 plates from Merck were used. HPLC were carried out using a Shimadzu System LC-10AD pump equipped with a model SPD-10Avp UV detector (Shimadzu, Tokyo, Japan), and an Optima Pak® C18 column (10 × 250 mm, 10 μm particle size, Shiseido Fine Chemicals, Tokyo, Japan). 2.2. Plant material The stem of Eleutherococcus senticosus was collected in Jilin, Jilin province, People's Republic of China, and authenticated by Professor Gao Li (College of Pharmacy, Yanbian University). A voucher specimen of the plant (No. 20141121) was deposited at the College of Pharmacy, Beihua University, Jilin, People's Republic of China.
2.4. Spectroscopic data 2.3. Extraction and isolation 2.4.1. Erythro-7′E-4-hydroxy-3,3′-dimethoxy-8,5′-oxyneoligna-7′-ene7,9-diol-9′-al (1) Yellow amorphous powder; [α]D25-4.4 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 291, 225, 203 nm; IR (KBr) νmax 3421, 1604, 1509, 1461, 976 cm− 1; 1H NMR (500 MHz, acetone-d6) and 13C NMR (125 MHz,
The stems of E. senticosus (10.0 kg) were extracted with MeOH at room temperature for 2 weeks and the MeOH solution was concentrated to get a crude extract. This extract was suspended in H2O, partitioned successively with n-hexane, EtOAc and BuOH to afford n-hexane, EtOAc 59
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constant J7,8 = 6.0 Hz, along with the observed NOESY correlations between H-2 with H-7, H-7 with H-8 and H-9 suggested the H-7 and H-8 were in erythro-configuration [16]. On the basis of the above data, the structure of 1 was elucidated as erythro-7′E-4-hydroxy-3,3′-dimethoxy8,5′-oxyneoligna-7′-ene-7,9-diol-9′-al. Compound 5 was obtained as brown amorphous powder; The molecular formula was established as C18H20O6 based on HREIMS at m/z 332.1263 for the [M]+ (calcd. for C18H20O6, 332.1260). The 1H NMR spectrum (Table 1) of 5 indicated three methoxy groups at δ 3.68 (6H, s, 1′/3′-OCH3), δ 3.72 (3H, s, 3-OCH3), a stereochemistry of the dihydrobenzofuran ring at δ 4.41 (1H, d, J = 9.0 Hz, H-7), δ 3.00 (1H, m, H8), δ 3.87 (1H, m, H-9b) and δ 4.01 (1H, m, H-9a). And two sets of isolated aromatic protons at δ 6.69 (1H, d, J = 1.0 Hz, H-2), δ 6.64 (1H, d, J = 8.0 Hz, H-5), δ 6.60 (1H, dd, J = 1.0, 8.0 Hz, H-6) and δ 6.33 (2H, s, H-2′/6′) arising from 1,3,4-trisubstituted and 1′,3′,4′,5′-tetrasubstituted aromatic ring systems respectively. The 13C NMR spectrum (Table 1) of 5 revealed the essence of twelve aromatic carbons, a hydroxymethyl carbon at δ 65.9 (C-9), three methoxy carbons at δ 56.8 (3OCH3) and δ 57.1 (1′/3′-OCH3). The structure of 5 was further demonstrated by analysis of HMBC spectra (Fig. 2). The HMBC correlations from H-2 to C-3, C-4, C-6, C-7, from H-7 to C-1, C-2, C-6, C-9 and C-5′, and from H-2′/6′ to C-8, C-1′, C-3′, C-4′, C-5′. In addition, two methoxy groups at δ 3.68 were located at C-1′ and C-3′ by the HMBC correlations from 1′/3′-OCH3 to C-2′/6′ and C-1′/3′, the methoxy group at δ 3.72 was located at C-3 by the HMBC correlation from 3-OCH3 to C3. The relative stereochemistry in the furan ring was defined as cis H-7/ 8, because NOE cross peaks of H-2 to H-7 and H-8, of H-7 to H-8 and H9 were observed in the NOESY spectrum (Fig. 2) [17]. Accordingly, compound 5 was elucidated as (7S,8S)-4-hydroxy-3,1′,3′-trimethoxy4′,7-epoxy-8,5′-neolign-9-ol. Compound 6 was obtained as brown amorphous powder; The molecular formula C20H22O6, as determined by the HREIMS at m/z 358.1415 for the [M]+ (calcd. for C20H22O6, 358.1416). The 1H NMR data (Table 1) of 6 indicated the presence of five aromatic protons at δ 7.35 (2H, s, H-2/6′), δ 7.25 (2H, s, H-4/6) and δ 7.17 (1H, s, H-2′), two methoxy groups at δ 3.68 (3H, s, 3-OCH3) and δ 3.88 (3H, s, 3′-OCH3), a trans double bond at δ 6.60 (1H, dt, J = 5.5, 15.5 Hz, H-8′) and δ 6.94 (1H, d, J = 15.5 Hz, H-7′), two oxymethylene groups at δ 4.27 (2H, m, H-9) and δ 4.61 (2H, d, J = 4.0 Hz, H-9′). The 13C NMR spectrum (Table 1) of 6 exhibited eighteen resonances attributed to the core structure, which included twelve aromatic carbons, two methines at δ 55.3 (C-8) and δ 89.3 (C-7) and two oxymethylenes at δ 63.6 (C-9′) and δ 64.8 (C-9). The above data suggested that compound 6 was similar to those of (7R,8S)-dehydrodiconiferyl alcohol [18] except that the NMR resonances of the 3-methoxy-4-hydroxyphenyl group in (7R,8S)-dehydrodiconiferyl alcohol was replace by those attributed to 3-methoxy-5hydroxy-phenyl in 6. The suggestion was refined by 2D NMR data (Fig. 2) analysis of 6. Particularly, in the HMBC spectrum, correlations from H-7′ to C-1′ and H-8′ to C-1′, C-2′ and C-6′ indicated the double bond attached to C-1′, from H-2 to C-3 and C-6, from H-7 to C-2, C-6, C8, C-4′ and C-5′, from H-8 to C-6′, from H-2′ to C-3′, C-4′ C-6′ and C-7′. The location of the methoxy group at δ 3.68 was established at C-3 by the correlations from 3-OCH3 to C-2 and C-3, another methoxy group at δ 3.88 was located at C-3′ by the HMBC correlations from 3′-OCH3 to C2′ and C-3′. In addition, a coupling constant J7,8 = 7.0 Hz, along with the observed NOESY correlations between H-7 to H-8 and H-9 confirmed the configuration of H-7/8 was cis [19]. On the basis of the above data, the structure of 6 was elucidated as (7S,8S,7′E)-5-hydroxy3,3′-dimethoxy-4′,7-epoxy-8,5′-neolign-7′-ene-9,9′-diol. Compound 7 was obtained as brown amorphous powder; The HREIMS data indicated a molecular formula of C20H22O6 based on the [M]+ ion signal at m/z 358.1412. The MS and NMR spectroscopic data (Table 1) of compound 7 were very similar to those of compound 6 except the 9′-OH in compound 6 were replaced by 9′-OCH3 in compound 7. The methoxy group at δ 3.40 located at C-9′ by the HMBC correlations from H-9′ to C-7′ and C-8′ and 9′-OCH3, from 9′-OCH3 to C-
acetone-d6) data are presented in Table 1. HREIMS m/z: 374.1369 [M]+(calcd. for C20H22O7, 374.1366). 2.4.2. (7S,8S)-4-Hydroxy-3,1′,3′-trimethoxy-4′,7-epoxy-8,5′-neolign-9-ol (5) Brown amorphous powder; [α]D25-2.1 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 292, 221 nm; IR (KBr) νmax 3396, 1600, 1501, 954 cm− 1; 1 H NMR (500 MHz, acetone-d6) and 13C NMR (125 MHz, acetone-d6) data are presented in Table 1. HREIMS m/z: 332.1263 [M]+(calcd. for C18H20O6, 332.1260). 2.4.3. (7S,8S,7′E)-5-Hydroxy-3,3′-dimethoxy-4′,7-epoxy-8,5′-neolign-7′ene-9,9′-diol (6) Brown amorphous powder; [α]D25-17.2 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 271, 226, 213 nm; IR (KBr) νmax 3411, 1612, 1508, 966, 934 cm− 1; 1H NMR (500 MHz, pyridine-d5) and 13C NMR (125 MHz, pyridine-d5) data are presented in Table 1. HREIMS m/z: 358.1415 [M]+(calcd. for C20H22O6, 358.1416). 2.4.4. (7S,8S,7′E)-5-Hydroxy-3,3′,9′-trimethoxy-4′-7-epoxy-8,5′-neolign7′-ene-9-ol (7) Brown amorphous powder; [α]D25-12.2 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 290, 222, 202 nm; IR (KBr) νmax 3513, 1602, 1532, 1140, 926 cm− 1; 1H NMR (500 MHz, chloroform-d) and 13C NMR (125 MHz, chloroform-d) data are presented in Table 1. HREIMS m/z: 358.1412 [M]+(calcd. for C20H22O6, 358.1416). 2.5. PTP1B, PP1and VHR assay The enzyme activity of PTP1B and PP1 were measured using p-nitrophenyl phosphate (pNPP) as described previously [15]. Dual-specificity protein tyrosine phosphatase (DS-PTP) was assayed with the VHR expressed in Escherichia coli [15]. 3. Results and discussion Compound 1 was obtained as yellow amorphous powder; The HREIMS of 1 exhibited a [M]+ ion at m/z 374.1369 suggesting the molecular formula of C20H22O7 (calcd. for C20H22O7, 374.1366). The 1H NMR spectrum (Table 1) of 1 exhibited an aldehyde signal at δ 9.64 (1H, d, J = 8.0 Hz, H-9′), trans-conformational olefine signals at δ 6.68 (1H, dd, J = 8.0, 15.5 Hz, H-8′) and δ 7.58 (1H, d, J = 15.5 Hz, H-7′), which were assigned to a propenal moiety and six aromatic protons at δ 7.38 (1H, s, H-6′), δ 7.22 (2H, s, H-2′/4′), δ 7.12 (1H, d, J = 1.5 Hz, H2), δ 6.91 (1H, dd, J = 1.5, 8.0 Hz, H-6) and δ 6.77 (1H, d, J = 8.0 Hz, H-5). Also evident were two methoxy groups at δ 3.82 (3H, s, 3-OCH3) and δ 3.94 (3H, s, 3′-OCH3), oxygenated methine and methylene signals at δ 4.92 (1H, d, J = 6.0 Hz, H-7), 4.45 (1H, m, H-8), δ 3.58 (1H, m, H9a) and δ 3.76 (1H, dd, J = 4.0, 12.0 Hz, H-9b). The 13C NMR spectrum (Table 1) of 1 revealed the presence of an aldehyde carbon at δ 194.4 (C-9′), a hydroxymethyl carbon at δ 62.5 (C-9), two carbon atoms connected to an oxygen at δ 74.3 (C-7) and δ 86.0 (C-8), two methoxy carbons at δ 56.7 (3-OCH3) and δ 56.9 (3′-OCH3), and twelve aromatic carbons. In addition, compound 1 exhibited spectroscopic data (Table 1) almost identical to those of the known compound erythro-7E4′,9′-dihydroxy-4,5′-dimethoxy-5,8′-oxyneolign-7-en-9-al [16]. The structure of 1 was further demonstrated by analysis of HMBC spectra (Fig. 2). The HMBC correlations from H-8′ to C-1′ and C-9′, from H-7′ to C-1′, C-2′, C-6′, C-9′ indicated the propenal moiety attached to C-1′, and from H-7 to C-1, C-2, C-6, from H-8 to C-1, C-5′. In addition, from H-2′ to C-3′, C-7′, from H-6′ to C-2′, C-4′, C-5′, C-7′, from H-7′ to C-1′, C-2′, C6′, C-9′, and from 3′-OCH3 to C-2′, C-3′ indicated the aromatic protons at δ 7.38 and δ 7.24 attached to C-6′ (δ 124.5) and C-2′ (δ 112.8) respectively, and also exhibited the methoxy group at δ 3.94 was located at C-3′. The other methoxy group at δ 3.82 was located at C-3 by the HMBC correlations from 3-OCH3 to C-2 and C-3. In addition, a coupling 60
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Fig. 1. Structures of compounds 1–8.
9′. In addition, the aromatic protons at δ 6.92 was located at C-2 by the HMBC correlations from H-2 to C-3, C-6, C-7, and in the HMQC spectrum, correlations from H-2 to C-2. Another methoxy at δ 3.87 was located at C-3 by the HMBC correlations from 3-OCH3 to C-2 and C-3. In addition, the configuration of H-7/8 was cis by the coupling constant J7,8 = 7.0 Hz and NOESY correlations from H-7 to H-8 and H-9 [19]. On the basis of the above data, the structure of compound 7 was elucidated as (7S,8S,7′E)-5-hydroxy-3,3′,9′-trimethoxy-4′-7-epoxy-8,5′neolign-7′-ene-9-ol. Along with four new compounds (1, 5–7), four known compounds (2–4, 8) (Fig. 1) were obtained from Eleutherococcus senticosus. The known compounds were identified as erythro-Guaiacylglycerol-β-coniferyl aldehyde ether (2) [20], 1,3-propanediol-2-O-4′-sinapyl ether (3) [21], (2-glyceryl)-O-coniferaldehyde (4) [22], dehydrodiconiferyl alcohol (8) [23]. Moreover, all the compounds were isolated from Eleutherococcus senticosus for the first time. All isolated compounds were assayed their inhibitory activity against PTP1B using an in vitro assay. (Table 2) The known PTP1B inhibitor, RK-682 (IC50 = 5.4 ± 1.1 μM) [15], was used as positive control in this assay. Compounds 1–4 and 6–8 were found to exhibit selective inhibitory activity on PTP1B with IC50 values ranging from 17.2 ± 1.6 to 32.7 ± 1.2 μM. Among the above isolates, compound 4 (IC50 = 17.2 ± 1.6 μM) which have propenal moiety at C-4 exhibited better inhibition activity than compound 3 (IC50 = 27.4 ± 1.4 μM), on which had the same side chain connected on C-1. In addition,
compound 1 (IC50 = 19.2 ± 1.2 μM) and 2 (IC50 = 20.6 ± 1.1 μM) which have propenal moiety displayed high selective inhibitory activity against PTP1B. This result suggested that the propenal moiety might have more active effect on PTP1B inhibitory activity. Furthermore, compound 8 (IC50 = 29.1 ± 1.4 μM) which have alkyl clain at C-1′ showed better inhibition activity than compound 5 (IC50 > 60) which have methoxy group at C-1′. It is indicating that alkyl clain displayed better PTP1B inhibitory activity than methoxy group. Compound 6 (IC50 = 25.1 ± 1.1 μM) containing a hydroxyl group in C-9′ showed more inhibitory activity than compound 7 (IC50 = 32.7 ± 1.2 μM) with methoxy group in C-9′, indicated that a hydroxyl group was more significance than methoxy group on PTP1B inhibitory. This data presented here may provide a basis for the development of new PTP1B inhibitors that are potentially useful in the treatment of type-2 diabetes as well as obesity. Conflict of interest The authors declare no conflict of interest. Conflict of interest statement We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any 61
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Fig. 2. 2D–NMR correlations of compounds 1, 5–7.
[2] S. Zhang, Z.Y. Zhang, PTP1B as a drug target: recent developments in PTP1B inhibitor discovery, Drug Discov. Today 12 (2007) 373–381. [3] E. Panzhinskiy, J. Ren, S. Nair, Pharmacological inhibition of protein tyrosine phosphatase 1B: a prom-ising strategy for the treatment of obesity and type 2 diabetes mellitus, Curr. Med. Chem. 20 (2013) 2609–2625. [4] D. Popov, Novel protein tyrosine phosphatase 1B inhibitors: interaction requirements for improved intracellular efficacy in type 2 diabetes mellitus and obesity control, Biochem. Biophys. Res. Commun. 410 (2011) 377–381. [5] Z.Y. Zhang, S.Y. Lee, PTP1B inhibitors as potential therapeutics in the treatment of type 2 diabetes and obesity, Expert Opin. Investig. Drugs 12 (2003) 223–233. [6] T.O. Johnson, J. Ermolieff, M.R. Jirousek, Protein tyrosine phosphatase 1B inhibitors for diabetes, Nat. Rev. Drug Discov. 1 (2002) 696–709. [7] L. Cui, M. Na, H. Oh, E.Y. Bae, D.G. Jeong, S.E. Ryu, S. Kim, B.Y. Kim, W.K. Oh, J.S. Ahn, Protein tyrosine phosphatase 1B inhibitors from Morus root bark, Bioorg. Med. Chem. Lett. 16 (2006) 1426–1429. [8] L. Cui, H.S. Lee, D.T. Ndinteh, J.T. Mbafor, Y.H. Kim, T.V.T. Le, P.H. Nguyen, W.K. Oh, New prenylated flavanones from Erythrina abyssinica with protein tyrosine phosphatase 1B (PTP1B) inhibitory activity, Planta Med. 76 (2010) 713–718. [9] S. Koren, I.G. Fantus, Inhibition of the protein tyrosine phosphatase PTP1B: potential therapy for obesity, insulin resistance and type-2 diabetes mellitus, Best Pract. Res. Clin. Endocrinol. Metab. 21 (2007) 621–640. [10] K.H. Leem, S. Lee, H.K. Kim, Extrusion process enhances the anti-inflammatory effect of Acanthopanax senticosus leaves, Food Sci. Biotechnol. 23 (2014) 911–916. [11] J.Y. Park, H.D. Ji, W.M. Lee, E.Y. Park, K.S. Jeong, H.K. Kim, J.H. Cho, S.O. Baik, M.H. Rhee, Anti-diabetic effects of fermented Acanthopanax senticosus extracts on rats with streptozotocin-induced type Ӏ diabetic mellitus, J. Med. Plant Res. 7 (2013) 1994–2000. [12] J.S. Choi, T.J. Yoon, K.R. Kang, K.H. Lee, W.H. Kim, Y.H. Suh, J. Song, M.H. Jung, Glycoprotein isolated from Acanthopanax senticosus protects against hepatotoxicity induced by acute and chronic alcohol treatment, Biol. Pharm. Bull. 29 (2006) 306–314. [13] Q. Lu, W.J. Liu, L. Yang, Y.G. Zu, B.S. Zu, M.H. Zhu, Y. Zhang, X. Zhang, R. Zhang, Z. Sun, J. Huang, X. Zhang, W. Li, Investigation of the effects of different organosolv pulping methods on antioxidant capacity and extraction efficiency of lignan, Food Chem. 131 (2012) 1313–1317. [14] N. Li, H.S. Lee, N. Zhang, Y.N. Sun, J.L. Li, S.S. Xing, J.G. Chen, L. Cui, Two new diphenyl ethers from Acanthopanax senticosus (Rupr. & Maxim.) Harms with PTP1B inhibitory activity, Phytochem. Lett. 13 (2015) 286–289. [15] L. Cui, M.K. Na, H. Oh, E.Y. Bae, D.G. Jeong, S. Ryu, S. Kim, B.Y. Kim, W.K. Oh, J.S. Ahn, Protein tyrosine phosphatase 1B inhibitors from Morus root bark, Bioorg. Med. Chem. Lett. 16 (2006) 1426–1429. [16] J.L. Li, N. Li, H.S. Lee, S.S. Xing, S.Z. Qi, Z.D. Tuo, L. Zhang, X.B. Wang, L. Cui, Four new sesqui-lignans isolated from Acanthopanax senticosus and their diacylglycerol acyltransferase (DGAT) inhibitory activity, Phytochem. Lett. 15 (2016) 147–151. [17] X. Liu, M.H. Yang, X.B. Wang, S.S. Xie, Z.R. Li, D.H. Kim, J.S. Park, L.Y. Kong, Lignans from the root of Paeonia lactiflora and their anti-β-amyloid aggregation activities, Fitoterapia 103 (2015) 136–142. [18] M.S.M. Yuen, F. Xue, T.C.W. Mak, H.N.C. Wong, On the absolute structure of optically active neolignans containing a dihydrobenzo[b] furan skeleton, Tetrahedron 54 (1998) 12429–12444.
Table 2 Inhibitory effects of compounds 1–8 (μM) on PTP1B, VHR and PP1. Compound
1 2 3 4 5 6 7 8 RK-682b
IC50a PTP1B
VHR
PP1
19.2 ± 1.2 20.6 ± 1.1 27.4 ± 1.4 17.2 ± 1.6 > 60 25.1 ± 1.1 32.7 ± 1.2 29.1 ± 1.4 5.4 ± 1.1
> 60 > 60 > 60 > 60 > 60 > 60 > 60 > 60 10.2 ± 1.4
> 60 > 60 > 60 > 60 > 60 > 60 > 60 > 60 NTc
a
IC50 values were calculated and expressed as means ± SD in micromolar. Positive control. The samples were tested for PTP1B, VHR and PP1 inhibitory activity in three independent experiments. c Not tested. b
nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled. Acknowledgments This research was supported by a grant from Technological Developing Scheme of Jilin Province of People's Republic of China (20150101225JC). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.fitote.2017.06.025. Reference [1] C.J. Zhou, S. Huang, J.Q. Liu, S.Q. Qiu, F.Y. Xie, H.P. Song, Y.S. Li, S.Z. Hou, X.P. Lai, Sweet tea leaves extract improves leptin resistance in diet-induced obese rats, J. Ethnopharmacol. 145 (2013) 386–392.
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[19] S. Lee, I.H. Song, J.H. Lee, W.Y. Yang, K.B. Oh, J. Shin, Sortase A inhibitory metabolites from the roots of Pulsatilla koreana, Bioorg. Med. Chem. Lett. 24 (2014) 44–48. [20] D.Y. Lee, M.C. Song, K.H. Yoo, M.H. Bang, I.S. Chung, S.H. Kim, D.K. Kim, B.M. Kwon, T.S. Jeong, M.H. Park, N.I. Baek, Lignans from the fruits of Cornus kousa Burg, and their cytotoxic effects on human cancer cell lines, Arch. Pham. Res. 30 (2007) 402–407.
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Four new neolignans isolated from Eleutherococcus senticosus and their protein tyrosine phosphatase 1B inhibitory activity (PTP1B)
MARK
Le Zhanga,1, Ban-Ban Lia,1, Hao-Ze Lia, Xiao Menga, Xin Lina, Yi-Yu Jianga, Jong-Seog Ahnb, Long Cuia,⁎ a b
College of Pharmacy, Beihua University, 3999 Binjiangdong Road, Jilin City, Jilin Province 132013, People's Republic of China Chemical Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Chungbuk 363-883, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords: Eleutherococcus senticosus PTP1B Obesity Neo-lignans
Four new compounds, erythro-7′E-4-hydroxy-3,3′-dimethoxy-8,5′-oxyneoligna-7′-ene-7,9-diol-9′-al (1), (7S,8S)4-hydroxy-3,1′,3′-trimethoxy-4′,7-epoxy-8,5′-neolign-9-ol (5), (7S,8S,7′E)-5-hydroxy-3,3′-dimethoxy-4′,7-epoxy8,5′-neolign-7′-ene-9,9′-diol (6) and (7S,8S,7′E)-5-hydroxy-3,3′,9′-trimethoxy-4′-7-epoxy-8,5′-neolign-7′-ene-9ol (7). Along with four known compounds (2–4, 8) were isolated from the EtOAc-soluble extract of Eleutherococcus senticosus. Their structures were elucidated on the basis of spectroscopic and physicochemical analyses. All the compounds were evaluated for in vitro inhibitory activity against PTP1B, VHR and PP1. Among them, compounds 1–4 and 6–8 were found to exhibit selective inhibitory activity on PTP1B with IC50 values ranging from 17.2 ± 1.6 to 32.7 ± 1.2 μM.
1. Introduction In physiologic condition, insulin and leptin are two crucial hormones for long-term energy storage. Insulin controls the pathways responsible for glucose uptake and lipogenesis, and leptin, that regulates food intake and energy expenditure. Resistance to insulin and leptin is the common hallmark of type 2 diabetes mellitus and obesity [1–3]. It is worth to mention that two pathologies are continuously increasing in prevalence worldwide. Insulin resistance is obvious in hepatocytes, skeletal muscle and adipocytes by defects of insulin receptor (IR) and post-receptor signaling, accompanied with the increase in expression or activity of protein tyrosine phosphatase 1B (PTP1B) [4]. PTP1B, a member of Protein tyrosine phosphatases (PTPs), is an enzyme that catalyze protein tyrosine dephosphorylate. Moreover, PTP1B has been considered as a negative regulator of insulin signaling. Although several PTPs such as PTP-α, SH2-domain-containing phosphotyrosine phosphatase (SHP2) and leukocyte antigen-related tyrosine phosphatase (LAR) have been found to implicate in the regulation of insulin signaling, there are a mass of evidences supporting PTP1B as the critical PTP-controlling insulin signaling pathway [5–6]. Overexpression of PTP1B inhibit the insulin receptor (IR) signaling cascade, whereas reducing the level of PTP1B, augments insulin-initiated signaling [7]. Furthermore, recent evidences have showed that leptin signaling pathway can be attenuated by PTPs and PTP1B also played a key role in
⁎
1
Corresponding author. E-mail address: [email protected] (L. Cui). These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.fitote.2017.06.025 Received 24 May 2017; Received in revised form 30 June 2017; Accepted 30 June 2017 Available online 01 July 2017 0367-326X/ © 2017 Elsevier B.V. All rights reserved.
this process [8–9]. Therefore, it suggested that the inhibitor of PTP1B can not only be used for the treatment of Type 2 diabetes but also obesity. Because of the severe situation that many current anti-diabetic and anti-obesity chemical agents have some limitations and even some severe adverse-effects, making effort to discover novel PTP1B inhibitors with suitable pharmacological properties is essential. Eleutherococcus senticosus is a shrub belonging to the Araliaceae, which is commonly distributed in China, Korea, Japan and Russia. It has been traditionally used as folk medicine for the treatment of rheumatism, diabetes, and hepatitis. The roots and stems of E. senticosus exhibit anti-inflammatory [10], anti-diabetic [11], hepatoprotective [12], anti-oxidant [13], and other such activities. Previous phytochemical and biological investigations found its roots and stem barks include lignans, triterpenoids, phenylpropanoids, flavonoids and diphenyl ethers [14]. During the course of our search for protein tyrosine phosphatase 1B (PTP1B) inhibitors from natural sources, E. senticosus was investigated. Four new neolignans were isolated from an EtOAc extract of the stems of E. senticosus along with four known compounds, and the evaluation of their PTP1B inhibitory activity.
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Table 1 1 H (500 MHz) and Position
13
1
C (125 MHz) NMR data of compounds 1 and 5–7 (δ in ppm, J in Hz).
a
δC 1 2 3 4 5 6 7 8 9a 9b 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 3-OCH3 1′-OCH3 3′-OCH3 9′-OCH3 a b c
134.6 112.1 148.4 147.2 115.6 121.1 74.3 86.0 62.5
5a δH
δC
7.12, d (1.5)
6.77, d (8.0) 6.91, dd (1.5,8.0) 4.92, d (6.0) 4.45, m 3.58, m 3.76,dd (4.0,12.0)
129.4 112.8 152.1 117.9 152.7 124.5 154.0 128.2 194.4 56.7
7.38, 7.58, 6.68, 9.64, 3.82,
56.9
3.94, s
7.22, s 7.22, s s d (15.5) dd (8.0,15.5) d (8.0) s
133.2 112.4 148.4 147.2 115.5 122.0 87.9 56.6 65.9 148.7 108.2 148.7 135.9 132.2 108.2
56.8 57.1 57.1
6b δH
δC
6.69, d (1.0)
6.64, 6.60, 4.41, 3.00, 4.01, 3.87,
d (8.0) dd (1.0,8.0) d (9.0) m m m
6.33, s
6.33, s
3.72, s 3.68, s 3.68, s
134.1 111.4 149.4 117.1 148.8 120.3 89.3 55.3 64.8
7c δH
δC
7.35, s 7.25, s 7.25, s 6.10 d (7.0) 4.00, m 4.27, m
132.4 112.2 145.5 149.4 131.2 116.5 130.6 129.5 63.6 56.4
7.35, 6.94, 6.60, 4.61, 3.68,
56.8
3.88, s
7.17, s
s d (15.5) dt (5.5,15.5) d (4.0) s
133.1 109.0 146.9 114.6 145.9 119.7 88.5 53.8 64.2
δH
6.92, s 6.90, s 6.90, 5.58, 3.62, 3.96, 3.92,
131.1 110.7 144.7 148.6 128.3 115.0 132.8 124.1 73.4 56.2
6.89, 6.56, 6.16, 4.08, 3.87
56.2 58.1
3.91 3.40
s d (7.0) m m d (5.0)
6.89, s
s d (16.0) dt (6.0,16.0) d (6.0)
NMR data were measured in acetone-d6. NMR data were measured in pyridine-d5. NMR data were measured in chloroform-d.
2. Experimental
and BuOH soluble fractions, respectively. A portion of the EtOAc-soluble fraction (81.0 g) was subjected to a silica gel column using a gradient of CH2Cl2/MeOH (from 100/1, 50/1, 30/1, 10/1, 5/1 to 1/1), and separated into 9 fractions (Fr.1–Fr.9). Fr.6 (39.5 g) was chromatographed over silica gel, eluted with a stepwise gradient of CH2Cl2/ MeOH (from 100/1, 50/1, 20/1, 10/1, 5/1, to 2/1) to afford 8 subfractions (Fr.6.1–Fr.6.8). Further purification of Fr.6.2 (11.8 g) was subjected to RP-18 column eluted with MeOH/H2O (20–100%) to get 24 fractions, after which the Fr.6.2.5 (511.5 mg) was subjected to the silica gel column and eluted with CH2Cl2/MeOH (from 100/0, 100/1, 50/1 to 25/1) to afford 9 fractions (Fr.6.2.5.1-Fr.6.2.5.9). Fr.6.2.5.6 (171.8 mg) was separated by HPLC, using a gradient solvent system of 35–40% MeOH in H2O over 70 min yielded compounds 1 (7.2 mg, tR = 51.0 min) and 3 (12 mg, tR = 48.6 min). Further purification of Fr.6.2.4 (251.7 mg) via semi-preparatory HPLC using an isocratic solvent system of 17% ACN in H2O over 120 min yielded compounds 2 (5.4 mg, tR = 85.2 min), 4 (13.9 mg, tR = 37.8) and 5 (21.6 mg, tR = 46.1 min). Fr.6.2.7 (736.9 mg) was subjected to the silica gel column using a gradient of CH2Cl2/MeOH (from 100/1, 50/1, 30/1, 10/1, 5/1 to 0/100), and separated into 12 fractions (Fr.6.2.7.1–Fr.6.2.7.12). Fr.6.2.7.2 (39.7 mg) was separated by HPLC, using an isocratic solvent system of 55% MeOH in H2O over 60 min yielded compound 6 (3.9 mg, tR = 30.0 min). In addition, Fr.6.2.7.1 (31.4 mg) via semi-preparatory HPLC using a gradient solvent system of 55% MeOH in H2O over 70 min yielded compound 7 (9.5 mg, tR = 61.3 min). Further purification of Fr.6.2.7.6 (131.7 mg) by semipreparative HPLC eluting with an isocratic solvent system of 45% MeOH in H2O over 65 min gave compound 8 (35.5 mg, tR = 41.9 min).
2.1. General Optical rotations were determined on a JASCO P-1020 polarimeter using a 100-mm glass microcell (JASCO, Tokyo, Japan). UV spectra were recorded in MeOH using a Shimadzu spectrometer (Shimadzu, Tokyo, Japan). IR spectra were recorded with a JASCO FT-IR 620 spectrophotometer (JASCO Corporation, Tokyo, Japan). Nuclear magnetic resonance (NMR) spectra were obtained from a Varian Unity Inova 500 MHz spectrometer (Varian Unity Inova, Phoenix, USA) using TMS as the internal standard. Mass spectra were obtained on a QTOF2 high resolution mass spectrometer (Micromass, Wythenshawe, UK). Column chromatography was conducted using silica gel 60 (200 μm particle size, Yantai Xinde Chemical Co., Ltd., Yantai, China) and RP-18 (150–63 μm particle size, Merck, Darmstadt, Germany). Thin-layer chromatography precoated TLC silica gel 60 F254 plates from Merck were used. HPLC were carried out using a Shimadzu System LC-10AD pump equipped with a model SPD-10Avp UV detector (Shimadzu, Tokyo, Japan), and an Optima Pak® C18 column (10 × 250 mm, 10 μm particle size, Shiseido Fine Chemicals, Tokyo, Japan). 2.2. Plant material The stem of Eleutherococcus senticosus was collected in Jilin, Jilin province, People's Republic of China, and authenticated by Professor Gao Li (College of Pharmacy, Yanbian University). A voucher specimen of the plant (No. 20141121) was deposited at the College of Pharmacy, Beihua University, Jilin, People's Republic of China.
2.4. Spectroscopic data 2.3. Extraction and isolation 2.4.1. Erythro-7′E-4-hydroxy-3,3′-dimethoxy-8,5′-oxyneoligna-7′-ene7,9-diol-9′-al (1) Yellow amorphous powder; [α]D25-4.4 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 291, 225, 203 nm; IR (KBr) νmax 3421, 1604, 1509, 1461, 976 cm− 1; 1H NMR (500 MHz, acetone-d6) and 13C NMR (125 MHz,
The stems of E. senticosus (10.0 kg) were extracted with MeOH at room temperature for 2 weeks and the MeOH solution was concentrated to get a crude extract. This extract was suspended in H2O, partitioned successively with n-hexane, EtOAc and BuOH to afford n-hexane, EtOAc 59
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constant J7,8 = 6.0 Hz, along with the observed NOESY correlations between H-2 with H-7, H-7 with H-8 and H-9 suggested the H-7 and H-8 were in erythro-configuration [16]. On the basis of the above data, the structure of 1 was elucidated as erythro-7′E-4-hydroxy-3,3′-dimethoxy8,5′-oxyneoligna-7′-ene-7,9-diol-9′-al. Compound 5 was obtained as brown amorphous powder; The molecular formula was established as C18H20O6 based on HREIMS at m/z 332.1263 for the [M]+ (calcd. for C18H20O6, 332.1260). The 1H NMR spectrum (Table 1) of 5 indicated three methoxy groups at δ 3.68 (6H, s, 1′/3′-OCH3), δ 3.72 (3H, s, 3-OCH3), a stereochemistry of the dihydrobenzofuran ring at δ 4.41 (1H, d, J = 9.0 Hz, H-7), δ 3.00 (1H, m, H8), δ 3.87 (1H, m, H-9b) and δ 4.01 (1H, m, H-9a). And two sets of isolated aromatic protons at δ 6.69 (1H, d, J = 1.0 Hz, H-2), δ 6.64 (1H, d, J = 8.0 Hz, H-5), δ 6.60 (1H, dd, J = 1.0, 8.0 Hz, H-6) and δ 6.33 (2H, s, H-2′/6′) arising from 1,3,4-trisubstituted and 1′,3′,4′,5′-tetrasubstituted aromatic ring systems respectively. The 13C NMR spectrum (Table 1) of 5 revealed the essence of twelve aromatic carbons, a hydroxymethyl carbon at δ 65.9 (C-9), three methoxy carbons at δ 56.8 (3OCH3) and δ 57.1 (1′/3′-OCH3). The structure of 5 was further demonstrated by analysis of HMBC spectra (Fig. 2). The HMBC correlations from H-2 to C-3, C-4, C-6, C-7, from H-7 to C-1, C-2, C-6, C-9 and C-5′, and from H-2′/6′ to C-8, C-1′, C-3′, C-4′, C-5′. In addition, two methoxy groups at δ 3.68 were located at C-1′ and C-3′ by the HMBC correlations from 1′/3′-OCH3 to C-2′/6′ and C-1′/3′, the methoxy group at δ 3.72 was located at C-3 by the HMBC correlation from 3-OCH3 to C3. The relative stereochemistry in the furan ring was defined as cis H-7/ 8, because NOE cross peaks of H-2 to H-7 and H-8, of H-7 to H-8 and H9 were observed in the NOESY spectrum (Fig. 2) [17]. Accordingly, compound 5 was elucidated as (7S,8S)-4-hydroxy-3,1′,3′-trimethoxy4′,7-epoxy-8,5′-neolign-9-ol. Compound 6 was obtained as brown amorphous powder; The molecular formula C20H22O6, as determined by the HREIMS at m/z 358.1415 for the [M]+ (calcd. for C20H22O6, 358.1416). The 1H NMR data (Table 1) of 6 indicated the presence of five aromatic protons at δ 7.35 (2H, s, H-2/6′), δ 7.25 (2H, s, H-4/6) and δ 7.17 (1H, s, H-2′), two methoxy groups at δ 3.68 (3H, s, 3-OCH3) and δ 3.88 (3H, s, 3′-OCH3), a trans double bond at δ 6.60 (1H, dt, J = 5.5, 15.5 Hz, H-8′) and δ 6.94 (1H, d, J = 15.5 Hz, H-7′), two oxymethylene groups at δ 4.27 (2H, m, H-9) and δ 4.61 (2H, d, J = 4.0 Hz, H-9′). The 13C NMR spectrum (Table 1) of 6 exhibited eighteen resonances attributed to the core structure, which included twelve aromatic carbons, two methines at δ 55.3 (C-8) and δ 89.3 (C-7) and two oxymethylenes at δ 63.6 (C-9′) and δ 64.8 (C-9). The above data suggested that compound 6 was similar to those of (7R,8S)-dehydrodiconiferyl alcohol [18] except that the NMR resonances of the 3-methoxy-4-hydroxyphenyl group in (7R,8S)-dehydrodiconiferyl alcohol was replace by those attributed to 3-methoxy-5hydroxy-phenyl in 6. The suggestion was refined by 2D NMR data (Fig. 2) analysis of 6. Particularly, in the HMBC spectrum, correlations from H-7′ to C-1′ and H-8′ to C-1′, C-2′ and C-6′ indicated the double bond attached to C-1′, from H-2 to C-3 and C-6, from H-7 to C-2, C-6, C8, C-4′ and C-5′, from H-8 to C-6′, from H-2′ to C-3′, C-4′ C-6′ and C-7′. The location of the methoxy group at δ 3.68 was established at C-3 by the correlations from 3-OCH3 to C-2 and C-3, another methoxy group at δ 3.88 was located at C-3′ by the HMBC correlations from 3′-OCH3 to C2′ and C-3′. In addition, a coupling constant J7,8 = 7.0 Hz, along with the observed NOESY correlations between H-7 to H-8 and H-9 confirmed the configuration of H-7/8 was cis [19]. On the basis of the above data, the structure of 6 was elucidated as (7S,8S,7′E)-5-hydroxy3,3′-dimethoxy-4′,7-epoxy-8,5′-neolign-7′-ene-9,9′-diol. Compound 7 was obtained as brown amorphous powder; The HREIMS data indicated a molecular formula of C20H22O6 based on the [M]+ ion signal at m/z 358.1412. The MS and NMR spectroscopic data (Table 1) of compound 7 were very similar to those of compound 6 except the 9′-OH in compound 6 were replaced by 9′-OCH3 in compound 7. The methoxy group at δ 3.40 located at C-9′ by the HMBC correlations from H-9′ to C-7′ and C-8′ and 9′-OCH3, from 9′-OCH3 to C-
acetone-d6) data are presented in Table 1. HREIMS m/z: 374.1369 [M]+(calcd. for C20H22O7, 374.1366). 2.4.2. (7S,8S)-4-Hydroxy-3,1′,3′-trimethoxy-4′,7-epoxy-8,5′-neolign-9-ol (5) Brown amorphous powder; [α]D25-2.1 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 292, 221 nm; IR (KBr) νmax 3396, 1600, 1501, 954 cm− 1; 1 H NMR (500 MHz, acetone-d6) and 13C NMR (125 MHz, acetone-d6) data are presented in Table 1. HREIMS m/z: 332.1263 [M]+(calcd. for C18H20O6, 332.1260). 2.4.3. (7S,8S,7′E)-5-Hydroxy-3,3′-dimethoxy-4′,7-epoxy-8,5′-neolign-7′ene-9,9′-diol (6) Brown amorphous powder; [α]D25-17.2 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 271, 226, 213 nm; IR (KBr) νmax 3411, 1612, 1508, 966, 934 cm− 1; 1H NMR (500 MHz, pyridine-d5) and 13C NMR (125 MHz, pyridine-d5) data are presented in Table 1. HREIMS m/z: 358.1415 [M]+(calcd. for C20H22O6, 358.1416). 2.4.4. (7S,8S,7′E)-5-Hydroxy-3,3′,9′-trimethoxy-4′-7-epoxy-8,5′-neolign7′-ene-9-ol (7) Brown amorphous powder; [α]D25-12.2 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 290, 222, 202 nm; IR (KBr) νmax 3513, 1602, 1532, 1140, 926 cm− 1; 1H NMR (500 MHz, chloroform-d) and 13C NMR (125 MHz, chloroform-d) data are presented in Table 1. HREIMS m/z: 358.1412 [M]+(calcd. for C20H22O6, 358.1416). 2.5. PTP1B, PP1and VHR assay The enzyme activity of PTP1B and PP1 were measured using p-nitrophenyl phosphate (pNPP) as described previously [15]. Dual-specificity protein tyrosine phosphatase (DS-PTP) was assayed with the VHR expressed in Escherichia coli [15]. 3. Results and discussion Compound 1 was obtained as yellow amorphous powder; The HREIMS of 1 exhibited a [M]+ ion at m/z 374.1369 suggesting the molecular formula of C20H22O7 (calcd. for C20H22O7, 374.1366). The 1H NMR spectrum (Table 1) of 1 exhibited an aldehyde signal at δ 9.64 (1H, d, J = 8.0 Hz, H-9′), trans-conformational olefine signals at δ 6.68 (1H, dd, J = 8.0, 15.5 Hz, H-8′) and δ 7.58 (1H, d, J = 15.5 Hz, H-7′), which were assigned to a propenal moiety and six aromatic protons at δ 7.38 (1H, s, H-6′), δ 7.22 (2H, s, H-2′/4′), δ 7.12 (1H, d, J = 1.5 Hz, H2), δ 6.91 (1H, dd, J = 1.5, 8.0 Hz, H-6) and δ 6.77 (1H, d, J = 8.0 Hz, H-5). Also evident were two methoxy groups at δ 3.82 (3H, s, 3-OCH3) and δ 3.94 (3H, s, 3′-OCH3), oxygenated methine and methylene signals at δ 4.92 (1H, d, J = 6.0 Hz, H-7), 4.45 (1H, m, H-8), δ 3.58 (1H, m, H9a) and δ 3.76 (1H, dd, J = 4.0, 12.0 Hz, H-9b). The 13C NMR spectrum (Table 1) of 1 revealed the presence of an aldehyde carbon at δ 194.4 (C-9′), a hydroxymethyl carbon at δ 62.5 (C-9), two carbon atoms connected to an oxygen at δ 74.3 (C-7) and δ 86.0 (C-8), two methoxy carbons at δ 56.7 (3-OCH3) and δ 56.9 (3′-OCH3), and twelve aromatic carbons. In addition, compound 1 exhibited spectroscopic data (Table 1) almost identical to those of the known compound erythro-7E4′,9′-dihydroxy-4,5′-dimethoxy-5,8′-oxyneolign-7-en-9-al [16]. The structure of 1 was further demonstrated by analysis of HMBC spectra (Fig. 2). The HMBC correlations from H-8′ to C-1′ and C-9′, from H-7′ to C-1′, C-2′, C-6′, C-9′ indicated the propenal moiety attached to C-1′, and from H-7 to C-1, C-2, C-6, from H-8 to C-1, C-5′. In addition, from H-2′ to C-3′, C-7′, from H-6′ to C-2′, C-4′, C-5′, C-7′, from H-7′ to C-1′, C-2′, C6′, C-9′, and from 3′-OCH3 to C-2′, C-3′ indicated the aromatic protons at δ 7.38 and δ 7.24 attached to C-6′ (δ 124.5) and C-2′ (δ 112.8) respectively, and also exhibited the methoxy group at δ 3.94 was located at C-3′. The other methoxy group at δ 3.82 was located at C-3 by the HMBC correlations from 3-OCH3 to C-2 and C-3. In addition, a coupling 60
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Fig. 1. Structures of compounds 1–8.
9′. In addition, the aromatic protons at δ 6.92 was located at C-2 by the HMBC correlations from H-2 to C-3, C-6, C-7, and in the HMQC spectrum, correlations from H-2 to C-2. Another methoxy at δ 3.87 was located at C-3 by the HMBC correlations from 3-OCH3 to C-2 and C-3. In addition, the configuration of H-7/8 was cis by the coupling constant J7,8 = 7.0 Hz and NOESY correlations from H-7 to H-8 and H-9 [19]. On the basis of the above data, the structure of compound 7 was elucidated as (7S,8S,7′E)-5-hydroxy-3,3′,9′-trimethoxy-4′-7-epoxy-8,5′neolign-7′-ene-9-ol. Along with four new compounds (1, 5–7), four known compounds (2–4, 8) (Fig. 1) were obtained from Eleutherococcus senticosus. The known compounds were identified as erythro-Guaiacylglycerol-β-coniferyl aldehyde ether (2) [20], 1,3-propanediol-2-O-4′-sinapyl ether (3) [21], (2-glyceryl)-O-coniferaldehyde (4) [22], dehydrodiconiferyl alcohol (8) [23]. Moreover, all the compounds were isolated from Eleutherococcus senticosus for the first time. All isolated compounds were assayed their inhibitory activity against PTP1B using an in vitro assay. (Table 2) The known PTP1B inhibitor, RK-682 (IC50 = 5.4 ± 1.1 μM) [15], was used as positive control in this assay. Compounds 1–4 and 6–8 were found to exhibit selective inhibitory activity on PTP1B with IC50 values ranging from 17.2 ± 1.6 to 32.7 ± 1.2 μM. Among the above isolates, compound 4 (IC50 = 17.2 ± 1.6 μM) which have propenal moiety at C-4 exhibited better inhibition activity than compound 3 (IC50 = 27.4 ± 1.4 μM), on which had the same side chain connected on C-1. In addition,
compound 1 (IC50 = 19.2 ± 1.2 μM) and 2 (IC50 = 20.6 ± 1.1 μM) which have propenal moiety displayed high selective inhibitory activity against PTP1B. This result suggested that the propenal moiety might have more active effect on PTP1B inhibitory activity. Furthermore, compound 8 (IC50 = 29.1 ± 1.4 μM) which have alkyl clain at C-1′ showed better inhibition activity than compound 5 (IC50 > 60) which have methoxy group at C-1′. It is indicating that alkyl clain displayed better PTP1B inhibitory activity than methoxy group. Compound 6 (IC50 = 25.1 ± 1.1 μM) containing a hydroxyl group in C-9′ showed more inhibitory activity than compound 7 (IC50 = 32.7 ± 1.2 μM) with methoxy group in C-9′, indicated that a hydroxyl group was more significance than methoxy group on PTP1B inhibitory. This data presented here may provide a basis for the development of new PTP1B inhibitors that are potentially useful in the treatment of type-2 diabetes as well as obesity. Conflict of interest The authors declare no conflict of interest. Conflict of interest statement We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any 61
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Fig. 2. 2D–NMR correlations of compounds 1, 5–7.
[2] S. Zhang, Z.Y. Zhang, PTP1B as a drug target: recent developments in PTP1B inhibitor discovery, Drug Discov. Today 12 (2007) 373–381. [3] E. Panzhinskiy, J. Ren, S. Nair, Pharmacological inhibition of protein tyrosine phosphatase 1B: a prom-ising strategy for the treatment of obesity and type 2 diabetes mellitus, Curr. Med. Chem. 20 (2013) 2609–2625. [4] D. Popov, Novel protein tyrosine phosphatase 1B inhibitors: interaction requirements for improved intracellular efficacy in type 2 diabetes mellitus and obesity control, Biochem. Biophys. Res. Commun. 410 (2011) 377–381. [5] Z.Y. Zhang, S.Y. Lee, PTP1B inhibitors as potential therapeutics in the treatment of type 2 diabetes and obesity, Expert Opin. Investig. Drugs 12 (2003) 223–233. [6] T.O. Johnson, J. Ermolieff, M.R. Jirousek, Protein tyrosine phosphatase 1B inhibitors for diabetes, Nat. Rev. Drug Discov. 1 (2002) 696–709. [7] L. Cui, M. Na, H. Oh, E.Y. Bae, D.G. Jeong, S.E. Ryu, S. Kim, B.Y. Kim, W.K. Oh, J.S. Ahn, Protein tyrosine phosphatase 1B inhibitors from Morus root bark, Bioorg. Med. Chem. Lett. 16 (2006) 1426–1429. [8] L. Cui, H.S. Lee, D.T. Ndinteh, J.T. Mbafor, Y.H. Kim, T.V.T. Le, P.H. Nguyen, W.K. Oh, New prenylated flavanones from Erythrina abyssinica with protein tyrosine phosphatase 1B (PTP1B) inhibitory activity, Planta Med. 76 (2010) 713–718. [9] S. Koren, I.G. Fantus, Inhibition of the protein tyrosine phosphatase PTP1B: potential therapy for obesity, insulin resistance and type-2 diabetes mellitus, Best Pract. Res. Clin. Endocrinol. Metab. 21 (2007) 621–640. [10] K.H. Leem, S. Lee, H.K. Kim, Extrusion process enhances the anti-inflammatory effect of Acanthopanax senticosus leaves, Food Sci. Biotechnol. 23 (2014) 911–916. [11] J.Y. Park, H.D. Ji, W.M. Lee, E.Y. Park, K.S. Jeong, H.K. Kim, J.H. Cho, S.O. Baik, M.H. Rhee, Anti-diabetic effects of fermented Acanthopanax senticosus extracts on rats with streptozotocin-induced type Ӏ diabetic mellitus, J. Med. Plant Res. 7 (2013) 1994–2000. [12] J.S. Choi, T.J. Yoon, K.R. Kang, K.H. Lee, W.H. Kim, Y.H. Suh, J. Song, M.H. Jung, Glycoprotein isolated from Acanthopanax senticosus protects against hepatotoxicity induced by acute and chronic alcohol treatment, Biol. Pharm. Bull. 29 (2006) 306–314. [13] Q. Lu, W.J. Liu, L. Yang, Y.G. Zu, B.S. Zu, M.H. Zhu, Y. Zhang, X. Zhang, R. Zhang, Z. Sun, J. Huang, X. Zhang, W. Li, Investigation of the effects of different organosolv pulping methods on antioxidant capacity and extraction efficiency of lignan, Food Chem. 131 (2012) 1313–1317. [14] N. Li, H.S. Lee, N. Zhang, Y.N. Sun, J.L. Li, S.S. Xing, J.G. Chen, L. Cui, Two new diphenyl ethers from Acanthopanax senticosus (Rupr. & Maxim.) Harms with PTP1B inhibitory activity, Phytochem. Lett. 13 (2015) 286–289. [15] L. Cui, M.K. Na, H. Oh, E.Y. Bae, D.G. Jeong, S. Ryu, S. Kim, B.Y. Kim, W.K. Oh, J.S. Ahn, Protein tyrosine phosphatase 1B inhibitors from Morus root bark, Bioorg. Med. Chem. Lett. 16 (2006) 1426–1429. [16] J.L. Li, N. Li, H.S. Lee, S.S. Xing, S.Z. Qi, Z.D. Tuo, L. Zhang, X.B. Wang, L. Cui, Four new sesqui-lignans isolated from Acanthopanax senticosus and their diacylglycerol acyltransferase (DGAT) inhibitory activity, Phytochem. Lett. 15 (2016) 147–151. [17] X. Liu, M.H. Yang, X.B. Wang, S.S. Xie, Z.R. Li, D.H. Kim, J.S. Park, L.Y. Kong, Lignans from the root of Paeonia lactiflora and their anti-β-amyloid aggregation activities, Fitoterapia 103 (2015) 136–142. [18] M.S.M. Yuen, F. Xue, T.C.W. Mak, H.N.C. Wong, On the absolute structure of optically active neolignans containing a dihydrobenzo[b] furan skeleton, Tetrahedron 54 (1998) 12429–12444.
Table 2 Inhibitory effects of compounds 1–8 (μM) on PTP1B, VHR and PP1. Compound
1 2 3 4 5 6 7 8 RK-682b
IC50a PTP1B
VHR
PP1
19.2 ± 1.2 20.6 ± 1.1 27.4 ± 1.4 17.2 ± 1.6 > 60 25.1 ± 1.1 32.7 ± 1.2 29.1 ± 1.4 5.4 ± 1.1
> 60 > 60 > 60 > 60 > 60 > 60 > 60 > 60 10.2 ± 1.4
> 60 > 60 > 60 > 60 > 60 > 60 > 60 > 60 NTc
a
IC50 values were calculated and expressed as means ± SD in micromolar. Positive control. The samples were tested for PTP1B, VHR and PP1 inhibitory activity in three independent experiments. c Not tested. b
nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled. Acknowledgments This research was supported by a grant from Technological Developing Scheme of Jilin Province of People's Republic of China (20150101225JC). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.fitote.2017.06.025. Reference [1] C.J. Zhou, S. Huang, J.Q. Liu, S.Q. Qiu, F.Y. Xie, H.P. Song, Y.S. Li, S.Z. Hou, X.P. Lai, Sweet tea leaves extract improves leptin resistance in diet-induced obese rats, J. Ethnopharmacol. 145 (2013) 386–392.
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