Article pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Adiponectin-Secretion-Promoting Phenylethylchromones from the Agarwood of Aquilaria malaccensis Sungjin Ahn,†,∥ Chi Thanh Ma,‡,∥ Jung Min Choi,† Seungchan An,† Moonyoung Lee,† Thi Hong Van Le,‡ Jeong Joo Pyo,† Joochang Lee,†,⊥ Min Sik Choi,§ Sung Won Kwon,† Jeong Hill Park,† and Minsoo Noh*,†
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Natural Products Research Institute, College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea ‡ Department of Pharmacognosy, University of Medicine and Pharmacy at Ho Chi Minh City, Ho Chi Minh City, 700000, Vietnam § College of Pharmacy, Dongduk Women’s University, Seoul 02748, Korea S Supporting Information *
ABSTRACT: The therapeutic potential of adiponectin regulation has received interest because of its association with diverse human disease conditions, such as diabetes, obesity, atherosclerosis, and cancer. Phenylethylchromone derivatives from Aquilaria malaccensis-derived agarwood promoted adiponectin secretion during adipogenesis in human bone marrow mesenchymal stem cells, and 5,6dihydroxy-2-(2-phenylethyl)chromone (1) was identified as a new chromone derivative. A target identification study with the most potent adiponectin-secretion-promoting phenylethylchromones, 6-methoxy-2-(2-phenylethyl)chromone (3) and 7-methoxy-2-(2-phenylethyl)chromone (4), showed that they are PPARγ partial agonists. Therefore, the diverse therapeutic effects of agarwood are associated with a PPARγ-mediated adiponectin-secretion-promoting mechanism.
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sclerosis in mice.6 The administration of adiponectin also inhibited pathogenic fibrosis in mice, inducing alcoholic and nonalcoholic fatty liver diseases.7 Therefore, novel adiponectin secretion regulators are being sought actively for the development of new drugs, because adiponectin has great therapeutic potential in various human diseases, such as obesity, type 2 diabetes, atherosclerosis, and fatty liver.8 It is advantageous to use cell-based phenotypic assays when screening for and studying adiponectin-secretion-promoting compounds, because adiponectin secretion is regulated by various molecular mechanisms. Phenotypic assays for the discovery of adiponectin secretion modulators have been developed using 3T3-L1 preadipocytes or human mesenchymal stem cells (hMSCs).9,10 Adipogenesis in an hMSC-based assay system includes earlier differentiation processes such as the lineage commitment of MSCs to preadipocytes, in contrast to that of murine preadipocyte cell lines, because MSCs can give rise to cells of multiple differentiation lineages.11 In this regard, phenotypic assays using hMSCs have more molecular target coverage for the discovery of novel compounds to promote adiponectin secretion.
garwood is a fragrant resin-containing natural product that is formed during plant defense responses against pathogen-induced injuries in the bark of Aquilaria species, which taxonomically belong to the family Thymelaeaceae. Agarwood has been used in traditional medicine systems of Asian countries, including mainland China, India, Korea, and Japan, to relieve the symptoms of various pathologies, such as gastrointestinal problems, asthma, pain, and fever. The diverse potential therapeutic effects of agarwood extracts or agarwoodderived compounds have been suggested to include potential antidiabetic, anticancer, antiallergic, antinociceptive, and sedative activities.1 However, the pharmacological mechanisms of these diverse therapeutic activities have not yet been fully elucidated. Adiponectin, an adipocyte-secreted hormone, plays an important role in cellular metabolism and has attracted therapeutic attention because of its significant association with various diseases such as obesity, diabetes, atherosclerosis, and cancer.2,3 In general, the serum adiponectin levels in this patient population are lower compared to those of a healthy population. In obese and diabetic patients, the serum ratio of adiponectin to leptin is lower than that of healthy individuals. Moreover, insulin sensitivity was improved when exogenous adiponectin was administered to diabetic mice.4,5 Adiponectin, in addition, improved the pathogenic outcome of athero© XXXX American Chemical Society and American Society of Pharmacognosy
Received: July 31, 2018
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DOI: 10.1021/acs.jnatprod.8b00635 J. Nat. Prod. XXXX, XXX, XXX−XXX
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confirmed the two hydroxy groups as being located at C-5 (δC 135.6) and C-6 (δC 135.0). Thus, the structure of compound 1 was elucidated as 5,6-dihydroxy-2-(2phenylethyl)chromone. On the basis of the physical and spectroscopic data of reference compounds reported in the literature, 14 known 2(2-phenylethyl)chromone derivatives were identified, namely, 2-(2-phenylethyl)chromone (2), 1 2 6-methoxy-2-(2phenylethyl)chromone (3),13 7-methoxy-2-(2-phenylethyl)chromone (4),14 2-[2-(4-methoxyphenyl)ethyl)]chromone (5),12 6,7-dimethoxy-2-(2-phenylethyl)chromone (6),13 6methoxy-2-[2-(3-methoxyphenyl)ethyl]chromone (7),13 5-hydroxy-6-methoxy-2-(2-phenylethyl)chromone (8),15 6-hydroxy-2-[2-(4-methoxyphenyl)ethyl]chromone (9),16 6-hydroxy-2-(2-phenylethyl)chromone (10),13 8-hydroxy-2-(2phenylethyl)chromone (11),17 2-[2-(2-hydroxyphenyl)ethyl]chromone (12),16 8-chloro-6-hydroxy-2-(2-phenylethyl)chromone (13),18 rel-(1aR,2R,3R,7bS)-1a,2,3,7b-tetrahydro2,3-dihydroxy-5-(2-phenylethyl)-7H-oxireno[f ][1]benzopyran-7-one (14),19 and 5-hydroxy-2-(2-phenylethyl)chromone (15).20 Among the known chromone compounds 9 and 11−13 were isolated from A. malaccensis-derived agarwood for the first time, while compound 15 has not been found in the genus Aquilaria previously. In the hBM-MSC differentiation model, 12 phenylethylchromone compounds at 30 μM significantly increased adiponectin secretion during adipogenesis compared to that induced by the IDX control (Figure 2A). However, compounds 9, 11, and 12, did not change adiponectin production in hBM-MSCs. Among 12 bioactive compounds, the concentration-dependent effects of the four most potent bioactive compounds were evaluated (Figure 2B). To calculate effective concentration 50 (EC50) values, pioglitazone, a clinically available peroxisome proliferator-activated receptor γ (PPARγ) agonist, was used as a reference agonist to determine the 100% response value. In dose−response curve analysis, the EC50 values of compounds 2, 3, and 4 were 25.3, 16.2, and 20.3 μM, respectively. Compound 1 upregulated significantly adiponectin production by 1.97-fold compared to that of the IDX control. However, the adiponectin-secretionpromoting activity of 1 by 60 μM was 48% of the maximal pioglitazone-induced response (Figure 2B). In mammalian adipocytes, adiponectin secretion is increased primarily upon the activation of PPARγ.21 In addition, other nuclear hormone receptors such as PPARα, PPARδ, glucocorticoid receptor (GR), and liver X receptor (LXR) have been known to induce adiponectin secretion.11,22 Chromone compounds 2−4 were tested for their ability to directly bind to GR, PPARα, PPARδ, PPARγ, or LXR. In a
In a preliminary screen of various natural products, the methanol extract of an agarwood formed in Aquilaria malaccensis Lam. promoted adiponectin production during adipogenesis in hBM-MSCs (Figure S1A, Supporting Information). Since an A. malaccensis-derived agarwood methanol extract increased adiponectin production, pharmacological mechanisms that explain the traditional uses of an agarwood in diverse human metabolic and inflammatory diseases may be associated with its adiponectin-secretionpromoting activity. Herein, a bioactivity-guided isolation study of adiponectin-secretion-promoting compounds using A. malaccensis-derived agarwood was performed (Figure S1B, Supporting Information).
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RESULTS AND DISCUSSION A novel chromone derivative, compound 1, was obtained as a yellowish oil. The positive-ion HRESIMS displayed an ion peak [M + H]+ at m/z 283.0958 (calcd for C17H15O4, 283.0970), consistent with the molecular formula C17H14O4, implying 11 degrees of unsaturation (Figure S2, Supporting Information). The IR spectrum of 1 showed absorptions for hydroxy (3402 cm−1) and α,β-unsaturated carbonyl groups (1704 cm−1) (Figure S3, Supporting Information). The 1H NMR spectrum of 1 revealed the presence of four consecutive aromatic protons (δH 7.20−7.31), two methylenes (δH 3.06, 2.98), a singlet olefinic proton (δH 6.08), and the signal of a pair of ortho-coupled aromatic protons (δH 7.11, 6.66, each d, J = 8.8 Hz) (Figure S4, Supporting Information). The 13C NMR spectrum displayed 17 signals, including a ketocarbonyl group (δC 183.1) and four oxygenated carbons (δC 169.1, 153.3, 143.7, 135.0) (Figure S5, Supporting Information). These data supported compound 1 as being a 2-(2-phenylethyl)chromone derivative with two hydroxy groups. In the HMBC spectrum (Figures 1 and S6, Supporting Information), the singlet signal of proton H-3 showed correlations with carbons C-2/C-4/C10/C-8′, together with correlations observed from H-7 to C-5/ C-6/C-9 and from H-8 to C-4/C-6/C-9/C-10, which
Figure 1. HMBC, 1H−1H COSY, and NOESY correlations of compound 1. B
DOI: 10.1021/acs.jnatprod.8b00635 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 2. Adiponectin-secretion-promoting and PPARγ-binding activities of phenylethylchromone compounds purified from A. malaccensis-derived agarwood. (A) Phenylethychromone compounds (30 μM) were added to IDX medium when adipogenesis was induced in hBM-MSCs. On the third day of the differentiation process, adipogenic media with the phenylethylchromone compounds were exchanged. On the fifth day, cell culture supernatants were harvested and adiponectin concentrations were measured. (B) The concentration dependency of phenylethylchromine compounds 1−4 on adiponectin-secretion-promoting activity was determined. (C) TR-FRET competitive binding assays with phenylethylchromone compounds 2−4 were performed for target deconvolution against GR, PPARα, PPARγ, and PPARδ. The receptor coactivation analysis for LXRα was analyzed. (D) TR-FRET-based competitive binding activities of 15 phenylethylchromone compounds to PPARγ were measured. (E) Pearson’s correlation coefficient (r2) between the PPARγ-binding affinities and relative adiponectin-secretion-promoting activities of phenylethylchromone compounds was calculated. Values represent means ± SD (n = 3); *p ≤ 0.05 and **p ≤ 0.01. IDX, adipogenesis-inducing media consisting of insulin, dexamethasone and 3-isobutyl-1-methylxanthine; Asp, aspirin; Gli, glibenclamide; Pio, pioglitazone.
time-resolved fluorescence resonance energy transfer (TRFRET)-based receptor binding assay, compounds 2−4 significantly replaced the binding of the labeled PPARγ ligand by 37.7%, 52.8%, and 50.3%, respectively (Figure 2C). Phenylethylchromone compounds 2−4 had no significant effects on GR, PPARα, PPARδ, or LXRα. Next, the PPARγbinding activity of the 12 active chromone compounds was measured (Figure 2D). In the Pearson correlation analysis between the level of competitive binding to PPARγ and adiponectin-secretion-promoting activity at 30 μM for each chromone compound, the r2 value was 0.73 (p < 0.01) (Figure 2E). Regarding the correlation coefficient, the adiponectinsecretion-promoting activity of the chromones isolated from A. malaccensis-derived agarwood was significantly associated with PPARγ binding affinity. In the concentration-dependent PPARγ binding study, compounds 2−4 bound PPARγ in a concentration-dependent manner with Ki values of 54.0, 18.1, and 14.9 μM, respectively; however, these compounds were not as potent as the clinically available PPARγ agonist pioglitazone (Ki = 0.062 μM) or the PPARγ-binding sulfonylurea antidiabetic drug glibenclamide (Ki = 0.66 μM) (Figure 3A). The value for compound 1 was not calculated because it replaced only 41.1% of the labeled PPARγ ligand binding at 60 μM, which was the highest concentration tested in the TR-FRET-based concentration-dependency assay. Both the correlation coefficient and concentration−response were consistent with the chromone compounds promoting adiponectin secretion during adipogenesis in hBM-MSCs via a PPARγ-dependent mechanism.23 To further understand the binding mode of phenylethylchromones, the molecular docking simulation of compound 4 to the PPARγ ligand binding domain (LBD) was performed using a rivoglitazone-bound crystal structure of
PPARγ (PDB code 5TWO).24 The docking model of compound 4 to the PPARγ-LBD was compared to that of pioglitazone, a full PPARγ agonist. The binding free energy of the pioglitazone docking model was −9.1 kcal/mol in the AutoDock Vina analysis and −39.0 kcal/mol for the CDOCKER energy methods (Figure 3B). The free energy scores of the docking models for purified 15 phenylethylchromone compounds in the AutoDock Vina analysis showed a similar tendency to their adiponectin-secretionpromoting activity (Figure S11A, Supporting Information). When correlated to the receptor binding affinity, the free energy scores for compound 4 were −8.4 and −23.7 kcal/mol for the AutoDock and CDOCKER analyses, respectively (Figure 3C). The ligand binding pocket (LBP) of PPARγ has been described as having three branches that give rise to a Y-shaped cavity with the potential to accommodate chemically diverse endogenous or exogenous ligands.25 Similar to the rivoglitazone-bound 5TWO crystal structure, pioglitazone adopts a horseshoe-shaped binding conformation that is centered around helix 3 (H3) of the PPARγ-LBD (Figure 3B). Pioglitazone forms hydrogen bonds and hydrophobic interactions with various amino acid residues, such as isoleucine (Ile) 262 (near Ω-loop), lysine (Lys) 263 (Ωloop), cysteine (Cys) 285 (H3), phenylalanine (Phe) 287 (H3), arginine (Arg) 288 (H3), histidine (His) 323 (H5), leucine (Leu) 330 (H5), Ile341 (β-sheet s3), and tyrosine (Tyr) 473 (H12), in both the hydrophilic and hydrophobic branches of the PPARγ LBP (Figure 3B). The docking model showed that the thiazolidinedione group of pioglitazone and the Tyr 473 residue in H12 formed a π−sulfur interaction as well as hydrogen bonding. Ligand interactions with Tyr473 (H12) are known to induce the stabilization of the coactivator protein interaction surface of PPARγ, which is known as the C
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activation function-2 (AF-2) surface and is important in the recruitment of transcriptional coactivators.24,26 In contrast to the pioglitazone-PPARγ-LBD docking model, compound 4 did not interact with Tyr473 in H12. The docking models with the lowest favorable free energy level showed that compound 4 occupies the hydrophobic PPARγ-LBP. In the internal hydrophobic LBP, compound 4 forms hydrogen bonds with serine 289 residue in H3 and also assumes hydrophobic interactions with Cys285 (H3), alanine (Ala) 292 (H3), Ile326 (H5), Leu330 (H5), and Ile341 (s3) (Figure 3C). These results indicated that compound 4 occupied the region between H3 and the β-sheet where most PPARγ partial agonists are found to reside in ligand-PPARγ LBD structures.10,27,28 Like compound 4, the docking model of compound 3 with the lowest favorable free energy showed a similar pattern to those of PPARγ partial agonists (Figure S11D, Supporting Information). Although the docking model implied the PPARγ partial agonism of phenylethylchromones, experimental validation was needed. To validate the PPARγ partial agonism, pioglitazone was coadministered with compound 4 during adipogenesis in hBM-MSCs. By definition, a partial agonist plays the role as an antagonist against a full agonist.10 When adiponectin production was measured in hBM-MSCs, compound 4 functionally antagonized the effects of pioglitazone on adiponectin production during adipogenesis, demonstrating that these adiponectin-secretion-promoting chromone compounds are PPARγ partial agonists (Figure 3D). Compound 4 promoted adiponectin production, and simultaneously it antagonized the effect of a PPARγ full agonist. Compound 3 also showed a PPARγ partial agonist effect on the pioglitazone-treated hBM-MSCs (Figure S12A, Supporting Information). Conclusively, phenylethylchromone compounds purified from A. malaccensis-derived agarwood promote adiponectin secretion during adipogenesis in hBMMSCs through PPARγ partial agonism. Thiazolidinedione (TZD) PPARγ full agonists such as rosiglitazone, troglitazone, and pioglitazone are PPARγ full agonists that can improve insulin sensitivity. The adverse effects of TZD PPARγ full agonists that ultimately led to market withdrawal include weight gain, renal fluid retention, hepatitis, and an increased risk of cardiovascular events. Major endogenous PPARγ ligand candidates such as fatty acids and prostanoids have a far weaker agonistic activity than TZD PPARγ full agonists. Recently, selective PPARγ modulators like PPARγ partial agonists that are as potent as endogenous PPARγ ligands have been suggested as possible new therapeutic drugs that may exhibit reduced adverse effects. In this regard, the adiponectin-secretion-promoting phenylethylchromones identified from A. malaccensis-derived agarwood provide a pharmacophore for selective PPARγ modulators, which could mitigate or avoid the adverse effects of therapeutic PPARγ full agonists.
Figure 3. Evaluation of the PPARγ partial agonism of phenylethylchromone compounds. (A) TR-FRET-based competitive binding activities of phenylethylchromone compounds to PPARγ were determined. Ki values for compounds 2−4 were calculated with the Cheng and Prusoff equation. Molecular docking simulations of pioglitazone (B) and 4 (C) to the structure of PPARγ-LBD (PDB 5TWO) were performed using Autodock Vina version 1.1.2. Free energy scores were calculated using both Autodock Vina and Accelrys Discovery Studio software. Helix numbering follows the convention used for PPARγ-LBD.25 (D) Experimental validation of the PPARγ partial agonism of compound 4. hBM-MSCs were differentiated in IDX media and incubated with compound 4 in the presence of pioglitazone. On the third day of the induction process, the adipogenic media containing the compounds were exchanged. On the fifth day, cell culture supernatants were harvested and adiponectin levels in supernatants were measured by ELISA. Values represent means ± SD (n = 3); *p ≤ 0.05 and **p ≤ 0.01. Pio, pioglitazone; Gliben; glibenclamide.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotation values were measured on a JASCO P-2000 polarimeter (Easton, MD, USA) at 20 °C. Ultraviolet (UV) spectra were measured on a Chirascan plus (Applied Photophysics Leatherhead, UK). FT-IR spectra were recorded on a JASCO FT/IR-4200 spectrometer. NMR spectra were recorded on a Bruker AVANCE (500 MHz) and Bruker ASCENE (800 MHz) FT-NMR spectrometer (Hamburg, Germany); NMR solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA). HRESIMS were measured on an Agilent 6350 Q-TOF mass spectrometer (Agilent, Santa Clara, CA, USA). LC analyses were D
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(MeOH/H2O, 80:20, flow rate 2 mL min−1, v/v) to yield compounds 11 (14.1 mg) and 13 (32.1 mg). Fractions EA2.12 and EA2.13 were crystallized from MeOH to yield compound 10 (1484 mg). Compound 6 (328 mg) was obtained from fraction EA2.14 by crystallization in MeOH. Fraction EA2.15 (549.4) was applied to a Sephadex LH-20 column to afford four fractions (EA2.15.1− EA2.15.4), and compound 9 (20.2 mg) was isolated from EA2.15.4 by crystallization in MeOH. Fraction EA5 was subjected to passage over a Sephadex LH-20 column (MeOH), to yield four fractions (EA5.1−EA5.5). Compound 14 (11.0 mg) was purified from fraction EA5.1 (275 mg) by semipreparative RP-HPLC under isocratic conditions (MeOH/H2O, flow rate 2 mL min−1, 65:35, v/v). 5,6-Dihydroxy-2-(2-phenylethyl)chromone (1): yellowish oil; [α]20D −2.7 (c 0.27, MeOH); UV (MeOH) λmax (log ε) 235 (4.99), 297 (4.31); IR (film) νmax 3402, 2929, 2871, 1704, 1656, 1619, 1594, 1454, 1376, 1315, 1233, 1186, 1157, 1124, 1036, 848, 749 cm−1; 1H NMR (800 MHz, CDCl3) δ 11.62 (1H, brs, OH-5), 7.31 (2H, t, J = 7.2, H-3′ and H-5′), 7.25 (1H, t, J = 7.2, H-4′), 7.20 (2H, d, J = 8.0, H-2′ and H-6′), 7.11 (2H, d, J = 8.8, H-7), 6.66 (1H, d, J = 8.8, H-8), 6.08 (1H, s, H-3), 3.06 (2H, t, J = 7.2, H-7′), 2.98 (2H, t, J = 7.2, H-8′); 13C NMR (200 MHz, CDCl3) δ 183.1 (C-4), 169.1 (C-2), 153.3 (C-9), 143.7 (C-5), 139.5 (C-1′), 135.6 (C-6), 128.8 (C-3′ and C-5′), 128.3 (C-2′ and C-6′), 126.9 (C-4′), 121.6 (C-7), 110.6 (C-8), 110.3 (C-10), 109.2 (C-3), 35.8 (C-8′), 33.3 (C7′); HRSEIMS m/z 283.0958 [M + H]+ (calcd for C17H15O4, 283.0970). Cell Culture, Oil Red O Staining, and Adiponectin Measurements. The Oil Red O staining and adiponectin enzyme linked immunosorbent assay (ELISA) were performed using a previously reported method with hBM-MSCs.11 In this assay, pioglitazone, glibenclamide, and aspirin were used as a positive control.30 Nuclear Receptor (NR) Assays. The TR-FRET-based NR binding assay was performed as previously described.31 Total RNA Isolation and Quantitative Real-Time PCR (Q-RTPCR). Total RNA samples for Q-RT-PCR were prepared as previously described. Q-RT-PCR was performed using a previously reported method.31 Q-RT-PCR primer sets (Applied Biosystems, Foster City, CA, USA) were used to determine the transcript levels of PPARG (Hs00234592_m1) and FABP4 (Hs00609791_m1). The housekeeping gene human glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 4333764F) was used to normalize sample variations. Molecular Docking Model Study. Molecular docking models of phenylethylchromones to PPARγ-LBD were generated using both AutoDock Vina 1.1.2 software (The Scripps Research Institute, La Jolla, CA, USA) and Accelrys Discovery Studio (Dassault Systems, BIODIVA Corporation, San Diego, CA, USA). The protein structural coordinates of PPARγ were obtained from the Protein Data Bank (PDB code number 5TWO) for the rivoglitazone-bound structure.24 Molecular docking was performed as previously described.10 Statistical Analysis. Statistical analyses were performed with RStudio for Windows (RStudio Inc., Boston, MA, USA). Means ± standard deviation (SD) were used to describe experimental values (n = 3 or 4). One-way analysis of variance (ANOVA) and post hoc tests were used for statistical tests. The correlation coefficient was performed based on Pearson’s correlation. The significance level was set at *p ≤ 0.05 and **p ≤ 0.01.
carried out using an Agilent 1260 Infinity system with a Phenomenex Luna 100 RP-C18 (250 × 4.6 mm i.d., 5 μm) column equipped with an autosampler, diode array detector (DAD), and column thermostat. Semipreparative high-performance liquid chromatography (HPLC) was performed using a Gilson 321 pump and Gilson UV/vis-155 detector system (Gilson, Middleton, WI, USA) with a Phenomenex Luna 100 RP-C18 (250 × 10 mm i.d., 5 μm) column. Column chromatography was performed using silica gel 60 (0.040−0.063 mm; Merck, Darmstadt, Germany) and a Sephadex LH-20 column (Amersham Bioscience AB, Uppsala, Sweden) as stationary phases. TLC was carried out on silica gel 60 F254 plates (Merck). All solvents used for isolation were purchased from Duksan Pure Chemical Co. (Gyoenggido, South Korea). Solvents for HPLC were purchased from J. T. Baker (Philipsburg, NJ, USA). Plant Material. Agarwood chips of A. malaccensis were purchased from Industrial Plantation Co. (Vientiane, Laos) in January 2010. A voucher specimen (AM-2010-01) was identified by Professor Jeong Hill Park at College of Pharmacy, Seoul National University, Seoul, Korea, and deposited at the herbarium of the Natural Products Research Institute, Seoul National University, Seoul, Korea.29 Extraction and Isolation. Agarwood chips of A. malaccencis (9.0 kg) were ground and extracted with MeOH 70% under reflux (20 L × 3 h, 3 times). The solvent was evaporated under reduced pressure to obtain a MeOH extract (864 g), which was suspended in water and successively partitioned with diethyl ether, ethyl acetate, and nbutanol, yielding 325, 211, and 135 g of residue, respectively. As a result, the diethyl ether fraction exhibited the most potent insulinsensitizing effects of adiponectin, which was again subjected to column chromatography to obtain pure compounds. The diethyl ether fraction (30 g) was chromatographed on a silica gel column (230−400 mesh, 300 g) and eluted with n-hexane/EtOAc (40:1 → 1:1, v/v) to obtain seven fractions (DE1−DE7). A crystalline precipitate of a methanol solution of fraction DE2 was filtered to yield compound 2 (2544 mg). The noncrystalline residue of fraction DE2 was separated by silica gel CC (n-hexane/EtOAc, 20:1 → 8:2, v/ v), to yield four fractions (DE2.1−DE2.4). Compound 2 (1716 mg) was continuously obtained from fraction DE2.2 by crystallization in MeOH. Fraction DE3 (3.36 g) was further subjected to a silica gel CC (230−400 mesh, 100 g), using n-hexane/EtOAc as the mobile phase (95:5 → 7:3, v/v) to yield nine subfractions (DE3.1−DE3.9). Fraction DE3.5 was subjected to Sephadex LH-20 CC to yield five subfractions (DE3.5.1−DE3.5.5). Fraction DE3.5.4 (87.2 mg) was separated by semipreparative RP-HPLC (MeOH/H2O, 65:35, v/v) to yield compound 15 (40.8 mg). Fraction DE4 (7.6 g) was applied to open silica gel CC (230−400 mesh, 200 g), eluted with n-hexane/ EtOAc (95:5 → 1:1, v/v), to yield 10 subfractions (DE4.1−DE4.10). Compound 8 (22.5 mg) was obtained from fraction DE4.4 by semipreparative RP-HPLC (MeOH/H2O, 65:35, flow rate 2 mL min−1, v/v). Fractions DE4.5 (1.45 g) and DE4.6 (781 mg) were crystallized in MeOH to generate compound 3 (1099 mg), and the residue of fraction DE4.5 was further separated by semipreparative RP-HPLC (MeOH/H2O, 65:35, flow rate 2 mL min−1, v/v) to yield compound 5 (86.4 mg). The rest of fraction DE4.6 was isolated and purified by semipreparative RP-HPLC (MeOH/H2O, 65:35, flow rate 2 mL min−1, v/v) to yield compound 1 (8.6 mg). Compound 7 (518 mg) was obtained from subfraction DE4.7 (814.5 mg) by recrystallization in MeOH, and the residue of this fraction was separated using semipreparative RP-HPLC (MeOH/H2O, 65:35, flow rate 2 mL min−1, v/v) to yield compounds 4 (24.6 mg) and 12 (13.1 mg). Fraction DE4.8 (430 mg) was continuously isolated by semipreparative RP-HPLC (MeOH/H2O, 65:35, flow rate 2 mL min−1, v/v) to yield compound 12 (68.4 mg). The EtOAC fraction (51.5 g) was chromatographed over a silica gel column (230−400 mesh, 300 g), eluted with n-hexane/EtOAc (200:1 to 1:1, v/v), to yield five fractions (EA1−EA5). Fraction EA2 (20 g) was fractionated by column chromatography on a silica gel column (230−400 mesh, 200 g), using a solvent system of n-hexane/EtOAc (95:5 to 1:1, v/v), to generate 17 fractions (EA2.1−EA2.17). Fraction EA2.11 was crystallized in MeOH to obtain a mixture of compounds 11 and 13 (65.3 mg), which then was subjected to semipreparative RP-HPLC
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00635. Detailed spectral data for compound 1, a novel chromone derivative, supportive pharmacological data of A. malaccensis-derived agarwood extracts and phenylethylchromone compounds, and supportive molecular docking simulation data (PDF) E
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AUTHOR INFORMATION
Corresponding Author
*Tel: +82-2-880-2481. Fax: +82-2-762-8322. E-mail:
[email protected]. ORCID
Jeong Hill Park: 0000-0003-3077-7673 Minsoo Noh: 0000-0002-4020-5372 Present Address ⊥
Department of Computational Biology, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States.
Author Contributions ∥
S. Ahn and C. T. Ma contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was supported by a grant from the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (Grant No. HN14C0088), a National Research Foundation of Korea (NRF) grant (2015R1A2A2A01008408), the MRC grant through NRF Korea (NRF-2018R1A5A2024425), and a grant by the Promising-Pioneering Researcher Program through Seoul National University (SNU) in 2015.
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DOI: 10.1021/acs.jnatprod.8b00635 J. Nat. Prod. XXXX, XXX, XXX−XXX
