Antioxidant and Anti-inflammatory Active Dihydrobenzofuran

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Antioxidant and Anti-inflammatory Active Dihydrobenzofuran Neolignans from the Seeds of Prunus tomentosa Qing-Bo Liu,†,‡ Xiao-Xiao Huang,†,‡ Ming Bai,†,‡ Xiao-Bing Chang,§ Xin-Jia Yan,†,‡ Tao Zhu,†,‡ Wei Zhao,†,‡ Ying Peng,# and Shao-Jiang Song*,†,‡ †

School of Traditional Chinese Materia Medica, ‡Key Laboratory of Structure-Based Drug Design and Discovery (Ministry of Education), and #School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China § Quality Research Department, ABA Chemicals Corporation, Shanghai 201203, People’s Republic of China S Supporting Information *

ABSTRACT: Prunus tomentosa seeds were researched for antioxidant and anti-inflammatory constituents. By activity-guided fractionation of P. tomentosa seed extract, six new dihydrobenzofuran neolignans, prunustosanans AI-IV (1−4) and prunustosanansides AI and AII (5 and 6), together with 10 known compounds (7−16) were isolated from bioactive fraction. The structures were determined by spectroscopic analyses, especially NMR, HRESIMS, and CD spectra. The antioxidant activity was greatest for 5, 10, and 12 against DPPH radical and for 8, 9, and 13 against ABTS radical. Moreover, compounds 7 and 11 exhibited much stronger inhibitory activity on nitric oxide (NO) production in murine microglia BV-2 compared with positive control minocycline (IC50 = 19.7 ± 1.5 μM). The results show that P. tomentosa seeds can be regarded as a potential source of antioxidants and inflammation inhibitors. KEYWORDS: Prunus tomentosa, food byproduct, seeds, neolignans, antioxidant, anti-inflammatory

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INTRODUCTION Inflammation is an important physical response to infection, injury, and irritation. At the injury site, an increase in the permeability of the blood vessel wall is followed by migration of immune cells, which can lead to edema during inflammation. However, excessive inflammation can trigger several acute and chronic diseases including atherosclerosis, obesity, metabolic syndrome, diabetes,1 neurodegenerative diseases,2 and even several types of cancers.3 During inflammation, large amounts of reactive oxygen species (ROS) are released to kill or destroy invading microorganisms or to degrade damaged tissues. Recent studies have produced substantial evidence that ROS are intimately involved in the pathogenesis of inflammatory processes.4 There is a link between antioxidant-scavenging of ROS and anti-inflammatory effects, and antioxidants play an important role in the treatment of inflammatory diseases.5 Several studies have established that fruits and their byproducts (peels, rinds, seeds) have potent antioxidant and antiinflammatory activities.6−12 Recently, an increasing number of researchers have become interested in fruits and their byproducts, and many novel antioxidant and anti-inflammatory agents have been discovered. Prunus tomentosa (commonly called Nanking cherry) belongs to the Prunus subgenus Lithocerasus in the family Rosaceae13 and is widely distributed in China, Japan, and Korea. The dried roots, fruits, and leaves of P. tomentosa have been used in traditional Chinese medicine to expel wind, drain dampness, treat frostbite, and alleviate pain.14,15 The fruit is usually eaten fresh or in the form of processed foods such as juice, jam, and wine.16,17 During the process of manufacturing P. tomentosa drinks, significant quantities of byproducts are generated, such as stems, peels, and seeds. Most of them are currently discarded © 2014 American Chemical Society

as industrial solid waste or underutilized as animal feed or fertilizer. Thus, their potential use is attracting more attention because of their considerable economic benefits and due to concern about protecting the environment. Among these byproducts, P. tomentosa seeds have anti-inflammatory and antioxidant activities.18,19 Therefore, the use of P. tomentosa seeds as a potential source of novel antioxidant and antiinflammatory agents has been an ongoing project in our laboratory. The bioassay-guided fractionation of P. tomentosa seeds extract led to the isolation of 6 new neolignans (1−6) and 10 known neolignans (7−16). In this paper, the structural determination of the new compounds and the antioxidant and anti-inflammatory activities of all 16 compounds are described. The results obtained in this study will help increase our knowledge about the bioactive components of P. tomentosa seeds and help in the use of P. tomentosa seeds to improve human health.

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MATERIALS AND METHODS

Chemicals and Reagents. Silica gel (200−300 mesh, Qingdao Marine Chemistry Ltd., Qingdao, China), polyamide (80−120 mesh, Qingdao Marine Chemistry Ltd.), and Cosmosil octadecyl silane (ODS) (40−80 mm, Nacalai Tosoh, Inc., Uetikon, Switzerland) were used for column chromatography (CC). TLC was conducted on silica gel GF254 (Qingdao Marine Chemistry Ltd.). Minocycline (mino), lipopolysaccharide (LPS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 6hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox), 2,2′azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), and 3-(4,5Received: Revised: Accepted: Published: 7796

May 8, 2014 July 8, 2014 July 14, 2014 July 14, 2014 dx.doi.org/10.1021/jf502171z | J. Agric. Food Chem. 2014, 62, 7796−7803

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Table 1. 1H and 13C NMR Data for 1−3 (CD3OD) 1a position

δC

1 2 3 4 5 6 7 8 9

132.4 109.2 147.8 146.5 114.8 118.4 89.0 53.2 63.1

1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′

129.1 112.9 144.2 153.0 131.3 118.7 198.2 40.5 57.5

3-OCH3 3′-OCHH3

2b

55.0 55.3

δH 6.96 (1H, d, 1.6)

6.79 6.85 5.66 3.61 3.88

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

d, 8.0) dd, 8.0, 1.6) d, 6.4) m) m)

7.57 (1H, br s)

7.66 (1H, br s) 3.21 (1H, t, 6.0) 3.97 (2H, t, 6.0) 3.85 (3H, s) 3.93 (3H, s)

3b

δC

δH

132.4 109.3 147.8 146.5 114.9 118.4 89.0 53.1 63.1

δC

6.97 (1H, d, 1.8)

6.80 6.86 5.67 3.60 3.90 3.86

128.7 112.9 144.4 153.4 129.3 119.0 198.2 74.2 64.8

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

d, 8.1) dd, 8.1, 1.8) d, 6.4) m) m) m)

7.59 (1H, br s)

7.67 (1H, br s) 5.15 3.93 3.79 3.84 3.93

55.0 55.4

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

t, 5.2) m) m) s) s)

132.4 109.2 147.8 146.5 114.9 118.4 89.1 53.1 63.1 128.7 112.9 144.2 153.4 129.3 119.0 198.2 74.2 64.8 55.0 55.4

δH 6.97 (1H, d, 1.8)

6.79 6.85 5.66 3.61 3.90 3.86

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

d, 1.8) dd, 8.1, 1.8) d, 6.4) m) m) m)

7.59 (1H, br s)

7.67 (1H, br s) 5.14 3.92 3.79 3.83 3.93

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

t, 5.2) m) m) s) s)

a

NMR spectroscopic data were recorded at 400 MHz (1H NMR) and 100 MHz (13C NMR). bNMR spectroscopic data were recorded at 300 MHz ( H NMR) and 75 MHz (13C NMR). 1

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell culture medium [Dulbecco’s modified Eagle’s medium (DMEM)], penicillin/ streptomycin, fetal bovine serum (FBS), and all other materials required for culturing cells were purchased from Gibco BRL, Life Technologies (Grand Island, NY, USA). Apparatus. Optical rotations were measured on a JASCO P-1020 polarimeter (Jasco Co., Tokyo, Japan). The UV spectra were measured on a Shimadzu UV-1700 spectrometer. The CD spectra were obtained using an MOS 450 detector from BioLogic. The FT-IR spectra were recorded using a Bruker IFS-55 spectrometer. HRESIMS experiments were performed on a MicroTOF spectrometer (Bruker Co., Karlsruhe, Germany). 1D NMR (1H and 13C) spectra were recorded on Bruker ARX-300, Bruker DPX-400, and Bruker AV-600 instruments (Bruker Co., Billerica, MA, USA); 2D NMR (HMBC, HSQC, and NOESY) spectra were recorded on Bruker DPX-400 and Bruker AV-600 instruments. High-performance liquid chromatography (HPLC) preparation was performed on a Waters 1525 instrument (UV 2549 detector) using a YMC C18 column (250 mm × 10 mm, 5 μm). GC was carried out using an Agilent 7890A gas chromatograph (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with an HP-5 capillary column (30 m × 320 mm × 0.25 μm). Plant Material. The seeds of P. tomentosa were collected from Liaoning province of China on July 18, 2012. The plant material was identified by Prof. Jin-Cai Lu (Department of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University). A voucher specimen (no. 20120718) has been deposited in the Herbarium of Shenyang Pharmaceutical University, Liaoning, People’s Republic of China. Extraction and Isolation. The dried and powdered seeds of P. tomentosa (15 kg) were refluxed three times 70% EtOH. Then, the aqueous alcohol solution was evaporated under reduced pressure to obtain the EtOH extract (650 g). This extract was suspended in H2O and partitioned successively with petroleum ether, EtOAc, and nBuOH. All fractions were tested in antioxidant (scavenging activities on DPPH and ABTS•+) and anti-inflammatory assays, and the ethyl acetate extract was found to exhibit the strongest activities of all these fractions. The ethyl acetate fraction (70 g) was subjected to a

polyamide CC and eluted successively with MeOH/H2O (30:70, 50:50, 70:30, 100:0, v/v) to yield four fractions (fractions 1−4). Fraction 1 (15 g) was separated on an ODS CC (4.0 × 50.0 cm) by gradient elution with MeOH/H2O (30:80−100:0, v/v) to give four fractions (fractions 1-1−1-4). Fraction 1-1 (4 g) was subjected to a silica gel CC (2.5 × 50.0 cm) and eluted using a gradient of CH2Cl2/ MeOH (40:1−3:1) to give five fractions (fractions 1-1-1−1-1-5). Fraction 1-1-4 was isolated by preparative HPLC using CH3CN/H2O (20:80, v/v; 8 mL/min) to afford 6 (17.4 mg, tR = 15.8 min), 5 (18.5 mg, tR = 18.1 min), 15 (70.4 mg, tR = 25.6 min), and 16 (30.9 mg, tR = 27.4 min). Fraction 1-2 (5 g) was loaded on a silica gel column CC (2.5 × 50.0 cm) using CH2Cl2/MeOH (50:1−5:1, v/v) as eluant to give six fractions (fractions 1-2-1−1-2-6). Fraction 1-2-2 was chromatographed on a Sephadex LH-20 column eluting with MeOH/H2O (1:1) and then purified by semipreparative HPLC using MeOH/H2O (34:66, v/v; 2.5 mL/min) to obtain 14 (13.3 mg, tR = 15.7 min), 2 (9.3 mg, tR = 22.5 min), 3 (8.9 mg, tR = 22.7 min), 12 (14.1 mg, tR = 24.7 min), and 13 (9.8 mg, tR = 27.0 min). Fraction 1-2-3 was separated on an ODS CC (4.0 × 50.0 cm) with MeOH/ H2O (10:90−100:0, v/v) to give three fractions (fractions 1-2-3-A−12-3-C). Fraction 1-2-3-A was further isolated by semipreparative HPLC using MeOH/H2O (40:60, v/v; 3.0 mL/min) to obtain 4 (12.7 mg, tR = 23.0 min), 7 (25.2 mg, tR = 25.7 min), 1 (18.3 mg, tR = 29.2 min), and 11 (14.3 mg, tR = 31.7 min). Fraction 1-2-3-B was purified by semipreparative HPLC using CH3CN/H2O (25:75, v/v; 3.5 mL/ min) to obtain 10 (50.5 mg, tR = 19.3 min), 8 (24.9 mg, tR = 30.1 min), and 9 (36.4 mg, tR = 30.2 min). Prunustosanan AI (1): colorless oil; [α]20 D −16.5 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 228 (0.53), 287 (0.30); CD [MeOH, nm (Δε)] 223 (−3.43), 281 (−2.61); IR (KBr) Vmax 3451, 1641, 1600, 1510, 1464, 1384, 1326, 1272, 1162, 1119, 1032 cm−1; HRESIMS at m/z 397.1257 [M + Na]+ (calcd for C20H22O7Na, 397.1263); 1H and 13 C NMR, see Table 1. Prunustosanan AII (2): colorless oil; [α]20 D −19.7 (c 0.17, MeOH); UV (MeOH) λmax (log ε) 232 (0.23), 287 (0.14); CD [MeOH, nm (Δε)] 224 (−4.73), 282 (−1.31); [Mo2(OAc)4]-induced CD [DMSO, nm (Δε)] 310 (−0.94); IR (KBr) Vmax 3441, 1641, 1600, 1516, 1464, 1430, 1384, 1329, 1125, 1034 cm−1; HRESIMS at m/z 413.1218 [M + 7797

dx.doi.org/10.1021/jf502171z | J. Agric. Food Chem. 2014, 62, 7796−7803

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Table 2. 1H and 13C NMR Data for 4−6 (CD3OD) 4a position 1 2 3 4 5 6 7 8 9 2‴ 1‴,3‴ 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 3-OCHH3 5-OCHH3 3′-OCHH3 Glc-1″ Glc-2″ Glc-3″ Glc-4″ Glc-5″ Glc-6″

5b

δC 133.4 109.2 147.7 146.1 114.7 118.3 87.5 54.1 63.3

134.1 112.8 143.8 146.8 128.9 116.3 50.6 63.6 63.7 55.0 55.4

δH 6.96 (1H, d, 2.0)

6.78 6.84 5.54 3.50 3.85

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

d, 8.0) dd, 8.0, 2.0) d, 6.4) m) m)

6.80 (1H, br s)

6.80 2.93 3.77 3.86 3.85

(1H, (1H, (1H, (2H, (3H,

br s) m) m) m) s)

3.89 (3H, s)

δC

6b δH

132.7 102.9 147.9 135.0 147.9 102.9 87.8 52.0 71.1

δC

6.72 (1H, br s)

6.72 5.61 3.65 4.23 3.76

135.6 112.8 143.8 146.1 127.7 116.8 31.5 34.4 60.8 55.3 55.3 55.4 103.2 73.8 76.9 70.1 76.7 61.4

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

br s) d, 6.0) m) dd, 10.8, 6.0) m)

6.73 (1H, br s)

6.78 2.62 1.81 3.55 3.81 3.81 3.86 4.39 3.23 3.35 3.28 3.29 3.87 3.67

(1H, (2H, (2H, (2H, (3H, (3H, (3H, (1H, (1H, (1H, (1H, (1H, (1H, (1H,

br s) t, 7.8) m) t, 6.6) s) s) s) d, 7.8) m) m) oc) oc) m) m)

138.4 102.6 153.3 134.8 153.3 102.6 87.4 52.2 71.2 83.4 60.7 135.8 112.9 143.9 146.0 127.7 116.8 31.5 34.4 60.8 55.4 55.4 55.5 103.2 73.8 76.9 70.2 76.7 61.4

δH 6.80 (1H, br s)

6.80 5.68 3.65 4.27 3.75 4.02 3.74

(1H, (1H, (1H, (1H, (1H, (1H, (4H,

br s) d, 6.0) m) dd, 9.6, 6.0) m) t, 4.8) dd, 4.8, 1.2)

6.74 (1H, br s)

6.75 2.62 1.81 3.55 3.82 3.82 3.87 4.39 3.22 3.35 3.28 3.28 3.84 3.65

(1H, (2H, (2H, (2H, (3H, (3H, (3H, (1H, (1H, (1H, (1H, (1H, (1H, (1H,

br s) t, 7.8) m) t, 6.6) s) s) s) d, 7.8) m) m) oc ) m) m) m)

a NMR spectroscopic data were recorded at 400 MHz (1H NMR) and 100 MHz (13C NMR); bNMR spectroscopic data were recorded at 600 MHz (1H NMR) and 150 MHz (13C NMR); c“o” refers to overlapped signals.

Na]+ (calcd for C20H22O7Na, 413.1207); 1H and 13C NMR, see Table 1. Prunustosanan AIII (3): colorless oil; [α]20 D −6.3 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 227 (0.32), 285 (0.18); CD [MeOH, nm (Δε)] 223 (−5.15), 287 (−2.54); [Mo2(OAc)4]-induced CD [DMSO, nm (Δε)] 309 (+1.48); IR (KBr) Vmax 3442, 1641, 1512, 1463, 1384, 1327, 1268, 1131, 1031 cm−1; HRESIMS at m/z 413.1206 [M + Na]+ (calcd for C20H22O7Na, 413.1207); 1H and 13C NMR, see Table 1. Prunustosanan AIV (4): colorless oil; [α]20 D −14.1 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 231 (1.24), 280 (0.34); CD [MeOH, nm (Δε)] 228 (−4.23), 281 (−3.18); [Mo2(OAc)4]-induced CD [DMSO, nm (Δε)] 310 (−0.39); IR (KBr) Vmax: 3396, 1605, 1513, 1464, 1424, 1384, 1268, 1157, 1133, 1030 cm−1; HRESIMS at m/z 399.1431 [M +Na]+ (calcd for C20H24O7Na, 399.1414); 1H and 13C NMR, see Table 2. Prunustosananside AI (5): colorless oil; [α]20 D −7.5 (c 0.19, MeOH); UV (MeOH) λmax (log ε) 230 (1.54), 281 (0.57); CD [MeOH, nm (Δε)] 226 (−0.96), 274 (0.83); IR (KBr) Vmax 3419, 1606, 1515, 1499, 1463, 1384, 1327, 1272, 1211, 1126, 1075, 1032 cm−1; HRESIMS at m/z 575.2089 [M +Na]+ (calcd for C27H36O12Na, 575.2099); 1H and 13C NMR, see Table 2. Prunustosananside AII (6): colorless oil; [α]20 D −19.3 (c 0.21, MeOH); UV (MeOH) λmax (log ε) 231 (0.48), 279 (0.18); CD [MeOH, nm (Δε)] 230 (−3.48), 283 (−2.57); IR (KBr) Vmax 3406, 1605, 1515, 1499, 1463, 1430, 1384, 1327, 1272, 1211, 1125, 1074, 1032 cm−1; HRESIMS at m/z 649.2464 [M +Na]+ (calcd for C20H22O7Na, 649.2467); 1H and 13C NMR, see Table 2.

Acid Hydrolysis and Derivatization of Compounds 5 and 6. Each compound (4.0 mg) was hydrolyzed with 2 M HCl (5.0 mL), heated for 4 h at 95 °C, and extracted with EtOAc (3 × 4.0 mL). Then, the aqueous layer was concentrated in vacuo to appropriate volume, the solution was examined by TLC (EtOAc/BuOH/H2O/ HOAc, 4:4:1:1) and compared with authentic samples, and glucose was detected. The remaining aqueous layer was evaporated to dryness to give a residue, which was dissolved in pyridine (1.0 mL), and then Lcysteine methyl ester hydrochloride (2.0 mg) was added to the solution. The mixture was heated at 60 °C for 2 h, and 0.5 mL of tremethylsilyimidazole (TMSI) was added, followed by heating at 60 °C for 2 h. The reaction product was subjected to GC analysis with flame ionization detection. The column temperature was raised from 120 to 280 °C at the rate of 8 °C/min, and the carrier gas was N2 (1.4 mL/min); the injection temperature was 250 °C and the injection volume, 1 mL. The absolute configuration of the monosaccharide were confirmed to be D-glucose by comparison of the retention times of its Me3Si ethers with that of standard sample (tR = 20.1 min). DPPH Radical Scavenging Assay. The DPPH radical scavenging activity of samples was measured using a published method20 with some modifications. The samples with different concentrations (0.1− 100 μM) in EtOH (100 μL) were added to 0.2 mM DPPH in EtOH (150 μL). The mixture was shaken vigorously and then immediately incubated in darkness for 1 h. The absorbance of the reaction solution was determined in a Varioskan Flash (Thermo Scientific, Waltham, MA, USA) at 515 nm. Trolox, a stable antioxidant, was used as a positive reference. The DPPH radical scavenging activity in terms of the percentage of sample was calculated as follows: DPPH scavenging 7798

dx.doi.org/10.1021/jf502171z | J. Agric. Food Chem. 2014, 62, 7796−7803

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Figure 1. Structures of compounds 1−16. activity (%) = [1 − (S − SB)/(C − CB)] × 100%, where S, SB, C, and CB are the absorption of the sample, the blank sample, the control, and the blank control, respectively. Tests were performed in triplicate. ABTS Radical Scavenging Activity Assay. The total antioxidant capacity was evaluated using an improved ABTS radical cation (ABTS•+) decolorization assay with some modifications.21 ABTS•+ was produced by reacting 7 mM stock solution of ABTS•+ with 2.45 mM potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 16 h before use. The ABTS•+ solution was diluted with ethanol, to an absorbance of 0.7 ± 0.02 at 734 nm. An ethanolic solution (100 μL) of different concentrations of samples (0.1−100 μM) or trolox standard at various concentrations was mixed with 150 μL of diluted ABTS•+ solution. After reaction at room temperature for 10 min, the absorbance was measured at 734 nm using a Varioskan Flash (Thermo Scientific). The ABTS•+ scavenging ability was calculated using the formula ABTS•+ scavenging activity (%) = [1 − (S − SB)/(C − CB)] × 100, where S, SB, C, and CB are the absorption of the sample, the blank sample, the control, and the blank control, respectively. Tests were performed in triplicate. Cell Culture and Viability Assay. BV-2 cells, a mouse microglia cell line, were cultured in DMEM containing 5% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin. BV-2 cells were plated at a density of 4 × 105/mL and maintained at 37 °C in a humidified atmosphere containing 5% CO2. MTT assays were used to determine cell viability. Briefly, BV-2 cells were treated with the various concentrations of neolignan samples (0.1−100 μM) for 1 h and then stimulated with LPS (100 ng/mL) for 24 h. Then, the cells were incubated with a solution of 0.25 mg/mL MTT followed by

incubation for 4 h at 37 °C and 5% CO2. Supernatant was removed, and the formation of formazan was observed by monitoring the signal at 540 nm using a Varioskan Flash (Thermo Scientific). NO-Releasing Assay. NO produced by the cells was determined by assaying the levels of NO2− using the Griess reagent. BV-2 cells were preincubated in 96-well plates at a density of 4 × 105 cells/well overnight and were then treated with different concentrations of the isolated compounds (0.1−100 μM) for 30 min. After 100 ng/mL LPS stimulation for 24 h, 100 μL of culture supernatant was mixed with the same volume of Griess reagent (1% sulfanilamide/0.1% N-(1naphthyl)ethylenediamine dihydrocholoride/2.5% phosphoric acid). The absorbance was recorded at 540 nm on a Varioskan Flash (Thermo Scientific). NO concentration was calculated with reference to a standard curve of sodium nitrite produced using known concentrations.22

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RESULTS AND DISCUSSION Compound 1 was obtained as a colorless oil. The HRESIMS at m/z 397.1257 [M + Na]+ indicated the molecular formula of C20H22O7. The UV spectrum showed absorbances at λmax 228 and 287 nm, and the IR spectral data indicated the presence of OH (3451 cm−1), CO (1641 cm−1), and aromatic rings (1600, 1510, and 1464 cm−1).23 The 1H NMR spectrum of 1 showed the presence of a 1,3,4-trisubstituted benzene ring [δ 6.96 (1H, d, J = 1.6 Hz), 6.85 (1H, dd, J = 8.0, 1.6 Hz), and 6.79 (1H, J = 8.0 Hz)], a 1,3,4,5-tetrasubstituted benzene ring [δ 7.66 (1H, br s) and 7.57 (1H, br s)], and a sequence of methine−methine− methylene [CH(O)−CH(Ph)−CH2O] successively coupled in 7799

dx.doi.org/10.1021/jf502171z | J. Agric. Food Chem. 2014, 62, 7796−7803

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Figure 2. Key HMBC and NOESY correlations of compounds 1−6.

2.27 Additionaly, the absolute configuration of the vicinal diol (8′-OH and 9′-OH) moiety in 2 was determined by the induced CD spectra of its in situ complex with Mo2(OAc)4 in DMSO solution (Snatzke’s method).28,29 On the basis of the empirical helicity rule relating the sign of the Cotton effect of the diagnostic O−C−C−O moiety, the negative Cotton effect at 310 nm indicated an 8′R configuration. Accordingly, the structure of 2 was identified as (7R,8S,8′R)-3,3′,5-trimethoxy4′,7-epoxy-8,5′-neolignan-7′-one-4,9,8′,9′-tetraol and named prunustosanan AII. Compound 3 was proposed to have the molecular formula C20H22O8 on the basis of HRESIMS. The IR and NMR spectroscopic data were in good agreement with those of 2 (Table 1), suggesting that the planar structure and relative configuration of 3 was the same as that of compound 2. The CD spectrum showed a negative Cotton effect (Δε287 = −2.54), which indicated the absolute configurations of 3 to be 7R,8S configuration.27 The 8′S configuration was supported by a positive Cotton effect at 310 nm in the Mo2(OAc)4-induced CD spectrum of 3.28,29 Thus, compound 3 was defined as an optical isomer of 2 and assigned as (7R,8S,8′S)-3,3′,5trimethoxy-4′,7-epoxy-8,5′-neolignan-7′-one-4,9,8′,9′-tetraol, named prunustosanan AIII. Compound 4 gave the positive HRESIMS m/z 399.1431 [M + Na]+ (calcd for C20H24O7Na, 399.1414), consistent with the molecular formula C20H24O7. The 13C NMR spectral data of 4 were very similar to those of 3, except for the reduction of a carbonyl group (δC 198.2) on C-7′ in 4. This difference was supported by the HMBC correlations (Figure 2) of H-7′/C-1′, C-2′, C-6′, and C-8′. The relative configurations of compound 4 were defined by the J7,8 value (6.4 Hz) and NOESY spectrum (Figure 2).26,27 The positive Cotton effect at 281 nm in the CD spectrum of 4 justified a 7R,8S configuration27 as shown in Figure 1. In addition, the configuration of 8′-OH was confirmed

this order [5.66 (1H, d, J = 6.4 Hz), 3.61 (1H, m) and 3.88 (2H, m)] in addition to two methoxyl groups at δ 3.93 (3H, s) and 3.85 (3H, s). The 13C NMR spectrum of 1 (Table 1) showed 20 carbon signals including 2 methoxy groups, 12 aromatic units, a carbonyl carbon, and 5 aliphatic carbons. Careful comparison of the NMR data of 1 (Table 1) with those of known neolignans24,25 revealed that compound 1 is a dihydrobenzofuran neolignan with oxidation of C-7′, which was confirmed by HMBC correlations (Figure 2) of H-8′ at δ 3.21 with C-7′, C-9′ and of H-9′ at δ 3.97 with C-7′ and C-9′. The relative stereochemistry of H-7 and H-8 was established as a trans relationship based on the J7,8 coupling constant (6.4 Hz)26 and NOESY cross peaks (Figure 2) H-7/H2-9, H-8/H-2, and H-8/H-6.27 As a negative Cotton effect at 281 nm (Δε = −2.61) was observed, 7R and 8S configurations were indicated.27 On the basis of the above evidence, the structure of 1 was established as (7R,8S)-3,3′,5-trimethoxy-4′,7-epoxy8,5′-neolignan-7′-one-4,9,9′-triol and named prunustosanan AI. Compound 2 was also obtained as a colorless oil, with C20H22O8 as its molecular formula, indicated by HRESIMS at m/z 413.1218 [M + Na]+. The IR spectroscopic data of 2 showed absorptions for hydroxy (3441 cm−1), carbonyl (1641 cm−1), and aromatic groups (1600, 1516, and 1464 cm−1).23 Detailed comparison of 13C NMR spectral data of 2 with those of 1 (Table 1) revealed signals at δC 40.5 and 57.5, moving downfield by δC 33.7 and 7.3, which were assigned to C-8′ and C-9′, respectively, indicating that one proton of H-8′ was substituted by a hydroxyl group. HMBC correlations (Figure 2) confirmed the structure and allowed the unambiguous assignment of its NMR data. The coupling constant J7,8 (6.4 Hz) in the 1H NMR of 2, as well as the NOESY correlations (Figure 2) of H-7/H2-9, H-8/H-2, and H-8/H-6, suggested a relative trans configuration.26,27 In the CD spectrum, a negative Cotton effect at 282 nm indicated the 7R,8S configuration for 7800

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(2α,3β)-7-O-methylcedrusin (9),24 (+)-7R,8S-5-methoxydihydrodehydroconiferyl alcohol (10),36 ficusal (11),23 phenylcoumaran-α′-aldehyde (12),37 2-(4-hydroxy-3-methoxyphenyl)-3-methyl-5-hydroxymethyl-7-methoxycoumaran (13),38 sakuraresinol (14),39 dihydrodehydrodiconiferyl alcohol 9-O-β-Dglucopyranoside (15),40 and dihydrodehydrodiconiferyl alcohol 9′-O-β-D-glucopyranoside (16)40 by analyses of their 1H and 13 C NMR and ESI-MS data in combination with the comparison of their data with the reported values. Neolignans are a class of secondary metabolites produced by oxidative dimerization of two phenylpropanoid units. Neolignans exhibit a wide range of pharmacological effects, particularly antioxidant and anti-inflammatory activities.41−43 The antioxidant activities of 1−16 were evaluated by their ability to scavenge DPPH and ABTS radicals (Table 3).

using Snatzke’s method: the negative Cotton effect at 310 nm in the induced CD spectrum of the complex of 4 with Mo2(OAc)4 indicated an 8′R configuration.28,29 Consequently, the structure of 4 was undoubtedly elucidated as (7R,8S,8′R)3,3′,5-trimethoxy-4′,7-epoxy-8,5′-neolignan-4,9,8′,9′-tetraol, named prunustosanan AIV. The molecular formula of 5 was determined as C27H36O12 by the [M + Na]+ ion peak at m/z 575.2089 in the HRESIMS. The IR spectrum of 5 showed absorption for hydroxyl (3419 cm−1), aromatic (1606 and 1515 cm−1), and glycosidic (1075 and 1032 cm−1) functions.23,30 The NMR data of 5 (Table 2) were very similar to those of a known neolignan glucoside.31 The anomeric proton signal at δH 5.62 (1H, d, J = 7.0 Hz, H1″) in the 1H NMR spectrum and six sugar carbon signals at δC 103.2 (C-1″), 73.8 (C-2″), 76.9 (C-3″), 70.1 (C-4″), 76.7 (C5″), and 61.4 (C-6″) demonstrated that the sugar was β-Dglucose.32,33 Furthermore, acid hydrolysis of 5 gave D-glucose. The position of the glucoside linkage was confirmed at C-9 by HMBC experiments (Figure 2), which showed long-range correlations between the 1″-proton and 9-carbon. The relative configuration of compound 5 was suggested to be relative trans according to the J7,8 value of 6.0 Hz. This was verified by NOE correlations (Figure 2) of H-7/H2-9 and H-8/H-6 and H-8/H2 in the NOESY spectrum of 5.26,27 In addition, the CD spectrum of 5 showed a positive Cotton effect at 274 nm (Δε = 0.83), indicating that the absolute configuration of 5 was the (7S,8R) configuration.31 Therefore, 5 was proposed to be (7S,8R)-3,3′,5-trimethoxy-4′,7-epoxy-8,5′-neolignan-7′-one4,9,9′-triol 4-O-β-D-glucopyranoside, a neolignan glucoside prunustosananside AI. Compound 6 showed the [M + Na]+ ion peak at m/z 649.2464 in the positive-ion mode HRESIMS, indicating the molecular formula C30H42O14. The IR spectrum of 6 showed the presence of aromatic (1605 and 1515 cm−1) and glycosidic groups (1074 and 1032 cm−1).23,30 Comparison of its NMR data with those of 5 (Table 2) disclosed their extreme similarity. The main difference observed in the 13C NMR spectrum of 6 was due to the presence of a 1,2,3-propanetriol moiety [δC 83.4 (C-2‴) and 60.7 (C-1‴and C-3‴)], which was confirmed by HMBC correlations (Figure 2) from H-2‴ to C4, C-1‴, and C-3‴. Acid hydrolysis of 6 afforded glucose. Combined with its 1H and 13C NMR data, the configurations of the anomeric carbons of sugars were determined to be β-D for glucose.32,33 The absolute configuration of glucose was further determined by acid hydrolysis and comparison with an authentic sample. The position of the glucoside linkage was confirmed at C-9 by HMBC experiments (Figure 2), which showed long-range correlations between the 1″-proton and 9carbon. The trans relationship between H-7 and H-8 was inferred from their coupling constant (J7,8 = 6.0 Hz), which was further verified by the NOE correlations (Figure 2) between H7 and H2-9 and between H-8 and H-2,6.26,27 The CD spectrum showed a negative Cotton effect (283 nm, Δε = −2.57), which suggested a 7R,8S configuration.27 According to the accumulated evidence above, the structure of 6 was designated (7R,8S)-3,3′,5-trimethoxy-4-(1‴,3‴-dihydroxy-2‴-propyloxyl)4′,7-epoxy-8,5′-neolignan-7′-one-4,9,9′-triol 4-O-β-D-glucopyranoside and had been accorded the trivial name prunustosananside AII. The other known compounds were identified as (E)-3[(2R,3S)-2,3-dihydro-3-hydroxymethyl-7-methoxy-2-(4′-hydroxy-3′-methoxyphenyl)-1-benzo[b]furan-5-yl]-2-propen-1-ol (7),34 (−)-7R,8S-dihydrodehydroconiferyl alcohol (8),35 rel-

Table 3. Antioxidant and Anti-inflammatory Activities of Compounds 1−16 compd

DPPH (IC50, μM)a

ABTS (IC50, μM)a

anti-inflammatory activity (IC50, μM)a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 trolox mino

>100 >100 >100 >100 42.6 ± 1.8 >100 >100 >100 >100 57.0 ± 2.3 >100 59.5 ± 3.5 >100 >100 >100 >100 39.2 ± 1.2 ndb

47.1 ± 2.7 46.4 ± 3.1 48.4 ± 4.1 57.1 ± 2.9 74.5 ± 3.9 >100 40.4 ± 1.8 33.7 ± 2.6 31.5 ± 2.3 43.0 ± 3.8 45.9 ± 2.8 56.2 ± 2.3 28.1 ± 1.1 >100 67.5 ± 5.9 51.4 ± 3.1 38.4 ± 0.8 ndb

>100 >100 >100 58.7 ± 3.0 34.7 ± 2.1 >100 4.4 ± 0.5 50.3 ± 2.6 48.4 ± 1.7 60.1 ± 4.9 4.8 ± 0.7 26.4 ± 1.4 44.3 ± 1.9 >100 >100 >100 ndb 19.7 ± 1.5

IC50 values represent the means ± SD of three parallel measurements. bnd, not determined.

a

Compounds 5, 10, and 12 showed moderate DPPH radical scavenging activities with IC50 values of 42.6 ± 1.8, 57.0 ± 2.3, and 59.5 ± 3.5 μM, respectively, whereas the other compounds (IC50 > 100 μM) did not display significant DPPH radical scavenging activity (Table 3). These results are comparable to those of previously isolated neolignan in other studies.44,45 Zhao et al. reported that two new neolignan glycosides (IC50 > 100 μM) from Pittosporum glabratum were inactive in the DPPH radical scavenging assay.46 A comparison of the structures of 5, 10, and 12 with those of 15, 8, and 11 indicated that additional methoxy groups at C-5 positions appeared to increase the antioxidant activity of the dihydrobenzofuran neolignans. In the ABTS assay, most of the isolated compounds showed significant antioxidant activity, with compounds 8 (IC50 = 33.7 ± 2.6 μM), 9 (IC50 = 31.5 ± 2.3 μM), and 13 (IC50 = 28.1 ± 1.1 μM) being the most potent, whereas compounds 1−4, 7, 10−12, 15, and 16 also showed moderate ABTS radical scavenging activities, comparable with that of the standard compound trolox with an IC50 value of 38.4 ± 0.8 μM (Table 3). Comparison of the structure of 6 with that of 5 or the structure of 10 with that of 14 showed 7801

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Notes

that the antioxidant activity declined sharply when a hydroxyl group on the benzene ring was replaced by a 1,2,3-propanetriol group. This fact indicated that the free phenolic hydroxyl was crucial for the antioxidant activity, which was consistent with previous observations.47,48 In this investigation, we further evaluated the antiinflammatory activity of the isolated compounds 1−16 using exposure to LPS-induced NO production in murine microglia BV-2 cell line. NO is a gaseous signaling molecule that plays key roles in immune and inflammatory responses and neuronal transmission in the brain.49 Under normal conditions, NO has neuroprotective and antioxidant effects. However, excess production of NO from activated microglia causes a number of neurodegenerative diseases.50 Inhibitors of NO production can be considered as potential anti-inflammatory agents. As shown in Table 3, compounds 4, 5, and 7−13 reduced NO levels in LPS-stimulated BV-2. In particular, the activities of 7 and 11 were more potent than that of the positive control, mino (IC50 = 19.7 ± 1.5 μM), in inhibiting NO production with IC50 values of 4.4 ± 0.5 and 4.8 ± 0.7 μM, respectively. The observed NO inhibitory activities appear to be somewhat correlated to their structures. For example, with regard to the results for compounds 8, 9 and 11−13, it appeared that shortening of the side chain might be important for the higher activity. Interestingly, comparing the structures and inhibitory activities of compound 7 with those of compounds 8 and 9, it appeared that the double bond at C-7 may increase the inhibition of NO production. In addition, the inflammatory activity of one pair of isomers, 8 and 9, was similar, which led us to conclude that the absolute configuration of the compounds might have little or no inhibitory effect on NO production. The cytotoxicity of compounds 1−16 to BV-2 cells was measured by an MTT assay. The results obtained indicated that the compounds exerted no cytotoxic effect at concentrations up to 100 μM and that the cell viability was >95% (see Table S1 in the Supporting Information), suggesting that compounds 1−16 do not affect normal cell growth. In conclusion, this study reports the structures of 16 neolignans isolated from the active fraction of P. tomentosa seeds, along with their antioxidant and anti-inflammatory activities. Compounds 5, 10, and 12 showed moderate activity against DPPH, and compounds 8, 9, and 13 exhibited significant ABTS free radical scavenging activity (IC50 = 28.1−33.7 μM). Furthermore, compounds 7 and 11 exhibited remarkable NO inhibition with IC50 values of 4.4 ± 0.5 and 4.8 ± 0.7 μM, respectively, compared with the standard antiinflammatory drug, mino, at 19.7 ± 1.5 μM. The results obtained in our study provide a potential justification for the use of the seeds from P. tomentosa industrial byproducts as a valuable source of raw material for new antioxidant and antiinflammatory agents.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Wen Li and Yi Sha of Shenyang Pharmaceutical University for recording the NMR spectra. ABBREVIATIONS USED HPLC, high-performance liquid chromatography; GAS, gas chromatograph; NMR, nuclear magnetic resonance spectrometry; COSY, correlation spectroscopy; HSQC, heteronuclear single-quantum coherence; HMBC, heteronuclear multiplebond correlation; NOESY, nuclear Overhaser effect spectroscopy; ESI, electrospray ionization; MS, mass spectrometry; UV, ultraviolet spectrometry; CD, electronic circular dichroism; IR, infared absorption spectrum

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ASSOCIATED CONTENT

* Supporting Information S

UV, CD, IR, NMR, and HRESIMS data for compounds 1−6 and effect of compounds 1−16 on growth of BV-2 cells. This material is available free of charge via the Internet at http:// pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*(S.J.S.) Phone: +8621 23986510. Fax: +8621 23986510. Email: [email protected]. 7802

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