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Isolation of Lignan and Fatty Acid Derivatives from the Grains of Echinochloa utilis and Their Inhibition of Lipopolysaccharide-Induced Nitric Oxide Production in RAW 264.7 Cells Duc Hung Nguyen,†,⊥ Bing Tian Zhao,† Duc Dat Le,† Young Ho Yoon,# Jee Youn Ko,# Koan Sik Woo,# Do Youn Jun,§ Young Ho Kim,§ and Mi Hee Woo*,† †

College of Pharmacy, Catholic University of Daegu, Gyeongsan 38430, Republic of Korea Functional Cereal Crop Research Division, Department of Functional Crop, NICS, RDA, Milyang 50426, Republic of Korea § Laboratory of Immunobiology, School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 39061, Republic of Korea ⊥ Phutho College of Pharmacy, Viettri City, Phutho Province 290000, Vietnam #

S Supporting Information *

ABSTRACT: Two new fatty acid derivatives, echinochlorins A (8) and B (9) and a racemic lignan, (±)-anti-1-(4-hydroxy-3methoxyphenyl)-2-{4-[(E)-3-acetoxypropen-1-yl]-2-methoxyphenoxy}propan-1,3-diol 3-acetate (1), were isolated from Echinochloa utilis grains, along with six known lignans (2−7) and two fatty acid derivatives (10, 11). Their structures were established by spectroscopic data analyses (IR, UV, HR-FABMS, GC-MS, and 1D and 2D NMR). The configuration of 1 was determined by Mosher’s method. Compound 5 displayed potential inhibitory activity on lipopolysaccharide-induced NO production in macrophage RAW 264.7 cells with an IC50 value of 4.8 ± 0.5 μM. These isolated compounds in crude EtOH extract were also quantitated by HPLC. KEYWORDS: Echinochloa utilis, NO inhibitory activity, lignans, echinochlorins A and B





INTRODUCTION

Echinochloa utilis, also known as Echinochloa esculenta (A. Braun), is a small-seeded subsistence cereal crop belonging to the Poaceae family. This plant is cultivated in Korea, Japan, and the northeastern part of China on a small scale at present.1 The grains of this plant are used as traditional food and can be cooked in a manner similar to rice (Oryza sativa L.). Japanese barnyard millet grains have been used for treatments of allergic diseases, such as atopic dermatitis.1 Some previous studies indicated that the grains contain high levels of proteins, lipids, amino acids, vitamin B1, and vitamin B2 and possess a high germination rate.2 The phytochemical study of this plant showed only three phenolic compounds (N-(p-coumaroyl)serotonin, luteolin, and tricin), which were isolated from Japanese barnyard millet grains with a strong antioxidant activity.3 However, there is no report for anti-inflammatory activity from the constituents of this plant until now. Hence, a bioassay-guided isolation of ethanol extract from the grains of E. utilis using chromatographic separation led to the isolation of seven lignans (1−7) and four fatty acid derivatives (8−11). In this paper, we describe the isolation, structural elucidation, and biological investigation of the isolates from the grains of E. utilis on LPS-induced NO production inhibition. Furthermore, the quantification of isolated compounds (1−11) in the crude EtOH extract was accomplished using high-performance liquid chromatography (HPLC) analysis. This study could be important to promote the development of market intelligence for agricultural commodities with anti-inflammatory function. © 2016 American Chemical Society

MATERIALS AND METHODS

General Procedures. The optical rotations were measured on a JASCO DIP-370 digital polarimeter. The infrared (IR) spectra were measured on a Mattson Polaris FT/IR-300E spectrophotometer. UV spectra were recorded in MeOH and CHCl3 using a Shimadzu spectrophotometer. The nuclear magnetic resonance (NMR) spectra were recorded in methanol-d4 and chloroform-d on an Oxford AS 400 MHz instrument (Varian, Palo Alto, CA, USA) or a 600 MHz instrument (Agilent, Santa Clara, CA, USA). High-resolution fast-atom bombardment mass spectrometry (HR-FAB-MS) data were collected on a Quattro II mass spectrometer. Column chromatography was performed on silica gel (Merck, Darmstadt, Germany; 63−200 μm particle size), RP-18 (Merck, 150 μm particle size), and Sephadex LH20 (Pharmacia Co. Ltd.). Fractions were monitored by TLC, and spots were visualized by spraying with 10% H2SO4 in ethanol, followed by heating. Thin layer chromatography (TLC) was performed on silica gel 60 F254 plates (Merck) with the following developing solvents: nhexane, methylene chloride, and ethyl acetate. Reversed phase (RP) TLC plates (Merck) were employed with the following developing solvents: methanol, acetonitrile, and water. The preparative HPLC runs were carried out using a Gilson system with an UV detector and an Optima Pak C18 column (10 × 250 mm, 10 μm particle size, RS Tech. Corp., Korea). The quantitative analyses were conducted on an HPLC chromatograph (Waters, Houston, TX, USA) and a Kinetex C18 column (4.6 × 250 mm, 5 μm particle size; Phenomenex, Torrance, CA, USA). Two chromatographic methods for quantitation Received: Revised: Accepted: Published: 425

November 28, 2015 December 29, 2015 January 2, 2016 January 2, 2016 DOI: 10.1021/acs.jafc.5b05638 J. Agric. Food Chem. 2016, 64, 425−432

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Journal of Agricultural and Food Chemistry

ε) 203 (4.23), 259 (3.84); IR (KBr) νmax cm−1 3666, 3416, 2971, 2337, 1736, 1598, 1510, 1477, and 1054; 1H and 13C NMR data (Table 1); HR-FAB-MS m/z 460.1731 (calcd for C24H28O9, m/z 460.1733).

of isolated compounds (1−11) were used as follows: 0−20 min (35% MeOH in H2O), 20−45 min (35−40% MeOH in H2O), 45−80 min (40−70% MeOH in H2O), and ultraviolet (UV) detection at 264 nm for lignan compounds (1−7); 0−50 min (62% THF in H2O) and UV detection at 258 nm for fatty acid derivatives (8−11). The flow rates for the mobile phase were set at 1.0 mL/min. (S)- and (R)-(−)-αmethoxy-α-(trifluoromethyl)phenylacetyl chlorides were purchased from Sigma-Aldrich. All other chemicals and solvents were of analytical grade and used without further purification. Plant Material. The grains of E. utilis were provided by the National Institute of Crop Science of Miryang in February 2013. A voucher specimen (EU.2013002) has been deposited at the College of Pharmacy, Catholic University of Daegu, Republic of Korea. Isolation Procedure of E. utilis Grains. The dried grains of E. utilis (30 kg) were extracted with standing 80% ethanol (20 L × 4 times) at 80 °C for 4 h. The combined ethanol extract was filtered and concentrated under reduced pressure to give the ethanol extract (1.1 kg). The concentration extract was then suspended in H2O (6 L) and partitioned successively between H2O and n-hexane (6 L × 7 times, 305 g), methylene chloride (6 L × 7 times, 68 g), ethyl acetate (6 L × 7 times, 35 g), n-butanol (9 L × 7 times, 130 g), and H2O-soluble fractions, respectively. The n-hexane, methylene chloride, ethyl acetate, n-butanol, and H2O-soluble fractions were tested on the NO production inhibition. Among them, the CH2Cl2 and EtOAc fractions exhibited strong activity. These fractions were further analyzed by TLC, and the result showed both the CH2Cl2 and EtOAc fractions to be quite similar. Therefore, they were combined for isolation. The CH2Cl2 and EtOAc fractions were chromatographed by using a silica gel column (63−200 μm particle size, 10 × 120 cm), eluting with a nhexane/EtOAc/MeOH gradient system (1:0:0 to 1:1:0 and 1:1:0.1 to 1:1:1, each 5 L), to give 14 fractions (A−N) according to their TLC patterns. These fractions were assayed again for NO production and DPPH assays. Fractions G and H demonstrated both strong antiinflammatory and antioxidant activities. Fraction H (2.4 g) was then subjected to chromatography over Sephadex LH-20 (2.5 × 120 cm) with methanol, to afford five subfractions (H-1−H-5). Subfraction H-3 (623 mg) was further subjected to RP-18 column chromatography (2.5 × 60 cm), eluted with MeOH/H2O (1:1, 2:1, 4:1, 8:1 and 12:1) to afford six fractions (H-3-1−H-3-6). Subfraction H-3-2 (386 mg) was chromatographed on silica gel (63−200 μm, 2.5 × 50 cm) eluted with CH2Cl2/EtOAc (4:1, 3 L) to yield compound 1 (15.0 mg). Subfraction H-3-5 (52.1 mg) was subjected to silica gel (63−200 μm, 2.5 × 50 cm) eluted with CH2Cl2/EtOAc (6:1, 3 L) to yield compound 8 (22.0 mg). Fraction H-4 (323 mg) was chromatographed on RP-18 with MeOH/H2O (3:2) to give six subfractions (H-4-1−H4-6). Subfaction H-4-3 (203 mg) was purified by RP-18 (4.5 × 50 cm, eluted with 66% MeOH in H2O, 4 L) to afford compound 2 (35.6 mg). Subfraction H-4-6 (32.1 mg) was chromatographed on RP-18 with MeOH/H2O (3:2) to yield compound 4 (12.0 mg). Fraction G (2.8 g) was subjected to RP-18 column chromatography with MeOH/ H2O (1:1, 2:1, 4:1, 8:1, 12:1) to afford 14 subfractions (G-1−G-14). Subfraction G-2 (98.7 mg) was subjected to semipreparative HPLC eluted with MeOH/H2O (50:50, 2 mL/min, UV 254 nm) to afford 5 (6 mg). Subfraction G-3 (183 mg) was chromatographed on silica gel (63−200 μm, 2.5 × 50 cm) eluted with CH2Cl2/acetone (10:1, 4 L) to afford two subfractions (G-3-1 and G-3-2). Subfaction G-3-1 (63 mg) was further purified by semipreparative HPLC eluted with MeOH/ H2O (56:44, 2 mL/min, UV 210 nm) to yield compounds 6 (6.7 mg) and 7 (16.6 mg), respectively. Subfraction G-7 (97 mg) was subjected to chromatography over Sephadex LH-20 eluted with MeOH to give compound 3 (37 mg). Subfraction G-8 (25 mg) was also purified using Sephadex LH-20 eluted with MeOH to yield 9 (8.2 mg). Subfraction G-13 (54.5 mg) was chromatographed on RP-18 (4.5 × 50 cm, eluted with 30% acetonitrile in H2O, 6 L) to give 10 (23 mg). Subfraction G-14 (180 mg) was also subjected to RP18 column chromatography (4.5 × 50 cm, eluted with 50% acetonitrile in H2O, 6 L) to yield 11 (61 mg). (±)-anti-1-(4-Hydroxy-3-methoxyphenyl)-2-{4-[(E)-3-acetoxypropen-1-yl]-2-methoxyphenoxy}propane-1,3-diol 3-acetate (1): colorless oil; [α]20D 0° (c 0.05, MeOH); UV (MeOH) λmax nm (log

Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Data for 1 (CD3OD) δH (J in Hz)

position 1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′a 9′b OCH3 OCH3 OCOCH3 OCOCH3 OCOCH3 OCOCH3

6.87 (1H, dd, 1.9, 8.3) 6.91 (1H, d, 8.3)

7.01 6.55 6.18 4.64

(1H, (1H, (1H, (2H,

d, 1.9) d, 15.9) dt, 6.5, 15.9) dd, 1.1, 6.5)

6.82 (1H, dd, 1.8, 8.3) 6.72 (1H, d, 8.3)

6.98 4.81 4.52 4.18 3.97 3.78 3.88 1.87 1.88

(1H, (1H, (1H, (1H, (1H, (3H, (3H, (3H, (3H,

d, 1.8) d, 5.8) m) dd, 3.7, 11.9) dd, 6.2, 11.9) s) s) s) s)

δC 132.6 121.1 118.7 149.6 149 111.7 135 123.2 64.9 133.4 121 116 147.5 152 111.9 74.5 83.4 66.4 56.5 56.7 21 20.7 172.6 172.9

Echinochlorin A (8): white amorphous powder; [α]20D + 20.2° (c 0.005, CHCl3); UV (CHCl3) λmax nm (log ε) 244 (4.55), 292 (4.52); IR (KBr) νmax cm−1 3399, 2916, 2850, 1715, 1630, 1596, 1516, 1466, 1267, and 1170; 1H and 13C NMR data (Table 2); HR-FAB-MS at m/ z 503.2595 [M + Na]+ (calcd for C26H40O8Na+, m/z 503.2621). Echinochlorin B (9): pale amorphous powder; UV (MeOH) λmax nm (log ε) 203 (4.39), 278 (4.64); IR (KBr) νmax cm−1 3441, 2928, 1676, 1666, 1583, 1407, 1240, 1076, 996, and 990; 1H and 13C NMR data (Table 2); HR-FAB-MS at m/z 309.2063 [M + H]+ (calcd for C26H41O8+, m/z 309.2066). Preparation of Mosher Esters. A previously described method was used.6,7 To 0.5 mg of compound 1 in 0.5 mL of CH2Cl2 were added sequentially 5 drops of pyridine, 0.5 mg of 4-(dimethylamino)pyridine, and 12 mg of (R)-(−)-α-methoxy-α-(trifluoromethyl)phenylacetyl (MTPA) chloride. The mixture was stirred until completely dissolved, and then it was left at room temperature overnight and subsequently purified over a microcolumn (6 × 0.6 cm) using silica gel (230−400 mesh) eluted with 3−4 mL of n-hexane/ CH2Cl2 (1:2). Then the eluate was dried, CH2Cl2 (5 mL) was added, and the solution was washed using 1% NaHCO3 (5 mL × 3) and H2O (5 mL × 2); the washed solution was dried in vacuo to yield the S Mosher ester (1S) of 1. The treatment of 1 (0.5 mg) using (S)(+)-MTPA chloride as mentioned above yielded the corresponding R Mosher ester (1R). Cell Culture. The RAW 264.7 cells were obtained from the Korean Cell Line Bank (KCLB, Chongno-gu, Seoul, Korea) and maintained in Dulbecco’s modified essential medium (DMEM). These cells were grown at 37 °C in DMEM supplemented with 10% heat-inactivated fetal bovine serum and penicillin (100 U/mL)/streptomycin (100 μg/ mL) in a humidified atmosphere of 5% CO2. Cells were seeded at an initial density of 5 × 104 cells/well in a 6-well tissue culture plate and pre-incubated with 12.5, 25.0, and 50.0 μg/mL of each test compound for 60 min. Cells were stimulated with LPS (0.1 μg/mL) and 426

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Table 2. 1H (600 MHz) and 13C (150 MHz) NMR Data for 8 (CDCl3), and 1H (400 MHz) and 13C (100 MHz) NMR Data for 9 (CD3OD) 8

9

position

δH (J in Hz)

δC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ OCH3 1″ 2″ 3″a 3″b

4.32−4.40 (2H, overlapped) 1.83 (2H, m) 1.90 (2H, m) 1.46−1.60 (2H, m) 1.46−1.60 (2H, m) 1.46−1.60 (2H, m) 1.46−1.60 (2H, m) 1.46−1.60 (2H, m) 1.46−1.60 (2H, m) 1.46−1.60 (2H, m) 1.83 (2H, m) 2.55 (2H, t, 6.1)

64.8 29.8 29.7 26.2 28.9 29.3 29.4 29.6 29.7 29.8 25.1 34.3 174.5

δH (J in Hz)

δC

2.20 (2H, t, 7.4) 1.50−1.55 (2H, m) 1.22−1.29 (2H, m) 1.22−1.29 (2H, m) 1.50−1.55 (2H, m) 2.56−2.61 (2H, m) 6.48−6.52 7.20−7.24 7.20−7.24 6.48−6.52

(1H, (1H, (1H, (1H,

m) m) m) m)

2.56−2.61 (2H, m) 1.50−1.55 (2H, m) 1.22−1.29 (2H, m) 1.22−1.29 (2H, m) 0.83 (3H, t, 6.9) 7.12 (1H, d, 8.0) 7.27 (1H, d, 8.0) 7.24 (1H, br s) 7.81 (1H, d, 15.8) 6.50 (1H, d, 15.8) 4.13 (3H, overlapped) 4.32−4.40 (2H, overlapped) 4.13 (1H, overlapped) 3.80 (1H, dd, 4.9, 9.8) 3.90 (1H, br d, 9.8)

177.8 35.0 26.1 30.1 30.0 25.0 41.8 202.8 137.5 141.0 141.0 137.5 202.7 41.8 25.1 32.7 23.7 14.4

148.2 115.0 123.2 127.2 109.6 147.0 144.9 115.8 167.7 56.1 65.3 70.4 63.6

incubated at 37 °C for 24 h. Cell supernatants were used for determination of nitrite concentration; cells were used for measurement of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions. Determination of NO Production and the Cell Viability Assay. Detection of accumulated nitrites in the cell supernatants was performed using Griess reagent as described previously.4 Briefly, the RAW 264.7 cells (5 × 104 cells/mL) were stimulated with or without 0.1 μg/mL of LPS for 24 h in the presence or absence of the test compounds (12.5, 25.0, and 50.0 μg/mL). The culture supernatant was used for nitric dioxide determination using Griess reagent.5 Equal volumes of culture supernatant and Griess reagent were mixed, and the absorbance was determined at 570 nm. A cell viability test was performed based on the reduction of [3-(4,5-demethylthiazol-2-yl)2,5-diphenyltetrazolium bromide] (MTT) (Sigma Chemical Co., St. Louis, MO, USA) reagent into an insoluble, dark purple formazan product in viable cells (5 × 104 cells/mL) incubated with test compounds (12.5, 25.0, and 50.0 μg/mL) for 24 h. Then, 50 μL of 2 mg/mL MTT reagent was added to the culture plates and further incubated at 37 °C for 2 h, and absorbance was determined at 570 nm.5 Immunoblot Analysis. Proteins were extracted from cells in icecold lysis buffer (50 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 1.0 mM EDTA, 1.0 mM phenylmethanesulfonyl fluoride, 1.0 μg/mL leupeptin, 1.0 mM sodium vanadate, 150 mM NaCl). Cell samples were loaded into wells in the gel, and then 50 μg of protein per lane was separated by sodium dodecyl sulfate−polyacrylamide gel electrophoresis and followed by transfer to a polyvinylidene difluoride membrane

(Millipore, Bedford, MA, USA). The membranes were blocked at 25 °C for 30 min with phosphate-buffered saline containing 5% skim milk and then incubated with the corresponding antibody. Antibodies against iNOS and COX-2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). After binding of an appropriate secondary antibody coupled to horseradish peroxidase, proteins were detected by enhanced chemiluminescence according to the instructions of the manufacturer (Amersham Pharmacia Biotec, Buckinghamshire, UK). Statistical Analysis. Tests were conducted in triplicate, with data reported as the mean ± standard deviation. The significance level of differences in means was detected using a two-way ANOVA test. Statistics were analyzed using Graphpad Prism software (version 5). Statistical significances were defined at p ≤ 0.05.



RESULTS AND DISCUSSION To investigate the active components with inhibitory activity against LPS-induced NO production, the methylene chloride and ethyl acetate fractions were subjected to a succession of chromatographic procedures, including silica gel chromatography, Sephadex LH-20, RP-C18, and semipreparative HPLC, to afford 11 compounds, including a racemic lignan (1) and 2 new fatty acid derivatives (8 and 9) along with 8 known compounds corresponding to 6 lignans (2−7) and 2 fatty acid esters (10 and 11). The structures of the known lignan and fatty ester compounds were identified as schisphenlignan I (2),8 427

DOI: 10.1021/acs.jafc.5b05638 J. Agric. Food Chem. 2016, 64, 425−432

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Figure 1. Chemical structures of compounds 1−11 isolated from the grains of E. utilis.

and aromatic substituents (1598, 1510, and 1477 cm−1). The H NMR spectrum for 1 showed two aromatic ABX spin systems [at δH 6.87 (1H, dd, J = 1.9, 8.3 Hz), 6.91 (1H, d, J = 8.3 Hz), and 7.01 (1H, d, J = 1.9 Hz) and 6.82 (1H, dd, J = 1.8, 8.3 Hz), 6.72 (1H, d, J = 8.3 Hz), and 6.98 (1H, d, J = 1.8 Hz)] and a pair of trans-olefinic protons [at δH 6.55 (1H, d, J = 15.9 Hz) and δH 6.18 (1H, dt, J = 6.5, 15.9 Hz)]. The 13C NMR spectrum for 1 displayed 24 carbon signals containing 2 carbonyls, 2 methoxyl groups, 2 oxymethylenes, and 2 oxymethines, supported by gHMQC spectrum. Additionally, the 1H−1H gCOSY spectrum for 1 indicated correlations between olefinic proton (δH 6.18) and oxymethylene proton (δH 4.64). Other correlations of oxymethine proton (δH 4.52) to oxymethine proton (δH 4.81) and oxymethylene proton (δH 4.18) were also observed. These data suggested that 1 was a dimeric coniferyl alcohol acetate derivative, which was further confirmed by gHMBC experiments (Figure 3). The gHMBC

dimeric coniferyl acetate (3),9 (+) syringaresinol (4),10 lucidenal (5),11 9,9′-bisacetyl-neo-olivil (6),12 (2S,3S,4S,5S)3,4-furandimethanol, tetrahydro-2,5-bis(4-hydroxy-3-methoxyphenyl)-3,4-diacetate (7),12 2′,3′-dihydroxypropyl nonadecanoate (10),13 and (8E,11E)-2′,3′-dihydroxypropyl octadeca8,11-dienoate (11) by comparing their NMR data with those in the literature or gas chromatography−mass spectrometry analysis data (Figure 1). All isolated compounds here are reported for the first time from E. utilis. In addition, we also report the results of the analysis to determine the isolated compounds (1−11) in the total 80% EtOH extract of the E. utilis grains using representative HPLC (Figure 2). Structural Elucidation. Compound 1 was obtained as a colorless oil with an optical rotation of [α]20D 0° (c 0.05, MeOH). The HR-FAB-MS peak indicated that the molecular formula for 1 was C24H28O9. The IR spectrum showed typical absorption bands of hydroxyl (3416 cm−1), ester (1736 cm−1),

1

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Figure 2. HPLC chromatograms of 80% EtOH extract for E. utilis grains at (A) 264 nm (1−7) and (B) 258 nm (8−11). (±)-anti-1-(4-Hydroxy-3methoxyphenyl)-2-{4-[(E)-3-acetoxypropen-1-yl]-2-methoxyphenoxy}propane-1,3-diol 3-acetate (1); schisphenlignan I (2); dimeric coniferyl acetate (3); (+)-syringaresinol (4); lucidenal (5); 9,9′-bisacetyl-neo-olivil (6); (2S,3S,4S,5S)-3,4-furandimethanol, tetrahydro-2,5-bis(4-hydroxy-3methoxyphenyl)-3,4-diacetate (7); echinochlorin A (8); echinochlorin B (9); 2′,3′-dihydroxypropyl nonadecanoate (10); (8E,11E)-2′,3′dihydroxypropyl octadeca-8,11-dienoate (11).

Figure 3. 1H−1H COSY (black, bold line) and 1H−13C key HMBC (red arrow line) correlations established for compound 1 and new compounds 8 and 9.

Table 3. Characteristic 1H NMR Data of Mosher Esters (1S and 1R) of Compound 1 (600 MHz, CDCl3)

spectrum showed the correlations of H-8′ to C-5 and C-7′ and of H-8 to C-1. Therefore, compound 1 was identified as 1-(4hydroxy-3-methoxyphenyl)-2-{4-[(E)-3-acetoxypropen-1-yl]-2methoxyphenoxy}propane-1,3-diol 3-acetate. Because the C-7′ position of 1 is a carbinol center, Mosher’s method was applied to determine the absolute configuration. Compound 1 was treated with (R)- and (S)-MTPA chlorides to give (S)- and (R)-MTPA esters, 1S and 1R, respectively. Differences in the chemical shifts of protons H-8′, H-7′, and H9′ab were observed between the two MTPA esters of 1 (ΔδSR = δS − δR), which may reveal the absolute configuration at C-7′. The characteristic 1H NMR data of 1S and 1R (Table 3) showed the same pattern in which the signal of H-7′ was shifted downfield and separated into two signals at δH 6.35 (d, J = 6.2 Hz) and 6.27 (d, J = 6.2 Hz) with an approximate 1:1 ratio. Similarly, the signals of H-8′ appeared as two signals at δH 4.68

1S

1R

position

δH (J in Hz)

δH (J in Hz)

7′ 8′ 9′a

6.35 (d, 6.2) or 6.27 (d, 6.2) 4.68 (m) or 4.64 (m) 4.20 (dd, 4.5, 12.0) or 4.13 (dd, 4.5, 12.0) 3.90 (dd, 5.2, 12.0) or 3.85 (dd, 5.2, 12.0)

6.27 (d, 6.2) or 6.35 (d, 6.2) 4.64 (m) or 4.68 (m) 4.13 (dd, 4.5, 12.0) or 4.20 (dd, 4.5, 12.0) 3.85 (dd, 5.2, 12.0) or 3.90 (dd, 5.2, 12.0)

9′b

(m) and 4.64 (m). In addition, H-9′a showed two signals at δH 4.20 (dd, J = 4.5, 12.0 Hz) and 4.13 (dd, J = 4.5, 12.0 Hz), and H-9′b exhibited two signals at δH 3.90 (dd, J = 5.2, 12.0 Hz) and 3.85 (dd, J = 5.2, 12.0 Hz). This observation suggested that 1 was a pair of (7′S)- and (7′R)-isomers in a 1:1 mixture. In a 429

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Journal of Agricultural and Food Chemistry

0.83). The 13C NMR spectrum of 9 showed 18 carbon resonances for a carboxylic acid, 2 carbonyl, 4 olefinic methine, and 10 methylene groups, which were supported by HSQC. The 1H and 13C NMR spectra for 9 were similar to those of (10E,12E)-9,14-dioxo-10,12-octadecadienoic acid, except for C16 and C-6 of two methylene groups in 9 at δC 32.7 and 25.0 but δC 27.3 and 30.4 in (10E,12E)-9,14-dioxo-10,12-octadecadienoic acid, respectively.15 The 1H−1H COSY spectrum for 9 indicated the correlations of H-2 to H-3 and of H-17 to H-18 (Figure 3). The key HMBC exhibited the correlations from H2 (δH 2.20) to C-1 (δC 177.8), C-3 (δC 26.1), and C-4 (δC 30.1). Significant correlations between H-8 and C-17 (δC 23.7)/C-16 (δC 32.7) and between H-14 (δH 2.56−2.61) and C-15 (δC 25.1)/C-16 (δC 32.7) were also observed (Figure 3). On the basis of the above data, the structure of compound 9 was defined as (9E,11E)-8,13-dioxooctadeca-9,11-dienoic acid, a new natural product, and named echinochlorin B. HPLC Analysis. We studied the HPLC profile of the 80% EtOH extract of E. utilis grains and quantitatively identified the 11 peaks by comparing the retention times with the reference compounds isolated from the CH2Cl2 and EtOAc fractions. The chromatogram for lignans 1−7 (Figure 2A) exhibited the retention times of (±)-anti-1-(4-hydroxy-3-methoxyphenyl)-2{4-[(E)-3-acetoxypropen-1-yl]-2-methoxyphenoxy}propane1,3-diol 3-acetate (1), schisphenlignan I (2), dimeric coniferyl acetate (3), (+)-syringaresinol (4), lucidenal (5), 9,9′-bisacetylneo-olivil (6), (2S,3S,4S,5S)-3,4-furandimethanol, and tetrahydro-2,5-bis(4-hydroxy-3-methoxyphenyl)-3,4-diacetate (7). The chromatogram for fatty acid derivatives 8−11 (Figure 2B) displayed the retention times of echinochlorin A (8), echinochlorin B (9), 2′,3′-dihydroxypropyl nonadecanoate (10), and (8E,11E)-2′,3′-dihydroxypropyl octadeca-8,11-dienoate (11). According to our results (Table 4), the

comparison of the proton resonances of 1 with those of a pair of the syn- and anti-isomers of 1-(4-hydroxy-3-methoxyphenyl)2-{4-[(E)-3-acetoxypropen-1-yl]-2-methoxyphenoxy}propane1,3-diol 3-acetate, the 1H and 13C NMR data of 1 were most consistent with the anti-isomer. The difference was the coupling constant at H-7′ (J = 5.8 Hz) for 1 in our result, whereas the J values were 3.6 Hz for the syn-form and 7.2 Hz for the anti-form in the study of Valcic et al.9 In addition, irradiation of H-7′ caused signal enhancement of H-8′; thus, no NOE effect was observed between H-7′ and H-8′, indicating an anti-position between H-7′ and H-8′ in compound 1. Therefore, compound 1 was suspected to be a racemic mixture of the anti-isomers, 1a (7′S,8′S) and 1b (7′R,8′R) (Figure 1), which was further confirmed by [α]20D 0°. Thus, compound 1 was established as a racemic mixture (1a and 1b) of anti-1-(4hydroxy-3-methoxyphenyl)-2-{4-[(E)-3-acetoxypropen-1-yl]-2methoxyphenoxy}propane-1,3-diol 3-acetate. Compound 1 was first isolated as a racemic mixture from Chilean propolis as threo (anti) and erythro (syn) configurations, but their absolute stereochemistries had not been determined.9 Compound 8 was obtained as a white amorphous powder with an optical rotation of [α]20D +20.2° (c 0.005, CHCl3). The HR-FAB-MS peak at 503.2595 [M + Na]+ (calculated for C26H40O8Na+, m/z 503.2621) indicated that the molecular formula of 8 was C26H40O8. The IR spectrum showed typical absorption bands of hydroxyl (3399 cm−1), carbonyl (1715 cm−1), and aromatic substituents (1596, 1516, and 1466 cm−1). The 1H NMR spectrum for 8 displayed an ABX spin system at δH [7.12 (1H, d, J = 8.0 Hz), 7.27 (1H, d, J = 8.0 Hz), 7.24 (1H, br s)], a pair of trans-olefinic protons at δH 7.81 (1H, d, J = 15.8 Hz) and δH 6.50 (1H, d, J = 15.8 Hz), and a methoxyl proton at δH 4.13 (3H, overlapped), indicating the ferulic acid moiety. In addition, the 1H NMR spectrum of 8 showed an oxymethine and two oxymethylene groups, which were assigned to a glycerol moiety. The 13C NMR spectrum for 8 showed two carbonyl, three oxymethylene, an oxymethine, and several methylene groups, which were supported by the gHMQC spectrum. On the basis of these spectroscopic data, compound 8 was a saturated fatty ester conjugated with ferulic acid and glycerol moiety. Detailed correlations of 8 were confirmed together with 1H−1H gCOSY and gHMBC experiments. The gHMBC spectrum revealed the significant correlations of H-1″ to C-13 and H-1 to C-9′ (Figure 3). The carbon signals at C-1″ (δC 65.3), C-2″ (δC 70.4), and C-3″ (δC 63.6) of the 2,3-dihydroxypropyl moiety were similar to those of (2R)-2,3-dihydroxypropyl butyrate.14 Hence, the configuration of C-2″ position was R. Therefore, compound 8 was identified as (2″R)-2″,3″-dihydroxypropyl 13-((4′-(1′-hydroxy6′-methoxyphenyl)acryloyl)oxy)tridecanoate, a new natural product, named echinochlorin A. Compound 9 was obtained as a pale amorphous powder. The HR-FAB-MS peak at 309.2063 [M + H]+ (calculated for C26H41O8+, m/z 309.2066) indicated that the molecular formula of 9 was C26H40O8. The IR spectrum showed typical absorption bands of hydroxyl (3441 cm−1) and carbonyl (1676 and 1666 cm−1). The 1H NMR spectrum for 9 showed four olefinic methine protons resonating at δH 7.20−7.24 (2H, m) and 6.48−6.52 (2H, m), which suggested the presence of two conjugated enone systems. These proton signals may be interpreted as the AA′BB′ system of a conjugated diene−dione. The 1H NMR for 9 demonstrated the signals of 10 methylene groups at δH [2.20 (2H, t, J = 7.4), 1.50−1.55 (6H, m), 1.22− 1.29 (8H, m), 2.56−2.61 (4H, m)] and a methyl group (δH

Table 4. Inhibition of LPS-Induced NO Production of Isolated Compounds 1−11 in Macrophage RAW 264.7 Cells and Contents of 1−11 in Total 80% EtOH Extract forE. utilis Grains compound

IC50 valuea (μM)

80% EtOH extract (mg/g)

1 2 3 4 5 6 7 8 9 10 11 dexamethasoneb

101.7 ± 7.6 51.9 ± 4.7 36.0 ± 4.7 >200 4.8 ± 0.5 27.2 ± 4.3 >200 >200 20.8 ± 1.6 >200 60.1 ± 2.8 1.0 ± 0.1

0.06 1.23 0.94 0.24 0.16 0.27 0.20 0.32 0.11 0.15 0.23

a

The inhibitory effects are represented as the concentration (μM) giving 50% inhibition (IC50) relative to the control. bDexamethasone was used as a positive control. These data represent the average values of three repeated experiments.

quantification of isolated compounds in EtOH extract indicated that compound 2 was obtained at the highest concentration of 1.23 mg/g. Other compounds (1 and 3−11) have been determined as 0.06, 0.94, 0.24, 0.16, 0.27, 0.20, 0.32, 0.11, 0.15, and 0.23 mg/g, respectively. 430

DOI: 10.1021/acs.jafc.5b05638 J. Agric. Food Chem. 2016, 64, 425−432

Article

Journal of Agricultural and Food Chemistry Effects of Isolated Compounds (1−11) on LPSInduced NO Production and iNOS and COX-2 Expression in RAW 264.7 Cells. The isolated compounds from E. utilis were tested for their cytotoxic activities against RAW 264.7 macrophages by MTT method and then tested for inhibitory activities against LPS-induced NO production in this cell line under the concentration ranges from 12.5 to 50.0 μg/ mL (Figure 4). The results showed that the cell viability effect

Figure 5. Inhibition of LPS-induced iNOS and COX-2 expression in RAW 264.7 cells by (A) positive control, dexamethasone (DM), and (B) compounds 1, 2, 3, 5, 6, and 9. The results are representatives of at least three independent experiments.

Figure 4. Inhibitory effect of compounds 1, 2, 3, 5, 6, 9, and 11 on the LPS-induced NO production in RAW 264.7 macrophages. RAW 264.7 macrophages were pretreated with different concentrations (12.5, 25.0, and 50.0 μg/mL) of the tested compounds for 1 h, then with LPS (0.1 μg/mL), and incubated for 24 h. Control value was obtained in the absence of LPS and compounds. Dexamethasone (DM) was used as a positive control. These data are expressed as the mean ± SD (n = 3). (∗) p < 0.05, (∗∗) p < 0.01, and (∗∗∗) p < 0.001 are compared with the LPS-induced group.

reduced the LPS-induced iNOS expression. Therefore, these compounds could suppress LPS-induced iNOS expressions at the transcription level. In addition, pre-incubation of cells with compounds 2, 3, 5, and 6 significantly suppressed the LPSinduced expression of COX-2 protein (Figure 5). We have also evaluated the iNOS and COX-2 protein expression for compound 11. However, Western blot assay result indicated that the expressions of iNOS and COX-2 proteins at each treatment concentration level remained the same as the LPSinduced control. These data suggest that the NO inhibitory effect of compound 11 may be produced via a mechanism other than iNOS and COX-2 mechanism, such as COX-1, TNF-α, or IL-6. When the structure−activity relationships were investigated, some conclusions were deduced. Compound 3, which possessed an acetyl group, afforded stronger inhibitory activity (IC50 value of 36.0 ± 4.7 μM) against NO production than compound 2 with a hydroxyl group (IC50 value of 51.9 ± 4.7 μM). Comparison with the IC50 values of compounds 6 and 7, a pair of lignan isomers, suggested that the (2R,3S,4S,5R)isomer, 6, possessed much stronger inhibitory activity (IC50 value of 27.2 ± 4.3 μM) than the (2S,3S,4S,5S)-isomer, 7, form (IC50 value > 200 μM) (Table 4). The anti-inflammatory activities of lignan derivatives were attributed to the hydroxyl and phenol groups in the molecules, and these groups are also essential for the inhibition of prostaglandin synthetase and for leucotriene synthesis.18 Compound 11 with two double bonds had stronger activity (IC50 value of 21.3 ± 1.0 μM) than compound 10 with no double bonds (IC50 value > 200 μM).

of these compounds was >95% at the treated concentrations (12.5, 25.0, and 50.0 μg/mL). In the anti-NO production assay, compound 5, a rare secolignan, exhibited potential inhibitory effect with a half-maximal inhibitory concentration (IC50) value of 4.8 ± 0.5 μM. The α-methylene group of 5 may be responsible for the anti-inflammatory activity, and compound 5 might be considered as a lead compound for anti-inflammatory agents. Tsutsui et al.16 also reported that the α-methylene group in the structures of the other secolignans (peperomin E and 2,6-didehydropeperomin B) is important for IκB inhibitory degradation upon stimulation with TNF-α or interleukin-1 in the common NF-κB signaling pathway.16 Compounds 3, 6, and 9 exhibited significant inhibition with IC50 values of 36.0 ± 4.7, 27.2 ± 4.3, and 20.8 ± 1.6 μM, respectively. Compounds 2 and 11 showed moderate effect (IC50 values of 51.9 ± 4.7 and 60.1 ± 2.8 μM). Compound 1 displayed weak effect (IC50 value of 101.7 ± 7.6 μM), whereas compounds 4, 7, 8, and 10 were very weak or inactive (IC50 values > 200 μM) (Table 4). Dexamethasone, an anti-inflammatory drug, was used as a positive control (IC50 value of 1.0 ± 0.1 μM).17 Western blot analyses were performed to determine the inhibitory effects of active compounds on pro-inflammatory mediators related to the modulation of iNOS and COX-2 expression. As shown in Figure 5, compounds 1, 2, 3, 5, 6, and 9 dose-dependently 431

DOI: 10.1021/acs.jafc.5b05638 J. Agric. Food Chem. 2016, 64, 425−432

Article

Journal of Agricultural and Food Chemistry

Sun, H. D. Six new lignans from the leaves and stems of Schisandra sphenanthera. Fitoterapia 2013, 86, 171−177. (9) Valcic, S.; Montenegro, G.; Timmermann, B. N. Lignans from Chilean propolis. J. Nat. Prod. 1998, 61, 771−775. (10) Das, B.; Venkataiah, B.; Kashinatham, A. (+)-Syringaresinol from Parthenium hysterophorus. Fitoterapia 1999, 70, 101−102. (11) Sriyatep, T.; Chakthong, S.; Leejae, S.; Voravuthikunchai, S. P. Two lignans, one alkaloid, and flavanone from the twigs of Feroniella lucida. Tetrahedron 2014, 70, 1773−1779. (12) Chaurasia, V. N.; Wichtl, M. Phenylpropane und lignane aus der Wurzel von Urtia dioica L. Dtsch. Apoth. Ztg. 1986, 126, 1559−1563. (13) Gong, X. M.; Wang, S.; Zhou, X. L.; Zhou, D. D.; Dai, H.; Deng, J. G. Study on chemical constituents of Citrullus vulgaris Schrad vine (II). J. Chin. Med. Mater. 2013, 36, 1614−1616. (14) Lokhande, M. N.; Chopade, M. U.; Bhangare, D. N.; Milind, D. N. Asymmetric synthesis of propranolol, naftopidil and (R)monobutyrin using a glycerol desymmetrization strategy. J. Braz. Chem. Soc. 2013, 24, 409−413. (15) Amakura, Y.; Kondo, K.; Akiyama, H.; Ito, H.; Hatano, T.; Yoshida, T.; Maitani, T. Conjugated ketonic fatty acid from Pleurocybella porrigens. Chem. Pharm. Bull. 2006, 54 (8), 1213−1215. (16) Tsutsui, C.; Yamada, Y.; Ando, M.; Toyama, D.; Wu, J.; Wang, L.; Taketani, S.; Kataoka, T. Peperomins as anti-inflammatory agents that inhibit the NF-κB signaling pathway. Bioorg. Med. Chem. Lett. 2009, 19, 4084−4087. (17) Kim, D. H.; Chung, J. H.; Yoon, J. S.; Ha, Y. M.; Bae, S.; Lee, E. K.; Jung, K. J.; Kim, M. S.; Kim, Y. J.; Kim, M. K.; Chung, H. Y. Ginsenoside Rd inhibits the expressions of iNOS and COX-2 by suppressing NF-kB in LPS-stimulated RAW 264.7 cells and mouse liver. J. Ginseng Res. 2013, 37 (1), 54−63. (18) Iwakami, S.; Shibuya, M.; Tseng, C. F.; Hanaoka, F.; Sankawa, U. Inhibition of arachidonate 5-lipoxygenase by phenolic compounds. Chem. Pharm. Bull. 1986, 34, 3960−3963. (19) Araujo, C. A.; Alegrio, L. V.; Gomes, D. C.; Lima, M. E.; Gomes, C. L.; Leon, L. L. Studies on the effectiveness of diarylheptanoids derivatives against Leishmania amazonensis. Mem. Inst. Oswaldo Cruz 1999, 94, 791−794. (20) Kim, D. S.; Park, S. Y.; Kim, J. K. Curcuminoids from Curcuma longa L. (Zingiberaceae) that protect PC12 rat pheochromocytoma and normal human umbilical vein endothelial cells from βA (1−42) insult. Neurosci. Lett. 2001, 303, 57−61.

Compounds 9 and 11 possess two double bonds, which are responsible for their anti-inflammatory abilities.19 The presence of a diene ketone system in the new compound (9) provides lipophilicity to the compound and, thus, probably provides better skin penetration.20 Our results suggest that the isolated compounds partially contribute to the anti-inflammatory effect of the E. utilis grains and are expected to be suitable for applications as supplemental and/or functional foods with beneficial effects against various inflammatory-related diseases.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b05638. NMR spectral data and HR-FAB-MS spectra of compounds 1, 8, and 9 (PDF)



AUTHOR INFORMATION

Corresponding Author

* (M.H.W.) Phone: +82-53-850-3620. Fax: +82-53-359-6731. E-mail: [email protected]. Funding

This work was supported by the Cooperative Research Program for Agriculture Science & Technology Development (Project PJ009865), Rural Development Administration, Republic of Korea. Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS We are grateful to Korea Basic Science Institute (KBSI) for mass spectral measurements. REFERENCES

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DOI: 10.1021/acs.jafc.5b05638 J. Agric. Food Chem. 2016, 64, 425−432