Fitoterapia 92 (2014) 188–193
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Anti-inflammatory activity of sulfate-containing phenolic compounds isolated from the leaves of Myrica rubra Han Hyuk Kim, Myeong Hwan Oh, Kwang Jun Park, Jun Hyeok Heo, Min Won Lee ⁎ Department of Pharmacognosy, College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea
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Article history: Received 25 July 2013 Accepted in revised form 10 October 2013 Available online 19 October 2013 Keywords: Myrica rubra Diarylheptanoid Flavonoid Sulfate Inflammation RAW 264.7 cells
a b s t r a c t Three sulfated phenolic compounds, juglanin B (11R)-O-sulfate (1), myricetin 3´-O-sulfate (2), and ampelopsin 3´-O-sulfate (3), were isolated from the leaves of Myrica rubra. Compound 1 was a new sulfated lignan, 2 was a new sulfated flavone, and 3 was a known sulfated flavone. The structures of the new compounds (1 and 2) were determined by acid hydrolysis and spectroscopic methods, including IR, FAB-MS, 1D and 2D NMR. The inhibitory activities of compounds 1–3 and their hydrolysates (1a–3a) against LPS-induced cytokine (TNF-α, IL-1β, and IL-6) production in macrophage RAW 264.7 cells were evaluated. The 2 new compounds (1 and 2) and their aglycones (1a and 2a) significantly reduced LPS-induced expression of iNOS and COX-2 proteins. © 2013 Published by Elsevier B.V.
1. Introduction Inflammation is a biological response to harmful stimuli, such as pathogens, physical injury, or damaged cells. Activated macrophages play an important role in inflammatory diseases related to overproduction of pro-inflammatory cytokines, including interleukin (IL)-1β; IL-6; tumor necrosis factor (TNF)-α; and inflammatory mediators, including reactive oxygen species (ROS), nitric oxide (NO), and prostaglandin E2 (PGE2). These inflammatory mediators are generated by activated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Overproduction of these macrophage mediators is involved in many diseases including rheumatoid arthritis, atherosclerosis, asthma, and pulmonary fibrosis. Thus, inhibiting the production of these macrophage mediators is an important target in treating inflammatory diseases [1,2]. Previously, we reported that flavonoid constituents from the leaves of Myrica rubra had potent COX-2 inhibitory
⁎ Corresponding author at: College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea. Tel.: +82 2 820 5602; fax: +82 2 822 9778. E-mail address: [email protected] (M.W. Lee). 0367-326X/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.fitote.2013.10.007
activities in the inflammatory response by RAW 264.7 macrophages [3]. As part of our continuing study of this plant, we isolated two new sulfate-containing compounds and a known compound and evaluated their anti-inflammatory activities. 2. Experimental 2.1. General Sephadex LH-20 (10–25 μm, GE Healthcare Bio-Science AB, Uppsala, Sweden) was used for open column chromatography. Reverse-phase medium pressure liquid chromatography (MPLC) using a rope system equipped with a Gilson Miniplus was carried out on ODS columns (Ultra pack ODS-SM-50B, 300 × 26 mm i.d., 230/70 mesh, Yamazen, Osaka, Japan) with a MeOH–H2O solvent system. TLC was carried out on a pre-coated, silica gel 60 F254 plate (Merck, Darmstadt, Germany), and spots were detected by UV radiation (254 nm) after spraying with FeCl3 solution or 10% H2SO4 and heating. FT-IR spectra were recorded on a Nicolet 6700 (Thermo Scientific, USA), and NMR spectra were recorded on a Varian VNS (1H-NMR, 600 MHz; 13 C-NMR, 150 MHz). HR-FAB-MS was measured with a
H.H. Kim et al. / Fitoterapia 92 (2014) 188–193
189
892, 843, 805, 747, 717, 623, 585, 535, 466; CD (MeOH) [θ]297 − 2.68, [θ]240 − 5.71, [θ]200 23.22; HR-FAB-MS m/z: 445.0719 [M-H]− (calculated for C20H22O7SK, 445.0723); 1H and 13C NMR: see Table 1. Myricetin 3´-O-sulfate (2): pale yellow, amorphous powder; −1 [α]25 : 3295, 1656, D +10° (c = 0.01, MeOH); IR (KBr) cm 1603, 1521, 1434, 1318, 1199, 1113, 1025, 937, 895, 792, 765, 730, 644, 587; HR-FAB-MS m/z 434.9431 [M-H]−, (calculated for C15H8O11SK, 434.9424); 1H and 13C NMR: see Table 1.
JMS-AX505WA, and elemental analysis was recorded on a Flash1112, Flash2000 (CE Instrument, Italy). CD spectra were measured on a Chirascan plus (Applied Photophysics, U.K.), and optical rotation data were recorded on an Autopol III, #A7214 (Rudolph Research, U.S.A.). 2.2. Plant material M. rubra leaves were collected from the Korea National Arboretum in Pocheon, Gyeong-gi, Korea in August 2010. The plant was identified by M.S. Kim Sung-Sik (Korea National Arboretum). The voucher specimen (MR 2010-08) has been deposited at the herbarium of the College of Pharmacy, Chung-Ang University.
2.4. Acid hydrolysis of 1–3 Each compound (20 mg) in pyridine (5 mL) and dioxane (5 mL) was heated at 100 °C for 1 h. The pyridine-dioxane was evaporated and the residue was diluted with MeOH (10 mL). The MeOH solution was passed through an ODS column (230/70 mesh, 26 × 300 mm) in reverse-phase MPLC system (5 mL/min, 280 nm), eluted with MeOH–H2O gradients (v/v, 0–100%). The eluate was concentrated and subjected to TLC in a CHCl3/MeOH/H2O (v/v/v, 70:30:4). The hydrolysates in the residue were identified on the basis of 1H and 13C NMR spectroscopic data and comparison of spectral data with the literature [4–6].
2.3. Extraction and isolation The air-dried leaves of M. rubra (2.5 kg) were extracted with 80% acetone at room temperature. The extract was then suspended in H2O and successively partitioned with n-Hexane and EtOAc to afford n-Hexane (28.0 g), EtOAc (34.8 g), and H2O (124.7 g) extracts after removing the solvent in vacuo. The H2O layer (50 g) was subjected to Sephadex LH-20 (10–25 μm, 2 kg, 10 × 80 cm) column chromatography using MeOH–H2O gradients (v/v, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20) as eluent to give 5 fractions (Fr. 1–5). Fr. 2 (640 mg) was further chromatographed over an ODS column (230/70 mesh, 320 g, 5 × 40 cm) in reverse-phase MPLC system (5 mL/min, 280 nm), eluted with MeOH/H2O (v/v, 40:60) to furnish 3 (753.1 mg), and 2 (78 mg), while eluted with MeOH/H2O (v/v, 60:40) to yield 1 (51.6 mg). Juglanin B (11R)-O-sulfate (1): off-white, amorphous −1 powder; [α]25 : D − 16.9° (c = 0.01, MeOH); IR (KBr) cm 3373, 2936, 1600, 1508, 1463, 1414, 1380, 1242, 1042, 931,
2.5. Cell culture Mouse macrophage RAW 264.7 macrophages were purchased from the Korean Cell Line Bank. These cells were grown at 37 °C in a humidified atmosphere (5% CO2) in Dulbecco's Modified Eagle's Medium (DMEM) (Sigma, St. Louis, MO, USA) containing 10% fetal bovine serum, 100 IU/mL penicillin G, and 100 mg/mL streptomycin (Gibco BRL, Grand Island, NY, USA).
Table 1 1 H and 13C NMR data for 1, 1a, 2, and 2a (in CD3OD). No.
1
1a a
b
δH (mult; J in Hz) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 4-OMe a1
δc
6.73 (d, 1.8) 2.94 2.48 2.24 1.71 2.07 1.81 4.76 2.17 3.03 2.76
(d, 16.8) (overlapped) (m), 1.78 (m) (m), 1.47 (m) (dd, J = 12.0, 1.8) (m) (t, 9.6) (m), 1.88 (m) (t, 14.4) (d, 18.0)
25.6 22.6 36.1 77.4 32.9 26.2 130.8 129.2 115.8 150.6 133.1 125.9 55.4
7.03 (dd, 8.4, 2.4) 6.79 (d, 8.4) 7.17 (d, 1.8) 6.85 (d, 1.8) 3.85 (s)
H NMR measured at 600 MHz,
125.5 125.5 140.2 148.8 111.1 131.2 30.1
b 13
δH (mult; J in Hz)
6.75 (d, 1.8) 2.88(m) 2.46 (m) 1.93(m), 1.65 (m) 1.81 (m), 1.43 (m) 1.77 (m) 1.53 (m) 3.99 (t, 9.6) 2.21 (m), 1.70(m) 2.91 (dd, 2.1, 17.1) 2.80 (dd, 2.1, 17.1) 7.01 (dd, 8.4, 2.4) 6.82 (d, 8.4) 7.21 (d, 2.4) 6.77 (d, 2.4) 3.83 (s)
No. δc 125.6 125.7 140.4 148.2 111.3 130.9 30.2 26.4 22.8 39.5 67.5 34.6 26.6
2 δH (mult; J in Hz)
2 3 4 5 6 7 8 9 10 1´ 2´ 3´ 4´ 5´ 6´
6.20 (d, 1.5) 6.45 (d, 1.5)
7.79 (d, 1.5)
7.64 (d, 1.5)
2a δc 146.1 136.1 176.0 160.6 98.1 163.9 93.4 156.7 103.2 121.8 114.0 140.1 139.6 146.0 112.2
130.7 129.4 116.2 151.4 133.7 125.4 55.7
C NMR measured at 150 MHz; obtained in CD3OD with TMS as an internal standard.
δH (mult; J in Hz)
6.18(d, 2.1) 6.38 (d, 2.1)
7.34 (s)
7.34 (s)
δc 146.6 135.5 175.8 160.9 97.8 164.1 93.0 156.7 103.1 121.7 107.1 145.2 135.9 145.2 107.1
190
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2.6. Measurement of cell viability Cell viability was measured by using a 3-(4,5-dimeth ylthiazol-2-yl)-2,5-diphenyltetra-zoliumbromide (MTT) assay, which is based on the reduction of MTT to formazan by mitochondrial dehydrogenase. Briefly, the cells (3 × 104/200 μL medium) were treated with each sample, and incubated for 24 h at 37 °C. After the MTT reagent (0.5 mg/mL) (Sigma, St. Louis, MO, USA) was added to the medium and incubated for an additional 4 h, the medium was then removed and the MTT-formazan was dissolved in 200 μL dimethylsulfoxide (DMSO). The extent of the reduction of MTT to formazan was quantified by measuring the absorbance at 540 nm using a microplate reader. 2.7. Inflammatory cytokine assay Cytokine concentrations in the supernatants of macrophage cultures were determined by enzyme-linked immunosorbent assay (ELISA) using an antibody from R&D system, according to the manufacturer's instruction. The cells were incubated with LPS in the presence of different concentrations of each compound for 18 h. The supernatants were collected and stored at −80 °C before analysis. Standards were prepared from recombinant mouse cytokines separately (R&D system, Minneapolis, MN). 2.8. Western blotting Each compound in deionized distilled water (6.25, 12.5, and 25 μM) was added to medium containing RAW 264.7 macrophages and incubated for 18 h. The control used only deionized distilled water. The cells were harvested by washing with PBS, lysed with 100 μL of RIPA buffer (50 mM Tris, 0.1% SDS, 50 mM NaCl, 1% NP-40, 1 mM PMSF, 10 μg/mL aprotinin, and 10 μg/mL leupeptin, pH 7.4), and centrifuged at 10,000 ×g for 20 min. The protein content in the supernatant was quantified using a Bio-Rad protein assay kit. Samples containing equal amounts of protein (20 μg) were analyzed using 10% SDS-PAGE. Proteins in the SDS-PAGE gel were transferred to PVDF membranes (Bio-Rad) using the Trans-Blot apparatus (Bio-Rad). After transfer, the membranes were blocked in a solution of 5% nonfat dried milk (w/v) in TBS-T (24.8 mM Tris, 137 mM sodium chloride, 2.7 mM potassium chloride, and 0.05% Tween 20, pH 7.4) for 1 h at room temperature. After 6 washes with TBS-T, the membranes were
incubated with primary iNOS and COX-2 polyclonal antibody from Santa Cruz Biotechnology (Santa Cruz, CA, US) and diluted (1:1000) with TBS-T for 12 h at 4 °C. Membranes were washed and incubated at room temperature for 2 h with mouse anti-rabbit IgG HRP from Santa Cruz Biotechnology (diluted 1:1000). After washing, proteins were detected in a dark room using an Enhanced Chemiluminesence kit (Amersham Bioscience UK, Ltd., Little Chalfont, UK). The detection reagent was poured onto the membrane and incubated for 1 min, and the band density was quantified with an LAS4000 image analyzer (Fujifilm Life Science, Tokyo, Japan).
3. Results and discussion M. rubra leaves were extracted with 80% acetone and fractionated with n-hexane, EtOAc, and water. Three sulfatecontaining compounds (1–3) were isolated from the water layer using combined chromatographic separations. The compounds included a new diarylheptanoid sulfate (1), a new flavonol sulfate (2), and a known flavonol sulfate (3) (Fig. 1). Compound (3) was identified on the basis of spectroscopic analysis and comparison of spectral data with the literature [7]. Compound 1 was obtained as an off-white, amorphous powder on thin layer chromatography (TLC) had a bluishgreen color when heated after spraying with 10% H2SO4 and a light brown color when sprayed with FeCl3 solution. The 1H NMR spectrum (Table 1) of 1 showed 2 sets of an aromatic ABX spin systems at δH 7.03 (1H, dd, J = 8.4, 2.4 Hz, H-15), 6.79 (1H, d, J = 8.4 Hz, H-16), and 7.17 (1H, d, J = 1.8 Hz, H-18) and a meta-coupled doublets at δH 6.73 (1H, d, J = 1.8 Hz, H-5) and 6.85 (1H, d, J = 1.8 Hz, H-19), indicating the presence of two aromatic ring with different substitution patterns. The spectrum also included signals attributable to six aliphatic methylenes at δH 2.48 (1H, overlapped, H-7), 2.94 (1H, d, J = 16.8 Hz, H-7), 1.78 (1H, m, H-8), 2.24 (1H, m, H-8), 1.47 (1H, m, H-9), 1.71 (1H, m, H-9), 1.81 (1H, m, H-10), 2.07 (1H, dd, J = 12.0, 1.8 Hz, H-10), 1.88 (1H, m, H-12), 2.17 (1H, m, H-12), 2.76 (1H, d, J = 18.0 Hz, H-13) and 3.03 (1H, t, J = 14.4, H-13), one methoxyl group at δH 3.83 (3H, s, 4-OMe), and one oxymethine at δH 4.76 (1H, t, J = 9.6 Hz, H-11). The 13C NMR spectrum of 1 also showed two sets of aromatic rings at δC 111.1 (C-5), 115.8 (C-16), 125.5 (C-1, 2), 125.9 (C-19), 129.2 (C-15), 130.8 (C-14), 131.2 (C-6), 133.1 (C-18), 140.2 (C-3), 148.8(C-4), 150.6 (C-17), six aliphatic carbons at δC 30.1 (C-7), 25.6 (C-8), 22.6
Fig. 1. Structures of compounds 1–3 and their hydrolysates (1a–3a).
H.H. Kim et al. / Fitoterapia 92 (2014) 188–193
(C-9), 36.1 (C-10), 32.9 (C-12), 26.2 (C-13), one methoxyl carbon at δC 55.4 (4-OMe), and one oxymethine carbon at δC 77.4 (C-11). These data closely resembled the spectra of juglanin B [16-Methoxytricyclo[12.3.1.12,6]nonadeca-1(18),2,4,6 (19),14,16-hexaene-3,9,17-triol] [4]. In the HMBC spectrum, 1 showed an HMBC correlation of H-7/C-5, C-6, C-19 and H-13/ C-14, C-15, C-18 between the aliphatic chain and two aromatic rings, indicating that the aliphatic chain was attached at two aromatic rings (Fig. 2). Furthermore, the presence of a signals for a hydroxyl groups δC 140.2 and 150.6, which were determined to be at C-3 and C-17 by the HMBC correlations of the H-5 (δH 6.73) and H-19 (δH 6.85) correlated with C-3, and the H-15 (δH 7.03) and H-18 (δH 7.17) correlated with C-17, respectively. One of the aromatic protons (1H, δH 6.73, H-5) correlated with the carbon signal at δC 148.8, which was assigned to the methoxy bearing C-4 based on long-distance correlations with O-methyl protons. The negative HR-FAB-MS and elemental analysis of 1 showed that the molecular formula of 1 was C20H23O7SK (anal. C 50.46, H 5.57, O 27.30, S 6.38%) with a pseudomolecular ion peak at m/z 445.0719 [M-H]− (calculated for C20H22O7SK, 445.0723). The IR spectrum had an absorption band at 1242 cm−1 ascribable to the S〓O vibration of a sulfate group. These results suggested the presence of a sulfate group in 1. In addition, acid hydrolysis [8] of 1 with pyridine-dioxane (1:1) afforded a desulfated product, juglanin B (1a), which is C20H24O4 [4]. In the view of HR-FAB-MS data, 1 was shown to be 118 (SO3K) mass units larger than 1a, compound 1 was presumed to be a sulfate derivative of 1a. Comparison of the 1H NMR spectra of 1 and 1a showed a downfield shift (+δH 0.77) for a proton (H-11) attached to an oxymethine carbon, in similarity to data reported earlier for steroid and carbohydrate sulfates [9,10]. Consistently, in the 13 C NMR spectrum of 1, C-11 (δC 77.4) shifted downfield (+δC 9.9) compared with 1a (δC 67.5). This sulfation shift was in good agreement with that published for sulfates of secondary alcohols [10]. These features suggested 1 as the 11-O-sulfate of 1a. The absolute configuration at C-11 of 1 was determined as R from its specific rotation ([α]25 D −16.9, MeOH), which had the same sign as of (R)-myricanol ([α]25 D −65.6, CHCl3) [11]. Based on these results, the structure of compound 1 was assigned to juglanin B (11R)-O-sulfate. Compound 2 was obtained as a pale yellow, amorphous powder. Compound 2 had a yellow color with 10% H2SO4
191
after heating and a dark green color with FeCl3 solution on TLC. The 1H NMR spectrum (Table 1) of 2 showed a meta-coupled two doublets at δH 6.20 (J = 1.5 Hz) and 6.45 ( J = 1.5 Hz) due to the C-6 and C-8 protons of a flavonoid skeleton. Two meta-coupled doublets at δH 7.64 (1H, d, J = 1.5 Hz, H-6´) and 7.79 (1H, d, J = 1.5 Hz, H-2´) arising from a flavonoid B-ring, suggested that this ring was an unasymmetrical 3´,4´,5´-trisubstitution pattern. The 13C NMR spectrum of 2 revealed 15 carbon signals, including δc 146.1 (C-2), 136.1 (C-3), and 176.0 (C-4) which are characteristic features of flavonol moiety. The chemical shifts of 2 were in good agreement with its hydrolysate, myricetin (2a), except those of B-ring [δc 111.8 (C-1´), 114.0 (C-2´), 140.1 (C-3´), 139.6 (C-4´), 146.0 (C-5´), and 112.2 (C-6´)]. The structure of 2 was suggested to be a sulfated myricetin by negative HR-FAB-MS data [m/z 434.9431 (calculated for C15H8O11SK, 434.9424)], elemental analysis (anal. C 41.55, H 3.36, O 40.85, S 4.93%), and IR absorption at 1199 cm−1 ascribable to the S〓O vibration of a sulfate group. In the 13C NMR spectrum, the effect of O-sulfate as an electron withdrawing group was well-known [12]. The carbon atoms ortho to the sulfate group experience a decreased electron density resulting in a downfield shift by 5.1–6.9 ppm. On the other hand, the carbon atom carrying the sulfate group showed an increased electron density resulting in an upfield shift by 3.7 ppm. The chemical shifts of these carbon atoms agree with those previously calculated for quercetin 3´-sulfate and phloroglucinol sulfates [13,14]. Based on these results, the structure of 2 was assigned to myricetin 3´-O-sulfate. Sulfated compounds are known for their universal occurrence in higher plants. But, the physiological significance of sulfates of secondary constituents in plant tissue is not clear. Apart from their possible involvement in neutralization of reactive hydroxyl groups, their accumulation in plants growing under saline conditions suggests a role in the sequestering of sulfate ions [15]. A large number of flavonoids are known to occur in plants in sulfated form [12,15]. Recent examples of other types of phenolic sulfates are the lignan, (+)-isolarisiresinol 3α-O-sulfate [16], and the coumarin sulfates [17], both containing sulfated alcohol groups. To demonstrate the anti-inflammatory activity of compounds 1–3 and their aglycones (1a–3a), we evaluated their
Table 2 Inhibitory effects of the compounds against LPS-stimulated expression of TNF-α, IL-1β, and IL-6. Compound
IC50 (μM)a TNF-α
1 1a 2 2a 3 3a PDTCb Quercetinc
1
a
± ± ± ± ± ± ± ±
2.14 1.57 2.45 2.47 1.50 3.04 2.13 0.81
22.9 17.9 20.2 19.2 N25 N25 18.0 16.9
± ± ± ±
IL-6 0.75 0.50 1.42 1.86
± 1.74 ± 0.34
22.7 20.3 22.2 23.9 N25 N25 16.8 16.8
± ± ± ±
1.61 1.42 1.14 1.73
± 2.40 ± 0.13
IC50 values were determined by regression analysis and are expressed as mean ± SD of 3 separate experiments. Used as positive control.
b,c
Fig. 2. Key HMBC correlations of 1.
20.1 19.5 19.9 19.7 20.3 21.9 16.8 13.6
IL-1β
192
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Fig. 3. Inhibitory effects of 1, 1a, 2, and 2a on LPS-induced expression of iNOS and COX-2 protein in RAW 264.7 cells. Cells were pretreated with 1, 1a, 2, and 2a for 1 h following LPS stimulation (1 μg/mL).
ability to inhibit LPS-induced cytokine (TNF-α, IL-1β and IL-6) production in RAW 264.7 cells. RAW 264.7 cells were treated with various concentrations (6.25, 12.5, and 25 μM) of the each compound and cell viability was measured by MTT assay. No compounds displayed significant cytotoxicity up to 25 μM, indicating that the anti-inflammatory effects of these compounds were not due to cytotoxicity (data not shown). The biphenyl-type diarylheptanoids (1 and 1a) and 3-hydroxyflavones (2 and 2a) significantly inhibited TNF-α, IL-1β, and IL-6 secretion in a concentration-dependent manner, whereas the 2,3-dihydroflavonols (3 and 3a) did not inhibit IL-1β or IL-6 production. A parent alcohol of the sulfate-containing diarylheptanoid, juglanin B (1a) had the most potent inhibitory activity with IC50 values of 19.5 ± 1.57, 17.9 ± 0.50 and 20.3 ± 1.42 μM, respectively (Table 2). Activated macrophages play an important role in inflammatory diseases related to overproduction of TNF-α, IL-1β, and IL-6, which are generated as a result of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) activation. The effects of compounds 1, 1a, 2, and 2a on LPS-induced expression of iNOS and COX-2 were investigated (Fig. 3). RAW 264.7 cells were stimulated with 1 μg/mL of LPS for 18 h in increasing concentrations of compounds 1, 1a, 2, and 2a, and iNOS and COX-2 protein levels were determined by western blotting. Compounds 1, 1a, 2, and 2a (6.25-25 μg/mL) dose-dependently reduced LPS-induced expression of iNOS and COX-2 but did not change β-actin expression. This result indicates that these compounds (1, 1a, 2, and 2a) suppress LPS-induced expression of iNOS and COX-2 at the transcriptional level. 4. Conclusion Two new sulfate-containing phenolic compounds (1 and 2) were isolated from M. rubra leaves and their structures
were determined from IR, FAB-MS, 1D and 2D NMR spectroscopic data. All isolated phenolic compounds (1–3) and their derivatives (1a–3a) were evaluated for anti-inflammatory potencies in reducing cytokine expression. The biphenyl-type diarylheptanoids (1 and 1a) and 3-hydroxyflavones (2 and 2a) significantly inhibited the secretion of TNF-α, IL-1β, and IL-6 and LPS-induced expression of iNOS and COX-2. Acknowledgment This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0022929). References [1] Moncada S, Palmer MJ, Higgs DA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 1992;43:109–34. [2] Lee HJ, Hyun EA, Yoon WJ, Kim BH, Rhee MH, Kang HK, et al. In vitro antiinflammatory and anti-oxidative effects of Cinnamomum camphora extracts. J Ethnopharmacol 2006;103:208–16. [3] Kim HH, Kim DH, Kim MH, Oh MH, Kim SR, Park KJ, et al. Flavonoid constituents in the leaves of Myrica rubra Sieb. et Zucc. with antiinflammatory activity. Arch Pharm Res 2013 [in press]. [4] Liu JX, Di DL, Wei XN, Han Y. Cytotoxic diarylheptanoids from the pericarps of walnuts (Juglans regia). Planta Med 2008;74:754–9. [5] Matsuda H, Higashino M, Chen W, Tosa H, Iinuma M, Kubo M. Studies of cuticle drugs from natural sources. III. Inhibitory effect of Myrica rubra on melanin biosynthesis. Biol Pharm Bull 1995;18:1148–50. [6] Lee SI, Yang JH, Kim DK. Antioxidant flavonoids from the twigs of Stewartia koreana. Biomol Ther 2010;18:191–6. [7] Nonaka GI, Muta M, Nishioka I. Myricatin, a galloyl flavanonol sulfate and prodelphinidin gallates from Myrica rubra. Phytochemistry 1983;22:237–41. [8] Ishida H, Miyamoto H, Kajino T, Nakayasu H, Nukaya H, Tsuji K. Study on the bile salt from megamouth shark. I. The structures of a new bile alcohol, 7-deoxyscymnol, and its new sodium sulfates. Chem Pharm Bull 1996;44:1289–92. [9] Goto J, Kato H, Hasegawa F, Nambara T. Synthesis of monosulfates of unconjugated and conjugated bile acids. Chem Pharm Bull 1979;27:1402–11.
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Anti-inflammatory activity of sulfate-containing phenolic compounds isolated from the leaves of Myrica rubra Han Hyuk Kim, Myeong Hwan Oh, Kwang Jun Park, Jun Hyeok Heo, Min Won Lee ⁎ Department of Pharmacognosy, College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea
a r t i c l e
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Article history: Received 25 July 2013 Accepted in revised form 10 October 2013 Available online 19 October 2013 Keywords: Myrica rubra Diarylheptanoid Flavonoid Sulfate Inflammation RAW 264.7 cells
a b s t r a c t Three sulfated phenolic compounds, juglanin B (11R)-O-sulfate (1), myricetin 3´-O-sulfate (2), and ampelopsin 3´-O-sulfate (3), were isolated from the leaves of Myrica rubra. Compound 1 was a new sulfated lignan, 2 was a new sulfated flavone, and 3 was a known sulfated flavone. The structures of the new compounds (1 and 2) were determined by acid hydrolysis and spectroscopic methods, including IR, FAB-MS, 1D and 2D NMR. The inhibitory activities of compounds 1–3 and their hydrolysates (1a–3a) against LPS-induced cytokine (TNF-α, IL-1β, and IL-6) production in macrophage RAW 264.7 cells were evaluated. The 2 new compounds (1 and 2) and their aglycones (1a and 2a) significantly reduced LPS-induced expression of iNOS and COX-2 proteins. © 2013 Published by Elsevier B.V.
1. Introduction Inflammation is a biological response to harmful stimuli, such as pathogens, physical injury, or damaged cells. Activated macrophages play an important role in inflammatory diseases related to overproduction of pro-inflammatory cytokines, including interleukin (IL)-1β; IL-6; tumor necrosis factor (TNF)-α; and inflammatory mediators, including reactive oxygen species (ROS), nitric oxide (NO), and prostaglandin E2 (PGE2). These inflammatory mediators are generated by activated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Overproduction of these macrophage mediators is involved in many diseases including rheumatoid arthritis, atherosclerosis, asthma, and pulmonary fibrosis. Thus, inhibiting the production of these macrophage mediators is an important target in treating inflammatory diseases [1,2]. Previously, we reported that flavonoid constituents from the leaves of Myrica rubra had potent COX-2 inhibitory
⁎ Corresponding author at: College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea. Tel.: +82 2 820 5602; fax: +82 2 822 9778. E-mail address: [email protected] (M.W. Lee). 0367-326X/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.fitote.2013.10.007
activities in the inflammatory response by RAW 264.7 macrophages [3]. As part of our continuing study of this plant, we isolated two new sulfate-containing compounds and a known compound and evaluated their anti-inflammatory activities. 2. Experimental 2.1. General Sephadex LH-20 (10–25 μm, GE Healthcare Bio-Science AB, Uppsala, Sweden) was used for open column chromatography. Reverse-phase medium pressure liquid chromatography (MPLC) using a rope system equipped with a Gilson Miniplus was carried out on ODS columns (Ultra pack ODS-SM-50B, 300 × 26 mm i.d., 230/70 mesh, Yamazen, Osaka, Japan) with a MeOH–H2O solvent system. TLC was carried out on a pre-coated, silica gel 60 F254 plate (Merck, Darmstadt, Germany), and spots were detected by UV radiation (254 nm) after spraying with FeCl3 solution or 10% H2SO4 and heating. FT-IR spectra were recorded on a Nicolet 6700 (Thermo Scientific, USA), and NMR spectra were recorded on a Varian VNS (1H-NMR, 600 MHz; 13 C-NMR, 150 MHz). HR-FAB-MS was measured with a
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892, 843, 805, 747, 717, 623, 585, 535, 466; CD (MeOH) [θ]297 − 2.68, [θ]240 − 5.71, [θ]200 23.22; HR-FAB-MS m/z: 445.0719 [M-H]− (calculated for C20H22O7SK, 445.0723); 1H and 13C NMR: see Table 1. Myricetin 3´-O-sulfate (2): pale yellow, amorphous powder; −1 [α]25 : 3295, 1656, D +10° (c = 0.01, MeOH); IR (KBr) cm 1603, 1521, 1434, 1318, 1199, 1113, 1025, 937, 895, 792, 765, 730, 644, 587; HR-FAB-MS m/z 434.9431 [M-H]−, (calculated for C15H8O11SK, 434.9424); 1H and 13C NMR: see Table 1.
JMS-AX505WA, and elemental analysis was recorded on a Flash1112, Flash2000 (CE Instrument, Italy). CD spectra were measured on a Chirascan plus (Applied Photophysics, U.K.), and optical rotation data were recorded on an Autopol III, #A7214 (Rudolph Research, U.S.A.). 2.2. Plant material M. rubra leaves were collected from the Korea National Arboretum in Pocheon, Gyeong-gi, Korea in August 2010. The plant was identified by M.S. Kim Sung-Sik (Korea National Arboretum). The voucher specimen (MR 2010-08) has been deposited at the herbarium of the College of Pharmacy, Chung-Ang University.
2.4. Acid hydrolysis of 1–3 Each compound (20 mg) in pyridine (5 mL) and dioxane (5 mL) was heated at 100 °C for 1 h. The pyridine-dioxane was evaporated and the residue was diluted with MeOH (10 mL). The MeOH solution was passed through an ODS column (230/70 mesh, 26 × 300 mm) in reverse-phase MPLC system (5 mL/min, 280 nm), eluted with MeOH–H2O gradients (v/v, 0–100%). The eluate was concentrated and subjected to TLC in a CHCl3/MeOH/H2O (v/v/v, 70:30:4). The hydrolysates in the residue were identified on the basis of 1H and 13C NMR spectroscopic data and comparison of spectral data with the literature [4–6].
2.3. Extraction and isolation The air-dried leaves of M. rubra (2.5 kg) were extracted with 80% acetone at room temperature. The extract was then suspended in H2O and successively partitioned with n-Hexane and EtOAc to afford n-Hexane (28.0 g), EtOAc (34.8 g), and H2O (124.7 g) extracts after removing the solvent in vacuo. The H2O layer (50 g) was subjected to Sephadex LH-20 (10–25 μm, 2 kg, 10 × 80 cm) column chromatography using MeOH–H2O gradients (v/v, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20) as eluent to give 5 fractions (Fr. 1–5). Fr. 2 (640 mg) was further chromatographed over an ODS column (230/70 mesh, 320 g, 5 × 40 cm) in reverse-phase MPLC system (5 mL/min, 280 nm), eluted with MeOH/H2O (v/v, 40:60) to furnish 3 (753.1 mg), and 2 (78 mg), while eluted with MeOH/H2O (v/v, 60:40) to yield 1 (51.6 mg). Juglanin B (11R)-O-sulfate (1): off-white, amorphous −1 powder; [α]25 : D − 16.9° (c = 0.01, MeOH); IR (KBr) cm 3373, 2936, 1600, 1508, 1463, 1414, 1380, 1242, 1042, 931,
2.5. Cell culture Mouse macrophage RAW 264.7 macrophages were purchased from the Korean Cell Line Bank. These cells were grown at 37 °C in a humidified atmosphere (5% CO2) in Dulbecco's Modified Eagle's Medium (DMEM) (Sigma, St. Louis, MO, USA) containing 10% fetal bovine serum, 100 IU/mL penicillin G, and 100 mg/mL streptomycin (Gibco BRL, Grand Island, NY, USA).
Table 1 1 H and 13C NMR data for 1, 1a, 2, and 2a (in CD3OD). No.
1
1a a
b
δH (mult; J in Hz) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 4-OMe a1
δc
6.73 (d, 1.8) 2.94 2.48 2.24 1.71 2.07 1.81 4.76 2.17 3.03 2.76
(d, 16.8) (overlapped) (m), 1.78 (m) (m), 1.47 (m) (dd, J = 12.0, 1.8) (m) (t, 9.6) (m), 1.88 (m) (t, 14.4) (d, 18.0)
25.6 22.6 36.1 77.4 32.9 26.2 130.8 129.2 115.8 150.6 133.1 125.9 55.4
7.03 (dd, 8.4, 2.4) 6.79 (d, 8.4) 7.17 (d, 1.8) 6.85 (d, 1.8) 3.85 (s)
H NMR measured at 600 MHz,
125.5 125.5 140.2 148.8 111.1 131.2 30.1
b 13
δH (mult; J in Hz)
6.75 (d, 1.8) 2.88(m) 2.46 (m) 1.93(m), 1.65 (m) 1.81 (m), 1.43 (m) 1.77 (m) 1.53 (m) 3.99 (t, 9.6) 2.21 (m), 1.70(m) 2.91 (dd, 2.1, 17.1) 2.80 (dd, 2.1, 17.1) 7.01 (dd, 8.4, 2.4) 6.82 (d, 8.4) 7.21 (d, 2.4) 6.77 (d, 2.4) 3.83 (s)
No. δc 125.6 125.7 140.4 148.2 111.3 130.9 30.2 26.4 22.8 39.5 67.5 34.6 26.6
2 δH (mult; J in Hz)
2 3 4 5 6 7 8 9 10 1´ 2´ 3´ 4´ 5´ 6´
6.20 (d, 1.5) 6.45 (d, 1.5)
7.79 (d, 1.5)
7.64 (d, 1.5)
2a δc 146.1 136.1 176.0 160.6 98.1 163.9 93.4 156.7 103.2 121.8 114.0 140.1 139.6 146.0 112.2
130.7 129.4 116.2 151.4 133.7 125.4 55.7
C NMR measured at 150 MHz; obtained in CD3OD with TMS as an internal standard.
δH (mult; J in Hz)
6.18(d, 2.1) 6.38 (d, 2.1)
7.34 (s)
7.34 (s)
δc 146.6 135.5 175.8 160.9 97.8 164.1 93.0 156.7 103.1 121.7 107.1 145.2 135.9 145.2 107.1
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2.6. Measurement of cell viability Cell viability was measured by using a 3-(4,5-dimeth ylthiazol-2-yl)-2,5-diphenyltetra-zoliumbromide (MTT) assay, which is based on the reduction of MTT to formazan by mitochondrial dehydrogenase. Briefly, the cells (3 × 104/200 μL medium) were treated with each sample, and incubated for 24 h at 37 °C. After the MTT reagent (0.5 mg/mL) (Sigma, St. Louis, MO, USA) was added to the medium and incubated for an additional 4 h, the medium was then removed and the MTT-formazan was dissolved in 200 μL dimethylsulfoxide (DMSO). The extent of the reduction of MTT to formazan was quantified by measuring the absorbance at 540 nm using a microplate reader. 2.7. Inflammatory cytokine assay Cytokine concentrations in the supernatants of macrophage cultures were determined by enzyme-linked immunosorbent assay (ELISA) using an antibody from R&D system, according to the manufacturer's instruction. The cells were incubated with LPS in the presence of different concentrations of each compound for 18 h. The supernatants were collected and stored at −80 °C before analysis. Standards were prepared from recombinant mouse cytokines separately (R&D system, Minneapolis, MN). 2.8. Western blotting Each compound in deionized distilled water (6.25, 12.5, and 25 μM) was added to medium containing RAW 264.7 macrophages and incubated for 18 h. The control used only deionized distilled water. The cells were harvested by washing with PBS, lysed with 100 μL of RIPA buffer (50 mM Tris, 0.1% SDS, 50 mM NaCl, 1% NP-40, 1 mM PMSF, 10 μg/mL aprotinin, and 10 μg/mL leupeptin, pH 7.4), and centrifuged at 10,000 ×g for 20 min. The protein content in the supernatant was quantified using a Bio-Rad protein assay kit. Samples containing equal amounts of protein (20 μg) were analyzed using 10% SDS-PAGE. Proteins in the SDS-PAGE gel were transferred to PVDF membranes (Bio-Rad) using the Trans-Blot apparatus (Bio-Rad). After transfer, the membranes were blocked in a solution of 5% nonfat dried milk (w/v) in TBS-T (24.8 mM Tris, 137 mM sodium chloride, 2.7 mM potassium chloride, and 0.05% Tween 20, pH 7.4) for 1 h at room temperature. After 6 washes with TBS-T, the membranes were
incubated with primary iNOS and COX-2 polyclonal antibody from Santa Cruz Biotechnology (Santa Cruz, CA, US) and diluted (1:1000) with TBS-T for 12 h at 4 °C. Membranes were washed and incubated at room temperature for 2 h with mouse anti-rabbit IgG HRP from Santa Cruz Biotechnology (diluted 1:1000). After washing, proteins were detected in a dark room using an Enhanced Chemiluminesence kit (Amersham Bioscience UK, Ltd., Little Chalfont, UK). The detection reagent was poured onto the membrane and incubated for 1 min, and the band density was quantified with an LAS4000 image analyzer (Fujifilm Life Science, Tokyo, Japan).
3. Results and discussion M. rubra leaves were extracted with 80% acetone and fractionated with n-hexane, EtOAc, and water. Three sulfatecontaining compounds (1–3) were isolated from the water layer using combined chromatographic separations. The compounds included a new diarylheptanoid sulfate (1), a new flavonol sulfate (2), and a known flavonol sulfate (3) (Fig. 1). Compound (3) was identified on the basis of spectroscopic analysis and comparison of spectral data with the literature [7]. Compound 1 was obtained as an off-white, amorphous powder on thin layer chromatography (TLC) had a bluishgreen color when heated after spraying with 10% H2SO4 and a light brown color when sprayed with FeCl3 solution. The 1H NMR spectrum (Table 1) of 1 showed 2 sets of an aromatic ABX spin systems at δH 7.03 (1H, dd, J = 8.4, 2.4 Hz, H-15), 6.79 (1H, d, J = 8.4 Hz, H-16), and 7.17 (1H, d, J = 1.8 Hz, H-18) and a meta-coupled doublets at δH 6.73 (1H, d, J = 1.8 Hz, H-5) and 6.85 (1H, d, J = 1.8 Hz, H-19), indicating the presence of two aromatic ring with different substitution patterns. The spectrum also included signals attributable to six aliphatic methylenes at δH 2.48 (1H, overlapped, H-7), 2.94 (1H, d, J = 16.8 Hz, H-7), 1.78 (1H, m, H-8), 2.24 (1H, m, H-8), 1.47 (1H, m, H-9), 1.71 (1H, m, H-9), 1.81 (1H, m, H-10), 2.07 (1H, dd, J = 12.0, 1.8 Hz, H-10), 1.88 (1H, m, H-12), 2.17 (1H, m, H-12), 2.76 (1H, d, J = 18.0 Hz, H-13) and 3.03 (1H, t, J = 14.4, H-13), one methoxyl group at δH 3.83 (3H, s, 4-OMe), and one oxymethine at δH 4.76 (1H, t, J = 9.6 Hz, H-11). The 13C NMR spectrum of 1 also showed two sets of aromatic rings at δC 111.1 (C-5), 115.8 (C-16), 125.5 (C-1, 2), 125.9 (C-19), 129.2 (C-15), 130.8 (C-14), 131.2 (C-6), 133.1 (C-18), 140.2 (C-3), 148.8(C-4), 150.6 (C-17), six aliphatic carbons at δC 30.1 (C-7), 25.6 (C-8), 22.6
Fig. 1. Structures of compounds 1–3 and their hydrolysates (1a–3a).
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(C-9), 36.1 (C-10), 32.9 (C-12), 26.2 (C-13), one methoxyl carbon at δC 55.4 (4-OMe), and one oxymethine carbon at δC 77.4 (C-11). These data closely resembled the spectra of juglanin B [16-Methoxytricyclo[12.3.1.12,6]nonadeca-1(18),2,4,6 (19),14,16-hexaene-3,9,17-triol] [4]. In the HMBC spectrum, 1 showed an HMBC correlation of H-7/C-5, C-6, C-19 and H-13/ C-14, C-15, C-18 between the aliphatic chain and two aromatic rings, indicating that the aliphatic chain was attached at two aromatic rings (Fig. 2). Furthermore, the presence of a signals for a hydroxyl groups δC 140.2 and 150.6, which were determined to be at C-3 and C-17 by the HMBC correlations of the H-5 (δH 6.73) and H-19 (δH 6.85) correlated with C-3, and the H-15 (δH 7.03) and H-18 (δH 7.17) correlated with C-17, respectively. One of the aromatic protons (1H, δH 6.73, H-5) correlated with the carbon signal at δC 148.8, which was assigned to the methoxy bearing C-4 based on long-distance correlations with O-methyl protons. The negative HR-FAB-MS and elemental analysis of 1 showed that the molecular formula of 1 was C20H23O7SK (anal. C 50.46, H 5.57, O 27.30, S 6.38%) with a pseudomolecular ion peak at m/z 445.0719 [M-H]− (calculated for C20H22O7SK, 445.0723). The IR spectrum had an absorption band at 1242 cm−1 ascribable to the S〓O vibration of a sulfate group. These results suggested the presence of a sulfate group in 1. In addition, acid hydrolysis [8] of 1 with pyridine-dioxane (1:1) afforded a desulfated product, juglanin B (1a), which is C20H24O4 [4]. In the view of HR-FAB-MS data, 1 was shown to be 118 (SO3K) mass units larger than 1a, compound 1 was presumed to be a sulfate derivative of 1a. Comparison of the 1H NMR spectra of 1 and 1a showed a downfield shift (+δH 0.77) for a proton (H-11) attached to an oxymethine carbon, in similarity to data reported earlier for steroid and carbohydrate sulfates [9,10]. Consistently, in the 13 C NMR spectrum of 1, C-11 (δC 77.4) shifted downfield (+δC 9.9) compared with 1a (δC 67.5). This sulfation shift was in good agreement with that published for sulfates of secondary alcohols [10]. These features suggested 1 as the 11-O-sulfate of 1a. The absolute configuration at C-11 of 1 was determined as R from its specific rotation ([α]25 D −16.9, MeOH), which had the same sign as of (R)-myricanol ([α]25 D −65.6, CHCl3) [11]. Based on these results, the structure of compound 1 was assigned to juglanin B (11R)-O-sulfate. Compound 2 was obtained as a pale yellow, amorphous powder. Compound 2 had a yellow color with 10% H2SO4
191
after heating and a dark green color with FeCl3 solution on TLC. The 1H NMR spectrum (Table 1) of 2 showed a meta-coupled two doublets at δH 6.20 (J = 1.5 Hz) and 6.45 ( J = 1.5 Hz) due to the C-6 and C-8 protons of a flavonoid skeleton. Two meta-coupled doublets at δH 7.64 (1H, d, J = 1.5 Hz, H-6´) and 7.79 (1H, d, J = 1.5 Hz, H-2´) arising from a flavonoid B-ring, suggested that this ring was an unasymmetrical 3´,4´,5´-trisubstitution pattern. The 13C NMR spectrum of 2 revealed 15 carbon signals, including δc 146.1 (C-2), 136.1 (C-3), and 176.0 (C-4) which are characteristic features of flavonol moiety. The chemical shifts of 2 were in good agreement with its hydrolysate, myricetin (2a), except those of B-ring [δc 111.8 (C-1´), 114.0 (C-2´), 140.1 (C-3´), 139.6 (C-4´), 146.0 (C-5´), and 112.2 (C-6´)]. The structure of 2 was suggested to be a sulfated myricetin by negative HR-FAB-MS data [m/z 434.9431 (calculated for C15H8O11SK, 434.9424)], elemental analysis (anal. C 41.55, H 3.36, O 40.85, S 4.93%), and IR absorption at 1199 cm−1 ascribable to the S〓O vibration of a sulfate group. In the 13C NMR spectrum, the effect of O-sulfate as an electron withdrawing group was well-known [12]. The carbon atoms ortho to the sulfate group experience a decreased electron density resulting in a downfield shift by 5.1–6.9 ppm. On the other hand, the carbon atom carrying the sulfate group showed an increased electron density resulting in an upfield shift by 3.7 ppm. The chemical shifts of these carbon atoms agree with those previously calculated for quercetin 3´-sulfate and phloroglucinol sulfates [13,14]. Based on these results, the structure of 2 was assigned to myricetin 3´-O-sulfate. Sulfated compounds are known for their universal occurrence in higher plants. But, the physiological significance of sulfates of secondary constituents in plant tissue is not clear. Apart from their possible involvement in neutralization of reactive hydroxyl groups, their accumulation in plants growing under saline conditions suggests a role in the sequestering of sulfate ions [15]. A large number of flavonoids are known to occur in plants in sulfated form [12,15]. Recent examples of other types of phenolic sulfates are the lignan, (+)-isolarisiresinol 3α-O-sulfate [16], and the coumarin sulfates [17], both containing sulfated alcohol groups. To demonstrate the anti-inflammatory activity of compounds 1–3 and their aglycones (1a–3a), we evaluated their
Table 2 Inhibitory effects of the compounds against LPS-stimulated expression of TNF-α, IL-1β, and IL-6. Compound
IC50 (μM)a TNF-α
1 1a 2 2a 3 3a PDTCb Quercetinc
1
a
± ± ± ± ± ± ± ±
2.14 1.57 2.45 2.47 1.50 3.04 2.13 0.81
22.9 17.9 20.2 19.2 N25 N25 18.0 16.9
± ± ± ±
IL-6 0.75 0.50 1.42 1.86
± 1.74 ± 0.34
22.7 20.3 22.2 23.9 N25 N25 16.8 16.8
± ± ± ±
1.61 1.42 1.14 1.73
± 2.40 ± 0.13
IC50 values were determined by regression analysis and are expressed as mean ± SD of 3 separate experiments. Used as positive control.
b,c
Fig. 2. Key HMBC correlations of 1.
20.1 19.5 19.9 19.7 20.3 21.9 16.8 13.6
IL-1β
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Fig. 3. Inhibitory effects of 1, 1a, 2, and 2a on LPS-induced expression of iNOS and COX-2 protein in RAW 264.7 cells. Cells were pretreated with 1, 1a, 2, and 2a for 1 h following LPS stimulation (1 μg/mL).
ability to inhibit LPS-induced cytokine (TNF-α, IL-1β and IL-6) production in RAW 264.7 cells. RAW 264.7 cells were treated with various concentrations (6.25, 12.5, and 25 μM) of the each compound and cell viability was measured by MTT assay. No compounds displayed significant cytotoxicity up to 25 μM, indicating that the anti-inflammatory effects of these compounds were not due to cytotoxicity (data not shown). The biphenyl-type diarylheptanoids (1 and 1a) and 3-hydroxyflavones (2 and 2a) significantly inhibited TNF-α, IL-1β, and IL-6 secretion in a concentration-dependent manner, whereas the 2,3-dihydroflavonols (3 and 3a) did not inhibit IL-1β or IL-6 production. A parent alcohol of the sulfate-containing diarylheptanoid, juglanin B (1a) had the most potent inhibitory activity with IC50 values of 19.5 ± 1.57, 17.9 ± 0.50 and 20.3 ± 1.42 μM, respectively (Table 2). Activated macrophages play an important role in inflammatory diseases related to overproduction of TNF-α, IL-1β, and IL-6, which are generated as a result of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) activation. The effects of compounds 1, 1a, 2, and 2a on LPS-induced expression of iNOS and COX-2 were investigated (Fig. 3). RAW 264.7 cells were stimulated with 1 μg/mL of LPS for 18 h in increasing concentrations of compounds 1, 1a, 2, and 2a, and iNOS and COX-2 protein levels were determined by western blotting. Compounds 1, 1a, 2, and 2a (6.25-25 μg/mL) dose-dependently reduced LPS-induced expression of iNOS and COX-2 but did not change β-actin expression. This result indicates that these compounds (1, 1a, 2, and 2a) suppress LPS-induced expression of iNOS and COX-2 at the transcriptional level. 4. Conclusion Two new sulfate-containing phenolic compounds (1 and 2) were isolated from M. rubra leaves and their structures
were determined from IR, FAB-MS, 1D and 2D NMR spectroscopic data. All isolated phenolic compounds (1–3) and their derivatives (1a–3a) were evaluated for anti-inflammatory potencies in reducing cytokine expression. The biphenyl-type diarylheptanoids (1 and 1a) and 3-hydroxyflavones (2 and 2a) significantly inhibited the secretion of TNF-α, IL-1β, and IL-6 and LPS-induced expression of iNOS and COX-2. Acknowledgment This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0022929). References [1] Moncada S, Palmer MJ, Higgs DA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 1992;43:109–34. [2] Lee HJ, Hyun EA, Yoon WJ, Kim BH, Rhee MH, Kang HK, et al. In vitro antiinflammatory and anti-oxidative effects of Cinnamomum camphora extracts. J Ethnopharmacol 2006;103:208–16. [3] Kim HH, Kim DH, Kim MH, Oh MH, Kim SR, Park KJ, et al. Flavonoid constituents in the leaves of Myrica rubra Sieb. et Zucc. with antiinflammatory activity. Arch Pharm Res 2013 [in press]. [4] Liu JX, Di DL, Wei XN, Han Y. Cytotoxic diarylheptanoids from the pericarps of walnuts (Juglans regia). Planta Med 2008;74:754–9. [5] Matsuda H, Higashino M, Chen W, Tosa H, Iinuma M, Kubo M. Studies of cuticle drugs from natural sources. III. Inhibitory effect of Myrica rubra on melanin biosynthesis. Biol Pharm Bull 1995;18:1148–50. [6] Lee SI, Yang JH, Kim DK. Antioxidant flavonoids from the twigs of Stewartia koreana. Biomol Ther 2010;18:191–6. [7] Nonaka GI, Muta M, Nishioka I. Myricatin, a galloyl flavanonol sulfate and prodelphinidin gallates from Myrica rubra. Phytochemistry 1983;22:237–41. [8] Ishida H, Miyamoto H, Kajino T, Nakayasu H, Nukaya H, Tsuji K. Study on the bile salt from megamouth shark. I. The structures of a new bile alcohol, 7-deoxyscymnol, and its new sodium sulfates. Chem Pharm Bull 1996;44:1289–92. [9] Goto J, Kato H, Hasegawa F, Nambara T. Synthesis of monosulfates of unconjugated and conjugated bile acids. Chem Pharm Bull 1979;27:1402–11.
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