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Two new lignans with antioxidative activities from Jatropha curcas ab
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Xiaofan Li , Ling Li , Jue Wang , Tiejie Wang & Liyan Wang
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Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences, Shenzhen University, Shenzhen 518060, P.R. China b
Key Lab for New Drugs Research of Traditional Chinese Medicine in Shenzhen, Research Institute of Tsinghua University in Shenzhen, High-Tech Industrial Estate Nanshan, Shenzhen 518057, P.R. China
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c
Shenzhen Institute for Drug Control, Shenzhen 518057, P.R. China Published online: 28 May 2014.
To cite this article: Xiaofan Li, Ling Li, Jue Wang, Tiejie Wang & Liyan Wang (2014) Two new lignans with antioxidative activities from Jatropha curcas, Natural Product Research: Formerly Natural Product Letters, 28:22, 1985-1991, DOI: 10.1080/14786419.2014.919284 To link to this article: http://dx.doi.org/10.1080/14786419.2014.919284
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Natural Product Research, 2014 Vol. 28, No. 22, 1985–1991, http://dx.doi.org/10.1080/14786419.2014.919284
Two new lignans with antioxidative activities from Jatropha curcas
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Xiaofan Liab, Ling Lib, Jue Wangc, Tiejie Wangc and Liyan Wanga* a Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences, Shenzhen University, Shenzhen 518060, P.R. China; bKey Lab for New Drugs Research of Traditional Chinese Medicine in Shenzhen, Research Institute of Tsinghua University in Shenzhen, High-Tech Industrial Estate Nanshan, Shenzhen 518057, P.R. China; cShenzhen Institute for Drug Control, Shenzhen 518057, P.R. China
(Received 26 February 2014; final version received 25 April 2014) Activity-guided isolation of dried seeds of Jatropha curcas L. led to the isolation of two new lignans along with eight known compounds. These compounds were determined by spectroscopic analysis to be jatrophasin C (1), jatrophasin D (2), bsitosterol (3), jatrophasin A (4), daucosterol (5), isoamericanol A (6), (^)-3,30 bisdemethylpinoresinol (7), 70 -epi-sesamin-dicatechol (8), isoprincepin (9) and americanol A (10), of which 1 and 2 were new compounds. The antioxidative activities along with peroxisome proliferator-activated receptor gamma exciting activity of these compounds were also determined. Keywords: Jatropha curcas; Euphorbiaceae; antioxidative activity; lignan
1. Introduction Jatropha curcas L. (Euphorbiaceae) is a multipurpose plant with many attributes and considerable potential. It is widely grown in Mexico, Nicaragua, Thailand and India, and is now being promoted in southern Africa, Brazil, Mali and Nepal. Seeds of this plant have been reported to show antineoplastic, desinsection and anti-oxidative activities (Openshaw et al. 2001). The oil from its seeds has been widely used as insecticide, for soap production and most importantly as a fuel substitute (Gubitz et al. 1999). Reuse of the seed cake after extraction of oil would be an income-generating and environmental saving process. Previous constituent research of the seed reported the isolation of phorbol esters, curcin and curcain which were tumour promoting and toxic (Stirpe et al. 1976; Lumia et al. 1988). However, few reports of the chemical investigation with other activities were found. In this study, an activity-guidedisolation was performed on the 95% ethanol extract of J. curcas L. seeds. With antioxidative activity screening, two new lignans along with eight known compounds were isolated and their structures were elucidated by spectral and chemical methods. Antioxidative activity of the isolated compounds was examined using DPPH radical scavenging test. Cytotoxicity and peroxisome proliferator-activated receptor gamma (PPARg) exciting activity of the new compounds were also examined. PPAR mainly controls expression of genes participating in fat metabolism or differentiation of adipocyte, and has been shown to regulate the expression of genes by binding to DNA sequences of peroxisome proliferators response element (PPREs) (Vamecq & Latrue 1999; Corton et al. 2000). The activation of PPARg can lead to increase of pre-adipocyte differentiation, promote insulin sensitivity and elevate glucose uptake (Lehmann
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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et al. 1995; Okuno et al. 1998). Therefore, ligands of PPARg can act as medicine for treating non-insulin-dependent diabetes mellitus (type 2 diabetes mellitus) caused by insulin resistance.
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2. Results and discussion The EtOH extract of dried seeds of J. curcas L. was partitioned successively with petroleum ether and n-BuOH to yield three layers, of which the BuOH layer showed a potent antioxidative effect (80.4% radical scavenging at 25 mg/mL). This layer was then separated by repeated column chromatography (CC) and preparative HPLC to yield compounds 1 –10, consisted of two new lignans, jatrophasin C (1) and jatrophasin D (2); six known lignans, jatrophasin A (4) (Li et al. 2010), isoamericanol A (6) (Waibel et al. 2003), (^ )-3,30 -bisdemethylpinoresinol (7) (Waibel et al. 2003), 70 -epi-sesamin-dicatechol (8) (Toyomori & Urada 2009), isoprincepin (9) (Waibel et al. 2003) and americanol A (10) (Waibel et al. 2003); and two sterols, b-sitosterol (3) (Hai et al. 2004) and daucosterol (5) (Zhao et al. 2010). The structure of the known compounds was elucidated by comparison with spectral data of the reported values. Jatrophasin C (1) was obtained as a brown powder and shown to have the molecular formula C19H20O7 on the basis of HR-ESI-MS data (m/z 359.1142, calcd for 359.1136, [M 2 H]2), which indicated 10 degrees of unsaturation. The UV (ultra violet) (MeOH) spectrum exhibited absorption bands lmax (log e ) at 330.0 (3.31) and 260.4 (3.91) nm, indicating the presence of benzene ring. The 1H NMR spectrum (Table 1) showed signals of a methoxyl group at d 3.84 (3H, s), two sets of oxymethylene protons at d 3.68 (dd, J ¼ 12.3, 2.3 Hz), 3.48 (dd, 12.3, 4.5) and d 4.18 (2H, d, 5.7). The low-field resonances at d 6.96 (d, J ¼ 1.7 Hz), 6.88 (d, J ¼ 8.3 Hz) and 6.92 (dd, J ¼ 1.7, 8.3 Hz) indicated a 1,3,4-substituted benzene ring; d 6.58, 6.56 (d, J ¼ 1.6 Hz) suggested another meta-substituted benzene ring; d 6.48 (d, J ¼ 15.8 Hz) and 6.20 (dt, J ¼ 15.8, 5.7 Hz) indicated a trans-olefin group. The 13C NMR and DEPT135 spectra showed 19 signals: 1 methoxyl group (d 56.8), 2 oxymethines (d 80.0, 77.9), 2 O-bearing quaternary carbons (d 63.8, 62.1) and 14 sp2 methines (d 104.1 – 149.8). The 1H – 1H COSY cross-peaks of H7/H8, H8/H9a and H8/H9b indicated a propane segment; while cross-peaks of H70 /H80 and H80 /H90 indicated a propylene segment. These segments along with the two benzene rings accounted for nine degrees of unsaturation and suggested the 1,4-benzodioxane-type lignan skeleton. HMBC correlations from H7 to C2/C6 and H20 /H60 to C70 indicated the connection of each segment. In the NOESY spectrum, weak but significant cross-peak could be observed between H-20 and H-7, indicating the structure as shown in Figure 1. The relationship of H-7 and H-8 was assigned as trans according to the coupling constant (J7,8 ¼ 8.0). Compound 1 showed no optical rotation or Circular Dichroism (CD) cotton curve, indicating a racemic mixture. Table 1. DPPH radical scavenging activity (IC50) of isolated compounds, with resveratrol as positive control. Compound
IC50 (mM)
Resveratrol 1 2 4 6 7 8 9 10
40.67 42.98 9.71 22.85 5.28 10.49 9.18 7.23 8.28
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Natural Product Research
Figure 1. Structures (left), as well as COSY, key HMBC ( compounds 1 2 isolated from J. curcas.
) and NOESY (
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Jatrophasin D (2) was obtained as a brown powder and shown to have the molecular formula C19H20O7 on the basis of HR-ESI-MS data (m/z 383.1106, calcd for 383.1101, [M þ Na]þ). The UV (MeOH) spectrum exhibited absorption bands lmax (log e) at 222.5 (4.60) and 274.5 (4.23) nm, indicating the presence of benzene ring. The 1H and 13C NMR spectra showed signals (Table 1) similar to those of jatrophasin C (1), indicating 1,4-benzodioxane-type lignan skeleton. The key HMBC correlation of d 3.87 (H-100 ) to d 150.2(C-50 ) suggested that the methyl group linked to C-50 position. All of the 1H and 13C NMR spectral signals of 2 were properly assigned based on the HMQC, HMBC and 1H – 1H COSY spectra (Table 1). The relationship of H-7 and H-8 was assigned as trans according to the coupling constant (J7,8 ¼ 8.0). Compound 2 showed no optical rotation or CD cotton curve, indicating a racemic mixture. All of the isolated compounds as well as resveratrol (used as positive control) were tested for antioxidative activity using DPPH scavenging assay. As shown in Table 2, compound 6 showed comparatively strong activity, compounds 2, 7– 10 showed medium activity, while compounds 3 and 5 showed almost no activity (, 20% DPPH radical scavenging up to 100 mM), suggesting that phenyl dihydroxy groups were important for these compounds to show strong activity. The new compounds 1 and 2 were also examined for PPARg exciting activity, with rosiglitazone (Ros) as positive control. As shown in Figure 2, PPARg activation was observed by both green fluorescent protein (GFP) and luciferase reporter assays on 1 and 2. The cytotoxicity of the two compounds was examined against 293T cell line, using resveratrol as positive control. The cytotoxic effect was weak and the IC50 values are shown in Table 2. Several reports of neolignans such as macelignan and flaxseed lignan secoisolariciresinol diglucoside (Fukumitsu et al. 2008) had been reported to reduce hyperlipaemia, hypercholesterolaemia, hyperinsulinaemia and hyperleptinaemia in diet-induced obesity in mice. These lignans were indicated to regulate adipogenesis-related gene expressions through an increase in PPARg DNA binding activity. In addition, FS-60, a lignan-rich fraction (Kwon et al. 2011), was reported to increase glucose disposal rates and enhance hepatic insulin sensitivity by working as a PPARg agonist in type-2 diabetic rats. These facts suggested the rationality of our Table 2. Cytotoxicity (IC50) of 1 and 2 against 293T cells. Compound
IC50 (mM)
Resveratrol 1 2
. 200 103.7 115.3
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Figure 2. PPARg exciting activity of compounds 1 and 2. The 293T cells were transiently transfected with PPRE reporter constructs, or co-transfected with PPRE and PPARg constructs. For (B), pRL-CMV plasmids were also co-transfected for normalisation. After transfection, cells were incubated in the presence of solvent (DMSO) alone, Ros (10 mM, positive control) or 1 (100 mM) and 2 (100 mM) for 24 h. For (A), the level of GFP expression represents the PPARg activation of each experiment. For (B), the firefly luciferase activity of PPRE reporter divided by the renilla luciferase activity of pRL-CMV reporter represents the PPARg activation of each experiment (presented as the relative luciferase activity).
results mentioned before. Although with diverse structures, the reported neolignans had bisphenol skeleton in common. Jatrophasin C (1) and D (2) revealed a new type of neolignan with this activity. Further research on the anti-diabetic effects and this type of compounds will be executed. 3. Experimental 3.1. General UV spectra were measured from ethanolic solutions on a Shimadzu UV-2401 spectrophotometer (Shimadzu, Kyoto, Japan). Optical rotations were obtained by using JASCO-P-1020 polarimeter (JASCO, Tokyo, Japan). The H and C NMR spectra were recorded on a Bruker AV400 spectrometer (Bruker Daltonics Inc., Switzerland) operating at 400 MHz (H) and 100 MHz (C), MS spectra were measured on Bruker Esquire 2000 mass spectrometer (Bruker Daltonics Inc., Switzerland). Absorption of DPPH was determined by Molecular spectra max 340 pc microplate spectrometer (Molecular Devices, Sunnyvale, CA, USA). Fluorescence and luminescence were detected by Beckman DTX880 multimode detector (Beckman Coulter Inc., CA, USA). GFP reporter was observed by Olympus U-LH100HG fluorescence microscopy (Olympus, Tokyo, Japan). 3.2. Plant material The seeds of J. curcas L. were collected from Yunnan Province, China, and identified by chief apothecary Xiong Ying, Shenzhen Institute for drug control, Shenzhen, China. A voucher specimen (No. JC2009-3) has been deposited in Shenzhen Research Center of Traditional Chinese Medicines and Natural Products, Shenzhen, China. 3.3. Extraction and isolation The dried seeds of J. curcas L. (4.0 kg) were crushed and refluxed for 2 h with 10.0 L of 95% ethanol for three times to yield 0.8 kg of ethanol extract . The extract was suspended in 5% methanol – 95% water (8.0 L) and partitioned successively with petroleum ether (8.0 L) and nBuOH (8.0 L) to yield a petroleum ether layer (482.2 g), n-BuOH layer (48.3 g) and H2O layer (205.1 g). The 47.0 g of n-BuOH layer was chromatographed on silica gel column (80 mm £ 500 mm, 200– 300 mesh, Qingdao Haiyang Chemical, Qingdao, China), eluted with 5.0 L of
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CHCl3 – MeOH ¼ 100:0, 95:5, 90:10, 85:15, 80:20, 75:25, 50:50, 0:100 to give 15 fractions (Fr. 1 –Fr. 15). Fr. 2 (153.2 mg, 50.1% radical scavenging at 10 mg/mL, eluted by CHCl3 –MeOH 100:0) was recrystalised in MeOH to afford 3 (100 mg). Fr. 6 (280.7 mg, 43.3% radical scavenging at 10 mg/mL, eluted by CHCl3 – MeOH 85:15) was subjected to Sephadex LH-20 CC (10X70 mm) using CHCl3 – MeOH 80:20 to give Frs 6.1 –6.7, of which Fr. 6.5 (68.5 mg) was purified by repeated ODS –HPLC (Shim-pack PREP-ODS, w20 mm £ 250 mm, eluted by 35% MeOH – H2O) to give 4 (3 mL/min, tR ¼ 11.0 min, 1.2 mg), 2 (tR ¼ 18.5 min, 5.9 mg) and 1 (tR ¼ 21.5 min, 7.7 mg). Fr. 7 (5.6 g, 67.1% radical scavenging at 10 mg/mL, eluted by CHCl3 – MeOH 80:20) was separated by Sephadex LH-20 CC (40 mm £ 280 mm, eluted by CHCl3 – MeOH 80:20) to give Fr. 7.1 (4.8 g) and Fr. 7.2 (305.0 mg). Fr. 7.1 was recrystalised in MeOH to afford 5 (200.0 mg), Fr. 7.2 was separated by ODS –HPLC (Shim-pack PREP-ODS, w20 mm £ 250 mm, eluted by 35% MeOH – H2O) to give 6 (3 mL/min, tR ¼ 19.0 min, 180.0 mg). Fr. 8 (4.7 g, 33.6% radical scavenging at 10 mg/mL, eluted by CHCl3 – MeOH 80:20) was separated by Sephadex LH-20 CC (40 mm £ 280 mm, eluted by CHCl3 – MeOH 80:20), followed by repeated ODS –HPLC (Shim-pack PREP-ODS, w20 mm £ 250 mm, eluted by 30% MeOH – H2O to 100% MeOH, 70 min, linear gradient) to give 7 (3 mL/ min, tR ¼ 11.5 min, 350.0 mg), 8 (tR ¼ 18.6 min, 21.0 mg), 9 (tR ¼ 24.3 min, 4.0 mg) and 10 (tR ¼ 21.4 min, 16.4 mg). All of the isolated compounds were re-analysed by HPLC to ensure their purity to be . 95% 3.4. DPPH radical scavenging assay Samples and resveratrol (as positive control, purchased from Santa Cruz Biotech, Santa Cruz, CA, USA purity .¼ 99%) were dissolved in DMSO and diluted with a mixture of ethanol and 0.4 M HOAc –NaOAc buffer (3:1) to working concentrations. DPPH was dissolved in ethanol to make 1 mg/5 mL solution. Then, 160 mL of the sample followed by 40 mL of DPPH solution was added into a 96-well transparent plate and incubated in the dark for 30 min. The absorption at 517 nm was finally determined. DPPH scavenging activity of each sample was calculated according to the formula: scavenging % ¼ 100 £ (1 –(AS – AB)/(AC –AB)). In this formula, AS is the absorption of samples AB the absorption of blank and AC the absorption of controls, which means that ethanol and 0.4 M HOAc – NaOAc buffer were substituted for samples. 3.5. Cell culture The 293T cells were purchased from ATCC, cultured in Dulbecco’s modified eagle medium (Hyclone) with 10% FBS. Cultures were maintained in a humidified incubator at 378in 5% CO2. 3.6. Transfection and reporter assay Transient transfection was performed using Lipofectamine 2000 (Invitrogen Ltd., Paisley, UK). In brief, 1 £ 105 cells (293T) were seeded into 24-well plates. After 24 h, cells were transfected with 0.5 mg of the luciferase reporter construct (pGL-PPRE3-CMVm), 0.2 mg of the PPARg expression construct (pCM-PPARg) and 0.025 mg of pRL-CMV plasmid (Promega Ltd., Southampton, UK) for normalisation. At 3 h post-transfection, compounds and Ros (as positive control, purchased from National Institutes for Food and Drug Control, China, purity .¼ 98%) were added with DMEM containing 10% FBS. After 24 h of incubation, cells were lysed in passive lysis buffer (Promega) and luciferase activity was measured with a Dual-glo luciferase assay system (Promega). For GFP reporter assays, cells were transfected with 0.5 mg of the GFP reporter construct (pGL-PPRE3-CMVm-gfp) along with 0.2 mg of pCM-PPARg plasmid. The cell harvest and compounds treatment procedures were the same as mentioned before. The GFP fluorescence was photographed by fluorescence microscopy (Olympus).
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3.7. Cytotoxicity (fluorometric microculture cytotoxicity assay) The 293T cells (6 £ 103) were split into 96-well plates and incubated for 24 h, followed by treatment of compounds. After 24 h of incubation, cells were treated with Fluorescein diacetate (Sigma Ltd., Dorset, UK) in PBS buffer (10 mg/mL), and after 1 h of incubation, fluorescence (exitation at 485 nm and emission at 538 nm) were detected. Assays were performed at least in triplicate.
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3.8. NMR data of compounds 1 and 2 Compound 1, 1H NMR (400 MHz, CD3OD): 6.58 (1H, d, J ¼ 1.6 Hz; H-2), 6.56 (1H, d, J ¼ 1.6 Hz; H-6), 4.80 (1H, d, J ¼ 8.0 Hz; H-7), 4.00 (1H, ddd, J ¼ 8.0, 4.5, 2.3 Hz; H-8), 3.48 (1H, dd, J ¼ 12.3, 4.5 Hz; H-8), 3.68 (1H, dd, J ¼ 12.3, 2.3 Hz; H-9), 3.84 (s; 3-OCH3), 6.96 (1H, d, J ¼ 1.7 Hz; H-20 ), 6.88 (1H, d, J ¼ 8.3 Hz; H-50 ), 6.92 (1H, dd, J ¼ 8.3, 1.7 Hz; H-60 ), 6.48 (1H, d, J ¼ 15.8 Hz; H-70 ), 6.20 (1H, dt, J ¼ 15.8, 5.7 Hz; H-80 ), 4.18 (1H, d, J ¼ 5.7 Hz; H-90 ); 13C NMR (100 MHz, CD3OD): 128.8 (C-1), 104.1 (C-2), 149.8 (C-3), 135.9 (C-4), 146.8 (C-5), 109.4 (C-6), 77.9 (C-7), 80.0 (C-8), 62.1 (C-9), 56.8 (OCH3), 132.1 (C-10 ), 115.6 (C-20 ), 145.3 (C-30 ), 144.6 (C-40 ), 117.9 (C-50 ), 120.9 (C-60 ), 131.4 (C-70 ), 128.2 (C-80 ), 63.8 (C-90 ). Compound 2, 1H NMR (400 MHz, CD3OD): 6.84 (1H, d, J ¼ 1.9 Hz; H-2), 6.80 (1H, d, J ¼ 8.1 Hz; H-5), 6.75 (1H, dd, J ¼ 8.1, 1.9 Hz; H-6), 4.80 (1H, d, J ¼ 8.0 Hz; H-7), 3.96 (1H, ddd, J ¼ 8.0, 4.7, 2.5 Hz; H-8), 3.49 (1H, dd, J ¼ 12.4, 4.7 Hz; H-8), 3.69 (1H, dd, J ¼ 12.4, 2.5 Hz; H-9), 3.87 (s; 50 -OCH3), 6.61 (1H, d, J ¼ 1.9 Hz; H-20 ), 6.67 (1H, d, J ¼ 1.9 Hz; H-20 ), 6.67 (1H, d, J ¼ 1.9 Hz; H-60 ), 6.47 (1H, brd, J ¼ 15.9; H-70 ), 6.23 (1H, dt, J ¼ 15.9, 5.8 Hz; H80 ), 4.18 (1H, d, J ¼ 5.8 Hz; H-90 ); 13C NMR (100 MHz, CD3OD): 129.6 (C-1), 115.6 (C-2), 146.3 (C-3), 147.2 (C-4), 116.4 (C-5), 120.4 (C-6), 77.5 (C-7), 80.2 (C-8), 62.1 (C-9), 56.8 (OCH3), 134.3 (C-10 ), 109.2 (C-20 ), 145.8 (C-30 ), 131.2 (C-40 ), 150.2 (C-50 ); 104.1 (C-60 ); 131.6 (C-70 ), 128.6 (C-80 ), 63.7 (C-90 ). Supplementary material Supplementary material relating to this article is available online, alongside Figures S1 – S11. Acknowledgements This research was partly supported by the National Natural Science Foundation of China (No. 81001611) and Basic research projects of Shenzhen (No. 2011168). We would like to thank Jing-Hui Huang (Key Lab for New Drugs Research of TCM, Research Institute of Tsinghua University in Shenzhen, China) for recording the NMR data.
References Corton JC, Anderson SP, Stauber AC. 2000. Central role of peroxisome proliferator-activated receptors in the actions of peroxisome proliferators. Annu Rev Pharmacol Toxicol. 40:491–518. Fukumitsu S, Aida K, Ueno N, Ozawa S, Takahashi Y, Kobori M. 2008. Flaxseed lignan attenuates high-fat diet-induced fat accumulation and induces adiponectin expression in mice. Br J Nutr. 100(3):669–676. Gubitz GM, Mittelbach M, Trabi M. 1999. Exploitation of the tropical oil seed plant Jatropha curcas L. Bioresour Technol. 67:73–82. Hai L, Zhang Q, Zhao Y, Liang H, Du N. 2004. Studies on chemical constituents in the root of Hedysa rum polybot rys. China J Chin Mater Med. 29:432– 434. Kwon D, Kim D, Yang H, Park S. 2011. The lignan-rich fractions of Fructus Schisandrae improve insulin sensitivity via the PPAR-g pathways in in vitro and in vivo studies. J Ethnopharmacol. 135(2):455– 462. Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM, Kliewer SA. 1995. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor gamma (PPAR gamma). J Biol Chem. 270(22):12953– 12956.
Downloaded by [University of Newcastle, Australia] at 14:26 07 January 2015
Natural Product Research
1991
Li L, Li X, Wu H, Li R, Wang N. 2010. Chemical constituents of anti-oxidative activities from seeds of Jatropha curcas. Chin Tradit Herb Drugs. 41:1932–1936. Lumia M, Sulla jit M, Suguri H, Endo Y, Shudo K, Wongchai V, Hecker E, Fujiki H. 1988. A new tumor promoter from the seed oil of Jatropha curcas L., an intramolecular diester of 12-deoxy-16-hydroxyphorbol. Cancer Res. 48(20):5800– 5804. Okuno A, Tamemoto H, Tobe K, Ueki K, Mori Y, Iwamoto K, Umesono K, Akanuma Y, Fujiwara T, Horikoshi H, et al. 1998. Troglitazone increases the number of small adipocytes without the change of white adipose tissue mass in obese Zucker rats. J Clin Invest. 101(6):1354 –1361. Openshaw K. 2001. A review of Jatropha curcas: an oil plant of unfulfilled promise. Biomass Bioenergy. 19:1–15. Stirpe F, Pession Brizzi A, Lorenzoni E, Strochi P, Montanaro L, Sperti S. 1976. Studies on the proteins from the seeds of Croton tiglium and of Jatropha curcas. Toxic properties and inhibition of protein synthesis in vitro. Biochem J. 156(1):1–6. Toyomori N, Urada H. 2009. One-pot preparation of catechol group-introduced dioxabicyclo [3.3.0] octanes. JP patent 13174. Vamecq J, Latrue N. 1999. Medical significance of peroxisome proliferator-activated receptors. Lancet. 354 (9173):141–148. Waibel R, Benirschke G, Benirschke M. 2003. Sesquineolignans and other constituents from the seeds of Joannesia princeps. Phytochemistry. 62(5):805–811. Zhao H, Fan M, Shi J, Wang A, Li J. 2010. Isolation and structure identification of chemical constituents from seeds of Lepidium apetalum. Chin Tradit Herb Drugs. 41:14–18.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Two new lignans with antioxidative activities from Jatropha curcas ab
b
c
c
Xiaofan Li , Ling Li , Jue Wang , Tiejie Wang & Liyan Wang
a
a
Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences, Shenzhen University, Shenzhen 518060, P.R. China b
Key Lab for New Drugs Research of Traditional Chinese Medicine in Shenzhen, Research Institute of Tsinghua University in Shenzhen, High-Tech Industrial Estate Nanshan, Shenzhen 518057, P.R. China
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c
Shenzhen Institute for Drug Control, Shenzhen 518057, P.R. China Published online: 28 May 2014.
To cite this article: Xiaofan Li, Ling Li, Jue Wang, Tiejie Wang & Liyan Wang (2014) Two new lignans with antioxidative activities from Jatropha curcas, Natural Product Research: Formerly Natural Product Letters, 28:22, 1985-1991, DOI: 10.1080/14786419.2014.919284 To link to this article: http://dx.doi.org/10.1080/14786419.2014.919284
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
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systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 Vol. 28, No. 22, 1985–1991, http://dx.doi.org/10.1080/14786419.2014.919284
Two new lignans with antioxidative activities from Jatropha curcas
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Xiaofan Liab, Ling Lib, Jue Wangc, Tiejie Wangc and Liyan Wanga* a Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences, Shenzhen University, Shenzhen 518060, P.R. China; bKey Lab for New Drugs Research of Traditional Chinese Medicine in Shenzhen, Research Institute of Tsinghua University in Shenzhen, High-Tech Industrial Estate Nanshan, Shenzhen 518057, P.R. China; cShenzhen Institute for Drug Control, Shenzhen 518057, P.R. China
(Received 26 February 2014; final version received 25 April 2014) Activity-guided isolation of dried seeds of Jatropha curcas L. led to the isolation of two new lignans along with eight known compounds. These compounds were determined by spectroscopic analysis to be jatrophasin C (1), jatrophasin D (2), bsitosterol (3), jatrophasin A (4), daucosterol (5), isoamericanol A (6), (^)-3,30 bisdemethylpinoresinol (7), 70 -epi-sesamin-dicatechol (8), isoprincepin (9) and americanol A (10), of which 1 and 2 were new compounds. The antioxidative activities along with peroxisome proliferator-activated receptor gamma exciting activity of these compounds were also determined. Keywords: Jatropha curcas; Euphorbiaceae; antioxidative activity; lignan
1. Introduction Jatropha curcas L. (Euphorbiaceae) is a multipurpose plant with many attributes and considerable potential. It is widely grown in Mexico, Nicaragua, Thailand and India, and is now being promoted in southern Africa, Brazil, Mali and Nepal. Seeds of this plant have been reported to show antineoplastic, desinsection and anti-oxidative activities (Openshaw et al. 2001). The oil from its seeds has been widely used as insecticide, for soap production and most importantly as a fuel substitute (Gubitz et al. 1999). Reuse of the seed cake after extraction of oil would be an income-generating and environmental saving process. Previous constituent research of the seed reported the isolation of phorbol esters, curcin and curcain which were tumour promoting and toxic (Stirpe et al. 1976; Lumia et al. 1988). However, few reports of the chemical investigation with other activities were found. In this study, an activity-guidedisolation was performed on the 95% ethanol extract of J. curcas L. seeds. With antioxidative activity screening, two new lignans along with eight known compounds were isolated and their structures were elucidated by spectral and chemical methods. Antioxidative activity of the isolated compounds was examined using DPPH radical scavenging test. Cytotoxicity and peroxisome proliferator-activated receptor gamma (PPARg) exciting activity of the new compounds were also examined. PPAR mainly controls expression of genes participating in fat metabolism or differentiation of adipocyte, and has been shown to regulate the expression of genes by binding to DNA sequences of peroxisome proliferators response element (PPREs) (Vamecq & Latrue 1999; Corton et al. 2000). The activation of PPARg can lead to increase of pre-adipocyte differentiation, promote insulin sensitivity and elevate glucose uptake (Lehmann
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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et al. 1995; Okuno et al. 1998). Therefore, ligands of PPARg can act as medicine for treating non-insulin-dependent diabetes mellitus (type 2 diabetes mellitus) caused by insulin resistance.
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2. Results and discussion The EtOH extract of dried seeds of J. curcas L. was partitioned successively with petroleum ether and n-BuOH to yield three layers, of which the BuOH layer showed a potent antioxidative effect (80.4% radical scavenging at 25 mg/mL). This layer was then separated by repeated column chromatography (CC) and preparative HPLC to yield compounds 1 –10, consisted of two new lignans, jatrophasin C (1) and jatrophasin D (2); six known lignans, jatrophasin A (4) (Li et al. 2010), isoamericanol A (6) (Waibel et al. 2003), (^ )-3,30 -bisdemethylpinoresinol (7) (Waibel et al. 2003), 70 -epi-sesamin-dicatechol (8) (Toyomori & Urada 2009), isoprincepin (9) (Waibel et al. 2003) and americanol A (10) (Waibel et al. 2003); and two sterols, b-sitosterol (3) (Hai et al. 2004) and daucosterol (5) (Zhao et al. 2010). The structure of the known compounds was elucidated by comparison with spectral data of the reported values. Jatrophasin C (1) was obtained as a brown powder and shown to have the molecular formula C19H20O7 on the basis of HR-ESI-MS data (m/z 359.1142, calcd for 359.1136, [M 2 H]2), which indicated 10 degrees of unsaturation. The UV (ultra violet) (MeOH) spectrum exhibited absorption bands lmax (log e ) at 330.0 (3.31) and 260.4 (3.91) nm, indicating the presence of benzene ring. The 1H NMR spectrum (Table 1) showed signals of a methoxyl group at d 3.84 (3H, s), two sets of oxymethylene protons at d 3.68 (dd, J ¼ 12.3, 2.3 Hz), 3.48 (dd, 12.3, 4.5) and d 4.18 (2H, d, 5.7). The low-field resonances at d 6.96 (d, J ¼ 1.7 Hz), 6.88 (d, J ¼ 8.3 Hz) and 6.92 (dd, J ¼ 1.7, 8.3 Hz) indicated a 1,3,4-substituted benzene ring; d 6.58, 6.56 (d, J ¼ 1.6 Hz) suggested another meta-substituted benzene ring; d 6.48 (d, J ¼ 15.8 Hz) and 6.20 (dt, J ¼ 15.8, 5.7 Hz) indicated a trans-olefin group. The 13C NMR and DEPT135 spectra showed 19 signals: 1 methoxyl group (d 56.8), 2 oxymethines (d 80.0, 77.9), 2 O-bearing quaternary carbons (d 63.8, 62.1) and 14 sp2 methines (d 104.1 – 149.8). The 1H – 1H COSY cross-peaks of H7/H8, H8/H9a and H8/H9b indicated a propane segment; while cross-peaks of H70 /H80 and H80 /H90 indicated a propylene segment. These segments along with the two benzene rings accounted for nine degrees of unsaturation and suggested the 1,4-benzodioxane-type lignan skeleton. HMBC correlations from H7 to C2/C6 and H20 /H60 to C70 indicated the connection of each segment. In the NOESY spectrum, weak but significant cross-peak could be observed between H-20 and H-7, indicating the structure as shown in Figure 1. The relationship of H-7 and H-8 was assigned as trans according to the coupling constant (J7,8 ¼ 8.0). Compound 1 showed no optical rotation or Circular Dichroism (CD) cotton curve, indicating a racemic mixture. Table 1. DPPH radical scavenging activity (IC50) of isolated compounds, with resveratrol as positive control. Compound
IC50 (mM)
Resveratrol 1 2 4 6 7 8 9 10
40.67 42.98 9.71 22.85 5.28 10.49 9.18 7.23 8.28
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Figure 1. Structures (left), as well as COSY, key HMBC ( compounds 1 2 isolated from J. curcas.
) and NOESY (
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Jatrophasin D (2) was obtained as a brown powder and shown to have the molecular formula C19H20O7 on the basis of HR-ESI-MS data (m/z 383.1106, calcd for 383.1101, [M þ Na]þ). The UV (MeOH) spectrum exhibited absorption bands lmax (log e) at 222.5 (4.60) and 274.5 (4.23) nm, indicating the presence of benzene ring. The 1H and 13C NMR spectra showed signals (Table 1) similar to those of jatrophasin C (1), indicating 1,4-benzodioxane-type lignan skeleton. The key HMBC correlation of d 3.87 (H-100 ) to d 150.2(C-50 ) suggested that the methyl group linked to C-50 position. All of the 1H and 13C NMR spectral signals of 2 were properly assigned based on the HMQC, HMBC and 1H – 1H COSY spectra (Table 1). The relationship of H-7 and H-8 was assigned as trans according to the coupling constant (J7,8 ¼ 8.0). Compound 2 showed no optical rotation or CD cotton curve, indicating a racemic mixture. All of the isolated compounds as well as resveratrol (used as positive control) were tested for antioxidative activity using DPPH scavenging assay. As shown in Table 2, compound 6 showed comparatively strong activity, compounds 2, 7– 10 showed medium activity, while compounds 3 and 5 showed almost no activity (, 20% DPPH radical scavenging up to 100 mM), suggesting that phenyl dihydroxy groups were important for these compounds to show strong activity. The new compounds 1 and 2 were also examined for PPARg exciting activity, with rosiglitazone (Ros) as positive control. As shown in Figure 2, PPARg activation was observed by both green fluorescent protein (GFP) and luciferase reporter assays on 1 and 2. The cytotoxicity of the two compounds was examined against 293T cell line, using resveratrol as positive control. The cytotoxic effect was weak and the IC50 values are shown in Table 2. Several reports of neolignans such as macelignan and flaxseed lignan secoisolariciresinol diglucoside (Fukumitsu et al. 2008) had been reported to reduce hyperlipaemia, hypercholesterolaemia, hyperinsulinaemia and hyperleptinaemia in diet-induced obesity in mice. These lignans were indicated to regulate adipogenesis-related gene expressions through an increase in PPARg DNA binding activity. In addition, FS-60, a lignan-rich fraction (Kwon et al. 2011), was reported to increase glucose disposal rates and enhance hepatic insulin sensitivity by working as a PPARg agonist in type-2 diabetic rats. These facts suggested the rationality of our Table 2. Cytotoxicity (IC50) of 1 and 2 against 293T cells. Compound
IC50 (mM)
Resveratrol 1 2
. 200 103.7 115.3
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Figure 2. PPARg exciting activity of compounds 1 and 2. The 293T cells were transiently transfected with PPRE reporter constructs, or co-transfected with PPRE and PPARg constructs. For (B), pRL-CMV plasmids were also co-transfected for normalisation. After transfection, cells were incubated in the presence of solvent (DMSO) alone, Ros (10 mM, positive control) or 1 (100 mM) and 2 (100 mM) for 24 h. For (A), the level of GFP expression represents the PPARg activation of each experiment. For (B), the firefly luciferase activity of PPRE reporter divided by the renilla luciferase activity of pRL-CMV reporter represents the PPARg activation of each experiment (presented as the relative luciferase activity).
results mentioned before. Although with diverse structures, the reported neolignans had bisphenol skeleton in common. Jatrophasin C (1) and D (2) revealed a new type of neolignan with this activity. Further research on the anti-diabetic effects and this type of compounds will be executed. 3. Experimental 3.1. General UV spectra were measured from ethanolic solutions on a Shimadzu UV-2401 spectrophotometer (Shimadzu, Kyoto, Japan). Optical rotations were obtained by using JASCO-P-1020 polarimeter (JASCO, Tokyo, Japan). The H and C NMR spectra were recorded on a Bruker AV400 spectrometer (Bruker Daltonics Inc., Switzerland) operating at 400 MHz (H) and 100 MHz (C), MS spectra were measured on Bruker Esquire 2000 mass spectrometer (Bruker Daltonics Inc., Switzerland). Absorption of DPPH was determined by Molecular spectra max 340 pc microplate spectrometer (Molecular Devices, Sunnyvale, CA, USA). Fluorescence and luminescence were detected by Beckman DTX880 multimode detector (Beckman Coulter Inc., CA, USA). GFP reporter was observed by Olympus U-LH100HG fluorescence microscopy (Olympus, Tokyo, Japan). 3.2. Plant material The seeds of J. curcas L. were collected from Yunnan Province, China, and identified by chief apothecary Xiong Ying, Shenzhen Institute for drug control, Shenzhen, China. A voucher specimen (No. JC2009-3) has been deposited in Shenzhen Research Center of Traditional Chinese Medicines and Natural Products, Shenzhen, China. 3.3. Extraction and isolation The dried seeds of J. curcas L. (4.0 kg) were crushed and refluxed for 2 h with 10.0 L of 95% ethanol for three times to yield 0.8 kg of ethanol extract . The extract was suspended in 5% methanol – 95% water (8.0 L) and partitioned successively with petroleum ether (8.0 L) and nBuOH (8.0 L) to yield a petroleum ether layer (482.2 g), n-BuOH layer (48.3 g) and H2O layer (205.1 g). The 47.0 g of n-BuOH layer was chromatographed on silica gel column (80 mm £ 500 mm, 200– 300 mesh, Qingdao Haiyang Chemical, Qingdao, China), eluted with 5.0 L of
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CHCl3 – MeOH ¼ 100:0, 95:5, 90:10, 85:15, 80:20, 75:25, 50:50, 0:100 to give 15 fractions (Fr. 1 –Fr. 15). Fr. 2 (153.2 mg, 50.1% radical scavenging at 10 mg/mL, eluted by CHCl3 –MeOH 100:0) was recrystalised in MeOH to afford 3 (100 mg). Fr. 6 (280.7 mg, 43.3% radical scavenging at 10 mg/mL, eluted by CHCl3 – MeOH 85:15) was subjected to Sephadex LH-20 CC (10X70 mm) using CHCl3 – MeOH 80:20 to give Frs 6.1 –6.7, of which Fr. 6.5 (68.5 mg) was purified by repeated ODS –HPLC (Shim-pack PREP-ODS, w20 mm £ 250 mm, eluted by 35% MeOH – H2O) to give 4 (3 mL/min, tR ¼ 11.0 min, 1.2 mg), 2 (tR ¼ 18.5 min, 5.9 mg) and 1 (tR ¼ 21.5 min, 7.7 mg). Fr. 7 (5.6 g, 67.1% radical scavenging at 10 mg/mL, eluted by CHCl3 – MeOH 80:20) was separated by Sephadex LH-20 CC (40 mm £ 280 mm, eluted by CHCl3 – MeOH 80:20) to give Fr. 7.1 (4.8 g) and Fr. 7.2 (305.0 mg). Fr. 7.1 was recrystalised in MeOH to afford 5 (200.0 mg), Fr. 7.2 was separated by ODS –HPLC (Shim-pack PREP-ODS, w20 mm £ 250 mm, eluted by 35% MeOH – H2O) to give 6 (3 mL/min, tR ¼ 19.0 min, 180.0 mg). Fr. 8 (4.7 g, 33.6% radical scavenging at 10 mg/mL, eluted by CHCl3 – MeOH 80:20) was separated by Sephadex LH-20 CC (40 mm £ 280 mm, eluted by CHCl3 – MeOH 80:20), followed by repeated ODS –HPLC (Shim-pack PREP-ODS, w20 mm £ 250 mm, eluted by 30% MeOH – H2O to 100% MeOH, 70 min, linear gradient) to give 7 (3 mL/ min, tR ¼ 11.5 min, 350.0 mg), 8 (tR ¼ 18.6 min, 21.0 mg), 9 (tR ¼ 24.3 min, 4.0 mg) and 10 (tR ¼ 21.4 min, 16.4 mg). All of the isolated compounds were re-analysed by HPLC to ensure their purity to be . 95% 3.4. DPPH radical scavenging assay Samples and resveratrol (as positive control, purchased from Santa Cruz Biotech, Santa Cruz, CA, USA purity .¼ 99%) were dissolved in DMSO and diluted with a mixture of ethanol and 0.4 M HOAc –NaOAc buffer (3:1) to working concentrations. DPPH was dissolved in ethanol to make 1 mg/5 mL solution. Then, 160 mL of the sample followed by 40 mL of DPPH solution was added into a 96-well transparent plate and incubated in the dark for 30 min. The absorption at 517 nm was finally determined. DPPH scavenging activity of each sample was calculated according to the formula: scavenging % ¼ 100 £ (1 –(AS – AB)/(AC –AB)). In this formula, AS is the absorption of samples AB the absorption of blank and AC the absorption of controls, which means that ethanol and 0.4 M HOAc – NaOAc buffer were substituted for samples. 3.5. Cell culture The 293T cells were purchased from ATCC, cultured in Dulbecco’s modified eagle medium (Hyclone) with 10% FBS. Cultures were maintained in a humidified incubator at 378in 5% CO2. 3.6. Transfection and reporter assay Transient transfection was performed using Lipofectamine 2000 (Invitrogen Ltd., Paisley, UK). In brief, 1 £ 105 cells (293T) were seeded into 24-well plates. After 24 h, cells were transfected with 0.5 mg of the luciferase reporter construct (pGL-PPRE3-CMVm), 0.2 mg of the PPARg expression construct (pCM-PPARg) and 0.025 mg of pRL-CMV plasmid (Promega Ltd., Southampton, UK) for normalisation. At 3 h post-transfection, compounds and Ros (as positive control, purchased from National Institutes for Food and Drug Control, China, purity .¼ 98%) were added with DMEM containing 10% FBS. After 24 h of incubation, cells were lysed in passive lysis buffer (Promega) and luciferase activity was measured with a Dual-glo luciferase assay system (Promega). For GFP reporter assays, cells were transfected with 0.5 mg of the GFP reporter construct (pGL-PPRE3-CMVm-gfp) along with 0.2 mg of pCM-PPARg plasmid. The cell harvest and compounds treatment procedures were the same as mentioned before. The GFP fluorescence was photographed by fluorescence microscopy (Olympus).
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3.7. Cytotoxicity (fluorometric microculture cytotoxicity assay) The 293T cells (6 £ 103) were split into 96-well plates and incubated for 24 h, followed by treatment of compounds. After 24 h of incubation, cells were treated with Fluorescein diacetate (Sigma Ltd., Dorset, UK) in PBS buffer (10 mg/mL), and after 1 h of incubation, fluorescence (exitation at 485 nm and emission at 538 nm) were detected. Assays were performed at least in triplicate.
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3.8. NMR data of compounds 1 and 2 Compound 1, 1H NMR (400 MHz, CD3OD): 6.58 (1H, d, J ¼ 1.6 Hz; H-2), 6.56 (1H, d, J ¼ 1.6 Hz; H-6), 4.80 (1H, d, J ¼ 8.0 Hz; H-7), 4.00 (1H, ddd, J ¼ 8.0, 4.5, 2.3 Hz; H-8), 3.48 (1H, dd, J ¼ 12.3, 4.5 Hz; H-8), 3.68 (1H, dd, J ¼ 12.3, 2.3 Hz; H-9), 3.84 (s; 3-OCH3), 6.96 (1H, d, J ¼ 1.7 Hz; H-20 ), 6.88 (1H, d, J ¼ 8.3 Hz; H-50 ), 6.92 (1H, dd, J ¼ 8.3, 1.7 Hz; H-60 ), 6.48 (1H, d, J ¼ 15.8 Hz; H-70 ), 6.20 (1H, dt, J ¼ 15.8, 5.7 Hz; H-80 ), 4.18 (1H, d, J ¼ 5.7 Hz; H-90 ); 13C NMR (100 MHz, CD3OD): 128.8 (C-1), 104.1 (C-2), 149.8 (C-3), 135.9 (C-4), 146.8 (C-5), 109.4 (C-6), 77.9 (C-7), 80.0 (C-8), 62.1 (C-9), 56.8 (OCH3), 132.1 (C-10 ), 115.6 (C-20 ), 145.3 (C-30 ), 144.6 (C-40 ), 117.9 (C-50 ), 120.9 (C-60 ), 131.4 (C-70 ), 128.2 (C-80 ), 63.8 (C-90 ). Compound 2, 1H NMR (400 MHz, CD3OD): 6.84 (1H, d, J ¼ 1.9 Hz; H-2), 6.80 (1H, d, J ¼ 8.1 Hz; H-5), 6.75 (1H, dd, J ¼ 8.1, 1.9 Hz; H-6), 4.80 (1H, d, J ¼ 8.0 Hz; H-7), 3.96 (1H, ddd, J ¼ 8.0, 4.7, 2.5 Hz; H-8), 3.49 (1H, dd, J ¼ 12.4, 4.7 Hz; H-8), 3.69 (1H, dd, J ¼ 12.4, 2.5 Hz; H-9), 3.87 (s; 50 -OCH3), 6.61 (1H, d, J ¼ 1.9 Hz; H-20 ), 6.67 (1H, d, J ¼ 1.9 Hz; H-20 ), 6.67 (1H, d, J ¼ 1.9 Hz; H-60 ), 6.47 (1H, brd, J ¼ 15.9; H-70 ), 6.23 (1H, dt, J ¼ 15.9, 5.8 Hz; H80 ), 4.18 (1H, d, J ¼ 5.8 Hz; H-90 ); 13C NMR (100 MHz, CD3OD): 129.6 (C-1), 115.6 (C-2), 146.3 (C-3), 147.2 (C-4), 116.4 (C-5), 120.4 (C-6), 77.5 (C-7), 80.2 (C-8), 62.1 (C-9), 56.8 (OCH3), 134.3 (C-10 ), 109.2 (C-20 ), 145.8 (C-30 ), 131.2 (C-40 ), 150.2 (C-50 ); 104.1 (C-60 ); 131.6 (C-70 ), 128.6 (C-80 ), 63.7 (C-90 ). Supplementary material Supplementary material relating to this article is available online, alongside Figures S1 – S11. Acknowledgements This research was partly supported by the National Natural Science Foundation of China (No. 81001611) and Basic research projects of Shenzhen (No. 2011168). We would like to thank Jing-Hui Huang (Key Lab for New Drugs Research of TCM, Research Institute of Tsinghua University in Shenzhen, China) for recording the NMR data.
References Corton JC, Anderson SP, Stauber AC. 2000. Central role of peroxisome proliferator-activated receptors in the actions of peroxisome proliferators. Annu Rev Pharmacol Toxicol. 40:491–518. Fukumitsu S, Aida K, Ueno N, Ozawa S, Takahashi Y, Kobori M. 2008. Flaxseed lignan attenuates high-fat diet-induced fat accumulation and induces adiponectin expression in mice. Br J Nutr. 100(3):669–676. Gubitz GM, Mittelbach M, Trabi M. 1999. Exploitation of the tropical oil seed plant Jatropha curcas L. Bioresour Technol. 67:73–82. Hai L, Zhang Q, Zhao Y, Liang H, Du N. 2004. Studies on chemical constituents in the root of Hedysa rum polybot rys. China J Chin Mater Med. 29:432– 434. Kwon D, Kim D, Yang H, Park S. 2011. The lignan-rich fractions of Fructus Schisandrae improve insulin sensitivity via the PPAR-g pathways in in vitro and in vivo studies. J Ethnopharmacol. 135(2):455– 462. Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM, Kliewer SA. 1995. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor gamma (PPAR gamma). J Biol Chem. 270(22):12953– 12956.
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1991
Li L, Li X, Wu H, Li R, Wang N. 2010. Chemical constituents of anti-oxidative activities from seeds of Jatropha curcas. Chin Tradit Herb Drugs. 41:1932–1936. Lumia M, Sulla jit M, Suguri H, Endo Y, Shudo K, Wongchai V, Hecker E, Fujiki H. 1988. A new tumor promoter from the seed oil of Jatropha curcas L., an intramolecular diester of 12-deoxy-16-hydroxyphorbol. Cancer Res. 48(20):5800– 5804. Okuno A, Tamemoto H, Tobe K, Ueki K, Mori Y, Iwamoto K, Umesono K, Akanuma Y, Fujiwara T, Horikoshi H, et al. 1998. Troglitazone increases the number of small adipocytes without the change of white adipose tissue mass in obese Zucker rats. J Clin Invest. 101(6):1354 –1361. Openshaw K. 2001. A review of Jatropha curcas: an oil plant of unfulfilled promise. Biomass Bioenergy. 19:1–15. Stirpe F, Pession Brizzi A, Lorenzoni E, Strochi P, Montanaro L, Sperti S. 1976. Studies on the proteins from the seeds of Croton tiglium and of Jatropha curcas. Toxic properties and inhibition of protein synthesis in vitro. Biochem J. 156(1):1–6. Toyomori N, Urada H. 2009. One-pot preparation of catechol group-introduced dioxabicyclo [3.3.0] octanes. JP patent 13174. Vamecq J, Latrue N. 1999. Medical significance of peroxisome proliferator-activated receptors. Lancet. 354 (9173):141–148. Waibel R, Benirschke G, Benirschke M. 2003. Sesquineolignans and other constituents from the seeds of Joannesia princeps. Phytochemistry. 62(5):805–811. Zhao H, Fan M, Shi J, Wang A, Li J. 2010. Isolation and structure identification of chemical constituents from seeds of Lepidium apetalum. Chin Tradit Herb Drugs. 41:14–18.
