Soluble Epoxide Hydrolase Inhibitory and Anti-inflammatory

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Soluble epoxide hydrolase inhibitory and anti-inflammatory components from the leaves of Eucommia ulmoides Oliver (Duzhong) Meng-Meng Bai, wei Shi, Jun-Mian Tian, Ming Lei, Young Ho Kim, Ya Nan Sun, Jang Hoon Kim, and Jin-Ming Gao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00055 • Publication Date (Web): 13 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Journal of Agricultural and Food Chemistry

Soluble Epoxide Hydrolase Inhibitory and Anti-inflammatory Components from the Leaves of Eucommia ulmoides Oliver (Duzhong) Meng-Meng Bai,†,‖ Wei Shi,†,‖ Jun-Mian Tian,† Ming Lei,† Jang Hoon Kim,‡ Ya Nan Sun,‡ Young Ho Kim,* ‡ and Jin-Ming Gao*† †

Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Science,

Northwest A&F University, Yangling 712100, Shaanxi, P. R. China ‡

College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea

* Corresponding author: Prof. Young Ho Kim, Ph.D. College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea. Tel.: +8242-821-5933. Fax: +82-42-823-6566. E-mail: [email protected] Prof. Jin-Ming Gao, Ph.D. Tel.: +86-29-87092515, E-mail: [email protected] ‖

These authors contributed equally to this work.

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Abstract: Eucommia ulmoides leaves have been used as a functional food and drink in China. The purpose of this study was to identify the bioactive constituents with soluble epoxide hydrolase (sEH) inhibitory activity and anti-inflammatory properties. Twenty-seven known compounds 1–27 were isolated from the leaves of E. ulmoides Oliver, and their structures were identified by NMR and ESIMS analysis, three of which 2,5-dimethoxy-3glucopyranosyl cinnamic alcohol (11), foliasalacioside E2 (26) and icariside F2 (27) were obtained from this plant for the first time. Compounds 1–7 exhibited soluble epoxide hydrolase (sEH) inhibitory activity at 100 M, among them quercetin (1) and kaempferol (5) displayed potential activities with IC50 values of 22.5±0.9 and 31.3±2.6 M with noncompetitive inhibition mode, respectively. Nuclear factor-kappa B (NF-κB) inhibitory activity of the isolated compounds was evaluated by the NF-κB liciferase assay in HepG2 cells. Compounds (1, 9, 20, and 27) displayed potent NF-κB inhibitory effects with IC50 values of 15.14 ± 2.29, 15.23 ± 2.34, 16.88 ± 2.17, and 16.25 ± 2.19 M, respectively, while other compounds showed weak inhibition of NF-κB transcriptional activity ranging from 17.54 to 92.6 µM. A structure-activity relationship of the flavonoids 1–9 was also discussed. The results obtained in this work might contribute to the understanding of pharmacological activities of E. ulmoides leaves and further investigation on its potential application values for food and drug. Keywords: Eucommia ulmoides; flavonoid; megastigmane; eucommioside-I; soluble epoxide hydrolase; NF-κB inhibitory effects

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INTRODUCTION Soluble epoxide hydrolase (sEH) is an important enzyme in mammal with potent biological activities on vascular and renal systems.1 It can hydrolyze epoxyeicosatrienoic acids (EETs), an metabolites of arachidonic acid generated by epoxygenase CYP enzymes. EETs are endothelium-derived hyperpolarizing factors (EDHFs) which act as regulators of vascular function.2 The cardiovascular effects of EETs include vasodilation, antimigratory actions on vascular smooth muscle cells and anti-inflammatory actions.3 However, sEH can convert EETs to its corresponding diols (dihydroxyeicosatrienoic acids; DHETs). Thus, inhibition of sEH becomes an effective pathway to treat cardiovascular disease. Inflammation contributes to the pathogenesis of cancer, cardiovascular diseases, and type2 diabetes mellitus.4,5 The nuclear factor kappaB (NF-κB) is a key regulator of many proinflammatory pathways.4,5 NF-κB normally resides in the cytoplasm, where it is retained by association with the endogenous inhibitor of kappa B (IκB) protein.6

However, when

activated by inflammatory stimuli such as tumor necrosis factor alpha (TNF-α), injury, or other stress-related stimuli, NF-κB is released, translocates to the nucleus and binds to DNA resulting in the transcription of proinflammatory target genes.7 Therefore, inhibitors targeting NF-κB signaling are considered as potential candidates for both prevention and therapy of inflammation. Eucommia ulmoides Oliver. (Duzhong), a well-known and the sole species of the genus Eucommia (Eucommiaceae family), has been traditionally used in various indigenous systems of medicines. Many studies have shown that Duzhong has numerous pharmacological effects, such as antihypertension,8 hepatoprotection,9 strengthening muscle10 and bone,11 and antiobesity.12 In addition, the male flowers or leaves have been utilized as health tea in food and beverage. E. ulmoides is a valuable traditional Chinese medicine, 3



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whose cortices have long been used as medicines. Due to the scarcity of its resources, people began using its leaves instead of cortexes in medicines. However, the main constituents for reducing blood pressure remain unclear due to lack of evidence. Previous phytochemical studies on this plant have afforded flavonoids, lignans and iridoids. Further bioactive studies showed pinoresinol diglucoside13 were thought to be responsible for the antihypertensive effects in E. ulmoides. Activity in anesthetized spontaneous hypertensive rat showed that some iridoids and lignans could also reduce blood pressure.14 Besides, considering the fact that flavonoids in Ginkgo biloba caused a potent vasorelaxation,15 they also exist in E. ulmoides in a large amount. It seems that plant flavonoids also act as antihypertension agents. In the course of our continuous investigation into the bioactive components from important edible and medicinal plants in the Qinba Mountains,16,17 we have carried out a phytochemical study on the leaves of E. ulmoides and on both sEH and potential NF-κB inhibition for sustainable utilization of bioresources. In this study, we report here isolation and structure identification of 27 metabolites from the leaves of E. ulmoides, as well as their biological functions, including inhibitory effects on soluble epoxide hydrolase and NF-kB activity. Based on the action mechanism of sEH, the work could highlight the group of natural compounds in E. ulmoides which are responsible for its cardiovascular effects. Therefore, this research might be helpful in a better understanding of their potential contribution to the pharmacological activities of E. ulmoides leaves and lay a significant basis for development and utilization of food and drug. MATERIALS AND METHODS General Experimental Procedures. UV spectra were obtained using a Thermo Scientific Evolution 300 UV-vis Spectrophotometer. 1H and

13

C NMR spectra were recorded on 400

MHz and 500 MHz NMR instruments (Bruker Daltonics Inc., Bremen, Germany). Chemical 4


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shifts are reported using solvent residual peak as the internal standard. ESI-MS spectra were performed on a Thermo Fisher LTQ Fleet instrument spectrometer (Thermo Fisher Scientific Inc., MA, U.S.). Column chromatography (CC) was performed on silica gel (90−150 μm) (Qingdao Marine Chemical Inc., Qingdao, China), Sephadex LH-20 (40−70 μm) (Amersham Pharmacia Biotech AB, Uppsala, Sweden), and Lichroprep RP-18 gel (40−63 μm; Merck, Darmstadt, Germany). GF254 plates (Qingdao Marine Chemical Inc., Qingdao, China) were used for thin-layer chromatography (TLC). Preparative TLC (PTLC) was carried out on silica gel 60 GF254 (Qingdao Marine Chemical, Ltd., Qingdao, China); semi-preparative HPLC (Waters Corp., USA) was performed by a Thermo BDS Hypersil column (250 × 4.6 mm and 250 × 10 mm, 5 μm) (Thermo Fisher Scientific Inc., MA, U.S.). Reagents. 3-Phenyl-cyano(6-methoxy-2-naphthalenyl)methyl ester-2-oxiraneacetic acid (PHOME),

12-[[(tricyclo[3.3.1.13,7]dec-1-ylamino)carbonyl]amino]-dodecanoic

acid

(AUDA), and purified recombinant sEH were purchased from Cayman (Cayman, Michigan, USA). The other chemical reagents and standard compounds were purchased from SigmaAldrich (St. Louis, MO, USA). AB-8 macroporous resin was purchased from the Chemical Plant of Nankai University (Tianjin, China), which was cross-linked polystyrene copolymer. All used chemicals (methanol, chloroform, ethyl acetate, petroleum ether and n-butanol etc.) were purchased from Kelong chemical engineering (Kelong, Chengdu, China). HPLC methanol were purchased from Tedia company (Tedia Inc., Fairfield, USA). Ultrapure water were made in our lab. Plant Materials. The green leaves of E. ulmoides were collected from Lveyang county, Shaanxi Province of China on August 25, 2012. The plant was taxonomically identified by one of authors (K.J.Z.). A voucher specimen (LXY-01234) has been deposited at the Herbarium of College of Life Sciences, Northwest A&F University, P. R. China. 5



Extraction, Isolation and Purification.

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The dried and powdered leaves of E. ulmoides (1

Kg) were extracted with 50% methanol. All the ﬁltrates were combined and concentrated under reduced pressure to give a crude extract. The crude extract was suspended in water and then extracted 3 times with n-butanol to obtain water layer and n-butanol fraction. Water layer was subjected to silica gel CC and eluted with CHCl3-MeOH with increasing polarities: 5:1, 5:2, 5:3 and 5:5 to give four fractions (Fr.1-Fr.4). Fr.1 was separated by Sephadex LH-20 (LH-20, MeOH) to obtain three fractions (Fr.11-Fr.1-3). Fr.1-1 was purified by PTLC (CHCl3-MeOH, 5:1), then HPLC (20% MeOH, flow rate: 2 mL/min;  = 254 nm) to yield 10 (15 mg, tR = 18.6 min). Fr.1-2 was separated by CC on LH-20 (MeOH) and RP-18 (20% MeOH) to give 21 (10 mg), and Fr.1-3 was chromatographed on silica gel ( CHCl3-MeOH, 9:1) to give 22 (21 mg). Fr.2 was subjected to AB-8 macroporous resin CC and sequentially eluted with water, 15% MeOH, and MeOH to provide three fractions (Fr.2-1-Fr.2-3). Fr.2-1 was separated by CC on LH-20 (MeOH), RP18 CC (20% MeOH), and then silica gel with (CHCl3-MeOH 20:1) to afford 19 (13 mg). Fr.2-2 was separated by LH-20 (MeOH) to yield two fractions (Fr.2-2-1 and Fr.2-2-2). Fr.22-1 was purified by RP-18 CC (40% MeOH), and then by HPLC (32% MeOH, flow rate: 2 mL/min;  = 254 nm) to afford 16 (45 mg). Fr.2-2-2 was submitted to silica gel CC and eluted with CHCl3-MeOH with increasing polarities (9:1-5:1) to give four fractions (Fr.2-2-21 to Fr.2-2-2-4). Fr.2-2-2-1 was separated by RP-18 CC (20% MeOH) and then by HPLC (30% MeOH, flow rate: 2 mL/min;  = 254 nm) to obtain 11 (32 mg, tR = 18.9 min). Fr.2-22-2 was subjected to CC on RP-18 using 20% to 30% MeOH, and then purified by HPLC (30% MeOH, flow rate: 2 mL/min;  = 254 nm) to afford 27 (11 mg, tR = 15.6 min) and 15 (20 mg, tR = 21.3 min). Fr.3 was loaded to an AB-8 column and successively eluted with water, 15% MeOH and MeOH to yield three fractions (Fr.3-1 to Fr.3-3). Purification of Fr.36


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2 by CC over LH-20 (MeOH) and silica gel (CHCl3-MeOH, 3:1) yielded 20 (60 mg), and Fr.3-3 by LH-20 (MeOH) and HPLC (40% MeOH; flow rate: 2 mL/min;  = 254 nm) afforded 4 (76 mg, tR = 14.0 min) and 3 (18 mg, tR = 22.0 min). n-Butanol fraction was separated by CC over AB-8 macroporous resin with water and then increasing concentrations of MeOH to provide five fractions A-E. Fr.C, eluted with 50% MeOH, was separated by silica gel CC using EtOAc-MeOH (20:1 to 5:1) as eluent to afford five fractions (Fr.C-1 to Fr.C-5). Fr.C-2 was separated by CC on silica gel (CHCl3-MeOH, 5:1), sephadex LH-20 (MeOH), and then RP-18 (30% MeOH) to give 2 (18 mg). Purification of Fr.C-3 by LH-20 (MeOH) CC followed by HPLC (35% MeOH, flow rate: 2 mL/min) provided 7 (26 mg, tR = 18.5 min). Fr.C-4 was purified successively by CC over LH-20 (MeOH), silica gel CC (CHCl3-MeOH, 10:1) and RP-18 to give 23 (10 mg). Fr.D, eluted with 75% MeOH, was subjected to silica gel CC with EtOAc-MeOH (20:1 to 5:1) to afford four fractions (Fr.D-1 - Fr.D-4). Fr.D-1 was submitted to silica gel CC (CHCl3-MeOH, 12:1) to obtain three fractions (Fr.D-1-1 - Fr.D-1-3). Purification of Fr.D-1-1 by LH-20 (MeOH) and RP-18 CC (40% MeOH) to yielded 12 (86 mg). Fr.D-1-2 was fractionated by LH-20 (MeOH) and RP-18 CC (40% MeOH) to afford 1 (21 mg) and 18 (13 mg). Fractionation of Fr.D-1-3 by LH-20 (MeOH) CC gave five fractions (Fr.D-1-3-1 to Fr.D-1-3-5). Further purification of Fr.D-1-3-2 and Fr.D-1-3-3 by RP-18 CC (30% MeOH) afforded 14 (50 mg) and 8 (8 mg), respectively, while Fr.D-1-3-5 by LH-20 (MeOH) CC produced 9 (10 mg). Fr.D-2 was separated by CC on silica gel CC (CHCl3-MeOH, 10:1)and LH-20 (MeOH) to furnish 6 (16 mg). Fr.D-3 was fractionated by silica gel CC (CHCl3-MeOH, 20:1 to 10:1) to give three fractions (Fr.D-3-1 - Fr.D-3-3), and purification of Fr.D-3-1 and Fr.D-3-2 by CC over RP-18 (30 % MeOH) and LH-20 (MeOH) afforded 17 (7 mg) and 13 (10 mg), respectively. Fr.E, eluted with 100 % MeOH, was fractionated by silica gel CC using EtOAc-MeOH (30:1 to 7



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10:1) to afford four fractions (Fr.E-1 to Fr.E-4). Purification of Fr.E-1 by CC on silica gel (CHCl3-MeOH, 15:1), LH-20 (MeOH), and RP-18 (30 % MeOH) gave 5 (10 mg). Fr.E-2 was separated by LH-20 (MeOH) and silica gel CC (CHCl3-MeOH, 20:1) to obtain 24 (11 mg). Fractionation of Fr.E-3 by silica gel CC (CHCl3-MeOH, 15:1) afforded two fractions (Fr.E-31 and Fr.E-3-2). Fr.E-3-1 was further purified by LH-20 (MeOH) and then silica gel CC (CHCl3-MeOH, 10:1) to afford 25 (17 mg), while Fr.E-3-2 by LH-20 (MeOH) gave 26 (18 mg). 2,5-Dimethoxy-3-glucopyranosyl cinnamic alcohol (11). C17H24O9, colorless solid; UV (MeOH) max 229, 267 nm; 13C NMR (125 MHz, DMSO-d6) δ 152.7 (C-1, C-4), 104.5 (C-2, C-6), 133.9 (C-3), 132.6 (C-5), 128.4 (C-7), 130.2 (C-8), 61.4 (C-9), 102.6 (C-1'), 77.2 (C-2'), 76.5 (C-3'), 70.0 (C-4'), 74.2 (C-5'), 60.9 (C-6'), 56.4 (2×OCH3); 1H NMR (500 MHz, DMSO-d6) δ 6.73 (2H, s, H-2, H-6), 6.47 (1H, d, J = 15.9 Hz, H-7), 6.34 (1H, dt, J = 15.9, 5.0 Hz, H-8), 4.11 (2H, m, H-9), 4.91 (1H, d, J = 7.0 Hz, H-1'), 3.04-3.60 (6H, m, H-2'-H-6'), 3.77 (6H, s, 2×OCH3); ESI-MS m/z 395.24 [M+Na]+. Foliasalacioside E2 (26). C24H42O11, amorphous solid; UV (MeOH) max 206 nm;13C NMR (125 MHz, MeOH-d4) δ 38.8 (C-1), 49.5 (C-2), 65.7 (C-3), 42.9 (C-4), 125.4 (C-5), 138.4 (C6), 25.4 (C-7), 39.0 (C-8), 76.3 (C-9), 19.8 (C-10), 29.0 (C-11), 30.4 (C-12), 20.1 (C-13), 102.2 (C-1'), 75.1 (C-2'), 78.1 (C-3'), 71.8 (C-4'), 76.9 (C-5'), 69.7 (C-6'), 105.2 (C-1''), 72.4 (C-2''), 74.2 (C-3''), 69.4 (C-4''), 66.6 (C-5''); 1H NMR (500 MHz, MeOH-d4) δ 1.72 (1H, m, H-2a), 1.41 (1H, dd, J = 11.6, 11.6 Hz, H-2b), 3.87 (1H, m, H-3), 2.20 (1H, m, H-4a), 1.95 (1H, m, H-4b), 2.24 (1H, m, H-7a), 2.03 (1H, m, H-7b), 1.59 (2H, m, H-8a, 8b), 3.91 (1H, m, H-9), 1.23 (3H, d, J = 6.1 Hz, H-10), 1.07 (3H, s, H-11), 1.09 (3H, s, H-12), 1.68 (3H, s, H13), 4.37 (1H, d, J = 7.7 Hz, H-1'), 4.38 (1H, d, J = 6.6 Hz, H-1''); ESI-MS m/z 505.29 [MH]-. 8


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Icariside F2 (27). C18H26O10, amorphous powder; UV (MeOH) max 211 nm;

13

C NMR (125

MHz, MeOH-d4) δ 139.0 (C-1), 129.3 (C-2), 129.3 (C-3), 128.7 (C-4), 129.3 (C-5), 129.3 (C6), 71.8 (C-7), 103.2 (C-1'), 75.1 (C-2'), 78.0 (C-3'), 71.8 (C-4'), 77.0 (C-5'), 68.7 (C-6'), 111.0 (C-1''), 78.0 (C-2''), 80.5 (C-3''), 75.0 (C-4''), 65.6 (C-5''); 1H NMR (500 MHz, MeOHd4) δ 7.28 – 7.70 (5H, m, H-2, 3, 4, 5, 6), 4.69 (1H, d, J = 11.8 Hz, H-7a), 4.93 (1H, d, J = 11.8 Hz, H-7b), 4.37 (1H, d, J = 7.7 Hz, H-1'), 5.09 (1H, d, J = 2.5 Hz, H-1''); ESI-MS m/z 425.33 [M+Na]+. sEH inhibitory activity. sEH inhibitory activity was determined using a hydrolysis reaction of PHOME in the presence of the sEH enzyme. Briefly, 130 L of sEH (62.5 ng/mL) in 25 mM Bis-Tris buffer (including 0.1% bovine serum albumin, pH 7.0) and 20 L of ligands diluted in MeOH were mixed in 96 well plate. 50 L of PHOME (40 M) in buffer was added as substrate. After starting the sEH reaction at 37 °C, products were measured using a Geniosmicroplate reader (Tecan, Mannedorf, Switzerland) during an hour at excitation and emission wavelengths of 320 and 465 nm, respectively. sEH inhibitory activity for each sample was calculated as follows: sEH inhibitory activity (%) = 100-[(S60-S0/C60-C0] × 100 where C60 and S60 were the fluorescence of control and inhibitor after 60 min, S0 and C0 is the fluorescence of control and inhibitor at zero min. Cell viability assay. Cell viability was evaluated by the Cell-Counting Kit (CCK)-8 methods. Briefly, CCK-8 was added to the cell culture medium for 1 h. The supernatant was removed and the formazan crystals were dissolved in dimethyl sulfoxide (DMSO). Absorbance was measured at 450 nm. The percentage of dead cells was determined relative to the control group. 9



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All compounds were tested for cytotoxic activity against HepG2 cell lines. All cell lines were maintained in Dulbecco's modified Eagles' medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37 C with 5% CO2. Each tested compound was dissolved with DMSO to obtain concentrations (0.1, 1, and 10 µM). They were incubated with the cells for 72 h. The negative control received the same amount of DMSO (0.1% in the highest concentration). Doxorubicin (0.1-0.58 mg/mL) was used as a positive control. The cell viability was determined by reduction of the yellow dye 3(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to a blue formazan product as described by Mosmann.18 NF-kB inhibitory activity Assay.

As nuclear factor-kappa B (NF-κB) is essential for

promoting inflammation-associated hepatic cancer, it is a potential target for hepatic cancer prevention in chronic hepatic inflammatory diseases.19 We examined the inhibitory effect of samples on NF-κB activated by TNF-α in hepatocellular carcinoma (HCC) cells such as HepG2 cells. The luciferase vector was first transfected into human hepatocarcinoma HepG2 cells. After a limited amount of time, the cells were lysed, and luciferin, the substrate of luciferase, was introduced into the cellular extract along with Mg2+ and an excess of ATP. Under these conditions, luciferase enzymes expressed by the reporter vector could catalyze the oxidative carboxylation of luciferin. Cells were seeded at 2 × 105 cells per well in 12-well plates and grown. After 24 h, cells were transfected with inducible NF-κB luciferase reporter and constitutively expressing Renilla reporter. After 24 h of transfection, medium was changed to assay medium (Opti-MEM + 0.5% FBS + 0.1 mM NEAA + 1 mM sodium pyruvate + 100 units/mL penicillin + 10 µg/mL streptomycin) and cells were pretreated for 1 h with either vehicle (DMSO) and compounds, followed by 1 h of treatment with 10 ng/mL TNF-α for 20 h. Unstimulated cells were used as a negative control (−), apigenin was used as 10


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a positive control. Dual luciferase assay was performed 48 h after transfection, and promoter activity values are expressed as arbitrary units using a Renilla reporter for internal normalization. Statistical Analysis. All data represent the mean ± S.D. of at least three independent experiments performed in triplicates. Statistical significance is indicated as determined by one-way ANOVA followed by Dunnett’s multiple comparison test, P

Soluble Epoxide Hydrolase Inhibitory and Anti-inflammatory

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