Fitoterapia 101 (2015) 46–50
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Two new lignans from Saururus chinensis and their DGAT inhibitory activity Na Li a, Zhen-Dong Tuo a, Shi-Zhou Qi a, Shan-Shan Xing a, Hyun-Sun Lee b, Jian-Guang Chen a, Long Cui a,⁎ a
College of Pharmacy, Beihua University, Jilin City, Jilin Province 132013, People's Republic of China Molecular Cancer Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 685-1 Yangcheongri, Ochangeup, Cheongwongun, Chungbuk 363-883, Republic of Korea
b
a r t i c l e
i n f o
Article history: Received 9 November 2014 Accepted in revised form 16 December 2014 Accepted 22 December 2014 Available online 28 December 2014
a b s t r a c t Two new lignans were isolated from Saururus chinensis, along with eight known compounds. Their structures were elucidated on the basis of spectroscopic and physico-chemical analyses. All the isolates were evaluated for in vitro inhibitory activity against DGAT1 and DGAT2. Among them, compounds 2, 3, 5 and 7 were found to exhibit selective inhibitory activity on DGAT1 with IC50 values ranging from 44.3 ± 1.5 to 87.5 ± 1.3 μM. © 2014 Elsevier B.V. All rights reserved.
Keywords: Saururus chinensis Saururaceae DGAT Lignan
1. Introduction Obesity is one of the leading metabolic diseases worldwide and is closely associated with diabetes, hypertension and cardiovascular disease [1]. There have been a great number of studies on the treatment of obesity, but more efficient therapeutic strategies need to be explored imminently. One of potential therapeutic methods involves inhibiting triacylglycerol (TG) synthesis. TG is a major form of energy storage in eukaryotic organisms. And the excess supply of TG in a tissue could lead to obesity [2]. A key enzyme in TG synthesis is acylcoenzyme A (CoA): diacylglycerol acyltransferase (DGAT), which catalyzes the final step of the TG synthesis pathway in mammalian cells by using diacylglycerol and fatty acyl CoA as substrates. Two isoforms of the DGAT enzyme are presently known namely, DGAT1 and DGAT2 [3,4]. Although both enzymes utilize the same substrate, there is no homology between DGAT1 and DGAT2: DGAT1 belongs to the acyl-CoA: ⁎ Corresponding author. Tel./fax: +86 432 64608281. E-mail address: [email protected] (L. Cui).
http://dx.doi.org/10.1016/j.fitote.2014.12.011 0367-326X/© 2014 Elsevier B.V. All rights reserved.
cholesterol acyltransferase (ACAT) gene family and DGAT2 is a member of a distinct and independent gene family. DGAT1 deficient mice are resistant to diet-induced obesity, have lower plasma glucose levels associated with an increase of insulin and leptin sensitivity and are also protected against diet-induced hepatic steatosis [5]. Previous studies reported that DGAT1 played a major role in modulating signals of energy homeostasis and a minor role in bulk TG synthesis [6]. Inhibition of DGAT1 might be an effective target for therapy of obesity and other related diseases. To date, several DGAT1 inhibitors (JTT-553, PF-04620110, AZD7687, LCQ908) have entered clinical trials in multiple pharmaceutical companies [7]. These DGAT1 inhibitors are widely available, but they have a great many limitations, including adverse effects and tolerability. Thus, efforts to discover novel, selective, orally bioavailable DGAT1 inhibitors have been intensified. Saururus chinensis (Lour.) Baill. (Saururaceae), a perennial herb, is widely cultivated in China and southern Korea. It has been traditionally used as folk medicine for the treatment of inflammation, jaundice and gonorrhea [8]. Previous chemical studies of S. chinensis have revealed the presence of lignans [9],
N. Li et al. / Fitoterapia 101 (2015) 46–50
47
HEK293 Tet-on cells were obtained from Clontech. Fatty acid-free BSA and sn-1,2-dioleoylglycerol were obtained from Sigma-Aldrich. [14C]-oleoyl CoA, were purchased from PerkinElmer, Inc.
flavonoids [10] and alkaloids [11]. Sauchinone, a lignan isolated from S. chinensis with unique structure, exhibited a great many pharmacological activities including anti-inflammatory [12], hepatoprotective [13], antidiabetic [14], and antioxidant effects [15] in various cell types. As a part of our exploration on the traditional medicine, S. chinensis was studied, which resulted in the isolation of two new sauchinone analogues (1–2) along with eight known compounds (3–10) (Fig. 1). Herein we reported the isolation, structural elucidation of two new lignans and evaluation of DGAT inhibitory activity about these isolates.
2.2. Plant material The root of S. chinensis was collected in Jinan, Shandong province, People's Republic of China, and authenticated by Professor Gao Li (College of Pharmacy, Yanbian University). A voucher specimen of the plant (No. 20100806) was deposited at the College of Pharmacy, Beihua University, Jilin, China.
2. Experimental
2.3. Extraction and isolation
2.1. General experimental procedures
The root (5.0 kg) of S. chinensis was extracted with MeOH at room temperature for 2 weeks and the solution was concentrated to obtain a crude extract (183.0 g, 67% inhibition at 30.0 μg/mL). This extract was suspended in H2O, partitioned successively with CHCl3, EtOAc and BuOH, and then the organic solvents were removed. A portion of the CHCl3-soluble fraction (18.0 g, 78% inhibition at 30.0 μg/mL) was chromatographed over a silica gel column using a gradient of CHCl3–MeOH (from 1:0, 100:1 to 5:1), and was separated into 10 fractions (Fr.1– Fr.10). Fr.2 (CHCl3–MeOH 100:1, 1.8 g, 73% inhibition at 30.0 μg/mL) was chromatographed over silica gel, eluted with a stepwise gradient of n-Hexane/CHCl3 (from 1:1, 1:2, 1:3 to 0:1) to afford 9 subfractions (Fr.21–Fr.29). The most active fraction, Fr.24 (260.0 mg, 81% inhibition at 30.0 μg/mL) was subjected to an RP-18 column and was eluted with MeOH–H2O (8:10, 9:10 to 100% MeOH) to yield five fractions (Fr.241– Fr.245). Fr.242 (58.0 mg) was separated by HPLC, using a gradient of 60% MeCN in H2O as the mobile phase to produce compounds 3 (4.9 mg) and 5 (9.7 mg). Fr.244 (48.0 mg) was purified by semipreparative HPLC using a gradient solvent
UV spectra were taken in MeOH using a Shimadzu spectrophotometer (Shimadzu, Tokyo, Japan). Nuclear magnetic resonance (NMR) spectra were obtained from a Varian Unity Inova 400 MHz spectrometer (Varian Unity Inova, Phoenix, USA) using TMS as the internal standard. All accurate mass experiments were performed on a Micromass QTOF (Micromass, Wythenshawe, UK) mass spectrometer. CD spectrum was recorded with a Jasco CD-2095-plus circular dichroism detector (JASCO Corporation, Tokyo, Japan). Column chromatography was conducted using silica gel 60 (40–63 and 63–200 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 was 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).
9' 9
O O
6
8
8'
H
7'
O 6' H H 3' O 3 R OCH3 1 R= OH 2 R= OH 1
7
1'
3
O
O
4
O
O
O
O
O
6
O
O
O
OH
O
OH OCH3
H3 CO
HO
OCH3
O H3CO
O
7
OH
OCH3 H3 CO 8
O H
OH
OH
O
O
H OO
OCH3
5
O
O
OCH3 H3CO HO
O
H
O
O HO 9
Fig. 1. Structures of compounds 1–10.
OCH3
10
OCH3
48
N. Li et al. / Fitoterapia 101 (2015) 46–50
system of 70% MeOH in H2O over 60 min to yield compounds 7 (4.4 mg) and 8 (6.7 mg). Fr.5 (CHCl3–MeOH 20:1, 2.3 g, 68% inhibition at 30.0 μg/mL) was chromatographed over silica gel, eluted with a stepwise gradient of CHCl3/MeOH (from 1:0, 100:1, 50:1 to 0:1) to afford 8 subfractions (Fr.51–Fr.58). Fr.53 (234 mg, 67% inhibition at 30.0 μg/mL) was subjected to an RP18 column and was eluted with MeOH–H2O (7:10, 8:10 to 100% MeOH) to yield six fractions (Fr.531–Fr.536). Fr.533 (45.0 mg) was separated by HPLC, using a gradient of 55% MeCN in H2O as the mobile phase to produce compounds 2 (4.9 mg) and 9 (7.2 mg). Purification of Fr.56 (90.0 mg, 55% inhibition at 30.0 μg/mL) was subjected to an RP-18 column and was eluted with H2O–MeOH (6:10, 7:10, to 100% MeOH) to yield four fractions (F.561–F.564). Fr.561 (32.0 mg) was purified by semipreparative HPLC using a gradient solvent system of 55% MeOH in H2O over 50 min to yield compounds 1 (4.7 mg) and 4 (6.8 mg). Fr.563 (28.0 mg) was purified by preparative HPLC using an isocratic solvent system of 90% MeOH in H2O over 45 min to obtain compounds 6 (6.1 mg) and 10 (7.8 mg). 2.3.1. 5′α-Hydroxy-4′-methoxy-sauchinone (1) Brown amorphous powder; [α]25 D -16.0° (c 0.1, CHCl3); UV (MeOH) λmax: 299, 248 nm; 1H (400 MHz) and 13C NMR (100 MHz) data in CDCl3, see Table 1; HREIMS m/z 358.1420 [M]+ (calcd for C20H22O6, 358.1416). 2.3.2. 5′β-Hydroxy-4′-methoxy-sauchinone (2) Brown amorphous powder; [α]25 D -22.69° (c 0.1, CHCl3); UV (MeOH) λmax: 299, 253 nm; 1H (400 MHz) and 13C NMR (100 MHz) data in CDCl3, see Table 1; HREIMS m/z 358.1418 [M]+ (calcd for C20H22O6, 358.1416). 2.3.3. Sauchinone (3) 1 Colorless powder; C20H20O6; [α]25 D -140.0° (c 1, CHCl3); H NMR (400 MHz, CDCl3) δ: 6.85 (1H, s), 6.40 (1H, s), 5.93 (1H, s),
Table 1 NMR data of compounds 1 and 2 in CDCl3 (1H: 400 MHz, 13C: 100 MHz). Position
1 δCa
1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′eq 7′ax 8′ 9′ 4,5-OCH2O 4′-OCH3 5′-OH a
116.3 142.5 99.6 146.5 146.5 106.7 34.5 34.9 21.5 32.9 192.2 97.7 183.2 92.1 39.2 26.4 33.6 21.3 101.1 s 56.8 4.95, br s
2 δH, mult. (J in Hz)
6.38, s
6.85, s 3.11, d, (4.8) 2.45, m 1.25, d, (7.2) 2.33, m 5.49, s
2.47, m 1.84, m 1.62, m 1.84, m 0.80, d (7.2) 5.87, 5.92, d (1.6) 3.78, s
δC 116.3 142.8 99.2 146.5 145.4 106.7 34.3 38.3 21.3 38.4 200.3 101.9 170.7 93.4 35.1 25.2 33.2 21.1 101.2 56.8 4.03, br s
δH, mult. (J in Hz)
6.39, d (1.2)
6.86, d (1.2) 3.13, m 2.44, m 1.23, d (7.2) 2.39, m 5.42, s
2.53, m 1.84, m 1.66, m 1.85, m 0.73, d (7.2) 5.91, 5.92, d (1.6) 3.87, s
Chemical shifts in ppm relative to TMS; coupling constants (J) in Hz.
5.89 (1H, s), 5.68 (1H, s), 5.62 (1H, s), 5.52 (1H, s), 3.05 (1H, d, J = 4.8 Hz), 2.55 (1H, td, J = 2.8, 12.0 Hz), 2.50 (1H, d, J = 5.6 Hz), 2.45 (1H, m), 1.93 (1H, m), 1.90 (1H, m), 1.65 (1H, m), 1.22 (3H, d, J = 7.6 Hz), 0.73 (3H, d, J = 7.6 Hz); 13C NMR (100 MHz, CDCl3) δ: 199.7, 168.7, 146.7, 145.0, 143.3, 115.8, 106.6, 101.4, 101.4, 100.4, 99.3, 98.7, 35.1, 34.8, 21.3, 37.6, 37.6, 25.3, 33.5, 20.9; EIMS m/z 356 [M]+. 2.4. DGAT1 and DGAT2 assays DGAT activity assays were carried out as described previously [16]. Briefly, DGAT activity in total membranes prepared from DGAT2- or DGAT1-overexpressing Sf-9 and HEK293 Tet-on cells was determined by measuring the formation of [14C]-triacylglycerol from [14C]-oleoyl CoA. The reaction mixture for the DGAT1 assay contained 175 mM Tris (pH 7.5), 100 mM MgCl2 (5 mM MgCl2 for DGAT2 assay), 200 μM sn-1,2-diacylglycerol, 20 μM [1-14C]-oleoyl CoA (5.5 μCi), 2 mg/mL BSA, and 32 μg of the membrane protein. The mixture was incubated for 20 min at 37 °C and then the reaction was stopped by the addition of 1.5 mL of stop solution [2-propanol–heptane–water (80:20:2, v/v/v)] and vortexed with 1.0 mL of heptane and 0.5 mL of water. The top heptane phase was collected and washed with 2.0 mL alkaline ethanol solution [ethanol–0.5 N NaOH–water (50:10:40, v/v/v)]. The radioactivity of the top phase was determined by liquid scintillation counting (Tri-Carb 2900TR Liquid Scintillation Analyzer, PerkinElmer, Inc.). 3. Results and discussion Compound 1 was obtained as brown amorphous powder, with the molecular formula C20H22O6, as determined by the HREIMS at m/z 358.1420 for the [M]+ (calcd. for C20H22O6, 358.1416). The 1H NMR spectrum of 1 (Table 1) displayed signals due to two methyl groups (δH 0.80 and 1.25, each d, J = 7.2 Hz), a methoxy group (δH 3.78), a hydroxyl group (δH 4.95), an olefinic proton (δH 5.49) and two aromatic protons (δH 6.38 and 6.85). Furthermore, the 1H NMR signals at δH 2.45 (H-8), 2.33 (H-1′), 2.47 (H-6′), and 1.84 (H-8′) showed the presence of four methine protons. In addition, a methylenedioxy group (δH 5.87, 5.92 and δC 101.1) which attached to an aromatic ring was also observed in the 1H NMR spectrum of 1. The 13C NMR signals at δC 142.5 (C-2) and 146.5 (C-4 and C-5) indicated that, in addition to a methylenedioxy group connected to an aromatic ring, there is an additional oxygen attached to the aromatic ring. The 13C NMR signal at δC 192.2 (C-2′) was attributed to a carbonyl group of an enone, while the 13C NMR signal at δC 183.2 (C-4′) indicated the presence of an oxygen atom attached to the β-carbon of an enone. In particular, the 1H and 13C NMR data of 1 were in close agreement with those of sauchinone (3) [17]. However, the positions of a methylenedioxy group (C-4′ and C-5′) in 3 were substituted by a hydroxyl group (δH 4.95) and a methoxy group (δH 3.78) in 1. The methoxy group (δH 3.78) was connected to C-4′ as deduced from the HMBC correlation (Fig. 2) of δH 3.78 (4′-OCH3) with δC 183.2 (C-4′), and the hydroxyl group (δH 4.95) was located at C-5′ respectively. These NMR data indicated that 1 was a derivative of 3. The relative configurations of 1 were mainly established by a series of the selective NOESY experiments (Fig. 3). From the NOESY spectrum (measured in CDCl3), the correlations
N. Li et al. / Fitoterapia 101 (2015) 46–50
O
O O
49
O
O O
O HO
O HO
OCH3
Fig. 2. Key HMBC correlations of 1 and 2.
from δH 3.11 (H-7)/δH 1.25 (9-CH3) and δH 2.47 (H-6′); from δH 2.33 (H-1′)/δH 1.84 (Heq-7′); δH 1.84 (Heq-7′)/δH 0.80 (9′-CH3); and from δH 2.47 (H-6′)/δH 3.11 (H-7), δH 1.25 (9-CH3) and δH 1.62 (Hax-7′), suggested that the configurations of H-7, H-6′ and H-1′ were a cis–trans form by comparison with the cis–trans form in 3. In addition, the NOESY correlation between δH 4.95 (5′-OH) and δH 1.84 (Heq-7′) established that 5′-OH was α-oriented. Therefore, the structure of 1 was designated as 5′α-hydroxy-4′-methoxy-sauchinone. Compound 2 was obtained as brown amorphous powder, with the molecular formula C20H22O6, as determined by the HREIMS at m/z 358.1418 for the [M]+ ion (calcd. for C20H22O6, 358.1416). Comparison of 1H and 13C NMR spectra (Table 1) between 2 and 1 suggested that these two compounds were stereoisomer. In the 1H NMR spectrum, the signal of 5′-OH (δH 4.03) of 2 was shifted somewhat upfield compared to the signal of 5′-OH (δH 4.95) of 1. Their 13C NMR spectra were largely in agreement except for the carbon shifts of C-2′ and C-4′. From the NOESY spectrum data (Fig. 3), the NOEs from δH 3.13 (H-7)/ δH 1.23 (9-CH3) and δH 2.53 (H-6′); from δH 2.39 (H-1′)/δH 1.84 (Heq-7′) and δH 0.73 (9′-CH3), and from δH 2.53 (H-6′)/δH 3.13 (H-7), δH 1.23 (9-CH3) and δH 1.66 (Hax-7′) indicated that the configurations of H-7, H-6′ and H-1′ were a cis–trans form by comparison with the cis–trans form in 1. However, the NOESY correlation from δH 4.03 (5′-OH)/δH 1.66 (Hax-7′) revealed that 5′-OH was β-oriented. Based on the above analysis, the structure of 2 was elucidated as 5′β-hydroxy-4′-methoxysauchinone. Along with two new lignans, eight known compounds (3–10) (Fig. 1) were obtained from S. chinensis. The known compounds were identified as sauchinone (3) [17],
H H
Hax
H O
O HO
H
Heq
H
O
machilin A (4) [18], meso-dihydroguaiaretic acid (5) [19], (−)-chicanin (6) [20], rel-(8R,8′R)-dimethyl-(7S,7′R)-bis(3,4methylenedioxyphenyl)tetrahydrofuran (7) [21], macelignan (8) [18], otobaphenol (9) [22] and saucerneol C (10) [23] based on the NMR data. Compounds (1–10) were assayed to evaluate their inhibitory activity against DGAT1 using an in vitro assay (Table 2). In order to determine the selectivity of these compounds, we examined their effects on the activity of DGAT2. The known DGAT inhibitor, kuraridine, was used as positive control [24]. The result showed that most isolates exhibited potential inhibitory activity against DGAT1 but displayed moderate DGAT2 inhibitory activity. Among them, compounds 2, 3, 5 and 7 exhibited selective effects to DGAT1 in a dose-dependent manner with IC50 values ranging from 44.3 ± 1.5 to 87.5 ± 1.3 μM. However, compound 2 (IC50 = 87.5 ± 1.3 μM) showed a higher level of activity than 1 (IC50 = 109.5 ± 1.3 μM), which indicated that the similar position of the β-hydroxyl substituent in C-5′ may have had more active effect on the increase of DGAT1 inhibitory activity. In addition, sauchinone (3) (IC50 = 66.5 ± 1.1 μM) with two methylenedioxy groups at C-4,5 and C-4′,5′ exhibited strong inhibitory activity, while the sauchinone analogues 1 (IC50 = 109.5 ± 1.3 μM) and 2 (IC50 = 87.5 ± 1.3 μM) with one methylenedioxy group at C-4,5 displayed weaker inhibitory effect against DGAT1. This result indicated that the activity of sauchinone analogues seems to be associated with the methylenedioxy group. In this study, DGAT inhibitory activity of sauchinone analogues was reported for the first time. This data presented here may provide a basis for the development of new DGAT1 inhibitors based on sauchinone analogues and it
O H
H
Hax
O H O
OCH3 Fig. 3. Key NOESY correlations of 1 and 2.
Heq
H
O HO
O H OCH3
50
N. Li et al. / Fitoterapia 101 (2015) 46–50
Table 2 Inhibitory effects of compounds 1–10 on DGAT1 and DGAT2 (expressed as IC50 values). Compound
1 2 3 4 5 6 7 8 9 10 Kuraridinea
IC50 (μM) DGAT1
DGAT2
109.5 ± 1.3 87.5 ± 1.3 66.5 ± 1.1 N200 63.9 ± 1.1 141.5 ± 1.3 44.3 ± 1.5 N200 169.8 ± 1.2 162.3 ± 1.7 10.3 ± 1.2
N200 N200 N200 120.8 ± 1.2 N200 108.3 ± 1.6 N200 173.5 ± 1.3 135.6 ± 1.1 N200 18.2 ± 1.0
a Positive control. The samples were tested for DGAT inhibitory activity in three independent experiments.
is valuable for further investigation and optimization of this type lignans. Acknowledgments This research was financially supported by the Project Sponsored by the State Education Ministry, Key Laboratory of Natural Resources of Changbai Mountain & Functional Molecules (201305, Yanbian University, Ministry of Education of the People's Republic of China), Special Funds of Medical Programmes of Jilin Province (YYZX201240), the Development and Reform Commission of Jilin Province (20111439), and Jilin Shizandra Development & Industrialization Engineering Research Center (2013G020). Appendix A. Supplementary data The NMR spectral data of compounds 1 and 2 are available as Supporting Information. Supplementary data associated with this article can be found, in the online version, at http:// dx.doi.org/10.1016/j.fitote.2014.12.011. References [1] Raj M, Kumar RK. Obesity in children & adolescents. Indian J Med Res 2010;132:598–607. [2] Turchetto-Zoletn AC, Maraschin FS, de Morais GL, Cagliari A, Andrade CM, Margis-Pinheiro M, et al. R BMC Evol Biol 2011;11:263. [3] Cases S, Smith SJ, Zheng YW, Myers HM, Lear SR, Sande E, et al. Identification of a gene encoding an acyl CoA: diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis. J Proc Natl Acad Sci USA 1998;95: 13018–23.
[4] Lardizabal KD, Mai JT, Wagner NW, Wyrick A, Voelker T, Hawkins DJ. DGAT2 is a new diacylglycerol acyltransferase gene family: purification, cloning, and expression in insect cells of two polypeptides from Mortierella ramanniana with diacylglycerol acyltransferase activity. J Biol Chem 2001; 276:38862–9. [5] Yamaguchi K, Yang L, McCall S, Huang J, Yu XX, Pandey SK, et al. Diacylglycerol acyltranferase 1 anti-sense oligonucleotides reduce hepatic fibrosis in mice with nonalcoholic steatohepatitis. Hepatology 2008;47: 625–35. [6] Cui L, Kim MO, Seo JH, Kim IS, Kim NY, Lee SH, et al. Abietane diterpenoids of Rosmarinus officinalis and their diacylglycerol acyltransferase-inhibitory activity. Food Chem 2012;132:1775–80. [7] DeVita RJ, Pinto S. Current status of the research and development of diacylglycerol O acyltransferase 1 (DGAT1) inhibitors. J Med Chem 2013; 56:9820–5. [8] Chung BS, Shin MG. Dictionary of Korean folk medicine. Seoul: Young Lim Sa; 1990 813. [9] Gao X, He J, Wu XD, Peng LY, Dong LB, Deng X, et al. Further lignans from Saururus chinensis. Planta Med 2013;79:1720–3. [10] Sung SH. A new dineolignan from Saururus chinensis root. Fitoterapia 2006;277:487–8. [11] Kim SR, Sung SH, Kang SY, Koo KA, Kim SH, Ma CJ, et al. Aristolactam BII of Saururus chinensis attenuates glutamate-induced neurotoxicity in rat cortical cultures probably by inhibiting nitric oxide production. Planta Med 2004;70:391–6. [12] Hwang BY, Lee JH, Jung HS, Kim KS, Nam JB, Hong YS, et al. Sauchinone, a lignan from Saururus chinensis, suppresses iNOS expression through the inhibition of transactivation activity of RelA of NF-kappaB. Planta Med 2003;12:1096–101. [13] Jeong GS, Li B, Lee DS, Kwon JW, Lee HS, Kwon TO, et al. Hepatoprotective constituents of Saururus chinensis roots against tacrine-induced cytotoxicity in human liver-derived Hep G2 cells. Korean J Pharmacogn 2007;38: 176–80. [14] Hwang JY, Zhang J, Kang MJ, Lee SK, Kim HA, Kim JJ, et al. Hypoglycemic and hypolipidemic effects of Saururus chinensis Baill in streptozotocininduced diabetic rats. Nutr Res Pract 2007;1:100–4. [15] Jung JY, Lee KY, Lee MY, Jung D, Cho ES, Son HY. Antioxidant and antiasthmatic effects of saucerneol D in a mouse model of airway inflammation. Int Immunopharmacol 2011;11:698–705. [16] Kim MO, Lee SU, Lee HJ, Choi K, Kim H, Lee S, et al. Identification and validation of a selective small molecule inhibitor targeting the diacylglycerol acyltransferase 2 activity. Biol Pharm Bull 2013;36:1167–73. [17] Sung SH, Kim YC. Hepatoprotective diastereomeric lignans from Saururus chinensis herbs. J Nat Prod 2000;63:1019–21. [18] Woo WS, Shin KH, Wagner H, Lotter H. The structure of nacelignan from Myristica fragrans. Phytochemistry 1987;5:1542–3. [19] Yang S, Min KN, Jang JP. Inhibition of protein tyrosine phosphatase 1B by lignans from Myristica fragrans. Phytother Res 2006;20:680–2. [20] Sadhu SK, Okuyama E, Fujimoto H, Ishibashi M. Separation of Leucas aspera, a medicinal plant of Bangladesh, guided by prostaglandin inhibitory and antioxidant activities. Chem Pharm Bull 2003;5:595–8. [21] Herath HMTB, Priyadadarshan AMA. Two lignans and an aryl alkanone from Myristica dactyloides. Phytochem 1996;5:1439–42. [22] Duan L, Tao HW, Hao XJ, Gu QQ, Zhu WM. Cytotoxic and antioxidative phenolic compounds from the traditional Chinese medicinal plant, Myristica fragrans. Planta Med 2009;11:1241–5. [23] Sung SH, Huh MS, Kim YC. New tetrahydrofuran-type sesquilignans of Saururus chinensis root. Chem Pharm Bull 2001;9:1192–4. [24] Chung MY, Rho MC, Ko JS, Ryu SY, Jeune KH, Kim K, et al. In vitro inhibition of diacylglycerol acyltransferase by prenylflavonoids from Sophora flavescens. Planta Med 2004;70:258–60.
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Two new lignans from Saururus chinensis and their DGAT inhibitory activity Na Li a, Zhen-Dong Tuo a, Shi-Zhou Qi a, Shan-Shan Xing a, Hyun-Sun Lee b, Jian-Guang Chen a, Long Cui a,⁎ a
College of Pharmacy, Beihua University, Jilin City, Jilin Province 132013, People's Republic of China Molecular Cancer Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 685-1 Yangcheongri, Ochangeup, Cheongwongun, Chungbuk 363-883, Republic of Korea
b
a r t i c l e
i n f o
Article history: Received 9 November 2014 Accepted in revised form 16 December 2014 Accepted 22 December 2014 Available online 28 December 2014
a b s t r a c t Two new lignans were isolated from Saururus chinensis, along with eight known compounds. Their structures were elucidated on the basis of spectroscopic and physico-chemical analyses. All the isolates were evaluated for in vitro inhibitory activity against DGAT1 and DGAT2. Among them, compounds 2, 3, 5 and 7 were found to exhibit selective inhibitory activity on DGAT1 with IC50 values ranging from 44.3 ± 1.5 to 87.5 ± 1.3 μM. © 2014 Elsevier B.V. All rights reserved.
Keywords: Saururus chinensis Saururaceae DGAT Lignan
1. Introduction Obesity is one of the leading metabolic diseases worldwide and is closely associated with diabetes, hypertension and cardiovascular disease [1]. There have been a great number of studies on the treatment of obesity, but more efficient therapeutic strategies need to be explored imminently. One of potential therapeutic methods involves inhibiting triacylglycerol (TG) synthesis. TG is a major form of energy storage in eukaryotic organisms. And the excess supply of TG in a tissue could lead to obesity [2]. A key enzyme in TG synthesis is acylcoenzyme A (CoA): diacylglycerol acyltransferase (DGAT), which catalyzes the final step of the TG synthesis pathway in mammalian cells by using diacylglycerol and fatty acyl CoA as substrates. Two isoforms of the DGAT enzyme are presently known namely, DGAT1 and DGAT2 [3,4]. Although both enzymes utilize the same substrate, there is no homology between DGAT1 and DGAT2: DGAT1 belongs to the acyl-CoA: ⁎ Corresponding author. Tel./fax: +86 432 64608281. E-mail address: [email protected] (L. Cui).
http://dx.doi.org/10.1016/j.fitote.2014.12.011 0367-326X/© 2014 Elsevier B.V. All rights reserved.
cholesterol acyltransferase (ACAT) gene family and DGAT2 is a member of a distinct and independent gene family. DGAT1 deficient mice are resistant to diet-induced obesity, have lower plasma glucose levels associated with an increase of insulin and leptin sensitivity and are also protected against diet-induced hepatic steatosis [5]. Previous studies reported that DGAT1 played a major role in modulating signals of energy homeostasis and a minor role in bulk TG synthesis [6]. Inhibition of DGAT1 might be an effective target for therapy of obesity and other related diseases. To date, several DGAT1 inhibitors (JTT-553, PF-04620110, AZD7687, LCQ908) have entered clinical trials in multiple pharmaceutical companies [7]. These DGAT1 inhibitors are widely available, but they have a great many limitations, including adverse effects and tolerability. Thus, efforts to discover novel, selective, orally bioavailable DGAT1 inhibitors have been intensified. Saururus chinensis (Lour.) Baill. (Saururaceae), a perennial herb, is widely cultivated in China and southern Korea. It has been traditionally used as folk medicine for the treatment of inflammation, jaundice and gonorrhea [8]. Previous chemical studies of S. chinensis have revealed the presence of lignans [9],
N. Li et al. / Fitoterapia 101 (2015) 46–50
47
HEK293 Tet-on cells were obtained from Clontech. Fatty acid-free BSA and sn-1,2-dioleoylglycerol were obtained from Sigma-Aldrich. [14C]-oleoyl CoA, were purchased from PerkinElmer, Inc.
flavonoids [10] and alkaloids [11]. Sauchinone, a lignan isolated from S. chinensis with unique structure, exhibited a great many pharmacological activities including anti-inflammatory [12], hepatoprotective [13], antidiabetic [14], and antioxidant effects [15] in various cell types. As a part of our exploration on the traditional medicine, S. chinensis was studied, which resulted in the isolation of two new sauchinone analogues (1–2) along with eight known compounds (3–10) (Fig. 1). Herein we reported the isolation, structural elucidation of two new lignans and evaluation of DGAT inhibitory activity about these isolates.
2.2. Plant material The root of S. chinensis was collected in Jinan, Shandong province, People's Republic of China, and authenticated by Professor Gao Li (College of Pharmacy, Yanbian University). A voucher specimen of the plant (No. 20100806) was deposited at the College of Pharmacy, Beihua University, Jilin, China.
2. Experimental
2.3. Extraction and isolation
2.1. General experimental procedures
The root (5.0 kg) of S. chinensis was extracted with MeOH at room temperature for 2 weeks and the solution was concentrated to obtain a crude extract (183.0 g, 67% inhibition at 30.0 μg/mL). This extract was suspended in H2O, partitioned successively with CHCl3, EtOAc and BuOH, and then the organic solvents were removed. A portion of the CHCl3-soluble fraction (18.0 g, 78% inhibition at 30.0 μg/mL) was chromatographed over a silica gel column using a gradient of CHCl3–MeOH (from 1:0, 100:1 to 5:1), and was separated into 10 fractions (Fr.1– Fr.10). Fr.2 (CHCl3–MeOH 100:1, 1.8 g, 73% inhibition at 30.0 μg/mL) was chromatographed over silica gel, eluted with a stepwise gradient of n-Hexane/CHCl3 (from 1:1, 1:2, 1:3 to 0:1) to afford 9 subfractions (Fr.21–Fr.29). The most active fraction, Fr.24 (260.0 mg, 81% inhibition at 30.0 μg/mL) was subjected to an RP-18 column and was eluted with MeOH–H2O (8:10, 9:10 to 100% MeOH) to yield five fractions (Fr.241– Fr.245). Fr.242 (58.0 mg) was separated by HPLC, using a gradient of 60% MeCN in H2O as the mobile phase to produce compounds 3 (4.9 mg) and 5 (9.7 mg). Fr.244 (48.0 mg) was purified by semipreparative HPLC using a gradient solvent
UV spectra were taken in MeOH using a Shimadzu spectrophotometer (Shimadzu, Tokyo, Japan). Nuclear magnetic resonance (NMR) spectra were obtained from a Varian Unity Inova 400 MHz spectrometer (Varian Unity Inova, Phoenix, USA) using TMS as the internal standard. All accurate mass experiments were performed on a Micromass QTOF (Micromass, Wythenshawe, UK) mass spectrometer. CD spectrum was recorded with a Jasco CD-2095-plus circular dichroism detector (JASCO Corporation, Tokyo, Japan). Column chromatography was conducted using silica gel 60 (40–63 and 63–200 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 was 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).
9' 9
O O
6
8
8'
H
7'
O 6' H H 3' O 3 R OCH3 1 R= OH 2 R= OH 1
7
1'
3
O
O
4
O
O
O
O
O
6
O
O
O
OH
O
OH OCH3
H3 CO
HO
OCH3
O H3CO
O
7
OH
OCH3 H3 CO 8
O H
OH
OH
O
O
H OO
OCH3
5
O
O
OCH3 H3CO HO
O
H
O
O HO 9
Fig. 1. Structures of compounds 1–10.
OCH3
10
OCH3
48
N. Li et al. / Fitoterapia 101 (2015) 46–50
system of 70% MeOH in H2O over 60 min to yield compounds 7 (4.4 mg) and 8 (6.7 mg). Fr.5 (CHCl3–MeOH 20:1, 2.3 g, 68% inhibition at 30.0 μg/mL) was chromatographed over silica gel, eluted with a stepwise gradient of CHCl3/MeOH (from 1:0, 100:1, 50:1 to 0:1) to afford 8 subfractions (Fr.51–Fr.58). Fr.53 (234 mg, 67% inhibition at 30.0 μg/mL) was subjected to an RP18 column and was eluted with MeOH–H2O (7:10, 8:10 to 100% MeOH) to yield six fractions (Fr.531–Fr.536). Fr.533 (45.0 mg) was separated by HPLC, using a gradient of 55% MeCN in H2O as the mobile phase to produce compounds 2 (4.9 mg) and 9 (7.2 mg). Purification of Fr.56 (90.0 mg, 55% inhibition at 30.0 μg/mL) was subjected to an RP-18 column and was eluted with H2O–MeOH (6:10, 7:10, to 100% MeOH) to yield four fractions (F.561–F.564). Fr.561 (32.0 mg) was purified by semipreparative HPLC using a gradient solvent system of 55% MeOH in H2O over 50 min to yield compounds 1 (4.7 mg) and 4 (6.8 mg). Fr.563 (28.0 mg) was purified by preparative HPLC using an isocratic solvent system of 90% MeOH in H2O over 45 min to obtain compounds 6 (6.1 mg) and 10 (7.8 mg). 2.3.1. 5′α-Hydroxy-4′-methoxy-sauchinone (1) Brown amorphous powder; [α]25 D -16.0° (c 0.1, CHCl3); UV (MeOH) λmax: 299, 248 nm; 1H (400 MHz) and 13C NMR (100 MHz) data in CDCl3, see Table 1; HREIMS m/z 358.1420 [M]+ (calcd for C20H22O6, 358.1416). 2.3.2. 5′β-Hydroxy-4′-methoxy-sauchinone (2) Brown amorphous powder; [α]25 D -22.69° (c 0.1, CHCl3); UV (MeOH) λmax: 299, 253 nm; 1H (400 MHz) and 13C NMR (100 MHz) data in CDCl3, see Table 1; HREIMS m/z 358.1418 [M]+ (calcd for C20H22O6, 358.1416). 2.3.3. Sauchinone (3) 1 Colorless powder; C20H20O6; [α]25 D -140.0° (c 1, CHCl3); H NMR (400 MHz, CDCl3) δ: 6.85 (1H, s), 6.40 (1H, s), 5.93 (1H, s),
Table 1 NMR data of compounds 1 and 2 in CDCl3 (1H: 400 MHz, 13C: 100 MHz). Position
1 δCa
1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′eq 7′ax 8′ 9′ 4,5-OCH2O 4′-OCH3 5′-OH a
116.3 142.5 99.6 146.5 146.5 106.7 34.5 34.9 21.5 32.9 192.2 97.7 183.2 92.1 39.2 26.4 33.6 21.3 101.1 s 56.8 4.95, br s
2 δH, mult. (J in Hz)
6.38, s
6.85, s 3.11, d, (4.8) 2.45, m 1.25, d, (7.2) 2.33, m 5.49, s
2.47, m 1.84, m 1.62, m 1.84, m 0.80, d (7.2) 5.87, 5.92, d (1.6) 3.78, s
δC 116.3 142.8 99.2 146.5 145.4 106.7 34.3 38.3 21.3 38.4 200.3 101.9 170.7 93.4 35.1 25.2 33.2 21.1 101.2 56.8 4.03, br s
δH, mult. (J in Hz)
6.39, d (1.2)
6.86, d (1.2) 3.13, m 2.44, m 1.23, d (7.2) 2.39, m 5.42, s
2.53, m 1.84, m 1.66, m 1.85, m 0.73, d (7.2) 5.91, 5.92, d (1.6) 3.87, s
Chemical shifts in ppm relative to TMS; coupling constants (J) in Hz.
5.89 (1H, s), 5.68 (1H, s), 5.62 (1H, s), 5.52 (1H, s), 3.05 (1H, d, J = 4.8 Hz), 2.55 (1H, td, J = 2.8, 12.0 Hz), 2.50 (1H, d, J = 5.6 Hz), 2.45 (1H, m), 1.93 (1H, m), 1.90 (1H, m), 1.65 (1H, m), 1.22 (3H, d, J = 7.6 Hz), 0.73 (3H, d, J = 7.6 Hz); 13C NMR (100 MHz, CDCl3) δ: 199.7, 168.7, 146.7, 145.0, 143.3, 115.8, 106.6, 101.4, 101.4, 100.4, 99.3, 98.7, 35.1, 34.8, 21.3, 37.6, 37.6, 25.3, 33.5, 20.9; EIMS m/z 356 [M]+. 2.4. DGAT1 and DGAT2 assays DGAT activity assays were carried out as described previously [16]. Briefly, DGAT activity in total membranes prepared from DGAT2- or DGAT1-overexpressing Sf-9 and HEK293 Tet-on cells was determined by measuring the formation of [14C]-triacylglycerol from [14C]-oleoyl CoA. The reaction mixture for the DGAT1 assay contained 175 mM Tris (pH 7.5), 100 mM MgCl2 (5 mM MgCl2 for DGAT2 assay), 200 μM sn-1,2-diacylglycerol, 20 μM [1-14C]-oleoyl CoA (5.5 μCi), 2 mg/mL BSA, and 32 μg of the membrane protein. The mixture was incubated for 20 min at 37 °C and then the reaction was stopped by the addition of 1.5 mL of stop solution [2-propanol–heptane–water (80:20:2, v/v/v)] and vortexed with 1.0 mL of heptane and 0.5 mL of water. The top heptane phase was collected and washed with 2.0 mL alkaline ethanol solution [ethanol–0.5 N NaOH–water (50:10:40, v/v/v)]. The radioactivity of the top phase was determined by liquid scintillation counting (Tri-Carb 2900TR Liquid Scintillation Analyzer, PerkinElmer, Inc.). 3. Results and discussion Compound 1 was obtained as brown amorphous powder, with the molecular formula C20H22O6, as determined by the HREIMS at m/z 358.1420 for the [M]+ (calcd. for C20H22O6, 358.1416). The 1H NMR spectrum of 1 (Table 1) displayed signals due to two methyl groups (δH 0.80 and 1.25, each d, J = 7.2 Hz), a methoxy group (δH 3.78), a hydroxyl group (δH 4.95), an olefinic proton (δH 5.49) and two aromatic protons (δH 6.38 and 6.85). Furthermore, the 1H NMR signals at δH 2.45 (H-8), 2.33 (H-1′), 2.47 (H-6′), and 1.84 (H-8′) showed the presence of four methine protons. In addition, a methylenedioxy group (δH 5.87, 5.92 and δC 101.1) which attached to an aromatic ring was also observed in the 1H NMR spectrum of 1. The 13C NMR signals at δC 142.5 (C-2) and 146.5 (C-4 and C-5) indicated that, in addition to a methylenedioxy group connected to an aromatic ring, there is an additional oxygen attached to the aromatic ring. The 13C NMR signal at δC 192.2 (C-2′) was attributed to a carbonyl group of an enone, while the 13C NMR signal at δC 183.2 (C-4′) indicated the presence of an oxygen atom attached to the β-carbon of an enone. In particular, the 1H and 13C NMR data of 1 were in close agreement with those of sauchinone (3) [17]. However, the positions of a methylenedioxy group (C-4′ and C-5′) in 3 were substituted by a hydroxyl group (δH 4.95) and a methoxy group (δH 3.78) in 1. The methoxy group (δH 3.78) was connected to C-4′ as deduced from the HMBC correlation (Fig. 2) of δH 3.78 (4′-OCH3) with δC 183.2 (C-4′), and the hydroxyl group (δH 4.95) was located at C-5′ respectively. These NMR data indicated that 1 was a derivative of 3. The relative configurations of 1 were mainly established by a series of the selective NOESY experiments (Fig. 3). From the NOESY spectrum (measured in CDCl3), the correlations
N. Li et al. / Fitoterapia 101 (2015) 46–50
O
O O
49
O
O O
O HO
O HO
OCH3
Fig. 2. Key HMBC correlations of 1 and 2.
from δH 3.11 (H-7)/δH 1.25 (9-CH3) and δH 2.47 (H-6′); from δH 2.33 (H-1′)/δH 1.84 (Heq-7′); δH 1.84 (Heq-7′)/δH 0.80 (9′-CH3); and from δH 2.47 (H-6′)/δH 3.11 (H-7), δH 1.25 (9-CH3) and δH 1.62 (Hax-7′), suggested that the configurations of H-7, H-6′ and H-1′ were a cis–trans form by comparison with the cis–trans form in 3. In addition, the NOESY correlation between δH 4.95 (5′-OH) and δH 1.84 (Heq-7′) established that 5′-OH was α-oriented. Therefore, the structure of 1 was designated as 5′α-hydroxy-4′-methoxy-sauchinone. Compound 2 was obtained as brown amorphous powder, with the molecular formula C20H22O6, as determined by the HREIMS at m/z 358.1418 for the [M]+ ion (calcd. for C20H22O6, 358.1416). Comparison of 1H and 13C NMR spectra (Table 1) between 2 and 1 suggested that these two compounds were stereoisomer. In the 1H NMR spectrum, the signal of 5′-OH (δH 4.03) of 2 was shifted somewhat upfield compared to the signal of 5′-OH (δH 4.95) of 1. Their 13C NMR spectra were largely in agreement except for the carbon shifts of C-2′ and C-4′. From the NOESY spectrum data (Fig. 3), the NOEs from δH 3.13 (H-7)/ δH 1.23 (9-CH3) and δH 2.53 (H-6′); from δH 2.39 (H-1′)/δH 1.84 (Heq-7′) and δH 0.73 (9′-CH3), and from δH 2.53 (H-6′)/δH 3.13 (H-7), δH 1.23 (9-CH3) and δH 1.66 (Hax-7′) indicated that the configurations of H-7, H-6′ and H-1′ were a cis–trans form by comparison with the cis–trans form in 1. However, the NOESY correlation from δH 4.03 (5′-OH)/δH 1.66 (Hax-7′) revealed that 5′-OH was β-oriented. Based on the above analysis, the structure of 2 was elucidated as 5′β-hydroxy-4′-methoxysauchinone. Along with two new lignans, eight known compounds (3–10) (Fig. 1) were obtained from S. chinensis. The known compounds were identified as sauchinone (3) [17],
H H
Hax
H O
O HO
H
Heq
H
O
machilin A (4) [18], meso-dihydroguaiaretic acid (5) [19], (−)-chicanin (6) [20], rel-(8R,8′R)-dimethyl-(7S,7′R)-bis(3,4methylenedioxyphenyl)tetrahydrofuran (7) [21], macelignan (8) [18], otobaphenol (9) [22] and saucerneol C (10) [23] based on the NMR data. Compounds (1–10) were assayed to evaluate their inhibitory activity against DGAT1 using an in vitro assay (Table 2). In order to determine the selectivity of these compounds, we examined their effects on the activity of DGAT2. The known DGAT inhibitor, kuraridine, was used as positive control [24]. The result showed that most isolates exhibited potential inhibitory activity against DGAT1 but displayed moderate DGAT2 inhibitory activity. Among them, compounds 2, 3, 5 and 7 exhibited selective effects to DGAT1 in a dose-dependent manner with IC50 values ranging from 44.3 ± 1.5 to 87.5 ± 1.3 μM. However, compound 2 (IC50 = 87.5 ± 1.3 μM) showed a higher level of activity than 1 (IC50 = 109.5 ± 1.3 μM), which indicated that the similar position of the β-hydroxyl substituent in C-5′ may have had more active effect on the increase of DGAT1 inhibitory activity. In addition, sauchinone (3) (IC50 = 66.5 ± 1.1 μM) with two methylenedioxy groups at C-4,5 and C-4′,5′ exhibited strong inhibitory activity, while the sauchinone analogues 1 (IC50 = 109.5 ± 1.3 μM) and 2 (IC50 = 87.5 ± 1.3 μM) with one methylenedioxy group at C-4,5 displayed weaker inhibitory effect against DGAT1. This result indicated that the activity of sauchinone analogues seems to be associated with the methylenedioxy group. In this study, DGAT inhibitory activity of sauchinone analogues was reported for the first time. This data presented here may provide a basis for the development of new DGAT1 inhibitors based on sauchinone analogues and it
O H
H
Hax
O H O
OCH3 Fig. 3. Key NOESY correlations of 1 and 2.
Heq
H
O HO
O H OCH3
50
N. Li et al. / Fitoterapia 101 (2015) 46–50
Table 2 Inhibitory effects of compounds 1–10 on DGAT1 and DGAT2 (expressed as IC50 values). Compound
1 2 3 4 5 6 7 8 9 10 Kuraridinea
IC50 (μM) DGAT1
DGAT2
109.5 ± 1.3 87.5 ± 1.3 66.5 ± 1.1 N200 63.9 ± 1.1 141.5 ± 1.3 44.3 ± 1.5 N200 169.8 ± 1.2 162.3 ± 1.7 10.3 ± 1.2
N200 N200 N200 120.8 ± 1.2 N200 108.3 ± 1.6 N200 173.5 ± 1.3 135.6 ± 1.1 N200 18.2 ± 1.0
a Positive control. The samples were tested for DGAT inhibitory activity in three independent experiments.
is valuable for further investigation and optimization of this type lignans. Acknowledgments This research was financially supported by the Project Sponsored by the State Education Ministry, Key Laboratory of Natural Resources of Changbai Mountain & Functional Molecules (201305, Yanbian University, Ministry of Education of the People's Republic of China), Special Funds of Medical Programmes of Jilin Province (YYZX201240), the Development and Reform Commission of Jilin Province (20111439), and Jilin Shizandra Development & Industrialization Engineering Research Center (2013G020). Appendix A. Supplementary data The NMR spectral data of compounds 1 and 2 are available as Supporting Information. Supplementary data associated with this article can be found, in the online version, at http:// dx.doi.org/10.1016/j.fitote.2014.12.011. References [1] Raj M, Kumar RK. Obesity in children & adolescents. Indian J Med Res 2010;132:598–607. [2] Turchetto-Zoletn AC, Maraschin FS, de Morais GL, Cagliari A, Andrade CM, Margis-Pinheiro M, et al. R BMC Evol Biol 2011;11:263. [3] Cases S, Smith SJ, Zheng YW, Myers HM, Lear SR, Sande E, et al. Identification of a gene encoding an acyl CoA: diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis. J Proc Natl Acad Sci USA 1998;95: 13018–23.
[4] Lardizabal KD, Mai JT, Wagner NW, Wyrick A, Voelker T, Hawkins DJ. DGAT2 is a new diacylglycerol acyltransferase gene family: purification, cloning, and expression in insect cells of two polypeptides from Mortierella ramanniana with diacylglycerol acyltransferase activity. J Biol Chem 2001; 276:38862–9. [5] Yamaguchi K, Yang L, McCall S, Huang J, Yu XX, Pandey SK, et al. Diacylglycerol acyltranferase 1 anti-sense oligonucleotides reduce hepatic fibrosis in mice with nonalcoholic steatohepatitis. Hepatology 2008;47: 625–35. [6] Cui L, Kim MO, Seo JH, Kim IS, Kim NY, Lee SH, et al. Abietane diterpenoids of Rosmarinus officinalis and their diacylglycerol acyltransferase-inhibitory activity. Food Chem 2012;132:1775–80. [7] DeVita RJ, Pinto S. Current status of the research and development of diacylglycerol O acyltransferase 1 (DGAT1) inhibitors. J Med Chem 2013; 56:9820–5. [8] Chung BS, Shin MG. Dictionary of Korean folk medicine. Seoul: Young Lim Sa; 1990 813. [9] Gao X, He J, Wu XD, Peng LY, Dong LB, Deng X, et al. Further lignans from Saururus chinensis. Planta Med 2013;79:1720–3. [10] Sung SH. A new dineolignan from Saururus chinensis root. Fitoterapia 2006;277:487–8. [11] Kim SR, Sung SH, Kang SY, Koo KA, Kim SH, Ma CJ, et al. Aristolactam BII of Saururus chinensis attenuates glutamate-induced neurotoxicity in rat cortical cultures probably by inhibiting nitric oxide production. Planta Med 2004;70:391–6. [12] Hwang BY, Lee JH, Jung HS, Kim KS, Nam JB, Hong YS, et al. Sauchinone, a lignan from Saururus chinensis, suppresses iNOS expression through the inhibition of transactivation activity of RelA of NF-kappaB. Planta Med 2003;12:1096–101. [13] Jeong GS, Li B, Lee DS, Kwon JW, Lee HS, Kwon TO, et al. Hepatoprotective constituents of Saururus chinensis roots against tacrine-induced cytotoxicity in human liver-derived Hep G2 cells. Korean J Pharmacogn 2007;38: 176–80. [14] Hwang JY, Zhang J, Kang MJ, Lee SK, Kim HA, Kim JJ, et al. Hypoglycemic and hypolipidemic effects of Saururus chinensis Baill in streptozotocininduced diabetic rats. Nutr Res Pract 2007;1:100–4. [15] Jung JY, Lee KY, Lee MY, Jung D, Cho ES, Son HY. Antioxidant and antiasthmatic effects of saucerneol D in a mouse model of airway inflammation. Int Immunopharmacol 2011;11:698–705. [16] Kim MO, Lee SU, Lee HJ, Choi K, Kim H, Lee S, et al. Identification and validation of a selective small molecule inhibitor targeting the diacylglycerol acyltransferase 2 activity. Biol Pharm Bull 2013;36:1167–73. [17] Sung SH, Kim YC. Hepatoprotective diastereomeric lignans from Saururus chinensis herbs. J Nat Prod 2000;63:1019–21. [18] Woo WS, Shin KH, Wagner H, Lotter H. The structure of nacelignan from Myristica fragrans. Phytochemistry 1987;5:1542–3. [19] Yang S, Min KN, Jang JP. Inhibition of protein tyrosine phosphatase 1B by lignans from Myristica fragrans. Phytother Res 2006;20:680–2. [20] Sadhu SK, Okuyama E, Fujimoto H, Ishibashi M. Separation of Leucas aspera, a medicinal plant of Bangladesh, guided by prostaglandin inhibitory and antioxidant activities. Chem Pharm Bull 2003;5:595–8. [21] Herath HMTB, Priyadadarshan AMA. Two lignans and an aryl alkanone from Myristica dactyloides. Phytochem 1996;5:1439–42. [22] Duan L, Tao HW, Hao XJ, Gu QQ, Zhu WM. Cytotoxic and antioxidative phenolic compounds from the traditional Chinese medicinal plant, Myristica fragrans. Planta Med 2009;11:1241–5. [23] Sung SH, Huh MS, Kim YC. New tetrahydrofuran-type sesquilignans of Saururus chinensis root. Chem Pharm Bull 2001;9:1192–4. [24] Chung MY, Rho MC, Ko JS, Ryu SY, Jeune KH, Kim K, et al. In vitro inhibition of diacylglycerol acyltransferase by prenylflavonoids from Sophora flavescens. Planta Med 2004;70:258–60.
